Alternative Therapies

ALTERNATIVE THERAPIES IN HEALTH AND MEDICINE

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A PEER-REVIEWED JOURNAL • SEPTEMBER/OCTOBER 2017 • VOL. 23, NO. 5 • $14.95 Electric Nutrition: Health and Healing Benefits of Biological Grounding • Use of Bioceramic Undershirt for Patients With Fibromyalgia • Efficacy of Fascial Distortion Model Treatment for Acute, Nonspecific Low-Back Pain • Antidiabetic, Antioxidant, and Hypolipidemic Potential of Sonchus asper Hill • Proapoptotic and Growth-inhibitory Effects of Plumbagin on Human Gastric Cancer Cells • An Overcome Gagging Through Acupressure for an Edentulous Patient • Commentary—Natural Cancer Therapy and Prevention Based on the Cytochrome P45O Enzyme CYP1B1

SEPTEMBER/OCTOBER 2017 Vol.23 No.4

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SEPTEMBER/OCTOBER 2017, VOL. 23, NO. 5

TABLE OF CONTENTS ORIGINAL RESEARCH 8

Electric Nutrition: The Surprising Health and Healing Benefits of Biological Grounding (Earthing)

Stephen T. Sinatra, MD, FACC; James L. Oschman, PhD; Gaétan Chevalier, PhD; Drew Sinatra, ND, LAc

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Reduction in Pain After Use of Bioceramic Undershirt for Patients With Fibromyalgia

María Aparecida Santos e Campos, PhD; Felipe García Pinillos, PhD; Pedro Ángel Latorre Román, PhD

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Efficacy of Fascial Distortion Model Treatment for Acute, Nonspecific Low-Back Pain in Primary Care: A Prospective Controlled Trial

Detlef Richter; Matthias Karst, MD; Hartmut Buhck, MD; Matthias G. Fink, MD

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Antidiabetic, Antioxidant, and Hypolipidemic Potential of Sonchus asper Hill

Rahmat Ali Khan, PhD

Proapoptotic and Growth-inhibitory Effects of Plumbagin on Human Gastric Cancer Cells Via Suppression of Signal Transducer and Activator of Transcription 3 and Protein Kinase B

Jing Li, PhD; Jia Li, PhD; Guowei Cai, PhD; Lin Shen; PhD; Furong Lu, PhD

PERSPECTIVES 50

Natural Cancer Therapy and Prevention Targeted on Cancer Cells and Cancer Stem Cells Based on the Cytochrome P45O Enzyme CYP1B1: A Commentary

William R. Ware, PhD

CASE REPORT 60

An Innovative Approach to Overcome Gagging Through Acupressure for an Edentulous Patient: A Case Report

Saravanan Thirumalai Thangarajan, MDS; Ramasamy Chidambaram, MDS; Padmanabhan Thallam Veeravalli, MDS; Kasim Mohamed, MDS

Table of Contents

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 3

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editor in chief Andrew W. Campbell, MD CONTRIBUTING EDITORS

Michael Balick, PhD • Mark Hyman, MD • Jeffrey Bland, PhD, FACN, FACB • Roberta Lee, MD • Tieraona Low Dog, MD Editorial Board Sidney MacDonald Baker, MD ◆ Autism Research Institute

Gerard Mullin, MD ◆ Johns Hopkins University

Mark Blumenthal ◆ American Botanical Council

John Neely, MD ◆ Pennsylvania State University

David M. Brady, ND, DC, CCN ◆ University of Bridgeport Kelly Brogan, MD, ABIHM ◆ George Washington University

Paula J. Nenn, MD, ABIHM ◆ Optimal Health and Prevention Research Foundation Garth L. Nicolson, PhD ◆ The Institute for Molecular Medicine

Jeffrey Dach, MD ◆ TrueMedMD, Davie, Florida

Xie Ning, PhD ◆ Heilongjiang University of Traditional Chinese Medicine

James Dillard, MD, DC, LAc ◆ Integrative Pain Medicine Jeanne Drisko, MD ◆ University of Kansas

Thomas O’Bryan, DC, CCN, DACBN ◆ Institute for Functional Medicine, National University of Health Sciences Dean Ornish, MD ◆ Preventive Medicine Research Institute

Andrea Girman, MD, MPH ◆ Genova Diagnostics

Nicole Pietschmann, PhD ◆ Berlin, Germany

Ajay Goel, PhD ◆ Baylor Research Institute

Joseph E. Pizzorno, ND ◆ Seattle, WA

Garry F. Gordon, MD, DO ◆ Gordon Research Institute

Lawrence A. Plumlee, MD ◆ Chemical Sensitivity Disorders Association

Junfeng He, PhD ◆ Hunan University of Traditional Chinese Medicine

Keith S. Rayburn, MD ◆ Dignity Health Medical Group – Dominican

Yuxin He, LAc, PhD ◆ Academy of Oriental Medicine at Austin

William J. Rea, MD ◆ Environmental Health Center – Dallas

Robert Hedaya, MD, DLFAPA ◆ National Center for Whole Psychiatry

Sandeep Saluja, MD ◆ Saran Ashram Hospital, Dayalbagh

Leila Jabbour, PhD ◆ Franklin Pierce University

Eric R. Secor Jr, PhD, ND, MPH, MS, LAc ◆ Helen & Harry Gray Cancer Center, Hartford Hospital Ellen Kamhi, PhD, RN, AHG, AHN-BC ◆ Stony Brook University Stephen T. Sinatra, MD, FACC, FACN, CNS ◆ University of Connecticut School of Medicine Anup Kanodia, MD, MPH ◆ Ohio State University Martha Stark, MD ◆ Harvard Medical School, Massachusetts Mental Health Center Datis Kharrazian, DC, DHSc, MS, MNeuroSci ◆ Harvard Medical School; Alex Vasquez, DC, ND, DO ◆ University of Texas Massachusetts General Hospital Günver Kienle, DrMed ◆ Institute for Applied Epistemology Aristo Vojdani, PhD, MSc, CLS ◆ Immunosciences Lab, Inc James B. Lago, EMT, DDS, BA ◆ Chicago Dental Health

Roeland van Wijk, PhD ◆ International Institute of Biophysics

Changzheng Li, PhD ◆ Department of Chinese Internal Medicine, Nanfang James M. Whedon, DC, MS ◆ Southern California University of Health Sciences Hospital, Southern Medical University, China Erqiang Li, PhD ◆ East West College of Natural Medicine Shi Xian, MD, PhD ◆ General Hospital of the Chinese People’s Liberation Army Holly Lucille, ND, RN ◆ Humility Inc Bill Manahan, MD ◆ University of Minnesota

Arthur Yin Fan, CMD, PhD, LAc ◆ McLean Center for Complementary and Alternative Medicine Qinhong Zhang, MD, PhD ◆ Stanford University

Pamela Miles, Reiki Master ◆ New York, NY

Shun Zhongren, PhD ◆ Heilongjiang University of Traditional Chinese Medicine

Daniel A. Monti, MD ◆ Thomas Jefferson University

Managing Editor, CRAIG GUSTAFSON • Creative Director, RANDY PALMER • Associate Editor, MICHAEL MILLER • Science Editor, PEGGY WRIGHT • E-mail: ATHM@innovisionhm.com Web: www.alternative-therapies.com

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REVIEW ARTICLE

Electric Nutrition: The Surprising Health and Healing Benefits of Biological Grounding (Earthing) Stephen T. Sinatra, MD, FACC; James L. Oschman, PhD; Gaétan Chevalier, PhD; Drew Sinatra, ND, LAc

ABSTRACT Context • Modern biomedicine has discovered that many of the most debilitating diseases, as well as the aging process itself, are caused by or associated with chronic inflammation and oxidative stress. Emerging research has revealed that direct physical contact with the surface of the planet generates a kind of electric nutrition, with surprisingly potent and rapid anti-inflammatory and antioxidant effects.

Objectives • The objective of this study was to explain the potential of grounding to clinicians as a simple strategy for prevention, therapy, and improving patient outcomes.

The research summarized here has pursued the goal of determining the physiological and clinical significance of biological grounding.

Design • The research team has summarized more than 12 peer-reviewed reports. Where appropriate, blinded

Stephen T. Sinatra, MD, FACC, is an assistant clinical professor of medicine at the School of Medicine, University of Connecticut in Farmington, Connecticut. James L. Oschman, PhD, is president of Nature’s Own Research Association in Dover, New Hampshire. Gaétan Chevalier, PhD, is a visiting scholar in the Department of Family Medicine and Public Health, School of Medicine, University of California, San Diego in San Diego, California. Drew Sinatra, ND, LAc, is a naturopathic doctor of integrative medicine at the CLEAR Center for Health in Mill Valley, California.

Corresponding author: James L. Oschman, PhD E-mail address: joschman@aol.com

H

ealth experts describe a sharp rise in noncommunicable diseases as a major challenge and barrier to global development in the 21st century. The diseases include cardiovascular, respiratory, neurodegenerative, and autoimmune diseases; type 2 diabetes; chronic kidney disease; and some cancers.

8 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

studies examined in this paper were conducted using a variety of statistical procedures.

Interventions • In all cases, the intervention examined conductive contact between the surface of Earth and the study’s participants, using conductive bed sheets, floor or desk pads, and electrode patches, such as those used in electrocardiography.

Results • All studies discussed revealed significant physiological or clinical outcomes as a result of grounding.

Conclusion • This body of research has demonstrated the potential of grounding to be a simple, natural, and accessible clinical strategy against the global epidemic of noncommunicable, degenerative, inflammatory-related diseases. (Altern Ther Health Med. 2017;23(5):8-16)

These conditions affect all nationalities and classes and are reaching epidemic proportions worldwide, accounting for approximately 40 million deaths annually.1 The costs in terms of human suffering and the economics of health care are staggering. The former director general of the World Health Organization (WHO) has described the situation as an impending disaster, commenting that the “root causes of these diseases are not being addressed.”2 Modern biomedicine has discovered that many of the most debilitating diseases, as well as the aging process itself, are caused by or associated with chronic inflammation and oxidative stress, intimately related pathologies. Cells of the immune system release various reactive species, known as free radicals, at sites of inflammation, promoting escalating oxidative stress, intracellular signaling cascades, and proinflammatory gene expression. Both chronic-inflammation and oxidative-stress processes can be simultaneously in play in many chronic disorders.3 Age is another factor; people are living longer. Researchers have documented the deterioration of the immune system as humans age, referring to it as immune-senescence. A prominent Sinatra—Electric Nutrition

medical theory, repeatedly confirmed, states that the general deterioration during aging is caused by oxidative stress and results in inflammation and injury to cells and DNA and protein cross-linking that creates wrinkles in the skin and impairs enzymatic functioning. As an example, chronic, low-grade inflammation is recognized as one of the major risk factors underlying brain aging.4 As of the end of May 2017, approximately 202 298 studies have correlated inflammation with various diseases, led by cancer, with 66 155 citations.5 Inflammation is defined as a localized response to trauma or infection that can wall off damaged tissues until the immune system removes foreign matter, damaged cells, and bacteria. The wall, or inflammatory barricade, serves the function of preventing bacteria or other pathogens or debris resulting from an injury from penetrating nearby heathy tissues. The problem is that the barricade can also be relatively impermeable to circulating antibiotics and antioxidant molecules, slowing the healing response and preventing regenerative cells from entering the region that needs repair.

Because of the barrier, interactions between inflammation and oxidative stress can continue—long after the injury—as a vicious cycle created by the immune system’s relentless and unsuccessful efforts to complete the healing process. A low level of chronic inflammation, often referred to as silent inflammation, can thus continue for years, damaging and compromising the functioning of adjacent healthy tissues and unnecessarily weakening the immune system.

These situations pose a challenge to the physician because a small pocket of chronic inflammation from an old injury can release toxins into the body fluids that gradually compromise distant organs. The difficulty for the physician is finding the source of the problem, which can be a pocket of inflammation from a long-forgotten trauma, an unrecognized dental issue seemingly (but not) resolved, or a walled-off tissue injury, to mention a few.

This inflammation process has been summarized in a recent publication6 and was first described by Selye7 in his classic 1956 book, The Stress of Life, and in his various journal articles. Selye’s work tied inflammatory responses to stress, cortisol secretion, and adaptation. Based on Selye’s work, the current research team has proposed that the inflammatory barricade is formed by a coagulation of debris that has been produced by collateral damage from highly reactive free radicals, which have been leaking beyond the injury site and damaging previously healthy tissue.

Selye’s histological studies showed that the inflammatory barricade is composed of connective tissue. Selye developed a model for his studies in which he injected air under the skin to create an artificial inflammatory pocket, which came to be known as a Selye or granuloma pouch. The method has been used in nearly 2000 studies of inflammation. Selye injected various irritants or pathogens into such pouches to research the ways cortisol and related hormones affected them.

Sinatra—Electric Nutrition

One of these studies examined the effects of the bacteria that cause rheumatic fever. Gundry,8 a cardiologist, has described the mechanism. Strep throat, caused by β-hemolytic Streptococcus, can lead to rheumatic fever, which can lead to rheumatic heart disease and the slow destruction of the heart valves. The immune system recognizes proteins on the surface of the bacteria and attacks and destroys them. Unfortunately, cells comprising the heart valves contain a very similar protein, and the immune system mistakenly identifies them as Streptococcus and then silently, painlessly, and systematically attacks the valves. Eventually, the destruction involved may require valve replacement. This process is a classic example of how an old inflammatory condition can slowly lead to damage to a distant organ or tissue.

Today, clinicians routinely deal with the manifestations of inflammation by prescribing anti-inflammatory drugs.

Such drugs are important clinical agents in their ability to reduce inflammation-related pain, but they have substantial side effects. Practitioners of alternative medicine bring other factors into the picture, such as a noninflammatory diet, targeted supplementation, and mind-body techniques.

Many studies have investigated the potential of nutritional antioxidants to prevent or ameliorate diseases such as cardiovascular disorders, cancer, diabetic complications, and Alzheimer’s disease. Results of these antioxidant trials are mixed in humans. Some studies indicate positive health effects; others show no effects or even harmful effects. Among the reasons for lack of consistent findings could be that the studies have not selected agents that target inflammation and oxidative stress simultaneously or use agents that block some of the oxidative and/or inflammatory pathways but exaggerate others.3 The best dietary antioxidant or combination of nutrients for reducing cancer risks is unknown.9 The current research team sees the inflammatory barricade as a barrier that prevents circulating antibiotics, anti-inflammatory drugs, and nutritional supplements from reaching the sites where they are needed. As a wall of connective tissue, the barricade is relatively impenetrable to these molecules. However, connective tissue is primarily composed of collagen molecules, and research has shown that collagen is a semiconductor, which is a class of materials providing electron conductance between an insulator and a conductor. Hence, the barricade, with its role in isolating chronic inflammation from nearby healthy tissue, can prevent antioxidants from reaching the free radicals in the inflammatory pouches and completing the inflammatory process. Electrons, however, can cross the barrier by semiconduction.

One overlooked element in the human environment— the surface of Planet Earth, including its landmasses and bodies of water—may provide a potent and surprising natural remedy for this challenge and the alarming rise in chronic inflammatory-related diseases. The current study intended to evaluate the physiological and clinical significance of biological grounding.

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THE MISSING LINK: PLANET EARTH In the late 19th century, a back-to-nature movement in Germany claimed many health benefits from being barefoot or sleeping naked on the ground outdoors, even in cold weather.10 In the 1920s, G. S. White, an American medical doctor, investigated the practice of sleeping grounded after being informed by some individuals that they could not sleep properly unless they were on the ground or connected to the ground in some way, such as with copper wires attached to water, gas, or radiator pipes that were grounded to Earth. He reported improved sleep using these techniques.11 However, these ideas never caught on in mainstream society.

At the end of the 20th century, Ober12 in the United States and Sokal and Sokal13 in Poland independently found distinct physiological and health benefits with the experimental use of a variety of indoor conductive arrangements—bed pads, mats, EKG and TENS-type electrode patches, and copper plates— connected to Earth outside.

Ober, a pioneer in the cable-television industry, recognized a similarity between the stabilizing effects of ground energy on television cable systems specifically and on electrical systems in general and its effects on the human body.12 Sokal and Sokal,13 meanwhile, concluded that grounding the human body represents a universal regulating factor in nature that strongly influences bioelectrical, bioenergetic, and biochemical processes and appears to offer a significant modulating effect on chronic illnesses.

It is a well-known scientific fact that Earth possesses a slightly negative electric charge, the result of countless lightning strikes and solar radiation, among other factors. This planetary attribute is based on a virtually limitless, unseen, and continuously renewed reservoir of free electrons, which are negatively charged subatomic particles.14,15 We use the terms free or mobile electrons to distinguish them from charged ions, which diffuse much more slowly through tissue fluids.

Throughout the world, electrical systems are connected to Earth’s surface and its negative charge to maintain stability and safety. These systems, from large grids and power stations to homes, buildings, and factories as well as the machinery and appliances that are operated by electricity, are thus said to be grounded or earthed.

Herein lies the surprise. Research conducted for more than a decade has demonstrated that Earth’s charge and storehouse of electrons represent a major natural resource of health and healing. Research on biological grounding is now suggesting that this very same electric charge on the planet’s surface plays a governing and nurturing role for both the animal and plant kingdoms—a form of electric nutrition, so to speak. It appears to have the potential to restore, normalize, and stabilize the internal environment of the human body’s countless bioelectrical systems that govern the functions of organs, tissues, cells, and biological rhythms.16,17 Significant benefits—such as better sleep, reduced inflammation and pain, and improved blood flow—result from walking barefoot outdoors or sitting, working, or sleeping indoors in contact with conductive sheets, pads,

mats, bands, and patches that are connected to Earth. Such contact is believed to transfer Earth’s free or mobile electrons from the ground into the body, a transfer resulting in rapid, sometimes instant, and significant physiological changes now documented in multiple published studies, most of them peer reviewed.18 Throughout history, humans mostly walked barefoot and slept on the ground, or they used footwear and bedding fashioned from animal skins that become permeated with body perspiration or ground moisture and thus permitted transfer of Earth’s electrons into the electrically conductive body. Through this mechanism, every part of the body could equalize with the electric potential of Earth.

Modern lifestyles, however, have increasingly created a barrier between humans and a natural conduction of Earth’s electrons into the body. Since the 1950s, for example, humans have increasingly worn insulating rubber or plastic soled shoes, instead of traditional leather, fashioned from hides and largely conductive. Obviously, humans also no longer sleep in conductive contact with the ground as they did in times past.

The results of grounding research raise an important question. Does the current disconnect with Earth’s electrons represent a critically important and overlooked contribution to physiological dysfunction and to the alarming global rise in inflammatory-related chronic diseases? The research, together with global anecdotal feedback, suggests that Earth’s electric charge is fundamental for maintaining health and promoting healing.

Figure 1 shows a possible relationship between the shoe-driven disconnection from Earth’s natural electric charge and diabetes, an inflammatory-related disease. Sales of shoes with synthetic soles have soared in the United States since the 1950s. The curve of increase is similar to that of diabetes. In the 1950s, 95% of shoes were made with leather soles, many of which were conductive. Currently, 95% of shoes have synthetic, nonconductive soles. The question arises as to whether this observation represents a correlation or a coincidence. Is the loss of our electrical roots a factor in the rise of diabetes and other inflammatory diseases, together with the usual suspects of sedentary living and overconsumption of calorie-rich, unnatural, nutrient-poor food loaded with sugar and high-fructose corn syrup sweeteners?18

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FUNCTION OF ELECTRONS IN THE BODY Humans are bioelectrical beings, with hearts, brains, and immune and endocrine systems being regulated by internal bioelectrical signals. On this bioelectrical stage, electrons are marquee performers. They are intimately involved in the body’s countless processes. They make it possible for atoms to bond with other atoms and form molecules. Redox reactions involve the transfer of one or more electrons from one atom or molecule to another.

Oxygen and reactive oxygen species—free radicals—are powerful oxidizing agents. When they are secreted at an injury site by white blood cells, they literally rip electrons out

Figure 1. Possible Relationship Between a Shoe-driven Disconnection From Earth’s Natural Electric Charge and Diabetes, an Inflammatory-related Disease

Abbreviations: CDC, Centers for Disease Control; SGMA, Sporting Goods Manufacturers Association. of molecules, which is the way that damaged cells and other debris from an injury are destroyed to clear the repair field so that regenerative cells can move in and restore structures and functions. In the oxidative process, oxygen becomes more stable, while the attacked molecules and cellular debris disintegrate to be phagocytized by other white blood cells.

Although oxygen is essential to metabolism and life, the oxygen molecule itself is extremely toxic and the body uses a variety of antioxidant processes to keep the concentration of oxygen low in the tissues. Too much oxygen creates oxidative stress. One of the key reactions in living cells, of course, is the electron transport chain in mitochondria that produces adenosine triphosphate (ATP), the energy source for all living processes. Studies have shown that providing electrons to animals dramatically increases ATP production and protein synthesis, 2 processes that are essential for wound healing.19 Electrons from Earth serve as a potent neutralizer or quencher of electron-seeking free radicals. The term electron deficiency may be appropriate to describe the largely ungrounded status of most of humanity. As noted, the modern lifestyle, notably the wearing of shoes with synthetic soles, has severed us from our electric roots, our connection with Earth and its natural supply of electrons.

It is interesting to note that one of the side effects of therapies involving touch, such as massage, is that the therapist can become depleted. Is this a result of donating their electrons to patients with a great deal of inflammation?

The patient feels better; the therapist gets burned out.21

The current team’s research indicates that the immune system may not function efficiency in an organism that has an insufficiency of free electrons. Grounding provides the reinforcements, like the cavalry coming to the rescue.

The manner by which grounding produces rapid and measurable improvements in whole-body physiology provides support for a concept first introduced by Nobel Laureate Albert Szent-Györgyi in 1941 and again in the 1980s. His remarkable insight was that proteins are semiconductors, rather than insulators, as had been thought previously.21-23 Moreover, when many proteins are organized in parallel, into arrays, as in the crystal lattices found throughout the human body (eg, in connective tissues, myofascia, tendons, cell membranes, bones, and muscles) some electrons will cease to belong to particular atoms or molecules and will be free to move from place to place within the organism, which is what the current research team means when it uses the terms free or mobile to describe these electrons. These revolutionary concepts are never discussed in conventional medicine but must now be reconsidered because they provide the best explanation for the rapid effects of Earthing. For a review, see Oschman, Chevalier, and Brown.6 The contemporary research of Gerald Pollack at the University of Washington features exclusion zone (EZ) water.24 EZ water refers to the highly organized state of water within cells discovered to have a vital influence on every biochemical process, including the way by which proteins

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carry out their functions. EZ water is negatively charged and, according to Pollack, grounding contributes negatively charged electrons, which further builds EZ water, enhancing protein folding and providing healthier functioning.

CLINICAL RESULTS OF GROUNDING The grounding studies conducted to date indicate multiple and rapid effects achieved by what appears to be the body’s broad uptake and use of electrons received from Earth. Findings among the documented results include (1) reduced inflammation and pain, (2) improved blood flow and reduced blood viscosity, (3) reduced stress, (4) better sleep, (5) improved energy, and (6) improved response to trauma and injuries and accelerated wound healing.

Reduction in Inflammation and Pain Grounding reduces or even prevents the cardinal signs of inflammation following injury: redness, heat, swelling, pain, and loss of function. Most pains, including the most severe, are due to inflammation and typically respond rapidly to Earthing. Healing-related pains usually lessen, often significantly, in intensity and duration.25-29 Improved Blood Flow and Reduced Blood Viscosity Thick, sludgy, and clumped blood is a hallmark of cardiovascular disorders and diabetes. Two studies investigated the effects of Earthing on blood viscosity. The first involved participants relaxing for 2 hours, with blood viscosity determined before and after by measuring the zeta potential of red blood cells. The greater the zeta potential, the greater is the negative charge on the red cells, pushing them apart and lowering blood viscosity.30 The second study used a commercial blood viscometer to measure viscosity of individuals practicing yoga on a grounded yoga mat.31 Both studies found that Earthing significantly reduced blood viscosity.

Two additional studies showed that blood flow regulation and circulation to the head, face, torso, and extremities were enhanced within a 1-hour session of grounding in a chair.32,33 These studies involved the use of laser speckle contrast imaging and thermography. Together, these studies found significant systemic benefits, affecting overall and local health, and suggest that grounding may represent an effective preventive and therapeutic strategy against diabetes and cardiovascular disease.

Consequently, any natural method for relieving stress has enormous potential to prevent or decrease the negative effects of most diseases. In multiple studies,25,29,34-36 grounding has been documented to exert a beneficial effect on stress, a likely result of systemic influences in the body, including (1) a normalizing influence on cortisol, the stress hormone25; (2) a calming impact on the electrical activity of the brain34; (3) a normalization of muscle tension34; (4) a rapid shift from a typically overactive expression of the sympathetic nervous system, associated with stress, into a parasympathetic, calming mode within the autonomic nervous system (ANS) that regulates heart and respiration rates, digestion, perspiration, urination, and even sexual arousal35; and (5) within the ANS, an improvement in heart rate variability (HRV)—the miniscule variations in the heart’s beat-to-beat interval—that serves as an accurate reflector of stress.38 Low HRV is associated with stress-related disorders, cardiovascular disease, diabetes, mental health issues, and reduced lifespan.

Grounding improves HRV to a degree far beyond mere relaxation.

In a recent study, researchers at Pennsylvania State University documented immediate and significant increases in HRV measurements that indicated improved vagus-nerve transmission among premature babies.37 Such an effect could potentially enhance stress and inflammatory regulatory mechanisms.

Better Sleep This effect is one of the most common responses from people, including many insomniacs, after they start grounding. In a 2004 study, 12 participants slept grounded for 8 weeks.25 Eleven reported that they fell asleep faster. All reported waking fewer times during the night. While different people respond differently, for many grounded sleep reduces pain from inflammation and thereby helps them get better rest.

Improved Energy Sleeping grounded provided more morning energy as well as vitality throughout the day.12,25,29 In the 2004 study, 9 of 12 participants reported a decrease in fatigue (ie, they were more refreshed and less fatigued).25 Better, deeper sleep, due to improvement in the day/night cortisol rhythm, is one explanation.

Reduced Stress Cortisol, a mediator and marker of stress, is associated with emotional and physiological stress, inflammation, and sleep dysfunction. Chronic elevation of cortisol from stress can lead to a disruption of the body’s circadian rhythms and contribute to sleep disorders, hypertension, cardiovascular disease, decreased bone density, decreased immune response, mood disturbances, autoimmune diseases, and abnormal glucose levels. Emotional and physical stress are aggravating factors for pain and psychological disorders.

Improved Response to Trauma and Injuries and Accelerated Wound Healing Faster-than-normal wound and surgical healing has been reported frequently through the years. Accelerated wound healing, whether involving injury, surgery, or burn, may result from a combination of reduced inflammation as well as improved circulation—vasodilation and lower blood viscosity—facilitating the delivery of healing factors to the site.26,29 In the domain of exercise and sports, significant changes in immune function responses and markers have been found among grounded—but not among nongrounded—

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Figure 2. Accelerated Improvement of an 8-month-old, Nonhealing, Open Wound by the Ankle, Suffered by an 84-year-old Woman With Diabetesa

The pictures in the right column represent closeups of the photos to the left. The top row shows the open wound and a pale-gray hue to the skin before grounding. The middle row shows the marked level of healing and improvement in circulation, as indicated by the skin color, after 1 week of grounding. The bottom row shows the wound healed over and the skin’s color looking dramatically healthier after 2 weeks of grounding.

a

individuals. In 2 studies of grounding on delayed onset muscle soreness, grounded participants had less pain, little inflammation, and a shorter recovery time.28,38 Researchers have also found that grounding during cycling exercise significantly reduced the level of blood urea, indicating less muscle and protein breakdown. These findings represent a major recovery benefit for training athletes.39 In competitive sports, dramatic examples of accelerated healing were reported by chiropractor Jeff Spencer,40,41 assigned to promote recovery from exertion and injury among American cyclists during several Tour de France

competitions. It is well known that at this extreme level of grueling performance, cyclists tend to experience slow wound healing after injury, threatening their ability to continue competing. Spencer found that grounding produced very rapid healing, together with only minimal classical signs of inflammation.

The series of photographs in Figure 2 above demonstrate vividly the potential for accelerated wound healing, in this case a typical open, nonhealing wound on the foot of an elderly diabetic woman. Within one week of grounding, signs of healing are already obvious.

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The cause of the wound was a poorly fitted boot that had resulted in a blister that developed into a resistant open wound. The sole treatment was a daily, 30-minute grounding session with an electrode patch while the woman was seated comfortably. The patient had undergone various treatments at a specialized wound center with no results. Vascular imaging of her lower extremities revealed poor circulation.

When first seen, she had a mild limp and was in pain. After the first 30 minutes of grounding, the patient described a noticeable lessening of pain. After 1 week, a reduction in pain of approximately 80% had occurred. At that time, she showed no evidence of a limp. After 2 weeks of daily grounding, she said the pain had disappeared totally.19 DISCUSSION Grounding and Biological Rhythms In explaining the dynamics of grounding, it is important to mention that the electrically active surface of Earth is a source of not only electrons, but also key rhythmic processes vital to normal biological rhythms. The electric field of Earth is not steady but varies from moment to moment in a rhythm known as the Schumann resonance. Behind the world that we can see with our senses lies a fantastic web of powerful but invisible energies and forces that affect us every moment of every day and that can be referred to as geophysical fields— the invisible energies of Earth’s gravity, magnetism, electricity, and electromagnetism. Human knowledge of these fields comes from centuries of detailed study in a variety of scientific fields: biology, physics, geophysics, atmospheric physics, astronomy, astrophysics, and cosmology.

Relationships with these geophysical rhythms are absolutely vital for health. Human physiology has more than 100 biological rhythms that are timed and coordinated with rhythms in the environment.42 These rhythms are especially important for women.

Female physiology and reproduction are regulated by an array of hormones, the concentrations of which vary from moment to moment in relation to rhythmic changes in the environment.

When a woman’s body rhythms are properly synchronized with her geophysical environment, she feels fine. When she becomes disconnected from the environmental pace-setting rhythms, her hormonal systems can become chaotic and can make themselves known with a variety of symptoms, some of which are very uncomfortable. The invisible silent pulses of nature give rise to normal hormonal regulations. Disconnection from the environment can disrupt these regulations, and grounding provides a simple way to restore balance.

One of the key elements in the grounding package is connection with the Schumann resonance, a standing wave made of electromagnetic fields vibrating at 7.83 Hz— vibrating approximately 8 times per second. It also has harmonics at higher frequencies, such as 14.3 Hz, 20.8 Hz, 27.3 Hz, and 33.8 Hz, produced collectively by lightning strikes throughout the world.

Have you ever stood in a hallway or stairwell or a large room and noticed that your voice creates a sort of echo or 14 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

reverberation? You are creating standing waves. In physics, a standing wave is caused by the presence of 2 waves traveling in opposite directions. Common examples of standing waves are provided by musical instruments, such as organ pipes or violin strings. Waves traveling in a pipe or along a string will reflect back when they reach the ends. The back-and-forth waves join to create a resonant tone or frequency that is characteristic of the geometry of the space. Pressing a guitar or violin string against different regions on the fret or fingerboard changes the effective length of the string and, therefore, the resonant frequency of the standing waves that can be produced.

In 1952, the German physicist Professor W. O. Schumann of the Technical University of Munich predicted that electromagnetic standing waves would be established in the atmosphere, within the giant resonant space between the surface of Earth and the ionosphere. The space or cavity between the ionosphere and Earth’s surface is now used in wireless information transfer over long distances. Radio signals bounce back and forth between the 2 surfaces. This skip phenomenon has been widely studied because it is the basis for long-distance radio communications.

Similarly, cloud-to-Earth lightning bolts pump energy into the cavity, creating standing electromagnetic waves that travel around Earth at the speed of light, circumnavigating the entire planet on average 7.83 times per second. A person standing on Earth at any point will be exposed to these Schumann frequencies. To use physics terminology, lightning pumps electromagnetic energy into the cavity, which causes that electromagnetic energy to vibrate or resonate at the resonant frequency of the cavity in the extremely low-frequency range (7.83 Hz). At the same time, lightning bolts bring electrons from the ionosphere to the surface of Earth. Multiple lightning strikes produce complex standing waves.

Just as organs use pipes of different sizes to produce different notes and different frequencies of standing waves, the frequency of the Schumann resonance varies as the ionosphere breathes in and out due to the atmospheric tides.

Many scientists have recognized the similarity of the Schumann signal and the alpha brainwave measured with an electroencephalogram. It has been suggested that the Schumann resonance has been ingrained into all life. Many biologists have concluded that the frequency overlap of Schumann resonances and biological fields is not accidental but is the culmination of a close interplay between these fields over evolutionary time.

Grounding and the Immune System Scientists believe that our immune system evolved in the course of millions of years of barefoot contact with the surface of Earth. One can assume that protective antioxidant and anti-inflammatory electrons from Earth were readily obtained by previous cultures during this vast stretch of time as a result of ordinary existence. Life involved direct contact with Earth, which is no longer the case.

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Did the immune system begin weakening as we started wearing shoes with insulating soles during the 1950s, leading to the ever-rising unwellness plaguing the world today?

It is widely suspected that causal factors include overload from environmental toxicants, chronic antibiotic use, sedentary living, emotional stress, and ingestion of unnatural, processed foods, genetically modified-GMOs among them.

Nobody thinks of their shoes, however, as a cause of chronic inflammation, but grounding research certainly points an incriminating finger at footwear and the resulting disconnection with Earth’s healing energy.

Grounding research is at an early stage, but the evidence compiled thus far consistently demonstrates that a great frontier of health and healing potential has been opened— right beneath our noses, or, more specifically, right under our feet. Humans have abandoned nature in many ways.

Grounding reconnects humans to one aspect that has been largely lost and overlooked.

The Clinical Prospects of Grounding For clinicians, grounding offers the prospect of a practical and simple strategy to help restore health, relieve patients’ aches and pains, and serve both preventive and therapeutic goals. Either by itself or in conjunction with conventional strategies, it can greatly improve patient outcomes with little effort.

Some ways to integrate grounding as a clinical strategy include (1) recommending outdoor barefoot sessions, (2) holding grounding sessions in the clinic, (3) offering grounding products to patients, and (4) using a grounding mat on the treatment table to reduce practitioners’ typical energy burnout.

Barefoot Sessions. Recommend outdoor barefoot sessions to patients, weather and conditions permitting. Ober has observed that going barefoot for approximately 30 or 40 minutes daily can significantly reduce pain and stress.18 Barefoot grounding outside is free; however, many people will neither have the time nor the inclination to add such a routine to their lives. For these and other people interested in pursuing outdoor grounding, conductive footwear is commercially available.

Grounding in the Clinic. Ground patients in the clinic with 30-minute or 1-hour sessions using grounding products, such as conductive chairs, mats, and patches.

Grounding Products. Offer grounding products to patients or direct them to vendors. A variety of grounding systems are available for indoor use while sleeping, working, or relaxing.

Grounding Mat. For physicians or their assistants using touch for palpating or adjusting patients, a grounding mat placed on the treatment table has been reported by practitioners to reduce typical energy burnout at the end of the day.20 One caveat exists. Because of the broad physiological effects generated by grounding, medication dosages may be impacted. This clinically relevant issue is discussed in the Earthing book18 and on the Earthing Institute’s Web site.43 As Sinatra—Electric Nutrition

an example, the combination of earthing and warfarin (Coumadin) has the potential to exert a compounded blood thinning effect and must be supervised by a physician.

Anecdotes of an elevated international normalized ratio (INR) have been reported. INR is a widely used measurement of coagulation. Earthing may also improve thyroid function and glucose metabolism and possibly require an adjustment in medication dosages.18 CONCLUSIONS New research indicates that grounding the body generates broad, beneficial, and significant physiological changes. The source of these effects is believed to be the mobile electrons omnipresent on the surface of Earth, which are responsible for the planet’s negative charge. Lifestyle changes have disconnected most humans from this primordial health and healing resource, creating what may be an unrecognized electron deficiency in the body, an overlooked cause or contributor to chronic inflammation and common chronic and degenerative diseases.

When Earth connection is restored through grounding, electrons flood throughout the body, reducing inflammation and oxidative stress while also reinforcing the body’s own defense mechanisms. Electron transfers are the basis of virtually all antioxidant and anti-inflammatory activity. And Earth may very well be the ultimate supplier! When the supply is restored, humans have the potential to thrive.

ACKNOWLEDGEMENTS

The authors wish to thank Martin Zucker for assistance in writing this paper.

AUTHOR DISCLOSURE STATEMENT

James L. Oschman, Stephen T. Sinatra, and Gaétan Chevalier own shares of EarthingFX, the company that manufactures indoor grounding systems.

REFERENCES

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Accessed August 23, 2017.

2. Chan M. The worldwide rise of chronic non-communicable diseases: A slowmotion catastrophe. Speech of the Director General of the World Health Organization (WHO), 2011. WHO Web site. http://www.who.int/dg/ speeches/2011/ministerial_conf_ncd_20110428/en/. Accessed August 23, 2017.

3. Biswas SB. Does the interdependence between oxidative stress and inflammation explain the antioxidant paradox? Oxid Med Cell Longev. 2016;2016:1.

4. Corbi G, Conti V, Davinelli S, et al. Dietar y phytochemicals in neuroimmunoaging: A new therapeutic possibility for humans? Front Pharmacol.

2016;7:364.

5. Pub Med, National Library of Medicine.

6. Oschman J, Chevalier G, Brown R. The effects of grounding (earthing) on inflammation, the immune response, wound healing, and prevention and treatment of chronic inflammatory and immune diseases. J Inflamm Res. 2015;8:83-96.

7. Selye H. The Stress of Life. 2nd ed. New York, NY: McGraw-Hill Education; 1978.

8. Gundry S. The Plant Paradox. New York, NY: Harper Wave; 2017.

9. Wu X, Cheng J, Wang X. Dietary antioxidants: Potential anticancer agents. Nutr Cancer. 2017;69(4):521-533.

10. Just A. Return to Nature: The True Natural Method of Healing and Living and The True Salvation of the Soul. New York, NY: B. Lust; 1903.

11. White G. The Finer Forces of Nature in Diagnosis and Therapy. Albuquerque, NM: Sun Publishing; 1981.

12. Ober C. Grounding the human body to neutralize bioelectrical stress from static electricity and EMFs. ESD Journal Web site. http://www.esdjournal.com/articles/ cober/ground.htm. Published January 2000. Accessed August 23, 2017.

13. Sokal K, Sokal P. Earthing the human body influences physiologic processes. J Altern Complement Med. 2011;17(4):301-308.

14. Williams E, Heckman S. The local diurnal variation of cloud electrification and the global diurnal variation of negative charge on the Earth. J Geophysical Research. 1993;98(D3):5221-5234.

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15. Anisimov S, Mareev E, Bakastov S. On the generation and evolution of aeroelectric structures in the surface layer. J Geophys Res. 1999;104(D12):1435914367.

16. Oschman J. Perspective: Assume a spherical cow: The role of free or mobile electrons in bodywork, energetic and movement therapies. J Bodywork Movement Ther. 2008;12:40-57.

17. Oschman J. Charge transfer in the living matrix. J Bodywork Movement Ther.

2009;13:215-228.

18. Ober C, Sinatra S, Zucker M. Earthing. 2nd ed. Laguna Beach, CA: Basic Health Publications; 2014.

19. Cheng N, Van Hoof H, Bockx E, et al. The effects of electric currents on ATP generation, protein synthesis, and membrane transport in rat skin. Clin Orthop Relat Res. 1982;171:264-272.

20. Oschman J. A new perspective on the cause and prevention of therapist burnout.

Massage & Bodywork Magazine. March/April, 2016:75-81.

21. Szent-Györg yi A. The study of energ y levels in biochemistr y.

Nature.1941;148:157-159.

22. Szent-Györgyi A. Towards a new biochemistry? Science. 93(2426):609-611.

23. Gascoyne P, Pethig R, Szent-Györgyi A. Water structure-dependent charge transport in proteins. Proc Natl Acad Sci. 1981:78:261-265.

24. Pollack G. The fourth phase of water: A central role in health. Presentation delivered at the International Breath of Life Conference; May 14, 2017; London, United Kingdom.

25. Ghaly M, Teplitz D. The biologic effects of grounding the human body during sleep as measured by cortisol levels and subjective reporting of sleep, pain, and stress. J Altern Complement Med. 2004;10(5):767-776.

26. Amalu W. Medical thermography case studies. http://162.214.7.219/~earthio0/ wp-content/uploads/2016/07/Medical-Thermographics.pdf. Accessed August 23, 2017.

27. Chevalier G, Mori K, Oschman J. The effect of Earthing (grounding) on human physiology, Pt. II: Electrodermal measurements. Subtle Energ Energ Med. 2007;18(3):11-34.

28. Brown R, Chevalier G, Hill M. Pilot study on the effect of grounding on delayedonset muscle soreness. J Altern Complement Med. 2010;16(3):265-273.

29. Oschman J, Chevalier G, Ober AC. Biophysics of Earthing (Grounding) the human body. In: Rosch P, ed. Bioelectromagnetic and Subtle Energy Medicine. 2nd ed. New York, NY: CRC Press; 2015:427-450.

30. Chevalier G, Sinatra S, Oschman J, Delany RM. Earthing (grounding) the human body reduces blood viscosity: A major factor in cardiovascular disease. J Altern Complement Med. 2013;19(2):102-110.

31. Brown R, Chevalier G. Grounding the human body during yoga exercise with a grounded yoga mat reduces blood viscosity. Open J Prev Med. 2015;5:159-168.

32. Chevalier G. Grounding the human body improves facial blood flow regulation: Results of a randomized, placebo controlled pilot study. J Cosmetic Dermatol Sci App. 2014;4(6):293-308.

33. Chevalier G, Melvin G, Barsotti T. One-hour contact with the Earth’s surface (grounding) improves inflammation and blood flow: A randomized, doubleblind, pilot study. Health. 2015;7:1022-1059.

34. Chevalier G, Mori K, Oschman J. The effect of Earthing (grounding) on human physiology, Pt. I. Electrodermal measurements. Euro Biol Bioelectromagnetic.

2006;2(1):600-621.

35. Chevalier G. Changes in pulse rate, respiratory rate, blood oxygenation, perfusion index, skin conductance, and their variability induced during and after grounding human subjects for 40 minutes. J Altern Complement Med. 2010;16(1):1-7.

36. Chevalier G, Sinatra S. Emotional stress, heart rate variability, grounding, and improved autonomic tone: Clinical applications. Integr Med Clin J. 2011;10(3):1621.

37. Passi R, Doheny KK, Gordin Y, Hinssen H, Palmer C. Electrical grounding improves vagal tone in preterm infants. Neonatology. 2017;112 (2):187-192.

38. Brown R, Chevalier G, Hill M. Grounding after moderate eccentric contractions reduces muscle damage. Open Access J Sports Med. 2015;5(6):305-317.

39. Sokal P, Jastrzębski Z, Jaskulska E, et al. Differences in blood urea and creatinine concentrations in earthed and unearthed subjects during cycling exercise and recovery. Evid Based Complement Alternat Med. 2013(2013):1-6.

40. Spencer J, quoted in Ober C, Sinatra ST, Zucker M. Earthing. 2nd ed. Laguna Beach, CA: Basic Health Publications; 2014:221-229.

41. Spencer J. Earthing speeds athletic recovery and healing. Earthing Institute Web site. http://www.earthinginstitute.net/earthing-speeds-athletic-recovery-andhealing/. Accessed August 23, 2017.

42. Palmer, JD. The Living Clock: The Orchestrator of Biological Rhythms. Oxford, United Kingdom: Oxford University Press; 2002.

43. Earthing Institute. http://www.earthinginstitute.net/. Accessed August 23, 2017.

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ORIGINAL RESEARCH

Reduction in Pain After Use of Bioceramic Undershirt for Patients With Fibromyalgia María Aparecida Santos e Campos, PhD; Felipe García Pinillos, PhD; Pedro Ángel Latorre Román, PhD

ABSTRACT Context • The treatment of pain in fibromyalgia (FM) has centered on pharmacotherapy and nonpharmacological treatment. The nondrug treatments for FM include the effects of far infrared radiation (FIR). Currently, FIR-emitting heat lamps and garments made of filaments (fibers) impregnated with FIR-emitting nanoparticles are being used to deliver thermal radiation effects.

Objective • The study intended to determine the benefits of a fabric coated with bioceramics for reducing pain in women with FM. Design • The research team designed a randomized, controlled pilot study.

Setting • The study was conducted in the laboratory of the Department of Corporal Expression, University of Jaén (Jaén, Spain).

Participants • Thirty-nine female participants diagnosed with FM took part in the study.

Intervention • Participants were randomly assigned to an experimental (intervention) group (EG), n = 20, or a placebo group (PG), n = 19. The EG wore a shirt impregnated with bioceramics, and the PG dressed in a shirt without bioceramics. Both groups wore the shirts for 8 h/d for 60 d.

María Aparecida Santos e Campos, PhD; Felipe García Pinillos, PhD; and Pedro Ángel Latorre Román, PhD, are all in the Department of Corporal Expression, University of Jaén, in Jaén, Spain.

Corresponding author: Pedro Ángel Latorrre Román, PhD E-mail address: platorre@ujaen.es

18 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Outcome Measures • Participants were evaluated twice, at baseline and postintervention. The tests used were (1) a visual analogue scale (VAS) for pain, (2) the FM Impact Questionnaire (FIQ), and (3) the Short Form-12 health survey. The research team measured each participant’s weight, height, and body mass index and evaluated their tender points. The research team also tracked participants’ daily use of pain medications as a daily tablet intake.

Results • Postintervention, the women in the EG showed a significant reduction in pain: (1) on the VAS (P < .001), (2) on the measurement of tender points (P < .001), and (3) on the algometer score (P < .001), as well as (4) a significant reduction in FM symptoms on the FIQ (P = .005) and (5) a reduction in daily tablet intake (P < .001). No significant changes in the PG group were found. Significant differences existed between the groups for the VAS, the FIQ, tender points, and the algometer score in posttest evaluation (P < .05).

Conclusions • Regular use of garments impregnated with a bioceramic solution (1%) can be beneficial in reducing pain and the impact of FM for patients. The low cost and comfort of the clothing make the therapy easy to be applied as a complementary tool in the treatment of FM. (Altern Ther Health Med. 2017;23(5):18-22)

F

ibromyalgia (FM) is a chronic condition characterized by widespread pain, muscle stiffness, reduced physical fitness,1 fatigue,2 poor sleep, anxiety, cognitive difficulties,3 reduced physical capacity,4 and the presence of specific points sensitive to palpation and pressure in the skeletal muscle system, called tender points.5 FM is associated with physical disabilities in daily activities, such as walking, lifting, and transporting objects.6 Patients with FM have a low level of physical activity in comparison with healthy people.4,7 In addition, they show a level of functional capacity and physical condition similar to that of older adults8,9 and are prone to falls.10 Aparecida Santos e Campos—Bioceramic Undershirt and Reduced FM

Jeschonneck et al11 found that patients with FM show a reduction in local circulation with vasoconstriction and a reduction in blood flow (local hypoxia) and body temperature in the pain-sensitive areas. Pienimaki et al12 have shown that cold exposure increases musculoskeletal pain, and Larson et al13 have indicated that the pain from FM gets worse with cold and stress but is alleviated with heat. Patients with FM show lower body temperatures and lower metabolic rates in response to stress.13 Some researchers14,15 have demonstrated the benefits of physical training in a heated pool to reduce pain in patients with FM and chronic fatigue. Thus, heat provides an important action in reducing the pain of FM. The treatment of pain in FM has centered on pharmacotherapy and nonpharmacological treatments, such as educating patients, encouragement of physical activity, and educational–psychological programs shown to be effective in the relief of the symptoms of FM.16 The nondrug treatments for FM include the effects of far infrared radiation (FIR). FIR (λ = 3 – 100 μm) is a subdivision of the electromagnetic spectrum that has been investigated for biological effects.

The biological effects of FIR include vasodilatation, an increase in body temperature, and analgesic and anti-inflammatory actions.17 A recent study18 has demonstrated the analgesic effects of FIR on patients during the postoperative period in total knee arthroplasty.

York and Gordon19 have shown that socks soaked in polyethylene terephthalate, which incorporates optically active particles, have a beneficial effect in reducing chronic foot pain, and other researchers have shown an improvement in pain at the site of an amputation.20 Liau et al21 tested the therapeutic effects of a far-infrared ray-emitting belt (FIRB) in the management of primary dysmenorrhoea in female patients, and the application of an FIRB appears to alleviate dysmenorrhea.

Conrado and Munin23 reported that one class of such infrared emitters consists of ceramics (also known as bioceramics) in a powdered form, which allows for its incorporation into creams and fabrics, such ceramic materials emit in the FIR when subjected to body temperature.

Ko and Berbrayer22 found a reduction in patients’ pain when they wore gloves impregnated with such bioceramics.

Furthermore, Conrado and Munin23 tested the effects on reducing women’s body mass by increasing their body temperature through the use of a garment soaked in bioceramics for 30 days and they found that the experimental data showed a reduction in body measurements, which may be a consequence of an increment in microcirculation and peripheral blood flow, and these changes might promote improved general health.

Currently, FIR-emitting heat lamps and garments made of filaments (fibers) impregnated with FIR-emitting ceramic nanoparticles are being used to deliver thermal-radiation effects in medical applications, and FIR’s effects have been found to be beneficial for health.24 FIR is biologically active and, in live organisms, induces a rise in body temperature and elevates the mobility of tissue fluids.25 Use of

FIR-impregnated fabrics has been found to provide an effective treatment on chronic foot pain.19 Literature about use of bioceramics for treatment of pain is scarce, and most of the work uses subjective analyses.

Additional investigations are necessary to understand the pain relief that bioceramics provide, including for people with FM. Therefore, the main purpose of the current study was to analyze the effects of FIR using fabric coated in bioceramics on pain in patients with FM.

Aparecida Santos e Campos—Bioceramic Undershirt and Reduced FM

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 19

METHODS Participants A randomized, controlled pilot study study was carried out on women with FM who belonged to the Jaén Association of FM (AFIXA) in Jaén, Spain. Participants were selected by convenience, having been diagnosed with FM by doctors in the rheumatology sector of the public health care system. They also met criteria for FM set by Wolfe et al.26 Initially, 60 women were identified and of those, 39 met the inclusion criteria.

Criteria to participate in the study included (1) being a member of AFIXA and (2) having no other disease that could interfere with the results of the study, such as other rheumatic diseases and/or serious psychiatric or somatic disorders (eg, cancer, severe coronary heart disease, or schizophrenia).

None of the participants had been taking part in exercise programs before the study or receiving any other type of therapy.

A research assistant randomly assigned participants either to the experimental (intervention) group (EG), n = 20, or to the placebo group (PG), n = 19, according to a random number table, by simple random sampling, and assigned a code number to each participant. Another research assistant analyzed the data.

After receiving detailed information about the objectives and process of the study, each participant signed a consent form to participate in it. The study was completed in accordance with the norms of the Declaration of Helsinki (2013 version) and following the directives of the European Union on Good Clinical Practice (111/3976/88 of July 1990), as specified in the Spanish legal framework for human clinical research (Royal Decree 561/1993 on clinical essays).

Procedures The participants were evaluated on 2 occasions, at baseline and postintervention. The patients completed (1) a visual analogue scale (VAS) for pain, (2) the FM Impact Questionnaire (FIQ), and (3) the Short Form-12 (SF-12) health survey. The research team measured each participant’s weight, height, and body mass index (BMI) and evaluated their tender points. The research team also tracked participants’ daily use of pain medications as a daily table. All patients were examined by the same group of researchers to reduce errors in the measurements.

Intervention For 60 days, participants in the EG wore a T-shirt impregnated with a 1% bioceramic solution, called FirBiotech (FIVE 710 SL, Jaén, Andalusia, Spain). This bioceramic tissue

is able to emit FIR into the body, increasing the body temperature.

Participants in the PG wore a T-shirt provided by the same manufacturer without bioceramics, both for at least 8 hours per day. The intervention occurred in accordance with the protocol of Conrado and Munin.23 The T-shirts were visually indistinguishable and were made specifically for the study.

The participants were blinded as to the shirt’s type.

During the study, the participants received no other type of therapy. Table 1 shows the far-infrared emissions in the fabric provided by the supplier, and Figure 1 shows in detail the deposits of the bioceramics on the fibers of the fabric.

Outcome Measures Visual Analog Scale. A VAS was used to measure each participant’s subjective intensity of pain, which was based on a line measuring 10 cm, where 0 was the absence of pain, and 10 was the worst pain possible.

FM Impact Questionnaire. The FIQ was used to evaluate a participant’s symptoms related to FM and has been validated in a Spanish population with FM.27 The point range of the FIQ goes from 0 to 100, and a higher score indicates a greater impact for the illness.

Short Form-12. To evaluate each participant’s health and quality of life, the SF-12 scale was used,28 which sums the results for 12 items on subscales of physical health and mental health. The score varies between 0 and 100, with higher scores indicating better health.

Height and Weight. To measure height (m) and weight, a stadiometer (Seca 220, Hamburg, Germany) and a Seca 634 weighing scale (Hamburg, Germany) were used, respectively.

Body Mass Index. The BMI was calculated by dividing the mass (kg) by the square of the height (m) of a participant.

Tender Points. Sensitivity to pain was measured with a Wagner FPK digital algometer (Greenwich, CT, USA) for 18 tender points, in accordance with the classification criteria of FM of the American College of Rheumatology.26 The tender point was positive when a participant noted pain at a pressure equal to or lower than 4 kg/cm2. The total number of positive tender points was registered for each patient, and a score was calculated. An algometer score was calculated as the sum of the minimum pain-pressure values obtained for each tender point.

Daily Tablet Intake. Personal diaries kept by the participants logged their daily intake of pain medication.

Statistical Analysis Descriptive statistics are represented as means, standard deviations, and percentages. Tests for normality and homogeneity of variances (Shapiro–Wilk and Levene’s, respectively) were conducted on all data before analysis. The Student t test for continuous variables and the χ2 test for categorical variables were used to analyze the homogeneity of groups in relation to the sociodemographic characteristics.

The pretest–posttest comparison was performed using an analysis of variance of repeated measures. The level of significance was P < .05. Data analysis was performed using SPSS, version 21 (SPSS, Armonk, NY, USA).

20 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Table 1. Estimated Emissions of Far Infrared Radiation From the Fabric Area of Infrafred Spectrum

% of Total Emissionsa

<600 cm-1

12.3

<400 cm-1

4.5

This indicates the total emissions relative to those it receives at all infrared wavelengths.

a

Figure 1. Detail of the Bioceramic Deposits on the Fabric Used in the Study

Table 2. Sociodemographic Variables at Baseline P Value

PG

EG

BMI, kg/m2, mean (SD)

26.07 (3.92)

27.58 (4.82)

.292

Years since diagnosis, mean (SD)

8.11 (5.60)

10.30 (5.51)

.233 .341

Marital status, n (%) -

-

Married

Single

13 (72.2)

16 (80)

Separated/Divorced

4 (22.2)

2 (10)

Widowed

1 (5.6)

2 (10)

Work, n (%) Yes

4 (21.1)

4 (20)

No

15 (78.9)

16 (80)

1 (5.3)

3 (15)

.614

Level of education, n (%) Uneducated Primary

10 (52.6)

3 (15)

Secondary

8 (42.1)

11 (55)

University

0 (0.0)

3 (15)

.107

Abbreviations: BMI, body mass index; SD, standard deviation; PG, placebo group; EG, experimental group.

Aparecida Santos e Campos—Bioceramic Undershirt and Reduced FM

Table 3. Changes in the Variables From Baseline to Postintervention for the EG and PG PG EG Mean (SD) Mean (SD) Effect Baseline Postintervention Δ Size P Value Baseline Postintervention Δ -1.50 Pain scale (VAS), 0-10 7.08 (1.88) 7.17 (1.84) 0.08 0.004 .689 6.94 (1.46) 5.43 (1.81)a -8.96 FIQ (0-100) 74.25 (10.51) 74.76 (10.38) 0.51 0.001 .870 66.58 (14.62) 57.62 (15.61)a SF-12, physical health (0-100) 24.52 (3.99) 24.35(4.25) -0.16 <0.001 .957 27.33 (6.10) 32.48 (14.89) 5.15 SF-12, mental health (0-100) 33.69(7.94) 33.46 (7.45) -0.23 <0.001 .927 39.04 (12.39 37.30 (12.88) -1.74 -2.00 TP, 0-18 17.42 (1.12) 17.47 (1.12) 0.05 0.001 .824 17.50 (1.43) 15.50 (1.79)a 12.88 Algometer score 32.18 (10.01) 32.39 (9.92) 0.20 <0.001 .904 39.84 (15.40) 52.72 (13.42)a Daily tablet intake 6.26 (2.25) 5.68 (2.58) -0.57 0.052 .170 11.05 (8.62) 9.42 (8.07) -1.63 a

Effect Size P Value 0.582 <.001 0.191 .005 0.077 .087 0.13 .484 0.673 <.001 0.625 <.001 0.302 <.001

Significant differences (P < .05) between the EG and the PG.

Abbreviations: EG, experimental group; PG, placebo group; SD, standard deviation; Δ, change; VAS, visual analogue scale for pain; FIQ, Fibromyalgia Impact Questionnaire; SF-12, Short Form-12; TP, tender point.

RESULTS Thirty-nine women participated in the study. With a mean age of 51.51 ± 11.66 years and a mean BMI of 26.85 ± 4.41 kg/m2, they had suffered from FM for a mean of 9.26 ± 5.59 years before the start of the study.

The descriptive statistics of participants’ sociodemographic variables are shown in Table 2. No significant differences existed between the EG and the PG. Table 3 shows the changes for all variables from baseline to postintervention. For the EG, the study found a significant reduction in pain on the VAS (P < .001) and for tender points (P < .001) and algometer score (P < .001). In addition, a significant reduction in FM symptoms occurred as evaluated using the FIQ (P = .005) as well as a reduction in participants’ daily use of pain medications as calculated as the daily tablet intake (P < .001). No significant changes occurred for the PG for any of the measures. Significant differences existed between the groups for the VAS, the FIQ, tender points, and the algometer score in posttest evaluation (P < .05).

The findings of this investigation should be compared with those of other previous studies. However, to the research team’s knowledge, the current study is the first that analyzes the effects of FIR on pain in patients with FM. However, the findings of the current study support the previous research in relation to the effectiveness of FIR in the treatment of other pathologies and painful processes such as dysmenorrheal, obesity, and foot pain.20,21,23 Moreover, in accordance with this study, Chen et al29 showed that near-infrared therapy reduces pain and partially increases pressure pain threshold in patients with chronic neck pain.

Last, and in relation to perceived health, the research team observed a nonsignificant improvement in physical health in the current study. To substantiate the current study’s results, the team considers it necessary for future studies to measure participant’s pain for a period after the use of the fabric stops. In addition, a larger representative sample of patients should be tested. New research should address the 2 limitations.

DISCUSSION The main purpose of the current study was to analyze the effects of FIR using fabric coated in bioceramics on pain in patients with FM. The principal finding in the current study indicates that the habitual use of fabrics impregnated with bioceramics can produce a reduction in pain and have a positive impact on the symptoms of women with FM and a reduction in pain medication use, quantified as daily tablets intake, with no side effects from the use of the fabric.

Therefore, the improvement of the painful clinic found in this study is related to the increase of body temperature due to the action of FIR. Consistent with Chen et al,29 although the exact mechanisms underlying the therapeutic effects of FIR on pain, it is not clear yet, those findings showed that the pain reduction values following the application of FIR are encouraging. A possible explanation for improvements specific to the use of FIR is due to heating deeper tissues than ordinary heating methods.30

CONCLUSIONS Regular use of garments impregnated with a bioceramic solution (1%) can be beneficial in reducing pain and the impact of FM for patients. The low cost and comfort of the clothing make the therapy easy to be applied as a complementary tool in the treatment of FM.

Aparecida Santos e Campos—Bioceramic Undershirt and Reduced FM

ACKNOWLEDGEMENTS

The authors thank Association of FM (AFIXA).

AUTHOR DISCLOSURE STATEMENT

The authors state that they have no conflicts of interest related to the current study.

REFERENCES

1. Carbonell-Baeza A, Aparicio VA, Sjostrom M, Ruiz JR, Delgado-Fernandez M. Pain and functional capacity in female fibromyalgia patients. Pain Med. 2011;12(11):1667-1675.

2. Maquet D, Croisier JL, Renard C, Crielaard JM. Muscle performance in patients with fibromyalgia. Jt Bone Spine. 2002;69(3):293-299.

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3. Wilson HD, Robinson JP, Turk DC. Toward the identification of symptom patterns in people with fibromyalgia. Arthritis Rheum. 2009;61(4):527-534.

4. Heredia Jiménez JM, Aparicio García-Molina VA, Porres Foulquie JM, Delgado Fernández M, Soto Hermoso VM. Spatial-temporal parameters of gait in women with fibromyalgia. Clin Rheumatol. 2009;28(5):595-598.

5. Häuser W, Zimmer C, Felde E, Köllner V. What are the key symptoms of fibromyalgia? Results of a survey of the German Fibromyalgia Association.

Schmerz. 2008;22:176-183.

6. Verbunt JA, Pernot DHFM, Smeets RJEM. Disability and quality of life in patients with fibromyalgia. Health Qual Life Outcome. 2008;6:8.

7. Mengshoel AM, Forre O, Komnaes HB. Muscle strength and aerobic capacity in primary fibromyalgia. Clin ExpRheumatol. 1990;8(5):475-479.

8. Pedro Ángel LR, Campos MAS e, Mejía Meza JA, Delgado Fernández M, Heredia JM. Análise das capacidades físicas de mulheres com fibromialgia segundo o nível de gravidade da enfermidade. Rev Bras Med do Esporte. 2012;18(5):308-312.

9. Panton LB, Kingsley JD, Toole T, et al. A comparison of physical functional performance and strength in women with fibromyalgia, age- and weightmatched controls, and older women who are healthy. Phys Ther.

2006;86(11):1479-1488.

10. Góes SM, Leite N, Shay BL, Homann D, Stefanello JMF, Rodacki ALF. Functional capacity, muscle strength and falls in women with fibromyalgia. Clin Biomech.

2012;27(6):578-583.

11. Jeschonneck M. Abnormal microcirculation and temperature in skin above tender points in patients with fibromyalgia. Rheumatology. 2000;39(8):917-921.

12. Pienimaki T, Karppinen J, Rintamaki H, et al. Prevalence of cold-related musculoskeletal pain according to self-reported threshold temperature among the Finnish adult population. Eur J Pain. 2014;18(2):288-298.

13. Larson AA, Pardo J V, Pasley JD. Review of overlap between thermoregulation and pain modulation in fibromyalgia. Clin J Pain. 2014;30(6):544-555.

14. Latorre P, Santos M, Heredia-Jiménez J, et al. Effect of a 24-week physical training programme (in water and on land) on pain, functional capacity, body composition and quality of life in women with fibromyalgia. Clin Exp Rheumatol.

2013;31(6 suppl 79):72-80.

15. Masuda A, Kihara T, Fukudome T, Shinsato T, Minagoe S, Tei C. The effects of repeated thermal therapy for two patients with chronic fatigue syndrome. J Psychosom Res. 2005;58:383-387.

16. Carbonell-Baeza A, Aparicio V a., Ortega FB, et al. Does a 3-month multidisciplinary intervention improve pain, body composition and physical fitness in women with fibromyalgia? Br J Sports Med. 2011;45(15):1189-1195.

17. Lin C-C, Liu X-M, Peyton K, et al. Far infrared therapy inhibits vascular endothelial inflammation via the induction of heme oxygenase-1. Arterioscler Thromb Vasc Biol. 2008;28(4):739-745.

18. Wong CH, Lin LC, Lee HH, Liu C-F. The analgesic effect of thermal therapy after total knee arthroplasty. J Altern Complement Med. 2012;18(2):175-179.

19. York RMB, Gordon IL. Effect of optically modified polyethylene terephthalate fiber socks on chronic foot pain. BMC Complement Altern Med. 2009;9:10.

20. Huang C-Y, Yang R-S, Kuo T-S, Hsu K-H. Phantom limb pain treated by far infrared ray. Conf Proc IEEE Eng Med Biol Soc. 2009;2009:1589-1591.

21. Liau B, Leung T, Ou M, et al. Inhibitory effects of far-infrared ray-emitting belts on primary dysmenorrhea. Int J Phytoenergy. 2012;2012:1-6.

22. Ko GD, Berbrayer D. Effect of ceramic-impregnated “thermoflow” gloves on patients with Raynaud’s syndrome: Randomized, placebo-controlled study.

Altern Med Rev. 2002;7(4):327-335.

23. Conrado LA, Munin E. Reduction in body measurements after use of a garment made with synthetic fibers embedded with ceramic nanoparticles. J Cosmet Dermatol. 2011;10(1):30-35.

24. Vatansever F, Hamblin MR. Far infrared radiation (FIR): Its biological effects and medical applications. Photonics Lasers Med. 2012;4:255-266.

25. Inoue S, Kabaya M. Biological activities caused by far-infrared radiation. Int J Biometeorol. 1989;33(3):145-150.

26. Wolfe F, Smythe HA, Yunus MB, et al. The American College of Rheumatology 1990 Criteria for the Classification of Fibromyalgia: Report of the Multicenter Criteria Committee. Arthritis Rheum. 1990;33(2):160-172.

27. Rivera Redondo J, González Hernández T. The Fibromyalgia Impact Questionnaire: A validated Spanish version to assess the health status in women with fibromyalgia. Clin Exp Rheumatol. 2004;22(5):554-560.

28. Vilagut G, María Valderas J, Ferrer M, Garin O, López-García E, Alonso J. Interpretación de los cuestionarios de salud SF-36 y SF-12 en España: Componentes físico y mental. Med Clin (Barc). 2008;130(19):726-735.

29. Chen SC, Lin SH, Lai MJ, Peng CW, Lai CH. Therapeutic effects of near-infrared radiation on chronic neck pain. J Exp Clin Med. 2008;5(4):131-135.

30. Ervolino F, Gazze R. Far infrared wavelength treatment for low back pain: Evaluation of a non-invasive device. Work. 2016;53(1):157-162.

22 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Aparecida Santos e Campos—Bioceramic Undershirt and Reduced FM

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ORIGINAL RESEARCH

Efficacy of Fascial Distortion Model Treatment for Acute, Nonspecific Low-Back Pain in Primary Care: A Prospective Controlled Trial Detlef Richter; Matthias Karst, MD; Hartmut Buhck, MD; Matthias G. Fink, MD

ABSTRACT Context • Low-back pain (LBP) is a prevalent and potentially crippling condition for which treatment is often unsatisfactory from the perspectives of physicians, patients, and payers. The application of the fascial distortion model (FDM), an integrated concept for the diagnosis and manipulative treatment of musculoskeletal disorders, is conceptually promising for LBP but has not been investigated systematically.

Objective • The study intended to provide proof of concept to establish the noninferiority of the FDM treatment as opposed to the therapy recommended by the German National Disease Management Guideline (NDMG) for acute LBP. Design • The study was a prospective, nonrandomized, controlled, parallel-group trial.

Setting • The study took place in a private practice for surgery and orthopedics.

Participants • Seventy-seven outpatients with acute LBP with an average age of 42.6 ± 13.5 y, 50.6% of whom were male, took part in the study.

Detlef Richter, is a resident surgeon in surgical-orthopedic private practice in Alfeld, Germany. Matthias Karst, MD, is a professor in the Interdisciplinary Pain Clinic, Department of Anesthesia and Intensive Care, Medical School Hanover, in Hanover, Germany. Hartmut Buhck, MD, is the owner and scientific director of Medical-Scientific Consultancy in Berlin, Germany. Matthias G. Fink, MD, is a professor in the Department of Rehabilitation Medicine, Medical School Hanover in Hanover, Germany.

Corresponding author: Hartmut Buhck, MD E-mail address: hartmut@medcommtools.de

24 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Intervention • Participants in the intervention group (FDM group) received osteopathic manipulative treatments according to the FDM, whereas the control group (NDMG group) received an active control treatment following the NDMG. Outcome Measures • Comparing the FDM group (n = 39) and the NDMG group (n = 38), the study measured pain (visual analog scale, patient diary), functional (FFbH-R) and self-reported vocational status, and use of medication (patient diary) at baseline and after 1, 4 and 12 wk of treatment.

Results • The study found marked improvements of the symptoms in both groups, with a faster onset of efficacy and significantly less medication under the FDM treatment.

Conclusions • FDM appears to be effective with regard to pain relief and functional improvement for LBP. (Altern Ther Health Med. 2017;23(5):24-32)

N

onspecific low-back pain (LBP) is a prevalent and potentially disabling condition that causes a substantial economic burden due to direct and indirect costs.

Irrespective of regional and social differences, expenditures for diagnosis and treatment as well as loss of productivity and disability payments are significant,1 and the indirect costs outweigh the direct costs by a substantial margin, rendering more effective treatment modalities economical even if they are more expensive in the short term.2,3 A broad range of treatment modalities for acute and chronic LBP is available, comprising a variety of pharmaceutical, physical, interventional, surgical, and psychological methods, and, clearly, one therapy that fits all patients does not exist. The broad choice of treatment options does not remotely imply that an effective and tolerable, let alone economical, choice is available for everybody; on the contrary, many patients deal Richter—FDM Treatment for Low-Back Pain

Table 1. Definitions of Fascial Distortions and Their Symptoms and Signs Distortion Trigger band Herniated trigger point Continuum distortion Folding distortion

Cylinder distortion Tectonic fixation

Definition Twisted, banded fascia Protrusion of tissue through a fascial layer Alteration of a transition zone between bone and tendon or ligament Three-dimensional alteration of fascial tissue, mostly joint capsule, interosseous membrane, or intermuscular septum

Torsion of circular superficial fasciae of the cylinder fascia 1. Loss of sliding ability of fascial surfaces 2. Loss of quantity and quality of synovia

Body Language Finger sweeping along a painful line Finger, thumb, or knuckle pressed into the affected area Finger points at a painful spot Extremities: 1. Clasping of a joint with the hand or placing a hand on the joint (unfolding) 2. Sweeping motion with hand or finger across joint (refolding) Trunk: Laying on of the hand 1. Repeated kneading or massaging of the affected tissue 2. Broad sweeping of the hand over a larger painful area Low-back pain: 1. Placing hands over iliac crest 2. Twisting or jerking torso

Subjective Description Burning/dragging pain Dull pain Pain at a circumscribed point Pain deep within the spinal column

Deep-seated pain, tingling, numbness, or temperature sensations Stiffness

In general: Moving joint with great force

with severe and crippling pain despite the best efforts of their physicians and a sizeable armament of diagnostic and therapeutic measures.

The choice of the most appropriate treatment depends on the patient’s age, history of illness, and somatic and psychological comorbidities as well as the personal preferences of the patient and therapist; however, the treatment should not be chosen arbitrarily but should be based on the best available evidence.

The assessment of the available evidence, however, is a major task in itself; a plethora of randomized controlled trials (RCTs) and systematic reviews exist, and little robust evidence is available beyond the efficacy of analgesics and strengthening physical therapy. Therefore, treatment algorithms should be derived from evidence- and consensus-based guidelines, such as that provided in the German National Disease Management Guidelines (NDMGs)4; the joint clinical practice guideline from the American College of Physicians and the American Pain Society5; and the European guidelines for the management of acute, nonspecific LBP in primary care.6 The results of novel treatment modalities should be evaluated using the guidelineadherent therapy as a benchmark.

Osteopathy and manipulative treatment of LBP has gained some interest in the past decade7-12; however, it is difficult to judge the merits of the approach due to the variety of methods and the overall scanty evidence. Therefore, the therapist needs to establish the efficacy and safety of a given method by appropriate research.

One relatively novel method of manipulative diagnosis and treatment is the fascial distortion model (FDM), developed by the American osteopath Stephen Typaldos.13-16 It is based on the concept of the fasciae as sensitive, pain-permitting organs17-19 and uses the patient’s body language

as an indicator of the underlying functional disturbance as well as guidance for the appropriate manipulative treatment. The FDM decodes categorized manual gestures (ie, pain-related body language) to 6 pathophysiological mechanisms involved in the etiology of pain (Table 1).16 The treatment is subsequently guided by those nonverbal expressions.

The intervention mainly consists of high-velocity and low-amplitude manipulations, so-called thrusts, of the affected joints, and specific massaging procedures at the surrounding connective tissue.20 Those procedures are based on the concept of a maladaptation of the crosslinks along the subcutaneous fasciae and a subsequent tension-free readaptation after manipulation.

Two of the most prevalent gestures in patients with LBP indicate distortions, called the herniated trigger point and the trigger band.

Herniated Trigger Point. The patient’s symptom is a dull pain in the gluteal region or the trigonum lumbale. Typically, as the sign of the condition, the patient presses his or her thumb or the knuckle of a finger into the area affected by the pain, and that pressure causes some relief, which, however, is only transient. The therapeutic solution is to follow the patient’s body language and apply increased pressure in the same area. The pressure is held until the patient experiences relief (ie, after approximately 2 min). The relief is often lasting, and the patient may not require further treatment.

Trigger Band. As the sign of the condition, the patient makes a sweeping motion with 1 or more fingers along an imaginary, painful, linear pathway. In LBP, that pathway typically starts in the paravertebral region of the thoracolumbar spine and extends over the gluteal region to the lateral or dorsal thigh. The fascial band is treated with a sweeping motion of the tip of the thumb that follows the imaginary line

Richter—FDM Treatment for Low-Back Pain

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 25

shown by the patient. The intervention typically loosens the restrictions of motion often associated with LBP. Patients often perceive the intervention as rather painful but report quick and substantial relief shortly afterward.

Systematic clinical trials of the FDM are scarce13,21,22 but indicate efficacy that is superior to that of a placebo. The only comparative study published so far found the FDM to be more efficacious than conventional manipulative treatment in patients with frozen shoulders.21 The application of the FDM in LBP is therefore promising but has not been systematically investigated. The present study is a prospective, nonrandomized, controlled, parallel-group trial intended to provide proof of concept to establish the noninferiority of the FDM treatment compared to the NDMG-guided therapy for acute LBP. The study’s nonrandomized design is due to the complex nature of the patient-therapist interactions in FDM, as in all other manipulative methods of treatment. A reliable randomization would have required sample stratification according to a number of criteria, and, consequently, the required sample size would have been prohibitive for an early-phase evaluation of the method. A successful demonstration of noninferiority against the conventional guideline-adherent treatment might be a rational basis for a future RCT. MATERIALS AND METHODS Participants The study took place in a private practice for surgery and orthopedics of 1 member of the research team. The participants were recruited at that private practice between July 2012 and December 2013.

To be included, prospective participants needed to be (1) seeking treatment at the investigator’s private practice for acute, nonspecific, LBP of up to 2 weeks in duration; (2) aged older than 18 years; (3) able and willing to understand the trial’s purpose and to communicate with the investigator; and (4) willing to provide written informed consent for participation after detailed information about the purpose and methods of the study.

Prospective participants were excluded if they (1) showed signs of inflammatory and/or rheumatic spinal diseases; (2) showed signs of anatomical alterations such as spondylarthritis or hereditary malformations; (3) had had traumata or operations of the pelvis, hip, or spine within the 3 months prior to enrollment; (4) had neurological diseases or signs of nerve compression; (5) had spinal hypermobility or instability; (6) had clinically significant arthritis; (7) had malignancies; (8) had coagulation disorders; or (9) were pregnant.

Upon visiting the clinic, eligible patients were informed about the study’s design and about the potential benefits and risks of the 2 treatment alternatives. Only patients who did not express a preference between the FDM- and the NDMG-based treatment were enrolled.

Of 483 patients who called the practice for an appointment during this period, 224 were excluded because 26 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

of complaint duration in excess of the prespecified 2-week threshold. A total of 181 of the remaining 259 patients met 1 of the exclusion criteria, most frequently because of longer pain duration upon close scrutiny (n = 80) or signs of specific causes of back pain (eg, inflammatory or rheumatic signs, disc herniation, lumbar fractures; n = 67). One patient was lost to follow-up after the first intervention, leaving 77 patients for evaluation.

The choice of treatment by the investigator was sequential (ie, the first 38 patients who were eligible and provided informed consent to participate were treated according to NDMG and the latter half according to FDM).

Patients were stratified for age, gender, and crude prognosis assessment (ie, the presence of LBP in the 12 months before enrollment and their functional capacity)24 to provide sufficient sample sizes for pertinent subgroup analyses.

The study’s materials and methods are reported according to the Transparent Reporting of Evaluations with Nonrandomized Designs (TREND) statement.23 The study’s protocol was approved by the ethics committee of the Medical School Hanover (approval No. 5846).

Procedures Patients’ statuses were evaluated at baseline and after 1, 4, and 12 weeks of treatment. At baseline only, the research team recorded the anamnestic duration and intensity of symptoms for the patient and his or her previous incapacitation(s) related to work. At 1, 4, and 12 weeks of treatment, the research team measured the primary, coprimary, and secondary outcome endpoints described in the following.

The primary endpoint for the efficacy analysis was the percentage of patients who were completely free of symptoms at the final examination. The coprimary endpoint was the percentage of responders at 1, 2, and 12 weeks after treatment onset.

The following parameters were analyzed as secondary endpoints: (1) pain-free range of motion of the spine—fingerfloor distance and lateral flexibility; (2) incapacitation related to work in days; (3) use of analgesics, including frequency and dosage; (4) frequency of specialist consultations; and (5) pain scores according to the patient diaries Interventions Patients in the intervention group (FDM group) were treated according to the FDM once per week for a prespecified maximum of 4 weeks, which was, however, not fully exploited due to early treatment response. In addition to the FDM treatment, the patients were allowed to receive all required guideline-conformant modalities, such as analgesic medications, topical applications, and active exercises. To provide specific information on the FDM’s efficacy, all concomitant interventions were meticulously recorded, and both treatment arms were statistically controlled for possible confounding effects of those interventions. Patients with a persistent treatment demand beyond the 4 weeks of the intervention or with 6 weeks of symptoms were classified as nonresponders.

Richter—FDM Treatment for Low-Back Pain

The active control treatment was performed according to the German NDMG4 and included all modalities recommended therein with the exception of FDM treatment. The recommendations herein are derived from international guidelines and are mostly identical with those given in pertinent American5 or European6 guidelines. Accordingly, the treatment included the following interventions in varying, patient- and investigator-driven combinations: (1) analgesic and anti-inflammatory pharmacotherapy, (2) active exercises, instead of immobilization, (3) thermotherapy, and (4) relaxation/psychoeducation.

Like for the intervention group, all treatments were recorded, and patients with persisting treatment demands beyond the 4 weeks of therapy or 6 weeks of symptoms were deemed nonresponders.

Outcome Measures The study protocol and study report include a number of outcome criteria for further analysis (eg, subjective experience of the treatment, current complaints, FABQ26) that are not referred to in this study.

Absence From Work. This measurement was self-reported and expressed in days of work missed by the participant. Each reporting opportunity collected only those days missed since the participant’s most recent visit to the clinic.

Physical Examination. In the examination, the research team measured the finger-floor distance (in maximal painfree anteflexion) and the Schober sign, both in centimeters.

Hanover Functional Ability Questionnaire. The questionnaire provided a score of a patient’s functional capacity. It was specifically developed for LBP.25 Pain Intensity Questionnaire. The study measured pain on a 10-cm visual analogue scale (VAS) with a range between 0 (no pain at all) and 10 (excruciating, unbearable pain). A reduction of 33% in pain intensity was employed as a threshold for response analyses.

Patient Diaries. These included pain, medication, interference with activities of daily living and sleep. Patients recorded pain intensity (VAS) and any intake of analgesic agents (with substance and dosing) as well as interference of their back pain with activities of daily living and sleep in a diary throughout study duration.

Sample-size calculations were based on that wider range of noninferiority and yielded a minimum of 34 patients per group, with α = .05 and a power of 80%. Statistical testing of metric and discrete parameters for significance was performed employing established nonparametric methods, with P < .05.

The outcomes for the intervention were statistically tested for noninferiority in comparison with the therapy that followed the NDMG.4 Efficacy limits for primary and secondary endpoints were chosen according to published studies versus a placebo, and statistical testing for noninferiority was performed according to the Consolidated Standards of Reporting Trials (CONSORT) methodical principles.30,31 RESULTS Baseline Seventy-seven outpatients, 38 females (49.4%) and 39 males (50.6%), participated in the trial. Their mean age was 42.6 ± 13.5 years, with a range from 19 to 75 years, and the patients were on average slightly overweight, with a mean body mass index (BMI) of 26.4 ± 5.2 and a range from 18.5 to 43.9. Patients in the 2 treatment groups showed no significant differences with respect to sociodemographic and baseline data (Table 2).

After the start of treatment, follow-up examinations were performed at approximately 1 week, with a mean of 7.5 ± 2.9 days (T2); at approximately 4 weeks, with a mean of 30.4 ± 4.2 days (T3); and at approximately 12 weeks, with a mean of 86.7 ± 6.7 days (T4) after the initial evaluation, and no significant group differences existed regarding the time between treatment and assessment. The slight deviations of the actual assessment date from exactly 1, 4, and 12 weeks were owing to practical considerations (public holidays, appointment availabilities, etc) in the private practice study setting.

Table 2. Participants’ Baseline Data All Participants Parameter N = 77 Age, y Range 19 to 75 42.6 ± 13.5 Mean ± SD Gender, n (%) Male 39 (50.6%) Female 38 (49.4%) BMI Range 18.5 to 43.9 26.4 ± 5.2 Mean ± SD Duration of acute pain, days Range 1 to 18 6.9 ± 4.4 Mean ± SD Pain within the past 63 (81.8%) 12 mo, n (%)

FDM Group N = 39

NDMG Group N = 38

19 to 75 40.3 ± 14.8

20 to 69 45.0 ± 11.8

20 (51.3%) 19 (48.7%)

19 (50.0%) 19 (50.0%)

18.5 to 37.3 25.5v± 4.7

19.4 to 43.9 27.4 ± 5.6

1 to 18 6.6 ± 4.3 34 (87.2%)

1 to 14 7.3 ± 4.5 29 (76.3%)

Statistical Analyses The required sample size for the present trial was calculated based on a noninferiority hypothesis with respect to the endpoint of chronification of the acute LBP (ie, persistence of symptoms beyond 3 months after the onset of treatment). A difference of 10% regarding that endpoint was considered clinically meaningful and socioeconomically relevant29 and, thus, was chosen as the predefined difference (D) for the statistical testing for noninferiority.

However, due to the very low risk of side effects for the FDM treatment in the previous experience of the research team, an extension of the noninferiority margin to 20% was considered acceptable and approved by the ethics committee.

Abbreviations: FDM, fascial distortion model; NDMG, German National Disease Management Guideline; SD, standard deviation; BMI, body mass index

Richter—FDM Treatment for Low-Back Pain

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 27

Figure 1. Results of Statistical Testing for Noninferiority of the Primary Endpoint

NDMGM Better

Odds Ratio

Table 3. Outcome of Primary and Selected Secondary Endpoints All Participants FDM Group NDMG Group N = 77 N = 39 N = 38 Mean ± SD Mean ± SD Mean ± SD 38 (49.4%) 20 (51.3%) 18 (47.4%)

Parameter Freedom from symptoms at 12 wk after treatment onset, n (%) Response after, n (%) 1 wk 71 (92.2%) 4 wk 76 (98.7%) 3 mo 75 (97.4%) Finger-floor distance (cm) baseline 35.4 ± 21.5 after 1 wk 13.9 ± 12.2 after 4 wk 09.0 ± 10.0 after 3 mo 08.9 ± 12.5 Schober sign (cm) baseline 1.92 ± 0.66 after 1 wk 3.00 ± 0.73 after 4 wk 3.22 ± 0.74 after 3 mo 3.88 ± 0.80 FFbHR (%) baseline 60.5 ± 22.9 after 1 wk 78.6 ± 17.7 after 4 wk 85.5 ± 16.0 after 3 mo 87.7 ± 15.6 Inability to work (d) baseline 1.81 ± 3.34 after 1 wk 2.51 ± 4.68 after 4 wk 2.99 ± 5.84 after 3 mo 3.26 ± 5.91

FDM Better

Note: The endpoint had a prespecified Δ requiring 10% or 20% of patients to be completely symptom free at the end of observation, respectively. If the lower 95% confidence interval would have been within the green area (depicting the more “generous” 20% interval), noninferiority would have been demonstrated. Both intervals were prespecified and tested, but only the 20% interval is shown in the figure.

Abbreviations: NDMG, German National Disease Management Guideline; FDM, fascial distortion model.

Primary Endpoint Overall, 38 of the 77 patients (49.4%) were completely free of symptoms at the time of the final examination at 12 weeks after initiation of the treatments (Table 3). The primary endpoint was met in 20 of the 39 patients (51.3%) in the FDM group and in 18 of the 38 patients (47.4%) in the NDMG group (odds ratio [OR], 1.29; 95% confidence interval [CI], 0.52 to 3.21), with the difference not being statistically significant. Statistical testing for noninferiority with a prespecified Δ of 20% yielded an inconclusive result with regard to the primary endpoint (Figure 1).

According to the primary response criterion of ≥33% pain reduction on the VAS, the FDM group consistently scored slightly better than controls, but the difference was statistically not significant throughout the observations.

37 (94.9%) 39 (100.0%) 39 (100.0%)

34 (89.5%) 37 (97.4%) 36 (94.8%)

34.1 ± 22.8 13.9 ± 13.9 08.7 ± 12.0 10.1 ± 12.6

36.7 ± 20.4 13.9 ± 10.4 09.3 ± 7.6 07. 7 ± 12.4

1.95 ± 0.65 2.87 ± 0.66 3.21 ± 0.74 3.18 ± 0.68a

1.89 ± 0.69 3.13 ± 0.78 3.23 ± 0.75 3.59 ± 0.86a

61.5 ± 20.6 77.2 ± 19.4 85.7 ± 17.3 86.3 ± 16.2

59.5 ± 25.3 80.0 ± 15.9 85.3 ± 14.6 89.1 ± 15.0

1.41 ± 3.23 1.97 ± 4.51 2.38 ± 5.09 2.69 ± 5.23

2.21 ± 3.44 3.05 ± 4.85 3.61 ± 6.52 3.84 ± 6.56

P < 0.05 for comparison between groups

a

Abbreviations: FDM, Fascial Distortion Model; NDMG, German National Disease Management Guideline; SD, standard deviation; FFbHR, Hanover Functional Ability Questionnaire Table 4. Number of Participants Taking NSAIDs and Ibuprofen Doses During the First Week

Parameter Ibuprofen Intake Dose, mg/7 d, mean ± SD Additional analgesic or anti-inflammatory medication for the 67 patients taking ibuprofen Other analgesic or anti-inflammatory medication for the 10 patients not taking ibuprofen

All Participants N = 77 N (%)

FDM Group N = 39 N (%)

NDMG Group N = 38 N (%)

P Value

67 (87.0%) 33 (84.6%) 34 (89.5%) 7746 ± 3245 6872 ± 3523 8594 ± 2813

.737 .052

10 (14.9%)

3 (9.1%)

7 (20.6%)

.305

3 (30.0%)

2 (33.3%)

1 (25.0%)

1.000

Secondary Endpoints The evaluation of participants’ pain diaries showed that their complaints—pain intensity and frequency, impairment of activities of daily living, and interference with sleep— declined in both groups, but the improvements occurred noticeably faster under FDM when compared to the NDMGguided treatment. The differences were not statistically different and diminished beyond the first week, so that toward then end of observation, both groups showed more or less identical behavior. A slow further improvement of the aforementioned complaints occurred until approximately week 6, after which the patients’ statuses remained largely

Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs; FDM, fascial distortion model; NDMG, German National Disease Management Guideline; SD, standard deviation.

28 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Richter—FDM Treatment for Low-Back Pain

Figure 2. Pain Intensity on a VAS During Observation of Use of FDM Therapy and an NDMG-adherent Control Treatment No significant differences between treatment groups Statistical significance for noninferiority (P < .05)

Statistical significance for noninferiority (P < .05)

Control

Pain Intensity (VAS, mean ± SD)

Treatment

Abbreviations: VAS, visual analogue scale; FDM, fascial distortion model; NDMG, German National Disease Management Guideline; SD, standard deviation; FFbHR, Hanover Functional Ability Questionnaire.

Figure 3. Percentage of Patients With Analgesic Medication During the First Week of FDM Therapy and an NDMG-adherent Control Treatment

Δ 28% - 32% P = .05 NNT = 3.2 to 3.5

all but 2 points of observation, with the latter 2 points of observation being inconclusive.

Analysis of further secondary endpoints such as analgesics demand confirmed the assessment derived from the remission/response and pain analyses.

The faster onset of effects under FDM therapy became evident when the analgesic medication was studied (Table 4). Between day 3 and week 2, significantly fewer patients in the FDM group required analgesics and/or nonsteroidal antiinflammatory drugs (NSAIDs). The difference was consistently in the order of magnitude of one third (ie, if 3 to 3.5 additional patients had been treated with FDM, 1 of them would have been able to cease medical treatment completely). See Figure 3. More specifically, Table 4 shows the number of patients under ibuprofen medication, a standard NSAID in NDMGguided treatment, with the administered doses. The proportion of patients under medication was largely equivalent, with the patients under FDM requiring an appreciably smaller amount, and the difference of approximately 1700 mg/week only just failing to be statistically significant.

Functional parameters in Table 3 confirmed the tendency toward slightly better results for the FDM, largely without statistical significance. However, the consistent observation of a difference of approximately 1 day of incapacitation related to work might be economically, if not statistically, significant.

Influence of Baseline on Outcomes Because of the very high percentage of patients who met the coprimary endpoint of response to treatment, the analysis of possible confounding factors Abbreviations: FDM, fascial distortion model; NDMG, German National Disease did not promise to be informative and was therefore omitted. The results of the Management Guideline; NNT, number needed to treat. logistic regression for the primary unchanged. No recurrence of complaints was observed endpoint of freedom from symptoms at 12 weeks after within the observation period. treatment are shown in Table 5. None of the baseline As an example, Figure 2 shows the VAS values for pain parameters showed a significant influence on the outcome in intensity in both groups. Between days 2 and 5, an appreciable, the multifactorial testing, and in particular, no tendency albeit insignificant, difference was found in favor of the existed at all indicating a detrimental influence for patients patients under FDM treatment that then evened out from day with more severe symptoms at baseline. The tendency toward 6 onward. More important than that slight difference was the a slightly better outcome after FDM treatment was confirmed, fact that statistical testing yielded results showing noninferiority with a 14% difference in the OR, and younger patients, with regard to pain intensity, with a prespecified Δ of 1 cm, for females, and patients without pain within the 10 months Richter—FDM Treatment for Low-Back Pain

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 29

Table 5. Influence of Baseline Parameters on Treatment Outcome, the Primary Endpoint Factor Treatment modality Gender Age Duration of pain Pain with the prior 10 mo Pain upon diagnosis FFbHR upon diagnosis

Unifactorial Evaluation Group (unit) Odds Ratio (95 % CI) P value FDM treatment 1.16 (0.47 to 2.86) .73 Male 2.20 (0.88 to 5.55) .093 Per year 0.96 (0.93 to 1.00) .048 Per day 0.97 (0.88 to 1.08) .59 None 3.13 (0.87 to 11.2) .081 Per unit VAS 0.96 (0.75 to 1.23) .73 Per % 1.01 (0.99 to 1.03) .57

Multifactorial Evaluation Odds Ratio (95 % CI) P value 1.14 (0.40 to 3.26) .80 2.28 (0.80 to 6.51) .12 0.97 (0.93 to 1.01) .12 0.99 (0.88 to 1.12) .93 3.10 (0.74 to 12.9) .12 0.95 (0.71 to 1.28) .75 1.00 (0.97 to 1.02) .77

Abbreviations: CI, confidence interval; FDM, fascial distortion model; FFbHR, Hanover Functional Ability Questionnaire. prior to enrollment fared slightly better than their respective counterparts.

DISCUSSION The unmet needs in the treatment of acute LBP are undeniable and substantial, both with regard to the individual patient’s well-being and from a payers’ perspective,5 and, therefore, exploratory research such as presented in the present trial is well justified. Before the discussion of the present trial’s results, the proof-of-concept character of the study has to be emphasized. The FDM as such has scarcely been investigated systematically,21 and no evidence had been published on its application in LBP as of October 2015.

Therefore, the current research team decided to gather some solid, albeit preliminary, evidence from a nonrandomized trial before launching a much more expensive RCT. Against that background, the results of the present trial appear very conclusive, and the fact that the CONSORTconformant test for noninferiority was inconclusive should not be given too much weight. In fact, although the primary endpoint could technically not be shown to be noninferior, the pain intensity for the intervention group on the VAS was noninferior at all but one points of assessment; moreover, the observation was confirmed with noticeably lower doses of analgesic medication being taken by the FDM patients. That observation is all the more remarkable because the patients were self-dosing the medication, (ie, taking it ad libitum) and the FDM intervention as such is rather painful in the short term. That pain might have prompted the patient to take the NSAID after treatment to alleviate the immediate effects.

Overall, the current research team’s observations showed that the 2 examined interventions yielded a largely equivalent effect over the entirety of the observation period, but the beneficial effects occurred noticeably earlier in patients treated according to the FDM. It is remarkable that the same results—efficacy equivalent to established treatment but

earlier improvement under FDM therapy—were observed in a study on use of the FDM for a frozen shoulder.21 Even under full consideration of the methodological constraints of early exploratory research, that finding strongly indicates the ability of FDM treatment to exploit the individual patient’s existing potential for quick and substantial improvement.

Moreover, the validity of those observations was underlined by a reduced demand for analgesic medication and by the fact that patients under the FDM treatment returned to work one day before their NDMG-treated counterparts. Although the difference was not statistically significant, again owing to the current study using a proof-ofconcept design resulting in low statistical power, its economic effect may potentially be substantial. It is well established that absence form work causes the majority of the overall economic burden of LBP,1,32 and a gross expenditure of €150 to €300 per sick-leave day can be estimated from data of insurers, hospital operating companies, and published pertinent evidence.33 German health surveillance is notoriously sketchy and unreliable, and pain syndromes especially are therefore difficult to quantify.34 However, the data of the largest payer in Germany showed approximately one million sick-leave cases, at approximately 14 days of absence, in 2008.35 According to those figures, a savings of €150 million per year would be attainable in that insured population alone, even based on the lower margin of the aforementioned estimate, and that is an order of magnitude in which a payer should certainly be interested. From an economic standpoint, the systematic application of FDM instead of NDMG-guided treatment might save substantial amounts if the data in the present trial could be validated by a further study, and funding of pertinent research could be in the best interests of all stakeholders involved.

The major principle of FDM is the common reflection of the course of injury and the therapist’s evaluation of the patient’s body language and clinical symptoms, which altogether can lead to a diagnosis and an exactly defined therapeutic consequence. Therefore, the patient participates as a director of her or his own treatment.

The exact mechanism of action of the FDM method is currently elusive. The originator of the method assumed an

30 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Richter—FDM Treatment for Low-Back Pain

Adverse Events In addition to the known collateral effects of FDM treatment that were part of the consent form, such as pain and subcutaneous hematomas, no adverse events occurred in either group.

influence on fibroblast activity, and, consequently, alterations of the extracellular connective-tissue matrix.13-16 Whereas that model is reasonably plausible to explain a slowing of the progress of inflammatory alterations in the periarticular tissue of the spinal column, it is hardly a sufficient concept for the quick pain relief and mobility gains observed in the current study’s participants as well as those with a frozen shoulder in the previously published study.21 A leading characteristic of the FDM treatment is the significant discomfort/pain that patients experience during the manipulation. In contrast to some simplistic assumptions, inflicting pain as such is not an effective means to treat it36; however, a body of evidence is emerging concerning the modulation of pain-signal transmission and processing through counterstimulation.37,38 Results of clinical trials have indicated that so-called sham interventions that inflict physical stimuli without following proposed therapeutic principles can show some efficacy in LBP and other painful musculoskeletal syndromes39-43 and, therefore, a powerful counterstimulation that activates gate-control mechanisms needs to be considered as one potential mode of action for the FDM treatment.

The results of the present trial require confirmation by further study and are thus to be treated as preliminary for the time being. It also needs to be pointed out that no sufficient basis exists for treatment recommendations that divert from those in the German NDMG or other international guidelines.

However, the current research team believes that the results of the present trial provide a sufficient basis for open-ended discussions with patients who actively seek alternatives to the mainly pharmacotherapy-driven principles of guideline-adherent treatment.

To further solidify the current research team also considers the present results to be a sufficient basis for the planning of an RCT that compares the same treatment groups as the present trial did. Because it is virtually impossible to establish a credible placebo modality, with a proven lack of efficacy, for FDM treatment and because the present trial established its efficacy, the step of placebo-controlled efficacy testing can and should be omitted.

At the same time, neither patient nor physician can be blinded against the treatment given to a participant, and blinded evaluation by a noninvolved investigator can only partly eliminate potential pitfalls in RCT evaluation.

Therefore, a future RCT needs to include potentially confounding parameters as stratification variables for randomization to avoid bias and yield meaningful results.

Together with the required statistical power to demonstrate noninferiority or superiority compared to established treatment standards, an appropriate study design must involve a sizeable patient population and prolonged recruitment time due to stratification of randomization.

Feasibility of such a study can and will be assessed based on the data from the present trial.

The present study is only the third clinical trial involving the FDM, and, therefore, its evidence is limited to a proof-of-

concept of the method’s efficacy. Future studies should reach beyond that point and elucidate the mechanism of action of the method more specifically. Presently, it is open to debate as to the extent that the efficacy of the FDM, which has been convincingly demonstrated in the present trial, is owing to the specific methodological principles of FDM treatment.

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ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 31

CONCLUSIONS FDM was shown to be efficacious with regard to specific pain relief and functional improvement in patients with LBP, and its results were comparable with guideline-adherent treatment. The potential of FDM to become part of the standard armament of treatment modalities for LBP should be elucidated in further RCTs. AUTHOR DISCLOSURE STATEMENT

The research team received no funding in relation to the study and has no conflicts of interest.

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26. Pfingsten M, Kröner-Herwig B, Leibing E, Kronshage U, Hildebrandt J. Validation of the German version of the Fear-Avoidance Beliefs Questionnaire (FABQ). Eur J Pain. 2000;4(3):259-266.

27. Pfingsten M, Lueder S, Luedtke K, Petzke F, Hildebrandt J. Significance of physical performance tests for patients with low back pain. Pain Med. 2014;15(7):1211-1221.

28. Semrau J, Hentschke C, Buchmann J, et al. Long-term effects of interprofessional biopsychosocial rehabilitation for adults with chronic non-specific low back pain: A multicentre, quasi-experimental study. PLoS One. 2015;10(3):e0118609.

29. Valat JP, Goupille P, Rozenberg S, et al. Acute low back pain: Predictive index of chronicity from a cohort of 2487 subjects: Spine Group of the Société Française de Rhumatologie. Joint Bone Spine. 2000;67(5):456-461.

30. Piaggio G, Elbourne DR, Altman DG, Pocock SJ, Evans SJ. Reporting of noninferiority and equivalence randomized trials: An extension of the CONSORT statement. JAMA. 2006;295(10):1152-1160.

31. Piaggio G, Elbourne DR, Pocock SJ, Evans SJ, Altman DG. Reporting of noninferiority and equivalence randomized trials: Extension of the CONSORT 2010 statement. JAMA. 2012;308(24):2594-2604.

32. Plass D, Vos T, Hornberg C, et al. Trends in disease burden in Germany: Results, implications and limitations of the Global Burden of Disease study. Dtsch Arztebl Int. 2014;111(38):629-638.

33. Ekman M, Johnell O, Lidgren L. The economic cost of low back pain in Sweden in 2001. Acta Orthop. 2005;76(2):275-284.

34. Häuser W, Neugebauer E, Petzke F. Versorgungsforschung zu Schmerz in Deutschland: Eine Bestandsaufnahme. Schmerz. 2015;29(5):469-478.

35. Gesundheit Statistik Gesundheitsberichterstattung des Bundes. Federal statistical health reporting. https://www.gbe-bund.de/. Accessed June 14, 2017.

36. Winters JC, Sobel JS, Groenier KH, Arendzen HJ, Meyboom-de Jong B. Comparison of physiotherapy, manipulation, and corticosteroid injection for treating shoulder complaints in general practice: randomised, single blind study.

BMJ. 1997;314(7090):1320-1325.

37. Gadsby JG, Flowerdew MW. Transcutaneous electrical nerve stimulation and acupuncture-like transcutaneous electrical nerve stimulation for chronic low back pain. Cochrane Database Syst Rev. 2000(2):Cd000210.

38. Kumar K, Rizvi S. Historical and present state of neuromodulation in chronic pain. Curr Pain Headache Rep. 2014;18(1):387.

39. Bronfort G, Haas M, Evans RL, Bouter LM. Efficacy of spinal manipulation and mobilization for low back pain and neck pain: A systematic review and best evidence synthesis. Spine J. 2004;4(3):335-356.

40. Liu L, Skinner M, McDonough S, Mabire L, Baxter GD. Acupuncture for low back pain: An overview of systematic reviews. Evid Based Complement Alternat Med. 2015;2015:328196.

41. Dickenson AH. Gate control theory of pain stands the test of time. Br J Anaesth.

2002;88(6):755-757.

42. Fink MG, Kunsebeck H, Wipperman B, Gehrke A. Non-specific effects of traditional Chinese acupuncture in osteoarthritis of the hip. Complement Ther Med. 2001;9(2):82-89.

43. Witte W. Schmerz und Anästhesiologie Aspekte der Entwicklung der modernen Schmerztherapie im 20: Jahrhundert. Anaesthesist. 2011;60(6):555-566.

32 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Richter—FDM Treatment for Low-Back Pain

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ORIGINAL RESEARCH

Antidiabetic, Antioxidant, and Hypolipidemic Potential of Sonchus asper Hill Rahmat Ali Khan, PhD

ABSTRACT Context • The recently growing occurrence of diabetes mellitus (DM) in the world is of immense concern.

Sonchus asper Hill (SA) is traditionally used in the treatment of diabetes and urinary disorders in Pakistan.

Objective • The present study intended to evaluate the antidiabetic activity of a Sonchus asper methanol extract (SAME) in rats.

Design • Thirty male, Sprague-Dawley rats, from 180 to 190 g each, were randomly divided into 5 groups of 6 rats each and received the following treatments: (1) group 1 (untreated control group) administered distilled water only; (2) group 2 administered 200 mg/kg of SAME only; (3) group 3 (diabetic control group) administered 55 mg/kg body weight (BW) of streptozotocin (STZ) in distilled water to induce diabetes; (4) group 4 administered 55 mg/kg BW of STZ in distilled water to induce diabetes and given 100 mg/kg of SAME; and (5) group 5 administered 55 mg/kg BW of STZ to induce diabetes and administered 200 mg/kg of SAME. Setting • The study was conducted in the Department of Biotechnology Bannu (Bannu, Pakistan).

Outcome Measures • The research team performed (1) a serum analysis for glucose, lipase, and insulin; (2) a measurement of the protein concentration of the Rahmat Ali Khan, PhD, is an assistant professor in the Biotechnology Department, University of Science and Technology, in Bannu, Pakistan.

supernatant from pancreatic tissue; (3) an assay of catalase activity; (4) an assay of superoxide activity; (5) an assessment of lipid peroxidation enzymes; (6) an assay of glutathione-S-transferase activity; (7) an assay of glutathione reductase activity; (8) an assay of glutathioneperoxidase activity; and (9) an assay of lipid peroxidation (TBARS) activity.

Results • The administration of STZ caused significant alterations in the blood glucose levels, the lipase activity, insulin secretions, cholesterol profiles, serum marker enzymes, antioxidant enzymes, and contents of thiobarbituric acid reactive substances (TBARS) in the rats (P < .01). On oral administration, the SAME showed statistically significant effects by improving the activity of the antioxidant enzymes, TBARS contents, and cholesterol profile of the diabetic rats (P < .01). In treatment, the glucose and insulin levels of the SAME rats were significantly lower than the diabetic rats on day 21 (P < .01).

Conclusions • The findings suggest that the significant antidiabetic potential of the SAME in ameliorating the diabetic conditions in diabetic rats might be due to the presence of the bioactive constituents in the extract.

(Altern Ther Health Med. 2017;23(5):34-40)

iabetes mellitus (DM) is a hormonal, metabolic, and chronic disorder associated with the dysfunction and failure of various organs in the body, such as the kidneys, nerves, heart, and pancreas.1,2 It is caused by partial or complete insulin deficiency, which produces inadequate blood glucose control and leads to acute and chronic

complications.3 Hyperglycemia, increased water intake, and polyurea are the main indications of DM.3 Streptozotocin (STZ) is produced by Streptomyces achromogenes, has been widely used to induce DM in experimental animals, and causes necrosis of pancreatic β-cells.4,5 The induction of STZ leads to an increase in the generation of reactive oxygen species (ROS), inhibits freeradical scavengers,6,7 and causes lipid peroxidation and DNA damage.8,9 The ROS produced by the STZ react with enzymes and reduce their activity. Antioxidant defense systems include the enzymes superoxide dismutase (SOD), catalase (CAT), seleniumdependent glutathione peroxidase, glutathione reductase (GSR), nonenzymatic glutathione (GSH), and vitamin E.10

34 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Khan—Sonchus asper Hill

Corresponding author: Rahmat Ali Khan, PhD E-mail address: rahmatgul_81@yahoo.com

D

In Pakistan, medicinal plants play an important role in diabetic control and reduction of glucose levels.11 Currently, several hundred medicinal plants have reportedly been used in the cure of diabetes.12,13 Sonchus asper (SA) has been used as a treatment for (1) wound healing14; (2) liver and kidney disease15,16; (3) cough, bronchitis, and asthma17,18; (4) malaria19; (5) erectile dysfunction20; (6) fever; (7) constipation; (8) diabetes; (9) scabies; and (10) heart disease,21 and it has been used as a diuretic and an antiseptic.17 Chemical studies have indicated that SA contains phenolic compounds, vitamin C (ascorbic acid), carotenoids, and omega-3 fatty acids.22 Phenolic compounds, which are secondary metabolites in plants, are one of the most widely occurring groups of phytochemicals that exhibit a wide range of physiological properties, such as antioxidant, antiallergenic, antimicrobial, antidiabetic, antithrombotic, anti-inflammatory, vasodilatory, and cardioprotective effects.23 Many phenolics, such as flavonols and flavonoids, are of particular importance because they have been found to possess antioxidant properties and hypoglycemic activity that are much stronger than those of vitamins C and E.24,25 The recently growing occurrence of DM in the world is of immense concern. Thus, the current research team has thought it sensible to investigate the amelioration of experimentally induced DM with SA. Therefore, the core objectives of the present study were to (1) evaluate whether the pancreas is subjected to oxidative damage during diabetes and (2) examine the accompanying changes in antioxidant status to understand the role of antioxidants in the pathogenesis of DM. The team has also probed whether SA protects pancreatic β-cells against STZ-induced injury.

Antidiabetic Activity. The groups of rats were divided as follows: (1) group 1 (untreated control group)— administered distilled water only; (2) group 2—administered 200 mg/kg of SAME only; (3) group 3 (diabetic control group)—administered 55 mg/kg body weight (BW) of STZ to induce diabetes; (4) group 4—administered 55 mg/kg BW of STZ to induce diabetes and given 100 mg/kg of SAME; and (5) group 5—administered 55 mg/kg BW of STZ to induce diabetes and administered 200 mg/kg of SAME. The STZ was given in distilled water according to the standard procedure of Winter et al,26 with some modifications.

Those rats that developed diabetes were afterward given the standard rat chow for 21 days. The nondiabetic rats were given a normal diet and drinking water and 12 hours per day of night-light cycle, and they were kept at room temperature.

Procedures Plant Material. SA at maturity was identified and collected from District Bannu in Pakistan. A specimen was submitted to the Herbarium of Pakistan at Quaid-i-Azam University (Islamabad, Pakistan) for future reference. Aerial parts of the plant—leaves, stem, flowers, and seeds—were shade-dried at room temperature for 2 weeks, chopped, and ground mechanically to a mesh size of one mm.

Preparation of Plant Extract. A total of 2 kg of a dried sample of SA was extracted twice with 6 liters of 80% methanol at 25°C for 48 hours to produce SA methanol extract (SAME).

The extract was filtered through No. 1 filter paper from Whatman Labware products and concentrated using a rotary evaporator (Panchun Scientific Co, Kaohsiung, Taiwan) under reduced pressure at 40°C. The dried extract was stored at 4ºC for the antidiabetic and hypoglycemic investigations.

Outcome Measures Serum Analysis. The serum analyses for glucose, lipase, and insulin were carried out through via a spectrophotometer UV-2800 (Biotechnology Medical Services, Pointe-Claire, Quebec, Canada), while the serum insulin was found through an RIA gamma counter (Biotechnology Medical Services, Pointe-Claire, Quebec, Canada). The lipid profiles and serummarker enzymes were estimated via a spectrophotometer UV-2800 (Biotechnology Medical Services, Pointe-Claire, Quebec, Canada) using kits. All the kits were purchased from Sigma-Aldrich (St Louis, MO, USA).

Antioxidant Enzymes. Pancreatic tissue was homogenized in 10 volumes of a 100-mmol KH2PO4 buffer containing 1 mmol of ethylene diamine triacetic acid (EDTA), at pH 7.4, and centrifuged at 12 000 × g for 30 minutes at 4°C. The supernatant was collected and used for the determination of protein and enzymatic studies as described later. The protein concentration of the supernatant of tissue was determined using crystalline BSA as the standard.

CAT Assay. CAT activities were determined with a reaction solution containing 2.5 mL of 50-mmol phosphate buffers at pH 5.0, 0.4 mL of 5.9-mmol H2O2, and 0.1 mL of enzyme extract.

Changes in the absorbance of the reaction solution at 240 nm were determined after 1 minute. One unit of CAT activity was defined as an absorbance change of 0.01 as units/minute.27 SOD Assay. SOD activity was estimated by the Kakkar et al28 method. The reaction mixture contained 0.1 mL of 186-µmol phenazine methosulphate, 1.2 mL of 0.052-mmol sodiumpyrophosphate buffer at pH 7.0, and 0.3 mL of supernatant after centrifugation at 1500 × g for 10 minutes and mixed for 15 minutes. An enzyme reaction was initiated by adding 0.2 mL of 780-µmol NADH and stopped after 1 minute by adding 1 mL of glacial acetic acid. The amount of chromogen formed was measured by recording its color intensity at 560 nm. The results were expressed in units per mg of protein.

Assessment of Lipid Peroxidation Enzymes. The induction of lipid peroxidation by carbon chloride (CCl4) and its protection by the SAME were determined by the estimation of various enzyme activities and the contents of thiobarbituric acid reactive substances (TBARS).

Khan—Sonchus asper Hill

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 35

MATERIALS AND METHODS Animals Thirty male, Sprague-Dawley rats, from 180 to 190 g each, were provided by the National Institute of Health in Islamabad, Pakistan, and divided into 5 groups of 6 rats each.

The study was conducted in the Department of Biotechnology Bannu (Bannu, Pakistan).

RESULTS Body Weight Figure 1 shows that the STZ free-radical caused lipid peroxidation and led to a significantly decreased body weight postintervention for the diabetic control rats, group 3, compared with the untreated rats, group 1. Administration of the SAME significantly increased the body weight of the rats from baseline to postintervention, with P < .01, whereas no significant changes were produced in the untreated rats.

Figure 1. Increase in BW in the groups. Group 1 was the untreated control group. Group 2 received the SAME only. Group 3 was the diabetic control group that received STZ to induce diabetes. Group 4 received 100 mg/kg BW of the SAME + STZ. Group 5 received 200 mg/kg BW of the SAME + STZ. Body Weight (g)

225

a

a

a

220

a,b

215 b

210 205 200

1

2

3

4

Groups

5

Indicates significance compared with the diabetic control group, group 3, at P < .01. b Indicates significance compared with the untreated control group, group 1, at P < .01. a

Abbreviations: BW, body weight; SAME, Sonchus asper methanol extract; STZ, streptozotocin.

Figure 2. Serum glucose concentration in the groups. Group 1 was the untreated control group. Group 2 received the SAME only. Group 3 was the diabetic control group that received STZ to induce diabetes. Group 4 received 100 mg/kg BW of the SAME + STZ. Group 5 received 200 mg/kg BW of the SAME + STZ. Serum Glucose (mg/dL)

GST Assay. Glutathione-S-transferase (GST) activity was assayed by the Habig et al29 method. The reaction mixture consisted of 1.475 mL of 0.1-mol phosphate buffer at pH 6.5, 0.2 mL of 1-mmol reduced glutathione, 0.025 mL of 1-mmol 1-chloro-2,4-dinitrobenzene (CDNB), and 0.3 mL of homogenate in a total volume of 2.0 mL. The changes in the absorbance were recorded at 340 nm, and the enzymatic activity was calculated as nmol of CDNB conjugate formed/min/mg protein, using a molar extinction coefficient of 9.6 × 103M-1cm-1.

GSR Assay. GSR activity was determined using the Carlberg and Mannervik30 method. The reaction mixture consisted of 1.65 mL of 0.1-mol phosphate buffer at pH 7.6, 0.1 mL of 0.5-mmol EDTA, 0.05 mL of 1-mmol oxidized glutathione, 0.L ml of 0.1-mmol nicotinamide adenine dinucleotide phosphate (NADPH), and 0.1 mL of homogenate in a total volume of 2 mL. Enzymatic activity was quantified at 25°C by measuring the disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M-1cm-1.

GPx Assay. Glutathione peroxidase (GPx) activity was assayed using the Mohandas et al31 method. The reaction mixture consisted of 1.49 mL of 0.1-mol phosphate buffer at pH 7.4, 0.1 mL of 1-mmol EDTA, 0.1 mL of 1-mmol sodium azide, 0.05 mL of 1-IU/mL glutathione reductase, 0.05 mL of 1-mmol GSH, 0.1 mL of 0.2-mmol NADPH, 0.0L ml of 0.25-mmol H2O2, and 0.1 mL of homogenate in a total volume of 2 mL. The disappearance of NADPH at 340 nm was recorded at 25°C. Enzymatic activity was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M-1cm-1.

TBARS Assay. The TBARS assay for lipid peroxidation was carried out following the Iqbal et al32 modified method.

The reaction mixture in a total volume of 1.0 mL contained 0.58 mL of 0.1 mol phosphate buffer at pH 7.4, 0.2 mL of homogenate sample, 0.2 mL of 100-mmol ascorbic acid, and 0.02 mL of 100-mmol ferric chloride. The reaction mixture was incubated at 37°C in a shaking water bath for 1 hour. The reaction was stopped by the addition of 1.0 mL of 10% trichloroacetic acid. Following the addition of 1.0 mL of 0.67% thiobarbituric acid, all the tubes were placed in a boiling-water bath for 20 minutes and then shifted to a crushed-ice bath before centrifuging at 2500 × g for 10 minutes. The amount of TBARS formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm, using a spectrophotometer UV-2800 (Biotechnology Medical Services, Pointe-Claire, Quebec, Canada) against a reagent blank. The results were expressed as nmol TBARS/min/mg tissue at 37°C using a molar extinction coefficient of 1.56 × 105 M-1cm-1.

400 350 300 250 200 150 100 50 0

b

a a

1

a

a

2

3

Groups

4

5

Indicates significance compared with the diabetic control group, group 3, at P < .01. b Indicates significance compared with the untreated control group, group 1, at P < .01. a

Statistical Analysis To determine the treatment effects, a 1-way analysis of variance was carried out using the computer software SPSS 13.0 (IBM, Armonk, NY, USA). The least significant difference at a 0.05% level of probability determined the level of significance among the various treatments.

Abbreviations: SAME, Sonchus asper methanol extract; STZ, streptozotocin; BW, body weight.

36 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Khan—Sonchus asper Hill

225 220

a

Figure 4. Serum insulin concentration in the groups. Group 1 was the untreated control group. Group 2 received the SAME only. Group 3 was the diabetic control group that received STZ to induce diabetes. Group 4 received 100 mg/kg BW of the SAME + STZ. Group 5 received 200 mg/kg BW of the SAME + STZ. Serum Insulin (mg/dL)

Lipase Activity (mg/dL)

Figure 3. Serum lipase enzyme activity in the groups. Group 1 was the untreated control group. Group 2 received the SAME only. Group 3 was the diabetic control group that received STZ to induce diabetes. Group 4 received 100 mg/kg BW of the SAME + STZ. Group 5 received 200 mg/kg BW of the SAME + STZ.

a

a a,b

215 210

b

205 200

1

2

3

Groups

4

225 220

a

a

a a,b

215 210

b

205 200

5

1

2

3

Groups

4

5

a

Indicates significance compared with the diabetic control group, group 3, at P < .01. b Indicates significance compared with the untreated control group, group 1, at P < .01.

a

Indicates significance compared with the diabetic control group, group 3, at P < .01. b Indicates significance compared with the untreated control group, group 1, at P < .01.

Abbreviations: SAME, Sonchus asper methanol extract; STZ, streptozotocin; BW, body weight.

Abbreviations: SAME, Sonchus asper methanol extract; STZ, streptozotocin; BW, body weight.

Table 1. Effects of SAME on Lipid Profile at 1 Week After STZ

Treatment (n = 6 each) Untreated controls, group 1 200 mg/kg of SAME only, group 2 55 mg/kg of STZ, group 3 100 mg/kg of SAME + STZ, group 4 200 mg/kg of SAME + STZ, group 5

Total High-density Low-density Triglycerides cholesterol Lipoprotein Lipoprotein (mg/dL) (mg/dL) (mg/dL) (mg/dL) Mean ± SE Mean ± SE Mean ± SE Mean ± SE 17.8 ± 0.45a

5.1 ± 0.25a

3.6 ± 0.21a

2.48 ± 0.32a

18.86 ± 0.50a

5.54 ± 0.22a

3.5 ± 0.20a

2.58 ± 0.28a

31.3 ± 0.58

12.2 ± 0.23

2.8 ± 0.18

8.4 ± 0.17b

22.0 ± 0.41a,b

7.7 ± 0.21a,b

3.08 ± 0.09a

4.2 ± 0.21a,b

18.3 ± 0.18a

5.4 ± 0.27a

3.5 ± 0.20a

2.53 ± 0.35a

b

b

b

Indicates significance compared with the diabetic control group, group 3, at P < .01. b Indicates significance compared with the untreated control group, group 1, at P < .01. a

Abbreviations: SAME, Sonchus asper methanol extract; STZ, streptozotocin.

Glucose Level and Lipase Activity Figure 2 and Figure 3 show that the serum glucose levels and lipase activity of the diabetic control animals, group 3, increased and decreased, respectively, from baseline to postintervention. In contrast, after 21 days of treatment with the SAME, the serum-glucose levels and lipase activity of the diabetic rats in groups 4 and 5 were significantly reduced and increased, respectively, in comparison with the diabetic control rats, with P < .01.

Khan—Sonchus asper Hill

Secretion of Insulin Insulin is a hormone that is central to regulating energy and glucose metabolism in the body. Administration of the STZ in the rats significantly decreased the insulin secretion from baseline to postintervention for group 3, with P < .01 (Figure 4). The secretion was significantly improved by administration of 100 mg/kg and 200 mg/kg BW of the SAME for groups 4 and 5, with P < .01. However, no significant changes were found in the untreated controls, group 1, and the rats receiving the SAME only, group 2.

Lipid Profile Table 1 shows the levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, highdensity lipoprotein (HDL) cholesterol, and triglycerides 1 week after the STZ injection.

Significant changes were found in the serum lipid profiles of the diabetic control rats in group 3 from baseline to postintervention, with P < .01.

After treatment, the serum cholesterol profile of the SAME-treated diabetic rats in groups 4 and 5 significantly decreased in comparison with the diabetic control rats, with P < .01. The reductions in the blood cholesterol profile that were elicited by the SAME were sustained throughout the course of treatment.

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 37

Serum Marker Enzymes Table 2 shows the levels of free ROS that had been produced during hyperglycemia. The activity of the serum marker enzymes had increased significantly from baseline to postintervention, with P < .01. The free radicals produced during DM in the diabetic control rats in group 3 had significantly increased the activity of the serum marker enzymes, with P < .01, and the activity was significantly reduced after treatment with the SAME, in a dependent manner, with P < .01.

Table 2. Effects of SAME on Serum Marker Enzymes During Hyperglycemia Treatment (n = 6 each) Untreated controls, group 1 200 mg/kg of SAME only, group 2 55 mg/kg of STZ, group 3 100 mg/kg of SAME + STZ, group 4 200 mg/kg of SAME + STZ, group 5

ALT (U/L) Mean ± SE 52.0 ± 2.12a

AST (U/L) Mean ± SE 76.0 ± 2.74a

ALP (U/L) Mean ± SE 178 ± 3.93a

γ-GT (U/L) Mean ± SE 105.5 ± 2.2a

51.0 ± 2.05a

77.6 ± 2.75a

169.6 ± 3.68a

106.3 ± 2.0a

111.0 ± 3.42b

234.0 ± 4.27b

470.3 ± 6.49b

154.3 ± 3.2b

91.0 ± 3.23a,b

136 ± 4.91a,b

247.7 ± 10.7a,b

128 ± 2.7a,b

56.0 ± 1.35a

97.5 ± 2.90a,b

197.8 ± 3.91a

108 ± 3.05a

Oxidative Stress Table 3 shows antioxidant activities such as aIndicates significance compared with the diabetic control group, SOD, CAT, GPx, GSR, GST, and TBARS in the group 3, at P < .01. various groups of rats. Administration of the bIndicates significance compared with the untreated control group, STZ in the rats significantly reduced the activity group 1, at P < .01. of antioxidant enzymes as well as increased the TBARS contents compared to the untreated Abbreviations: SAME, Sonchus asper methanol extract; control group, group 1, with P < .01. The STZ, streptozotocin. treatment with the SAME markedly improved the oxidative Table 3. Effects of SAME on Antioxidant Enzymes stress that have been induced by STZ, with P < .01. For the TBARS SOD GST GPH-Px GSR TBARS CAT U/mg nM/min/mg nM/min/ nM/min/mg (nM/min/ content, no significant changes Treatment U/min Protein Protein mg Protein Protein mg protein) in antioxidant enzymes was (n = 6 each) Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE found for the diabetic control Untreated controls, group, group 3.

22.7 ± 1.18a 192.0 ± 2.49a 84.2 ± 5.9a 127.3 ± 10.5a 28.33 ± 1.12a group 1 4.8 ± 0.10a 200 mg/kg of SAME

only, group 2 4.9 ± 0.09a 22.4 ± 0.62a 193.0 ± 3.09a 82.0 ± 6.8a 131.2 ± 12.4a 29.17 ± 1.22a DISCUSSION 55 mg/kg of STZ, Phenolic and polyphenolic group 3 2.9 ± 0.06b 11.9 ± 0.38b 104.0 ± 2.4b 46.0 ± 3.6b 62.33 ± 9.28b 52.17 ± 2.61 flavonoids possess a variety of 100 mg/kg of biological behaviors and have SAME + STZ 4.04 ± 0.09a,b 16.2 ± 0.38a,b 142.0 ± 2.6a,b 61.0 ± 6.6a,b 90.2 ± 11.3a,b 43.0 ± 2.76a,b been found to be the main 200 mg/kg constituents in medicinal plants.

SAME + STZ 4.8 ± 0.125a 20.4 ± 0.37a,b 190.0 ± 2.9a 75.0 ± 4.2a 112.3 ± 6.5a 30.0 ± 1.77a Ksouri et al33 found that Tamarix gallica analysis had revealed the presence of bioactive polyphenolic aIndicates significance compared with the diabetic control group, group 3, at P < .01. compounds. Kamalakkannan and bIndicates significance compared with the untreated control group, group 1, at P < .01.

Stanely34 found that an HPLC chromatogram had revealed the Abbreviations: SAME, Sonchus asper methanol extract; CAT, catalase, A; SOD, superoxide presence of hyperoside and dismutase; GST, glutathione S-transferase; GPH-Px, glutathione peroxidase; quercetin, which are putatively GSR, glutathione reductase; TBARS, thiobarbituric acid reactive substances; responsible for the hypoglycemic STZ, streptozotocin. behavior of the SAME. Zu et al35 have reported the presence of the same photochemical during by dried extracts of the SAME. The blood-glucose levels HPLC characterization in medicinal plants. decreased in the animals treated with the SAME after 21 days STZ-induced hyperglycemia, at 60 mg/kg BW, has been of treatment. Previously, an investigation by Lino et al37 described as a useful experimental model for studying showed that a methanol extract of Bauhinia forficate that had hypoglycemic activity.36 In that study, the hypoglycemic been administered daily for 7 days in diabetic rats at doses of effects of different dried extracts of the SA were verified in 100 and 200 mg/kg BW could decrease blood glucose.

STZ-induced diabetic rats using a single dose of 200 mg/kg In diabetic rats, Bollen and Stalmans38 had attributed BW of the corresponding extract. high glucose levels and a decrease in the hepatic glycogen The increased levels of plasma glucose in the and lipid and cholesterol contents to the lower availability of STZ-induced diabetic rats in the current study were lowered the active form of the enzyme glycogen synthetase.

38 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Khan—Sonchus asper Hill

Earlier studies have shown that insulin deficiency in STZ-induced diabetic rats is associated with hypercholesterolemia and hypertriglyceridemia. Insulin deficiency may be responsible for dyslipidemia, because insulin has an inhibitory action on 3-hydroxy-3methylglutaryl coenzyme A reductase, a key enzyme that is rate-limiting in the metabolism of cholesterol-rich LDL particles.39,40 The mechanisms that are responsible for the development of hypertriglyceridemia in uncontrolled diabetes in humans, and possibly in insulin-deficient STZ-diabetic rats, cause a number of metabolic abnormalities that occur sequentially.

Acute insulin deficiency initially causes an increase in mobilization of free fatty acids from adipose tissue, resulting in an increased secretion of very-low-density lipoproteins and triglycerides from the liver.41 In diabetic rats, a decrease in lipoprotein lipase activity42 occurs, resulting in an impaired clearance of LDL and chylomicrons from plasma.43 In the current study, treatment with the SAME significantly decreased both serum cholesterol and triglyceride levels in diabetic rats. Significant improvements in serum LDL and HDL levels also occurred with treatment by the SAME compared to the diabetic control animals.

Oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. Free-radical production caused by hyperglycemia can occur via at least 3 different routes: (1) nonenzymatic glycation, (2) auto-oxidation of glucose, and (3) intracellular activation of the polyol pathway.44 High free-radical levels and a simultaneous decrease in the antioxidant-enzyme levels can lead to cell damage, inactivation of enzymes, and lipid peroxidation. In the present study, the diabetic control rats showed a significant increase in the TBARS levels and a decrease in the levels of SOD, CAT, GPx, GSR, and GSH in the rat-liver homogenates compared with the normal rats, indicating a dysfunction in the antioxidant defensive system in DM. Treatment with the SAME reduced the TBARS levels compared with those of the diabetic controls. On the other hand, it also increased the SOD and CAT levels in diabetic rats. Similar investigations have been reported in other studies by Minami et al.45 The mechanism of STZ-induced diabetes has been the theme of many investigations, and it is generally accepted that free radicals can trigger the damage that ultimately leads to β-cell death.46,47 Therefore, the pancreas is especially liable to the action of chemically induced free-radical damage.

Many substances have been shown to ameliorate the diabetogenicity of STZ in animals, and they protect by reacting with the free radicals formed from a chemical during its interaction with the β-cell, or they prevent radical formation.42 The current study has indicated that the SAME produced antihyperglycemic effects in experimental diabetes by providing a regenerative modification against the damage caused by STZ to endocrine cells of the pancreas as well as by delivering anti-inflammatory activity. The hypoglycemic mechanisms function by stimulating glucose uptake by the Khan—Sonchus asper Hill

peripheral tissues, by inhibiting insulinase activity in both the liver and the kidney,48,49 by inhibiting endogenous glucose production, or by inhibiting renal glucose reabsorption.

Similar results have been reported during investigation of the protective role of isolated flavonoids in plants against chemically induced DM.50 CONCLUSIONS The findings suggest that the significant antidiabetic potential of the SAME in ameliorating the diabetic conditions in diabetic rats might be due to the presence of the bioactive constituents in the extract. A total of 100 mg/kg to 200 mg/kg BW of SA are recommended as an herbal medicine for DM therapy. Its activities might be due to the presence of bioactive flavonoids and phenolic compounds. Purification and isolation of those bioactive constituents are in progress.

AUTHOR DISCLOSURE STATEMENT The author declares no conflicts of interest.

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2. Chua DY, Bakris GL. Diabetes and chronic kidney disease: Tragedy and challenge. Blood Purif. 2004;22:130-135.

3. White DA, Baxter M. Diabetes mellitus: In: White DA, Baxter M, eds. Hormones and Metabolic Control. 2nd ed. London, United Kingdom: Arnold; 1994:79-92.

4. Uchigata Y, Yamamoto H, Kawamura A, Okamoto H. Protection by superoxide dismutase, catalase and poly (ADP-ribose) sunthetase inhibitors against alloxanand streptozotocin-induced islet DNA strand breaks and against the inhibition of proinsulin synthesis. J Biol Chem. 1982;257:6084-6088.

5. Merzouk H, Madani S, Chabane D, Prost J, Bouchenak M, Belleville J. Timecourse of changes in serum glucose, insulin, lipids and tissue lipase activities in macrosomic offspring of rats with streptozotocin-induced diabetes. Clin Sci. 2000;98:21-30.

6. Kroncke KD, Fehsel K, Sommer A, Rodriguez ML, Kolb-Bachofen V. Nitric oxide generation during cellular metabolization of the diabetogenic N-methyl-nitrosourea: Streptozotocin contributes to islet cell DNA damage. Biol Chem.

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7. Chen YT, Zheng RL, Jia JZ, Ju Y. Flavonoids as superoxide scavengers and antioxidants. Free Radic Biol Med. 1990;19:19-20.

8. Tiedge M, Lortz S, Drinkgern J, Lenzen S. Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells.

Diabetes. 1997;46:1733-1742.

9. Matkovics B, Kotorman M, Varga IS, Hai DQ, Varga C. Oxidative stress in experimental diabetes induced by streptozotocin. Acta Physiol Hung. 1998;85:29-38.

10. Nadler JL, Winter L. Free radicals, nitric oxide and diabetic complications. In Roith DLE, Taylor SI, Olefsky JM, eds. Diabetes Mellitus. Philadelphia, PA: Lippincott-Raven Publishers; 1996:840-847.

11. Bailey CJ, Day C. Traditional plant medicines as treatment for diabetes. Diabetes Care. 1989;12:553-564.

12. Kar A, Choudhary BK, Bandyopadhyay NG. Comparative evaluation of hypoglycemic activity of some Indian medicinal plants in alloxan diabetic rats.

J Ethnopharmacol. 2003;84:105-108.

13. Srinivasan K. Plant foods in the management of diabetes mellitus: Spices as beneficial antidiabetic food adjuncts. Intl J Food Sci Nutrit. 2005;6:399-414.

14. Rehman EU. Indigenous knowledge on medicinal plants, village Barali Kass and its allied areas, District Kotli Azad Jammu and Kashmir, Pakistan. Ethno Leaflets.

2006;10:254-264.

15. Ahmad M, Khan MA, Manzoor S, Zafar M, Sultana S. Check list of medicinal flora of Tehsil Isakhel, District Mianwali Pakistan. Ethno Leaflets. 2006;10:41-48.

16. Koche DK, Shirsat RP. Imran S. Nafees M, Zingare AK, Donode KA. Ethnomedicinal Survey of Nageria Wild Life Sanctuary, District Gondia (M.S.).

India- Part II. Ethnob Leaflets. 2008;12:532-537.

17. Rivera ND, Oben DCC. Ethnopharmacology of Murcia (SE Spain). Conference internationale Ethnomedecine. Heidelb2e.4rg-2, 7 mars 1993.

18. Zabihullah Q, Rashid A, Akhter N. Ethanobotnical survey in kot Manzary baba valley malakand agency, Pakistan. Pakistan J Plant Sci. 2006;12:115-121.

19. Manan Z, Sirajuddin RA, Islam M, Ikramullah M. Diversity of medicinal plants in Wari sub division district Upper Dir, Pakistan. Pakistan J Plant Sci. 2007;3:21-28.

20. Kareru PG, Kenji GM, Gachanja AN, Keriko JM, Mungai G. Traditional medicine among the Embu and Mbeere peoples of Kenya. African J Trad Compl Alter Med. 2007;4:75-86.

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21. Sabeen M, Ahmad AA. exploring the folk medicinal flora of Abbotabad City Pakistan. Ethno Leaflets. 2009;13:810-833.

22. Guil-Guerrero JS, Gimenez-Gimenez A, Rodrguez-Garc I, Torija-Isasa ME. Nutritional composition of Sonchus species (S. asper L., S. oleraceus L., S. tenerrimus L.). J Sci Food Agric. 1998;76:628-632.

23. Middleton Jr E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer.

Pharmacol Reviews. 2000;52:673-751.

24. Balasundram N, Sundram K, Samman S. Phenolic com-pounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses.

Food Chem. 2006;99:191-203.

25. Zhou T, Luo D, Li X, Luo Y. Hypoglycemic and hypolipidemic effects of flavonoids from lotus (Nelumbo nuficera Gaertn) leaf in diabetic mice. J Med Plants Res. 2009;3:290-293.

26. Winter CA, Risley EA, Nuss CW. Carrageenan-induced oedemas in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med. 1962;111:544-547.

27. Chance B, Maehly AC. Assay of catalase and peroxidases. Methods in Enzymol.

1955;11:764-775.

28. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 1984;21:130-132.

29. Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-transferases: The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130-7139.

30. Carlberg I, Mannervik EB: Glutathione level in rat brain. J Biol Chem. 1975;250: 4475-4480.

31. Mohandas J, Marshal JJ, Duggin GG, Horvath JS, Tiller DJ. Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney, possible implications in analgesic nephropathy. Biochem Pharmacol.

1984;33:1801-1807.

32. Iqbal M, Sharma SD, Zadeh HR, Hasan N, Abdulla M, Athar M. Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate (Fe-NTA) mediated hepatic injury. Redox Repor. 1996;2:385-391.

33. Ksouri R, Falleh H, Megdiche W, et al. Antioxidant and antimicrobial activities of the edible medicinal halophyte Tamarix gallica L. and related polyphenolic constituents. Food Chem Toxicol. 2009;47:2083-2091.

34. Kamalakkannan N, Stanely P. Rutin improves the antioxidant status in streptozotocin-induced diabetic rat tissues. Mol Cell Biochem. 2006,;293:211-219.

35. Zu Y, Fu Y, Liu Y, Hou C, Kong Y. Simultaneous determination of four flavonoids in Pigeonpea [Cajanus cajan (L.) Millsp.] leaves using RP-LC-DAD. Chromatographia. 2006;63:9-10.

36. Frode TS, Medeiros YS: Animal models to test drugs with potential antidiabetic activity. J Ethnopharmacol. 2008;115:173-183.

37. Lino Cde S, Diogenes JPL, Pereira BA, et al. Antidiabetic activity of Bauhinia forficata extracts in alloxan-diabetic rats. Biol Pharm Bull. 2004;27:125-127.

38. Bollen M, Stalmans W. The hepatic defect in glycogen synthesis in chronic diabetes involves the G-component of synthase phosphatase. Biochem J. 1984;217:427-434.

39. Gold AH. The effect of diabetes and insulin on liver glycogen synthetase activation. J Biol Chem. 1970;245:903-905.

40. Thakkar NV, Patel JA. Pharmacological evaluation of “Glyoherb”: A polyherbal formulation on streptozotocin-induced diabetic rats. Intl J Diabetes. 2010;30:1-7.

41. Balasse EO, Bier DM, Havel RJ. Early effects of anti-insulin serum on hepatic metabolism of plasma free fatty acids in dogs. Diabetes. 1972;21:280-284.

42. Nikkila EA, Huttunen JK, Ehnholm C. Postheparin plasma lipoprotein lipase and hepatic lipase in diabetes mellitus. Diabetes. 1977;26:11-21.

43. Bagdade JD, Porte DJ, Bierman EL. Acute insulin withdrawal and the regulation of plasma triglyceride removal in diabetic subjects. Diabetes. 1968;17:127-130.

44. Ceriello A. Oxidative stress and glycemic regulation. Metabolism. 2000;49:27-29.

45. Minami T, Shimuzu M, Tanaka H, Okazaki Y, Charian MG. Metallothionein does not protect mouse endocrine cells from damage induced by alloxan injection. Toxicology. 1999;132:33-41.

46. Vancoa J, Svajlenova O, Ramanska E, Muselik J, Valentova J. Antibacterial activity of different copper (II) Schiff base complexes and their effect on alloxan-induced diabetes. J Traces Element Med Biol. 2004;18:155-161.

47. Jorns A, Tiedge M, Lenzen S, Munday R. Effect of Super oxide dimutase, catalase, chelating agents and free radical scavangers on toxicity of alloxan to isolated pancreatic islets in vitro. Free Radic boil Med. 1999;26:1300-1304.

48. Achrekar B, Kakij GS, Pote MS, Kelkar H. Hypoglycemic activity of Eugenia jambolana and Ficus bergaensis. Invivo. 1999;5:143-147.

49. Niranjani PS, Singh D, Prajapati K, Jaini SK. Antidiabetic activity of ethanolic extract of Dalbergia sisso L. leaves extract in alloxan induced diabetic rats. Intl J Curr Pharm Res. 2010;2:24-27.

50. Coskun MK, Korkmaz A, Oter S. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and β-cell damage in rat pancreas. Pharmacol Res. 2005;51:117-123.

40 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Khan—Sonchus asper Hill

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ORIGINAL RESEARCH

Proapoptotic and Growth-inhibitory Effects of Plumbagin on Human Gastric Cancer Cells Via Suppression of Signal Transducer and Activator of Transcription 3 and Protein Kinase B Jing Li, PhD; Jia Li, PhD; Guowei Cai, PhD; Lin Shen; PhD; Furong Lu, PhD ABSTRACT Context • Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer-related deaths in the world. The current treatments include surgery and chemotherapy, either alone or in combination with radiotherapy, but the prognosis for patients with GC is usually poor. A safe and effective chemopreventive treatment for this malignant disease is urgently needed.

Objective • The study intended to investigate the effects and underlying mechanisms of plumbagin, a quinonoid constituent that is derived from the roots of the medicinal plant Plumbago zeylanica, which exhibits potent anticancer properties against a number of cancers.

Design • The in vitro study used the human GC cell line SGC-7901.

Setting • All experiments were conducted at the Hubei University of Chinese Medicine and Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).

Intervention • SGC-7901 cells were cultured in 30-mm dishes and treated with plumbagin at concentrations of 0, 5, 10, to 20 μmol/L. The cells were incubated with 10 μmol/L plumbagin for different amounts of time (0, 2, 4, 8, 12, and 24 h) in contact with the cancer cells.

Outcome Measures • The cell viability was examined using a cell counting kit-8 viability assay, and the cell proliferation rate was determined using a 5-ethynyl-2’deoxyuridine incorporation assay. The cell cycle distribution was assessed by flow cytometry using propidium iodide staining, and Western blotting was used to assess the expression of BAX, BCL-2, and caspase-3 and to identify any downregulation in the activation of transcription 3 (STAT3), protein kinase B (Akt), and extracellular signal-regulated kinase (ERK1/2).

Results • The plumbagin concentrations of 5–20 mmol/L reduced the viability of the GC cells in a dependent manner.

Plumbagin suppressed the expression of BAX, BCL-2, pro-caspase-3, and cleaved-caspase-3. It also restrained the expression and phosphorylation of STAT3 and decreased the phosphorylation of Akt1 but did not change the total protein or phosphorylation levels of ERK1/2.

Conclusions • Plumbagin inhibits cell apoptosis in human GC cells, and that effect may be related with its ability to suppress phosphorylation of STAT3 and Akt. Given those 2 effects, plumbagin may be a promising agent in the treatment of gastric cancer. (Altern Ther Health Med. 2017;23(5):42-48)

Jing Li, PhD, is a physician in the Department of Acupuncture, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), in Wuhan, China. Guowei Cai, MD, is a physician in the Department of Acupuncture, Union Hospital, Tongji Medical College, HUST. Jia Li, PhD, is a lecturer at Hubei University of Chinese Medicine and Hubei Provincial Collaborative Innovation Center of Preventive Treatment

by Acupuncture and Moxibustion, in Wuhan, China.

Lin Shen, PhD, is a physician in the Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, HUST. Furong Lu, PhD, is a physician in the Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, HUST.

astric cancer (GC) is the fourth most common cancer and the second leading cause of cancerrelated death in the world.1 Approximately 934 000 new cases of and 700 000 deaths from GC occur annually.2 The current major treatments for GC include surgery and chemotherapy, either alone or in combination with radiotherapy, but the prognosis for patients with GC is usually poor. A safe and effective chemopreventive treatment for this malignant disease is urgently needed.

42 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

Corresponding author: Furong Lu, PhD E-mail address: furonglulove@163.com

G

Natural products derived from traditional therapies have been serving humans as treatments for cancer and other ailments for thousands of years. A quinonoid constituent, plumbagin—5-hydroxy-2methyl-1,4-naphthquinone—is one such agent. The root of the medicinal plant Plumbago zeylanica, a major source of plumbagin, has been safely used for centuries in traditional Ayurvedic and Chinese medicines.3 Plumbagin has been shown to possess hepatoprotective,4 neuroprotective,5 antimicrobial, antiatherosclerotic, and anticancer effects.6 Growing evidence suggests that plumbagin exerts anticancer activities against a variety of tumor cell lines and animal models of cancer.7,8 Powolny et al3 reported that plumbagin significantly induced apoptosis by modulating the cellular redox status and generating reactive oxygen species (ROS) in human prostate cancer cells. Plumbagin also induced G2-M cell cycle arrest through the inhibition of protein kinase B (Akt) in breast cancer cells.9 Additional reports have shown that plumbagin can inhibit the invasion and migration of liver cancer and lung cancer cells by suppressing matrix metalloproteinase-2 and urokinase-plasminogen activator.10,11 Plumbagin has also been shown to induce apoptosis through ROS and c-Jun N-terminal kinase in melanoma cells.12 Different intracellular signaling pathways play important roles in cancer development and therapy. Among them, the signal transducer and activator of transcription 3 (STAT3) is now thought to play a major role in tumor formation. STAT3 is constitutively activated in various tumors and can promote cellular proliferation, survival, and transformation.13,14 Other research has shown that constitutively activated STAT3 supports survival of GC cells in association with increased expression.15,16 Altered expression of mitogen-activating protein kinase and extracellular signal-regulated kinase (ERK) can promote effects ranging from apoptosis to malignancy in different cell types. In addition, the phosphoinositide 3-kinase (PI3K)/Akt pathway has been extensively investigated, and its activation promotes the proliferation and survival of cancer cells.17,18 Moreover, activation of Akt has been suggested to be associated with mortality in aggressive GCs.19 The 3 signaling molecules play critical roles in control of the cell cycle, survival, proliferation, and migration of tumor cells and may be related to cancer development as well as prognosis.

In the current study, the research team investigated the antiproliferative potential of plumbagin on GC cells. The effects of plumbagin on phosphorylation of STAT3, ERK, and Akt— 3 crucial regulators of growth and apoptosis—were also analyzed.

The plumbagin was initially dissolved in dimethyl sulfoxide (DMSO) at a concentration of 200 mM, stored at -20°C in a dark-colored bottle, and then thawed and diluted in cell culture medium before use. The antibodies against BAX, BCL-2, ERK, phospho-ERK, STAT3, phospho-STAT3 (Ser 727), and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Pro-caspase-3, cleaved-caspase-3, Akt, and phospho-Akt antibodies were provided by Epitomic, Inc (Burlingame, CA, USA). The goat antimouse-HRP conjugate and goat antirabbit-HRP conjugate were purchased from Cell Signaling Technology (Beverly, MA, USA). Penicillin, streptomycin, RPMI 1640 medium, and FBS were obtained from GIBCO BRL Life Technologies (Grand Island, NY, USA).

Intervention SGC-7901 cells were cultured in 30-mm dishes and treated with plumbagin at various concentrations (0, 2.5, 5, 10, 20, and 40 μmol/L). However, we selected only the representative obvious changes of cell growth from 0, 5, 10, to 20 μmol/L. As for time dependent design, SGC-7901 cells were incubated with 10 μmol/L plumbagin for different amounts of time (0, 2, 4, 8, 12, and 24 hours) in contact with the cancer cells.

Figure 3A shows results for the first gap period (G1) and the second gap period (G2), and the synthesis period (S) while treated with plumbagin at various concentrations from 0, 5, 10, and 20 μmol/L (corresponding images left to right).

Procedures Plumbagin, ribonuclease (RNAse) and propidium iodide (PI) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Outcome Measures Cell Viability Assay. The inhibition of cell viability was measured by the cell counting kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan).20 Briefly, SGC-7901 cells were grown overnight in 96-well culture plates, with 10 000 cells per well. Then, the cells were incubated with or without different concentrations of plumbagin for the indicated periods. Thereafter, 10 μL of the kit reagent 2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonyl)-2Htetrazolium (WST-8) was added to each well and cocultured for 4 hours at 37°C. Finally, the optical density was detected at a 450-nm wavelength using a 96-well, multiscanner autoreader (Thermo Electron Corp, Waltham, MA, USA).

Cell Proliferation Assay. The 5-ethynyl-2’-deoxyuridine (EdU) assay that was used to stain the proliferating cells was the Cell-Light EdU DNA Cell Proliferation Kit (RIBOBio Co, Guangzhou, China).21 The cells were seeded in medium into 96-well, cell-culture plates, grown overnight, and then exposed to media with or without plumbagin. Cells were then exposed to 50 μM of EdU for 4 hours at 37°C and fixed with 4% paraformaldehyde for 15 minutes. After being treated with 0.5% Triton X-100 for 20 minutes, the cells were washed with phosphate-buffered saline (PBS) 3 times.

Thereafter, the cells were incubated with 100 μL of a 1× Apollo reaction cocktail for 30 minutes and identified by Hoechst 33342 at 5 μg/mL for nuclei staining for 30 minutes.

Images were captured using a fluorescent microscope (Olympus, Tokyo, Japan).

Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 43

METHODS Cell Culture The GC cell line SGC-7901 was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium containing 1 g/L of 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin in 5% CO2 at 37°C.

Figure 1. The effects of plumbagin on the morphological characteristics and viability of the SGC-7901 Cells. Figure 1A shows the dose-dependent effect of plumbagin treatment for 24 hours on the SGC-7901 cells. Figure 1B shows the time course of the effects of 10 μΜ of plumbagin on the SGC-7901 cells for the indicated time periods (0–24 hours), as determined by a cell counting kit-8 assay. The data shown are the means calculated using data from 3 independent experiments. Figure 1C shows SGC-7901 cells that were treated with the indicated concentrations of plumbagin for 24 hours (400× magnification).

A. Cell Inhibitory (%)

80 60 40 20 0

2.5

5

10

20

40

12

24

100 80 60 40 20 0

C.

0

Plumbagin (μM)

B.

Statistical Analysis All quantitative data are presented as mean ± standard deviation (SD). The Student t test was used for estimating significance among groups. P < .05 was considered statistically significant. All statistical analyses were performed using SPSS for Windows, version 13.0 (IBM, Armonk, NY, USA).

RESULTS Viability The cytotoxicity of plumbagin on the SGC-7901 cells was calculated from the loss of cell viability, using the CCK-8 assay. The research team observed that cellular growth was suppressed by plumbagin in a dose- and time-dependent manner in the cells (Figure 1A and Figure 1B). At 20 μM of plumbagin, a significant loss of viability was detected after 24 hours of treatment. After the same period, plumbagin also caused the SGC-7901 cells to undergo a change in shape, including shrinkage, rounding, flattening, and partial detachment (Figure 1C), thus demonstrating the cytotoxic effects of plumbagin on the cells.

100

Cell Inhibitory (%)

Cell Cycle Analysis. The cell cycle distribution was assessed by flow cytometry using PI staining.22 The SGC-7901 cells were collected by trypsinization after treatment with or without plumbagin at various concentrations for 24 hours. The cells were washed and fixed with 70% ethanol overnight at -20°C. Then the cells were resuspended in PBS containing 250 μg/mL of RNAse and 50 μg/mL of PI, incubated for 30 minutes in the dark at room temperature and analyzed with a fluorescence-activated cell sorter Vantage flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA, USA).

Western Blot Analysis.23,24 The cells were plated in 10-cm dishes and incubated overnight prior to the addition of plumbagin. After incubation with plumbagin for different periods, the cells were washed with PBS. Afterward, the total cell extracts were prepared using the M-PER mammalian protein extraction reagent (Pierce, Appleton, WI, USA) and protease inhibitors. The protein concentrations were determined using a bicinchoninic acid assay from Pierce.

Equal amounts of protein were electrophoresed on an 8% to 12% sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. Blots were first blocked with 5% nonfat dry milk for 1 hour and incubated with respective primary antibodies overnight at 4°C. Then the membranes were extensively washed and incubated for 1 hour with secondary antibodies at room temperature. The protein was visualized using the enhanced chemiluminescence system from the Beyotime Institute of Biotechnology (Jiangsu, China).

0

2

4

8

Time (h)

Proliferation To detect the antiproliferative effects of plumbagin, the research team used the EdU incorporation assay, a more sensitive and specific method. The team found that the number of EdU-positive cells in all plumbagin-treated groups 44 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

Figure 2. The effects of plumbagin on DNA replication in the SGC-7901 cells. Plumbagin decreased the number of proliferating cells. The cells were incubated with 5 μM of plumbagin for 12 hours. The EdU-labeled replicating cells were examined under a fluorescent microscope (200× magnification).

Hiechst 33342

EdU

Merged

Control

Plumbagin

Abbreviation: EdU, 5-ethynyl-2’-deoxyuridine.

Figure 3. The effects of plumbagin on the cell cycle of the SGC-7901 cells. Figure 3A shows that plumbagin induced cell cycle arrest. The SGC-7901 cells were treated with 0, 5, 10, and 20 μΜ plumbagin for 24 hours (left to right). The distribution of cells in each phase of the cell cycle was detected by PI staining and flow cytometry. Figure 1B shows that the cell-cycle distribution was quantified for each treatment group. Data shown are the mean ± SD of 3 individual experiments. “%” means percentage of apoptosis (vs corresponding G0/G1, S, or G2/M in 0 μM group).

a a a

a

a a

a

a

P < .05.

a

Abbreviations: PI, propidium iodide; SD, standard deviation; G0, G0 gap period; G1, first gap period; G2, second gap period; S, synthesis period; M, mitosis period. was significantly reduced compared to the control cells, indicating that the plumbagin had an antiproliferative effect against GC cells (Figure 2).

Cell Cycle Arrest To determine whether plumbagin could induce cell cycle arrest, PI-stained cells were analyzed by flow cytometry.

Treatment with plumbagin increased the percentage of cells in the S and G2/M phases in a dose-dependent manner, as compared with the controls (Figure 3). At the same time, the proportion of G0/G1 phase cells was decreased gradually in a dose-dependent manner (Figure 3). The data suggest that plumbagin can inhibit cell growth by inducing cancer cells to undergo a S-G2/M phase arrest.

Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 45

Figure 4. The effects of plumbagin on the expression of apoptosisrelated proteins. Plumbagin suppressed the expression of BAX, BCL-2, pro-caspase-3, and cleaved-caspase-3. The SGC-7901 cells were incubated with 10 μM of plumbagin for different time periods (0, 2, 4, 8, 12, and 24 hours). Whole-cell extracts were prepared and analyzed by Western blotting with antibodies against the indicated proteins.

0

2

Time (h) 4 8

12

24 BAX BCL-2

Figure 5. The effects of plumbagin on the expression and phosphorylation of STAT3, Akt1, and ERK1/2 in the SGC-7901 cells. Figure 5A shows that plumbagin suppressed the expression and phosphorylation of STAT3, and Figure 5B shows that it decreased the phosphorylation of Akt1. Figure 5C shows that plumbagin did not, however, change the total protein or phosphorylation levels of ERK1/2. The SGC-7901 cells were incubated with 10 μM of plumbagin for different periods (0, 2, 4, 8, 12, and 24 hours). Whole-cell extracts were prepared and analyzed by Western blotting with the indicated antibodies.

A.

2

Time (h) Ser 473 4 8 12

24

STAT3

cleaved-caspase-3

β-actin

β-actin

STAT3, Akt1, and ERK1/2 The overall hypothesis underlying the current study was that plumbagin blocks the signaling pathways in GC cells that enhance their proliferation and survival. To address that hypothesis, the research team analyzed the activation of STAT3, Akt, and ERK, all of which have been shown to enhance tumorcell survival and proliferation.26,27 Plumbagin significantly decreased the phosphorylation of STAT3 (Figure 5A) and the phosphorylation of Akt1 (Figure 5B) after 12 and 24 hours of treatment. However, the phosphorylation of ERK1/2 displayed only a very weak decrease at 12 hours (Figure 5C), which was not significantly different from the controls.

Time (h) Ser 727 4 8 12

p-STAT3

pro-caspase-3

Apoptosis-related Proteins Because the plumbagin had promoted the induction of apoptosis in the SGC-7901 cells,25 the research team assessed the effects of plumbagin on cellular, apoptosis-related proteins. The plumbagin treatment of the cells resulted in an increased expression of the proapoptotic protein BAX and decreased expression of the antiapoptotic protein BCL-2 in a timedependent manner (Figure 4). The team also analyzed the effects of plumbagin on the caspase-related pathway by measuring the marker proteins pro-caspase-3 and cleaved-caspase-3. As shown in Figure 4, the treatment with plumbagin markedly decreased the protein levels of pro-caspase-3 and increased the amount of cleaved-caspase-3 in a time-dependent manner.

2

0

B.

0

24

p-Akt1 Akt1 β-actin C. 0

2

Time (h) Tyr 204 4 8 12 24

p-ERK1/2 ERK1/2 β-actin Abbreviations: STAT3, transcription 3; Akt1, protein kinase B; ERK1/2, extracellular signal–regulated kinase.

DISCUSSION To improve understanding of the mechanisms behind the anticancer properties of plumbagin in GC, the current research team investigated the effects of the compound on SGC-7901 cells. The team found that plumbagin treatment inhibited cell growth, induced an S-G2/M phase arrest, and

induced apoptosis in a dose- and time-dependent manner, accompanied by typical apoptotic morphological changes.

Furthermore, the team observed that the expression of the proapoptotic protein BAX was upregulated and expression of the anti-apoptotic protein BCL-2 was downregulated by plumbagin in a time-dependent manner. Those effects were associated with suppressed activation of 2 key intracellular signaling molecules, Akt and STAT3. On the other hand, plumbagin did not affect the activation of ERK1/2, despite its well-established role in cell growth and survival.

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Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

Plumbagin, an analogue of vitamin K, has shown efficacy against several preclinical models of human cancer, including prostate carcinoma,28 lung and breast cancers,29,30 and melanoma.31 The current study’s cell viability assay confirmed similar effects for plumbagin on the SGC-7901 cells in a dose- and time-dependent manner. The effective concentration of plumbagin for the induction of GC cell death was comparable with those of existing natural products that have been used against human GC cells, such as wogonin and curcumin.32,33 DMSO is widely used to dissolve hydrophobic drugs in both in vitro and in vivo studies. DMSO has anticancer properties but no effective standard exists for its use. In addition, the plumbagin was bioavailable to the GC cells. In a previous study that the current research team had performed,25 the cellular uptake of plumbagin for an anticancer effect was found. A side-by-side comparison of the plumbagin prepared in absolute DMSO or a DMSO solution showed precisely that the anticancer properties of the plumbagin were fully maintained, regardless of the transcription factor nuclear factor κB (NF-κB) signaling pathway.

The mitochondrial pathway is one of the major pathways for apoptosis, and the proapoptotic protein BAX and the antiapoptotic protein BCL-2 are key players in that pathway.34 The current study showed that the plumbagin treatment led to an upregulated expression of BAX and a downregulated expression of BCL-2, which resulted in a significant increase in the BAX/BCL-2 ratio in the GC cells. Therefore, it is likely that the imbalance of BAX/BCL-2 contributed to the apoptosis-promoting activity of plumbagin. Moreover, plumbagin activated the downstream effector caspase-3, another key player in the mitochondrial apoptosis pathway.

To explore the underlying mechanism further, the current research team analyzed 3 key intracellular signaling proteins, STAT3, Akt, and ERK, in GC cells treated with plumbagin. Those proteins have been extensively studied in regard to their roles in cancer. STAT3 is a known oncogene in many human cancers. The constitutive activation of STAT3 not only supports the survival of GC cells but is also a molecular prognostic marker for the GC patient’s outcome.35 The current study’s data showed that plumbagin inhibited the phosphorylation of STAT3 at the Ser727 site, which is an important site related to cellular apoptosis.

Akt is a well-established growth and survival factor and a central player in tumorigenesis.36 Akt activation promotes the cell cycle and proliferation and enhances the survival of cancer cells. In addition, under some conditions, Akt can activate the prosurvival NF-κB through the phosphorylation of I-κB kinase.36 The current research team has shown that plumbagin can inhibit the activation of NF-κB in GC cells,25 which can be mediated by inhibition of Akt activation.

With regard to therapeutic approaches for human cancer, the PI3K/Akt pathway plays an important role in the chemoresistance of GC cells. Therefore, the phytochemical plumbagin can be used in combination with chemotherapy to decrease the development and growth of resistant cancer

cells. Contrary to the current research team’s expectations, plumbagin did not affect the expression or phosphorylation of ERK1/2, although it has been reported that plumbagin can diminish the activation of ERK1/2 in A549 cells.37 That discrepancy may be due to the different cellular contexts because the ERK pathway is responsive to multiple upstream signals, only some of which may be inhibited by plumbagin.

One of the limitations of this study is that although plumbagin can inhibit the activation of STAT3 and Akt rather than ERK1/2 in human GC cells with dose dependent, we still need further evidence to reveal the causes of the results.

Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 47

CONCLUSIONS The current results have demonstrated that plumbagin exerts potent effects on the induction of apoptosis, growth inhibition, and G2/M cell cycle arrest in SGC-7901 cells in vitro. Those effects are likely to be due, at least partly, to suppressed activation of STAT3 and Akt. Based on that evidence, plumbagin should be strongly considered as a basis for the development of novel pharmaceutical agents for the treatment of gastric cancer, either alone or in combination with chemotherapy.

ACKNOWLEDGEMENTS

The current work was partially supported by the Natural Science Foundation of China (NSFC No. 81403485). The funding sources provided relevant information on the compound, reviewed the manuscript before submission, and provided key influences on the study’s design, data collection and analysis, decision to publish, and preparation of the manuscript.

AUTHOR DISCLOSURE STATEMENT

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted

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3. Powolny AA, Singh SV. Plumbagin-induced apoptosis in human prostate cancer cells is associated with modulation of cellular redox status and generation of reactive oxygen species. Pharm Res. 2008;25(9):2171-2180.

4. Ruangnoo S, Itharat A, Sakpakdeejaroen I, et al. In vitro cytotoxic activity of Benjakul herbal preparation and its active compounds against human lung, cervical and liver cancer cells. J Med Assoc Thai. 2012;95(Suppl 1):S127-S134.

5. Son TG, Camandola S, Arumugam TV, et al. Plumbagin, a novel Nrf2/ARE activator, protects against cerebral ischemia. J Neurochem. 2010;112(5):1316-1326.

6. Eldhose B, Gunawan M, Rahman M, et al. Plumbagin reduces human colon cancer cell survival by inducing cell cycle arrest and mitochondria-mediated apoptosis. Int J Oncol. 2014;45(5):1913-1920.

7. Sandur SK, Pandey MK, Sung B, et al. 5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: Potential role in chemosensitization. Mol Cancer Res. 2010;8(1):107-118.

8. Ono T, Ota A, Ito K, et al. Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines. Oral Dis. 2015;21(4):501-511.

9. Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther. 2006;5(12):3209-3221.

10. Shih YW, Lee YC, Wu PF, et al. Plumbagin inhibits invasion and migration of liver cancer HepG2 cells by decreasing productions of matrix metalloproteinase-2 and urokinase- plasminogen activator. Hepatol Res. 2009;39(10):998-1009.

11. Shieh JM, Chiang TA, Chang WT, et al. Plumbagin inhibits TPA-induced MMP-2 and u-PA expressions by reducing binding activities of NF-kappaB and AP-1 via ERK signaling pathway in A549 human lung cancer cells. Mol Cell Biochem. 2010;335(1-2):181-193.

12. Wang CC, Chiang YM, Sung SC, et al. Plumbagin induces cell cycle arrest and apoptosis through reactive oxygen species/c-Jun N-terminal kinase pathways in human melanoma A375.S2 cells. Cancer Lett. 2008;259(1):82-98.

13. Kannappan R, Gupta SC, Kim JH, et al. Tocotrienols fight cancer by targeting multiple cell signaling pathways. Genes Nutr. 2012;7(1):43-52.

14. Besser D, Bromberg JF, Darnell JE Jr, et al. A single amino acid substitution in the v-Eyk intracellular domain results in activation of Stat3 and enhances cellular transformation. Mol Cell Biol. 1999;19(2):1401-1409.

15. Jackson CB, Giraud AS. STAT3 as a prognostic marker in human gastric cancer. J Gastroenterol Hepatol. 2009;24(4):505-507.

16. Yang Y, Zhao Q, Cai Z, et al. Fas signaling promotes gastric cancer metastasis t h rou g h STAT 3 - de p e nde nt upre gu l at ion of fas ci n. PL o S O ne .

2015;10(5):e0125132.

17. Wu H, Fan F, Liu Z, et al. Norcantharidin combined with EGFR-TKIs overcomes HGF-induced resistance to EGFR-TKIs in EGFR mutant lung cancer cells via inhibition of Met/PI3k/Akt pathway. Cancer Chemother Pharmacol.

2015;76(2):307-315.

18. Zhang XY, Kuang JL, Yan CS, et al. NRSN2 promotes non-small cell lung cancer cell growth through PI3K/Akt/mTOR pathway. Int J Clin Exp Pathol.

2015;8(3):2574-2581.

19. Hao NB, Tang B, Wang GZ, et al. Hepatocyte growth factor (HGF) upregulates heparanase expression via the PI3K/Akt/NF-κB signaling pathway for gastric cancer metastasis. Cancer Lett. 2015;361(1):57-66.

20. Kang MH, Kim JS, Seo JE, et al. BMP2 accelerates the motility and invasiveness of gastric cancer cells via activation of the phosphatidylinositol 3-kinase (PI3K)/ Akt pathway. Exp Cell Res. 2010;316(1):24-37.

21. Park HH, Lee KY, Kim SH, et al. L-DOPA-induced neurotoxicity is reduced by the activation of the PI3K signaling pathway. Toxicology. 2009;265(3):80-86.

22. Lv L, Xiao XY, Gu ZH, et al. Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells. Mol Cell Biochem. 2011;346(1-2):11-21.

23. Xiao H, Zhang Q, Lin Y, et al. Combination of atorvastatin and celecoxib synergistically induces cell cycle arrest and apoptosis in colon cancer cells. Int J Cancer. 2008;122(9):2115-2124.

24. Arcaro A, Daga M, Cetrangolo GP, et al. Generation of adducts of 4-hydroxy-2nonenal with heat shock 60 kDa protein 1 in human promyelocytic HL-60 and monocytic THP-1 cell lines. Oxid Med Cell Longev. 2015;2015:296146.

25. Li J, Shen L, Lu FR, et al. Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway. Acta Pharmacol Sin. 2012;33(2):242-249.

26. Park E, Park J, Han SW, et al. NVP-BKM120, a novel PI3K inhibitor, shows synergism with a STAT3 inhibitor in human gastric cancer cells harboring KRAS mutations. Int J Oncol. 2012;40(4):1259-1266.

27. Qu JL, Qu XJ, Zhao MF, et al. Gastric cancer exosomes promote tumor cell proliferation through PI3K/Akt and MAPK/ERK activation. Dig Liver Dis. 2009;41(12):875-880.

28. Aziz MH, Dreckschmidt NE, Verma AK. Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormonerefractory prostate cancer. Cancer Res. 2008;68(21):9024-9032.

29. Hsu YL, Cho CY, Kuo PL, et al. Plumbagin (5-hydroxy-2-methyl-1,4naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo. J Pharmacol Exp Ther. 2006;318(2):484-94.

30. Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther. 2006;5(12):3209-3221.

31. Wang CC, Chiang YM, Sung SC, et al. Plumbagin induces cell cycle arrest and apoptosis through reactive oxygen species/c-Jun N-terminal kinase pathways in human melanoma A375.S2 cells. Cancer Lett. 2008;259(1):82-98.

32. Zhao Q, Wang J, Zou MJ, et al. Wogonin potentiates the antitumor effects of low dose 5-fluorouracil against gastric cancer through induction of apoptosis by down-regulation of NF-kappaB and regulation of its metabolism. Toxicol Lett.

2010;197(3):201-210.

33. Cai XZ, Wang J, Li XD, et al. Curcumin suppresses proliferation and invasion in human gastric cancer cells by downregulation of PAK1 activity and cyclin D1 expression. Cancer Biol Ther. 2009;8(14):1360-1368.

34. Qiao L, Wong BC. Targeting apoptosis as an approach for gastrointestinal cancer therapy. Drug Resist Update. 2009;12:55-64.

35. Jackson CB, Giraud AS. STAT3 as a prognostic marker in human gastric cancer. J Gastroenterol Hepatol. 2009;24(4):505-507.

36. Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis. Proc Natl Acad Sci U S A. 2001;98(20):10983-10985.

37. Shieh JM, Chiang TA, Chang WT, et al. Plumbagin inhibits TPA-induced MMP-2 and u-PA expressions by reducing binding activities of NF-kappaB and AP-1 via ERK signaling pathway in A549 human lung cancer cells. Mol Cell Biochem. 2010;335(1-2):181-193.

48 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Li—Proapoptotic and Growth-inhibitory Effects of Plumbagin on GC Cells

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PERSPECTIVES

Natural Cancer Therapy and Prevention Targeted on Cancer Cells and Cancer Stem Cells Based on the Cytochrome P45O Enzyme CYP1B1: A Commentary William R. Ware, PhD ABSTRACT The importance of the P450 enzyme CYP1B1 in both cancer therapy and prevention are reviewed and evidence is discussed, which provides strong biological plausibility for the therapeutic merits of this approach. The significant resistance to chemotherapy among common cancers, and the realization that in many cases chemotherapy leaves behind drug-resistant cancer cells that eventually cause recurrence and metastatic disease, has recently prompted the search for natural compounds that would circumvent this problem. There is also the important question of therapies that will kill cancer stem cells (CSCs) because they appear, in many cases, to be resistant to conventional therapy. The repeated observation that resveratrol has the ability kill CSCs in a variety of cancers suggests that this natural product may act through a mechanism that is cancer-type independent. There are a few other natural products that have this property and all act through a well-understood mechanism involving CYP1B1, which is known to be overexpressed at the protein level in ordinary cancer cells but not in noncancer cells. These natural products toxic to ordinary cancer cells serve as substrates for this enzyme and the metabolites cause apoptosis or cell

cycle arrest. This small group includes resveratrol. The observation that resveratrol also has this same action on CSCs independent of cancer type suggests the hypothesis that these cells also overexpress CYP1B1 and offer the same target as ordinary cancer cells. A number of case histories are reviewed that involve a variety of cancers from different sites and grades or stages treated with a proprietary mixture of CYP1B1 substrates extracted from fruit. They illustrate therapeutic effectiveness with even late-stage cancers put into durable remission. This implies combined ordinary cancer cell and CSC toxicity. In addition, a recent proposed mechanism for CYP1B1 overexpression only in cancer cells are described. Recently developed blood tests for CYP1B1, its cancer active substrates, and the resultant metabolites are reviewed as they add significantly to the biological plausibility of the CYP1B1 mechanism. Thus, a targeted therapy many researchers are actively seeking already exists with evidence of human efficacy and compelling biological plausibility. (Altern Ther Health Med. 2017;23(5):50-58)

INSERT AD

William R. Ware, PhD, is emeritus professor in the Faculty of Science at the University of Western Ontario in London, Ontario, Canada.

he US Centers for Disease Control and Prevention announced in August 2016 that cancer now surpasses cardiovascular disease as the top cause of death in nearly half of the United States. From 2011 to 2014, cancer mortality increased by 2.6% with almost 600 000 deaths in the last year of that period. The recent book The Death of Cancer1 may have been somewhat premature aside from its

focus on blood related cancers. Solid adult tumors and metastatic disease continue to present a huge challenge.

The conventional and widely held view for 5 decades is that the disease cause is genetics, but the genetic picture that has emerged in the last few years suggests this view is burdened by impossible complexity. Human genome studies have revealed the presence of approximately 250 oncogenes and 700 tumor suppressors. Cancer in this view represents approximately 1 million different genotypes. In the Cancer Genome Atlas project, nearly 1 million or more tumor samples have also been studied. Great enlightenment was expected. Instead millions of mutations have been discovered as well as 10 000 to 50 000 single nucleotide variants. When tumor cells are compared to adjacent normal tissue, tumor cells are like a huge genetic train wreck. With rare exceptions,

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Wareâ&#x20AC;&#x201D;Perspectives

Corresponding author: William R. Ware, PhD E-mail address: warewr@ogers.com

T

using genetic fingerprints to guide drug design appears to be a daunting challenge or worse.2 An alternative view that has become a serious contender only in the last decade is that cancer is a metabolic disease.

This theory is supported by the fact that most oncogenes and tumors suppressors appear to be associated with cancer cell metabolism. There appear to be only 3 major metabolic pathways affected. Thus, the basic metabolic disease model seems to be remarkably simple.3 The metabolic theory of carcinogenesis focuses on the mitochondria. As we age, not only do our cells lose mitochondria, but those that remain can become damaged and dysfunctional. Cellular energy generation involves the mitochondria, normally with glucose as the fuel. The instability in tumor genetics is considered as a downstream effect of the initial disturbance of cellular energy production.

In this theory, unspecified conditions cause damage and impair cellular mitochondrial energy production capacity (respiration) and initiate the path to malignant cancer. One result is the cytoplasmic environment changes from suppressing to promoting cancer. These events can be triggered by inflammation, carcinogens, radiation, low oxygen levels, new mutations, viral infections, and age. One result is a large increase in glucose needs to satisfy energy demands, a hallmark of cancer cells first pointed out by Warburg years ago. This change in mitochondrial function suggests important therapeutic interventions such as ketogenic diets because cancer cells cannot use ketones as an alternative fuel. In addition, hyperbaric oxygen treatments, which eliminate the low oxygen environment that encourages cancer cell proliferation, appear to have merit.4-6 During the past 2 decades, it has been recognized that the complex interplay of genetic diversity, epigenetic mechanisms, and tumor microenvironment have contributed to tumor heterogeneity and drug resistance, which some view as responsible for the failure of current therapies to significantly halt or reverse progression or increase patient survival.2 This heterogeneity includes cancer stem cells (CSCs), which are a subset of cancer cells with the ability to self-renew, differentiate, and drive tumor growth and metastasis.7-9 Thus, they are receiving increasing attention in the context of both cancer progression and metastasis, and they are thought to be involved in the initial stages of carcinogenesis.10 Cancer therapy must consider this important cancer cell type in order not to ignore a central feature of the problem being addressed, namely finding a therapy that works.11-13 Thus, important unresolved issues remain. The complexity of cancer appears to require multiplicity of therapies. Multidrug resistance to chemotherapy is a serious challenge as are radiation resistant tumors. This problem is so serious that it has prompted considerable activity associated with the search for natural products, which might induce cancer cell death or slow progression but not involve mechanisms prone to treatment resistance.8,14-16 The longterm plan probably involves identifying specific naturally

occurring chemicals, slightly modifying them while retaining their therapeutic value, and, after patenting, proceeding with clinical trials. This pushes an approved therapy quite a way into the future.

In what follows, a natural therapy both targeted on cancer cells and successful in preliminary cancer therapy investigations will be examined in the context of the viewing tumors as likely containing a subset of CSCs. It is based on targeting a single cytochrome P450 CYP1B1.

CSCs, TUMOR HETEROGENEITY, AND NEW OPPORTUNITIES FOR EFFECTIVE THERAPY Intensive chemotherapy is now thought to leave treatment resistant cancer cells to proliferate and metastasize, a so-called evolutionary or Darwinian effect,17 and new treatment protocols are being suggested.18,19 The theory that all cancer cells can be killed by intensive conventional chemotherapy has come under increased scrutiny. These residual cells likely include CSCs, which are notorious for being multidrug resistant. In some, remission may mean the burden of cancer cells has been reduced to the point where renewed proliferation will not yield symptomatic disease, localized or metastatic, during the remaining lifetime of the patient. However, there are many exceptions and with cancers that occur in younger adults this is less likely to be the case. Although in general chemotherapy and radiation do not appear to be killing all cancer cells, some surgical approaches succeed in accomplishing this simply because they are securely encapsulated or localized with no lymph node involvement.

Heterogeneity of tumor tissue is increasingly associated with the failure of conventional therapies.8 This heterogeneity is thought to be caused by a combination of genetic and functional diversity. Normal tissues derive from heterogeneous cell types that are derived from tissue stem cells, a process that is mostly epigenetically controlled. This view involving heterogeneity avoids some of the problems associated with theories, based merely on a stochastic accumulation of genetic mutations and, instead, emphasizes the combination of genetic and epigenetic mechanisms. However, tumors also contain CSCs. CSCs are viewed as undifferentiated cancer cells with the ability to self-renew, have high tumorigenic potential, and are characterized by multilineage differentiation capabilities.

The combination of a slow cell cycle, the ability to detoxify or facilitate the efflux of cytotoxic agents, a resistance to oxidative stress, and rapid DNA damage repair all lead to these cells possessing significant therapeutic resistance. It is thus becoming increasingly evident that CSCs are an important issue in carcinogenesis and progression and, importantly, the treatment of cancer.11,12,20 Truly effective therapy appears to require removal or killing of all the CSCs along with ordinary cancer cells.

However, the fact that most conventional treatments appear unsuccessful in destroying or removing all CSCs is well known. This has prompted considerable interest in therapies finding naturally occurring substances that when used against CSCs either induce apoptosis, cell cycle arrest, or

Wareâ&#x20AC;&#x201D;Perspectives

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 51

interfere with pathways with the result of interrupting proliferation.8,15 Mostly cell culture experiments appear to have been reported. One of the most studied natural products is resveratrol, a polyphenol found in a few foods and red wine. The induction of apoptosis or cell division arrest with cellular exposure to resveratrol has been observed in culture studies of CSCs associated with the following cancer types: medulloblastoma,21 glioblastoma,22 colon,23 acute myeloid leukemia,24 pancreas,25 thyroid,26 head and neck,27 nasophyryngel,28 breast,29 ovary,30 melanoma,31 and liver.32 It is unusual for a single natural molecule to produce the same significant end result for a wide range of cancer types, because one might expect variations related to the cancer type and there is a considerable variation in CSCs especially because there are large differences of the degree of differentiation, the local environment and nature of the niche in which some exist, the cell surface receptors, and other factors. Studies of natural products included the examination of a number of postulated mechanisms, mostly involving the interference of one or more pathways or the impact of one or more surface receptors.14,33 The apparent cancer-typeindependent toxicity found in the effect of resveratrol on CSCs encourages the search for a general mechanism, especially because resveratrol itself, even taken in very large amounts as a supplement, has no adverse effects and, thus, the potential therapeutic benefit appears specific to CSCs but is not tumor-type specific. No general systemic effects would be expected as seen with many chemotherapeutic agents.

These observations are of particular interest because resveratrol was the first of a small number of natural products identified that kill ordinary cancer cells independent of the cancer type with no adverse effects on normal cells.34 An attractive mechanism that would be consistent with the large number of CSCs from different tumors targeted by resveratrol and resulting in cell death or inhibition of cell division is that resveratrol is involved in a process in the cytoplasm of these particular cells that produces from resveratrol a substance that acts as a prodrug. This implies a process of converting this polyphenol to a chemical with the ability to induce these fatal changes independent of the cancer cell type, but only in CSCs. It is well established that the P450 CYP1B1 enzyme, which is overexpressed in ordinary cancer cells, does exactly this, with resveratrol acting as a substrate and producing a known metabolite, a toxic chemical piceatannol, which results in apoptosis and/or cell division arrest and, thus, cell death.34 This enzyme’s overexpression appears to be a unique phenomenon common only to cancer cells and it is clearly a specific target because resveratrol is otherwise innocuous. This is consistent with the well-established fact that normal cells do not express the enzyme CYP1B1 except in minute and totally insignificant amounts.35 The most reasonable inference from the large amount of data involving resveratrol cited previously is that CSCs, at least at and beyond some stage in development, also overexpress CYP1B1 as the enzyme. The therapeutic significance of this inference is clear and must ultimately be

verified experimentally. A common assertion one sees in the literature is that cancer cannot be cured without killing all the CSCs. A natural product operating through the previously described mechanism appears to have the capability of accomplishing this.

CANCER CELLS OVEREXPRESSING CYP1B1 An incomplete list of ordinary tumor cells that have been shown to overexpress CYP1B1 includes brain,37,38 breast,39-42 colorectal,38,40,41,43,44 kidney,38,41,42 lung,41,45,46 liver,41 lymph nodes,38 lymphoma,40 melanoma,40 multiple myeloma,40 non-Hodgkin’s lymphoma,38,41 esophageal,38,40,41 acute lymphocytic leukemia,40 acute myeloid leukemia,40,41 bladder,38,40,41,47 osteoscarcoma,48 ovarian,40,41,49 prostate,40,42,47 skin,38 stomach,38,41 testicular,38,41 and uterine.38,41 These studies were carried out mostly prior to the advent of interest in and appreciation of the importance of CSCs and it is highly likely that some of the cells positive for the enzyme were indeed CSCs given the procedures employed in the studies cited previously would not have in most cases separated the 2 types.

Because resveratrol is a salvestrol, it appears plausible that CSCs overexpress CYP1B1 and would represent a significant advance over the natural products currently described in the literature as having anti-CSC properties based on experimental studies but operating by different

52 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Ware—Perspectives

SUBSTRATES FOR CYP1B1-INDUCED CELL DEATH The belief among some researchers studying this subject was that resveratrol was probably not the only important substrate in this context and this prompted a search for other substrates in fruits and vegetables.36 A small number were found, mostly in fruits. In addition, an interesting observation was that the levels were dependent on whether the produce was organically grown and if the variety had been altered by recent hybridization, especially to remove bitterness, a common characteristic of anti-cancer-active substrates for CYP1B1. This led to the hypothesis that these particular substrates served a protective purpose in plants against pests and predators and consumption of the fruits or vegetables, especially before the advent of modern agriculture, conferred natural protection from cancer in humans.

Cancer cell culture methods were used to judge the effectiveness of the various extracts screened as potential cancer therapeutic agents and eventually of the best several were selected and extraction procedures perfected to prepare a dry, oral formulation suitable for use as a nutritional supplement. This selected substrate formulation was given the trade name Salvestrol after the Latin word salve, meaning “to save,” although researchers also use the term to describe any substrate of CYP1B1 that induces cell death only through the action of a metabolite. The final formulation had vastly more power to kill cancer cells in cultures than resveratrol and was able to do so in a dose dependent manner in contrast with resveratrol, which would start to inhibit CYP1B1 as the dose was increased.

mechanisms as compared to that involving CYP1B1.14 Assuming CSCs do indeed overexpress the enzyme CYP1B1, one might reasonably expect similar effects of active substrates as are seen in ordinary cancer cells.50,51 Other evidence supporting the notion that CSCs overexpress CYP1B1 will be presented later in the context of observations from case histories of cancer patients treated with active CYP1B1 substrates.

An additional critical observation in all these studies of CYP1B1 overexpression was that normal cells, even those adjacent to tumors, did not express this enzyme at the detection limits at that time. Unfortunately, simultaneously there arose considerable confusion because some investigators equated of finding CYP1B1 messenger RNA (mRNA) expression with the unverified assumption of the existence of the enzyme in the cytoplasm. This is not correct because the CYP1B1 mRNA is found in most human cells but transcription is turned off. This is of course a very important issue because a chemical agent interacting with an enzyme unique to cancer cells while metabolizing this chemical as a substrate to produce a substance responsible for cell death is exquisitely targeted with no systemic toxicity. Confusing finding CYP1B1 mRNA and jumping to the incorrect conclusion that this implies the presence of the actual cytoplasmic enzyme has been a severe impediment to the appreciation of the merits of this natural intervention. Nevertheless, this appears to be a widely held misconception.

The initial observation concerning the overexpression of CYP1B1 only in cancer cells is now more than 15 years old.

Additional verification inspired by this observation and summarized previously has ignored the therapeutic implications and investigators in this field still appear unaware of the clinical implications. Aside from the mRNA confusion, one explanation for this research being largely ignored is that publication of clinical benefits discussed later has been in low-impact journals,52 most of which are not indexed by PubMed, or has been published in books written for the general public.36,51 Interest in CYP1B1 appears mostly to be that it can metabolize endogenous and exogenous substances such as estrogen and aromatic hydrocarbons to produce carcinogens. Because only cancer cells have significant cytoplasmic levels of the enzyme, the generation of one of a very limited number of carcinogens in patients who already have diagnosed cancer, therefore, does not appear to be significant. It seems somewhat bizarre, given the unique therapeutic potential of this enzyme and as discussed later and its suggested role in the natural defense against human cancer, that interest is almost entirely focused on inhibiting rather than exploiting it.

Table 1. Types of Cancer, Stages, and Number of Cases Treated With Salvestrols Where Adequate Information Was Available Along With Follow-up Primary Site Lung: Squamous-cell carcinoma Melanoma Prostate Breast Bladder Liver Colon Hodgkin’s lymphoma Anus: Squamous cell carcinoma Lymphocytic leukemia Primary peritoneal carcinoma Pancreas Benign prostatic hyperplasia

Stage II to III IV III III II IIIB

Cases 1 1 3 2 1 1 1 1 1 1 1 1 1

Modified and reproduced by permission.51

a

implicated in the downregulation of miR-27b. This posttranscriptional regulation has also been attributed to epigenetic factors involved in the methylation of DNA in an area critical to these processes.53,54 Research concerning this issue is extremely limited. However, significant downregulation appears restricted to cancer cells.

WHAT TRIGGERS THE OVEREXPRESSION OF THE ENZYME CYP1B1 ONLY IN CANCER CELLS? This issue has been recently addressed and evidence presented that a microRNA (miR-27b) controls the expression of the enzyme, and when the miR-27b level is low, this allows the overexpression to proceed. Interleukin 6 has also been

BIOLOGICAL PLAUSIBILITY, CANCER DETECTION, AND DISEASE TRACKING PROVIDED BY BLOOD TESTS If CYP1B1 is functioning as believed in cancer cells, then either tissue or blood tests for the enzyme, substrate, and metabolite become important to confirm the proposed mechanism. Although tissue methods proved troublesome, blood tests based on state-of-the-art analytical techniques were satisfactory.35 CYP1B1 was indeed found in large quantities in cancer cells and in minute quantities in normal cells that may have been a manifestation of adventitious cancer cells or the presence of very early-undetected cancer.

Analytical techniques allowed the tracing of the time lines for the metabolism. In individuals without evidence of cancer, the substrate as expected was not metabolized because of only minute amounts of the enzyme present. For cancer patients, the substrate deceased in concentration and cell destruction released the metabolite into the circulation, which grew in proportion to the decrease in substrate. Thus, the absence of CYP1B1 in an individual’s serum strongly suggested the absence of cancer, and in cancer patients it was found that the higher the cancer grade or larger the tumor, the faster the substrate was metabolized with very advanced cancers having a greatly increased ratio of metabolite to substrate.35 This would appear to be adequate evidence concerning the nature of the mechanism at work and provides strong biological plausibility for the therapeutic application.

Ware—Perspectives

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 53

Finding a high level of CYP1B1 in an individual’s blood of course does not indicate the location of the cancer or cancers and in some cases it might remain mysterious if the stage was early or the cancer asymptomatic. However, therapy with active substrates such as salvestrols does not require knowledge of the identity or location of the cancer, although location can influence the degree of exposure to the substrate in special cases such as is suspected for prostate cancer (B. Schaefer, personal communication, September 2016).

An active area of current research worldwide involves searching for blood markers for cancer. The experimental results described previously appear to indicate that serum CYP1B1 levels are an attractive candidate for this application and in fact offer a rather simple solution and apparently a universal marker.

CASE HISTORIES: THE EVIDENCE OF BENEFIT FOR CANCER PATIENTS For cancer patients, how well does this novel natural optimized therapy using salvestrols work and do the results provide any insight as to how well this therapy might be working on CSCs? Can we go beyond the argument that resveratrol is a salvestrol and known to kill CSCs and, therefore, CSCs overexpress CYP1B1? Given that the high potency substrate formulation was still merely a natural product, a mixture of 4 fruit extracts, it could not be patented, and formal clinical trials are generally financially prohibitive.

Thus, reliance had to be placed on case histories of effectiveness and, to be convincing, they had to be backed by clinical evidence of the presence of the cancers and evidence for remission when it occurred. Although such anecdotal evidence is not acceptable and even ignored in modern evidence based medicine, it is nevertheless the common starting point in the development of many drugs and interventions.

At this writing, 16 case studies of therapy with salvestrols with satisfactory documentation have been accumulated and published.51,52 The results are given in Table 1.51 The age range was 36 to 94 years, 59% male. Regarding conventional treatment prior to starting therapy, 35% had surgery, 35% chemotherapy, 6% radiation, and 18% had 2 different treatments and 23% had no conventional treatment. The time from treatment to remission, time to remission followed by relapse, and variation in time to remission are of considerable interest. The results can be summarized as follows: 1. For these 16 cases, average time to observed remission from start of treatment was 6 months (range, 1-18 mo). However, 15 reached remission within 1 year with a prostate cancer case taking 18 months.

2. Average time in remission and still in remission, 41 months (range, 8-82 mo), 12 cases.

3. Average time in remission followed by recurrence, 45 months (range, 19-60 mo), 5 cases.

4. Average time to remission for rapid responders, 2.1 months (range, 1-3 mo), 6 cases.

54 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

5. Mortality: 2 deaths, 1 from Alzheimer’s disease, 1 of unknown cause. Both were in remission.

6. All who had recurrences abandoned treatment after remission and did not change diet or lifestyle.

7. For those still in remission, 10 of 12 continued to take maintenance doses.

8. No adverse side effects were reported.

9. In the cases where the attending oncologist estimated life expectancy associated with conventional treatment, most patients exhibited a large and unexpected increase in survival due to salvestrol therapy.

It will be noted that Table 1 includes benign prostatic hyperplasia (BPH), normally not considered cancer. However, BPH tissue contains cells that overexpress CYP1B1 as the enzyme,55 and thus it is not surprising that the disease appears to respond to this treatment. Consistent with this, a recent study rather strongly links BPH to increasing the risk of both prostate and bladder cancer.56 The effect on BPH is important because conventional treatments for BPH have unpleasant side effects and treatment with salvestrols might prevent progression to prostate cancer and as well bladder cancer. There is urgent need to examine (1) the same question in benign precancer in the breast, cervix, and other tissues; (2) the question of salvestrol preventing BPH from progressing to prostate or bladder cancer; and (3) salvestrol’s utility in decreasing or eliminating the urinary dysfunction associated with BPH. In Table 1, some of the cases involved advanced disease and some included metastasis.51 Given that CSCs are implicated as being significantly involved in advanced and metastatic disease, but remission was still achieved in the majority of cases described, this suggests that CSCs were being effectively impacted. The case histories also revealed that sustained remission was generally only achieved when a maintenance dose was continued to be taken, again suggesting that residual cancer cells were still present and it is reasonable to assume that some were CSCs. Some case histories showed that salvestrol treatment when used as an adjunct to chemotherapy by patient choice resulted in totally unexpected beneficial results.51 This could be interpreted as salvestrols killing treatment resistant cells and CSCs. Two of the case histories are particularly suggestive of a significant impact on CSCs.51 One involved stage IV melanoma. This implies a population of CSCs within the tumors, because CSCs are known to be significantly involved in melanoma.57 The patient, 94 years of age, clearly had actively progressing metastatic melanoma and refused conventional treatment. Using only salvestrol therapy, she was free of any signs of cancer at 12 months and remained in remission for an additional 8 months prior to death from noncancer causes. All the various locations where the progressing melanoma was evident were free of external signs of the disease within 12 months.51 At diagnosis, she had been given 2 weeks beyond which the family was told that morphine would be required for pain control.

Ware—Perspectives

A second untreated case was a 55-year-old man who went from bladder cancer to also developing kidney cancer and then also pancreatic cancer. Five months after starting salvestrol treatment, he was pronounced cancer free. At the end of follow-up, he had been in remission for 60 months, well beyond the typical survival rate for pancreatic cancer.

Here again the participation of stem cells is indicated because of the presumed active metastasis. These 2 case histories that were well documented by modern methods suggest that CSCs not only overexpress CYP1B1 but respond to salvestrol metabolites that cause cell death. Furthermore, both pancreatic CSCs58 and melanoma CSCs31,33 are known to respond to resveratrol with cell apoptosis and cell cycle arrest, which again encourages consideration of a CYP1B1 driven mechanism, which includes CSCs because resveratrol is a classic salvestrol. The duration of remission for the pancreatic cancer survivor would also suggest that salvestrols had killed virtually all of the cancer cells including the CSCs because typical life expectancy is only 4 and 8 months.

Due to the widespread use of salvestrols worldwide, many case histories are currently being informally collected in patient records, but there appears to be no systematic organization or reporting of the data, which is unfortunate.

Excellent results are being reported anecdotally including success with pediatric brain cancers. This is a particularly important area and a small informal clinical trial involving 20 children with various brain cancers is underway in New Zealand. Success has been achieved not only with pediatric brain cancer but also anecdotal reports of success with adult brain cancer implies that one or more of the salvestrols in the commercial formulation crosses the blood brain barrier.

Although it is understood that these human studies are merely anecdotal evidence, they add to the experimental blood studies in establishing that there may be significant merit in this approach to cancer therapy. They also highlight the importance of the implication based on the previously described body of evidence that CSCs may also overexpress CYP1B1 independent of the cancer type and they are also killed by a metabolite of the substrates contained in the commercial product, as they are by resveratrol and, in fact, killed much more effectively due to the increased effectiveness of the commercial formulation of substrates compared to resveratrol.

The clinical results described previously are admittedly very limited, especially with regard to long-term follow-up.

The need for large and systematic clinical studies seems urgent given the very slowly improving 5-year survival rates for many solid adult tumors and poor prognosis of pediatric and adult brain cancer.

THERAPEUTIC FAILURE The previously described case histories are not meant to imply the absence of failures. However, there are simple explanations for failures. CYP1B1 can be inhibited by a number of commonly encountered substances; chemicals found in food, medications, household items, and supplements can act as inhibitors. Resveratrol found in red Wareâ&#x20AC;&#x201D;Perspectives

wine and also as a dietary supplement is an example. The extract is highly concentrated and can effectively participate in competitive inhibition. The amount normally consumed in red wine is insufficient to interfere with therapy based on extracts such as salvestrols. The following are contraindicated.

All inhibit the action of the enzyme on the substrates being used therapeutically.59 1. Fungicides: Present in agrochemicals, dandruff shampoos, and antifungal creams and cleaning agents used in cleaning ductwork and as prescription drugs.

2. Resveratrol: Only in high doses as found in supplements.

3. B17: Laetrile, amygdalin, cyanogenic compounds.

4. Fruit and vegetable juices: Only excessive consumption.

5. Grapefruit.

6. Cannabis.

7. Gingko biloba.

8. Ginseng: Asian or North American.

9. Metformin.

10. Carbon monoxide (smoking).

11. Calcium D-glucarate supplement.

12. Artificial sweeteners.

As is normal with enzyme mediated chemical reactions, CYP1B1 does not operate in isolation and cofactors are needed. For CYP1B1, these have been found to include biotin, magnesium, niacin, riboflavin, iron, vitamin C, and unrefined fatty acids (Equazen and Efalex).60 Thus, a poor diet can result in a deficiency in some of the cofactors and cause the therapy to either fail or achieve poor response.

It is important to emphasize that the evidence thus far accumulated is of necessity anecdotal, with the exception of the blood analysis work and the extensive experimental work reported on the presence or absence of CYP1B1 in cancer and normal cells. Nevertheless, this evidence consistently indicates the validity of the picture suggesting these fruit based substrates appear to be effective independent of the cancer type and the fact that even high grade cancers including those of metastatic origin with low patient life expectancy have on treatment resulted in remission. Although many, but not all, of the remission times so far observed are obviously short compared with the expected time for recurrence for cancers that are not cured but are merely metastasizing or growing back at the original site, they suggest the possibility that CSCs have also been killed because some of these patients had advanced, metastatic, and, in some cases, treatment-resistant disease and remained in remission for an extended period.

ROLE OF CYP1B1 IN PRIMARY PREVENTION The number of cancer cells needed for cancer to become symptomatic depends on the type and the detection protocol, but it could be as high as 109 or more. This represents a huge ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 55

range where cancer cells are present but the individual is merely siting back and waiting for symptoms to appear, totally oblivious of the impending disaster. It can be argued that salvestrols are the ideal preventive agent. They appear to target all cancer cells, should kill cancer cells very early in the natural history of the disease, have compelling biological plausibility, and may also target benign hyperplasic tissue and some benign tumors. They are also theoretically able to kill individual cells or small clumps of cells that are years away from yielding diagnosable cancer. Mutations from cosmic rays and natural radioactivity, replication errors, and epigenetic factors related to environmental and metabolic factors all potentially yield cancer cells that would be expected to be destroyed sooner or later by circulating salvestrols, which appear to permeate the whole body. The same would apply to tumors of significant size but too small to detect by any present conventional procedures.

Furthermore, the blood tests discussed previously appear capable of detecting subclinical cancer and even determine the success of salvestrols in eradicating it, all without the patient or physician having any idea about the cancer type, location, or stage of development. All that is required is for each cancer cell to encounter a salvestrol molecule, which enters the cell cytoplasm. This approach is simple and apparently totally safe. The active chemicals are obtained from fruits that have been in the human diet for eons: strawberries, blackberries, blueberries, and tangerines.

Furthermore, one is not trying to zero-in on one cancer but all cancers. One can seemingly ignore the huge literature on preventing this or that cancer, generally with only modest success. An important point is that there are too many cancer types to make targeting individual cancers for prevention a reasonable approach unless there is a strong genetic predisposition to a certain cancer. Given that salvestrols are effective on diagnosed tumors, some with very large numbers of cancer cells, the dose for prevention would be expected to be low in the context of prevention. But there is a risk of inadequate low doses for cancers that are about to make their presence known, and these can include large silent tumors such as are common in ovarian cancer.

Although follow-up studies using low doses of salvestrols for prevention would be very informative and desirable, they require large cohorts and long-term follow-up, and they would need to be financed by nonindustry sources, which is unlikely because of the bias against natural approaches to solve problems in mainstream medicine. Nevertheless, salvestrols offer a prevention option that appears significant.

continues for a long period, but in other cases the patient goes home with the message “we think we got it all” and then waits for signs of recurrence. The fear for surgery to “not get all the cancer” prompts adjunctive chemotherapy and radiation therapy or both. Still, recurrence occurs. The word salvage entered the vocabulary of oncology some time ago.

In prostate cancer, it is called biochemical failure, which translates into prostate-specific antigen levels moving up from the postsurgical or postradiation nadir. Surgeons must balance the benefits of wide surgical margins against the collateral damage involved, especially in the brain.

Salvestrols would seem to offer a solution to the problem of secondary prevention because exactly the same principles are involved as in primary prevention and actual treatment.

Someone electing conventional treatment has the opportunity to attempt to prevent recurrence with no apparent risk and with an approach that is independent of the nature of the potential recurrence or primary site. This is an important in the context of metastatic cancer. In addition, in many cancers, the use of salvestrols as the only therapy is not appropriate due to the urgency of relieving symptoms caused by the tumor, such as is seen for example in colorectal, brain, ovarian, and thyroid cancers.

It was recently suggested that a search should be made for a CYP1B1 substrate that yielded a fluorescent metabolite, which would then help identify surgical margins and the extent of invasion of neighboring tissue by the primary cancer field with many applications.60 All that is needed is a light beam of the appropriate wavelength. With an appropriate fluorescent metabolite, the signal to background florescence should provide very high sensitivity (signal to noise), simply because CYP1B1 is cancer-cell specific.

CYP1B1 IN SECONDARY PREVENTION It is well known among physicians and among the general public that cancer recurrence, or, put another way, treatment failure, is common. Surgical margins are sometimes not well defined, lymph nodes containing cancer cells are missed, and, as mentioned previously, chemotherapy leaves a subset of cancer cells resistant to treatment that contribute to local recurrence and metastasis. In some cases, therapy at some level

CONCLUSION The frequent appearance of the polyphenol resveratrol in the literature concerning natural ways to target and kill CSCs and the large number of cancer types where apoptosis or cell cycle arrest has been observed in cell cultures after exposure to this polyphenol suggests that the same mechanism might be operating here as is seen clearly in ordinary cancer cells and is well understood. In ordinary cancer cells, a mechanism operates independent of cancer type or stage and involves a unique phenomenon, an enzyme of the P450 family present only in cancer cells at the protein level. Among the substrates that this enzyme metabolizes are a limited number of naturally occurring chemicals that yield metabolites that are capable of killing the cancer cell. Because resveratrol is one of these substrates, and it is well established to be operating by this mechanism, it thus might be inferred that this same mechanism is in operation based on observations in numerous CSC culture experiments reported. Thus, it can be inferred with reasonable level of confidence that CSCs overexpress this same enzyme as a protein and would offer a highly significant therapeutic target, especially because substrates vastly more effective than resveratrol have been discovered and made available as dry extracts for human use as a dietary supplement.

56 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Ware—Perspectives

This is what many researchers have been looking for and it is suggested that it already has been found, perfected, and tried on human subjects with success. In the context of ordinary cancer cells, it has indeed been clear but underappreciated for more than a decade that salvestrols represent the long-sought-after magic bullet. It appears that this can be generalized to CSCs and would represent a significant advance in cancer therapy, especially because CSCs are resistant to conventional therapy and implicated in proliferation and metastatic processes. The importance of this is underscored by the view held by some that if one cannot remove or kill all the CSCs, then the cancer will sooner or later recur unless the patient dies first. The broad hint provided by all the work done with resveratrol on CSCs should not be taken lightly or ignored. Of the various natural products thus far examined in this context, resveratrol appears to be the only chemical that qualifies as meeting the definition of a salvestrol.

Although examples are very limited, it appears that when advanced cancer, which probably is composed of significant numbers of CSCs and responsible for the rapid proliferation, is treated with the use of selected CYP1B1 substrates, remission and no detectable residual cancer has been the result. This suggests, but of course does not prove, that CSCs have been targeted successfully. The development of stem cell research techniques and cellular enzyme detection protocols have now reached the point that this should be a relatively straight forward undertaking. It seems very important that definitive experiments be conducted to prove that CSCs overexpress CYP1B1 at the protein level.

Finally, it would be unfortunate if there is little interest in the issues raised because natural products that cannot be patented are involved, and thus there may never be clinical trials acceptable to evidence based medicine. It should be pointed out that the early resveratrol results prompted the synthesis of a prodrug Stilserene, which CYP1B1 metabolized to yield a chemical that was capable of killing cancer cells.61 This drug has recently been licensed to a pharmaceutical company for clinical trials.36 Meanwhile, an entirely adequate solution for cancer patients to consider exists in the form of a carefully formulated natural product mixture that is simply marketed as a dietary supplement and incidentally has never been observed to induce adverse side effects, which is not surprizing for an extract of 4 common fruits.

AUTHOR DISCLOSURE STATEMENT

The Author declares that there is no conflict of interest, nor was there any financial support for the preparation of this paper.

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Wareâ&#x20AC;&#x201D;Perspectives

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An Innovative Approach to Overcome Gagging Through Acupressure for an Edentulous Patient: A Case Report Saravanan Thirumalai Thangarajan, MDS; Ramasamy Chidambaram, MDS; Padmanabhan Thallam Veeravalli, MDS; Kasim Mohamed, MDS

ABSTRACT The gag reflex is a physiological reaction, which safeguards the airway from foreign bodies. In some people, this response is exaggerated to the extent that the acceptance/provision of dental treatment becomes impossible. Management of patients with gagging

Saravanan Thirumalai Thangarajan, MDS, is a senior lecturer; Ramasamy Chidambaram, MDS, is a senior lecturer; Padmanabhan Thallam Veeravalli, MDS, is a professor and head of the Department of Prosthodontics, and Kasim Mohamed, MDS, is a professor, Faculty of Dental Sciences, at Sri Ramachandra University in Porur, Tamil Nadu, India.

Corresponding author: Saravanan Thangarajan Thirumalai, MDS E-mail address: docsharan86@yahoo.co.in

G

agging is the perplexing situation that dentists commonly encounter during dental procedures while making maxillary impressions. Gagging is a physiological reaction that safeguards the airway from foreign bodies. Patients with a mild gagging sensation are managed successfully by minor procedural modifications.

However, in certain situations, the response can be exaggerated to the extent that the acceptance or provision of dental treatment becomes impossible. It tends to compromise the dental treatment and becomes distressing for both the patient and the clinician.

The apprehension developed toward the dental appointments prevents patients with a gag reflex from seeking regular oral care. Consequently, the patients can have poor dental hygiene and seek dental treatment only when in pain, or they may request treatment under general anesthesia.1,2 Several approaches exist for managing those patients, and it is the dentist’s responsibility to choose the appropriate method for managing each case. Of the many approaches available, acupressure is highly successful in controlling the gag reflex.

60 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

involves many different approaches. The aim of this report is to illustrate the role of acupressure in controlling gagging, because it is a safe, affordable, quick, and relatively noninvasive technique. (Altern Ther Health Med. 2017;23(5):60-61)

Acupressure is a method of traditional Chinese medicine that is based on the same principles as acupuncture is, and it uses finger pressure to stimulate the pressure points. The first acupressure point for anxiety is called Yintang, or “Hall of Impression.” The point lies midway between the medial ends of the eyebrow, in the area designated by many cultures as the third eye. Yintang3 has a powerful action in calming the mind, and because of that action, it is almost always used in the treatment of insomnia, anxiety, and agitation. The aim of the current study was to illustrate the benefits of acupressure in controlling the gag reflex, providing a safe, economical, quick, and relatively noninvasive technique.

PATIENT INFORMATION A female patient, aged 38 years, visited the Department of Prosthodontics at Sri Ramachandra University (Porur, Chennai, India) with the chief complaint of a vomiting sensation upon wearing a maxillary complete denture. The patient had a history of complete edentulism (ie, toothlessness) of both the maxillary and the mandibular arches for the 2 years prior to her visit, and she had been wearing dentures in the same areas for the prior 1.5 years, with no relevant medical history.

PHYSICAL EXAM AND DIAGNOSTIC ASSESSMENT Upon oral examination of the patient, the clinician determined that the patient’s maxillary and mandibular, residual alveolar ridge were rounded and well formed. The severity of the patient’s gagging was assessed using a Gagging Severity Index (GSI).4 The GSI is a scale with 5 grades: (1) grade 1—very mild, occasional, and controlled by the patient; (2) grade 2—mild but requiring control by the Thirumalai—Gagging and Acupressure

Figure 1. Maxillary Impression Made With Mandibular, Edentulous, Perforated Stock Trays

Figure 2. The Acupressure Point (Yintang Point)

INTERVENTION The primary impression of the maxillary and mandibular, edentulous residual ridge was made with irreversible, hydrocolloid impression material (Alginoplast, Heraeus Kulzer GmbH, Hanau, Holland). A maxillary impression (Figure 1) was made with mandibular, edentulous, perforated stock trays.

During the impression procedure, the acupressure point, Yintang, was pressed with a fingertip to apply prolonged pressure. The point lies midway between the medial ends of the eyebrow. In the procedure, it is ideal to apply pressure (Figure 2) that is gradual, steady, and penetrating, for approximately 3 minutes. As a general rule, the pressure applied at the acupressure point should be such that it “hurts good” (ie, the pressure should be somewhere between a firm, pleasant pressure and outright pain).5 The effectiveness of the acupressure in preventing or reducing gagging was assessed during the dental treatment with the help of the Gagging Prevention Index (GPI).4 The GPI is a scale with 5 grades: (1) grade 1—an obtunded gag reflex; treatment was successful; (2) grade 2—a partially controlled gag reflex; all treatment was possible; (3) grade 3—a partially controlled gag reflex but frequent gagging; simple treatment was possible; (4) grade 4—an inadequately controlled gag reflex; simple treatment was unable to be completed; and (5) grade 5—the gag reflex was severe; no treatment was possible.

The assessment during the dental treatment found the gagging to be reduced from grade 3 to grade 2, resulting in a partially controlled gag reflex and allowing treatment to be possible.

From the primary cast (Figure 3), the denture base and occlusal rims were fabricated directly, omitting some clinical procedures, such as border molding and secondary impression. A U-shaped, maxillary denture base with an occlusal rim (Figure 4) was fabricated to relieve the distal extension of the denture base into the soft palatal region.

After the process of tentative jaw relation, teeth setting and a wax trial of the denture were performed. During both the tentative jaw relation and the try-in procedures, acupressure was applied to the Yintang point in a firm, gentle manner.

During the try-in procedure, a functional impression was made with the medium-body, Hydrosil polyvinyl siloxane impression material (Dentsply/Caulk, Milford, DE, USA), and the final dentures were processed and inserted (Figure 5).

patient with reassurance from the dental team; (3) grade 3— moderate and consistent, creating limits on treatment options; (4) grade 4—severe, with treatment being impossible; and (5) grade 5—very severe, affecting patient behavior and dental attendance and making treatment impossible. The patient’s gagging was found to be grade 3. After considering the various treatment modalities, the clinician decided that acupressure would be the safest and most reliable technique that could be adopted for the patient.

Outcomes The reduction of the patient’s gag reflex resulted in the successful processing and insertion of the final dentures. The final prostheses were clinically assessed and were found to be successful and satisfactory, because the roofless maxillary dentures were fabricated using mandibular impression trays that also aided in reducing the gag reflex. Because the roofless dentures did not extend into the posterior palatal seal region, no contact occurred with the acrylic denture base in that region, thereby reducing the ongoing incidence of gag reflex for the patient.

Thirumalai—Gagging and Acupressure

ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5 61

Figure 3. The Denture Base and Occlusal Rims Fabricated Directly From a Primary Cast

Figure 4. The Fabricated, U-Shaped, Maxillary Denture Base With Occlusal Rim

Figure 5. The Final Dentures as Processed and Inserted A.

B.

DISCUSSION The etiology of gagging is complex and not fully understood to date. The etiology is somatic, psychogenic, or a combination of both. Sometimes gagging during dental procedures seems to be related to a heightened sensitivity to the taste or smell of products, and it may be triggered even without contact with the palate (oral cavity). Many techniques have been reported in the literature to overcome the problem of gagging during impression making. However, no single technique is available that is suitable for every patient.6 Psychological approaches involve a prolonged duration of the procedures with highly cooperative patients to obtain successful results.7 Surgery is not highly recommended and is not suitable for all cases.8 Singer’s marble technique9 requires the motivation of the patient. However, that approach presents definite medico-legal risks of the patient’s aspiration of some of the marbles during its practice.

On the other hand, drugs have a limited effect on mild cases and seem to stimulate gagging for severe cases.2 Topical anesthetics tend to increase nausea and vomiting.10 That result is due to a sense of numbness produced in the sensitive

palate and pharyngeal areas. Centrally acting drugs, such as antihistamines, sedatives, tranquilizers, and parasympathetic and central nervous system depressants offer only a shortterm solution, especially for some severe cases.11 Complicated techniques have been employed in severe cases. Hypnosis and behavioral therapy have been used in hysteric patients.12 Keeping in mind that the patient’s chief complaint was a vomiting sensation upon wearing a maxillary denture, acupressure was chosen as the appropriate treatment in the current case study because it provided the ability to avoid the disadvantages of the aforementioned techniques. The technique proposed in the current study was the application of acupressure at a particular site to relieve the patient’s stress and, thereby, reduce the incidence of gag reflex.

Acupressure, although based on the principles of acupuncture, provides a strong advantage in that it is completely noninvasive and does not require any instruments, like needles. The acupressure technique is simple compared to many of those discussed previously.

In the current case, the suppression of gagging is presumed to have resulted from the relaxation effects of the

62 ALTERNATIVE THERAPIES, SEP/OCT 2017 VOL. 23 NO. 5

Thirumalai—Gagging and Acupressure

acupressure. With the personal participation of the patient, the impression-making process was smooth and effortless because it gave the patient confidence and control of the situation. If a more pronounced effect is required for some patients, the needling of Yintang and of additional points such as PC-6 Neiguan and CV-24 Chengjiang have been reported to reduce the gag reflex significantly.

According to the philosophy of Chinese medicine, the liver is responsible for creating a smooth flow of Qi (energy) throughout the body and for smoothing the patient’s emotions. Anger, irritability, and frustration are all signs that the Qi is not flowing smoothly. That circumstance is referred to as liver Qi stagnation. That stagnation might be managed through points such as LV-3 and LI-4.

The current case study has shown that the acupressure technique can be successful and appropriate, irrespective of the severity of gagging reflex. Considering the advantages of the present technique, such as its being noninvasive, efficient, economical, and unhazardous, the current research team concludes that acupressure can be a novel method in managing patients with gag reflex.

CONCLUSIONS Gag reflex is experienced in various stages of dental procedures. It is highly crucial to make a clear-cut distinction between the general or local causes of gag reflex and the psychological component involved in it. No universal remedy seems to exist for the successful management of the gagging patient. Wide ranges of management strategies have been explained in the current article, and they should be tailored to suit the needs of the individual patient. However, the skill and patience of the clinician is greatly responsible for controlling such a situation to carry out the treatment with satisfactory results for the patient. The simple technique of combining acupressure with selective impression-making procedures to alleviate the problem of gagging during impression-making was helpful irrespective of the severity of the reflex and the type of impression material.

REFERENCES

1. Wright SM. Medical history, social habits, and individual experiences of patients who gag with dentures. J Prosthet Dent. 1981;44:74-78.

2. Conny DJ, Tedesco LA. The gagging problem in prosthodontic treatment- part I: Description and causes. J Prosthet Dent. 1983;49:601-606.

3. Litscher G. Shenting and Yintang: Quantification of cerebral effects of acupressure, manual acupuncture, and laserneedle acupuncture using high-tech neuromonitoring methods. Med Acupunct. 2006;16(3):1.

4. Dickinson C. Gagging Problems in Dental Patients: Literature Review for the Diploma in Dental Sedation. London, England: GKT Dental Institute of King’s College London; 2000.

5. Gach MR. How to apply pressure to acupressure points. www.acupressure.com 6. Conny DJ, Tedesco LA. The gagging problem in prosthodontic treatment. Part 1: Identification and causes. J Prosthet Dent. 1983;49:601.

7. Kovats JJ. Clinical evaluation of the gagging denture patient. J Prosthet Dent.

1971;36(3):613-619.

8. Leslie SW. A new operation to overcome gagging as an aid to denture construction. J Can Dent Assoc. 1940;6:291.

9. Singer lL. The marble technique: A method for treating the “hopeless gagger” for complete dentures. J Prosthet Dent. 1973;29(21):146-150.

10. Schole ML. Management of the gagging patient. J Prosthet Dent. 1959;9:578.

11. Saunders WH. Intravenous valium for the gaggy patient. Trans Acad Opthalmol Otolaryngol. 1973;77(6):ORL411.

12. Epstein LH, Hershen M. Behavioral control of hysterical gagging. J Clin Psychol.

1974;30(1):102-104.

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