Biochemical investigation of The plant Terminalia arjuna ( Arjun) General Introduction The study of disease and their treatment have been existing since the beginning of human civilization. Norman R. Farnsworth of the University of Illinois declared that, for every disease that affect mankind there is a treatment and cure occurring naturally on the earth. Plant kingdom is one of the major search areas for effective works of recent days. The importance of plants in search of new drugs is increasing with the advancements of medical sciences. For example, ricin, a toxin produced by the beans of Ricinus communis, has been found to be effectively couple to tumor targeted monoclonal antibiotics and has proved to be a very potent antitumor drug (Spalding, 1991; Gupta, 1992). Further have the HIV inhibitory activity has been observed in some novel coumarins (complex angular pyranocoumarins) isolated from Calophyllum lanigerum (Kashman et al., 1992) and glycerrhizin (from Glycerrhiza species). Hypericin from Hypercium species) is an anticancer agent. Taxol is another example of one of the most potent antitumor agent found from Taxus bravifolia. In fact, plants are the important sources of a diverse range of chemical compounds. Some of these compounds possessing a wide range of pharmacological activities are either impossible or to difficult to synthesize in the laboratory. A phytochemist uncovering these resources is producing useful materials for screening programs for drug discovery. Emergence of newer disease also leading the scientists to go back to nature for newer effective molecules. Recently developed genetic engineering in plants has further increased their importance in the field of medicine, for example in the production of antibiotics by expression of an appropriate gene in the plant. By using these techniques it is possible to modify the activity or regulate the properties of the key enzymes responsible for the production of secondary metabolites. Thus by knowing the potential resources it is possible to increase the content of the important active compounds (Owen et al., 1992) and in the future, genes responsible for very specific biosynthetic processes may be encoded into host organism to facilitate difficult synthetic transformation. Thus plants are considered as are of the most important and interesting subjects that should be explored for the discovery and development of newer and safer drug candidates 1.1 Definition of medicinal plants The plants that possess therapeutic properties or exert beneficial pharmacological effects on the animal body are generally designated as Medicinal plants. Although there are no apparent morphological characteristics in the medicinal plants that make them distinct from other plants growing with them, yet they possess some special qualities or virtues that make them medicinally important. It has now been established that the plants, which naturally synthesize and accumulate some secondary metabolites like alkaloids, glycosides, tannins and volatile oils and contain minerals and vitamins, possess medicinal properties. Accordingly the WHO consultative group on medicinal plants has formulated a definition of medicinal plants in the following way- “ A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs.� 1.2 History of medicinal plants
From historical records, it is apparent that most of the early peoples like Assyrians, Babyloins Egyptians & ancient Hebrews, were familiar with the properties of many medicinal plants. Babyloins were aware a large number of medicinal plants & their properties. Historical records of Assyria & Babyloins indicate that by 650 BC there were about 250 plant medicines in use in that region. As evident from the Papyrus Ebers, the ancient Egyptians possessed a good knowledge of medicinal properties of hundreds of plants. Many of the presents day important plant drug like henbane (Hyoscymus spp.), mandrake (Mandragora officinarum). The great treatise supplies various information on medicinal use of the plants in the Indian subcontinent. It noted that the Indo-Aryans used the soma plant (Amaniat muscaria a narcotic & hallucinogenic mushroom) as medicinal agents. The practice of the medicine using medicinal plants flourished most during the Greek civilization when historical personalities like Hippocrates and Theophrastus practice herbal medicine. The materia medica of the Greek physician Hippocrates consists of some 300-400 medicinal plants which include opium, mint, rosemary, sage and verbena. The Arabian Muslims physicians like AlRazi and Ibn Sina, brought about the revolution in the history of medicine by brings new drug plants(Ghani, 2003). The current list of the medicinal plants growing around the world includes more than a thousands items. 1.3 Contribution of medicinal plants to modern medicine Medicinal plant have been serving as the major sources of medicine for maintenance of health and wellbeing of the human beings from the very beginning of their existence on earth. These medicinal plants were used by the early man, as are done now, in a variety of forms, such as in the entire form, and as powders, pastes, juices, infusions and decoctions for the treatment of their various disease and ailments. These various converted forms of the medicinal plants may thus be very conveniently and genuinely called medicinal preparations or medicaments. This way, the medicinal plants formed an integral part of the health management practices and constituted important items of medicines from the very early days of human civilization. In the course of their traditional uses, the medicinal plants have contributed substantially to the gradual development of medicines to their present state. As therapeutic uses of plants continued with the progress of civilization and development of human knowledge, scientists endeavored to isolate different chemical constituents from plants, put them to biological and pharmacological tests and thus have been able to prepared. In this way, ancient uses of Datura plants have led to the isolation of hyoscine, hyoscyamine, atropine and tigloidine, Cinchona baek to quinine and quinidine, Rauvilfia serpentineto reserpine and rescinnamine, Digitalis purpurea to digitoxin and digoxin, papaver somniferum to morphine and codaine, Ergotamine and ergometrine, Senna to sennosides. Catharanthus roseus to vinblastine and vincristine – to mention a few. Isolation of the natural analgesic drug morphin from papaver somniferumcapsules, in 1804 is probably the first most important example of naturadrug which plants have directly contributed. 2. Plants Preiew
In the project we have used the methanolic extract of Terminalia arjuna bark. 2.1 Common Name: Arjuna(Bengali) , Arjun Myrobalan (English). 2.2 Scientification classification[5] Kingdom:Plantae Division:Magnoliophyta Class:Magnoliopsida Order:Myrtales Family:Combretaceae Genus:Terminalia Species: T. arjuna Binomial name Terminalia arjuna http://en.wikipedia.org/wiki/Terminalia_arjuna 2.4 Terminalia arjuna Terminalia arjuna is a medicinal plant of the genus Terminalia, widely used by ayurvedic physicians for its curative properties in organic/functional heart problems including angina, hypertension and deposits in arteries. According to Ayurvedic texts it also very useful in the treatment of any sort of pain due a fall, ecchymosis, spermatorrhoea and sexually transmitted diseases such as gonorrhoea. Arjuna bark (Terminallia arjuna) is thought to be beneficial for the heart. This has also been proved in a research by Dr. K. N. Udupa in Banaras Hindu University's Institute of Medical Sciences, Varanasi (India). In this research, they found that powdered extract of the above drug provided very good results to the people suffering from Coronary heart diseases Research suggests that Terminalia is useful in alleviating the pain of angina pectoris and in treating heart failure and coronary artery disease. Terminalia may also be useful in treating hypercholesterolemia [22]. The cardio protective effects of terminalia are thought to be caused by the antioxidant nature of several of the constituent flavonoids and oligomeric proanthocyanidins, while positive inotropic effects may be caused by the saponin glycosides. In addition to its cardiac effects, Terminalia may also be protective against gastric
ulcers, such as those caused by NSAIDs. [23]. The leaves of this tree are also fed on by the Antheraea paphia moth which produces the tassar silk (Tussah), a form of wild silk of commercial importance [24]. 2.4.1 Description Terminalia arjuna is a deciduous tree found Throughout India growing to a height of 60-90 feet. The thick, white-to-pinkish-gray bark has been used in India’s native Ayurvedic medicine fo Over three centuries, primarily as a cardiac tonic Clinical evaluation of this botanical medicine Indicates it can be of benefit in the treatment of Coronary artery disease, Heart failure, and possibly Hypercholesterolemia. It has also been found to be Antiviral and antimutagenic.
2.4.2 Botany Tree up to 25 meter hight; bark grey, smooth; leaves sub-opposite, 5–14 × 2–4.5 cm., oblong or elliptic oblong, glabrous, often inequilateral, margin often crenulate, apex obtuse or subacute, base rounded or sometimes cordate; petioles 0.5–1.2cm; glands usually two. Flowers small, white. Fruit 2.3–3.5 cm long, fibrous woody, glabrous with 5 hard wings, striated with numerous curved veins. Flowering time April–July in Indian conditions. Seeds hard germination 50–76 days (50–60%). 2.4.3 Useful Parts [9] Every parts useful medicinal properties Arjun holds a reputedpositionin both Ayurvedic and Yunani Systems of medicine.According to Ayurveda it is alexiteric, styptic, tonic, anthelmintic, and useful in fractures, uclers, heart diseases, biliousness, urinary discharges, asthma, tumours, leucoderma, anaemia, excessive prespiration etc. According to Yunani system of medicine, it is used both externally and internally in gleet and urinary discharges. It is used as expectorant, aphrodisiac, tonic and diuretic. 2.4.4 Traditional uses [10] Every part of the tree has useful medicinal properties. Arjun holds a reputed position in both Ayurvedic and Yunani Systems of medicine. According to Ayurveda it is alexiteric, styptic, tonic, anthelmintic, and useful in fractures, uclers, heart diseases, biliousness, urinary discharges, asthma, tumours, leucoderma, anaemia, excessive prespiration etc. According to Yunani system of medicine, it is used both externally and internally in gleet and urinary discharges. 2.4.5 Therapeutic properties and Uses [11] The bark constituents an important crude drug, which is esteemed as a cardiac tonic. It reduces blood pressure and cholesterol levels. Powdered bark relives hypertension. It has a diuretic and general tonic effect in case of liver cirrhosis. The bark also acts as an astringent and febrifuge, and is used in the treatment of red and swollen the mouth, tongue and gums. It stops bleeding and pus formation in the gums, and is useful in asthma, dysentery, menstrual
problems, pain, leucorrhoea, wounds and skin eruptions. (Mohammed Ali, 2nd edition) Cardiomyopathy like Myocardial infraction, angina, coronary artery disease, heart failure, hypercholesterolemia, hypertension. In case of heart attack though it can not act against, like streptokinase or eurokinase, but regular use of it after just recovering from heart attack, reduces the chance of further attack to a great level. Besides no such Toxicity or side effects has so far be found it can be advocated to use in regular basis for a strong and well functioning heart. Arjuna reduces angina episodes much better than nitroglycerin - In one study, angina episodes were cut in half by the Arjuna, with none of the nasty side effects. Plus, it can be used as long as you like, without fear it'll stop working. Arjuna has been shown to help reverse hardening of the arteries. 2.4.6 Chemical constituents [12] Arjuna bark contains tannins (12%), β-sitosterol, triterpenoid saponins(arjunic acid, arjunolic acid, arjungenin, arjunglycosides), essential oil, reducing sugars, calcium salts and traces of aliminium and magnisun salts. flavonoids (arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomeric proanthocyanidins (OPCs), phytosterols, calcium, Magnesium, zinc, and copper.
COCH
HO
HO CH2 HO
Arjunolic Acid 2.4.7 Mechanisms of Action [13] Improvement of cardiac muscle function and subsequent improved pumping activity of the heart seems to be the primary benefit of Terminalia. It is thought the saponin glycosides might be responsible for the inotropic effect of Terminalia, while the flavonoids and OPCs provide free radical antioxidant activity and vascular strengthening.3 A dose-dependent decrease in heart rate and blood pressure was noted in dogs given Terminalia intravenously. Recently, two new cardenolide cardiac glycosides were isolated from the root and seed of Terminalia. The main action of these cardenolides is to increase the force of cardiac contraction by means of a rise in both intracellular sodium and calcium. 2.5 Clinical Indications [14]
2.5.1 Angina Pectoris An open study of Terminalia use in stable and unstable angina demonstrated a 50-percent Reduction of angina in the stable angina group after three months (p<0.01). A significant reduction was also found in systolic blood pressure in these patients (p<0.05). During treadmill testing, both the onset of angina and the appearance of ST-T changes on ECG were significantly delayed in the stable angina group (p<0.001), indicating an improvement in exercise tolerance. The unstable angina group did not experience significant reductions in angina or systolic blood pressure. Both groups showed improvements in left ventricular ejection fraction. Evaluation of overall clinical condition, treadmill results, and ejection fraction showed improvement in 66% of stable angina patients and 20 percent of unstable angina patients after three months. In this study Terminalia was also associated with a lowering of systolic blood pressure. Two clinical studies found similar results when Terminalia arjuna was compared to isosorbide mononitrate in stable angina patients.8,9 Both studies showed a similar reduction in the number of anginal episodes, as well as improvements in stress tests. In one study 58 males with chronic stable angina with evidence of ischemia on treadmill testing received Terminalia arjuna (500 mg every eight hours), isosorbide mononitrate (40 mg daily), or a matching placebo for one week each, separated by a wash-out period of at least three days in a randomized, double-blind, crossover design. Terminalia therapy was associated with a significant decrease in the frequency of angina and need for isosorbide dinitrate. Treadmill parameters improved significantly during therapy with Terminalia compared to those with placebo. Similar improvement in clinical and treadmill parameters were observed with isosorbide mononitrate compared to placebo therapy. No significant differences were observed in clinical or treadmill parameters when Terminalia arjuna and isosorbide mononitrate therapies were compared. 2.5.2 Congestive Heart Failure A double-blind, placebo-controlled, two-phase trial of Terminalia extract in 12 patients with severe refractory heart failure (NYHA Class IV) was conducted, in which either 500 mg Terminalia bark extract or placebo was given every eight hours for two weeks, in addition to the patientsâ&#x20AC;&#x2122; current pharmaceutical medications (digoxin, diuretics, angiotensinconvertingenzyme inhibitors, vasodilators, and potassium supplementation). All patients experienced dyspnea at rest or after minimal activity at the start of the trial. Dyspnea, fatigue, edema, and walking tolerance all improved while patients were on Terminalia therapy. Treatment with Terminalia was also associated with significant improvements in stroke volume and left ventricular ejection fraction, as well as decreases in end-diastolic and endsystolic left ventricular volumes compared to placebo. In the second phase of the study, patients from phase I continued on Terminalia extract for two years. Improvements were noted in the ensuing 2-3 months, and were maintained through the balance of the study. After four monthsâ&#x20AC;&#x2122; treatment, nine patients improved to NYHA Class II and three improved to NYHA Class III. 2.5.3 Cardiomyopathy/Post-Myocardial Infarction A study was conducted on 10 post-myocardial-infarction patients and two ischemic cardiomyopathy Patients, utilizing 500 mg Terminalia extract every eight hours for three months, along with conventional treatment. Significant reductions in angina and left ventricular mass, in addition to improved left ventricular ejection fraction, were noted in the Terminalia group; whereas, the control group taking only conventional drugs experienced decreased angina only. The two patients with cardiomyopathy improved from NYHA Class III to NYHA Class I during the
study. 2.5.4 Hyperlipidemia Animal studies suggest Terminalia might reduce blood lipids. Rabbits made hyperlipidemic on an atherogenic diet were given an oral Terminalia extract, and had a significant, doserelated decrease in total- and LDL-cholesterol, compared to placebo (p<0.01) However, the amounts used (100 mg/kg and 500 mg/kg body weight) were very large, and it remains to be seen if similar changes will be observed in humans taking relatively smaller oral doses. In a similar study of rats fed cholesterol (25 mg/kg body weight) alone or along with Terminalia bark powder (100 mg/kg) for 30 days, Terminalia feeding caused a smaller increase in blood lipids and an increase in HDL cholesterol, compared to the cholesterol-only group. The researchers concluded that inhibition of hepatic cholesterol biosynthesis, increased fecal bile acid excretion, and stimulation of receptor-mediated catabolism of LDL cholesterol were responsible for Terminaliaâ&#x20AC;&#x2122;s lipid-lowering effects. In another study, rabbits were fed a cholesterol-rich diet in combination with three indigenous Terminalia species; Terminalia arjuna, T. belerica, and T. chebula. Upon histological Examination, the rabbits fed the diet and T. arjuna exhibited the most potent hypolipidemic effect, with partial inhibition of atheroma. In a randomized, controlled trial, Terminalia bark was compared to vitamin E. One hundredAnd-five patients with coronary heart disease (CHD) were matched for age, lifestyle, and diet variables, as well as drug treatment status. None of the patients were previously on Lipidlowering medications. Placebo, vitamin E (400 IU), and Terminalia (500 mg) were administered. Results showed no significant changes in the placebo group or the vitamin E group. The Terminalia group had a significant decrease in total cholesterol and LDL cholesterol. Lipid peroxidase levels decreased significantly in both vitamin E and Terminalia groups; however, there was a greater decrease in the vitamin E group 2.6 Other Clinical Indications [15] Terminalia bark harbors constituents with promising antimutagenic and anticarcinogenic potential that should be investigated further.16-19 In vitro studies have also shown Terminalia to possess anti-herpes virus activity. 2.7 Botanical-Drug Interactions [16] Terminalia arjuna extracts have been used in clinical studies concomitantly with standard Heart medications, including digoxin, diuretics, angiotensin-converting-enzyme inhibitors, And vasodilators, with no reported adverse effects. Simultaneous use of Terminalia with other Cardiac medications should be undertaken with caution. 2.8 Dosage and Toxicity [17] A typical dose of dried bark is 1-3 grams daily, while 500 mg bark extract four times per day has been used in congestive heart failure. No toxicity has been documented. 2.9 Pharmacology [18] The bark is acrid, and credited with styptic, tonic, febrifugal and antidysenteric properties. In fractures and contusions, with excessive ecchymosis, the powdered bark is taken with milk.
The powdered bark seemed to give relief in symptomatic complaint in hypertension; it appearently had a diuretic and a general tonic effect in cases of cirrhosis of the liver. A decoction of the bark is used as a wash in ulcers.The alcoholic extract of bark contained: CaO, 0.33; MgO, 0.078; and Al2O3, .076%.The fruit is tonic and deobstruent. The juice of the fresh leaves is used in earache.These extracts also inhibited carotid occlusion response. Hypotension and bradycardia were also observed and were mainly of central origin. Arjuna bark is popularly used as a cardiac tonic. The diuretic property has also been observed,Which is because of a saponin 'The bark is useful in diseases of the heart, allays thirst and relieves fatigue. 2.10 Literature review 1. A standardized protocol for genomic DNA isolation from Terminalia arjuna for genetic diversity analysis (Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.9 No.1, Issue of January 15, 2006 © 2006 by Pontificia Universidad Católica de Valparaíso -- Chile Received May 2, 2005 / Accepted July 19, 2005) 2. In Vitro and In Vivo Anthelmintic Activity of Terminalia arjuna Bark (INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560– 8530; ISSN Online: 1814–9596) 3. Drug designing for ring finger protein 110 involved in adenocarcinoma (human breast cancer) using casuarinin Extracted from Terminalia arjuna (Indian Journal of Science and Technology Vol.2 No 2 (Feb. 2009) ISSN: 0974- 6846 Research article) 4. Antidyslipidemic and antioxidant activities of different fractions of terminalia arjuna stem bark (Indian Journal of Clinical Biochemistry, 2004, 19 (2) 141-148) 5. RP-LC determination of oleane derivatives in Terminalia arjuna (D.V. Singh, R.K. Verma, S.C. Singh, M.M. Gupta * Analytical Biophysical Chemistry Di_ision, Central Institute of Medicinal and Aromatic Plants, P.O.-CIMAP, Lucknow 226 015, India Received 3 August 2001; received in revised form 20 September 2001; accepted 30 September 2001) 6. anti- inflammatory, immunodulatory and antinociceptive activity 0f terminalia arjuna Roxb bark powder in mice & rats (Indian Journal of experimental Biology. Vol.47, july 2009, pp577 – 583) 3.1 Present Study Protocol Our present study was designed to isolate pure compounds as well as to observe pharmacological activities of the isolated pure compounds with crude extracts of the plant Terminalia arjuna. The study protocol consisted of the following steps: Extraction of the powdered bark of the plant with distilled methanol.
Filtration of the crude methanolic extract by using the Marking cotton cloth and subsequently through the filter paper and solvent evaporation.
Elucidation of the structure of the isolated compounds with the help of spectroscopic
method. Brine shrimp lethality bioassay and determination of LC50 of crude extract. Investigation of in vitro antimicrobial activity of crude extract.
determination of antioxidant activity of crude extract
4.1 Selected plants The fresh plants of Terminalia arjuna were selected for chemical investigation. 4.1.2 Collection of the plant parts The fresh barks of the plant were collected in the month of January 2010 from the area of Uttara Dhaka and were taxonomically identified by the National Herbarium of Bangladesh. These are familiar plant and widely distributed in all over Bangladesh. 4.1.3 Drying of the plant parts The collected of barks of plant were made from unwanted materials. They were cut into small pieces and sun dried for seven days. 4.1.4 Storage and Preservation of plant parts Plant parts from desired plants undergo a period of storage before they are finally used for research purpose in the laboratory. During this period many undesirable changes may occur in the plant parts if they are not properly stored and preserved against the reabsoption of moisture, oxidation, excessive heat or humidity, direct sunlight, growth of moulds and bacteria and infestation by insects and rodents. Proper storage and preservation of plant parts are thus very important factors in maintaining a high degree of quality in them. All efforts towards proper storage should be geared to protect the drugs from all the above deteriorating factors and agents. 4.1.5 Grinding of the plant parts The dried small pieces of barks were ground into small powder by a grinder machine from Depart of Pharmacy, Southeast University, and Dhaka. Then the powders were preserved in separate airtight container. 4.1.6 Extraction of the Plant Material The plant powdered materials 100gm were extracted with 350ml methanol in a flat bottom glass container, through occasional shaking and stirring for 20 days. The extract was then filtered through filter paper. The filtrates were concentrated at 50º C under reduce pressure to afford a radish mass of extracts. 4.2 Antioxidant [19] 4.2.1 Principles The free radical scavenging activities (antioxidant capacity) of the plant extracts on the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were estimated by the method of BrandWilliams et al., 1995. 2.0 ml of a methanol solution of the extract at different concentration were mixed with 3.0 ml of a DPPH methanol solution (20μg/ml). The antioxidant potential was assayed from the bleaching of purple colored methanol solution of DPPH radical by the
plant extract as compared to that of tert-butyl-1-hydroxytoluene (BHT) by UV spectrophotometer.
N N
N
.
O2N
+RH NO2
NO2
*DPPH (oxidized form)
+R.
NH O2N
NO2
NO2
DPPH (reduced form)
* DPPH = 1, 1-diphenyl-2-picrylhydrazyl 4.2.2 Materials 1. 1,1-diphenyl-2-picrylhydrazyl 2. Distilled water 3. Methanol 4. UV-spectrophotometer 5. Beaker (100 & 250ml) 6. Test tube 7. Light-proof box 8. Pipette (5ml) 9. Micropipette (10-100 Âľl) 10. Amber reagent bottle DPPH was used to evaluate the free radical scavenging activity (antioxidant potential) of various compounds and medicinal plants (Choi et al., 2000; Desmarchelier et al., 2005). So, DPPH is used as reagents. DPPH is a stable free radicals potentially reactive with substance able to donate a hydrogen atom and thus useful to asses compounds antioxidant activity of specific of specific compounds of extracts (Dinis, Maderia, Almeida, 1994). Because of its odd electron, DPPH has a strong absorption band at 517 nm. Since this electron becomes paired in the presence of a free radical scavenger, the absorption decreases stoichiometrically with respect to the number of electrons taken up. This change in absorbance produced by this reaction has been widely used to test the ability of several molecules to act as free radical scavengers (Dinis, Maderia, Almeida, 1994). The absorbance was taken by UV-spectrophotometer and methanol was taken for extraction and as a solvent. Ascorbic acid was as a standard.
4.2.3 Method 2.0 ml of a methanol solution of the extract at different concentration (500 to 0.977 μg/ml) were mixed with 3.0 ml of a DPPH methanol solution (20 μg/ml). After 30 min reaction period at room temperature in dark place the absorbance was measured against at 517 nm against methanol as blank by UV spetrophotometer. Inhibition free radical DPPH in percent (I%) was calculated as follows: (I%) = (1 – Asample/Ablank) X 100 Where Ablank is the absorbance of the control reaction (containing all reagents except the test material). Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotted inhibition percentage against extract concentration. BHT was used as positive control. Tests carried out in triplicate and average value was taken. DPPH in methanol-3.0 ml
Extract in methanol-2.0 ml
(conc.- 20 μg/ml)
(conc.- 500 to 0.977 μg/ml)
Purple colour
+ Reaction allowed for 30 minutes in absence of light at room temperature
Absorbance measured at 517 nm using methanol as blank
Calcualtiion of IC50 value from the graph plotted inhibition percentage against extract concentration
Fig.1: Schematic representation of the method of assaying free radical scavenging activity 4.3 Cytotoxicity Test (Brine Shrimp Lethality Bioassay) [20] 4.3.1 Introduction Brine shrimp lethality bioassay (Meyer et.al, 1982; Persoone, 1980) is rapid general bioassay for the bioactive compound of the natural and synthetic origin. Bioactive compounds are almost always toxic at high dose. Pharmacology is simply toxicology at a lower dose or
toxicology is simply pharmacology at a higher dose. Brine shrimp lethality bioassay is a bench top bioassay method for evaluating anticancer, anti-microbial and pharmacological activities of natural products and it is a recent development in the bioassay for the bioactive compounds. By this method, natural product extracts, fractions as well as the pure compounds can be tested for their biosphere- activity. Here, in vivo lethality in a simple zoologic organism (Brine shrimp nauplii) is used as a convenient monitor for screening and in the discovery of new bioactive natural products. This bioassay is indicative cytotoxicity and a wide range of pharmacological activity of the compounds (McLaughlin, 1990; Persoone , 1980). Brine shrimp lethality bioassay stands superior to other cytotoxicity testing procedures because it is a rapid method utilizing only 24 hours, inexpensive and requires no special equipment. Unlike other methods, it does not require animal serum. Furthermore, it utilizes a large number of organisms for statistical validation and a relatively small amount of sample. 4.3.2 Principle Brine shrimp eggs are hatched in simulated sea water to get nauplii. Sample solutions are prepared by dissolving the test materials in pre-calculated amount of DMSO. Ten nauplii are taken in vials containing 5 ml of simulated sea water. The samples of different concentrations are added to the premarked vials with a micropipette. The assay is performed using three replicates. Survivors are counted after 24 hours. These data are processed in a simple program to estimate LC50 values. Table 1: Amount taken of Test Samples of Experiment
Type of sample
Measured Amount (mg)
Terminalia arjuna bark extract 4.00
4.3.3 Materials a. b. c. d. e. f. g. h. i.
Artemia salina leaches (Brine shrimp eggs) Sea salt (NaCl) Small tank with electric air bubbler Lamp to attract shrimp. Pipettes Micropipette Glass vials Magnifying glass Test Samples of experimantal plant.
Procedure 4.3.4 Preparation of simulated sea water (Brine water) Since the lethality test involves the culture of brine shrimp nauplii that is the nauplii should be grown in sea water. Sea water contains 3.8% of sodium chloride & hence 3.8% salt solution should be needed for this purpose. Accordingly 3.8% of sodium chloride solution
was made by dissolving sodium chloride (38 gm) in distilled water (1000 ml) & was filtered. 4.3.5 Hatching of Shrimps Sea water was kept in a small tank & shrimps eggs were taken into the divided tank, constant oxygen supply was carried out & constant temperature (37ºC) was maintained. Two days were allowed for the shrimp to hatch and mature as nauplii. These nauplii were taken for bioassay. 4.3.6 Preparation of test solutions Measured amount (4.00 mg) of each sample was dissolved in100 µl of DMSO. A series of solutions of lower concentrations were prepared by serial dilution with DMSO. From each of these test solutions 50 µl were added to premarked glass test tubes containing 5 ml of seawater and 10 shrimp nauplii. So, the final concentration of samples in the test tubes were 4ooµg/ml, 200 µg/ml,100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.125 µg/ml, 1.56 µg/ml, 0.78125µg/ml for 10 dilutions and 100-0.78125µg/ml for 8 dilutions respectively. 4.3.7 Preparation of controls Vincristine sulphate served as the positive control. 0.2mg of vincristine sulphate was dissolved in DMSO to get an initial concentration of 20 µg/ml from which serial dilutions were made using pure dimethyl sulfoxide DMSO to get 10 µg/ml, 5 µg/ml, 2.5µg/ml, 1.25 µg/ml, 0.625 µg/ml, 0.3125 µg/ml, 0.15625 µg/ml, 0.078125 µg/ml, 0.0390 µg/ml. The control groups containing 10 living brine shrimp nauplii in 5 ml simulated sea water received the positive control solutions. As for negative control, 30 µl of DMSO was added to each of three premarked glass vials containing 5 ml of simulated seawater and 10 shrimp nauplii. The test was considered invalid if the negative control showed a rapid mortality rate and therefore conducted again 4.3.8 Counting of nauplii and analysis of data After 24 hours, the vials were inspected using a magnifying glass and the number of survivors were counted. The percent (%) mortality was calculated for each dilution. The concentration-mortality data were analyzed statistically by using probit analysis and linear regression using a simple IBM-PC program. The effectiveness or the concentration-mortality relationship of plant product is usually expressed as a median lethal concentration (LC50) value. This represents the concentration of the chemical that produces death in half of the test subjects after a certain exposure period. 4.4 Antibacterial Activity Test [21] 4.4.1 Introduction of Antimicrobial Screening Any chemical substance or biological agent that destroys or suppresses the growth of microorganism is called antimicrobial agent. Antimicrobial screening of a crude extract or pure compound isolated from natural sources is essential to ascertain its activity against various types of pathogenic organisms. Antimicrobial activity of any plant can be detected by observing the growth response of various microorganisms to the plant extract, with is placed in contact with them. In general Antimicrobial screening is undertaken in two phages: a primary qualitative assay to detect the presence or absence of activity and a secondary assay which quantities the relative potency, expressed as Minimum Inhibitory Concentration (MIC) value, of a pure compound, an
important method in the further development of a new Antimicrobial compound. The primary assay can be done in three ways as: a. Diffusion method b. Dilution method c. Bioautographic method Among these methods, the disc Diffusion method is widely acceptable for the preliminary evaluation of Antimicrobial activity. Disc Diffusion is essentially a qualitative or semiqualitative test indicating the sensitivity or resistance of microorganisms to the test materials. However no distinction between bacteriostatic or bacteriosidal activity can be made by this method. 4.4.2 Principle of Disc Diffusion Method Diffusion is based on the ability of a drug to a diffuse from a confined source through the nutrient agar medium and creates concentration gradient. If agar is seeded with a sensitive organism, a zone of inhibition will result where the concentration exceeds the minimum concentration (MIC) for that particular organism. In this method, measured amount of the test samples are dissolved in definite volumes of solvent to give solutions of known concentration (µg/ml). Then sterile filter paper discs (5 mm diameters) are impregnated with known amounts of the test substances and dried. The dried discs are places on plates (Petri dishes, 120mm diameters) containing a suitable medium (nutrient agar) seeded with the test organisms. These plates are kept at low temperature (4ºC) for 24 hours to allow maximum diffusion. A number of events take place simultaneously which include• The dried discs absorb water from the agar medium and the material under test is dissolved. • The test material diffuses from the discs to the surrounding medium according to the physical law that controls the diffusion of molecules through agar gel. • There is a gradual change of taste material concentration on the agar surrounding each disc. The plates are then kept in an incubator (37ºC) for 12-18 hours to allow the growth of microorganism. If the test material has antimicrobial activity, it will inhibit the growth of the microorganism, giving a clear, distinct zone called “Zone of Inhibition”. The antimicrobial activity of the test agent is determined in term of millimeter by measuring the diameter of the zone of inhibition. The greater the zone of inhibition the greater the activity of the test material against the test organism. The principles factors which determine the size of the zone of inhibition are – • Intensive antimicrobial susceptibility of the test sample. • Growth rate of the test organism. • Diffusion rate of the test sample which is related to its water solubility. • Concentration of the test organisms incubated in the medium. • Concentration of the test sample per disc. • Thickness of the test medium in the Petri discs. 4.4.3 Test Materials Of terminalia arjuna • Methanolic extract of bark 4.4.4 Test Organisms
Both Gram-positive and Gram-negative stains of bacteria we used as the test organism to observe the anti-bacterial activity of the compounds. The bacterial steins used for this investigation are listed in the Table. These organisms were collected from the Microbiology research laboratory, Department of Pharmacy, Southeast University of Dhaka. The pure of which was previously collected from the Microbiology Department of Dhaka University. Table 2: List of pathogenic bacterial strains Gram-positive 1. Bacillus megaterium 2. Bacillus subtilis 3. stayphylococcus aures 4. sareina lutea
Gram-negative 1. Salmonella typhi 2. Escherichia coli 3. Shigella dysenteriae 4. Vibrio minicus 5. Vibrio parahemolyticus 6. Shigella boydii 7. salmonellaparatyphi 8. pseudomonas auregonosa
4.4.5 Apparatus and Reagents 1. Filter paper discs (5mm in diameter) 2. Petri dishes 3. Refrigerator 4. Test tubes 5. Sterile forceps 6. Sterile cotton 7. Incubating loop 8. Bunsen burner 9. Micropipette(10-100µl) 10. Laminar air flow unit (Biocraft’s Scientific Industries, India) 11. Autoclave(|YX-280B 18L) 12. Incubator(OSK-9636, Japan) 13. Nutrient agar media(DIFCD) 14. Ethanol 15. Vials 16. Standard disc(Kanamycin 30 µg/disc) 4.4.6 Culture Medium The main requirement for the growth of bacteria were as followsa. Source of energy such as carbohydrate, protein and nucleic acid. b. Essential trace elements e.g. Mg, Mn, Fe, and Co c. Optimum pH of media and d. Optimum temperature for incubation A number of culture media are available to demonstrate the antibacterial activity. This area. Nutrient agar media b. Nutrient broth media c. Mueller-Hinton agar media d. Tryptic soy broth(TSB)
Among these, nutrient agar media is most frequently used and its composition is shown. Table 3: Composition of nutrient agar media Ingredient Amount(g/l) Peptic digest of animal tissue 5.0 Sodium chloride 5.0 Beef extract 1.5 Yeast extract 1.5 Agar 15.0 Distilled water 100 ml pH 7.5 ±1.0 at 25ºC 4.4.6 Experimental Procedure 4.4.7 Preparation of the Medium The instant nutrient agar media was accurately weighted and then reconstituted with distilled water in a conical flask according to specification (4% w/v). It was then heated with water bath to dissolve the agar and a transparent solution was obtained. The prepared media was then transferred in 9ml and 5ml in a number of clean test tubes, respectively to prepare plates and slants. The slants were used for making sub-culture of microorganism, which in turn use for sensitivity tests. The test tubes were then plugged with cotton and sterilized in an autoclave ate temperature of 126ºc and pressure of Ib/sq inch for 15 minutes. 4.4.8 Sterilization Procedure In order to avoid any type of contamination and cross contamination by the test organisms the antimicrobial screening was done in Laminar Hood and all types of precautions were highly maintained. UV light was switched on one hour before working in the Laminar Hood. Petri dishes and other glassware were sterilized by autoclaving at a temperature of 1210C and a pressure of 15-lbs/sq. inch for 20 minutes. Micropipette tips, cotton, forceps, blank discs etc. were also sterilized. 4.4.9 Preparation of Subculture With the help of a inoculating lop, the test organisms were transferred from the pure culture to the agar slants in a laminar airflow unit. The incubated slants were then incubated at 37º for 18-24 hours to ensure the growth of test organisms. This culture was used for sensitivity test. 4.4.10 Preparation of the Test Plate The test organism was transferred from the subculture to the test tube containing 9ml autoclaved medium with the help of an incubating loop in aseptic area. The test tube was shaken by rotation to get a uniform suspension of the organism. The bacterial suspensions were immediately transferred to the sterile petridshes in an aseptic area and were rotated several times, first clockwise and anticlockwise to ensure homogeneous dispersion of the organism into the medium. The depth of media into each petridsh was approximately 4mm. After plates were cooled to room temperature, it stored in a refrigerator at 4ºC.
4.4.11 Preparation of Discs Three types of discs were used for antibacterial screening. These werea. Sample discs b. Standard discs c. Blank/control discs a. Sample discs: Sterilized filters discs (5 mm in diameter) were taken in a blank Petri dish. Sample solution of the desired concentration was applied on the discs with the help of a micropipette in an aseptic condition. These discs were left for a few minutes in aseptic condition for complete removal of solvent. b. Standard discs: these were used to compare to the antibacterial activity of test material. In our investigation Kanamycin (30 µg/disc) was used as a reference. c. Blank discs: Only solvent was applied to the disc to determine the antibacterial effects of the solvent used. 4.4.12 Application of the Test Samples Standard Kanamycin (30 µg/disc) discs were used as positive control to ensure the activity of standard antibiotic against the test organisms as well as for comparison of the response produced by the known antimicrobial agent with that of produced by the test sample. Blank discs were used as negative controls which ensure that the residual solvents (left over the discs even after air-drying) and the filter paper were not active themselves. 4.4.13 Diffusion and Incubation The sample disc and standard antibiotic disc were placed gently on the solidified agar plates freshly seeded with the organism with the help of a sterile forceps to ensure complete contact with medium surface. The arrangement of the disc was such that the discs were no closer than 15 mm to the plate to prevent overlapping the zone of inhibition. The plates were then inverted and kept in a refrigerator for about 24 hours at 4ºC. This was sufficient time for the material to diffuse to a considerable area of the medium. Finally, the plates were incubated at 37ºC for 24 hours. 4.4.13 Determination of Antimicrobial Activity by Measuring the Zone Of Inhibition: After 24 hour incubation, the antimicrobial activities of the test materials were determined by measuring the diameter of the zones of inhibition in millimeter with transparent scale.
(A)
(B)
Fig 2: (A) Clear zone of inhibition and (B) Determination of clear zone of inhibition
5.1 Result of Antibacterial Activity Test of the Crude Extract One of the most important and effective in vitro antibacterial screening of crude drugs is disc diffusion method. The crude extract or pure compounds having antibacterial activity inhibit bacterial growth in the surrounding area gives a clear, distinct zone, known as zone of inhibition. The result can be calculated by measuring the diameter (mm) of the zone of inhibition. The larger the zone of inhibition the greater the activity of the applied sample. The methanolic extracts of Terminalia arjuna bark were tested for their antibacterial activity against the number of Gram positive and Gram negative bacteria. Standard antibiotic discs of Kanamycin (30µg/disc) for bacterial species. In this antibacterial screening of crude extracts were used at a concentration of 500µg/disc. Evaluation of Antimicrobial Activity The results of antibacterial activity of crude extracts against a number of Gram positive and Gram negative bacteria are given below in the tableTable 4: Diameter of the zone of inhibition of different plant extracts Name of Bacteria Methanolic extract Kanamycin of bark of 30µg/disc Terminalia arjuna Gram positive Staphylococus aereus
19
25
Bacillus megaterium
15
30
Bacillus subtilis Sarcina lutea Gram negative Salmonella paratyphi Salmonella typhi Escherichia coli Shigella dysenteriae Sheigella boydii
15 13
32 28
15 17 16 13 13
30 33 32 28 36
Vibro parahemolytica Vibro minius Pseudomonas auregonosa
12 15 15
26 30 30
The control disc containing the solvent had no zone of inhibition, so their data are omitted from the above data. Discussion From the above data, it is evident that Mehanolic extract are showed 13-19 mm zone of inhibition against mentioned gram positive bacteria. And 13-17 mm zone of inhibition against mentioned gram negative bacteria. 5.2 Result of Antioxidant (DPPH)
Table 5: LC50 values of crude methanolic extract of Terminalia arjuna SL.NO. 1 2 3 4 5 6 7 8 9 10
Concentration (µg/ml) 200 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39
Asorbance of Absorbance extract of blank 0.061 0.405 0.061 0.405 0.075 0.405 0.076 0.405 0.110 0.405 0.122 0.405 0.187 0.405 0.262 0.405 0.299 0.405 0.299 0.405
% inhibition 84.93 84.93 81.48 81.23 72.83 69.87 53.83 35.30 26.17 26.17
LC50 (µg/ml)
7.03
120 y = -7.6135x + 103.55 R2 = 0.8978
% Inhibition
100 80
% Inhibition
60
Linear (% Inhibition) Linear (% Inhibition)
40 20
0.39
0.78
1.56
3.13
6.25
12.5
25
50
100
200
0
Concentration(µg/ml)
Fig 3: LC50 values of crude methanolic Bark extract of Terminalia arjuna. Table 6: LC50 values of Ascorbic acid SL.NO .
Concentrat Absorbance of Absorbance % ion(µg/ml) extract of blank inhibition
1
500
0.053
0.42
86.75
2
250
0.099
0.42
75.25
3
125
0.0154
0.42
61.5
4
62.5
0.166
0.42
58.5
5
31.25
0.179
0.42
55.25
6
15.625
0.198
0.42
50.5
LC50 (µg/ml)
6.964
7
7.8125
0.207
0.42
48.25
8
3.90625
0.213
0.42
46.75
9
1.953125
0.227
0.42
43.25
10
0.976563
0.238
0.42
40.5
Fig 4: Graphical presentation of inhibition (%) against concentration of Ascorbic acid. Discussion In antioxidant activity test, the methanolic extract exhibited significant antioxidant activities with the LC 50 values of 7.03µg/ml respectively. 5.3 Result and Discussion of Brine Shrimp Lethality Bioassay Brine Shrimp Lethality Bioassay is a recent development in the bioassay for bioactive compounds which indicates toxicity as well as a wide range of pharmacological activities (i.e. anticancer, antiviral, insecticidal & pesticidal etc.) of the compounds. Bioactive compounds are almost always toxic in high dose. Pharmacology is simply toxicology at lower doses and toxicology is simply Pharmacology at higher doses. Natural product extracts and pure compounds can be tested for their bioactivity by this method. Here, in-vivo lethality in a simple zoological organism (Brine Shrimp nauplii) is used as a convenient monitor for screening and fractionation in the discovery of new bioactive natural products. In this bioassay, methanol extract showed positive results indicating that the compounds are biologically active. From this experiment, it was revealed that each of the test samples showed different mortality rates at different concentrations. The mortality rate of brine shrimp was found to be decreased with the increase of concentration of samples, and a plot of log of concentration versus percent morality on the graph produced an approximate linear correlation between them. From the graph the concentration at which 50% mortality (LC50) of the brine shrimp nauplii occurred and was determined. Table 7: Result of methanolic extract of bark of Terminalia arjuna Concentration Log C No. of No. of No. of % of LC50 (C) µg/ml Nauplii Nauplii Nauplii morality µg/ml
400
2.602
taken 10
alive 0
dead 10
100
200
2.301
10
3
7
70
100
2.000
10
3
7
70
50
1.699
10
3
7
70
25
1.398
10
4
6
60
12.50
1.097
10
5
5
50
6.25
0.796
10
5
5
50
3.125
0.495
10
6
4
40
1.562
0.194
10
6
4
40
0.781
-0.107
10
8
2
20
Fig 5: Effect of Bark of Terminalia arjuna on brine shrimp nauplii. Table 8: Effect on vincristin Sulphate on Brine Shrimp Nauplii Conc.(C) (µg/ml)
LogC
20 10 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 0.0390
1.30103 1 0.69897 0.39794 0.09691 -0.20142 - 0.50515 - 0.80616 - 1.10721 - 1.40894
% Mortelity LC50 (Vincristin (µg/ml) Sulphate) 100 90 90 80 70 0.46875 60 40 30 30 20
0.97
Fig 6: Effect of Vincristin sulphate on brine shrimp nauplii. Discussion In brine shrimp lethargy bioassay, the methanol extract exhibited significant cytotoxic activities with the LC 50 values of 0.97µg/ml respectively Reference 1. Kirsty O'Shea 1990 00.36.15 Katie Spalding 1991 00.36.54. Issn:4562-67, Vol:9, p 426-34. [1] 2. Ghani. A (2003), Medicinal Plants of Bangladesh with chemical constituents and Uses “2nd " Edition. Asiatic society of Bangladesh, ISBN: 984-512-348-I. [2,4] 3. Anthocephalus chinensis (Lam.) Rich. Ex Walp. In: Part II: Species descriptions. In Tropical Tree Seed Manual. 2003, ed. Vozzo JA. United States Department of Agriculture. [3] 4. Alternative Medicine Review Monographs.Page:411-414. [6,7,8,9,10,11,13,14,15,16,17,18] 5. Text book of Pharmacognosy. 2nd Edition. Mohammed Ali. [12] 6. Chakravarty, A. K., Das et al, 1996 In vitro study of antioxidant activity of Syzygium cumini fruit ; Pharmacognosy Research Laboratory, Department of Botany; Issue:4; pp: 727-733; vol:90; [19] 7. Meyer, B. N., Ferringni, N. R., Puam, J. E., Lacobsen, L. B., Nichols, D. E. and McLaughlin, J. L. (1982) ‘Brine shrimp: a convenient general bioassay for active constituents.’ Planta Medica. [20] 8. Persoone, G.(1980) Procending of the InternationalSymposium on Brine Shrimp. 9. Ram,AJ, Raju, V.R.R. and Bakshu, L.M. (2003) In Vitro antimicrobial activity of certain medicinal plants from eastern Ghats, India, used for skin diseases. J Enthopharmacol: 353-357. [21] 10. Miller AL (1998). "Botanical influences on cardiovascular disease". Altern Med Rev 3 (6): 422–31. PMID 9855567. [22, 24] 11. Devi RS, Narayan S, Vani G, Shyamala Devi CS (2007). "Gastroprotective effect of Terminalia arjuna bark on diclofenac sodium induced gastric ulcer.". Chem
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