GJRMI - VOlume 3, Issue 5, May 2014

Indexing links of GJRMI GJRMI has been indexed in the Following International Databases

Google Scholar, ProQuest, DHARA online; DOAJ; Index Copernicus; NewJour; ScienceCentral; getCITED; RoMEO; Geneva Foundation for Medical Education & Research ; Catalog ebiblioteca; Ayurbhishak; Medicinal plants (Dravya Guna); Indianscience.in; Necker; Hong Kong University of Science and Technology Library; University of Zurich; University of Kansas; Western Theological Seminary; CaRLO; Mercyhurst University; University Library of Regensberg; WZB; Jadoun science; University of California, San Fransisco (UCSF Library); University of Washington; University of Saskatchewan; University of Winnipeg; Universal Impact Factor; Global Impact factor, Ulrich’s Periodicals Directory, New York Public Library, WISE, Cite factor, DRJI, Miami University Libraries, AYUSH RESEARCH PORTAL - Department of AYUSH, Ministry of Health & Family welfare, Govt. of India -

Chakradatta Ayurveda Chikitsalaya, Mysore.

(Panchakarma & Netra Roga Chikitsa Kendra)

Get treated through Ayurveda, at our Hospital.

(Exclusive Panchakarma Therapy available with accommodation) Address: Beside Vikram Jyothi Hospital, Temple Road, V V Mohalla, Mysore – 12, Karnataka, India.

Contact: Mobile: +919980952358, +919035087999 E- mail: raviamrita.kumar9@gmail.com

Consultant Physician: Dr. Ravi Kumar. M. (Specialized in different types of Keraliya Ayurvedic treatments especially in ENT & Eye diseases)

Arudra Ayurveda, Bangalore (A PANCHAKARMA TREATMENT CENTRE)

All types of Keraliya Ayurvedic treatments available for all the diseases) Ayurvedic Treatments in the following diseases: Eye diseases, Asthma, Skin diseases, Joint diseases, Diseases of the nervous system, Gynaecological & Obstetric diseases, Obesity, Asthma, Stress, Anxiety, Insomnia, Depression, Loss of Memory & Concentration, Piles, digestive tract diseases, Infertility etc.

Address: No. 40, IInd cross, KV Pai Layout, Konanakunte, Near Silicon city school, Bangalore – 62, Karnataka, India.

Contact: Mobile: +919480748861

An International, Peer Reviewed, Open access, Monthly E-Journal

ISSN 2277 – 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

Managing Editor Dr. Shwetha Hari

Administrator & Associate Editor Miss. Shyamala Rupavahini

Advisory Board Prof. Rabinarayan Acharya Dr. Dinesh Katoch Dr. S.N.Murthy Dr. Mathew Dan Mr. Tanay Bose Dr. Nagaraja T. M. Prof. Sanjaya. K. S. Dr. Narappa Reddy

Editorial board Dr. Kumaraswamy Dr. Madhu .K.P Dr. Sushrutha .C.K Dr. Ashok B.K. Dr. Janardhana.V.Hebbar Dr. Vidhya Priya Dharshini. K. R. Mr. R. Giridharan Mr. Sriram Sridharan

Honorary Members - Editorial Board Dr Farhad Mirzaei Mr. Harshal Ashok Pawar Dr. Sabarinath Subramaniam Dr. Yogitha Bali

INDEX – GJRMI - Volume 3, Issue 5, May 2014 MEDICINAL PLANTS RESEARCH Pharmacology ANTIBACTERIAL, ANTI-SWARMING POTENTIAL OF ETHANOL EXTRACTS OF PHYSALIS MINIMA L. WHOLE PLANT AND URENA LOBATA L. ROOT ON CEPHALOSPORIN RESISTANT PROTEUS SPECIES Mamunur Roshid, Aktar Uzzaman Chouduri

184–195

Bio-Technology CATECHIN DETECTION IN CALLUS AND IN VITRO CULTURES OF THE EASTERN STRAWBERRY TREE, ARBUTUS ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN PALESTINE Zahra Aljabari, Jawad Alzeer, Rami Arafeh

196–205

Review Article ANDROGRAPHIS PANICULATA A TRADITIONAL HERB WITH PHARMACOLOGICAL PROPERTIES: A REVIEW Nishan Chatterjee, Sunipa Biswas, Nimai Chandra Saha, Surjyo Jyoti Biswas

206–214

INDIGENOUS MEDICINE Ayurveda – Dravya Guna PHARMACOGNOSTICAL EVALUATION ON TANNIN CONTENT IN HARITAKI LEAVES (TERMINALIA CHEBULA RETZ. - COMBRETACEAE) BEFORE AND AFTER FLOWERINGFRUITING Patil Sunny C, Harisha C R, Baghel A S, Dwivedi R R

215–224

Ayurveda – Dravya Guna ANTIDYSLIPIDAEMIC EFFECT OF THE STEM BARK OF CHIRABILWA (Holoptelea integrifolia Planch.) - A CLINICAL TRIAL Sinimol T P, Shahul Hameed A

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – TENDER LEAVES OF TERMINALIA BELLIRICA (GAERTN.) ROXB., OF THE FAMILY COMBRETACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

225–231

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research Article ANTIBACTERIAL, ANTI-SWARMING POTENTIAL OF ETHANOL EXTRACTS OF PHYSALIS MINIMA L. WHOLE PLANT AND URENA LOBATA L. ROOT ON CEPHALOSPORIN RESISTANT PROTEUS SPECIES Mamunur Roshid1, Aktar Uzzaman Chouduri2* 1,2

Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh *Corresponding author: Email: auchow5@yahoo.com, auchow5.pharm@ru.ac.bd; Phone: +88-0721-711110 (Office), +88-01712792350 (Cell); Fax: +88-0721750064

Received: 01/04/2014; Revised: 25/04/2014; Accepted: 02/05/2014

ABSTRACT Swarming of Proteus bacteria has been implicated in pathogenesis. In previous study, eleven Proteus strains isolated from municipal water were found to be resistant to cephalosporins and four isolates, 11(Pv), 661(Pp), 911(Pm), and 912(Pm), were resistant to normal human serum. The increasing evidence of antibiotic resistance necessitates medicinal plants to develop alternative strategies of treatment. This study aimed to search medicinal plants with high antibacterial potentials in order to manage antibiotic resistant uropathogens. Twelve specimens of nine medicinal plants which are available locally were analyzed for their anti-infective properties against resistant uropathogens using disc diffusion method. Remarkable antibacterial activities of ethanol extract of Physalis minima whole plant followed by Azadirachta indica leaf, Asparagus racemosus root, Phyllanthus emblica fruit, Urena lobata root and Tamarindus indica bark were found against eleven test bacteria and eleven resistant Proteus isolates. Physalis minima extract showed the highest zone of inhibition but it had no anti-swarming effect. Interestingly complete inhibition of swarming was found by Urena lobata root extract at 500 µg/ml concentration although its antibacterial activity was very low or nil. Thus, the mixture of two extracts would be a powerful anti-infective agent to combat UTI and/or wound infection caused by resistant Proteus bacteria. The extracts could be further analyzed for the drug development.

KEY WORDS: Urena lobata L., Physalis minima L., antibacterial and anti-swarming activities, cephalosporin resistant Proteus bacteria.

ABBREVIATIONS: UTI-Urinary tract infection, CAUTI-Catheter associated urinary tract infection, ESBL- Extended spectrum β-lactamase, NHS- Normal human serum.

Cite this article: Mamunur Roshid, Aktar Uzzaman Chouduri (2014), Antibacterial, anti-swarming potential of ethanol extracts of Physalis minima L. whole plant and Urena lobata L. root on cephalosporin resistant Proteus species, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 184–195

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

INTRODUCTION Thirty-one species of medicinal plants were reported by traditional healers as being used for UTIs, including leucorrhea, frequent or infrequent urination, cloudy urination, and burning sensations during urination (Hossan et al., 2010). The major parts (flower, bark, root, leaves) of one of these medicinal plants, Urena lobata Linn are used as folk medicine for UTIs (Nandwani et al., 2008; Hossan et al., 2010).

U. lobata Linn (common name Ceasar weed) is native to China but it is available in many tropical countries including Bangladesh, India, South America, Africa, Australia, and the United States. Especially U. lobata roots have been shown to bear a broad-spectrum antibacterial activity against Gram-positive and Gram-negative microorganisms (Mazumder et al., 2001).

The Proteus pathogens are thought to be the principal cause of UTI, CAUTI and wound infections. We isolated pathogenic Proteus bacteria from municipal tap water (Wadud and Chouduri, 2013) that were multi-antibiotic resistant especially to cephalosporins (Chouduri and Wadud, 2013) and several pathogenic features of those isolates have already been reported (Chouduri et al., 2014; Chouduri and Wadud, 2014). Although the pharmacological industries have produced a number of new antibiotics in the last four decades, resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents (Cohen, 1992). The increasing evidence of antibiotic resistance among bacterial pathogens necessitates medicinal plants as an alternate therapy in restricting the resistant infectious organisms.

Previously it had been reported that recently the extensive use of cephalosporins for the treatment of infectious diseases allows pathogens to be resistant to the antibiotics of cephalosporin group. Therefore, an urgent need is to search new antibiotic or an alternate therapy of infectious diseases. This study aimed to manage the emergence of antibiotic resistance by phytochemicals of selective medicinal plants. To serve the purpose here

nine medicinal plants (Table 1) having potential antimicrobial properties have been selected that are traditionally used as folk medicine for urological disorders.

Nwodo et al. (2011) found the significant antimicrobial activities in aqueous and alcoholic extract of Tamarindus indica bark.

Fruit of Phyllanthus emblica Gaertn is commonly known as Indian gooseberry or amla. The alcoholic extract of Phyllanthus emblica exhibited strong and broad spectrum antibacterial activity against various pathogenic bacteria and numerous biological activities has also been reported (Ahmad et al., 1998; Khan, 2009; Khosla and Sharma, 2012). The root extract of Asparagus racemosus showed antibacterial activity against resistant uropathogens isolated from patients having UTI (Narayanan et al., 2011). The alcoholic extract of Azadirachta indica leaf showed potential antimicrobial activities including Proteus mirabilis (Yasmeen et al., 2012). Leaves of Abroma augusta Linn has been widely investigated and its antibacterial potentials have been reported by researchers (Saikot et al., 2012; Zulfiker et al., 2013). The extract of Mimosa pudica Linn root is an alternative wound healing agent widely used as folk medicine in Indian subcontinent for the treatment of vaginal and uterine complications.

It is very useful in diarrhea, amoebic dysentery, bleeding piles and urinary infections (Joseph et al., 2013). The ethanol extract of Coccinia grandis leaves exhibited antimicrobial activity against biofilm and ESBL producing uropathogenic Escherichia coli strains UPEC17 and -82 (Poovendran et al., 2011).

The general acceptance of traditional medicine for health care and the development of microbial resistance to several available antibiotics have led researchers to investigate the activity of medicinal plants against infectious diseases (Low et al., 2002; Yarnell, 2002). Therefore, the aim of this study was to evaluate the role of ethanolic fractions of the medicinal plants to interfere with the growth and virulence of multi-antibiotic, especially cephalosporin resistant uropathogenic Proteus bacteria isolated in our previous study.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Table 1: List of medicinal plants tested Sl 1 2 3 4 5 6 7 8 9 10 11 12

Scientific name Tamarindus indica Phyllanthus emblica Physalis minima Asparagus racemosus Urena lobata Urena lobata Urena lobata Urena lobata Azadirachta indica Coccinia grandis Abroma augusta Mimosa pudica

Family Leguminosae Phyllanthaceae Solanaceae Asparagaceae Malvaceae Malvaceae Malvaceae Malvaceae Meliaceae Cucurbitaceae Malvaceae Leguminosae

Local name Tetul Amloki/Amla Bontepari/Potka Shotomuli Bonokra Bonokra Bonokra Bonokra Neem Telakucha Ulotcombol Lojjaboti

Plant part Bark Fruit Whole plant Root Root Leaf Fruit Bark Leaf Whole plant Leaf Root

Abbreviation Ti-b Pe-f Pm-w Ar-r Ul-r Ul-l Ul-f Ul-b Ai-l Cg-w Aa-l Mp-r

Plant parts were collected from the medicinal plant garden, Department of Pharmacy, University of Rajshahi and around Rajshahi City area, Bangladesh on Nov 2013, and duly identified by a plant taxonomist Mr. Arshed Alom, Department of Botany, University of Rajshahi, Bangladesh where a specimen voucher (75/05.07.2008) was recorded in the department herbarium for future reference. Twelve specimens of nine medicinal plants enlisted in table 1 were air-dried under shade. A representative image of two plants and plant parts has been shown in figure 1.

Once dried, the plant material was ground, extracted by maceration for more than 72 hrs with ethanol, filtered (Paper Whatman No. 3) and the solvent was vacuum evaporated in a Soxhlet apparatus (Rotary Evaporator, RE 300, Bibby Sterilin Ltd, UK). Then solutions were evaporated to dryness and further dilutions were made in the same solvent to obtain the required extract concentrations for the different assays.

Streptococcus agalactiae, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, and six Gram negative bacteria, Pseudomonas aeruginosa, Shigella flexneri, Shigella dysenteriae, Escherichia coli, Shigella sonnei, Agrobacterium species, were used for antibacterial activity assay of the plant extracts.

Eleven Proteus strains of four species: P. vulgaris (hereafter termed as Pv), P. mirabilis (Pm), P. hauseri (Ph), and P. penneri (Pp) named as 11(Pv), 661(Pp), 662(Ph), 663(Pp), 664(Pp), 665(Pp), 666(Pp), 667(Pp), 668(Pp), 911(Pm) and 912(Pm) isolated from municipal tap water (Rajshahi City, Bangladesh) in our previous study (Wadud and Chouduri, 2013) have been used. Those strains were multidrug resistant to broad spectrum antibiotics and possessed several pathogenic features including swarming motility, urease production, extracellular proteases, biofilm formation as reported earlier (Chouduri and Wadud, 2013; Chouduri et al., 2013; Chouduri and Wadud, 2014). Strains stored at −40ºC in Luria-Bertani (LB) broth supplemented with 12% (v/v) glycerol were freshly grown at 37ºC to carry out this study.

Bacterial strains

Growth media and culture conditions

From our laboratory stock five Gram positive bacteria, Staphylococcus aureus,

Nutrient agar media purchased from Difco, USA was used for antibacterial activity assay

MATERIALS AND METHODS Plant material

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

of the plant extracts. The bacterial strains were incubated at 37°C for overnight as described elsewhere (Nesa et al., 2013; Chouduri and Wadud, 2013). Fresh cell culture in nutrient broth media prepared on water bath (Advantec Lab-Thermo Shaker, TS-20, Toyo Kaisha Ltd) with mild shaking at 37°C was used to test the swarming motility of Proteus strain.

Test for antibacterial activity Plant extracts were tested for antibacterial and specifically anti-Proteus activity using disk diffusion method on nutrient agar media as reported elsewhere (Dash et al., 2005; Parvin et al., 2014). The extracts were separately dissolved in 1 ml of ethanol and the filter paper discs (6 mm diameter) were impregnated with known amounts of test substances and prepared disc with various potencies, 25 μg to 1 mg/disc.

Discs were placed on pre-seeded bacterial culture plates and then kept at low temperature (4°C) overnight to allow maximum diffusion of the components. The plates were then allowed to incubate at 37°C for 18 hrs. Then the diameter (in millimeter) of zone of inhibition for each extract against tested microorganisms was noted. Reference standard discs of cefixime (5 µg), ceftazidime (30 µg), kanamycin (30 µg) (Hi-media, India) were used as positive control and blank disc as negative control.

Swarming motility test Proteus strains were grown overnight in 10 ml of LB broth medium (1% Tryptone, 0.5% Yeast extract, and 0.5% NaCl) at 37ºC with shaking (200 rpm). Then 5 μl of fresh cell culture was spotted at the center of LB agar plates (LB medium containing 1.5% agar) previously dried to remove water drops from the surface of the agar medium as described in other reports (Kwil et al., 2013) and incubated at 37ºC for 24 hrs unless it is mentioned otherwise. Then the mean diameters of swarming zones measured in millimeter at three different directions were used for analysis.

Inhibition of swarming motility The effects of plant extracts on swarming motility of Proteus strains were assessed as described in other report (Liaw et al., 2000; Roshid et al., 2014). Briefly, an overnight bacterial culture (5 μl) was inoculated centrally onto the surface of dry LB agar plates prepared with extracts at various concentrations which were then incubated at 37ºC for 24 hrs. The perimetric distance of swarming motility was assayed by measuring the fronts of swarming areas in three different directions.

Data analysis For data processing, the software Microsoft Excel 2007 was used. Results of triplicate experiments were averaged, and means ± standard deviations were calculated.

RESULTS Antibacterial activities of plant extracts The ethanol extracts of the plant specimens were tested for their antibacterial activities on five Gram-positive and six Gram-negative bacteria from our laboratory stock. The extracts named Ti-b, Pe-f, Pm-w, Ar-r, Ul-r, and Ai-l showed remarkable antibacterial activities with a wide zone of inhibition whereas Ul-l, Ul-f, Ul-b, Cg-w, and Mp-r were inactive in antibacterial activities (Table 2). However, the antibacterial potentials of the test extracts based on their zone of inhibition were evaluated where Pm-w was the best one showing 15–22 mm clear zone on culture plate followed by Ai-l (18–20 mm), Ar-r (10–19 mm), Pe-f (10– 16 mm), Ul-r (8–14 mm) and Ti-b (9–13 mm).

The antibacterial potentials of the extracts Pmw and Ai-l against three Gram-positive bacteria, S. agalactiae, B. megaterium, B. subtilis, and two Gram-negative bacteria, P. aeruginosa, S. flexneri, were very close and comparable to that of reference antibiotic kanamycin (Table 2).

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Figure 1: Effective medicinal plant species to cephalosporin resistant Proteus.

A: whole plant of Urena lobata L. (image courtesy- www.google.com.bd), B: leaf specimen of U. lobata L. washed with water, C: fruits specimen of U. lobata L. under shade drying, D: whole plant of Physalis minima L. (image courtesy- www.google.com.bd).

Table 2: Antibacterial activities of plant extracts on bacterial pathogens.

Test strains

Diameter of zone of inhibition (mm) of test extracts (1 mg/disc) Ti-b

Pe-f

Pm-w Ar-r

Kan

Ul-r Ul-l Ul-f Ul-b Cg-w Ai-l Aa-l Mp-r

Gram-positive S. aureus S. agalactiae B. cereus B. megaterium B. subtilis

12±0.3 10±0.4 12±0.9 9±0.4 11±0.4

12±0.6 12±0.3 10±0.7 12±0.7 11±0.5

22±0.3 19±0.2 20±0.3 22±0.4 21±0.5

P. aeruginosa S. flexneri S. dysenteriae E. coli S. sonnei A. species

12±0.4 13±0.2 13±0.3 12±1.1 12±0.4 11±0.7

14±1.0 16±0.8 11±1.1 14±0.8 10±0.5 10±0.3

19±0.6 20±0.2 18±0.9 21±0.3 17±0.7 15±0.6

14±0.4 13±0.2 − 12±0.2 12±0.9 − 14±0.6 12±0.4 − 11±0.9 11±0.2 − 10±0.3 13±0.5 − Gram-negative 11±0.4 14±0.5 − 13±0.6 13±0.7 − 14±0.7 12±0.7 − 19±0.8 8±0.6 − 14±0.9 8±0.5 − 14±0.4 8±0.4 −

− − − − −

− − − − −

− − − − −

− − − − −

− − 8±0.6 − −

34±1.2 23±1.3 34±0.6 23±0.5 22±0.8

− − − − − −

− − 18±0.3 − − − 19±0.8 − − 7±0.2 18±0.1 − − 8±0.1 19±0.1 − − − 20±0.6 − − − 19±0.4 −

− − 8±0.8 − 7±0.4 7±0.6

22±0.9 25±1.5 31±1.4 40±0.8 31±1.1 29±0.9

18±0.1 19±0.1 20±0.3 18±0.5 20±0.4

(−) sign indicates no activity. Values were expressed as mean ± SD (n=3). Ti-b: Tamarindus indica bark, Pe-f: Phyllanthus emblica fruit, Pm-w: Physalis minima whole plant, Ar-r: Asparagus racemosus root, Ul-r: Urena lobata root, Ul-l: Urena lobata leaf, Ul-f: Urena lobata fruit, Ul-b: Urena lobata bark, Cg-w: Coccinia grandis whole plant, Ai-l: Azadirachta indica leaf, Aa-l: Abroma augusta leaf, Mp-r: Mimosa pudica root, Kan: Kanamycin (30 µg/disc).

Screening of plant extracts for their abilities to inhibit Proteus Next our efforts aimed to search medicinal plants to combat these strong cephalosporin resistant Proteus isolates to control and manage UTI caused by these bacteria. To do so, twelve specimens of nine medicinal plants as enlisted in table-1 were selected based on their reported information. The extracts exhibiting high antibacterial activities on several Gram-positive and Gram-negative bacteria were used to test whether they have any inhibitory effect on multi-antibiotic resistant Proteus strains

isolated in our previous study (Wadud and Chouduri, 2013). The extract Pm-w showed remarkable zone of inhibition of Proteus strains (22 mm) whereas no clear zone of inhibition was observed for reference antibiotic cefixime (Figure 2). A representative image has been shown in figure 2. The extract Ul-r showed clear zone of inhibition of Proteus strains but relatively higher inhibition was found for the strain 11(Pv). Then the extracts were screened for their effects on swarming motility of the test strains since swarming is one of the crucial pathogenic factors of Proteus bacteria.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Figure 2: Antibacterial activities of extracts on cephalosporin resistant Proteus isolates.

Extracts at 1 mg/disc concentration were used. The diameters of zone of inhibition in isolate 11(Pv): 8 mm (Ul-b), 15 mm (Ul-r), 22 mm (Pm-w), 11 mm (Cg-w); in 662(Ph): 13 mm (Ul-r), 21 mm (Pm-w); in 666(Pp): 23 mm (Ai-l), 15 mm (Ar-r), 12 mm (Ti-b), 12 mm (Pe-f), 29 mm (Pm-w). Reference standard discs Cfx: cefixime (5 µg) and Cfd: Ceftazidime (30 µg).

Figure 3: Effects of extracts on Proteus swarming.

Three top swarmer strains, A: 911(Pm), B: 912(Pm), C: 662(Ph) were subjected to a test for swarming motility on LB agar plate in the presence of U. lobata bark (■), leaf (▲), fruit (●), and root (○) extract at 500 µg/ml concentration and the absence of extract (♦). The U. lobata root extract strongly inhibited the swarming of all test strains.

Effects of plant extracts on swarming motility of Proteus strain Eleven Proteus isolates found to be strongly resistant to cephalosporin by disc diffusion method as reported earlier (Chouduri and Wadud, 2014) were subjected to a bactericidal activity assay by NHS where four isolates 11(Pv), 661(Pp), 911(Pm), and 912(Pm) were found to be resistant to NHS (unpublished data). Isolates 912(Pm), 911(Pm) and 662(Ph) were strong swarmer on LB agar media (Chouduri et al., 2014), therefore, these isolates were undertaken to a test of swarming in the presence of various concentrations of plant extracts especially U. lobata extracts (Figure 3)

since major parts of this plant are used as folk medicine for UTI (Nandwani et al., 2008; Hossan et al., 2010). No noticeable effects of the extracts except Ul-r were found on the swarming motilities of the test strains (Figure 3). The extract Ai-l accelerated the swarming of Proteus isolates about 2 fold. However, interestingly complete inhibition of swarming was found by the extract Ul-r at 500 µg/ml concentration (Figure 3) although its antibacterial activity was nil or very low by disc diffusion method (Figure 2, Table 3). The lag phase of swarming continued up to 4 hrs of incubation and the basal swarming starts after 4 hrs of incubation in the presence of the extracts.

The zigzag pattern of swarming curves was a

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

consequence of swarming-plus-consolidation cycle of the strains. However, anti-swarming effect of U. lobata root extract can be of interest to develop phytomedicine for the management and control of UTI and/or wound

infection caused by antibiotic resistant Proteus bacteria. Moreover, the extract of Physalis minima had no anti-swarming effect although its antibacterial activity was stronger than that of others.

Table 3: Antibacterial activities of extracts on cephalosporin resistant Proteus isolates.

Proteus Diameter of zone of inhibition (mm) of test extracts (1 mg/disc) Cfx strains Ti-b Pe-f Pm-w Ar-r Ul-r Ul-l Ul-f Ul-b Cg-w Ai-l Aa-l Mp-r 11(Pv) 7±0.6 16±0.8 22±0.2 16±0.4 15±0.2 − − 661(Pp) 15±0.3 14±0.4 22±0.5 17±0.1 −

− 8±0.2 11±0.1 18±0.3 − − − − 21±0.3 −

− −

− −

− − −

− − −

662(Ph) 8±0.2 13±0.3 21±0.3 663(Pp) 9±0.4 10±0.5 12±0.3 664(Pp) 13±0.3 17±0.5 18±0.6 665(Pp) 9±0.5 11±0.7 13±0.5 666(Pp) 12±0.6 12±1.1 29±0.4

14±0.9 13±0.3 − 12±0.2 − − 15±0.4 − −

− − −

− − −

− − −

15±0.9 − 14±0.5 − 19±0.3 −

13±0.4 − 15±0.5 7±0.3

− −

− −

− −

− −

15±0.7 − − − 23±0.1 − 10±0.4 13±0.6

667(Pp) 11±0.4 668(Pp) 12±0.6 911(Pm) 8±0.4 912(Pm) 8±0.3

15±0.3 8±0.4 14±0.5 − 15±0.2 − 14±0.9 −

− − − −

− − − −

− − − −

− − − −

18±0.6 27±0.5 15±0.8 17±1.2

8±0.2 15±1.1 14±0.6 18±0.4 11±1.0 − 11±0.7 −

− − − −

7±0.4 − − − − 14±0.6 − 28±0.7

(−) sign indicates no activity. Values were expressed as mean ± SD (n=3). Ti-b: Tamarindus indica bark, Pe-f: Phyllanthus emblica fruit, Pm-w: Physalis minima whole plant, Ar-r: Asparagus racemosus root, Ul-r: Urena lobata root, Ul-l: Urena lobata leaf, Ul-f: Urena lobata fruit, Ul-b: Urena lobata bark, Cg-w: Coccinia grandis whole plant, Ai-l: Azadirachta indica leaf, Aa-l: Abroma augusta leaf, Mp-r: Mimosa pudica root, Cfx: Cefixime (5 µg/disc).

Proteus Extracts strain 911(Pm) Control Ul-b Ul-l Ul-f Ul-r 912(Pm) Control Ul-b Ul-l Ul-f Ul-r 662(Ph) Control Ul-b Ul-l Ul-f Ul-r

Table 4: Kinetics of swarming motility Rate of swarming (mm/h) 0–3 h 3–4 h 4–5 h 5–6 h 6–7 h 1.03 1.62 11.50 4.00 3.50 0.98 1.25 10.00 2.00 2.04 1.02 1.50 17.50 2.50 2.35 0.95 1.62 20.50 1.50 1.45 0.89 1.25 − − − 0.97 1.50 13.50 5.00 7.00 1.10 1.62 21.50 2.00 1.50 1.09 1.12 14.50 3.00 2.96 0.94 1.37 16.00 5.50 6.00 0.95 1.12 − − − 1.03 1.25 2.75 3.00 2.00 0.92 1.37 9.50 3.50 6.50 1.17 1.87 13.50 3.00 2.88 0.88 1.13 7.50 7.00 6.95 1.11 2.00 − − −

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

7–8 h 3.50 8.00 9.00 10.00 − 9.00 10.00 14.00 9.50 − 4.00 13.00 10.50 10.50 −

8–9 h 5.50 7.00 9.50 9.50 − 6.50 12.00 6.50 2.50 − 5.50 4.89 5.50 9.50 −

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Kinetics of U. lobata extract-induced swarming motility Three Proteus isolates exhibiting enhanced swarming motility were assessed for their abilities to swarm onto the LB agar plate in the presence of U. lobata extracts. The velocities of swarming (mm/h) of the test strains in the presence of U. lobata root extract were found to be zero after the lag phase (4 hrs) (Table 4) and the swarming velocities for other extracts were instantaneously accelerated just after the lag phase (4 hrs) that were the maximal velocities up to 7 hrs of incubation period. The similar duration of lag phase (4 hrs) of clinical isolates of P. mirabilis was reported by Rauprich et al. (Rauprich et al., 1996). The rapid onset of swarming after lag phase is possibly due to the formation of elevated number of flagella on bacteria or multinucleation in the first generation of swarmer cells. The cycle time of swarming-plusconsolidation phase found by Rauprich et al., (1996) is about 3.5 hrs at 37°C on 1.5% agar plate. In this study, the high swarming velocities after lag phase at 4–5 hrs and the subsequent gradual decline of the velocity up to 7 hrs indicated the first cycle of swarming-plusconsolidation phase and the following high velocities at 7–8 hrs indicated the second cycle of swarming-plus-consolidation phase resembling the findings of Rauprich et al.

(1996). However, only the U. lobata root extract showed significant inhibition of swarming of the test strains and no noticeable effects of other test extracts on swarming were observed.

DISCUSSION Among the most common infections UTI is affecting humans and represent a serious health problem for millions of people each year.

Proteus is an important opportunistic uropathogen, frequently isolated from catheterized patients or individuals with structural abnormalities of the urinary tract (Khalid et al., 2013; Hoban et al., 2012; Alves et al., 2014) although it does not commonly cause UTI in the normal host. UTI is

commonly managed with antibiotic therapy but the increasing evidence of antibiotic resistance is restricting the therapeutic option. Thus the acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available antibiotics have led researchers to investigate the antimicrobial activity of herbal extracts.

The World Health Organization reported that about 80% of the world’s population depends primarily on traditional medicine that mainly involves the use of plant extracts (Low et al., 2002). The screening of plant extracts and plant products has shown that medicinal plants represent a potential source of new antiinfective agents. For instance, cranberry has long been of interest for its beneficial effects in preventing UTI (Ahuja et al., 1998; Howell et al., 1998; Howell and Foxman 2002; McCall et al., 2013). Plants containing flavonoids, terpenoids, steroids, phenolic compounds and alkaloids have been reported to have antimicrobial activity. Three compounds (kaempferol, quercetin, tiliroside) isolated from ethyl acetate fraction of U. lobata leaf showed strong antimicrobial activities against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Bacillus polyxyma and Candida albicans (Adewale et al., 2007). But in this study, the ethanol extract of U. lobata leaf showed no antibacterial activities against test bacterial pathogens including Proteus. In contrast, U. lobata root had a significant antiswarming effect on Proteus isolates although its antibacterial activity was very low. It has been reported that U. lobata root has no significant toxic effects on serum total proteins, albumin and globulins (Omonkhua and Onoagbe, 2011). Therefore, U. lobata root can be used as a source of alternative anti-infective agent for the treatment of UTI and wound infection caused by antibiotic resistant bacteria.

The chloroform extract of P. minima exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (Leong et al., 2011). The strong antibacterial

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

activity of ethanol extract of P. minima leaf has been reported (Gavimath et al. 2012). In this study, we found strong inhibition of antibiotic resistant Proteus isolates by the treatment of ethanol extract of P. minima whole plant. Thus, P. minima can also be the plant of interest for the treatment and control of antibiotic resistant uropathogens.

Howell et al., (1998) determined that proanthocyanidins isolated from the cranberry fruit inhibit P-fimbrial adhesion in vitro, and thus may be the compounds responsible for the beneficial effect on UTI prevention (Howell et al., 1998). The urine of humans who consumed cranberry juice cocktail also exhibited antiadhesion activity (Howell and Foxman, 2002), which suggests that a certain level of absorption occurred and that bioactive proanthocyanidins and/or their metabolites have been excreted in the urine to inhibit adhesion. The bactericidal activities of anacardic acid and totarol (a diterpene extracted from the totara tree) on methicillin resistant strains of S. aureus and the synergistic effect of these compounds associated with methicillin have been reported (Muroi and Kubo, 1996). Therefore, more studies pertaining to the use of plants as therapeutic agents should be emphasized, especially those related to the control of antibiotic resistant microbes.

CONCLUSION The ethanol extract of Physalis minima whole plant showed strong antibacterial activities against cephalosporin resistant uropathogen Proteus and the extract of Urena lobata root showed strong anti-swarming effect on Proteus. Therefore, a mixture of two extracts would be a powerful anti-infective agent to combat UTI caused by antibiotic resistant Proteus. This study could offer scientific basis for the in-depth evaluation of ethanol extract of P. minima whole plant and U. lobata root. The phytochemical(s) in P. minima and U. lobata extracts having the potential antibacterial activities and antiswarming effect are remain to be identified and are required to go through the toxicity analyses before they can be safely applied.

ACKNOWLEDGEMENTS Authors wish to thank the Department of Pharmacy, University of Rajshahi, Bangladesh for providing laboratory facilities to carry out the entire experiments. We thank the Ministry of Science and Technology, Government of the People's Republic of Bangladesh for the NST fellowship provided to author MR to carry out the research.

REFERENCES Adewale AO, David AA, Abiodun OO, Craig OA (2007). Studies on antimicrobial, antioxidant and phytochemical analysis of Urena lobata leave extract. J. Phys.

Nat Sci. 1(2): 12–20.

Ahuja S, Kaak B, Roberts J (1998). Loss of fimbrial adhesion with the addition of Vaccinium macrocarpon to the growth medium of P-fimbriated Escherichia coli. J. Urol. 159: 559–562.

Ahmad I, Mehmood Z, Mohammad F (1998).

Screening of some Indian medicinal plants for their antimicrobial properties.

J. Ethnopharmacol. 62: 183–193.

Alves MJ, Barreira JC, Carvalho I, Trinta L, Pereira L, Ferreira IC, Pintado M (2014).

Propensity for biofilm formation by clinical isolates from urinary tract infections: developing a multifactorial predictive model to improve the antibiotherapy. J. Med. Microbiol., 63: 471–477.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Chouduri AU, Wadud A (2013). Strong cephalosporin resistant uropathogen, Proteus mirabilis, in urban tap water harbors a risk to public health, Bangladesh. Glob. Adv. Res. J. Microbiol. 2: 164–171.

Chouduri AU, Wadud A, Islam AU (2014).

Extended spectrum multi-drug resistance versus pathogenic factorsswarming, proteases, and urease- of Proteus species. Int. Res. J. Microbiol.

5: 8–15.

Chouduri AU, Wadud A (2014). Twitching motility, biofilm communities in cephalosporin resistant Proteus spp and the best in vitro amoxicillin susceptibility to isolates. Am. J. Microbiol. Res. 2: 8–15.

Cohen ML (1992). Epidemiology of drug resistance: implications for a postantimicrobial era. Science 257: 1050–1055.

Dash S, Nath LK, Bhise S, Bhuyan N (2005).

Antioxidant and antimicrobial activities of Heracleum nepalense D Don root.

Trop. J. Pharm. Res. 4: 341–347.

Hoban DJ, Lascols C, Nicolle LE, Badal R, Bouchillon S, Hackel M, Hawser S (2012). Antimicrobial susceptibility of Enterobacteriaceae, including molecular characterization of extended-spectrum beta-lactamase–producing species, in urinary tract isolates from hospitalized patients in North America and Europe: results from the SMART study 2009– 2010. Diagnostic Microbiol. Infec.

Dis., 74: 62–67.

Hossan MS, Hanif A, Agarwala B, Sarwar MS, Karim M, Rahman MTU, Jahan R, Rahmatullah M (2010). Traditional use of medicinal plants in Bangladesh to treat urinary tract infections and sexually transmitted diseases. Ethnobot.

Res. Appl. 8: 61–74.

Howell AB, Vorsa N, der Marderosian A (1998). Inhibition of the adherence of Pfimbriated Escherichia coli to uroepithelial-cell surfaces by proanthocyanidin extracts from cranberries. New England J. Med. 339: 1085–1086.

Howell AB, Foxman B (2002). Cranberry juice and adhesion of antibiotic-resistant uropathogens. JAMA 287: 3082–3083.

Joseph B, George J, Mohan J (2013).

Pharmacology and traditional uses of Mimosa pudica. Int. J. Pharm. Sci. Drug Res. 5: 41–44.

Khalid MIH, Teh LK, Lee LS, Zakaria ZA, Salleh MZ (2013). Genome sequence of Proteus mirabilis strain PR03, isolated from a local hospital in Malaysia. Genome Announ., 1: e003 27–13.

Khan MA, Khan H, Khan S, Mahmood T, Khan PM, Jabar A (2009). Antiinflammatory, analgesic and antipyretic activities of Physalis minima Linn. J. Enz. Inhib. Med. Chem. 24: 632–637.

Khosla S, Sharma S (2012). A short description on pharmacogenetic properties of Phyllanthus emblica. Spatula DD. 2: 187–193.

Kwil I, Kazmierczak D, Rozalski A (2013).

Swarming growth and resistance of Proteus penneri and Proteus vulgaris strains to normal human serum. Adv. Clin. Exp. Med. 22: 165–175.

Leong OK, Muhammad TST, Sulaiman SF (2011). Cytotoxic activities of Physalis minima L. chloroform extract on human lung adenocarcinoma NCI-H23 cell lines by induction of apoptosis.

Evidence-Based Compl. Alter. Med. 2011: 1–10.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Liaw SJ, Lai HC, Ho SW, Luh KT, Wang WB (2000). Inhibition of virulence factor expression and swarming differentiation in Proteus mirabilis by pnitrophenylglycerol. J. Med. Microbiol.

49: 725–731.

Low CAM, Regnier TJC, Korsten L (2002).

Medicinal bulbous plants of South Africa and their traditional relevance in the control of infectious diseases. J. Ethnopharmacol. 82: 147–154.

Mazumder UK, Gupta M, Manikandan L, Bhattacharya S (2001). Antibacterial activity of Urena lobata roots.

Fitoterapia 72: 927–929.

McCall J, Hidalgo G, Asadishad B, Tufenkji N (2013). Cranberry impairs selected behaviors essential for virulence in Proteus mirabilis HI4320. Can. J. Microbiol. 59: 430–436.

Muroi H, Kubo I (1996). Antibacterial activity of anacardic acids and totarol, alone and in combination with methicillin, against methicillin-resistant Staphylococcus aureus. J. Appl. Bacteriol. 80: 387–394.

Nandwani D, Calvo JA, Tenorio J, Calvo F, Manglona L (2008). Medicinal plants and traditional knowledge in the Northern Mariana Islands. J. Appl.

Biosci. 8: 323–330.

Narayanan AS, Raja SSS, Ponmurugan K, Kandekar SC, Natarajaseenivasan K, Maripandi A, Mandeel QA (2011).

Antibacterial activity of selected medicinal plants against multiple antibiotic resistant uropathogens: a study from Kolli hills, Tamil Nadu, India. Beneficial Microbes 2: 235–243.

Nesa L, Salam A, Islam AU, Chouduri AU (2013). Multi-drug resistant Neisseria gonorrhea among hotel-based sex workers in Rajshahi, Bangladesh. Int. J. Microbiol. Res. 4: 167–176.

Nwodo UU, Obiiyeke GE, Chigor VN, Okoh AI (2011). Assessment of Tamarindus indica extracts for antibacterial activity.

Int. J. Mol. Sci. 12: 6385–6396.

Omonkhua AA, Onoagbe IO (2011).

Evaluation of the long-term effects of Urena lobata root extracts on blood glucose and hepatic function of normal rabbits. J. Toxicol. Env. Health Sci. 3: 204–213.

Parvin

S, Kader MA, Chouduri AU, Rafshanjani MAS, Haque ME (2014).

Antibacterial, antifungal and insecticidal activities of the n-hexane and ethyl-acetate fractions of methanolic extract of the leaves of Calotropis gigantea Linn. J. Pharma.

Phytochem. 2: 47–51.

Poovendran P, Vidhya N, Murugan S (2011).

Antimicrobial activity of Coccinia grandis against biofilm and ESBL producing uropathogenic E. coli. Glob.

J. Pharmacol. 5: 23–26.

Rauprich O, Matsushita M, Weijer CJ, Siegert F, Esipov SE, Shapiro JA (1996).

Periodic phenomena in Proteus mirabilis swarm colony development. J. Bacteriol. 178: 6525–6538.

Roshid M, Wadud A, Chouduri AU (2014).

Potent inhibition of swarming in uropathogenic Proteus bacteria by ethanol extracts of Phyllanthus emblica fruit and Tamarindus indica bark. Int. Res. J. Biol. Sci. 3: 1–7.

Saikot FK, Khan A, Hasan MF (2012).

Antimicrobial and cytotoxic activities of Abroma augusta Lnn. leaves extract.

Asian Pac. J. Trop. Biomed. 2: S1418– S1422.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184–195

Wadud A, Chouduri AU (2013). Microbial safety assessment of municipal water and incidence of multi-drug resistant Proteus isolates in Rajshahi, Bangladesh. Curr. Res. Microbiol.

Biotechnol. 1: 189–195.

Yarnell E (2002). Botanical medicines for the urinary tract. World J. Urol. 20: 285– 293.

Source of Support: National Technology (NST) Fellowship, of Bangladesh

Science and Government

Yasmeen R, Hashmi AS, Anjum AA, Saeed S, Muhammad K (2012). Antibacterial activity of indigenous herbal extracts against urease producing bacteria. J. Anim. Plant Sci. 22: 416–419.

Zulfiker AHM, Roy PP, Momin MAM, Khan MS, Bulbul IJ, Ahmed T, Rana MS (2013). Investigation of antioxidant and antimicrobial potential of chloroform and petroleum ether extracts of selected medicinal plants of Bangladesh. British J. Med. Medical Res. 3: 4.

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research Article CATECHIN DETECTION IN CALLUS AND IN VITRO CULTURES OF THE EASTERN STRAWBERRY TREE, ARBUTUS ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN PALESTINE Zahra Aljabari1, Jawad Alzeer2, Rami Arafeh1* 1, 3

Biotechnology Research Center. Palestine Polytechnic University. P.O. Box 198, Hebron, Palestine.

Department of Applied Chemistry, Faculty of Applied Sciences. Palestine Polytechnic University, P.O.Box 198 Hebron, Palestine. *Corresponding author: E-mail: arafeh@ppu.edu; Tel: +970-22231921 – ext. 137; Fax: +970-2231921 – ext. 119 2

Received: 25/03/2014; Revised: 15/04/2014; Accepted: 20/04/2014

ABSTRACT The Eastern Strawberry tree, Arbutus andrachne L., is a medicinal evergreen small tree naturally distributed from Eastern Mediterranean to the Northern Black Sea region. In Palestine, the tree is known for its high medicinal value and recently has been included within the endangered species.

For conservation and utilization of A. andrachne we investigated the presence of catechin, an antioxidant and active flavonoid in the ethylacetate fraction in leaves of wild plant material and also in the extract of callus and the in vitro grown vegetative tissues. HPLC analysis of catechin revealed 0.063% in callus extract, 2.5% in the in vitro growing tissues and 0.5% in wild growing plants. In vitro propagation and callus culture are promising approaches for the secondary metabolites production in the case of A. andrachne.

KEY WORDS: Arbutus andrachne L., callus culture, catechin, medicinal plant, secondary metabolites.

Cite this article: Zahra Aljabari, Jawad Alzeer, Rami Arafeh (2014), CATECHIN DETECTION IN CALLUS AND IN VITRO CULTURES OF THE EASTERN STRAWBERRY TREE, ARBUTUS ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN PALESTINE, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 196–205

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

INTRODUCTION The Eastern Strawberry Tree, Arbutus andrachne L. (Ericaceae), is one of the important medicinal trees in the Eastern Mediterranean region. It is a medium evergreen tree grows in mountainous rocky habitats with alkaline soil. The plant parts are known to have valuable medicinal values due to its content in antioxidants and natural pigments that makes it a multiple uses plant. In the traditional folk medicine, it is used as astringent and for urinary antiseptic treatments, treatment for aching joints and wounds and against some cancer types (Said et al., 2002; Serce et al., 2010). Furthermore, a cosmetic value of the dried leaves' powder as skin whitening agent in face masks has been described by Issa et al.

(2008). Recently, the plant gained higher attention after being listed in databases of the endangered species in Palestine (Eshtayeh & Jamous, 2002) and Israel (ROTEM, 2002). The species is progressively encountering genetic erosion after being collected for its medicinal and cosmetic uses. The unplanned expansion in agricultural activities, overgrazing and collection for fire contribute in the genetic erosion of A. andrachne. Following to the continuous increase in demand for the plant during the last few years several attempts have addressed the propagation and conservation of A. andrachne in which some successes have been achieved, examples are discussed in (Bertsouklis & Papafotiou, 2009; Karam & AlSalem, 2001; Kose, 1998; Mostafa, et al., 2010; Tilki & Guner, 2007 ).

Catechins are polyphenolic flavonoids that can be found in wide range of natural sources including leaves of herbs like green tea Camellia sinensis, fruits like apples Malus pumila, fruit skin, juice and oil seed of grapes Vitis vinifera, wood and bark of trees in the genus Acacia reviewed in (Ruidavets, et al., 2000) and (Iacopini, Baldi, Storchi, & Sebastiani, 2008). Catechin exists in different chemical forms like −epicatechin, −epicatechin-3-gallate, −epigallocatechin, −epigallocatechin-3-gallate, +catechin and +gallocatechin. They exhibit many biological

activities accountable for their medicinal values. They play an antioxidant, anti-cancer, anti-angiogenic, anti-mutagenic, hypocholesterolemic, anti-ageing, anti-diabetic, antibacterial, anti-HIV and anti-inflammatory effects (Al-Hanbali, et al., 2009; Ivanov, et al., 2011; Sakar, et al., 1991; Suzuki, et al., 2005; Zaveri, 2006). Saker et al. (1991) have described some chemical constituents present in A. andrachne, namely +catechin, −epicatechin and arbutin in addition to other constituents in the tree bark such as monotropein and unidoside.

Recently, A. andrachne is being overexploited and attempts are focused to conserve it and supply sufficient material for propagation and utilization. In this study, we present the use of A. andrachne material obtained by in vitro culture for the production of secondary metabolites, particularly catechin, as a representative of flavonoids in comparison to its presence in the leaves of wild trees.

MATERIALS AND METHODS Plant material: Seeds of A. andrachne were collected in November 2007 from wild growing trees West of Hebron city [N:31°32′00″, E:35°05′42″].

Plant characterization was carried out by Dr. Rami Arafeh and a voucher specimen of the sampled plant was deposited in the Biotechnology Research Center at Palestine Polytechnic University. Ripe fruits were soaked in tap water for 72 h before the seeds were separated manually and washed from the fruit pulp. Seeds were stored at room temperature at 21±2°C to be used for experimental work.

Callus induction and maintenance: Since A. andrachne seeds exhibit physiological dormancy (Karam & Al-Salem, 2001; Mostafa, et al., 2010), a pretreatment for the seeds was carried out by soaking for 24 h in a solution of 5.0 mg/l GA3 at room temperature.

Seeds then were surface sterilized in 5% v/v solution of commercial bleach for 20 min then washed 3X with autoclaved deionized water.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

Callus tissue was initiated from germinating seeds that cultured on the surface of solid Gamborg’s B5 medium with vitamins (Gamborg et al., 1968) and supplemented with 1.0 mg/l 2,4−D. The medium was also supplemented with 3.0% w/v sucrose in addition to 0.1% w/v polyvinylpyrrolidone (PVP) as antioxidant to prevent tissue browning. Seeds were inoculated on 6.0 cm Petri dishes in the growth room for six weeks in full darkness. Induced callus clumps were transferred to WP media supplemented with 2.0 mg/l TDZ, 0.5 mg/l NAA, and 1.0 g/l PVP. Five calli pieces (~ 5.0 mm diameter) were placed on the surface of the media under cold fluorescent light at 45–50 µmol.m-2.sec-1.

Culture growth conditions were adjusted as described in Shatnawi et al., (2010).

Total crude extract Three different sources of plant tissues were used for the total crude extraction and later for catechin detection; a) leaves from a wild tree harvested in mid September 2008, b) in vitro vegetative parts cultured on WP medium for four months. The medium was supplemented with 6.0 mg/l zeatin; c) callus that have been grown and maintained for 4 months on WP medium supplemented with 2.0 mg/l TDZ + 0.5 mg/l NAA. Plant material was air dried at room temperature then ground to a fine powder with mortar and pestle. One gram of powder was soaked in 100.0 ml ethylacetate (EtOAc) or 5% methanol. The mixture was shaked for 48 h at 100 rpm then centrifuged for 15 min at 5000 rpm. The supernatant was separated then air dried under fume hood. The percentage yield of the dry extracts was calculated for both solvents as described by Alzeer et al., (2014).

Qualitative and quantitative analyses of catechin Analytical TLC For the qualitative detection of phenolic compounds particularly +catechin, TLC analysis was performed from the procedure described in Wagner & Bladt (2009) with the

following modifications; the TLC analysis was run on a precoated TLC plates (MachereyNagel, Dueren, Germany, Cat# 818133). The plates were covered with silica gel layer of 0.20 mm, 60 F254 with UV indicator. The mobile phase composed of 50 ml of chloroform: acetone: acetic acid at 65:21.5:13.5% v/v/v.

TLC plates were run for three consecutive times in the mobile phase and after each run the plates were air dried. The detection of phenolic compounds was visualized with a UV lamp at 254 nm. Finally, Plates were sprayed with FeCl3 solution (1.0 g of FeCl3 dissolved in 100.0 ml water:methanol at 50:50% v/v for visualization of total phenolic compounds.

Retention factor (Rf) value for catechin was measured by using the formula: distance spot moves Rf = distance solvent moves Analytical HPLC: HPLC analysis was conducted in the Center for Chemical and Biological Analyses at AlQuds University. The HPLC setting as described in Bramati et al., (2002) was followed; quantitative analysis of catechin was performed on HPLC (Alliance, Waters 2692 separation module) using the analytical column RP18 Waters Symmetry Shield TM, (5.0 µm, 4.6×250 nm). Samples for the analysis were prepared by dissolving 100.0 mg of the dried ethanol extract obtained from in vitro, wild material leaves, and callus in 0.7 ml HPLCgrade 8% methanol. Total run time was adjusted to 21 min using the following gradient elution, 98% A, 2% B (0–19) min, 80%A, 20%B at 20 min, then back to 98% A, 2% B at 21 min, A: buffered water 1% H3PO4, B: acetonitrile. Flow rate was adjusted at 1.0 ml/min, UV detection was adjusted at 280 nm and the injection volume was 40 µl. Serial reference standard solutions of +catechin (0, 1, 20, 40, 60, 80, and 100 ppm) were prepared by dissolving catechin in a HPLC-grade 99.9% methanol to construct the calibration curve.

Percentage yield of catechin from different sources was calculated in mg per 100 mg of plant dried material against external catechin standard using the following equation: W/W%

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

= (C× FV×D×100%)/W, where C is catechin concentration in the sample (mg/ml) extrapolated from the calibration curve linear regression, FV is the final volume of the sample in milliliters, D is the dilution factor, and W is the sample weight in milligrams.

RESULTS In vitro seed germination High germination percentage (70%) followed by callus growth was observed after the seeds have been pretreated with GA3 and cultured on B5 medium supplemented with 1.0 mg/l 2,4-D. Callus from germinated seeds, cotyledons and roots were successfully grown on WP medium supplemented with 2.0 mg/l

TDZ, 0.5 mg/l NAA and 1.0 g/l PVP (Figure 1).

Crude extract yield One gram of dried leaves’ powder from wild growing tree, leaves from in vitro plants and dried callus was immersed in EtOAc or in 5% methanol in order to collect total crude extracts. Results indicate higher yield of crude extract in the in vitro grown tissues than the wild material or from callus tissue (Table 1).

Furthermore, the EtOAc fraction was almost double amount than the 5% methanol fraction in the three tissues used (Table 1). For the TLC and HPLC analysis the fraction derived from EtOAc was used.

Figure 1. Callus induction after six weeks from (a) cotyledons,(b) seeds, and (c) roots on B5 media supplemented with 1.0 mg/l 2,4-D under full dark, (d) subcultured callus on WP media with 2.0 mg/l TDZ and 0.5 mg/l NAA under light condition.

Table 1. Yield percentage of crude extract in 1.0 g of dried callus from different tissues.

Catechin yield in EtOA fraction calculated by HPLC analysis.

Type of explant In vitro leaves

EtOAc Methanol Catechin % in mg/100g 5% a 43.3 17.7 a 2.5a

Wild plant leaves

28.3b

13.4b

0.5b

Seeds derived callus 20.0c

10.0c

0.063c

Figures with different letters are statistically different at p<0.05.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

Qualitative and quantitative detection of catechin TLC analysis of catechin: Extracts derived from wild tree leaves, in vitro growing vegetative parts and callus tissue were investigated by TLC analysis for the detection of catechin. Catechin in the 5% methanol fraction was not detected whereas significant amount was revealed in the EtoAC extract particularly in both in vitro and wild material. Catechin identification was first visualized with a UV lamp then stained with FeCl3 solution. FeCl3 provided an excellent mean to selectively visualize phenolic flavonoids (Yadav & Agarwala, 2011). The Rf values of the major spot in wild and in vitro leaves were dominantly observed in accordance to the reference values of catechin at Rf = 0.4 (Figure 2). Slightly less polar compounds were also observed at lower Rf values. According to the results revealed by TLC analyses, extract from callus tissue of A. andrachne did not show any traces of catechin (Figure 2, lane 5). After three consecutive runs of the TLC plate, mix

spot was not separated during the three runs.

This clearly indicates the presence catechin from in vitro leaves match well with reference catechin (Figure 2). Catechin from in vitro material showed darker and higher concentration spot than the wild material.

HPLC analysis of catechin HPLC analysis was carried out on the EtOAc extract of in vitro, wild plant leaves and callus of A. andrachne. Under the chromatographic conditions described in the methodology, the retention time of catechin was 12.563 to 12.615 min. The chromatograms for catechin in the wild leaves, in vitro vegetative tissues and callus extract of A. andrachne are shown in (Figure 3, 4 and 5).

Percentage yield of catechin from different sources was calculated in mg per 100 mg of plant dried material against external catechin standard (Table 1).

Figure 2. TLC plate spotted with 3.0 µl of EtOAc extract from different sources of A. andrachne after being sprayed with FeCl3.

1.0 µl of (R) Catechin standard, (1) in vitro grown A. andrachne, (2) mix 1 and R, (3) in vivo grown A. andrachne, (4) mix 3 and R, (5) callus extract of A. andrachne and (6) mix 5 and R.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196â&#x20AC;&#x201C;205

Figure 3. HPLC chromatogram for catechin detection in A. andrachne in vitroâ&#x20AC;&#x201C;grown leaves.

Figure 4. HPLC chromatogram for catechin detection in the leaves of wild grown A. andrachne plant.

Figure 5. HPLC chromatogram for catechin detection in A. andrachne callus extract.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196â&#x20AC;&#x201C;205

Results indicate congruency between TLC and HPLC analysis. The revealed TLC pattern indicated catechin in both wild and in vitro material with a higher concentration regarding the in vitro grown material. TLC could not reflect any traces of catechin in callus tissue, however, the HPLC analysis of callus extract showed catechin at a retention time of 12.563 min (Figure 5). Furthermore, HPLC chromatograms showed increase in concentrations of other chemical compounds from in vitro grown material at the retention time of 7.816, 8.295 and 15.507 min. The presence of catechin in A. andrachne grown under in vitro conditions indicates that the plant tends to produces catechin under controlled environment in higher concentration than the wild material of September harvest.

DISCUSSION Based on a recent literature review, this is the first study that addressed the detection of a flavonoid compounds in the in vitro grown tissues of A. andrachne. Our results indicated that this plant species, if grown under in vitro conditions, produces higher concentration of catechin compared to the wild growing plant.

In studies on other medicinal plants, higher secondary metabolites content was detected in the in vitro growing material compared to wild ones (Arafeh et al., 2006); Narula et al. 2004).

Higher yield of volatile oils (camphor and borneol) were detected in Salvia fruticosa in vitro microshoots more than greenhouse growing plants (Arikat et al., 2004). Karam et al. (2003) also reported that the yield of rosmarinic acid in the in vitro grown S. fruticosa was higher (2.1â&#x20AC;&#x201C;5.1 mg/100 mg dry weight) than in the leaves or roots (0.21 or 0.72 mg/100 mg dry weight, respectively) of greenhouse-grown plants.

It is well documented in literature that under controlled environment some plants tend to produce higher yield of secondary metabolites than their naturally growing counterparts. Example is in the study of Arikat, et al. (2004) and the review of Karuppusamy, (2009) and Hussain et al. (2012). The likely

reason behind the difference was attributed to the presence of plant growth regulators and elicitors and precursors of the secondary metabolites in the growth media.

Mostafa et al., (2010) studied the arbutin (a hydroquinone found in leaves of A. andrachne) content and compared the yield between in vitro-grown material and samples from wild plant material collected in August, October and December. They reported higher arbutin yield in the three wild samples compared to the in vitro-grown one. In our work as revealed by HPLC analysis, the catechin content in the in vitro growing material was nearly three times more than the wild grown material. Some plants tend to accumulate certain secondary metabolites when planted under in vitro conditions in higher quantities than the wild material, examples were documented in Hashimoto et al. (1993).

Seeds treated prior to the in vitro culture showed comparable results (70% germination) with the finding of Mostafa et al. (2010) (at least 80%), however, in their work, they used agar-gelled water supplemented with GA3 at 2.0 mg/l.

In the present study, the total catechin content measured in the in vitro vegetative parts was 2.5 mg/100 mg, which is five times more than the material in the wild 0.5 mg/100 mg and 40 times in callus tissue.

This catechin concentration in callus was not surprising since callus is undifferentiated mass of cells and it is proved that secondary metabolism in plant cells is tightly linked to its differentiation state. When cells are completely undifferentiated, secondary metabolite pathways are partially shut off. A similar conclusion is observed in the in vivo leaf tissues of tobacco, when they are differentiated cells they are able to synthesis enzymes like chalcone synthase which is involved in flavonoids biosynthesis pathway. However, callus generated from the leaf tissues, which contains partially or totally undifferentiated cells are unable to produce as much of this

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

enzyme like their growing tissues in natural conditions.

metabolites production via in vitro culture techniques.

CONCLUSION

ACKNOWLEDGEMENTS

This study demonstrated that in vitro growing tissues particularly callus, accumulated the antioxidant flavonoid catechin.

This would eventually decrease the pressure on a threatened wild plant in nature and provide new alternative source for secondary

We would like to thank the Deanship of Graduate Studies and Scientific Research for its support in this project. We also would like to thank Dr. Muhannad Quree’ from the Faculty of Pharmacy at Al-Quds University for helping in the HPLC analysis and Mr. Zaid Al-Taradeh for his kind technical help in the laboratory.

REFERENCES Al-Hanbali, M., Ali, D., Bustami, M., AbdelMalek, S., Al-Hanbali, R., Alhussainy, T., et al. (2009). Epicatechin suppresses IL-6, IL-8 and enhances IL-10 production with NF-kappaB nuclear translocation in whole blood stimulated system. Neuro Endocrinol Lett, 30 (1), 131–138.

Alzeer, J., Vummidi, B, R., Arafeh, R., Rimawi, W., Saleem, H. & Luedtke, N. W. (2014). The influence of extraction solvents on the anticancer activities of Palestinian medicinal plants. Journal of Medicinal Plant Research. 8, (8), 408– 415, Arafeh R.M, Shibli R.A, Mahmoud M.A, & M.A, S. (2006). Callusing, cell suspension culture and secondary metabolites production in Persian oregano (Origanum vulgare L.) and oregano (O. syriacum L.). Jordan J Agric Sci, 2, 274–282.

Arikat, N. A., Jawad, F. M., Karam, N. S., & Shibli, R. A. (2004). Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.).

Sientia Hort, 100, 193–202.

Bertsouklis, K. F., & Papafotiou, M. (2009). In vitro propagation of Arbutus andrachne L. . Acta Hortic. , 813, 477–480

Bramati, L., Minoggio, M., Simonetti, P., Mauri, P., & Pietta, P. (2002).

Quantitative Characterization of Flavonoid Compounds in Rooibos Tea (Aspalathus linearis) by LC−UV/DAD. Journal of Agricultural and Food Chemistry, 50 (20), 5513–5519.

Eshtayeh M.S, & Jamous, R. M. (2002). The BERC red list of threatened plants of the West Bank and Gaza Strip. BERC publications.

Gamborg O.L, Miller R.A, & K, O. (1968).

Nutrient requirements of suspension culture of soybean root cells.

Experimental Cell Research, 50, 15– 158.

Hashimoto, T., Yun, D.J, & Yamada, Y. (1993). Production of tropane alkaloids in genetically engineered root cultures.

Pyhtochemistry, 32, 713–718.

Hussain, M. S., Fareed, S., Ansari, S., Rahman, M. A., Ahmad, I. Z., & Saeed, M. (2012). Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci, 4(1), 10–20.

Iacopini, P., Baldi, M., Storchi, P., & Sebastiani, L. (2008). Catechin, epicatechin, quercetin, rutin and resveratrol in red grape: Content, in vitro antioxidant activity and

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

interactions.

Journal of Food Composition and Analysis, 21, 589– 598.

Issa, R., Afifi, F., & Amro, B. (2008). Studying the anti-tyrosinase effect of Arbutus andrachne L. extracts. Int. J. Cosmet.

Sci, (30), 271–276.

Ivanov, S. A., Nomura, A., Malfanov, I. L., Sklyar, I. V., & Ptitsyn, L. R. (2011).

Isolation of a novel catechin from Bergenia rhizomes that has pronounced lipase-inhibiting and antioxidative properties. Fitoterapia, (82), 212–218.

Karam N.S., Fawzia M.J., Arikat, N. A., & Shibli, R. A. (2003). Growth and rosmarinic acid accumulation in callus, cell suspension, and root cultures of wild Salvia fruticosa. Plant Cell, Tissue and Organ Culture, (73), 117–121.

Karam, N. S., & Al-Salem, M. M. (2001).

Breaking dormancy in Arbutus andrachne L. seeds by stratification and gibberellic acid. Seed Science and Technology, 29, 51–56.

Karuppusamy, S. (2009). A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. Journal of Medicinal Plants Research, 3(13), 1222–1239.

Kose, H. (1998). Studies on the germination of some woody ornemental plants existing in Turkish Flora. 1. Arbutus unedo L. and Arbutus andrachne L. Anadolu. .

Journal of Aegean Agricultural Research Istitute, 8, 55–65.

Mostafa S.E., Karam N.S., Shibli R. A., & Alali, F. Q. (2010). Micropropagation and production of arbutin in oriental strawberry tree (Arbutus andrachne L.).

Plant Cell Tissue and Organ Culture, 103(111–121).

Narula, A., Kumar, S., Bansal, K. C., & Srivastava, P. S. (2004).

Biotechnological approaches towards improvement of medicinal plants. In: Srivastava, P. S., Narula, A., Srivastava, S. eds. Plant Biotechnology and Molecular Markers.

Anamaya Publishers. New Delhi., 78–116.

ROTEM. (2002). Constructing Red Number for endangered plant species. Israel flora as a test case.

Ruidavets, J. B., Teissedre, P. L., Ferrie`res, J., Carando, S., Bougard, G., & Cabanis, J. C. (2000).

Catechin in the Mediterranean diet: vegetable, fruit or wine?. Atherosclerosis, 153(107–117).

Said, O., Khalil, K., Fulder S., & Azaizeh, H. (2002). Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region.

Journal of Ethnopharmacology, 83, 251–265.

Sakar, M. K., Berkman, M. Z., Calis, I., & Ruedi, P. (1991). Constituents of Arbutus andrachne L. Fitoterapia, 62(2), 176–177.

Serce, S., Ozgen M, Torun, A.A, & Ercis, L.S. (2010).

Chemical composition, antioxidant activities and total phenolic content of Arbutus andrachne L. (Fam. Ericaceae) (the Greek strawberry tree) fruits from Turkey. Journal of Food Composition Analysis, 23(6), 619–623.

Shatnawi, M., Al-Fauri, A., Megdadi, H., AlShatnawi, M.K., Shibli, R., AbuRomman, S. & Al-Ghzawi, A. (2010).

In Vitro multiplication of Chrysanthemum morifolium Ramat and it is responses to NaCl induced salinity.

Jordan Journal of Biological Sciences, 3, (3), 101–110.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 196–205

Suzuki, T., Someya, S., Hu, F., & M., T. (2005). Comparative study of catechin compositions in five Japanese persimmons (Diospyros kaki). . Food Chemistry, 93, 149–152.

Tilki, F., & Guner, S. (2007). Seed germination of three provenances of Arbutus andrachne L in response to different pretreatments, temperature and light.

Propagation of Ornamental Plants 7(4), 175–179.

Source of Support: Deanship of Graduate Studies and Scientific Research, Palestine

Yadav, R. N. S., & Agarwala, M. (2011).

Phytochemical analysis of some medicinal plants. Journal of Phytology, 3, 10–14.

Wagner H, & Bladt S, (2009). Plant Drug Analysis: A Thin Layer nd Chromatography Atlas. 2 ed., Springer-Verlag, Berlin Heidelberg.

Zaveri, N. T. (2006). Green tea and its polyphenolic catechins: Medicinal uses in cancer and noncancer applications.

Life Sciences, 78, 2073–2080.

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206â&#x20AC;&#x201C;214 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review Article ANDROGRAPHIS PANICULATA A TRADITIONAL HERB WITH PHARMACOLOGICAL PROPERTIES: A REVIEW Nishan Chatterjee1, Sunipa Biswas2, Nimai Chandra Saha3, Surjyo Jyoti Biswas4* 1,2,4

Post Graduate Department of Zoology, Midnapore College, Midnapore, West Bengal, India-721101.

Education Directorate, Government of West Bengal, Bikash Bhavan, Salt Lake, Kolkata, West Bengal India700091. *Corresponding author: E-mail: surjyo@rediffmail.com 3

Received: 14/03/2014; Revised: 20/04/2014; Accepted: 30/04/2014

ABSTRACT Andrographis paniculata (Kalmegh) is herb belonging to family Acanthaceae and cultivated widely in India. This plant is known for its wide range of pharmacological properties and various traditional uses. Considerable efforts are being made by various scientists to validate its utility through scientific and pharmacological screening. The reported biological activities are antibacterial, antifungal, antiviral, anthelmintic, anticancer, hyperglycemic, anti-inflammatory, antivenomic, antiasthmatic, hepatoprotective and used to treat cold and fever. This review intends to integrate traditional knowledge, summarizes modern scientific findings and suggests areas where further research can be conducted.

KEYWORDS: Andrographis paniculata, Pharmacological properties, Kalmegh

Cite this article: Nishan Chatterjee, Sunipa Biswas, Nimai Chandra Saha, Surjyo Jyoti Biswas (2014), ANDROGRAPHIS PANICULATA A TRADITIONAL HERB WITH PHARMACOLOGICAL PROPERTIES: A REVIEW, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 206â&#x20AC;&#x201C;214

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206–214

INTRODUCTION Andrographis paniculata (AP) commonly known as Kalmegha in Hindi, Kalamegha in Sanskrit and Kalmegh in Bengali is an erect herb belonging to family Acanthaceae which grows in many South east Asian countries and in India. Other vernacular names of AP have been listed in table 1. The plant is highly praised for its therapeutic potential in Indian phytotherapy and traditional medicine. Both crude and alcoholic extracts of AP have been reported to have wide variety of pharmacological activities viz. antibacterial, antifungal, antiviral, anthelmintic, anticancer, hyperglycemic, anti-inflammatory, antivenomic, in alleviation of upper respiratory tract infections, hepatoprotective, preventive effects against cold (Dey et al., 2013, Datta et al., 2012, Coon and Ernst, 2004, Akbar, 2011).

The herb grows upto 3–4 feet in height, the leaves are lanceolate and 2–3 inches long. The

flowers are small, solitary and flowering time is from September to December. Traditionally the leaves of this herb are used for bronchitis, worm infestation, influenza and dyspepsia. The expressed juice of the leaves is a domestic remedy in flatulence and diarrhoea. This plant is also known as ‘King of bitters’ which was used for centuries both in Indian and Chinese systems of medicines where both fresh and dried leaves and roots were used as folkloric medicines and traditional household remedies.

Traditionally this plant was used as powder, raw juice, decoction either singly or in combination with other plant extracts for various types of ailments. There is need for critical evaluation since few scientists have reported side effects of AP (Akbar, 2011). This review is intended to give a view mainly on the biological activities of AP, the compounds isolated, their pharmacological properties, clinical and laboratory investigations and their safety evaluation.

Table 1: Vernacular names of Andrographis paniculata Sl.No.

Vernacular Names

1.

Sanskrit:

Bhunimba

2.

Hindi:

Kirayat

3.

Bengali:

Kalmegh

4.

Telugu:

Naelavemu

5.

Tamil:

Nilavaembu, Siriyanangai

6.

Oriya:

Bhuinimba

7.

Chinese

ChuanxinLian

8.

Kannada

Nelabevu

9.

Thai

FaThalai Chon

10.

Malay

HempeduBumi

11.

Punjabi

Chooraita

Phytochemical studies: It has been reported by various investigators that AP contains lactones, diterpenes, alkanes, ketones and aldehydes and flavonoids. Though flavonoids are mainly

found in roots but the aerial parts are predominant in alkanes, aldehydes and ketones.

The intense bitter taste of the leaves is due to presence of large amounts of kalmeghin and andrographolide. Deoxyandrographolide, 19β-

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206–214

D-glucoside has been isolated from leaves of AP (Weiming and Xiaotian, 1982). 12 new flavonoids and 14 new diterpenoids have been isolated from the aerial parts of AP (Chen et al., 2006a, Chen et al., 2006b). Li et al., (2007) isolated andrographic acid which is a new diterpenoid and two new ent-labdane diterpenoid glycosides from the aerial parts of the plant. On the other hand 1, 8-dihydroxy-3, 7-dimethoxyxanthone, 4,-8-dihydroxy-2, 7dimethoxyxanthone, 1, 2-dihydroxy-6,8 dimethoxyxanthone and 3-7- 8 trimethoxy-1hydroxyxanthone were isolated from roots ( Dua et al., 2004, Akbar, 2011).

Medical formulations There are many formulations commercially available in the market both nationally and internationally, where the major constituent is Andrographolide. They are Bhunimbadi khada, kalansundarras, kalpataruras, Andrographis 200 and 400, Liv 52, andrographis 60V caps, Ilogen excel etc (www.iherbs.com, www.ayurvedicherbs.com, www.paradiseherbs.com).

Pharmacological activities Antimicrobial effect: It has been experimentally proved that crude powder from the aerial parts of the plant shows no antimicrobial activity but it’s aqueous extract of leaves exhibit significant antimicrobial activity against Gram positive S. aureus, and Gram negative Pseudomonas aeruginosa. Mishra et al., (2009) found that the IC50 of methanol extract was 7.2microgram/ml against Plasmodium falciparum.

Andrographolide, neoandrographolide, 14deoxy-11, 12-didehydro andrographolide showed antiviral activity against herpes simplex virus or HSV1 (Wiart et al., 2005).

Antiprotozoal and antihelmenthic property Dua et al., (2009) reported antiprotozoal activity of some xanthones isolated from roots of AP, xanthones reduced the growth of T. brucei, T. cruzi and L. infantum. Further, it has been reported by other workers that water

extract of the leaves of AP showed significant filaricidal properties in canines (Dutta and Sukul 1982). Kaleysa 1975 reported significant anti-helminthic action of AP against Ascaris lumbricoides.

Immunomodulatory properties Puri et al., (1993), reported several immuno-stimulatory agents from AP. According to Puri intragastric administration of ethanolic extract of aerial parts of AP at a rate of 1 mg/kg body weight to mice stimulated antibody production, it was also responsible for delayed hypersensitivity reaction in sheep red blood cells. The whole extract of AP was more effective than andrographolide or neo andrographolide alone suggesting other constituents may involve in the immunostimulant response/process. Sheeja et al., (2006) showed that administration of cyclophosphamide increased cytokine TNFα which was considerably reduced with administration of AP extract.

Anti-inflammatory activities Chiou et al., (2000) reported suppression of inducible NO synthase (iNOS) by andrographolide. Other investigators reported inhibitory effects of neoandrographolide on prostaglandin E2 and NO production in LPS stimulated murine macrophage (Liu et al., 2007a, Liu et al., 2007b, Abu-Ghefreh et al., 2009). Suppression of NO production in activated macrophages in vitro and ex vivo by neoandrographolide isolated from AP was also reported by Batkhuu et al., (2002). Methanolic extract of this plant inhibited formation of ROS (reactive oxygen species) which completely inhibited carrageenan induced inflammation as reported by Sheeja et al., (2006).

Antioxidant properties Since this plant contains higher flavonoid and phenolic content, this might attribute to its antioxidant capabilities. It has been reported that suppression of rat neutrophil ROS production by diterpenoid lactone andrographolide (Shen et al., 2000, Shen et al., 2002). Verma and Vinayak (2008) reported

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206â&#x20AC;&#x201C;214

antioxidant action of AP on lymphoma. It has been reported that activities of mitochondrial electron transport chain complexes were decreased in different brain parts by pretreatment with water and ethanolic extracts of AP (Das et al., 2009). Ojha (2009) reported antioxidant activity of AP in ischemic myocardium of rats. It might be that the neoandrographolide present in the plant extract take up the free oxygen radical due to the donating activity of allylic hydrogen of the unsaturated lactone ring.

Effects on Reproductive system The therapeutic efficacy of AP needs critical evaluation because there are many reports that the extract of this plant affects the reproductive system. Some studies reported that AP extract may be used as an antifertility agent. When dry leaf powder were administered to male albino rats there was a decrease in spermatogenesis, increased abnormal sperm head morphology, decreased sperm motility, regression of cells of Leydig, degeneration of epididymis and prostrate (Akbarsha et al., 1990). It has also been reported that intra-peritoneal injection of aerial parts of AP to female albino mice caused abortion and also it prevented implantation of embryos. Panossian et al., (1999) reported that treatment with extract of AP did not alter progesterone levels in pregnant rats. Since experimental investigations contradicted each other there is ample scope of research in this direction.

Cardiovascular activity The aqueous extract of AP lowers the systolic blood pressure of spontaneously hypertensive rats possibly by reducing circulating angiotensin converting enzyme in the plasma as well as by reducing free radical levels in the kidneys (Zhang and Tan 1996). A hypotensive activity of AP in rats was also reported by Yu et al., (2003). Further, studies

by Zhang et al., (1998) and Zhang and Tan (1997) on cardiovascular activity of 14deoxyandrographolide and 14-deoxy-11, 12didehydroandrographolide revealed that AP

induced relaxation of the isolated rat thoracic aortae. They also reported that 14deoxyandrographolide and 14-deoxy-11,12didehydroandrographolide significantly decreased the mean arterial pressure and heart rate of anaesthetized rats.

Antivenomic activities It has been reported by Chang and But (1987) that 10 cases of viper bites were cured within 3-5 days by a formula which has AP as primary constituent. It has been demonstrated

by Premendran et al., (2011) that AP has cobra venom neutralizing activity at dose of 2 g/kg.

Further, methanolic extracts of AP has potent Daboia russelli venom neutralizing activity and could be used as snakebite evenomation (Meenatchisundaram et al., 2009). Kale et al., 2013 reported anti-scorpion venom activity of AP when treated with 1g/Kg dose.

Effect on respiratory infections Upon literature survey it was revealed that there were contradictory reports of AP administration against respiratory infections.

Thamlikitkul et al., (1991) administered AP extract to cure cough and sore throat and compared it with paracetamol administration; they found that 6g AP powder administration for 3 days reduced sore throat and fever considerably. In a double blind study conducted

by Caceres et al., (1997) revealed that common cold was prevented by 4% andrographolide.

Effect on hepatic enzymes A crude extract of A. paniculata was recently found to induce hepatic CYP1A1 and CYP2B expression, based on observations of significant increases in ethoxyresorufin Odealkylase (EROD) activity and pentoxyresorufinO-dealkylase activities (Jarukamjorn et al.,2006). Interestingly, andrographolide plus typical CYP1A inducers, including-naphthoflavone, TCDD, and benz[a]anthracene, synergistically induced CYP1A1 expression in mouse hepatocytes in primary culture, and the synergism was blocked by an AhR antagonist, resveratrol (Jaruchotikamol et al.,2007).

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206â&#x20AC;&#x201C;214

Antidiabetic activity It has been reported by Umamaheswari et al., (2009) that oral administration of an ayurvedic formulation of AP considerably reduces streptozotocin induced diabetes mellitus. Further ethanolic leaf extract of this plant reduces oxidative stress and also have antihyperglycaemic properties.

The antihyperglycaemic properties have been attributed to andrographolide which is present in leaves of AP in high amounts (Yu et al., 2003). Antidiabetic property of aqueous extract of AP was further confirmed by Borhanuddin et al., (1994) and Husen et al., (2004).

Insecticidal activity The ethanolic extracts of AP have been reported to have insecticidal property which was mainly attributed to andrographolides, homoandrographolide and andrographin. A study conducted by Elango et al., (2010) revealed that hexane fractions of AP have potent larvicidal, ovicidal and insect repellent properties.

Anticancerous property Zhou et al., (2006) reported that andrographolide induced cell death which is proapoptotic via Bcl2. Andrographolide induced DNA fragmentation and enhanced percentage of apoptotic cells has been reported

by Harjotaruno et al., (2007), in TD-47 human breast cancer cell line by increasing expression of p53, Bax and caspase 3 in a concentration dependent manner and also lowering the expression of Bcl2. Satyanarayana et al., (2004) reported cell cycle inhibition in MCF-7 cells which are human breast cancer cell line by decreasing the expression of p27 and also cyclin dependent kinase. Zhou et al., (2010) demonstrated that Andrographolide is able to significantly suppress both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Such inhibition is found to be achieved through suppression of Janus activated kinase (JAK)1/2 and interaction between STAT3 and gp130. They also

investigated the effect of andrographolide on doxorubicin-induced apoptosis in human cancer cells. Their observation revealed that andrographolide could be a potential candidate for treatment of cancer with combination with other chemotherapeutic agents.

Adverse health effects It has been reported that high doses of AP extract causes gastric discomfort, vomiting, head reeling and anaphylactic effects. When being administered with anticoagulants, there may be increased risk of bruising and bleeding since Andrographis itself inhibits the platelet aggregation and prevents blood clots (Calabrese et al., 2000).

CONCLUSION Kalmegh was used in Indian systems of medicines for centuries. Literature survey showed that extensive works has already been done worldwide with regard to its efficacy against various types of ailments. However it would be prudent to investigate its constituents singly and in combination against various other diseased states, how they modulate pathological changes and which form is more potent or effective in treating disease. AP showed consistent hepatoprotective effects in animal models against various types of induced hepatotoxicity however inconsistency was found against bacterial investigations. This might be due to collection of plant materials at different time intervals, place of collection, seasonal variation, different extraction procedures, and its storage which might affect its active compounds both quantitatively and qualitatively. Antihyperglycemic activity of AP extracts (both water and alcohol) was found to be even better than commonly available antidiabetic drugs in animal models. Further, it shows a profound impact on blood pressure thereby making it a good candidate for understanding of its constituents on blood pressure and its regulation. Effect of AP extracts on reproductive system has been carried out in animal model by some investigators however till date its efficacy on human reproductive system or on nervous

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206–214

system has not been carried out, so focus can be generated on these aspects for better drug development. The information summarizes here concerning Andrographis paniculata is intended to serve as a reference and update researchers with adequate information involved in ethno-pharmacological research.

ACKNOWLEDGEMENT Grateful acknowledgements are made to Professor A. R Khuda-Bukhsh, Department of Zoology, University of Kalyani and Dr. Prabir De, Scientist, CCMB, Hyderabad for inspiration and advice.

REFERENCES Abu-Ghefreh AA, Canatan H, Ezeamuzie I (2009). In vitro and in vivo antiinflammatory effects of andrographolide.

International Immunopharmacol. 9: 313–18.

Calabrese C, Berman SH, Babish JG (2000). A phase I trial of andrographolide in HIV positive patients and normal volunteers.

Phytother Res 14:333–8.

Akbar S (2011). Andrographis paniculata: A review of pharmacological activities and clinical effects.

Altern. Med. Review 16 (1): 66–77.

Chandrasekaran CV, Gupta A, Agarwal A (2010). Effect of an extract of Andrographis paniculata leaves on inflammatory and allergic mediators in vitro. J Ethnopharmacol.129 203–7.

Akbarsha MA, Manivannan B, Hamid K S, Vijayan B (1990). Antifertility effect of Andrographis paniculata (nees) in male albino rat. Ind J. Exp. Biol 28: 421–6.

Chang HM, But PPH (1986). Pharmacology and applications of Chinese material medica. World Scientific Publishing Co. Ple. Ltd, Singapore, 1, 918–928.

Batkhuu J, Hattori K, Takano F, Fushiya S, Oshiman K, Fujimiya Y (2002).Suppression of NO production in activated macrophages in vitro and ex vivo by Neoandrographolide isolated from Andrographispaniculata. Biol.

Pharm. Bull. 25 (9) 1169–74.

Chen LX, Qu GX, Qiu F (2006a). Studies on diterpenoids from Andrographis paniculata. Zhongguo Zhong Yao Za Zhi 31: 1594–7.

BorhanuddinM, Shamsuzzoha M, Hussain AH (1994). Hypoglycaemic effects of Andrographis paniculata Nees on nondiabetic rabbits. Bangladesh Med. Res. Council Bull 20:24–6.

Caceres DD, Hancke JL, Burgos RA, Wikman GK (1997). Prevention of common colds with Andrographis paniculata dried extract: A pilot double blind study. Phytomed. 4: 101–4.

Chen LX, Qu GX, Qiu F. (2006b). Studies on flavonoids of Andrographis paniculata.

Zhongguo Zhong Yao ZaZhi 31: 391–5.

Chiou,

WF, Chen CF, Lin JJ (2000).

Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells by andrographolide.

British J Pharmacol. 129: 1553–60.

Coon JT, Ernst E (2004). Andrographis paniculata in the treatment of upper respiratory tract infections: A systematic review of safety and efficacy. Planta Med. 70: 293–8.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206–214

Das S, Gautam N., Dey SK, Maiti T, Roy S (2009). Oxidative stress in the brain of nicotine-induced toxicity: protective role of Andrographis paniculata Nees and vitamin E. Applied Physiol. Nutr.

Metabolism 34(2): 124–35.

Dua VK, Ojha VP, Roy R (2004). Antimalarial activity of some xanthones isolated from the roots of Andrographis paniculata. J Ethnopharmacol 95:24751.

Dua VK, Verma G, Dash AP (2009). In vitro antiprotozoal activity of some xanthones isolated from the roots of Andrographis paniculata. Phytother Res. 23:126-8.

Dey YN, Kumari S, Ota S, Srikanth N (2013).

Phytopharmacological review of Andrographis paniculata (Burm.f) Wall. exNees. Int. J. Nutr. Pharmacol.

Neurol. Diseases . 3(1): 3–10.

Dutta

A, Sukul NC (1982). Filaricidal properties of a wild herb, Andrographis paniculata .J Helminthol 56 : 81–4.

ElangoG, RahamanAA, Kamaraj C, Zahir AA, BagavanA (2010). Studies on effects of indigenous plant extracts on filarial vector Culex tritaeniorhynchus Giles.

Parasitol. Res 107: 167–176 Ghosh BK, Datta AK, Mandal A, Dubey PK, Haldar S (2012). An overview on Andrographis paniculata. Int J Res Ayur Pharm. 3(6) 752–60.

Harjotaruno S, Widyawaruyanti A, Sismindari, Zaini NC (2007). Apoptosis inducing effect of Andrographolide on TD-47 Human breast cancer cell line. Afr J. Trad. Complement Altern Med. 4(3): 345–51.

Husen R, Pihie AH, Nallappan M (2004).

Screening for antihyperglycaemic activity in several local herbs of Malaysia. J Ethnopharmacol 95:205-8.

Jaruchotikamol A, Jarukamjorn K, Sirisangtraku W, Sakumaa T, Kawasakia Y, Nemotoa N (2007).

Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes. Toxicol.

Applied Pharmacol 224(2):156–62 Kale RS, Bahekar S E, Nagpure SR, Salwe KJ (2013). Anti-scorpion venom activity of Andrographis paniculata: A combined and comparative study with antiscorpion serum in mice. Ancient Sci Life 32: 156–60 Kaleysa RR. (1975). Screening of indigenous plants for anthelmintic action against human Ascarislumbricoides . Part I. Indian J Physiol Pharmacol 19: 47-9.

Li J, Huang W, Zhang H, Wang X, Zhou H (2007). Synthesis of andrographolide derivatives and their TNF-alpha and IL6 expression inhibitory activities.

Bioorganic Medicinal Chem. Letters 17: 6891–4.

Liu J, Wang ZT, Ji LL (2007). In vivo and in vitro anti-inflammatory activities of neoandrographolide. Am J. Chin. Med 35: 317-28 Meenatchisundaram S, Parameswari G, Subbraj T, Suganya T, Michael A (2009).

Medicinal and pharmacological activities of Andrographispaniculata – Review.Ethnobotanical Leaflets 1:6.

Mishra K, Dash AP, Swain BK, Dey N (2009). Anti-malarial activities of Andrographis paniculata and Hedyotiscorymbosa extracts and their combination with curcumin. Malar J. 8:26.

Ojha SK, Nandave M., Kumari S. Arya D.S. (2009) Antioxidant Activity of Andrographis paniculata in ischemic myocardium of rats Global J.Pharmacol 3 (3): 154–7.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206–214

Panossian A, Kochikian A, Gabrielian E, Muradian R, Stepanian H, Arsenian F, Wagner H (1999).Effect of Andrographis paniculata extract on progesterone in blood plasma of pregnant rats. Phytomed. 6(3): 157-61.

Thamlikitkul V, Dechatiwongse T, Theerapong S, Chantrakul C, Boonroj P, Punkrut W (1991). Efficacy of Andrographis paniculata, Nees for pharyngotonsillitis in adults. J Med. Assoc. Thai. 74: 437–42.

Premendran SJ, Salwe KJ, Pathak S, Brahmane R, Manimekalai K (2011). Anti-cobra venom activity of plant Andrographis paniculata and its comparison with polyvalent anti-snake venom. J Nat Sci. Biol. Med. 2(2):198-204.

Umamaheswari S, Joseph LD, Srikanth J, Lavanya RD, Reddy C, Uma Maheswara C. (2009) Anti-diabetic activity of a polyherbal formulation (DIABET). Int J Pharmaceut Sci. 2: 18– 22.

Puri A, Saxena R, Saxena RP, Saxena KC, Srivastava V, Tandon JS (1993).

Immunostimulant agents from Andrographis paniculata. J. Nat. Prod 56: 995-9.

Verma N, Vinayak M. (2008). Antioxidant action of Andrographis paniculata on lymphoma. Mol Biol Rep. 35(4): 535– 40.

Satyanarayana C, Dhanavanthri S Deevi, Rajagopalan R, Srinivas N, Rajagopal S (2004). DRF 3188 a novel semisynthetic analog of andrographolide: cellular response to MCF 7 breast cancer cells. BMC Cancer 4:26 doi:10.1186/1471-2407-4-26 Sheeja K, Kuttan G. (2006). Ameliorating effects of Andrographis paniculata extract against cyclophosphamideinduced toxicity in mice. Asian Pac J Cancer Prev 7: 609-14.

Sheeja K, Shihab PK, Kuttan G. (2006).

Antioxidant and anti-inflammatory activities of the plant Andrographis paniculata Nees. Immunopharmacol Immunotoxicol 28: 129-40.

Shen YC, Chen CF, Chiou WF (2002).

Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect.

British J Pharmacol. 135: 339–406.

Weiming C and Xiaotian L (1982).

Deoxyandrographolide 19-beta-Dglucoside from the leaves of Andrographis paniculata.Planta Med. 45:245-46 Wiart C, Kumar K, Yusof MY, Hamimah H, Fauzi ZM, Sulaiman M (2005) Antiviral properties of ent-labdenediterpenes of Andrographis paniculata Nees, inhibitors of herpes simplex virus type 1. Phytother Res 19:1069–70.

Yu BC, Hung CR, Chen WC (2003). Antihyper glycemic effect of andrographolide in streptozotocin-induced diabetic rats.

Planta Med. 69(12): 1075–1079 Zhang CY, Kuroyangi M, Tan BK (1998) Cardiovascular activity of 14-deoxy11,12-didehydroandrographolide in the anaesthetized rat and isolated rat atria.

Pharmacol Res 38: 413–7.

Zhang CY, Tan BK (1996) Hypotensive activity of aqueous extract of Andrographis paniculata in rats. Clin Exp Physiol Pharmacol 23: 675–678.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 206–214

Zhang CY, Tan BK (1997) Mechanisms of cardiovascular activity of Andrographis paniculata in the anaesthetized rat. J Ethnopharmacol 56: 97–101 Zhou J, Ong C, Hur GM, Shen HM (2010).

Inhibition of the JAK-STAT3 pathway by andrographolide enhances chemosensitivity of cancer cells to

Source of Support: NIL

doxorubicin. Biochemical Pharmacol 79: 1242–50.

Zhou J, Zhang S, Ong CN, Shen HM (2006).

Critical role of pro-apoptotic Bcl-2 family members in andrographolide induced apoptosis in human cancer cells. Biochem Pharmacol. 72: 132–44.

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215–224 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research Article PHARMACOGNOSTICAL EVALUATION ON TANNIN CONTENT IN HARITAKI LEAVES (TERMINALIA CHEBULA RETZ. -COMBRETACEAE) BEFORE AND AFTER FLOWERING-FRUITING Patil Sunny C1*, Harisha C R2, Baghel A S3, Dwivedi R R4 1

PG Scholar, Department of Basic Principles, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 361 008 2 Head, Pharmacognosy Lab, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 361 008 3 Associate Professor, Department of Basic Principles, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 361 008 4 Professor and Head, Department of Basic Principles, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 361 008 *Corresponding Author: Email: sunnypatil125@gmail.com Mob: +919067965954

Received: 14/03/2014; Revised: 25/04/2014; Accepted: 02/05/2014

ABSTRACT Haritaki (Terminalia chebula Retz., Fam. Combretaceae) is one of the most abundantly used plant for various ailments in Ayurvedic classics. In folklore practice, it is used in the treatment of asthma, hiccough, sore throat, vomiting, bleeding piles, diarrhea, dysentery, heart and bladder diseases etc. Though whole plant of Haritaki possesses high medicinal value, fruit is the part which is used most in therapeutics. The medicinal efficacy of Haritaki can be attributed to various chemical constituents present in it. Tannin is one of these, which is responsible for various therapeutic properties of Haritaki. The present work is carried out to study the tannin content and its localization in Haritaki leaves before and after flowering–fruiting in which it was observed that leaves of Haritaki before flowering-fruiting are having more concentration of tannin than after floweringfruiting leaves. It was also noted that fruit of Haritaki which is a totally new entity formed in the plant shows recognizable amount of tannin content in it. This work may provide incentive for proper evaluation of the chemical constituents which exist there in the leaves and later get transported to the fruits.

KEY WORDS: Haritaki, Medicinal value, Pharmacognosy, Tannin, Terminalia chebula Retz.

Cite this article: Patil Sunny C, Harisha C R, Baghel A S, Dwivedi R R (2014), PHARMACOGNOSTICAL EVALUATION ON TANNIN CONTENT IN HARITAKI LEAVES (TERMINALIA CHEBULA RETZ. -COMBRETACEAE) BEFORE AND AFTER FLOWERING-FRUITING, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 215–224

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215–224

INTRODUCTION Medicinal plants are being used for combating various human ailments since the dawn of civilization. Ayurveda is an applied science related to medicine in which foremost emphasis has been given to the treatment of diseases using various medicinal herbs.

Haritaki (Terminalia chebula Retz., Fam. Combretaceae) is one of the most extensively used plants described in most of the ancient classics of Ayurveda. Owing to their natural origin, cost effectiveness and lesser side effects, there has been growing interest in exploiting the biological activities of different Ayurvedic medicinal herbs since last one or two decades.

T. chebula has been extensively used not only in Ayurveda but also in Siddha (S. Aruna et al., 2014), Unani, Homoeopathic medicine and has become a cynosure of modern medicine (Anwesa Bag et al., 2013). Because of its extraordinary power of healing, it is known as ‘King of Medicines’ (Kshirod K. Gupta et al., 2013) and is always listed at the top of the list of ‘Ayurvedic Materia Medica’.

Therapeutic efficacy of this plant can be attributed to the large number of different types of phyto-constituents present in it. It has been demonstrated to possess multiple pharmacological and medicinal activities, such as antioxidant, antimicrobial, antidiabetic, antiarthritic, anti-inflammatory, antimutagenic, antiproliferative, hepato-protective, radioprotective, cardio-protective, gastrointestinal motility and wound healing activity (Anwesa Bag et al., 2013). Fruit of T. chebula, commonly known as ‘Harad’ in Hindi, is the part used for medicinal purpose which possesses diverse health benefits. It has been used as traditional medicine for household remedy against various human ailments since antiquity. The observed health benefits of this fruit may be credited to the presence of various phytochemicals like tannin, polyphenols,

terpenes, anthocyanins, flavonoids, alkaloids and glycosides (Anonymous, 2010).

Tannin is one of the important constituents responsible for medicinal efficacy of the fruit.

Thus in this study a detailed pharmacognostical examination of T. chebula leaves before and after flowering–fruiting has been carried out with the hypothesis that the leaves of Haritaki before flowering would have more concentration of tannin than after floweringfruiting leaves. This study may offer immense opportunity for researchers engaged in validation of the active ingredients present in the medicinal plant and also contribute to know the actual plant physiology about the transportation of secondary metabolites.

MATERIALS AND METHODS Collection and preservation of the sample Fresh leaves of Terminalia chebula Retz. were collected from the botanical garden of Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurveda University (GAU), Jamnagar in the month of April 2013 and November 2013 before and after flowering-fruiting respectively as per collection standards (API, 2001). Fruits were also collected from the same location. The plant specimen was authenticated by the Pharmacognosist, GAU, Jamnagar, India. A sample specimen was deposited to Pharmacognosy Lab. (Specimen No.- Phm. 6123/2013) for future references. Leaves and fruits were separated, shade dried, pulverized, sieved through 80 no. mesh and preserved in an air tight glass bottle.

Macroscopic study Macroscopic characters of leaves before and after flowering–fruiting and those of fruit were analyzed systematically and their morphological characters like size, shape etc. were noted down.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215–224

Organoleptic study of dried powders Dried powders of both the sample leaves and of fruit were evaluated for organoleptic characters like texture, taste, odour and colour etc.

Microscopic study Free hand transverse sections of leaves through midrib including its petiole and of fruit were taken and washed with chloral hydrate solution. Sections were first observed in distilled water then stained with phloroglucinol and concentrated HCl (Khandelwal K.R et al., 1996).

Histochemical analysis Histochemical analysis was carried out to determine the tannin content and its localization in both the samples of leaves as well as in fruit (Krishnamurty KV, 1988).

Powder microscopy Dried powders of leaves and fruit were studied following standard procedures (Trease GE et al., 2002). The micro photographs were taken by Carl zeiss trinocular microscope.

RESULTS Macroscopical study Leaves Macroscopic investigation of both the leaf samples showed similar characteristics. Leaves were 10–20 cm long, sub-opposite, simple; exstipulate; petiolate; laminae broadly elliptic to elliptic-oblong, rarely ovate, the bases obtuse, the margins entire, the tips acute, glabrescent, with two large glands at the top of the petiole. (Plate A, a–b) Fruit Single seeded drupe of Haritaki was glabrous, sub globose to ellipsoid in shape, smooth and golden yellow in colour. It became ridged, wrinkled and brown when dried. (Plate A, c–d) Organoleptic study of dried powders of leaves and fruit Organoleptic study of powders of both the leaf samples was carried out to see any difference in the organoleptic characters of leaf before and after flowering-fruiting. Characters of fruit powder were also observed. Results are tabulated in Table-1.

TABLE 1: Organoleptic characters of dried powders of T. chebula fruit and leaves before and after flowering-fruiting

Sr. No. 1 2

Parameter Colour Odour

Leaf: before flowering-fruiting Light greenish Characteristic

Leaf: after flowering-fruiting Light yellowish Characteristic

3 4

Taste Texture

Astringent Rough

Less astringent Rough

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Fruit Yellowish brown Aromatic, slightly astringent Astringent, sweet Rough

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215â&#x20AC;&#x201C;224

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215â&#x20AC;&#x201C;224

Microscopical study Histological and histochemical evaluation of petiole before flowering-fruiting T.S of the petiole was more or less circular in shape, showed epidermis, cortex, vascular bundles and central pith. (Plate C) Epidermis was single layered, made up of barrel shaped cells arranged compactly and

covered with thick cuticle. Hypodermis was present just beneath the epidermis, made up of compactly arranged single layered collenchyma cells. Hypodermal cells were rich in tannin content filled with brownish material. Cortex consisted of parenchymatous cortical cells enriched by chlorophyll pigments and rosette crystals of calcium oxalate and some of the cortical cells were filled with brown content

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215–224

(tannin). Endodermis was single layered, consisted of some rosette crystals. Continuous bands of pericyclic fibers were present beneath the endodermis followed by distinct vascular bundles. Adjacent cells of the vascular bundles were found to be greatly filled with tannin content. Vascular bundles were open and collateral in which phloem was present beneath the pericyclic fibers consisting of sieve elements and phloem fibers while xylem was radially arranged beneath the phloem along with xylem parenchyma and xylem fibers.

Centrally located large pith showed presence of parenchymatous cells showing some starch grains and also filled with some tannin content.

Histological and histochemical evaluation of leaf before flowering-fruiting T.S of the leaf through mid rib showed distinct upper and lower epidermis and centrally located vascular bundles (Plate C).

Upper and lower epidermis was single layered, covered with the cuticle; lower epidermis was interrupted by stomata. Beneath the upper epidermis, 2–3 layers of pallisade parenchyma and beneath the lower epidermis 6–7 layers of spongy parenchyma were present.

Upper epidermis i.e. Pallisade cells were strongly filled with the tannin content as compared to the lower spongy parenchyma.

The mesophyll tissue consisted of large quantity of oil globules and prismatic crystals.

Mesophyll, upper pallisade parenchyma and lower spongy parenchyma were separated by vascular trends. Vascular bundles were situated at the centre of the midrib, made up of phloem situated towards lower epidermis, xylem situated towards upper epidermis along with few elements of xylem parenchyma cells.

Ground tissue of the midrib portion i.e. parenchyma cells were strongly filled with tannin content, heavily at adjacent cells of the vascular bundles. Some of the central pith parenchyma cells were enriched by tannin content.

Histological and histochemical evaluation of petiole after flowering-fruiting T.S. of the petiole was more or less circular in shape, showed epidermis, cortex, vascular bundles and central pith. (Plate C) Epidermis was single layered, made up of barrel shaped cells arranged compactly and covered with thick cuticle. Hypodermis was present just beneath the epidermis, made up of compactly arranged single layered collenchyma cells. Hypodermal cells were showing less localization of tannin. Cortex consisted of parenchymatous cortical cells enriched by chlorophyll pigments and rosette crystals of calcium oxalate. Very poor amount of brown content (tannin) was noted in the cortical cells.

Endodermis was single layered consisting of some rosette crystals. Continuous bands of pericyclic fibers were present beneath the endodermis followed by distinct vascular bundles. Adjacent cells of the vascular bundles showed little concentration of tannin content.

Vascular bundles were open and collateral in which phloem was present beneath the pericyclic fibers consisting of sieve elements and phloem fibers, while xylem was radially arranged beneath the phloem along with xylem parenchyma and xylem fibers. Centrally located large pith was having parenchymatous cells showing some starch grains and also filled with some tannin content which was not so prominent.

Histological and histochemical evaluation of leaf after flowering-fruiting T.S of the leaf through mid rib showed distinct upper and lower epidermis and centrally located vascular bundles (Plate C).

Upper and lower epidermis was single layered, covered with the cuticle in which lower epidermis was interrupted by stomata.

Beneath the upper epidermis 2–3 layers of pallisade parenchyma and beneath the lower epidermis 6–7 layers of spongy parenchyma were present. Upper epidermis i.e. pallisade cells were showing slightly more tannin content as compared to the lower spongy parenchyma.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215â&#x20AC;&#x201C;224

The mesophyll tissue consisted large quantity of oil globules and prismatic crystals.

Mesophyll, upper pallisade parenchyma and lower spongy parenchyma were separated by vascular trends. Vascular bundles were situated at the centre of the midrib, made up of phloem situated towards lower epidermis, xylem

situated towards upper epidermis along with few elements of xylem parenchyma cells.

Ground tissue of the midrib portion i.e. parenchyma cells were with tannin content, which was not so prominent. Some of the central pith parenchyma cells were showing little tannin content.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215â&#x20AC;&#x201C;224

Histological and histochemical evaluation of fruit Transverse section of the fruit showed the outer epicarp, middle mesocarp and inner endocarp. (Plate A, e-f) Outer epicarp was made up of single layer of rectangular to barrel shaped epidermal cells compactly arranged without intercellular spaces with cuticle. On histochemical evaluation, epidermal cells were seen filled with the tannin content. Largely occupied mesocarp cells were made up of parenchyma cells interrupted by groups of sclereids, stone cells, fibers and vascular bundles. Histochemical evaluation also revealed sclereids and adjacent vascular tissues i.e. xylem parenchyma and fibers which were thickly enriched with the tannin content.

Endocarp attached with the seed was showing cells having somewhat little isolated tannin content.

Powder microscopy of leaf before floweringfruiting The diagnostic characters of the powder of leaf before flowering-fruiting revealed epidermal cells with rich tannin content, more quantity of tannin in the fragments of pallisade

parenchyma, trichomes heavily filled with tannin content and lower fragments of spongy parenchyma cells rich in tannin content. Oil globules, stomata, prismatic and rosette crystals, were the other characters noted in the powder microscopy. (Plates D1 and D2) Powder microscopy of leaf after floweringfruiting The diagnostic characters of the powder of leaf after flowering-fruiting revealed epidermal cells with less tannin content, less quantity of tannin in the fragments of pallisade parenchyma, trichomes poorly filled with tannin content and lower fragments of spongy parenchyma cells showing somewhat absence of the tannin content. Oil globules, stomata, prismatic and rosette crystals, were the other characters noted in the powder microscopy.

(Plates D1 and D2) Powder microscopy of fruit On organoleptic study, fruit powder of Haritaki showed yellow brown colour, aromatic and slightly astringent odour, astringent taste which later ends in sweet and rough texture.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215â&#x20AC;&#x201C;224

Diagnostic characters of the powder revealed presence of sclereids out of which some were pitted with wide lumen, mesocarp cells, pitted stone cells with wide lumen, fibers, tannin contents, simple and compound starch grains and vascular strands (Plate B).

DISCUSSION Macroscopic examination of leaves before and after flowering-fruiting did not show any prominent difference in their characteristics while their organoleptic evaluation showed recognizable difference in the colour and taste parameters. Histological and histochemical evaluation of petiole before flowering-fruiting revealed hypodermal cells, parenchymatous cortical cells and adjacent cells of vascular bundles filled with rich tannin content whereas in the evaluation of petiole after floweringfruiting, these areas showed poor tannin content or somewhat absence of tannin content in the cells. Histological and histochemical evaluation of leaf before flowering-fruiting revealed upper epidermis i.e. pallisade cells, ground tissue of the midrib portion i.e. parenchyma cells and adjacent cells of vascular bundles having rich tannin content whereas the evaluation of leaf after flowering-fruiting showed very poor localization of tannin content in the cells of these areas. On histological and histochemical evaluation of fruit, it was observed that sclereids and adjacent vascular tissues i.e. xylem parenchyma and fibers were thickly enriched with the tannin content. Cells of endocarp attached with the seed and epidermal cells were also seen filled with the tannin

content. In powder microscopy, leaf before flowering-fruiting revealed trichomes, epidermal cells, spongy and palisade parenchyma cells which were filled with rich amount of tannin content whereas powder microscopy of leaf after flowering-fruiting showed very poor tannin content in these parts of the leaf. Powder microscopy of fruit showed the characters like sclereids, stone cells, fibers, simple and compound starch grains, mesocarp cells with tannin, vascular strands and tannin contents. Thus histological, histochemical and powder microscopical examinations suggest presence of abundant tannin content in the leaf of Haritaki before flowering-fruiting and its recognizable absence or less localization in the leaf after flowering-fruiting. Histochemical and powder microscopical examinations of fruit reveals noticeable presence of tannin content in it. Thus there is scope to the possibility that tannin content in the leaves before fruiting got transported to fruit and thus leaves after fruiting are showing comparatively less tannin content.

CONCLUSION Detailed pharmacognostical evaluation of T. chebula leaves before and after floweringfruiting suggests that the leaves of Haritaki before flowering-fruiting are having more concentration of tannin than after floweringfruiting leaves. Fruit which is a totally new entity formed in the plant shows recognizable amount of tannin content in it. This work may provide incentive for the future research works carried out for the evaluation of variations in the chemical constituents of the plant.

REFERENCES Anonymous, (2010) Quality Standards of Indian medicinal Plants, Vol. 1, ICMR, New Delhi, p. 205 Anwesa Bag, Subir Kumar Bhattacharyya, Rabi Ranjan Chattopadhyay, Asian Pac J Trop Biomed. 2013 March; 3(3): p.

244â&#x20AC;&#x201C;252

Govt.

of India, (2001) The Ayurvedic pharmacopoeia of India. New Delhi: Government of India Ministry of Health and Family Welfare Department of Indian System of Medicine & Homoeopathy; p. 47

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 215–224

Khandelwal K.R., Kokate C.K., Gokhale S.B. (1996).

Practical Pharmacognosy Techniques and Experiments. Nirali Prakashan, Pune, p. 10–39 Krishnamurty K.V., (1988) Methods in the Plant Histochemistry.

Madras: Vishwanandan Pvt, Limited; p. 1–74

Source of Support: NIL

Kshirod Kumar Gupta, Girish Chandra Joshi, AYU, 2013; Jul-Sep; 34(3): p. 331–334 S.

Aruna, L.V. Nandakishore, Scholars Academic Journal of Biosciences, 2014; 2(2): p. 132–136

Trease G.E., Evans WC (2002), Trease and Evans Pharmacognosy, Harcourt brace & Co. 1999, p. 512, 547

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225â&#x20AC;&#x201C;231 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research Article ANTIDYSLIPIDAEMIC EFFECT OF THE STEM BARK OF CHIRABILWA (Holoptelea integrifolia Planch.) - A CLINICAL TRIAL Sinimol T P1*, Shahul Hameed A2 1

Lecturer, Department of Dravyagunavijnan, Ahalia Ayurveda Medical College, Palakkad, Kerala, India Associate Professor, Department of Dravyagunavijnan,Govt. Ayurveda College, Kannur, Kerala, India *Corresponding author: Email: drsinitp@gmail.com; Mob: 9446519427 2

Received: 15/03/2014; Revised: 20/04/2014; Accepted: 05/05/2014

ABSTRACT The powder of stem bark of Chirabilwa (Holoptelea integrifolia Planch.) was clinically evaluated to find out its effect in altered serum lipid levels and signs & symptoms of dyslipidaemia.

The statistical analysis of the change before and after treatment in values of Lipid profile and signs & symptoms of dyslipidaemia were the assessment criteria. It was found that the study drug Chirabilwa (Holoptelea integrifolia Planch.) was effective in reducing Total Cholesterol, Low Density Lipoprotein, Very Low Density Lipoprotein & Triglycerides and in increasing High Density Lipoprotein. Chirabilwa (Holoptelea integrifolia Planch.) was also effective in reducing the symptoms of dyslipidaemia such as difficulty in physical activity, breathlessness on slight exertion, excess sweat, dizziness and lassitude.

KEYWORDS: Dyslipidaemia, Chirabilwa, Holoptelea integrifolia Planch., Cholesterol

Cite this article: Sinimol T P, Shahul Hameed A (2014), ANTIDYSLIPIDAEMIC EFFECT OF THE STEM BARK OF CHIRABILWA (Holoptelea integrifolia Planch.) - A CLINICAL TRIAL, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 225â&#x20AC;&#x201C;231

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225â&#x20AC;&#x201C;231

INTRODUCTION Non communicable diseases (NCDs) are the major killer diseases in the world today, causing more deaths than all other diseases combined. The first WHO (World Health Organisation) global status report on non communicable diseases of year 2010 confirms that 36.1 million people died from non communicable diseases in 2008 (Margaret Chan, 2011). World Health Organisation predicts that non communicable diseases will cause over three quarters of all deaths in 2030 (Fuster V, Kelly BB, 2010). India is also passing through an epidemiological and demographic transition leading to the emergence of non communicable diseases as major health problem, estimated to account for 53% of all deaths and 44% of disability in 2005 (Samu.K, 2012).

Dyslipidaemia, a major non communicable disease is a major health problem in both developed and developing nations (Margaret Chan, 2011). It is an extremely important condition, principally because of its contribution to atherogenesis and as it is an independent and modifiable risk factor for Cardio Vascular Disease (CVD), together with Hypertension, smoking and sedentary habits (Poss J et al., 2011). The ever increasing epidemic nature of the disease prompts the scientific researchers to understand the dimension of the condition (Li YP et al, 2012).

Modern medications available to dyslipidaemia are costly, giving only temporary relief and are potentially hazardous (Robert. K. Murray et al., 2000). Traditional Ayurvedic approaches to health have become all the more relevant in the present century in the context of universal rise of NCDs (non communicable diseases).

Ayurvedic approach to these conditions is holistic, which is a combination of diet, exercise, awareness of environmental influences and the use of medications.

From Ayurvedic classics, an exact correlation of dyslipidaemia to any disease cannot be made. Scientific treatment can be adopted by analyzing dosha dooshya vikriti

(imbalance of functional and structural entities of our body).

Unhealthy diet and activities that vitiate doshas (functional entities of our body) leads to vitiation of srotases (channels in the body). It is the root cause for all diseases. The abnormality in dhatus (tissues) brings about abnormality in srotases (channels) and vice-versa. This leads to impairment of agni (digestive fire) and formation of ama (undigested food). This is followed by faulty dhatuparinama (formation of tissues) and vitiated medodhatu (lipids) is formed (Yadavji Trikamji, 2006).

Chirabilwa (Holoptelea integrifolia Planch.) is a large decidous tree distributed throughout the greater parts of India upto an altitude of 2000 feet. It is a drug which has been mentioned by various Ayurvedic books as an ingredient in many formulations indicated for the management of derangement of meda (body fat). It possesses tikta rasa (bitter taste), kashaya rasa (astringent taste), laghu (easily digestible property), rooksha guna (dry property), ushna veerya (hot potency) and katu vipaka (pungent post metabolic effect) (Chunekar K.C, 1995). Chirabilwa is included in many medohara (lipid curtailing) groups by various Ayurvedic text books. But no research has been conducted yet to clinically evaluate its antidyslipidaemic effect as a single drug. Hence a clinical study to evaluate the antidyslipidaemic effect of the stem bark of Chirabilwa was taken up.

MATERIALS AND METHODS For the clinical study, a total of 32 patients fulfilling the diagnostic criteria of dyslipidaemia approaching the outpatient department of Government Ayurveda Medical College and Hospital, Trivandrum were selected for the study. The National Institutes of Health's National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) guidelines were used as the diagnostic criteria. The patients were assigned into a single group taking into consideration the inclusion and exclusion criteria for the

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225–231

interventional study.

Inclusion criteria: Patients of both sexes in the age group of 16–70 years, diagnosed to have dyslipidaemia.

Exclusion criteria: Pregnant and lactating woman, patients undergoing other treatments for dyslipideamia, patients taking any long term medication, known cases of Hypothyroidism, Coronary Artery Disease (CAD), liver disease, renal disease.

Preparation of the drug The stem bark of Chirabilwa (Holoptelea integrifolia Planch.) was collected from the herbal garden of Pharmacognosy Unit, Govt.

Ayurveda College, Trivandrum, India during January 2011. The plant identification was done by Mr. G.R. Jayakumar, Senior Research officer, Pharmacognosy Unit, Govt. Ayurveda College, Trivandrum. Voucher specimens were preserved in the Herbarium at the Department of Dravyaguna vijnana, Ahaliya Ayurveda College, Kerala, India. The collected stem barks of Chirabilwa (Holoptelea integrifolia Planch.) were washed well to remove mud and soil contaminants, dried in shade and powdered using a micropulveriser to the mesh size 80.

60g of fine powder was weighed out and sealed in polythene packets. 6 gm of powder was given orally, twice daily before food with luke warm water for 30 days.

Clinical Trial The Clinical research design was approved by the Institutional ethics committee, Government Ayurveda Medical College, Trivandrum, India.

Investigations: Routine Urine examination and biochemical examination with Serum Total Cholesterol (S. TC), Serum High Density Lipoprotein (S. HDL), Serum Low Density Lipoprotein (S. LDL), Serum Very Low

Density Lipoprotein (S. VLDL) and Serum Triglycerides were performed before and after the study.

Assessment Criteria: The patients were assessed based on the clinical parameters explained in Table 2 and laboratory findings.

The clinical parameters were graded as follows.

All the symptoms except Incidence of hypersomnia & dizziness were graded as: Grade 0- not noted /not present, Grade 1 –mild, Grade 2- moderate, Grade 3-severe. Incidences of hypersomnia & dizziness were graded as: Grade 0- never, Grade 1 –less frequent, Grade 2- moderately frequent and Grade 3- Highly frequent.

Statistical analysis: Values obtained were statistically evaluated using paired‘t’ test and Wilcoxon’s signed rank test. In the entire statistical test, the P-values less than 0.05 were considered to be statistically significant.

RESULTS 46.88% of cases were distributed among the age group of 50–59 years, which showed this age group is more prone to the development of dyslipidaemia. 68.75% had vatakapha prakriti (body constitution), vata and kapha are the main doshas involved in the pathogenesis of dyslipidaemia. 87.5% of cases belonged to middle class. 71.88% were residing in urban area indicating the high prevalence of the disease in urban area. 90.63% were taking both vegetarian & non vegetarian food showing the role of non-vegetarian diet in causing the disease. 81.25% patients had regular pattern of eating, 78.13% patients used only coconut oil for cooking, 84.38% had moderate appetite, 59.38% were stout, 59.38% had weight between 65–75 kg, 96.88% of patients never smoked, 63% of patients never consumed alcohol, 37.5% patients had less sleep. 59.38% patients had no exercise, lack of exercise is considered to be a main cause of the disease.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225–231

Table-01. Analysis of effect of treatment on Serum Lipid Profile Mean

SD

KS

P

MD

Z or t

TC-BT

241.56

35.80 0.167 0.024* -30.50 4.581$

TC-AT

211.06

26.42 0.158 0.040*

HDL-BT

50.50

9.90 0.105

0.200

HDL-AT

56.16

11.33 0.127

0.200

LDL-BT

158.75

28.00 0.153 0.056

LDL-AT

128.41

26.51 0.188 0.005*

VLDL-BT

32.25

20.53 0.233 0.000*

VLDL-AT

26.53

15.78 0.216 0.001*

5.66 2.643#

P 0.001*

0.013*

30.34

4.33 $ 0.0001*

5.72

4.18 $ 0.0004*

TG-BT

161.44 102.57 0.234 0.001* -27.19 4.339$

TG-AT

134.25

0.001*

87.35 0.242 0.001*

*: Significant at 5% level (P<0.05), KS: Kolmogorov Smirnov test, $: Wilcoxon’s Signed Rank test, #: Paired t test

Table - 02. Analysis of signs & symptoms of dyslipidaemia

3 % 12.5

Before treatment Grade 2 1 0 % % % 12.5 12.5 62.5

3 % 0

9.375

6.25

9.375

75

0

0

12.5

3.125

0

0

96.875

0

3.125

0

3.125 0 0 12.5 12.5 31.25 0

0 9.375 3.125 9.375

93.75 84.375 90.625 68.75 62.5 34.375 93.75

0 0 0 0 0 0 0

3.125

25 0

3.125 6.25 6.25 9.375 9.375 9.375 6.25

0 0

0 0

Signs & Symptoms

Difficulty in physical activity Breathless-ness on slight exertion Breathlessness without known reason Shallow breathing Excess thirst Excess hunger Excess sweat Dizziness Lassitude Incidence of Hypersomnia Bromhid-rosis Decrease in sexual vigour

3.125 3.125

15.625

96.875 0 96.875 3.125

After treatment Grade 2 1 0 % % % 3.125 12.5 84.375

0 0 0 6.25 6.25 0 0 0

Wilcoxon’s test Z P 3.145

0.002*

87.5

2.460

0.014*

96.875

1.000

0.317

0 96.875 3.125 96.875 3.125 96.875 15.625 84.375 12.5 81.25 25 68.75 6.25 93.75

1.414 1.518 1.000 2.598 3.025 4.086 1.342

0.157 0.129 0.317 0.009* 0.002* 0.001* 0.180

1.342 1.342

1.000 1.000

0 0

* Significant at 5% level (P<0.05)

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

100 96.875

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225â&#x20AC;&#x201C;231

Effect of treatment 1. Effect of treatment on Serum Lipid Profile The drug was statistically effective (P<0.05) in reducing Total Cholesterol, LDL, VLDL & Triglycerides and in increasing HDL (Table No. - 1).

2. Effect of treatment on Symptoms of dyslipidaemia

Signs

&

The drug was statistically effective (P<0.05) in reducing difficulty in physical activity, breathlessness on slight exertion, excess sweat, dizziness and lassitude. The drug was statistically not (P>0.05) effective in reducing breathlessness without known reason, shallow breathing, excess thirst, excess hunger, Incidence of hypersomnia, bromhidrosis, decrease in sexual vigour (Table No. - 2).

DISCUSSION Dyslipidaemia is a term used to denote abnormal fractions of circulating lipids or lipoproteins in blood. Dyslipidaemia as such is not mentioned in Ayurvedic classical texts. In Ayurvedic context, Dyslipidaemia can be considered as a particular condition of increased medodhatu (lipids) associated with increased kapha dosha and deranged kleda (unhealthy secretions) in the body. Agnisada (decreased digestive fire) and increased kapha dosha lead to the formation of ama (undigested food). Due to obstruction of nourishment pathways to other dhatus (tissues), only medodhatu (lipids) gets nourished at the cost of other dhatus (tissues). Meda (lipids) with its high picchila (sticky) property causes lepana (coating) of srotases (channels of body), gradually blocking them. As a result of srotorodha (obstruction in channels of our body), the activities of vata dosha also get increased resulting in the symptoms of dyslipidaemia (Srikantha Murthy K.R, 2005).

Probable mode of action of the drug One basic principle of Ayurveda is that similar factors cause increase and dissimilar

factors cause decrease of our body constituents.

Based on this principle, mode of action can be explained. As stated earlier, dyslipidaemia is caused by the excessive indulgence of factors causing increase of medodhatu (lipids), ie. substances having madhura rasa (sweet taste), seeta veerya (cold potency), guru guna (difficult to digest property), snigdha gunas (unctuous property) and sedentary life style.

(Acharya Yadunandana Upadhyaya, 2005).

Thus treatment should be with drugs having tika katu kashaya rasa (bitter, pungent and astringent taste), ushna veerya (hot potency) and laghu rooksha guna (which are easy to digest and dry). The study drug Chirabilwa is having tika kashaya rasa (bitter and astringent taste), laghu rooksha guna (which is easily digestible and dry), ushna veerya (hot potency) and katu vipaka (pungent post digestive effect), thus possessing all the above properties (Chunekar K.C, 1995).

Drugs having katu rasa (pungent taste), ushna veerya (hot potency) and laghu guna (easily digestible) can increase the strength of agni (digestive fire) which may act on improperly digested food. These properties can pacify kapha and dessicate vitiated meda (lipids) and kleda (unhealthy secretions), the major contributing factors for the condition.

Thus it can remove the obstruction of channels.

Laghu rooksha guna (which is easy to digest and dry) and katu rasa (pungent taste) can act as lekhana (dessicate and scrape away the morbid tissues especially meda and kapha).

Chirabilwa is included in lekhana mahakashaya group (group of drugs that dessicate and scrape away the morbid tissues especially meda and kapha) of Caraka Samhita (Yadavji Trikamji, 2006); Shleshma samsamana (groups of drugs which decrease kapha), several kaphamedohara groups (groups of drugs which decrease meda and kapha) like Saalasaradi, Varanadi, Aragwadadi, Asanadi and Arkadi of Susruta Samhita (Yadavji Trikamji, 2006); Asanadi, Varanadi, Aragwadadi and Arkadi groups of Ashtanga Hridaya (Hari Sadasiva Sastri Paradakara, 2005). Drugs having these properties and ushna

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225–231

veerya can also act as pramathi (drugs that expel cumulative mala from channels). Hence the mode of action of Chirabilwa can be attributed principally to its laghu rooksha guna (which is easy to digest and dry), ushna veerya (hot potency), tikta kashaya rasa (bitter and astringent taste) and katu vipaka (pungent post digestive effect), with which it decreases meda (lipids) by means of lekhana (dessicate and scrape away the morbid tissues especially meda and kapha) and pramathi (drugs that expel cumulative excretory products from channels) modes. β-sitosterol is a chemical constituent of Chirabilwa (Ram.P.Rastogi. & Mehrotra B.N, 1970). It is to be noted that β-sitosterol is used as a dietary supplement to lower blood cholesterol. β-Sitosterol inhibits cholesterol absorption in the intestine. When the sterol is absorbed in the intestine, it is transported by lipoproteins and incorporated into the cellular membrane. Phytosterols and phytostanols both inhibit the uptake of dietary and biliary cholesterol, decreasing the levels of LDL and serum total cholesterol. Because the structure of β-sitosterol is very similar to that of

cholesterol, β-sitosterol takes the place of dietary and biliary cholesterol in micelles produced in the intestinal lumen. This causes less cholesterol absorption in the body (Fauci & Longo, 2008).

CONCLUSION Chirabilwa (Holoptelea integrifolia Planch.) is a classical drug possessing lekhana (dessicate and scrape away the morbid tissues especially lipids and kapha dosha) action. The study drug is effective in raising Serum High Density Lipoprotein and decreasing Serum Total Cholesterol, Serum Low Density Lipoprotein, Serum Very Low Density Lipoprotein and Triglycerides. The drug reduces some signs and symptoms of dyslipidaemia like difficulty in physical activity, breathlessness on slight exertion, excess sweat, Dizziness and lassitude.

Therefore it can be concluded that the study drug, the powder of the stem bark of Chirabilwa (Holoptelea integrifolia Planch.), Family -Ulmaceae] is effective in dyslipidaemia.

REFERENCES Betteridge D.J and Morrell J.M. (1998), Clinician’s guide to Lipids and Coronary heart disease. London: Hodder Arnold Publications.

Chunekar K.C. (Editor). (1995), Bhavaprakasa Nighantu.

10th Ed. Varanasi: Chowkhambha Krishnadas Academy Publications.

Margaret Chan, (2011), New WHO report: deaths from noncommunicable diseases on the rise, with developing world hit hardest. [Online] Available from: http://www.who.int/mediacentre/news/r eleases/2011/ncds_20110427/en/.

[Accessed: 19th June 2012].

Fauci and Longo. (Editors). (2008). Harrison’s Internal Medicine. 17th Ed. U.S.A: The Mc Graw-Hill Companies Publications Fuster V, Kelly BB (2010). Promoting Cardiovascular Health in the Developing World: A Critical Challenge to Achieve Global Health.

[Online] Available from: http://www.ncbi.nlm.nih.gov/books/NB K45688/. [Accessed: 9th June 2012].

Hari Sadasiva Sastri Paradakara, (2005), Acharya Vagbhata. Astanga Hridaya.

Reprint edition.

Varanasi: Chowkhambha Sanskrit Sansthan Publications.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225–231

Li YP, Ma RL, Zhang M, Liu JM, Ding YS, Guo H, Zhang JY, Li SG, Sun F, Guo SX (2012), Epidemic features of dyslipidemia among Uygur, Kazakh, and Han adults in Xinjiang, China in 2010. [Online] Available from: http://www.ncbi.nlm.nih.gov/pubmed/2 4378138. [Accessed: 24th June 2012] Poss J, Custodis F, Werner C, Weingartner O, Bohm M, Laufs U. (2011), Cardiovascular disease and Dyslipidemia: beyond LDL. [Online] Available from: http://www.ncbi.nlm.nih.gov/pubmed/2 1418031. [Accessed: 24th January 2011] Ram.P.Rastogi. and Mehrotra B.N. (1970), Compendium of Indian Medicinal plants, New Delhi: Published by Central Drug Research Institute.

Robert Murray, David Bender, Kathleen M. Botham, Peter J. Kennelly, Victor Rodwell, P. Anthony Weil (2000).

Source of Support: NIL

Harper’s biochemistry, Connecticut: Appleton Publications

25th Ed. & Lange

Samu.K. (2012), Health/Epidemics. [Online] Available from: http://www.isidelhi.org.in/hrnews/HR_ THEMATIC_ISSUES/Health/Health2012.pdf. [Accessed: 29th September 2012] Srikantha Murthy K.R. (2005), Astanga Sangraha. Reprint edition. Varanasi: Chowkhambha Orientalia Publications.

Yadavji Trikamaji, (2005), Susruta Samhita.

Reprint edition.

Varanasi: Chowkhambha Krishnadas Academy Publications.

Yadavji Trikamaji, (2006), Charaka Samhitha, 6th Ed. Varanasi: Chowkhamba Sanskrit Samsthan.

Yadunandana Upadhyaya, (2005), Madhava Nidana.

7th Ed. Varanasi: Chowkhambha Orientalia Publications.

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Call for Papers â&#x20AC;&#x201C; Vol. 3, Issue 7, July 2014 Submit your manuscripts (Research articles, Review articles, Short Communications, Letters to the Editor, Book Reviews) to Global Journal of Research on Medicinal plants & Indigenous medicine â&#x20AC;&#x201C; GJRMI Submit it online through www.gjrmi.com or mail it to submitarticle@gjrmi.com on or before June 10th 2014.

To advertise on the Flip book Cover page freely, write to chiefeditor@gjrmi.com or editorinchief@gjrmi.com Or Call - +919590574495