Bangladesh Journal of Pharmacology Research Article Antimicrobial, anti-oxidant and cal- cium channel blocking activities of Amberboa divaricata BJP Introduction The genus Amberboa belongs to family compositae and is represented by six species, one of which is Amberboa divaricata, an erect, stiff, dichotomously branched annu- al herb (Vardhana, 2008). Locally this plant is known as Birumdundi, Badaward or Daaba (Qureshi and Bhatti, 2008). Previous phytochemical investigation revealed that A. divaricata contains fructose, triacontane, campes- terol, sigmasterol, sitosterol, jaceosidine, cynaropicrin, cycloartane type triterpenoids, guaianolides, sesquiter- penoids, lupeol, flavonol glycosides, green essential oils, acid resins, organic acids, gums, fatty matter and alkaloids (Ibrahim et al., 2010). Traditionally, this herb is used as tonic, aperient, deobstruent, febrifuge, anti- diarrheal, and anti-periodic, it is used in coughs, fever and general debility. It has cytotoxic and antibacterial properties. Seeds are antidote, astringent and resolvent (Bhattacharjee, 2005). For skin irritation plant is boiled in water and bath is taken. About two gram of plant is given in malaria and continued for three days to treat fever. For blood purification juice of fresh plant is used with black pepper (Qureshi and Bhatti, 2008). Beside a number of traditional uses, this herb was not previously evaluated pharmacologically to validate its use by traditional healers. So this study was designed to check its possible spasmolytic, antibacterial and anti-oxidant effects. Materials and Methods Plant material and extraction The whole plant of A. divaricata was purchased from the local herbal market of Bahawalpur. It was authenticated by a botanist at Govt. S.E. College Bahawalpur, and sample of plant material was submitted at herbarium of pharmacology section Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur, Pakistan, having a voucher number AD- WP-03-10-004. The plant material (500 mg) was cleaned A Journal of the Bangladesh Pharmacological Society (BDPS); www.bdps.info Bangladesh J Pharmacol 2014; 9: 29-36 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract Traditional healers in Pakistan use the herb Amberboa divaricata as tonic, aperiant, deobstruent, febrifuge, anti-diarrheal, antiperiodic, antipyretic, anti- cough and in skin disorders. In vitro tissue experiments were carried out on rabbit jejunum to elucidate the possible mechanism of its prescribed effects on gastrointestinal tract, while antibacterial and anti-oxidant experiments were performed to provide pharmacological evidence of its traditional use in skin disorders. The 70%methanolic crude extract of A. divaricata produced dose- dependent relaxation in isolated rabbit jejunum tissue in a concentration range of 0.1–3.0 mg/mL (n=5). Calcium response curves were constructed at concentration of 0.03 and 0.1 mg/mL (n=5), which produced rightward shift in a pattern similar to that of verapamil, confirming the calcium channel blocking activity. Agar disc diffusion assay at a concentration of 10 mg crude extract/disc showed clear zones of inhibition. Article Info Received: 12 November 2013 Accepted: 15 December 2013 Available Online: 21 January 2014 DOI: 10.3329/bjp.v9i1.16989 Cite this article: Iqbal SM, Mushtaq A, Jabeen Q. Anti- microbial, anti-oxidant and calcium channel blocking activities of Amber- boa divaricata. Bangladesh J Pharma- col. 2014; 9: 00-00. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Antimicrobial, anti-oxidant and calcium channel blocking activities of Amberboa divaricata Shahid Muhammad Iqbal, Aamir Mushtaq and Qaiser Jabeen Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur, Bahawalpur 631 00, Pakistan. from dirt and extraneous matter and then coarsely powdered. The powdered material was soaked in sufficient volume of 70%methanol with occasional stirring for three days, and then it was filtered initially through muslin cloth followed by filteration through Whatman qualitative grade 1 filter paper. The plant material was subjected to this procedure three times, the filtrate was collected, pooled together and evaporated at 40-50°C in rotary evaporator (Laborota 4000-efficient Heidolph, Germany). After the removal of solvent a thick semi solid mass of yellowish brown color was obtained with a percentage yield of about 11%. The crude extract was stored in air tight container at -20°C. Chemicals and animals All the chemicals used were of analytical grade, acetyl- choline chloride, atropine sulfate, verapamil hydrochlo- ride, Folin-Ciocalteu reagent and DPPH were purchas- ed from Sigma Chemicals Company, St. Louis, MO, USA. Sodium chloride, potassium chloride, magnesium chloride, calcium chloride, D-glucose, Sodium dihydro- gen phosphate, sodium bi carbonate, EDTA, Mueller Hinton agar, nutrient broth and methanol were pur- chased from Merck, Germany. Animals used in this study like rabbits (1-1.5 kg), Swiss albino mice (20-25 g) were obtained from National Institute of Health Pakistan, and kept at animal house of Faculty of Pharmacy and Alternative Medicine, The Islamia Uni- versity of Bahawalpur. The animals were allowed to feed and drink water but prior to experiment they were kept fasting for 24 hours. On the day of experiment rabbit was killed by a blow on back of the head and jejunum was excised out, which was immediately placed in Tyrode solution previously prepared, aerated and maintained at 37°C. Three microbial strains were used for antimicrobial activity, out of which two were gram positive i.e. Staphylococcus aureus, Bacillus subtilis and one was gram negative i.e. Escherichia coli. The bacterial strains were obtained from the Department of Biochemistry and Biotechnology, The Islamia Univer- sity of Bahawalpur. Preliminary phytochemical analysis Phytochemical analysis of crude extract of A. divaricata (AD.Cr) was performed by methods as previously described (Usman et al., 2009). Briefly the presence of alkaloids was detected when orange precipitates appear -ed on treating with Dragendorff’s reagent. Appearance of brownish green precipitates on treating with FeCl3 indicate the presence of Tannins. Formation of persisted froth on vigorously shaking in water exhibited the presence of saponins. Appearance of cherry red color on performing modified Borntrager’s test, reveal the presence of anthraquinones. The presence of glycosides was observed by performing Keller-killiani test. Flavo- noids were detected by treating the aqueous solution of extract with KOH solution which results in dark yellow color and steroids were determined by mixing the extract with acetic acid and then addition of concentrated H2SO4 in ice cooled above solution which results in the formation of violet-blue color. Carbohydrates were determined by performing Molish test while Barfoed and Seliwanoff’s test showed the presence of monosaccharides and keto-nes respectively. Appearance of red color on treating with acidified solution of phloroglucinol, indicate the presence of pentoses. In vitro tissue experiments In vitro tissue experiments were performed as previously described (Jabeen et al., 2009). Briefly several pieces of about 2 cm were cut from the excised jejunum and cleaned from the fatty matter, and then they were mounted in 50 mL tissue organ bath containing Tyrode solution maintained at 37°C. The tissue was continually aerated with carbogen gas (95% O2 + 5% CO2) and equilibrated for 30 min before addition of any drug or extract. Intestinal responses were recorded with the help of kymograph (MTA-786/1024). After 30 min of equilibration, jejunal tissue was stabilized with sub maximal concentration of acetyl-choline i.e. (0.3 µM). After stabilization intestinal tissue started to exhibit uniform spontaneous rhythmic contractions, and then the extract was added in cumulative manner to check its activity. The calcium channel blocking activity was explored by depolarizing the jejunal tissue with high K+ (80 mM) as previously described (Farre et al., 1991). Addition of high K+ caused sustained contraction in intestinal tissue, and then extract was added in cumulative manner to check its inhibitory effect on K+ induced contractions. To confirm the calcium channel blocking activity of crude extract, tissue was stabilized in Tyrode solution. The Tyrode solution was then replaced with calcium free Tyrode solution that contained 0.1 mM EDTA, and tissue was allowed to stand for 30 min in order to remove the calcium from tissue. Then calcium free Tyrode solution was replaced with potassium rich and calcium free Tyrode solution containing 0.1 mM EDTA. The tissue was again allowed to stand for 30 min and then controlled calcium response curves were constructed and re constructed with Ca2+ until two super imposable curves are obtained. Then tissue was pretreated with different concentrations of plant extract for 60 min before constructing calcium response curves, and then compared with calcium response curve of verapamil a standard calcium channel blocker. Antibacterial activity The antibacterial activity of crude extract of A. divaricata was performed by methods with little modifications as previously described (Salama and Marraiki, 2010). Agar disc diffusion assay- The antibacterial activity of crude extract of A. divaricata was determined by using 30 Bangladesh J Pharmacol 2014; 9: 29-36 Mueller Hinton agar. Pre-adjusted bacterial culture of 0.5 Mcfarland standards was spread on 15 cm diameter petri plates, then sterile filter paper discs each containing 10mg of crude extract were placed. After placing the discs, plates were covered and incubated at 37°C for 24 hours. The antibacterial activity was deter- mined by measuring the clear zone around the filter paper discs. Gentamicin and ampicillin discs were used as standards, and the experiment was carried out in triplicate. Determination of MIC and MBC- Minimum inhibitory concentration was determined by microdilution assay as previously described (Sahin et al., 2003). The inocu- lums of bacteria were prepared and adjusted to 0.5 McFarland standards. The dried extract was reconstituted to the maximum concentration to be tested i.e. 20 mg/mL and then a serial 2-fold dilutions were made in a concentration ranging 10-0.078 mg/mL with sterile nutrient broth. Then 95 µL sterile nutrient broth and 5 µL of inocula were dispensed in sterile 96 well plates. A 100 µL of crude plant extract (20 mg/mL) was added into first well. Then 100 µL of each of serial dilutions were added to the next wells. The final volume in each well was 200 µL. Ampicillin/ Gentamicin were used as standard antibiotics, pure bacterial culture, nutrient broth served as positive and negative control respectively. One plate was used for each bacterial strain to prevent the cross contamination. Then sterile plate sealer was placed on each plate and incubated at 37°C for 24 hours. Microbial growth was observed and confirmed by plating 5 µL samples from clear wells on nutrient agar medium at 37°C for 24 hours. The MIC was described as minimum concentration of the crude extract which inhibited the growth of microorganism. The lowest concentration that showed no growth after this sub culture was taken as Minimum bactericidal concentration (Reddy et al., 2008). Each experiment was performed in triplicate. Determination of total phenolic contents- Total phenolic contents of crude extract of A. divaricata were determi- ned as described elsewhere (Ksouri et al., 2009). Briefly in a small test tube 125 µL of properly diluted crude extract was added to 500 µL of water and then 125 µL of Folin-Ciocalteu reagent was added and mixed. Then added 1250 µL of Na2CO3 (7%) and made-up the final volume to 3 mL with distilled water, and mixed it again thoroughly. After incubating for 90 min at 23°C in dark, the absorbance was taken at 760 nm. Total phenolic content of crude extract was determined by using stan- dard gallic acid curve (0-500 µg/mL), and expressed as mg gallic acid equivalents per gram of dry weight of extract. The experiment was performed in triplicate. Determination of total anti-oxidant capacity The total anti-oxidant activity of the crude extract of A. divaricata was determined by the method as described elsewhere (Niciforovic, 2010). Briefly 0.3 mL of sample extract was mixed with 3 mL of reagent solution (4 mM ammonium molybdate 0.6 M sulfuric acid and 28 mM sodium phosphate), then incubated at 95°C for 90 min. The absorbance of the solution was measured at 695 nm after proper cooling of reaction mixture to room temper -ature. The total anti-oxidant capacity was expressed as milligrams of ascorbic acid per gram of the dry weight of extract. The experiment was performed in triplicate. DPPH radical scavenging assay- DPPH radical scaven- ging assay was performed as previously described (Raja -Kannan et al., 2010), with little modifications. Briefly in a 96-well plate, 10 µL of test substance and 90 µL of 100 µM methanolic solution of diphenylpicryl-hydrazyl (DPPH) was added and mixed. The contents were incu- bated at room temperature for 30 min in the dark. Then absorbance was taken by using 96-well plate reader Synergy UT, Bioteck instrument USA at 517 nm. Quer- citin was used as standard anti-oxidant. DPPH scaven- ging activity was determined by the given formula. The assay was performed in triplicate. %Scavenging activity = 100–[Absorbance of test compound/Absorbance of control] × 100 Acute toxicity testing Acute toxicity test was performed on mice as previously described (Gilani et al., 2008). Animals were randomly assigned into four groups, and each group contained five mice. Group Ι, ΙΙ, and ΙΙΙ received the increasing doses of extract; i.e. 300, 1000 and 3000 mg/ kg per oral respectively. Group ΙV served as negative control and received 10 mL/kg normal saline. The animals were kept under observation for 6 hours, allowed feed and drink water ad libitum. After 24 hours, they were observed for any lethality. Statistical analysis The statistical analysis was performed by using the software GraphPad Prism 5.01, while the data was expressed as mean of three experiments ± standard error of mean (n = no of experiments). Results The crude extract of A. divaricata was found to contain alkaloids, tannins, saponins, glycosides, flavonoids, steroidal compounds, carbohydrates, ketones, pentoses and soluble starch while coumarins and anthraqui- nones were absent. The effect of crude extract of A. divaricata was checked on spontaneously contracting rabbit jejunal preparation in a concentration range of 0.1–3 mg/mL. It decreased spontaneous contractions of rabbit jejunum and on further increasing the concentration up to 3 mg/mL, it completely blocked the contractions as represented in Bangladesh J Pharmacol 2014; 9: 29-36 31 Figure 1. The spasmolytic effect was concentration- dependent and tissue regained spontaneous contrac- tions after 5-10 min of washing with Tyrode solution. The spasmolytic effect was further elaborated when crude extract of A. divaricata relaxed the pre-contracted rabbit jejunum with high K+ i.e. (80 mM). Again the effect was concentration dependent and at concentra- tion of 3 mg/mL, it completely relaxed the contracted tissue as shown in Figure 2. As these preliminary experiments performed on isolated rabbit jejunum tissue indicated the Ca+2 channel blocking activity of crude extract of A. divaricata, so calcium response curves were made in the presence and absence of crude extract of A. divaricata. The calcium response curves constructed at concentrations of 0.03 and 0.1 mg/mL showed right ward shift in a pattern similar to that of verapamil as shown in Figure 3, which confirms that the crude extract of A. divaricata contains calcium channel blocking activity. The aqueous methanolic crude extract of A. divaricata (Ad.Cr) was checked at the concentration of 10 mg/disc for its antibacterial activity against three bacterial strains. The extract inhibited the growth of all three bacterial strains. The diameter of zone of inhibitions for S. aureus, B. subtilis and E. coli were 13.3 ± 0.6, 12.0 ± 1.0, 10.7 ± 1.2 mm respectively. Agar disc diffusion assay exhibited the sensitivity of all three strains against crude extract of A. divaricata as shown in Figure 4. So micro dilution assay was performed which revealed the MIC values for B. subtilis and S. aureus was 2.5 mg/mL, while for E. coli it was 5 mg/mL. Minimum bactericidal 32 Bangladesh J Pharmacol 2014; 9: 29-36 0.1 0.3 1 3 mg/mL A B Figure 1: Representative tracing showing the (A) normal contractions of isolated rabbit jejunum tissue and (B) spasmolytic effect of crude extract of A. divaricata Ad.Cr when introduced in a cummulative manner Figure 2: Concentration-dependent inhibitory effect of the crude extract of A. divaricata (Ad.Cr) on spontaneous and K+-induced contractions in isolated rabbit jejunum preparations. Values shown are mean ± S.E.M. of 3-5 observations Ad.Cr mg/mL % O f s po nt an eo us c on tra ct io ns 100 75 50 25 0 0.1 1 10 Spontaneous K+ -ind contraction concentration is the minimum concentration that kills bacteria. The MBC values for all three strains were same i.e. 5 mg/mL. Anti-oxidant capacity of A. divaricata crude extract was determined by calculating total phenolic contents, total anti-oxidant capacity and DPPH radical scavenging assay. The total phenolic contents, of A. divaricata crude extract were found to be 40.5 ± 2.5 (mg GAE/g DW), while the total anti-oxidant capacity was 22.4 ± 2.4 mg AAE/g DW, which showed its anti-oxidant potential. At the concentration of 0.1 mg/mL A. divaricata crude extract scavenged 31.4 ± 1.1% of DPPH radical, while at the same concentration quercetin caused 95.6 ± 0.02% inhibition as shown in Figure 5. Acute toxicity test was performed on mice, and up to 3 g/kg, the crude extract of A. divaricata did not exhibited any adverse effect on the animals. So, the crude extract of A. divaricata was rendered safe up to the dose of 3 g/ kg. Discussion Gastrointestinal motility is a complex physiological fun- ction. The two main determining factors of gastrointes- tinal motility are the smooth muscles and the enteric nervous system (Roman and Gonella, 1987). So, the enteric neurons and intestinal smooth muscle cells are two promising targets for drugs. The contraction of Smooth muscles basically depends on increased concen -tration of free cytosolic calcium, that may be either due to the extracellular entry of Ca2+ through calcium channels or by the release of Ca2+ ions from intracellular stores (Pietrobon et al., 1990). Ca2+ ions are continually exchanged between intracellular and extra-cellular Ca2+ stores which results in cyclic depolarization and repolarization of intestinal tissue that accounts for its involuntary contractions (Ali et al., 2009). Tradi- tionally, the herb A. divaricata has been used in over- active disease of gastrointestinal tract such as abdomi- nal spasm and diarrhea. The crude extract of A. divaricata exhibited spasmolytic effect on rabbit’s jejunal preparation. A number of previous studies have men- Bangladesh J Pharmacol 2014; 9: 29-36 33 -4.5 -3.5 -2.5 -1.5 0 25 50 75 100 AD.Cr 0.03mg/ml AD.Cr 0.1mg/ml Control Log [Ca++] M % o f C o n tr o l M ax . -4.5 -3.5 -2.5 -1.5 0 25 50 75 100 Control Verapamil 0.03 M Verapamil 0.1 M Log [Ca++] M % o f C o n tr o l M ax . Figure 3: Concentration–response curves of Ca2+ in the absence and presence of different concentrations of (A) crude extract of A. divaricata (Ad.Cr). (B) Verapamil in isolated rabbit jejunum preparations. Values shown are mean ± S.E.M. of 4–5 observations A B S. a ur eu s B . s ub til is E. c ol i 0 10 20 30 40 Ad Cr 10 mg/disc Ampicillin 25 /disc Gentamicin 10 /disc I n h ib it io n Z o n e D ia m e te r ( m m ) Figure 4: Comparison of Inhibition Zone Diameter against bacterial species by the crude extract of A. divaricata (Ad.Cr), ampicillin and gentamicin. Values are mean ± S.D. of three replicates tioned that spasmolytic effect of medicinal plants is usually due to calcium channel blockade (Gilani et al., 1999), so the crude extract of A. divaricata was checked on tissue preparation pretreated with high potassium, where it relaxed the contracted tissue. High concentra- tion of K+ in extracellular space acts as non-receptor spasmogen and depolarizes the smooth muscles follow- ed by contraction (Karaki et al., 1997) in which voltage dependent Ca+2 channels are involved (Gharib et al., 2008). The phytochemical tests of the crude extract of A. divaricata showed that it contains various classes of bio- active compounds e.g. flavonoids, glycosides, saponins, steroids tannins and phenolic compounds. Previously it has been described that phenolic compounds and some flavonoids have effects on intestinal motility both in vivo and in vitro (Di carlo et al., 1999). Flavonoids such as quercitin has well documented antispasmodic effects on gastrointestinal smooth muscles and some resear- chers also conclude that this antispasmodic effect is related to calcium release from intracellular stores and/ or interference with calcium entry through calcium channels (Capasso et al., 1991). A much recent study described that flavonoids contained in hexane extract of Syzygium Samarangense exhibited relaxant activity which was due to the blockade of calcium influx (Ghayur et al., 2006). So, it can be considered that the antispasmodic activity of A. divaricata may be due to its flavonoids and various phenolic compounds. Antimicro -bial agents can be derived from medicinal herbs and more than 1000 herbs have exhibited antimicrobial effects (Nychas, 1995). A. divaricata is used in skin irrita- tions and as antimicrobial by traditional healers, so A. divaricata crude extract was evaluated against common bacterial species E. coli, B. subtilis and S. aureus. Plants are capable of synthesizing number of phenolic com- pounds and their derivatives which are considered as secondary metabolites (Schultes, 1978). Most of these secondary metabolites are part of plant defense mecha- nisms and protect them against the invasion of herbi- vores, insects and microorganisms. Phytochemicals, isolated from medicinal plants exhibit toxicity to micro- organisms by different mechanisms. The mechanism underlying phenolic compound toxicity to microorgani- sms is thought to be mediated by enzyme inhibition possibly through interaction with sulfhydryl groups and with different proteins (Mason and Wasserman, 1987). Tannins are polymeric phenols which stimulate immune system and are considered to have wide range of anti infective activities (Haslam, 1996). They make complexes with proteins so they have ability to inactivate microbial enzymes, adhesion and transport proteins (Stern et al., 1996). Scalbert, listed 33 studies that described antimicrobial activities of tannins against fungi, bacteria and yeasts (Scalbert, 1991). Similarly flavonoids are the compounds which are synthesized in response to microbial infection in plants (Dixon et al., 1983), and are found to be effective against wide range of microorganisms (Cowan, 1999). They disrupt microbial membranes, make complex with bacterial cell wall, extracellular and soluble proteins (Tsuchiya et al., 1996). Phenolic compounds present in plants have gained considerable importance due to their anti- oxidant activities (Pan et al., 2008). Total phenolic contents of crude extract of A. divaricata exhibited its anti-oxidant potential, so the crude extract was subjected to further anti-oxidant assays. Phosphomolybdenum method was used to determine the total anti-oxidant capacity of A. divaricata which is a quantitative assay because it described the number of equivalents of ascorbic acid per gram of dry extract (Prieto et al., 1999). Plant extracts are frequently analyzed for their anti-oxidant activity by DPPH radical scavenging assay. DPPH is a stable free radical which interacts with compounds that can give an electron or a hydrogen atom and such compounds are considered as anti-oxidants (Singh et al., 2002). Conclusion From the results of different experiments performed on the crude extract of A. divaricata that it has spasmolytic, antibacterial and anti-oxidant effects, which provide a solid reason for its use in traditional medicine. These effects of crude extract are attributed due to the presence of different phytochemical compounds like flavonoids, tannins, saponins and different phenolic compounds. The spasmolytic activity provides rationale for its use in overactive bowl disorders while antibacterial and anti-oxidant properties give reason for its use in skin disorders. 34 Bangladesh J Pharmacol 2014; 9: 29-36 Figure 5: DPPH radical scavenging activities of Amberboa divar- icata (Ad.Cr) and quercetin. Values are shown mean ± SEM of 3 values Ad.Cr 0.1 mg/ml Quercitin 0.1 mg/ml 0 20 40 60 80 100 % I n h ib it io n Acknowledgements The authors of this publication are thankful to the Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur, Pakistan for providing all the necessities used in this research. References Ali N, Ahmad B, Bashir S, Shah J, Azam S, Ahmad M. Calcium channel blocking activities of Withania coagulans. Afr J Pharm Pharmacol. 2009; 3: 39-442. Bhattacharjee SK, De LC. Medicinal herbs and flowers. 1st ed. Jaipur, India, Aavishkar Publishers, 2005, p 36. Capasso A, Pinto A, Mascolo N, Autore G, Capasso F. Reduction of agonist induced contractions of guinea-pig isolated ileum by flavonoids. Phytotherapy Res. 1991; 5: 85- 87. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999; 12: 546-82. Di Carlo G, Mascolo N, Izzo AA, Capasso F. Flavonoids: Old and new aspects of a class of natural therapeutic drugs. Life Sci. 1999; 65: 337-53. Dixon RA, Dey PM, Lamb CJ. Phytoalexins: Enzymology and molecular biology. Adv Enzymol. 1983; 55: 1-69. Farre AJ, Columbo M, Fort M, Gutierrez B. Differential effects of various Ca++ antagonists. Gen Pharmacol. 1991; 22: 177- 81. Gharib, Naseri MK, Mohammadian M, Gharib Z. Antispas- modic effect of Physalis alkekengi fruit extract on rat uterus. Iran J Reprod Med. 2008; 6: 193-98. Ghayur MN, Gilani AH, Khan A, Amor EC, Villasenor IM, Choudhary MI. Presence of calcium antagonist activity explains the use of Syzygium samarangense in diarrhoea. Phytotherapy Res. 2006; 20: 49-52. Gilani AH, Jabeen Q, Khan, AU, Shah AJ. Gut modulatory, blood pressure lowering, diuretic and sedative activities of cardamom. J Ethnopharmacol. 2008; 115: 463-72. Gilani AH, Shaheen F, Zaman M, Janbaz KH, Shah BH, Akhtar MS. Studies on antihypertensive and antispasmodic activities of methanolic extract of Acacia nilotica. Phytotherapy Res. 1999; 13: 665-69. Haslam E. Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. J Nat Prod. 1996; 59: 205-15. Ibrahim M, Khan R, Malik A. Two new guaianolides from Amberboa ramosa. Nat Prod Commun. 2010; 5: 1865-68. Jabeen Q, Bashir S, Lyoussi B, Gilani AH. Coriander fruit exhibits gut modulatory, blood pressure lowering and diuretic activities. J Ethnopharmacol. 2009; 122: 123-30. Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano MS, Harada KI. Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev. 1997; 49: 157-230. Ksouri R, Falleh H, Megdiche W, Trabelsi N, Mhamdi B, Chaieb K, Bakrouf A, Magne C, Abdelly C. Anti-oxidant and antimicrobial activities of the edible medicinal halophyte Tamarix gallica L. and related polyphenolic constituents. J Food Chem Toxicol. 2009; 47: 2083-91. Mason TL, Wasserman BP. Inactivation of red beet betaglucan synthase by native and oxidized phenolic compounds. Phytochemistry 1987; 26: 2197-2202. Niciforovic N, Mihailovic V, Maskovic P, Solujic S, Stojkovic A, Muratspahic DP. Anti-oxidant activity of selected plant species: Potential new sources of natural anti-oxidants. Food Chem Toxicol. 2010; 48: 3125-30. Nychas GJE. Natural antimicrobials from plants. In: New methods of food preservation. Gould GW. (ed). London Blackie Academic and Professional, 1995, pp 58-89. Pan Y, Wang K, Huang S, Wang H, Mu X, He C, Ji X, Zhang J, Huang F. Anti-oxidant activity of microwave-assisted extract of longan (Dimocarpus longan Lour.) peel. Food Chem. 2008; 106: 1264-70. Pietrobon D, Virgilio F, Pozzan T. Structural and functional aspects of calcium homeostasis in eukaryotic cells. Eur J Biochem. 1990; 193: 599-622. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of anti-oxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E1. Analyt Biochem. 1999; 269: 337-41. Qureshi R, Bhatti GR. Ethnobotany of plants used by the Thari people of Nara Desert, Pakistan. Fitoterapia 2008; 79: 468-73. Raja-Kannan RR, Arumugam R, Anantharaman P. In vitro anti- oxidant activities of ethanol extract from Enhalus acoroides (L.F.) Royle. Asian Pacific J Tropical Med. 2010; ??: 898-901. Reddy BS, Reddy RKK, Naidu VGM, Madhusudhana K, Agwane SB, Ramakrishna S, Diwan PR. Evaluation of antimi-crobial, anti-oxidant and wound healing potentials of Holoptelea integrifolia. J Ethnopharmacol. 2008; 115: 249-56. Roman C, Gonella J. Extrinsic control of digestive tract motility. In: Physiology of the gastrointestinal tract. Johnson LR (ed). 2nd ed. New York. Raven, 1987, pp 507-53. Sahin F, Karaman I, Gulluce M, Ogutcu H, Sengul M, Adiguzel A, Ozturk S, Kotan R. Evaluation of antimicrobial activities of Satureja hortensis L. J Ethnopharmacol. 2003; 87: 61-65. Salama HMH, Marraiki N. Antimicrobial activity and phytoche-mical analyses of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt. Saudi J Bio Sci. 2010; 17: 57-63. Scalbert A. Antimicrobial properties of tannins. Phytochemis- try 1991; 30: 3875-83. Schultes RE. The kingdom of plants. In: Medicines from the earth. Thomson WAR (ed). New York, McGraw-Hill Book Co., 1978, p 208. Singh RP, Murthy KNC, Jayaprakasha GK. Studies on the anti- oxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J Agricul Food Chem. 2002; 50: 81-86. Stern JL, Hagerman AE, Steinberg PD, Mason PK. Bangladesh J Pharmacol 2014; 9: 29-36 35 Phlorotannin-protein interactios. J Chem Ecol. 1996; 22: 1887 -99. Tsuchiya H, Sato M, Miyazaki T, Fujiwara S, Tanigaki S, Ohyama M, Tanaka T, Linuma M. Comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant Staphylococcus aureus. J Ethnopharmacol. 1996; 50: 27-34. Usman H, Abdulrahman FI, Usman A. Qualitative phytochemi -cal screening and in vitro antimicrobial effects of methanol stem bark extract of Ficus Thonningii (Moraceae). Afr J Tradit Complement Altern Med. 2009; 6: 289-95. Vardhana R. Direct uses of medicinal plants and their identifi- cation. 1st ed. New Delhi, India, Sarup and Sons, 2008, p 360. 36 Bangladesh J Pharmacol 2014; 9: 29-36 Author Info Aamir Mushtaq (Principal contact) e-mail: aamir_mushtaq@hotmail.com ADCr 003mgml: undefined: undefined_2: undefined_3: undefined_4: undefined_5: undefined_6: undefined_7: DatePrinted: This article was downloaded by you on: Sep 15, 2018