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INDEX Medicinal Plant Research Pharmacology HYPOCHOLESTEROLEMIC ACTIVITIES OF AQUEOUS AND ETHANOL EXTRACTS OF EQUISETUM HIEMALE L. Jun Yang, Yan Zhang, Zuozhao Wang, Jingbo Liu………………………………… ……. 87-96 Natural Sciences & Life INVENTORY OF MEDICINAL PLANTS USED FOR TRADITIONAL TREATMENT OF ECZEMA IN THE REGION OF HODNA (M’SILA ALGERIA) Madani SARI, Noui HENDEL, Amel BOUDJELAL and Djamel SARRI……………….. 97-100 Bio-Chemistry PHYSICOCHEMICAL COMPOSITION OF INDONESIAN VELVET BEAN (MUCUNA PRURIENS L.) Ratnaningsih Eko Sardjon, Iqbal Musthapa, Hayat Sholihin, Rizal Pauzan Ramdhani….....101-108 Microbiology ANTIBACTERIAL ACTIVITY OF SEEDS OF MUCUNA PRURIENS L. AGAINST ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS Deshwal Vishal Kumar…………………………………………………………….............. 109-113 Bio-chemistry PHYTOCHEMICAL SCREENING OF ANTHOCLEISTA GRANDIFLORA GILG. STEM BARK Odeghe Othuke Bensandy, Uwakwe Augustine A, Monago Comfort…………………….. 114-122 Bio-Technology REVIEW ON DATURA METEL: A POTENTIAL MEDICINAL PLANT Khaton Monira and Shaik Munan………………………………………………………….. 123-132
Indigenous Medicine Ayurveda AN AROMATIC PLANT OF BASMATI FLAVOR: PANDANUS AMARYLLIFOLIUS ROXB. Jyothi T, Niranjan Y, Harisha CR………………………………………………………… 133-139 COVER PAGE PHOTOGRAPHY : DR. HARI VENKATESH K R, PLANT ID – FLOWER OF GLORIOSA SUPERBA L. PLACE – KOPPA, CHIKMAGALUR DISTRICT, KARNATAKA, INDIA Global Journal of Research on Medicinal Plants & Indigenous Medicine
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GJRMI, Volume 1, Issue 4, April 2012, 87 - 96
Original Research Article HYPOCHOLESTEROLEMIC ACTIVITIES OF AQUEOUS AND ETHANOL EXTRACTS OF EQUISETUM HIEMALE L.
Jun Yang1, Yan Zhang2, Zuozhao Wang3, Jingbo Liu4﹡
1,2,3,4
Laboratory of Nutrition and Functional Food, Jilin University, Xi’an Road 5333#, Changchun 130062, P.R. China ﹡ Corresponding author - E-mail: ljb168@sohu.com; jingboliu@yahoo.cn. Tel: +86-431-87836351. Fax: +86-431-87835760
Received: 22/02/2012;
Revised: 12/03/2012;
Accepted: 16/03/2012;
ABSTRACT The main purpose of this study was to evaluate the hypo-cholesterolemic effect of aqueous (AE) and ethanol (EE) extracts of Equisetum hiemale L. stems in diet-induced hyper-lipidemic rats. The oral administration of AE and EE at the dose of 10, 20, and 40 gbw/day were found to ameliorate the hyper-lipidemic condition by dose-dependently reducing serum concentrations of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), coronary heart disease (CHD) risk ratio and by dose-dependently increasing serum high-density lipoprotein cholesterol (HDL-C) content. Further, the oral administration of AE and EE dose-dependently reduced liver triglyceride and cholesterol content. Additionally the AE and EE treatment dosedependently increased fecal bile acids, triglyceride and cholesterol excretion. The results suggest that both the aqueous and ethanol extracts of Equisetum hiemale L. had triglyceride- and cholesterollowering effect. One of their mechanisms of hypo-cholesterolemic action may be due to the promotion of fecal cholesterol and bile acids excretion.
Key words: Equisetum hiemale L., aqueous and ethanol extracts, hypo-cholesterolemic effect
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INTRODUCTION Coronary heart disease (CHD) is the single largest cause of death in the world, accounting 12.8% of deaths (WHO, 2011). There are many risk factors associated with CHD. The major risk factors are tobacco use, alcohol use, high blood pressure, high cholesterol, obesity, physical inactivity and unhealthy diets. One of the major modifiable risk factors for CHD is abnormal blood lipid levels, that is high total cholesterol (TC), high levels of triglycerides (TG), high levels of low-density lipoprotein cholesterol (LDLC) or low levels of high-density lipoprotein cholesterol (HDL-C) (Mackay et al., 2004). Various drugs were developed to maintain normal lipid homeostasis, such as statins, bile acid sequestrants, nicotinic acid and fibrates. But most of them have severe side effects, including myopathy, hyperglycemia, hyperuricemia, hepatotoxicity, cholesterol gallstones and gastrointestinal complaints (NCEP, 2002). Thus the plants with potential hypocholesterolemic activity and favorable safety profiles have recently attracted considerable attention, such as garlic (Yeh and Liu, 2001; Ginter and Simko, 2010), curcumin (Wongcharoen and Phrommintikul, 2009), Artichoke (Pittler et al., 2009), fenugreek (Vijayakumar et al., 2010), coconut (Zakaria et al., 2010; Nevin and Rajamohan, 2009) and Butea monosperma (Lamk.) Taub. (Akhtar et al., 2010; Bavarva and Narasimhacharya, 2008). Equisetum hiemale L., commonly known as “scouring rush”, is a traditional Chinese medicine widely distributed in the world (USDA, NRCS). In herbal medicine, the
roots and stems are used to treat “cloudy eye” (Pharmacopoeia of P.R. China, 2010) and also act as a diuretic agent (Perez Gutierrez et al., 1985). In our previous study, E. hiemale L. was found to exhibit potential cholesterol-lowering effect, thus the present study was carried out to validate the hypocholesterolemia effect of E. hiemale L. MATERIALS AND METHODS Ethics The protocol of this study was approved by the institutional internal review board. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (National Research Council, 1996). Plant material and extraction procedure Equisetum hiemale L. was harvested in early October 2010 from Changbai Mountain in eastern China, identified by Professor Dacheng Jiang (engaged in Pharmacognosy) in Changchun University of Chinese Medicine. Fresh E. hiemale L. stems were dried at 40°C in a hot air oven, ground in a blender to obtain a fine powder (particle diameter size: 250– 850 µm) followed by extraction using reflux for 3.0 h with liquid to raw material ratio of 30 at 90°C (for water extract) or 70°C (for ethanol extract). The extracts were filtered, centrifuged and concentrated followed by lyophilization. The dried extracts were dissolved in double-distilled water and the water extract of E. hiemale L. was assigned as AE, whereas, ethanolic extract as EE.
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Plate 1. The Plant: Equisetum hiemale L.
Plate 2. Equisetum hiemale L. in its Habitat
Plate 3. Crude drug used for the study
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Chemicals Kits for analytical determination of total cholesterol, HDL-C, LDL-C, triglycerides, and bile acids were obtained from Chang Chun Huili Biotech Co., Ltd (Changchun, China). All other reagents and chemicals used were of analytical grade and purchased from Beijing Chemical Works (Beijing, China). Animals and diets Eighty male Wistar rats (480±10 g, SPF) after physical and behavioral examination were obtained from Experimental Animal Centre of Jilin University (Changchun, China), then individually housed in polypropylene cage (320×215×170 mm) with stainless steel cover, containing dust-free sterilized softwood shaving as bedding material, which was renewed every three days. Animals were given 2 weeks of acclimation before feeding experiment. The feeding procedure was carried out in a room under following conditions: temperature of 23±2°C, relative humidity 50– 60%, ventilation frequency ≥10 times/h, and a 12 h light/dark cycle. Laboratory animal diets were provided by Experimental Animal Centre of Jilin University. Normal diet was produced according to Chinese national standards (GB 14924-2001). High-cholesterol diet (HCD) was produced according to the following formula: 90% commercial powdered diet (GB 14924-2001) supplemented with 2% cholesterol, 0.5% bile salts and 7.5% beef tallow. Animal treatment Eighty male Wistar rats were randomly divided into 8 groups: Group 1 (Control group): fed on normal diet (ND) and orally administered normal sodium. Group 2 (Model group): fed on highcholesterol diet (HCD) and orally administered normal sodium (1 ml/100 gbw/day). Group 3 (AE-L group): fed on HCD and orally administered AE (10 mg/100 gbw/day).
Group 4 (AE-M group): fed on HCD and orally administered AE (20 mg/100 gbw/day). Group 5 (AE-H group): fed on HCD and orally administered AE (40 mg/100 gbw/day). Group 6 (EE-L group): fed on HCD and orally administered EE (10 mg/100 gbw/day). Group 7: (EE-M group) fed on HCD and orally administered EE (20 mg/100 gbw/day). Group 8: (EE-H group) fed on HCD and orally administered EE (40 mg/100 gbw/day). After 4 weeks of treatment, animal blood, liver, and feces were collected to determine triglycerides (TG), cholesterol (TC, LDL-C, and HDL-C) and bile acid content. Measurement of serum, liver, and fecal lipids Serum TG, TC, HDL-C, and LDL-C levels were measured enzymatically using a Beckman CX-9 auto-analyzer (Beckman Coultes Inc, Fullerton, CA, USA). The coronary heart diseases (CHD) risk ratio was later calculated as the ratio of TC/HDL-C. Lipids in liver and excrement were extracted, purified and solubilized in water using the method described by Folch et al. (1957) and Carr et al. (1993), measured by the same procedures used to determine serum lipids. Fecal bile acid was obtained according to the procedure described by Huang et al. (2010) and determined enzymatically on Beckman CX-9 auto-analyzer. Statistical analysis All data presented are means ± standard deviation (SD). Statistical analyses were calculated using one-way analysis of variance (ANOVA). The Ducan multiple rank test was performed to determine statistical significance among groups using SPSS software version 11.5 (SPSS Inc, Chicago, IL).
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RESULTS Body weight gain of experimental rats The body weight increased in all groups throughout the treatment without any significant differences between them (figure 1). Serum lipid and lipoprotein cholesterol The concentrations of TG, TC, HDL-C, and LDL-C in the rats of model group showed significant increase (P<0.01) after 4 weeks of treatment as compared to the control (Table 1). A significant increase of CHD risk ratio of rats in the model group was also observed (P<0.05). Both the administration of aqueous extract of E.
hiemale L. (AE) and ethanol extract of E. hiemale L. (EE) ameliorated the hyperlipidemic condition by reducing serum concentrations of TG, TC, LDL-C, CHD risk ratio and increasing serum HDL-C content in a dose-dependent way. And the hyperlipidemiaameliorating effects reached significant levels when higher concentrations of AE and EE (20, 40 mg/100 gbw/day) were administered to the rats fed on HCD compared with the rats in the model group (P<0.05). It can be seen from table 1 that the same concentrations of AE and EE exhibited similar hypo-cholesterolemic activity.
Table 1. Serum lipid and lipoprotein cholesterol concentrations in rats fed on different diets at the end of experiment. Group Control Model AE-L AE-M AE-H EE-L EE-M EE-H
TG (mg/L) 1.05 ± 0.22a 2.66 ± 0.22b 1.95 ± 0.18c 1.28 ± 0.46ad 1.18 ± 0.35af 2.47 ± 0.30be 1.89 ± 0.54cde 1.74 ± 0.44cdf
TC (mg/L) 1.73 ± 0.13a 2.78 ± 0.10b 2.50 ± 0.11ce 2.30 ± 0.20cd 2.19 ± 0.15d 2.65 ± 0.23bef 2.41 ± 0.14cf 2.30 ± 0.25cdf
HDL-C (mg/L) 0.81 ± 0.09a 0.93 ± 0.09bd 0.88 ± 0.13ab 1.22 ± 0.13c 1.28 ± 0.18c 0.94 ± 0.12ab 1.14 ± 0.08cd 1.24 ± 0.18cd
LDL-C (mg/L) 0.51 ± 0.07a 1.20 ± 0.24b 0.90 ± 0.17b 0.60 ± 0.10a 0.55 ± 0.06a 0.94 ± 0.14b 0.61 ± 0.11a 0.57 ± 0.16a
CHD risk ratio 2.16 ± 0.22a 3.02 ± 0.33b 2.90 ± 0.40b 1.89 ± 0.20ac 1.73 ± 0.22cd 2.83 ± 0.32b 2.12 ± 0.15a 1.88 ± 0.27ad
Values are expressed as mean ± SEM of 5 rats per group. abcdefValues sharing a common letter in the same row are not significantly different (P<0.05).
a
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Figure 1. Growth curves of experimental rats during the 4-week feeding period. Each bar represents mean Âą SD of 10 rats.
Liver weight and lipids Liver weight index was significantly different in the control group and all other groups fed on HCD (figure 2). Among different doses of EE, the administration of high dose of EE (40 mg/100 gbw/day) significantly reduced the liver weight index of rats compared to the rats in model group (P<0.05). Both TG and TC concentrations in liver were significantly increased by the HCD (P<0.05). The AE and EE treatment dosedependently reduced liver concentrations of TG and TC. The liver TG and TC-lowering effect reached significant levels when high concentrations of AE and EE (40 mg/100 gbw/day) were administered to the rats fed on HCD (AE-H and EE-H group) compared with
the rats in model group (P<0.05). Same concentrations of AE and EE showed similar liver TG and TC-lowering effect. Fecal lipids Fecal excretion levels of bile acids, triglyceride and cholesterol of rats fed on HCD showed a significant increase compared with rats in the control group (P<0.01). The administration of AE and EE dose-dependently increased fecal concentrations of bile acids, TG and TC. High dose of AE and EE (40 mg/100 gbw/day) significantly increased fecal extraction of bile acids, fecal triglycerides and cholesterol compared with the rats in model group (P<0.05). Same concentrations of AE and EE showed similar fecal bile acids, TG and TC-lowering effect.
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Figure 2. Liver weight index, triglyceride and cholesterol levels of experimental rats. Values are expressed as mean Âą SEM of 5 rats per group. abcdeValues sharing a common letter in the same group of bars are not significantly different (P<0.05).
Figure 3. Fecal bile acids, triglyceride and cholesterol of rats fed on experimental diet. Values are expressed as mean Âą SEM of 5 rats per group. abcde Values sharing a common letter in the same group of bars are not significantly different.
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DISCUSSION AND CONCLUSION The present study investigated the effect of aqueous and ethanol extract of E. hiemale L. on the lipid profiles in rats fed a high cholesterol diet. The result showed that both aqueous and ethanol extracts of E. hiemale L. reduced hypertriglyceridemia and hypercholesterolemia. Rats in the model group had higher concentrations of TG, TC, LDL-C, and CHD risk ratio in serum than in those of the control group, indicating that the hypercholesterolemic model was successfully established. In addition, rats fed on HCD developed a fatty liver with a high triglyceride and cholesterol content. This could be due to the continuous intake of cholesterol and beef tallow. It is reported that dietary cholesterol and fatty acids stimulate each other's biosynthesis in the liver of rats (Fungwe et al., 1994). The higher doses of aqueous and ethanol extracts of E. hiemale L. (20, 40 mg/100 gbw/day) significantly lowered serum TG, TC, LDL-C and increased serum HDL-C in the rats fed on HCD diet. It is widely acknowledged that elevated serum TC, LDL-C and low HDL-C are predictors for atherosclerosis and CHD or even death from CHD (Roberts, 1995; Pekkanen, et al., 1990). The Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program considered elevated serum LDL cholesterol level as a major cause of CHD, decreased serum HDL cholesterol content as an independent risk factor for CHD while serum TG and TC/HDL-C as emerging risk factors (NCEP, 2002). Thus the extracts with hypocholesterolemic effect have potential protective effect against CHD.
The TC content in the liver of rats fed on HCD was remarkably lowered by administration of higher dose of aqueous and ethanol extracts of E. hiemale L. (20, 40 mg/100 gbw/day), it could be due to the increased fecal bile acids and cholesterol excretion. The increase in excretion of bile acids and cholesterol would activate cholesterol 7Îą-hydroxylase, which is the rate determining enzyme in the conversion of cholesterol to bile acids. The increase in cholesterol 7Îąhydroxylase can enhance the conversion of liver cholesterol to bile acids for excretion, which is the major pathway of cholesterol elimination (Yang and Koo, 2000; Chiang, 2009), which results in a reduction of hepatic cholesterol content. Since the expression of the LDL receptor is controlled by feedback inhibition of intracellular cholesterol, the reduction of hepatic cholesterol content in turn up-regulate the expression of LDL receptors. This results in an increase in clearance of cholesterol from the circulation by LDL receptor and thus lowers blood cholesterol (Brown and Goldstein, 1986). The present study demonstrated that both the aqueous and ethanol extracts of E. hiemale L. had hypo-cholesterolemic effect. They were found to reduce the total and LDL cholesterol, improve HDL cholesterol, and lower the CHD risk ratio in the rats fed a HCD. One of their mechanisms of hypo-cholesterolemic action may be due to the promotion of fecal cholesterol and bile acids excretion.
ACKNOWLEDGEMENTS Financial help from Science and Technology Development Program of Jilin Province Science and Technology Committee is gratefully acknowledged.
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REFERENCES Akhtar MS, Naeem F, Muhammad F, Bhatty N (2010). Effect of Butea monosperma (Lamk.) Taub. (Palas papra) fruit on blood glucose and lipid profiles of normal and diabetic human volunteers. Afr. J. Pharm. Pharmacol. 4:539– 544.
(FPS) prevents hypercholesterolemia in Rats. Lipids Health Dis. 9:9.
Bavarva JH, Narasimhacharya AVRL (2008). Preliminary study on antihyperglycemic and antihyperlipaemic effects of Butea monosperma in NIDDM rats. Fitoterapia 79:328–331.
National Cholesterol Education Program (NCEP) (2002). Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation 106: 3143– 3421.
Brown MS, Goldstein JL (1986). A receptormediated pathway for cholesterol homeostasis. Science 232:34–47. Carr TP, Andresen CJ, Rudel LL (1993). Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin Biochem. 26:39–42. Chiang JYL (2009). Bile acids: regulation of synthesis. J. Lipid Res. 50:1955–1966.
Mackay, J. and G. A. Mesah (2004). The atlas of heart disease and stroke. Geneva: World Health Organization, pp. 24–25.
Nevin KG, Rajamohan T (2009). Wet and dry extraction of coconut oil: impact on lipid metabolic and antioxidant status in cholesterol coadministered rats. Can. J. Physiol. Pharmacol. 87:610–616.
Committee for the Pharmacopoeia of P.R. China, 2010 Committee for the Pharmacopoeia of P.R. China (2010), Pharmacopoeia of P.R. China vol. I, China Medicinal Science Press, Beijing, pp. 58.
Pekkanen J, Linn S, Heiss G, Suchindran CM, Leon A, Rifkind BM, Tyroler HA (1990). Tenyear mortality from cardiovascular disease in relation to cholesterol level among men with and without preexisting cardiovascular disease. N. Engl. J. Med. 322:1700–1707.
Folch J, Lees M, Sloane Stanley GH (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–509.
Perez Gutierrez RM, Laguna GY, Walkowski A (1985). Diuretic activity of Mexican equisetum. J. Ethnopharmacol. 14:269–272.
Fungwe TV, Fox JE, Cagen LM, Wilcox HG, Heimberg M (1994). Stimulation of fatty acid biosynthesis by dietary cholesterol and of cholesterol synthesis by dietary fatty acid. J. Lipid Res. 35:311–318. Ginter E, Simko V (2010). Garlic (Allium sativum L.) and cardiovascular diseases. Bratisl Lek Listy. 111:452–456. Huang XQ, Tang J, Zhou Q, Lu HP, Wu YL, Wu WK (2010). Polysaccharide from Fuzi
Pittler MH, Thompson CO, Ernst E (2002). Artichoke leaf extract for treating hypercholesterolaemia. Cochrane Database Syst Rev. CD003335. Roberts WC (1995). Preventing and arresting coronary atherosclerosis. Am. Heart. J. 130:580–600. USDA, NRCS. 2004. The PLANTS Database, Version 3.5 (http://plants.usda.gov), National Plant Data Center, Baton Rouge, LA, USA.
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Vijayakumar MV, Pandey V, Mishra GC, Bhat MK (2010). Hypolipidemic effect of fenugreek seeds is mediated through inhibition of fat accumulation and upregulation of LDL receptor. Obesity 18:667–674. WHO (World Health Organization) (2011). The 10 leading causes of death by broad income group (2008). http://www.who.int/mediacentre/factsheets/f s310/en/index.html. Wongcharoen W, Phrommintikul A (2009). The protective role of curcumin in cardiovascular diseases. International Int. J. Cardiol. 133:145– 151.
Source of Support: Nil
Yang TTC, Koo MWL (2000). Chinese green tea lowers cholesterol level through an increase in fecal lipid excretion. Life Sci. 66:411–423. Yeh YY, Liu LJ (2001). Cholesterol-lowering effect of garlic extracts and organosulfur compounds: Human and animal studies. J. Nutr. 131:989S–993S. Zakaria ZA, Ahmad Z, Somchit MN, Arifah AK, Khairi HM, Sulaiman MR, Teh LK, Salleh MZ, Long K (2010). Antihypercholesterolemia property and fatty acid composition of mardiproduced virgin coconut oils. Afr. J. Pharm. Pharmacol. 4:636–644.
Conflict of Interest: None Declared
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Short Communication INVENTORY OF MEDICINAL PLANTS USED FOR TRADITIONAL TREATMENT OF ECZEMA IN THE REGION OF HODNA (M'SILA ALGERIA) Madani SARI1*, Noui HENDEL1, Amel BOUDJELAL1 and Djamel SARRI1 1
M'sila University, Faculty of Sciences, Department of Natural Sciences and Life, Ichbilia. BP. 166 28000, M'sila. (Algeria) *Corresponding Author E-mail: Mad_sari@yahoo.fr Received: 29/02/2012;
Revised: 17/03/2012;
Accepted: 21/03/2012;
ABSTRACT The ethno botany study in the region of Hodna helped to highlight the different traditional uses of plants by the villagers. The goal is to make an inventory of plants from traditional medicine that treats Eczema. 1900 question cards were established in order to obtain information on medicinal plants in the area of study that deals with Eczema, those targeted were herbalists, healers and villagers. The total numbers of people surveyed were 35 whose age was between 20 and 80. The result was the identification of 25 species distributed in 18 botanical families with a dominance of especially Lamiaceae, Liliaceae, Asteraceae and Oleaceae. Keywords: Medicinal plants, Traditional treatment, Eczema, Hodna, Algeria
INTRODUCTION Eczema is one among the group of skin diseases, causing inflammation, dry skin and itching. The two types of eczema are the most common atopic dermatitis and contact dermatitis. The first is an inflammation that occurs because to family history related to the affection, the second is an inflammation that occurs from exposure to allergens and irritant substances (Elwina, 2008). There is no known cure for eczema, thus treatments aim to control
the symptoms i e to reduce inflammation and relieve itching. Eczema is also treated in the field of herbal medicine. In order to discover the medicinal plants used in Eczema, an Ethnobotanical survey was conducted to promote traditional medicine, which is widely practiced in the region of Hodna. To enlist all the plants that heal the disease of contact dermatitis is the case in our study.
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MATERIALS AND METHODS Study area
Ethnobotanical surveys
The region of M'sila, occupies a privileged position in the central part of Northern Algeria. As a whole, it is part of the central highlands. It covers an area of 18,718 km2 and it is located at an altitude of 500 meters, situated between 35째 42' 07" N 4째 32' 49"E (W.G.S., 84). The climate of the investigation area is continental, due in part to the Saharan influences. Summer is hot and dry while winter is very cold, with low and irregular rainfall; it is of the order of 100 to 250 mm /year (Seltzer, 1946 and Le Houerou, 1995). Morphology and its geographical position gives this region a unified ecological aspect represented by the predominance of the steppe, which covers 1.2 million hectare (63% of the total area) of the state. The areas used for agriculture accounts for 20% of the total area devoted mainly to cereals, to arboriculture and market gardening.
The Ethnobotanical surveys were conducted from February 2006 to June 2010, information was collected on traditional uses of wild plants and also those cultivated. Using the 1900 questionnaire that have been developed, we conducted Ethnobotanical surveys of the entire M'sila region in order to have as much information regarding the traditional use of medicinal plants by local people because of their knowledge in ethno medicine. All investigations described the information about (Babba Aissa, 1999): - Date, - Research area (district/village), - Informants (name/age/sex/educational level), - Scientific name of plant, - Local name of plant, part of the plant used, - Usage purpose of the plant, - Dosage, - How to use it (decoction, infusion, etc.), - Usage period of the plant - Side effects of the plant.
Table 1: Age group of people surveyed
Age group 20-30 30-40 40-50 50-60 60-70 70-80 Total
Men 4 15 6 2 1 1 29
Women
1 1 0 3 0 1 6
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Total 5 16 6 5 1 2 35
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Table 2: Plants traditionally used to treat Eczema in the region of Hodna (M'sila-Algeria) Botanical name / Family
Local name
Number of Informants
Parts used
Preparation
Ajuga iva (L.) Schreb. (Lamiaceae) Allium cepa L. (Liliaceae) Allium sativum L. (Liliaceae) Anthemis nobilis L. (Asteraceae) Artemisia herba alba Asso. (Asteraceae) Atriplex halimus L. (Chénopodiaceae) Colocynthis vulgaris (L.) Lud. (Cucurbitaceae) Fraxinus excelsior L. (Oleaceae) Globularia alypum L. (Globulariaceae) Juniperus phoenicea L. (Cupressaceae) Lavandula stoechas L. (Lamiaceae) Nerium oleander L. (Apocynaceae) Olea europaea L. (Oleaceae) Pistacia lentiscus L. (Anacardiaceae) Quercus ilex L. (Fagaceae) Retama retam Webb. (Fabaceae) Ricinus communis L (Euphorbiaceae) Rosmarinus officinalis L. (Lamiaceae) Ruta chalepensis L. (Rutaceae) Salvia officinalis L. (Lamiaceae) Teucruim polium L. (Lamiaceae) Thapsia garganica L. (Apiaceae) Thymelaea hirsuta Endl. (Thymelaeaceae) Viola odorata L. (Violaceae) Ziziphus lotus (L) Desf. (Rhamnaceae)
Chendgoura
2
Leaf
Decoction, maceration
El Basla
1
Pulp
Unction
Thoum
1
Fruit
Cataplasm
Babounej
4
Flower
Infusion, decoction, lotion, poultice
Chih
1
Aerial part
Decoction, lotion, maceration
G’taf
1
Leaf
Lotion
Hadj
1
Flower
Decoction, massage.
Dardar
1
Leaf
Infusion, powdered
Tesselgha
1
Whole plant
Decoction
Ara-aar
2
Aerial part
Lotion, infusion, decoction
Khozama
1
Aerial part
Decoction, maceration
Defla
2
Leaf
Cinder, decoction
Zitoune
1
Fruit
Oil (unction)
Dharou
1
Fruit
Oil (unction)
Balout
1
Leaf
Powdered, decoction
R’tem
1
Aerial part
Lotion
Kharoua
1
Fruit
Decoction, Oil
Iklil el djabal
1
Aerial part
Infusion
Fidjel
1
Aerial part
Decoction, infusion, powdered
Swak Nbi
1
Aerial part
Infusion
Khayata
1
Aerial part
Decoction, maceration
Bounafaa
1
Aerial part
Cataplasm
Methnane
2
Aerial part
Infusion, decoction, maceration
Banafsadj
1
Flower
Extract (compress)
Sedra
1
Leaf
Lotion, infusion
RESULTS AND DISCUSSION With the help of flora of Quezel and Santa (1962–1963), Ozenda (1983) and Maire (1952– 1987) and the herbarium of the Department of
Natural Sciences and Life, the University of M'sila, we determined the species collected in the field to compile a complete list of medicinal
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species identified in the study area, parallel to the results of surveys of villagers « 35 people: 6 women and 29 men of different ages, the informants age ranges are between 20 to 80 years » (Table 1). The results showed that medicinal species used in treating Eczema were 25 which belong to 18 families and 24 genera (Table 2). It was noted that species from Lamiaceae family was high (20%). The plant parts most commonly used were the aerial parts (42.30%), leafs (26.92%), flowers (11.74%). Instructions of plant usage varied. The most common form of preparation used was decoction (28.89%), followed by infusion (15.56%), lotion (13.34%), and maceration (11.11%). CONCLUSION This study has shed light on the different traditional uses of plants by villagers in the region of Hodna (M'sila) to treat Eczema. This work provides a list of classification of medicinal plants that treat Eczema in the M'sila region. We identified 25 medicinal species distributed in 18 botanical families with a dominance especially of Lamiaceae. Thus, the purpose of this study was an inventory of traditional uses of medicinal plants in the Hodna and to provide baseline data for future pharmacological and phytochemical studies. ACKNOWLEDGEMENTS We are very much grateful to all the local herbalists, healers and villagers who shared their knowledge on the use of medicinal plants with us. Without their contribution, this study would have been impossible. We would also like to thank the CNEPRU for the financial support to conduct this study.
Source of Support: Nil
REFERENCES Babba Aïssa F. (1999) Encyclopedia useful plants. Flora of Algeria and the Maghreb. Vegetable substances from Africa, East and the West. Modern Library Rouiba, EDAS, Algiers, Algeria. Elwina (2008) Http://www.consoglobe.com/solutionseczema-2676-cg Le Houerou HN. (1995) Bioclimatology and Biogeography of the arid steppes of North Africa. biodiversity, sustainable development and desertisation. Mediterranean optional, Serial B, 10. Maire R. (1952_1987) Flora of North Africa (Morocco, Algeria, Tunisia, Tripolitania, Cyrenaica and the Sahara). 16 vol., Le Chevalier Publisher, Paris. Ozenda P. (1983) Flora of the Northern Sahara. CNRS, Paris. Quezel P. and Santa S. (1962_1963) New flora of Algeria and Southern desert regions. 2 Tomes, CNRS, Paris. Seltzer P. (1946) The climate of Algeria. Inst. Weath. and Phys. Algiers University Press. WGS 84 (1984) World Geodetic System, Wikipedia, map of M'sila region.
Conflict of Interest: None Declared
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Original Research Article PHYSICOCHEMICAL COMPOSITION OF INDONESIAN VELVET BEAN (MUCUNA PRURIENS L.) Ratnaningsih Eko Sardjon1, Iqbal Musthapa*1, Hayat Sholihin1, Rizal Pauzan Ramdhani1 1
Biological Chemistry Group, Chemistry Department, Indonesia University of Education Corresponding Author: *iqbalmust@yahoo.com
Received: 10/02/2012;
Revised: 19/03/2012;
Accepted: 22/03/2012;
ABSTRACT Mucuna pruriens var. utilis or Velvet bean is one of the native plants of tropical regions, including Indonesia. It has been used as a traditional food in some countries. The metal and microbial analysis showed that both seed and shell of Indonesian velvet bean can be consumed safely, because their concentration was below the maximum level required. Phytochemical analysis indicated the presence of alkaloids, tannins, saponins and steroids in both seeds and shells. The high performance liquid chromatography analysis showed that the seeds contained higher amount (7.56%) of 3,4-dihydroxy-L-phenylalanine (L-DOPA) than that of shells (3.89%).
Key words: Mucuna pruriens, physico-chemical, L-DOPA
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INTRODUCTION Mucuna pruriens (L.) DC. Var. utilis (Wall.ex Wight) Bak. ex Burck.), the Mucuna bean or karabenguk (Indonesia) is also known as velvet bean. Among the wild legumes, the genus Mucuna is widespread in tropical and sub-tropical regions of the world and considered as an alternative protein source. M. pruriens is an under-utilized legume species grown predominantly in Asia, Africa and in some parts of the America (Vadivel and Janardhanan, 2000). Traditionally in Indonesia, the mature seeds of Mucuna bean are consumed by a Central Java tribe, which is boiled repeatly and fermented to prepare a traditional recipe, called tempe. The demand on these seeds
increased after the discovery that Mucuna seeds contain (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid or 3,4-dihydroxy-Lphenylalanine (L-DOPA) an anti-parkinsons disease drug (Farooqi et al., 1999; Mohan and Kala, 2010). Physicochemical properties of the velvet bean from different locations has been studied (Teixeira et al., 2003; Mohan and Kala, 2010; Ujowundu et al. 2010; Raju et.al, 2010) which provided diverse compositions. However, there is no report on the physicochemical properties of velvet bean from Indonesia. In this paper, we have reported the physicochemical composition and determine the level of L-DOPA from shells and seeds of Indonesian velvet bean.
Figure 1. Structure of (S)-2-amino-3-(3, 4-dihydroxyphenyl)-propanoic acid (L-DOPA) O HO HO
MATERIALS AND METHODS Collection of seed samples The samples of velvet bean, Mucuna pruriens (L.) were collected from Bantul district area, Yogyakarta, Indonesia. After thoroughly drying under sun, the shells and seeds were separated and pulverized with a grinding machine. Determination of metals and microbial contaminant Determination of metal contaminant conducted to determine the level of Lead (Pb), Cadmium (Cd), Tin (Sn), Mercury (Hg) and Arsenic (As) in shells and seeds of velvet bean. Metal contaminant procedure refers to the Indonesian National Standard (SNI) 01-28961998 and 01-4866-1998. On the other hand, determination of microbial contamination was conducted to determine E. coli, Salmonella,
OH NH2
fungi, Yeast, C. perfringens and Bacillus cereus and the procedure refers to ISO 28972008. Those determination were carried out at Balai Besar Industri Agro, Agro Based Industry Calibration and Analytical Laboratories (ABICAL), Bogor, Indonesia. Phytochemical screening Seeds and shells of velvet bean (M. pruriens) were extracted with solution of ethanol:water (1:1) at pH=3 (adjusted with citric acid), using maceration for 3x24 h. This test was performed to determine the secondary metabolites groups contained in the velvet bean extract. Alkaloid test: Alkaloid test was carried out by adding few drops of Mayer's reagent to the solution of 0.5 g in 1 ml of chloroform. Formation of white precipitate indicates presence of alkaloids. Reagent Mayer was prepared by 1 gram KI dissolved in 20 ml of
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distilled water, and 0.271 g HgCl2 was added to a solution of KI Tannin test: Test was done by weighing 0.1 g of extract dissolved in 2 ml of water and then adding a few drops of 1% FeCl3. The appearance of dark blue color indicates the presence of tannin (phenolic).
prepared by dissolving 10 mg extract in 10 ml of mobile phase. The mixture was homogenized with ultrasonic for 10 minutes and filtered through 0.45 µm membrane. 20 µl the solution was injected into HPLC. RESULTS AND DISCUSSION Morphological Characteristics
Saponin test: Saponin test was conducted by dissolving 0.1 g of extract in 3 ml of water, then shaken vigorously for 10 minutes. The emergence of foam indicates the presence of saponin. Steroid test: Steroids test was conducted by dissolving 0.1 g of extract in 1 ml of water, then adding 1 ml of CH3COOH and 1 ml of concentrated H2SO4 to it. The emergence of the blue or purple color indicates presence of steroids. Flavonoids test: Flavonoid test was conducted by dissolving 0.1 g of extract in 3 ml of water and then adding 0.1 g of Mg powder and 1 ml of concentrated HCl. Yellow color indicates the presence of flavonoids. Determination of L-DOPA levels using HPLC method The HPLC system consisted of peak series Shimadzu and isocratic pump with UV visible detector. Analysis of L-DOPA was carried out at 280 nm by using a chromosil C18 reverse phase column of 250 x 4.6 mm. The mobile phase consisted of water, methanol and phosphoric acid in the ratio of 97 ml : 20 ml : 1 ml or 82.20:16.95:0.85 and flow rate of 1 ml/min. Standard and Sample Solutions A stock solution containing 0.1 mg/ml of LDOPA was prepared in the mobile phase. Using appropriate aliquots, different dilutions of standard solution in the range of 25–50 µg/ml were prepared. A sample solution was
The morphological characteristics of seeds and shells of Indonesian velvet bean are shown in Figure 2. The beans were sorted, cleaned, separated between seeds and shells and dried. Grinding the dried bean gave seed and shells powder in ratio 86.2:13.8. The separation between seeds and shells was carried out to determine the physicochemical characteristics of each part of the beans. The morphological characteristics of Indonesian velvet bean are shown in Table 1. The beans were homogenous in shape, diameter, length, weight, smell, and taste, with various colours, i.e. white, black or white with black dots. Colour variation was characteristic of velvet beans, and it would not be an difficult to standardize the sample. Moisture and Ash Content The amount of water content in the material is associated with the purity and the presence of contaminants in the simplicia. Determination of ash content provides an overview of internal and external mineral content derived from the initial process to obtain good simplicia and extracts derived from natural plants and contaminants during the process. The number of maximum allowable ash content is associated with purity and contamination. Water and ash content contained in shells and seeds of Indonesian velvet beans are shown in Table 2. The results indicated that seed and shell contained moisture and ash contents below the maximum level. Therefore, the seed and shell of Indonesian velvet are safe to be used as a drug or food.
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Figure 2. The physical performance: (a) Indonesian velvet beans (b) Seeds (c) shells
(a)
(b)
(C)
Table 1. The morphology characteristics of Indonesian velvet bean The morphology parameter Shape Diameter Lenght Weight Colour Smell Taste
Indonesian velvet beans Oval and flat 0.682 cm 1.491 cm 0.963 g White, black, white with black dots Characteristic of velvet bean bitter
Table 2. Moisture and ash content of shells and seeds of Indonesian velvet bean Parameter
Shell
Seed
Moisture content (%) 9.14 10.80 Ash content (%) 2.18 3.04 *Indonesia Ministry of Health Regulations no 661/MENKES/SK/VII/1994
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Standard* (Maximum Level) 10 10
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Metals contaminants Metal measurements were performed to determine how many metal contaminants were contained in the sample. The measurements includes that of Lead (Pb), Cadmium (Cd), Tin (Sn), Mercury (Hg) and Arsenic (As). The analysis was based on SNI (Standard National Indonesia) 7387:2009 about maximum limit of metal contaminants in food (Table 3).
Table 3 shows that shells and seeds of Indonesian velvet bean contain metal contamination of lead, cadmium, tin, mercury and arsenic below the safe limit for consumption, except the lead content of shells. Therefore, the velvet beans could be considered safe to be used as a food or medicine after the shells are removed.
Table 3. Metal contaminat on shells and seeds of Indonesian velvet bean (mg/kg) Parameters
seeds
shells
Lead (Pb) Cadmium (Cd) Tin (Sn) Mercury (Hg) Arsen (As)
< 0.048 < 0.003 < 0.8 < 0.005 < 0.003
1.66 < 0.003 < 0.8 < 0.005 < 0.003
*
Standard* (Maximum level) 0.5 0.2 0.03 1.00
SNI 7387:2009 = Maximum level of metal contaminants on food in Indonesia
Microbial Contaminants
cereus. The test results of microbial contamination are enlisted in Table 4.
Microbial contamination measurement was performed to quantify the microbial contaminants contained in the sample. The microbial contamination is a crucial parameter for food and medicinal products. The microbe determination includes, E. coli, Salmonella, fungi, yeast, C. perfringens and Bacillus
Based on the results comparison with standards, contaminations in the shell karabenguk were within the consumption as food or for purposes.
obtained and the microbial and seeds of safe limits for pharmaceutical
Table 4. Microbial contaminantion of shells and seeds of Indonesian velvet bean Parameters
Seeds
Total Plate Count (30oC, 72 h) 4.5 x 104 (coloni/g) E. coli (MPN2/g) <3 Salmonella (25g-1) 0 fungi (coloni/g) < 10 yeast (coloni/g) < 10 C. perfringens (g-1) 0 Bacillus cereus (coloni/g) 0 1 2
shells
Standard1 (Maximum level)
5.4 x 104
1 x 106
<3 0 < 10 < 10 0 0
10 Negative 1 x 104 1 x 104 Negative 1 x 104
SNI 7388:2009 about Maximum level of microbial contaminant on food MPN (The Most Probable Number) = the estimate number of microbes present in samples
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Phytochemical screening This test was performed to determine the class of compounds present in the extracts. The extracts were examined for the presence of alkaloid, tannins, saponins, steroids, and flavonoids.
The phytochemical test results have been summarized in Table 5, which indicates that velvet bean contained alkaloids, tannins, saponins, and steroids, while flavonoid was absent.
Table 5. Phytochemical screening results for shell and seed of Indonesian velvet bean Parameter Seed Alkaloids √ Tannins √ Saponins √ Steroids √ Flavonoids Note: (√) = present; (-) = absent L-DOPA Content Determination of L-DOPA was carried out using HPLC. L-DOPA has a chromophoric group, so it can be detected with the aid of UV detector in HPLC at λ = 250 nm. Mobile phase used was a mixture of water : methanol : phosphoric acid pH 2.5 with a ratio of 97:20:1 or 82.20:16.95:0.85. Analysis was carried out using C18 column and UV detection at λ = 250 nm with a flow rate of 1 ml/min, isocratic separation, at a temperature of 27oC. The LDOPA standard chromatograms are shown in Figure 3. Figure 3 shows that the peak of L-DOPA standard appears at the retention time (RT) 2.47 minutes. The same procedures were also applied into sample of seed and shell of Indonesian velvet bean extracts. Their chromatogram can be shown in Figure 4a and 4b. Figures 4a and 4b shows that both the seeds and shells of Indonesian velvet bean gave each peak at retention time of 2.47. It is meant that both the sample contain L-DOPA. The intensity of peak in the chromatogram of seed was higher than the shell. The concentration of L-
Shell √ √ √ √ -
DOPA was calculated by comparing with a standard calibration curve. The results are shown in Table 6. The observation made in the present study shows that both seed and shell of Indonesian velvet bean were still safe for consumption as the metal and microbial contaminants were below the maximum level. The alkaloids, tannins, saponins and steroids were also present in both the samples, nevertheless the L-DOPA levels were different. CONCLUSION The present study indicated that Indonesian velvet bean could be used as a source of LDOPA which is used for the preparation of antiparkinson drugs. The metal and microbial analysis showed that both seed and shell of Indonesian velvet bean were still safe for consumption, they are below the maximum level. Phytochemical analysis showed that both the samples contain alkaloid, tannin, saponin and steroid. The data indicated that seed extract of velvet bean contained higher L-DOPA (7.56%) while shells contain only 3.89%.
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Table 6. L-DOPA Content Sample Seeds Shells
L-DOPA Content (%) 7.56 3.89
Figure 3. Standard L-DOPA Chromatogram (Column Length: 25 cm; concentration: 1000 ppm)
Figure 4 Chromatogram of seeds (a) and shell (b) of Indonesian velvet beans extract
(a)
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(b) ACKNOWLEDGEMENTS The authors acknowledge Rector of Universitas Pendidikan Indonesia for funding the study. They also acknowledge the Balai Besar Industry Agro, Agro Based Industry Calibration and Analytical Laboratories (ABICAL), Bogor for providing metal and microbial contaminant evaluation. REFERENCES Farooqi AA, Khan MM, Asundhara M. (1999). Production technology of medicinal and aromatic crops, Natural Remedies Pvt. Ltd., Bangalore, pp 26–28. Mohan MR, Kala BK (2010). Chemical Composition and Nutritional Evaluation of Lesser Known Pulse of the Genus, Mucuna. Adv. Biores. 1(2):105–106.
Levodopa. International Journal of Research in Pharmaceutical and Biomedical Sciences.1:23–26 Teixeira AA, Rich EC, Szabo NJ (2003). Water Extraction of L-Dopa from Mucuna Bean. Tropical and Subtropical Agroecosystem. 1:159–171. Ujowundu CO, Kalu FN, Emejulu AA, Okafor OE, Nkowonta CG, Nwonsunjoku E.C. (2010). Evaluation of The Chemical Composition of Mucuna utils Leaves used in Herbal Medicine in Southeastern Nigeria. Afr. J. Pharm. Pharmacol. 4(11):811–816. Vadivel V, Janardhanan K (2000). Nutritional and anti-nutritional composition of velvet bean: An under-utilized food legume in South India. Inter. J. Food Sci. Nutri. 51: 279–287
Raju RR, Babu NB, Rao PS (2010) RP-HPLC Method Development and Validation of
Source of Support: Nil
Conflict of Interest: None Declared
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Original Research Article ANTIBACTERIAL ACTIVITY OF SEEDS OF MUCUNA PRURIENS L. AGAINST ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS Deshwal Vishal Kumar1 1
Department of Microbiology, Doon (P.G.) Paramedical College, Dehradun-248001, Uttarakhand, India 1 Corresponding Author: email ID: vishal_deshwal@rediffmail.com Contact number: +919897538555 Received: 26/02/2012; Revised: 14/03/2012; Accepted: 25/03/2012;
ABSTRACT The present study was carried out to evaluate the antibacterial activity of different extracts of Mucuna pruriens L. against Escherichia coli and Staphylococcus aureus. Three extracts such as aqueous extract, ethanol extract and chloroform were prepared for the present study. Agar well diffusion method was used for evaluating antibacterial activity against the above said pathogens. Results suggested that aqueous extract of Mucuna pruriens showed more antibacterial activity when compared to ethanol extract, chloroform extract & norfloxacin against E. coli and S. aureus. In case of E. coli, Maximum inhibition zone was observed in aqueous solution of the medicinal plant (30mg/ml) which was 17.07% more as compared to norfloxacin (30mg/ml). Similarly, aqueous extract (30mg/ml) of Mucuna pruriens showed 17.36% more inhibition zone when compared to norfloxacin (30mg/ml). Study confirmed that different extracts of Mucuna pruriens were effective against E. coli and S. aureus. Key words: Mucuna pruriens, antibacterial, E. coli, S. aureus
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INTRODUCTION Plants are being used as medicines by mankind since the ancient times and they are being taken as a good source of drugs (Deshwal and Siddiqui, 2011a). Development of antibiotic resistance strains has forced scientists to search for new antimicrobial substances from various sources as novel antimicrobial chemotherapeutic agents. Use of medicinal plant may be effective for protection against various bacterial, viral and other diseases (Kumari et al., 2012). Recently, Makhloufi et al. (2012) reported antibacterial activity of essential oil, aqueous and ethanol extract of Artemisia herba-alba Asoo. Much attention is being paid recently to the biologically active compounds derived from plants used in herbal medicine (Shai et al., 2008; Firas and Bayati, 2008; Abiramasundari et al., 2011). Urinary tract infection (UTI) is a nonspecific term and refers to bacterial invasion into the urologic system. UTI can be divided into two anatomically distinct categories: lower tract infection, including urethritis and cystitis, and upper tract infections, such as urethritis, pyelitis (upper collecting system) and pyelonephritis (renal parenchyma) (Shigemura et al., 2005). Urinary tract infections (UTIs) are serious problems affecting millions of people each year. UTI in women are more common compared to men. In United States each year, physicians write approximately 11.3 million prescriptions for adult women with UTIs (Foxman et al., 2000). Different pathogens are associated with urinary tract infection and these are Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Enterobacter spp., Proteus mirabilis, Klebsiella oxytoca, Enterobacter faecalis, Staphylococcus aureus, Streptococcus, Streptococcus epidermidis, Enterococcus faecium, and other Pseudomonas species (Deshwal and Vig, 2011a). Significant bacteria is defined as the persistent isolation of 105 colony forming unit (CFU) of bacteria per ml of clean voided, midstream urine specimens plated within 6h of collection (Ojo and Anibijuwon, 2010).
Mucuna pruriens L. belongs to the family Fabaceae. It has some medicinal value and it is also food - feed crop. The roots are bitter, sweet, thermogenic, emollient, stimulant, purgative, aphrodisiac and diuretic. The seeds are aphrodisiac, astringent, laxative, Anthelmintic, alexipharmic and tonic (Taylor, 2005). A clinical study confirmed the efficacy of the seeds of Mucuna pruriens in the management of Parkinsonâ&#x20AC;&#x2122;s disease by virtue of their L-DOPA content (Manyam et al., 1995). Salau and Odeleye (2007) mentioned that methanol extract of M. pruriens showed broad-spectrum antimicrobial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Proteus mirabilis. So, the present study was carried out to evaluate the antimicrobial activity of Mucuna pruriens L. against Escherichia coli and Staphylococcus aureus. MATERIALS AND METHODS Isolation of microorganism: Urine sample was collected in a sterilized container from a patient suffering from urinary tract infection. The mid-stream urine was collected after carefully cleaning the genitalia and mid-stream urine was collected because the first portion of urine may contain most of the contaminants. Pathogen was isolated and counted by standard plate method and MacConkey agar without crystal violet and Blood agar medium was used for isolation of various pathogens. The plates were incubated at 37oC for 24â&#x20AC;&#x201C;48h. The pathogens were calculated on the observation that, when the bacterial count was greater than 105 cfu /ml, it was taken as threshold and the sample was confirmed as UTI positive. Characterization of pathogens: Pathogens were characterized according to Holt et al. (1994).
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Preparation extracts:
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and
selection
of
different
Three extracts such as aqueous extract, Ethanol extract and Chloroform were selected for the present study. These extracts were prepared according to Deshwal (2012). Sterilization and preparation of different concentration of extracts:
Dried extracts and liquid extracts were sterilized according to Ekwenye and Elegalam, (2005) and Deshwal (2012) respectively. Norfloxacin antibiotic worked as a control drug. Antibacterial activity of Mucuna pruriens by well diffusion method: Antibacterial activity was performed according to Deshwal (2012).
Table 1: In vitro antibacterial activity of different extracts of Mucuna pruriens L. on the growth of E. coli by well diffusion test. Extract
Inhibition zone (mm) Concentration a
15 mg/ml 20 mg/mla 25 mg/mla 30 mg/mlb 16.7±0.4 17.8±0.4 19.4±0.5 21.7±0.5 Aqueous 16.0±0.6 17.1±0.5 18.7±0.5 21.0±0.5 Ethanol 15.5±0.5 16.6±0.5 18.1±0.3 20.3±0.5 Chloroform 15.0±0.4 16.1±0.5 17.2±0.5 18.5±0.5 Norfloxacin Values are mean of 4 replicate ±SD; a, Significant at 0.05 level of ANOVA; b Significant at 0.01 level of ANOVA
Table 2: In vitro antibacterial activity of different extracts of Mucuna pruriens L. on the growth of Staphylococcus aureus by well diffusion test. Extract
Inhibition zone (mm) Concentration
15 mg/mlb 20 mg/mlb 25 mg/mlb 30 mg/mlb 17.3±0.3 18.4±0.4 20.1±0.4 22.5±0.5 Aqueous 16.5±0.4 17.7±0.3 19.3±0.5 21.7±0.4 Ethanol 16.2±0.3 17.4±0.6 18.9±0.4 21.3±0.3 Chloroform 15.5±0.4 16.6±0.5 17.7±0.4 19.2±0.4 Norfloxacin Values are mean of 4 replicate ±SD; b Significant at 0.01 level of ANOVA RESULTS AND DISCUSSION On the basis of microbial count in urine sample, 7 out of 15 suspects were UTI positive. Isolated strains were characterized on the basis of Biochemical test and founded that strains
were of Escherichia coli and Staphylococcus aureus. As previously stated that isolates were characterized on the basis of Bergey’s manual of determinative bacteriology (Holt et al., 1994).
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Different extract of Mucuna pruriens showed antibacterial activity against E. coli and Staphylococcus aureus. In case of E. coli, maximum inhibition zone was observed in aqueous extract of Mucuna pruriens when compared to ethanol, chloroform, norfloxacin (control drug). Aqueous solution of the medicinal plant (30mg/ml) showed 17.07% more inhibition zone when compared to norfloxacin (30mg/ml). Similar results were observed in ethanol (30mg/ml) and chloroform (30mg/ml) extracts by 13.1% and 9.66% respectively when compared to norfloxacin (30mg/ml) (Table-1). In case of Staphylococcus aureus, maximum inhibition zone was observed in aqueous extract of Mucuna pruriens when compared to other extracts like ethanol, chloroform and norfloxacin (control drug). Aqueous solution of the medicinal plant (30mg/ml) showed 17.36% more inhibition zone when compared to norfloxacin (30mg/ml). Similar results were observed in ethanol (30mg/ml) and chloroform extracts (30mg/ml) by 13.1% and 10.9% respectively when compared to norfloxacin (30mg/ml) (Table-2).
Results suggested that inhibition zone was increased as the concentration of Mucuna pruriens was increased. Inhibition zones of the medicinal plant were always more when compared to norfloxacin. These results suggest that seeds of Mucuna pruriens exhibit good antibacterial property. Similarly, Salau and Odeleye (2007) mentioned that high concentrated (160mg/ml) extract of leaf of M. pruriens showed broad-spectrum antimicrobial activity against bacterial pathogens. Recently, Korir et al. (2012) observed that two medicinal plant species Senna didymobotrya, Cyathula polycephala showed antimicrobial activity. Deshwal and Siddiqui (2011a, b) screened the anti-microbial activity in Tylophora indica, Cassia sophera, Coleus forskohlii and Stevia rebaudiana. Similarly, Hindumathy et al. (2011) also reported that alcohol and water extracts of Cymbopogon citratus effectively inhibited the growth of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus. Our study revealed that different extract of seeds of Mucuna pruriens inhibits the growth of pathogenic Escherichia coli and Staphylococcus aureus.
REFERENCES Abiramasundari P, Priya V, Jeyanthi GP, and Gayathri Devi S (2011). Evaluation of the Antibacterial activity of Cocculus hirsutus. Hyg. J. Drug Medicin. 3(2): 26–31.
Deshwal VK and Siddiqui MMM (2011b). Screening and evaluation of antimicrobial activity in Coleus forskohlii and Stevia rebaudiana. J. Plant Dev. Sci. 3(2): 95–101.
Deshwal VK (2012). Antibacterial activity of Piper nigrum Linn. against E. coli causing Urinary tract infection. Int. J. Pharmaceutical Inven. 2(2): 1–7.
Deshwal VK and Vig K (2011a). Isolation and characterization of Urinary tract infection (UTI) causing pathogens and their comparative study in different genders. Dev. Microbiol. Mol. Biol. 2(2): 113– 116.
Deshwal VK and Siddiqui MMM (2011a). Screening and Evaluation of Antimicrobial Activity in Tylophora indica and Cassia sophera. Bioch. Cell. Arch. 11(2):461–464.
Deshwal VK and Vig K (2011b). Screening for Antibacterial activity of seeds of Tribulus terrestris L. growing in Uttarakhand
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activities of essential oil and crude extracts from Artemisia herba-ALBA ASOO, growing wild in bechar, south west of Algeria. Global J. Res. Medicinal Plants & Indigen. Med. 1(1): 7–13.
(INDIA). Int. J. Pharmaceutical Inven. 1(1): 42–46. Ekwenye UN and Elegalam NN (2005). Antibacterial Activity of Ginger (Zingiber Officinale Roscoe and Garlic (Allium Sativum L.) Extracts on Escherichia coli and Salmonella typhi. Int. J. Mol. Med. and Adv. Sci. 1(4): 411–416. Firas A and Bayati A (2008). Synergistic antibacterial activity between Thymus vulgaris and Pimpinella anisum essential oils and methanol extracts. J. Ethnopharmacol. 116: 403–406. Foxman B, Barlow R, D’Arcy H, Gillespie B and Sobel JD (2000). Urinary tract infection: self-reported incidence and associated costs. Ann. Epidemiol. 10:509–515. Hindumathy CK (2011). In vitro study of antibacterial activity of Cymbopogon citratus. World Acad. Sci., Engineer. Tech. 74: 193–197. Holt JG, Krieg NR, Sneath PHA, Staley JT and Williams ST (1994). Bergey’s manual of determinative bacteriology, 9th edn. Baltimore: Williams and Wilkins press. Korir RK, Mutai C, Kiiyukia C and Bii C (2012). Antimicrobial activity and safety of two Medicinal Plants traditionally used in Bomet District of Kenya. Res. J. Med. Plant. 6(5): 370–382. Kumari R, Tiwary BK, Prasad A and Ganguly S (2012). Study on the immunomodulatory effect of herbal extract of Asparagus racemosus willd. in broiler chicks. Global J. Res. Medicinal Plants & Indigen. Med. 1(1): 1–6. Makhloufi A, Benlarbi L, Mebarki L and Akermi MM (2012). Antimicrobial
Source of Support: Nil
Manyam BV, Dhanasekaran M and Hare TA (1995). An Alternative Medicine Treatment for Parkinson's disease: Results of a multicenter clinical trial. J. Alternative Compl. Medicin. 1(3): 249– 255. Ojo OO and Anibijuwon II (2010). Urinary tract infection among female students residing in the campus of the University of Ado Ekiti, Nigeria. Afr. J. Microbiol. Res. 4 (12):1195–1198. Salau
AO and Odeleye OM (2007). Antimicrobial activity of Mucuna pruriens on selected Bacteria. Afr. J. Biotechnol. 6(18): 2091–2092.
Shai LJ, McGawa MA, Aderogbaa LK and Eloff JN (2008). Four pentacyclic triterpenoids with antifungal and antibacterial activity from Curtisia dentata (Burm. f) leaves. J. Ethnopharmacol. 119: 238–244. Shigemura K, Tanaka K, Okada H, Nakano Y, Kinoshita S, Gotoh A, Arakawa S and Fujisawa M (2005). Pathogen occurrence and antimicrobial susceptibility of Urinary tract infection cases during a 20year period (1983–2002) at single institution in Japan. Jpn. J. Infect. Dis. 58: 303–308. Taylor L (2005). The Healing Power of Rainforest Herbs: A Guide to Understanding and Using Herbal Medicinals (Eds), Square One Publishers, NY.
Conflict of Interest: None Declared
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Original Research Article PHYTOCHEMICAL SCREENING OF ANTHOCLEISTA GRANDIFLORA GILG. STEM BARK Odeghe Othuke Bensandy1*, Uwakwe Augustine A1, Monago Comfort C1 1
Department of Biochemistry, University of Port Harcourt, Rivers State. Nigeria. *
Correponding author: bensandym@yahoo.com, +2348066768804
Received: 06/03/2012; Revised: 21/03/2012; Accepted: 31/03/2012;
ABSTRACT The phytochemical studies carried out in this research indicated that the stem bark of Anthocleista grandiflora Gilg. plant contains alkaloids, flavonoids, terpenes, saponins, glycosides and protease inhibitor. The total alkaloid composition was 7.91g/100g of which akuammidine had the highest amount of 4.84g/100g while camptothecin had the lowest amount of 0.0014g/100g. Flavonoid had a total composition of 2.1506g/100g and quercetin in major amount 1.4912g/100g and the lowest quantity of 0.0029g/100g in Isorhamnetin. Saponin had a total amount of 5.87g/100g in which Avenacin-A1 had the highest amount while Avenacin-B2 had the lowest concentration of 3.01g/100g and 0.19g/100g respectively. Glycoside total amount was 0.22g/100g with the highest amount of 0.089g/100g in Ouabain and the lowest amount of 0.0029g/100g in digitoxin. Terpenes total amount was 0.032g/100g with bauerenol acetate having the highest amount of 0.0076g/100g and β-amyrin with the lowest amount of 0.0044g/100g. The total composition of protease inhibitor was 0.017g/100g of which Cysteine had the highest amount of 0.0093g/100g while lysine had the lowest amount of 0.0078g/100g. This research work indicates that, the stem bark of this plant may be useful in the treatment of malaria. The efficacy of the plant material as a potent trado-medical resource is thus confirmed. Keywords: Alkaloids, flavonoids, glycosides, protease inhibitor, saponins, terpenes, Anthocleista grandiflora
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INTRODUCTION Anthocleista grandiflora Gilg., commonly known as the forest fever tree is a large tree of moist forests in the eastern and south-eastern African tropics and the comores. It is a member of the family Gentianaceae and a small genus of only 14 species. It is a tall, slender tree grows up to 30m with a preference for forests in high rainfall areas. The leaves are very large up to 100cm x 50cm and in terminal clusters. It is an evergreen plant. (http://www.napreca.net/publications/11sympo sium/word/C-20-26-Mulholland.doc). The tree is sometimes epiphytic with auxiliary spines or tendrils, leaves opposite, occasionally alternate, rarely venticillate, fascicute or in a whorl, stipules usually present, often reduced to lines connecting petiole bases. The flowers are in cymes these are often grouped into thyrses; sometimes umbel-like, scorpioid or reduced to single flower bracts usually small. The flowers are usually bisexual and cream coloured. It is not edible as food but possesses root, stems, bark, leaves and flowers which are claimed to have medicinal properties (Palmer and Pitman; 1972). . In Southern Africa, bark decoctions are used traditionally to treat malaria (Palmer and Pitman; 1972). Regionally, preparations of the bark has also found use as an anthelmintic specifically for roundworms (Githers 1949), antidiarrhoeal (Watt and Bieyer-Brandwijk 1962; Mabogo 1990) and to treat diabetes, high blood pressure and venereal diseases (Mabogo 1990). Furthermore, in the northern continent, epilepsy is remedied with the aid of the stem bark decoction (Newinger 2000). MATERIALS AND METHODS Collection and identification of medicinal Plants Samples of fresh Anthocleista grandiflora Gilg. stem bark were collected in March, 2011, at Choba area, Port Harcourt, Rivers State. The plant specimen was identified and authenticated by Dr. I.K. Agbagwa, a taxonomist in the
Department of Plant Science and Biotechnology, University of Port Harcourt, Rivers State, Nigeria. After due identification, the plant stem barks were sorted to eliminate any dead matter and other unwanted particles and stored for subsequent use. All reagents used were GC-grade purity. Determination of Alkaloid Composition The method reported by Tram et al. (2002) was adopted. 30 g of the ground sample was added to 250 ml of boiling deionized water and allowed to soak for 30 min, before filtration. The filtrate was acidified to pH 4 with acetic acid, before extracting with 30 ml of petroleum spirit and chloroform. The acidic aqueous phase was made alkaline (PH 9), with 25% aqueous ammonia, and then extracted three times with 30ml of chloroform. The chloroform extract was concentrated to 1.0ml, before chromatographic analysis. Chromatographic analysis was carried out on an HP 6890 (Hewlett Packard, Wilmington, DE, USA), GC apparatus, fitted with a flame ionization detector (FID) (range scanned: 220–500 nm), and powered with HP Chemstation Rev A 09.01 (1206) software, to quantify and identify compounds. The column was a ZP-5 Column (30 m × 0.32 mm × 0.25 µm film thickness). Injections were accomplished with a 20 µl fixed loop. Prior to GC analysis, all solutions were filtered through 0.45 µm membranes filter and then degassed in an ultrasonic bath for 30 min. Determination of the Saponin Composition The method of Hanafy and Lobna (2007) was adopted. The pulverized sample was defatted with petroleum ether at 40-600C for 3 h. After filtering the petroleum ether, the sample was extracted with methanol for 3 h, with mild heating. The methanol extract was concentrated and re-extracted with methanol/acetone (1:5) mixture. The precipitate obtained was dried under vacuum, which turned to a whitish amorphous powder after complete drying. It was eluted on a silica gel (230–400 mesh) column, with chloroform/methanol/water (7:3:1).
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The first fraction collected was air dried at room temperature and the residue obtained was treated as pure saponin. The residue was dissolved in methanol for chromatographic analysis. Determination of Glycosides Composition The method of AOAC (2006) was adopted. 1.0 g of the pulverized sample was extracted by pouring 10ml of ethanol/water (7:3) mixture on it, and allowing it to stand for 2 h. The mixture was filtered with Whatman filter paper no. 1 and the extract was purified by washing with lead acetate. The purified extract was further purified by adding sodium hydrogen phosphate, before concentrating to 1ml, for chromatographic analysis. Determination of Flavonoid Composition The method of Millogo-Kone et al. (2009) was adopted. The dried and pulverized sample was made to be free of water by ensuring constant weight for a period of time in the laboratory. 10.0g of the sample was weighed into the 250ml conical flask capacity with addition of 100ml of distilled water and boiled for 10 min. The crude methanolic extract was obtained by pouring 100ml of the boiling methanol: water (70:30) onto 10.0g of the plant materials. The mixture was allowed to macerate for 18 h, and then filtered with Whatman filter paper no. 1. The filtrate was concentrated to 5ml for gas chromatography analysis. Determination of Terpenes Composition The method of Popa et al. (2009) was adopted.
The sample was pulverized and the terpenes constituents were extracted with redistilled chloroform. The terpenes were removed with 10ml for 15 minutes. The extract was filtered and concentrated to 1 ml in the vial for gas chromatography analysis and 1Âľl was injected into the injection port of GC. Determination of Protease Inhibitor The modified method of AOAC (2006) was adopted. The dried and pulverized sample was made to be free of water by ensuring constant weight for a period of time in the laboratory. 10.0g of the sample was weighed into the 250ml conical flask capacity. The sample was defatted by extracting the fat content of the sample with 30ml of the petroleum spirit three times with soxhlet extractor that was equipped with thimble. The sample was hydrolyzed by using 30ml of deionized water three times. The amino acid group of the sample was recovered by extraction with 30ml of the methylene chloride thrice and the cysteine and lysine were recovered by selective elution and later concentrated to 1ml for gas chromatography analysis. RESULTS The result of the phytochemical screening of the aqueous plant extract of A.grandiflora stem bark using gas chromatography indicated the presence of different classes of secondary metabolites that are essential in herbal medicine. Among the phytochemicals obtained were alkaloids, glycosides, protease inhibitors, saponins, terpenes and flavonoids.
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Table 3.1: Alkaloid Composition of Anthocleista grandiflora Stem Bark S.No 1. 2 3 4 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
Component Choline Trigonelline Augustifoline Sparteine Ellipcine Lupanine 13-Îąhydrorhombifoline 9-Octadecenamide Dihydro-oxo-demethoxyhaemanthamine Augustamine Oxoassoanine Cinchonidine Crinane -3alpha-01 Buphanidrine Indicine-N-oxide Undulatine Powerlline Ambelline 6-Hydoxybuphanidrine Acronycin Monocrotaline 6-Hydroxypowerlline Nitidine Crinamidine Ibeta, 2 beta-Epoxyambelline 6-Hydroxyundulatine Epoxy-3,7-dimethyoxycrinane-II-one Echitammidine Akuammidine Voacangine Mitraphylin Camptothecin Echitamine Colchicine Emetine Tetrandrine Thalicarpin Paclitaxel Total Alkaloid Composition
Composition (g/100g) 0.0012 ______ 0.0575 0.0020 0.0024 0.0463 0.0020 0.0017 0.0023 0.0147 0.0020 0.0035 0.0047 0.0022 0.0018 0.0088 0.0083 0.0083 0.0032 0.0026 0.0030 0.0067 0.0021 0.0133 0.0033 0.0028 0.0045 0.0368 4.8401 2.7115 0.0047 0.0011 0.1361 0.0092 0.0034 0.0092 0.0020 0.0015 7.9140g/100g
Retention Time (Min) 7.056 7.641 7.722 8.953 9.733 11.044 11.936 12.936 14.151 14.918 15.396 16.246 16.368 16.489 16.669 17.566 18.587 18.661 19.771 20.468 21.125 21.323 21.817 22.358 23.964 24.612 24.788 25.478 26.734 26.827 27.059 27.427 28.206 28.634 28.921 29.574 29.757 30.585 727.938 Min
Table 3.2: Glycoside Composition of Anthocleista grandiflora Stem Bark S.No 1. 2. 3. 4. 5. 6.
Component (g/100g) Arbutin Salicin Amygydalin Ouabain Digitoxin Digoxin Total Glycoside Composition
Composition (g/100g) 0.0081 0.0138 0.0359 0.0891 0.0028 0.0702 0.2198g/100g
Retention Time (Min) 17.357 18.834 19.513 20.469 21.436 23.110 120.719 min
Table 3.3: Protease Inhibitor Composition of Anthocleista grandiflora Stem Bark S.No 1 2
Component (g/100g) Cysteine Lysine Total Protease Inhibitor Composition
Composition (g/100g) 0.0093 0.0078 0.0172g/100g
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Retention Time (Min) 14.156 16.459 30.615 min
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Table 3.4: Saponin Composition of Anthocleista grandiflora Stem Bark S.No 1. 2. 3. 4.
Component (g/100g) Avenacin-A1 Avenacinâ&#x20AC;&#x201C;B1 Avenacin-A2 Avenacin-B2 Total Saponin Composition
Composition (g/100g) 3.0060 2.4753 0.1959 0.1893 5.8671g/100g
Retention Time (Min) 21.439 23.118 24.790 26.350 95.697 min
Table 3.5 Terpene Composition of Anthocleista grandiflora Stem Bark S.No 1. 2. 3. 4. 5.
Component (g/100g) Taraxerol Lpha-amyrin Beta-amyrin Lupeol Bauerenol acetate Total Terpene Composition
Composition (g/100g) 0.0064 0.0075 0.0044 0.0069 0.0076 0.0328g/100g
Retention Time (Min) 19.521 20.470 21.438 23.117 24.790 109.336 min
Table 3.6: Flavonoid Composition of Anthocleista grandiflora Stem Bark S.No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Component Catechin Gallocatechin Genistein Daidzein Apigenin Butein Naringenin Biochanin Luteolin Kaemferol Epicatechin Epigallocatechin Quercetin Epicatechin-3-gallate Epigallocatechin-3gallate Isorhamnetin Robinetin Ellagic Acid Myricetin Baicalein Nobiletin Baicalin Silymarin TOTAL FLAVONOID COMPOSITION
Composition (g/100g) 0.0168 0.0059 0.0053 0.0162 0.2973 0.0173 0.0269 0.0210 0.0045 0.0894 0.0528 0.0096 1.4912 0.0076 0.0047
Retention Time (Min) 9.345 9.457 10.243 10.568 11.234 12.125 12.456 12.967 13.234 13.689 14.358 14.678 15.235 16.395 16.587
0.0029 0.0166 0.0229 0.0064 0.0155 0.0174 0.0082 0.0081 2.1506g/100g
16.856 17.322 17.654 17.811 18.276 18.867 19.276 20.456 339.179 min
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Figure 1: Displaying Anthocleista grandiflora in its Habitat DISCUSSION From the results, there were different classes of secondary metabolites that are essential in herbal medicine. The total alkaloid components obtained after the gas chromatographic separation of alkaloid fraction were 38 having a total composition of 7.9140g/100g of which Akuammidine had the highest concentration of 4.8401g/100g while Camptothecin had the lowest concentration of 0.0014g/100g as shown in table 1.1. The total alkaloid content of Sansevieria liberica leaves was lower than that of Anthocleista grandiflora stem bark. (Ikewuchi et al, 2011). The medicinal plants that are moderately rich in alkaloids and tannis have potential health promoting effects. (Ikewuchi and Ikewuchi, 2008); (Jigam et al, 2010). The presence of alkaloids in high quantity Anthocleista grandiflora confirms the findings of (Ajaiyeoba et al 2006) that the traditional use of the plants for the treatment of malaria was due to the presence of alkaloids.
Glycosides were also found to contain 6 constituents with a total composition of 0.2198g/100g with the greatest composition of 0.089g/100g in Ouabain and the lowest concentration of 0.0029g/100g in Digitoxin. Glycosides and saponins are reported to possess broad range of pharmacological properties. Ouabain is a cardiotonic steroid. (Dmitrieva and Doris 2002) and the cardiac glycosides are used for treating heart problems that may result from severe malaria attack. (Fatoba et al, 2003). On the other hand, protease Inhibitor component of the gas chromatographical separation indicated two types of constituents with a total composition of 0.017g/100g of which Cysteine had the highest concentration of 0.0093g/100g while Lysine had the lowest concentration of 0.0078g/100g.
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In addition, a total of four saponins were obtained from the gas chromatographic isolation of saponin fraction with a total composition of 5.87g/100g in which AvenacinA1 had the highest concentration of 3.01g/100g while Avenacin-B2 had the lowest concentration of 0.19g/100g. Saponins have anti carcinogenic properties and other health benefits. They may also play a significant role in antimalarial activity of plants. (Adesokan and Akanji 2010). Avenacins have antimicrobial properties (Armah et al, 1999) Saponins are reported to have broad range of pharmacological properties (Soetan 2008). Avenacins have antimicrobial properties (Armah et al, 1999); (Mert Türk et al, 2005). Terpenes with five types of components indicated a total composition of 0.032g/100g with Bauerenol acetate having the highest amount of 0.0076g/100g whereas β-amyrin were present in lowest amount of 0.0044g/100g. The gas chromatographic isolation of the plant extract (A. grandiflora) yielded twenty three flavonoid components with a total composition of 2.1506g/100g in which quercetin had the highest concentration of 1.4912g/100g and the lowest concentration of 0.0029g/100g in isorhamnetin. Flavonoid has been reported to posssess several pharmacological properties, including anti-inflammatory activity, oestrogenic activity, enzyme inhibition, antimicrobial activity. (Havsteen, 1983); (Harbone and Baxter,1999), antiallergic activity, antioxidant activity Middleton and Chithan, (1993); vascular activity and cytotoxic
antitumour activity (Harborne and Williams, 2000). Flavonoid-rich plant extracts from Hyperium spp. (Dall’Agnol et al, 2003), (Capsella and Chromolaena El-Abyad et al 1990) have been reported to possess antibacterial activity. The phytochemical screening revealed that Anthocleista grandiflora stem bark is highly rich in alkaloids, moderately rich in saponins and flavonoids with little content of glycosides, protease inhibitor and terpenes. These compounds have been reported to have health promoting effects. Basu et al (2007). CONCLUSION In conclusion, this investigation showed that Anthocleista grandiflora stem barks are rich in alkaloids. Therefore, the presence of some of the identified phytochemicals confers medicinal properties on the plant. It was concluded that the presence of these phytochemically-active components in the plant sample might be responsible for their therapeutic activity as antimalarial plants. This finding supports their use as medicinal plant products and potential sources of useful drugs for treating some diseases. ACKNOWLEDGEMENT The authors acknowledged the assistance from the World Bank and the federal Republic of Nigeria with the World Bank step B.
REFERENCES Adesokan AA. and Akanji, MA, (2010). Antimalarial Bioactivity of Enantia chlorantha stem bark. Medical Plants: Phytochemistry Pharmacology and Therapeutics 4(1): 441–447. Ajaiyeoba E. Falade M, Ogbole O, Okpako L and Akinboye D. (2006). In vivo antimalarial and Cytotoxic properties of Annona senegalensis extract. African
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saponins. Can J. Animal Sci 58:783– 789 Armah CN, Mackie AR, Roy C, Prince K, Osbourn AE, Bowyer P and Ladha S. (1999). The Membrane Permeabilizing Effect of Avenacin A-1 involves the Reorganization of Bilayer cholesterol. Biophysical journal, 76: 281–290 Basu SK, Thomas JE. and Acharya SN. (2007). Prospects for Growth in Global Nutraceutical and Functional Food Markets: A Canadian Perspective. Aust J Basic Appl Sci, 1(4): 637–649 Dmitrieva RI and Doris PA. (2002). “Cardiotonic Steroids: Potential Endogenous Sodium Pump Ligands with Diverse Function”. Experimental Biology and Medicine. 227(8):561–569. El-Abyad MS, Ismail IK. and Rizk MA (1982). Ecological studies on the Rhizosphers of some Egyptian halophylic plants. Egypt. J. Bot., 25:91–109 Fatoba
PO, Omojasola PF, Awe S. and Ahmed FG. (2003).Phytochemical screening of some selected tropical African Mosses. Nigerian Society for Experimental Biology (NISEB) Journal 3(2): 49–52.George, P. M. Encyclopedia of Food. Vol.1. Humane Press, Washington. 526p.
Githens TS (1949). Drug plants of Africa. African handbooks: 8. University of Pennsylvania Press, Philadelphia, USA Hanafy
MS and Lobna MAS. (2007). “Saponins Production in Shoot and Callus Cultures of Gypsophila Paniculata”. Journal of Applied Science. Research. 3(10): 1045–1049.
Harborne JB and Williams CA. (2000): Advances in flavonoid research since 1992. Phytochemistry 55, 481–504.
Havsteen B. (1983): Flavonoids, a class of natural products of high pharmacological potency. Biochem. Pharmacol. 32:1141–1148. Ikewuchi CC and Ikewuchi JC (2008). Chemical profile of Pleurotus tuberregium (Fr) Sing’s Sclerotia.The Pacific Journal of Science and Technology 10(1): 28–30. Ikewuchi CC, Ayalogu EO, Onyeike EN and Ikewuchi JC (2011). “Study on the Alkaloid, Allicin, Glycoside and Saponin Composition of the Leaves of Sansevieria liberica Gérôme and Labroy by Gas Chromatography”. Pacific Journal of Science and Technology. 12(1):367–373. Mabogo DEN (1990). The ethnobotany of the Vhavenda. Unpublished M.Sc thesis. University of Pretoria. Middleton E and Chithan K (1993): The impact of plant flavonoids on mammalian biology: implications for immunity, inflammation and cancer. In : The Flavonoids: Advances in research since 1986 (Ed. Harborne, J. B.). Chapman and Hall, London. 619–652. Millogo-Kone H, Lompo M, Kini F, Asimi S, Guissou IP and Nacoulma O. (2009). Evaluation of Flavonoids and Total Phenolic Contents of Stem Bark and Leaves of Parkia biglobosa (Jacq.) Benth. (Mimosaceae)-Free Radical Scavenging and Antimicrobial Activities. Research Journal of Medical Sciences, 3: 70–74. Neuwinger HD (2000). African traditional medicine. A dictionary of plant use and applications. Medpharm Scientific Publishers, Stuttgart Pp 45 Palmer, E and Pitman N. (1972). Trees of southern Africa. Volume 3. Cape Town: pp. 1845–1847
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Popa NC, Tamba – Berehoiu R, Popescu S, Varga M, Codină G, Gabriela (2009): Predictive model of the alveografic parameters in flours obtained from Romanian grains, Romanian Biotechnological Letters Vol. 14, No. 2, pp. 4234–4242. Soetan KO. (2008). “Pharmacological and Other Beneficial Effects of Antinutritional Factors in Plants - A review”. African Journal of Biotechnology. 7(25):4713–4721.
Source of Support: Nil
Tram NTC, Mitova M, Bankova N, Handjieva V and. Popov SS (2002). “GC-MS of Crinum latifolium L. Alkaloids”. Z. Naturforsch. 57c: 239–242. Watt JM and Breyer-Brandwijk MG. (1962). The medicinal and poisonous plants of southern and eastern Africa. E. & S. Livingstone Ltd., Edinburgh and London.
Conflict of Interest: None Declared
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Review Article REVIEW ON DATURA METEL: A POTENTIAL MEDICINAL PLANT Khaton M Monira1, Shaik M Munan2* 1,2
Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, 7003, Bangladesh. *
Corresponding Author: munanbt2004@gmail.com, 0088-01712518103
Received: 02/03/2012; Revised: 07/04/2012; Accepted: 10/04/2012
ABSTRACT Plants have been an exemplary source of medicine. Ayurveda, traditional medicine, tribal medicine and other Bangladeshi literatures mention the use of plants in the treatment of various human ailments. Bangladesh has about 6,000 plant species and among them, around five thousands have been claimed to possess medicinal properties. Researches conducted in the last few decades on exploring plants mentioned in ancient literature or used traditionally for treating diseases is increasing. Datura metel is well known for its insecticidal, herbicidal, anti-fungal, anti-bacterial, anti-cancer, anti-inflammatory and anti-rheumatoid activity. Datura is also rich in Alkaloidal compounds.
The present
paper summarizes
the phyto-chemistry,
traditional
uses
and
pharmacological actions of the plant Datura metel. Keywords: Datura metel, Medicinal Plants, Alkaloid compounds, Phyto-chemistry, Pharmacology, Antifungal activity, Anti-cancer activity.
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INTRODUCTION Datura metel L., with local bengali name “Dhutura”, is an erect shrub with spreading branches. A perennial herbaceous plant, belonging to the Solanaceae family can reach a height of 1.5m. Leaves are simple, alternate, dark green, broadly ovate, shallowly lobed and glabrous. Flowers are large, solitary, and trumpet-shaped with a sweet fragrance usually appreciated in the mornings and evenings, with a wide range of colours, ranging from white to yellow and light to dark purple. The flowers are hermaphrodite and are pollinated by insects. The fruit is in the form of a capsule covered with short spines. Datura can tolerate average soil but prefers soil which is rich and moist or even very alkaline soil but hardly survives under shade. It prefers a warm temperature and is distributed in warmer regions of the world (Drake et al., 1996). Datura probably is of American origin and widely cultivated in all tropical and subtropical regions for its beautiful flowers (Glotter et al., 1973). D. metel can also be found in East Asia or India, and is used in traditional Bangladeshi herbal medicine. In Traditional Chinese Medicine, the flowers of D. metel are known as baimantuoluo and used for skin inflammation and Psoriasis (Wang et al., 2008). In Ayurvedic medicine, seeds of D. metel are used to treat Skin rashes, Ulcers, Bronchitis, Jaundice and Diabetes (Agharkar et al., 1991). In Brazil, seeds are used for tea making which would serve as a sedative and flowers are dried and smoked as cigarettes (Agra et al., 2007). There are various species of Datura which are now cultivated for the production of secondary metabolites. Phytochemistry Many different Alkaloids are found in the whole plant of Datura, which increased gradually with increase in age of the plant (Afsharypuor et al., 1995). Main constituents of the Datura plant are a huge number of tropane alkaloids (hyoscyamine, hyoscine, littorine, acetoxytropine, valtropine, fastusine, fastusinine), a number of withanolides and various trigloyl esters of tropine and pseudotropine (Table 1). Calystegines, the
nortropane alkaloids with glycosidase inhibitory activity, have also been found in various Datura species (Ghani, 2003). The root contains higher amount of atropine compared to the other parts. The aerial parts usually accumulated relatively higher amounts of scopolamine and relatively lower amounts of atropine as compared to the root of the plant (Afsharypuor et al., 1995). Pharmacological action D. metel contains tropane alkaloids and are used as sedative, anti-spasmodic and mydriatic agents (Nuhu, 2002). The whole plant, but especially the leaves and seed, have anaesthetic, hallucinogenic, anti-asthmatic, anti-spasmodic, anti-tussive, narcotic, bronchodilator, anodyne, hypnotic and mydriatic effects. Leaves are used as a local application for rheumatic swellings of the joints, Lumbago, Sciatica, Neuralgia, painful Tumors, Scabies, Eczema, Allergy and glandular Inflammations, such as Mumps; used externally for earache and smoked to relieve spasmodic Asthma. Seeds are also used externally for piles (Yusuf et al., 2009). Seeds, leaves and roots are used in insanity, fever with catarrh, diarrhea, skin diseases and cerebral complications. Arthritis Treatment Gout is a disease that results from an overload of uric acid in the body, leads to the formation of tiny crystals of monosodium urate monohydrate that deposit in tissues of the body, especially the joints (Virsaladze et al., 2007), also called gouty arthritis (Kamienski, and Keogh, 2006). The xanthine oxidase inhibitory activity was assayed for D. metel which is traditionally used for the treatment of gout (Umamaheswari, 2007). More than 50% xanthine oxidase inhibitory activity (in vitro) was seen in the methanolic extracts of D. metel which was comparable with the standard antigout drug, allopurinol which showed 93.21% inhibition at 100 µg/mL concentration with an IC50 value of 6.75µg/mL. The methanolic extract was also screened for in vivo hypouricaemic activity against potassium
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oxonate-induced hyperuricaemia in mice and the extract was found effective (Umamaheswari, 2007). Hyoscyamine and scopolamine are the two commercially important anesthetic, anti-
spasmodiac and anticholinergic drugs, also the two most important alkaloids produced in roots and then translocated to the aerial parts of Datura plants (Hashimoto and Yamada, 1994; Zhang et al., 2004).
Table 1. Alkaloidal constituents of D. metel. Plant Parts
Leaves
Alkaloid al content (%) 0.426-
Seeds
0.426
Roots
0.35
Flowers
-
Fruit (Pericarp) Cultured callus In vitro propagate d shoots
Main Constituents
References
Atropine, hyoscyamine and scopolamine, l-oxo21,24S-epoxy-(20S,22S-witha-2,5,25-trienolide, pyrrole derivative (2'-(3,4-dimethyl-2,5-dihydro1Hpyrrol-2-yl)-1'-methylethyl pentanoate)
Dabur et al., 2005; Rastogi and Mehrotra, 1993, Siddiqui et al., 1987 Hyoscyamine, daturanolone and fastusic acid Ghani, 2003 and many other tropane alkaloids Hyoscyamine, 3α, 6β-Ditigloyloxytropane, 3α, Ghani, 2003 6β-ditigloyloxytropan-7β-ol, tigloidine, apohyoscine, hyoscine, 3α-tigloyloxytropane, norhyoscine, meteloidine, hyoscyamine, cuscohygrine and tropine Withanolide (baimantuoluoline A, B, and C and Agharkar, 1991; withafastuosin E and withametelin C), Manickam et withametelins I, J, K, L, M, N, O, P, 12β- al., 1993; Yang hydroxy-1,10-seco-withametelin B and 1,10- et al., 2010a seco-withametelin B β-sitosterol, triterpene, daturanolone daturadiol Cholesterol and 5α-pregnane3β,20β-diol
and Ghani, 2003 De, 2003
C28 sterol 3β,24ξ-dihydroxy-ergosta-5,25- De, 2003 dienolide and the withanolide 12deoxywithastramonolide.
Insecticidal Activity Different percentage (at 2.5, 5.0, 7.5 and 10.0%) of methanolic extract of Datura metel seeds, were tested against Helicoverpa armigera (Hubner), the cotton bollworm, is a moth, the larvae of which fed on a wide range
of plants, including many important cultivated crops. The 1.5 and 2.0% fractions of methanolic extract showed significant adverse effects on various biological parameters viz. larval survival, weight and duration, pupal period, % of pupation and adult emergence (Singh and Singh 2008).
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Herbicidal Activity
Antifungal Activity
Aqueous and organic solvent (methanol and nhexane) 0, 5, 10 and 15% (w/v) extracts of shoot and root of Datura metel L. (Syn, Datura alba Nees.) were studied against Phalaris minor Retz., one of the most problematic weeds of wheat. 5-15% of methanol and 15% nhexane root extract significantly reduced the germination, shoot and root length was significantly suppressed by all the employed concentrations of aqueous as well as organic solvent extracts in fact reduce the biomass a lot (Javaid et al., 2008).
Different polar and nonpolar solvent extracts of D. metel showed significant antifungal activity against many different fungal species. The hexane, chloroform, acetone and methanolic fractions of Datura metel L. were investigated for antifungal properties using pathogenic species of Aspergilli. The chloroform fraction was found to be endowed with antifungal activity. The minimum inhibitory concentration (MIC) of chloroform fraction of D. metel L. was 625.0 mg/ml against all the three species of Aspergilli, (A. fumigatus, A. flavus and A. niger) by microbroth dilution and percent spore germination inhibition assays (Sharma, 2002). The MIC by disc diffusion assay was observed to be 12.5 mg disc. Methanolic extracts of Datura metel were analyzed against pathogenic Aspergilli, (A. flavus and A. niger) and in vitro MICs were found to be 1.25–2.50 mg/ml by both microbroth dilution and percent spore germination assays (Dabur et al., 2004; Khan and Nasreen, 2010). A novel compound 2-(3,4dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1methylethyl pentanoate was isolated from Datura metel L. and in vitro activity of this dihydropyrrole derivative against Aspergillus and Candida species was evaluated. The compound was found to be active against all the species tested, namely Candida albicans, C. tropicalis, A. fumigatus, A. flavus and A. niger (Dabur et al., 2005). The post-antifungal effect (PAFE) of the antifungal compound 2-(3,4dimethyl-2,5-dihydro-1Hpyrrol-2-yl)-1methylethyl pentanoate (DHP) on A. fumigatus was investigated and the secretory protein inhibited by DHP was identified but mechanism whereby DHP inhibit these proteins is unknown (Dabur et al., 2007). Aqueous and methanolic extracts of Datura metel was evaluated in vitro against Ascochyta rabiei, (the causal agent of chickpea blight), 21-34% and 20-40% reduction in growth of A. rabiei with aqueous and methanol extracts of shoot of D. metel respectively and 21-34% and 15-25% and 11-29% reduction was reported with root extracts (Bajwa et al., 2008).
Anticancer Activity
Activity
and
Antiproliferative
Nitrogen-containing polyhydroxylated heterocyclic compounds are competitive inhibitors of various glycosidases, found most effective against various diseases including diabetes, cancer, and viral infections, along with additional activities, such as immunomodulatory properties and inhibition of glycolipid synthesis (Jacob, 1995). Withanolides was isolated from D. metel which are a group of steroidal lactones, many of these compounds exhibit a variety of biological activities, including anti-inflammatory, antioxidant, antitumor, and immunosuppressive properties. Withanolides can inhibit tumor cell proliferation and angiogenesis and induce the phase II enzyme quinone reductase (Pan et al., 2007). Calystegines have been isolated in several Solanaceous species and found to occur in different genera: including Datura (Nash et al., 1993). Three withanolide glycosides named daturametelins, together with two known ones, daturataturin and 7,27-dihydroxy-1-oxowitha2,5,24-trienolide, were isolated from the methanolic extract of the aerial parts of Datura metel L. All compounds were tested for their antiproliferative activity towards the human colorectal carcinoma (HCT-116) cell line. The nonglycosidic compound exhibited the highest activity of the tested withanolides, with an IC50 value of 3.2±0.2 µM (Ma et al., 2006).
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Antibacterial Activity Crude aqueous and ethanol extracts of leaf, stem bark and roots of D. metel were investigated against eight clinical bacterial isolates (Streptococcus betahemolytic, S. dysenteriae Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus cereus and Salmonella typhi). The leaf and stem bark extracts was antagonistic against the test bacteria species with inhibitory zones and Staph. aureus was the most inhibited majorly with the ethanol extract (Akharaiyi, 2011). Hypoglycemic Activity The seeds of D. metel were investigated for hypoglycemic and antihyperglycemic activities in normal Wistar albino rats and diabetic rats. D. metel seed powder was suspended in 1% sodium CMC and given to normal (in the form of mucilage), diabetic rats, blood glucose levels above 300 mg/dL (orally) at doses of 25, 50 and 75 mg/kg body weight. Blood sampling in different time frame within 24h and dose dependent hypoglycemia was observed in animals treated. The dose dependent antihyperglycemic activity was also observed with D. metel in alloxan-induced diabetic rats. Seed powder of D. metel possessed blood glucose lowering effect in normoglycemic and in alloxan-induced hyperglycemic rats. Thus, the folk usage of the seeds of D. metel for controlling diabetes may be validated by this study and the seeds offer promise for the development of potent phytomedicine for diabetes (Murthy et al., 2004). Free radical scavenging activity D. metel seeds were analysed for the fatty acids and fat-soluble bioactive compounds. The amount of total lipid in D. metel seeds was 55g/kg in weight and mainly linoleic acid followed by oleic, palmitic and stearic acids. The crude n-hexane extract was characterized by a relatively high amount of phytosterols along with stigmasterol, β-sitosterol, lanosterol, ∆5-avenasterol and sitostanol. In this extract, γtocopherol was the major component present
accounting for more than 80% of total tocopherols detected. n-hexane extract of D. metel seeds was able to quench only 40 % of DPPH radical. D. metel seeds contain a considerable amount of oil and may be a good source of essential fatty acids and lipid-soluble bioactives. The presence of tocopherols and sterols may have medicinal importance for human being (Ramadan et al., 2007). Antioxidant Activity The aqueous extracts of leaf, stem bark and roots of D. metel showed phytochemical and antioxidant activities. The aqueous extract of the plant displayed antioxidant activity of between 49.30-23.82% and can consider the plant as a natural source of antioxidants (Akharaiyi, 2011). Toxicities and Cytotoxicity Activity All of the plant parts of Datura are poisonous. Even a small dose is very poisonous because of the presence of toxic tropane alkaloid or the presence of anticholinergic substances such as scopolamine, hyoscyamine and atropine can cause neural toxicity (Ko, 1999). The toxicity sign and symptoms include acute confusion, fever, tachycardia, hot flushed dry skin, dilated pupils, dry mouth, urinary retention, hallucinations, headache, delirium, rapid and weak pulse, convulsions, and coma and even death (Kam and Liew 2002; Ko, 1999). Using the MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] assay cytotoxic activity of withametelins I, J, K, L, M, N, O, P, 12-β-hydroxy-1,10-secowithametelin B and 1,10-seco-withametelin B isolated from methanolic extracts of D. metel were investigated. The withametelins I, K, L and N exhibited cytotoxic activities against A549 (lung), BGC-823 (gastric), and K562 (leukemia) cancer cell lines, with IC50 values ranging from 0.05 to 3.5 µM. Withamilin J showed moderate cytotoxic activity against BGC-823 and K562 but less cytotoxicity against A549 (Pan et al., 2007).
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Table 2. Bioactive compound and fraction with activity. Name of the Compound
Extraction Plant Activity References procedure parts Baimantuoluoline A 50% EtOH eluate Flower Exhibiting Yang et al., 2007 fraction activity for psoriasis Withanolides 50% EtOH eluate Flower Exhibiting Yang et al., 2007 fraction activity for psoriasis Withafastuosin 50% EtOH eluate Flower Exhibiting Yang et al., 2007 fraction activity for psoriasis (E)-methyl 4-(3-(4 Extracted Flower Treatment of Yang et al., 2010a hydroxyphenyl)-Nwith 70% EtOH psoriasis methylacrylamido) butanoate 6,7-dimethyl-1-D-ribitylExtracted Flower Treatment of Yang et al., 2010a quinoxaline-2,3(1H,4H)with 70% EtOH psoriasis dione-5′-O-βDglucopyranoside (5α,6α,7β,22R)-5,6,7,27Extracted Flower Treatment of Yang et al., 2010 tetrahydroxy-1-oxowithawith 70% EtOH psoriasis 2,24-dien-27-O-βD-glucopyranoside (5α,6β,7α,22R)-5,6,7,27Extracted Flower Treatment of Yang et al., 2010 tetrahydroxy-1-oxowithawith 70% EtOH psoriasis 2,24-dien-27-O-β-Dglucopyranoside (5α,6β,7α,12β,22R)Extracted Flower Treatment of Yang et al., 2010 5,6,7,12,27-pentahydroxy-1- with 70% EtOH psoriasis oxowitha-2,24-dien-27-O-βD-glucopyranoside (5α,6β,22R)-5,6,27Extracted Flower Treatment of Yang et al., 2010 trihydroxy-1-oxowitha-2,24- with 70% EtOH psoriasis dien-27-O-β-Dglucopyranoside Withametelins Methanol extract Flower Cytotoxic Pan et al., 2007 1, 10-seco-withametelin B Methanol extract Flower Cytotoxic Pan et al., 2007 12 β -hydroxy-1,10-seco- Methanol extract Flower Cytotoxic Pan et al., 2007 withametelin B alkaloid datumetine p-methoxybenzoic Leaves Antispasmodic Siddiqui et al., acid drug 1986 2-(3,4-dimethyl-2,5Leaves Antifungal Dabur et al., 2005 dihydro-1H-pyrrol-2-yl)-1activity methylethyl pentanoate Serotonin Methanol Flower Induced during Murch et al., 2009 stress Melatonin Methanol Flower Cold stress Murch et al., 2009
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Traditional use
(Duttagupta et al., 1980) but after redifferentiation of organ like shoots or roots in the dark alkaloids synthesized (Duttagupta et al., 1984). A hairy root line of Datura metel was reported following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was optimized with reducing salt to half in B5 liquid medium and adding 8.7% to 70% permeabilizing agent Tween 20 and obtained a better biomass yield (2.3mg/l/day) and secreted scopolamine (0.84 mg/l/day) (Cusido et al., 1999).
Datura has a wide range of traditional applications, including the treatment of epilepsy, hysteria, insanity, heart diseases, and for fever with catarrh, diarrhea and skin diseases. Crushed leaves are used to relieve pain. In China, the plant is used in the treatment of asthma. In Vietnam, the dried flowers and leaves are cut into small chips and used in antiasthmatic cigarettes. About 3 to 5g of the flower extract can be used as an anesthetic through oral consumption that produces general anesthesia within 5 minutes, which lasted for about 5 to 6 h. The flower of the D. metel is used in the treatment of pain, chronic bronchitis and asthma (Kam and Liew 2002; Ko, 1999). In Bangladesh, leaves are used for scabies, eczema and allergy (Chowdhury et al., 1996). Dried whole plant powder is used to smoke to cure excessive or abnormal breathing, applied around the eyes to enlarge pupils. Application or drinking of leaf juice relieves pain and swelling. Leaf juice is mixed with a little opium and applied to the affected area to reduce swelling of gums or base of ears. Leaf juice is mixed with lime and turmeric and applied to the breasts to reduce breast pain (Rahmatullah et al., 2010). The flowers of Datura metel have been used in traditional Bangladeshi medicine for the treatment of asthma, convulsions, pain, and rheumatism for centuries.
Plants are a potential source of a large number of valuable secondary metabolites. Studying valuable secondary metabolites isolated from medicinal plants can open new possibilities to find bioactive alternatives to synthetic chemical. A number of alkaloids including hyoscine, hyoscyamine, meteloidine, scopolamine, tigloidine, tropine, withametelline and datumetine etc. have been reported from Datura species (Rastogi et al., 1998). Some of these alkaloids have found application in health care. Some of the bioactive compounds or fractions with their suggested activity from D. metel are summarized in Table 2.
In-vitro production of trophane alkaloids
CONCLUSION
Adventitious shoots that generated from young leaves of D. metel and shoot buds developed on MS medium with 2 mg/l benzylaminopurine (BAP) and elongated on hormone-free solid basal medium. The micro shoots failed to produce alkaloids (De, 2003). In unorganized plant tissue culture of Datura failed to produce tropane alkaloids scopolamine and hyoscyamine due to the suppression of alkaloid biosynthesis in dedifferentiated cells (Savary and Dougall, 1990). In callus tissues of D. metel, alkaloids were not present
D. metel L. is a medicinal plant used as phyto-medicine to treat traditionally a wide range of health complications. This plant can be explored further as per its diversity of traditional uses and on the basis of wide range of chemical compounds reported to be present in various parts of the plant. In the present investigation, Phyto-chemistry, Pharmacology and traditional uses of D. metel has been reviewed. Furthermore, the undocumented knowledge on this plant species has to recorded and should be explored widely so that it could serve the Humanity.
Compounds isolated from Datura metel l.
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Source of Support: Nil
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Conflict of Interest: None Declared
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Regular Article PHARMACOGNOSTIC EVALUATION OF AN AROMATIC PLANT OF BASMATI FLAVOR: PANDANUS AMARYLLIFOLIUS ROXB. Jyothi T1*, Niranjan Y2, Harisha CR3 1*
Research assistant, ALN Rao Memorial Ayurvedic Medical College, Koppa, Chikmagalur District, Karnataka, INDIA 2 Lecturer, Dept. of PG studies in Kayachikitsa, ALN Rao Memorial Ayurvedic Medical College, Koppa, Chikmagalur District, Karnataka INDIA 3 Head, Pharmacognosy Laboratory, IPGT& RA, Gujarat Ayurved University, Jamnagar- 361008 Gujarat, INDIA
Received: 03/03/2012; Revised: 31/03/2012; Accepted: 10/04/2012
ABSTRACT Many herbs are incorporated into food for their aromatic flavor like Lemon grass, Ocimum etc. One among them is Pandanus amaryllifolius Roxb. from the family Pandanaceae, cultivated in Southern India which is used for imparting aroma to rice preparations. Basmati rice is considered best due to its unique flavor. It is consumed by many as principal food, but is very expensive and unaffordable to the common man. Leaves of Pandanus amaryllifolius are widely used to flavor ordinary rice with the characteristic Basmati aroma. 2-acetyl-1-pyrroline is said to be the compound which gives this characteristic aroma. Besides its flavoring property, it also possesses therapeutic utilities and is used to treat Rheumatism and Diabetes. The whole plant is considered diuretic, sedative and having wound healing properties. Though with such wide range of uses, the plant has been least explored pharmacognostically. This study was undertaken to fulfill this aspect of the plant by throwing light upon the Microscopic, Powder microscopic & macroscopic characters. Key Words: Pandanus amaryllifolius, morphology, microscopic studies, powder microscopy
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INTRODUCTION
Macroscopic study:
Pandanus amaryllifolius Roxb. (Pandanaceae) is a shrub, native to Malaysia (West Indonesia, Malaysia, Philippines and Ceylon) 1 growing in marshy lands and also cultivated in Southern India especially in the states of Kerala and Karnataka. It shows perpetuated sucker roots, aerial stem 0.5–1m in height, 2–5 cm in diameter, bearing long aerial descending roots often penetrating in the soil like prop roots. Leaves are simple.2
Macroscopic characters such as size, shape, margin, apex, surface, colour, odour, taste, nature and texture were studied.
In South India, people prefer to grow this plant in home garden to procure its leaves in fresh condition, whenever required, to use as a flavoring agent especially to impart its pleasant Basmati flavor to the ordinary rice during cooking process. 2-acetyl-1-pyrroline is said to be the compound which gives this characteristic aroma 3 . Besides the flavoring property the leaves of the plant are also known to possess number of therapeutic properties like anti-rheumatism, anti-diabetic, diuretic, sedative and wound healing4, 5. In spite of all these reputed uses, the leaves have not yet been investigated fully which led us worth to evaluate them systematically with an objective to study the leaves of P. amaryllifolius for its macroscopic and microscopic characteristics including powder microscopy.
Microscopic study: Free hand transverse sections of leaves were used for microscopic evaluation. Surface preparation was done by placing wet leaf on the glass slide and tissues were scrapped off with the sharp edge of razor blade with utmost care. Water was slowly and continuously added and scrapping was done till transparent and colorless epidermis was exposed7. The powder microscopy of dried leaves was carried out with and without staining8, 9. The photomicrographs were taken by Carl Zeiss binocular microscope. Histochemical test: Free hand sections of leaves were taken, cleared with chloral hydrate and then stained with phloroglucinol and hydrochloric acid to observe the lignified elements. Other reagents were also used separately to stain fixed oil globules (Sudan III), Tannin (ferric chloride), starch grains (IKI) 10 etc. Glycerin was used for slide mounting. RESULTS
MATERIALS AND METHODS
Macroscopic Characteristics:
Fresh plants of P. amaryllifolius were collected from the site of cultivation i.e. North Malabar area of Kerala and herbal garden of Institute for Post Graduate Teaching And Research in Ayurveda, Gujarat Ayurved University, Jamnagar. Correct authentification of the plant was done 6 and with the help of experts of the institute. Leaves were separated from the plant, cleaned by washing in fresh water and dried in shade. Macroscopic and microscopic characters of the leaf were studied using fresh leaves.
Leaf simple, alternate, linear, lanceolate, 30–40 cm long, 2–4 cm wide, apex acute, base symmetrical, margin entire except at the apex where it becomes minutely spiny upto 1–1.3 cm of its length. Venation is parallel; midrib is prominent located in the channel formed by two longitudinally running veins each on its either side, other veinlets are obscure and run in parallel direction. Leaf surface is glabrous and smooth. (Fig No. 1, 2). Upper surface is dark green, lower surface is pale green in color. Taste bland. Odor is pleasant especially after crushing.
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Microscopic characteristics of the leaves: Diagrammatic representation of transverse section (Fig. No. 3)) of the leaf blade passing through the midrib, shows protrusion sion conically at the lower side and narrowly grooved on the upper side, also shows discontinued patches of sclerenchymatous cells under the upper epidermis and rows of meristele of various sizes and shapes above the lower epidermis, the one located in the midrib being biggest in size. The meristeles located in lamina extensions is distantly placed and a few connect both the epidermis. Collenchyma cells are present in midrib just above the lower epidermis. idermis. Detailed T.S (Fig. No.4) passing through midrib shows two layers of upper and lower epidermis covered with thin cuticle, embedded at places with stomata. The cells of the outer layer of the upper epidermis are small and uniform in size, unlike the cells lying underneath it, which are tangentially elongated and tubular in shape (Fig No. 5.1). The cells of lower epidermis are identical with those of upper epidermis but are highly papillose (Fig.No. 5.2). .2). The cells lying underneath upper epidermis are not uniform throughout and show 5–77 small cells alternating with big size sized cells (bulliform motor cells) (Fig.No. 5.1);
underneath them lies groups of 2–8 2 thick walled non lignified sclerenchymatous cells (Fig. No. 5.4). The meristele located in the midrib b is bigger in size than the other meristeles of the leaf blade and is composed of 2–3 3 centrally located metaxylem and radially running 3–4 4 protoxylem (Fig. (Fig No. 5.5 and 5.6), protected dorsiventrally by sclerenchymatous fibers embedded at places with tracheids and parenchyma. Occasionally a layer of ill developed endodermis is also seen encircling the meristele. Two to three rows of Collenchyma cell bands are also als located underneath the cells of lower epidermis (Fig No. 5.7). Remaining cells of the ground tissue of the midrib runs radially; and is of different sizes and shapes. The cells of the mesophyll consist of spongy parenchyma; but at places they also run radially; dially; prismatic and acicular crystals of calcium oxalate and oil globules are embedded throughout the parenchymatous cells of the section (Fig. No. 5.8, 5.9 and 5.10). Histochemical tests: The histochemical tests (Table No.1) revealed the presence of lignified lig tissue, oil globules and calcium oxalate crystals. Tannins and starch were absent.
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GJRMI, Volume 1, Issue 4, April 2012, 133–139 Table No.1. Histochemical tests
Sl. No 1 2 3 4 5
Reagent used
Findings
Color
Result
Phloroglucinol and Hydrochloric acid Sudan red III Ferric chloride IKI Hydrochloric acid
Lignified tissue
Pink
Present
Oil globules Red Tannin Blue Starch Blue Calcium oxalate Effervescence/Soluble
Organoleptic characters: The dried leaf powder was dark green in color without any characteristic taste, odor pleasent. Powder Microscopy: The diagnostic characters of the powder showed overlapping fragments of outer and inner epidermis in surface view the cells of the outer epidermis being hexagonal, thick walled unlike those lying underneath it, which are narrow and runs tangentially; fragments of annular and spiral vessels; sclerenchymatous cells in surface view, fibers with thick walled and narrow lumen. Lower epidermis showing papillae in surface view and in sectional view,
REFERENCES 1. http://www.igardendigest.com/screw_pi ne.htm accessed on 15-04-12 2. http://219.93.41.233/wapi/mctweb.dll/g etobject?MID=MEDICINALPLANT& ObjID=576 accessed on 13-08-2010 3. Wongpornchai S, Sriseadka T, Choonvisase S, (2003). “Identification and quantitation of the rice aroma compound, 2-acetyl-1-pyrroline, in bread flowers (Vallaris glabra Ktze.)”.J. Agri. Food. Chem.51 (2): 457–
Present Absent Absent Present
transversely cut fragments of lamina, prismatic crystals of calcium oxalate and oil globules scattered as such or embedded in the cells of the epidermis and mesophyll. Stomata embedded in the upper and lower epidermis in surface view. CONCLUSION Present study defines the identity of Pandanus amaryllifolius Roxb. in terms of macroscopic, microscopic, histochemical parameters which were lacking till date. It is already known that 2-acetyl-1-pyrroline compound is the flavoring principle. Other chemical constituents and therapeutic/ toxicological evaluations are to be carried out in order to fulfill the complete scientific profile of the plant.
462.doi:10.1021/jf025856x.PMID 12517110. 4. http://www.buzzle.com/articles/pandanleaves.html accessed on 13-08-2010 5. Li J. and Ho S.H (2003). Pandan leaves (Pandanus amaryllifolius. Roxb) as a natural cockroach repellent. Proceedings of the 9th National Undergraduate Research Opportunities Programme. 6. Green A, (2006), Field guide to herbs & spices, Philadelphia, Quirk Books. P. 80-1
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7. Khandelwal K R, (2008), Practical Pharmacognosy, Techniques & Experimental, Pune, Nirali Prakashan. p. 51 8. Wallis, T.E., 1985. Text book of Pharmacognosy, 5th Ed, CBS Publishers, New Delhi, 571–578
Source of Support: Nil
9. Trease, G.E., Evans, W.C. (1983). Pharmacognosy, 12th Ed. Bailliere Tindall, Eastbourne. U.K. 95-99, 512– 547 10. Khandelwal K R, (2008), Practical Pharmacognosy Techniques & Experimental, Pune, Nirali Prakashan. p. 183–4
Conflict of Interest: None Declared
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