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INDEX MEDICINAL PLANT RESEARCH Health Sciences EFFECT OF RUELLIA PRAETERMISSA EXTRACTS ON ERYTHROPOIESIS IN PREGNANT WOMEN Salah A. Martin, Njunda L. Anna……………………………………………………………..309–314 Nature & Life Sciences PLACE OF LABIATES IN FOLK MEDICINE OF THE AREA OF M’SILA (ALGERIA). Noui HENDEL, Larbi LAROUS, Madani SARI, Amel BOUDJELAL and Djamel SARRI....315–322 Botany FATTY ACID PROFILE OF FRUITS OF LUFFA ACUTANGULA VAR. AMARA C.B.CLARKE Jadhav Santosh Jaysingrao, Chavan Niranjana Sunil………………………………………….323–327 Veterinary Science INTRODUCTION OF FIVE WELL-KNOWN AYURVEDIC MEDICINAL PLANTS AS FEED ADDITIVE ON LIVESTOCK’S PERFORMANCE: A REVIEW Mirzaei F, Hari Venkatesh K R………………………………………………………………..328–339 Pharmacology EVALUATION OF ANTI ULCER ACTIVITY OF FICUS PUMILA L. LEAF EXTRACT IN ALBINO RATS. Muhammed Ashraf V K, Thamotharan G, Sengottuvelu S, Haja Sherief S, Sivakumar T…...340–351 Himalayan Floral Research HIERARCHICAL CLUSTER ALGORITHMS OF OCCURRENCES AND ANTIMICROBIAL ACTIVITY OF ROOT EXTRACT OF AN ETHNOMEDICINALLY IMPORTANT PLANT: SIDA CORDIFOLIA LINN. Kumari Priti, Joshi Girish Chandra, Singh Bibhesh Kumar…………………………………...352–363
Indigenous Medicine Ayurveda PHARMACOGNOSTIC EVALUATION OF STEPHANIA JAPONICA (Thumb.) Miers Savitha H, Sanjaya K.S, Brijesh K, Hari Venkatesh K.R, Jyothi T…………………………...364–371 PHYTOCHEMICAL AND ANTIFUNGAL STUDIES ON ROOT OF IPOMOEA SEPIARIA KOENIG EX. ROXB Majumder Sayani, Ashok B.K., Nishteswar K………………………………………………..372–380
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FRUITS OF VATA VRUKSHA, FICUS BENGHALENSIS L., MORACEAE PLACE – UMBLEBYLU, SHIMOGA DISTRICT, KARNATAKA, INDIA
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Original Research Article EFFECT OF RUELLIA PRAETERMISSA EXTRACTS ON ERYTHROPOIESIS IN PREGNANT WOMEN Salah A. Martin1*, Njunda L. Anna2 1
Department of Zoology and Animal Physiology, Faculty of Science, University of Buea, P.O. Box 63 Buea, Cameroon. 2 Department of Medical Laboratory Sciences, Faculty of Health Science, University of Buea, Cameroon. *
Corresponding Author: E-mail: salahmartin@yahoo.fr
Received: 14/06/2012; Revised: 22/07/2012; Accepted: 24/07/2012
ABSTRACT The effect of the extracts of Ruellia Praetermissa Schweinf. ex Lindau. on hemoglobin (Hb), Hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV). mean corpuscular hemoglobin concentration (MCHC), red blood cell count (RBC), was investigated in 50 Pregnant women attending prenatal clinic in Belo maternity. The women were assigned to 5 groups of 10 women per group. The first group was the control and the other 4 were the experimental groups. The control was administered daily, 0.5 ml of saline solution while the experimental groups were administered daily oral doses of the plant extract in concentrations of 200 mg/kg, 400 mg/kg, 800 mg/kg and 1,600 mg/kg respectively for 16 days. Blood samples were collected on the 17th day and analyzed. The extracts contain flavonoids aglycones (luteolin and apigenin) and their respective, glycosides and a high concentration of triterpenes (campesterol, stigmasterol, -sitosterol, lupeol) and iridoid glycosides. It was also found to be rich in vitamins and minerals. The extracts increased the values in a dose dependent manner Hb (P < 0.05), RBC (P < 0.05), hematocrit (P < 0.05). It however showed no remarkable effect on the values of MCH and MCHC (P > 0.05) but with a dose depending decreasing effect on MCV (P < 0.05). The active principles of this plant drug stimulate erythropoiesis which leads to increase in circulating RBCs with slightly microcytic sizes (MCV), normochromic weight, (MCH) normochromic hemoglobin contents (MCHC). The result of this study thus supports the traditional use of Ruellia Praetermissa in pregnancies threatened with miscarriage and as a remedy for anemia. KEYWORDS: Ruellia Praetermissa, Pregnant Women, Blood
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INTRODUCTION Gestational problems are a potential source of mortality for both gestation mothers and their developing foetuses in the underdeveloped world in general and Africa in particular. There is great need for effective safe prenatal drugs from natural sources which are available and affordable with respect to modern medicines. Plant drugs have effects haematological parametrs (Nnamdi et. al., 2012) which influence reproductive health. Ruellia praetermissa Schweinf. ex Lindau., is a wild herb and indigenous to central and south eastern Asia and also widespread in tropical and subtropical Africa. In the North West Region of Cameroon it enjoys a folk reputation as blood and pregnancy medicine. In other south Eastern African countries; it is widely applied to relieve pain (Gelfand et al., 1985). The Plant drug exerts estrogenic and cholinergic effects (Salah et al., 2002). The extracts regularize pregnancies threatened with miscarriages in early stages. This is due to its ability to mimic 17β-estradiol. It stimulates the growth of the uterine endometrium. This is by the proliferation and the development of the cells of the uterine endometrium as it upregulates estrogen, luteinizing hormone LH and progesterone receptors on the uterine muscles at the beginning of gestation and to excite the uterine myometrium at term. (Salah et al., 2002). In addition, the extracts of this plant drug has a stimulatory effect on the motility of the gastrointestinal tract (Salah et al, 2000) and antihypertensive effect by the inhibition of Angiotensin-converting enzyme (ACE) activity (Salah et al, 2001). These biological effects are of particular interest since high blood pressure and indigestion frequently characterize gestation period. This herbal drug is rich in flavonoids such as luteolin, quercetin and apigenin. (Wagner and Bladt, 1998). The favonoids also have an antispasmodic effect on uterine smooth muscle. (Salah 2001). The plant extract has 5-Lipoxygenase inhibition and antisplasmodic effects (Salah, 1999). The plant drug promotes implantation, and stabilizes the uterine endometrium in female rats (Salah and Wagner, 2009). The present study is aimed at
finding out the scientific basis of the use of Ruellia praetermissa as a prenatal herbal drug in the North-Western region of Cameroon. MATERIALS AND METHODS Plant Material The plant material was collected in Belo, North West Province of Cameroon in September 2011. The specimen was verified and authenticated as the one earlier identified by Kofany of the Cameroon National Herbarium Obili Yaounde under the voucher specimen number 43596 deposited in 1996. Extraction and sample preparation Sun dried leaves of the plant were pulverized and 250 g was extracted using the sohxlet for 12 h in each case progressively with 2 l of n-hexane, chloroform, ethyl acetate, and methanol. The extracts (160, 140, 250 and 300 mg, respectively) were recovered by rotavaporization. The chaffs were boiled in water at 85oC for 6 h and 225 mg of extract was recovered by lyophilization. The procedure was repeated 20 times to obtain the plant drug necessary for the whole investigation. Plant Drug Analysis a) TLC The ethyl acetate extract (5 mg) was cochromatographed with flavonoid test samples (rutin, chlorogenic acid, hyperoside and isochlorogenic acid), luteolin and luteolin-7glucoside, quercetin, isoquercetin, delphinidin and caffeic acid using ethyl acetate-formic acid –glacial acetic acid-water (100:11:11:26) as the mobile phase and precoated silica gel 60 F254 (20 x 20 thickness 0.25 mm Merck, Darmstadt, Germany) as the stationary phase. The plate was first observed at UV254 nm then with natural products-polyethylene glycol reagent (NP/PEG) and evaluated at UV366 nm. b) HPLC This was carried out with an HP 1090. A liquid chromatography and an HP 1040 photodiode array with a Hewlett Packard detector as a LiChrospher 100 RP 18 (5 µm) column 125 x 4 mm (Merck, Darmstadt,
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Germany), and a precolumn LiChrospher RP18 4-4 mm (5 µm) (Merck, Darmstadt, Germany) with detection at wavelength 210 nm, 254 nm, 280 nm and 366 nm. The mobile phase used for the separation was HPLC graded water (Solvent A) and acetonitrile (Solvent B), all acidified with 33 µL of phosphoric acid (85%). It was started at 10% of solvent B and a linear gradient of 10–30% MeCN during 20 min for a total run of 30 min, at a flow of 1 mL/min, and a volume of 12.5 µL of 1 mg/mL of ethyl acetate extract of R. praetermissa was injected. Experimental Subjects
The study was conducted in Boyo, and Bamenda in the North West Region of Cameroon from December 2011 to April 2012. A total of 50 pregnant women between 20 and 40 years of age attending routine prenatal clinics in Belo Maternity Center were recruited for the study. They gave their informed consent to participate in the study which was approved by the ethics commission of our institution. All participants were interviewed to assess their physical wellness, nutritional factors, gestational history and hematological records. The women were assigned to 5 groups of 10 each. The first group was the control which received daily doses of 0.5 ml of saline solution. The second, third, fourth and fifth groups received 200 mg/kg, 400 mg/kg, 800 mg/kg and 1,600 mg/kg respectively in daily oral doses for sixteen days. Blood samples (0.5 ml) were obtained on the 17th day from each of the women. This was done with needles and
syringes in 10 ml tubes using venous puncture by drawing specimens from a superficial vein in the antecubital fossa of the arm. Ethylenediamine tetraacetic acid (EDTA) was used to prevent blood from clotting. These samples were analyzed in the Diagnostic Laboratory in in Bamenda for the determination of blood parameters under investigation: Hematocrit (Hct), Hemoglobin Concentration (Hb), and Red blood Cell Counts (RBC)
Blood Analysis The Hct was measured by the percentage of the total blood volume that is made up of RBCs. The height of the RBCs column was measured after centrifugation and compared to the column of the total whole blood. The ratio of the height of the RBC column compared with the original total blood column was multiplied by 100% and that was recorded as the Hct value. The haemoglobin value and the red blood cell count were done by an automated cell counter. The values of RBC, hematocrit, and haemoglobin test were used to calculate the RBC indices (MCV, MCH and MCHC). Statistical Analysis Data was analysed using Sigma Plot 11 for Windows and the significance was set at the 5% level. The results were expressed as mean ± SEM, differences between means analysed using Student’s‘t’ test. P values of 0.05 or less and 0.001 were taken as being statistically significant. RESULTS AND DISCUSSION The results of the chemical analysis of the four extracts revealed that the plant drug contains flavonoids aglycones (luteolin and apigenin) and their respective, glycosides. The extract also contains a high concentration of triterpenes (campesterol, stigmasterol, βsitosterol, lupeol) and iridoid glycosides. The effect of various doses of the extract of R. Praetermissa on the mean hematological parameters are shown in Tables 1 & 2 below. Hematocrit (Hct): Increasing the dosage concentration of the plant drug had an effect of increasing the Hematocrit (Hct) in all the treatment groups. Hematocrit (Hct) value at 200 mg/kg 43.90 ± 2.06 % was however not significantly different from that of the control group 43.35 ± 1.82 %. At high doses of the extract, 800 mg/kg and 1,600 mg/kg, there was a significant increase from 47.75 ± 1.5% to 48.25 ± 1.49 % respectively (P < 0.05).
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Hemoglobin Concentration (Hb): Increase in hemoglobin concentration occurred with increased concentration of applied extract. The hemoglobin value of 13.26 ± 1.5 g/dL of the control group increased to 15.22 ± 0.19 g/dL at 800 mg/kg and 16.10 ± 0.07 g/dL at 1,600 mg/kg respectively. All these values are significantly different from the control group (P < 0.05). Significant change in hemoglobin concentration also occurred between the two high doses i.e. 800 and 1,600 mg/kg.
Red Blood Cell Count: Women that received 200 and 400 mg/kg body weight of the extract had significantly lower red cell value P < 0.05 than those who received 800 and 1,600 mg/kg body weight of the extract. There was no significant difference between the red cell count of those that received 200 mg/kg dose of extract and the normal saline in the control group.
Table 1: Effect of the Extract of Ruellia Praetermissa on the Hemtaocrit (Hct) haemoglobin concentration and red blood cell count in the pregnant women blood Treatment with Ruellia Extract 0.5 ml Saline 200 mg/kg 400 mg/kg 800 mg/kg 1600 mg/kg
(Hct) (%) ± S.E. 43. 35 ± 1.82 43.90 ± 2.06 46.62 ± 1.60 47.75 ± 1.50 48.25 ± 1.49
Hb(g/dL) ± S.E. 13.26 ± 1.5 13.34 ± 1.7 14.75 ± 0.14 15.22 ± 0.19 16.10 ± 0.07
RBC(million/mm3) ± S.E. 4.10 ± 0.23 4.34 ± 0.18 4.80 ± 1.03 5.33 ± 0.03 5.53 ± 1.1
Table 2: Effect of the Extract of Ruellia Praetermissa on the Blood indices (mean corpuscular volume [MCV], Mean corpuscular haemoglobin [MCV], and Mean corpuscular haemoglobin concentration [MCHC]). Treatment with Ruellia Extract 0.5 ml Saline 200 mg/kg 400 mg/kg 800 mg/kg 1600 mg/kg
MCV ± S.E. (mm3) 105.68 ± 2.92 101.15 ± 0.50 96.26 ± 1.06 89.58 ± 1.72 87.25 ± 1.35
MCH ± S.E. (Pg) 32.34 ± 1.4 30.73 ± 1.73 36.56 ± 0.25 28.55 ± 0.53 29.11 ± 1.39
MCHC ± S.E. (g/dL) 30.58 ± 1.1 30.38 ± 1.7 31.63 ± 1.5 32.87 ± 2.43 33.36 ± 1.14
MCV, MCH AND MCHC Women in the control group who received only normal saline had significantly higher MCV values (P < 0.05) than those recorded for all the groups of women who received the extract in various doses. The MCV values decreased in the dose dependent manner. The MCH and MCHC showed no significant effect (P > 0.05) as the plant drugs were administered in different concentrations.
The result of this study showed that the aqueous extract of the leaf of Ruellia Praetermissa increased the overall hemoglobin concentration, red cell count and packed cell volume in Pregnant women in a dose dependent manner. Extract of sorghum has been demonstrated to present similar effects on rats. (Ogwumike, 2002). However, during pregnancy, there is a decrease in hematocrit, red blood cell count and hemoglobin values due
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to increase ECF volume that increase plasma volume resulting in hemodilution (Sembulingam and Prema, 2010). This presents a potential source of anaemia for the gestation mother which can result in miscarriage. Gestational state also causes physiological polycythemia due to the increased emotional conditions and demand for proton buffering. Hypoxia due to the high demand of respiratory gases for both the gestation mother and the developing fetus also stimulates erythropoietin secretion (Strand, 1998). Erythropoietin or erythrocyte stimulating factor is the main hormone responsible for erythropoiesis in bone marrow, liver and spleen. The plant extract inhibits Angiotensin converting enzymes (ACE) activity and consequently stimulates the secretion of erythropoietin by peritubular capillaries of the nephron.(Vander et. al., 1994). Ruellia is a vegetable rich in Vitamin B12 (cyanocobalamin) and minerals such as Iron and copper. (Duke, J., 2010). Cyanocobalamin is the major maturation factor necessary for erythropoisis, iron is necessary for the formation of the heme part of the haemoglobin and copper is important for the absorption of iron from the gastrointestinal tract. (Sembulingam and Prema, 2010). The iridoid glycosides isolated from this plant drug exert an inotropic effect of the myocardial muscles (Salah, 1999) and hence increase the cardiac output during pregnancy to satisfy the demands of the developing fetus (Constanzo, 2004). Increase cardiac output increases
erythropoietic activity resulting in more than usual number of erythrocytes. The active principles of this plant drug stimulate erythropoiesis which leads to increase in circulating RBCs with slightly microcytic sizes (MCV), normochromic weight, (MCH) normochromic hemoglobin contents (MCHC). The increase in numbers of erythrocytes containing the same amount and concentration of haemoglobin will fulfil the physiological needs of the pregnant mother with the growing fetus. CONCLUSION This study suggests that administration of Ruellia praetermissa enhances the process of erythropoiesis in gestation mothers and hence counters the anemic effects of pregnancy in a dose dependent manner. In combination with our previous studies on the effects of Ruellia on ovulation, growth of the uterine endometrium, implantation and the estrogenic effects. These findings provide the pharmacological basis for the traditional use of this plant for prenatal care in the North West Region of Cameroon. ACKNOWLEDGEMENT The authors would like to express their gratitude to the Health Board of the Belo Maternity, the ethical committee for the administrative and ethical clearances and all the gestation mothers who accepted to participate in this study.
REFERENCES Constanzo L. S. Physiology.(2004) Sounders – An imprint of Elsevier. 2nd Ed. p Duke, J. (2010) Medicinal Plants of the World. In Computer Index with more than 85,000 entries. 3 vols. Gelfand M. Mavi S, Drummond R. B. and Ndemera B. (1985) The traitional medical practictioner in Zimbabwe: His principles of practice and Pharmacopoeia. MamboPress 411p,
Nnamdi Chinaka C, Uwakwe A A, and Chuku L C (2012). Effects of Aqueous and Ethanolic Extracts of Dandelion (Taraxacum officinale F. H. Wigg) Leaves and Roots on Some Haematological Parameters on Normal and STZ –Induced Diabetic Wister Rats. Global J. Res. Med. Plants & Indigen. Med., Volume 1, Issue 5, May 2012, 172–180
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Ogwumike o. O. (2002). Hemopoietic effect
of aqueous extract of the leaf sheath of sorghum bicolor in albino rats African J. of Biomed Res, Vol. 5, No. 1-2, pp. 69–71 Salah A. M. (1999) Anti-inflammatory and cardiovascular effects of some chemically active principles from Ruellia praetermissa. Doctorate Thesis: 75–93, Salah A. M., Dongmo A. B, .Kamanyi A., Bopelet M. and Wagner H.:(2001) Angiotensin-Converting Enzyme Inhibitory Effects by Ruellia Praetermissa. Pharmaceutical Biology Vol 39, No. 1 pp 16–19, Salah A. M., Dongmo A. B, .Kamanyi A., Bopelet M. Vierling W., and Wagner H.: (2000) In vitro purgative effect of Ruellia Praetermissa Scenf.ex.Lidau (Acanthaceae). J. of Ethnopharmacol. 72: 269–272, Salah A. M., and H. Wagner H. (2009), : Effects of Ruellia praetermissa extract on ovulation, implantation, and the uterine endometrium of female rat. J. of Medicinal Plants Research Vol. 3(9), pp. 641–645.
Source of Support: Nil
Salah A. M., Gathumbi J. Vierling W and Wagner H.:(2002) Estrogenic and cholinergic properties of the methanol extract of Ruellia praterissa Sceinf.ex.Lindau (Acanthaceae) in female rats. Phytomedicine 9: 52–55. Salah
A. Martin: (2001) Chemical and pharmacological investigation of Ruellia praetermissa, Hibiscus Sabdaruiffa and Commelina congesta extracton tracheal, ileal and uterine smoot and papillary mscle of guinea pig: Ph.D. Thesis.
Sembulingam S., and Prem S. (2010) Essentials of Medical Physiology. Jaypee brothers Medical Publishers (P) LTD, 5th Ed. pp 64–91 Strand
L. Fleur(1998). Physiology – A regulatory system approach. MacMillan Publishing Co., INC . 4th Ed. pp 268– 288.
Vander A. J., Sherman J. H., and Luciano S. D, (1994). Human Physiology – Mechanism of Body Function. McGraw-Hillm INC 6th Ed. pp 515– 560 Wagner H., and Bladt S., (1998) Plant Drug Analysis, 2nd Ed, Springer Verlag.
Conflict of Interest: None Declared
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Original Research Article PLACE OF LABIATES IN FOLK MEDICINE OF THE AREA OF M’SILA (ALGERIA). Noui HENDEL1*, Larbi LAROUS 2, Madani SARI3, Amel BOUDJELAL4and Djamel SARRI5.
1, 3, 4, 5
Faculty of Sciences. Department of Nature and Life Sciences, University of M’sila. BP166, Rue Ichbilia. ALGERIA 2 Faculty of Nature and Life Sciences. Department of Microbiology, University of FERHAT Abbas, 19000. Setif. ALGERIA *Corresponding Author E-mail: hendel_n@yahoo.fr
Received: 12/06/2012; Revised: 15/07/2012; Accepted: 20/07/2012
ABSTRACT The aim of this study was the inventory and the identification of labiates among the medicinal plants used in traditional medicine by the community of M’sila, North center-East of Algeria. Ethnobotanical data from local population were collected by direct interviews and a semi-structured questionnaire carried out by interviewees belonging to the population. Various data on informants were noted. From 82 plant species belonging to 33 families listed, Lamiaceae species represent 22% of the total plants listed and 48.6% of the frequency of use. Their botanical and vernacular names, medicinal part used and uses are given. Among diseases and ailments treated by labiates are stomach troubles and colic, diabetes, rheumatism, hypertension, eczema, anemia and cancer. Medicinal plants are frequently employed for treatment of ailments and illness in the community of M’sila in spite of the availability of the prescribed drugs. The documentation of this ethno-medicinal knowledge is important. The evaluation of the pharmacological activity for promising medicinal plants is suggested.
KEYWORDS: labiates, medicinal plants, Lamiaceae, ethno-botany, traditional medicine, Hodna, Algeria.
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INTRODUCTION The World Health Organization (WHO) estimates that 80% of the populations living in rural areas in developing countries depend on traditional medicine for their health care needs. WHO defines traditional medicine as "the total combination of knowledge and practices, whether explicable or not, used in diagnosing, preventing or eliminating physical, mental or social diseases and which may rely exclusively on past experience and observation handed down from generation to generation, verbally or in writing" (WHO, AFR/RC50/9, 2000) Algeria, concerning its geographical position, presents a broad range of climatic stages inducing a biodiversity with an avalanche of much of plants used like grass, natural food and for therapeutic aims. It is primarily due to its vast geography. The drug companies in this country, but also doctors and chemists seek to better knowing the inheritance of the spontaneous species used in traditional medicine. Their modes of use, their indications in various pathologies thus the active ingredients are studied since tens of years (Djebaili, 1984; Baba Aissa, 1991; 1999. Abdelguerfi, 2003). For this reason, one of our priorities is to conserve diversity, including uses made of plants by local inhabitants. MATERIALS AND METHODS Study area M’sila state, named capital of the HODNA (fig.1), occupies a privileged position in the central part of northern Algeria; between the Tell and the Sahara. Its climate is continental, semi-arid with an average temperature of 35°C in summer and of 07°C in winter and irregular rainfall of the order 100 to 300 mm/year. It covers an area of 18,718 km2 situated at an altitude of 500 meters between 35° 42' 07" N 4° 32' 49"E . (Le Houerou, 1995; Moreau et al., 2005).
The area is characterized by an ecological diversity represented by two principal ecosystems: steppe and forest ecosystems. Of vocation primarily agro-pastoral, with an estimated population of 991846, the principal activity of the population of rural areas is breeding sheep and caprine. Agriculture areas account for 20% of the total area devoted mainly to cereals. Rustic arboriculture is marked by the predominance of the apricot followed by the olive tree (Feliachi et al., 2003; Behlouli et al., 2008; T.A.D - CONSULT; 2008). Traditional medicine is largely applied in this area and particularly to the south; Biomedical facilities and prescription medications are available in the towns of the state in addition to the herbalist shops, confirm its interest. Data collection To carry out this work, ethno-botanical information on the plants used by the population were obtained by visiting Traditional Medical Practitioners (TMP’s), herb sellers and connoisseurs in selected localities. The use of semi-structured questionnaire and oral interviews were adopted to obtain the relevant ethno-botanical data. The questionnaires are divided into 03 sections: (1) was about botanical and vernacular names, plant parts used and medicinal uses. (2). concerns personal information on informer as age, sex educational level and duration of practice. Section (3) contains code or/and collected specimen. The questionnaires were administered by trained interviewees belonging to the population. In some cases, samples were bought in order to get the information. Dried plant samples collected were identified using flora of Quézel and Santa (1962–1963), Ozenda (1983) and Maire (1952– 1987); verified, characterized and confirmed by professional botanists of the department. Voucher specimens were deposited in the Herbarium. In parallel, in order to know the herb diversity of the study zone and confirm availability of local plants used, field research was carried out with the aid of forest executives
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of the district. Plant samples were collected between March and June 2010 and 2011. Identity of collected plants and comparison
with herb seller samples was carried out by botanists of the department.
Fig.1 A map of Algeria showing the location of M’sila in North center-East
Fig.2 Predominant plant families of trado-medicinal use in M’sila.
RESULTS AND DISCUSSION Personal information on informants A number of 89 people were interviewed in this study: 18% of traditional medical practitioners (TMP’s), 34% of herb sellers and 48% of connoisseurs. The age of 56% of them exceeds 30 years, 38% have no educational level and 70% were women. In this area, all inhabitants were concerned with traditional use of plants. However, women had more knowledge on the medicinal species when compared to men. These results indicate the widespread use of plants because of knowledge
transmittance between generations and plant availability compared to prescribed medications expensive and non-available sometimes, particularly in rural agglomerations. It was noted that a small number of people particularly some herb sellers refused to contribute for personal reasons or others related to their trade.
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Fig.3 labiates used in traditional medicine in M’sila
Medicinal plants A total of eighty-two plant species and seventy-one genera distributed over thirty-three families with a total frequency 663 were listed. The dominant families were Lamiaceae, Apiaceae, Leguminosae and Liliaceae. Lamiaceae was the most dominant (fig 2). Lamiaceae species represent 22% of the total plants listed and 48.6% of the frequency of use. People of the study area use frequently plants as remedies for treatment of widespread diseases and ailments; important number of herb sellers and clear frequentation of women to plant shops, availability of spontaneous or cultivated plants in rural areas will explain this situation. In addition high level contribution of women and educational level largely confirm the socioeconomic situation of the region. Table 1 summarizes the stated relationship between medicinal labiate plants and pathologies for which they are used. The major illnesses treated by the indigenous people include digestive disorders, bronchopulmonary problems, skin disease, diabetes, vascular system disorders, and other diverse diseases.
Predominance of 04 families as cited in Fig.2 particularly Lamiaceae (Fig.3) is probably due to the availability of these plants in the region. These results indicate the importance of Lamiaceae family members and their broad use as source of traditional drugs. Some of them listed above are popular species around the world; species belonging to genera Teucrium, Marrubium, Origanum, Rosmarinus, Mentha, Thymus, Ajuga, Salvia, Lavandula are used in the treatment of common illnesses like digestive disorders, abscesses, gout, conjunctivitis, menstrual disorders, cholecystitis, hepatitis, inflammations, liver diseases and in stimulation of fat and cellulite decomposition (Ivancheva and Stantcheva, 2000; Rokaya et al., 2010; Stankovic et al., 2010). 20% of medicinal plants recorded to treat eczema in the study area are labiates (Sari et al., 2012). Some Thymus species are widely used in Algerian folk medicine for their antitussive, antiseptic, expectorant, antihelmintic and antispasmodic properties (Hazzit et al., 2009).
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Tab. 1 Medicinal plants, plant parts used, preparation and medicinal uses by local people of M’sila, Algeria Plant name (local name)
Part used
Preparation
Medicinal uses
Ajuga iva L. Shreb (Chendgoura)
Whole plant
Infusion, Powder + honey
Stomachic; diabetes; hypertension; tonic; cephalic; diarrhea; fever.
Lavandula multifida L. (Khzama)
Aerial parts, leaf
Infusion, Ocular drip, Cataplasm for head
Sedative; antispasmodic; cancer; astringent; stomachic.
Lavandula stoechas L. (Halhal)
Aerial parts, leaf
Infusion, Decoction
Asthma, influenza, wounds,
Marjorana majorana L. (Madqouch)
Flowered tops, leaf
Infusion, Decoction
Regulation; sedative; cramp
Marrubium vulgare L. (Mariouet)
Aerial parts, leaf
Cataplasm for the head
Diabetes; febrifuge; vermifuge; diarrhea.
Marrubium supinum L. (Mariouet)
Aerial parts, leaf
Infusion , Ocular drips
Fever, otitis, slimming, hypertension, eczema
Melissa officinalis L. (Teronjene)
Aerial parts
Infusion, Powders, Bath
Sedative; hypotension; rheumatism.
Mentha spicata L. (Naânaâ)
Aerial parts
Infusion
Hypotension; sedative; disinfectant; astringent
Mentha pulgium L. (Fliou)
Aerial parts
Infusion
Antispasmodic, hypotension, stomach pains and stomachic
Ocimum basilicum (Hbaq)
Flower, leaf
Infusion
Tension; stomachic; sedative; aerophagia.
Origanum glandulosum Desf. (Zaâter)
Leaf, flower, flowered tops, aerial parts, roots
Infusion, Powder + olive oil
Hypotension, antispasmodic; astringent.
Origanum majorana L. ( Zaâter el moulk)
Aerial parts
Evaporation of powder + olive oil
Tension; stomachic; sedative; respiratory tract
Rosmarinus officinalis L. (Klil)
Aerial parts, leaf
Infusion, Compress
Pain abdominal; tonic; rheumatism; circular disorder.
Salvia officinalis L. (Miramia)
Aerial parts, leaf
Infusion, Powder + honey, Powder
Diabetes; stomachic; stomach pain, choleraic
Salvia verbenaca L. (Khayata)
Aerial parts, leaf
Infusion, Decoction
Aromatic, stomachic, tonic, vulnerary, disinfectant, antispasmodic, antisudoral, astringent (diarrhea), carminative,
Teucrium polium L. (Djaїda)
Aerial parts, leaf
Infusion, Powder
Disinfectant; stomachic; hemorrhoids; weakens
Thymus algeriensis B. et R. (Zâaitra)
Aerial parts, leaf
Infusion, Decoction
Hypertension, carminative, diabetes, vermifuge, cholesterol, anginas, aromatic
Thymus ciliatus (Tourn.) L. (Djertil)
Aerial parts, leaf
Infusion, Decoction
Antibiotic vermifuge; carminative; tonic
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Laboratory studies report the anti-diabetic activity of aqueous extracts of some plants of the study area; oral administration of 200 and 300 mg/kg body weight of aqueous extract the Marrubium vulgare induced a significant antidiabetic effect and anti-hyperlipidemic effect. (Boudjelal et al., 2012). The Antioxidant effect of Ajuga iva aqueous extract and other biological activities of some Algerian medicinal plants extracts have also been cited (Djeridane et al., 2006; Taleb-Senouci et al., 2009). Methanolic extracts of Teucrium polium and Ajuga iva present antibacterial activity against some pathogenic bacteria (Zerroug et al., 2011).
Mentha spicata L., Origanum glandulosum Desf., Thymus ciliatus (Tourn.) L. and Teucrium polium L. The preservation of the traditional knowledge is an essential requirement for maintaining continuity and transmission of traditional medicine. It should be noted that these results indicate that population of the area of M’sila uses medicinal plants for the treatment of current ailments and very important diseases. Labiates have a significant role and are applied as infusion of the aerial parts or as powder mixed with honey or olive oil. The indigenous population also uses folk-medicines derived from mineral and animal origin.
CONCLUSION Traditional medicine is usually exerted by people because the use of medicinal plants decreases the disadvantages of the chemical drugs. This study was aimed at the Lamiaceae plants being used by the local people of M’sila. These plants are used routinely for treatment of gastrointestinal problems, hypertension and particularly for some diseases like diabetes, asthma and eczema on the other hand. The main plants are; Rosmarinus officinalis L., Ajuga iva L. Shreb, Marrubium vulgare L.,
Many valuable drugs have found their way into the contemporary medicine as a result of studies on folk medicine remedies. The present study is also an eye opener for the Modern researchers to explore much more from the so called Medicinal plant world. ACKNOWLEDGEMENTS The authors are grateful to CNEPRU (Algeria) for financial support, to forest sector of Hammam Dalâa and all contributors to the completion of this work: interviewer’s, herbalists and TMP’s of the area.
REFERENCES Abdelguerfi A. Summary report on «Biodiversity Important for Agriculture in Algeria » MATE-GEF/PNUD: Project ALG/97/G31. 2002/2003. ‟French version” Baba Aissa F. (1991). Medicinal plants in Algeria. Identification, description of active ingredient properties and traditional use of common plants in Algeria. (Bouchène and Ad. Diwan) Algiers. 181 p. ‟French version”
Maghreb. vegetable substances of Africa, of East and Occident. EDAS. Algiers. 368 p. ‟French version” Behlouli F., Tiaiba A. and Slamani A. (2008). Study of the various methods of apricot drying, outline on the traditional methods of drying in the area of Hodna, State of M’Sila. Review of Renewable energies .SMSTS’08 Algiers (2008) 61–66. ‟French version”
Baba Aissa F. (1999). Encyclopedia of the useful plants. Flora of Algeria and
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Beloued A. (1998). Medicinal plants of Algeria. U.P.O. Algiers. 277p. ‟French version” Milan Boudjelal A., Henchiri Ch., Siracusa L., Sari M. and Ruberto G. (2012). Compositional analysis and in vivo anti-diabetic activity of wild Algerian Marrubium vulgare L. infusion. Fitoterapia 83 (2012) 286–292. Djebaili S., 1984 - Algerian steppe. Phytosociology and ecology. U.P.O. Ed., Ben-Aknoun, Algiers, 177 p. ‟French version” Djeridane A., Yousfi M., Nadjemi B., Boutassouna D., Stocker P., Vidal N. (2006). Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds. Food Chemistry, 97:654–660. Feliachi K. (2003). National Commission (AnGR). National report on the Animal Genetic resources in Algeria. 46p. ‟French version” Hazzit M., Baaliouamer A, Veríssimo A.R., Faleiro M.L., Miguel M.G. (2009).Chemical composition and biological activities of Algerian Thymus oils. Food Chemistry, doi: 10.1016/j.food.chem. 2009.03.018. Ivancheva S., Stantcheva B. (2000). Ethnobotanical inventory of medicinal plants in Bulgaria. Journal of Ethnopharmacology, 69:165–172. Le Houerou HN. (1995): Bioclimatology and Biogeography of the arid steppes of the North of Africa. Biological diversity, sustainable development and desertisation. Mediterranean Options. Series. B; 10. ‟French version” Maire R. (1952–1987): Flora of North Africa (Morocco, Algeria, Tunisia; Tripolitania, Cyrenaica and the Sahara,
S. Stankovic, Marina Topuzovic, Slavica Solujic and Vladimir Mihailovic. 2010. Antioxidant activity and concentration of phenols and flavonoids in the whole plant and plant parts of Teucrium chamaerdys L. var. glanduliferum Haussk. J. Med. Plant. Res. 4(20): 2092–2098.
Moreau S., Benziene A.S., Boudjadja A., Gaouar A., Kaabeche M., Moali A. and Sellami, D. (2005) Plan of management of site of Mergueb. Wilaya of M’sila (Algeria). PROJECT DGF/GEF/PNUD-ALG/G35/2005. ‟French version” Rokaya M. B., Münzbergová Z., and Timsinac B. (2010). Ethnobotanical study of medicinal plants from the Humla district of western Nepal. Journal of Ethnopharmacology 130: 485–504. Sari M., Hendel N., Boudjelal A., and Sarri D.(2012). Inventory of medicinal plants used for traditional treatment of eczema in the region of Hodna (M'sila Algeria). Global J. Res. Med. Plants & Indigen. Med., 1(4): 97–100. Stankovic M. S., Topuzovic M., Solujic S. and Mihailovic V. (2010). Antioxidant activity and concentration of phenols and flavonoids in the whole plant and plant parts of Teucrium chamaerdys L. var. glanduliferum Haussk. J. Med. Plant. Res., 4(20): 2092–2098. T.A.D
- CONSULT– EURL. Territory. Arrangement. Development. (2008). Office of Engineering and technical studies. Relative study with the characterization and the delimitation of mountainous areas and mountainous solid masses “Massif of the Hodna Stage II” ‟French version”
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Taleb-Senouci D., Ghomari H., Krouf D., Bouderbala J., Prost J., Lacaille-Dubois M.A., Bouchenaka M. (2009). Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats. Phytomedicine, doi:10.1016/j.phymed.2008.12.004D. WHO
(World Health Organization). Regional Committee for Africa. AFR/RC50/9. 2000. Promoting the
Source of Support: Nil
role of traditional medicine in health systems: a strategy for the African region. Ouagadougou, Burkina Faso. 28 August-2 September 2000. Zerroug M.M., Zouaghi M., Boumerfeg S., Baghiani A., Nicklin J. and Arrar L. (2011). Antibacterial Activity of Extracts of Ajuga Iva, and Teucrium Polium. Advances in Environmental Biology, 5(2): 491–495.
Conflict of Interest: None Declared
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Original Research Article FATTY ACID PROFILE OF FRUITS OF LUFFA ACUTANGULA VAR. AMARA C.B.CLARKE Jadhav Santosh Jaysingrao1*, Chavan Niranjana Sunil 2
1, 2
Department of Botany, Shivaji University, Kolhapur, Maharashtra, India. Corresponding Author: Email: Santosh.jadhav318@gmail.com ; Mobile: +91- 9823012218
Received: 03/07/2012; Revised: 24/07/2012; Accepted: 26/07/2012
ABSTRACT The lipid profile of fruits of Luffa acutangula var. amara C.B.Clarke was analyzed from methanolic extract using Gas Chromatography with Flame Ionization Detector technique. The results showed that the extract contain saturated fatty acids, monosaturated fatty acids and polyunsaturated acids at 66.40%, 13.50%, and 20.00 % respectively. The major fatty acids of fruit are pentadeconic acid and palmitic acid.
KEYWORDS: Luffa acutangula var. amara, fruit, Methanolic extract, lipid profile.
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INTRODUCTION
MATERIAL AND METHODS
The family Cucurbitaceae comprises members that are cultivated throughout the world as source of food, fiber and indigenous medicines (Nayar NM and More TA 1998) Luffa acutangula (L.) Roxb. (Syn: Luffa acutangula var. amara C.B.Clarke) (Fig. 1, 2, 3) belonging to family Cucurbitaceae is an annual herb found in all parts of India, especially along the costal lines (Chakravarty HL 1982). The plant is used as a laxative, carminative. It is used to cure Vata, Kapha, liver complaints, leucoderma, piles etc. (Chopra et.al., 1986). It is used as a bitter tonic (Biswas and Ghosh 1973). Fatty acids are important constituents of healthy diet as they help to move oxygen through blood stream to all parts of the body. They aid cell membrane development, strength and functions of tissues and organs. There are different types of fatty acids such as Omega 3, Omega 6, and Omega 9. Omega 3 and Omega 6 are considered as essential fatty acids. Omega 3 family comes from α linoleic acid, Omega 6 from linoleic acid (Dulf FV et.al., 2006).Omega 9 series particularly originates from oleic acid. Besides significance in human nutrition the lipids have number of applications in various industries (Ikhuoria EV and Maliki M 2007). The present study deals with fatty acid profile of the fruits of Luffa acutangula (L.) Roxb. The figures showing fresh fruits,dry fruit and seeds and habit of plant.
Plant material:
Fig: 1 Fresh fruits of L.acutangula var.amara
Mature fresh fruits of Luffa acutangula var.amara were collected from village, Aachara in the Sindhudurga district of Maharashtra. The plant was authenticated by Prof. Dr.S.R.Yadav, Department of Botany, Shivaji University, Kolhapur. The fruits were washed thoroughly to remove adhered dust and other particles. Fruits are shade dried and powdered. Methanolic extract of fruit powder was prepared. Esterification of lipids was carried out by boron trifluoride method. 0.5 ml of plant extract was mixed with 5 ml of NaOH in methanol. The mixture was boiled for 2 min and cooled. 5 ml boron trifluoride was added. Then 5 ml n-heptane was added. The resultant mixture was injected for Gas Chromatography with flame ionization detector (GC-FID).Fatty acid methyl esters were analyzed on Chemito 1000 GC unit. A gas chromatograph with flame ionization detector (FID) was used. Fatty acid methyl esters were separated on CP-SIL 88, 30 m, 0.25 ID, 0.25 film thickness capillary column using column temperature 100°C for 5 min; then temperature was increased by 1.5°C up to 198°C and held for 9 min.with the following conditions, injector temperature 250oC and carrier gas nitrogen. The flow rate was 1 ml/min. The identification of fatty acids was done by standard library provided with the instrument. Fig: 2 Dry fruit with seeds
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TABLE:1 Fatty acid composition of fruits of Luffa acutangula var. amara C.B.Clarke Sl.no.
Fatty acid
Abbreviation
Area percentage
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Caproic acid Caprylic acid Capric acid Undecanoic acid Myristic acid Pentadecanoic acid Palmitic acid Stearic acid Arachidic acid Oleic acid Cis-10-pentadecanoic acid Cis-11-Eicosenoic acid Cis-10-Heptadecanoic acid Linolelaidic acid Linoleic acid Linoleic acid Cis-5,8,11,14,17Eicosapentaenoic acid Cis-4,7,10,13,16,19Docosahexaenoic acid Total Saturated Fatty Acid Total Monosaturated Fatty Acid Total Polyunsaturated Acid Total Trans Fat
C6: O C8: O C10: O C11: O C14: O C15: O C16: O C18: O C20: O C18:ln9c C15:1 C20:1 C17:1 C18:2n6t C18:2n6c C18:3n3 C20:5n3 C22:6n3
0.40% 0.40% 0.30% 0.40% 6.10% 33.40% 20.10% 4.70% 0.60% 10.70% 0.40% 1.90% 0.50% 0.60% 17.10% 0.50% 1.70% 0.10% 66.40% 13.50% 20.00% Not detected
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RESULTS AND DISCUSSION The fatty acid composition of fruit of Luffa acutangula var.amara has been depicted in Table 1. Total 19 fatty acids were detected. Pentadecanoic acid (33.40%) is the major monosaturated fatty acid followed by palmitic acid (20.10%) and Linoleic acid (17.10%). The fruit comprises of 66.40% of total saturated fatty acids, 13.50% of monosaturated fatty acids and 20.00% of polysaturated acids. Total Trans fatty acids were absent. Cucurbit seeds are generally rich in oils. The total lipid content of seeds of pumpkin, water melon, musk melon and cucumber vary between 38–45%. The major fatty acids present in them are palmitic acid, stearic acid, Oleic acid, linoleic acid and linolenic acids which together constitute more than 80% of the total fatty acid content (More et.al., 1981, Madan et.al., 1982). Seeds of several species are used for their taste (in bakery preparations and savouries) and richness of oils and proteins( up to 40% each) (Nayar NM, More TA 1998). Several derivatives of oleanolic acid have also been isolated from Luffa acutangula. The triterpene, mogrosideV found in Cucurbitaceae is intensely sweet. Bryonolic acid from Luffa seeds has been shown to be antiallergic in nature (Tanaka et.al., 1991). More than 150 fatty acids have been reported to be present in
the seed oil of Cucurbitaceae (Seshadri 1986). Fatty acids play important role in functions of skin (Elias PM 1983). The number of fatty acids have specific functions in restoring the permeability barriers (Elias PM and Puniras M., 1983). Gas liquid chromatography analysis of whole seeds of Cucumis melo revealed that oil content of seeds ranged from 12.5 to 39.1 percent, the linoleic, oleic, palmitic, and stearic acids were present. Unsaturated fatty acids constituted 64.6 to 88.2 percent of the total fatty acids. While Cucumis and Cucurbita have common saturated and unsaturated fatty acids. (More et.al., 1981, Madan et.al., 1982). Momordica and Trichosanthes seeds contain about 30% oil. Punicic acid is the major constitute of seed oil of T. bracteata (41.8%) and T. nervifolia (51.7%) (Laxminarayana et. Al., 1988). The present investigation justifies the use of fruits of Luffa acutangula var amara. Recently the species have been received attention but the biochemical profiling of species is not done so far. Thus here an attempt is made for quantitative analysis of fatty acids of fruits. The ecological and pharmacognostic study of species is under progress.
ACKNOWLEDGEMENTS The authors are thankful to Head of the Department of Botany, Shivaji University, Kolhapur for providing the laboratory facilities.
REFERENCES: Azeemoddin G.and Rao STD (1967) seed fat of Momordica tuberose and Luffa tuberosa Curr.sci.20:100 Biswas KP and Ghosh AK (1973) Bharatiya Banaushadhi 2 vols. Calcutta Univ.Calcutta-73 Chopra RN, Nayar SL and Chopra IC (1986) Glossary of Indian medicinal plants.CSIR, New Delhi.
Dulf FV, Bele C, Spinean S,Veronicas, Chdea G and Zegrean CS (2006) comparitives studies on fatty acid finger print from total lipids and phytistorol esters of some edible plant oils. bulletein USAMVCN,62:225–230 Dyerberg J (1986) Linolenate derived polyunsaturated fatty acids and prevention of atherosclerosis. Nutrition Reviews, 44:125–134
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PM, J Invest J Dermatol.(1983) Epidermal lipids barrier functions and desquamation, suppl:44s–49s
Halder T and Gadgil VN (1983) Fatty acids of callus tissues of 6 species of Cucurbitaceae.Phytochemistry 22:1965–1968 Ikhuoria EU and Maliki M (2007) Characterization of avocado pear (Persea americana)and Africian pear (Dacyodes edulis) extracts. African Journal of Biotechnology. 6 (7), 950– 952 Lakshminarayan and Sunder Rao K,Kittur MH and Mahajanshetty CS (1988) occurrence of punicic acid in T. bracteata and T.nervifolia seed oils J Amer.oil chem.soc 65:347–348 Madan TR and Lal BM (1984) some studies on chemical composition of cucurbit kernels and their seed coats Qual. plant 34:81–86
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Madan T R, More T A, Lal B M and Seshadri V S (1982) A study of seed of Muskmelon, A Lesser known sourceof edible oil J sci food Agri 30:973–978 More TS and Sheshadri V S (1984) Association of fruit quality with seed characters and oil and protein content of muskmelon seeds. Cucurbit Genet.coop.Rept7:4648 Nayar N M and More T A (1998) cucurbits , oxford and IBH publishing, Delhi.. Puniras
M, Precis de cosmetologie, dermatologigue, Masson Eds. Paris, PP 220, 222
Sheshadri V S (1986) cucurbits 91-164 PP in Vegetable crops in India(Eds. Bose T k and Som M G) Naya Prokash, Calcutta. Tanaka Y, Ephrussi B, Haborn E, Hagberg A, Kamp T, Nachtsheim H, Pontecorvo G and Rhoades AM (1957): Rep. interncomm. Genetic symbols and Nomenclature intern. Union Biol. Sci. Collogues B 30:1–6
Conflict of Interest: None Declared
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Review article INTRODUCTION OF FIVE WELL-KNOWN AYURVEDIC MEDICINAL PLANTS AS FEED ADDITIVE ON LIVESTOCKâ&#x20AC;&#x2122;S PERFORMANCE: A REVIEW Mirzaei F, Hari Venkatesh K R Senior Scientist, Department of Livestock Production and Management, Animal Science Research Institute of Iran, Karaj, 31585, Iran. Lecturer / Scientist Incharge - Animal House, A.L.N Rao Memorial Ayurvedic Medical College, Koppa- 577126, Chikmagalur District, Karnataka, India. Corresponding Author: E-mail address: farmir2003203@yahoo.com Received: 20/06/2012; Revised: 12/07/2012; Accepted: 24/07/2012
ABSTRACT According to the FAO, the lack of drugs to treat diseases and infections causes losses of 30 to 35% in the breeding sector of many developing countries, where poor animal health remains a major constraint to breeding. There are two principle reasons behind the changes in legislation on the use of in-feed antibiotic growth promoters. The first is to try to combat the development of microbial resistance to antibiotic drugs and the consequences on human health. The second is a response to consumer pressures to eliminate the use of all non-plant xenobiotic agents from the diets of animals, so natural resources of medicine like phyto-medicines can help smallholders in rural areas to manage their only income resources from diseases and mortality. This review introduces five most common ethno-veterinary plants named; Asparagus racemosus willd. (Shatavari), Leptadenia reticulata (Retz.) Wight & Arn. (Jivanti), Cuminum cyminum Linn. (Jeeraka), Nigella sativa L. (Kalajaji) and Pueraria tuberosa (Willd.) DC. (Vidarikanda) for eco-efficiently livestock production management to meet climate smart agriculture practices.
KEYWORDS: Medicinal plants, livestock, clean production, Asparagus racemosus, Leptadenia reticulata, Cuminum cyminum, Nigella sativa, Pueraria tuberosa
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INTRODUCTION It is estimated that there are 2, 50,000 species of higher plants on earth of which more than 80,000 are medicinal (Kumar and Joshi 1987). Recent studies have shown that medicinal plants (their extracts) with lower concentration of plant secondary metabolites are good candidates for achieving one or more beneficial impacts on ruminantâ&#x20AC;&#x2122;s performance (Teferedegne, 2000). Medicinal plants are generally considered as safe for human consumption (FDA, 2004) and these can be employed to modify rumen microbial fermentation. The extracts of leaves, fruits or roots of various plants are traditionally used as medicine and also have galactogouge property and recently these have been recognized as having antimicrobial and anti-methanogenic properties (Davidson and Naidu, 2000, Alexander, 2005, Malik, 2007) due to the presence of plant secondary metabolites such as saponins, terpenoids, phenylpropanoids, tannins and essential oils. Extensive grazing system is being practiced chiefly by the resource poor farmers. Under this system inaccessibility to the timely veterinary health care and the cost of treatment are the main constraints, affecting the productivity and sometimes viability of the system. Under these conditions it becomes essential to utilize the locally available resources as ethno-veterinary medicines to ensure general well-being and welfare of the animals (Mirzaei, 2010). World health organization has recognized the necessity for investigation and mobilization of ancient medicinal practice to fulfil the primary health care of the animals and realizes that the traditional system of medicine may play an important role in the development of livestock of the third world countries (WHO, 2008). There is a renewed interest, especially in developed countries, in using plants to treat livestock, pets, and humans (Cornel University, 2009). Some of the herbal plants are reported to have beneficial effect on the production, reproduction and health of the animals (Medplant, 2008). Many herbs and plant extracts have antimicrobial activities against a wide range of bacteria, yeasts, and molds (Thompson, 1986; Voda et al., 2003). A knowledge of the chemical
constituents of plants is desirable, not only for the discovery of therapeutic agents, but also because such information may be of value in disclosing new sources of such economic materials as tannins, oils, gums, precursors for the synthesis of complex chemical substances, etc. Therefore keeping in mind aforesaid constraints and possible benefits, the present review was planned with the following objectives: To study the effect of herbal supplementation on feed intake and feed conversion efficiency of ruminant production management. To study the effect of medicinal plants supplementation on production performance of ruminant production management. 1. Herbals and their mode of action The first world medical conference under the patronage of Anoushiravan, the King of ancient Iran was convened at 550 Christian Era in Cteciphon city. Hundreds of physicians from other countries were in attendance in this congress. Ferdowsi, Iranian Poet has versified this historical event in Shah Nameh , Ferdowsiâ&#x20AC;&#x2122;s book. Khosrow dispatched the famous Iranian physician, Borzoya (Borzouyeh) to India, who brought medical and scientific books, chess, herbal plants and Indian doctors with him (Mirzaei, 2010). Herbs are more compatible with body because of their normal nature and having medicine homologues components together; and usually lack unwanted side effects; therefore they are most suitable, especially in cases of long consumption as well as in chronic diseases (Borimnejad, 2008). One of the most important determinants of research conducted is the availability of funding. There are three areas in which it is most likely that appreciable goat research funding opportunities will be available in the foreseeable future: biotechnology, medical research and food safety. For example there may be genetic engineering techniques developed for inserting specific genes into animals to overcome many human health problems or to study specific disease problems.
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Though there is still need for further research on traditional topics such as relating to levels and efficiencies of production. Unfortunately it is doubtful that there will be greater support in the future and lower levels of funding seem more likely (Sahoo et al., 2001). Ethno-veterinary is a science that involves the popular practical knowledge used to treat and prevent animal diseases. Although a number of ethno biological inventories concerning the use of medicinal plants and animals in human health have been realized, the ethno-veterinary medicine is poorly described. This scarce description of the ethno-veterinary medicine resources is in stark contrast to the problems of livestock rearing, where the lacking regular access to essential medicines greatly hampers productivity. According to the FAO (Mirzaei, 2010), the lack of drugs to treat diseases and infections causes losses of 30 to 35% in the breeding sector of many developing countries, where poor animal health remains the major constraint to breeding (Daba and Abdel-Rahman, 1998). Several forms of organic farming are being successfully practiced in diverse climate, particularly in rain fed, tribal, mountains and hill areas of the India. Much of the Natural plant products with economic importance like herbs, medicinal plants by default come under this category (Mirzaei, 2010). Livestock raisers and healers everywhere have traditional ways of classifying, diagnosing, preventing and treating common animal diseases. Many of these "ethno-veterinary" practices offer viable alternatives or complement to conventional, western style veterinary medicine especially where the latter is unavailable or inappropriate.The non-nutrient bioactive principles in plants are essentially the secondary metabolites. They are differing from the ubiquitous primary metabolites (i.e., carbohydrates, proteins, fats, nucleic acids) in that their distribution is limited. Plant secondary metabolites are a natural resource that is largely unexploited in conventional animal production system. Essential oils are complex mixture of secondary metabolites consisting of low-boiling point, phenylpropenes and terpenes. They are
particularly associated with plants defined as herbs and spices (Greathead, 2003). Recent and continuing changes to legislation controlling the use of animal feed additives have stimulated interest in bioactive secondary metabolites as alternative performance enhancers. There are two principle reasons behind the changes in legislation on the use of in-feed antibiotic growth promoters. The first is to try to combat the development of microbial resistance to antibiotic drugs and the consequences on human health. The second is a response to consumer pressures to eliminate the use of all non-plant xenobiotic agents from the diets of animals. Some herbal essential oils like carvacrol and thymol oils have been reported to inhibit the growth of E. coli, decrease the intracellular ATP concentration of E. coli, and while simultaneously increase the extracellular ATP concentration. It is the lipophilic character of essential oils that enables them to disrupt cell walls, as they consequently accumulate in membranes (Greathead, 2003). The rumen protozoa are proteolitic and actively ingest rumen bacteria. They are considered to be the most important cause of bacterial protein turnover in the rumen and therefore can have a major effect on the efficiency of rumen N metabolism (Wallace and McPherson, 1994). In general, gram-positive bacteria appear to be more susceptible to inhibition by plant essential oil compounds compared with gram-negative bacteria (Davidson and Naidu, 2000). This effect has been related to the presence of an outer membrane on gram-negative organisms, which endows them with a hydrophilic bacterial surface that acts as a strong impermeability barrier (Nikaido, 1994). Essential oils are steam-volatile or organic-solvent plant extracts, used traditionally by man for many centuries for the pleasant odour of the essence, its flavour or its antiseptic and/or preservative properties. The word â&#x20AC;&#x2DC;saponinâ&#x20AC;&#x2122; is derived from the Latin word sapo (soap) and traditionally saponin-containing plants have been utilized for washing. Saponins, like essential oils, cover a wide variety of chemical compounds and, also like essential oils, man has made use of their properties for centuries (Hostettmann and Marston, 1995; Wallace, 2004).
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The sensitivity of ciliate protozoa towards saponins may be explained by the presence of sterols in protozoal, but not in bacterial, membranes (Williams and Coleman, 1992).Thus, the sterol-binding capability of saponins most probably causes the destruction of protozoal cell membranes (Hostettmann and Marston, 1995). Plants and their extracts have important potential as manipulators of rumen fermentation for productivity and health benefits (JimĂŠnez-Peralta et al., 2011; Salem et al., 2011). They have specific effects on members of the rumen microflora and fauna that can be beneficial to animal productivity and health (Wallace, 2004). Gastrointestinal parasitism has been classified as a major health and welfare problem for ruminants. Parasitism, especially by helminth parasites, impairs health by causing inappetance, diarrhea, and anemia and in severe cases, death (Athanasiadou et al., 2001). The complexity and the bioactivity of plant secondary metabolites have the potential to reduce the likelihood that microorganisms or parasites will develop resistance, and their effectiveness is such that concentrations as low as 0.1 g/kg feed may be sufficient (Salem et al., 2010). In some cases they may already be components of feedstuffs nevertheless, issues such as toxicity, photosensitivity, residues, taint, allergenicity and cost effectiveness still need to be addressed before these compounds will gain widespread acceptance in the agricultural industries. Furthermore their use as prepared compounds will need to be agreed by the registration authorities within the countries in which they will be used or in which the products from livestock will be sold and consumed (Acamovic and Brooker, 2005). Scientific validation and use of ethnoveterinary medicine can play a role in poverty reduction by improving productivity of animals through convenient, accessible and economical use of their practices. The fact that medicinal plants are predominantly harvested in an unregulated manner undermines the whole industry and yield from the wild is wholly unpredictable. Supplies are at the mercy of the weather, pests, and other uncontrollable variables. Farming these species would help even out the
supply, regularize the trade, provide certifiable products of uniform quality, and make available to rural areas new sources of income. This would also indirectly help in poverty alleviation (Iqbal et al., 2005). 2. Distribution, use and effects of herbals bioactive 2.1. Shatavari (Figure 1) Shatavari, Asparagus racemosus Willd. (Asparagaceae) is recommended in Ayurvedic (is the system of traditional medicine native to India) texts for prevention and treatment of gastric ulcers, dyspepsia and as a galactogogue. A. racemosus has also been used successfully by some Ayurvedic practitioners for nervous disorders, inflammation, liver diseases and certain infectious diseases. A study of ancient classical Ayurvedic literature claimed several therapeutic attributes for the root of A. racemosus (Sanskrit:Shatavari) and has been specially recommended in cases of threatened abortion and as a galactogogue. Root of A. racemosus has been referred as bitter-sweet, emollient, cooling, nervine tonic, constipating, galactogogue, aphrodisiac, diuretic, rejuvenating, carminative, stomachic, antiseptic and as tonic. Beneficial effects of the root of A. recemosus are suggested in nervous disorders, dyspepsia, diarrhoea, dysentry, tumors, inflammations, hyperdipsia, neuropathy, hepatopathy, cough, bronchitis, hyperacidity and certain infectious diseases (Goyal et al., 2003). The genus Asparagus (with about 300 species) is a rich source of sapogenins and saponin, from various parts of the plant (LacailleDubois, 2000). It has been reported that Shatavari supplementation induced leucocytosis with predominant neutrophil associated with stimulation of phagocytic and bactericidal capacity of neutrophils and macrophages (Thatte and Dhanukar, 1989). Shatavari root has growth promoter property. Calves supplemented with Shatavari root decoction at the rate of 100 mg/kg for a varying period of 4 weeks to 8 months showed 81.19 % weight gain as compared to 67.9% in control. It did not have any adverse
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effect on the progeny of the treated animals. The growth promoting effect can be ascribed to its adaptogenic property (Sharma, 1996). Supplementation of Shatavari root powder at 5 and 10 % level reduces the plasma and hepatic Fig.1 Tubers of Asparagus racemosus Willd.
lipid (cholesterol) levels and also decreases lipid peroxidation, also Shatavari supplementation increases dry matter intake significantly in lactating crossbred cows (Tiwari et al., 1993; Barhane and Singh, 2002). Fig.2 Habit of L. reticulata (Retz.) Wight & Arn
Fig.3 & 4 Seeds and Habit of Nigella sativa L.
Fig.5 Fruits of Cuminum cyminum Linn.
Fig. 6 Tubers of Pueraria tuberose (Willd.) DC.
Photo Courtesy: Dr. Harisha C R (Fig.1 & 6); Dr. Hari Venkatesh K R (Fig.3, 4, 5); ayurvedamaruthuvam.blogspot.com (Fig.2)
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Supplementation of Shatavari fresh root of at the rate of 500 g per day with concentrate at the time of milking significantly increased milk yield of buffaloes (Patel and Kanitkar, 1969). Feeding herbal formulation containing 25% Shatavari enhanced milk production (25.1%) over control group, also significant improvement in daily milk yield in buffaloes and crossbred cows, but response of supplementation of Shatavari in buffaloes is higher than cows, but the reason was not explained (Mahantra et al., 2003; Somkuwar et al., 2005; Tanwar et al., 2008). The dose of Shatavari in dairy animal based on body weight or dry matter intake is not well standardized. Supplementation of Shatavari 100 g/day/animal and 50 g/day/animal irrespective of body weight have been found to increase milk production in crossbred cows (Tanwar et al., 2008; Mishra et al., 2008). However, It was found that supplementation of Shatavari (100 g on alternate day/ animal) in freshly calved crossbred cows did not improved milk production. Similar result was also in lactating goats. In addition, supplementation of Shatavari root powder 100 g plus 10 g Aloe dried pulp powder to cows following artificial insemination per animal/day improved conception rate (Barhane and Singh, 2002; Hedge et al., 2002; Vihan, 1988). The presence of saponin in Shatavari root has been written up (Jadhav and Bhutani, 2006). Besides saponin, root extract of Shatavari contain flavonoids (6.7 mg/100ml), polyphenol including tannin (88.2 mg/100ml) and Vitamin-C (42.4 mg/100ml), the presence of phytocomponents in Shatavari root such as phytosterols (0.79%), saponin (8.833%), polyphenols (1.692%), flavonoids (0.476%) and total ascorbic acid (0.762%) were also estimated (Velavan et al., 2007; Visavadiya and Narasimhacharya, 2007) . Herbal formulations containing extracts of A. racemosus may be recommended for use as positive immuno-modulator in normal and immuno-compromized broiler chicks. It also indicated the determinative roles of herbal feed additives in effective augmentation of humoral and cell mediated immune responses providing better protection level against infections (Kumari et al., 2012a). Another study showed that herbal preparations of A. racemosus root extract can be
beneficially used as an effective feed supplement in poultry for its encouraging results in relation to total body weight gain and feed conversion efficiency. It can also be used potentially before mass vaccination of the chicks for its property of immuno-modulation like levamisole. (Kumari et.al., 2012b) 2.2. Jivanti (Fig. 2) Jivanti, Leptadenia reticulata (Retz.) Wight & Arn., (Apocynaceae), well known for its tonic, restorative and stimulant property in the Indian system of medicine. This plant is distributed in the Southern parts of India. L. reticulata is a muchbranched twining shrub. The bark is yellowish brown, corky, deeply cracked; the leaves are ovate-cordate, coriaceous glabrous above, more or less finely pubescent beneath; the flowers are in many-flowered cymes, greenish yellow; the follicles are sub-woody and turgid. The main constituents reported are stigmasterol, βâ&#x20AC;&#x201C;itosterol, flavonoids, pregnane glycosides and proteins. Presence of triterpenes and steroids were also reported. Aerial parts of L. reticulata are reported to contain tocopherol and possess several pharmacological activities such as galactogogue, antimicrobial and anti-inflammatory activity (Sathiyanarayanan et al., 2007). Jivanti is considered a stimulant and tonic in Ayurvedic system (is the system of traditional medicine native to India). Its medicinal use dates back to 4500 to 1600 B.C, as mentioned in the Atharvaveda. Its lactogenic effect has been reported in various domestic animals (Dadarkar et al., 2005). Jivanti and a cocktail of herbs namely galog a proprietary product of Indian Herbs Research & Supply Co, are known for their lactogenic properties since the times of Atharva veda Charak Samhita (old literature in India). L. reticulata has been shown to increase milk production without affecting milk composition (Anjaria and Gupta, 1967). Galog is an herbal preparation which contains L. reticulata as the main component and it may be attributed to certain metabolic changes in the body tissues and the mammary gland, where the absorbed nutrients are utilized more effectively (Thakur, 1977).
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2.3. Kalajaji (Kalonji) (Fig. 3 & 4) Kalajaji, Nigella sativa L. (Ranunculaceae), commonly known as black seed or black cumin, is used in folk medicine as a natural remedy for a number of diseases such as asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness and gastrointestinal disturbances. Furthermore, modern pharmacological and toxicological studies have demonstrated that crude extracts of the seeds and some of its active constituents (volatile oil and thymoquinone) might have protective effect against nephrotoxicity and hepatotoxicity induced by either disease or chemicals. N. sativa oil has also antipyretic, analgesic, anti-inflammatory, antimicrobial, and antineoplastic activity (Ali and Blunden, 2003). Thymoquinone, active constituent of N. sativa seeds, is a pharmacologically active quinone, which possesses several properties including analgesic and anti-inflammatory actions (Houghton et al., 1995). Protection against chemical induced carcinogenesis (Hassan and El-Dakhakhny, 1992; Worthen et al., 1998), and the inhibition of eicosanoids generation (Houghton et al., 1995). Moreover, it has been reported that thymoquinone prevents oxidative injury in hepatocytes induced by carbon tetrachloride or tert-butyl hydroperoxide in various in vitro (Daba and Abdel-Rahman, 1998) and in vivo (Mansour et al., 2001; Nagi et al., 1999) hepatotoxicity models, as well as acetic acid-induced colitis in rats (Mahgoub, 2001). It has been suggested that thymoquinone may act as an antioxidant agent and prevent the membrane lipid peroxidation in hepatocytes (Mansour et al., 2002). Ethanolic extract of Artemisia herba-alba (Makhloufi A et al., 2012), N. Sativa, Punica granatum possessed the most outstanding in vitro antibacterial activity, with maximum inhibition zone of 18–22.4 mm. The lowest minimum inhibitory concentration value was measured in Punica granatum as 0.01 mg/ml against methicillin-resistant Staphylococcus aureus. The results showed that the ethanolic extract had better antibacterial effect than the aqueous extract and the anti-staphylococal activity of the ethanolic extract of plants against methicillin-resistant S.
aureus, was better than methicillin-sensitive S. aureus strains (Dadarkar et al., 2005). Treated Fe (III) solution with extract of N. sativa as well as two other active biological reductants, hydroquinone and hydroxyl ammonium chloride and found that N. sativa is a stronger reducing agent than hydroxyl ammonium chloride and weaker than hydroquinone (Tawab and Fatima, 2006). Treatment protocols in rats with N. sativa inhibited reactive oxygen species production induced by experimental autoimmune encephalomyelitis indicated by diminished levels of malondialdehyde of both brain and medulla spinalis tissues and nitric oxide in brain only. When N. sativa was given alone to the rats, no changes were shown in brain, medulla spinalis, and serum oxidant/antioxidant parameters. In conclusion, N. sativa may protect brain and medulla spinalis tissues against oxidative stress induced by the experimental autoimmune encephalomyelitis. In addition, N. sativa display its antioxidant and regulatory effects via inflammatory cells rather than the host tissue (brain and medulla spinalis) for the experimental autoimmune encephalomyelitis in rats (Ozugurlu et al., 2005). 2.4. Jeeraka (Jeera ) (Fig. 5) Jeeraka, Cuminum cyminum Linn. (Apiaceae) is a wild grassy plant with 15–50 cm height, widely used ingredient in Indian food. It has been used for a very long time in traditional medicine in the treatment of diarrhea, dyspepsia and gastric disorders, and as an antiseptic agent. C. cyminum, originates from the Greek kyminon (Sayyah, 2002; Banerjee and Mukhopadhyay, 2003). Cumin has been used as a preservative in spicy foods and other food products. It is known to inhibit the growth of some fungi in the putrefaction of foods and to control mildew disease. The plant possibly originates from the Mediterranean area, perhaps Egypt and Syria. Nowadays it grows extensively in Turkey and Iran. It seems to have been cultivated in Palestine for a very long time. Cumin bears fruit two months after seeding and is harvested when the plants begin to whiten and the seeds lose their
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yellow coloring (Banerjee and Mukhopadhyay, 2003).
have been isolated and characterized from the species (Devaiah and Venkatasubramanian, 2008).
2.5. Vidarikanda (Fig. 6) CONCLUSION Vidarikanda, Pueraria tuberosa (Willd.) DC. (Leguminosae), one of the important plants used in Indian medicine, is commonly known as Vidarikand. The tuberous roots of P. tuberosa are used to relieve symptoms of dysmenorrhoea (often begin immediately following ovulation), dysfunctional uterine bleeding and menopausal syndrome. It possesses spasmolytic, antiinflammatory, anti-implantation, antihyperglycaemic, oestrogenic and contraceptive properties. Studies have been conducted on the chemistry and therapeutic effect of various parts of the plant. Phyto-compounds like β-sitostreol, stigmasterol, daidzein, puerarin, puerarone and coumestan, isoflavone C-glycoside-4,6-diacetylpuerarin (root), pterocarpintuberosin (roots and tubers), puetuberosanol and hydroxytuberosone
It is concluded that Ayurveda has been practiced for thousands of years in India with great success. Uses of medicinal plant for human being is already well documented and support its therapeutic use as a multi-purpose medicinal agent. But, only a few studies have been done on dairy animals particular for dairy goat, these herbs which are of pharmacological and chemical type. But, growing human awareness and demand of chemical residues free and clean milk, there is a need to carry bio-chemical work and more detailed studies on dairy animals. Utilization of phytomedicines will not only improve the reproductive efficiency and health of our animals but also support the farmer’s income through production of more milk per animal.
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growth performance of lambs. Anim. feed Sci. Tech. 170, 27– 34. Salem, A.Z.M., Mejía, H.P., Ammar, H., Tinoco, J.L., Camacho, L.M., Rebollar, S.R. 2010. Efficacy of tree leaves extracts on controlling some gastrointestinal parasites species in growing lambs. In: 61st Annual Meeting of the European Association for Animal Production (EAAP), 23rd to 27th of August 2010, Heraklion, Greece. pp. 220 (abstract). Sathiyanarayanan L., Arulmozhi S. and Chidanbaranathan N., 2007, Anticarcinogenic activity of Leptadenia reticulata against Dalton’s Ascitic Lymphoma, Iranian J. Pharmacol. Therapeutics. 6, 133–135. Sayyah, M., Peirovi, A., Kamalinejad, M., 2002. Anti-nociceptive effect of the fruit essential oil of Cuminum cyminum L. in rat, Iranian Biomed. J. 6, 141–145. Sharma, S., Dahanukar, S., Karandikar, S.M., 1996. Effects of long-term administration of the roots of Ashwagandha (Withania somnifera) and Shatavari (Asparagus racemosus) in rats. Indian Drugs. 23, 133–139. Somkuwar, A.P., Khadtare, C.M., Pawar, S.D. and Gatne, M.M.2005. Influence of Shatavari feeding on milk production in buffaloes. Pashudhan. 31. 3. Tanwar, P.S., Rathore, S.S., Kumar, Y., 2008. Effect of Shatavari (Asparagus recemosus) on milk production in dairy animals. Indian J. Anim. Res. 42, 232233. Tawab, I.A., Fatima, N., 2006. Black seeds (N. sativa); A source of iron and antioxidants. Int. J. Biol. Biotech., 3, 151–155. Teferedegne, B. 2000. New perspectives on the use of tropical plants to improve ruminant nutrition. Proc. Nutr. Soc. 59, 209–214.
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Thompson, D.P.,1986. Effect of essential oils on spore germination of Rhizopus, Mucor and Aspergillus species. Mycologia. 78, 482–485. Tiwari S.P., Lal, R., Arora, S.P., Narang, M.P., 1993. Effect of feeding Anifeed-a herb combination on milk production in crossbred cows. Indian J. Anim. Nutr. 10,115–117. Velavan, S., Nagulendran, K., Mahesh, R. and Begum, V.M.H., 2007. In vitro antioxidant activity of Asparagus racemosus root. Pharmacognosy Magazine. 3, 26–33. Vihan, V.S., 1988. Immunoglobulin levels and their effect on neonatal survival in sheep and goats. Small Rumin. Res. 1, 135–144. Visavadiya, N. P. and Narasimhacharya A.V.R.L. 2007. Asparagus root regulates cholesterol metabolism and improves antioxidant status in hypercholesteremic rats. Evid. Based Complement. Altern.Med. 1–8 Voda, K., Boh, B., Vrtacnik, M., Pohleven, F., 2003. Effect of the antifungal activity of oxygenated aromatic essential oil compounds on the white-rot Tramates versicolor and the brown-rot Coniophora putana. Int. Biodeter. Biodegrad 51, 51– 59. WHO.2008.http://who.int/dg/speeches/2008/2008 1107/en/index.html
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Wallace, R.J., McPherson, C.A., 1994. Factors affecting the rate of breakdown of bacterial protein in rumen fluid. Br. J. Nutr. 58,313–323 Wallace, R J., 2004. Antimicrobial properties of plant secondary metabolites, Proc. Nutr. Soc. 63, 621–629.
Source of Support: Nil
Williams, A., G., Coleman, G.S., 1992. The Rumen Protozoa. Springer, Verlag Verlag, New York. pp. 441. Worthen, D.R., Ghosheh, O.A., Crooks, P.A., 1998. The in vitro anti-tumor activity of some crude and purified components of black seed Nigella sativa L. Anticancer Res. 18. 1527–1532.
Conflict of Interest: None Declared
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Original Research Article EVALUATION OF ANTI ULCER ACTIVITY OF FICUS PUMILA L. LEAF EXTRACT IN ALBINO RATS. Muhammed Ashraf V K1, Thamotharan G2*, Sengottuvelu S3, Haja Sherief S4, Sivakumar T5 1,2,3,4,5
Department of Pharmacology, Nandha College of Pharmacy and Research Institute, Erode - 52, Tamilnadu, India *Corresponding author: Mob:-+91 90 2526 5999; Email: jthams0309@gmail.com
Received: 22/06/2012; Revised: 28/07/2012; Accepted: 30/07/2012
ABSTRACT The objective of this study was to investigate the gastro protective activity of ethanolic extract of leaves of Ficus pumila L. (Moraceae) in different experimental models of gastric ulcer in rats. Two doses (200 mg/kg and 400 mg/kg) of the extracts were used for the study. The experimental ulcers were induced by different models such as pyloric ligation, ethanol and cold restrained ulcer models. Omeprazole (10 mg/kg) and sucralfate 100 mg/kg) were used as the standard drugs. All drugs were administered by the oral route. The ulcer index and percentage of ulcer protection were measured. Phytochemical tests and various parameters such as free acidity, total acidity, total hexose, Hexosamine, Fucose and total protein were measured. The result showed that administration of the extract of Ficus pumila L. to the Albino rats significantly decreases the ulcer index with increase in the percentage of ulcer protection in a dose dependent manner when compared with control groups. Phyto-chemical analysis revealed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. It could be concluded that the ethanolic extract of leaves of Ficus pumila possess significant, dose dependent anti ulcer activity.
KEYWORDS: Anti-ulcer, Ficus pumila L., pyloric ligation, ethanolic extract, ethanol, cold restraint Ulcer.
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INTRODUCTION The ulcer is a lesion of the gastric mucous membrane and it occurs probably due to an imbalance between the aggressive (acid, pepsin, bile and H. pylori) and the defensive (gastric mucus and bicarbonate secretion, prostaglandins, nitric oxide, innate resistance of the mucosal cells) factors (Rao et al., 2004). Gastric and duodenal ulcers are the two types of ulcer distinguished, which affects a considerable number of people in the world (Ineu et al., 2008). The infection with Helicobacter pylori and the use of non-steroidal anti-inflammatory drugs (NSAIDs) are the two essential causes for the development of gastric ulcer (Oâ&#x20AC;&#x2122;Malley 2003). Outwards of these well identified causes certain other factors can increase the ulcer development risk such as alcohol consumption, cigarette smoking, psychological stress and dietary factors. It has been suggested that reactive oxygen species, primarily super-oxide anions, hydroxyl radicals, and lipid peroxides are the harmful species known to cause gastric ulcer development (Smith et al., 1996). Plant extracts, containing a wide variety of antioxidants such as phenolic and flavonoid compounds are some of the most attractive sources of new drugs and have been shown to produce promising results in the treatment of gastric ulcers (Hiruma-Lima et al., 2001). Herbal medicine is fast emerging as an alternative treatment to synthetic drugs for treatment of ulcer possibly due to lower costs, availability, fewer adverse effects and perceived effectiveness (Thirunavukkarasu et al., 2009) & plants are more potent healers because they promote the repair mechanisms in the natural way (Veda Vidya T et al., 2012). In the regions of Tamil Nadu, many Ficus species like F. religiosa & F. virens are considered as the abode of spirits and worshiped in many forms accrediting them as the sthalavrikshas. (Umavathi R et al., 2012). In the present study one another plant from the Genus Ficus, named Ficus pumila L. of the Moraceae family was selected, which is a scandent shrub with evergreen coriaceous leaves that is normally
grown between the trees as well as on fragmented surface. The leaves of the plant has been traditionally consumed by some Okinawan elders either as a beverage or used as an invaluable medicinal herb by the folks to treat diabetes, dizziness, high blood pressure, and neuralgia (Mitsuhashi, 1988; Tobinaga, 1989). Several studies have been performed on the composition of Ficus pumila L.; phytochemical analysis was performed and confirmed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. The important constituents isolated in the previous study were apigenin, luteolin, rutin, genistein, hesperidin, astragalin, isoquercitrin, and chrysin (Abraham et al., 2008). Although many other species of this genus such as Ficus deltoidea (Fathim Zahra et al., 2009), Ficus bengalenesis (Mayank Krishna et al., 2011), Ficus nervosa ( Rama Devi et al., 2012), Ficus religiosa (Sarmistha Saha et al., 2010), Ficus arnottiana (Gregory Marslin et al.,2009), Ficus indica (L.)Mill (Galati et al., 2002), Ficus hispida (Sivaraman et al., 2010), Ficus trichopoda (Balogun et al.,2011), Ficus glomerata (Rao et al., 2008), Ficus asperifolia (Raji Y et al., 2011) has been reported for anti-ulcer activity. However, the anti ulcer activity of Ficus pumila L. had never been investigated, thus the present study was initiated to evaluate the anti ulcer activity of ethanolic extract of leaves of Ficus pumila L. in Albino rats. MATERIALS AND METHODS Collection of Plant material and preparation of extract The leaves of Ficus pumila L. were collected from the campus of Nandha college institutionErode (Tamilnadu). The plant was identified and authenticated by the taxonomist Dr. G.V.S. Murthy, Botanical Survey of India, Tamilnadu Agricultural University Campus (TNAU), Coimbatore. The voucher specimen (BSI/SRC/5/23/2012-13/Tech-448) has been deposited in the herbarium of TNAU for future reference. The leaves were shade dried, powdered and were extracted using 70% ethanol as the solvent in a soxhlet apparatus until complete extraction. Solvent evaporation
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under reduced pressure was carried out to get semisolid extract which was used for the studies. Experimental animal The study was conducted on Wistar Albino rats of 150–200 g and was maintained under standard conditions (room temperature 240C– 270C and humidity 60–65%). Animals were provided with standard rodent pellet (M/s. Hindustan Lever Ltd, Mumbai) and had free access to water. Rats of either sex were selected and grouped in to four of six animals each. All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics committee (688/2/C-CPCSEA) of Nandha College of Pharmacy, (Proposal NoNCP / IAEC / No: 9/2012-13) and were in accordance with the guidelines of the IAEC. Phytochemical screening: The freshly prepared crude ethanolic extract of Ficus pumila L. was qualitatively tested for the presence of major phytochemical constituents according to standard methods (Trease, G.E., Evans, W.C. -1996)
Anti ulcer studies a) Pyloric ligation induced ulcers: Four groups of albino Wister male rats (n = 6) were selected. In this model, Group 1 served as normal control (vehicle) received 0.5% Carboxy methyl cellulose (CMC), p.o, and group 2 Omeprazole (10 mg/kg, p.o), whereas groups 3 and 4 animals received ethanolic extract of leaves of F. pumila L. (200
and 400 mg/kg, p.o. respectively) daily for 3 days. Animals were fasted overnight prior to starting the experiment and water ad libitum. Pyloric ligation was performed by ligating the pyloric end of the stomach of rats on 3rd day under Phenobarbital anaesthesia (35 mg/kg i.p), after 30 min of administration of ethanolic extract of F. pumila or Omeprazole. Animals were allowed to recover and stabilize in individual cages and were deprived of water during postoperative period (Ramaswami et al., 2010). After 4 h of pyloric ligation, rats were sacrificed, stomach was removed and gastric juice was collected for performing gastric secretion study and for ulcer scoring. The gastric juice was collected and centrifuged. The volume and pH was recorded and subjected to bio-chemical estimations like free acidity and total acidity (Hawk et al.1947), total proteins (Lowry et al., 1951), total hexoses (Winzler et al., 1958), hexosamine (Dische et al .,1950) and fucose (Dische et al., 1948), ulcer was scored as follows. Mean ulcer score for each animal will be expressed as ulcer index. The percentage of ulcer protection was determined as follows: Ulcer index (UI) was measured by using following formula
UI = UN + US + UP ×10-1 Where, UI = Ulcer Index; UN = Average number of ulcers per animal; US = Average number of severity score; UP = Percentage of animals with ulcers.
The following Table indicating Ulcer Score and Descriptive Observation. 0
Normal coloured stomach
0.5
Red colouration
1
Spot ulceration
1.5
Haemorrhagic streak
2
Ulcers ≥3 but ≤ 5 mm.
3
Ulcers > 5 mm
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Gastric Secretion Study The gastric juice was collected 4 h after pyloric ligation and the gastric volume and pH were measured. The collected gastric juice was centrifuged for 5 min at 2000 rpm. The volume of the supernatant was expressed as ml/100 g body weight. Free acidity and total acidity (Hawk et al., 1947) were determined by titrating with 0.01 M NaOH using Toepferâ&#x20AC;&#x2122;s reagent and phenolphthalein as indicator. Dissolved mucosubstances like total hexoses (Winzler et al., 1958), hexosamine (Dische et al., 1950), fucose (Dische et al.,1948) and total protein (Lowry et al., 1951) also were estimated. b) Ethanol-induced ulcer The overnight fasted rats were divided into four groups of six each. All the groups of rats were given treatments as follows by oral route: group 1 received 1 ml/kg of 5% CMC (control group), group 2 received 100 mg/kg sucralfate, group 3 received 200 mg/kg, and group 4 received 400 mg/kg of ethanolic extract of F. pumila L. Thirty minutes later, ulcers were induced by administering 1 ml of absolute ethanol (99%) to each rat. One hour later all the rats were sacrificed and the stomach were excised, cut along the greater curvature and gently rinsed under tap water. The stomachs were stretched on a corkboard and a magnifying glass (10X magnification) was used to spot and count the craters (Ukwe et al., 2010). The ulcer index was obtained by the sum of a groupâ&#x20AC;&#x2122;s crater score and divided by magnification. Ulcer inhibition (UI) was calculated as above. c) Cold restraint induced ulcers Four groups of Wistar Albino rats (n = 6) were selected. In this model, Group 1 served as Normal control (vehicle) received 0.5% CMC,
p. o., and Group 2 Omeprazole (10 mg/kg, p.o), whereas Groups 3 and 4 animals received ethanolic extract of F. pumila (200 and 400 mg/kg, p.o. respectively) daily for 3 days. Animals were fasted overnight prior to start of the experiment, and water ad libitum. On day 3, after 30 min of administration of ethanolic extract of F. pumila or Omeprazole, rats were immobilized in a stress cage and were placed at 4â&#x20AC;&#x201C;60C in an environmental cage (Ramaswami et al., 2010) The animals were sacrificed 2 h later and ulcer index was calculated following the method as described earlier. Statistical analysis The values were expressed as mean Âą SEM. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Dunnetâ&#x20AC;&#x2122;sâ&#x20AC;&#x2DC;tâ&#x20AC;&#x2122; - test. P values < 0.05 were considered significant. RESULTS a) Phytochemical analysis The phytohemical analysis of the ethanolic extract of F. pumila L. revealed the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. b) Pylorus ligation induced model The antiulcer activity of ethanolic extract of F. pumila L. was carried out by pylorus ligation induced ulcer model (Table 1). F. pumila L. showed significant dose dependent antiulcer effect at the dose of 200 mg/kg and 400mg/kg (p.o) with an ulcer index of (1.68 Âą 0.14), (3.43 Âą 0.09) and % ulcer protection (72.91), (79.56) respectively. This shows the decrease in ulcer index and increase in % of ulcer protection. It was compared with the standard drug Omeprazole. The bio-chemical parameter showed that there was a decrease in gastric volume, free acidity, total acidity and total
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protein while an increase in total hexose, hexosamine and fucose (Table 2 and 3). c) Ethanol induced model The antiulcer activity of ethanolic extract of F. pumila L. was carried out by ethanol induced ulcer model (Table 4). F. pumila L. treated groups were compared with sucralfate (P < 0.01). Pre-treatment of rats with F. pumila L. extracts produced a dose dependent protection in the ethanol induced ulceration model as compared to control group. F. pumila L. showed significant antiulcer effect at the dose of 200 mg/kg and 400 mg/kg (p.o) dose. 200 mg/kg and 400 mg/kg dose produced an ulcer index of (11.12 ± 0.32), (6.69 ± 0.41) and % protection (50.22), (70.85) respectively. This shows the decrease in ulcer index and increase in % of ulcer protection. It was compared with the standard drug sucralfate (81.15).
d) Cold restraint induced model The effect of ethanolic extract of F. pumila L. in cold restraint stress ulcer model was shown in Table 5. Hypothermic and Immobilization stress produced considerable ulcero-genicity in rats. The ulcers were in the form of hemorrhagic mucosal lesions in the stomach, which were confined to the rugae of glandular segment. The parameters studied included ulcer index and % of ulcer protection. F. pumila L. treated groups were compared with Omeprazole (P < 0.01). F. pumila L. showed a significant dose dependent antiulcer effect at the dose of 200 mg/kg and 400 mg/kg (p.o).200 mg/kg and 400 mg/kg were produced an ulcer index of (6.46 ± 0.25) and (3.80 ± 0.29) and % ulcer protection of (68.30) and (81.35) respectively. This shows significant decrease in ulcer index and increase in % ulcer protection. It was compared with the standard drug Omeprazole which showed significant % of ulcer protection (86.90).
Table 1: Effect of Ficus pumila L. on different ulcer models Indicating Ulcer index & Percentage of Ulcer Protection. Treatment
Pylorus Ligation Induced Model
Ethanol Induced Ulcer Model
Cold Restraint Stress Ulcer Model
Ulcer Index
% Protection
Ulcer Index
% Protection
Ulcer Index
% Protection
Solvent Control (0.5% CMC)
16.78 ± 0.75
-
22.34 ± 0.57
-
20.38 ± 1.04
-
F Pumila L (200mg/kg)
4.56 ± 0.13**
72.91
11.12 ± 0.32**
50.22
6.46 ± 0.25**
68.30
F Pumila L (400mg/kg)
3.43 ± 0.09**
79.56
6.69 ± 0.41**
70.85
3.80 ± 0.29**
81.35
Standard
1.68 ± 0.14**
89.99
4.21 ± 0.40**
81.15
2.67 ± 0.41**
86.90
(Results are mean ± S.E.M. (n = 6) Statistical comparison was performed by using ANOVA followed by Dunnet’t’ test. * P < 0.05, **P < 0.01,***P < 0.001 were consider statistically significant when compared to control group.)
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Table 2: Effect of Ficus pumila L. on Pylorus ligation indicating biochemical parameters. Sl.No
Treatment
Gastric
pH
volume(ml) Solvent control
1
6.55 ± 0.51
Free acidity (µeq/ml/100 g)
2.63 ± 0.19
42.80 ± 3.01
Total Acidity (µeq/ml/100 g) 73.47 ± 2.42
(0.5% CMC 1 ml/kg) Omeprazole
2
2.35 ± 0.24** 4.65 ± 0.29* 18.27 ± 1.71** 21.88 ± 1.41**
(10 mg/kg)
Extract of F.pumila
3
3.47 ± 0.19** 3.50 ± 0.19**26.43 ± 1.10** 53.51 ± 1.57**
(200 mg/kg) Extract of F.pumila
4
2.65 ± 0.24** 4.15 ± 0.22**23.18 ± 1.49** 31.38 ± 2.72**
(400 mg/kg) Results are mean ± S.E.M.(n = 6) Statistical comparison was performed by using ANOVA followed by Dunnet’t’ test. *P < 0.05, **P < 0.01,***P < 0.001 were considered statistically significant when compared to control group. Table 3: Effect of Ficus pumila L. on Pylorus Ligated (Shay) Rat Model Indicating Total Hexose, Hexosamine, Fucose & Total Protein. SL.
Treatment
No 1
Solvent
Total Hexoses (µg/ml)
Hexosamine (µg/ml)
Fucose (µg/ml)
Total Protein (µg/ml)
268.05 ± 12.43
263.14 ± 11.52
66.11 ± 7.95
340.83 ± 18.87
control (0.5% CMC 1 ml/kg) 2
Omeprazole
394.95 ± 12.83** 493.28 ± 19.85** 174.13 ± 13.43**
180.5 ± 8.83**
(10 mg/kg) 3
Exract of
329.46 ± 6.92**
414.43 ±14.51**
118.56 ± 7.61**
273.33 ± 14.49**
371.94 ± 11.52** 460.26 ± 13.58** 150.15 ± 10.65**
174.5 ± 10.99**
F.pumila (200 mg/kg) 4
Extract of F.pumila (400 mg/kg)
Results are mean±S.E.M.(n=6) Statistical comparison was performed by using ANOVA followed by Dunnet’t’ test. *P < 0.05, **P < 0.01,***P < 0.001 were consider statistically significant when compared to control group.
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Graph.1: Effect of Ficus pumila on Pylorus Ligated (Shay) Rat Model indicating Ulcer index and % ulcer Protection. 100 90 80 70 60 50 40 30 20 10 0 % ulcer protection
ulcer index
vehicle control(1ml/kg)
omeprazole(10mg/kg)
F.pumila(200mg/kg)
F.pumila(400mg/kg)
Graph 2: Effect of Ficus pumila on Ethanol Induced Ulcer Model Indicating Ulcer index & % Ulcer Protection. 90 80 70 60 50 40 30 20 10 0 ulcer index vehicle control(1ml/kg)
% ulcer protection sucralfate(100mg/kg)
F.pumila(200mg/kg)
F.pumila(400mg/kg)
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Graph 3: Effect of Ficus pumila on cold restraint induced Ulcer Model Indicating Ulcer index and % Ulcer Protection. 100 90 80 70 60 50 40 30 20 10 0 ulcer index
% ulcer protection vehicle control(1ml/kg)
omeprazole(10mg/kg)
F.pumila(200mg/kg)
F.pumila(400mg/kg)
DISCUSSION The etiology of peptic ulcer is unknown in most of the cases, yet it is generally accepted that it results from an imbalance between aggressive factors and defensive factors ((Akah et al., 1998).. To regain the balance, different therapeutic agents including plant extracts may be used (Gurbaz et al.,2003, Sairam et al., 2003). The causes of gastric ulcer ulceration by pyloric ligation are believed to be due to stress induced increase in gastric hydrochloric acid secretion and/or stasis of acid, and the volume of secretion is also an important factor in the formation of ulcer due to exposure of the unprotected lumen of the stomach to the accumulating acid (Raju et al .,2009). 2009). Pylor Pyloric ligation induces ulcers by auto digestion of the gastric mucosa and breakdown of the gastric mucosal barrier. These factors are associated with the development of upper gastrointestinal damage including lesions, ulcers and life threatening perforation and haemorrhage. Gastric wall mucus, an obligatory component of which is hexosamines, is thought to play an important role as a defensive factor aga against gastrointestinal damage (Davenport Davenport 1968). The determined gastric wall mucus was used as an
indicator for gastric mucus secretion, while mucosal hexosamine content was used as an indicator for gastric wall mucus synthesis (Lukie et al.,1972). ). In the present study, gastric wall mucus and hexosamine amine contents in ethanol induced ulcerated rats were markedly lowered when compared to those of the non-ulcerated non group. It was found that pre-treatment pre with extract of F. pumila L. increased both gastric mucus and Hexosamine contents significantly. This finding ding indicates that the extract of F. pumila L. can preserve both gastric mucus synthesis and secretion in the experimental rats. rats Ethanol has also been reported to cause disturbances in gastric secretion, damage to the mucosa, alterations in the permeability, gastric mucus depletion and free radical production. This is attributed to the release of superoxide anion and hydroperoxy free radicals, radicals during metabolism of ethanol as oxygen derived free radicals has been found to be involved in the mechanism of acute and chronic ulceration in the gastric mucosa (Jude Jude et al., 2009). Ethanol induced gastric lesion formation may be due to stasis in gastric blood flow which contributes to the development of the haemorrhage and necrotic aspects of tissue injury. Alcohol rapidly penetrates the gastric mucosa
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apparently causing cell and plasma membrane damage leading to increased intra cellular membrane permeability to sodium and water. The massive intracellular accumulation of calcium represents a major step in the pathogenesis of gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium (Raju et al., 2009). It was observed in this study that the extract significantly reduced ethanol induced ulcer. This may be due to cyto-protective effect of the extract via antioxidant effects. The extract shows protection against characteristic lesions produced by ethanol administration, this antiulcer effect of F. pumila L. may be due to both reductions in gastric acid secretion and gastric cyto-protection. The antiulcer property of F. pumila L. in pyloric ligation model is evident from its significant reduction in free acidity, total acidity, number of ulcers and ulcer index. F. pumila L. treated animals significantly inhibited the formation of ulcers in the pylorus ligated rats and also decreased both the concentration and increased the pH, which suggests that F. pumila L. can suppress gastric damage induced by aggressive factors.. The significant increase in the antiulcer activity of F. pumila L. could be attributed to the presence of carbohydrate, glycosides, sterols, flavonoids and triterpenes. Flavonoids are among the cytoprotective materials for which anti-ulcerogenic efficacy has been extensively confirmed. It suggests that, these active compounds would be able to stimulate mucus, bicarbonate and the prostaglandin secretion and counteract with the deteriorating effects of reactive oxidants in gastrointestinal lumen
(Sakat et al .,2009). So the antiulcer activity of F. pumila L. may be attributed to its flavonoid content. The results of the present study suggest that the ethanolic extract of F. pumila L. leaves may be beneficial in the treatment of gastric lesions. Further studies are required to identify the active constituents and elucidation of the mechanism of action. Cold restrained stress provides both emotional stress as well as physiological stress to the animal, which induces ulcer due to the auto digestion of gastric mucosal barrier, accumulation of HCl and generation of free radicals . The role of the free radicals in gastric ulcerations is well-documented (Cochran et al., 1983) CONCLUSION From this study, we can conclude that the ethanolic extract of F. pumila L. leaf extract have significant anti-ulcer activity in animal models. It has muco-protective activity and gastric anti-secretary when compared with that of the reference drug Omeprazole and Sucralfate. The anti-ulcer activity is probably due to the presence of flavonoids. Further studies are being carried out to characterize and explore the biological activity of the compounds present in the extract. ACKNOWLEDGEMENT We are deeply thankful to Mr. Nandha Kumar Pradeep, Secretary, Nandha College of Pharmacy and Research Institute for support and institutional facilities.
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Alimi, H Hfaiedh, N Bouoni, Z.Hfaiedh, M Sakly, M., Zourgui, L Ben Rhouma, K, (2010). Antioxidant and antiulcerogenic activities of Opuntia ficus indica f. inermis root extract in rats. Phytomedicine 17, 1120–1126. Balogun S.O., J.K. Tanayen,A.M. Ajayi, A. Ibrahim,J.O.C. Ezeonwumelu, A.A. Oyewale,O.J. Oloro, A.D.T. Goji,D.M. Kiplagat and B. Adzu(2011): Preliminary Evaluation of AntiDiarrheal, Ulcer-Protective and Acute Toxicity of Aqueous Ethanolic Stem Bark Extract of Ficus trichopoda in Experimental Rodents, Asian Journal of Medical Sciences 3(1): 37–42 Chi-Ren Liao, Chun-Pin Kao, Wen-Huang Peng, Yuan-Shiun Chang, Shang-Chih Lai, and Yu-Ling Ho (2012). Analgesic and Anti-Inflammatory Activities of Methanol Extract of Ficus pumila L. in Mice. Evidence-Based Complementary and Alternative Medicine. 2012;2012:340141. Cochran T, Stefanko J, Moore C, Saik R. Dimethyl sulphoxide protection against gastric stress ulceration. Current Surgery. 1983; 40: 435–437. Davenport HW. Destruction of the gastric mucosal barrier by detergents and urea. Gastroentero. 1968; 54: 175–180. Dische Z and Barenfreund E (1950), A spectrophotometric method for the microdetermination of hexosamines. J BioChem, 184: 517. Dische Z and Schettles LB (1948), A specific colour reaction for methyl pentoses and spectrophotometric micro method for determination. J Biochem, 175: 595– 603. Fathima M.A.S. Zahra, A.A.Mahmood, M.M.Hapipah, M.N.sunita & I.salmah (2009):anti-ulcerogenic activity of aqueous extract of Ficus deltoidea against ethanol induced gastric mucosal
injury in rats. Medical research journal of science 3(2) 42–46. Galati E.M, S. Pergolizzi, N. Miceli, M.T. Monforte, M.M. Tripodo (2002): Study on the increment of the production of gastric mucus in rats treated with Opuntia ficus indica (L.) Mill. cladodes. J Ethnopharmacol 83:229– 233 Gregory Marslin, Vithalrao K. P, Franklin G & Kalaichelavan V (2009). Antiulcer (ulcer-preventive) activity of Ficus arnottiana Miq (Moraceae) leaf methanolic extract.” American journal of pharmacology and toxicology; 4: (3) 89–93. Gurbaz I,Ustan O., Yesilada E., Sezik E. and Kutsal O. Anti-ulcerogenic activity of some plants used as folk remedy in Turkey. J Ethnopharmacol. 88 :93-97 (2003). Hawk PB, Oser BL and Summerson WH (1947), Practical Physiological Chemistry. Mc Graw- Hill Book Company, New York, 375. Hichem Alimi, Najla Hfaiedh, Zouhour Bouonia, Mohsen Sakly, Khémais Ben Rhouma (2011) Evaluation of antioxidant and antiulcerogenic activities of Opuntia ficus indica f. inermis flowers extract in rats. Environmental toxicology and pharmacology (32) 406–416 Hiruma-Lima, C.A Gracioso, J.S Toma, W Almeida, A.B Paula, A.C Brasil (2001): Gastro protective effect of aparisthman, a diterpene isolated from Aparisthmium cordatum on experimental gastric ulcer models in rats and mice. Phytomedicine 8, 94– 100. Ineu RP, Pereira ME, Aschner M, Nogeueira, CW, Zeni G, Rocha JBT (2008). Diphenyl diselenide reverses gastric lesions in rats: involvement of oxidative
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O’Malley (2003). Gastric ulcers and GERD: the new “plagues” of 21st century update for the clinical nurse specialist. Clin. Nurse Spec. 17, 286–289. Pistelli L, E. E. Chiellini, and I. Morelli (2000) “Flavonoids from Ficus pumila,” Biochemical Systematics and Ecology, vol. 28, no. 3, pp. 287–289 Raji Y, Oyeyemi WA, Shittu ST, Bolarinwa AF (2011). Gastro-protective effect of methanol extract of Ficus asperifolia bark on indomethacin-induced gastric ulcer in rats. Niger J Physiol Sci.Nov 23;26(1):43–8. Raju D, Ilango K, Chidra V, Ashish K, Evaluation of Anti-ulcer activity of methanolic extract of Terminalia chebula fruits in experimental rats. J. Pharm. Sci. & Res. 2009; 3:101–107.
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Thirunavukkarasu P, Ramkumar L, Ramanathan T (2009) Anti-ulcer Activity of Excoecaria agallocha bark on NSAID-induced gastric ulcer in Albino Rats. Global journal of pharmacology 3(3), 123–126.
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Source of Support: Nil
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Conflict of Interest: None Declared
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Original Research Article HIERARCHICAL CLUSTER ALGORITHMS OF OCCURRENCES AND ANTIMICROBIAL ACTIVITY OF ROOT EXTRACT OF AN ETHNOMEDICINALLY IMPORTANT PLANT: SIDA CORDIFOLIA LINN. Kumari Priti1*, Joshi Girish Chandra2, Singh Bibhesh Kumar3 1, 2
Regional Research Institute of Himalayan Flora, Tarikhet-263663 (Uttarakhand) India Department of Chemistry, Govt. Postgraduate College, Ranikhet-263645(Uttarakhand) India *Corresponding Author; E-mail: pritiksingh78@yahoo.co.in; Tel/ Fax: +91-5966-220151 3
Received: 26/06/2012; Revised: 27/07/2012; Accepted: 31/07/2012
ABSTRACT The recent knowledge of the importance of biodiversity highlights an under-appreciated truth although society is dependent on natural and managed ecosystems for goods and services that are essential for human survival, we know all too little about how ecosystems work. Human domination of earth's ecosystems is markedly reducing the diversity of species within many habitats worldwide, and is accelerating extinction. Sida cordifolia Linn. is one of the important ethno medicinal plants, of Ayurvedic importance and have trade value as well. Root extract of Sida cordifolia Linn. represents a source mixture of antibacterial constituents that can be as effective as modern medication Becillus subtillis, Pseudomonas aereuguinosa, Staphylococcus aureus and Escherchia coli bacteria were shown to be proactive. For the reason of over exploitation and habitat degradation, Sida cordifolia Linn. having gradually low frequency and they are now turning into a rare species. Their occurrence too was found less as compared to other species.
KEYWORDS: Antibacterial activity, Ethno-medicine, Hierarchical Cluster Algorithms, Occurrence, Sida cordifolia Linn.
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INTRODUCTION The uniqueness and extraordinary natural value of the Himalaya is well known worldwide. It is known for its richness in plant diversity. Indian Himalayas is the most beautiful mountain chain of the world. Phytogeographically, these are divided into three main regions the North Western Himalaya, the Western Himalaya and Eastern Himalaya. Western Himalaya comprises the parts of Jammu & Kashmir, Himachal Pradesh and Kumaun & Garhwal Himalaya (Uttarakhand). During the past two decades major degradation of the mountain ecosystems in the Western Himalaya has been witnessed. The exact extent of loss of forest cover is difficult to assess. Human interaction with environment involves exploitation of natural resources implying thereby a certain degree of environmental destruction which is unavoidable. Studies have been carried out to explore and identify the threatened plants of Indian Himalaya (Pangtey & Samant 1988; Samant et al., 1993, 1996 a & b, 1998 a & b; Samant 1994; Pandey &Well 1997; Kala et al., 1998; Kumari et al., 2011). Plants are used medicinally in different countries and are a source of many potent and powerful drugs (Srivastava et al., 1996). Sida cordifolia Linn. (Figure 1) commonly known as Denusha (Kumauni) is a herb that is used as a ethno-medicine in the Almora district. This herb is extensively used as a common herbal drug in the Indian subcontinent. The water extract of the leaves was reported to possess analgesic and anti-inflammatory activities in animal models (Gunatilaka et al., 1980). It is used in Ayurvedic medicine. (Pole et al., 2006 and Sebastian, 2006). It has been investigated as an anti-inflammatory (Franzotti et al., 2000) for treating cancer (Jenny et al., 2005), for encouraging liver re-growth (Silva et al., 2006) and antibacterial growth (Isman et al., 2003). Medicinal plants represent a rich source of antimicrobial agents. Due to its ephedrine content, it possesses psychostimulant properties, affecting the central nervous system and also the heart (Adam et al., 2006). It is also used as a fat-burning
supplement. It has a depressive effect on the central nervous system (Franco et al., 2005). Moreover, previous phytochemical studies on the roots had shown the presence of ephedrine, vasicinol, vasicinone and N-methyl tryptophan (Franzotti, 2000). Recent analysis has revealed that ephedrine and pseudoephedrine constitute the major alkaloids from the aerial parts of the plant, which also show traces of sitosterol and palmitic, stearic and hexacosanoic acids. From seed oil sterculic, malvalic and coronaric acids are isolated along with other fatty acids. Present study deals with the assessment of occurrences of Sida cordifolia L. and its antimicrobial activity of an ethno-medicinally important plant from Almora district of Uttarakhand. In recent years, multiple drug resistance has developed due to indiscriminate use of existing antimicrobial drugs in the treatment of infectious diseases. Antimicrobial resistance is a threat to mankind because most of the infection causing bacteria has become multidrug resistant. Antibiotic resistant bacteria may keep people sick longer, and sometimes people are unable to recover at all. Because of the concern about the side effects of conventional medicine, the use of natural products as an alternate to conventional treatment in healing and treatment of various diseases has been on the rise in the last few decades. The knowledge and expertise in folk remedies conserved by remote hilly area people needs to be documented and investigated for modern drug therapeutics (Kumari et al., 2011 & Joseph et al., 2011). Due to lack of modern medical facilities, expensive drugs and poor transportation, patients of these localities normally suffers for a long. In these unfavorable situations traditional herbal healers of this remote locality play a vital role to provide them as an alternate source of therapeutic facility for their primary healthcare. Subsequently, with the advancements in the techniques of phyto-chemistry and pharmacology, a number of active principles of medicinal plants were isolated and introduced as valuable drugs in modern system of medicine.
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GJRMI, Volume 1, Issue 8, August 2012, 352–363 Figure 1: Sida cordifolia Linn.
Figure 2: Map of Almora District
The manuscript describes distribution and local/traditional uses of the medicinal flora and identifies the plants that need conservation and protection. EXPERIMENTAL Study area The district of Almora lies between 29o30’N to 30o20’N latitudes and 79o20’ E to 80o20’E longitudes. It is located in the central part of Kumaun. Its boundary is extended over to Nainital in the south, south-west in Champawat, east in Pithoragarh, north in Bageshwar and Chamoli districts, whereas western boundary is confined to area covering Pauri district. The Almora district covers area of 46 Km in north-south in length and 86 Km east-west in width. The area of study covers the whole 3629.66 sq. Km. According to 2011
census, Almora district has a total population 621927, in which 290414 are male and 331513 are female. The Almora district consists of three subdivisions- Bhikhiasen, Ranikhet and Almora. Presently, Syaldey, Sult, Bhikiyasen, Chukhutia, Dwarahat, Tarikhet, Hawalbagh, Takula, Bhasiyachana, Dhowladevi and Lamgarha are its developmental blocks. Almora, Ranikhet and Dwarahat are consisting of its urban areas (Figure 2: Map of Almora District). Methodology of occurrence assessment The study was conducted in Tropical forest of Almora district of Kumaun region of Uttarakhand covering an altitude of 500 m to 1200 m above the sea level (ASL). Total seven sites (Figure 3: Google map of study site) were located on the basis of the occurrence of sida cordifolia L. and thirty-five areas were selected
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in Almora district on account of typical topography. Out of total seven sites, each site having five areas, each area have three locations and each location have ten quadrates of 1×1. The sites were selected on each and every accessible aspect and habitat in study area. Degrees of dispersion of an individual Frequency (%) =
species in an area are usually expressed in terms of percentage occurrence. It was studied by sampling the study area at several places at random and recorded the name of the species that occurred in each sampling units. It was calculated by the equation:
Number of quadrates in which the species occurred × 100 Total number of quadrates studied
In the present study NCSS 2007 software was used for cluster analysis and scatter plot. Figure 3: Different study sites in Almora district
Antibacterial studies The collected plants were identified at Regional Research Institute of Himalayan flora, Tarikhet, Uttarakhand, India (Accession No. RKT 20326).The root parts collected from the study area were dried in shade for two weeks and then powdered with the help of electric grinder. 50 g of shed-dried powder was extracted with ethanol in solvent extractor for 72 h. After filtration solvent was removed under reduced pressure in a rotary evaporator at 40°C and the pure extract was kept in airtight bottle in refrigerator until the moment of analysis.
In vitro antibacterial activity of the infusion (test compound) against Becillus subtillis, Pseudomonas aereuguinosa, Staphylococcus aureus and Escherchia coli were carried out using paper disc diffusion method (Saxena et al., 1993). The nutrient agar medium (peptone, beef extract, NaCl and agar-agar) and 5 mm diameter paper discs of Whatmann No.1 were used. The test compound was dissolved in ethanol in 0.1–0.4% concentrations. The paper discs were soaked in different solutions of the compound, dried and the placed in the Petri plates (9 mm diameter) previously seeded with
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the test organisms, Becillus subtillis, Pseudomonas aereuguinosa, Staphylococcus aureus and Escherchia coli. The plates were incubated for 24–30 h at 27 ± 1°C and the inhibition zones (mm) were measured around each disc. As the organism grows, it forms a turbid layer, except in the region where the concentration of antibacterial agent is above the minimum inhibitory concentration, and a zone of inhibition is seen. The size of the inhibition zone depends upon the culture medium, incubation conditions, rate of diffusion and the concentration of the antibacterial agent.
RESULTS AND DISCUSSION Frequency is a measure of the uniformity of the distribution of a species; thus a low frequency indicates that a species is either irregularly distributed or rare in a particular stand or forest. Frequency distributions of plant density, cover, biomass per unit area, and height, as measures for expressing biological abundance and biological dominance of vegetation, have been used to describe species composition and spatial patterns of vegetation in different plant communities (Chen et al., 2008) In the present study, the documented species based on frequency (%) in different sampling sites is depicted in Figure 4.
Figure-4 Frequency (%) of all thirty-five areas of seven sites under three locations (A=Location-1, B=Location-2, C=Location-3) A B C
84
80
78
75
72
Frequency(%)
85
30
A B C
65
80
60
72
55
64
Frequency(%)
50 45 40 35
56 48 40 32
30
24
25
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18
17
15
14
13
12
11
16 16
Frequency(%)
A B C
20
10
40
20
36 9
45 5
42
4
50
3
48
2
55
19
54
8
60
60
7
65
66
6
70
1
Frequency(%)
A B C
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25
24
23
22
21
12
40
40 35 30
35
25
30
20
25
15
20
10
Frequency of this species in all thirty five areas is ranges from 10 - 80%. Location - A of area - 3, 9, 10 and 17 having 80% of frequency, Location - B of area 9 is also having 80% frequency. Whereas the lowest mean frequency is 16.7 of area 19 and 31. On the basis of above frequency % of seven sites of thirty-five area in three locations, the hierarchical clustering algorithms have been formed as double dendrograms. Double dendrograms are dendrograms that cluster both the rows (thirtyfive areas) and the variables (three locations) in a single graph. In this cluster analysis the Group Average clustering method have been chosen in which the distance between two groups is defined as the average distance between each of their members. The clustering details are presented in Figure 5. On the other
hand Euclidean distance type clustering method with Standard deviation scale type was used for the cluster analysis. All thirty-five areas have been grouped into three clusters as cluster 1, 2 and 3 when clustering rows shown in Figure 6. Value of Cophenetic Correlation is 0.708252 in clustering rows whereas the value of Cophenetic Correlation in variables is 0.627467. Total 20 linkage sections have been seen in clustering rows. The highest distance is seen in linkage number one with value 1.752355. The details of distance value and distance bars are presented in table - 1. Cluster 1 is the biggest cluster having 23 areas where cluster 3 is smaller than cluster 1 with seven areas; on the other hand cluster 2 is the smallest cluster with 5 areas shown in figure 5.
Table-1: Linkage Section when Clustering Rows Links 1
Distance values 1.752355
Distance bars |IIIIIIIIIIIIIIIIIIIIIIIIIIIIII
2
1.482099
3
35
18
45
34
24
50
31
30
Frequency(%)
36
45
30
42
50
55
29
48
55
60
28
Frequency(%)
54
65
27
60
26
66
Frequency(%)
A B C
A B C
33
A B C
32
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Links 11
Distance values 0.572448
|IIIIIIIIII
|IIIIIIIIIIIIIIIIIIIIIIIII
12
0.545926
|IIIIIIIII
1.046278
|IIIIIIIIIIIIIIIIII
13
0.542146
|IIIIIIIII
4
0.895959
|IIIIIIIIIIIIIII
14
0.513023
|IIIIIIIII
5
0.86791
|IIIIIIIIIIIIIII
15
0.470551
|IIIIIIII
6
0.855035
|IIIIIIIIIIIIIII
16
0.451959
|IIIIIIII
7
0.770754
|IIIIIIIIIIIII
17
0.443067
|IIIIIIII
8
0.762105
|IIIIIIIIIIIII
18
0.433402
|IIIIIII
9
0.682087
|IIIIIIIIIIII
19
0.414827
|IIIIIII
10
0.638065
|IIIIIIIIIII
20
0.414827
|IIIIIII
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GJRMI, Volume 1, Issue 8, August 2012, 352–363 Figure 5: Clustring details when clustring rows
There are two clusters has been seen with three variables that is location 1, 2 and 3 on the side of clustring variables. The two links have been seen with 0.627467 cophenenetic correlation. The distance value of link two is 1.434285 whereas the value of link one is 1.371726. On the basis of occurance of Sida in thirty - five area in three locations, seven groups have been seen in figure 6. Occurrence of Sida in area 1, 13, 27, 34, 7, 22 of location one, area 15, 16, 27, 11, 28 of location two and area 1, 2, 33, 15, 16, 6, 24, 13, 30 of location three, which is represented with colour green in dendrograme are similar in study area. Area 2, 33, 8, 5, 15, 16, 11, 32, 21, 25 of location one, area 1, 2, 33, 8, 6, 18, 12, 25, 10 of location two and area 14, 27, 21, 22, 10 and 17 of location three which is represented by yellow in figure 6 having similar occurrence in study area. Area 6, 24, 12, 28, 4 of location one, area 24, 7, 21, 22, 3, 4, 17, of location two and area 7, 18, 12, 25, 28, 3, 9 of location three, represented by orange in figure 6 was occurred similarly in study area. All the patterns of similarity in occurrence in different study sites are presented as colored matrix in figure 6. Area 30, 14, 18, 26, 29 of location one, area 13, 30, 14, 32, 34, 20, 35, 23, 31 of location two and area 32, 34, 29 of location three which are represented by color aqua are similar in their amount of occurrence. Area 3, 9, 10, 17 of location one, area 9 of location two and area 4 of location three which are represented by color red are similar in their occurrence with same amount. Area 19, 20, 35 of location one, area
19 in location two and area 19 & 31 of location three with light blue are similar in their occurrence with amount alike. Area 23, 21 of location one, area 26 & 29 of location two and area 20, 35, 26 of location three which are represented by dark blue in colored matrix (figure 6) are similar in their occurrence with same amount. On the basis of frequency percentage it was found that, occurrence of Sida cordifolia Linn. is near to the ground in this area. Reason of having gradually low frequency is over exploitation and habitat degradation and they are now turning into a rare species. Their occurrence too was found less as compared to other species. On the basis of mean frequency distribution in different study areas scatter plot has been plotted in figure 7. Scatter plot shows the relationship between the mean frequency and the areas by displaying data point on a two dimensional graph. The imaginary drawing of scatter plot is a straight line so it ‘fit’ as well as possible. The more the points cluster closely around the imaginary line of best ‘fit’ the negative stronger relationship that exists between the areas and mean frequency. The data points of the graph are much more spread out, although they definitely follow a downward pattern. Therefore, it would be a good guess to say that, this is roughly a − 0.3 correlation. So the linear low negative correlation has been seen between the frequencies of identified species in different areas of Almora district.
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GJRMI, Volume 1, Issue 8, August 2012, 352â&#x20AC;&#x201C;363 Figure 6: Double dendrograme with frequency (%)
Rows
Amount 80.00
Distance 2.00 1.50
60.00
1.00
50.00
0.50
10.00
0.00
Location-1
Location-2
Variables
Location-3
70.00
1.50
1.13
0.75
0.38 0.001
2 33 8 5 15 16 6 24 13 30 14 27 11 32 34 7 21 22 18 12 25 28 3 9 4 10 17 19 20 35 23 31 26 29
Distance
Cluster-1
Cluster-2
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GJRMI, Volume 1, Issue 8, August 2012, 352â&#x20AC;&#x201C;363 Figure 7: Scatter Plot of Mean Frequency Frequency Distributions in Different Study area
M ean Frequency
80.0
53.3
26.7
0.0
1.0
12.3
Areas
23.7
35.0
Figure 8 Antibacterial studies of Sida cordifolia Linn.
The biological activity of the S. cordifolia Linn. and chloroamphenicol (as a standard drug) were tested against bacteria because bacteriums can achieve resistance to antibiotics through biochemical and morphological modifications. The organisms used in the present investigations included Becillus subtillis, Staphylococcus aureus (as gram positive bacteria) and Pseudomonas aereuguinosa and Escherchia coli (as gram negative bacteria). The diffusion agar technique was used to evaluate the antibacterial activity of the S. cordifolia Linn. The Sida has moderate activity in comparison with gram positive bacteria (Becillus subtillis, Staphylococcus aureus) and less active in comparison with gram negative bacteria
(Pseudomonas aereuguinosa and Escherchia coli). The activity of the Sida increases as the concentration (0.1â&#x2C6;&#x2019;0.4%) increases because it is a well known fact that concentration plays a vital role in increasing the degree of inhibition shown in figure 8. CONCLUSION Fast growing urbanization of life, people are being attracted towards use of natural products and as such utility of such plants are gaining ground. Quality of Life (QoL) is an important outcome of healthcare measures. It is an emerging domain of interest which measures the missing dimension of heath especially in chronic disabling conditions (Niranjan et al., 2012). Ayurveda gives immense magnitude for
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the improvement and assessment of QoL as an outcome of treatment (Charaka). Thus the need is to protect, promote and conserve such natural resources to have advantage of biodiversity conservation. It can’t be denied that in ancient days when medical facilities were not so developed, people were depend on traditional knowledge systems. The dormant knowledge during the ancient times definitely was solid and was helpful in curing different diseases but that is now not in vogue. It now needs revival research and further development to make herbal medicine knowledge savvy. The result shows negative correlation between the frequencies of identified species in different areas of Almora district. This commotion in mean frequency shows the rarity of Sida cordifolia Linn. In particular forest stands. The bio-efficacy of Sida cordifolia Linn. has been examined against the growth of bacteria in vitro to evaluate their anti-microbial
potential. On the basis of results, it can be concluded that root extract of Sida cordifolia Linn. represents a source mixture of antibacterial constituents that can be as effective as modern medication to combat pathogenic micro-organisms. The results can be compared with a lot of ethno-medicinal plants showing antimicrobial properties (Silva et al., 2011; Umar et al., 2011; Kumari et al., 2011; Höferl et al., 2009). Therefore conservation of traditional knowledge systems is considered necessary so that a fund of disciplined knowledge savvy system be created and a continuum of furtherance of preservation and strengthen this knowledge system. ACKNOWLEDGEMENT One of the authors (B K Singh) is thankfully acknowledged. Council of Scientific and Industrial Research (CSIR), New Delhi and University Grants Commission (UGC) New Delhi, India for financial assistance.
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Höferl M, Buchbauer G, Jirovetz L, Schmidt E, Stoyanova A, Denkova Z, Slavchev A & Geissler M, (2009), Correlation of Antimicrobial Activities of Various Essential Oils and Their Main Aromatic Volatile Constituents, Journal of Essential Oil Research, 21, Issue 5.
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Ueberall, F (2005), (Free full text). Journal of carcinogenesis 4:15. Joseph B, Ajisha A.U., Kumari S. and Sujatha S. (2011), Effect of Bioactive Compounds and its Pharmaceutical Activities of Sida cordifolia (Linn.), International Journal of Biological & Medical Research, 2(4), 1038–1042. Kala C.P, Rawat, G.S & Uniyal, V.K. (1998), Ecology and conservation of the valley of flowers Nanital Park, Garhwal Himalaya. Wildlife Institute of India, Dehradun. Kumari P, Joshi GC, Tewari LM. (2011), Assessment of Availability of Traditionally used Flora in Curing Jaundice in Central Himalayan Region, Journal of Phytology 3(9), 26−32 . Kumari P, Joshi GC, Tewari LM, Singh BK. (2011), Quantitative assessment and antibacterial activity of Origanum vulgare L. Journal of Phytology 3(12) 15−21. Kumari P, Joshi G C, Tewari LM. (2011), Diversity and status of ethno-medicinal plants of Almora district in Uttarakhand, India, International Journal of Biodiversity and Conservation 3(7), 298−326. Kumari P, Joshi GC, Tewari LM. (2011), Contribution of indigenous anti-diabetic flora in Almora district, Uttarakhand, India, Current Botany 2 (8), 01−07. Mehta PM Eds. Charaka Samhita, Vimana Sthana, Rogabhishagjiteeya Vimana Adhyaya, 8/89-90, Vol. 2, First edition. Jamnagar; Shree Gulabkunverba Ayurvedic Society. 1949 : pp. 916–7. Niranjan Y, Santwani M A, Baghel M S. (2012), Quality of Life Consequences in Diabetic Polyneuropathy, Global J. Res. Med. plants & Indigen. Med., Volume 1 (7), 295–300.
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Sanctuary. Int. J. Sustain. Dev. & World Ecol. 5, 194−203. Samant S.S., Dhar, U. & palni, L.M.S. (1998b), Medicinal plants of Indian Himalaya: Diversity Distribution Potential Values. Gyanodaya Prakashan, Nanital. Saxena C, Sharma DK, Singh RV (1993), Phosphorous Sulfur Silicon 85 : 9. Silva Rl., Melo, GB., Melo, VA., Antoniolli, AR.,Michellone, PR., Zucoloto, S., Picinato, MA., Franco, CF (2006), "Effect of the aqueous extract of Sida cordifolia on liver regeneration after partial hepatectomy." .Acta cirurgica brasileira / Sociedade Brasileira para Desenvolvimento Pesquisa em Cirurgia 21 1: 37–9.
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Silva N C C, Barbosa L, Seito L N & Fernandes A J (2011), Antimicrobial activity and phytochemical analysis of crude extracts and essential oils from medicinal plants, Natural Product Research, DOI: 10.1080/14786419.2011.564582. Srivastava J, Lambert J, Vietmeyer N. (1996), Medicinal Plants: An expanding role in development, World Bank Technical Paper No. 320. Umar M I, Javeed A, Ashraf M, Riaz A, Mukhtar M M, Afzal S & Altaf R (2011), Polarity Based Solvents Extraction of Opuntia Dillenii and Zingiber Officinale for In-vitro Antimicrobial Activities, International Journal of Food Properties, DOI: 10.1080/10942912.2010.517886.
Conflict of Interest: None Declared
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Original Research Article PHARMACOGNOSTIC EVALUATION OF STEPHANIA JAPONICA (Thumb.) Miers â&#x20AC;&#x201C; A PLANT SOURCE FOR THE DRUG PATHA Savitha H1*, Sanjaya K S2, Brijesh K3, Harivenkatesh K R4, Jyothi T5 1
P G Scholar, Post Graduate Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa, Karnataka, India. 2 Principal, ALN Rao Memorial Ayurvedic Medical College, Koppa, Karnataka, India 3, 4 Lecturer, ALN Rao Memorial Ayurvedic Medical College, Koppa, Karnataka, India 5 Research assistant, ALN Rao Memorial Ayurvedic Medical College, Koppa, Karnataka, India *Corresponding Author: e-mail: savithadr08@gmail.com Received: 19/06/2012; Revised: 20/07/2012; Accepted: 30/07/2012
ABSTRACT Stephania japonica (Thumb.) Miers (Syn. Stephania hernandifolia Walp.) belonging to family Menispermaceae is a slender twining shrub growing wild in the forests of Western Ghats. Traditionally, whole plant, root, stem, leaf & flowers are used medicinally by the folk medicinal practitioners of Bangladesh in various diseases like fever, diarrhoea, jaundice, urinary problems etc. Scientifically, though the plant has gained its importance recently, there is a need for the pharmacognostic evaluation. Hence, the study was undertaken to fulfill macroscopic and microscopic aspect of root, stem & leaf of Stephania japonica which includes microscopic characters of root powder. Study reveals, root with more starch grains, bigger vascular bundles than that of the stem, patches of stone cells at places in cortex region. But stone cells are absent in stem. Pith of stem is bigger than that of the root. In leaf, two bands of sclerenchymatous cells, which are wide, angular, having thickened lignified walls, are seen on the upper and lower side of the vascular bundle. KEYWORDS: Stephania japonica, Macroscopy, Microscopy, powder microscopy.
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INTRODUCTION: Patha is an important Ayurvedic drug used in several formulations & diseases. Cissampelos pareira Linn., Cyclea peltata (Lam.) J.Hooker & Thoms., and Stephania japonica (Thumb.) Miers are the source plants for Patha in different regions among which Cissampelos pareira Linn., is the accepted source (API, 2001). But Cyclea peltata (Lam.) J.Hooker & Thoms., is used as Patha in Kerala (P.K Warrier et.al., 2006). The tubers of S. japonica are also commonly used and adulterated for C. peltata in Kerala. They are available in plenty in the market. The properties of these roots are more or less the same as that of Patha. Since the tubers of both these plants are almost similar in shape and size it is very difficult to distinguish between these two (P.K Warrier et.al., 2006). The roots of C. pareira are sometimes found adulterated with the roots of Stephania glabra (Y.K Sarin,1996), a North Indian species. Hence we can infer that, S. glabra is used as an adulterant of Patha in North India while its counterpart S. japonica is used as an adulterant of Patha in South India. In spite of the differences in the market samples, S. japonica is considered as a potential alternate source for Patha (K.C Chunekar, 2010; K.K Hullatti, 2011) which is distributed in the forests of the Western Ghats, commonly from Coorg to Tinnevaly, upto 6000 ft. (J.S Gamble, 2008). There have been many studies conducted regarding C. pareira and C. peltata while S. japonica is least explored pharmcognostically & hence it was thought worth to study in detail the plant which would be helpful for correct identification of the drug in the market samples. This paper highlights macroscopic & microscopic characteristics of leaves, stem and root including powder microscopy of root. Stephania japonica (Thumb.) Miers (Syn. Stephania hernandifolia Walp.) is a slender twining shrub. Leaves peltate, thinly papyraceous, glabrous above, paler or glaucous below, with the nerves below floccosepuberulous, ovate, rounded at base, acute at
apex or rarely obtuse, margin entire, 4–15 cm long, and 4–12.5 cm broad. Inflorescence axillary, compound umbelifer cymes. Fruit drupe, 6 mm long, 4 mm broad. (J.S Gamble, 2008; K.R Kirtikar & B.D Basu, 2008). S. japonica is a folklore medicine administered by the traditional medicinal practitioners of Bangladesh (known as Kavirajas) for the treatment of various ailments. The whole plant is used in the treatment of dysentry, fever, diabetes, sexual weakness, gynaecological problems & leprosy. Leaves are used to cure fever, diarrhoea in children, skin diseases, whitish discharge & burning micturation. Stem is used in arthritis, joint displacement, bone fracture, fever & leucorrhea. Flowers are used as blood purifier & in problems related to ovary. Leaves and roots (in combination) are used to treat fever, diarrhoea & urinary problems. (Rownak Jahan, et.al., 2010). Phyto-chemically, S. japonica contains Bisbenzylisoquinoline type of alkaloids and its methanolic root extracts has been proven for its moderate diuretic activity (K.K Hullatti, 2011). MATERIALS AND METHODS Collection of drug: Fresh plant specimen for the proposed study were collected from a coffee estate at Kachekal, Koppa (Tq.), Chikkamagalur (Dt.), Karnataka state, India. The plant material was identified, authenticated by Prof. Radhakrishna Rao, botanist, A.L.N Rao Memorial Ayurvedic Medical College, Koppa using various floras. (Balakrishna Gowda, 2004; J.S Gamble, 2008) and voucher specimen was placed in the department for future reference (Voucher Ref no.PGDG/VHM - 101). The matured roots were separated from aerial parts, cut into small pieces & dried in shade; coarsely powdered drug was used for powder microscopy. Macroscopic & microscopic characters of root, stem & leaf were studied using fresh specimen.
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GJRMI, Volume 1, Issue 8, August 2012, 364â&#x20AC;&#x201C;371 Fig. 1 Stephania japonica- Plant and its parts
1.1. Plant, 1.2 and 1.4 Flowers, 1.3. Stem.
Fig 2: Stephania japonica- Plant parts with their measurements
2.1 Leaf, 2.2 Stem, 2.3 Rhizome, 2.4 Root, 2.5 Root powder, 2.6 Herbarium, 2.7 Cross section of root
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GJRMI, Volume 1, Issue 8, August 2012, 364â&#x20AC;&#x201C;371 Fig. 3: Stephania japonica - T.S of Root Cork Cortex Sclerenchymatous bands Tracheids Vascular bundles
Pith
Fig. 4: Stephania japonica - Root characters
4.1 Cork, cortex and stone cells, 4.2 Tracheids, 4.3 Pith, 4.4 Vascular bundles, 4.5 Medullary rays, 4.6 Sclerenchymatous band, 4.7 Simple and compound starch grains with hilum
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GJRMI, Volume 1, Issue 8, August 2012, 364â&#x20AC;&#x201C;371 Fig 5: Stephania japonica - Root powder
5.1 Cork in surface view, 5.2 Simple and compound starch grains with hilum, 5.3 Sclereids, 5.4 Fibers.
Fig. 6: Stephania japonica - T.S of Stem
Fig.7: Stephania japonica - Stem characters
7.1 Epidermis and cortex, 7.2 Sclerenchymatous band, 7.3 Tracheids, 7.4 & 7.6 Vascular bundles, 7.5 Pith with starch grains
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GJRMI, Volume 1, Issue 8, August 2012, 364–371 Fig .8: Stephania japonica - T.S of Leaf midrib Upper epidermis Palisade parenchyma Spongy parenchyma Vascular bundle
Sclerenchymatous band
Lower epidermis
Organoleptic evaluation:
RESULTS AND DISCUSSION:
The color, odor and taste of root powder were recorded separately.
Result and discussion on the root: Macroscopic characters:
Microscopic evaluation:
Roots are cylindrical in shape, often tortuous; cut pieces 30 cm in length, 0.5–1 cm breadth. Externally dark brown and internally light brown in color. Surface is rough and at places rugged due to transverse wrinkles (Fig. 2.4). Fracture short and splintery. Odor is characteristic, bitter in taste. Texture fibrous.
Surface preparation of leaf was done by placing wet leaf on a glass slide & tissues were scrapped off with the sharp edge of razor blade with utmost care. Water was slowly & continuously added & scrapping was done till transparent & colorless epidermis was exposed. Free hand transverse sections of root, stem & leaves were used for microscopic evaluation. The powder microscopy of the root was carried out. Sections & powder diagnostic characters were drawn with camera lucida. The photomicrographs were taken by Carl Zeiss binocular microscope. (Khandelwal K.R, 2008; Jyothi T, et.al., 2012) Histochemical evaluation: Histochemical tests were done for lignified tissue, starch grains and crystals. (Khandelwal K.R, 2008)
Microscopic characters: Transverse section of the root showed (Fig.3), 2–3 layers of thin walled rectangular cork cells, followed by cortex of 6–10 layered oval shaped parenchymatous cells. Stone cells were scattered in the cortex (Fig. 4.1). At the end of cortex region, 3–5 layers of half moon shaped sclerenchymatous bands were seen which were attached to each other by tracheids forming a circle (Fig. 4.2 and 4.6). Below the cortex, stelar region consists of xylem, xylem fibers, phloem, phloem fibers, medullary rays and pith. Biseriate to mutiseriate medullary rays (Fig. 4.5) appear to be very wide at a number of places due to addition of delignified xylem parenchymatous cells. Around 8–10 vascular bundles (Fig. 4.4) were arranged radially in circular manner. Plenty of simple
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and compound starch grains with hilum (Fig. 4.7) were spread throughout the section. Powder microscopy of root: Powder microscopy showed fragments of cork, simple and compound starch grains with hilum, stone cells, fibers and sclereids. (Fig. 5) Result and discussion on the stem: Macroscopic characters: Stems are cylindrical in shape, thin, cut pieces 30 cm in length and 0.2–0.6 cm in breadth. Externally greenish when it is young and it turns brown on maturity, but some of the young stem shows purplish color and internally creamish yellow in color (Fig. 2.2). Surface is smooth with longitudinal striations, fracture short and splintery. Odor is characteristic, bitter in taste, texture fibrous. Microscopic characters: Transverse section of the stem showed, (Fig. 6) single layer of thin walled round shaped epidermis, followed by cortex of 6–8 layered oval shaped parenchymatous cells (Fig. 7.1). At the end of cortex region, 3–5 layers of half moon shaped sclerenchymatous bands (Fig. 7.2) were seen which were attached to each other by tracheids (Fig. 7.3) forming a circle. Below the cortex, stelar region consists of xylem, xylem fibers, phloem, phloem fibers and pith. Around 8–10 vascular bundles were arranged radially in circular manner. Size of the vascular bundle is smaller than that of the root (Fig. 7.4 and 7.6). Pith (Fig. 7.5) is bigger than that of the root. In pith tannin can be observed at places. Simple and compound starch grains with and without hilum were spread throughout the section. Result and discussion on the leaf: Macroscopic characters: Leaves are simple, alternate, and ovate in shape, size is 4–15 cm long and 4–12.5 cm broad, apex is acute or rarely obtuse, rounded at base, margin entire, reticulate venation, glabrous above and paler or glaucous below (Fig 1 and 2.1) Odour characteristic, texture thinly papyraceous.
Microscopic characters: Cross section of midrib showed upper epidermis with thin walled cells, rectangular in shape without cuticle layer. Lower epidermis was with thin walled bulliform cells. Collenchymatous cells were absent. Two bands of sclerenchymatous cells, which were wide, angular, having thickened lignified walls, were seen on the upper and lower side of the vascular bundle. Single vascular bundle is located in the centre of the midrib which is collateral. Rest of the midrib is filled with thin walled parenchymatous cells. The lamina was with upper epidermis, lower epidermis and mesophyll tissue. Upper epidermis was rectangular and lower epidermis was bulliform. Mesophyll tissue has two types of parenchymatous cells; single layered elongated, compact palisade parenchyma in the upper portion and 5–7 layered, loosely arranged spongy parenchyma with intercellular spaces in the lower portion (Fig 8). CONCLUSION Identification and authentication of raw material is the major problem in the herbal Pharmaceutical Industry. S. japonica though is itself a potential medicinal drug and a source for Patha, is usually considered & found to be used as an adulterant to the roots of Cyclea peltata in South India while its counterpart S. glabra is found to be used as an adulterant to the roots of Cissampelos pariera in North India. So, here an attempt had been made for correct identification of Stephania japonica (Thumb.) Miers., through its macroscopic and microscopic characteristics, which could serve as a potential reference material to the Pharmacognosists to compare it with the genuine plant material of Patha in the herbal pharmaceutical industry. In the flow of time, S. japonica may emerge as a potential plant drug in treatment, sometimes in future, when the present study would play a vital part to help researchers in proper identity of the genuine sample. Further comparative studies on C. pariera and S. japonica would be a Milestone in the standardization of Patha.
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REFERENCES Anonymous (2001), The Ayurvedic Pharmacopoeia of India, Govt. of India, Part 1, Vol 1, p.no 122,123. Gamble J.S (2008), Bishen Singh Mahendra pal Singh, Flora of the presidency of Madras, published under the authority of the secretary of state for Indiain council, Dehradun, Vol 1, p.no 29.
K.K Hullatti, M.S Sharada and I.J Kuppasth, Studies on diuretic activity of three plants from Menispermaceae family, Der Pharmacia Sinica, 2011, Vol 2, Issue 1, p.no 129–134.
Khandelwal K.R (Oct 2008), Practical Pharmacognosy, Nirali prakashan, Pune, p.no 10–19.
Gowda Balakrishna (2004), Vanaspathi Kosha, Plant wealth of Sringeri, Karnataka, Kalpatharu Research Academy, Bangalore, 1st Edition, p.no 127, 128.
Kirtikar K.R & Basu B.D (2008), Indian Medicinal Plants, International book distributers, Dehra-Dun, 2nd Edition, Vol I, p.no 92, 93.
Jyothi
T, Niranjan Y, Harisha C.R, Pharmacognostic evaluation of an aromatic plant of basmati flavor: Pandanus amaryllifolius roxb., Global J. Res. Med. Plants & Indigen. Med., Vol 1, Issue 4, p.no 133–139.
P.K Warrier, V.P.K Nambier, C Ramankutty (2006), Indian Medicinal Plants, a compendium of 500 species, Orient Longman Private Limited, Arya Vaidya Sala, Kottakkal, Vol 2, p.no 276–280.
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Chunekar (2010), Bhavaprakasha Nighantu, Chaukhamba Bharati Academy, Varanasi, p.no 382.
Source of Support: Nil
Rownak Jahan, Mst. Afsana Khatun, Nusratun Nahar, Farhana Israt Jahan, Anita Rani Chowdhury, Aynun Nahar, Syeda Seraj, Mostafi Jumrut Mahal, Zubaida Khatun, Mohammed Rahmatullah (2010), Use of Menispermaceae family plants in folk medicines of Bangladesh, Adv. In Nat. Appl. Sci., Vol 4, Issue 1, p.no 1– 9.
Conflict of Interest: None Declared
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Original Research Article PHYTOCHEMICAL AND ANTIFUNGAL STUDIES ON ROOT OF IPOMOEA SEPIARIA KOENIG EX. ROXB. Majumder Sayani1*, Ashok B K2 Nishteswar K3 1
M.Pharm (Ayu.) Scholar, Department of Dravyaguna Research Assistant, Pharmacology Laboratory 3 Professor and Head, Department of Dravyaguna, Institute for Post Graduate Teaching & Research in Ayurveda, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat, India 2
*
Corresponding author: Email: sayani1988sayani@gmail.com; Mob: +917878582855
Received: 28/06/2012; Revised: 25/07/2012; Accepted: 27/07/2012
ABSTRACT Plants have been one of the major sources of medicines since the beginning of human civilization. Ipomoea sepiaria Koenig Ex. Roxb. is a medicinal plant considered as a source plant of the classical Ayurvedic drug Lakshmana. The root of this plant is known for the treatment of leucorrhoea and infertility. This study was undertaken to know antifungal effect of methanolic extract of root against the fungal strain Candida albicans which is responsible for leucorrhoea. The inhibitory effect was assessed by agar well diffusion method. The phytochemical profile was also carried out through primary phyto-chemical screening and HPTLC analysis. The result shows that the root of Ipomoea sepiaria is having phyto-constituents like carbohydrates, alkaloids, glycosides, flavonoids, phenolic compounds, tannin and saponin. The methanolic extract of root was found to possess significant antifungal activity and this supports the use of root in the treatment of leucorrhoea.
KEY WORDS: Lakshmana, Ipomoea sepiaria, Candida albicans, leucorrhoea, antifungal activity.
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INTRODUCTION Ipomoea sepiaria Koening ex. Roxb., is a source of the classical Ayurvedic medicinal plant Lakshmana (Kamat SD, 2006). It is a glabrous or occasionally pubescent or hirsute, slender twinning climber with a slightly thickened or tuberous perennial root (Aiyer KN et. al., 1957). The root system consists of a fairly long, somewhat thickened taproot and several slightly thinner or slender branches, arising from its base with very few wiry rootlets (Duthie JF, 1994). Leaves are simple alternate, entire, blotched with brownish patches towards the middle (Sivarajan VV, 2004). Flowers are delicate purple or white with a purple eye, along with short to long peduncles and short pedicels (Haunes HH, 1988) In folklore practice this herb is known as a good antidote to arsenic poisoning, uterine tonic, aphrodisiac and anti-ulcer drug (Kirtikar KR and Basu BD, 1960). It is also used as diuretic, deobstruent and tonic. It is reported to be used in burning sensation, strangury, general debility and sterility in women (Prajapati ND et. al., 2003).The literatures further specify the use of root in case of diabetes (Jain SK, 1991) and constipation (Venkataswamy R et. al., 2010). In one of the Ayurvedic texts Basavarajeeyam (18th Century) it is mentioned that the root powder in the dose of 1 teaspoon is administered with rice water for leucorrhoea (Nishteswar K, 2003). Leucorrhoea is a common condition in women of reproductive age. It refers to a whitish secretion from vagina which acts as a moisturizer and a protective coating over the vaginal wall. However, if vagina is infected by microorganisms, the normal vaginal florae become stickier, odorous and the whitish secretion becomes yellowish or greenish depending on the microbes that cause the infection. These symptoms are collectively termed as pathological leucorrhoea. These usually present with other symptoms like vaginal or vulval pruritus, dyspareunia or pelvic pain. Candida albicans, an yeast like fungal strain is generally responsible for this
pathological leucorrhoea. Moreover, sometimes Gardnerella vaginilis and Tricomonas vaginalis a bacterial and a protozoal strain respectively are also known to produce leucorrhoea (Rentz AM, 1998). As I. sepiaria plant is used in the treatment of leucorrhoea, it should possess some antimicrobial property. Hence in the present study antifungal activity of the root has been evaluated, also preliminary phytochemical analysis was carried out as a standardization perspective. MATERIALS AND METHODS Collection of plants: The whole plant of Ipomoea sepiaria (photo slides 1 & 2) was collected from the campus of Gujarat Ayurved University, Jamnagar, Gujarat in month of November. The collected samples were authenticated by the Botanical Survey of India, Office of the Scientist-‘F’, Central National Herbarium, Botanic Garden, Howrah, West Bengal, specimen No.- CNH/104/2011/Tech.II/581. From the plants the roots were separated and washed properly with water and shade dried and made into a fine powder using a mechanical grinder and was sieved with mesh no. 40, and stored in an air-tight container. Phytochemical screening: The root powder was divided into two parts- one extracted with methanol by Soxhlet apparatus and another was extracted with water by maceration process. These extracts were subjected for the preliminary phytochemical screening for functional groups by following standard methods (Khandelwal KR, 2004). HPTLC study: Sample preparation: For the HPTLC studies following samples were prepared by maceration process. The samples were titled as Track-1, Track-2 & Track-3. Track-1- Methanolic extract Track-2- Chloroform extract Track-3- Petrolium ether extract
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Mobile phase: Toluene: Ethyl acetate: Glacial acetic acid (6.5:3.5:0.2) v/v. Detection: Spray with Vanillin-sulphuric acid
Chamber saturation: 30 min. Development time: 30 min. Development distance: 7 cm. Scanner: Camag Scanner III. Detection: Deuterium lamp, Tungsten lamp Data System: Win cats software The developed plate was scanned to obtain densitogram in visible range from 600 nm to 800 nm with 100 nm interval.
Chromatographic conditions Application mode: Camag Linomat V Development Chamber: Camag Twin trough Chamber. Plates: Precoated Silica Gel GF254 Plates. Photo slides 1 & 2 Plant profile and part used
ANTIFUNGAL ACTIVITY EVALUATION Determination of the effects: The antifungal activity of methanolic extract of I. sepiaria root against C. albicans was determined by using Microdilution Broth Assay (Andrews JM, 2001; Thongson C et. al., 2004) and Well Diffusion Method. Nystatin (a synthetic antifungal drug) served as positive control. Culturing of Candida albicans: A pure culture of C. albicans was purchased from Microbial Type Culture Center (MTCC) of Chandigarh, India (MTCC No.227) and sub culture was made using nutrient agar plate and stored in 4°C for future use.
Macro-dilution technique for inhibitory concentration determination:
minimum (MIC)
The varying concentration of the extracts 7, 8, 25, 50, 75, 100 mg/ml were prepared and 1 ml of each concentration was added to each 9 ml of broth containing 0.1 ml of standardized test organism of C. albicans. The tubes were then incubated at 37oC for 24 h. After incubation period was over, the minimum inhibitory concentration was determined at the concentration in which the fungal growth was inhibited and which was clearly visible at naked eye observation (Atata RF et. al., 2003). Well diffusion method for determination of zone of Inhibition: Antifungal potential of the plant extract was tested by Agar well diffusion method (Schillinger U and Lucke F, 1989). In brief, for
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fungi Malt Extract Agar plates were used. To prepare nutrient agar plates, at first 100 ml of distilled water with 2.8 g of Nutrient Agar (Himedia) was taken in a 250 ml flask. Then a cotton plug was applied to the flask and Petri plates were wrapped according to requirement. Heat was given to dissolve the constituents in the flask and kept in autoclave at 15 lbs pressure for 20 min. for sterilization. Petri plates were removed and poured the medium quickly under aseptic condition. To prepare the Malt Extract Agar plates 2 g of Malt Extract Powder (Himedia) and 2 g of Agar Agar were taken in 100 ml distilled water in a 250 ml conical flask, dissolved properly and then sterilized and Malt Agar plates were prepared. To check the antifungal potentiality, 100 µl of fungal cultures were sprayed on their respective culture medium using a glass rod spreader. Then wells were prepared by cork borer and 50 µl of root extracts were added in each well separately. Then the plates were incubated in Incubator at 37°C. The plates were observed after 72 h of incubation. Then the
zone of inhibitions by plant extracts were measured and tabulated. RESULTS The phytochemical screening of root samples for various functional groups revealed the presence of carbohydrates, alkaloids, glycosides, triterpenoids, saponins and tannin [Table 1]. In HPTLC analysis, at short UV 254 nm [Fig - 1, Fig – 2, Fig - 3) and after derivatization [Fig - 7, Fig – 8, Fig - 9), different spots were found in all three extracts i.e. methanol extract, chloroform extract and petroleum ether extract [Table-2] which indicates the presence of different phytocomponents [Fig – 10, Fig – 11, Fig – 12, Fig 16]. Presence of one common Rf value (0.01) in all three samples, indicate the presence of one common component in all three extracts. At long UV 366 nm [Fig - 4, Fig – 5, Fig – 6], methanol extract, chloroform extract and petroleum ether extract showed 7, 9 and 5 spots respectively [Table-3] [Fig – 13, Fig – 14, Fig – 15, Fig 17].
Table-1: Preliminary qualitative analysis of methanolic and water extracts of Ipomoea sepiaria root for the presence of various functional groups Material Methanolic extract
Reagent/ Test Kellar kiliani
Functional group Carbohydrates
Fehling’s solution
Carbohydrates
Dragendroff’s reagent Alkaloids
Water extract
Observation Result Reddish brown Present ring Red precipitate Present
Legal’s test Brontrager’s test Alkaline reagent test FeCl3 Ammonia test
Glycosides Glycosides Flavonoids Tannin Phenolic compounds
Orange brown Present precipitate Pink Present Pink Present Colourless Present Green Present Yellow Present
Heat test Biuret test Salkowski test Resin test Froth formation test
Proteins Proteins Steroids & terpenoids Resin Saponin
No coagulation No violet colour Red colour White precipitate Forth formation
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Absent Absent Present Present Present
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GJRMI, Volume 1, Issue 8, August 2012, 372–380 Table 2:- Rf value in Short UV (254 nm) of all three extracts
NO 1 2
SAMPLE ME CE
NO. OF SPOTS 4 8
Rf VALUES 0.01, 0.06, 0.27, 0.77 0.01, 0.07, 0.10, 0.13, 0.26, 0.35, 0.48, 0.77 PE 4 0.01, 0.33, 0.80, 0.91 3 ME-methanolic extract; CE-chloroform extract; PE - petrolium ether extract Table 3:- Rf value in Long UV (366 nm) of all three extracts SL NO 1
SAMPLE ME
NO. OF SPOTS 7
RF VALUE 0.01, 0.08, 0.10, 0.26, 0.40, 0.80, 0.94 CE 9 0.01, 0.07, 0.10, 0.13, 0.24, 2 0.35, 0.43, 0.78, 0.94 PE 5 0.01, 0.30, 0.37, 0.82, 0.91 3 ME-methanolic extract; CE-chloroform extract; PE - petrolium ether extract Table-4: Zone of inhibition of methanolic extracts of Ipomoea sepiaria root and standard Concentration (µg /ml) Methanolic Zone of extract of root inhibition (mm) Nystatin
Fig-1
Fig-2
Fig-3
25 11.93 ± 0.36
50 13.35 ± 0.55
75 14.49 ± 1.19
100 15.06 ± 0.44
21
24
NA
25
Fig-4
Fig-5
Fig-6
Fig-7
Fig-8
Fig-9
Fig-1--- Track 1 (254 nm); Fig-2 ---Track 2 (254 nm); Fig-3 --- Track 3 (254 nm) Fig-4 ---Track 1 (366 nm); Fig-5 ---Track 2 (366 nm) ; Fig-6 --- Track 3 (366 nm) Fig-7 --- Track 1 (After spraying with Vanillin sulfuric acid); Fig-8 --- Track 2 (After spraying with Vanillin sulfuric acid) ; Fig-9 --- Track 3 (After spraying with Vanillin sulfuric acid) Global Journal of Research on Medicinal Plants & Indigenous Medicine
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Fig-10. 254nm peak display Track-11
Fig-13. 366nm peak display Track-1
Fig-11. 254nm peak display Track-2
Fig-14. 366nm peak display Track-2
Fig-16. Multiple Trackss (254 nm)
Fig-12. 254nm peak display Track-3
Fig-15. 366nm peak display Track-3
Fig-17. Multiple le Tracks Trac (366 nm)
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GJRMI, Volume 1, Issue 8, August 2012, 372–380
Fig-18. U.V. Spectral comparison Rf 0.36 of T-1,2,3
Fig-19. U.V. Spectral comparison Rf 0.86 of T-1, 2, 3
Fig-20 U.V. Spectral comparison Rf 0.96 of T-1, 2, 3
Photo slide 3 ---Zone of inhibition of the sample
From the above mentioned spectral comparison [Fig-18, Fig-19, Fig-20] some identical Rf value were found in case of all three samples i.e. 0.36, 0.86 and 0.96, which indicates presence of same phyto-component in all the three extracts. The results of the Microdilution Broth Assay showed that the MIC of methanolic extract of root was found to be 8 µg/ml. The zone of inhibition was calculated in four concentrations i.e. 25, 50, 75, and 100 µg/ml and compared with the standard (Nystatin) [Table 4 & Photo slide -3]. DISCUSSION Phytochemical screening in primary stages was done by performing qualitative tests. The root contains carbohydrates, alkaloids,
glycosides, flavanoids, phenolic compounds, steroids, terpenoids, tannin, saponins and resin. The extract showed positive response with Kellar Kiliani test, which indicates the presence of deoxy sugar and positive response with Fehling test showed the presence of reducing sugars. The organism tested for antifungal activity under initial screening, appeared to be susceptible to methanolic extract of root. While comparing the zone of inhibition with the standard (Nystatin 21 mm at 25 µg/ml conc.) the sample showed 11.93 mm zone of inhibition at 25 mg/ml concentration against C. albicans. This confirms the earlier research by Vijayan Mini et. al., (2010) in which they showed that acetone extract of Ipomoea sepiaria has significant anti-fungal activity
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against Candida glabrata. Thus the presence of one or more phyto-constituents like carbohydrates, alkaloids, glycosides, flavonoids, phenolic compounds, tannin and saponin may be responsible for observed activity profile. However this needs further detailed research. CONCLUSION The methanolic extract of root of Ipomoea sepiaria is having very good antifungal activity and this supports the use of root in the
treatment of leucorrhoea. Further researches are needed to explore the phyto constituents particularly responsible for the observed activity profile. ACKNOWLEDGMENTS We acknowledge Mr. J. N. Pandey, Principal of Bengal Institute of Pharmaceutical Sciences (BIPS), a joint Venture Institute with Government of West Bengal, India for giving the necessary permission to carry out the research work in the Institute.
REFERENCES Aiyer
KN, Namboodiri AN, Kolammal M.(1957). Pharmacognosy of Ayurvedic Drugs, Series-I, Number-3, The central research institute, University of Travancore, Trivandrum. pp. 57–63
Andrews JM (2001). BSAC standardized disc susceptibility testing method, J. Antimicrob, Chemother. 1: 48–57. Atata RF, Sani H (2003). Effect of stem bark extracts of Enantia chloranta on some clinical isolates, Biokemistri. 15: 84–92. Duthie JF (1994). Flora of Upper Gangetic Plain and of the Adjacent Siwalik & Sub-Himalayan Tracts, Vol-II, Bishen Singh Mahendra Pal Singh, Dehradun, pp114. Haunes HH (1988). The Botany of Bihar & Orissa, Part-III-IV, Bishen Singh Mahendra Pal Singh, Dehradun. pp 598.
Khandelwal KR (2004). Practical th Pharmacognosy, 12 edition, Nirali Prakasan, Pune. pp 149–153. Kirtikar KR and Basu BD (1960). Indian medicinal plants, Vol.3 Part-1, International book distributor, Dehradun, pp. 1702–1712. Nishteswar K (2003). Herbs in Basavarajeeyam, Chaukhamba Surbharati Prakasan, Vijayawada (A.P). pp. 86. Prajapati ND., Purohit SS., Sharma AK., Kumar T. (2003). A handbook of Indian Medicinal Plants, A complete Source Book, Agrobios, India. pp. 291–292. Rentz AM (1998). The impact of candidemia on length of hospital stay, outcome, and overall cost of illness, Clinic. Infect. Dis. 27: 781–88.
Jain SK (1991). Dictionary of Indian Folk Medicine and Ethnobotany, Deep Publication, New Delhi. pp108.
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Schillinger U, Lucke F (1989). Antimicrobial activity of Lactobacillus sake isolated from meat. J Appl Environ Microbiol. 55:1901–1906.
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Thongson C., Davidson PM., Mahakarrchanakul W, Weiss J. (2004). Antimicrobial activity of ultrasoundassociated solvent extracted species, Lett. Appl. Microbiol. 39: 401–06. Venkataswamy R., Mubarack HM., Doss A., Ravi TK., Sukumar M. (2010). Ethnobotanical Study of Medicinal plants used by Malasar tribals in Coimbatore District of Tamil Nadu
Source of Support: Nil
(South India), Asian J. Exp. Biol. Sci. 387–392. Vijayan Mini N., Barreto Ida, Dessai Seema, Dhuri Shital, D’ Silva Riva, Rodrigues Astrida. (2010). Antimicrobial activity of ten common herbs, commonly known as ‘Dashapushpam’ from Kerala, India. African Journal of Microbiology Research. 4(22): 2357–62.
Conflict of Interest: None Declared
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