UJPAH Vol. I No. 12 June 2012
Dr. S. Farooq Dr. G.K. Pathak Dr. I.P. Saxena Dr. I.P. Pandey Chief Editor Patron Editor Treasurer Advisory Board
Dr. S. Farooq - Chief Editor, Universities' Journal of Phyto-Chemistry and Ayurvedic Heights Dr. I. P. Saxena - Editor, Universities' Journal of Phyto-Chemistry and Ayurevedic Heights,India Former Principal DAV (PG) College, Dehradun, EX. V. C., H. N. B. Garhwal University, Srinagar Dr. Maya Ram Uniyal - Ex-Director, Ayurved (Govt. of India), Advisor, Aromatic and Medicinal Plants (Govt of Uttarakhand)
Dr. G. K. Pathak - Former H.O.D, Chemistry Deptt, D. A.V. (PG) College, Dehradun Dr. Himmat Singh - Former Sr. Scientist, I.I.P.; Principal A.I.S.T., Dehradun B. B. Raizada - Former Principal, D. B. S. College Dehradun, U.K., India Dr. B. L. Khandelwal - Emeritus Scientist, CSIR, Director, Engg. College, Chattisgarh Dr. Iqbal Ahmed - Reader, Deptt of Agricultural Microbiology, A.M.U. Aligarh Ms. Alka Shiva - President and Managing Director, Centre of Minor Forest Products (COMFORPTS), Dehradun, UK., India Dr. I. P. Pandey - Eminent Scientist, Dehradun, Uttarakhand Dr. Sanjay Naithani - Head, Pulp & Paper Division, FRI, Dehradun, Uttarakhand Dr. Umar Farooq - Associate Professor, Shoolini University, Solan, H.P.
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UJPAH Vol. I No. 12 June 2012 Contents
Editorial 03
Silymarin-Phospholipid Complex: A Drug Delivery System in NDDS 04-13 Devendra Singh Rawat, Bandana K. Thakur, M. Semalty, A. Semalty, M. S. M. Rawat
Mechanism of Poly phenols from Bitter Gourd ,Cinnamon ,Basil and 14-20 B. Berries in Controlling High Blood Sugar Level Anil Kumar Gupta
Phytochemical Evaluation of Ingredients and Role of Bioactive Markers 21-28 in Hepatoprotective Property of Liv 52. Zafar Mehmood and Suman Chhimwal
Identification and Estimation of Bioactive Marker in 29-32 Bacopa monnieri using HPTLC Technique Jyoti Chhimwal and Mayaram Uniyal
Study of Antimicrobial Activities and Optimization of Plant Extract 33-39 Preparation of Mangifera indica; The King of Fruits Chandan prasad, Sudhir K. srivastava , R.P.Mishra Deepanjali Saxena, Omkar Shukla and Digvijay Singh
Evaluation of Toxic Metals in Hepato Protective Herbs Kasni ,Bhringraj , 40-43 Makoi,Kutki and Kalmegh used in Ayurvedic Medicines Arunesh Kumar Dixit and Shalini Pandey
Chemical Constituents from bark of Euonymus tingen (Celastraceae) 44-46 S.C. Sati, Maneesha, D. Sati, Amita Sharma and I. P. Pandey
An in Vitro Antimicrobial Study of Punica Granatum Peel Against Diarrhoeal Diseases 47-51 Dinesh Kumar, R.K.Singh
Evaluation of Antimicrobial Activity of Some Medicinal Plants 52-57 Extract on Pathogenic Fungal Strains M.K.Tripathi and I P Pandey
Phytochemical and Pharmacological Attributes of Phaseolus trilobus 58-63 (A Major Herbal Ingredient of Pharma Products) Navpreet Kaur, Rashmi and Y.C. Tripathi
About Taxus wallichiana Zucc (Talispatra) on Front Cover 64 (Potential Anticancer Herbal Drug) Forthcoming Events 65 Instructions to Contributors 66-67
UJPAH Vol. I No. 12 June 2012
Editorial
Indian Herbal Pharmaceuticals Industry and R & D
India is a powerful force in the international pharmaceuticals market having one lakh formulations of around 1,100 basic modern drugs in the market in which share of Ayurvedic or Ayush Pharma in international market is nil where as the research and development efforts of the Indian pharmaceuticals is also almost nil though the modern pharma data also confirms that out of 10000 molecules synthesized only 20 reach the pre-clinical stage and 10 to the clinical trial stage and ultimately only one gets the approval for marketing, therefore the Scientists have synthesized 1/10th of 1% of the potential medicine that could be made though the number of undiscovered drugs is infinite. It is also estimated that the whole process takes about 15 years and the investment is one billion Dollars or 5.6 Kharab Rupees. Therefore the graph of research which was rising in 90s has dipped down since 2005. Most modern pharma MNC,s are not interested in developing molecules and have shut down for molecule research. Where as R&D on Indian Herbs stood to almost nil. States have Drug Policies without a Reaearch and Development Policy for the herbs found in their states which lead to an engineered destruction. Therefore to promote our indigenous system of medicine, it is our moral duty to explore all possibilities to involve young Indian scientists, the real strength to pick up research on Indian herbs by way of invitro studies and scientific documentation to bring acceptability of Indian herbs in the national and international markets. UJPAH's endeavour is also to make more and more involvement of young scientists to take up this task. This will generate employability, and consumption which will lead change in traditional cultivation of just grains which we grow more than its demand this will help suffering farmers and give better paying crop opportunity to cultivate herbs which will lead to manufacturing of affordable herbal medicines for our indigenous health care system. Scientifically well documented Indian herbs will increase our foreign exchange, name, fame and bring prosperity to our country.
I, conclude by thanking Dr. Rajendra Dobhal, Director General-UCOST, Tasmia & The Indian Cambridge School for their monetary help in bringing out this issue and to all the Board members of UJPAH, contributors of research papers, participants, eminent scientists and not the least, all staff members of UCOST for associating themselves for the release of UJPAH, Vol. I, No.-12, 2012 in UCOST auditorium. Dr S. Farooq Chief Editor
03 UJPAH Vol. I No. 12 June 2012
Silymarin-Phospholipid Complex: A Drug Delivery System in NDDS
Devendra Singh Rawat1 , Bandana K. Thakur1 , M. Semalty2 , A. Semalty2 , M. S. M. Rawat*1 1Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal) 246 174, UK., India 2Department of Pharmaceutical Sciences, HNB Garhwal University, Srinagar (Garhwal) 246 174, Uttarakhand, India *E mail: [email protected]
Abstract - The fruits of the milk thistle plant (S. antioxidant and protein restoring activities [11]. It marianum) contain flavanoids known for is a powerful polyphenolic antioxidant with wide hepatoprotective effect. Silymarin is a strong range of biological effects [12-15] that cumulatively hepatoprotective bioactive and has been shown make its use as an attractive treatment for liver to have positive effects in treating liver diseases of dysfunction. various kinds, hepatitis, cirrhosis, fatty infiltration Because the oxidative/nitrosative stress is now of the liver (chemical and alcohol induced) and known to play in pathophysiology, the inflammation of the bile duct. The poor water flavonolignans in silymarin with wide range of its solubility of the silymarin led to the formation of potential application have received increasing more soluble complex with the phospholipid attention in treating or preventing of various which might improve solubility, dissolution and forms of cancer [16-18], angiogenicity [19], bioavailability of the active constituents. Silymarin diabetes [20], inhibitory effects on multi-drug (as a drug) phospholipid solid system was resistance proteins [21] and potentiating effects prepared by chemical refluxing method. The solid- with chemotherapeutic agents [22]. state study of the complex was done. The Many of the flavonoids are poorly absorbed by the formation of the complex with phospholipid in the solid-state was confirmed by infrared body [23]. The poor absorption of flavonoids is spectroscopy, differential scanning calorimetry, X- mainly due to the multiple ring molecules too large ray diffractometry and scanning electron to be absorbed by simple diffusion, and due to microscopy (SEM). poor miscibility with oils and other lipids, severely Keywords - Silymarin, Phospholipid, FTIR, limiting their ability to pass across the lipid-rich Differential Scanning Calorimetry, X-ray Powder outer membranes of the enterocytes of the small Diffractometry, Scanning Electron Microscopy. intestine. Silymarin is poorly soluble in water and its Introduction oral absorption is only about 23-47 %, leading to its The bioactive silymarin, from the milk thistle low bioavailability. The poor solubility and (Silybum marianum) have received much attention bioavailability of silymarin led to the development in clinical and basic research assessing effects and mechanisms [1]. Silymarin a flavonolignans, as of enhanced formulation. The phospholipid silybin, isosilybin, silydianin, and silychristin, with (phosphatidylcholine) is an integral part of the cell silybin the major active component [2-4] have membrane exists in zwitterionic form and its demonstrated hepatoprotective [5-10] action in amphiphilic nature has received much attention the organisms due to its positive effect on owing to their good biocompatibility and metabolism and physiology of liver cells, biodegradability [24]. Its emulsifying action influencing their regenerative capacity by the provides an effective medium for increased
04 UJPAH Vol. I No. 12 June 2012 absorption of the bioactive which dramatically each individual sample in a covered sample pan enhances bioavailability and delivers faster and under nitrogen gas flow. The investigations were improved absorption in the gastrointestinal tract. carried out over the temperature range 25-300 ºC The present work deals with the study of with a heating rate of 10 ºC min-1. preparation and physicochemical evaluation of X-ray powder diffraction (X-RPD) - The crystalline silymarin-phospholipid complex by means of IR state of rutin in the different samples was spectroscopy, Differential Scanning Calorimetry, evaluated with X-ray powder diffraction. X-ray Powder Diffractometry and Scanning Diffraction patterns were obtained on a Bruker Electron Microscopy, in order to modify the Axs-D8 Discover Powder X-ray diffractometer physicochemical nature of silymarin for improved (Germany). The X-ray generator was operated at solubility and bioavailability. 40 KV tube voltages and 40 mA of tube current, Materials and methods - Silymarin was purchased using the Ká lines of copper as the radiation from Sigma Aldrich, Mumbai (In dia), with 97% source. The scanning angle ranged from 5 to 50 o purity. Soya phosphatidylcholine (LIPOID S-80) of 2è in step scan mode (step width 1o /min). was obtained as a gift sample from LIPOID, Scanning electron microscopy (SEM) - SEM Germany. All other chemical reagents were of imaging of the complex was performed using a analytical grade. Scanning Electron Microscope (JEOL JSM 5600). Preparation (Silymarin-Phospholipid Complex) The silymarin-phospholipid complex (Sil-PC) was Results and discussion prepared by refluxing silymarin and I.R. studies - The possible interaction between phosphatidylcholine. The equimolar silymarin and phosphatidylcholine (PC) in the concentration of phospholipid and silymarin were phospholipid complex was studied by IR placed in 100 ml round bottom flask in 20 ml of spectroscopy and presented in Fig 1. In IR spectra dichloromethane. The reaction proceeded by the hydroxyl stretching frequency at 3435 cm-1, C- refluxing the reaction mixture in a magnetic stirrer H stretching band of long fatty acid chain at 2918 at 45-500 C for 8-10 hr. Thereafter the volume of cm-1 and 2850 cm-1 , carbonyl stretching band at resulting solution concentrated to 2-3 ml and then 1738 cm-1 in the fatty acid ester, P=O stretching the sufficient amount of n-hexane was added to band at 1236 cm-1 and P-O-C stretching band at get precipitation. The precipitate filtered, washed, 1091 cm-1 and N+(CH3)3 stretching at 970 cm-1 in dried under vacuum and stored in an air tight phosphatidylcholine. In case of silymarin the O-H stretching at 3447 cm- container until further use as the complex. Infrared Spectroscopy - The IR spectra were 1, C=O stretching at 1638 cm-1, C=C vibrations in recorded on a Perkin Elmer FT-IR, RX-1 the benzene ring at 1508 cm-1, O-H bending at 1272 spectrophotometer in KBr pellets. cm-1, C-C stretching in the ring at 824.06 cm-1 , 792.35 cm-1. Differential scanning calorimetry (DSC) - The FTIR of the complex showed the significant Thermogrames of silymarin, phosphatidylcholine changes in the spectrum, and the absorption and the Sil-PC complex were recorded on peaks of hydroxyl (O-H) group of the silymarin differential scanning calorimeter (2910 Modulated have remarkable broadning, whereas, the P=O DSC V4.4E, TA Instruments, US). The thermal absorption band of the phosphatidylcholine also behavior was studied by heating 3.0 ± 0.2 mg of 05 UJPAH Vol. I No. 12 June 2012
remarkably broadened. The spectrum of the of both the constituents. Therefore, the physical mixture is quite different from the spectroscopic changes, showed that, the spectrum of the complex and showed same broadening of hydroxyl group frequency of vibrational frequencies as that of the individual silymarin accounts for the interaction to polar end components and seemed to be only a summation of the phospholipid.
(a)
6 5 . 6 6 0
5 5
5 0
4 5
4 0 % T 8 2 4 .1 1 3 5
3 0
1 0 3 2 .4 3
2 5 3 4 4 7 .9 5 1 5 0 8 .5 9 1 0 8 3 .5 9
2 0
1 1 6 4 .6 3 S i ly m a r in 1 2 7 2 .4 4 1 4 . 4 4 0 0 0 . 0 3 6 0 0 3 2 0 0 2 8 0 0 2 4 0 0 2 0 0 0 1 8 0 0 1 6 0 0 1 4 0 0 1 2 0 0 1 0 0 0 8 0 0 6 0 0 4 5 0 . 0 c m - 1 (b)
3 0 . 0
2 8
2 6
2 4
2 2
2 0
1 8
1 6
% T 1 4
1 2
8 2 6 .2 7 1 0
8
6
4 1 0 8 6 .0 5
2 1 4 6 7 .6 2 Sil-Com plex -1 1 6 3 8 .3 3 2 9 1 8 .3 3 1 7 4 0 .7 7 0 . 0 4 0 0 0 . 0 3 6 0 0 3 2 0 0 2 8 0 0 2 4 0 0 2 0 0 0 1 8 0 0 1 6 0 0 1 4 0 0 1 2 0 0 1 0 0 0 8 0 0 6 0 0 4 5 0 . 0 c m - 1 (c) 06 UJPAH Vol. I No. 12 June 2012
(d) Fig.1- IR spectra: (a) Phospholipid, (b) Silymarin, (c) Silymarin- phospholipid complex, (d) Physical mixture
DSC studies - Differential scanning calorimetry The non-polar hydrocarbon tail of phospholipids (DSC) is a fast and reliable method to detect drug- may be melted during this phase, yielding a sharp excipient compatibility to provide maximum peak. This melting might have occurred in two information regarding the possible interactions. In phases which subsequently gave another peak DSC, an interaction is concluded by elimination of (107.90°C) which is relatively less sharp. endothermic peaks, appearance of new peaks, Silymarin showed two sharp endothermic peaks at change in peak shape and its onset, peak 86.58 and 149.35° C. On the other hand Sil-PC temperature/melting point and relative peak area complex (Fig. 2c) showed a single sharp peak at or enthalpy. The thermal curves of pure 60.17° C, which is different from the peaks of the components (silymarin), phospholipid and of the individual components of the complex. Moreover drug-phospholipid system are shown in Fig 2. the onset temperature is low in the prepared Phospholipid showed two major peaks at 83.21° C complex. It is evident that the original peaks of and 107.90° C and a small peak at 64.45° C. The first silymarin and phospholipids disappear from the one peak of phospholipids is mild peak (at 64.45° thermogram of the complex and the phase C), which is probably due to the hot movement of transition temperature is lower than that of phospholipids polar head group. The second phospholipids indicate the interaction of silymarin (83.21° C) peak is very sharp and it appears due to with phospholipid. phase transition from gel to liquid crystalline state.
07 UJPAH Vol. I No. 12 June 2012 phase transition temperature is lower than that of with phospholipid. phospholipids indicate the interaction of silymarin
(a)
(b)
08 UJPAH Vol. I No. 12 June 2012
(c) Fig.2- DSC Thermograms: (a) Phospholipid, (b) Silymarin and (c) Silymarin-Phospholipid Complex
XRD studies - Fig.3 shows the X-ray diffraction characterized only by large diffraction peaks in patterns of the silymarin, phospholipid and the which it is no longer possible to distinguish the complex. In the X-ray diffractogram silymarin characteristic peaks of the drug. The results, showed intense diffraction peaks of crystallinity at confirmed that silymarin is no longer present as a a diffraction angle of 2è and suggested that the crystalline material and its phospholipid complex drug is present as a crystalline material, whereas exist in the amorphous state. Thus, XRD data the phospholipid showed a single diffraction peak. supports the DSC studies which indicated the A total drug amorphization was induced by interaction between the individual components of complex formation where X-ray diffraction the complex and well supported by previous patterns of silymarin-phospholipid complex were literature [25, 26].
(a) 09 UJPAH Vol. I No. 12 June 2012
(b)
(c) Fig.3- X-ray diffraction patterns at 2è -scale (a) Silymarin, (b) Phospholipid and (c) Silymarin-Phospholipid Complex
SEM studies - The scanning electron micrographs particles in the phospholipid complex and of silymarin and the complex are given in Fig. 4. appeared as fluffy, irregular and rough surface, The silymarin was characterized as smaller size revealing an apparent interaction in the solid-state and regular shape of crystals with an apparently which might have resulted in the enhanced smooth surface. In contrast, the changes has been dissolution rate as compared to pure drug. found in the morphology and shape of the
10 UJPAH Vol. I No. 12 June 2012
Fig.4- Scanning Electron Micrographs: Magnification: 100 X, 400 X, 1 KX (a) Silymarin (b) Sil-Pc complex
Conclusions References The prepared complex showed interaction of Wellington K. and Jarvis B. Silymarin: A review of its silymarin with phosphatidylcholine. The FT-IR, clinical properties in the management of hepatic DSC, X-ray diffraction and SEM studies confirmed disorders. BioDrugs. 2001, 15, 465-489. the formation of the complex. As the amphiphilic drug-lipid complexes have been reported to be [1]. Quaglia MG, Bossu E, Donati E, Mazzanti G stable and more bioavailable, the phospholipid and Brandt A. Determination of complex of silymarin may serve as a value added silymarine in the extract from the dried herbal drug delivery system. silybum marianum fruits by high performance liquid chromatography and Acknowledgements capillary electrophoresis. Journal of The authors are thankful to the DST Research Pharmaceutical and Biomedical Analysis. grant (SRSO/HS/72/2006). The authors 1999, 19, 435-442. acknowledge LIPOID GmbH Germany for [2]. Skottova N, Krecman V and Simanek V. providing the gift sample of phosphatidylcholine Activities of silymarin and its for the research work. Facilities provided by the flavanolignans upon low density UGC-DAE Consortium for Scientific Research, lipoprotein oxidizability in vitro. Indore (India), are gratefully acknowledged. Phytotherapy Research. 1999, 13, 535-537. [3]. Hikino H, Kiso Y, Wagner H, Fiebig M. 11 UJPAH Vol. I No. 12 June 2012
Antihepatotoxic actions of [11]. Crocenzi FA, Pellegrino JM, Sanchez Pozz, flavonolignans from Silybum marianum EJ, Mottino AD, Garay EA and Roma MG. fruits. Planta Med., 1984, 50, 248-250. Effect of silymarin on biliary bile salt [4]. Kvasnicka F, Biba B, Sevcik R, Voldrich M secretion in the rat. Biochemical and Kratka J. Analysis of the active Pharmacology. 2000, 59, 1015-1022. components of silymarin. Journal of [12]. Sonnenbichler J and Zetl I. Mechanism of Chromatography. A, 2003, 990, 239-245. action of silibinin. V. Effect of silibinin on [5]. Pietrangelo A, Montosi G, Garuti C, Contri the synthesis of ribosomal RNA, mRNA M, Giovannini F, Ceccarelli D and Masini A. and tRNA in rat liver in vivo. Hoppe- Iron-induced oxidant stress in Seyler's Zeitschrift Fur Physiologische nonparenchymal liver cells: mitochondrial Chemie. 1984, 365, 555-566. derangement and fibrosis in acutely iron- [13]. Sonnenbichler J and Zetl, I. Biochemical dosed gerbils and its prevention by effects of the flavanolignane silibinin on silybin. Journal of Bioenergetics and RNA, protein and DNA synthesis in rat Biomembranes. 2002, 34, 67-79. livers. Progress in Clinical and Biological [6]. Mourelle M, Muriel P, Favar, L and Franco Research. 1986, 213, 319-331. T. Prevention of CCL4-induced liver [14]. Sonnenbichler J, Goldberg M, Hane L, cirrhosis by silymarin. Fundamental & Madubuny, I, Vogl S and Zetl I. Stimulatory Clinical Pharmacology. 1989, 3, 183-191. effect of silibinin on the DNA synthesis in [7]. Muriel P, Garciapina T, Perez-Alvarez V partially hepatectomized rat livers: non- and Mourelle M. (Silymarin protects response in hepatoma and other malign against paracetamol-induced lipid cell lines. Biochemical Pharmacology. peroxidation and liver damage. Journal of 1986, 35, 538-541. Applied Toxicology. 1992, 12, 439-442. [15]. Kren V. & Walterova, D. (2005) Silybin and [8]. Vogel G, Tuchweber B, Trost W and Mengs silymarin - new effects and applications. U. Protection by Silymarin against Biomedical Papers of the Medical Faculty Amanita phalloides intoxication in of the University Palacky´ , Olomouc, beagles. Toxicology and Applied Czechoslovakia, 149, 29-41. Pharmacology. 1984, 73, 355-362. [16]. Singh, RP, and Agarwal R. Flavonoid [9]. Paulova J, Dvorak M, Kolouch F, Vanova L antioxidant silymarin and skin cancer. and Janeckova L. Verification of the Therapy and Toxicology. 2002, 30, 134-138. hepatoprotective and therapeutic effect [17]. Singh RP and Agarwal R. Mechanisms and of silymarin in experimental liver injury preclinical efficacy of silibinin in with tetrachloromethane in dogs. preventing skin cancer. European Journal Veterinarni Medicina. 1990, 35, 629-635. of Cancer. 2005, 41, 1969-1979. [10]. Sonia Abrol, Aman Trehan and Om [18]. Jiang C, Agarwal R and Lu J. Anti- Parkash Katare. Comparative Study of angiogenic potential of a cancer Different Silymarin Formulations: chemopreventive flavonoid antioxidant, Formulation,Characterisation and In silymarin: inhibition of key attributes of Vitro/In Vivo Evaluation. Current Drug vascular endothelial cells and angiogenic Delivery. 2005, 2, 45-51. 12 UJPAH Vol. I No. 12 June 2012
cytokine secretion by cancer epithelial DU145 cells. Cell Cycle. 2002, 1, 137-142. cells. Biochemical and Biophysical [22]. Manach C, Scalbert A, Morand C, et al., Research Communications. 2000, 276, Polyphenols: food sources and 371-378. bioavailability. Am J Clin Nutr., 2004, 79, [19]. Soto C, Mena R, Luna J, Cerbon M, Larrieta 727-747. E, Vital P, Uria E, Sanchez M, Recoba R, [23]. Kidd PM, Ph D. Phosphatidylcholine: a Barron H, Favari L and Lara A. Silymarin superior protectant against liver damage. induces recovery of pancreatic function Altern. Med. Rev., 1996, 1(4), 258-274. after alloxan damage in rats. Life [24]. Babu GVMM, Prasad CDS, Murthy KVR. Sciences. 2004, 75, 2167-2180. Evaluation of modified gum karaya as [20]. Chung SY, Sung MK, Kim NH, Jang JO, Go carrier for the dissolution enhancement EJ and Lee HJ. Inhibition of P-glycoprotein of poorly watersoluble drug nimodipine. by natural products in human breast Int. J. Pharm. 2002, 234, 1-17. cancer cells. Archives of Pharmacal [25]. Moneghini M, Kikie I, Voinovich D, Research. 2005, 28, 823-828. Perissutti B and Grcic JF. Processing of [21]. Tyagi A, Agarwal C and Agarwal R. The carbamazepine-PEG 4000 solid cancer preventive flavonoid silibinin dispersions with supercritical carbon causes hypophosphorylation of Rb/p107 dioxide: preparation, characterization and Rb2/ p130 via modulation of cell cycle and in vitro dissolution. Int. J. Pharm. regulators in human prostate carcinoma 2001, 222, 129-138.
13 UJPAH Vol. I No. 12 June 2012
Mechanism of Poly phenols from Bitter Gourd ,Cinnamon ,Basil and B. Berries in Controlling High Blood Sugar Level
Anil Kumar Gupta Associate Professor, Department of Chemistry D.A.V. College, 208001 C.S.J.M. University, Kanpur (India) E-mail: [email protected]
Abstract - Generally, two types of insulin are patients are treated by oral anti diabetic drugs and found. One is endogenous which is secreted insulin, which are exogenous amino derivatives inside the body by pancreas and other is (xenobiotics), they are not completely effective in exogenous which is actually given to a diabetic controlling blood glucose level. patient as a medicine when the secretion of Due to diabetes, sometime post meal blood endogenous insulin is inefficient or insufficient. In glucose1 rises high and remain uncontrolled even a non diabetic person, it is the endogenous on taking the oral drugs and exogenous insulin, insulin which metabolizes the blood glucose but which can lead to a series of complications in brain, contrary to that, in case of diabetic patient, it is kidneys, heart, eyes, nerves and major arteries. the blood glucose which is utilized for Some of the known oral antidiabetic drugs have biotransformation of oral anti-diabetic drugs been Tolbutamide, Tolazamide, Glipizide, (given in type-2) and exogenous insulin (given in Glyburide, Chloropropamide, Glimepride, type-1 diabetes) and thus glucose level can be Repaglinide, Nateglinide, Rosiglitazone, reduced upon taking these medicines, however, Troglitazone, Metomorphin hydrochloride, their monitoring is not possible and glucose level Acrobose and Miglitol. Just like exogenous insulin, in such patients always remain higher than all of them contain amino functional group. normal level due to under dose (hyperglycemia) It has been very difficult to control the lows and or lower than normal level due to over dose highs of sugar level despite use of these drugs and (hypoglycemia). For a patient optimum dose of people were not found to be judicious about these these drugs are quite necessary which seems to medicines and kept oscillating between oral drugs be practically impossible. In the present study we and exogenous insulin. Therefore, a newer have solved this problem by inventing a newer treatment was constantly needed. treatment based on herbal composition An attempt has been made to solve the mystery containing bitter gourd, cinnamon, basil and blue behind how these drugs containing amino berries. functional group, lower the blood glucose and Keywords - Diabetes, Endogenous & Exogenous herbal powder composition which contains insulin, Hyper &hypoglycemia, Uridine hydroxyl functional group and controls blood diphosphate glucoronic acid, Entero hepatic sugar without any side effects. circulation. Now patients don't have to spend days and weeks anxiously waiting for blood glucose to come Introduction normal. The herbal powder composition is good An estimated 180 million people now suffer from choice in case of high post - meal blood glucose rise diabetes around the world. Currently, diabetic that remain uncontrolled even on oral drugs and
14 UJPAH Vol. I No. 12 June 2012 exogenous insulin. glucose concentration falls to less than 45 mg/dl. Herbal powder composition also carries a negative We have overcome this key problem of lows and risk of hypoglycemia due to entero hepatic highs of blood sugar by using herbal powder circulation. composition which contains various poly phenolic In a non - diabetic person blood glucose is compounds having hydroxyl functional group as metabolized by endogenous insulin produced byâ phermacophoric moiety instead of - NH - group - cells of islet of langerhans of pancreas, there is no present in oral drugs and exogenous insulin. problem of uncontrolled glucose level. There are dramatic and exciting results. The But in diabetic patients, production of biggest advantage the herbal powder offers is that endogenous insulin is insufficient or ineffective. To it controls high sugar level without any risk of such patients, oral anti diabetic drugs and hypoglycemia. exogenous insulin are given. These drugs utilize When herbal powder composition is used, the glucose indirectly for their bio transformation and UDPGA conjugates with hydroxy compounds of stimulate glucose utilization in form of uridine the herbal composition through hydroxyl diphosphate glucuronic acid (UDPGA). functional group in presence of glucuronyl The mechanism of biotransformation of these transferase to form O - glucuronide and in case of drugs takes place by glucuronic acid path way. overdose the O - glucuronide is accumulated in the In the glucoronic acid pathway of glucose gallbladder and released into the small intestine utilization, first of all glucose from blood enters and can be hydrolyzed by bacterial enzyme to give into the liver cells by insulin independent transport back UDPGA. system and enzyme glucokinase convert glucose UDPGA is reabsorbed and returned to the liver. to glucose 6- phosphate. Phospho glucomutase, Thus UDPGA is recycled to prevent further catalyzes the conversion of glucose 6 - phosphate conversion of glucose to UDPGA, or fall of glucose to glucose 1 - phosphate. Now glucose 1 - level (hypoglycemia) phosphate and UTP, form Uridine di phosphate Recycling of UDPGA is entero hepatic circulation glucose (UDPG) in presence of UDP glucose (fig.2). pyrophosphorylase. The glucose portion of UDPG is oxidized to yield uridine diphosphate glucuronic Methods acid (UDPGA). The UDPGA conjugates with oral Polyphenols are soluble in lime water. Therefore, drugs and exogenous insulin through their - NH - in a beaker dry powder of bitter gourd, cinnamon, group in presence of Glucurnoyl transferase to basil and blue berries are heated with lime of form the polar compound by the process known as desired density (nine degree baume) and filtered. glucuronidation2. The polar compound called N - The filtrate was treated with hydrogen sulphide glucuronide is more easily cleared from blood by gas to remove calcium sulphide by filtration. The kidneys and excreted in the urine. Thus glucose is filtrate so obtained is boiled till concentrated to a eliminated in the form of N - glucuronide (Fig.-1) solid mass and then dried and grinded to powder. Most common complications of oral drugs and At least 1.36 part of powder is mixed with 0.64 part exogenous insulin in diabetic patient is of amla powder. Herbal powder composition so hypoglycemia which is caused due to overdose at obtained contain mixture of various poly phenolic the time of managing blood glucose level. This is compounds like Catechin, Quercetin, Gingerol , particularly observed in patients who are on Rosmarinic Acid gallic Acid, hydroxyl tyrosol and intensive treatment regime. emblicanin. Symptoms of hypoglycemia appears when blood At least two to five grams of the herbal powder composition can be orally taken with water to see 15 UJPAH Vol. I No. 12 June 2012 the result. Herbal powder composition may also be orally, the excess of glucose present in blood is used in type 2 and type 1 diabetic patients taking converted into more glucuronic acid which then oral anti diabetic drugs and insulin. In such cases form uridine di phosphate glucuronic acid hydroxyl derivatives of the composition and - NH - (UDPGA). Mean while free hydroxyl group of derivatives of oral drugs & insulin are found to pharmacophore present in herbal powder interact with an additive effect3 as they present composition conjugates with UDPGA and form the same pharmacodynamic action to give glucuronide in presence of enzyme glucuronyl significant results. The wt% composition of herbal transferase to control glucose level in the blood powder composition is measured by acetylation within two hour of application irrespective of over method.4 or under dose due to entero hepatic circulation. The reading of blood glucose is measured by A fall of nearly 80 - 120 mg/dl of post prandial and glucometer. fasting plasma glucose takes place within two Discussion hours after the oral intake (five grams) of herbal Hence Oral herbal powder composition may be powder composition in the patients not taking any treated as best choice of medicine in both type of amino derivatives, Table 1 In the patients, taking diabetes (Type- I and type - II), is more cost amino derivatives, only two grams of the herbal effective with negligible side effects due to powder composition offer additive effect and presence of hydroxyl functional group in glucose level is controlled (Table 2 & 3). comparison to sulphonamides (Oral drugs) and Minimum value of fasting plasma glucose and post exogenous insulin, both of which contain amino prandial glucose do not go below 70 to 100 mg/dl, functional group as pharmacophoric moiety. due to entero hepatic circulation hence there is When the herbal powder composition is taken negative risk of hypoglycemia.
Table 1 Reading of Post Prandial & Fasting Glucose Using Herbal powder composition alone .
Post Prandial Plasma Glucose Fasting Plasma Glucose (mg./dl) (mg./dl.) Month/Year Before use No. of After use Before No.of grams After use S.No. grams use 1. 6/2011 230 05 102 170 05 75 2. 9/2011 213 05 100 157 05 72 3. 10/2011 336 05 216 206 05 86 4. 11/2011 275 05 155 146 05 76 5. 12/2011 297 05 177 185 05 105 6. 1/2012 279 05 159 193 05 113 7. 2/2012 270 05 150 171 05 91 8. 4/2012 280 05 160 190 05 110
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Table 2 Reading of post prandial glucose using herbal powder composition together with oral anti diabetic drugs.
Before Massage After Massage Patient Post Prandial Plasma No. of Post Prandial Plasma Glucose Glucose mg/dl. grams mg./dl.
A 220 2 140 B 334 2 225 C 273 2 233 D 254 2 130
Table 3 Reading of Post Prandial glucose using herbal powder composition together with oral drugs and insulin
Before use After use Patient Post Prandial Plasma Glucose No. of Post Prandial Plasma Glucose mg/dl. grams mg./dl. A 387 2 190 B 310 2 270 C 442 2 268
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N-Glucuronid formation, which does not undergo hydrolysis and causes hypoglycemia Fig.-1
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O -Glucuronide formation, which undergoes hydrolysis and Entero hepatic circulation. Fig.-2
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Conclusion described here, a massage herbal oil composition in controlling gestational and 1. Treatment of diabetes using herbal powder juvenile diabetes was also prepared. composition is a surprising invention. 2. It is good news for patients who have Acknowledgements uncontrolled sugar despite maximum doses I am grateful to principle D.A.V.College, Dr. Smt. of oral drugs & insulin. Rekha Sherma for encouraging me to carry 3. The biggest advantage with herbal powder on my research work. composition is that it causes negative risk of hypoglycemia on account of entero hepatic References circulation. 1. Gupta, A.K. Merits and demerits of blood 4. The cost of 100 grams of herbal powder glucose present in the body of teenagers and composition is much affordable hence it also young adults. Jigyasa. 4, 87-89(2010) reduces financial burden of diabetes on the 2. David L. Nelson & Michael M. Cox. Lehninger common man. Principle of Bio - Chemistry, Third addition, 5. Other existing medicines have binding to be Page No. 743, Macmillan Worth Publisher. taken daily but herbal powder composition is 3. Andrejus Korolkovas. Essentials of medicinal only required when blood glucose level is chemistry, second addition, page No. 15, A uncontrolled. Wiley - Inter Science Publication. 6. Herbal powder composition can be used by 4. Gupta, A.K. Invention of acetylation method both types of diabetics (Types I & Types II). for measurement of fuel ethanol percent in 7. Based on the mechanism of polyphenols in fermented liquid. Proceedings of sugar controlling high blood sugar level as technologists' association of India. 70, 35- 39(2009).
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Phytochemical Evaluation of Ingredients and Role of Bioactive Markers In Hepatoprotective Property of Liv 52.
Zafar Mehmood and Suman Chimwal E-mail: [email protected]
Abstract - Liver is an organ of paramount any hepatotoxin. importance, which plays a pivotal role in regulating Liver cell injury caused by various toxic chemicals various physiological processes in the body. It has (certain anti-biotic, chemotherapeutic agents, great capacity to detoxify toxic substances and carbon tetrachloride (CCl4), thioacetamide (TAA) synthesize useful principles. The damage to the etc.), excessive alcohol consumption and liver caused by hepatotoxic agents is of grave microbes is well-studied. consequences. In spite of tremendous scientific advancement in the field of hepatology in recent In spite of tremendous strides in modern medicine, years, liver problems are in rise. Jaundice and there are hardly any drugs that stimulate liver hepatitis are two major hepatic disorders. Natural function, offer protection to the liver from remedies from medicinal plants are considered to damage or help regeneration of hepatic cell. be effective and safe alternative treatment for Herbal medicines have been used in the treatment liver diseases. Herbs play a major role in the management of of liver diseases for a long time. The present review various liver disorders. The present study was is aimed at compiling data on promising conducted to review the hepatoprotective phytochemicals from medicinal plants that have potential of a polyherbal formulation Liv 52. been tested in hepatotoxicity models using Some of the ingredients as well as the tablets of Liv modern scientific system. 52 were tested for the bioactive markers. It was Now a days there are different marketed concluded from the study that Liv 52 has more formulations available for treating liver diseases significant hepatoprotective activity. and amongst them Liv-52 is one of them.
Keywords- Phytochemical, Hepatoprotective Liv-52 is an indigenous multiherbal hepatotonic activity, Polyherbal formulations that has been widely used as an hepatoprotective Introduction agent in various liver disorders. Liver plays a major role in the detoxification and excretion of many endogenous and exogenous The present study was undertaken to review the compounds; and type of injury (due to systemic hepatoprotective activity of Liv 52. Sapakal et al, (2009 ) reported CCl4-induced drugs, food preservatives, agrochemicals and hepatotoxicity and hepatoprotective role of Liv 52 addiction to alcohols) or impairment of its in rats. functions may lead to many complications in ones health. There is a no rational therapy available for Some of the ingredients of Liv-52 as well as the Liv liver disorder, and it is a still challenge to modern 52 tablets were analysed for active biomarker medicine. Hepatic injury can be life threatening compounds. when the entirely or most of the liver is exposed to
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Material & Methods
Phytochemical evaluation of some ingredients and tablets of Liv 52 The following herbs were analysed for bioactive markers
S.No. Item Medicinal activity References
1. Amla Hepatoprotective The wealth Of India Rich source of (1992) vitamin C,antioxidants 2. Bhringraj Hepatoprotective The wealth Of India (1992) 3. Bhoomiamla Hepatoprotective The wealth Of India (1992) 4. Kasni Hepatoprotective Ahmed et al(2003)
5. Makoi Hepatoprotective Prashanth et al(2001)
6. Liv 52 Tablets Hepatoprotective Sapakal et al(2009)
Estimation of Flavonoids 4. Take 1 ml each of test solution in 10 ml 1. Extract the finely powdered plant volumetric flask and follow the same material in methanol on a water bath at 80 procedure as that of standard. C for 30 minutes and filter into a 50 ml 5. Prepare standard and sample blank volumetric flask. Make up the volume solutions by diluting 1 ml of standard or with methanol. sample to 10 ml with ethanol and read the 2. Weigh 100 mg of standard rutin in 100 ml optical density at 410 nm. standard flask and make up the to volume with methanol. Pipette out 5ml of the Estimation of bitters above solution in 100ml standard flask and make up the volume with methanol. 1. Weigh accurately 5 g of the sample and 3. Take 1 ml of the standard solution in 10 ml add 100ml of methanol and reflux for one volumetric flask.Add 1 ml of aluminium hour at 80 C. chloride reagent and add ethanol 2. Filter the extract and residue is further required to make the volume to 10 ml. extracted with 50 ml X two times and 25 Measure the optical density of the ml X two times of methanol till the solution against the reagent blank (1 ml of complete extraction of bitters is affected. aluminium chloride made up to 10 ml with 3. Concentrate the combined methanol ethanol) at 410 nm exactly after 15 extract to a thick paste form and dissolve minutes after the addition of reagent. the residue in 50 ml of hot water.
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3. Shake the aqueous extract repeatedly found to possess potent hepatoprotective activity with 50 ml X three times and 25 ml X two against CCl4, paracetamol (in vivo) and times of Ethylacetate. thioacetamide, galactosamine (in vitro) induced 4. Collect the combined Ethylacetate hepatotoxicity. extracts and evaporate on a water bath in Al-Said et al. demonstrated the strong anti- a tarred china dish and dry at 100 C for one inflammatory activity of Capparis spinosa, which hour. was comparable to oxyphenbutazone. Bonina et al. documented a significant antioxidant Results and Discussion activity of Capparis spinosa and also identified Test results of flavonoids & bitters are depicted in flavonols (kaempferol and quercetin derivatives) table-1 that showed the presence of flavonoids and hydroxycinnamic acids (caffeic acid, ferulic and bitters in the tested ingredients as well as in acid, p-cumaric acid, and cinnamic acid) as major the Liv 52 tablets revealed the hepatoprotective antioxidants from Capparis spinosa. activity of the formulation. In another study, Germano et al. observed the The results of the study conducted by Sapakal et al antioxidant activity of Capparis spinosa using tests are shown in Table-2. CCl4 such as lipid peroxidation, bleaching of free intoxication in normal rats significantly elevated radicals and autoxidation of iron ions. the serum levels of SGOT,SGPT,bilirubin(Total and Mahasneh et al. observed potent antimicrobial direct) and total proteins, whereas there was a and antifungal activity of Capparis spinosa. significant decrease in level of total proteins that He et al. isolated 2,3,4,9-tetrahydro-1H-pyrido- indicated acute hepatocellular damage and biliary (3,4-b) indole-3-carboxylic acid, azelaic acid and obstruction. The rats treated with Liv-52 showed daucosterol as the major constituents of significant reduction in all biochemical Cichorium intybus. parameters, whereas increase in the level of total Du et al. identified the other chemical constituents protein was observed in comparison to that of the as alpha-amyrin, taraxerone, baurenyl acetate and standard group (silmaryn treated). beta-sitosterol. The study of Girish et al(2009) confirms the Aktay et al. observed the hepatoprotective effect efficacy of polyherbal formulations in (confirmed by histopathological examination of hepatoprotection in biochemical, pathological and Cichorium intybus against CCl4-induced pharmacological parameters against PCM induced hepatotoxicity and reported significant hepatotoxicity in mice. The polyherbal prevention of the elevation of malondialdehyde hepatoprotective formulation Liv 52 was most formation (plasma and hepatic) and enzyme levels effective at normal doses used in this study (AST and ALT). justifying its use as a hepatoprotective agent. Ahmed et al. screened Cichorium intybus for antihepatotoxic activity and measured the degree Overall compliance to the drug treatment was of protection using biochemical parameters (AST, found to be excellent. These beneficial clinical ALT, ALP and TP). Potent antihepatotoxic activity efficacies of Liv 52 DS in acute viral hepatitis might (comparable to the silymarin) was observed with be due to the synergistic action of its ingredients, almost complete normalization of the tissues (as which had been well documented in various neither fatty accumulation nor necrosis was experimental and clinical studies by various observed on histopathological study). researchers. Kim et al. studied the effects of Cichorium intybus Khanfar et al. isolated and identified the active on the immunotoxicity of ethanol and reported ingredients of Capparis spinosa as beta- significant increase in the number of circulating sitosterylglucoside-6'-octadecanoate and 3- leukocytes, the weights of concerned organs methyl-2-butenyl-beta-glucoside. P-Methoxy (liver, spleen and thymus), number of splenic benzoic acid isolated from Capparis spinosa was plaque forming cells, hemagglutination titers and 23 UJPAH Vol. I No. 12 June 2012 the secondary IgG antibody response. There were activity of aminopyrine N-demethylase, uridine also significant increases in delayed-type diphosphate glucuronyltransferase and hypersensitivity reaction, phagocytic activity, glutathione S-transferase, without any alteration natural killer cell activity, cell proliferation and in levels of ALP, ALT and gamma- interferon gamma secretion. glutamyltransferase levels in the serum. Sultana et al. reported that the presence of Son et al. reported Solanum nigrum as a potent Cichorium intybus in the reaction mixture scavenger of hydroxyl radicals and (containing calf thymus DNA and free radical diphenylpicrylhydrazyl radicals. generating system) protects DNA against Prashanth Kumar et al. tested in vitro Solanum oxidative damage to its deoxyribose sugar moiety. nigrum for its cytoprotection (against gentamicin- All these studies suggest that the observed induced toxicity) and observed significant hepatoprotective effect of Cichorium intybus inhibition of cytotoxicity, alongwith hydroxyl might be due to its ability to suppress the oxidative radical scavenging potential, which might be the degradation of DNA in the tissue debris. mechanism of cytoprotection. El et al. and Papetti et al. documented the Similarly, Akhtar et al. observed gastric mucosal antioxidative activity (radical scavenging effects, cytoprotection offered by Solanum nigrum inhibition of hydrogen peroxide, and iron against aspirin-induced gastric ulcers. chelation) of Cichorium intybus. Upadhyay et al. identified arjunetoside, oleanolic Gurbuz et al. observed significant cytoprotection and arjunic acids as active ingredients from against ethanol-induced damage and these results Terminalia arjuna. were further confirmed by using histopathological Munasinghe et al. reported the potent antioxidant techniques. activity of Terminalia arjuna, which might be due to Amirghofran et al. reported the capacity of its effects on lipid peroxidation. Cichorium intybus to enhance the proliferation of Ali et al. demonstrated that arjunaphthanoloside lymphocytes after stimulation with the allogenic from Terminalia arjuna inhibits nitric oxide cells. production and terminoside A isolated from Kim et al. investigated the effect of Cichorium Terminalia arjuna decreases inducible nitric oxide intybus on mast cell-mediated immediate type synthase levels in lipopolysaccharide-stimulated allergic reactions and observed inhibition of peritoneal macrophages. systemic anaphylactic reaction, reduction in the Jafri et al. reported significant hepatoprotective plasma histamine level. effects of Cassia occidentalis in chemically induced Ikeda et al. identified saponins (nigrumnins I and liver damage. Bin-Hafeez et al. showed that Cassia II) as the active ingredients of Solanum nigrum. occidentalis modulates hepatic enzymes and Solanum nigrum was investigated for its provides hepatoprotection against induced hepatoprotective activity against CCl4-induced immunosuppression. hepatic damage and Raju et al. observed Harnyk et al. documented the clinically beneficial remarkable hepatoprotective activity confirmed effects of Achillea millefolium in the treatment of by evaluated biochemical parameters (AST, ALT, chronic hepatitis. Krivenko et al. reported similar ALP and TB). clinical improvements in chronic Sultana et al. demonstrated that Solanum nigrum hepatocholecystitis and angiocholitis with Achillea protect DNA against the oxidative damage and the millefolium. Lin et al. observed anti-hepatoma results suggest that the observed activity of Achillea millefolium. hepatoprotective effect of Solanum nigrum might Candan et al. and Bezic et al. reported the be due to the ability to suppress the oxidative antioxidant and antimicrobial activities of Achillea degradation of DNA in the tissue debris. millefolium Moundipa et al. studied the effects of Solanum Devarshi et al. studied Mandura bhasma for the nigrum on hepatotoxicity and reported increased hepatoprotective property in hepatitis induced by
24 UJPAH Vol. I No. 12 June 2012
CCl4 and observed prevention of CCl4 mediated actions (hepatoprotective, antimicrobial, changes in the enzyme activities, which suggest antioxidant and anti-inflammatory) exhibited by the hepatoprotective role of Mandura bhasma. the ingredients of Liv.52 DS might explain the Therefore, as discussed above, these synergistic beneficial mechanism of action of Liv 52 DS in acute viral hepatitis.
Table 1 S.No. Item Flavonoids Bitters
1. Amla 0.94% 12.15%
2. Bhringraj 0.31% ----
3. Bhoomiamla 0.67% ----
4. Kasni 0.18% ----
5. Makoi 0.088% ----
6. Liv-52 Tablets 0.32 5.70
Table 2- Effect of Liv 52 on CCL4 -induced Hepatotoxicity
25 UJPAH Vol. I No. 12 June 2012
References Jordanian origin. Nat. Prod. Res. 2003; 17(1): 9-14 1.Sapakal,V.D; Ghadge, R.V; Anaik, R.S; Naikwade,N.S and Magdum, C.S.(2009) 11. Gadgoli C, Mishra SH. Antihepatotoxic activity Comparative hepatoprotective activity of Liv52 of p-methoxy benzoic acid from Capparis spinosa. and livomyn against carbon tetrachloride-induced J. Ethnopharmacol. 1999; 66(2): 187-192. hepatic injury in rats.international journal of Green Pharmacy.79-82. 12. Al-Said MS, Abdelsattar EA, Khalifa SI, el-Feraly 2.Girish,C;Koner,B.C;Jayanthi,S;Rao,K.R;Rajesh,B FS. Isolation and identification of an anti- and Pradhan, S.C. (2009) Hepatoprotective inflammatory principle from Capparis spinosa. activity of six polyherbal formulations in Pharmazie. 1988; 43(9): 640-641. paracetamol induced liver toxicity in mice.Indian J Med Res 129,569-578 13. Ageel AM, Parmar NS, Mossa JS, Al-Yahya MA, 3.The wealth Of India: A Dictionary of Indian Raw Al-Said MS, Tariq M. Anti-inflammatory activity of Materials and Industrial Products- Raw Materials some Saudi Arabian medicinal plants. Agents Series, Publications & Information Directorate Actions 1986; 17(3-4): 383-384. ,CISIR, New Delhi, Revised Ser, 1992; vol-3(Ca-Ci); pp.215-216. 14.Bonina F, Puglia C, Ventura D, Aquino R, Tortora S, Sacchi A. et al. In vitro antioxidant and in vivo 4. Krugman S, Ward R, Giles JP. The natural history photoprotective effects of a lyophilized extract of of infectious hepatitis Am. J. Med. 1962; 32: 717-728 Capparis spinosa L. buds. J. Cosmet. Sci. 2002; 53(6): 321-335. 5. Simons JN, Pilot-Matias TJ, Leary TP, et al. Identification of two flavivirus-like genomes in the 15. Germano MP, De Pasquale R, D'Angelo V, GB hepatitis agent. Proceedings of the National Catania S, Silvari V, Costa C. Evaluation of extracts Academy of Sciences U.S.A. 1995; 92: 3401-3405. and isolated fraction from Capparis spinosa L. buds as an antioxidant source. J. Agric. Food 6. Oshiba, Okamoto H, Mishiro S. Detection of the Chem. 2002; 50(5): 1168-1171. GBV-C hepatitis virus genome in serum from patients with fulminant hepatitis of unknown 16.Mahasneh AM. Screening of some indigenous aetiology. Lancet 1995; 346: 1131-1132. Qatari medicinal plants for antimicrobial activity. Phytother. Res. 2002; 16(8): 751-753. 7. Ikawa T, Sugai Y, Okamoto H. Hepatitis G infection in drug abusers with chronic hepatitis C 17.Ali-Shtayeh MS, Abu Ghdeib SI. Antifungal (letter). New Engl. J. Med. 1996; 334: 195-196. activity of plant extracts against dermatophytes. Mycoses 1999; 42(11-12): 665-672. 8. Innen J, Wages J, Jr. Zhang-Keck et al. Molecular cloning and disease association of hepatitis G virus: 18. He Y, Guo YJ, Gao YY. Studies on chemical A transfusion-transmissible agent. Science 1996; constituents of root of Cichorium intybus. 271: 505-508. Zhongguo. Zhong. Yao. Za. Zhi. 2002; 27(3): 209- 210. 9. Asuko K, Mitsui T, Iwano K, et al. Infection with hepatitis GB virus C in patients on maintenance 19.Du H, Yuan S, Jiang P. Chemical constituents of hemodialysis. New Engl. J. Med. 1996; 334: 1485- Cichorium intybus L. Zhongguo. Zhong. Yao. Za. 1490. Zhi. 1998; 23(11): 682-683, 704.
10. Khanfar MA, Sabri SS, Zarga MH, Zeller KP. The 20. Aktay G, Deliorman D, Ergun E, Ergun F, chemical constituents of Capparis spinosa of Yesilada E, Cevik C. Hepatoprotective effects of 26 UJPAH Vol. I No. 12 June 2012
Turkish folk remedies on experimental liver oligoglycosides from Solanum nigrum. Chem. injury. J. Ethnopharmacol. 2000; 73(1-2): 121-129. Pharm. Bull. (Tokyo) 2000; 48(7): 1062-1064. 30. Raju K, Anbuganapathi G, Gokulakrishnan V, Rajkapoor B, Jayakar B, Manian S. Effect of dried 21. Ahmed B, Al-Howiriny TA, Siddiqui AB. fruits of Solanum nigrum LINN against CCl4- Antihepatotoxic activity of seeds of Cichorium induced hepatic damage in rats. Biol. Pharm. Bull. intybus. J. Ethnopharmacol. 2003; 87(2-3): 237- 2003; 26(11): 1618- 1619. 240. 31. Moundipa PF, Domngang FM. Effect of the leafy 22. Mun JH, Woo YJ, Jeon WH, An NH, Park JS. vegetable Solanum nigrum on the activities of Effects of the ethanol extract of Cichorium intybus some liver drug-metabolising enzymes after on the immunotoxicity by ethanol in mice. Int. aflatoxin B1 treatment in female rats. Br. J. Nutr. Immunopharmacol. 2002; 2(6): 733-744. 1991; 65(1): 81-91.
23. Sultana S, Perwaiz S, Iqbal M, Athar M. Crude 32. Son YO, Kim J, Lim JC, Chung Y, Chung GH, Lee extracts of hepatoprotective plants, Solanum JC. Ripe fruit of Solanum nigrum L. inhibits cell nigrum and Cichorium intybus inhibit free radical- growth and induces apoptosis in MCF-7 cells. mediated DNA damage. J. Ethnopharmacol. 1995; Food Chem. Toxicol. 2003; 41(10): 1421-1428. 45(3): 189-192. 33. Prashanth Kumar V, Shashidhara S, Kumar MM, 24. El SN, Karakaya S. Radical scavenging and iron- Sridhara BY. Cytoprotective role of Solanum chelating activities of some greens used as nigrum against gentamicin-induced kidney cell traditional dishes in Mediterranean diet. Int. J. (Vero cells) damage in vitro. Fitoterapia 2001; Food Sci. Nutr. 2004; 55(1): 67-74. 72(5): 481-486.
25. Papetti A, Daglia M, Gazzani G. Anti- and pro- 34. Akhtar MS, Munir M. Evaluation of the gastric oxidant activity of water soluble compounds in antiulcerogenic effects of Solanum nigrum, Cichorium intybus var. silvestre (Treviso red Brassica oleracea and Ocimum basilicum in rats. chicory). J. Pharm. Biomed. Anal. 2002; 30(4): 939- J. Ethnopharmacol. 1989; 27(1-2): 163-176. 945. 35. Qureshi S, Rai MK, Agrawal SC. In vitro evaluation of inhibitory nature of extracts of 18- 26. Gurbuz I, Ustun O, Yesilada E, Sezik E, Akyurek plant species of Chhindwara against 3- N. In vivo gastroprotective effects of five Turkish keratinophilic fungi. Hindustan Antibiot. Bull. 1997; folk remedies against ethanol-induced lesions. 39(1-4): 56-60. J. Ethnopharmacol. 2002; 83(3): 241-244. 36. Upadhyay RK, Pandey MB, Jha RN, Singh VP, 27. Amirghofran Z, Azadbakht M, Karimi MH. Pandey VB. Triterpene glycoside from Terminalia Evaluation of the immunomodulatory effects of arjuna. J. Asian. Nat. Prod. Res. 2001; 3(3): 207- five herbal plants. J. Ethnopharmacol. 2000; 212. 72(1-2): 167-172. 37. Munasinghe TC, Seneviratne CK, Thabrew MI, 28. Kim HM, Kim HW, Lyu YS, Won JH, Kim DK, Lee Abeysekera AM. Antiradical and YM. et al. Inhibitory effect of mast cell-mediated antilipoperoxidative effects of some plant immediate-type allergic reactions by Cichorium extracts used by Sri Lankan traditional medical intybus. Pharmacol. Res. 1999; 40(1): 61-65. practitioners for cardioprotection. Phytother. Res. 2001; 15(6): 519-523. 29. Ikeda T, Tsumagari H, Nohara T. Steroidal 27 UJPAH Vol. I No. 12 June 2012
38. Ali A, Kaur G, Hayat K, Ali M, Ather M. A novel Ethnopharmacol. 1999; 68(1-3): 193-203. naphthanol glycoside from Terminalia arjuna with antioxidant and nitric oxide inhibitory activities. 47. Caceres A, Lopez B, Juarez X, del Aguila J, Pharmazie. 2003; 58(12): 932-934. Garcia S. Evaluation of antifungal activity of seven American plants. J. Ethnopharmacol. 1993; 39. Ali A, Kaur G, Hamid H, Abdullah T, Ali M, Niwa 40(3): 207-213. M. et al. Terminoside A, a new triterpene glycoside 48. Graham JG, Zhang H, Pendland SL, Santarsiero from the bark of Terminalia arjuna inhibits nitric BD, Mesecar AD, Cabieses F. et al. oxide production in murine macrophages. J. Asian. Antimycobacterial Naphthopyrones from Nat. Prod. Res. 2003; 5(2): 137-142. Senna obliqua. J. Nat. Prod. 2004; 67(2): 225-227.
40. Cheng HY, Lin CC, Lin TC. Antiherpes simplex 49. Harnyk TP. The use of preparations of plant virus type 2 activity of casuarinin from the bark of origin in treating and rehabilitating elderly patients Terminalia arjuna Linn. Antiviral. Res. 2002; 55(3): with chronic hepatitis. Lik. Sprava. 1999; (7-8): 447-455. 168-170.
41. Perumal Samy R, Ignacimuthu S., Sen A. 50. Krivenko VV, Potebnia GP, Loiko VV. Screening of 34 Indian medicinal plants for Experience in treating digestive organ diseases antibacterial properties. J. Ethnopharmacol. with medicinal plants. Vrach. Delo. 1989; (3): 76- 1998; 62(2): 173-182. 78.
42. Jafri MA, Jalis Subhani M, Javed K, Singh S. 51. Lin LT, Liu LT, Chiang LC, Lin CC. In vitro anti- Hepatoprotective activity of leaves of Cassia hepatoma activity of fifteen natural medicines occidentalis against paracetamol and ethyl from Canada. Phytother. Res. 2002; 16(5): 440- alcohol intoxication in rats. J. Ethnopharmacol. 444. 1999; 66(3): 355-361. 52. Candan F, Unlu M, Tepe B, Daferera D, Polissiou 43. Bin-Hafeez B, Ahmad I, Haque R, Raisuddin S. M, Sokmen A. et al. Antioxidant and antimicrobial Protective effect of Cassia occidentalis L. on activity of the essential oil and methanol cyclophosphamide-induced suppression of extracts of Achillea millefolium Subsp. millefolium humoral immunity in mice. J. Ethnopharmacol. Afan. (Asteraceae). J. Ethnopharmacol. 2003; 2001; 75(1): 13-18. 87(2-3): 215-220.
44. Samy RP, Ignacimuthu S. Antibacterial activity 53. Bezic N, Skocibusic M, Dunkic V, Radonic A. of some folklore medicinal plants used by tribals in Composition and antimicrobial activity of Achillea Western Ghats of India. J. Ethnopharmacol. 2000; clavennae L. essential oil. Phytother. Res. 2003; 69(1): 63-71. 17(9): 1037-1040.
45. Perez C, Anesini C. In vitro antibacterial activity 54. Devarshi P, Kanase A, Kanase R, Mane S, Patil S, of Argentine folk medicinal plants against Varute AT. Effect of Mandura bhasma on lipolytic Salmonella typhi. J. Ethnopharmacol. 1994; activities of liver, kidney and adipose tissue of 44(1): 41-46. albino rat during CCl4-induced hepatic injury. J. Biosciences. 1986; 10(2): 227-234. 46. Tona L, Ngimbi NP, Tsakala M, Mesia K, Cimanga K, Apers S et al. Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo. J. 28 UJPAH Vol. I No. 12 June 2012
Identification and Estimation of Bioactive Marker in Bacopa monnieri using HPTLC Technique
*Jyoti Chhimwal and Mayaram Uniyal *Department of Pharmaceutical Sciences,H.N.B.Garhwal University, Srinagar,Grahwal
Abstract - The analysis of medicinal plants is properties or capable of improving mental ability challenging because plant based material are and memory (2). B.Monnieri is widely used in complex mixtures of compound which exhibit indigenous system of medicine. It helps in natural variability. Identification methods must be concentration, comprehension, recall and specific to distinguish the presence of wrong alertness(3). Brahmi is particularly beneficial as it species. Bacoside A, triterpenoid saponins, is a aid in categorizing information in brain and its major constituent isolated from the plant Bacopa subsequent expression. Bacopa is also called as a monnieri Linn. This substance appears to have natural antioxidant which may give details its antioxidant and liver protective potential. neuroprotective role seen in the memory centers Bacoside A is thought to be the active ingredient in for the brain (4). Triterpenoid saponins, the major Bacopa herb along with bacoside B. High components in Brahmi (5), were reported to be performance thin layer chromatography is a responsible for the cognitive enhancing activity of valuable tool for reliable identification and Brahmi. Major component of bacopa are saponins bacoside A and B. the sample were analyzed for estimation procedures by using HPTLC for the the content of bacoside A and B using HPTLC determination of Bacosides A and B, because it can technique (6-8). provide chromatographic fingerprints that can be visualized and stored as electronic image. Among Material and Methods the Complex mixtures of biologically active Plant material compound in the plant Bacopa, Bacosides can be Fresh aerial parts of B.monnieri were collected used as an analytical marker to determine the from the different local (Dehradun) area. The plant quality of plant material. materials were authenticated by Dr. Mayaram Keywords - Bacopa monnieri, BscosidesA and B, Uniyal, Advisor, Medicinal plants, Uttarakhand HPTLC, Identification, Quantification Chromatogram layer Introduction TLC plates silica gel 60 F254, 10 x 20cm (Merck) The use of medicinal plants has increased substantially; use of chromatography of medicinal Chemicals plant was accepted as a strategy for identification Butanol, Acetic acid, methanol, vanillin and and estimation of the quality of plant material (1). sulphuric acid (Merck) purchased from The amount of the same constituent in different H.V.technology, Dehradun sample of the same pant may vary substantially, depending on geographic region, climate, soil and Standard preparation (1mg/ml): Weigh accurately other factors. It is a fact that sufficient bioactive about 5mg of standard containing Bacoside A and phytochemical constituent is crucial for B in a 5ml volumetric flask. Dissolve and make the therapeutic effects. Quality control of medicinal volume up to the mark with methanol. plant is therefore highly desired to determine the quality of medicinal plant material. Bacopa monnieri Linn is an important medicinal plant Sample preparation belonging to family scrophulariaceae. B.monnieri Weigh accurately 500mg of the powder of Brahmi is a medicinal plant which has proven brain tonic into 250ml round bottomed flask. Add about 50ml 29 UJPAH Vol. I No. 12 June 2012 methanol by refluxing on a water bath for reagent followed by heating at 110o C for 5 min in 30minutes at 800C. Allow the residue to settle and oven. The image of the plate can be captured at decant the dissolved extract into 100ml volumetric white light. flask. Repeat the extraction with 25ml of methanol until the methanol extract to become colorless. Result and Discussion Make up the volume to 100ml with methanol and Thin layer chromatographic analysis of B. Monnieri filter the extract and use the filtrate for the Methanolic extract showed the presence of two analysis. distinct spots using n-Butanol: Acetic acid : Water ( 4: 1: 1 v/v/v ) as a solvent system and cleared as a Sample application violet coloured zone after derivatization by Vanillin Band wise with Linomat 5, band length 12mm, sulphuric acid reagent. The spots with Rf value distance from lower edge 8 mm and distance from 0.38 and 0.53 was observed out of which the spot left edge 20mm. Apply 10µl of sample and standard with Rf= 0.38 was matched with standard solution in a distance of 12mm from the bottom of Bacoside B & the spot with Rf= 0.53 was matched a pre coated silica plate. with standard bacoside A spot. Sample preparation and development of suitable Chromatography mobile phase or solvent system are two important Use Camag Twin Trough Chamber (20 x 20 cm), stages in development of the analytical using wetted filter paper with Butanol: acetic acid: procedures, which become more significant for water (4.0: 1.0: 1.0 v/v/v) used as a mobile phase medicinal plants because of their complexity of and migration distance 12cm. the chemical compounds and their affinity towards different solvent systems. By using Densitometry various mobile phase compositions a better Scan the plate densitometrically at 400nm (White resolution of bacoside A and B with symmetrical light) after dipping with vanillin sulphuric acid and reproducible peaks was achieved with n- reagent. Butanol: Acetic acid: Water (4: 1: 1 v/v/v). The identification of the peak was confirmed by adding Post Chromatographic Derivatization the standard compound to the extract The For visualization of bacosides A and B, the plate chromatograms of standard and test sample are was manually dipped in Vanillin sulphuric acid shown in fig.1.
Fig.1- After derivatization with vanillin sulphuric acid reagent A, B, C à Samples of Bacopa monnieri Sà Standard Containing 84.02% Bacoside A and B 30 UJPAH Vol. I No. 12 June 2012
Table 1- Percentage of Bacoside A and B in Different samples of B.monnieri
Bacopa monnieri
Bacopa monnieri
Bacopa monnieri
The bacoside A and B were quantified in three similar HPTLC fingerprints and the percentage of different samples of Bacopa monnieri. Bacoside bacoside A and B was found almost similar in all content in sample-1, sample-2 and sample-3 of samples. Therefore this method can be B.monnieri was found to be 18.28 %, 18.34% and successfully used for the routine analysis of 17.70% w/w respectively. bacoside A and B in B. monnieri, which can be explored for quality control of raw material and Conclusion herbal products of traditional system of medicine Aerial parts of Brahmi collected from three places containing leaves of B. monnieri one of the in Dehradun were analyzed. All samples gave the ingredient.
Fig.2 HPTLC Chromatogram of Standard Fig.3 HPTLC Chromatogram of B.Monnieri-Sample1
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Bacoside A and B
Fig.4 HPTLC Chromatogram of B.Monnieri-Sample2 Fig.5 HPTLC Chromatogram of B.Monnieri-Sample3
Acknowledgement Consistent Cognitive Decline- an Ayurvedic We are highly thankful to Dr. Mayaram Uniyal for Pharmacological Management. Indian J.of identifying and authenticating B. monnieri .The Gerontology, 2006;20(4): 317. Himalaya Drug Company, Dehradun (UK) for 4) Bhutani, K.K.,Fingerprinting of Ayurvedic providing the standard of bacoside and lab Drugs. The Eastern Pharmacist,2000; 43: 21. facilities for this research. 5) Agarwal,H., Kaul,N., Paradkar,A.R. and Mahadik, K.R., Separation of Bacoside A and References Bacopaside II, Major Triterpenoid saponins in 1) WHO/PHARM/92.559, Quality control Bacopa monnieri, by HPTLC and SFC.Acta. methods for Medicinal Plant Material, Chromatography,2006; 17: 125. WHO/PHARM,Geneva,1992. 6) Harbone, J.B., In Phytochemical methods: A 2) Roodenrys,S. Booth,D. and Bulzomi, S., Guide to Modern Technique of Plant Analysis, Chronic Effects of Brahmi (B. monnieri) on Champman and Hall, 1973,Pg 4-7. H u m a n m e m o r y . 7) Kurth,R., In Thin Layer Chromatography, Neuropshycopharmacology,2002; 27(2): 279- Acedemic Press, London,1964. 281. 8) Stahl, E., In Thin Layer Chromatography, 3) Agarwal,A.,Sharma, A., and Dubey,G.P., Age George Allen an Unwin Ltd,2nd ed.1969,1-16.
32 UJPAH Vol. I No. 12 June 2012
Study of Antimicrobial Activities and Optimization of Plant Extract Preparation of Mangifera indica; The King of Fruits
*Chandan Prasad1 , Sudhir K. Srivastava1 , R.P.Mishra2 Deepanjali Saxena3 ,Omkar Shukla1 and Digvijay Singh1 1Department of Chemistry, DAV college Kanpur, 2 R & D Division, M.R.D. Life science, Lucknow, 3 D.Y. Patil University ,Mumbai.
Abstract - The cold water, hot water, methanol Java, Sumatra and the Malay Peninsula. The most- suspension, Methanol column & methanol soxhelt cultivated Mangifera species, M. indica (mango) extract of Mangifera indica peel powder was has its origins in India and Myanmar. Chemical subjected to antimicrobial bioassay. Antibacterial constituents of MI are always of an interest. The activity was assayed against three pathogens different chemical constituents of the plant are namely Pseudomonas aeruginosa.E.coli and especially the polyphenolics, flavonoids, S.aureus for aqueous and methanolic extract of triterpenoids, Mangiferin a xanthone glycoside dry powder of M.indica peel. Among all hot water (major bio-active constituent) isomangiferin, extract showed Maximum activity against tannins & gallic acid derivatives. The bark is S.aureus with 2.20 cm of zone of inhibition. reported to contain protocatechic acid, catechin, Methanol suspension extract was best giving large mangiferin, alanine, glycine, ã -aminobutyric acid, zone of inhibitation against all the pathogens. The kinic acid, shikimic acid and the tetracyclic MIC of cold water was 4.28 mg/ml against triterpenoids cycloart-24-en-3â ,26diol, 3- Pseudomonas aeruginosa followed by methanol ketodammar-24 (E )-en-20S,26-diol, C-24 epimers column was 10.71 mg/ml against E.Coli and of cycloart-25 en 3â , 24, 27-triol and cycloartan-3â , S.aureus . 24, 27-triol.(Chen et. al, 2004). Indicoside A and B, Keywords - Mangifera indica , Antimicrobial manghopanal, mangoleanone, friedelin, cycloartan-3â -30-diol and derivatives, mangsterol, Introduction manglupenone, mangocoumarin, n-tetacosane, n- Mangifera indica (MI) also known as mango, aam, heneicosane, n-triacontane and mangiferolic acid has been an important tree in the Ayurvedic and methyl ester and others isolated from stem bark of indigenous medical systems for over 4,000 years. MI. Mangostin, 29-hydroxy mangiferonic acid and Mangoes belong to genus Mangifera which mangiferin have been isolated from the stem bark consists of about 30 species of tropical fruiting together with common flavonoids. The flower trees in the flowering plant family Anacardiaceae. yielded alkyl gallates such as gallic acid, ethyl According to Ayurveda, varied medicinal gallate, methyl gallate, n-propyl gallate, n-pentyl properties are attributed to different parts of gallate, n-octyl gallate, 4-phenyl gallate, 6-phenyl- mango tree. Mango is one of the most popular of n-hexyl gallate and dihydrogallic acid. Root of all tropical fruits. Mangiferin, being a polyphenolic mango contains the chromones, 3-hydroxy-2-(4'- antioxidant and a glucosyl xanthone, has strong methylbenzoyl)-chromone and 3-methoxy-2-(4'- antioxidant, anti lipid peroxidation, methyl benzoyl)-chromone. The leaf and flower immunomodulation, cardiotonic, hypotensive, yield an essential oil containing humulene, wound healing, antidegenerative and antidiabetic elemene, ocimene, linalool, nerol and many activities (Shankarnarayanan et. al, 1979). The others. The fruit pulp contains vitamins A and C,â - genus Mangifera originates in tropical Asia with carotene and xanthophylls. An unusual fatty acid, the greatest number of species found in Borneo, cis-9, cis-15-octadecadienoic acid was isolated
33 UJPAH Vol. I No. 12 June 2012 from the pulp lipids of mango. Phenolic Bacterial cultures of Staphylococcus aureus (Gram antioxidants, free sugars and polyols isolated and positive), Pseudomonas aeruginosa (Gram analyzed from Mango (MI) stem bark. negative) and Escherichia coli (Gram negative) Quantitative analysis of the compounds has been were obtained from IMTECH, Chandigarh, and performed by HPLC and mangiferin was found to were sub-cultured on to petri plate containing be the predominant component. The natural C- nutrient agar media. Single colony was transferred glucoside xanthone mangiferin [2-C-â -Dgluco- in sterile 50 ml of nutrient broth and incubated at pyranosyl-1, 3, 6, 7-tetrahydroxyxanthone] C19 H 37 o C in shaker incubator at 140 rpm for 14 hrs. 18 O 11 ; Mw, 422.35; anhydrous has been reported Bacterial cells were recovered by centrifugation in various parts of MI leaves, fruits, stem bark, and were suspended in sterile distilled water; heartwood and roots (Andreas et. al., 2000). The concentration of pathogens was optimized by presence of a phenolic compound from leaves of maintaining OD to 0.1 at 600 nm before use. MI was named as homomangifirin. The extract of The antimicrobial activity of Mangifera indica peel mango showed a powerful scavenging activity of powder was determined by agar well diffusion hydroxy radicals and acted as a chelator of iron. It method against Staphylococcus aureus, also showed a significant inhibitory effect on the Pseudomonas aeruginosa and Escherichia coli. peroxidation of rat brain phospholipid and 10.0 ml nutrient agar media was poured in a sterile prevented DNA damage caused by bleomycin or petri dish, 100 µl of test organisms were spread on copper-phenenthroline system (Maxwell, 1997). the surface of media, wells were prepared with In vitro, antioxidant and free radical scavenging help of sterile borer, which were aseptically filled properties of a stem bark aqueous extract of by 30µl plant extracts with positive (Tetracycline; mango tree (MI), whose formulations are used in 50µg/ml) and negative control (autoclaved Cuba as food supplements under the brand name distilled water). Plates were incubated aerobically of Vimang. The potential anti-diarrhoeal activity of at 37 0 C for 14 hrs. The diameters of zones of methanolic (MMI) and aqueous (AMI) extracts of inhibition were measured. seeds of MI has been evaluated in experimental Active extracts obtained by agar well diffusion diarrhoea, induced by castor oil and magnesium assay were further subjected to determine the sulphate in mice. The results illustrate that the Minimum Inhibitory Concentration (MIC) required extracts of MI have significant anti-diarrheal for the bacterio-static effects by standard micro- activity and part of the activity of MMI may be dilution agar double layer methodology. This is attributed to its effect on intestinal transit. carried out by double agar gradient plate method. Nutrient agar (5.0 ml) was poured into sterilized Material and Methods petri dishes, leaving the plate in slanted position. Peels of Mangifera indica were purchased from After setting the media, another 5.0 ml of nutrient the local market of Gomti Nagar, Lucknow. The agar (along with plant extract; 5.0 mg/ml) was mango peels were dried in the incubator and added to the plates to make the level unity; thus grinded in electronic blender to fine powder and the plate contained an increasing concentration of were subjected to bioactive compounds plant extract along the diameter of the plate. Now extraction. The extract was obtained with help of the 70µl of prepared inoculums of cultures were various methods and solvents such as hot water spread. Plates were incubated in upright position extract, cold water extract, organic suspension at 37 o C for 14 hrs. Concentration gradient along extract, organic column extract and organic with the diameter was calculated for each mm. Soxhlet extract. All the extracts were dried up to visible colonies were observed, distance was crystal form and were dissolved in distilled water measured from top end and concentration of the at concentration of 500mg/ml. compound was calculated as MIC.
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Results Extraction was carried out using hot water extraction, cold water extraction, Methanol column, Methanol suspension and Methanol Soxhlet.
Extraction Method Yield obtained Color of the (grams) extract obtained
Hot water extraction 0.610 Dark yellow Cold water extraction 1.138 Dark brown Methanol suspension 0.610 Dark yellow Methanol column 1.329 Blackish brown Methanol Soxhlet 3.447 Dark brown Table 1- Yield of each extract of Mangifera indica peel powder
Antibacterial activities of all the extracts were assayed against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus by agar well diffusion method.
Organisms P. aeruginosa E. coli S. aureus Tetracycline(avg) 1.88 cm 2.20 cm 2.50 cm Extracts (500mg/ml) Hot water extraction 1.40 cm 1.30 cm 2.20 cm Cold water extraction 1.60 cm 1.80 cm 1.50 cm Methanol suspension 2.00 cm 2.10 cm 2.00 cm Methanol column 1.80 cm 1.70 cm 1.90 cm Methanol Soxhlet 1.70 cm 1.60 cm 1.50 cm Distilled water 0.00 cm 0.00 cm 0.00 cm
Table 2- Antibiogram of aqueous and methanolic extract against P. aeruginosa, E. coli and S. aureus.
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Extracts found to have inhibitory effects were tested for determination of MIC by agar double layer method against susceptible bacterial species.
Test organisms/ Minimum Inhibitory Concentration (in mg/ml) Extracts(500mg/ml) P. aeruginosa E. coli S. aureus
Hot water extraction 16.71 15.00 17.57 Cold water extraction 4.28 12.85 12.00 Methanol column 17.14 10.71 10.71 Methanol Soxhlet 15.00 25.00 19.28
Table 3- MIC (in mg/ml) of Mangifera indica peel extract (aqueous and methanolic) against P. aeruginosa, E. coli and S. aureus.
Discussion than that of the standard antibiotic tetracycline. Results of present investigation reveal the Tetracycline showed average zone of inhibition of antibacterial nature of methanolic and aqueous 2.50cm against all the test organism. extract of peel of Mangifera indica. The peel of References Mangifera indica was extracted in the dry powder Abubakar, M. E. 2009. Antibacterial efficacy of form using aqueous and methanol solvent. Data of stem bark extracts of Mangifera indica against Table 1 demonstrates that maximum yield was some bacteria associated with respiratory tract obtained by using methanol Soxhlet extraction infections. Sci. Res. & Essay 4 (10): 1031-1037 method. Ajila, C.M.; Leelavathi, K.; & Prasada Rao, U.J.S. Antibacterial activity was assayed against all three 2007. Improvement of dietary fiber content and pathogens namely, P. aeruginosa, E. coli and S. antioxidant properties in soft dough biscuits with aureus for aqueous and organic extracts of M. the incorporation of mango peel powder. J. Cereal indica peel and among all, hot water extract Sci 48(2): 319-326 proved maximum activity against S. aureus with Ashoush, I.S.& Gadallah, M.G.E. 2011. Utilization of 2.20cm of zone of inhibition. While methanol Mango Peels and Seed Kernels Powders as suspension extract was best extraction procedure Sourcesof Phytochemicals in Biscuit. World J. Dairy by giving large zone of inhibitions against all the & Food Sci. 6 (1): 35-42, pathogens selected in the study (Table 2, fig 3, fig Carvalho, A.A.T.; Vanetti, M.C.D.; Mantovani , H.C. 4, fig 5). The Minimum Inhibitory Concentration of 2008. Bovicin HC5 reduces thermal resistance of hot water extract, cold water extract, methanol Alicyclobacillus acidoterrestrisin acidic mango column and methanol soxhlet was carried out. pulp. J. App. Microbiol. 104(6): 1685-1691 Cold water recorded MIC of 4.28mg/ml against P. Chanda, S.; Baravalia, Y.; Kaneria, M. & Rakholiya, aeruginosa followed by methanol column with K. 2010. Fruit and vegetable peels - strong natural MIC of 10.71 mg/ml against E. coli and S. aureus. source of antimicrobiolbics. Int. J. Antimicrobiol. Sterile distilled water which was used as negative Agents: 444 - 450. control showed no zone of inhibition in any test Engels, C.; Knödler, M.; Zhao, YY.; Carle, R.; Gänzle, organism. The efficacies of all extracts were less MG.; Schieber, A. 2009. Antimicrobiolbial Activity
36 UJPAH Vol. I No. 12 June 2012 of Gallotannins Isolated from Mango (Mangifera Mirghani, E.S.; Yosuf, F.; kabbashi, N.A.; Vejayan, J. indica L.) Kernels. J. Agric. Food Chem 57(17):7712- & Yosuf, Z.B.M. 2009. Antibacterial Activity of 7718 Mango Kernel Extracts. Journal of Applied Garrido, G.; González, D.; Lemus, Y.; García, D.; Sciences, 9: 3013-3019. Lodeiro, L.; Quintero, G.; Delporte, C.; Núñez- Nikhal, S.; Mahajan, S.D. 2010. Evaluation of Sellés, AJ.; Delgado, R. 2004. In vivo and in vitro antibacterial and antioxidant activity of Mangifera anti-inflammatory activity of Mangifera indica L. indica (leaves). J. Pharm. Sci. & Res. 2(1): 45-47 extract (VIMANG). 50(2): 143-149 Ravi, V.; Prabhu, M.; Subramanyam, D. 2001. Gupta, Charu; Garg .P, Amar; & Uniyal, Ramesh. Isolation of bacteriocin producing bacteria from 2008. Antibacterial activity of Amchur (Dried Pulp mango pulp and its antimicrobiolbial activity. J. of Unripe Mangifera indica) extracts on some food Microbiol. & Biotech. Res. 1(2): 54-63 borne bacteria. J. Pharm. Res. 1(1): 54-57 Sairam, K.; Hemalatha, S.; Kumar, A.; Srinivasan, T.; Juliana, A. S.; Zeila, P. L.; Hélio, K.; Alba, R.; Ganesh, J.; Shankar, M.; Venkataraman, S. 2003. Monteiro, S. B.; Lourdes, C. d. S.; Wagner, V. & Evaluation of anti-diarrhoeal activity in seed Clélia, A. H. L. 2009. Molecules.14: 1098 - 1110 extracts ofMangifera indica. J. Ethanopharma. Khammuang, S. & Sarnthima, R. 2011. Antioxidant 84(1): 11-15 and antibacterial activities of selected varieties of Sowmiya, S.; Soundarapandian, P.; & Rajan, S. thai mango seed extract. Afr. J. biomed. Res. 21(1): 2009. Bioactive Studies of Mangifera indica 37 - 42 aganist Bacteria Isolated from Urine Samples. Mahida, Y. & Mohan, J. S. S. 2007. Screening of Current Res. J. Bio.Sci. 1(3): 139-143 plants and their potential antibac activity against Susy Mary Souto de Oliveir; 1 Vivyanne S. Falcão- staphylococcus and salmonella spc. Natural Prod. Silva; 2 José P.Siqueira-Junior; 2 Maria José de Radiance. 6(4): 301-305 Carvalho Costa; 3 Margareth de Fátima F. de Melo Masibo, M & He, Q. 2009. In vitro antimicrobiolbial Diniz. 2011. Modulation of drug resistance in activity and the major polyphenol in leaf extract of Staphylococcus aureus by extract of mango Mangifera indica L. Malaysian J. Microbiol. 5(2): 73- (Mangifera indica) peel, Brazilian J. 80 Pharmacognosy. 21(1): 190-193.
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Evaluation of Toxic Metals in Hepato Protective Herbs Kasni , Bhringraj ,Makoi,Kutki and Kalmegh used in Ayurvedic Medicines
Arunesh Kumar Dixit and Shalini Pandey E-mail: [email protected]
Abstract - Exposure to toxic metal has become an increasingly recognized source of illness in human Introduction beings world wide. Lead and Mercury are naturally Exposure to toxic metal has become an occurring elements in the earth's crust and are increasingly recognized source of illness in common environmental Contaminants. Use of human beings world wide (1). Liver is a vital herbs and herbal medicines to relieve and treat organ among the vertebrate bodies involved in many human diseases (liver infection, kidney at least 500 body functions is also not Cough, and ascites etc) is increasing around the untouched with exposure of toxic metals leads world due to their mild features and low side to liver disorders. The heart and Kidney are effects. WHO estimates that about three quarters now a day being transplanted but the of the world population currently use herbs and transplantation of liver is still at large. other form of traditional medicines to treat their Advanced civilization and luxury in life are diseases. The Ayurvedic system of medicine bringing a lot of testing and trails to our body recommends various crude drugs for treatment of inform of pollution. Even the innocent human hepatic disorders. Therefore it is important to have being without taking toxic substances in the good quality control and good agriculture and food or water ,their blood contents are collection practices for medicinal herbs to protect enriched with pollutants like Pb,As,Cd, and Hg the consumers from potential contamination of through the inhalation of polluted atmosphere heavy metals present in our environment due to triggered by industrial exhaust, cosmetics, their toxicities to human. automobiles etc (Brag and Bettega 1990 A study has been done on accumulation of toxic willians 1984, Muril etal 2001) (2)(3)(4). Use of metals, namely Pb, Cd, Hg and As in herbs Eclipta herbs and herbal medicines to relieve and treat alba (Bhrinraj), Solenum nigrum (Makoi), many human diseases (liver infection, kidney Andrographis paniculata (Kalmegh), Picrorhiza Cough, and ascites etc) is increasing around kurroa (Kutki) and Cichorium intybus (Kasni). the world due to their mild features and low These plants are recommended as side effects. WHO estimates that about three hepatoprotective in Ayurvedic system of quarters of the world population currently use medicine. herbs and other form of traditional medicines Heavy metals ( Pb, Cd, Hg & As) become toxic to treat their diseases. The Ayurvedic system of medicine recommends large number of plants when they are not metabolized by the body and and formulations to have hepatoprotective accumulate in the soft tissues leads to liver activity. Nearly 160 phytoconstituents from toxicity. Heavy metal level was estimated in Kasni, 101plants have been claimed to possess liver Bhringraj, Makoi,Kutki and Kalmegh collected protecting activity .In India, more than 87 from wild and traders. plants are used in 33 patented and proprietary Keywords - Hepatoprotective herbs, Eclipta alba muti-ingradient plant formulations ( handa (Bhrinraj), Solenum nigrum (Makoi), Andrographis etal., 1986)(5) for treatment of hepatic paniculata (Kalmegh), Picrorhiza kurroa (Kutki) disorders. The presence of excess heavy and Cichorium intybus (Kasni).Toxic Metals, metals in Ayurvedic drug in JAMA article ( Atomic Absorption Spectrophotometer (GFAAS & Saper etal,2004) it is important to have good HydrEA mode). 40 UJPAH Vol. I No. 12 June 2012 quality control and good agriculture and collection paniculata ( Kalmegh), Picrorhiza kurroa ( Kutki) practices for medicinal herbs to protect the and Cichorium intybus (Kasni) plants are consumers from potential contamination of heavy commonly used in polyherbal formulation for metals present in environment due to their treatment of liver disorders. toxicities to human. Therefore, we are selecting (Table 1- shows the medicinal uses of Kasni,, some hepatoprotective herbs to analyse their Bhringraj, Makoi,Kutki and Kalmegh for the heavy metal contents. Eclipta alba (Bhrinraj), treatment of hepatic disorders and other diseases) Solanum nigrum ( Makoi), Andrographis Table 1 Sr.No Name of the plant Part used Medicinal uses Reference
1.0 Kasni, Seeds jaundice, liver Nadeem Naseem enlargement, gout and (et al 2009)(6) rheumatism. 2.0 Bhringraj Aerial part Edema, hepatitis, The wealth Of India rheumatic joint pains (1992)(7)
3.0 Kutki root Hepatoprotective Anandan (etal 1999 (8) 4.0 Makoi Aerial part Hepatoprotective Prashanth et al(2001) (9) 5.0 Kalmegh Aerial part Hepatoprotective Abhishek et al(2010) (10)
Material and Methods Sample digestion Sample collection 1.0 gm of powdered samples were ashed inside All five herbs Eclipta alba (Bhrinraj), Solanum the muffle furnace at temperature 450±50 degree nigrum ( Makoi), Andrographis paniculata ( C and digested in 10 ml of conc. HNO3 and Sulfuric Kalmegh), Picrorhiza kurroa ( Kutki) and Cichorium acid (4:1) mixture until a clear solution was intybus (Kasni) are obtained from different obtained. Now filter the digested samples into 100 sources of wild and trader. ml conical flask and make the volume upto 100ml by adding double distilled demonized water. Sample preparation Kutki (Part used -root) was washed thoroughly Atomic absorption spectrophotometer with de-ionized distilled water, dried in a The lead and cadmium content were estimated shade..Dust and dirt were removed from Aerial with GFAAS) and As and Hg were estimated part of Bhringraj, Makoi, Kalmegh and seeds of HydrEA technique of Analytik jena (novAA 400). kasni through sifter. All five herbs are compressed The instrument was calibrated with standard into a powder separately with the help of a grinder solutions using the different concentration mode. (Zurera et al., 1987)(11).
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Results and Discussion automobile emission, municipal incinerators etc. Heavy metals as environmental contaminants are This paper deals with the heavy metal not a recent phenomenon. They are ubiquitous in concentration were estimated Eclipta alba trace concentrations in soil and vegetation. In fact (Bhrinraj), Solanum nigrum (Makoi), Andrographis many are required by plants and animals as paniculata (Kalmegh), Picrorhiza kurroa (Kutki) micronutrients. The man made sources of metal and Cichorium intybus (Kasni) are obtained from contamination are mainly associated with certain different sources of wild and trader. industrial activities, agriculture practices, Table 2
Name of Part Pb content Cd content As content Hg content (ppm) (ppm) (ppm) (ppm the herb used Source Source Source Source
Wild Trader Wild Trader Wild Trader Wild Trader
Kasni, Seeds 1.18 2.88 0.16 0.28 0.96 0.88 0.12 0.25
Aerial Bhringraj 2.42 3.85 0.11 0.22 0.48 1.16 0.32 0.35 part
Kutki Root 3.32 4.51 0.15 0.35 0.39 0.45 0.22 0.30
Makoi Aerial 2.05 2.55 0.23 0.40 0.58 0.41 0.26 0.40 part
Kalmegh Aerial 2.07 1.96 0.1 8 0.42 0.41 0.22 0.22 0.28 part
Lead (Pb) immune system ( Heyes,1997)(13). Among the investigated hepatoprotetive plants Picrorhiza kurroa ( Kutki) collected from trader Cadmium (Cd) exhibited higher Pb concentration that is 4.51 ppm In studied plant, Cd concentration ranged between and Cichorium intybus (Kasni) collected from wild 0.11 ppm in Eclipta alba (Bhrinraj) and 0.42 ppm in possess minimum concentration of Pb that is 1.18. Andrographis paniculata ( Kalmegh). The permissible (Table -2). The permissible limit set by FAO/WHO limit set by FAO/WHO (1984) (12) in edible plants (1984)(12) in edible plants was 0.43 ppm. However was 0.21 ppm. However for medicinal plants limit for medicinal plants limit was 10 ppm set by was 0.3ppm set by China,Malaysia,Thialant and China,Malaysia,Thialant and WHO. In India the WHO. In India the permissible limit of Pb as per PFA is permissible limit of Pb as per PFA is 10ppm. Pb 1.0ppm. Cd causes both acute and chronic poisoning causes both acute and chronic poisoning and also and also poses adverse effect on kidney, liver poses adverse effect on kidney,liver vascular and vascular and immune system (Heyes,1997)(13). 42 UJPAH Vol. I No. 12 June 2012
Arsenic (As) Toxicol.Pharmacol.,130:219-226 The range of As in studied plant was high with a 4 Williams,R.P.J., 1984. Zinc: What is its role in minimum of 0.22 ppm in Andrographis paniculata biology? Endeavour.New Series,8: 65-70. (Kalmegh) and maximum of 1.16 in Eclipta alba 5 Handa SS, Sharma A, Chakraborti KK. Natural (Bhrinraj) The permissible limits of heavy metals in products and plants as liver protecting drugs. Ayurvedic drugs as per WHO and FDA Arsenic (As) is Fitoterapia 1986, 57(5): 307-352. 3 10 ng/g (Radhika Singh-2008)(14) 6 Nadeem Naseem,Malik Shah Zaman Latif ,Munir Tahir , Abdul Khalique Mercury (Hg) Naveed,Maqbool Hassan,S A Malik The range of Hg varied with value between 0.12ppm Hepatoprotective effect of Cichorium Intybus in Cichorium intybus (Kasni) and 0.40 ppm in linn (Kasni) Extracts against Carbon Solanum nigrum ( Makoi) The permissible limits of Tetrachloride induced Liver Damage. heavy metals in Ayurvedic drugs as per WHO and Journal of Rawalpindi Medical College (JRMC); FDA for mercury is 1 ì g/g(Radhika Singh-2008)(14) 2009;13(2):53-55 7 The wealth Of India: A Dictionary of Indian Conclusion Raw Materials and Industrial Products- Raw This study reveled that investigated Materials Series, Publications & Information hepatoprotective plants are exhibited the toxic Directorate ,CISIR, New Delhi, Revised Ser, metal contamination in range of permissible limit 1992; vol-3(Ca-Ci); pp.215-216. except in arsenic whose main reason is the industrial 8 Anandan,R.,R. Deepq Rekha and T.Devaki, pollution and contaminated water. It is also clear 1999. Protective effect of Picrorrhiza. Kurroa from this study that the plants collected from the on mitochondrial glutathione antioxidant trader shown higher concentration of toxic metal system in D-galactosamine induced hetatitis content than collected wild. As uses of in mice. Cur.Sci.,76:1543-1545 hepatoprotective herbs are increasing regularly. 9 Prashanth Kumar V, Shashidhara S, Kumar Medicinal plants today are cultivated commercially MM, Sridhara BY. Cytoprotective role of in the contaminated environment, due to the Solanum nigrum against gentamicin- industrialization resulting in contaminating soil, induced kidney cell (Vero cells) damage in water and air and finally to the cultivated plant. It is vitro. Fitoterapia 2001; 72(5): 481-486. clear from this paper; the plant which is collected 10 Abhishek Niranjan, S.KTewari , Alok Lehri: from the wild has less concentration of heavy metal Biological activities of Kalmegh (Andrographis content than plants collected from trader. paniculata Nees) and its active principles-A Therefore today's need is to promote medicinal reviw.Indian Journal of Natural Products and plant cultivation followed by good agricultural and Resources Vol.1(2),June2010,pp.125-135 collection practices ( GACP) and have good quality 11 Zurera, G.; Estrada, B. and Rincon, F. (1987): control practices for herbal medicines screening in Lead and Cadmium contamination levels in order to protect consumers from liver toxicity. edible vegetables. Bull Environ Contam Toxicol 38: 805-812. References 12 FAO/WHO (1984). Contaminants. In Codex 1 ND Lyn Patrick. Alt. Med. Rev, 2003, 8, 106- Alimentarius, vol. XVII, 128. Edition 1. FAO/WHO, Codex Alimentarius 2 Bray,T.M. and W.J. better,1990.The Commision, Rome physiological role of zinc as an antioxidant. 13 Heyes RB (1997). The Carcinogenicity of Free Radic.Biol.Med.,8:281-291 metals in humans. Cancer 3 Murill,P.,N. Alba, V.M. Perez-Alvarez,M. Causes Control. 8: 371-385. Shibayama and V.K. Tsutsumi.2001.Kupffer 14 Radhika Singh Toxic metal analysis in cells inhibitor prevents hepatic lipid Ayurvedic drug system. peroxidation and damage induced by carbon tetrachloride.Comp.Biiochem.physiol.C. XXXII National systems Conference,NSC 2008 17-19,2008. 43 UJPAH Vol. I No. 12 June 2012
Chemical Constituents from bark of Euonymus tingen (Celastraceae) S.C. Sati, Maneesha, D. Sati, Amita Sharma and I. P. Pandey* Department of Chemistry, H.N.B.Garhwal University, Srinagar, UK., India, Department of Chemistry DAV (PG) College, Dehradun, UK., India E-mail: [email protected]
Abstract - Quaillic acid (1) and 6-hydroxy 5-methyl Extraction and isolation 3',4',5'-trimethoxy aurone 4-O-á -L- Shade dried and coarsely powdered stem bark of rhamnopyranoside (2) were isolated from bark of Euonymus tingen (4 kg) was extracted thrice with Euonymus tingen. The structures of these 95% ethanol (5L) at 50°C (15 hrs) on a heating compounds were characterized by means of mantle. The reaction mixture was filtered off and chemical and spectral methods including the filtrate was concentrated under reduced advanced 2D NMR studies. These compounds pressure to yield brownish residue (440 g). This were first time isolated from this species. residue was fractionated with EtOAc (repeatedly Keywords- Euonyus tingen, Celastraceae, Quaillic 3-4 times), yield EtOAc soluble and insoluble acid, aurone fraction. EtOAc soluble portion (310 g) with n- hexane: Chloroform (93:7) as eluting solvent with Introduction increasing polarity of CHCl3 afforded two Euonymus tingens (Roxb.), vern. Kumkum, compounds in pure form tentatively designed as belongs to family Celastraceae is a evergreen tree compound 1 and 2 respectively. These compounds or large shrubs. The bark paste of the plant is were purified by recrystallization. useful in eye diseases. Number of Tingenone, hydroxytingenone, triterpenoid quinine methides Results and discussion has been isolated from Euonymus tingen [1,2,3] Compound 1 was obtained as an amorphous powder, m.p. 206-207°C, positive to Liebermann- Material and Methods Burchard and Molish reagent. The molecular General experimental procedure formula was determined as C30H46O5 by the MPs were incorrected, UV spectra were taken in LCMS at m/z 486 [M]+. Other fragmentation peaks MeOH using AlCl3 as shift reagent, IR recorded in were observed at 442, 414, 398 etc. The mass KBr on a Perkin Elmer FT-IR Spectrophotometer. spectrum of compound also exhibits Retro-Diels 1H-NMR were run at 300MHz using TMS as internal Alder fragmentation pattern, thereby furnishes standard and C5D5N and DMSO as solvent. 13 C- the peaks at m/z 246 and 240. The IR spectrum NMR recorded in 125MHz, using C5D5N and DMSO revealed absorption band at 3419 cm-1 (OH), 1724 as solvent, FAB-MS on JOEL. JMS700 Mstaion cm-1 (C=O) and 1678 cm-1 (C=C) of compound Spectrophotometer. showed the presence of thirty carbon signals in the broad band spectrum. In the 13C NMR spectrum, 6 Plant material methyl carbons at ä 10.2, 15.4, 17.2, 27.1, 33.2 and Stem bark (6 kg) of Euonymus tingen was collected 24.5 together data with 1H NMR spectrum. A from Bheti, Nand Prayag, District Chamoli during broad signal at ä 5.24 (H-12) in the 1H NMR is due to October 2009 and identified by taxonomist in the the presence of an olefenic proton which was Department of Botany, H.N.B. Garhwal University further supported by 13C NMR values atä 123.60 (C- Srinagar. A voucher specimen (GUH-8327) of the 12) and 145.02 (C-13). Moreover, two downfield plant is deposited at the Herbarium of signals at ä 174.8 (C-28) and 206.4 (C-23) were Department. clearly indicative to carbonyl carbons [4]. 44 UJPAH Vol. I No. 12 June 2012
Compound 1 has been identified as Quaillic acid showed that three methoxyl groups were present (Fig.1) on the basis of spectral data [IR, 13C, 1H- in the ring B, two hydroxyl and one methyl group NMR and Mass] and reported literature [5]. in the ring A. The positions of hydroxyl were Compound 2 was crystallized as yellow crystals shown to be at C-4 and C-6 of glycone by UV from CHCl3:MeOH, m.pt: 192-193°C. The LCMS of spectral shifts were C-5 and C-7 for attachment of compound showed molecular ion peak at m/z 520 methyl group in the ring A. C-5 position for methyl [M]+, which corresponds to the molecular formula group was confirmed by 1H NMR and 13C-NMR . C25H28O12. The other fragmentation peaks were Attachment of the sugar at C-4 of the aglycone observed at m/z 358, 357, 341, 330, 192, 138 and 92. was determined by comparing the 13C NMR The aglycone C19H18O7 was found to be aurone spectra of the glycoside with that of aglycone. No on the basis of colour reaction and UV absorption change in ëmax with the addition of AlCl3, band ëmax (nm) at 260, 330, 405 [6]. Aglycone was confirmed that it was a 4-O-glycoside. The analysed for three methyoxyl groups and their hydrolysis of the glycoside with B The sugar was presence was confirmed by a singlet at ä 3.63 (9H). identified as rhamnose by paper co- NMR showed presence of methyoxyl groups at 3', chromatography with authentic sample and by 1H- 4' and 5' position. 1H-NMR studies of the aglycone NMR spectral study of glycoside, a doublet at ä 1.10 showed the presence of three aromatic protons corresponding to 3H of rhamnosyl -CH3, broad suggesting a hexa-substituted aurone. A multiplet signal at ä 3.5-3.8 for 4H and a singlet at ä 4.2 due to at ä 7.88-8.00 (2H) was due to C-2' and C-6' protons -C-1" protons of rhamnose. This led to the and a singlet at ä 6.23 (1H) for C-7 protons. A singlet formulation of the compound B-5 as 6-hydroxy- at ä 1.52 (3H) and ä 6.75 (1H) was assignable to 3H of 5,3',4',5'-tetramethoxy aurone 4-O-á -L- Me group and benzyne proton (-CH-) respectively. rhamnopyranoside. The sugar Structure of The above position of various groups was further compound B-5 was also confirmed by comparative confirmed by 13C NMR spectral analysis. study with the authentic sample (Kesari et al., On acetlyation aglycone formed diacetate, m.p. 2004). The compound has been identified as 6- 104°C indicating the presence of two hydroxyl hydroxy 5-methyl 3',4',5'-trimethoxy aurone 4-O- groups. Mass spectral data showed a molecular á-L-rhamnopyranoside (Fig.2). ion peak at 520. Two fragments of m/z 192 and 166
OCH HO 3 O
CH OCH3
H3C COOH OCH3 O O OH H3C O HO HO CHO OH Fig. 1 OH Fig. 2
45 UJPAH Vol. I No. 12 June 2012
13 1 Table- C (125 MHz) and H NMR (300 MHz) data of compound 1 and 2 in Pyridine (C5D5N) and DMSO-d6
References Wall (F.Celastraceae). Tetrahedron Lett. 23, 1. Gaur, R.D. Flora of District Garhwal, 1047-1050, 1962. Transmedia Srinagar Garhwal, 1999, 330. 4. Chen, Q.; Luo, J.G. and Kong, L.Y. 2. Brown,P.M.; Moir, M.; Thomson, R.H., King, Triterpenoid saponins from Gypsophilaa T.J.; Krishnamoorthy, V.; Seshadri,T. R. altissima L. Chem. Pharm. Bull. 58(3), 412- Tingenone and hydroxytingenone, 414, 2010. triterpenoid quinine methides from 5. Frechet D.; Christ, B.; Du Sorbier, B.M.; E.tingen. J. Chem. Soc., Perkin Trans. 1, 2721- Fischer, H. and Vuilhogne, M. 2725, 1973. Phytochemistry, 30, 927-931, 1991. 3. Krishnamoorthi,V.; Ramanathan,J.D. and 6. Mabry, T.J. and Markhom, K.R.; In Seshadri,T.R. Constitution of Tingenone, A Harbone,J.B.; Mabry,T.J. H. (Eds), The component of the stembark of E.Tingens flavonoids, Chapman Hall, London 7&9, 1970. 46 UJPAH Vol. I No. 12 June 2012
An in Vitro Antimicrobial Study of Punica Granatum Peel Against Diarrhoeal Diseases Dinesh Kumar*, R.K.Singha *Department of Biochemistry (Faculty of life sciences) Uttrakhand Technical University, Dehradun, Uttrakhand, aHead, Deptt of Biochemistry, SGRR institute of Medical and Health Sciences, Dehradun, *E mail: [email protected]
Abstract - Punica granatum (Punicaceae) is gastrointestinal disorders such as cholera, and commonly used in India as a traditional medicine dysentery. However, several of them have not for the treatment of pathogenic bacteria. The been investigated from a pharmacological point of present study investigates the antibacterial view to demonstrate their antibacterial activity of Pomegranate rind extracts (alcoholic properties, which could support their use as and aqueous) against various enteric pathogens. antidiarrhoeal remedies in traditional medicine Both Standard strains and clinical isolates of Vibrio (27). Multi drug resistance among cholerae, Entero toxigenic E. coli, Entero enteropathogens in various geographic regions pathogenic E. coli, Entero aggregative E.coli, present as a major threat in the control of diarrhea Salmonella and Shigella species along with few [8,9]. The rise in antibiotic-resistant pathogens has strains of Candida were used in the study. led to the development of new therapeutic agents Antimicrobial susceptibility testing was performed that are effective against these bacteria. Recently, following standard procedure by the Punch well there has been considerable interest in the use of technique. The results obtained were encouraging various plant materials as an alternative medicine as the ethanolic extract showed greater zones of to treat some of the enteric infections and many inhibition against the various enteric pathogens compounds of plant products have been tested in comparison with the aqueous extract. specifically targeted against resistant pathogenic Most significant inhibitory effect was seen against bacteria [10].Punica granatum is one of the oldest Shigella flexneri and Aeromonas hydrophila. known edible fruits [8] . In addition to its ancient historical uses, pomegranate is used in several Keywords - Punica granatum peel, antimicrobial systems of medicine for a variety of ailments [12]. activity, enteric pathogens. However, to date, very few studies have been conducted on the antimicrobial activity of P. Introduction granatum peels. Therefore, the present study was Diarrhea is a major public health problem in aimed to evaluate the antimicrobial activity of the developing countries and is said to be endemic in ethanolic (EtOH ) and aqueous (Aq) extract of P. many regions of Asia and is the leading cause of a granatum peel against various enteric pathogens high degree of morbidity and mortality in vitro. ,contributing to the death of 3.3 to 6 million children annually (19). Multidrug resistance among Material and Methods enteropathogens in various geographic regions Plant collection present as a major threat in the control of diarrhea Fresh fruit peels of Punica granatum were [8,9]. However, antibiotic resistance is a major collected from the Himalaya Drug Company, clinical cause of concern in treating infections Dehradun, and Uttrakhand, India. The collected caused by these microorganisms. Many readily fruit peels were washed under running tap water, available plants in India are used as traditional air dried, homogenized to a coarse powder and folklore medicine for the treatment of stored in air-tight bottles at 4oC.
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inhibition > 8 mm were considered to be sensitive. Preparation of the plant extract Experiments were performed in triplicates for Coarse powdered Pomegranate peel was each combination of extract and the bacterial extracted by ethanol using Soxhlet apparatus strain. Aqueous extract was prepared by steeping plant Sensitivity of bacteria to standard antibiotics material in chloroform-water mixture in the ratio The sensitivity pattern of the reference strains of 1:99 for 5-7 days by Maceration process. bacteria were compared with the five commonly employed antibiotics, viz. Ampicillin, Cefixime, Bacterial strains used in the study Chloramphenicol, Co-trimoxazole, Gentamicin Bacterial strains used in the study were the isolates obtained from clinical samples and standard Results and Discussion strains from dept of microbiology, Mahant The results of the present study were encouraging Indiresh Hospital, Dehradun, Uttrakhand. All the as ethanolic peel extract showed significant bacterial cultures, viz. Vibrio cholera ;Vibrio antibacterial activity against many enteric parahemolyticus, species of Shigella, Salmonella, pathogens tested in comparison with the aqueous Escherichia. coli, Aeromonas hydrophila, and few extract. The results of this study strengthen the Candida spp were used in the study. The reference claims of the available published data on this strains of bacteria were maintained on nutrient useful drug. Alcoholic extracts of Punica granatum agar slants [16], sub cultured regularly (every 30 appeared to be the most effective with the zone of days) and stored at 4°C. inhibition sizes ranging from 15 to 30 mm. The different bacterial cultures responded to standard antibiotics and aqueous/alcoholic extract in a Determination of antibacterial activity variable manner, resulting in zones of inhibition of Antibacterial activity was tested on Muller-Hinton 9-38 mm. Statistical analysis revealed that Agar (MHA) plates by employing Punch well aqueous /alcoholic extracts of Punica granatum method. Varying concentrations of the were better/equally effective to that of standerd pomegranate rind extracts were prepared antibiotics used in the experiment against some of (alcoholic and aqueous) by dissolving in Dimethyl the bacterial strains. Some enteric pathogens like Sulphoxide (DMSO) [20] to obtain a final E.coli, Shigella flexneri were more susceptible to concentration of 1%, 3%, 5%, 7% and 9%. against the alcoholic extracts than to cefixime, test organisms. The test inoculums were then chloramphenicol or cotrimoxazole. Shigella, swabbed uniformly onto the MHA plates and wells Salmonella species and E. coli were all inhibited to of diameter 6mm were punched out in each plate. a considerable extent by the peel extracts of 50ì l of each of these extracts were pipetted out Punica granatum. Considerable significant activity into these wells, the plates incubated upright at of ethanolic extract was observed on Aeromonas 370 C overnight. The sensitivity of different hydrophila, Shigella flexneri. and Candida albicans bacterial strains to aqueous and alcoholic extracts and that of aqueous extract on Aeromonas of the peel of Punica granatum was calculated by hydrophila and Shigella flexneri. Ethanolic extract measuring the diameter (in millimeters) of had shown considerable activity against Shigella inhibition zone. Readings were taken at the end of flexneri even with the use of 1%. Our study also 24hrs and 48hrs. Bacteria showing a clear zone of revealed that as the increase in concentration of the extract increased the inhibitory zone size.
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Antibacterial zone produced by aqueous and ethanol extracts of pomegranate peel against few medically important enteric pathogens
Sr. No. Test strains Extracts Concentration Of Extracts
1 % 3 % 5 % 7 % 9 % 1. Vibrio cholera Aq 10 12 15 16 16 Et 11 14 16 18 18 2. Vibrio parahemolyticus Aq 11 13 16 16 16 Et 9 11 11 14 14
3. Aeromonas hydrophila Aq 13 16 19 19 19 Et 15 19 20 22 23
4. E coli Aq 10 12 14 15 15 Et 15 15 15 18 20 5. Salmonella Aq 10 11 12 14 14 typhimurium Et 11 13 15 20 20
6. Shigella flexneri Aq 15 17 21 21 22 Et 22 26 27 29 29 7. Shigella dysentriae Aq 9 11 11 13 14 Et 10 12 13 13 14 8. Shigella sonnei Aq 10 11 11 12 12 Et 12 13 13 16 16 9. Shigella boydii Aq 9 10 13 16 17 Et 13 13 15 15 17 10. Candida albicans Aq 9 9 12 13 14 Et 15 18 20 24 27
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Conclusion extract using in vivo models. 2002, J Agric Traditional medicinal practice has been known for Food Chem, , 50, 4791-5. centuries in many parts of the world for the 3. Ricci D.; Giamperi .; Bucchini A.; Fraternale D.; treatment of various human ailments [21] The use Antioxidant activity of Punica granatum of antibiotics has revolutionized the treatment of fruits. 2006, Fitoterapia, 77, 310-12. various enteric bacterial infections. However, their 4. Sanchez .; Fonseca G.; Fuentes J.L.; Cozzi R.; indiscriminate use has led to an alarming increase Cundari E.; Fiore M.; et al. Assessment of the in antibiotic resistance among microorganisms, genotoxic risk of Punica granatum L. thus necessitating the need for development of (Punicaceae) whole fruit extracts. 2008, J novel antimicrobials. Recent years have witnessed Ethnopharmacol, 115, 416-22. a renewed interest in exploring natural resources 5. Related A.; LinksHeber D.; Seeram N.P.; for developing such compounds [22,24]. Medicinal Wyatt H.; Henning S.M.; Zhang Y.; et al. Safety plants are relied upon by 80% of the world's and antioxidant activity of a pomegranate population, and in India, the use of plants as ellagitannin-enriched polyphenol dietary therapeutic agents remains an important supplement in overweight individuals with component of the traditional medicinal system. A increased waist size. 2007, J Agric Food number of plants have been documented for their Chem, 55, 10050-4. biological and antimicrobial properties. The 6. Parmar H.S.; Kar A.; Medicinal values of fruit aqueous extracts of pomegranate peel have peels from Citrus sinensis, Punica granatum shown to have antidiarrhoeal activity as tested in and Musa paradisiaca with respect to animal models [25]. Most of the above-mentioned alterations in tissue lipid peroxidation and microorganisms are developing resistance to serum concentration of glucose, insulin and commonly employed antibiotics and are a thyroid hormones. 2008.; J Medicinal common cause of many enteric infections [26]. Food,11, 376-81. Thus the antimicrobial activity of the medicinal 7. Aviram M.; Rosenblat M.; Gaitini D.; Nitecki S.; plant reported in the present study is noteworthy Hoffman A.; Dornfeld L.; et al. Pomegranate considering the havoc created by these enteric juice consumption for 3 years by patients pathogens. In conclusion, peel of the Punica with carotid artery stenosis reduces common granatum exhibited significant antibacterial carotid intima-media thickness, blood activity. The peel of this plant has been in use for pressure and LDL oxidation. 2004,Clin Nutr, , many years as decoctions or infusions prepared in 23, 423-33. water to treat various other ailments. This study 8. Parmar H.S.; Kar A.; Protective role of Citrus thus provides a scientific basis for the use of these sinensis, Punica granatum and Musa aqueous /alcoholic plant extracts as home-made paradisiaca peels against diet induced remedies and their possible application in treating atherosclerosis. 2007, Nutrition Res, 27, 710- gastrointestinal disorders. Further studies may 18. lead to their use as safe alternatives to synthetic 9. Braga L.C.; Shupp J.W.; Cummings C.; Jett M.; antimicrobial drugs. Takahashi J.A.; Carmo L.S.; et al. Pomegranate extract inhibits References Staphylococcus aureus growth and 1. Farooq S. 555 Medicinal Plants : Field and subsequent enterotoxin production2005, J Laboratory Manual, (Identification with its Ethnopharmacol, 96, 335-9. Phytochemical and in vitro studies data.), 10. Naz S.; Siddiqi R.; Ahmad S.; Rasool S.A.; 2005, 144-145 Sayeed S.A. Antibacterial activity directed 2. Chidambaram M.K.N.; Jayaprakasha G.K.; isolation of compounds from Punica Singh R.P. Studies on antioxidant activity of granatum. 2007, J Food Sci, 72, 341-5. pomegranate (Punica granatum) peel 11. Zhang J.; Zhan B.; Yao X.; Gao Y.; Shong J. 50 UJPAH Vol. I No. 12 June 2012
Antiviral activity of tannin from the pericarp V.;Traditional Medicinal Knowledge in India - of Punica granatum L. against genital Herpes An Appraisal. 2010, Socio-Economic virus in vitro. 1995, Zhongguo Zhong Yao Za voices.report. Zhi, 20, 556- 22. Natural products and resources repository; 12. Olapour S.; Najafzadeh H.; 2010, Asian 2010, National Institute of Science Journal of medical Sciences 2(6) 266-270 Communication And Information 13. Julie Jurenka, MT (ASCP). 2008, Alternative Resources,CSIR,New Delhi; vol 1 pp 1-66 Medicine Review, Number 2, Review Article, 23. Silva N.C.C.; Fernandes Júnior A.; 2010The 13 (128), Journal of Venomous Animals and Toxins 14. Beginners Guide To Soxhlet Extraction. including Tropical Diseases, 16 , (3) ,pp 402- Anabolicminds Archive. April 2003 413 15. Singh J.; Maceration, Percolation and 24. Das1 K.; Tiwari R. K. S.; and. Shrivastava D. K.; Infusion Techniques of Extraction of 2010Journal of Medicinal Plants Medicinal and Aromatic Plants (MAPs). Research, Vol. 4(2), pp 104-111 Central Institute of Medicinal and Aromatic 25. Qnais E.Y.; 2007, Pharmaceutical Biology, Vol. Plants (CIMAP).Lucknow (India) 45, No. 9, pp. 715-720 16. Maintenance of Bacterial Strains. Science In 26. Jackson H. O. Onyuka.; 2011World Academy The Real World. Microbes In Action,1999, of Science, Engineering and Technology75 pp1-3 27. NCCLS. Methods for determining bactericidal 17. Richard S.;, Lynn steele-moore.; Goodwin A. activity of antimicrobial agents. Approved C.; Antimicrobial susceptibility testing guideline, 1999; M26-A. National Committee for protocols Clinical Laboratory Standards, Wayne,Pa. 18. Lalitha M.K.; Manual on Antimicrobial 28. Ahmet D.; Duman.;, Mustafa Ozgen.; Kenan Susceptibility Testing; 2004, Christian S. 2009 Dayisoylu , Nurcan Erbil and Medical College. Coskun Durgac Molecules. 19. online at: www.who.int/drugresistance/en 29. Bradshaw, L.J. Laboratory Microbiology,.; 20. Robert V.; Dimethyl Sulfoxide (DMSO)-A 1992, Saunders College Publishing: New "New" Clean, Unique, Superior Solvent; York, USA, 2000, American Chemical Society Annual 30. Daljit Singh Arora.; Gurinder Jeet Kaur.; 2007, Meeting. Journal Of Natural Medicines, Vol. 61(3). 21. Indira priyadarsini G.; Sowbhagya rani
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Evaluation of Antimicrobial Activity of Some Medicinal Plants Extract on Pathogenic Fungal Strains
M.K.Tripathi1 and I P Pandey2 1 SPUC, Central Institute of Agricultural Engineering, Bhopal, India 2 Department of Chemistry, DAV College Dehradun, India 1Email: [email protected]
Abstract - Medicinal plants are source of great Rhizopus arrhizus, in humans, it has caused economic value in Indian subcontinent. Thousands cutaneous zugomycosis in a surgical wound as well of plant species are known to have medicinal value as mucormycosis. Penicillium notatum also known and that the use of their different parts for cure of as Penicillium chrysogenum is a mold commonly specific ailments has been in vogue since ancient found in most homes. Penicillium is known as the times. We attempt here to isolate some active blue-green mould on bread, fruits, and nuts. compounds from plant materials and demonstrate their antimicrobial activity. Isolation of alkaloids Material and Methods from different sources; characterization of their Collection of Plant Material antimicrobial activity by disc agar method and MIC Plant material i.e. leaves of Neem, Clove oil, Ginger was determined by incorporating various and Garlic for purpose of alkaloid extraction were concentrations of extracts (1x, 2 x, 3x, 4x) in Potato collected from the different area of Dehradun. Dextrose broth (spectrophotometric method). Alkaloids were extracted by using different Keywords - Fungus, Antifungal, Medicinal plants. extraction methods.
Introduction Preparation of Neem Leaf Extract In India, medicinal plants are widely used by all The ethanolic extraction of neem leaf was sections of people either directly as folk remedies prepared according to the procedure described by or in different indigenous systems of medicine or Chattopadhyay 1998. The leaves were cut into indirectly in the pharmaceutical preparations of pieces, dried in an oven at 60oC for 9 hrs. Air dried modern medicines. Rig-Veda mentions 67 plants powder (1Kg) of Azadirachta indica leaves was having therapeutic effects, Yajurveda lists 81 mixed with 3L of 70% ethyl alcohol. This sample was plants and Atharveda 290 plants (Nab Chandra and extracted at a soxhlet solvent ratio of 1:10 for 6 hrs Manjula, 1992). In fact, plants produce a diverse in a sachet extractor. The solvent (70% ethyl range of bioactive molecules, making them rich alcohol) was then evaporated at a constant source for curing diseases. In the present study, temperature until a very concentrated extract was we have used Neem (Azadirachta indica), Garlic obtained. (Allium sativum), Ginger (Zingier officinale) and Clove oil (Eugenia caryophyllata) against different Preparation of Ginger, Garlic Extract fungus. Microsporum gypseum is a geophilic Dry pieces of ginger and garlic were put in oven at fungus with a world-wide distribution which may 50°C. By using electric blender or pestle or mortal cause infections in animals and humans, they were ground. Soaked the powder of garlic particularly children and rural workers during and ginger (20g) in 100 ml of sterile (Autoclaved) warm humid weather. Usually it produces a single water and allowed it to stand for 72 hours. After inflammatory skin or scalp lesion. Aspergillus that, the sample was filtered in other flask with the niger is one of the most common and easily help of filter paper and after filtration, the filtrate identifiable species of the genus Aspergillus with was stored in the cold room at 4°C and the its white to yellow mat later bearing black conidia. . remaining sample was again dissolved in the 52 UJPAH Vol. I No. 12 June 2012 respective solvents and left for the re-extraction degree of sensitivity was determined by from the sample. measuring the zones of inhibition of growth around the discs. Growth was inhibited in the area Preparation of Clove Oil of the disc to which the fungus was susceptible Clove oil was taken from market with and not around the area to which it was resistant. concentration, 3g/ml (U.K Pharma 4/2611, Behari Colony Delhi - 110032). Minimum Inhibitory Concentration (MIC) By Spectrophotometer Method Storage of Extracts MIC was determined by incorporating various All the plant extracts, ethanolic and water extracts concentrations of extracts (1x, 2 x, 3x, 4x) in Potato were stored at 4°C to bring them out at the time of Dextrose broth. 100µl of standard fungal inoculum putting antifungal activity. was added to each tube and incubated at room temperature for overnight. Suitable control was Fungal Strains Used also included. Potato Dextrose broth with 100µl of Fungal strains- C.albicans, M.gypseum, A.flavus, inoculum served as positive control. Potato P.crysoganum, R.arrhizus, A.niger and Dextrose broth alone served as negative control. P.griseafulvum were used for testing. The tubes for each extract were incubated overnight at room temperature for overnight. The Maintenance of Fungal Strains MIC was regarded as lowest concentration of the All the seven fungal strains in the laboratory were extract that did not show any viable growth cultivated on the surface of Potato Dextrose Agar overnight incubation (compared with control). slant and preserved in the refrigerator at 4°C and The spectrophotometric analysis method was brought out at room temperature whenever used at wavelength 650 nm. required for the study. Results and Discussion Anti bacterial activity of plant extract The seven microorganism strains (fungus) were Disk Diffusion Method used for the characterization of antifungal activity The disc diffusion method (Lorian, 1996)-filter of the extract from above samples, neem, garlic paper discs, 6.0 mm in diameter were charged with ginger, clove oil. The strains were, Candida albican appropriate concentrations of the plant extracts. [I], Microsporum gypseum [II], Aspergillus flavus The discs were stored dry in the cold. They may be [III], Penicillium chrysogenum [IV],Rhizopus prepared in the laboratory or purchased arrhizus [V], Aspergillus niger [VI], Penicillium commercially. After overnight incubation, the griseofulvum [VII].
Table 1- Inhibition zone (mm) of Neem extract at various concentration on some microorganisms.
Strains I II III IV V VI VII Conc.
1x 13 17 -- -- 10 -- 18 2x 10 ------10
3x 09 ------09
4x 07 ------
53
UJPAH Vol. I No. 12 June 2012
Graph 1- MIC values of Neem extract on various fungal strains
MIC values for Neem extract
0.4 0.35 I 0.3 II 0.25 III 0.2 IV 0.15 V 0.1 VI 0.05 VII Optical Density (650nm) 0 1x 2x 3x 4x Concentration
By using disc and agar well method, antimirobial fungal strains. Optical density measured by activity of ethanolic extract of Neem is found more spectrophotometer analysis was found to be very effective for C.albicans, M.gypseum, R.arrhizus, less for 1x concentration in case of Candida which P.griseofulvum for 1x dilution upto 4x shows the higher effect on growth of fungus concentration for C.albicans, P.griseofulvum strains.
Table 2- Inhibition zone (mm) of Garlic extract at various concentration on some micro organisms.
Strains
I II III IV V VI VII
Conc.
1x 17 12 15 15 15 14 18
2x 15 10 -- 13 15 10 --
3x 12 09 -- 10 12 -- -- 4x -- 07 -- 09 09 -- --
54 UJPAH Vol. I No. 12 June 2012
Graph 2- MIC values of Garlic extract on various fungal strains
MIC values of Garlic extract
0.6 I 0.5 II 0.4 III 0.3 IV
0.2 V VI 0.1 VII Optical Density (650nm) 0 1x 2x 3x 4x Concentration
Garlic extract shows greater hydrolyzing zone for 1x spectrophotometric method was found in M. concentration by diffusion methods, found to be gypseum, P. chrysogenum, R. arrhizus strains of effective in almost all the strains and upto 4x dilution fungus by using 1x concentration of garlic extract in case of C. albicans, M. gypseum, P. chrysogenum, which shows the minimum inhibitory concentration R. arrhizus strains. Decrease in O.D. by
Table 3- Inhibition zone (mm) of Ginger extract at various concentration on some micro organisms.
Strains I II III IV V VI VII
Conc.
1x 13 21 -- -- 12 -- 16
2x -- 19 -- -- 10 -- 14
3x ------08 -- 11
4x ------06 -- 10
55 UJPAH Vol. I No. 12 June 2012
Graph 3- MIC values of Ginger extract on various fungal strains
MIC value of Ginger extract
0.4 0.35 I 0.3 II 0.25 III 0.2 IV 0.15 V 0.1 VI 0.05 VII Optical Density (650nm) 0 1x 2x 3x 4x Concentration
Antimicrobial activity of ginger extract is found to this extract. Decrease in O.D. was observed upto be effective in case of C. albicans, M. gypseum, R. 4x concentration in R. arrhizus and P.griseofulvum arrhizus and P.griseofulvum fungal strains by disc strains (fungus) by using spectrophotometric and well diffusion method for 1x concentration of analysis.
Table 4- Inhibition zone (mm) of Clove oil extract at various concentration on some micro organisms.
Strains I II III IV V VI VII
Conc.
1x 34 20 47 36 30 43 20
2x 28 15 39 30 25 38 19
3x 25 13 28 23 22 27 17
4x 20 11 21 21 -- 20 15
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Graph 4- MIC values of Clove oil extract on various fungal strains
MIC values of Clove Oil extract
0.35 0.3 I 0.25 II 0.2 III 0.15 IV 0.1 0.05 V 0 VI
Optical Density (650nm) 1 2 3 4 VII Concentration
Clove oil extract was found to be most effective for References all the fungal strains having greatest hydrolyzing 1. Ayres, J.C.; Mundt, J.O. and Sandine, W.E. zone characterized by diffusion methods and (1980). Microbiology of Foods. San effective upto 4x (diluted) concentration. Francisco: WH Freeman and Company. Decrease in O.D. was seen in all the strains at 1x 2. Chattopadhyay, R.R. (1998). Possible concentration of alkaloids of clove extract by using biochemical mode of antiinflammatory spectrophotometric analysis. action of Azadirachta indica A Juss in rats. From this study we have concluded that the Indian J Exp Biol, 36, 418-20. antimicrobial activity of neem, garlic, ginger and 3. Lorian, V. 1996. Antibiotics in Laboratory clove alkaloids were effective for most of these Medicine, fourth ed. Williams and above pathogenic fungal strains. Clove extract Wilkins,Baltimore. Indust. Res., 62: 623. was found to be most effective for all fungal 4. Zaika, L.L. (1988). Spices and herbs: their strains and value of minimum inhibitory antimicrobial activity and its determination. concentration was also found to be low. J. Food Safety 9:97-118.
57 UJPAH Vol. I No. 12 June 2012 Phytochemical and Pharmacological Attributes of Phaseolus trilobus A Major Herbal Ingredient of Pharma Products Navpreet Kaur, Rashmi* and Y.C. Tripathi Chemistry Division, Forest Research Institute, P.O. New Forest, Dehradun, India *E-mail: [email protected]
Abstract - Phaseolus trilobus Ait. (Family: flavones and carotenoid pigments occurring in Fabaceae) commonly known as Ranmoong or different colour genotypes of garden plants Mudgparni, is found throughout the tropical and (Ambasta, 1986; Anon, 1985). Plants can be used as warm temperate regions of the world. It is an taxonomic markers for discovery of new important gradient of a well known ayurvedic therapeutic compounds (Agarwal, 1986; Raman, health promotive, diseases preventive product 2006). Herbal medicines have been used by the 'Chyavanprash'. It is widely used in ayurvedic mankind since time immemorial. Ayurveda, the system of medicine to treat jaundice, diarrhoea, oldest traditional system of India, reveals that haemorrhoids, dyspepsia, sedative, coolant, ancient Indians had a rich knowledge of medicinal anthelmintic and good for eyes. The plant is value of different plants. India has been endowed reported to possess beneficial effects as a tonic, with a very rich flora owing to the extreme diuretic, hepatoprotective, antioxidant and variations in climate and geographical conditions antimicrobial. It is also found to induce defence prevalent in the country (Galani et al., 2010; mechanisms against the exposure of UV-B Nandkarni, 1982). radiations. A wide range of active components Phaseolus is a genus of annual or perennial herbs including flavonoids, isoflavonoids, tannins, or twiners found throughout the tropics stigmasterol, vitamin K, vitamin C and proteins (Ambasta, 1986) and warm temperate regions of have been isolated from the different parts of the the world. About five species occur in India, three plant. The present article assesses the botanical of them are cultivated throughout the world description, traditional medicinal uses, mainly for their edible pods. Phaseolus trilobus Ait. phytochemical and pharmacological information syn Vigna trilobata (L) Verdc. (figure 1) popularly on Phaseolus trilobus. called as African gram or jungle mat bean belongs Keywords - Phaseolus trilobus, fabaceae, to family Fabaceae, is a medicinal plant widely used flavonoids, isoflavonoids, antioxidant activity, in Indian traditional system of medicine for curing traditional medicinal uses. jaundice, diarrhoea, haemorrhoids, dyspepsia, sedative, coolant and anthelmintic. Phaseolus Introduction trilobus (Anon, 1985; Vaidyaratnam et al., 1996) is The plant kingdom is a virtual goldmine of native to Asia and found throughout India, in potential drug targets and other active molecules forests at low altitudes and in open rich soil and on awaiting to be discovered. It has been estimated Himalayas up to 7000 ft. It also grows in Africa, that only 10-15% of the 7, 50,000 existing species of Australia, Madagascar, Mauritius and South higher plants have been surveyed for biologically America (Bhattacharya et al., 1998; Drury, 1958). active compounds (Agarwal, 1986). Phytochemistry has an important role in plant tissues in the detection and analysis of the products of secondary metabolism that may be formed in such tissues. In the past, phytochemistry has contributed to higher plant genetics in providing the means of identifying anthocyanin,
58 UJPAH Vol. I No. 12 June 2012
Tribe : Phaseoleae Subtribe : Phaseolinae Also placed in Papilionaceae Genus : Phaseolus Species : Trilobus
Morphology Primarily a regenerating annual (occasionally perennial) herb with (often) reddish, glabrous or rarely pubescent stems. It is a regenerating annual or perennial herb having numerous stems 0.6-0.9 m from a woody rootstock, long, prostate, wiry, slender, not at all twining, glabrous or more or less hairy. Leaves are alternate and trifoliate, petiole 3.8-7.5 cm. long, grooved, glabrous or with a few scattered hairs; stipules variable in length, 4-16 mm. long, ovate-oblong, subacute, attached above the base, ciliate, leaflets 1.3-2.5 cm. long, usually as broad as long, commonly 3-lobed (the middle lobe the largest and oblong, broadly spathulate, obtuse, the lateral lobes often oblong or more or less spathulate, obtuse or subacute), all pale green, membranous, glabrous or with few Vernacular names: In different parts of India hairs on the nerves, base sub acute; petiolules 1.5- Phaseolus trilobus is known by different names 2.5 mm. long, hairy; staples small, lanceolate, (Anon, 1985). acute, deciduous; bracteoles beneath the calyx English: Three-lobed Kidney Bean, Wild Kidney 3mm. long, glabrous or sparingly hairy, slightly Bean recurved. Flowers are medium sized, under 2cm, Hindi: Ranmoong, Mudgparni, Mugani, reddish or bluish on auxiliary racemose peduncles, Mugawana, Mungani, Trianguli linear; Pods cylindrical, 1.5-5 cm long, 2.5-3 mm Sanskrit: Aranyamudga, Hrasva, Kakamunga, wide, glabrous to sparingly pubescent with short Karanjika. Koshila, Kshudrasaha, Kurangika, ad-pressed hairs, black when ripe seeds are 6-12 Mudgaparni, Shimbi, Vanamudga, Vanya and are uniform with a dark brown shining seed Bengla: Mugani coat (Ravindran et al., 2008; Ghosh, 2000; Hajra et Kanarese: Kohasaru al., 1995; Jadhav, 2008). Gujarati: Adabaumagi, Adavada, Magavala Marathi: Arkamath, Jangalimath, Ranamuga, Distribution Ranmath Phaseolus trilobus is native to Asia found in Tamil: Naripayar, Panipayar Bhutan, India, Indonesia, Burma (Myanmar), Telugu: Pillippersara Pakistan, Sri Lanka, Taiwan and Vietnam. It is Habitat: Paleotropics; Wild creepers similar to naturalized in Africa, Australia, Madagascar, green gram; Used as fodder. Mauritius and South America. It is found Used Part: Whole plant and Fruits. throughout India, in forests at low altitudes and in open rich soil and on Himalayas up to 7000 ft. The Taxonomic classification (Anon, 1985): plant occurs in grassland, on road verges, irrigated Family : Fabaceae land, drain edges and banks of irrigation channels. (alt. Leguminosae) It is also grown in India, Pakistan and Sudan as a Subfamily : Faboideae 59 UJPAH Vol. I No. 12 June 2012 short-term pasture and green manure crop Quercitin, kaempferol, vitexin, isovitexin have (Murray, 1984; Duthie, 1960; Kirtikar et al., 1981; been reported (Ambasta, 1986; Verma et al., 1993). Hutchinson, 1964). The fruit of this plant is found to contain proteins, minerals and vitamin K and C (Fursule et al.,2010; Phytochemistry of Phaseolus trilobus Galani et al., 2010). The plant also contains Phaseolus trilobus due to their numerous friedelin, epifriedelin, stigmasterol and tannins. medicinal uses have attracted the attention of The bean contains methionine, tryptophan and chemists the world over and thus subjected to tyrosine. The seed protein contained lysine, valine, thorough chemical examination. The plant has leucine and phenylalanine. Molecular structures of been investigated for its various biologically active some of the important chemical constituents are chemical constituents. From roots, seed, leaves depicted below (Drury, 1958; Alston, 1964; and seed coat are dalbergioidin, kievitone, Ambasta, 1986; Williams et al., 1995): phaseollidin and flavonoid glycosides viz.
[Quercetin] [Vitexin]
[Kaempferol] [Dalbergioidin]
60 UJPAH Vol. I No. 12 June 2012
H3CCH3
HO O
H OHO HO OH [Kievitone] [Friedelin]
HO O
O
OH
[Phaseollidin] [Methionine]
[Tryptophan] [Tyrosine] 61 UJPAH Vol. I No. 12 June 2012
Traditional medicinal uses contributing to the maintenance of the structural It is grown in India, Pakistan and Sudan as a short- integrity of the leaf cell components (Raman, term pasture and green manure crop. Pods are 2006). eaten as a vegetable and cooked seeds reported to be used as delicious food (Bhattacharya et al., Market value 1998) by tribal of Chattisgarh. Phaseolus trilobus is A significant number of health and therapeutic extensively used by tribal people of Nandurbar products with Phaseolus as such and as one of the district of Maharashtra, in the treatment of major ingredients are available in the market as: jaundice and other liver disorders (Fursule et al., a. Chyavanprash: It is an important ingredient of a 2010). Plant is used as febrifuge in Bihar (Jadhav, well known Ayurvedic health-promotive and 2008). Leaves are considered as sedative, coolant, diseases -preventive product Chyavanprash. antibilious (Kumar, 2001) tonic and they are used in Chyavanprash is a powerful free radical scavenger cataplasms for week eyes (Agarwal, 1986). Leaves formulation and when taken regularly, claimed to are also applied in the form of paste to the eyes to build immunity, decreases fatigue, strengthens improve the sight, and also in ophthalmia and in digestive power, and keeps the memory bright hemorrhoids (Murray, 1984). Fruit is aphrodisiac, and the lungs clear (Anon, 2005). astringent, styptic, anthelmintic and good for b. Gold-360: Leaves of Phaseolus trilobus are used eyes. Its consumption cures inflammation, fever, as one of the ingredients in preparation of this burning sensation, thirst, piles, dysentery, cough, rejuvenative tonic. Beneficial health effects of this gout and biliousness (Chopra et al., 2002; Khare, formulation include whole body rejuvenation, anti- 2007). In Ayurveda, the juice of the plant is aging, hormone balance and immunity. prescribed in rat- bite fever. Roots are used for curing fever, cough, diarrhea, haemorrhoids, Conclusion ophthalmology and dyspepsia (Shukla et al., 2007). The present article describes the usefulness of Phaseolus trilobus plant which has a great impact Pharmacological aspect with regard to multidirectional pharmacological The plant has been pharmacologically assessed for applications in indigenous system of medicine. its hepatoprotective and antioxidant properties. Moreover, Phaseolus trilobus is a rich source of a Methanol and aqueous extracts of the plant variety of organic compounds of varying structural possess hepatoprotective activity comparable to patterns and due to their natural distribution are Silymarin. Antioxidant activity of the plant thus highly relevant not only for medicinal but also extractives has been confirmed using in vitro and for chemotaxonomic studies. The traditional in vivo antioxidant models viz. anti-lipid knowledge with its holistic and systematic peroxidation assay, superoxide radical scavenging approach supported by experimental base can assay and glutathione estimation in liver and found serve as an innovative and powerful discovery to be comparable with ascorbic acid (Fursule et al., engine for newer, safer and affordable medicines. 2010; Daniel et al., 2006). Studies were conducted Possibilities are still there for finding out further to determine the role of defense mechanism in biologically active constituents. Looking to the various parameters subjected to UV- B radiation in versatile traditional uses of Phaseolus trilobus and Phaseolus trilobus. In general, ultraviolet-B modern pharmacological reports the plant claims radiation can have negative impact on the growth immense potential for its utilisation in and development of plants. Here, in this study it pharmaceutical sector and thus its chemistry is was shown that exposure of Phaseolus trilobus to being widely studied. UV-B radiations activated several defense mechanisms in morphology, anatomy, UV References absorbing compounds and also induced the Agarwal, V.S (1986). Economic Plants of India, antioxidant defence mechanisms altogether Kailash Prakashan, Calcutta, pp282. 62 UJPAH Vol. I No. 12 June 2012
Alston, R.E (1964). Biochemistry of Phenolic Pradesh, Daya Publishing House, pp238-239. Compounds (J.B. Harborne, J.B.ed.) Academic Khare, C.P (2007). Indian Medicinal Plants: an press, London, pp171. Illustrated Dictionary, Springer Pvt. Ltd. (India), Ambasta, S.P (1986). The Useful Plants Of India, pp477. Publications and Information Directorate Council Kirtikar, K.R and B. D. Basu (1981). Indian Medicinal of Scientific and Industrial Research (CSIR), New Plants, Bishan Singh Mahinder Pal Singh, Delhi, pp677-678. Dehradun, 2nd ed., I: 794-795. Anon (2005). Medicinal Plant Kumar, S (2001). Flora of Haryana, Bishen Singh Resources of South-West Bengal, Mahinder Pal Singh, Dehradun, pp130. Research Wing, Directorate of Forests. Lindley, J (1981). Flora Medica, Ajay Book Service, Saraswaty Press Ltd. (Government of West New Delhi, pp253. Bengal Enterprise), Kolkata, pp182. Murray, J.A (1984). Plants and Drugs of Sind, Anonymous (1985). The Wealth of India: A Periodical Expert Book Agency, New Delhi, pp126. dictionary of Indian Raw Material and Industrial Nandkarni, A.K (1982). Indian Materia Medica, Products, Raw Materials, New Delhi, VIII: Ph- Re, Popular Prakashan, Mumbai, 3rd ed, I:pp 942. pp3-5. Raman, N (2006). Pharmaceutical Techniques, Bhattacharya, B and B.M. Johri (1998). Flowering (Raman, N. ed.) New India Publishing Agency, New Plants Taxonomy & Phylogeny, Narosa Publishing Delhi, pp1. Home, New Delhi, pp255. Ravindran, K.C, A. Indrajith, V. Balakrishnan, K. Chopra, R.N, S.L. Nayar and I.C. Chopra (2002). Venkatesan and G. Kulandaivelu (2008). Glossary of Indian Medicinal Plants. First ed. Determination of defence mechanism in National Institute o f S c i e n c e Phaseolus trilobus ait. Seedlings treated under UV- communication and Information Resources B radiation, African Crop Science Journal, 16 (2): 111 (CSIR), New Delhi, pp189. - 118. Daniel, J.B, H. Martin and L. Reinhard (2006). Shukla, P.K and O.P. Chaubey (2007). Threatened Phenotypic plasticity of cyanogenesis in lima bean Wild Medicinal Plants: Assessment, Conservation Phaseolus lunatus-activity and activation of â - and Management, Anmol Publications Pvt. Ltd., glucosidase, J. Chem. Ec.o, 32 (2): 261-75. New Delhi, pp107. Drury, H (1858). Useful Plants of India, Asylium Vaidyaratnam, P.S and A.V. Kottakar (1996). Indian Press, Mount Road, Madras, pp337. Medicinal Plants: a Compendium Of 500 Species, Duthie, J.F (1960). Flora of the Upper Gagnetic Orient Longman Ltd., Madras, V: pp370-373. Plain, Botanical Survey of India, 1: pp207. Verma, B.D, N. P. Balakrishnan and R.D. Dixit Fursule, R.A and S. D. Patil (2010). (1993). Flora of Madhya Pradesh, Botanical Survey Hepatoprotective and antioxidant activity of of India, 1: pp409. Phaseolus trilobus, Ait on bile duct ligation induced Williams, C.A, J. C. Onyilagha and J. B Harborne liver fibrosis in rats, J Ethnopharm., 416-419. (1995). Flavonoid profiles in leaves, flowers and Galani, V.J, B. G. Patel and D. G Rana (2010). stems of forty-nine members of the Sphaeranthus indicus Lin.: A Phytophamacological Phaseolinae, Bio. System. and Eco., 23 (6): 655-667. review, Int. J Ayur. Res., 1(4): 247-253. Ghosh, G.K (2000). Herbs of Manipur, A.P.H. Publishing Corporation, New Delhi, 1: 465-466. Hajra, P.K and B. Balodi (1995). Plant Wealth of Nanda Devi Biosphere Reserve, Botanical Survey of India, Calcutta, pp106. Hutchinson, J (1964). The Genera of Flowering Plants, Clarendon Press, Oxford, I: pp436-437. Jadhav, D (2008). Medicinal Plants of Madhya 63 UJPAH Vol. I No. 12 June 2012
About Taxus wallichiana Zucc (Talispatra) on Front Cover (Potential Anticancer Herbal Drug)
Hindi Name : Talispatra, Thunair English Name : Himalayan Yew Unani Name : Zarnab Constituents Botanical Name : Taxus wallichiana Zucc Yew contains a mixture of alkaloids Synonym : Taxus baccata Linn known as taxine and also diterpenses Family : Taxaceae (including taxol in some varieties), Description lignans, tannin and resin. Ever green tree, slow growing reaching The plant is the source of buccatin, one of 25mm in height. Has rust-red bark, dark the taxanes used as precursor of green needle- like leaves. The female plant paclitaxel (trade name-taxol) which has produces fleshy red cup-like fruit. proved active against ovarian, breast and colon cancer. It is also used in small doses Habitat to treat rheumatic and urinary problems. Yew grows throughout northern It has emenogogue and sedative temperate zones. More often found in properties. It is also used in hysteria, cultivation then in the wild. Lime rich soil is epilepsy and nervousness. favourable for its growth. Dr Maya Ram 64 UJPAH Vol. I No. 12 June 2012 Forthcoming Events
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Instructions to Contributors
The Universities' Journal of Phyto-Chemistry And the authors (First author) free of charge orders Ayurvedic Heights is a bi-annual Journal dealing for additional reprint should be in multiple of 25 with subjects viz., medicinal plants, natural and should be made along with the manuscript. products, phytochemicals beneficial in Human They will be charged for the manuscript, Health Protection, Nutrition and Plant Defence. postage and packing. Field Botany, Natural History, Holistic Approach, 7. It is further emphasised that the manuscripts Ayurveda, Toxicological, Therapeutical and Clinical should be neatly typed double spaced Aspects. It is open to research workers in India and throughout including tables, on pages of abroad. Original articles, Research articles, short uniform size with at least 2.5 cms margin on all communications and Letters to the Editor on sides. Author(s) should not break or hyphenate relevant topics are published in English. the words. Authors are also required to include: The copyright lies with the publisher. Publication 1. The chemical structure, formula and proprietary of papers will be on clear understanding that they name of novel or ill defined compounds. have not been submitted to another Journal for 2. The w/w yield of prepared extracts in terms of publication. starting crude material. The Journal has a Helping Board of Editors which 3. Complete formulation details of all crude drug would assist young research workers free of mixtures. charge for necessary correction and in preparation 4. The herbarium specimen number of the plant of proper format for the publication of the paper. (s) studied in case of less well known plants be In that event such paper/s may be addressed to cited using the collector and collection number 'The Director, Helping Board of Editors. (eg. Doe 123) as well as indicating the name of Universities' Journal of Phytochemistry and the herbarium institution where it has been Ayurvedic Heights. deposited. The papers are accepted for publication on the 8. Key words: Authors are requested to assign 3-6 advice of the referees. keywords to the manuscript preferabley taken 1. The typed manuscript should not exceed 10 from index Medicus of Excerpta Medica Index pages and tables & graphs should not exceed for abstraction and indexing purposes. They 10% of the typed manuscript. should be mentioned after abstract and before 2. Manuscript should be typed on one side of the introduction. paper with double space. Two copies of the 9. References to literature are to be arranged manuscript should be submitted. alphabetically and placed at the end of the 3. The usual format of a manuscript comprises of article, and referred to by name and year abstract (not exceeding 150 words). chronologically. Standard abbreviations listed Introduction, Keywords, Material and in the World list of Science Periodicals should be Methods, Observations/ Results, Discussion, used. A sample citation is given here: Singh, G.; Acknowledgments, References, Tables and Dhar, U. And Singhal, A.K.(1976). Origin of Figures. Kashmir Saffron - a possible clue from weeds 4. Tables and Figures should be double spaced on Sci.Cult., 42:485-487. separate pages, numbered consecutively in 10.For speedy publication, contributors are Roman numerals and placed at the end of the requested to submit their manuscript as manuscript. electronic files with two hard copies. The 5. Those students who want their papers to be storage medium required CDs. The software for evaluated, corrected and formatted may text should preferably be of MS word for address to the Director Helping Board only. Windows version 6.0 onwards or word perfect 6. The reprint of their articles will be supplied to version 5.1 or upwards. Coral Draw or any
66 UJPAH Vol. I No. 12 June 2012
compatible software or as picture in MS word [email protected] version 6 onwards may be used for giving 12.The publisher or editors have no responsibility illustrations. Image and PDF files are not for the statements, opinions and views acceptable. expressed by contributors in the Journal and in 11. All correspondence pertaining to Journal may case of any dispute. New Delhi shall be the place be addressed to the Chief Editor, E-mail: of jurisdiction for all legal purposes.
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