Publication Ref No.: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-9/NOV/006 ISSN

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1 PHARMACOGNOSTIC EVALUATION AND PHYTOCHEMICAL ANALYSIS OF THE TUBER OF AMORPHOPHALLUS PAEONIIFOLIUS Yadu Nandan Dey *1, Ajoy Kumar Ghosh 2 Yadu Nandan Dey 1 Central Council for research in Ayurveda and Siddha, Janakpuri, New Delhi, India 1 Gupta college of Technological Sciences, Ashram More, Asansol, West Bengal, India yadunandan132@gmail.com ABSTRACT The present research work discusses the development of a UV spectrophotometric method for Metronidazole. Simple, accurate and cost efficient spectrophotometric method has been developed for the estimation of Metronidazole (MND) in Tablet dosage form. The optimum conditions for the analysis of the drug were established. The maximum wavelength (λ max) was found to be 277nm. The percentage recovery of Metronidazole was in the 98.2± Beers law was obeyed in the concentration range of 1-10µg/ml. Calibration curves shows a linear relationship between the absorbance and concentration. The line equation y=0.0283x with r 2 of was obtained. Validation was performed as ICH guidelines for Linearity, accuracy, precision, LOD and LOQ. The sample solution was stable up to 36 hours. The proposed method may be suitable for the analysis of MND in tablet formulation for quality control purposes. Key Words: Phytochemical analysis, Amorphophallus paeoniifolius, Ash value, Physical constants Extractive values, INTRODUCTION In recent years, the use of herbal medicines worldwide has provided an excellent opportunity to India to look for therapeutic lead compounds from our ancient system of therapy, i.e. Ayurveda, which can be utilized for development of new drug. Over 50% of all modern drugs are natural product origin and they play an important role in drug development programs of the pharmaceutical industry 1. Ash values are used to determine quality and purity of a crude drug. Ash contains inorganic radicals like phosphates, carbonates, silicates of sodium, potassium, magnesium, calcium etc. Sometimes, inorganic variables like calcium 44

2 oxalate, silica, carbonate content of the crude drug affects Total ash value. Such variables are then removed by treating with acid like Hydrochloric acid and then acid insoluble ash value is determined. Extractive values are useful for the evaluation of the drug. It gives idea about the nature of the chemical constituents present in a crude drug. It is useful for the estimation of specific constituents, soluble in that particular solvent used for extraction. Loss on drying is the loss in weight in % w/w resulting from water and volatile matter of any kind that can be driven off under specified conditions. 2,3 The tubers of wild plants are highly acrid and cause irritation in throat and mouth due to excessive amount of calcium oxalate present in the tubers. The tubers are anodyne, antiinflammatory, antihaemorrhoidal, haemostatic, expectorant, carminative, digestive, appetizer, stomachic, anthelmintic, liver tonic, aphrodisiac, emmenagogue, rejuvenating and tonic. They are traditionally used in arthralgia, elephantiasis, tumors, inflammations, hemorrhoids, hemorrhages, vomiting, cough, bronchitis, asthma, anorexia, dyspepsia, flatulence, colic, constipation, helminthiasis hepatopathy, splenopathy, amenorrhea, dysmenorrhoea, seminal weakness, fatigue, anemia and general debility 4. The tuber is reported to have antiprotease activity 5, analgesic activity 6, cytotoxic activity 7, anti-inflammatory activity 8 and CNS depressants activity. 9 In view of this, an attempt has been made to study the pharmacognostic evaluation and phyto-chemical analysis of the different extacts of the tuber of Amorphophallus paeoniifolius. The objective of the present research is to prove the traditional uses of the plant A. paeoniifolius due to its active bioactive phytocontituents. Further it is planned to isolate the active phytoconstituents by using biologically action guided extraction procedure. MATERIALS AND METHODS Plant The tuber of Amorphophallus paeoniifolius (Dennst.) Nicolson var.campanulatus (Decne) (Areaceae), was collected from Asansol, West Bengal, India. The tuber of Amorphophallus paeoniifolius (Dennst.) Nicolson var.campanulatus (Decne) Sivadasan (Areaceae), was identified with the Herbarium of Botanical Survey of India, Botanic Garden, Howrah with ref no. CNH/I-I/ (272)/ 2008/ Tech. II/ 314. Preparation of extract The tuber of the plant was dried in shade and made to fine powder using a laboratory mill. The dried powder is stored in dessicator. The dry powder is extracted with methanol and petroleum ether using soxhlet extractor. PHARMACOGNOSTIC EVALUATION OF THE PLANT A. Ash value and extractive values The dried powder of the tubers of the plant were taken and the parameters like moisture content, total ash, acid-insoluble ash, alcohol(90%ethanol) and water soluble extractive values were determined. 10 B. Acid neutralizing capacity The Acid-Neutralizing capacity was carried out as per USP 31. All tests were conducted at temperature 37± 3 C. A ph meter was standardized using the 0.05M potassium biphthalate buffer. Magnetic stirrer was used to produce stirring rate 300± 30 rpm. 0.5 g of ash of tuber of Amorphophallus paeoniifolius was transferred to 250 ml beaker and 70 ml distilled water was added. It was mixed with magnetic stirrer for 1 min. Then 30 ml 1.0 N Hcl was added to the test solution with continuous stirring for 15 min. Excess Hcl was titrated with 0.5 N NaOH to attain a stable ph of 3.5. The number of meq of acid consumed was calculated by formula: Total meq = (30 N Hcl ) (V NaOH N NaOH ) 45

3 Where N Hcl and N NaOH are normalities of hydrochloric acid and sodium hydroxide respectively and V NaOH is volume of sodium hydroxide and the result were expressed as total meq per gm of substance. 11 C. Loss on drying The loss on drying procedure was carried out as per Indian Pharmacopoeia (1996) Method A. Weigh a glass-stoppered, shallow weighing bottle.transfer to the bottle 1 g of the sample, cover it and accurately weigh the bottle and the contents. Distribute the sample as evenly as practicable by gentle sideways shaking to a depth not exceeding 10 mm.place the loaded bottle in a hot air oven at 105 C for 1 hour with the stopper remains open. After 1 hour, close the stopper and allow it to cool to room temperature in desiccators before weighing. Weigh the bottle and the contents. D. Morphology of the Plant: A stout herbaceous plant with underground hemispherical depressed dark brown corm; leaves compound,large,solitary, petiole stout, mottled, cm long, leaflets cm long of variable width, obovate or oblong, acute, strongly and many nerved; male and female inflorescences contiguous, neuters absent, appendage of spadix sub-globose or amorphous,equally or longer than the fertile region, spathe campanulate, pointed,strongly,closely veined, greenish-pink externally,base within purple,margins recurved,undulate and crisped, male inflorescence subturbinate, female 7.5 cm or more long; fruits obovoid 2-3 seeded red berries. 12,13, PHYTOCHEMICAL TESTS The qualitative chemical tests for various phytoconstituents were carried out for methanolic and petroleum ether extracts of Amorphophallus paeoniifolius.the phyto chemical tests were performed for the presence of Alkaloids (Mayer s test, Dragendorff s test, Wagner's test, Tannic acid test), Glycosides(Free sugar, Raymond s test, Legal s test, Bromine water Test), specific glycosides like Saponin Glycosides(Froth Test, Hemolysis test), Anthrauinone Glycosides(Borntrager's test, Modified Borntrager's test), Cardiac Glycosides(Kedde s test, Keller killiani test), Cyanogenetic Glycosides, tannins & Phenolic Compounds(Gelatin test, Ferric chloride test, Vanillin Hydrochloride test, Alkaline reagent test, Mitchell s test), Flavonoids (Shinoda test, Zinc Hydrochloride reduction test, Alkaline reagent test), Proteins & Amino Acids(Millons test, Ninhydrin test), Sterols & Triterpenoids (.Libermann-Buchard test, Salkowski test), Carbohydrates (Molisch's test, Benedict's test, Barfoed's test, Camnelisation, Selwinoff s test, Tollen's test, Bromine water test, Fehling's test), Fats & Fixed Oils (Stain test, Saponification test) 14,15,16,17,18 RESULTS ( Refer Table No. 01, 02 and 03 ) Table 1 The physical properties of the extracts of the tuber Table 2 The Pharmacognostic parameters of the extracts of the tuber Table 3 Qualitative Phyto-Chemical Test Phytochemical screening of methanolioc extract indicated the presence of steroids, flavonoids, alkaloids and carbohydrates. Phytochemical screening of Petroleum ether extract indicates the presence of steroids, fats and fixed oil. DISCUSSION: From the above study it was found that the ash value of the dried tuber was found to be 7.35%. The water soluble ash and the acid insoluble ash were 3.09 and 1.47 respectively. The acid neutralizing capacity of the plant was

4 meq of 1 N Hcl and the loss on drying was found to be 5.1%. Phytochemical screening of methanolioc extract indicated the presence of steroids, flavonoids, alkaloids and carbohydrates.phytochemical screening of Petroleum ether extract indicates the presence of steroids, fats and fixed oil.the methanolic extract was found to be reddish in colour having characteristic odor viscous consistency while the petroleum ether extract was found to be pale yellow in color havind viscous consistency. The percentage yield of the methanolic and petroleum ether extracts were found to be 9.81% and 6.23% respectively. REFERENCES: 1. Baker JT, Borris RP, Carte B, Cordell GA, Soejarto DD. Natural product drug discovery and development: New perspective on international collaboration. J. Natl. Prod. 1995; 58, Kokate CK, Purohit AP, Gokhale SB.5 Pharmacognosy. 32 nd ed. Nirali Prakashan; Evans WC, Trease GE. Trease and Evans pharmacognosy. W.B. Saunders Ltd. London Pp 32, 33, 95-99, 512, Nair RV. Indian Medicinal Plants 3, Madras: Orient Longman; p Prathibha S, Nambisan B, Leelamma S. Plant Foods. Hum Nutr, 48: 247, (1995). 6. Shilpi JA, Ray PK, Sardar MM, Uddin SJ. Analgesic activity of Amorphophallus campanulatus tuber. Fitoterapia 2005;76: Angayarkanni J, Ramkumar KM, Poornima T, Priyadarshini U. Cytotoxic activity of Amorphophallus paeniifolius tuber extracts in vitro. American-Euresian J Agric Environ Sci 2007;2: Dey YN, De S, Ghosh AK, Anti-inflammatory activity of methanolic extracts of Amorphophallus paeoniifolius and its possible mechanism. International J. of Pharma and Biosciences 2010;1(3), Dey YN, Das SS, Sen M, De S and Ghosh AK. Effects of Petroleum Ether Extract of Amorphophallus paeoniifolius Tuber on Central Nervous System in Mice. Indian J. Pharm. Sci., 2009, 71 (6): British Pharmacopoeia 11: Ash values, Acid insoluble Ash, Water soluble and alcohol soluble extractive. Appendix XI. (1980). Her Majesty s Stationery office, London. A108, A Khandelwal KK. Practical pharmacognosy; Techniques and experiments. 7 th ed.: Nirali prakashan; Nair RV. Indian Medicinal Plants 3, Madras: Orient Longman; p Agrawal DP. Complementary and alternative medicine: an overview. Curr. Sci. 2002; 82, Kokate CK, Purohit AP, Gokhale SB.5 Pharmacognosy. 32 nd ed. Nirali Prakashan; Evans WC, Trease GE. Trease and Evans pharmacognosy. W.B. Saunders Ltd. London Pp 32, 33, 95-99, 512, Brain KR and Turner TD. Practical evaluation of phytopharmaceuticals. Wright- Scientechnica, Bristol. 1 st Ed Ciulei I. Methodology for analysis of vegetable drugs. UNIDO Romania Harborne JB. Phytochemical methods. A guide to modern technique of plant analysis. Chapman and Hill, London

5 TABLES AND FIGURES: Table 1 The physical properties of the extracts of the tuber Sl. No. Physical Characteristics Methanolic Extract Petroleum ether Extract 1. Percentage yeild 9.81% 6.23% 2. Color Reddish Pale yellow 3. Odor Characteristic Characteristic 4. Consistency Viscous Viscous Table 2 The Pharmacognostic parameters of the extracts of the tuber Sl. No. Physical Constants Value Ash Value Water Soluble Ash Acid Insoluble Ash Acid Neutralizing Capacity Loss on drying 7.35 % 3.09 % 1.47 % ±0.8 meq of 1 N Hcl 5.1 % Table 3 Qualitative Phyto-Chemical Test Phytochemical Methanolic Petroleum ether Extract Extract Mayer s Test +ve ve Dragendorff s Test +ve ve Alkaloids Wagner s Test +ve ve Hager s Test +ve ve Tannic Acid Test +ve ve Raymond s Test NA NA Glycosides Legal s Test ve ve Bromine Water Test ve ve Saponin Glycosides Froth Test ve ve Hemolysis Test ve ve 48

6 Borntrager s Test ve ve Anthraquinone Test Modified Borntrager s Test ve ve Cardiac Glycosides Kedde s Test ve ve Keller Killiani Test ve ve Cyanogenetic Glycosides ve ve Gelatin Test ve ve Ferric Chloride Test ve ve Tannins and Phenolic Vanillin Hydrochloride Compounds NA NA Test Alkaline Reagent Test ve ve Shinoda Test +ve ve Flavonoids Zinc Hydrochloride Reduction Test +ve ve Alkaline Reagent Test +ve ve Proteins and Amino Millon s Test ve ve Acids Ninhydrin Test ve ve Libermann-Buchard Steroids and +ve +ve Test Triterpenoids Salkowski Test +ve +ve Molisch s Test +ve ve Carbohydrates Benedict s Test +ve ve Tollen s Test +ve ve Fehling s Test +ve ve Fats and Fixed Oils Stain Test ve +ve Saponification Test ve +ve +ve- Presence of phytocontituents confirmed by the phytochemical test -ve- Absence of phytoconstituents by the phytochemical test 49