Phytochemical Analysis of Medicinal Plants PDF

Journal of Phytology 2011, 3(12): 10-14 ISSN: 2075-6240 Available Online: http://journal-phytology.com/ Phytochemical analysis of some medicinal plants RNS Yadav* and Munin Agarwala Centre For Studies In Biotechnology, Dibrugarh University, Dibrugarh, Assam-786004, India Abstract...

Journal of Phytology 2011, 3(12): 10-14 ISSN: 2075-6240 Available Online: http://journal-phytology.com/ Phytochemical analysis of some medicinal plants RNS Yadav* and Munin Agarwala Centre For Studies In Biotechnology, Dibrugarh University, Dibrugarh, Assam-786004, India Abstract North-eastern India has been known for its rich biological diversity. For this study, seven medicinal plants such as Bryophyllum pinnatum, Ipomea aquatica, Oldenlandia corymbosa, Ricinus communis, Terminalia bellerica, Tinospora cordifolia, and Xanthium strumarium, were selected. The aim of the present study was to investigate the presence of phytochemicals and to determine the total phenolic and flavonoid contents of the selected medicinal plants. Soxhlet apparatus was used for the organic solvent extraction. Solvents used were water, methanol, ethanol, and acetone. Total phenolic contents of the aqueous extracts of the plants were determined by the Folin-Ciocalteus reagent method whereas total flavonoid contents of the aqueous extracts were determined by the Aluminium Chloride method. Proteins, carbohydrates, phenols, tannins, flavonoids, saponins, were detected in all of the plants tested. Total phenolic contents obtained were 18.4mg/gm, 18.8mg/gm, 11.6mg/gm, 29.2mg/gm, 29.6mg/gm, 40.8mg/gm, 12.8mg/gm, 71.6mg/gm of the extract and total flavonoid contents obtained were 8.4mg/gm, 37.6mg/gm, 4.4mg/gm, 6mg/gm, 42.8mg/gm, 18mg/gm, 6mg/gm, 28.8mg/gm of the extract for the plants Bryophyllum pinnatum (Leaves), Ipomea aquatica (Leaves), Oldenlandia corymbosa (Whole plant), Ricinus communis (Roots), Terminalia bellerica (Leaves), Tinospora cordifolia (Leaves), Tinospora cordifolia (Stem), and Xanthium strumarium (Leaves) respectively. Our findings provided evidence that crude aqueous and organic solvent extracts of these tested plants contain medicinally important bioactive compounds and it justifies their use in the traditional medicines for the treatment of different diseases. Keywords: Bryophyllum pinnatum, Ipomea aquatica, phytochemicals, phenols, tannins, flavonoids, etc. INTRODUCTION organisms. Secondary metabolites are chemically and taxonomically The importance of plants is known to us well. The plant extremely diverse compounds with obscure function. They are widely kingdom is a treasure house of potential drugs and in the recent used in the human therapy, veterinary, agriculture, scientific research years there has been an increasing awareness about the importance and countless other areas.A large number of phytochemicals of medicinal plants. Drugs from the plants are easily available, less belonging to several chemical classes have been shown to have expensive, safe, and efficient and rarely have side effects. The inhibitory effects on all types of microorganisms in vitro. plants which have been selected for medicinal use over thousands of Plant products have been part of phytomedicines since time years constitute the most obvious choice of examining the current immemorial. This can be derived from barks, leaves, flowers, roots, search for therapeutically effective new drugs such as anticancer fruits, seeds. Knowledge of the chemical constituents of plants is drugs , antimicrobial drugs , antihepatotoxic compounds. desirable because such information will be value for synthesis of According to World Health Organization (WHO), medicinal plants complex chemical substances [9,10,11]. would be the best source to obtain variety of drugs. About 80% of In the present work, qualitative and quantitative individuals from developed countries use traditional medicines, which phytochemical analysis were carried out in seven plants, has compounds derived from medicinal plants. However, such plants Bryophyllum pinnatum, Ipomea aquatica, Oldenlandia corymbosa, should be investigated to better understand their properties, safety, Ricinus communis, Terminalia bellerica, Tinospora cordifolia, and and efficiency. Xanthium strumarium, of Northeastern region of India. Medicinal plants contain some organic compounds which provide definite physiological action on the human body and these MATERIALS AND METHODS bioactive substances include tannins, alkaloids, carbohydrates, Collection of plant materials terpenoids, steroids and flavonoids [4,5]. These compounds are synthesized by primary or rather secondary metabolism of living Fresh parts of seven medicinal plants, Bryophyllum pinnatum (Leaves), Ipomea aquatica (Leaves), Oldenlandia corymbosa (Whole Received: Oct 12, 2011; Revised: Nov 24, 2011; Accepted: Dec 07, 2011. plant), Ricinus communis (Roots), Terminalia bellerica (Leaves), Tinospora cordifolia (Leaves/Stem), and Xanthium strumarium *Corresponding Author (Leaves) were collected from different regions of Sonitpur and RNS Yadav Dibrugarh districts of Assam. The plant materials were taxonomically Centre For Studies In Biotechnology, Dibrugarh University, Dibrugarh, identified and authenticated by The Department Of Life Science, Assam-786004, India Dibrugarh University, Dibrugarh, Assam. The plant materials were shade dried until all the water molecules evaporated and plants Tel: +91-9435032590; Fax: +91-373-2370323; became well dried for grinding. After drying, the plant materials were Email: [email protected] Journal of Phytology 2011, 3(12): 10-14 11 ground well using mechanical blender into fine powder and the mixture was shaken properly. After that, 2ml of concentrated transferred into airtight containers with proper labeling for future use. H2SO4 was poured carefully along the side of the test tube. Appearance of a violet ring at the interphase indicated the presence Preparation of plant extracts of carbohydrate. Hot water extraction Iodine test 5gm of dried finely powdered plant material was taken in a beaker and 200ml of distilled water was added. The mixture was Crude extract was mixed with 2ml of iodine solution. A dark heated on a hot plate with continuous stirring at 30º-40ºC for 20 blue or purple coloration indicated the presence of the carbohydrate. minutes. Then the water extract was filtered through filter paper and the filtrate was used for the phytochemical analysis. The water Test for phenols and tannins extract was kept in refrigerator when not in use. Crude extract was mixed with 2ml of 2% solution of FeCl3. A Solvent extraction blue-green or black coloration indicated the presence of phenols and tannins. Crude plant extract was prepared by Soxhlet extraction method. About 20gm of powdered plant material was uniformly Test for flavonoids packed into a thimble and extracted with 250ml of different solvents Shinoda test separately. Solvents used were methanol, ethanol, and acetone. The process of extraction continues for 24 hours or till the solvent in Crude extract was mixed with few fragments of magnesium siphon tube of an extractor become colorless. After that the extract ribbon and concentrated HCl was added drop wise. Pink scarlet was taken in a beaker and kept on hot plate and heated at 30-40ºC colour appeared after few minutes which indicated the presence of till all the solvent got evaporated. Dried extract was kept in flavonoids. refrigerator at 4ºC for their future use in phytochemical analysis. Alkaline reagent test Qualitative phytochemical analysis Crude extract was mixed with 2ml of 2% solution of NaOH. An The extract was tested for the presence of bioactive intense yellow colour was formed which turned colourless on compounds by using following standard methods [12,13,14]. addition of few drops of diluted acid which indicated the presence of flavonoids. Test for proteins Millon’s test Test for saponins Crude extract when mixed with 2ml of Millon’s reagent, white Crude extract was mixed with 5ml of distilled water in a test precipitate appeared which turned red upon gentle heating that tube and it was shaken vigorously. The formation of stable foam was confirmed the presence of protein. taken as an indication for the presence of saponins. Ninhydrin test Test for glycosides Liebermann’s test Crude extract when boiled with 2ml of 0.2% solution of Ninhydrin, violet colour appeared suggesting the presence of amino Crude extract was mixed with each of 2ml of chloroform and acids and proteins. 2ml of acetic acid. The mixture was cooled in ice. Carefully concentrated H2SO4 was added. A colour change from violet to blue Test for carbohydrates to green indicated the presence of steroidal nucleus, i.e., glycone Fehling’s test portion of glycoside. Equal volume of Fehling A and Fehling B reagents were Salkowski’s test mixed together and 2ml of it was added to crude extract and gently boiled. A brick red precipitate appeared at the bottom of the test tube Crude extract was mixed with 2ml of chloroform. Then 2ml of indicated the presence of reducing sugars. concentrated H2SO4 was added carefully and shaken gently. A reddish brown colour indicated the presence of steroidal ring, i.e., Benedict’s test glycone portion of the glycoside. Crude extract when mixed with 2ml of Benedict’s reagent and Keller-kilani test boiled, a reddish brown precipitate formed which indicated the presence of the carbohydrates. Crude extract was mixed with 2ml of glacial acetic acid containing 1-2 drops of 2% solution of FeCl3. The mixture was then Molisch’s test poured into another test tube containing 2ml of concentrated H2SO4. A brown ring at the interphase indicated the presence of cardiac Crude extract was mixed with 2ml of Molisch’s reagent and glycosides. 12 RNS Yadav and Munin Agarwala Test for steroid was measured at 765nm. Gallic acid was used as standard (1mg/ml). All the tests were performed in triplicates. The results were Crude extract was mixed with 2ml of chloroform and determined from the standard curve and were expressed as gallic concentrated H2SO4 was added sidewise. A red colour produced in acid equivalent (mg/g of extracted compound). the lower chloroform layer indicated the presence of steroids. Another test was performed by mixing crude extract with 2ml of Total flavonoid content chloroform. Then 2ml of each of concentrated H2SO4 and acetic acid were poured into the mixture. The development of a greenish Aluminium chloride colorimetric method was used with some coloration indicated the presence of steroids. modifications to determine flavonoid content. 1ml of sample plant extract was mixed with 3ml of methanol, 0.2ml of 10% aluminium Test for terpenoids chloride, 0.2ml of 1M potassium acetate and 5.6ml of distilled water and remains at room temperature for 30 minutes. The absorbance Crude extract was dissolved in 2ml of chloroform and was measured at 420nm. Quercetin was used as standard (1mg/ml). evaporated to dryness. To this, 2ml of concentrated H2SO4 was All the tests were performed in triplicates. Flavonoid contents were added and heated for about 2 minutes. A grayish colour indicated the determined from the standard curve and were expressed as presence of terpenoids. quercetin equivalent (mg/g of extracted compound). Test for alkaloids RESULTS Crude extract was mixed with 2ml of 1% HCl and heated The phytochemical characteristics of seven medicinal plants gently. Mayer’s And Wagner’s reagents were then added to the tested were summarized in the table-1. The results revealed the mixture. Turbidity of the resulting precipitate was taken as evidence presence of medically active compounds in the seven plants studied. for the presence of alkaloids. From the table, it could be seen that, proteins, carbohydrates, phenols and tannins, flavonoids and saponins were present in all the Quantitative phytochemical analysis plants. Glycosides were absent only from the leaves of Tinospora Total phenolic content cordifolia. Steroids were absent only in the leaves of Xanthium strumarium while terpenoids were absent in the leaves of Ipomea The amount of phenol in the aqueous extract was determined aquatica, roots of Ricinus communis and also in the leaves of by Folin-Ciocalteu reagent method with some modifications. 2.5ml of Xanthium strumarium. Alkaloids were absent in the roots of Ricinus 10% Folin-Ciocalteu reagent and 2ml of 2% solution of Na2CO3 were communis, leaves of Terminalia bellerica and also in the leaves of added to 1ml of plant extract. The resulting mixture was incubated Tinospora cordifolia. for 15 minutes at room temperature. The absorbance of the sample Table 1. Phytochemical constituents of seven medicinal plants studied. Phenols/Tannins Carbohydrates Terpenoids Glycosides Flavonoids Saponins Alkaloids Proteins Steroids Plants Bryophyllum pinnatum (leaves) + + + + + + + + + Ipomea aquatica (leaves) + + + + + + + - + Oldenlandia corymbosa (leaves) + + + + + + + + + Ricinus communis (leaves) + + + + + + + - - Terminalia bellerica (leaves) + + + + + + + + - Tinospora cordifolia (leaves) + + + + + - + + - Tinospora cordifolia (stems) + + + + + + + + + Xanthium strumarium (leaves) + + + + + + - - + Total phenolic contents obtained were 18.4mg/gm, plants Bryophyllum pinnatum (Leaves), Ipomea aquatica (Leaves), 18.8mg/gm, 11.6mg/gm, 29.2mg/gm, 29.6mg/gm, 40.8mg/gm, Oldenlandia corymbosa (Whole plant), Ricinus communis (Roots), 12.8mg/gm, 71.6mg/gm of the extract and total flavonoid contents Terminalia bellerica (Leaves), Tinospora cordifolia (Leaves), obtained were 8.4mg/gm, 37.6mg/gm, 4.4mg/gm, 6mg/gm, Tinospora cordifolia (Stem), and Xanthium strumarium (Leaves) 42.8mg/gm, 18mg/gm, 6mg/gm, 28.8mg/gm of the extract for the respectively. Journal of Phytology 2011, 3(12): 10-14 13 Fig 1. It shows the total phenolic contents of Bryophyllum pinnatum (Leaves), Ipomea aquatica (Leaves), Oldenlandia corymbosa (Whole plant), Ricinus communis (Roots), Terminalia bellerica (Leaves), Tinospora cordifolia (Leaves), Tinospora cordifolia (Stem), and Xanthium strumarium (Leaves) respectively. Fig.2. It shows the total flavonoid contents of Bryophyllum pinnatum (Leaves), Ipomea aquatica (Leaves), Oldenlandia corymbosa (Whole plant), Ricinus communis (Roots), Terminalia bellerica (Leaves), Tinospora cordifolia (Leaves), Tinospora cordifolia (Stem), and Xanthium strumarium (Leaves) respectively. DISCUSSION anticancer activities [22,23,24]. The plant extracts were also revealed to contain saponins Phytochemical analysis conducted on the plant extracts which are known to produce inhibitory effect on inflammation. revealed the presence of constituents which are known to exhibit Saponins has the property of precipitating and coagulating red blood medicinal as well as physiological activities. Analysis of the plant cells. Some of the characteristics of saponins include formation of extracts revealed the presence of phytochemicals such as phenols, foams in aqueous solutions, hemolytic activity, cholesterol binding tannins, flavonoids, saponins, glycosides, steroids, terpenoids, and properties and bitterness [26,24]. Steroids have been reported to alkaloids. have antibacterial properties and they are very important The phenolic compounds are one of the largest and most compounds especially due to their relationship with compounds such ubiquitous groups of plant metabolites. They possess biological as sex hormones. Alkaloids have been associated with properties such as antiapoptosis, antiaging, anticarcinogen, medicinal uses for centuries and one of their common biological antiinflammation, antiatherosclerosis, cardiovascular protection and properties is their cytotoxicity. Several workers have reported improvement of endothelial function, as well as inhibition of the analgesic [30,14], antispasmodic and antibacterial [31,32] angiogenesis and cell proliferation activities.Several studies properties of alkaloids. Glycosides are known to lower the blood have described the antioxidant properties of medicinal plants which pressure according to many reports. The results obtained in this are rich in phenolic compounds [18,19]. Natural antioxidant mainly study thus suggest the identified phytochemical compounds may be come from plants in the form of phenolic compounds such as the bioactive constituents and these plants are proving to be an flavonoid, phenolic acids, tocopherols etc..Tannins bind to increasingly valuable reservoir of bioactive compounds of substantial proline rich protein and interfere with protein synthesis. Flavonoids medicinal merit. are hydroxylated phenolic substances known to be synthesized by plants in response to microbial infection and they have been found to CONCLUSION be antimicrobial substances against wide array of microorganisms in vitro. Their activity is probably due to their ability to complex with The results revealed the presence of medicinally important extracellular and soluble proteins and to complex with bacterial cell constituents in the plants studied. Many evidences gathered in wall. They also are effective antioxidant and show strong earlier studies which confirmed the identified phytochemicals to be 14 RNS Yadav and Munin Agarwala bioactive. Several studies confirmed the presence of these antioxidant potential of ethyl acetate extract/fractions of Acacia phytochemicals contribute medicinal as well as physiological auriculiformis A. Cunn. Fod Chem. Toxicol., 45: 1216-1223. properties to the plants studied in the treatment of different ailments. Han, X., Shen, T., Lou, H. 2007. Dietry polyphenols and their Therefore, extracts from these plants could be seen as a good biological significance. Int. J. Mol. Sci., : 950-988. source for useful drugs. The traditional medicine practice is recommended strongly for these plants as well as it is suggested that Brown, J.E., Rice-Evans, C.A. 1998. 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