INTRODUCTION:

The use of herbs as medicine is the oldest form of healthcare known to humanity and has been used in all cultures throughout history¹. The medicinal value of these plants can be observed from the chemical agents they possess which may alter certain physiological actions in the human body. The most important of these bioactive constituents of plants are terpenes, alkaloids, flavonoids and phenolic compounds². Nearly 80% of the world’s population relies on traditional medicines for primary health care, most of which involve the use of plant extracts³. In India, almost 95% of the prescriptions were plant based in the traditional systems of Unani, Ayurveda, Homeopathy and Siddha⁴. The study of plants continues principally for the discovery of novel secondary metabolites. Around 80% of products were of plant origin and their sales exceeded US $65 billion in 2003⁵.

Tinospora cordifolia (Thunb.) Miers, (Guduchi) is one of the important dioecious plants belongs to the family Menispermaceae. In Hindi, the plant is commonly known as Giloe⁶ which is a Hindu mythological term that refers to the heavenly elixir that has saved celestial beings from old age and kept them eternally young. In Ayurveda, it is designated as Rasayana drug recommended to enhance general body resistance, promote longevity and as antistress and adaptogen^7-8.

The fact that it is called “Amrita” signifies its use for revitalization and its importance in Ayurveda. This significant plant is also mentioned in important Pharmacopoeias^9-10. Almost all the parts of the plant are documented to be useful in ethnobotanical surveys conducted by ethnobotanists^11-14. Phytochemistry of T. cordifolia belongs to different classes such as alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds and polysaccharides^15-16.

Three major groups of compounds; protoberberine alkaloids, terpenoids, and polysaccharides are considered as putative active constituents of this plant^17-18. Details of various important aspects such vernacular names of T. cordifolia and its important ethnobotanical, Ayurvedic properties, pharmacological and phytochemistry have already been published¹⁹.

In view of therapeutic importance of this plant, the present research work aim to evaluate the maximum extract yield and phytochemical screening of different parts of T. cordifolia for the presence of important phytochemicals, so that full pharmacological potential of the plant could be exploited.

MATERIALS AND METHODS:

Collection and sample:

Fresh parts of T. cordifolia leaves, stem and aerial roots were collected from the Aligarh. The samples were identified at Department of Botany, Aligarh Muslim University, Aligarh with plant specimen number A.M.U/31337/2010.

Extraction:

The material was washed thoroughly 2-3 times with running tap water, and then air dried under shade after complete shade drying. Dried material was grinded in mixer; the powder was kept in small plastic bags with paper labelling. Assembly was arranged and thimble was prepared. 10 gram of air dried powdered drug was extracted with Hexane for 3 days, than extract solution was collected and concentrate under vacuum using Rota-vapour. Then the plant material was again collected and air dried. When completely dried it was again packed back in the thimble. Same method was repeated for chloroform, alcohol and water. Finally the dried extracts were collected in pre-weighted glass vials and post-weight for each vial was taken. Extracts for leaf, stem and root of T. cordifolia were collected and finally the percentage yield was calculated for all the extracts of all the parts

Phytochemical Screening:

The extract was tested for the presence of bioactive compounds by using following standard methods ^21-23

Steriods:

Crude extract was mixed with 2ml of chloroform and concentrated H₂SO₄ was added sidewise. A red colour produced in the lower chloroform layer indicated the presence of steroids. Another test was performed by mixing crude extract with 2ml of chloroform. Then 2ml of each of concentrated H₂SO₄ and acetic acid were poured into the mixture. The development of a greenish coloration indicated the presence of steroids.

Terpenoids:

Crude extract was dissolved in 2ml of chloroform and evaporated to dryness. To this, 2ml of concentrated H₂SO₄ was added and heated for about 2 minutes. A grayish colour indicated the presence of terpenoids.

Fatty Acids:

0.5 ml of extract was mixed with 5 ml of ether. These extract was allowing it for evaporation on filter paper and dried the filter paper. The appearance of transparency on filter paper indicates the presence of fatty acids.

Tannins:

2 ml of extract was added to few drops of 1% lead acetate. A yellowish precipitate indicated the presence of tannins.

Test for phenolic compounds:

Formation of intense green, purple, blue or black colours with addition of 1% ferric chloride solution to the extract.

Test for alkaloids:

200mg plant extract is dissolved in 10ml methanol and then filtered. In 1ml filtrate 6 drops of Dragondroff’s reagent is added. Appearance of orange precipitate indicates presence of alkaloids.

Test for flavonoides:

5ml of dilute ammonia solution was added to the filtrate followed by concentrated sulphuric acid. A yellow colour observed indicates the presence of flavonoides.

Tests for proteins and amino acids:

Test solution with 2ml of Millons reagent (Mercuric nitrate in nitric acid containing traces of nitrous acid), white precipitate appears, which turns red upon gentle heating.

Test for fats and fixed Oils:

Add a few drops of 0.5N of alcoholic potassium hydroxide to small quantities of various extracts along with a drop of Phenolphthalein separately and heat on a water bath for 1-2 hrs. The formation of soap or partial neutralization of alkali indicates the presence of Fixed oils and Fats.

Saponins:

5 ml of extract was mixed with 20 ml of distilled water and then agitated in a graduated cylinder for 15 minutes. Formation of foam indicates the presence of saponins.

RESULTS:

The extracts yield obtained by extracting of plant material by various solvents is shown in (Figure 1). Highest percentage of yield was found in aqueous extract of leaf (26.3 % w/w), followed by stem (24.1% w/w) while lowest in aerial root (20.6% w/w) while in alcoholic extract aerial root showed slightly higher (21.4%w/w) value than aqueous extract of aerial root (20.6% w/w). Lowest percentage of extract was reported in n-hexane in which stem showed maximum concentration (5.17% w/w), followed by leaf (5.01% w/w), and least in aerial root (4.31% w/w). The phytochemical screening of leaves, stem and aerial root showed presence of various phytochemical, however their concentration varied in different parts of the plants (Table 1). Phytochemical analysis revealed the presence of steroids, alkaloids, flavonoids, phenolics, tannins and saponins in leaves, stem and aerial root. Leaf extract indicated the presence of all tested compounds; however stem and aerial root showed absence of fixed oil. Absence of steroids was observed in chloroform aerial root extract but hexane aerial root extract showed the presence. Similarly in case, of proteins and amino acid which showed presence in aqueous aerial root extract not in methanolic aerial root extract.

DISCUSSION:

Hexane were found to be the least effective solvents in extracting phytochemicals, which could be due lesser amount of compounds in the plant, which could be dissolved in this solvents. The phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites²⁴. They possess biological properties such as antiapoptosis, antiaging, anticarcinogen, antiinflammation, antiatherosclerosis, cardiovascular protection and improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities²⁵. Natural antioxidant mainly come from plants in the form of phenolic compounds such as flavonoid, phenolic acids, tocopherols etc ²⁶. Recently, flavonoids have attracted interest due to the discovery of their pharmacological activities^27-28. Saponins have the property of precipitating and coagulating red blood cells and having antifungal properties²⁹. Some of the characteristics of saponins include formation of foams in aqueous solutions, hemolytic activity, cholesterol binding properties and bitterness³⁰. Steroids have been reported to have antibacterial properties³¹and they are very important compounds especially due to their relationship with compounds such as sex hormones³². Alkaloids have been coupled with medicinal uses for centuries and one of their common biological properties is their cytotoxicity³³. Several workers have reported the antispasmodic and antibacterial³⁴properties of alkaloids. Glycosides are known to lower the blood pressure according to many reports³⁵. The results obtained in this study thus suggest the identified phytochemical compounds may be the bioactive constituents and these plants are proving to be an increasingly valuable reservoir of bioactive compounds of substantial medicinal merit.

Table1. Showing the phytochemical screening in different parts of Tinospora cordifolia.

Extracts	Chemical constituents	Leaf	Stem	Aerial root
n-hexane	Steroids	++	+	+
	Fixed oil	+	-	-
Chloroform	Alkaloids	++	+++	++
	Steroids	+	++	+
Alcohol	Flavanoids	+++	++	+
	Tannin	++	++	++
	Phenolics	++	++	+
	Alkaloids	+	++	+
	Proteins and amino acids	+	+	-
Aqueous	Tannins	+++	+++	+
	Flavanoids	++	++	++
	Phenolics	+++	++	+++
	Proteins and amino acids	++	+	+
	Saponins	++	+	+

Figure1. Showing the yield percentage of different solvent extract.

CONCLUSION:

The medicinal plants appear to be rich in, widely used in traditional medicine to combat and heal different ailments. Aqueous and alcohol were considered to be the best solvent in extracting metabolites in this study. The anti-inflammatory, antispasmodic, antianalgesic and antidiuretic can be attributed to their high steroids, tannins, terpenoids and saponins. Exploitation of these pharmacological properties involves additional research of these active ingredients by carrying out techniques of extraction, purification, separation, crystallization and identification.

REFRENCES:

1 Barnes J, Anderson LA and Phillipson JD. Herbal medicine. 3rd Edition, Pharmaceutical Press, London. 2007; 1-23.

2 Gurib-Fakim A. Medicinal plants: Tradition of yesterday and drugs of tomorrow. Review article. Mol. Aspects Med. 2006; 27 (1):1-93.

3 Sandhya B, Thomas S, Isabel W and Shenbagarathai R. Ethnomedicinal plants used by the valaiyan community of Piranmalai hills (Reserved forest), Tamil Nadu, India – A pilot study. Afr J Tradit Complement Altern Med. 2006; 3(1): 101-114.

4 Satyavati GV, Gupta AK and Tandon N. Medicinal plants of India, Indian Council of Medical Research, New Delhi, India. 1987.

5 Patwardhan B, Vaidhya ADB and Chorghade M. Ayurveda and Natural products drug discovery. Curr Sci. 2004; 86: 789-799.

6 Bhandari C.Vanaushadhi Chandroda (Chaukhamba Sanskrit Sansthan, Varanasi). 2006; 86.

7 Patwardhan B and Gautam M. Botanical immunodrugs: scope and opportunities, Drug Discov Today. 2005; 10: 495-502.

8 Patil M, Patki P, Kamath HV and Patwardhan B. Antistress activity of Tinospora cordifolia (wild) Miers., Indian Drugs. 1997; 34: 211-215.

9 Anonymous. Indian Pharmacopoeia, Guduchi (Published by Indian Pharmacopoeia Commission, Govt. Of India). 2007; 2037-2034.

10 Anonymous. The Ayurvedic Pharmacopoeia of India, (The Controller of Publications, Delhi). 1st Edn, 2001; 41- 42.

11 Jain SK. Dictionary of Folk Medicine and Ethnobotany, (Deep Publishers, New Delhi).1991; 179 -80.

12 Lalramnghinglova H. Ethno-Medicinal Plants of Mizoram, (Bishen Singh Mahendra Pal Singh, Dehradun). 2003; 283.

13 Singh V and Pandey RP. Ethnobotany of Rajasthan India, (Scientific publisher, Jodhpur, India). 1998; 203 - 231.

14 Sood SK, Parmar S and Lakhanpal TN. Ethnic plants of India used in cancer cure, (Bishen Singh Mahendra Pal Singh, Dehradun). 2005; 235.

15 Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B and Ghosh AC. Chemistry and medicinal properties of Tinospora cordifolia (Guduchi), Indian J Pharm. 2003; 35: 83 - 91.

16 Anonymous. The Ayurvedic Pharmacopoeia Of India (Department Of AYUSH, Ministry Of Health And Family Welfare, Government Of India, New Delhi). 2009; 70-72.

17 Chintalwar G, Jain A, Sipahimalani A, Banerji A, Sumariwalla P, Ramakrishnan R and Sainis K. An immunologically active arabinogalactan from Tinospora cordifolia, Phytochemistry. 1999; 52:1089 - 1093.

18 Bisset N and Nwaiwu J. Quaternary alkaloids of Tinospora species, Planta Med. 1983; 48: 275 - 279.

19 Choudhary N, Siddiqui MB, Azmat S and Khatoon S. Tinospora cordifolia: ethnobotany, phytopharmacology and phytochemistry aspects, IJPSR. 2013; 4: 891-899.

20 Harbone JB. Phytochemicals methods. London. Chapman and Hill. 1973.

21 Kokate CK, Purohit AP and Gokhale SB. Pharmacognosy. Nirali Prakashan. 2009; 6.16-6.17.

22 Evans WC and Trease GE. Trease and Evans pharmacognosy. W.B. Saunders, China. 2002; 193-407.

23 Khandelwal KR. Practical Pharmacognosy. Nirali Prakashan. 1995; 149-155

24 Singh R, Singh SK and Arora S. Evaluation of antioxidant potential of ethyl acetate extract/fractions of Acacia auriculiformis A. Cunn. Fod Chem. Toxicol. 2007; 45: 1216-1223.

25 Han X, Shen T and Lou H. Dietry polyphenols and their biological significance. Int. J. Mol. Sci. 2007; 950-988.

26 Ali SS, Kasoju N, Luthra A, Singh A, Sharanabasava H, Sahuand A and Bora U. Indian medicinal herbs as source of antioxidants. Food Res. Int. 2008; 41: 1-15.

27 Pietta P. Flavonoids as Antioxidants. J. Nat. Prod. 2000; 63: 1035-1042.

28 Brahmachari G and Gorai D. Progress in the research on naturally occurring flavones and flavonols: An overview. Curr. Org. Chem. 2006; 10: 873-898.

29 Osuagwu GGE, Okwulehie IC and Emenike JO. Phytochemical and Mineral content of the leaves of four Nigerian Pterocarpus species. Int. J. Mol. Med. Adv. Sci. 2007; 3(1): 6 – 11.

30 Sodipo OA, Akiniyi JA and Ogunbamosu JU. Studies on certain characteristics of extracts of bark of Pansinystalia macruceras (K schemp) picrre Exbeille. Global J. Pure Appl. Sci.2000; 6: 83-87.

31 Raquel FE. Bacterial lipid composition and antimicrobial efficacy of cationic steroid compounds. Biochemica et Biophysica Acta. 2007; 2500-2509.

32 Okwu DE. Evaluation of chemical composition of medicinal plants belonging to Euphorbiaceae. Pak Vet. J. 2001; 14: 160-162.

33 Nobori T, Miurak k, Wud J, Takabayashik LA and Crson DA. Deletion of the cyclin-dependent kinase -4 inhibitor gene in multiple human cancers. Nature. 1994; 368(6473): 753-756.

34 Okwu DE and Okwu ME. Chemical composition of Spondias mombin linn. plant parts. J. Sustain. Agric. Environ. 2004; 6(2): 140-147.

35 Nyarko AA and Addy ME. Effects of aqueous extract of Adenia cissampeloides on blood pressure and serum analyte of hypertensive patients. Phytotherapy Res. 1990; 4(1): 25-28.

Received on 23.12.2013 Modified on 13.01.2014

Res. J. Pharmacology & P’dynamics. 6(1): Jan.-Mar. 2014; Page 01-04