Scientific Validation ofTinospora cordifolia using various Analytical Techniques PROJECT REPORT Submitted to University of Rajasthan, Jaipur As a part of curriculum of BECHELOR OF SCIENCE IN BIOTECHNOLOGY (2008-09) by SUMIT KUMAR B.Sc IInd Yr. Integrated Department of Biotechnology Mahatma Gandhi Institute of applied Sciences, Jaipur CERTIFICATE This is to certify that Mr. Sumit Kumar S/O Mr. Shankar lal student of B.Sc. part II (Biotechnology Integrated) has undergone 8 weeks training under the supervision of Dr. Ekta Menghani at Mahatama Gandhi Institute of Applied sciences, Jaipur. The student has completed his work in Biotechnology laboratory and undertaken the training as per the curriculum of University of Rajasthan, Jaipur. Dr. Ekta Menghani, Department of Biotechnology, Mahatma Gandhi Institute of Applied Sciences, Jaipur. Prof. C. K. Ojha, Director (Academics) Place: Jaipur Date: UNDERTAKING I Sumit Kumar a student of B.Sc. Part-II (Biotechnology Integrated) hereby affirm that I have undergone 8 week’s short term training at Mahatma Gandhi Institute of Applied Sciences, Jaipur where I am a regular student. I have undertaken the training in Biotechnology laboratory of the Institute under the guidance of Dr. Ekta Menghani. Place : Jaipur Date : Sumit kumar B. Sc IInd Yr. Integrated ACKNOWLEDGEMENT Behind every success there is certainly an unseen power of almighty God. Success is attained with the assistance of predecessors, teachers, family members & friends. It is my privilege to acknowledge people who have supported me throughout the training work & helped me to complete the process of investigation. I sincerely extend my gratitude to Prof. C. K. Ojha, Honorable Director (Academics), MGiaS, Jaipur for getting me permission to complete my project work. I would also wish to thank Dr. Ekta Menghani for her excellent guidance & support throughout this study. I am highly grateful for her exemplary supervision. I am indebted to my friends and all those people who have rendered me direct or indirect help in completing this project work. Sumit Kumar B.Sc. Part-II Biotechnology(Int.) CONTENTS 1. INTRODUCTION OF Tinospora cordifolia 2. MATERIALS AND METHOD I. Microscopic Studies II. Thin layer chromatography [TLC] of successive test extracts for finger printing Analysis. III. Bioefficacies of successive extracts against selected bacterial species. 3. RESULTS AND DISCUSSION 4. CONCLUSION 1.Introduction: Guduchi (Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms) is a large glabrous, deciduous climbing shrub belonging to the family Menispermaceae. It is distributed throughout tropical Indian subcontinent and China, ascending to an altitude of 300m. In Hindi the plant is commonly known as Giloya, which is a Hindu mythological term that refers to the heavenly elixir that have saved celestial being from old age and kept them eternally young. Guduchi is one of the most versatile rejuvenate herbs. It works on all the tissue elements in the body. The Sanskrit name guduchi means the one, which protects the body. It is also called as amrta or nectar, as it is extremely useful in strengthening the immune system of the body and keeping the functions of its various organs in harmony. It possesses various synonyms like jvaranasi febrifuge, vayastha – promotes longevity, rasayan- a rejuvenative, tikta – bitter etc. Maharsi Caraka has categorized it as stanya sodhana – relieve burning sensation of the skin, trptighna – anti saturative, vayah sthapana – promote longevity and as medhya nervine tonic. Susurta has mentioned it as sukra sodhana – sperm purifier. Sarngadhara has classified it as samsamana – pacifier and rasayana – a rejuvenator. It possesses properties like jvaraghna – antipyretic, kamala nasaka – cures jaundice, amavataghna – mitigates gout, gandamala nasaka – alleviates glandular swellings. Guduchi is recommended as a drug of the choice in the treatment of gout. Different constituents that it includes are bitter principles. A glucoside, alkaloide etc. The glycoside – giloin and a non-glucoside – gilenin and gilosterol have been found. The alkaloid tinosporin, tinosporic acid and tinosporol have been identified in the leaves. Tinosporidine and sitosterol isolated from stem, cordifol, heptacosanol and octacosonal from leaves a new furanoid diterpene – tinosporide – isolated from stems and its structure determined. Guduchi is bitter, pungent and astringent in taste, sweet in the post digestive effect and hot in potency. It alleviates all the dosas. It possesses light and oily attributes. It has a special potency as an anti-toxin. The root, stem, leaves and satva (starch) of guduchi are used for medicinal purpose, externally; the medicated oil of the plant is effectively used to reduce the pain and oedema, in gout and skin diseases. In filariasis, the paste of guduci, karuka, sunthi, devadara and vidanga works well when applied externally. Internally, guduchi is one of the most effective rasayanas- rejuvenatives. It works well on all the seven dhatus tissues and keeps the systems in balance. The rasayana accords longevity, enhances memory, improves health, and bestows youth, betters complexion, voice, energy and luster of the skin. In the diseases due to vata dosa it is given with ghrta, in pitta dosa with sugar and kapha dosa with honey. It is immensely helpful in the digestive ailments like hyperacidity, colitis, worm infestations, and loss of appetite, abdominal pain, excessive thirst, vomiting and liver disorders like hepatitis. Guduchi is one of the best bitter tonics useful in fevers especially of pitta origin. It alleviates body heat, thirst, burning sensation to the skin and vomiting if any, due to pitta. In tubercular fever, it combines well with ativisa for decoction. The juices of guduchi, amalaki and haridra act synergistically in urinary problems. In hepatitis, the fresh juice of guduchi given with rock candy, hastens the recovery. It particularly helps in diseases like raktapitta, anaemia, cardiac debility, diabetes, sexual debility and splenic disorders, due to vitiation of pitta. Guduchi is the drug of choice amongst all the remedies in treating gout vatarakta. The decoction of guduchi and sunthi is a very effective combination for the treatment of gout and rheumatic disorders. Its medicinal ghee with kantakari is beneficial in cough Guduchi juice works well with cow’s milk or lodhra in leucorrhea and with cumin seeds in burning sensation due to pitta. The starch (sattva) of guduchi is traditionally used as a household remedy, for chronic fever, to alleviate it as well as to reduce burning sensation and to increase the energy and appetite. It is beneficial in tuberculosis and general debility also. Guduchi is recommended in the skin diseases and its decoction with nimba and vasa effectively relieves the itching and oozing. It also works well in the cutaneous rashes and condylomata, in the secondary stage of syphilis. Guduchi should be always used fresh for good results and the twiner which grows on nimba tree is said to have better results. So in the present project attempts have been made to made a complete study on Guduchi Satva where its authentication and validation was worked out in a series of manner. In standardization of Guduchi satva the extract of the Tinospora cordifolia stem were extracted from authenticated stem and simultaneously Market sample of Giloy satva were also performed and the characterization of authentic and market sample were performed and results were discussed. The validation of guduchi satva and gilov market samples were simultaneously screened and the results proved that this can be used as a Monograph for any authentic Validate Pharmacopoeia where the various thin layer chromatograms, bioefficacies results and microscopic structures will be aid as an identification tools. These characterizations will be further enhance the quality of the market sample of giloy and their justification as the potent use as antidiabetic agent even their use as anticancerous agent. Tinospora cordifolia Scientific classification Kingdom Planti Division: Magnoliophyta Class: Magnoliopsida Order: Ranunculales Family: Menispermaceae Tinospora cordifolia Genus: Tinospora Species: Cordifolia Common name Guduchi, Giloy (H) Sanskrit Guduchi, Amrita, Chakralakshana Latin: Tinospora cordifolia – Caulis (Menispermaceae) Guduchi is described as the one who protects the body against disease. It is one of the most versatile rejuvenating herbs, it promotes longevity hence called Vayastha, Sanskrit name Guduchi itself means the one which protects our body. Another Sanskrit name, Amrita means that originating from divine nectar as it is extremely useful in strengthening the immunity of body and keeping the functions of various organs of the body in harmony. This is a virile creeper that grows throughout the forests of India. Those growing up Neem trees are said to be the best as synergy between these two bitter plants enhances Guduchi’s efficacy. Its therapeutic strength lies in its rejuvenating and strengthening properties while also detoxifying and cleansing the whole system, specifically via Liver.(Anonymous, 1976). Tinospora cordifolia, also called Guduchi is an herbaceous vine of the family Menispermaceae indigenous to the tropical areas of India, Myanmar and Sri Lanka. Synonyms: Guduchi , amrita (Sanskrit), giloe , gulancha (Bengali), giloya (Hindi), gado, galo (Gujarati), duyutige , teppatige (Telugu), heartleaf moonseed (English). According to the 1911 British Pharmaceutical Codex, "Tinospora or Gulancha consists of the dried stem of Tinospora cordifolia, Miers (N.O. Menispermaceae), a climbing shrub indigenous to tropical India. The stems are collected in the hot season and dried. The drug occurs in straight or twisted cylindrical pieces and in slices, averaging about 2 centimeters in diameter, some pieces being much smaller. Externally, they are covered with a thin, papery, brown cork, bearing the raised scars of numerous lenticels. The cork readily exfoliates and discloses a greenish cortex longitudinally wrinkled and marked with lenticels. The fracture is fibrous and the transverse section exhibits a yellowish wood with radically arranged wedge-shaped wood bundles, containing large vessels, separated by narrower medullary rays. The odour is not characteristic, but the taste is bitter."( Nadkarni KM, Nadkarni AK, 1976). Guduchi herb - Immune boost Guduchi or Tinospora Cordifolia is a large, climbing shrub. Guduchi is an important part of the Ayurvedic category of Rasayanas, or rejuvenative tonics. Human studies conducted on Guduchi's immune-boosting ability show it to be linked to enhancing the function of protective cells called macrophages. The plant is used to improve the immune system and the body's resistance to infections. The stem is used in dyspepsia, fevers, and urinary diseases. Its principle constituents are tinosporine, tinosporide, tinosporaside, cordifolide, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin and §-sitosterol. The active principles and juice of the fresh plant possess a number of pharmacological activities. It is bitter, stomachic, antiperiodic, aphrodisiac. Guduchi is useful in chronic diarrhea, to remove urinary stones (Calculi), as a diuretic, CNS depressant, hypoglycemic, antibacterial, antipyretic, anti-inflammatory, anti-rheumatic, antiallergic, analgesic, hepatoprotective, and to reduce blood urea. The plant is used in Ayurvedic rasayanas to improve the immune system and the body's resistance against infections. It is used as an immunomodulator in immunosuppression of obstructive jaundice, hepatic fibrosis, peritonitis and sepsis. The plant has been found effective in preventing fibrous changes and promotes regeneration of the liver against CCl4 induced hepato toxicity. Guduchi has been proven to be effective in inhibiting the growth of bacteria and enhancing the buildup of immune resistance. Scientific research is now providing clues to Guduchi's immune-boosting ability to fight diseases. In a study using human white blood cells, Guduchi increased the killing ability of macrophages, the immune cells responsible for fighting invaders. CONSTITUENTS The active adaptogenic constituents are diterpene compounds including tinosporone, tinosporic acid, cordifolisides A to E, syringen, the yellow alkaloid, berberine, Giloin, crude Giloininand, a glucosidal bitter principle as well as polysaccharides, including arabinogalactan polysaccharide (TSP). Its principal constituents are tinosporine, tinosporide, tinosporaside, cordifolide, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin and b-sitosterol. A verity of constituents have been isolated from Tinospora cordifolia plant and their structures were elucidated. They belong to different classes such as alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds and polysaccharides. Leaves of this plant are rich in protein (11.2%) and are fairly rich in calcium and phosphorus. Studies on the physical characteristic and chemical composition of starch obtained from Guduchi Satwa (extract) were carried out and the polysaccharide was found to consist chiefly of 1g4 linked glucan with occasionally branched points. Its similarities and differences from amylase were elucidated. An arabinogalactan had been isolated from the dried stems of T. cordifolia . PARTS USED Roots, Stem, Leaves and satva (starch) MEDICINAL PROPERTIES & USES Giloy or Guduchi is an evergreen perennial climber found throughout tropical India. Stem of Guduchi is used in Ayurveda for medicinal purpose. It is one of the multi purpose and most widely used herb of Ayurvedic system of medicine, which is used fresh. Guduchi is an appetizer, digestive, carminating and controls hyperacidity by controlling secretion of gastric juices. Amrita is used for revitalization and is a Rasayana herb according to Ayurvedic concept. It is cardio protective and blood purifying herb. It has immunomodulating properties and thus is used in immunological disorders and controls weakness in debilitating diseases. It is used in formulations for cardiac weakness, anemia, oligospermia, chronic fever, liver disorders and for skin diseases due to toxicity. It is Ant periodic, Antipyretic, Diuretic and Anti-inflammatory. It is a constituent of several compound preparations. It is used in fever, urinary disorders, dyspepsia, torpidity of the liver, skin diseases, secondary syphilis, rheumatism, constipation, tuberculosis, leprosy and general debility. It is also used in treatment of rheumatism and jaundice. It is a blood purifier and may be useful in AIDS and other immune diseases also. It is also being proposed for cancer patients before and after chemotherapy. • Guduchi acts as a diuretic and found to be effective against Renal obstruction like calculi and other urinary disorders. Guduchi acts as a memory booster, develops inteligence, promotes mental clarity. It is described as one of the Medhya Rasayana (mental rejuvenative) in the Charak Samhita (The oldest and most potent book of Ayurvedic Medicine). • • • Guduchi is regarded as a liver protector. Guduchi is considered helpful in eye disorders as a tissue builder and promotes mental clarity. The stem of guduchi is used in general debility, dyspepsia and urinary diseases. • • Guduchi is anti-pyretic and act as a tonic after fever, also has action against alternative fever like Malaria. According to the 1918 United States Dispensatory edited by Joseph Remington, Horatio Wood et al.: Tinospora. Br. Add. 1900.—"The dried stem of Tinospora cordifolia Miers (Fam. Menispermaceae), collected in the hot season." Br. Add., 1900. Tinospora has long been used in India as a medicine and in the preparation of a starch known as gilae-ka-sat or as palo. It is said to be a tonic, antiperiodic, and a diuretic. Flückiger obtained from it traces of an alkaloid and a bitter glucoside. The Br. Add., 1900, recognized an infusion (Infusum Tinosporae Br. Add., 1900, two ounces to the pint), dose one-half to one fluidounce (15-30 mils); a tincture (Tinctura Tinosporae Br. Add., 1900, four ounces to the pint), dose, one-half to one fluidrachm (1.8-3.75 mils); and a concentrated solution [Liquor Tinosporae Concentratus Br. Add., 1900), dose, one-half to one fluidrachm (1.83.75 mils). Tinospora crispa Miers (more), which is abundant in the Philippines, is used freely by the natives under the name of makabuhay (that is, "You may live"), as a panacea, especially valuable in general debility, in chronic rheumatism, and in malarial fevers. It may be prepared in the same way and given in the same doses as Tinospora cordifolia. Tinospora cordifolia is used in Ayurvedic herbal medicine as a hepatoprotectant, protecting the liver from damage that may occur following exposure to toxins. Recent research has demonstrated that a combination of T. cordifolia extract and turmeric extract is effective in preventing the hepatotoxicity which is otherwise produced as a side effect of conventional pharmaceutical treatments for tuberculosis using drugs such as isoniazid and rifampicin. The stem of Tinospora cordifolia is one of the constituents of several ayurvedic preparations used in general debility, dyspepsia, fever and urinary disease. The stem is bitter, stomachic, diuretic, stimulates bile secretion, causes constipation, allays thirst, burning sensation, vomiting, enriches the blood and cures jaundice. The extract of its stem is useful in skin diseases. The root and stem of T. cordifolia are prescribed in combination with other drugs as an anti-dote to snake bite and scorpion sting. Dry barks of T. cordifolia has anti-spasmodic, antipyretic, anti-allergic, anti-inflammatory and antileprotic properties. The aqueous extract of the stem antagonizes the effect of agonists such as 5hydroxytriptamine, histamine, bradykinine and prostaglandins E1 and E2 on the rabbit smooth muscle, relaxes the intestinal, uterine smooth muscle and inhibits the constrictor response of histamine and acetylcholine on smooth muscle. In travenous exposure to aqueous extract of T. cordifolia in doses of 5.0, 10.0 and 15.0 mg/kg body weight produces a temporary but marked fall in blood pressure and bradycardia in anaes-thetized dogs. It is reported that the daily administration of either alcoholic or aqueous extract of T. cordifolia decreases the blood glucose level and increases glucose tolerance in rodents. Aqueous extract also caused a reduction in blood sugar in alloxan induced hyperglycemia in rats and rabbits in the dose of 400 mg/kg. However, histological examination of pancreas has not revealed any evidence of regeneration of b-cells of islets of Langerhans and the possible mode of action of the plant is through glucose metabolism. The aqueous extract has also exhibited some inhibitory effect on adrenaline-induced hyperglycemia. Ethyl acetate extract of its roots has afforded a pyrrolidine derivative with hypoglycemic activity in rabbits. Another study has also revealed significant hypoglycemic effect of extract of leaves in normal and alloxan diabetic rabbits. However, the extract had no significant effect on total lipid levels in normal or treated rabbits. T. cordifolia is reported to benefit the immune system in a variety of ways. The alcoholic and aqueous extracts of T. cordifolia have been tested successfully for immunomodulatory activity. Pre-treatment with T. cordifolia was to impart protection against mortality induced by intra-abdominal sepsis following coecal ligation in rats. It has also significantly reduced the mortality from E. coli induced peritonitis in mice. In a clinical study, it has afforded protection in cholestatic patients against E. coli infection. These activities are not due to its anti-bacterial activity as shown by the negative in-vitro antibacterial activity of the plant extract. It is reported that the treatment in rats had resulted in significant leucocytosis and predominant neutrophilia. It has been also observed that it stimulates the macrophages as evidenced by an increase in the number and % phagocytosis of S.aureaus by peritoneal macrophages in rats. Other workers have also supported these observations. The phagocytic and Intra-cellular killing capacity of polymorphs in rats, tested at 3.5 h after E. coli infection were significant. The anti-stress and tonic property of the plant was clinically tested and it was found that it brought about good response in children with moderate degree of behaviour Disorders and mental deficit. It has also significantly improved the I.Q. levels. The hepatoprotective action of T. cordifolia was reported in one of the experiment in which goats treated with T. cordifolia have shown significant clinical and hematobiochemical improvement in CCl4 induced hepatopathy. Extract of T. cordifolia has also exhibited in vitro inactivating property against Hepatitis B and E surface antigen. The aqueous extract of T. cordifolia exerted a significant anti-inflammatory effect on cotton pellet granuloma and formalin induced arthritis models. Its effect was comparable with Indomethacin and its mode of action appeared to resemble that of a non-steroidal anti-inflammatory agent. The dried stem of T. cordifolia produced significant antiinflammatory effect in both acute and subacute models of inflammation. T. cordifolia was found to be more effective than acetylsalicylic acid in acute inflammation. But in subacute inflammation, the drug was inferior to phenylbutazone. In a clinical evaluation, a compound preparation 'Rumalaya' containing T. cordifolia was reported to significantly reduce the pain in patients suffering from rheumatoid arthritis. The aqueous extract of roots of T. cordifolia has shown the anti-oxidant action in alloxan diabetes rats. The administration of the extract of T. cordifolia roots (2.5, 50 mg/kg body weight) for 6 weeks resulted in a significant reduction of serum and tissue cholesterol, phospholipids and free fatty acids in alloxan diabetic rats. Jagetia et al., have found that guduchi killed the HeLa cells very effectively in vitro and thus it indicates that guduchi needs attention as an anti-neoplastic agent. In this study exposure of HeLa cells to 0, 5, 10, 25, 50 and 100 mg/ml of guduchi extract (methanol, aqueous and methylene chloride) resulted in a dose dependent but significant increase in cell killing when compared to non drug treated controls. Ether extract of the stem distillate of aerial part of cordifolia has inhibited the in vitro growth Mycobacterium tuberculosis at 1:50,000 dilution. Ethanolic extract has exhibited significant antipyretic activity in experimental rats. 'Septilin' syrup, compound preparation containing T. cordifolia (7.82% in 5 ml of syrup) was found to elicit good clinical response in children suffering from upper respiratory tract infection and chronic otitis media. The Ayurveda literature reports that it can cause constipation, if taken regularly in high doses; it has no side effect and toxicity. Yet the safety and the potential indications in human beings have to established using modern methods. INDICATIONS FOR USE OF Tinospora cordifolia HERB POWDER 1. It is a very useful treatment Lichen for gout planus and high and uric acid. others. 2. It is a natural blood purifier and is very useful for skin problems like Acne, Psoriasis, eczema, 3. It is an immunomodulator and is useful for immunity deficient conditions like AIDS and other auto-immune disorders. PHARMACOLOGICAL ACTION Cholagogue, detoxicant, immune modulator, anti-inflammatory, diuretic, anthelminthic, nervine tonic. The aqueous extract of guduchi stem has shown the presence of arabinogalactan that showed immunological activity. The stem is used in dyspepsia, fevers and urinary diseases. The bitter principle present shows antiperiodic, antispasmodic, anti-inflammatory and antipyretic properties. Guduchi has been reported to treat throat cancer in humans. Dichloromethane extract is the most promising one as far as cytotoxic effect is concerned. These preliminary studies were done to establish the cytotoxic effect of guduchi at higher doses. Extracts of Tinospora cordifolia (TCE) have been shown to possess anti-tumor properties and was shown to upregulate antitumor activity of tumor-associated macrophages (TAM). Evidence has shown that an alcoholic extract of Tinospora cordifolia (ALTC) enhances the differentiation of TAM to dendritic cells (DC) in response to granulocyte/macrophage-colony-stimulating factor, interleukin4, and tumour necrosis factor. INDICATION Liver: Liver damage, viral hepatitis or toxicity from alcohol, chemicals and medicinal drugs. Useful in repairing fibrosis and regenerating liver tissue. Applied in all conditions of aggravated ranjakapitta and pitta in the blood. Joints: Gout (vatarakta), arthritis (amavata) and other inflammatory joint conditions. It acts by clearing pitta toxins and uric acid via the urinary system that have accumulated in raktavahasrotas. It also removes ama toxins from the system without destabilising any of the other dosha. Immunity: All auto-immune diseases causing inflammation. Applicable in degenerative diseases such as cancer, AIDS and arthritis as it boosts the immune system. Use to offset the ulcerative and toxic effects of chemo-radiotherapy. Skin: Supperative and inflammatory skin conditions such as eczema, psoriasis, Systemic Lupus Erythmatosus. Useful when there is high tejas and pitta that has burnt immune protecting ojas away resulting in inflammatory skin conditions. Skin problems from excessive alcohol, recreational drug and pharmaceutical drug use may indicate the use of Guduchi. Specific for burning sensations on the skin (daha). GIT: Guduchi heals a bowel affected with constipation, intestinal bleeding, haemorrhoids or dysentery. Useful at redressing intestinal floral imbalance with candida-like symptoms (krimi, grahani) such as bloating, flatulence and malabsorption. Its bitter yet heating qualities are used to stimulate raktadhatvagni and strengthen digestion in pitta types. Metabolic: It regulates blood sugar levels via its direct effect on rakta and medas-dhatu thus benefiting diabetes and hypoglycaemia. Guduchi is very calming to vata and the nervous system via its unctuous nature soothing nervous irritation. Reproductive: Its ability to clear heat is applied when sexual dysfunction is caused by a hyper-heat condition. It is often used in formulas for male sexual dysfunction caused by pitta imbalance as its sweet post-digestive effect nourishes shukradhatu. Rejuvenating Properties The term "Rasayana" accords to longevity, enhances memory, better complexion, voice energy and luster of skin thus bestows youth. Anupana (Carrier of Drug) Internally it can be given with Ghee, Sugar and honey in Vata, Pitta and Kapha Dosha respectively.( Nayampalli S, Ainapure SS, Nadkarni PM, 1982). REFERENCE 1. Anonymous. Wealth of India: Raw materials, Vol X. New Delhi: CSIR; 1976. 2 . Nadkarni KM, Nadkarni AK, editors. Indian Materia Medica, Vol 1. 3rd ed. Mumbai: M/S Popular Prakasan Pvt. Ltd;1976. 3. Kirtikar KR, Basu BD, editors. Indian Medicinal Plants, Vol 1. 2nd ed. New Connaught Place, Dehra Dun: M/S Bishen Singh, Mahendra Pal Singh; 1975. 4. Chopra RN, Nayar SL, Chopra IC, editors. Glossary of Indian Medicinal plants. New Delhi: CSIR; 1956. 5. Chopra RN, Chopra LC, Handa KD, Kapur LD, editors. Indigenous Drugs of India. 2nd ed. Kolkata: M/S Dhar VN & Sons; 1982. 6 Zhao TF, Wang X, Rimando AM, Che C. Folkloric medicinal plants: Tinospora sagittata var. cravaniana and Mahonia bealei. Planta Med 1991;57:505. 7. Nayampalli S, Ainapure SS, Nadkarni PM. Study of antiallergic acid Bronchodilator effects of Tinospora cordifolia. Indian J Pharm 1982;14:64-6. 2. Materials and Method: 2.1. Collection: Authentic samples: Various market samples of Guduchi (Tinospora cordifolia) were procured from Chunnilal Attar Ayurvedic Store, Ghat Gate, Jaipur in the month of October, 2008. 2.2. Identification: All the samples were authenticated and were given identification number. The identification were as follows: • • Tinospora cordifolia(Powdered Stem) Giloy satva (White stem extract) These samples were authenticated and submitted in Department of Biotechnology, MGiaS, Jaipur (Rajasthan). 2.3. Processing of plant materials: During the course of the study each sample was screened for its foreign matter and milled, before use. 2.4. Experimental details: Present studies were performed on Guduchi, Giloy satva, for the following studiesI. II. III. Microscopic Studies Thin layer chromatography [TLC] of successive test extracts for finger printing analysis. Bioefficacies of successive extracts against selected bacterial species. I. Microscopic methods Microscopical evaluation deals with the identification of various characters of tissues, cells and cell contents by microscope. Methods of preparing specimens of crude materials for microscopical studies vary depending on the part used like leaf, stem, root, bark, flower, fruit and also on the nature of the material i.e. entire, cut or powdered. Preparation of sections: Drugs which are hard to cut, are boiled for 20 to 30 min in water. Cross section or transverse section with a razor blade was taken. Thin materials such as leaves, slender stems or flat seeds are placed in a potato slit and sections were taken with ordinary blade. The section was placed on a slide, clear with chloral hydrates, covered with a cover glass and observed under microscope. Powder microscopy Likewise, powder of the selected species and their adulterants were subjected to microscopic analysis and the structures of various evaluations were drawn. For examination of powder characteristics, sufficient amount of powdered drug (fruit or leaves) in chloral hydrate on a slide was warmed over a low flame or on a hot-plate for a short time, covered it with a cover glass and observed under microscope (4, 10, 20 or 45 X). Disintegration of hard and woody tissues: The material was cut into pieces and few pieces transferred to a test tube containing 4 ml of nitric acid and heated to boiling. Later, powdered potassium chlorate was gently added, warmed and allowed to react leading to disintegration of the tissue. When completely bleached, pressure was applied with glass rod for complete disintegration of the tissues. The material was allowed to settle down, decant the liquid and the settled material washed repeatedly with water until the acidity is removed. The material was transferred on to a slide, a drop of glycerol added, covered with a cover glass and observed under microscope. Microscopic test 1 Starch – For examining the presence of starch, the specimen was taken in I2 wherein starch turns blue. 2 Aleurone grains – For examining the presence of aleurone grains a specimen was prepared in I2 and aleurone grains stained yellow. 3 Fixed oil – For examining the presence of fixed oil, specimen was stained with sudan red resulting in the droplet of fixed oil to become pink coloured. II. Phytochemical profile [TLC] II.1. Extraction procedure For TLC profile of selected species each of the dried and powdered (100 gm.) test samples of Guduchi and Giloy satva were Soxhlet extracted successively in petroleum ether, Benzene, chloroform, ethyl acetate and methanol for 6 h. These extracts were filtered, evaporated to dryness and weighed. Each extract (10 mg) was dissolved in 10 ml to make a concentration of 1mg/ml used for further studies. II.2. TLC plates Each extract was applied on silica gel G Thin Layer Chromatography[TLC] coated plates (Merck: 20x20 cm; with thickness 0.2-0.3mm) which were activated at 100°C for 30 minutes and brought to room temperature, just before use. Each extract of various species was applied 1cm above the edge of the chromatographic plates along with the reference compounds and developed in air-tight chamber already saturated with~200 ml of various solvent systems (Harborne, 1973). II.3. TLC spraying reagents Various extracts of test samples were subjected to different solvent systems for identification of any significant biomolecules. After having used different solvent systems, on the basis of better resolution of spots for generating “Thin Layer Chromatography [TLC] fingerprints” for chemical libraries of the test drugs following solvent systems were used in the present study- Pet ether :Benzene (1:3) for Petroleum ether extracts, Benzene :Ethyl acetate (1:2) for Benzene extracts, Chloroform :methanol (1:2) for Chloroform extracts, Benzene :Ethyl acetate (2:1) for Ethyl acetate extracts, and Chloroform : Methanol (2:1) for Methanol extracts. II.4. TLC spraying reagents During the work of present studies, different visualizing reagents i.e. 10% sulphuric acid {10 ml conc. Sulphuric acid dissolved in 100ml absolute alcohol}, I2 vapour (Saturated iodine chamber) and Drangendroff reagent were used. II.5. Qualitative TLC Thin glass plates were coated (0.2-0.3 mm) with silica gel G (30 g/60 ml distilled water) and dried at room temperature. The coated plates were activated in an oven at 100°C for 30 minutes and cooled. The plates were then placed in developing tanks having ~ 150 ml of an organic solvent mixture of pet ether: benzene (1:3). The lid of the developing tanks was sealed with vacuum grease. The plates were removed after making the solvent front and were air-dried. The dried plates were sprayed with Dragendroff reagent (8 g bismuth subnitrate dissolved in 25 ml 30% HNOз and further addition of 28 g KI and 1 ml of 6 N HCl) and alkaloid positive spot ( Rf value) was calculated. III. Bioefficacies – antibacterial efficacies III. 1 Sources of test organisms Bacteria – Pure culture of all test organisms, namely Escheriachia coli ( Gram negative), Pseudomonas aeruginosa ( Gram negetuve) and Bacillus subtillis ( Gram positive), Staphylococcus aureus ( Gram positive), the human pathogens, were obtained through the courtesy of Microbiology Lab, Mahatma Gandhi Institute of applied Science ( MGiaS), Jaipur, which were maintained on Nutrient broth media. III.2 Culture of test microbes For the cultivation of bacteria, Nutrient Agar Medium (NAM) was prepared using 20 gm Agar, 5 gm Peptone, 3 gm beef extract and 3 gm NaCl in one litre distilled water and sterilized at 15 lbs pressure and 121°C temperature for 25-30 minutes. Agar test plates were prepared by pouring approximately 15 ml of NAM into the Petri dishes (10 mm) under aseptic conditions. A saline solution was prepared (by mixing 0.8 % NaCl) in distilled water, followed by autoclaving and the bacterial cultures were maintained on this medium by regular sub-culturing and incubation at 37°C for 24 hours. To prepare the test plates, in bacteria, 10-15 ml of the respective medium was poured into the Petri plates and used for screening. For assessing the bactericidal efficacy, a fresh suspension of the test bacteria was prepared in saline solution from a freshly grown Agar slant. III.3 Preparation of test extracts Crushed powder (50 g) of all the species were successively soxhlet extracted with petroleum ether (60°-80°C), Benzene, Chloroform, Ethyl acetate, Methanol and Distilled water. Later, each of the homogenates was filtered and the residue was re-extracted twice for complete exhaustion, the extracts were pooled individually. Each filtrate was concentrated to dryness in vitro and re dissolved in respective solvents, out of which 80 mg/ 10 disc i.e. 8mg/disc concentration were stored at 4°C in a refrigerator, until screened for antibacterial activity. III.4 Bactericidal assay For both, bactericidal in vitro Disc diffusion method was adopted (Gould and Bowie, 1952), because of reproducibility and precision. The different test organisms were proceeded separately using a sterile swab over previously sterilized culture medium plates and the zone of inhibition were measured around sterilized dried discs of Whatman No.1 paper(6 mm in diameter), which were containing 8 mg of the text extracts, its control (of the respective solvent) and tetracycline as reference drugs separately. Such treated discs were air-dried at room temperature to remove any residual solvent, which might interfere with the determination, sterilized and inoculated. These plates were initially placed at low temperature for 1 h so as to allow the maximum diffusion of the compounds from the test disc into the agar plate and later, incubated at 37°C for 24 h in case of bacteria, after which the zones of inhibition could be easily observed. Five replicates of each test extract were examined and the mean values were then referred. The inhibition zones in each case were recorded and the activity index (AI) was calculated as compared with those of their respective standard reference drugs (AI = Inhibition Zone of test sample / Inhibition zone of standard). 3. Results & Discussion: I. Microscopic Identification: Various slides were prepared to screen the Tinospora cordifolia and Giloy satva and the results were marvelous that even in market sample of Baidhynath the genuine sample was not present. The structure of starch grain present in stem powder of Tinospora cordifolia are somewhat oval with concentric rings and hilum eccentric but it is astonished to see the slides of market sample which resembles to the starch grain of Zea mays a cheap adulterant and hence we can say safely that if we are not using the genuine drug in the Ayurvedic preparation how can we get the better results rather we can say that no results its time to cure the human with Authentic Ayurvedic preparations so before use these durgs must be validate which is not only safe but also increase the efficacy and potency of the drug. Simultaneously, various starch grains were also screened for their structure and their shape and sizes were outlined for better characterization II. TLC Profile: TLC has been regarded as a simple, rapid and inexpensive method for the separation, identification and semi-quantification of a wide variety of substances by scanning these chromo-strips with or without detecting reagents, under visible or UV light. The resultant differential chromatographic “finger prints” can actually be used as “markers” in the standardization of each extract in a particular solvent system separating compounds at specific Rf values are simple, reproducible and thus, reliable marker to check the purity of the crude drugs v/s adulterant, on which account the data can be scientifically useful at quarantines. In view of this, an attempt was made to compare various extracts of Tinospora cordifolia and Giloy satva by running the chromo plates in different solvents so as to generate spots which can later be useful in its standardization. Extract-wise results of TLC profile are as follows II.1 Tinospora cordifolia Petroleum Ether extract of Tinospora cordifolia when performed in solvent system – Pet ether: Benzene (1:3) showed three spots on exposure to 10% Sulphuric acid were seen at Rf value of 0.09(purple), 0.21(light pink) and 0.34(pink). Benzene extract of Tinospora cordifolia when performed in solvent system – Benzene : Ethyl acetate(1:2) showed seven spots on exposure to 10% Sulphuric acid were seen at Rf value of 0.06 (Greenish brown), 0.14(Pinkish brown), 0.20&0.30(Dark pink), 0.40(Dark pinkish brown), 0.57 (Pinkish green) & 0.99(Dark green). Chloroform extract of Tinospora cordifolia when performed in solvent system – Chloroform: Methanol (1:2) showed nine spots on exposure to 10% Sulphuric acid were seen at Rf value of 0.04 (Dark green), 0.11(Yellowish green), 0.22(Light yellowish green), 0.36(Parrot green), 0.45(Light parrot green), 0.59(Light green), 0.63(Very light green), 0.82(Light brownish green) & 0.91(Dark brownish green). Fig B: Thin layer chromatography of various extracts of Tinospora cordifolia Ethyl acetate extract of Tinospora cordifolia when performed in solvent system – Benzene: Ethyl acetate (2:1) showed two spots on exposure to 10% Sulphuric acid were seen at Rf value of 0.09 (Very light yellow) & 0.93(Light yellow). Methanol extract of Tinospora cordifolia when performed in solvent system – Chloroform: Methanol (2:1) showed four spots on exposure to Dragendroff reagent were seen at Rf value of 0.09(Buff orange), 0.60(Light Buff orange), 0.85(Buff orange) & 0.97(Dark Buff orange). II.2 Giloy satva Petroleum Ether extract of Giloy satva when performed in solvent system – Pet ether: Benzene (1:3) showed no spots on exposure to 10% Sulphuric acid. Benzene extract of Giloy satva when performed in solvent system – Benzene: Ethyl acetate (1:2) showed no spot on exposure to 10% Sulphuric acid. Chloroform extract of Giloy satva when performed in solvent system – Chloroform : Methanol(1:2) showed one spot on exposure to 10% Sulphuric acid were seen at Rf value of 0.95(Light yellow). Fig C : Thin layer chromatography of various extracts Giloy satva Ethyl acetate extract of Giloy satva when performed in solvent system – Benzene: Ethyl acetate (2:1) showed no spot on exposure to 10% Sulphuric acid. Methanol extract of Giloy satva when performed in solvent system – Chloroform: Methanol (2:1) showed no spot on exposure to 10% Sulphuric acid. III. BIOEFFICACY-ANTIMICROBIAL POTENTIALS For the last several decades, enormous interest has been generated in the remarkable features of medicinal plant research for their biological activities, though a no. of plants still remain neglected. Several classes of natural products have been identified for potential pharmacological efficacy but emphasis on bioactivity relationship of the plant constituents has attended much lesser to expectancy. In more recent times, microbial metabolites have been utilized in medicine, however, natural products are considered as chemical models or templates for designing the synthesis of many compounds used as drugs. One of the successful strategies for the investigation of medicinal agents from higher plants includes pharmacological screening of plant extracts followed by a bioactivity-guided fractionation leading to the isolation of pure constituents. From the purified extracts, a wide variety of new substances as useful pharmacoactive agents have been isolated from the fractions that shared activity efficiency due to specific group of compounds present in various herbal drugs which otherwise could be missing from other plant(s) used as adulterants. By such doings, there is an increase in the biomass of the herbal drugs for gaining profit economically but eventually resulting in poor quality in terms of its drug efficacy and might be responsible at times for its toxicity. Various sequential extracts of Guduchi (Tinospora cordifolia) and Giloy satva were screened for various test microbes and their inhibition zones and activity indexes were calculated. These results are given on the following pages. III.1. Bactericidal efficacy of Tinospora cordifolia – When antibacterial activity of Tinospora cordifolia was performed against Escherichia coli, the maximum efficacy was exhibited by Distilled water extract (IZ – 24mm) and the minimum efficacy was shown by Pet ether extract (IZ – 6mm). While, mediocre efficacies were shown by benzene, chloroform, ethyl acetate, methanol extracts having inhibition zone – 10mm, 10mm, 16mm & 11mm respectively. When antibacterial activity of Tinospora cordifolia was performed against Bacillus subtilis, the maximum efficacy was exhibited by Methanol extract (IZ – 11mm) and the minimum efficacy was shown Chloroform extract (IZ – 0mm). While, mediocre efficacies were shown by pet ether, benzene, ethyl acetate and distilled water extracts having inhibition zone- 10mm, 9mm, 8mm & 7mm respectively. When antibacterial activity of Tinospora cordifolia was performed against Pseudomonas aeruginosa, the maximum efficacy was exhibited by Methanol extract (IZ – 12mm) and the minimum efficacy was shown by Distilled water extract (IZ – 0mm). While, mediocre efficacies were shown by pet ether, benzene, chloroform and ethyl acetate extracts having inhibition zone- 8mm, 8mm, 6mm & 10mm respectively. Fig D: Inhibition zone of Methanol extract of Tinospora cordifolia against Pseudomonas aeruginosa When antibacterial activity of Tinospora cordifolia was performed against Staphyllococcu aureuss, the maximum efficacy was exhibited by Distilled water extract (IZ – 17mm) and the minimum efficacy was shown by Methanol extract (IZ – 7mm). While, mediocre efficacies were shown by pet ether, benzene, chloroform and ethyl acetate extracts having inhibition zone- 12mm, 9mm, 8mm & 12mm respectively. Successive series Of Tinospora cordifolia (Graphical presentation) 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 ne e l ha no M et D e ce ha no D ist M et ille d w at er ta t l ist ille th nz e ce ro f te w d at er er or m hl o ro f or m hl o ta t la Et hy la Et hy Activity Index Pe Be er Be nz e Fig E: Antibacterial activity of Tinospora cordifolia against E. coli 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 te th ne C Activity Index Fig F: Antibacterial activity of Tinospora cordifolia against B. subtilis Pe C 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 ne rm he r at e no ze of o et ha ce t et D ist ill w at e r l M Ethyl acetate Activity Index Be n C hl or Pe t Fig G: Antibacterial activity of Tinospora cordifolia against P. aeruoginosa 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Pet Ether Benzene Chloroform Methanol Distill water Activity Index Fig H: Antibacterial activity of Tinospora cordifolia against S. aureus Et hy la Results in Tabulated and graphical form are as follows: Table 1: Bioefficacy of Tinospora cordifoli in terms of Inhibition Zone and Activity Index Successive series 1.Petroleum ether 2.Benzene 3.Choloroform 4.Ethyl acetate 5.Methanol 6.Distilled water (Standard tetracycline) I.Z. = Inhibition Zone of growth of Microorganisms A.I. = Activity Index A.I. = I.Z. of Successive / I.Z. of Standard Escherichia coli IZ 6 10 10 16 11 24 33 Pseudomonas Bacillus Staphylococcus AI 0.48 0.36 0.32 0.48 0.28 0.68 1.0 aeruginosa subtilis aureus AI IZ AI IZ AI IZ 0.18 8 0.22 10 0.33 12 0.30 8 0.30 6 0.48 10 0.33 12 0.72 1.0 36 0.22 9 0.16 0.27 8 0.33 11 7 1.0 30 0.30 9 8 0.26 12 0.36 7 0.23 17 1.0 25 III.2. Bactericidal efficacy of Giloy satva – When antibacterial activity of Giloy satva was performed against Escherichia coli, the maximum efficacy was exhibited by Distilled water extract (IZ – 22mm) and the minimum efficacies were shown by pet ether, benzene, chloroform and ethyl acetate (IZ – 0mm). While, mediocre efficacy was shown by methanol extract having inhibition zone – 9mm. When antibacterial activity of Giloy satva was performed against Bacillus subtilis, the maximum efficacy was exhibited by Methanol extract (IZ – 14mm) and the minimum efficacies were shown by pet ether, benzene, chloroform and ethyl acetate (IZ – 0mm). While, mediocre efficacy was shown by distilled water extract having inhibition zone – 10mm. Fig I : Inhibition zone of successive extracts of Giloy satva against Pseudomonas aeruginosa where E=Benzene(IZ=0mm), F=Chloroform(IZ=0mm), G=Ethyl acetate(IZ=0mm) & H=Methanol(IZ=11mm). When antibacterial activity of Giloy satva was performed against Pseudomonas aeruginosa, the maximum efficacy was exhibited by Methanol extract (IZ – 11mm) and the minimum efficacies were shown by pet ether, benzene, chloroform and ethyl acetate (IZ – 0mm). While, mediocre efficacy was shown by distilled water extract having inhibition zone – 9mm. When antibacterial activity of Giloy satva was performed against Staphyllococcus aureus, the maximum efficacies were exhibited by Methanol and Distilled water (IZ – 13mm) and the minimum efficacies were shown by pet ether, benzene, chloroform and ethyl acetate (IZ – 0mm). Successive serious of Giloy satva (Graphical presentation) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Pet Ether Benzene Chloroform Ethyl acetate Methanol Distill water Activity Index Fig J : Antibacterial activity of Giloy satva against E. coli 0.5 Activity Index 0.4 0.3 0.2 0.1 0 Pet Ether Benzene Chloroform Ethyl acetate Methanol Distill water Fig K: Antibacterial activity of Giloy satva against B. subtilis 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Pet Ether Benzene Chloroform Ethyl acetate Methanol Distill water Activity Index Fig L: Antibacterial activity of Giloy satva against P. aeruoginosa 0.6 Activity Index 0.5 0.4 0.3 0.2 0.1 0 Pet Ether Benzene Chloroform Ethyl acetate Methanol Distill water Fig M: Antibacterial activity of Giloy satva against S. aureus Results in Tabulated and graphical form are as follows: Table 2: Bioefficacy of Giloy satva in terms of Inhibition Zone and Activity Index Successive series 1.Petroleum Escherichia coli IZ Pseudomonas Bacillus subtilis IZ AI 0.43 0.31 1.0 Staphylococcus aureus IZ 13 13 24 AI 0.54 0.54 1.0 aeruginosa AI IZ AI 0.24 11 0.59 9 1.0 32 ether 2.Benzene 3.Choloroform 4.Ethyl acetate 5.Methanol 9 6.Distilled 22 water (Standard tetracycline) 37 0.34 14 0.28 10 1.0 32 I.Z. = Inhibition Zone of growth of Microorganisms A.I. = Activity Index A.I. = I.Z. of Successive / I.Z. of Standard 4.Conclusions: Standardization of ayurvedic drugs and plant materials is the need of the day. Several pharmacopoeias containing monographs on plant materials describe only the physicochemical parameters. Hence, modern methods describing the identification and quantification of active compounds in the plant material may be useful for proper standardization of herbs and their formulation. Tinospora cordifolia (Menispermaceae) is one such plant which is widely used in indigenous system of medicine. The stems have been used for centuries, in ayurvedic preparations for the treatment of jaundice, diabetics, skin diseases and anemia. The plant has been shown to possess anti-inflammatory and anti-allergic properties. More recently, the immunomodulatory properties , antioxidant and antineoplastic activities have been reported. Among the complex mixture of biologically active compounds in the plant, microscopic identification, TLC, and bio efficacies can be used as an analytical characterization to determine the quality of plant material of different sources. It is very surprising that the simple tests which are not useful in identification sometimes become very beneficial for the characterization and validation of important Ayurvedic drugs. Hence, the microscopic characterization is simply a authentic and easy tool for validation of Guduchi satva. The use of safe drug is the motto of our healthy being so if we use the correct parameters for the identification of the drug it will be very useful for Ayurvedic drugs to not only enhance their quality and efficacy but also their therapeutic potentials as drugs.