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, Prof. C. K. Ojha,

Department of Biotechnology, Director (Academics)

Mahatma Gandhi Institute of

Applied Sciences, Jaipur.

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 Sumit kumar

Date : 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

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

Kingdom Planti

Division: Magnoliophyta

Class: Magnoliopsida

Order: Ranunculales

Family: Menispermaceae

Genus: Tinospora

Species: Cordifolia

Tinospora cordifolia

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 (1.8-

3.75 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 anti-

leprotic properties.

The aqueous extract of the stem antagonizes the effect of agonists such as 5-

hydroxytriptamine, 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 immuno-

modulatory 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 anti-

bacterial 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 hemato-

biochemical 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 anti-

inflammatory 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 for gout and high uric acid.

2. It is a natural blood purifier and is very useful for skin problems like Acne,

Psoriasis, eczema, Lichen planus and others.

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, interleukin-

4, 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 studies-

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.

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)

Fig E: Antibacterial activity of Tinospora cordifolia against E. coli

Fig F: Antibacterial activity of Tinospora cordifolia against B. subtilis

Fig G: Antibacterial activity of Tinospora cordifolia against P. aeruoginosa

Fig H: Antibacterial activity of Tinospora cordifolia against S. aureus

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

Escherichia

coli

Pseudomonas

aeruginosa

Bacillus

subtilis

Staphylococcus

aureus

IZ AI IZ AI IZ AI IZ AI

1.Petroleum

ether

6 0.18 8 0.22 10 0.33 12 0.48

2.Benzene 10 0.30 8 0.22 9 0.30 9 0.36

3.Choloroform 10 0.30 6 0.16 - - 8 0.32

4.Ethyl acetate 16 0.48 10 0.27 8 0.26 12 0.48

5.Methanol 11 0.33 12 0.33 11 0.36 7 0.28

6.Distilled

water

24 0.72 - - 7 0.23 17 0.68

(Standard

tetracycline)

33 1.0 36 1.0 30 1.0 25 1.0

I.Z. = Inhibition Zone of growth of Microorganisms

A.I. = Activity Index

A.I. = I.Z. of Successive / I.Z. of Standard

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)

Fig J : Antibacterial activity of Giloy satva against E. coli

Fig K: Antibacterial activity of Giloy satva against B. subtilis

Fig L: Antibacterial activity of Giloy satva against P. aeruoginosa

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

Escherichia

coli

Pseudomonas

aeruginosa

Bacillus

subtilis

Staphylococcus

aureus

IZ AI IZ AI IZ AI IZ AI

1.Petroleum

ether

- - - - - - - -

2.Benzene - - - - - - - -

3.Choloroform - - - - - - - -

4.Ethyl acetate - - - - - - - -

5.Methanol 9 0.24 11 0.34 14 0.43 13 0.54

6.Distilled

water

22 0.59 9 0.28 10 0.31 13 0.54

(Standard

tetracycline)

37 1.0 32 1.0 32 1.0 24 1.0

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 physico-

chemical 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.