Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi) – validation of the Ayurvedic pharmacology through experimental and clinical studies

T. cordifolia (Guduchi) is a large, glabrous, perennial, deciduous, climbing shrub of weak and fleshy stem found throughout India. It is a widely used plant in folk and Ayurvedic systems of medicine. The chemical constituents reported from this shrub belong to different classes, such as alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds and polysaccharides. Various properties of T. cordifolia, described in ancient texts of Ayurveda, like Rasayana, Sangrahi, Balya, Agnideepana, Tridoshshamaka, Dahnashaka, Mehnashaka, Kasa-swasahara, Pandunashaka, Kamla-Kushta-Vataraktanashaka, Jwarhara, Krimihara, Prameha, Arshnashaka, Kricch-Hridroganashak, etc., are acquiring scientific validity through modern research adopting "reverse pharmacological" approach. Potential medicinal properties reported by scientific research include anti-diabetic, antipyretic, antispasmodic, anti-inflammatory, anti-arthritic, antioxidant, anti-allergic, anti-stress, anti-leprotic, antimalarial, hepato-protective, immuno-modulatory and anti-neoplastic activities. This review brings together various properties and medicinal uses of T. cordifolia described in Ayurveda, along with phytochemical and pharmacological reports.


INTRODUCTION
Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi) is a large, glabrous, deciduous climbing shrub belonging to the family Menispermaceae. [1][2][3] It is distributed throughout the tropical Indian subcontinent and China, ascending to an altitude of 300 m. In Hindi, the plant is commonly known as Giloe, [4] which is a Hindu mythological term that refers to the heavenly elixir that has saved celestial beings from old age and kept them eternally young. Other common names and synonyms are Guduchi, Amrita, Amritavalli, Madhuparni, Guduchika, Chinnobhava, Vatsadani, Tantrika, Kundalini, Chakralakshanika (Sanskrit), Gulancha (Bengali), Gurcha (Hindi), Garo, Galac (Gujarati), Thippateega (Telugu), Amrutavalli (Kannada), Amrita, Gilo (Kashmiri), Chittamrutu (Malayalam), Gulvel (Marathi), Guluchi (Oriya), Gilo (Punjabi), Seendal, Seendil Kodi (Tamil), Siddhilata, Amarlata (Assamese) Heartleaf Moonseed, Tinospora (English). [5] Guduchi, the Sanskrit name, means one which protects the entire body. The term amrita is attributed to its ability to impart youthfulness, vitality and longevity. The stems of T. cordifolia are rather succulent with long fi liform fl eshy aerial roots from the branches. The bark is creamy white to gray, deeply left spiraily the space in between being spotted with large rosette-like lenticels. The leaves are membranous and cordate. The fl owers are small and yellow or greenish yellow. In axillary and terminal racemes or racemose panicles, the male fl owers are clustered and female fl owers are usually solitary. The drupes are ovoid, glossy, succulent, red and pea sized. The seeds are curved. Fruits are fl eshy and single seeded. Flowers grow during summer; and fruits, during winter. [6,7] Stem of the T. cordifolia appears in varying thicknesses, ranging from 0.6 to 5 cm in diameter; young stems are green with smooth surfaces and swelling at nodes, while the older ones show a light brown surface marked with warty protuberances due to circular lenticels; transversely smoothened surface shows a radial structure with conspicuous medullary rays traversing porous tissues; tastes bitter [ Figure 1]. [5] ETHNOBOTANICAL, FOLK AND TRIBAL USES OF T. CORDIFOLIA (GUDUCHI) There are over 400 different tribal and other ethnic groups in India. Each tribal group has its own tradition, folk language, beliefs and knowledge about use of natural resources as medicines. [22] T. cordifolia fi nds a special mention for its use in tribal or folk medicine in different parts of the country. Some of the important uses reported in the literature [22] are listed in the Table 3. Almost all the parts of the plant are documented to be useful in ethnobotanical surveys conducted by ethnobotanists. [23][24][25][26]

PHYTOCHEMICAL CHARACTERIZATION
A large number of chemicals have been isolated from T. cordifolia, belonging 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%), calcium and phosphorus. [27] Four new clerodane furano diterpene glucosides (amritosides A, B, C and D) have been isolated as their acetates from stems. The structures of these compounds were established on the basis of spectroscopic studies. [28] The glycosyl component of a polysaccharide from T. cordifolia has been isolated, purifi ed, methylated, hydrolyzed, reduced and acetylated. The partially methylated alditol acetate (PMAA) derivative thus obtained have been subjected to Gas Chromatography-Mass Spectrometry (GC-MS) studies. The following types of linkages were reported: terminal-glucose, 4-xylose, 4-glucose, 4, 6-glucose and 2, 3, 4, 6-glucose. [29,30] Callus and cell suspension cultures have been established from the stem explants of the plant. Accumulation of berberine and jatrorrhizine (protoberberine alkaloids) was observed in both callus and cell suspension cultures. [31] The signaling mechanism of the novel (1, 4)-alpha-D-glucan (RR1) isolated from T. cordifolia was investigated in macrophages to evaluate its immunostimulating properties. [32] An arabinogalactan has been isolated from the dried stems and examined by methylation analysis, partial hydrolysis and carboxyl reduction. Purifi ed polysaccharide showed polyclonal mitogenic activity against B-cells; their proliferation did not require macrophages. [33] Detailed chemical constitution of T. cordifolia is given in Table  4. [34] Phytochemical characterization includes a test for one of  the phytochemical components, namely, tinosporaside (limits, 0.03% to 0.04%). [35,36]

REVERSE PHARMACOLOGICAL AND CLINICAL CORRELATES
Indian contributions to the therapeutic revolution through reverse pharmacology will have to eventually integrate stateof-the-art high-throughput screening, combinatorial chemistry and effects of the old or novel compounds/ plants on human gene expression and proteomics. [37] Much work has been done on T. cordifolia to validate its effects and this section describes some of these studies.
T. cordifolia has been used in Ayurvedic preparations for the treatment of various ailments throughout the centuries. It is used as a rasayana to improve the immune system and body resistance against infections. The whole plant is used medicinally; however, the stem is approved for use in medicine as listed by the Ayurvedic Pharmacopoeia of India. [5] This is due to higher alkaloid content in the stems than in the leaves. It is a traditional belief that Guduchi satva obtained from the Guduchi plant growing on neem tree (Azadirachta indica) is more bitter and more effi cacious and is said to incorporate the medicinal values of neem. [38]

EFFECTS ON STRESS, LEARNING AND MEMORY
T. cordifolia is known as a medhya rasayana (learning and memory enhancer) in Ayurveda. It is also described to be useful for treatment of bhrama (Vertigo) in various Ayurvedic texts. Signifi cant response has been found in children with moderate degree of behavior disorders and mental defi cit, along with improvement in IQ levels. [34] The root of T. cordifolia is known to be used traditionally for its anti-stress activity. [22] In a 21-day randomized, doubleblind placebo-controlled study, the pure aqueous extract of the root was found to enhance verbal learning and logical memory. [39] T. cordifolia has also been shown to enhance cognition (learning and memory) in normal rats and reverse cyclosporine-induced memory defi cit. Both the alcoholic and aqueous extracts of T. cordifolia produced a decrease in learning scores in Hebb William maze and retention memory, indicating enhancement of learning and memory.
The histopathological examination of hippocampus in cyclosporine-treated rats showed neurodegenerative changes, which were protected by T. cordifolia. [40] Various extracts of the T. cordifolia exhibited comparable anti-stress activity in mice. [41,42]

ANTI-INFLAMMATORY, ANTI-ARTHRITIC AND ANTI-OSTEOPOROTIC ACTIVITIES
T. cordifolia is mentioned to treat vatarakta (gouty arthritis) and daha (burning sensation) in various Ayurvedic texts [ Table 2]. It is traditionally used in compound formulations for the treatment of rheumatoid arthritis. [43] The alcoholic extract of T. cordifolia has been found to exert anti-infl ammatory actions in models of acute and subacute infl ammation. [44] The water extract of the stem of neem-giloe [The T. cordifolia that grow on Azadirachta indica (neem)] signifi cantly inhibited acute infl ammatory response evoked by carrageenin in a dose of 50 mg/100 g given orally and intraperitoneally. A signifi cant inhibition of primary and secondary phases of infl ammation was observed in a model of adjuvant-induced arthritis. It also signifi cantly inhibited antibody formation by typhoid "H" antigen. A mild analgesic effect of its own as well as potentiation of morphine analgesia has been reported. [45] In another study aqueous extract of T. cordifolia showed a signifi cat antiinfl ammatory effect in the cotton pellet granuloma and formalin induced arthritis model, it's effect was comparable with indomethacin and its mode of action appeared to resemble that of nonsteroidal antiinflamatory ageant. The dried stem of T. cordifolia produced signifi cant anti-infl ammatory effect in both acute and subacute models of infl ammation. T. cordifolia was found to be more effective than acetylsalicylic acid in acute infl ammation, although in subacute infl ammation, the drug was inferior to phenylbutazone. [46] The aqueous extract of stem was reported to exert a signifi cant anti-infl ammatory effect in both cotton pellet-induced granuloma (1, 250 and 500 mg/kg given orally) and formalin-induced arthritis (1 mg/kg given orally) rat models. [47,48]  Twak-roga (Skin disease) Decoction of stem is administered orally.
Mundas of Chhota Nagpur Fracture Paste of whole plant used as plaster.
In certain parts of India Bites of poisonous insects and venomous snake, eye disorders.
The paste of Guduchi is applied to the part bitten and administered internally through mouth at intervals of half an hour. Juice or decoction of the root is poured into the eyes.

ANTI-ALLERGIC ACTIVITY
T. cordifolia is used for the treatment of kasa (cough) and swasa (asthma), which is described in various texts of Ayurveda [ Table 2]. T. cordifolia is traditionally used for the treatment of asthma, and the juice is also employed for the treatment of chronic coughs. [49] In a clinical study, 100% relief was reported from sneezing in 83% of the patients on treatment with T. cordifolia,. Similary, there was relief from nasal discharge was reported in 69%; from nasal obstructions 61% and from nasal pruritus, in 71%. In placebo group, there was relief from sneezing only in 21% patients; from nasal discharge, in 16.2%; from nasal obstruction, in 17%; and from nasal pruritus, in 12%. Thus, T. cordifolia signifi cantly decreased all symptoms of allergic rhinitis and was well tolerated. [50] The anti-allergic and bronchodilator properties of an aqueous extract of the stem evaluated on histamine-induced bronchospasm in guinea pigs, capillary permeability in mice and mast cell disruption in rats showed that it signifi cantly decreased bronchospasm induced by 5% histamine aerosol, decreased capillary permeability and reduced the number of disrupted mast cells. [51,52]

ANTIOXIDANT ACTIVITY
T. cordifolia is mentioned as vishaghni, vishahara and tridoshashamaka in various texts of Ayurveda [ Table 2]. A signifi cant increase in the concentration of thiobarbituric acidreactive substances (TBARS) in brain, along with its decrease in heart, was observed in diabetic rats. Tinospora cordifolia treatment decreased the concentrations of glutathione reductase (GSH) and decreased activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in the tissues of diabetics rats. Alcoholic extract of the root of T. cordifolia (TCREt) administered at a dose of 100 mg/kg orally to diabetic rats for 6 weeks normalized the antioxidant status of heart and brain. The effect of T. cordifolia root extract was better than glibenclamide (600 µ/kg) although Insulin (6 units/kg) restored all the parameters to normal status. [53,54] T. cordifolia has also been reported to elevate GSH levels, expression of the gammaglutamylcysteine ligase and Cu-Zn SOD genes. The herb also exhibited strong free radical-scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. [55] Aqueous extract of T. cordifolia inhibited Fenton (FeSO4) reaction and radiationmediated 2-deoxyribose degradation in a dose-dependent fashion, with an IC50 value of 700 µ/mL for both Fenton and radiation-mediated 2-DR degradation. Similarly, it showed a moderate but dose-dependent inhibition of chemically generated superoxide anion at 500 µ/mL concentration and above, with an IC50 value of 2000 µ/mL. [56] In various studies, T. cordifolia was found effective in iron-mediated lipid damage and gamma-ray-induced protein damage, [57] amelioration of cyclophosphamide-induced toxicity, [58] alteration of lethal effects of gamma rays, [59] induction of enzymes of carcinogen/ drug metabolism and inhibition of lipid peroxidation in mice, [60] free radical generation and lipid peroxidation during oxygen-glucose deprivation, [61] and nitric oxide scavenging effetcs. [62] The extract of T. cordifolia has demonstrated antioxidant action in the alloxan induced diabetes model as well. [63]

ANTINEOPLASTIC AND RADIO-PROTECTIVE ACTIVITY
Intraperitoneal injection of the alcoholic extract of T. cordifolia has been shown to Dalton's lymphoma (DL) bearing mice e stimulated macrophage functions likephagocytosis, antigen-presenting ability and secretion of Interleukin-1 (IL-1), tumour necrosis factor (TNF) and Reference Nutrient Intake (RNI) as well as slowed tumor growth and increased lifespan of the tumor-bearing host. [64] T. cordifolia was has beem shown effective in several other tumour models including Ehrlich ascites carcinoma (EAC) in mice. [65] It induces proliferation and myeloid differentiation of bone marrow precursor cells in a tumorbearing host, [66] activates tumor-associated macrophagesderived dendritic cells, [67] is effective against various cancers, [68] killing the cancer cells very effectively in vitro [69,70] inhibits skin carcinogenesis in mice, [71] and inhibits experimental metastasis. [72] T. cordifolia may offer an alternative treatment strategy for cancer in combination with gamma radiation. [73,74]

ANTIPYRETIC AND ANTI-INFECTIVE ACTIVITY
Traditionally T. cordifolia is known for its jwarahara activity (antipyretic activity), as mentioned in Table 2. The watersoluble fraction of 95% ethanolic extract of T. cordifolia plant has shown signifi cant antipyretic activity. [75] In another experimental study, antipyretic effects have been reported in the hexane-and chloroform-soluble portions of T. cordifolia stems. [76] Various studies show remarkable anti-infective and antipyretic properties of T. cordifolia. [77,78] Pre-treatment with T. cordifolia was shown to impart protection against mortality induced by intra-abdominal sepsis following coecal ligation in rats and signifi cantly reduced mortality from induced by E. coli-induced peritonitis in mice. [79]

HEPATO-PROTECTIVE ACTIVITY
Various Ayurvedic preparations of T. corfi folia are indicated in pandu (anemia) and kamla (jaundice). A clinical study has shown that Guduchi plays an important role in normalization of altered liver functions (ALT, AST). [80] The antihepatotoxic activity of T. cordifolia has been demonstrated in CCl 4 induced liver damage, normallising liver function as assessed by morphological, biochemical (SGPT, SGOT, serum alkaline phosphatase, serum bilirubin) and functional (pentobarbitone sleep time) tests. T. cordifolia revealed hepatoprotective action in goats. [81] A signifi cant increment in the functional capacities of rat peritoneal macrophages was observed following T. cordifolia treatment. [82] Addition of extract for the fi rst 6 weeks to chloroquine showed regression of spleen by 37% to 50% after 6 weeks and 45% to 69% after 6 months from the start of treatment. Likewise, decrease in IgM and increase in Hb, as well as wellbeing (Karnofsky performance scale), were observed. [83] T. cordifolia prevents antitubercular drugs [84,85] and bile salts [86] induced hepatic damage, x and obstructive jaundice. [87] The extract has also exhibited in vitro inactivating property against hepatitis B and E surface antigens in 48 to 72 hours. [88]

ANTI-HYPERGLYCEMIC ACTIVITY
T. cordifolia is widely used in Ayurveda for treating diabetes mellitus. [53,58,63] Various studies demonstrate amelioration of experimental diabetic neuropathy and gastropathy in rats, [89] reduction of blood sugar in alloxan-induced hyperglycemic rats and rabbits, [90] signifi cant reduction in blood glucose and brain lipids, [91] increase in glucose tolerance in rodents, [92,93] increase in glucose metabolism, [93] inhibitory effect on adrenalineinduced hyperglycemia by pyrrolidine derivative, [94,95] and signifi cant hypoglycemic effect in normal and alloxan diabetic rabbits [96] following administration of T. cordifolia.

IMMUNOMODULATORY ACTIVITY
In Ayurveda T. cordifolia is believed to have rasayana (rejuvenating), balya (tonic), vayah-sthapana (antiaging), aayushyaprada (increases the lifespan), vrishya (aphrodisiac) and chakshusya (useful in eye disorders) properties [ Table 2]. The alcoholic and aqueous extracts of T. cordifolia are reported to have benefi cial effects on the immune system [81,87] and have been tested successfully for their immunomodulatory activity. [97][98][99][100][101][102][103] The degradation of proteins due to photosensitization as assessed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was effectively reduced by simultaneous treatment with G1-4A/ PPI (partially purifi ed immunomodulator) from T. cordifolia during photosensitization. [104] The novel (1,4)-alpha-D-glucan derived from the plant activates the immune system through the activation of macrophages via TLR6 signaling, NFkappaB translocation and cytokine production. [32,105] Tinospora cordifolia differentially regulate elevation of cytokines as evidenced by the increased production of antiangiogenic agents IL-2 and tissue inhibitor of metalloprotease-1 (TIMP-1) in the B16F10-injected, extract-treated animals. The observed antiangiogenic activity of the plant T. cordifolia is related to the regulation of the levels of cytokines and growth factors in the blood. [106] The aqueous extract of T. cordifolia was found to enhance phagocytosis in vitro. The aqueous and ethanolic extracts also induced an increase in antibody production in vivo. [107] T. cordifolia extract (TCE) treatment caused signifi cant reduction in eosinophil count and improved hemoglobin in HIV patients. Sixty percent patients receiving TCE and 20% on placebo reported decrease in the incidence of various symptoms associated with disease. [108] Diabetic patients with foot ulcers on T. cordifolia as an adjuvant therapy showed signifi cantly better fi nal outcome with improvement in wound healing. [109]  ]. RTc treatment thus suggesting its radio-protective mechanism. [110] Ten days of treatment with T. cordifolia (100 mg/kg/d) induced a signifi cant (P < 0.01) increase in the number of (Colony Forming Units of ranulocyte-macrophage series (CFU-GM; 255 ± 49.32 vs. 38.51 ± 9.98). This suggests that activation of macrophages by T. cordifolia leads to increase in GM-CSF, which leads to leukocytosis and improved neutrophil function. [111]

DIURETIC ACTIVITY
T. cordifolia has been described as useful in mutrakriccha (urinary trouble) separately and in the form of various formulations in Ayurveda, as mentioned in Table 2. In a scientifi c study on rats and human volunteers, T. cordifolia was found to have diuretic effects. [112] It was also found effective in modulation of morphology and some gluconeogenic enzymes activity in diabetic rat kidney. [113]

CARDIOPROTECTIVE ACTIVITY
Bhavprahash Nighantu and Shaligram Nighantu describe T. cordifolia to have hrudya (cardioprotective) properties and is useful in hridroga (cardiac disorders) ( Table 2). A dosedependent reduction in infarct size and in serum and heart lipid peroxide levels were observed with prior treatment with T. cordifolia in ischemia-reperfusion-induced myocardial infarction in rats. [114] The stem extract has been normalize alterations in the lipid metabolism caused by diabetes mellitus in streptozotocin-induced diabetic rats indiectly benefi ting the heart. [115] Administration of the extract of T. cordifolia roots (2.5 and 5.0 g/kg body weight) for 6 weeks resulted in a signifi cant reduction in serum and tissue cholesterol, phospholipids and free fatty acids in alloxan diabetic rats. [116] ANTI-LEPROTIC ACTIVITY T. cordifolia is used for its kushtahara (anti-leprotic) properties, along with wide use in kandu and visarpa (types of skin disorders) and has been shown to exert anti-leprotic activity in a combination formulation. [117] GASTROINTESTINAL AND ANTI-ULCER ACTIVITY Ayurvedic properties of T. cordifolia include sangrahi, arshahara, aruchinashaka, dipana, agnidipana, chardihara, trishnahara, trishnanashaka and hikkahara. Treatment with a formulation containing T. cordifolia has been shown to reduce ulcer index total acidity,with an increase in the pH of gastric fl uid in pylorus-ligated rats and in the ethanol-induced gastric mucosal injury in rats. [118] ANTIFERTILITY ACTIVITY Oral administration of 70% methanolic extract of T. cordifolia stem to male rats at a dose level of 100 mg/d for 60 days did not cause body weight loss but decreased the weight of testes, epididymis, seminal vesicle and ventral prostate in a signifi cant manner. [119] OSTEOPROTECTIVE ACTIVITY Rats treated with T. cordifolia (10 mg/kg body weight) showed an osteoprotective effect, as the bone loss in tibiae was slower than that in controls. Serum osteocalcin and cross-laps levels were signifi cantly reduced. This study demonstrates that extract of T. cordifolia has the potential for being used as antiosteoporotic agent. [120]

SAFETY PHARMACOLOGY
It is a common misconception that Ayurvedic medicines are always safe. In fact, they also pose serious health risks either in the form of adverse reactions or in the form of drug interactions. In a clinical study, T. cordifolia has been shown to be at a dose of 500 mg/d for a period of 21 days in healthy individuals. [80] It has also been shown not to exert any remarkable adverse effects on the cardiovascular system, [121,122] renal system, [112,122] central nervous system [39][40]45,121,123] and gastrointestinal system. [49,101,124]

CONCLUSION
The pharmacological actions attributed to Tinospora cordifolia in Ayurvedic texts have been validated by a remarkable body of modern evidence suggesting that this drug has immense potential in modern pharmacotheraoeutics.