Research Journal of Chemical Sciences ________________________________________ ISSN 2231-606X Vol. 1(6), 71-79, Sept. (2011) Res.J.Chem.Sci. International Science Congress Association 71 Assessment of the Multifaceted Immunomodulatory Potential of the Aqueous Extract of Tinospora CordifoliaUpadhyaya R., Pandey R.P., Sharma V. and Verma Anita K. 1Nanobiotech Lab, Dept. of Zoology, K. M. College, University of Delhi, Delhi- 110007, INDIA 2 Dept. of Botany, MKR Government College, Ghaziabad, Uttar Pradesh, INDIAAvailable online at: www.isca.in (Received 1st August 2011, revised 14th August 2011 accepted 23rd August 2011)Abstract Immunomodulation relates to potentiation or suppression of the immune responses of the host, depending on the requirement of the situation. Tinospora cordifolia (T.C.), an indispensable medicinal plant, has been used for the treatment of various diseases and has been recommended for improving the immune system. In the current study, we have evaluated the cytotoxicity and immunomodulatory activity of the aqueous extract of T.C. using in vitro and ex vivo models. Results show that there was dose-dependant cytotoxicity in B16F10 mouse melanoma cells. ~90% cytotoxicity was observed 72 hours post-treatment with 100µg/ml of T.C. extract. Interestingly, a low dose of 1µg/ml also showed ~60% cytotoxicity. Further, the aqueous extract of T.C. exhibited boosting of phagocytic ability of macrophages, remarkable enhancement in nitric oxide production by stimulation of splenocytes and macrophages at 1mg/kg dosage of the extract. Immunomodulatory ability of a compound can be determined by its capability to influence the cytokine production, mitogenicity, stimulation and activation of immune effector cells. The extracts have been investigated for their effect on the cytokine profile (IL-6) by ELISA. Splenocytes cultured in low concentration of T.C, as low as 1.56 g/ml, produced significantly higher levels of IL-6 as compared to un-stimulated cells. The present study reveals the multifaceted immunomodulatory potential of T.C.. Keywords:Tinospora cordifolia, cytokines-IL-6, cytotoxicity, Immuno-modulation. IntroductionAlternative medicine is now recognized as an invaluable resource even by the most intransigent clinicians of advanced countries. Plant extracts have been widely investigated for their possible immunomodulatory properties1-4. Natural products are generally known to be efficacious with minimal side effects, but some natural compounds have been reported to act as mutagens and/or carcinogens6-8. These genotoxic insults often lead to cancer, Alzheimer’s disease10 and other chronic degenerative diseases, such as atherosclerosis and heart diseases, which are the leading cause of death in human and animal populations11. There is great interest in development of new drugs from traditionally used medicinal plants like Tinospora cordifolia [TC] Ayurveda refers to Tinospora cordifolia as Amrita' or the ‘Nectar of Immortality’. The term 'Amrita' is attributed to this drug in recognition of its ability to impart youthfulness, vitality and longevity. Immunomodulation can be determined by the capacity of the compounds to influence the cytokine production, mitogenicity, stimulation and activation of immune effector cells 12Tinospora cordifolia, a rasayana drug is widely used in the Ayurvedic system of medicine as an anti-inflammatory, anti-arthritic, anti-allergic, anti-malarial and as an anti-diabetic13 drug. The extract from the stem of T. cordifoliais often used in dyspepsia, fever and urinary infections14. The anti-tumor promoting activity of Tinospora extract in a two-stage skin carcinogenesis model has already been explored 15. Owing to its ability to impact a plethora of diseases, the present study was undertaken to understand the effect of TC on the production of IL-6 in mice. IL-6 - being a pleiotropic cytokine involved in a variety of normal processes including acute reactions to injury, inflammation, activation of cytotoxic T-cells, and differentiation of B-cells - has generated interest as a new drug molecule against a variety of diseases. IL-6 may be of use in activating antitumor responses since increased levels of circulating IL-6 have been observed following systemic treatment of patients or animals with endotoxin 17. Many other factors such as adhesion molecules affect metastasis, and it is possible that IL-6 interacts with them18. Hence, we went ahead to investigate molecular drugs from medicinal plants that could modulate the immune response. The therapeutic potential of Tinospora cordifolia, commonly known as ‘Guduchi’, was studied to assess the immunomodulatory activity using in-vitro as well as ex-vivo models. Material and MethodsSodium dodecyl sulphate, N,N,N’,N’ tetramethyl-ethylenediamine, acrylamide, bisacrylamide, bromophenol blue, mercaptoethanol, ethylenediaminetetra acetic acid, trypan blue and MTT were procured from Sigma St Louis. 3,5-Dinitrosalicylic acid, potassium sodium tartarate, sodium hydroxide, dinitrosalicylic acid, sulphanilamide, N-1 naphthylene diamine, Coomassie brilliant blue R-250, ammonium per sulphate, glycine, glycerol, acetic acid and methanol were purchased from MERCK. RPMI-1640 Research Journal of Chemical Sciences _______________________________________________________ ISSN 2231-606X Vol. 1(6), 71-79, Sept. (2011) Res.J.Chem.Sci. International Science Congress Association 72 medium and foetal calf serum were purchased from Gibco, BRL. Lipopolysaccharide LPS, E coli 055-B5 and Sodium thioglycollate were procured from Difco and Micro BCA protein assay reagent kit was purchased from Pierce. Preparation of Aqueous Extract of T. cordifolia (TC):Dried 60gm fine powder from stem of Tinospora cordifoliawas soaked in 600ml of water. It was kept at room temperature (RT) for 48 hours with intermittent mixing. Aqueous extract of Tinospora cordifolia obtained after 48 hours of soaking was filtered using Whatman paper. Filtrate was stored at 4C. 90% Ammonium sulphate saturation was performed for protein precipitation. Precipitate of 90% ammonium sulphate saturation, stored at 4C, was centrifuged at 10000 rpm for 30 minutes. Supernatant was decanted off and pellet was resuspended in PBS 19. Protein Purification and Quantitation: The protein content of the dialyzed sample was estimated by BCA Method20. The BCA protein assay combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartarate. In the second step of the color development reaction, bicinchoninic acid (BCA) reacts with the reduced (cuprous) cation that was formed in step one. The intense purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is a water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the pale blue color of the first reaction. The molecular weight of the protein obtained from the aqueous extract of Tinosporacordifolia was determined by Sodium dodecyl sulphate - Polyacrylamide Gel Electrophoresis (SDS-PAGE 21. HPLC-LC/MS: The HPLC of TC extract was run in Agilent 1200 series XBD C-18 column (4.6x150mm) (USA). The injection volume was 5µl. The sample was run in gradient solvent (90% water 10% ACN & 90% ACN 10% water) at the UV 254nm. For LC/MS, the sample was run in Agilent technologies 6310 Ion trap LCMS. Fourier transform infrared spectroscopy [FTIR]: The transmission FTIR spectrum of the lyophilized powder of TC extract was done in KBr pellet by Perkin Elmer, UK RX1 model (Resolution 4cm-1; KBr Beamspliter; Source MIR; Detector LiTaO). NMR: For the H NMR experiments, lyophilized samples were dissolved in DO and the experiments were carried out at 25±0.05C, using a Bruker Spectrospin, operating at 300MHz and 7.05T with an inverse 5mm probe using PULSPROG. Chemical shifts were measured in parts per million (ppm). In-vitro Cytotoxicity Assay: MTT Assay was carried out in accordance with previously published protocol22. Briefly, B16F10 melanoma cells (5x10 cells/well) were cultured on a flat-bottomed 96 well plate. After 48 hours incubation, 20µl of MTT stock solution was added to each well of the assay plate, which was then incubated for 2 hours at 37şC. After incubation, the formazan crystals formed by the reduction of tetrazolium salt by the mitochondria of living cells were dissolved in DMSO. The plates were read in ELISA plate reader at wavelength of 540nm. Nitric Oxide [NO] Assay: The peritoneal exudates from mice were collected after 72 hours of an i.p. injection of 4% sodium thioglycolate solution. The exudate was centrifuged at 1100 rpm for 10 minutes at 4şC. The pellet was resuspended in RPMI . The cells [~0.5 million/well] were incubated for 3 hours at 37şC in the presence of 5% CO and then washed with RPMI. Cells with and without LPS (5mg/ml) in RPMI were incubated for 48 hours for production and estimation of NO, since nitric oxide is highly reactive and converted into sodium nitrite to form a stable end product of the reaction. Equal volumes of supernatant collected after incubation of macrophage culture was incubated with freshly prepared Griess reagent at room temperature for 5 minutes. Absorbance at 550 nm was measured by using ELISA reader. Nitrite was quantified by comparison with NaNO as a standard. Immunization of Mice: Equal volumes of collagen solution (in acetic acid) and complete Freund’s adjuvant (Chondrax) were taken in separate glass syringes. Emulsion was prepared by continuous passage between two syringes connected by a tube. The process was carried out in cold under ice. Emulsion formation was confirmed by pouring a drop on water surface. Prepared emulsion was dispensed in 1ml syringe and used for immunization of mice. Mice (Balb/c) were immunized by subcutaneous injection of 100 l emulsion using 26g needle. Simultaneously, the mice were given i.p. injection with 180 l of crude protein (0.6mg/ mice). Results and DiscussionThe aqueous extract of Tinospora cordifolia was precipitated with 90% ammonium sulfate, dialyzed against water and dried in vacuum to obtain crude T.C. (table-1). This crude TC was subjected to Q-Sepharose ion-exchange chromatography, resulting in the isolation of TC extract (figure-1). Its protein concentration was determined by BCA method (figure-1), and molecular weight was determined by SDS-PAGE (figure-2). Research Journal of Chemical Sciences ______ Vol. 1(6), 71-79, Sept. (2011) International Science Congress Association Flow chart for purifying protein from aqueous Purified Protein Pellets of Protein suspended in minimum amount of Phosphate Buffer Centrifugation Protein Precipitate Aqueous crude extract TC dried stem Figure-1 Q- Sepharose anion exchange chromatographic separation of different fractions from aqueous of Tinospora cordifolia ______ _________________________________ ______________ International Science Congress Association Flow chart for purifying protein from aqueous Tinospora cordifolia 220 97. 4 66 46 30 21. 5 14. 3 MW: KDa Purified Protein Pellets of Protein suspended in minimum amount of Phosphate Buffer Centrifugation Dialysis Protein Precipitate Aqueous crude extract TC dried stem 90% Ammonium Sulphate Precipitatio n water Sepharose anion exchange chromatographic separation of different fractions from aqueous extract Tinospora cordifolia Figure SDS PAGE indicating the molecular weight of the unknown protein ______________ __ ISSN 2231-606X Res.J.Chem.Sci. 73 Tinospora cordifolia n Figure -2 SDS PAGE indicating the molecular weight of the unknown protein Research Journal of Chemical Sciences ______ Vol. 1(6), 71-79, Sept. (2011) International Science Congress Association LC/M ______ _________________________________ ______________ International Science Congress Association HPLC-LC/MS Figure-3 HPLC of the aqueous extract of TC Figure- 4 LC/M S Scan of the aqueous extract of TC ______________ __ ISSN 2231-606X Res.J.Chem.Sci. 74 Research Journal of Chemical Sciences ______ Vol. 1(6), 71-79, Sept. (2011) International Science Congress Association The Proton NMR Chemical Shifts of Fourier Transform Infra Red Spectroscopy Dose Dependent Cytotoxicity of aqueous extract of T Satisfactory retention times and good resolution of extract was achieved using reverse phase C eluted with acetonitrile- water (10:90 v/v) at a flow rate of 102030405060708090100100Percent Cytotoxicity (%) ______ _________________________________ ______________ International Science Congress Association NMR Figure- 5 The Proton NMR Chemical Shifts of the aqueous extract of TC Figure- 6 Fourier Transform Infra Red Spectroscopy of the aqueous extract of TC Figure-7 Dose Dependent Cytotoxicity of aqueous extract of T inospora in B- 16 mouse melanoma cell line Satisfactory retention times and good resolution of the TC was achieved using reverse phase C -18 column water (10:90 v/v) at a flow rate of 0.6 ml/min. A sharp and symmetric peak with good baseline resolution and minimal tailing, thus facilitating the accurate measurement of peak area. 501010.1Dose (ug/ml) 24 h 48 h 72 h ______________ __ ISSN 2231-606X Res.J.Chem.Sci. 75 the aqueous extract of TC of the aqueous extract of TC 16 mouse melanoma cell line 0.6 ml/min. A sharp and symmetric peak was obtained, with good baseline resolution and minimal tailing, thus facilitating the accurate measurement of peak area. Two Research Journal of Chemical Sciences _______________________________________________________ ISSN 2231-606X Vol. 1(6), 71-79, Sept. (2011) Res.J.Chem.Sci. International Science Congress Association 76 peaks were found, one at 3.001 minute along with another one minor peak at 25.638 minute. In LC/MS Scan also two peaks were found among which one coincided with HPLC peak and the second peak after 25 minute was prominent in LC/MS. The sample clearly has more than one compound with expecting more ionic compound elution after 25 minute figure-3. In LC/MS the four different mass of 751.6, 354.5, 406.2 260 were found in between 2.1 to 3.6 minute & 25.3 to 30.5 minute. It conclude that the sample have the mixture of at least 4 different compound figure- 4. TheProton NMR chemical shifts of the TC extract was very sharp in between 3.2ppm to 3.6ppm and 4.7ppm to 4.3ppm, which represents the amide containing aliphatic compound in the samples (figure- 5). In FTIR, the prominent transmission peak was found at 3417.18cm-1, 1654.15cm-1 1398.89cm-1. The peak value corresponds to the presence of amide N-H and carboxylic stretch containing compound (figure- 6). The aqueous extract of T. cordifolia mainly contained polysaccharides, phenolic compounds and bitter principles 23, 24. The cytotoxic effect of TC increased in a dose- dependent manner in B-16F10 melanoma cell line in a 72 hours assay. 90% cytotoxicity was observed at 100µg/ml dose after 72 hours. The cytotoxic action of ‘guduchi’ could be attributed to the presence of alkaloids, di- terpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds or polysaccharides. Interestingly, ~60% cytotoxicity was observed at a low dose of 1µg/ml of the purified extract (figure-7). Results show that there is increase in nitric oxide [NO] production by the macrophages obtained from the aqueous extract of TC treated mice when stimulated with LPS (figure-8). 1mg/kg of extract shows remarkable enhancement in NO production. NO has been considered to be a key molecule in the regulation of the immune response to tumors. We measured the production of NO from the peritoneal murine macrophages and observed that when macrophages were treated with the aqueous extract of TC (1mg/kg), there was dose-dependent enhancement in NO production. This increase in the NO production is supposed to have been due to the cascade of the signaling pathway. These data further strengthens the role of macrophage in producing NO by direct lysis of B16F10 melanoma cells in vitro. Figure-8a and 8b In vitro effect of varying concentrations of aqueous extract of Tinospora on NO production by peritoneal macrophages [a] and splenocytes [b] Figure-9 Dose dependent IL-6 production by Aq extract of TC. The values are means of triplicates at each point and the error bars represent SD. Data are representative of five different experiments The data reveals immune modulation and cytotoxicity against tumor cells. NO has been recognized as the major effector molecule concerned in the demolition of tumor cells by activated macrophages and our data confirms the possibility that the extract of TC might be an immunomodulator and the possibility that the induction of nitric oxide may contribute to tumoricidal activities. Tinospora cordifolia has also been found to contain an immunomodulator arabinogalactan in polar fraction25. Research Journal of Chemical Sciences _______________________________________________________ ISSN 2231-606X Vol. 1(6), 71-79, Sept. (2011) Res.J.Chem.Sci. International Science Congress Association 77 Although scientific studies have been done on a large number of Indian medicinal plants, a considerably smaller number of marketable drugs or phyto-chemical entities have entered the evidence-based therapeutics26. It has been reported that aqueous extract of TC has potent immunomodulatory properties. However, the mechanism of immunomodulation remains ambiguous. Since plant extracts have been reported to reveal various immunomodulating activities, we focused on the activation of splenocytes and lymphocytes by the TC extract, to augment the production of cytokines. The induction of cytokine is one of the methods to measure the augmentation activity of the immune system that plays a fundamental role in controlling the homeostasis of the whole organism. IL-6 is vital for B-cell stimulation and regulation of acute-phase responses and inflammation 27. In this study, we have shown that TC is a potent regulator of IL-6 production and plays a key role in the cytokine network. The finding that IL-6 was up-regulated might provide a decisive key to the understanding of the mechanism of action for clinical relevance and the rationale for TC treatment. IL-6 deficiency is associated with enhanced susceptibility to, and diminished clearance of, a wide range of pathogens including extracellular and intracellular bacteria 28–31, parasites 32-35 and fungi36. IL-6- deficient mice also exhibit impaired wound healing and tissue regeneration subsequent to injury37-40, altered nociceptive responses and higher tumor progression 41. Recently, it was shown that IL-6 induces hepcidin in hepatocytes which decreases the availability of iron by inhibition of macrophage iron release and intestinal iron absorption42. These results represent an approach to determine immunomodulatory effects and can be used for specific and differential immunomodulation. The key finding of our experiments was the impact of TC extract on IL-6 release (figure-9). It has opened a new window for investigation of the signaling pathway. The era is of regenerative and alternative medicine for treatment of various diseases. But, there are a lot of gaps to be filled by new investigations. There is a great interest in development of new drugs from traditionally used medicinal plants; Tinospora cordifolia may provide new lead molecules for the development of alternative drugs. ConclusionThe results of this study confirm the earlier claims that Tinospora cordifolia has immunomodulatory potential and further demonstrates that this activity is mainly due to the aqueous extract of TC, a glycoprotein isolated from the stem of Tinospora cordifolia. This aqueous extract primarily acts on the macrophages and stimulates the production of effector molecules like nitric oxide and cytokine mediators and also augments the antigen specific recall response indicating its ability to enhance antigen presenting ability of macrophages. Acknowledgement RPP is grateful to ICMR for a fellowship. References1. Amirghofran Z., Azadmehr A., Javidnia K., Haussknechtia elymatica: A plant with immunomodulatory effects, Iran J Immunol., 126-31 (2007) 2. Kapur P., Pereira B.M., Wuttke W., and Jarry H., Androgenic action of Tinospora cordifolia ethanolic extract in prostate cancer Cell line LNCaP, Phytomedicine, 16(6-7), 679-82 (2008) 3. 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