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ORIGINAL ARTICLE
Year : 2012  |  Volume : 4  |  Issue : 1  |  Page : 15-21  

Black tea (Camellia sinensis) decoction shows immunomodulatory properties on an experimental animal model and in human peripheral mononuclear cells


1 KPC Medical College and Hospital, 1F, Raja S.C. Mullick Road, Kolkata-700 032, West Bengal, India
2 Department of General Medicine, NRS Medical College and Hospital, 138, AJC Bose Road, Kolkata-700 014, India
3 Department of Pharmacology, Institute of Post-Graduate Medical Education and Research and Seth Sukhlal Karnani Memorial Hospital, 244, Acharya Jagadish Chandra Bose Road, Police Station-Bhowanipore, Kolkata-700 020, West Bengal, India
4 Department of Pharmacology, KPC Medical College and Hospital, 1F, Raja S.C. Mullick Road, Kolkata-700 032, West Bengal, India

Date of Submission01-Jul-2011
Date of Decision22-Aug-2011
Date of Web Publication21-Dec-2011

Correspondence Address:
Chandan Chattopadhyay
358, NSC Bose Road, Kolkata-700047
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8490.91029

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   Abstract 

Background: Black tea (Camellia sinensis) has been used as a daily beverage for time immemorial. Immunomodulatory effects of tea are recognized as it stimulates the proliferation of cultured human peripheral blood mononuclear cells. Anti-inflammatory effects of tea have also been depicted in the available literature. Therefore, we designed this study to examine the potential immunemodulatory and anti-inflammatory activities of black tea in a rat model and in human peripheral mononuclear cells. Aims: The purpose of the study was to determine (1) evaluation of anti-inflammatory effects of black tea on rats, (2) evaluation of immunemodulator effects of black tea on rats, and (3) evaluation of immunemodulator effects of black tea on human peripheral mononuclear cells. Materials and Methods: Black tea decoction (10% and 20%) was prepared. Acute anti-inflammatory activity of tea decoction was evaluated using carrageenan and dextran whereas chronic anti-inflammatory (Immunomodulatory) effects were evaluated in a complete Freunds' adjuvant-induced arthritis model. Immunostimulatory role was evaluated in cultured human (in vitro) peripheral mononuclear cells (T-lymphocytes) by using methyl thioazolyl tetrazolium (MTT) and Trypan blue assay. Study Design: An experimental study was designed. Results: Black tea decoction (10% and 20%) strength has shown significant anti-inflammatory effects (64.8% and 77% reduction, respectively), on carrageenan-induced acute inflammatory models (rat paw edema) which can be comparable with the standard drug indomethacin (89.1%). In a chronic anti-inflammatory model, black tea decoction (10% and 20%) has shown significant suppressive effects on rat paw edema (38.56% and 69.53%) observed on 21 st day. Lymphoproliferative action of tea was evaluated on human peripheral mononuclear cells using an MTT assay where the number of living cells were expressed in terms of optical density at 570 nm. An experiment has shown that black tea increases the maximum number of T-lymphocytes at 72 h with a maximum strength of 20%. Maximum number of viable cells (T-lymphocytes) was observed with black tea at 20% strength at 72 h. The results were expressed as mean ± SD, and the significance was evaluated by Student's t-test versus control, with P < 0.05 implying significance. Conclusions: Taken together, our data indicate that black tea has potential anti-inflammatory and immunomodulatory action and this corroborates with the current trend of tea being promoted as a 'health drink'.

Keywords: Anti-inflammatory, black tea, immunomodulation


How to cite this article:
Chattopadhyay C, Chakrabarti N, Chatterjee M, Mukherjee S, Sarkar K, Chaudhuri A R. Black tea (Camellia sinensis) decoction shows immunomodulatory properties on an experimental animal model and in human peripheral mononuclear cells. Phcog Res 2012;4:15-21

How to cite this URL:
Chattopadhyay C, Chakrabarti N, Chatterjee M, Mukherjee S, Sarkar K, Chaudhuri A R. Black tea (Camellia sinensis) decoction shows immunomodulatory properties on an experimental animal model and in human peripheral mononuclear cells. Phcog Res [serial online] 2012 [cited 2019 Nov 15];4:15-21. Available from: http://www.phcogres.com/text.asp?2012/4/1/15/91029


   Introduction Top


The search for anti-inflammatory agents with analgesic effects coupled with minimal side effects led the attention of Indian workers towards another treasure of remedies-indigenous system of medicine. The importance and utility of medicinal plants in the treatment of any chronic disease is well perceived as they have the additional advantage of being cheap, and may be used for a prolonged period. Chopra pioneered the usefulness of many indigenous drugs in inflammatory conditions. [1] Thereafter, various medicinal plants have been selected for scientific testing and screening of anti-inflammatory action.

Tea (Camellia sinensis) has been used as a daily beverage for several years. The active components of tea responsible for such biological effects are known to be catechins (known as polyphenols), which constitute seven forms including epigalocatechingallate (EGCg). EGCg is a major catechin compound present in tea extracts and is also the most active form in a variety of biological activities.

Inflammation is the response of a tissue and its microcirculation to pathogenic injury. It is characterized by the generation of inflammatory mediators and movement of fluid and leukocytes from the blood into extravascular tissues which gives rise to the four cardinal signs of inflammation, namely rubor (redness), calor (heat), tumor (swelling), and dolor (pain) as described by Anlus Celsus, the Roman encyclopedist, in the second century AD. Inflammation may be classified into acute, sub-acute, and chronic or immunological. There are various mediators for these types of inflammation in different stages, which are histamine, 5-hydroxytryptamine, bradykinin, prostaglandins, leukotrienes, etc. However, the immunomodulatory effects of tea are slowly being recognized as it is now known that EGCg stimulates the production of Interleukin 1α, IL1β, TNF-α in cultured human peripheral blood mononuclear cells (PBMCs). It protects against UV radiation induced immunosuppression and tolerance induction by reducing IL-10 production and increasing IL-12 production in epidermal and dermal cells. However, detailed immunomodulatory effects of tea on immune cells are currently an area of active research.

This study was undertaken to examine the potential immune-modulator and anti-inflammatory activity of black tea in the rat model and in human peripheral mononuclear cells.

Objectives

  1. Evaluation of anti-inflammatory effects of black tea on rats.
  2. Evaluation of immune-modulator effects of black tea on rats.
  3. Evaluation of immune-modulator effects of black tea on in vitro human peripheral T-lymphocytes.

   Materials and Methods Top


Animals

The entire study was carried out in a post-graduate teaching institute in eastern India, as a part of post-graduate dissertation using Sprague-Dawley adult rats of either sex, weighing between 150 and 200 g. The animals were maintained under standard laboratory conditions with free access to commercial pellet feed and water ad libitum. The animals were housed for a period of 7 days for acclimatization prior to the commencement of experimental work at a room temperature of 27°C under fixed 12-h alternate light and darkness cycle. The protocol was approved and carried out after the permission of Institutional Animal Ethics Committee.

Preparation of the plant product

Black tea leaves were commercially obtained from P and A Arse, Rajdhani Apt. BIK-I Rg Barua Road, Ganeshguri, Guwahati 781 006. For the preparation of tea extract, tea leaves (100 gm) were extracted with ethyl acetate using soxhlet assembly. The extract was concentrated in a rotary flash evaporator under reduced pressure to semisolid mass. Black tea decoction (10% and 20%) was prepared by soaking 10 and 20 g of black tea in 100 ml boiled water separately, soaked for 2 min and thereafter filtered. This filtrate was designated as black tea decoction. The dose of this decoction orally administered to each rat was 0.1 ml/10 g of body weight. An initial pilot study suggested that 20% of this preparation has given best results. Therefore, we have decided to set forward our study with 10% and 20% black tea decoction.

Models for evaluation of anti-inflammatory activity

In all models, the animals were grouped as follows: [Table 1]
Table 1:

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Carrageenan-induced rat paw edema

We followed the method adopted by Winter et al, subsequently modified by Ghosh et al.[2],[3] Forty-eight rats were taken and divided into four groups of 12 rats each as indicated above.

A fresh solution of 1% carrageenan was prepared by dissolving 50 mg of carrageenan powder in 5 ml of normal saline (0.9% NaCl). All animals were starved for 18 h before starting of the experiment. Animals of groups III and IV were pretreated with the test drug in the doses mentioned above 30 min prior to the carrageenan injection. All orally administered drugs were given via. a rat feeding cannula. Subsequently 30 min after the respective treatments, 0.1 ml of 1% carrageenan was injected subcutaneously into the subplantar region of right hind paw of all animals to induce edema. The paw had been previously marked with ink at the level of the lateral malleolus, and the paw volume was measured plethysmographically. [4] The paw volume was measured initially at 0 h and subsequently at 1, 2, and 3 rd hour after the carrageenan injection. The mean increase in the paw volume was measured hourly and at every time point, the percentage inhibition of edema was calculated. [5]

Mean increase in paw volume of Mean increase in paw volume of



Statistical analysis

The results were expressed as mean ± SD, and the significance was evaluated by Student's t-test versus control, with P < 0.05 implying significance.

Dextran-induced rat paw edema

In this model, dextran was used as the phlogistic agent and the methodology of Rowley et al,[6] was used with slight modifications. [3] Forty-eight rats were taken and divided into four groups of 12 rats each. The four groups were treated as per the protocol mentioned in the previous model. Indomethacin and tea decoction were administered 30 min prior to the injection of dextran. A volume of 0.1 ml of 2% dextran solution in normal saline was injected into the subplantar tissue of the right hind paw of each rat (Vogel, 1997a). The paw volume was recorded by a plethysmometer initially at 0 h and subsequently hourly at 1, 2, and 3 h after the dextran injection. The percentage inhibition of edema at every time point was calculated as per Vetrichelvan and Jegadeesan. [5]

Chronic or immunological induced inflammation

Adjuvant-induced arthritis

The method of adjuvant arthritis in rats as described by Pearson et al. exhibits many similarities to human rheumatoid arthritis and was accordingly followed as a model of chronic or immunologically induced inflammation. [7] Male Sprague-Dawley rats with an initial body weight of 130-150 g were used. Sixty rats were taken and divided into five groups of 12 rats each. The groups were treated as follows: [Table 2].
Table 2:

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On day 1, 0.1 ml of complete Freund's adjuvant was injected into the subplantar region of the left hind paw of each rat. (Each milliliter of complete Freund's adjuvant contains 1 mg Mycobacterium tuberculosis (H37RA, ATCC 25177), heat killed and dried 0.85 ml mineral oil and 0.15 ml Mannide monooleate). Dosing with a test compound or the standard was started on the same day and continued daily orally for 12 days. Paw volumes of both legs and body weight were recorded on the day of injection. On day 5, the volume of the injected paw was measured again, indicating the primary lesions and the influence of therapeutic agents on this paw. The severity of the adjuvant-induced disease was followed by measurement of the non-injected paw (secondary lesions) with a plethysmometer. On day 21, the body weight was determined again and the severity of the secondary lesions was evaluated visually and graded according to the following scheme.

Arthritic index [8][Table 3]
Table 3: Arthritic index

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Evaluation

  1. For primary lesions: the percent inhibition of paw volume of the injected left paw over control was measured at day 5.
  2. For secondary lesions: The percent inhibition of paw volume of the non-injected right paw over control was measured at day 21.
  3. An arthritic index was calculated as the sum of the scores as indicated above for each animal. The average of the treated animals was compared with the control group.
Lymphoproliferative (Blastogenesis) assay with tea decoction

Separation of peripheral blood mononuclear cells from human blood

Human blood was collected in heparin and layered gently upon Ficoll Hypaque solution, the ratio of blood: Ficoll being 5:3. The suspension was centrifuged at 1000 rpm for 45 min. At the interphase of plasma and Ficoll, a buffy coat/ring of cells was identified. This layer was collected in 10 ml PBS and centrifuged at 2000 rpm for 10 min. This procedure was repeated thrice to remove any contaminating Ficoll. The cell pellet was finally resuspended in a 1.0 ml medium (RPMI 1640) supplemented with 10% fetal calf serum and pencillin/streptomycin. The cells were then diluted 1:50 and counted in a hemocytometer. The cell count was then adjusted to 1 × 105 cells per ml. The viability of cells was judged with the trypan blue exclusion test.

Quantification of peripheral blood mononuclear cells

The stock solution of cell is diluted with PBS, and 10 μl of cell suspension is taken on a hemocytometer for a cell counter. Two microliters of 0.2% trypan blue solution is added to it and number of viable and non-viable cells are counted, based on the principal that trypan blue enters only non-viable cells.

Methyl thioazolyl tetrazolium assay

The methyl thioazolyl tetrazolium (MTT) was weighed and then dissolved in 50 μl DMSO. Then, the RPMI medium was added so as to make a final solution of 10 mg/ml. Ten microliters of MTT was then added to each well; i.e., 100 μl was present per well. It was then incubated for 4 h at 37 °C in the presence of 5% CO 2 in a carbon dioxide incubator. After 4 h, the plates were gently tilted and the supernatant was removed, and placed in a microcentrifuge tube. To the remaining sample in the well, 100 l of DMSO was added. The tubes were subjected to centrifugation at 2000 rpm for 10 min. After centrifugation, the supernatant was discarded and 100 μl of solution DMSO was added to the centrifuge tube and transferred to the well. The reading of each well was then taken in an ELISA reader at OD-570 nm.


   Results Top


Black tea decoction (10% and 20%) has shown significant anti-inflammatory effects on carrageenan and dextran-induced acute inflammatory models which can be comparable with the standard drug indomethacin [Table 4] and [Table 5]. In chronic anti-inflammatory model, black tea decoction (10% and 20%) has shown significant suppression of rat paw edema on 21st day [Table 6] and [Table 7]. Lymphoproliferative action of tea is evaluated by using [Figure 1] the MTT assay where the number of living cells are expressed in terms of optical density at 570 nm. Different concentrations of tea ranging from 20% to 1% were used. In controls, tea was not added. Subsequently with lower concentrations, lesser degree of proliferation was observed. Two time points, i.e. 72 and 96 h were selected to decide the optimum assay duration. An experiment has shown that tea increases maximum number of T-lymphocytes at 72 h with a maximum strength of 20% [Figure 1]. Lymphoproliferative action of tea was also evaluated by using the Trypan blue exclusion test at 72 and 96 h where the viability of cells (Lymphocytes) were judged. Maximum number of viable cells was observed with tea at 20% strength at 72 h. All other strengths of tea showed almost equal efficacy at 72 and 96 h.
Figure 1: Evaluation of lymphoproliferative action of black tea using MTT assay

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Table 4: Effects of black tea decoction on carrageenan-induced rat paw edema

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Table 5: Effects of black tea decoction on dextran-induced rat paw edema

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Table 6: Effects of black tea decoction on Freund's adjuvant induced arthritis in rats

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Table 7: Effect of black tea on the arthritic index in a model of Freund's adjuvant induced arthritis

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   Discussion Top


Plant products are in use for a long time in Ayurvedic and folklore medicine for the cure of ailments with minimal side effects and comparable efficacy. The plant kingdom has been explored for drugs relieving pain and inflammation as well. Chopra et al, explored usefulness of indigenous drugs in inflammatory arthritic conditions. In our laboratory, Nag et al, and many other workers have screened active principles of a number of plants for this purpose. [1],[9] Accordingly, a study was undertaken to assess the immunomodulatory activity of tea decoction in the rat model. The results obtained in the anti-inflammatory models in this study shows that tea possesses pronounced activity against the acute experimental model of inflammation. In the model of acute inflammation as exemplified by the carrageenan-induced paw edema, a significant anti-inflammatory action of the test drug was observed [Table 4]. Carrageenan is a sulfated mucopolysaccharide derived from Irish sea moss, Chondrus. It was found to be a superior phlogistic agent over other varieties such as brewer's yeast, formalin, dextran, and egg albumin. Carrageenan-induced rat paw edema is found to be biphasic, the initial phase is due to release of histamine, 5-hydroxytryptamine (5-HT) and bradykinin in the first hour after the administration of carrageenan and a more pronounced second phase is attributed to the release of prostaglandin, bradykinin, protease, and lysosome-like substances in 2-3 h. [10],[11] The later phase is reported to be sensitive to most of the clinically effective anti-inflammatory agents. [12] It can therefore be deduced that the anti-inflammatory activity may be mediated through inhibition of any of these mediators either alone or in combination. Thus, it can be concluded that the anti-inflammatory action of black tea in a carrageenan-induced edema model is by inhibition of histamine, 5-HT, bradykinin, and prostaglandin, individually or in combination. In future, to more precisely determine the chemical mediators that are being inhibited by tea, individual mediators can be injected and the study repeated in the same fashion. In the dextran-induced rat paw edema model, tea showed significant anti-inflammatory activity. Dextran-induced edema is mediated mainly by histamine and 5-HT and dextran is known to be a powerful liberator of histamine. [6],[13] Black tea (20% and 10%) showed an inhibition of 82% and 64.8% [Table 5], respectively, in comparison to the standard drug indomethacin that showed an inhibition of 91.89%. A similar kind of study by Ratnasooriya and Fernando showed statistically significant anti-inflammatory activity of black tea by multiple mechanisms. [14] In our study also black tea had shown statistically significant anti-inflammatory effects like indomethacin.

Histamine released from mast cells and 5-HT released from platelets also can elicit pain, but they are 50 times less potent than bradykinin. [15] Prostaglandins sensitize the nerve endings to the effects of bradykinin and other algogens. [16] In the immunologically mediated chronic inflammatory model of Freund's adjuvant-induced arthritis, considered as the best available model of rheumatoid arthritis, tea showed a profound degree of anti-inflammatory activity in both the primary and secondary phase. [17] This procedure has been proposed by several authors to differentiate between anti-inflammatory and immunosuppressive activity. [18] It has been proposed that those anti-inflammatory compounds capable of inhibiting secondary lesions can be considered as immunosuppressive agents. [19] In this model of immunologically mediated chronic synovial inflammation and arthritis, macrophages play a central role. After activation, they are capable of synthesizing mediators such as PGE 2 and cytokines such as TNF-α and IL-1. In turn, these synthetic products induce the production of variety of enzymes which initiate cartilage and bone destruction. [20] Indomethacin and black tea 20% produced a significant inhibition of 51.2% and 45.26%, respectively, on 5 th day, which indicates its action in the primary phase [Table 6]. Dexamethasone showed most significant inhibition on 5 th and 21 st day [Table 6] and [Table 7]. Black tea (10%, 20%) decoction at above-mentioned doses showed a maximum inhibition of 38.56%, and 69.53%, respectively, and was statistically significant [Table 6]. Therefore, tea definitely has some effects on the immunological and systemic secondary phase of adjuvant arthritis.

Finally, to examine another aspect of its immune-modulatory action, a lymphoproliferative assay was undertaken with black tea decoction. Stock solution was made to 20%. The degree of proliferation was measured by two approaches, namely the MTT assay and Trypan blue dye exclusion assay [Figure 1] and [Figure 2]. MTT is methyl thiazolyl tetrazolium, a yellow compound, which in the presence of mitochondiral dehydrogenase is converted into formazan which is purple in color. As only living cells produce this enzyme, by performing this assay, the amount of living cells may be assessed. Hence, the greater the amount of enzyme, the larger will be the amount of formazan produced, more will be the optical density (O.D.). This represents a greater number of living cells. Different concentrations of tea ranging from 20% to 1% were used. As is evident from [Figure 1], maximum proliferation was observed at the maximum concentration used, i.e. 20%. Subsequently with lower concentrations, the degree of proliferation was less. From this experiment, we can firmly conclude that tea has stimulatory effects on T lymphocytes, and in 72h gave the best time kinetics. Due to economic constraints, we could not use a mitogen in this study.
Figure 2: Evaluation of lymphoproliferative action of black tea using the Trypan blue exclusion test

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The results obtained by the MTT assay were corroborated with a cell count assay using Trypan blue, based on the known principle that Trypan blue enters only nonviable, i.e. dead cells. The cell count assay showed a similar profile in which maximum proliferation was observed at 72 h [Figure 2] using the highest concentration of tea. Black tea (20%) decoction displayed maximum immunomodulatory (immunostimulatory) properties in our in vitro system. [21]

Taken together, our data indicate that black tea has potential acute and chronic anti-inflammatory action. This corroborates with the current trend of tea being promoted as 'health drink'. Our results support the idea that tea has a beneficial effect. Significant antiarthritic activity was observed with regular administration of black tea 10% and 20% in the Freund's adjuvant-induced model of arthritis. Roy et al, stated that chronic treatment with Black tea (in arthritic rats) resulted in a decrease of paw diameter and tissue lipid peroxidation, along with a restoration of GSH, catalase, and superoxide dismutase levels. [22] Ratnasooriya and Fernando showed that black tea possessed strong, oral gastric ulcer healing activity which is mediated via multiple mechanisms. [23]

However, such a study would be difficult to undertake in humans as the majority of the population are 'tea consumers'. Hence, we propose that after a 'wash out' period of 4 weeks, preliminary studies can be undertaken with normal healthy volunteers.

 
   References Top

1.Chopra RN, Chopra IC, Handa KK, Kapoor LD. In: Indigenous drugs in India. 2 nd ed, Calcutta: M/s U. N. Dhar and Sons Ltd; 1958.   Back to cited text no. 1
    
2.Winter CA, Risley EA, Nuss GW. Carrageenin induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Expt Biol Med 1962;111:544-7.  Back to cited text no. 2
    
3.Ghosh MN, Singh H. Inhibitory effects of a pyrizolidine alkaloid, crotalburin on rat paw edema and cotton pellet granuloma. Br J Pharm 1974;51:503.  Back to cited text no. 3
    
4.Bhatt KR, Mehta RK, Shrivastava PN. A simple method for recording anti-inflammatory effects on rat paw oedema. Indian J Physiol Pharmacol 1977;21:399.   Back to cited text no. 4
    
5.Vetrichelvan T, Jegadeesan M. Effect of alcoholic extract of achyranthes bidentata blume on acute and subacute inflammation. Indian J Pharmacol 2002;34:115.  Back to cited text no. 5
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6.Rowley DA, Benditt EP. 5-Hydoxytryptamine as mediators of vascular injury provided by agents which damage most cells in rats. J Exp Med 1956;103:399.  Back to cited text no. 6
    
7.Pearson CM, Wood FD. Studies on polyarthritis and other lessions induced in rats by injection of mycobacterium adjuvant, I: General clinic and pathological characteristics and some modifying factors. Arthris Rheum 1959;2:440.   Back to cited text no. 7
    
8.Schleyerbach R. Antiarthrotic and immunmodulatory activity. In: Vogel GH, editors. Drug discovery and evaluation. 2 nd ed. Berlin, Germany; Springer; 2002. p. 802.   Back to cited text no. 8
    
9.Nag BN, Dutta MK, Sikdar S. Anti-inflammatory effect of Vitex negundo. Indian J Pharmacol 1978;9:32.  Back to cited text no. 9
    
10.Brooks PM, Day RO. Non-steroidal anti-inflammatory drugs: Difference and similarities. N Engl J Med 1991;324:1716.   Back to cited text no. 10
    
11.Castro J, Sasame H, Sussman H, Buttette P. Diverse effect of SKF52 and antioxidants on CCl 4 induced changes in liver microsomal P-450 content and ethyl morphine metabolism. Life Sci 1968;7:129.   Back to cited text no. 11
    
12.Smucker E, Arrhenius E, Hulton T. Alteration in microsomal electron transport, oxidative N-demethylation and azo-dye cleavage in CCl 4 and dimethyl nitrosamine induced liver injury. Biochem 1967;103:55.   Back to cited text no. 12
    
13.Ghosh MN, Banerjee RH, Mukherjee SK. Capillary permeability increasing property of hyaluronidase in rat. Indian J Physiol Pharmacol 1963;7:17.  Back to cited text no. 13
    
14.Anti-inflammatory activity of Sri Lankan black tea (Camellia sinensis0 L.) in rats. Pharmaconog Res 2009;1:11-20.  Back to cited text no. 14
    
15.Garcia LJ. Bradykinin-system. In: Vane JR, Ferreira SH, editors. Inflammation. Berlin: Springer-Verlag; 1978. p. 464.  Back to cited text no. 15
    
16.Moncada S, Ferreira SH, Vane JR. Pain and inflammatory mediators. In: Vane JR, Ferreira SH, editors. Inflammation. Berlin: Springer-Verlag; 1978. p. 588-616.  Back to cited text no. 16
    
17.Waltz DT, DiMartino MJ, Kuch JH, Zuccarello W. Adjuvant-induced arthritis in rats - Temporal relationship of drug effects on physiological, biochemical and haematological parameters. Pharmacologist 1969:11:266.  Back to cited text no. 17
    
18.Perper RJ, Alvarej B, Colombo C, Schroder H. The use of a standardized adjuvant arthritis assay to differentiate between anti-inflammatory and immunosuppressive agents. Proc Soc Exp Biol Med 1971;137:506.  Back to cited text no. 18
    
19.Middleton E Jr. Effect of plant flavonoids on immune and inflammatory cell function. Adv Exp Med Biol 1998;439:175.  Back to cited text no. 19
    
20.Hopkins SJ. Cytokines and eicosanoids in rheumatic diseases. Ann Rheum Dis 1991;49:207.  Back to cited text no. 20
    
21.Zvetkova E, Wirleitner B, Tram NT, Schennach H, Fuchs D. Aqueous extracts of Crinum latifolium (L.) and Camellia sinensis show immunomodulatory properties in human peripheral blood mononuclear cells. Int Immunopharmacol 2001;1:2143-50.  Back to cited text no. 21
    
22.Roy DK, Kumar KT, Zothanpuia, Karmakar S, Pal S, Samanta SK, et al. Pharmacological studies on Indian black tea (leaf variety) in acute and chronic inflammatory conditions. Phytother Res 2008;22:814-9.   Back to cited text no. 22
    
23.Ratnasooriya. Gastric ulcer healing activity of Sri Lankan black tea (Camellia sinensis L.) in rats. Pharmacogn Mag 2009;5: 260-5.  Back to cited text no. 23
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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