Anti-inflammatory activity of Seabuckthorn (Hippophae rhamnoides) leaves

International Immunopharmacology 5 (2005) 1675 – 1684 www.elsevier.com/locate/intimp

Anti-inflammatory activity of Seabuckthorn (Hippophae rhamnoides) leaves Lilly Ganjua,T, Yogendra Padwada, Richa Singha, Dev Karana, Sudipta Chandaa, Mohinder Kumar Choprab, Parul Bhatnagarb, Ravi Kashyapb, Ramesh Chandra Sawhneya a

Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi-110054, India b Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timarpur, Delhi-110054, India Received 20 December 2004; received in revised form 2 February 2005; accepted 17 March 2005

Abstract Immunomodulatory activity of Seabuckthorn (SBT) leaf extract was evaluated in adjuvant induced arthritis (AIA) rat model. Inflammation was induced by injecting Complete Freund’s Adjuvant (CFA) in the right hind paw of rats. SBT extract was administered intraperitoneally to treat the inflammation. The extent of inflammation and treatment response was evaluated by clinical analysis, scintigraphic visualization using technitium-99m-glutathione (Tc99m-GSH) and lymphocyte proliferation. Serial evaluation was carried out on days 1, 7, 14, 21 and 28 after creation of inflammation. The Tc99m-GSH uptake in the inflamed leg was compared with the normal contralateral leg of the same animal. The measurements were done by obtaining scintigraphic images using gamma camera and an online computer. Both qualitative and quantitative evaluation of radiotracer accumulation was considered to evaluate the anti-inflammatory response. The lymphocyte proliferation study revealed cellular immunosuppression during the early phase of the disease. Administration of SBT extract on the same day or 5 days prior to inflammatory insult into the joint, significantly reduced the inflammation as compared to the untreated animals in a dose dependent manner. These observations suggest that the SBT leaf extract has a significant anti-inflammatory activity and has the potential for the treatment of arthritis. D 2005 Elsevier B.V. All rights reserved. Keywords: Seabuckthorn; Arthritis; Inflammation; Immunosuppression; Scintigraphy

1. Introduction T Corresponding author. Immunomodulation Laboratory, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi-110054, India. Tel.: +91 11 23985033; fax: +91 11 23914790. E-mail address: [email protected] (L. Ganju). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.03.017

Immunomodulation using medicinal plants can provide an alternative to conventional chemotherapy for a variety of diseases, especially when host defence mechanism has to be activated under the

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conditions of impaired immune responsiveness. Seabuckthorn (SBT) (Hippophae rhamnoides, Family—Elaeagnaceae) is the popular medicinal plant used in various parts of the world for preparation of vitamin and nutrient products [1,2]. All parts of the plant are rich in various biologically active compounds. Its fruits are used for commercial scale production of medically important fatty oil (Oleum hippophae) [3–5] and the leaf extracts have been reported to have marked anti-bacterial, anti-viral and anti-tumor activities [6,7]. Leaf drugs, containing flavanoids, increase the wound healing effect after chemical burns and plain wounds. SBT leaves have also been reported to have significant anti-oxidant and immunomodulatory property [8]. However, its anti-inflammatory activity remains to be determined. Therefore, the present study was undertaken to determine the anti-inflammatory effect of SBT in adjuvant induced arthritis (AIA) rat model leading to a severe inflammatory joint disease which primarily affects synovial membrane of affected joints, with clinical and laboratory features representing a valid model for human rheumatoid arthritis (RA) [9–11]. Inflammatory response in such models has been studied by clinical analysis [12], by measuring the physical swelling of the hind paw, offering a simple and reproducible method. Ercan et al. [13] in 1978 had introduced the use of technitium-99m labeled glutathione (Tc99m-GSH) for evaluation of inflammation. GSH is a natural tripeptide and is seen to accumulate at site of inflammation in proportion to the inflammatory response. Kourounakis and Kapusta [14] demonstrated the response at cellular level by evaluating the response of splenic lymphocytes from rats with AIA to phytohemagglutinin and Con-A, T-cell mitogen, was diminished during the active phase of the disease, and returned to normal with a remission. This suggested a possible relationship between the T-cell function of splenic lymphocyte and AIA. Sakaguchi et al. [15] described the histological analysis of swollen joints with severe synovitis, massive subsynovial infilteration of mononuclear cells, pannus formation and cartilage erosion. The present study is undertaken to determine the anti-inflammatory property of SBT using clinical, cellular, Tc99m-GSH sequential scintigraphy and histopathological analysis in AIA rat model.

2. Materials and methods 2.1. Plant extract Leaves of SBT were collected from hilly regions of Western Himalayas, India and dried under shade. The leaf extraction was carried out using powdered leaf material with 70% ethanol overnight. The extract was dried at 50 8C and then dissolved in 70% alcohol. Our preliminary investigations with extracts of SBT revealed that the immune stimulating principle is confined in the aqueous fraction and after the final extraction was obtained as a puffy solid. The dilutions of extract were carried out in sterile double distilled water. 2.2. Animals The study was approved by the institute’s animal ethical committee and confirmed to national guidelines on the care and use of laboratory animals. Both male and female albino rats (Sprague Dawley strain), 10–12 weeks old, weighing 125–150 g were used for the study. The animals were maintained at 25 F 2 8C in the institute’s animal house with food and water ad libitum. Animals were divided into eight groups of eight each. Group I was normal control which received phosphate buffer saline (PBS), Group II received Complete Freund’s Adjuvant (CFA) (Sigma, USA) only, Group III received CFA + SBT, Group IV received CFA + dexamethasone (Ranbaxy, India) used as positive control. Groups III and IV were treated from the day of administration of CFA. Groups V and VI received CFA + SBT and CFA + dexamethasone, respectively, for 5 days prior to the induction of AIA. Group VII received SBT + CFA and Group VIII received CFA + dexamethasone 10 days after induction of AIA. 2.3. Development and evaluation of arthritis The AIA was induced in rats by injecting 300 Al of CFA intradermally into the foot pad. Evaluation of arthritis was done by measuring the thickness of ossicular tissue using a dial gauge caliper assessed degree of arthritis. Severity of inflammation was according to increasing extent of erythema and edema of the periarticular tissue.

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2.4. Treatment To determine the optimum dose, animals were initially treated with 50, 100, 200 and 400 mg/kg body weight of SBT. Intraperitoneal administration of 200 mg/kg body weight of SBT leaf extract was found to be the optimum dose for anti-inflammatory property, hence used for further studies. Intraperitoneal injection was given till the end of the experiment on every alternate day. Dexamethasone was used as a positive control at a dose of 4 mg/kg body weight of the commercially available drug. All the animals received SBT and dexamethasone for 28 days.

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progression of the disease both in treated and untreated animals till 28 days. 2.7. Image evaluation Scintigraphic images were evaluated to study the inflamed ankle joints. Radiotracer accumulation in inflamed joint was compared with contralateral normal ankle joint of the animal. A region of interest (ROI) was placed to quantify the radiotracer accumulation in inflamed joint and its comparison with contralateral normal joint. Data were expressed as ratio of counts accumulated in the two identical ROI on ankles in the scintigrams.

2.5. Preparation of Tc99m-GSH 2.8. Lymphocyte proliferation assay The Tc99m-GSH was prepared by the method of Mishra et al. [16]. In brief, GSH (65 AM) and stannous chloride dehydrate (1 AM) were dissolved in 1 ml of nitrogen purged water. The final pH of the mixture was adjusted to 5.5 with 0.5 N NaOH. The contents were filtered through 0.22 Am membrane filter into a sealed sterile vial and stored at 70 8C until use. At the time of use, frozen content of the vial was thawed, brought to room temperature and mixed with requisite amount of Tc99m-pertechnetate (20 mci), freshly extracted, in 1–2 ml volume. The reaction was allowed to proceed for a further period of 10 min at room temperature to obtain the optimum yield. The yield of labeled product was determined chromatographically using ITLC-SGStrip (Gelman, USA). 2.6. Intravenous injection and imaging Animals were immobilized in a restrainer. A 500 Aci Tc99m-GSH was slowly injected intravenously through the tail vein. After 3 h, animals were anaesthetized using ketamine (80 mg/kg body weight) and atropine (2 mg/kg body weight) and immobilized. Scintigraphy of rat to study biodistribution of Tc99m-GSH after 3 h of radiotracer administration was performed using a gamma camera (DIACAM, Siemens, Germany). A low energy high resolution collimator was used to obtain an image for 10 min. The data was acquired in an online ICON computer in 256  256 matrix size. The procedure was repeated every seventh day to determine the

Lymphocyte proliferation assay was performed using mitogen concanavaline A (Con-A) (Sigma, USA). Both the treated and untreated animals were sacrificed to remove the spleen in RPMI-1640 medium (Sigma, USA). Spleen was crushed and cell suspension was washed with plain medium. Cell lysis was done with 0.9% ammonium chloride and resuspended in complete medium with 10% fetal bovine serum (FBS) (Sigma, USA). Cells were then cultured at a final concentration of 3  105 cells/100 Al/well in triplicate in flat bottom microtitre tissue culture plates (Tarson, India). Viability of cells was measured by trypan blue dye exclusion method. After 3 days of incubation at 37 8C under humidified air supplemented with 5% CO2, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to measure the response of spleen cells exposed to Con-A stimulation. Cultured cells were treated with 10 Al of MTT solution and incubated for 4 h at 37 8C. The formation of formazan crystals in viable cells were solubilised with 100 Al lysis buffer (10% SDS in 0.01M HCl) for 1 h. The optical density (OD) of each well was read at 540 nm. 2.9. Histology of joint tissues Rats were sacrificed to remove the swollen joints. Specimens were fixed for 24 h in 2% glutaraldehyde in phosphate buffer saline, dissected, decalcified and returned to 2% glutaraldehyde and submitted for routine paraffin embedding. Tissue

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sections were stained using haematoxyline and eosin stain. The histological findings were graded on the basis of synovial hyperplasia and mononuclear cell infiltration. 2.10. Statistics The data was analysed using SPSS 10 software. All data are presented as mean F SD. Analysis of variance for repeated measurement were used where applicable. The post-hoc test was done with Student’s Newman Keuls test where appropriate. Significant level was set at P b 0.05.

tarsal and interphalangeal inflammation. Peak severity in swelling occurred within 1–2 days after the onset of inflammation. Weight bearing on the affected limb was poorly tolerated by the rats and swelling usually persisted 3–4 weeks, gradually culminated in a deformed joint. In SBT and dexamethasone treated animals edema began to subside gradually and showed a significant reduction ( P b 0.05) in swelling as compared to the untreated animals (Fig. 2). The reduction in swelling in SBT treated groups was at par with dexamethasone treated animals. After 28 days of treatment, the swelling in treated animals was reduced almost equal to normal controls, whereas it persisted longer in untreated animals.

3. Results

3.2. Scanning of inflamed joints

3.1. Clinical analysis

In gamma camera images obtained 3 h after intravenous administration of Tc99m-GSH, the radioactivity was found to be accumulated more in inflamed as compared to the non-inflamed areas (Fig. 3). All other organs showed low uptake values except kidneys and the urinary bladder. Fig. 4 shows Tc99m-GSH counts ratio in inflamed and noninflamed area in different treatment groups. On day one, the Tc99m-GSH uptake in SBT and dexamethasone treated groups (Group III and IV) was signifi-

All the animals which received CFA, developed severe inflammation, typically hind limb became severely red and edematous within 16–24 h period such that inflammation score was 8.0 F 0.4 mm (mean F SEM) for a group of eight rats evaluated on the day of onset of arthritis from age matched untreated, treated and normal controls (Fig. 1). Involvement was predominantly distal with ankle,

Fig. 1. Swollen hind paw (↑) with swollen toes; and an intact paw of 2 days old adjuvant induced arthritic rats. A=only CFA; B=CFA + Seabuckthorn leaf extract treated and C=CFA + dexamethasone treated.

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9

Control

CFA

Dexamethasone

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SBT

Swelling (mm)

8 7 6

*

*

5

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14

16

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2 1 0 2

4

6

8

10

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22

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days Fig. 2. Effect of Seabuckthorn leaf extract treatment on swelling of hind paw in adjuvant induced arthritic rats. (CFA=Complete Freund’s Adjuvant; SBT=seabuckthorn).

cantly lower ( P b 0.05) than in untreated group (Group II). Whereas the radioactivity uptake in dexamethasone treated group (Group IV) was significantly lower ( P b 0.05) than the SBT treated group (Group III). However, in groups treated 5 days prior to CFA administration, the Tc99m-GSH uptake in SBT treated animals (Group V) was not significantly different from the untreated animals (Group II). Whereas dexamethasone treated group (Group VI) showed a significantly lower ( P b 0.05) uptake. On day seven, again there was significantly low ( P b 0.05) accumulation of Tc99m-GSH in both SBT and dexamethasone treated animals (Groups III and IV) than in untreated animals (Group II). There was no significant difference between SBT and dexame-

thasone treated animals. However, animals treated 5 days prior to CFA administration showed significant difference ( P b 0.05) in uptake of radioactivity both in SBT as well as in dexamethasone treated groups (Groups V and VI). On day 14, the Tc99m-GSH uptake in groups III and IV i.e. SBT and dexamethasone treated animals on the same day of CFA administration, was significantly lower ( P b 0.05) than the untreated group (Group II). There was no significant difference in radioactivity uptake in animals treated 5 days before (Group V and VI) or 10 days after (Groups VII and VIII) CFA administration from that of the untreated group (Group II). From day 21 onwards, in SBT treated animals, whether on the same day (Group III) or 5 days prior (Group V) or 10 days after (Group VII) CFA administration, the accumulation of Tc99m-GSH was identical to that of the untreated animals (Group II). But animals treated with dexamethasone on the same day of CFA administration showed a significant difference ( P b 0.05) from untreated animals (Group II). 3.3. Cell mediated immunity

Fig. 3. Typical scintigram of adjuvant induced arthritic rats showing accumulation of the radiopharmaceutical in inflamed regions 3 h post intravenous injection of Tc99m-GSH. A=only CFA; B=Seabuckthorn leaf extract + CFA; C=dexamethasone + CFA.

Lymphocytes are the key effector cells of mammalian immune system and Fig. 5 represents the effect of SBT in AIA rats on the mitogenic response of splenic lymphocytes to Con-A. Lymphocytes were obtained from normal and AIA rats on days 7, 14, 21 and 28 post adjuvant administration and subjected to proliferation assay. On day seven, SBT and dexamethasone

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CFA

CFA+SBT

CFA+Dexa

CFA+Dexa 5

CFA+SBT 10

CFA+Dexa 10

CFA+SBT 5

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Ratio (counts)

3 2.5 2 1.5

a

a

b

a a

a

a

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a

a

a

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1

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0.5 0 1

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Days Fig. 4. Ratio of Tc99m-GSH counts between inflamed and non-inflamed limbs following treatment with Seabuckthorn leaf extract and dexamethasone. a vs. untreated ( P b 0.05); b vs. SBT ( P b 0.05). (CFA=Complete Freund’s Adjuvant; SBT=Seabuckthorn; Dexa=dexamethasone; CFA + Dexa 5=treatment started 5 days prior to CFA administration; CFA + Dexa 10=treatment started 10 days after CFA administration).

treated animals (Group III, IV, V, VI), showed a significant immunosuppression ( P b 0.05) in lymphocyte proliferation as compared to untreated animals (Group II) and controls (Group I). There was a significant difference ( P b 0.05) between the SBT

and dexamethasone treated animals. Animals treated 5 days prior to CFA administration (Group V and VI) showed maximum suppression. Where as untreated animals (Group II) failed to show any suppression as compared to controls (Group I).

1.8

Control

CFA

CFA+SBT

CFA+Dexa

1.6

CFA+SBT 5

CFA+Dexa 5

SFA+SBT 10

CFA+Dexa 10

1.4

Optical density

a

1.2 1

a

0.8 0.6 0.4

** * * * * * *

*

* a

b a

b a

*

*

* c a

*

a a

*

* *

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*

b a

*

a a

b a

c a

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0.2 0 7

14

21

28

days Fig. 5. Con-A responses of splenic lymphocytes from normal controls and adjuvant induced arthritis untreated and treated rats. *vs. control ( P b 0.05); a vs. untreated CFA ( P b 0.05); b vs. SBT ( P b 0.05); c vs. Dexa ( P b 0.05). (CFA=Complete Freund’s Adjuvant; SBT=Seabuckthorn; Dexa=dexamethasone; CFA + Dexa 5=treatment started 5 days prior CFA administration; CFA + Dexa 10=treatment started 10 days after CFA administration).

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On day 14, again SBT and dexamethasone treated animals (Groups III, IV, V, VI, VII and VIII) showed significant ( P b 0.05) suppression in lymphocyte proliferation than in untreated animals (Group II) and normal controls (Group I). Amongst the treated animals again maximum suppression was observed in animals treated 5 days prior to CFA administration (Group V and VI). There was no difference between the untreated animals (Group II) and controls (Group I). On day 21, all the AIA rats whether treated or untreated showed significant ( P b 0.05) suppression as compared to controls (group I). There was no difference between the treated and untreated animals. On day 28, as the clinical phase was over and animals had entered into the chronic phase, there was no difference in the lymphocyte proliferation of controls (Group I), SBT and dexamethasone treated (Group III

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and IV) and SBT treated 5 days prior to CFA administration (Group V). But animals treated with dexamethasone 5 days prior to CFA administration (Group VI) and SBT and dexamethasone treated animals 10 days after the CFA administration (Group VII and VIII) continued to show suppression like untreated animals (Group II). 3.4. Histopathological analysis Results of histopathological examination are shown in Fig. 6. Joint sections were made after 15 days of CFA administration. All rats injected with CFA developed pronounced edema and exhibited marked synovial hyperplasia with significant infiltration of mononuclear cells (B), whereas in animals treated with SBT there was less cartilage erosion, bone destruction and mild synovial hyper-

Fig. 6. Knee joint sections of treated and untreated AIA animals stained with hematoxylin–eosin. Sections were made 15 days after CFA administration. A=normal joint; B=untreated CFA rat with cartilage erosion, severe synovial hyperplasia and infiltration of mononuclear cells; C, D=Seabuckthorn leaf extract and dexamethasone treated on the same day of CFA administration; E, F=Seabuckthorn leaf extract and dexamethasone treated 5 days prior to CFA administration, showing mild synovial hyperplasia, infiltration of fewer mononuclear cells and less bone destruction; G, H=Seabuckthorn leaf extract and dexamethasone treated 10 days after CFA administration showing infiltration of mononuclear cells and bone and cartilage erosion.

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plasia with very few mononuclear cell infiltration. This marked difference was observed in groups treated with SBT on the same day (C) or 5 days prior (E) to CFA administration. But in groups treated 10 days after (G and H) not much difference was observed when compared to untreated animals (B). The dexamethasone treated animals showed mild synovial hyperplasia (D).

4. Discussion Mammalian immune system recognizes age, pathogens and non-self molecules and trigger defence mechanism by the activation or suppression of immune competent cells. The present study demonstrated that the administration of CFA containing killed M. tuberculosis developed AIA in rats. Treatment of these AIA rats with SBT showed decline in inflammation which was comparable to dexamethasone treated groups, considered as positive control. For a true assessment of the responsiveness of a model of arthritis to drugs, the treatment schedules should parallel, as closely as possible to those currently being used in the clinical practice. Therefore, we have used dose ranges which span the clinically used dose and schedule. Furthermore, for our initial study, it was decided to commence treatment at a time when animals had established erosive disease since most patients are probably in this condition when they commence second-line of antiarthritic or anti-inflammatory therapy. Such an approach may obviously reduce the sensitivity of the model to drug treatment, particularly since this model is characterized by very rapid erosive disease [17]. This appears to be the case with SBT, on comparing the dynamic changes in lymphocyte proliferation and accumulation of Tc99m-GSH and their histopathological changes among all the treated groups, the values of SBT treatment on the same day and 5 days prior to CFA administration were significantly different than the groups treated after 10 days of CFA administration. This indicates that SBT treatment on the same day or 5 days prior to CFA administration caused optimal reduction in inflammation. There was a clinically significant reduction in swelling of inflamed area in SBT and dexamethasone treated animals. The ability of SBT extract to reduce

the response to AIA is consistent with the findings of earlier studies on the effect of commercially available drugs or cytokines or herbal preparations [18–22], as SBT is as effective as dexamethasone, a traditionally used immunosuppressant for arthritis, at least in the early phase of the disease. The dosing regimen used in these experiments covered the clinically relevant stage of the immune response. There was no toxicity, anorexia or weight loss observed in the treated animals. Tc99m-GSH imaging showed sufficient accumulation of radiopharmaceutical in experimental models of inflammation for scintigraphic visualization. The accumulation of radiopharmaceutical in the inflamed joints could be better visualized in untreated rats than in SBT treated animals. It is believed that enhanced permeability is the main underlying mechanism of localization in inflammatory lesion for Tc99m-GSH. Since GSH is a small molecule, it diffuses more rapidly into the inflamed tissues and achieves high concentration ratios. GSH plays a critical role in detoxification reactions by reducing H2O2 formed as a by-product of oxygen requiring metabolism [23]. There is an increase in the demand for GSH in cell injury during inflammation or infection [23,24]. In our study, accumulation of higher amount of Tc99m-GSH in untreated animals, confirms the observation, that there is enhanced permeability in the inflamed area, whereas treatment with SBT prevented the permeability up to some extent, hence did not allow the Tc99m-GSH to get accumulated in higher amounts in the early phase of the disease. The substantial reduction in swelling of inflamed joints indicates the beneficial effects of SBT leaves in AIA. The rate of lymphocyte proliferation of SBT treated groups at many time points were significantly lower than that of untreated groups which revealed that SBT could suppress the proliferation rate and regress the inflammation. The Con-A response of splenic lymphocytes was diminished during the early phase of the disease, and returned to normal during late phase. Earlier Rawson and Huang [25] have also reported that Con-A response of blood lymphocytes from patients with rheumatoid arthritis is diminished, possibly due to the action of suppressor cells. Selective modulation of T-cell response in autoimmunity has been achieved convincingly in experimental model of autoimmunity and in that regard

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AIA has proven to be a suitable model to elaborate new strategies of immunotherapy. AIA is T-cell dependent as was demonstrated in transfer experiments and by the failure to induce arthritis in nu/nu mice [26]. It has been shown that in vivo depletion of most ah TCR+ cells markedly suppressed both the induction and the progression of AIA [27]. It has also been reported that administration of cyclosporine A and FK 506 [28] effectively inhibited the development of arthritis through T cells. In our study it was observed that SBT used for treatment of AIA in rats resulted in immunosuppression during the acute phase of the disease. It selectively inhibited T-cell activation which was clearly indicated by decreased lymphocyte proliferation. This observation supports the T-cell directed immunosuppression by SBT leaf extract and confirm the observation of earlier investigators using immunomodulators of plant origin [29–31]. The anti-inflammatory effect of SBT was also associated with disappearance of follicular aggregation of lymphocytes from the inflamed synovium. Particularly remarkable was the significant reduction of synovitis in joints, which was evident from the histology of the inflamed tissues. This suggests that the T-cell suppression response to AIA in spleen cells of CFA administered rats demonstrate that the anti-inflammatory response is not limited to the synovium but reaches to the systemic region. Treatment of rheumatoid arthritis includes drugs to control inflammation e.g. nonsteroidal anti-inflammatory drugs (NSAIDs) and slow acting anti-inflammatory drugs (SAARDs) which probably induce regression or arrest progression of the disease and also have tissue protective effects. The limitations of SAARDs therapy, however are their well-known toxicity and the variation in clinical efficacy [32]. Hence, there has been an interest in using an alternative to conventional chemotherapy, which could provide the protection and prevention for such type of diseases where host immune response is impaired or a selective immunosuppression is required. The possible future clinical implication of the study is the observation that therapeutical dosing with SBT leaf extract inhibited established arthritis in rats. Leaves of SBT are claimed to be a rich source of many antioxidant substances, like flavanoids and other polyphenolic compounds. The main antioxidant components of leaves are flavanoids, isorhamnetin,

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quercetin and flavonols like epicatechin and leucoanthocyanidins [33]. The immunosuppressive property of SBT without exerting any side effects or cytotoxic effects, may make it a potent biodefence agent against arthritis. The extract proved to be as useful as dexamethasone in the treatment of AIA, and can be used as an adjunct to the existing chemotherapy. The plant derived immunomodulators thus have tremendous future potential for developing new pharmaceutical products. Phytochemical analysis of SBT extract through fractionation guided immunoactivity evaluation is in progress to identify bioactive molecules responsible for immunomodulatory properties in the plant.

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