Tamarind Seed (Tamarindus indica) Extract Ameliorates Adjuvant-Induced Arthritis via Regulating the Mediators of Cartilage/Bone Degeneration, Inflammation and Oxidative Stress

Abstract

Medicinal plants are employed in the treatment of human ailments from time immemorial. Several studies have validated the use of medicinal plant products in arthritis treatment. Arthritis is a joint disorder affecting subchondral bone and cartilage. Degradation of cartilage is principally mediated by enzymes like matrix metalloproteinases (MMPs), hyaluronidases (HAase), aggrecanases and exoglycosidases. These enzymes act upon collagen, hyaluronan and aggrecan of cartilage respectively, which would in turn activate bone deteriorating enzymes like cathepsins and tartrate resistant acid phosphatases (TRAP). Besides, the incessant action of reactive oxygen species and the inflammatory mediators is reported to cause further damage by immunological activation. The present study demonstrated the anti-arthritic efficacy of tamarind seed extract (TSE). TSE exhibited cartilage and bone protecting nature by inhibiting the elevated activities of MMPs, HAase, exoglycosidases, cathepsins and TRAP. It also mitigated the augmented levels of inflammatory mediators like interleukin (IL)-1β, tumor necrosis factor-α, IL-6, IL-23 and cyclooxygenase-2. Further, TSE administration alleviated increased levels of ROS and hydroperoxides and sustained the endogenous antioxidant homeostasis by balancing altered levels of endogenous antioxidant markers. Overall, TSE was observed as a potent agent abrogating arthritis-mediated cartilage/bone degradation, inflammation and associated stress in vivo demanding further attention.

Figure 1. HPLC chromatogram of TSE.

(A) Total ion current (TIC) chromatogram from HPLC separation of compounds present in the crude TSE extract and (B) HPLC-UV-Vis chromatogram of TSE at 280 nm.

Figure 2. Influence of TSE on arthritis induced physical changes.

(A) Measurement of paw edema by mercury displacement method. (B) Arthritis induced changes in body weight. (C) Macroscopic observation of severity of inflammation at paw joints. FCA injected day was considered as day zero, paw edema and body weight were measured until last day of treatment with an interval of every four days. From day eleven of FCA injection arthritic rats were treated with oral administration of TSE and Ibuprofen independently for fifteen days. Results are presented as mean ± SEM. a-compared with saline control and b-compared with arthritic group, * p < 0.05.

Figure 3. TSE alleviated cartilage degradation on arthritic rats.

(A) Determination of hyaluronidase activity by zymography: (i) Serum hyaluronidase, (ii) Ankle bone joint hyaluronidase. Arrow head indicates the activity bands. M represents the molecular weight markers in kDa. (B) Determination of MMPs activity by zymography: (i) Serum MMPs, (ii) Ankle bone joint MMPs and the gels in this figure are cropped, full length gels are presented in supplementary figure S7. M indicates molecular weight markers in kDa. (C) Protein expression levels of serum MMPs measured by (i) Immunoblotting and their corresponding densitograms of (ii) MMP-13, (ii) MMP-3 and (iii) MMP-9. β-actin, GAPDH and rat serum albumin (RSA) were used as loading controls. Gel in this figure is cropped and the full length gel is presented in supplementary figure S7. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented. Serum sample are as follows, Lane I – Saline treated, Lane II – Arthritic, Lane III – Arthritic rats treated with Ibuprofen (10 mg/kg), Lane IV – TSE treated arthritic rats (25 mg/kg), Lane V – TSE treated arthritic rats (50 mg/kg) and Lane VI – TSE alone (50 mg/kg). Results are presented as mean ± SEM. a-compared with saline control and b-compared with arthritic group, * p < 0.05.

Figure 4. Histological analysis of Hematoxylin-eosin and Safranin-O stained right hind paw ankle joints from control and experimental groups of animals.

Tissue sections from each group were stained with H & E (A–D) the arrows indicates, (A) Normal joint space with smooth and monolayer synovial cells lining of saline control joint. (B) Reduced joint space with inflammatory cells infiltration and moderate pannus formation of arthritic joint. (C) Moderately recovered joint space and pannus formation with inflammatory cells infiltration of ibuprofen treated joint. (D) Normal joint space with smooth synovial cell lining of TSE treated joint. Further, the glycosaminoglycan content in the knee joints of control and experimental groups were evaluated by Safranin-O staining (E–H), (E) Thick stained normal joints indicating high glycosaminoglycans content. (F), (G) Light stained arthritic and ibuprofen joints indicating deteriorated glycosaminoglycans and (H) Thick stained joints indicating the inhibition of glycosaminoglycans deterioration by TSE treatment. Original magnification 100x.

Figure 5. Effect of TSE on arthritis induced bone resorption.

The right hind paw ankle bone joint homogenate of control and experimental animals were assessed for the bone resorption and degeneration. (A) Cathepsin K activity as determined by zymography. M indicates molecular weight markers in kDa. Arrow head indicates activity band (~37 kDa). The gel in this figure is cropped, full length gel is presented in supplementary figure S8. (B) Exoglycosidases activity. (C) Cathepsin D activity and (D) Alkaline phosphatase (ALP), acid phosphatase (ACP) and tartrate resistant acid phosphatase (TRAP) activities. Paw ankle joint sample are as follows, Lane I – Saline treated, Lane II – Arthritic, Lane III – Arthritic rats treated with Ibuprofen (10 mg/kg), Lane IV – TSE treated arthritic rats (25 mg/kg), Lane V – TSE treated arthritic rats (50 mg/kg) and Lane VI – TSE alone (50 mg/kg). Results are presented as mean ± SEM. a-compared with saline control and b-compared with arthritic group, * p < 0.05.

Figure 6. Protective role of TSE on inflammatory cytokines of control and experimental arthritic rats.

Serum pro- and anti-inflammatory cytokines of experimental rats were quantified using ELISA kits as per manufacturer’s instructions. (A) TNF-α, (B) IL-1β, (C) IL-6 and (D) IL-10. Results are presented as mean ± SEM. a- compared with saline control and b-compared with arthritic group, * p < 0.05.

Figure 7. Influence of TSE on inflammatory mediators of control and experimental arthritic rats.

Figure represents the protein expression levels of serum inflammatory mediators and corresponding densitograms of TNF-α, IL-1β, IL-6, COX-2, IL-23 and IL-10. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented. β-actin, GAPDH and rat serum albumin (RSA) were used as loading controls. The gel in this figure is cropped, full length gel is presented in supplementary figure S9. Results are presented as mean ± SEM. a- compared with saline control and b-compared with arthritic group, * p < 0.05.

Figure 8. Effect of TSE on arthritis-triggered oxidative stress in control and experimental animals.

(A) Influence of TSE on arthritis altered serum stress markers: (i) ROS, (ii) Hydroperoxides and (iii) GSH levels. (B) Influence of TSE on arthritis altered serum levels of endogenous antioxidant enzymes: (i) Superoxide dismutase, (ii) Catalase and (iii) Glutathione-S-transferase. Results are presented as mean ± SEM. a- compared with saline control and b-compared with arthritic group, * p < 0.05.

Figure 9. Proposed mechanism of action of anti-arthritic efficacy of Tamarind seed extract (TSE).

TSE is demonstrated to block arthritis-mediated cartilage degeneration, bone resorption, inflammation and oxidative stress by modulating the respective enzymatic and non-enzymatic parameters. The rat picture used in the figure is captured by Mr. MS Sundaram (first author) while performing animal experiment for the present study.