The anti-arthritic activity of total glycosides from Pterocephalus hookeri, a traditional Tibetan herbal medicine

Abstract

Context: Pterocephalus hookeri (C. B. Clarke) Hock., a traditional Tibetan herbal medicine rich in glycosides, has been used to treat several diseases including rheumatoid arthritis.

Objective: To evaluate the anti-arthritic activity of total glycosides from P. hookeri, and its possible mechanisms of action.

Materials and methods: Anti-arthritic activity of total glycosides from P. hookeri (oral administration for 30 days at 14-56 mg/kg) was evaluated using paw swelling, arthritis scores and histopathological measurement in adjuvant-induced arthritis (AA) Sprague-Dawley rats. The NF-κB p65 expression in synovial tissues, and serum superoxide dismutase (SOD) activity, malondialdehyde (MDA) and nitric oxide (NO) levels was measured in AA rats, respectively. Further assessment of anti-inflammatory and analgesic activities of these glycosides were carried out using inflammation and hyperalgesia models induced by xylene, carrageenan, agar and acetic acid, respectively.

Results: Total glycosides (56 mg/kg) decreased the paw swelling (38.0%, p < 0.01), arthritis scores (25.3%, p < 0.01) and synovial inflammation in AA rats. The glycosides significantly (p < 0.05-0.01) attenuated the inflammation induced by xylene, carrageenan, acetic acid and agar, increased the pain threshold in acetic acid-induced writhing in mice and mechanical stimuli-induced hyperalgia in AA rats. The glycosides (14, 28, 56 mg/kg) also suppressed the NF-κB p65 expression (33.1-78.2%, p < 0.05-0.01), reduced MDA (21.3-35.9%, p < 0.01) and NO (20.3-32.4%, p < 0.05-0.01) levels, respectively, enhanced the SOD activity (7.8%, p < 0.05) at 56 mg/kg in AA rats.

Discussion and conclusion: Our findings confirmed the anti-arthritic property of the total glycosides from P. hookeri, which may be attributed to its inhibition on NF-κB signalling and oxidative stress.

Keywords: NF-κB; Pterocephalus hookeri (C. B. Clarke) Hock; Tibetan herbal medicine; adjuvant-induced arthritis; anti-arthritic activity; anti-inflammatory and analgesic activities; oxidative stress; total glycosides.

Figure 1.

UPLC chromatogram of total glycosides from P. hookeri. Four compounds were deduced by comparing individual peak retention times with those of the standard substances. The extracts were analyzed by Waters Acquity UPLC-photodiode array (Waters, Milford, MA, USA). The chromatographic separation was carried out on Acquity UPLCR BEH C18 (2.1 mm ×50 mm, 1.7 μm) with the column temperature at 35 °C. The mobile phase was acetonitrile (A) with 0.2% phosphoric acid solution (B) in a gradient mode which was described as follows: 0–4 min, 10% A and 90% B; 5–10 min, 15% A and 85% B; 11–14 min, 20% A and 80% B. The flow rate was 0.5 mL/min and the PDA UV wavelength was 273 nm. The four main compounds of total glycosides from P. hookeri were identified, including sweroside (peak 1), loganin (peak 2), sylvestroside I (peak 3) and cantleyoside (peak 4).

Figure 2.

Effect of total glycosides from P. hookeri on acetic acid-induced abdominal writhing in mice. Total glycosides (28, 56 and 112 mg/kg) or indomethacin (25 mg/kg) or vehicle was administered 30 min prior to acetic acid injection. The latency time (A) and reduced number of writhing (B) were recorded for 15 min. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 3.

Effect of total glycosides from P. hookeri on pain threshold in adjuvant-induced arthritis rats. Arthritis was induced by intradermal injection of 0.1 mL of complete Freund’s adjuvant (CFA). Total glycosides (14, 28 and 56 mg/kg) or nimesulide (33.33 mg/kg) or vehicle was administered 1 h after CFA injection and their daily treatment continued until 30 days after CFA challenge. The pain threshold (pressure pain, g) was assessed on 7, 14, 21 and 30 days after CFA injection via algesimeter. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 4.

Effect of total glycosides from P. hookeri on hot-plate test in mice. Mice were pretreated with total glycosides or morphine (3.33 mg/kg) or vehicle for 3 days. Mice were subjected to the hot-plate test, and the latency time was then determined at 15, 30, 60, 90 and 120 min after administration of drug. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 5.

Effect of total glycosides from P. hookeri on carrageenan-induced acute paw swelling in rats. Total glycosides (14, 28 and 56 mg/kg) or indomethacin (12.5 mg/kg) or vehicle was administered for 7 days. One hour after last administration, rats were injected with 1% carrageenin into the right hind paw, paw volume was then measured at 1, 2, 3 and 4 h after carrageenin injection. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 6.

The anti-inflammatory effect of total glycosides from P. hookeri on several acute and chronic inflammation models. (a) Xylene-induced ear oedema, (b) acetic acid-induced peritoneal capillary permeability and (c) agar-induced granuloma in mice. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 7.

Effect of total glycosides from P. hookeri on paw swelling in AA rats. The left hind paw of rats was injected intradermally with 0.1 ml of CFA. Total glycosides (14, 28, 56 mg/kg), or nimesulide (33.33 mg/kg) or vehicle was pretreated for 3 days before CFA injection and then administered 1 h after CFA injection and their daily treatment continued until 30 days after CFA challenge. The primary swelling was measured at 6, 12, 24 36, 48 and 72 h after CFA injection. And secondary paw swelling was assessed on 6, 12, 18, 24 and 30 days after CFA injection. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 8.

Effect of total glycosides from P. hookeri on arthritis score in AA rats. Arthritis score was evaluated on 12, 18, 24 and 30 days after CFA injection. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 9.

Effect of total glycosides from P. hookeri on the expression of NF-κB p65 in synovium of AA rats. The expression of NF-κB p65 in synovial tissues were measured by immunohistochemistry. (a) Quantitative results of NF-κB p65 expression are depicted as integral optical density. All data are represented as mean ± SD, n = 6, *p < 0.05, **p < 0.01 vs control. (b–g) Typical images of NF-κB p65 expression in synovial tissues were obtained by the microscope (400× ), (b) normal, (c) control, (d) nimesulide (33.33 mg/kg), (e) 14 mg/kg of total glycosides, (f) 28 mg/kg of total glycosides, (g) 56 mg/kg of total glycosides. Arrows indicate the positive expression.

Figure 10.

Antioxidant effects of total glycosides from P. hookeri in AA rats. (a) Activity of serum SOD and (b) serum levels of MDA and (c) NO are depicted. All data are represented as mean ± SD, n = 10, *p < 0.05, **p < 0.01 vs control.

Figure 11.

Effect of total glycosides from P. hookeri on histopathology of synovium in AA rats. (a) Normal synovial tissue, (b) synovial tissue of AA rats treated with vehicle showed obvious inflammatory cell infiltration (**) and synovial hyperplasia (##), (c) synovial tissue of AA rats treated with nimesulide (33.33 mg/kg), (d–f) synovial tissue of AA rats treated with 14, 28 and 56 mg/kg of total glycosides from P. hookeri, respectively. Panels c, d, e and f show reduction in inflammatory cell infiltration and synovial hyperplasia (original magnification  400×).