Extract of the Chinese herbal formula Huo Luo Xiao Ling Dan inhibited adjuvant arthritis in rats

Abstract

Ethnopharmacological relevance: The herbal formula Huo Luo Xiao Ling Dan (HLXL) and its modifications have been used in traditional Chinese medicine for about one hundred years to alleviate pain and inflammation.

Aim: To investigate the effects of HLXL on complete Freund's adjuvant (CFA)-induced multiple-joint arthritis in rats.

Materials and methods: Male Lewis rats, 190-210 g, were immunized subcutaneously at the base of the tail with 200 microl of heat-killed Mycobacterium tuberculosis in mineral oil (5 mg/ml). HLXL (2.30 and 4.60 g/kg) or vehicle control (n=8 per group) was administered orally (i.g.) once a day between days 16 and 25 post-CFA injection. The rats were observed for signs of arthritis with arthritic changes (erythema, edema, induration) being scored on a scale of 0-4 of increasing severity using a standard scoring system. The maximum arthritis score per rat was 16. A plethysmometer was used to measure edema volume in each paw. Adverse effects of HLXL were monitored by closely observing the animals for unusual behavioral changes. Levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in local tissue were measured by enzyme-linked immunosorbent assay on day 25 post-CFA.

Results: HLXL significantly decreased arthritis scores between days 23-25 in the 2.30 g/kg group and 21-25 in the 4.60 g/kg group (p<0.05). It reduced paw edema on days 22 and 24 in the 2.30 g/kg group and on days 20, 22 and 24 in the 4.60 g/kg group compared to control (p<0.05). Local tissue TNF-alpha and IL-1beta levels on day 25 post-CFA injection were significantly (p<0.05) lower in rats treated with HLXL than in control rats. No observable adverse effects were found.

Conclusion: The data suggest that HLXL produces significant anti-arthritic effects that may be mediated by suppressing pro-inflammatory cytokines, and it appears to be safe.

Fig. 1

HPLC trace of the HLXL extract. The major representative peaks of herbs in the formula are marked. The numbers in parentheses are the numbers assigned to the herbs as listed in Table One. The second number indicates the marker number for the respective herb. For example, (1)-3 represents the third marker of the first herb (Boswellia carterii Birdw.) listed in the HLXL formula. The HPLC conditions were YMC C-18 analytical column (4.6 mm × 15 cm); 100% Methanol at 1.0 ml/min; UV monitor at 210 nm. Retention times of purified HLXL are delineated directly on this tracing.

Fig. 2

Effect of HLXL on arthritis as assessed with arthritic scores (Mean ± S.E., n = 8/group) in rats. Note that both 2.30g/kg and 4.60g/kg (daily, i.g.) of HLXL significantly decreased the arthritic score and suppressed peak arthritic severity compared to vehicle control, *p<0.05 vs. vehicle control, at the same time point.

Fig. 3

Effect of HLXL on arthritis assessed with paw volume (Mean ± S.E., n = 8/group) in rats. Data showed that paw volume increased remarkably following arthritis formation and peaked around day 18 in the control group. HLXL (daily, i.g.) significantly decreased arthritic paw edema volume and suppressed peak arthritic edema in both 2.30 g/kg and 4.60g/kg groups, *p<0.05 and **p<0.01 vs. vehicle control, at the same time point.

Fig. 4

Effect of HLXL on IL-1β and TNF-α levels (pg/mg protein, Mean ± S.E.) 25 days post-CFA injection. Tissue was obtained from four groups of rats: group N (no arthritis + vehicle treatment, n=4), group H4.6 (arthritis + HLXL treatment at 4.60g/kg/day, n=7), group H2.3 (arthritis + HLXL treatment at 2.30g/kg/day n=6), and group C (arthritis + vehicle treatment n= 6). Data showed that both IL-1β and TNF-α increased significantly in local tissue following development of arthritis. However, after HLXL treatment, local tissue IL-1β and TNF-α decreased significantly, *p<0.05 and **p<0.01, compared to the vehicle control (Group C).