Anti-osteoarthritic effects of ChondroT in a rat model of collagenase-induced osteoarthritis

Adult male Sprague–Dawley rats weighing 170–180 g were housed in a room with constant temperature (24–26 °C) and humidity (40%–60%). Food (Pellet, GMO, Korea) and water were available ad libitum. Animals were acclimated to the laboratory environment for 1 week before commencement of the experiment, and all procedures were approved by the Institutional Animal Care and Use Committee of the Dongshin University (DSU-2015-1003-01). After the experiment, all the rats were euthanized using Carbon dioxide (CO2) in accordance with AVMA guidelines.

The five herbal medicines constituting ChondroT were purchased from Omniherb Co. (Yeongcheon, Korea), and their origin was taxonomically confirmed by Professor Jong-Kil Jeong at the Department of Herbology, College of Oriental Medicine, Dongshin University.

The five herbs (OK, LJ, AG, CM, and PA) were combined in a 6:4:4:4:3 ratio (Table 1). ChondroT was prepared by carrying out water extraction once, using 10-fold solvent at 100 °C for 3 h, and then filtering it (180 mesh). The water extract solution was concentrated using a continuous vacuum evaporator (approximately 55–60 °C, 670 mmHg), and this was followed by vacuum drying using a vacuum drier (720 mmHg) for 8 h. The yield was approximately 29.4%. Voucher specimens (No. GHJTY 501) of the collected herb samples were deposited in the herbarium of Jung Woo Shin Yak (Asan, Korea).

Chlorogenic acid (1) and berberine chloride (2) were purchased from U.S. Pharmacopeial Convention (Rockville, MD, USA), and Decursin (3) was purchased from ChemFaces Biochemical Co. Ltd. (Wuhan, China). The purities of the reference compounds 1–3 were 97.3%, 81.0%, and 99.4%, respectively, measured using HPLC analysis. The chemical structures of these compounds have been shown in Fig. 1a. HPLC-grade methanol, acetonitrile and water were obtained from J.T. Baker (Phillipsburg, NJ, USA), while analytical grade formic acid was purchased from Sigma–Aldrich (St. Louis, MO, USA).

For quality control of the ChondroT sample, all experiments were performed using a Shimadzu Prominence LC-20A Series (Shimadzu, Kyoto, Japan) equipped with a solvent delivery unit (LC-20AT), online degasser (DGU-20A3R), column oven (CTO-20 AC), auto-sampler (SIL-20 AC), and UV-VIS detector (SPD-20A). Data acquisition and processing were conducted using Lab solution software (version 5.73 SP3, Kyoto, Japan). Compounds 1–3 were separated using a Waters SunFire C18 column (4.6 × 250 mm; 5 μm, Milford, MA, USA) maintained at 40 °C. The mobile phases consisted of (A) 0.1% (v/v) aqueous formic acid and (B) 0.1% (v/v) formic acid in acetonitrile, and the gradation conditions were optimized as follows: a duration range of 0–10 min, 10%–35% B; 10–30 min, 35%–65% B; 30–35 min, 65%–80% B; 35–40 min, 80% B; 40–41 min, 80%–10% B; and 41–50 min, 10% B. The flow rate and injection volume were 1.0 mL/min and 10 μL, respectively. For HPLC analysis, lyophilized ChondroT (1 g) was dissolved in 100 mL of 70% methanol and extracted for 60 min by sonication. The extracted solution was centrifuged at 3000 rpm for 10 min, and then passed through a 0.45-μm syringe filter before HPLC analysis.

Blood samples were collected, and 100 μL of it was used to measure leukocytes using a Multispecies Hematology Analyzer (950, Hemavet, USA). The rest of the blood sample was used to measure aspartate aminotransferase (AST), alanine transaminase (ALT), albumin, BUN, and creatinine levels with the help of a high-speed centrifuge (VS-6000CFi, Korea) at 3000 rpm for 20 min.

TNF-α was measured using a Rat TNF-α kit (Invitrogen, USA), IL-1β was assessed using a Rat IL-1β kit (R&D Systems, USA) and IL-6 was evaluated using a Rat IL-6 kit (Invitrogen, USA). The optical densities (OD) of all samples were measured at 450 nm using Spectramax (M2, Molecular Devices, USA).

The left knee joint was removed and fixed in Bouin solution for > 24 h. Decalcification was performed using a 2.5% nitric acid solution, which was changed once a day for 7 days. The removed tissue was dehydrated using a Tissue Processor (Tissue-Tek^® II, Japan). After deparaffinization and staining with H&E (Muto, Japan), the samples were observed under an optical microscope (Nikon, Japan).

After deparaffinization, the left knee joint was treated with Weigert’s Iron Hematoxylin solution (Sigma, USA) for 10 min and stained with 0.001% Fast Green solution (Sigma, USA) for 5 min. The sample was then incubated with 1% acetate solution for 10 s and stained with 0.1% safranin O solution (Sigma, USA) for 5 min. Thereafter, the tissue was dehydrated and observed under an optical microscope (Nikon, Japan).

Quality assessment of ChondroT was performed using three marker compounds and HPLC. The selected compounds were as follows: compound 1 (Lonicerae Folium), compound 2 (Phellodendri Cortex), and compound 3 (Angelicae Gigantis Radix). All analytes were separated within 40 min, and the typical chromatogram of a 70% methanol extract of ChondroT has been shown in Fig. 1b. Quantification was performed by UV-VIS detection at 330 nm based on retention time. The retention times of components 1–3 were 9.25, 11.35, and 34.51 min, respectively. Under optimized chromatography conditions, the concentrations of the marker compounds 1–3 in ChondroT were 3.67 ± 0.08, 2.41 ± 0.22, and 1.87 ± 0.18 mg/g, respectively (Table 2, Fig. 1).

With regard to the effect of the amount of ChondroT administered on anti-inflammatory and inflammation-inducing agents in serum, a significant increase in the level of TNF-α was observed as compared to the intact group. The indomethacin, Joins tab, ChondroT100, and ChondroT200 groups also showed a significant increase compared with the control group.

With regard to the level of IL-1β, the control group showed a significant increase as compared to the intact group, while the indomethacin, Joins tab, ChondroT100, and ChondroT200 groups showed a significant decrease as compared to the control group. In the case of the level of IL-6, the control group showed a significant increase as compared to the intact group, while the indomethacin, Joins tab, ChondroT50, ChondroT100, and ChondroT200 groups showed a significant decrease as compared to the control group. and in ChondroT100 and ChondroT200 groups, TNF-α, IL-1β, and IL-6 were equivalent as compared with those in the indomethacin and Joins tab (Table 3, Fig. 2).

Histopathologically, a small portion of synovial membrane was seen to be destroyed in the control group, while the indomethacin and Joins tab groups clearly exhibited synovial membrane cells of cartilage and synovial cavity compared to the control group. In addition, the ChondroT100 and ChondroT200 groups exhibited synovial membrane cells, synovial cavity, and cartilage cells similar to the intact group (Fig. 3).

Safranin O-fast staining showed that the positive reaction of proteoglycans in articular cartilage in the control group was lower than that of the intact group. Also, in the ChondroT200 group, the deep layer of cartilage showed a higher positive reaction for proteoglycans as compared to the control group (Fig. 4).

With regard to the effect of ChondroT administration on leukocyte, eosinophils in the control group exhibited a significant decrease compared with those in the intact group, whereas WBC, neutrophils, lymphocytes, and monocytes exhibited no significant differences. WBC and lymphocytes exhibited a significant decrease in ChondroT100 and ChondroT200 groups compared with those in the control group (Table 4, Fig. 5).

The collagenase induced control group exhibited a significant increase in AST compared to the intact group, while the indomethacin, Joins tab, ChondroT50, ChondroT100, and ChondroT200 groups showed a significant decrease compared with the control group. In the case of ALT, a decreasing tendency was observed, although this was not statistically significant (Table 5, Fig. 6).

The ChondroT100 and ChondroT200 groups showed a significant decrease in albumin as compared to the control group while there were no significant differences with regard to BUN and creatinine (Table 6, Fig. 7).