Quantitative evaluation of in vitro effects and interactions of active fractions in a Chinese medicinal formula (Yaotongning Capsule) on rat chondrocytes

Yaotongning Capsule (YTNC) is a Traditional Chinese Medicinal (TCM) formula that has been demonstrated to be effective for osteoarthritis (OA) treatment in clinical use. Many compounds and 10 component medicinal materials (CMMs for short, i.e., the fundamental elements used in TCM formulas) in YTNC are challenging to study the pharmacological effects and interactions of the CMMs. Besides, it is difficult to know whether the YTNC formula is reasonable, and if YTNC formula could be improved without comparing YTNC with other TCM formulas of treating OA. Based on different combinations of the active fractions from the 10 CMMs of YTNC and eight additional herbs frequently used in the TCM formulas of treating OA, the present study evaluated systematically the in vitro effects of these active fractions and the interactions among the active fractions from YTNC on rat chondrocytes to find possible solutions of the above questions.Based on the formulation of YTNC and the concept of combinatorial chemistry, the active fractions were applied to form the whole YTNC prescription (i.e., the combination of all YTNC active fractions and the extract of YTNC׳s vehicle), five disassembled formulas of YTNC (i.e., the combinations of some active fractions in YTNC) and 21 TCM samples consisted of different kinds of active fractions. The degenerated chondrocytes were induced with interleukin-1β (IL-1β), and then the half-effective concentration (EC50) value of the proliferation activity was analyzed to evaluate the 27 TCM samples. Nine samples were screened for the following evaluation on glycosaminoglycan (GAG) synthesis. Rat articular cartilage was obtained from six Sprague-Dawley rats (seven days of age), and then chondrocytes were isolated through enzymatic digestion with 0.2% Collagenase II. Proliferations of chondrocytes were examined through Cell Counting Kit-8 assay, when the intracellular levels of GAG were detected by 1,9-Dimethylmethylene blue staining. The interactions between the active fractions in YTNC were evaluated by comparing experimental EC50 values of the YTNC formulas with their additive EC50 values. The effects of every active fraction were estimated by comparing the EC50 values of the TCM sample containing the active fraction with that of the initial sample without the active fraction.The whole formula of YTNC was very good at promoting the proliferation and GAG synthesis among all the 27 TCM samples. The vehicle of YTNC (Chinese rice wine) strengthened the two activities of YTNC. Refer to promoting the proliferation in chondrocytes, Davallia mariesii flavonoids (not belong to YTNC) were more potent than Glycyrrhiza uralensis flavonoids in YTNC, while the saponins, volatile oils and polysaccharides of YTNC were more potent than those from the eight additional herbs. Some samples including fewer active fractions were as good as YTNC. The YTNC formula and its disassembled formulas exhibited good activities both in promoting the proliferation and GAG synthesis, and the whole formula was most potent among the six YTNC formulas.The YTNC formula is reasonable and has advantage in promoting the proliferation and GAG synthesis in IL-1β induced chondrocytes. YTNC׳s vehicle Chinese rice wine plays an important role in strengthening the activity of YTNC. YTNC may have the potential activity on treating chondrocytes degeneration caused by OA. However, the formula still can be simplified based on the combination of alkaloids, flavonoids and 50% of saponins from Glycyrrhiza uralensis to improve its quality controllability and safety. The present study can be a quite purposeful work for developing new YTNC-based formulas with maximal therapeutic efficacy and minimal adverse effects.