181 sorts of herbal products were screened in pharmacological experiment in vitro.The natural product, containing efficacy indicating material of KH-207, was found among the tested natural products. The natural product is similar to KH-207 in term of pharmacological effect. We call the natural product the KD-30. I. Anti-inflammatory effect Formation of NO2- is pivotal factor in inflammation process.
To evaluate anti-inflammatory effects of KH-207 and KD - 30, NO2- was examined in mouse macrophage RAW 264.7 cells and chondrocyte of rabbit. This agent dramatically exhibited formation of NO2- compared with control group. Also, KH-207 and KD-30 effectively suppressed inflammation response of the carrageenan-induced edema. II. Analgesic effect In analysis of analgesic effects of KH-207 and KD-30, this agent suppressed production of prostaglandin E2 (PDE2), which is inflammatory compound, in RAW 264.7 cells and chondrocytes.
It also inhibited cyclooxygenase-2 (COX-2) expression to normal level, which is enzyme for generation of PDE2. Together, the agent has excellent effect in formalin and writhing test . III. Cartilage protection KH-207 and KD-30 prevented not only hydrolysis of proteoglycan but also MMP-1, MMP-3, and MMP-13 gene expression. And this agent inhibited MMP-9 activity, which is the enzyme involvedin hydrolysis of cartilage. IV. Pharmacological effect test involved in osteoarthritis After an administration of KH-207 and KD-30 for four weeks, synovial fluid was collected in the osteoarthritis-induced New Zealand white rabbit. The synovial fluid is used to pharmacological experiments. Pharmacological experiments included total protein (anti-inflammation), proteoglycan (protection of cartilage), prostaglandin E2 (analgesic effect), and volume of synovial fluid (anti-inflammation).
As a result of experiment, it demonstrated that KH-207 and KD-30 has excellent effect related to osteoarthritis treatments. Particularly, regeneration of cartilage is observed in histopathological and gross finding. V. Comparison of KD-30 with commercially available drugs -Anti-inflammation effect: To compare anti-inflammatory effect, NO2- generation of drugs was measured in the chondrocyte of rabbit. The KD-30 had the excellent efficacy of inflammation in all drugs, next to KH-207 and Diclofenac. -Analgesic effect: The Piroxicam and Ketoprofen had the excellent inhibition effect of PGE2, however, its drugs were not related to anti-inflammation and cartilage protection. The KH-207 showed the similar effect to the Diclofenac in the activity of PGE2 inhibition. The KD-30 as herbal extract showed the good effect than CM (negative control), and showed the similar effect to JOINS, which the new drug in Korea, in the activity of PGE2 inhibition. -Cartilage protection: KH-207 and KD-30 showed the excellent inhibition of proteoglycan decomposition next to the Celecoxib in the DMB assay using the rabbit chondrocyte, Especially, KH-207 and KD-30 were the excellent inhibitor of MMP-9, which is the enzyme involved in hydrolysis of cartilage.
These results mean that KH-207 and KD-30 have the excellent protection of cartilage. VI.Cytotoxicity test (KGLP Chemon Co., Ltd.) Acutic Toxicity of KD-30 did not observe in all animal models (female and male). The LD50 was more than 5.0 g/kg.To determine volume of chronic toxicity test of KD-30, 4 weeks repeated toxicity test was performed in animal models. As results of toxicity test, maximal volume is determined at 1,000 mg/kg. To date, toxicity and side effect were not found in all animal models in 26 weeks repeated toxicity test. The chronic toxicity test will be finished on August 2006.