Study On The Optimal Compatibility Of Baicalin, Icarrin And Astragalus Saponin Ⅰ In Chinese Herbal Compound Biminne

Background:Allergic rhinitis(AR) is chronic inflammation disease in nasal mucosa, suffered by 10-25% population in the world-wide range with its incidence increasing year after year. It also has become one of the most important risk factors of asthma. Traditional Chinese medicine has a long history and indicates distinct values in prevention and treatment of AR. Chinese herbal compound Biminne is an effective prescription of AR that is designed by our institiute, consisting of eleven herbs including Astragalus, Scutellaria, epimedium, Saposhnikovia, Schisandra, ebony meat, ginkgo leaf, psoralen, Angelica, Rehmannia, Polygonatum. An large-scale, randomized, double-blind, and placebo-controlled clinical researches conducted in Australia confirmed that Biminne is able to improve patients'clinical symptoms and decrease the IgE level in serum. Experimental study elucidated the mechanisms further and found that Biminne exerted the therapeutical effects through regulating the endocrine and immune function. Our early studies also suggested that the major effective ingredients of Biminne are baicalin (BA), icarrin (ICA) and astragalus saponinⅠ(ASI). Chinese herbal formula was based on multiple principles to combine each herb and emphasizes the proportion of each herb. The proper proportion of BA, ICA, AS is not clear. As a result, it is necessary to clarify the optimal compatibility among BA, ICA and ASI. The target of this study is to elucidate this and lay a foundation for enriching contents of the model of new drug development based on the combination of multiple pure compounds.Objective:To study on the optimal dose combination of Baicalin, Icarrin and Astragalus saponinⅠderived from Chinese herbal formula BiminneMethods:Firstly, allergic rhinitis mice model was established by intraperitoneal interjection of ovalbumin (OVA) and aluminum hydroxide gel suspension. On day 1, day 7 and day 14, the mice were sensitized by injecting intraperitoneallly with 10% OVA, and on day 21 to 28 the sensitized mice were subjected to 5%OVA inhalation challenge. After the model established, the mice were sacrificed and the spleen cells were isolated. The isolated spleen cells were cultured in 96-well plates in the presence of concanavalin A (ConA) or BA/ICA/ASI with different concentrations or culture medium only. The effective dosage range of BA, ICA and ASI was detected by XTT method. Secondly,10 groups of dose combinations assembled by U*10(108) form were administered to the ConA stimulating cells so that spleen cells were diveded into the control group, ConA group and different dose combinations groups. The inhibitory effects on the splenocyte proliferation were analyzed and the regression equation was calculated by statistic method in order to make the optimal dose combination clear. Finally, the levels of interleukin-4 (IL-4), IL-5 and IFN-gamma (IFN-γ) in the supernatants of cell culture medium were analyzed by enzyme-linked immunosorbent assay and the proliferative activity of splenocyte were tested between each effective position group and the optimized dose combination group so as to verify the above result.Results:In the study of determining the effective dosage range of three effective components, the OD value of ConA group was obviously higher than the control group (P<0.01). When BA and ASI at the extrame low concentration (0.003-0.016μM), the OD values of them were higher than ConA group (P<0.05 or P<0.01); while when BA or ICA were in the range of 2-10μM and ASI in the range of 1-10μM, the OD values of them were lower than ConA group (P<0.05 or P<0.01). This indicated that 2-10μM,2-10μM and 1-10μM were the effective dosage ranges of suppressing the splenocyte proliferation of BA, ICA and ASI respectively.In the study of searching the optimized compatibility of three effective positions, it was found that the regression equation fitted by the stepwize regression was statistically meaningful (P<0.01), the equation is as follows:OD=-59.59659 +7.73626×BA+13.97243×ICA-0.23033×BA2-0.75713×ICA2+0.00957×ASI2-0.79014×BA×ICA-0.01608×BA×ASI. Based on this equation, we calculated the values for proportion of baicalin to icarrin to astragalus saponin I when OD reached the minimum. So we deduced that when the proportion of baicalin to icarrin to astragalus saponin I was 1:2.14:2.65, the inhibitory effect was most remarkable. The partial derivatives of BA, ICA and ASI were calculated, and it was suggested that when the three effective positions were combined applied, ICA played a major role, and BA took the second place, while ASI showed the weakest effect.Further research confirmed that in the study of the proliferative activity of splenocyte, the OD value of ConA group was apparantly higher than the control group (P<0.01). BA group, ICA group and ASI group took as many dosage as the optimized dose combination group, while the inhibitory effect in the optimized dose combination group is superior to each effective component group (P<0.05 or P<0.01). In the study of analyzing the levels of IL-4, IL-5 and IFN-gamma, the levels in ConA group was higher than the control group (P<0.01); the levels of IL-4 and IL-5 of each effective component group and the optimized group were lower than that of ConA group, and the level of IFN-gamma higher than that of it (P<0.01); the level of IL-4 of the optimized dose combination is lower than that of each effective dose combination group (P<0.05 or P<0.01), and the level of IFN-gamma higher than that of them (P<0.01), and the level of IL-5 lower than that of ICA and ASI groups (P<0.01).Conclusion:A optimized dose combination makes the major effective ingredients in Chinese herbal compound Biminne most effectively suppress the proliferation of splenocyte from sensitized mice and regulate the imbalance of the Thl/Th2 cytokines caused by allergy so that pave the way for the further study of this herbal formula based on the combination of multiple pure compounds.