Identification Of The Components Of Honeysuckle Efficacy Study

Objective: Find the chemical identification mark of Lonicera japonica, related to its pharmacodynamic action, to establish an identification method which can present its efficacy, on the basis of ascertaining the antiviral part of Lonicera japonica. The identification method should possess extensive applicability to be used to identify the dried medical herb, the active part (or the extract) and the preparation of Lonicera japonica.Methods: Ascertain the active part by the antivirus test in the vitro; screen the abstraction technology by the orthogonal design; handle the data by the analysis of variance and direct-viewing; enrich the active part by the macroporous resin adsorptive process; investigate each parameter of macroporous resin adsorbing by one-factor design; determine the contents of the active compounds alignment by HPLC and spectrophotometry; research the identification mark by HPLC and HPLC-MS.Results: 1 the preparative method of the active antivirus part of Lonicera japonicaThe optimal extraction technology was A1B3C1 that the Lonicera japonica was extracted by 70% ethanol (8 times of the dried medical herb) 3 times, 1h per time. The parameter of macropotous resin adsorptive process was as followed: the extract (the concentration of the solution was 100mg dried medical herb per milliliter, pH=3) was subjected to macropotous resin column (diameter/high=1:11) with the adsorbing current velocity 0.8mL/min, the ratio of loading capacity to resin volume was 1:3. After eluted with water (twice of resin volume) until the effluent was colorless, the resin was eluted by 50% ethanol under the flow rate 1mL/min until the effluent was colorless, and the effluent eluted by 50% ethanol was the active part. Prepare 3 samples as the method above, and the average content of Chlorogenic acid was 20.23%±0.37%, the total flavonoids 75.15%±1.55%.2 The HPLC fingerprint of dried medical herb of Lonicera japonicaThe chromatographic condition: Waters Spherisorb ODS2 (5μtm, 4.6×250mm) as Analytical Column; ACN-0.35%H₃PO₄ (11.5:88.5→21:79, gradient elution for 60min) as mobile phase; wave length for determination 240nm; flow rate 1.5mL/min; and sample size 5μl. Under the condition above, RSD of the average rate of the relative retention time to the peak area was below 3% in the tests of precision, stability and reproducibility. The result of the stability test showed that the solution was stable in 22h. 14 common peaks were found after analyzing 10 samples, and the relative retention time,calculated with Chlorogenic acid peak as the reference peak,were respectively 0.2432±0.0059, RSD=2.44%; 0.2879±0.0068, RSD=2.37%; 0.5187±0.0035, RSD=0.67%; 0.6594±0.0122, RSD=1.85%; 0.7344±0.0085, RSD=1.15%; 0.8209±0.0157, RSD=1.92%; 1.0000±0.0000, RSD=0%; 1.0913±0.0118, RSD=1.08%; 1.4691±0.0156, RSD=1.06%; 1.5444±0.0256, RSD=1.66%; 4.2805±0.0721, RSD=1.68%; 4.7019±0.1054, RSD=2.24%; 5.0998±0.0899, RSD=1.76%; 6.1591±0.1209, RSD=1.96%.There were 3 single peaks whose relative area were all above 10%, and the proportion of their relative areas were respectively 0.4930±0.0403, RSD=8.18%;1.0000±0, RSD=0%; 0.4955±0.0313, RSD=6.31%。3 The identification method for Lonicera japonica and its preparations5 common peaks were found after contrasting the HPLC chromatograms of the medical material, the active part and the praeparatum. The relative retention time, calculated with Chlorogenic acid peak(peak 2) as the reference peak, were respectively 0.62824±0.0063, RSD=1.00%; 1±0.0, RSD=0%; 1.37544±0.0154, RSD=1.12%; 4.39244±0.0935, RSD=2.13%; 4.92004±0.0347, RSD=0.70%.4 common peaks were found after contrasting the HPLC-MS chromatograms of the medical material, the active part and the praeparatum. The relative retention time, calculated with Chlorogenic acid peak(peak 2) as the reference peak, were respectively 0.70164±0.0099, RSD=1.40%; 1.0±0.00, RSD=0.0%; 1.52514±0.0084, RSD=0.55%; 4.80724±0.0474, RSD=0.99%. The final presumption was that possibly the common peak 1 was Loganin, the common peak 2 was Chlorogenic acid, the common peak 3 was Kingiside and the common peak 4 was 3, 5-Dicaffeoyl-quinic acid or 3, 4-Dicaffeoyl-quinic acid.Conclusion: The identification method established was based on research of the drug efficacy. The commonness characteristics of Lonicera japonica were brought up from the dried medical herb, the active part and many kinds of the preparations on the market. So the method was able to present the efficacy of Lonicera japonica and could be used to identify the dried medical herb, the active part (or the extract) and the preparations of Lonicera japonica with extensive applicability.