| Kudingcha is a traditional Chinese folk medicine and edible plants,isochlorogenic acids are the one of the main active components,the results of the present study showed that the isochlorogenic acids from Ilex kudingcha has a good anti-inflammatory effect.However,the less anti-rheumatoid arthritis effect was observed.In this study,the extraction and purification of isochlorogenic acids and the pharmacological action of anti-rheumatoid arthritis will be studied in depth.In order to determine the content of isochlorogenic acids in Ilex kudingcha,the high performance liquid chromatography(HPLC)was used.The chromatographic conditions were as follows: the flow rate was 1.0 mL / min;the column temperature was 40℃;the injection volume was 10 μL;the detection wavelength was 325 nm.The results showed that the sample volume of three kinds of isochlorogenic acids in 0.02 μg ~ 2.00 μg showed a good linear relationship,Meanwhile,the results of precision,repeatability and sample recovery were preferably,in accordance with the requirements of quantitative analysis.The test method established is accurate,reliable and reproducible,which provided the basis for the further study and application of isochlorogenic acids in Ilex kudingcha.In order to optimize the extraction process of isochlorogenic acids in Ilex kudingcha,the orthogonal test was conducted to optimize the water extraction conditions for three isochlorogenic acids.The L9(34)orthogonal experiment was carried out to optimize the water extraction conditions.Using the total content of isochlorogenic acids as an indicator,the amount of total content of isochlorogenic acids was determined by HPLC.The results showed the optimal parameters: first supplement of 183% water absorption after adding 10 times the amount of water,soaking 2 h,the rest of each time with 10 times the amount of water,a total of 3 times,each boiling 1 h.To purify the isochlorogenic acids in kudingchas from Ilex kudingcha,the purification process parameters were optimized.The MCI-GEL HP50 SS resin was optimized as the best one by the static adsorption and desorption experiments.Then the adsorption and desorption experiments were carried out to study the different purification processes.The optimal parameters were as follows: the wet sample,sample column temperature of 30℃,the sample flow rate was 3 BV/h,the sample concentration was 4.32 mg/mL,the sample solution was 10 mL,elution velocity was 3 BV/h 5 BV,first with water to remove impurities,and then 25% ethanol elution 10 BV.The results showed that the flow rate was 3 BV/h,the concentration of sample solution was 4.32 mg/mL,the amount of sample solution was 10 mL,the elution flow rate was 3 BV/h,washed with 5 BV water miscellaneous,and then washed with 10 BV of 30% ethanol,the isochlorogenic acids from Ilex kudingcha can be better purified.The purity of the isochlorogenic acids was 82.30%,the purification efficiency was about 9.53,and the retention rate was 73.40%.To study the pharmacological effects of anti-rheumatoid arthritis of isochlorogenic acids from Ilex kudingcha,a model of rheumatoid arthritis in mice was prepared by injecting Complete Freund’s adjuvant into the toe.The tail vein was injected with different doses of isochlorogenic acid and dexamethasone.Dexamethasone was used as positive control drug.The pharmacological effects of isochlorogenic acids from Ilex kudingcha on rheumatoid arthritis were evaluated by comparing the indexes of rheumatoid arthritis with other groups.According to the results of the mice toes swelling inhibition rate,the mice in normal control group does not appear in the process of the whole experimental arthritis symptoms,and the mice in other groups have different degrees of joint swelling symptoms.The experimental results showed that the mice in control group,DCQ-15 and DCQ-20 groups have obvious inhibitory effect on foot swelling in mice,which have the significant difference compared to that of model group(P<0.01).The foot swelling inhibition rate in mice were as follows: 77.33%,57.11%,79.51%;The mice in DCQ-5 group has inhibitory effect on foot swelling,which have the difference compared to that of model group(P<0.05).In addition,the swelling inhibition rate was 9.85%.The results of histopathological examination showed that the biopsy bone structure of the vechile control group is complete,any tissue swelling and pathological changes were not observed.Compared with the mice in normal control group,the mice in model group diffused inflammation,with a large number of inflammatory cells infiltration in subcutaneous tissue.Compared with the mice in model group,reduced inflammation and cells infiltration in different level the mice in drug group and control group were observed.The mice in high dose group has more significant effect compared with that of in the control group.The treatment thoroughly prevented foot tissue pathological change in mice.The results show that the isochlorogenic acids has a definite therapeutic effect,inhibiting the inflammatory cells infiltration effectively.It have a positive improvement and protection on the destruction of connective tissues.The result of serological indexes showed that the serum NO level in mice of control group,DCQ-15 and DCQ-20 groups were significantly lower,compared to that of the model group(P<0.05);Serum PGE2 levels in mice of control group and DCQ-15 and DCQ-20 groups were significantly lower,compared to that of the model group(P<0.05).Through the research of the extraction and purification in isochlorogenic acids from Ilex kudingcha,we obtained the high purity of Isochlorogenic acids.We found that isochlorogenic acids from Ilex kudingcha has good anti-rheumatoid arthritis pharmacological effects by an animal experimental study using adjuvant arthritis mice.The results provided a good foundation and scientific basis for further study on the isochlorogenic acids from Ilex kudingcha. |
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