Study On The Contents Of Active Components And Pharmacological Effects Of The Combination Of Shiwei And Kiwifruit Root

Objective:Shiwei and Kiwifruit root have the functions of clearing away heat and diuresis in clinic.They are often used in combination in the folk for the treatment of inflammation and cancer.In order to provide reference for the compatible application of Shiwei and Kiwifruit root,we studied the dissolution changes of effective components and their synergistic effects on anti-oxidation,anti-tumor and anti-inflammatory through pharmacological experiments in vivo and in vitro.Methods:1.35 kinds of extracts were obtained from Shiwei and Kiwifruit root by 7 different weight ratios and 5 different solvent extraction methods,and the active substance content and pharmacological action were studied.2.The contents of protocatechuic acid,chlorogenic acid and caffeic acid in 35 kinds of extracts were determined by HPLC,and the effect of compatibility of Shiwei and Kiwifruit root on the dissolution of active components was investigated.3.The scavenging capacity of 35 extracts on DPPH free radical,ABTS free radical and hydroxyl free radical,lipid antioxidant capacity and total reducing capacity were studied in vitro.The results were verified by L02 cell experiment,and the best compatibility and preparation method of Shiwei and Kiwifruit root were preliminarily screened.4.The toxic effects of drugs on tumor cells Bel7402,Hep G2,Hep2 and A549 were studied by cell experiment,and the effect of Shiwei and Kiwifruit root on tumor treatment was explored.5.The toxicity of 35 extracts on macrophage RAW264.7 was determined to screen the dosage of anti-inflammatory experiment;The content of inflammatory factor no in RAW264.7 was determined after the optimal dosage of the drug was given to study the inhibitory effect of the drug on LPS induced inflammatory cells;In vivo experiments were carried out to determine the inhibitory effect of the drug on ear swelling in mice and to screen the best anti-inflammatory extract.6.The results were analyzed and compared to determine the best compatibility ratio and extraction solvent of Shiwei and Kiwifruit root.Results:1.In this experiment,we found that the best comprehensive effect was obtained when the weight ratio of Shiwei and Kiwifruit roots was2:1 and the extraction solvent was 50%ethanol.2.The effective components of Kiwifruit root and Pyrrosia chinensis are protocatechuic acid,chlorogenic acid and caffeic acid.The separation degree and peak shape of the peak are good when the mobile phase condition is methanol-0.1%formic acid water=0.168:0.632（Total flow rate was 0.8m L/min）.By measuring the dissolution of the components to be determined,it was found that when extracted with different solvents,the content range of the protocatechuic acid in extract of Pyrrosia fortunei（weight ratio 1:0）was2.1942-3.5481 mg/g,chlorogenic acid was 7.3110-13.6211 mg/g and caffeic acid was 0.4521-0.7320 mg/g;and When the protocatechuic acid was extract from the root of Actinidia chinensis（weight ratio 0:1）,the content was0.0104-0.0325 mg/g.The content of chlorogenic acid and affeic acid were0.0865-0.3652 mg/g and 0.1110-0.2449 mg/g,respectively.The dissolution of protocatechuic acid,chlorogenic acid and caffeic acid from the roots of Pyrrosia chinensis was significantly higher than that from the roots of Actinidia chinensis.After compatibility of Shiwei and Kiwifruit root,the dissolution trend of protocatechuic acid fluctuated greatly with the increase of the weight ratio of Kiwifruit root.The dissolution reached the peak when the weight ratio was 2:1,and the peak was weak at 1:2;the contents of chlorogenic acid and caffeic acid showed a downward trend,and their downward trend was basically the same.After the roots of Pyrrosia fortunei and Actinidia chinensis were extracted with five different combined solvents of water and ethanol,the dissolution of protocatechuic acid and chlorogenic acid was the highest in 50%ethanol extraction,and the dissolution of caffeic acid was the highest in ethanol extraction.When the compatibility ratio of Shiwei and Kiwifruit root was 2:1 and the extraction solvent was 50%ethanol,the synergistic dissolution of protocatechuic acid was the strongest,about4.3965 mg/g.When the ratio of chlorogenic acid and caffeic acid was 1:0 and the extraction solvent was 50%ethanol and ethanol respectively,the highest dissolution rates of chlorogenic acid and caffeic acid were 13.6211 mg/g and0.7144 mg/g,respectively.3.In vitro,the antioxidant capacity of the compatibility of Shiwei and Kiwifruit root was tested.The results showed that when different solvents are used for extraction,DPPH radical scavenging ability was from large to small:50%alcohol extraction>first alcohol then water extraction>first water then alcohol extraction>water extraction>alcohol extraction.In the different compatibility ratio,the antioxidant effect was the best with the weight ratio was 3:1.HE-3-1 had the strongest scavenging capacity with EC₅₀=275.7μg/m L.ABTS free radical scavenging ability and lipid antioxidant ability were significantly different in different solvent extraction,shows 50%alcohol extraction>alcohol extraction before water>alcohol extraction>water extraction>water extraction,50%alcohol extraction>alcohol extraction before water>alcohol extraction>water extraction>water extraction after alcohol extraction,respectively.The optimum extraction conditions were 50%alcohol extraction and 2:1 weight ratio（HE-2-1）,with the EC₅₀ were 208.5μg/m L and 186.5μg/m L.The scavenging ability of hydroxyl radical was 50%alcohol extraction>water extraction≈alcohol extraction>first alcohol then water extraction>first water then alcohol extraction.The scavenging ability was stronger in 1:0 and 2:1,and the HE-2-1 was the strongest,EC₅₀=4.7mg/m L.The results showed that the total reducing power was 50%alcohol extraction>alcohol extraction before water extraction>alcohol extraction>water extraction>water extraction before alcohol extraction,when the compatibility ratio is 1:0,the ability of scavenging free radicals is the strongest.HE-1-0 had the strongest reducing ability,followed by EW-1-0.The reducing ability of HE-1-0 was similar to that of 0.1 mg/m L VC at the concentration of 1 mg/m L.The results of cell experiment were affected by the combination of solvent and compatibility ratio,and the effect of solvent was more significant.More than half of the extracts were non-toxic at the concentration of 2 mg/m L,80%were non-toxic at the concentration of1mg/m L,and all the extracts were non-toxic at the concentration of 0.5mg/m L.L02 cells,injured by H₂O₂,were pretreated with the extract of Shiwei-Kiwifruit root（0.5 mg/m L）.The results showed that 11 kinds of extracts had different antioxidant capacity.Among them,50%alcohol extraction had the most antioxidant effect,followed by water extraction,50%alcohol extract had the most antioxidant effect,followed by water extract,while alcohol extract followed by water and alcohol extract did not show antioxidant effect,and the damage was aggravated.Under different evaluation methods,the antioxidant activity of different solvent extracts was different,but the 50%ethanol extract had the highest antioxidant activity.4.In the concentration range of 0.125～4 mg/m L,the extracts of Shiwei and Kiwifruit root have inhibitory effect on four kinds of tumor cells,which are Hep G2>Bel7402>A549>Hep2.The anti-tumor effect of Kiwifruit root is better than Shiwei,which shows a certain synergistic anti-tumor effect,and the 50%ethanol extract sample has the best effect.5.The results of RAW264.7 avirulence test showed that the samples of Shiwei-Kiwifruit extracted by ethanol had a certain degree of damage to the cells,and the other solvent extracts were basically avirulent at the concentration of 1 mg/ml,which could be used for further research.The results showed that the extracts of the other 28 groups showed different degrees of anti-inflammatory effect on LPS induced inflammatory injury except the drugs extracted with ethanol solvent.The anti-inflammatory effect of HE-2-1 was the best,and the scavenging ability of NO was the strongest（14.22±2.12）.The extract HE-2-1 was used to study the inhibition of ear swelling in rats.It was found that the ear swelling degree of mice in high-dose group（200 mg/kg）and middle dose group（150 mg/kg）was significantly alleviated,and the effect of high-dose group was significantly better than that of positive control group（Diclofenac sodium,15 mg/kg）.Therefore,it was determined that the extract of Shiwei Kiwifruit root in different proportions and different solvents could reduce the inflammatory reaction and exert the anti-inflammatory effect.The best ratio was 2:1,50%alcohol extraction.Conclusion:The results showed that 50%ethanol extract had high content of active components,which showed good antioxidant,anti-tumor and anti-inflammatory effects.Among them,HE-2-1（weight ratio 2:1,50%ethanol extraction）has significant pharmacological activity,which can provide reference for the compatible extraction of Shiwei and Kiwifruit root.