| Objective:In this study M.pubescens have been chosen as the research object for its good efficacy and weak studies on chemical composition and pharmacological activity,to trace and sift the active parts and their components of M.pubescens and expound the physical basis of their activity,whilst eastablishing feasible quality standards to ensure the effectiveness and the controllability of the herbal medicine.Methods:1 Studies on chemical components of M.pubescens10 kg of M.pubescens were extracted by 80%ethyl alcohol,using heating reflux extraction method and the extracted material was further extracted by petroleum ether,acetic ether and n-butyl alcohol,accordingly.As a result,120g of extractum,extracted by acetic ether,was obtained.Monomeric compounds were obtained through silicagel column,Sephadex LH-20 and the recrystallization purification process,and were identified on the basis of spectral analysis and physico-chemical properties.2 Pharmacodynamics studyUse dimethybenzene-induced mouse ear edema model and carrageenan-induced rat paw edema model to evaluate the anti-inflammation effect of the water extract and different portions extracted from the water extracts of M.pubescens.In vivo experiments,use dimethybenzene-induced mouse ear edema model to evaluate the anti-inflammation effect of Mussaenoside,while use the cell pattern with RAW264.7 cells activated by lipopolysaccharides(LPS)in vitro experiment.Analgesic activity of Mussaenoside has been observed by mice writhing,induced by acid and hot-plate method.Antibacterial effects of Mussaenoside.have been studied with 7 common bacteria.Bacteriostatic ring was observed by Disk diffusion and serial dilution method was adopted to determine the minimum inhibitory concentration(MIC).3 Initial studies on pharmacokinetics of Mussaenoside in rats.HPLC(High Performance Liquid Chromatography),with gardenoside as the internal standard,was established to determine the content of Mussaenoside in the blood plasma in rats.Furthermore,the pharmacokinetics of Mussaenoside in mice has been initially studied with this method.Compartment model fitting of average plasma concentration in mice-time data has been conducted and relevant parameters have been calculated after Mussaenoside is given to mice through disposable meridian injection,with the help of DAS 2.0 pharmacokinetics intelligence analysis software.4 Studies on quality control of M.pubescens.The study established the criteria of quality control of M.Pubescens,for it was not consummated in the former criterion which had only morphological and histological identification.The quality standard was incomplete which no TLC and content determination presented.Thin-layer chromatographic identification method was established,with Mussaenoside and scopoletin as reference substance.The methods of HPLC were established to determine the content of Mussaenoside and scopoletin in M.pubescens herbs.Determination method of total iridoid glycoside content in M.pubescens herbs has been established,with the adoption of UV-visible spectrophotometry.Quantitative analysis of total organic acid has been conducted using back potentiometric titration method,with salicylic acid as reference substance.HPLC fingerprint of M.pubescens herbs was established,with the chromatographic peak of Mussaenoside as reference peak.Results:1 Studies on chemical components of M.pubescensNine monomeric compounds were obtained through silicagel column,Sephadex LH-20 and the recrystallization purification process,of which 8 compounds were identified.One iridoid compound:Mussaenoside(1);3 triterpenes&sterol compounds:Ursolic acid(2);?-Sitosterol(3);aplysterol(4);1 coumarin compound:scopoletin(5);3 alcohol,acid and ester compounds:salicylic acid(6);2-hydroxy-5-(3-hydroxy-1-methoxy-1-oxopropan-2-yl)-2-methyl-cyclopentane carboxylic acid(7);Long chain fatty alcohol(8).2 Pharmacodynamics studyStudies on anti-inflammatory acti-vity of the water extract show that medium to high dosage of the water extract(16.8,33.6g/Kg)can obviously reduce the xylene-induced ear swelling in mice,whilst the results are insignificant with the small dosage(8.4g/Kg).The results are significant with medium and high dosage(12,24g/Kg)on reducing the swelling of carrageenan-induced hind paw edema in rats(Compared with the blank control group,P<0.05),whilst the results are insignificant with small dosages(6g/Kg).Results show that portions extracted through Ethyl acetate and 1-Butanol,as well as water-soluble portion can significantly reduce both the xylol-induced ear swelling in mice and the carrageenan-induced hind paw swelling in rats,however,the portion extracted through petroleum ether has insignificant effect.Observation of in vitro anti-inflammatory activity of Mussaenoside in the experiment of xylol-induced ear swelling in mice shows that the medium to high dosage of the Mussaenoside(0.10,0.15g/Kg)can significantly reduce the xylol-induced ear swelling in mice(Compared with the blank control group,p<0.05)and the high dosage has a better effect than Aspirin.Results show that Mussaenoside can obviously reduce the production and release of NO(RAW264.7),which is induced by LPS.In writhing test,the results suggest that each dosage of Mussaenoside can significantly inhibit the number of writhings and groups with high dosages have better effects than positive control Aspirin groups.In the hot-plate experiment,Mussaenoside can significantly delay thermal stimulus induced mice's reaction to pain,when 2 dosages have been increased.The Disk diffusion method test results indicate that,when concentration of Mussaenoside solution is 0.1g/mL.the inhibition zone diameter for staphylococcus aureus is 17.4±0.55 and 11.4±0.89,20.8±0.84,15.4±0.55mm for pseudomonas aeruginosa,Bacillus dysteriae,Aerobacter cloacae,Bacillus typhi,K.pneumoiae and E.coli,respectively.When the concentration is 0.2g/mL,the results are:18.0±0.71,19.2±0.45,26.4±0.55,20.4±0.55,12.6±0.55,22.4±0.55,18.6±0.55mm,respectively.The MIC test results suggest that Mussaenoside has antibacterial effect on the bacterium mentioned above and the MIC are 2.5,10,0.3125,1,25,1.25,10,0.625mg/mL,respectively.3 Initial studies on pharmacokinetics of Mussaenoside in rats.The experimental result shows that the plasma concentration,using 2-comparment model meridian injection calculation method,corresponds well to that of actually measured(weighting:1)and it proves superior to other models.The distribution half-life time(t1/2?)is 9.892min and the elimination half-life time(t1/2?)is 55.384min.The compartment transfer constant(K12)is 0.021min-1 from central compartment to peripheral compartment and it is 0.04mim-1 conversely.4 Studies on quality control of M.pubescensThin-layer chromatographic identification method was established,with Mussaenoside as reference substance.Extractions with 2 grams of her'b powder were conducted by using methanol ultrasonic extraction method and further extractions were done with ethyl acetate,when concentrated.This sample test solution was visually examined when it was heated to 105?,with ethyl acetate-methanol-glacial acetic acid(16:4:1)as developing agent and 10%sulphoacid ethanol solution as color-developing agent.These results show that both the test sample and the reference substance displayed the spots of the same color at the corresponding positions on each of the chromatography.Thin-layer chromatographic identification method was established,with scopoletin as reference substance.Extractions with 2 grams of herb powder were conducted by using methanol ultrasonic extraction method and further extractions were done with chloroform,when concentrated.This sample test solution was visually examined under the UV lamp(365nm)with petroleum ether-ethyl acetate(16:4:1)as developing agent.These results show that both the test sample and the reference substance displayed the fluorescent spots of the same color at the corresponding positions on each of the chromatography.Content determination of Mussaenoside:Contents of Mussaenoside ange from 0.9%-1.6%of the 10 batches of herbs from different origins and it ranges from 0.64%-1.67%for different months.Different organs have diffent contents,foliage>leaf>root>tender stem>medium stem>old stem>fruit,from high to low in order and the difference is great,from 0.99%to 8.99%.Determination of total iridoid glycoside content:Contents of total iridoid glycoside range from 5.29%to 8.97%for different origins and 4.67%to 8.92%for different months.Determination of content of total organic acid:Among the 10 batches of herbs from different origins,contents of the total organic acid range from 2.75%to 7.21%,and 2.10%to 8.65%for those from different months.Determination of content of scopoletin:Among the 10 batches of herbs from different origins,scopoletin content ranges from 0.00151%to 0.00450%,from 0.00141%to 0.00446%for those from different months and from 0.00176%to 0.00465%for different organs.Through analysis of the HPLC fingerprints of the 10 batches of M.pubescens herbs,11 peaks have been specified as common peaks,which constitutes the characteristics of the chromatographic fingerprint of M.pubescens Herbs.And,with Mussaenoside as the reference,most parameters,such as,relative retention time;relative peak area and the relative areas of the non-common peaks,of other chromatographic peaks,have been calculated.11 Peaks were selected as the common fingerprint peaks and the common fingerprint has been established.The relative standard deviations were less than 3%in the precision test,stability test and repeated test.And the similarity of 10 batches of samples were no less than 0.9 to the common fingerprint.Conclusion:1 Studies on chemical components of M.pubescens In our systematical study on the chemical constituents of M.pubescens,9 compounds were obtained from the portion extracted through Ethyl acetate of the 80%ethanol extract of this plant through various chromatographic techniques.8 of their structures were elucidated on the basis of physic-chemical property and spectroscopic analysis.This study will provide the reference for the further use of M.pubescens Among those compounds extracted,#7 might be one that is newly found.We can only deduce its 2-dimensional structure due to the minor quantity extracted,thus we are uncertain of its comformation.Compound#4,#5,#6,#7 are all first obtained through this extraction experiment.2 Pharmacodynamics studyStudies on anti-inflammatory activity of the water extract suggest that medium to high dosage of the water extract of M.pubescens can inhibit the acute inflammation.Studies on anti-inflammatory activity based on different portions extracted from the water extracts of M.Pubescens suggest that portions extracted both through Ethyl acetate and 1-Butanol,together with the water-soluble portion have inhibitory effects on acute inflammation.In vivo experiments,the results suggest that medium to high dosage of Mussaenoside have good effect on inhibition of acute inflammation.In vitro experiments,the results suggest that Mussaenoside has effect on relief of inflammation reaction.Both the writhing test and the hot-plate test can suggest that Mussaenoside has significant analgesic effect and high dosage has the same effect as Aspirin.The results of Disk diffusion and serial dilution method suggest that Mussaenoside solution has strong inhibitory effect on the 7 bacterium tested.This study demonstrated that the active portions of the anti-inflammatory in M.pubescens,confirmed Mussaenoside has significant anti-inflammatory,analgesic,antibacterial activity,which is the main pharmacodynamic basis for Mussaenda herbs,providing the theoretical basis for Mussaenda further development and utilization.3 Initial studies on pharmacokinetics of Mussaenoside in rats.In order to elucidate the absorption and pharmacokinetic profile of mussaenoside,a rapid,sensitive,and reliable analytical HPLC method was developed and fully validated to determine Mussaenoside in biological samples.The intra-day and inter-day precision(relative standard deviation)was generally good((10%)at low,medium and high concentrations.The over all recovery of the analytes was over 80%.These results indicate that the method was efficinet with a simple preparation procedure and a short running time for Mussaenoside.With its high selectivity,acceptable accuraey,precision and sensitivity,this validated HPLC method was successfully used to support the pharmacokinetics study of Mussaenoside.The assay was then used to determine the Pharmaeokinetie and absolute bioavailability of zingerone in rats.This test concerned with a study of the pharmacokinetics of Mussaenoside in rats,so as to support the metabolism and Pharmacokinetic study in human and to offer the reference for clinical efficacy and safety.4 Studies on quality control of M.pubescensTLC identification methods were established,with Mussaenoside and scopoletin as reference substance respectively.Both the test sample and the reference substance displayed the spots of the same color at the corresponding positions on each of the chromatography.Two methods are simple and special in operation,the results are stable with a good repeatality.These can be well applied to control the quality of M.Pubescens.The methods of content determination established in this study were accurate,reliable,good reproducibility,strong characteristic and can be used for the quality estimation about M.Pubescens and can provide the scientific basis of development of the quality standard and reasonable application.Establishment of this fingerprint has provided a certain basis for differentiation and quality evaluation of M.Pubescens.Evaluating the quality of M.pubescens with the method of HPLC fingerprint can greatly improve the level of quality evaluation.The method is reliable and can be helpful on the quality control of M.pubescens... |
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