| ObjectiveThrough the study of chemical composition,virtual screening,pharmacological activity,content,fingerprint of the flavonoids from Herba Abri,to dig out the new chemical composition,to screen the most active potential chemical composition by molecular docking technology and carry out the related activity test,so as to clarify the effective material basis of Herba Abri.To further establish the exclusive,stable,feasible quality evaluation techniques of the active compositions,and evaluate their quality.The results would provide the basis and new ideas and new methods for the improvement of medicinal value,quality assessment,quality optimization,development and utilization of the genuine medicine Herba Abri in Guangdong province.MethodsUsing solvent extraction technology with column chromatography for enrichment,separation and purification of the chemical compositions,their structures were identified by the determination of physicochemical properties and spectroscopy analysis of UV,MS,TLC,HPLC and NMR.The computer simulation software of molecular docking technology was applied to analyze the optimal active potential compositions by virtual screening of chemical constituents docked with related diseases active receptor.DPPH and phosphomolybdenum complex methods were applied to detect the antioxidant activity of Herba Abri flavonoids.The hypolipemic effect of flavonoids was detected in vitro by measuring the activity of pancreatic lipase.The hypolipemic and hepatoprotective effects of flavonoids were evaluated in vivo with hyperlipidemia mouse model.The inhibited effect of apigenin and its glycosides on B16 cell(melanoma cell of mouse)proliferation was detected by MTT method.The absorption and tissue distribution of Abrusamide A in mice was detected in vivo by oral gavage,and Abrusamide A and Abrusamide B were tested for their hepatoprotective activities on CCl1-induced injury of Human L-02 cells by MTT assay.HPLC was applied to establish a method for determination of Abrusamide A.UV was applied to explore the principle of different chromogenic agents on different configurations of flavonoids and establish the exclusive determination of apigenin and its glycosides by TEA-UV method.L9(34)orthogonal method was applied to optimize the extraction technology of total flavonoids,HPLC was applied to establish the external standard method(ESM)and quantitative analysis of multi-components by singlemarker(QAMS)for the determination of multiple flavonoids components.HPLC was applied to establish the fingerprint analysis and evaluation technology of Herba Abri flavonoids.ResultsTwelve chemical compounds were isolated and their structures were identified respectively,they were respectively ?-sitosterol(1),quebrachitol(3),proline(4),3,6-dimethyl-2,5-piperazinedione(5),abrine(6),hypaphorine(7),6-C-glycopyranosyl-8-C-glycopyranosyl apigenin(8),6-C-arabinosyl-8-C-glycopyranosyl apigenin(9),6-C-glycopyranosyl-8-C-arabinosyl apigenin(10),N-(4-hydroxycinnamoyl)tyrosine(11),Abrusamide B(12)and Abrusamide A(13).ADMET prediction and molecular docking results suggested that negative CDOCKER ENERGY(-KJ/mol)of Herba Abri chemical compounds in hypolipemic and hepatoprotective activities were Amide compounds>flavonoids>alkaloids and other types of compounds>saponins,the most active potential compounds were respectively Abrusamide B,Abrusamide A,V-(4-hydroxycinnamoyl)tyrosine,abrine,apigenin and luteolin and their glycosides,chalcone,protocatechuic acid,isolariciresinol,Emodin and Physcion.The negative CDOCKER ENERGY of Abrusamide B and Abrusamide A were higher than that of the positive drug adefovir dipivoxil,they could not combine with CYP2D6 and plasma protein which prompted to play pharmacodynamics with unbound state in bloodstream.The hypolipemic active potential of amides and flavonoids is better than that of emodin and physcion.Herba Abri flavonoids had certain antioxidant activity,the IC50 of DPPH eleminating rate was respectively 0.116 5(6-C-glycopyranosyl-8-C-glycopy-ranosyl apigenin),0.120 3(6-C-arabinosyl-8-C-glycopyranosyl apigenin),0.125 4(6-C-glycopyranosyl-8-C-arabinosyl apigenin),0.465 6(total flavono-ids),but all are weaker than Vc(0.009 1).The DPPH removal rate and the total antioxidant activity increased with the increasing of the concentration of flavonoids.Flavonoids can enhance the activity of lipase in certain concentration,and enhanced with the concentration increased.The result suggested a certain role in promoting activity of lipase in vitro.After giving mice the medicine by intragastric administration for 14 days,the results of hypolipemic and hepatoprotective test in vivo showed that TC,TG,LDL-C and liver index in model group were significantly higher than that in normal group,with HDL-C significantly lower than that in normal group.Compared with model group,above each index in each group could be significantly improved.Especially in TG,the reduction effect of total flavonoids was better than that of Xuezhikang group.The liver pathological observation showed that model group had mild steatosis with partial fat droplet accumulation phenomenon.Compared with model group,each group had some improved effect on fatty liver with the best effect of high dose of ZHT group..The inhibited results of Herba Abri flavonoids on B16 cell(melanoma cell of mouse)proliferation showed that blank control group and DMSO group had no inhibitory effect;the inhibition rate of total flavonoids was 26%in 200? mol/L.The inhibition rates of apigenin were about 17%in 12.5 ? mol/L,about 47%in 25 ? mol/L,about 90%in 50 ? mol/L.The inhibitory rate of apigenin on B16 increased significantly with increasing of concentration,it could speculate that the IC50 of apigenin B16 cell was about 28 ?mol/L according to inhibited concentration curve.Apigenin had greater inhibitory activities in 25 ? mol/L and 50 ? mol/L than total flavonoids(P<0.01),and it showed obvious differences in B16 cell inhibitory activity between apigenin and its two glycosidesAbrusamide A went into the blood and various tissues at the prototype state after oral gavage,and the drug concentration was highest in the blood,followed by liver,pancreas and kidney.Abrusamide A and Abrusamide B had significant promote proliferative effects on L-02 cells at concentrations of 50,100,200 and 400 ?g/mL(P<0.01),but had weak inhibitory effects with the survival rate of about 86%at concentrations of 800 ?g/mL.It showed very weak cytotoxicity of Abrusamide A and Abrusamide B with IC50 respectively at 1078 ?g/mL and 1068 ?g/mL.In the results of hepatoprotective test by CCl4-injury,the survival rates of Abrusamide A and Abrusamide B at concentrations of 500,250 and 125 ?g/mL were significantly higher than that of CCl4-injury group(P<0.01,P<0.01,P<0.05),and at high concentrations were also higher than that of bifendatatum.All above results showed that Abrusamide A and Abrusamide B had significant hepatoprotective activities.The determination results of Abrusamide A showed that the content range of Abrusamide A was 0.038%-0.146%in Abrus cantoniensis Hance(AC),and its content range was 0.08-0.12%in A.mollis Hance(AM).The results of the relationship study between flavonoids structure and chromogenic method displayed that different chromogenic method had significant influence on the absorption spectrum of different flavonoid structure.The changes of UV absorption spectra had a good correlation with the position and number of 4 carbonyl,hydroxyl,hydroxy substituted groups of flavonoids structure,but has nothing to do with 0-diphenols hydroxy.The TEA method could make the apigenin flavonoids to produce characteristic absorption at 400 nm.The presence and concentration of water had significant influence on TEA chromogenic method of apigenin flavonoids.When the water reached a certain concentration,its influence would achieve the best balance condition.The content of total apigenin flavonoids of Herba Abri could be determined with TEA-UV method.The solvent of sample solution preparation was 50%ethanol for extraction and constant volume.The solvent of control solution preparation was 50%ethanol for apigenin and its glycosides or 95%ethanol for rutin.The value for evaluation was absorption difference between Acolor and Acolor front at 400 nm.6-C-glycopyranosyl-8-C-glycopyranosyl apigenin,apigenin and rutin can all be used for the evaluation of the content of total flavonoids in Herba Abri with fixed relationship among the three.The content deviation of his conversion between apigenin and rutin was too large at the low absorption difference of Acolor-Acolor front.When the absorption difference was more than 0.50,the content value was close among three conversions.The determination results showed that the size of total flavonoids content in different parts of Herba Abri was leaf>stem>root.The content of total flavonoids in leaf was in-5%,up to 4.8%.The content of total flavonoids in stem was 0.1-1%.The content of total flavonoids in root was 0.01-0.1%.The content of total flavonoids in different samples of Herba Abri was different which might be affected by different place of origin or different content of medicinal parts.Those influenceing factors of ultrasonic extraction arranged in order were as follows:ultrasonic extracting time>ethanol concentration>extracting times>solid-liquid ratio,and ultrasonic extracting time Ultrasonic time was the significant influence factors.A2B1C3D2 was optimum ultrasonic extraction technology,which was ultrasonic extracted 2 times with 20 times the amount of 50%ethanol by 45 min each times.The results of determination of 6-C-glycopyranosyl-8-C-glycopyranosyl apigenin(1),6-C-arabinosyl-8-C-glycopyranosyl apigenin(2),6-C-glycopyr-anosyl-8-C-arabinosyl apigenin(3)by ESM and QAMS showed that the content error of leaves could be controlled within 5%,that of stems could mostly be controlled within 8%with some more than 8%,that of roots was biggest.The content error of extraction process could also be controlled within 7%.In AC,the content.range of 1 in stems was 0.0050-0.095%with 0-0.083%of 2 and 0.010-0.14%of 3,the content range of 1 in roots was 0.002 0-0.006 3%with 0.006 0-0.013%of 2 and 0.003 9-0.014%of 3,the content range of 1 in leaves was 0.013-0.54%with 0.031-0.85%of 2 and 0.019-0.88%of 3.In AM,the content range of 1 in stems was 0.006 5-0.033%with 0.015-0.033%of 2 and 0.014-0.033%of 3,the content range of 1 in leaves was0.34-1.95%with 0.59-1.31%of 2 and 0.51-1.22%of 3.HPLC fingerprints evaluation results of Herba Abri flavonoids showed that there were significant differences in flavonoids chromatographic peaks of different medicinal parts(root,stem and leaf).There were strong and stable characteristic chromatographic peaks and more information with a smooth line in the HPLC fingerprints of leaves.There were strong and stable characteristic chromatographic peaks and more information with a smooth baseline in the HPLC fingerprints of leaves.The HPLC fingerprints of stems were similar to the leaves with lower peaks and uneven baseline.This suggested the leaves were the most suitable parts for analysis of HPLC fingerprints.Through the analysi s and comparison of 15 batches of HPLC fingerprints of Herba Abri,ten strong characteristic common peaks of flavonoids were signed with their peaks area more than 95%of all peaks area,and the sum of all known peaks area was more than 45%of total peak area of 10 flavonoids.NO.1 peak(6-C-glycopyranosyl-8-C-glycopyranosyl apigenin)was denoted as the reference peak(S).All common peaks had good stabile retention time with different peak area.Through similarity evaluation and cluster analysis by computer software,15 batches of HPLC fingerprints could be divided into three types:The first type was AC with nearby place of origin,their HPLC fingerprints similarity was relatively close with above 0.93 of correlation coefficient.The second type was also AC with relatively far place of origin and lower similarity with correlation coefficient between 0.30 and 0.88.The third category was AM close to the first category with slightly lower similarity with correlation coefficient between 0.83 and 0.98.Through application of HPLC fingerprints method by 20 batches of Herba Abri pieces,HPLC fingerprint similarity was influenced by place of origin,the closer the place of origin,the higher the similarity.Conclusion12 compounds were identified.Proline,3,6-dimethyl-2,5-piperazine-dione and N-(4-hydroxycinnamoyl)tyrosine were isolated for the first time;Abrusamide B and Abrusamide A were new compounds.ADMET and molecular docking prediction showed that amide compounds and flavonoids might be the main hypolipemic and hepatoprotective bioactive components in Herba Abri.Active experimental studies had shown that the total flavonoids of Herba Abri had some antioxidant activity and promoting lipase activity in vitro,had hypolipemic and hepatoprotective effect in vivo on hyperlipidemia mice.The results suggested that flavonoids could be the effective parts.Total flavonoids also had the inhibitory effect on B16cells with structure-activity relationships between apigenin and its C-glycosides.The inhibitory effect of apigenin was significantly stronger than its C-glycosides.The above experimental results are consistent with the results of computer molecular docking simulation.The results of cell activity and ADMET prediction indicated the prototype of Abrusamide A could produce therapeutic effect in the body.Abrusamide A and Abrusamide B displayed significant promote effects on the proliferation of L-02 cells with low cytotoxicity and had significant hepatoprotective effects on CCl4-induced injury of L-02 cells.The results suggested that amide compounds could be the new hepatoprotective effective parts of Herba Abri and had great potential drug development.The HPLC method has been established to determine the content of Abrusamide A in the leaves of Herba Abri.The method is stable,accurate,and feasible.Different chromogenic methods have correlation with the absorption spectrum of different flavonoid structures.The TEA-UV method is exclusive,stability and suitable for the determination of apigenin flavonoids of Herba Abri.TEA and water are the key factor to affect color characteristic absorption wavelength of apigenin component.Apigenin glycosides,apigenin or rutin can be used as an index for the determination of total flavonoids in Herba Abri by TEA-UV.The evaluation content with an index of apigenin glycosides is good,accurate and true.The evaluation result with an index of apigenin can only reflect the aglycone content.The index of rutin can be used as preliminary indirect evaluation.The optimized extraction process of total flavonoids was stable and available,convenient,and suitable for extraction of total flavonoids in Herba Abri.QAMS is stable and feasible with good correction factors.The error ofmeasured value is in the reasonable scope between ESM and QAMS.ESM and QAMS are also suitable for determination and quality evaluation of flavonoids components in Herba Abri.The evaluation result of total flavonoids by HPLC is consistent with that by TEA-UV.Medicinal parts and place of origin have a significant effect on the content of total flavonoids and 3 flavonoid glycosides in AC and AM.The content of flavonoid glycoside in leaves is highest,second in stems,lowest in roots.The flavonoid content of AM was slightly higher than that of AC.The results suggest that it is preferred for Herba Abri medicine with complete root,stem and leaf.The place of origin and medicinal parts have a significant effect on HPLC fingerprints of Herba Abr,the closer the place of origin,the more similar,the more likely HPLC fingerprint clustered into the same category.The HPLC fingerprints of leaves of Herba Abr have good similarity with most correlation coefficent>0.90.The HPLC fingerprints of Herba Abr pieces have similarity poor,with most correlation coefficent<0.90.The results suggest that the leaves of Herba Abr are the best parts for analysis of HPLC fingerprints.The HPLC fingerprint method is accurate,stable,feasible and suitable for analysis and evaluation for Herba Abr flavonoids.Herba Abr is Geoherbs in Guangdong province with new hepatoprotective active site in the leaves.We should strengthen its development and utilization to improve its medicinal value and technology content. |
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