| OBJECTIVE: Herpes simplex virus(HSV)often has long-term latency and recurrent attacks after invading in human body,causing a variety of diseases and serious complications.Clinical treatment drugs are mainly nucleoside analogs such as acyclovir(ACV).For severe and frequent relapse cases,large doses are required for long-term administration,which is prone to drug resistance.Our previous studies found that the crude extract of Prunella vulgaris was not only effective to HSV standard strain,but also had significant activities to ACV resistant virus strain.However,its active principle is still unclear.Therefore,this dissertation conducts a study on the chemical characterization of the active principles from Prunella vulgaris against HSV,and carried out the in‐vitro and in‐vivo safety and efficacy evaluation.METHODS:(1)Different solvents were used to prepare Prunella vulgaris extract;the in vitro CCK-8 colorimetric method was used to detect the anti-HSV activity of Prunella vulgaris extract with different solvents;Rat,guinea pig and rabbit models were used to carry out in vivo studies on acute toxicity,oral and vaginal mucosal irritation,skin irritation,and skin allergies of the water extract of Prunella vulgaris.(2)Different concentrations of ethanol were used to prepare gradient ethanol precipitation samples of Prunella vulgaris water extract;Identification reaction,UPLCDAD-QTOF-MS/MS,HPGPC were used to chemical characterized and analyzed the difference from each extract and grade ethanol precipitation parts;CCK-8 colorimetry was used to test the anti-HSV activities of each gradient ethanol precipitation sample,and the 30% ethanol precipitation named PVE30 was been found with the best antiHSV activities among the gradient ethanol precipitation samples.Chemical characterization of PVE30 was carried out by analytical method such as identification reaction,HPLC,UV,automatic amino acid analyzer.The CCK-8 colorimetric method was further used to detect the inhibition effect of PVE30 on ACV resistant virus strains;at the same time,guinea pig skin infection model was established to studied the antiHSV-1 effect of PVE30 in vivo;mouse vaginal infection model was established to studied the anti-HSV-2 effect of PVE30 in vivo.(3)PVG,the most active part from PVE30,was obtained by several separation methods such as column chromatography,ion complexation,savage,trichloroacetic acid precipitation and salting out,combined with the anti-HSV activities assay.Chemical characterization of PVG was performed by observed its physical and chemical properties observation;identified the major components by identification reaction andIR and UV spectrometry;analyzed the molecular weight range of PVG by HPGPC;the composition and content of monosaccharides in PVG were determined by complete acid hydrolysis and pre-column derivatization;the types and content of amino acids in PVG was determined by automatic amino acid analyser;the type and unit of phenolic components were analyzed by the ion fragments on UPLC-DAD-QTOF-MS/MS after mild acid hydrolysis.Furthermore,the inhibitory effect of PVG on HSV standard strains and ACV resistant virus strains was investigated by the CCK-8 colorimetric method.RESULTS:(1)The in vitro anti-HSV activities assay showed that the water extract of Prunella vulgaris had better anti-HSV1 and HSV-2 activities,with the EC50 of 83.60±2.96 μg/m L and 85.46±1.38 μg/m L respectively,and the selectivity index SI was greater than 5.98 and 5.85,respectively.However,90% and 70% ethanol extract had no significant anti-HSV activity.Safety evaluation results showed that the water extract of Prunella vulgaris had no acute toxicity,no obvious irritation and sensitization to oral mucosa and vaginal mucosa.(2)The water extract of Prunella vulgaris was segmented by graded ethanol precipitation.Chemical characterization and comparative analysis were carried out with the water extract,ethanol extract and each graded ethanol precipitations of Prunella vulgaris.The results showed that the ethanol extract and the ethanol precipitation supernatant of water extract is mostly composed by small molecules with the molecular weight less than 2.0 k Da.Meanwhile,the ethanol precipitation parts were mostly composed of macromolecule components.It was found that there was no linear relationship between the molecular weight of each alcohol precipitation part and ethanol concentration.The CCK-8 colorimetry showed that the anti-HSV activities of PVE30 were better than other graded ethanol precipitations.The EC50 of PVE30 against HSV-1 and HSV-2 were 45.62±3.37 μg/m L and 57.14±2.37 μg/m L,respectively;the selectivity index SI was greater than 10.96 and 8.75,respectively.The anti-HSV activity of PVE30 was about twice stronger as that of the water extract.Chemical characterization of PVE30 showed that it mainly contained carbohydrates,proteins and polyphenols.Combined with the above results and according to the UVspectrophotometric method of 2020 edition of the Chinese Pharmacopoeia,the method for determining total carbohydrates content,protein content and polyphenols content were established.The results showed that PVE30 contains 37.23±1.19% of total carbohydrates,14.36±1.96% of proteins and 17.94±0.68% of polyphenols.PVE30 was subjected to acid hydrolysis and derivatization,and its monosaccharide composition was determined by HPLC.The molar percentage of PVE30 was calculated as follows: galacturonic acid(Gal UA): galactose(Gal): glucose(Glc): arabinose(Ara): xylose(Xyl): Rhamnose(Rha): Mannose(Man): Ribose(Rib): Glucuronic acid(Glc UA): Fucose(Fuc)= 57.68:9.79:7.03:5.66:5.49:5.07:4.28: 3.03:1.02:0.94,in which galacturonic acid content was the highest,up to 208.10 mg/g.The protein component in PVE30 was measured by the automatic amino acid analyzer.The results showed PVE30 was consisted of more than 15 common amino acids,of which the acidic amino acid like glutamine and aspartic acid had higher content among others,which was 7.70 mg/g and 3.20 mg/g,respectively.In vitro anti-drug resistance test results showed that PVE30 has stable and good inhibitory effects on drug-resistant strains HSV-1/Blue and clinically drug-resistant strains HSV-1/106 and HSV-1/153,with EC50 of 54.54±2.05 μg/ m L,53.86±4.66 μg/m L and 39.39±0.63 μg/m L.In vivo experiments also show that PVE30 can significantly inhibit the skin lesions of guinea pigs caused by HSV-1infactions,and it was dose-dependent.The inhibitory activity of the high and medium dose groups had better efficacy than that of the ACV group.For mice vaginal infections caused by HSV-2,the survival rate of mice in the PVE30 intervention group was higher than that of the virus control group.The onset and death time were delayed by 1-2 days,and the average survival days of the animals along with the virus infection rate were reduced by 25%.The above results showed that PVE30 can not only improve the lesions of HSV-1 infected on guinea pigs’ back,but can also reduce the symptoms of mice vaginal infection caused by HSV-2.In other words,PVE30 can relieve the lesions cased by HSV-1 and HSV-2 infactions,and can significantly reduce the infection rates and mortalities of the animals,extend the life span of the HSV infected animals.(3)Separation methods such as column chromatography,ion complexation,savage,trichloroacetic acid precipitation,and salting-out were used to further search for the anti-HSV active principles from PVE30.The samples were prepared by each separation route and the anti-HSV activities were tested in vitro.It was found that and the antiHSV activities of PVG was improved,which was obtained by salting out method.Chemical characterization of PVG revealed that PVG was a water-soluble macromolecular substance with molecular weight of 41.69 k Da for Mw and 2.40 k Da for Mn,and contained 50.37 ± 1.84% total sugar,19.29 ± 3.03% protein,and 21.38 ± 0.23% polyphenols.For the carbohydrate components in PVG,the composition and content of each monosaccharide measured by complete acid hydrolysis and pre-column derivatization were as follows: galacturonic acid(Gal UA)100.25 mg/g,xylose(Xyl)90.83 mg/g,arabinose(Ara)66.71 mg/g,galactose(Gal)64.11 mg/g,glucose(Glc)44.82 mg/g,mannose(human),27.71 mg/g,rhamnose(Rha)17.01 mg/g,fucose(Fuc)8.24 mg/g,glucuronic acid(Glc UA)5.74 mg/g,ribose(Rib)4.73 mg/g,converted into a molar ratio that is Xyl: Gal UA: Ara: Gal : Glc: Man: Rha: Fuc: Rib: Glc UA = 23.83: 20.34: 17.5: 14.01: 9.8: 6.06: 4.08: 1.98: 1.24: 1.16.For the proteins in PVG,it was proved that the free state protein was not existed through savage method,after the acid hydrolysates of PVG,it was measured by an automatic amino acid anlyzer.Results showed the protein from PVG is composed of more than 15 common amino acids.Among them,the acidic amino acids like glutamic acid and aspartic acid have a higher content,which are 3.90 mg/g and 2.50 mg/g respectively.For the polyphenols in PVG,extracted by ethyl acetate after mild acid hydrolysis,analyzed the fraction of ethyl acetate extraction by UPLC-DAD-QTOF-MS/MS,which identified the polyphenols in PVG was a type of dibenzylbutyrolactone-type lignans polymers.The in vitro anti-HSV test showed PVG’s EC50 were 23.25±2.12 μg/m L and 30.83±0.49 μg/m L for HSV-1 and HSV-2 standard virus strains respectively,and its SI was greater than 21.51 and12.88,respectively.Compared with PVE30,the anti-HSV activities of PVG was doubled;compared with water extract of Prunella vulgaris the anti-HSV activities was increased by about three times.At the same time,for the resistant strain HSV-1/Blue,the EC50 of ACV was 38.74±1.16 μg/m L,while the EC50 of PVG was 25.58±1.93 μg/m L,and its anti-HSV activities were better than ACV;for clinically resistant strain HSV-1/153,the EC50 of ACV was 32.99±.032 μg/m L,while the EC50 of PVG was 25.58±1.93 μg/m L,and the anti-HSV activities were also better than ACV.And for drug-resistant virus strains,the ACV concentration needs to be increased by about 300 times to exert an inhibitory effect equivalent to that of the anti-HSV standard strain,while PVG has the same inhibitory effect on the HSV standard virus strain and the drug-resistant virus strain.The above results indicate that PVG not only had a good inhibitory effect on the standard HSV strain,but also had a significant anti-HSV activity against the ACVresistant HSV strain,and the inhibitory effect on the standard strain and the resistant strain is equivalent.CONCLUSION:(1)The water extract of Prunella vulgaris had good anti-HSV activities,had no obvious irritation and allergic reaction to the oral cavity,vaginal mucosa,skin,etc.,and had good safety.(2)The anti-HSV active principles of Prunella vulgaris were mostly concentrated in the ethanol precipitations parts,the 30% ethanol precipitations part named PVE30 had the best activities of all;while the ethanol extract and the ethanol precipitation supernatants of water extract were mostly consisted of components whose molecular weight were lesser than 2.0 k Da,which included the rosmarinic acid,which is the index component of Prunella vulgaris in the Chinese Pharmacopoeia.But this kind of small molecule substance is not the main active principles of Prunella vulgaris against HSV.Chemical characterized was performed on PVE30,which has the best anti-HSV activities among other graded ethanol precipitations.Content determination methods conforming to the Pharmacopoeia was established.It was determined that PVE30 contained 37.23±1.19% of carbohydrates,14.36±1.96% of proteins,and 17.94±0.68% of polyphenols.Among them,uronic acid and acidic amino acids had relatively higher contents.The study on the anti-HSV activities of PVE30 in vivo and in vitro tests found that PVE30’s in vitro antiHSV activities was doubled compared with the water extract,and it also had similar inhibitory effect on ACV-resistant strains and standard strains.At the same time,in vivo experiments have shown that PVE30 had a good effect both on skin and genital herpes caused by HSV-1 and HSV-2 infections.(3)The PVG fractions separated from PVE30 by salting-out method had an anti-HSV activities twice as much as that of PVE30,and four times as much as that of the water extract,and it also had a significant inhibitory effect on ACV-resistent virus strains.Chemical characterization showed PVG was defined as a water-soluble substance with a relatively large molecular weight as 41.69 k Da for Mw and 2.40 k Da for Mn,which contained components of 50.37±1.84% total carbohydrates,19.29±3.03% proteins and 21.38±0.23% polyphenols.The proteins components of PVG does not exist in the free state,and there are at least 15 amino acids that make up the proteins,among them the acidic amino acids like glutamic acid and aspartic acid have a higher content.Xylose(Xyl)and galacturonic acid(Gal UA)had higher contents compared with other monosaccharides in PVG.The composition of polyphenols in PVG were mainly a kind of polymer of dibenzylbutyrolactone-type lignans.The above research provides new ideas and scientific basis for correcting the active principles of Prunella vulgaris against HSV... |
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