| Dryopteris crassirhizoma Nakai also known as dongbeiguanzhong,for Dryopteridaceae plants of Dryopteris crassirhizoma(Dryopteris crassirhizoma Nakai)the dried rhizome and petiole residues,bitter taste,slightly cold,to the stomach,gallbladder,a small drug,with sterilization,detoxification,insecticide poison temperature spots and other effects.It is mainly used in the treatment of influenza virus,fungus infection and other diseases.However,there are few studies on the inhibition of fungi and bacteria,especially in clinical common skin tinea bacteria.The preliminary studies in our laboratory showed that the phloroglucinol derivatives in Dryopteris fragrans had better antibacterial effect.Dryopteris crassirhizoma Nakai and Dryopteris fragrans are congeneric plants,rich in phloroglucinol derivatives.Therefore,this paper on the Dryopteris crassirhizoma Nakai phloroglucinol derivatives enrichment process was optimized,and then screen antibacterial activity of total phloroglucinol derivatives and fractions.The separation of chemical components from the effective fractions was studied,and the inhibition of fungal and bacterial compounds was studied.The main research contents were as follows:1: The enrichment of phloroglucinol derivatives.The extraction and purification technology of total phloroglucinol derivatives was screened by orthogonal method with the content of total phloroglucinol derivatives as the standard.2: The M38-A2 microdilution method was used to screen inhibition.Screening of antibacterial activity of extracts and fractions.The screening of Trichophyton rubrum,Microsporum canis,Gypsum microspore bacteria,Trichophyton mentagrophytes,4 species of fungi,and the antibacterial activity of Staphylococcus aureus Rosenbach,Staphylococcus epidermidis,P.aeruginosa,Escherichia coli,4 strains of bacteria,determination of minimum inhibitory concentration(MIC).3: The effective fractions were separated and purified by silica gel,gel column chromatography,semi preparative liquid phase,recrystallization,and so on.According to the physical and chemical properties,spectral data and color reaction of the monomer,the structure of compounds were determined.4: The M38-A2 microdilution method was used to screen inhibition.Separation of the active site,the 10 compounds of the phloroglucinol derivatives were obtained.The screening of Trichophyton rubrum,Microsporum canis,Gypsum microspore bacteria,Trichophyton mentagrophytes,4 species of fungi,and the antibacterial activity of Staphylococcus aureus Rosenbach,Staphylococcus epidermidis,P.aeruginosa,Escherichia coli,4 strains of bacteria,determination of minimum inhibitory concentration(MIC).The results were as follows:1: The best enrichment process: using the ratio of material to liquid 1:15,3 times,each time 24 h,extraction.The concentration of 1:8 was 2.5,and pH was acidified.2: The antibacterial activity of the total and each fraction: total extract>B1>D2> D1> F> C > B2> A,G,H invalid.3: Through the physical and chemical properties,color rendering and data analysis,we identified 10 compounds of phloroglucinol derivatives.Respectively: 1-(2,4,6-Thihydroxyphenyl)Butan-1-one(Y1),1-(2,4,6-Thihydroxy phenyl-3-methyl)Butan-1-one(Y2),1-(3-Acetyl-2,4,6-thihydroxy phenyl-5-methyl)Butan-1-one(Y3),1-(3-Acetyl-2,4,6-thihydroxy-phenyl)-1-Butanone(Y4),flavaspidic acid AA(Y7),flavaspidic acid PB(Y5),Norflavaspidic acid AB(Y6),filixic acid ABA(Y8),flavaspidic acid AB(Y9),filixic acid ABP.The compounds 1-(2,4,6-Thihydroxyphenyl)Butan-1-one,1-(2,4,6-Thihydroxy phenyl-3-methyl)Butan-1-one,1-(3-Acetyl-2,4,6-thihydroxy phenyl-5-methyl)Butan-1-one,1-(3-Acetyl-2,4,6-thihydroxy-phenyl)-1-Butanone for the first time from Dryopteris crassirhizoma Nakai.4: The M38-A2 method was used to test the antibacterial activity of 10 kinds of phloroglucinol derivatives.The order of inhibition activity of Trichophyton rubrum: Filixic acid ABA > Flavaspidic acid PB?Norflavaspidic acid AB.Others invalid.The order of inhibition activity of Trichophyton mentagrophytes: Filixic acid ABA > Norflavaspidic acid AB?Flavaspidic acid AA.Others invalid?The order of inhibition activity of Gypsum microspore bacteria: Filixic acid ABA > Flavaspidic acid AA > 1-(3-Acetyl-2,4,6-thihydroxy-phenyl)-1-Butanone,Others invalid?The order of inhibition activity of Microsporum canis: Filixic acid ABA > Norflavaspidic acid AB ?1-(3-Acetyl-2,4,6-thihydroxy phenyl-5-methyl)Butan-1-one> Flavaspidic acid AA> Flavaspidic acid PB> Flavaspidic acid AB.Others invalid?The order of inhibition activity of Staphylococcus aureus Rosenbach: Filixic acid ABA> Norflavaspidic acid AB ? Filixic acid ABP> Flavaspidic acid PB > 1-(3-Acetyl-2,4,6-thihydroxy phenyl-5-methyl)Butan-1-one?1-(2,4,6-Thihydroxy phenyl-3-methyl)Butan-1-one > Flavaspidic acid AB.Others invalid.The order of inhibition activity of Staphylococcus epidermidis: Filixic acid ABA> Filixic acid ABP >1-(3-Acetyl-2,4,6-thihydroxy phenyl-5-methyl)Butan-1-one?Norflavaspidic acid AB >1-(3-Acetyl-2,4,6-thihydroxy-phenyl)-1-Butanone ? Flavaspidic acid AA ? 1-(2,4,6-Thihydroxy phenyl-3-methyl)Butan-1-one.Others invalid.10 phloroglucinol derivatives had no effect on Escherichia coli and P.aeruginosa.Filixic acid ABA had better inhibitory effect on fungi.The inhibition of Trichophyton rubrum MIC was 40 ?g/mL.The inhibition of Trichophyton mentagrophytes MIC was 20 ?g/m L.The inhibition of Gypsum microspore bacteria MIC was 40 ?g/m L.The inhibition of Microsporum canis MIC was 10 ?g/m L.The effect of Filixic acid ABP and Filixic acid ABA on bacteria was better.Filixic acid ABP inhibition of Staphylococcus aureus Rosenbach MIC was 20 ?g/mL.The inhibition of Staphylococcus epidermidis MIC was 20 ?g/m L.Filixic acid ABA inhibited the MIC of Staphylococcus aureus Rosenbach was 2.5 ?g/m L,the inhibition of Staphylococcus epidermidis MIC was 2.5 ?g/m L.Positive drug cefoxitin inhibitied the MIC of Staphylococcus aureus Rosenbach was 4? g/m L,the inhibition of Staphylococcus epidermidis MIC was 16 ?g/mL,The effect of Filixic acid ABA on Staphylococcus aureus Rosenbach and Staphylococcus epidermidis was better than that of positive drug.The conclusions were as follows:To optimize the enrichment process of total phloroglucinol derivatives in Dryopteris crassirhizoma Nakai.10 phloroglucinol derivatives were isolated from the effective fractions,and 4 of them were isolated for the first time in Dryopteris crassirhizoma Nakai.Filixic acid ABA,a better inhibitor of fungi and bacteria,was screened out to lay the foundation for further development of new drugs. |
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