| Objective:Elsholtzia rugulosa Hemsl.,a traditional medicine of the Yi ethnic group,has the effects of clearing heat and detoxifying,dissipating food and resolving accumulation,and hemostasis and analgesia.The preliminary research of the research group found that the flavonoid content in the ethyl acetate extracted fraction(EAF)of the crude extract was 184.91±3.86 mg/g,which showed significant α-glucosidase inhibitory activity,stronger than that of the positive drug acarbose.In addition,it had antioxidant,and acetylcholinesterase and butyrylcholinesterase inhibitory activities.In order to further improve the content of total flavonoid of E.rugulosa,this study optimized the extraction process of total flavonoid in crude extract by using ultrasound assisted extraction method,enriched them by using macroporous adsorption resin,and studied the chemical composition of the obtained total flavonoid fraction,to provide a theoretical basis for the in-depth development of E.rugulosa.Methods:1.The effects of ethanol volume fraction,solid-liquid ratio,extraction time,and ultrasound power on the extraction rate of total flavonoid of E.rugulosa were investigated using ultrasound assisted extraction method through single factor and Plackett Burma experiments.Furthermore,the Box Behnken design scheme in response surface optimization was used to explore the relationship between various factors,in order to predict the optimal extraction conditions for the total flavonoid in E.rugulosa and experimental verification was conducted.2.The extract of E.rugulosa obtained under the optimal extraction process conditions was separated using AB-8,HPD600,D101,and ADS-17 macroporous adsorption resins,and was eluted with different proportions of ethanol and water to obtain different elution parts.The total flavonoid content of the extract was determined to select the most suitable macroporous adsorption resin for enriching the flavonoid of E.rugulosa.3.Various enzyme inhibition and free radical scavenging activities were measured on different parts separated by D101 macroporous adsorption resin,and Pearson correlation analysis was used to analyze the correlation between total flavonoid content and biological activities.4.The chemical components of 50%ethanol elution site(D50 site)of the D101 macroporous adsorption resin were separated by modern separation technology.The structures of isolated compounds were identified through nuclear magnetic resonance,mass spectrometry data and comparison with those in literature.Results:1.The single factor experimental results indicated that the extraction rate of total flavonoid varied with the fluctuation of factor levels under different extraction conditions.Through the Plackett Burma experimental design,it was found that the four extraction factors had significant effects on the extraction rate of total flavonoid of E.rugulosa,with the order of significance from highest to lowest being:ethanol volume fraction>material liquid ratio>extraction time>ultrasound power.Further response surface analysis and experimental verification were conducted to obtain the optimal extraction process conditions:ethanol volume fraction 51%,ultrasound power 400 W,solid-liquid ratio 1:35,extraction time 57 minutes.The extraction rate of total flavonoid was 11.81 ± 0.06%.According to the optimal extraction process,the total flavonoid content of the extract(RSM)obtained was 308.52±5.23 mg/g.2.The content determination results showed that the flavonoid content increased to varying degrees after enrichment with macroporous adsorption resin,compared with the total flavonoid content of RSM.Among them,the 50%elution site of D101 macroporous adsorption resin had the highest total flavonoid content,which was 436.86±9.59 mg/g.3.The α-glucosidase inhibitory activities of water,20%,50%,and 70%ethanol elution sites of D101 macroporous adsorption resin were stronger than that of the positive control drug acarbose.Among them,the α-glucosidase inhibitory activity of D50 part was the strongest,with an IC50 value of 3.17 ±5.31 μg/mL,which is lower than the IC50 value of the ethyl acetate site(7.05±0.63 μg/mL).All extraction sites did not have α-amylase inhibitory activities.The cholinesterase inhibitory activity of D50 site was higher than that of ethyl acetate site,and the DPPH free radical scavenging activity of this site was the strongest.The IC50 value was only 5.42 ± 0.06 μg/mL,which was equivalent to the positive control drug Vc(IC50 value was 3.73± 1.41 μg/mL).Pearson correlation analysis showed that the total flavonoid content was negatively correlated to the IC50 values of α-glucosidase inhibitory activity,cholinesterase inhibitory activity and free radical scavenging activity.4.Nine compounds were isolated from the D50 part,namely:Stigmasterol(1),4’,5dihydroxy-7-methoxyflavone(2),luteolin(3),tilianin(4),(-)-syringaresinol-4-O-β-Dglucopyranoside(5),methyl rosmarinate(6),β-sitosterol(7),betulin(8),and obtusalin(9).Among them,betulinol and obtusalin are the first pentacyclic triterpenoids isolated from E.rugulosa.Conclusion:This study optimized the extraction process and enriched with macroporous adsorption resin to obtain the total flavonoid fraction D50,which had a higher total flavonoid content than the ethyl acetate fraction obtained by the extraction method.The inhibitory activities of αglucosidase and cholinesterase were also improved.Pearson correlation analysis showed that the total flavonoid content was positively correlated to the α-glucosidase inhibitory activity,cholinesterase inhibitory activity and free radical scavenging activity.D50 part contained various components such as flavonoids,terpenoids,steroids,and phenylpropanoids.This study provides a theoretical basis for the development of new drugs based on the effective site of E.rugulosa. |
