Study On The Biological Activity Of Phoebe Zhennan Leaves And Extraction And Identification Of Their Active Component

Phoebe zhennan S.K.Lee&F.N.Wei is a plant of Lauraceae.It has certain medical value.As early as in the traditional Chinese pharmaceutical books’syndrome herbs’,’Taiping Shenghui Fang’,’Puji Fang’,etc.,the application of P.zhennan in infectious diseases,medical diseases and dermatological diseases was clearly recorded.As a Chinese specialty tree species,P.zhennan is mainly distributed in South China,Hubei,Jiangxi,Zhejiang,Guizhou and other places.Its wood material is very good and has a special aroma.It was a royal imperial material in the Ming and Qing Dynasties.P.zhennan has been listed as a national second-class protected plant in China because of its serious deforestation and slow growth.Although its wood development and utilization are limited,its potential utilization value needs to be developed urgently because of its short-term renewable leaves.In order to further study the biological activity of P.zhennan leaves and fully develop its value,this study first determined the biological activity of the ethanol extract of P.zhennan leaves by in vitro antioxidant test and in vitro bacteriostatic test.Secondly,the extraction process of ultrasonic-assisted ethanol extraction of active components from P.zhennan leaves was optimized by single factor test combined with response surface method.Finally,the active components of P.zhennan leaves in five different polar solvents were analyzed by headspace-solid phase microextraction and gas chromatography-mass spectrometry（HS-SPME-GC-MS）.The main results are as follows:The first part is the antioxidant experiment:the antioxidant activity of the ethanol extract of P.zhennan leaves was determined by 1,1-diphenyl-2-picrylhydrazyl（DPPH）free radical scavenging test,3-oxo-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxygen（PTIO）free radical scavenging test,iron ion reduction ability（FRAP）and other evaluation methods.The results showed that the scavenging rate of DPPH was 94.67%at the concentration of 2 mg/m L,and the EC₅₀value was 0.674 mg/m L.The EC₅₀value of PTIO free radical was 5.405 mg/m L,the maximum scavenging rate was（47.79±0.04）%,and the Trolox equivalent of scavenging ability was 0.33 mg TE/m L.When the concentration was 0.20 mg/m L,the metal ion reduction ability reached 84.80%,and the Trolox equivalent of iron ion reduction ability was 0.03 mg TE/m L.The second part is in vitro antibacterial experiment.It was found that the ethanol extract of P.zhennan leaves had antibacterial activity against the tested strains of Myxoderma compacta,Phanerochaete chrysosporium,Chaetomium globosum,Arthrobotrys aculeatus,Cucurbitaceae races,Alternaria tenuissima.The EC₅₀values were 1.348 mg/m L,2.74 mg/m L,1.798 mg/m L,2.316 mg/m L,5.643 mg/m L and 1.969 mg/m L,respectively.The third part is the extraction process optimization experiment:the leaves of P.zhennan were crushed and sieved,and the ethanol extract was extracted by ultrasonic assisted ethanol solution.The effects of liquid-solid ratio,ultrasonic time,ethanol concentration and ultrasonic power on the extraction rate of ethanol extract from P.zhennan leaves were investigated.The optimal level interval of four single factors was screened by single factor test.The mathematical model was established by Box-Behnken center combination principle,and the optimal extraction process was optimized by response surface method.The results showed that under the conditions of liquid-solid ratio 9.144,ultrasonic time 31.78min,ethanol concentration 78.936%and ultrasonic power 164.552W,the highest extraction rate was 11.21%.Considering the accuracy and cost of the actual operation,the optimum conditions were determined as follows:liquid-solid ratio 9.1,ultrasonic time 31.8min,ethanol concentration 78.9%,ultrasonic power 160W.The extraction rate was（11.18±0.11）%,and the relative error with the analysis results was less than5%.The results showed that the response surface method could better optimize the extraction process of ethanol extract from P.zhennan leaves.The results of this study had positive guiding significance for the subsequent extraction process of P.zhennan leaves.The fourth part is the analysis of the active components of P.zhennan leaves:In order to explore the main active components of P.zhennan leaves,the leaves were extracted by several commonly used solvents,and then the active components of P.zhennan leaves were analyzed and identified by HS-SPME-GC-MS analysis technology.The main results are as follows:（1）A total of 17 common compounds（15 terpenes,1 alkane,1 aromatic）and 185 non-common compounds were analyzed from methanol,anhydrous ethanol,ethyl acetate,n-hexane and petroleum ether extracts.The components with the highest relative content in the five solvent extracts wereα-Copaene,which were methanol（13.81%）,anhydrous ethanol（17.05%）,ethyl acetate（9.84%）,n-hexane（14.56%）,and petroleum ether（10.51%）.（2）In this paper,the biological activity of the ethanol extract of P.zhennan leaves should be the result of the combination of compounds with relative content of more than 1%,such asα-Copaene,gamma-muurolene,d-Cadinene and trans-Caryophyllene,and compounds with relative content of less than 1%,such as Linalool,alpha-Curcumene and（-）-.β.-Bourbonene.（3）The results of component analysis showed that the leaves of P.zhennan had potential medicinal value,and many of its active components were also widely found in many other medicinal plants.In the future,the biological activities of the other four solvent extracts could be explored in order to reveal the effective mechanism of its active components and expand the application field of P.zhennan leaves.This study proved that the ethanol extract of P.zhennan leaves has strong antioxidant activity and antibacterial activity.After optimizing the process of ethanol extract of P.zhennan leaves,a higher extraction rate can be obtained,and the different polar solvents of P.zhennan leaves were preliminarily explored.The extract contains a large number of the same and different active substances,which lays a scientific theoretical foundation for the development and utilization of P.leaves.