Optimization Of Extraction Process And Biological Activity Evaluation Of Mulberry Leaves And Mulberry Branches Extracts By Response Surface Methodology

The resources of mulberry leaves and branches are abundant in China,and their traditional development and utilization methods are mainly to use mulberry leaves for feed in the silkworm industry.Mulberry branches are mostly used as agricultural and forestry wastes.However,the medicinal value and various effects of mulberry leaves and branches have also made their related by-products a focus of development in recent years.At the same time,with the improvement of people’s living standards and health standards,clean and harmless natural plant products are also receiving more attention.Efficient and rapid extraction of natural products with good utilization value from mulberry leaves and twigs,as well as chemical composition analysis and efficacy evaluation,will provide theoretical support for the development of the mulberry leaf and twig-related by-product processing industry,while also conducive to reducing resource waste and protecting the environment.Using the content of totally natural products in mulberry leaves and branches as an indicator,the effects of four factors,namely ethanol concentration,extraction temperature,extraction time,and liquid-solid ratio,on the content of totally natural products in mulberry leaves and branches studied using ethanol ultrasonic-assisted extraction.Firstly,the optimal range of each extraction condition was explored through a single factor test,and the optimal conditions of ethanol concentration,extraction temperature,extraction time,and liquid-solid ratio were selected as the zero points for response surface optimization design,Optimization results of extraction process:（1）Mulberry leaves:ethanol concentration 70.382%,extraction temperature 60.11℃,extraction time 59.101 min,liquid-solid ratio 25.158:1;（2）Mulberry branch:ethanol concentration 69.874%,extraction temperature 60.609℃,extraction time46.087 min,liquid material ratio 35.541:1.According to actual production and experimental verification,the final determination process is as follows:（1）Mulberry leaves:ethanol concentration of 70%,extraction temperature of 60℃,extraction time of 59 min,liquid-solid ratio of 25:1;（2）Mulberry branch:ethanol concentration 70%,extraction temperature 60℃,extraction time 46 min,liquid material ratio 35:1.Under these conditions,the contents of total natural products,total flavonoids,total polyphenols,and total polysaccharides in the mulberry leaf extract were 184.67 mg/g,15.586 mg/g,8.623 mg/g,and 13.261 mg/g,respectively,while those in the mulberry branch extract were 183.26 mg/g,13.749 mg/g,7.145 mg/g,and 10.752mg/g.The antioxidant efficacy of mulberry leaf ethanol extract（EUML）and mulberry branch ethanol extract（EUMB）was evaluated.The experimental results showed that both had good antioxidant capacity,and the concentration of the extract was positively correlated with total reducing power,DPPH·free radical scavenging rate,and·OH free radical scavenging rate.In the total reduction ability test and·OH free radical scavenging test,under the concentration range of this experiment,the total reduction ability and·OH free radical scavenging ability of EUML are both stronger than those of EUMB,and the IC₅₀ of EUML is 1.304mg/m L;In the DPPH·free radical scavenging ability test,in the range of 0.2 mg/m L to 0.564 mg/m L,the DPPH·free radical scavenging rate of EUML was higher than that of EUMB,and in the range of 0.564 mg/m L to 1.0 mg/m L,the DPPH·free radical scavenging rate of EUMB was higher than that of EUML.However,at the lowest concentration of 0.2 mg/m L in this experiment,the DPPH·free radical scavenging ability of both reached a higher level（80.56%for EUML and78.12%for EUMB）.In terms of total reduction capacity,EUML has better stability（R²=0.9919）;EUMB has better stability in terms of DPPH·free radical and·OH free radical scavenging abilities（R² values of 0.9526 and 0.9836,respectively）.Three kinds of wheat（Triticum aestivum L.）,Chinese cabbage（Brassica pekinensis（Lour.）Rupr.）,and radish（Raphanus sativus L.）seeds were selected to study the effects of EUML and EUMB on the germination process of three crop seeds under the optimal extraction process.Within the experimental concentration range,EUML and EUMB showed significant inhibitory effects on the 3-day and 7-day germination rates of three crop seeds at low dilution ratios,The inhibitory effect decreases with the increase of dilution ratio.From the perspective of germination index,EUML and EUMB diluted by 100 times inhibited the germination rate of the three seeds,and with the increase of dilution times,the growth rate of the seeds also increased.The inhibitory effect of EUML on seed germination is generally stronger than that of EUMB.In subsequent experiments,mulberry leaves were selected and analyzed for volatile components using gas chromatography mass spectrometry.The results showed that the volatile components of mulberry leaves contain a large number of alkanes and a variety of chemical components with high application potential.