Study On The Main Medicinal Components Of The New Resource Protein Mulberry Leaves And Their In Vitro Hypoglycemic Activity Evaluation

MORI FOLIUM is the dry leaves of Morus alba L.,which are of high medicinal value in traditional Chinese medicine,modern research shows that mulberry leaves have many biological activities such as hypoglycemic and lipid-lowering,antioxidant,anti-tumor,etc.,and are often used as drugs for the treatment of diabetes.Mulberry leaves contain 1-deoxynojirimycin(DNJ)and flavonoids,polysaccharides which are known as ?-glycosidase inhibitors.As a medicinal and edible resource approved by the ministry of health,mulberry has always been favored by people,so its resource utilization and development is one of the hot spots in modern research.Based on the wide application value of mulberry leaves,in recent years,Chinese researchers have cultivated a stress-resistant and strain-protein mulberry through manual selection and cross breeding,which has the characteristics of drought resistance,cold tolerance,barren tolerance,developed root system,and cutting resistance.It has strong adaptability and has been widely promoted in planting,and has broad development prospects.At present,the research on protein mulberry at home and abroad mainly focuses on the nutritional value,such as carbohydrates,proteins,fatty acids,cellulose,vitamins and mineral elements,etc.,and its medicinal value research is still in the preliminary stage and lack of systematically reported.In this experiment,the new resource protein mulberry leaf was used as the research object,and the mulberry leaves of Tong Ren Tang and National Institutes for Food and Drug Control were used as the control medicinal materials to study the main medicinal components and their hypoglycemic activity in vitro,in order to provide a scientific basis for the new resource protein mulberry.The main research results are as follows:(1)Total protein,total flavonoids,total polysaccharides,and total alkaloids in protein mulberry of 26 batches from two varieties of Guisangyou No.12 and Guisangyou No.62 from four producing areas and traditional mulberry leaves were determined,analyze the differences between different origins.Based on the single-factor test,this study conducted an orthogonal test on the extraction method of total flavonoids from mulberry leaves,screened out the optimal extraction process,and this method was used to extract.The experimental results are as follows:the protein content of protein mulberry leaves in Hunan and Jiangsu production areas is high,ranging from 210.43 to 301.51 mg·g-1.Among them,Guisangyou 12 in Hunan production area has the highest protein content;The total flavonoid content of protein mulberry leaves in Gansu production areas is high,ranging from 48.53 to 64.42 mg·g-1.Among them,Guisangyou 62 in Hunan production areas has the highest total flavonoid content;Hunan and Gansu production areas has the highest total flavonoid content.The total polysaccharide content of protein mulberry leaves is high,ranging from 33.58 to 48.50 mg·g-1.Among them,the total polysaccharide content of Guisangyou 12 mulberry leaves in Gansu production area is the highest;the content of total alkaloids from the protein mulberry leaves in Hunan and Gansu production areas is high,ranging from 5.80 to 7.83 Mg·g-1.Among them,Guisangyou 62,the mulberry leaf in Hunan production area,has the highest total flavonoid content.In summary,the two protein mulberry varieties in the Hunan and Jiangsu producing areas are both suitable as an advantageous resource for the development of total protein and medicinal parts.(2)The determination of chlorogenic acid and flavonoids based on ultra-high performance liquid phase(UPLC)was established to qualitatively and quantitatively analyze different batches of protein mulberry leaves and traditional mulberry leaves.The liquid phase conditions are as follows:chromatographic column Waters ACQUITY UPLC BEH-C18 column(2.1 nm×100 mm,1.7 ?m);mobile phase A phase is 0.3%formic acid aqueous solution and phase B is acetonitrile.Chromatographic gradient elution and elution procedure as:0.0?1.0 min,4%B;1.0?2.5 min,4%?0%B;2.5?7.5 min,10%?20%B;7.5?8.5 min,20%?48%B;8.5?12.0 min,48%?60%B;12.0?13.0 min,60%?90%B;13.0?14.0 min,90%B.The flow rate was 0.4 mL·min-1,the injection volume was 2?L,the detection wavelength was 380 nm,and the column temperature was 35?.This method can simultaneously identify 4 active ingredients of chlorogenic acid,rutin,isoquercitrin and astilin,and the analysis time is shortened to 15 min,which is simple and fast.The results shows that the chlorogenic acid content of protein mulberry leaves ranged from 2.53 to 6.48 mg·g-1,and the content of Guisangyou 12 in Gansu production area is the highest;the content of rutin in mulberry leaves in Hunan and Gansu production areas is obviously higher than other sources of mulberry leaves,whose content is ranging from 0.59 to 0.93 mg·g-1.Among them,the content of rutin in Guisangyou 62 is higher than that of Guisangyou 12;the content of isoquercitrin in protein mulberry leaves is 0.32?1.60mg·g-1,among which Guisangyou No.62 in Hunan production area has the highest content;the two varieties of protein mulberry in Gansu production area have higher content of astilin,ranging from 0.40 to 0.74 mg·g-1.(3)The high-performance liquid chromatography-evaporative light scattering detector combined technology(HPLC-ELSD)was used to establish the content analysis method of alkaloid 1-deoxynojirimycin(DNJ)for different batches of protein mulberry and traditional mulberry leaves.The method was investigated and the optimal chromatographic conditions were:InertSustain C18 column(250 mm × 4.6 mm,5 ?m),the mobile phase was acetonitrile-0.3%formic acid-0.1%triethylamine gradient elution gradient:0.0?2.0 min,2%;2.0?4.0 min,2%?10%;4.0-8.0 min,10%?29%;8.0-15.0 min,29%?40%;15.0?20.0 min,50%.Flow rate:0.8 mL·min-1;column temperature 45?;injection volume 5 ?L.ELSD drift tube temperature:100? carrier gas flow rate 3 mL·min-1.The DNJ content of protein mulberry leaves is between 1.89 and 3.70 mg·g-1,of which Guisangyou 12 is the highest in Hunan producing area.(4)Analyse of ?-glycosidase inhibitory ability and antioxidant activity of protein mulberry and traditional medicine mulberry leaf alcohol extracts.In order to comprehensively compare the antioxidant properties of mulberry leaf extracts of new resources and traditional resources,this study uses three methods,DPPH,FRAP and ABTS.The results show that the protein mulberry leaves have good ?-glycosidase inhibitory ability and antioxidant activity,and the activity varies with the place of origin and sampling time.Comprehensive evaluation shows that the two protein mulberry leaves samples from Gansu and Hunan production areas have strong antioxidant activity in the three evaluation systems.In addition,the protein mulberry leaf samples from these two production areas have stronger ?-glycosidase inhibitory ability than the other two production areas.Correlation analysis shows that the antioxidant capacity of mulberry leaves in vitro and the in vitro ?-glycosidase inhibitory ability shows a very significant positive correlation(P<0.001),and the correlation coefficients are all greater than 0.9.(5)Multivariate statistical analysis was performed for all measured parameters(the total protein content,total flavonoid content,total alkaloid content,total polysaccharide content,chlorogenic acid content,rutin content,isoquercitrin content,asperin content,DNJ content,antioxidant activity and glycosides).Take further research on the internal relationship between different parameters to provide ideas and reliable basis for resource development and utilization of protein mulberry.In summary,the content of the main medicinal components of the new resource protein mulberry leaves is higher than that of the traditional medicine mulberry,and the in vitro?-glycosidase inhibitory capacity and antioxidant capacity are also higher than those of the traditional medicine mulberry.The protein mulberry from two production areas of Hunan and Gansu is significantly superior than the traditional medicinal mulberry in terms of the content of the medicinal parts and the in vitro activity,showing great development and utilization value.This article temporarily divides the protein mulberry into four major categories according to their respective characteristics:functional type,edible type,feeding type and ecological type,in order to realize the diversified development and utilization of protein mulberry resources and expand and enhance emerging functional uses.In this study,based on the results of chemical qualitative and quantitative results and in vitro biological activity,combine with the method of multivariate statistical analysis,the intrinsic quality and medicinal value of the new resource protein mulberry leaves are comprehensively and systematically evaluate,and the key technology to the quality control and evaluation index of protein mulberry leaves is established,and provide a scientific basis for the quality evaluation of new resource protein mulberry leaf.