Study On Accumulation Of Phenolic Substances In Raspberry Leaves And Optimization Of Extraction And Purification Process

Raspberry(Rubus idaeus L.)is a plant of the Rosaceae family in the genus Rubus,widely distributed in temperate and cold regions.It is mainly cultivated in Europe,with lower cultivation in Asia,but has great development potential in China.Studies have shown that raspberry leaves contain a large amount of active ingredients such as polyphenols,flavonoids,tannins,etc.,but as the main waste product in the raspberry production process,they have not been effectively developed and utilized.In order to thoroughly study and evaluate the value of raspberry leaves,this study first investigated the contents of chlorophyll,carotenoids,total phenolics content(TPC),total flavonoids content(TFC),tannins,and saponins in raspberry leaves of different varieties,months,and leaf positions,and analyzed the changes in monomeric phenolic compounds using HPLC-MS,providing a basis for the selection of raspberry leaves.Secondly,the extraction and purification process of phenolic compounds in raspberry leaves was optimized,and the differences in phenolic compounds before and after purification were compared.Finally,the effects of steam explosion on the content and composition of phenolic compounds were investigated,and the effects of antioxidant and enzyme inhibition before and after purification were also studied.The main research contents and results are as follows:(1)Using Heritage,Australian red,Tulameen,and Reveille raspberry leaves as materials,the study investigated the effects of different growing seasons(May to October)and leaf positions(top,middle,and bottom)on the accumulation of secondary metabolites in raspberry leaves.The results showed that the total content of secondary metabolites was highest in Heritage,followed by Australian red,Tulameen,and Reveille.TPC,TFC,quercetin-3-Oglucuronide(Q3GR),kaempferol-3-O-rutinoside(K3R),and ellagic acid(EA)mainly accumulated in the top leaves,while condensed tannins and saponins mainly accumulated in the bottom leaves.From May to October,the photosynthetic pigments showed a trend of increase followed by a decrease,while TPC,TFC,hydrolyzable tannins,condensed tannins,and saponins showed a gradual increase.Compared with variety and leaf position,the growing season had a greater influence on the accumulation of secondary metabolites in raspberry leaves.This study provides a theoretical basis for the harvesting of raspberry leaves.Using Heritage,Australian red,Tulameen,and Reveille raspberry leaves as materials,the study investigated the effects of different growing seasons(May to October)and leaf positions(top,middle,and bottom)on the accumulation of secondary metabolites in raspberry leaves.The results showed that the total content of secondary metabolites was highest in Heritage,followed by Australian red,Tulameen,and Reveille.TPC,TFC,quercetin-3-O-glucuronide(Q3GR),kaempferol-3-Orutinoside(K3R),and ellagic acid(EA)mainly accumulated in the top leaves,while condensed tannins and saponins mainly accumulated in the bottom leaves.From May to October,the photosynthetic pigments showed a trend of increase followed by a decrease,while TPC,TFC,hydrolyzable tannins,condensed tannins,and saponins showed a gradual increase.Compared with variety and leaf position,the growing season had a greater influence on the accumulation of secondary metabolites in raspberry leaves.This study provides a theoretical basis for the harvesting of raspberry leaves.(2)In order to further improve the utilization of phenolic compounds in raspberry leaves,the process of steam explosion(SE)and high-speed homogenization(H)extraction of phenolic compounds in raspberry leaves were optimized.The optimal SE process was found to be 25 min at 115℃ with a liquid-to-material ratio of 29 m L/g,resulting in a TPC value of113.86 mg/g,which is close to the theoretical value.The optimal high-speed homogenization(H)process was found to be 1 min at 4000 r/min,resulting in a TPC value of 101.96 mg/g.When SE and H were combined with the existing ultrasound process(U),it was found that the TPC content of SEHU and SEUH was between 136.30±6.08 mg/g and 140.51±1.67 mg/g,which was higher than that of single conditions and other combined processes.This indicates that SE pretreatment efficiently destroyed the internal structure of the plant,making it easier for U+H to release the phenolic compounds in the plant,increasing the TPC in raspberry leaves and providing a novel method of extraction.(3)In order to purify phenolic compounds from raspberry leaves,XDA-6,a moderately polar macroporous resin,was selected as the best resin among six types of macroporous resins.Static adsorption experiments showed that the maximum adsorption capacity of the resin was 69.22 mg/g,the desorption amount was 60.41 mg/g,and the desorption ratio reached 87.46%.The adsorption isotherm fitted better with the Langmiur model,indicating that the adsorption tended towards monolayer adsorption.The adsorption kinetics simulation showed that there were multiple factors affecting the adsorption of phenolic compounds from raspberry leaves by the resin,which fitted better with the pseudo-second-order model.The optimal adsorption and desorption conditions were as follows: sample concentration of 1.76 mg/m L,sample p H of 2,sample flow rate of 3 BV/h,and sample volume of 19 BV,the most suitable ethanol concentration for desorption was 60%,flow rate was 5BV/h,and elution volume was 3 BV.After the first purification(RLP-1),the TPC value reached738.98 mg/g,which was 5.26 times higher than that of the unpurified sample(RLE).After the second purification(RLP-2),the TPC value was 905.27 mg/g,which was 6.44 times higher than that of RLE.The content of the main phenolic compounds,Q3 GR,K3R,Procyanidin B1(Proc B1),and EA,and the contents of RLP-1 were 3.78,2.37,3.46 and 12.91 times higher than those of RLE,respectively.These results indicated that high polyphenol extracts could be obtained stably under this process condition,which was suitable for the purification of phenolic compounds from raspberry leaves.(4)The effect of SE on the content and composition of phenolic compounds in the extracts and purified materials was studied,and the effect of SE on the activity before and after purification was also studied.The results showed that at 115℃,SE treatment decreased TPC value,and only slightly increased at 20 min compared with 10 min.However,the contents of EA and GA increased after 20 min of SE treatment,indicating that appropriate Se treatment could degrade the high phenolic compounds.Moreover,the antioxidant activity and enzyme inhibition rate were improved after purification.