Study On Extraction, Identification, Separation, Purification And Activity Of Crude Extracts From Raspberry Leaves

Rubus idaeus L.belongs to the Rubus plant of Rosaceae,also known as raspberry,bramble,Rubus corchorifolius,etc.It is a small berry of color mellow,with high nutritional value and economic value.The roots,stems and leaves of raspberry can be used as medicine,sweet flavor and mild-natured,tonifying the kidney and controlling nocturnal emission with astringent drugs,tonifying liver and improving acuity of vision.The stem and leaf decocting water can be used to relieve itching,cure frostbite and treat blister.In addition,raspberry can be used for the maintenance of barren hills and slopes,with certain ecological value.In this paper,the extraction,identification,separation,purification and activity of secondary metabolites from raspberry leaves were studied.The main findings are as follows:Extraction of raspberry leaves by ultrasonic assisted ethanol extraction.By the response surface experiment,the optimum extraction conditions of the main antioxidant active substances(total flavonoids and SOD)in the leaves of raspberry were 60%ethanol,1:40 g/mL solid-liquid ratio,120 W assisted extraction of 30 min.Under these conditions,the total flavonoids in the leaves of raspberry were 51.74 mg/g,the total activity of SOD was 5828.96 U,and the total protein content was 17.20 mg/g.Based on this optimum process,comparsion antioxidants in leaves from 9 varieties including Australian raspberry,black raspberry,Tulameen,Nova(spiny),Nova,Heriatge,Malahat,Reville and Autumn Bliss,the results showed that antioxidant contents were higher in black raspberry,Nova(spiny)and Nova and lower in poor Reville and Heriatg.Compared the content of total flavonoids in 9 kinds of raspberry and blackberry with the uniriped dried fruit of Rubus chingii Hu.and 16 herbs leaves(Scutellaria barbata D.Don,Nelumbo nucifera Gaertn,Artemisia argyi Levl.et Vant,Isatis indigotica Fort,Eriobotrya japonica Thunb,Lycopus lucidus Turcz.vat.hirtus Rege,Platycladus orientalis(L.),Lysimachia christinae Hanceetc.),the results showed that the total flavonoids content of 10 variety leaves were higher than the Rubus chingii.Hu(33.293 mg/g);The total flavonoids content of blackberry and black raspberry were 91.682 mg/g and 74.391 mg/g respectively,only lower than that lotus(95.729 mg/g),but higher than those of other herb leaves.25 compounds were identified from Australian red and blackberry alcohol extract by HPLC-MS.For example 8 polyphenols:chlorogenic acid,ellagic acid,ellagic acid derivatives,etc,11 flavonoids:(-)-epicatechin,rutin,quercetin-3-O-glucuronide,quercetin-3-O-glucoside,kaempferol-3-O-glucuronide,etc,and 6 triterpenoids:polygalic acid,asiatic acid and its derivatives.Chlorogenic acid,ellagic acid,(-)-epicatechin,quercetin-3-O-glucuronide(Q3GA)and asiatic acid Australian red and blackberry in July and October respectively by HPLC-MS quantitative analysis.The results showed that the total amount of 5 compounds in the Australian red leaves decreased nearly 2 times as the time changed,while the Blackberry increased by 1 time.The quantitative results of chlorogenic acid showed that there was a significant difference in the content among varieties,and the Australian raspberry was 70times higher than the blackberry at the same time.The content of(-)-epicatechi was not only different in variety,but also in time difference,Australia red(-)-epicatechin content is lower than the Blackberry,and with the change of time,(-)-epicatechin in the two leaves were increased by 18 times and 1 time respectively.The content of ellagic acid decreased dramatically in Australian red and blackberries over time.The proportion of Q3GA in total content is the highest and with the change of time,the Q3GA content of Australian red is 2times less than that in blackberry and 2 times in blackberry;asiatic acid in two varieties was accumulated over time and increased 4 times and 3 times respectively.Other tirterpenoids,such as and the polygalic acid,asiatic acid derivatives,all had a trend to accumulate over time..Separated and purificated by polyamide chromatography column.compounds 1 and 2(637 mg)were isolated from 5 g petroleum ether.And compound 2 was identified as polygala acid,based on the[M-H]~-m/z 487.3056.The proportion of eluant is methanol:chloroform=13:3(2%formic acid).Activity studies showed that triterpenoids such as Polygalic acid and their derivatives in raspberry leaves has a certain scavenging effect on DPPH free radicals,which a rate of scavenging was 10.07%when the concentration was 0.5 mg/m L.It has bacteriostasis against to the Staphylococcus aureus,when the concentration was 1.6 mg/m L,the rate of bacteriostasis of Staphylococcus aur eus was 53.39%.In summary,the raspberry leaves contain flavonoids,SOD,and triterpenoids,which compounds have high medicinal and edible values.Their content varies greatly between varieties and harvest time.Especially triterpenoids,it can accumulate significantly over time.Thoes results provide the data to support for breeding good varieties of raspberry leaves,to support the comprehensive development and use of raspberries.