Isolation And Purification Of Secondary Ginsenosides From Panax Ginseng And Its Protective Effects On Heat Stress-induced Scrotal Injury In Mice

Panax ginseng C.A.Meyer is a unique Chinese herbal medicine with the characteristics of both food and medicine.Its ginsenosides is regarded as the main active components of ginseng,accounting for 2~3% of the total mass of ginseng.Red ginseng(RG)is a cooked product of ginseng.During the steaming process,the original ginsenosides will produce the secondary saponins by the glycosylation and the C-20 dehydration isomerization under elevated temperature.Because it rich in secondary saponins such as ginsenoside Rg3,it has a warmer effect and stronger activity.It is widely used in processed products and related dosage forms,such as typifying qi and blood,improving sperm and soothing,and enhancing immune function.In order to further develop ginseng and processed products,in this study firstly uses the different processing time and temperature to influence the secondary saponins production in RG.Based on the determining of the best optimal conditions for the production of secondary ginsenosides,the saponin components in RG are extracted,isolation and investigation of its pharmacodynamics.1.Effects of different processing time and temperature on the production of secondary saponins in RG.In this study,the effects of processing time on secondary saponin production were compared by steaming for 2h,3h and 4h,and the changes of secondary saponin content at different drying temperatures were further investigated by combining 30°C,50°C and 70°C.The experimental results show that after steaming for 2h,3h,4h,they are placed at 30°C,50°C,70°C for 48 h,after pulverization,The sample is extracted by methanol ultrasonically,and HPLC is used for determination of 15 ginsenosides content in RG samples.According to the quantitative analysis,after being steamed for 2h,3h and 4h,under different drying temperatures,the content of secondary saponins such as Rk3,Rh4,Rg3,Rk1 and Rg5 increased obviously within a certain range,and the secondary saponins content will also change correspondingly under different temperature drying conditions.This experiment also shows from the side that the influence degree of each factor on the processing technology of RG is different,and the order of influence is roughly: steaming time > drying temperature.Therefore,it is preliminaries determined that the steaming temperature of 120 °C,steaming for 4h,drying temperature of 50 °C can make the total content of secondary saponins in RG reach a maximum,this study provides reference for RG quality control research,also the depth of RG development provides a material basis.2.Extraction,isolation and purification of secondary saponins from RG.The RG is associated with conventional chromatographic separation and semi-preparative high performance liquid chromatography by means of conventional plant extraction means through macroporous resin,silica gel column chromatography,ODS column and the like.A total of 11 monomers were isolated from red ginseng samples,and their chemical structures were further identified by modern spectroscopy.The spectroscopy analysis showed that 20(S)-ginsenoside Rg2,20(R)-ginsenoside Rg2,Rh1,Rg6,F4,Rk3,Rh4,20(R)-ginsenoside Rg3,Rk1,and Rg5.3.Ginsenoside Rg3 is represented component of ginseng secondary saponins and one of the index components in the quality control of RG.This study further explored the pharmacokinetics effects of G-Rg3 on mouse testicular injury induced by scrotal heat stress(HS).The mice were randomly divided into 4 groups(8 mice per group): Control group,HS group(42 °C,18 min),HS+G-Rg3(5,10mg/kg).G-Rg3 was suspended in 0.05%(w/v)sodium carboxymethylcellulose(CMC-Na),and the administration group was administered by continuous intravenous administration for 14 days.The control group and the HS group were given 0.9 % saline in the same manner.One hour after the administration on the 7th day,the mice were modeled,and the lower abdomen of each mouse was placed in a 42 °C water baths for 18 minutes except the control group.The results showed that compared with the model group,G-Rg3 can significantly inhibit the decrease of serum testosterone content,and low and high doses can inhibit the increase of malondialdehyde(MDA)level and the decrease of superoxide dismutase(SOD)and glutathione(GSH)levels in tissue homogenate.Meantime,by histopathological H&E staining and further analysis of heme oxygenase 1(HO-1),heat shock protein(HSP70)immunofluorescence and Western blot analysis,G-Rg3 can significantly inhibit the expression of oxidized protein,improve oxidative stress to alleviate testicular injury.TUNEL staining,immunohistochemistry and Western blot analysis showed that G-Rg3 also has inhibiting apoptosis.Thus,it is fully revealed that G-Rg3 can exert anti-HS damage through ROS-mediated oxidative stress and apoptosis through MAPK signaling pathway.In summary,this study determines the optimal conditions for the production of secondary saponins in RG by different processing time and temperature,and further separates them to obtain(S)-Rg2,(R)-Rg2,(S)-Rh1,Rg6,F4,Rk3,Rh4,(R)-Rg3,Rk1,Rg5 ten kinds of monomers,At the same time,G-Rg3 is rich contents in RG,can protects against multiple organs such as liver and kidney,have high medicinal value.Based on above pharmacological research,G-Rg3 explores the pharmacodynamics of Rg3 and proves G-Rg3 has obvious protective effect on testicular injury induced by scrotal heat stress in mice,and provides new ideas for further development of secondary saponins and related processed products.It also provides a theoretical basis for clinical treatment of male infertility and guiding reproductive health care.