Chemical Constituents Of Malonyl Ginsenosides From Stems And Leaves Of Panax Ginseng C. A. Meyer And Their Protective Effects On Acetaminophen-Induced Liver Injury In Mice

The toxicity of acetaminophen（APAP）after ingestion is the main cause of drug-induced liver failure.At present,the drug for the treatment of APAP poisoning is N-acetylcysteine（NAC）.NAC must be used within 8 hours after overdose of APAP in order to achieve the best effect.However,the toxic reaction of APAP is sometimes not obvious,and the side effects of NAC are serious,so it is worth exploring to find a safe and effective drug for the prevention and treatment of liver injury.In China,Panax ginseng C.A.Meyer is a traditional Chinese herbal medicine,which has been used for thousands of years and has a wide range of biological activities.Ginsenosides are the main active components of ginseng,including neutral ginsenosides and malonyl ginsenosides.In recent years,a large number of animal experiments and clinical experiments have shown that ginseng and ginsenosides can improve the protective effect of APAP-induced liver injury in mice.However,whether malonyl ginsenosides can prevent and treat liver injury has not been reported.Malonyl ginsenosides（MGR）exist in different parts of ginseng,such as roots,stems and leaves,flowers and fruits.Previous studies have shown that malonyl ginsenosides account for about 50%of the total ginsenosides in ginseng root and are the main active components of ginseng root.It has the effects of reducing blood sugar,lowering blood lipids,improving insulin resistance,promoting long-term potentiation of dentate gyrus and neurite growth in rats.However,due to the strong water solubility of malonyl ginsenosides,it is difficult to prepare,so there are few reports on the chemical and pharmacological studies of malonyl ginsenosides at home and abroad.In addition,previous studies mainly focused on the traditional medicine parts of Panax ginseng,but ignored the study of malonyl ginsenosides in the stems and leaves of Panax ginseng.In this paper,malonyl ginsenosides from the stems and leaves of Panax ginseng were extracted,isolated,identified and determined,and their protective effects on liver injury in mice were studied.In this experiment,fresh ginseng stems and leaves were extracted by ethanol cold soaking method.The impurities such as pigments in stems and leaves were removed by alcohol extraction and water precipitation,and then separated and purified by macroporous resin column chromatography,silica gel column chromatography and ODS column chromatography.The components were prepared by HPLC.Five monomer saponins（MRb₁,MRb₂,MRb₃,MRc,MRd）were isolated and identified from the stems and leaves of Panax ginseng by ESI-MS,¹³C NMR,¹H NMR and 2DNMR（HSQC,HMBC,¹H-¹HCOSY,NOESY）.Among them,malonyl ginsenoside Rb₃was isolated from stems and leaves of P.ginseng for the first time.Its structure is 3-O–[β–D–glucopyranosyl–（1→2）-β–D-glucopy ranosyl]–20-O–[β-D–xylopyranosyl–（1→6）-β-D–glucopyranoside]-dammar-24-ene-3β,12β,20S-triol.The contents of 13 main ginsenosides in the stems and leaves of Panax ginseng were determined by HPLC.The contents of malonyl ginsenosides and total ginsenosides ranged from 0.65%to 0.84%,and 2.9%to 4.2%,respectively.These results indicated that malonyl ginsenosides and neutral saponins in the stems and leaves of Panax ginseng were relatively high,which can be used as a reliable source of ginsenosides.This experiment mainly studied the therapeutic effect of malonyl ginsenoside extract from the stems and leaves of Panax ginseng on APAP-induced acute liver injury in mice.Forty SPF male ICR mice were randomly divided into blank（Normal）group,APAP model group and MGR（300mg/kg and 150mg/kg）+APAP group.MGR was administered continuously for 7 days.One hour after the last administration,APAP with the concentration of 250mg/kg was injected intraperitoneally to induce liver injury in mice.After 24 hours,the serum levels of glutamic oxaloacetic transaminase（AST）and glutamic pyruvic transaminase（ALT）were measured to evaluate the changes of liver function,and the levels of reduced glutathione（GSH）,superoxide dismutase（SOD）,malondialdehyde（MDA）,tumor necrosis factorα（TNF-α）and interleukin 1β（IL-1β）were measured to evaluate the level of oxidative stress and inflammation in the liver of mice.In addition,the pathological changes of liver tissue were observed by hematoxylin-eosin staining.The expression of apoptotic protein in PI3K/AKT signal pathway was detected by Western Blot technique.The results showed that MGR could significantly reduce the levels of AST,ALT,IL-1βand TNF-αin serum,and increase the level of GSH and decrease the level of MDA in liver.At the same time,it was found that the degree of liver injury in mice was well improved by hematoxylin-eosin staining.Western Blot results show that MGR can prevent liver injury caused by apoptosis by activating PI3K/AKT signal pathway.To sum up,MGR has a protective effect on acute liver injury induced by APAP in mice,and its mechanism may be related to the improvement of oxidative stress and the prevention of apoptosis.