| Ginseng(Panax ginseng C.A.Meyer),a perennial herb plant belonging to the family Araliaceae,contains a variety of biological and pharmacological ingredients.It is a widely used phytomedicine at home and abroad,and is also a famous raw material plant for functional food and dietary supplements.The main edible part of ginseng plant is the root,thus leading to a fact that the research on ginseng focuses also mainly on its root,further resulting in the waste and even discard of its aboveground parts(such as stems,leaves,flowers and fruits).Ginseng plants require strict growth environment and grow slowly.Even for the garden ginseng under intensive cultivation,it usually takes 5-10 years to become harvestable.Coupled with the unique growth environment,the production area is narrow,the yield is low,and the supply is short of demand.Ginsenosides are recognized as the main active components in ginseng plants,but the cost of isolating and purifying individual ginsenosides from ginseng roots is high,and the yield is limited.Therefore,shoots that can be harvested every year could become an ideal resource to obtain ginsenosides.Under certain steaming conditions,the polar ginsenosides originally existing in ginseng plants will be transformed into less-polar ginsenosides with higher activity.For instance,due to the production of less-polar ginsenosides such as Rg3,Rg5,Rk1 and Rh2,steamed ginseng exerts stronger anticancer activity;in addition,the red ginseng produced after steaming not only has protective effect on microbial infection and a positive effect on memory,but also can reduce the oxidative stress conditions related to chronic diseases and improve peripheral circulation disorders.However,the changes of ginsenoside composition,the mechanism of ginsenoside transformation and the biological and pharmacological activities of extracts from ginseng shoots before and after steaming are still unclear.This study was conducted for the first time to evaluate changes in the compositions,contents and transformation mechanisms of ginsenosides in ginseng shoot extracts(GSE0,GSE2,GSE4,and GSE6)prepared with different steaming times(0,2,4,and 6 h)at 120 ℃,as well as the changes of total phenols and flavonoids contents and antioxidant activities.The anti-inflammatory activities of GESs were further compared by lipopolysaccharide(LPS)-induced RAW264.7 mouse macrophages(RAW264.7 cells).And the extract with the highest content of less-polar ginsenosides,total phenols and flavonoids,and with the strongest antioxidant and anti-inflammatory abilities was selected.Furthermore,this selected extract was further utilized to explore the anti-inflammatory mechanism in vivo;its anti-inflammatory and hepatoprotective functions were further confirmed by mouse acute liver injury model;and its molecular mechanism of hepatoprotective effect was further explored by transcriptomics and metabolomics.The main conclusions of these studies are as follows:(1)A total of 35 ginsenosides were identified by UPLC-Q-TOF-MS/MS,except for the 15 ginsenosides originally existing in GSE0,the remaining 20 less-polar ginsenosides were all formed after steaming.With the aid of authentic standards of 18 ginsenosides,the changes and contents of ginsenosides in GSE0,GSE2,GSE4 and GSE6 were determined by high performance liquid chromatography(HPLC).It was found that the ginsenosides HPLC profiles of GSE changed greatly after steaming at 120 ℃.The 11 polar ginsenosides at retention times between 25-90 minutes gradually decreased or even disappeared,from 1.338 ± 0.083 to 0.290 ± 0.013 mg/g;while the lesspolar ginsenosides between 115-140 minutes gradually increased and reached the maximum value,from 0.006 ± 0.000 to 0.455 ± 0.203 mg/g.Thus,the optimal conditions for obtaining the highest contents of less-polar ginsenosides were determined.The total phenols measured by Folin-Ciocalteu method increased from 2666.01 ± 38.92 to 4733.00 ± 178.23 mg GA eq/100 g D.W.;the total flavonoids measured by aluminum chloride colorimetric method increased from 7771.40 ± 168.49 to 11610.52 ± 260.46 mg RT eq/100 g D.W.;the antioxidant capacity measured by DPPH and ABTS also increased with the duration of steaming time.DPPH radical scavenging capacity increased from3174.06 ± 121.69 to7492.75 ± 1276.25 μmol Trolox eq/100 g.d.w..The free radical scavenging capacity of ABTS increased from 3607.81 ± 563.76 to 6937.50 ± 590.89 μmol Trolox eq/100 g.d.w.(2)The anti-inflammatory activity of GSEs was compared by a LPS-induced inflammation model in RAW264.7 cells.MTT assay shows that the three concentrations(25,50,or 100 μg)of GSEs exhibited no toxicity to cells;GSEs showed inhibitory effect on the release of inflammatory mediators such as NO,IL-6 and TNF-α,and the inhibition degree was enhanced with the increase of steaming time and concentration,which was further confirmed by the m RNA expression levels of inflammatory genes i NOS,COX-2,IL-6 and IL-1β,and GSE6 exhibited the strongest antiinflammatory activity.Correlation analysis shows that higher contents of less-polar ginsenosides,total phenols and total flavonoids endowed GSE6 stronger antioxidant and anti-inflammatory activities.(3)To further explore the anti-inflammatory effect of ginseng shoots extract in vivo,five different concentrations(50,100,150,200,250 mg/kg)of ginseng shoots extract(GSE)steamed at120 ℃ for 6 h were pretreated in mice by gavage for 7 days,and then injected with acetaminophen to establishe a liver injury model.By detecting transaminase(AST and ALT),antioxidant enzymes(GSH and SOD),lipid peroxidases(MDA and 4-HNE),inflammatory factors(TNF-α,IL-1β,COX-2,and i NOS)and apoptosis-related proteins(Bcl-2,Bax,Caspase3-8-9),it is found that the mice pretreated with GSE could prevent acetaminophen-induced liver injury.Accordingly,the optimal dose and liver protection mechanism were also determined.GSE could prevent liver injury through antioxidant,anti-inflammatory,and anti-apoptosis.(4)Steamed ginseng shoots extracts(GSE)exterted strong hepatoprotective effect,but the potential molecular and metabolic regulation is still unclear.Through the combined analysis of metabonomics and transcriptomics,it is found for the first time that GSE could up-regulate genes and metabolites related to bile secretion,retinol metabolism,and amino acid metabolism pathway,of which the genes and metabolites are correlated with antioxidant,anti-inflammatory,antiapoptotic,and liver protection effects.Fifteen new metabolic markers and 25 new transcriptional markers were identified,which provided a new perspective for the deep application of ginseng shoots and screening hepatoprotection biomarkers and therapeutic strategy for APAP-induced liver injury. |
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