| Ginseng(Panax Ginseng C.A.Meyer)is a perennial herb with Panax paniculata.It is a famous traditional Chinese herb and widely used in health and medical care industries.The main functional active ingredients of ginseng are ginsenosides.After the hydrolysis of ginsenosides,a large amount of major ginsenosides lose some saccharide ligands,and rare ginsenosides with stronger biological activity can be obtained.However,given that the content of rare ginsenosides in nature is extremely low,their development and application are limited.Cordyceps,a fungus in the Ergodicaceae family,is called cordyceps,a complex of larvae and carcasses that hosts the subsets of the bat moth larvae.The major ginsenosides can be transformed into rare ginsenosides by Cordyceps sinensis,and bacteriological products containing rare ginsenosides,cordycepin,cordycepic acid,and dual efficacy components can be obtained.In this study,total ginsenoside was extracted with an ultrasonic-aided composite enzymatic method.After the separation and purification of ginsenoside monomers Rb1,Rg1,and Re,the transformation pathway of ginsenosides Rb1,Rg1,and Re and conversion rate by Isaria fumosorosea ZZ and Cordyceps sinensis Y3 were investigated,the ginsenoside that exerted antihyperlipidemia effect was extracted using a high-cholesterol zebrafish model,and the antihyperlipidemia effects of Rb1 and Rg3 and related possible molecular mechanism were explored.The main contents were as follows:(1)Total ginsenoside was extracted using an ultrasonic-aided composite enzymatic method.Technological conditions were optimized,and the Box-behnken design was subjected to response surface analysis based on the results of a single-factor experiment.The optimum technological conditions were as follows: addition amount of cellulase,0.20%;addition amount of pectinase,0.25%;material liquid ratio,1:20(g/m L);ultrasonic time,25 min;enzymolysis temperature,45 °C;enzyme solution p H,5;enzymolysis time,54.2 min;ultrasonic power,500 W;and extraction rate of total ginsenosides,4.53% ± 0.03%.Ginsenoside monomers Rb1,Rg1,and Re were separated and purified with a macroporous adsorption resin through silica gel chromatography.Three kinds of ginsenosides were determined using TLC,infrared spectroscopy,and HPLC.The purity of ginsenoside was over 95%.(2)Isaria fumosorosea ZZ and Cordyceps sinensis Y3 were obtained through screening and identification with the p-nitrophenol-D-grapevine(p NPG)method.The stable specimen strains of wild Cordyceps fungus were collected in the original forest of Changbai Mountain.Optimal culture conditions were determined by studying the production rules of β-glucosidase and cordycepic acid under different conditions.In the ZZ strain,the optimal carbon source of enzymes was cellobiose,nitrogen source was corn pulp powder,and the initial p H of the medium was 8.The optimal carbon source for the β-glucosidase production of strain Y3 was cellobiose,the nitrogen source was beef extract,and the initial p H of the culture medium was 8.Isaria fumosorosea ZZ of cellobiose and sucrose proteomic data were analyzed using a quantitative proteomics method based on tandem quality label labeling technology.The results were as follows: 519 differential proteins(P>1.5),205 up-regulated proteins,and 314 down-regulated proteins.In the differential proteins,the processes related to biological processing are mainly “cell metabolism” and “organic metabolism,” followed by “primary metabolism” and “nitrogen compound metabolism.” The number of proteins related to “intracellular organelles” and “membrane-bound organelles” was first in rank in the classification of cell components.Differences in protein mainly belongs to one of the most abundant protein in the “ring of organic compounds,” “heterocyclic compounds,” and “hydrolase and transferase activity” classes.It not only affected the related protein expression of carbohydrates and amino acids;lipid metabolism;and genetic information after treatment with cellobiose but also influenced the process of modification after translation and energy metabolism.A total of 60 glycosidase-related proteins were identified,30 of which were differentially expressed(four up-regulated and 26 down-regulated).The production of β-glucosidase and chitinase was not inhibited in the presence of cellobiose,whereas that of other glucosidases was inhibited.(3)UPLC-MS was used in studying the pathway of transformation of Rb1,Rg1,and Re by ZZ and Y3.The results show that one of the pathway for ZZ and Y3 to transform Rb1 was Rb1 →Rd → Rg3,and the other pathway was Rb1 → Rd → F2 → CK.In the pathway of transformation of Rg1,the pathway was Rg1→Rh1.After fermentation by ZZ for 192 h,the conversion rate of Rb1 into Rd was 46.93%,whereas the conversion rates of F2,CK,and Rg3 were 45.71%,53.07%,and 21.98%,respectively.After 192 h of fermentation by Y3,the conversion rate of Rb1 was F2 87.48%,CK 17.34% and Rg3 21.58%.After fermentation by Isaria fumosorosea ZZ for 192 h,the conversion rate of Rg1 into Rh1 was 37.15%.The conversion rate of Cordyceps sinensis Y3 into Rh1 after 192 h was Rh1 36.42%.Strain ZZ and Y3 had no transformation effect on Re.(4)Ginsenosides were screened on the basis of the anti-hyperlipidemia effect on zebrafish larvae with a high-cholesterol animal model.Zebrafish of 5 dpf(Days Post fertilization)was fed with 12 kinds of ginsenoside-containing fluorescent probes.After 12 days of continuous feeding,fluorescent inverted microscope was used in observing cholesterol accumulation in whole and local bodies,and relative fluorescence density and survival rates were calculated.The results show that Rg3,PPD,Re,Rh2,Rb1,and Rg1 had certain lipid-lowering effects.The ratios of local trunk fat reduction were as follows: Re(7.54%),Rh2(1.11%),Rb1(14.22%),and Rg1(2.60%).A zebrafish adult high-cholesterol model was adopted and fed with Rb1 and Rg3 with 25,50,and 100 μg/kg dosage.After 40 days of feeding,the levels of TC and TG in the zebrafish blood samples of the Rb1 and Rg3 treatment groups were reduced compared with those in the control group.Hematoxylin-eosin(HE)and oil red O staining was used in the histopathological examination of the liver in each dose group.The antihyperlipidemia effects of Rb1 and Rg3 were then verified.The key genes related to lipid metabolism in the liver was detected by Real Time-PCR.The results showed that Rb1 down-regulated the m RNA expression of HMGCR,LDLR,and MTP m RNA in a dose-dependent manner,and the expression levels of LXR,ABCG1,NPC1L1,and CYP7A1 m RNA were up-regulated.Rg3 treatment up-regulated the m RNA expression levels of LXR,SREBP2 ABCG1,NPC1L1,and CYP7A1 and down-regulated the m RNA levels of HMGCR and LDLR.These results indicate that the mechanism of antihyperlipidemia effect is related to the up-regulation of cholesterol transport and bile acid synthesis and to the inhibition of cholesterol synthesis and lipoprotein assembly or secretion. |
