| Objective:We explored the pharmacological effects of Maca from the protection of alcoholic liver injury,defecation,intestinal flora and other aspectsto provide test basis for Maca to declare new medicinal herbs.We evaluated the immunity function of health food formula Maca(Maca Ginseng granules)to provide the basis for its research and development.In addition,network pharmacology was used to predict the mechanism of Maca action and provide reference for further research of Maca.Methods and Results:1.Research on the protective effect of alcoholic liver injuryMethods:50% alcohol gavage was used to cause alcoholic liver injury in mice to study the liver function of Maca water extraction,alcohol extraction and Maca powder on alcoholic liver injury mice(ALT,AST,ALP),inflammatory factors(MCP-1,TNF-a,IL-1β),lipid metabolism index(TG)and lipid peroxidation index(GSH,MDA,SOD).Results:Compared with the normal group,the ALT,AST and ALP in the serum of the model group were significantly increased(P<0.01 or P<0.001),and the lipid metabolism index TG in the mouse serum was significantly increased(P<0.01).The content of lipid peroxidation index MDA in liver tissue was significantly increased(P<0.05),and the content of GSH was significantly decreased(P<0.05),indicating that the alcoholic liver injury mouse model was successfully modeled.The liver function test results showed that compared with the model group,after 14 days of administration,the activity of ALT and AST in the serum of mice in the medium-dose Maca water extract group decreased significantly(P<0.05),and the activity of ALP in the serum of the low-dose Maca water extract group was significantly decreased(P<0.05);the activity of AST and ALP in the serum of the high-dose Maca alcohol extract group was significantly decreased(P<0.05 or P<0.01).The test results of inflammatory factors showed that compared with the model group,after 14 days of administration,the Maca water extract high and low dose groups and the Maca alcohol extract high,medium and low dose groups could significantly reduce the content of MCP-1 in the liver tissues of mice(P<0.05 or P<0.01).Maca water extract(high,medium)dose groups,Maca alcohol extract low dose group and Maca powder group could significantly inhibit the content of inflammatory cytokine TNF-A in serum of mice(P<0.05 or P<0.01).The Maca test group could reduce the content of TNF-a in the liver tissue to varying degrees,and the high-dose Maca alcohol extract group could significantly reduce the content of inflammatory factor TNF-a in the liver tissue of mice(P<0.05).The Maca test group could reduce the IL-1β content in the serum of mice to varying degrees.Lipid metabolism test results showed that compared with the model group,the Maca test substance group could reduce the content of TG in the serum of mice to varying degrees.The Maca alcohol extract high,medium and low dose groups and the Maca powder group could significantly reduce the content of TG in mouse serum.(P<0.05 or P<0.01).Lipid peroxidation index test results showed that compared with the model group,the Maca water extract high,medium and low dose groups,the Maca alcohol extract high,medium and low dose groups and the Maca powder group could significantly increase the content of GSH in mouse liver tissue(P<0.05 or P<0.01);the medium-dose Maca water extract group and the low-dose Maca alcohol extract group could reduce the MDA level in the liver tissue of mice.The high dose of Maca water extract was better than that of Maca powder(P < 0.05)in reducing inflammatory cytokine TNF-α.Compared with powder group,the effects of Maca water extract and alcohol extract on other indexes of alcoholic liver injury mice were not significantly different.2.Research on laxative functionMethods:We used small intestine exercise experiment and laxative experiment which used gavage loperamide hydrochloride to establish mouse intestinal peristalsis inhibition model and mouse constipation model,and observed the effect of Maca water extraction,alcohol extraction and powder on the ink advancing rate of the mouse small intestinal peristalsis inhibition model,the time of first defecation in constipated mice,the number of defecation particles and the weight of defecation within 5 hours.Results:Small intestine exercise experiment: Loperamide hydrochloride was used to induce a mouse small intestinal peristalsis inhibition model.The intestinal ink advancing rate of the model group was significantly lower than that of the normal group,and there was a significant difference compared with the normal group(P<0.01),indicating that the model was successful.Compared with the model group,the Maca water extract high-dose group and the Maca alcohol extract high-dose group could significantly increase the small intestine propulsion rate of mice after 4 days of administration(P<0.01 or P<0.05).Laxative experiment: Loperamide hydrochloride was used to cause a mouse constipation model.In the model group,the first defecation time of mice was significantly prolonged,and the number of feces and fecal weight in mice within 5 hours were significantly reduced,indicating that the model was successful.Compared with model group,the high and medium dose of Maca water extract tended to shorten the first defecation time of mice,the high and medium dose of Maca water extract groups and the medium dose of Maca alcohol extract tended to increase the number of defecation grains in mice within 5 h,and the high dose of Maca water extract tended to increase the weight of defecation in mice within 5 hours.Compared with Maca powder,Maca water extract and Maca alcohol extract had no significant difference in the defecative effect on mice.3.Research on regulating the function of intestinal floraMethods:Mice were administered with Maca water extraction by gavage for 30 days,16 S r DNA technology was used to determine the intestinal flora of the cecal content,and total DNA was extractioned with DNA extractionion kit.The 16SV4 region of the extractioned DNA was amplified by PCR,and the amplification primer was 338F-806 R.The amplified product was used for library construction,and the library was subjected to high-throughput sequencing using the Miseq platform.Bioinformatics analysis such as OTU clustering and species annotation on the obtained data and analyze the significance of species abundance at the level of phylum and genuswere carried out.Results:The results of species composition analysis showed that at the phylum level,from high to low the relative abundance of the mouse intestinal flora were Firmicutes,Bacteroidetes,Actinobacteria,Saccharibacteria and Proteobacteria and Firmicutes and Bacteroidetes had absolute advantages.Compared with the blank group,Peruvian Maca water extract high dose group,Yunnan Maca water extract high,medium and low dose groups could reduce the abundance of Firmicutes;Peru Maca water extract high dose group,Yunnan Maca water extract high and low dose groups could increase the abundance of Bacteroidetes.At the genus level,Lactobacillus and norank＿f＿＿Bacteroidales＿S24-7＿group hadthe highest relative abundance,and the other abundance bacterial groups were norank＿f＿＿Lachnospiraceae,Lachnospiraceae＿NK4A136＿group,unclassified＿f＿＿Lachnospirace 、Bifidobacterium and Alloprevotella.Compared with the blank group,the middle dose group of Yunnan Maca water extraction increased the abundance of Lactobacillus;the middle and low dose groups of Yunnan Maca water extraction increased the abundance of Alloprevotella.The results of community structure changes showed that compared with the blank group,there were significant differences in the intestinal microbial communities in the high,medium,and low dose groups of Maca water extraction.Species difference analysis results showed that at the genus level,compared with the blank group,the Maca water extract high and low dose groups could significantly increase the abundance of Alipipes(P<0.05);Maca water extract medium dose group could significantly reduce the abundance of Desulfovibrio and Eubacterium＿brachy＿group(P<0.01);Maca water extract low-dose group could significantly reduce the abundance of Desulfovibrio and norank＿f＿＿Peptococcaceae(P<0.01).Metabolic function prediction results showed that compared with the blank group,the Maca water extract high and low dose groups reduced RNA processing and modification,cell movement,extracellular structure,and cytoskeleton items,and increased other metabolic functions;Maca water extraction in the middle-dose group decreased cell movement and cytoskeleton items and increased other metabolic functions.4.Prediction of Maca’s mechanism of action based on network pharmacologyMethods:Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine 2.0(TCMIP V2.0)was used to predict the targets and pathways between Maca and inflammation.Results:Target prediction for drugs and inflammatory diseases: there were 318 targets for Maca and 63 targets for inflammatory diseases.Drug and disease core target network analysis: The common targets between Maca and inflammatory diseases were adenosine receptor 2a(ADORA2A),adenosine receptor A1(ADORA1),and tumor necrosis factor(TNF).Enrichment analysis of core target pathways: There were 21 key pathways between Maca and inflammatory diseases.Network visualization analysis: The key genes involved in the inflammation pathway were nuclear transcription factor kappa B1(NF-κB1/p50),nuclear transcription factor kappa B1(NF-κB2/p52),conserved Helix-Loop-Helix Ubiquitous Kinase(CHUK),inhibitor kappa B kinase β(IKKβ/IKBKB),inhibitor kappa B kinase ε(IKKε/IKBKE),glucocorticoid receptor gene(NR3C1),dual specificity phosphatase 1(DUSP1).5.Research on immune enhancement function of Maca Ginseng granulesMethods:According to Technical Specification for Health Food Inspection and Evaluation(2003 Edition)to enhance immunity function evaluation method,adopt delayed-type allergy test,serum hemolysin test,antibody-forming cells test,mouse carbon clearance test,mouse abdominal cavity macrophage swallows the chicken red blood cell test,Con A-induced mouse lymphocyte transformation test and NK cell activity determination were adopted to evaluate the effect of Maca Ginseng granules on immune function.Results:Delayed allergy test in mice: Compared with the blank group,the high-dose and medium-dose Maca Ginseng granules could significantly increase the swelling of the toes of mice(P<0.05).Mouse lymphocyte transformation test: Compared with the blank group,the high,medium and low doses of Maca Ginseng granules had no obvious effect on the proliferation ability of mouse lymphocytes.Antibody-forming cell test: Compared with the blank group,the high-dose Maca Ginseng granules could significantly increase the number of hemolytic plaques in mice(P<0.01).Serum hemolysin test: Compared with the blank group,the high,medium and low doses of Maca Ginseng granules had no significant effect on half of the hemolysis value of mice.Mouse carbon clearance test: Compared with the blank group,the Maca Ginseng granules high,medium and low dose groups could significantly improve the carbon clearance ability of mice(P<0.05 or P<0.01).Test of mouse abdominal cavity macrophage swallows the chicken red blood cells:Compared with the blank group,the high,medium and low dose Maca Ginseng granules could significantly increase the phagocytic rate of mouse macrophages phagocytosing chicken red blood cells(P<0.05 or P<0.01)and phagocytic index(P<0.01).NK cell activity test: Compared with the blank group,the group Maca Ginseng granules had no significant effect on mouse NK cell activity.According to the criterion for the function of enhancing immunity,the Maca Ginseng granules had the function of enhancing immunity.Conclusion:1.Maca water extraction and alcohol extraction had a protective effect on alcoholic liver injury.Maca water extraction and alcohol extraction could improve liver function,abnormal lipid metabolism,lipid peroxidation,and reduce the content of inflammatory factors.2.Maca had the function of promoting small intestine motility.Maca water extraction and Maca alcohol extraction could significantly increase the ink advancing rate of small intestinal peristalsis inhibition model mice.3.Maca water extraction had the effect of regulating the intestinal flora.At the phylum level,the Maca water extraction could increase Bacteroides phylum in the intestinal flora.At the genus level,the Maca water extraction could increase the content of Lactobacillus and Prevotella.Screening of significant differences in abundance at the genus level showed that Maca water extraction could significantly increase the abundance of Alipipes,and significantly reduce the abundance of Desulfovibrio and Eubacterium＿brachy＿group and norank＿f＿＿Peptococcaceae.4.Network pharmacology analysis showed that Maca may act on targets such as ADORA2 A,ADORA1,TNF and NF-κB1(p50),NF-κB2(p52),CHUK,IKKβ(IKBKB),IKKε(IKBKE),NR3C1,DUSP1 and other genes to inhibit inflammation.5.The Maca Ginseng granules had the function of enhancing immunity.The Maca Ginseng granules could increase the swelling of the toes of mice,increase the number of hemolytic plaques in mice,increase mouse carbon clearance capacity and phagocytosis rate of chicken red blood cells by mouse macrophages. |
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