Study On The Properties Of The Foreign Traditional Chinese Medicine Maca Gan Wen Jian Spleen And Its Material Basis And Mechanism Of Action

1 BackgroundSince ancient times,China has the great tradition of absorbing excellent overseas herbal medicine,and we always have a welcome attitude for them.Maca is native to Peru and has been introduced into China due to its unique biological activities.And it is widely used in combination with traditional Chinese medicine.However,due to the lack of clear Chinese medicine medicinal properties,this compatibility application lacks the theoretical support of traditional Chinese medicine.As a result,research on the Chinese medicine properties of Maca is a key issue that needs to solve in urgently.In the early stage,our research team carried out literature research,theoretical discussion and preliminary pharmacological experiment which verified the hypothesis of Chinese medicine properties of Maca.Maca is bit warm,sweet,spicy,kidney,belongs to liver,spleen,and kidney channel,and has the function of nourishing the kidney and strengthening the essence,soothing the liver and strengthening the spleen.Maca's function of relieving physical fatigue and enhancing immunity is popular in China,while fatigue and immunity are closely related to spleen deficiency syndrome.Therefore,based on the establishment of a mouse model of spleen deficiency syndrome,this study selects the traditional Chinese medicine properties of Warm,Sweet and Strengthening Spleen of Maca.We will give it a preliminary verification,and then study the material basis of its properties,and discusses its mechanism of promotion of energy metabolism and immune regulation.2 AimsEstablish two kinds of spleen deficiency syndrome model,and verify the traditional Chinese medicine properties of Maca of Warm,Sweet and Strengthening Spleen,(2)Investigate and compare the pharmacological bias of Maca powder and its water extract and alcohol extract,and trace the main active ingredients of Maca.(3)Defining the material basis of Maca's TCM propertis and discussing its mechanism of promoting energy metabolism and immune regulation in the spleen deficiency syndrome.3 Methods(1)Establishing an animal model of spleen deficiency syndrome with complex factor modeling method(double factors of diet loss and fatigue),investigating the effect of Maca powder on the material and energy metabolism of mice with spleen deficiency syndrome,and verifying the Chinese medicine properties of Maca of Warm,Sweet and Strengthening Spleen.(2)Establishing an animal model of spleen deficiency syndrome induced by chemical factors(cyclophosphamide),and investigating the effects of maca on energy metabolism and immune regulation in mice spleen deficiency syndrome,and verifying the Chinese medicine properties of Maca of Warm,Sweet and Strengthening Spleen.(3)The spleen deficiency syndrome model established by cyclophosphamide induction was selected to investigate the effect of maca powder,maca water extract and alcohol extract on spleen deficiency syndrome mice,and trace the maca the main active ingredient of its properties.(4)Further study on the method of extracting and refining maca polysaccharides,analyzing the composition and pharmacological effects of maca polysaccharides.(5)Study the effects of maca polysaccharide on energy metabolism in mice with spleen deficiency syndrome.The contents of Na+-K+-ATPase,Ca2+-Mg2+-ATPase and mitochondrial FOFi-ATPase in liver tissue of mice with spleen deficiency were detected.The gene expression levels of NADH,SDH and VDAC,which were the key enzymes of energy metabolism in the Krebs cycle were detected by RT-qPCR method.The lvel of VDAC protein were measured by Western Blot.The mechanism of the metabolism of Maca polysaccharide in mice with spleen deficiency was clarified.(6)To study the effects of maca polysaccharide on the immune regulation of mice with spleen deficiency syndrome.The peripheral blood lymphocyte cycle and cell subsets of spleen-deficiency mice were measured by flow cytometry.The serum immunocytokines IFN-y and IL-2,IL-4,TNF-? contents were measured by enzyme immunoassay.The gene expressions of transcription factors T-bet,GATA-3 were detected by RT-qPCR,spleen lymphocyte proliferation was detected by MTT,spleen cell apoptosis was observed by TUNEL fluorescence staining,The expression levels of apoptotic factors Caspase-3,BAX and Bcl-2 in spleen cells were detected by Western Blot and immunofluorescence assay.The mechanism of maca polysaccharide on immune regulation in mice with spleen deficiency was determined.4 Results(1)Maca powder can alleviate the physical fatigue of mice with spleen deficiency syndrome established by the two-factors method.And Maca could increase the time of exhaustive swimming,promote storaging MG and LG,inhibit the production of metabolites of LA,FFA,CREA and UREA,promotes the activities of energy metabolism enzymes like LDH,CK and liver ATPases,and increase the ratio of cAMP/cGMP in serum.Maca can promote the material and energy metabolism of mice with spleen deficiency,and reduce the accumulation of metabolites(2)Maca powder can alleviate the symptoms of spleen deficiency established by cyclophosphamide induction method.Maca could reduce the temperature ratio of cold zone in mice,protect the immune organs,increase WBC count,up-regulate IL-2,IFN-? expression levels,and enhance energy metabolism Na+-K+-ATPase and Ca 2+-Mg 2+-ATPase activities.Maca can enhance the energy metabolism of mice with spleen deficiency,promote the immune regulation of the body(3)Using ginseng as a control,the same dose of maca powder,its aqueous extract and alcohol extract to cyclophosphamide-induced spleen-deficiency mice model were further compared.The cold and hot trends of and immune regulation Maca water extract on spleen-deficiency mice were the most obvious,better than the alcohol extract and powder,it is the most prominent.(4)Extracting maca polysaccharide by water extraction and alcohol precipitation method,and purifying maca polysaccharide by separating protein and resin,Maca polysaccharide can be obtained more than 75%.Which is mainly composed of arabinose(Ara),glucose(Glc),galactose(Gal)and mannose(Man).The material basis of Maca's TCM properties is maca polysaccharide(5)Maca polysaccharide can alleviate the symptoms of spleen deficiency in mice,increase body weight,prolong the swimming time of spleen-deficiency mice,increase the temperature-dependent proportion of cold zone in mice,and increase the serum cAMP/cGMP ratio of spleen-deficient mice.The contents of the body ATPase Na+-K+-ATPase and Ca2+-Mg2+-ATPase ion pump and the FOF1-ATPase activities were enhanced.Maca polysaccharide can promote the synthesis of ATP,and increase the expression of NADH and SDH,and enhance the activity of VDAC in the liver tissue of mice with spleen deficiency.The decomposition of three major nutrients to form pyruvate into mitochondria were promoted,and then oxidative phosphorylation reaction happened to produce ATP and storage energy,which has revealed the mechanism of maca polysaccharide of promoting energy metabolism in mice with spleen deficiency syndrome(6)Maca polysaccharide inhibits the atrophy of immune organs,increases the white blood cell counts of peripheral blood,enhances the immune regulation,and exerts the properties of Warm,Sweet and Strengthening Spleen,Its mechanism of action is letting lymphocytes which were staying in G0/G1 phase enter into S phase and G2/M phase,increase the proportion of CD4+ T lymphocytes;increase the contents of IFN-y,TNF-?,IL-2,and decrease the content of IL-4,increase the expression level of transcription factor T-bet gene,and decrease the expression level of GATA-3 gene,transform Th0 cells into Th1 cells,and inhibit the activation of Th2 cells,and restore the balance of Thl/Th2 cell subsets.It inhibited the apoptosis of spleen lymphocytes,decreased the expression of proapoptotic factors BAX and Caspase-3,and increased the expression of apoptosis inhibitor Bcl-2 protein.5 ConclusionThe traditional Chinese medicine properties of Warm,Sweet and Strengthening Spleen of Maca powder were verified based on the two mice models of spleen deficiency syndrome.It was mainly related to promoting the energy metabolism of the body and enhancing the immune regulation.The material basis of Maca to playing important property is maca polysaccharide.The mechanism of action of maca polysaccharide on enhancing energy metabolism of the body is promoting the activities of Na+-K+-ATPase,Ca2+-Mg2+-ATPase andF0F1-ATPase.It increased the gene expression of NADH and SDH and VDAC as well as its protein levels in liver tissue of mice with spleen deficiency.The mechanism of maca polysaccharides promoting immune regulation is to regulate the expression of T-bet and GATA-3 by regulating the levels of immune cytokines IFN-?,TNF-?,IL-2 and IL-4,and maintain the body Th1./Th2 cell subsets balance;reduce the expression of proapoptotic factors BAX,Caspase-3 protein,increase the expression level of apoptosis inhibitor Bcl-2 protein,inhibit the apoptosis of spleen lymphocytes,thereby playing an immunomodulatory role.