| Objective:In order to explore the optimal ratio and extraction method of the ginseng poria cocos drug pair with the best anti aging activity,screening was conducted based on the Saccharomyces cerevisiae system as a model,and a preliminary study was conducted on its activity and mechanism of action from the perspective of antioxidant stress and energy metabolism.After selecting the best ginseng poria cocos compatibility with anti aging activity,a fingerprint of ginseng poria cocos was established using high-performance liquid chromatography,and the content of its common components was determined to analyze the spectrum effect relationship.To explore the anti aging effect of ginseng tuckahoe on aging rats with spleen deficiency;A preliminary study was conducted on the protective mechanism of Ginseng Poria cocos medicinal pair on the niche of spleen matrix in aging rats.Methods:1.Screening and mechanism study on anti aging activity of ginseng poria cocosUsing Saccharomyces cerevisiae as a model organism,the growth curve of yeast was plotted using MTT method to determine the growth cycle;Screening was conducted on the compatibility ratio,dosing concentration,and dosing time.Colorimetry was used to detect antioxidant related enzyme activities,ROS,and MDA content in yeast cells.Scanning electron microscopy was used to observe the morphology of yeast cells,and plate sampling method was used to carry out resistance experiments.The changes of MMP were detected by flow cytometry.Real-time fluorescence quantitative PCR was used to detect the expression level of antioxidant and energy metabolism related genes in Saccharomyces cerevisiae.2.Analysis of anti aging components of ginseng poria cocos and study on spectrum effect relationshipUsing HPLC to optimize the chromatographic conditions of 10 batches of Ginseng Poria cocos and complete methodological investigations,establish HPLC fingerprints,import the fingerprints into the "Chinese herbal chromatographic fingerprint similarity evaluation system software" to evaluate the similarity of the fingerprints,use high-performance liquid chromatography to analyze the optimal composition of the selected Ginseng Poria cocos drug pairs,and determine the content of the identified components,Compare the different components of different batches of samples.The relationship between antioxidant activity and characteristic components was analyzed through correlation analysis using spectral efficiency relationship research methods.3.Study on the anti aging effect of ginseng tuckahoe medicine on aging rats with spleen deficiencyThe spleen and stomach aging model was established using D-galactose combined with irregular diet,and the external behavior characteristics and weight changes of rats were recorded.The pathological morphology of the spleen was analyzed through behavioral tests,organ index,Elisa detection of serum biochemical indicators,and HE staining β-Galactosidase staining was used to analyze the proportion of positive cells and evaluate the anti aging efficacy of the extract with the best ratio of ginseng tuckahoe drug pair.4.Study on the regulatory effect and mechanism of ginseng tuckahoe medicine on the niche of spleen matrixTo establish a cell injury model induced by hydrogen peroxide(H2O2),the appropriate concentrations of Raw264.7 and 3T3-L1 to induce oxidative stress were selected using the CCK-8 method.CCK-8 method confirmed the appropriate concentration of Ginseng Poria drug pair that has a promoting effect on 3T3-L1 cell proliferation;Elisa measured the changes of antioxidant enzyme activity,ROS,MDA content in Raw264.7 cells before and after administration;The phagocytic capacity of Raw264.7 cells was measured by neutral red staining method;Western blot was used to detect the effects of ginseng poria cocos on the expression of iron metabolism related proteins HO-1,VCAM1,and SLC40A1 in spleen tissue and Raw264.7 cells,phagocytosis related protein CD163,and WT1 in CSF1 and3T3-L1 cells.Scanning electron microscopy was used to observe the effect of ginseng tuckahoe on the ultrastructure of spleen,and Prussian blue staining was used to analyze the effect of ginseng tuckahoe on the iron metabolism function of spleen.Immunofluorescence assay was used to analyze the effect of Ginseng Poria cocos on the expression of F4/80,WT1 and the red pulp reticular structure.Results:1.Screening and mechanism study on anti aging activity of ginseng poria cocosAfter screening,the anti-aging ability of the 1:4 aqueous extract of Ginseng Poria cocos is superior to other formulations,and the determined dose concentration is 220 μg/m L,and the dose time is 28 hours.Ginseng Poria cocos 1:4 aqueous extract can significantly enhance the antioxidant related enzyme activities of SOD,CAT,and POD in Saccharomyces cerevisiae,and reduce the content of ROS and MDA.According to flow cytometry,the 1:4 aqueous extract of Ginseng Poria cocos can effectively reduce the downward trend of mitochondrial membrane potential,while significantly increasing the ATP content in yeast cells.At the q PCR level,the ginseng poria cocos 1:4 aqueous extract significantly downregulated the antioxidant related genes SOD1 in Saccharomyces cerevisiae,and upregulated the expression of GSH1 and CTT1 genes;Significant downregulation of ATP1 and CDC19 and upregulation of MRS1 gene expression were observed for energy metabolism pathway related genes.According to the electron micrograph,it can be clearly seen that the ginseng poria cocos 1:4 water extract has an anti aging effect on yeast cells.According to the resistance test results,the ginseng poria cocos1:4 water extract can enhance the growth ability of yeast in the face of environmental stress.2.Analysis of anti aging components of ginseng poria cocos and study on spectrum effect relationshipThe HPLC method was used to examine the methodology,and the results showed that the relative retention time of the common peak(RSD)was less than 1%,and the RSD of the relative peak area was less than 3%.A 1:4 water extract fingerprint of 10 batches of ginseng poria cocos was established,and the similarity between each batch was>0.900,indicating good similarity.Determine the content of the identified common peak and calculate its content.Through correlation analysis,there is a good linear relationship between the common peak area and antioxidant activity of fingerprints of different batches of samples,while ginsenosides Rg1,Rb1,Rg3,and poric acid have significant correlation coefficients for antioxidant activity.3.Study on the Protection of Ginseng Poria Pill on Spleen and Stomach Functions in Senile Rats with Spleen DeficiencyGinseng Poria cocos 1:4 water extract can increase the weight of aging rats with spleen deficiency,enhance their autonomous activity ability,and increase the number of times to cross,stand,and modify;Improve spleen organ index and improve spleen atrophy caused by aging.According to Elisa testing,the 1:4 aqueous extract of ginseng poria cocos increased the activity of antioxidant enzymes in rat serum,decreased the content of ROS,AGEs,and MDA,and alleviated oxidative stress;Increase the content of Ig G and IL-2 in rat serum and reduce TNF-α、IL-6 content regulates immune function;It can repair spleen damage caused by D-galactose,restore normal morphology,and have a clear boundary between red and white pulp;At the same time,the proportion of aging positive cells was reduced.4.Study on the regulatory effect and mechanism of ginseng tuckahoe medicine on the niche of spleen matrixGinseng Poria cocos 1:4 aqueous extract can increase the phagocytic ability of RAW264.7 to neutral red and increase the expression of CD163 in cells and spleen tissues,proving that it can restore the phagocytic function of macrophages.Ginseng Poria cocos 1:4aqueous extract can promote the proliferation of 3T3-L1 cells,increase the expression of WT1 in cells and spleen tissue,and maintain the normal function of fibroblasts.Ginseng Poria cocos 1:4 aqueous extract can avoid iron accumulation in the spleen of aging rats,increase the expression of VCAM1,HO-1,and SLC40A1 in cells and spleen tissues,and regulate the iron metabolism function of the spleen.At the same time,it improves the ultrastructure of the spleen in aging rats and restores the normal internal structure of the spleen.Ginseng Poria cocos 1:4 aqueous extract can also increase the expression of F4/80 and WT1,maintain the number of splenic red pulp macrophages and reticulofibroblasts,and stabilize their structures.Conclusion:By using Saccharomyces cerevisiae as a model tool to screen the anti aging activity of Ginseng Poria cocos drugs,it is concluded that the best activity is the 1:4 water extract of Ginseng Poria cocos.By using HPLC technology to analyze the components of the 1:4water extract of Ginseng Poria cocos and establish a fingerprint,it was found that the most influential factors on anti aging activity were Rg1,Rb1,Rg3,and Poria cocos acid.Ginseng Poria Cocos 1:4 aqueous extract can alleviate oxidative stress,improve immune function,restore normal spleen morphology,and reduce aging spleen cells to achieve anti aging effects.Ginseng Poria cocos 1:4 aqueous extract can maintain a stable structure between splenic red pulp macrophages and splenic red pulp fibroblasts,regulate macrophage phagocytosis and iron metabolism,avoid iron accumulation,and restore the spleen’s blood filtration function,thereby achieving the homeostasis of the spleen matrix niche.The research results have clarified the protective effect of ginseng tuckahoe on aging rats with spleen deficiency,providing theoretical and experimental basis for the development of ginseng tuckahoe related drugs and health food. |
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