CN115887485A - Application of pachyman in regulating intestinal microbial structure and metabolite of obese organism - Google Patents
Application of pachyman in regulating intestinal microbial structure and metabolite of obese organism Download PDFInfo
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Abstract
The research of the invention shows that the pachymaran has the effects of preventing and treating the weight gain amplitude, fat accumulation, hyperlipidemia, intestinal microorganism imbalance, short-chain fatty acid, bile acid, fat droplet accumulation in liver and cell damage of obese mice caused by high-fat diet, and provides a new visual angle for the fields of biomedicine, health care products and the like in the treatment of obesity, hyperlipidemia, non-alcoholic fatty liver disease and chronic metabolic disorder.
Description
The technical field is as follows:
the invention belongs to the field of biological medicines and health care products, and particularly relates to an application of pachyman in regulating intestinal microbial structures and metabolites thereof of obese organisms.
Background art:
with the development of society, the increase of living standard, and the increase of fat accumulation in human body caused by poor dietary habits, environment, lack of motion, unstable mood and other factors, thereby causing obesity. In recent years, the prevalence rate of obesity in China is obviously increased, and the obesity is a serious public health problem. Obesity not only simply causes weight gain, but its severity is such that it induces diseases such as heart disease, diabetes, hypertension, renal failure, etc., and is a "source of constant morbidity. Hyperlipidemia is one of the complications of obesity, and is manifested by high levels of Triglyceride (TG), total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), and the elevation of these lipid markers further leads to lipid metabolism disorder, inducing fatty liver. While fatty liver adversely affects fat metabolism, aggravates hyperlipidemia and obesity, and thus forms a vicious circle. Therefore, effective prevention and treatment of obesity is a hot issue for research in metabolic disorders.
The edible fungus polysaccharide has good pharmacological action and almost no toxic or side effect on human bodies. In recent years, a large number of researches show that the edible fungus polysaccharide can reduce blood fat by inhibiting the synthetic pathway of total cholesterol and triglyceride, activating an AMPK signal pathway, regulating intestinal flora and the like, improve lipid metabolism disorder and oxidative stress reaction of obese mice and achieve the effects of relieving insulin resistance, resisting inflammation and resisting tumors. Such as ganoderma lucidum polysaccharide, coriolus versicolor polysaccharide, auricularia auricula polysaccharide, polyporus umbellatus polysaccharide and the like, can reduce the level of total cholesterol and triglyceride, and can play roles in reducing blood fat and improving metabolic disorder by regulating the expression of genes related to intestinal flora, short-chain fatty acid and glycolipid.
Poria is the dry sclerotium of Poria cocos (Schw.) Wolf of Polyporaceae, is rich in beta-pachymaran, triterpenoids, ergosterol, amino acids and other active ingredients, is one of the commonly used traditional Chinese medicine adjuvants in China, and has obvious medicinal effects of promoting urination, protecting digestive system and tranquilizing nervous system. In vitro cell experiments prove that the pachyman can effectively inhibit hepatitis B virus and protect liver tissues; in vivo animal experiment shows that pachyman can enhance the functions of lymphocyte and macrophage to achieve the effect of enhancing organism immunity. At present, the effects of pachymaran on improving diabetes, hypertension, non-alcoholic fatty liver disease and the like are reported. However, the effects of pachyman on intestinal microorganisms, short-chain fatty acids and bile acids in the body during the treatment of obesity have not been reported.
The invention content is as follows:
the invention aims to explore the prevention and treatment effects of pachyman on weight gain amplitude, fat accumulation, hyperlipidemia, intestinal microbial imbalance, short-chain fatty acid, bile acid, fat drop accumulation in liver and cell destruction of fat mice caused by high-fat diet, and provide a new perspective for the treatment of obesity, hyperlipidemia, non-alcoholic fatty liver disease and chronic metabolic disorder in the fields of biomedicine, health products and the like.
In order to achieve the purpose, the technical scheme of the invention is as follows:
use of pachyman as a medicament or food for increasing the content of beneficial microorganisms and decreasing the content of harmful microorganisms in the intestinal tract of an obese organism for the modulation of the microbial structure of the intestinal tract of an obese organism.
In a further improvement, the beneficial microorganisms include microorganisms of the genera Ackermansia, bacteroides, enterobacter murinus, achillea, and Bifidobacterium; the harmful microorganisms include microorganisms of the genera faecalis, bacillus, lactobacillus, roche and lactobacillus.
In a further improvement, the pachyman is used as a medicament or food for reducing the ratio of firmicutes microorganisms to bacteroidetes microorganisms in the intestinal tract of obese organisms.
Use of pachyman for regulating metabolites of an obese body as a medicament or food for increasing glucose tolerance levels and lowering serum triglyceride, total cholesterol and low density lipoprotein cholesterol levels in an obese body.
Use of pachyman for the regulation of metabolites in an obese body as a medicament or food for reducing the diameter of epididymal adipocytes in an obese body.
Use of pachyman for regulating metabolites of an obese body as a medicament or food for the treatment of non-alcoholic fatty liver disease.
In a further improvement, the pachyman is used as a medicine or food for an obese body to increase the total short chain fatty acid, propionic acid, butyric acid, valeric acid and isobutyric acid content.
In a further improvement, the pachyman is used as a medicament or food for reducing the fatty acid content in the liver of an obese organism.
In a further improvement, the fatty acids include 7-ketodeoxycholic acid, 3 beta-glycocholic acid, cholic acid 7 sulfate, glycochenodeoxycholic acid 3 sulfate disodium salt, ursolic acid and glycoursodeoxycholic acid 3 sulfate sodium salt.
In a further improvement, the tuckahoe is used as a medicine or food for improving the morphology of liver cells, slowing down the accumulation of lipid in the liver, down-regulating the activity of liver glutamic pyruvic transaminase and improving the pathological degree of liver fat vacuole.
The invention has the following advantages:
(1) The pachyman can resist the weight increase (9.26%) of obese mice caused by high-fat feeds, obviously reduce the relative weight increase rate (21.64%) of the obese mice, the Lee's index (5.24%), the adipose tissue coefficient and the size of epididymis fat cells, and further achieve good weight-losing effect.
(2) The pachyman can obviously improve glucose tolerance abnormity (5.40-19.67%) of obese mice, reduce triglyceride (20.57%), total cholesterol (26.47%) and low-density lipoprotein cholesterol (41.38%) in serum, and further achieve the purpose of reducing blood fat.
(3) The pachyman can remarkably reduce the activity of glutamic-pyruvic transaminase of the liver (43.22%), improve the pathological change degree of fat vacuole of the liver and further achieve the effect of protecting the liver.
(4) The pachymaran can remarkably reduce the abundance of unfavorable flora of the genera of coprobacterium, bacillus, lactobacillus, ralstonia and lactobacillus and improve the abundance of beneficial flora of the genera akkermansia, bacteroides, enterobacter, corynebacterium and bifidobacterium.
(5) The pachyman provided by the invention can obviously improve the content of total short-chain fatty acid, propionic acid, butyric acid, valeric acid and isobutyric acid.
(6) The pachyman obviously reduces the content of 7-ketodeoxycholic acid, 3 beta-glycocholic acid, cholic acid 7 sulfate, glycochenodeoxycholic acid 3 sulfate disodium salt, ursolic acid and glycoursodesoxycholic acid 3 sulfate sodium salt.
Drawings
FIG. 1 is a graph showing the effect of pachyman on glucose tolerance in hyperlipidemic obese mice. Control group: a common feed group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; lower case, significant difference (P < 0.05); capital letters, very significant differences (P < 0.01).
FIG. 2 shows the effect of pachymaran on body weight (A), body weight gain (B), body weight gain rate (C) and Lee's coefficient (D) in hyperlipidemic obese mice. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; ns, no significant difference; * Significant difference (P < 0.05); * Very significant differences (P < 0.01).
FIG. 3 shows the effect of pachymaran on the weight and morphology of epididymal adipose tissue (A), inguinal adipose tissue (B), and brown adipose tissue (C) in hyperlipidemic and obese mice. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; ns, no significant difference; * Significant difference (P < 0.05); * Very significant differences (P < 0.01).
FIG. 4 is a pathological section (200X) of mouse epididymal adipocytes observed by HE staining under an optical microscope. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group.
FIG. 5 shows the effect of pachyman on triglyceride (A), total cholesterol (B) and low density lipoprotein cholesterol (C) in high fat obese mice. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; ns, no significant difference; * Very significant differences (P < 0.01).
FIG. 6 is a photograph of a stained section of the liver pathology of a mouse (200X) under an optical microscope and the effect of pachyman on the liver-related index of a hyperlipidemic obese mouse. A, influence of pachymaran on glutamic-pyruvic transaminase activity in mouse serum; b, influence of pachymaran on liver coefficients of obese mice; c, mouse liver HE staining pathology section (200 ×); d, mouse liver oil red O stained section (200X). Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group.
FIG. 7 Effect of pachymaran on gut microbiota levels in hyperlipidemic obese mice. A, different treatment groups have relative horizontal content; b, influence of pachymaran on the relative content of firmicutes in the high-fat obese mice; c, influence of pachymaran on relative content of Bacteroides bacteroidetes in high-fat obese mice; d, influence of pachymaran on relative content ratio of firmicutes and bacteroidetes in the mice with high fat obesity. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; ns, no significant difference; * Significant difference (P < 0.05); * Very significant differences (P < 0.01).
FIG. 8 Effect of pachymaran on the level of gut microbiota in hyperlipidemic obese mice. A, the relative content of different treatment groups at the same level; b, influence of pachymaran on relative content of different genera in the firmicutes of the high fat obese mice; and C, influence of pachymaran on relative content of Chinese genuses of Bacteroides, microbactria wartii and Actinomycetes in the high fat obese mouse. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; ns, no significant difference; * Significant difference (P < 0.05); * Very significant differences (P < 0.01).
FIG. 9 Effect of pachyman on the short chain fatty acid content in high fat obese mice. Control group: normal diet group; high fat group: a group of high fat diets; polysaccharide group: high fat diet + pachyman group; ns, no significant difference; * Significant difference (P < 0.05); * Very significant differences (P < 0.01).
Figure 10 thermograph and fold-difference analysis of bile acid content of hyperlipidemic obese mice with pachymaran.
Detailed Description
The present invention will be described in detail with reference to specific examples. The experimental methods used in the examples are all conventional experimental methods unless otherwise specified. Materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Pachyman is purchased from Jing Zhou Tongtian pharmacy.
The SPF male C57BL/6J mice are provided by Shenzhen Seibano Gene technology, inc., with license number: SCXK (Jing) 2019-0008.
Example 1
Grouping, modeling and dosing of mice, which comprises the following specific steps:
1. grouping mice: the experimental animals are SPF male C57BL/6J mice, the age of 7 weeks, the weight of 19-21 g, and 90 animals are raised in the center of the experimental animals of the institute of microbiology, guangdong province, the environmental temperature is 21 +/-2 ℃, the humidity is 55 +/-10%, and the light and the dark are alternated for 12 hours. After 1 week of acclimatization, the groups were randomly divided into a control group, a high fat group and a pachyman group, with 10 individuals per group per 3 groups treated.
2. Molding and administration: the control group mice were fed with normal feed (Jiangsu province cooperative medical biotechnology, llc), the high-fat group and pachyman group mice were fed with 60% high-fat feed (D12492 mass ratio: casein 200, l-cystine 3, sucrose 68.8, maltodextrin 12.5, cellulose 50, soybean oil 25, lard 245, mineral 10, vitamin 10, choline tartrate 2, potassium citrate monohydrate 16.5, calcium carbonate 5.5, calcium hydrogen phosphate dihydrate 13, energy ratio: carbohydrate 20, protein 20, fat 60, changzhou mice-mouse two-biotechnology, ltd.). After 8 weeks of feeding, the control group and the high fat group mice were gazed with 0.8% physiological saline (100. Mu.L/d), and the polysaccharide group mice were gazed with 400mg/kg/d pachymaran solution, during which the mice freely ate and drink water, and the weight and food intake of the mice were recorded weekly. When the mice were gavaged for 14 weeks, the weight of the mice in the control group and the pachyman group was significantly different from that in the high fat group.
Example 2
Measuring relevant indexes such as weight loss and observing pathological liver slices.
1. Effect of pachymaran on glucose tolerance in obese mice:
two weeks before collecting samples, the patient takes no food for 12h, blood is taken by sterilized surgical tail cutting, and the fasting blood glucose value of the mouse is tested by a glucometer to be used as the initial value (0 min) of the oral glucose tolerance test OGTT. Mice were subjected to intragastric administration with a glucose solution (1 g/kg body weight), and blood glucose values were measured 15min, 30min, 60min, 90min and 120min after intragastric administration. Specific data are shown in table 1.
TABLE 1 influence of pachyman on glucose tolerance in obese mice
Note: a, b, c, significance difference (P < 0.05); a, B, C, very significant difference (P < 0.01).
As shown in fig. 1 and table 1, after the pachyman intervention for 12 weeks, compared with the control mice, the OGTT was 15min, 30min, 60min and 90min, the glucose concentration in the blood of the high-fat mice and the pachyman mice was significantly increased (17.70% -40.90%), and the glucose concentration in the blood of the polysaccharide mice was significantly decreased (5.40% -19.67%) compared with the high-fat mice, indicating that the pachyman could effectively improve the glucose tolerance of the obese mice.
2. Effect of pachyman on body weight and related indices of obese mice:
the mice gavaged for 14 weeks were fasted for 24 hours, the body weights were weighed, the body lengths (distance from the tip of the nose to the anus) were measured, and the weight gain weight (final weight-adaptation to 1-week-weight) and the weight gain rate = (final weight-adaptation to 1-week-weight) were calculatedWeekly weight)/adaptation to 1 week weight, lee's index = (body weight) 1/3 X 10/body length. Specific data are shown in table 2.
TABLE 2 influence of pachyman on body weight and related indices in obese mice
Note: a, b, c, very significant difference (P < 0.01).
As shown in fig. 2 and table 2, after the pachyman intervention for 14 weeks, the body weight, body weight gain rate and Lee's index of the mice in the high fat group were significantly increased by 16.02%, 55.36%, 55.53% and 6.51%, respectively, compared with those in the control group, while the body weight and Lee's index of the mice in the pachyman group were not significantly different; compared with the high-fat group, the body weight correlation coefficient of the mice in the pachyman group has very significant difference, the body weight is reduced by 9.26%, the body weight increase is reduced by 20.95%, and the Lee's index is reduced by 5.24%, which indicates that the pachyman can resist the weight increase of the mice caused by high-fat feed.
3. Effect of pachyman on adipose tissue in obese mice:
the mice after 14 weeks of gastric lavage were fasted for 24h with CO 2 After anesthesia, cervical dislocation was sacrificed, brown fat, inguinal fat and epididymal adipose tissue were taken, the weight was recorded, and the adipose tissue correlation coefficient was calculated. Specific data are shown in table 3.
TABLE 3 influence of pachyman on adipose tissue-related index in obese mice
Note: a, b, c, very significant difference (P < 0.01).
As shown in table 3 and fig. 3, after 14 weeks of pachyman intervention, the epididymal fat coefficient and inguinal fat coefficient of the mice in the high fat group were significantly increased (53.51% and 39.28%), while the inguinal fat coefficient of the mice in the polysaccharide group was significantly increased only (28.26%); compared with the high-fat mice, the fat factors of the epididymis and the inguinal fat factor of the polysaccharide mice are obviously reduced by 34.69% and 15.36%, respectively (A and B in figure 3); in addition, the brown fat index of the mice in the high-fat group and the polysaccharide group is obviously lower than that of the mice in the control group (reduced by 40.75%), and the coefficient of the mice in the polysaccharide group is significantly different between the mice in the control group and the high-fat group and is increased by 51.60% (C in figure 3). From the adipose tissue morphology (D in fig. 3), pachyman decreased the length and width of epididymal fat and inguinal fat in obese mice. The results show that pachyman can effectively reduce the fat of the rest part of the body of the high-fat-induced obese mouse.
4. Observation of fat mouse epididymis fat cell HE staining pathological section:
fixing the epididymis adipose tissue in 10% paraformaldehyde solution, dehydrating with 80%, 90%, 95%, and 100% ethanol in sequence, performing xylene transparency treatment, embedding in paraffin, and slicing; the slices are decolorized in xylene for 20min, absolute ethyl alcohol for 5min, 75% ethyl alcohol for 5min and tap water in sequence, and then placed in hematoxylin for dyeing for 3-5 min; and after eosin dyeing for 5min, sequentially decoloring and transparentizing in absolute ethyl alcohol for 5min and xylene for 5min, and finally sealing by using neutral gum. The pathological features of epididymal adipocytes were observed by electron microscopy (Leica, DM6/DMC 4500).
According to the attached figure 4, after the pachymaran intervention for 14 weeks, the diameter of epididymal fat cells of the mice in the high fat group is obviously increased, and the number of fat cells in the same visual field range is obviously reduced compared with the control group of mice. Compared with the mice in the high fat group, the diameter of the epididymal fat cells of the mice in the polysaccharide group is obviously reduced, and the number of the fat cells in the same visual field range is obviously increased. The above results indicate that pachyman can significantly reduce the diameter of epididymal adipocytes.
5. Influence of pachyman on serum-related indices in obese mice:
mice gavaged for 14 weeks were fasted for 24h with CO in closed cages 2 Anaesthetizing, taking blood from abdominal cavity, standing at room temperature for 1h, centrifuging at 3000rpm for 15min, and collecting upper layer serum. Use the kit (purchase in Nanjing to build up bioengineering research)In serum, triglyceride, total cholesterol and low density lipoprotein cholesterol levels are determined, and the specific data are shown in table 4.
TABLE 4 influence of pachymaran on mouse serum index
Note: a, b, c, very significant difference (P < 0.01).
As can be seen from table 4 and fig. 5, after 14 weeks of pachyman intervention, the serum content of triglyceride (a in fig. 5), total cholesterol (B in fig. 5) and low-density lipoprotein cholesterol (C in fig. 5) in the mice in the high-fat group was significantly increased (increased by 17.56%, 42.61% and 37.75%) compared with the control mice, while the serum content of triglyceride and low-density lipoprotein cholesterol in the mice in the polysaccharide group was not significantly different, and the blood lipid index of 3 blood lipids in the serum of the mice in the polysaccharide group was significantly lower than that of the mice in the high-fat group, and was respectively reduced by 20.57%, 26.47% and 41.38%. The results show that the pachymaran can effectively reduce the relevant indexes of hyperlipidemia in the serum of fat mice induced by high-fat diet.
6. The influence of pachyman on related indexes of the liver of an obese mouse and pathological section observation thereof are as follows:
the gavage 14-week mice were fasted for 24h with CO 2 Anaesthetizing, taking blood from the abdominal cavity, dislocation and killing the cervical vertebra, taking liver tissues, weighing, and calculating the liver correlation coefficient (%) = the weight of the liver tissues/the weight of the mouse. The liver was cut into two portions, and one portion was soaked in 4% paraformaldehyde tissue fixative. Slicing the liver tissue, and observing an HE stained section in the synchronous step 4; simultaneously, carrying out oil red O infection for 8-10 min (keeping out of the sun), taking out the slices, staying for 3s, immersing in 60% isopropanol for differentiation for 2 times for 3s and 5s, and then sequentially immersing in pure water for immersion washing for 2 times, each for 10s; hematoxylin staining is carried out for 3-5min, 60% ethanol is differentiated for 2-8 s, distilled water is washed for 10s, bluing liquid is bluing for 1s, tap water is used for immersion washing for 10-15 s, and finally, glycerol gelatin is used for sealing tablets and sealing tablets. Changes in lipid droplets in the liver were observed by electron microscopy (Leica, DM6/DMC 4500).
According to a in fig. 6, the glutamic-pyruvic transaminase activity (related to the degree of liver cell destruction) was significantly reduced (47.86% and 43.22% reduced) in the sera of the control group and the polysaccharide group mice, compared with the high fat group mice, without significant difference therebetween. The liver coefficient of the control group mouse was significantly higher than that of the high-fat group and polysaccharide group mice, and the liver coefficient of the polysaccharide group mouse was significantly higher than that of the high-fat group mice by 16.41% (B in fig. 6).
The HE staining result under an optical microscope shows that (the cell nucleus is blue, the cytoplasm is red), liver cells of mice in a high-fat group have obvious lipid vacuole pathological changes, the area of total lipid vacuoles is large, and the cell boundary and the cell shape are fuzzy; while the liver cell boundaries and morphology of the control and polysaccharide mice were clear and regular with less lipid vacuolar lesions (C in fig. 6). The observation result of an oil red O staining microscope shows that (D in figure 6), compared with the control group of mice, the liver of the high-fat group of mice obviously contains more orange fat drops, and more expanded bubbles exist among cells; the livers of the polysaccharose mice are very light orange red, and the cells have fewer expanded air bubbles. The results show that the pachyman can effectively improve the cell morphology of the liver, slow down the accumulation of lipid (such as lipid droplets) in the liver and improve the fatty liver caused by high-fat feed.
Example 3
Determination of intestinal microorganisms, short-chain fatty acids and bile acids
1. Effect of Pachymaran on intestinal flora of obese mice
And (3) extracting the DNA of the mouse feces of the groups of mice by a paramagnetic particle method, and diluting the sample to 1 ng/mu L by using sterile water after the quality is qualified. And (3) taking the diluted genome DNA as a template, carrying out PCR by adopting V3-V4 region primers (515F and 806R) and high-efficiency high-fidelity enzyme, and recovering a target band by adopting a test heaven root kit. Use of
The DNA PCR-Free Sample Preparation Kit library construction Kit (Illumina) is used for constructing a library, the constructed library is quantified by Qubit and Q-PCR, and after the library is qualified, novaSeq6000 is used for on-machine sequencing. Adopting software FLASH and QIIME to obtain effective data, and making alpha moreSample, PCA and intestinal microbial differential analysis.
Intestinal tract microbial sequencing results show that the relative content of firmicutes in the high fat obese mice is 60.98 percent, the relative content of bacteroidetes is 18.71 percent, and the relative content of verrucomicrobia is 10.00 percent; in the control group and the polysaccharide group, the relative content of firmicutes was 37.94% and 10.33%, the relative content of bacteroidetes was 40.21% and 34.42%, and the relative content of verrucomicrobia was 15.79% and 14.44%, respectively (a-C in fig. 7). In addition, the ratio of firmicutes and bacteroidetes in the high-fat group was significantly higher than that in the control group and polysaccharide group (D in fig. 7). A large number of researches show that the abundance of firmicutes in intestinal microorganisms of obese mice is obviously improved, and the abundance of bacteroidetes is obviously reduced, which is consistent with the research results of the inventor. At the genus level, the relative content of faecalis, bacillus, xenobacter, lactobacillus, rochlia, rombobacillus, citrobacter and lactobacillus in the intestinal tract of hyperlipidemic obese mice increased, while the relative content of akmansia, bacteroides, jejunus, cladosporium and bifidobacterium decreased. After the gastric pachyman is infused, the relative content of the bacillus, lactobacillus, rochlya and lactobacillus is obviously reduced, and the relative content of the akkermansia, bacteroides, enterobacter, corynebacterium and bifidobacterium is obviously increased. Research shows that Ackermans, bacteroides, enterobacter murinus, mycobacterium and Bifidobacterium can obviously reduce the body weight, hyperlipidemia and glucose resistance of high-fat induced obese mice. The results suggest that pachymaran can improve intestinal microbial disturbance of high fat induced obese mice, increase the content of beneficial microorganisms and reduce the content of harmful microorganisms.
2. Influence of pachyman on short-chain fatty acid in feces of high-fat-induced obese mice
Weighing 20mg of mouse feces, adding 1mL of 0.5% phosphoric acid (v/v) buffer solution, adding a small steel ball, performing ball milling for 10s under 20Hz, repeating for 2 times, performing vortex for 10min, and performing ultrasonic treatment for 5min under ice bath (maximum vortex frequency). Centrifugation was carried out at 12000rpm for 10min at 4 ℃ to obtain 100. Mu.L of supernatant, 500. Mu.L of MTBE solvent containing the internal standard was added thereto, and vortexed for 3min. And (3) performing ultrasonic treatment for 5min in ice bath, centrifuging for 10min at 4 ℃ and 12000rpm, and taking supernatant for GC-MS/MS analysis. GC-MS/MS analysis was carried out using a gas chromatograph Agilent 7890B in combination with a mass spectrometer 7000D, with a column (DB-FFAP, 30 m. Times.0.25 mm. Times.0.25 μm) with helium as carrier gas, a flow rate of 1.2mL/min and a sample size of 2 μ L. The temperature of the oven is kept at 90 ℃, after 1min, the temperature is increased to 150 ℃ at the speed of 20 ℃/min, the temperature is kept for 0.6min, the temperature is increased to 200 at the speed of 25 ℃/min, and the temperature is kept for 0.5min. All samples were analyzed in a multiple reaction monitoring mode with inlet and transfer line temperatures of 200 ℃ and 230 ℃ respectively.
According to the formula 9, the content of total short-chain fatty acids in the excrement of the high-fat induced obese mouse is remarkably reduced, and the content of propionic acid, butyric acid and valeric acid is remarkably reduced; after the pachyman is infused into the stomach, the content of total short-chain fatty acid, propionic acid, butyric acid, valeric acid and isobutyric acid is obviously increased. Therefore, pachyman can reduce hyperlipidemia and liver damage caused by obesity by regulating multiple short chain fatty acids.
3. Influence of pachyman on bile acid in liver of high fat-induced obese mice
Taking a frozen liver sample of 20mg, adding a steel ball, adding 10 mu L of internal standard mixed working solution with the concentration of 1 mu g/mL and 200 mu L of 20% methanol acetonitrile for homogenate; shaking at 2500rpm for 10min, and placing in a refrigerator at-20 deg.C for 10min; centrifuging at 12000rpm for 10min at 4 deg.C, collecting supernatant, concentrating in a concentrator, re-dissolving with 100 μ L50% methanol water, and performing ultra performance liquid chromatography (ExionLC) TM AD) and tandem Mass Spectrometry (
6500 +) to be detected. And (3) chromatographic column: waters ACQUITY UPLC HSS T3C 18 column (1.8 μm,100 mm. Times.2.1 mm); mobile phase: phase A is ultrapure water (containing 0.01% acetic acid and 5mmol/L ammonium acetate), and phase B is acetonitrile (containing 0.01% acetic acid); the flow rate was 0.35mL/min, the column temperature was 40 ℃ and the amount of sample was 3. Mu.L. The fold change is greater than 2 or less than 0.5 and VIP ≥ 1 and P<0.05 bile acid as differential bile acid.
According to fig. 10, the abundance of bile acids in the livers of the polysaccharose mice was closer to the abundance of bile acids in the livers of the control mice. High fat diet induced significant increase of bile acid content in liver of obese mice, and significant up-regulation of 18 kinds of primary, secondary or tertiary bile acid content, wherein 7, 12-diketolithocholic acid, 7-ketodeoxycholic acid, 3 beta-deoxycholic acid, ursocholic acid and isocynodeoxycholic acid respectively increased by 14.87, 9.14, 4.59, 4.16 and 4.00 times, and only 6, 7-diketolithocholic acid appeared significantly down-regulated. After the gavage pachyman is added, the content of bile acid in 10 livers of high-fat diet obese mice is obviously reduced, wherein the content of 7-ketodeoxycholic acid is reduced to 4.86 percent, and the content of 3 beta-glycocholic acid, cholic acid 7 sulfate, glycochenodeoxycholic acid 3 sulfate disodium salt, ursolic acid and glycoursodeoxycholic acid 3 sulfate sodium salt is reduced to the level similar to that of a control group of mice. The content of bile acid in the liver of an obese mouse is increased, so that energy metabolism abnormality such as carbohydrate metabolism, lipid metabolism and the like in the liver is caused, the accumulation of cholesterol and fat is further caused, and liver cells are damaged; the pachyman can obviously reduce the content of bile acid in the liver, and accumulate lipid drops in the liver and damage liver cells.
In conclusion, the invention aims to research the influence of pachyman on glucose tolerance, weight gain amplitude, adipose tissue, serum blood lipid index, liver tissue, intestinal microorganisms, short-chain fatty acid and bile acid of high-fat-induced obese mice, and finally proves that the intervention of pachyman can obviously reduce the weight, the weight gain rate, the Lee's index, the epididymal fat and inguinal fat coefficient, the triglyceride content, the total cholesterol content, the low-density lipoprotein cholesterol content and the glutamic-pyruvic transaminase activity, and obviously increase the brown adipose tissue coefficient, the liver coefficient and the glucose tolerance; increasing the abundance of beneficial bacterial populations of Ackermans, bacteroides, enterobacter murinus, achillea species and Bifidobacterium, and decreasing the abundance of undesirable bacterial populations of Achnum, bacillus, lactobacillus and Roseburia; increasing the content of total short-chain fatty acid, propionic acid, butyric acid, valeric acid and isobutyric acid, reducing the content of various bile acids in the liver, and further playing a role in protecting the liver.
Therefore, the pachyman has positive effects of relieving obesity, reducing blood fat, improving intestinal microbial flora of obese organisms, increasing short-chain fatty acid, reducing liver bile acid and protecting liver. The invention provides the application of pachyman in weight reduction, lipid reduction and liver protection, and provides a new resource for the development of related products in the fields of biological medicines and health care products.
The above list is only a few embodiments of the present invention. However, the present invention is not limited to the above embodiments, and there are many variations. All modifications which can be derived or suggested by the person skilled in the art from the present disclosure are to be considered within the scope of the present invention.
Claims (10)
1. The application of pachyman in regulating the intestinal microbial structure of obese organisms is characterized in that the pachyman is used as a medicine or food for increasing the content of beneficial microorganisms and reducing the content of harmful microorganisms in the intestinal tracts of the obese organisms.
2. The use of pachyman for the modulation of the intestinal microbial structure of an obese body as claimed in claim 1, wherein said beneficial microorganisms comprise microorganisms of the genera akkermansia, bacteroides, enterobacter murinus, corynebacterium and bifidobacterium; the harmful microorganisms include microorganisms of the genera faecalis, bacillus, lactobacillus, roche and lactobacillus.
3. The use of pachyman for the modulation of the intestinal microbial structure of an obese body according to claim 1, wherein said pachyman is used as a medicament or food for reducing the ratio of firmicutes microorganisms and bacteroidetes microorganisms in the intestinal tract of an obese body.
4. Use of pachyman for the regulation of metabolites in obese organisms, for the preparation of a medicament or food for increasing glucose tolerance levels and lowering serum levels of triglycerides, total cholesterol and low density lipoprotein cholesterol in obese organisms.
5. Use of pachymaran for the regulation of metabolites in an obese body as a medicament or food for reducing the diameter of epididymal adipocytes in an obese body.
6. Use of pachyman for the regulation of metabolites in obese organisms, wherein said pachyman is used as a medicament or food for the treatment of non-alcoholic fatty liver disease.
7. Use of pachyman for the modulation of metabolites in an obese body as claimed in claim 6, wherein said pachyman is used as a medicament or food for the obese body to increase the total short chain fatty acid, propionic acid, butyric acid, valeric acid and isobutyric acid content.
8. Use of pachyman for the regulation of the metabolites of the obese body as claimed in claim 6, wherein said pachyman is used as a medicament or food for reducing the fatty acid content of the liver of the obese body.
9. The pachymaran use for the modulation of metabolites in obese organisms, according to claim 8, wherein the fatty acids comprise 7-ketodeoxycholic acid, 3 β -glycocholic acid, cholic acid 7 sulfate, glycochenodeoxycholic acid 3 sulfate disodium salt, ursolic acid and glycoursodeoxycholic acid 3 sulfate sodium salt.
10. The use of pachyman for regulating the metabolic products of an obese body according to claim 6, wherein said poria cocos wolf is used as a medicine or food for improving the cellular morphology of the liver, slowing down the accumulation of lipids in the liver, down-regulating the activity of hepatic glutamic-pyruvic transaminase, improving the degree of hepatic fatty vacuolization.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116870101A (en) * | 2023-06-25 | 2023-10-13 | 汕头大学 | Application of monascus fermentation product in regulating lipid metabolism |
| CN116903760A (en) * | 2023-05-25 | 2023-10-20 | 浙江大学 | Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116903760A (en) * | 2023-05-25 | 2023-10-20 | 浙江大学 | Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity |
| CN116903760B (en) * | 2023-05-25 | 2024-09-13 | 浙江大学 | Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity |
| CN116870101A (en) * | 2023-06-25 | 2023-10-13 | 汕头大学 | Application of monascus fermentation product in regulating lipid metabolism |
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