CN114524857B - Cholic acid derivative and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to preparation of a natural medicine/prodrug for controlling metabolic abnormalities such as blood sugar, blood fat and the like. The invention discloses a cholic acid derivative, which has the following chemical structural formula:

wherein R is

Or (b)

Description

Cholic acid derivative and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to preparation of a natural medicine/prodrug for controlling metabolic abnormalities such as blood sugar, blood fat and the like.

Background

In recent years, along with the change of life style of people and the popularity of high-fat and high-sugar western diet, the prevalence of common metabolic chronic diseases such as hyperglycemia, hyperlipidemia, nonalcoholic fatty liver and the like rapidly rises, and the prevalence of the metabolic chronic diseases becomes a non-negligible national health threat.

Because most metabolic diseases belong to chronic diseases, the traditional Chinese medicine has the characteristics of long medicine taking period and the like, the currently clinically common blood sugar and blood fat reducing means mainly comprise oral medicines, the common blood sugar reducing medicines comprise sulfonylurea and biguanide oral medicines, and the main medicine for reducing blood fat is statin medicines. Most of these drugs are synthetic in structure rather than naturally occurring in the host; and the long-term administration of the medicines can cause side effects with different degrees, for example, biguanides can cause abnormal reactions of digestive tracts or lactic acidosis; long-term administration of statins can lead to hyperglycemia, cognitive impairment, liver and muscle damage, and the like. Therefore, how to find safe and effective natural products for long-term drug development of chronic metabolic diseases such as hyperglycemia and hyperlipidemia has been attracting attention.

Disclosure of Invention

A first object of the present invention is to provide cholic acid derivatives having the following chemical formula:

wherein R is

The invention also provides a preparation method of the cholic acid derivative, which comprises the following steps: culturing SJ-2 strain in improved GAM liquid culture medium for 24-48 hr to obtain culture solution; inoculating the culture solution into an improved GAM liquid culture medium for culturing for 24-48 hours according to the inoculum size of 1-5% by volume, and then centrifuging under anaerobic condition to obtain a collection solution; resuspension of the harvest broth with fermentation medium and adjustment of the biomass of SJ-2 bacteria in the fermentation medium to OD _600nm About 1 to obtain a heavy suspension; culturing the heavy suspension at 37 deg.C for 12-24 hr under anaerobic condition, and centrifuging to collect supernatant to obtain cholic acid derivative.

In a specific embodiment of the invention, the preparation method of the improved GAM liquid medium comprises the following steps: 10 g of casein peptone, 3 g of soybean peptone, 15g of tryptone, 13.5 g of digestive serum, 5g of yeast extract, 2 g of beef powder, 1.2 g of beef liver extract, 3 g of glucose, 0.3 g of soluble starch, 0.5 g of L-cysteine hydrochloride, 0.5 g of L-arginine, 0.3 g of L-tryptophan, 2 g of sodium bicarbonate, 2.5 g of potassium dihydrogen phosphate, 3 g of NaCl, 0.15 g of sodium thioglycolate, 2.46g of sodium acetate, 0.01g of hemin, 0.001g of resazurin, 10% (v/v) of clarified tumor gastric juice, and adding distilled water to 1L, and sterilizing at 115 ℃ for 25 minutes.

In a specific embodiment of the invention, the fermentation medium is prepared by adding a cholate solution with a final concentration of 1g/L and a small molecule acid mixture with a final concentration of 10mM into a modified GAM liquid medium.

In the specific embodiment of the invention, the preparation method of the small molecule acid mixed solution comprises the following steps: acetic acid, propionic acid, butyric acid and valeric acid were mixed in equimolar ratio and the pH was adjusted to 7.0.

The cholic acid derivative provided by the invention has better technical effects in preparing an antagonist for inhibiting FXR activation, preparing a hypoglycemic medicament, preparing a hypolipidemic medicament and preparing a medicament for treating fatty liver.

Description of the drawings:

FIG. 1A morphology of SJ-2 colonies.

FIG. 2 shows a diagram of SJ-2 cell morphology.

FIG. 3 is a graph showing the results of LC-MS detection of ACA compounds produced by SJ-2 bioconversion.

FIG. 4 is a graph showing the results of LC-MS detection of pure ACA compounds by liquid chromatography.

FIG. 5 ace-CA ¹ H NMR chart.

FIG. 6 ace-CA ¹³ C NMR chart.

FIG. 7 Pro-CA ¹ H NMR chart.

FIG. 8 Pro-CA ¹³ C NMR chart.

FIG. 9 But-CA ¹ H NMR chart.

FIG. 10 But-CA ¹³ C NMR chart.

FIG. 11 Val-CA ¹ H NMR chart.

FIG. 12 Val-CA ¹³ C NMR chart.

Fig. 13, liver tissue section of mice.

The specific embodiment is as follows:

examples

1. Obtaining, culturing and identifying production strains for producing ACA compounds by fermentation

Obtaining, separating and identifying production strains:

the strain was obtained by the patent inventor on day 17 of 2019 from anaerobic separation from fresh feces provided after the informed consent was signed by the stool donor himself.

Colony morphology: the colonies were grown rapidly in modified GAM broth medium, grown for 2-3 days at 37℃under anaerobic conditions, and were 1-2mm in diameter, white, smooth, and devoid of water-soluble pigments (FIG. 1). Under transmission electron microscopy, the cell morphology was observed to be short rod-like or shuttle-like, approximately 1.2-1.6 microns long and 0.6-0.8 microns wide (FIG. 2).

The modified GAM broth medium contained per liter: 10 g of casein peptone, 3 g of soybean peptone, 15g of tryptone, 13.5 g of digestive serum, 5g of yeast extract, 2 g of beef powder, 1.2 g of beef liver extract, 3 g of glucose, 0.3 g of soluble starch, 0.5 g of L-cysteine hydrochloride, 0.5 g of L-arginine, 0.3 g of L-tryptophan, 2 g of sodium bicarbonate, 2.5 g of potassium dihydrogen phosphate, 3 g of NaCl, 0.15 g of sodium thioglycolate, 2.46g of sodium acetate, 0.01g of hemin, 0.001g of resazurin, 10% (v/v) of clarified tumor gastric juice, and distilled water to 1L, and sterilizing at 115 ℃ for 25 minutes. An additional 15g of agar was added to the solid medium.

The molecular identification method comprises the following steps:

the 16S rRNA is subjected to full sequence analysis, the identification classification is named Christensenella minuta, the corresponding Chinese is named as Klebsiella and the strain is named as SJ-2, and the strain is preserved in China general microbiological culture Collection center (CGMCC) on the 4 th month 2 of 2021, and the preservation number is: CGMCC No.22122, the preservation unit address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Namely, the deposit number of the patent strain is: CGMCC No.22122; the homology of the sequence with 16SrRAN marked by the microkresoxim is 99 percent, and the sequence is shown as SEQ ID NO.1 in a sequence table.

The culture and preservation method of the strain comprises the following steps:

klebsiella minutissima Teng Senshi (Christensenella minuta) SJ-2 was activated 1 time in modified GAM broth medium supplemented with 10% clarified rumen fluid. Then inoculating the culture medium into modified GAM liquid culture medium with the same components in an inoculum size of 0.1-10% by volume ratio, and performing anaerobic culture for 1-7 days at 37 ℃.

The method for long-term storage of the bacteria comprises the following steps:

the bacterial liquid is centrifuged at 10000rpm at 4 ℃ for 2 minutes to collect the precipitated cells, and after washing with PBS buffer solution, the cells are resuspended in a solution containing 15% -25% skim milk powder at a ratio of 5:1, and the cells are stored in a sealed manner after freeze-drying or in a storage solution containing 15% glycerol and 85% serum after liquid nitrogen freezing at-80 ℃.

2. Method for producing ACA by converting cholic acid by SJ-2 bacteria

Culturing SJ-2 strain in improved GAM liquid culture medium for 24-48 hr to obtain culture solution;

the preparation method of the improved GAM liquid culture medium (g/L) comprises the following steps: 10 g of casein peptone, 3 g of soybean peptone, 15g of tryptone, 13.5 g of digestive serum, 5g of yeast extract, 2 g of beef powder, 1.2 g of beef liver extract, 3 g of glucose, 0.3 g of soluble starch, 0.5 g of L-cysteine hydrochloride, 0.5 g of L-arginine, 0.3 g of L-tryptophan, 2 g of sodium bicarbonate, 2.5 g of potassium dihydrogen phosphate, 3 g of NaCl, 0.15 g of sodium thioglycolate, 2.46g of sodium acetate, 0.01g of hemin, 0.001g of resazurin, 10% (v/v) of clarified tumor gastric juice, and adding distilled water to 1L, and sterilizing at 115 ℃ for 25 minutes. An additional 15g of agar was added to the solid medium.

After SJ-2 bacteria grow into the late logarithmic phase, inoculating the culture solution into an improved GAM liquid culture medium for culturing for 24-48 hours according to the inoculum size with the volume ratio of 1-5%, and centrifuging and collecting the SJ-2 bacteria entering the late logarithmic phase under anaerobic conditions to obtain an collected liquid;

resuspension of the harvest broth with fermentation medium and adjustment of the biomass of SJ-2 bacteria in the fermentation medium to OD _600nm About 1 to obtain a heavy suspension;

the fermentation medium is prepared by adding a cholate solution with a final concentration of 1g/L and a small molecule acid mixed solution with a final concentration of 10mM (acetic acid, propionic acid, butyric acid and valeric acid are mixed in an equimolar ratio) into a modified GAM liquid medium, and regulating the pH to 7.0.

Culturing the heavy suspension at 37 ℃ for 12-24 hours under anaerobic condition, and centrifuging to collect supernatant to obtain ACA, which is used for experiments such as detection and purification of ACA conversion.

LCMS detection method of ACA Compounds

Sample preparation: 1ml of fermentation supernatant was filtered with a 0.22 μm filter, centrifuged at 1.5 ten thousand rpm for 30 minutes in a high-speed centrifuge, and then directly loaded.

The conditions of the liquid chromatography were: c8 chromatographic column, column length 100mm, diameter 2.1mm, particle size 1.7 μm, flow rate 0.2mL/min, column temperature 35 ℃, sample volume 5 μl (suitable adjustment and reduction considering volume effect); liquid chromatography mobile phase A phase 10mM NH ₄ HCO ₃ Aqueous, phase B pure acetonitrile, was eluted with a gradient, initially 25% B, after 0.5min, followed by a linear increase to 40% B over 12 min. The rise to 90% b was continued for 3 minutes, and the recovery to 25% b was continued for 0.5 minutes, with an equilibration time of 2.5 minutes.

The conditions for mass spectrometry were: and adopting an ESI source anion mode, wherein the spraying voltage is 3kV, and the capillary temperature is set to 300 ℃. The sheath gas and the auxiliary gas are both nitrogen, the flow rates are 45and 10 (arbitrary units), and the heating temperature of the auxiliary gas is 350 ℃. The method adopts a full scanning mode, the resolution is set to 120000, and the scanning range is set to m/z 73.4 to 1100.

Calculation method of efficiency of conversion of SJ-2 Strain to ACA:

it is known that under conversion of SJ-2 bacteria, all cholic acid added to the medium is converted into ACA compound of C2-C5 acyl group, and thus the total conversion efficiency of ACA is calculated as a ratio of reduction of peak area in LCMS detection peak plot of cholic acid added at time 0 after 24 hours of conversion catalysis: (cholic acid peak area at time 0-24 hours cholic acid peak area)/cholic acid peak area at time 0 is 100%.

5. Separation and purification method of bile acid acylated derivative ACA

Adding equal volume of ethyl acetate into the supernatant of the fermentation broth after 24 hours of conversion, carrying out ultrasonic extraction for 3 times, wherein the ultrasonic time is 3X 0.5 hours, collecting ethyl acetate extract by a separating funnel, and concentrating by a vacuum rotary evaporator to obtain a crude extract. The crude extract was dissolved in 10mL of methanol and separated using a C18 prep column.

The liquid phase conditions are as follows: c18 preparing a column, wherein the length of the column is 250mm, the diameter of the column is 20mm, the flow rate is 10ml/min, and the sample injection volume is 100 mu L; the phase A of the liquid chromatography mobile phase is 0.01% of aqueous solution of trifluoro acid, the phase B is pure methanol, 70% of phase B+30% of phase A is adopted for equal elution, the corresponding component of the target peak appearing at the wavelength of A205nm is picked up, the purity of the separated component is detected by an LCMS method, and the separated component is dried by nitrogen and then weighed for standby.

6. Experimental results

LCMS detection results and SJ-2 conversion of different carbon chain length acyl cholic acids

SJ-2 cells transformed cholic acid 0 and 24 hour supernatants, respectively, were sampled and assayed by LCMS. As shown in FIG. 3, the sample at time 0 shows the results of a and b (CA+SA_0h) in FIG. 3, and the results after 24 hours of conversion show c-h (CA+SA_24h) in FIG. 3. The results showed that only the characteristic peak of cholic acid (b in FIG. 3) having a mass-to-charge ratio (m/z) of 407.3 was detected at time 0, the peak area after extraction of 407.3m/z was 93413948.47, and 4 new peaks appeared clearly at 24 hours of conversion, the m/z of the mass spectrum thereof was 449.3,463.3,477.3 and 491.3, corresponding to acetocholic acid, propionylcholic acid, butyryl cholic acid and valeryl cholic acid, the peak areas after peak extraction were 18071866.68,20406632.73,22769156.54 and 8969173.10, respectively, and the peak area of the peak extracted by remaining cholic acid (407.3) after reaction was 45837961.92, and the conversion rate of cholic acid into ACA-series compounds by SJ-2 bacteria was 50.9% by calculation.

Separation and purification of microbiologically transformed ACA products

The separation and purification experiment of ACA conversion products is carried out by utilizing a C18 semi-preparation column through a liquid chromatography, and the LCMS detection result after evaporating and redissolving the methanol by utilizing the collected characteristic peak components is shown in fig. 4, so that four ACA compounds can be effectively separated and purified through the liquid chromatography. Four pure products of unknown cholic acid derivatives having molecular weights of 450.3, 464.3, 478.3 and 492.3, respectively, were isolated and then examined by nuclear magnetic resonance for the following spectra: one-dimensional 1H spectrum, one-dimensional 13C spectrum experiment, two-dimensional 1H-1H correlation spectrum (adjacent hydrocarbon coupling relation), two-dimensional 1H-13C correlation spectrum (directly-bonded hydrocarbon coupling relation), two-dimensional NOESY correlation spectrum (distance of protons in molecular stereo space structure), two-dimensional 1H-13C remote correlation experiment (remote hydrocarbon relation, dieber secondary tertiary carbon).

TABLE 1 ¹³ C NMR Data(δC)for Compounds 1-4 in CDCl3

TABLE 2 ¹ H NMR Data[δH,mult(J in Hz)]for Compounds 1-4 in CDCl3

The chemical structure is finally determined, wherein R groups are acetyl, propionyl, butyryl and valeryl.

The structural formula of the compound is as follows:

wherein R is

7. Use and effect of the compound and its preparation:

luciferase reporter gene detection cell assay:

the farnesyl ester derivative X receptor (Farnesoid X receptor, FXR) is a bile acid receptor, plays an important role in bile acid metabolism and cholesterol metabolism, and is expected to become a therapeutic target for reducing cholesterol and treating certain cardiovascular diseases and liver diseases.

24 hours after transfection of the luciferase reporter system of FXR, the cells were exposed to either 20mmol/LFXR agonist chenodeoxycholic acid CDCA (positive control), or to a treatment group of 20mmol/L CDCA and 50mmol/L acetocholic acid (Ace-CA), propionylcholic acid (Pro-CA), butyryl cholic acid (But-CA) and valeryl cholic acid (Val-CA), respectively; cells were not treated as negative controls.

Luciferase assays were performed using a commercial dual luciferase assay system (Promega).

Firefly and Renilla luciferase activity was measured using a Veritas microplate luminometer (Turner Biosystems).

To quantify the dose response inhibition of FXR by different ACA compounds, cells were exposed to Ace-CA, pro-CA, but-CA and Val-CA in a concentration range of 5mmol/L to 200mmol/L (upper solubility limit) for 8 hours, fluorescence was measured and the semi-inhibitory concentration IC of the compound was calculated using GraphPad Prism 6 in "log (inhibitor) and normalized response-variable slope" mode ₅₀ 。

As a result of cell experiments with luciferase reporter gene, ACA series compounds can inhibit FXR activation induced by CDCA and act as antagonists.

ACA compounds were dosed for FXR inhibition with Ace-CA, pro-CA, but-CA and Val-CA, with IC 50's of 35.52, 29.61,17.87 and 11.18. Mu.M, respectively.

TABLE 3 relative fluorescence intensity values of FXR inhibition effect of four Acylcholic acids on double fluorescein fluorescence System

TABLE 4 relative fluorescence intensity values of FXR inhibition effect of different concentrations of Acetylcholic acid on double-fluorescein fluorescence System

TABLE 5 relative fluorescence intensity values of FXR inhibition effect of different concentrations of butyrylcholic acid on the double-fluorescein fluorescence System

TABLE 6 relative fluorescence intensity values of FXR inhibition effect of different concentrations of propionylcholic acid on the double fluorescein fluorescence System

TABLE 7 relative fluorescence intensity values of FXR inhibition effect of different concentrations of valeryl cholic acid on bifluorescent fluorescence systems

Animal experiment design:

experiments induced obese C57 mice (DIO mice), male, 15 weeks old with a high fat diet. The gastric lavage frequency is once a day, and ACA groups (3) irrigate with Ace-CA, pro-CA, but-CA and Val-CA pure physiological saline solution of 50mg/kg body weight each day; the preparation group (3) is filled with ACA mixture (mass mixture of ethyl, propyl, butyl and valeryl cholic acid) with body weight of 20mg/kg per day in improved GAM culture medium; the control group (3) was filled with the same amount of physiological saline every day. The total experimental period was 8 weeks.

The modified GAM medium was (g/L): 10 g of casein peptone, 3 g of soybean peptone, 15g of tryptone, 13.5 g of digestive serum, 5g of yeast extract, 2 g of beef powder, 1.2 g of beef liver extract, 3 g of glucose, 0.3 g of soluble starch, 0.5 g of L-cysteine hydrochloride, 0.5 g of L-arginine, 0.3 g of L-tryptophan, 2 g of sodium bicarbonate, 2.5 g of potassium dihydrogen phosphate, 3 g of NaCl, 0.15 g of sodium thioglycolate, 2.46g of sodium acetate, 0.01g of hemin, 0.001g of resazurin, 10% (v/v) of clarified tumor gastric juice, and distilled water to 1L, pH 7.2+/-0.1, and sterilizing at 115 ℃ for 25 minutes

Endpoint sampling: a. plasma samples (supernatant collected by anticoagulation centrifugation), B, liver and other substantive organs (liver photographing and weighing, paraformaldehyde fixation is carried out on the left and right middle leaves respectively, paraffin embedding and frozen slicing are carried out, red oil staining and HE staining are carried out after slicing, and the residual liver is accurately weighed and 100mg of liquid nitrogen quick-frozen 3 tubes are used for other index detection, C, intestinal tract and content.

Tissue sample detection index:

a) Blood glucose related index: free blood glucose, fasting blood glucose, glucose tolerance (OGTT), insulin resistance (ITT) test of venous blood;

b) Blood lipid related index: total cholesterol and total triglycerides content in plasma;

c) Liver function-related: h & E staining and oil red staining, and the expression levels of the cholic acid synthesis rate-limiting enzyme cholesterol 7α -hydroxylase (Cyp 7a 1) and gluconeogenesis rate-limiting enzyme glucose-6-phosphatase (G6 pase) in liver tissue.

The blood glucose related index is a blood glucose meter, and other physiological and biochemical indexes except blood glucose are detected by using a commercial ELISA kit after carrying out crude protein extraction on blood or homogenized liver tissue.

And (3) statistical inspection:

whether the data of each treatment group has statistical difference compared with the data of the control group or not, the significance test is carried out by adopting a double-sided new complex polar difference method (Dunnett-t test), the control group is taken as a control class, and the difference is considered to be significant when the P value is less than 0.05.

Experimental results:

ACA compounds and related formulations reduce free blood glucose in model mice:

the data in Table 8 shows that ACA compounds and related formulations have significant free blood glucose lowering effects, alleviating hyperglycemia symptoms.

TABLE 8 free blood glucose concentration (mmol/L) of venous blood after 8 weeks of model mouse intervention

ACA compounds and related formulations reduce fasting blood glucose in model mice:

the data in Table 9 shows that ACA compounds and related formulations have significant effects in reducing fasting blood glucose in model mice and alleviating hyperglycemia symptoms.

TABLE 9 fasted blood glucose concentration (mmol/L) after 8 weeks of model mouse intervention

ACA compounds and related formulations reduce glucose tolerance in model mice:

the results in table 10 show that ACA compounds and related formulations can significantly increase the glucose tolerance of model mice, alleviating impaired glucose tolerance.

TABLE 10 area under the curve of glucose tolerance (AUC) after 7 weeks of model mouse intervention

ACA compounds and related formulations reduce model mouse insulin resistance:

the results in table 11 show that ACA compounds and related formulations can significantly reduce insulin resistance symptoms in model mice.

TABLE 11 area under the curve (AUC) of insulin resistance 8 weeks after model mouse intervention

ACA compounds and related formulations reduce total triglyceride content in the blood of model mice:

the results in Table 12 show that ACA compounds and related formulations can significantly reduce the total triglyceride content in the blood of model mice and alleviate hyperlipidemia symptoms.

TABLE 12 endpoint blood total triglyceride concentration (mmol/L) after 8 weeks of model mouse intervention

ACA compounds and related formulations reduce total cholesterol content in the blood of model mice:

the results in Table 13 show that ACA compounds and related formulations can significantly reduce the total cholesterol content in the blood of model mice and alleviate the high cholesterol symptoms.

TABLE 13 Total cholesterol concentration (mmol/L) at endpoint blood after 8 weeks of model mouse intervention

ACA compounds and related formulations reduce liver fat accumulation and hepatic cell damage, among other pathological indicators:

as can be seen from fig. 13, the fat accumulation of the liver of the mice treated with the ACA and the preparation was significantly reduced, fat vacuoles were rarely seen in the tissue sections, and the large vesicular fat became substantially lost. After model mouse intervention, liver lobular structural disorder and clear limit are corrected, and hepatocyte network structure is reduced or basically vanished, normal morphology, nuclear circle and centering. The phenotype shows that the symptoms of non-alcoholic fatty liver and liver injury of the disease model mice are greatly relieved.

ACA compound and related preparation for up-regulating expression level of Cyp7a1 in liver tissue and down-regulating expression level of G6pase

The results of detecting the expression levels of cholic acid synthesis rate-limiting enzyme cholesterol 7 alpha-hydroxylase (Cyp 7a 1) (table 14) and gluconeogenesis rate-limiting enzyme glucose-6-phosphatase (G6 pase) (table 15) in liver tissues of two mice in the treatment groups show that ACA compounds and related preparations can play a role in reducing cholesterol by up-regulating the expression of Cyp7a1 in the liver so as to accelerate the conversion of cholesterol into cholic acid and discharging the cholic acid into intestinal tracts; meanwhile, the G6pase is regulated downwards to inhibit gluconeogenesis in the liver, slow down the rise of blood sugar and play a role in regulating blood sugar.

TABLE 14 Cyp7a1 protein content (pg/mg) in liver-milled samples 8 weeks after model mouse intervention

TABLE 15G 6pase protein content (pg/mg) in liver-milled samples 8 weeks after model mouse intervention

Reference is made to:

1.Tahrani AA,Barnett AH,Bailey CJ.Pharmacology and therapeutic implications of current drugs for type 2 diabetes mellitus.Nat Rev Endocrinol.2016；12:566-92.

2. liang Li, qiao Hua, wang Ting, alfava, metformin hydrochloride adverse reaction; 21:652-4.

3. Wen Yina the side effects of statins cardiovascular disease control knowledge (popular science edition) 2014;10:24-6.

4. Li Jing and Lv Qianzhou "statin" 4 people "muscle" side effects are great; 000:68.

5.Wu JY,Wang K,Wang XM,Pang YL,Jiang CT.The role of the gut microbiome and its metabolites in metabolic diseases.Protein Cell.2020,10.1007/s13238-020-00814-7.

6.Wahlstrom A,Sayin SI,Marschall HU,Backhed F.Intestinal Crosstalk between Bile Acids and Microbiota and Its Impact on Host Metabolism.Cell Metab.2016；24:41-50.

7.Wang K,Liao M,Zhou N,Bao L,Ma K,Zheng Z,et al.Parabacteroides distasonis Alleviates Obesity and Metabolic Dysfunctions via Production of Succinate and Secondary Bile Acids.Cell reports.2019；26:222-35.e5.

8.Kliewer SA,Mangelsdorf DJ.Bile Acids as Hormones:The FXR-FGF15/19 Pathway.Digest Dis.2015；33:327-31.

9.Fiorucci S,Mencarelli A,Palladino G,Cipriani S.Bile-acid-activated receptors:targeting TGR5 and farnesoid-X-receptor in lipid and glucose disorders.Trends Pharmacol Sci.2009；30:570-80.

10.Zheng XJ,Chen TL,Jiang RQ,Zhao AH,Wu Q,Kuang JL,et al.Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism.Cell Metab.2021；33:791-+.

11.Sun LL,Xie C,Wang G,Wu Y,Wu Q,Wang XM,et al.Gut microbiota and intestinal FXR mediate the clinical benefits of metformin.Nat Med.2018；24:1919-+.

12.Tahrani AA,Barnett AH,Bailey CJ.Pharmacology and therapeutic implications of current drugs for type 2 diabetes mellitus.Nat Rev Endocrinol.2016；12:566-92.

13. Liang Li, qiao Hua, wang Ting, alfava, metformin hydrochloride adverse reaction; 21:652-4.

14. Wen Yina the side effects of statins cardiovascular disease control knowledge (popular science edition) 2014;10:24-6.

15. Li Jing and Lv Qianzhou "statin" 4 people "muscle" side effects are great; 000:68.

16.Wu JY,Wang K,Wang XM,Pang YL,Jiang CT.The role of the gut microbiome and its metabolites in metabolic diseases.Protein Cell.2020,10.1007/s13238-020-00814-7.

17.Wahlstrom A,Sayin SI,Marschall HU,Backhed F.Intestinal Crosstalk between Bile Acids and Microbiota and Its Impact on Host Metabolism.Cell Metab.2016；24:41-50.

18.Wang K,Liao M,Zhou N,Bao L,Ma K,Zheng Z,et al.Parabacteroides distasonis Alleviates Obesity and Metabolic Dysfunctions via Production of Succinate and Secondary Bile Acids.Cell reports.2019；26:222-35.e5.

19.Kliewer SA,Mangelsdorf DJ.Bile Acids as Hormones:The FXR-FGF15/19 Pathway.Digest Dis.2015；33:327-31.

20.Fiorucci S,Mencarelli A,Palladino G,Cipriani S.Bile-acid-activated receptors:targeting TGR5 and farnesoid-X-receptor in lipid and glucose disorders.Trends Pharmacol Sci.2009；30:570-80.

21.Zheng XJ,Chen TL,Jiang RQ,Zhao AH,Wu Q,Kuang JL,et al.Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism.Cell Metab.2021；33:791-+.

22.Sun LL,Xie C,Wang G,Wu Y,Wu Q,Wang XM,et al.Gut microbiota and intestinal FXR mediate the clinical benefits of metformin.Nat Med.2018；24:1919-+.

Sequence listing

<110> institute of microorganisms at national academy of sciences

<120> A strain of Klebsiella minutissima Teng Senshi strain SJ-2 and application thereof

<141> 2022-02-22

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1391

<212> DNA

<213> Small kris Teng Senshi bacterium (Christensenella minuta)

<400> 1

gctctctcct tacggttaag ccactggctt cgggtgctcc caacttccgt ggtgtgacgg 60

gcggtgtgta caaggcccgg gaacgcattc accgcgacat gctgattcgc gattactagc 120

aactccgact tcatgtgggc gggttgcagc ccacaatccg aactgggacc ggctttttga 180

gattcgcttc cccttacggg ttcgctgccc tttgtaccgg ccattgtagc acgtgtgtag 240

cccaagacat aaggggcatg atgatttgac gtcgtcccca ccttcctccg agttgtcccc 300

ggcagtctca ctagagttcc cgcctttacg cgctggcaac tagcaataag ggttgcgctc 360

gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420

ctctctgccc cgaagggaaa ctgtatctct acagtcgtca gaggatgtca agccttggta 480

aggttcttcg cgttgcttcg aattaaacca catgctccgc tgcttgtgcg ggcccccgtc 540

aattcctttg agtttcaacc ttgcgatcgt actccccagg cgggatactt aatgcgtttg 600

cttcggcacg gaaccctatc gggccccaca cctagtatcc atcgtttacg gcgtggacta 660

ccagggtatc taatcctgtt tgctccccac gctttcgtgc ctcagtgtca gttacagtcc 720

agaaagtcgc cttcgccact ggtgttcctc ctaatatcta cgcatttcac cgctacacta 780

ggaattccac ttccctctcc tgtactcaag tcacacagtt tcaaatgcaa ccccggggtt 840

aagccccggt ctttcacatc tgacttacat gaccacctac gcacccttta cgcccagtaa 900

ttccggacaa cgcttgctcc ctacgtatta ccgcggctgc tggcacgtag ttagccggag 960

cttcctccta tggtaccgtc atttctttcg tcccatagga caaaggttta caatccgaag 1020

accttcttcc ctcacgcggc gttgctgggt cagggtttcc cccattgccc aatattcccc 1080

actgctgcct cccgtaggag tctggaccgt gtctcagttc cagtgtggcc gatcaccctc 1140

tcaggtcggc tacccatcgt tgacttggtg ggccgttacc tcaccaacta tctaatggga 1200

cgcgagccca tcctgcatcg aataaatcct tttacctcaa aaccatgcgg tttcgtggtc 1260

tcatgcggta ttagcagtcg tttccaactg ttgtcccccg ttgcagggca ggttgctcac 1320

gcgttactca cccgtccgcc actcggtata cccacagttc ctcccgaagg attcacaaag 1380

ggcaacctcg t 1391

Claims (1)

1. Application of cholic acid derivatives in preparing antagonists for inhibiting FXR activation, hypoglycemic drugs, hypolipidemic drugs and/or drugs for treating fatty liver; the method is characterized in that the acyl cholic acid has the following chemical structural formula:

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wherein R is

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