A composition for regulating liver circadian rhythm, preparation method and application

Abstract

The application provides a composition for regulating liver circadian rhythm, a preparation method and application. The composition significantly increases the liver biological rhythm amplitude by up-regulating the expression of core genes Clock, Per1, Per2 and Bmal1 of liver biological rhythm, and then improves the fat accumulation, abnormal increase of body weight, liver function damage caused by liver rhythm disorder and restores the liver circadian rhythm, so as to achieve the purpose of treating the liver circadian rhythm disorder and abnormal lipid metabolism caused by high-fat and high-fructose diet.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a composition, preparation method and application for regulating liver circadian rhythm. Background Technology

[0002] Factors such as high-fat diet, high-fructose diet, and abnormal eating times can disrupt the liver's peripheral biological clock, leading to liver rhythm disorders.

[0003] Liver rhythm disorder is mainly manifested by a decrease in the diurnal expression peak-to-trough difference of core biological clock genes such as Clock, Bmal1, Per1 and Per2, and abnormal expression peaks or phases, leading to an imbalance between lipid synthesis and decomposition, accumulation of liver triglycerides and cholesterol, abnormal weight gain, hepatocyte steatosis and abnormal liver function indicators.

[0004] Currently, lipid metabolism abnormalities caused by liver rhythm disorders are typically treated with dietary control, regular sleep patterns, exercise, timed eating, and lipid-lowering, blood sugar-lowering, or liver-protecting methods. However, adherence to lifestyle interventions is poor, and existing drugs mainly target blood lipids, blood sugar, or liver damage indicators, lacking the ability to directly improve the abnormal diurnal expression of core liver circadian rhythm genes. Summary of the Invention

[0005] In view of the above problems, the purpose of this invention is to provide a composition for regulating liver rhythm, a preparation method thereof, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In one aspect, this application provides a composition for regulating the liver's circadian rhythm, comprising wolfberry oligosaccharide and puerarin.

[0007] Secondly, this application provides a method for preparing a composition for regulating the liver's circadian rhythm, comprising: mixing wolfberry oligosaccharide, puerarin, pregelatinized starch, microcrystalline cellulose, croscarmellose sodium, silicon dioxide and magnesium stearate, followed by dry granulation and tableting to obtain the composition for regulating the liver's circadian rhythm.

[0008] Thirdly, this application provides the use of a composition for regulating the liver's circadian rhythm, namely, for preparing a drug for regulating the liver's circadian rhythm.

[0009] Fourthly, this application provides the use of a composition for regulating the liver's circadian rhythm, namely, for preparing a drug to improve abnormal lipid metabolism.

[0010] The present invention has the following beneficial effects: In this application, the composition containing wolfberry oligosaccharide and puerarin significantly increases the amplitude of liver circadian rhythm by upregulating the expression of the core genes Clock, Per1, Per2 and Bmal1, thereby improving fat accumulation, abnormal weight gain, liver function damage and repairing liver diurnal rhythm caused by liver rhythm disorder, and achieving the purpose of treating liver diurnal rhythm disorder and lipid metabolism abnormality caused by high-fat and high-fructose diet. Detailed Implementation

[0011] In a first aspect, this application provides a composition for regulating the liver's circadian rhythm, comprising lycopene oligosaccharide and puerarin, wherein the total mass of lycopene oligosaccharide and puerarin is 60% of the total mass of the composition.

[0012] The core genes of liver circadian rhythms include the clock gene Clock, cyclins Per1 and Per2, and Bmal1, a protein similar to the nuclear transporter of aryl hydrocarbon receptors in brain and muscle tissue. The Lycium barbarum oligosaccharides and puerarin in this application can significantly increase the amplitude of liver circadian rhythms by upregulating the expression of these core genes, thereby improving fat accumulation, abnormal weight gain, and liver function damage caused by liver rhythm disorders. Therefore, the composition containing Lycium barbarum oligosaccharides and puerarin can exert good effects on weight loss and reducing fatty liver, or improve liver damage caused by excessive intake of high-fat, high-fructose diets.

[0013] In this application, the mass ratio of Lycium barbarum oligosaccharide to puerarin is 1:(0.1-2), the average molecular weight of Lycium barbarum oligosaccharide is 0.5-2.0 kDa, and the purity of puerarin is ≥95%; more preferably, the mass ratio of Lycium barbarum oligosaccharide to puerarin is 1:(0.25-1), the average molecular weight of Lycium barbarum oligosaccharide is 0.71.0 kDa, and the purity of puerarin is ≥98%.

[0014] In this application, *Lycium barbarum* oligosaccharides are low-molecular-weight sugar components prepared from polysaccharides derived from *Lycium barbarum* fruit through enzymatic hydrolysis, acid hydrolysis, membrane separation, and freeze-drying. Specifically, the preparation method of *Lycium barbarum* oligosaccharides in this application includes: S01: The dried wolfberries were pulverized, defatted with petroleum ether, and extracted with purified water to obtain the extract.

[0015] After pulverizing the dried goji berries, pass them through a 40-mesh sieve and add them to petroleum ether. Defatting is carried out at 25°C for 30 minutes at a material-to-liquid ratio of 1:10 g / ml, repeated three times each time. The defatted dried goji berry powder is then added to purified water and extracted at 90°C for 2 hours. After filtration, the extract is obtained.

[0016] S02: The extract was concentrated under reduced pressure, precipitated with ethanol, deproteinized, dialyzed once, and freeze-dried once to obtain wolfberry polysaccharide.

[0017] After filtering the extract through a 150-mesh filter cloth, it was concentrated under reduced pressure at -0.085 MPa and 60℃ to 1 / 4-1 / 6 of the extract volume to obtain a concentrated solution. Ethanol was added to the concentrated solution to make the final volume fraction of ethanol in the mixture 75%. After standing at 4℃ for 12 hours to precipitate, the precipitate was collected by centrifugation to obtain crude Lycium barbarum polysaccharide.

[0018] The crude polysaccharide of Lycium barbarum was dissolved in purified water, and Sevag reagent (1 / 4 volume of the crude polysaccharide) was added. The mixture was deproteinized at 25°C for 20 min, followed by centrifugation at 3000 rpm for 10 min at 25°C. The deproteinized crude polysaccharide was then added to a dialysis bag with a molecular weight cutoff of 3500 Da, placed in purified water, and dialyzed at 4°C for 36 h. Finally, it was freeze-dried at -60°C for 36 h to obtain the Lycium barbarum polysaccharide.

[0019] S03: After the wolfberry polysaccharide is reconstituted, hydrolyzed by Pronase E, and inactivated, it is degraded by sulfuric acid, neutralized by sodium carbonate, and centrifuged to obtain the supernatant.

[0020] Lycium barbarum polysaccharides were reconstituted in purified water, and Pronase E was added for enzymatic hydrolysis at 37°C for 24 hours. After inactivating the enzyme by heating to 80°C, the polysaccharides were initially degraded using 40 mmol / L sulfuric acid. Following the initial degradation, the mixture was neutralized with 0.25 mol / L sodium carbonate, and the supernatant was obtained after centrifugation.

[0021] S04: The supernatant was subjected to secondary dialysis, trifluoroacetic acid degradation, membrane filtration purification, and secondary freeze-drying to obtain wolfberry oligosaccharide.

[0022] The supernatant was added to a dialysis bag with a molecular weight cutoff of 1000 Da, placed in purified water, and dialyzed at 4°C for 36 h. After dialysis, a secondary degradation was performed using 0.5 mmol / L trifluoroacetic acid to obtain a Lycium barbarum oligosaccharide solution. The Lycium barbarum oligosaccharide solution was placed in a fume hood to evaporate and remove the trifluoroacetic acid, then passed through a membrane filter and purified by filtration at 25°C and 0.5 MPa using a membrane with a molecular weight cutoff of 500 Da. Oligosaccharide fractions with a molecular weight greater than 500 Da were collected, and finally, freeze-dried at -60°C to obtain Lycium barbarum oligosaccharides.

[0023] In addition, the composition for regulating liver diurnal rhythm provided in this application also includes pharmaceutically acceptable excipients, including but not limited to one or more of fillers, binders, disintegrants, flow aids, and lubricants. The fillers are selected from one or more of pregelatinized starch, lactose, mannitol, sucrose, corn starch, potato starch, calcium phosphate, and calcium carbonate; the binders are selected from one or more of microcrystalline cellulose, starch paste, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, methylcellulose, and gelatin; the disintegrants are selected from one or more of croscarmellose sodium, sodium carboxymethyl starch, croscarmellose, dry starch, and low-substituted hydroxypropyl cellulose; the flow aids are selected from one or more of silica, micronized silica gel, talc, magnesium stearate, and tricalcium phosphate; and the lubricants are selected from one or more of magnesium stearate, calcium stearate, sodium stearate fumarate, polyethylene glycol, hydrogenated vegetable oil, and sodium lauryl sulfate.

[0024] In this application, the total mass of filler, binder, disintegrant, flow aid, and lubricant is 40% of the composition mass, and the amount of each excipient added can be determined according to actual production conditions. Preferably, the mass ratio of wolfberry oligosaccharide, filler, binder, disintegrant, flow aid, and lubricant is 1:0.3:0.6:0.075:0.015:0.01.

[0025] Secondly, this application provides a method for preparing a composition for regulating the liver's circadian rhythm. The preparation method includes: mixing wolfberry oligosaccharide, puerarin, filler, binder, disintegrant, glidant and lubricant, followed by dry granulation and tableting to obtain the composition for regulating the liver's circadian rhythm.

[0026] Thirdly, this application provides the use of a composition for regulating the liver's circadian rhythm, namely, for preparing a drug for regulating the liver's circadian rhythm.

[0027] Fourthly, this application provides the use of a composition for regulating the liver's circadian rhythm, namely, for preparing a drug to improve abnormal lipid metabolism.

[0028] The technical solution of the present invention will be further explained and illustrated below through specific embodiments. Puerarin uses commercially available puerarin raw materials with a purity of not less than 98%.

[0029] Example 1 This application provides a method for preparing wolfberry oligosaccharides, the method comprising: S101: After pulverizing the dried wolfberries, pass them through a 40-mesh sieve and add them to petroleum ether. Defatt the mixture at 25°C for 30 minutes at a material-to-liquid ratio of 1:10 g / ml, repeating this process three times. Add the defatted dried wolfberry powder to purified water and extract at 90°C for 2 hours. After filtration, obtain the extract.

[0030] S102: After filtering the extract through a 150-mesh filter cloth, the solution was concentrated under reduced pressure at -0.085 MPa and 60℃ to 1 / 4-1 / 6 of its original volume to obtain a concentrated solution. Ethanol was added to the concentrated solution to make the final volume fraction of ethanol in the mixture 75%. After standing at 4℃ for 12 hours to precipitate, the precipitate was collected by centrifugation to obtain crude Lycium barbarum polysaccharide. The crude Lycium barbarum polysaccharide was dissolved in purified water, and Sevag reagent was added at 1 / 4 of the volume of the solution. The solution was deproteinized at 25℃ for 20 minutes and centrifuged at 25℃ and 3000 rpm for 10 minutes. The deproteinized Lycium barbarum polysaccharide was added to a dialysis bag with a molecular weight cutoff of 3500 Da and placed in purified water for dialyzing at 4℃ for 36 hours. Then, it was cold-dried at -60℃ to obtain Lycium barbarum polysaccharide.

[0031] S103: Lycium barbarum polysaccharides were reconstituted in purified water, and Pronase E was added. Enzymatic hydrolysis was performed at 37°C for 24 hours. After inactivating the enzyme by heating to 80°C, primary degradation was carried out using 40 mmol / L sulfuric acid. Following primary degradation, neutralization was achieved using 0.25 mol / L sodium carbonate. The supernatant was obtained after centrifugation.

[0032] S104: The supernatant was added to a dialysis bag with a molecular weight cutoff of 1000 Da, placed in purified water, and dialyzed at 4°C for 36 h. After dialysis, a secondary degradation was performed using 0.5 mmol / L trifluoroacetic acid to obtain a Lycium barbarum oligosaccharide solution. The Lycium barbarum oligosaccharide solution was placed in a fume hood to evaporate and remove the trifluoroacetic acid, then passed through a membrane filter and purified by filtration at 25°C and 0.5 MPa using a membrane with a molecular weight cutoff of 500 Da. Oligosaccharide fractions with a molecular weight greater than 500 Da were collected. Finally, the oligosaccharides were freeze-dried at -60°C to obtain Lycium barbarum oligosaccharides.

[0033] Example 2 This application provides a composition for regulating the liver's circadian rhythm. The composition comprises 200 mg of Lycium barbarum oligosaccharide and 100 mg of puerarin, wherein the Lycium barbarum oligosaccharide is the Lycium barbarum oligosaccharide prepared in Example 1. 1:(0.1-2) Example 3 This application provides a composition for regulating the liver's circadian rhythm. The composition includes mg of Lycium barbarum oligosaccharide and mg of puerarin, wherein the Lycium barbarum oligosaccharide is the Lycium barbarum oligosaccharide prepared in Example 1.

[0034] Example 4 This application provides a composition for regulating the liver's circadian rhythm. The composition includes mg of Lycium barbarum oligosaccharide and mg of puerarin, wherein the Lycium barbarum oligosaccharide is the Lycium barbarum oligosaccharide prepared in Example 1.

[0035] Example 5 This application provides a composition for regulating the liver's circadian rhythm. The composition includes mg of Lycium barbarum oligosaccharide and mg of puerarin, wherein the Lycium barbarum oligosaccharide is the Lycium barbarum oligosaccharide prepared in Example 1.

[0036] Example 6 This application provides a composition for regulating the liver's diurnal rhythm. The composition includes 200 mg of Lycium barbarum oligosaccharide, 100 mg of puerarin, 60 mg of pregelatinized starch, 120 mg of microcrystalline cellulose, 15 mg of croscarmellose sodium, 3 mg of silicon dioxide, and 2 mg of magnesium stearate. The Lycium barbarum oligosaccharide is the Lycium barbarum oligosaccharide prepared in Example 1.

[0037] The method for preparing the composition for regulating the liver's diurnal rhythm provided in this application includes: After mixing wolfberry oligosaccharide, puerarin, pregelatinized starch, microcrystalline cellulose, croscarmellose sodium, silicon dioxide and magnesium stearate, the mixture was dry-granulated and tableted to obtain a composition for regulating the liver's diurnal rhythm.

[0038] To verify the ameliorative effect of the composition provided in this application on liver rhythm disorder, fat accumulation, and liver damage induced by a high-fat, high-fructose diet, animal experiments were conducted using the composition prepared in Example 6. The experimental results were statistically analyzed using SPSS 22.0 software, and the experimental data are expressed as mean ± variance. One-way ANOVA was used for comparisons among multiple groups, and Tukey's method was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant. The diurnal variation in clock gene expression was also assessed in each group. The specific experimental procedure is as follows: Four-week-old SPF-grade male C57BL / 6J mice, weighing 18-22g, were housed in an environment with a temperature of 25±2℃, relative humidity of 65±5%, and a 12-hour light / 12-hour dark cycle, with free access to food and water. The acclimatization period was defined as ZT0 (lights on at 08:00) and ZT12 (lights off at 20:00). After one week of acclimatization, they were randomly divided into three groups of 24 mice each: Control group: fed with standard XTCON50H feed; Model group: fed with high-fat XTHF45 diet and drinking water with fructose concentration of 15g / 100mL; Intervention group: fed with high-fat XTHF45 diet and drinking water with fructose concentration of 15g / 100mL.

[0039] After one week of feeding according to the above grouping, the intervention group mice were administered the composition prepared in Example 6 once daily by gavage at ZT6 (14:00±30min), with a dose of 200 mg / kg body weight for Lycium barbarum oligosaccharide and 100 mg / kg body weight for puerarin, at a volume of 10 mL / kg body weight. The control and model groups were administered the same volume of the drug delivery carrier by gavage at the same time. This drug delivery carrier consisted of all components in Example 6 except for Lycium barbarum oligosaccharide and puerarin. After 9 weeks of feeding according to the above administration, mouse body weight was recorded. After fasting for 12 hours, blood and liver tissue were collected to measure weight gain rate and liver function indicators. Simultaneously, liver tissue was collected from 6 mice in each group at four time points (ZT0, ZT6, ZT12, and ZT18) to detect the relative mRNA expression levels of core circadian rhythm genes and analyze the relative peak-to-trough difference in diurnal expression.

[0040] 1. Weight growth rate detection The weight gain rate of each group of mice was calculated based on all mice in each group, as shown in Table 1.

[0041] Table 1: Body weight and weight gain rate of mice in each group Note: a indicates comparison with the control group, P<0.05; b indicates comparison with the model group, P<0.05.

[0042] As shown in Table 1, compared with the control group, the weight gain rate of the model group mice was significantly increased, indicating that the high-fat, high-fructose mouse model was successfully established. After 9 months of intervention, compared with the model group, the weight gain rate of the intervention group mice decreased from 61.5% to 43.2%, indicating that the composition prepared in Example 6 of this application can effectively reduce the weight of high-fat, high-fructose mice.

[0043] 2. Liver function test Commercial ALT, AST, TG, and TC assay kits were used to detect the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, as well as the levels of triglycerides (TG) and total cholesterol (TC) in the liver. The results are shown in Tables 2 and 3.

[0044] Table 2: ALT and AST activities in mice of each group Note: a indicates comparison with the control group, P<0.05; b indicates comparison with the model group, P<0.05.

[0045] Table 3: TG and TC content in mice of each group Note: a indicates comparison with the control group, P<0.05; b indicates comparison with the model group, P<0.05.

[0046] As shown in Table 2, compared with the control group, the serum ALT and AST activities of mice in the model group were increased, indicating that the high-fat, high-fructose diet led to abnormal liver function in the mice. After 9 months of intervention, the ALT and AST activities of mice in the intervention group were significantly decreased compared with the model group.

[0047] As shown in Table 3, compared with the control group, the liver TG and TC levels of mice in the model group were increased, indicating that a high-fat, high-fructose diet led to lipid accumulation in the liver. After 9 months of intervention, the liver TG and TC levels of mice in the intervention group were significantly decreased compared with the model group.

[0048] This indicates that the composition prepared in Example 6 of this application can effectively reduce the activity of ALT and AST in the serum of high-fat, high-fructose mice and the content of TG and TC in the liver, thereby improving the lipid accumulation and abnormal liver function in mice induced by a high-fat, high-fructose diet.

[0049] 3. Detection of relative mRNA expression levels of core circadian rhythm genes The relative mRNA expression levels of the following core circadian clock genes in liver tissue were detected by RT-qPCR using the SYBR Green assay: clock gene (Clock), Bmal1 (a protein similar to the nuclear transporter of aryl hydrocarbon receptors in brain and muscle tissues), and cyclins Per1 and Per2. The results are shown in Tables 4-7. GAPDH was used as an internal reference gene, and 2... (-ΔΔCt) The relative expression level was calculated using the method; the control group at time point ZT0 was used as the calibration group, and its relative expression level was set to 1.

[0050] Table 4: Relative expression levels of Clock mRNA Note: a indicates that compared with the control group at the same time point, P<0.05; b indicates that compared with the model group at the same time point, P<0.05.

[0051] As shown in Table 4, the liver clock mRNA expression levels in the control group, model group, and intervention group all exhibited intraday fluctuations at different time points, and all remained at relatively high levels at ZT6. Compared to the control group, the liver clock mRNA expression levels in the model group decreased at different time points; compared to the model group, the liver clock mRNA expression levels in the intervention group increased at different time points, with little difference from the control group.

[0052] Table 5: Relative expression levels of Bmal1 mRNA Note: a indicates that compared with the control group at the same time point, P<0.05; b indicates that compared with the model group at the same time point, P<0.05.

[0053] As shown in Table 5, the hepatic Bmal1 mRNA expression levels in the control group, model group, and intervention group all exhibited intraday fluctuations at different time points, with relatively high levels at ZT0, ZT6, and ZT0, respectively. Compared to the control group, the hepatic Bmal1 mRNA expression level in the model group decreased at all time points; compared to the model group, the hepatic Bmal1 mRNA expression level in the intervention group increased at ZT0 and ZT18, and abnormal expression decreased at ZT12.

[0054] Table 6: Relative expression levels of Per1 mRNA Note: a indicates that compared with the control group at the same time point, P<0.05; b indicates that compared with the model group at the same time point, P<0.05.

[0055] As shown in Table 6, in the control group, hepatic Per1 mRNA expression was low at ZT0, then increased at ZT6, reached a relative peak at ZT12, and declined at ZT18. This expression pattern showed a relatively staggered relationship with Bmal1. Compared to the control group, the expression level of hepatic Per1 mRNA in the model group decreased at all time points, and the expression pattern was the same as that in the control group. Compared to the model group, the expression level of hepatic Per1 mRNA in the intervention group increased at all time points, and the difference from the control group was small.

[0056] Table 7: Relative expression levels of Per2 mRNA Note: a indicates that compared with the control group at the same time point, P<0.05; b indicates that compared with the model group at the same time point, P<0.05.

[0057] As shown in Table 7, in the control group, hepatic Per2 mRNA expression was low at ZT0, gradually increased, and maintained relatively high expression at ZT18. Compared with the control group, the model group showed increased hepatic Per1 mRNA expression at ZT0, and decreased expression at ZT6, ZT12, and ZT18. Compared with the model group, the abnormally high expression of Per1 and Per2 at ZT0 was reduced in the intervention group mice.

[0058] As can be seen from the above, the composition prepared in Example 6 of this application can improve the fluctuation of core circadian rhythm gene expression in mice induced by a high-fat, high-fructose diet.

[0059] 4. Analysis of the relative peak-to-trough difference in diurnal expression of core biological clock genes Based on the relative gene expression levels at four time points (ZT0, ZT6, ZT12, and ZT18), the diurnal expression fluctuation range of each core circadian clock gene was evaluated using the relative peak-to-trough difference method, as shown in Table 8. The calculation formula is: Relative peak-to-trough difference = (peak value - trough value) / average value. A larger value indicates a more significant difference in gene expression levels over 24 hours, and a more pronounced diurnal fluctuation.

[0060] Table 8: Relative peak-to-trough difference in expression for each genome As shown in Table 8, compared with the control group, the relative peak-to-trough differences of Clock, Bmal1, Per1, and Per2 in the liver of mice in the model group were all reduced, with the most significant decrease in the relative peak-to-trough difference of Bmal1. After 9 months of intervention, compared with the model group, the relative peak-to-trough differences of Clock, Bmal1, Per1, and Per2 in mice in the intervention group showed an increase and recovery.

[0061] The above experimental results indicate that a high-fat, high-fructose diet can lead to abnormal weight gain, massive lipid accumulation in the liver, hepatocyte damage, and significant downregulation of the expression of core liver clock genes in mice. The composition provided in this application can increase the amplitude of circadian rhythms by upregulating the expression of core genes Clock, Per1, Per2, and Bmal1, thereby effectively inhibiting weight gain, significantly improving liver damage, significantly reducing hepatic lipid accumulation, and restoring the liver's diurnal rhythm.

[0062] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A composition for regulating the circadian rhythm of the liver, characterized in that, Including wolfberry oligosaccharides and puerarin.

2. The composition for regulating the liver's circadian rhythm according to claim 1, characterized in that, The mass ratio of the wolfberry oligosaccharide to the puerarin is 1:(0.1-2).

3. The composition for regulating the liver's circadian rhythm according to claim 1, characterized in that, The average molecular weight of the wolfberry oligosaccharide is 0.5-2.0 kDa, and the purity of the puerarin is ≥95%.

4. The composition for regulating the liver's circadian rhythm according to claim 1, characterized in that, The preparation method of the wolfberry oligosaccharide includes: The dried wolfberries were pulverized, defatted with petroleum ether, and extracted with purified water to obtain an extract. The extract was concentrated under reduced pressure, precipitated with ethanol, deproteinized, dialyzed once, and freeze-dried once to obtain wolfberry polysaccharide; After the Lycium barbarum polysaccharide was reconstituted, hydrolyzed by Pronase E, and inactivated, it was degraded by sulfuric acid, neutralized by sodium carbonate, and centrifuged to obtain the supernatant. The supernatant was subjected to secondary dialysis, trifluoroacetic acid degradation, membrane filtration purification, and secondary freeze-drying to obtain wolfberry oligosaccharides.

5. The composition for regulating the liver's circadian rhythm according to claim 4, characterized in that, Concentrate under reduced pressure to 1 / 4-1 / 6 of the original volume; ethanol precipitation temperature is 4℃, time is 12h; deproteinization reagent is Sevag reagent, temperature is 25℃, time is 20min, the amount added is 1 / 4 of the volume of crude wolfberry polysaccharide solution; one dialysis temperature is 4℃, time is 36h; one freeze-drying temperature is -60℃, time is 36h.

6. The composition for regulating the liver's circadian rhythm according to claim 4, characterized in that, The protease E enzyme hydrolysis was carried out at 37℃ for 24 hours; followed by two dialysis cycles; and a second freeze-drying was performed at -60℃ for 36 hours.

7. The composition for regulating the circadian rhythm of the liver according to any one of claims 1-6, characterized in that, The composition further includes one or more of fillers, binders, disintegrants, flow aids, and lubricants.

8. A method for preparing the composition for regulating the liver's circadian rhythm according to any one of claims 1-7, characterized in that, include: After mixing wolfberry oligosaccharides, puerarin, fillers, binders, disintegrants, glidants, and lubricants, the mixture is dry-granulated and tableted to obtain a composition that regulates the liver's diurnal rhythm.

9. The use of the composition for regulating hepatic circadian rhythm according to any one of claims 1-7 in the preparation of a medicament for regulating hepatic circadian rhythm.

10. The use of the composition for regulating hepatic circadian rhythm according to any one of claims 1-7 in the preparation of a medicament for improving lipid metabolism disorders.

Images