Fermentation composition of curcumin and probiotics and application of fermentation composition in improvement of drug-induced liver injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a curcumin and probiotic fermentation composition and application thereof in improvement of drug-induced liver injury. The curcumin and probiotic fermented composition provided by the invention is prepared by the following steps: co-fermenting and culturing curcumin and probiotics, and extracting the cultured supernatant; and the probiotics are parabacteroides dieldrii. According to the invention, through co-culture of curcumin and parabacteroides dieldrii, the extracted fermentation supernatant composition can significantly improve the activity of hepatocytes. Experimental results show that the fermentation supernatant composition of the curcumin and the parabacteroides dieldrii provided by the invention continuously shows an effect of remarkably improving the cell viability and has a remarkable promoting effect on the liver cell viability. The invention provides a new thought and technical means for developing safer and more effective liver protection medicines, and is expected to play an important role in the field of medicines in the future.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a fermentation composition of curcumin and probiotics and its application in improving drug-induced liver injury. Background Art

[0002] Drug-Induced Liver Injury (DILI) mainly refers to the stimulation of the liver caused by the drug itself and its metabolites, resulting in abnormal liver function, or an allergic reaction caused by its metabolites, which further leads to a drug adverse reaction of immune system damage. Drug-induced liver injury affects a large number of patients globally. Oxaliplatin, as the third-generation platinum-based anticancer drug, belongs to the platinum compounds of diamminocyclohexane. Similar to other platinum drugs, it exerts its anticancer effect by destroying the proliferation of cancer cells and is commonly used to treat malignant tumors such as colorectal cancer, ovarian cancer, and lung cancer. However, while treating tumors, Oxaliplatin is also accompanied by a series of adverse reactions such as neurotoxicity, myelosuppression, and hepatotoxicity. Research shows that among patients receiving chemotherapy with Oxaliplatin-containing regimens, the incidence of hepatic sinusoidal injury is relatively high. Therefore, Oxaliplatin-induced liver injury is the main factor restricting the use of Oxaliplatin chemotherapy in patients with colorectal liver metastases.

[0003] Currently, effective treatment methods for Oxaliplatin hepatotoxicity are very limited. Clinically, the methods of reducing or stopping the drug are usually adopted, resulting in the delay of treatment for cancer patients. Therefore, how to solve the problem of Oxaliplatin hepatotoxicity has become a major challenge in its clinical application. Finding hepatotoxicity protectants has important practical significance for the clinical safe application of Oxaliplatin.

[0004] Curcumin is a natural phenolic antioxidant extracted from the rhizomes of Zingiberaceae plants (such as Curcuma longa, Curcuma phaeocaulis, Curcuma aromatica, etc.), with the chemical formula C 21 H 20 O 6 . Curcumin has a powerful antioxidant effect, can scavenge free radicals in the body, prevent oxidative damage to cells, delay aging, and prevent a variety of chronic diseases. Research shows that curcumin has various activities such as anti-inflammatory, antioxidant, and free radical scavenging, and has good therapeutic effects on inflammatory diseases such as arthritis and enteritis. Curcumin also has the function of protecting the liver, can reduce the damage of alcohol and drugs to the liver, and the combined application of Oxaliplatin and curcumin shows potential therapeutic prospects. However, there are still many problems in the application of curcumin. Its functions are complex. During the research process of the present invention, it was found that after the combination of curcumin and Oxaliplatin, it may increase the cytotoxicity of Oxaliplatin. After experimental verification, the combination of the two will further reduce the activity of normal hepatocytes.

[0005] It can be seen that how to effectively exert the anti-inflammatory and antioxidant activities of curcumin in patients with oxaliplatin-induced drug-induced liver injury to improve liver injury, while avoiding the enhancement of the cytotoxicity of oxaliplatin by curcumin and further exacerbating liver injury, has become a key problem to be solved urgently. Therefore, developing curcumin health products with good liver protection activity has significant practical value and important significance. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a fermentation composition of curcumin and probiotics and its application in improving drug-induced liver injury. The fermentation composition of curcumin and probiotics provided by the present invention can significantly improve the activity of hepatocytes and effectively avoid the problem of increased cytotoxicity and further exacerbation of liver injury after the combined use of curcumin and oxaliplatin.

[0007] The technical solution of the present invention is as follows:

[0008] A fermentation composition of curcumin and probiotics, wherein the composition is obtained by co-fermenting curcumin and probiotics and extracting the supernatant after cultivation; the probiotic is Parabacteroides distasonis.

[0009] Further, the strain number of the Parabacteroides distasonis is BNCC354946.

[0010] Further, the specific preparation method of the fermentation composition of curcumin and probiotics is as follows:

[0011] S1. Resuscitate and purify Parabacteroides distasonis to prepare a Parabacteroides distasonis seed solution;

[0012] S2. Inoculate the Parabacteroides distasonis seed solution obtained in step S1 into a bacterial liquid medium containing curcumin, perform anaerobic cultivation to obtain a fermentation broth, and then extract the supernatant to obtain the product.

[0013] Even further, the preparation method of the Parabacteroides distasonis seed solution in step S1 is as follows:

[0014] Activate the Parabacteroides distasonis strain using a bacterial solid medium, anaerobically cultivate it in an incubator at 37°C for 48 h, then inoculate 2 loops of the activated strain into 8 mL of a bacterial liquid medium, and anaerobically cultivate it at 37°C for 24 h to obtain a Parabacteroides distasonis seed solution.

[0015] Further, the preparation method of the solid bacterial culture medium is as follows: Weigh 33 g of Wilkins-Chalgren anaerobic broth medium and 15 g of agar powder, add 1 L of distilled water, stir evenly, autoclave at 121 °C for 15 min, and invert the plate and wait for it to solidify for standby.

[0016] Further, the preparation methods of the seed liquid and the bacterial liquid culture medium in step S2 are as follows: Weigh 33 g of Wilkins-Chalgren anaerobic broth medium, add 1 L of distilled water, stir evenly, and autoclave at 121 °C for 15 min.

[0017] Furthermore, the formula of the Wilkins-Chalgren anaerobic broth medium is as follows: Casein peptone 10.0 mg / ml, peptone 10.0 mg / ml, yeast extract powder 5.0 mg / ml, sodium chloride 5.0 mg / ml, glucose 1.0 mg / ml, L-arginine 1.0 mg / ml, sodium pyruvate 1.0 mg / ml, hemin 0.005 mg / ml, vitamin K1 0.0005 mg / ml.

[0018] Furthermore, the inoculation amount of the Bacteroides paradiacae seed liquid in step S1 is 1-2% of the mass of the bacterial liquid culture medium.

[0019] Furthermore, the bacterial culture medium in step S2 also contains 10-20 μM of curcumin.

[0020] Furthermore, the temperature during anaerobic culture in step S2 is 37 °C, and the culture time is 24-48 h.

[0021] Further, the method for extracting the supernatant is as follows: Centrifuge or filter and separate the fermentation broth in step S2 to obtain the supernatant.

[0022] Furthermore, the rotation speed during centrifugation is 10000-12000 rpm, and the centrifugation time is 10-15 min.

[0023] Furthermore, filtration is carried out using a 0.22 μm microporous membrane for sterilization.

[0024] In addition, the present invention also provides an application of the above fermentation composition of curcumin and probiotics in the preparation of a preparation for improving drug-induced liver injury.

[0025] Further, the drug-induced liver injury is caused by platinum-based chemotherapeutic drugs.

[0026] Furthermore, the platinum-based chemotherapeutic drugs.

[0027] Furthermore, the preparation is an oral preparation or an injection preparation, comprising an effective amount of the fermentation composition of curcumin and probiotics prepared above and pharmaceutically acceptable excipients.

[0028] The present invention uses Parabacteroides distasonis as a starting strain, creatively extracts the supernatant after culture through a co-culture system of curcumin and Parabacteroides distasonis, and obtains a fermentation supernatant composition with hepatoprotective activity. The metabolic conversion effect of probiotics is utilized to not only enhance the original hepatoprotective effect of curcumin, but also effectively avoid the side effect of curcumin enhancing the cytotoxicity of oxaliplatin.

[0029] Experimental data show that in an environment containing oxaliplatin, the use of curcumin alone will lead to increased cytotoxicity and thus reduce cell survival rate. However, when the fermentation supernatant composition of the present invention is used, it continues to show the ability to significantly improve liver cell activity, which shows that the composition can not only effectively protect the liver from damage, but also overcome the negative effects that traditional curcumin may cause in some cases. In addition, this new composition also shows good biocompatibility and low toxicity risk, making it a potential treatment option, especially for patients who need long-term medication or have a high risk of liver damage.

[0030] Specifically, the key technical points of the present invention include the following aspects: 1. Selection of the co-culture system: The probiotic bacterium Parabacteroides distilogenes is creatively selected as the co-culture object with curcumin, which can effectively metabolize curcumin and produce a series of beneficial metabolites. 2. Optimization of the fermentation process: By carefully adjusting the fermentation conditions (such as temperature, pH value, culture time, etc.), it is ensured that the active ingredients are obtained to the maximum extent. 3. Optimization of dosage and ratio: According to the experimental results, the ratio of curcumin to the fermentation supernatant is accurately adjusted to find the best combination to achieve the best therapeutic effect.

[0031] Compared with the prior art, the fermentation composition of curcumin and probiotics provided by the present invention and its application in improving drug-induced liver injury have the following advantages:

[0032] The present invention co-cultivates curcumin and Parabacteroides distiliensis, and the fermentation supernatant composition extracted can significantly improve the activity of hepatocytes. Experimental results show that the fermentation supernatant composition of curcumin and Parabacteroides distiliensis provided by the present invention continues to show the effect of significantly improving cell activity and has a significant promoting effect on hepatocyte activity. The present invention provides new ideas and technical means for the development of safer and more effective liver protection drugs, and is expected to play an important role in the future medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1To investigate the effects of orally administered Parabacteroides distasonis and Bifidobacterium pseudolongum on the body weight of mice with liver injury;

[0034] Figure 2 To investigate the effects of orally administered Parabacteroides distasonis and Bifidobacterium pseudolongum on the serum ALT and AST levels of mice with liver injury;

[0035] Figure 3 To investigate the effects of orally administered Parabacteroides distasonis and Bifidobacterium pseudolongum on the serum inflammatory factor levels of mice with liver injury;

[0036] Figure 4 To investigate the effects of orally administered Parabacteroides distasonis and Bifidobacterium pseudolongum on the oxidative stress level of liver tissues in mice with liver injury;

[0037] Figure 5 To investigate the effects of the doses of curcumin and oxaliplatin on the proliferation of AML12 cells, where a is the effect of the curcumin dose on the proliferation of AML12 cells; b is the effect of the oxaliplatin dose on the proliferation of AML12 cells and IC20;

[0038] Figure 6 To investigate the effect of the combined administration of curcumin and oxaliplatin on the proliferation of AML12 cells;

[0039] Figure 7 To investigate the effects of curcumin and oxaliplatin on the ALT and AST levels of AML12 cells;

[0040] Figure 8 To investigate the effects of curcumin and oxaliplatin on the TNF-α and IL-6 levels of AML12 cells;

[0041] Figure 9 To investigate the effects of curcumin and oxaliplatin on the SOD and MDA levels of AML12 cells;

[0042] Figure 10 To investigate the effects of bacterial culture supernatant and oxaliplatin on the survival rate of mouse hepatocyte AML12, where a is the effect of the bacterial culture supernatant concentration on the proliferation of AML12 cells; b is the effect of the combined administration of bacterial supernatant and oxaliplatin on the proliferation of AML12 cells;

[0043] Figure 11 To investigate the effects of bacterial culture supernatant on the AST and ALT levels of AML12 cells;

[0044] Figure 12 To investigate the effects of bacterial culture supernatant on the SOD and MDA levels of AML12 cells;

[0045] Figure 13 To investigate the effects of bacterial culture supernatant on the TNF-α and IL-6 levels of AML12 cells. Detailed implementation methods

[0046] The present invention will be further described below through the description of specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are all within the protection scope of the present invention.

[0047] In the following examples and comparative examples, reagents not specifically mentioned are conventional reagents and can be purchased from conventional reagent production and sales companies. The methods used, unless otherwise specified, are all prior arts.

[0048] Example 1

[0049] 1. Cultivation and extraction of Parabacteroides distasonis and Bifidobacterium pseudolongum

[0050] (1) Preparation of culture medium

[0051] Bacterial liquid medium: Weigh 33 g of Wilkins-Chalgren anaerobic broth medium, add 1 L of distilled water, stir evenly, and autoclave at 121 °C for 15 min.

[0052] Bacterial solid medium: Weigh 33 g of Wilkins-Chalgren anaerobic broth medium and 15 g of agar powder, add 1 L of distilled water, stir evenly, and autoclave at 121 °C for 15 min. Invert the plate and wait for it to solidify for later use.

[0053] The formula of the Wilkins-Chalgren anaerobic broth medium is as follows:

[0054] Modified MRS liquid medium: Weigh 10 g of peptone, 10 g of beef extract, 4 g of yeast powder, 20 g of D(+)-glucose, 0.2 g of magnesium sulfate, 5 g of sodium acetate, 2 g of trisodium citrate, 2 g of dipotassium hydrogen phosphate, 0.05 g of manganese sulfate, 1 g of Tween 80, 0.5 g of L-cysteine, add 1 L of distilled water, stir evenly, and autoclave at 121 °C for 15 min.

[0055] Modified MRS solid medium: Weigh 10 g of peptone, 10 g of beef extract, 4 g of yeast powder, 20 g of D(+)-glucose, 0.2 g of magnesium sulfate, 5 g of sodium acetate, 2 g of trisodium citrate, 2 g of dipotassium hydrogen phosphate, 0.05 g of manganese sulfate, 1 g of Tween 80, 0.5 g of L-cysteine, and 15 g of agar powder, add 1 L of distilled water, stir evenly, and autoclave at 121 °C for 15 min. Invert the plate and wait for it to solidify for later use.

[0056] (2) Preparation of Parabacteroides distasonis seed solution

[0057] Bacteroides paravasculorum (purchased from Beina Biology, strain number: BNCC354946) was activated using a bacterial solid medium and anaerobically cultured in an incubator at 37°C for 48 h. Then, two loops of the activated strain were inoculated into 8 mL of a liquid medium and anaerobically cultured at 37°C for 24 h to obtain a Bacteroides paravasculorum seed solution.

[0058] (3) Preparation of Bifidobacterium pseudolongum seed solution

[0059] Bifidobacterium pseudolongum (purchased from the Shanghai Culture Collection of Microorganisms, strain number: SHBCC D51582) was activated using a modified MRS solid medium and anaerobically cultured in an incubator at 37°C for 48 h. Then, two loops of the activated strain were inoculated into 8 mL of a modified MRS liquid medium and anaerobically cultured at 37°C for 24 h to obtain a Bifidobacterium pseudolongum seed solution.

[0060] (4) Extraction method of Bacteroides paravasculorum and Bifidobacterium pseudolongum

[0061] The two kinds of cultured bacterial solutions in (2) and (3) were centrifuged at 12,000 rpm for 10 min, the supernatant was discarded, and they were resuspended using sterile and anaerobic phosphate-buffered saline (PBS).

[0062] 2. Gavage of bacteria to liver injury mice:

[0063] Forty male BALB / cJ mice (6 - 8 weeks old) were randomly divided into 6 groups after 5 days of adaptive feeding with normal feed. The groups were: Control group, Oxaliplatin group (OXA), Live Bacteroides paravasculorum treatment group (OXA + Live P. distasonis), Live Bifidobacterium pseudolongum treatment group (OXA + Live B. pseudo), Heat-killed Bacteroides paravasculorum treatment group (OXA + Dead P. distasonis), and Heat-killed Bifidobacterium pseudolongum treatment group (OXA + Dead B. pseudo). There were 5 mice in the Control group and 7 mice in each of the other groups. Each week, the mice in each group were intraperitoneally injected with 8 mg / kg OXA, and the mice in the Control group were injected with 5% glucose solution instead. Each treatment group was gavaged with the corresponding bacteria 30 minutes before and 3 days after the intraperitoneal injection of oxaliplatin, 2×10 8 CFU per mouse, and the mice in the Control group were gavaged with PBS instead, for 7 consecutive weeks.

[0064] 3. Evaluation of the therapeutic effect of gavage with bacteria:

[0065] The body weight of mice was recorded weekly. After the last administration, all mice were fasted overnight with free access to water. Blood was collected by orbital enucleation, and then the mice were sacrificed. The liver was dissected to detect the biochemical markers of liver injury, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the inflammatory factors, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in the serum. The oxidative stress indexes in liver tissue, catalase (CAT), malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) were detected.

[0066] 4. Experimental results:

[0067] The effects of Parabacteroides distasonis and Bifidobacterium pseudolongum on liver injury in mice are shown as Figures 1 to 4 follows. Among them, OXA+Live P.distasonis: oral live Parabacteroides distasonis treatment group, OXA+Live B.pseudo: oral live Bifidobacterium pseudolongum treatment group, OXA+Dead P.distasonis: oral inactivated Parabacteroides distasonis treatment group, OXA+Dead B.pseudo: oral inactivated Bifidobacterium pseudolongum treatment group, PBS: healthy control group, OXA: drug-induced liver injury model group.

[0068] As Figure 1 shown in the live bacteria gavage experiment, both live Parabacteroides distasonis and Bifidobacterium pseudolongum alleviated the body weight trend of mice, and the former had a more obvious effect, while the inactivated bacteria lost the improvement effect. As Figure 2 shown, all 4 treatment groups reduced the contents of ALT and AST enzymes in the serum of liver injury mice to a certain extent, but only live Parabacteroides distasonis had the most significant improvement effect. Figure 3 It was shown that in the serum, live Parabacteroides distasonis significantly reduced the release of the two inflammatory factors, while live Bifidobacterium pseudolongum significantly improved the release of TNF-α in the serum of mice, and the improvement effect of the inactivated bacteria was not significant. As Figure 4 shown, live Parabacteroides distasonis significantly reduced the levels of various oxidative stresses in the liver of liver injury mice, and live Bifidobacterium pseudolongum also showed certain antioxidant activity. Live Parabacteroides distasonis played a significant alleviating role in most indexes and was superior to live Bifidobacterium pseudolongum. It was confirmed the therapeutic potential of Parabacteroides distasonis for liver injury.

[0069] Example 2

[0070] 1. Preparation of drug solution:

[0071] (1) Preparation of AML12 complete medium containing curcumin: Weigh 2.947 mg of curcumin powder, dissolve it in 100 μL of DMSO, and then add it to 10 mL of cell culture medium. Vortex to mix evenly to obtain a curcumin cell culture medium with a concentration of 800 μM, and then serially dilute it to curcumin cell culture media with concentrations of 0, 5, 10, 20, and 40 μM.

[0072] The composition of the AML12 complete medium is 89% DMEM-H / F-12 + 10% FBS + 1% ITS Liquid Media Supplement + 40 ng / ml Dexamethasone, purchased from Beina Biotechnology, product number: BNCC361109.

[0073] (2) Preparation of cell culture medium containing oxaliplatin: Weigh 2.56 mg of oxaliplatin powder, dissolve it in 2 mL of cell culture medium, and vortex to mix evenly to obtain an oxaliplatin cell culture medium with a concentration of 1280 μg / mL, and then serially dilute it to curcumin cell culture media with concentrations of 0, 0.5, 1, 2, 4, 8, 16, and 32 μg / mL.

[0074] 2. Method for detecting the viability of normal mouse hepatocytes by CCK8 assay:

[0075] Normal mouse hepatocytes AML12 grow in AML12 complete medium and are cultured in a cell culture incubator at 37 °C, 5% CO 2 and fully saturated humidity. The medium is changed every other day to maintain a good growth state. Logarithmic growth phase cells are seeded at 5×10 3 cells / well in a 96-well plate, with 100 μL of cell suspension seeded in each well. Five replicates are set for each time point. Add 200 μL of sterile PBS buffer to the edge wells to prevent medium evaporation. After culturing in the incubator for 12 h, add 100 μL of cell culture medium containing different concentrations of curcumin and oxaliplatin to each well.

[0076] After culturing in the incubator until the corresponding time point, aspirate the medium, wash three times with PBS, add 100 μL of fresh medium containing 10% CCK8 solution, and incubate at 37 °C for 1 h. Measure the absorbance value of each well sample at a wavelength of 450 nm using a microplate reader. The experimental results are expressed as mean ± standard deviation, and a bar graph is made with time as the abscissa and the percentage value of cell viability as the ordinate.

[0077] 3. Detection of the viability of normal mouse hepatocytes under co-culture conditions:

[0078] According to the dose exploration results, the cell viability under the conditions of 20 μM curcumin and 1.932 μg / mL oxaliplatin was explored. A control group (Con), a curcumin group (Cur), an oxaliplatin group (OXA), and a combined group (OXA+Cur) were set up. The control group was only added with AML12 complete medium, the curcumin group was added with 20 μM curcumin, the oxaliplatin group was added with 1.932 μg / mL oxaliplatin, and the combined group was added with 20 μM curcumin and 1.932 μg / mL oxaliplatin. The remaining cell viability measurement methods were the same as above.

[0079] 4. Detection of cell inflammatory factors and oxidative stress levels:

[0080] Biochemical markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in cells were detected; inflammatory factors tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6); oxidative stress indicators catalase (CAT), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were detected.

[0081] 5. Experimental results:

[0082] The experimental results of the effects of curcumin and oxaliplatin on the viability, oxidative stress, and inflammatory factor levels of mouse hepatocytes AML12 are as Figures 5 to 9 shown, where Con: blank group, Cur: curcumin group, OXA: oxaliplatin group, OXA+Cur: combined administration group.

[0083] The cell viability results are as Figure 5 shown in a and b, indicating that curcumin at a certain concentration has a proliferative effect on hepatocytes. The IC20 of oxaliplatin for hepatocytes is 1.932 μg / mL. Figure 6 It shows that under the condition of co-culturing curcumin and oxaliplatin, the cytotoxicity is increased and the cell viability is reduced. Figure 7 , 8, 9 indicate that curcumin does not improve the cell damage caused by oxaliplatin and does not exert anti-inflammatory and antioxidant activities. This indicates that there may be potential risks in the combined use of curcumin alone and oxaliplatin.

[0084] Example 3

[0085] 1. Preparation of fermentation supernatant composition:

[0086] Bacterial liquid medium: Weigh 6.6 g of Wilkins-Chalgren anaerobic broth medium, add 200 mL of distilled water, stir evenly, and autoclave at 121 °C for 15 min.

[0087] Bacterial solid medium: Weigh 6.6 g of Wilkins-Chalgren anaerobic broth medium and 3 g of agar powder, add 200 mL of distilled water, stir evenly, and autoclave at 121 °C for 15 min. Invert the petri dish and wait for it to solidify for later use.

[0088] Preparation of bacterial liquid medium containing curcumin: Weigh 3.684 mg of curcumin powder, dissolve it in 500 μL of DMSO, and then add it to 50 mL of bacterial liquid medium. Vortex to mix evenly to obtain a curcumin bacterial medium with a concentration of 200 μM, and then dilute it to a curcumin bacterial medium with a concentration of 20 μM.

[0089] Cultivation method of Parabacteroides distasonis: Activate the strain using solid medium and anaerobically culture it in an incubator at 37 °C for 48 h. Then, take 2 loops of the activated strain and inoculate it into the liquid medium, and anaerobically culture it at 37 °C for 24 h to obtain the seed liquid. The seed liquid is inoculated into 20 mL of liquid medium with or without 20 μM curcumin at an inoculation amount of 1%, and anaerobically cultured for 48 h. Then, centrifuge at 12,000 rpm for 10 min to extract the supernatant, and obtain the co-fermentation and single-fermentation supernatants respectively for later use.

[0090] 2. Detection of the effect of supernatant compositions at different doses on hepatocyte viability by CCK8 method:

[0091] The dose gradient of the fermentation supernatant composition is 0, 2.5, 5, 10, 20 μL / well, and the remaining cell viability measurement methods are the same as above.

[0092] 3. Detection of the viability of normal mouse hepatocytes under different culture conditions:

[0093] Set up cell culture medium groups (DMEM) with or without 20 μM curcumin, bacterial medium groups (WC), and supernatant composition groups (Supernatant). According to the dose exploration results, explore the effect of different groups on cell viability under the condition of co-culturing with 10 μL / well concentration and 1.932 μg / mL oxaliplatin. The remaining cell viability measurement methods are the same as above.

[0094] 4. Detection of cell inflammatory factors and oxidative stress levels:

[0095] Detect biochemical markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in cells; inflammatory factors tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6); detect oxidative stress indicators catalase (CAT), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH).

[0096] 5. Experimental results:

[0097] The experimental results of the effects of bacterial culture supernatant and oxaliplatin on the survival rate, oxidative stress and inflammatory factor levels of mouse hepatocytes AML12 are as follows Figures 10 to 13 shown. Among them, DMEM: cell culture medium. WC: bacterial culture medium. Supernatant: bacterial culture supernatant.

[0098] The results of cell survival rate are as follows Figure 10 shown in a. Except for the dose of 20 μL / well, the other supernatant composition dose groups had a significant promoting effect on the proliferation of hepatocytes. Figure 10 b shows that only the supernatant composition continuously showed a significant improvement in cell activity induced by oxaliplatin at 12 h, 24 h, 36 h, and 48 h, and was superior to the effect of single fermentation supernatant, while the other negative controls did not show an improvement effect. Figure 11 , 12, 13 show that the co-fermentation composition of Parabacteroides distasonis and curcumin significantly improved the hepatocyte damage, oxidative stress and inflammation caused by oxaliplatin, showed good hepatoprotective activity, and was superior to the single fermentation supernatant.

[0099] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A fermentation composition of curcumin and probiotics, characterized in that: The composition is prepared by co-fermentation and culture of curcumin and probiotics, and extracting the supernatant after culture; the probiotics are Parabacteroides distasonis.

2. The fermented composition of curcumin and probiotics according to claim 1, characterized in that: The strain number of the Parabacteroides distasonis is BNCC 354946.

3. The fermented composition of curcumin and probiotics according to claim 1 or 2, characterized in that: The specific preparation method of the fermented composition of curcumin and probiotics is as follows: S1, resuscitating and purifying Parabacteroides distiliensis to prepare Parabacteroides distiliensis seed solution; S2, the Parabacteroides dieldii seed solution obtained in step S1 is inoculated into a bacterial liquid culture medium containing curcumin, and anaerobically cultured to obtain a fermentation liquid, and then the supernatant is extracted to obtain the product.

4. The fermented composition of curcumin and probiotics according to claim 3, characterized in that: In the step S1, the inoculation amount of the Parabacteroides distiliensis seed solution is 1-2% of the mass of the bacterial culture medium.

5. The fermented composition of curcumin and probiotics according to claim 3, characterized in that: The bacterial liquid culture medium in step S2 is Wilkins-Chalgren anaerobic broth medium, and the specific formula is as follows: 10.0 mg / ml tryptic peptone, 10.0 mg / ml peptone, 5.0 mg / ml yeast extract powder, 5.0 mg / ml sodium chloride, 1.0 mg / ml glucose, 1.0 mg / ml L-arginine, 1.0 mg / ml sodium pyruvate, 0.005 mg / ml hemin, and 0.0005 mg / ml vitamin K1.

6. The fermented composition of curcumin and probiotics according to claim 3, characterized in that: The bacterial culture medium in step S2 also contains 10-20 μM curcumin.

7. The fermented composition of curcumin and probiotics according to claim 3, characterized in that: The temperature during anaerobic culture in step S2 is 37° C., and the culture time is 24 to 48 hours.

8. Use of the fermented composition of curcumin and probiotics according to any one of claims 1 to 7 in preparing a preparation for improving drug-induced liver injury.

9. The use according to claim 8, characterized in that: The drug-induced liver injury is caused by platinum chemotherapy drugs.

10. The use according to claim 8, characterized in that: The preparation is an oral preparation or an injection preparation, comprising an effective amount of the fermentation composition of curcumin and probiotics prepared above and pharmaceutically acceptable excipients.

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