Gardenia jasminoides ellis and radix scutellariae compound granules with liver protection effect as well as preparation method and application thereof

Abstract

The invention discloses fructus gardeniae and radix astragali compound granules with a liver protection effect. The fructus gardeniae and radix astragali compound granules are prepared from the following raw materials in parts by weight: 800 to 1200 parts of fructus gardeniae, 800 to 1200 parts of radix astragali and 800 to 1200 parts of fructus crataegi. The composition further comprises cyclodextrin and disintegrating starch. The invention further discloses a preparation method of the gardenia jasminoides ellis and radix astragali compound granules with the liver protection effect, and the preparation method specifically comprises the following steps: (1) weighing the raw materials, and averagely dividing radix astragali into two parts; (2) performing alcohol extraction on the fructus gardeniae; (3) performing water extraction on the first part of astragalus membranaceus; (4) performing alcohol extraction on the second part of radix astragali; (5) carrying out alcohol extraction on the hawthorn; and (6) uniformly mixing the gardenia extract, the astragalus membranaceus water extract, the astragalus membranaceus alcohol extract, the hawthorn extract, cyclodextrin and disintegrating starch, and granulating to obtain the traditional Chinese medicine composition. The three medicines are combined to achieve the effects of protecting the liver and enhancing immunity, and liver injury, immunosuppression and oxidative stress caused by AFB1 can be effectively relieved.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine health care, and more particularly to a Zhihuang compound granule with liver protection effect and a preparation method and application thereof. Background Art

[0002] Aflatoxins (AF) are a class of difuranocoumarin compounds with similar structures, mainly produced by the metabolism of toxin-producing parasitic Aspergillus (Aspergillus parasiticus) and Aspergillus flavus (Aspergillus flavus). From the chemical structure, aflatoxin contains a oxazinone and a difuran ring. The former is related to carcinogenesis, and the latter is the basic toxin structure. The former can enhance the toxicity of the latter.

[0003] At present, the most common AFs are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2). The first four are usually naturally present, while the latter two are formed by metabolic hydroxylation of AFB1 and AFB2 in the body. Among them, AFB1 is the main form and has the highest toxicity. It was classified as a human carcinogen by the International Agency for Research on Cancer (IARC) as early as 1993. AFB1 is widely present in the environment, soil and food crops, and seriously threatens the health of humans, poultry and livestock, especially it is highly toxic to the liver.

[0004] Studies have shown that liver damage caused by AFB1 can be divided into three aspects. First, AFB1 produces AFBO through CYP450-mediated hepatotoxicity. AFB1 is extremely toxic and can cause hepatocellular necrosis, bile duct hyperplasia, liver fibrosis and even liver cancer. It is recognized as one of the two important factors closely related to the occurrence of primary hepatocellular carcinoma. Second, AFB1 has immunosuppressive toxicity, which can reduce the body's resistance to secondary infections of bacteria, fungi and parasites. It mainly acts on the body's cellular immune system and can induce thymic dysplasia. AFB1 reduces lymphocyte function and number, inhibits phagocytic ability, complement activity and natural killer cell lysis. Third, AFB1 will generate a large amount of ROS after metabolism by CYP450, which causes oxidative stress damage. Based on the above analysis, it can be seen that the effects caused by AFB1 mainly include liver damage, immunosuppression and oxidative stress.

[0005] Therefore, how to protect the liver is an urgent problem to be solved by technicians in this field. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a Zhihuang compound granule with liver protection effect and a preparation method and application thereof, so as to solve the deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The invention discloses a Zhihuang compound granule with liver protection effect, comprising the following raw materials in parts by weight: 800-1200 parts of Gardenia, 800-1200 parts of Astragalus and 800-1200 parts of Hawthorn; and also comprising cyclodextrin and disintegrated starch.

[0009] Furthermore, the above-mentioned Zhihuang compound granules with liver-protecting effect include the following raw materials in parts by weight: 1000 parts of gardenia, 1000 parts of astragalus and 1000 parts of hawthorn; and also include cyclodextrin and disintegrated starch.

[0010] A method for preparing Zhihuang compound granules having a liver-protecting effect, specifically comprising the following steps:

[0011] (1) Weigh the raw materials according to the weight of the Zhihuang Fufang Granules, and divide the Astragalus into two equal parts;

[0012] (2) adding ethanol to gardenia, performing reflux extraction, and concentrating the extract to dryness under reduced pressure to obtain a gardenia extract;

[0013] (3) adding water to the first portion of Astragalus, decocting, and freeze-drying to obtain an Astragalus water extract;

[0014] (4) adding ethanol to the second portion of Astragalus, refluxing for extraction, and concentrating the extract to dryness under reduced pressure to obtain an Astragalus alcohol extract;

[0015] (5) adding ethanol to hawthorn, performing reflux extraction, and concentrating the extract to dryness under reduced pressure to obtain a hawthorn extract;

[0016] (6) The gardenia extract, astragalus water extract, astragalus alcohol extract, hawthorn extract, cyclodextrin and disintegrated starch are mixed evenly and granulated to obtain the gardenia and huang compound granules with liver protection effect.

[0017] Furthermore, in the above step (2), the mass percentage of ethanol is 75%, and the amount used is 8 times the weight of the gardenia; the number of reflux extraction is 2 times, and the time is 90 minutes.

[0018] Furthermore, in the above step (3), the amount of water used is 8 times the weight of the first portion of Astragalus; and the decoction time is 90 minutes.

[0019] Furthermore, in the above step (4), the mass percentage of ethanol is 75%, and the amount used is 8 times the weight of the second portion of Astragalus; the number of reflux extractions is 2 times, and the time is 90 minutes.

[0020] Furthermore, in the above step (5), the mass percentage of ethanol is 75%, and the amount used is 10 times the weight of hawthorn; the number of reflux extraction is 2 times, and the time is 90 minutes.

[0021] Furthermore, in the above step (6), the crude drug content of the Zhihuang Fufang granules is 1 g / g.

[0022] The present invention also seeks to protect the use of the Zhihuang Compound Granules or the Zhihuang Compound Granules prepared by the above preparation method in the preparation of liver protection drugs.

[0023] Furthermore, the above-mentioned liver protection drug is a drug that alleviates liver damage, immunosuppression and oxidative stress caused by AFB1.

[0024] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] According to traditional Chinese medicine theory and modern pharmacological effects, Gardenia has the effects of purging fire and relieving restlessness, clearing heat and removing dampness, cooling blood and detoxifying. Modern pharmacological studies have shown that it has the effects of protecting the liver, promoting bile secretion, lowering blood pressure, calming, stopping bleeding, and reducing swelling. It is often used in the treatment of icteric hepatitis, sprains, hypertension, diabetes and other diseases in clinical Chinese medicine. However, Gardenia is bitter and cold and hurts the stomach. It is not suitable for people with spleen deficiency and loose stools. Therefore, it is suitable for long-term use with tonic medicine. The main effects of Astragalus are to replenish qi and raise yang, consolidate the surface and stop sweating, promote diuresis and reduce swelling, promote fluid and nourish blood, relieve stagnation and relieve arthritis, support toxins and discharge pus, and astringe sores and promote muscle regeneration. Modern pharmacological studies have shown that it can enhance the body's immune function, protect the liver, promote diuresis, resist aging, resist stress, reduce blood pressure and have a wide range of antibacterial effects, but it should not be taken for patients with strong surface evil, qi stagnation and dampness obstruction, food stagnation, carbuncle at the beginning or after ulceration, and patients with yin deficiency and yang hyperactivity. Fried hawthorn can strengthen the spleen and appetite, and relieve food stagnation. At the same time, modern pharmacological research shows that the total polyphenols and total flavonoids it contains can scavenge DPPH free radicals and ABTS free radicals, and has a strong antioxidant effect.

[0026] Therefore, the present invention utilizes the "liver-protecting and bile-promoting" effect of gardenia, the effect of astragalus on replenishing qi and raising yang and enhancing immunity, and the effect of hawthorn on strengthening the spleen and appetizing. At the same time, hawthorn can alleviate the harmful effect of the bitter and cold nature of gardenia on the spleen and stomach, and astragalus can overcome the bitter and cold nature of gardenia. The three medicines are used together to protect the liver and enhance immunity, which can effectively alleviate liver damage, immunosuppression and oxidative stress caused by AFB1. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the HPLC chromatogram of Zhihuang Fufang Granules;

[0028] Figure 2 To study the effect of Zhihuang Fufang Granules on blood cells of mice poisoned by aflatoxin;

[0029] Figure 3To study the effect of Zhihuang Fufang Granule on serum AST and ALT in patients with aflatoxin poisoning;

[0030] Figure 4 To study the effect of Zhihuang Fufang Granule on the liver tissue structure of mice poisoned by aflatoxin.

[0031] Figure 5 To study the effect of Zhihuang Fufang Granules on serum IgG and IgM in mice with aflatoxin poisoning;

[0032] Figure 6 To study the effect of Zhihuang Fufang Granules on the intestinal flora structure of mice poisoned by aflatoxin.

[0033] Figure 7 Zhihuang Fufang Granules changed the composition of intestinal flora in mice poisoned by aflatoxin;

[0034] Figure 8 Screening of core functional bacteria that enable Gardenia Compound Granules to exert its efficacy. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] The Zhihuang compound granules with liver protection effect include the following raw materials in parts by weight: 1kg of Gardenia, 1kg of Astragalus and 1kg of Hawthorn; and also include cyclodextrin and disintegrated starch;

[0038] The preparation method of the Zhihuang compound granules having liver protection effect specifically comprises the following steps:

[0039] (1) Weigh the raw materials according to the weight of the Zhihuang Fufang Granules, and divide the Astragalus into two equal parts;

[0040] (2) adding 8 kg of 75% ethanol by mass to the gardenia, performing reflux extraction twice for 90 min, and concentrating the extract to dryness under reduced pressure to obtain a gardenia extract;

[0041] (3) adding 8 kg of water to the first portion of Astragalus, boiling for 90 min, and freeze-drying to obtain an Astragalus water extract;

[0042] (4) adding 8 kg of 75% by mass ethanol to the second portion of Astragalus, reflux extraction was performed twice for 90 min, and the extract was concentrated to dryness under reduced pressure to obtain an Astragalus alcohol extract;

[0043] (5) adding 10 kg of 75% by mass ethanol to the hawthorn, performing reflux extraction twice for 90 min, and concentrating the extract to dryness under reduced pressure to obtain a hawthorn extract;

[0044] (6) The gardenia extract, astragalus water extract, astragalus alcohol extract, hawthorn extract, cyclodextrin and disintegrated starch are mixed evenly and granulated to obtain the hepatoprotective Zhihuang compound granules with a crude drug content of 1 g / g.

[0045] Example 2

[0046] The Zhihuang compound granules with liver protection effect include the following raw materials in parts by weight: 800g of Gardenia, 800g of Astragalus and 1200g of Hawthorn; and also include cyclodextrin and disintegrated starch;

[0047] The preparation method of the Zhihuang compound granules having liver protection effect specifically comprises the following steps:

[0048] (1) Weigh the raw materials according to the weight of the Zhihuang Fufang Granules, and divide the Astragalus into two equal parts;

[0049] (2) adding 8 kg of 75% ethanol by mass to the gardenia, performing reflux extraction twice for 90 min, and concentrating the extract to dryness under reduced pressure to obtain a gardenia extract;

[0050] (3) adding 8 kg of water to the first portion of Astragalus, boiling for 90 min, and freeze-drying to obtain an Astragalus water extract;

[0051] (4) adding 8 kg of 75% by mass ethanol to the second portion of Astragalus, reflux extraction was performed twice for 90 min, and the extract was concentrated to dryness under reduced pressure to obtain an Astragalus alcohol extract;

[0052] (5) adding 10 kg of 75% by mass ethanol to the hawthorn, performing reflux extraction twice for 90 min, and concentrating the extract to dryness under reduced pressure to obtain a hawthorn extract;

[0053] (6) The gardenia extract, astragalus water extract, astragalus alcohol extract, hawthorn extract, cyclodextrin and disintegrated starch are mixed evenly and granulated to obtain the hepatoprotective Zhihuang compound granules with a crude drug content of 1 g / g.

[0054] Example 3

[0055] The Zhihuang compound granules with liver protection effect include the following raw materials in parts by weight: 1200g of Gardenia, 1200g of Astragalus and 800g of Hawthorn; and also include cyclodextrin and disintegrated starch;

[0056] The preparation method of the Zhihuang compound granules having liver protection effect specifically comprises the following steps:

[0057] (1) Weigh the raw materials according to the weight of the Zhihuang Fufang Granules, and divide the Astragalus into two equal parts;

[0058] (2) adding 8 kg of 75% ethanol by mass to the gardenia, performing reflux extraction twice for 90 min, and concentrating the extract to dryness under reduced pressure to obtain a gardenia extract;

[0059] (3) adding 8 kg of water to the first portion of Astragalus, boiling for 90 min, and freeze-drying to obtain an Astragalus water extract;

[0060] (4) adding 8 kg of 75% by mass ethanol to the second portion of Astragalus, reflux extraction was performed twice for 90 min, and the extract was concentrated to dryness under reduced pressure to obtain an Astragalus alcohol extract;

[0061] (5) adding 10 kg of 75% by mass ethanol to the hawthorn, performing reflux extraction twice for 90 min, and concentrating the extract to dryness under reduced pressure to obtain a hawthorn extract;

[0062] (6) The gardenia extract, astragalus water extract, astragalus alcohol extract, hawthorn extract, cyclodextrin and disintegrated starch are mixed evenly and granulated to obtain the hepatoprotective Zhihuang compound granules with a crude drug content of 1 g / g.

[0063] Performance Testing

[0064] To explore the protective effect of Zhihuang Fufang Granules on mice with aflatoxin-induced liver damage and its effects on intestinal flora and metabolism.

[0065] 1. Introduction

[0066] Based on the theoretical basis of traditional Chinese medicine, the present invention combines Gardenia, Astragalus and Crataegus to investigate their protective effects on mice with liver damage caused by aflatoxin, and analyzes the changes in their intestinal flora and metabolites to explore their possible mechanism of action.

[0067] 2. Materials and Methods

[0068] 2.1 Test materials and reagents

[0069] Gardenia, Astragalus and Crataegus were purchased from a Chinese herbal medicine company in Bozhou.

[0070] 95% ethanol, chromatography-grade acetonitrile, formic acid, and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0071] Double distilled water was prepared in the laboratory.

[0072] Aflatoxin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0073] Silybin meglumine tablets (National Medicine Standard No. H32026145) were purchased from Jiangsu Zhongxing Pharmaceutical Co., Ltd.

[0074] ALT (C009-1-1), AST (C010-1-1), MDA (C009-1-1), and SOD (C010-1-1) biochemical index detection kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0075] Mouse IgG and IgM enzyme-linked immunosorbent assay kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0076] Blood routine test reagents were purchased from Shenzhen Dima Biotechnology Co., Ltd.

[0077] Hematoxylin-eosin stain was purchased from Solebio Biotechnology Co., Ltd.

[0078] 2.2 Preparation of test drugs

[0079] Take the Zhihuang Fufang Granules (ZHLG) with liver protection effect prepared in Example 1 and set aside.

[0080] 2.3 Fingerprint of Zhihuang Fufang Granules

[0081] Weigh Zhihuang Fufang granules, dissolve in 50% methanol, pass through a microporous filter with a pore size of 0.22μm, and set aside. Use Waters 2695 high performance liquid chromatograph, full wavelength diode array detector, and Diamond C18 column (250mm×2.0mm, 4.5μm). Gradient elution was performed with 0.1% formic acid aqueous solution and acetonitrile, where the change of acetonitrile concentration over time was as follows: 0-20min, 5%→10%, 20-20.1min, 10%→90%, 20.1-25min, 90%, 25.0-25.1min, 90%→5%, 25.1-28min, 5%. Column temperature was 30℃. The injection volume was 10μL.

[0082] The results are as follows Figure 1 As shown, among them, 1. Chlorogenic acid, 2. Gardenia glycoside, A: test sample fingerprint; B: mixed standard solution fingerprint; C: Gardenia glycoside fingerprint; D: Chlorogenic acid fingerprint.

[0083] 2.4 Experimental Animal Grouping

[0084] 48 mice were randomly divided into 6 groups, namely, control group (Control, 8 mice), model group (Model, 8 mice), positive control group (Positive, 8 mice), ZHLG low-dose group (GGCA-Ⅰ, 8 mice), ZHLG medium-dose group (GGCA-Ⅱ, 8 mice), and ZHLG high-dose group (GGCA-Ⅲ, 8 mice). On the 1st to 3rd day of the experiment, except for the control group, the other groups were given AFB1 modeling at 450μg / kg for 3 consecutive days. The control group was gavaged with normal saline every day, and the administration volume was 0.2mL / 10g. On the 1st to 7th day of the experiment, the ZHLG low-dose group, ZHLG medium-dose group, and ZHLG high-dose group were gavaged with Zhihuang Fufang granules at 1.5g / kg, 3g / kg, and 6g / kg, respectively, and the positive control group was given Silybin Meglumine Tablets at a dose of 150mg / kg.

[0085] 2.4 Sample collection

[0086] On the 8th day of the experiment, the mice were collected for anticoagulated blood, non-anticoagulated blood, liver tissue, and cecal contents. After the anticoagulated blood was allowed to stand at 25°C for 2 hours, it was centrifuged at 3500rpm to separate the serum for later use; the non-anticoagulated blood was directly used for blood cell analysis; the liver tissue was used for HE staining to observe the liver tissue structure; and the cecal contents were used for 16s intestinal flora analysis and non-targeted metabolome analysis.

[0087] 2.5 HE staining to observe the pathological structure of liver tissue

[0088] Collect liver tissue, fix it with 4% paraformaldehyde, then fix the tumor tissue with paraffin, cut it into small pieces of 5μm thickness, spread it evenly on the slide, and put the slices into a drying oven at 66℃ for 20-30min. Pass through 3 layers of xylene in sequence, 5min each. Pass through 3 layers of (100%-95%-80%) ethanol in sequence, 3min each. Put the slices in a beaker and rinse slowly with running water to wash away the ethanol until the slices are clean and transparent. Slice into hematoxylin for 2-5min, rinse with running water. Differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with running water. Blue with saturated lithium carbonate solution for a few seconds, rinse with running water. Dehydrate the slices in 95% ethanol for 2min, shake up and down several times. Slice into eosin stain (alcohol soluble) for a few seconds. Pass through 2 layers of 95% ethanol in sequence, 5-10s each, the time should not be too long. Dehydrate through 2 layers of 100% ethanol in sequence, 1min each. Transparent the slides with phenol-xylene, xylene I, and II, each for 2 min. Seal the slides with neutral gum and observe the results under a microscope.

[0089] 2.6ALT and AST testing

[0090] ALT (C009-1-1) and AST (C010-1-1) biochemical index detection kits were used to detect the serum ALT and AST levels of mice in each group. The specific steps are as follows: take a 96-well plate, add 5 μL of sample to the detection well, use a spray gun to add 25 μL of matrix solution, stick the plate seal, shake slightly to mix, and put it in a 37°C incubator for incubation for 30 minutes; then, use a spray gun to add 2,4-dinitrophenylhydrazine 25 μL, add 5 μL of sample to the control well (only the control well), re-stick the plate seal, shake slightly to mix, and put it in a 37°C incubator for incubation for 20 minutes; then, add 240 μL of 0.4M NaOH solution to each well. At a wavelength of 550nm, detect the OD value of each well, and calculate the ALT and AST levels of each sample according to the instructions.

[0091] 2.7 Analysis of blood routine indicators

[0092] The percentages of neutrophils, monocytes, lymphocytes, basophils and eosinophils in the blood of mice were detected using the DiMed five-differentiation blood routine cell analyzer.

[0093] 2.8 Serum IgG and IgM testing

[0094] The ELISA method was used to detect the IgG and IgM antibody content in the serum of each group of mice. The specific steps are as follows: add 100 μL of serum or antibody standard to the pre-coated 96-well ELISA plate, incubate at 25°C and 300 rpm for 2.0 h, and wash each well with 300 μL of washing solution 6 times. Then, add 100 μL of the corresponding detection antibody labeled with horseradish peroxidase, continue incubation at 25°C and 300 rpm for 1.0 h, wash 3 times, add the color developing solution, and color at 25°C in the dark for 15 minutes. Add 100 μL of stop solution to each well to complete the reaction. Use an ELISA instrument to measure the OD value at wavelengths of 450nm and 630nm to calculate the IgG and IgM antibody content in the serum of each group of mice.

[0095] 2.9 MDA and SOD detection

[0096] The MDA (C009-1-1) and SOD (C010-1-1) biochemical index detection kits were used to detect the serum MDA and SOD levels of each group of mice. The specific steps are as follows: Take a 96-well plate, add 5 μL of sample (only the detection well) according to the following plate arrangement (Table 1), use a spray gun to add 25 μL of matrix solution, stick the plate seal, shake slightly to mix, and put it in a 37°C incubator for incubation for 30 minutes; then, use a spray gun to add 2,4-dinitrophenylhydrazine 25 μL, add 5 μL of sample to the control well (only the control well), re-stick the plate seal, shake slightly to mix, and put it in a 37°C incubator for incubation for 20 minutes; then, add 240 μL of 0.4M NaOH solution to each well. At a wavelength of 550nm, detect the OD value of each well, and calculate the MDA and SOD levels of each sample according to the instructions.

[0097] 2.10 Analysis of intestinal flora

[0098] QIAam DNA Stool Mini Kit was used to extract DNA from mouse cecum contents, and the concentration and purity were analyzed by ultra-trace nucleic acid detector, and the DNA was stored at -80℃ for future use. Hangzhou Lianchuan Biological Company used Illumina NovaSeq platform to sequence the DNA of each group of mice, and amplified the specific sequence of the V3-V4 region of the 16S rRNA gene with primer sequences of 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3'). The sequencing data were spliced, quality controlled, and chimera filtered to obtain valid data. The above data were clustered and correlated with OTUs with a consistency of 97%. The Alpha diversity of the bacterial community was expressed by Ace, Chao1 and Shannon index, and the Beta diversity was expressed by principal coordinates analysis (PCoA), reflecting the differences between samples in different groups. The smaller the distance between the scattered points, the closer the colony composition structure between the samples represented by the scattered points. The three groups of bacteria with significant differences at each classification level were further screened out by linear discriminant analysis effect size (LEFSe). In order to count the intestinal flora categories with significant differences between the groups, linear discriminant analysis (LDA) was performed between the groups, the data was reduced in dimensionality, and the influence of species with significant differences (LDA score) was evaluated.

[0099] Alpha diversity mainly reflects the species diversity of a single sample. The Ace and Chao1 indices reflect the species richness of the microbiota in the sample. The larger the value, the higher the abundance of the microbiota. The Shannon index reflects the species diversity of the microbiota. The larger the value, the higher the diversity of the microbiota.

[0100] 2.11 Statistical analysis

[0101] The experimental data were analyzed using SPSS22.0 software. Quantitative data were expressed as mean ± standard deviation. The t-test was used for comparison between groups. P < 0.05 indicated significant differences, a indicated P < 0.05, b indicated P < 0.01, and c indicated P < 0.001.

[0102] 3. Results

[0103] 3.1 Effect of Zhihuang Fufang Granules on the Blood Cell Composition of Aflatoxin-Intoxicated Mice

[0104] The results are as follows Figure 2 As shown, A: percentage of different blood cells; B: scatter plot of blood cells of mice in each group; compared with the model group, a means P < 0.05, b means P < 0.01, and c means P < 0.001.

[0105] Depend on Figure 2 It can be seen that the percentage of neutrophils and monocytes in the model group increased significantly, indicating that aflatoxin may cause a certain inflammatory response in the body. In addition, after the intervention of Zhihuang Fufang Granules, the percentage of neutrophils and monocytes in the blood of mice in the ZHLG low-dose group and ZHLG medium-dose group were significantly lower than those in the model group, indicating that Zhihuang Fufang Granules can help alleviate the inflammatory response caused by aflatoxin. The percentage of blood lymphocytes and eosinophils in mice in the model group was significantly lower than that in the control group (P < 0.05), and the percentage of blood lymphocytes and eosinophils in mice in the ZHLG low-dose group was significantly higher than that in the model group (P < 0.05, P < 0.01). The above results indicate that aflatoxin can cause a decrease in the immunity of mice, and Zhihuang Fufang Granules can effectively enhance the immunity of mice poisoned by aflatoxin.

[0106] 3.2 Zhihuang Fufang Granules Effectively Improve Liver Function

[0107] The results are as follows Figure 3 As shown, compared with the model group, a represents P < 0.05, b represents P < 0.01, and c represents P < 0.001.

[0108] Depend on Figure 3 It can be seen that the serum AST and ALT in the model group were significantly higher than those in the control group (P < 0.01, P < 0.001), indicating that the aflatoxin liver injury model was successfully established. The serum AST and ALT of mice in the positive control group and the low-dose ZHLG group were significantly lower than those in the model group (P < 0.05, P < 0.01). The above results suggest that Zhihuang Fufang Granules can effectively reverse the liver damage caused by aflatoxin.

[0109] 3.3 Zhihuang Fufang Granules Effectively Improved Indicators Related to Oxidative Stress

[0110] The results showed that the serum MDA and SOD in the model group were significantly higher than those in the control group (P < 0.01, P < 0.001), indicating that aflatoxin can cause oxidative stress in mice. The serum MDA and SOD in the positive control group and the low-dose ZHLG group were significantly lower than those in the model group (P < 0.05, P < 0.01). The above results indicate that Zhihuang Fufang Granules can effectively reverse the oxidative stress caused by aflatoxin.

[0111] 3.4 Zhihuang Fufang Granules can improve liver tissue structure

[0112] The results are as follows Figure 4 As shown, compared with the model group, a represents P < 0.05, b represents P < 0.01, and c represents P < 0.001.

[0113] Depend on Figure 4 It can be seen that the liver lobule structure of the mice in the control group was intact, and radial hepatic cords and hepatic sinusoids were observed around the central vein, while the liver lobule structure of the mice in the model group almost disappeared, and the hepatic cords were disordered. The above results once again confirmed that aflatoxin can cause significant damage to liver tissue. The structural characteristics of the hepatic lobule can be clearly observed in the mice in the low-dose ZHLG group. Although the ZHLG medium-dose group and the ZHLG high-dose group were improved compared with the model group, the structural characteristics of the hepatic lobule were not obvious. The above results suggest that high-dose Zhihuang Fufang Granules may be detrimental to the efficacy of its drug.

[0114] 3.5 Effect of Zhihuang Fufang Granules on Serum IgG and IgM Antibodies

[0115] The results are as follows Figure 5 As shown, compared with the model group, a represents P < 0.05, b represents P < 0.01, and c represents P < 0.001.

[0116] Depend on Figure 5 It can be seen that the serum IgG and IgM of mice in the model group were significantly lower than those in the control group (P < 0.01, P < 0.001), indicating that aflatoxin can cause low immune function in mice. The serum IgG and IgM levels of mice in the positive control group and the low-dose ZHLG group were significantly higher than those in the model group (P < 0.05, P < 0.01). The above results indicate that Zhihuang Fufang Granules can effectively improve the low immune function caused by aflatoxin.

[0117] 3.6 Zhihuang Fufang Granules can effectively improve the intestinal flora structure of mice with aflatoxin poisoning

[0118] like Figure 6 As shown, A: Venn diagram; B: α diversity analysis; C: PCoA Analysis. Figure 6It can be seen that the number of unique OTUs in the control group, model group, ZHLG low-dose group, ZHLG medium-dose group, and ZHLG high-dose group were 91, 68, and 222, respectively. At the same time, the number of OTUs in the ZHLG low-dose group, ZHLG medium-dose group, and ZHLG high-dose group was more than that in the model group and the control group, indicating that Zhihuang Fufang granules would change the intestinal flora structure of mice poisoned by aflatoxin. In addition, the Shannon, Simpson, and Chao1 indexes all showed that the flora richness and species diversity of mice treated with Zhihuang Fufang granules were higher than those in the control group and the model group. The PCoA analysis results showed that the control group, model group, ZHLG low-dose group, ZHLG medium-dose group, and ZHLG high-dose group were significantly separated, and there was a certain aggregation phenomenon within each sample group. The first principal component (PCoA1) and the second principal component (PCoA2) explained 63.69% and 21.96% of the total variables, respectively. The above results show that aflatoxin poisoning can cause changes in the intestinal flora structure of mice.

[0119] 3.7 Effect of Zhihuang Fufang Granules on the Composition of Intestinal Microflora in Mice Intoxicated by Aflatoxin

[0120] The results are as follows Figure 7 As shown, A: Relative abundance stacked bar chart of sample; B: clustering heat map of groups; C: clustering heat map of sample; D: Bubble Plot of abundance in different groups.

[0121] Depend on Figure 7It can be seen that aflatoxin poisoning can lead to a decrease in the abundance of HT002, Bacteroides, Lachnospiraceae, Clostridiales, Colidextribacter, Eubacterium, Lachnospiraceae_NK4A136_group, Peptococcaceae_unclassified, Firmicutes_unclassified, Ruminococcaceae_unclassified, Anaerotignum, Streptococcus, Monoglobus, Clostridium, Eubacterium_xylanophilum Ligilactobacillus, Muribaculaceae, and Clostridia_UCG-014 genera in mice, while after the intervention of Zhihuang Fufang Granules, the abundance of the above-mentioned bacterial flora increased significantly. In addition, the abundance of Desulfovibrio, RF39_unclassified, Alistipes, Erysipelotrichaceae_unclassified, Ruminococcus, Alloprevotella, Mucispirillum, Erysipelatoclostridium, Enterorhabdus, Candidatus, Saccharimonas, Lactobacillus, and Akkermansia in the intestines of mice poisoned by aflatoxin increased significantly. After intervention with Zhihuang Fufang Granules, except for Mucispirillum and Erysipelatoclostridium, the abundance of the above-mentioned flora decreased. The above results indicate that Zhihuang Fufang Granules can effectively restore the intestinal flora disorder of mice caused by aflatoxin poisoning. In addition, the results of cluster analysis also show that the intestinal flora of mice in the Zhihuang Fufang Granules intervention group is more similar to that of the control group.

[0122] 3.8 Screening of core functional flora

[0123] The results are as follows Figure 8 As shown, A: Bubble Plot of indicator analysis; B: LEfSe analysis; C: Cluster heat map of correlation between flora and indicators; * indicates significant correlation, ** indicates extremely significant correlation.

[0124] Depend on Figure 8It can be seen that the LEfSe analysis results show that HT002, Eubacteriaceae, Eubacterium, uncultured_Eubacterium, Lachnospiraceae_unclassified, Olsenella, Atopobiaceae, Coriobacteriales , Olsenella_sp__F0206, Ileibacterium, Ileibacterium_unclassified, Clostridium_sensu_stricto_1_unclassified, Moraxellaceae, Acinetobacter, Clostri dium_sensu_stricto_1, Mesorhizobium_sp_, Dubosiella, Mesorhizobium, Dubosiella_unclassified, Acinetobacter_baylyi, and Phyllobacteriaceae were the dominant bacteria in the Zhihuang Fufang granule intervention group, and Methylobacterium_Methylorubrum_unclassified, Chloroplast_unclassified, Chloroplast, Cyanobacteriia, and Chloroplast_unclassified were the dominant bacteria in the mice poisoned by aflatoxin. The top 30 bacterial communities with the highest abundance were selected for correlation analysis with AST, ALT, IgG and IgM. The results showed that AST was significantly negatively correlated with Lachnospiraceae_NK4A136_group (P < 0.01, and significantly positively correlated with Lactobacillus (P < 0.01). ALT was significantly positively correlated with Desulfovibrio and Lactobacillus (P < 0.05). IgG and IgM levels were also significantly correlated with Lachnospiraceae_NK4A136_group, Desulfovibrio and Lactobacillus. The above analysis results showed that Lachnospiraceae_NK4A136_group, Desulfovibrio and Lactobacillus may be the key bacterial communities for the efficacy of Zhihuang Fufang Granules, which should be further studied in the next step of research.

[0125] 4 Discussion

[0126] Aflatoxin poisoning can cause a variety of adverse symptoms including immunosuppression with liver damage as a typical symptom. The present invention combines the traditional Chinese medicine compatibility theory and the results of modern pharmacological research, and uses Gardenia, Crataegus and Astragalus to make the above Chinese medicine into Zhihuang Compound Granules by solvent extraction to investigate its protective effect on aflatoxin poisoned mice. According to the results of blood routine, liver tissue sections, biochemical indicators and serum immunoglobulin detection, it is confirmed that Zhihuang Compound Granules can significantly increase the percentage of lymphocytes and eosinophils in the blood of AFB1 poisoned mice, reduce the serum AST, ALT, SOD and MDA levels of AFB1 poisoned mice, and improve the liver tissue structure, that is, Zhihuang Compound Granules can effectively enhance the immunity of aflatoxin poisoned mice, improve oxidative stress and liver damage.

[0127] In recent years, the establishment of the concept of "gut-liver axis" has given people a new understanding of the relationship between intestinal flora and liver disease. First of all, the liver and intestine are closely related. They have the same embryonic origin. 70% of the blood supply of the liver comes from the intestine. The intestinal mucosa is an important barrier in the human body. When the mucosa is normal, it can prevent most intestinal bacteria and their products from shifting [Chen Jiao, Zhang Yinglin, Liu Zuojin. Research progress on the mechanism of liver-gut axis [J]. Modern Medicine and Health, 2014, 30 (22): 3405-3408.]. These blood usually contain digestive tract metabolites and intestinal microbial products. Once the integrity of the intestine is destroyed, the intestinal flora is imbalanced, and the intestinal bacteria grow excessively, it can cause damage to the intestinal barrier function. The intestinal bacteria products enter the liver through the portal system and cause liver damage. Studies have shown that bacterial overgrowth, changes in bacterial species, and bacterial translocation in the intestine will change the permeability and immune status of the intestine, making it easier for bacteria and their products to enter the liver through the portal vein, affecting the normal physiological function of the liver and leading to the occurrence and development of a series of liver diseases [Fukui H. Gut Microbiota and Host Reaction in Liver Diseases [J]. Microorganisms (Basel), 2015, 3 (4): 759-791.]. Intestinal flora imbalance is an important common pathway for the occurrence and development of autoimmune liver disease, alcoholic liver disease, metabolic-related fatty liver disease, viral hepatitis, cirrhosis, and liver cancer.

[0128] In order to explore the protective mechanism of Zhihuang Fufang Granules on mice poisoned by aflatoxin, the present invention used 16SrDNA sequencing to analyze the intestinal flora structure of the cecal contents of mice in the control group, model group, and Zhihuang Fufang Granule intervention group. The results showed that Zhihuang Fufang Granules would change the intestinal flora structure of mice poisoned by aflatoxin, and the flora richness and species diversity of mice treated with Zhihuang Fufang Granules were higher than those in the control group and model group. The results of relative abundance analysis of bacterial flora showed that aflatoxin poisoning could lead to significant changes in the intestinal flora structure of mice, including HT002, Bacteroides, Lachnospiraceae, Clostridiales, Colidextribacter, Eubacterium, Lachnospiraceae_NK4A136_group, Peptococcaceae_unclassified, Firmicutes_unclassified, Ruminococcaceae_unclassified, Anaerotignum, Streptococcus, Monoglobus, Clostridium, Eubacterium]_xylanophilumLigilactobacillus, Muribacula The abundance of the genera ae and Clostridia_UCG-014 decreased, while the abundance of the genera Desulfovibrio, RF39_unclassified, Alistipes, Erysipelotrichaceae_unclassified, Ruminococcus, Alloprevotella, Mucispirillum, Erysipelatoclostridium, Enterorhabdus, Candidatus, Saccharimonas, Lactobacillus, and Akkermansia increased significantly. After intervention with Zhihuang Fufang Granules, the relative abundance of the above-mentioned flora was significantly adjusted back, that is, Zhihuang Fufang Granules can effectively restore the intestinal flora disorder of mice caused by aflatoxin poisoning. The cluster analysis results also showed that the intestinal flora of the mice in the Zhihuang Fufang Granules intervention group was more similar to that of the control group. We further found through LEFSe analysis and correlation analysis that Lachnospiraceae_NK4A136_group, Desulfovibrio, and Lactobacillus may be the key flora for Zhihuang Fufang Granules to exert its efficacy.

[0129] Lachnospira belongs to the phylum Firmicutes, family Lachnospiraceae. It is present in the intestines of most healthy people and may be a potential beneficial bacterium. It is involved in the metabolism of various carbohydrates, especially pectin (a complex dietary fiber and prebiotic) in fruits and vegetables. Fermentation leads to the production of acetic acid and butyric acid, which provide the main source of energy for the host.

[0130] Intestinal bacteria, including Desulfovibrio, are the main producers of hydrogen sulfide in the gastrointestinal tract, and the diffusion of hydrogen sulfide released by bacteria into the subepithelial area is controlled by enterocytes and colonocytes. This interaction is important for regulating mucosal function and the intestinal environment. 2 The role of H in intestinal inflammation is complex and sometimes contradictory. Some experimental and clinical data suggest that H 2 S is associated with chronic colon disease and inflammation of the large intestine. Similarly, the presence of some Desulfovibrio species has been associated with chronic periodontitis, cell death, and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, or at least an increased risk of relapse. However, there are also studies showing that H 2 S can directly promote angiogenesis, and its beneficial effects on gastrointestinal ulcer healing may be partly due to enhanced mucosal blood flow at the ulcer edge, which is crucial for ulcer repair.

[0131] Although the present invention confirms the efficacy of Zhihuang Fufang Granules, since Gardenia, Astragalus, and Hawthorn all have extremely complex effective ingredients, the present invention has not been able to carry out in-depth work on the scientific basis of the formula, the key ingredients of the formula, how these ingredients regulate changes in the intestinal flora, and the verification of the function of the intestinal flora. After that, it is planned to verify through proteomics, metabolomics, component analysis, combined with cell model construction, so as to further clarify the mechanism and target of Zhihuang Fufang Granules in the treatment of aflatoxin poisoning, and lay the foundation for its further in-depth development.

[0132] 5. Conclusion

[0133] Zhihuang Fufang Granules may be a promising liver protection drug by regulating the intestinal flora structure, protecting liver damage caused by aflatoxin, improving the body's immunity, and inhibiting oxidative stress.

[0134] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Zhihuang Fufang granule with liver protection effect, characterized in that: The invention comprises the following raw materials in parts by weight: 800-1200 parts of gardenia, 800-1200 parts of astragalus and 800-1200 parts of hawthorn; Also included are cyclodextrins and disintegrated starches.

2. The Zhihuang Fufang granules with liver protection effect according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 1000 parts of gardenia, 1000 parts of astragalus and 1000 parts of hawthorn; Also included are cyclodextrins and disintegrated starches.

3. A method for preparing Zhihuang Fufang granules with liver protection effect, characterized in that: The specific steps include: (1) Weigh the raw materials according to the weight proportions of the Zhihuang Fufang Granules according to claim 1 or 2, and divide the Astragalus membranaceus into two equal parts; (2) adding ethanol to gardenia, performing reflux extraction, and concentrating the extract to dryness under reduced pressure to obtain a gardenia extract; (3) adding water to the first portion of Astragalus, decocting, and freeze-drying to obtain an Astragalus water extract; (4) adding ethanol to the second portion of Astragalus, refluxing for extraction, and concentrating the extract to dryness under reduced pressure to obtain an Astragalus alcohol extract; (5) adding ethanol to hawthorn, performing reflux extraction, and concentrating the extract to dryness under reduced pressure to obtain a hawthorn extract; (6) The gardenia extract, the astragalus water extract, the astragalus alcohol extract, the hawthorn extract, the cyclodextrin and the disintegrated starch are uniformly mixed and granulated to obtain the gardenia and huang compound granules with liver protection effect.

4. The method for preparing the Zhihuang Fufang granules having a liver-protecting effect according to claim 3, characterized in that: In step (2), the mass percentage of ethanol is 75%, and the amount used is 8 times the weight of gardenia; the number of reflux extraction is 2 times, and the time is 90 minutes.

5. The method for preparing the Zhihuang Fufang granules with liver protection effect according to claim 3, characterized in that: In step (3), the amount of water used is 8 times the weight of the first portion of Astragalus; and the decoction time is 90 minutes.

6. The method for preparing the Zhihuang Fufang granules having liver protection effect according to claim 3, characterized in that: In step (4), the mass percentage of the ethanol is 75%, and the amount used is 8 times the weight of the second portion of Astragalus; the reflux extraction is performed twice for 90 minutes.

7. The method for preparing the Zhihuang Fufang granules with liver protection effect according to claim 3, characterized in that: In step (5), the mass percentage of ethanol is 75%, and the amount used is 10 times the weight of hawthorn; the number of reflux extraction is 2 times, and the time is 90 minutes.

8. The method for preparing the Zhihuang Fufang granules with liver protection effect according to claim 3, characterized in that: In step (6), the crude drug content of the Zhihuang Fufang granules is 1g / g.

9. Use of the Zhihuang Fufang Granules as claimed in claim 1 or 2 or the Zhihuang Fufang Granules prepared by the preparation method as claimed in any one of claims 3 to 8 in preparing a liver-protecting drug.

10. The use according to claim 9, characterized in that: The liver protection drug is a drug that alleviates liver damage, immunosuppression and oxidative stress caused by AFB1.

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