Traditional Chinese medicine composition of self-simulation Sijunzi powder addition and subtraction prescription and application of traditional Chinese medicine composition

Abstract

The invention discloses a self-simulation Sijunzi powder addition and reduction prescription traditional Chinese medicine composition and application thereof, the traditional Chinese medicine composition is composed of Codonopsis pilosula, fried bighead atractylodes rhizome, Poria cocos, hawthorn and honey-fried licorice root according to a weight ratio of 2-3: 2-3: 2-3: 2-3: 1-1.5. Compared with the Sijunzi powder, the self-simulated Sijunzi powder addition and reduction prescription has the advantages that the effects of reducing the liver cell injury of the dairy cow and improving the immunity of the dairy cow are proved through a mode of combining an in-vitro experiment and an in-vivo experiment for the first time; the traditional Chinese medicine composition has good health-care and treatment values in enhancing rumen fermentation capability, regulating rumen flora structure, improving blood immune globulin level, slowing down lipid mobilization and regulating blood metabolism capability. Meanwhile, the formula and the preparation method are simple, can be easily converted into a modern industrial production process, and have great development potential, so that good economic and social benefits can be obtained.

Description

Technical Field

[0001] The invention relates to the field of veterinary technology, and in particular to a self-made Sijunzi powder modified prescription Chinese medicine composition and application thereof. Background Art

[0002] As a precious resource unique to my country, Chinese medicine is taken from natural plants, animals or minerals. Under the guidance of traditional Chinese medicine theory, it has undergone thousands of years of clinical evidence and has left a deep imprint in the development of Chinese civilization. Feeding natural plants not only has the advantages of being natural and residue-free, but also has the dual functions of medicine and nutrition. The classic Chinese medicine prescription Sijunzi Decoction is composed of ginseng, Atractylodes macrocephala, Poria cocos, and licorice. It has the effects of replenishing qi, invigorating qi and strengthening the spleen. It is mainly used to treat spleen and stomach qi deficiency, less food, and thin body. Spleen and stomach qi deficiency is caused by lack of transportation and transformation; the spleen and stomach are the foundation of the acquired constitution and the source of qi and blood biochemical transformation. If the spleen and stomach qi are deficient, the reception and transportation are weak, and the diet is reduced; spleen and stomach qi deficiency, dampness and turbidity are generated internally, spleen and stomach transportation is unfavorable, qi and blood biochemical transformation is insufficient, and the body is thin, which manifests as a sub-healthy state of low immunity. In the recipe of Sijunzi Decoction, ginseng is the main ingredient, which is sweet and warm, replenishes qi, strengthens the spleen and nourishes the stomach; white atractylodes is bitter and warm, which strengthens the spleen and eliminates dampness, and strengthens the power of replenishing qi and promoting circulation; sweet and mild poria is used as an auxiliary, which strengthens the spleen and eliminates dampness. The combination of poria and atractylodes can strengthen the spleen and eliminate dampness. Roasted licorice is used to replenish qi and harmonize the stomach, and harmonize the other medicines. The combination of the four medicines can achieve the effect of replenishing qi and strengthening the spleen.

[0003] However, the Sijunzi powder composed of Codonopsis pilosula, Atractylodes macrocephala, Poria cocos and Licorice still has room for further improvement in regulating the immunity of postpartum dairy cows. To this end, China's authorized patent CN114209769B discloses a natural plant composition and application that enhances animal immunity, which is added to the traditional Chinese medicine prescription Sijunzi Decoction by adding Astragalus, Pollen Typhae, Allium sativum, Cherry Laevigata, and Fagopyrum truncatum; the natural plant feed composition has the effects of invigorating qi and strengthening spleen, invigorating qi and raising yang, strengthening the body and eliminating evil, detoxifying and astringing, and can accelerate the metabolism of various toxins and harmful substances in the body, and quickly improve the body's immunity and health level. For another example, Chinese patent application CN103041231A is a Chinese medicine composition with the function of invigorating qi and strengthening spleen, wherein the components and weight ratios of the Chinese medicine composition are: 1-8 parts of Codonopsis pilosula; 1-8 parts of Atractylodes macrocephala (fried); 1-8 parts of Poria cocos; 1-5 parts of Licorice; 1-5 parts of Hawthorn; 1-5 parts of Malt; 1-5 parts of Cinnamon. However, traditional Sijunzi powder is composed of Codonopsis pilosula, Atractylodes macrocephala, Poria cocos and Radix Glycyrrhizae Preparata. The existing Sijunzi powder modified prescription uses a wide variety of Chinese medicines, and generally adds 2 to 5 Chinese medicines on the basis of the original prescription. Some of the Chinese medicines are expensive, which greatly increases the pharmaceutical cost and is not conducive to the promotion and application of the prescription in animal husbandry. In particular, the Sijunzi powder modified prescription disclosed in the above prior art has complex ingredients, and its efficacy still needs further improvement. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an application of a self-prepared modified Sijunzi powder prescription in reducing liver cell damage in dairy cows; regulating the structure of rumen flora; enhancing rumen fermentation capacity; improving immunoglobulin levels and slowing down fat mobilization.

[0005] The first aspect of the present invention provides a self-made Sijunzi powder modified prescription, a preparation method and use thereof.

[0006] The self-made Sijunzi powder plus or minus prescription in the present invention is improved according to the Sijunzi powder in the "Chinese Veterinary Pharmacopoeia". In the present invention, the self-made Sijunzi powder plus or minus prescription is composed of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root, and the weight ratio is 2-3:2-3:2-3:2-3:1-1.5. In a preferred embodiment, the preparation method of the self-made Sijunzi powder plus or minus prescription includes:

[0007] S1. Wash the raw medicinal materials Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root with clean water, and then dry them.

[0008] S2. Prepare fried Poria cocos: Place the Poria cocos slices gently in the pot, then heat slowly over low heat, and continue to stir-fry until the surface turns slightly yellow. During this process, stir-fry evenly to prevent burning.

[0009] S3. Prepare roasted licorice: Carefully add an appropriate amount of refined honey to the licorice slices, then slowly stir-fry over low heat until they turn yellow to dark yellow. When the licorice slices are no longer sticky, take them out and set them aside to cool.

[0010] S4. Grind the entities of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root into powder respectively by machine.

[0011] S5. The ground medicinal material powder is passed through a 100-mesh sieve to remove impurities, ensure the purity and quality of the medicinal material, make the medicinal composition more uniform, and thus improve the efficacy.

[0012] S6. Accurately mix the sieved medicinal powders of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and Glycyrrhiza uralensis in a preferred ratio of 2:2:2:2:1, and mix them thoroughly to achieve the best efficacy.

[0013] The inventors have proved through experiments that the self-prepared Sijunzi Powder modified prescription prepared by the above-mentioned ratio and method can effectively reduce in vitro dairy cow liver cell damage; enhance in vitro rumen fermentation capacity; regulate the composition and quantity of in vitro rumen flora, increase blood immunoglobulin levels and slow down lipid metabolism; thereby playing a role in alleviating the negative energy balance of dairy cows after delivery.

[0014] The second aspect of the present invention provides a novel self-made Sijunzi powder modified prescription for use in reducing in vitro dairy cow liver damage and related products, and compares it with the traditional Sijunzi powder prescription.

[0015] The products include but are not limited to health products and medicines.

[0016] Beneficial technical effects of one or more of the above technical solutions:

[0017] In the technical scheme of the present invention, Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root are used as raw materials and mixed according to the preferred ratio of 2:2:2:2:1. Through in vitro cell experiments, it was confirmed for the first time that the self-prepared prescription was superior to the traditional Sijunzi powder in reducing dairy cow hepatocytes, demonstrating its outstanding value in the fields of health care and treatment.

[0018] The third aspect of the present invention provides a novel self-made Sijunzi powder modified prescription for use in enhancing in vitro rumen fermentation capacity and related products.

[0019] The products include but are not limited to health products and medicines.

[0020] Beneficial technical effects of one or more of the above technical solutions:

[0021] In the technical scheme of the present invention, Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root are used as raw materials and mixed according to the preferred ratio of 2:2:2:2:1. Through in vitro fermentation experiments, it is confirmed for the first time that the self-prepared prescription has a significant effect in enhancing the fermentation capacity of the rumen, demonstrating its outstanding value in the fields of health care and treatment.

[0022] The fourth aspect of the present invention provides a novel self-made Sijunzi powder modified prescription for use in regulating the composition and quantity of in vitro rumen flora and related products.

[0023] The products include but are not limited to health products and medicines.

[0024] Beneficial technical effects of one or more of the above technical solutions:

[0025] In the technical scheme of the present invention, codonopsis, stir-fried atractylodes, poria, hawthorn and roasted liquorice are used as raw materials and mixed according to the preferred ratio of 2:2:2:2:1. Through in vitro experiments, it is confirmed for the first time that the prescription has a significant effect in regulating the structure and quantity of rumen flora, showing its excellent value in the field of health care and treatment.

[0026] The fifth aspect of the present invention provides a novel self-prepared Sijunzi powder modified prescription for improving the level of immunoglobulin in postpartum dairy cows, slowing down lipid mobilization and regulating blood metabolism, and its application in related products.

[0027] The products include but are not limited to health products and medicines.

[0028] Beneficial technical effects of one or more of the above technical solutions:

[0029] In the above technical scheme, codonopsis, stir-fried atractylodes, poria, hawthorn and roasted liquorice are used as raw materials, and are mixed according to a ratio of 2:2:2:2:1. At the same time, animal experiments have proven for the first time that the self-prepared Sijunzi powder plus or minus prescription has good health and therapeutic value in improving the level of blood immunoglobulin in dairy cows, slowing down lipid mobilization and regulating blood metabolism. At the same time, the preparation method of the present invention is simple, easy to be converted into a modern industrial production process, and has great development potential, so it can achieve good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Base Peak diagram of the QC sample of the self-prepared Sijunzisan modified prescription in positive and negative ion modes. (A) Positive ion mode; (B) Negative ion mode.

[0031] Figure 2 Effects of self-prepared Sijunzi powder on liver function indexes of dairy cow hepatocytes (n=3). (A) AST; (B) ALT; (C) TG. Different letters on the shoulders indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05), as shown in the figure below.

[0032] Figure 3 Effect of self-made Sijunzi powder on the content of apolipoprotein in dairy cow hepatocytes (n=3). A) MTTP; (B) BovineApo-B100.

[0033] Figure 4 Effects of different doses of self-prepared Sijunzi powder on rumen gas production in dairy cows (n=5).

[0034] Figure 5 Effects of self-made Sijunzi powder on rumen fermentation parameters of dairy cows (n=5). (A) pH; (B) NH3-N; (C) MCP.

[0035] Figure 6 Effects of self-made Sijunzi powder on short-chain fatty acids in the rumen of dairy cows (n=5). (A) SCFAs; (B) Aceticacid; (C) Propionic acid; (D) N-butyric acid; (E) Isobutyric acid; (F) Valeric acid; (G) N-valeric acid.

[0036] Figure 7Effects of self-prepared Sijunzi powder on the diversity of rumen flora in dairy cows. (A) Venn diagram of OUT of two groups of rumen microorganisms; (B) Specaccum species accumulation curve; (C) Abundance level curve; (D) Alpha diversity analysis, Chao1 index is used to evaluate the total number of OUTs in the community; Simpson index is used to evaluate the evenness of the community; Shannon index is used to comprehensively evaluate the richness and evenness of the community; (E) Principal coordinate analysis (PCoA) of two groups of rumen microorganisms; (F) Non-metric multidimensional scaling (NMDS) analysis of two groups of rumen microorganisms.

[0037] Figure 8 Effects of self-made Sijunzi powder on the composition of rumen flora of dairy cows and analysis of differential flora. (A) Species composition abundance bar chart based on TOP20 at the phylum level; (B) Species composition abundance bar chart based on TOP20 at the genus level; (C) STAMP species difference analysis chart based on the phylum level; (D) STAMP species difference analysis chart based on the genus level.

[0038] Fig. 9 Spearman correlation heat map. (A) Correlation heat map between rumen microorganisms and SCFAs; (B) Correlation heat map between active ingredients of self-made Sijunzi powder and rumen microorganisms.

[0039] Fig.10 Effects of self-made Sijunzi powder on immune indexes of postpartum dairy cows (n=8). (A) IgA; (B) IgG; (C) IgM.

[0040] Fig.11 Effects of self-made Sijunzi powder on blood fat metabolism indices of postpartum dairy cows (n=8). (A) KET; (B) NEFA; (C) TG; (D) MTTP.

[0041] Fig.12 Effects of self-prepared Sijunzi powder on the blood metabolome of postpartum dairy cows. (A) Principal component analysis (PCA); (B) OPLS-DA score plot; (C) OPLS-DA permutation test plot; (D) volcano plot; (E) differential metabolite clustering heat map.

[0042] Fig.13 KEGG pathway enrichment analysis of differential metabolites. KEGG enrichment analysis bubble chart. DETAILED DESCRIPTION

[0043] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, technical solutions, features and effects of the present invention application are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.

[0044] Example 1: Preparation of self-prepared Sijunzisan modified prescription and detection of main active ingredients

[0045] The preparation method of the self-made Sijunzi powder prescription is as follows: the entities of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Hawthorn fruit and roasted Licorice root are ground into powder, and then mixed according to the ratio of Codonopsis pilosula: stir-fried Atractylodes macrocephala: Poria cocos: Hawthorn fruit: roasted Licorice root (2:2:2:2:1).

[0046] Detection of the main active ingredients of the self-prepared Sijunzi Powder plus or minus prescription: weigh 100 mg of the self-prepared Sijunzi Powder plus or minus prescription, add 1 mL of ultrapure water to dissolve, shake for 1 min, add 3 mL of ethanol (analytical grade) and let stand for 24 h, centrifuge at 3000 rpm to obtain the supernatant, use nitrogen to blow dry the ethanol, and obtain a refined solution. LC-MS / MS (Liquid chromatography-massspectrometry) was used to identify its main active ingredients. The refined solution was filtered through a 0.22 μm filter membrane, 10 μL was taken for injection, and repeated 3 times. The chromatographic acquisition used an ultra-high pressure liquid chromatograph (Nexera X2 LC-30, Shimadzu), and the chromatographic column selected was ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) (Waters, Milford, MA, USA). The column temperature was set at 40°C and the flow rate was set at 0.3 mL / min. The chromatographic mobile phase A was 0.1% formic acid (111670, Millipore, USA) in water, and B was 0.1% formic acid in acetonitrile. The chromatographic gradient elution program was as follows: 0-5 min, 0% B; 5-20 min, B changed linearly from 0% to 100%; 20-25 min, B maintained at 100%; 25-25.1 min, B changed linearly from 100% to 0%; 25.1-30 min, B maintained at 0%. After UPLC separation, the sample was analyzed by mass spectrometry using a QE Plus mass spectrometer (Q Exactive Plus, Thermo Scientific), using a HESI source for ionization, and the mass spectrometry acquisition time was 30 min. The collected mass spectrometry raw data were used to perform peak alignment, retention time correction and peak area extraction using MSDIAL software, and finally the main active ingredients of the self-prepared Sijunzi Powder were obtained.

[0047] The results of the main active ingredients of the self-prepared Sijunzi powder showed that 13 main active ingredients were detected by LC-MS / MS technology, including Luteolin, Glycitein, Pachymic acid, Poricoic acid A, Poricoic acid B, Dehydroeburicoic acid, Atractylenolide III, Formononetin, Calycosin, Licochalcone B, Liquoric acid, Quercetin and Vestitol ( Figure 1 A and B).

[0048] A self-prepared modified Sijunzi powder prescription was prepared and used in Examples 2-6.

[0049] Example 2: Self-prepared Sijunzi powder plus minus prescription reduces liver cell damage in dairy cows

[0050] The experiment used sodium oleate at a concentration of 0.25mmol / L to establish a fat accumulation model of dairy cow hepatocytes. 5 / well were inoculated into six-well plates and placed in a carbon dioxide incubator (37°C, CO25%) to culture until the cell confluence was about 70%. The complete culture medium (FBS10% and penicillin & streptomycin 1%) was replaced with DMEM basal culture medium, and the cells were starved for 12 hours. The complete culture medium was set as the CON group; the DMEM basal culture medium containing 0.25mmol / L sodium oleate was set as the MOD group; the traditional Sijunzi powder (Codonopsis pilosula: Atractylodes macrocephala: Poria cocos: Licorice root = 2:2:2:1) containing 0.25mmol / L sodium oleate was set as the OSJZS group; the traditional Sijunzi powder containing 0.25mmol / L sodium oleate and 40g / L was set as the SJZS group. Three replicates were set for each group. After replacing the cell culture medium, the culture was continued in the incubator for 9 hours. After the culture was completed, the cells were collected for the determination of subsequent indicators.

[0051] (1) Determination of liver function indicators

[0052] Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and triglyceride (TG) microplate detection kits (C010-2-1, C009-2-1 and A110-1-1, Nanjing Jiancheng Bioengineering Institute, China) were used to measure the contents of AST, ALT and TG in hepatocytes.

[0053] (2) Determination of apolipoprotein content

[0054] Microsomal triglyceride transfer protein (MTTP) and apolipoprotein B100 (BovineApo-B100) ELISA kits (NWLT and JYM0040Bo, Shanghai ELISA Biotechnology Co., Ltd. and Wuhan Gene Beauty Biotechnology Co., Ltd., China) were used to measure the levels of MTTP and BovineApo-B100 in hepatocytes.

[0055] The results of liver function indexes showed that compared with the model group, both traditional Sijunzi powder and self-prepared Sijunzi powder with modified formula significantly reduced the contents of AST, ALT and TG in hepatocytes (P < 0.05), among which the levels of AST, ALT and TG in hepatocytes of the self-prepared Sijunzi powder group were closer to those of normal hepatocytes. Figure 2 A to C. This indicates that both traditional Sijunzi powder and self-prepared Sijunzi powder plus or minus prescription can reduce liver injury induced by 0.25mmol / L sodium oleate, but the self-prepared Sijunzi powder plus or minus prescription has a better effect.

[0056] The results of hepatocyte apolipoprotein content showed that compared with the model group, both traditional Sijunzi powder and self-prepared Sijunzi powder with modifications significantly increased the content of apolipoprotein MTTP and BovineApo-B100 (P < 0.05), among which the MTTP and BovineApo-B100 levels in the hepatocytes of the self-prepared Sijunzi powder group were closer to those of normal hepatocytes. Figure 3 A and B. This shows that both traditional Sijunzi Powder and self-prepared Sijunzi Powder plus or minus prescription can increase the content of apolipoprotein in liver cells, enhance the ability of liver cells to transport lipids, and reduce the accumulation of lipids in liver cells, but the self-prepared Sijunzi Powder plus or minus prescription is more effective.

[0057] Example 3: Self-prepared Sijunzi powder modified prescription enhances in vitro rumen fermentation capacity

[0058] The rumen fluid of postpartum dairy cows was collected through the rumen fistula, filtered through 4 layers of gauze, and quickly placed in a preheated insulation bucket, and CO2 was continuously introduced to make it completely anaerobic. The buffer solution was prepared according to the method of Menke et al., placed in a preheated culture bottle (1000 mL) with CO2, and the rumen fluid and buffer solution were evenly mixed in a volume ratio of 1:2 to make a mixed fermentation liquid, which was then transferred to a 39°C constant temperature water bath for storage.

[0059] The experiment was divided into a control group (CONT) and a group of different doses of self-made Sijunzi powder (SJZS). The doses of the self-made Sijunzi powder group were 100, 200, 300, 400 and 500 mg, respectively, with 5 replicates in each group. Different doses of self-made Sijunzi powder were added to the syringe, and then 30 mL of the mixed fermentation liquid was added to the syringe. The air inside was expelled, and the syringe was sealed and placed in a 39°C constant temperature shaker for fermentation for 48 hours.

[0060] (1) Determination of rumen gas production

[0061] After the fermentation is completed, observe the changes in the gas volume in the syringe and record the gas production of rumen fermentation at 0, 3, 6, 9, 12, 24 and 48 h according to the scale of the syringe.

[0062] (2) Determination of rumen fermentation parameters

[0063] After 48 h of fermentation, pH, NH3-N and microbial protein (MCP) were measured. The NH3-N content was determined according to the method of Broderick & Kang (1980); the MCP content was determined by the Coomassie Brilliant Blue method.

[0064] (3) Determination of rumen short-chain fatty acid content

[0065] After 48 h of fermentation, the SCFAs content in the rumen fluid was determined by gas chromatograph (HP-INNOwax, Shimadzu Corporation, Japan). First, the rumen fluid of each group was centrifuged at 5400 rpm for 10 min to remove impurities, 1 mL of the supernatant was taken in a 1.5 mL centrifuge tube, and 0.2 mL of 25% metaphosphoric acid solution containing the internal standard 2EB (80079718, Shanghai McLean Biochemical Technology Co., Ltd., China) was added and mixed, placed in an ice water bath for 30 min, and centrifuged again at 10000 rpm for 10 min, and the supernatant was taken for determination. Gas chromatograph parameters: SK-FFAP column, column flow rate 2.0 mL / min, 1 μL injection volume; temperature program 120℃ for 3 min, then 10℃ every 1 min, 180℃ for 1 min; injection port temperature 220℃; detection instrument parameters: hydrogen flow 36 mL / min; air flow 450 mL / min; instrument temperature 250℃; column flow ± tail gas flow 40 mL / min. Standard acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid (A628, P1386, 129542, B103500, 11754 and V9769, Shanghai McLean Biochemical Technology Co., Ltd., China) were used for detection. The SCFAs standard curve was drawn with the peak area of ​​the standard as the ordinate and the concentration of the standard as the abscissa, and the linear regression equation of SCFAs was obtained (Table 1). Finally, the SCFAs content of the sample was calculated based on the standard curve.

[0066] Table 1 Linear regression equation information of SCFAs

[0067] Metabolites Retention time Linear regression equation Correlation coefficient Component RetentionTime(min) Equation R Aceticacid 2.598 y=13621.2*x+7627.47 0.988 Propionic acid 3.168 y=21560.2*x+5898.78 0.993 Isobutyricacid 3.350 y=34442.7*x+4097.11 0.988 N-butyricacid 3.842 y=30191.6*x+9869.68 0.994 Isovaleric acid 4.155 y=39200.4*x+2177.58 0.995 N-valeric acid 4.733 y=37176.9*x+6648.87 0.993

[0068] The results of rumen gas production showed that within 48 hours, the rumen gas production of the control group and the groups with different doses of self-prepared Sijunzi powder gradually increased with the extension of fermentation time. Among them, the rumen gas production increased sharply during the period of 0 to 12 hours, while the rumen gas production gradually became stable during the period of 12 to 48 hours. At the same fermentation time point, the gas production of the groups with different doses of self-prepared Sijunzi powder was significantly higher than that of the control group. Within the dosage range of 200 to 500 mg, the gas production of self-prepared Sijunzi powder at 3, 6, 9, 12, 24 and 48 hours was significantly higher than that of the control group (P < 0.05), and showed an obvious dose-dependent relationship. Figure 4 .

[0069] The results of rumen fermentation parameters showed that the rumen pH and NH3-N of the different doses of self-made Sijunzi powder groups were significantly lower than those of the control group (P < 0.05), and the MCP content was significantly higher than that of the control group (P < 0.05). Compared with the different doses of self-made Sijunzi powder, the pH value and NH3-N decreased significantly with the increase of dose (P < 0.05), and the MCP content increased significantly (P < 0.05). The pH, NH3-N and MCP values ​​showed a good dose-dependent relationship. (See Figure 5 A~C). This indicates that the self-prepared Sijunzi powder can significantly promote the rumen fermentation capacity of dairy cows, and the best effect is achieved with 500 mg.

[0070] The results of rumen short-chain fatty acids showed that the rumen SCFAs, acetic acid, propionic acid, and N-butyric acid in the different doses of self-prepared Sijunzi powder groups were significantly higher than those in the control group (P < 0.05), and a good dose-dependent relationship was shown between the dose groups. The rumen Isobutyric acid, N-valeric acid, and Isovaleric acid contents in different doses of self-prepared Sijunzi powder were significantly higher than those in the control group (P < 0.05), and there was no significant difference between the doses of 100 mg to 300 mg (P > 0.05), and a good dose-dependent relationship was shown only in the dose range of 300 mg to 500 mg. The above results indicate that self-prepared Sijunzi powder has a good promoting effect on the production of rumen SCFAs, with 500 mg having the best effect (see Figure 6 A~G).

[0071] Example 4: Self-prepared Sijunzi powder modified prescription regulates the composition and quantity of rumen flora in vitro

[0072] The optimal dose of 500 mg of self-made Sijunzi powder was selected. After fermentation for 48 hours, the composition and quantity of the flora in the rumen fluid of each group were determined by 16S rRNA high-throughput sequencing technology. First, the rumen fluid DNA was extracted by magnetic beads, and then the extracted DNA was quantitatively analyzed by Nanodrop, and the quality of the extracted DNA was detected by 1.2% agarose gel electrophoresis. Then the DNA was diluted to 20 ng / uL, and the primers of standard bacteria (upstream: ACTCCTACGGGAGGCAGCA, downstream: GGACTACHVGGGTWTCTAAT) and amplification polymerase were added to amplify the V3-V4 region of bacterial 16S. The PCR amplification recovery product was fluorescently quantified. According to the fluorescence quantitative results and the sequencing amount requirements of each sample, the samples were mixed in the corresponding proportions. The sequencing library was prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit for high-throughput sequencing.

[0073] The QIIME2 (2019.4) software was used to first annotate the rumen microbial 16S rRNA gene sequence for species taxonomy, and then perform Alpha diversity analysis and Beta diversity analysis. Finally, the online website (https: / / www.cloudtutu.com / # / login) was used to perform intergroup difference analysis based on the relative abundance of the flora in the samples. Finally, the Kidio Cloud Platform (https: / / www.omicshare.com / ) was used to draw the Spearman correlation heat map of the main active ingredients, rumen microorganisms, and SCFAs of the optimal dose of self-prepared Sijunzi powder.

[0074] The results of rumen flora diversity showed that a total of 14,286 OUTs were obtained from the rumen fluid of the control group and the self-made Sijunzi powder group, of which 9,057 OUTs were found in the self-made Sijunzi powder group and 7,490 OUTs were found in the control group. The two groups had a total of 2,261 OUTs, accounting for 15.83% of the total number of OUTs ( Figure 7 A). As the number of samples increases, the species accumulation curve gradually becomes stable ( Figure 7 B). The abundance level curve decreases slowly, indicating that the sample has high diversity ( Figure 7 C). The results of alpha diversity analysis showed that the Simpson index of the self-prepared Sijunzi powder group was significantly higher than that of the control group (P<0.05) ( Figure 7 D), indicating that the self-made Sijunzi powder can significantly increase the richness and diversity of rumen flora. The results of Beta diversity analysis showed that the two groups of rumen flora were clearly separated in the PCoA and NMDS diagrams ( Figure 7 E and 7F), indicating that the self-made Sijunzi powder significantly changed the composition and quantity of rumen flora in dairy cows.

[0075] The results of rumen flora differences showed that at the phylum level, the dominant phyla in both groups were Bacteroidota and Firmicutes, accounting for 56.0% and 26.9% in the control group, and 59.4% and 25.6% in the self-prepared Sijunzi powder group, respectively. Figure 8 A). Compared with the control group, the relative abundance of Bacteroidetes in the self-made Sijunzi powder group increased, while the relative abundance of Firmicutes decreased. At the genus level, the rumen flora of the two groups was mainly composed of F082, Rikenellaceae_RC9_gut_group and Prevotella, accounting for 22.9%, 16.9% and 3.18% in the control group, and 12.7%, 16.6% and 18.6% in the self-made Sijunzi powder group, respectively. Figure 8 B). The results of STAMP species difference analysis showed that the relative abundance of Synergistota, Elusimicrobiota and Desulfobacterota in the self-made Sijunzisan group was significantly higher than that in the control group (P<0.05), while the relative abundance of Proteobacteria and Patescibacteria was significantly lower than that in the control group (P<0.05) ( Figure 8 C). The relative abundance of Succiniclasticum and Prevotella genera in the self-prepared Sijunzi powder group was significantly higher than that in the control group (P<0.05), while the relative abundance of Christensenellaceae_R-7_group, Muribaculaceae, UCG-005, Comamonas and F082 genera was significantly lower than that in the control group (P<0.05) ( Figure 8 D).

[0076] The results of the correlation analysis between differential microorganisms and rumen SCFAs and the active ingredients of the self-prepared Sijunzi powder showed that the Spearman correlation heat map showed that the rumen flora Succiniclasticum and Prevotella genera were significantly positively correlated with rumen SCFAs (P < 0.05), while UCG-005, Comamonas and F082 genera were significantly negatively correlated with rumen SCFAs (P < 0.05), and Muribaculaceae and Christensenellaceae_R-7_group genera were not significantly correlated with rumen SCFAs (P > 0.05) ( Fig. 9 A).

[0077] The active ingredients Luteolin and Glycitein of the self-prepared Sijunzi powder were significantly positively correlated with the genus Prevotella of the rumen flora (P < 0.05), the active ingredients Poricoic acid B and AtractylenolideⅢ were significantly positively correlated with the genus Christensenellaceae_R-7_group of the rumen flora (P < 0.05), and the active ingredient Licochalcone B was significantly positively correlated with the genus UCG-005 of the rumen flora (P < 0.05). The active ingredients Liquoric acid and Licochalcone B were significantly negatively correlated with the genus Comamonas (P < 0.05) ( Fig. 9 B).

[0078] Example 5: Self-prepared Sijunzi powder modified prescription improves postpartum immunoglobulin level in dairy cows, slows lipid mobilization and regulates blood metabolism

[0079] Sixteen healthy Holstein cows weighing 600±30kg, with a body condition score of 3.0-3.5 and parity 2-3 were randomly divided into a control group (CONT) and a self-made Sijunzi powder group (SJZS) in a large intensive dairy farm in Ningxia, China. The cows in the two groups were fed the same TMR mixed diet and had free access to water. Before morning feeding every day, 300g of self-made Sijunzi powder was mixed with 5L drinking water and then gavaged in the self-made Sijunzi powder group, and the cows in the control group were gavaged with the same volume of drinking water for 7 consecutive days. Before morning feeding on the 8th day, 6mL of blood was collected from the tail vein with a 10mL anticoagulant blood collection tube containing EDTA, centrifuged at 3000r / min for 20min at 4℃, and the supernatant was aspirated into a 1.5mL cryotube and stored at -80℃ for subsequent detection of biochemical indicators and blood metabolome.

[0080] (1) Determination of immune indicators

[0081] The levels of IgA, IgG, and IgM in the blood were determined using bovine immunoglobulin A (IgA), bovine immunoglobulin G (IgG), and bovine immunoglobulin M (IgM) ELISA kits (YJ542063, YJ330698, and YJ627279, Shanghai ELISA Biotechnology Co., Ltd., China).

[0082] (2) Determination of lipid metabolism indicators

[0083] The blood ketone level was measured by blood ketone meter (AHM001P, Kelibo, Wuhan, China); the NEFA and TG levels in blood were measured by nonesterified fatty acid (NEFA) assay kit and triglyceride (TG) test kit (A042-2-1 and A110-1-1, Nanjing Jiancheng Bioengineering Institute, China). The MTTP level in blood was measured by bovine microsomal triglyceride transfer protein (MTTP) kit (NWLT, Shanghai ELISA Biotechnology Co., Ltd., China).

[0084] (3) Blood metabolomics analysis

[0085] UHPLC-MS / MS (Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry) was used to perform non-targeted metabolomics analysis of plasma samples. After 100 μL of plasma sample was added to a 1.5 mL PE tube, 400 μL of pre-cooled high-concentration methanol (1.06007.4008, millipore, USA) and acetonitrile (1.00030.4008, millipore, USA) solution (v / v, 1:1) was added and mixed, then treated in an ice-water bath for 1 h, centrifuged at 14000g for 20 min at 4°C, and the supernatant was collected for detection. Chromatographic acquisition: Waters ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 μm), flow rate 0.3 mL / min, mobile phase including A solution: 0.1% formic acid (111670, Millipore, USA) and B solution: 100% acetonitrile. The elution gradient was 0% buffer B within 2 min, the buffer B content increased linearly to 48% within 4 min, and then the content continued to increase to 100% and maintained for 2 min, then the buffer B content decreased to 0% within 0.1 min gradient, and then equilibrated for 3 min. Mass spectrometry acquisition: Electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry data acquisition, respectively. HESI source conditions were set as follows: spray voltage: 3.8 kV (positive) and 3.2 kV (negative); capillary temperature: 320 ° C; sheath gas (nitrogen) flow rate: 30 arb; auxiliary gas flow rate: 30 arb; auxiliary gas flow rate: 5 arb; probe heater temperature: 350 ° C; S-Lens RF level: 50.

[0086] After UHPLC-MS / MS data preprocessing, principal component analysis (PCA) of blood metabolites was performed using R (version 4.0.3) and R software package to observe the variation between and within sample groups. An OPLS-DA (Orthogonal partial least squares discriminant analysis, OPLS-DA) model was constructed to obtain the VIP value (Variable importance for the projection) of blood metabolites. The FC value (Foldchange) and P value of metabolites obtained by univariate analysis were combined to screen differential metabolites, and pathway enrichment analysis of differential metabolites was performed using MetaboAnalyst.

[0087] The results of immune indexes showed that compared with the control group, the blood IgA, IgG and IgM levels of postpartum dairy cows in the self-prepared Sijunzi powder group were increased, among which IgA and IgM increased extremely significantly (P < 0.01). Fig.10 A to C. This indicates that the self-prepared Sijunzi powder increases the level of blood immunoglobulin in postpartum dairy cows.

[0088] The results of lipid metabolism indicators showed that compared with the control group, the KET content of postpartum dairy cows in the self-prepared Sijunzi powder group was significantly reduced (P < 0.05), the NEFA and TG contents were extremely significantly reduced (P < 0.01), and the MTTP content was extremely significantly increased (P < 0.01). Fig.11 A to D. This indicates that the self-prepared Sijunzi powder has a certain slowing effect on fat mobilization in postpartum dairy cows.

[0089] The results of differential metabolite analysis showed that non-targeted metabolomics analysis of plasma samples was performed using UHPLC-MS / MS to observe the effect of self-prepared Sijunzi powder on the blood metabolome of postpartum dairy cows. A total of 283 blood metabolites were detected in positive and negative ion modes, including 80 organic acids and derivatives, 72 lipids and lipid-like molecules, 39 organic heterocyclic compounds, 33 organic oxygen compounds, 31 benzene compounds, 9 organic nitrogen compounds, 8 phenylpropanoids and polyketides, 6 nucleosides, nucleotides, and analogues, and 5 other categories.

[0090] The PCA results showed that there was a separation trend between the self-made Sijunzi powder group and the control group (PC1 16.3%, PC2 14.7%), indicating that the self-made Sijunzi powder changed the blood metabolic profile of postpartum dairy cows to a certain extent, and there were differences in the metabolite composition and concentration between the two groups of samples ( Fig.12 A). The OPLS-DA model was constructed to further observe the importance of metabolites for classification. The OPLS-DA permutation test graph showed that the intercept of Q2 and the Y axis was less than zero (R2 = 0.79, Q2 = -0.44), indicating that the constructed OPLS-DA model was reasonable and did not appear overfitting ( Fig.12 C). The OPLS-DA score plot shows that the two groups of blood samples are clearly separated ( Fig.12 B). Metabolites with VIP≥1 were selected for further univariate analysis to obtain the FC value and P value of each metabolite ( Fig.12 D). With VIP≥1, FC≤0.83 or FC≥1.2 and P<0.05 as the standard, a total of 21 differential metabolites were screened, of which 5 were significantly upregulated and 16 were significantly downregulated. The Rt(min), Mean.SJZS, Mean.CONT, VIP, FC, P-value and FDR of the differential metabolites are shown in Table 2. The cluster heat map showed that the 21 differential metabolites screened had good intra-group repeatability and obvious distinction between groups ( Fig.12 E), indicating that the screened differential metabolites are representative to a certain extent.

[0091] Table 2 Blood differential metabolites and related parameters

[0092]

[0093]

[0094] Note: a) Variable importance for the projection of differential metabolites calculated based on orthogonal partial squares discriminant analysis. b) Fold change of differential metabolites calculated based on relative peak intensity. ↑ indicates significant upregulation, ↓ indicates significant downregulation.

[0095] Pathway enrichment analysis results showed that KEGG pathway enrichment analysis was performed on 21 differential metabolites. Seven differential metabolites were hit during enrichment, which could affect linoleic acid metabolism (P < 0.05), steroid hormone biosynthesis (P < 0.05), glycerophospholipid metabolism, steroid biosynthesis, primary bile acid biosynthesis in lipid metabolism pathways and beta-alanine metabolism (beta-Alanine metabolism), propionate metabolism (Propanoate metabolism), inositol phosphate metabolism (Inositol phosphate metabolism), arginine and proline metabolism (Arginine and prolinemetabolism), valine, leucine and isoleucinedegradation (Valine, leucine and isoleucinedegradation) in amino acid metabolism pathways. Fig.13 The differential metabolites included 12(13)-EpOME, cholesterol, cholesterol 3-sulfate, malonic semialdehyde, phosphorylcholine, 4-aminobutyraldehyde and 2-Methyl-3-oxopropanoic acid. The metabolic pathways, P-value, impact and relevant metabolites are shown in Table 3.

[0096] Table 3 Metabolic pathways and related parameters of differential metabolites

[0097]

[0098]

[0099] The above disclosure is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the above claims.

Claims

1. A Chinese medicinal composition of Sijunzi powder with modifications, which is composed of codonopsis pilosula, stir-fried atractylodes macrocephala, tuckahoe, hawthorn and roasted liquorice, with a weight ratio of 2-3:2-3:2-3:2-3:1-1.

5.

2. The Chinese medicine composition according to claim 1, wherein the weight ratio of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root is 2:2:2:2:

1.

3. A method for preparing the Chinese medicine composition according to claim 1 or 2, comprising the following steps: S1. Wash the raw medicinal materials, such as Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and Licorice root, with clean water, and then dry them; S2. Prepare fried Poria cocos: gently place the Poria cocos slices in a pot, then slowly heat over a low heat, and continue to stir-fry until the surface turns slightly yellow; S3. Prepare roasted licorice: Carefully add an appropriate amount of refined honey to the licorice slices, and then slowly fry them over a low fire until they turn yellow to dark yellow. When the licorice slices are no longer sticky, take them out and set them aside to cool. S4, grinding the entities of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root into powders by machine respectively; S5, passing the ground medicinal material powder through a 100-mesh sieve to remove impurities; S6. Mix the sieved medicinal powders of Codonopsis pilosula, stir-fried Atractylodes macrocephala, Poria cocos, Crataegus pinnatifida and roasted Licorice root according to the proportions and mix them thoroughly.

4. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of health products or medicines for reducing liver cell damage in dairy cows.

5. Use of the Chinese medicine composition according to claim 1 or 2 in preparing health products or medicines for improving the rumen fermentation ability of dairy cows.

6. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of health products or medicines for regulating the composition and quantity of rumen flora of dairy cows.

7. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of health products or medicines for increasing immunoglobulin levels in postpartum dairy cows, slowing down lipid mobilization and regulating blood metabolism.

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