Sijunzi powder compressed tablet and quality detection method thereof

Abstract

The invention discloses a Sijunzi powder compressed tablet and a quality detection method thereof, and the Sijunzi powder compressed tablet is prepared by the following method: (1) taking Sijunzi powder, adding 52-58v / v% ethanol with the weight being 8-12 times that of the Sijunzi powder, carrying out heating reflux extraction twice for 1.4-1.6 hours and 0.8-1.2 hours respectively, filtering, and carrying out vacuum concentration to recover the ethanol so as to obtain an alcohol extract; (2) adding 8-12 times by weight of water into the medicine residues, decocting for two times, respectively extracting for 1.4-1.6 hours and 0.8-1.2 hours, combining filtrates to obtain a water extract, combining the water extract with the alcohol extract, and freeze-drying by using a freeze-drying machine to obtain an extract; and (3) pressing the extract into traditional Chinese medicine tablets by using a Chinese herbal medicine tablet press. Different active parts of the Sijunzi powder are separated through an alcohol-water double extraction method, and the component utilization rate is effectively increased; the extract is prepared into tablets, the dosage of the medicine is reduced through volume compression, and the medicine is convenient to store and transport.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary medicine, and particularly to a Sijunzi powder compressed tablet and a quality detection method thereof. Background Art

[0002] In recent years, with the continuous expansion of the production scale of China's breeding industry, the yields of major livestock and poultry breeds such as pigs, cattle, sheep, and poultry have continued to rise. According to statistical data, the yields of products such as pork, eggs, and milk rank among the top in the world, and the total output value of the animal husbandry industry has increased steadily. However, with the country's restrictions on the use of antibiotics, the use of traditional high-dose antibiotics has been strictly restricted, and natural alternatives have gradually become the main choice in the breeding industry.

[0003] Traditional Chinese medicine, as a precious resource unique to China, is derived from natural plants, animals, or minerals. Under the guidance of traditional Chinese medicine theory and through thousands of years of clinical evidence, it has left a deep mark on the development of Chinese civilization. Feed natural plants not only have the advantages of being natural and residue-free, but also have the dual functions of medicine and nutrition. The classic traditional Chinese medicine formula Sijunzi Decoction is composed of ginseng, atractylodes macrocephala, poria cocos, and licorice, and has the effects of replenishing qi and strengthening the spleen and stomach, and is mainly used to treat spleen and stomach qi deficiency syndrome, poor appetite, and emaciation. Spleen and stomach qi deficiency is caused by weak transportation; the spleen and stomach are the foundation of acquired constitution and the source of qi and blood generation. When there is spleen and stomach qi deficiency, the function of acceptance and transportation is weak, resulting in reduced food intake; when there is spleen and stomach qi deficiency, endogenous dampness turbidity is generated, the transportation and transformation of the spleen and stomach are unfavorable, and the generation of qi and blood is insufficient, leading to emaciation and a sub-healthy state with low immunity. In the Sijunzi Decoction formula, ginseng is the monarch drug, which is sweet and warm in nature, replenishes qi, and strengthens the spleen and stomach; atractylodes macrocephala, which is bitter and warm, is used as the minister drug to strengthen the spleen and dry dampness, enhancing the power of replenishing qi and promoting transportation; poria cocos, which is sweet and light, is used as the assistant drug to strengthen the spleen and promote diuresis. When combined with atractylodes macrocephala, the effect of strengthening the spleen and removing dampness is more prominent; roasted licorice is used as the envoy drug to replenish qi and harmonize the middle, and to reconcile all the drugs. The four drugs are combined to achieve the effect of strengthening the spleen and replenishing qi.

[0004] In dairy cattle breeding, Sijunzi powder, as a classic formula for replenishing qi and strengthening the spleen, is widely used to treat spleen and stomach qi deficiency. However, the traditional use method of Sijunzi powder has the following problems:

[0005] (1) High dose: In the production of ruminants, due to the large body weight of ruminants such as dairy cattle and the limitations of the drug dose-effect relationship, the application of traditional Chinese medicine often requires the use of large doses of drugs. This makes the use, storage, and transportation of drugs inconvenient in the actual production process.

[0006] (2) Inconvenient administration: Currently, the main method for ruminants to use traditional Chinese medicine formulas is rumen intubation and gavage, which not only increases the workload of veterinarians, but also may endanger the lives of animals, easily cause stress reactions, and lead to secondary diseases, thus bringing economic losses to the ranch. In addition, the impact of this method on animal welfare cannot be ignored.

[0007] (3) Low drug efficacy utilization rate: The utilization rate of the decoction is not high and the volume is large, affecting the convenience of actual operation.

[0008] (4) Difficult to control product quality: The extraction of active ingredients by the decoction is limited, affecting the actual drug efficacy.

[0009] Therefore, developing a comprehensive solution from the extraction process, quality control to optimizing the administration method is a key issue that modern animal husbandry urgently needs to solve. Summary of the Invention

[0010] In view of the deficiencies of the prior art, the present invention provides a Sijunzi Powder compressed tablet and its quality detection method. In order to achieve the invention purpose of the present invention, the following technical solutions are proposed:

[0011] In the first aspect of the present invention, a Sijunzi Powder compressed tablet is provided, which is prepared by the following method:

[0012] (1) Take Sijunzi Powder, add 8 - 12 times the weight of 52 - 58 v / v% ethanol, heat and reflux for extraction twice, with the extraction times being 1.4 - 1.6 hours and 0.8 - 1.2 hours respectively. After filtration, concentrate under reduced pressure to recover ethanol to obtain an ethanol extract;

[0013] (2) Add 8 - 12 times the weight of water to the medicinal residues and decoct twice, with the extraction times being 1.4 - 1.6 hours and 0.8 - 1.2 hours respectively. Combine the filtrates to obtain a water extract. Combine the water extract with the ethanol extract and use a freeze dryer for freeze drying to obtain an extract;

[0014] (3) Subsequently, use a Chinese herbal medicine tablet press to press the extract into Chinese medicine tablets.

[0015] (4) Wrap the Chinese medicine tablets with edible rice paper into a columnar body to form a Chinese medicine stick.

[0016] In a preferred embodiment of the present invention, it is characterized in that 10 times the weight of 55 v / v% ethanol is added in step (1).

[0017] In a preferred embodiment of the present invention, it is characterized in that in step (3), the volume of the tablet is further reduced to less than one - third of the original volume.

[0018] In a preferred embodiment of the present invention, it is characterized in that after step (3), it further includes: in step (4), the Chinese medicine tablets are wrapped with edible rice paper into a columnar body to form a Chinese medicine stick.

[0019] On the other hand, the present invention also relates to a quality detection method for the above - mentioned Sijunzi Powder compressed tablet, which includes the following steps:

[0020] (1) Extract with chloroform, ethyl acetate, and n-butanol in sequence to obtain extracts of 4 different extraction parts. After evaporating the organic solvents respectively and freeze-drying, the chloroform part of S5, the ethyl acetate part of S6, the n-butanol part of S7, and the remaining part of S8 are obtained;

[0021] (2) Weigh the chloroform part of S5, the ethyl acetate part of S6, the n-butanol part of S7, and the remaining part of S8 into EP tubes respectively, add pre-cooled methanol-aqueous solution (1:1, v / v), and mix well to redissolve the samples; after centrifuging the samples, take the supernatant for instrumental analysis;

[0022] (3) Analyze the samples by high performance liquid chromatography-mass spectrometry.

[0023] In a preferred embodiment of the present invention, the conditions of the high performance liquid chromatography are as follows: during the whole analysis process, the samples are placed in a 4°C autosampler, and a C18 chromatographic column is used; the injection volume is 4 μL, the column temperature is 40°C, and the flow rate is 300 μL / min; the chromatographic mobile phase A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution; the chromatographic gradient elution program is as follows: 0 - 1 min, B is maintained at 2%; 1 - 5 min, B linearly changes from 2% to 48%; 5 - 7 min, B linearly changes from 48% to 80%; 7 - 11 min, B linearly changes from 80% to 100%; 11 - 13 min, B is maintained at 100%; 13 - 13.01 min, B linearly changes from 100% to 2%, 13.01 - 15 min, B is maintained at 2%. These chromatogram conditions are helpful for the separation of active ingredients, and at the same time can ensure that the separation time is maintained within 18 min, which is helpful for improving the detection accuracy and detection efficiency.

[0024] The present invention realizes the following innovations by extracting different active ingredients of Sijunzi Powder and combining the Chinese herbal medicine tabletting technology and optimizing the administration process:

[0025] (1) Optimization of the extraction process: By the alcohol-water double extraction method, different active parts of Sijunzi Powder are separated, effectively improving the utilization rate of ingredients;

[0026] (2) Tabletting technology: The extracts are made into tablets, and the drug usage amount is reduced by volume compression, which is convenient for storage and transportation;

[0027] (3) Optimization of the administration method: The tablets are wrapped with edible rice paper to make columnar Chinese medicine rods, and are administered through a calcium rod injector, avoiding the risk of accidental inhalation into the trachea during gavage, reducing the stress response of animals, and improving the operation efficiency and animal welfare.

[0028] (4) Optimize the quality inspection method: Further perform non-targeted metabolomics analysis on the above-mentioned extracts by sequentially extracting with organic solvents to identify its specific components and establish a traditional Chinese medicine fingerprint for providing a reliable basis for product quality inspection. Description of the Drawings

[0029] Figure 1 Rumen gas production

[0030] Figure 2 pH value

[0031] Figure 3 Rumen microbial protein

[0032] Figure 4 Volatile fatty acids

[0033] Figure 5 TIC chromatogram of QC sample in positive ion mode

[0034] Figure 6 TIC chromatogram of QC sample in negative ion mode

[0035] Figure 7 Scatter plot of the first and second principal components of PCA

[0036] Figure 8 Hierarchical clustering heat map of differential metabolites

[0037] Figure 9 Stacked plot of traditional Chinese medicine fingerprint

[0038] Figure 10 Waterfall plot of traditional Chinese medicine fingerprint Detailed implementation manners

[0039] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following takes preferred embodiments to describe in detail the specific implementation manners, technical solutions, features and their effects of the application according to the present invention. The specific features, structures or characteristics in the following descriptions of multiple embodiments can be combined in any suitable form.

[0040] Example 1:

[0041] 1. Extraction process

[0042] 1. Take four portions of 250 g of Sijunzi Powder and add 10 times the amount of 55 v / v% ethanol, 75

[0043] v / v% ethanol, 95 v / v% ethanol and purified water were used. Heating under reflux extraction was performed 2 times using a rotary evaporator, with extraction times of 1.5 hours and 1 hour respectively. After filtration, ethanol was recovered by reduced pressure concentration. The medicinal residues were added with 10 times the amount of water and decocted 2 times, with extraction times of 1.5 hours and 1 hour respectively. The filtrates were combined to obtain the water extract. Then, the ethanol in the ethanol extract was recovered under reduced pressure, and the water extracts were combined and freeze-dried using a vacuum freeze dryer to obtain different extracts, namely S1 (55 v / v% ethanol fraction), S2 (75 v / v%

[0044] ethanol fraction), S3 (95 v / v% ethanol fraction), and S4 (water fraction).

[0045] 2. The alcohol-water double extraction method was used to further extract the medicinal components. Specifically, 250 g of Sijunzi Powder was taken and added with 6 times the amount of 55 v / v% ethanol. Reflux extraction was performed 2 times in a rotary evaporator, with extraction times of 1.5 hours and 1 hour. The filtrates were combined to obtain the ethanol extract. The medicinal residues were added with 10 times the amount of water and decocted 2 times, with extraction times of 1.5 hours and 1 hour respectively. The filtrates were combined to obtain the water extract. Then, the ethanol in the ethanol extract was recovered under reduced pressure, and the water extracts were combined and concentrated under reduced pressure to obtain the medicinal liquid. Extraction was performed successively with chloroform, ethyl acetate, and n-butanol to obtain extracts of 4 different extraction parts. After evaporating the organic solvents and freeze-drying, S5 (chloroform fraction), S6 (ethyl acetate fraction), S7 (n-butanol fraction), and S8 (remaining fraction) were obtained.

[0046] 3. Further non-targeted metabolomics analysis was performed on the above extracts to identify their specific components and establish a fingerprint of traditional Chinese medicine, providing a reliable basis for subsequent drug research.

[0047] 2. Tablet Preparation

[0048] Preparation of Tablets from the Extract of the 55 v / v% Ethanol Fraction of Sijunzi Powder and the Administration Rod Coated with Rice Paper

[0049] 98.445 g of extract was obtained by extracting 250 g of traditional Chinese medicine Sijunzi Powder using the alcohol-water double extraction method with 55 v / v% ethanol, and the volume of the extract was reduced by 2.54 times. Subsequently, a traditional Chinese medicine tablet with a diameter of 1.9 cm and a thickness of 5 mm was pressed from the extract using a traditional Chinese medicine tablet press, and the volume of the tablet was further reduced to one-third of the original volume, and the final total volume was reduced to 7.62 times. According to the provisions of the Pharmacopoeia of the People's Republic of China for Veterinary Drugs, each dairy cow needs to be administered 300 g of Sijunzi Powder per day. Through this method, 118.134 g of extract can be obtained, which can be made into about 21 traditional Chinese medicine tablets. The common ratio of the water for administering drugs is 1:5 to 1:10, that is, 5 to 10 ml of water is added to 1 g of the drug. Pastures usually choose to dissolve 300 g of Sijunzi Powder in 2500 ml of water, with a volume of 2500 cm 3However, after 300 grams of Sijunzi Powder is made into traditional Chinese medicine tablets by the water extraction method, the volume is only 39.37 cm 3 , which is 68.73 times smaller than that of the solution administered by rumen gavage. The tablet form not only helps to reduce the dosage of the drug, greatly simplifies the administration process and reduces the workload of ranch veterinarians, but also effectively prevents the stress response caused by gavage in dairy cows, thereby reducing the risk of stress-related diseases.

[0050] At the same time, further optimize the administration of the traditional Chinese medicine stick by wrapping the tablet with edible rice paper into a columnar body. The effect is good when administered through a calcium stick dispenser. This method not only reduces the dosage of the drug, but also simplifies the administration process, avoids the risk of accidental entry into the trachea caused by intubation and gavage. Skilled workers can complete the administration within 1 minute, reducing the workload of veterinarians and reducing the stress response of dairy cows caused by gavage, greatly improving the operation efficiency and animal welfare.

[0051] 3. Optimize the administration method

[0052] (1) Preparation of columnar traditional Chinese medicine stick: Make an edible rice paper bag according to the diameter of the tablet, and fill the tablet into the edible rice paper bag to ensure that the traditional Chinese medicine stick contains a sufficient amount of the drug.

[0053] (2) Administration process:

[0054] · Administration for small animals: For small animals, since the dosage of drugs for small animals is small, the traditional Chinese medicine tablets can be taken orally directly.

[0055] · Administration for large animals: For ruminants such as dairy cows, the administration can be completed through a calcium stick dispenser, which is convenient to operate and avoids accidental entry into the trachea and animal stress caused by gavage.

[0056] 4. Experimental results

[0057] 4.1 Obtain traditional Chinese medicine extracts: Through the above extraction methods, traditional Chinese medicine extracts from different parts were obtained. The weight of the extracts was reduced to varying degrees compared with the volume of the original drug, reducing the dosage of drugs in clinical practice and subsequent research.

[0058] Drug Name Weight after extraction (g) S1 (55% ethanol) 98.445 S2 (75% ethanol) 61.467 S3 (95% ethanol) 45.274 S4 (aqueous fraction) 101.217 S5 (chloroform fraction) 0.404 S6 (ethyl acetate fraction) 14.618 S7 (n-butanol fraction) 7.924 S8 (remaining aqueous fraction) 17.171

[0059] 4.2 Effects of traditional Chinese medicine extracts from different parts on rumen fermentation parameters of dairy cows.

[0060] Short-term artificial rumen culture experiments were carried out using different extracts (S1 to S8), and the rumen fermentation parameters of each group were measured and analyzed, including the changes in gas production, pH value, microbial protein (MCP), and volatile fatty acids (VFA). By comparing these fermentation parameters, the regulatory effects of each extract on rumen fermentation can be evaluated.

[0061] Rumen fermentation parameters in dairy cows are key indicators for evaluating the microbial fermentation process and its efficiency in the rumen, which can reflect the status of rumen function, nutrient digestion and utilization rate, and microbial activity. In research, these parameters are usually used to evaluate the effects of feed ingredients or feed additives (such as traditional Chinese medicine extracts) on the rumen function of dairy cows. The main rumen fermentation parameters include gas production, pH value, microbial protein content, and volatile fatty acids, etc.

[0062] 4.2.1 Experimental methods

[0063] (1) Preparation of rumen fluid culture bags

[0064] The rumen fluid culture bag consists of a rubber stopper and a nylon sealed bag. By tightly sealing the nylon sealed bag with the rubber stopper and using a syringe to extract the air inside the bag through the rubber stopper, an anaerobic environment required for in vitro rumen fermentation can be effectively created. Compared with the traditional method using a glass syringe, this method is more simple, accurate, and has a lower experimental cost. In addition, the rubber stopper provides better sealing performance, thus improving the accuracy of experimental data.

[0065] (2) Preparation of rumen buffer solution

[0066] Table 1. Preparation of rumen buffer solution

[0067]

[0068] (3) Collection of rumen fluid

[0069] Six healthy Holstein dairy cows were selected as rumen fluid donor cows, and the rumen contents of the dairy cows were collected through rumen intubation before morning feeding. The collected rumen fluid was mixed in equal volume, filtered through four layers of gauze and mixed evenly, and quickly placed in a thermos flask filled with carbon dioxide and brought back to the laboratory for subsequent in vitro rumen fermentation culture. The collected rumen fluid was divided into a blank control group and 9 experimental groups. The blank control group was not added with Sijunzi Powder and its extracts. In the first 8 experimental groups, 160 mg of traditional Chinese medicine samples (S1 - S8) were added respectively, and the last group was added with an equal amount of Sijunzi Powder. Each group was set with 3 replicates.

[0070] (4) Preparation and culture of mixed culture solution

[0071] The prepared rumen fluid was quickly added to the buffer solution pre-saturated with CO 2 and preheated to 39 °C in a ratio of 1:2 (rumen fluid to buffer solution) to prepare a mixed culture solution. A few drops of 0.1% resazurin solution were added to the mixed culture solution as an anaerobic indicator (it turns red in the presence of free oxygen and colorless in the absence of oxygen). At the same time, while heating the mixed culture solution, it was stirred with a magnetic stirrer, and CO 2, until the solution becomes colorless. Subsequently, add about 30 mL of mixed culture medium to each culture bag, place the culture bag vertically, seal the bag mouth with a rubber stopper, and evacuate the gas inside the bag. Place the culture bag in a water bath shaker or artificial rumen incubator preheated to 39 °C, start the equipment for cultivation, and control the shaker speed at 80 - 90 revolutions per minute.

[0072] 4.2.2 Experimental detection sample collection

[0073] At 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, and 48 h after the fermentation experiment, draw the gas inside the bag with a syringe, and record the gas production volume according to the scale value (mL) of the syringe piston. Accurately take 4 mL of fermentation culture medium and add 1 mL of 25% metaphosphoric acid into a 5 mL centrifuge tube for determining the content of volatile fatty acids (VFA), and store it frozen in a -20 °C refrigerator. Take another part of the fermentation culture medium and quickly place it in a liquid nitrogen tank and transfer it to a -80 °C refrigerator for preservation, which is used for rumen microbial protein and metagenome detection.

[0074] 4.2.3 Experimental results

[0075] (1) Gas production

[0076] Gas is produced during rumen fermentation, such as carbon dioxide (CO 2 ) and methane (CH 4 ). The production of these gases can reflect the activity of rumen fermentation, and the reduction of methane production helps to reduce the carbon footprint of dairy cows. According to the experimental results (as Figure 1 shown), except for the extract from the S5 part, the gas production of the extracts from the other parts was significantly up-regulated compared with the blank control group, and the gas production of the extracts from each part was significantly down-regulated compared with the Sijunzi powder group.

[0077] (2) pH value

[0078] The pH value of the rumen directly affects the activities and metabolic processes of the microbial community. Usually, the rumen pH value of healthy dairy cows is between 5.5 and 7.0. Being too acidic (below 5.5) may lead to rumen acidosis, while being too alkaline may inhibit certain fermentation activities. The experimental results (as Figure 2 shown) indicate that except for the S5 group, the pH values of the other groups were lower than those of the blank group and the differences were not significant, and all were within the normal range.

[0079] (3) Rumen microbial protein (MCP)

[0080] MCP is a biological protein synthesized by rumen microbiota using proteins and ammonia nitrogen in the rumen. Dairy cows obtain nitrogen and amino acids by digesting these microbial proteins. The amount of MCP reflects the protein synthesis efficiency of rumen fermentation. The experimental results (as Figure 3As shown, the content of bacterial protein in the experimental groups of extracts from different parts is higher than that in the blank group and the Sijunzi Powder group, which proves that the Chinese herbal medicine extracts from different parts have promoting effects on rumen fermentation and protein synthesis of dairy cow rumen microorganisms.

[0081] (4) Volatile Fatty Acids (VFA)

[0082] VFA are the main products when rumen microorganisms ferment carbohydrates (such as fiber and starch), mainly including acetic acid, propionic acid and butyric acid. VFA are the main source of energy for dairy cows, and their ratio will affect the milk yield and milk quality of dairy cows. The experimental results (as Figure 4 shown) indicate that the VFA content in the experimental groups of Chinese herbal medicine extracts from other parts except the S5 part is higher than that in the blank group, and at the same time, the S1, S2 and S3 parts are higher than the Sijunzi Powder group.

[0083] (5) Comprehensive evaluation:

[0084] Comprehensive evaluation of the efficacy indexes of Sijunzi Powder by entropy weight - TOPSIS (calculated by entropy weight method)

[0085] 1. Steps of entropy weight - TOPSIS comprehensive evaluation:

[0086] (1). Standardized matrix:

[0087] o Standardize the indexes and convert the data to the interval [0, 1].

[0088] (2). Calculate entropy value:

[0089] o Calculate the entropy value Ej of each index. The entropy value reflects the information entropy distribution of the index.

[0090] Ej = -k∑i = 1mPijln(Pij)

[0091] where Pij = Rij∑i = 1mRij, Rij is the proportion of the index value, k = 1ln(m).

[0092] (3). Calculate weight:

[0093] o Calculate the weight wj of each index according to the entropy value.

[0094] $w_j=\frac{1 - E_j}{\sum_{j = 1}^n(1 - E_j)}$

[0095] (4). Construct the weighted normalization matrix:

[0096] o Weight the normalized index values with weights.

[0097] (5). Calculate the positive ideal solution and the negative ideal solution:

[0098] o The positive ideal solution is the maximum value of each index, and the negative ideal solution is the minimum value of each index.

[0099] (6). Calculate the Euclidean distance from the ideal solution:

[0100] o Calculate the Euclidean distance of each solution from the positive ideal solution ($S^+$) and the negative ideal solution ($S^-$).

[0101] (7). Calculate the comprehensive score:

[0102] (7). Calculate the comprehensive score:

[0103] o Calculate the comprehensive score of each solution based on the distance.

[0104] $C_i=\frac{S_i^-}{S_i^+ + S_i^-}$

[0105] The larger the comprehensive score, the better the solution.

[0106] 2. Calculation results

[0107] (1). The data table after normalization

[0108] Table 2: The data table after normalization

[0109] Grouping VFA Gas production pH MCP Blank group 0.000 0.115 0.442 0.000 S1 0.865 0.780 0.928 0.610 S2 0.969 0.733 0.935 0.534 S3 1.000 0.841 0.949 0.394 S4 0.733 0.908 1.000 0.498 S5 0.113 0.000 0.000 0.625 S6 0.653 0.929 0.913 0.610 S7 0.582 0.655 0.993 1.000 S8 0.412 0.931 0.920 0.622 Sijunzi Powder 0.449 1.000 0.967 0.271

[0110] (2). Index weights

[0111] ● VFA: 31.29%

[0112] · Gas production: 26.82%

[0113] · pH: 18.49%

[0114] · MCP: 23.40%

[0115] (3). Comprehensive score ranking

[0116] Table 3: Comprehensive score ranking table

[0117] Ranking Grouping Comprehensive score 1 S1 0.777 2 S2 0.761 3 S3 0.745 4 S4 0.732 5 S6 0.731 6 S7 0.709 7 S8 0.636 8 Sijunzi Powder 0.596 9 S5 0.256 10 Blank group 0.157

[0118] (4). Conclusion

[0119] 1. In the case of extracting with low-concentration ethanol with a lower usage cost, the S1 group has the highest score and is optimal in both comprehensive performance and cost.

[0120] 2. The score of Sijunzi Powder is 0.596, showing average performance but still superior to the blank group.

[0121] 3. The S5 group and the blank group have the lowest scores, indicating that these two groups are at a disadvantage in multiple indicators.

[0122] The research results show that the traditional Chinese medicine extracts from different parts obtained by this method have different effects on the rumen fermentation parameters of dairy cows, proving that this method is conducive to further in-depth research on the traditional Chinese medicine formula. By extracting the traditional Chinese medicine components from different parts with different solvents, the comprehensive analysis and detection of the components of the traditional Chinese medicine formula can be achieved.

[0123] 4.3 Identification results of the components of each active part of Sijunzi Powder

[0124] Take the traditional Chinese medicine extracts of eight groups of S1-S8, and each group is subjected to non-targeted metabolomics analysis with three replicates. To ensure the accuracy of the experimental results and the stability of the instrument, QC samples (quality control samples) were prepared, which were composed of equal amounts of all experimental samples. The QC samples were used to balance the chromatographic-mass spectrometry system and evaluate the stability of the system during the experiment. Before the experiment, the samples to be measured need to be thawed at 4°C, and 10 mg of each sample is weighed into an EP tube. Subsequently, 1500 μL of pre-cooled methanol-aqueous solution (1:1, v / v) was added, and the samples were fully mixed to redissolve the samples. The samples were centrifuged at 20000 g for 15 minutes at 4°C, and then the supernatant was taken for instrumental analysis. This process ensured the accuracy of metabolite extraction and mass spectrometry analysis, providing a reliable basis for the metabolic profile study of different traditional Chinese medicine extracts.

[0125] 4.3.1 Instruments and reagents

[0126] Table 4, Instruments and reagents for metabolomics

[0127]

[0128] 4.3.2 LC-MS / MS analysis

[0129] (1) Chromatographic separation: During the entire analysis process, the sample was placed in an autosampler at 4°C. The sample was analyzed using a Shimadzu Nexera X2 ultra-high performance liquid chromatography system (UHPLC) with a C18 (2.1x100 mm, 1.6 μm) (Waters, Milford, MA, USA) chromatographic column. The injection volume was 4 μL, the column temperature was 40°C, and the flow rate was 300 μL / min. The chromatographic mobile phase A was 0.1% formic acid aqueous solution, and B was 0.1% formic acid acetonitrile solution. The chromatographic gradient elution program was as follows: 0 - 1 min, B was maintained at 2%; 1 - 5 min, B linearly changed from 2% to 48%; 5 - 7 min, B linearly changed from 48% to 80%; 7 - 11 min, B linearly changed from 80% to 100%; 11 - 13 min, B was maintained at 100%; 13 - 13.01 min, B linearly changed from 100% to 2%, and 13.01 - 15 min, B was maintained at 2%.

[0130] (2) Mass spectrometry acquisition: Each sample was detected in positive ion (+) and negative ion (-) modes using electrospray ionization (ESI). Mass spectrometry analysis was performed on a mass spectrometer (AB SCIEX) in the information-dependent basis (IDA) mode for both positive and negative ions.

[0131] (3) Mass spectrometry parameters: The instrument was set to perform TOF MS scans in the m / z range of 60 - 1200 Da and product ion scans in the m / z range of 25 - 1200. The accumulation time for TOF MS scans was set to 0.08 seconds / spectrum, and for product ion scans it was 0.03 seconds / spectrum. Product ion scans were performed using the information-dependent acquisition (IDA) mode, and the high-sensitivity mode was selected. In each cycle, 15 precursor ions were selected for fragmentation, with a collision energy (CE) of 30 V and an extended range of ±15.

[0132] (4) Ion source parameters: Positive ion mode: Source temperature 550°C, Ion source gas 1 (GAS1): 50, Ion source gas 2 (GAS2): 50, Curtain gas (CUR): 50, Ion spray voltage (ISVF) 5500 V.

[0133] Negative ion mode: Source temperature 500°C, Ion source gas 1 (GAS1): 50, Ion source gas 2 (GAS2): 50, Curtain gas (CUR): 50, Ion spray voltage (ISVF) -4500 V.

[0134] 4.3.3 Data preprocessing

[0135] The original data was subjected to peak alignment, retention time correction, and peak area extraction using the MSDIAL software. For metabolite structure identification, the Chinese medicine library was searched by means of accurate mass number matching (mass tolerance < 10 ppm) and secondary spectrum matching (mass tolerance < 0.01 Da). For the extracted data, ion peaks with > 50% missing values within the group were deleted and not involved in subsequent statistical analysis; the total peak areas of positive and negative ion data were normalized separately, the positive and negative ion peaks were integrated, and pattern recognition was performed using Python software. After the data was preprocessed by Unit variance scaling (UV), subsequent data analysis was carried out.

[0136] 4.3.4 Experimental Results

[0137] (1) Experimental Quality Evaluation

[0138] The system stability of this project experiment was evaluated and analyzed by adopting two strategies: comparing the mass spectrometry TIC maps of QC samples and performing PCA statistical analysis on the overall samples. The total ion current chromatograms (TIC) of the QC samples in the positive and negative ion detection modes were respectively subjected to spectral display and comparison, as shown in Figure 5 and Figure 6 . The results showed that the response intensities and retention times of each chromatographic peak were basically consistent, indicating that the variation caused by instrument error during the entire experimental process was small and the data quality was reliable.

[0139] (2) Screening of Differential Components

[0140] Principal component analysis (PCA) of the overall samples: The MSDIAL software was used to extract metabolite ion peaks, and a total of 60,258 ion peaks were collected in the positive and negative ion modes. The peaks extracted from all experimental samples and QC samples were subjected to PCA analysis after UV treatment, and a PCA model was obtained through 7-fold cross-validation. As shown in the figure, each sample was closely clustered together, indicating good repeatability of this project experiment, and the discreteness between groups was obvious, indicating that there were significant differences in the components of traditional Chinese medicine extracts from different parts.

[0141] (3) Hierarchical Clustering Heat Map of Differential Metabolites

[0142] To evaluate the rationality of candidate metabolites and more comprehensively and intuitively display the relationships between samples and the differences in the expression patterns of metabolites in different samples, we used the expression levels of qualitatively significant differential metabolites to perform hierarchical clustering on each group of samples, thereby assisting us in accurately screening biomarker metabolites and studying the changes in related metabolic processes. The clustering results of the samples can test the expression stability of the selected target metabolites in the samples within the group. At the same time, metabolites clustered in the same cluster have similar expression patterns and may be in relatively close reaction steps during the metabolic process. The following figure shows the hierarchical clustering results of differential metabolites in the comparison groups. The redder the color, the higher the relative expression level, and the bluer the color, the lower the relative expression level. As Figure 8 shown, there are significant differences in the components among the traditional Chinese medicine extracts from different parts.

[0143] (4) Construction of traditional Chinese medicine fingerprint

[0144] By analyzing the chemical components in traditional Chinese medicinal materials or their preparations using techniques such as high-performance liquid chromatography (HPLC), a series of representative peaks of traditional Chinese medicine extracts are obtained, and the "fingerprint" spectra of each group of traditional Chinese medicine extracts are drawn using Origin 2024 software (as Figure 9 and 10 shown). The traditional Chinese medicine fingerprint can reveal the main chemical components in the medicinal materials and their relative contents, thereby helping to identify the types and sources of medicinal materials and judge the component differences among the extracts of the Sijunzi Powder in each group.

[0145] Non-targeted metabolomics studies have shown that the traditional Chinese medicine extracts from different parts obtained by using this method have relatively significant differences, which is beneficial for subsequent in-depth research on the Sijunzi Powder formula, such as exploring the effects of traditional Chinese medicine extracts from a certain part on the rumen fermentation parameters of dairy cows and studying the most important pharmacodynamic components of the Sijunzi Powder during the process of strengthening the spleen.

[0146] 5. Beneficial effects

[0147] (1) Reduction in drug dosage and volume: Through the extraction process of the present invention, traditional Chinese medicine extracts from different parts are obtained, and the volume of the original drug is significantly reduced, reducing the clinical dosage of the drug, which is beneficial for improving the drug efficacy and reducing the production cost. Taking the water-extracted tablets of the Sijunzi Powder as an example, after the water-extracted part extract of the Sijunzi Powder is made into tablets, its total volume is reduced by 7.62 times compared with the medicinal materials and 68.73 times compared with the volume of the gavage solution used clinically, which is convenient for storage, transportation and use;

[0148] (2) Comprehensive separation of components: By using different solvents to extract the drug, more comprehensive separation of the drug components can be achieved, which is convenient for identifying the components of the drug and further research.

[0149] (3)Improve drug efficacy: Different extracts have different effects on the rumen fermentation parameters of dairy cows, which can improve the rumen function, nutrient digestibility and microbial activity of dairy cows, improve the health and production performance of dairy cows, and have important application value.

[0150] (4)Simplify clinical application: The Chinese herbal medicine extract is made into tablets by using a tablet press, and further the tablets are wrapped into a columnar body with edible rice paper to optimize into a Chinese medicine stick for administration. The medicine is administered through a calcium stick applicator, and the effect is good. This method not only reduces the dosage of the drug, but also simplifies the administration process, avoids the risk of accidental entry into the trachea caused by intubation and gavage. Skilled workers can complete the administration within 1 minute, reducing the workload of veterinarians and reducing the stress response of dairy cows caused by gavage, greatly improving the operation efficiency and animal welfare.

[0151] The specific embodiments of the present invention disclosed above are only for illustration, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the above-mentioned claims.

Claims

1. A Sijunzisan compressed tablet, which is prepared by the following method: (1) taking Sijunzi powder, adding 8-12 times the weight of 52-58 v / v% ethanol, heating and refluxing for extraction twice, the extraction time is 1.4-1.6 hours and 0.8-1.2 hours respectively, filtering and concentrating under reduced pressure to recover ethanol to obtain an alcohol extract; (2) adding 8-12 times the weight of the residue into water and boiling twice, with the extraction time being 1.4-1.6 hours and 0.8-1.2 hours respectively, combining the filtrates to obtain a water extract, combining the water extract with the alcohol extract, and freeze-drying the mixture using a freeze dryer to obtain an extract; (3) The extract is then compressed into Chinese medicine tablets using a Chinese medicine tablet press.

2. The Sijunzisan compressed tablet according to claim 1, characterized in that In step (1), 10 times the weight of 55 v / v% ethanol was added.

3. The Sijunzisan compressed tablet according to claim 1, characterized in that In step (3), the volume of the tablet is further reduced to less than one third of the original volume.

4. The Sijunzisan compressed tablet according to claim 1, characterized in that The method further comprises the following steps after step (3): step (4) wrapping the Chinese medicine tablets into a columnar body by using edible glutinous rice paper to form a Chinese medicine stick.

5. The quality inspection method of the Sijunzisan compressed tablets according to any one of claims 1 to 4, comprising the following steps: (1) extracting with chloroform, ethyl acetate, and n-butanol in sequence to obtain extracts from four different extraction parts, evaporating the organic solvents and freeze-drying them to obtain the chloroform part of S5, the ethyl acetate part of S6, the n-butanol part of S7, and the remaining part of S8; (2) Weigh the chloroform fraction of S5, the ethyl acetate fraction of S6, the n-butanol fraction of S7, and the remaining fraction of S8 into EP tubes respectively, add pre-cooled methanol-water solution (1:1, v / v), and mix thoroughly to re-dissolve the samples; the samples are centrifuged and the supernatant is taken for instrumental analysis; (3) Analyze the samples using high performance liquid chromatography-mass spectrometry.

6. The quality detection method according to claim 5, wherein the conditions of the HPLC are as follows: during the entire analysis process, the sample is placed in an automatic sampler at 4°C, and a C18 chromatographic column is used; wherein the injection volume is 4 μL, the column temperature is 40°C, and the flow rate is 300 uL / min; the chromatographic mobile phase A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution; The chromatographic gradient elution program was as follows: 0-1 min, B was maintained at 2%; 1-5 min, B changed linearly from 2% to 48%; 5-7 min, B changed linearly from 48% to 80%; 7-11min, B changes linearly from 80% to 100%; 11---13min, B was maintained at 100%; 13---13.01min, B changed linearly from 100% to 2%; 13.01---15min, B was maintained at 2%.

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