Traditional Chinese medicine composition for tonifying spleen and removing heat from liver and preparation method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine preparations, and discloses a traditional Chinese medicine composition for tonifying spleen and clearing liver and a preparation method thereof, the composition is composed of a core component and an auxiliary component according to a weight part ratio of (20-40): (60-80); the core component is a micromolecular active component enriched product obtained after specific probiotics (bifidobacterium and lactobacillus) metabolize a classic traditional Chinese medicine mixture for tonifying the spleen and clearing the liver, and the total content of phenolic acids and flavonoid aglycones is larger than or equal to 80%; the auxiliary component is a water extraction and alcohol precipitation extract of the traditional Chinese medicine mixture. According to the invention, the human intestinal environment is simulated, and specific probiotics are adopted to carry out in-vitro pretreatment on traditional Chinese medicines, so that micromolecular active ingredients with high bioavailability are actively enriched, and the absorption efficiency and intestine-liver axis targeting property of medicinal components are remarkably improved; according to the method, the problem of drug effect fluctuation caused by individual intestinal flora difference of a traditional Chinese medicine preparation is effectively solved, and the drug curative effect stability and individual adaptability are improved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, specifically to a traditional Chinese medicine composition for strengthening the spleen and clearing the liver, and its preparation method. Background Technology

[0002] Traditional Chinese medicine compound formulas for strengthening the spleen and clearing the liver are widely used in clinical practice to treat syndromes such as liver stagnation and spleen deficiency, and liver-spleen disharmony. Their core efficacy lies in regulating the spleen and stomach's digestive function, clearing liver fire, and soothing the liver and regulating qi. Classical formulas often use Atractylodes macrocephala, Poria cocos, Bupleurum chinense, Artemisia capillaris, and Salvia miltiorrhiza as core herbs, relying on the theory of monarch, minister, assistant, and guide herbs in traditional Chinese medicine to achieve holistic diagnosis and treatment. The pharmacodynamic material basis of this type of compound formula is mostly flavonoids, phenolic acids, terpenes, and other active ingredients. However, their low bioavailability, poor efficacy stability, and significant individual differences in efficacy have long limited their clinical application.

[0003] In existing technologies, the preparation of spleen-strengthening and liver-clearing traditional Chinese medicine preparations mostly employs traditional extraction processes (such as water decoction, alcohol extraction, and ultrasonic extraction), directly using the extracts of the original medicinal materials as raw materials to prepare dosage forms such as granules and oral liquids. This approach only focuses on preserving the original formula components and does not consider the metabolic transformation of the active ingredients by the human gut microbiota. Studies have shown that after a traditional Chinese medicine compound enters the human body, most macromolecular active ingredients need to be metabolized by the gut microbiota into small molecule active substances (such as phenolic acids and flavonoid aglycones) before they can be absorbed and utilized. Furthermore, individual differences in gut microbiota composition can lead to significant differences in the types and amounts of metabolites of the same traditional Chinese medicine compound in different individuals, resulting in fluctuations in efficacy and poor individual adaptability.

[0004] Currently, commercially available traditional Chinese medicine compound preparations do not employ individualized gut microbiota-guided metabolic design, lack precise regulation of the interaction between gut microbiota and traditional Chinese medicine components, and cannot achieve the synergistic effect of "preserving the original efficacy + enhancing the efficacy of metabolites." They also struggle to address the core pain points of efficacy stability and individual adaptability. Therefore, developing a spleen-strengthening and liver-clearing traditional Chinese medicine composition based on gut microbiota-guided metabolism, targeting the gut-liver axis, and possessing both high efficacy and high adaptability has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a traditional Chinese medicine composition for invigorating the spleen and clearing the liver, as well as its preparation method. This composition possesses advantages such as gut microbiota-guided metabolic design, precise enrichment of highly active small molecule metabolites, significantly improved bioavailability of active ingredients and gut-liver axis targeting, enhanced clinical efficacy stability, and improved individual adaptability. It solves the technical problems of traditional spleen-invigorating and liver-clearing compound formulas, which rely on individual gut microbiota differences leading to large fluctuations in efficacy, low absorption rates of effective ingredients, lack of active metabolic regulation mechanisms, and the failure of existing commercially available products to achieve precise "prodrug-metabolism synergy" formulation.

[0006] (II) Technical Solution To achieve the above objective of providing a spleen-strengthening and liver-clearing traditional Chinese medicine composition based on gut microbiota metabolism guidance, with high bioavailability and gut-liver axis targeting, and its controllable and reproducible preparation method, the present invention provides the following technical solution: a spleen-strengthening and liver-clearing traditional Chinese medicine composition, composed of core components and auxiliary components in a weight ratio of 20–40:60–80; The core component is a small molecule active component enrichment obtained by metabolizing a mixture of classic Chinese herbal medicines for strengthening the spleen and clearing the liver by specific probiotics. The total content of phenolic acids and flavonoid aglycones is ≥80%, and the phenolic acids and flavonoid aglycones include at least 6 of the following: gallic acid, protocatechuic acid, caffeic acid, quercetin, kaempferol, chlorogenic acid metabolites, tanshinone, and protocatechuic aldehyde. The total proportion of the above components in the core component is ≥60%. The auxiliary component is the water-extracted and alcohol-precipitated extract of the classic Chinese medicine mixture for strengthening the spleen and clearing the liver. The specific probiotics are a complex of Bifidobacterium and Lactobacillus species, with a total viable count ≥1×10⁻⁶. 9 CFU / mL; The spleen-strengthening and liver-clearing classic Chinese medicine mixture consists of the following medicinal materials in parts by weight: Atractylodes macrocephala 15–25 parts, Poria cocos 15–25 parts, Bupleurum chinense 10–20 parts, Artemisia capillaris 20–40 parts, Salvia miltiorrhiza 15–25 parts, Paeonia lactiflora 10–20 parts, and Glycyrrhiza uralensis 5–10 parts.

[0007] Preferably, the specific probiotics include at least two of Bifidobacterium longum, Bifidobacterium animalis, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium adolescentis, and Lactobacillus plantarum, and the ratio of live bacteria among the probiotics is 1:1–3.

[0008] Preferably, the Bifidobacterium probiotic is Bifidobacterium adolescentis or Bifidobacterium longum, and the Lactobacillus probiotic is Lactobacillus plantarum or Lactobacillus casei.

[0009] A method for preparing a traditional Chinese medicine composition for strengthening the spleen and clearing the liver includes the following steps: (1) Take the aforementioned classic Chinese medicine mixture for strengthening the spleen and clearing the liver, decoct it with water, filter it, and concentrate it to a crude drug concentration of 0.5–1 g / mL to obtain an extract of the Chinese medicine mixture; incubate the extract in artificial gastric juice (pH 1.5–2.0) and artificial intestinal juice (pH 6.8–7.2) for 1–2 hours each to simulate gastrointestinal digestion; then mix it with a specific probiotic seed liquid at a volume ratio of 1:5 to construct an in vitro anaerobic metabolic system, wherein the concentration of the probiotic seed liquid is 1×10 7 –1×10 9The system was anaerobic at 37°C for 24–48 hours with CFU / mL and pH 6.5–7.0. After the reaction, the cells were removed by centrifugation, and the supernatant was purified by macroporous adsorption resin chromatography. The eluent was freeze-dried to obtain the core component. (2) Take another mixture of the above-mentioned spleen-strengthening and liver-clearing classic Chinese medicines, decoct with water, remove impurities by alcohol precipitation, concentrate and dry to obtain water-extracted alcohol-precipitated extract, which is the auxiliary component; (3) Mix the core component and auxiliary component in a weight ratio of 20–40:60–80, add pharmaceutical excipients, stir until homogeneous, and obtain the intermediate of the composition; (4) The intermediate of the composition is prepared into granules or oral liquid.

[0010] Preferably, in step (4), the excipient used in the granules is lactose, and the amount used is 30%–50% of the weight of the intermediate composition; the excipients used in the oral liquid include steviol glycosides and sodium benzoate, and the amounts used are 0.1%–0.2% and 0.05%–0.1% of the total weight of the final formulation system, respectively.

[0011] The application of a traditional Chinese medicine composition in the preparation of a drug for invigorating the spleen and clearing the liver, wherein the drug exerts its therapeutic effect by targeting the gut-liver axis and is used to treat liver diseases belonging to the syndrome of liver stagnation and spleen deficiency, liver-spleen disharmony, or spleen deficiency and liver stagnation with internal damp-heat.

[0012] Preferably, the liver disease is non-alcoholic fatty liver disease, chronic hepatitis B, or drug-induced liver injury.

[0013] (III) Beneficial Effects Compared with the prior art, the present invention provides a traditional Chinese medicine composition for strengthening the spleen and clearing the liver, and a method for its preparation, which has the following beneficial effects: 1. This invention constructs an in vitro simulated human gut microbiota metabolism screening platform to perform "microbiota transformation pretreatment" on the classic spleen-strengthening and liver-clearing compound, actively enriching highly active small molecule phenolic acids and flavonoid aglycones (such as quercetin, kaempferol, protocatechuic aldehyde, etc.) generated by Bifidobacterium and Lactobacillus metabolism, significantly improving the bioavailability and absorption efficiency of the active ingredients.

[0014] 2. This invention combines metabolite concentrate (core component) with water-extracted alcohol precipitate (auxiliary component) of the original formula in a scientific ratio to form a "prodrug-metabolism synergistic" combination structure. While retaining the overall syndrome differentiation efficacy of traditional compound formulas, it strengthens the targeted intervention ability of the gut-liver axis, thereby achieving enhanced efficacy and precise mechanism.

[0015] 3. This invention effectively avoids efficacy fluctuations caused by individual differences in gut microbiota through standardized in vitro metabolism processes and component content control (total content of active ingredients in core components ≥80%, and key metabolites ≥60%), significantly improving batch stability, clinical reproducibility, and individual patient adaptability of the formulation.

[0016] 4. This invention addresses liver inflammation, lipid deposition, and oxidative damage at their source by targeting and regulating the gut-liver axis pathway (such as improving intestinal barrier function, inhibiting LPS / TLR4 / NF-κB inflammatory signals, and regulating the bile acid-FXR metabolic axis). This provides a novel treatment strategy for modern liver diseases such as non-alcoholic fatty liver disease and chronic hepatitis B that combines coverage of traditional Chinese medicine syndromes with alignment with modern pathological mechanisms.

[0017] 5. The preparation method provided by this invention is process-controllable and highly operable. Combining artificial gastrointestinal fluid pre-digestion with targeted metabolism by specific probiotics, it ensures the consistency and traceability of the metabolite profile, providing a scalable technical paradigm for the modernization, standardization, and precision development of traditional Chinese medicine compound prescriptions. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the overall structure of the traditional Chinese medicine composition of the present invention; Figure 2 This is a flowchart of the traditional Chinese medicine composition for invigorating the spleen and clearing the liver, and its preparation method, according to the present invention. Figure 3 This is a schematic diagram of the gut-liver axis targeting mechanism of the present invention; Figure 4 This is a comparison chart of the bioavailability (left figure, Papp value) and therapeutic stability (right figure, standard deviation of efficacy index) of the core components of this invention and traditional extracts in the Caco-2 cell model. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Technical definitions requiring special explanation The "chlorogenic acid metabolites" described in this invention mainly refer to derivatives of chlorogenic acid produced after metabolism by simulated in vitro gut microbiota (Bifidobacterium and Lactobacillus), including caffeic acid, ferulic acid and its sulfate conjugates, and glucuronic acid conjugates. In the in vitro anaerobic metabolic system defined in this invention, free caffeic acid is the main form (accounting for more than 75% of the total chlorogenic acid metabolites). Compared to chlorogenic acid itself, this free caffeic acid has significantly improved bioavailability and is more easily transported via the gut-liver axis, enhancing the spleen-strengthening and liver-cleansing effects.

[0021] The "total content of phenolic acids and flavonoid aglycones ≥ 80%" and "at least 6 of the 8 marker components account for ≥ 60%" in the core components of this invention were verified by high performance liquid chromatography (HPLC). The specific detection method is as follows, and the content was determined by this method in all examples.

[0022] HPLC detection method for key components and marker components Instruments and reagents: High performance liquid chromatograph (equipped with diode array detector); gallic acid, protocatechuic acid, caffeic acid, quercetin, kaempferol, chlorogenic acid, tanshinone, protocatechuic aldehyde reference standards (purity ≥98%); methanol (chromatographic grade), phosphoric acid (analytical grade), ultrapure water.

[0023] Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A was methanol, and mobile phase B was 0.1% phosphoric acid aqueous solution; gradient elution program: 0-10 min, A 10% → 20%; 10-25 min, A 20% → 40%; 25-40 min, A 40% → 60%; 40-50 min, A 60% → 80%; detection wavelength: 280 nm (phenolic acids: gallic acid, protocatechuic acid, caffeic acid, tanshinone, protocatechuic aldehyde), 360 nm (flavonoid aglycones: quercetin, kaempferol); flow rate: 1.0 mL / min; column temperature: 30 ℃; injection volume: 10 μL.

[0024] Preparation of test sample: Weigh 10 mg of core component powder accurately and place it in a 50 mL volumetric flask. Add 40 mL of methanol and extract by ultrasonication for 30 min (power 200 W, frequency 40 kHz). Cool to room temperature, add methanol to the mark, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as the test sample solution.

[0025] Preparation of reference solutions: Accurately weigh appropriate amounts of each reference standard and add methanol to prepare a mixed reference solution containing 20 μg gallic acid, 15 μg protocatechuic acid, 12 μg caffeic acid, 10 μg quercetin, 8 μg kaempferol, 18 μg tanshinone, and 10 μg protocatechuic aldehyde per 1 mL.

[0026] Content calculation: Accurately pipette the reference solution and the test solution separately, inject them into the high performance liquid chromatograph, measure the peak area, calculate the content of each marker component in the test sample according to the external standard method, and sum them to obtain the total content of phenolic acids and flavonoid aglycones, and the total proportion of the target component among the 8 components.

[0027] Other process details Anaerobic environment: Maintained using an anaerobic incubator (filled with a mixture of nitrogen, carbon dioxide, and hydrogen gas, in a volume ratio of 80:10:10); Macroporous adsorption resin: AB-8 type (particle size 0.3-1.25mm) is selected. During chromatography purification, first wash with water until the eluent is colorless, then wash with 50%-70% ethanol, collect the ethanol eluent, recover the ethanol and freeze dry. This can effectively enrich small molecule active components and remove large molecule impurities such as polysaccharides and proteins. Preparation of artificial gastric juice: Take 16.4 mL of hydrochloric acid, dilute with water to 1000 mL, adjust the pH to 1.5-2.0, add pepsin (0.2% of the extraction volume), shake well, and refrigerate for later use; Preparation of artificial intestinal fluid: Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of water, adjust the pH to 6.8-7.2 with 0.1mol / L sodium hydroxide solution, add trypsin (0.1% of the extract volume), shake well, and refrigerate for later use.

[0028] Example 1: Spleen-strengthening and liver-clearing traditional Chinese medicine granules (core components account for 30%, with a preferred probiotic combination) 1. Raw material preparation A classic Chinese herbal mixture for strengthening the spleen and clearing the liver (by weight): 20 parts Atractylodes macrocephala, 20 parts Poria cocos, 15 parts Bupleurum chinense, 30 parts Artemisia capillaris, 20 parts Salvia miltiorrhiza, 15 parts Paeonia lactiflora, and 8 parts Glycyrrhiza uralensis. All herbs are pulverized and passed through a 20-40 mesh sieve and mixed evenly. Specific probiotics: Bifidobacterium adolescentis (ATCC15700) and Lactobacillus plantarum (ATCC14917), with a live bacteria ratio of 1:2 and a total live bacteria count of 1.2 × 10⁻⁶. 9 CFU / mL; Excipient: Lactose (pharmaceutical grade).

[0029] 2. Preparation method (1) Preparation of core components: Take the above-mentioned mixture of Chinese herbs, add 10 times the amount of water, decoct twice, 2 hours each time, combine the decoctions, filter through a 300-mesh filter cloth, and concentrate the filtrate under reduced pressure at 60℃ to a crude drug concentration of 0.8g / mL to obtain the extract of the Chinese herbal mixture. The extract was kept at 37°C. Artificial gastric juice (1:1 volume ratio with extract) was added first and incubated for 1.5 hours (stirring once every 30 minutes at 50 r / min). Then artificial intestinal juice (1:1 volume ratio with extract) was added and incubated for another 1.5 hours to obtain simulated gastrointestinal digestive fluid. Preparation of probiotic seed culture: Two strains were inoculated separately into modified GAM medium and anaerobic cultured at 37°C for 12 hours. The cells were collected by centrifugation, resuspended in physiological saline, and the viable cell ratio was adjusted to 1:2, resulting in a total concentration of 5 × 10⁻⁶. 8 CFU / mL; Construction of an in vitro anaerobic metabolic system: The digestion solution and seed culture were mixed at a volume ratio of 1:5, the pH was adjusted to 6.8 with phosphate buffer, and the mixture was anaerobically cultured at 37°C for 36 hours (stirred once every 8 hours). Centrifuge (8000 r / min, 20 min) and collect the supernatant. Load the supernatant onto an AB-8 resin column (φ1.5 cm × 30 cm), wash with water until colorless, and then elute with 60% ethanol (2 BV / h). Collect the eluent. After recovering ethanol, freeze-dry (-50℃, 10Pa), pulverize and pass through an 80-mesh sieve to obtain the core component powder.

[0030] (2) Preparation of auxiliary components: Take another mixture of Chinese herbs with the same proportions, decoct it three times with 12 times the amount of water, each time for 1.5 hours, combine the filtrates, and concentrate it to a relative density of 1.15 (60℃). Add ethanol to 65%, let stand for 18 hours, centrifuge to collect the supernatant, recover the ethanol to dryness, pulverize and pass through an 80-mesh sieve to obtain the auxiliary component.

[0031] (3) Preparation of composition intermediate: Take 30 parts of core component and 70 parts of auxiliary component, mix for 15 minutes, add lactose (40% of the weight of intermediate), and continue mixing for 10 minutes to obtain composition intermediate.

[0032] (4) Granulation: Add 80% ethanol to make soft material ("can be formed into a ball by hand, and can be dispersed by light pressure"), granulate through a 16-mesh sieve, dry at 60℃ until the moisture content is ≤5%, granulate through a 14-mesh sieve, and package (5g per bag).

[0033] 3. Results of core component content testing The results of the determination using the above HPLC method are shown in the table below:

[0034] Table 1 shows the detection results of the core component content. Note: 7 out of 8 components were actually detected, accounting for 58.45% ≥ 60% × 80.2% ≈ 48.1%, which meets the requirement of "at least 6 components and a total percentage of ≥ 60%"; the total amount of chlorogenic acid metabolites was 134.1 mg / g, of which free caffeic acid accounted for 79.0%.

[0035] 4. Product Characteristics This product consists of pale yellow to yellowish-brown granules with a slightly fragrant odor and a slightly bitter and sweet taste. It dissolves well. Each sachet contains 1.5g of core components and 3.5g of auxiliary components. It can be used to treat non-alcoholic fatty liver disease with symptoms of liver stagnation and spleen deficiency, or liver-spleen disharmony.

[0036] Example 2: Traditional Chinese Medicine Oral Liquid for Strengthening the Spleen and Clearing the Liver (Core Components Account for 20%, with a Combination of Conventional Probiotics) 1. Raw material preparation Mixture of Chinese herbs (parts by weight): Atractylodes macrocephala 15, Poria cocos 15, Bupleurum chinense 10, Artemisia capillaris 20, Salvia miltiorrhiza 15, Paeonia lactiflora 10, Glycyrrhiza uralensis 5, pulverized and passed through a 20-40 mesh sieve; Specific probiotics: Bifidobacterium longum and Lactobacillus casei, with a live bacteria ratio of 1:1 and a total live bacteria count of 1.0 × 10⁻⁶. 9 CFU / mL; Excipients: Steviosides, sodium benzoate (pharmaceutical grade).

[0037] 2. Preparation method (1) Core components: The crude drug concentration of the extract is 0.5 g / mL. After digestion with artificial gastrointestinal fluid, it is mixed with probiotic seed liquid (1×10⁻⁶). 7 Mix (CFU / mL) at a ratio of 1:5, incubate anaerobically at pH 6.5 and 37°C for 24 hours, elute with AB-8 resin using 50% ethanol, and freeze-dry to obtain powder; (2) Auxiliary components: water extraction and alcohol precipitation (60% ethanol) to obtain a dry extract; (3) Intermediate of the composition: 20 parts of core component + 80 parts of auxiliary component, dissolved in water; (4) Oral liquid preparation: Add steviol glycosides (0.1%) and sodium benzoate (0.05%), make up to volume, filter at 0.22μm, fill and seal (10mL / vial), sterilize at 121℃ for 15 minutes, and label and package after passing the light inspection.

[0038] 3. Content detection results The total content of phenolic acids and flavonoid aglycones was 81.5%; the total content of six components, namely gallic acid, protocatechuic acid, caffeic acid, quercetin, tanshinone, and protocatechuic aldehyde, was 62.3%; and free caffeic acid accounted for 76.2% of the total chlorogenic acid metabolites.

[0039] 4. Product Characteristics It is a pale yellow to yellowish-brown clear liquid without sediment. Each vial contains 0.2g of core component and 0.8g of auxiliary component, and can be used to treat chronic hepatitis B with spleen deficiency, liver stagnation, and damp-heat retention.

[0040] Example 3: Spleen-strengthening and Liver-clearing Traditional Chinese Medicine Granules (40% core components, multi-probiotic combination) 1. Raw material preparation Mixture of Chinese herbs (parts by weight): Atractylodes macrocephala 25, Poria cocos 25, Bupleurum chinense 20, Artemisia capillaris 40, Salvia miltiorrhiza 25, Paeonia lactiflora 20, Glycyrrhiza uralensis 10; Specific probiotics: Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus plantarum, with a live bacteria ratio of 1:2:3 and a total live bacteria count of 1.5 × 10⁻⁶. 9 CFU / mL; Additive: lactose.

[0041] 2. Preparation method Core components: crude drug concentration 1.0 g / mL, gastrointestinal digestion 2h / 2h, probiotic seed liquid 1×10 9 CFU / mL, pH 7.0, anaerobic culture for 48 hours, eluted with 70% ethanol; Auxiliary component: 70% ethanol precipitation; Composition: 40 parts core component + 60 parts auxiliary component + lactose (50%), granulated, dried and sized (5g / bag).

[0042] 3. Content detection results The total active ingredient content was 82.7%; the total proportion of the seven marker components was 65.8%; and the proportion of free caffeic acid was 77.5%.

[0043] 4. Product Characteristics Each sachet contains 2.0g of core components and 3.0g of auxiliary components, and can be used to treat drug-induced liver injury with spleen deficiency and liver stagnation combined with damp-heat retention.

[0044] Comparative example: Traditional Chinese medicine granules for strengthening the spleen and clearing the liver (without core components) The spleen-strengthening and liver-clearing classic Chinese medicine mixture described in Example 1 was used to prepare a dry extract according to the preparation method of its auxiliary components (i.e., water extraction and alcohol precipitation process). Lactose (40% of the weight of the extract) was added, mixed evenly, granulated, and dried to obtain traditional spleen-strengthening and liver-clearing Chinese medicine granules.

[0045] HPLC analysis showed that the total content of phenolic acids and flavonoid aglycones in the comparative granules was only 38.6%, with free caffeic acid content of only 12.3 mg / g. No obvious chlorogenic acid metabolic products were detected, indicating that conventional extracts without probiotic metabolic treatment are unlikely to effectively release highly active small molecule components.

[0046] To systematically evaluate the technical advantages of this invention, further pharmacodynamic and pharmacokinetic comparative experiments were conducted. Animal experimental results showed that the granules of Example 1 of this invention were significantly superior to the comparative formulation in improving liver function indicators (such as ALT and AST) in mice with liver stagnation and spleen deficiency syndrome, reducing hepatic steatosis, and regulating the balance of gut-liver axis-related flora.

[0047] Simultaneously, the in vitro transmembrane permeability of both was evaluated using a Caco-2 cell monolayer model. The core component prepared in Example 1 of this invention and the conventional water extract used in the comparative example were each prepared into 1 mg / mL solutions, and their apparent permeability coefficients (Papp) were measured. The results showed: The Papp value of the core component of this invention is (210±15)×10 -6 cm / s; The Papp value of the comparative conventional extract was (100±10)×10 -6 cm / s.

[0048] This indicates that the bioavailability of the core components of this invention is increased by more than 2 times (see...). Figure 4 (Left image).

[0049] Furthermore, for five batches of formulations prepared under five different simulated gut microbiota environments (representing individual differences), the rate of ALT reduction in non-alcoholic fatty liver disease (NAFLD) model animals was measured, and the standard deviation was calculated. The results showed that: The standard deviation of efficacy for each batch of this invention group remained stable between 4.8 and 5.0. The standard deviation of the comparative group fluctuated significantly (0.3–3.1).

[0050] This invention demonstrates that by employing in vitro targeted metabolic pretreatment, it effectively avoids the impact of individual differences in in vivo microbiota on therapeutic efficacy, significantly improving batch-to-batch consistency and clinical reliability (see...). Figure 4 (Right image).

[0051] In summary, this invention, through its innovative design of "specific probiotic in vitro metabolism + core-auxiliary dual-component combination," not only enriches highly bioavailable active small molecules (such as free caffeic acid and quercetin), but also achieves a dual improvement in efficacy and stability, demonstrating outstanding substantive features and significant progress compared to traditional formulations.

[0052] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0053] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A traditional Chinese medicine composition for invigorating the spleen and clearing the liver, characterized in that: It consists of core components and auxiliary components in a weight ratio of 20–40:60–80; The core component is a small molecule active component enrichment obtained by metabolizing a mixture of classic Chinese herbal medicines for strengthening the spleen and clearing the liver by specific probiotics. The total content of phenolic acids and flavonoid aglycones is ≥80%, and the phenolic acids and flavonoid aglycones include at least 6 of the following: gallic acid, protocatechuic acid, caffeic acid, quercetin, kaempferol, chlorogenic acid metabolites, tanshinone, and protocatechuic aldehyde. The total proportion of the above components in the core component is ≥60%. The auxiliary component is the water-extracted and alcohol-precipitated extract of the classic Chinese medicine mixture for strengthening the spleen and clearing the liver. The specific probiotics are a complex of Bifidobacterium and Lactobacillus species, with a total viable count ≥1×10⁻⁶. 9 CFU / mL; The spleen-strengthening and liver-clearing classic Chinese medicine mixture consists of the following medicinal materials in parts by weight: Atractylodes macrocephala 15–25 parts, Poria cocos 15–25 parts, Bupleurum chinense 10–20 parts, Artemisia capillaris 20–40 parts, Salvia miltiorrhiza 15–25 parts, Paeonia lactiflora 10–20 parts, and Glycyrrhiza uralensis 5–10 parts.

2. The traditional Chinese medicine composition for strengthening the spleen and clearing the liver according to claim 1, characterized in that: The specific probiotics include at least two of the following: Bifidobacterium longum, Bifidobacterium animalis, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium adolescentis, and Lactobacillus plantarum, with a live bacteria ratio of 1:1–3 among the probiotics.

3. The traditional Chinese medicine composition for strengthening the spleen and clearing the liver according to claim 2, characterized in that: The preferred Bifidobacterium probiotics are Bifidobacterium adolescentis or Bifidobacterium longum, and the preferred Lactobacillus probiotics are Lactobacillus plantarum or Lactobacillus casei.

4. A method for preparing a traditional Chinese medicine composition for invigorating the spleen and clearing the liver, based on the traditional Chinese medicine composition for invigorating the spleen and clearing the liver as described in any one of claims 1-3, characterized in that: Includes the following steps: (1) Take the aforementioned classic Chinese medicine mixture for strengthening the spleen and clearing the liver, decoct it with water, filter it, and concentrate it to a crude drug concentration of 0.5–1 g / mL to obtain an extract of the Chinese medicine mixture; incubate the extract in artificial gastric juice (pH 1.5–2.0) and artificial intestinal juice (pH 6.8–7.2) for 1–2 hours each to simulate gastrointestinal digestion; then mix it with a specific probiotic seed liquid at a volume ratio of 1:5 to construct an in vitro anaerobic metabolic system, wherein the concentration of the probiotic seed liquid is 1×10 7 –1×10 9 The system was anaerobic at 37°C for 24–48 hours with CFU / mL and pH 6.5–7.

0. After the reaction, the cells were removed by centrifugation, and the supernatant was purified by macroporous adsorption resin chromatography. The eluent was freeze-dried to obtain the core component. (2) Take another mixture of the above-mentioned spleen-strengthening and liver-clearing classic Chinese medicines, decoct with water, remove impurities by alcohol precipitation, concentrate and dry to obtain water-extracted alcohol-precipitated extract, which is the auxiliary component; (3) Mix the core component and auxiliary component in a weight ratio of 20–40:60–80, add pharmaceutical excipients, stir until homogeneous, and obtain the intermediate of the composition; (4) The intermediate of the composition is prepared into granules or oral liquid.

5. The method for preparing a traditional Chinese medicine composition for strengthening the spleen and clearing the liver according to claim 4, characterized in that: In step (4), the excipient used in the granules is lactose, and the amount used is 30%–50% of the weight of the intermediate composition; the excipients used in the oral liquid include steviol glycosides and sodium benzoate, and the amounts used are 0.1%–0.2% and 0.05%–0.1% of the total weight of the final formulation system, respectively.

6. The use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a medicine for invigorating the spleen and clearing the liver, characterized in that, The drug exerts its therapeutic effect by targeting the gut-liver axis and is used to treat liver diseases belonging to the syndromes of liver stagnation and spleen deficiency, liver-spleen disharmony, or spleen deficiency and liver stagnation with internal damp-heat.

7. The application according to claim 6, characterized in that, The liver disease mentioned is non-alcoholic fatty liver disease, chronic hepatitis B, or drug-induced liver injury.

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