A traditional Chinese medicine composition and its application, and a method for preparing a preparation containing the same

Abstract

The present invention belongs to the field of traditional Chinese medicine technology and discloses a Chinese medicine composition, its application, and a preparation method of a preparation containing the same. The preparation comprises the following steps: mixing 400-550 parts of gentian and 590-840 parts of red peony root, extracting twice with 70% ethanol, and concentrating the filtrate under reduced pressure to obtain a thick paste; adding silicon dioxide to the thick paste, drying, and crushing to obtain a fine powder; mixing 30-40 parts of tangerine peel oil and 25-35 parts of mentha oil, adding silicon dioxide, adsorbing completely, and sieving to obtain an adsorbate; mixing the fine powder and the adsorbate to form capsules or tablets. The Chinese medicine composition of the present invention is used to improve cholestatic liver damage and has good effects in soothing the liver and promoting bile secretion, relieving heat and jaundice, and preventing and treating chronic cholestatic liver disease.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and more particularly to a traditional Chinese medicine capsule and tablet for treating cholestatic liver disease and a preparation method thereof. Background Art

[0002] Cholestasis occurs in approximately 35% of patients with chronic liver disease. Patients often experience symptoms such as jaundice, yellow urine, itching, and lipid metabolism disorders. Cholestasis can occur in people of all ages, and its incidence tends to increase with age. Studies have shown that cholestasis can be caused by a variety of factors, including drug-induced liver injury, hereditary conditions (progressive familial intrahepatic cholestasis), and diseases (cholelithiasis, sepsis, and cholangitis). Persistent cholestasis can progress to liver fibrosis, cirrhosis, and even liver cancer. The goal of cholestasis treatment is to reduce the retention of bile acids and other bile components in hepatocytes. Currently, drug treatments for cholestasis are very limited, with ursodeoxycholic acid (UDCA) being the first-line drug in clinical practice. UDCA (Ultra-Derived Cholangitis) (UDCA) has a poor response in 40% of patients with primary biliary cholangitis (PBC). Its efficacy is limited to the early stages of PBC, and high-dose UDCA can exacerbate the condition and increase the risk of cirrhosis and esophageal varices. For patients who do not respond well to UDCA, another cholestatic drug recently approved by the US Food and Drug Administration is obeticholic acid (OCA). OCA can significantly improve biochemical markers, slow disease progression, and improve survival. However, OCA treatment is associated with severe side effects such as pruritus and abdominal pain. Excessive use of OCA can worsen liver damage, and its high cost limits widespread clinical use. Therefore, identifying new targets and clinically effective drugs with minimal side effects is crucial for the treatment of hepatostatic liver diseases.

[0003] In recent years, Traditional Chinese Medicine (TCM), alone or in combination with UDCA, has demonstrated significant progress in the treatment of cholestatic liver disease, demonstrating its unique advantages in improving biochemical response rates, ameliorating clinical symptoms, combating liver fibrosis, promoting anti-inflammatory and choleretic effects, and regulating immune imbalance. While fundamental research has provided a robust theoretical basis for TCM treatment of cholestatic liver disease, numerous challenges remain, including the lack of robust and effective animal and cell models to elucidate the efficacy and mechanisms of TCM treatment for cholestatic liver disease, the lack of clarity regarding the primary active ingredients and mechanisms of action of TCM for this purpose, and the prevalence of clinical reports primarily based on decoctions, inconsistent efficacy standards, small sample sizes, and limited availability of marketed TCM patent medicines. Therefore, the urgent need for those skilled in the art to address the problem of providing a TCM patent medicine formulation with defined chemical composition, controlled quality, safety, and efficacy for the prevention and treatment of cholestatic liver disease (gallbladder stagnation and heat syndrome) to reduce patient medication costs and improve medication compliance has become a pressing technical challenge. Summary of the Invention

[0004] In view of this, the present invention provides a Chinese medicine composition for treating cholestatic liver disease, its use, and methods for preparing capsules and tablets containing the same. The present invention also provides a Chinese patent medicine preparation for preventing and treating cholestatic liver disease (gallbladder heat syndrome) by soothing the liver and promoting bile secretion, relieving heat and alleviating jaundice, with the aim of reducing medication costs and improving medication compliance for patients.

[0005] One of the purposes of the present invention is to provide a traditional Chinese medicine composition, which comprises, by weight, 400 to 540 parts of gentian, 590 to 840 parts of red peony root, 30 to 40 parts of tangerine peel oil, and 25 to 35 parts of mentha oil.

[0006] Preferably, the Chinese medicinal components include: 500 parts of gentian, 840 parts of red peony root, 34 parts of tangerine peel oil, and 25 parts of mentha oil.

[0007] Further preferably, the medicinal part of the red peony root of the present invention and the medicinal part of the gentian are the roots and rhizomes. The two medicines need to be extracted twice with 70% ethanol to completely extract the effective ingredients; peppermint oil is a variety included in the 2020 edition of the "Chinese Pharmacopoeia", with a relative density of 0.888~0.908, an optical rotation of -17°~-24°, and a refractive index of 1.456~1.466; the relative density of tangerine peel oil is 0.830~0.843, the optical rotation is +80°~+90°, the refractive index is 1.460~1.476, and the limonene content is ≥85%.

[0008] A second object of the present invention is to provide a method for preparing a capsule preparation or tablet for treating cholestatic liver disease, comprising the following steps:

[0009] (1) Weighing Chinese medicinal raw materials according to the ratio of the Chinese medicinal composition and setting aside;

[0010] (2) Mix gentian and red peony root, extract twice with 70% ethanol, filter, and concentrate the filtrate under reduced pressure to obtain a thick paste for later use;

[0011] (3) Add silicon dioxide to the thick paste, stir well, vacuum dry, and crush to obtain fine powder 1, which is set aside;

[0012] (4) Mixing tangerine peel oil and peppermint oil, adding silicon dioxide, adsorbing completely, sieving, and obtaining mixture 1 for standby use;

[0013] (5) mixing the fine powder 1 and the mixture 1, granulating, and drying to obtain granules;

[0014] (6) The granules are directly loaded into capsules to obtain capsule preparations; or the granules are mixed with pregelatinized starch and sodium starch glycolate, compressed into tablets, and film-coated to obtain tablets.

[0015] Preferably, in step (2), the ethanol extraction is performed by adding 6-8 times the mass of the mixed Chinese medicinal raw materials, and the decoction and extraction are performed for 1.0 to 1.5 hours each time.

[0016] Preferably, the ethanol extraction is as follows: adding 8 times the mass of ethanol of the mixed Chinese medicinal raw materials for the first time and extracting for 1.5 hours; adding 6 times the mass of ethanol of the mixed Chinese medicinal raw materials for the second time and extracting for 1 hour.

[0017] Preferably, in step (2), the vacuum degree of the reduced pressure concentration is -0.07 to -0.08 MPa, and the relative density of the thick paste measured at 70° C. to 80° C. is 1.20 to 1.25.

[0018] Preferably, in step (3), the vacuum drying temperature is 65 to 70° C., the vacuum degree is -0.06 to -0.08 MPa; the amount of silicon dioxide used is 35 g to 45 g, and the pulverization condition is fine powder.

[0019] Preferably, in step (4), the amount of silicon dioxide added is 18 g to 22 g, and the mixture is passed through a No. 3 sieve.

[0020] Preferably, in step (5), a No. 2 sieve is used for granulation, and the granules are dried at a temperature of 35° C. to 40° C.

[0021] Preferably, in step (6), granulation is performed using a No. 2 sieve, the amount of the pregelatinized starch is 10 g, the amount of sodium starch glycolate is 5 g, and the granules are dried at 35° C. to 40° C.

[0022] It is worth noting that the Chinese medicine capsule preparation disclosed in the present invention is a compound preparation composed of four Chinese medicinal herbs: gentian, red peony root, tangerine peel oil, and peppermint oil. The principle of its formula is as follows: gentian is bitter and cold, enters the liver and gallbladder meridians, and has the functions of clearing heat and drying dampness, purging liver and gallbladder fire, and directly suppressing damp-heat evil toxins. It is an important medicine for treating jaundice, so it is the monarch drug. Red peony root is bitter and slightly cold, enters the liver meridian, clears heat and cools blood, disperses blood stasis and relieves pain. When used together with the monarch drug, it can clear damp-heat from the liver and gallbladder, and dissipate blood stasis and dredge the meridians, and is the minister drug. Peppermint is pungent and cool, enters the liver and lung meridians, and has the functions of soothing the liver and relieving depression, dispelling heat evil, and promoting light and clear upward movement. It helps gentian and red peony root reach the liver and gallbladder meridians, and is an adjuvant. Tangerine peel is bitter, pungent, and warm, enters the spleen and lung meridians, and has the functions of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and preventing gentian and red peony root from damaging the stomach due to their bitter and cold nature. It can also promote qi circulation and relieve depression, and help bile to flow out, and is also an adjuvant. It is used for cholestatic liver disease with gallbladder heat syndrome. Symptoms: Bright yellow body and eyes, pain in the upper abdomen and right flank extending to the shoulders and back, persistent fever, bitter taste in the mouth and dry throat, nausea and vomiting, dark yellow urine, constipation, or even pale stools. Red tongue with a dry yellow coating, and a rapid and wiry pulse.

[0023] A third object of the present invention is to provide a use of a traditional Chinese medicine composition in the preparation of a medicament for treating cholestatic liver disease.

[0024] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a Chinese medicine capsule preparation and tablet for treating cholestatic liver disease, comprising four Chinese herbs: gentian, red peony root, tangerine peel oil, and mentha oil. The Qingdanshugan capsule can improve the basic physiological condition of rats with cholestasis, increase bile volume flow, reduce serum ALT, AST, and γ-GT activity, and TBIL, DBIL, IBIL, and TBA levels, and improve liver pathological changes in rats. It improves cholestatic liver damage and has excellent effects in soothing the liver and promoting bile secretion, relieving heat and alleviating jaundice, and preventing and treating chronic cholestatic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0027] Figure 1 This is the HPLC spectrum of the test sample in Experiment 1.

[0028] Figure 2 This is the HPLC spectrum of the gentiopicroside reference substance in Experiment 1.

[0029] Figure 3 This is the HPLC spectrum of the paeoniflorin reference substance in Experiment 1.

[0030] Figure 4 This is the HPLC spectrum of the paeoniflorin reference substance in Experiment 1.

[0031] Figure 5 This is the HPLC spectrum of the loganine acid reference substance in Experiment 1.

[0032] Figure 6 This is the GC spectrum of the Qingdanshugan Capsule test sample in Experiment 1.

[0033] Figure 7 This is the GC spectrum of the limonene reference substance in Experiment 1.

[0034] Figure 8 This is the GC spectrum of the γ-terpinene reference substance in Experiment 1.

[0035] Figure 9 This is the GC spectrum of the menthol reference substance in Experiment 1.

[0036] Figure 10 This is the GC spectrum of the menthone reference substance in Experiment 1.

[0037] Figure 11 Pathological changes of liver tissues of rats in the normal group, model group, ursodeoxycholic acid group, and capsule preparation group of the present invention in Experiment 2 (HE, 10X) DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] Research has shown that:

[0040] Gentiana scabra extract (GA) can alleviate the abnormal bile accumulation and reflux caused by α-naphthyl isothiocyanate (ANIT) through FXR and its target genes, thereby reducing jaundice and improving liver function. GA pharmacologically stimulates upregulation of FXR expression. Enhanced FXR expression, through SHP, feedback inhibits the expression of the bile acid synthase CYP7αl, reducing bile acid synthesis, activating the transcription of BSEP and MRP2 on the hepatocyte membrane, and accelerating bile acid efflux from hepatocytes to bile canaliculi. It also downregulates NTCP expression, reducing bile acid uptake. Gentiopicroside, a compound found in gentiana scabra, significantly ameliorates liver damage and fibrosis in rats with CCl4-induced liver fibrosis and inhibits hepatocyte apoptosis and oxidative stress. These effects may be related to activation of the Nrf2 antioxidant pathway. Gentiopicroside downregulated serum AST, ALT, TBA, and TBil levels in mice with cholestatic liver injury (P < 0.05) and improved liver lesions. Gentiopicroside upregulated PPAR-α mRNA levels in liver tissue and cells of mice with cholestatic liver injury and decreased mRNA levels of the proinflammatory cytokines NF-κB, TNF-α, and IL-1β. Gentiopicroside upregulated PPAR-α, CYP3A4, and CPT2 protein levels in liver tissue of mice with cholestatic liver injury and decreased CAR protein levels. PPAR-α inhibitors attenuated the upregulation of PPAR-α in cholestatic liver injury cells by gentiopicroside and further affected PPAR-α's regulatory effects on CYP3A4, CPT2, and CAR proteins, thereby exerting an anti-cholestatic effect. Loganinic acid in gentian exerts its anti-liver injury effects through anti-inflammation, modulation of the Nrf2 pathway to inhibit oxidative stress, and inhibition of cell apoptosis.

[0041] Red peony root extract can reduce serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), and total bile acid (TBA) in rats with α-naphthyl isothiocyanate (ANIT)-induced hepatic stasis (P < 0.01), and significantly increase bile flow (P < 0.05). Paeoniflorin activates nuclear factor erythroid-related factor 2 (Nrf2) through a phosphatidylinositol 3-hydroxykinase (PI3k) / protein kinase B (Akt)-dependent pathway, increasing glutathione (GSH) synthesis and significantly inhibiting ANIT-induced changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBL), and γ-glutamyl transpeptidase (γ-GT). Histological examinations revealed that paeoniflorin significantly alleviated liver injury and bile duct proliferation in rats. Paeoniflorin may treat hepatic stasis by intervening in bile acid metabolism targets, promoting the expression of these proteins (MAP2K1, MAPK1, ILBP, ABCB1, and LTA4H). Paeoniflorin can regulate bile acid pool composition, reduce toxic bile acid levels, upregulate FXR and BSEP activity, restore bile acid pool homeostasis, and improve ANIT-induced cholestasis. Paeoniflorin alleviates CCl4-induced liver fibrosis in mice by upregulating heme oxygenase-1, modulating oxidative stress, inflammation, and hepatic stellate cell activation. Paeoniflorin and biloba lactone glycosides increase serum and cerebral cortical β-EP levels and reduce cerebral cortical PGE2 production, resulting in analgesic effects. Biloba lactone glycosides alleviate neuropathic pain by inhibiting spinal cord NLRP3 activation. Biloba lactone glycosides significantly inhibit the proliferation and induce apoptosis of SMMC-7721 hepatocellular carcinoma cells.

[0042] Limonene in tangerine peel oil can help expel gallstones and inhibit cholesterol synthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Limonene not only reduces the levels of related enzymes in bile and serum but also dissolves gallstones in bile, making it effective in treating calculous cholecystitis and chronic cholecystitis caused by cholestasis. γ-terpinene can protect the activity of several antioxidant enzymes, including SOD, GSH-Px, and CAT, restoring cell damage caused by hydrogen peroxide and exerting an antioxidant effect. In vivo, γ-terpinene can protect the activity of several antioxidant enzymes in serum and liver, exerting its antioxidant effect.

[0043] Menthol, primarily containing menthone and menthol, has antispasmodic, analgesic, choleretic, litholytic, antibacterial, antiviral, and anti-inflammatory effects. It exerts its antispasmodic effect by inhibiting calcium ion channels in the smooth muscle of the digestive tract. It also promotes choleresis and lowers total bile cholesterol levels by upregulating the messenger RNA (mRNA) expression of cholesterol 7α-hydroxylase (CYP7A1) and farnesoid X receptor (FXR).

[0044] The above studies show that the multiple active ingredients in the composition of the present invention produce synergistic effects through multiple targets and multiple pathways to treat cholestatic liver diseases.

[0045] Example 1 A method for preparing capsules and tablets for treating cholestatic liver disease:

[0046] Recipe: Gentiana 400g, Red Peony Root 600g, Tangerine Peel Oil 30g, Menthol Oil 35g

[0047] Preparation method:

[0048] (1) Weigh 400 g of gentian, 600 g of red peony root, 30 g of tangerine peel oil, and 35 g of peppermint oil, and set aside;

[0049] (2) extracting the dandelion and red peony root with 70% ethanol twice, filtering, concentrating the filtrate under reduced pressure, and concentrating the medicinal liquid into a thick paste for standby use; wherein

[0050] The first decoction time is 1.5 hours, and the second decoction time is 1 hour; the amount of alcohol added is 6 to 8 times the weight of the medicinal material; the relative density of the concentrated paste of the medicinal liquid measured at 70°C to 80°C is 1.2 to 1.25; and the vacuum degree during reduced pressure concentration is -0.07 to -0.08 MPa;

[0051] (3) adding silicon dioxide to the thick paste obtained in step (2), mixing, drying, and crushing the dry paste into fine powder to obtain fine powder 1, which is set aside;

[0052] The drying temperature of the thick paste is 65-70°C and the vacuum degree is -0.07-0.08Mpa;

[0053] (4) Add appropriate amount of silicon dioxide to tangerine peel oil and peppermint oil for adsorption, sieve the adsorbate, and set aside the adsorbate 1;

[0054] (5) Combine the fine powder 1 from step (3) and the adsorbent 1 from step (4), mix well, granulate, dry, and encapsulate the granules into capsules to prepare 1000 g granules.

[0055] (6) Alternatively, the granules obtained in step (5) are mixed with pregelatinized starch and sodium starch glycolate, pressed into tablets, and film-coated to prepare 1000 tablets.

[0056] Example 2 Preparation method of a capsule for treating cholestatic liver disease:

[0057] Recipe: Gentiana 450g, Red Peony Root 590g, Tangerine Peel Oil 34g, Menthol Oil 25g

[0058] Preparation method:

[0059] (1) Weigh 450 g of gentian, 590 g of red peony root, 34 g of tangerine peel oil, and 25 g of peppermint oil, and set aside;

[0060] (2) extracting the dandelion and red peony root with 70% ethanol twice, filtering, concentrating the filtrate under reduced pressure, and concentrating the medicinal liquid into a thick paste for standby use; wherein,

[0061] The first decoction time is 1.5 hours, and the second decoction time is 1 hour; the amount of alcohol added is 6 to 8 times the weight of the medicinal material; the relative density of the concentrated paste of the medicinal liquid measured at 70°C to 80°C is 1.2 to 1.25; and the vacuum degree during reduced pressure concentration is -0.07 to -0.08 MPa;

[0062] (3) adding silicon dioxide to the thick paste obtained in step (2), mixing, drying, and crushing the dry paste into fine powder to obtain fine powder 1, which is set aside;

[0063] The drying temperature of the thick paste is 65-70°C and the vacuum degree is -0.07-0.08Mpa;

[0064] (4) Add appropriate amount of silicon dioxide to tangerine peel oil and peppermint oil for adsorption, sieve the adsorbate, and set aside the adsorbate 1;

[0065] (5) Combine the fine powder 1 from step (3) and the adsorbent 1 from step (4), mix well, granulate, dry, and encapsulate the granules into capsules to prepare 1000 g granules.

[0066] (6) Alternatively, the granules obtained in step (5) are mixed with pregelatinized starch and sodium starch glycolate, pressed into tablets, and film-coated to prepare 1000 tablets.

[0067] Example 3

[0068] Recipe: Gentiana 500g, Red Peony Root 840g, Tangerine Peel Oil 34g, Menthol Oil 25g

[0069] Preparation method:

[0070] (1) Weigh 500 g of gentian, 840 g of red peony root, 34 g of tangerine peel oil, and 25 g of peppermint oil, and set aside;

[0071] (2) extracting the dandelion and red peony root with 70% ethanol twice, filtering, concentrating the filtrate under reduced pressure, and concentrating the medicinal liquid into a thick paste for standby use; wherein,

[0072] The first decoction time is 1.5 hours, and the second decoction time is 1 hour; the amount of alcohol added is 6 to 8 times the weight of the medicinal material; the relative density of the concentrated paste of the medicinal liquid measured at 70°C to 80°C is 1.2 to 1.25; and the vacuum degree during reduced pressure concentration is -0.07 to -0.08 MPa;

[0073] (3) adding silicon dioxide to the thick paste obtained in step (2), mixing, drying, and crushing the dry paste into fine powder to obtain fine powder 1, which is set aside;

[0074] The drying temperature of the thick paste is 65-70°C and the vacuum degree is -0.07-0.08Mpa;

[0075] (4) Add appropriate amount of silicon dioxide to tangerine peel oil and peppermint oil for adsorption, sieve the adsorbate, and set aside the adsorbate 1;

[0076] (5) Combine the fine powder 1 from step (3) and the adsorbent 1 from step (4), mix well, granulate, dry, and encapsulate the granules into capsules to prepare 1000 g granules.

[0077] (6) Alternatively, the granules obtained in step (5) are mixed with pregelatinized starch and sodium starch glycolate, pressed into tablets, and film-coated to prepare 1000 tablets.

[0078] Example 4

[0079] Recipe: Gentiana 550g, Red Peony Root 800g, Tangerine Peel Oil 40g, Menthol Oil 30g

[0080] Preparation method:

[0081] (1) Weigh 550 g of gentian, 800 g of red peony root, 34 g of tangerine peel oil, and 25 g of peppermint oil, and set aside;

[0082] (2) extracting the dandelion and red peony root with 70% ethanol twice, filtering, concentrating the filtrate under reduced pressure, and concentrating the medicinal liquid into a thick paste for standby use; wherein,

[0083] The first decoction time is 1.5 hours, and the second decoction time is 1 hour; the amount of alcohol added is 6 to 8 times the weight of the medicinal material; the relative density of the concentrated paste of the medicinal liquid measured at 70°C to 80°C is 1.2 to 1.25; and the vacuum degree during reduced pressure concentration is -0.07 to -0.08 MPa;

[0084] (3) adding silicon dioxide to the thick paste obtained in step (2), mixing, drying, and crushing the dry paste into fine powder to obtain fine powder 1, which is set aside;

[0085] The drying temperature of the thick paste is 65-70°C and the vacuum degree is -0.07-0.08Mpa;

[0086] (4) Add appropriate amount of silicon dioxide to tangerine peel oil and peppermint oil for adsorption, sieve the adsorbate, and set aside the adsorbate 1;

[0087] (5) Combine the fine powder 1 from step (3) and the adsorbent 1 from step (4), mix well, granulate, dry, and encapsulate the granules into capsules to prepare 1000 g granules.

[0088] (6) Alternatively, the granules obtained in step (5) are mixed with pregelatinized starch and sodium starch glycolate, pressed into tablets, and film-coated to prepare 1000 tablets.

[0089] The following tests were carried out using the capsule preparation prepared in Example 2.

[0090] Test 1

[0091] Determination of the content of the main components of Qingdanshugan Capsule (tentative name)

[0092] [Content Determination] Paeoniflorin, gentiopicroside, paeoniflorin and loganinic acid were determined according to liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0512).

[0093] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; and a detection wavelength of 230 nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 2000.

[0094] Preparation of reference solution

[0095] Preparation of reference solution: Take appropriate amount of paeoniflorin, gentiopicroside, paeoniflorin ester and loganinic acid reference substances, weigh them accurately, and add methanol to make solutions containing 0.5, 0.2, 0.1 and 0.1 mg per mL respectively.

[0096] Preparation of test solution

[0097] Take an appropriate amount of this product, crush it, take about 0.5g, accurately weigh it, put it in a stoppered conical flask, accurately add 25mL of methanol, weigh it, and ultrasonically treat it (power 240W, frequency 45kHz) for 30 minutes. Let it cool, weigh it, add methanol to make up for the loss in weight, shake it well, filter it, and take the filtrate to obtain it.

[0098] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0099] Table 1 Content determination results of 3 batches of Qingdanshugan capsules

[0100]

[0101] [Content Determination] Volatile oil was determined according to gas chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0521).

[0102] Determination of chromatographic conditions

[0103] A capillary column with modified polyethylene glycol as the stationary phase (column length, 30 m, inner diameter, 0.25 mm, film thickness, 0.25 μm) was used. The column temperature was programmed: initial temperature 60°C, hold for 4 min, increase at 3°C / min to 150°C, and then increase at 5°C / min to 200°C. The FID detector temperature was 220°C; the injection port temperature was 220°C. Split injection was performed with a split ratio of 30; the flow rate was 1 mL / min; and the injection volume was 1 μL. The number of theoretical plates calculated based on the limonene peak should be no less than 10,000.

[0104] Sample preparation

[0105] Preparation of reference solution: Take appropriate amounts of limonene, γ-terpinene, menthone, and menthol reference substances, accurately weigh them, and add anhydrous ethanol to prepare solutions containing 1.5, 0.7, 3.5, and 4.4 mg per mL, respectively.

[0106] Preparation of test solution: Take about 3 g of the product, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of anhydrous ethanol, weigh the weight, and ultrasonically treat it (power 240 W, frequency 45 kHz) for 30 minutes. Let it cool, weigh it, add anhydrous ethanol to make up for the loss in weight, shake it well, filter it through a 0.45 μm microporous filter membrane, and take the filtrate for GC analysis.

[0107] Table 2 Content determination results of 3 batches of Qingdanshugan capsules

[0108]

[0109] Experiment 2: Effect of the Chinese medicine composition of the present invention (capsules prepared in Example 2) on rat hepatobiliary stasis liver disease model

[0110] 1. Purpose of the experiment

[0111] The present invention adopts an α-naphthylisothiocyanate (ANIT) model to observe the therapeutic effect of Longshao Qingdan Shugan Capsule on a rat cholestatic liver disease model by comparing the general condition of the ANIT model, calculating organ indexes, comparing the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutamyl transpeptidase (γ-GT) in serum; detecting the levels of total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acid (TBA); and performing liver tissue pathology examination.

[0112] 2. Experimental Materials

[0113] 2.1 Pharmaceuticals

[0114] 2.1.1 Test article

[0115] Longshao dry extract powder, a brown extract powder with a content of 2.43 g (crude drug) / g (extract powder), was provided by the Institute of Innovative Drugs, Hunan Academy of Traditional Chinese Medicine. The corresponding concentrations of the drug solution were prepared using physiological saline.

[0116] 2.1.2 Positive control drug

[0117] Ursodeoxycholic acid, specification: 250 mg, batch number: L24092A, produced by Losan Pharma GmbH, Germany.

[0118] 2.2 Experimental animals

[0119] Sixty SD rats, SPF grade, male, weighing 180–220 g, were purchased from Hunan Slake Jingda Animal Co., Ltd. (production license number SCXK (Xiang) 2019-004). The animals were housed in the SPF barrier system of the Medical Experimental Animal Center of Hunan Academy of Traditional Chinese Medicine (use license number SYXK (Xiang) 2024-0015).

[0120] 2.3 Main reagents

[0121] α-Naphthylisothiocyanate (ANIT) (Batch number: K2425449, Shanghai Aladdin Biotechnology Co., Ltd., prepared into a suspension with olive oil, used immediately after preparation, and stored in a sealed container).

[0122] 2.4 Main instruments

[0123] Paraffin rotary slicer (Leica, Germany, model: RM2235), fully automatic tissue embedding machine (Leica, Germany, model: EG1150), fully automatic sealing dehydration machine (Leica, Germany, model: HistoCore PEARL). Biochemical analyzer (Trilogy II, DREW).

[0124] 3 Experimental methods

[0125] 3.1 Grouping, drug administration, and modeling

[0126] After 3 days of adaptive feeding, SD rats were weighed and randomly divided into a normal control group, a model group, a high-, medium-, and low-dose Qingdanshugan capsule group (1.01, 2.02, and 4.04 crude drug / kg), and an ursodeoxycholic acid (60 mg / kg) group using a random number table, with 10 rats in each group. Qingdanshugan capsules and ursodeoxycholic acid capsules (shelled) were prepared with normal saline to the corresponding concentrations before daily administration. The rats were administered 10 mL / kg orally once daily for 7 consecutive days. On the 4th day of administration, all groups except the normal control group were given 60 mg / kg of α-isothiocyanate ANIT by gavage to induce a cholestasis model. The normal control group was given an equal volume of olive oil by gavage. Body weight changes in the rats were recorded before and 7 days after administration.

[0127] 3.2 Index detection

[0128] 3.2.1 Sample Collection

[0129] After the final dose, rats were anesthetized with 2.5% pentobarbital, and blood, liver, bile, and fecal samples were collected. Blood samples were centrifuged at 3000 rpm for 10 min, serum was separated, and stored at -80°C. A portion of the liver sample was fixed in 4% paraformaldehyde solution for subsequent histopathological observation, and the other portion was divided into enzyme-free cryopreservation tubes and stored at -80°C for proteomics analysis. Under sterile conditions, 1-3 g of rat feces was directly collected and placed in a sterile EP tube, placed in a dry ice box, and then immediately transferred to a -80°C low-temperature freezer for storage.

[0130] 3.2.2 Organ Index Calculation

[0131] The rat liver weight was weighed and recorded using an analytical balance, and the organ index of each rat was calculated according to the formula: Organ index = liver weight / body weight × 100%.

[0132] 3.2.3 Bile volume flow detection

[0133] After intraperitoneal anesthesia, rats were fixed in the supine position, and the abdomen was opened under the xiphoid process. The common bile duct was cannulated and bile was collected. To ensure accurate measurement, bile was sampled for 20-30 minutes after 5 minutes of cannulation and stored at -80°C until analysis. The ratio of bile collection volume to outflow time was the bile volume flow rate.

[0134] 3.2.4 Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyl transpeptidase (γ-GT) activities and total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acid (TBA) levels

[0135] After anesthesia, rats were blooded from the abdominal aorta. After standing at room temperature for 4 h, the blood was centrifuged at 4°C and 3000 rpm for 10 min. The serum was separated and stored at 0°C. The activities of AST, ALT, and γ-GT and the levels of DBIL, TBIL, IBIL, and TBA in the serum were determined according to the kit instructions.

[0136] 3.2.5 Rat liver tissue pathology

[0137] The livers of rats in each group were fixed in 4% paraformaldehyde for 48 h, dehydrated, embedded in paraffin, and routinely sectioned. The livers were stained with hematoxylin-eosin (HE), and the pathological changes of the livers were observed under a microscope.

[0138] 3.3 Metering settings

[0139] The proposed adult clinical dosage of Qingdanshugan Capsule is 13.1g of crude drug per day. Based on a 70kg body weight, the daily human dosage is 13.1g / 70kg = 0.19g crude drug per kg. The rat dosage, converted per kg body weight, is 0.19*5.4 = 1.01g crude drug per kg. This study used 1, 2, and 4 times the rat clinical equivalent dose as the low, medium, and high doses for the rat experiment, i.e., 1.01, 2.02, and 4.04 crude drug per kg (0.41, 0.82, and 1.64g dry paste per kg).

[0140] The clinical equivalent dose of ursodeoxycholic acid in rats is 60 mg / kg. See Table 3 for details.

[0141] Table 3 Trial grouping and dosage design

[0142]

[0143] 3.3 Statistical methods

[0144] Statistical analysis was performed using SPSS 16.0, with statistical significance set at P < 0.05. Measurement data were expressed as mean ± standard deviation (±s). Leven's test was used to test normality and homogeneity of variance. If normality and homogeneity of variance were met, statistical analysis was performed using one-way ANOVA and post hoc LSD. If normality and heterogeneity of variance were not met, the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P < 0.05), Dunnett's test (nonparametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0145] 4 Experimental results

[0146] 4.1 Comparison of body weight of rats in each group before and 7 days after administration

[0147] As shown in Table 4, compared with the normal group, the model group rats had a significant weight loss after 7 days (P < 0.01). Compared with the model group, the rats in the high-, medium-, and low-dose Qingdanshugan Capsule groups and the ursodeoxycholic acid group had a statistically significant weight gain (P < 0.05, P < 0.01). Qingdanshugan Capsule can improve the basic physiological conditions of rats with cholestasis.

[0148] Table 4 Comparison of body weight of rats in each group before and 7 days after administration (g, )

[0149]

[0150]

[0151] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0152] 4.2 Comparison of organ indices of rats in each group

[0153] As shown in Table 5, compared with the normal group, the liver index of the rats in the model group was significantly increased, which was statistically significant (P < 0.01); compared with the model group, the liver index of the rats in the high- and medium-dose Qingdanshugan capsule groups was significantly decreased, the liver index of the low-dose Qingdanshugan capsule group did not decrease significantly, and the liver index of the rats in the ursodeoxycholic acid group was significantly decreased, which was statistically significant (P < 0.05).

[0154] Table 5 Comparison of liver indexes of rats in each group

[0155]

[0156] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0157] 4.2 Comparison of organ indices of rats in each group

[0158] As shown in Table 6, compared with the normal group, the liver index of the rats in the model group was significantly increased, which was statistically significant (P < 0.01); compared with the model group, the liver index of the rats in the high- and medium-dose Qingdanshugan capsule groups was significantly decreased, the liver index of the low-dose Qingdanshugan capsule group did not decrease significantly, and the liver index of the rats in the ursodeoxycholic acid group was significantly decreased, which was statistically significant (P < 0.05).

[0159] Table 6 Comparison of liver indexes of rats in each group

[0160]

[0161] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0162] 4.3 Comparison of bile volume flow in rats of each group

[0163] As shown in Table 7, compared with the normal group, the bile volume flow of rats in the model group was significantly decreased, which was statistically significant (P < 0.01); compared with the model group, the bile volume flow of rats in the high-, medium- and low-dose Qingdanshugan capsule groups was significantly increased, which was statistically significant (P < 0.01); with the increase of the dose, the effect was not significantly enhanced; the ursodeoxycholic acid group could also increase the bile volume flow of rats, which was statistically significant (P < 0.05, P < 0.01).

[0164] Table 7 Comparison of bile volume flow rate in rats of each group (μL / min, )

[0165]

[0166] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0167] 4.4 Comparison of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamyl transpeptidase (γ-GT) activities in rats of each group

[0168] As shown in Table 8, compared with the normal group, the serum ALT, AST, and γ-GT activities of the rats in the model group were significantly increased (P < 0.01); compared with the model group, the serum ALT, AST, and γ-GT activities of the rats in the high- and medium-dose Qingdanshugan capsule groups were significantly decreased, and the serum ALT and γ-GT activities of the rats in the low-dose Qingdanshugan capsule group were significantly decreased, with statistical significance (P < 0.05). The serum ALT, AST, and γ-GT activities of the rats in the ursodeoxycholic acid group were also significantly decreased, with statistical significance (P < 0.05, P < 0.01).

[0169] Table 8 Comparison of serum ALT, AST and γ-GT activities in rats of each group (U / L, )

[0170]

[0171] Note: Compared with the normal group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0172] 4.5 Comparison of serum levels of total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acid (TBA) in rats of each group

[0173] As shown in Table 9, compared with the normal group, the serum TBIL, DBIL, IBIL, and TBA levels of the rats in the model group were significantly increased (P < 0.01); compared with the model group, the serum TBIL, DBIL, IBIL, and TBA levels of the rats in the high- and medium-dose Qingdanshugan capsule groups were significantly decreased, with statistical significance (P < 0.05), and the serum DBIL, IBIL, and TBA levels of the rats in the low-dose Qingdanshugan capsule group were significantly decreased, with statistical significance (P < 0.05). The ursodeoxycholic acid group could also reduce the serum TBIL, DBIL, IBIL, and TBA levels of rats with cholestasis, with statistical significance (P < 0.05, P < 0.01).

[0174] Table 9 Comparison of TBIL, DBIL, IBIL and TBA levels in rats of each group (μmol / L, )

[0175]

[0176] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0177] 4.6 Pathological changes of liver tissues in rats in each group

[0178] like Figure 11As shown, the normal group had clear liver tissue structure, with neatly arranged hepatocytes in cords. No significant pathological changes such as inflammatory cell infiltration, hepatocyte degeneration, necrosis, fibrosis, or cholestasis were observed. In the model group, varying degrees of central vein dilatation, hepatocyte swelling and ballooning, hepatocyte nuclear shrinkage, apoptosis, and necrosis were observed. Hyperplasia of portal vessels and perivascular edema were observed. Small bile duct hyperplasia, inflammatory cell infiltration, and fibrosis were observed. Biliary dilatation, swelling and degeneration of the bile duct epithelium and vascular walls were observed. Arterial wall thickening was observed, and the bile ducts and arteries were infiltrated with numerous eosinophils. In the ursodeoxycholic acid group, the liver tissue structure was relatively intact. Although some central vein dilatation, periluminal hepatocyte ballooning, a small amount of hepatocyte apoptosis, and interstitial edema, inflammatory cells, and fibrosis were observed, the degree of liver damage was significantly alleviated. The pathological structures of liver tissues of rats in the high-, medium- and low-dose groups of Qingdanshugan Capsule were alleviated to varying degrees. The cord-like arrangement of liver cells was relatively neat, the number of apoptotic cells decreased, a small amount of inflammatory cell infiltration in the portal area, partial bile duct dilatation, and periductal inflammatory cell infiltration, as well as a small amount of ballooning of liver cells around the portal area and central vein were observed.

[0179] 5 Experimental Conclusions

[0180] In summary, the Qingdanshugan capsule prepared by the present invention can improve the basic physiological conditions of rats with cholestasis, increase bile volume flow, reduce serum ALT, AST, γ-GT activities and TBIL, DBIL, IBIL, and TBA levels, and improve cholestatic liver injury. It also improves the liver pathological conditions of rats.

[0181] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Chinese medicine composition for treating cholestatic liver disease, characterized in that: The traditional Chinese medicine composition is prepared by weight from 400 to 550 parts of gentian, 590 to 840 parts of red peony root, 30 to 40 parts of tangerine peel oil, and 25 to 35 parts of mentha oil. The tangerine peel oil has a relative density of 0.830-0.843, an optical rotation of +80°-+90°, a refractive index of 1.460-1.476, and a limonene content of ≥85%; The relative density of the peppermint oil is 0.888-0.908, the optical rotation is -17°--24°, and the refractive index is 1.456-1.

466.

2. A method for preparing a preparation for treating cholestatic liver disease, characterized in that: The following steps are involved: (1) Weigh the Chinese medicinal raw materials according to the Chinese medicinal composition of claim 1 and set aside; (2) Mix Gentiana and Paeonia lactiflora, extract twice with ethanol, filter, and concentrate the filtrate under reduced pressure to obtain a thick paste for later use; (3) Add silicon dioxide to the thick paste, vacuum dry, and crush to obtain fine powder 1 for later use; (4) Mix tangerine peel oil and peppermint oil, add silicon dioxide, adsorb completely, sieve, and obtain adsorbate 1, which is set aside; (5) mixing the fine powder 1 and the adsorbent 1, granulating, and drying to obtain granules; (6) The granules are directly loaded into capsules to obtain capsule preparations; or the granules are mixed with pregelatinized starch and sodium starch glycolate, compressed into tablets, and film-coated to obtain tablets.

3. The method for preparing a preparation for treating cholestatic liver disease according to claim 2, characterized in that: In step (2), the ethanol is 70% ethanol, and the amount of ethanol added for the ethanol extraction is 6-8 times the mass of the gentian and red peony root, and each decoction extraction is performed for 1.0-1.5 hours.

4. The method for preparing a preparation for treating cholestatic liver disease according to claim 2, characterized in that: In step (2), the vacuum degree of the reduced pressure concentration is -0.07 to -0.08 MPa, and the relative density of the thick paste at 70°C to 80°C is 1.20 to 1.

25.

5. The method for preparing a preparation for treating cholestatic liver disease according to claim 2, characterized in that: In step (5), a No. 2 sieve is used for granulation, and the granules are dried at a temperature of 35°C to 40°C.

6. Use of the traditional Chinese medicine composition according to claim 1 in preparing a medicament for treating cholestatic liver disease.

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