Traditional Chinese medicine composition, application and preparation method of preparation containing traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and discloses a traditional Chinese medicine composition, application and a preparation method of a preparation containing the traditional Chinese medicine composition. Comprising the following steps: mixing 400-550 parts of radix gentianae and 590-840 parts of radix paeoniae rubra, extracting twice with 70% ethanol, and concentrating filtrate under reduced pressure to obtain thick paste; adding silicon dioxide into the thick paste, drying and crushing to obtain fine powder; uniformly mixing 30-40 parts of pericarpium citri reticulatae oil and 25-35 parts of dementholized peppermint oil, adding silicon dioxide, completely adsorbing, and sieving to obtain an adsorbate; and uniformly mixing the fine powder with the adsorbate to prepare capsules or tablets. The traditional Chinese medicine composition disclosed by the invention is used for improving cholestasis type liver injury, and has good effects in soothing liver-gallbladder, clearing heat, removing jaundice and preventing and treating chronic cholestasis type liver diseases.

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, tablet for treating cholestatic liver disease and a preparation method thereof. Background Art

[0002] The incidence rate of cholestasis in chronic liver diseases is about 35%. Patients often present symptoms such as jaundice, yellow urine, pruritus, lipid metabolism disorders, etc. People of any age group are likely to have cholestasis, and with the increase of age, the incidence rate shows an upward trend. Research shows that various factors such as drug-induced liver injury, heredity (progressive familial intrahepatic cholestasis), and diseases (cholelithiasis, sepsis, cholangitis) can all lead to cholestasis. Persistent cholestasis will further develop into liver fibrosis, cirrhosis, and even liver cancer. The treatment goal of cholestasis is to reduce the retention of bile acids and other components of bile in hepatocytes. At present, the drug treatment for cholestasis is very limited. The first-line drug in clinical practice is Ursodeoxycholic acid (UDCA). 40% of the patients have poor responses during the treatment of Primary biliary cholangitis (PBC) with UDCA, and its efficacy is only limited to the early stage of PBC. Moreover, high-dose UDCA will aggravate the condition and increase the risks of cirrhosis, esophageal varices, etc. For patients with poor responses to UDCA treatment, in recent years, another drug approved by the US Food and Drug Administration for treating cholestasis is Obecholic acid (OCA). OCA can significantly improve biochemical indicators, delay the progression of the disease, and improve the survival rate, but serious side effects such as pruritus and abdominal pain will occur during the treatment with OCA. Overuse of OCA will aggravate liver injury, and at the same time, its expensive price limits its widespread clinical use. Therefore, it is very important to find new targets and drugs that are clinically effective and have few side effects for treating cholestatic liver disease.

[0003] In recent years, the efficacy of traditional Chinese medicine alone or in combination with UDCA in the treatment of cholestatic liver disease has made significant progress, and it is particularly distinctive in improving biochemical response rate, improving clinical symptoms, anti-liver fibrosis, anti-inflammatory and choleretic, and regulating immune imbalance. Basic research results provide a substantial theoretical basis for the treatment of cholestatic liver disease with traditional Chinese medicine, but there are still many problems to be improved and solved, such as the lack of stable and effective animal models and cell models to illustrate the efficacy and mechanism of traditional Chinese medicine in the treatment of cholestatic liver disease, the main active ingredients and main mechanisms of action of traditional Chinese medicine in the treatment of cholestatic liver disease are still unclear, and clinical reports are mainly based on decoctions, the efficacy standards are not unified, the sample size is small, and there are few Chinese patent medicines on the market. Therefore, how to provide a Chinese patent medicine preparation with clear chemical composition, controllable quality, safety, and effectiveness for the prevention and treatment of cholestatic liver disease (gallbladder stagnation and heat syndrome) to reduce the patient's medication costs and improve medication compliance has become a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] In view of this, the present invention provides a Chinese medicine composition for treating cholestatic liver disease, its application, and a method for preparing a capsule preparation and a tablet containing the same. A Chinese patent medicine preparation is provided for preventing and treating cholestatic liver disease (gallbladder viscera heat syndrome) by soothing the liver and promoting bile secretion, relieving heat and relieving jaundice, in order to reduce the patient's medication cost and improve medication compliance.

[0005] One of the purposes of the present invention is to provide a 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 peppermint oil.

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

[0007] Further preferably, the medicinal parts of the red peony root of the present invention and the medicinal parts 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] The 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 proportion of the Chinese medicinal composition and setting them aside;

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

[0011] (3) Add silicon dioxide to the thick paste, stir evenly, dry under vacuum, and pulverize to obtain fine powder 1 for standby.

[0012] (4) Mix tangerine peel oil and volatile oil of mentha, add silicon dioxide, adsorb completely, and sieve to obtain mixture 1 for standby.

[0013] (5) Mix fine powder 1 and mixture 1, granulate, and dry to obtain granules.

[0014] (6) Directly load the granules into capsules to obtain capsule preparations; or mix the granules with pregelatinized starch and sodium carboxymethyl starch, tableting, and film coating to obtain tablets.

[0015] Preferably, in step (2), the ethanol extraction is carried out with an ethanol addition amount of 6 - 8 times the mass of the mixed traditional Chinese medicine raw materials, and each decoction extraction is carried out for 1.0 - 1.5 h.

[0016] Preferably, the ethanol extraction is carried out by adding ethanol with a mass 8 times that of the mixed traditional Chinese medicine raw materials for the first time and extracting for 1.5 h; adding ethanol with a mass 6 times that of the mixed traditional Chinese medicine raw materials for the second time and extracting for 1 h.

[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 temperature of the vacuum drying is 65 - 70°C, and the vacuum degree is -0.06 to -0.08 Mpa; the dosage of silicon dioxide is 35 g - 45 g, and the pulverization condition is fine powder.

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

[0020] Preferably, in step (5), granulation is carried out with a No. 2 sieve, and the drying condition of the granules is 35°C - 40°C.

[0021] Preferably, in step (6), granulation is carried out with a No. 2 sieve, the dosage of pregelatinized starch is 10 g, the dosage of sodium carboxymethyl starch is 5 g, and the drying condition of the granules is 35°C - 40°C.

[0022] It should be noted that the traditional Chinese medicine capsule preparation disclosed in the present invention is a compound preparation composed of four traditional Chinese medicines, namely gentiana, red peony root, tangerine peel oil, and peppermint oil. The formula principle is as follows: Gentiana is bitter and cold, belonging to the liver and gallbladder meridians. It functions to clear heat and dry dampness, purge the excessive fire of the liver and gallbladder, and directly reduce the pathogenic dampness and heat toxins. It is a key medicine for treating jaundice, so it is the monarch drug. Red peony root is bitter and slightly cold, belonging to the liver meridian. It clears heat and cools blood, disperses stasis and relieves pain. Combined with the monarch drug, it jointly exerts the effects of clearing the dampness and heat of the liver and gallbladder, and promoting blood circulation to remove stasis and dredge collaterals, so it is the ministerial drug. Peppermint is pungent and cool, belonging to the liver and lung meridians. It functions to soothe the liver and relieve depression, disperse heat pathogens, and rise gently upwards. It helps the gentiana and red peony root to reach the liver and gallbladder meridians, so it is the adjuvant drug; Tangerine peel is bitter, pungent and warm, belonging to the spleen and lung meridians. It functions to regulate qi and strengthen the spleen, dry dampness and resolve phlegm, prevent the bitter and cold properties of gentiana and red peony root from damaging the stomach; It can also promote qi movement and relieve depression, and assist in the drainage of bile, so it is also the adjuvant drug. It is used for the syndrome of stagnation of gallbladder qi and heat in cholestatic liver disease. Symptoms include: the whole body and eyes are yellow, the yellow color is bright, the pain in the upper abdomen and the right hypochondrium radiates to the shoulder and back, the body heat does not subside, the mouth is bitter and the throat is dry, nausea and vomiting, the urine is yellow and red, the stool is constipated, and even the color is white. The tongue is red, the tongue coating is yellow and dry, and the pulse is stringy and rapid.

[0023] The third object of the present invention is to provide the application of the traditional Chinese medicine composition in the preparation of drugs for treating cholestatic liver disease.

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

[0025] The traditional Chinese medicine capsule preparation and tablet provided by the present invention for treating cholestatic liver disease are composed of four traditional Chinese medicines, namely gentiana, red peony root, tangerine peel oil, and peppermint oil. The Qingdan Shugan Capsule can improve the basic physiological conditions of cholestatic rats, increase the bile volume flow rate, reduce the activities of serum ALT, AST, γ-GT and the levels of TBIL, DBIL, IBIL, TBA, and improve the liver lesions of rats. It can improve cholestatic liver injury and has good effects in soothing the liver and promoting bile flow, clearing heat and removing jaundice, and preventing and treating chronic cholestatic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0027] Figure 1 It is the HPLC chromatogram of the test sample in Test 1.

[0028] Figure 2 It is the HPLC chromatogram of the gentiopicroside reference substance in Test 1.

[0029] Figure 3 It is the HPLC chromatogram of the paeoniflorin reference substance in Test 1.

[0030] Figure 4 It is the HPLC chromatogram of albiflorin reference substance in Experiment 1.

[0031] Figure 5 It is the HPLC chromatogram of loganin acid reference substance in Experiment 1.

[0032] Figure 6 It is the GC chromatogram of the test sample of Qingdan Shugan Capsule in Experiment 1.

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

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

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

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

[0037] Figure 11 It is the pathological changes of liver tissues of rats in the normal group, model group, ursodeoxycholic acid group, and the capsule preparation group of the present invention in Experiment 2 of the present invention (HE, 10X) Detailed implementation manners

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] Research proves that:

[0040] Gentiana extract (GA) can alleviate the abnormal accumulation of bile and the lesion of bile reflux into the blood caused by α-naphthyl isothiocyanate (ANIT) through FXR and its target genes, thereby reducing jaundice and improving liver function; the drug-induced upregulation of FXR expression by GA, and the enhanced FXR inhibits the expression of bile acid synthase CYP7αl through SHP feedback, reduces the synthesis of bile acids, activates the transcription of BSEP and MRP2 on the liver cell membrane, and accelerates the efflux transport of bile acids from hepatocytes to bile ducts; downregulates the expression of NTCP and reduces the intake of bile acids. Gentiopicroside in Gentiana can significantly improve the liver injury and fibrosis of rats with CCl4-induced liver fibrosis, and inhibit hepatocyte apoptosis and oxidative stress response, and its effect may be related to the activation of the Nrf2 antioxidant pathway. Gentiopicroside can downregulate the contents of AST, ALT, TBA, and TBil in the serum of mice with cholestatic liver injury (P<0.05) and improve the liver lesions of mice; gentiopicroside can upregulate the PPAR-α mRNA level in the liver tissue and model cells of mice with cholestatic liver injury model, and reduce the mRNA levels of pro-inflammatory cytokines NF-κB, TNF-α, and IL-1β; gentiopicroside can upregulate the protein levels of PPAR-α, CYP3A4, and CPT2 in the liver tissue of mice with cholestatic liver injury and reduce the CAR protein level; the PPAR-α inhibitor can reduce the upregulation of PPAR-α by gentiopicroside in cholestatic liver injury cells and further affect the regulatory effect of PPAR-α on the proteins of CYP3A4, CPT2, and CAR, thereby playing an anti-cholestatic liver injury role. Loganic acid in Gentiana plays an anti-liver injury role through three aspects: anti-inflammatory, regulating the Nrf2 pathway to inhibit oxidative stress, and inhibiting apoptosis.

[0041] The extract of Paeonia lactiflora Pall. can reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), and total bile acid (TBA) in the serum of rats with obstructive liver disease induced by α-naphthylisothiocyanate (ANIT) (P < 0.01), and significantly increase bile flow (P < 0.05). Paeoniflorin activates nuclear factor erythroid 2-related factor 2 (Nrf2) through the phosphatidylinositol-3-kinase (PI3k) / protein kinase B (Akt)-dependent pathway, increases the synthesis of glutathione (GSH), and significantly inhibits the changes in serum ALT, AST, ALP, TBIL, DBIL, total bile acid (TBL), and γ-glutamyl transpeptidase (γ-GT) induced by ANIT. Histological examination showed that paeoniflorin had an obvious alleviating effect on liver injury and bile duct proliferation in rats. Paeoniflorin can promote the expression of these proteins (MAP2K1, MAPK1, ILBP, ABCB1, and LTA4H) by intervening in the related targets of bile acid metabolism and treat obstructive liver disease. Paeoniflorin can regulate the composition of the bile acid pool, reduce the content of toxic bile acids, up-regulate the activities of FXR and BSEP, restore the homeostasis of the bile acid pool, and improve ANIT-induced cholestasis. Paeoniflorin regulates oxidative stress, inflammation, and hepatic stellate cell activation by up-regulating heme oxygenase-1 to alleviate CCl4-induced liver fibrosis in mice. Paeoniflorin and albiflorin can increase the level of β-EP in the serum and cerebral cortex and reduce the production of PGE2 in the cerebral cortex, having an analgesic effect. Albiflorin relieves neuropathic pain by inhibiting the activation of NLRP3 in the spinal cord. Albiflorin can significantly inhibit the proliferation of hepatocarcinoma SMMC-7721 cells and induce apoptosis of hepatocarcinoma SMMC7721 cells.

[0042] Limonene in tangerine peel oil can expel stones and inhibit cholesterol synthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme for cholesterol synthesis. Limonene can not only reduce the content of related enzymes in bile and serum, but also dissolve gallstones in bile, showing good therapeutic effects on cholecystitis with stones and chronic cholecystitis caused by cholestasis. γ-Terpinene can protect the activities of several antioxidant enzymes such as SOD, GSH-Px, and CAT, restore cell damage caused by hydrogen peroxide, and play an antioxidant role; in vivo, γ-terpinene can protect the activities of several antioxidant enzymes in serum and liver and play an antioxidant role.

[0043] Peppermint essence mainly contains menthone and menthol, and has pharmacological effects such as spasmolysis and pain relief, cholagogic and litholytic, antibacterial, antiviral, and anti-inflammatory. It exerts a spasmolytic effect by inhibiting the calcium channels of the digestive tract smooth muscle; it exerts cholagogic and the effect of reducing the total cholesterol level in bile by upregulating the messenger RNA (mRNA) expression of cholesterol 7α-hydroxylase (CYP7A1) and farnesoid X receptor (FXR receptor).

[0044] The above research shows that multiple active ingredients in the composition of the present invention produce a synergistic effect through multiple targets and multiple pathways to treat cholestatic liver diseases.

[0045] Example 1 A preparation method for capsules and tablets for treating cholestatic liver diseases:

[0046] Formula: 400 g of gentian, 600 g of red peony root, 30 g of tangerine peel oil, 35 g of peppermint essence oil

[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 essence oil, and set aside;

[0049] (2) Extract the gentian and red peony root twice with 70% ethanol, filter, and concentrate the filtrate under reduced pressure. The medicinal liquid is concentrated into a thick paste and set aside; among them

[0050] The first decoction time is 1.5 h, the second decoction time is 1 h; and the amount of added alcohol is 6 - 8 times the mass of the medicinal materials; the relative density of the thick paste obtained by concentrating the medicinal liquid is measured to be 1.2 - 1.25 at 70℃ - 80℃; and the vacuum degree during reduced pressure concentration is -0.07 - -0.08 Mpa;

[0051] (3) Add silicon dioxide to the thick paste obtained in step (2), mix evenly, dry, and crush the dry paste into fine powder to obtain fine powder 1, and set aside;

[0052] Among them, the drying temperature of the thick paste is 65 - 70℃, and the vacuum degree is -0.07 - -0.08 Mpa;

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

[0054] (5) Combine the fine powder 1 in step (3) and the adsorbate 1 in step (4), mix evenly, granulate and dry, and fill the granules into capsules to make 1000 g of granules. That's it.

[0055] (6) Or mix the granules prepared in step (5) with pregelatinized starch and sodium carboxymethyl starch, press into tablets, and coat with a film coating to make 1000 tablets. That's it.

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

[0057] Formulation: 450 g of Gentiana scabra, 590 g of Paeonia lactiflora, 34 g of tangerine peel oil, 25 g of peppermint oil

[0058] Preparation method:

[0059] (1) Weigh 450 g of Gentiana scabra, 590 g of Paeonia lactiflora, 34 g of tangerine peel oil, and 25 g of peppermint oil, and set aside;

[0060] (2) Extract Gentiana scabra and Paeonia lactiflora twice with 70% ethanol, filter, and concentrate the filtrate under reduced pressure. The medicinal liquid is concentrated into a thick paste and set aside; among them,

[0061] The first decoction time is 1.5 h, the second decoction time is 1 h; and the amount of added ethanol is 6 - 8 times the mass of the medicinal materials; the relative density of the thick paste obtained by concentrating the medicinal liquid is measured at 1.2 - 1.25 at 70℃ - 80℃; and the vacuum degree during reduced pressure concentration is -0.07 - -0.08 Mpa;

[0062] (3) Add silicon dioxide to the thick paste obtained in step (2), mix well, dry, and crush the dried paste into fine powder to obtain fine powder 1, and set aside;

[0063] Among them, the drying temperature of the thick paste is 65 - 70℃, and the vacuum degree is -0.07 - -0.08 Mpa;

[0064] (4) Add tangerine peel oil and peppermint oil, adsorb with an appropriate amount of silicon dioxide, sieve the adsorbent, and obtain adsorbent 1, and set aside;

[0065] (5) Combine fine powder 1 obtained in step (3) and adsorbent 1 obtained in step (4), mix well, granulate and dry, and fill the granules into capsules to make 1000 g of granules. That's it.

[0066] (6) Or mix the granules prepared in step (5) with pregelatinized starch and sodium carboxymethyl starch, tableting, and film coating to make 1000 tablets. That's it.

[0067] Example 3

[0068] Formulation: 500 g of Gentiana scabra, 840 g of Paeonia lactiflora, 34 g of tangerine peel oil, 25 g of peppermint oil

[0069] Preparation method:

[0070] (1) Weigh 500 g of Gentiana scabra, 840 g of Paeonia lactiflora, 34 g of tangerine peel oil, and 25 g of peppermint oil, and set aside;

[0071] (2) Extract Gentiana scabra and Paeonia lactiflora twice with 70% ethanol, filter, and concentrate the filtrate under reduced pressure. The medicinal liquid is concentrated into a thick paste and set aside; among them,

[0072] The first decocting time is 1.5 h, and the second decocting time is 1 h; the amount of added alcohol is 6 to 8 times the mass of the medicinal materials; the relative density of the thick paste obtained by concentrating the medicinal liquid is measured to be 1.2 to 1.25 at 70 °C to 80 °C; and the vacuum degree during vacuum concentration is -0.07 to -0.08 Mpa;

[0073] (3) Add silicon dioxide to the thick paste obtained in step (2), mix well, dry, and crush the dry paste into fine powder to obtain fine powder 1 for standby;

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

[0075] (4) Add appropriate amount of silicon dioxide to adsorb orange peel oil and peppermint oil, sieve the adsorbent, and keep the adsorbent 1 for standby;

[0076] (5) Combine the fine powder 1 in step (3) and the adsorbent 1 in step (4), mix well, granulate and dry, and load the granules into capsules to make 1000 g of granules. Thus obtained.

[0077] (6) Alternatively, mix the granules prepared in step (5) with pregelatinized starch and sodium carboxymethyl starch, press into tablets, and coat with a film coating to make 1000 tablets. Thus obtained.

[0078] Example 4

[0079] Formula: 550 g of gentiana, 800 g of red peony root, 40 g of orange peel oil, 30 g of peppermint oil

[0080] Preparation method:

[0081] (1) Weigh 550 g of gentiana, 800 g of red peony root, 34 g of orange peel oil, and 25 g of peppermint oil for standby;

[0082] (2) Extract gentiana and red peony root twice with 70% ethanol, filter, and concentrate the filtrate under reduced pressure to obtain a thick paste of the medicinal liquid for standby; among them,

[0083] The first decocting time is 1.5 h, and the second decocting time is 1 h; the amount of added alcohol is 6 to 8 times the mass of the medicinal materials; the relative density of the thick paste obtained by concentrating the medicinal liquid is measured to be 1.2 to 1.25 at 70 °C to 80 °C; and the vacuum degree during vacuum concentration is -0.07 to -0.08 Mpa;

[0084] (3) Add silicon dioxide to the thick paste obtained in step (2), mix well, dry, and crush the dry paste into fine powder to obtain fine powder 1 for standby;

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

[0086] (4) Adsorb orange peel oil and peppermint oil with an appropriate amount of silica, sieve the adsorbent, and reserve Adsorbent 1.

[0087] (5) Combine the fine powder 1 from step (3) and Adsorbent 1 from step (4), mix evenly, granulate and dry, and fill the granules into capsules to make 1000 g of granules. That's it.

[0088] (6) Alternatively, mix the granules obtained in step (5) with pregelatinized starch and sodium carboxymethyl starch, tablettenize, and coat with a film coating to make 1000 tablets. That's it.

[0089] Use the capsule preparation obtained in Example 2 for the following tests.

[0090] Test 1

[0091] Determination of the content of the main components of Qingdan Shugan Capsules (tentative name)

[0092]

Content determination

[0093] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile - 0.1% phosphoric acid solution (14:86) as the mobile phase; the detection wavelength is 230 nm. The number of theoretical plates calculated based on the paeoniflorin peak should be not less than 2000.

[0094] Preparation of reference substance solution

[0095] Preparation of reference substance solution Weigh appropriate amounts of paeoniflorin, gentiopicroside, albiflorin, and loganin reference substances accurately, and dissolve them in methanol to prepare solutions containing 0.5, 0.2, 0.1, and 0.1 mg per mL respectively. That's it.

[0096] Preparation of test solution

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

[0098] Determination method Accurately pipette 10 μL of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph for determination. That's it.

[0099] Table 1 Results of content determination of 3 batches of Qingdan Shugan Capsules

[0100]

[0101]

Content Determination

[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); the column temperature was programmed temperature rise, and the programmed temperature rise conditions were: initial temperature 60°C, hold for 4 min, rise to 150°C at a rate of 3°C / min, and then rise to 200°C at a rate of 5°C / min; the temperature of the FID detector was 220°C; the injection port temperature was 220°C. Split injection was used, with a split ratio of 30; the flow rate was 1 mL / min; the injection volume was 1 μL. The number of theoretical plates calculated by the limonene peak should be not less than 10000.

[0104] Sample Preparation

[0105] Preparation of reference substance solution Weigh appropriate amounts of reference substances of limonene, γ-terpinene, menthone, and menthol accurately, and prepare solutions containing 1.5, 0.7, 3.5, and 4.4 mg per mL with absolute ethanol respectively, and you will get it.

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

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

[0108]

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

[0110] 1. Experimental purpose

[0111] The present invention uses the α-naphthylisothiocyanate (ANIT) model. By comparing the general conditions of the ANIT model, calculating the organ index, the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transpeptidase (γ-GT) in the serum; detecting the levels of total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acid (TBA), and performing histopathological examination of the liver tissue, the therapeutic effect of Longshao Qingdan Shugan capsules on the rat model of cholestatic liver disease was observed.

[0112] 2. Experimental materials

[0113] 2.1 Drugs

[0114] 2.1.1 Test articles

[0115] Longshao dry extract powder, which is a brown extract powder with a content of 2.43 g (raw medicine) / g (extract powder), was provided by the Institute of Innovative Drugs, Hunan Academy of Chinese Medicine. During the experiment, it was prepared into a corresponding concentration of liquid medicine with normal saline.

[0116] 2.1.2 Positive control drugs

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

[0118] 2.2 Experimental animals

[0119] 60 SPF-grade male SD rats, weighing 180 - 220 g, were from Hunan Slack Jingda Animal Co., Ltd. (production license number: SCXK(Xiang)2019 - 004), and the animals were housed in the SPF-grade barrier system of the Medical Experimental Animal Center of Hunan Academy of Chinese Medicine (usage license number: SYXK(Xiang)2024 - 0015).

[0120] 2.3 Main reagents

[0121] Alpha-naphthylisothiocyanate (ANIT) (batch number: K2425449, Shanghai Aladdin Biochemical Technology Co., Ltd.), which was prepared into a suspension with olive oil, used immediately after preparation, and stored sealed.

[0122] 2.4 Main instruments

[0123] Paraffin rotary microtome (Leica, Germany, model: RM2235), automatic tissue embedding machine (Leica, Germany, model: EG1150), automatic enclosed dehydration machine (Leica, Germany, model: HistoCore PEARL). Biochemical analyzer (model: TrilogyII, DREW).

[0124] 3 Experimental methods

[0125] 3.1 Grouping, drug administration and model establishment

[0126] After 3 days of adaptive feeding, the body weights of SD rats were measured, and they were randomly divided into a normal control group, a model group, high, medium, and low-dose Qingdan Shugan Capsule (1.01, 2.02, 4.04 crude drugs / kg) groups, and an ursodeoxycholic acid (60 mg / kg) group, with 10 rats in each group. Before drug administration every day, Qingdan Shugan Capsule and ursodeoxycholic acid capsules (after removing the shells) were formulated into corresponding concentrations with normal saline according to the dose, and used immediately after preparation. The rats were given intragastric administration at 10 mL / kg, once a day, for 7 consecutive days. On the 4th day of drug administration, except for the normal control group, the other groups were given intragastric administration of α-isothiocyanato-β-phenylethane (ANIT, 60 mg / kg) to induce a cholestasis model, and the normal control group was given intragastric administration of an equal volume of olive oil, and the changes in the body weights of the rats before and 7 days after drug administration were recorded.

[0127] 3.2 Index detection

[0128] 3.2.1 Sample collection

[0129] After the last drug administration, the rats were anesthetized with 2.5% pentobarbital, and blood, liver, bile, and fecal samples of the rats were collected. The blood samples were centrifuged at 3000 r / min for 10 min to separate the serum, which was stored at -80°C. Part of the liver samples were fixed in 4% paraformaldehyde solution for subsequent histopathological observation, and the other part was aliquoted into enzyme-free cryotubes and stored at -80°C for proteomics detection. 1 - 3 g of rat feces were directly taken under sterile conditions and placed in a sterile EP tube, put into a dry ice box, and then immediately transferred to a -80°C low-temperature refrigerator for storage.

[0130] 3.2.2 Calculation of organ index

[0131] The liver weights of the rats were measured and recorded with 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 Detection of bile volume flow

[0133] After intraperitoneal anesthesia of the rats, they were fixed in the supine position, laparotomy was performed under the xiphoid process, and a bile duct intubation operation was carried out to collect bile. To ensure accurate measurement, bile was collected and sampled 20 - 30 min after intubation for 5 min, and stored at -80°C until analysis. The ratio of the bile collection volume to the outflow time was the bile volume flow.

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

[0135] After anesthetizing the rats, blood was collected from the abdominal aorta. After standing at room temperature for 4 h, it was centrifuged at 3000 r / min for 10 min at 4°C to separate the serum, which was stored at 0°C. The activities of AST, ALT, γ-GT and the levels of DBIL, TBIL, IBIL, and TBA in the serum were measured according to the kit instructions.

[0136] 3.2.5 Histopathological examination of rat liver tissues

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

[0138] 3.3 Dosage setting

[0139] The clinically proposed dosage of Qingdan Shugan Capsule for adults: 13.1 g of crude drug per day. Calculated based on a body weight of 70 kg, the daily clinical dosage for humans is 13.1 g / 70 kg = 0.19 g of crude drug / kg. The dosage for rats converted according to the dosage per kilogram of body weight is: 0.19 * 5.4 = 1.01 g of crude drug / kg. In this experiment, 1, 2, and 4 times the clinically equivalent dose for rats were used as the low, medium, and high doses for the rat experiment, namely 1.01, 2.02, and 4.04 g of crude drug / kg. (0.41, 0.82, 1.64 g of dry extract / kg)

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

[0141] Table 3 Experimental groupings and dosage designs

[0142]

[0143] 3.3 Statistical methods

[0144] SPSS 16.0 was used for statistical analysis. The level of statistical significance was set at P < 0.05. Measurement data were expressed as mean ± standard deviation (±s). The Leven’s test method was used to test normality and homogeneity of variance. If they met the normality and homogeneity of variance, one-way analysis of variance (One-way ANOVA) and post Hoc LSD were used for statistical analysis; if they did not meet the normality and heterogeneity of variance, the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P < 0.05), Dunnett’s Test (non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered during evaluation.

[0145] 4 Experimental results

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

[0147] As shown in Table 4, compared with the normal group, the body weight of the rats in the model group significantly decreased after 7 days (P < 0.01); compared with the model group, the body weights of the rats in the high, medium, and low dose groups of Qingdan Shugan Capsule and the Ursodeoxycholic Acid group significantly increased, with statistical significance (P < 0.05, P < 0.01). Qingdan Shugan Capsule can improve the basic physiological conditions of rats with cholestasis.

[0148] Table 4 Comparison of body weights 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 significantly increased, with statistical significance (P < 0.01); compared with the model group, the liver indices of the rats in the high and medium dose groups of Qingdan Shugan Capsule significantly decreased, the decrease in the liver index of the low dose group of Qingdan Shugan Capsule was not obvious, and the liver index of the rats in the Ursodeoxycholic Acid group significantly decreased, with statistical significance (P < 0.05).

[0154] Table 5 Comparison of liver indices 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 significantly increased, with statistical significance (P < 0.01); compared with the model group, the liver indices of the rats in the high and medium dose groups of Qingdan Shugan Capsule significantly decreased, the decrease in the liver index of the low dose group of Qingdan Shugan Capsule was not obvious, and the liver index of the rats in the Ursodeoxycholic Acid group significantly decreased, with statistical significance (P < 0.05).

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

[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 of rats in 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, with statistical significance (P < 0.01); compared with the model group, the bile volume flow of rats in the high, medium, and low dose groups of Qingdan Shugan Capsule was significantly increased, with statistical significance (P < 0.01); as the dose increased, the effect was not significantly enhanced; the Ursodeoxycholic Acid group could also increase the bile volume flow of rats, with statistical significance (P < 0.05, P < 0.01).

[0164] Table 7 Comparison of bile volume flow of rats in 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 the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) in the serum of rats in each group

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

[0169] Table 8 Comparison of the activities of ALT, AST, and γ-GT in the serum of rats in 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 the levels of total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acid (TBA) in the sera of rats in each group

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

[0174] Table 9 Comparison of the levels of TBIL, DBIL, IBIL, and TBA in rats in each group (umol / 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 in the liver tissues of rats in each group

[0178] As Figure 11As shown, the hepatic tissue structure of the normal group was clear, the hepatocytes were arranged in neat cords, and no obvious pathological changes such as inflammatory cell infiltration, hepatocyte degeneration, necrosis, fibrosis, and cholestasis were observed. In the model group, varying degrees of central vein dilation, hepatocyte swelling, and ballooning degeneration of hepatocytes were visible. The hepatocyte nuclei were shrunken, with apoptotic necrosis. There was hyperplasia of blood vessels in the portal area, perivascular edema, hyperplasia of small bile ducts, inflammatory cell infiltration combined with fibrous tissue hyperplasia, bile duct dilation, swelling and degeneration of bile duct epithelium and blood vessel walls, thickening of the arterial wall, and a large number of eosinophil infiltrations around the bile ducts and arteries. The hepatic tissue structure of the ursodeoxycholic acid group was relatively complete. Although partial central vein dilation, ballooning degeneration of hepatocytes around the lumen, as well as a small number of hepatocyte apoptosis, interstitial edema in the portal area, inflammatory cells, and fibrous tissue hyperplasia were visible, the degree of liver injury was significantly alleviated. In the high, medium, and low dose groups of Qingdan Shugan Capsule, the pathological structure of the rat liver tissue was alleviated to varying degrees. The hepatocytes were arranged in relatively neat cords, the number of apoptotic cells decreased, there was a small amount of inflammatory cell infiltration in the portal area, partial bile duct dilation, periductal inflammatory cell infiltration, and ballooning degeneration of a small number of hepatocytes around the portal area and central vein.

[0179] 5 Experimental Conclusions

[0180] In summary, the Qingdan Shugan Capsule prepared by the disclosed method of the present invention can improve the basic physiological conditions of cholestatic rats, increase the bile volume flow rate, reduce the activities of serum ALT, AST, γ-GT and the levels of TBIL, DBIL, IBIL, TBA, and improve cholestatic liver injury. It can also improve the liver lesions in rats.

[0181] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Chinese medicine composition, characterized in that: Calculated by weight, the Chinese medicinal components include: 400-550 parts of gentian, 590-840 parts of red peony root, 30-40 parts of tangerine peel oil, and 25-35 parts of peppermint oil.

2. A Chinese medicine composition according to claim 1, characterized in that: The relative density of the 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%.

3. A Chinese medicine composition according to claim 1, characterized in that: 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.

4. A method for preparing a preparation for treating cholestatic liver disease, characterized in that: The following steps are involved: (1) Weighing the Chinese medicine raw materials according to the Chinese medicine composition according to any one of claims 1 to 3 and setting aside; (2) Mix gentian and red peony root, extract twice with ethanol, filter, and concentrate the filtrate under reduced pressure to obtain a thick paste for later use; (3) adding silicon dioxide to the thick paste, vacuum drying, and crushing to obtain fine powder 1 for later use; (4) Mixing tangerine peel oil and peppermint oil, adding silicon dioxide, adsorbing completely, sieving, and obtaining adsorbate 1 for standby use; (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.

5. The method for preparing a preparation for treating cholestatic liver disease according to claim 4, 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 1.0-1.5 hours.

6. The method for preparing a preparation for treating cholestatic liver disease according to claim 4, 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.

7. The method for preparing a preparation for treating cholestatic liver disease according to claim 4, characterized in that: In step (3), the amount of silicon dioxide used is 35 to 45 g; In step (4), the amount of silicon dioxide added is 18 g to 22 g, and the sieving is performed using a No. 3 sieve.

8. The method for preparing a preparation for treating cholestatic liver disease according to claim 4, characterized in that: In step (5), a No. 2 sieve is used for granulation, and the granules are dried at 35° C. to 40° C.

9. The method for preparing a preparation for treating cholestatic liver disease according to claim 4, characterized in that: In step (6), the amount of pregelatinized starch is 10 g and the amount of sodium carboxymethyl starch is 5 g.

10. Use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for treating cholestatic liver disease.

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