Soothing gallbladder and eliminating stone granules and preparation method and use thereof

Abstract

The invention discloses a gallbladder-clearing and stone-removing granule and its preparation method and use, which is mainly composed of the following effective ingredients: 500-700 parts by weight of Lysimachia chinensis, 250-350 parts by weight of Curcuma aromatica, 250-350 parts by weight of Artemisia capillaris, 120-180 parts by weight of White Peony Root, 120-180 parts by weight of Scutellaria baicalensis, 80-110 parts by weight of Gentiana scabra, and 80-110 parts by weight of Radix Rhei. The present invention uses Lysimachia chinensis with the effects of removing dampness and jaundice, diuresis and stranguria, detoxification and swelling as the monarch medicine, and is matched with Curcuma aromatica and Artemisia capillaris with the effects of clearing liver and promoting gallbladder, relieving heat and toxicity, promoting gallbladder and jaundice as the minister medicine; White Peony Root and Scutellaria baicalensis with the effects of clearing heat and relieving acute pain are used as adjuvants; Gentiana scabra and Rhubarb with the effects of clearing heat and drying dampness, purging heat and toxicity, breaking stagnation, and promoting blood stasis are used as the guiding medicine, and the compatibility of all medicines produces the effect of synergistic effect.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine, which is a medicine for treating gallstones and a preparation method and application thereof. Background Art

[0002] There are many drugs for treating gallstones clinically. However, the efficacy of drugs, especially those for cholesterol-induced gallstones, needs to be further improved, and the medical community is constantly seeking new effective drugs. Summary of the invention

[0003] The purpose of the present invention is to provide a gallbladder-soothing and stone-removing granule which can effectively treat cholesterol-induced gallbladder stones, and a preparation method and use thereof.

[0004] The technical solution of the present invention is:

[0005] A gallbladder-soothing and stone-dispelling granule is characterized in that it is mainly composed of the following effective ingredients: 500-700 parts by weight of Lysimachia chinensis, 250-350 parts by weight of Curcuma aromatica, 250-350 parts by weight of Capillaris scoparia, 120-180 parts by weight of White Peony Root, 120-180 parts by weight of Scutellaria baicalensis, 80-110 parts by weight of Gentiana scabra and 80-110 parts by weight of Rhubarb.

[0006] The described gallbladder-soothing and stone-removing granules are mainly composed of the following effective ingredients: 600 parts by weight of Lysimachia chinensis, 300 parts by weight of Curcuma aromatica, 300 parts by weight of Capillaris ternata, 150 parts by weight of White Peony Root, 150 parts by weight of Scutellaria baicalensis, 100 parts by weight of Gentiana scabra and 100 parts by weight of Rhubarb.

[0007] A preparation method of the described gallbladder-soothing and stone-removing granules is characterized by: grinding gentian and rhubarb into powders respectively, sieving through 100 meshes for standby use; placing five medicinal materials of Lysimachia chinensis, Curcuma aromatica, Artemisia capillaris, White Peony Root and Scutellaria baicalensis in a sealed container and boiling according to the formula ratio, adding 6 times of water for the first time, boiling and extracting for 1.5 hours and filtering, adding 4 times of water for the second time, boiling and extracting for 1 hour and filtering, combining the filtrates, and concentrating the filtrates to an appropriate amount; adding the above powders and an appropriate amount of edible aspartame essence, mixing evenly, making granules, drying, and obtaining the finished product.

[0008] A use of the soothing gallstone-eliminating granules in preparing medicine for treating cholesterol-induced gallstones.

[0009] The invention discloses a compound Chinese medicine preparation, which is prepared with Herba Lysimachiae with the effects of removing dampness and jaundice, promoting diuresis and relieving stranguria, and removing toxins and swelling as the main drug (clinically used for damp-heat jaundice, gallbladder distension and hypochondriacal pain, stranguria with stones, stranguria with heat, painful urination, etc.), and with Radix Curcumae and Capillaris with the effects of clearing the liver and promoting bile secretion, removing heat and toxicity, promoting bile secretion and removing jaundice as the assistant drugs; Radix Paeoniae Alba and Radix Scutellariae with the effects of clearing heat and relieving acute pain as the adjuvant drugs; and Radix Gentianae and Radix Rhei with the effects of clearing heat and drying dampness, purging heat and toxicity, breaking up stagnation and promoting blood stasis as the guiding drugs, and the combination of the drugs produces the effect of synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0011] Figure 1 This is a schematic diagram of the effect of Shudanxiaoshi Granule on reducing serum inflammatory factors in guinea pigs with cholesterol-induced gallstones.

[0012] Figure 1 Compared with the Model group and the Control group, △ P<0.01; SXG high / low dose group / UAC group, compared with Model group, *P<0.05, **P<0.05, compared with Control group, # P<0.05, n=12.

[0013] Figure 2 This is a schematic diagram of how Shudanxiaoshi Granules enhances the expression of cholecystokinin receptor gene (CCK-AR mRNA) in the gallbladder smooth muscle tissue of guinea pigs with cholesterol-induced gallstones.

[0014] Figure 2 Compared with the Model group and the Control group, △ P<0.01; SXG high / low dose group / UAC group, compared with Model group, *P<0.05, **P<0.05, compared with Control group, # P<0.05, n=12. DETAILED DESCRIPTION

[0015] A gallbladder-soothing and stone-dissolving granule is mainly composed of the following effective ingredients: 600 grams of Lysimachia chinensis, 300 grams of Curcuma aromatica, 300 grams of Artemisia capillaris, 150 grams of White Peony Root, 150 grams of Scutellaria baicalensis, 100 grams of Gentiana scabra, and 100 grams of Rhubarb.

[0016] Preparation method: grind gentian and rhubarb into powder respectively, sieve through 100 mesh for later use; put five medicinal materials of Lysimachia chinensis, Curcuma aromatica, Artemisia capillaris, White Peony Root and Scutellaria baicalensis into a sealed container and boil according to the formula ratio, add 6 times the amount of water for the first time, boil and extract for 1.5 hours and filter, add 4 times the amount of water for the second time, boil and extract for 1 hour and filter, combine the filtrate, and concentrate the filtrate to an appropriate amount (relative density of 1.25-1.30 at 80°C); add the above powder and an appropriate amount of edible aspartame essence, mix well, make into granules, dry, and make 1000g to obtain the finished product.

[0017] Properties: This product is brown to brown granules with sweet taste.

[0018] Usage and Dosage: Take with boiled water, 10g at a time, 3 times a day.

[0019] Specification: 10g per bag.

[0020] Note: Pregnant women should not take this medicine.

[0021] A use of the soothing gallstone-eliminating granules in preparing medicine for treating cholesterol-induced gallstones.

[0022] Modern pharmacology efficacy research: Effect of Shudanxiaoshi granules on cholesterol-induced gallstones in guinea pigs.

[0023] 1. Materials and Methods

[0024] 1.1 Experimental animals: guinea pigs of either sex, 250-280 g, provided by the Experimental Center of Nantong University, license number: SYXK(Su)2017-0046.

[0025] 1.2. Establishment of cholesterol-induced guinea pig gallstone model

[0026] 1.2.1. Prepare cholesterol stone-forming feed (formula: 40% alfalfa meal, 15% corn meal, 10% soybean meal, 10% bran, 10% flour, 5% sucrose, 3% fish meal, 2% casein, 2% lard, 1% cholesterol, 1% yeast powder, 1% salt).

[0027] 1.2.2. Animal grouping and dosing: 60 guinea pigs were randomly divided into 5 groups, with 12 in each group and 5 in each cage.

[0028] ① Normal control group: 12 guinea pigs, fed 40g of ordinary feed daily;

[0029] ② Model group: 12 guinea pigs, each fed with 40g cholesterol stone-forming feed daily;

[0030] ③ Shudan Xiaoshi Granules (SXG) low-dose group: 12 guinea pigs were fed 40g cholesterol stone-forming feed daily and given 3ml SXG (containing 0.5g) by gavage twice a day;

[0031] ④ High-dose SXG group: 12 guinea pigs were fed 40g cholesterol stone-forming feed daily and given 3ml SXG (containing 1.0g) by gavage twice a day;

[0032] ⑤ Ursodeoxycholic acid capsules (UAC) group: 12 guinea pigs were fed 40g cholesterol stone-forming feed daily and given 3ml UAC (containing 10mg) by gavage once a day;

[0033] 1.3. Sample collection: The above guinea pigs were continuously fed with the drug for 45 days. After fasting for 24 hours, the guinea pigs were killed by intraorbital injection of air. The chest-abdominal cavity was cut open with a scalpel to expose the heart or gallbladder. A. About 5 ml of blood from the heart was extracted with a syringe; B. The bile duct was blocked with a vascular clamp and all the bile was extracted (the bile and blood were placed at room temperature for 30 minutes, and then centrifuged at 2000r / pm for 10 minutes, and the bile supernatant or serum was respectively extracted and stored in a -200C refrigerator for testing); C. The gallbladder was removed and dissected, and the number and rate of gallbladder stones in each group of guinea pigs were calculated, and the stones were taken for qualitative analysis of gallbladder stone formation; D. Preparation of gallbladder smooth muscle tissue samples: The intact gallbladder was taken, cut open longitudinally, and the bile sediment was collected. The bile was cleaned with filter paper, and the mucosa and serosa were removed. Then the gallbladder balance muscle tissue was placed in a cryovial and stored in a liquid nitrogen tank.

[0034] 1.4. Qualitative analysis of gallstones: Take 10 mg of stone powder and place it in a test tube, add 10 ml of chloroform, mix thoroughly for 15 minutes, filter, take 3 ml of the filtrate and place it in another test tube, add 1 ml of acetic anhydride and 0.2 ml of concentrated sulfuric acid respectively, mix, place in a dark place for 10 minutes to observe, and those that show blue-green color are positive for cholesterol stones.

[0035] 1.5. Biochemical index detection: 752 UV-visible spectrophotometer (manufactured by Shanghai Third Analytical Instrument Factory) was used to measure total cholesterol, total bilirubin, calcium ion, direct bilirubin, bile acid and content concentration.

[0036] 1.5.1. Determination of total cholesterol content by enzymatic colorimetry: According to the instructions of the total cholesterol kit, the absorbance value was determined by a spectrophotometer to prepare a cholesterol standard curve. The measurement conditions were: wavelength 500nm, optical microscope 1.0cm, reaction temperature 37°C, and calculation: total cholesterol content (mg / dl) = absorbance of the measurement tube / absorbance of the standard tube x concentration of the standard solution.

[0037] 1.5.2. Determination of total bilirubin content by chemical colorimetry: According to the instructions of the total bilirubin kit, use a spectrophotometer to determine the absorbance value and prepare a total bilirubin standard curve. The determination conditions are: wavelength 550nm, optical microscope 1.0cm, reaction temperature 25°C, calculation: total bilirubin content (mg / dl) = (absorbance of the determination tube - absorbance of the blank tube) x standard solution factor. The wavelength is 550nm and the factor is 23.7.

[0038] 1.5.3. Determination of direct bilirubin content by chemical colorimetry: According to the instructions of the direct bilirubin kit, use a spectrophotometer to determine the absorbance value and prepare a direct bilirubin standard curve. The determination conditions are: wavelength 546nm, optical microscope 1.0cm, reaction temperature 25℃, calculation: direct bilirubin content (mg / dl) = (absorbance of the determination tube - absorbance of the blank tube) x standard solution factor. The wavelength is 546nm, and the factor is 13.2.

[0039] 1.5.4. Determination of bile acid content by enzymatic colorimetry: According to the instructions of the bile acid kit, the absorbance value was determined by a spectrophotometer to prepare a bile acid standard curve. The measurement conditions were: wavelength 540 nm, optical microscope 1.0 cm, reaction temperature 37°C, and calculation: bile acid content (mg / dl) = absorbance of the measurement tube / absorbance of the standard tube x concentration of the standard solution.

[0040] 1.5.5. Determination of calcium concentration using sodium ethylenediaminetetraacetic acid method: According to the calcium test kit instructions, the absorbance value was determined by spectrophotometer to prepare the calcium standard curve. The determination conditions were: wavelength 500nm, optical microscope 1.0cm, reaction temperature 37℃, EDTANa 2 The solution is titrated until the solution changes from purple-red to purple-blue to light blue, and the amount of EDTANa eliminated is recorded. 2 The amount of solution is calculated as follows: calcium content (mg / dl) = consumption of the measuring tube / consumption of the standard tube×10.

[0041] 1.6ELISA method for detection of serum inflammatory factors

[0042] (1) Preparation of serum: The whole blood was placed in a heparin-pretreated EP tube, allowed to stand for 30 min, centrifuged at 3500 rpm for 15 min, and the supernatant was collected for later use.

[0043] (2) Preparation of standard: Add ddH according to the amount marked on the test tube. 2 O to dissolve the powder and vortex to mix.

[0044] (3) Preparation of standard curve: Take the standard solution and add an equal volume of standard diluent as the highest concentration of the standard curve. Take the standard diluted in the previous level and add an equal volume of standard diluent to dilute it step by step. The standard diluent is zero concentration.

[0045] (4) Soak the ELISA plate in the washing solution for 30 seconds, discard the washing solution and absorb the water.

[0046] (5) Add 100 μL of 2-fold diluted standard as the standard well and 100 μL of standard dilution as the blank well.

[0047] (6) Add 90 μL of 1× assay buffer and 10 μL of serum sample to the sample well.

[0048] (7) Add 50 μL of detection antibody diluted 1:100 to all other wells.

[0049] (8) Seal the plate with film and incubate on a shaker at 300 rpm for 90 min at room temperature.

[0050] (9) Discard the sample and primary antibody and wash the ELISA plate five times.

[0051] (10) Add 100 μL of HRP-labeled secondary antibody diluted 1:100 to all other wells;

[0052] (11) Seal the plate with sealing film, place on a shaker at 300 rpm, and incubate at room temperature for 30 min.

[0053] (12) Discard the secondary antibody and wash the ELISA plate five times.

[0054] (13) Add 100 μL of TMB to each well of the ELISA plate and incubate at room temperature in the dark for 5-30 min.

[0055] (14) Add 100 μL of stop solution to each well of the ELISA plate.

[0056] (15) A multifunctional microplate reader (BioTek Co, USA) was used to set the absorbance value at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm for detection and calculation of the results.

[0057] 1.7. Detection of CCK-AR mRNA in Gallbladder Smooth Muscle Tissue

[0058] (1) ① Trizol reagent (Technology, USA); ② Access RT-PCR System (Promega, USA); ③ internal reference actin primers: upstream (625-649, 24 bp): 5'-ctg act gac tacctc atg aag act-3', downstream (872-852, 21 bp): 5'-gga agg aag get gga alga gtg-3'. PCR product length: 248 bp. Guinea pig CCK-AR primers: upstream (367-386, 20 bp): 5'-tea tee cca ate tgc tcaag-3', downstream (658-639, 20 bp): 5'-cac ata ttc gcg gtctgg tt-3', PCR product length: 292 bp. Total RNA was extracted from gallbladder smooth muscle tissue, and RNA was measured by UV spectrophotometer;

[0059] (2) CCK-AR and internal reference p-actin primers: designed according to standard methods;

[0060] (3) RT-PCR amplification: A one-step RT-PCR kit was used. The PCR products of the RT-PCR products were subjected to electrophoresis, gel imaging of the amplified fragments was performed, and absorbance scanning was performed using an image recognition system to calculate the CCK-AR / p-actin integral ratio. The light absorbance integral ratio was used as the gene expression of gallbladder smooth muscle cholecystokinin receptor (CCK-AR).

[0061] Statistical analysis

[0062] The experimental data were expressed as mean ± standard error of the mean (SEM), and GraphPad Prism software was used to process and analyze the experimental data. One-way ANOVA and Student-Newman-Keuls (SNK) test were used for statistical analysis of data among groups. When P < 0.05, it was considered to be statistically significant.

[0063] 2. Results

[0064] 2.1. Shudanxiaoshi Granule reduces cholesterol-induced gallstone formation in guinea pigs

[0065] Drugs were given to prevent and treat cholesterol gallstone formation in guinea pig gallbladders. The gallstone formation rates in guinea pigs in the high-dose and low-dose groups of Shudanxiaoshi Granules and ursodeoxycholic acid capsules were 16.66%, 33.33%, and 25%, respectively, while that in the gallstone model group was as high as 83.33%. Shudanxiaoshi Granules significantly reduced the cholesterol-induced gallstone formation rate in guinea pigs, as shown in Table 1.

[0066] Table 1. Drugs for the prevention and treatment of cholesterol gallstones in guinea pigs

[0067]

[0068]

[0069] Compared with Teb.1.Mode group and SXG high / low dose group / UAC group and Model group, *P<0.05, **P<0.05.

[0070] 2.2. Shudanxiaoshi Granules Reduce Total Cholesterol, Bile Acid and Calcium Contents in the Bile of Guinea Pigs with Cholesterol-Induced Gallstones

[0071] In guinea pigs with cholesterol-induced gallstones, the contents of total cholesterol (TC), bile acid (BA), and calcium (Ca) in the bile of the high / low dose groups of Shudanxiaoshi Granule (SXG) and the ursodeoxycholic acid capsule (UAC) group were significantly decreased (*P<0.05) (see Table 2).

[0072] Table 2. Analysis of TC, BA, and Ca content (ng / dl) in bile of guinea pigs with cholesterol gallstones treated with drugs

[0073]

[0074] Teb.2. Comparison between Mode group and Control group, △ P<0.01; SXG high / low dose group / UAC group, compared with Model group, *P<0.05, **P<0.05, compared with Control group, # P<0.05, n=12.

[0075] 2.3. Shudanxiaoshi Granules Reduce Total Bilirubin and Direct Bilirubin in the Bile of Guinea Pigs with Cholesterol-Induced Gallstones

[0076] In guinea pigs with cholesterol-induced gallstones, the levels of total bilirubin (TBIL) and direct bilirubin (DBIL) in the bile of the high / low dose groups of Shudanxiaoshi Chongji (SXG) and the ursodeoxycholic acid capsule (UAC) group were significantly decreased (*P<0.05) (see Table 3).

[0077] Table 3. Analysis of TBIL and DBIL content (ng / dl) in bile of guinea pigs with cholesterol gallstones treated with drugs

[0078]

[0079] Teb.3.Comparison between Model group and Control group, △ P<0.01; SXG high / low dose group / UAC group, compared with Model group, *P<0.05, **P<0.05, compared with Control group, # P<0.05, n=12.

[0080] 2.4. Shudanxiaoshi Granule reduces serum inflammatory factors in guinea pigs with cholesterol-induced gallstones

[0081] The levels of inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) were measured in guinea pig serum. The results showed that Shudanxiaoshi Chongji significantly reduced the levels of IL-6 and NF-α in guinea pig serum with cholesterol-induced gallstones (P<0.05). Figure 1 Tip: Shudanxiaoshi Granule has anti-inflammatory effect.

[0082] 2.5. Shudanxiaoshi Granule Enhances CCK-AR mRNA Expression in Gallbladder Smooth Muscle Tissue of Guinea Pigs with Cholesterol-Induced Gallstones

[0083] The gallbladder smooth muscle tissue of guinea pigs was taken and the expression of CCK-AR mRNA was detected by RT-PCR. The results showed that Shudanxiaoshi Chongji significantly enhanced the expression of cholecystokinin receptor gene in the gallbladder smooth muscle tissue of guinea pigs with cholesterol-induced gallstones (P<0.05). Figure 2 .

[0084] 3. Conclusion

[0085] The above research results show that Shudanxiaoshi granules can significantly reduce the cholesterol-induced gallstone formation rate in guinea pigs, and its effect is positively correlated with the dose; for cholesterol-induced guinea pig gallstones, Shudanxiaoshi granules can significantly reduce the total cholesterol, bile acid and calcium content in the gallbladder bile of guinea pigs; at the same time, the total bilirubin and direct bilirubin content in the bile also decrease significantly; Shudanxiaoshi granules can also reduce the serum IL-6 and NF-α inflammatory factors in guinea pigs with cholesterol-induced gallstones, indicating that Shudanxiaoshi granules have anti-inflammatory effects; molecular biology studies have shown that Shudanxiaoshi granules can enhance the gene expression of cholecystokinin receptor in gallbladder smooth muscle tissue. Therefore, Shudanxiaoshi granules have the effects of soothing the liver and promoting gallbladder, soothing the gallbladder and regulating qi, promoting gallbladder and removing stones, and promoting qi and relieving pain.

Claims

1. Use of a gallbladder-soothing and stone-removing granule in the preparation of a medicament for treating cholesterol-induced gallstones, characterized in that: The soothing gallbladder and eliminating stone granules are composed of the following effective ingredients: 500-700 parts by weight of Lysimachia chinensis, 250-350 parts by weight of Curcuma aromatica, 250-350 parts by weight of Artemisia capillaris, 120-180 parts by weight of White Peony Root, 120-180 parts by weight of Scutellaria baicalensis, 80-110 parts by weight of Gentiana scabra, and 80-110 parts by weight of Rhubarb. The soothing gallbladder and eliminating stone granules are prepared by the following method: grinding Gentiana scabra and Rhubarb into powder respectively, sieving through 100 mesh for standby use; placing Lysimachia chinensis, Curcuma aromatica, Artemisia capillaris, White Peony Root and Scutellaria baicalensis into a sealed container and boiling according to the formula ratio, adding 6 times of water for the first time, boiling and extracting for 1.5 hours and filtering, adding 4 times of water for the second time, boiling and extracting for 1 hour and filtering, combining the filtrates, and concentrating the filtrates to an appropriate amount; adding the above powders and an appropriate amount of edible aspartame essence, mixing evenly, making granules, drying, and obtaining the finished product.

2. The use of the Shudanxiaoshi granules according to claim 1 in the preparation of a medicament for treating cholesterol-induced gallstones, characterized in that: The described gallbladder-soothing and stone-removing granules are composed of the following effective ingredients: 600 parts by weight of Lysimachia chinensis, 300 parts by weight of Curcuma aromatica, 300 parts by weight of Capillaris ternata, 150 parts by weight of White Peony Root, 150 parts by weight of Scutellaria baicalensis, 100 parts by weight of Gentiana scabra, and 100 parts by weight of Rhubarb.

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