A Chinese medicine composition and preparation method thereof

Abstract

The present invention relates to the field of Chinese medicine preparation and processing, specifically a Chinese medicine composition and a preparation method thereof, which is made of the following components in parts by weight: 0.1-0.2 parts of ursodeoxycholic acid, 450-560 parts of auxiliary Chinese medicinal materials A, 50-65 parts of auxiliary Chinese medicinal materials B, 5-10 parts of auxiliary Chinese medicinal materials C, 14000-17000 parts of auxiliary materials and 0.07-0.16 parts of supplements. The present invention utilizes a coenzyme self-circulation system to economically and efficiently catalyze CDCA to synthesize UDCA, realizes the large-scale process production requirements, uses synthetic ursodeoxycholic acid to replace the natural bear bile medicinal materials in the important formula Xiongdan Pills, and optimizes and updates the existing prescriptions, thereby greatly reducing the production cost while ensuring the efficacy of the Chinese medicine composition.

Description

Technical Field

[0001] The invention relates to the field of preparation and processing of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition and a preparation method thereof. Background Art

[0002] Bear Bile Pills have the effects of clearing away heat and dispersing wind, relieving pain and removing cataracts. They are mainly used clinically to treat symptoms such as red and swollen eyes, photophobia and excessive tearing caused by wind-heat or damp-heat in the liver meridian. This product is an oral Chinese patent medicine containing ingredients such as Rehmannia, Gentiana, Plantago, Cassia, bear bile, borneol, etc., which can effectively inhibit inflammatory reactions and relieve inflammatory symptoms. In addition, it can also inhibit the growth and reproduction of various bacteria and play an antibacterial role.

[0003] For 13 centuries, bear bile has been regarded as a precious and effective medicine in traditional Chinese medicine. However, the practice of raising bears to extract bile is controversial. From the perspective of modern medicine, the main component of bear bile is ursodeoxycholic acid (UDCA), which can be obtained through industrial synthesis. It is not difficult to obtain this drug through synthesis, and the cost is not too high, which well solves the controversial issue of traditional bear farming to extract bile. The main recognized effect of UDCA is to dissolve gallstones, which is probably related to what traditional medicine calls "choleretic". It can inhibit the reabsorption of cholesterol in the intestine and reduce the secretion of cholesterol into bile, thereby reducing the saturation of cholesterol in bile. A large part of gallstones is formed by the precipitation of cholesterol in bile due to supersaturation. Conversely, after the cholesterol concentration in bile is reduced, the precipitated cholesterol can be redissolved. This is the principle of ursodeoxycholic acid dissolving stones. For patients with gallstones who are unwilling or unable to undergo surgery, ursodeoxycholic acid is a viable option. In addition, ursodeoxycholic acid also has a certain degree of effect on improving liver function, so it is also used to treat various hepatobiliary diseases, such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver and viral hepatitis.

[0004] As far as the above uses are concerned, synthetic ursodeoxycholic acid can completely replace bear bile, and it has more advantages than natural bear bile. The reason is simple. Bear bile is a complex mixture. It not only contains bile acid, bile pigment and other main ingredients, but also contains waste products excreted by the bear's liver metabolism, which may contain harmful substances. Not only that, affected by many factors, the content of active ingredients in bear bile will also fluctuate. The same amount of bear bile cannot be guaranteed to contain the same amount of active ingredients, so the efficacy is also uncertain. In contrast, synthetic products not only have fewer impurities and a certain content of active ingredients, but are also cheaper and easier to obtain.

[0005] Therefore, it is of great significance to use synthetic ursodeoxycholic acid to replace the bear bile in bear bile pills to produce synthetic bear bile pills. In the prior art, the synthesis methods of synthetic ursodeoxycholic acid mainly consist of chemical synthesis and biotransformation. The routes of chemical synthesis of UDCA are mostly cumbersome, highly polluting, and involve dangerous steps such as high temperature and high pressure; while the biotransformation method usually uses lithocholic acid (LCA), chenodeoxycholic acid (CDCA) or cholic acid (CA) as substrates, and uses whole cell conversion method or enzyme method to synthesize UDCA. The whole cell conversion method mainly adds substrates such as chenodeoxycholic acid (CDCA) or lithocholic acid (LCA) during microbial culture, and uses microorganisms to convert them into UDCA; the enzyme method generally uses CDCA or cholic acid (CA) as substrates, and uses the specificity of biological enzymes to produce stereoisomerism at specific positions of the substrate to complete the two-step oxidation and reduction reaction to generate UDCA. Compared with other preparation methods, the biotransformation method has significant advantages such as short route, high conversion rate, low production cost, and green safety, and has gradually become the mainstream research direction for UDCA preparation.

[0006] However, during the production research, we found that the substrate feed concentration in the whole-cell conversion system is generally very low. Once the substrate concentration is increased, the microbial growth will be significantly inhibited. In addition, the whole-cell conversion of microorganisms requires a high purity of the substrate. Therefore, it is difficult to put it into industrial production. In comparison, enzymatic synthesis is easier to put into industrial production.

[0007] The most common enzymatic synthesis substrates in the prior art are LCA, CDCA and CA. However, the biosynthesis process of UDCA still has some limitations in actual production. First, the key enzymes in the enzymatic reaction (especially 7β-HSDH) are unstable, and the enzyme activity will decrease rapidly in the reaction system. Secondly, the enzyme's tolerance to substrate concentration is still very low, and substrate or product inhibition is serious.

[0008] The most common existing Bear Gall Pill formula is: 1013g of Gentiana, 608g of Alisma (salt), 760g of Rehmannia, 608g of Angelica, 608g of Gardenia, 608g of Chrysanthemum, 608g of Plantago (salt), 608g of Cassia, 608g of Bupleurum, 608g of Saposhnikovia, 608g of Scutellaria, 608g of Equisetum, 608g of Coptis Powder, 62.29g of Menthol, 1031g of Rhubarb, 76g of Borneol, and 12.66g of Bear Gall. Preparation method: Grind the above 17 ingredients, Bear Gall, Menthol, and Borneol into fine powder respectively, grind with Coptis Powder, sieve, and mix. The remaining thirteen herbs, including gentian, are decocted twice with water, the first time for 2 hours and the second time for 1 hour, the decoctions are combined, filtered, the filtrate is concentrated into a thick paste, dried, crushed, sieved, mixed with the above powder and appropriate auxiliary materials, sieved, mixed, and capsuled to make 10,000 capsules. The Bear Bile Pill is a professional treatment drug for glaucoma, early cataracts, bruised eyes, vitreous opacities, floaters, keratitis, conjunctivitis, and fundus hemorrhage caused by internal fire ascending and external wind evil.

[0009] On the basis of the existing technology, we have carried out a large number of experimental studies and method improvements, replaced bear bile with synthetic ursodeoxycholic acid in the existing public bear bile pill formula, and then improved the existing formula in order to provide a synthetic production process for Chinese medicine compositions suitable for industrial production, breaking the existing process limitations, meeting market demand, and effectively reducing production costs. Summary of the invention

[0010] The purpose of the present invention is to provide a Chinese medicine composition and a preparation method thereof, which utilizes a coenzyme self-circulation system to economically and efficiently catalyze CDCA to synthesize UDCA, thereby achieving large-scale process production requirements, using synthetic ursodeoxycholic acid to replace the natural bear bile medicinal material in the important formula of bear bile pills, and optimizing and updating the existing prescription, thereby greatly reducing the production cost while ensuring the efficacy of the Chinese medicine composition.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] A Chinese medicine composition is prepared from the following components in parts by weight: 0.1-0.2 parts of ursodeoxycholic acid, 450-560 parts of auxiliary Chinese medicinal materials A, 50-65 parts of auxiliary Chinese medicinal materials B, 5-10 parts of auxiliary Chinese medicinal materials C, 14000-17000 parts of auxiliary materials and 0.07-0.16 parts of supplements;

[0013] The auxiliary Chinese medicinal material A is composed of the following components in parts by weight: 80-120 parts of galangal, 80-120 parts of rhubarb, 40-80 parts of scutellaria, 40-80 parts of atractylodes, 40-80 parts of chrysanthemum, 55-95 parts of rehmannia, and 40-80 parts of equisetum;

[0014] The auxiliary Chinese medicinal material B is made by grinding coptis root and oyster in a weight ratio of 1:1;

[0015] The auxiliary Chinese medicinal material C is borneol;

[0016] The supplement comprises 0.05-0.1 parts of vitamin B1 and 0.02-0.06 parts of vitamin B2.

[0017] Preferably, the auxiliary material is starch.

[0018] The method for preparing a Chinese medicine composition according to any one of the above items specifically comprises the following steps:

[0019] S1, synthesis of ursodeoxycholic acid by enzymatic method;

[0020] S2, accurately weighing the auxiliary Chinese medicinal material A for preparing the Chinese medicine composition according to the formula, decocting and concentrating to obtain a concentrated solution, and then spray-drying to obtain component A;

[0021] S3, accurately weighing the auxiliary Chinese medicinal material B for preparing the Chinese medicine composition according to the formula, sterilizing and crushing to obtain component B;

[0022] S4, accurately weighing auxiliary Chinese medicinal materials C for preparing the Chinese medicine composition according to the formula, and grinding to obtain component C;

[0023] S5, mixing component B and component C obtained in steps S3 and S4 uniformly and sieving them, then mixing them with component A obtained in step S2 and ursodeoxycholic acid synthesized in step S1 in proportion, adding a supplement and mixing them uniformly again to obtain a drug powder;

[0024] S6, adding auxiliary materials to the medicine powder obtained in step S5, mixing and making pills, and packaging after passing the inspection.

[0025] Preferably, the enzymatic synthesis of ursodeoxycholic acid in step S1 specifically comprises the following steps:

[0026] S11, preparation of purified enzyme solution: synthesizing the artificially synthesized double mutant of 7α-hydroxysteroid dehydrogenase of Clostridium salvinii and the double mutant of Ruminococcus sphaeroides into an Escherichia coli expression vector to obtain pSZD-6×His-7α-HSDH and pSZD-6×His-7β-HSDH plasmids, amplifying the plasmids and extracting the plasmids, and then transforming the extracted plasmids to obtain recombinant Escherichia coli for expanded culture expression, homogenizing and breaking the packages to obtain two-component dehydrogenase: 7α-HSDH and 7β-HSDH;

[0027] S12, preparing a reaction system: dissolving chenodeoxycholic acid in an ethanol solution, adding the two-component dehydrogenase, cosolvent and coenzyme obtained in step S11, and then adding a sodium hydroxide solution to adjust the pH value of the system to 7.5-8.5, placing the reaction system on a shaker at 28-32° C. for catalytic reaction to synthesize ursodeoxycholic acid;

[0028] S13, adding sodium hydroxide and water to the system to precipitate ursodeoxycholic acid into sodium salt under strong alkali, separating ethanol, adding water to ursodeoxycholic acid sodium salt, recovering the residual ethanol under reduced pressure, adjusting the pH value to 2-3 with dilute hydrochloric acid, precipitating ursodeoxycholic acid crystals, separating and drying to obtain ursodeoxycholic acid.

[0029] Preferably, the addition ratio of 7α-HSDH to 7β-HSDH in the system is 1:1-2.

[0030] Preferably, the cosolvent is 0.4% Tween 80.

[0031] Preferably, the coenzyme is 0.5mmol / L NADP + .

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The Chinese medicine composition has been studied on reducing the existing technology. The research results show that compared with the formula of the existing technology, the raw materials of the Chinese medicine composition are simpler. The use of galangal and atractylodes can achieve the same therapeutic effect as the original bear bile pill formula, and can also effectively solve the side effects of the original formula, and optimize the defects of the original formula that may cause eye irritation and allergic reactions. It has been found that the Chinese medicine composition can also enhance the body's absorption of pills, maintain blood drug stability, and effectively reduce the amount of treatment drugs.

[0034] The preparation method of the traditional Chinese medicine composition adopts an enzymatic method to synthesize ursodeoxycholic acid, artificially synthesizes 7α-HSDH and 7β-HSDH genes, and successfully expresses them in Escherichia coli. The mutated 7α / β-hydroxysteroid dehydrogenase with high enzyme activity economically and efficiently catalyzes chenodeoxycholic acid (CDCA) to synthesize ursodeoxycholic acid (UDCA) by utilizing the coenzyme self-circulation system, with a conversion rate of up to 98.6%, which greatly improves the production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the synthesis principle of ursodeoxycholic acid of the present invention; DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0037] Bear bile: clears away heat and detoxifies, soothes the liver and improves eyesight, kills insects and stops bleeding.

[0038] Gentian: The Chinese medicinal material Gentian has the effects of clearing away heat and dampness, and purging liver and gallbladder fire; Gentian is very bitter and cold, and can easily damage the stomach, so the dosage should not be too large, and people with weak spleen and stomach should not take it.

[0039] Alisma (Salt): Salted Alisma is fried with salt. Since salt can enter the kidney, it can increase the effect of Alisma entering the kidney. It is sweet, light, slightly cold in nature and flavor. It enters the kidney and bladder meridians. It can be used to treat water retention, edema, urination difficulties, and dizziness caused by phlegm and fluid. It is often used in combination with Poria cocos and Atractylodes macrocephala. It can also be used for damp-heat leucorrhea and short and red urine. It can be used in combination with Plantago asiatica and Smilax glabra. Salted Alisma is also used for kidney yin deficiency and hyperactivity of virtual fire. It can be used in combination with drugs such as Rehmannia glutinosa and Paeonia suffruticosa that nourish yin and purge fire. Because salted Alisma has diuretic effects, lowers blood cholesterol and has anti-atherosclerotic effects, it is a commonly used drug for the treatment of hypertension, hyperlipidemia and hypercholesterolemia.

[0040] Rehmannia root: It has the functions of clearing away heat, cooling blood, nourishing yin and promoting body fluid; Rehmannia root is cold, slippery and stagnant, so it is not suitable for people with spleen deficiency, poor appetite, loose stools, and dampness and fullness in the middle.

[0041] Angelica: Its main effects are to tonify blood and activate blood circulation, regulate menstruation and relieve pain, and moisten the intestines and promote bowel movements. Angelica is sweet, warm and moistening, so it is not suitable for people with dampness, fullness in the middle, and diarrhea.

[0042] Gardenia jasminoides: It has the effects of purging fire and relieving restlessness, clearing away heat and dampness, cooling blood and detoxifying; it can be used externally to reduce swelling and relieve pain. This product is bitter and cold and can damage the stomach. It is not suitable for people with yin and blood deficiency, spleen deficiency and loose stools.

[0043] Chrysanthemum: The main effects are to dispel wind-heat, calm liver yang, clear the liver and improve eyesight, clear away heat and detoxify. It is used to treat wind-heat cold, the initial stage of febrile disease, liver yang hyperactivity, headache and dizziness, red and swollen eyes, blurred vision, sores, carbuncle and swelling; Chrysanthemum tastes spicy, sweet, bitter, and slightly cold. It enters the lung and liver meridians. It is used to treat wind-heat cold, the initial stage of febrile disease, liver yang hyperactivity, headache and dizziness, red and swollen eyes, blurred vision, sores, carbuncle and swelling.

[0044] Plantago seed (prepared with salt): Plantago seed prepared with salt can clear away heat and dampness, and has a strong effect of purging heat and promoting diuresis. It can promote diuresis without damaging yin, can clear the liver, and has the effect of clearing heat and improving eyesight. It is used for blurred vision and decreased vision.

[0045] Cassia seed: Cassia seed has the effects of clearing away heat, improving eyesight, moistening the intestines and promoting bowel movements; Cassia seed is a moisturizing and laxative agent, so those with spleen deficiency and loose stools should take it with caution.

[0046] Bupleurum: Bupleurum has the effects of dispersing and reducing fever, soothing the liver and relieving depression, and raising the Yang Qi; Bupleurum has a promoting effect, so it is not suitable for people with symptoms of true Yin deficiency and rising liver Yang.

[0047] Saposhnikovia divaricata: Saposhnikovia divaricata has the effects of dispelling wind and relieving exterior symptoms, eliminating dampness and relieving pain, and stopping convulsions; the flavor of Saposhnikovia divaricata is pungent and slightly warm, which can damage yin and blood and help fire, so people with blood deficiency and convulsions as well as yin deficiency and excessive fire should take it with caution.

[0048] Scutellaria baicalensis: Scutellaria baicalensis has the functions of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus; Scutellaria baicalensis is bitter, cold, and drying, and can suppress the vital energy, so people with weak spleen and stomach, poor appetite, and loose stools should not take it.

[0049] Horsetail: Sweet, bitter, neutral in nature. Enters the lung, liver, and gallbladder meridians. It has the effects of dispelling wind and heat, relieving muscles, and removing cataracts. It is often used for eye cataracts, tearing in the wind, intestinal wind bleeding, bloody dysentery, rectal prolapse, malaria, sore throat, carbuncle, etc.

[0050] Coptis powder: The main ingredients are coptis root and oyster. It is mainly used to treat blister sores.

[0051] Menthol: Used for nasal congestion, insect bites, chapped skin, minor burns, abrasions, sunburn and skin itching, etc.

[0052] Rhubarb: It has the functions of purging qi and removing accumulation, clearing away heat and purging fire, cooling blood and detoxifying, removing blood stasis and dredging menstruation, and promoting dampness and removing jaundice. Rhubarb is bitter and cold, and is good at purging qi and removing blood stasis, so women during pregnancy, menstruation, and lactation should take it with caution or avoid taking it.

[0053] Borneol: invigorates the mind, clears away heat and relieves pain; people with Yin deficiency and Yang hyperactivity, slow cold in children, spleen deficiency and diarrhea, liver and kidney deficiency, and eye diseases should not take borneol. Pregnant women should be cautious when taking borneol.

[0054] Alpinia officinalis: used for cold pain in the abdomen, vomiting due to stomach cold, belching and acid reflux; it should not be used alone by those with weak constitution.

[0055] Atractylodes: It has the effects of drying dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. Atractylodes is pungent, pungent, warm and dry, so it is not suitable for people with yin deficiency, internal heat, and qi deficiency and excessive sweating.

[0056] Vitamin B1: It can maintain normal sugar metabolism and the functions of the nervous and digestive systems. Its deficiency will affect the body's energy supply and cause symptoms related to the nervous system, cardiovascular system and digestive system.

[0057] Vitamin B2: It is used to prevent and treat vitamin B2 deficiency, such as angular cheilitis, chapped lips, glossitis, scrotitis, conjunctivitis, seborrheic dermatitis, etc., and can relieve migraine symptoms. In addition, it can also relieve symptoms caused by malnutrition and progressive weight loss.

[0058] The invention studies the efficacy and contraindications of each component, attempts to use galangal and atractylodes to replace gentian, plantain seed, cassia seed, siler and gardenia in the original formula, so that the galangal and atractylodes become the assistant drugs for assisting the bear bile to exert its therapeutic effect, and then uses synthetic ursodeoxycholic acid to replace the natural bear bile, which has a higher component purity and is used as the main drug of the formula, and finally vitamin B1 and vitamin B2 are added to stimulate the absorption of the drug by the human intestine, so that the bear bile pill of the prescription can be released and absorbed to the maximum extent in the human body.

[0059] Example 1

[0060] Synthetic ursodeoxycholic acid (synthetic principle as Figure 1 shown):

[0061] S11, preparation of purified enzyme solution:

[0062] The double mutants Q255L / C260S of 7α-hydroxysteroid dehydrogenase (7α-HSDH) of Clostridium sardinii (GenBank accession number: AET80685.1) and T189V / V207M of Ruminococcus ATCC35915 (GenBank accession number: CBL26204) were artificially optimized and synthesized by Nanjing GenScript Biotechnology Co., Ltd., and synthesized into the vector pSZD to obtain pSZD-6×His-7α-HSDH and pSZD-6×His-7β-HSDH plasmids. The synthesized plasmids were transferred into E. coli DH5α for plasmid amplification and plasmid extraction. The plasmids obtained above were transformed into E. coli BL21 (DE3) competent cells, and positive transformants were screened by kanamycin resistance plates. The recombinant E. coli was cultured in a shaking incubator at 37°C until OD600 was about 0.6-0.8, then the temperature was lowered to the induction temperature, 0.4 mmol / L isopropylthiogalactoside (IPTG) was added and placed at 20°C, 30°C, and 37°C to induce expression, and then the bacterial solution after culturing for 20 hours was centrifuged at 4°C and 12000 r / min for 10 min, the upper culture medium was discarded, and an appropriate amount of 50 mmol / L Tris-HCl (pH 8.0) was added for resuspending, and the cell lysate was collected by ultrasonic disruption, and centrifuged at 12000 r / min for 10 min to obtain the supernatant (S) and precipitate (P) of the lysate, and the protein expression level was detected by SDS-PAGE. The protein expression conditions were as follows: 30°C, 200r / min induction for 20h, followed by expanded culture expression and homogenized cell disruption to obtain the two-component dehydrogenase: 7α-HSDH and 7β-HSDH.

[0063] S12, prepare the reaction system: dissolve 80g of chenodeoxycholic acid in 120mL of 78% ethanol solution, add 0.02-0.06g of the two-component dehydrogenase obtained in step S11, wherein the addition ratio of 7α-HSDH to 7β-HSDH is 1:1-2; add 0.8-1.8g of cosolvent: 0.4% Tween 80; add 0.08-0.14g of 0.5mmol / L NADP + As a coenzyme. Then add 60~70mL of 10mol / L sodium hydroxide solution to adjust the pH value of the system to 7.5~8.5, place the reaction system in an enzyme reaction tank at 28~32℃ for catalytic reaction to synthesize ursodeoxycholic acid;

[0064] S13, adding 15-20 g of sodium hydroxide and 8-13 g of water to the system, precipitating ursodeoxycholic acid into sodium salt under strong alkali, separating ethanol, and continuing to add 80-100 g of water to the sodium salt of ursodeoxycholic acid, after the residual ethanol is completely recovered under reduced pressure, adjusting the pH value to 2-3 with dilute hydrochloric acid, precipitating ursodeoxycholic acid crystals, separating, and drying to obtain ursodeoxycholic acid.

[0065] Test Example 1

[0066] 1. The addition ratio of 7α-HSDH and 7β-HSDH was set to 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, and 1:2, and other reaction conditions were kept unchanged. The test showed that the conversion rate of synthesized ursodeoxycholic acid was the highest when the addition ratio was between 1:1.6 and 1:1.8.

[0067] 2. The addition ratio of 7α-HSDH and 7β-HSDH was set to 1:1.7, and the dosage of two-component dehydrogenase in the reaction system was set to 0.02g, 0.03g, 0.04g, 0.05g, and 0.06g. Other reaction conditions were kept unchanged. The test showed that the conversion rate of synthesized ursodeoxycholic acid was the highest when the dosage of two-component dehydrogenase in the reaction system was between 0.04g and 0.05g.

[0068] 3. Set the addition ratio of 7α-HSDH and 7β-HSDH to 1:1.7, and change the coenzyme NADP in the reaction system to + The dosage was set to 0.08g, 0.09g, 0.10g, 0.11g, 0.12g, 0.13g, and 0.14g, and other reaction conditions were kept unchanged. The test showed that the coenzyme NADP in the reaction system + The conversion rate of synthesized ursodeoxycholic acid is highest when the dosage is between 0.11g and 0.12g.

[0069] The orthogonal experiment was designed and the results showed that the optimal dosage of the above components was 0.05g of two-component dehydrogenase (the addition ratio of 7α-HSDH and 7β-HSDH was set to 1:1.8) for every 80g of chenodeoxycholic acid, 1.4g of cosolvent: 0.4% Tween 80, and 0.11g of 0.5mmol / L NADP. + As a coenzyme. Then add 67mL of 10mol / L sodium hydroxide solution to adjust the pH value of the system to 8.0, place the reaction system in an enzyme reaction tank at 31°C for catalytic reaction, and the conversion rate of synthesizing ursodeoxycholic acid is the highest, reaching 99.64%.

[0070] The above ratio was expanded for large-scale industrial production, and the test results showed that the conversion rate of ursodeoxycholic acid produced by the above method was 96.89% (the process loss was within a controllable range).

[0071] Example 2

[0072] Weigh 0.1g of ursodeoxycholic acid; auxiliary Chinese medicinal materials A: 80g of Alpinia officinalis, 80g of Rhubarb, 40g of Scutellaria baicalensis, 40g of Atractylodes lancea, 40g of Chrysanthemum, 55g of Rehmannia glutinosa, 40g of Equisetum arvense; auxiliary Chinese medicinal materials B: 50g of Coptis chinensis powder; auxiliary Chinese medicinal materials C: 5g of borneol; supplements: 0.05g of vitamin B1 and 0.02g of vitamin B2.

[0073] Perform laboratory synthesis:

[0074] The auxiliary Chinese medicinal material A for preparing the Chinese medicine composition is accurately weighed according to the formula, decocted and concentrated to obtain a concentrated solution, and then spray-dried to obtain component A; the auxiliary Chinese medicinal material B for preparing the Chinese medicine composition is accurately weighed according to the formula, sterilized and crushed to obtain component B; the auxiliary Chinese medicinal material C for preparing the Chinese medicine composition is accurately weighed according to the formula, ground to obtain component C; component B and component C are evenly mixed and sieved, and then mixed with component A and synthetic ursodeoxycholic acid in proportion, and a supplement is added and mixed again to obtain a medicine powder; auxiliary materials are added to the obtained medicine powder, and mixed to make pills.

[0075] Example 3

[0076] Weigh 0.15g of ursodeoxycholic acid; auxiliary Chinese medicinal materials A: 100g of Alpinia officinalis, 100g of Rhubarb, 60g of Scutellaria baicalensis, 60g of Atractylodes lancea, 60g of Chrysanthemum, 75g of Rehmannia glutinosa, 60g of Equisetum arvense; auxiliary Chinese medicinal materials B: 58g of Coptis chinensis powder; auxiliary Chinese medicinal materials C: 7.5g of borneol; supplements: 0.08g of vitamin B1 and 0.04g of vitamin B2.

[0077] Perform laboratory synthesis:

[0078] The auxiliary Chinese medicinal material A for preparing the Chinese medicine composition is accurately weighed according to the formula, decocted and concentrated to obtain a concentrated solution, and then spray-dried to obtain component A; the auxiliary Chinese medicinal material B for preparing the Chinese medicine composition is accurately weighed according to the formula, sterilized and crushed to obtain component B; the auxiliary Chinese medicinal material C for preparing the Chinese medicine composition is accurately weighed according to the formula, ground to obtain component C; component B and component C are evenly mixed and sieved, and then mixed with component A and synthetic ursodeoxycholic acid in proportion, and a supplement is added and mixed again to obtain a medicine powder; auxiliary materials are added to the obtained medicine powder, and mixed to make pills.

[0079] Example 4

[0080] Weigh 0.2g of ursodeoxycholic acid; auxiliary Chinese medicinal materials A: 120g of Alpinia officinalis, 120g of Rhubarb, 80g of Scutellaria baicalensis, 80g of Atractylodes lancea, 80g of Chrysanthemum, 95g of Rehmannia glutinosa, 80g of Equisetum arvense; auxiliary Chinese medicinal materials B: 65g of Coptis chinensis powder; auxiliary Chinese medicinal materials C: 10g of borneol; supplements: 0.1g of vitamin B1 and 0.06g of vitamin B2.

[0081] Perform laboratory synthesis:

[0082] The auxiliary Chinese medicinal material A for preparing the Chinese medicine composition is accurately weighed according to the formula, decocted and concentrated to obtain a concentrated solution, and then spray-dried to obtain component A; the auxiliary Chinese medicinal material B for preparing the Chinese medicine composition is accurately weighed according to the formula, sterilized and crushed to obtain component B; the auxiliary Chinese medicinal material C for preparing the Chinese medicine composition is accurately weighed according to the formula, ground to obtain component C; component B and component C are evenly mixed and sieved, and then mixed with component A and synthetic ursodeoxycholic acid in proportion, and a supplement is added and mixed again to obtain a medicine powder; auxiliary materials are added to the obtained medicine powder, and mixed to make pills.

[0083] Comparative Example 1

[0084] Natural bear bile was used to replace the synthetic ursodeoxycholic acid in Example 3, and its dosage was adjusted according to the content of ursodeoxycholic acid in natural bear bile. 1.5 g of ursodeoxycholic acid was weighed; auxiliary Chinese medicinal materials A: 100 g of Alpinia officinalis, 100 g of Rhubarb, 60 g of Scutellaria baicalensis, 60 g of Atractylodes lancea, 60 g of Chrysanthemum, 75 g of Rehmannia glutinosa, and 60 g of Equisetum arvense; auxiliary Chinese medicinal materials B: 58 g of Coptis chinensis powder; auxiliary Chinese medicinal materials C: 7.5 g of borneol; supplements: 0.08 g of vitamin B1 and 0.04 g of vitamin B2.

[0085] Perform laboratory synthesis:

[0086] The auxiliary Chinese medicinal material A for preparing the Chinese medicine composition is accurately weighed according to the formula, decocted and concentrated to obtain a concentrated solution, and then spray-dried to obtain component A; the auxiliary Chinese medicinal material B for preparing the Chinese medicine composition is accurately weighed according to the formula, sterilized and crushed to obtain component B; the auxiliary Chinese medicinal material C for preparing the Chinese medicine composition is accurately weighed according to the formula, ground to obtain component C; component B and component C are evenly mixed and sieved, and then mixed with component A and synthetic ursodeoxycholic acid in proportion, and a supplement is added and mixed again to obtain a medicine powder; auxiliary materials are added to the obtained medicine powder, and mixed to make pills.

[0087] Comparative Example 2 (Existing Commercially Available Formula)

[0088] 100g of Gentiana, 60g of Alisma (salt), 76g of Rehmannia, 60g of Angelica, 60g of Gardenia, 60g of Chrysanthemum, 60g of Plantago (salt), 60g of Cassia, 60g of Bupleurum, 608g of Saposhnikovia, 60g of Scutellaria, 60g of Equisetum, 60g of Coptis powder, 6.2g of Menthol, 100g of Rhubarb, 7.6g of Borneol, 1.2g of Bear Gallbladder. Grind the above 17 ingredients, Bear Gallbladder, Menthol and Borneol into fine powder respectively, grind with Coptis powder, sieve and mix. Add water to the remaining 13 ingredients, such as Gentiana, and boil twice, the first time for 2 hours and the second time for 1 hour. Combine the decoction, filter, concentrate the filtrate into a thick paste, dry, crush, sieve, mix with the above powder and appropriate auxiliary materials, sieve, mix, put into capsules, and make granules.

[0089] Test Example 2

[0090] After the drug safety test was performed on the Chinese medicine compositions synthesized in Examples 2-4 and Comparative Example 1, the efficacy was tested. The test standard was "Ministry of Health Drug Standard Chinese Medicine Formula Preparation Volume 2", and the test results were in compliance with the requirements by a third-party agency.

[0091] Test Example 3

[0092] Clinical trial of a Chinese medicine composition for treating red, swollen and painful eyes, photophobia and excessive tearing caused by wind-heat or damp-heat in the liver meridian.

[0093] 1. Materials and methods

[0094] 1.1 General Information

[0095] 1.1.1 A total of 240 patients diagnosed with red, swollen and painful eyes, photophobia and excessive tearing caused by wind-heat or damp-heat in the liver meridian were selected and randomly divided into control group 1, control group 2 and observation group, with 80 cases in each group. In control group 1, there were 80 patients, including 45 males and 35 females, aged 22 to 78 years old, with an average age of (45.27±15.46) years old. The course of disease was 3 months to 1 year, with an average course of disease of (4.47±1.64) years. In control group 2, there were 80 patients, including 47 males and 33 females, aged 23 to 76 years old, with an average age of (44.57±13.42) years old. The course of disease was 3 months to 1.5 years, with an average course of disease of (4.47±1.64) years. There were 80 patients in the observation group, including 48 males and 32 females, aged 25-74 years old, with an average age of (46.29±16.87) years old. The course of disease was 3 months to 1 year, with an average course of disease of (4.69±2.05 years. There was no statistically significant difference in basic data such as age, gender, and course of disease between the two groups (P>0.05), and the two groups were comparable.

[0096] 1.1.2 Diagnostic criteria: Western medicine diagnostic criteria: acute conjunctivitis, pseudoconjunctivitis and epidemic keratitis. Traditional Chinese medicine diagnostic criteria: refer to the relevant diagnostic criteria in the "Guidelines for Clinical Research of New Chinese Medicines", red, swollen and painful eyes, photophobia and excessive tearing caused by wind-heat or damp-heat in the liver meridian.

[0097] 1.1.3 Criteria for inclusion of cases

[0098] (1) Aged 18-75;

[0099] (2) The clinical diagnosis conforms to the diagnostic standards of traditional Chinese and Western medicine;

[0100] (3) The subjects were informed of the study and signed the informed consent form.

[0101] 1.1.4 Exclusion criteria

[0102] (1) Those under 18 years old (not including 18 years old) or over 75 years old (not including 75 years old); (2) Pregnant or lactating women; (4) Those whose condition is critical and whose efficacy or safety cannot be judged; (5) Those taking hormonal drugs at the same time; (6) Those with allergic constitution or those who are allergic to the drugs in the test drug prescription.

[0103] 1.2 Methods

[0104] 1.2.1 Treatment methods

[0105] The observation group was given the Chinese medicine composition prepared in Example 3 of the present invention, orally, 1 pill at a time, 1-2 times a day, or as prescribed by a doctor.

[0106] The control group 1 was orally administered the Chinese medicine composition of comparative example 1.

[0107] The control group 2 was orally administered with the Bear Bile Pill of comparative example 2. Public information shows: The drug [Function and Indications]: Clears away heat and dissipates wind, relieves pain and removes cataracts. It is used for red, swollen and painful eyes, photophobia and excessive tears caused by wind-heat or damp-heat in the liver meridian. The functions and indications are similar to those of the experimental drug, and it is a legal drug of the same type with relatively certain efficacy in China, which conforms to the generally recognized principles of effectiveness, safety and comparability. One course of treatment is 2 weeks.

[0108] 1.2.2 Efficacy observation indicators

[0109] 1) Symptoms and signs: recorded using the integral method, with none, mild, moderate, and severe being scored as 0, 1, 2, and 3 points respectively.

[0110] 2) Safety indicators: three routine tests, renal function and electrocardiogram.

[0111] 1.2.3 Criteria for determining efficacy

[0112] Referring to the "Guidelines for Clinical Research of New Chinese Medicines", it is divided into cure, remarkable effect, effectiveness and ineffectiveness.

[0113] Recovery: Clinical symptoms and signs of TCM are significantly improved, and the syndrome score is reduced by ≥90%;

[0114] Markedly effective: clinical symptoms and signs of TCM are significantly improved, and the syndrome score is reduced by ≥70%;

[0115] Effective: TCM clinical symptoms and signs are improved, and the syndrome score is reduced by ≥30%;

[0116] Ineffective: There is no significant improvement in TCM clinical symptoms and signs, or they may even get worse, and the syndrome score is reduced by less than 30%.

[0117] 1.2.4 Statistical analysis

[0118] SPSS16.0 software was used to perform statistical analysis on the data, and t-test was used for counting data. P < 0.05 was considered to be significantly different, indicating statistical significance.

[0119] 2 Results

[0120] 2.1 Comparison of clinical efficacy among the three groups

[0121] After treatment, the symptom scores of the three groups were significantly reduced compared with those before treatment, and the difference was statistically significant (P < 0.05); the degree of reduction in the observation group was significantly better than that in control group 1 and control group 2, and the difference was statistically significant (P < 0.05); the clinical marked efficacy and total efficacy of the observation group were significantly higher than those in control group 1 and control group 2, and the difference was statistically significant (P < 0.05), see Tables 1 and 2.

[0122] Table 1 Comparison of syndrome scores among the three groups before and after treatment

[0123]

[0124] Note: * indicates P < 0.05 compared with before treatment; # indicates P < 0.05 compared with the control group after treatment.

[0125] The above test results show that the formula of Example 2 can still achieve the therapeutic effect of the existing bear bile pills after reducing the formula and adjusting the existing technology, but it can use artificially synthesized ursodeoxycholic acid instead of natural bear bile ingredients, which is more in line with market development needs.

[0126] Table 2 Comparison of clinical efficacy (cases)

[0127]

[0128] Note: * indicates P < 0.05 compared with the control group.

[0129] The above test results show that the formula of Example 2 can still achieve the therapeutic effect of the existing bear bile pills after reducing and adjusting the existing technology, and is better than the control group in terms of curative effect. In addition, the observation group can use artificially synthesized ursodeoxycholic acid instead of natural bear bile ingredients, which is more in line with market development needs.

[0130] Test Example 4

[0131] The first method of dissolution determination method in Appendix XC of Part II of the 2015 edition of the Chinese Pharmacopoeia was adopted, with 500 mL of ethanol solution as the dissolution medium, the rotation speed was 50 rpm, and the method was operated according to this method. 3 mL of solution was taken out at 0.25 h, 0.75 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, and 10 h (automatic rehydration), and immediately passed through a 0.22 μm microporous filter membrane. 4 μL of the filtrate was injected into a high performance liquid chromatograph for determination. The cumulative release of ursodeoxycholic acid in the traditional Chinese medicine composition and Xiongdan Pills was used as the investigation index. The results are shown in Table 3 below:

[0132] Table 3 Cumulative release of ursodeoxycholic acid in traditional Chinese medicine composition

[0133]

[0134] As shown in Table 3, the Chinese medicine composition of the present invention can also maintain a stable blood drug concentration, thereby reducing the number of medication times.

[0135] Example 5

[0136] According to the above formula ratio and process method, industrial production is carried out.

[0137] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways without departing from the scope defined by the spirit of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: The invention is prepared from the following components in parts by weight: 0.1-0.2 parts of ursodeoxycholic acid, 450-560 parts of auxiliary Chinese medicinal material A, 50-65 parts of auxiliary Chinese medicinal material B, 5-10 parts of auxiliary Chinese medicinal material C, 14000-17000 parts of auxiliary materials and 0.07-0.16 parts of supplements; The auxiliary Chinese medicinal material A is composed of the following components in parts by weight: 80-120 parts of galangal, 80-120 parts of rhubarb, 40-80 parts of scutellaria, 40-80 parts of atractylodes, 40-80 parts of chrysanthemum, 55-95 parts of rehmannia, and 40-80 parts of equisetum; The auxiliary Chinese medicinal material B is made by grinding coptis root and oyster in a weight ratio of 1:1; The auxiliary Chinese medicinal material C is borneol; The supplement comprises 0.05-0.1 parts of vitamin B1 and 0.02-0.06 parts of vitamin B2.

2. A Chinese medicine composition according to claim 1, characterized in that: The auxiliary material is starch.

3. A method for preparing a Chinese medicine composition according to any one of claims 1-2, characterized in that: The specific steps include: S1, synthesis of ursodeoxycholic acid by enzymatic method; S2, accurately weighing the auxiliary Chinese medicinal material A for preparing the Chinese medicine composition according to the formula, decocting and concentrating to obtain a concentrated solution, and then spray-drying to obtain component A; S3, accurately weighing the auxiliary Chinese medicinal material B for preparing the Chinese medicine composition according to the formula, sterilizing and crushing it to obtain component B; S4, accurately weighing auxiliary Chinese medicinal materials C for preparing the Chinese medicine composition according to the formula, and grinding to obtain component C; S5, mixing component B and component C obtained in steps S3 and S4 uniformly and sieving them, then mixing them with component A obtained in step S2 and ursodeoxycholic acid synthesized in step S1 in proportion, adding a supplement and mixing them uniformly again to obtain a drug powder; S6, adding auxiliary materials to the medicine powder obtained in step S5, mixing and making pills, and packaging after passing the inspection; The enzymatic synthesis of ursodeoxycholic acid in step S1 specifically includes the following steps: S11, preparation of purified enzyme solution: synthesizing the artificially synthesized double mutant of 7α-hydroxysteroid dehydrogenase of Clostridium salvinii and the double mutant of Ruminococcus sphaeroides into an Escherichia coli expression vector to obtain pSZD-6×His-7α-HSDH and pSZD-6×His-7β-HSDH plasmids, amplifying the plasmids and extracting the plasmids, and then transforming the extracted plasmids to obtain recombinant Escherichia coli for expanded culture expression, homogenizing and breaking the packages to obtain two-component dehydrogenase: 7α-HSDH and 7β-HSDH; S12, preparing a reaction system: dissolving chenodeoxycholic acid in an ethanol solution, adding the two-component dehydrogenase, cosolvent and coenzyme obtained in step S11, and then adding a sodium hydroxide solution to adjust the pH value of the system to 7.5-8.5, placing the reaction system on a shaker at 28-32° C. for catalytic reaction to synthesize ursodeoxycholic acid; S13, adding sodium hydroxide and water to the system to precipitate ursodeoxycholic acid into sodium salt under strong alkali, separating ethanol, adding water to ursodeoxycholic acid sodium salt, recovering the residual ethanol under reduced pressure, adjusting the pH value to 2-3 with dilute hydrochloric acid, precipitating ursodeoxycholic acid crystals, separating, and drying to obtain ursodeoxycholic acid; In the above step S12, the addition ratio of 7α-HSDH and 7β-HSDH in the system is 1:1~2; The cosolvent is 0.4% Tween 80; The coenzyme is 0.5mmol / L NADP + .

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