CN110507669B

CN110507669B - Refined bear gall powder and its use for treating cholecystitis gall-stone and improving gall-bladder function

Info

Publication number: CN110507669B
Application number: CN201910902372.6A
Authority: CN
Inventors: 傅金荣; 付金洪
Original assignee: Jiangxi Tianyuan Pharmaceutical Co ltd
Current assignee: Jiangxi Tianyuan Pharmaceutical Co ltd
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2023-05-02
Anticipated expiration: 2039-09-24
Also published as: CN110507669A

Abstract

The invention relates to refined bear gall powder and application thereof in treating cholecystitis gall-stone and improving gall-bladder function. In particular, one aspect of the present invention relates to the use of a compound of formula I for the preparation of a product for the prevention or treatment of gallstone diseases, for the prevention or treatment of cholecystitis, and for improving gallbladder function: the compound uses Cu-K alpha radiation, and diffraction peaks are formed at 8.53+/-0.20 degrees, 10.96+/-0.20 degrees, 12.03+/-0.20 degrees, 13.14+/-0.20 degrees, 14.82+/-0.20 degrees, 17.26+/-0.20 degrees, 22.53+/-0.20 degrees, 24.21+/-0.20 degrees, 26.68+/-0.20 degrees, 29.42 +/-0.20 degrees and 31.24+/-0.20 degrees in a powder X-ray diffraction spectrum expressed by a 2 theta angle. The compound exhibits excellent biological properties such as excellent bioavailability and can exert the same physiological activity as bear gall powder or tauroursodeoxycholic acid. For example, it can be used for preventing or treating cholelithiasis, preventing or treating cholecystitis, and improving gallbladder function.

Description

Refined bear gall powder and its use for treating cholecystitis gall-stone and improving gall-bladder function

Technical Field

The invention belongs to the technical field of medicines, and relates to a bear gall powder product and a preparation method thereof. The bear gall powder product exhibits excellent biological effects, and can be used for preventing or treating gall-stone diseases, preventing or treating cholecystitis, and improving gall-bladder function, for example.

Background

Bear gall has been used for over one thousand years, and has the reputation of gold in medicine, namely four major animal medicines bear gall, tiger bone, bezoar and musk. At present, 153 Chinese patent medicines prepared by single bear gall powder and compound Chinese patent medicine preparations containing bear gall powder components relate to 183 families of medicine production enterprises. The comprehensive efficacy of the traditional Chinese medicine can not be replaced by other medicines. Among 660 Chinese medicinal books (containing more than 8 ten thousand traditional prescriptions) from Han dynasty to Qing dynasty, 366 books are about recorded on the efficacy application of bear gall and prescription compatibility. Mainly comprises a drug property wheel, a general prescription, a Tang materia Medica, a Qianjin prescription, a materia Medica schema pickup, etc. The nature and flavor of bear gall powder can be summarized as bitter in flavor and cold in nature. The meridian of liver, gallbladder, heart, lung, spleen, stomach and large intestine. Bear gall is bitter and cold in nature, mainly enters liver and gall meridian, can clear liver fire, improve vision and remove nebula, and is mainly used for treating conjunctival congestion and nebula obstacle; clear damp-heat in liver and gallbladder, promote bile flow and alleviate jaundice, and is mainly indicated for icterus and reddish urine. Entering heart meridian, clearing heart fire, promoting blood circulation and relieving pain, and mainly treating heart pain; it can clear heat, induce resuscitation, stop wind and stop spasm after entering heart and liver meridians, and is mainly indicated for qi-heat diseases, with unconsciousness, infantile convulsions and epilepsy convulsion. It enters spleen and stomach meridians, can remove food retention and remove food stagnation, kill parasites and treat infantile malnutrition, and is indicated for food stagnation abdominal pain and infantile fever. It enters large intestine meridian and can clear heat and remove dampness, and is indicated for diarrhea, dysentery, hemorrhoids and sore. All sores and ulcers belong to heart, enter heart meridian, and can clear heat, cool blood and detoxify, and are mainly indicated for furuncle, malignant boil, blood accumulation and blood stranguria. Entering lung meridian, it can clear lung-heat, relieve sore throat, resolve phlegm and relieve cough, and is indicated for sore throat and cough due to phlegm-heat. Modern pharmacological researches have proved that bear gall powder has various pharmacological effects, and the effects of protecting liver, promoting bile flow, dissolving gall stone, resisting liver fibrosis, tranquilizing, relieving spasm, resisting convulsion, relieving pain, tonifying heart, lowering blood pressure, resisting thrombosis, resisting atherosclerosis, reducing blood lipid, relieving cough, eliminating phlegm, relieving asthma, resisting tumor, resisting inflammation, relieving fever, inhibiting bacteria and the like are summarized. The characteristics of multi-component and multi-target pharmacodynamics of the bear gall powder are reflected, and scientific basis is provided for the clinical treatment of liver and gall, cardiovascular and cerebrovascular diseases, infectious diseases and other serious diseases of the bear gall powder. Pharmacological studies carried out by the current academy on bear gall powder are mainly carried out from the following aspects: liver and gall system, central nervous system, cardiovascular and cerebrovascular system, digestive system, respiratory system, antiinflammatory, antibacterial, antiviral etc., ophthalmic, otorhinolaryngology etc.

The Chinese pharmacopoeia of multiple versions all collect medicinal materials of bear gall and/or bile secreted by bear gall or preparations containing bear gall. For example, in 2005 edition, the dry gall bladder of bear is obtained from bear as bear black Selenarctos thibetanus Cuvier or palm Ursus arctos Linnaeus, which are attached to page 24 of Chinese pharmacopoeia. The dried product obtained by draining bile of bear gall powder which is a pandas black bear Selenarctos thibetanus Cuvier of the pandas through gall bladder surgery is received in an annex 27 page of Chinese pharmacopoeia of 2010 edition; the preparation is prepared from fel Ursi powder as raw material, and comprises fel Ursi capsule, fel Ursi heart-relieving pill/pill, fel Ursi hemorrhoid ointment, etc. A Chinese pharmacopoeia of 2015 edition contains fel Ursi capsule, fel Ursi heart-saving pill, fel Ursi hemorrhoid ointment, and fel Ursi hemorrhoid suppository etc. which are prepared from fel Ursi powder.

At present, the dried product of the artificial drainage bear gall bile is taken as a substitute of the natural bear gall and is approved to be marketed, and is named as bear gall powder, and is a new drug approved by the national ministry of health. The fel Ursi powder is a dry product obtained by draining bile from fel Ursi of Urberaceae by gallbladder operation, has cold nature and bitter taste, and has effects of clearing heat, suppressing hyperactive liver, improving eyesight by entering liver, gallbladder, spleen, stomach and large intestine. Modern researches have found that bear gall powder has relatively complex chemical composition and mainly contains conjugated ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), cholic Acid (CA), deoxycholic acid (DCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), cholesterol, bile pigments, amino acids, proteins, peptides, fatty acids, trace elements and the like. Ursodeoxycholic acid is an important characteristic component. The traditional Chinese medicine composition is mainly applied to liver and gall diseases such as gall-stone, fatty liver, cholecystitis, viral hepatitis, chronic hepatitis B and the like, eyelid herpes zoster, hemorrhoids and the like clinically.

Zhanghua (Zhanghua, et al, HPLC fingerprint method for determining bile acid component in fel Ursi capsule, J.Hua.Western medicine, 2009,24 (4): 402-403) describes that fel Ursi capsule is made of low temperature dried fel Ursi drained bile, has cold property and bitter taste, has effects of clearing heat, removing toxic substances, promoting bile flow, relieving spasm and improving eyesight, and contains active components mainly tauro ursodeoxycholic acid and tauchenodeoxycholic acid.

CN1060337C (chinese patent application No. 93116933X, title of the invention bear gall powder enteric capsule and preparation process thereof) records that bear gall powder is composed of three parts: the first part is combined bile acid accounting for about 50% of the total weight of bear gall powder, and comprises tauroursodeoxycholic acid, tauroursodeoxycholic acid and the like, wherein the content of the tauroursodeoxycholic acid is the greatest, and the content of the tauroursodeoxycholic acid accounts for about 20% of the total weight of bear gall powder; the second part is water-soluble protein, amino acid, inorganic salt and other components which are the main reasons for generating moisture absorption, and the second part has no direct physiological activity and usually only plays a nutritional role; the third is the component of fat-soluble bile substances such as bilirubin, cholesterol, steroids, etc., which are not physiologically active, and even cholesterol, for example, is often the component that needs to be avoided as much as possible because they often cause some cardiovascular and cerebrovascular diseases.

Accordingly, those skilled in the art have spent great efforts in further purifying bear gall powder in an effort to remove unnecessary and even harmful components. For example, CN103520210a (201310523280. X, cheng Yuantang) discloses a bear gall powder purification method comprising the following steps performed in the following order: (1) Adding polyvinylpyrrolidone (PVP) and sterile distilled water into freshly extracted bear bile, shaking, mixing, standing, and clarifying to obtain supernatant; (2) Adding 2 times of absolute ethyl alcohol into the supernatant obtained in the step (1) to prepare bear gall alcohol extract; (3) Adding the bear gall purified product obtained in the step (2) into a macroporous silica gel column, washing the silica gel column for 3 times by using 2 times of 75% absolute ethyl alcohol by volume, and collecting washing liquid; (4) Diluting the washing liquid obtained in the step (3) by using sterile distilled water so that the concentration of the absolute ethyl alcohol is 16.5%; (5) Adding the supernatant of bear bile containing 16.5% absolute ethyl alcohol obtained in the step (4) into a cellulose powder CF11 column, carrying out vortex oscillation and uniform mixing, carrying out bear bile separation, centrifuging for 5min under the condition of 5000rpm, adding an equal volume of sterile distilled water into the cellulose powder CF11 column after the centrifugation is finished, and centrifuging for 5min under the condition of 5000 rpm; (6) Collecting the supernatant obtained in the step (5), adding 1/10 volume of 3mol/L NaAc and equal volume of isopropanol, uniformly mixing, precipitating at 20 ℃, centrifuging at 12000rpm for 30min at 4 ℃ after the precipitation is completed, discarding the supernatant, and collecting the precipitate; (7) Washing the precipitate collected in the step (6) by adding 75% absolute ethyl alcohol, centrifuging at 12000rpm for 30min at 4 ℃ after washing, discarding the supernatant, and collecting the precipitate; (8) Dissolving the precipitate obtained in the step (7) in distilled water, stirring for 1-3 hours to prepare bear gall solution, regulating the pH value to 2-8, heating to 50 ℃ and keeping the temperature, adding compound protease, inactivating enzyme after enzymolysis is finished, performing ultrafiltration membrane filtration, and performing spray drying on the obtained supernatant to obtain a finished bear gall powder product. The bear gall powder obtained by the technology of the invention is high in effective content, low in pigment content, free of fishy smell and improved in medicinal value.

CN105147729a (201510650516.5, dianchuan) discloses a preparation method of bear gall powder, comprising the following steps: (A), filtering: taking fresh drained bear bile, and filtering to obtain filtrate; (B), sterilization: adding high-concentration ethanol into the filtrate, wherein the ethanol content in the liquid medicine is 75-85% after the ethanol is added, stirring, standing for 20-40 minutes, and standing for 24-72 hours; (C), concentrating: concentrating under reduced pressure to remove ethanol, concentrating under reduced pressure at 35-45 ℃ to obtain sterilized bear gall solution; (D), freeze drying: the method for freeze-drying the sterilized bear gall liquid comprises the following steps: cooling the sterilized bear gall liquid to 35-40 ℃ at a cooling rate of 1-2 ℃/min, keeping the temperature for 0.5-3.5 h, vacuumizing to 1-15 Pa, and then heating to room temperature at a heating rate of 1-3 ℃/min. The invention is believed to utilize the characteristic that fresh bear bile is liquid, adopts the miscibility of ethanol and bile, utilizes the killing effect of ethanol on viruses and pathogenic bacteria, and controls the content and time of ethanol in the liquid medicine, so as to not destroy active ingredients in the bear bile, kill various pathogenic bacteria and viruses, ensure the safety and effectiveness of clinical medication, overcome the defect of traditional production of bear gall powder, and the obtained bear gall powder is golden yellow in appearance, has transparent luster and greatly improves the quality of bear gall powder.

CN106386659a (201610755739.2, shiyou) discloses a method for industrial production of bear gall powder, which comprises the steps of selecting fine variety of black bear, raising, taking gall, feeding Chinese herbal medicine, and ultra-low temperature freeze drying, and specifically comprises the following steps: (1) selection of black bear fine breeds: black bear with strong body, obvious variety characteristics, strong feeding power, good production performance, strong physique, strong limb and hoof and over 3 years old is selected as the cultured bear; (2) feeding: feeding the cultured bear by adopting a compound feed, wherein the compound feed comprises the following components in percentage by mass: 50-60 parts of corn, 3-5 parts of fish meal, 10-15 parts of barley, 10-15 parts of fried soybean, 3-5 parts of meat and 3-5 parts of silkworm chrysalis; feeding until the weight of the black bear reaches 90 kg or more, and taking the gallbladder; (3) taking the liner: adopting an animal autologous catheter painless drainage method to harvest the gall bladder, specifically, utilizing the tissue of the black bear to manufacture an annular sphincter at the abdominal wall end between the abdominal wall and the gall bladder of the black bear, closing the lumen when the sphincter contracts, opening the lumen when the sphincter relaxes, allowing the sphincter to be subjected to the regulation of vegetative nerves or hormone, using the characteristic of the sphincter to feed nutrient solution to the black bear when the gall bladder is harvested, opening the lumen, sterilizing the opened lumen passage by using an alcohol cotton ball, probing the sterilized stainless steel hollow probe into the lumen passage, automatically flowing the gall into a cup for receiving the gall along the hollow probe, and pulling out the probe after the gall is harvested; (4) feeding Chinese herbal medicines: after the gallbladder is taken, the black bear is fed with the Chinese herbal medicine added with syrup water, and the black bear is continuously fed for 10 days; the Chinese herbal medicine formula comprises the following components: 20-30 parts of magnolia officinalis, 20-30 parts of green tangerine peel, 20-30 parts of cinnamon and 20-30 parts of spinach; 20-30 parts of polygonum multiflorum, 20-30 parts of liquorice, 20-30 parts of dyers woad leaf and 20-30 parts of radix isatidis; 20-30 parts of corn gluten meal, 20-30 parts of soybean meal, 20-30 parts of walnut meal and 20-30 parts of peanut cypress; 40-50 parts of glossy privet fruit, 40-50 parts of coix seed, 5-10 parts of dried orange peel and 5-10 parts of ginger; pulverizing the above Chinese herbal medicines, mixing, adding syrup water, and feeding black bear; (5) ultra-low temperature freeze drying: freezing the obtained fel Ursi at-60deg.C or below, and vacuum sublimating the water to obtain dry fel Ursi powder. The bear gall powder prepared by the method has good quality, high yield and no harm to the body of a black bear.

CN102114044a (201010617773.6, kuzhentang) discloses a method for extracting bear bile, which comprises cutting a wound on the gall bladder of bear, forming a fistula with muscle tissue, and fastening; after healing, the fistula is extruded to collect bile. The method can be used for discontinuous, timed and quantitative collection and long-term collection, and can not influence the utilization of bile of bear. The invention discloses a high-quality bear gall powder and a preparation method thereof, wherein the bear gall powder presents bright yellow fluorescence under a 365nm ultraviolet lamp, and has 3 chromatographic peaks under 198 nm; the preparation method comprises standing bile, separating, drying, pulverizing, and sieving. The bear gall powder prepared by the invention is believed to have pure appearance and color, high content of tauroursodeoxycholic acid, no moisture absorption and easy storage, transportation and processing.

CN103040869a (201310027917.6, kang Ao) discloses artificial bear gall powder, which is prepared by taking poultry gall as a raw material and adding sodium tauroursodeoxycholate, wherein the weight ratio of the sodium tauroursodeoxycholate to the poultry gall is as follows: 20-40 parts of bezoar ursodeoxycholic acid sodium and 80-60 parts of fowl gall. The invention also provides a preparation method of the artificial bear gall powder. The invention has the advantages of scientific and unique formula, easily obtained raw materials, simple process, low cost, quality assurance, curative effect assurance and wild animal protection, and the internal quality and the appearance are almost similar to those of the natural bear gall, so that the artificial bear gall powder which is simple, convenient, economical, safe and environment-friendly, has the quality and the appearance very similar to those of the natural bear gall is provided, and is an ideal substitute for the natural bear gall.

CN1311002a (00102073.0, dan Lixia) discloses refined bear gall powder and a preparation method thereof, which belong to the field of pharmacy, the common bear gall powder is easy to absorb moisture and agglomerate, has tooth sticking feeling, extremely fishy smell, extremely bitter, more impurities, slow absorption, unstable property and easy putrefaction, has low content of effective components, limits the application range of the bear gall powder, and is often added with flavoring agents for oral administration, thereby relieving fishy smell. The invention adopts an ethanol extraction method, an ethanol extraction active carbon decoloration method and an ethyl acetate separation method to purify and refine bear gall powder, and the refined bear gall powder has light and fine color, no sticky tooth feel, no fishy smell, light bitter taste, difficult putrefaction, difficult moisture absorption and caking, stable property and average content of active ingredients increased to more than 2 times. The storage time is prolonged, the application range is enlarged, and the curative effect of the medicine is improved.

CN106038601a (2016610368003. X, large and easy) discloses a preparation method of bear gall powder with high content, high purity and low fishy smell, which comprises the steps of fully mixing bear gall powder, active carbon and filter aid, extracting the mixture with ethanol solution until cholanic acid in the extracting solution is negative, collecting ethanol extracting solution, concentrating and recovering ethanol, collecting concentrated solution, and drying; the filter aid is a silicate mineral that is insoluble in water and alcohol. The method is simple, has better operability and controllability, the content of the prepared bear gall powder calculated by the tauroursodeoxycholic acid can reach more than 45 percent, the recovery rate is nearly 100 percent, the recovery rate calculated by the weight can reach more than 85 percent, the method is suitable for industrialized mass production, the cost is low, and the method can be used for preparing the high-quality high-content high-purity low-fishy smell bear gall powder required by special preparation enterprises.

However, there is still a need in the art for new methods for preparing bear gall powder having superior properties, in particular for obtaining bear gall powder containing highly pure conjugated bile acids in high yields, and for medical and health care uses of such bear gall powder, for example, for preventing or treating gall stone diseases, for preventing or treating cholecystitis, and for improving gall bladder function.

Disclosure of Invention

The present invention aims to provide a novel process for producing bear gall powder having excellent properties, and in particular to a process for obtaining bear gall powder containing high purity conjugated bile acid in high yield. The aim of the invention is achieved by the following scheme.

In a first aspect, the present invention provides a method for preparing refined bear gall powder, comprising the steps of:

(1) Diluting collected bear bile with water (for example, diluting with 2-3 times volume of water, for example, diluting with 2.5 times volume of water), filtering with a 80-mesh screen, (optionally, adjusting the pH of the filtrate obtained by filtering with a 80-mesh screen to be 3.0-3.5 such as pH=3.3 by using a 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride such as 1.2% sodium chloride to the filtrate) to obtain crude bear gall (the content of tauroursodeoxycholic acid and tauchenodeoxycholic acid in the process intermediate can be determined);

(2) Using a tangential flow ultrafiltration system (such as a Shibi pure KR2i type tangential flow ultrafiltration system), flushing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall obtained in the step (1) to obtain filtrate and 5-15 times (such as 10 times) concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);

(3) Adjusting the filtrate obtained in the previous step to have a ph=6.5 to 7.0, for example, ph=6.8, using a 1M sodium hydroxide solution, then adding arginine (in an amount 2 to 3 mol times, for example, 2.5 mol times, the amount of tauroursodeoxycholic acid in the filtrate), stirring at a temperature of 40 to 50 ℃ for 2 to 3 hours, for example, 44 to 46 ℃ for 2.5 hours, filtering the precipitate, then adding 1 to 2 times, for example, 1.5 times volume of ethyl acetate to the filtrate, standing for 2 to 4 hours, for example, 3 hours, precipitating a precipitate, filtering, discarding the filtrate to obtain a precipitate;

(4) Adding ethanol to the precipitate obtained in the previous step (for example, adding ethanol in a volume ratio of 1g: 3-5 ml of ethanol to 1g:4ml of the precipitate), stirring at room temperature for 0.5 hours, standing for 2-4 hours, for example, 3 hours, and filtering out the precipitate to obtain filtrate;

(5) Adding 1-2 times of ethyl acetate-diethyl ether (5:1) mixed solution with the volume of 1-2 times of the volume of the filtrate obtained in the previous step, standing for 5-8 hours, such as 6 hours, separating out precipitate, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined bear gall powder.

The method according to the first aspect of the present invention, wherein in step (1), the filtrate obtained by filtering the 80 mesh sieve is adjusted to have a ph=3.0 to 3.5 using a 1M hydrochloric acid solution.

The method according to the first aspect of the present invention, wherein in step (1), after adjusting the filtrate to have ph=3.0 to 3.5, 1.0 to 1.5% sodium chloride is further added to the filtrate. It has surprisingly been found that by adjusting the filtrate to a ph=3.0-3.5 and adding a specified amount of sodium chloride thereto, it is possible to let the tauroursodeoxycholic acid into the filtrate fraction when subsequently subjected to tangential flow ultrafiltration, while the other bound cholic acids are mostly fed into the concentrated reflux.

The process according to the first aspect of the present invention, wherein in step (2), taurochenodeoxycholic acid is 0 to 5%, preferably 0 to 3%, preferably 0 to 2% by weight of taurochenodeoxycholic acid in the filtrate obtained by subjecting to tangential flow ultrafiltration. That is, in the obtained filtrate, taurochenodeoxycholic acid was substantially absent.

The process according to the first aspect of the present invention, wherein in step (2), the tauroursodeoxycholic acid is 0 to 8%, preferably 1 to 5%, preferably 1 to 3% by weight of tauroursodeoxycholic acid in the reflux liquid obtained by subjecting to tangential flow ultrafiltration. That is, there was substantially no residual tauroursodeoxycholic acid in the resulting reflux liquid. The taurochenodeoxycholic acid and tauroursodeoxycholic acid may be separated by tangential flow ultrafiltration, which is performed in step (2) above.

The process according to the first aspect of the present invention, wherein the content of tauroursodeoxycholic acid in the refined bear gall powder obtained in step (5) is more than 70%, e.g. 70-74%, in particular more than 71%, e.g. 71-74%, in particular more than 72%, e.g. 72-74%.

The method according to the first aspect of the present invention, wherein the molar ratio of tauroursodeoxycholic acid to arginine in the refined bear gall powder obtained in the step (5) is 1:0.98 to 1.02, in particular 1:0.99 to 1.01.

The method according to the first aspect of the present invention, wherein the total amount of tauroursodeoxycholate and arginine in the refined bear gall powder obtained in step (5) is more than 95%, e.g. 95-100%, in particular more than 96%, e.g. 96-100%, in particular more than 97%, e.g. 97-100%, in particular more than 98%, e.g. 98-100% of the total amount of the refined bear gall powder.

The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in the step (5) has a melting point of 187 to 189 ℃. From the above results, it was confirmed that the refined bear gall powder obtained by the present invention was an arginine salt of tauroursodeoxycholate having a melting point of 187 to 189 ℃.

The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in step (5) has diffraction peaks at about 8.53 °, about 10.96 °, about 12.03 °, about 13.14 °, about 14.82 °, about 17.26 °, about 22.53 °, about 24.21 °, about 26.68 °, about 29.42 °, about 31.24 ° in a powder X-ray diffraction pattern expressed in terms of 2θ using Cu-kα radiation.

The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in the step (5) has diffraction peaks at 8.53±0.20°, 10.96±0.20°, 12.03±0.20°, 13.14±0.20°, 14.82±0.20°, 17.26±0.20°, 22.53±0.20 °, 24.21±0.20 °, 26.68±0.20°, 29.42 ±0.20°, 31.24±0.20° in a powder X-ray diffraction pattern expressed in terms of 2θ using cu—kα radiation.

The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in the step (5) has diffraction peaks at 8.53±0.10°, 10.96±0.10°, 12.03±0.10°, 13.14±0.10°, 14.82±0.10°, 17.26±0.10°, 22.53±0.10 °, 24.21±0.10 °, 26.68±0.10°, 29.42 ±0.10°, 31.24±0.10° in a powder X-ray diffraction pattern expressed in terms of 2θ using cu—kα radiation.

The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in the step (5) has a powder X-ray diffraction pattern shown in fig. 1 using Cu-ka radiation.

Further, the second aspect of the present invention provides a refined bear gall powder which is arginine salt of tauroursodeoxycholate having a melting point of 187-189 ℃.

The refined bear gall powder according to the second aspect of the present invention, wherein the content of tauroursodeoxycholic acid is more than 70%, e.g. 70-74%, in particular more than 71%, e.g. 71-74%, in particular more than 72%, e.g. 72-74%.

The refined bear gall powder according to the second aspect of the present invention, wherein the molar ratio of tauroursodeoxycholic acid to arginine is 1:0.98 to 1.02, in particular 1:0.99 to 1.01.

The refined bear gall powder according to the second aspect of the present invention, wherein the percentage of total amount of tauroursodeoxycholic acid to total amount of arginine is more than 95%, e.g. 95-100%, in particular more than 96%, e.g. 96-100%, in particular more than 97%, e.g. 97-100%, in particular more than 98%, e.g. 98-100% of the total amount of the refined bear gall powder.

The refined bear gall powder according to the second aspect of the present invention, which uses Cu-ka radiation, has diffraction peaks at about 8.53 °, about 10.96 °, about 12.03 °, about 13.14 °, about 14.82 °, about 17.26 °, about 22.53 °, about 24.21 °, about 26.68 °, about 29.42 °, about 31.24 ° in a powder X-ray diffraction pattern expressed in terms of 2θ angle.

The refined bear gall powder according to the second aspect of the present invention, which uses Cu-ka radiation, has diffraction peaks at 8.53±0.20°, 10.96±0.20°, 12.03±0.20°, 13.14±0.20°, 14.82±0.20°, 17.26±0.20°, 22.53±0.20°, 24.21±0.20°, 26.68±0.20°, 29.42 ±0.20°, 31.24±0.20° in a powder X-ray diffraction pattern expressed in terms of 2θ angle.

The refined bear gall powder according to the second aspect of the present invention, which uses Cu-ka radiation, has diffraction peaks at 8.53±0.10°, 10.96±0.10°, 12.03±0.10°, 13.14±0.10°, 14.82±0.10°, 17.26±0.10°, 22.53±0.10°, 24.21±0.10°, 26.68±0.10°, 29.42 ±0.10°, 31.24±0.10° in a powder X-ray diffraction pattern expressed in terms of 2θ angle.

The refined bear gall powder according to the second aspect of the present invention, which uses Cu-ka radiation, has a powder X-ray diffraction pattern shown in fig. 1.

The refined bear gall powder according to the second aspect of the invention is prepared according to the method comprising the following steps:

The refined bear gall powder according to the second aspect of the present invention, wherein in the step (1), the filtrate obtained by filtering the 80 mesh sieve is adjusted to have a ph=3.0 to 3.5 using a 1M hydrochloric acid solution.

The refined bear gall powder according to the second aspect of the present invention, wherein in step (1), 1.0 to 1.5% sodium chloride is further added to the filtrate after the filtrate is adjusted to have ph=3.0 to 3.5. It has surprisingly been found that by adjusting the filtrate to a ph=3.0-3.5 and adding a specified amount of sodium chloride thereto, it is possible to let the tauroursodeoxycholic acid into the filtrate fraction when subsequently subjected to tangential flow ultrafiltration, while the other bound cholic acids are mostly fed into the concentrated reflux.

The refined bear gall powder according to the second aspect of the present invention, wherein in the filtrate obtained by subjecting to tangential flow ultrafiltration in the step (2), taurochenodeoxycholic acid is 0 to 5%, preferably 0 to 3%, preferably 0 to 2% by weight of taurochenodeoxycholic acid. That is, in the obtained filtrate, taurochenodeoxycholic acid was substantially absent.

The refined bear gall powder according to the second aspect of the present invention, wherein in the reflux liquid obtained by subjecting to tangential flow ultrafiltration in the step (2), tauroursodeoxycholic acid is 0 to 8%, preferably 1 to 5%, preferably 1 to 3% by weight of tauroursodeoxycholic acid. That is, there was substantially no residual tauroursodeoxycholic acid in the resulting reflux liquid. The taurochenodeoxycholic acid and tauroursodeoxycholic acid may be separated by tangential flow ultrafiltration, which is performed in step (2) above.

Further, a third aspect of the present invention provides a compound of formula I:

the compound according to the third aspect of the invention, which has a melting point of 187-189 ℃.

The compound according to the third aspect of the invention, wherein the tauroursodeoxycholic acid content is more than 70%, e.g. 70-74%, in particular more than 71%, e.g. 71-74%, in particular more than 72%, e.g. 72-74%.

The compound according to the third aspect of the invention, wherein the molar ratio of tauroursodeoxycholic acid to arginine is 1:0.98 to 1.02, in particular 1:0.99 to 1.01.

The compound according to the third aspect of the present invention, which uses Cu-ka radiation, has diffraction peaks at about 8.53 °, about 10.96 °, about 12.03 °, about 13.14 °, about 14.82 °, about 17.26 °, about 22.53 °, about 24.21 °, about 26.68 °, about 29.42 °, about 31.24 ° in a powder X-ray diffraction pattern expressed in terms of 2θ angle.

The compound according to the third aspect of the present invention, which uses cu—kα radiation, has diffraction peaks at 8.53±0.20°, 10.96±0.20°, 12.03±0.20°, 13.14±0.20°, 14.82±0.20°, 17.26±0.20°, 22.53±0.20°, 24.21±0.20°, 26.68±0.20°, 29.42 ±0.20°, 31.24±0.20° in a powder X-ray diffraction pattern expressed in terms of 2θ angle.

The compound according to the third aspect of the present invention, which uses cu—kα radiation, has diffraction peaks at 8.53±0.10°, 10.96±0.10°, 12.03±0.10°, 13.14±0.10°, 14.82±0.10°, 17.26±0.10°, 22.53±0.10°, 24.21±0.10°, 26.68±0.10°, 29.42 ±0.10°, 31.24±0.10° in a powder X-ray diffraction pattern expressed in terms of 2θ.

The compound according to the third aspect of the present invention, which uses Cu-ka radiation, has a powder X-ray diffraction pattern as shown in fig. 1.

The compound according to the third aspect of the invention, which is prepared according to a process comprising the steps of:

(5) Adding 1-2 times volume of ethyl acetate-diethyl ether (5:1) mixed solution, standing for 5-8 hours, such as 6 hours, to the filtrate obtained in the previous step, precipitating, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain the compound of the formula I.

The compound according to the third aspect of the present invention, wherein in step (1), the filtrate obtained by filtering the 80-mesh sieve is adjusted to have a ph=3.0 to 3.5 using a 1M hydrochloric acid solution.

The compound according to the third aspect of the present invention, wherein in step (1), 1.0 to 1.5% sodium chloride is further added to the filtrate after the filtrate is adjusted to have ph=3.0 to 3.5. It has surprisingly been found that by adjusting the filtrate to a ph=3.0-3.5 and adding a specified amount of sodium chloride thereto, it is possible to let the tauroursodeoxycholic acid into the filtrate fraction when subsequently subjected to tangential flow ultrafiltration, while the other bound cholic acids are mostly fed into the concentrated reflux.

The compound according to the third aspect of the present invention, wherein in step (2), taurochenodeoxycholic acid is 0 to 5%, preferably 0 to 3%, preferably 0 to 2% by weight of taurochenodeoxycholic acid in the filtrate obtained by subjecting to tangential flow ultrafiltration. That is, in the obtained filtrate, taurochenodeoxycholic acid was substantially absent.

The compound according to the third aspect of the present invention, wherein in step (2), tauroursodeoxycholic acid is 0 to 8%, preferably 1 to 5%, preferably 1 to 3% by weight of tauroursodeoxycholic acid in the reflux liquid obtained by subjecting to tangential flow ultrafiltration. That is, there was substantially no residual tauroursodeoxycholic acid in the resulting reflux liquid. The taurochenodeoxycholic acid and tauroursodeoxycholic acid may be separated by tangential flow ultrafiltration, which is performed in step (2) above.

Further, the fourth aspect of the present invention provides the use of the refined bear gall powder of any one of the second aspect of the present invention or the compound of the third aspect for the preparation of a product for preventing or treating gall-stone diseases, preventing or treating cholecystitis, and improving gall-bladder function.

Tauroursodeoxycholic acid (tauroursodeoxycholic acid, TUDCA), chemical name 2- [ [ (3α,5β,7β) -3, 7-dihydroxy-24-oxocholestan-24-yl ] amino ] ethanesulfonic acid dihydrate, also expressed as 3α,7β -dihydroxycholanoyl-N-taurine, CAS No:14605-22-2. The chemical structural formula of tauroursodeoxycholic acid is as follows:

the molecular formula: C26H45NO6S, molecular weight: 499.7

TUDCA is found from fel Ursi in 1902, and is the main bile acid in fel Ursi, and has spasmolytic, anticonvulsant, antiinflammatory and cholelithiasis dissolving effects. Tauroursodeoxycholic acid is an active ingredient of bear bile, is developed by the pharmaceutical factory of Bedsidi in Italy, is marketed in Italy for the first time in 1991, is approved to be sold in China under the trade name taurolite (taurocolite) in 2007, and is clinically mainly used for treating cholecystolithiasis, primary sclerosing cholangitis, primary biliary cirrhosis, chronic viral hepatitis C and the like. Clinical researches show that compared with ursodeoxycholic acid, the tauroursodeoxycholic acid has the advantages of high litholytic speed, high total dissolution rate and no obvious adverse reaction.

The invention carries out finish machining treatment on the bear gall powder, and the obtained refined bear gall powder presents one or more excellent performances.

Drawings

FIG. 1 is a typical powder X-ray diffraction pattern of the refined bear gall powder of the present invention.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. The following examples further illustrate the invention, but do not limit it.

In the present invention, if it is used to A) the refined bear gall powder of the present invention of example 1 (i.e., arginine salt of tauroursodeoxycholate in the form of T), B) the refined bear gall powder of group 2 (arginine salt of tauroursodeoxycholate), C) taurolite, when their dosages are described, all the dosages are converted into the amount of tauroursodeoxycholate, unless otherwise specified; for oral administration, as not otherwise stated, the powder was ground to a fine powder that could pass through an 80 mesh screen prior to administration, and suspended in 2% sodium carboxymethyl cellulose at a concentration of 2% tauroursodeoxycholate prior to administration.

The method for measuring the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in various materials comprises the following steps: reference Zhanghua (Zhanghua, et al, HPLC fingerprint method for determination of bile acid component in bear gall bladder, J.Hua.Western journal, 2009,24 (4): 402-403) describes HPLC method described in "experimental section", and external standard method of tauroursodeoxycholic acid control and tauchenodeoxycholic acid control (both purchased from Chinese food and drug administration institute) was used to calculate peak area. When determining the arginine salt of tauroursodeoxycholate, the arginine salt of tauroursodeoxycholate dissociates the free tauroursodeoxycholate in the mobile phase and the retention time is consistent with that of a reference substance, and the arginine does not influence the determination of other substances in the HPLC method after determination.

The method for measuring the arginine content in various materials comprises the following steps: reference Liu Rui (Liu Rui, et al, HPLC method for determining arginine content in ibuprofen injection, J.North-west pharmaceutical journal, 2013, 28 (4): 361) section 2.1.4 to section 2.1.10) and the specificity, linearity, precision, repeatability, stability, recovery rate meet the requirements of common analytical methods, and tauroursodeoxycholic acid does not affect the determination of arginine.

Example 1: preparing refined bear gall powder

(1) Diluting (2.5 times volume of water is added) collected fel Ursi (when it is subjected to reduced pressure drying to obtain powder, determining the content of taurochenoxycholic acid and taurochenoxycholic acid in the powder to be 22.63% and 16.31% respectively, wherein the data are the percentage content of two substances in solid matters of fel Ursi) with water, filtering with a 80-mesh screen, regulating the pH value of the filtrate obtained by filtering with a 1M hydrochloric acid solution to be 3.3, adding 1.2% sodium chloride into the filtrate to obtain crude fel Ursi solution (the crude fel Ursi solution is subjected to reduced pressure drying to obtain powder, the content of taurochenoxycholic acid and taurochenoxycholic acid in the powder is determined to be 28.47% and 20.33% respectively, which shows that the ratio of the two substances is basically unchanged from the solid matters before filtration, but the two substances are concentrated due to the filtration;

(2) Using a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system), flushing a pipeline with ultrapure water, installing a 1kD midi Kros filter, and filtering the crude bear gall solution obtained in the step (1) to obtain filtrate and 10 times of concentrated reflux liquid (measuring the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate or powder obtained by decompression drying);

(3) Adjusting the ph=6.8 of the filtrate obtained in the previous step by using a 1M sodium hydroxide solution, then adding arginine (the amount of which is 2.5 mol times of the amount of tauroursodeoxycholic acid in the filtrate), stirring at 44-46 ℃ for 2.5 hours, filtering to remove the precipitate, then adding ethyl acetate with the volume being 1.5 times of that of the filtrate, standing for 3 hours, precipitating the precipitate, filtering, removing the filtrate to obtain the precipitate;

(4) Adding ethanol into the precipitate obtained in the previous step (for example, adding ethanol in a volume ratio of 1g to 4ml of the weight of the precipitate), stirring at room temperature for 0.5 hour, standing for 3 hours, and filtering out the precipitate to obtain filtrate; (As known to those skilled in the art, when referring to ethanol, unless indicated to the concentration, it refers to 98% ethanol

(5) Adding ethyl acetate-diethyl ether (5:1) mixed solution with the volume of 2 times to the filtrate obtained in the previous step, standing for 6 hours, separating out precipitate, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined fel Ursi powder.

In the above process, it is determined that:

in step (2), taurochenodeoxycholic acid is 0.76% by weight of taurochenodeoxycholic acid in the filtrate obtained by tangential flow ultrafiltration, indicating that taurochenodeoxycholic acid is substantially absent in the obtained filtrate;

in the step (2), the content of tauroursodeoxycholic acid in the filtrate obtained by tangential flow ultrafiltration is measured, and the mass of tauroursodeoxycholic acid is calculated by combining the volume of the filtrate, and compared with the mass of tauroursodeoxycholic acid in the bear bile fed in the step (1), the recovery rate is 98.26%, which shows that the tauroursodeoxycholic acid after tangential flow ultrafiltration has extremely high recovery rate;

in the step (2), the filtrate obtained by tangential flow ultrafiltration is subjected to partial decompression and drying to remove the solvent to obtain a powder solid, and the determination shows that the tauroursodeoxycholic acid in the solid accounts for 83.68 percent of the weight of the solid, so that the tauroursodeoxycholic acid can be remarkably enriched in the filtrate by tangential flow ultrafiltration;

in the step (2), tauroursodeoxycholic acid is 1.14% of the weight of tauroursodeoxycholic acid in the reflux liquid obtained by tangential flow ultrafiltration, which shows that the reflux liquid has no tauroursodeoxycholic acid residue basically; subjecting to tangential flow ultrafiltration in step (2) above, taurochenodeoxycholic acid and tauroursodeoxycholic acid may be separated;

In the refined bear gall powder obtained in the step (5), the content of tauroursodeoxycholic acid is 73.62 percent;

in the refined bear gall powder obtained in the step (5), the mol ratio of the tauroursodeoxycholic acid to the arginine is 1:1.004;

in the refined bear gall powder obtained in the step (5), the total amount of tauroursodeoxycholic acid and arginine is 99.27 percent of the total amount of the refined bear gall powder;

compared with the quality of tauroursodeoxycholic acid in the bear bile fed in the step (1), the refined bear gall powder obtained in the step (5) has 94.51 percent of recovery rate of tauroursodeoxycholic acid in the whole process from the step (1) to the step (5), which shows that the method has very high recovery rate of tauroursodeoxycholic acid;

the melting point of the refined bear gall powder obtained in the step (5) is 187-189 ℃.

From the above results, it was confirmed that the refined bear gall powder obtained by the present invention is an arginine (1:1) salt of tauroursodeoxycholate having a melting point of 187 to 189 ℃.

The diffraction pattern of the crystals was determined using the following powder X-ray diffraction analysis method: rigaku Dmax/2400 powder X diffractometer; cu-K alpha radiation, a graphite monochromator, a 40kV/40mA scanning range of 2 theta is 5-40 degrees, the scanning speed is 4 degrees/min, and the step size is 0.01 degrees; the scanning mode is continuous scanning; slit arrangement: exit slit DS:1/2 ° anti-scatter slit: SS 1/2 °; RS is 0.3mm.

The powder X-ray diffraction pattern of the refined bear gall powder obtained in the step (5) of the present example 1 is shown in fig. 1, and the partial typical diffraction angle data of fig. 1 are as follows: powder X-ray diffraction patterns expressed in terms of 2θ have diffraction peaks at 8.53 ° (22.0%, relative abundance, the same applies below), 10.96 ° (100.0%), 12.03 ° (39.5%), 13.14 ° (19.7%), 14.82 ° (45.3%), 17.26 ° (33.3%), 22.53 ° (69.3%), 24.21 ° (24.6%), 26.68 ° (46.8%), 29.42 ° (61.2%), 31.24 ° (49.5%). This shows that the refined bear gall powder obtained in the step (5) of the present example 1 shows a typical crystalline form, which may be referred to as a T-form or may be referred to as (i.e., T-form tauroursodeoxycholate arginine salt, etc. is called as a T-form in the present invention).

The refined bear gall powder (i.e. T crystal form) obtained in the step (5) of the present example 1 is essentially a compound shown as the following formula I, namely tauroursodeoxycholate arginine salt (1:1):

example 2: preparing refined bear gall powder

Steps (1) to (4) were carried out as in example 1; in the step (5), the ethyl acetate-diethyl ether (5:1) mixed solution is changed into ethyl acetate, and the precipitate is dried under reduced pressure to remove the solvent, so as to obtain the refined bear gall powder. The melting point of the refined bear gall powder is 162-163 ℃ through measurement, and the difference of the melting points shows that the refined bear gall powder has different crystal forms from the refined bear gall powder of the example 1.

Example 3: preparing refined bear gall powder

Steps (1) to (4) were carried out as in example 1; in the step (5), the ethyl acetate-diethyl ether (5:1) mixed solution is changed into diethyl ether, and the precipitate is dried under reduced pressure to remove the solvent, so as to obtain the refined bear gall powder. The melting point of the refined bear gall powder is 173-175 ℃ by measurement, and the difference of the melting points shows that the refined bear gall powder has different crystal forms from the refined bear gall powder of the example 1.

The results of examples 2 and 3 above show that the use of ethyl acetate-diethyl ether (5:1) mixture in step (5) results in a T-form having a melting point of 187-189℃which is different from the crystals obtained without using this mixture.

The three kinds of refined bear gall powder obtained in the examples 1-3 are placed at the temperature of 45 ℃ for 3 months, and then the melting points of the refined bear gall powder are measured, so that the melting points of the three batches of refined bear gall powder are 188-189 ℃, 168-174 ℃ and 180-185 ℃ respectively, and the result shows that the product of the example 1 is stable, while the melting points of the products of the examples 2-3 show great change and the melting range is large, thus indicating that the stability of the product has defects.

Example 4: preparing refined bear gall powder

(1) Diluting (2.5 times volume of water is added) collected bear bile (content of taurochenoxycholic acid and taurochenoxycholic acid in solid matters is respectively 22.17% and 17.15%) by adding water, filtering by using a 80-mesh screen, and adding 1.2% sodium chloride into filtrate to obtain crude bear gall (content of taurochenoxycholic acid and taurochenoxycholic acid in solid matters is respectively 28.23% and 20.07%);

(2) Using tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system), flushing pipeline with ultrapure water, installing 1kD MidiKros filter, and filtering the crude bear gall solution obtained in step (1) to obtain filtrate and 10 times concentrated reflux liquid.

The recovery rate of the filtrate obtained by the tangential flow ultrafiltration in the step (2) is 42.53% compared with the mass of the tauroursodeoxycholic acid in the bear bile fed in the step (1), which shows that the recovery rate of the tauroursodeoxycholic acid after the tangential flow ultrafiltration is quite low. Because of this very low recovery, this example does not continue with subsequent operations.

Example 5: preparing refined bear gall powder

(1) Diluting (2.5 times volume of water is added) collected bear bile (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the solid matters is 22.43% and 16.84%) by adding water, filtering by using a 80-mesh screen, and regulating the pH value of the filtrate obtained by filtering by using a 1M hydrochloric acid solution to be 3.3 to obtain crude bear gall liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the solid matters is 28.72% and 20.65% respectively);

The recovery rate of the filtrate obtained by the tangential flow ultrafiltration in the step (2) is 56.14% compared with the mass of the tauroursodeoxycholic acid in the bear bile fed in the step (1), which shows that the recovery rate of the tauroursodeoxycholic acid after the tangential flow ultrafiltration is quite low. Because of this very low recovery, this example does not continue with subsequent operations.

In addition, it was determined that in the filtrates obtained by subjecting step (2) of examples 4 and 5 to tangential flow ultrafiltration, taurochenodeoxycholic acid was 34.52% and 41.17% by weight of taurochenodeoxycholic acid, respectively, indicating that a large proportion of taurochenodeoxycholic acid entered the filtrate.

From the results of example 4 and example 5, it can be seen that in performing tangential flow ultrafiltration, the solution adjustment of ph=3.3 while adding sodium chloride thereto is beneficial for improving recovery of tauroursodeoxycholic acid. In addition, the present inventors have not explained that although taurochenodeoxycholic acid and tauroursodeoxycholic acid and other conjugated bile acids may have similarities in chemical structure or performance, they exhibit significantly different filtration behaviors in tangential flow ultrafiltration processes, and it seems that such differences in filtration behaviors have a certain correlation with ph=3.3 and treatment of sodium chloride, and although these results do not affect the contribution of the present invention to the prior art, the present inventors have still expected to be able to explain the above phenomena in the near future with advances in technology.

Experimental example 1: oral bioavailability of refined bear gall powder

Test article: refined fel Ursi powder obtained in example 1, refined fel Ursi powder obtained in example 2, and commercially available tauroursodeoxycholic acid capsule (taurolite, H20150398, 250 mg/granule). The weight of the powder in each capsule of taurolidine is measured to be about 250mg, which indicates that no auxiliary material or only a trace of auxiliary material is added. Examples 1 and 2 refined bear gall powder and taurolite were each ground to a fine powder which was able to pass through an 80 mesh sieve prior to administration, and each was suspended in 2% sodium carboxymethyl cellulose at a concentration of 2% prior to administration.

Animals: 24 male Japanese white rabbits (supplied by the experimental animal science center of Jiangxi university of Chinese medicine) are divided into three groups at random, wherein the group A is provided with refined bear gall powder in example 1, the group B is provided with refined bear gall powder in example 2, and the group C is provided with taurolite.

Administration and blood sampling and blood sample treatment: 1ml of blood (auricular vein) was taken as a blood sample at 0 time before administration; the administration dosage is calculated by tauroursodeoxycholic acid, 50mg tauroursodeoxycholic acid is administrated by stomach irrigation at the dosage of 50mg tauroursodeoxycholic acid/kg body weight, and blood is taken from the patient in the same way for 0.5h, 1h, 2h, 4h, 6h, 10h, 15h, 21h and 30h respectively; the blood sample is centrifuged to obtain 0.5mL of plasma, then the blood sample is extracted by shaking with methanol, the precipitate is removed by centrifugation, the methanol extraction is repeated three times, and the methanol is volatilized after the methanol solution is combined for measurement.

Reference Yang Xinxin (Yang Xinxin, et al, HPLC method, HPLC chromatographic conditions for slow release eye drop gel for bear gall rabbit eye pharmacokinetic study, time-series national medicine, 2017, 28 (7): 1634): the chromatographic column is Di-Ma C18 (5 μm,4.6mm×250 mm), the mobile phase is sodium dihydrogen phosphate buffer salt solution (0.03 mol/L, pH value 4.4) methanol=62:38, the flow rate is 1.0ml/min, the detection wavelength is 210nm, the column temperature is 30 ℃, and the sample injection amount is 20 μl. Tauroursodeoxycholic acid control was purchased from Sigma-Aldrich, and the control and blood sample treatments were dissolved in methanol and diluted with mobile phase for HPLC determination. The specificity, linearity, precision, stability, detection limit, quantitative limit and recovery rate of the HPLC method all meet the analysis requirements of common biological samples.

Blood samples at each time point were measured, and based on the blood concentration at each time point, curve data at the time of blood concentration was processed by 3P87 software and an open two-chamber model to obtain an area under the curve peak AUC (0- > +), a maximum blood concentration Cmax and a fitted peak time Tmax, and the relative bioavailability of each sample to the commercial product was calculated by AUC, and the results were shown in the following table (expressed as mean.+ -. Sd):

sample of	Tmax(h)	Cmax(μg/mL)	AUC(μg/mL/h)	Relative bioavailability (%)
					Example 1	2.83±0.38	934.26±56.81	126 937.63	151.7
Example 2	3.11±0.62	742.63±47.52	79 348.57	94.9
					Taurolite	3.28±0.33	717.59±67.28	83 648.93	100

From the above results, it can be seen that the bioavailability of the product of example 1 is far higher than that of the commercially available product and the product of example 2 with a different crystal form.

In view of the fact that the arginine tauroursodeoxycholate of the present invention is in a dissociated state, i.e., free tauroursodeoxycholate, after absorption into the blood circulation, will exert the due biological effects of its active ingredients as taurolite. For example, the product of the invention, tauroursodeoxycholate arginine salt, can be used for dissolving diseases such as cholesterol calculus and the like.

Example 2: hypolipidemic effect of refined bear gall powder

1. Material

Test article: refined fel Ursi powder obtained in example 1, refined fel Ursi powder obtained in example 2, and commercially available tauroursodeoxycholic acid capsule (taurolite, H20150398, 250 mg/granule). Examples 1 and 2 refined bear gall powder and taurolite were each ground to a fine powder which was able to pass through an 80 mesh sieve prior to administration, and each was suspended in 2% sodium carboxymethyl cellulose at a concentration of 2% prior to administration.

Cholesterol was purchased from Sigma-Aldrich company under product number C8667 with purity > 99%; cholic acid was purchased from Sigma-Aldrich company under the product number C1129 with purity not less than 98%; methyl thiouracil (commercially available tablet, H32022717).

Animals: male mouse of Kunming species with weight of 18-22 g, wistar male rat with weight of 180-220 g, and experimental animal science center of Jiangxi university.

2. Effects on blood lipid in normal rats

Rats were taken 40, randomly divided into 5 groups of 8.

Blank control group: 2% sodium carboxymethylcellulose (the volume is the same as that of the refined bear gall powder of example 1),

positive control group: the medicine is compound three-dimensional linoleic acid capsule I (clinically used hypolipidemic) 0.6 granules/kg (equivalent to 10 times of the clinical common dosage),

example 1 group: example 1 refined bear gall powder (i.e. T crystal form) 50mg/kg (equivalent to 10 times of the clinical normal dose, the same applies below),

example 2 group: example 2 refined bear gall powder 50mg/kg,

taug Luo Tezu: taurolite 50mg/kg.

Each of the above groups was administered 1 time a day, 14d. Blood was collected 4 hours after the last administration, and parameters such as serum Total Cholesterol (TC), triglyceride (TG), and High Density Lipoprotein (HDL) were measured by an enzymatic colorimetric method, whereby low density lipoprotein (LDL, ldl=tc- (hdl+tg)/5), and arteriosclerosis index (AI, ai= (TC-HDL)/HDL) were calculated, and the group comparison was performed. In the result of the influence of the refined bear gall powder on the blood fat of normal rats, partial data such AS HDL/TC ratio, arteriosclerosis Index (AI) and the like which can accurately reflect the relation between the blood fat level and AS, CHD are listed in the following table (mean value + -S).

Group of	TC(mmol/L)	HDL/TC(％)	AI(Tc—HDL)/HDL
				Blank control	0.671±0.036	63.72±15.33	0.569±0.143
Positive control	0.614±0.025*(-8.5％)	85.26±20.24***	0.173±0.086***
				Taurolite	0.583±0.041**(-13.1％)	81.03±14.73**	0.234±0.077**
Example 2	0.576±0.032**(-14.2％)	77.24±13.28**	0.294±0.092**
				Example 1	0.528±0.038***(-21.3％)	88.53±16.52***	0.130±0.084***

Comparison to the blank, <0.05, <0.01, <0.001; the percentage in brackets in the TC column is the percentage decrease in TC value compared to the blank control.

3. Effects on hyperlipidemia mice blood lipid

60 mice were randomly divided into 5 groups of 12 mice each. Each group was fed with a high fat diet consisting of 4% cholesterol, 1% cholic acid, 5% lard, 0.2% methylthiopyrim and 93.3% normal diet for 7d. The drug dose, method of administration, day of administration, assay and data calculation for each group were as described above for the test for normal rats. Taking out eyeball to collect blood after the last administration for 4 hours, measuring the various indexes, and comparing among groups. In the result of the influence of refined bear gall powder on the blood fat of the hyperlipidemic mice, the partial data such AS HDL/TC ratio, arteriosclerosis Index (AI) and the like which can accurately reflect the relation between the blood fat level and AS, CHD are listed in the following table (mean value + -S).

Comparison to the blank, <0.05, <0.01, <0.001; compared with the positive control group, # <0.05, # <0.01, # # <0.001; the percentage in brackets in the TC column is the percentage decrease in TC value compared to the blank control.

Hyperlipidemia is a common and frequently occurring disease. Elevated serum cholesterol (TC) is an important factor in the induction of Atherosclerosis (AS) and Coronary Heart Disease (CHD). CHD is one of the leading causes of death in the western world population. In recent years, the incidence of CHD in China also has a growing trend. The incidence rate of CHD of people over 35 years old in China is 3% -5%, and cardiovascular diseases are increased to 1-2 from the past 3-7 in the leading cause of death of people. HDL is inversely related to morbidity. HDL inhibits the uptake of LDL by cells, prevents cholesterol from accumulating in cells, and transports excess cholesterol out in the form of esters, thus preventing arteriosclerosis from occurring. Therefore, the HDL/TC ratio and the Arteriosclerosis Index (AI) can more accurately reflect the relation between the blood lipid level and AS and CHD, and in general, the larger the HDL/TC ratio and the smaller the Arteriosclerosis Index (AI) are, the better the blood lipid reducing effect of the medicine is shown [ Guo Hong, journal of China circulation, 1992,7 (1): 86 ]. The normal animal has lower sensitivity to the drug, if the total cholesterol in the serum of the normal rat can be reduced by 20 percent after the drug is taken, the drug can be considered to have cholesterol reducing effect [ Li Yikui, etc., the traditional Chinese medicine pharmacology experimental methodology, shanghai science and technology publishing company, 1991, 397 ] and the tauroursodeoxycholate arginine salt has the blood lipid reducing effect according to the blood lipid reducing result of the drug on the normal animal. The above results show that compared with the positive medicine and other tauroursodeoxycholic acid, the T-crystal form tauroursodeoxycholic acid arginine salt can obviously reduce serum cholesterol of rats in normal rats and mice with a hyperlipidemia model, has stronger hypolipidemic effect than the positive medicine and other tauroursodeoxycholic acid, and can obviously increase HDL/TC ratio and reduce Arteriosclerosis Index (AI). These results suggest that the tauroursodeoxycholate arginine salt of the T crystal form has obvious functions of reducing blood fat and resisting arteriosclerosis.

Example 3: refined bear gall powder for treating cholelithiasis, cholecystitis and cholecystitis

1. Test for dissolving gallstones in vitro

Reagent: a) refined bear gall powder (namely T-crystal form tauroursodeoxycholate arginine salt) in the embodiment 1, B) refined bear gall powder (tauroursodeoxycholate arginine salt) in the embodiment 2 group and C) taurolite are adopted as research reagents, and D) redistilled water.

Calculus: the 3 kinds of human stones obtained from the attached medicine of Nanchang university are cholelithiasis, cholesterol calculus and mixed calculus, respectively, which are quantitatively analyzed for the type of the stones by acetic anhydride method, classified, washed and dried to constant weight.

And (3) preparing a reagent: the redistilled liquid is used for preparing the liquid medicine of three reagents (A group, B group and C group) respectively, and the liquid medicine is converted into the high concentration of 0.5 percent of tauroursodeoxycholic acid. Then weighing a certain weight (1-1.2 g) of the calculus, soaking the calculus in 10ml of the test liquid with different concentrations, changing the test liquid for 1 time every 2 days, and simultaneously setting a control group (the distilled water is used for replacing the test liquid, and the group D) for comparative observation. The stones were removed, washed, dried and constant weights were recorded for Yu Dan weight on day 15 and day 45, respectively, and the litholytic rate was calculated using the following formula: calculus dissolution rate (%) = (initial weight of calculus-weight after dissolution)/(initial weight of calculus x 100%). The results were as follows:

And (3) injection: group a and group C of the same stone compare, P <0.01.

From the table above, group A (T-form tauroursodeoxycholate arginine salt) has significantly better litholytic effect on three stones than the commercial product, P <0.01; whereas the arginine salt of example 2 was substantially indistinguishable from the commercial product. The T-crystal form tauroursodeoxycholate arginine salt has excellent in-vitro litholytic effect.

2. In vivo anti-calculus test

The test examines the anti-calculus effect of the T-crystal tauroursodeoxycholate arginine salt in vivo through a test for inhibiting the formation of the bait cholesterol calculus of rabbits.

Taking 66 healthy New Zealand male white rabbits (supplied by experimental animal science center of Jiangxi university), randomly dividing the male white rabbits into 5 groups, wherein the group A adopts the refined bear gall powder (namely T-crystal tauroursodeoxycholate arginine salt) in the embodiment 1 of the invention, the group B adopts the refined bear gall powder (tauroursodeoxycholate arginine salt) in the embodiment 2, the group C adopts taurolite, the group D adopts blank control groups and the group E adopts model groups; group D10 animals, with 14 animals per group for each remaining group. Examples 1 and 2 refined bear gall powder and taurolite were each ground to a fine powder that was able to pass through an 80 mesh screen prior to administration, and each was suspended in 2% sodium carboxymethyl cellulose at a 2% tauroursodeoxycholate concentration prior to administration.

Except for normal feed feeding in group D of the blank group, each of the other groups was fed daily with a 1% cholesterol composition Dan Siliao, and was given by intragastric administration once every 2 days, each dose: A. the B, C three doses are all equivalent to 50mg/kg of tauroursodeoxycholate and the E group model group is given an equal volume of 2% sodium carboxymethylcellulose. After 45d observation, the pellets were cut and tested for the rate of stone formation, and the results were shown in the following table.

Note that: * Total weight of calculus refers to the total weight of calculus removed from the body of each animal in the group from which the calculus has been formed.

Observations from each group of animals: active health, vigorous appetite and fur luster among all animal experiments in group D; during the feeding process, the E group rabbits gradually have the problems of reduced ingestion, listlessness, unclean fur, no weight increase and death in sequence, and 3 rabbits die after the laparotomy, and the occurrence of stones in biliary tract is found (the stones are counted as Dan Dongwu); the other three groups of rabbits were better than group E in state, and none of the three groups had died. When the gallbladder of a blank control group is normal and the gallbladder of rabbits of other groups to which the stone-forming feed is added is observed visually after the 45d section, the gallbladder of rabbits of other groups to which the stone-forming feed is added is proliferated, so that the model group has the most serious pathological changes, the shape is irregular, and the inflammatory thickening of the capsule wall is obvious. After the model group is cut, 11 rabbits in the model group are checked to form stones, wherein 7 stones are in the gall bladder, 2 stones are in the common hepatic duct, 2 stones are both in the common hepatic duct and the gall bladder, and the stone forming rate is as high as 78.6%; all animals in the blank control group are not littered, and comparison of the stone conditions of the model group and the blank control group shows that the molding is successful after being fed by the littering feed for 45 days; the A, B, C component stone fraction was significantly lower than in group E, while group a had significantly lower stone formation than in groups B and C; the total stone weight data and the stone formation rate show the same difference trend among groups. The results show that the T-crystal form tauroursodeoxycholate arginine salt has the function of inhibiting the occurrence of gall-stone, and further has the function of preventing the occurrence of the gall-stone in vivo.

3. Improving the effect of the ingredients of the lithogenic bile

It is known that the bile components (i.e. the lithogenic bile components) of patients with cholecystitis and gall stones are obviously different from those of normal people, and the main surfaces of the patients are in a saturated state with high cholesterol content, and the concentration of bile acid is reduced due to the increase of reabsorption of bile acid caused by the change of biliary tract inflammation (i.e. cholecystitis); the mucus (glycoprotein) of the gall bladder of a patient is hypersecretion, and the mucus is a nucleation promoting factor, so that not only the viscosity of the bile is increased, but also the cholesterol in a saturated state is easy to form crystals, and the mucus has an important influence on the formation of cholesterol stones. The test examines the effect of the arginine salt of the T crystal form in improving the ingredients of the lithogenic bile.

Taking 66 healthy New Zealand male white rabbits, and randomly dividing the rabbits into 5 groups, wherein the group A adopts the refined bear gall powder (namely T-type tauroursodeoxycholate arginine salt) in the embodiment 1, the group B adopts the refined bear gall powder (tauroursodeoxycholate arginine salt) in the embodiment 2, the group C adopts Talarote, the group D adopts blank control group and the group E adopts model group; group D10 animals, with 14 animals per group for each remaining group. Examples 1 and 2 refined bear gall powder and taurolite were each ground to a fine powder that was able to pass through an 80 mesh screen prior to administration, and each was suspended in 2% sodium carboxymethyl cellulose at a 2% tauroursodeoxycholate concentration prior to administration.

Except for the normal feed feeding given to group D, each of the remaining groups was fed with a 1% cholesterol composition Dan Siliao, and was given once daily by intragastric administration, each dose: A. the B, C three doses are all equivalent to 50mg/kg of tauroursodeoxycholate and the E group model group is given an equal volume of 2% sodium carboxymethylcellulose.

45d later, the test is carried out, 1g/kg of pentobarbital sodium is used for intravenous injection anesthesia of the ear margin, the anesthesia is carried out, the fixation is carried out after the anesthesia, the abdomen is opened, the common bile duct is exposed along the first section of the duodenum, the cannula is respectively inserted into the liver side and the intestinal side, and the catheter is led out of the body to suture the abdominal wall after the ligation fixation. The following assays were performed with bile taken separately: bilirubin was measured immediately by azo method, cholesterol by ferric sulfate chromogenic method, bile acid by fluorescence spectrophotometry, mucus content by Alcian Blue method, and the results are shown in the following table.

Group of	Bilirubin (mg/dl)	Cholesterol (mg/ml)	Total bile acid (mg/ml)	Mucus (mg/ml)
					Group A	1.51±0.46**	0.26±0.10***	0.43±0.11***	1.63±0.42**
Group B	1.94±0.58*	0.45±0.14*	0.31±0.07**	2.18±0.36*
					Group C	2.07±0.73	0.41±0.11*	0.29±0.09**	2.43±0.62
Group D	1.21±0.69	0.19±0.07	0.23±0.12	0.62±0.34
					Group E	2.37±0.71##	0.61±0.23###	0.14±0.07#	3.06±0.87###

Note that: each drug group was compared with group E by P <0.05, P <0.01, P <0.001; group E compares #P <0.05, #P <0.01 with group D.

Compared with the blank control group, the content of bilirubin, cholesterol and mucus is obviously increased by P <0.01 or P <0.001, the content of total bile acid is reduced, and the significant P <0.05 shows that the modeling is successful. Compared with the model group, the three reagents have different degrees of reduction in bilirubin, cholesterol and mucus, and especially the group A has obvious reduction effect compared with the other two reagents; the three reagents can also improve the concentration of the total bile acid to different degrees, and especially the effect of the T-shaped crystal group is most obvious. It is known that bile acids such as tauroursodeoxycholic acid have a calculus-dissolving action and inhibit the activity of hydroxymethylglutaryl-CoA reductase, which is the rate-limiting enzyme for synthesizing cholesterol by the liver and thus reduces endogenous cholesterol synthesis, thereby reducing cholesterol content, inhibiting cholesterol 7α -dehydrogenase and thus inhibiting synthesis of other bile acids, and also effectively inhibiting absorption of cholesterol in the intestinal tract and promoting reduction of cholesterol content in bile, and also can bring cholesterol bile in a saturated state into an unsaturated state, and the reduction of cholesterol concentration in bile can not only prevent formation of cholesterol calculus but also promote its resolubilization. The results show that the T-shaped arginine salt has the effects of reducing bilirubin, cholesterol and mucus content in bile and increasing the content of total bile acid, and the indexes show that the T-shaped arginine salt can obviously improve the lithogenic bile component, and further can be used for preventing or treating cholelithiasis, preventing or treating cholecystitis and improving the function of the gall bladder.

All references cited in this specification, including but not limited to all papers, publications, patents, patent applications, briefs, textbooks, reports, manuscripts, brochures, books, internet articles, journal articles, periodicals, and the like, are hereby incorporated by reference in their entireties into this specification. The discussion of references herein is intended merely to summarize assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

Although embodiments of the present disclosure have been described using specific terms, devices, and methods, such description is for illustrative purposes only. The words used are words of description rather than limitation. It should be understood that variations and changes may be made by those of ordinary skill in the art without departing from the spirit and scope of the present disclosure as set forth in the following claims. Additionally, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein.

Claims

1. Use of a compound of formula I:

Wherein,,

the compound has a melting point of 187-189 ℃;

the compound uses Cu-K alpha radiation, and diffraction peaks are arranged at 8.53 degrees, 10.96 degrees, 12.03 degrees, 13.14 degrees, 14.82 degrees, 17.26 degrees, 22.53 degrees, 24.21 degrees, 26.68 degrees, 29.42 degrees and 31.24 degrees in a powder X-ray diffraction pattern expressed in terms of 2 theta angles;

the compound is prepared by a method comprising the following steps:

(1) Diluting the collected bear bile with 2-3 times of water, filtering with a 80-mesh screen, regulating the pH value of the filtrate with 1M hydrochloric acid solution to be 3.0-3.5, and then adding 1.2% sodium chloride into the filtrate to obtain crude bear bile liquid;

(2) Using a tangential flow ultrafiltration system, flushing a pipeline with ultrapure water, installing a 1kD midi Kros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain filtrate and 5-15 times of concentrated reflux liquid;

(3) Adjusting the pH value of the filtrate obtained in the previous step to be=6.8 by using a 1M sodium hydroxide solution, then adding arginine into the filtrate, wherein the amount of the arginine is 2-3 times of the amount of tauroursodeoxycholic acid in the filtrate, stirring the filtrate at the temperature of 40-50 ℃ for 2.5 hours, filtering to remove precipitate, then adding ethyl acetate with the volume of 1-2 times into the filtrate, standing for 2-4 hours, separating out precipitate, filtering, and removing the filtrate to obtain precipitate;

(4) Ethanol is added into the sediment obtained in the previous step, and the weight of the sediment is as follows: the volume ratio of the ethanol is 1g: 3-5 ml, stirring for 0.5 hours at room temperature, standing for 3 hours, and filtering out sediment to obtain filtrate;

(5) Adding ethyl acetate-diethyl ether with the volume ratio of 1-2 times to the filtrate obtained in the previous step to 5:1, standing for 6 hours, separating out a precipitate, filtering to obtain the precipitate, and drying under reduced pressure to remove the solvent to obtain the compound shown in the formula I.

2. Use according to claim 1, said compound having a powder X-ray diffraction pattern expressed in terms of 2Θ angles, shown in figure 1, using Cu-ka radiation.

3. The use according to claim 1, wherein in step (2), the shi must pure KR2i type tangential flow ultrafiltration system is used, the line is flushed with ultrapure water, a 1kD midi kros filter is installed, and the crude bear gall solution obtained in step (1) is filtered to obtain a filtrate and a 10-fold concentrated reflux solution.

4. The use according to claim 1, wherein in step (3) the amount of arginine is 2.5 molar times the amount of tauroursodeoxycholic acid in the filtrate.

5. The use according to claim 1, wherein in step (3), the precipitate is filtered off by stirring at a temperature of 44 to 46 ℃ for 2.5 hours, then 1.5 times the volume of ethyl acetate is added to the filtrate, left for 3 hours, the precipitate is precipitated, filtered off, and the filtrate is discarded to give a precipitate.

6. Use according to claim 1, wherein in step (4) ethanol is added to the precipitate obtained in the previous step, the weight of the precipitate: the volume ratio of the ethanol is 1g:4ml, stirred at room temperature for 0.5 hours, allowed to stand for 3 hours, and the precipitate was filtered off to obtain a filtrate.

7. Use according to claim 1, wherein in step (2), taurochenodeoxycholic acid is 0 to 2% by weight of tauroursodeoxycholic acid in the filtrate obtained by subjecting to tangential flow ultrafiltration.

8. Use according to claim 1, wherein in step (2) the tauroursodeoxycholic acid is 1-3% by weight of tauroursodeoxycholic acid in the reflux liquid obtained by tangential flow ultrafiltration.