CN110548037A - Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer - Google Patents

Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer Download PDF

Info

Publication number
CN110548037A
CN110548037A CN201910902326.6A CN201910902326A CN110548037A CN 110548037 A CN110548037 A CN 110548037A CN 201910902326 A CN201910902326 A CN 201910902326A CN 110548037 A CN110548037 A CN 110548037A
Authority
CN
China
Prior art keywords
filtrate
degrees
filtering
tauroursodeoxycholic acid
powder
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910902326.6A
Other languages
Chinese (zh)
Other versions
CN110548037B (en
Inventor
傅金荣
付金洪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianyuan Jiangxi Pharmaceutical Co Ltd
Original Assignee
Tianyuan Jiangxi Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianyuan Jiangxi Pharmaceutical Co Ltd filed Critical Tianyuan Jiangxi Pharmaceutical Co Ltd
Priority to CN201910902326.6A priority Critical patent/CN110548037B/en
Publication of CN110548037A publication Critical patent/CN110548037A/en
Application granted granted Critical
Publication of CN110548037B publication Critical patent/CN110548037B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C277/00Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C277/08Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J41/00Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C07J41/0061Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07BGENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13Crystalline forms, e.g. polymorphs

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Steroid Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

the present invention relates to refined bear gall powder and its application in building up body, treating and preventing tumor and cancer. In particular, the invention relates to a compound shown in a formula I in preparation of a medicine for preventing or treating tumors and cancers: the compound 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 by 2 θ using Cu-Kalpha radiation. The compound exhibits excellent biological properties such as excellent bioavailability and can exert physiological activities as well as bear gall powder or tauroursodeoxycholic acid. For example, it can be used for the prophylaxis or treatment of tumors and cancers.

Description

Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer
Technical Field
the invention belongs to the technical field of medicines, and relates to bear gall powder products and a preparation method thereof. The bear gall powder product has excellent biological effect, and can be used for preventing or treating tumors and cancers.
Background
The bear gall has been used as a medicine for more than one thousand years, is really reputed as 'gold in medicine', and is the first of four animal medicinal materials, namely bear gall, tiger bone, bezoar and musk. At present, 153 kinds of Chinese patent medicines prepared by using bear gall powder alone and compound Chinese patent medicine preparations containing bear gall powder components relate to 183 families of medicine production enterprises. The comprehensive efficacy of the medicine cannot be replaced by other medicines. In 660 classic traditional Chinese medicine (containing more than 8 ten thousand traditional prescriptions) from the Han Dynasty to the Qing Dynasty, about 366 writings recorded the efficacy and compatibility of the prescriptions of bear gall. It mainly includes the prescriptions of herbs, Puji Fang, Tang Ben Cao, Qian jin Fang, Ben Cao gang mu and Ben Cao gang mu Shi Yi. The property and flavor of bear gall powder can be summarized as bitter and cold in nature. It enters liver, gallbladder, heart, lung, spleen, stomach and large intestine meridians. Bear gall is bitter and cold in property, mainly enters liver and gallbladder meridians, can clear liver fire, improve eyesight and remove nebula, and is mainly used for treating conjunctival congestion, nebula and cataract; clear damp-heat in liver and gallbladder, promote bile flow and relieve jaundice, and is indicated for jaundice and dark urine. Entering heart meridian, clearing heart fire, dredging collaterals and relieving pain, mainly treating cardialgia; entering heart and liver meridians, it can clear heat and induce resuscitation, extinguish wind and stop convulsions, and is indicated for coma, infantile convulsions and epilepsy and convulsions due to chronic infectious disease convulsive seizure. Entering spleen and stomach meridians, it can remove food stagnation and resolve stagnation, kill parasites and cure malnutrition, and is mainly indicated for abdominal pain due to food stagnation and fever due to malnutrition stagnation. Entering large intestine meridian, it can clear heat and dry dampness, and is indicated for dysentery due to damp-heat, hemorrhoids and skin ulcer. All sores and itching relieving herbs belong to the heart, enter the heart meridian, clear heat, cool blood and remove toxicity, and are mainly used for treating furunculosis, malignant sores, blood accumulation and blood stranguria. Entering lung meridian, it can clear lung heat, relieve sore throat, resolve phlegm and stop cough, and is indicated for sore throat, pharyngitis, phlegm-heat and cough. Modern pharmacological research proves that bear gall powder has various pharmacological effects, and summary mainly include the effects of protecting liver, benefiting gall, dissolving gallstone, resisting hepatic fibrosis, calming, spasmolysis, resisting convulsion, relieving pain, strengthening heart, reducing blood pressure, resisting thrombus, resisting atherosclerosis, reducing blood fat, relieving cough, eliminating phlegm, relieving asthma, resisting tumor, resisting inflammation, relieving fever, inhibiting bacteria and the like. Embodies the pharmacodynamic action characteristics of multiple components and multiple targets of the bear gall powder, and provides scientific basis for clinically treating serious diseases such as liver and gall, cardiovascular and cerebrovascular diseases, infectious diseases and the like by the bear gall powder. At present, pharmacological research aiming at bear gall powder in academic circles is mainly carried out from the following aspects: liver and gall system, central nervous system, cardiovascular and cerebrovascular system, digestive system, respiratory system, anti-inflammatory, antibacterial, antiviral, ophthalmic, otorhinolaryngological, etc.
The Chinese pharmacopoeia of multiple editions all contain medicinal materials of bear gall and/or bile secreted by the bear gall or preparations containing the medicinal materials. For example, in the appendix 24 page of the first part of the Chinese pharmacopoeia 2005 edition, the bear gall is the dried gall bladder of the bear Selenarctisthimbetanus Cuvier or the bear Ursus arctus Linnaeus, which are animals of the family Ursidae. The 2010 version of Chinese pharmacopoeia, first part of appendix 27, contains fel Ursi powder, which is a dried product obtained by draining bile from a bear of the family Ursidae through a gallbladder operation; the first pharmacopoeia also contains the preparation of bear gall capsule, bear gall heart-saving pill, bear gall hemorrhoid treating ointment, etc. as well as bear gall powder. In the first part of the 2015 edition of Chinese pharmacopoeia, there are prepared bear gall capsule, bear gall heart-saving pill, bear gall hemorrhoid ointment, bear gall hemorrhoid suppository, etc. which are prepared from bear gall powder.
At present, the dry product of artificially drained bear gall is used as a substitute of natural bear gall and approved to be put into the market, is named as bear gall powder and is a new drug approved by the national ministry of health. The fel Ursi powder is a dried product obtained by draining bile from black bear of Uridae by gallbladder operation, has cold nature and bitter taste, enters liver, gallbladder, spleen, stomach and large intestine channels, and has effects of clearing heat, suppressing hyperactive liver, and improving eyesight. Modern researches find that the chemical components of the bear gall powder are relatively complex and mainly contain bound ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), Cholic Acid (CA), deoxycholic acid (DCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), cholesterols, cholesterins, amino acids, proteins, peptides, fatty acids, trace elements and the like. Wherein ursodeoxycholic acid is an important characteristic component. At present, the traditional Chinese medicine composition is mainly applied to liver and gall diseases such as gallstones, fatty liver, cholecystitis, viral hepatitis, chronic hepatitis B and the like, and diseases such as eyelid herpes zoster, hemorrhoids and the like clinically.
Zhang 36191; (Zhang 36191;, Hua, et al, HPLC fingerprint chromatogram method for determination of bile acid components in bear bile capsule; Huaxi pharmaceutical journal, 2009, 24 (4): 402-403) records that the bear bile capsule is prepared from low-temperature dried product of black bear drainage bile, has cold and bitter properties of bear bile, has the functions of clearing heat, removing toxicity, benefiting bile, relieving spasm and improving eyesight, and the active components of the bear bile capsule mainly comprise tauroursodeoxycholic acid and tauroursodeoxycholic acid.
CN1060337C (chinese patent application No. 93116933X, title of the invention: bear bile powder enteric capsule and its preparation process) records that bear bile powder is composed of three parts: the first part is combined bile acid which accounts for about 50 percent of the total weight of the bear gall powder and comprises tauroursodeoxycholic acid, tauroursodeoxycholic acid and the like, wherein the content of the tauroursodeoxycholic acid is the maximum and accounts for about 20 percent of the total weight of the bear gall powder; the second part is water-soluble protein, amino acid, inorganic salt and other components which are main reasons for generating moisture absorption, mostly have no direct physiological activity and generally only play a nutritional role; the third part is a fat-soluble bile element such as bilirubin, cholesterol, steroids, etc., which are not physiologically active, even cholesterol, for example, is generally an ingredient that is desirably avoided from intake, because it usually causes cardiovascular and cerebrovascular diseases.
Therefore, those skilled in the art have spent great efforts to further purify bear gall powder in order to remove unnecessary and even harmful components. For example, CN103520210A (201310523280.X, shengnao) discloses a method for purifying bear gall powder, comprising the following steps in the following order: (1) adding polyvinylpyrrolidone (PVP) and sterile water distilled water into freshly extracted bear bile, shaking, mixing, standing, and clarifying to obtain supernatant; (2) adding 2 times volume of absolute ethyl alcohol into the supernatant obtained in the step (1) to prepare the bear gall alcohol extract; (3) adding the purified bear gall 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 to ensure that the concentration of the absolute ethyl alcohol is 16.5%; (5) adding the bear bile supernatant containing 16.5% of absolute ethyl alcohol obtained in the step (4) into a cellulose powder CF11 column, performing vortex oscillation and uniform mixing, performing bear bile separation, centrifuging for 5min at 5000rpm, after the centrifugation is finished, adding sterile distilled water with the same volume into the cellulose powder CF11 column, and centrifuging for 5min at 5000 rpm; (6) collecting the supernatant obtained in the step (5), adding 3mol/LNaAc with the volume of 1/10 and isopropanol with the same volume, uniformly mixing, precipitating at 20 ℃, centrifuging for 30min at 12000rpm under the condition of 4 ℃ after the precipitation is finished, removing the supernatant, and collecting the precipitate; (7) adding 75% absolute ethyl alcohol to wash the precipitate collected in the step (6), centrifuging for 30min at 12000rpm in an environment at 4 ℃ after washing is finished, removing 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, adjusting the pH to 2-8, heating to 50 ℃ and keeping the temperature, adding compound protease, inactivating enzyme after enzymolysis is finished, filtering with an ultrafiltration membrane, and spray drying the obtained supernatant to obtain the finished bear gall powder. The bear gall powder obtained by the technology is believed to have high effective content, low pigment content, no fishy smell and improved medicinal value.
CN105147729A (201510650516.5, chun) discloses a preparation method of bear gall powder, which comprises the following steps: (A) and filtering: collecting fresh drained bear bile, and filtering to obtain filtrate; (B) and sterilizing: adding high-concentration ethanol into the filtrate, wherein the ethanol content in the liquid medicine after the ethanol is added is 75-85%, stirring, standing for 24-72 hours, wherein the stirring time is 20-40 minutes; (C) and concentrating: concentrating under reduced pressure to remove ethanol, concentrating under reduced pressure at 35-45 deg.C, and concentrating under reduced pressure to obtain sterilized fel Ursi solution; (D) and (3) freeze drying: freeze-drying the sterilized bear gall liquid, wherein the freeze-drying method comprises the following steps: and (3) 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 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 miscible of ethanol and bile, utilizes the killing effect of ethanol on viruses and pathogenic bacteria, does not destroy the active ingredients in the bear bile by controlling the content and time of the ethanol in the liquid medicine, kills various pathogenic bacteria and viruses, ensures the safety and effectiveness of clinical medication, overcomes the defects of traditional bear bile powder production, and greatly improves the quality of the bear bile powder, and the obtained bear bile powder has golden yellow appearance and transparent luster.
CN106386659A (201610755739.2, tianyou) discloses a method for industrially producing bear gall powder, which comprises the steps of selecting black bear fine breeds, raising, taking gall, feeding Chinese herbal medicines, and freeze-drying at ultra-low temperature, and specifically comprises the following steps: (1) selecting black bear fine varieties: selecting black bears with strong body, obvious variety characteristics, strong feed intake, good production performance, strong physique, strong limb hoof and over 3 years old as culture bears; (2) feeding: the compound feed is adopted for feeding the cultured bears, and 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 black bears till the weight reaches 90 kg and more, and taking out the gall when the black bears are fed; (3) taking a liner: the method comprises the following steps of adopting an animal self-tube-making painless drainage method to collect gall bladder, wherein a ring sphincter is manufactured at the abdominal wall end by utilizing the tissue of a black bear between the abdominal wall and the gall bladder of the black bear, a tube cavity can be closed when the sphincter contracts, the tube cavity is opened when the sphincter expands, the sphincter is controlled by vegetative nerves or regulated by hormones, by utilizing the characteristic, a nutrient solution is fed to the black bear when the gall bladder is collected, the sphincter of the black bear is expanded, the tube cavity is opened, an open tube cavity passage opening is sterilized by an alcohol cotton ball, a sterilized stainless steel hollow probe is inserted into a tube cavity passage, the gall automatically flows into a cup connected with the gall along the hollow probe, and the probe is pulled out after the gall bladder is collected; (4) the Chinese herbal medicines are taken: after the gallbladder is taken, feeding the black bear with the Chinese herbal medicine added with syrup water, and continuously feeding for 10 days; the Chinese herbal medicine formula comprises: 20-30 parts of mangnolia 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 folium isatidis and 20-30 parts of isatis root; 20-30 parts of corn protein powder, 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 medicinal materials, mixing, adding syrup water, and feeding black bear; (5) and (3) ultralow temperature freeze drying: freezing the extracted fel Ursi at-60 deg.C or below, and vacuum-drying to sublimate water to obtain dried fel Ursi powder. The bear gall powder prepared by the method is good in quality, high in yield and harmless to the body of the black bear.
CN102114044A (201010617773.6, return to zhentang) discloses a method for extracting bear bile, which comprises cutting a wound on the gall bladder of a bear, forming a fistula with muscle tissues and tightening; squeezing the fistula after healing to collect bile. The method can be used for discontinuous, fixed-time, quantitative and long-term collection, and can not influence the utilization of bile by the bear. The invention discloses a high-quality bear gall powder and a preparation method thereof, wherein the bear gall powder shows 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 method is pure in appearance and color, high in tauroursodeoxycholic acid content and free of moisture absorption, and is beneficial to storage, transportation and processing.
CN103040869A (201310027917.6, kang ao) discloses an artificial bear gall powder, which is prepared by adding sodium tauroursodeoxycholate into fowl gall as a raw material, wherein the weight ratio of the sodium tauroursodeoxycholate to the fowl gall is as follows: 20-40 parts of sodium tauroursodeoxycholate and 80-60 parts of poultry 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 guarantee and wild animal protection, has the internal quality and appearance characters almost similar to those of natural bear gall, provides the artificial bear gall powder which is simple, convenient, economic, safe and environment-friendly and has the quality and appearance extremely similar to those of the natural bear gall, and is an ideal substitute of the natural bear gall.
CN1311002A (00102073.0, Shilixia) discloses a refined bear gall powder and a preparation method thereof, belonging to the field of pharmacy, the common bear gall powder is easy to absorb moisture and agglomerate, has sticky tooth feel, extremely fishy smell, extremely bitter taste, more impurities, slow absorption, unstable property, easy decay and low content of effective components, and limits the application range of the bear gall powder. The invention adopts an ethanol extraction method, an ethanol extraction activated carbon decolorization method and an ethyl acetate separation method to purify and refine the bear gall powder, and the refined bear gall powder has light and exquisite color, no sticky tooth feeling, no fishy smell, light bitter taste, difficult decay, difficult moisture absorption and agglomeration, stable property and average content of effective components increased to more than 2 times. Prolong the storage time, expand the application range and improve the curative effect of the medicine.
CN106038601A (201610368003.X, facile) discloses a preparation method of bear gall powder with high content, high purity and low fishy smell, which comprises mixing bear gall powder, activated carbon and filter aid, extracting the mixture with ethanol solution until cholanic acid in the extractive solution is negative, collecting ethanol extractive solution, concentrating and recovering ethanol, collecting concentrated solution, and drying; the filter aid is a water and alcohol insoluble silicate mineral. The method is simple, has better operability and good controllability, the content of the prepared bear gall powder calculated by tauroursodeoxycholic acid can reach more than 45 percent at most, the recovery rate is nearly 100 percent, and the recovery rate calculated by weight can reach more than 85 percent.
However, there is still a need in the art for new methods for preparing bear bile powder with excellent properties, especially for obtaining bear bile powder containing high purity conjugated bile acids in high yield, and medical and health uses of such bear bile powder, e.g. for the prevention or treatment of tumors and cancers.
Disclosure of Invention
the present invention aims to provide a novel method for preparing bear gall powder with excellent properties, particularly to provide a method for obtaining bear gall powder containing high-purity conjugated bile acid with high yield. The object of the invention is achieved by the following scheme.
in a first aspect of the present invention, there is provided a method for preparing a refined bear gall powder, comprising the steps of:
(1) Diluting the collected bear bile with water (for example, diluting with 2-3 times of water, for example, diluting with 2.5 times of water), filtering with an 80-mesh sieve, (optionally, filtering the filtrate with the 80-mesh sieve to obtain a filtrate, adjusting the pH of the filtrate to 3.0-3.5, for example, pH 3.3, using 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride, for example, 1.2% sodium chloride to the filtrate) to obtain a crude bear bile (the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate can be measured);
(2) Using a tangential flow ultrafiltration system (such as Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 5-15 times (such as 10 times) of concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);
(3) Adjusting the pH of the filtrate obtained in the previous step to 6.5-7.0, for example, 6.8, using 1M sodium hydroxide solution, adding arginine (in an amount of 2-3, for example, 2.5, times the amount of tauroursodeoxycholic acid in the filtrate), stirring at 40-50, for example, 44-46, for 2-3, for example, 2.5 hours, filtering to remove the precipitate, adding 1-2, for example, 1.5 times the volume of ethyl acetate to the filtrate, standing for 2-4, for example, 3 hours, precipitating the precipitate, filtering, and removing the filtrate to obtain a precipitate;
(4) adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1 g: 3-5 ml, for example, 1 g: 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 2-4 hours, for example, 3 hours, and filtering off the precipitate to obtain a filtrate;
(5) adding 1-2 times volume of ethyl acetate-diethyl ether (5: 1) mixture into the filtrate obtained in the previous step, standing for 5-8 hr, such as 6 hr, precipitating, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined fel Ursi powder.
The method according to the first aspect of the present invention, wherein in the step (1), the filtrate obtained by filtering the 80-mesh screen is adjusted to pH 3.0 to 3.5 using 1M hydrochloric acid solution.
The method according to the first aspect of the present invention, wherein in the step (1), after the pH of the filtrate is adjusted to 3.0 to 3.5, 1.0 to 1.5% of sodium chloride is further added to the filtrate. It has been surprisingly found that by adjusting the filtrate to a pH of 3.0 to 3.5 and adding a specified amount of sodium chloride to the filtrate, the tauroursodeoxycholic acid can be made to enter the filtrate fraction when subsequently subjected to tangential flow ultrafiltration, while the other bound cholic acids are mostly made to enter the concentrated reflux.
The method according to the first aspect of the present invention, wherein in the step (2), the filtrate obtained by the tangential flow ultrafiltration, taurochenodeoxycholic acid is 0 to 5%, preferably 0 to 3%, preferably 0 to 2% by weight of tauroursodeoxycholic acid. That is, in the resulting filtrate, taurochenodeoxycholic acid was substantially absent.
the method according to the first aspect of the present invention, wherein in the reflux obtained by the tangential flow ultrafiltration in the step (2), the tauroursodeoxycholic acid accounts for 0-8%, preferably 1-5%, and preferably 1-3% of the weight of the tauroursodeoxycholic acid. That is, in the obtained reflux liquid, substantially no tauroursodeoxycholic acid remained. The taurochenodeoxycholic acid and tauroursodeoxycholic acid can be separated by the tangential flow ultrafiltration of the step (2).
The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in step (5) has a tauroursodeoxycholic acid content of more than 70%, such as 70 to 74%, particularly more than 71%, such as 71 to 74%, particularly more than 72%, such as 72 to 74%.
The method according to the first aspect of the present invention, wherein the refined bear gall powder obtained in step (5) has a molar ratio of tauroursodeoxycholic acid to arginine of 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 percentage of the total amount of tauroursodeoxycholic acid and arginine in the refined bear gall powder obtained in step (5) is more than 95%, such as 95 to 100%, particularly more than 96%, such as 96 to 100%, particularly more than 97%, such as 97 to 100%, particularly more than 98%, such as 98 to 100% of the total amount of the refined bear gall powder.
the method according to the first aspect of the present invention, wherein 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 purified bear gall powder obtained in the present invention is tauroursodeoxycholic acid arginine salt 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 °, and about 31.24 ° in a powder X-ray diffraction pattern expressed in terms of 2 θ using Cu — K α radiation.
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 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 °, and 31.24 ± 0.20 ° in a powder X-ray diffraction pattern expressed by 2 θ using Cu — K α radiation.
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 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 by an angle of 2 θ using Cu — K α 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 by using Cu-Ka radiation.
Further, the invention provides a refined bear gall powder, which is tauroursodeoxycholic acid arginine salt with a melting point of 187-189 ℃.
The refined bear gall powder according to the second aspect of the invention, wherein the tauroursodeoxycholic acid content is more than 70%, such as 70-74%, particularly more than 71%, such as 71-74%, particularly more than 72%, such as 72-74%.
The refined bear gall powder according to the second 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 refined bear gall powder according to the second aspect of the present invention, wherein the percentage of the total amount of tauroursodeoxycholic acid and arginine to the total amount of the refined bear gall powder is greater than 95%, such as 95-100%, particularly greater than 96%, such as 96-100%, particularly greater than 97%, such as 97-100%, particularly greater than 98%, such as 98-100%.
the refined bear gall powder according to the second aspect of the invention 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 °, and about 31.24 ° in a powder X-ray diffraction pattern expressed in terms of 2 θ using Cu — K α radiation.
According to the second aspect of the present invention, the refined bear gall powder 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 by 2 θ using Cu-Ka radiation.
According to the second aspect of the present invention, there is provided a purified bear gall powder having 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 by an angle of 2 θ using Cu-Ka radiation.
The refined bear gall powder according to the second aspect of the invention has a powder X-ray diffraction pattern shown in figure 1 by using Cu-Ka radiation.
the refined bear gall powder according to the second aspect of the invention is prepared by a method comprising the following steps:
(1) Diluting the collected bear bile with water (for example, diluting with 2-3 times of water, for example, diluting with 2.5 times of water), filtering with an 80-mesh sieve, (optionally, filtering the filtrate with the 80-mesh sieve to obtain a filtrate, adjusting the pH of the filtrate to 3.0-3.5, for example, pH 3.3, using 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride, for example, 1.2% sodium chloride to the filtrate) to obtain a crude bear bile (the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate can be measured);
(2) Using a tangential flow ultrafiltration system (such as Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 5-15 times (such as 10 times) of concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);
(3) Adjusting the pH of the filtrate obtained in the previous step to 6.5-7.0, for example, 6.8, using 1M sodium hydroxide solution, adding arginine (in an amount of 2-3, for example, 2.5, times the amount of tauroursodeoxycholic acid in the filtrate), stirring at 40-50, for example, 44-46, for 2-3, for example, 2.5 hours, filtering to remove the precipitate, adding 1-2, for example, 1.5 times the volume of ethyl acetate to the filtrate, standing for 2-4, for example, 3 hours, precipitating the precipitate, filtering, and removing the filtrate to obtain a precipitate;
(4) adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1 g: 3-5 ml, for example, 1 g: 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 2-4 hours, for example, 3 hours, and filtering off the precipitate to obtain a filtrate;
(5) adding 1-2 times volume of ethyl acetate-diethyl ether (5: 1) mixture into the filtrate obtained in the previous step, standing for 5-8 hr, such as 6 hr, precipitating, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined fel Ursi powder.
the purified bear gall powder of the second aspect of the invention, wherein in the step (1), the pH of the filtrate obtained by filtering the bear gall powder with an 80-mesh screen is adjusted to 3.0-3.5 by using 1M hydrochloric acid solution.
In the purified bear gall powder according to the second aspect of the invention, in the step (1), the pH of the filtrate is adjusted to 3.0 to 3.5, and then 1.0 to 1.5% of sodium chloride is further added to the filtrate. It has been surprisingly found that by adjusting the filtrate to a pH of 3.0 to 3.5 and adding a specified amount of sodium chloride to the filtrate, the tauroursodeoxycholic acid can be made to enter the filtrate fraction when subsequently subjected to tangential flow ultrafiltration, while the other bound cholic acids are mostly made to enter the concentrated reflux.
The refined bear gall powder according to the second aspect of the invention, wherein in the filtrate obtained by the tangential flow ultrafiltration in the step (2), the taurochenodeoxycholic acid is 0-5%, preferably 0-3%, and preferably 0-2% of the weight of the tauroursodeoxycholic acid. That is, in the resulting filtrate, taurochenodeoxycholic acid was substantially absent.
The refined bear gall powder according to the second aspect of the invention, wherein in the reflux liquid obtained by the tangential flow ultrafiltration in the step (2), the tauroursodeoxycholic acid accounts for 0-8%, preferably 1-5%, preferably 1-3% of the weight of the tauroursodeoxycholic acid. That is, in the obtained reflux liquid, substantially no tauroursodeoxycholic acid remained. The taurochenodeoxycholic acid and tauroursodeoxycholic acid can be separated by the tangential flow ultrafiltration of the step (2).
further, the third aspect of the present invention provides a compound represented by the following formula I:
The compound according to the third aspect of the present invention has a melting point of 187 to 189 ℃.
the compound according to the third aspect of the present invention, wherein the tauroursodeoxycholic acid content is more than 70%, such as 70 to 74%, particularly more than 71%, such as 71 to 74%, particularly more than 72%, such as 72 to 74%.
A 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.
a compound according to the third aspect of the present invention, which 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 degrees 2 Θ using Cu-ka radiation.
The compound according to the third aspect of the present invention 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 by an angle of 2 θ using Cu — K α radiation.
the compound according to the third aspect of the present invention 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 by an angle of 2 θ using Cu — K α radiation.
A compound according to the third aspect of the invention, which uses Cu-ka radiation, has a powder X-ray diffraction pattern as shown in figure 1.
A compound according to the third aspect of the present invention, which is prepared by a process comprising the steps of:
(1) Diluting the collected bear bile with water (for example, diluting with 2-3 times of water, for example, diluting with 2.5 times of water), filtering with an 80-mesh sieve, (optionally, filtering the filtrate with the 80-mesh sieve to obtain a filtrate, adjusting the pH of the filtrate to 3.0-3.5, for example, pH 3.3, using 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride, for example, 1.2% sodium chloride to the filtrate) to obtain a crude bear bile (the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate can be measured);
(2) Using a tangential flow ultrafiltration system (such as Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 5-15 times (such as 10 times) of concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);
(3) Adjusting the pH of the filtrate obtained in the previous step to 6.5-7.0, for example, 6.8, using 1M sodium hydroxide solution, adding arginine (in an amount of 2-3, for example, 2.5, times the amount of tauroursodeoxycholic acid in the filtrate), stirring at 40-50, for example, 44-46, for 2-3, for example, 2.5 hours, filtering to remove the precipitate, adding 1-2, for example, 1.5 times the volume of ethyl acetate to the filtrate, standing for 2-4, for example, 3 hours, precipitating the precipitate, filtering, and removing the filtrate to obtain a precipitate;
(4) Adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1 g: 3-5 ml, for example, 1 g: 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 2-4 hours, for example, 3 hours, and filtering off the precipitate to obtain a filtrate;
(5) Adding a mixture of ethyl acetate and ethyl ether (5: 1) in an amount of 1-2 times, for example, 2 times, the volume of the filtrate obtained in the previous step, standing for 5-8 hours, for example, 6 hours, precipitating, filtering to obtain a precipitate, and drying under reduced pressure to remove the solvent to obtain the compound of formula I.
the compound according to the third aspect of the present invention, wherein in the step (1), the filtrate obtained by filtering through an 80-mesh screen is adjusted to pH 3.0 to 3.5 using 1M hydrochloric acid solution.
The compound according to the third aspect of the present invention, wherein in the step (1), after the pH of the filtrate is adjusted to 3.0 to 3.5, 1.0 to 1.5% of sodium chloride is further added to the filtrate. It has been surprisingly found that by adjusting the filtrate to a pH of 3.0 to 3.5 and adding a specified amount of sodium chloride to the filtrate, the tauroursodeoxycholic acid can be made to enter the filtrate fraction when subsequently subjected to tangential flow ultrafiltration, while the other bound cholic acids are mostly made to enter the concentrated reflux.
The compound according to the third aspect of the present invention, wherein in the filtrate obtained by the tangential flow ultrafiltration in the step (2), taurochenodeoxycholic acid is 0 to 5%, preferably 0 to 3%, and preferably 0 to 2% by weight of tauroursodeoxycholic acid. That is, in the resulting filtrate, taurochenodeoxycholic acid was substantially absent.
The compound according to the third aspect of the present invention, wherein in the reflux obtained by the tangential flow ultrafiltration in step (2), the tauroursodeoxycholic acid accounts for 0-8%, preferably 1-5%, and preferably 1-3% of the weight of the tauroursodeoxycholic acid. That is, in the obtained reflux liquid, substantially no tauroursodeoxycholic acid remained. The taurochenodeoxycholic acid and tauroursodeoxycholic acid can be separated by the tangential flow ultrafiltration of the step (2).
Further, the fourth aspect of the present invention provides the use of the refined bear gall powder of the second aspect of the present invention or the compound of the third aspect of the present invention in the preparation of products for preventing or treating tumors and cancers.
Tauroursodeoxycholic acid (TUDCA), with the chemical name 2- [ [ (3 α,5 β,7 β) -3, 7-dihydroxy-24-oxocholestan-24-yl ] amino ] ethanesulfonic acid dihydrate, can also be expressed as 3 α,7 β -dihydroxycholanyl-N-taurine, CAS No: 14605-22-2. The chemical structural formula of tauroursodeoxycholic acid is as follows:
The molecular formula is as follows: C26H45NO6S, molecular weight: 499.7
TUDCA, which is the main bile acid in bear gall and has the functions of spasmolysis, anticonvulsant, anti-inflammatory and cholelithiasis dissolving, was discovered in 1902 from bear gall. Tauroursodeoxycholic acid is an effective component of bear bile, is developed by Italy Besidi pharmaceutical factory, is firstly marketed in Italy in 1991, is approved to be sold in China in 2007 under the name of taurolite (taurolite), and is mainly used for treating cholecystolith calculus, primary sclerosing cholangitis, primary biliary cirrhosis, chronic viral hepatitis C and the like in clinic. Clinical research shows that compared with ursodeoxycholic acid, tauroursodeoxycholic acid has the advantages of higher stone dissolving speed, higher total dissolution rate and no obvious adverse reaction.
The refined bear gall powder obtained by carrying out finish machining treatment on the bear gall powder has 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. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it.
In the present invention, if a) the refined bear gall powder of example 1 of the present invention (i.e., tauroursodeoxycholic acid arginine salt of T crystal form), B) the refined bear gall powder of example 2 (tauroursodeoxycholic acid arginine salt of C) taurolite, were used as the study reagents, when describing their dosages, the dosages were converted into the amount of tauroursodeoxycholic acid, if not otherwise stated; for oral administration, as not otherwise specified, all are ground to a fine powder capable of passing through a 80 mesh sieve prior to administration, and suspended in 2% sodium carboxymethylcellulose at a concentration of 2% tauroursodeoxycholic acid prior to administration.
The method for determining the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in various materials comprises the following steps: referring to the HPLC method described in the experimental part of Zhang 36191; (Zhang 36191;, Hua et al, HPLC fingerprint chromatogram method for measuring bile acid components in bear gall capsules, Waxi pharmaceutical journal 2009, 24 (4): 402-403), the peak areas of the HPLC method are calculated by using the external standard methods of a tauroursodeoxycholic acid reference substance and a tauroursodeoxycholic acid reference substance (both purchased from China food and drug testing institute). When the tauroursodeoxycholic acid arginine salt is measured, free tauroursodeoxycholic acid is dissociated from the tauroursodeoxycholic acid arginine salt in a mobile phase and the free tauroursodeoxycholic acid is kept for the same time as a control product, and arginine does not influence the measurement of other substances in the HPLC method.
The method for determining the content of arginine in various materials comprises the following steps: the method is carried out by referring to Liu Rui literature (Liu Rui, et al, HPLC method for measuring arginine content in ibuprofen injection, journal of Western North pharmacy, 2013, 28(4):361) from 2.1.4 to 2.1.10 sections, and the specificity, linearity, precision, repeatability, stability and recovery rate meet the requirements of a common analysis method, and the tauroursodeoxycholic acid does not influence the measurement of arginine.
Example 1: preparation of refined bear gall powder
(1) diluting the collected bear bile (22.63% and 16.31% when dried under reduced pressure to obtain powder, which is the percentage of the two substances in the bear bile solid) with water (diluted by 2.5 times the volume of water), filtering with 80 mesh sieve, adjusting pH to 3.3 with 1M hydrochloric acid solution, adding 1.2% sodium chloride to the filtrate to obtain crude bear bile (drying the crude bear bile under reduced pressure to obtain powder, measuring the content of the tauroursodeoxycholic acid and tauroursodeoxycholic acid in the powder to be 28.47% and 20.33%, respectively, which shows that the ratio of the two is substantially the same as that before filtration, but the bear bile is concentrated due to the removal of impurities, and optionally concentrating the bear bile according to the percentage results and the percentage results, Calculating the mass of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the materials according to the volume of the crude bear gall liquid; the mass recovery rate of the two substances in the bear bile and the crude bear bile liquid is expressed in terms of percentage obtained by dividing the mass of the tauroursodeoxycholic acid in the crude bear bile liquid by the mass of the tauroursodeoxycholic acid in the bear bile and multiplying the mass by 100 percent, the recovery rate of the tauroursodeoxycholic acid is 99.78 percent, and the recovery rate of the tauroursodeoxycholic acid is 99.43 percent;
(2) Using a tangential flow ultrafiltration system (Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 10 times of concentrated reflux liquid (measuring the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate or the powder obtained by reduced pressure drying);
(3) Adjusting the pH value of the filtrate obtained in the previous step to 6.8 by using 1M sodium hydroxide solution, adding arginine (the amount of arginine is 2.5 mol times of the amount of tauroursodeoxycholic acid in the filtrate), stirring at the temperature of 44-46 ℃ for 2.5 hours, filtering and discarding precipitates, adding 1.5 times of ethyl acetate in volume to the filtrate, standing for 3 hours, precipitating precipitates, filtering, discarding the filtrate to obtain precipitates;
(4) adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1g to 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 3 hours, and filtering out the precipitate to obtain a filtrate; (As known to those skilled in the art, ethanol refers to 98% ethanol when ethanol is mentioned, unless concentrations are noted)
(5) Adding 2 times volume of ethyl acetate-diethyl ether (5: 1) mixture into the filtrate obtained in the previous step, standing for 6 hr to precipitate, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined fel Ursi powder.
In the process, the determination:
In the filtrate obtained by the tangential flow ultrafiltration in the step (2), the taurochenodeoxycholic acid is 0.76 percent of the weight of the tauroursodeoxycholic acid, which indicates that the taurochenodeoxycholic acid is basically not contained in the obtained filtrate;
in the step (2), the filtrate obtained by the tangential flow ultrafiltration is measured, the content of the tauroursodeoxycholic acid in the filtrate is measured, the mass of the tauroursodeoxycholic acid is calculated by combining the volume of the filtrate, and compared with the mass of the tauroursodeoxycholic acid in the bear bile fed in the step (1), the recovery rate is 98.26 percent, which shows that the tauroursodeoxycholic acid after the tangential flow ultrafiltration has extremely high recovery rate;
In the step (2), a part of the filtrate obtained by tangential flow ultrafiltration is taken to be dried under reduced pressure to remove the solvent to obtain a powder solid, and the solid is determined to contain 83.68% of tauroursodeoxycholic acid in the weight of the solid, which indicates that the tauroursodeoxycholic acid can be obviously enriched in the filtrate by the tangential flow ultrafiltration;
in the reflux liquid obtained by the tangential flow ultrafiltration in the step (2), the tauroursodeoxycholic acid accounts for 1.14 percent of the weight of the tauroursodeoxycholic acid, which shows that the obtained reflux liquid basically has no residue of the tauroursodeoxycholic acid; the taurochenodeoxycholic acid and the tauroursodeoxycholic acid can be separated by the tangential flow ultrafiltration in the step (2);
In the refined bear gall powder obtained in the step (5), the content of tauroursodeoxycholic acid is 73.62%;
In the refined bear gall powder obtained in the step (5), the molar ratio of tauroursodeoxycholic acid to arginine is 1: 1.004;
In the refined bear gall powder obtained in the step (5), the percentage of the total amount of tauroursodeoxycholic acid and arginine to the total amount of the refined bear gall powder is 99.27 percent;
Compared with the mass of the tauroursodeoxycholic acid in the bear bile fed in the step (1), the recovery rate of the tauroursodeoxycholic acid in the whole process from the step (1) to the step (5) is 94.51 percent, which shows that the method has very high recovery rate for the 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 purified bear gall powder obtained in the present invention is an arginine tauroursodeoxycholate (1: 1) salt 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 type powder X-ray diffractometer; Cu-Kalpha radiation, a graphite monochromator, 40kV/40mA, a 2 theta scanning range of 5-40 degrees, a scanning speed of 4 degrees/minute and a step length of 0.01 degrees; the scanning mode is continuous scanning; slit setting: and (4) emergent slit DS: 1/2 DEG anti-scatter slit: SS 1/2 degrees; RS is 0.3 mm.
the powder X-ray diffraction pattern of the purified bear gall powder obtained in the step (5) of the example 1 is shown in FIG. 1, and the data of the typical diffraction angles of the part of FIG. 1 are as follows: a powder X-ray diffraction pattern expressed in terms of 2 θ has diffraction peaks at 8.53 ° (22.0%, relative abundance, the same 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 indicates that the refined bear gall powder obtained in step (5) of this example 1 is in a typical crystal form, which can be referred to as "T crystal form" or "T crystal form" (i.e., tauroursodeoxycholic acid arginine salt, etc.).
in this example 1, the refined bear gall powder (i.e. T crystal form) obtained in step (5) is essentially a compound represented by the following formula I, i.e. tauroursodeoxycholic acid arginine salt (1: 1):
Example 2: preparation of refined bear gall powder
The steps (1) to (4) were carried out as in example 1; and (5) changing the ethyl acetate-diethyl ether (5: 1) mixed solution into ethyl acetate, and drying the precipitate under reduced pressure to remove the solvent 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 a different crystal form from the refined bear gall powder in the example 1.
Example 3: preparation of refined bear gall powder
The steps (1) to (4) were carried out as in example 1; in the step (5), the ethyl acetate-ether (5: 1) mixed solution is changed into 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 ℃ through determination, and the difference of the melting points shows that the refined bear gall powder has different crystal forms from the refined bear gall powder in the example 1.
The results of examples 2 and 3 above show that the use of the ethyl acetate-diethyl ether (5: 1) mixture in step (5) results in the formation of crystal form T having a melting point of 187-189 deg.C, unlike the crystals obtained without the use of this mixture.
The three refined bear gall powders obtained in the examples 1 to 3 are placed at the temperature of 45 ℃ for 3 months, and then the melting points of the three refined bear gall powders are measured, so that the melting points of the three batches of refined bear gall powders are 188-189 ℃, 168-174 ℃ and 180-185 ℃, respectively, and the results show that the product of the example 1 is stable, while the melting points of the products of the examples 2 to 3 show large change and large melting range, which shows that the stability has defects.
Example 4: preparation of refined bear gall powder
(1) Diluting bear bile (containing tauroursodeoxycholic acid and tauroursodeoxycholic acid respectively 22.17% and 17.15% in solid content) with water (diluting with 2.5 times volume of water), filtering with 80 mesh screen, adding 1.2% sodium chloride into filtrate to obtain crude bear bile solution (containing tauroursodeoxycholic acid and tauroursodeoxycholic acid respectively 28.23% and 20.07% in solid content);
(2) And (2) using a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 10 times of concentrated reflux liquid.
through determination, the recovery rate of the filtrate obtained by the tangential flow ultrafiltration in the step (2) compared with the mass of the tauroursodeoxycholic acid in the bear bile fed in the step (1) is 42.53%, which indicates that the recovery rate of the tauroursodeoxycholic acid after the tangential flow ultrafiltration is quite low. Due to this very low recovery rate, the present example did not continue with the subsequent operation.
Example 5: preparation of refined bear gall powder
(1) Diluting bear bile (containing tauroursodeoxycholic acid and tauroursodeoxycholic acid respectively 22.43% and 16.84% in solid content) with water (diluting with 2.5 times volume of water), filtering with 80-mesh sieve, and adjusting pH of filtrate obtained by filtering with 80-mesh sieve to 3.3 with 1M hydrochloric acid solution to obtain crude bear bile solution (containing tauroursodeoxycholic acid and tauroursodeoxycholic acid respectively 28.72% and 20.65% in solid content);
(2) and (2) using a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 10 times of concentrated reflux liquid.
Through determination, the recovery rate of the filtrate obtained by the tangential flow ultrafiltration in the step (2) is 56.14 percent compared with the mass of the tauroursodeoxycholic acid in the bear bile fed in the step (1), which indicates that the recovery rate of the tauroursodeoxycholic acid after the tangential flow ultrafiltration is quite low. Due to this very low recovery rate, the present example did not continue with the subsequent operation.
Additionally, it was determined that taurochenodeoxycholic acid was 34.52% and 41.17&, respectively, by weight of tauroursodeoxycholic acid, in the filtrate obtained by subjecting step (2) of examples 4 and 5 to tangential flow ultrafiltration, indicating that a greater proportion of tauroursodeoxycholic acid entered the filtrate.
From the results of examples 4 and 5, it can be seen that when tangential flow ultrafiltration is performed, it is advantageous to adjust the pH of the solution to 3.3 while adding sodium chloride thereto to improve the recovery of tauroursodeoxycholic acid. In addition, limited to the prior art, the present inventors have not yet explained that although taurochenodeoxycholic acid and bound bile acids such as tauroursodeoxycholic acid may have similarities in chemical structure or performance, they exhibit significantly different filtration behaviors in the tangential flow ultrafiltration process, and it seems that such a difference in filtration behavior has some correlation with the treatment of pH 3.3 and sodium chloride, although these results do not affect the contribution of the present invention to the prior art, the present inventors still expect to be able to explain the above phenomenon in the near future with the progress of the technology, although it is not.
Experimental example 1: oral bioavailability of refined bear gall powder
And (3) testing the sample: the refined bear gall powder obtained in example 1, the refined bear gall powder obtained in example 2, and the commercially available tauroursodeoxycholic acid capsules (taurulite, H20150398, 250 mg/granule). Taurulite determines a powder weight of about 250mg per capsule, indicating that no or only very minor amounts of excipients were added. Examples 1 and 2 the refined bear bile powder and taurulate were ground to a fine powder capable of passing through a 80 mesh sieve prior to administration, and were suspended in 2% sodium carboxymethylcellulose at a concentration of 2% prior to administration.
Animals: 24 male big-ear white Japanese rabbits (supplied by the scientific and technological center for experimental animals of Jiangxi Chinese medicinal university) weigh 2.0-2.5 kg, and are randomly divided into three groups, wherein the group A is administered with the refined bear bile powder of the example 1, the group B is administered with the refined bear bile powder of the example 2, and the group C is administered with taurolite.
Administration and blood sampling and blood sample processing: 1ml of blood (ear marginal vein) was taken before administration as a 0-hour blood sample; the administration dosage is calculated by tauroursodeoxycholic acid form, and the administration is performed by intragastric administration at the dosage of 50mg tauroursodeoxycholic acid/kg body weight, and blood is taken by the same method for 0.5h, 1h, 2h, 4h, 6h, 10h, 15h, 21h and 30h respectively; centrifuging blood sample to obtain 0.5mL of plasma, extracting with methanol under shaking, centrifuging to remove precipitate, repeating the methanol extraction for three times, mixing methanol solutions, and volatilizing methanol to obtain the final product.
referring to the HPLC method of Yanxinxin literature (Yanxinxin, et al, bear bile sustained-release eye-drop gel rabbit ophthalmic pharmacokinetics research, Shizhen national medicine, 2017, 28(7):1634), HPLC chromatographic conditions: the column was dimales C18(5 μm, 4.6 mm. times.250 mm), the mobile phase was sodium dihydrogen phosphate buffered saline (0.03mol/L, pH 4.4) and methanol 62:38, flow rate 1.0ml/min, detection wavelength 210nm, column temperature 30 ℃, sample size 20 μ L. The tauroursodeoxycholic acid control was purchased from Sigma-Aldrich, and the control and the blood sample treatment were dissolved in methanol and diluted with mobile phase for HPLC assay. The specificity, linearity, precision, stability, detection limit, quantification limit and recovery rate of the HPLC method all meet the analysis requirements of general biological samples.
The blood concentration data of blood samples at each time point were measured according to the blood concentration at each time point, and the curve data were processed by 3P87 software and an open-type two-chamber model to obtain the area AUC (0- ∞) under the peak of the curve and the maximum blood concentration Cmax and the fitted peak reaching time Tmax, and the relative bioavailability of each sample relative to the commercial product was calculated by AUC, and the results are shown in the following table (expressed as mean. + -. sd):
Sample (I) 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 (Taurolite) 3.28±0.33 717.59±67.28 83 648.93 100
As can be seen from the above results, the bioavailability of the product of example 1 is much higher than that of the commercially available product and the product of example 2 having a different crystal form.
In view of the fact that the tauroursodeoxycholic acid arginine salt of the present invention is in a dissociated state after being absorbed into blood circulation, i.e. in a free form, will exert the due biological effects of its active ingredients as well as taurulate. For example, the tauroursodeoxycholic acid arginine salt of the invention can be used for dissolving diseases such as cholesterol calculus and the like.
experimental example 2: blood lipid reducing effect of refined fel Ursi powder
1. Material
And (3) testing the sample: the refined bear gall powder obtained in example 1, the refined bear gall powder obtained in example 2, and the commercially available tauroursodeoxycholic acid capsules (taurulite, H20150398, 250 mg/granule). Examples 1 and 2 the refined bear bile powder and taurulate were ground to a fine powder capable of passing through a 80 mesh sieve prior to administration, and were suspended in 2% sodium carboxymethylcellulose at a concentration of 2% prior to administration.
The cholesterol is purchased from Sigma-Aldrich company, and has the product number C8667, and the purity is more than or equal to 99 percent; cholic acid is purchased from Sigma-Aldrich company, product number C1129, and purity is more than or equal to 98%; methyl thiouracil (commercially available tablet, H32022717).
Animals: male Kunming mice with the weight of 18-22 g, male Wistar rats with the weight of 180-220 g and experimental animal science and technology center of Jiangxi traditional Chinese medicine university.
2. Influence on blood fat of normal rat
40 rats were taken and randomly divided into 5 groups of 8 rats each.
Blank control group: 2% sodium carboxymethylcellulose (same volume as the refined fel Ursi powder in example 1),
Positive control group: the medicine is compound three-dimensional linoleic acid capsule I (a clinically used hypolipidemic medicine) with 0.6 grain/kg (equivalent to 10 times of the clinically used dose),
Example 1 group: example 1 bear gall powder (namely T crystal form) 50mg/kg (which is 10 times of the clinical common dosage, the same below) is refined,
example 2 group: example 2 bear gall powder 50mg/kg,
Taurolite group: taurolite 50 mg/kg.
The above groups were administered 1 time daily for 14 days. 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 the enzymatic colorimetric method, whereby low density lipoprotein (LDL, LDL ═ TC — (HDL + TG)/5) and arteriosclerosis index (AI, AI ═ TC — HDL)/HDL) were calculated and compared among groups. In the results of the influence of the refined bear gall powder on the blood fat of normal rats, partial data such AS parameters HDL/TC ratio, Arteriosclerosis Index (AI) and the like which can accurately reflect the relation between the blood fat level and AS and 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 (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***
(ii) 0.05, <0.01, < 0.001; the percentage in parentheses in the column TC is the percentage reduction in TC values compared to the blank control.
3. influence on blood lipid of hyperlipidemic mouse
60 mice were taken and randomly divided into 5 groups of 12 mice each. Each group was fed with high fat diet consisting of 4% cholesterol, 1% cholic acid, 5% lard, 0.2% methyl thiouracil and 93.3% normal diet for 7 days. The drug dose, administration method, administration days, assay and data calculation for each group were the same as those in the above test for normal rats. Blood is collected from the eyeball 4h after the last administration, and the above various indexes are measured for comparison among groups. In the results of the influence of the refined bear gall powder on the blood fat of the hyperlipidemic mouse, partial data such AS parameters HDL/TC ratio, Arteriosclerosis Index (AI) and the like which can accurately reflect the relation between the blood fat level and AS and CHD are listed in the following table (mean value +/-S).
(ii) 0.05, <0.01, < 0.001; compared with the positive control group, the # # <0.05, # <0.01, # # # < 0.001; the percentage in parentheses in the column TC is the percentage reduction in TC values compared to the blank control.
Hyperlipidemia is a common and frequently encountered 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 population of western countries. In recent years, CHD incidence in China also tends to increase. The CHD incidence rate of people over 35 years old in China is 3% -5%, and cardiovascular diseases are increased from 3-7 to 1-2 in the past in the antegrade of the death cause of people. HDL is inversely related to morbidity. HDL inhibits the uptake of LDL by cells, prevents the accumulation of cholesterol in cells, and transports excess cholesterol out in the form of ester, thereby preventing arteriosclerosis. Therefore, the relationship between blood lipid level and AS, CHD can be reflected more accurately by HDL/TC ratio and Arteriosclerosis Index (AI), and generally speaking, the larger the HDL/TC ratio and the smaller the Arteriosclerosis Index (AI), the better the blood lipid-lowering effect of the medicine is (Guohong, J. circulatory system of China, 1992, 7 (1): 86 ]. The sensitivity of the blood fat of normal animals to drugs is low, if the total cholesterol in the serum of normal rats is reduced by 20% after the drugs are taken, the drugs can be considered to have the function of reducing the cholesterol (Liyiqu, et al, traditional Chinese medicine pharmacology experimental methodology, Shanghai science and technology publishing Co., 1991, 397). The results show that compared with the positive medicament and other tauroursodeoxycholic acid, the T crystal form tauroursodeoxycholic acid arginine salt can obviously reduce the serum cholesterol of rats in normal rats and hyperlipidemic model mice, has stronger hypolipidemic action than the positive medicament and other tauroursodeoxycholic acid, and can obviously increase the HDL/TC ratio and reduce the Arteriosclerosis Index (AI) through the T crystal form. These results suggest that tauroursodeoxycholic acid arginine salt of the T crystal form has obvious effects of reducing blood fat and resisting arteriosclerosis.
Experimental example 3: function of refined bear gall powder on gall stone, cholecystitis and gall bladder function
1. Test for in vitro dissolution of gallstones
Reagent testing: A) the research reagent is prepared by using the refined bear gall powder (namely T crystal form tauroursodeoxycholic acid arginine salt) in the embodiment 1 of the invention, B) the refined bear gall powder (tauroursodeoxycholic acid arginine salt) in the embodiment 2 and C) tauroult, and D) re-distilled water.
calculus: the 3 kinds of human calculi from the attached medicine of Nanchang university are cholechrome calculi, cholesterol calculi and mixed calculi, which are classified and washed and dried to constant weight after being analyzed for the types of calculi by acetic anhydride.
preparing a reagent: the test solutions of the three reagents (group A, group B and group C) are prepared respectively by redistilled water, and the high concentration of the test solutions is 0.5 percent calculated by the conversion of the test solutions into tauroursodeoxycholic acid. Then weighing a certain weight (1-1.2 g) of the calculus, soaking in 10ml of the test liquid medicine with different concentrations, newly replacing the test liquid medicine for 1 time every 2 days, and simultaneously setting a control group (replacing the test liquid medicine with heavy distilled water, and D group) for comparative observation. On day 15 and day 45, respectively, the stones were removed, washed, dried and constant weight, the weight of the remaining stones was recorded, and the litholysis rate was calculated by the following formula: the stone dissolution rate (%) - (initial weight of stone-weight after dissolution) ÷ initial weight of stone × 100%. The results are as follows:
Note: group a of the same stones compared to group C,. P < 0.01.
As can be seen from the table above, the litholytic effect of the group A (T crystal form tauroursodeoxycholic acid arginine salt) on three kinds of stones is obviously superior to that of the commercial product, and P is less than 0.01; in example 2, the arginine salt is substantially the same as the commercially available product. The T crystal form tauroursodeoxycholic acid arginine salt has excellent in-vitro litholytic effect.
2. In vivo stone prevention test
The test examines the in vivo anti-calculus effect of the T crystal form tauroursodeoxycholic acid arginine salt through the experiment of inhibiting the formation of bait-feeding cholesterol calculus of the rabbits.
66 healthy New Zealand male white rabbits (supplied by the Experimental animal science and technology center of Jiangxi Chinese medicinal university) were randomly divided into 5 groups, wherein group A used refined bear gall powder (namely T crystal form tauroursodeoxycholic acid arginine salt) in example 1 of the invention, group B used refined bear gall powder (tauroursodeoxycholic acid arginine salt) in example 2, group C tauroux, blank control group in group D and model group E; group D10 animals, the remaining groups 14 animals per group. Examples 1 and 2 the refined bear bile powder and taurulate were ground to a fine powder capable of passing through a 80 mesh sieve prior to administration, and were suspended in 2% sodium carboxymethylcellulose at a concentration of 2% tauroursodeoxycholic acid prior to administration.
Except for the blank control group D which was fed with normal feed, the other groups were fed with 1% cholesterol-containing stone-forming feed every day, and were gavaged every 2 days at doses: A. b, C the dosages of the three groups are all equal to 50mg/kg of tauroursodeoxycholic acid, and the E group model group is given 2% sodium carboxymethylcellulose with equal volume. After 45 days, the animals were killed, and the stone formation rate was measured and compared among groups, and the results are shown in the following table.
Note: total stone weight refers to the total weight of stones removed from each stone-forming animal in the group.
Observations in each group of animals: all animals in group D were lively, healthy, appetizing, shiny; all rabbits in group E gradually suffered from decreased food intake, lassitude, unclean skin, no weight increase and 3 death in sequence during the feeding process, and calculus occurred in biliary tract after laparotomy (all of them are counted as stone-forming animals); the state of the rest three groups of rabbits is superior to that of the E group, and no death occurs in the three groups. After 45d, the gallbladder of only a blank control group is normal, the gallbladder of other rabbits given the stone-forming feed is proliferated to different degrees, the pathological changes of a model group are the most serious, the shapes are irregular, and the inflammatory thickening of the gallbladder wall is obvious. After the killing, the examination shows that 11 rabbits in the 14 rabbits in the model group form stones, wherein 7 stones are in the gall bladder, 2 stones are in the common hepatic duct, and 2 stones are 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 do not form stones, and the comparison of the stone forming conditions of the model group and the blank control group shows that the molding is successful after the animals are fed with the stone forming feed for 45 d; a, B, C showed significantly lower stone formation rates than group E, while group A also showed significantly lower stone formation rates than groups B and C; the total stone weight data and the stone forming rate show the same difference trend among groups. The results show that the T crystal form tauroursodeoxycholic acid arginine salt has the function of inhibiting gallstone generation, and further has the function of preventing gallstones in vivo.
3. Improving stone-forming bile component
As is known, the bile components (namely, lithogenesis bile components) of patients with cholecystitis and cholelithiasis are obviously different from the bile components of normal people, the cholesterol content of the patients is high on the main surface, the patients are in a saturation state, and the concentration of bile acid is reduced due to the fact that the bile acid reabsorption is increased when the biliary tract inflammation is changed (namely, the cholecystitis); the secretion of gall bladder mucus (glycoprotein) of a patient is hyperfunction, the mucus is a nucleation promoting factor, the viscosity of bile is increased, cholesterol in a saturated state is easy to form crystals, and the cholecystolithiasis has important influence on the formation of cholesterol calculus. The test examines the effect of the arginine salt of the crystal form T in improving the stone-forming bile component.
Taking 66 healthy New Zealand male white rabbits, and randomly dividing the rabbits into 5 groups, wherein group A adopts the refined bear gall powder (namely T-crystal form tauroursodeoxycholic acid arginine salt) in the embodiment 1 of the invention, group B adopts the refined bear gall powder (tauroursodeoxycholic acid arginine salt) in the group 2 of the embodiment, group C taurocholate, a blank control group in the group D and a model group in the group E; group D10 animals, the remaining groups 14 animals per group. Examples 1 and 2 the refined bear bile powder and taurulate were ground to a fine powder capable of passing through a 80 mesh sieve prior to administration, and were suspended in 2% sodium carboxymethylcellulose at a concentration of 2% tauroursodeoxycholic acid prior to administration.
Except for group D, which was fed with normal diet, the other groups were fed with 1% cholesterol in stone-forming diet, and were gavaged once a day at doses: A. b, C the dosages of the three groups are all equal to 50mg/kg of tauroursodeoxycholic acid, and the E group model group is given 2% sodium carboxymethylcellulose with equal volume.
After 45 days, performing experiment, performing intravenous injection anesthesia on the rabbit by using 1g/kg sodium pentobarbital, fixing after anesthesia, performing laparotomy, exposing common bile duct along the first segment of duodenum, respectively inserting tubes to the liver side and the intestine side, and after ligation and fixation, leading the catheter out of the body and suturing the abdominal wall. The following measurements were performed for the inoculated bile: bilirubin was immediately measured by azo method, cholesterol by ferric sulfate color development, bile acid by fluorescence spectrophotometry, mucus content by Alcian Blue method, and the results are shown in the table below.
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: comparing P <0.05, P <0.01, P <0.001 for each group of medicines with group E; group E compared with group D, # P <0.05, # P < 0.01.
Compared with the blank control group, the contents of bilirubin, cholesterol and mucus in the model group are all obviously increased by P <0.01 or P <0.001, the content of total bile acid is reduced, and the significant meaning P <0.05 shows that the model is successfully made. Compared with a model group, the three reagents are reduced in bilirubin, cholesterol and mucus to different degrees, and particularly, the group A has obvious reduction effect compared with the other two reagents; the three reagents can also improve the concentration of total bile acid to different degrees, and especially the effect of the T crystal form group is most obvious. It is known that bile acids such as tauroursodeoxycholic acid and the like have the function of dissolving stones, can inhibit hydroxymethylglutaryl coenzyme A reductase activity (the enzyme is a rate-limiting enzyme for synthesizing cholesterol by the liver and reduces the synthesis of endogenous cholesterol), thereby reducing the content of cholesterol, inhibiting cholesterol 7 alpha-dehydrogenase, inhibiting the synthesis of other bile acids, effectively inhibiting the absorption of cholesterol in intestinal tracts, promoting the reduction of the content of cholesterol in bile, enabling the cholesterol bile in a saturated state to be in an unsaturated state, and promoting the cholesterol concentration in the bile to be reduced, thereby not only preventing the formation of cholesterol stones, but also promoting the redissolution of the cholesterol stones. The results show that the T crystal form arginine salt has the effects of reducing the content of bilirubin, cholesterol and mucus in bile and increasing the content of total bile acid, and the indexes show that the T crystal form arginine salt can obviously improve stone-forming bile ingredients, and further can be used for preventing or treating cholelithiasis, cholecystitis and gallbladder function improvement.
Experimental example 4: hepatic fibrosis inhibiting effect of refined bear gall powder
1. Material
The experimental animal is a male SD rat (supplied by scientific and technological center of Experimental animals of pharmaceutical university in Jiangxi), and the weight of the experimental animal is 170-195 g.
Reagent: dimethylnitrosamines were purchased from Sigma-Aldrich, Direct Red (Direct Red-80) from Sigma-Aldrich, immunohistochemical monoclonal antibody ED1 from Santa Cruz, USA, and α -SMA from Danish Dako. Reagent testing: A) the research reagents of the refined bear gall powder (namely T crystal form tauroursodeoxycholic acid arginine salt) in example 1, the refined bear gall powder (tauroursodeoxycholic acid arginine salt) in example 2 and the refined bear gall powder (C) tauroursoder are adopted, the refined bear gall powder and the refined bear gall powder are ground into fine powder capable of passing through a 80-mesh sieve before administration, and the fine powder is suspended in 2% sodium carboxymethylcellulose at the concentration of 2% tauroursodeoxycholic acid before administration.
2. Method of producing a composite material
Reference is made to the Matsuda literature [ Matsuda Y, et al, predictive and therapeutic effects in rates of adipocyte growth factor in fusion on liver fi brosis/cirrhosis.hepatology, 1997; 26:81-89] and various reagents are administered with reference to the model. The reagents for injection were prepared in a concentration of 1mL/kg using physiological saline for injection. The 50 animals were randomly divided into 5 groups, group a (10): 1mL/kg of 10g/L dimethyl nitrosamine (prepared by normal saline) is continuously injected in an abdominal cavity for 3 days/week for 4 weeks, and simultaneously 50mg/kg/d of tauroursodeoxycholic acid arginine salt of T crystal form is perfused in a stomach for 4 weeks; group B (10): 10g/L of 1mL/kg of dimethyl nitrosamine (prepared by normal saline) is continuously injected in an abdominal cavity for 3 days/week for 4 weeks, and simultaneously 50mg/kg/d of tauroursodeoxycholic acid arginine salt in the embodiment 2 is perfused in a stomach for 4 weeks; group C (10): 1mL/kg of 10g/L dimethylnitrosamine (in physiological saline) for 3 consecutive days/week, 4 weeks for intraperitoneal injection, and 4 weeks for intragastric gavage with taurulite 50 mg/kg/d; group D, normal control group (10): 1mL/kg of physiological saline for injection is continuously injected for 3 days/week, the intraperitoneal injection is carried out for 4 weeks, and meanwhile, 2% sodium carboxymethylcellulose is used for equal-volume intragastric gavage as the intragastric gavage of the group A for 4 weeks; group E, model group (10): 10g/L dimethyl nitrosamine (in normal saline) 1mL/kg for 3 consecutive days/week, 4 weeks for intraperitoneal injection, and 4 weeks for isovolumetric gavage with 2% sodium carboxymethylcellulose as in the gavage of group A. After the experiment was completed, the animals were weighed, anesthetized with ether, and centrifuged to collect blood from the heart.
And (3) detecting serum biochemical indexes: the ALT and AST activity of serum is detected by Reiman's method, the total protein content is detected by Biuret method, all the kits are purchased from Japan Rongyan chemical company, the operation is strictly carried out according to the kit instruction, the absorbance is measured by a spectrophotometer, and the AST and ALT values and the total protein content are calculated by contrasting a standard curve.
And (3) pathological examination: the liver was weighed immediately after blood sampling and the liver/body weight percentage was calculated. And (3) pathological examination: two liver tissues are taken, fixed by 40g/L neutral formaldehyde, embedded by conventional paraffin, sliced by HE and directly red-stained (1g/L directly red picric acid saturated solution), and pathological changes of the liver tissues and the proliferation degree of fibrous tissues are observed under a light microscope. The sections directly stained in red were subjected to quantitative analysis of collagen fibers using an Aperio digital pathology scanner and analysis system (Aperio AT2 model, come card microsystems), and the observation conditions were: and (3) the objective lens is 10 times, 5 fields are randomly selected for each slice, and the ratio of the total area of the target to the total area of the statistical field is obtained through image acquisition, segmentation processing and parameter statistical analysis. Immunohistochemical staining: the slice thickness is 4-5 mu m, dewaxing is carried out to water conventionally, immunohistochemical staining is carried out by using an SP method, the working concentration of ED1 is 1: 500, and the working concentration of alpha-SMA is 1: 50; the number and distribution of KC and HSC in liver tissues were observed. Statistical processing statistical analysis was performed using SPSS 13.0 software.
3. Results
The trial group (e.g. group a) compared to the model group: the change trends of the parameters show that the T crystal form arginine salt has the biological effect of inhibiting hepatic fibrosis. The detection results of the indexes at the end of the 4 th week of the experiment are shown in the following table. The results of 5 parameters are compared statistically, and the p of the group E is less than 0.001 or less than 0.01 compared with the group D, which indicates that the molding is successful.
compared to group E, p <0.05, p <0.01, p < 0.001. Compared to group A, # p <0.05, # p < 0.01.
From the above data, it can be seen that A, B, C each group exhibited statistical differences from the model group; group a differed significantly more significantly than the B, C two groups, and group a was significantly better than the B, C two groups. Pathological observations showed that: the hepatic lobule structure of group D is normal, the hepatic cells have no degeneration and necrosis, and the central venous wall and the manifold area of the hepatic lobule have a small amount of collagen fibers; group E hepatic lobule structural disorder, liver cell degeneration, focal or lamellar necrosis, massive fibrous tissue hyperplasia in the zone of confluence, and thick fibrous interval formed and extending into the liver tissue, and diffuse liver cirrhosis is mostly formed; the degeneration and necrosis of the hepatic cells in the group A are lighter than those of the model group, the fibrous tissues in the region of the sink area are reduced, the fibrous intervals become thinner or disappear, a small amount of fine fibrous intervals stretch into the hepatic tissues, and only a small part of the fine fibrous intervals form diffuse cirrhosis; B. the observations in group C showed that it was better than group E but not as good as group a. Immunohistochemical staining results: group D alpha-SMA has a small amount of positive expression on the central vein wall of the hepatic lobule, positive expression on various blood vessel walls in the manifold area, and no positive cells among hepatic cells, ED1 has a small amount of positive cell distribution around the central vein of the hepatic lobule, in the manifold area and in the liver parenchyma; the E group is characterized in that a large number of liver KC (ED1+) and HSC (alpha-SMA +) are distributed in hyperplastic fibrous tissues and intervals in a diffuse way, and liver parenchyma is distributed in a scattered way; the distribution of liver KC and HSC in group a was similar to that of the model group, but the number of both cells was significantly reduced; B. the observations in group C showed that it was better than group E but not as good as group a.
Some documents report that tauroursodeoxycholic acid can resist the increase of mouse serum glutamic pyruvic transaminase caused by carbon tetrachloride CCl4, has a certain protection effect on liver pathological tissue change caused by CCl4, can relieve liver steatosis caused by high-fat and high-calorie diet, and is effective on acute and chronic hepatitis, icteric hepatitis and cirrhosis. The experimental serum detection result shows that the T crystal form arginine salt has a good enzyme reducing effect, wherein the AST activity is obviously reduced, the total serum protein content and the liver/body mass ratio are increased, and the area density of the collagen fiber of the liver tissue is obviously reduced, so that the T crystal form has a good effect of inhibiting DMN (dimethyl formamide) to induce rat hepatic fibrosis, and further has the effect of protecting the liver.
Experimental example 5:drug efficacy test using human colon cancer HCT116 cells:
And (3) testing the sample: the refined bear gall powder obtained in example 1, the refined bear gall powder obtained in example 2, and the commercially available tauroursodeoxycholic acid capsules (taurulate, H20150398, 250 mg/granule) were all ground into fine powder capable of passing through a 80-mesh sieve before administration, and were suspended in 2% sodium carboxymethylcellulose at a concentration of 2% tauroursodeoxycholic acid before administration.
McCoy's 5a medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin and 2mM glutamine at 37 ℃ in 5% CO2The incubator of (a) to culture HCT116 cells. The initial concentration of the cell culture was 1X 106One cell/ml, and bottle-divided passage after full cell growth every 3 to 4 days. Tumor cells in logarithmic growth phase were used for in vivo tumor inoculation. HCT116 tumor cells resuspended in serum-free McCoy's 5a medium at 5X 106The test animals were inoculated in 100. mu.l subcutaneously in the right flank. When the tumor grows to 120mm3Select tumors from left and rightthe animals with more uniform volume were dosed in groups of 5, 10 animals each.
measurement and experimental indices of tumors: the tumor is measured twice a week by using a vernier caliper, the long diameter and the short diameter of the tumor are measured, and the volume calculation formula is as follows: volume is 0.5 x long diameter x short diameter2. Relative Tumor Volume (RTV) and relative tumor volume increase ratio (%, T/C) were calculated from the tumor volume. RTV-Vt/V0, where Vt is mean tumor volume on day t after group administration and V0 is mean tumor volume on day t after group. T/C ═ TRTV/CRTV × 100, where TRTV is the treatment group RTV and CRTV is the solvent control group RTV. The tumor growth inhibition (%, TGI) was calculated as follows: (1-T/C). times.100%.
Tumor volumes and changes in body weight over time were determined for the following 5 groups: 1. solvent control group (2% sodium carboxymethylcellulose, once daily); 2. irinotecan 60mg/kg (i.p. every three days) in the positive control group; 3. example 1 group 50mg/kg (once daily); 4. example 2 group 50mg/kg (once daily); 5. taurolite group 50mg/kg (once daily); the dosages of the three test medicines are converted into the dosage of the tauroursodeoxycholic acid. Each group was dosed continuously and tumor volume, tumor volume (mm) was measured at the specified time3Mean) results are given in the table below.
Time/d solvent control group Positive control group EXAMPLE 1 group EXAMPLE 2 group taurolite group
8 132 127 134 122 128
12 203 218 194 187 221
15 483 187 182 174 193
18 738 158 173 193 202
22 1143 179 169 207 224
26 1838 149 187 256 268
29 2527 179 221 421 378
33 - 164 362 728 693
36 - 186 469 1042 936
39 - 218 627 1374 1187
43 - 232 961 1726 1594
47 - 273 1137 2392 1948
50 - 336 1363 - 2461
Note: -means that at least half of the animals died without further measurement.
As can be seen from the data in the above table, although the tumor volume increased faster in the group of example 1 than in the positive control group, the rate of increase was significantly lower than in the group of example 2 and taurulate, indicating that the antitumor activity of arginine form T of the present invention was stronger than that of example 2.
Experimental example 6: antitumor Activity test
And (3) testing the sample: the refined bear gall powder obtained in example 1, the refined bear gall powder obtained in example 2, and the commercially available tauroursodeoxycholic acid capsules (taurulate, H20150398, 250 mg/granule) were all ground into fine powder capable of passing through a 80-mesh sieve before administration, and were suspended in 2% sodium carboxymethylcellulose at a concentration of 2% tauroursodeoxycholic acid before administration.
the test method comprises the following steps: reference is made to the Cantonella (Cantonella, et al, 20(s) -protopanaxadiol, ginsenoside Rh2And ginsenoside Rg3Comparison of antitumor effects, journal of senile science in china, 2014, stage 17), study of antitumor effects of each test sample, and randomly grouping mice (each tumor model) by establishing three transplanted tumor animal models of mouse liver cancer H22, Lewis lung cancer and melanoma B1612-15 per group), 2% sodium carboxymethylcellulose is given to the control group, 20mg/kg of Cyclophosphamide (CTX) is given to the positive drug group, and the daily dose of the three test samples is 50mg/kg calculated by tauroursodeoxycholic acid. The positive medicine is administrated by intraperitoneal injection every other day for 1 time, and the rest groups are administrated by intragastric administration for 1 time every day for 25 days continuously. The weight of the mice is recorded every day, and the weight of the mice and the weight of the tumor are weighed after the last dose is taken, and the tumor inhibition rate is calculated. The results of the tumor inhibition (%, mean. + -. SD) for each group are shown in the following table.
model (model) Positive drug/% Taurolite/%) Example 2/%) Example 1/%)
Liver cancer H22 48.24±3.22 24.27±2.82***# 21.63±3.36***## 37.42±7.4l*
lewis lung carcinoma 44.67±6.39 17.53±3.76**## 19.31±2.51**## 38.16±4.83*
Melanoma B16 34.83±5.38 13.92±5.24***## 11.87±5.38***### 26.24±6.28*
In each model, p <0.05, p <0.01, p < 0.001; in each model, # p <0.05, # p <0.01, # p <0.001, compared to the group of example 1.
All references cited in this specification, including without limitation all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, internet articles, journal articles, periodicals, and the like, are hereby incorporated by reference into this specification in their entirety. The discussion of the references herein is intended merely to summarize the 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 is to be understood that variations and modifications may be effected by one of ordinary skill in the art without departing from the spirit and scope of the disclosure as set forth in the appended 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 (10)

1. The use of a compound of formula I:
2. Use according to claim 1, said compound having a melting point of 187-189 ℃.
3. Use according to claim 1, of a compound which, using Cu-Ka radiation, in a powder X-ray diffraction pattern expressed in degrees 2 θ,
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 °; or
Diffraction peaks exist 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; or
diffraction peaks exist at 8.53 +/-0.10 degrees, 10.96 +/-0.10 degrees, 12.03 +/-0.10 degrees, 13.14 +/-0.10 degrees, 14.82 +/-0.10 degrees, 17.26 +/-0.10 degrees, 22.53 +/-0.10 degrees, 24.21 +/-0.10 degrees, 26.68 +/-0.10 degrees, 29.42 +/-0.10 degrees and 31.24 +/-0.10 degrees; or
Has the powder X-ray diffraction pattern shown in figure 1.
4. Use according to claim 1, said compound being prepared according to a process comprising the steps of:
(1) Diluting the collected bear bile with water (for example, diluting with 2-3 times of water, for example, diluting with 2.5 times of water), filtering with an 80-mesh sieve, (optionally, filtering the filtrate with the 80-mesh sieve to obtain a filtrate, adjusting the pH of the filtrate to 3.0-3.5, for example, pH 3.3, using 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride, for example, 1.2% sodium chloride to the filtrate) to obtain a crude bear bile (the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate can be measured);
(2) using a tangential flow ultrafiltration system (such as Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 5-15 times (such as 10 times) of concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);
(3) adjusting the pH of the filtrate obtained in the previous step to 6.5-7.0, for example, 6.8, using 1M sodium hydroxide solution, adding arginine (in an amount of 2-3, for example, 2.5, times the amount of tauroursodeoxycholic acid in the filtrate), stirring at 40-50, for example, 44-46, for 2-3, for example, 2.5 hours, filtering to remove the precipitate, adding 1-2, for example, 1.5 times the volume of ethyl acetate to the filtrate, standing for 2-4, for example, 3 hours, precipitating the precipitate, filtering, and removing the filtrate to obtain a precipitate;
(4) adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1 g: 3-5 ml, for example, 1 g: 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 2-4 hours, for example, 3 hours, and filtering off the precipitate to obtain a filtrate;
(5) Adding a mixture of ethyl acetate and ethyl ether (5: 1) in an amount of 1-2 times, for example, 2 times, the volume of the filtrate obtained in the previous step, standing for 5-8 hours, for example, 6 hours, precipitating, filtering to obtain a precipitate, and drying under reduced pressure to remove the solvent to obtain the compound of formula I.
5. Use according to claim 1, wherein:
in the step (1), filtering the filtrate obtained by filtering the 80-mesh screen by using a 1M hydrochloric acid solution to adjust the pH value of the filtrate to 3.0-3.5;
In the step (1), after the pH of the filtrate is adjusted to 3.0-3.5, 1.0-1.5% of sodium chloride is added into the filtrate;
In the step (2), in the filtrate obtained by the tangential flow ultrafiltration, the taurochenodeoxycholic acid accounts for 0-5%, preferably 0-3%, and preferably 0-2% of the weight of the tauroursodeoxycholic acid;
In the reflux liquid obtained by the tangential flow ultrafiltration in the step (2), the tauroursodeoxycholic acid accounts for 0-8% of the weight of the tauroursodeoxycholic acid, preferably 1-5%, and preferably 1-3%.
6. The application of the refined bear gall powder in preparing products for preventing or treating tumors and cancers is characterized in that the refined bear gall powder is tauroursodeoxycholic acid arginine salt with a melting point of 187-189 ℃.
7. The use according to claim 6, wherein the refined bear gall powder:
the content of tauroursodeoxycholic acid is more than 70%, such as 70-74%, particularly more than 71%, such as 71-74%, particularly more than 72%, such as 72-74%;
The molar ratio of tauroursodeoxycholic acid to arginine is 1: 0.98 to 1.02, in particular 1: 0.99 to 1.01;
The percentage of the total content of the tauroursodeoxycholic acid and the arginine to the total content of the refined bear gall powder is more than 95 percent, such as 95 to 100 percent, particularly more than 96 percent, such as 96 to 100 percent, particularly more than 97 percent, such as 97 to 100 percent, particularly more than 98 percent, such as 98 to 100 percent;
which uses Cu-Ka radiation and 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 2 θ;
The Cu-Kalpha radiation is used, and diffraction peaks exist 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 pattern expressed by 2 theta angles;
the Cu-Kalpha radiation is used, and diffraction peaks exist at 8.53 +/-0.10 degrees, 10.96 +/-0.10 degrees, 12.03 +/-0.10 degrees, 13.14 +/-0.10 degrees, 14.82 +/-0.10 degrees, 17.26 +/-0.10 degrees, 22.53 +/-0.10 degrees, 24.21 +/-0.10 degrees, 26.68 +/-0.10 degrees, 29.42 +/-0.10 degrees and 31.24 +/-0.10 degrees in a powder X-ray diffraction pattern expressed by a 2 theta angle;
Using Cu-ka radiation, having the powder X-ray diffraction pattern shown in figure 1.
8. The use according to claim 6, wherein said refined bear gall powder is prepared by a method comprising the steps of:
(1) Diluting the collected bear bile with water (for example, diluting with 2-3 times of water, for example, diluting with 2.5 times of water), filtering with an 80-mesh sieve, (optionally, filtering the filtrate with the 80-mesh sieve to obtain a filtrate, adjusting the pH of the filtrate to 3.0-3.5, for example, pH 3.3, using 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride, for example, 1.2% sodium chloride to the filtrate) to obtain a crude bear bile (the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate can be measured);
(2) Using a tangential flow ultrafiltration system (such as Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 5-15 times (such as 10 times) of concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);
(3) Adjusting the pH of the filtrate obtained in the previous step to 6.5-7.0, for example, 6.8, using 1M sodium hydroxide solution, adding arginine (in an amount of 2-3, for example, 2.5, times the amount of tauroursodeoxycholic acid in the filtrate), stirring at 40-50, for example, 44-46, for 2-3, for example, 2.5 hours, filtering to remove the precipitate, adding 1-2, for example, 1.5 times the volume of ethyl acetate to the filtrate, standing for 2-4, for example, 3 hours, precipitating the precipitate, filtering, and removing the filtrate to obtain a precipitate;
(4) adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1 g: 3-5 ml, for example, 1 g: 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 2-4 hours, for example, 3 hours, and filtering off the precipitate to obtain a filtrate;
(5) Adding 1-2 times volume of ethyl acetate-diethyl ether (5: 1) mixture into the filtrate obtained in the previous step, standing for 5-8 hr, such as 6 hr, precipitating, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined fel Ursi powder.
9. Use according to claim 8, wherein:
In the step (1), filtering the filtrate obtained by filtering the 80-mesh screen by using a 1M hydrochloric acid solution to adjust the pH value of the filtrate to 3.0-3.5;
In the step (1), after the pH of the filtrate is adjusted to 3.0 to 3.5, 1.0 to 1.5% of sodium chloride is further added to the filtrate;
in the step (2), in the filtrate obtained by the tangential flow ultrafiltration, the taurochenodeoxycholic acid accounts for 0-5%, preferably 0-3%, and preferably 0-2% of the weight of the tauroursodeoxycholic acid;
in the reflux liquid obtained by the tangential flow ultrafiltration in the step (2), the tauroursodeoxycholic acid accounts for 0-8% of the weight of the tauroursodeoxycholic acid, preferably 1-5%, and preferably 1-3%.
10. A process for the preparation of a compound for use according to claims 1 to 9 or a purified bear gall powder comprising the steps of:
(1) diluting the collected bear bile with water (for example, diluting with 2-3 times of water, for example, diluting with 2.5 times of water), filtering with an 80-mesh sieve, (optionally, filtering the filtrate with the 80-mesh sieve to obtain a filtrate, adjusting the pH of the filtrate to 3.0-3.5, for example, pH 3.3, using 1M hydrochloric acid solution, and then adding 1.0-1.5% sodium chloride, for example, 1.2% sodium chloride to the filtrate) to obtain a crude bear bile (the content of tauroursodeoxycholic acid and taurochenodeoxycholic acid in the process intermediate can be measured);
(2) using a tangential flow ultrafiltration system (such as Shibipure KR2i type tangential flow ultrafiltration system), washing a pipeline with ultrapure water, installing a 1kD MidiKros filter, and filtering the crude bear gall liquid obtained in the step (1) to obtain a filtrate and 5-15 times (such as 10 times) of concentrated reflux liquid (the content of tauroursodeoxycholic acid and tauroursodeoxycholic acid in the process intermediate can be measured);
(3) adjusting the pH of the filtrate obtained in the previous step to 6.5-7.0, for example, 6.8, using 1M sodium hydroxide solution, adding arginine (in an amount of 2-3, for example, 2.5, times the amount of tauroursodeoxycholic acid in the filtrate), stirring at 40-50, for example, 44-46, for 2-3, for example, 2.5 hours, filtering to remove the precipitate, adding 1-2, for example, 1.5 times the volume of ethyl acetate to the filtrate, standing for 2-4, for example, 3 hours, precipitating the precipitate, filtering, and removing the filtrate to obtain a precipitate;
(4) Adding ethanol (for example, ethanol is added according to the volume ratio of the weight of the precipitate to the volume of the ethanol of 1 g: 3-5 ml, for example, 1 g: 4 ml) into the precipitate obtained in the previous step, stirring at room temperature for 0.5 hour, standing for 2-4 hours, for example, 3 hours, and filtering off the precipitate to obtain a filtrate;
(5) Adding 1-2 times volume of ethyl acetate-diethyl ether (5: 1) mixture into the filtrate obtained in the previous step, standing for 5-8 hr, such as 6 hr, precipitating, filtering to obtain precipitate, and drying under reduced pressure to remove solvent to obtain refined fel Ursi powder.
CN201910902326.6A 2019-09-24 2019-09-24 Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer Active CN110548037B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910902326.6A CN110548037B (en) 2019-09-24 2019-09-24 Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910902326.6A CN110548037B (en) 2019-09-24 2019-09-24 Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer

Publications (2)

Publication Number Publication Date
CN110548037A true CN110548037A (en) 2019-12-10
CN110548037B CN110548037B (en) 2023-06-02

Family

ID=68741225

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910902326.6A Active CN110548037B (en) 2019-09-24 2019-09-24 Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer

Country Status (1)

Country Link
CN (1) CN110548037B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110507669A (en) * 2019-09-24 2019-11-29 江西天元药业有限公司 Refined bear gall powder and its treatment cholecystitis gallstones disease improve the purposes of gallbladder function
CN110507670A (en) * 2019-09-24 2019-11-29 江西天元药业有限公司 Refined bear gall powder and prophylactic treatment hepatopathy liver fibrosis improve the purposes of liver function
CN110559303A (en) * 2019-09-24 2019-12-13 江西天元药业有限公司 Refined bear gall powder for reducing blood fat, preventing and treating cardiovascular and cerebrovascular diseases and atherosclerosis
CN111393501A (en) * 2020-05-06 2020-07-10 黑龙江中医药大学 Hemiketal structure of taurine-3-dehydro-chenodeoxycholic acid and preparation method of taurine-3-dehydro-chenodeoxycholic acid

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101048164A (en) * 2004-11-01 2007-10-03 柳署弘 Methods and compositions for reducing neurodegeneration in amyotrophic lateral sclerosis
CN101547699A (en) * 2006-09-15 2009-09-30 柳署弘 Bile preparations for colorectal disorders
US20100069654A1 (en) * 2007-04-23 2010-03-18 Massimo Parenti Process for the preparation of tauroursodesoxycholic acid
CN102712672A (en) * 2009-08-25 2012-10-03 林重庆 Polyhydroxylated bile acids for treatment of biliary disorders
CN102836175A (en) * 2012-05-23 2012-12-26 杭州宝积生物科技有限公司 Artificial bear bile
CN102958531A (en) * 2010-04-22 2013-03-06 耶路撒冷希伯来大学伊森姆研究发展公司 High affinity leptins and leptin antagonists
CN103919787A (en) * 2014-04-17 2014-07-16 厦门大学 Pharmaceutical application of tauroursodeoxycholic acid and acceptable salts thereof
CN107028954A (en) * 2015-07-24 2017-08-11 武汉朗来科技发展有限公司 Ornithine aspartate is preparing the purposes in preventing and treating fatty liver medicament
CN108578434A (en) * 2018-07-14 2018-09-28 齐光伟 Prevent or treat method and the bear gall powder used of eye conjunctivitis and eyelid herpes zoster
CN108785328A (en) * 2018-07-14 2018-11-13 齐光伟 Prevent or treat method and the bear gall powder used of entity tumor and hematological system tumor
CN110507669A (en) * 2019-09-24 2019-11-29 江西天元药业有限公司 Refined bear gall powder and its treatment cholecystitis gallstones disease improve the purposes of gallbladder function

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101048164A (en) * 2004-11-01 2007-10-03 柳署弘 Methods and compositions for reducing neurodegeneration in amyotrophic lateral sclerosis
CN101547699A (en) * 2006-09-15 2009-09-30 柳署弘 Bile preparations for colorectal disorders
US20100069654A1 (en) * 2007-04-23 2010-03-18 Massimo Parenti Process for the preparation of tauroursodesoxycholic acid
CN102712672A (en) * 2009-08-25 2012-10-03 林重庆 Polyhydroxylated bile acids for treatment of biliary disorders
CN102958531A (en) * 2010-04-22 2013-03-06 耶路撒冷希伯来大学伊森姆研究发展公司 High affinity leptins and leptin antagonists
CN102836175A (en) * 2012-05-23 2012-12-26 杭州宝积生物科技有限公司 Artificial bear bile
CN103919787A (en) * 2014-04-17 2014-07-16 厦门大学 Pharmaceutical application of tauroursodeoxycholic acid and acceptable salts thereof
CN107028954A (en) * 2015-07-24 2017-08-11 武汉朗来科技发展有限公司 Ornithine aspartate is preparing the purposes in preventing and treating fatty liver medicament
CN108578434A (en) * 2018-07-14 2018-09-28 齐光伟 Prevent or treat method and the bear gall powder used of eye conjunctivitis and eyelid herpes zoster
CN108785328A (en) * 2018-07-14 2018-11-13 齐光伟 Prevent or treat method and the bear gall powder used of entity tumor and hematological system tumor
CN110507669A (en) * 2019-09-24 2019-11-29 江西天元药业有限公司 Refined bear gall powder and its treatment cholecystitis gallstones disease improve the purposes of gallbladder function

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110507669A (en) * 2019-09-24 2019-11-29 江西天元药业有限公司 Refined bear gall powder and its treatment cholecystitis gallstones disease improve the purposes of gallbladder function
CN110507670A (en) * 2019-09-24 2019-11-29 江西天元药业有限公司 Refined bear gall powder and prophylactic treatment hepatopathy liver fibrosis improve the purposes of liver function
CN110559303A (en) * 2019-09-24 2019-12-13 江西天元药业有限公司 Refined bear gall powder for reducing blood fat, preventing and treating cardiovascular and cerebrovascular diseases and atherosclerosis
CN110507670B (en) * 2019-09-24 2023-04-07 江西天元药业有限公司 Refined bear gall powder and application of refined bear gall powder in preventing and treating liver diseases, liver fibrosis and improving liver function
CN110507669B (en) * 2019-09-24 2023-05-02 江西天元药业有限公司 Refined bear gall powder and its use for treating cholecystitis gall-stone and improving gall-bladder function
CN111393501A (en) * 2020-05-06 2020-07-10 黑龙江中医药大学 Hemiketal structure of taurine-3-dehydro-chenodeoxycholic acid and preparation method of taurine-3-dehydro-chenodeoxycholic acid
CN111393501B (en) * 2020-05-06 2021-02-09 黑龙江中医药大学 Preparation method of taurine-3-dehydro-chenodeoxycholic acid

Also Published As

Publication number Publication date
CN110548037B (en) 2023-06-02

Similar Documents

Publication Publication Date Title
CN110548037B (en) Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer
CN110511261B (en) Refined bear gall powder with improved bioavailability and preparation method thereof
RU2700793C2 (en) Pharmaceutical composition containing silybin, vitamin e and l-carnitine
EP3434285A1 (en) Pharmaceutical composition and use thereof
CN108339000B (en) Panax plant extract, and pharmaceutical composition and application thereof
CN108578434A (en) Prevent or treat method and the bear gall powder used of eye conjunctivitis and eyelid herpes zoster
CN108785328A (en) Prevent or treat method and the bear gall powder used of entity tumor and hematological system tumor
TWI300352B (en) Water soluble extract from plant of solanum genus and the preparation process thereof, and pharmaceutical composition containing the water soluble extract
CN108743622A (en) Immunity of organisms and muscle power are improved using bear gall powder and alleviate the method for organism fatigue
CN108635375A (en) Prevent or treats hepatopathy and liver fibrosis and improve the bear gall powder and purposes of liver function
CN108670979A (en) Adjust-blood lipid norcholesterol prevents the bear gall powder of cardiovascular and cerebrovascular diseases and atherosclerosis
CN108578433A (en) For preventing or treating gall stone cholecystitis and improve the bear gall powder of gallbladder function
CN108653332A (en) Inhibition thrombosis and platelet aggregation and the bear gall powder and purposes for preventing cerebral ischemia
CN110507669B (en) Refined bear gall powder and its use for treating cholecystitis gall-stone and improving gall-bladder function
CN110559303B (en) Refined bear gall powder for reducing blood fat and preventing and treating cardiovascular and cerebrovascular diseases and atherosclerosis
CN110507670B (en) Refined bear gall powder and application of refined bear gall powder in preventing and treating liver diseases, liver fibrosis and improving liver function
CN111228319A (en) Brucea javanica extract, preparation method thereof and application thereof in preventing and treating breast cancer
CN109568419B (en) Morinda officinalis total iridoid glycoside and preparation method and application thereof
CN106890189A (en) Application of the chonglou saponin in antineoplastic sensitizer is prepared
CN110882286A (en) Application of wall-removed ganoderma lucidum spore powder
JP4087114B2 (en) Hepatitis B and cirrhosis treatment composition
JPS647048B2 (en)
CN105012382B (en) Walnut meal extract and preparation method and application thereof
CN111297849B (en) Pharmaceutical composition for treating laryngeal cancer, preparation method and application thereof
CN111298061B (en) Traditional Chinese medicine composition for clearing heat and detoxicating, removing stasis and resolving masses and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant