阅读说明：本技术 精制熊胆粉及其增强体质治疗和预防肿瘤和癌症的用途 (Refined bear gall powder and its use for strengthening physique, treating and preventing tumor and cancer ) 是由 傅金荣 付金洪 于 2019-09-24 设计创作，主要内容包括：本发明涉及精制熊胆粉及其增强体质治疗和预防肿瘤和癌症的用途。具体的,本发明一方面涉及式I所示化合物在制备用于预防或治疗肿瘤和癌症：该化合物,其使用Cu-Kα辐射,在以2θ角度表示的粉末X-射线衍射图谱中,在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°处有衍射峰。该化合物呈现优异的生物学性能例如具有优异的生物利用度,并且可以发挥与熊胆粉或牛磺熊去氧胆酸一样的生理学活性。例如其可用于预防或治疗预防或治疗肿瘤和癌症。(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.)

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.

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.