CA3050146C - Traditional chinese medicine herb composition, making thereof, and application thereof - Google Patents
Traditional chinese medicine herb composition, making thereof, and application thereof Download PDFInfo
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Abstract
The present disclosure belongs to the field of traditional Chinese medicine herb, and specifically relates to a traditional Chinese medicine herb composition capable of protecting liver and benefiting gallbladder and preparation and application thereof. The medicinal component raw materials comprise: rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei. The traditional Chinese medicine herb composition and a traditional Chinese medicine herb extract provided in the present disclosure can significantly reduce serum ALT, AST, ALP, T-Bil, D-Bil and TBA, significantly reduce hepatocyte necrosis, improve hepatocyte enlargement, cytoplasmic looseness, ballooning degeneration and feathery degeneration conditions, and alleviate inflammatory cell infiltration, thus preventing or treating cholestasis.
Description
Title Traditional Chinese medicine herb composition, making thereof, and application thereof Background of the Present Disclosure Field of Disclosure The present disclosure belongs to the field of traditional Chinese medicine herb, and specifically relates to a traditional Chinese medicine herb composition capable of protecting liver and gallbladder, making thereof, and application thereof.
Description of Related Arts Cholestasis is clinically manifested as jaundice with elevated levels of serum bilirubin, bile salts, alkaline phosphatase, etc., and histologically often manifested as an accumulation of bile components in a liver. The cholestasis usually causes changes in related hepatic parenchymal cells, especially the bile acid detergency can cause feathery degeneration of hepatic cells.
Bile secretion is one of the most important functions of the liver. Bilirubin and bile acid are the main components of bile, and the metabolic states of the two can reflect the liver's ability to transport organic anions. An increase in T-Bil with a marked increase in D-Bil indicates a presence of cholestatic jaundice. When the cholestasis occurs, the bile secretion decreases and the distribution of storage amount of the bile acid is rapidly changed, resulting in a significant increase in concentration of the bile acid in serum and urine. A variety of organs contain ALT and AST, the ALT content of the liver is the highest, most of which exists in the cytoplasm. The AST content of liver is second only to the AST
content of a heart, which mainly exists in the liver cell mitochondria. These two enzymes generally do not rise during the cholestasis, and the ALT and the AST enter the blood only during hepatocyte necrosis or permeability transition caused by the cholestasis, resulting in an increase in activity of enzymes in the blood. ALP is an enzyme of a group of hydrolyzed phosphate monoester compounds in an alkaline environment. Serum ALP is mainly from the liver and skeletons. Pathological conditions such as the cholestasis, hepatocyte damage and liver occupying can induce an increase in a production of ALP and an increase in activity of the serum ALP when a capillary bile duct pressure is increased. Elevated serum ALP is the most characteristic early manifestation of cholestasis.
It is significant to research and develop drugs which have a good therapeutic effect on cholestasis.
Summary of the Present Disclosure The present disclosure provides a traditional Chinese medicine herb composition capable of protecting liver and benefiting gallbladder and preparation and application thereof.
In particular, the present disclosure provides a traditional Chinese medicine herb composition for preventing and/or treating liver damage and/or cholestasis, wherein medicinal component raw materials of the traditional Chinese medicine herb composition consist of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei.
There is also provided a traditional Chinese medicine herb extract, obtained by extracting a traditional Chinese medicine herb composition disclosed herein as a raw material by an extraction method, wherein the extraction method is selected from boiling, alcohol extraction, water extraction and alcohol precipitation, alcohol extraction and water precipitation, and a salting out method.
Description of Related Arts Cholestasis is clinically manifested as jaundice with elevated levels of serum bilirubin, bile salts, alkaline phosphatase, etc., and histologically often manifested as an accumulation of bile components in a liver. The cholestasis usually causes changes in related hepatic parenchymal cells, especially the bile acid detergency can cause feathery degeneration of hepatic cells.
Bile secretion is one of the most important functions of the liver. Bilirubin and bile acid are the main components of bile, and the metabolic states of the two can reflect the liver's ability to transport organic anions. An increase in T-Bil with a marked increase in D-Bil indicates a presence of cholestatic jaundice. When the cholestasis occurs, the bile secretion decreases and the distribution of storage amount of the bile acid is rapidly changed, resulting in a significant increase in concentration of the bile acid in serum and urine. A variety of organs contain ALT and AST, the ALT content of the liver is the highest, most of which exists in the cytoplasm. The AST content of liver is second only to the AST
content of a heart, which mainly exists in the liver cell mitochondria. These two enzymes generally do not rise during the cholestasis, and the ALT and the AST enter the blood only during hepatocyte necrosis or permeability transition caused by the cholestasis, resulting in an increase in activity of enzymes in the blood. ALP is an enzyme of a group of hydrolyzed phosphate monoester compounds in an alkaline environment. Serum ALP is mainly from the liver and skeletons. Pathological conditions such as the cholestasis, hepatocyte damage and liver occupying can induce an increase in a production of ALP and an increase in activity of the serum ALP when a capillary bile duct pressure is increased. Elevated serum ALP is the most characteristic early manifestation of cholestasis.
It is significant to research and develop drugs which have a good therapeutic effect on cholestasis.
Summary of the Present Disclosure The present disclosure provides a traditional Chinese medicine herb composition capable of protecting liver and benefiting gallbladder and preparation and application thereof.
In particular, the present disclosure provides a traditional Chinese medicine herb composition for preventing and/or treating liver damage and/or cholestasis, wherein medicinal component raw materials of the traditional Chinese medicine herb composition consist of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei.
There is also provided a traditional Chinese medicine herb extract, obtained by extracting a traditional Chinese medicine herb composition disclosed herein as a raw material by an extraction method, wherein the extraction method is selected from boiling, alcohol extraction, water extraction and alcohol precipitation, alcohol extraction and water precipitation, and a salting out method.
2 Date Recue/Date Received 2021-06-11 There is further provided a method for preparing the traditional Chinese medicine herb extract as disclosed herein, comprising the following steps: extracting the above-mentioned traditional Chinese medicine herb composition as the raw material by an extraction method disclosed herein.
There is also provided a use of the traditional Chinese medicine herb composition, or a use of the traditional Chinese medicine herb extract, in preparation of a drug for preventing and/or treating liver damage.
There is further provided a use of the traditional Chinese medicine herb composition, or a use of the traditional Chinese medicine herb extract, in preparation of a drug for preventing and/or treating cholestasis.
There is also provided a use of the traditional Chinese medicine herb composition, or a use of the traditional Chinese medicine herb extract, in preparation of a drug having any of the following efficacies: (1) reducing a serum alanine aminotransferase level; (2) reducing an aspartic acid aminotransferase level; (3) reducing a serum alkaline phosphatase level; (4) reducing a serum total bilirubin level; (5) reducing a direct bilirubin level;
(6) reducing a total bile acid level; (7) reducing hepatocyte necrosis; (8) improving conditions of hepatocyte enlargement, hepatocyte cytoplasmic looseness, hepatocyte ballooning degeneration and hepatocyte feathery degeneration; and (9) alleviating inflammatory cell infiltration.
There is further provided a traditional Chinese medicine herb preparation, comprising a therapeutically effective amount of the traditional Chinese medicine herb composition, or the traditional Chinese medicine herb extract, wherein the therapeutically effective amount is 1.00-2.65 g/kg.
In a first aspect of the present disclosure, a traditional Chinese medicine herb composition is provided, the medicinal component raw materials comprise:
rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei.
In one embodiment, the medicinal component raw materials of the traditional Chinese medicine herb composition comprise rhizoma polygoni cuspidati, pericarpium citri 2a Date Recue/Date Received 2021-06-11 reticulatae viride, fructus crataegi and radix et rhizoma rhei.
In one embodiment, the range of a ratio of the parts by weight of rhizoma polygoni 2b Date Recue/Date Received 2021-06-11 cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei is as follows: 1-30 parts of rhizoma polygoni cuspidati, 1-12 parts of pericarpium citri reticulatae viride, 1-30 parts of fructus crataegi and 0.1-10 parts of radix et rhizoma rhei.
In one embodiment, the rhizoma polygoni cuspidati is 1-15 parts by weight.
In one embodiment, the rhizoma polygoni cuspidati is 15-30 parts by weight.
In one embodiment, the rhizoma polygoni cuspidati is 15 parts by weight.
In one embodiment, the pericarpium citri reticulatae viride is 1-6 parts by weight.
In one embodiment, the pericarpium citri reticulatae viride is 6-12 parts by weight.
In one embodiment, the pericarpium citri reticulatae viride is 6 parts by weight.
In one embodiment, the fructus crataegi is 1-15 parts by weight.
In one embodiment, the fructus crataegi is 15-30 parts by weight.
In one embodiment, the fructus crataegi is 15 parts by weight.
In one embodiment, the radix et rhizoma rhei is 0.1-1 part by weight.
In one embodiment, the radix et rhizoma rhei is 1-10 parts by weight.
In one embodiment, the radix et rhizoma rhei is 1 part by weight.
In one embodiment, the range of the ratio of the parts by weight of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei is as follows: 15 parts of rhizoma polygoni cuspidati, 6 parts of pericarpium citri reticulatae viride, 15 parts of fructus crataegi and 1 part of radix et rhizoma rhei.
Further, in a second aspect of the present disclosure, a traditional Chinese medicine herb extract is provided and obtained by extracting active components from the above traditional Chinese medicine herb composition as a raw material.
In the present disclosure, each raw material and its formula in the traditional
There is also provided a use of the traditional Chinese medicine herb composition, or a use of the traditional Chinese medicine herb extract, in preparation of a drug for preventing and/or treating liver damage.
There is further provided a use of the traditional Chinese medicine herb composition, or a use of the traditional Chinese medicine herb extract, in preparation of a drug for preventing and/or treating cholestasis.
There is also provided a use of the traditional Chinese medicine herb composition, or a use of the traditional Chinese medicine herb extract, in preparation of a drug having any of the following efficacies: (1) reducing a serum alanine aminotransferase level; (2) reducing an aspartic acid aminotransferase level; (3) reducing a serum alkaline phosphatase level; (4) reducing a serum total bilirubin level; (5) reducing a direct bilirubin level;
(6) reducing a total bile acid level; (7) reducing hepatocyte necrosis; (8) improving conditions of hepatocyte enlargement, hepatocyte cytoplasmic looseness, hepatocyte ballooning degeneration and hepatocyte feathery degeneration; and (9) alleviating inflammatory cell infiltration.
There is further provided a traditional Chinese medicine herb preparation, comprising a therapeutically effective amount of the traditional Chinese medicine herb composition, or the traditional Chinese medicine herb extract, wherein the therapeutically effective amount is 1.00-2.65 g/kg.
In a first aspect of the present disclosure, a traditional Chinese medicine herb composition is provided, the medicinal component raw materials comprise:
rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei.
In one embodiment, the medicinal component raw materials of the traditional Chinese medicine herb composition comprise rhizoma polygoni cuspidati, pericarpium citri 2a Date Recue/Date Received 2021-06-11 reticulatae viride, fructus crataegi and radix et rhizoma rhei.
In one embodiment, the range of a ratio of the parts by weight of rhizoma polygoni 2b Date Recue/Date Received 2021-06-11 cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei is as follows: 1-30 parts of rhizoma polygoni cuspidati, 1-12 parts of pericarpium citri reticulatae viride, 1-30 parts of fructus crataegi and 0.1-10 parts of radix et rhizoma rhei.
In one embodiment, the rhizoma polygoni cuspidati is 1-15 parts by weight.
In one embodiment, the rhizoma polygoni cuspidati is 15-30 parts by weight.
In one embodiment, the rhizoma polygoni cuspidati is 15 parts by weight.
In one embodiment, the pericarpium citri reticulatae viride is 1-6 parts by weight.
In one embodiment, the pericarpium citri reticulatae viride is 6-12 parts by weight.
In one embodiment, the pericarpium citri reticulatae viride is 6 parts by weight.
In one embodiment, the fructus crataegi is 1-15 parts by weight.
In one embodiment, the fructus crataegi is 15-30 parts by weight.
In one embodiment, the fructus crataegi is 15 parts by weight.
In one embodiment, the radix et rhizoma rhei is 0.1-1 part by weight.
In one embodiment, the radix et rhizoma rhei is 1-10 parts by weight.
In one embodiment, the radix et rhizoma rhei is 1 part by weight.
In one embodiment, the range of the ratio of the parts by weight of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei is as follows: 15 parts of rhizoma polygoni cuspidati, 6 parts of pericarpium citri reticulatae viride, 15 parts of fructus crataegi and 1 part of radix et rhizoma rhei.
Further, in a second aspect of the present disclosure, a traditional Chinese medicine herb extract is provided and obtained by extracting active components from the above traditional Chinese medicine herb composition as a raw material.
In the present disclosure, each raw material and its formula in the traditional
3 Chinese medicine composition are the most critical. After knowing the traditional Chinese medicine formula, those skilled in the art can use various conventional traditional Chinese medicine active component extraction methods to extract the active components, such as conventional boiling, an alcohol extraction method, a water extraction and alcohol precipitation method, an alcohol extraction and water precipitation method, and a salting out method. The above extraction methods can obtain the main medicinal components in the above traditional Chinese medicine formula, and therefore the above extraction methods can have certain prevention and treatment effects on the cholestasis.
The water extraction and alcohol precipitation method is a method of first extracting the medicinal material active components by using water as a solvent, and then removing impurities in an extracting solution by precipitation using different concentrations of ethanol. When an ethanol content is 50 to 60% (weight to volume ratio, i.e., g/100 mL) generally, the impurities such as starch can be removed. When the alcohol content is 70%
or more, except for a few inactive components such as tannins and water-soluble pigments, most of the remaining impurities can be removed by precipitation.
The alcohol extraction and water precipitation method is a method of first extracting medicinal material components with a suitable concentration of ethanol, and then removing the impurities in the extracting solution by using water.
After extracting the active components, the traditional Chinese medicine composition can be concentrated, purified or dried by a conventional step to obtain a traditional Chinese medicine extract. The concentration methods comprise, but are not limited to, atmospheric pressure evaporation, reduced-pressure evaporation, thin film evaporation, and multi-effect evaporation. The purification methods comprise, but are not limited to, salting out purification, macroporous resin adsorption purification, ethanol precipitation purification, ion exchange resin purification, flocculation precipitation purification, membrane separation purification, polyamide adsorption purification, silica gel
The water extraction and alcohol precipitation method is a method of first extracting the medicinal material active components by using water as a solvent, and then removing impurities in an extracting solution by precipitation using different concentrations of ethanol. When an ethanol content is 50 to 60% (weight to volume ratio, i.e., g/100 mL) generally, the impurities such as starch can be removed. When the alcohol content is 70%
or more, except for a few inactive components such as tannins and water-soluble pigments, most of the remaining impurities can be removed by precipitation.
The alcohol extraction and water precipitation method is a method of first extracting medicinal material components with a suitable concentration of ethanol, and then removing the impurities in the extracting solution by using water.
After extracting the active components, the traditional Chinese medicine composition can be concentrated, purified or dried by a conventional step to obtain a traditional Chinese medicine extract. The concentration methods comprise, but are not limited to, atmospheric pressure evaporation, reduced-pressure evaporation, thin film evaporation, and multi-effect evaporation. The purification methods comprise, but are not limited to, salting out purification, macroporous resin adsorption purification, ethanol precipitation purification, ion exchange resin purification, flocculation precipitation purification, membrane separation purification, polyamide adsorption purification, silica gel
4 adsorption chromatography purification and the like. The drying methods comprise, but are not limited to, a drying method, a drum drying method, a belt drying method, a moisture absorption drying method, a boiling drying method, a spray drying method, a reduced pressure drying method, a freeze drying method, an infrared drying method, a microwave drying method and the like.
In one embodiment, the extraction is performed using an ethanol extraction method. For example, the extraction can be performed using 60% to 80% ethanol (weight to volume ratio, i.e., g/100 mL).
In one embodiment, the extraction is performed using a water extraction method.
In a third aspect of the present disclosure, a method for preparing the above-mentioned traditional Chinese medicine herb extract is provided, comprising the following steps: extracting active components from the above-mentioned traditional Chinese medicine herb composition as a raw material.
In one embodiment, the preparation method comprises the steps: taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, and fructus crataegi according to a ratio, extracting with ethanol, concentrating and drying an extracting solution to obtain a dry extract, then adding radix et rhizoma rhei powder, and evenly mixing.
In one embodiment, the ethanol is selected from 60% to 80% ethanol, preferably, 70% ethanol.
The radix et rhizoma rhei powder is obtained by pulverizing the radix et rhizoma rhei.
In one embodiment, the preparation method comprises the steps: taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei according to the ratio, and concentrating and drying an extracting solution to obtain a dry extract.
In one embodiment, the ethanol is selected from 60% to 80% ethanol, preferably, 70% ethanol.
In one embodiment, the preparation method comprises the steps: taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei according to the ratio, extracting with water, filtering, concentrating and drying.
In a fourth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of drugs for preventing and/or treating liver damage are provided.
In a fifth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of drugs for preventing and/or treating the cholestasis.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can prevent and/or treat intrahepatic cholestasis.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can prevent and/or treat acute intrahepatic cholestasis.
In a sixth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of an ALT reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of ALT in serum.
In a seventh aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of an AST reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of AST in serum.
In an eighth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of an ALP reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of ALP in serum.
In a ninth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a T-Bil reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of T-Bil.
In a tenth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a D-Bil reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of D-Bil.
In an eleventh aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a TBA reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce the level of TBA.
In a twelfth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a drug having any of the following efficacies: (1) reducing hepatocyte necrosis; (2) improving hepatocyte enlargement, cytoplasmic looseness ballooning degeneration and feathery degeneration conditions; and (3) alleviating inflammatory cell infiltration are provided.
In a thirteenth aspect of the present disclosure, a traditional Chinese medicine herb preparation is provided, comprising a therapeutically effective amount of the above-mentioned traditional Chinese medicine extract.
The traditional Chinese medicine herb preparation preferably comprises one or more conventional pharmaceutically acceptable excipients.
The pharmaceutically acceptable excipients comprise (but are not limited to):
pharmaceutically acceptable carriers, diluents, fillers, binders, and other excipients.
Therapeutically inert inorganic or organic carriers known to those skilled in the art comprise (but are not limited to) lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyhydroxy compounds such as polyethylene glycol, water, sucrose, ethanol, glycerin, and the like, various preservatives, lubricants, dispersants and flavoring agents. Humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added therein, which are used as needed to aid in the stability of the formula or help to enhance activity or bioavailability of the formula or produce an acceptable mouthfeel or odor in the case of oral administration. The traditional Chinese medicine herb preparation such as granules and ointments can be prepared by a conventional method. The pharmaceutical composition provided in the present disclosure may also be used with other therapeutic agents.
Preferably, the traditional Chinese medicine herb preparation is an oral preparation, comprising, but not limited to, granules, pills, tablets, capsules, syrups, sprays and the like.
After extracting the active components in the traditional Chinese medicine composition, the above-mentioned pharmaceutical composition can be prepared by using conventional pharmaceutical excipients and additives.
The therapeutically effective dose of the pharmaceutical composition provided in the present disclosure is as follows: the orally safe and effective amount is usually 1.00 to 2.65 g/kg by body weight based on the total weight of raw medicinal materials.
Certainly, the specific dose should also consider factors such as a route of administration, a state of health of the patient, etc., which is within the skill of a skilled physician.
Compared with the prior art, the present disclosure has the following beneficial effects:
The traditional Chinese medicine composition and the traditional Chinese medicine extract provided in the present disclosure can significantly reduce serum ALT, AST, ALP, T-Bil, D-Bil and TBA, significantly reduce hepatocyte necrosis, improve hepatocyte enlargement, cytoplasmic looseness, and ballooning degeneration and feathery degeneration conditions, and alleviate inflammatory cell infiltration, thus preventing or treating cholestasis.
Brief Description of the Drawings Fig. 1: Effects of extract A, extract B, extract C and UDCA on serum ALT, AST, ALP, T-Bil, D-Bil and TBA in ANIT-induced cholestasis mice; compared with a Vehicle group, *P <0.05; compared with an ANIT model group, #P<0.05.
Fig. 2: Analysis of pathological sections of liver in each group (H&E, x400), A:
normal control group; B: ANIT model group; C: extract A drug protection group;
D: extract B drug protection group; E: extract C drug protection group; F: positive drug UDCA
protection group.
Detailed Description of the Preferred Embodiments Before the specific embodiments of the present disclosure are further described, it is to be understood that the scope of protection of the present disclosure is not limited to the specific embodiments described below; it is also to be understood that the terms used in the embodiments of the present disclosure are intended to describe particular specific embodiments and are not intended to limit the scope of protection of the present disclosure.
The test methods in which the specific conditions are not specified in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by each manufacturer.
When a numerical range is given by the embodiments, it is to be understood that unless otherwise stated, two endpoints of each numerical range and any one numerical value between the two endpoints can be selectively used. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the embodiments, any method, device, and material in the prior art similar or equivalent to the methods, devices, and materials described in the embodiments of the present disclosure may also be used according to the mastery of the prior art and the description of the present disclosure by those skilled in the art to accomplish the present disclosure.
Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present disclosure are all conventional techniques in the art.
Embodiment 1 I. Experimental materials 1.1 Drugs a-naphthyl isothiocyanate (ANIT; Sigma, MO, USA); ursodeoxycholic acid (UDCA; TCI Japan, Japan); radix et rhizoma rhei powder (obtained by pulverizing radix et rhizoma rhei, 95% of medicinal powder passes through a 100-mesh sieve, the remaining powder needs to pass through a 80-mesh sieve), radix et rhizoma rhei, rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, and fructus crataegi are provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.
1.2 Reagents ALT kit, AST kit, ALP kit, T-Bil kit, D-Bil kit, TBA kit (Nanjing Jiancheng, Shanghai, China); olive oil (Sinopharm Group, Shanghai, China); sodium carboxymethyl cellulose (CMC-Na; Sinopharm Group, Shanghai, China); normal saline, and neutral tissue fixative.
1.3 Main instruments Automatic biochemical analyzer; optical microscope; Thermo Scientific Varioskan Flash full-wavelength scanning multi-functional reader (Thermo Fisher Scientific, Germany); Eppendorf 5424R cryogenic high-speed centrifuge (Eppendorf, Germany);
SANYO ice machine (SANYO Electric Co., Ltd., Japan); Millipore water purifier (Millipore, Bedford, MA, USA).
1.4. Experimental animals C57BL/6 mice, weighing (20 2) g, male, clean grade, a number of 60, purchased from Shanghai Slac Laboratory Animal Co., Ltd. The animals were raised in an animal room shifting between 12 hours day and night at a temperature of (22 1) C, and a humidity of (65 10)%, and were fed adaptively for 3 days before the experiment.
II. Experimental methods 2.1 Drug preparation ANIT: an appropriate amount of powder was taken, and ultrasonically dissolved in olive oil to prepare 10 mg/mL of a product until the powder was completely dissolved.
UDCA: dissolved to 9 mg/mL with 0.5% sodium carboxymethyl cellulose (CMC-Na) and evenly mixed well before gavage.
Extract A: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 70% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract A.
Extract B: 24 g of radix et rhizoma rhei, 360 g of rhizoma polygoni cuspidati, g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 70% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract as an extract B.
Extract C: 24 g of radix et rhizoma rhei, 360 g of rhizoma polygoni cuspidati, g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and decocted and extracted with water, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract as an extract C.
2.2 Animal experiment method Sixty C57BL/6 mice were randomly divided into a normal control group, an ANIT
model group, three extract drug protection groups and a positive drug UDCA
control group, with 10 mice in each group. All drugs were dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) to the desired concentration. The normal control group and the model group were given the corresponding CMC-Na solution. The mice in the other groups were intragastrically administered with the same reagent at a dose of 10 mL/kg per day (i.g.), qd, for 5 consecutive days. Specifically, the administration dose of the extract A
drug protection group was 2.69 g/kg each time, the administration dose of the extract B drug protection group was 2.34 g/kg, the administration dose of the extract C drug protection group was 2.13 g/kg, and the administration dose of the positive drug UDCA control group was 0.09 g/kg. After the fifth administration for 12 h, for the normal control group, 10 mL/kg of olive oil was intragastrically administered, for the other groups, the mice were intragastrically administered with an ANIT olive oil solution at a dose of 10 mL/kg for molding. After molding for 12 h and 36 h, the administration was continued twice. 12 h before the end of the experiment, the animals were fasted and not deprived of water.
All animals were anesthetized with ether 48 h after the administration of ANIT
(i.e.
12 h after the last administration), and enucleation of eyeballs were carried out and blood was drawn for determination of serum biochemical indicators. The liver tissue (left lobe of liver) of the same part was excised for pathological examination and fixed with 10% neutral formalin for later use.
2.3 Determination of serum biochemical indicators The mice were anesthetized with ether and then the enucleation of eyeballs were carried out and blood was drawn, the whole blood was allowed to stand at room temperature for 3 h, and centrifuged for 15 min at 3500 rpm/min, and the upper serum was taken. The serum alanine aminotransferase (ALT), aspartic acid aminotransferase (AST), serum alkaline phosphatase (ALP) activity, serum total bilirubin (T-Bil), and direct bilirubin (D-Bil) contents were determined by the fully automatic biochemical analyzer.
The content of total bile acid (TBA) was determined and calculated by the method as described in the kit.
2.4. Liver tissue pathology observation After tissue fixation, the tissue was pruned, paraffin-embedded, cut into 4 pm slices, subjected to xylene dewaxing, gradient ethanol dehydration, hematoxylin-eosin staining (H&E staining), ethanol dehydration, xylene permeabilization and resin sealing, and observed under an optical microscope.
2.5. Statistical analysis The experimental data measurement data were expressed as mean standard deviation (x SD). The comparison of the two means was analyzed by one-way ANOVA in statistical software. P<0.05 indicated that the difference was statistically significant.
III. Experimental results 3.1 Evaluation of serum biochemical indicators having protective effects on ANIT-induced cholestasis in mice As shown in Table 1, Table 2 and Fig. 1, the six serological indicators of mice in the ANIT model group were significantly higher than the six serological indicators in the blank group (P< 0.05), indicating successful modeling. Compared with the ANIT
model group, after the continuous administration of the extract A, the increase in serum indicators caused by ANIT could be significantly reduced, and the difference was statistically significant (P<0.05), wherein the serum AST and ALP in mice have no significant difference compared with the normal control group (P>0.05). The extract B
could significantly reduce the levels of ALT, AST, T-Bil and D-Bil in mice serum (P<0.05), and the extract C only could significantly reduce the levels of ALT and AST
(P<0.05). The positive control drug UDCA could significantly reduce the other five serological indicators except for the AST (P<0.05). The above results indicate that extract A has protective effects on the liver damage in ANIT-induced cholestasis mice, and is superior to UDCA
in reducing ALT and AST indicators.
Table 1 Effects of extract A, extract B, extract C and UDCA on serum ALT, AST
and ALP in ANIT-induced cholestasis mice Group ALT AST ALP
Vehicle 34.1 3.635 155.1 20.78 540 149.2 ANIT 2831 848.3* 6459 1738* 1665 518.1*
ANIT+A 782.3 481.9*# 1800 1383# 904.8 456.2#
ANIT+B 1638 547.9*# 3890 1363*# 1482 547.8*
ANIT+C 1453 627.6*# 3972 1851*# 1448 182.5*
ANIT+UDCA 2831 200.9*# 4828 683.7* 1064 154.9*#
Note: Compared with the Vehicle group, *P<0.05; compared with the model group, #P<0.05 Table 2 Effects of extract A, extract B, extract C and UDCA on serum T-Bil, D-Bil and TBA in ANIT-induced cholestasis mice Group T-Bil D-Bil TBA
Vehicle 1.219 0.6141 0.3 0.3197 18.34 3.745 ANIT 269.8 72.74* 216.7 59.96* 1109 256.4*
ANIT+A 123.2 78.97" 104.2 60.7" 788.0 337.0"
ANIT+B 194.9 44.84*# 156.2 35.61*# 992.6 144.7*
ANIT+C 220.3 29.21* 180.1 22.44* 952.1 129.8*
ANIT+UDCA 130.1 39.54*# 114.1 35.74*# 845.2 78.62*#
Note: Compared with the Vehicle group, *P<0.05; compared with the model group, #P<0.05 3.2. Pathological analysis of liver having protective effects on ANIT-induced cholestasis in mice As shown in Fig. 2, it can be seen under the optical microscopy that the liver cells Date Recue/Date Received 2020-08-05 in the normal control group have normal morphology and are tightly arranged, no hepatic parenchymal cell lesions are seen, the hepatic lobule structure is complete, hepatic cell cords are radially arranged from the terminal venules, there is no obvious change in inflammatory cell infiltration and fibrous tissue hyperplasia in a portal area, and the bile capillaries have no cholestasis. In the ANIT model group, hepatocytes are enlarged, the cytoplasm is loosened, and some of the cells are degenerated in a ballooning or feathery shape, the focal necrosis can be seen, the outline of hepatic lobules is unclear, there is massive inflammatory cell infiltration in the portal area, and the capillary bile duct structure is destructed or lost. In the extract A protection group, compared with the model group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, the inflammatory cell infiltration is alleviated, and bile capillaries with normal structure are occasionally seen. In the extract B protection group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, and the inflammatory cell infiltration is alleviated. In the extract C protection group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are not significantly improved, the area of focal necrosis is reduced, and the inflammatory cell infiltration is alleviated. After UDCA
administration protection, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, and the inflammatory cell infiltration in the portal area is alleviated.
IV. Discussion ANIT-induced intrahepatic cholestasis in mice can also cause liver cell damage. Its therapeutic matrix for hepatotoxicity in animals is pathologically and physiologically very similar to human drug-induced hepatitis and is widely used to replicate intrahepatic cholestasis models. The toxication of ANIT is related to its metabolites.
Under the action of liver P450 enzyme, the sulfur oxide as a toxic product in the ANIT structure attacks the intrahepatic bile duct endothelial cells, resulting in the occurrence of hyperbilirubinemia and decreased bile secretion. At the same time, the sulfur oxide also causes a membrane lipid peroxidation, resulting in degeneration and necrosis of hepatocytes, and causing acute intrahepatic cholestasis.
In the research of the present disclosure, the ANIT-induced intrahepatic cholestasis model of mice is used to evaluate the efficacies of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei compound liver protecting and gallbladder benefiting drugs obtained by different extraction methods. The experimental results show that the extract A can significantly reduce an increase in serum ALT, AST, ALP, T-Bil, D-Bil and TBA in ANIT-induced mice. Compared with the positive drug UDCA, the extract A can effectively reduce the two serological indicators of ALT and AST. The pathological sections of liver show that in the extract A protection group, compared with the model group, the hepatocyte necrosis is obviously reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, and the inflammatory cell infiltration is alleviated.
In summary, the extract A has good protective effects on the liver of ANIT-induced cholestasis model mice.
In addition, referring to the preparation method of the extract A, the followings are respectively prepared:
Extract M-1: 24 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-1.
Extract M-2: 720 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-2.
Extract M-3: 360 g of rhizoma polygoni cuspidati, 24 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-3.
Extract M-4: 360 g of rhizoma polygoni cuspidati, 288 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-4.
Extract M-5: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 24 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
In one embodiment, the extraction is performed using an ethanol extraction method. For example, the extraction can be performed using 60% to 80% ethanol (weight to volume ratio, i.e., g/100 mL).
In one embodiment, the extraction is performed using a water extraction method.
In a third aspect of the present disclosure, a method for preparing the above-mentioned traditional Chinese medicine herb extract is provided, comprising the following steps: extracting active components from the above-mentioned traditional Chinese medicine herb composition as a raw material.
In one embodiment, the preparation method comprises the steps: taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, and fructus crataegi according to a ratio, extracting with ethanol, concentrating and drying an extracting solution to obtain a dry extract, then adding radix et rhizoma rhei powder, and evenly mixing.
In one embodiment, the ethanol is selected from 60% to 80% ethanol, preferably, 70% ethanol.
The radix et rhizoma rhei powder is obtained by pulverizing the radix et rhizoma rhei.
In one embodiment, the preparation method comprises the steps: taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei according to the ratio, and concentrating and drying an extracting solution to obtain a dry extract.
In one embodiment, the ethanol is selected from 60% to 80% ethanol, preferably, 70% ethanol.
In one embodiment, the preparation method comprises the steps: taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei according to the ratio, extracting with water, filtering, concentrating and drying.
In a fourth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of drugs for preventing and/or treating liver damage are provided.
In a fifth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of drugs for preventing and/or treating the cholestasis.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can prevent and/or treat intrahepatic cholestasis.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can prevent and/or treat acute intrahepatic cholestasis.
In a sixth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of an ALT reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of ALT in serum.
In a seventh aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of an AST reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of AST in serum.
In an eighth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of an ALP reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of ALP in serum.
In a ninth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a T-Bil reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of T-Bil.
In a tenth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a D-Bil reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract can be used to reduce the level of D-Bil.
In an eleventh aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a TBA reducing agent are provided.
In one embodiment, the above-mentioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce the level of TBA.
In a twelfth aspect of the present disclosure, uses of the above-mentioned traditional Chinese medicine herb composition or traditional Chinese medicine herb extract in preparation of a drug having any of the following efficacies: (1) reducing hepatocyte necrosis; (2) improving hepatocyte enlargement, cytoplasmic looseness ballooning degeneration and feathery degeneration conditions; and (3) alleviating inflammatory cell infiltration are provided.
In a thirteenth aspect of the present disclosure, a traditional Chinese medicine herb preparation is provided, comprising a therapeutically effective amount of the above-mentioned traditional Chinese medicine extract.
The traditional Chinese medicine herb preparation preferably comprises one or more conventional pharmaceutically acceptable excipients.
The pharmaceutically acceptable excipients comprise (but are not limited to):
pharmaceutically acceptable carriers, diluents, fillers, binders, and other excipients.
Therapeutically inert inorganic or organic carriers known to those skilled in the art comprise (but are not limited to) lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyhydroxy compounds such as polyethylene glycol, water, sucrose, ethanol, glycerin, and the like, various preservatives, lubricants, dispersants and flavoring agents. Humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added therein, which are used as needed to aid in the stability of the formula or help to enhance activity or bioavailability of the formula or produce an acceptable mouthfeel or odor in the case of oral administration. The traditional Chinese medicine herb preparation such as granules and ointments can be prepared by a conventional method. The pharmaceutical composition provided in the present disclosure may also be used with other therapeutic agents.
Preferably, the traditional Chinese medicine herb preparation is an oral preparation, comprising, but not limited to, granules, pills, tablets, capsules, syrups, sprays and the like.
After extracting the active components in the traditional Chinese medicine composition, the above-mentioned pharmaceutical composition can be prepared by using conventional pharmaceutical excipients and additives.
The therapeutically effective dose of the pharmaceutical composition provided in the present disclosure is as follows: the orally safe and effective amount is usually 1.00 to 2.65 g/kg by body weight based on the total weight of raw medicinal materials.
Certainly, the specific dose should also consider factors such as a route of administration, a state of health of the patient, etc., which is within the skill of a skilled physician.
Compared with the prior art, the present disclosure has the following beneficial effects:
The traditional Chinese medicine composition and the traditional Chinese medicine extract provided in the present disclosure can significantly reduce serum ALT, AST, ALP, T-Bil, D-Bil and TBA, significantly reduce hepatocyte necrosis, improve hepatocyte enlargement, cytoplasmic looseness, and ballooning degeneration and feathery degeneration conditions, and alleviate inflammatory cell infiltration, thus preventing or treating cholestasis.
Brief Description of the Drawings Fig. 1: Effects of extract A, extract B, extract C and UDCA on serum ALT, AST, ALP, T-Bil, D-Bil and TBA in ANIT-induced cholestasis mice; compared with a Vehicle group, *P <0.05; compared with an ANIT model group, #P<0.05.
Fig. 2: Analysis of pathological sections of liver in each group (H&E, x400), A:
normal control group; B: ANIT model group; C: extract A drug protection group;
D: extract B drug protection group; E: extract C drug protection group; F: positive drug UDCA
protection group.
Detailed Description of the Preferred Embodiments Before the specific embodiments of the present disclosure are further described, it is to be understood that the scope of protection of the present disclosure is not limited to the specific embodiments described below; it is also to be understood that the terms used in the embodiments of the present disclosure are intended to describe particular specific embodiments and are not intended to limit the scope of protection of the present disclosure.
The test methods in which the specific conditions are not specified in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by each manufacturer.
When a numerical range is given by the embodiments, it is to be understood that unless otherwise stated, two endpoints of each numerical range and any one numerical value between the two endpoints can be selectively used. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the embodiments, any method, device, and material in the prior art similar or equivalent to the methods, devices, and materials described in the embodiments of the present disclosure may also be used according to the mastery of the prior art and the description of the present disclosure by those skilled in the art to accomplish the present disclosure.
Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present disclosure are all conventional techniques in the art.
Embodiment 1 I. Experimental materials 1.1 Drugs a-naphthyl isothiocyanate (ANIT; Sigma, MO, USA); ursodeoxycholic acid (UDCA; TCI Japan, Japan); radix et rhizoma rhei powder (obtained by pulverizing radix et rhizoma rhei, 95% of medicinal powder passes through a 100-mesh sieve, the remaining powder needs to pass through a 80-mesh sieve), radix et rhizoma rhei, rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, and fructus crataegi are provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.
1.2 Reagents ALT kit, AST kit, ALP kit, T-Bil kit, D-Bil kit, TBA kit (Nanjing Jiancheng, Shanghai, China); olive oil (Sinopharm Group, Shanghai, China); sodium carboxymethyl cellulose (CMC-Na; Sinopharm Group, Shanghai, China); normal saline, and neutral tissue fixative.
1.3 Main instruments Automatic biochemical analyzer; optical microscope; Thermo Scientific Varioskan Flash full-wavelength scanning multi-functional reader (Thermo Fisher Scientific, Germany); Eppendorf 5424R cryogenic high-speed centrifuge (Eppendorf, Germany);
SANYO ice machine (SANYO Electric Co., Ltd., Japan); Millipore water purifier (Millipore, Bedford, MA, USA).
1.4. Experimental animals C57BL/6 mice, weighing (20 2) g, male, clean grade, a number of 60, purchased from Shanghai Slac Laboratory Animal Co., Ltd. The animals were raised in an animal room shifting between 12 hours day and night at a temperature of (22 1) C, and a humidity of (65 10)%, and were fed adaptively for 3 days before the experiment.
II. Experimental methods 2.1 Drug preparation ANIT: an appropriate amount of powder was taken, and ultrasonically dissolved in olive oil to prepare 10 mg/mL of a product until the powder was completely dissolved.
UDCA: dissolved to 9 mg/mL with 0.5% sodium carboxymethyl cellulose (CMC-Na) and evenly mixed well before gavage.
Extract A: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 70% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract A.
Extract B: 24 g of radix et rhizoma rhei, 360 g of rhizoma polygoni cuspidati, g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 70% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract as an extract B.
Extract C: 24 g of radix et rhizoma rhei, 360 g of rhizoma polygoni cuspidati, g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and decocted and extracted with water, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract as an extract C.
2.2 Animal experiment method Sixty C57BL/6 mice were randomly divided into a normal control group, an ANIT
model group, three extract drug protection groups and a positive drug UDCA
control group, with 10 mice in each group. All drugs were dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) to the desired concentration. The normal control group and the model group were given the corresponding CMC-Na solution. The mice in the other groups were intragastrically administered with the same reagent at a dose of 10 mL/kg per day (i.g.), qd, for 5 consecutive days. Specifically, the administration dose of the extract A
drug protection group was 2.69 g/kg each time, the administration dose of the extract B drug protection group was 2.34 g/kg, the administration dose of the extract C drug protection group was 2.13 g/kg, and the administration dose of the positive drug UDCA control group was 0.09 g/kg. After the fifth administration for 12 h, for the normal control group, 10 mL/kg of olive oil was intragastrically administered, for the other groups, the mice were intragastrically administered with an ANIT olive oil solution at a dose of 10 mL/kg for molding. After molding for 12 h and 36 h, the administration was continued twice. 12 h before the end of the experiment, the animals were fasted and not deprived of water.
All animals were anesthetized with ether 48 h after the administration of ANIT
(i.e.
12 h after the last administration), and enucleation of eyeballs were carried out and blood was drawn for determination of serum biochemical indicators. The liver tissue (left lobe of liver) of the same part was excised for pathological examination and fixed with 10% neutral formalin for later use.
2.3 Determination of serum biochemical indicators The mice were anesthetized with ether and then the enucleation of eyeballs were carried out and blood was drawn, the whole blood was allowed to stand at room temperature for 3 h, and centrifuged for 15 min at 3500 rpm/min, and the upper serum was taken. The serum alanine aminotransferase (ALT), aspartic acid aminotransferase (AST), serum alkaline phosphatase (ALP) activity, serum total bilirubin (T-Bil), and direct bilirubin (D-Bil) contents were determined by the fully automatic biochemical analyzer.
The content of total bile acid (TBA) was determined and calculated by the method as described in the kit.
2.4. Liver tissue pathology observation After tissue fixation, the tissue was pruned, paraffin-embedded, cut into 4 pm slices, subjected to xylene dewaxing, gradient ethanol dehydration, hematoxylin-eosin staining (H&E staining), ethanol dehydration, xylene permeabilization and resin sealing, and observed under an optical microscope.
2.5. Statistical analysis The experimental data measurement data were expressed as mean standard deviation (x SD). The comparison of the two means was analyzed by one-way ANOVA in statistical software. P<0.05 indicated that the difference was statistically significant.
III. Experimental results 3.1 Evaluation of serum biochemical indicators having protective effects on ANIT-induced cholestasis in mice As shown in Table 1, Table 2 and Fig. 1, the six serological indicators of mice in the ANIT model group were significantly higher than the six serological indicators in the blank group (P< 0.05), indicating successful modeling. Compared with the ANIT
model group, after the continuous administration of the extract A, the increase in serum indicators caused by ANIT could be significantly reduced, and the difference was statistically significant (P<0.05), wherein the serum AST and ALP in mice have no significant difference compared with the normal control group (P>0.05). The extract B
could significantly reduce the levels of ALT, AST, T-Bil and D-Bil in mice serum (P<0.05), and the extract C only could significantly reduce the levels of ALT and AST
(P<0.05). The positive control drug UDCA could significantly reduce the other five serological indicators except for the AST (P<0.05). The above results indicate that extract A has protective effects on the liver damage in ANIT-induced cholestasis mice, and is superior to UDCA
in reducing ALT and AST indicators.
Table 1 Effects of extract A, extract B, extract C and UDCA on serum ALT, AST
and ALP in ANIT-induced cholestasis mice Group ALT AST ALP
Vehicle 34.1 3.635 155.1 20.78 540 149.2 ANIT 2831 848.3* 6459 1738* 1665 518.1*
ANIT+A 782.3 481.9*# 1800 1383# 904.8 456.2#
ANIT+B 1638 547.9*# 3890 1363*# 1482 547.8*
ANIT+C 1453 627.6*# 3972 1851*# 1448 182.5*
ANIT+UDCA 2831 200.9*# 4828 683.7* 1064 154.9*#
Note: Compared with the Vehicle group, *P<0.05; compared with the model group, #P<0.05 Table 2 Effects of extract A, extract B, extract C and UDCA on serum T-Bil, D-Bil and TBA in ANIT-induced cholestasis mice Group T-Bil D-Bil TBA
Vehicle 1.219 0.6141 0.3 0.3197 18.34 3.745 ANIT 269.8 72.74* 216.7 59.96* 1109 256.4*
ANIT+A 123.2 78.97" 104.2 60.7" 788.0 337.0"
ANIT+B 194.9 44.84*# 156.2 35.61*# 992.6 144.7*
ANIT+C 220.3 29.21* 180.1 22.44* 952.1 129.8*
ANIT+UDCA 130.1 39.54*# 114.1 35.74*# 845.2 78.62*#
Note: Compared with the Vehicle group, *P<0.05; compared with the model group, #P<0.05 3.2. Pathological analysis of liver having protective effects on ANIT-induced cholestasis in mice As shown in Fig. 2, it can be seen under the optical microscopy that the liver cells Date Recue/Date Received 2020-08-05 in the normal control group have normal morphology and are tightly arranged, no hepatic parenchymal cell lesions are seen, the hepatic lobule structure is complete, hepatic cell cords are radially arranged from the terminal venules, there is no obvious change in inflammatory cell infiltration and fibrous tissue hyperplasia in a portal area, and the bile capillaries have no cholestasis. In the ANIT model group, hepatocytes are enlarged, the cytoplasm is loosened, and some of the cells are degenerated in a ballooning or feathery shape, the focal necrosis can be seen, the outline of hepatic lobules is unclear, there is massive inflammatory cell infiltration in the portal area, and the capillary bile duct structure is destructed or lost. In the extract A protection group, compared with the model group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, the inflammatory cell infiltration is alleviated, and bile capillaries with normal structure are occasionally seen. In the extract B protection group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, and the inflammatory cell infiltration is alleviated. In the extract C protection group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are not significantly improved, the area of focal necrosis is reduced, and the inflammatory cell infiltration is alleviated. After UDCA
administration protection, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, and the inflammatory cell infiltration in the portal area is alleviated.
IV. Discussion ANIT-induced intrahepatic cholestasis in mice can also cause liver cell damage. Its therapeutic matrix for hepatotoxicity in animals is pathologically and physiologically very similar to human drug-induced hepatitis and is widely used to replicate intrahepatic cholestasis models. The toxication of ANIT is related to its metabolites.
Under the action of liver P450 enzyme, the sulfur oxide as a toxic product in the ANIT structure attacks the intrahepatic bile duct endothelial cells, resulting in the occurrence of hyperbilirubinemia and decreased bile secretion. At the same time, the sulfur oxide also causes a membrane lipid peroxidation, resulting in degeneration and necrosis of hepatocytes, and causing acute intrahepatic cholestasis.
In the research of the present disclosure, the ANIT-induced intrahepatic cholestasis model of mice is used to evaluate the efficacies of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei compound liver protecting and gallbladder benefiting drugs obtained by different extraction methods. The experimental results show that the extract A can significantly reduce an increase in serum ALT, AST, ALP, T-Bil, D-Bil and TBA in ANIT-induced mice. Compared with the positive drug UDCA, the extract A can effectively reduce the two serological indicators of ALT and AST. The pathological sections of liver show that in the extract A protection group, compared with the model group, the hepatocyte necrosis is obviously reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, and the inflammatory cell infiltration is alleviated.
In summary, the extract A has good protective effects on the liver of ANIT-induced cholestasis model mice.
In addition, referring to the preparation method of the extract A, the followings are respectively prepared:
Extract M-1: 24 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-1.
Extract M-2: 720 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-2.
Extract M-3: 360 g of rhizoma polygoni cuspidati, 24 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-3.
Extract M-4: 360 g of rhizoma polygoni cuspidati, 288 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-4.
Extract M-5: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 24 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
5.
Extract M-6: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 720 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
Extract M-6: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 720 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 24 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
6.
Extract M-7: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 2.4 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
Extract M-7: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 60% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 2.4 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
7.
Extract M-8: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 240 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
Extract M-8: 360 g of rhizoma polygoni cuspidati, 144 g of pericarpium citri reticulatae viride, and 360 g of fructus crataegi were taken, and extracted with 80% ethanol, a solvent was recovered from an extracting solution under reduced pressure, thick paste was obtained, the thick paste was placed at a temperature of 100 C, and a pressure of -0.1 MPa and dried for 5 h, and cooled to room temperature to obtain a dry extract, and then 240 g of radix et rhizoma rhei powder was added and evenly mixed to obtain an extract M-
8.
Referring to the animal experiment method of the second part 2.2, an extract M-protection group with an administration dose of 2.69 g/kg, an extract M-2 protection group with an administration dose of 2.69 g/kg, an extract M-3 protection group with an administration dose of 2.69 g/kg, an extract M-4 protection group with an administration dose of 2.69 g/kg, an extract M-5 protection group with an administration dose of 2.69 g/kg, an extract M-6 protection group with an administration dose of 2.69 g/kg, an extract M-7 protection group with an administration dose of 2.69 g/kg and an extract M-8 protection group with an administration dose of 2.69 g/kg were set. Other operations were the same as the animal experiment method of the second part 2.2.
The results show that: the extract M-1 protection group, the extract M-2 protection group, the extract M-3 protection group, the extract M-4 protection group, the extract M-5 protection group, the extract M-6 protection group, the extract M-7 protection group and the extract M-8 protection group can significantly reduce the increase in serum indicators caused by ANIT, and the difference is statistically significant (P<0.05).
Compared with the normal control group, the serum AST and ALP in mice have no significant difference (P>0.05), but the effects of the extract A protection group are the best. The pathological sections of liver show that in the extract M-1 protection group, the extract M-2 protection group, the extract M-3 protection group, the extract M-4 protection group, the extract M-5 protection group, the extract M-6 protection group, the extract M-7 protection group, and the extract M-8 protection group, compared with the model group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, the inflammatory cell infiltration is alleviated, and bile capillaries with normal structure are occasionally seen, but the effects of the extract A protection group are the best.
The above description of the detailed embodiments is only the preferable embodiments provided in the present disclosure, and it is not any formal and substantive limitation of the present disclosure. It should be noted that for those with ordinary skill in the art, without departing from the method of the present disclosure, a plurality of improvements and supplements can be made, which should be viewed as the protection range of the present disclosure. Those skilled in the art can make some changes, modifications, variations and other equivalent changes by using the technological content disclosed above without departing from the spirit and scope of the present disclosure, which are the equivalent embodiments of the present disclosure; meanwhile, all changes, modifications and variations completed by those with ordinary skill in the art according to the substantive technology should fall within the scope of the technical scheme provided in present disclosure.
Referring to the animal experiment method of the second part 2.2, an extract M-protection group with an administration dose of 2.69 g/kg, an extract M-2 protection group with an administration dose of 2.69 g/kg, an extract M-3 protection group with an administration dose of 2.69 g/kg, an extract M-4 protection group with an administration dose of 2.69 g/kg, an extract M-5 protection group with an administration dose of 2.69 g/kg, an extract M-6 protection group with an administration dose of 2.69 g/kg, an extract M-7 protection group with an administration dose of 2.69 g/kg and an extract M-8 protection group with an administration dose of 2.69 g/kg were set. Other operations were the same as the animal experiment method of the second part 2.2.
The results show that: the extract M-1 protection group, the extract M-2 protection group, the extract M-3 protection group, the extract M-4 protection group, the extract M-5 protection group, the extract M-6 protection group, the extract M-7 protection group and the extract M-8 protection group can significantly reduce the increase in serum indicators caused by ANIT, and the difference is statistically significant (P<0.05).
Compared with the normal control group, the serum AST and ALP in mice have no significant difference (P>0.05), but the effects of the extract A protection group are the best. The pathological sections of liver show that in the extract M-1 protection group, the extract M-2 protection group, the extract M-3 protection group, the extract M-4 protection group, the extract M-5 protection group, the extract M-6 protection group, the extract M-7 protection group, and the extract M-8 protection group, compared with the model group, the hepatocyte necrosis is reduced, the hepatocytes are enlarged, the cytoplasm is loosened, the ballooning degeneration and feathery degeneration conditions are improved, the inflammatory cell infiltration is alleviated, and bile capillaries with normal structure are occasionally seen, but the effects of the extract A protection group are the best.
The above description of the detailed embodiments is only the preferable embodiments provided in the present disclosure, and it is not any formal and substantive limitation of the present disclosure. It should be noted that for those with ordinary skill in the art, without departing from the method of the present disclosure, a plurality of improvements and supplements can be made, which should be viewed as the protection range of the present disclosure. Those skilled in the art can make some changes, modifications, variations and other equivalent changes by using the technological content disclosed above without departing from the spirit and scope of the present disclosure, which are the equivalent embodiments of the present disclosure; meanwhile, all changes, modifications and variations completed by those with ordinary skill in the art according to the substantive technology should fall within the scope of the technical scheme provided in present disclosure.
Claims (10)
1. A traditional Chinese medicine herb composition for preventing and/or treating liver damage and/or cholestasis, wherein medicinal component raw materials of the traditional Chinese medicine herb composition consist of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei.
2. The traditional Chinese medicine herb composition as defined in claim 1, wherein a range of a ratio of the parts by weight of rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei is as follows:
1-30 parts of rhizoma polygoni cuspidati, 1-12 parts of pericarpium citri reticulatae viride, 1-30 parts of fructus crataegi and 0.1-10 parts of radix et rhizoma rhei.
1-30 parts of rhizoma polygoni cuspidati, 1-12 parts of pericarpium citri reticulatae viride, 1-30 parts of fructus crataegi and 0.1-10 parts of radix et rhizoma rhei.
3. A traditional Chinese medicine herb extract, obtained by extracting the traditional Chinese medicine herb composition according to claim 1 or 2 as a raw material by an extraction method, wherein the extraction method is selected from boiling, alcohol extraction, water extraction and alcohol precipitation, alcohol extraction and water precipitation, and a salting out method.
4. A method for preparing the traditional Chinese medicine herb extract as defined in claim 3, comprising the following steps: extracting the traditional Chinese medicine herb composition according to claim 1 or 2 as the raw material by the extraction method defined in claim 3.
5. The preparation method as defined in claim 4, wherein the preparation method is selected from any one of the following: (1) taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, and fructus crataegi according to a ratio of the parts by weight, Date Recue/Date Received 2021-06-11 extracting with ethanol, concentrating and drying an extracting solution to obtain a dry extract, then adding radix et rhizoma rhei powder, and evenly mixing; (2) taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei according to a ratio of the parts by weight, extracting with ethanol, and concentrating and drying an extracting solution to obtain a dry extract; and (3) taking rhizoma polygoni cuspidati, pericarpium citri reticulatae viride, fructus crataegi and radix et rhizoma rhei according to a ratio of the parts by weight, extracting with water, filtering, concentrating and drying.
6. The preparation method as defined in claim 5, wherein the ethanol is selected from 60% to 80% ethanol.
7. A use of the traditional Chinese medicine herb composition according to claim 1 or 2, or a use of the traditional Chinese medicine herb extract as defined in claim 3, in preparation of a drug for preventing and/or treating liver damage.
8. A use of the traditional Chinese medicine herb composition according to claim 1 or 2, or a use of the traditional Chinese medicine herb extract as defined in claim 3, in preparation of a drug for preventing and/or treating cholestasis.
9. A use of the traditional Chinese medicine herb composition according to claim 1 or 2, or a use of the traditional Chinese medicine herb extract as defined in claim 3, in preparation of a drug having any one of the following efficacies: (1) reducing a serum alanine aminotransferase level; (2) reducing an aspartic acid aminotransferase level; (3) reducing a serum alkaline phosphatase level; (4) reducing a serum total bilirubin level; (5) reducing a direct bilirubin level; (6) reducing a total bile acid level; (7) reducing hepatocyte necrosis; (8) improving conditions of hepatocyte enlargement, hepatocyte cytoplasmic Date Recue/Date Received 2021-06-11 looseness, hepatocyte ballooning degeneration and hepatocyte feathery degeneration; and (9) alleviating inflammatory cell infiltration.
10. A traditional Chinese medicine herb preparation, comprising a therapeutically effective amount of the traditional Chinese medicine herb composition according to claim 1 or 2, or the traditional Chinese medicine herb extract as defined in claim 3, wherein the therapeutically effective amount is 1.00-2.65 g/kg.
Date Recue/Date Received 2021-06-11
Date Recue/Date Received 2021-06-11
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| CN201810748766.6A CN108567843B (en) | 2018-07-10 | 2018-07-10 | Traditional Chinese medicine composition and preparation and application thereof |
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| PCT/CN2018/100211 WO2020010664A1 (en) | 2018-07-10 | 2018-08-13 | Traditional chinese medicine composition, and preparation and use thereof |
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