Method for preparing consistent decoction and consistent decoction granules prepared by same

Abstract

The method comprises the following steps: (1) weighing a proper amount of radix glehniae, radix ophiopogonis, radix angelicae sinensis, radix rehmanniae, fructus lycii and szechwan chinaberry fruit, adding water for the first time for soaking, decocting with strong fire for a period of time, then decocting with slow fire for a period of time, and filtering to obtain filter residues and first filtrate; adding water for the second time into the filter residue, decocting with strong fire for a period of time, then decocting with slow fire for a period of time, and filtering to obtain a second filtrate; (2) combining the first filtrate and the second filtrate, and fixing the volume to obtain a consistent decoction water extract; and (3) adding beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin into the consistent decoction water extract, stirring, standing, concentrating to obtain a concentrated solution, and drying the concentrated solution to obtain the consistent decoction dry paste powder.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for preparing Yi Guan Jian and Yi Guan Jian granules prepared thereby. Background Art

[0002] Yi Guan Jian has good clinical efficacy, but the clinical dosage form is a decoction, which is not convenient for adjustment, storage and transportation, and the patient compliance is not high. The "Principles for the Verification of Key Information of Ancient Classic Prescriptions (Draft for Comments)" and "Requirements for the Application of Traditional Chinese Medicine Compound Preparations of Ancient Classic Prescriptions (Draft for Comments)" issued by the State Administration of Traditional Chinese Medicine propose that the process route, route of administration and dosage form of the classic prescriptions should be consistent with the key information of the ancient classic prescriptions released by the state and the records in ancient medical classics, and the decoctions can be made into granules.

[0003] The present invention takes the classic prescription Yi Guan Jian as the research object, refers to the requirements for the development and application of classic prescriptions, and on the basis of the research on the benchmark sample of Yi Guan Jian completed in the early stage of the present invention, carries out the preparation of Yi Guan Jian granules, including the research on intermediates and granules, to lay the foundation for its stable clinical efficacy.

[0004] The prior art does not disclose the method for preparing the Yi Guan Jian of the present invention and the Yi Guan Jian granules prepared thereby. Summary of the invention

[0005] Based on this, the present invention provides a method for preparing Yi Guan Jian Gan Gao Powder, the method comprising the following steps:

[0006] (1) Weighing appropriate amounts of Adenophora adenophora, Ophiopogon japonicus, Angelica sinensis, Rehmannia root, Lycium barbarum and Toosendan fruit, adding first water for soaking, first decocting over high heat for a period of time, then decocting over low heat for a period of time, filtering to obtain a filter residue and a first filtrate; adding second water to the filter residue, first decocting over high heat for a period of time, then decocting over low heat for a period of time, filtering to obtain a second filtrate;

[0007] (2) combining the first filtrate and the second filtrate, fixing the volume, and obtaining a Yi Guan Jian water extract; and

[0008] (3) adding β-cyclodextrin or hydroxypropyl-β-cyclodextrin to the Yiguanjian water extract, stirring, standing, and concentrating to obtain a concentrated solution, and drying the concentrated solution to obtain a Yiguanjian dried paste powder.

[0009] Further, in step (1), the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is (1-10):(1-10):(1-10):(5-15):(5-15):(1-10).

[0010] Furthermore, the mass ratios of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan are (4-7):(4-7):(4-7):(9-12):(9-12):(6-8).

[0011] Furthermore, the mass ratios of the Adenophora adenophora, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia root, the Lycium barbarum and the Toosendan fruit are (5.32-5.88):(5.32-5.88):(5.32-5.88):(10.6305-11.7495):(10.6305-11.7495):(7.087-7.833).

[0012] Further, the mass ratio of the Adenophora adenophora, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia glutinosa, the Lycium barbarum and the Toosendan fruit is about 5.6: about 5.6: about 5.6: about 11.19: about 11.19: about 7.46.

[0013] Furthermore, the Rehmannia root is raw Rehmannia root.

[0014] Further, in step (1), the mass / volume (mg / ml) ratio of the total mass of the Adenophora adenophora, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia root, the Lycium barbarum and the Toosendan fruit to the volume of the first water is 60-100, for example, about 83.3.

[0015] Furthermore, the soaking time is 60 to 120 minutes, for example, about 90 minutes.

[0016] Furthermore, the frying pot is a casserole or a stainless steel pot.

[0017] Furthermore, the decocting is performed with a cover.

[0018] Furthermore, the voltage of the high fire is 400-600W, for example, about 500W.

[0019] Furthermore, the high heat decocting time is high heat decocting until boiling.

[0020] Furthermore, the voltage of the slow fire is 150-350W, for example, about 250W.

[0021] Furthermore, the slow-cooking time is 10 to 60 minutes, for example, about 30 minutes.

[0022] Furthermore, the filtration is performed while hot.

[0023] Furthermore, the hot filtration is performed by filtering the mixture in a funnel using gauze while the mixture is hot.

[0024] Furthermore, the volume / volume (mL / mL or L / L) ratio between the volume of the first water and the volume of the second water is 1 to 2, such as about 1.2.

[0025] Furthermore, in step (3), the mass / mass (mg / mg or g / g) ratio of the mass of the β-cyclodextrin to the total mass of the Adenophora adenophora, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia root, the Fructus Lycii and the Fructus Toosendan is 15% to 25%, for example, about 10.5%.

[0026] Furthermore, the stirring is magnetic stirring.

[0027] Furthermore, the stirring temperature is 20-40°C, for example, about 30°C.

[0028] Furthermore, the stirring speed is 300-500 r / min, for example, about 400 r / min.

[0029] Furthermore, the stirring time is 20 to 40 minutes, for example, about 30 minutes.

[0030] Furthermore, the standing time is 1 h to 3 h, for example, about 2 h.

[0031] Furthermore, the concentration is concentration under reduced pressure.

[0032] Furthermore, the temperature of the reduced pressure concentration is 40-60°C, for example, about 50°C.

[0033] Furthermore, the pressure of the reduced pressure concentration is -0.1 MPa to -0.08 MPa, for example, about -0.09 MPa.

[0034] Furthermore, the density of the concentrate is 1.15±0.05 g / cm 3 .

[0035] Furthermore, the drying is spray drying.

[0036] Furthermore, the inlet air temperature of the spray drying is 160-200°C, for example, about 180°C.

[0037] Furthermore, the atomization pressure of the spray drying is 0.1-0.2 MPa, for example, about 0.13 MPa.

[0038] Furthermore, the needle pressure of the spray drying is 0.1-0.2 MPa, for example, about 0.15 MPa.

[0039] Furthermore, the rotation speed of the spray drying peristaltic pump is 10-20 r / min, for example, about 15 r / min.

[0040] According to another aspect of the present invention, provided is a Yiguanjian water extract prepared by the above method or a preparation containing the Yiguanjian water extract.

[0041] According to another aspect of the present invention, provided is a Yi Guan Jian dried paste powder prepared by the above method or a preparation comprising the Yi Guan Jian dried paste powder.

[0042] According to another aspect of the present invention, a method for preparing a Yi Guan Jian preparation is provided, the method comprising uniformly mixing the Yi Guan Jian dried paste powder with auxiliary materials to obtain the Yi Guan Jian preparation.

[0043] Furthermore, the method further comprises a step of dry granulation.

[0044] Furthermore, the feed rate of the dry granulation is 5 to 20 r / min, for example, about 10 r / min.

[0045] Furthermore, the hydraulic station pressure of the dry granulation is 50-90 bar, for example, about 70 bar.

[0046] Furthermore, the speed of the roller of the dry granulation is 1 to 15 r / min, for example, about 5 r / min.

[0047] Furthermore, the feeding speed of the dry granulation is 1-10 r / min, for example, about 5 r / min.

[0048] Furthermore, the granulation speed of the dry granulation is 60-100 r / min, for example, about 80 r / min.

[0049] According to another aspect of the present invention, a Yi Guan Jian preparation prepared by the above method is provided.

[0050] Furthermore, the preparation is an oral preparation such as granules.

[0051] Furthermore, the auxiliary material is maltodextrin and / or lactose.

[0052] Furthermore, the ratio of the maltodextrin to the lactose is (0.1-3):(0.1-3), for example, about 1:about 1.

[0053] Furthermore, the mass / mass (mg / mg or g / g) ratio of the Yi Guan Jian dried paste powder to the auxiliary material is (0.1-3):(0.1-3), for example, about 1:about 1.

[0054] Furthermore, the mass of the granules is 8-16 g / bag, for example, about 12 g / bag.

[0055] According to another aspect of the present invention, there is provided a use of the above-mentioned preparation in the preparation of a medicament for preventing and / or treating liver fibrosis.

[0056] Furthermore, the preparation prevents and / or treats liver fibrosis in one or more of the following ways: reducing the level of serum markers, improving liver tissue pathological abnormalities, and reducing liver tissue hydroxyproline content.

[0057] Furthermore, the serum marker is selected from aspartate aminotransferase and / or alanine aminotransferase.

[0058] Furthermore, the liver tissue pathological abnormality is selected from one or more of the following: inflammatory cell infiltration, tissue necrosis, collagen deposition and increased Sirius red staining positive area.

[0059] Beneficial effects of the present invention:

[0060] The present invention adopts a method of adding an auxiliary material with inclusion function before concentration, and prepares a Yi Guan Jian intermediate through reduced pressure concentration and spray drying, which solves the technical bottleneck that the ligustilide component cannot be retained to a certain extent, and on this basis, selects suitable auxiliary materials, granulation methods and auxiliary material ratios, adopts orthogonal experiments to determine the preparation process of Yi Guan Jian granules, and evaluates the therapeutic effect. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0062] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0063] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0064] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0065] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0066] As described in the background technology section, the prior art does not have the Yi Guan Jian preparation method of the present invention and the Yi Guan Jian granules prepared therefrom. In order to solve the above problems, the present invention provides a method for preparing Yi Guan Jian dried paste powder, the method comprising the following steps:

[0067] (1) Weighing appropriate amounts of Adenophora adenophora, Ophiopogon japonicus, Angelica sinensis, Rehmannia root, Lycium barbarum and Toosendan fruit, adding first water for soaking, first decocting over high heat for a period of time, then decocting over low heat for a period of time, filtering to obtain a filter residue and a first filtrate; adding second water to the filter residue, first decocting over high heat for a period of time, then decocting over low heat for a period of time, filtering to obtain a second filtrate;

[0068] (2) combining the first filtrate and the second filtrate, fixing the volume, and obtaining a Yi Guan Jian water extract; and

[0069] (3) adding β-cyclodextrin or hydroxypropyl-β-cyclodextrin to the Yiguanjian water extract, stirring, standing, and concentrating to obtain a concentrated solution, and drying the concentrated solution to obtain a Yiguanjian dried paste powder.

[0070] In the present invention, when mass, ratio, time, power, temperature, rotation speed, pressure, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "1 to 10" is disclosed, the described range should be interpreted as including the range "1 to 10", "1 to 8", "1 to 6", "1 to 4", "1 to 2", "3 to 10", "3 to 8", "3 to 6", "3 to 4", "5 to 10", "5 to 8", "5 to 6", "7 to 10", "7 to 8", "9 to 10", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0071] In a preferred embodiment, in step (1), the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is (1-10):(1-10):(1-10):(5-15):(5-15):(1-10).

[0072] In a preferred embodiment, the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is (4-7):(4-7):(4-7):(9-12):(9-12):(6-8).

[0073] In a preferred embodiment, the mass ratios of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan are (5.32-5.88):(5.32-5.88):(5.32-5.88):(10.6305-11.7495):(10.6305-11.7495):(7.087-7.833).

[0074] In a preferred embodiment, the mass ratio of the Adenophora adenophora, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia glutinosa, the Fructus Lycii and the Fructus Toosendan is about 5.6: about 5.6: about 5.6: about 11.19: about 11.19: about 7.46.

[0075] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.6" includes ±5% of 5.6, or from 5.32 to 5.88; "about 11.19" includes ±5% of 11.19, or from 10.6305 to 11.7495; "about 7.46" includes ±5% of 7.46, or from 7.087 to 7.833.

[0076] In a preferred embodiment, the Rehmannia root is raw Rehmannia root.

[0077] In a preferred embodiment, in step (1), the mass / volume (mg / ml) ratio between the total mass of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan to the volume of the first water is 60-100, for example, about 83.3.

[0078] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 83.3" includes ±5% of 83.3, or from 79.135 to 87.465.

[0079] In a preferred embodiment, the soaking time is 60 to 120 minutes, such as about 90 minutes.

[0080] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 90" includes ±5% of 90, or from 85.5 to 94.5.

[0081] In a preferred embodiment, the frying pot is a casserole or a stainless steel pot.

[0082] In a preferred embodiment, the decocting is covered decocting.

[0083] In a preferred embodiment, the voltage of the high fire is 400-600W, for example, about 500W.

[0084] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0085] In a preferred embodiment, the high-fire decocting time is high-fire decocting until boiling.

[0086] In a preferred embodiment, the voltage of the slow fire is 150-350W, for example, about 250W.

[0087] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0088] In a preferred embodiment, the slow-cooking time is 10 to 60 minutes, such as about 30 minutes.

[0089] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0090] In a preferred embodiment, the filtration is hot filtration.

[0091] In a preferred embodiment, the hot filtration is hot filtration using gauze in a funnel.

[0092] In a preferred embodiment, the volume / volume (mL / mL or L / L) ratio between the volume of the first water and the volume of the second water is 1 to 2, such as about 1.2.

[0093] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26.

[0094] In a preferred embodiment, in step (3), the mass / mass (mg / mg or g / g) ratio of the mass of the β-cyclodextrin to the total mass of the Glehnia littoralis, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia root, the Fructus Lycii and the Fructus Toosendan is 15% to 25%, for example, about 10.5%.

[0095] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10.5%" includes ±5% of 10.5%, or from 9.975% to 11.025%.

[0096] In a preferred embodiment, the stirring is magnetic stirring.

[0097] In a preferred embodiment, the stirring temperature is 20-40°C, such as about 30°C.

[0098] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0099] In a preferred embodiment, the stirring speed is 300-500 r / min, for example, about 400 r / min.

[0100] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 400" includes ±5% of 400, or from 380 to 420.

[0101] In a preferred embodiment, the stirring time is 20 to 40 minutes, for example, about 30 minutes.

[0102] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0103] In a preferred embodiment, the standing time is 1 h to 3 h, such as about 2 h.

[0104] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0105] In a preferred embodiment, the concentration is concentration under reduced pressure.

[0106] In a preferred embodiment, the temperature of the reduced pressure concentration is 40-60°C, such as about 50°C.

[0107] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0108] In a preferred embodiment, the pressure of the reduced pressure concentration is -0.1 MPa to -0.08 MPa, for example, about -0.09 MPa.

[0109] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about -0.09" includes ±5% of -0.09, or from -0.0945 to -0.0855.

[0110] In a preferred embodiment, the density of the concentrate is 1.15±0.05 g / cm 3 .

[0111] In a preferred embodiment, the drying is spray drying.

[0112] In a preferred embodiment, the inlet air temperature of the spray drying is 160-200°C, such as about 180°C.

[0113] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 180" includes ±5% of 180, or from 171 to 189.

[0114] In a preferred embodiment, the atomization pressure of the spray drying is 0.1-0.2 MPa, for example, about 0.13 MPa.

[0115] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.13" includes ±5% of 0.13, or from 0.1235 to 0.1365.

[0116] In a preferred embodiment, the needle pressure of the spray drying is 0.1-0.2 MPa, such as about 0.15 MPa.

[0117] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.15" includes ±5% of 0.15, or from 0.1425 to 0.1575.

[0118] In a preferred embodiment, the rotation speed of the spray drying peristaltic pump is 10-20 r / min, such as about 15 r / min.

[0119] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0120] According to another aspect of the present invention, provided is a Yiguanjian water extract prepared by the above method or a preparation containing the Yiguanjian water extract.

[0121] According to another aspect of the present invention, provided is a Yi Guan Jian dried paste powder prepared by the above method or a preparation comprising the Yi Guan Jian dried paste powder.

[0122] According to another aspect of the present invention, a method for preparing a Yi Guan Jian preparation is provided, the method comprising uniformly mixing the Yi Guan Jian dried paste powder with auxiliary materials to obtain the Yi Guan Jian preparation.

[0123] In a preferred embodiment, the method further comprises a step of dry granulation.

[0124] In a preferred embodiment, the feed rate of the dry granulation is 5-20 r / min, such as about 10 r / min.

[0125] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0126] In a preferred embodiment, the hydraulic station pressure of the dry granulation is 50-90 bar, such as about 70 bar.

[0127] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 70" includes ±5% of 70, or from 66.5 to 73.5.

[0128] In a preferred embodiment, the roller speed of the dry granulation is 1-15 r / min, such as about 5 r / min.

[0129] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0130] In a preferred embodiment, the feeding speed of the dry granulation is 1-10 r / min, such as about 5 r / min.

[0131] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0132] In a preferred embodiment, the granulation speed of the dry granulation is 60-100 r / min, such as about 80 r / min.

[0133] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0134] According to another aspect of the present invention, a Yi Guan Jian preparation prepared by the above method is provided.

[0135] In a preferred embodiment, the preparation is an oral preparation such as granules.

[0136] In a preferred embodiment, the auxiliary material is maltodextrin and / or lactose.

[0137] In a preferred embodiment, the ratio of the maltodextrin to the lactose is (0.1-3):(0.1-3), for example, about 1:about 1.

[0138] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0139] In a preferred embodiment, the mass / mass (mg / mg or g / g) ratio of the Yi Guan Jian dried paste powder to the auxiliary material is (0.1-3):(0.1-3), for example, about 1:about 1.

[0140] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0141] In a preferred embodiment, the mass of the granules is 8-16 g / bag, for example, about 12 g / bag.

[0142] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0143] According to another aspect of the present invention, there is provided a use of the above-mentioned preparation in the preparation of a medicament for preventing and / or treating liver fibrosis.

[0144] In a preferred embodiment, the preparation prevents and / or treats liver fibrosis in one or more of the following ways: reducing the level of serum markers, improving liver tissue pathological abnormalities, and reducing liver tissue hydroxyproline content.

[0145] In a preferred embodiment, the serum marker is selected from aspartate aminotransferase and / or alanine aminotransferase.

[0146] In a preferred embodiment, the liver tissue pathological abnormality is selected from one or more of the following: inflammatory cell infiltration, tissue necrosis, collagen deposition and increased Sirius red staining positive area.

[0147] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0148] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0149] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0150] Example

[0151] 1. Experimental instruments, materials and reagents

[0152] 1.1 Experimental instruments

[0153] Table 1 Experimental instruments

[0154]

[0155]

[0156] 1.2 Experimental Materials and Reagents

[0157] Table 2 Experimental materials and reagents

[0158]

[0159]

[0160] 2. Experimental methods for intermediates

[0161] 2.1 Concentration process

[0162] Based on the experimental results of the previous stage of the present invention, the vacuum concentration was selected. In the process of research, it was found that the content of ligustilide was lost in large quantities during the concentration process and was greatly affected by temperature, so the method of retaining ligustilide and the temperature of vacuum concentration were studied.

[0163] 2.1.1. Preparation of reference solution

[0164] Accurately weigh appropriate amounts of p-coumaric acid, ferulic acid, rutin, verbascoside, ligusticolide I, ligusticolide H, ligustilide, rehmannia glutinosa D, toosendanin and betaine reference substances, dissolve them in methanol to prepare reference substance stock solutions with mass concentrations of 1.03 mg / mL, 0.97 mg / mL, 0.10 mg / mL, 0.97 mg / mL, 0.22 mg / mL, 0.27 mg / mL, 0.17 mg / mL, 0.47 mg / mL, 0.10 mg / mL and 0.10 mg / mL, respectively, and store them in a 4°C refrigerator for later use.

[0165] 2.1.2. Preparation of accompanying sample solution

[0166] Weigh appropriate amounts of Radix Rehmanniae (batch number: 20210310), Fructus Lycii (batch number: 20210304), Radix Angelicae Sinensis (batch number: 20210313), Radix Ophiopogonis (batch number: 20210306), Radix Adenophorae (batch number: 20210307) and Fructus Toosendan (batch number: 20210402), add 12 times the amount of water of the decoction pieces, soak for 90 minutes, decoct for 30 minutes, filter, add 10 times the amount of water to the residue, decoct for 30 minutes, filter, combine the two filtrates, shake well, and obtain the Yi Guan Jian accompanying sample solution. Subsequent experimental results are all based on the accompanying sample.

[0167] 2.1.3 Preparation of test solution

[0168] Prepare the accompanying sample solution according to item "2.1.2". Take 5 mL of the accompanying sample solution, dilute it to 10 mL with methanol, centrifuge it at 12000 r / min for 5 min, take the supernatant and filter it through a 0.45 μm microporous filter membrane. Inject the filtrate into the HPLC system to collect data.

[0169] 2.1.4 Detection Method

[0170] (1) Multi-index component detection method

[0171] The method established in the previous stage of the present invention was used, and the instrument was Agilent 1260Ⅱ high performance liquid chromatograph; the chromatographic column was Agilent ZORBAX Eclipse XDB-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase: 0.1% phosphoric acid water (A)-acetonitrile (B) gradient elution (0-5 min, 12% B; 5-38 min, 12-18% B; 38-53 min, 18%-28% B; 53-65 min, 28%-90% B; 65-72 min, 90% B) detection wavelength is 332 nm (0-22.01 min, p-coumaric acid) , 278nm (22.01~27.21min, ferulic acid, rutin), 332nm (27.21~37min, verbascoside), 278nm (37~52min, ligustilide I, ligustilide H), 332nm (52~72min, ligustilide); flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 20μL; data processing software: Chemstation.

[0172] (2) Detection method of rehmannia glutinosin D

[0173] Instrument: Thermo Ultimate 3000 ultra-high performance liquid chromatograph; Waters Atlantis TM T 3Chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol (A)-pure water (B) gradient elution (0-36 min, 7% A; 36-40 min, 7%-90% A; 40-46 min, 90% A), volume flow rate of 1.0 mL / min; detection wavelength: 210 nm; injection volume: 10 μL; data processing software: Chromeleon 7.

[0174] (3) Toosendanin detection method

[0175] Instrument: Agilent 1260Ⅱ High Performance Liquid Chromatograph; Using Shiseido CAPCELL PAK C 18 Chromatographic column (4.6mm×250mm, 5μm); mobile phase is acetonitrile (A)-0.1% formic acid aqueous solution (B) gradient elution (0-8min, 10%-30% A; 8-18min, 30%-50% A; 18-20min, 50%-90% A; 20-25min, 90% A), volume flow rate is 1.0mL / min; detection is by evaporative light scattering detector; nitrogen flow rate is 1.6L / min; evaporator temperature is 80℃; nebulizer temperature is 75℃; injection volume is 10μL; data processing software: Chemstation.

[0176] (4) Betaine detection method

[0177] The instrument was an Agilent 1260Ⅱ high performance liquid chromatograph; a Waters Xbridge Amide column (4.6mm×250mm, 3.5μm) was used; the mobile phase was acetonitrile (A)-0.1% triethylamine aqueous solution (B) gradient elution (0-20min, 89% A; 20-22min, 89%-65% A; 22-25min, 65% A), with a volume flow rate of 1.0mL / min; detection was by evaporative light scattering detector; the nitrogen flow rate was 1.6L / min; the evaporator temperature was 80℃; the nebulizer temperature was 75℃; the injection volume was 5μL; data processing software: Chemstation.

[0178] 2.1.5. Investigation of factors affecting the content of main ingredients by adding auxiliary materials before concentration

[0179] (1) Screening of auxiliary materials

[0180] Prepare the Yi Guan Jian sample solution according to item "2.1.2". Take 300 mL of the solution in a 500 mL conical flask, add 14% of the crude drug amount of β-cyclodextrin, HP-β-cyclodextrin and maltodextrin respectively, stir magnetically at 40°C and 600 r / min for 1 h, let it stand for 24 h, and then concentrate under reduced pressure at 60°C and -0.09 MPa to a relative density of 1.15±0.05 g / cm3 , dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-index component detection method under "2.1.4".

[0181] (2) Amount of auxiliary materials

[0182] Prepare Yi Guan Jian sample solution according to item "2.1.2". Take 300 mL of solution in a 500 mL conical flask, add 3.5%, 7.0%, 10.5% and 14.0% β-CD respectively, stir at 40℃ and 600r / min for 1h, let stand for 24h, and then concentrate under reduced pressure at 60℃ and -0.09MPa to a relative density of 1.15±0.05g / cm 3 , dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-index component detection method under "2.1.4".

[0183] (3) Stirring temperature

[0184] Prepare the Yi Guan Jian sample solution according to item "2.1.2". Take 300 mL of the solution in a 500 mL conical flask, add 10.5% of the crude drug β-CD, stir at 600 r / min for 1 h at 30°C, 40°C, 50°C, and 60°C, let stand for 24 h, and then concentrate under reduced pressure at 60°C and -0.09 MPa to a relative density of 1.15 ± 0.05 g / cm 3 , dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-index component detection method under "2.1.4".

[0185] (4) Mixing time

[0186] Prepare the Yi Guan Jian sample solution according to item "2.1.2". Take 300 mL of the solution in a 500 mL conical flask, add 10.5% of the crude drug β-CD, stir at 30°C with magnetic stirring at 600 r / min for 0.5 h, 1 h, 2 h, and 4 h, respectively. After standing for 24 h, concentrate under reduced pressure at 60°C and -0.09 MPa to a relative density of 1.15 ± 0.05 g / cm 3, dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-index component detection method under "2.1.4".

[0187] (5) Stirring speed

[0188] Prepare the Yi Guan Jian sample solution according to item "2.1.2". Take 300 mL of the solution in a 500 mL conical flask, add 10.5% of the crude drug β-CD, stir magnetically at 400 r / min, 600 r / min, 800 r / min, and 1000 r / min for 0.5 h at 30 °C, let stand for 24 h, and then concentrate under reduced pressure at 60 °C and -0.09 MPa to a relative density of 1.15 ± 0.05 g / cm 3 , dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-index component detection method under "2.1.4".

[0189] (6) Standing time

[0190] Prepare the Yi Guan Jian sample solution according to item "2.1.2". Take 300 mL of the solution in a 500 mL conical flask, add 10.5% of the crude drug β-CD, stir at 400 r / min for 0.5 h at 30 °C, let stand for 0 h, 2 h, 6 h, 12 h and 24 h respectively, and then concentrate under reduced pressure at 60 °C and -0.09 MPa to a relative density of 1.15 ± 0.05 g / cm 3 , dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-index component detection method under "2.1.4".

[0191] 2.1.6. Concentration temperature investigation

[0192] (1) Sample processing for multi-component and betaine detection

[0193] Prepare the Yi Guan Jian accompanying sample solution according to item "2.1.2", take 300 mL of the solution into a 500 mL conical flask, treat the accompanying sample solution under the optimal conditions investigated under item "2.1.5", and concentrate under reduced pressure to a relative density of 1.15 ± 0.05 g / cm at 50 °C, 60 °C, and 70 °C, with a vacuum degree of -0.09 MPa. 3 , dilute the concentrate to 100mL, take 5mL into a 15mL volumetric flask, add methanol, extract by ultrasonic for 30min, let it cool to room temperature, dilute to 15mL with methanol, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm microporous membrane, and test it according to the multi-component and betaine test method under "2.1.4".

[0194] (2) Sample processing for detection of rehmannia glutinosa and toosendanin

[0195] Prepare the Yi Guan Jian accompanying sample solution according to item "2.1.2", take 300 mL of the solution into a 500 mL conical flask, treat the accompanying sample solution under the optimal conditions investigated under item "2.1.5", and concentrate under reduced pressure to a relative density of 1.15 ± 0.05 g / cm at 50 °C, 60 °C, and 70 °C, with a vacuum degree of -0.09 MPa. 3 , dilute the concentrate to 50mL, extract by ultrasonic for 30min, cool to room temperature, shake well, centrifuge at 12000r / min for 5min, take the supernatant and filter through 0.45μm microporous membrane, and detect according to the detection method of rehmannia glutinosa and toosendanin under "2.1.4".

[0196] 2.2 Drying process

[0197] The present invention has previously investigated the drying methods, including freeze drying, spray drying and vacuum concentration drying. The results show that freeze drying retains the chemical components better, but the yield is low and the moisture content is high; vacuum concentration drying has a greater impact on the content of each chemical component, and the loss of components is large; spray drying has a smaller impact on each chemical component, and has a high yield and low moisture content. Therefore, spray drying was selected based on the experimental results of the present invention. In the process of experimental research, it was found that the content of ligustilide was greatly affected by temperature, so the inlet air temperature of spray drying was investigated.

[0198] 2.2.1. Preparation of reference solution

[0199] Same as Section 2.1.1.

[0200] 2.2.2. Preparation of accompanying sample solution

[0201] Weigh appropriate amounts of 6 medicinal pieces of Rehmannia root (batch number: 20230101), Fructus Lycii (batch number: 20230101), Angelica sinensis (batch number: 20230102), Ophiopogon japonicus (batch number: 20230101), Glehnia littoralis (batch number: 20230106) and Fructus Toosendan (batch number: 20230102), put them in a round-bottom flask, add 12 times the amount of water of the medicinal pieces, soak for 90 minutes, decoct for 30 minutes, filter, add 10 times the amount of water to the residue, decoct for 30 minutes, filter, combine the two filtrates, shake well, and obtain the Yi Guan Jian accompanying sample solution.

[0202] 2.2.3. Preparation of test solution

[0203] Same as Section 2.1.3.

[0204] 2.2.4 Detection Method

[0205] Same as Section 2.1.4.

[0206] 2.2.5 Investigation of spray drying inlet air temperature

[0207] The relative density is 1.15±0.05g / cm 3 The concentrated liquid was subjected to an atomization pressure of 0.13MPa, a needle pressure of 0.15MPa, and a peristaltic pump speed of 15r / min. The influence of different inlet air temperatures (140℃, 160℃, and 180℃) on the drying results was investigated. The dry paste powder yield was calculated, and the moisture content of the dry paste powder and the content of each index component were determined.

[0208] 2.3 Effects of different intermediates on CCl 4 Comparison of intervention effects on induced liver fibrosis in mice

[0209] 2.3.1. Information on different intermediates

[0210] The intermediate dry paste powder (ZJT-A) prepared without adding β-cyclodextrin during concentration and the intermediate dry paste powder (ZJT-B) prepared according to the optimal intermediate preparation process.

[0211] The preparation method of ZJT-A is as follows: weigh an appropriate amount of 6-flavor slices of Yi Guan Jian, add 12 times the amount of water, soak for 90 minutes, boil for 30 minutes, filter, add 10 times the amount of water to the residue, boil for 30 minutes, filter, combine the two filtrates, and concentrate under reduced pressure at 50°C and -0.09MPa to a density of 1.15±0.05g / cm 3 The concentrated liquid was spray dried under the conditions of atomization pressure of 0.13 MPa, needle pressure of 0.15 MPa, peristaltic pump speed of 15 r / min, and air inlet temperature of 180°C to obtain the intermediate ZJT-A.

[0212] Preparation method of ZJT-B: Weigh an appropriate amount of 6-flavor slices of Yi Guan Jian, add 12 times the amount of water, soak for 90 minutes, decoct for 30 minutes, filter, add 10 times the amount of water to the residue, decoct for 30 minutes, filter, combine the two filtrates, add 10.5% β-cyclodextrin, stir at 30°C and 400r / min for 0.5h, let stand for 2h, and then concentrate under reduced pressure at 50°C and -0.09MPa to a density of 1.15±0.05g / cm 3 The concentrated liquid was spray dried under the conditions of atomization pressure of 0.13 MPa, needle pressure of 0.15 MPa, peristaltic pump speed of 15 r / min, and air inlet temperature of 180°C to obtain the intermediate ZJT-B.

[0213] 2.3.2 Animal testing and testing

[0214] (1) Animal grouping: Male C57 mice were randomly divided into 5 groups: control group, CCl 4 Model group, sorafenib positive group (10 mg / kg), ZJT-A group (9.33 mg crude drug / kg), and ZJT-B group (9.33 mg crude drug / kg).

[0215] (2) Modeling and administration: 2 mL / kg ip 15% CCl 4 -Olive oil, injected 3 times a week (Monday, Wednesday and Friday), modeling for 6 weeks in total, and the body weight of the animals was recorded once a week. From the first day of the fourth week of modeling, the ZJT-A group and the ZJT-B group were orally administered with the corresponding solution prepared with 0.3% CMC-Na solution at a dose of 9.33 mg (raw drug) / kg body weight, the positive group was orally administered with 10 mg / kg of sorafenib prepared with 0.3% CMC-Na solution, and the control group and the model group were administered with the same volume of 0.3% CMC-Na solution, once a day.

[0216] (3) Animal treatment and sampling methods: Each group of mice was fasted for 12 hours after the last administration at the end of the 6th week of modeling. After weighing the weight of each group of mice, 3% sodium pentobarbital was injected at a rate of 0.1 mL / 10 g body weight for anesthesia. After successful anesthesia, blood was collected from the abdominal aorta. The whole blood was placed at 4°C and then centrifuged at 4000 r / min for 15 minutes. The supernatant was placed in a -80°C refrigerator for later use. The liver and spleen of the mouse were then removed, and the blood was absorbed with filter paper, and then photographed, weighed, and recorded. Then, liver tissue of about 1 cm × 1 cm was cut from the same part of the mouse liver lobe and fixed in 10% neutral formaldehyde buffer to prepare pathological sections.

[0217] (4) Serum alanine transaminase (ALT) and aspartate transaminase (AST) detection: Serum liver function was detected by fully automated analysis. The serum samples were thawed at room temperature, shaken and mixed, and placed in the fully automated analyzer of the laboratory department of Shuguang Hospital in the order of the labels. The corresponding ALT and AST reagents were added to the reagent bottles of the analyzer, and the ALT and AST activities were detected according to the steps in the kit instructions.

[0218] (5) HE staining and picrosirius red staining to observe liver histological changes. Liver tissue from the same part of the mouse liver lobe was taken, fixed in 10% neutral formaldehyde buffer, dehydrated in an automatic dehydrator, embedded in paraffin, and sliced. HE and picrosirius red staining, the sealed slides were automatically scanned with a data scanning slicer to observe liver cell damage, inflammatory infiltration, bile duct reaction, collagen fiber proliferation and deposition. The image analysis software Leica LAS ImageAnalysis was used to quantitatively analyze the picrosirius red staining positive area. Positive staining area percentage = positive staining area / total image area

[0219] (6) Detection of hydroxyproline (HYP) content in liver tissue by alkaline hydrolysis method The HYP content in liver tissue was detected by alkaline hydrolysis method. 50 mg of liver tissue was weighed, hydrolyzed, and the pH was adjusted to 6.0-6.8. The detection steps were carried out according to the instructions of the kit.

[0220] 3. Experimental results of intermediates

[0221] 3.1 Concentration process

[0222] 3.1.1. Investigation of factors affecting the content of main ingredients by adding auxiliary materials before concentration

[0223] (1) Screening of auxiliary materials

[0224] The results of the excipient screening are shown in Table 3. The results show that except for ligustilide, the average contents of the other chemical components have no significant changes. When β-CD and HP-β-CD are selected as excipients, the content of ligustilide is the highest and almost the same. Since the price of HP-β-CD is about 10 times that of β-CD, β-CD is selected as the excipient from the perspective of economic benefits.

[0225] Table 3 Effect of different excipients on the average content of each chemical component (n=3)

[0226]

[0227] (2) Amount of auxiliary materials

[0228] The results of the investigation on the dosage of excipients are shown in Table 4. The amount of β-CD added has no obvious effect on the components except ligustilide. When 14.0% of the crude drug amount of β-CD is added to the benchmark sample, the average content of ligustilide is the highest, but the difference is small compared with the results of the investigation on the excipients with an addition of 10.5%. In addition, other excipients will be added in the subsequent preparation research process. Therefore, the amount of β-CD excipient is reduced as much as possible in this step to leave room for the amount of excipients in the subsequent preparation research. Therefore, the excipient with an addition of 10.5% of the crude drug amount is selected.

[0229] Table 4 Effect of different amounts of auxiliary materials on the average content of each chemical component (n=3)

[0230]

[0231]

[0232] (3) Stirring temperature

[0233] The results of the stirring temperature investigation (Table 5) show that the stirring temperature has a significant effect on the average content of ligustilide. When the stirring temperature is controlled at 30°C, the content of ligustilide is the highest, so the stirring temperature is selected to be 30°C.

[0234] Table 5 Effect of different stirring temperatures on the average content of each chemical component (n=3)

[0235]

[0236] (4) Mixing time

[0237] According to the results of the stirring time investigation (Table 6), stirring for 0.5 h to 4 h had little effect on the average contents of the seven chemical components, so the stirring time was selected as 0.5 h.

[0238] Table 6 Effect of different stirring times on the average content of each chemical component (n=3)

[0239]

[0240]

[0241] (5) Stirring speed

[0242] By analyzing the results of the stirring speed investigation (Table 7), it was found that when the rotation speed reached 1000r / min, the average contents of p-coumaric acid and ferulic acid were significantly reduced, and the average contents of the other five components were relatively stable. Therefore, the stirring speed can be controlled at 400r / min~800r / min. Referring to the literature on volatile oil inclusion, a stirring speed of 400r / min was selected.

[0243] Table 7 Effect of different stirring speeds on the average content of each chemical component (n=3)

[0244]

[0245] (6) Standing time

[0246] The results of the standing time investigation showed (Table 8) that the length of the standing time had a great influence on the content of ligustilide. When the standing time was between 2h and 24h, the content of ligustilide was relatively stable. Considering the time cost, 2h of standing time was chosen.

[0247] Table 8 Effect of different standing time on the average content of each chemical component (n=3)

[0248]

[0249] 3.1.2. Concentration temperature investigation

[0250] The reduced pressure concentration temperature was investigated according to the method in "2.1.6", and the results are shown in Table 9. Except for ferulic acid, ligustilide I and ligustilide H, the average contents of the remaining chemical components decreased with the increase of concentration temperature, among which the content of ligustilide decreased most significantly, so the concentration temperature of 50℃ was the best.

[0251] Table 9 Effect of different vacuum concentration temperatures on the average content of each chemical component (n=3)

[0252]

[0253]

[0254] 3.2 Drying process

[0255] It can be seen from Tables 10 and 11 that the yield of dry paste powder increases with the increase of inlet air temperature, and the moisture content decreases with the increase of inlet air temperature, but different inlet air temperatures have little effect on the content of each index component. Therefore, it is more appropriate to choose an inlet air temperature of 180°C.

[0256] Table 10 Investigation results of spray drying inlet air temperature

[0257] Inlet air temperature 140℃ 160℃ 180℃ Concentrate / g 309.50 319.80 311.07 Solid content / g 94.34 97.48 94.81 Dry paste powder / g 77.43 82.69 83.50 Yield / % 82.08 84.83 88.07 Moisture / % 2.13 1.51 1.38

[0258] Table 11 Effect of different inlet air temperatures on the average content of each chemical component (n=3)

[0259]

[0260]

[0261] The final preparation method of the intermediate was as follows: weigh an appropriate amount of the six pieces of Yi Guan Jian, add 12 times the amount of water, soak for 90 minutes, decoct for 30 minutes, filter, add 10 times the amount of water to the residue, decoct for 30 minutes, filter, combine the two filtrates, add 10.5% of the crude drug amount of β-cyclodextrin, stir at 30°C and 400r / min for 0.5h, let stand for 2h, and then concentrate under reduced pressure at 50°C and -0.09MPa to a density of 1.15±0.05g / cm 3 The concentrated liquid was spray dried under the conditions of atomization pressure of 0.13 MPa, needle pressure of 0.15 MPa, peristaltic pump speed of 15 r / min, and air inlet temperature of 180°C to obtain an intermediate.

[0262] 3.3 Effects of different intermediates on CCl 4 Comparison of intervention effects on induced liver fibrosis in mice

[0263] (1) Effect on serum liver function

[0264] The serum biochemical results are shown in Table 12. Compared with the normal group, the serum ALT and AST of the mice in the model group were significantly increased (P < 0.01). Compared with the model group, the ZJT-A group showed no significant difference, while the serum ALT and AST of the ZJT-B group and the positive group were significantly decreased (P < 0.01, P < 0.05).

[0265] Table 12 for CCl 4 Effects of serum biochemistry on mice with induced liver fibrosis (mean ± sd, n = 10)

[0266] Group Number of animals ALT(U / L) AST(U / L) Normal group 10 30.78±5.34 165.77±25.13 Model Group 10 207.89±31.12## 500.02±56.72## Positive group 10 97.01±21.34** 254.32±37.29** ZJT-A Group 10 189.01±36.25 400.98±63.43 ZJT-B Group 10 154.71±21.09* 372.62±31.21*

[0267] Note: Compared with the normal group: ##P<0.01; compared with the model group: *P<0.05, **P<0.01.

[0268] (2) CCl 4 Effect of HYP content on liver histopathology and liver fibrosis in mice

[0269] HE staining results showed that the liver lobule structure of the control group was clear and there was no inflammatory cell infiltration; the liver lobule structure of the mice in the model group was severely damaged, and a large number of inflammatory cell infiltration and tissue necrosis were observed; compared with the model group, the ZJT-A group did not show obvious changes, while the ZJT-B group and the positive drug group showed significant improvement. SR staining results showed that only a small amount of collagen fiber deposition was observed in the portal area of ​​the liver tissue of the mice in the control group; a large amount of collagen deposition appeared in the liver tissue of the mice in the model group, which was distributed in a divergent manner with the portal area as the center; compared with the model group, the ZJT-A group did not show obvious changes, while the ZJT-B group and the positive drug group showed significant improvement. 。

[0270] The semi-quantitative results of HYP and SR are shown in Table 13. Compared with the control group, the HYP content in the liver tissue of the model group increased significantly (P < 0.01); compared with the model group, the ZJT-A group did not show significant changes, while the ZJT-B group and the positive drug group had significant improvement effects (P < 0.01, P < 0.05). The semi-quantitative results of Sirius red staining for collagen showed that compared with the normal group, the positive staining area of ​​the liver tissue of the mice in the model group increased significantly (P < 0.01), the ZJT-A group did not show significant changes, while the ZJT-B group and the positive drug group had significant improvement effects (P < 0.01, P < 0.05).

[0271] Table 13 Semi-quantification of HYP content and collagen-positive area in mouse liver tissue (mean ± sd, n = 10)

[0272] Group Number of animals HYP (μg / mL) SR positive staining area (%) Normal group 10 324.55±41.23 1.65±0.35 Model Group 10 700.01±112.14## 6.34±1.27## Positive group 10 444.51±70.14** 3.34±0.79** ZJT-A Group 10 589.89±102.12 5.13±0.92 ZJT-B Group 10 476.34±64.54* 3.78±0.88*

[0273] Note: Compared with the normal group: ##P<0.01; compared with the model group: *P<0.05, **P<0.01.

[0274] 4. Experimental methods for granules

[0275] 4.1. Granulation method selection

[0276] 4.1.1 Moisture determination

[0277] Determine the moisture content (%) according to the second method (drying method) of the moisture determination method (General Chapter 0832) of the 2020 edition of the "Chinese Pharmacopoeia".

[0278] 4.1.2 Solubility test

[0279] Weigh 10 g of the homemade Yi Guan Jian shaped granule sample respectively, add 200 mL of hot water to dissolve, stir for 5 minutes, and observe immediately. Refer to the solubility inspection item of soluble granules under General Chapter 0104 Granules of the 2020 edition of the "Chinese Pharmacopoeia" to examine the solubility of the shaped granules.

[0280] 4.1.3 Prescription screening

[0281] The evaluation indicators used were particle forming rate, dissolution rate, moisture absorption rate, angle of repose and moisture, and the impact of each parameter in particle preparation was combined to assign points to evaluate the prepared granules.

[0282] (1) Determination of particle forming rate

[0283] According to the particle size inspection method under General Chapter 0104 Granules of the 2020 edition of the "Chinese Pharmacopoeia", the granules are screened according to the second binary method. The granules that can pass through the No. 1 sieve but cannot pass through the No. 5 sieve are qualified granules, and the granule forming rate is calculated.

[0284] Particle forming rate (%) = qualified particle mass / total mass of particles obtained × 100%

[0285] (2) Determination of dissolution rate

[0286] According to the 2020 edition of the "Chinese Pharmacopoeia" and related literature reports, the solubility of granules was determined. 10 g of qualified granules were added with 200 mL of hot water and stirred for 5 minutes. 20 mL of supernatant was taken by centrifugation and placed in an evaporating dish with constant weight. After evaporation in a water bath, it was dried at 105°C to constant weight. The weight was weighed and the solubility rate was calculated.

[0287] Dissolution rate (%) = dissolved particle mass / particle mass × 100%

[0288] (3) Determination of moisture absorption

[0289] Place the weighing bottle in a glass desiccator (with a supersaturated NaCl solution at the bottom and a relative humidity of 75%) and equilibrate at 25°C for 24 hours. Take 2g of qualified particles (W1) and place them in a flat weighing bottle (open the flat weighing bottle). When the weight no longer changes, weigh it (W2) and calculate the moisture absorption rate.

[0290] Moisture absorption rate (%) = (W2-W1) / W1×100%

[0291] (4) Determination of the angle of repose

[0292] Using the fixed funnel method, take a watch glass with a radius R of 4 cm, fix the funnel above the watch glass (the watch glass is placed on a horizontal table), and the lower end of the funnel faces the center of the watch glass. Slowly add homemade particles (which can pass through the No. 1 sieve but not the No. 5 sieve) from the top of the funnel and let it flow down naturally. When the edge of the cone formed by the particles at the lower end of the funnel coincides with the edge of the watch glass, measure the particle height H and calculate the angle of repose (α).

[0293] α=arc tg(H / R)

[0294] (5) Evaluation method

[0295] The above four indicators are important factors that determine whether the particle preparation is successful. The weights of particle forming rate, dissolution rate and moisture absorption rate are 0.3 respectively; the weight of repose angle is 0.1.

[0296] Comprehensive score / %=[(molding rate / maximum molding rate+minimum moisture absorption rate / moisture absorption rate+melting rate / maximum melting rate)×0.3+(minimum angle of repose / angle of repose)×0.1]×100%.

[0297] 4.1.4. Screening of auxiliary materials

[0298] Take the intermediate ZJT-B and the auxiliary materials (microcrystalline cellulose, soluble starch, maltodextrin, mannitol, lactose) in a ratio of 3:2 and mix them evenly. Use wet granulation, add an appropriate amount of wetting agent (85% ethanol) to make a soft material, sieve through a 14-mesh sieve, dry in an oven (55°C), and granulate. Determine the moisture content of the granules and the four evaluation indicators, make a comprehensive evaluation, and select the best auxiliary materials.

[0299] 4.1.5 Wet granulation

[0300] Take the intermediate ZJT-B of Yiguanjian and the auxiliary materials in a ratio of 3:2, mix them evenly, add an appropriate amount of wetting agent (85% ethanol) to make a soft material, sieve through a 14-mesh sieve to make granules, put them in an oven (55°C) to dry, and granulate them. Determine the moisture content of the granules and four evaluation indicators.

[0301] 4.1.6 Dry granulation

[0302] Take the intermediate ZJT-B of Yi Guan Jian and the auxiliary materials in a ratio of 3:2, mix them evenly, pour them into the dry granulator, and make granules under the conditions of feeding speed of 5 rpm, feeding speed of 10 rpm, hydraulic station pressure of 65 bar, roller speed of 5 rpm and granulation speed of 80 rpm. The temperature in the whole granulation process is controlled at 20℃±2℃. Determine the moisture content of the granules and 4 evaluation indicators.

[0303] 4.2. Investigation of the proportion of auxiliary materials

[0304] 4.2.1. Moisture determination

[0305] Same as Section 4.1.1.

[0306] 4.2.2 Solubility test

[0307] Same as Section 4.1.2.

[0308] 4.2.3 Preparation of intermediates

[0309] Same as Section 4.1.3.

[0310] 4.2.4 Prescription Screening

[0311] Same as Section 4.1.4.

[0312] 4.2.5. Investigation of the proportion of auxiliary materials

[0313] (1) Investigation of the ratio of two auxiliary materials

[0314] The intermediate and the auxiliary material were mixed evenly in a ratio of 1:1, and the ratio of the two auxiliary materials was 1:1, 2:1, and 3:1, respectively, and poured into a dry granulator. Granules were obtained under the conditions of a feeding speed of 5 rpm, a feeding speed of 10 rpm, a hydraulic station pressure of 65 bar, a roller speed of 5 rpm, and a granulation speed of 80 rpm. The temperature during the entire granulation process was controlled at 20°C ± 2°C. The four evaluation indicators of the granules were measured and a comprehensive evaluation was performed.

[0315] (2) Drug-excipient ratio study

[0316] The Yi Guan Jian intermediate and auxiliary materials were mixed evenly in the ratio of 2:1, 3:2 and 1:1, respectively, and poured into the dry granulator. Granules were obtained under the conditions of feeding speed of 5 rpm, feeding speed of 10 rpm, hydraulic station pressure of 65 bar, roller speed of 5 rpm and granulation speed of 80 rpm. The temperature during the whole granulation process was controlled at 20℃±2℃. The four evaluation indicators of granules were measured and comprehensively evaluated.

[0317] 4.3 Granule Process Research

[0318] 4.3.1. Moisture determination

[0319] Same as Section 4.1.1.

[0320] 4.3.2 Solubility test

[0321] Same as Section 4.1.2.

[0322] 4.3.3 Preparation of intermediates

[0323] Same as Section 4.1.3.

[0324] 4.3.4 Prescription Screening

[0325] Same as Section 4.1.4.

[0326] 4.3.5 Granule Process Investigation

[0327] Using L 9 (3 4 ) orthogonal experiment, feed speed (A), hydraulic station pressure (B) and roller speed (C) were selected as investigation factors, and 3 levels were selected for each factor. On this basis, the molding process of granules was investigated. The factor levels are shown in Table 14.

[0328] Table 14 Orthogonal test factor level table

[0329]

[0330] 4.3.6 Verification Experiment

[0331] In order to verify the reliability of the granule forming process, this experiment prepared 5 batches of granules in parallel according to the determined granule preparation process method, and measured the granule moisture content, forming rate, dissolution rate, moisture absorption rate and repose angle.

[0332] 4.4 Effects of different granules on CCl 4 Comparison of intervention effects on induced liver fibrosis in mice

[0333] 4.4.1. Preparation of different granules

[0334] Including the wet granulation samples (KL-A) and dry granulation samples (KL-B) in 4.1 formulation method selection, as well as the first batch of samples (KL-C) in the granule verification experiment after the preparation process was optimized, and the intermediate dry paste powder (ZJT-B) prepared according to the optimal intermediate preparation process was used as a control.

[0335] Preparation method of KL-A: Take the intermediate ZJT-B of Yi Guan Jian and lactose in a ratio of 3:2, mix them evenly, add an appropriate amount of wetting agent (85% ethanol) to make a soft material, sieve through a 14-mesh sieve to granulate, put them in an oven (55°C) to dry, and granulate them to obtain granular KL-A.

[0336] Preparation method of KL-B: Take the Yi Guan Jian intermediate ZJT-B and lactose in a ratio of 3:2, mix them evenly, pour them into a dry granulator, and prepare granules KL-B under the conditions of a feeding speed of 5 rpm, a feeding speed of 10 rpm, a hydraulic station pressure of 65 bar, a roller speed of 5 rpm and a granulation speed of 80 rpm. The temperature during the entire granulation process is controlled at 20°C±2°C.

[0337] Preparation method of KL-C: Take an appropriate amount of intermediate ZJT-B, add auxiliary materials (maltodextrin: lactose is 1:1) in a ratio of 1:1, shake well, pour into a dry granulator, and use dry granulation to obtain granules KL-C under the conditions of a feeding speed of 5 rpm, a feeding speed of 10 rpm, a hydraulic station pressure of 70 bar, a roller speed of 5 rpm and a granulation speed of 80 rpm. The temperature during the entire granulation process is controlled at 20°C±2°C.

[0338] Preparation method of ZJT-B: weigh an appropriate amount of 6-flavor slices of Yi Guan Jian, add 12 times the amount of water, soak for 90 minutes, decoct for 30 minutes, filter, add 10 times the amount of water to the residue, decoct for 30 minutes, filter, combine the two filtrates, add 10.5% β-cyclodextrin in the amount of crude drug, stir at 30°C and 400r / min for 0.5h, let stand for 2h, and then concentrate under reduced pressure at 50°C and -0.09MPa to a density of 1.15±0.05g / cm 3The concentrated liquid was spray dried under the conditions of atomization pressure of 0.13 MPa, needle pressure of 0.15 MPa, peristaltic pump speed of 15 r / min, and air inlet temperature of 180°C to obtain the intermediate ZJT-B.

[0339] 4.4.2 Animal testing and testing

[0340] (1) Animal grouping

[0341] Male C57 mice were randomly divided into 7 groups, namely control group, CCl 4 Model group, sorafenib positive group (10 mg / kg), KL-A group (9.33 mg crude drug / kg), KL-B group (9.33 mg crude drug / kg), KL-C group (9.33 mg crude drug / kg), and ZJT-B group (9.33 mg crude drug / kg).

[0342] (2) Modeling and drug administration

[0343] 2 mL / kg ip 15% CCl 4 -Olive oil, injected 3 times a week (Monday, Wednesday and Friday), modeling for 6 weeks in total, and the body weight of the animals was recorded once a week. From the first day of the fourth week of modeling, the KL-A, KL-B, KL-C and ZJT-B groups were orally administered with the corresponding solution prepared with 0.3% CMC-Na solution at a dose of 9.33 mg (raw drug) / kg body weight, the positive group was orally administered with 10 mg / kg of sorafenib prepared with 0.3% CMC-Na solution, and the control group and the model group were administered with the same volume of 0.3% CMC-Na solution, once a day.

[0344] (3) Animal processing and sampling methods

[0345] Each group of mice fasted for 12 hours after the last administration at the end of the 6th week of modeling. After weighing the weight of each group of mice, 3% sodium pentobarbital was injected at 0.1mL / 10g body weight for anesthesia. After successful anesthesia, blood was collected from the abdominal aorta, and the whole blood was placed at 4℃ and centrifuged at 4000r / min for 15min. The supernatant was placed in a -80℃ refrigerator for later use. The liver and spleen of the mouse were then removed, and the blood was absorbed with filter paper, and photos were taken, weighed and recorded. Then, liver tissue of about 1cm×1cm in size was cut from the same part of the mouse liver lobe and fixed in 10% neutral formaldehyde buffer to make pathological sections.

[0346] (4) Serum alanine transaminase (ALT) and aspartate transaminase (AST) testing

[0347] Fully automated analysis method for testing serum liver function: Thaw the serum samples at room temperature, shake and mix, and place them in the fully automated analyzer of the laboratory department of Shuguang Hospital in order of labels. Add the corresponding ALT and AST reagents to the reagent bottles of the analyzer, and detect ALT and AST activities according to the steps in the kit instructions.

[0348] (5) Observation of liver histological changes by HE staining and picrosirius red staining

[0349] Liver tissues from the same part of the liver lobe of mice were fixed in 10% neutral formaldehyde buffer, dehydrated in an automatic dehydrator, embedded in paraffin, and sliced. HE and sirius red staining were performed, and the sealed slices were automatically scanned with a data scanning slicer to observe the degree of liver cell damage, inflammatory infiltration, bile duct reaction, collagen fiber proliferation and deposition. The image analysis software Leica LASImage Analysis was used to quantitatively analyze the positive area of ​​sirius red staining.

[0350] Positive staining area percentage = positive staining area / total image area

[0351] (6) Detection of hydroxyproline (HYP) content in liver tissue by alkaline hydrolysis method

[0352] The HYP content in the liver tissue was detected by alkaline hydrolysis method. 50 mg of liver tissue was weighed, hydrolyzed, and the pH was adjusted to 6.0-6.8. The detection steps were carried out according to the instructions of the kit.

[0353] 5. Experimental results of granules

[0354] 5.1. Selection of preparation method

[0355] (1) Screening of auxiliary materials

[0356] The experimental results are shown in Table 15. The molding rate of microcrystalline cellulose is far lower than the pharmacopoeia standard, and the solubility of microcrystalline cellulose and soluble starch is poor, which does not meet the solubility standard of soluble particles in the pharmacopoeia, so microcrystalline cellulose and soluble starch are excluded as excipients for the preparation of Yi Guan Jian granules. Mannitol is not mixed evenly with the intermediate, resulting in a large number of white dots on the appearance of the molded granules. Therefore, mannitol is excluded as an excipient for the preparation of Yi Guan Jian granules based on the appearance. Maltodextrin has strong hygroscopicity and lactose is expensive. From the perspective of economic benefits and excipient properties, it is clear that the subsequent preparations will use maltodextrin and lactose as double excipients for research.

[0357] Table 15 Results of screening experiments on auxiliary materials for forming particles

[0358] Excipient name Moisture / % Forming rate / % Solubility Microcrystalline Cellulose 2.96 79.60 Not completely dissolved, with a lot of precipitation at the bottom Maltodextrin 3.30 88.53 All melted, no obvious turbidity Soluble starch 3.70 95.96 Not completely dissolved, with sediment at the bottom Mannitol 2.80 94.53 All melted, no obvious turbidity lactose 2.48 89.09 All melted, no obvious turbidity

[0359] (2) Selection of preparation method

[0360] By comparing the effects of wet granulation and dry granulation on the moisture content, forming rate, dissolution rate, moisture absorption rate and repose angle of the granules, it was found that the moisture content of the granules examined by the two preparation methods was less than 8%, which met the requirements of the pharmacopoeia; the differences between the dissolution rate, moisture absorption rate and repose angle were small; since dry granulation can be granulated twice, the difference in forming rate between the two preparation methods has no comparative significance. In addition, dry granulation does not involve the problem of drying during the experiment, avoiding further loss of volatile components. After comprehensive consideration, dry granulation was selected as the preparation method, and the results are shown in Table 16.

[0361] Table 16 Preparation method selection experimental results

[0362] Granulation method Moisture / % Forming rate / % Dissolution rate / % Moisture absorption rate / % Angle of repose° Wet granulation 2.08 88.51 93.51 20.75 34.99 Dry granulation 4.27 59.97 92.52 21.58 32.01

[0363] 5.2. Investigation of the proportion of auxiliary materials

[0364] (1) Investigation of the ratio of two auxiliary materials

[0365] The analysis results showed that when the ratio of maltodextrin to lactose was 1:1, the highest comprehensive evaluation was 97.67%. Therefore, the optimal ratio of the two auxiliary materials of Yi Guan Jian granules was maltodextrin to lactose of 1:1, as shown in Table 17.

[0366] Table 17 Investigation results of the ratio of double auxiliary materials for forming granules

[0367] Auxiliary material ratio Moisture / % Forming rate / % Dissolution rate / % Moisture absorption rate / % Angle of repose° Comprehensive evaluation / % 1:1 4.36 62.50 92.21 21.24 30.96 97.67 2:1 4.03 58.45 91.11 21.74 30.96 94.73 3:1 3.99 59.80 91.89 22.31 30.96 94.94

[0368] (2) Drug-excipient ratio study

[0369] The investigation results are shown in Table 18. The comprehensive evaluation of all results of drug-excipient ratios are all above 95%. Among them, when the drug-excipient ratio is 1:1, the highest comprehensive evaluation is 99.56%, so the drug-excipient ratio is selected as 1:1.

[0370] Table 18 Experimental results of drug-excipient ratio of formed granules

[0371] Drug-excipient ratio Moisture / % Forming rate / % Dissolution rate / % Moisture absorption rate / % Angle of repose° Comprehensive evaluation / % 2:1 3.78 62.15 89.40 22.52 30.96 95.02 3:2 4.36 62.50 92.21 21.24 30.96 97.67 1:1 4.07 62.28 92.72 19.71 32.01 99.56

[0372] 5.3 Granule Process Research

[0373] (1) Granule Processing Investigation

[0374] The results of the orthogonal test are shown in Tables 19 and 20. The variance analysis results show that factors A, B, and C have no significant effect, and the order of influence of each factor on the molding process is C>A>B, that is, roller speed>feeding speed>hydraulic station pressure. According to the variance and range analysis results, the numerical intuitive analysis shows that A1>A3>A2, B3>B1>B2, C2>C1>C3, and the dry preparation process is determined to be A. 1 B 3 C1 , that is, the feed speed is 10rpm, the hydraulic station pressure is 70bar, and the pressure roller speed is 5rpm.

[0375] Table 19 Variance analysis table

[0376]

[0377]

[0378] Table 20 Dry granulation orthogonal test results

[0379]

[0380] (2) Verification experiment

[0381] The results of the five batches of validation experiments are shown in Table 21. The granules have uniform color, an average one-time molding rate of 69.64%, an average melting rate of 96.01%, an average moisture absorption rate of 22.15%, an average repose angle of 33.02°, and RSDs of all less than 3%. The validation results show that this method is stable and reliable and can be used as a molding process for Yi Guan Jian granules.

[0382] Table 21 Results of 5 batches of particle verification experiments

[0383]

[0384]

[0385] The final method for preparing granules was determined as follows: take an appropriate amount of the intermediate, add auxiliary materials in a 1:1 ratio (maltodextrin: lactose is 1:1), shake well, pour into a dry granulator, and use dry granulation to prepare granules under the conditions of a feeding speed of 5 rpm, a feeding speed of 10 rpm, a hydraulic station pressure of 70 bar, a roller speed of 5 rpm and a granulation speed of 80 rpm. The granule specification is 12 g / bag.

[0386] 5.4 Effects of different granules on CCl 4 Comparison of intervention effects on induced liver fibrosis in mice

[0387] (1) Effect on serum liver function

[0388] The serum biochemical results are shown in Table 22. Compared with the normal group, the serum ALT and AST of the mice in the model group were significantly increased (P < 0.01). Compared with the model group, the serum ALT and AST of the KL-A and KL-B groups did not show significant changes, while the serum ALT and AST of the KL-C group, ZJT-B group and positive group decreased significantly (P < 0.01, P < 0.05).

[0389] Table 22 for CCl 4Effects of serum biochemistry on induced liver fibrosis in mice (mean ± SD, n = 10)

[0390] Group Number of animals ALT(U / L) AST(U / L) Normal group 10 32.45±5.43 176.23±22.65 Model Group 10 <![CDATA[208.43±30.25 ## ]]> <![CDATA[415.12±45.76 ## ]]> Positive group 10 100.12±18.76** 260.45±34.29** KL-A group 10 187.26±34.23 387.23±44.98 KL-B Group 10 189.11±36.76 388.45±41.73 KL-C Group 10 157.89±18.97* 314.84±34.25* ZJT-B Group 10 154.65±19.01* 316.77±31.68*

[0391] Note: Compared with the normal group: ## P<0.01; compared with the model group: *P<0.05, **P<0.01.

[0392] (2) CCl 4 Effect of HYP content on liver histopathology and liver fibrosis in mice

[0393] HE staining results showed that the liver lobule structure of the control group was clear, without inflammatory cell infiltration; the liver lobule structure of the mice in the model group was severely damaged, with a large number of inflammatory cell infiltration and tissue necrosis; compared with the model group, the inflammatory cell infiltration and tissue necrosis in the KL-A group and the KL-B group did not show obvious changes, while the improvement effect in the KL-C group and the ZJT-B group was significant.

[0394] SR staining results showed that only a small amount of collagen fiber deposition was visible in the portal area of ​​the liver tissue of the control group mice; a large amount of collagen deposition was observed in the liver tissue of the model group mice, which was distributed divergently from the portal area to the surrounding areas; compared with the model group, the KL-A group and the KL-B group did not show obvious changes, while the effects of the KL-C group and the ZJT-B group were significant.

[0395] The semi-quantitative results of HYP and SR are shown in Table 23. Compared with the control group, the HYP content in the liver tissue of the model group increased significantly (P < 0.01); compared with the model group, the KL-A group and the KL-B group did not show significant changes, while the KL-C and ZJT-B groups showed significant improvement (P < 0.01). The semi-quantitative results of Sirius red staining of collagen showed that compared with the normal group, the collagen area of ​​the liver tissue of the mice in the model group increased significantly (P < 0.01); compared with the model group, the KL-A group and the KL-B group did not show significant changes, while the KL-C and ZJT-B groups showed significant improvement (P < 0.01).

[0396] Table 23 Semi-quantification of HYP content and collagen-positive area in mouse liver tissue (mean ± sd, n = 10)

[0397] Group Number of animals HYP (μg / mL) SR positive staining area (%) Normal group 10 325.63±40.01 1.63±0.34 Model Group 10 690.01±101.37## 6.29±1.30## Positive group 10 450.34±69.72** 3.32±0.81** KL-A group 10 578.23±103.43 4.89±1.10 KL-B Group 10 581.23±101.89 4.96±1.12 KL-C Group 10 487.12±77.23* 3.75±0.78* ZJT-B Group 10 495.21±80.23* 3.87±0.82*

[0398] Note: Compared with the normal group: ## P<0.01; compared with the model group: *P<0.05, **P<0.01.

[0399] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing a dried paste powder, characterized in that: The method comprises the following steps: (1) Weighing appropriate amounts of Adenophora adenophora, Ophiopogon japonicus, Angelica sinensis, Rehmannia root, Lycium barbarum and Toosendan fruit, adding first water for soaking, first decocting over high heat for a period of time, then decocting over low heat for a period of time, filtering to obtain a filter residue and a first filtrate; adding second water to the filter residue, first decocting over high heat for a period of time, then decocting over low heat for a period of time, filtering to obtain a second filtrate; (2) combining the first filtrate and the second filtrate, fixing the volume, and obtaining a Yiguanjian water extract; and (3) adding β-cyclodextrin or hydroxypropyl-β-cyclodextrin to the water extract of Yiguanjian, stirring, standing, and concentrating to obtain a concentrated solution, and drying the concentrated solution to obtain a dried paste powder of Yiguanjian.

2. The method according to claim 1, characterized in that In step (1), the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is (1-10):(1-10):(1-10):(5-15):(5-15):(1-10); Preferably, the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is (4-7):(4-7):(4-7):(9-12):(9-12):(6-8); More preferably, the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is (5.32-5.88):(5.32-5.88):(5.32-5.88):(10.6305-11.7495):(10.6305-11.7495):(7.087-7.833); Preferably, the mass ratio of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan is about 5.6: about 5.6: about 5.6: about 11.19: about 11.19: about 7.46; Particularly preferably, the Rehmannia root is raw Rehmannia root; Particularly preferably, in step (1), the mass / volume (mg / ml) ratio of the total mass of the Radix Adenophorae, the Radix Ophiopogonis, the Radix Angelicae Sinensis, the Radix Rehmanniae, the Fructus Lycii and the Fructus Toosendan to the volume of the first water is 60 to 100, for example, about 83.3; Particularly preferably, the soaking time is 60 to 120 minutes, for example, about 90 minutes; Particularly preferably, the frying pot is a casserole or a stainless steel pot; Particularly preferably, the decocting is covered decocting; Particularly preferably, the voltage of the high fire is 400-600W, for example, about 500W; Particularly preferably, the high-fire decocting time is high-fire decocting until boiling; Particularly preferably, the voltage of the slow fire is 150 to 350 W, for example, about 250 W; Particularly preferably, the slow-cooking time is 10 to 60 minutes, for example, about 30 minutes; Particularly preferably, the filtration is hot filtration; Particularly preferably, the hot filtration is hot filtration in a funnel using gauze; Particularly preferably, the volume / volume (mL / mL or L / L) ratio between the volume of the first water and the volume of the second water is 1 to 2, such as about 1.

2.

3. The method according to claim 1, characterized in that In step (3), the mass / mass (mg / mg or g / g) ratio of the mass of the β-cyclodextrin to the total mass of the Adenophora adenophora, the Ophiopogon japonicus, the Angelica sinensis, the Rehmannia root, the Fructus Lycii and the Fructus Toosendan is 15% to 25%, for example, about 10.5%; Preferably, the stirring is magnetic stirring; Preferably, the stirring temperature is 20-40°C, for example about 30°C; Preferably, the stirring speed is 300-500 r / min, for example, about 400 r / min; Preferably, the stirring time is 20 to 40 minutes, for example, about 30 minutes; More preferably, the standing time is 1 h to 3 h, for example about 2 h; Also preferably, the concentration is reduced pressure concentration; Preferably, the temperature of the reduced pressure concentration is 40-60°C, for example, about 50°C; Preferably, the pressure of the reduced pressure concentration is -0.1 MPa to -0.08 MPa, for example, about -0.09 MPa; Preferably, the density of the concentrate is 1.15±0.05 g / cm 3 ; Particularly preferably, the drying is spray drying; Particularly preferably, the inlet air temperature of the spray drying is 160-200°C, for example about 180°C; Particularly preferably, the atomization pressure of the spray drying is 0.1 to 0.2 MPa, for example, about 0.13 MPa; Particularly preferably, the needle pressure during spray drying is 0.1-0.2 MPa, for example, about 0.15 MPa; Particularly preferably, the rotation speed of the spray drying peristaltic pump is 10-20 r / min, such as about 15 r / min. 4 . An aqueous extract of Yiguanjian prepared by the method according to claim 1 , or a preparation comprising the aqueous extract of Yiguanjian.

5. A Yi Guan Jian Gan Gao Powder prepared by the method according to any one of claims 1 to 3 or a preparation comprising the Yi Guan Jian Gan Gao Powder.

6. A method for preparing a Yi Guan Jian preparation, characterized in that: The method comprises mixing the dried paste powder of Yi Guan Jian with auxiliary materials to obtain the Yi Guan Jian preparation.

7. The preparation method according to claim 6, characterized in that: The method further comprises the step of dry granulation; Preferably, the feed rate of the dry granulation is 5 to 20 r / min, for example, about 10 r / min; Preferably, the hydraulic station pressure of the dry granulation is 50 to 90 bar, for example, about 70 bar; Preferably, the roller speed of the dry granulation is 1 to 15 r / min, for example, about 5 r / min; Preferably, the feeding speed of the dry granulation is 1 to 10 r / min, for example, about 5 r / min; Preferably, the granulation speed of the dry granulation is 60-100 r / min, such as about 80 r / min.

8. A Yi Guan Jian preparation prepared by the method according to claim 6 or 7.

9. The preparation according to claim 4, 5 or 8, characterized in that The preparation is an oral preparation such as a granule; Preferably, the auxiliary material is maltodextrin and / or lactose; More preferably, the ratio of the maltodextrin to the lactose is (0.1-3):(0.1-3), for example, about 1:about 1; More preferably, the mass / mass (mg / mg or g / g) ratio of the Yi Guan Jian dry paste powder to the auxiliary material is (0.1-3):(0.1-3), for example, about 1:about 1; Also preferably, the mass of the granules is 8-16 g / bag, for example, about 12 g / bag.

10. Use of the preparation according to claim 4, 5, 8 or 9 in the preparation of a medicament for preventing and / or treating liver fibrosis; Preferably, the effect of the preparation in preventing and / or treating liver fibrosis is through one or more of the following ways: reducing the level of serum markers, improving liver tissue pathological abnormalities, and reducing liver tissue hydroxyproline content; More preferably, the serum marker is selected from aspartate aminotransferase and / or alanine aminotransferase; More preferably, the liver tissue pathological abnormality is selected from one or more of the following: inflammatory cell infiltration, tissue necrosis, collagen deposition and increased Sirius red staining positive area.

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