Traditional Chinese medicine compound extract as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a traditional Chinese medicine compound extract. The preparation method comprises the following steps: mixing clear pastes extracted from the raw materials to obtain a clear paste mixed solution; adding water for dilution to obtain a diluent, and centrifuging the diluent to obtain a centrifugal clear liquid and a centrifugal precipitate; filtering by adopting a ceramic membrane to obtain clear liquid and trapped liquid; loading, leaching, eluting and regenerating the clarified liquid to obtain loading flow-through liquid, leacheate, eluent and regenerated liquid in sequence; respectively concentrating and freeze-drying the centrifugal precipitate, the trapped fluid, the loading flow-through fluid, the leacheate, the eluent and the regenerated fluid; then performing centrifugal precipitation, and mixing trapped fluid and regenerated fluid; or mixing the loading flow-through liquid with the leacheate; or eluting to obtain the traditional Chinese medicine compound extract. The traditional Chinese medicine compound extract can better reduce damage to the spleen, thymus and uterus; the levels of inflammatory factors, transforming growth factors and vascular endothelial growth factors in serum are reduced, and a better effect is achieved in the aspect of improving gynecological diseases such as chronic pelvic inflammation.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and more specifically, relates to a traditional Chinese medicine compound extract and a preparation method and application thereof. Background Art

[0002] The Chinese magnolia is native to China and is now distributed in parts of South China, Central China, Southwest China and Taiwan. It has the effects of dispelling wind and dampness, treating pain in the hands and feet, rheumatism in the waist, treating injuries, and relaxing muscles and activating blood circulation. The Chinese magnolia is the stem or stem bark of the broad-leaved magnolia and fine-leaved magnolia of the Berberidaceae family. It has the effects of clearing heat, drying dampness, detoxifying, and treating lung heat cough. Andrographis paniculata was first recorded in "Lingnan Collection of Medicinal Herbs". It tastes bitter and is cold in nature. It has the effects of clearing heat and detoxifying, reducing inflammation and swelling. It can treat pharyngitis and dysentery, and can also detoxify snake venom. It has high medicinal value. The single-sided needle is the root, root bark, stem and leaf of the thorny shell Zanthoxylum bungeanum of the Rutaceae family. It has the effects of digesting food and promoting transportation, promoting qi and relieving pain. It is often used for spleen dysfunction, anorexia, abdominal distension, and abdominal qi stagnation and pain. Millettia reticulata and Angelica sinensis have the effects of nourishing blood and activating blood circulation. Codonopsis can invigorate Qi and strengthen the spleen, promote the transformation of water and dampness and stop leukorrhea. Golden cherry root is the root or root bark of the Rosaceae plant Golden cherry, which has the effect of consolidating essence and astringing intestines, and is mainly used to treat spermatorrhea, enuresis, dysentery and diarrhea.

[0003] The above-mentioned Chinese medicines are often used to prepare various Chinese medicine compound preparations because of their many functions and therapeutic effects, such as Fuke Qianjin Capsule, Sanjin Tablet, Jinji Capsule and its tablets. Among them, Fuke Qianjin Capsule is composed of the above-mentioned Qianjinba, Jinyinggen, Andrographis paniculata, Mahonia, Single-sided needle, Angelica, Millettia reticulata and Codonopsis pilosula, and is mainly used for chronic pelvic inflammatory disease, endometritis, chronic cervicitis, etc.

[0004] However, the preparation methods of existing Chinese medicine compound preparations containing the above-mentioned various Chinese medicines are generally extensive. For example, the preparation and extraction of various Chinese medicine components of Fuke Qianjin Capsule are mainly based on simple water extraction or alcohol extraction, such as extracting Angelica sinensis with ethanol percolation to make a clear paste; extracting Andrographis paniculata with ethanol reflux to make a clear paste; extracting Chinese magnolia vine and Chinese needle needle into a clear paste, etc. This method is relatively limited in the extraction of effective ingredients and the removal of impurities in Chinese medicine raw materials. Therefore, how to improve the effective active ingredients in the above-mentioned various Chinese medicine compound extracts by optimizing the process, remove impurities in the raw materials, and achieve the refining of the extract components, while maintaining the stability of the preparation process, has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a method for preparing a Chinese medicine compound extract, which can effectively remove impurities in various Chinese medicine raw material extracts, significantly improve the effective active ingredients in the Chinese medicine compound extract and the solid yield of each preparation step; and the preparation method has good stability.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0007] A method for preparing a Chinese medicine compound extract comprises the following steps:

[0008] S1. Mixing: The raw materials Qian Jinba, golden cherry root, andrographis paniculata, angelica, Mahonia, single needle, Millettia reticulata and Codonopsis pilosula are used as a combination, and the raw materials are extracted from the clear paste to obtain a clear paste mixture;

[0009] S2. Dilution and centrifugation: dilute the clear paste mixture with water to obtain a dilution, centrifuge the dilution to obtain a centrifugal clear liquid and a centrifugal precipitate;

[0010] S3. Clarification and filtration: The centrifuged clear liquid is filtered using a ceramic membrane, followed by washing with an alcohol solvent, the filtered liquid is collected to obtain a clarified liquid, and the unfiltered liquid is collected to obtain a retentate;

[0011] S4. Enrichment and capture are performed using a high performance preparative liquid chromatography system: the clarified liquid is used for sample loading, and the sampled liquid is collected to obtain a sampled flow-through liquid; then pure water is used for elution, and the liquid eluted with pure water is collected to obtain an eluent; and then an alcohol solvent is used for elution and regeneration in sequence to obtain an eluent and a regeneration liquid;

[0012] S5. respectively concentrating and freeze-drying the centrifugal precipitate, the retentate, the sample flow-through, the eluent, the elution liquid, and the regeneration liquid;

[0013] The centrifugal precipitate after freeze-drying, the retentate and the regeneration solution are mixed to obtain the Chinese medicine compound extract;

[0014] or mixing the freeze-dried sample flow-through and the eluent to obtain the Chinese herbal compound extract;

[0015] Or the eluate after freeze-drying is the Chinese medicine compound extract.

[0016] Preferably, in step S2, the centrifugation is butterfly centrifugation, the rotation speed of the butterfly centrifugation is 8000-10000 r / min, and the outlet pressure is controlled at 0.05 MPa-0.1 MPa.

[0017] Preferably, in step S3, the pore size of the ceramic membrane is 30-1000 nm. More preferably, the pore size of the ceramic membrane is 300-500 nm; most preferably, the pore size of the ceramic membrane is 500 nm.

[0018] Preferably, in step S4, the concentration of the alcohol solvent used for elution is 50-70%. Further preferably, the concentration of the alcohol solvent used for elution is 60%.

[0019] Preferably, in step S4, the concentration of the alcohol solvent used in the regeneration is 80-90%. The concentration of the alcohol solvent used in the regeneration is 90%.

[0020] Preferably, in step S4, the detection conditions of the high performance preparative liquid chromatography system are: preparation column: C18YE, 30 μm, 1500 g; flow rate: 280-350 mL / min; membrane pressure of 1.45-8.2 MPa; detection wavelength of 254 nm, 280 nm.

[0021] Preferably, in step S5, the freeze-drying temperature is ≤-40°C.

[0022] Preferably, in step S5, the concentration temperature is 35-75°C.

[0023] Further preferably, the concentration temperature is 35-45°C, and after the alcohol solvent is concentrated and evaporated, the temperature is raised to 50-60°C and the evaporation is performed in small amounts and multiple times. Under this preferred condition, the loss of effective ingredients (such as ligustilide components) in the Chinese medicine compound extract can be effectively reduced.

[0024] Preferably, in step S1, the clear paste of the raw material is extracted by the following steps:

[0025] Angelica paste: Angelica is extracted by ethanol percolation to make a paste;

[0026] Andrographis Paniculata Paste: Andrographis Paniculata is extracted by ethanol reflux method to prepare a paste;

[0027] Chinese mahonia and Chinese nymphaeaceae clear paste: Chinese mahonia and Chinese nymphaeaceae are boiled and extracted twice, and the filtrates are combined after filtration to make a clear paste;

[0028] Clear paste of Eleutherodactyla oxyphylla, root of golden cherry, Millettia spatholobi and Codonopsis pilosula: Boil Eleutherodactyla oxyphylla, root of golden cherry, Millettia spatholobi and Codonopsis pilosula in water once for extraction and filter. Boil the filtered residues with the residues of Andrographis paniculata and Angelica sinensis together. Combine the two filtrates, filter and concentrate to make a clear paste.

[0029] The angelica paste, andrographis paste, magnolia officinalis, needle paste, kelp, golden cherry root, millettia reticulata, and codonopsis paste are mixed to obtain a paste mixture. More specifically, the extraction method of the raw material paste can refer to the preparation method of gynecological Qianjin capsule in the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0030] Further preferably, the present invention also provides another method for preparing a clear paste mixture, wherein the clear paste of the raw material is extracted by the following steps:

[0031] Angelica clear paste: Angelica is extracted by ethanol percolation, and the extract is centrifuged to obtain the Angelica supernatant, which is the clear paste;

[0032] Andrographis paniculata clear paste: extract Andrographis paniculata with ethanol reflux method, centrifuge the extract to obtain Andrographis paniculata supernatant, which is the clear paste;

[0033] The preparation of the Chinese magnolia, smilax glabra, and the smilax glabra, golden cherry root, Millettia spatholobi, and Codonopsis pilosula paste is as described above. Mix the angelica paste, andrographis paniculata paste, Chinese magnolia, smilax glabra, smilax glabra, golden cherry root, Millettia spatholobi, and Codonopsis pilosula paste to obtain a paste mixture.

[0034] In the above two kinds of clear paste mixtures, the dosage of Angelica sinensis, Codonopsis pilosula, Andrographis paniculata and Lycopodiella ciliata is 9% of the total amount of medicinal materials respectively, and the dosage of Cherry Blossom Root, Millettia spatholobi, Mahonia lauraceae and Eleutherodactyla scabra is 16% of the total amount of medicinal materials respectively.

[0035] Furthermore, the present invention also seeks to protect the Chinese herbal compound extract prepared by the preparation method of the above-mentioned Chinese herbal compound extract.

[0036] Furthermore, the present invention also seeks to protect the use of the above-mentioned Chinese medicine compound extract in the preparation of drugs for treating anti-inflammatory or gynecological diseases.

[0037] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for preparing a Chinese medicine compound extract, the method can effectively remove impurities in various Chinese medicine raw material extracts, significantly improve the effective active ingredients in the Chinese medicine compound extract and the solid yield of each preparation step; and the preparation method has good stability. Compared with the effect of the existing gynecological Qianjin capsule, the Chinese medicine compound extract provided by the present invention can not only better reduce the damage to the spleen, thymus and uterus; it can also better reduce the levels of inflammatory factors, transforming growth factors and vascular endothelial growth factors in serum, and has a better effect in improving gynecological diseases such as chronic pelvic inflammatory disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the mixing of the original sample in step (2) of Example 1, and the centrifugation of the supernatant and the centrifugal precipitate in step (3).

[0039] Figure 2 Schematic diagram of the centrifuged supernatant, clarified liquid and retentate in Example 1.

[0040] Figure 3 Schematic diagram of the sample flow-through and eluent in Example 1.

[0041] Figure 4 Schematic diagram of the eluent and regeneration solution in Example 1.

[0042] Figure 5 This is a high performance preparative liquid chromatogram of the sample flow-through liquid, eluent, eluting liquid and regeneration liquid of Example 1.

[0043] Figure 6 This is the integrated peak diagram after injection of each test sample in Example 1.

[0044] Figure 7 These are the chromatograms of the original mixed sample, the centrifuged clear liquid, the centrifuged precipitate diluted with water, and the centrifuged precipitate diluted with 60% alcohol in Example 1.

[0045] Figure 8 It is a chromatogram of the mixed original sample, centrifuged clear liquid, centrifuged precipitate, clarified liquid, retentate, sample flow-through liquid, eluent, eluent, regenerated liquid, concentrated liquid, and lyophilized powder in Example 1.

[0046] Fig. 9 It is the fingerprint of the diluent, centrifuged supernatant, clarified liquid, eluate and lyophilized powder in Example 1.

[0047] Fig.10 Schematic diagram of the similarity results of the diluent, centrifuged supernatant, clarified liquid, eluate and lyophilized powder in Example 1.

[0048] Fig.11 The characteristic peak quantity transfer results are for the analysis and comparison of the mixed original sample of Example 1 and the combined original sample of Example 6.

[0049] Fig.12 This is the chromatogram of the original sample, the centrifuged supernatant, the centrifuged precipitate diluted with water, and the centrifuged precipitate diluted with 60% alcohol in Example 2.

[0050] Fig.13 The chromatograms of the centrifuged supernatant, clarified liquid and retentate in Example 2 are shown in FIG.

[0051] Fig.14 It is the chromatogram of the whole process in Example 2.

[0052] Fig.15 This is the fingerprint of the combined original sample, centrifuged supernatant, clarified liquid, eluate and lyophilized powder in Example 2.

[0053] Fig.16 The similarity results of the combined original sample, centrifuged supernatant, clarified liquid, eluate and lyophilized powder in Example 2 are shown.

[0054] Fig.17 These are observation diagrams of the uterine morphology of rats in different experimental groups.

[0055] Fig.18 This is the effect of different experimental groups on the spleen and uterine indexes of CPID model rats.

[0056] Fig.19 The results of HE staining of the uterus of rats in different experimental groups.

[0057] Fig. 20This is the effect of different experimental groups on the concentration of inflammatory factors in the serum of CPID model rats.

[0058] Fig.21 This is the effect of different experimental groups on the levels of TGF-β1 and VEGF in the serum of CPID model rats.

[0059] Fig. 22 This is the fingerprint analysis diagram of the whole process in Comparative Example 1.

[0060] Fig.23 Schematic diagram of the first needle of the capture spectrum in Comparative Example 1.

[0061] Fig.24 Schematic diagram of the second needle for capturing the spectrum in Comparative Example 1.

[0062] Fig.25 It is the chromatogram of total sample, supernatant, centrifugal precipitate, clarified liquid, loading flow-through liquid, eluent, elution liquid and regeneration liquid.

[0063] Fig.26 It is a comparison chart of the fingerprint spectra of the diluent, supernatant, permeate, eluate, regeneration solution, concentrate and dry powder. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0065] Example 1 Preparation method of a Chinese medicine compound extract

[0066] (1) The preparation method of the gynecological Qianjin capsule in the 2020 edition of the "Pharmacopoeia of the People's Republic of China" was used to prepare Angelica sinensis paste, Andrographis paniculata paste, Mahonia lactiflora and Smilax glabra paste, Eleutherodactyla scabra, Chrysanthemum officinale root, Millettia spatholobi, and Codonopsis pilosula paste.

[0067] (2) Mixing and dilution: Take the angelica paste, andrographis paste, magnolia officinalis paste, scutellaria baicalensis paste, radix scutellariae, root of golden cherry, millettia spatholobi, and codonopsis paste in step (1) and mix them to obtain 8501.5 ml of the original mixed sample. According to the standard of 3% solid content, 1 / 3 of the mixed sample (the amount of 2 needles) 2833.83 ml of the sample is diluted with water to 37.0 L to obtain a dilution, and 100 ml of the sample is retained. The total solid mass is measured to be 1087.8 g. The dilution process and the relevant information of the dilution are shown in Table 1 below.

[0068] Table 1

[0069]

[0070] (3) Butterfly centrifugation: Take 37L of the dilution solution with batch number QJJN-HH-210224 in step (2), and perform butterfly centrifugation at 9500r / min and outlet pressure controlled at 0.05Mpa to 0.1Mpa to obtain centrifuged clear liquid (QJJN-HH-LXQY-210224) and centrifuged precipitate (QJJN-HH-LXCD-210224). The centrifugation process and the relevant information of the centrifuged liquid are shown in Table 2 below.

[0071] Figure 1 Schematic diagram of the original mixed sample, the centrifuged supernatant and the centrifuged precipitate (from left to right in the figure are the original mixed sample, the centrifuged supernatant and the centrifuged precipitate). Figure 1 As shown, the mixed original liquid is light brown, has low transmittance, is turbid throughout, contains a large amount of insoluble matter, and has low clarity. The centrifuged clear liquid is yellowish brown, has low transmittance, and low clarity.

[0072] Table 2

[0073]

[0074] (4) Clarification and filtration: Take 26.32 L (solid content 17.2 mg / ml) of the centrifuged supernatant (QJJN-HH-LXQY-210224) in step (3) and perform clarification and filtration to obtain a clarified liquid (i.e., a permeate, QJJN-HH-CQY-210224) and a retentate (QJJN-HH-JLY-210224); the operating conditions for clarification and filtration are: sample solvent: pure water; membrane parameters: 500 nm ceramic membrane; flushing: adding 4 L of 60% ethanol; discharge: 32.85 L of permeate after passing through the membrane; the process parameters and specific equipment are shown in Table 3 below.

[0075] Figure 2 Schematic diagram of centrifugal supernatant, clarified liquid and retentate (from left to right in the figure are centrifugal supernatant, clarified liquid and retentate). Figure 2 As shown, the centrifuged clear liquid is yellow-brown, with low transmittance and low clarity. The clarified liquid is brown-yellow, with high transmittance and high clarity. The retentate is dark brown, with low transmittance, turbidity, a large amount of insoluble matter, and low clarity.

[0076] Table 3

[0077]

[0078]

[0079] The volume proportion of ethanol in the clarified liquid was 7.31%, and no precipitation occurred after the alcohol-containing permeate was added with water. No precipitation occurred after the alcohol-containing permeate was mixed with the ordinary permeate. The relevant information of the clarified liquid and the retentate is shown in Table 4 below.

[0080] Table 4

[0081]

[0082] (5) Enrichment and capture: 32.85 L of the clarified liquid with batch number QJJN-HH-CQY-210224 in step (4) was taken and liquid phase preparation was performed using a high performance preparative liquid chromatography system. The process parameters and equipment information of the liquid phase preparation are as follows and shown in Tables 5 and 6.

[0083] ①Balance: pure water, 4BV;

[0084] ② Sample loading: 32.85 L of the clarified liquid of QJJN-HH-CQY-210224 was loaded to obtain the loading flow-through liquid (QJJN-HH-SYY-210224);

[0085] ③ Elution: Use pure water for elution, 2BV, to obtain the eluent (QJJN-HH-LXY-210224);

[0086] ④ Elution: 60% food grade ethanol was used for elution, 4 BV, and the eluent (QJJN-HH-XTY-210224) was obtained;

[0087] ⑤ Regeneration: Use 90% food grade ethanol for regeneration, 3BV, to obtain regeneration liquid (QJJN-HH-ZSY-210224);

[0088] The capture conditions were as follows: B pump loading for 116 minutes.

[0089] Table 5

[0090] Time (minutes) A％ B％ 0 0 100 116 0 100 117 0 100 127 0 100 128 0 100 148 0 100 149 0 100 164 0 100

[0091] Table 6

[0092]

[0093] The relevant information of the sample flow-through solution, eluent, elution solution and regeneration solution is shown in Table 7 below.

[0094] Table 7

[0095]

[0096] Figure 3 Schematic diagram of the sample flow-through and eluent (from left to right in the figure are the sample flow-through and eluent respectively), Figure 4 Schematic diagram of eluent and regeneration liquid (from left to right in the figure are eluent and regeneration liquid respectively), Figure 5 The high performance preparative liquid chromatograms are for the sample flow-through, eluent, elution and regeneration liquid. Figure 3 and 4As shown, the loading flow-through liquid is light yellow, with high transmittance and clarity; the eluent liquid is brown, with high transmittance and clarity; the elution liquid is brown, with high transmittance and clarity; and the regeneration liquid is colorless, with high transmittance and clarity.

[0097] (6) Concentration under reduced pressure: 8.0 L of the retentate of batch number QJJN-HH-JLY-210224, 34.8 L of the sample flow-through of QJJN-HH-SYY-210224, 3.29 L of the eluent of QJJN-HH-LXY-210224, 6.17 L of the eluent of batch number QJJN-HH-XTY-210224, and 4.75 L of the regeneration liquid of batch number QJJN-HH-ZSY-210224 were placed in a 20 L rotary evaporator at 70 ° C and a vacuum degree of -0.1 mbar, and concentrated to 0.9 L, 1.23 L, 1.02 L, 1.8 L, and 0.1 L, respectively, to obtain the respective concentrated solutions. The relevant information of each concentrated solution is shown in Table 8 below.

[0098] Table 8

[0099]

[0100] (7) Freeze drying: The sample flow-through, retentate, eluent, elution, regeneration solution, and centrifugal precipitate are separately packaged in 1000 ml rotary evaporator bottles and 500 ml freezer boxes. Run the freeze dryer until the temperature is below -40°C and turn on the vacuum pump; when the vacuum becomes stable, hang the packaged samples outside the freeze dryer and ventilate. After the samples are completely freeze-dried, take them out and weigh them, and combine the same samples and seal them for storage.

[0101] The freeze-dried centrifugal precipitate, the retentate and the regeneration solution are mixed to obtain the Chinese medicine compound extract.

[0102] The concentrated and freeze-dried sample flow-through and eluent are mixed to obtain the Chinese herbal compound extract.

[0103] The eluate is concentrated and freeze-dried to obtain the Chinese herbal compound extract.

[0104] 1. Determination of total solid mass of Chinese herbal compound extracts

[0105] (1) Total solid mass determination method:

[0106] ① Preparation method of diluent / centrifugal clear liquid / centrifugal sedimentation / clarified liquid / retentate / sample flow-through liquid / rinsing liquid / elution liquid / regeneration liquid test sample

[0107] Before sampling, fully mix the above liquids, accurately measure 10 mL, place in an evaporating dish that has been dried to constant weight, evaporate to dryness in a 100°C water bath, dry at 105°C for 30 min, cool in a desiccator for 30 min, quickly and accurately weigh, measure twice in parallel, and calculate the paste rate.

[0108] ② Preparation method of concentrated solution test sample

[0109] Before sampling, fully mix the concentrated solution of Qianjin extract, accurately measure 2 mL of the concentrated solution, place it in an evaporating dish that has been dried to constant weight, evaporate it in a 100°C water bath, dry it at 105°C for 3 hours, cool it in a desiccator for 30 minutes, quickly and accurately weigh it, measure it twice in parallel, and calculate the paste rate.

[0110] ③Calculation formula for the paste yield:

[0111]

[0112]

[0113] Note: W3: mass of evaporating dish + dry paste (g); W2: constant weight of evaporating dish (g); W1: mass of medicinal slices (g); V1: total volume of extract (mL); V2: volume of 5mL extract (mL); V3: total volume of concentrate (mL); V4: volume of 5mL concentrate (mL); V5: volume of extract after sample retention (mL).

[0114] (2) Total solid mass test results

[0115] The total solid mass was measured according to the method for measuring the total solid mass in step (1) above. The results are shown in Table 9 below.

[0116] Table 9

[0117]

[0118]

[0119] As shown in Table 9 above, the solid mass yield of the centrifuged clear liquid is 83.64%, the total solid mass yield is 95.39%, and the centrifugation process is stable. The total solid mass yield of the clarified liquid reaches 95.78%, and the clarification process is relatively stable. The total solid mass yield of each fraction in the preparation process is 96.29%, and the preparation process is relatively stable. The total solid mass yield of the concentration process is 92.63%, and the concentration process is stable. The total solid mass yield of the drying process is 111.43%, and the yield is stable.

[0120] 2. Process control of the preparation method of Chinese herbal compound extracts

[0121] (1) Characteristic peak value transfer sample preparation method

[0122] ①Test sample preparation method

[0123] Clarified liquid test sample: take 2 mL of the clarified liquid with batch number QJJN-HH-CQY-210224 and filter it through a 0.45 um microporous membrane.

[0124] Centrifuged clear liquid test sample: Accurately pipette 1.60 mL of the centrifuged clear liquid with batch number QJJN-HH-LXQY-210224, dilute to 2 mL with pure water, and filter through a 0.45 um microporous filter membrane.

[0125] Centrifugal precipitate test sample: Accurately weigh 7.78 mg of the centrifugal precipitate with batch number QJJN-HH-LXCD-210224, dilute to 2 mL with pure water and 60% ethanol respectively, and filter through a 0.45 um microporous filter membrane to obtain the product.

[0126] Mix the original sample: Accurately pipette 1.13 mL of the mixed solution of Qianjin extract with batch number QJJN-HH-210224, dilute it to 2 mL with pure water, and filter it through a 0.45 um microporous filter membrane.

[0127] Retentate test sample: Accurately pipette 487uL of Qianjin extract retentate with batch number QJJN-HH-JLY-210224, dilute to 2mL with pure water, and filter through 0.45um microporous filter membrane.

[0128] Sample flow-through liquid test sample: Pipette 2 mL of the sample flow-through liquid with batch number QJJN-HH-SYY-210224 and pass it through a 0.45 um microporous filter membrane.

[0129] Eluent test sample: Accurately pipette 188.81uL of eluent with batch number QJJN-HH-LXY-210224, dilute to 2mL with pure water, and filter through a 0.45um microporous filter membrane.

[0130] Eluent test sample: Accurately pipette 354.1uL of the eluent with batch number QJJN-HH-XTY-210224, dilute to 2mL with 60% alcohol, and filter through a 0.45um microporous filter membrane.

[0131] Regeneration liquid test sample: Accurately pipette 272.6uL of regeneration liquid with batch number QJJN-HH-ZSS-210224, dilute to 2mL with pure water, and filter through 0.45um microporous membrane.

[0132] Concentrated liquid test sample: Accurately pipette 103.1uL of the concentrated liquid of QJJN-HH-XTNSY-210224, dilute to 2mL with 60% alcohol, and filter through a 0.45um microporous membrane.

[0133] Eluent lyophilized powder test sample: Accurately weigh 2.2 mg of lyophilized powder of QJJN-HH-XTDGF-210224, dilute to 2 mL with 60% alcohol, and filter through a 0.45 um microporous membrane.

[0134] ②Chromatographic conditions

[0135] Instrument: Waters Alliance, e2695-2998 (HPLC-007);

[0136] Chromatographic column: Kromasil 100-5-C18 (250×4.6 mm id, 5 μm);

[0137] Mobile phase: A: acetonitrile; B: 0.1% phosphoric acid / water;

[0138] Flow rate: 1.0 mL / min;

[0139] Column temperature: 30°C;

[0140] Injection volume: 50 μL;

[0141] Detection wavelength: 254, 280nm;

[0142] Elution conditions:

[0143] Time (min) Phase A (%) Phase B (%) 0 14 86 10 14 86 25 17 83 50 20 80 58 31 69 78 55 45 85 55 45

[0144] ③ Characteristic peak value transfer results

[0145] Figure 6 It is the integrated peak graph after each test sample is injected. Figure 6 As shown in Table 10, the integral peaks of the characteristic peaks and the quantitative transfer of the whole process of the preparation technology of the Chinese herbal compound extract.

[0146] Table 10

[0147]

[0148]

[0149] It can be seen that the transfer rates of each characteristic peak in the mixed dilution-butterfly centrifugation process are all greater than 90% (except for peak 7 ligustilide (oil layer), it is speculated that the oil layer may have been passed through the membrane).

[0150] The chromatographic peak transfer rate of the clarification filtration process is above 93%, among which the yield of the latter fat-soluble peak is relatively high. The reason is that the addition of alcohol top washing in the clarified liquid further enriches the fat-soluble components slightly higher than that in the centrifuged liquid. The overall clarification yield is qualified.

[0151] Except for peak 7 ligustilide, the transfer rates of the characteristic peaks in the elution preparation process were 88%-96%, indicating that the efficient preparation process was generally stable.

[0152] Except for peak 7 ligustilide (peak 7 was lost during both the concentration and freeze-drying processes), the transfer rates of the characteristic peaks in the concentration-freeze-drying process were 88%-144%, indicating that the concentration-freeze-drying process was generally stable. In addition, according to the results of the concentration process, the concentration temperature of the concentration process can be adjusted to 40°C for rotary evaporation of ethanol, and the temperature after the alcohol is rotary evaporated is adjusted to 55°C and the rotary evaporation is performed in small amounts and multiple times. The adjusted process can effectively reduce the loss of ligustilide components.

[0153] From the above characteristic peak value transfer results, it can be seen that the clarification filtration process is stable, the high-efficiency preparation process is stable, and the concentration-freeze-drying process is stable.

[0154] ④ Characteristic peak value transfer results

[0155] Figure 7 The chromatograms are of the original mixed sample, the centrifuged supernatant, the centrifuged precipitate diluted with water, and the centrifuged precipitate diluted with 60% alcohol in Example 1. Figure 8 It is a chromatogram of the mixed original sample, centrifuged clear liquid, centrifuged precipitate, clarified liquid, retentate, sample flow-through liquid, eluent, eluted liquid, regenerated liquid, concentrated liquid, and lyophilized powder of Example 1.

[0156] Depend on Figure 7 , Figure 8 As shown in Table 11 below, the precipitate was dissolved in water and analyzed. There were some fat-soluble components (components of Angelica sinensis and Andrographis paniculata) in the insoluble matter. The precipitate was dissolved in 60% alcohol. 60% alcohol was able to dissolve all the precipitate. Therefore, the insoluble precipitate should still contain a lot of Angelica sinensis and Andrographis paniculata components.

[0157] Table 11

[0158]

[0159]

[0160] 3. Establishment of fingerprint

[0161] (1) Preparation method and chromatographic conditions of fingerprint, wherein the preparation of the sample and the chromatographic conditions are the same as those in the above-mentioned "2. Process control of the preparation method of the Chinese herbal compound extract, characteristic peak value transfer sample preparation method in step ①". The liquid phase conditions are the same as those in "2. Process control of the preparation method of the Chinese herbal compound extract, step ②".

[0162] (2) The fingerprints of the diluent, centrifuged supernatant, clarified solution, eluate and lyophilized powder in Example 1 are as follows Fig. 9 The similarity results are shown in Fig.10 As shown in Table 12.

[0163] Table 12

[0164] Dilution Centrifuge Clarified liquid Eluent Lyophilized powder Dilution 1 0.955 0.938 0.911 0.925 Centrifuge supernatant 0.955 1 0.986 0.976 0.982 Clarified liquid 0.938 0.986 1 0.992 0.986 Eluent 0.911 0.976 0.992 1 0.991 Lyophilized powder 0.925 0.982 0.986 0.991 1

[0165] It can be seen from Figure and Table 12 that in the process of dilution-centrifugation-clarification-preparation-lyophilization, the fingerprint similarity is greater than 0.9.

[0166] Example 2 Preparation method of a Chinese medicine compound extract

[0167] The difference between this embodiment and embodiment 1 is that in step (1), after preparing the angelica paste and the Andrographis paste, the Andrographis paste and the Angelica paste are centrifuged to separate the Andrographis precipitate and the Angelica oil layer to obtain the Andrographis and Angelica supernatant. Then the Andrographis and Angelica supernatant are mixed with the Herba Lycopodii, the Herba Chrysanthemi, the Herba Millettiae, the Herba Codonopsis, the Herba Mahoniae, and the Herba Lycopodii to obtain the combined original. The other steps are the same as those in embodiment 1.

[0168] (1) Total solid mass determination: The total solid mass determination method in Example 1 was used for testing, and the results are shown in Table 13 below.

[0169] Table 13

[0170]

[0171] It can be seen from Table 13 above that the solid yield of the clarified liquid obtained by the clarification and filtration process is 97.8%, the total solid mass yield is 115.94%, and the clarification process is stable; the total solid mass yield of each fraction in the liquid phase preparation process is 98.01%, and the liquid phase preparation process is relatively stable; the total solid mass yield of the concentration process is 97.41%, and the concentration process is stable; the total solid mass yield of the drying process is 96.11%, and the freeze-drying process is stable.

[0172] (2) Process control of the preparation method of Chinese herbal compound extracts

[0173] ① The test was conducted using the sample preparation method and chromatographic conditions of "2. Process control of the preparation method of Chinese herbal compound extract, characteristic peak value transfer sample preparation method in step ①" in Example 1.

[0174] The characteristic peak value transfer results of the mixed original sample of Example 1 and the combined original sample of Example 6 are as follows: Fig.11 As shown, the value transfer of the entire preparation process is shown in Table 14 below.

[0175] Table 14

[0176]

[0177]

[0178] Depend on Fig.11It can be seen that the transfer rates of each characteristic peak of the dilution-centrifugation process are all greater than 90%; the transfer rate of the chromatographic peak of the clarification filtration process is about 100%, and the transfer rate of peak 1 is 86%. Peaks 1 and 2 contain more flavonoid components (tannins), and the ceramic membrane may be adsorbed. The content of peak 1 in the retentate is about 25%; the transfer rate of each characteristic peak of the liquid phase preparation process is greater than 90%, and the high-efficiency preparation process is generally stable; the transfer rate of each characteristic peak of the concentration-freeze-drying process; the transfer rate of the concentration-freeze-drying peak is all above 95%, and the process is stable. From the results in Table 14 above, it can be seen that the membrane filtration process is stable, the high-efficiency liquid phase preparation process is stable, and the concentration-freeze-drying process is stable.

[0179] ②Centrifugal process analysis

[0180] Fig.12 The chromatograms are the combined original sample, centrifuged supernatant, centrifuged precipitate diluted with water, and centrifuged precipitate diluted with 60% alcohol.

[0181] Depend on Fig.12 As shown in Table 15 below, when the precipitate is dissolved in water, there is no main component in the insoluble matter, and the precipitate content when dissolved in 60% ethanol is also relatively small, so it can be considered that the precipitate has no main component.

[0182] Table 15

[0183]

[0184] ③ Clarification process analysis

[0185] Fig.13 It is the chromatogram of centrifugal supernatant, clarified liquid and retentate. Fig.14 is the chromatogram of the whole process. Fig.13 As shown in Table 16 below, the proportion of chromatographic peaks at 11-15 min after clarification is different. This part belongs to the flavonoid component, and the ceramic membrane may have an effect on this part of the components.

[0186] Table 16

[0187] Peak Centrifuged supernatant (%) Clarified liquid (%) Retentate (%) 1 100.00 86.60 24.12

[0188] (3) Fingerprint of the preparation method of Chinese herbal compound extract

[0189] The preparation method and chromatographic conditions of the fingerprint are the same as those in Example 1.

[0190] The fingerprints of the original sample, centrifuged supernatant, clarified solution, eluate and lyophilized powder were combined as follows: Fig.15 The similarity results of the combined original sample, centrifuged supernatant, clarified solution, eluate and lyophilized powder are shown in Fig.16 and as shown in Table 17.

[0191] Table 17

[0192] Merge as is Centrifuge Clarified liquid Eluent Dry powder Merge as is 1 0.980 0.824 0.891 0.887 Centrifuge 0.98 1 0.880 0.929 0.916 Clarified liquid 0.824 0.880 1 0.985 0.948 Eluent 0.891 0.929 0.985 1 0.966 Lyophilized powder 0.887 0.916 0.948 0.966 1

[0193] The similarity of the fingerprints of the dilution-centrifugation-clarification-preparation-lyophilization process is 0.82-0.9. Some small peaks are difficult to correspond, so the similarity does not reach above 0.9.

[0194] Example 3 Study on screening effective components of Chinese herbal compound extracts for treating chronic pelvic inflammatory disease

[0195] (1) Experimental Materials

[0196] 183 SPF female SD rats, weighing 160-200 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number SYXK (Xiang) 2019-0009. Animal breeding environment: temperature 22-24 °C, humidity 60%-65%, day and night alternation, free food and water.

[0197] Gynecological Qianjin Capsule (0.4g*48 tablets, national medicine standard Z20020024), purchased from Zhuzhou Qianjin Pharmaceutical Co., Ltd.; Kangfuyan Capsule (0.4g*48 tablets, national medicine standard Z20055634), purchased from Shandong Buchang Shenzhou Pharmaceutical Co., Ltd.; Cefaclor Dispersible Tablets (0.125g*10 tablets, national medicine standard H20041126), purchased from Anhui Anke Hengyi Pharmaceutical Co., Ltd.; Chinese medicine compound extract, prepared and provided by Zhuzhou Qianjin Pharmaceutical Co., Ltd. according to the preparation method of Example 1 and Example 2. Each drug is made into a certain concentration of aqueous solution for intragastric administration with ultrapure water.

[0198] Estradiol benzoate (2ml:4mg, veterinary drug number 220412511), Sichuan Jinke Pharmaceutical Co., Ltd.; PBS phosphate buffer (Cat.NO.P1010, PH 7.2-7.4, Solarbio), each bag was dissolved in 2L ultrapure water; Chloral hydrate (lot#:C11916119, Macklin), prepared into a 10% aqueous solution with ultrapure water, 0.5 McFarland turbidimeter tube (Yuezhu Medical No. 20140010, Zhuhai Beso Biotechnology Co., Ltd.), hemostatic sponge (Gan Medical No. 20182640140, Guangzhou Kuaikang Medical Equipment Co., Ltd.); Rat IL-18 ELISA Kit (ZC-36389), IL-6 ELISA Kit (ZC-36404), VEGF ELISA Kit (ZC-37412), TGF-β1 ELISA ELISA kits (ZC-37645), TNF-α ELISA Kit (ZC-37624), and IL-1β ELISA Kit (ZC-36391) were purchased from Zhuocai Biotechnology Co., Ltd.; 4% paraformaldehyde universal tissue fixative (21295584, biosharp).

[0199] High-speed refrigerated centrifuge (Microfuge 20R, Beckman Coulter, Inc., USA), single-channel pipette (10-1000ul, Eppendorf), multi-function microplate reader (2021005559, BIOTEK / Synergy).

[0200] (2) Experimental methods

[0201] ①Establishment of chronic pelvic inflammatory disease (CPID) model

[0202] After one week of adaptive feeding, rats were subcutaneously injected with 0.2 mL of estradiol injection (estradiol benzoate injection for animals) once a day for 5-7 consecutive days. During the injection period, attention was paid to observe whether the vaginal opening of the rats was congested, red and swollen, and the estrus period was observed. After inducing the estrous cycle of the rats, the lower abdomen of the rats was disinfected with 75% alcohol under anesthesia, and a 2 cm longitudinal incision was made in the lower abdomen to expose the uterus. The uterus of the rats was scraped with a curette. Two weeks later, 0.3 mL of Ureaplasma urealyticum + Staphylococcus aureus + Escherichia coli (concentration 1*10 8 cfu / mL), and fill the needle hole with a piece of sterile hemostatic sponge (0.2*0.2cm), and insert a piece of sterile hemostatic sponge into the vagina to prevent the bacterial fluid in the abdominal cavity from flowing out.

[0203] ② Grouping and drug administration

[0204] The successfully modeled rats were divided into model group, Chinese herbal compound extract (1-1, 1-2, 1-3, 1-4, 2-1, 2-2, 2-3, 2-4) group, Kangfuyan capsule (KFY) group, and Fuke Qianjin capsule (FKQJ) group according to the random number table method.

[0205] Construction of sham-operated group (JSS group) rats: The construction of sham-operated group rats was the same as the operation of "model rat construction" in 2.1 above, the difference being that the sham-operated group rats were not infected with Mycoplasma + Staphylococcus aureus + Escherichia coli, and the inflammation model was not constructed.

[0206] Blank group: The blank group used normal mice without treatment.

[0207] in:

[0208] The Chinese medicine compound extract (small polarity part group, 1-1, 52.542 mg·kg) obtained by mixing the centrifugal precipitate, the retentate and the regeneration liquid after concentration and freeze-drying in Example 1 -1 ·d -1 ) for subsequent efficacy experiments.

[0209] The concentrated and freeze-dried sample flow-through and eluent in Example 1 were mixed to obtain a Chinese medicine compound extract (large polar part group, 1-2, 172.204 mg kg -1 ·d -1 ) for subsequent efficacy experiments.

[0210] The eluate in Example 1 was concentrated and freeze-dried to obtain a Chinese medicine compound extract (eluate group, 1-3, 20.304 mg kg -1 ·d -1 ) for subsequent experiments.

[0211] The above eluate group was taken and 1.5 times the dose was administered to obtain the eluate 1.5 times dose group (1-4, 30.457 mg kg -1 ·d -1 ) for subsequent efficacy experiments.

[0212] The Chinese medicine compound extract (2-1, 69.174 mg kg -1 ·d -1 ) for subsequent efficacy experiments.

[0213] The concentrated and freeze-dried sample flow-through and eluent in Example 2 were mixed to obtain a Chinese medicine compound extract (2-2, 209.626 mg kg -1 ·d -1 ) for subsequent efficacy experiments.

[0214] The eluate in Example 2 was concentrated and freeze-dried to obtain a Chinese medicine compound extract (2-3, 46.487 mg kg -1 ·d -1 ) for subsequent experiments.

[0215] The eluate group of Example 2 was taken and 1.5 times the dose was administered to obtain a 1.5 times dose group (2-4, 69.731 mg·kg -1 ·d -1 ) for subsequent efficacy experiments.

[0216] Kangfuyan capsule group: dosage 324.002 mg·kg -1 ·d -1 The model rats were given Kangfuyan Capsules.

[0217] Gynecological Qianjin Capsule Group: dosage: 216.001 mg·kg -1 ·d -1 The model rats were given Fuke Qianjin Capsule.

[0218] Model group: Model rats were given distilled water, and the amount of distilled water was calculated based on the body surface area of ​​the rats.

[0219] Sham operation group: Rats in the sham operation group were given the same amount of distilled water as the model group.

[0220] Blank group: The blank group was given the same amount of distilled water as the model group.

[0221] Each group was continuously administered with drug (or distilled water) for 21 days.

[0222] ③Sample collection

[0223] After continuous administration for 21 days, the rats were fasted but not watered for 24 h. After anesthesia with 10% chloral, blood was collected from the abdominal aorta and allowed to stand at room temperature. After the blood was clearly stratified, the blood was rotated at 3000 r·min. -1 Centrifuge for 15 minutes, take the supernatant as serum, store in -80℃ refrigerator for later use, and detect the levels of TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF in the serum of rats in each group according to the instructions of the kit. Take the uterine tissue of rats in each group, and observe the morphology and color of the uterine tissue with naked eyes. Take about 200 mg of uterine tissue from the same side of rats in each group and fix it in 4% paraformaldehyde for later use.

[0224] ④ Body weight and organ index of rats

[0225] During the experiment, the body weight of rats in each group was recorded weekly. After the rats were killed, the uterine tissue and spleen tissue of the rats were removed, cleaned with PBS phosphate buffer, dried with filter paper, and weighed to calculate the organ index, which was organ index = organ mass (g) / rat body mass (g) × 100.

[0226] ⑤ Gross morphology and histopathological examination of rat uterus

[0227] The bilateral uterine tissues of rats were taken for macroscopic morphological observation; the right uterine tissues were taken, sliced ​​after dehydration, wax immersion, embedding, and baking at 60℃ for 3h, and the slices were dewaxed by xylene, dehydrated by alcohol gradient, and HE stained. After sealing with neutral gum, the pathological changes of rat uterine tissues were observed under a microscope. CaseViewer software was used to observe the results of HE staining of each uterus, the integrity of the endometrium, the state and arrangement of each cell, and the edema and inflammatory cell infiltration.

[0228] ⑥ Detection of TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF levels in rat serum

[0229] Serum samples were taken, and the levels of serum TNF-α, IL-1β, IL-18, IL-6, TGF-β1 and VEGF in rats in each group were detected according to the instructions of the kit.

[0230] ⑦Statistical analysis

[0231] SPSS 25.0 statistical software was used to organize the data. The measurement data were expressed as x±s. The LSD test was used for pairwise comparison. The difference was statistically significant when P < 0.05. Note: "**" indicates P < 0.01, and "*" indicates P < 0.05.

[0232] (3) Experimental results

[0233] ①Observation of rat uterus morphology

[0234] 21 days after modeling, the uteri of rats in the blank group, sham operation group, and model group were randomly selected and the morphology of the uteri was observed with the naked eye. Fig.17 The uterine morphology of rats in each group is observed. Fig.17 The results showed that there was no difference between the left and right sides of the uterus in the sham operation group, and the size, shape and color were normal. The uterus of the model group was thick, swollen and had residual fluid, the fallopian tubes and ovaries were dilated, and there was fat tissue adhesion at the pinhole; there was also adhesion in the pelvic cavity, indicating that the model was successful.

[0235] After the treatment, the abdominal cavity was opened and the pelvic condition was observed. The blank group showed no obvious abnormalities in the uterus and pelvic cavity. Although some rats in the sham group had a small amount of adhesion between the pelvic organs and the surrounding tissues, there was no bleeding; there was no statistical difference between the blank group and the sham group. The model group showed swelling due to pus accumulation inside the uterus, and the uterine cavity tended to be transparent and edematous, and adhered to the surrounding tissues into a mass, which was difficult to separate. Compared with the model group, each drug group could significantly improve the pelvic adhesion of rats with pelvic inflammatory disease.

[0236] ②Effects on spleen, thymus and uterus indexes in CPID model rats

[0237] There was no statistical difference in the organ index between the sham operation group and the blank group (P>0.05), which proved that the surgical process had no effect on the experimental results. Compared with the blank group, the spleen, thymus and uterus indexes of the rats in the model group were significantly increased, indicating that the CPID model rats had certain immune damage (spleen and thymus enlargement), accompanied by uterine effusion and swelling. Except for the positive control drug, 1-1, 1-2, 1-3 and 1-4 can reduce the spleen and uterine index of the model rats to a certain extent. From the overall situation, 1-2 has the best effect (P<0.05). 2-1, 2-2 and 2-3 all have certain effects, with no significant difference from FKQJ, while 2-4 is better than 2-1, 2-2 and 2-3 (P<0.01), especially in terms of spleen index, see Fig.18 .

[0238] ③ Results of HE staining of rat uterus

[0239] HE staining results showed that the endometrium in the blank group was smooth and structurally intact, with only a small amount of neutrophil infiltration. Similarly, the uterine wall of the sham-operated group was clearly layered; the endometrial glands were regular, the muscle fiber texture was clear; and the epithelial cells were arranged in a single layer of columns. In the model group, obvious neutrophil infiltration was observed in the endometrium, with severe tissue edema, and the endometrium became thinner and thicker, with inflammatory cells infiltrating, some of which penetrated deep into the muscle layer and accumulated throughout the entire layer. The endometrial epithelial cells in each drug-treated group proliferated or fell off slightly, and the inflammatory cell infiltration was significantly reduced. The specific results of HE staining of the rat uterus are shown in Fig.19 .

[0240] 4) Effects on the concentration of inflammatory factors in the serum of CPID model rats

[0241] Compared with the blank group, there were no significant differences in the levels of serum factors in the sham-operated group (P>0.05), while the levels of serum IL-6, TNF-α, IL-1β and IL-18 in the model group were significantly increased (P<0.01).

[0242] Except for the positive control drug, compared with the model group, 1-1, 1-2, 1-3, and 1-4 can significantly reduce the concentrations of IL-6, TNF-α, IL-1β, and IL-18 in the serum of model rats, indicating that they have a significant effect on improving the inflammatory response of pelvic inflammatory disease model rats. In general, except for IL-18 and IL-6, 1-2 is superior to 1-1, 1-3, and 1-4 (P < 0.05). In terms of IL-1β, 1-2 is significantly superior to FKQJ (P < 0.01). In terms of IL-18, the modified group 1 (1-1, 1-2, 1-3, 1-4) is significantly superior to FKQJ (P < 0.01). In general, the effect of modified group 2 (2-1, 2-2, 2-3, 2-4) is weaker than that of modified group 1, among which there is no significant effect on IL-6 (P > 0.05). Except for IL-18, in the modified group 2, 2-4 had the best effect. In terms of reducing IL-1β and TNF-α, 2-4 had the best effect (P < 0.01). The specific effects of each group on the concentration of inflammatory factors in the serum of CPID model rats are shown in Fig. 20 The comparison between the test groups is shown in Table 18 below.

[0243] Table 18

[0244]

[0245]

[0246]

[0247] Note: “**” indicates P < 0.01, “*” indicates P < 0.05, and “ns” indicates P > 0.05

[0248] ⑤Effects on the levels of TGF-β1 and VEGF in serum of CPID model rats

[0249] Transforming growth factor TGF-β1 and vascular endothelial growth factor (VEGF) are important factors regulating cell growth and differentiation. They can promote increased vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis, and are highly correlated with pelvic adhesions and tissue hyperplasia.

[0250] Compared with the blank group, there was no significant difference in the levels of TGF-β1 and VEGF in the serum of the rats in the sham operation group (P>0.05), while the levels of TGF-β1 and VEGF in the serum of the rats in the model group were significantly increased (P<0.01). Except for the positive control drug, compared with the model group, 1-1, 1-2, 1-3, and 1-4 could significantly reduce the levels of TGF-β1 and VEGF in the serum of the model rats; among them, the effects of 1-1 and 1-2 were significantly better than those of 1-4, and 1-2 had the best effect, which was significantly better than FKQJ (P<0.01). 2-1 and 2-2 had a certain reducing effect (P<0.01). However, there was no significant difference between the effect of 2-1 and FKQJ (P>0.05). Interestingly, 2-3 and 2-4 had a very significant effect of reducing the levels of TGF-β1 and VEGF (P<0.01). Especially 2-4, which was significantly better than other groups as a whole (P<0.01). The specific effects on the levels of TGF-β1 and VEGF in the serum of CPID model rats are shown in Fig.21 The comparison between the test groups is shown in Table 19 below.

[0251] Table 19

[0252]

[0253]

[0254] Note: “**” indicates P < 0.01, “*” indicates P < 0.05, and “ns” indicates P > 0.05

[0255] Comparative Example 1 Optimization of Preparation Process of Chinese Herbal Compound Extract

[0256] 1. Use ceramic membrane clarification + preparation process

[0257] The difference between this process and Example 1 is that in step (2), the extract is diluted according to 5% solid content; step (3) butterfly centrifugation is not performed; in step (5), the chromatographic column uses 30μm-C18YE; in step (6), the temperature of reduced pressure concentration is 60°C; in step (7), spray drying is used to dry the rotary evaporation liquid. The results of this process are shown in Table 20 below.

[0258] Table 20

[0259]

[0260]

[0261] (2) Total solid mass determination and process control

[0262] The raw material liquid of each step in the above step (1) was tested using the total solid mass determination method, process control method and full process fingerprint method in Example 1. Fig. 22 Table 21 is the total solid mass of the whole process; Table 22 is the value transfer of the whole process.

[0263] Table 21

[0264]

[0265] Table 22

[0266]

[0267]

[0268] It can be seen from Table 21 that the total solid mass yield of Qianjin Capsule mixed dilution-clarification filtration process is 90.14%, and the clarified liquid yield is 75.47%, with large losses; the total solid mass yield of the clarification filtration-liquid phase preparation process is 97.59%, and the high-efficiency liquid phase preparation process is stable; the total solid mass yield of the concentration process is 86.35%, and the concentration process is relatively stable; the total solid yield of the spray drying process is 64.89%, and the total solid loss is large during the spray drying process.

[0269] It can be seen from Table 22 that the transfer rates of each characteristic peak in the dilution-clarification filtration process are all greater than 80%, except for peaks 3, 4, and 5, which have poor separation; the transfer rates of each characteristic peak in the clarification filtration-liquid phase preparation process are between 55% and 130%, among which peak 1 is lost, and the high-performance liquid phase preparation process is unstable; the transfer rates of each characteristic peak in the concentration process are greater than 80%, except for peak 8, which has reduced response, and the concentration process is relatively stable; the transfer rates of each characteristic peak in the spray drying process are lower than 70%, and the spray drying process has large losses.

[0270] 2. Automatic centrifugation using butterfly centrifuge

[0271] This process uses a butterfly centrifuge for process optimization. The specific operation process is: under 9500r / min, 22L of Qianjin extract mixed solution (i.e. the mixed original sample in Example 1) and 22L of Qianjin extract combined solution (the combined original sample in Example 2) are automatically centrifuged using a butterfly centrifuge, and finally 5L of water is added to each flushing system, and the centrifuged clear liquid and centrifuged precipitate are collected, and 100ml of each sample is retained. Comparison of original sample and centrifuged sample: Take the original sample of the extract and its corresponding centrifuge liquid, and perform a small test centrifugation on them. The solid mass and yield are shown in Table 23 below.

[0272] Table 23

[0273]

[0274]

[0275] It can be seen from Table 23 that when a butterfly centrifuge is used for centrifugation, the centrifugation process is stable and the centrifugal yield is high, which can be used for process optimization of Chinese medicine compound extracts.

[0276] 3. Butterfly centrifugation + plate frame + preparation process (40% elution)

[0277] (1) Preparation of Angelica sinensis paste, Andrographis paniculata paste, Mahonia lactiflora, Atractylodes lancea paste, Rhizoma Coptidis, Radix Chrysanthemi Indici, Millettia spatholobi, and Codonopsis pilosula paste: Same as in Example 1

[0278] (2) Mixing and dilution

[0279] The same operation as in Example 1 was used to obtain the original mixed sample, and the volume of the liquid prepared by 2 injections was 2834 ml (thawed and shaken evenly before taking), and diluted to 22 L with water at 5% solid content according to the solid content to obtain a dilution, and 100 ml of the sample was retained. The relevant information of the dilution is shown in Table 24 below.

[0280] Table 24

[0281]

[0282] (3) Butterfly centrifuge

[0283] Under the condition of 9500r / min, 22L of Qianjin extract mixed solution was automatically centrifuged, and finally 5L of water was added to the system for flushing, and the centrifuged clear liquid (batch number is QJJN210118-HH-LXSQ) and centrifuged precipitate (batch number is QJJN210118-HH-LXCD) were collected, and 100ml of each sample was retained. The relevant information of the centrifugation process and the centrifuged liquid is shown in Table 25 below.

[0284] Table 25

[0285]

[0286] (4) Clarification and Filtration

[0287] Take 25L of the centrifuged clear liquid with batch number QJJN210118-HH-LXSQ, install 10 pieces of filter paper (source: Shenyang Great Wall Filter Paper Co., Ltd., model: 321) for filtration, add 5L of pure water for top washing after about 3 minutes of filtration, collect 321 filtrate, and keep 100mL of sample;

[0288] Install 10 filter paper sheets (source: Shenyang Great Wall Filter Paperboard Co., Ltd., model: 335), filter the 321 filtrate, and collect the 335 filtrate; then reinstall 16 335 filter paper sheets, circulate the filtrate in the equipment for 10 minutes and then filter again. After about 2 minutes of filtration, add appropriate amount of water for top washing, collect the final 335 filtrate, and obtain a clarified liquid with a volume of 28.4L, and keep 100mL of the sample. The specific equipment information used for clarification filtration and the related information of the clarified liquid are shown in Tables 26 and 27 below.

[0289] Table 26

[0290]

[0291] Table 27

[0292]

[0293] (5) Enrichment and capture

[0294] 28.3L of clarified liquid with batch number QJJN210120-HH-CQY was taken and divided into two batches (divided into the first injection and the second injection) for liquid phase preparation. The specific equipment information, process parameters and capture liquid related information used for enrichment and capture are shown in Tables 28 and 29 below.

[0295] (1) Balance: pure water, 4 BV;

[0296] (2) Sample loading: The clarified liquid with batch number QJJN210120-HH-CQY was loaded to obtain the load flow-through liquid, 14.15 L;

[0297] (3) Elution: pure water, 2 BV, to obtain the eluent;

[0298] (4) Elution: 40% food grade ethanol, 4 BV, to obtain eluent;

[0299] (5) Regeneration: 90% food grade ethanol, 3 BV, to obtain regeneration solution;

[0300] Table 28

[0301]

[0302] Table 29

[0303]

[0304] Capture conditions: Pump B loading for 48 minutes.

[0305] Time (minutes) A％ B％ 0 0 100 48 0 100 58 0 100 78 0 100 93 0 100

[0306] Note: The 40% elution and 90% elution at 48min to 93min in the gradient are prepared ethanol solutions, and the whole process is injected with a single pump.

[0307] Flow rate: 300mL / min;

[0308] Sample loading pressure: ①2.20MPa~4.73MPa; ②3.35MPa~4.45MPa

[0309] Washing pressure: ①4.09MPa~4.40MPa; ②4.24MPa~4.45MPa

[0310] Elution pressure: ①4.77MPa~4.78MPa; ②4.50MPa~5.21MPa

[0311] Regeneration pressure: ①3.66MPa~3.88MPa; ②4.42MPa~4.73MPa

[0312] Capture the first shot of the spectrum Fig.23 As shown, the second needle of the capture spectrum is as follows Fig.24 shown.

[0313] (5) Vacuum concentration

[0314] 18.5 L of the combined solution of batch numbers QJJN210120-HH-1-SYY and QJJN210120-HH-1-LXY was placed in a 20 L rotary evaporator at 60°C and a vacuum degree of 120 mbar, and concentrated to 1.0 L to obtain the product. 18.8 L of the combined solution of batch numbers QJJN210120-HH-2-SYY and QJJN210120-HH-2-LXY was placed in a 20 L rotary evaporator at 60°C and a vacuum degree of 120 mbar, and concentrated to 1.0 L to obtain the product. Take 9.1L of the combined solution of batch numbers QJJN210120-HH-1-XTY and QJJN210120-HH-1-ZSY and place it in a 20L rotary evaporator at 55°C and a vacuum degree of 120mbar, and concentrate to 0.8L to obtain the product. Take 9.1L of the combined solution of batch numbers QJJN210120-HH-2-XTY and QJJN210120-HH-2-ZSY and place it in a 20L rotary evaporator at 55°C and a vacuum degree of 120mbar, and concentrate to 1.0L to obtain the product. Reserve 50ml of each of the four concentrated solutions. The relevant information of each concentrated solution is shown in Tables 30 and 31 below.

[0315] Table 30

[0316]

[0317] Table 31

[0318]

[0319] (6) Spray drying

[0320] Four batches of concentrated liquids, QJJN210121-HH-1-SY+LX-NSY, QJJN210121-HH-1-XT+ZS-NSY, QJJN210121-HH-2-SY+LX-NSY, QJJN210121-HH-2-XT+ZS-NSY, were spray dried in a spray dryer at a feed rate of 14-14.5 rpm, an inlet temperature of 120°C, an atomization pressure of 2 MPa, and a fan frequency of 50 Hz. The dry powder was collected at an outlet temperature of 97-100°C to obtain the obtained product. The equipment and process parameters for spray drying are shown in Table 32 below.

[0321] Table 32

[0322]

[0323] (7) Total solid mass determination

[0324] 1) The method for determining the total mass is the same as the test method in Example 1.

[0325] 2) Overall quality results

[0326] Table 33

[0327]

[0328]

[0329] The total mass results are shown in Table 33 above. It can be seen from the table that the total solid mass yield of the clarified liquid in the centrifugal process is 75.53%, and the total solid mass of the plate and frame filtration is 80.72%. The centrifugal and plate and frame processes are unstable.

[0330] The total solid mass yield of the two-injection preparation process was 90.62%; 95.34%, and the average preparation solid recovery rate was 92.98%. There was some solid loss in the preparation process; the solid recovery in the concentration process was normal.

[0331] The total solid mass yields of the two drying processes were 76.98% and 80.77%, and the drying process was unstable.

[0332] 3) The determination method of process control is the same as the test method in Example 1.

[0333] 4) Characteristic peak value transfer results.

[0334] 5) The preparation method and chromatographic conditions of the fingerprint are the same as those in Example 1.

[0335] Table 34

[0336]

[0337] The results of the quantity transfer of the entire preparation process are shown in Table 34 above. Fig.25 It is the chromatogram of total sample, supernatant, centrifugal precipitate, clarified liquid, loading flow-through liquid, eluent, elution liquid and regeneration liquid. Fig.26 It is a comparison chart of the fingerprint spectra of the diluent, supernatant, permeate, eluate, regeneration solution, concentrate and dry powder.

[0338] As can be seen from the above table and figure, the transfer rates of each characteristic peak in the dilution-centrifugation process are all greater than 80% (except peaks 1, 2, and 6). The transfer rates of each characteristic peak in the plate and frame filtration process 1, 2, 3, 4, and 5 are significantly reduced, and the peak shape of this polarity segment has also changed significantly. The peak area of ​​characteristic peaks 6, 7, and 8 has increased, and the high-efficiency preparation process is unstable; the transfer rates of each characteristic peak in the concentration are less than 80% (except peaks 6 and 7), the concentration process is unstable and there is peak loss; the spray drying loss is large.

[0339] From the results of characteristic peak value transfer, it can be seen that the centrifugal filtration process is unstable, the high-efficiency preparation process is unstable, the concentration process is unstable, and the drying process is unstable.

[0340] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A method for preparing a Chinese medicine compound extract, characterized in that: The following steps are involved: S1. Mixing: The raw materials Qian Jinba, golden cherry root, andrographis paniculata, angelica, Mahonia, single needle, Millettia reticulata and Codonopsis pilosula are used as a combination, and the raw materials are extracted from the clear paste to obtain a clear paste mixture; S2. Dilution and centrifugation: dilute the clear paste mixture with water to obtain a dilution, centrifuge the dilution to obtain a centrifugal clear liquid and a centrifugal precipitate; S3. Clarification and filtration: The centrifuged clear liquid is filtered using a ceramic membrane, followed by washing with an alcohol solvent, the filtered liquid is collected to obtain a clarified liquid, and the unfiltered liquid is collected to obtain a retentate; S4. Enrichment and capture are performed using a high performance preparative liquid chromatography system: the clarified liquid is used for sample loading, and the sampled liquid is collected to obtain a sampled flow-through liquid; then pure water is used for elution, and the liquid eluted with pure water is collected to obtain an eluent; and then an alcohol solvent is used for elution and regeneration in sequence to obtain an eluent and a regeneration liquid; S5. respectively concentrating and freeze-drying the centrifugal precipitate, the retentate, the sample flow-through, the eluent, the elution liquid, and the regeneration liquid; The centrifugal precipitate after freeze-drying, the retentate and the regeneration solution are mixed to obtain the Chinese medicine compound extract; or mixing the freeze-dried sample flow-through and the eluent to obtain the Chinese herbal compound extract; Or the eluate after freeze-drying is the Chinese medicine compound extract.

2. The preparation method according to claim 1, characterized in that: In step S2, the centrifugation is butterfly centrifugation, the rotation speed of the butterfly centrifugation is 8000-10000 r / min, and the outlet pressure is controlled at 0.05 MPa-0.1 MPa.

3. The preparation method according to claim 1, characterized in that: In the step S3, the pore size of the ceramic membrane is 30 to 1000 nm.

4. The preparation method according to claim 1, characterized in that: In the step S4, the concentration of the alcohol solvent used for elution is 50-70%.

5. The preparation method according to claim 1, characterized in that: In the step S4, the concentration of the alcohol solvent used for the regeneration is 80-90%.

6. The preparation method according to claim 1, characterized in that: In step S4, the detection conditions of the high performance preparative liquid chromatography system are: preparation column: C18YE, 30 μm, 1500 g; flow rate: 280-350 mL / min; membrane pressure of 1.45-8.2 MPa; detection wavelength of 254 nm, 280 nm.

7. The preparation method according to claim 1, characterized in that: In the step S5, the freeze-drying temperature is ≤-40°C.

8. The preparation method according to any one of claims 1 to 7, characterized in that: In step S1, the clear paste of the raw material is extracted by the following steps: Angelica sinensis clear paste: Angelica sinensis is extracted by ethanol percolation to prepare a clear paste; or Angelica sinensis is extracted by ethanol percolation, and the extract is centrifuged to obtain the Angelica sinensis supernatant, which is the clear paste; Andrographis paniculata clear paste: extract Andrographis paniculata by ethanol reflux method to prepare clear paste; or extract Andrographis paniculata by ethanol reflux method, centrifuge the extract to obtain Andrographis paniculata supernatant, which is the clear paste; Chinese mahonia and Chinese nymphaeaceae clear paste: Chinese mahonia and Chinese nymphaeaceae are boiled and extracted twice, and the filtrates are combined after filtration to make a clear paste; Clear paste of Eleutherodactyla oxyphylla, root of golden cherry, Millettia spatholobi and Codonopsis pilosula: Boil Eleutherodactyla oxyphylla, root of golden cherry, Millettia spatholobi and Codonopsis pilosula in water once for extraction and filter. Boil the filtered residues with the residues of Andrographis paniculata and Angelica sinensis together. Combine the two filtrates, filter and concentrate to make a clear paste.

9. The Chinese herbal compound extract prepared by the method for preparing the Chinese herbal compound extract according to any one of claims 1 to 8.

10. Use of the Chinese herbal compound extract according to claim 9 in the preparation of drugs for treating anti-inflammatory or gynecological diseases.

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