A detection method for an anti-fuyan drug composition

Abstract

The present invention relates to a detection method for an anti-gynecitis pharmaceutical composition. The HPLC wavelength conversion method is used to establish a fingerprint of the anti-gynecitis pharmaceutical composition. Chemical pattern recognition analysis is simultaneously performed, and the contents of matrine, phellodendronine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride are determined, providing a reference for the overall quality evaluation and improvement of the quality control standards of the anti-gynecitis pharmaceutical composition.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a detection method for an anti-gynecitis drug composition. Background Art

[0002] Kangfuyan Capsules are a Guizhou-based, premium Miao medicinal preparation derived from a proven Miao recipe. They contain Sophora flavescens, Radix Angelicae Sinensis, Phellodendron amurense, Forsythia suspensa, Leonurus japonicus, Adzuki beans, Artemisia argyi, Radix Angelicae Sinensis, and Linderae sibiricum. They are used to treat gynecological inflammatory conditions, including damp-heat-induced pelvic inflammatory disease, vaginitis, chronic cervicitis, leucorrhea, vaginal itching, bleeding, and dysmenorrhea, with remarkable efficacy. The main ingredient in the formula, Sophora flavescens, clears heat and dampness, acts as an insecticide and diuretic, and the matrine it contains has broad-spectrum antibacterial properties. The main ingredient, Radix Angelicae Sinensis, clears heat and detoxifies, and the rutin it contains has anti-inflammatory, antibacterial, and antioxidant properties. The combination of these two herbs clears heat and dampness, detoxifies, and stops leucorrhea, specifically targeting the main symptoms of lower abdominal pain, leucorrhea, and vaginal itching. The ministerial herbs Phellodendron amurense and Forsythia suspensa clear heat, while Leonurus japonicus promotes blood circulation and regulates menstruation. The phellodendronine and berberine in Phellodendron amurense have the effects of purging heat and detoxifying, reducing fever and relieving phlegm; forsythiaside A in Forsythia suspensa has antipyretic and anti-infective pharmacological activities. The combined use of these three herbs enhances the monarch herbs' heat-clearing and detoxifying, dampness-relieving and leukorrhea-relieving properties, and can also dissipate blood stasis, relieve pain, and promote diuresis and reduce swelling. The adjuvant herbs Angelica sinensis promotes blood circulation and replenishes blood, while Linderae odoratum promotes qi circulation and relieves pain. Red beans promote diuresis and reduce swelling, detoxify and discharge pus, while Artemisia argyi dispels cold and relieves pain, warms the middle, and regulates menstruation. The combined use of these four herbs enhances the effects of promoting qi circulation, removing blood stasis, relieving pain, and promoting diuresis and reducing swelling. Angelica sinensis, Artemisia argyi, and Linderae odoratum all have a warming nature and can assist the monarch and ministerial herbs in the formula to prevent stagnation of qi and blood. The combination of these herbs, with their cold and warm properties, works harmoniously and effectively, relieving all symptoms.

[0003] Kangfuyan Capsules are a patented Miao medicine preparation and have been included in the National Medical Insurance Catalogue. However, existing quality standards only cover thin-layer chromatography identification of properties and quantitative determination of matrine in Sophora flavescens, which cannot fully evaluate the overall quality of Kangfuyan Capsules. Traditional Chinese medicine fingerprints offer advantages in evaluating the quality of traditional Chinese medicines and ensuring their consistent quality, making them a viable technique for evaluating their overall quality. Principal component analysis in chemical pattern recognition allows for rapid and visual data identification, while cluster analysis can determine sample batch stability. Combined with fingerprints, these methods can reveal patterns among the complex components of traditional Chinese medicines.

[0004] In response to the above-mentioned shortcomings of the existing technology, the invention team conducted a large number of experiments and used HPLC fingerprint analysis to determine the HPLC fingerprint of the anti-gynecitis drug composition. At the same time, chemical pattern recognition analysis was performed, and the contents of matrine, phellodendronine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride were determined to provide a reference for the overall quality evaluation and improvement of quality control standards of the anti-gynecitis drug composition. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for establishing a fingerprint of an anti-gynecitis drug composition.

[0006] Another object of the present invention is to provide a method for chemical pattern recognition analysis of anti-gynecitis pharmaceutical compositions.

[0007] Another object of the present invention is to provide a method for determining the content of an anti-gynecitis pharmaceutical composition.

[0008] The prescription of the anti-gynecitis pharmaceutical composition of the present invention is: 250g of Sophora flavescens, 250g of Radix Angelicae Sinensis, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Red Bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae strychnos.

[0009] The preparation method of the anti-gynecitis pharmaceutical composition of the present invention is as follows: taking 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Red Bean, and 15g of Forsythia suspensa from the above nine ingredients, grinding them into fine powder, sieving, and setting aside; adding water to the remaining Sophora flavescens, Phellodendron amurense, Red Bean, Forsythia suspensa and the other five ingredients including Leonurus japonicus, decocting twice, the first time for 2 hours and the second time for 1 hour, combining the decoctions, filtering, and concentrating the filtrate to a thick paste with a relative density of 1.31 to 1.35 at 60 to 80°C, adding the above fine powder, mixing, drying, and grinding into fine powder to obtain the anti-gynecitis pharmaceutical composition.

[0010] The detection method of the present invention includes fingerprint establishment, chemical pattern recognition analysis and content determination.

[0011] The method for establishing the fingerprint is:

[0012] (1) Preparation of test solution

[0013] Take 0.2-0.8 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 5-15 mL of 60-80% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 10-60 minutes. Let it cool, weigh it again, make up the reduced mass with 60-80% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0014] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test solution;

[0015] (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively;

[0016] (4) Preparation of mixed reference solution

[0017] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0018] (5) Chromatographic conditions and system suitability test

[0019] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.05-0.15% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0020] (6) Determination method

[0021] Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

[0022] Preferably, the fingerprint is established by:

[0023] (1) Preparation of test solution

[0024] Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 30 minutes. Let it cool, weigh it again, make up the reduced mass with 70% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0025] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the same preparation method as the test solution;

[0026] (3) Preparation of negative sample solution: prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae in the same manner as the test sample solution;

[0027] (4) Preparation of mixed reference solution

[0028] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0029] (5) Chromatographic conditions and system suitability test

[0030] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.1% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0031] (6) Determination method

[0032] Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

[0033] The fingerprint spectrum contains 29 common peaks, and a total of 5 peaks are identified, namely matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0034] Among the 29 common peaks, peak 3 is matrine; peak 14 is phellodendrine hydrochloride; peak 20 is rutin; peak 22 is forsythiaside A; and peak 28 is berberine hydrochloride.

[0035] The chemical pattern recognition analysis method includes cluster analysis and principal component analysis.

[0036] The cluster analysis method is as follows: 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 are imported into statistical software, and an inter-group linkage system clustering method is adopted to perform cluster analysis with squared Euclidean distance as the measure. When the distance is 5, the 11 batches of samples can be clustered into 2 categories: among them, samples numbered S1 and S9 are clustered into one category, and samples numbered S10 and S11 are clustered into one category.

[0037] The principal component analysis method is as follows: 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 are imported into analysis software for principal component analysis, and the initial eigenvalue of the principal component is greater than 1 as the judgment basis for the quality evaluation of the anti-gynecitis drug composition. The initial eigenvalues of the first four principal components are greater than 1, and the cumulative variance contribution rate is 93.259%, indicating that these four principal components can cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples; the initial eigenvalue of principal component 1 is the largest, which is 17.377, and the information covered is the richest, corresponding to components 3, 5-8, 11-16, and 18-29; the initial eigenvalue of principal component 2 is 5.643, corresponding to Components 1, 2, 4, and 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the peaks with larger score coefficients of principal component 1 were matrine 3, phellodendrine hydrochloride 6, 8, and 14, forsythiaside A 15, 18, and 22, and berberine hydrochloride 25, 26, 27, and 28. It was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into two categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the results of cluster analysis.

[0038] The method for the content determination is:

[0039] (1) Preparation of test solution

[0040] Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 30 minutes. Let it cool, weigh it again, make up the reduced mass with 70% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0041] (2) Preparation of mixed reference solution

[0042] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0043] (3) Chromatographic conditions and system suitability test

[0044] Column: ACE C 18-AR, chromatographic column specifications: 250mm×4.6mm, 5μm; mobile phase: 0.1% phosphoric acid as mobile phase A, mobile phase B is acetonitrile, gradient elution; the gradient elution is specifically: 0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15 %B→20%B; 65-85 min, 20%B→35%B; 85-95 min, 35%B→70%B; Column temperature: 30°C; Detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; Flow rate: 1 ml / min; Resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000.

[0045] (4) Determination method

[0046] Accurately pipette 10 μl of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0047] The fingerprint spectrum is used for detecting anti-gynecitis drug compositions and preparations containing Radix Sophorae Flavescentis, Radix Angelicae Sinensis, Radix Phellodendri and Fructus Forsythiae.

[0048] Anti-gynecitis drug composition: the batch numbers are 20200503, 20200603, 20210108, 20210302, 20191208, 20191109, 20191023, 20200101, 20200202, 20200118, and 20200621, and the numbers are "S1-S11" respectively.

[0049] Beneficial effects:

[0050] 1. The present invention investigates the effects of different extraction methods (reflux, ultrasound), different volumes of methanol solution (30%, 50%, 70%, 100%) and different extraction times (30, 45, 60, 90, 120 min) on the extraction effect. The results show that 70% methanol ultrasound for 30 min is the optimal extraction condition for the preparation.

[0051] 2. The present invention investigated the separation effects of various mobile phase systems, including methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, methanol-0.1% formic acid, and acetonitrile-0.1% formic acid. The results showed that the separation effect was best when the mobile phase system was acetonitrile-0.1% phosphoric acid.

[0052] 3. The present invention performed a full-wavelength scan on the sample components and finally determined the optimal elution procedure and detection wavelength (220nm: 0~80min; 254nm: 81~95min) by observing the number, peak shape and response degree of each chromatographic peak in the 3D spectrum.

[0053] 4. The present invention established a fingerprint of an anti-gynecitis pharmaceutical composition using HPLC wavelength conversion, identifying 29 common peaks and 5 chemical components. Comparison of the fingerprints of 11 sample batches with the control fingerprint revealed similarities greater than 0.99, indicating that the preparation had relatively stable quality. Cluster analysis results showed that, at a distance of 5, the 11 sample batches could be clustered into two categories: S1 and S9 in one category, and S10 and S11 in the other. The principal component analysis results were consistent with the cluster analysis results, with the cumulative variance contribution of the first four principal components being 93.259%, indicating that these four principal components encompass 93.259% of the information from the 29 common components. Peaks 3 (matrine), 6, 8, 14 (corticosteroid hydrochloride), 15, 18, 22 (forsythiaside A), and 25, 26, 27, and 28 (berberine hydrochloride) exhibited significant principal component 1 scores, suggesting that the presence of multiple components synergistically influences the quality of the anti-gynecitis pharmaceutical composition. The main components of S10 and S11 are different from those of other batches, indicating that in the production process of anti-gynecitis drug compositions, attention should be paid not only to the quality of the control agents but also to the quality control of the raw medicinal materials, such as standardizing the origin of the medicinal materials, the harvesting season, and the quality control standards of the medicinal material slices. At the same time, it is necessary to optimize the control parameters of the preparation production process to ensure the stability of the quality of the anti-gynecitis drug compositions and the consistency of the clinical efficacy.

[0054] 5. This study demonstrates that matrine has anti-inflammatory and antioxidant effects, phellodendrine hydrochloride has antibacterial and oxidative stress protection effects, rutin has anti-inflammatory and immunomodulatory effects, forsythiaside A has significant anti-inflammatory and antiviral activities, and berberine hydrochloride has antibacterial and anti-inflammatory effects. The pharmacological effects of these five ingredients are closely related to the functions and indications of the anti-gynecitis drug composition. Therefore, this study identified these five ingredients as the target assay components of the preparation. Content determination results showed differences in sample content between different batches. It is recommended that these five active ingredients be included in the quality standards for the anti-gynecitis drug composition to improve its overall quality.

[0055] 6. The HPLC fingerprint and content determination methods established in this study are stable and reliable. Combined with chemical pattern recognition analysis, they can provide a reference basis for improving the quality standards of anti-gynecitis drug compositions and provide applications in the detection of preparations containing Sophora flavescens, Radix Angelicae Dahuricae, Phellodendron amurense, and Forsythia suspensa. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 HPLC comparison chart of different extraction methods;

[0057] Figure 2 HPLC comparison chart of different methanol volumes;

[0058] Figure 3 Comparison of investigation results at different extraction times;

[0059] Figure 4 Comparison chart of HPLC with different mobile phases (water as aqueous phase);

[0060] Figure 5 Comparison chart of HPLC with different mobile phases (using 0.1% formic acid water as the aqueous phase);

[0061] Figure 6 HPLC comparison chart of different mobile phases (using 0.1% phosphoric acid water as the aqueous phase);

[0062] Figure 7 Full wavelength scan diagram;

[0063] Figure 8 HPLC fingerprints (S1-S11) and control fingerprints (R) of 11 batches of anti-gynecitis drug compositions;

[0064] Figure 9 HPLC chart of test solution (3. matrine; 14. phellodendronine hydrochloride; 20. rutin; 22. forsythiaside A; 28. berberine hydrochloride);

[0065] Figure 10 HPLC chart of mixed reference substances (3. matrine; 14. phellodendronine hydrochloride; 20. rutin; 22. forsythiaside A; 28. berberine hydrochloride);

[0066] Figure 11 HPLC profiles of single herbal medicine pieces and test sample solutions (A Sophora flavescens; B Radix Angelicae Sinensis; C Phellodendron amurense; D Forsythia suspensa; E Leonurus japonicus; F Angelica sinensis; G Linderae umbellatae; H Artemisia argyi; I Adzuki beans; J Sample);

[0067] Figure 12 Cluster analysis dendrogram of 11 batches of anti-gynecitis drug compositions;

[0068] Figure 13 Principal component analysis diagram of 11 batches of anti-gynecitis drug compositions;

[0069] Figure 14 Spectrum of mixed reference solution;

[0070] Figure 15 Spectrum of test solution (S1);

[0071] Figure 16 Lack of the spectrum of the negative control solution of Sophora flavescens;

[0072] Figure 17Spectrum of negative control solution lacking galangal;

[0073] Figure 18 The spectrum of the negative control solution of Phellodendron amurense is missing;

[0074] Figure 19 Lack of Forsythia suspensa negative control solution spectrum. DETAILED DESCRIPTION

[0075] Example 1

[0076] Prescription: 250g of Sophora flavescens, 250g of Radix Angelicae Sinensis, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Adzuki Bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae scutellariae; the preparation method of the anti-gynecitis pharmaceutical composition is: take 160g of Sophora flavescens, 90g of Phellodendron chinense, 15g of Adzuki Bean, and 15g of Forsythia suspensa from the above nine ingredients.

[0077] Preparation method: Take 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Adzuki Bean, and 15g of Forsythia suspensa, grind them into fine powder, sieve, and set aside; add water to the remaining Sophora flavescens, Phellodendron amurense, Adzuki Bean, Forsythia suspensa, and the remaining five herbs including Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 at 60-80°C, add the above-mentioned fine powders, mix well, dry, and grind into fine powder.

[0078] Fingerprint:

[0079] (1) Preparation of test solution

[0080] Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 30 minutes. Let it cool, weigh it again, make up the reduced mass with 70% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0081] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test solution;

[0082] (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively;

[0083] (4) Preparation of mixed reference solution

[0084] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0085] (5) Chromatographic conditions and system suitability test

[0086] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.1% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0087] (6) Determination method

[0088] Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

[0089] Chemical pattern recognition analysis:

[0090] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into statistical software, and cluster analysis was performed using the inter-group linkage system clustering method with the squared Euclidean distance as the measure. When the distance was 5, the 11 batches of samples could be clustered into two categories: samples numbered S1 to S9 were clustered into one category, and samples numbered S10 and S11 were clustered into one category.

[0091] Principal Component Analysis:

[0092] The peak areas of 29 common peaks of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into the analysis software for principal component analysis. The initial eigenvalue of the principal component>1 was used as the judgment basis for the quality evaluation of the anti-gynecitis capsule. The initial eigenvalues of the first four principal components were>1, and the cumulative variance contribution rate was 93.259%, indicating that these four principal components could cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples. The initial eigenvalue of principal component 1 was the largest, which was 17.377, and the information covered was the richest, corresponding to components 3, 5-8, 11-16, and 18-29. The initial eigenvalue of principal component 2 was 5.643, corresponding to components 1, 2, and 4. , 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the peaks with larger score coefficients of principal component 1 were matrine 3, phellodendrine hydrochloride 6, 8, 14, forsythiaside A 15, 18, 22, berberine hydrochloride 25, 26, 27, 28, and it was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into two categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the results of cluster analysis.

[0093] Assay:

[0094] (1) Preparation of test solution

[0095] Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 30 minutes. Let it cool, weigh it again, make up the reduced mass with 70% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0096] (2) Preparation of mixed reference solution

[0097] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0098] (3) Chromatographic conditions and system suitability test

[0099] Column: ACE C 18-AR, chromatographic column specifications: 250mm×4.6mm, 5μm; mobile phase: 0.1% phosphoric acid as mobile phase A, mobile phase B is acetonitrile, gradient elution; the gradient elution is specifically: 0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15 %B→20%B; 65-85 min, 20%B→35%B; 85-95 min, 35%B→70%B; Column temperature: 30°C; Detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; Flow rate: 1 ml / min; Resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000.

[0100] (4) Determination method

[0101] Accurately pipette 10ul of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0102] Example 2

[0103] Prescription: 250g of Sophora flavescens, 250g of Radix Angelicae Sinensis, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Adzuki Bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae scutellariae; the preparation method of the anti-gynecitis pharmaceutical composition is: take 160g of Sophora flavescens, 90g of Phellodendron chinense, 15g of Adzuki Bean, and 15g of Forsythia suspensa from the above nine ingredients.

[0104] Preparation method: Take 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Adzuki Bean, and 15g of Forsythia suspensa, grind them into fine powder, sieve, and set aside; add water to the remaining Sophora flavescens, Phellodendron amurense, Adzuki Bean, Forsythia suspensa, and the remaining five herbs including Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 at 60-80°C, add the above-mentioned fine powders, mix well, dry, and grind into fine powder.

[0105] Fingerprint:

[0106] (1) Preparation of test solution

[0107] Take 0.2 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 5 mL of 60% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 10 minutes. Let it cool, weigh it again, make up the reduced mass with 60% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0108] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test solution;

[0109] (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively;

[0110] (4) Preparation of mixed reference solution

[0111] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0112] (5) Chromatographic conditions and system suitability test

[0113] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0114] (6) Determination method

[0115] Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

[0116] Chemical pattern recognition analysis:

[0117] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into statistical software, and cluster analysis was performed using the inter-group linkage system clustering method with the squared Euclidean distance as the measure. When the distance was 5, the 11 batches of samples could be clustered into 2 categories: S1 and S9 were clustered into one category, and S10 and S11 were clustered into one category.

[0118] Principal Component Analysis:

[0119] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into the analysis software for principal component analysis. The initial eigenvalue of the principal component>1 was used as the judgment basis for the quality evaluation of the anti-gynecitis drug composition. The initial eigenvalues of the first four principal components were>1, and the cumulative variance contribution rate was 93.259%, indicating that these four principal components could cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples; the initial eigenvalue of principal component 1 was the largest, which was 17.377, and the information covered was the richest, corresponding to components 3, 5-8, 11-16, and 18-29; the initial eigenvalue of principal component 2 was 5.643, corresponding to components 1, 2, 4, 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the main component 1 had a larger score coefficient, including peak 3 for matrine, 6, 8, 14 for phellodendrine hydrochloride, 15, 18, 22 for forsythiaside A, and 25, 26, 27, 28 for berberine hydrochloride. It was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into 2 categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the cluster analysis results.

[0120] Assay:

[0121] (1) Preparation of test solution

[0122] Take 0.2 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 60% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 10 minutes. Let it cool, weigh it again, make up the reduced mass with 60% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0123] (2) Preparation of mixed reference solution

[0124] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0125] (3) Chromatographic conditions and system suitability test

[0126] Column: ACE C 18-AR, chromatographic column specifications: 250mm×4.6mm, 5μm; mobile phase: 0.05% phosphoric acid as mobile phase A, mobile phase B is acetonitrile, gradient elution; the gradient elution is specifically: 0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15 %B→20%B; 65-85 min, 20%B→35%B; 85-95 min, 35%B→70%B; Column temperature: 30°C; Detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; Flow rate: 1 ml / min; Resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000.

[0127] (4) Determination method

[0128] Accurately pipette 10 μl of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0129] Example 3

[0130] Prescription: 250g of Sophora flavescens, 250g of Radix Angelicae Sinensis, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Adzuki Bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae scutellariae; the preparation method of the anti-gynecitis pharmaceutical composition is: take 160g of Sophora flavescens, 90g of Phellodendron chinense, 15g of Adzuki Bean, and 15g of Forsythia suspensa from the above nine ingredients.

[0131] Preparation method: Take 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Adzuki Bean, and 15g of Forsythia suspensa, grind them into fine powder, sieve, and set aside; add water to the remaining Sophora flavescens, Phellodendron amurense, Adzuki Bean, Forsythia suspensa, and the remaining five herbs including Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 at 60-80°C, add the above-mentioned fine powders, mix well, dry, and grind into fine powder.

[0132] Fingerprint:

[0133] (1) Preparation of test solution

[0134] Take 0.8 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 15 mL of 80% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 60 min. Let it cool, weigh it again, make up the reduced mass with 80% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0135] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test solution;

[0136] (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively;

[0137] (4) Preparation of mixed reference solution

[0138] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0139] (5) Chromatographic conditions and system suitability test

[0140] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.15% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0141] (6) Determination method

[0142] Accurately pipette 10ul of the above-mentioned mixed reference solution, test solution, single medicinal sample solution and negative sample solution respectively and inject them into high performance liquid chromatography for determination, calibrate the common peaks and obtain the fingerprint of the anti-gynecitis drug composition.

[0143] Chemical pattern recognition analysis:

[0144] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into statistical software, and cluster analysis was performed using the inter-group linkage system clustering method with the squared Euclidean distance as the measure. When the distance was 5, the 11 batches of samples could be clustered into 2 categories: S1 and S9 were clustered into one category, and S10 and S11 were clustered into one category.

[0145] Principal Component Analysis:

[0146] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into the analysis software for principal component analysis. The initial eigenvalue of the principal component>1 was used as the judgment basis for the quality evaluation of the anti-gynecitis drug composition. The initial eigenvalues of the first four principal components were>1, and the cumulative variance contribution rate was 93.259%, indicating that these four principal components could cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples; the initial eigenvalue of principal component 1 was the largest, which was 17.377, and the information covered was the richest, corresponding to components 3, 5-8, 11-16, and 18-29; the initial eigenvalue of principal component 2 was 5.643, corresponding to components 1, 2, 4, 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the main component 1 had a larger score coefficient, including peak 3 for matrine, 6, 8, 14 for phellodendrine hydrochloride, 15, 18, 22 for forsythiaside A, and 25, 26, 27, 28 for berberine hydrochloride. It was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into 2 categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the cluster analysis results.

[0147] Assay:

[0148] (1) Preparation of test solution

[0149] Take 0.8 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 15 mL of 80% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 60 min. Let it cool, weigh it again, make up the reduced mass with 80% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0150] (2) Preparation of mixed reference solution

[0151] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0152] (3) Chromatographic conditions and system suitability test

[0153] Column: ACE C 18-AR, chromatographic column specifications: 250mm×4.6mm, 5μm; mobile phase: 0.15% phosphoric acid as mobile phase A, mobile phase B is acetonitrile, gradient elution; the gradient elution is specifically: 0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15 %B→20%B; 65-85 min, 20%B→35%B; 85-95 min, 35%B→70%B; Column temperature: 30°C; Detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; Flow rate: 1 ml / min; Resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000.

[0154] (4) Determination method

[0155] Accurately pipette 10 μl of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0156] Example 4

[0157] Prescription: 250g of Sophora flavescens, 250g of Radix Angelicae Sinensis, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Adzuki Bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae scutellariae; the preparation method of the anti-gynecitis pharmaceutical composition is: take 160g of Sophora flavescens, 90g of Phellodendron chinense, 15g of Adzuki Bean, and 15g of Forsythia suspensa from the above nine ingredients.

[0158] Preparation method: Take 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Adzuki Bean, and 15g of Forsythia suspensa, grind them into fine powder, sieve, and set aside; add water to the remaining Sophora flavescens, Phellodendron amurense, Adzuki Bean, Forsythia suspensa, and the remaining five herbs including Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 at 60-80°C, add the above-mentioned fine powders, mix well, dry, and grind into fine powder.

[0159] Fingerprint:

[0160] (1) Preparation of test solution

[0161] Take 0.3 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 8 mL of 65% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 20 minutes. Let it cool, weigh it again, make up the reduced mass with 65% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0162] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test solution;

[0163] (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively;

[0164] (4) Preparation of mixed reference solution

[0165] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0166] (5) Chromatographic conditions and system suitability test

[0167] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.08% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0168] (6) Determination method

[0169] Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

[0170] Chemical pattern recognition analysis:

[0171] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into statistical software, and cluster analysis was performed using the inter-group linkage system clustering method with the squared Euclidean distance as the measure. When the distance was 5, the 11 batches of samples could be clustered into 2 categories: S1 and S9 were clustered into one category, and S10 and S11 were clustered into one category.

[0172] Principal Component Analysis:

[0173] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into the analysis software for principal component analysis. The initial eigenvalue of the principal component>1 was used as the judgment basis for the quality evaluation of the anti-gynecitis drug composition. The initial eigenvalues of the first four principal components were>1, and the cumulative variance contribution rate was 93.259%, indicating that these four principal components could cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples; the initial eigenvalue of principal component 1 was the largest, which was 17.377, and the information covered was the richest, corresponding to components 3, 5-8, 11-16, and 18-29; the initial eigenvalue of principal component 2 was 5.643, corresponding to components 1, 2, 4, 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the main component 1 had a larger score coefficient, including peak 3 for matrine, 6, 8, 14 for phellodendrine hydrochloride, 15, 18, 22 for forsythiaside A, and 25, 26, 27, 28 for berberine hydrochloride. It was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into 2 categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the cluster analysis results.

[0174] Assay:

[0175] (1) Preparation of test solution

[0176] Take 0.3 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 8 mL of 65% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 20 minutes. Let it cool, weigh it again, make up the reduced mass with 65% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0177] (2) Preparation of mixed reference solution

[0178] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0179] (3) Chromatographic conditions and system suitability test

[0180] Column: ACE C 18-AR, chromatographic column specifications: 250mm×4.6mm, 5μm; mobile phase: 0.08% phosphoric acid as mobile phase A, mobile phase B is acetonitrile, gradient elution; the gradient elution is specifically: 0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15 %B→20%B; 65-85 min, 20%B→35%B; 85-95 min, 35%B→70%B; Column temperature: 30°C; Detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; Flow rate: 1 ml / min; Resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000.

[0181] (4) Determination method

[0182] Accurately pipette 10 μl of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0183] Example 5

[0184] Prescription: 250g of Sophora flavescens, 250g of Radix Angelicae Sinensis, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Adzuki Bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae scutellariae; the preparation method of the anti-gynecitis pharmaceutical composition is: take 160g of Sophora flavescens, 90g of Phellodendron chinense, 15g of Adzuki Bean, and 15g of Forsythia suspensa from the above nine ingredients.

[0185] Preparation method: Take 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Adzuki Bean, and 15g of Forsythia suspensa, grind them into fine powder, sieve, and set aside; add water to the remaining Sophora flavescens, Phellodendron amurense, Adzuki Bean, Forsythia suspensa, and the remaining five herbs including Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 at 60-80°C, add the above-mentioned fine powders, mix well, dry, and grind into fine powder.

[0186] Fingerprint:

[0187] (1) Preparation of test solution

[0188] Take 0.6 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 12 mL of 75% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 40 min. Let it cool, weigh it again, make up the reduced mass with 75% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0189] (2) Preparation of single slice sample solution: Prepare single slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test solution;

[0190] (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively;

[0191] (4) Preparation of mixed reference solution

[0192] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0193] (5) Chromatographic conditions and system suitability test

[0194] Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.12% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min;

[0195] (6) Determination method

[0196] Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

[0197] Chemical pattern recognition analysis:

[0198] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into statistical software, and cluster analysis was performed using the inter-group linkage system clustering method with the squared Euclidean distance as the measure. When the distance was 5, the 11 batches of samples could be clustered into 2 categories: S1 and S9 were clustered into one category, and S10 and S11 were clustered into one category.

[0199] Principal Component Analysis:

[0200] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 were imported into the analysis software for principal component analysis. The initial eigenvalue of the principal component>1 was used as the judgment basis for the quality evaluation of the anti-gynecitis drug composition. The initial eigenvalues of the first four principal components were>1, and the cumulative variance contribution rate was 93.259%, indicating that these four principal components could cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples; the initial eigenvalue of principal component 1 was the largest, which was 17.377, and the information covered was the richest, corresponding to components 3, 5-8, 11-16, and 18-29; the initial eigenvalue of principal component 2 was 5.643, corresponding to components 1, 2, 4, 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the main component 1 had a larger score coefficient, including peak 3 for matrine, 6, 8, 14 for phellodendrine hydrochloride, 15, 18, 22 for forsythiaside A, and 25, 26, 27, 28 for berberine hydrochloride. It was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into 2 categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the cluster analysis results.

[0201] Assay:

[0202] (1) Preparation of test solution

[0203] Take 0.6 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 12 mL of 75% methanol, weigh the mass, and sonicate at a power of 250 W and a frequency of 40 kHz for 40 min. Let it cool, weigh it again, make up the reduced mass with 75% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution;

[0204] (2) Preparation of mixed reference solution

[0205] Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively;

[0206] (3) Chromatographic conditions and system suitability test

[0207] Column: ACE C 18-AR, chromatographic column specifications: 250mm×4.6mm, 5μm; mobile phase: 0.12% phosphoric acid as mobile phase A, mobile phase B is acetonitrile, gradient elution; the gradient elution is specifically: 0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15 %B→20%B; 65-85 min, 20%B→35%B; 85-95 min, 35%B→70%B; Column temperature: 30°C; Detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; Flow rate: 1 ml / min; Resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000.

[0208] (4) Determination method

[0209] Accurately pipette 10 μl of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

[0210] In order to further verify the effectiveness of the present invention, the inventors conducted a series of verification experiments, which are excerpted as follows:

[0211] Experimental example

[0212] 1. Materials

[0213] 1.1 Main instruments

[0214] An Ultimate 3000 HPLC instrument (Thermo Fisher Scientific, China) was used, an MS105DU electronic analytical balance (Mettler Toledo, Switzerland) was used, and a KQ-250DA ultrasonic cleaner was used (Kunshan Ultrasonic Instrument Co., Ltd.).

[0215] 1.2 Drugs and Reagents

[0216] The anti-gynecitis drug composition (batch numbers are 20200503, 20200603, 20210108, 20210302, 20191208, 20191109, 20191023, 20200101, 20200202, 20200118, 20200621, numbered S1-S11) was purchased from Guizhou Yuancheng Pharmaceutical Co., Ltd.; Sophora flavescens, Radix Angelicae Dahuricae, Phellodendron amurense, Fructus Forsythiae, Leonurus japonicus, Adzuki bean, Folium Artemisiae Argyi, Radix Angelicae Sinensis, and Linderae Root slices were purchased from Guizhou Yuancheng Pharmaceutical Co., Ltd. Provided by Yuancheng Pharmaceutical Co., Ltd. and identified by Associate Professor Long Qingde of the School of Pharmacy of Guizhou Medical University; phellodendronine hydrochloride, forsythiaside A, matrine, berberine hydrochloride, and rutin reference substances (batch numbers wkq21031209, wkq21040902, wkq21030302, wkq21022004, and wkq20030203, with a purity of not less than 98%) were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd. Acetonitrile was chromatographically pure, other reagents were analytically pure, and water was purified water.

[0217] 2 Establishment of fingerprint method

[0218] [Prescription] Sophora flavescens 250g, Radix Angelicae Sinensis 250g, Phellodendron amurense 150g, Forsythia suspensa 50g, Leonurus japonicus 30g, Adzuki bean 30g, Artemisia argyi 30g, Angelica sinensis 30g, Linderae scutellariae 30g.

[0219] Preparation Method: Take 160g of Sophora flavescens, 90g of Phellodendron amurense, 15g of Adzuki Bean, and 15g of Forsythia suspensa from the above nine ingredients, grind them into fine powder, sieve, and set aside. Add water to the remaining Sophora flavescens, Phellodendron amurense, Adzuki Bean, Forsythia suspensa, and the remaining five ingredients including Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 (60-80°C). Add the above fine powder, mix well, dry, and grind into fine powder.

[0220] 2.1 Experimental investigation of fingerprint method

[0221] 2.1.1 Investigation of the preparation method of the test solution

[0222] 2.1.1.1 Investigation of extraction methods

[0223] Weigh 2 portions of 0.5 g of the anti-gynecitis drug composition in parallel, accurately weigh them, place them in a round-bottomed conical flask with a stopper, accurately add 10 mL of 70% methanol, weigh the mass, extract one sample by ultrasonic extraction (power 250 W, frequency 40 KHz) for 30 min, and extract the other by reflux extraction for 30 min. After taking out, cool it and weigh the mass again. Make up the reduced mass with 70% methanol, shake it well, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution. Take 10 μL of the test solution for sampling and determination respectively. The results of the investigation of the extraction method are shown in Figure 1 .

[0224] Result: By Figure 1 It can be seen that there is no significant difference in the number of peaks, peak separation, peak shape, etc. obtained by reflux extraction and ultrasonic extraction, but ultrasonic extraction is simpler to operate than reflux extraction, so ultrasonic extraction is used for extraction.

[0225] 2.1.1.2 Volume Study of Methanol Extraction Solvent

[0226] Weigh 0.5 g of 4 portions of anti-gynecitis drug composition in parallel, accurately weigh them, place them in a stoppered conical flask, accurately add 10 mL of 30%, 50%, 70%, and 100% methanol respectively, weigh the mass, use ultrasonic extraction (power 250W, frequency 40KHz) for 30 minutes, cool after taking out, weigh the mass again, and then make up the reduced mass with 30%, 50%, 70%, and 100% methanol respectively, shake well, filter with a 0.45μm microporous filter membrane, and take the filtrate to obtain the test solution. Take 10μL of the test solution for sampling and determination. The volume investigation results of the extraction solvent methanol are shown in Figure 2 .

[0227] Result: By Figure 2 It can be seen that the chromatographic peaks extracted by 30% and 100% methanol are fewer and the response values are not high. The number of chromatographic peaks extracted by 50% and 70% methanol is basically the same, but using 70% methanol as the extraction solvent can significantly improve the separation degree and response value of the chromatographic peaks (especially the chromatographic peaks of 40min to 60min), so 70% methanol is used as the best extraction solvent.

[0228] 2.1.1.3 Investigation of extraction time

[0229] Weigh 5 portions of 0.5 g of the anti-gynecitis drug composition in parallel, accurately weigh them, place them in a stoppered conical flask, accurately add 10 mL of 70% methanol to each, weigh the mass, and extract them by ultrasound (power 250 W, frequency 40 KHz) for 30, 45, 60, 90, and 120 min respectively. After taking them out and cooling them, weigh them again, and then make up the reduced mass with 70% methanol, shake them well, filter them with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution. Take 10 μL of the test solution for sampling and determination. The results of the investigation of the extraction time are shown in Figure 3 .

[0230] Result: By Figure 3 It can be seen that there is no significant difference in the number of chromatographic peaks, response values, peak areas and other parameters extracted at the five time points of 30, 45, 60, 90 and 120 minutes. In order to save cost and time, 30 minutes of extraction was selected as the optimal extraction time.

[0231] 2.1.1.4 Confirmation of the test solution preparation method

[0232] Based on the above experimental results, it was finally determined that the preparation of the test solution of the anti-gynecitis drug composition was as follows: 0.5 g of the anti-gynecitis drug composition was taken, accurately weighed, placed in a stoppered conical flask, 10 mL of 70% methanol was accurately added, the mass was weighed, ultrasonic (power 250 W, frequency 40 kHz) for 30 min, cooled, weighed again, and the reduced mass was supplemented with 70% methanol. Shake well, filter with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0233] 2.1.2 Investigation of mobile phase

[0234] In this study, water, 0.1% formic acid water, and 0.1% phosphoric acid water were used as aqueous phases, and methanol and acetonitrile were used as organic phases to investigate the mobile phase system. The results of mobile phase screening are shown in Figure 4 、 Figure 5 、 Figure 6 .

[0235] Result: By Figure 4 、 Figure 5 、 Figure 6 It can be seen that acetonitrile can extract more chromatographic peaks in the organic phase, and the baseline is relatively stable. Figure 5 From the above, the number of chromatographic peaks extracted with acetonitrile as the organic phase is relatively large, but the baseline cannot be guaranteed to be stable when 0.1% formic acid water is used as the aqueous phase. Figure 6 From the above analysis, it can be seen that using 0.1% phosphoric acid water as the aqueous phase can correct the phenomenon of unstable baseline, but the number of chromatographic peaks extracted using methanol as the organic phase is small. Therefore, acetonitrile-0.1% phosphoric acid solution is used as the optimal mobile phase system. Under this mobile phase, the chromatographic peak baseline is stable, the number of chromatographic peaks is large, the peak shape is good, and the chromatographic peak response value is high.

[0236] 2.1.3 Selection of detection wavelength

[0237] Take the test solution of the anti-gynecitis drug composition and use the DAD detector to perform full wavelength scanning at a wavelength of 200-400nm. Due to the complexity of the composition of traditional Chinese medicine, and the fact that the components may interact with each other after being made into preparations, making the composition more complex, different chemical components have maximum absorption wavelengths at different wavelengths. Combined with the chromatogram of the single medicinal material, the components in each medicinal material can be attributed to the greatest extent, and the main pharmacologically active ingredients should be presented as completely as possible. Based on the above comprehensive factors, the wavelength conversion method was used for determination, and the detection wavelength was finally determined to be (220nm: 0~80min; 254nm: 81~95min). The results are shown in Figure 7 .

[0238] Results: This study initially investigated the effects of different extraction methods (reflux, sonication), different volumes of methanol (30%, 50%, 70%, 100%), and different extraction times (30, 45, 60, 90, and 120 min) on the extraction efficiency. The optimal extraction conditions for this preparation were determined to be 70% methanol with sonication for 30 min. Furthermore, this study also investigated the separation of components using various mobile phase systems, including methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, methanol-0.1% formic acid, and acetonitrile-0.1% formic acid. Acetonitrile-0.1% phosphoric acid was found to provide the best separation. Furthermore, full-wavelength scanning was performed on the sample components. By observing the number, peak shape, and response of each chromatographic peak in the 3D chromatogram, the optimal elution schedule and detection wavelength (220 nm: 0-80 min; 254 nm: 81-95 min) were determined.

[0239] 2.2 Determination of fingerprint method

[0240] 2.2.1 Preparation of test solution

[0241] Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, ultrasonicate (power 250 W, frequency 40 kHz) for 30 min, let cool, weigh the mass again, make up the reduced mass with 70% methanol, shake well, filter with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0242] 2.2.2 Preparation of single herbal medicine slice solutions and negative sample solutions

[0243] Single-piece sample solutions and negative sample solutions lacking Sophora flavescens, Radix Angelicae Dahuricae, Phellodendron chinense, and Fructus Forsythiae were prepared in the same manner according to the prescription ratio (the components measured in the content determination were derived from Sophora flavescens, Radix Angelicae Dahuricae, Phellodendron chinense, and Fructus Forsythiae, respectively).

[0244] 2.2.3 Preparation of mixed reference solution

[0245] Accurately weigh appropriate amounts of matrine, phellodendronine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of the above five components of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively.

[0246] 2.2.4 Chromatographic conditions

[0247] ACE C 18-AR (250mm×4.6mm, 5μm) chromatographic column, acetonitrile (B)-0.1% phosphoric acid (A) as the mobile phase for gradient elution (0-4min, 5% B; 4-21min, 5% B→10% B; 21-35min, 10% B→11% B; 35-45min, 11% B→15% B; 45-65min, 15% B→20% B; 65-85min, 20% B→35% B; 85-95min, 35% B→70% B); flow rate: 1mL / min; detection wavelengths: 220 and 254nm; column temperature: 30℃; injection volume: 10μL.

[0248] 2.2.5 Establishment of fingerprints and identification of common peaks

[0249] Eleven batches of anti-gynecitis drug composition test sample solutions and mixed reference sample solutions were taken and continuously sampled and measured according to the chromatographic conditions under "2.2.4", and the chromatograms were recorded. The chromatographic data of the 11 batches of test samples were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The spectrum of the test sample S1 was used as the reference spectrum, the time window width was set to 0.2min, and after multi-point calibration, the full chromatographic spectrum peak matching was performed to generate superimposed fingerprints, and the median method was used to generate a reference spectrum. As a result, a total of 29 common peaks were calibrated for the 11 batches of samples, see Figure 8 Comparing the HPLC charts of the test solution with those of the mixed reference substance, a total of five peaks were identified, namely matrine (peak 3), phellodendronine hydrochloride (peak 14), rutin (peak 20), forsythiaside A (peak 22), and berberine hydrochloride (peak 28). Figure 9 、 Figure 10 .

[0250] 2.2.6 Similarity Evaluation

[0251] Using the reference fingerprint as a reference, 11 batches of anti-gynecitis drug compositions were evaluated for similarity using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)." The results showed that the similarities between the fingerprints of the 11 samples and the reference were 0.997, 0.998, 0.998, 0.999, 0.997, 0.997, 0.997, 1.000, 0.999, 0.999, and 0.999, respectively, indicating minimal differences in the quality of the preparations and a stable preparation process.

[0252] 2.2.7 Correlation between common peaks and individual herbal medicine pieces

[0253] Take the single-ingredient decoction piece solution under "2.2.1" and inject it according to the chromatographic conditions under "2.2.4" and record the chromatogram. Compare the chromatograms of the S1 sample with those of each single-ingredient decoction piece and assign the common peaks. The results are shown in Table 1. Figure 11 .

[0254] Table 1 Attribution of common peaks

[0255]

[0256] 2.3 Methodological investigation test

[0257] 2.3.1 Precision test

[0258] According to the method under "2.2", take the test solution and inject it continuously for 6 times. Taking forsythiaside A (peak 22) as the reference, calculate the relative retention time and relative peak area of each common peak.

[0259] The results showed that the RSD of the relative retention time of the 29 common peaks was less than 0.61%, and the RSD of the relative peak area was less than 2.97% (n=6), indicating that the method had good precision.

[0260] 2.3.2 Stability test

[0261] According to the method under "2.2", the test solution was injected and measured at 0, 2, 4, 8, 12, and 24 hours. The relative retention time and relative peak area of each common peak were calculated using forsythiaside A (peak 22) as a reference. The results showed that the RSD of the relative retention time of the 29 common peaks was less than 1.77%, and the RSD of the relative peak area was less than 3.41% (n=6), indicating that the test solution was stable at room temperature for 24 hours.

[0262] 2.2.4 Repeatability Test: Six portions of the anti-gynecitis drug composition (S6) were prepared according to the method in "2.1.1." Samples were injected and analyzed according to the chromatographic conditions in "2.2.1." The relative retention times and peak areas of the common peaks were calculated, using forsythiaside A (peak 22) as a reference. The RSDs for the relative retention times and relative peak areas of the 29 common peaks were <0.46% and <2.89% (n=6), respectively, indicating good repeatability.

[0263] 2.4 Chemical pattern recognition analysis

[0264] 2.4.1 Cluster Analysis

[0265] The 29 common peak areas of 11 batches of anti-gynecitis drug composition samples were imported into statistical software, and cluster analysis was performed using the inter-group linkage system clustering method and squared Euclidean distance as the measure. The results showed that when the distance was 5, the 11 batches of samples could be clustered into 2 categories: S1 to S9 were clustered into one category, and S10 and S11 were clustered into one category. Figure 12 .

[0266] 2.4.2 Principal Component Analysis

[0267] Principal component analysis (PCA) was performed using analysis software to analyze the peak areas of 29 common peaks from 11 batches of anti-gynecitis drug compositions. The quality of the compositions was evaluated based on an initial eigenvalue of >1 for the PCs. The results (Tables 2 and 3) show that the initial eigenvalues of the first four PCs were >1, with a cumulative variance contribution of 93.259%. This indicates that these four PCs encompass 93.259% of the information from the 29 common components, reflecting the key characteristics of the samples. PC 1 had the largest initial eigenvalue (17.377), encompassing the richest information, corresponding to components 3, 5-8, 11-16, and 18-29. PC 2 had an initial eigenvalue of 5.643, corresponding to components 1, 2, 4, and 9. PC 3 had an initial eigenvalue of 2.288, corresponding to component 10. PC 4 had an initial eigenvalue of 1.737, corresponding to component 17. The principal component 1 scores with large coefficients are peaks 3 (matrine), 6, 8, 14 (corticosteroid hydrochloride), 15, 18, 22 (forsythiaside A), 25, 26, 27, 28 (berberine hydrochloride), suggesting that multiple components exist that synergistically affect the quality of the anti-gynecitis drug composition. Eleven batches of anti-gynecitis drug compositions were classified into two categories: S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, while S10 and S11 were concentrated in the second quadrant and clustered into one category. The results are consistent with the cluster analysis results, as shown in the results. Figure 13 .

[0268] Table 2 Eigenvalues and variance contribution rates

[0269]

[0270] Table 3 Score coefficient matrix of 29 ingredients in 11 batches of anti-gynecitis drug compositions

[0271]

[0272]

[0273] 2.5 Content determination

[0274] 2.5.1 Chromatographic conditions

[0275] Same as item “2.2.4”.

[0276] 2.5.2 System suitability test

[0277] Take the test solution, negative sample solution under "2.2.1" and the mixed reference solution under "2.2.3", and perform the injection and determination according to the method under "2.2.4", and record the chromatogram.

[0278] Results: The separation of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride were all greater than 1.5, and the theoretical plate numbers were all greater than 9 000. The negative samples had no interference with the determination. Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 .

[0279] 2.5.3 Linear Relationship Investigation

[0280] Accurately measure 1, 2, 4, 6, and 8 mL of the mixed reference solution under "2.2.3" into 10 mL volumetric flasks, and dilute to volume with methanol to obtain a series of reference solution concentrations. Combine these reference solutions with the mixed reference solution under "2.1.2" and measure the concentrations according to the chromatographic conditions under "2.2.4." Record the peak areas. Perform linear regression using the peak area of each component as the ordinate (Y) and the mass concentration as the abscissa (X).

[0281] Results: The measured components showed good linear relationships within the corresponding mass concentration ranges. The results are shown in Table 4.

[0282] 2.5.4 Precision test

[0283] Take the mixed reference solution under "2.2.3" and inject it six times continuously according to the chromatographic conditions under "2.2.4" and record the peak area.

[0284] Results: The RSDs of the peak areas of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride were 2.72%, 2.47%, 2.76%, 2.66%, and 2.98% (n=6), respectively, indicating that the instrument had good precision.

[0285] 2.5.5 Stability test

[0286] The test solution in "2.2.1" was placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, and then injected and analyzed according to the chromatographic conditions in "2.2.4." The results showed that the RSDs of the peak areas for matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride were 0.64%, 0.80%, 1.01%, 0.95%, and 0.66%, respectively (n=6), indicating that the test solution had good stability within 24 hours of placement at room temperature.

[0287] 2.5.6 Repeatability test

[0288] Take an appropriate amount (S6) of the same batch of anti-gynecitis drug composition and prepare 6 test sample solutions in parallel according to the method under "2.2.1". Continuously inject and measure according to the chromatographic conditions under "2.2.4", record the peak area and substitute it into the regression equation to calculate the content.

[0289] Results: The RSDs of matrine, phellodendrine hydrochloride, rutin, forsythiaside A and berberine hydrochloride were 1.71%, 1.51%, 1.52%, 1.46% and 1.60% (n=6), respectively, indicating that the method had good reproducibility.

[0290] 2.5.7 Sample recovery test

[0291] Accurately weigh 0.25 g of sample (S6) of known content (6 portions) and add appropriate amounts of methanol solution of the reference substance (matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride, concentrations of 13.223, 0.2650, 0.854, 1.3590, and 2.1450 mg / mL, respectively). Prepare the test solution according to the method in "2.2.1" and inject the sample according to the chromatographic conditions in "2.2.4". Record the peak area and calculate the recovery. The results are shown in Table 5.

[0292] 2.5.8 Sample content determination

[0293] Take appropriate amounts of the anti-gynecitis drug composition from 11 batches and prepare test solutions according to the method in "2.2.1". Inject and measure according to the chromatographic conditions in "2.2.4". Record the peak area and substitute it into the regression equation to calculate the content. The results are shown in Table 6.

[0294] Table 4 Results of linear relationship investigation of five components including matrine

[0295]

[0296] Table 5. Recovery test results of matrine and other components (n=6)

[0297]

[0298]

[0299] Table 6 Determination of the content of matrine and other ingredients in the anti-gynecitis drug composition (mg / g)

[0300]

[0301] 3 Discussions

[0302] In this study, a fingerprint of an anti-gynecitis drug composition was established using HPLC wavelength conversion. A total of 29 common peaks were identified, and five chemical components were identified. Comparison of the fingerprints of 11 sample batches with the control showed similarities greater than 0.99, indicating that the quality of the preparation was relatively stable. Cluster analysis showed that, at a distance of 5, the 11 sample batches could be clustered into two clusters: S1 and S9 in one cluster, and S10 and S11 in the other. Principal component analysis results were consistent with cluster analysis, with the cumulative variance contribution of the first four principal components reaching 93.259%, indicating that these four principal components capture 93.259% of the information of the 29 common components. Peaks 3 (matrine), 6, 8, and 14 (phellodendronine hydrochloride), 15, 18, and 22 (forsythiaside A), and 25, 26, 27, and 28 (berberine hydrochloride) exhibited significant principal component 1 scores, suggesting that the presence of multiple components synergistically influences the quality of the anti-gynecitis drug composition. The main components of S10 and S11 are different from those of other batches, indicating that in the production process of anti-gynecitis drug compositions, attention should be paid not only to the quality of the control agents but also to the quality control of the raw medicinal materials, such as standardizing the origin of the medicinal materials, the harvesting season, and the quality control standards of the medicinal material slices. At the same time, it is necessary to optimize the control parameters of the preparation production process to ensure the stability of the quality of the anti-gynecitis drug compositions and the consistency of the clinical efficacy.

[0303] Studies have shown that matrine has anti-inflammatory and antioxidant effects, phellodendrine hydrochloride has antibacterial and oxidative stress protection effects, rutin has anti-inflammatory and immunomodulatory effects, forsythiaside A has significant anti-inflammatory and antiviral activities, and berberine hydrochloride has antibacterial and anti-inflammatory effects. The pharmacological effects of these five ingredients are closely related to the functions and indications of the anti-gynecitis drug composition. Therefore, this study identified these five ingredients as the target components for determination in this preparation. Content determination results showed variability among samples from different batches. It is recommended that these five active ingredients be included in the quality standards for the anti-gynecitis drug composition to improve its overall quality.

[0304] In summary, the HPLC fingerprint and content determination method established in this application are stable and reliable, and combined with chemical pattern recognition analysis, can provide a reference basis for improving the quality standards of anti-gynecitis drug compositions.

Claims

1. A detection method for an anti-gynecitis pharmaceutical composition, the prescription of the anti-gynecitis pharmaceutical composition is: 250g of Sophora flavescens, 250g of Radix Codonopsis pilosulae, 150g of Phellodendron chinense, 50g of Forsythia suspensa, 30g of Leonurus japonicus, 30g of Adzuki bean, 30g of Artemisia argyi, 30g of Angelica sinensis, and 30g of Linderae scutellariae; the preparation method of the anti-gynecitis pharmaceutical composition is as follows: take 160g of Sophora flavescens, 90g of Phellodendron chinense, 15g of Adzuki bean, and 15g of Forsythia suspensa from the above nine ingredients, grind them into fine powder, sieve them, and set aside; add water to the remaining Sophora flavescens, Phellodendron chinense, Adzuki bean, Forsythia suspensa and the remaining five ingredients of Leonurus japonicus, and boil them twice, the first time for 2 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.31-1.35 at 60-80°C, add the above fine powder, mix well, dry, and grind into fine powder, and obtain the product; characterized in that The detection method includes fingerprint establishment, chemical pattern recognition analysis and content determination; The method for establishing the fingerprint is: (1) Preparation of test solution Take 0.2-0.8 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 5-15 mL of 60-80% methanol, weigh the mass, and ultrasonicate at a power of 250 W and a frequency of 40 kHz for 10-60 minutes. Let it cool, weigh it again, make up the reduced mass with 60-80% methanol, shake it well, filter it with a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution; (2) Preparation of single-drug slice sample solution: Prepare single-drug slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the preparation method of the test sample solution; (3) Preparation of negative sample solution: According to the preparation method of the test solution, prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae respectively; (4) Preparation of mixed reference solution Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively; (5) Chromatographic conditions and system suitability test Column: ACE C 18 -AR, column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.05-0.15% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; Column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min; (6) Determination method Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition; The chemical pattern recognition analysis method includes cluster analysis and principal component analysis; The cluster analysis method is as follows: 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 are imported into statistical software, and cluster analysis is performed using the inter-group linkage system clustering method and squared Euclidean distance as the measure. When the distance is 5, the 11 batches of samples can be clustered into 2 categories: S1 to S9 are clustered into one category, and S10 and S11 are clustered into one category; The principal component analysis method is as follows: 29 common peak areas of 11 batches of anti-gynecitis drug composition samples numbered S1 to S11 are imported into analysis software for principal component analysis, and the initial eigenvalue of the principal component is greater than 1 as the judgment basis for the quality evaluation of the anti-gynecitis drug composition. The initial eigenvalues of the first four principal components are greater than 1, and the cumulative variance contribution rate is 93.259%, indicating that these four principal components can cover 93.259% of the information of the 29 common components and reflect the main characteristics of the samples; the initial eigenvalue of principal component 1 is the largest, which is 17.377, and the information covered is the richest, corresponding to components 3, 5-8, 11-16, and 18-29; the initial eigenvalue of principal component 2 is 5.643, corresponding to Components 1, 2, 4, and 9; the initial eigenvalue of principal component 3 was 2.288, corresponding to component 10; the initial eigenvalue of principal component 4 was 1.737, corresponding to component 17; the peaks with larger score coefficients of principal component 1 were matrine 3, phellodendrine hydrochloride 6, 8, and 14, forsythiaside A 15, 18, and 22, and berberine hydrochloride 25, 26, 27, and 28. It was speculated that there were multiple components that synergistically affected the quality of the anti-gynecitis drug composition; 11 batches of anti-gynecitis drug compositions were classified and divided into two categories: No. S1 and S9 were concentrated in the first and fourth quadrants and clustered into one category, and No. S10 and S11 were concentrated in the second quadrant and clustered into one category. The results were consistent with the results of cluster analysis.

2. The detection method according to claim 1, wherein The method for establishing the fingerprint is: (1) Preparation of test solution Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, and ultrasonicate at a power of 250 W and a frequency of 40 kHz for 30 min. Let it cool, weigh it again, make up the reduced mass with 70% methanol, shake it well, filter it through a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution; (2) Preparation of single-drug slice sample solution: Prepare the single-drug slice sample solution according to the prescription ratio of the anti-gynecitis drug composition and the same preparation method as the test sample solution; (3) Preparation of negative sample solution: Prepare negative sample solutions of Radix Sophorae Flavescentis, Radix Angelicae Dahuricae, Radix Phellodendri, and Fructus Forsythiae in the same manner as the test sample solution; (4) Preparation of mixed reference solution Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively; (5) Chromatographic conditions and system suitability test Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.1% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; Column temperature: 30°C, detection wavelength: 0-80 min, 220 nm; 81-95 min, 254 nm; flow rate: 1 ml / min; (6) Determination method Accurately pipette 10 μl of the mixed reference solution, test solution, single slice sample solution, and negative sample solution respectively and inject them into a high performance liquid chromatograph for determination, calibrate the common peaks, and obtain the fingerprint of the anti-gynecitis drug composition.

3. The detection method according to any one of claims 1 to 2, characterized in that The obtained fingerprint contained 29 common peaks, and 5 peaks were identified, namely matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

4. The detection method according to claim 3, characterized in that Among the 29 common peaks, peak 3 is matrine; peak 14 is phellodendrine hydrochloride; peak 20 is rutin; peak 22 is forsythiaside A; and peak 28 is berberine hydrochloride.

5. The detection method according to claim 1, wherein The method for the content determination is: (1) Preparation of test solution Take 0.5 g of the anti-gynecitis drug composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 70% methanol, weigh the mass, and ultrasonicate at a power of 250 W and a frequency of 40 kHz for 30 min. Let it cool, weigh it again, make up the reduced mass with 70% methanol, shake it well, filter it through a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution; (2) Preparation of mixed reference solution Accurately weigh appropriate amounts of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride reference substances, dissolve and dilute with methanol to prepare mixed reference substance solutions with mass concentrations of 3809.4 μg / mL, 84.5 μg / mL, 166.6 μg / mL, 420.0 μg / mL, and 596.2 μg / mL, respectively; (3) Chromatographic conditions and system suitability test Column: ACE C 18 -AR, chromatographic column specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: 0.1% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; the gradient elution is specifically as follows: 0-4 min, 5% B; 4-21 min, 5% B→10% B; 21-35 min, 10% B→11% B; 35-45 min, 11% B→15% B; 45-65 min, 15% B→20% B; 65-85 min, 20% B→35% B; 85-95 min, 35% B→70% B; Column temperature: 30°C; detection wavelength: 220 nm from 0 to 80 min; 254 nm from 81 to 95 min; flow rate: 1 ml / min; resolution of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride was greater than 1.5, and the number of theoretical plates was greater than 9000. (4) Determination method Accurately pipette 10 μl of the mixed reference solution and test solution into a high performance liquid chromatograph for determination and calculation to obtain the contents of matrine, phellodendrine hydrochloride, rutin, forsythiaside A, and berberine hydrochloride.

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