A method for preparing a fukekang composition and a detection method thereof

Abstract

The present application relates to a kind of preparation method of Fugunkang composition and its detection method, the preparation method is cut into section, first using red koji and saccharomyces cerevisiae combined fermentation extraction, dregs is extracted with water before boiling, concentrate and extract the fermentation liquor and water, it is obtained.The metabolic function of microorganism is fully utilized, promote the release and absorption of effective components of traditional Chinese medicine in vivo, so as to improve the curative effect of traditional Chinese medicine, reduce the side effect of traditional Chinese medicine.The detection method includes spray agent in just bed, parsnip medicine thin layer identification and the content detection of rutin, hypericin;The thin layer identification can identify just bed and parsnip medicine under the same chromatographic condition, the content detection increases the content detection of hypericin on the basis of original, and the content of rutin and hypericin can be determined simultaneously, save the detection cost, ensure the quality of raw material and finished product.

Description

Technical Field

[0001] This invention relates to the field of drug preparation and testing, specifically to a method for preparing and testing a gynecological skincare composition. Background Technology

[0002] Fufukang Spray, a product of Guizhou Liangji Pharmaceutical Co., Ltd., has the effects of clearing heat and detoxifying, promoting blood circulation and relieving pain, and killing parasites and relieving itching. It is used for fungal vaginitis, trichomonal vaginitis, bacterial vaginosis, vulvitis, and pruritus. It is composed of 120g of *Justicia procumbens*, 80g of *Senecio scandens*, and 20g of polysorbate 80. The preparation method is as follows: the above two herbs are soaked in water for 4-6 hours, then decocted three times: the first time for 2 hours, the second time for 1.5 hours, and the third time for 1 hour. The decoctions are filtered separately, the filtrates are combined and concentrated to a clear paste with a relative density of 1.13-1.15 (70-80℃). After cooling, ethanol is added to achieve an alcohol content of 90%. The mixture is allowed to stand for 24 hours, filtered, and the ethanol is recovered from the filtrate until no alcohol odor remains. The prescribed amount of polysorbate 80 is added, followed by water to the specified volume. The mixture is stirred well, filtered, and then bottled. The technical problems with this preparation method are: (1) low extraction and transfer rate; (2) low content of effective components in the finished product.

[0003] To address the problems with the above preparation methods, the inventors, combining research on modern Chinese medicine fermentation processes, conducted numerous experiments. They cut the medicinal materials into segments, first using a combined fermentation extraction with Monascus purpureus and Saccharomyces cerevisiae, then further extracting the residue by boiling it in water, combining the fermentation broth and the water extract for concentration. This fully utilizes the metabolic functions of microorganisms to promote the release and absorption of the effective components of Chinese medicine in the body, thereby improving the efficacy and reducing the side effects of Chinese medicine.

[0004] The Fufukang spray prepared by the improved preparation method was tested for quality according to the standard of the State Drug Administration (WS-10860(ZD-0860)-2002). Some test results showed the following problems: (1) The thin-layer identification spots of Acanthopanax were blurry and the reproducibility was poor, which affected the judgment of the test results; (2) During the thin-layer chromatography content determination of Senecio scandens, the main spot was blurry and showed tailing phenomenon, and the Rf value of the spot was small, which affected the judgment of the test results; (3) Acanthopanax and Senecio scandens were identified separately, which wasted the test cost; (4) The quality test lacked the detection of hyperoside content in Senecio scandens.

[0005] To address the aforementioned testing issues, effectively improve product quality, and enhance the quality standards of Fufukang spray, we conducted in-depth research and investigation into the original testing methods for Fufukang spray, referring to (WS-10860(ZD-0860)-2002) and pharmacopoeia standards. We optimized the extraction solvent and method for thin-layer chromatography identification, and changed the developing solvent. The results showed clear thin-layer spots with moderate Rf values, and the ability to simultaneously identify Justicia procumbens and Senecio scandens under the same chromatographic conditions. We also added the detection of hyperoside content in Senecio scandens, ensuring the quality of the medicinal materials and further guaranteeing product quality. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a gynecological skincare composition.

[0007] Another objective of this invention is to provide a method for detecting Fufukang spray.

[0008] The formula for the Fufukang spray of this invention is: 12 parts of Acanthopanax, 8 parts of Senecio scandens, and 2 parts of polysorbate 80.

[0009] The preparation method of the Fufukang spray of the present invention is as follows:

[0010] The preparation method of the spray is as follows:

[0011] (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into sections;

[0012] (2) Mix Acanthus sinensis and Senecio scandens evenly, add 5 to 8 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 30 to 40℃ for 2 to 3 days, centrifuge and filter to obtain fermentation extract;

[0013] (3) Take the fermented and extracted residue, add 5 to 8 times the amount of water and decoct twice for 1 hour each time. Combine the extracts to obtain the aqueous extract.

[0014] (4) After combining the fermentation extract and the water extract, vacuum concentration is carried out at a vacuum degree of -0.05Mpa to -0.08Mpa and a concentration temperature of 50 to 80℃ to obtain a clear extract with a relative density of 1.15 to 1.20; ethanol is added to make the alcohol content reach 90%, let stand for 24 hours, filter, recover the ethanol from the filtrate until there is no alcohol odor, add the prescribed amount of polysorbate 80, add water to 1000ml, stir well, filter, and fill into bottles to obtain the product.

[0015] Priority

[0016] The preparation method of the Fufukang drug is as follows:

[0017] (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into sections;

[0018] (2) Mix Acanthus sinensis and Senecio scandens evenly, add 6 to 8 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 35 to 40℃ for 2 to 3 days, centrifuge and filter to obtain fermentation extract;

[0019] (3) Take the fermented and extracted residue, add 6 to 8 times the amount of water and decoct twice, each time for 1 hour. Combine the extracts to obtain the aqueous extract.

[0020] (4) After combining the fermentation extract and the water extract, vacuum concentration was carried out at a vacuum degree of -0.06Mpa to -0.08Mpa and a concentration temperature of 60 to 80℃ to obtain a clear extract with a relative density of 1.15 to 1.18; ethanol was added to make the alcohol content reach 90%, and the mixture was allowed to stand for 24 hours. The mixture was then filtered, and the ethanol was recovered from the filtrate until there was no alcohol odor. The amount of polysorbate 80 in the prescription ratio was added, and water was added to 1000ml. The mixture was stirred, filtered, and then filled into bottles to obtain the final product.

[0021] Further priority,

[0022] The preparation method of the Fufukang drug is as follows:

[0023] (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into sections;

[0024] (2) Mix Acanthus and Senecio scandens evenly, add 6 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 35℃ for 3 days, centrifuge and filter to obtain fermentation extract;

[0025] (3) Take the fermented and extracted residue, add 6 times the amount of water and decoct twice for 1 hour each time. Combine the extracts to obtain the aqueous extract.

[0026] (4) After combining the fermentation extract and the water extract, the extract is concentrated under vacuum at a vacuum degree of -0.06 MPa and a concentration temperature of 60°C to a clear extract with a relative density of 1.15. Ethanol is added to make the alcohol content reach 90%, and the extract is allowed to stand for 24 hours. The extract is filtered, and the ethanol is recovered from the filtrate until there is no alcohol odor. Polysorbate 80 is added in the prescribed ratio, and water is added to 1000 ml. The extract is stirred, filtered, and bottled to obtain the final product.

[0027] The detection method for the Fufukang spray of the present invention includes the identification of Acanthopanax and Senecio scandens medicinal materials; and the detection of the content of rutin and hyperoside.

[0028] The identification method for the medicinal materials *Justicia procumbens* and *Senecio scandens* described in this invention is as follows: Take 30 ml of this product, add 30-60 ml of ethyl acetate, heat and reflux to extract for 2-4 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to obtain the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference medicinal materials, and prepare reference medicinal material solutions using the same method. Perform thin-layer chromatography, taking 5-10 μl of each of the above two solutions and spotting them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Use cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal materials.

[0029] Preferred,

[0030] The identification method for the medicinal materials *Justicia procumbens* and *Senecio scandens* described in this invention is as follows: Take 30 ml of this product, add 40-50 ml of ethyl acetate, heat and reflux to extract for 2-3 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to obtain the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference medicinal materials, and prepare reference medicinal material solutions using the same method. Perform thin-layer chromatography, taking 5-8 μl of each of the above two solutions and spotting them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Use cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal materials.

[0031] Further preferred,

[0032] The identification method for *Justicia procumbens* and *Senecio scandens* medicinal materials described in this invention is as follows: Take 30 ml of this product, add 50 ml of ethyl acetate, heat under reflux for 3 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to obtain the test solution. Take 2 g each of *Justicia procumbens* and *Senecio scandens* reference medicinal materials, prepare reference medicinal material solutions using the same method, and perform thin-layer chromatography. Apply 5 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Develop using cyclohexane-ethyl acetate-methanol (5:3:2), remove, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal materials.

[0033] The method for detecting the content of rutin and hyperoside described in this invention is as follows:

[0034] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0035] Preparation of the test solution: Accurately measure 2-5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 60-80% methanol, weigh it, heat under reflux for 0.5-1.5 hours, cool it, weigh it again, replenish the lost weight with 60-80% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0036] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.2–0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 mL / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows:

[0037]

[0038] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0039] Preferred,

[0040] The method for detecting the content of rutin and hyperoside described in this invention is as follows:

[0041] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0042] Preparation of the test solution: Accurately measure 4-5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 70-75% methanol, weigh it, heat under reflux for 0.5-1 hour, cool it, weigh it again, replenish the lost weight with 70-75% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0043] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.4–0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 mL / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows:

[0044]

[0045]

[0046] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0047] Preferred,

[0048] The method for detecting the content of rutin and hyperoside is as follows:

[0049] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0050] Preparation of the test solution: Accurately measure 5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 75% methanol, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0051] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should not be less than 5000; gradient elution was performed as follows:

[0052]

[0053] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0054] The "part" mentioned in this invention can be a conventional unit in the art, such as "g" or "kg".

[0055] Beneficial effects:

[0056] 1. This application addresses the preparation problem of Fufukang spray. The inventors, combining modern research on fermentation processes of traditional Chinese medicine, conducted extensive research and developed a method that involves cutting medicinal materials into segments, firstly fermenting them using Monascus purpureus and Saccharomyces cerevisiae, then extracting the residue by boiling it in water, and finally concentrating the combined extracts. This extraction method boasts a high transfer rate, fully utilizing the metabolic functions of microorganisms to promote the release and absorption of effective components of traditional Chinese medicine in the body, thereby improving the efficacy of traditional Chinese medicine and reducing its side effects.

[0057] 2. This invention obtains the optimal fermentation extraction method by selecting the fermentation strain, fermentation time, and fermentation temperature. Combined with the original preparation method, it obtains the optimal preparation method for Fufukang spray. Through comparative experiments on the rutin content before and after the optimization of the preparation method, the results show that the average transfer rate of this application is 36.13%, which is higher than the 30.40% before optimization; the average rutin content of 10 batches is 1.29 mg / ml, which is higher than the 1.15 mg / ml before optimization, further proving the feasibility of the extraction method of this scheme.

[0058] 3. The detection method in this application includes optimization of the thin-layer chromatography (TLC) identification of *Justicia procumbens* and *Senecio scandens* in Fufukang spray. This allows for the simultaneous identification of *Justicia procumbens* and *Senecio scandens* under the same chromatographic conditions, saving detection costs and improving detection efficiency. Furthermore, this application compares the TLC methods for *Justicia procumbens* and *Senecio scandens* before and after optimization. The results show that before optimization, the TLC results for *Justicia procumbens* and *Senecio scandens* were mostly characterized by blurred spots, tailing, and unsatisfactory resolution, with only a few batches meeting the requirements, affecting the judgment of product quality. After optimization, the TLC results for *Justicia procumbens* and *Senecio scandens* were all characterized by clear spots, satisfactory resolution, moderate Rf values, and good reproducibility.

[0059] 4. The detection method in this application includes the determination of rutin and hyperoside content in Fufukang spray. The method optimizes the rutin content determination in the original standard and adds the hyperoside content determination item, ensuring product quality and effectively controlling raw material quality. This content detection method can simultaneously detect the content of rutin and hyperoside, saving detection costs. Furthermore, this application also compared the rutin and hyperoside content determination methods before and after optimization. The results showed that the average rutin content of 10 batches of finished products after optimization (1.30 mg / ml) was higher than that before optimization (1.15 mg / ml). Method validation results showed that the linearity test indicated good linearity of rutin in the range of 0.1 mg / ml to 5.0 mg / ml; the hyperoside injection concentration also showed good linearity in the range of 40.0 μg / ml to 2000.0 μg / ml; the precision test showed that after six repeated injections, the relative standard deviation (RSD%) was calculated to be 1.81%, indicating good instrument precision; the specificity test showed that the sample solution chromatogram had corresponding chromatographic peaks at the corresponding positions of the reference solution chromatogram, and there was no negative interference.

[0060] 5. This application also determined the content of rutin in 10 batches of Fufukang spray samples. The results showed that the rutin content in all ten batches of samples was... 27 H 30 O 16 The average content of flavonoids is 1.28 mg / ml, which meets the requirements (each 1 ml of this product contains total flavonoids in the form of rutin (C 27 H 30 O 16(The concentration of hyperoside (C) in ten batches of samples shall not be less than 1.0 mg / ml). 21 H 20 O 12 The average content is 1.20 μg / ml, and the content of hyperoside (C) in the finished product Fufukang spray is tentatively determined to be... 21 H 20 O 12 The content standard is 1.15 μg / ml. Detailed Implementation

[0061] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0062] Example 1

[0063] Formula: 120g of Acanthopanax, 80g of Senecio scandens, 20g of polysorbate.

[0064] Preparation method: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into segments; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 6 times the amount of water, inoculate with Monascus purpureus and Saccharomyces cerevisiae at 5.2×100CFU / mL respectively, ferment at 35℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 6 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum reduce the concentration to a clear paste with a relative density of 1.15 at a vacuum degree of -0.06Mpa and a concentration temperature of 60℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add polysorbate 80 according to the prescription ratio, add water to 1000ml, stir well, filter, and fill to obtain the product.

[0065] Example 2

[0066] Formula: Same as Example 1.

[0067] Preparation method: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into segments; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 5 times the amount of water, inoculate with Monascus purpureus and Saccharomyces cerevisiae at 5.2×100CFU / mL respectively, ferment at 30℃ for 2 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 5 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum reduce the concentration to a clear paste with a relative density of 1.15 at a vacuum degree of -0.05Mpa and a concentration temperature of 50℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add polysorbate 80 according to the prescription ratio, add water to 1000ml, stir well, filter, and fill to obtain the product.

[0068] Example 3

[0069] Formula: Same as Example 1.

[0070] Preparation method: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into segments; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 8 times the amount of water, inoculate with Monascus purpureus and Saccharomyces cerevisiae at 5.2×100CFU / mL respectively, ferment at 40℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 8 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum reduce the concentration to a clear paste with a relative density of 1.20 at a vacuum degree of -0.08Mpa and a concentration temperature of 80℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add polysorbate 80 according to the prescription ratio, add water to 1000ml, stir well, filter, and fill into the package to obtain the product.

[0071] Example 4

[0072] Formula: Same as Example 1.

[0073] Preparation method: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into segments; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 6 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 32℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 6 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum reduce the concentration to a clear paste with a relative density of 1.18 at a vacuum degree of -0.06Mpa and a concentration temperature of 60℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add polysorbate 80 according to the prescription ratio, add water to 1000ml, stir well, filter, and fill to obtain the product.

[0074] Example 5

[0075] Formula: Same as Example 1.

[0076] The preparation method is as follows: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into segments; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 7 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 33℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 7 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum reduce the concentration to a clear paste with a relative density of 1.15 at a vacuum degree of -0.07Mpa and a concentration temperature of 70℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add the amount of polysorbate 80 in the prescription ratio, add water to 1000ml, stir well, filter, and fill to obtain the product.

[0077] Example 6

[0078] Formula: Same as Example 1.

[0079] The preparation method is as follows: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into segments; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 6 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 38℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 5 times the amount of water and decoct twice, each time for 1 hour, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum reduce the concentration to a clear paste with a relative density of 1.15 at a vacuum degree of -0.08Mpa and a concentration temperature of 65℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add the amount of polysorbate 80 in the prescription ratio, add water to 1000ml, stir well, filter, and fill to obtain the product.

[0080] The Fufukang sprays prepared in Examples 1-6 were tested according to the detection methods in Examples 7-12:

[0081] Example 7

[0082] [Identification]

[0083] Take 30 ml of this product, add 50 ml of ethyl acetate, heat under reflux for 3 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to obtain the test solution. Take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, prepare reference material solutions using the same method, and perform thin-layer chromatography. Apply 5 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, remove, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0084] Content determination

[0085] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0086] Preparation of the test solution: Accurately measure 4-5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 75% methanol, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0087] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should not be less than 5000; gradient elution was performed as follows:

[0088]

[0089] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0090] Example 8

[0091] [Identification]

[0092] Take 30 ml of this product, add 30 ml of ethyl acetate, heat under reflux for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to obtain the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, and prepare reference material solutions using the same method. Perform thin-layer chromatography, taking 5 μl of each of the above two solutions and spotting them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Use cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0093] Content determination

[0094] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0095] Preparation of the test solution: Accurately measure 2 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 60% methanol, weigh it, heat under reflux for 0.5 hours, cool it, weigh it again, make up the lost weight with 60% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0096] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.2% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows:

[0097]

[0098] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0099] Example 9

[0100] [Identification]

[0101] Take 30 ml of this product, add 60 ml of ethyl acetate, heat under reflux for 4 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to prepare the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, and prepare reference material solutions using the same method. Perform thin-layer chromatography, apply 10 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder, develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0102] Content determination

[0103] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0104] Preparation of the test solution: Accurately measure 5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 80% methanol, weigh it, heat under reflux for 1.5 hours, cool it, weigh it again, make up the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0105] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should not be less than 5000; gradient elution was performed as follows:

[0106]

[0107] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0108] Example 10

[0109] [Identification]

[0110] Take 30 ml of this product, add 50 ml of ethyl acetate, heat under reflux for 3 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to prepare the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, and prepare reference material solutions using the same method. Perform thin-layer chromatography, apply 8 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder, develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0111] Content determination

[0112] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0113] Preparation of the test solution: Accurately measure 3 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 65% methanol, weigh it, heat under reflux for 0.8 hours, cool it, weigh it again, make up the lost weight with 65% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0114] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.3% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows:

[0115]

[0116] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0117] Example 11

[0118] [Identification]

[0119] Take 30 ml of this product, add 45 ml of ethyl acetate, heat under reflux for 2.5 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to prepare the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, and prepare reference material solutions using the same method. Perform thin-layer chromatography, apply 7 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder, develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0120] Content determination

[0121] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0122] Preparation of the test solution: Accurately measure 3 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 75% methanol, weigh it, heat under reflux for 1.2 hours, cool it, weigh it again, make up the lost weight with 72% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0123] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.5% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows:

[0124]

[0125] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0126] Example 12

[0127] [Identification]

[0128] Take 30 ml of this product, add 55 ml of ethyl acetate, heat under reflux for 3 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to prepare the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, and prepare reference material solutions using the same method. Perform thin-layer chromatography, apply 6 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder, develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0129] Content determination

[0130] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0131] Preparation of the test solution: Accurately measure 2-5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 60-80% methanol, weigh it, heat under reflux for 1.2 hours, cool it, weigh it again, make up the lost weight with 78% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0132] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.3% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows:

[0133]

[0134]

[0135] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0136] To further verify the effectiveness of the present invention, the inventors conducted a series of verification experiments, some of which are excerpted below:

[0137] Experimental Example 1

[0138] 1. Comparative Study of Preparation Methods of Fufukang Spray

[0139] 1.1 Formula: 120g of Acanthopanax, 80g of Senecio scandens, and 20g of polysorbate.

[0140] 1.2 Evaluation criteria: Determination of transfer rate and rutin content.

[0141] 1.3 Judgment Criteria Source: Fufukang Spray (WS-10860(ZD-0860)-2002).

[0142] 1.4 Examination Content:

[0143] 1.4.1 Preparation method before optimization

[0144] (1) Soak the above two medicinal materials in water for 4 to 6 hours, then decoct them three times: the first time for 2 hours, the second time for 1.5 hours, and the third time for 1 hour. Filter them in batches, combine the filtrates and concentrate them to a clear extract with a relative density of 1.13 to 1.15 (70 to 80°C). Let it cool, add ethanol to make the alcohol content reach 90%, let it stand for 24 hours, filter it, recover the ethanol from the filtrate until there is no alcohol taste, add the amount of polysorbate 80 in the prescription, add water to the specified amount, stir well, filter, and fill into a container to obtain the product.

[0145] 1.4.2 Investigation into the optimization of preparation methods

[0146] 1.4.2.1 Investigation of fermentation strains

[0147] Based on research into modern Chinese medicine fermentation processes and the fermentation characteristics of bacteria and fungi, and in conjunction with literature review, this experiment selected *Lactobacillus bulgaricus*, *Bifidobacterium*, *Monascus purpureus*, and *Saccharomyces cerevisiae* as the key screening strains for the following experiments:

[0148] Five portions of Acanthopanax quinquefolius (120g) and Senecio scandens (80g) were weighed according to the formula ratio and cut into sections. Each portion was then inoculated with 6 times the total amount of water. The first portion was inoculated with Lactobacillus bulgaricus, the second with Bifidobacterium, the third with Monascus purpureus, the fourth with Saccharomyces cerevisiae, and the fifth with both Monascus purpureus and Saccharomyces cerevisiae. The inoculation amount was 5.2 × 100 CFU / mL. The mixture was fermented at 35℃ for 3 days, centrifuged, and filtered to obtain the fermentation extract. The extraction rate results are shown in Table 1.

[0149] Table 1 Comparison of fermentation results from different strains

[0150]

[0151] The experimental results showed that among the five selected strains, the rutin extraction rate was the highest when Monascus purpureus + Saccharomyces cerevisiae was fermented. Therefore, Monascus purpureus + Saccharomyces cerevisiae was selected as the preferred fermentation strain.

[0152] 1.4.2.2 Investigation of Fermentation Temperature

[0153] Five portions of 120g of Acanthopanax and 80g of Senecio scandens were weighed according to the formula ratio and cut into sections. Each portion was added with 6 times the total amount of water of the medicinal materials. The samples were inoculated with Monascus purpureus and Saccharomyces cerevisiae respectively, with an inoculum of 5.2×100 CFU / mL. The samples were fermented for 3 days at 25℃, 30℃, 35℃, 40℃ and 50℃ respectively. The samples were centrifuged and filtered to obtain the fermentation extract. The extraction rate results are shown in Table 2.

[0154] Table 2 Comparison of different fermentation temperatures

[0155]

[0156] The experimental results show that when different fermentation temperatures are selected, the rutin extraction rate is relatively high and the difference is not significant at 35-45℃. Therefore, "35℃" is the preferred fermentation temperature.

[0157] 1.4.2.3 Investigation of fermentation time

[0158] Five portions of 120g of Acanthopanax and 80g of Senecio scandens were weighed according to the formula ratio and cut into sections. Each portion was added with 6 times the total amount of water of the medicinal materials. The samples were inoculated with Monascus purpureus and Saccharomyces cerevisiae respectively, with an inoculum of 5.2×100 CFU / mL. The samples were fermented at 35℃ for 1 day, 2 days, 3 days, 4 days and 5 days respectively. After centrifugation and filtration, the fermentation extract was obtained. The extraction rate results are shown in Table 3.

[0159] Table 3 Comparison of different fermentation times

[0160]

[0161]

[0162] The experimental results show that when different fermentation times are selected, the rutin extraction rate is relatively high and the difference is not significant. Therefore, "3 days" is the preferred fermentation time.

[0163] 1.4.3 Confirmation of the optimized preparation method

[0164] Combining the above-mentioned preferred "fermentation method" and "preparation method before optimization", the optimized preparation method is as follows: (1) Take Justicia procumbens and Senecio scandens and cut them into segments respectively; (2) Mix Justicia procumbens and Senecio scandens segments evenly, add 6 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 35℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the residue after fermentation extraction, add 6 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain water extract; (4) Combine the fermentation extract and water extract, and vacuum decompression concentrate to a clear paste with a relative density of 1.15 at a vacuum degree of -0.06Mpa and a concentration temperature of 60℃; add ethanol to make the alcohol content reach 90%, let stand for 24 hours, filter, recover ethanol from the filtrate until there is no alcohol taste, add the prescribed amount of polysorbate 80, add water to 1000ml, stir well, filter, and fill to obtain the product.

[0165] 1.4.3.1 Comparison Experiment Before and After Optimization of Preparation Method

[0166] Ten batches of the spray prepared before and after optimization were taken respectively, and the rutin content was tested according to the method for determining rutin content in "Example 7". The results are shown in Table 4.

[0167] Table 4 Comparison of results before and after optimization of preparation method

[0168]

[0169]

[0170] Results: As shown in Table 4, the average transfer rate of the 10 batches of products after optimization was higher than that before optimization, and the average rutin content was also higher than that before optimization. Therefore, the feasibility, excellence and rationality of the optimized preparation method are proven.

[0171] Experiment Example 2

[0172] Comparison of thin-layer chromatography methods before and after optimization for Acanthopanax and Senecio scandens medicinal materials

[0173] 1.1 Thin-layer chromatographic method for Acanthopanax and Senecio scandens before optimization

[0174] (1) Take 30 ml of this product and extract it three times with 20 ml of ethyl acetate each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 2 ml of ethanol to obtain the test solution. Separately, take 2 g of *Justicia procumbens* reference material, add 20 ml of ethyl acetate, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 ml of ethanol to obtain the reference material solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2000 Edition, Part I, Appendix VI B). Apply 5-10 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Develop the plate using toluene-ethyl acetate-formic acid (6:3:1) as the developing solvent. Remove the plate, air dry, and examine it under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0175] (2) Take 50 ml of this product and extract it twice with 25 ml of ether each time. Combine the ether extracts, evaporate to dryness, and dissolve the residue in 1 ml of ethanol to obtain the test solution. Separately, take 10 g of Senecio scandens reference material, add 100 ml of 75% ethanol, heat under reflux for 1 hour, filter, evaporate the filtrate until there is no alcohol odor, add water to make up to 25 ml, filter, wash the filtrate four times with 15 ml of petroleum ether (60-90℃), discard the petroleum ether, extract the aqueous solution twice with 20 ml of ethyl acetate each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of ethanol to obtain the reference material solution. Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2000 Edition, Part I, Appendix VI B). Apply 5–10 μl of each of the two solutions described above to the same silica gel G thin-layer plate using sodium carboxymethyl cellulose as a binder. Develop the plate using toluene-ethyl acetate-formic acid (5:4:1) as the developing solvent. Remove the plate, air-dry it, spray it with 1% ferric chloride solution, and examine it under sunlight after 10 hours. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material.

[0176] 1.2 Optimized Thin-Layer Chromatography Method

[0177] Take 30 ml of this product, add 50 ml of ethyl acetate, heat under reflux for 3 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to obtain the test solution. Take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, prepare reference material solutions using the same method, and perform thin-layer chromatography. Apply 5 μl of each of the above two solutions to the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, remove, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials.

[0178] 1.3 Detection

[0179] Ten batches of spray were prepared according to the preferred method under "Experimental Example 1". The identification of Acanthus chinensis and Senecio scandens in the spray was tested according to the above method. The results are shown in Table 5.

[0180] Table 5 Comparison of Thin-Layer Identification Results of Acanthopanax and Senecio scandens Before and After Optimization

[0181]

[0182] Results: As shown in Table 5, before optimization, the thin-layer chromatography results of Acanthopanax and Senecio scandens showed that the spots were blurry, the separation was not satisfactory, and the reproducibility was poor, which affected the judgment of product quality. After optimization, the thin-layer chromatography results of both were clear, the separation met the requirements, and the Rf value was moderate, indicating good reproducibility.

[0183] 2. Methodological Validation

[0184] 2.1 Specificity

[0185] Take the Fufukang spray prepared according to "Example 1", take the reference medicinal materials of Acanthus chinensis and Senecio scandens, and negative samples lacking Acanthus chinensis and Senecio scandens. Prepare the test solution, reference medicinal material solution and negative test solution by the optimized method of "Experimental Example 2", spot them on the same silica gel G thin layer plate, develop with cyclohexane-ethyl acetate-methanol (5:3:2) as the developing solvent, take it out, dry it and examine it under ultraviolet light at 365nm. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference medicinal materials, and the spots are well separated, with no interference from the negative sample.

[0186] 2.2 Reproducibility

[0187] Ten batches of the optimized Fufukang spray were tested according to the optimized method in "Experimental Example 2". The results are shown in Table 6.

[0188] Table 6. Test methods, conditions, and reproducibility test results

[0189]

[0190] 2.3 Durability

[0191] 2.3.1 Comparison of different thin-layer plates

[0192] Ten batches of Fufukang spray under the "reproducibility" test were taken and tested according to the preferred method in "Experimental Example 2". The thin film thickness of the silicone G commercial plate and the high-efficiency silicone G commercial plate from Qingdao Ocean Chemical Plant were compared. The results are shown in Table 7.

[0193] Table 7. Durability Results of Different Thin-Layer Boards

[0194]

[0195] Results: The spots on the high-efficiency silica gel plate were clear, the separation met the requirements, and the Rf value was moderate; the spots on the silica gel G plate were also clear, the separation met the requirements, and the Rf value was moderate. Therefore, this method has good durability.

[0196] 2.3.2 Comparison of different humidity levels

[0197] Ten batches of Fufukang spray under the "Reproducibility" item were taken, and the high efficiency thin-layer plate development effect was compared under low humidity (30%) and high humidity (75%) conditions. The results are shown in Table 8.

[0198] Table 8. Development effect of high-efficiency thin-layer plate under low humidity (30%) and high humidity (75%) environments.

[0199]

[0200] Results: The spots were clear, the separation met the requirements, the Rf value was moderate, and good identification chromatograms could be obtained under both conditions.

[0201] The above methodological validation experiments showed that the optimized thin-layer chromatography method for identifying Acanthopanax and Senecio scandens under different humidity conditions, using high-efficiency thin-layer plates and silica gel G commercial plates, produced clear chromatographic spots, met the separation requirements, and had a moderate Rf value. The validation experiments demonstrated the reliability and reproducibility of the optimized method, and that it could simultaneously identify Acanthopanax and Senecio scandens under the same chromatographic conditions, saving detection costs and improving detection efficiency.

[0202] Experimental Example 3

[0203] 1. Comparison of the determination methods for rutin and hyperoside before and after optimization

[0204] Ten batches of Fufukang spray under the "reproducibility" test were taken, and the contents of rutin and hypericin were tested according to the following method. The results are shown in Table 9.

[0205] 1.1 Detection method before optimization

[0206] Preparation of the reference solution: Accurately weigh 20 mg of rutin reference standard dried to constant weight under reduced pressure at 120 °C, place it in a 100 ml volumetric flask, add 70 ml of methanol to dissolve it, and dilute to the mark. Shake well to obtain the solution. (Each 1 ml contains 0.2 mg of anhydrous rutin).

[0207] Preparation of the standard curve: Accurately pipette 0 ml, 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, 5.0 ml, and 6.0 ml of the reference solution into separate 25 ml volumetric flasks. Add water to each flask to a final volume of 6 ml. Add 1 ml of 5% sodium nitrite solution, mix well, and let stand for 6 minutes. Add 1 ml of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 ml of sodium hydroxide solution, then add water to the mark, shake well, and let stand for 15 minutes. Measure the absorbance at 500 nm using spectrophotometry (Chinese Pharmacopoeia 2000 Edition, Part I, Appendix VB). Plot the standard curve with absorbance on the ordinate and concentration on the abscissa.

[0208] For the assay, accurately measure 5 ml of the sample and extract four times with 20 ml of water-saturated n-butanol each time. Combine the extracts, evaporate to dryness, dissolve the residue in 5 ml of methanol in portions, and transfer to 10 ml volumetric flasks. Dilute to the mark with methanol, shake well, centrifuge, and use the supernatant as the test solution. Accurately measure 1 ml of the test solution and, following the method under the preparation of the standard curve, starting from "add water to 6 ml", determine the absorbance according to the procedure (take another 1 ml of the test solution, and operate according to the procedure except without adding sodium hydroxide test solution, as a blank solution for the test solution). Read the weight of rutin in the test solution from the standard curve and calculate the result.

[0209] 1.2 Optimized Detection Method

[0210] Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol.

[0211] Preparation of the test solution: Accurately measure 5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 75% methanol, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0212] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should not be less than 5000; gradient elution was performed as follows:

[0213]

[0214] The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0215] Table 9 Comparison of results before and after optimization of content detection method

[0216]

[0217]

[0218] As shown in the table above, the average rutin content of the ten batches of finished products after optimization was higher than that before optimization, indicating that the effective components were extracted more completely after optimization. In addition, the optimization process added the detection of hyperoside content, which ensured the quality of the medicinal materials and further guaranteed the quality of the products.

[0219] 2. Method Validation

[0220] 2.1 Linear Examination

[0221] 2.1.1 Linearity Study of Rutin

[0222] Accurately measured rutin reference solutions with concentrations of 0.1 mg / ml, 0.2 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, and 5.0 mg / ml were used. Regression analysis was performed on the peak areas of the rutin reference solutions at the standard concentrations, yielding the linear equation Y = 5617.1x + 205.29, R0. 2 =0.9982, indicating that the rutin concentration showed a good linear relationship in the range of 0.1 mg / ml to 5.0 mg / ml. The results are shown in Table 10.

[0223] Table 10. Examination of the linear relationship of rutin

[0224]

[0225] 2.1.1 Linearity study of hyperoside

[0226] Accurately measured hyperoside reference solutions with concentrations of 40.0 μg / ml, 80.0 μg / ml, 200.0 μg / ml, 400.0 μg / ml, 800.0 μg / ml, and 2000.0 μg / ml were used. Regression analysis was performed on the peak areas at the standard concentrations, yielding the linear equation Y = 28.523x - 115.29, R0. 2 =0.9999, indicating that hyperoside showed a good linear relationship in the range of 40.0 μg / ml to 2000.0 μg / ml. The results are shown in Table 11.

[0227] Table 11. Investigation of the linear relationship of hyperoside

[0228]

[0229] 2.2 Precision Test

[0230] Take the 0.1 mg / ml rutin reference solution prepared according to the above method, and inject it 6 times under the above chromatographic conditions. Calculate the relative standard deviation to verify the precision of the method. The calculated RSD% value is 1.81%, which indicates that the instrument has good precision. The measurement results are shown in Table 12.

[0231] Table 12 Precision Test

[0232]

[0233] 2.3 Exclusivity

[0234] The Fufukang spray prepared in "Example 1" and the negative sample were used to prepare sample solution and negative sample solution according to the test solution preparation method. Under the above chromatographic conditions, the reference solution, sample solution and negative sample solution were injected into the liquid chromatograph respectively. The chromatogram of the sample solution showed the corresponding chromatographic peak at the corresponding position of the chromatogram of the reference solution, and the negative sample solution had no interference.

[0235] 2.4 Content determination of ten batches of samples:

[0236] Ten batches of Fufukang spray were prepared according to the method in "Example 1" and tested according to the content detection method in "Example 7". The results of rutin content are shown in Table 13 and hyperoside content are shown in Table 11.

[0237] Table 13 Results of Rutin Content Determination in Ten Batches of Samples

[0238]

[0239]

[0240] Table 14 Results of Hyperoside Content Determination in Ten Batches of Samples

[0241]

[0242] Results: As shown in Table 13, the ten batches of samples contained rutin (C 27 H 30 O 16 The average content of flavonoids is 1.28 mg / ml, which meets the requirements (each 1 ml of this product contains total flavonoids in the form of rutin (C 27 H 30 O 16 (Calculated, not less than 1.0 mg / ml); As shown in Table 14, the ten batches of samples contained hyperoside (C 21 H 20 O 12 The average content is 1.20 μg / ml, and the content of hyperoside (C) in the finished product Fufukang spray is tentatively determined to be... 21 H 20 O 12 The content standard is 1.15 μg / ml.

[0243] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made to it based on the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a gynecological skin care composition, wherein the drug prescription is: 120g of Acanthopanax papyriferus, 80g of Senecio scandens, and 80-20g of polysorbate; characterized in that, The preparation method of the spray is as follows: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into sections; (2) Mix Acanthus and Senecio scandens evenly, add 5 to 8 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 30 to 40℃ for 2 to 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the fermented and extracted residue, add 5 to 8 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain the aqueous extract; (4) After combining the fermentation extract and the water extract, vacuum concentration is carried out at a vacuum degree of -0.05Mpa to -0.08Mpa and a concentration temperature of 50 to 80℃ to obtain a clear extract with a relative density of 1.15 to 1.20; ethanol is added to make the alcohol content reach 90%, let stand for 24 hours, filter, recover the ethanol from the filtrate until there is no alcohol taste, add the prescribed amount of polysorbate 80, add water to 1000ml, stir well, filter, and fill into bottles to obtain the product.

2. The preparation method according to claim 1, characterized in that, The preparation method of the Fufukang drug is as follows: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into sections; (2) Mix the Acanthus chinensis and Senecio scandens segments evenly, add 6 to 8 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 35 to 40℃ for 2 to 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the fermented and extracted residue, add 6 to 8 times the amount of water and decoct twice, each time for 1 hour, combine the extracts to obtain the aqueous extract; (4) After combining the fermentation extract and the water extract, vacuum concentration is carried out at a vacuum degree of -0.06Mpa to -0.08Mpa and a concentration temperature of 60 to 80℃ to obtain a clear extract with a relative density of 1.15 to 1.18; ethanol is added to make the alcohol content reach 90%, let stand for 24 hours, filter, recover the ethanol from the filtrate until there is no alcohol odor, add the prescribed amount of polysorbate 80, add water to 1000ml, stir well, filter, and fill into bottles to obtain the product.

3. The preparation method according to claim 1, characterized in that, The preparation method of the Fufukang drug is as follows: (1) Take Justicia procumbens and Senecio scandens according to the prescription ratio and cut them into sections; (2) Mix Acanthus and Senecio scandens evenly, add 6 times the amount of water, inoculate with 5.2×100CFU / mL Monascus purpureus and Saccharomyces cerevisiae respectively, ferment at 35℃ for 3 days, centrifuge and filter to obtain fermentation extract; (3) Take the fermented and extracted residue, add 6 times the amount of water and decoct twice, 1 hour each time, combine the extracts to obtain the aqueous extract; (4) After combining the fermentation extract and the water extract, vacuum concentration was carried out at a vacuum degree of -0.06Mpa and a concentration temperature of 60℃ to obtain a clear extract with a relative density of 1.15; ethanol was added to make the alcohol content reach 90%, and the mixture was allowed to stand for 24 hours. The extract was filtered, and the ethanol was recovered from the filtrate until there was no alcohol odor. Polysorbate 80 was added in the prescribed ratio, and water was added to 1000ml. The mixture was stirred, filtered, and filled into bottles to obtain the final product.

4. A method for detecting the Fufukang spray prepared by the method of claim 1 or 3, characterized in that, The testing methods for Fufukang spray include the identification of Acanthopanax and Senecio scandens medicinal materials; and the detection of the content of rutin and hyperoside. The identification methods for the medicinal materials Acanthopanax and Senecio scandens are as follows: Take 30 ml of this product, add 30-60 ml of ethyl acetate, heat under reflux for 2-4 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of ethanol to prepare the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference materials, and prepare reference material solutions using the same method. Perform thin-layer chromatography, taking 5-10 µl of each of the above two solutions and spotting them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as a binder. Use cyclohexane-ethyl acetate-methanol in a ratio of 5:3:2 as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference materials. The method for detecting the content of rutin and hyperoside is as follows: Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol. Preparation of the test solution: Accurately measure 2-5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 60-80% methanol, weigh it, heat under reflux for 0.5-1.5 hours, cool it, weigh it again, replenish the lost weight with 60-80% methanol, shake well, filter it, and take the filtrate to obtain the test solution. Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.2–0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 mL / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows: The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

5. The detection method according to claim 4, characterized in that, The identification method for the medicinal materials *Justicia procumbens* and *Senecio scandens* is as follows: Take 30 ml of this product, add 40-50 ml of ethyl acetate, heat and reflux to extract for 2-3 hours, filter, evaporate the filtrate to dryness, add 2 ml of ethanol to dissolve the residue, and use it as the test solution; then take 2 g each of *Justicia procumbens* and *Senecio scandens* reference medicinal materials, and prepare reference medicinal material solutions using the same method. Perform thin-layer chromatography test, take 5-8 µl of each of the above two solutions, and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as the binder. Use cyclohexane-ethyl acetate-methanol in a ratio of 5:3:2 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal materials.

6. The detection method according to claim 5, characterized in that, The identification method for the medicinal materials *Justicia procumbens* and *Senecio scandens* is as follows: Take 30 ml of this product, add 50 ml of ethyl acetate, heat and reflux for 3 hours to extract, filter, evaporate the filtrate to dryness, add 2 ml of ethanol to dissolve the residue to prepare the test solution, take 2 g each of *Justicia procumbens* and *Senecio scandens* reference medicinal materials, prepare reference medicinal material solutions in the same way, and perform thin-layer chromatography test. Take 5 µl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as binder. Use cyclohexane-ethyl acetate-methanol in a ratio of 5:3:2 as the developing solvent, develop, remove, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal materials.

7. The detection method according to claim 4, characterized in that, The method for detecting the content of rutin and hyperoside is as follows: Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol. Preparation of the test solution: Accurately measure 4-5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 70-75% methanol, weigh it, heat under reflux for 0.5-1 hour, cool it, weigh it again, replenish the lost weight with 70-75% methanol, shake well, filter it, and take the filtrate to obtain the test solution. Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.4–0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 mL / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows: The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

8. The detection method according to claim 7, characterized in that, The method for detecting the content of rutin and hyperoside is as follows: Preparation of reference solutions: Take an appropriate amount of rutin reference standard, accurately weigh it, and prepare a solution containing 0.1 mg per ml with 75% methanol; take an appropriate amount of hyperoside reference standard, accurately weigh it, and prepare a solution containing 40 μg per ml with 75% methanol. Preparation of the test solution: Accurately measure 5 ml of this product and place it in a stoppered conical flask. Accurately add 25 ml of 75% methanol, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the test solution. Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol as mobile phase A and 0.6% phosphoric acid solution as mobile phase B; the flow rate was 1.0 ml / min, the detection wavelength was 360 nm, and the theoretical plate number, calculated based on the rutin peak, should be no less than 5000; gradient elution was performed as follows: The assay involves precisely pipetting 20 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

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