A pharmaceutical composition for cervical and vaginal lesions, a preparation method thereof, and use thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine, and provides a pharmaceutical composition for cervical and vaginal lesions, and a preparation method and application thereof.The pharmaceutical composition comprises, by weight fraction, 0.01-1 part of astilbin, 7.5-30 parts of sophora, 10-35 parts of houttuynia cordata, 7.5-30 parts of honeycomb, 6-24 parts of jackinthepulp, 8-24 parts of cnidium and 2.5-5 parts of oleic acid.The pharmaceutical composition has outstanding therapeutic effect on vaginal or cervical lesions caused by HPV, especially on vaginitis, vaginal and cervical intraepithelial neoplasia.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and provides a pharmaceutical composition for cervical and vaginal lesions, its preparation method and application. Background Technology

[0002] Human papillomavirus (HPV) is a type of non-enveloped, double-stranded DNA virus with epithelial affinity. After infecting a host, it can replicate extensively in the basal cells of the epithelium, genitals, and oral mucosa. When the body's immunity is reduced, it can randomly integrate into the host genome, subsequently silencing the E2 gene, thereby reducing its inhibition of the E6 and E7 genes, leading to overexpression of E6 and E7 proteins, supplemented by the expression of E5 protein, thereby inducing unlimited proliferation of infected cells and even carcinogenesis, such as vaginal cancer, cervical cancer, and other gynecological diseases.

[0003] HPV infection can cause vulvar lesions such as condyloma acuminata (genital warts) and vulvar squamous intraepithelial lesions. Condyloma acuminata is a benign wart-like growth caused by low-risk HPV types (such as types 6 and 11). It appears as cauliflower-like, cockscomb-like, or papillary protrusions with a rough surface, and commonly occurs on the vulva, vaginal opening, and cervical surface; it rarely becomes cancerous. Squamous intraepithelial lesions, on the other hand, are abnormal proliferations of cervical or vaginal mucosal epithelial cells, presenting as cervical erosion, polyps, or no obvious abnormalities. They are precancerous lesions and are associated with persistent infection with high-risk HPV types (such as types 16 and 18).

[0004] Chinese invention patent application CN1840116A discloses a topical traditional Chinese medicine for treating skin and mucous membrane diseases caused by human papillomavirus. It is an ointment made from the traditional Chinese medicines Cnidium monnieri, Podophyllum hexandrum, Brucea javanica, Smilax glabra, Houttuynia cordata, Sophora flavescens, and Vespa nidus. The efficacy of the ointment in the treatment of condyloma acuminata has been verified, with a recurrence rate of only 4.01%.

[0005] Through in-depth research on the components of the aforementioned pharmaceutical preparation, the inventors discovered that, after further extraction, each traditional Chinese medicine component exhibited significant differences in its therapeutic effects on vaginal and cervical lesions caused by HPV under different active ingredient ratios. Among these, the raw material Smilax glabra in the aforementioned external traditional Chinese medicine possesses multiple effects, including antioxidant, antitumor, anti-inflammatory, antibacterial, immunomodulatory, skin disease treatment, and kidney protection. Its main active ingredients include flavonoids, terpenes, organic acids, sterols, and saponins. Therefore, the extraction of these active ingredients or their rational formulation to maximize their efficacy is one of the research directions for further improving the efficacy of the composition.

[0006] Existing technologies (Research Progress on Pharmacological Effects of Smilax glabra, Zhao Mengjia et al., Drug Information, 2021, 10(1), 46-53) have conducted statistical studies on the active components of Smilax glabra. They found that the active components of Smilax glabra mainly include flavonoids such as astilbene, piperidin, neoastilbene, and isoflavone; organic acids such as linoleic acid and oleic acid; and phenylpropanoids such as smilax glabra glycoside C, smilax glabra glycoside D, and smilax glabra glycoside E. However, the active components of Smilax glabra have not yet been fully identified, and the pharmacological mechanisms of individual active components have not yet been fully elucidated.

[0007] Therefore, further optimization of the proportions of each group and the active ingredients is expected to further improve the therapeutic effect of existing traditional Chinese medicine compositions on vaginal or cervical lesions caused by HPV virus. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides a pharmaceutical composition for cervical and vaginal lesions, a method for preparing the composition and its application.

[0009] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a pharmaceutical composition for cervical and vaginal lesions, the pharmaceutical composition comprising, by weight parts: 0.01-1 parts of astilbene, 7.5-30 parts of Sophora flavescens, 10-35 parts of Houttuynia cordata, 7.5-30 parts of bee honeycomb, 6-24 parts of Podophyllum hexandrum, 8-24 parts of Cnidium monnieri, and 2.5-5 parts of oleic acid.

[0010] Preferably, the pharmaceutical composition comprises, by weight parts: 0.1-0.5 parts of astilbene, 15-20 parts of Sophora flavescens, 15-30 parts of Houttuynia cordata, 15-20 parts of bee honeycomb, 10-15 parts of Podophyllum hexandrum, 15-22 parts of Cnidium monnieri, and 2.5-3.5 parts of oil.

[0011] More preferably, the pharmaceutical composition comprises, by weight parts: 0.2 parts astilbin, 17.5 parts Sophora flavescens, 20 parts Houttuynia cordata, 17.5 parts Vespa nidus, 12 parts Podophyllum hexandrum, 20 parts Cnidium monnieri, and 3.5 parts oleic acid.

[0012] More preferably, the pharmaceutical composition comprises, by weight parts: 0.1 parts astilbin, 15 parts sophora flavescens, 20 parts houttuynia cordata, 15 parts bee honeycomb, 12 parts nardostachys chinensis, 15 parts Cnidium monnieri, and 2.5 parts oleic acid.

[0013] Preferably, the mass ratio of astilbene glycoside to oleic acid is 1:5-40.

[0014] More preferably, the mass ratio of astilbin to oleic acid is 1:15-25.

[0015] More preferably, the mass ratio of astilbene glycoside to oleic acid is 1:25 or 1:17.

[0016] In some specific embodiments of the present invention In a second aspect, the present invention provides a method for preparing any of the above-described pharmaceutical compositions, comprising the following steps: (1) Raw material processing: Sophora flavescens, Houttuynia cordata, bee honeycomb, Podophyllum hexandrum and Cnidium monnieri are crushed and sieved respectively; (2) The crushed Cnidium monnieri and Podophyllum hexandrum were subjected to alcohol extraction with an ethanol solution containing a solubilizer, and the residue and alcohol extract were obtained by filtration. (3) Combine the crushed Sophora flavescens, Houttuynia cordata, and honeycomb with the filter residue from step (2), add them to water and decoct. After centrifugation and filtration, obtain the decoction. (4) Mix the alcohol extract and the water extract, add astilbene and oleic acid, mix evenly, and then concentrate to obtain the final product.

[0017] Furthermore, the concentration of the solubilizer in the ethanol solution is 1-5 wt.%; the concentration of the ethanol solution is 75-85 vol.%.

[0018] Furthermore, the solubilizer is a cyclodextrin and its derivatives, specifically one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin.

[0019] In some specific embodiments of the present invention, the alcohol extraction in step (2) is performed 1-3 times, and the alcohol extraction time is 0.5-2h each time; the amount of ethanol solution added each time is 5-10 times the weight of Cnidium monnieri and Podophyllum hexandrum.

[0020] In some specific embodiments of the present invention, in step (3), the decoction is performed 1-3 times, each time for 1.5-2.5 hours, and the amount of water added each time is 5-10 times the amount of Sophora flavescens, Houttuynia cordata and bee honeycomb.

[0021] Thirdly, the present invention also provides a pharmaceutical formulation comprising any of the pharmaceutical compositions described above or a pharmaceutical composition prepared by any of the preparation methods described above, and pharmaceutically acceptable excipients.

[0022] Furthermore, the dosage form of the pharmaceutical preparation includes any one of tincture, ointment, suppository, gel, effervescent tablet, granule, and injection.

[0023] Fourthly, the present invention also provides the use of any of the above-described pharmaceutical compositions or pharmaceutical compositions prepared by any of the above-described preparation methods, or the above-described pharmaceutical preparations in the preparation of drugs for treating and / or preventing vaginal and cervical lesions.

[0024] Furthermore, the vaginal and cervical lesions include any one of vaginitis, vaginal ulcer, cervicitis, cervical ulcer, vaginal squamous intraepithelial neoplasia, and cervical squamous intraepithelial neoplasia.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention has found that in this traditional Chinese medicine composition, the active ingredient astilbin in Smilax glabra and the common active ingredient oleic acid in Smilax glabra and Brucea javanica play the role of traditional Chinese medicine in improving vaginal and cervical lesions caused by HPV. Moreover, under a specific ratio, the synergistic effect is significant, and it has excellent anti-inflammatory effect, especially in effectively improving vaginal or cervical squamous intraepithelial lesions. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1 A pharmaceutical composition for cervical and vaginal lesions, comprising the following raw materials: 0.2 parts astilbin, 17.5 parts sophora flavescens, 20 parts houttuynia cordata, 17.5 parts bee honeycomb, 12 parts dwarf spatholobus, 20 parts cnidium monnieri, and 3.5 parts oleic acid.

[0028] The preparation method is as follows: (1) Raw material processing: Take Sophora flavescens, Houttuynia cordata, honeycomb, Podophyllum hexandrum and Cnidium monnieri, crush them and pass them through a 200-mesh sieve; (2) The crushed Cnidium monnieri and Podophyllum hexandrum were extracted three times with 80% ethanol solution. The first extraction was performed with 10 times the amount of ethanol solution for 1 hour, the second extraction was performed with 8 times the amount of ethanol solution for 1 hour, and the third extraction was performed with 6 times the amount of ethanol solution for 1 hour. The three ethanol extracts were combined and filtered to obtain the residue and ethanol extract. (3) Combine the crushed Sophora flavescens, Houttuynia cordata, and honeycomb with the filter residue from step (2) and decoct in water three times. The first time, add 10 times the amount of water and decoct for 1 hour; the second time, add 8 times the amount of water and decoct for 1 hour; the third time, add 6 times the amount of water and decoct for 1 hour. Combine the three water extracts, centrifuge and filter to obtain the water decoction. (4) Mix the alcohol extract and the water decoction, add astilbin and oleic acid, stir and mix thoroughly, and then concentrate to a relative density of 1.30-1.35 to obtain the final product.

[0029] Example 2 A pharmaceutical composition for cervical and vaginal lesions has the same raw material composition as in Example 1, except that an ethanol solution containing a solubilizer (1% hydroxypropyl-β-cyclodextrin) is used in the preparation process, as detailed below: (1) Raw material processing: Take Sophora flavescens, Houttuynia cordata, honeycomb, Podophyllum hexandrum and Cnidium monnieri, crush them and pass them through a 200-mesh sieve; (2) The crushed Cnidium monnieri and Podophyllum hexandrum were extracted three times with 80% ethanol solution (containing 1% hydroxypropyl-β-cyclodextrin). The first extraction was performed with 10 times the amount of ethanol solution for 1 hour, the second extraction was performed with 8 times the amount of ethanol solution for 1 hour, and the third extraction was performed with 6 times the amount of ethanol solution for 1 hour. The three ethanol extracts were combined and filtered to obtain the residue and the ethanol extract. (3) Combine the crushed Sophora flavescens, Houttuynia cordata, and honeycomb with the filter residue from step (2) and decoct in water three times. The first time, add 10 times the amount of water and decoct for 1 hour; the second time, add 8 times the amount of water and decoct for 1 hour; the third time, add 6 times the amount of water and decoct for 1 hour. Combine the three water extracts, centrifuge and filter to obtain the water decoction. (4) Mix the alcohol extract and the water decoction, add astilbin and oleic acid, stir and mix thoroughly, and then concentrate to a relative density of 1.30-1.35 to obtain the final product.

[0030] Example 3 A pharmaceutical composition for cervical and vaginal lesions, comprising the following raw materials: 0.02 parts astilbin, 15 parts Sophora flavescens, 20 parts Houttuynia cordata, 15 parts bee honeycomb, 12 parts Podophyllum hexandrum, 15 parts Cnidium monnieri, and 2 parts oleic acid.

[0031] The preparation method is the same as in Example 2.

[0032] Example 4 A pharmaceutical composition for cervical and vaginal lesions comprises: 0.1 parts astilbin, 15 parts sophora flavescens, 20 parts houttuynia cordata, 15 parts bee honeycomb, 12 parts nardostachys chinensis, 15 parts Cnidium monnieri, and 5 parts oleic acid.

[0033] The preparation method is the same as in Example 2.

[0034] Example 5 A pharmaceutical composition for cervical and vaginal lesions comprises: 0.5 parts astilbin, 7.5 parts sophora flavescens, 10 parts houttuynia cordata, 7.5 parts bee honeycomb, 24 parts nardostachys chinensis, 24 parts cnidium monnieri, and 2.5 parts oleic acid.

[0035] The preparation method is the same as in Example 2.

[0036] Example 6 A pharmaceutical composition for cervical and vaginal lesions comprises: 1 part astilbin, 20 parts sophora flavescens, 20 parts houttuynia cordata, 20 parts bee honeycomb, 8 parts nardostachys chinensis, 8 parts Cnidium monnieri, and 2.5 parts oleic acid.

[0037] The preparation method is as follows: (1) Raw material processing: Take Sophora flavescens, Houttuynia cordata, honeycomb, Podophyllum hexandrum and Cnidium monnieri, crush them and pass them through a 200-mesh sieve; (2) The crushed Cnidium monnieri and Podophyllum hexandrum were extracted twice with 80% ethanol solution (containing 3% β-cyclodextrin). The first extraction was carried out with 10 times the amount of ethanol solution for 1.5 h, and the second extraction was carried out with 6 times the amount of ethanol solution for 1.5 h. The two ethanol extracts were combined and filtered to obtain the residue and ethanol extract. (3) Combine the crushed Sophora flavescens, Houttuynia cordata, and honeycomb with the filter residue from step (2) and decoct twice in water. The first time, add 10 times the amount of water and decoct for 2 hours, and the second time, add 6 times the amount of water and decoct for 1 hour. Combine the two water extracts, centrifuge and filter to obtain the decoction. (4) Mix the alcohol extract and the water decoction, add astilbin and oleic acid, stir and mix thoroughly, and then concentrate to a relative density of 1.30-1.35 to obtain the final product.

[0038] Comparative Example 1 A water extract thick paste was prepared according to Example 2 of Patent 200610001690.8, as follows: A pharmaceutical composition for cervical and vaginal lesions comprises: 60 parts of Smilax glabra, 30 parts of Sophora flavescens, 40 parts of Houttuynia cordata, 30 parts of Vespa nidus, 24 parts of Podophyllum hexandrum, 30 parts of Cnidium monnieri, and 10 parts of Brucea javanica.

[0039] The preparation method is as follows: (1) Dry and crush the croton seeds through a 20-mesh sieve, put them into a supercritical CO2 extraction device, and extract them at 30 MPa and 60℃ for 100 min to obtain croton seed oil and the remaining residue; (2) Take Cnidium monnieri, Podophyllum hexandrum, and the residue remaining after extracting Brucea javanica oil and extract three times with 80% ethanol solution. The first extraction is with 10 times the amount of ethanol solution for 1 hour, the second extraction is with 8 times the amount of ethanol solution for 1 hour, and the third extraction is with 6 times the amount of ethanol solution for 1 hour. Combine the three ethanol extracts and filter to obtain the residue and ethanol extract. Recover the ethanol from the ethanol extract to obtain a thick ethanol extract. (3) Take Sophora flavescens, Houttuynia cordata, Smilax glabra, bee honeycomb and the above filter residue and decoct them in water three times. The first time, add 10 times the amount of water and decoct for 2 hours. The second time, add 8 times the amount of water and decoct for 1.5 hours. The third time, add 6 times the amount of water and decoct for 1 hour. Combine the three water extracts, centrifuge, filter and concentrate to a relative density of 1.30-1.35 to obtain a thick water extract. (4) Mix the alcohol extract, water extract and Brucea javanica oil evenly to obtain the final product.

[0040] Comparative Example 2 Compared with Example 2, the only difference is the ratio of each component, which is as follows: 0.5 parts of astilbene, 10 parts of Sophora flavescens, 40 parts of Houttuynia cordata, 17.5 parts of bee honeycomb, 12 parts of Podophyllum hexandrum, 7.5 parts of Cnidium monnieri and 3.5 parts of oleic acid.

[0041] Comparative Example 3 Compared with Example 2, the only difference is the amount of astilbene and oleic acid used, specifically: 1.5 parts of astilbene and 2.5 parts of oleic acid.

[0042] Comparative Example 4 Compared with Example 2, the only difference is that oleic acid is replaced with an equal amount of linoleic acid.

[0043] Comparative Example 5 Compared with Example 2, the only difference is that astilbene is replaced with an equal amount of taurine.

[0044] Test Example 1: Effects of the drug composition on the proliferation of HaHaT cells, HeLa cells, and A431 cells (1) Experimental drugs: The drug compositions prepared in the above examples and comparative examples are added to deionized water and gradually diluted to prepare test drug solutions with a concentration of 0.05-2.5 mg / mL.

[0045] (2) Experimental method: HaCaT cells in the logarithmic growth phase were cultured in DMEM medium to form a 0.5×10⁻⁶ cell line. 6 Cell suspension at 100 μL / mL was seeded into 96-well plates and randomly divided into 13 groups. The negative control group received 100 μL of deionized water. Groups 1-6 received experimental drug solutions prepared with the drug compositions of Examples 1-6 at their respective concentrations. Groups 1-5 received experimental drug solutions prepared with the drug compositions of Comparative Examples 1-5 at their respective concentrations. A blank control group received only 100 μL of LMEM culture medium (three replicates per group). The culture plates were incubated at 37°C with 5% CO2 and saturated humidity for 24 h. Then, MTT solution (5 mg / mL) was added, and the plates were incubated for another 4 h. Cell viability was calculated using an enzyme-linked immunosorbent assay (ELISA) detector, and the IC50 was calculated. 50 value.

[0046] The IC50 values ​​of HeLa cells and A431 cells for each drug composition were analyzed using the same method. 50 The values ​​were measured, with Hela cells cultured in DMEM medium and A431 cells cultured in F12 medium.

[0047] (3) Experimental results: The IC50 values ​​of the above-mentioned drug compositions for the proliferation of HaCaT cells, HeLa cells, and A431 cells are [not specified]. 50 The values ​​are shown in Table 1.

[0048] Table 1. IC50 of each drug composition on HaCaT cells, HeLa cells, and A431 cells. 50 value

[0049] As can be seen from the table above, the drug compositions in Examples 1-6 showed positive effects on the IC50 values ​​of HaCaT cells, HeLa cells, and A431 cells. 50 The values ​​were all lower than those of comparative groups 1-5, revealing that the pharmaceutical composition prepared in this invention has outstanding performance in inhibiting the expression of HPV-related oncoproteins (such as E6 / E7) and squamous cell carcinoma of the skin (SCC).

[0050] Test Example 2: Effects of the drug composition on rats with vulvar squamous intraepithelial lesions (1) Experimental method: 1.1 Experimental animals: SPF-grade female SD rats, 6-8 weeks old, weighing 160-180g.

[0051] 1.2 Experimental drugs: The drug compositions prepared in the above examples and comparative examples were mixed with gum arabic and distilled water in a ratio of 1g:1g:0.2mL to prepare ointments; gum arabic and distilled water were mixed in a ratio of 2g:0.2mL to prepare blank matrix.

[0052] 1.3 Grouping and Dosing: SD rats were used to establish a model of vulva infection. They were anesthetized via intraperitoneal injection of 0.6% sodium pentobarbital solution. After anesthesia, the skin was shaved and cleaned using an animal shaver to fully expose the vulva. The vulvar skin was then brushed with a stiff-bristled toothbrush until it became congested and red. A small amount of 0.5% DMBA acetone solution was then applied to the vulvar skin once with a fine-tipped pen. After air-drying, the rats were returned to their normal rearing cages. Treatment was administered at fixed times on Mondays, Wednesdays, and Fridays for 14 weeks, with observation continuing until week 18. SD rats that successfully developed the model (around week 10, exophytic neoplasms of varying sizes, pale pink in color but less than 5 mm in diameter, with clear boundaries from surrounding normal tissue; around week 14, the neoplasms further enlarged, with visible surface hemorrhage and necrosis; around week 18, obvious vulvar tumors were visible, with obvious surface hemorrhage and necrosis, and the necrotic tissue easily sloughed off upon touch) were randomly divided into 12 groups of 10 rats each: the model group, Examples 1-6 groups, and Comparative Examples 1-5 groups. Ten SD rats that did not undergo modeling were used as a blank control. The SD rats in Examples 1-6 and Comparative Examples 1-5 groups were administered 0.25 g of the drug composition ointment prepared in Examples 1-6 and Comparative Examples 1-5, respectively, intravaginally. The model group and blank control group were administered 0.25 g of the blank matrix. Administration was every other day for a total of 20 days. After each administration, a cotton ball was used to fix the ointment at the vaginal opening to prevent drug leakage.

[0053] 1.4 Test Indicators: After the administration of the drug, the SD rats in each group were anesthetized and 1% toluidine blue solution was applied to the vulvar skin. Then, the skin was decolorized with 1% acetic acid. The skin tissue of the SD rats was collected from the undecolorized areas using a skin drill. The tissue was fixed with 4% paraformaldehyde tissue fixative, dehydrated, embedded in paraffin, and serially sectioned at a thickness of 4 μm.

[0054] 1.4.1 Pathological observation of the slides under a microscope was performed, and the pathological morphological evaluation criteria are as follows: ① Mucosa: Slight thickening of squamous epithelium with only 2-3 layers in some areas is marked as "+", significant thickening is marked as "++", and extensive thickening that makes the mucosa uneven is marked as "+++". ② Some columnar epithelium may show hyperplasia and a tendency to squamous transformation, marked as "+", obvious squamous transformation is marked as "++", and extensive squamous transformation area is marked as "+++"; ③ Only a very small number of squamous epitheliums showed erosion, which was replaced by the hyperplasia of adjacent columnar epithelium, which was "+". The obvious replacement of squamous epithelium by columnar epithelium was "++". Large areas of squamous epithelium were shed and replaced by columnar epithelium, which was "+++". ④; "+" indicates only a very small area of ​​erosion that breaks through the basement membrane, "++" indicates obvious mucosal deformity and necrosis, and "++++" indicates mucosal ulceration with infection.

[0055] ⑤ Inflammatory cell infiltration: A small amount of inflammatory cell infiltration is seen in the mucosa and superficial submucosa ("+"), a moderate amount of inflammatory cell infiltration is seen in the mucosa and submucosa interstitium ("++"), and a large amount of extensive inflammatory cell infiltration is seen in the submucosa and deep interstitium ("++").

[0056] ⑥ Interstitial fibroblast proliferation: "+" indicates a slight increase in fibroblasts compared to the normal control group; "++" indicates significant fibroblast proliferation with a small number of collagen fiber bundles; and "++++" indicates fibroblast proliferation accompanied by the formation of a large number of collagen fiber bundles.

[0057] Based on the above assessment criteria, the degree of pathological changes in vulvar tissue is divided into the following four levels: Normal: Only a small amount of superficial inflammatory cell infiltration was observed in the six items.

[0058] Mild: Two or more of the six items are marked "+".

[0059] Moderate: Two or more of the six items are marked with "+", and two items are marked with "++".

[0060] Severe: One of the six items has "++", two items have "++", and three items have "+"; or three items have "++".

[0061] 1.4.2 Next, after dewaxing and washing the slides, antigen retrieval was performed. The slides were placed in 3% hydrogen peroxide solution at room temperature in the dark and incubated for 25 min. Then, they were washed three times with PBS (pH 7.4), 5 min each time. 3% BSA was added for blocking at room temperature for 30 min. Primary antibodies (anti-mtp53:DO-1 antibody, 1:1000; anti-VEGF:A-20 antibody, 1:800) were added to PBS and incubated overnight at 4°C with shaking. After washing three times with PBS, secondary antibodies (HRP-labeled secondary antibody, 1:5000) were added and incubated at room temperature for 50 min. After washing three times with PBS, DAB chromogenic solution was used for development. Finally, the slides were dehydrated and mounted. Microscopic examination was performed, and optical density analysis was performed using IPP 6.0 software. Five high-power fields were randomly selected from each slide under high power, and the average optical density value was calculated. The average optical density value represents the relative expression intensity of the proteins (Mtp53 and VEGF).

[0062] 1.5 Data Statistics: All data were processed using SPSS 22.0 statistical software. Quantitative data are expressed as mean ± standard deviation. Independent samples t-tests were used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0063] (2) Experimental results: Pathological observations and results of inflammation improvement are shown in Table 2. The relative expression of Mtp53 and VEGF proteins in the vulvar skin of rats in each group are shown in Table 3.

[0064] Table 2. Pathological observations and inflammatory cell data of rats in each group.

[0065] Note: Different letters indicate significant differences.

[0066] Table 3. Relative expression levels of Mtp53 and VEGF proteins in the vulvar skin of rats in each group.

[0067] Note: Different letters indicate significant differences.

[0068] As shown in Tables 2 and 3, the model group SD rats exhibited a higher number of severe pathological changes, and the number of inflammatory cells, the relative expression levels of Mtp53 and VEGF were significantly increased compared to the blank control group, indicating that the vulvar squamous intraepithelial lesion model was successfully established in SD rats. The drug compositions of Examples 1-6 of this invention can effectively improve the degree of vulvar squamous intraepithelial lesions, reduce the degree of inflammation in SD rats with vulvar squamous intraepithelial lesions, and effectively inhibit the expression of Mtp53 and VEGF proteins in the vulvar squamous intraepithelial lesion tissue of SD rats, showing potential application in improving vulvar or cervical squamous intraepithelial lesions.

[0069] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A pharmaceutical composition for cervical and vaginal lesions, said pharmaceutical composition being made from the following raw materials in parts by weight: astilbin 0.01-1 parts, sophora flavescens 7.5-30 parts, houttuynia cordata 10-35 parts, bee honeycomb 7.5-30 parts, nardostachys chinensis 6-24 parts, cnidium monnieri 8-24 parts, and oleic acid 2.5-5 parts.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is made from the following raw materials in parts by weight: 0.1-0.5 parts astilbin, 15-20 parts sophora flavescens, 15-30 parts houttuynia cordata, 15-20 parts bee honeycomb, 10-15 parts nardostachys chinensis, 15-22 parts cnidium monnieri, and 2.5-3.5 parts oleic acid.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition is made from the following raw materials in parts by weight: 0.2 parts astilbin, 17.5 parts sophora flavescens, 20 parts houttuynia cordata, 17.5 parts bee honeycomb, 12 parts nardostachys chinensis, 20 parts cnidium monnieri, and 3.5 parts oleic acid; Or, 0.1 parts of astilbene, 15 parts of sophora flavescens, 20 parts of houttuynia cordata, 15 parts of bee honeycomb, 12 parts of dwarf spatholobus, 15 parts of cnidium monnieri and 2.5 parts of oleic acid.

4. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of astilbene to oleic acid is 1:5-50.

5. The pharmaceutical composition according to claim 4, characterized in that, The mass ratio of astilbene to oleic acid is 1:15-25.

6. The pharmaceutical composition according to claim 5, characterized in that, The mass ratio of astilbene glycoside to oleic acid is 1:17 or 1:

25.

7. The method for preparing the pharmaceutical composition according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Raw material processing: Sophora flavescens, Houttuynia cordata, bee honeycomb, Podophyllum hexandrum and Cnidium monnieri are crushed and sieved respectively; (2) Add the crushed Cnidium monnieri and Podophyllum hexandrum to an ethanol solution containing a solubilizer for alcohol extraction, and filter to obtain the residue and alcohol extract; (3) Combine the crushed Sophora flavescens, Houttuynia cordata, and honeycomb with the filter residue from step (2), add them to water and decoct. After centrifugation and filtration, obtain the decoction. (4) Mix the alcohol extract and the water decoction, add astilbene and oleic acid, mix evenly, and then concentrate to obtain the final product.

8. The preparation method according to claim 7, characterized in that, The concentration of the solubilizer in the ethanol solution is 1-5 wt.%; the concentration of the ethanol solution is 75-85 vol.%.

9. The preparation method according to claim 7, characterized in that, The solubilizer is one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin.

10. The preparation method according to claim 7, characterized in that, In step (2), the alcohol extraction is performed 1-3 times, and the extraction time is 0.5-2h each time. The amount of ethanol solution added each time is 5-10 times the weight of Cnidium monnieri and Podophyllum hexandrum. In step (3), the decoction is performed 1-3 times, and the decoction time is 1.5-2.5h each time. The amount of water added each time is 5-10 times the weight of Sophora flavescens, Houttuynia cordata and Vespa nidus.

11. A pharmaceutical preparation, characterized in that, It includes a pharmaceutical composition and pharmaceutically acceptable excipients; the pharmaceutical composition is the pharmaceutical composition according to any one of claims 1-6 or the pharmaceutical composition prepared by any one of claims 7-10; the dosage form of the pharmaceutical preparation includes any one of tincture, ointment, suppository, gel, effervescent tablet, granule and injection.

12. The use of the pharmaceutical composition according to any one of claims 1-6, the pharmaceutical composition prepared by any one of claims 7-10, or the pharmaceutical preparation according to claim 11 in the preparation of a drug for treating and / or preventing vulvar squamous intraepithelial lesions.

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