Pharmaceutical composition for cervical and vaginal lesions as well as preparation method and application of pharmaceutical composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and provides a pharmaceutical composition for cervical and vaginal lesions as well as a preparation method and application of the pharmaceutical composition. The pharmaceutical composition is prepared from the following components in parts by weight: 0.01 to 1 part of astilbin, 7.5 to 30 parts of radix sophorae flavescentis, 10 to 35 parts of herba houttuyniae, 7.5 to 30 parts of honeycomb, 6 to 24 parts of dysosma versipellis, 8 to 24 parts of fructus cnidii and 2.5 to 5 parts of oleic acid. The pharmaceutical composition disclosed by the invention has a prominent treatment effect on vagina or cervical lesions, especially vaginitis and vagina and cervical squamous intraepithelial neoplasia caused by HPV (Human Papilloma Virus).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and provides a pharmaceutical composition for cervical and vaginal lesions, a preparation method and application thereof. BACKGROUND

[0002] Human papillomavirus (HPV) is a kind of double-stranded DNA virus without envelope and with epitheliophilic characteristics. After infecting the host, it can replicate in large quantities in the basal cells of the epidermis, genitalia, oral mucosa and other parts of the epithelium. When the body's immunity is reduced, it can randomly integrate into the host genome, then silence the E2 gene, thereby reducing the inhibition of E6 and E7 genes, making E6 and E7 proteins overexpress, and assisting the expression of E5 protein, thereby inducing infected cells to proliferate indefinitely and even cancer such as gynecological diseases such as vaginal cancer and cervical cancer.

[0003] HPV infection causes lesions of the vulva such as condyloma (condyloma acuminatum) and vulvar squamous intraepithelial lesion. Among them, condyloma (condyloma acuminatum) is a benign verrucous neoplasm caused by low-risk HPV (such as types 6 and 11), which appears as a cauliflower, cockcomb or papillary protrusion, with a rough surface, and is commonly found on the vulva, vaginal orifice, cervical surface, and rarely cancer. Squamous intraepithelial lesion is an abnormal proliferation of cervical or vaginal mucosal epithelial cells, cervical erosion, polyps or no obvious abnormalities, which is a precancerous lesion and is associated with persistent infection with high-risk HPV (such as types 16 and 18).

[0004] Chinese patent application CN1840116A discloses an external use traditional Chinese medicine for treating skin and mucosal diseases caused by human papillomavirus, which is an ointment prepared from traditional Chinese medicines such as fructus caryophylli, tripteros, calophyllum inophyllum, smilax glabra, houttuynia cordata, sophora flavescens, and honeycomb, and verifies its effect in the treatment of condyloma, with a recurrence rate of only 4.01%.

[0005] Through in-depth research by the inventors on each component in the above-mentioned pharmaceutical preparation, it is found that the further extraction of each traditional Chinese medicine component and the reasonable proportioning of different active ingredients can significantly differ in the treatment of vaginal and cervical lesions caused by HPV. Among them, the raw material smilax glabra in the above-mentioned external use traditional Chinese medicine has multiple effects such as antioxidant, antitumor, anti-inflammatory and antibacterial, immune regulation, treatment of skin diseases, and protection of the kidney, and the main active ingredients include flavonoids, terpenes, organic acids, sterols, saponins and the like. Therefore, how to extract the active ingredients or how to reasonably proportion them to maximize the efficacy is one of the research directions for further providing the efficacy of the composition.

[0006] The prior art (Research Progress on Pharmacological Effects of Smilax Glabra, Zhao Mengjia et al., Drug Information, 2021, 10(1), 46-53) statistically studies the active ingredients of S. glabra, and finds that the active ingredients of S. glabra mainly include flavonoids such as astilbin, taxifolin, new isohexandrin, isohexandrin, etc.; organic acids such as linoleic acid, oleic acid, etc.; phenylpropanoids such as smilaxin C, smilaxin D and smilaxin E, etc., but the active ingredients of S. glabra have not been fully identified at present, and the pharmacological mechanism of the single active ingredient has not been fully identified.

[0007] Therefore, by further optimizing the allocation ratio of each component and the active ingredient, it is expected to further improve the treatment effect of the existing traditional Chinese medicine composition on the lesions of the vagina or cervix caused by HPV virus. SUMMARY

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

[0009] The technical scheme of the present application is as follows: In a first aspect, the present application provides a pharmaceutical composition for cervical and vaginal lesions, and the pharmaceutical composition comprises, by weight fraction, 0.01-1 parts of astilbin, 7.5-30 parts of Sophora flavescens, 10-35 parts of Houttuynia cordata, 7.5-30 parts of honeycomb, 6-24 parts of Tripterospermum chinense, 8-24 parts of Cnidium monnieri and 2.5-5 parts of oleic acid.

[0010] Preferably, the pharmaceutical composition comprises, by weight fraction, 0.1-0.5 parts of astilbin, 15-20 parts of Sophora flavescens, 15-30 parts of Houttuynia cordata, 15-20 parts of honeycomb, 10-15 parts of Tripterospermum chinense, 15-22 parts of Cnidium monnieri and 2.5-3.5 parts of oil.

[0011] More preferably, the pharmaceutical composition comprises, by weight fraction, 0.2 parts of astilbin, 17.5 parts of Sophora flavescens, 20 parts of Houttuynia cordata, 17.5 parts of honeycomb, 12 parts of Tripterospermum chinense, 20 parts of Cnidium monnieri and 3.5 parts of oleic acid.

[0012] More preferably, the pharmaceutical composition comprises, by weight fraction, 0.1 parts of astilbin, 15 parts of Sophora flavescens, 20 parts of Houttuynia cordata, 15 parts of honeycomb, 12 parts of Tripterospermum chinense, 15 parts of Cnidium monnieri and 2.5 parts of oleic acid.

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

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

[0015] More preferably, the mass ratio of the astragalin and the oleic acid is 1:25 or 1:17.

[0016] In some embodiments of the present application, In a second aspect, the present application provides a preparation method of the pharmaceutical composition as described above, comprising the following steps: (1) Raw material processing: separately crushing Sophora flavescens, Houttuynia cordata, honeycomb, Tripterygium wilfordii and Cnidium monnieri through sieving; (2) Alcohol extraction of the crushed Cnidium monnieri and Tripterygium wilfordii by adding an ethanol solution containing a solubilizing agent, and filtering to obtain a filter residue and an alcohol extract; (3) Combining the crushed Sophora flavescens, Houttuynia cordata and honeycomb with the filter residue of step (2), adding water for decoction, and centrifuging and filtering to obtain a water decoction; (4) Mixing the alcohol extract and the water extract, adding astragalin and oleic acid, and uniformly mixing and concentrating to obtain the pharmaceutical composition.

[0017] Further, the concentration of the solubilizing agent in the ethanol solution is 1-5 wt.%; and the concentration of the ethanol solution is 75-85 Vol.%.

[0018] Further, the solubilizing agent is one or more of cyclodextrin and its derivatives, such as β-cyclodextrin, hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin.

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

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

[0021] In a third aspect, the present application further provides a pharmaceutical preparation, comprising the pharmaceutical composition as described above or prepared by the preparation method as described above, and a pharmaceutically acceptable excipient.

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

[0023] In a fourth aspect, the present application further provides the use of the pharmaceutical composition as described above or prepared by the preparation method as described above, or the pharmaceutical preparation as described above, in the preparation of a drug for treating and / or preventing vaginal and cervical lesions.

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

[0025] Compared with the prior art, the present application has the following beneficial effects: It is found in the present application that the active ingredient astilbin in Smilax glabra and the common active ingredient oleic acid in Smilax glabra and Calophyllum inophyllum play the role of traditional Chinese medicine in improving the vaginal and cervical lesions caused by HPV, and the synergistic effect is significant under a specific ratio, and the present application has excellent anti-inflammatory effect, and can effectively improve the squamous intraepithelial lesions of vagina or cervix. DETAILED DESCRIPTION

[0026] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0027] Embodiment 1 A pharmaceutical composition for cervical and vaginal lesions, the raw material composition comprises: astilbin 0.2 parts, Sophora flavescens 17.5 parts, Houttuynia cordata 20 parts, honeycomb 17.5 parts, Tripterospermum 12 parts, Cnidium 20 parts and oleic acid 3.5 parts.

[0028] The preparation method is as follows: (1) Raw material treatment: crush Sophora flavescens, Houttuynia cordata, honeycomb, Tripterospermum and Cnidium, and pass through a 200-mesh screen; (2) The crushed Cnidium, Tripterospermum and 80% ethanol solution are added to extract three times, wherein 10 times the amount of ethanol solution is added for the first time for 1 h, 8 times the amount of ethanol solution is added for the second time for 1 h, and 6 times the amount of ethanol is added for the third time for 1 h; the three times of alcohol extract are combined, and the filtrate and alcohol extract are obtained by filtration; (3) The crushed Sophora flavescens, Houttuynia cordata and honeycomb are combined with the filtrate of step (2) and added to water to decoct three times, wherein 10 times the amount of water is added for the first time for 1 h, 8 times the amount of water is added for the second time for 1 h, and 6 times the amount of water is added for the third time for 1 h; the three times of water extract are combined, and the water decoction is obtained by centrifugation and filtration; (4) The alcohol extract and the water decoction are mixed, astilbin and oleic acid are added, and then fully stirred and mixed uniformly, and concentrated to a relative density of 1.30-1.35, to obtain the product.

[0029] Embodiment 2 A pharmaceutical composition for cervical and vaginal lesions, which has the same composition of raw materials as in Example 1, except that an ethanol solution containing a solubilizing agent (1% hydroxypropyl-β-cyclodextrin) is used in the preparation process, and the preparation process is as follows: (1) Raw material processing: grind Sophora flavescens, Houttuynia cordata, honeycomb, Dysosma versipellis and Cnidium monnieri into powder and pass through a 200-mesh sieve; (2) Extract the ground Cnidium monnieri, Dysosma versipellis and 80% ethanol solution (containing 1% hydroxypropyl-β-cyclodextrin) three times, wherein 10 times the amount of ethanol solution is added for the first extraction for 1 hour, 8 times the amount of ethanol solution is added for the second extraction for 1 hour, and 6 times the amount of ethanol is added for the third extraction for 1 hour; combine the three alcohol extracts, filter to obtain the filter residue and the alcohol extract; (3) Combine the ground Sophora flavescens, Houttuynia cordata, honeycomb and the filter residue of step (2) and add water to decoct three times, wherein 10 times the amount of water is added for the first decoction for 1 hour, 8 times the amount of water is added for the second decoction for 1 hour, and 6 times the amount of water is added for the third decoction 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, mix thoroughly, and concentrate to a relative density of 1.30-1.35 to obtain the product.

[0030] Example 3 A pharmaceutical composition for cervical and vaginal lesions, which has the following composition: astilbin 0.02 parts, Sophora flavescens 15 parts, Houttuynia cordata 20 parts, honeycomb 15 parts, Dysosma versipellis 12 parts, Cnidium monnieri 15 parts, and oleic acid 2 parts.

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

[0032] Example 4 A pharmaceutical composition for cervical and vaginal lesions, which has the following composition: astilbin 0.1 part, Sophora flavescens 15 parts, Houttuynia cordata 20 parts, honeycomb 15 parts, Dysosma versipellis 12 parts, Cnidium monnieri 15 parts, and oleic acid 5 parts.

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

[0034] Example 5 A pharmaceutical composition for cervical and vaginal lesions, which has the following composition: astilbin 0.5 part, Sophora flavescens 7.5 parts, Houttuynia cordata 10 parts, honeycomb 7.5 parts, Dysosma versipellis 24 parts, Cnidium monnieri 24 parts, and oleic acid 2.5 parts.

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

[0036] Example 6 A pharmaceutical composition for cervical and vaginal lesions, which has the following composition: astilbin 1 part, Sophora flavescens 20 parts, Houttuynia cordata 20 parts, honeycomb 20 parts, Dysosma versipellis 8 parts, Cnidium monnieri 8 parts, and oleic acid 2.5 parts.

[0037] The preparation method is as follows: (1) Raw material treatment: grind Sophora flavescens, Houttuynia cordata, honeycomb, Tripterygium wilfordii and Cnidium monnieri into powder and pass through a 200-mesh screen; (2) Extract the ground Cnidium monnieri and Tripterygium wilfordii twice in 80% ethanol solution (containing 3% β-cyclodextrin), wherein 10 times the amount of ethanol solution is added for the first time and extracted for 1.5 h, and 6 times the amount of ethanol solution is added for the second time and extracted for 1.5 h; combine the two alcohol extracts, filter to obtain the filter residue and the alcohol extract; (3) Combine the ground Sophora flavescens, Houttuynia cordata, honeycomb and the filter residue of step (2) and add water to decoct twice, wherein 10 times the amount of water is added for the first time and decocted for 2 h, and 6 times the amount of water is added for the second time and decocted for 1 h; combine the two water extracts, centrifuge, filter, and obtain the water decoction; (4) Mix the alcohol extract and the water decoction, add maritimeroside and oleic acid, mix thoroughly, and concentrate to a relative density of 1.30-1.35 to obtain the product.

[0038] Comparative Example 1 The water extract thick paste is prepared according to Example 2 of patent 200610001690.8, and the specific process is as follows: A pharmaceutical composition for cervical and vaginal lesions, comprising: 60 parts of Smilax glabra, 30 parts of Sophora flavescens, 40 parts of Houttuynia cordata, 30 parts of honeycomb, 24 parts of Tripterygium wilfordii, 30 parts of Cnidium monnieri and 10 parts of Calophyllum inophyllum.

[0039] The preparation method is as follows: (1) Dry and grind Calophyllum inophyllum into powder and pass through a 20-mesh screen, and then put it into a supercritical CO2 extraction device, extract at 30 MPa and 60℃ for 100 min to obtain Calophyllum inophyllum oil and the remaining residue; (2) Take Cnidium monnieri, Tripterygium wilfordii, and the remaining residue after extracting Calophyllum inophyllum oil, and extract them three times with 80% ethanol solution, wherein 10 times the amount of ethanol solution is added for the first time and extracted for 1 h, 8 times the amount of ethanol solution is added for the second time and extracted for 1 h, and 6 times the amount of ethanol is added for the third time and extracted for 1 h; combine the three alcohol extracts, filter to obtain the filter residue and the alcohol extract; recover ethanol from the alcohol extract to obtain the alcohol extract thick paste; (3) Take Sophora flavescens, Houttuynia cordata, Smilax glabra, honeycomb and the above filter residue, add water to decoct three times, wherein 10 times the amount of water is added for the first time and decocted for 2 h, 8 times the amount of water is added for the second time and decocted for 1.5 h, and 6 times the amount of water is added for the third time and decocted for 1 h; combine the three water extracts, centrifuge, filter, and concentrate to a relative density of 1.30-1.35 to obtain the water extract thick paste; (4) Mix the alcohol extract thick paste, the water extract thick paste and Calophyllum inophyllum oil, and mix thoroughly to obtain the product.

[0040] Comparative Example 2 The difference compared with Example 2 is only that the amount of each component is different, specifically: 0.5 parts of astilbin, 10 parts of Sophora flavescens, 40 parts of Houttuynia cordata, 17.5 parts of honeycomb, 12 parts of Tripterygium wilfordii, 7.5 parts of Cnidium monnieri, and 3.5 parts of oleic acid.

[0041] Comparative Example 3 The difference compared with Example 2 is only that the amount of astilbin and oleic acid is different, specifically: 1.5 parts of astilbin and 2.5 parts of oleic acid.

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

[0043] Comparative Example 5 The difference compared with Example 2 is only that the astilbin is replaced by an equal amount of taxifolin.

[0044] Test Example 1: Effect of the pharmaceutical composition on the proliferation of HaCaT cells, Hela cells and A431 cells (1) Experimental drug: The pharmaceutical composition prepared in each of the above examples and comparative examples is added to deionized water and gradually diluted to prepare a test drug solution with a concentration of 0.05-2.5 mg / mL.

[0045] (2) Experimental method: Take HaCaT cells in the logarithmic growth phase, configure them into a cell suspension of 0.5×10 6 / mL with DMEM culture solution, inoculate 100 μL into each well of a 96-well plate, and randomly divide them into 13 groups. Among them, the negative control group is added with 100 μL of deionized water, the Example 1-6 groups are added with experimental drug solutions prepared by diluting the pharmaceutical composition of each concentration of Example 1-6, the Comparative Example 1-5 groups are added with test drug solutions prepared by diluting the pharmaceutical composition of each concentration of Comparative Example 1-5, and another blank control group is added with only 100 μL of DMEM culture solution (3 replicate wells are set for each group). Place the culture plate in a 37℃, 5% CO2 saturated humidity incubator for 24 h, then add MTT solution (5 mg / mL), incubate for another 4 h, use enzyme-linked immunoassay detector to colorimetrically determine the cell survival rate of each well, and calculate the IC 50 value.

[0046] The IC 50 values of each pharmaceutical composition on Hela cells and A431 cells are determined in the same way, wherein the culture solution of Hela cells is DMEM culture solution and the culture solution of A431 cells is F12 culture solution.

[0047] (3) Experimental results: The IC 50 values of each of the above pharmaceutical compositions on the proliferation rates of HaCaT cells, Hela cells and A431 cells are shown in Table 1.

[0048] Table 1 IC values of each pharmaceutical composition on HaCaT cells, Hela cells and A431 cells 50 values

[0049] From the above table, it can be seen that the IC values of the pharmaceutical compositions of Examples 1-6 on HaCaT cells, Hela cells and A431 cells are all lower than those of Comparative Examples 1-5, which reveals that the pharmaceutical compositions prepared in the present application have outstanding performance in inhibiting the expression of HPV-related cancer proteins (such as E6 / E7) and skin squamous cell carcinoma (SCC). 50

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

[0051] 1.2 Experimental drugs: The pharmaceutical compositions prepared in the above examples and comparative examples, gum arabic and distilled water are prepared into a paste at a ratio of 1 g: 1 g: 0.2 mL; and gum arabic and distilled water are prepared into a blank matrix at a ratio of 2 g: 0.2 mL.

[0052] 1.3 Grouping and administration: ​SD rats were modeled, anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital solution, and after anesthesia, the skin was shaved with an animal hair clipper and cleaned, the vulva was fully exposed, the vulva skin was brushed with a hard bristle toothbrush until the skin was congested and red; then a hook pen was used to dip an appropriate amount of 0.5% DMBA acetone solution and apply it to the vulva skin once, and after it was naturally dried, it was put back into the conventional feeding cage for feeding. Treatment was given every Monday, Wednesday, and Friday at a fixed time, for a total of 14 weeks, and observation was continued until 18 weeks. SD rats with successful modeling (around the 10th week, small papillary neoplasms of varying sizes were visible, pink in color, but the diameter was less than 5 mm, and the boundary with the surrounding normal tissue was clear; around the 14th week, the papillary neoplasms further increased in size, and surface bleeding and necrosis were visible; around the 18th week, obvious vulvar tumors were visible, with obvious surface bleeding and necrosis, and necrotic tissue was easily detached upon contact) were randomly divided into 12 groups, 10 rats in each group, namely the model group, Examples 1-6 groups, and Comparative Examples 1-5 groups; another 10 SD rats without modeling were used as a blank control. The SD rats in Examples 1-6 groups and Comparative Examples 1-5 groups were respectively given 0.25 g of the pharmaceutical composition ointment prepared in Examples 1-6 and Comparative Examples 1-5 in the vagina, the model group and the blank control group were given 0.25 g of the blank matrix, and the drug was given every other day, for a total of 20 days. After each administration, a cotton ball was fixed to the vaginal orifice to prevent the drug from flowing out.

[0053] 1.4 Test index: After administration, the SD rats in each group were anesthetized, and 1% toluidine blue solution was applied to the vulva skin, followed by decolorization with 1% acetic acid. The non-decolorized material was taken, and the SD rat vulva skin tissue was taken with a skin drill, the tissue was fixed with 4% paraformaldehyde tissue fixative, dehydrated, paraffin-embedded, and 4 μm thick continuous sections were prepared.

[0054] 1.4.1 Pathological observation of the sections under a microscope, and the pathological morphological evaluation criteria are as follows: ① Mucosa: squamous epithelial layer only locally exceeds 2-3 layers, slightly thickened as "+", significantly thickened as "++", and widely thickened and uneven as "+++"; ② Some columnar epithelium shows a tendency to squamous metaplasia as "+", obvious squamous metaplasia as "++", and extensive squamous metaplasia as "+++"; ③ Only a few areas of squamous epithelium show erosion, which is replaced by adjacent columnar epithelium as "+", columnar epithelium replacement of squamous epithelium as "++", and large areas of squamous epithelium replaced by columnar epithelium as "+++"; ④ Only a few areas of erosion break through the basement membrane as "+", obvious mucosal deformity necrosis as "++", and mucosal ulcer with infection as "+++".

[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 deep submucosa and 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 observation and inflammatory cell data of rats in each group

[0065] Note: Different letters represent significant differences.

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

[0067] Note: Different letters represent significant differences.

[0068] As can be seen from Tables 2 and 3, in the pathological observation of the model group SD rats, the number of severe pathological changes is relatively large, and the number of inflammatory cells, the relative expression of Mtp53 and VEGF are all significantly increased compared with the blank control group, indicating that the modeling of the squamous intraepithelial lesion of the vulva of the SD rats is successful. The pharmaceutical composition of the present application Example 1-6 can effectively improve the degree of squamous intraepithelial lesion of the vulva tissue, reduce the degree of inflammation of the squamous intraepithelial lesion of the vulva of SD rats, and effectively inhibit the expression of Mtp53 and VEGF proteins in the squamous intraepithelial lesion tissue of the vulva of SD rats, and has potential application in improving the squamous intraepithelial lesion of the vulva or cervix.

[0069] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A pharmaceutical composition for cervical and vaginal lesions, said pharmaceutical composition comprising, by weight parts: Astilbene 0.01-1 part, Sophora flavescens 7.5-30 parts, Houttuynia cordata 10-35 parts, Vespa nidus 7.5-30 parts, Podophyllum hexandrum 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 comprises, by weight parts: 0.1-0.5 parts of astilbin, 15-20 parts of sophora flavescens, 15-30 parts of houttuynia cordata, 15-20 parts of bee honeycomb, 10-15 parts of nardostachys chinensis, 15-22 parts of cnidium monnieri, and 2.5-3.5 parts of oil.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition comprises, by weight parts: 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 glycoside to oleic acid is 1:5-50; preferably 1:15-25; more preferably 1:17 or 1:

25.

5. The method for preparing the pharmaceutical composition according to any one of claims 1-4, 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) 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.

6. The preparation method according to claim 5, 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.%.

7. The preparation method according to claim 5, characterized in that, The solubilizer is cyclodextrin and its derivatives, specifically one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin.

8. The preparation method according to claim 5, 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.

9. 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 1-4 or the pharmaceutical composition prepared by any one of claims 5-8; the dosage form of the pharmaceutical preparation includes any one of tincture, ointment, suppository, gel, effervescent tablet, granule and injection.

10. The use of the pharmaceutical composition according to any one of claims 1-4, the pharmaceutical composition prepared by any one of claims 5-8, or the pharmaceutical formulation according to claim 9 in the preparation of drugs for treating and / or preventing vaginal and cervical lesions, characterized in that, The vaginal and cervical lesions include any one of the following: vaginitis, vaginal ulcer, cervicitis, cervical ulcer, vaginal squamous intraepithelial neoplasia, and cervical squamous intraepithelial neoplasia.

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