CN108704020A - A kind of drug and preparation method thereof for treating leukoplakia vulvae - Google Patents

A kind of drug and preparation method thereof for treating leukoplakia vulvae Download PDF

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CN108704020A
CN108704020A CN201810875265.4A CN201810875265A CN108704020A CN 108704020 A CN108704020 A CN 108704020A CN 201810875265 A CN201810875265 A CN 201810875265A CN 108704020 A CN108704020 A CN 108704020A
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parts
medicine
vulva
weight
treatment
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李淑玲
顾珠
顾一珠
王哲
万鹏飞
王钰童
李雅琳
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Abstract

The present invention relates to a kind of drugs and preparation method thereof for treating leukoplakia vulvae.The drug of the treatment leukoplakia vulvae is obtained by the root bark of shaggy-fruited dittany, nystatin, dyclonine hydrochloride, dexamethasone, borneol, phenergan, chlormycetin powder, hydrophily emulsifiable paste matrix II by special preparation step.The drug treating both manifestation and root cause of disease of the treatment leukoplakia vulvae, toxic side effect are few, put forward a new idea for treatment leukoplakia vulvae, new method, have extensive clinical value.

Description

Medicine for treating white lesions of vulva and preparation method thereof
Technical Field
The invention relates to a medicine for treating white lesions of vulva and a preparation method thereof.
Background
Vulvar white lesions, also called chronic vulvar dystrophy, are a group of chronic diseases of female vulvar skin and mucosa tissue degeneration and pigment change, and are mainly characterized by vulvar pruritus, vulvar skin and mucosa whitening, roughness and thickening, chapping and even ulcer, often causing severe and intolerable vulvar pruritus, vulvar atrophy after long-term illness, often accompanied by vaginal stenosis and dyspareunia, and seriously affecting physical and mental health of patients. The etiology of this disease is not well defined to date. In recent years, a lot of researchers have studied the cause and mechanism of the disease, and it is considered that many factors such as low or poor conditions, hormone changes, enzyme changes, autoimmune disorders and the like are the cause of the disease. At the same time, the pathological and histological manifestations of the group of diseases are complex, so that the diseases become intractable diseases, and part of patients become cancerous due to atypical hyperplasia.
Because the etiology of the white lesions of the vulva is unknown and the disease course is long, various treatment methods exist at present, but the curative effect is not ideal. As early as 70 s, scholars thought that white lesions of the vulva were a precancerous lesion, and most of them were treated by simple vulva excision or vulva partial excision. However, it is found that the white lesions of the vulva will not become cancerous unless there is atypical hyperplasia in the lesions, and after the operation, not only the vulva scar is contracture and deformed, but also the recurrence rate after the operation can reach 50%. Therefore, conventional surgical resection treatment has been abandoned for a long time, and surgical treatment is performed on patients with atypical hyperplasia for preventing cancer changes. Thereafter, the sex hormone medicines such as glucocorticoid, testosterone propionate and progesterone are applied locally at home and abroad or the physical treatments such as laser and freezing are used for conservative treatment. After the medicine is taken, the pruritus symptom can be immediately relieved and is welcomed by patients, but the medicine needs to be applied for a plurality of times every day, and the medicine needs to be repeatedly used for a long time, and the pruritus symptom is frequently relapsed after the medicine is stopped. Prolonged continuous use of highly potent steroid formulations can lead to local skin atrophy and sometimes masculine side effects such as increased hair or increased clitoris during treatment with testosterone propionate. Particularly uncomfortable for the patient is that the vulvar skin mucosa is always difficult to restore the normal form. In view of the disadvantages of western medicines for treating white lesions of vulva, there are cases of treatment with traditional Chinese medicines, however, due to the special characteristics of long treatment course and slow effect, the patients still suffer various discomforts for a long time. The composition has good therapeutic effect on leukoplakia vulvae by using hypocrellin ointment (discussing the clinical value of hypocrellin ointment for treating leukoplakia vulvae [ J ]. China sanitary standard management, 2017,8(20):108-109.), but the composition is known to be poor in public use.
Disclosure of Invention
The invention provides a medicine for treating white lesions of vulva and a preparation method thereof, which can treat both symptoms and root causes, has little toxic and side effects and does not generate medicine dependence and solve the problems in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the medicine for treating white lesions of vulva comprises the following active ingredients by weight: 440-460 parts of dittany bark, 140-160 parts of nystatin, 40-60 parts of dyclonine hydrochloride, 10-30 parts of dexamethasone, 8-12 parts of borneol, 4-6 parts of finasteride, 75-95 parts of chloramphenicol powder and 220-240 parts of hydrophilic cream matrix II.
The medicine for treating white lesions of vulva is preferably prepared from the following raw materials in parts by weight: 450 parts of cortex dictamni, 150 parts of nystatin, 50 parts of dyclonine hydrochloride, 20 parts of dexamethasone, 5 parts of borneol 10g of finasteride (promethazine hydrochloride tablet), 85 parts of chloramphenicol powder and 230 parts of hydrophilic cream matrix II.
The hydrophilic cream matrix II comprises the following raw materials in parts by weight:
113 parts of stearic acid, 70 parts of glycerin monostearate, 55 parts of glycerin, 85 parts of white vaseline, 10 parts of sodium dodecyl sulfate and 1 part of ethyl p-hydroxybenzoate, and purified water is added to the mixture to reach 1000 parts.
The preparation method of the medicine for treating white lesions of vulva comprises the following operation steps:
(1) according to the weight parts, nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and fenoac are taken, and proper amount of ethanol is added for dissolving to obtain liquid medicine; when the temperature of the hydrophilic cream matrix II is reduced to below 50 ℃, uniformly mixing the hydrophilic cream matrix II with the liquid medicine by adopting an increasing method to obtain an ointment, and cooling the ointment for later use;
(2) according to the weight parts, adding dilute ethanol which is 8 times of the weight of the dittany bark into the dittany bark, carrying out reflux extraction for 3 times, wherein the extraction time is 2 hours, 1.5 hours and 1.5 hours respectively, filtering, combining 3 times of ethanol extract, recovering ethanol under reduced pressure, and concentrating the ethanol extract to obtain thick paste with the relative density of 1.25-1.30 for later use;
(3) and (3) mixing the ointment prepared in the step (1) and the thick paste prepared in the step (2) and uniformly mixing to obtain the ointment.
The adding amount of the ethanol in the step (1) is 3 to 13 percent of the total weight of the nystatin, the dyclonine hydrochloride, the dexamethasone, the borneol, the chloromycetin powder and the fenoac.
The relative density in the step (2) is a relative density value measured at 50 ℃.
The invention has the beneficial effects;
the medicine for treating white lesions of vulva adopts a scheme of combined Chinese and western medicine treatment, and the nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder, finasteride and hydrophilic cream matrix II are matched with the alcohol extract of the cortex dictamni as a traditional Chinese medicine component, so that the medicine has the advantages of rigorous prescription, rich medicine sources, low price, convenient use, definite curative effect and no obvious toxic or side effect, most importantly, the medicine can avoid operative treatment for patients, fills the blank that no ideal externally-used Chinese patent medicine exists for treating the precancerous lesions of the white lesions of vulva at present, and is a good medicine for treating the precancerous lesions of the white lesions of vulva; the components are synergistic, so that the traditional Chinese medicine composition is easy to permeate into the dermis of the skin, quick in effect, lasting in effect and obvious and definite in clinical curative effect, can improve the immune function and the oxidation resistance of a patient, and is convenient to use. The medicine for treating white lesions of the vulva can effectively treat white lesions of the vulva with various degrees, is characterized by obviously relieving clinical symptoms, improving blood circulation of lesion parts, changing the ultrastructure of skin tissue cells, increasing mitochondria, reducing ribosome and changing lesion skin into normal, and is an ideal medicine for treating precancerous lesions of the white lesions of the vulva.
Compared with compound nystatin, the medicine for treating leukoplakia vulvae has obvious improvement on vulva pruritus, vulva hypopigmentation and atrophy, and has the function of regulating the immune function of an organism. The pathological and electron microscope observation results show that the curative effect of the medicine for treating white lesions of the vulva is consistent with the clinical curative effect, and the medicine for treating the white lesions of the vulva also has the curative effect of treating precancerous lesions, can effectively treat various types of white lesions of the vulva, has no toxic or side effect after long-term external use, and is characterized by obviously relieving the clinical symptoms, improving the blood circulation of lesion parts, changing the ultrastructure of skin tissue cells, increasing mitochondria, reducing the ribosome and changing the lesion skin to be normal, thereby being an ideal medicine for treating the white lesions of the vulva. The research of the medicine for treating white lesions of the vulva provides a new idea and a new method for treating white lesions of the vulva, and has wide clinical application value.
Detailed Description
In order to clearly illustrate the technical features of the present invention, the present invention is explained in detail by the following embodiments.
Example 1
The effective components of the medicine for treating white lesions of vulva are prepared from the following raw materials by weight: 450g of dittany bark, 150g of nystatin, 50g of dyclonine hydrochloride, 20g of dexamethasone, 10g of borneol, 5g of finasteride, 85g of chloromycetin powder and 230g of hydrophilic cream matrix II;
the hydrophilic cream matrix II is prepared from the following raw materials in parts by weight:
113g of stearic acid, 70g of glycerin monostearate, 55g of glycerin, 85g of white vaseline, 10g of sodium dodecyl sulfate and 1g of ethyl p-hydroxybenzoate, and purified water is added to reach 1000 g.
The preparation method of the medicine for treating white lesions of vulva comprises the following operation steps:
(1) according to the weight, nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and finasteride are taken, and 25.6g of ethanol is added to dissolve the nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and finasteride to obtain liquid medicine; taking 230g of hydrophilic cream matrix II, cooling to below 50 deg.C, mixing with hydrophilic cream matrix II by increasing method to obtain unguent, and cooling;
(2) according to the weight, adding diluted ethanol which is 8 times of the weight of the dittany bark into the dittany bark, carrying out reflux extraction for 3 times, wherein the extraction time is 2 hours, 1.5 hours and 1.5 hours respectively, filtering, combining 3 times of ethanol extract, recovering ethanol under reduced pressure, and concentrating to obtain thick paste with the relative density of 1.25-1.30 (measured at 50 ℃), for later use;
(3) and (3) mixing the ointment prepared in the step (1) and the thick paste prepared in the step (2), uniformly mixing, subpackaging, sterilizing and inspecting the quality to obtain the ointment.
Example 2
The effective components of the medicine for treating white lesions of vulva are prepared from the following raw materials by weight: 440g of dittany bark, 140g of nystatin, 40g of dyclonine hydrochloride, 10g of dexamethasone, 8g of borneol, 4g of finasteride, 75g of chloromycetin powder and 220g of hydrophilic cream matrix II;
the composition of the hydrophilic cream base II was the same as that of the hydrophilic cream base II of example 1.
The preparation method of the medicine for treating white lesions of vulva comprises the following operation steps:
(1) according to the weight, nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and finasteride are taken, and 8.31g of ethanol is added to dissolve the nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and finasteride to obtain liquid medicine; taking 220g of hydrophilic cream matrix II, cooling to below 50 deg.C, mixing the medicinal liquid with the hydrophilic cream matrix II by incremental method to obtain unguent, and cooling;
(2) according to the weight, adding diluted ethanol which is 8 times of the weight of the dittany bark into the dittany bark, carrying out reflux extraction for 3 times, wherein the extraction time is 2 hours, 1.5 hours and 1.5 hours respectively, filtering, combining 3 times of ethanol extract, recovering ethanol under reduced pressure, and concentrating to obtain thick paste with the relative density of 1.25-1.30 (measured at 50 ℃), for later use;
(3) and (3) mixing the ointment prepared in the step (1) and the thick paste prepared in the step (2), uniformly mixing, subpackaging, sterilizing and inspecting the quality to obtain the ointment.
Example 3
The effective components of the medicine for treating white lesions of vulva are prepared from the following raw materials by weight: 460g of dittany bark, 160g of nystatin, 60g of dyclonine hydrochloride, 30g of dexamethasone, 12g of borneol, 6g of finasteride, 95g of chloromycetin powder and 240g of hydrophilic cream matrix II;
the composition of the hydrophilic cream base II was the same as that of the hydrophilic cream base II of example 1.
The preparation method of the medicine for treating white lesions of vulva comprises the following operation steps:
(1) according to the weight, nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and finasteride are taken, and 47.2g of ethanol is added to dissolve the nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and finasteride to obtain liquid medicine; taking 240g of hydrophilic cream matrix II, cooling the hydrophilic cream matrix II to below 50 ℃, uniformly mixing the liquid medicine and the hydrophilic cream matrix II by adopting an increasing method to obtain an ointment, and cooling the ointment for later use;
(2) according to the weight, adding diluted ethanol which is 8 times of the weight of the dittany bark into the dittany bark, carrying out reflux extraction for 3 times, wherein the extraction time is 2 hours, 1.5 hours and 1.5 hours respectively, filtering, combining 3 times of ethanol extract, recovering ethanol under reduced pressure, and concentrating to obtain thick paste with the relative density of 1.25-1.30 (measured at 50 ℃), for later use;
(3) and (3) mixing the ointment prepared in the step (1) and the thick paste prepared in the step (2), uniformly mixing, subpackaging, sterilizing and inspecting the quality to obtain the ointment.
Pharmacodynamic study:
first, experimental material
1. Experimental animals: 50 Wistar rats with weight of 180-220 g and male sex; 50 Kunming mice, 18-22 g weight, male.
2. Medicine preparation: chloral hydrate (Hospital, Jinan Qianfushan, Shandong, lot number: 080812),
aminopyridine (Merck, batch No.: 0000817),
5% 5-Fluorouracil (5-FU) (Shandong U anti-medicine GmbH, Lot No. 0809221),
sodium sulfide (Tianjin Baishi chemical Co., Ltd., batch No. 20030811),
diethyl ether (Tianjin, Guanghua Chemicals Co., Ltd., batch No. 20060425),
penicillin (Shandong anti-medicine Co., Ltd., batch No. S070923),
the invention relates to a medicine for treating white lesions of vulva (hereinafter, the medicine is called the invention for short)
3. Laboratory instruments electronic balance (Shanghai balance instruments, FA1104),
oven (China republic of people Shanghai laboratory instruments general factory, 101A-Z)
Second, Experimental methods
(I) anti-inflammatory assay
50 rats are randomly divided into 5 groups, a control group is coated with 0.5 g/day of vaseline, a western medicine is coated with 5% of 5-fluorouracil (5-FU) for 0.5 g/day, a large-dose group is coated with 2 g/day of the medicine of the invention, a medium-dose group is coated with 1 g/day of the medicine of the invention, a small-dose group is coated with 0.5 g/day of the medicine of the invention, the medicine is continuously applied for 3 days, the rats are anesthetized after the third administration for 40min, the inguinal region is depilated by a blade, the iodine tincture is disinfected, the incision is 1cm, 30mg of sterile cotton balls are placed under the skin, the suture is carried out, and the rats are raised in a single. Dosing was continued on day four for up to day 7. After 2 hours of administration at 7d, the animals were anesthetized, the cotton balls were carefully separated and removed, oven-dried at 50 ℃ to obtain the total weight of the cotton balls, the total weight of the cotton balls was reduced by the original weight of the cotton balls, and then the granuloma dry weight of 100g body weight (g granuloma/100 g body weight) was calculated and the differences between the groups were compared.
(II) test for relieving itching
50 Kunming mice, 18 g-22 g, are randomly divided into 5 groups, the backs of the mice are unhaired by 8% sodium sulfide for 2cm multiplied by 2cm, the mice are respectively administrated, a control group is coated with vaseline for 0.5 g/day, a western medicine group is coated with 5% 5-fluorouracil (5-FU) for 0.5 g/day, a large dose group is coated with the medicine of the invention for 0.9 g/day, a medium dose group is coated with the medicine of the invention for 0.6 g/day, a small dose group is coated with the medicine of the invention for 0.3 g/day, the continuous administration time is 7 days, after the last administration for 40min, 4-aminopyridine lmg/kg is injected subcutaneously to cause itching, after 10s, the times of licking the body within 10min are recorded, and the difference between the groups is compared.
(III) results of the experiment
1. Compared with a control group, the large and medium dose groups of the drug have extremely obvious inhibition effect on rat cotton ball granuloma, while western drug groups have no obvious difference, which shows that the drug is superior to western drug 5% 5-fluorouracil (5-FU) and has better anti-inflammatory effect.
TABLE 1 Effect of the drugs of the present invention on granulation inhibition in rats
Group of Number of animals Dried weight of granulation (g/100g body weight)
Control group 8 1.9±0.29
Western medicine group 9 1.8±1.8
High dose group 8 1.32±0.25
Middle dose group 9 1.42±0.17
Low dose group 8 1.87±0.23
Compared with the control group, P is less than 0.01, and the difference is significant.
2. The antipruritic effect of the medicament is shown in table 2, and the large and medium dosage groups of the medicament have better prevention and treatment effects on the itch caused by the allergy caused by the 4-aminopyridine and have obvious dose-effect relationship. The large dose group of the medicine of the invention has similar results with the western medicine 5% 5-fluorouracil (5-FU) group.
TABLE 2 Effect of the drugs of the present invention on the cutaneous pruritus response in mice (X. + -. s)
Group of Number of animals Number of times of scratching
Control group 10 37.9±3.1
Western medicine group 10 16.3±3.7
High dose group 10 17±2
Middle dose group 10 24.7±3.1
Low dose group 10 38±3.6
Compared with the control group, P <0.01, there was a significant difference.
(IV) conclusion of the experiment
The pharmacodynamic experiment takes Wistar rats and Kunming mice as research objects, and the anti-inflammatory and antipruritic effects of the medicine are observed through a cotton ball granulation test and a 4-aminopyridine allergy itch-causing test, and the results show that:
1. the drug can obviously inhibit Wistar rat cotton ball granuloma and has better anti-inflammatory effect.
2. The medicine has better prevention and treatment effects on the itching caused by the allergy caused by the 4-aminopyridine, and has obvious itching relieving effect.
Experimental study:
the research aims are as follows: the influence of the medicine on the cellular immunity, the humoral immunity and the local skin tissue immunity change of a patient and the influence on the oxidation and oxidation resistance balance of an organism are researched, and the action mechanism of the medicine for treating the precancerous lesions of the white lesions of the vulva is discussed.
First, experimental reagent, instrument and method
(I) Primary reagent
SOD reagent box, purchased from Nanjing to build the bioengineering institute; the MAD kit is purchased from Nanjing to build a bioengineering institute; paraffin wax, commercial formaldehyde (about 40%), absolute ethanol, 95% ethanol, 85% ethanol, acetone, xylene, hematoxylin, eosin, neutral resin, all provided by the pathology department of the Qilu hospital, Shandong university. Mouse anti-human CD3, CD4, CD8, CD45R0, CD68, HLA-DR monoclonal antibody, PV-9000 immunohistochemical detection kit, DNA kit and PBS buffer were purchased from Beijing Zhongshan Jinqiao Biotechnology Co.
(II) Main instruments and apparatus
FACScalibur flow cytometer, Becton-Dickinson, USA
722 spectrophotometer, Shanghai Coli instruments Ltd
Low temperature refrigerator at-70 ℃ SANYO
Paraffin slicer, Leica instruments Co, Germany
Micropipette, Gilson Corp, USA
Vacuum oven (oven) for drying Shuangying, san Shui scientific instruments ltd, Tianjin
Electrothermal constant-temperature incubator, Shandong Weifang medicine group limited medical apparatus and instruments factory
DKS-28 digital display water bath, Hangzhou blue sky laboratory test Instrument factory
Microwave oven, U.S. Corp
Desk centrifuge, Shanghai Luxiang apparatus centrifuge Instrument Co Ltd
Small three-purpose water tank, Beijing Western-style district medical instrument factory
Dyeing jar, Tianjin City restricted liability company for science and technology of secondary products
CAT.NO.710 slide
Light microscope, photomicrograph, image analysis system, Olympus, japan.
(III) method of experiment
1. Detection of serum T cell subsets CD3+, CD4+, CD8+ and immunoglobulin IgG, IgA, lgM and SOD, MDA
(1) Collecting a specimen: venous blood of the study subjects was collected at 6mL on an empty stomach in the morning. After 3mL of the serum is subjected to anticoagulation treatment, the serum is sent to a central laboratory to detect T cell subsets CD3+, CD4+, CD8+ and immunoglobulin lgG, lgA and IgM, and after 3mL of the serum is separated, the serum is stored at 0-4 ℃ to be detected as SOD and MDA;
(2) detecting CD3+, CD4+ and CD8+ by an immunofluorescence flow type fine treadmill according to the instructions; the fluorescent dye is Propidium Iodide (PI) produced by Sigma company in America; the RNase is produced by Sigma company of America;
(3) detection of peripheral serum immunoglobulins lgG, lga.igm: the kit provided by Backman company of America is used for turbidimetric determination on an ARRAY 360 SYSTEM instrument;
(4) SOD activity determination: using nitrite method, the activity of which is based on nitrite unit (Nu/ml) in each milliliter of serum as activity unit, adopting S0D kit and S0D color immune plate purchased from Nanjing institute of bioengineering;
(5) MDA detection: by a thiobarbituric acid method (TBA method), MDA in the lipid peroxide degradation product can be condensed with thiobarbituric acid to form a red product, the maximum absorption peak is at 532nm, the content (mu mol/L) is measured by a 722 spectrophotometer, and the kit is provided by Nanjing institute of bioengineering, and strict eucalyptus illumination operation is performed;
2. detection of distribution and quantity of local tissues CD3, CD4, CD8, CD68, HLA-DR and observation of tissue structure change
(1) Collecting and processing a specimen: the selected 102 patients were subjected to vulvar skin biopsy before and after treatment at the same part, wherein the normal control group comprises 10 patients, the patients are from normal vulvar skin tissues of hand trees of female pudendum, the age is 26-68 years, the average age is 36.12 +/-7.35 years, all tissue samples are fixed by 10% formalin, embedded by conventional paraffin, and sliced continuously, and the slice thickness is 4 μm;
(2) HE staining
Baking the 1 set of slices in a constant temperature oven at 60 ℃ for 60min, and using a manually operated hematoxylin and eosin staining method, wherein the staining steps are as follows: xylene I dewaxing 10min → xylene II dewaxing 5min → absolute ethyl alcohol II1min → 95% ethyl alcohol 1min → 85% ethyl alcohol 1min → tap water washing 2min → distilled water washing lmin hematoxylin staining 5min → tap water washing lmin → 1% ethyl alcohol differentiation 20S → tap water washing lmin → dilute ammonia (1%) bluing 30S → tap water washing lmin → eosin staining 20S → tap water washing 30S → 85% ethyl alcohol dehydration 20S → 95% ethyl alcohol I lmin → 95% ethyl alcohol II1min → absolute ethyl alcohol I2min → absolute ethyl alcohol II2min → neutral gum sealing;
(3) immunohistochemical staining
Staining was performed by immunohistochemical staining in histopathological techniques, using monoclonal antibodies CD3, CD4, CD8, CD68, HLA-DR as lymphocyte subpopulation markers, and semi-quantitative analysis was performed. Positive control and negative control are set in the whole experiment process, and the experiment steps are as follows:
paraffin sections were dewaxed to water and washed three times with PBS (PH7.4) for five minutes each time;
secondly, performing antigen retrieval by using 0.1mol/L citric acid buffer solution with pH of 6.0, putting 500mL of retrieval solution into a 1000mL beaker, heating the beaker in a microwave oven until the solution boils slightly, putting the tissue slices on a high-temperature-resistant plastic frame, slowly putting the plastic frame into the beaker, heating the beaker for 5 minutes, taking out the tissue slices, naturally cooling the tissue slices to room temperature, taking out the tissue slices, and washing the tissue slices with PBS for three times, five minutes each time;
③ dropping a drop of 3% hydrogen peroxide solution to each section, incubating for 10min at room temperature to block the activity of endogenous peroxidase, then washing three times with PBS, five minutes each time.
dripping one drop of non-immune goat serum into each slice, incubating for 10 minutes at room temperature, and sealing non-specific antigen sites;
pouring out excessive serum, dripping a drop of primary antibody on the slice, placing the slice in a wet box, and standing overnight in a refrigerator at 4 ℃;
sixthly, after the temperature is returned to the room temperature, washing the solution with PBS for three times, and each time for five minutes;
seventhly, dropwise adding a reagent 1(polymer helper), incubating for 20 minutes at 37 ℃, washing for three times by PBS (phosphate buffer solution) for 5 minutes each time;
dropping reagent 2(polyperoxidase-anti-mouse IgG), incubating for 30 minutes at 37 ℃, washing for three times with PBS (phosphate buffer solution), and each time for five minutes;
⑨, DAB color development, observing for 3-10 minutes under a microscope, and stopping full water washing;
redyeing the haematoxylin at the red junction for 30 seconds, differentiating by hydrochloric acid alcohol for 3 seconds, and performing ammonia water anti-blue reaction for 15 seconds.
Immunohistochemical results determination and quantification methods: after DAB coloration, positive cells are calculated, cells with brown granular-like substances appearing on cytoplasm or cell membranes are taken as positive cells, signal expression is positive when the number of the positive cells is more than or equal to 10%, and expression is negative when the number of the positive cells is less than 10%.
3. Observing the change of the ultrastructure of the tissue before and after the treatment under an electron microscope
After the skin biopsy is isolated, the tissue is quickly washed with 0.1M Phosphate Buffer (PB) to remove contaminants such as blood around the tissue, 3% glutaraldehyde is added immediately, and the sample is trimmed to a cross section of 1mm after 5 minutes2Strip tissue of 2-3mm length was placed in fresh glutaraldehyde fixative and fixed at 4 ℃ for 2 hours, 1% osmic acid (OsO4 fixed for 1 hour, gradient ethanol and acetone dehydration, Epon812(SP I, USA) infiltration, embedding, blue staining of toluidine in semi-thin section, optical lens positioning of capillary vessels in basement membrane and dermis superficial layer, double staining of ultrathin section with lead and uranium, and H-800 transmission electron microscopy (Hitachi, Tokyo, Japan) for observation and recording.
4. Statistical method
The experimental data were processed using SPSS13.0 software and the comparison between the rates was examined using X2. Two groups of comparisons were performed using t-test and the inter-group comparisons were performed using analysis of variance. The grade data is represented by the sum of rank test and the measurement data is represented by the mean plus minus standard deviation. The difference is significant when P is less than 0.05.
Second, result in
1. The research shows that the patients with the precancerous lesions of the white vulva lesions have the cell immune function disorder, the CD3 and the CD4 are reduced, the CD8 is increased, the CD4/CD8 is reduced, and the difference is statistically significant compared with the normal value, wherein P is less than 0.05. The levels of CD3, CD4, CD8 and CD4/CD8 are not obviously different between two groups before treatment, and P is greater than 0.05 and is comparable. After treatment, the treatment group has the advantages of increased CD3 and CD4, reduced CD8 and increased CD4/CD8, the comparison difference before and after treatment has statistical significance, P is less than 0.05, and the comparison between the treatment group and a normal value has no significant difference, and P is more than 0.05. The indexes of the control group have no obvious difference before and after treatment, and P is more than 0.05. The comparative differences of CD3, CD4, CD8 and CD4/CD8 after the treatment of the two groups have statistical significance, and P is less than 0.05. Therefore, the following results can be obtained: after treatment, the treated group obviously improves the cell immune disorder of the patients, while the control group does not have the effect. As in table 3 below:
TABLE 3 comparison of T cell subset detection results before and after treatment for the treatment groups and the control group
Analysis of variance shows that the difference between the treatment group and the treatment group has statistical significance, P is less than 0.05; the difference compared to normal values was not statistically significant, P > 0.05. The control group had no statistical difference from the pre-treatment comparison,. P > 0.05; the difference compared to normal values is statistically significant, P < 0.05.
2. Comparison of serum IgG, IgA, and IgM results before and after treatment
The patients with the white lesions of the vulva and the precancerous lesions have the dysregulation of humoral immunity function, the IgG level is reduced, the difference compared with a normal control group has statistical difference, and the P is less than 0.05. There was no significant difference in lgG levels between the two groups before treatment, P >0.05, comparable. After treatment, IgG of the treatment group is increased, comparison difference before and after treatment has statistical significance, P is less than 0.05, and the comparison with normal value after treatment has no significant difference, and P is more than 0.05. The indexes of the control group have no obvious difference before and after treatment, and P is more than 0.05. (see Table 4)
TABLE 4 comparison of serum IgG, IgA, IgM results before and after treatment in the treatment group with those in the control group
Note: p <0.05 compared to normal; p <0.05 compared to pre-treatment.
3. Comparison of serum superoxide dismutase and malondialdehyde results before and after treatment in the treatment group and the control group
The research shows that the patient with white vulva lesion precancerous lesion has the catalpa rapae with oxidation and oxidation resistance, and compared with a normal control group, the patient before treatment has the statistical significance of the reduction of the serum SOD level, the increase of the MDA level and the difference (P is less than 0.05). The treated group can obviously increase the serum SOD level and reduce the MDA level, and the difference has statistical significance (P is small and dry 0.05). Compared with the control group before treatment, the differences of the serum SOD and MDA levels after treatment have no statistical significance (P is more than 0.05). See table 5:
TABLE 5 comparison of serum superoxide dismutase and malondialdehyde results before and after treatment in the treatment groups with those in the control group
Note that P is less than 0.05 before treatment compared to normal group, P is less than 0.05 after treatment compared to before treatment, and △ P is less than 0.05 compared to control group by U test.
4. Comparing the expression of CD3, CD4, CD8, CD68 and HLA-DR in the treatment group before and after treatment with that in the control group
The study also investigated the immune function imbalance in patients with precancerous lesions of white lesions of the vulva locally from lesions of the skin tissue. The characteristics of inflammatory infiltration of premalignant lesions of vulvar white lesions associated with skin lesions and the effects of treatment on this were studied using immunohistochemical methods.
A large number of CD4+, CD8+ lymphocytes were found to be present in the dermal inflammatory cell infiltration zone in nearly equal proportion, and a small number of CD4+, CD8+ lymphocytes were found near the epidermal dermal junction, occasionally in the sub-epidermal layer. A large number of cells stained with the mcro fine band marker CD68 were present in the inflammatory cell wetting zone and widely scattered in the hardened area of the dermis. While HLA-DR expression is increased in dermal inflammatory cell infiltration and perivascular. Some HLA-DR expression was seen around keratinocytes, suggesting that these keratinocytes may be involved in antigen presentation. The results of this study show that there are immune alterations in the skin layers, both epidermis and dermis, of the lesions in the outer-cloudy-white lesions. The medicine can be used for treating and regulating the immune function of the organism. In the experimental results, the difference between the expression conditions of CD3 and CD8 in the treatment group before and after treatment and the control group has statistical significance, and P is less than 0.05; the differences between the two groups compared after treatment were not statistically significant, P > 0.05. The difference between the expression of CD4 in the treated group before and after treatment and the expression of CD4 in the control group after treatment and the difference between the two groups after treatment have statistical significance, and P is less than 0.05. The comparative difference of the expression conditions of CD68 and HLA-DR before and after treatment in the treatment groups has statistical significance, and P is less than 0.05; the comparative difference of the expression conditions of CD68 and HLA-DR before and after treatment of the control group has no statistical significance, and P is more than 0.05.
And (3) clinical observation:
after 2 years of clinical effect observation and follow-up visit. During application, the medicine disclosed by the invention can effectively relieve pruritus vulvae, can restore normal elasticity of vulvar pathological skin and mucosal tissues, and can increase the pigmentation of vulva so that the white spot color gradually tends to be normal; the follow-up observation shows that the medicine of the invention has the functions of preventing and treating white lesions and precancerous lesions of vulva, and the medicine of the invention is externally used for a long time without any toxic and side effect. Through clinical observation of Qilu Hospital in Shandong province, Jinan City, affiliated Hospital of Shandong Chinese medicine university and Qianfoshan Hospital in Shandong province, the total effective rate is 89.78% on average.
Clinical and experimental researches prove that the medicament has obvious clinical curative effect on treating white lesions and precancerous lesions of vulva. Experimental research shows that the medicine has the functions of regulating the immune function of an organism and improving the balance of oxidation and oxidation resistance. The pathological and electron microscope observation results show that the curative effect of the medicine for treating white lesions of vulva is consistent with the clinical effect, and simultaneously show that the medicine has the curative effect of preventing precancerous lesions from further malignant changes. The medicine can effectively treat vulva white lesions of various degrees, is characterized by obviously relieving clinical symptoms, improving blood circulation of lesion parts, changing the ultrastructure of skin tissue cells, increasing mitochondria, reducing ribosome and normalizing lesion skin, and is an ideal medicine for treating precancerous lesions of the vulva white lesions. The medicine has the advantages of strict formula, rich medicine sources, low price, convenient use, definite curative effect and no obvious toxic or side effect, and most importantly, the medicine can avoid surgical treatment for patients, fills the blank that no ideal externally-applied Chinese patent medicine is available for treating the precancerous lesions of the white lesions of the vulva at present, and is a good medicine for treating the precancerous lesions of the white lesions of the vulva.
Comparative experiment:
the medicine of the invention is compared with the existing medicine for treating white lesions of vulva for observation of effect; counting according to the standards of healing, obvious effect, effectiveness and ineffectiveness; wherein,
the healing judgment standard is as follows: the symptoms disappear, and the color of vulvar skin mucous membrane is normal;
the judgment standard of the display effect is as follows: the symptoms disappear or the patient is occasionally itchy, the white lesion area is greatly reduced, the tissue becomes soft, and the adhesion is loosened;
the effective judgment standard is as follows: the symptoms are reduced, and the white lesion area is reduced;
the invalid judgment standard is as follows: no improvement in symptoms.
The existing medicine for treating white lesions of vulva is recorded as a control group, and the formula is as follows: the traditional Chinese medicine composition comprises 50g of cortex dictamni, fructus psoraleae and fructus cnidii powder, 5 million units of nystatin tablets, 3g of dyclonine, 0.3g of dexamethasone powder, 20g of sublimed sulfur, 2g of borneol, 3g of promethazine, 80g of urea, 4g of chloromycetin powder, 50g of glyceryl monostearate, 100g of stearic acid, 50g of white vaseline, 150g of liquid paraffin, 100g of glycerol, 2g of sodium dodecyl sulfate, 2mL of triethanolamine, 1g of ethylparaben and a proper amount of distilled water; the results are given in table 6 below:
TABLE 6
From the above table 6, the medicine of the present invention has a significant therapeutic effect on white lesions of vulva compared with the existing medicines.
The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.
The present invention is not described in detail, but is known to those skilled in the art.

Claims (6)

1. A medicine for treating white lesions of vulva is characterized in that: the active ingredients of the medicine are prepared from the following raw materials in parts by weight: 440-460 parts of dittany bark, 140-160 parts of nystatin, 40-60 parts of dyclonine hydrochloride, 10-30 parts of dexamethasone, 8-12 parts of borneol, 4-6 parts of finasteride, 75-95 parts of chloramphenicol powder and 220-240 parts of hydrophilic cream matrix II.
2. The medicine for treating white lesions of the vulva as claimed in claim 1, characterized in that: the active ingredients are prepared from the following raw materials in parts by weight: 450 parts of cortex dictamni, 150 parts of nystatin, 50 parts of dyclonine hydrochloride, 20 parts of dexamethasone, 10g of borneol, 5 parts of finasteride, 85 parts of chloromycetin powder and 230 parts of hydrophilic cream matrix II.
3. The medicament for treating white lesions of the vulva as claimed in claim 1 or 2, characterized in that: the hydrophilic cream matrix II comprises the following raw materials in parts by weight:
113 parts of stearic acid, 70 parts of glycerin monostearate, 55 parts of glycerin, 85 parts of white vaseline, 10 parts of sodium dodecyl sulfate and 1 part of ethyl p-hydroxybenzoate, and purified water is added to the mixture to reach 1000 parts.
4. A method for preparing a medicament for the treatment of leukoplakia vulvae as claimed in claim 1 or 2, characterized in that: the method comprises the following operation steps:
(1) according to the weight parts, nystatin, dyclonine hydrochloride, dexamethasone, borneol, chloromycetin powder and fenoac are taken, and proper amount of ethanol is added for dissolving to obtain liquid medicine; when the temperature of the hydrophilic cream matrix II is reduced to below 50 ℃, uniformly mixing the hydrophilic cream matrix II with the liquid medicine by adopting an increasing method to obtain an ointment, and cooling the ointment for later use;
(2) according to the weight parts, adding dilute ethanol which is 8 times of the weight of the dittany bark into the dittany bark, carrying out reflux extraction for 3 times, wherein the extraction time is 2 hours, 1.5 hours and 1.5 hours respectively, filtering, combining 3 times of ethanol extract, recovering ethanol under reduced pressure, and concentrating the ethanol extract to obtain thick paste with the relative density of 1.25-1.30 for later use;
(3) and (3) mixing the ointment prepared in the step (1) and the thick paste prepared in the step (2) and uniformly mixing to obtain the ointment.
5. The method for preparing a medicine for treating white lesions of vulva according to claim 4, characterized in that: the adding amount of the ethanol in the step (1) is 3 to 13 percent of the total weight of the nystatin, the dyclonine hydrochloride, the dexamethasone, the borneol, the chloromycetin powder and the fenoac.
6. The method for preparing a medicine for treating white lesions of vulva according to claim 4, characterized in that: the relative density in the step (2) is a relative density value measured at 50 ℃.
CN201810875265.4A 2018-08-03 2018-08-03 A kind of drug and preparation method thereof for treating leukoplakia vulvae Pending CN108704020A (en)

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CN113134085A (en) * 2020-01-17 2021-07-20 四川大学华西第二医院 Medicine for treating white lesions of vulva

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CN104258232A (en) * 2014-10-11 2015-01-07 青岛华仁技术孵化器有限公司 Pharmaceutical preparation for treating white lesions of vulva

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