CN108524917A - Application of the melittin in the infection for the treatment of high-risk HPV virus and uterine neck cancer drug - Google Patents

Abstract

The present invention relates to a kind of new applications of melittin, i.e., melittin is as the infection for the treatment of high-risk HPV virus and the active constituent of uterine neck cancer drug.Present invention discover that melittin has the expression for inhibiting two kinds of cervical carcinoma high-risk viral E6/E7 albumen of HPV16, HPV18 and inhibits cervical cancer cell growth, the effects that promoting its apoptosis, achieve the effect that anti-HPV viruse, so that it is can be applied to the prevention of the cervical carcinoma caused by HPV viruse and treatment, opens up a new application field of melittin.

Description

Application of the melittin in the infection for the treatment of high-risk HPV virus and uterine neck cancer drug

Technical field

The present invention relates to melittin answering in treatment high-risk human mammilla papillomavirus (HPV) infection and uterine neck cancer drug With.

Background technology

Cervical carcinoma is one of three big malignant tumour of female reproductive system, lethal for the fourth-largest high incidence of global women and height Rate tumour especially occupies second and third position respectively in developing country's incidence and lethality in female malignant (Torre, L.A.et al.Global cancer statistics, 2012.CA Cancer J Clin 65,87-108, doi：10.3322/caac.21262(2015)).In recent years, the incidence of cervical carcinoma is continuously improved, and nearly 3 times are promoted between 10 years, And rejuvenation situation is clearly, has seriously endangered the physical and mental health of women.According to incompletely statistics, the whole world new hair uterine neck in 2012 Cancer 528000, lethal 266000, the cervical cancer pathogenesis rate of East Asia crowd is 7.9/10 ten thousand (Torre, L.A.et Al.Global cancer statistics, 2012.CA Cancer J Clin 65,87-108, doi：10.3322/ Caac.21262 (2015), Koh, W.J.et al.Cervical Cancer, Version 2.2015.Journal of the National Comprehensive Cancer Network：JNCCN 13,395-404；quiz 404(2015)).China is every Year, cancer new cases occupied first place in the world, and wherein cervical carcinoma newly sends out more than 13.5 ten thousand people every year, accounted for the 1/3 of whole world morbidity sum.Palace The morbidity of neck cancer and high-risk human mammilla papillomavirus (HR-HPV) persistent infection are closely related, all aggressive cervical carcinomas and HPV (Vaccarella, S., Lortet- are can detect that in 95% Cervical intraepitheliaI neoplasia, carcinoma in situ pathological tissue Tieulent, J., Plummer, M., Franceschi, S.＆Bray, F.Worldwide trends in cervical cancer incidence：impact of screening against changes in disease risk Factors.Eur J Cancer 49,3262-3273, doi：10.1016/j.ejca.2013.04.024 (2013), Bosch, F.X.＆de Sanjose, S.The epidemiology of human papillomavirus infection and Cervical cancer.Dis Markers 23,213-227 (2007)).High-risk HPV is mainly HPV16 and HPV18, HPV Main tumorigenesis albumen E6, E7 of expression play key effect (Biological during developing into cervical carcinoma by HPV infection agents.Volume 100 B.A review of human carcinogens.IARC Monogr Eval Carcinog Risks Hum 100,1-441 (2012)).70~80% women infected HPV in all one's life, wherein 10% women is faced and held The precarious position of continuous property HPV infection.Early stage effectively inhibits the expression of HPV and virus protein E6, E7 to become and prevents having for cervical carcinoma Effect approach.

The prevention of China's cervical carcinoma at present is mainly realized by promoting HPV vaccines and universal HPV screenings, but these measures exist It produces little effect in a short time.There are following bottleneck problems in clinical diagnosis and treatment at present：1, in China, HPV vaccines have just emerged, and are applicable in people Group is relatively narrow, anti-HPV types are limited, and the expensive vaccine that hinders is promoted；Although 2, China's health policy is to cervical carcinoma screening Great support is given, but uterine neck cancerous precaution and early diagnosis covering are not forced to obtain in common people HPV detections, cervical carcinoma screening consciousness Rate is unsatisfactory；3, clinically the anti-HPV traditional treatments means of female genital tract are limited, mostly use the non-specific medicine such as interferon Object is treated, and the defects for the treatment of cycle is long, side effect is apparent, financial burden weight is presented；4, for treatment of human cervical cancer, major programme With a hook at the end or do not retain operative treatment, radiotherapy and the chemotherapy of reproductive function, but the clinical therapeutic efficacy of most schemes is not good enough, very To there is the still high phenomenon of HPV levels after preservative operation treatment.

Melittin is the main component of bee venom, accounts for the 45%-50% of bee venom dry weight.Bee venom effect is extensive, mainly have it is anti-inflammatory, Antitumor, antiviral, antibacterium, analgesia, alleviates the effects that rheumatism and anti-freezing at decompression.Melittin is that molecular weight is 2840 Polypeptide chain (sweet-different bright-sweet -propyl- figured silk fabrics-is bright-rely-bright-Su-Su-of figured silk fabrics-are sweet-bright-dried meat -propyl- bright-different bright-Si-colors-are different bright-relies- Essence-relies-essence-valley-valley), have because of its unique structure amphipathic：The ends N- (1-20aa) are hydrophobicity, the ends C- (21-26aa) It is hydrophily (Raghuraman, H.＆Chattopadhyay, A.Melittin：a membrane-active peptide With diverse functions.Biosci Rep 27,189-223, doi：10.1007/s10540-006-9030-z (2007)).When enhancing in alkaline solution or with ion concentration, self crosslinking of melittin is in alpha-helix tetramer structure, is had The feature of film binding peptide and memebrane protein transbilayer helix, thus it is not only water-soluble, but also can dissolve cells play cytotoxic activity (Andersson, A., Danielsson, J., Graslund, A.＆Maler, L.Kinetic models for peptide- induced leakage from vesicles and cells.European biophysics journal：EBJ 36, 621-635, doi：10.1007/s00249-007-0131-9 (2007), Bello, J., Bello, H.R.＆Granados, E.Conformation and aggregation of melittin：dependence on pH and Concentration.Biochemistry 21,461-465 (1982)).Compared with the antitumor drug of current clinical application, Melittin has molecular weight relatively small, and immunogenicity is slightly lower, is not likely to produce the advantages such as allergic reaction.Therefore, melittin conduct Antitumor drug is paid close attention in recent years, but application of the melittin in anti-HPV viruse and treatment of human cervical cancer is not found, There is research field such as gastric cancer (Mahmoodzadeh, A., Zarrinnahad, H., Bagheri, K.P., Moradia, A.＆ Shahbazzadeh, D.First report on the isolation of melittin from Iranian honey bee venom and evaluation of its toxicity on gastric cancer AGS cells.J Chin Med Assoc 78,574-583, doi：10.1016/j.jcma.2015.06.008 (2015)), liver cancer (Song, C.C.et al.[Effects of melittin on growth and angiogenesis of human hepatocellular Carcinoma BEL-7402 cell xenografts in nude mice] .Ai Zheng 26,1315-1322 (2007)), breast cancer (Jeong, Y.J.et al.Melittin suppresses EGF-induced cell motility and invasion by inhibiting PI3K/Akt/mTOR signaling pathway in breast cancer Cells.Food Chem Toxicol 68,218-225, doi：10.1016/j.fct.2014.03.022 (2014)), ovary Cancer (Jo, M.et al.Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/ STAT3 pathway.Toxicol Appl Pharmacol 258,72-81, doi：10.1016/j.taap.2011.10.009 (2012)) etc..

Invention content

The object of the present invention is to provide a kind of new applications of melittin, i.e., melittin is as treatment high-risk HPV virus sense The active constituent of dye and uterine neck cancer drug.

The high-risk HPV is the HPV18 for infecting Hela cells, infects the HPV16 of caski cells.

The high-risk HPV tumorigenesis albumen is E6, E7 albumen.

The cervical carcinoma comes from cervical cancer tumer line Hela (HPV18), Caski (HPV16).

The active constituent of the described melittin treatment high-risk HPV virus infection and uterine neck cancer drug can be made vagina to Medicine preparation, the preparation can be suppository, ointment, capsule, effervescent tablet, gelling agent, lotion, film or foaming agent.

Melittin proportion in the treatment high-risk HPV virus infection and uterine neck cancer drug as active constituent For 0.05-2 ‰.

The vagina administration preparation includes following ingredient and its content：Melittin 0.05-2 ‰；Carbomer 1.0- 6.0%；Triethanolamine 1.0-5%；Borneol 3-6%；Glucose 1-5%；Glycerine 0.5-5%；Remaining is water.

The borneol is selected from natural borneol and/or synthetic borneol.

For the bottleneck problem in current cervical carcinoma prevention process and the blank in melittin research, the present invention is according to bee venom The extensive biological characteristics such as peptide antiviral and antitumor find that melittin has and inhibit two kinds of cervical carcinomas of HPV16, HPV18 high-risk The expression of viral E6/E7 albumen and the effects that inhibit cervical cancer cell growth, promote its apoptosis, achievees the effect that anti-HPV viruse, So that it is can be applied to the prevention of the cervical carcinoma caused by HPV viruse and treatment, opens up a new application of melittin Field.

Description of the drawings

Fig. 1 is Hela cells after 4 concentration melittins are handled 24 hours, as bee venom peptide concentration increases, HPV18E7, HPV18E6 expression quantity decline；

Fig. 2 is Caski cells after 4 concentration melittins are handled 24 hours, as bee venom peptide concentration increases, HPV16E7, HPV16E6 expression quantity decline；

Fig. 3 is the expression by each administration group treated Hela cervical carcinoma tumor-bearing mice knurls E6, E7：With bee Phallotoxins concentration increases, and HPV16E7, HPV16E6 expression quantity decline, and E6 is particularly evident, and melittin 8mg/kg administration groups E6, E7 Expression quantity is significantly lower than interferon and cis-platinum group；

Fig. 4 is the expression by each administration group treated Caski cervical carcinoma tumor-bearing mice knurls E6, E7：With Bee venom peptide concentration increases, and HPV16E7, HPV16E6 expression quantity decline, and E6 is particularly evident, and melittin 8mg/kg administration groups E6, E7 Expression quantity be significantly lower than interferon and cis-platinum group；

Fig. 5 is Hela cells after 4 concentration melittins are handled 24 hours, as bee venom peptide concentration increases, cell number Amount is reduced, and gap increases, and volume-diminished, form shrinkage is rounded；

Fig. 6 is Caski cells after 4 concentration melittins are handled 24 hours, as bee venom peptide concentration increases, cell number Amount is reduced, and gap increases, and volume-diminished, form shrinkage is rounded.

Fig. 7 be different quality concentration (0,2,4,8,16ug/ml) melittin act on Hela, Caski for 24 hours, 48h.Its is right Hela, Caski growth inhibition effect increase with the increase of concentration and action time；Melittin acts on Hela, Caski IC50 when for 24 hours is respectively 5.008ug/ml, 4.327ug/ml；

Fig. 8 is that hela cells are handled for 24 hours by melittin, and with the increase of drug concentration, early apoptosis and middle and advanced stage wither It dies and increases；

Fig. 9 is that Caski cells are handled for 24 hours by melittin, and with the increase of drug concentration, early apoptosis and middle and advanced stage wither It dies and increases, and based on early apoptosis increase；

Figure 10 is under each group administration concentration, and cervical carcinoma tumor volume increases increase with time, melittin pair The increased inhibiting effect of tumor volume enhances in dose dependent, and wherein 8mg/kg melittins make the inhibition that tumor volume increases With being substantially better than cis-platinum.

Specific implementation mode

Below by the preferred forms of the embodiment description present invention.These embodiments are further intended to citing and illustrate this Invention, rather than it limit the invention in any way the range of accompanying claims.

Melittin is provided in the present invention by inhibiting the expression of high-risk HPV 16,18 type virus proteins to be controlled in anti-HPV Application in treatment and the application in preventing and treating the cervical carcinoma caused by HPV infection optionally prove that melittin inhibits high The expression of danger type HPV tumorigenesis albumen, and then provide melittin as active constituent and effectively inhibit cervical carcinoma growth, promote uterine neck The drug of cancer cell-apoptosis.

Test example 1：The influence that melittin expresses HPV18 HPV16 E6/E7

(1) cell culture and total protein extraction

A.Hela and Caski (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre) is respectively with being added to 10% tire The DMEM in high glucose and RPMI-1640 culture mediums of cow's serum are cultivated under the conditions of 5%CO2 and 37 DEG C.

B. it chooses and grows to Hela the and Caski cells that fusion rate reaches 80% or more, to its addition containing final concentration of The culture medium of 2ug/ml, 4ug/ml, 8ug/ml melittin, normal incubation medium, which is added, in control group continues in 5%CO2 and 37 DEG C of condition Lower culture 48 hours.

C. waste liquid is abandoned, PBS is rinsed 1 time, and each pancreatin of the addition containing EDTA is digested to cell rounding.Collect each group cell extremely Centrifuge tube, 1000rpm × 5min centrifugations.Supernatant is abandoned, precipitation is resuspended in PBS, centrifuges 1000rpm × 5min.Abandon supernatant.

DEG C b.4 at, with the total protein in the Protein Extraction Reagent extraction each group cell containing protease inhibitors.

(2) foundation of cervical carcinoma bearing mouse model and knurl total protein extraction.

A.Balb/c-nu nude mices, female, age of mouse 6-8 weeks, weight 18-22g are raised under SPF grades of environment, totally 60.It takes HeLa, Caski cell in exponential phase, with serum-free medium centrifuge washing 2 times after 0.25% trypsin digestion, It is 10^7/ml that serum-free medium adjustment cell concentration, which is used in combination,.It is extracted with the syringe of No. 6 syringe needles of band under aseptic condition 0.2m1 cell suspension inoculations are seeded to right side armpit skin in the nude mice by subcutaneous through 75% alcohol disinfecting, by Hela, Caski respectively Under.It is vaccinated nude mice by subcutaneous after about 4 days and tubercle occurs or to tumor tissue growth to about 150mm³When, Nude Mouse Model Establish

It is grouped as follows：It will be inoculated with Hela, the mouse of Caski is respectively set to A, and B groups, every group is respectively set following 6 groups, every group 5.

Blank control group：Physiological saline

Positive controls 1：Cis-platinum 2.5mg/kg

Positive controls 2：500,000 U/kg of interferon alpha-2

Low dose group：2mg/kg

Middle dose group：4mg/kg

High dose group：8mg/kg

B. by the melittin of above-mentioned each group dosage and control group reagent multi-point injection to each group tumor-bearing mice tumor locus, often It is administered once for 24 hours, continuous 20 days.After drug withdrawal for 24 hours, animal is put to death, tumor mass is removed.

C. clean scissors shreds tissue block.Lysate is split and (contains PMSF) in tissue pieces, 10min whirlpools concussion one It is secondary, after cracking 30min, lysate is moved in 1.5ml centrifuge tubes with pipettor, then 12000rpm centrifuges 5min at 4 DEG C, Supernatant is taken to be sub-packed in the preservation of 0.5ml centrifuge tubes.

(3) Western blot detect HPV18 E7, HPV16 E7 expression

A. the albumen concentration for using Pierce BCA determination of protein concentration kit measurements to extract.Calculate albumen and loading Buffer applied sample amounts.

B. sample solution is configured, 100 DEG C × 10min boils sample.

C.SDS-PAGE gel electrophoresises：4% concentration is selected to concentrate glue (80V × 30min), 12% concentration separation gel (120V × 60min) protein isolate.

D. transferring film：Destination protein and B-actin are transferred to pvdf membrane (300mA × 30min).

E.5% skimmed milk power closes 1h.

F. it is incubated：Primary antibody (mouse monoclonal HPV18 E7, HPV16 E7 primary antibodies, Santa Cruz；Rabbit polyclonal B- Actin primary antibodies, Suo Laibao) 4 DEG C overnight, secondary antibody (mouse and rabbit secondary antibody (Suo Laibao)) is incubated at room temperature 45min.

G.ECL methods develop the color.

The result is shown in Figure 1 is to Fig. 4, and Hela cells are after 4 concentration melittins are handled 24 hours, with bee venom peptide concentration liter Height, HPV18E7, HPV18E6 expression quantity decline.Caski cells are after 4 concentration melittins are handled 24 hours, with bee venom Peptide concentration increases, and HPV16E7, HPV16E6 expression quantity decline.By each administration group treated Hela cervical carcinoma tumors The expression of body E6, E7：As bee venom peptide concentration increases, HPV16E7, HPV16E6 expression quantity decline, and E6 is particularly evident, and The expression quantity of melittin 8mg/kg administration groups E6, E7 is significantly lower than interferon and cis-platinum group.By each administration group, treated The expression of Caski cervical carcinoma tumor-bearing mice knurls E6, E7：As bee venom peptide concentration increases, HPV16E7, HPV16E6 expression Amount declines, and E6 is particularly evident, and the expression quantity of melittin 8mg/kg administration groups E6, E7 is significantly lower than interferon and cis-platinum group.

HPV16, HPV18 to giving various dose melittin carry out in vitro study and In vivo study, with Western Blotting detects the expression quantity of tumorigenesis albumen E6, E7.Various dose is respectively negative control group, low dose group, middle dose group And high dose group.In vitro study is cervical cancer tumer line Hela (HPV18), Caski (HPV16) cell culture.In vivo study pair As for cervical carcinoma bearing mouse model.This method finds the increase with bee venom peptide concentration, HPV18 E6/E7 and HPV16 E6/ E7 expression quantity continuously decreases, and illustrates effectively inhibit high-risk HPV 18 and HPV16 by the drug of active constituent of melittin The expression of tumorigenesis albumen further prevents cervical carcinoma to achieve the effect that anti-HPV18, HPV16.

Test example 2：Influence of the melittin to cervical cancer cell Hela and Caski

(1) cell culture and morphological observation

A.Hela and Caski (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre) is respectively with being added to 10% tire The DMEM in high glucose and RPMI-1640 culture mediums of cow's serum are cultivated under the conditions of 5%CO2 and 37 DEG C.

B. it chooses and grows to Hela the and Caski cells that fusion rate reaches 80% or more, to its addition containing final concentration of The culture medium of 2ug/ml, 4ug/ml, 8ug/ml melittin, normal incubation medium, which is added, in control group continues in 5%CO2 and 37 DEG C of condition Lower culture 48 hours, is placed under inverted microscope and observes.

As a result see Fig. 5 to Fig. 6, Hela cells are after 4 concentration melittins are handled 24 hours, with bee venom peptide concentration liter Height, cell quantity are reduced, and gap increases, and volume-diminished, form shrinkage is rounded.Caski cells are handled by 4 concentration melittins After 24 hours, as bee venom peptide concentration increases, cell quantity is reduced, and gap increases, and volume-diminished, form shrinkage is rounded.

(2) cell culture and CCK-8 methods detect melittin to cervical cancer cell inhibited proliferation

A. Hela the and Caski cells 3.0*104/ml of logarithmic growth phase is added in 96 orifice plates, the holes 100ul/.

B. routine culture is divided into group after 24 hours, and every group of 3 parallel holes are separately added into the melittin of various concentration, make it Final concentration respectively reaches；Normal culture solution is added in control group, continues culture 48 hours.

C. CCK-8 solution 10ul are added into each hole, are protected from light incubation 1 hour, the extinction of 450nm wavelength is measured with microplate reader It spends (OD values) and calculates inhibitory rate of cell growth.

Growth inhibition ratio=(1- dosing group cell A values/cellular control unit A values) * 100% is tested in triplicate, with cell Growth inhibition ratio and drug concentration do curve.

As a result see Fig. 7, different quality concentration (0,2,4,8,16ug/ml) melittin acts on Hela, Caski for 24 hours, 48h. It increases Hela, Caski growth inhibition effects with the increase of concentration and action time；Melittin makees Hela, Caski With for 24 hours when IC50 be respectively 5.008ug/ml, 4.327ug/ml.

(3) cell culture Flow cytometry cervical carcinoma promotes cells apoptosis

A. with final concentration of 0ug/ml, 2ug/ml, 4ug/ml, the melittin processing Hela and Caski cells 48 of 8ug/ml Hour

B. waste liquid is abandoned, PBS is rinsed 1 time, and each pancreatin of the addition containing EDTA is digested to cell rounding.Collect each group cell extremely Centrifuge tube, 1000rpm × 5min centrifugations.Supernatant is abandoned, precipitation is resuspended in PBS, centrifuges 1000rpm × 5min, abandons supernatant, be repeated 2 times.

C. 100ul Binding Buffer suspension cells are drawn, are added after 5ul Annexin V/FITC are mixed in room temperature It is protected from light incubation 5 minutes, the propidium iodide solution (PI) of 10ul 20ug/ml is added, and add 400ul PBS, carries out streaming inspection at once It surveys.

As a result see that Fig. 8 and Fig. 9, hela cells are handled by melittin for 24 hours, with the increase of drug concentration, early apoptosis And middle and advanced stage apoptosis increases.Caski cells are handled for 24 hours by melittin, with the increase of drug concentration, early apoptosis and in Late apoptic increases, and based on early apoptosis increase

Test example 3：Influence of the melittin to cervical carcinoma tumor-bearing mice knurl

(1) foundation of cervical carcinoma bearing mouse model

Balb/c-nu nude mices, female, age of mouse 6-8 weeks, weight 18-22g are raised under SPF grades of environment, totally 60.Take place Serum-free medium centrifuge washing is used after HeLa, Caski cell of exponential phase, 0.25% trypsin digestion 2 times, and It is 10^7/ml with serum-free medium adjustment cell concentration.Under aseptic condition 0.2m1 is extracted with the syringe of No. 6 syringe needles of band Cell suspension inoculation in the nude mice by subcutaneous through 75% alcohol disinfecting, respectively by Hela, Caski be seeded to right side armpit it is subcutaneous.About 4 It is vaccinated nude mice by subcutaneous after it and tubercle occurs or to tumor tissue growth to about 150mm³When, Nude Mouse Model establishes

It is grouped as follows：It will be inoculated with Hela, the mouse of Caski is respectively set to A, and B groups, every group is respectively set following 6 groups, every group 5.

Blank control group：Physiological saline

Positive controls 1：Cis-platinum 2.5mg/kg

Positive controls 2：500,000 U/kg of interferon alpha-2

Low dose group：2mg/kg

Middle dose group：4mg/kg

High dose group：8mg/kg

(2) melittin administration detection tumor-bearing mice knurl variation.

A. by the melittin of above-mentioned each group dosage and control group reagent multi-point injection to each group tumor-bearing mice tumor locus, often It is administered once for 24 hours, continuous 20 days.

B. the every 4 days growing states for measuring knurl during calculate knurl product, draw knurl growth curve.

Volume V=major diameter * minor axis * indulges diameter

C. after being discontinued for 24 hours, animal is put to death, tumor mass, calculating of weighing are removed.

Calculate tumor-like hyperplasia (%)=(1- administration groups average knurl weight/blank control group average knurl weight) * 100%

The result is shown in Figure 10, under each group administration concentration, cervical carcinoma tumor volume increases increase with time, bee venom Peptide enhances the increased inhibiting effect of tumor volume in dose dependent, the suppression that wherein 8mg/kg melittins increase tumor volume It makes of being substantially better than cis-platinum.Tables 1 and 2 statistics indicate that, melittin tumor-like hyperplasia in dose dependent enhance, wherein 8mg/kg melittin tumor-like hyperplasias are significantly greater than cis-platinum and interferon.Cervical carcinoma to giving various dose melittin carries out body Outer research and In vivo study, various dose are respectively negative control group, low dose group, middle dose group and high dose group.It grinds in vitro Study carefully for cervical cancer tumer line Hela (HPV18), Caski (HPV16) cell culture, by being used in microscopically observation and CCK-8 Method detects the inhibited proliferation of melittin, passes through the apoptosis-promoting effect of Flow cytometry melittin.In vivo study pair The growth inhibition effect of melittin is detected as cervical carcinoma bearing mouse model, passing through tumor mass growth curve and tumor-like hyperplasia. This method finds effectively inhibit the proliferation of cervical cancer cell by the drug of active constituent of melittin, and it is promoted to wither It dies, further melittin has apparent antitumaous effect.

Table 1

The tumor-like hyperplasia of Hela tumor-bearing mices

Table 2

The tumor-like hyperplasia of Caski tumor-bearing mices

Prepare embodiment 1

Melittin uterine neck gel, mainly by melittin, carbomer, glycerine, triethanolamine, benzyl alcohol, ethylenediamine tetra-acetic acid Disodium (EDTA-2Na), water composition, the content of various composition are to contain melittin 0.05-1.0g, carbomer in every 100 grams of gels 10 grams, 12 grams of glycerine, 0.4 gram of triethanolamine, 0.2 gram of disodium ethylene diamine tetraacetate, 3 grams of benzyl alcohol, remaining is water.Carbomer is Gel main component forms gel；Glycerine is moisturizer, plays lubricating action；Triethanolamine is antalkali；Benzyl alcohol is Preservative plays disinfection antisepsis；Disodium ethylene diamine tetraacetate (EDTA-2Na) is antioxidant, prevents product oxidation from becoming Property.Preparation method：

1) the water for injection immersion swelling that carbomer is added 60% is weighed by formula ratio, stands 10-15 hours, is formed transparent Hydrogel adjusts PH5-6, matrix is made.

2) glycerine, triethanolamine, benzyl alcohol, the disodium ethylene diamine tetraacetate (EDTA-2Na) of formula ratio are added, is emulsified Stirring is added remaining water for injection and continues stirring 20-60 minutes after 1-2 hours, stir evenly, form carbomer gel.

3) gel obtained is solidifying according to melittin uterine neck is obtained after the weight progress filling and package of 5 grams every of specification requirement Glue product.

Prepare embodiment 2

The component content of melittin vagina effervescence is (/ 1000) respectively：Melittin 0.05-1.0g, lactose 100g, Chinese holly Rafter acid 100g, sodium bicarbonate 120g, lauryl sodium sulfate 5g, magnesium stearate 1g.Preparation method includes：

1) lactose and citric acid for pressing formula ratio, are sufficiently mixed, melittin and sodium bicarbonate, lauryl sodium sulfate Mixing.

2) plus ethanol solution is appropriate, wet granular processed, mixing, crosses 20 mesh sieve, and 42~45 DEG C of dryings are dried for 4~6 hours, Obtain effervescence granular.

3) solution is diluted to debita spissitudo by formula, crosses 80 mesh sieve.

4) it dries 4-6 hours for 42~45 DEG C；Moisture is surveyed, moisture should be 1% hereinafter, obtaining bee venom peptide particles.

5) magnesium stearate is added, tabletting, packaging obtain melittin vagina effervescence.

Prepare embodiment 3

The fundamental component of melittin irrigation is EDETATE SODIUM, glycerine, tasteless opacifiers, peppermint, borneol etc..

The lotion production procedure for preparing 1kg is described below.The weight ratio of the raw material is：Melittin 0.05-1.0g, eucalyptus Set oil 1g, EDETATE SODIUM 10g, glycerine 2.5g, tasteless opacifiers 2.5g, peppermint 0.1g, borneol 0.5g.

Production process：

1) melittin, EDETATE SODIUM, glycerine, tasteless opacifiers are added in the pure water of total amount 60% and are stirred evenly.

2) by eucalyptus oil, peppermint, borneol (borneol is first dissolved with 95% ethyl alcohol 1%), the pure of 80 DEG C of ＞ is added after mixing Upper liquid is added after stirring evenly in water 5%, fully dissolving.

3) being eventually adding can be filling after the total amount of liquid that pure water is prepared to requirement fully stirs evenly.

Prepare embodiment 4

Melittin bioprotein dressing, it is respectively melittin that ingredient, which includes (for preparing one kilogram of gel dressing), 0.05-1.0g, carbomer 20g, green-tea extract 10g, glycerine 25g, triclosan 2.5g.

Preparation method：

1) glycerine and green-tea extract are added in 800~900g pure water, carbomer is added after stirring and dissolving, with high speed Dispersion machine makes it fully dissolve.

2) it and then by the melittin crossed with 0.25 μm of membrane filtration is added in above-mentioned system, continuing stirring makes it uniformly.

3) triclosan is dissolved in a small amount of alkali and is added thereto, supplied one kilogram with pure water, stirred evenly.

4) obtain one kilogram of gel dressing is adjusted pH value to 4.5 to 6.5 by the NaOH solution for being 20% with mass concentration Between.

Prepare embodiment 5

Melittin ointment, ingredient include (per 100g)：Melittin 0.05-1.0g, propolis 15g, oil phase substrate 10g, emulsification Agent 5g, penetration enhancer 2g, preservative 0.05g and remaining be water；The oil phase substrate is by vaseline, polyethylene glycol 400 With propylene glycol by volume 0.5：1：2 compositions, the penetration enhancer is by borneol and menthol by weight 1：0.8 composition.

Preparation method：

1) it takes propolis raw material 10g~50g to crush, is put into 1000ml conical flasks, 95% ethanol solution is added and is put into ultrasound It is made it dissolve in wave, then is rinsed to propolis and be completely dissolved in ultrasonic wave with 95% ethanol solution, will again moved after supernatant liquid filtering Enter and evaporate solution in revolving bottle, remaining liq pours into evaporating dish, is put into vacuum drying chamber, and melittin 1.0- is added 2.0g completely removes residual ethanol, and it is spare to obtain melittin-propolis cement.

2) it takes oil phase substrate to be heated to 75 DEG C, penetration enhancer is added, stir to being completely dissolved, obtain oil phase liquid；

3) emulsifier is added to the water and is heated to 75 DEG C, stirred to being completely dissolved, obtain water phase liquid；

4) it will be mixed in melittin-propolis cement that step 1) obtains and the oil phase liquid 2) obtained and the water phase liquid 3) obtained It closes, is subsequently added into preservative, 50min is stirred at 1000rpm to get melittin ointment.

Prepare embodiment 6

Melittin suppository, (per 100g) it includes：Melittin 0.05-1.0g, alum 0.5g, boric acid 3g, camphor 0.5g, ice Piece 0.5g, polyoxyethylene stearate (40) ester 93.5g

Preparation method：

1) polyoxyethylene stearate (40) ester is set in material tank, is heated, stirring is until matrix is completely dissolved and maintains temperature At 76-84 DEG C；

2) (1) step gains step gains are transferred to after 200 mesh screens in blend tank；

3) melittin is taken, is added in blend tank, mixing, stirring keep ointment evenly dispersed, and sequentially add alum, boric acid, It is kept stirring；Camphor, borneol are added in blend tank together after being dissolved with a small amount of ethyl alcohol, stir evenly, homogeneous keeps ointment completely equal Even dispersion；

4) control fluid temperature is filling at 40-50 DEG C, freezes to get melittin suppository.

Clinical implementation is tested

This experiment is melittin Gel Examples 1 of the present invention to not treated with the HPV persistent infection persons of high-level cervical lesions The clinography of effect.Research object selects：For HPV persistent infections (HPV-DNA detections are positive, checked after 6 months still positive) And not with high-level cervical lesions.

One, inclusion criteria：

1 the past is without operation on cervix history

2 exclude the high-level lesion of uterine neck and canceration (row TCT checks, for patient that TCT results are ASCUS or more into One walking vaginoscopy)

3 without melittin contraindication

Antiviral drugs and immunomodulator are not used in 4 half a year

5 non-pregnancies, nursing period women

6 without acute inflammatory diseases such as acute vaginitis, acutes pelvitis of pelvic cavity

7 without irregular vagina bleeding

8 without immunologic hypofunction (malignant tumour, SLE, AIDS etc.)

9 have follow-up condition and partner treatment

10 all patients sign informed consent form, this clinical test of voluntary participation

Two, exclusion criteria：

1 immunologic hypofunction person, after tumor post-operation or Radiotherapy chemotherapy, AIDS and systemic loupus erythematosus etc.；

2 gestation and women breast-feeding their children；

3 suffer from acute genital inflammation, such as gonorrhoea, mycoplasma or choamydiae infection；

Control group：Without using any pharmaceutical intervention, patient is practised contraception using sheath during observation；

Treatment group：Before sleeping after cleaning vulva, drug is pushed into fornix portion using propeller, a 1g once a day connects With being discontinued after 10 days, the next menstrual cycle repeats.Menstrual period deactivates, and next period the starts medication for 3-5 days after clean period, HPV is checked after 3 menstrual cycles of continuous use, and the HPV patients that turn out cloudy discontinue medication, and the HPV patients that do not turn out cloudy are further continued for using bee venom 3 menstrual cycles of peptide gel：Forbid hip bath and sexual life during medication, is practised contraception using sheath in medication later six months.

Curative effect judgment method：

It turns out cloudy effective：HPV (-) when check；

Effectively：It is quantitatively reduced in check HPV infection, but not fully erased；

In vain：When check, HPV infection is quantitative without changing or quantitatively increasing.

As a result：It treats 3 Ge Yue treatment groups HPV clearance rates and effective percentage is higher than control group.After 6 months, treatment group HPV is clear Except rate and effective percentage are also higher than control group.The effect of checking treatment group and control group after medication after 3 months and 6 months comparison is detailed It see the table below 3.

Table 3

Note：Clearance rate=N turns out cloudy/and N total numbers of persons × 100% is efficient=(N turn out cloudy+N effective)/N total number of persons × 100%

It these results suggest that melittin of the present invention has significant curative effect to the treatment of people's HPV infection.

Claims (8)

1. the object of the present invention is to provide a kind of new applications of melittin, it is characterised in that：I.e. melittin is as treatment high-risk-type HPV viruse infects and the active constituent of uterine neck cancer drug.

2. a kind of new application of melittin as described in claim 1, it is characterised in that：The high-risk HPV is infection The HPV18 of Hela cells, the HPV16 for infecting caski cells.

3. a kind of new application of melittin as described in claim 1, it is characterised in that：The high-risk HPV tumorigenesis albumen For E6, E7 albumen.

4. a kind of new application of melittin as described in claim 1, it is characterised in that：The cervical carcinoma comes from cervical carcinoma Cell line Hela (HPV18), Caski (HPV16).

5. a kind of new application of melittin as described in claim 1, it is characterised in that：The melittin treats high-risk-type HPV viruse infects and vagina administration preparation can be made in the active constituent of uterine neck cancer drug, and the preparation can be suppository, soft Paste, capsule, effervescent tablet, gelling agent, lotion, film or foaming agent.

6. a kind of new application of melittin as described in claim 1, it is characterised in that：The treatment high-risk HPV virus Infection and uterine neck cancer drug in as active constituent melittin proportion be 0.05-2 ‰.

7. a kind of new application of melittin as described in claim 1, it is characterised in that：The vagina administration preparation includes such as Lower ingredient and its content：Melittin 0.05-2 ‰；Carbomer 1.0-6.0%；Triethanolamine 1.0-5%；Borneol 3-6%；Grape Sugared 1-5%；Glycerine 0.5-5%；Remaining is water.

8. a kind of new application of melittin as described in claim 1, it is characterised in that：The borneol is selected from natural borneol And/or synthetic borneol.