CN106474147B

CN106474147B - Dressing for preventing and controlling human papilloma virus infection and preparation method thereof

Info

Publication number: CN106474147B
Application number: CN201610896425.4A
Authority: CN
Inventors: 余乃绚
Original assignee: Shanghai Jielong Investment Management Co ltd
Current assignee: Shanghai Jielong Investment Management Co ltd
Priority date: 2016-10-14
Filing date: 2016-10-14
Publication date: 2022-07-05
Anticipated expiration: 2036-10-14
Also published as: CN106474147A

Abstract

The invention discloses a dressing for preventing and controlling human papilloma virus infection and a preparation method thereof, wherein the mass percentage of lentinan sulfate in the dressing is 0.01-0.1%. According to the invention, researches show that the lentinan sulfate has a good effect on clearing HPV viruses, and the sulfate modification rate of the lentinan sulfate has an important influence on the removal of HPV viruses by the lentinan sulfate. When the sulfate radical modification rate is low, the HPV virus removing capacity of the lentinan sulfate is enhanced along with the increase of the sulfate radical modification rate; when the sulfate modification rate reaches a certain level, about 20%, and then the sulfate modification rate is increased, the capability of the lentinan sulfate to eliminate HPV virus is reduced.

Description

Dressing for preventing and controlling human papilloma virus infection and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a dressing for preventing and controlling human papilloma virus infection and a preparation method thereof.

Background

Today, with the continuous advancement of technology, people still have no way to address cancer restraint. As scientists continue to strive for the pathogenesis of cancer remains unknown. However, one cancer pathogenesis has been identified by the world health organization-cervical cancer, which is caused by infection with human papilloma virus (HPV virus).

HPV virus is a papilloma vacuolatum virus A genus of papovaviridae family, is a spherical double-stranded DNA virus, and can cause squamous epithelial proliferation of human skin and mucosal epithelium. Various pathologies can arise, including warts, genital condyloma applanation, and the like. Different types of HPV viruses cause different disorders, and their distribution is tissue-specific. For example, HPV2 primarily causes skin warts. Through continuous research by scientists, more than 100 human-related HPVs have been discovered, and the nucleotide sequences of the human-related HPVs are substantially different and can be roughly divided into two types according to the differences of biology and survival time: an alpha species and a beta species; wherein the alpha species are only present in humans and primates and are closely related to the occurrence of cervical cancer, wherein 50% of cervical cancer occurrences are related to HPV16 and 20% of cervical cancer occurrences are related to HPV 18. Meanwhile, the alpha species are classified into 14 high-risk types of HPV, and the rest are low-risk types of HPV. High risk HPVs include HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. A study by the national cancer institute of america indicated that about 10% of women infected with HPV types 16 and 18 developed advanced pre-cervical cancer lesions within 3 years after infection (this is about 4% of women infected with other high-risk HPV types), and 20% developed advanced pre-cervical cancer lesions within 10 years (about 7% of other high-risk HPV types). Low risk types of HPV do not cause cervical cancer, but these HPV cause genital condyloma acuminata or very subtle cervical cellular changes, such as HPV-6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73 and 81. Among them, HPV6 and 11 are the most common low risk types of HPV, with which 90% of genital condyloma acuminata are associated. Therefore, high risk type HPV infection is the leading cause of cervical lesions, especially malignant lesions.

Currently, cervical cancer is the major killer of health in women. Research has shown that HPVDNA can be detected in more than 90% of cervical cancers, which is also the main evidence for the world health organization to confirm that cervical cancer is caused by HPV. The incidence rate of cervical cancer in China is second in the world and is in a youthful trend, nearly 15 ten thousand cases are generated each year, about 1/3 of patients in the world is occupied, and about 8 thousands of people die. While humans are the only host for HPV viruses, it takes at least ten years from infection with HPV viruses to the development of cervical cancer, where virus-cell contact is a necessary and prerequisite for HPV infection of the host; during the period, the wound is generated by infecting the mucous epithelium of the human body, the epithelium is damaged, the virus contacts and enters cells (the optimal period for preventing and eliminating), the virus is matured and releases and infects more tissue epithelium, the wound is further enlarged, the condition of the disease is aggravated and worsened, and the immune system can not eliminate and form wart, cervical erosion, cervical carcinoma and the like. At present, the most effective mode for preventing HPV virus is recognized at home and abroad to be injecting HPV vaccine, and large-area clinical experiments prove that the HPV vaccine can indeed prevent infection of some HPV viruses. Bivalent vaccines against HPV16 and HPV18 Kulansu Schke and nine-valent vaccines against HPV-6, 11, 16, 18, 31, 33, 45, 52, 58 default Sadong are available on the market. However, HPV vaccines do not prevent all types of HPV and are also expensive. In addition, there is currently no corresponding specific drug for the treatment of conditions caused by HPV viruses in therapy, most of which are treated with interferon and the like. The interferon drugs not only easily generate resistance, but also have the defects of unobvious curative effect, high price and the like. Therefore, there is an urgent need for a biological agent and a drug which can effectively remove HPV in human body and has no toxic action to human.

In recent years, research on anti-HPV drugs and biological agents has been rapidly developed. Wherein, the traditional Chinese medicine plays an important role in treating HPV virus, for example realgar, coptis chinensis and the like can be used for treating cervical epithelial neoplasia caused by persistent HPV virus. Other Chinese herbal compound has a function in HPV treatment, such as toxin clearing suppository. Natural products derived from animals and plants, such as polysaccharides, glycolipids, polypeptides and the like, have remarkable activity in resisting influenza viruses. The invention discovers that some polysaccharides can treat and prevent HPV infection, and experimental data show that the clearance rate of the polysaccharides reaches 100 percent. The invention solves the problem that the market lacks of effective biological preparations for resisting HPV virus, and brings good news to women siblings.

Lentinus edodes of the Agaricaceae class contains various bioactive substances, and Lentinus edodes polysaccharide is an important active ingredient. It plays an important role in resisting tumor, virus, oxidation and infection and improving human immunity. The lentinan is polymerized from glucose, and the side chain part also contains other glycosyl, protein and other groups which can improve the biological activity of the lentinan.

Disclosure of Invention

The invention provides an application of lentinus edodes polysaccharide sulfate in preparing a medicament for preventing and treating HPV virus infection. The lentinan sulfate has good effect of eliminating HPV virus.

Application of Lentinus Edodes polysaccharide sulfate in preparing medicine for preventing and treating HPV virus infection is provided. The lentinan reacts with sulfuric acid to generate lentinan sulfate. Because the polysaccharide carries sulfate groups, and the sulfate groups have negative charges, the surface of the mushroom polysaccharide sulfate has a large number of negative charges. The C end of the L1 region and the N end of the L2 region of the HPV viral protein coat carry positive charges, so that lentinan sulfate can competitively bind with the C end of the L1 region and the N end of the L2 region of the HPV protein coat to carry positive charge regions, and occupy positions to block the combination of HPV and a basement membrane receptor HSPG. Blocks the only way that HPV recognizes the host cell, thereby rendering the HPV virus unable to infect humans. Meanwhile, the modified lentinan does not affect the original functions of the lentinan, such as enhancing the immunity of a human body, inducing the generation of interferon in the body and the like through verification.

Experiments show that the modification rate of the sulfate radical of the lentinan sulfate has important influence on the elimination of HPV virus by the lentinan sulfate. When the sulfate radical modification rate is low, the HPV virus removing capacity of the lentinan sulfate is enhanced along with the increase of the sulfate radical modification rate; when the sulfate modification rate reaches a certain level, about 20%, and then the sulfate modification rate is increased, the capability of the lentinan sulfate to eliminate HPV virus is reduced. Preferably, the sulfate modification rate of the lentinan sulfate is not less than 10%. More preferably, the sulfate modification rate of the mushroom fungus polysaccharide sulfate is not less than 20%.

The invention also provides a mushroom fungus polysaccharide sulfate dressing, which comprises the following components in percentage by mass:

preferably, the mushroom fungus polysaccharide sulfate dressing comprises the following components in percentage by mass:

preferably, in the mushroom polysaccharide sulfate dressing, the sulfate modification rate of the mushroom polysaccharide sulfate is not less than 10%. More preferably, the sulfate modification rate of the mushroom fungus polysaccharide sulfate is not less than 20%.

Preferably, the preparation method of the mushroom fungus polysaccharide sulfate dressing comprises the following steps:

(1) dissolving lentinan sulfate in water, and performing membrane filtration to remove bacteria;

(2) fully swelling and dissolving carbomer in water;

(3) dissolving poloxamer, glycerol and triclosan in water, and adjusting pH until triclosan is completely dissolved;

(4) and (2) uniformly mixing the solution obtained in the steps (1), (2) and (3) and EDTA disodium, supplementing water and adjusting the pH to 4.5-6.5.

Preferably, in the preparation method, the mushroom fungus polysaccharide sulfate is prepared by adopting a chlorosulfonic acid-pyridine method, the volume ratio of chlorosulfonic acid to pyridine is 1: 1-5, the esterification reaction time is not less than 2 hours, and the esterification reaction temperature is 30-90 ℃.

According to the invention, researches show that the lentinan sulfate has a good effect on clearing HPV viruses, and the sulfate modification rate of the lentinan sulfate has an important influence on the removal of HPV viruses by the lentinan sulfate. When the sulfate radical modification rate is low, the HPV virus removing capacity of the lentinan sulfate is enhanced along with the increase of the sulfate radical modification rate; when the sulfate modification rate reaches a certain level, about 20%, and then the sulfate modification rate is increased, the capability of the lentinan sulfate to eliminate HPV virus is reduced.

Detailed Description

Examples 1 to 4

Lentinan is purchased from Shanghai-derived leaf Biotech Inc. (Cat. No. 37339-90-5).

Preparing mushroom fungus polysaccharide sulfate by using a chlorosulfonic acid-pyridine method, placing 10mL of anhydrous pyridine into a three-neck flask provided with a condenser pipe and a stirring device, cooling the mushroom fungus polysaccharide sulfate by using an ice salt bath, violently stirring, slowly dropwise adding 4mL of chlorosulfonic acid, continuously stirring for 30 minutes to obtain a large amount of light yellow solid, and finishing the reaction to obtain the esterification reaction reagent. Weighing refined 0.5g of lentinan, dissolving in 25mL of anhydrous formamide, vortex to prepare uniform suspension, pouring into a preheated flask of an esterification reagent, continuously stirring, maintaining the water bath temperature at 60 ℃, and carrying out esterification reaction for t hours. And after the reaction is finished, removing the flask, cooling to room temperature, adding 3 times of volume of absolute ethyl alcohol, standing, centrifuging, and precipitating to obtain the lentinan sulfate.

Dialyzing the precipitate against double distilled water for 3 days, against PBS for one day, and passing through BaCl₂-determination of the sulfate modification rate by the gelatin method; the final polysaccharide concentration was determined using a glucose standard curve method. As shown in Table 1, within a certain range, increasing the esterification reaction time is beneficial to increasing the sulfate radical modification rate of the obtained mushroom fungus polysaccharide sulfate, and the recovery rate of the polysaccharide is not influenced basically.

TABLE 1

Examples	Esterification reaction time t (h)	Sulfate group modification Rate (%)	Polysaccharide recovery (%)
				Example 1	1	8.9	63.4
Example 2	2	17.3	66.5
				Example 3	4	21.9	65.2
Example 4	6	25.2	64.5

Examples 5 to 7

Detecting the influence of different chlorosulfonic acid and pyridine ratios on the sulfate radical modification rate of the obtained mushroom fungus polysaccharide sulfate. The remaining parameters were the same as in example 3 except that the volume ratio of chlorosulfonic acid to pyridine was different and the amount of chlorosulfonic acid was 4mL, so the amount of pyridine was different. The sulfate modification rate and the polysaccharide recovery rate are respectively detected, and the experimental results are shown in table 2, wherein the sulfate modification rate of the obtained mushroom polysaccharide sulfate is reduced with the reduction of the volume ratio of chlorosulfonic acid to pyridine, but the polysaccharide recovery rate is increased.

TABLE 2

Examples	Volume ratio of chlorosulfonic acid to pyridine	Sulfate group modification Rate (%)	Polysaccharide recovery (%)
				Example 5	1∶1	25.3	43.4
Example 6	1∶2.5	19.2	66.5
				Example 7	1∶5	12.1	71.8

Examples 8 to 10

And detecting the influence of different esterification reaction temperatures on the sulfate radical modification rate of the obtained mushroom fungus polysaccharide sulfate. The remaining parameters were the same as in example 3, except that the esterification reaction temperature was different. The sulfate modification rate and the polysaccharide recovery rate are respectively detected, and the experimental results are shown in table 3, wherein the higher the esterification reaction temperature is, the higher the sulfate modification rate of the lentinan sulfate is, and the lower the polysaccharide recovery rate is.

TABLE 3

Examples	Temperature (. degree.C.) for esterification reaction	Sulfate group modification Rate (%)	Polysaccharide recovery (%)
				Example 8	30	18.7	59.5
Example 9	60	25.3	63.4
				Example 10	90	31.0	29.3

Example 11

1) Construction of HPV pseudovirus

Pseudoviruses are viruses in which a retrovirus is capable of integrating the envelope glycoprotein of another, different virus species to form an envelope with the exogenous virus, while the genome retains the genomic properties of the retrovirus itself. The infection capacity of the pseudovirus is not different from that of the normal virus, but the pseudovirus can not normally proliferate after entering cells and then can be killed automatically, so that the pseudovirus has no harm to the cells. Therefore, it is also recognized that the most effective way to mimic HPV viral infection. Buck et al reported (Buck, C.B., D.V.Passtrana, D.R.Lowy, and J.T.Schiller, effective intracellular assembly of pathogenic vectors.J Virol,2004.78(2): p.751-7.) that they constructed the optimized genes of HPV membrane structural proteins L1, L2 into eukaryotic expression vectors and reporter plasmids simultaneously transfected eukaryotic cells, and they found that L1, L2 can be efficiently expressed in eukaryotic cells and self-assembled to form pseudoviral particles with immunogenicity. By using the construction method of Buck et al, the method is not only convenient, but also the produced pseudovirus has higher titer which can reach 10⁷And TU. The pseudovirus packaged by the method is successfully applied to the production of HPV vaccines and drug screening. The invention constructs three HPV pseudovirus models of low-risk HPV6 and high-risk HPV16 and HPV18 by using the method. The specific construction mode is as follows:

and amplifying the optimized HPV gene L1L2 by using Polymerase Chain Reaction (PCR), cloning to a eukaryotic cell expression vector, and carrying out enzyme digestion and sequencing identification to form expression vectors of pHPV6, pHPV16 and pHPV18 which are stably expressed. The three expression vector plasmids and the reporter plasmid pSEAP (containing alkaline phosphatase reporter gene) which are sequenced and identified to be correct are massively amplified and are ready for use.

293FT cells were cultured, and passaged individually to 4 100mm plates at 37 ℃ with 5% CO₂When the cells are cultured to grow to 80% of fusion degree, 293FT cells are transfected by plasmids pHPV6, pHPV16, pHPV18 and pSEAP reporter plasmids respectively through lipoFilter transfection reagent, and only pSEAP reporter plasmids are transfected by a control group. The cells transfected with the plasmid were incubated at 37 ℃ with 5% CO₂After the incubator continues to culture for 12 hours, the fresh culture medium is replaced, after the incubator continues to culture for 72 hours, the culture medium is removed and washed 2 times by DPBS, cells are digested for 3 minutes by 0.25% of pancreatin, 10mL of the culture medium is added and transferred into a 15mL centrifuge tube, 1000g of the culture medium is centrifuged for 5 minutes to remove supernatant, cell lysate (0.3% Brij58, 0.2% Benzonase, DPBS of 0.2% plasmid safe ATP dependent DNase) with the same volume as the precipitate is added for 24 hours at 37 ℃, then the mixture is placed on ice for 5 minutes, 0.17 volume of 5M NaCl is added, the mixture is uniformly mixed by vortex, the mixture is placed on ice for 20 minutes, the mixture is centrifuged for 10 minutes at 5000g of 4 ℃, and the supernatant is placed at-80 ℃.

2) Viral titer assay

293FT cells were passaged to 96-well plates at 1X 10 cells per well⁵5% CO at 37 ℃₂Culturing for 8 h;

carrying out gradient dilution on HPV pseudovirus, removing culture medium in 96 holes, adding 50uL pseudovirus diluent, then adding 50uL normal culture medium, and carrying out 5% CO treatment at 37 DEG C₂Culturing for 72h, collecting 50uL culture supernatant, detecting with Clontech alkaline phosphatase detection kit to calculate TCID50, and when the ratio of the experimental group to the control group reaches 10⁶The pseudovirus can be collected.

3) Detection of inhibitory Activity of lentinan sulfate against HPV6, HPV16 and HPV18

gradient dilution of lentinan sulfate (prepared in example 3) and lentinan, removal of culture medium from 96-well plate, respectively adding 50uL of lentinan sulfate diluent and lentinan diluent, and adding 5% CO at 37 deg.C₂Culturing for 30 minutes, then adding 50uL 100TCID50 pseudovirus into a 96-well plate, continuously culturing for 12 hours, changing a fresh culture medium, after culturing for 72 hours, taking 50uL culture solution supernatant, detecting by using a Clontech alkaline phosphatase detection kit, and calculating IC50 (dosage when certain medicine with certain concentration induces apoptosis to be 50 percent) of lentinan sulfate and lentinan by using SigmaPlut, and the result shows that the lentinan sulfate has good removal rate on three HPV viruses and the removal rate of the lentinan on the three HPV viruses is poor in Table 4.

TABLE 4

Example 12

The mushroom polysaccharide sulfate contains different sulfate radical contents and has the influence on clearing HPV.

Only the sulfate modification rate is different, the other experimental methods are the same as those in example 11, and the experimental results are shown in Table 5, the shiitake mushroom polysaccharide sulfate with different sulfate modification rates has different HPV clearing capability, and the capability is enhanced along with the increase of the sulfate modification rate, but after the sulfate modification rate reaches a certain degree, the increase of the sulfate modification rate basically has no influence on the HPV clearing capability.

TABLE 5

Example 13

The effect of different kinds of polysaccharide sulfates on HPV clearance.

Lentinan sulfate, pachyman sulfate and ganoderan sulfate were prepared separately by the same method as in example 3 (parameters were the same), and then the sulfate modification rate and polysaccharide recovery rate were measured separately, and the results are shown in table 6.

TABLE 6

	Mushroom polysaccharide sulfate	Pachyman sulfate	Ganoderma lucidum polysaccharide sulfate
				Sulfate group modification Rate (%)	17.3	21.5	19.3
Polysaccharide recovery (%)	63.4	66.5	64.5

Then, the effect of different polysaccharide sulfates on HPV elimination was examined in the same manner as in example 11, and the results are shown in Table 7, wherein the ability of lentinan sulfate to eliminate HPV is stronger than that of ganoderan sulfate and pachyman sulfate under similar sulfate modification rate conditions.

TABLE 7

Example 14

Taking the preparation of 100kg of dressing as an example,

a) weighing 10g of lentinan sulfate, dissolving in 10kg of water, and vacuum filtering with 0.22um filter membrane;

b) weighing 2kg of carbomer, dissolving in 10kg of water, stirring at high speed, mixing uniformly, swelling overnight, and then autoclaving at 121 ℃ for 30 minutes for later use;

c) weighing 3kg of glycerol, 1kg of poloxamer and 100g of triclosan, dissolving the glycerol, the poloxamer and the triclosan in 10kg of water, dropwise adding 2M NaOH, stirring until the triclosan is completely dissolved, and then carrying out autoclaving at the temperature of 121 ℃ for 30 minutes for later use;

d) the solution obtained in a) b) c) and 10g of disodium EDTA are mixed together, stirred until homogeneous, the pH is adjusted to 4.5 and the total amount is made up to 100kg with sterile water.

Example 15

Taking the preparation of 100kg of dressing as an example,

a) weighing 50g of lentinan sulfate, dissolving in 10kg of water, and vacuum filtering with 0.22um filter membrane;

b) weighing 0.5kg of carbomer, dissolving in 10kg of water, stirring at high speed, uniformly mixing, swelling overnight, and then autoclaving at 121 ℃ for 30 minutes for later use;

c) weighing 3kg of glycerol, 5kg of poloxamer and 150g of triclosan, dissolving in 10kg of water, dropwise adding 2M NaOH, stirring until the triclosan is completely dissolved, and then carrying out autoclaving at 121 ℃ for 30 minutes for later use;

d) mixing the solution obtained in a) b) c) and 50g of disodium EDTA, stirring uniformly, adjusting the pH to 6.0, and simultaneously adding sterile water to 100 kg.

Example 16

Taking the preparation of 100kg of dressing as an example,

a) weighing 100g of lentinan sulfate, dissolving in 10kg of water, and vacuum filtering with 0.22um filter membrane;

b) weighing 3kg of carbomer, dissolving in 10kg of water, stirring at high speed, mixing uniformly, swelling overnight, and then autoclaving at 121 ℃ for 30 minutes for later use;

c) weighing 1kg of glycerol, 10kg of poloxamer and 300g of triclosan, dissolving in 10kg of water, dropwise adding 2M NaOH, stirring until the triclosan is completely dissolved, and then carrying out autoclaving at 121 ℃ for 30 minutes for later use;

d) mixing the solution obtained in a) b) c) with 100g disodium EDTA, stirring well, adjusting the pH to 6.5 while adding sterile water to 100 kg.

Example 17

And (5) performing dressing stability experiments.

The dressings prepared in the above examples (examples 14 to 16) were evaluated immediately after the preparation, and evaluated for morphological color, presence or absence of particulate matter, pH, and the like. Three of the three are respectively stored at 4 ℃, 25 ℃ and 37 ℃ for one month and observed every week. The final results show that the prepared dressing is well preserved under various temperature conditions and times, both morphologically and in terms of pH.

Time points are as follows: 0 week

Time points are as follows: 1 week

Time points are as follows: 2 weeks

Time points are as follows: 3 weeks

Time points are as follows: 4 weeks

Example 18

And (5) animal safety experiment.

The drug is administered to a specific part of an animal, and the influence of the drug on the part, for example, whether a stimulus response occurs or not, whether red swelling of the skin is caused or not, and the like are observed. Skin and vaginal parts are selected, and complete skin and damaged skin dressing and vaginal dressing are carried out, and dressing conditions are observed in a certain time, and the following results are obtained:

the dressing prepared in the embodiment 14-16 is verified to be good in safety by a GLP unit laboratory, and an animal safety experiment is as follows:

1. dressing the intact skin and damaged skin of mouse, and removing hair from the two side regions of spinal column at the application position (8% sodium sulfide removing hair with a depilating area of 30 cm)²) (ii) a The fine sand paper for damaged skin can be used for scratching the unhairing part, and the fine sand paper can be immediately applied for 2 times a day at a ratio of 5.0g/kg for four weeks, with no skin irritation reaction.

2. Dressing the intact skin and damaged skin of New Zealand rabbit repeatedly, and removing hair (8% sodium sulfide removing hair and 30cm area) from the two side regions of back spine by applying the dressing²) (ii) a The fine sand paper for damaged skin can be used for scratching the unhairing part, and the fine sand paper can be immediately applied for 2 times a day at a ratio of 5.0g/kg for four weeks, with no skin irritation reaction.

3. Meanwhile, the vaginal dressing of the mouse and the New Zealand rabbit is repeatedly applied for 2 times a day at a dose of 5.0g/kg for four weeks, and no vaginal irritation reaction is seen.

Claims

1. The application of lentinan sulfate in preparing medicine for preventing and treating HPV infection,

the medicine is a mushroom fungus polysaccharide sulfate dressing and comprises the following components in percentage by mass:

the sulfate radical modification rate of the lentinus edodes polysaccharide sulfate is not less than 10 percent and not more than 20 percent,

the preparation method of the mushroom fungus polysaccharide sulfate dressing comprises the following steps:

(1) dissolving lentinan sulfate in water, and performing membrane filtration sterilization;

(2) swelling and dissolving carbomer in water;

(4) uniformly mixing the solution obtained in the steps (1), (2) and (3) and EDTA disodium, supplementing water and adjusting the pH to 4.5-6.5;

the mushroom fungus polysaccharide sulfate is prepared by adopting a chlorosulfonic acid-pyridine method, wherein the volume ratio of chlorosulfonic acid to pyridine is 1: 1-5, the esterification reaction time is not less than 2 hours, and the esterification reaction temperature is 30-90 ℃.