Compound composition for treating HPV (human papillomavirus) infection as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a compound composition for treating HPV (human papillomavirus) infection as well as a preparation method and application thereof. The compound composition is prepared from the following raw materials in parts by weight: 100 to 300 parts of rhizoma smilacis glabrae, 50 to 180 parts of radix sophorae flavescentis, 60 to 250 parts of herba houttuyniae, 50 to 160 parts of honeycomb, 50 to 150 parts of dysosma versipellis, 1 to 3 parts of osthole and 2 to 8 parts of oleic acid. The composition prepared from specific components and specific dosage has a remarkable effect of treating HPV infection, and the effect is superior to that of each pair of components.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a compound composition for treating HPV infection, a preparation method and an application thereof. Background Art

[0002] Human papillomavirus (HPV) is a circular DNA virus that primarily infects epithelial cells, with humans as its sole reservoir. Current research has demonstrated that infection with high-risk HPV is the root cause of cervical lesions and cancer. Current treatments for HPV infection primarily include physical therapy, medication, and vaccines. Physical therapy, including ozone water cleaning, laser treatment, microwave therapy, and cryotherapy, can remove localized warts but carries the risk of traumatic infection and may cause side effects such as hyperpigmentation and scarring. Medication options include interferon vaginal suppositories and Baofukang suppositories. However, interferon primarily inhibits viral replication and is not effective in addressing HPV-induced issues such as gynecological inflammation and metabolic immunodeficiency. Baofukang suppositories also have a low transdermal absorption rate, and their effectiveness needs to be further improved. While vaccines can prevent HPV infection, they must be administered prior to infection, are costly, and face significant barriers to widespread adoption in my country.

[0003] For this reason, relevant researchers have turned their attention to pure Chinese herbal preparations for the treatment of HPV infection that are low in cost, have an overall regulatory effect on low immunity, and have few toxic and side effects. For example, Chinese invention patent application CN115227795A discloses a compound medicine for the treatment of persistent HPV infection, which comprises, by mass fraction, 50-100 parts of houttuynia cordata, 50-100 parts of motherwort, 50-100 parts of astragalus root, and 50-100 parts of glossy privet fruit. 100 parts, 50-100 parts of Ecliptae, 30-60 parts of Psoralea corylifolia, 30-60 parts of Morinda officinalis, 30-60 parts of Aconite root processed, 30-60 parts of Ginseng, 30-60 parts of Rhizoma Alismatis, 30-60 parts of Poria, 20-50 parts of Atractylodes, 20-50 parts of Scutellaria, 20-50 parts of Phellodendron, 30-60 parts of Lithospermum officinale, 30-60 parts of Cortex Moutan, 20-50 parts of Trigonosciadium japonicum, 20-50 parts of Curcuma, 10-30 parts of Radix Glycyrrhizae Preparata. The compound medicine has good preventive and therapeutic effects, has a therapeutic effect on persistent infection of cervical HPV caused by various reasons, can quickly relieve symptoms such as lower abdominal pain, red and white vaginal discharge caused by persistent infection of cervical HPV, and regulate the body's qi and blood status at the same time, treating both the symptoms and the root cause. However, the patent application has the problems that the raw material composition is complex, the compound medicine formula and the preparation method need to be further optimized. Chinese invention patent application CN1840116A discloses a topical Chinese medicine for treating skin and mucous membrane diseases caused by human papillomavirus. The compound preparation is made from the Chinese herbs Cnidium monnieri, Scutellaria baicalensis, Brucea javanica, Smilax glabra, Houttuynia cordata, Sophora flavescens, and Nigella cerevisiae. The preparation is preferably an ointment. Further research conducted by the inventors on the formulation of the aforementioned patent application indicates that the formulation needs to be further optimized to enhance the efficacy of the formulation. Summary of the Invention

[0004] In view of the existing technology, the present invention provides a compound composition for treating HPV infection, a preparation method and an application. By optimizing the raw material composition, ratio and preparation process, the compound composition provided by the present invention has the characteristics of small dosage, low cost and better effect in treating HPV infection.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: The first aspect of the present invention relates to a compound composition for treating HPV infection, which is prepared from the following raw materials by weight: 100-300 parts of Smilax glabra, 50-180 parts of Sophora flavescens, 60-250 parts of Houttuynia cordata, 50-160 parts of Honeycomb, 50-150 parts of Illicium verum, 1-3 parts of osthole, and 2-8 parts of oleic acid.

[0006] Preferably, the compound composition is prepared from the following raw materials by weight: 120-240 parts of Smilax glabra, 50-150 parts of Sophora flavescens, 70-120 parts of Houttuynia cordata, 50-120 parts of Honeycomb, 50-120 parts of Illegible Lotus, 1.2-2.4 parts of osthole, and 3-6 parts of oleic acid.

[0007] More preferably, the mass ratio of osthole to oleic acid is 0.4-0.8:1.

[0008] A second aspect of the present invention relates to a method for preparing the above-mentioned compound composition for treating HPV infection, comprising the following steps: (1) Pretreatment: Grind the raw materials of Smilax glabra, Sophora flavescens, Houttuynia cordata, Beehive, and Dioscorea obovata into coarse powder; (2) extracting the coarse powder from step (1) with an ethanol solution, filtering, and recovering ethanol from the filtrate to obtain an extract; (3) Add osthole and oleic acid to the extract and freeze-dry to obtain the extract.

[0009] Preferably, the ethanol solution extraction times in step (2) is 2-3 times.

[0010] Preferably, the volume concentration of the ethanol solution in step (2) is 60-85%, the amount of ethanol solution added each time is independently selected from the solid-liquid ratio of 1:6-10, and the time of each extraction is independently selected from 1-2 hours.

[0011] Preferably, the relative density of the extract in step (2) at 50° C. is 1.05-1.15.

[0012] The third aspect of the present invention relates to the use of the above-mentioned compound composition for treating HPV infection or the compound composition for treating HPV infection prepared according to the above-mentioned preparation method in the preparation of a drug for treating HPV infection.

[0013] Preferably, the drug is an internal preparation or an external preparation.

[0014] Further preferably, the external preparation is selected from liniments, creams, external gels, ointments, granules, lotions or tinctures.

[0015] Preferably, the application is specifically skin warts, condyloma acuminata, cervical squamous intraepithelial lesions and / or vaginal squamous intraepithelial lesions caused by HPV infection.

[0016] Beneficial effects of the present invention: The compound composition provided by the present invention is prepared by comprising specific components and specific dosages, and has the characteristics of small dosage, low cost and better effect in treating HPV infection. DETAILED DESCRIPTION

[0017] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0018] Example 1: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 120 parts of smilax glabra, 150 parts of sophora flavescens, 70 parts of houttuynia cordata, 120 parts of honeycomb, 50 parts of iris root, 2.4 parts of osthole, and 3 parts of oleic acid.

[0019] Example 2: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 240 parts of Smilax glabra, 50 parts of Sophora flavescens, 120 parts of Houttuynia cordata, 50 parts of Honeycomb, 120 parts of Illicium verum, 1.2 parts of Osthole, and 3 parts of oleic acid.

[0020] Example 3: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 180 parts of Smilax glabra, 80 parts of Sophora flavescens, 85 parts of Houttuynia cordata, 75 parts of Honeycomb, 70 parts of Illicium verum, 2 parts of osthole, and 4 parts of oleic acid.

[0021] The preparation method of the compound composition of the above-mentioned embodiment 1-embodiment 3 comprises the following preparation steps: (1) Pretreatment: Grind the raw materials of Smilax glabra, Sophora flavescens, Houttuynia cordata, Beehive, and Dioscorea obovata into coarse powder; (2) The crude powder of step (1) was extracted twice with an ethanol solution having a volume concentration of 75%, each time with 8 times the amount of ethanol solution for 1 hour, the filtrate was filtered and combined, and the ethanol was recovered to obtain an extract with a relative density of 1.1 at 50°C; (3) Add osthole and oleic acid to the extract and freeze-dry to obtain the extract.

[0022] Comparative Example 1: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 180 parts of Smilax glabra, 80 parts of Sophora flavescens, 85 parts of Houttuynia cordata, 75 parts of Honeycomb, 70 parts of Illicium verum, 2 parts of osthole, and 4 parts of linoleic acid.

[0023] The difference from Example 3 is that the raw material composition is different, and the raw material of this comparative example uses an equal amount of linoleic acid instead of oleic acid. Other components are the same as those of Example 3.

[0024] Preparation method: except that step (3) is changed to: "add osthole and linoleic acid to the extract, and freeze-dry to obtain the product", the remaining steps are the same as the preparation steps of Example 3.

[0025] Comparative Example 2: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 180 parts of Smilax glabra, 80 parts of Sophora flavescens, 85 parts of Houttuynia cordata, 75 parts of Honeycomb, 70 parts of Illicium verum, 2 parts of tea polyphenols (purchased from Zhejiang Oriental Tea Technology, EGCG ≥ 98%), and 4 parts of oleic acid.

[0026] The difference from Example 3 is that the raw material composition is different. In this comparative example, an equal amount of tea polyphenols is used instead of osthole.

[0027] Preparation method: except that step (3) is changed to: "add tea polyphenols and oleic acid to the extract, freeze-dry, and obtain", the remaining steps are the same as the preparation steps of Example 3.

[0028] Comparative Example 3: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 180 parts of Smilax glabra, 80 parts of Sophora flavescens, 85 parts of Houttuynia cordata, 75 parts of Honeycomb, 70 parts of Illicium verum, 4 parts of osthole, and 2 parts of oleic acid.

[0029] The difference from Example 3 is that the amounts of osthole and oleic acid in the raw materials and the ratio of the two are different. In Example 3, osthole is 2 parts and oleic acid is 4 parts, and the ratio of the two is 0.5:1, while in Comparative Example 3, osthole is 4 parts and oleic acid is 2 parts in the raw materials, and the ratio of the two is 2:1.

[0030] The preparation method is the same as Example 3.

[0031] Comparative Example 4: A compound composition for treating HPV infection is prepared from the following raw materials in parts by weight: 180 parts of Smilax glabra, 80 parts of Sophora flavescens, 85 parts of Houttuynia cordata, 75 parts of Honeycomb, 70 parts of Illicium verum, 1 part of osthole, and 5 parts of oleic acid.

[0032] The difference from Example 3 is that the amount of osthole and oleic acid in the raw materials and the ratio of the two are different. In Example 3, osthole is 2 parts and oleic acid is 4 parts, and the ratio of the two is 0.5:1, while in Comparative Example 4, osthole is 1 part and oleic acid is 5 parts in the raw materials, and the ratio of the two is about 0.2:1.

[0033] The preparation method is the same as Example 3.

[0034] Comparative Example 5: It was prepared according to the preparation method of Example 1 of Chinese invention patent application CN1840116A, specifically: Raw materials: Cnidium monnieri 300g, Illicium verum 240g, Brucea javanica 100g, Smilax glabra 600g, Houttuynia cordata 400g, Sophora flavescens 300g, Beehive 300g; Preparation of active pharmaceutical ingredients: Extract Brucea javanica oil by squeezing or supercritical extraction; extract Cnidium monnieri, Illicium verum, and Brucea javanica after oil extraction three times with 80% ethanol: the first time is 1 hour with 10 times the amount of ethanol, the second time is 1 hour with 8 times the amount of ethanol, and the third time is 1 hour with 6 times the amount of ethanol; combine the three alcohol extracts, filter, and recover ethanol from the filtrate to form a thick paste of alcohol extract, and set aside the residue. Add Sophora flavescens, Houttuynia cordata, Smilax glabra, and Beehives to the above-mentioned residues and decoct three times with water: the first time is 2 hours with 10 times the amount of water, the second time is 1.5 hours with 8 times the amount of water, and the third time is 1 hour with 6 times the amount of water; the combined decoctions are allowed to stand, centrifuged, and the supernatant is concentrated to a relative density of 1.30-1.35 to obtain a thick paste of water extract for later use; The product is obtained by mixing the Brucea javanica oil, alcohol extract and water extract.

[0035] Experimental Example 1: Cellular Efficacy Experiment Before the experiment, the drugs of Examples 1 to 3 and Comparative Examples 1 to 5 were dissolved in DMSO, filtered, and the filtrate was the test drug solution, which was stored at low temperature for future use.

[0036] 1. Effect on the proliferation rate of HaCaT cells HaCaT cells were cultured in DMEM medium to a volume of 0.5 × 10 6 The cell suspension of different density was inoculated into the culture plate, 0.1 mL per well, and 0.1 mL of test solution of different concentrations was added to each well. After culturing for 24 h, MTT was added and cultured for another 4 h. The colorimetry was performed using an enzyme-linked immunosorbent assay (ELISA) to calculate the cell proliferation rate of each well and obtain the IC values ​​of different test solutions. 50 Value (mg / mL).

[0037] 2. Effect on Hela cell proliferation rate HeLa cells were cultured in DMEM medium to a volume of 0.5 × 10 6 The cell suspension of different density was inoculated into the culture plate, 0.1 mL per well, and 0.1 mL of test solution of different concentrations was added to each well. After culturing for 24 h, MTT was added and cultured for another 4 h. The colorimetry was performed using an enzyme-linked immunosorbent assay (ELISA) to calculate the cell proliferation rate of each well and obtain the IC values ​​of different test solutions. 50 Value (mg / mL).

[0038] 3. Effect on the proliferation rate of A431 cells A431 cells were cultured in F12 medium to a volume of 0.5 × 10 6 The cell suspension of different density was inoculated into the culture plate, 0.1 mL per well. After culturing for 48 h, 0.1 mL of test solution of different concentrations was added to each well. After culturing for another 24 h, MTT was added and the culture was continued for 4 h. The colorimetry was performed using an enzyme-linked immunosorbent assay (ELISA) to calculate the cell proliferation rate of each well and obtain the IC values ​​of different test solutions. 50 Value (mg / mL).

[0039] The specific data results are shown in Table 1.

[0040] Table 1: Effects on cell proliferation rate

[0041] According to the above experimental results, whether it is HaCaT cells, Hela cells, or A431 cells, the IC 50 The concentration is much lower than the IC 50 concentration, so the effect of the embodiment is better than that of the comparative examples.

[0042] Experimental Example 2: Efficacy on Condyloma Acuminatum Animal Model Test Drug: Add equal proportions of PEG400, propanol, and PEG2000 to the compositions prepared in Examples 1-3 and Comparative Examples 1-5, respectively, and stir to obtain an ointment. Each gram of ointment is equivalent to 1 g of the crude drug, and set aside. Simultaneously, prepare a blank ointment using the same proportions of PEG400, propanol, and PEG2000.

[0043] Experimental animals: Female nude mice, 4 weeks old, weighing 15 ± 3 g, were randomly divided into a blank control group, a model control group, and a drug treatment group (Examples 1-3, Comparative Examples 1-5). The animals were housed in a room maintained at 18-26°C, with a humidity of 40-70%, and natural daylighting to ensure stable conditions. They were fed a pelleted diet specifically for mice (provided by Tianjin Aoyi De Laboratory Supplies Co., Ltd.) and had free access to food and water.

[0044] The blank control group consisted of healthy, normal nude mice. The model control and drug treatment groups were constructed using experimental animals for a condyloma acuminata animal model. The condyloma acuminata animal model was constructed as follows: warts obtained during clinical surgery were placed in an EP tube containing lactated Ringer's solution, placed in an ice box, and quickly transported to the laboratory. The warts were then placed in 4 mL of D-hanks solution with 10 mL of 100× penicillin-streptomycin and shaken well. The condyloma acuminata tissue was measured with a vernier caliper and cut into several 0.5 cm × 0.5 cm × 0.5 cm tissue blocks. The blocks were then soaked in the mixed solution for 45 minutes. The mice were anesthetized with 50 mg / kg of 1% sodium pentobarbital intraperitoneally. The right back of the mice was disinfected three times with 75% alcohol. A sterile scalpel blade was used to make an incision on the right back of the nude mice. The warts were grasped with sterile forceps, embedded in the skin, and secured. The warts were then sutured with surgical suture. Three weeks later, the condyloma acuminata animal model was successfully established. The drug treatment group received the experimental ointment applied to the dorsal implant site of nude mice, while the model control group received a blank ointment applied twice daily, 0.5 g each time. After two weeks of treatment, all animals were sacrificed, and total RNA was extracted from the wart tissue of each experimental group using an Amyjet Scientific total RNA extraction kit. The entire experimental procedure adhered to the kit's instructions. RNA concentration was determined by mixing the RNA stock solution and enzyme-free water in a cuvette at a ratio of 2 μL:48 μL. RNA concentration and A / OD ratio were measured spectrophotometrically. A value between 1.8 and 2.0 was considered high purity, and the detected RNA concentration was recorded. RNA samples were stored in a -80°C freezer or, after reverse transcription, at -20°C. The total mass of reverse-transcribed RNA was 3 μg, and the volume of RNA stock solution required for reverse transcription was calculated based on this mass. 13 μL of the fluorescent substrate 5× NI-RT Master Mix was added to each enzyme-free EP tube reaction in the order indicated. Finally, enzyme-free water was added to bring the volume up to 20 μL. All operations were performed on ice. The mixture in the centrifuge tube was then vortexed to mix thoroughly and briefly centrifuged to collect the solution on the tube walls. The reaction was then continued as follows: first at room temperature for 3 minutes, then at 37°C for 20 minutes, and finally at 80°C for 1 minute. After the reaction was complete, the reaction was briefly centrifuged and cooled on ice. The reverse-transcribed cDNA products were rapidly quantified using multiplex fluorescence quantitative PCR within 3 hours to detect HPV viral load (copies) in the reverse-transcribed cDNA samples. The results are shown in Table 2.

[0045] Table 2: Effects on viral load (copies)

[0046] Compared with the model control group, & P<0.05, && P < 0.01, &&& P<0.001; compared with Example 3, # P<0.05, ## P < 0.01, ### P<0.001.

[0047] According to the above experimental results, it can be seen that the drugs of Examples 1-3 and Comparative Examples 1-5 can reduce the viral load of condyloma acuminata animal model mice, but the effects of the Examples are better than those of the Comparative Examples.

[0048] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A compound composition for treating HPV infection, characterized in that: The compound composition is prepared from the following raw materials by weight: 100-300 parts of smilax glabra, 50-180 parts of sophora flavescens, 60-250 parts of houttuynia cordata, 50-160 parts of honeycomb, 50-150 parts of ophiopogon, 1-3 parts of osthole and 2-8 parts of oleic acid.

2. The compound composition for treating HPV infection according to claim 1, characterized in that: The compound composition is prepared from the following raw materials by weight: 120-240 parts of smilax glabra, 50-150 parts of sophora flavescens, 70-120 parts of houttuynia cordata, 50-120 parts of honeycomb, 50-120 parts of ophiopogon, 1.2-2.4 parts of osthole, and 3-6 parts of oleic acid.

3. The compound composition for treating HPV infection according to any one of claims 1 to 2, characterized in that: The mass ratio of osthole to oleic acid is 0.4-0.8:

1.

4. A method for preparing the compound composition for treating HPV infection according to any one of claims 1 to 3, comprising the following steps: (1) Pretreatment: Grind the raw materials of Smilax glabra, Sophora flavescens, Houttuynia cordata, Beehive, and Dioscorea obovata into coarse powder; (2) extracting the coarse powder from step (1) with an ethanol solution, filtering, and recovering ethanol from the filtrate to obtain an extract; (3) Add osthole and oleic acid to the extract and freeze-dry to obtain the extract.

5. The preparation method according to claim 4, characterized in that The number of extractions with the ethanol solution in step (2) is 2-3 times, the volume concentration of the ethanol solution is 60-85%, the amount of ethanol solution added per time is independently selected from a solid-liquid ratio of 1:6-10, the time for each extraction is independently selected from 1-2 hours, and the relative density of the extract at 50°C is 1.05-1.

15.

6. Use of the compound composition for treating HPV infection according to any one of claims 1 to 3 or the compound composition for treating HPV infection prepared according to the preparation method according to any one of claims 4 to 5 in the preparation of a drug for treating HPV infection.

7. The use according to claim 6, characterized in that The medicine is an internal preparation or an external preparation.

8. The use according to claim 7, characterized in that The external preparation is selected from liniments, creams, external gels, ointments, granules, lotions or tinctures.

9. The use according to claim 6, characterized in that The application is specifically for skin warts, condyloma acuminata, cervical squamous intraepithelial lesions and / or vaginal squamous intraepithelial lesions caused by HPV infection.

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