Use of a traditional Chinese medicine composition in the preparation of a medicine for inhibiting human papilloma virus

Abstract

The application relates to a use of a traditional Chinese medicine composition in preparation of a medicine for inhibiting human papilloma virus, which is prepared from raw medicine with the following weight parts: 70-100 parts of rhubarb, 70-100 parts of mirabilite, 70-100 parts of gallnut. The traditional Chinese medicine composition has excellent killing effect on human papilloma virus (HPV), low cost, small toxic and side effects, and has a good application prospect.

Description

Use of a traditional Chinese medicine composition in preparing a drug for inhibiting human papillomavirus Technical Field

[0001] The invention belongs to the field of antiviral Chinese medicine, and particularly relates to use of a Chinese medicine composition in preparing a medicine for inhibiting human papillomavirus. Background Art

[0002] Human papillomavirus (HPV) is a group of spherical, tiny, non-enveloped, circular, double-stranded DNA viruses belonging to the genus Papillomavirus in the family Papovaviridae. Based on their biological characteristics and carcinogenic potential, HPV is divided into high-risk and low-risk types. For example, HPV14 / 7 are low-risk cutaneous types, associated with common warts, flat warts, and plantar warts; HPV6 / 111 are low-risk types, closely associated with the development of genital warts; and HPV 16 / 18 are high-risk types, carrying the highest risk of inducing cancer. HPV infection can cause a range of symptoms and lesions, including genital warts, vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, and cervical cancer, posing a threat to human life and health.

[0003] Currently, there are no effective treatments worldwide for HPV infection, particularly high-risk HPV infection. Therefore, prevention is key. The most effective way to prevent cervical cancer is to prevent high-risk HPV infection and to detect and treat the infection early. HPV vaccination is an effective means of preventing infection. Maintaining personal hygiene and avoiding unsafe sex can also help reduce the risk of infection.

[0004] It takes a long time for HPV infection to develop into lesions such as cervical disease, or even malignant tumors, so suppressing HPV is particularly important. Finding safe and effective anti-HPV treatments has always been a research hotspot.

[0005] Chinese invention patent application CN 113559237 A provides a traditional Chinese medicine gel composition for preventing and inhibiting HPV. The composition includes recombinant human collagen peptides, marine fish oligopeptides, lithospermum officinale, phellodendron amurense, astragalus root, dandelion root, black plum, safflower, honeysuckle, poria, horsetail, cyperus rotundus, and polyhexamethylene biguanide. While the composition has some HPV inhibition efficacy, the reported composition contains a large number of medicinal ingredients, is costly, and its antibacterial efficacy needs to be further improved.

[0006] There is an urgent need for a compound gel with simple taste, low cost and excellent antibacterial effect.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a use of a traditional Chinese medicine composition in preparing a medicine for inhibiting human papillomavirus.

[0009] The present invention provides a use of a traditional Chinese medicine composition in preparing a drug for inhibiting human papillomavirus. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight:

[0010] 70-100 parts of rhubarb, 70-100 parts of Glauber's salt, and 70-100 parts of gallnut.

[0011] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight:

[0012] 80-90 parts of rhubarb, 80-90 parts of sodium sulfate, and 80-90 parts of gallnut.

[0013] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight:

[0014] 90 parts of rhubarb, 90 parts of sodium sulfate, and 90 parts of gallnuts.

[0015] Furthermore, the medicine is a preparation prepared with the powder of the raw material medicine of the traditional Chinese medicine composition, or the water or organic solvent extract of the raw material medicine as the active ingredient, and the addition of pharmaceutically acceptable excipients.

[0016] Furthermore, the medicine is a preparation prepared by adding 6 to 7 times the amount of water to the raw material of the traditional Chinese medicine composition and soaking it for 40 minutes, then boiling and extracting it for 30 minutes, concentrating the extract to half the amount of the original water, and adding pharmaceutically acceptable excipients.

[0017] Furthermore, the drug is a transdermal preparation.

[0018] Furthermore, the transdermal preparation is any one of a gel, an ointment, a cream, an unguent and a liquid preparation.

[0019] Furthermore, the gel is made of the following raw materials and excipients in parts by weight:

[0020] 70-100 parts of rhubarb, 70-100 parts of sodium sulfate, 70-100 parts of gallnut, and 0.5-10 parts of gel base; preferably, 90 parts of rhubarb, 90 parts of sodium sulfate, 90 parts of gallnut, and 1.8 parts of gel base.

[0021] Furthermore, the gel matrix is ​​xanthan gum, agar, alginic acid, pectin, and natural gum.

[0022] Furthermore, the human papillomavirus is a high-risk human papillomavirus;

[0023] Preferably, the human papillomavirus is HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-14, HPV-11, HPV-7 and / or HPV-6.

[0024] Furthermore, the medicine is a medicine for preventing and treating human papillomavirus infectious diseases.

[0025] Furthermore, the human papillomavirus infectious disease is condyloma acuminata, vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, cervical cancer, rectal cancer, oral cancer or tonsil cancer.

[0026] The "Sodium Sulfate" described in the present invention is a sulfate mineral, Sodium Sulfate Family Sodium Sulfate, which is a processed and refined crystal mainly containing sodium sulfate decahydrate and is a traditional Chinese medicine.

[0027] The traditional Chinese medicine composition of the present invention, which is composed of three herbs, rhubarb, sodium sulfate and gallnut, in a specific proportion, has an excellent killing effect on human papillomavirus. In vitro cell experiments have confirmed that the killing rate of human papillomavirus pseudoviruses (HPV6 / 7 / 11 / 14 / 16 / 18) including skin low-risk type 7, skin high-risk type 14, mucosal low-risk types 6 and 11, and mucosal high-risk types 16 and 18, reaches over 90%. The traditional Chinese medicine composition of the present invention has low cost and few toxic and side effects. It can eliminate human papillomavirus outside the human body by acting on the skin, cut off the transmission route of human papillomavirus infectious diseases, and effectively prevent and treat human papillomavirus infectious diseases. Specifically, it can prevent and treat genital warts, vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, cervical cancer, rectal cancer, oral cancer or tonsil cancer caused by human papillomavirus, and has good application prospects.

[0028] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0029] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. DETAILED DESCRIPTION

[0030] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0031] Example 1. Preparation of the compound gel preparation of the present invention

[0032] Recipe: 90g rhubarb, 90g Glauber's salt, and 90g gallnut.

[0033] Preparation method: Weigh the above medicinal ingredients according to the ratio, add 1800 mL of water and soak for 40 minutes, boil and keep boiling for 30 minutes, filter, concentrate the filtrate to 900 mL, add 1.8 g of xanthan gum, stir evenly, and divide into packages to obtain a gel.

[0034] Example 2: Preparation of the compound gel preparation of the present invention

[0035] Recipe: 70g rhubarb, 100g Glauber's salt, and 100g gallnut.

[0036] Preparation method: Same as Example 1

[0037] Example 3: Preparation of the compound gel preparation of the present invention

[0038] Recipe: 100g rhubarb, 70g Glauber's salt, and 70g gallnut.

[0039] Preparation method: Same as Example 1

[0040] Example 4. Preparation of the compound gel preparation of the present invention

[0041] Recipe: 70g rhubarb, 70g Glauber's salt, 100g gallnut.

[0042] Preparation method: Same as Example 1

[0043] Example 5. Preparation of the compound gel preparation of the present invention

[0044] Recipe: 80g rhubarb, 90g Glauber's salt, and 90g gallnut.

[0045] Preparation method: same as Example 1.

[0046] Example 6: Preparation of the compound gel preparation of the present invention

[0047] Recipe: 90g rhubarb, 80g Glauber's salt, 90g gallnut.

[0048] Preparation method: same as Example 1.

[0049] Example 7. Preparation of the compound gel preparation of the present invention

[0050] Recipe: 70g rhubarb, 100g Glauber's salt, 70g gallnut.

[0051] Preparation method: same as Example 1.

[0052] Example 8. Preparation of the compound gel preparation of the present invention

[0053] Recipe: 100g rhubarb, 70g Glauber's salt, and 100g gallnut.

[0054] Preparation method: same as Example 1.

[0055] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0056] Experimental Example 1: Experiment on the inhibition of HPV16 by the compound gel preparation of the present invention

[0057] 1. Equipment

[0058] 1. Experimental virus strain: Human papillomavirus type 16 pseudovirus (HPV16) (Henan Biaowu Biotechnology Co., Ltd.).

[0059] 2. Host cells: 293T human embryonic kidney cells.

[0060] 3. Cell maintenance medium, complete cell culture medium, fetal bovine serum, and sterile purified water.

[0061] 4. Carbon dioxide incubator (BPN-CH model) (Instrument No.: Y / QW-022).

[0062] 5. Biological safety cabinet (BSC-1100IIA2) (Instrument No.: Y / QW-027).

[0063] 6. Low temperature constant temperature bath (DC-0520 model) (Instrument No.: Y / QW-009).

[0064] 7. Inverted biological microscope (XD-202 model) (Instrument No.: Y / FW-029).

[0065] 8. Carrier: 1cm×1cm white plain cloth.

[0066] 9. Cell culture flasks, 96-well culture plates, and pipettes.

[0067] 2. Experimental Methods

[0068] Refer to "Technical Specifications for Disinfection" (2002 edition) 2.1.1.10.7.

[0069] 1.1. Preparation of virus suspension

[0070] The titer used in the experiment was 107TCID 50 The specific preparation process of HPV16 suspension is as follows:

[0071] (1) Remove the frozen host cells for testing from liquid nitrogen, thaw rapidly in warm water at 37°C, and transfer them to a cell tube containing cell maintenance medium using a capillary pipette. Pipet several times to mix, and immediately centrifuge (3000 rpm, 3 min). Remove the supernatant. Add the appropriate cell maintenance medium, pipette several times to mix, and centrifuge as above. Transfer the cells to a culture flask containing 10 ml of complete culture medium after the same centrifugation. Observe the cell growth daily. When the cells have grown into a complete monolayer, use them for disinfection testing.

[0072] (2) Remove the frozen test virus seed, thaw it in a 37°C water bath, dilute it 10-fold with cell maintenance medium, and then inoculate it into a cell bottle that has grown a monolayer of cells. Place it in a 37°C incubator to allow it to adhere to the cells and grow. Observe the pathological changes daily. When 3 / 4 of the cells show pathological changes, harvest the virus.

[0073] (3) Place the culture medium containing the virus and host cells in an ice bath and use ultrasound (or repeated freeze-thaw cycles) to disrupt the host cells and release the virus. Then, centrifuge as quickly as possible (6000 rpm, 15 minutes) to remove the precipitate (mainly cell debris). The supernatant is the desired virus suspension. Dispense 1.0 ml per tube into sterile centrifuge tubes (1.5 ml).

[0074] (4) Take one tube of virus suspension and determine its virus titer according to the virus titer determination method to make it reach 10 7 TCID 50 The rest were frozen and stored at -80℃ for future use.

[0075] 1.2 Quantitative testing of the inactivation ability of the compound gel preparation on HPV16 virus suspension

[0076] The compound gel prepared in Example 1 was used to perform a quantitative inactivation test of HPV16 virus suspension, and a positive control group and a negative control group were set up. The specific test operation procedures are as follows:

[0077] 1) Take the compound gel as the test sample of the test group and place it in a water bath at 20℃±1℃ for later use.

[0078] 2) Mix 100 μl of organic interfering substance (0.3% BSA) with 100 μl of virus stock solution and incubate in a 20°C ± 1°C water bath for 5 minutes. Add 0.8 ml of the sample to be tested, mix immediately, and record the time. After 8 hours, remove 0.1 ml and add to the neutralizer (lecithin) and mix thoroughly.

[0079] 3) In the positive (virus) control group, sterile deionized water was used instead of the test sample, and HPV16 was added for testing and culture according to the same steps as the test group to observe whether the HPV16 virus grew well.

[0080] 4) Use complete medium without HPV16 virus as a negative control, inoculate cells, and observe whether the medium is contaminated and whether the cells grow well; the specific method is as follows:

[0081] Take 0.1 ml of complete medium without HPV16 and add it dropwise to 4 wells of a 96-well plate (each well should already be covered with a monolayer of host cells). Incubate at 37°C for 1 to 2 hours to ensure that the complete medium is fully adsorbed on the cells. Remove the culture plate and replace the cell maintenance medium. Continue to culture in a carbon dioxide incubator (37°C, 5% CO2). Observe the cytopathic effect under a microscope daily for 5 consecutive days, observing and recording the cytopathic effect in each well.

[0082] 5) Virus titer of each group was determined using the endpoint dilution method.

[0083] 6) The experiment was repeated 3 times.

[0084] 7) Endpoint dilution procedure: First, perform a 10-fold serial dilution of the sample to be titrated using cell maintenance medium. Then, titrate the residual virus in each dilution in a 96-well culture plate. Perform four wells for each dilution (each well should be filled with a monolayer of host cells). Incubate at 37°C for 1–2 hours to ensure that all residual virus is adsorbed to the cells. Remove the plate and replace the cell maintenance medium. Continue incubating in a CO2 incubator (37°C, 5% CO2). Observe the cytopathic effect daily under a microscope for 5 consecutive days, observing and recording the cytopathic effect in each well.

[0085] Calculation of viral infection titer by endpoint dilution method: half cell infectious dose (TCID 50 )express.

[0086] The average inactivation logarithm value is calculated as follows: let the average virus infection titer (TCID50) of the positive (virus) control group be N0, and the average virus infection titer (TCID50) of the test (disinfection) group be Nx.

[0087] Average logarithmic inactivation value = log No - log Nx

[0088] 2. Experimental Results

[0089] The test results of the inactivation ability of the compound gel preparation of Example 1 on the HPV16 virus suspension are shown in Table 1.

[0090] Table 1 Human papillomavirus type 16 pseudovirus (HPV16) inactivation test results

[0091] Note: Negative control cells grew well and showed no cytopathic effects.

[0092] From the results of three repeated tests in Table 1, it can be seen that the compound gel prepared in Example 1 of the present invention has an average inactivation logarithm value of 3.17 for human papillomavirus type 16 pseudovirus (HPV16) after 8 hours of action, and an average killing rate of 99.93%, which has a strong efficacy in killing HPV16.

[0093] Experimental Example 2 Inhibition experiment of the compound gel preparation of the present invention on different types of human papillomavirus

[0094] The compound gel prepared in Examples 1 to 8 was used to inactivate six types of human papillomavirus, including skin low-risk type 7; skin high-risk type 14; mucosal low-risk types 6, 11; and mucosal high-risk types 16, 18. The procedure was specifically performed according to the method of Experimental Example 1. The compound gel in each test group was tested three times, and the average killing rate of the three times was calculated. A positive control group and a negative control group were also set.

[0095] Table 2 Results of inactivation tests of different types of human papillomavirus

[0096] Note: Negative control cells grew well and showed no cytopathic effects.

[0097] As shown in Table 2, the ratio of rhubarb, sodium sulfate, and gallnut in the compound gel is 70-100:70-100:70-100, and the killing rate against five types of human papillomavirus (cutaneous low-risk type 7, skin high-risk type 14, mucosal low-risk types 6, 11, and mucosal high-risk types 16, 18) reaches over 90%. In particular, when rhubarb, sodium sulfate, and gallnut are combined in equal proportions, the killing rate against each type of human papillomavirus reaches over 99%. The present invention produces a broad-spectrum killing effect on human papillomavirus by combining rhubarb, sodium sulfate, and gallnut in a specific ratio.

[0098] Experimental Example 3 Inhibitory Effect of Different Chinese Herbal Combinations in the Compound Preparation of the Present Invention on Different Types of Human Papillomavirus

[0099] 1. Test sample:

[0100] Sample ①: 270g of rhubarb, prepared according to the same preparation method as Example 1

[0101] Sample ②: 270g of Glauber's salt, prepared according to the same preparation method as Example 1

[0102] Sample ③: 270g of Gallnut, prepared in the same manner as in Example 1

[0103] Sample ④: 65g of rhubarb, 65g of sodium sulfate, and 140g of gallnut, prepared according to the same preparation method as in Example 1

[0104] Sample ⑤: 105g of rhubarb, 60g of sodium sulfate, and 105g of gallnut, prepared according to the same preparation method as in Example 1

[0105] Sample ⑥: 60g of rhubarb, 145g of sodium sulfate, and 65g of gallnut, prepared according to the same preparation method as in Example 1

[0106] Sample ⑦: 134g rhubarb, 68g sodium sulfate, 68g gallnut, prepared according to the same preparation method as Example 1

[0107] Sample ⑧: 60g of rhubarb, 105g of sodium sulfate, and 105g of gallnut, prepared according to the same preparation method as in Example 1

[0108] Sample 9: 1.8 g of xanthan gum was dissolved in water and stirred into a gel.

[0109] 2. Experimental methods

[0110] Samples ① to ⑨ were used to perform inactivation tests on six types of human papillomavirus: cutaneous low-risk type 7; cutaneous high-risk type 14; mucosal low-risk types 6, 11; and mucosal high-risk types 16, 18. The procedure was specifically performed according to the method of Experimental Example 1. The compound gel in each test group was tested three times, and the average value of the three times was calculated. Positive and negative control groups were also set up.

[0111] Table 3 Killing effects of the compound gel preparation on different types of human papillomavirus

[0112] Note: Negative control cells grew well and showed no cytopathic effects.

[0113] As can be seen from Table 3: the gel with Glauber's salt as the active ingredient and the gel made with only the auxiliary material xanthan gum have no killing effect on human papillomavirus; the gel with rhubarb as the active ingredient has no killing effect on human papillomavirus HPV-7 and HPV-18, and its killing ability on other human papillomaviruses is very low; the gel with gallnut as the active ingredient and the gel made by mixing gallnut, rhubarb and Glauber's salt in a ratio other than 70-100:70-100:70-100 as the active ingredient still have weak killing ability on human papillomavirus.

[0114] In summary, the gel prepared from the traditional Chinese medicine composition of rhubarb, sodium sulfate, and gallnut in specific proportions has been verified by in vitro cell experiments to have a killing rate of over 90% against human papillomavirus pseudoviruses (HPV6 / 7 / 11 / 14 / 16 / 18) including skin low-risk type 7, skin high-risk type 14, mucosal low-risk types 6 and 11, and mucosal high-risk types 16 and 18. The traditional Chinese medicine composition of the present invention has low cost and few toxic and side effects. It can eliminate human papillomavirus from the human body by acting on the skin, cut off the transmission pathway of human papillomavirus infectious diseases, and effectively prevent and treat human papillomavirus infectious diseases, thus having good application prospects.

Claims

1. Use of a traditional Chinese medicine composition in the preparation of a drug for inhibiting human papillomavirus, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 70-100 parts of Rheum officinale, 70-100 parts of mirabilite, 70-100 parts of Chinese gall.

2. The use according to claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 80-90 parts of Rheum officinale, 80-90 parts of mirabilite, 80-90 parts of Chinese gall.

3. The use according to claim 2, wherein The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 90 parts of Rheum officinale, 90 parts of mirabilite, 90 parts of Chinese gall.

4. The use according to claim 1, characterized in that, The drug is a preparation prepared by using the powder of the raw materials of the traditional Chinese medicine composition, or the water or organic solvent extract of the raw materials as the active ingredient, plus pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that The drug is a preparation prepared by soaking the raw materials of the traditional Chinese medicine composition in 6-7 times the amount of water for 40 minutes, then boiling and extracting for 30 minutes, concentrating the extract to half of the original amount of water, and then adding pharmaceutically acceptable excipients. The drug is a transdermal drug delivery preparation; The transdermal drug delivery preparation is any one of a gel, an ointment, a cream, an oil ointment and a liquid preparation.

6. The use according to claim 5, characterized in that, The gel is prepared from the following raw materials in parts by weight: 70-100 parts of Rheum officinale, 70-100 parts of mirabilite, 70-100 parts of Chinese gall, 0.5-10 parts of a gel matrix; preferably, 90 parts of Rheum officinale, 90 parts of mirabilite, 90 parts of Chinese gall, 1.8 parts of a gel matrix.

7. The use according to claim 6 or 7, characterized in that, The gel matrix is xanthan gum, agar, alginic acid, pectin, natural gum.

8. The use according to claim 6 or 7, characterized in that, The human papillomavirus is a high-risk human papillomavirus; preferably, the human papillomavirus is HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-14, HPV-11, HPV-7 and / or HPV-6.

9. The use according to any one of claims 1 to 8, characterized in that, The drug is a drug for preventing and treating human papillomavirus infectious diseases.

10. The use according to claim 9, characterized in that, The human papillomavirus infectious diseases are condyloma acuminata, vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, cervical cancer, rectal cancer, oral cancer or tonsillar cancer.