New application of fructus arctii essential oil and ganoderma lucidum spore oil, pharmaceutical composition for treating human papilloma virus infection as well as preparation method and application of pharmaceutical composition

Abstract

The invention provides application of fructus arctii essential oil in preparation of an external medicine for resisting human papilloma virus. The invention further provides application of the ganoderma lucidum spore oil in preparation of the external medicine for resisting the human papilloma virus. The invention provides a pharmaceutical composition for treating human papilloma virus infection. The pharmaceutical composition is prepared from the following raw materials in parts by weight: 3.2-4.8 parts of fructus arctii essential oil and 0.8-1.2 parts of ganoderma lucidum spore oil. The invention also provides a preparation method and application of the pharmaceutical composition. According to the invention, a compounding process is adopted, the formula of main medicines and auxiliary materials is determined, the compound ganoderma lucidum essential oil with good stability is successfully prepared, and a thin-layer chromatography method and a gas chromatography quality evaluation method for the main medicines in the compound are preliminarily established. The compound coriolus versicolor essential oil is administrated in a local smearing mode, in-vitro experiments show that the compound coriolus versicolor essential oil can interfere with virus replication and is high in antiviral effect, especially for HPV16, the effect is mild, no stimulation to skin is found through clinical observation, and adverse reactions are few.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition for treating human papillomavirus infection, its preparation method, and its use. Background Technology

[0002] Human papillomavirus (HPV) infection is a common sexually transmitted disease that can lead to malignant tumors such as cervical cancer. Currently, treatment for HPV infection mainly involves topical therapy, antiviral therapy, traditional Chinese medicine, and immune modulation, with topical preparations being particularly common in treatment.

[0003] Reishi spore powder, as a relatively good immunomodulator, can significantly improve human immune function. Zhou Yongli et al., "Study on the Anti-Cervical Human Papillomavirus Infection Effect of Reishi Spore Powder," *Chinese Journal of Medical Science*, January 2017, Vol. 7, No. 2, published the value of Reishi spore powder in the treatment of cervical human papillomavirus (HPV) infection. Other literature has also used Reishi in combination with Nucleoside Ionizing Tablets to treat cervical HPV infection (Kou Haimei et al., "Clinical Observation of Nucleoside Ionizing Tablets Combined with Reishi Dispersible Tablets in the Treatment of Cervical Human Papillomavirus Infection," *Chinese Journal of Clinical Research*, 2019, Vol. 11, No. 1). Among these, HPV16 is the most common and carcinogenic genotype in the HPV family, known as the "number one high-risk type."

[0004] CN201910193919.X, Invention Title: A Composition for the Prevention and Elimination of Human Papillomavirus (HPV), discloses a composition for the prevention and elimination of human papillomavirus (HPV), mainly composed of *Radix Amurensis*, *Radix Kalanchoe*, *Radix Sophorae Flavescentis*, *Fructus Cnidii*, *Fructus Chicorydalis*, *Radix Coptise*, and / or *Radix Arctii*, and / or *Semen Plantagoeae*, and / or toad venom. The composition of this invention can be used to prepare suitable formulations and is widely applied in the treatment of human papillomavirus (HPV) diseases.

[0005] There are currently no reports of using burdock seed and Ganoderma lucidum in combination for the treatment of human papillomavirus infection. Summary of the Invention

[0006] This invention provides a pharmaceutical composition for treating human papillomavirus infection, its preparation method, and its uses.

[0007] This invention provides the use of burdock seed essential oil in the preparation of topical medicines against human papillomavirus.

[0008] This invention provides the use of Ganoderma lucidum spore oil in the preparation of topical anti-human papillomavirus drugs.

[0009] This invention provides a pharmaceutical composition for treating human papillomavirus infection, which is prepared from the following raw materials in the indicated weight ratios: Burdock seed essential oil 1-4 parts, Ganoderma lucidum spore oil 1-4 parts.

[0010] Preferably, it is prepared from raw materials in the following weight ratio: Burdock seed essential oil 3-4 parts, Ganoderma lucidum spore oil 1-2 parts; Preferably, it is prepared from raw materials in the following weight ratio: Four parts burdock seed essential oil and one part Ganoderma lucidum spore oil.

[0011] It is a pharmaceutical preparation made from burdock seed oil and Ganoderma lucidum spore oil as active ingredients, with the addition of pharmaceutically acceptable excipients or auxiliary ingredients.

[0012] The preparation described herein is a topical preparation. It is applied topically and should be applied after cleaning and disinfecting the affected area before going to sleep at night. The medication should then be applied evenly to the surface of the affected area and gently massaged until fully absorbed.

[0013] The excipients of the topical preparation provided by this invention further include: a preservative and a penetration enhancer; the preservative is cinnamon oil and ethylhexylglycerin; the penetration enhancer is eucalyptus oil, and its weight percentage is: Burdock seed essential oil accounts for 0.4%. Ganoderma lucidum spore oil accounts for 0.1%, cinnamon essential oil accounts for 0.01%, ethylhexylglycerin accounts for 0.15%, and eucalyptus oil accounts for 1%.

[0014] This invention provides a pharmaceutical composition for treating human papillomavirus infection, comprising the following steps: a. Weigh the raw materials according to their respective weight ratios; b. Mix and sonicate to obtain the final product.

[0015] The present invention also provides the use of the pharmaceutical composition in the preparation of a medicament for treating human papillomavirus infection.

[0016] The pharmaceutical composition of this invention (hereinafter referred to as "Compound Arctium lappa Essential Oil") is an empirical formula from Director Jin Yishun of Fujian Provincial Hospital. The prescription consists of Arctium lappa fruit and Ganoderma lucidum, and has anti-HPV effects. The principal ingredient is Arctium lappa fruit, the dried, mature fruit of Arctium lappa L., a plant of the Asteraceae family. It is a commonly used traditional Chinese medicine, cold in nature, pungent and bitter in taste, and has the effects of dispersing wind-heat, clearing the lungs and promoting rash eruption, detoxifying and relieving sore throat. It is mainly used to treat wind-heat colds, lung-heat coughs, carbuncles and boils, etc. Studies have reported that arctiin and arctigenin do have anti-cancer and anti-tumor effects. Ganoderma lucidum is the dried fruiting body of the fungus Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang, belonging to the Polyporaceae family. Its main component is Ganoderma lucidum polysaccharide. Modern pharmacological studies show that Ganoderma lucidum polysaccharide can enhance the immune activity of human peripheral blood lymphocytes, thereby strengthening its anti-tumor effects. In recent years, cervical cancer has become the second most common malignant tumor among women, after breast cancer, and its age of onset is showing a significant trend towards younger ages. Therefore, effective intervention for precancerous lesions is crucial for preventing or reducing the incidence of cervical cancer. However, existing treatments still have limitations, making the development of new treatment plans and methods extremely important.

[0017] This invention employs a compound formulation process to determine the formula of the main drug and excipients, successfully preparing a stable compound Ganoderma lucidum essential oil. A preliminary thin-layer chromatography method and a gas chromatography quality evaluation method for the main medicinal ingredients in the compound were also established. The compound Ganoderma lucidum essential oil is administered topically. In vitro experiments show that it can interfere with viral replication, exhibiting strong antiviral activity, particularly against HPV16. Furthermore, its effects are mild, and clinical observations have revealed no skin irritation and few adverse reactions. Attached Figure Description

[0018] Figure 1 The preparation process route for compound Ganoderma lucidum essential oil; Figure 2 Image of the finished product of compound burdock seed essential oil; Figure 3 Thin-layer chromatography (365 nm) was used to identify burdock seeds (where 1, 2, and 3 are three batches of test samples, 4 is the burdock seed reference material, and 5 is the negative control). Figure 4 Thin-layer chromatography (365 nm) was used for the identification of Ganoderma lucidum (1, 2, and 3 are three batches of test samples, 4 is the reference material of Ganoderma lucidum, and 5 is the negative control). Figure 5 Thin-layer chromatography was used to identify camellia oil (where 1, 2, and 3 are three batches of test samples, 4 is camellia oil, and 5 is soybean oil). Figure 6 It contains 37 MIX fatty acid standards; Figure 7 This is the gas chromatogram of the test sample. Detailed Implementation

[0019] Example 1: Preparation of the Compound Ganoderma Lucidum Essential Oil of the Present Invention The main active ingredients in compound Ganoderma lucidum essential oil, such as arctigenin and fatty acids, have good lipid solubility and exhibit good anti-tumor effects. Among them, medium-chain fatty acid monoglycerides can extensively disrupt the lipid bilayer structure of enveloped viral particles, affecting their infectivity. They can also disrupt viral fusion with host cells, viral replication and recombination, and the protein structures required to protect viral RNA or DNA, leading to a weakening or loss of viral replication ability (viruses without replication ability act as antigens), thus stimulating the body to produce corresponding antibodies and thereby exerting an antiviral effect. Therefore, this invention, based on clinical experience, optimizes the proportions of excipients and active ingredients to prepare compound Ganoderma lucidum essential oil, aiming to solve the problem of its unstable quality.

[0020] 1. Instruments and reagents 1.1 Instruments Table 1. Instruments used in the experiment

[0021] 1.2 Drug Testing Table 2 Reagents used in the experiment

[0022] 2. Methods and Results 2.1 Raw material screening for compound sesame oil 2.1.1 Determination of the main drug When determining the main active ingredients, the potential irritation caused by essential oils was fully considered. Through rigorous analysis and scientific planning, the proportion of medicinal essential oils in the total essential oils was set at 0.5%. This prescription uses burdock seed and Ganoderma lucidum as the core active ingredients. According to the prescription ratio, burdock seed essential oil accounts for 0.4%, and Ganoderma lucidum spore oil accounts for 0.1%. Together, they constitute the main active ingredient of the compound burdock and Ganoderma lucidum essential oil.

[0023] 2.1.2 Determination of excipients In the process of screening excipients, commonly used excipients are referenced, and their evaluation and analysis are carried out by comprehensively considering multiple key dimensions such as formulation stability and skin safety.

[0024] Regarding the selection of preservatives, adding 0.01% cinnamon essential oil to the cream system, along with 0.15% of other preservatives, can effectively inhibit microbial growth while ensuring product quality stability, achieving an ideal preservative effect. Cinnamon essential oil, as a natural antibacterial component, works synergistically with the synthetic preservative ethylhexylglycerin, leveraging the advantages of natural ingredients while ensuring the reliability of the preservative system, providing dual protection for product quality and safety.

[0025] In the screening of penetration enhancers, a literature review revealed that eucalyptus oil, compared to the traditional classic penetration enhancer azone, is less irritating to the skin and possesses excellent penetration-enhancing properties. As a natural plant extract, eucalyptus oil not only effectively promotes the transdermal absorption of drug components but also reduces the potential risk of skin irritation, better meeting the dual requirements of modern skincare products for safety and effectiveness. Therefore, it was selected as an essential oil penetration enhancer.

[0026] Furthermore, considering the significant impact of the base oil on the overall performance of the essential oil, and after comprehensively considering the available resources in the laboratory and the physicochemical properties of the base oil, camellia oil was ultimately selected as the base oil. Camellia oil is rich in unsaturated fatty acids, vitamins, and other nutrients, possesses good skin affinity and stability, and can provide an ideal dispersion carrier for the medicinal components, ensuring uniform mixing of all ingredients and enhancing the product's user experience and efficacy. The preparation process route is as follows: Figure 1 .

[0027] 2.3 Formulation and Processing of Compound Ganoderma Lucidum Essential Oil 2.3.1 Prescription Table 3. Composition of Compound Ganoderma Lucidum Essential Oil Formula

[0028] 2.3.2 Manufacturing Method The above formulas are precisely measured into 1000 mL volumetric flasks, brought to a final volume, capped and sealed, and gently shaken to mix initially. The flasks are then sonicated (500 W power, 40 kHz frequency) for 30 min. After the sonication is complete, the volumetric flasks are removed. The 10 mL essential oil bottles are boiled to sterilize and dried. The essential oils in the volumetric flasks are then filled and sealed to obtain the final product.

[0029] 3. Summary and Discussion 3.1 Determination of the proportion of the main drug Considering the skin irritation of the main medicinal essential oil, this study decided that the content of the main medicinal ingredient accounted for 0.5% of the total essential oil. Since the ratio of burdock seed to Ganoderma lucidum in the prescription is 4:1, the burdock seed essential oil accounts for 0.4% of the total essential oil content, and the Ganoderma lucidum spore oil accounts for 0.1% of the total essential oil content.

[0030] 3.2 Screening of Preservatives The compound Ganoderma lucidum essential oil uses oil as the overall solvent system. Based on considerations of preservative solubility, this study specifically selected the oil-soluble preservative ethylhexylglycerin. After a period of time at room temperature, observation revealed that the compound Ganoderma lucidum essential oil system exhibited good stability, without layering, turbidity, or precipitation, demonstrating good compatibility between ethylhexylglycerin and the essential oil solvent system. Furthermore, a systematic literature review revealed that cinnamon essential oil not only possesses natural antibacterial and preservative activities and can be used as a natural preservative in essential oil products, but also produces a significant synergistic effect when used in combination with other preservatives. This synergistic effect can effectively reduce the dosage of chemical preservatives, minimizing potential skin irritation and improving product safety and gentleness, providing new ideas and directions for optimizing the formulation of compound Ganoderma lucidum essential oil.

[0031] 3.3 Screening of penetration enhancers Eucalyptus oil is often used as a guiding agent in topical Chinese medicine preparations to promote transdermal absorption and enhance efficacy. Modern research also shows that eucalyptus oil can act as a transdermal penetration enhancer to promote drug absorption. Experimental studies have shown that compared to the classic penetration enhancer azone, eucalyptus oil is less irritating to the skin. Therefore, this study selected eucalyptus oil, a natural plant essential oil, as a penetration enhancer for compound Ganoderma lucidum essential oil.

[0032] Example 2: Quality Evaluation of Compound Ganoderma Lucidum Essential Oil Essential oils, as highly concentrated products of natural active ingredients, are widely used in medicine and health care. However, essential oils have complex compositions, and their quality is easily affected by various factors. Therefore, establishing a comprehensive and scientific quality evaluation system is crucial to ensure the safety, stability, and efficacy of essential oils. Based on this, this study used thin-layer chromatography (TLC) for qualitative identification of the components of compound Ganoderma lucidum essential oil, combined with gas chromatography (GC) for quantitative analysis of fatty acids. Strictly adhering to the requirements of the *Pharmacopoeia of the People's Republic of China* (2020 edition) and the *National Standards of the People's Republic of China*, a systematic quality evaluation of compound Ganoderma lucidum essential oil was conducted, aiming to provide a scientific basis for the quality control and application of this essential oil.

[0033] 1. Instruments and reagents 1.1 Instruments Table 4. Instruments used in the experiment

[0034] 1.2 Drug Testing Table 5 Reagents used in the experiment

[0035] 2. Methods and Results 2.1 General Quality Evaluation of Compound Antrodia Chrysanthemum Essential Oil 2.1.1 Color and Aroma Take a sample and place it in a 25 mL colorimetric tube. Observe it at room temperature and in the absence of direct sunlight. The essential oil will appear as a bright yellow, transparent liquid. Figure 2 Place an appropriate amount of essential oil in a test tube. At room temperature, the aroma is primarily that of eucalyptus oil.

[0036] 2.1.2 Relative density Referring to the specific gravity bottle method under General Chapter 0601, Relative Density Determination, Sections 73-74 of the 2020 edition of the Chinese Pharmacopoeia (Part IV), take a clean, dry, and accurately weighed specific gravity bottle, fill it with the test sample (the temperature should be below 20℃ or the temperature specified under each variety), attach a thermometer (there should be no air bubbles in the bottle), and place it in a water bath at 20℃ (or the temperature specified under each variety) for several minutes to allow the temperature of the contents to reach 20℃ (or the temperature specified under each variety). Remove any liquid overflowing from the side tube with filter paper and immediately cover the bottle. Then remove the specific gravity bottle from the water bath, wipe the outside of the specific gravity bottle clean with filter paper, accurately weigh it, subtract the weight of the specific gravity bottle to obtain the weight of the test sample, pour out the test sample, wash the specific gravity bottle, fill it with freshly boiled and cooled water, and measure the weight of the water at the same temperature using the same method. Calculate the result using the following formula.

[0037]

[0038] The average value obtained from the measurements was then substituted into the formula to obtain the relative density of the compound sesame oil, which is 0.9297. The data is shown in Table 6.

[0039] Table 6. Results of relative density determination of compound Ganoderma lucidum essential oil 1 2 3 average value Test sample weight (g) 9.26 9.28 9.24 9.26 Water weight (g) 10.02 9.95 9.91 9.96 2.1.3 pH value Take 10 mL of the compound Ganoderma lucidum essential oil product and determine its pH value according to the pH determination method (General Chapter 0631 of the 2020 edition of the Chinese Pharmacopoeia). Before the determination, select three or two suitable standard buffer solutions to calibrate the instrument according to the provisions under each variety, so that the pH value of the test solution is within the range specified. First, use two standard buffer solutions to automatically calibrate the instrument, so that the slope is 90%~105% and the drift value is within 0±30 mV or ±0.5 pH units. Then, use a third standard buffer solution with a pH value between the two calibration buffer solutions and as close as possible to the test sample to verify the instrument value until the instrument reading does not differ from the specified value of the verification buffer solution by more than ±0.05 pH units. Alternatively, select two standard buffer solutions with pH values ​​approximately 3 pH units apart. First, use the first standard buffer solution with a pH value closer to that of the test sample solution to calibrate (position) the instrument so that the instrument reading is consistent with the value listed in the table. Then, use the second standard buffer solution to check the instrument reading, and the difference from the value listed in the table should not exceed ±0.02 pH units. If the difference is greater than this, the slope should be carefully adjusted to match the value listed in the table for the second standard buffer solution. Repeat the above positioning and slope adjustment operations until the instrument reading differs from the specified value of the standard buffer solution by no more than ±0.02 pH units. Otherwise, the instrument needs to be checked or the electrode replaced before calibration to meet the requirements.

[0040] Before each change of standard buffer solution or test solution, thoroughly wash the electrode with purified water, then wash it with the new standard buffer solution or test solution, or thoroughly wash the electrode with purified water and then remove all water. The final test results are shown in Table 7, indicating a weakly acidic pH. The pH value of human skin surface is usually between 4.5 and 5.5, which is also weakly acidic. This product can adapt to the acid-base environment of the skin, reducing skin irritation and keeping the skin in a healthy state.

[0041] Table 7. Results of pH value determination of compound Ganoderma lucidum essential oil 1 2 3 average value pH value 4.4 4.3 4.5 4.4 2.1.4 Stability Test Accurately measure the same test solution and place it at room temperature for 3, 5, 7 and 15 days to observe whether there are problems such as layering or emulsification. The results are shown in Table 8.

[0042] Table 8. Results of essential oil stability test Time (days) 3 5 7 15 result No layering observed No layering observed No layering observed No layering observed 2.2 Thin-layer chromatography identification of burdock seeds 2.2.1 Preparation of the test sample Take 7 g of this product, dilute to 15 mL with ethanol, sonicate (500 W power, 40 kHz frequency) for 20 min, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 3 mL of anhydrous ethanol to obtain the test solution.

[0043] 2.2.2 Preparation of the control herbal solution Take 0.5 g of burdock seed reference material, add 20 mL of anhydrous ethanol, sonicate (500 W power, 40 kHz frequency) for 30 min, filter, evaporate the filtrate to dryness, add 2 mL of ethanol to dissolve the residue, and use it as the reference material solution.

[0044] 2.2.3 Preparation of negative control solution Take the prescription without burdock seed essential oil, prepare the essential oil according to "Chapter 1 2.3.2", take 7 g, make up to 15 mL with anhydrous ethanol, sonicate (power 500 W, frequency 40 kHz) for 20 min, cool, filter, evaporate the filtrate to dryness, add 3 mL of anhydrous ethanol to dissolve the residue, and use it as a negative control solution.

[0045] 2.2.4 Expand Referring to relevant literature, 10 μL each of the above-mentioned test solution, negative control solution, and reference drug solution were spotted onto the same silica gel G thin-layer plate. Petroleum ether (60–90℃)-ethyl acetate (3:2) was used as the developing solvent. After development, the plate was removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible. The plates were then examined under a UV lamp (365 nm). The test solution and reference drug solution showed identical spots at the same positions, while the negative control showed no spots at these positions. Figure 3 .

[0046] 2.3 Thin-layer chromatography identification of Ganoderma lucidum 2.3.1 Preparation of the test sample Take 7 g of this product, dilute to 15 mL with anhydrous ethanol, sonicate (500 W power, 40 kHz frequency) for 20 min, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 3 mL of anhydrous ethanol to obtain the test solution.

[0047] 2.3.2 Preparation of control herbal solution Take 2 g of Ganoderma lucidum reference material, add 30 mL of anhydrous ethanol, heat under reflux for 30 min, filter, evaporate the filtrate to dryness, add 2 mL of methanol to dissolve the residue, and use it as the reference material solution.

[0048] 2.3.3 Preparation of negative control solution Take the prescription without Ganoderma lucidum spore oil, prepare the essential oil according to "Chapter 1 2.3.2", take 7 g, make up to 15 mL with anhydrous ethanol, sonicate (power 500 W, frequency 40 kHz) for 20 min, cool, filter, evaporate the filtrate to dryness, add 3 mL of anhydrous ethanol to dissolve the residue, and use it as a negative control solution.

[0049] 2.3.4 Expand Referring to relevant literature, 10 μL each of the above-mentioned test solution, negative control solution, and reference drug solution were spotted onto the same silica gel G thin-layer plate. The developing solvent was chloroform-acetonitrile-methanol-formic acid (13:1.5:0.2:0.2); the development method was three times, each time developing to 3, 6, and 8 cm, and drying at 50℃ for 8 min before each development; the colorimetric reagent was 3% ethyl sulfate solution; the color development was performed at 105℃, heated until the bands were clearly visible, and examined under a UV lamp (365 nm). The test solution and reference drug solution showed the same spots at the same positions, while the negative control showed no spots at this position. Figure 4 .

[0050] 2.4 Thin-layer chromatographic identification of camellia oil 2.4.1 Preparation of the test sample Take 7 g of this product, dilute to 15 mL with ethanol, sonicate (500 W power, 40 kHz frequency) for 20 min, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 3 mL of ethanol to obtain the test solution.

[0051] 2.4.2 Preparation of negative solution Take 7 g of camellia oil, dilute it to 15 mL with ethanol, sonicate it (500 W power, 40 kHz frequency) for 20 min, cool it, filter it, evaporate the filtrate to dryness, and dissolve the residue in 3 mL of ethanol.

[0052] Take 7 g of soybean oil, add ethanol to make up to 15 mL, sonicate (power 500 W, frequency 40 kHz) for 20 min, cool, filter, evaporate the filtrate to dryness, and add 3 mL of ethanol to dissolve the residue.

[0053] 2.4.3 Expand Take 10 μL each of the above-mentioned test solution, oleic acid control solution, and linoleic acid control solution, and spot them separately on the same silica gel G thin-layer plate. Develop the plate using acetone-petroleum ether (1:4) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under a UV lamp (365 nm). The test solution and control solutions show the same spots at the same positions. Figure 5 .

[0054] 2.5 Gas chromatography determination of fatty acid content 2.5.1 Chromatographic conditions and system suitability test The chromatographic column used was a Kromat PC-2560 (100 m × 0.25 mm × 0.20 μm) containing 100% dicyandipropyl polysiloxane from the USA. Nitrogen gas with a purity of 99.99% was used as the carrier gas. The column flow rate was 0.9 mL / min, the injection port temperature was 270℃, the split ratio was 20:1, the pressure was 698 kPa, the injection volume was 1 μL, the detector temperature was 280℃, and the heating rate was as follows: starting at 120℃, held for 1 min, then increased to 175℃ at a rate of 10℃ / min, held for 10 min, then increased to 220℃ at a rate of 5℃ / min, and held for 27 min.

[0055] 2.5.2 Preparation of Standard Solutions The standard solutions were prepared according to the acid hydrolysis-extraction method in GB5009.168-2016. **Undecanoic acid triglyceride internal standard solution (5.00 mg / mL):** Accurately weigh 2.5 g (accurate to 0.1 mg) of undecanoic acid triglyceride into a beaker, dissolve in methanol, transfer to a 500 mL volumetric flask, and dilute to volume with methanol. **Mixed fatty acid methyl ester standard solution:** Transfer an appropriate amount of the mixed fatty acid methyl ester standard to a 10 mL volumetric flask, dilute to volume with n-heptane, and store at -10°C or below. **Single fatty acid methyl ester standard solution:** Transfer individual fatty acid methyl esters from ampoules to 10 mL volumetric flasks, rinse the ampoules with n-heptane, and dilute to volume with n-heptane to obtain single standard solutions of different fatty acid methyl esters. Store at -10°C or below.

[0056] 2.5.3 Preparation of the test solution Sample pretreatment was performed according to the acid hydrolysis-extraction method in GB 5009.168-201: 8 mL of 2% sodium hydroxide methanol solution was added to the fat extract, and the mixture was refluxed in an 80°C water bath until the oil droplets disappeared. 7 mL of 15% boron trifluoride methanol solution was added, and reflux was continued for 2 min, followed by cooling. 20 mL of n-hexane was accurately measured, shaken, and then 10 mL of saturated sodium chloride solution was added, shaken, and allowed to stand for phase separation. 5 mL of the n-hexane layer was transferred to a centrifuge tube, 3 g of anhydrous sodium sulfate was added, shaken, and allowed to stand. The upper filter membrane was then used for centrifuge.

[0057] 2.5.4 Results of Sample Content Determination The mixed standards and samples of 37 fatty acids were analyzed by instrument, and the results are as follows: Figure 6 and Figure 7From left to right: methyl butyrate, methyl hexanoate, methyl octanoate, methyl caprylate, methyl undecanoate, methyl dodecanoate, methyl tridecanate, methyl tetradecanate, methyl cis-9-tetradecanoate, methyl pentadecanoate, methyl cis-10-pentadecananoate, methyl hexadecanoate, methyl cis-9-hexadecanoate, methyl heptadecanate, methyl cis-10-heptadecananoate, methyl octadecanoate, trans- 9-Octadecaenoic acid methyl ester, cis-9-octadecadecaenoic acid methyl ester, trans, trans-9,12-octadecadienoic acid methyl ester, cis, cis-9,12-octadecadienoic acid methyl ester, eicosate carbonate methyl ester, cis, cis-6,9,12-octadectrienoic acid methyl ester, cis-11-eicosatecaenoic acid methyl ester, cis, cis, cis-9,12,15-octadectrienoic acid methyl ester, methyl dodecanoate carbonate, cis, cis-11, Methyl 14-eicosadienoic acid, methyl docosinate, cis,cis,cis-8,11,14-eicostrienoic acid, methyl cis-13-docosahexaenoic acid, methyl cis-11,14,17-eicostrienoic acid, methyl cis-5,8,11,14-eicosatetraenoic acid, methyl tricarbonate, methyl cis-13,16-docosaadienoic acid, methyl tetracarbonate, methyl cis-5,8,11,14,17-eicosatepentanoic acid, methyl cis-15-docosahexaenoic acid, and methyl cis-4,7,10,13,16,19-docosahexaenoic acid were used. Based on the relative retention times of the peaks in the standard chromatograms, the detected fatty acid methyl esters in the samples were qualitatively analyzed. The relative content of fatty acids in the samples was quantitatively analyzed using the peak area of ​​undecanoic acid internal standard. The calculation formula is as follows, and the calculation results are shown in Table 7.

[0058] Result calculation:

[0059] In the formula: X i —The content of fatty acid methyl ester I in the sample, in grams per 100 grams (g / 100g); F i —Response factor of fatty acid methyl ester i; A i —The peak area of ​​fatty acid methyl ester i in the sample; A C11 —Peak area of ​​methyl undecanoate, the internal standard added to the sample; ρ C11 —Concentration of triglycerides undecanoate, in milligrams per milliliter (mg / mL); V C11 —The volume of undecylate triglyceride added to the sample is expressed in milliliters (mL); 1.0067 — Conversion factor for the conversion of triglycerides undecanoate to methyl undecanoate; m—mass of the sample, in milligrams (mg); 100 — A coefficient that converts the content to the content per 100g of sample.

[0060]

[0061] In the formula: F i —Response factor of fatty acid methyl ester i; ρ Si —The concentration of each fatty acid methyl ester i in the mixed standard, in milligrams per milliliter (mg / mL); A 11 —Peak area of ​​methyl undecanoate; A Si —Peak area of ​​fatty acid methyl ester i; ρ 11 —The concentration of methyl undecanoate in the mixed standard is expressed in milligrams per milliliter (mg / mL).

[0062] Table 9 Calculation results of fatty acid content in samples sequence Fatty acid (abbreviation) Fatty acid methyl esters Content (g / 100g) 1 C11:0 Methyl undecanoate 0.9583 2 C16:0 Methyl hexadecanoate 1.7922 3 C18:1n9c methyl cis-9-octadecenoate 23.9081 4 C18:2n6c methyl cis,cis-9,12-octadecadienoate 1.4706 3. Summary and Discussion 3.1 Selection and Optimization of Thin-Layer Chromatography Developing Reagent In the field of traditional Chinese medicine quality control, thin-layer chromatography (TLC) identification is a commonly used and important analytical method. The selection of the developing solvent is considered the core of this technique, directly affecting the separation of sample components. A suitable selection enables effective separation and rapid migration of components, improving separation efficiency. Simultaneously, it ensures the reproducibility of experimental results, laying the foundation for accurate qualitative and quantitative analysis and serving as a crucial guarantee for obtaining reliable chromatographic analysis results. Taking the TLC identification of burdock seed and Ganoderma lucidum as an example, the optimization process of the developing conditions fully demonstrates the key influence of adjusting the type and ratio of developing solvent on the chromatographic separation effect.

[0063] Referring to the 2020 edition of the Chinese Pharmacopoeia, the initial developing conditions for thin-layer chromatography identification of burdock seeds are chloroform-methanol-water (40:8:1). Under these conditions, the experiment found poor spot resolution in the chromatogram of the burdock seed test sample, with spots of different components failing to disperse effectively, leading to overlapping or tailing of adjacent spots. Simultaneously, the negative control solution chromatogram contained interfering spots in similar positions to the test sample, making it difficult to accurately distinguish whether the sample contained the target component based on spot location, severely affecting the accuracy of qualitative identification. To address this issue, this study, through literature review, adjusted the developing solvent system, replacing it with petroleum ether (60–90℃)-ethyl acetate (3:2). In the new system, petroleum ether, as a low-polarity solvent, provides basic elution capacity, while ethyl acetate enhances the dissolution and migration of the target component by adjusting polarity. After optimizing the ratio of the two, the spot resolution in the chromatogram of the burdock seed test sample was significantly improved, with each component spot clearly and independently, and no interfering spots at corresponding positions in the negative control solution chromatogram, fully meeting the requirements for qualitative analysis. Thin-layer chromatography (TLC) identification of Ganoderma lucidum also faces challenges related to development conditions. The 2020 edition of the Chinese Pharmacopoeia recommends petroleum ether (60–90℃)-ethyl formate-formic acid (15:5:1) as the developing solvent. However, in practical applications, the spots on the test sample chromatogram are extremely indistinct, pale in color, and have blurred outlines, making accurate identification difficult. Simultaneously, the chromatograms of the negative control solution show no significant difference from the test sample, failing to effectively eliminate interference through spot characteristics. Through in-depth analysis of the polarity characteristics of Ganoderma lucidum components, this study consulted references and adjusted the developing solvent to chloroform-acetonitrile-methanol-formic acid (13:1.5:0.2:0.2). In this system, chloroform, as the main solvent, provides strong elution power; acetonitrile and methanol improve the partition coefficient of the target components by fine-tuning their polarity; and formic acid reduces the polar interaction between the components and the stationary phase by adjusting the pH value, thereby significantly enhancing the clarity of the spots. After optimization, the spots in the chromatogram of the Ganoderma lucidum test sample were darker and had clearer outlines, showing a significant difference from the chromatogram of the negative control solution, which greatly improved the feasibility of the identification method.

[0064] In summary, the selection of the type and ratio of developing solvent is a core element of thin-layer chromatography separation. Its mechanism of action is mainly reflected in two aspects: Firstly, by adjusting parameters such as solvent polarity, acidity / alkalinity, and viscosity, the partition coefficients of each component in the sample between the stationary and mobile phases are altered, thereby achieving effective separation and rapid migration, and improving separation efficiency. Secondly, a suitable developing solvent system can reduce background interference, ensure the reproducibility of experimental results across different batches, and provide a reliable basis for accurate qualitative and quantitative analysis. Therefore, in the quality control research of traditional Chinese medicine, it is necessary to systematically optimize the developing conditions by considering the chemical properties, polarity characteristics, and impurity interference of the target components. This is not only an important guarantee for obtaining reliable chromatographic analysis results but also a key link in improving the scientific rigor and soundness of traditional Chinese medicine quality standards.

[0065] 3.2 Determination of fatty acid content Gas chromatography is a chromatographic separation and analysis method that uses gas as the mobile phase. Based on the peak times and order shown in the graph, qualitative analysis of compounds can be performed; based on the peak height and area, quantitative analysis can be performed. It features high efficiency, high sensitivity, strong selectivity, fast analysis speed, wide application, and simple operation. It is suitable not only for the qualitative and quantitative analysis of volatile organic compounds but also for the analysis of non-volatile liquids and solids.

[0066] In this study, gas chromatography combined with acid hydrolysis-extraction was used to analyze fatty acid methyl esters in the samples. This method demonstrates significant scientific validity and feasibility. Using a Kromat PC-2560 column from the USA, along with specific chromatographic conditions, 37 fatty acid methyl esters were effectively separated, laying the foundation for accurate qualitative and quantitative analysis.

[0067] From the perspective of standard solution preparation, a comprehensive standard system was constructed by preparing internal standard solutions, mixed standard solutions, and single standard solutions. Using triglycerides undecanoate as the internal standard, and considering its conversion coefficient, quantitative analysis was performed using the internal standard method, which effectively eliminated instrumental and operational errors and improved data accuracy. During the preparation of the test solution, acid hydrolysis, esterification, and extraction were strictly performed according to national standard methods to ensure standardized sample pretreatment and reduce interference factors.

[0068] The analysis successfully detected fatty acid methyl esters, including C11:0, C16:0, C18:1n9c, and C18:2n6c, in the sample. Overall, the analytical method established in this study is stable and reliable, and of great significance for the quality standard evaluation of compound Ganoderma lucidum essential oil. However, there is still room for optimization. Further research could focus on optimizing chromatographic conditions to improve separation efficiency; expanding the sample size and including different types of samples to enhance the research's generalizability; and thus providing more comprehensive technical support for fatty acid component analysis.

[0069] Through systematic experiments, the proportion of the main ingredient and the types and contents of excipients in the compound Ganoderma lucidum and Ganoderma lucidum essential oil were determined: the main ingredient content was 0.5%, of which burdock seed essential oil accounted for 0.4%, Ganoderma lucidum spore oil accounted for 0.1%, cinnamon essential oil accounted for 0.01%, ethylhexylglycerin accounted for 0.13%, eucalyptus oil accounted for 1%, and camellia oil was used as the base oil; general tests were performed on the compound Ganoderma lucidum and Ganoderma lucidum essential oil in accordance with GB / T 26516-2011; thin-layer chromatography was used to identify burdock seed, Ganoderma lucidum and camellia oil in the compound Ganoderma lucidum and Ganoderma lucidum essential oil in accordance with the 2020 edition of the Chinese Pharmacopoeia, and a thin-layer chromatography identification method for burdock seed and Ganoderma lucidum was established; fatty acid content was determined in accordance with GB5009.168-2016; all results were reliable.

[0070] The following pharmacological experiments demonstrate the beneficial effects of this invention.

[0071] Experimental Example 1: In vitro antiviral test of the compound Ganoderma lucidum essential oil of the present invention. 1. Viral amplification: In the experiment, HPV16 virus suspension was appropriately diluted and inoculated into HeLa cells, an appropriate amount of cell culture medium was added, and the cells were cultured at 37°C in a 5% CO2 environment. CPE was observed daily for 5 days. When cells are found to detach in large clumps from the culture flask, immediately freeze and thaw the cells three times, aspirate the virus suspension into a centrifuge tube, centrifuge at 6000 r / min for 10 min to remove the precipitate, remove the supernatant and store at -80℃. 2. Grouping: Virus + neutralizer + cell; Virus + disinfectant (i.e., essential oils in various proportions) + neutralizer + cell.

[0072] 3. Inactivation experiment: 3.1. Take the disinfectant to be tested, dilute it with sterile hard water to 1.25 times the required concentration, and keep it in a water bath at 20℃±1℃ for later use.

[0073] 3.2. Mix 100 μL of organic interfering substance with 100 μL of virus stock solution, incubate in a water bath at 20℃±1℃ for 5 min, add 0.8 mL of the disinfectant to be tested (if it is a suspension, vortex it evenly before aspirating), mix immediately and record the time. After the specified incubation time, immediately take out 0.1 mL and mix it with the neutralizing agent.

[0074] 3.3. The mixed sample was serially diluted 10-fold with cell maintenance medium, and samples were taken for virus titer (TCIDs0) testing.

[0075] 4. Test Results According to standard recommendations, the Reed-Muench two-stage method was used to test the viral titer TCIDso. The sampling data was repeated three times to ensure accuracy. The virus killing rate of each group of essential oils was calculated based on the test results.

[0076] Table 10 Results of in vitro antiviral activity test of the compound Ganoderma lucidum essential oil of the present invention

[0077]

[0078] Note: The total amount of burdock seed essential oil and Ganoderma lucidum spore oil in each experimental group was 0.5%. The ratio of the two differed. Among the experimental groups 1-7, the ratio of burdock seed essential oil to Ganoderma lucidum spore oil was 5:0; 4:1; 3:2; 1:1; 2:3; 1:4:0:5, respectively.

[0079] The above experiments demonstrate that both burdock seed oil and Ganoderma lucidum spore oil have good antiviral activity when used alone. When used in combination, they have a synergistic effect and significantly improved activity. In particular, the burdock seed oil: Ganoderma lucidum spore oil 4:1 ratio group has an antiviral activity rate of 99.31%.

Claims

1. Use of burdock seed essential oil in the preparation of topical anti-human papillomavirus drugs.

2. Use of Ganoderma lucidum spore oil in the preparation of topical anti-human papillomavirus drugs.

3. A pharmaceutical composition for treating human papillomavirus infection, characterized in that: It is prepared from the following raw materials in the following weight ratio: Burdock seed essential oil 1-4 parts, Ganoderma lucidum spore oil 1-4 parts.

4. The pharmaceutical composition for treating human papillomavirus infection according to claim 3, characterized in that: It is prepared from the following raw materials in the following weight ratio: Burdock seed essential oil 3-4 parts, Ganoderma lucidum spore oil 1-2 parts; Preferably, it is prepared from raw materials in the following weight ratio: Four parts burdock seed essential oil and one part Ganoderma lucidum spore oil.

5. The pharmaceutical composition for treating human papillomavirus infection according to claim 3 or 4, characterized in that: It is a pharmaceutical preparation made from burdock seed oil and Ganoderma lucidum spore oil as active ingredients, with the addition of pharmaceutically acceptable excipients or auxiliary ingredients.

6. The pharmaceutical composition for treating human papillomavirus infection according to claim 5, characterized in that: The preparation described is for external use.

7. The pharmaceutical composition for treating human papillomavirus infection according to claim 6, characterized in that: The aforementioned topical preparations include solutions, tinctures, liniments, sprays, ointments, creams, and gels.

8. The pharmaceutical composition for treating human papillomavirus infection according to claim 6 or 7, characterized in that: The excipients of the topical preparation also include: preservatives and penetration enhancers; the preservatives are cinnamon oil and ethylhexylglycerin; the penetration enhancer is eucalyptus oil, with the following weight percentage: Burdock seed essential oil accounts for 0.4%. Ganoderma lucidum spore oil accounts for 0.1%, cinnamon essential oil accounts for 0.01%, ethylhexylglycerin accounts for 0.15%, and eucalyptus oil accounts for 1%.

9. A pharmaceutical composition for treating human papillomavirus infection according to any one of claims 3-8, characterized in that: It includes the following steps: a. Weigh the raw materials according to their respective weight ratios; b. Mix and sonicate to obtain the final product.

10. Use of the pharmaceutical composition according to any one of claims 3-8 in the preparation of a medicament for treating human papillomavirus infection; preferably, the human papillomavirus is HPV16.

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