Antiviral properties of eugenia aromaticum, cymbopogon martinii, melaleuca viridiflora, and calophyllum inophyllum essential oils: modulation of e7 and e2 protein pathways in human papillomavirus (HPV) infection

Abstract

The invention is a composition for the treatment of HPV infections, which, with its components containing [3-caryophyllene, geranyl acetate, geraniol, and nerol, modulates the E7 and E2 protein pathways to provide a natural supportive therapeutic effect in managing HPV infections, exhibits antiviral, antibacterial, and immunomodulatory activity, significantly reduces viral replication, and suppresses protein expression, Contains Eugenia aromaticum (clove oil), which prevents viral replication by disrupting the structural integrity of cell membranes, exhibiting antimicrobial and antiviral activity with its high eugenol content, and Contains Cymbopogon martinii (palmarosa oil), which disrupts viral cell walls and inhibits the replication process, exhibiting antiviral and antibacterial properties with its high geraniol and citronellal content, and Contains Melaleuca viridiflora (niaouli oil), which strengthens cellular immune mechanisms to limit the intracellular spread of viruses and exhibits antiviral and immunomodulatory effects with its viridiflorol content.

Description

[0001] D E S C R I P T I O N

[0002] Antiviral Properties of Eugenia aromaticum, Cymbopogon martinii, Melaleuca viridiflora, and Calophyllum inophyllum Essential Oils: Modulation of E7 and E2 Protein Pathways in Human Papillomavirus (HPV) Infection

[0003] Technical Field

[0004] The invention relates to a composition used for the treatment of human papillomavirus (HPV) infections.

[0005] Prior Art

[0006] Currently, no direct treatment method targets HPV infections in the existing technology. Instead, methods such as surgery, cryotherapy (freezing), laser treatments, and immunomodulators are employed to treat diseases caused by HPV. Surgical methods and tissue removal are often invasive, painful, and carry a risk of recurrence for patients. Topical drugs such as podophyllin, imiquimod, and sinecatechins, commonly used in the treatment of genital warts, fail to completely eliminate warts and can cause irritation and discomfort during treatment. Although these methods provide short-term relief, they cannot entirely eradicate the virus, and the risk of recurrence remains high.

[0007] Vaccines play a crucial role in the early detection and prevention of HPV -related cancers. Vaccines such as Gardasil and Cervarix offer protection against the most common high-risk HPV types. However, these vaccines are only effective before exposure to the virus and lack therapeutic effects for individuals already infected with HPV. This limitation is particularly significant in low-income countries, where access to HPV screening and vaccination programs is limited, creating a substantial need for post-infection treatment options.

[0008] HPV infections, which commonly affect the mucosa of the cervix, anogenital region, and upper respiratory tract, are prevalent viral infections. Current treatments include surgical removal of infected tissues, cryotherapy, and drug therapies, but these methods carry recurrence risks and may sometimes be ineffective. While HPV vaccines are beneficial for prevention, they are insufficient for treating existing infections. Consequently, alternative and natural treatment methods for HPV infections have garnered significant attention.

[0009] Given the limitations of existing treatments, scientific research has increasingly focused on alternative treatment options. Natural compounds and herbal medicines, particularly essential oils (EOs), are central to these studies. Herbal therapies are generally cost-effective, exhibit broad biological activity, and present therapeutic alternatives with fewer side effects.

[0010] Purpose of the Invention

[0011] The purpose of this invention is to develop compositions utilizing the essential oils of Eugenia aromaticum, Cymbopogon martinii, Melaleuca viridiflora, and Calophyllum inophyllum for the treatment of HPV infections.

[0012] Another purpose of the invention is to inhibit viral replication through the combination of specific essential oils.

[0013] Another purpose of the invention is to prevent viral entry and binding to host cells using a combination of specific essential oils.

[0014] Another purpose of the invention is to utilize the terpenoids and phenolic compounds in specific essential oil combinations to block viral entry into the cell membrane and inhibit intracellular replication.

[0015] To achieve the mentioned these purposes, the developed composition includes:

[0016] Eugenia aromaticum (clove oil): Contains high amounts of eugenol, which exhibits antimicrobial and antiviral activity, and disrupts the structural integrity of cell membranes to inhibit viral replication. Cymbopogon martinii (palmarosa oil): Rich in geraniol and citronellal, offering antiviral and antibacterial properties by disrupting viral cell walls and inhibiting the replication process.

[0017] Melaleuca viridiflora (niaouli oil): Contains viridiflorol, which demonstrates antiviral and immunomodulatory effects by enhancing cellular immune mechanisms and limiting the intracellular spread of viruses.

[0018] Description of Figures

[0019] Figure 1: Chromatographic analysis graph of the essential oils.

[0020] Figure 2: Triangular plots showing the anti-HPV effect (IC50) under different ratios of essential oils.

[0021] Figure 3: Piepel graphs of the essential oils.

[0022] Figure 4: Optimization graphs of the anti-HPV mixture of essential oils.

[0023] Figure 5: Concentration graphs of HP V -related components.

[0024] Figure 6: Graphs of Jss and Papp measurements for the permeation properties of various components in healthy and HPV-infected skin and vaginal tissues.

[0025] Figure 7: Celloger Pro analysis of HPVCs treated with essential oils.

[0026] Figure 8: Cell cycle analysis of HPVCs treated with essential oils.

[0027] Figure 9: Analysis of the effects of various treatments on cell survival rates and HPV gene expression.

[0028] Figure 10: Heatmap of compound-protein-related interactions.

[0029] Detailed Explanation of the Invention

[0030] Human Papillomavirus (HPV) is a widespread sexually transmitted infection associated with numerous cancers, including genital, anal, and oropharyngeal cancers. HPV is a double-stranded DNA virus with over 200 distinct genotypes, categorized into low-risk and high-risk types. Low-risk HPV types typically cause benign lesions such as warts, while high-risk types (e.g., HPV-16 and HPV-18) can lead to cervical, anal, genital, and head and neck cancers.

[0031] By the age of 50, nearly 80% of the global population has been exposed to this virus. HPV infections can affect the mucosal membranes and skin surfaces in areas such as the anogenital region, upper respiratory tract, and head and neck. While HPV infections are often asymptomatic and usually cleared by the immune system within 6-12 months of onset, persistent infections can progress to cancer. Examples of malignant lesions caused by HPV include oral and respiratory squamous cell carcinomas, as well as cervical and anogenital cancers. HPV is responsible for a persistent 5% of human cancers. Additionally, it causes vaginal warts (Verruca vulgaris), which are benign epithelial proliferations associated with HPV infection.

[0032] Currently, there is no specific treatment for HPV infections, and therapeutic measures such as tissue ablation, chemotherapy, cryotherapy, and immunomodulation are insufficient to completely eradicate the virus. Two strategies offering protection against oncogenic HPV include vaccination and cervical screening. Unfortunately, in developing countries, there is no recommended regimen for immunization, prevention, testing, or treatment. Additionally, the standard treatment for warts includes topical applications such as salicylic acid, podophyllotoxin, trichloroacetic acid, formaldehyde, 5 -fluorouracil, and photodynamic therapy. Painful procedures like cryotherapy, laser ablation, electrocautery, and surgical excision carry a high likelihood of recurrence. Aromatherapy is an alternative / supportive treatment strategy for managing warts.

[0033] An alternative source of medicine may be required to counteract the undesirable side effects of existing treatments and to prevent organ damage. Natural products, such as traditional herbs, are highly effective in treating a wide range of diseases, including cancer. The applications of essential oils (EOs) in aromatherapy and phytomedicine have garnered significant attention over the past 0-40 years. Consequently, researchers have extensively studied essential oils, particularly for their anticancer, antioxidant, and antimicrobial properties. Essential oils are composed of tens to hundreds of distinct molecules, primarily a mix of terpenes and benzene derivatives produced by aromatic plants. The broad bioactivity of essential oils is attributed to these terpenes and benzene derivatives. These compounds are typically extracted using methods such as solvent extraction, supercritical fluid extraction, subcritical water extraction, steam distillation, or hydrodistillation. Essential oils are naturally occurring volatile liquids composed of complex molecules with distinctive scents. In nature, essential oils protect plants by suppressing the appetite of herbivores through their scent, thereby defending plants against pests.

[0034] Essential oils are biologically active compounds derived from plants and have been shown to possess antiviral, antibacterial, antifungal, and anti-inflammatory properties. Recent research has revealed that many essential oils have the potential to both inhibit viral replication and prevent viral entry and binding to host cells. Specifically, the terpenoids and phenolic compounds in essential oils can block viral entry into cell membranes and inhibit intracellular replication.

[0035] Approximately 17,500 higher plant species are known to contain essential oils. However, certain plant families, such as Zingiberaceae, Poaceae, Apiaceae, Asteraceae, Lamiaceae, Rutaceae, and Lauraceae, are particularly rich in essential oils. Essential oils consist of tens to hundreds of different chemical compounds, including alcohols, terpenes, hydrocarbons, aldehydes, and organic acids. Terpenes such as myrcene, thujane, farnesene, and azulene have been extensively studied for their medicinal benefits.

[0036] The antiviral properties of essential oils have been tested on various viruses, but studies on HPV remain limited. Nonetheless, some research has suggested that oils such as Eugenia aromaticum (clove oil), Cymbopogon martinii (palmarosa oil), Melaleuca viridiflora (niaouli oil), and Calophyllum inophyllum (tamanu oil) may possess broad-spectrum antiviral properties.

[0037] Four different essential oils — CLOV (Eugenia aromaticum), PALM (Cymbopogon martinii), NIAO (Melaleuca viridiflora), and TAMA (Calophyllum inophyllum) — have been comprehensively studied using various materials and methods.

[0038] For calibration curves, the essential oils and their components — CLOV, PALM, NIAO, and TAMA — were provided by Art de Huile (Istanbul, Turkey). Specific compounds such as 1,8-cineole (#27574), a-terpineol (#86483), a-pinene (#147524), limonene (#183164), viridiflorol (#489938), P-pinene (#402753), - caryophyllene (#W225208), eugenol (#E51791), eugenyl acetate (#35838), humulene (alpha) (#473758), methyleugenol (#W232001), geraniol (#163333), nerol (#282197), neral (#W274304), geranyl acetate (#163339), linalool (#L2602), oleic acid (#01008), linoleic acid (#L1376), palmitic acid (#P0500), stearic acid (#S4751), and a-linolenic acid (#L2376) were obtained from Sigma Aldrich (St. Louis, MO, USA).

[0039] The following were obtained from Thermo Fisher (Waltham, MA, USA): QIAwave RNA Mini Kit (#74534, QIAGEN, Hilden, Germany), CRP Human Instant ELISA™ Kit (#BMS288INST), HisProbe™-HRP conjugate (#15165), polyvinylidene difluoride (PVDF) membrane (#LC2002), Pierce™ dilution-free™ rapid gold BCA protein assay (#A55860), Pierce™ ECL western blot substrate (#32106), HPV pan-specific monoclonal antibody (MAb) (LI, #MA5-33266), HPV polyclonal antibody (L2, #BS8547R), phage coat protein (El, #MAl-34468), HPV protein MAb (E2, #MAl-75534), HPV Type 16 / 18 MAb (E6, #MA1- 46057), HPV Type 16 MAb (E7, #MA5-15822), GAPDH MAb (GA1R, #MA5-15738-D800), and TaqMan™ Fast Advanced Master Mix (#4444556).

[0040] HPV-infected cells (HPV-2A, ATCC-45022, ATCC, Manassas, VA, USA) derived from human keratinocytes (HEKa, PCS-200-011, ATCC) were provided by the Shwarz laboratory and verified through STR profiling. These cells were cultured on induced pluripotent stem cells (iPSCs) (ACS7010, ATCC, Manassas, VA, USA). Regular mycoplasma testing confirmed that all cell lines were negative. The cervical cancer cell line (DoTc2, CRL-7920™) was also obtained from ATCC. Dulbecco’s Modified Eagle Medium (DMEM), penicillin-streptomycin solution, trypsin-EDTA solution (0.25%), phosphate-buffered saline (PBS), fetal bovine serum (FBS), resazurin, and Hoechst dye were obtained from Thermo Fisher (Waltham, MA, USA).

[0041] Analytical samples were prepared by dissolving the extract and reference materials in a mixture of acetonitrile (ACN, A) and purified water containing 1 % formic acid (B) at an 85: 15 (v / v) ratio. The solution was filtered using a 0.45 pm nylon HPLC filter (HNWP04700, Millipore, Burlington, MA, USA). LC / MS-MS analysis was conducted using a QTRAP® 5500 system equipped with a Turbo V™ source and an ESI probe. Data were collected with MultiQuant™ software (v 3.69, Sciex Inc., Framingham, MA, USA). Chromatographic separation was achieved using an ACQUITY Premier BEH C18 column (186009453#, 100 mm * 2.1 mm, 1.7 pm) from Waters Corporation (Milford, MA, USA). A sample injection volume of 20 pL was used, with a flow rate maintained at 1.0 mL / min.The linear gradient elution profile was as follows: Initial conditions: A (85: 15, v / v) at 0 min, Transition to A (25:75, v / v) over 110 min, Return to A (85:15, v / v) at 111 min, maintaining this ratio until 135 min. Electrospray ionization (ESI) was performed with a capillary voltage of 4.2 kV, using nitrogen as the sheath and auxiliary gas, with the heated capillary temperature set at 500°C. MS / MS spectra were generated by collision-induced dissociation (CID) with a collision energy of 40 eV. Identification was based on m / z values and fragmentation patterns, while quantification followed the method described by Yang and his friends.

[0042] In CLOV, PALM, NIAO, and TAMA, 16 phenolic compounds, including flavonoids, flavonoid glycosides, phenolic acids, phenolic aldehyde, and coumarin, along with 6 fatty acids, were identified. The phenolic standards exhibited linearity within the ranges of 0.005-1 mg / L, 0.005-4 mg / L, and 0.025-10 mg / L (Supplementary File-S 1). Each calibration curve demonstrated a regression coefficient greater than 0.99. The method’s limit of detection (LOD) and limit of quantification (LOQ) were determined based on calibration curves derived from five measurements.

[0043] To optimize the combinations of four different essential oils — CLOV (Eugenia aromaticum), PALM (Cymbopogon martinii), NIAO (Melaleuca viridiflora), and TAMA (Calophyllum inophyllum) — a D- optimal mixture design was used (StateEase 360 v23.1.5, Stat -Ease Inc., Minneapolis, MN, USA).

[0044] The independent variables (XCLOV, XPALM, XNIAO, and XTAMA) were the proportions of each oil (CLOV, PALM, NIAO, and TAMA) in the mixture. These essential oils were initially selected based on their IC50 values (the concentration inhibiting 50% of viral activity) against HPV-infected cells, measured as 74.22, 89.71, 101.83, and 223.92 pg / mL. respectively. The dependent variable (Yanti-HPVCs) was the effectiveness of the oil combinations in inhibiting HPV-infected cells, measured through IC50 values (Table 1). The essential oils used in the initial mixture design included CLOV, PALM, NIAO, and TAMA. HPVCs were cultured under standard conditions (37°C, 5% CO2, and 95% humidity) in an appropriate growth medium (DMEM supplemented with 10% FBS, 100 U / mL penicillin, 100 pg / mL streptomycin, 2 mM L- glutamine, 0.1 mM NEAA, and 10 mM HEPES buffer), seeded into 96-well plates, and allowed to adhere overnight.

[0045] The oil mixtures, prepared according to the design model, were applied to the cells at concentrations of 20, 200, 2000, and 10000 pg / mL. After 48 hours of treatment, cell viability was assessed using an appropriate cell viability assay.

[0046] Table 1. D-optimal mixture design model w th independent and dependent variables

[0047] Based on the initial results (Table 1), the three essential oils with the most significant reductions in IC50 values (CLOV, PALM, and NIAO) were selected for further analysis. A new design was conducted with these three independent variables (ACLOV, BPALM, and CNIAO) to further refine the optimal combination. The optimized mixture of the three essential oils was then applied to HPV-infected cells (HPVCs), and posttreatment cell viability was assessed to confirm effectiveness (Table 2). All experiments were conducted in triplicate, with results presented as mean ± standard deviation. Statistical analysis was performed using ANOVA to determine the significance of differences between treatment groups, and the mixture design software provided regression models and contour plots to visualize the interactions between the oils and their effects on IC50 values. Table 2. Second mixture design for possible optimal oil mixture

[0048] In the preferred application of the invention, to achieve a stronger antiviral effect, the composition includes the following, as stated in Formula-9 in the table above:

[0049] 31.5% Eugenia aromaticum (clove oil),

[0050] 31.5% Cymbopogon martinii (palmarosa oil), and

[0051] 37% Melaleuca viridiflora (niaouli oil).

[0052] Ex- vivo permeation studies were also conducted for vaginal and transdermal applications. The study was performed on both HPV-infected and healthy vaginal tissues. Vaginal tissues (2 ' 4 cm2) were obtained from the vaginal walls of patients. All procedures adhered to the Washington University School of Medicine Ethical Committee Guidelines and received approval from the same committee (Approval No.: 2024-688). Informed consent was obtained from all participants in the study. The tissues were carefully prepared by removing connective tissue and fat layers and then washed with cold PBS. The prepared tissue was mounted between the donor and receptor compartments of a Franz diffusion cell (#4G -01-00-01-05, PermeGear Inc., Hellertown, PA, USA). The receptor compartment was filled with PBS containing 0.1% Na-azide and maintained at 37°C with constant stirring (120 rpm).

[0053] A known quantity of the test oil was applied to the tissue in the donor compartment, which was sealed to prevent evaporation. Samples were collected from the receptor compartment at predetermined intervals, and fresh medium was added to maintain sink conditions. These samples were analyzed using a validated LC / MS- MS method to quantify active components (flavonoids, flavonoid glycosides, phenolic acids, and phenolic aldehyde). Data were analyzed to calculate the cumulative amount of molecules permeated per unit area over time, and results were plotted to determine the permeation profile, steady-state flux (Jss), and permeability coefficient (Papp).

[0054] A similar ex-vivo permeation study was conducted using cadaver skin tissues. Preparations, Franz diffusion cell setups, and analysis methods consistent with those used in the vaginal permeation study were applied to allow for comparable results.

[0055] For viral activity analysis, IC50 values for each oil and the optimal mixture were determined. HPV -infected cells (HPVCs) were washed twice with PBS to remove FBS residues and then infected with the virus at a multiplicity of infection (MOI) of 0.01, representing a 1: 100 ratio of infectious viral particles to cells. The viral stock was prepared in a serum-free medium and incubated with the HPVCs for 60 minutes at 37°C.

[0056] The antiviral activity of the compounds against HPV was evaluated by measuring the inhibition of cytopathic effects (CPE) in cell culture using previously established methods. Cell cultures were infected with the virus and treated with essential oils (0.002-1% [v / v] = 20-10,000 pg / mL). Controls included untreated infected cells (DC_HPVCs) and untreated, uninfected cells (DC_mock). Optical density was measured for treated infected cells (DT_HPVCs), DC_HPVCs, and DC_mock. Antiviral activity was calculated using the following formula (Eql):

[0057] Antiviral Activity (%) = [(DT_HPVCs - DC_mock) / (DC_HPVCs - DC mock)] x 100 Eql

[0058] The IC50 value for each oil was determined (Tables 1 and 2), and the selectivity index was calculated as the ratio of TC50 to IC50 (n = 3). After determining IC50 values for each oil and the optimal mixture, the cells were carefully washed and seeded into a 35-mm imaging dish at a density of 1.2 x 106cells per quadrant. For nuclear viability assessment, cells were stained with Hoechst dye, which binds specifically to the DNA within the cell nuclei. This staining allowed visualization of nuclear morphology and identification of viable cells. Transmission and fluorescence images were captured at 15 -minute intervals over a 24-hour period using the Celloger Pro imaging system (Curiosis, Seoul, South Korea).

[0059] For flow cytometry analysis, fluorescence microscopy images displayed morphological changes and fluorescent marker uptake in HPVCs under different treatment conditions (treated with PALM, CLOV, and NIAO, compared to untreated HPVCs). For cell cycle analysis, HPVCs were seeded in 6-well plates and treated with the respective compounds for 24 hours. The cells were then washed with PBS, trypsinized, and fixed in 70% ethanol at 4°C for a minimum of 2 hours. Following fixation, the cells were washed with PBS and stained with a propidium iodide (PI) solution containing RNase A. The stained cells were analyzed using NovoCyte Flow Cytometers (Agilent, Santa Clara, CA, USA), and the percentages of cells in different cell cycle phases (G0 / G1, S, G2 / M) were calculated using FlowJo software (v.l 1.0.5, Framingham, MA, USA).

[0060] The cytotoxicity of the essential oils (both the optimal formulation and individual oils) was evaluated using a resazurin-based in vitro toxicology assay, following the manufacturer's guidelines. Briefly, cells (HPVCs: 140,000; DoTc2: 135,000; and HEKa: 160,000 cells / well) were cultured to 90% confluence and exposed to essential oil concentrations of 20-10,000 pg / mL for 24 hours. After incubation, cells were washed and then incubated with a medium containing 10% resazurin for 1 hour. The levels of the reduced form of resazurin were measured using a microplate reader in fluorescence mode (540 nm excitation and 595 nm emission; Synergy HT, BioTek Inc., Winooski, VT, USA). Data were analyzed using Gen5 Software (v.2.0.1, Agilent, Santa Clara, CA, USA), and cell viability was normalized to untreated controls under the same conditions (n=3).

[0061] An ELISA method was used to investigate changes in HPV proteins (LI, L2, El, E2, E6, E7) and the housekeeping gene GAPDH after treatment with essential oils. The procedure began by coating a 96-well ELISA plate with specific capture antibodies for each target protein, followed by overnight incubation at 4°C. The plate was washed and blocked with 5% BSA or non-fat dry milk for 1 hour at room temperature to prevent nonspecific binding. Diluted cell lysates or tissue homogenates from treated samples were added to the wells and incubated for 2 hours at room temperature or overnight at 4°C. After washing, primary antibodies specific to each HPV protein and GAPDH were added and incubated for 1-2 hours. Following another wash, HRP- or AP-conjugated secondary antibodies were added and incubated for 1 hour. The plate was washed again, and a substrate solution (TMB) was added, allowing color development in the dark at room temperature. For HRP, the reaction was stopped with 1 M HC1, while for AP, absorbance was measured directly. Absorbance was read at 450 nm using a plate reader. Data analysis included comparing absorbance values to standard curves and normalizing to GAPDH to account for sample variability.

[0062] The inhibition of HPV and GAPDH genes was assessed using a Western blot method. HPVCs were first cultured in DMEM containing 10% FBS and penicillin-streptomycin solution and treated with the IC50 values of each oil and the optimal oil mixture for 24 hours. After treatment, the cells were washed, harvested, and lysed in PBS with protease inhibitors. The lysate was centrifuged, and the supernatant containing the proteins was collected. Protein concentration was quantified using a BCA protein assay. Equal amounts of protein were separated via electrophoresis on a 4-20% gradient SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was blocked and incubated overnight with primary antibodies, followed by incubation with an HRP-conjugation kit. After washing, the proteins were detected using an ECL substrate kit and visualized with an iBright CL1500 Imaging System (Invitrogen, Waltham, MA, USA).

[0063] Quantitative PCR (qPCR) was employed to evaluate the gene expressions of LI, L2, El, E2, E6, E7, and GAPDH proteins before and after treatment with essential oils. HPVCs were cultured in media supplemented with 10% FBS and penicillin-streptomycin solution. HPVCs were treated with the IC50 values of each oil and the optimal oil mixture for 24 hours. After treatment, total RNA was extracted using an RNA extraction kit and reverse-transcribed into cDNA. Specific primers for HPV genes (LI, L2, El, E2, E6, E7) and the housekeeping gene GAPDH were used for qPCR amplification (Table 3). The reactions were set up in a 96- well plate using TaqMan qPCR master mix. The qPCR cycling conditions included an initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds, with a final melting curve analysis to confirm product specificity. The relative expression levels of HPV genes were calculated using the AACt method, normalized to GAPDH, and compared between treated and untreated samples (ProFlex™ PCR System, Applied Biosystem, Waltham, MA, USA). Statistical analysis was performed to determine the significance of changes in gene expression levels. Table 3. Primer sequences for HPV genes and housekeeping gene GAPDH

[0064] The anti-HPV activity of various molecules in the essential oils (EOs) was evaluated using COMPIO software (vl.0.7, Comparative Biosciences Inc., Cambridge, MA, USA) based on their interactions with HPV -related proteins and receptors. Initially, 3D structures of HPV proteins (LI, L2, El, E2, E6, and E7) were retrieved from the Protein Data Bank (PDB) and prepared by adding hydrogen atoms, optimizing structures, and removing unnecessary ligands. Similarly, 3D structures of oil molecules (e.g., oleic acid, linoleic acid, palmitic acid, stearic acid, alpha-linolenic acid, inocalophyllin B, inophyllum C, inophyllum E, inophyllum P, calophyllolide, and 12-oxocalanolide A) were imported and optimized. Molecular docking was performed by selecting the docking module within COMPIO, defining binding sites on HPV proteins, and using high- accuracy search algorithms. The resulting docking scores and binding poses were analyzed, and significant interactions, such as hydrogen bonds and hydrophobic interactions, were identified. Key residues involved in binding were noted, and the oil molecules were ranked based on their binding affinity and interaction profiles. Validation of the docking results was performed using additional docking software or existing experimental data.

[0065] The in-silico anti-HPV activity of various oil molecules was evaluated for molecular docking using PyMOL (v.3.0.2, San Diego, CA, USA). First, the 3D structures of HPV proteins (LI, L2, El, E2, E6, E7) were retrieved from the PDB, optimized, and unnecessary ligands were removed. Then, the 3D structures of oil molecules, including oleic acid, linoleic acid, palmitic acid, stearic acid, alpha-linolenic acid, inocalophyllin B, inophyllum C, inophyllum E, inophyllum P, calophyllolide, and 12-oxocalanolide A, were imported and optimized. Binding sites on HPV proteins were defined, and molecular docking was conducted. Docking scores and binding poses were analyzed, particularly focusing on hydrogen bonds and hydrophobic interactions. Oil molecules were ranked based on binding affinity and interaction profiles. By comparing these profiles, potential anti-HPV activity among the oil molecules was determined.

[0066] Chromatographic analysis of CLOV, PALM, NIAO, and TAMA revealed distinct chemical profiles with varying predominant components. Chromatogram A (CLOV) exhibited a major peak at 6.05 minutes, corresponding to P-caryophyllene, constituting 57.09% of the oil. This was significantly higher than other components, such as eugenol (12.47%) and eugenyl acetate (13.39%), emphasizing P-caryophyllene as the dominant compound (Figure 1A and Table 4). Chromatogram B (PALM) identified geranyl acetate as the highest component at 27.79%, followed by nerol at 25.55% and geraniol at 19.51%. These compounds formed the major portion of the oil, highlighting its richness in terpenoids (Figure IB and Table 4).

[0067] Chromatogram C (NIAO) revealed predominant constituents such as P-caryophyllene (19.51%) and viridiflorol (17.37%), significant for both fragrance and therapeutic properties. Other notable components included a-pinene (15.59%) and a-terpineol (9.29%), contributing to the oil’s aromatic profile (Figure 1C and Table 4). Chromatogram D (TAMA) showed linoleic acid as the most abundant compound, constituting 39.57% of the oil. Palmitic acid (14.02%) and a-linolenic acid (13.28%) were also significant, along with minor components such as calophyllolide (0.66%) and inophyllum (8.19%) (Figure ID and Table 4).

[0068] The variations in these compositions emphasize the diverse chemical nature of essential oils, influenced by factors such as plant source, extraction methods, and geographical conditions. Recent research highlights the biological activities of these major components. For example, P-caryophyllene, predominant in CLOV, is known for its anti-inflammatory and analgesic properties. Eugenol, another significant component of CLOV, has demonstrated antimicrobial and antioxidant effects. Linoleic acid, the primary component in TAMA, is renowned for its skin barrier repair properties. These findings underscore the therapeutic potential of essential oils, making them valuable in pharmacological and therapeutic applications.

[0069] Figure 1 presents the chromatographic analysis of essential oils. LC-MS / MS chromatograms of 0.25 pg / inL EOs are shown for (A) CLOV, (B) PALM, (C) NIAO, and (D) TAMA. Peaks correspond to the following compounds: a) Eugenol, b) Eugenyl acetate, c) Humulene, d) P-Caryophyllene*, e) Methyleugenol, ) Calophyllolide, k) Geraniol, 1) Nerol, m) Neral, n) Geranyl acetate, o) Linalool, 1,8-Cineole, a-Terpineol, a- Pinene, Limonene, Viridiflorol, -Pinene, P-Caryophyllene*, Inophyllum, x) Oleic acid, y) Linoleic acid, z) Palmitic acid, t) Stearic acid, and u) a-Linolenic acid.

[0070] * According to the Herbal Codex, the expected retention time (Rt) of P-caryophyllenes is after 6 minutes.

[0071] V

[0072] Table 4. Composition of the EOs

[0073] “ Retention time {mm).

[0074] 'Percentage of relative weight s According to the herbal codex, expected rt of p-caiyophylleiies after 6 mins

[0075] * statistical significant difference (p<0.0 )

[0076] Figure 2 presents triangular graphs showing the anti-HPV effects (IC50 values) of essential oils (EOs) at varying ratios. Figure 2A displays IC50 values when the essential oils are mixed with a minimal amount of TAMA, whereas Figure 2B shows IC50 values when mixed with the maximum amount of TAMA. The color gradient ranges from 91.78 to 108.64 pg / mL, indicating anti-HPV activity. Blue and green colors represent lower IC50 values (higher activity), while red and yellow colors indicate higher IC50 values (lower activity). Results demonstrate that TAMA does not significantly influence anti-HPV activity compared to other oils (p > 0.05). Higher amounts of TAMA are associated with increased IC50 values, indicating reduced efficacy. This finding highlights the potential to enhance anti-HPV effects by optimizing the ratios of different essential oils, regardless of the presence of TAMA.

[0077] Figure 2 shows triangular graphs of the anti-HPV effect (IC50) of essential oils at different ratios; A) Minimum amount of TAMA, and B) Maximum amount of TAMA.

[0078] Figure 3 presents piepel plots showing the anti-HPV effect (IC50 values) of EOs (essential oils) with varying amounts of TAMA. Figure 3A represents the minimum amount of TAMA, while Figure 3B represents the maximum amount of TAMA. The X-axis indicates deviations from the reference blend (L_Pseudo Units), and the Y -axis shows anti-HPV IC50 values (pg / mL). The plots for each EO indicate no significant difference in anti-HPV activity across varying levels of TAMA. Both graphs exhibit similar trends, suggesting that TAMA does not significantly influence the efficacy of essential oils against HPV. This finding aligns with recent studies indicating that TAMA’s contribution to anti-HPV activity is minimal compared to other essential oils.

[0079] Figure 3 illustrates the piepel plots of EOs: (A) Minimum amount of TAMA and (B) Maximum amount of TAMA.

[0080] Figure 4 demonstrates the optimization of EO blends for anti-HPV activity, with sub figures A and B representing a 3D response surface and an overlay plot, respectively. Figure 4A, the 3D response surface plot, depicts the anti-HPV effect (measured by IC50 values) based on varying proportions of EOs labeled as A, B, and C. The color gradient ranges from blue (53.91 pg / mL) to red (70.62 pg / mL), where lower IC50 values indicate higher anti-HPV activity. Figure 4B, the overlay plot, highlights the most effective blend regions in yellow.

[0081] Based on these design models, Formula 9 (F9), identified within the optimal region, was selected as the optimal blend. This optimal blend shows a significant reduction in IC50 values, suggesting stronger anti- HPV activity. Recent studies support the use of optimization models to enhance the efficacy of EO formulations for antiviral applications. These findings emphasize the importance of achieving the correct blend ratios to maximize therapeutic benefits. Figure 4 demonstrates the optimization of anti-HPV EO blends: A) 3D surface plot, and B) triangular overlay plots for clove, palmarosa, and niaouli oils.

[0082] In the permeation studies, essential oils (EOs) were evaluated both individually and as part of the optimal formulation. Over time, a notable reduction in specific components of the blend was observed in the basolateral compartment (for HPV -infected tissue samples), indicating its potential application in infection contexts (Figures 5B and 5D). Figure 5A illustrates the hydrophobic properties of geraniol and eugenol, which lead to significantly lower release rates compared to other active components (p<0.05), even in healthy skin tissue. Specifically, reductions were observed in cineole and P-caryophyllene from NIAO, geraniol and linalool from PALM, and eugenol and P-caryophyllene from CLOV (p<0.05). EOs are known for their broad spectrum of biological activities, including antiviral properties. Recent research highlights the antiviral efficacy of various EO components. Previous studies have demonstrated cineole and P-caryophyllene's antiviral activity against herpes simplex vims, indicating their potential for viral inhibition. Similarly, linalool and geraniol have shown significant antiviral effects against various viral strains. Furthermore, F9's release study revealed reductions in viridiflorol, limonene, P-pinene, and a-pinene, which are also components of NIAO (Figures 5C and 5D). These observations indicate that these specific molecules actively contribute to combating HPV in infected tissues and possess potential antiviral properties. Eugenol, the main component of CLOV, has shown promising results in inhibiting the replication of several vimses, including HSV and influenza. Additionally, the antiviral activities of components like limonene and pinene have been explored, further highlighting the potential of EOs in antiviral therapies. These findings support the observed reductions in the basolateral compartment of HPV -infected tissues and demonstrate the superior performance of the optimal blend (F9: 31.5% CLOV, 31.5% PALM, and 37% NIAO) in the release study. The results confirm that this specific ratio is the most effective in delivering and utilizing the necessary active components for combating HPV.

[0083] Figure 5 illustrates the concentration of HPV -related components: A) Healthy skin sample, B) HPV-infected skin sample, C) Healthy vaginal tissue sample, and D) HPV -infected vaginal tissue sample.

[0084] Figure 6 evaluates the permeation properties of various components in healthy and infected skin and vaginal tissues using Jss and Papp measurements. Overall, permeation values (both Jss and Papp) in healthy tissues are higher compared to infected tissues, indicating that infection hinders permeation. This can be attributed to the involvement of active components in the healing process, which alters the tissue environment and reduces permeability. During healing, immune cells such as macrophages and neutrophils migrate to the site of injury or infection, clearing debris and pathogens, promoting tissue repair, and alleviating inflammation. The influx of these immune cells and the associated inflammatory response can alter the structural and biochemical properties of the tissue, reducing permeability to external substances. Cineole and viridiflorol exhibited the highest permeation rates, with cineole achieving the highest flux (1 .5 pg / cm2h) in healthy skin tissue and viridiflorol reaching the peak value (13.0 pg / cm2h) in healthy skin tissue as well (Figure 6A). In contrast, components like geraniol and a-pinene showed significantly lower permeation, particularly in infected tissues (p<0.05). Skin tissues generally provided higher permeation compared to vaginal tissues; healthy skin tissue exhibited the highest Jss and Papp values across most components. These findings are critical for designing effective drug delivery systems targeting these tissues, emphasizing the impact of tissue health on component permeation and highlighting the superior permeability of skin tissues compared to vaginal tissues. Figure 6 compares the flux and permeability coefficients in various tissues under healthy and infected conditions: A) violin plots for Jss and B) box plots for Papp.

[0085] For anti-HPV activity and toxicity profiles, the provided images illustrate the effects of various treatments (F9, PALM, CLOV, and NIAO) on HPV-infected cells (HPVCs) compared to uninfected control cells (HEKa) after a 24-hour treatment period (Figure 7). The green dye in the images indicates viral replication within the cells. In untreated HPVCs, significant green fluorescence reflecting high levels of viral replication is observed. Each treated sample (HPVCs-F9, HPVCs-PALM, HPVCs-CLOV, and HPVCs-NIAO) exhibits a marked reduction in green fluorescence compared to the untreated control (p<0.05), indicating effective inhibition of viral replication within 24 hours. Among the treatments, HPVCs-NIAO and HPVCs-CLOV appear to be the most effective, as evidenced by minimal green fluorescence, suggesting a substantial reduction in viral replication. HPVCs-F9 and HPVCs-PALM (p<0.05) also show reduced viral activity compared to the untreated control, but not to the same extent as HPVCs-NIAO and HPVCs-CLOV.

[0086] Uninfected control samples show no significant green fluorescence, confirming that the green dye specifically indicates viral replication and that uninfected cells are not undergoing this process. These findings are consistent with recent studies highlighting the potential of natural compounds and novel antiviral agents in reducing HPV replication within infected cells over short treatment durations.

[0087] Figure 7 shows the Celloger Pro analysis of HPVCs treated with EOs. Cell cycle analysis of HPVCs treated with CLOV, NIAO, PALM, and F9 reveals significant alterations in cell cycle distribution compared to the control group (untreated HPVCs) (p<0.05) (Figure 8). In the control group, the majority of cells reside in the G0 / G1 phase, with moderate representation in the S phase and fewer cells in the G2 / M phase. Treatment with CLOV results in a noticeable reduction in G0 / G1 phase cells and an increase in both the S and G2 / M phases, suggesting enhanced DNA replication and cell division. NIAO treatment similarly increases the S phase, with a slight rise in the G2 / M phase, indicating active DNA synthesis. PALM treatment shows a significant shift towards the S phase, with a moderate increase in the G2 / M phase, highlighting a substantial impact on DNA replication. F9 treatment causes the most pronounced shift, with the highest increase in the S phase and a notable rise in the G2 / M phase, indicating robust DNA replication and cell cycle progression. Overall, these results suggest that the oils, particularly F9, strongly influence HPVC cell cycle dynamics, enhance DNA replication, and potentially promote cell division.

[0088] Figure 8 shows the cell cycle analysis of HPVCs treated with different essential oils. The provided data illustrate the effects of CLOV, NIAO, PALM, and F9 on HPVCs, focusing on cell survival and viral protein expression. Figure 9A depicts cell survival percentages, revealing that treatment with F9 significantly reduces cell survival in HPVCs and DoTc2 cells compared to the control and other treatments, indicating its potent cytotoxic effect. Figure 9B shows the expression levels of HPV genes (El, LI, L2, E2, E6, and E7) via Western blot analysis. F9 treatment results in a marked decrease in the expression of these viral proteins compared to the control, highlighting its inhibitory effect on viral replication and protein synthesis.

[0089] Figure 9C quantifies the relative protein levels, where F9-treated cells show the lowest levels of viral proteins across all targets, further emphasizing its effectiveness in suppressing HPV protein production. Finally, Figure 9D, presenting relative expression normalized to GAPDH, confirms that F9 treatment leads to the most substantial reduction in the expression of all viral proteins compared to other treatments. These findings suggest that F9 is significantly more effective than CLOV, NIAO, and PALM in reducing both cell survival and viral protein expression in HPVCs. This aligns with recent research on the effectiveness of novel therapeutic agents in targeting HPV infections, underscoring F9's potential as a powerful antiviral agent.

[0090] Figure 9 illustrates the impact of various treatments on cell survival and HPV gene expression: A) cell survival percentages of HPVCs, DoTc2, and HEKa cell lines, B) expression levels of HPV genes via Western blot, C) scatter plot representing relative protein levels of HPV genes, and D) graph showing relative expression of HPV genes. El, LI, L2, E2, E6, and E7 are HPV genes.

[0091] The heatmap analysis reveals significant correlations between various EG components and protein expressions (El, LI, L2, E2, E6, and E7) (Figure 10). Notably, cineole exhibits a strong correlation with El (0.8) and E6 (0.4), indicating its potential role in modulating these proteins' expressions, which aligns with recent findings on cineole's potent anti-inflammatory properties and therapeutic applications. Similarly, 0- caryophyllene shows moderate correlations with E2 (0.7) and El (0.6), supporting its role in modulating protein expressions in cancer cells. Geraniol's high correlation with E2 (0.7) and LI (0.6) is consistent with its documented effects on protein expression related to skin health. Linalool demonstrates balanced moderate correlations across all proteins, particularly E2 (0.7), reflecting its influence on immune response proteins. Eugenol shows strong positive correlations with E2 (0.9) and LI (0.8), indicative of its significant impact on cancer-related protein expressions. Viridiflorol stands out with the highest positive correlations, particularly with LI (0.9) and E2 (0.9), highlighting its bro ad -spectrum effects. In contrast, limonene and P-pinene show lower overall correlations, with notable impacts on E6 (0.9) and E7 (0.9), respectively, reflecting their unique roles in respiratory health and anti-inflammatory processes. Finally, a-pinene's strong correlations with L2 (0.9) and E6 (0.8) underscore its significant protein modulation effects, as detailed in recent studies (p<0.05). This comprehensive analysis underscores the nuanced roles of EOs in protein expression modulation, providing a robust foundation for further therapeutic exploration.

[0092] Figure 10. The heatmap of compound -protein interactions. The Lipinski Rule of Five (RO5) provides guidelines predicting dmg-like properties and oral bioavailability of chemical compounds. In the context of these rules, the analysis of essential oil components indicates that cineole, geraniol, linalool, eugenol, viridiflorol, limonene, P-pinene, and a-pinene generally comply with these criteria, demonstrating good potential for oral bioavailability (Table 5). Specifically, cineole, geraniol, linalool, eugenol, viridiflorol, limonene, and both P-pinene and a-pinene have molecular weights well below the 500 g / mol threshold. Their LogP values, indicating hydrophobicity, are mostly below the critical value of 5. However, P-caryophyllene has a LogP value of 6, suggesting potential bioavailability challenges due to excessive hydrophobicity. Furthermore, these compounds meet the criteria of having fewer than 5 hydrogen bond donors and fewer than 10 hydrogen bond acceptors, which favor absorption and permeability in the body. Therefore, except for P- caryophyllene, these essential oil components exhibit properties aligned with RO5, making them promising candidates for further development in pharmaceutical applications.

[0093] Table 5. RO5 of active components of EOs.

[0094] Component Molecular I ,ogP Hydrogen Hydrogen

[0095] Weight Bond Bond

[0096] (g / mol) Donors Acceptors

[0097] Binding studies of the active components of essential oils with the active site of HPV inhibitory proteins reveal varying binding affinities. Cineole exhibits the strongest interaction with the HPV inhibitory protein at the El site, with a binding score of -8.066. Following this, P-caryophyllene binds to the E2 site with a score of -5.551. Other significant interactions include Geraniol at the L2 site (-6.211), Linalool at the E6 site (-5.201), Eugenol at the E2 site (-5.195), and Viridiflorol at the LI site (-5.752). Limonene interacts at the E7 site with a score of -5.233, while P-pinene and a-pinene show interactions at the E6 and L2 sites with scores of -5.028 and -4.988, respectively. These findings suggest that cineole and P-caryophyllene have strong potential as HPV protein inhibitors due to their low binding scores, indicating higher binding affinity.

[0098] Table 6. Docking score and 3D representation of the interaction of EOs' active compounds.

[0099]

[0100] Eugenia aromaticum (CLOV), Cymbopogon martinii (PALM), Melaleuca viridiflora (NIAO), and Calophyllum inophyllum (TAMA) essential oils show high potential as effective antiviral agents against HPV infections. Ex-vivo permeation studies confirmed their enhanced penetration capabilities, supporting their applicability in targeted therapies. The optimal essential oil blend was determined using a D-optimal mixture design. Cytotoxicity assessments verified the safety of these oils at therapeutic concentrations in HPVC, DoTc2, and HEKa cells, while molecular docking studies provided insights into their mechanisms of action. The optimal essential oil mixture (31.5% CLOV, 31.5% PALM, and 37% NIAO), containing 0- caryophyllene, geranyl acetate, geraniol, and nerol, demonstrated its efficacy in modulating E7 and E2 protein pathways, as confirmed by ELISA, qPCR, and Western blot analyses, establishing its role as a natural supportive treatment for managing HPV infections.

[0101] The invention comprises a mixture of specific essential oils (clove oil, palmarosa oil, niaouli oil, and tamanu oil). These essential oils suppress HPV infections by modulating E7 and E2 protein pathways. Ex-vivo permeation studies demonstrated the penetration of these oils into infected tissues, confirming their safety in healthy tissues.

[0102] In particular, the specific blend of these essential oils has shown antiviral activity against HPV -infected cells and suppressed the expression of viral proteins. Additionally, both the individual oils and their combinations were found to inhibit viral replication in HPV -infected cells through antiviral activity.

[0103] Optimally, a mixture of 31.5% Eugenia aromaticum, 31.5% Cymbopogon martinii, and 37% Melaleuca viridiflora effectively treated HPV infections. These compositions exhibited antiviral activity confirmed by ELISA, qPCR, and Western blot analyses.

[0104] In conducted studies, the optimal oil blend (31.5% CLOV, 31.5% PALM, 37% NIAO) was applied to HPV - infected cells, significantly reducing viral replication by suppressing E2 and E7 protein pathways. Moreover, cytotoxicity analyses demonstrated that these oils are safe at therapeutic doses.

[0105] Clove oil (Eugenia aromaticum) is well-known for its antimicrobial and antiviral activities due to its high eugenol content. Eugenol can inhibit viral replication by disrupting the structural integrity of cell membranes.

[0106] Palmarosa oil (Cymbopogon martinii) contains high levels of geraniol and citronellal, components with potent antiviral and antibacterial properties. Palmarosa oil acts effectively by both disrupting viral cell walls and inhibiting replication processes.

[0107] Niaouli oil (Melaleuca viridiflora) demonstrates antiviral and immunomodulatory effects, particularly due to its viridiflorol content. This oil can strengthen cellular immune mechanisms and limit the intracellular spread of viruses.

[0108] Tamanu oil (Calophyllum inophyllum) is recognized for its anti-inflammatory and wound-healing properties. It also exhibits antimicrobial effects and has the potential to suppress viral replication within cells. Essential oils are suitable for use in drug delivery systems. These oils can be incorporated into topical creams, gels, ointments, or transdermal systems, facilitating the penetration of their active components into the skin or mucosal tissues. Particularly for mucosal infections such as HPV, the direct application of the antiviral components of essential oils to the infected area provides an effective treatment option.

[0109] In summary, given the limitations of current HPV treatment methods, the potential antiviral activities of essential oils are garnering significant attention. Synergistic combinations of clove, palmarosa, niaouli, and tamanu oils show promising results against HPV infections. Molecular docking studies and cytotoxicity analyses confirm the efficacy of these oils against HPV. Moreover, the use of essential oils offers an alternative treatment option, particularly for patients seeking natural and less invasive therapeutic methods.

[0110] The invention is a composition for the treatment of HPV infections. By modulating E7 and E2 protein pathways through components containing -caryophyllene, geranyl acetate, geraniol, and nerol, the composition demonstrates a natural supportive therapeutic effect with antiviral, antibacterial, and immunomodulatory activities. It significantly reduces viral replication and suppresses protein expression and is characterized by containing:

[0111] Eugenia aromaticum (clove oil): Rich in eugenol, possessing antimicrobial and antiviral activities, it inhibits viral replication by disrupting the structural integrity of cell membranes.

[0112] Cymbopogon martinii (palmarosa oil): Containing high levels of geraniol and citronellal, it exhibits antiviral and antibacterial properties by both disrupting viral cell walls and inhibiting the replication process.

[0113] Melaleuca viridiflora (niaouli oil): With its viridiflorol content, it strengthens cellular immune mechanisms and limits the intracellular spread of viruses, demonstrating antiviral and immunomodulatory effects.

Claims

C L A I M S1. The invention pertains to a composition for the treatment of HPV infections, characterized by its components containing P-caryophyllene, geranyl acetate, geraniol, and nerol, which modulate the E7 and E2 protein pathways, thereby providing a natural supportive therapeutic effect in managing HPV infections. This composition demonstrates antiviral, antibacterial, and immunomodulatory activities, significantly reduces viral replication, and suppresses protein expression. The composition contains:Eugenia aromaticum (clove oil), rich in eugenol, which disrupts the structural integrity of cell membranes to inhibit viral replication while exhibiting antimicrobial and antiviral activities,Cymbopogon martinii (palmarosa oil), rich in geraniol and citronellal, possessing antiviral and antibacterial properties by disrupting viral cell walls and inhibiting the replication process,Melaleuca viridiflora (niaouli oil), containing viridiflorol, which exhibits antiviral and immunomodulatory effects by enhancing cellular immune mechanisms and limiting the intracellular spread of viruses.

2. The composition mentioned in Claim 1 , characterized by containing any of the following weight / volume formulations;33.33% Eugenia aromaticum (clove oil), 33.33% Cymbopogon martinii (palmarosa oil), and 33.33% Melaleuca viridiflora (niaouli oil),35.1% Eugenia aromaticum (clove oil), 30.9% Cymbopogon martinii (palmarosa oil), and 33.0% Melaleuca viridiflora (niaouli oil),40% Eugenia aromaticum (clove oil), 30% Cymbopogon martinii (palmarosa oil), and 30% Melaleuca viridiflora (niaouli oil),45% Eugenia aromaticum (clove oil), 27.5% Cymbopogon martinii (palmarosa oil), and 27.5% Melaleuca viridiflora (niaouli oil),30% Eugenia aromaticum (clove oil), 40% Cymbopogon martinii (palmarosa oil), and 30% Melaleuca viridiflora (niaouli oil),30% Eugenia aromaticum (clove oil), 30% Cymbopogon martinii (palmarosa oil), and 40% Melaleuca viridiflora (niaouli oil),37% Eugenia aromaticum (clove oil), 31.5% Cymbopogon martinii (palmarosa oil), and 31.5% Melaleuca viridiflora (niaouli oil),31.5% Eugenia aromaticum (clove oil), 31.5% Cymbopogon martinii (palmarosa oil), and 37% Melaleuca viridiflora (niaouli oil),31.5% Eugenia aromaticum (clove oil), 37% Cymbopogon martinii (palmarosa oil), and 31.5% Melaleuca viridiflora (niaouli oil).

3. The composition mentioned in Claim 1 , characterized in that in the preferred embodiment of the invention, it contains by weight / volume;31.5% Eugenia aromaticum (clove oil),31.5% Cymbopogon martinii (palmarosa oil), and37% Melaleuca viridiflora (niaouli oil)4. The composition mentioned in Claim 1 , characterized by its application transdermally to skin with HPV infection and / or topically to mucosal tissues such as the vagina.

5. The composition mentioned in Claim 1, characterized by being in the form of a topical cream, ointment, or gel.

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