Low-cytotoxicity d-limonene nanoemulsion for preventing oral cancer and preparation method of low-cytotoxicity d-limonene nanoemulsion

Abstract

The invention discloses a low-cytotoxicity d-limonene nanoemulsion for preventing oral cancer. The low-cytotoxicity d-limonene nanoemulsion comprises the following components: 0.4-10% of d-limonene, 0.1-5% of tea tree oil, 0.25-5% of an oil phase, 1-15% of a surfactant, 1-5% of a cosurfactant and the balance of deionized water or distilled water, wherein the surfactant is a mixture of caprylic / capric polyethylene glycol glyceride and polyoxyethylene hydrogenated castor oil RH-40; and the cosurfactant is PEG (Polyethylene Glycol)-400. CCK8, Calcein-AM / PI cell live and dead double staining experiments and animal model verification prove that the nano-emulsion formed by the caprylic / capric polyethylene glycol glyceride / RH-40 / PEG-400 composite surfactant and the d-limonene / tea tree oil has lower cytotoxicity than the nano-emulsion prepared from Tween-80; the invention provides a safer and more effective novel preparation for preventing and treating oral precancerous lesions.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to natural drugs that inhibit or prevent the proliferation of cancer cells, specifically a low-cytotoxic drug for the prevention of oral cancer. d - Limonene nanoemulsion and its preparation method, using Labrasol ® (or Labrasol) ® ALF / RH-40 / PEG is a mixed surfactant used to construct a low-cytotoxic oral rinse formulation that can prevent / treat oral leukoplakia and thus prevent oral cancer. Background Technology

[0002] As the primary gateway for microorganisms to enter the human body, the oral cavity's complex internal environment makes it one of the most complex and numerous structures in the human body in terms of microbial community structure. Imbalance in the oral microbial ecosystem can trigger various oral diseases, such as stomatitis and oral ulcers, while recurrent chronic oral inflammation may induce precancerous lesions of the oral cavity.

[0003] Oral cancer is a general term for malignant tumors occurring in the oral cavity. It has complex causes, a poor prognosis, and a high recurrence rate. Currently, there are no drugs or treatments with minimal side effects, making prevention particularly important. Oral leukoplakia, as a common precancerous lesion, increases the risk of oral cancer. Effective prevention and treatment of oral leukoplakia are crucial for preventing the development of oral cancer. Currently, surgical treatment is the primary method for treating oral cancer; however, surgery has significant side effects, especially the postoperative changes in appearance, which are difficult for patients to accept. Therefore, there is a very urgent need for drug-based prevention / treatment of early-stage oral leukoplakia.

[0004] Drug delivery for treating oral mucosal diseases differs significantly from systemic administration. Its core objective is to achieve effective concentrations at the lesion site and maintain sufficient duration of action, while minimizing systemic absorption and side effects. Oral mucosal delivery requires drugs with rapid onset of action or those providing local treatment. Its successful application highly depends on the drug's physicochemical properties and the ingenious design of its dosage form. It serves as a crucial bridge between oral and injectable drug delivery in modern pharmaceutics.

[0005] A patent already exists for "a method to prevent oral cancer". d In the paper "Limonene Nanoemulsion Synergistic Composition and Preparation Method and Application" (CN114558052B), Tween-80 and EL-35 are used as surfactants to construct a nanoemulsion system, which to some extent solves the problem of... d-Stability and absorption issues of limonene. Pure Tween-80 is a colorless and transparent liquid. Although Tween-80 has been approved as a legal oral pharmaceutical excipient by drug regulatory agencies in China and many other countries (such as the FDA), and raw materials for injectable applications have also been developed, the quality of domestically produced Tween-80 has long been inconsistent due to differences in production processes. It often contains more impurities, has a darker color, and these impurities can remain in the final product, causing certain allergic reactions and toxicity.

[0006] Furthermore, although both EL-35 and Tween-80 achieve water solubility by linking polyoxyethylene chains (hydrophilic portions), their "oil-soluble tails" belong to different chemical structures. In most routine non-intravenous administration scenarios, especially considering the risks of hemolysis and metabolic interference, the toxicity risk of EL-35 increases significantly at high concentrations, during intravenous injection, or when used in combination with certain organic solvents (such as propylene glycol), requiring extreme caution.

[0007] Therefore, Tween-80 and EL-35 pose potential cytotoxicity risks. Long-term or high-concentration use may have toxic effects on oral mucosal cells, posing a risk of mucosal irritation and affecting the biosafety of formulations, thus limiting their application. Therefore, developing a low-toxicity, high-efficiency nanoemulsion delivery system has become an urgent problem to be solved. Summary of the Invention

[0008] In response to the existing d The present invention addresses the issue of high surfactant toxicity and insufficient safety in limonene nanoemulsion compositions. It aims to provide a formulation that prevents / inhibits the progression of precancerous oral lesions, exhibiting lower toxicity and better cellular safety compared to existing formulations. d - A limonene nanoemulsion synergistic composition and its preparation method, which is based on a composite nonionic surfactant, octanoic acid-decanoic acid-polyethylene glycol glycerol ester (Labrasol). ® / Labrasol ® A low-toxicity and high-efficiency nanoemulsion composition with ALF and polyoxyethylene hydrogenated castor oil RH-40 (kolliphor RH40) as the core significantly reduces cytotoxicity through formulation optimization, while improving the effect of preventing oral precancerous lesions.

[0009] This invention is achieved using the following technical solution: Firstly, a low-cytotoxic method for preventing oral cancer. d - Limonene nanoemulsion, comprising the following components: d - Limonene, tea tree oil, oil phase, surfactant, co-surfactant, and the remainder being deionized water or distilled water. The surfactant is PEG-C(ethyl octanoate) / (decanoate) / (poly(ethylene glycol) glycerol ester (Labrasol). ® / Labrasol ®ALF) and polyoxyethylene hydrogenated castor oil RH-40 (kolliphor RH40).

[0010] Further preferably, the d - Limonene is a natural extract with a purity of 90-99%, and tea tree oil is a natural extract that meets international standards (ISO 4730-2017); the oil phase is medium-chain triglycerides (MCT), olive oil, or soybean oil; the co-surfactant is polyethylene glycol, propylene glycol, or ethanol.

[0011] More preferably, the co-surfactant is PEG-400.

[0012] Further preferred, d - The weight percentage of limonene is 0.5~2%, the weight percentage of tea tree oil is 0.1~1%, the weight percentage of oil phase is 0.25~5%, the weight percentage of surfactant is 1~15%, and the weight percentage of co-surfactant is 1~5%.

[0013] Further preferred, d -Limonene is 0.4~10%, tea tree oil is 0.1~5%, surfactant is 3.75~10%, and co-surfactant is 1.25~3.33%.

[0014] A further preferred embodiment is that the mass ratio of caprylic / capric acid glyceride polyethylene glycol glycerol to polyoxyethylene hydrogenated castor oil RH-40 is (1~4):(9~1).

[0015] A further preferred option is the mass ratio of surfactant to co-surfactant, i.e., K. m The ratio is 7:1 to 1:1; the mass ratio of all surfactants to all oil phases (surfactant-oil ratio), i.e., the SOR value is 1:1 to 9:1.

[0016] Further preferred, d - Limonene 1%, tea tree oil 1%, MCT 0.5%, surfactant 4.375%, PEG-400 1.46%.

[0017] Further preferred, the mass ratio of octanoic acid / capric acid / polyethylene glycol glycerol ester and polyoxyethylene hydrogenated castor oil RH-40 is 3:7; the SOR value is 7:3, and the K... m The ratio is 3:1.

[0018] Secondly, a method for preparing a low-cytotoxic d-limonene nanoemulsion for the prevention of oral cancer includes the following steps: (1) First, d- Limonene, tea tree oil, oil phase, surfactant and co-surfactant are placed in a container in a certain weight ratio and stirred at 100~2000 r / min for 10~120 min to obtain a mixture; (2) Weigh out deionized water or distilled water according to the weight ratio, and slowly add the deionized water or distilled water dropwise to the above mixture under stirring conditions. After the addition is complete, stir for 30 to 120 minutes to prepare a nanoemulsion with a particle size of 10 to 100 nm, which is a low-cytotoxic agent for the prevention / treatment of oral precancerous lesions. d - Limonene nanoemulsion products.

[0019] Alternatively, it can be prepared using a high-pressure homogenization method: Each material is placed in a high-pressure homogenizer according to its weight ratio, and homogenized for 10-120 minutes at a working pressure of 200-1500 bar to produce a nanoemulsion with a particle size of 10-100 nm, which is a low-cytotoxic agent for the prevention / treatment of precancerous oral lesions. d - Limonene nanoemulsion products.

[0020] Nanoemulsions / microemulsions are a highly effective dosage form for the prevention and treatment of oral mucosal diseases. Surfactants are an indispensable core component in the preparation of nanoemulsions / microemulsions, and their importance is mainly reflected in the following aspects, which can be called the "soul" of nanoemulsions / microemulsions: (1) reducing interfacial tension is the driving force for the formation of nanoemulsions / microemulsions; (2) providing spatial or electrostatic barriers to stabilize the structure of nanoemulsions / microemulsions; (3) determining the type, physicochemical properties and functions of nanoemulsions / microemulsions. However, in practical applications, surfactants face significant safety challenges in the preparation of nanoemulsions / microemulsions, especially in food, cosmetics and injectable drugs, where it is necessary to select biocompatible, low-toxicity or non-toxic natural or synthetic surfactants (such as phospholipids, polysorbates, glycosides). In short, without suitable surfactants, it is impossible to obtain a stable, uniform, and applicable nanoemulsion / microemulsion system. Therefore, the selection and optimization of surfactants is the most critical and core link in the development of nanoemulsion / microemulsion formulations.

[0021] Caprylic / Capric Caprylic / Capric Polyethylene Glyceryl Acetate (trade name: Labrasol) ® / Labrasol ® ALF is a mixture of monoglycerides, diglycerides, triglycerides, and polyethylene glycol (molecular weight 400) containing monoesters and diesters. Labrasol... ® and Labrasol ® ALF is the same substance; ALF is a product with a low aldehyde value. Labrasol ® / Labrasol ®Both ALF and RH-40 are safer pharmaceutical excipients with low toxicity, high biocompatibility, and excellent solubilizing and mucosal penetration-enhancing abilities, which can significantly reduce formulation toxicity and improve bioavailability. They can be used to construct novel, low-toxicity, and highly stable nanoemulsion systems to address the potential toxicity issues of existing formulations, providing safer and more reliable formulations for the prevention of oral precancerous lesions. Meanwhile, no formulations using a mixed surfactant of PEGylated caprylic / capric acid glyceride / RH-40 have been observed. d - Reports on the use of limonene low-toxicity nanoemulsions for the effective prevention of oral cancer.

[0022] The screening and optimization process of surfactants, which is the core technology of this invention, was determined through the following experiments: Preliminary screening of surfactants: Candidate surfactants Labrasol were screened using a SOR ratio of 9:1. ® Labrafil ® 1944 CS, soybean lecithin, RH-40, sodium lauryl sulfate, S-1570, P-1570, and glyceryl monolaurate were screened to investigate their compatibility with... d - The compatibility of limonene with emulsifying agents. Results showed that RH-40 and Labrasol... ® These two surfactants were selected as candidates because they can form stable emulsions.

[0023] Table 1. Screening results of low-toxicity surfactants

[0024]

[0025] Surfactant ratio optimization: Based on preliminary screening, adjust the ratio of Labrasol... ® The mass ratio of RH-40 to emulsifier was found to be 3:7, which resulted in the best emulsification effect, uniform emulsion particle size, and optimal stability. Therefore, this ratio was determined.

[0026] Co-surfactants and K m Value optimization: Using PEG-400, 1,2-propanediol, and other candidate co-surfactants, the K values ​​were investigated under a surfactant ratio of 3:7. m The effect of surfactant value (co-surfactant) on emulsion stability. The results showed that only PEG-400 as a co-surfactant was effective in forming the nanoemulsion in this system. m At a ratio of 3:1, PEG-400 and RH-40 / Labrasol ® The combined effect resulted in good emulsification, hence this parameter was determined.

[0027] SOR value optimization: By adjusting the mass ratio of total emulsifier to oil phase, it was found that when SOR=7:3, the emulsification rate was the highest that the system could achieve, the system stability was the best, and the oil phase had the highest carrying capacity. Therefore, this ratio was determined.

[0028] The screening and optimization process was determined experimentally, and the results showed that Labrasol ® Both RH-40 and K can form a stable emulsion at a mass ratio of 3:7, therefore these two are identified as candidate surfactants. SOR=7:3, K m The emulsification rate is the optimal value that this system can achieve when the ratio is 3:1. Therefore, the above parameters are determined.

[0029] The above-mentioned low cytotoxicity for the prevention of oral cancer d - The preparation method of limonene nanoemulsion adopts the phase transition method: the raw materials are prepared by... d - Limonene, tea tree oil, MCT, RH-40 and Labrasol ® (Mixed surfactants), PEG-400 (co-surfactant), are accurately weighed according to the calculated proportions and placed in a container. The mixture is magnetically stirred at 28°C for 20 minutes to obtain a mixture. Continue stirring and add deionized water dropwise until the system changes from turbid to a transparent and homogeneous nanoemulsion. Continue stirring for 30 minutes to prepare a nanoemulsion with a particle size of 10-100 nm. This is the novel, low-toxicity, and highly effective agent for preventing / inhibiting the progression of precancerous oral lesions described in this invention. d - Limonene nanoemulsion synergistic composition products.

[0030] The inventors proved through experiments that the surfactant RH-40 used is compatible with Labrasol. ® Compared to the surfactants Tween-80 and EL-35 used in the existing formulation in invention CN114558052B, this invention exhibits lower toxicity, better cellular safety, and a more significant preventive effect against oral precancerous lesions (leukoplakia). Therefore, the present invention... d - Limonene nanoemulsion synergistic compositions can be used as safer drugs to prevent oral cancer or stop the progression of precancerous lesions of the oral cavity, as well as for the preparation of mouthwashes for the prevention of oral cancer or the prevention of precancerous lesions of the oral cavity in the context of oral health care and hygiene.

[0031] The significance of this invention lies in the fact that it is the first discovery and proof that the traditional surfactants Tween-80 and EL-35 used in existing nanoemulsion formulations have potential cytotoxicity risks, which limit their long-term clinical application. Through a series of experiments, a new type of low-toxic surfactant was screened and the required co-surfactants and the proportions of each component were determined. The invention also verifies that the novel low-toxicity synergistic nanoemulsion composition has a significant preventive effect on preventing oral precancerous lesions / stopping the progression of oral precancerous lesions.

[0032] This invention is scientifically and rationally designed. Through CCK8 and Calcein-AM / PI cell live / dead double staining experiments and animal model verification, it has been shown that the caprylic / capric acid / polyethylene glycol glycerol ester / RH-40 / PEG-400 composite surfactant... d The limonene / tea tree oil nanoemulsion exhibits lower cytotoxicity than the nanoemulsion prepared by Tween-80. This invention provides a safer and more effective novel formulation for the prevention and treatment of oral precancerous lesions, and has significant practical application value. Attached Figure Description

[0033] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This shows a comparison of the CCK8 results between Example 1 and Comparative Example 1.

[0036] Figure 2 This shows a comparison of the CCK8 results between Example 1 and Comparative Example 2.

[0037] Figure 3 The results of Calcein-AM / PI cell live / dead double staining are shown for Example 1 and Comparative Example 2 after HOK cells were treated with concentrations of 100 μg / mL, 1 μg / mL, and 0.1 μg / mL for 24 h and 48 h, respectively.

[0038] Figure 4 The results of the antibacterial performance test in Example 1 are statistically presented.

[0039] Figure 5 The results of the antibacterial performance test in Example 1 are shown.

[0040] Figure 6 The results of the animal experiment in week 8 are represented. (1) corresponds to group C, (2) corresponds to group M, (3) corresponds to group P1, and (4) corresponds to group P2. Detailed Implementation

[0041] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0042] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0043] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Example 1

[0044] A low-toxicity and high-efficiency d - Limonene nanoemulsion composition (2% effective content), formulation (weight percentage) is as follows: d -Limonene: 1%; Tea tree oil: 1%; Oil phase (MCT): 0.5%; Surfactant (Labrasol) ® : RH-40=3:7): 4.375%; Co-surfactant (PEG-400): 1.46%; The rest: deionized water.

[0045] Preparation method: The components were mixed in proportion and stirred at 28℃ for 20 min. Deionized water was then added dropwise until the system changed from turbid to a transparent and homogeneous nanoemulsion. Stirring was continued for another 30 min to obtain the nanoemulsion. The particle size of the emulsion exhibited a normal distribution, with parallel measurements showing a particle size of 23.5 ± 0.36 nm, a PDI of 0.2723 ± 0.0623, and a Zeta potential of 18.62 ± 5.33 mV.

[0046] Comparative Example 1: Preparation without [specific ingredient] according to the method of Example 1 d - Blank nanoemulsion samples of limonene and tea tree oil are provided.

[0047] Comparative Example 2: Prepared according to CN114558052B d - Comparison sample of limonene nanoemulsion composition (2% effective content): Formula (by weight percentage): d -Limonene: 1%; Tea tree oil: 1%; Oil phase (MCT): 0.5%; Surfactant (Tween-80 : EL-35 = 1:1): 4.375%; Co-surfactant (1,2-propanediol): 1.46%; The rest: deionized water.

[0048] Preparation method: Mix the components in proportion, stir at 28℃ for 20 min, add deionized water dropwise until the system changes from turbid to transparent and homogeneous nanoemulsion, continue stirring for 30 min to obtain nanoemulsion.

[0049] The above-mentioned new low-toxicity d -Limonene nanoemulsion and cytotoxicity of Comparative Examples 1 and 2 (Note: In Examples 1 and 2) d The evaluation methods and results for the prevention of oral precancerous lesions (with equal contents of limonene and tea tree oil) are as follows: First, cell safety evaluation (1) CCK8 experiment: with a cell density of 3×10 5 Cells / well seeding was performed, and the nanoemulsion concentration was set to 1×10⁻⁶. 3 μg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, and OD were measured after incubation for 24 h. 450 Calculate cell viability.

[0050] like Figure 1 As shown, compared with Comparative Example 1 (without essential oil): Cell viability in Example 1 was slightly lower than the control group at 1000 μg / mL and 100 μg / mL (difference within the error range); however, at 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, the viability was significantly higher than the control group, suggesting that low concentrations... d - Limonene is beneficial for cell proliferation and enhances safety.

[0051] like Figure 2 As shown, compared with Comparative Example 2, the survival rate of the novel low-toxicity nanoemulsion was significantly higher at 1000 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL and 0.01 μg / mL than that of the formulation in Comparative Example 2; the nanoemulsion in Comparative Example 2 had a higher survival rate at only 100 μg / mL (possibly related to the lower toxicity of Tween-80 / EL-35 at this concentration), and its overall safety was better.

[0052] (2) Calcein-AM / PI double staining experiment: Nanoemulsions of concentrations 100 μg / mL, 1 μg / mL, and 0.1 μg / mL from Example 1 were co-incubated with normal human oral keratinocytes (HOK cells) for 24 h and 48 h, respectively, and observed under a laser confocal microscope. Figure 3 As shown, the results indicate that both Example 1 and Comparative Example 2 slightly inhibited cell proliferation at the tested concentrations, and their effects were stable within the concentration range. The number of live cells in Example 1 was higher than that of nanoemulsions prepared with existing formulations, consistent with the results of CCK8, further confirming its superior safety.

[0053] Second, antibacterial performance testing Using PBS as a blank control, the antibacterial effects of Example 1 and Comparative Example 1 (2% effective content) against *Escherichia coli* and *Staphylococcus aureus* were tested. Figure 4 , Figure 5 The results show: Escherichia coli group: Example 1 colony count was 0 (inhibition rate 100%), while the control example 1 had a colony count of 2.6 × 10⁻⁶. 5 cfu / mL; Staphylococcus aureus group: Example 1, colony count 1.6 × 10⁻⁶ 5 cfu / mL, the blank comparative example 1 was 2.7 × 10⁻⁶. 5 cfu / mL; Example 1 showed better antibacterial performance than the blank control example 1, and its inhibitory effect on Escherichia coli was better than that on Staphylococcus aureus.

[0054] Third, animal experiments on the prevention of oral leukoplakia. Several Chinese hamsters aged 6-8 weeks and weighing 18-20g were selected, with half males and half females. 0.5% dimethylbenzanthracene (DMBA) was applied to the right cheek pouch of the Chinese hamsters three times a week using a cotton swab. The experiment lasted for 10 weeks (8 weeks of DMBA induction, followed by drug intervention until week 10). The grouping and results are shown in Table 2.

[0055] Table 2. Animal experimental groupings and results for the prevention of oral leukoplakia.

[0056] like Figure 6 As shown, the results indicate that Example 1 is significantly more effective than Comparative Example 1 in preventing DMBA-induced oral precancerous lesions, confirming its high efficiency. Example 2

[0057] A low-toxicity and high-efficiency d - Limonene nanoemulsion composition (2% effective content), formulation (weight percentage) is as follows: d -Limonene: 0.5%; Tea tree oil: 1.5%; Oil phase (MCT): 0.5%; Surfactant (Labrasol) ® : RH-40=8:2): 7.5%; Co-surfactant (PEG-400): 2.5%; The rest: deionized water.

[0058] The preparation method is the same as in Example 1, and the particle size of the emulsion is about 28 nm.

[0059] The product of this invention is mainly used to prevent oral cancer or to stop the progression of precancerous lesions in the oral cavity. It can be used directly or diluted as needed.

[0060] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the present invention. Although detailed descriptions have been provided with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments, and they should all be covered within the protection scope of the claims.

Claims

1. A low cytotoxicity oral cancer preventive composition comprising: d - limonene nanoemulsion comprising the following components: d - limonene, tea tree oil, oil phase, surfactant, co-surfactant, the rest being deionized water or distilled water; characterized in that: The surfactant is Capryol PGMC and polyoxyethylene hydrogenated castor oil RH-40.

2. A low cytotoxicity oral cancer-preventing composition according to claim 1, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized in that: The d - limonene is a natural extract with a purity of 90-99%, the tea tree oil is a natural extract; the oil phase is medium-chain triglycerides, olive oil or soybean oil; the co-surfactant is polyethylene glycol, propylene glycol or ethanol.

3. A low cytotoxicity oral cancer-preventing composition according to claim 2, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized in that: The co-surfactant is PEG-400.

4. A low cytotoxicity oral cancer-preventing composition according to claim 3, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized by the fact that: d - the weight percentage of limonene is 0.5-2%, the weight percentage of tea tree oil is 0.1-1%, the weight percentage of the oil phase is 0.25-5%, the weight percentage of the surfactant is 1-15%, and the weight percentage of the co-surfactant is 1-5%.

5. A low cytotoxicity oral cancer-preventing composition according to claim 4, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized by the fact that: d - limonene 0.4-10%, tea tree oil 0.1-5%, surfactant 3.75-10%, co-surfactant 1.25-3.33%.

6. A low cytotoxicity oral cancer-preventing composition according to claim 5, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized in that: The mass ratio of Capryol PGMC and polyoxyethylene hydrogenated castor oil RH-40 is (1~4):(9~1).

7. A low cytotoxicity oral cancer-preventing composition according to claim 6, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized in that: The mass ratio of surfactant to co-surfactant, i.e. K m values of 7:1 to 1 :1 ; the mass ratio of all surfactants to all oil phases, i.e. SOR values of 1 :1 to 9:

1.

8. A low cytotoxicity oral cancer-preventing composition according to claim 7, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized in that: d - limonene 1%, tea tree oil 1%, MCT 0.5%, surfactant 4.375%, PEG-400 1.46%.

9. A low cytotoxicity oral cancer-preventing composition according to claim 8, wherein the composition is a mouthwash. d - limonene nanoemulsion, characterized in that: The mass ratio of caprylic acid capric acid polyethylene glycol glyceride and polyoxyethylene hydrogenated castor oil RH-40 is 3:7; the SOR value is 7:3, K m value is 3:

1.

10. A low cytotoxicity oral cancer preventive composition according to any one of claims 1 to 9. d - Process for the preparation of a nanemulsion of limonene, characterized in that: Comprising the following steps: (1) First, the d - Limonene, tea tree oil, oil phase, surfactant and co-surfactant are placed in a container in a weight ratio, stirred at 100-2000 r / min for 10-120 min to obtain a mixture; (2) The deionized water or distilled water is weighed according to the weight ratio, and then slowly dripped into the above mixture under stirring. After the dripping is completed, stirring is carried out for 30-120 min, to prepare a nanoemulsion with a particle size of 10-100 nm, which is a low cytotoxicity nanoemulsion for preventing / treating precancerous lesions of oral cancer d - limonene nanoemulsion; Alternatively, by high pressure homogenization method: the material is placed in the high pressure homogenizer according to the weight ratio, under the working pressure of 200-1500 bar, homogenized for 10-120 min, to prepare nanoemulsion with particle size of 10-100 nm, which is low cytotoxicity d - limonene nanoemulsion.

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