Coenzyme Q10, also known as ubiquinone, is a fat-soluble natural vitamin that exists in many organisms. It is a natural antioxidant produced by the cells themselves, which can improve the immunity of the organism, and it is an indispensable active substance involved in the metabolism in the human body. In the present study, we selected suitable oil phase, emulsifier and co-emulsifier, and used pseudo-ternary phase diagram to prepare coenzyme Q10 nanoemulsion formulation, and evaluated its stability through relevant experiments, in order to explore the development of a coenzyme Q10 nanoemulsion formulation with stable quality and good solubility.
1 Material
1.1 Main reagents: Coenzyme Q10 (Zhejiang Xinchang Pharmaceutical Factory); Isopropyl myristate (Yongxin Chemical Co., Ltd.); Soy lecithin (Shanghai Avet Pharmaceutical Science and Technology Co., Ltd.); Polyglycerol fatty acid ester (Yangzhou Bosch Chemical Co., Ltd.); Polysorbate 80 (Guangzhou Hechengrong Chemical Co., Ltd.); Propylene Glycol (Guangzhou Chemical Industry Co., Ltd.); Methylene Blue (Ningbo Haicheng Chemical Industry Co., Ltd.); Sudan Red (Ningbo Haicheng Chemical Industry Co., Ltd.).
1.2 Main Instruments: BS214S Electronic Balance (Made in Germany); Zetasi - zer3000HS Grain Sizer (Malvern, UK); Constant Temperature Magnetic Stirrer (Hangzhou Instrument Factory)
2 Methodology and results
2.1 Determination of oil phase, emulsifier and co-emulsifier
It was confirmed that the solubility of coenzyme Q10 in isopropyl myristate was the largest among the oils with different carbon chain lengths, so isopropyl myristate was selected as the oil phase. In addition, three emulsifiers, namely soy lecithin, polyethylene glycol caprylate and polysorbate 80, as well as polyglycerol fatty acid ester and propylene glycol co-emulsifier, were taken separately, and mixed in the ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9 for each group, and then added into isopropyl myristate in the oil phase, and then vibrated.
When the binary solution becomes clear during the titration, the formation of nano-emulsions is determined by visual observation of emulsification and Zeta particle size determination. The amount of S/C titrant consumed for the formation of nano-emulsions is recorded, and the percentages of oil phase, water phase, emulsifier and co-emulsifier in the nano-emulsion system are calculated. The group that could form nano-emulsions and required less amount of emulsifier and co-emulsifier and the drug loading capacity for clinical application was selected. The results showed that the formation of nanoemulsions was possible in lesser quantities when polyethylene glycol glyceryl caprylate was used as emulsifier and polyglycerol fatty acid esters were used as co-emulsifiers.
2.2 Preparation of Coenzyme Q10 Nano-Emulsion
After determining the composition of the coenzyme Q10 nano-emulsion, polyethylene glycol glyceryl caprylate and polyglycerol fatty acid esters were mixed according to certain ratios to form a mixture, coenzyme Q10 was dissolved in isopropyl myristate as the oil phase, and the coenzyme Q10 was added into the mixture of emulsifier and co-emulsifier according to the determined ratios in the proportions of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and finally the distilled water was titrated gradually until the formation of coenzyme Q10 nano-emulsion, and the amount of each phase in the formation of the nano-emulsion was recorded, as shown in Table 1. 8, 1:9 and finally titrated distilled water until the formation of Coenzyme Q10 nano-emulsion and the amount of each phase during the formation of nano-emulsion was recorded as shown in Table 1.
Table 1 Recording the amount of each phase in the formation of nano-emulsion
experiment number | Oil phase %() | Aqueous phase %() | Emulsifier/co-emulsifier %() | in the end |
1 | 81.0 | 2.7 | 16.3 | turbidity |
2 | 67.2 | 7.1 | 25.7 | turbidity |
3 | 54.8 | 13.2 | 32.0 | turbidity |
4 | 43.7 | 18.2 | 38.1 | turbidity |
5 | 35.2 | 25.2 | 39.6 | turbidity |
6 | 31.1 | 27.2 | 41.7 | turbidity |
7 | 22.5 | 35.7 | 41.8 | turbidity |
8 | 11.3 | 40.5 | 48.2 | Formation of nano-emulsions |
9 | 4.3 | 34.6 | 61.1 | Formation of nano-emulsions |
10 | 1.7 | 32.8 | 65.5 | Formation of nano-emulsions |
2.3 Type identification of Coenzyme Q10 nano-emulsions
The nano-emulsions were identified as oil-in-water O(/W) or water-in-oil W(/O) by staining method, which was based on the diffusion speed of the red oil-soluble dye, Sudan IV, and the blue water-soluble dye, methylene blue, in the nano-emulsions. The results showed that the Coenzyme Q10 nano-emulsions were oil-in-water nano-emulsions.
2.4 Stability experiments
2.4.1 Light experiments:
Coenzyme Q10 nano-milk was placed in a flat glass bottle with frosted mouth and placed under 4500 lx light, samples were taken on the 5th and 10th day and the content of coenzyme Q10 was detected by high performance liquid chromatography (H(PLC)), the results showed that the appearance of coenzyme Q10 nano-milk was still light yellow and transparent, and there was no turbidity or sedimentation, and the difference of coenzyme Q10 content was not statistically significant, which indicated that coenzyme Q10 nano-milk was stable and stable. This indicates that the coenzyme Q10 nano-emulsion is stable. See Table 2.
Table 2 Results of light experiments
Stability test conditions | Time d() | exterior condition | Coenzyme Q10 Content %() |
Light 4 (500 lx) | 0 | Yellowish transparent | 83.7 |
5 | Yellowish transparent | 82.1 | |
10 | Yellowish transparent | 80.4 |
2.4.2 Temperature experiments:
The samples were taken at 4, 25, 40 and 60 ℃ on the 5th and 10th days, and the coenzyme Q10 content was detected by HPLC. It was found that the appearance of the coenzyme Q10 nano-emulsion was still light yellow and transparent, and no turbidity or precipitation was detected, and there was no statistically significant difference in the coenzyme Q10 content, which indicated that the coenzyme Q10 nano-emulsion had good stability. See Table 3.
Table 3 Results of temperature experiments
Stability test conditions | Time d() | exterior condition | Coenzyme Q10 Content %() |
4 °C | 0 | Yellowish transparent | 88.5 |
5 | Yellowish transparent | 87.4 | |
10 | Small amount precipitated | 85.3 | |
25 °C | 0 | Yellowish transparent | 88.6 |
5 | Yellowish transparent | 89.7 | |
10 | Yellowish transparent | 87.5 | |
40 °C | 0 | Yellowish transparent | 86.5 |
5 | Yellowish transparent | 85.8 | |
10 | Yellowish transparent | 83.2 | |
60 °C | 0 | Yellowish transparent | 91.1 |
5 | Yellowish transparent | 87.3 | |
10 | Yellowish transparent | 85.7 |
2.4.3 Air exposure test:
The coenzyme Q10 nano-emulsion was exposed to room temperature of 20~22 ℃ and relative humidity of 50%~75% for 10 days, and then the samples were taken on the 5th and 10th days for observation and detection of coenzyme Q10 content by HPLC. It was found that the appearance of coenzyme Q10 nano-emulsion was still light yellow and transparent, no turbidity or precipitation was found, and the difference of coenzyme Q10 content was not statistically significant, which indicated that the coenzyme Q10 nano-emulsion was stable. See Table 4.
Table 4 Air exposure test results
Stability test conditions | Time d() | exterior condition | Coenzyme Q10 Content %() |
Exposed air test | 0 | Yellowish transparent | 92.7 |
5 | Yellowish transparent | 89.1 | |
10 | Yellowish transparent | 86.2 |
2.4.4 Accelerated testing:
Coenzyme Q10 nano-emulsion was taken and placed in a centrifuge tube fitted with a high-speed centrifuge, and centrifuged at 13,000 r/min for 30 min. There was no delamination, indicating that Coenzyme Q10 nano-emulsion was stable.
3 Discussion
Nano-emulsion [1] is a colloidal dispersion system formed by dispersing emulsion droplets with a particle size of 10-100 nm in another liquid, and the droplets are mostly spherical, transparent or translucent. Nano-emulsion, also known as microemulsion M (E), consists of oil phase, water phase, surfactant and co-surfactant, as long as the composition of the four phases is appropriate, it can be formed into a homogeneous, transparent or slightly emulsified liquid, which is a thermodynamically stable system. According to the structure, they can be divided into oil-in-water, water-in-oil and bicontinuous nanoemulsions. Since nanoemulsions as carriers have good local drug delivery and transdermal properties, the study of nanoemulsions as transdermal drug delivery systems has become a hot spot of pharmacy research since the 1990s.
Nano-emulsions have several advantages over other carriers for transdermal drug delivery (including liposomes):
①It is an isotropic transparent liquid, which can not be stratified even after hot-pressure sterilization or centrifugation, and is a thermodynamically stable system;
② The process is simple, the preparation process does not require special equipment, can be formed spontaneously, nano-emulsion particle size is generally 10-100 nm;
③ It has slow release and targeting effects;
④ Many peptide drugs are formulated as nano-emulsions to provide a protective effect against the drug;
⑤ Blank nanoemulsion itself has antibacterial effect, so it has strong killing effect on Staphylococcus aureus and Pseudomonas aeruginosa on the surface of the skin.
In addition, the most prominent feature of nanoemulsion drug carriers compared with other systems is that they can improve the stability of drugs and solubilize drugs. 3] found that the drugs can be wrapped in nanoemulsion droplets, avoiding the contact with the continuous phase, which can protect the unstable drugs, and no matter it is water-soluble drugs or fat-soluble drugs, the solubilization of drugs in nanoemulsions can be achieved to a large extent, and solubilization in nanoemulsions can lead to a high concentration of drugs in the nanoemulsion droplets, which is very meaningful for the effective release and improvement of the bioavailability of drugs. The solubilization of drugs in nanoemulsions can make the concentration of drugs in small droplets of nanoemulsions very high, which is very meaningful for the effective release of drugs and the improvement of the bioavailability of drugs.
Coenzyme Q10 is easy to decompose and unstable when exposed to light, and the content of the drug decreases significantly under light conditions; in addition, Coenzyme Q10 is fat-soluble and difficult to dissolve in water, so it is difficult to be absorbed and utilized when added directly. The properties of nanoemulsion carrier provide us a good idea for the application of coenzyme Q10. Therefore, we prepared coenzyme Q10 nanoemulsion by using nanoemulsion as the delivery carrier of coenzyme Q10, which was confirmed to be oil-in-water nanoemulsion through the authentication.
In addition, various experiments have proved that the stability of coenzyme Q10 nano-emulsion has been greatly improved. After the nano-emulsion carrier treatment of coenzyme Q10, it is difficult for coenzyme Q10 to come into contact with the external environment, avoiding the influence of the external environment on coenzyme Q10, and avoiding the influence of light and temperature on coenzyme Q10, which greatly improves the stability of the whole system of the product.
In addition, the nanoemulsion technology used in this paper to prepare a nanoemulsion preparation of coenzyme Q10 can increase its solubility in the aqueous phase and the concentration of the active ingredient per unit volume, so as to increase the strength of the drug effect; moreover, the nanoemulsion of coenzyme Q10 nanoemulsion preparation has a small size, so that the dispersion of the contained active ingredient is increased, which is easily absorbed and utilized by the body, and thus the bioavailability of the components of coenzyme Q10 can be greatly improved. However, more basic research is needed to find emulsifiers and co-emulsifiers that can promote the formation of nanoemulsions with fewer toxic side effects in order to make coenzyme Q10 nanoemulsions more suitable for clinical application.
It is believed that with the development of pharmaceutical technology and the improvement of raw materials, we have prepared coenzyme Q10 nano-emulsions using nano-emulsions as the delivery vehicle of coenzyme Q10, which were confirmed to be oil-in-water nano-emulsions through the authentication. In addition, the stability of coenzyme Q10 nano-emulsions has been greatly improved by various experiments. The coenzyme Q10 treated with the nanoemulsion carrier makes it difficult for the coenzyme Q10 to come into contact with the external environment, thus avoiding the influence of the external environment on the coenzyme Q10, and the influence of light and temperature on the coenzyme Q10 can be avoided, which greatly improves the stability of the whole system of the product.
In addition, the nanoemulsion technology used in this paper to prepare a nanoemulsion preparation of coenzyme Q10 can increase its solubility in the aqueous phase and the concentration of the active ingredient per unit volume, so as to increase the strength of the drug effect; moreover, the nanoemulsion of coenzyme Q10 nanoemulsion preparation has a small size, so that the dispersion of the contained active ingredient is increased, which is easily absorbed and utilized by the body, and thus the bioavailability of the components of coenzyme Q10 can be greatly improved. However, in order to make Coenzyme Q10 nanoemulsions more clinically applicable, more basic research is needed to find emulsifiers and co-emulsifiers that can promote the formation of nanoemulsions with less toxic side effects. It is believed that with the development of pharmaceutical technology and the updating of raw materials, the nano-emulsion delivery system will be more and better applied in the pharmaceutical field.
References
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