Coenzyme Q10 nanoliposomes were prepared by ethanol injection ultrasonication. The encapsulation rate, retention rate, average particle size and the variation of average particle size were used as the response indicators, and the orthogonal test method was applied to optimize the formulation of coenzyme Q10 nanoliposomes and the process of preparation. The optimal formulation was phospholipid:cholesterol:Tween 80:Coenzyme Q10=2.5:0.4:1.8:1.2 (W/W), and the aqueous phase was 0.01 mol/L phosphate buffer (pH 7.4); the optimal preparation conditions were 1 ml of ethanol, stirring time of 10 min, hydration temperature of 45 ℃, and ultrasonic power of 450W.
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The optimized formulation and process produced liposomes with uniform morphology, particle size distribution in the range of 20 300 nm, the average particle size was 68 nm, the encapsulation rate was higher than 95%, and the particle size distribution did not change significantly after four months of storage at 4 ℃, the change of the average particle size was less than 10%, and the retention rate was higher than 90%. The optimized coenzyme Q 10 nanoliposomes had reasonable formulation, simple and feasible process, high encapsulation rate and good stability.
Coenzyme Q 10 is an important active substance in the human body involved in cellular metabolism, but also an important antioxidant and non-specific immune enhancer[1] . o Studies have shown that Coenzyme Q 10 has a good therapeutic and restorative effect on cardiovascular and cerebrovascular diseases such as Alzheimer's disease, diabetes mellitus, hypertension, stroke, arteriosclerosis, Parkinson's disease, etc. Although the total amount of Coenzyme Q 10 present in all human body organs does not exceed 2g, this indicator decreases with age, aging, and increasing diseases. Although Coenzyme Q 10 is present in all organs of the human body, the total amount of Coenzyme Q 10 is not more than 2g, and this indicator will decrease with age, aging of the body, and increase in diseases.
Therefore, both healthy people and patients with diseases need to supplement CoQ10 with healthy food to maintain youthfulness, health, reduce diseases and prolong life. However, due to the large molecular weight of CoQ10 and poor water solubility, the bioavailability of CoQ10 in the small intestine after oral administration of traditional tablets and capsules is relatively low, so how to effectively improve the bioavailability of CoQ10 has become a hot research topic in recent years[2-5] . Therefore, how to effectively improve the bioavailability of coenzyme Q 10 in vivo has become a hot research topic in recent years[2-5] .
In addition, due to the unsaturated double bond in the molecular structure of coenzyme Q10, it is extremely unstable, easily oxidized and decomposed by oxygen and light in the air. Its decomposition is accelerated by heat or metal ions, which often leads to the decrease of coenzyme Q10 content in the products, affecting the quality of the products and the effect of the actual use of the products. It is necessary to protect the coenzyme Q10. Because of the above reasons, new products such as sublingual liposome sprays, Q-gel, NutraDrops, NanoFlow, etc. have been developed overseas. In addition, self-emulsifying carriers [3-4] and nanoparticles [5] have only been reported in the literature.
Liposomes are hydrophilic vesicles composed of phospholipid bilayers. Liposomes have long been used in pharmaceutical and cosmetic applications, and in recent years, they have shown great potential for application in the food industry. This carrier is natural, non-toxic and biodegradable; it can improve the stability of the encapsulated substance, promote the absorption of the encapsulated substance, prolong the action time of the encapsulated substance, and have a targeting effect on the local lesion site; the particle size can be from dozens of nanometers to a few micrometers, and it has a very good dispersibility. When the average particle size of liposomes is controlled to be around 100 nm, they have the special effects of nanoparticles in terms of stability, absorption and in vivo distribution, and are therefore defined as nano-liposomes.
Studies have shown that orally administered nanoliposomes can increase the adsorption on intestinal epithelial cells to prolong the absorption time, increase the transport of drugs through the lymphatic system, and enter the circulation through phagocytosis by intestinal Payer's area M cells [6]. According to the characteristics of nanoliposomes, the use of this special microencapsulation technology for the embedding of nutrient coenzyme Q10 can enhance its stability and water solubility, thus promoting the absorption of coenzyme Q10. It has been reported that the bioavailability and therapeutic efficacy of coenzyme Q10 have been significantly improved after it was formulated into liposomal spray[7] . In China, Zhang Yangmei et al.[8] studied the quality evaluation method of Coenzyme Q10 liposomes. However, there is no report on the preparation method, loading and stability of coenzyme Q 10 nanoliposomes.
Therefore, in order to improve the water solubility and bioavailability of Coenzyme Q 10, the encapsulation rate (EE), retention rate (RR), average particle size (D0.5) and the variation of average particle size (△ D0.5) of the liposomes were used as the indexes in this paper, and the orthogonal experiment design was used to optimize the formulation and the preparation process of Coenzyme Q 10 nanoliposomes based on the one-way test. An orthogonal experimental design was used to optimize the formulation and preparation of coenzyme Q 10 nanoliposomes on the basis of a one-way test.
1 Materials and Methods
1.1 Reagents and Instruments
Egg yolk phospholipids (biochemical reagent) East China Normal University Chemical Plant Tween 80 (chemically pure) Zhejiang Wenzhou Huaqiao Chemical Reagent Co. Coenzyme Q10 (98.0% 101.0%) Nissin Pharmaceuticals Cholesterol (analytically pure) China Pharmaceuticals (Group) Shanghai Chemical Reagent Co.
ZX98-1 Rotary Evaporator Shanghai Instrument and Motor Factory; CL20-B
Freezing Centrifuge Shanghai Anting Scientific Instrument Factory; VCX50 Ultrasonic Processing
SONICS & MATERIALS, USA; UV-1100 Ultraviolet -
Visible Spectrophotometer Beijing Ruili Analytical Instrument Company; Nano-ZS90
Laser Light Scattering Instrument Malvern, UK.
1.2 Methodology
1.2.1 Determination of Factors and Levels of Influence in Orthogonal Experimental Designs
1.2.1.1 Formulation screening
Based on the results of the one-way test, the concentration of phospholipids was set at 12.5 mg/ml, and the four factors (ratio of cholesterol to phospholipids (A), ratio of Tween 80 to phospholipids (B), ratio of coenzyme Q 10 to phospholipids (C) and concentration of NaCl in the hydration medium (D)) that have a greater influence on the embedding effect were selected, and three levels of each factor were taken, and the orthogonal experimental design of the prescription was carried out by using the orthogonal table of L9 (34). The experimental factors and levels are shown in Table 1.
Table 1 Table of factor levels of orthogonal design of L9 (34 ) formulation
Table 1 Factors and levels of L9 (34 ) orthogonal array
formulation design
level (of achievement etc) | factor | |||
A(W/W) | B(W/W) | C(W/W) | D (mol/L) | |
1 | 0.3:2.5 | 1.6:2.5 | 1.2:2.5 | 0 |
2 | 0.4:2.5 | 1.8:2.5 | 1.7:2.5 | 0.07 |
3 | 0.5:2.5 | 2.0:2.5 | 2.1:2.5 | 0.15 |
1.2.1.2 Preparation process conditions
According to the results of the one-way test, the factors that have a greater influence on the embedding effect were selected.
The four factors were: ethanol dosage (a), hydration temperature (b), stirring time (c), ultrasound intensity (d), and each factor was taken at three levels, and the L9 (34) orthogonal table was used to design the orthogonal experiments for the preparation process. The experimental factors and levels are shown in Table 2.
Table 2 L9 (34) process orthogonal design factor level table
level (of achievement etc) | factor | |||
a(ml) | b ( ) | c(min) | d(W) | |
1 | 1 | 45 | 10 | 350 |
2 | 2 | 50 | 20 | 400 |
3 | 3 | 55 | 30 | 450 |
1.2.2 Preparation of coenzyme Q10 nanoliposomes by ethanol injection - ultrasound method
According to the formula, weighed the appropriate amount of phospholipids, cholesterol, Tween 80 and coenzyme Q10, added the appropriate amount of anhydrous ethanol, water bath to dissolve (30min, 55 ℃), and then injected into 20 ml of phosphate buffer (pH 7.4) with a syringe, hydrated for a certain period of time after the removal of ethanol by rotary evaporation, cooling, ultrasonication in iced water bath for 4min (1s/1s), freezing and centrifugation ( 11000 X g, 30min, 4 ℃) filled with N 2 sealed, stored in the refrigerator under refrigeration. After the reaction, the sample was cooled by rotary evaporation to remove ethanol, sonicated in ice water bath for 4 min (1s/1s), and then frozen by centrifugation (11000 X g, 30 min, 4 ℃). Since light has a certain effect on the stability of Coenzyme Q10, all operations should be protected from light.
1.2.3 Determination of Coenzyme Q 10 liposome particle size
A Nano-ZS90 laser light scattering instrument was used. The prepared samples were loaded into a cuvette, held at 2.5 °C, and measured. The average particle size (D0.5) and the polydispersity index (PDI) of the particle size distribution were recorded.
1.2.4 Testing of product quality
where: amount of embedded coenzyme Q 10 = total coenzyme Q 10 content free coenzyme Q 10 content.
The total content of coenzyme Q10 was determined by Tween 80 solubilization-ultraviolet spectrophotometry [9 ].
Determination of free coenzyme Q 10: Organic solvent washing and ultraviolet spectrophotometric method[9] .
1.2.5 Statistical processing
The results were substituted into S PSS software and analyzed by ANOVA test.
2 Results and analysis
2.1 Optimization of Coenzyme Q 10 nanoliposome formulation
The results of the orthogonal test of the formulation are shown in Table 3, and the analysis of polarity is shown in Fig. 1. By comparing the size of the R value of the polar deviation, it can be seen that different factors play different roles in the test, and by comparing the size of the K value, it is possible to know the extent of the influence of the factor at different levels of the test. Therefore, using the encapsulation rate and average particle size as the measurement index for visual analysis, within the selected test range, the core material coenzyme Q 10 (C factor) has the greatest influence on the amount of coenzyme Q 10, the higher the content, the lower the encapsulation rate, and the larger the average particle size.
Cholesterol (factor A) and Tween 80 (factor B) were the next most influential factors, with mean particle size increasing with increasing cholesterol percentage, encapsulation rate decreasing slightly with increasing cholesterol percentage, and Tween 80 having the opposite effect. For both retention and change in mean particle size, cholesterol (factor A) had the greatest effect, with higher proportions resulting in less change in mean particle size over the range selected, followed by NaCl concentration (factor D), with higher salt concentrations resulting in greater change in mean particle size.
The analysis of variance (ANOVA) is shown in Table 4. From the statistical results, it can be seen that the proportion of coenzyme Q 10 in the core material (factor C) had a significant effect on the mean particle size (p<0.1), and the proportion of cholesterol (factor A) had a significant effect on the degree of variation of the mean particle size (p<0.05), and the effects of these factors on the encapsulation rate and retention rate were not significant.
According to the viewpoint of ANOVA, the optimal level should be determined only for the significant factors, and the appropriate level can be determined for the other factors according to the actual needs. The other factors can be determined as necessary. Since the results of the encapsulation rate did not differ much, in order to obtain coenzyme Q10 nanoliposomes with appropriate particle size and good stability of the product (measured by the retention rate and the degree of change of the average particle size), the optimal formulation was C1A2B2D3, i.e., phospholipid:coenzyme Q10:cholesterol:tween 80=2.5:1.2:0.4:1.8 (W/W), and 0.01 mol/L phosphate buffer (pH 7.4) was used to determine the optimal level of the product. phosphate buffer (pH 7.4) as the hydration medium.
The core material, coenzyme Q 10, has a significant influence on the embedding effect and stability of the product. Since coenzyme Q 10 is insoluble in water, results to date indicate that it is mainly localized in the middle layer of the bilayer (synthetic phospholipid bilayer model) and partially in the aggregated state[10-11] . The volume of the middle layer of the bilayer is limited, so the encapsulation rate decreases with the increase of the proportion of coenzyme Q10 when the lipid bilayer is saturated with certain concentrations of the wall materials (e.g., phospholipids, cholesterol, and Tween 80). The results of extreme variance analysis also showed that the incorporation of coenzyme Q10 into the lipid bilayer led to an increase in the average particle size of liposomes.
The main reason for this trend is that Coenzyme Q 10 is a rigid crystal at room temperature, and when it is embedded in the liposome bilayer, it can enhance the mechanical strength of the system, so it is more difficult to break the liposomes to the same extent under the same process conditions. Liposomes are thermodynamically unstable system, and will be destabilized by aggregation and fusion during storage. It was found that the average particle size of blank liposomes increased from 117.5 nm to 260.9 nm after three months of storage at 4, whereas the average particle size of nano-liposomes embedded with coenzyme Q10 only increased by a few nanometers under the same storage conditions, which indicated that the core material coenzyme Q10 could effectively inhibit the aggregation and fusion of liposomes. This indicates that the core material coenzyme Q10 can effectively inhibit the aggregation and fusion of liposomes.
In addition, the malondialdehyde content of coenzyme Q10 nanoliposome system remained unchanged (~0.014 μg/mg PL) after three months of storage at 4, whereas the malondialdehyde content of the blank liposomes increased from (0.032±0.006) μg/mg PL to (0.082 0.007) g/mg PL, so that the antioxidant effect of coenzyme Q10 can be used as an effective antioxidant to prevent the oxidation of phospholipids, the main wall material in liposomes, and cholesterol as an effective auxiliary wall material to liposomes as a membrane fluidity regulator, which can inhibit the rotation of lipid molecules and prevent oxidation of phospholipids. Therefore, coenzyme Q10 has an antioxidant effect and can be used as an effective antioxidant to prevent the oxidation of phospholipids, which is the main wall material of liposomes, and cholesterol, as an effective auxiliary wall material, acts as a regulator of the membrane fluidity of liposomes, which can inhibit the rotational isomerization of lipid molecules, reduce the fluidity of membranes, and reduce the aggregation and fusion of liposomes during the storage period.
However, the introduction of cholesterol into the lipid bilayer enhances the rigidity of the system, and the decrease in the encapsulation rate due to the addition of too much cholesterol may be related to the mechanism of encapsulation of the core material in the lipid bilayer. Since the core material, coenzyme Q 10, is embedded between the bilayer membranes, the larger the membrane area is, the more the core material is encapsulated. The addition of cholesterol increases the rigidity of the membrane and reduces the curvature, thus the total surface area of the liposomes formed in the same mass of lipid decreases, which leads to a decrease in the encapsulation amount. The increased rigidity of the bilayer reduces the leakage of coenzyme Q10 and improves the retention rate.
Although the effect of Tween 80 on the quality index of CoQ 10 nanoliposomes was not significant at any of the levels selected in the orthogonal test, the results of the one-way test at a wide range of levels showed that the incorporation of Tween 80 into the preparation of liposomes not only reduced the particle size of CoQ 10 liposomes, but also improved the degree of change in the size of the liposomes during the storage process, and mainly inhibited the leakage of the core material, CoQ 10. Coenzyme Q 10 leakage from the core material was inhibited. Tween 80 has one long hydrophobic chain and three short hydrophilic chains. Tween 80 doped in the liposome bilayer is physically adsorbed on the surface of the lipid bilayer, and its polyoxyethylene group extends out from the lipid bilayer and densely covers the surface of the bilayer, forming a hydrophilic phase with a certain thickness.
Tween 80 adsorbed on the outer surface of the lipid bilayer promotes the increase of liposome curvature, while Tween 80 adsorbed on the inner surface of the lipid bilayer has the opposite effect [12]. Since more Tween 80 was adsorbed on the outer surface of the lipid bilayer, the incorporation of Tween 80 into the lipid bilayer helped to obtain liposomes with smaller particle size. When two stereospecific liposomes are in close proximity, the chemical potential energy between the liposomes decreases due to the presence of water-soluble chains, and therefore, due to osmosis, a large amount of water enters the liposomes and separates them[13] .
2.2 Optimization of Coenzyme Q 10 nanoliposome preparation process
The results of the process orthogonal test are shown in Table 5, and the analysis of extreme deviation is shown in Fig. 2. o From the extreme deviation R, it can be seen that, for the three indexes of retention rate, average particle size and the degree of change of average particle size, the ultrasonic intensity (d factor) had the greatest effect, increasing the ultrasonic intensity, the average particle size decreased significantly, the retention rate of the core was increased during the storage period, and the degree of change of the average particle size was reduced accordingly, and the effect of the ethanol dosage (a factor) was next to that, and both average particle size and the degree of change of average particle size during storage increased with the increase of ethanol dosage. The effect of ethanol dosage (factor a) was the second most important factor, the average particle size and the degree of change of the average particle size during storage increased with the increase of ethanol dosage, and the effects of hydration time (factor c) and hydration temperature (factor b) were less important. The effect of hydration time (factor c) and hydration temperature (factor b) was less significant. The effect of encapsulation rate was less significant, and decreased slightly with the increase of hydration time.
The ANOVA is shown in Table 6.
From the statistical results, it can be seen that the hydration temperature and hydration time (factors b and c) had a very significant effect on the encapsulation rate (p < 0.05), the ultrasonic power (factor d) had a very significant effect on the retention rate of coenzyme Q10 in the core material (p < 0.05), and the ethanol dosage and the ultrasonic power (factors a and d) had a significant effect on the mean particle size (p < 0.1), and none of these factors had a significant effect on the degree of change of the mean particle size. The effects of these factors on the degree of change in the mean particle size were not significant. Therefore, the optimal process conditions were determined as d3 a 1 c 1b 1 , i.e., ultrasonic power of 450 W, ethanol dosage of 1 ml, hydration time of 10 min and hydration temperature of 45 ℃.
Coenzyme Q 10 is insoluble in water and soluble in ethanol. After comparison, it was found that ethanol injection was a suitable method for the preparation of Coenzyme Q 10 nanoliposomes, with high encapsulation and retention rates, and the average particle size of liposomes was only about 157 nm even without sonication. However, ethanol interferes with the ordering of the liposome bilayer and improves the mobility, which may lead to the aggregation and fusion of liposomes, so most of the ethanol should be removed as much as possible. After the liposomes were broken by ultrasonication, the average particle size was significantly reduced to several tens of nanometers, and the stability was also improved.
The equipment used in the injection method is simple, easy to operate, reproducible and convenient for large-scale industrialized production. The prepared liposomes are small and homogeneous, with high encapsulation rate and good stability, in which the temperature has a great influence on the stability of the finished products, so low temperature storage is required.
2.3 Validation experiments
The encapsulation rate of three batches of coenzyme Q 10 nanoliposomes prepared according to the optimized formulation and process was (97 1.8)% After four months of light storage at 4, the average particle size increased by only a few nanometers, and the retention rate of coenzyme Q 10 in the core was more than 90% (see Table 7). After crushing, the average particle size was significantly reduced to a few tens of nanometers, and the stability was also improved. The equipment used in the injection method was simple, easy to operate, reproducible, and convenient for large-scale industrial production. The prepared liposomes were small and homogeneous, with high encapsulation rate and good stability, in which the temperature had a great influence on the stability of the finished products, so low temperature storage was required.
2.3 Validation experiments
The encapsulation rate of three batches of coenzyme Q 10 nanoliposomes prepared according to the optimized formulation and process was (97 1.8)% After four months of light storage at 4, the average particle size increased by only a few nanometers, the retention of coenzyme Q 10 in the core was greater than 90%, the distribution of the particles did not change significantly, and the degree of change in the average particle size was less than 10%, with retention rate higher than 90%.
3 CONCLUSIONS
The formulation and preparation process of coenzyme Q 10 nanoliposomes were optimized by orthogonal experiments. The optimal formulation was phospholipid:cholesterol:Tween 80:coenzyme Q10=2.5:0.4:1.8:1.2 (W/W), and the aqueous phase was 0.01 mol/L phosphate buffer (pH 7.4); the optimal preparation conditions were 1 ml of ethanol, stirring time of 10 min, hydration temperature of 45 ℃, and ultrasonic power of 450 W. The liposomes were homogeneous, with particle size between 20 and 300 nm. The optimized formulation and process produced liposomes with uniform morphology, particle size between 20 and 300 nm, average particle size of 68 nm, encapsulation rate higher than 95%, and stored at 4 for 3 months, the distribution of particle size did not change significantly, and the degree of change of the average particle size was less than 10%, and the retention rate was higher than 90%. The optimized Coenzyme Q 10 nanoliposomes were reasonably formulated, simple and feasible, with high encapsulation rate and good stability.
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