Preparation and in vitro performance evaluation of resveratrol for oral self-microemulsion

The purpose of this study was to improve the solubility of resveratrol (Res) by a self-microemulsifying drug-delivery system (SMEDDS). Through a solubility experiment, the pseudoternary phase diagram and ternary phase diagram were used to optimize the Res SMEDDS formula. The optimum formulation consisted of 5% IPM, 20% PEG400, and 65% Cremophor RH40. The water solubility, stability, in vitro release and antioxidant activity of the Res SMEDDS were characterized. The Res solubility in the SMEDDS was at least 1,000 times compared to that in water. The average droplet size of the microemulsion was 28.00±1.67 nm and uniform distribution. The Res SMEDDS should be stored at low temperature and in the dark to avoid light conditions. Res SMEDDS was able to improve the in vitro release rate of Res, and the in vitro release of Res from Res SMEDDS was significantly faster that of Res powder and unaffected by pH value of media. Antioxidant assays showed that antioxidant activities of Res in Res SMEDDS were unaffected compared to Res powder. Cytotoxicity study indicated that Res SMEDDS at the concentration of less than 100 μM was safe. These results demonstrated the potential use of Res SMEDDS for oral administration of Res.

Self-microemulsifying drug delivery systems (SMEDDS) are isotropic mixtures of oils, hydrophilic emulsifiers and co-emulsifiers. SMEDDS possess thermodynamic stability and are spontaneously emulsified into droplets of size in the range of 10-100 nm under slight stirring [13]. The SMEDDS is used for the improvement of the bioavailability of poorly soluble drugs based on high stability, low viscosity and simple preparation [14][15][16]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Characterization of the SMEDDS Appearance. The Res SMEDDS was serially tenfold diluted (10, 100, and 1000 times) with deionized water and then the concentration was assessed by comparing with the appearance of the same concentration of Res solution.
Morphology, size distribution and zeta potential. The blank SMEDDS and Res SMEDDS was diluted 100 times with deionized water to obtain the emulsion, and the particle size distribution was determined immediately by a Malvern laser particle size analyzer. TEM analysis was performed to determine the microstructure of the Res emulsion with the method reported by Chen Y et al [15].

Stability study
The light stability and thermal stability of the Res SMEDDS were studied with reference to the relevant provisions of Appendix XIXC "Stability Testing of Drug Substances and Products" of the Chinese Pharmacopoeia (2015 edition).
In vitro drug release. In vitro release of the Res SMEDDS and Res was tested by the method of Pineros et al [18] with some modifications. Briefly, a 900-mL solution composed of hydrochloric acid solution (pH = 1.2), phosphate buffer (pH = 6.8) and phosphate buffer (pH = 7.4) was used as dissolution medium with stirring at 50 r/min for 37˚C. An aliquot (5 mL) of the sample was collected at 5, 10, 20, 30, 45, and 60 min. At the same time, an equivalent volume (5 mL) of fresh dissolution medium was added to compensate for the removed volume. The sample was filtered through a 0.45-μm filter and the concentration of Res was measured by UV spectrophotometry. The release of the Res SMEEDS and Res at different pH values was examined with the same quantity of drug.
DPPH Free radical scavenging experiment. The DPPH free radical scavenging of the Res SMEEDS and Res was analyzed by the method of Pápay et al. with slight modifications [22]. Briefly, a 79-mg/L DPPH� ethanol solution was freshly prepared and protected from light. Solutions of Res SMEDDS and Res at various concentrations (200, 400, 600, 800, and 1000 μg/L) were prepared in ethanol, and 0.5 mL of each of these samples with different concentrations was mixed with 9.5 mL of DPPH� solution, and the mixture was shaken at 37˚C for 1 h in the dark. The absorbance at 517 nm was determined with a UV/Vis spectrophotometer, the DPPH� scavenging rate was calculated according to the following equation (Eq 1): Where A blank is the absorbance of the DPPH� solution, and A Sample is the absorbance of the sample. All measurements were performed in triplicate. ABTS Free radical scavenging experiment. The free radical scavenging activity was measured by the ABTS +� method as described previously with slight modifications [23]. The ABTS stock solution was prepared by dissolving it in water to a concentration of 3.84g/L. The ABTS free radical (ABTS +� ) was prepared by reacting the ABTS stock solution and 1.34 g/L potassium persulfate at a volume ratio of 1:1, and the mixture was stored in the dark at room temperature for 12 h before use. The blue-green ABTS +� solution was adjusted to an absorbance of 0.70±0.02 at 734 nm with additional water. Solutions of Res SMEDDS and Res at concentrations (40, 60, 80, 100, and 200 μg/L) were prepared in ethanol, 0.5 mL of each of these samples with different concentrations was added to 9.5mL of ABTS +� solution, and the mixture was shaken at 37˚C for 1h in the dark. The absorbance was determined at 734 nm on a UV/Vis spectrophotometer, the ABTS +� scavenging rate is calculated according to Eq 1.
Where A blank is the absorbance of the ABTS +� solution, and A Sample is the absorbance of the sample. All measurements were performed in triplicate. Cytotoxicity assays. The cytotoxicity of Res SMEDDS and Res was determined by a MTT kit according to the manufacturer's instructions. Briefly, 5×10 4 cell/mL of PC12 cells suspended in DMEM SH30022.01 medium (100μL) containing 10% fetal bovine serum and 5% FBS were seeded into 96-well plates. 100μL containing 10% MTT solution was added to each well, and the cells were incubated at 37˚C for 4 h. Carefully remove the culture solution in the well and add 100 μL of DMSO to each well to dissolve the crystals. The absorbance was then measured at 570 nm. The cytotoxicity of the Res SMEDDS and Res and was proceed through indirect contact testing according to MTT assay of ISO 10993-5-2009, Calculate the relative growth rate (RGR) of the sample according to Eq 3, and the cytotoxicity fraction of the Res SMEDDS and Res was evaluated according to Table 1.
Where OD Test is the absorbance of the experimental group, and OD Control is the absorbance of the control group. All measurements were performed in four times.

Preliminary screening of the SMEEDS formula components
The SMEEDS excipient should have excellent solubilization capacity for the drug, which is essential for allowing presentation of the drug in SMEEDS, and excellent compatibility between emulsifiers and co-emulsifiers was found to be beneficial to the formation of small particle size emulsion [18,19].
Compared with the mixture of oil, emulsifier, and co-emulsifier, Res exhibited a UV typical maximal absorption peak at 305 nm (Fig 1).The solubility of Res in the oil phases, emulsifier and co-emulsifier are listed in Table 2. There was no significant difference in the solubility of Res in the three oil phases measured (P>0.05), but IPM was more compatible with the Table 1. Cytotoxicity grades and corresponding relative growth rates.
Grades 0 and 1 were considered to be non-cytotoxic, grade 2 was mildly cytotoxic, grades 3 and 4 were moderately cytotoxic, and grade 5 was markedly cytotoxic.
https://doi.org/10.1371/journal.pone.0214544.t001 emulsifiers than corn germ oil and ethyl oleate (Table 3). Accordingly, IPM was selected as the oil phase. Compared with Tween 20 and Tween 60, Cremophor EL and Cremophor RH40 have a stronger emulsifying capacity due to their larger number of ethylene oxide (EO), thus they are more compatible with the three oil phases [24]. Besides, Res has high solubility in Cremophor EL and Cremophor RH40, therefore, Cremophor EL and Cremophor RH40 were chosen as the alternative emulsifiers for subsequent comparisons. Among the co-emulsifiers, Res has relatively high solubility in anhydrous ethanol, PEG400 and propylene glycol, therefore, they were selected as preliminary co-emulsifiers.

Screening of emulsifiers and co-emulsifiers
The co-emulsifier can increase the fluidity of the oil-water interface film and reduce the surface tension of the oil-water interface [25], which is beneficial to the formation of the microemulsion, the larger the microemulsion region is, the stronger the emulsifying ability is [26]. When IPM was used as the oil phase, the pseudoternary phase diagram of the different co-emulsifiers and emulsifiers are shown in Fig 2. Cremophor RH40 or Cremophor EL used as emulsifier, and PEG400 used as co-emulsifier (Fig 2A and 2D) with moderate molecular weight exhibited excellent ability to assist the emulsification of microemulsions compared to shorter anhydrous ethanol (Fig 2B and 2E) and propylene glycol (Fig 2C and 2F), Therefore, PEG400 was chosen as co-emulsifier. Although both Cremophor RH40 (hydrophilic-lipophilic balance (HLB) = 14-16) and Cremophor EL (HLB = 12-14) could form O/W microemulsions with the same co-emulsifier (PEG400), there was quite a difference in their emulsification ability. As shown in Fig 2A and  2D, the O/W microemulsion region with Cremophor RH40 (HLB = 14-16) is larger than that with Cremophor EL (HLB = 12-14), because the larger the HLB value of the emulsifier is, the stronger the emulsifying capacity is. Moreover, the toxicity of Cremophor RH40 is smaller [27]. Therefore, Cremophor RH40 was selected as the emulsifier. Based on the above data, the oil phase, emulsifier and co-emulsifier of SMEDDS formulation were IPM, Cremopher RH40 and PEG400, respectively.

Self-microemulsion phase diagram construction and formula optimization
Self-microemulsification efficiency refers to the capacity of the SMEDDS to spontaneously form or disperse into a homogeneous microemulsion when the SMEDDS were added to water under mild agitation. The self-emulsifying equilibrium time and droplet size were used to assess self-microemulsification efficiency [21]. As shown in Fig 3, the highest content of oil phase in the SMEEDS is up to 40%, but the content of oil can not be too close to the critical point of the SMEEDS, because the change of the external environment may cause the SMEEDS critical point to shrink, which may lead to instability of the SMEEDS. In addition, when the content of oil was more than 15%, the emulsifying equilibrium time exceeded 2 min, which indicated that the self-emulsifying efficiency had decreased (Fig 3). Therefore, it is reasonable to fix the oil percentage at 15%. The ratio of emulsifier to co-emulsifier was very effective for a stable and efficient SMEDDS formation. There was no significant difference for the solubility of Res in PEG400 and Cremophor RH40 (P>0.05) ( Table 2). Therefore, the change of the content of emulsifier and coemulsifier had no obvious effect on the drug loading of Res SMEDDS under the condition of fixed oil phase content, and the drug loading was approximately 9% (w/w). In order to prevent the precipitation of drugs in the storage process, the drug loading of Res SMEDDS was 5% (w/ w) according to previous studies [15]. The change of the percentage of emulsifier (Cremophor RH40) and co-emulsifier (PEG400) will affect the efficiency of self-emulsification. With the increase of the percentage of co-emulsifier (PEG400), the emulsification time was significantly reduced (P<0.05) ( Table 4). When the content of PEG400 in the SMEDDS increased from 5 to 20%, there were no obvious differences in droplet size. However, when the content of PEG400 increased from 20 to 35%, the droplet size increased from 27.69 nm to 812 nm. Excessive amount of co-emulsifier will  cause the system to become unstable due to its high hydrophilicity, moreover, the droplet size will increase due to the expansion of the interfacial film [28,29]. Therefore, 20% PEG400 should be chosen for the formulation. Based on the above results, the SMEDDS formulation was a mixture of 15% (w/w) IPM, 65% (w/w) Cremophor RH40 and 20% (w/w) PEG400. The Res drug loading was 5% (w/w).

Appearance
At room temperature, the Res SMEDDS (50 mg/g) was a viscous, transparent yellowish liquid. When the Res SMEDDS was serially tenfold diluted (10, 100 and 1000 times) with deionized water, the diluted solution becomes clear or slightly bluish. In contrast, under the same conditions, the same concentration of Res was suspended in water, even at a minimum concentration of 50 μg/g (Fig 4). The results indicated that the Res SMEDDS can improve the water solubility of Res, and the solubility of Res in SMEDDS was at least 1,000 times higher than that of the Res powder.

Size and morphology
Droplet size is one of the most important parameters of microemulsion diluted from SMEDDS that affects the release rate of drug and drug stability. Smaller droplets have a greater interfacial area that significantly enhances the release rate of drug. After the Res SMEDDS was diluted 100 times with deionized water, the droplet size, zeta potential and TEM image were shown in Fig 5. Compared with the particle size of the blank nano-emulsion of 26.23±1.56nm, the particle size of the Res microemulsion was 28.00±1.67 nm, there was no significant difference (P>0.05). Therefore, 5% drug loading of Res had no significant effect on the particle size of the microemulsion. The PDI of the blank nano-emulsion and the Res nano-emulsion were 0.169 and 0.213, respectively, which indicated that Res SMEDDS exhibited good dispersion properties. The zeta potentials of the blank microemulsion and Res microemulsion were -2.18 mv and -3.25mv, and higher than that reported by Chen Y [15], which indicated that the Res microemulsion was more stable. Because charged ions on the surface of these nanoparticles prevented the aggregation and fusion of the nanoparticles by electrostatic repulsion [30]. Previous studies have shown that the droplet size of the Res microemulsion is generally in the range of 50-200 nm [15,31,32], while the Res microemulsion prepared in this work had a smaller droplet size with an average droplet size of 28.00±1.67 nm, which was smaller that those that have been reported by Chen Y [15]. The smaller the particle size of the microemulsion is, the easier it is to be absorbed [33], therefore, Res SMEDDS prepared in this work may be exhibit better bioavailability. In addition, the TEM image revealed that the morphology of the Res emulsion was a regular circle with good dispersibility, which is consistent with the results of Chen Y [15]. Statistical analysis of the droplet size of the droplets in the TEM image with the NanoMeasure software also revealed that the average droplet size of the Res SMEDDS was about 28.81 nm, which was consistent with the droplet size measurement results.

In vitro stability of Res SMEDDS
The effect of the temperature on the stability parameters of the SMEEDS is shown in Table 5. There was no significant difference in the appearance, drug loading and droplet size of the Res SMEDDS at 4 and 40˚C in the dark for 10 days (P>0.05), which demonstrated that the Res SMEDDS had excellent stability. At the temperature of 60˚C for 10 days, the appearance color of the Res SMEDDS became darker. The reason is that the cloud point of Cremophor RH40 is 44˚C,when the temperature is higher than the cloud point of Cremophor RH40, irreversible phase separation occurs, which lead to become a darker solution. In addition, the Res content was reduced due to the instability of Res at 60˚C [34], The effect of exposure to light on the stability of the SMEDDS is shown in Table 6, under long-term illumination (4500Lx), the Res drug loading of the Res SMEDDS decreased on the 10th day due to the light instability of Res [35]. Therefore, the Res SMEDDS should be stored in the dark below 40˚C.

In vitro elution degree
The release profiles of Res powder drug and Res SMEDDS at different pH values were shown in Fig 6. The release percentage of Res from Res SMEDDS was more than 80% within 10 minutes, whereas the highest release percentage of Res powder drug was less than 55% within 60 minutes at the three different pH values. Statistically significant differences were observed between the two formulations at the same pH (P<0.05). The free energy required to form microemulsion is very low, spontaneous formation of microemulsion accelerate the dissolution of drug. In addition, smaller droplet size and larger interfacial area also advantageously increase the drug release rate [36], therefore, Res SMEDDS was able to enhance the in vitro release of Res. As shown in Fig 6, pH values of media had no effect on the in vitro release of Res from Res SMEDDS (P>0.05), which is consistent with release properties of phenol compounds Gingerol-SMEDDS reported by Xu Y et al [37]. However, the cumulative percentages of the Res powder at different pH values were 32.53±1.83 (pH = 1.2), 45.40±1.91 (pH = 6.8) and 50.87 ±1.87 (pH = 7.4), which was significant difference at the three different pH values (P<0.05). Release of Res powder is dependent on its solubility, the solubility of Res is sensitive to pH [38]. In addition, the major intermolecular interactions of Res powder are through hydrogen bonds, Each of the three oxygen atoms in the hydroxyl groups participates in two hydrogen bond intermolecular interactions [39], breakdown of intermolecular hydrogen bond may be promote dissolution of Res due to weak basic medium. Therefore, release of Res powder was affected by pH values of media.
In addition, the particle size of the Res SMEDDS did not change significantly and the distribution was uniform at different pH values (P>0.05) ( Table 7), which indicated that the Res SMEDDS was stable, and Res release of Res SMEDDS was unaffected by pH values of media. Because Res in Res SMEDDS was encapsulated and mainly located in the core of core/shell microemulsions [40], which led to stable release of Res from Res SMEDDS and no significant change of nanoparticle size and distribution.
These results suggested that the in vitro release of Res from Res SMEDDS was faster that of Res powder and unaffected by pH value of media.

In vitro antioxidant
There are many methods to evaluate the antioxidant activity of microemulsions in vitro, among which the DPPH free radical scavenging method and the ABTS free radical scavenging method are commonly used. The results revealed that the DPPH� and ABTS +� scavenging rate increased gradually with the increase of the Res concentration (Fig 7), which indicated that ABTS +� and DPPH�scavenging capacity of Res exhibited a certain dose-response relationship. However, there was no significant difference in the DPPH� and ABTS +� free radical scavenging rates between the Res SMEDDS and Res powder at the same Res concentration (P>0.05). This means that the Res SMEDDS and Res powder had similar scavenging activity.  Resveratrol self-microemulsion Cytotoxicity PC12 cells were treated with different concentrations of Res powder and Res SMEDDS,and their effects on the RGR of PC12 cells and toxicity grades were evaluated based on the detected OD(S1 File).
The MTT assay showed that the RGR of the cells decreased with the increase of drug concentration. When the drug concentration exceeded 20μM, compared with Res powder, the RGR of Res SMEDDS was smaller and statistically significant (P <0.05) ( Table 8), which demonstrated that Res SMEDDS was slightly higher cytotoxic to PC12 cells than that of Res powder due to slightly toxic of the components in SMEDDS. However, according to the RGR classification of ISO 10993-5-2009 standard, the cytotoxicity of each concentration group of Res SMEDDS belongs to "1" grade (Table 8), therefore, Res SMEDDS with a concentration below 100 μM was safe [41].

Conclusion
A fat-soluble drug Res is insufficiently absorbed by the human body due to its poor solubility. Accordingly, formulation strategies for enhancing solubility and dissolution rate of poorly water-soluble drugs are developed to improve the oral bioavailability. In this work, the developed Res SMEDDS system, composed of IPM (15%), PEG400 (20%), and Cremophor RH40 (65%), was found to significantly enhance the solubility of Res. Res SMEDDS needed to keep under 40˚C and avoid light exposure. The in vitro release of Res from Res SMEDDS was significantly faster that of Res powder and unaffected by pH value of media. There was no significant change for antioxidant activities of Res in presence of Res SMEDDS compared to Res powder. Res SMEDDS at the concentration of less than 100 μM was safe. These results demonstrated the potential use of Res SMEDDS for oral administration of Res.