Preparation Of A Ferulic Acid– Phospholipid Complex To Improve Solubility, Dissolution, And B16F10 Cellular Melanogenesis Inhibition Activity
Mar 29, 2023
Abstract
Background
Ferulic acid (FA; 4-hydroxy-3-methoxy cinnamic acid) is present in many foods, including wheat, rice, barley, oats, citrus fruits, and tomatoes [1]. FA has been shown to afford significant skin protection against UV-induced oxidative stress [2]. It reverts chronic UVB-induced oxidative damage in mice skin tumors by modulating the expression of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 [3]. It also modulates the expression of mutated p53, Bcl-2, and Bax in UVB-induced mice skin tumors [4]. Several studies have established that FA inhibits the expression of cytotoxic and inflammation-associated enzymes [5] and matrix metalloproteinases (MMPs), and attenuates the degradation of collagen fibers [6].
According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.
In addition, cistanche also has the function of promoting collagen production, which can increase the elasticity and luster of the skin and help repair damaged skin cells. Cistanche Phenylethanol Glycosides have a significant down-regulating effect on tyrosinase activity, and the effect on tyrosinase is shown to be competitive and reversible inhibition, which can provide a scientific basis for developing and utilizing the whitening ingredients in Cistanche. Therefore, cistanche has a key role in skin whitening. It can inhibit melanin production to reduce discoloration and dullness; and promote collagen production to improve skin elasticity and radiance. Due to the widespread recognition of these effects of cistanche, many skin whitening products have begun to infuse herbal ingredients such as Cistanche to meet consumer demand, thus increasing the commercial value of Cistanche in skin whitening products. In summary, the role of cistanche in skin whitening is crucial. Its antioxidant effect and collagen-producing effect can reduce discoloration and dullness, improve skin elasticity and luster, and thus achieve a whitening effect. Also, the wide application of Cistanche in skin whitening products demonstrates that its role in commercial value cannot be underestimated.
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Phospholipid complexes are widely used in the pharmaceutical industry. They have good permeability and safety and are receiving increasing attention for application in cosmetics. Because phospholipids are biofunctional surfactants with good solubilizing properties, they can be used as carrier systems for less soluble drugs [7], improving transdermal permeation and the cumulative penetration rate of topical drugs [8]. Transdermal permeation of drugs involves dissolution, distribution, and diffusion into the skin. Physical and chemical properties, especially the oil–water partition coefficient of the drug to be administered, affect this process [9].
Methods
Materials
Cell culture
Preparation of FA–PC using solvent evaporation
Screening for the optimal proportion of ferulic acid and phospholipids
FA and phospholipids at molar ratios of 2:1, 1:1, 1:2, 1:3, and 1:4 were added to 100 mL round-bottom flasks and dissolved in anhydrous ethanol (FA, 2.0 mg/mL). The mixtures were stirred constantly at 40 °C for 1 h, and then the anhydrous ethanol was removed by rotary evaporation. The dried FA–PC complexes were placed in a desiccator for 24 h.
Screening for optimal reaction temperature for FA–PC preparation
Screening for optimal reaction time for FA–PC preparation
Screening for optimal FA concentration
Characterization of FA–PC
Differential scanning calorimetry (DSC)
Solubility and oil-water partition coefficient
Solubility
The solubilities of powdered FA and FA–PC were determined by adding an excess of samples to 10.0 mL [10] of water or n-octanol and then shaking on a swing bed for 3 h at 37 °C. The mixtures were centrifuged at 15,000 rpm for 10 min to remove insoluble FA. Then, the supernatants were filtered through 0.45 µm membranes. Afterward, the filtrates were diluted tenfold with methanol, and the FA content was determined using a UV spectrophotometer (UV-3150; Shimadzu; Japan).
Samples (10 mL) of FA and FA–PC in water-saturated n-octanol were prepared and shaken. n-octanol–saturated water (10 mL) was added to each sample, and the miscible liquid was agitated for 24 h. Afterward, the samples were allowed to stand for layering. The FA concentration in each phase was determined by UV spectrophotometry (UV-3150; Shimadzu; Japan). Analyses were carried out in triplicate.
In vitro diffusion
In vitro diffusion studies were performed utilizing Franz diffusion cells (TK-20A; Shanghai Xie Kai Financial Information Service Co., Ltd.; China). In addition, we used Strat-M® membranes, which are synthetic membranes with diffusion characteristics that correlate more closely to human skin than animal skin models [11]. The membranes were clamped between the donor and receiver chambers of the vertical diffusion cells, and the receiver chambers were filled with phosphate-buffered saline (PBS; pH 7.4) to solubilize FA or FA–PC and ensure sink conditions.
Chromatographic separation was carried out using an Agilent 1260LC series system (Agilent Technologies, Palo Alto, CA, USA) equipped with an online vacuum degasser, quaternary pump, autosampler, thermostatted column compartment, and diode array detection (DAD). Agilent Technologies ChemStation software for liquid chromatography (LC; B.02.01) was used, and HPLC separation was performed using an Eclipse plus-C18 column (4.6 × 250 mm, 5 μm). The detection wavelength was 0.05% acetic acid (A) and methanol (B) (40:60, v/v). The flow rate was 1.0 mL/min. The column temperature was set at 30 °C. Cumulative corrections were made to ascertain the amount of FA released at each time interval. All measurements were performed in triplicate, and the percent of cumulative FA that permeated through the membrane (%Q) was plotted as a function of time.
Inhibition of melanogenesis
B16F10 cell viability assay
Cell viability and cell proliferation were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [2]. B16F10 cells were pretreated with 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL concentrations of FA and FA–PC. After incubation for 48 h, MTT solution (final concentration: 5 mg/mL) was added, and the cells were incubated for 3 h at 37 °C. Finally, the absorbance of each sample was measured on a microplate reader at 570 nm to obtain the percentage of viable cells.
Measurement of melanin content
Melanin content was measured as described previously [6] with some modifications. B16F10 melanoma cells were seeded (2 × 105 cells/well in 3 mL of medium) in six-well culture plates and incubated overnight to allow cells to adhere. At the end of the treatment, the cells were washed with PBS and lysed with 1 M NaOH containing 10% dimethyl sulfoxide (DMSO) for 30 min at 80 °C. The absorbance (optical density; OD) was measured at 475 nm using a microplate reader. Melanin content was calculated using the following formula:
Data analysis
The statistical significance of the differences between the mean measurements of each treated group and that of the control group was determined using Dunnett’s t-test. P values <0.05 were considered statistically significant.
Results and discussion
Optimal preparation of FA–PC
Solubility and oil–water partition coefficient of FA–PC
Table 1 shows the solubility of FA, PM, and FA–PC in water and in n-octanol. FA–PC exhibited good solubility in water and n-octanol (1.68 and 7.77 mg/mL, respectively), and the solubility of FA–PC in n-octanol was found to be significantly higher than that of FA (1.34 mg/ mL).
Lipophilicity is usually measured as a partition coefficient (P) between two immiscible phases. It is typically expressed as log P. The apparent partition coefficients of FA and FA–PC in the n-octanol/water system was determined. Results indicated that log P was higher for FA–PC (1.21) than for FA (0.99) when measured at pH 5.0. The slightly increased log P was related to the significantly improved n-octanol solubility of FA–PC compared to that of FA. The increased solubility of FA–PC in n-octanol may be explained by the amorphous characteristics of the FA–PC. As lipophilicity and permeability are well-correlated, these results suggest that the transdermal permeability of FA might be improved by applying it as a phospholipid complex.
DSC
DSC is a reliable method for screening drug-excipient compatibility and provides maximum information about possible interactions between the drugs and excipients [10]. The presence of interaction can be concluded from the elimination of endothermic peaks, the appearance of new peaks, changes in peak shape and onset, peak temperature/melting point, and relative area or enthalpy. Figure 1 shows the DSC thermograms of FA (Fig. 1a), PC (Fig. 1b), PM (Fig. 1c), and FA–PC (Fig. 1d). The thermal curve for pure FA has a typical sharp endothermic melting at about 172.7 °C, indicative of its anhydrous and crystalline state, while that of phospholipids exhibit a minor endothermal peak at 231.7–248.6 °C. The DSC curve for PM, consisting of the superimposed thermal profiles for FA and phospholipids, shows no significant changes except for a small shift to a higher temperature (175.9 °C), indicating no interactions between the components. FA–PC has one major peak at 158.2 °C, which differs from the FA peak, indicating an interaction between FA and PC. Our results suggest that different degrees of interaction and/or amorphization in different mixtures or complexes can be obtained depending on their preparation method and this is associated with the differences in solubility.
FTIR
FTIR spectroscopy can confirm the formation of FA–PC by comparing the FA–PC spectrum with that of pure FA. Figure 2 shows the FTIR spectra of FA, PC, PM, and FA–PC. The FA spectrum (Fig. 2a) shows a characteristic hydroxyl stretching band at 3436 cm−1. It all becomes a wide singlet in the spectrums FA–PC, PM, and phospholipid (Fig. 2b–d). The FA spectrum (Fig. 2a) exhibits characteristic peaks at 1620 cm−1 (C=C stretching) and 1450 cm−1 (C=C aromatic ring stretching). In the FA–PC spectrum (Fig. 2b), these two peaks are not visible, probably owing to weakening or removal, or shielding by the phospholipid molecule.
The FA spectrum (Fig. 2a) exhibits characteristic unsaturated carboxyl peaks at 1691 cm−1 (C=O stretching), and 1664 cm−1 (C=C stretching). In the FA–PC spectrum (Fig. 2b), these two peaks are not visible, probably owing to the attractive forces between the negative carboxyl charge in FA and the positive nitrogen charge in phospholipids. The phospholipid spectrum (Fig. 2d) has peaked at 1733 cm−1 (C=O stretching), 1238 cm−1 (P=O stretching), and 1087 cm−1 (P–O–C stretching). The
SEM
The surface morphologies of FA, PC, PM, and FA– PC were investigated using SEM (Fig. 3). In Fig. 3c, FA appears crystalline, almost rectangular, while FA–PC particles (Fig. 3a) appear irregular in shape with a smooth surface. FA–PC possesses a significantly different shape and surface topography compared to that of FA and PC (Fig. 3b). This probably owes to the complete miscibility of FA in PC. In the PM scan (Fig. 3d), both FA and phospholipids are easily distinguishable.
In vitro diffusion studies
Recently, the synthetic Strat-M® membrane was introduced as a substitute for human skin in vitro diffusion studies [11]. The Strat-M® membrane is composed of two layers of polyethersulfone that are resistant to diffusion. The polyethersulfone layers lie atop one layer of polyolefin, which is more open and diffusive. This synthetic membrane is characterized by low batch-to-batch variability, thus providing more consistent data. Moreover, it has been shown that diffusion data for Strat-M® membranes correlate well with those of human skin [11].
To evaluate the influence of PC on the in vitro diffusion properties of FA, the %Q of FA and FA–PC was plotted against time. The results in the present paper showed a trend that phospholipids significantly increased FA permeation into the Strat-M® membrane. In addition, FA–PC resided longer on the Strat-M® membrane than did FA (Fig. 4). Therefore, the incorporation of phospholipids into FA maybe increases its residence time in the stratum corneum and makes it more suitable for skin permeation. Regarding the permeation time, although Strat-M® membrane has been reported to have a good correlation with those of human skin [11], it also has textural differences compared with human skin. The influence factors of the permeation times may be included solvent, the concentration of the compounds, pH value, et al. More experiments should be done in future analysis.
Inhibition of melanogenesis
Effect of FA–PC on melanin synthesis
According to the literature, FA can inhibit cellular tyrosinase activity and melanogenesis in B16F10 melanoma cells through the downregulation of the cellular proteins c-kit and ERK1/2 [12]. In the present study, FA–PC decreased melanin content in B16F10 cells more obviously than FA at tested concentrations of 0.25, 0.5, and 1.0 mg/mL (Table 2). Thus, we determined that FA–PC is more effective than FA at inhibiting melanin synthesis in B16F10 cells.
Conclusion
Authors’ contributions
LL and LY have made substantial contributions to the conception and design, or acquisition of data, or analysis and interpretation of data; XY and WX have been involved in drafting the manuscript or revising it critically for important intellectual content; and ZJ and DY have given final approval of the version to be published. All authors read and approved the final manuscript.
Acknowledgments
This work was supported by the National Natural Science Foundations of China (31501402).
Competing interests
The authors declare that they have no competing interests.
Received: 27 December 2016 Accepted: 14 March 2017
Published online:22 March 2017
