As human lifespan has extended and we are entering an aging society, the interest in well aging and physiological aging of the human body has recently been increasing. As the human body ages, it is exposed to various diseases; in particular, oxidative stress is the main factor that accelerates the occurrence of disease. Oxidative stressors play an important role in the formation of reactive oxygen species (ROS) and cause various types of inflammation in the body. Various types of ROS exist, such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide [1,2]; these are produced during normal metabolic processes in the human body and play an important role in cell signaling and homeostasis [3]. In addition, active oxygen species catalyze the oxidation of tyrosine to dopamine and increase melanin synthesis, thereby causing hyperpigmentation, such as freckles and black mushrooms. Moreover, hyperpigmentation appears in the process of melanin pigment formation to protect the skin from excessive exposure to ultraviolet rays, which accelerate photoaging. Excessive exposure to ultraviolet rays causes active oxygen species in the skin, thus causing oxidative stress and destroying lipid peroxidation and transmembrane signaling pathways [4]. Antioxidants, which not only neutralize damage caused by oxidative stress but also protect cells, are necessary to suppress such active oxygen [5]. Commonly known antioxidants include enzyme-based antioxidants (such as superoxide dismutase, catalysts, glutathione redox, natural antioxidants of phenol compounds, flavone derivatives, ascorbic acid, carotenoids), and synthetic antioxidants (such as butylated hydroxytoluene and propyl gallate) [6].

Meanwhile, oxidative stress causes various reactions in the skin, and typical examples include inflammatory reactions, such as erythema and edema [7]. Inflammation is a human body defense mechanism against physical stimuli, such as chemicals or bacteria. Continuous inflammatory reactions cause pain, edema, and fever [8,9]. When macrophages, lymphocytes, obesity cells, neutrophils, and NK cells that are responsible for immune functions in the body are activated, inflammatory mediators are expressed and inflammatory reactions occur [10]. Typical dermatitis symptoms include atopic or acne-prone skin. Note that atopic dermatitis is associated with the secretion of various cytokines (interleukin [IL]-4 and IL-13), increased immunoglobulin (Ig) E, and infiltration of cells into tissues (CD4+, CD25+, eosinophils, or obese cells) [11]. The initial reactions to dermatitis are caused by histamine secreted by obese cells; histamine is best known among the substances secreted by the degranulation of obese cells that dilate blood vessels and cause scleroderma [12].

In this study, ferulic acid was separated from Ferula foetida as a white-light tan crystalline powder with no or slightly unusual odor. Generally, edible ingredients include wheat, rice, eggplant, wheat, oats, grains, leaves, fruits, beans, coffee, peanuts, tomatoes, spinach, broccoli, carrots, avocados, and pineapples [13]. The chemical structure is composed of functional groups such as a phenolic hydroxyl groups (-OH), double bonds, amd carboxyl groups (-COOH) (Fig. 1).

Figure 1. Chemical structure of ferulic acid.

In previous studies, Han et al. [14] demonstrated the toxic inhibitory effect of ferulic acid and related phenolic compounds on cancer cell lines; further, ferulic acid has been reported to have excellent antioxidant and anti-inflammatory effects [15,16]. Phenylpropanoid is known to have excellent antioxidant activities by removing active oxygen, known as harmful oxygen, and phenolic compounds have been studied in various fields of drugs, food, and cosmetics because of their excellent antioxidant activities [17]. Recently, as the interest in aging inhibition has increased, interest in natural plant-derived substances with excellent antioxidant properties has also increased. Therefore, by observing the antioxidant and anti-inflammatory effects of ferulic acid, a plant-derived ingredient that is safe for the human body, this study determines a method to utilize ferulic acid as a raw material for functional cosmetics.

Reagent and cell culture

Ferulic acid and 3-(4,5-dimethyliazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). 2’,7’-Dichlorofluorescin diacetate (DCF-DA) was purchased from Molecular Probe Co. (Eugene, OR, USA). RAW 264.7 cell and RBL2H3 macrophages were purchased from Seoul National University’s Cellular Bank. RAW 264.7 cell and RBL2H3 macrophages were grown at 37°C with 10% fetal bovine serum and 5% penicillin/streptomycin (100 IU/ 50 μg/ml).

Cytotoxicity measurement using MTT

To confirm the cytotoxicity of ferulic acid, the MTT method was applied. RAW 264.7 cell was used. In 96 well microplates, it was divided into 1×10⁴ cells per well, incubated for 24 hours, and then the sample was added by concentration and incubated at 37°C for 48 hours in CO₂ cubator. After 72 hours, the cultivation solution was removed, and 1 ml of 500 μg/ml MTT solution dissolved in Krebs solution (NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 0.4 mM, MgCl₂ 0.5 mM, HEPES [pH 7.4] 10 mM, CaCl₂ 1.8 mM, glucose 5 mM) was added to each well and cultivated for 4 hours in the dark. Next, the supernatant was removed and 200 μl of dimethyl sulfoxide was added to each well to dissolve the MTT formazan. After completely dissolving the MTT formazan for 10 minutes at room temperature, the absorbance was measured at 570 nm.

DPPH radical scavenging activity

First, 180 μl of 0.1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution dissolved in ethanol to 96 well microplates ferulic acid prepared in each concentration was added 20 μl each. Next, it was incubated at 37°C for 30 minutes in the dark, and absorbance measurements were processed to absorbance measurement at 517 nm using FL 600 spectro fluorometer (BioTek, Winooski, VT, USA).

DPPH radical scavenging activity (%)

=100-{(absorbance of added/absorbance of non-added)×100}

Intracellular oxidation stress measurement

The fluorescence of DCF-DA in RAW 264.7 cells was measured by the conversion of DCF, which is produced by oxidation and deacetylation with intracellular oxygen radicals (ROS), into a fluorescent substance. First, after suspending RAW 264.7 cells in 10 ml of Krebs buffer, 20 μM DCF-DA was added and incubated for 30 minutes in a light-shielded state. After washing once with Krebs buffer without DCF-DA, cells were extracted by centrifugation. Raw 264.7 cells was divided into 1×10⁴ cells/ml and pretreated with ferulic acid by concentration; next, 1 mg/ml of silica was added to it to induce H₂O₂ production for 30 minutes. After centrifugation, the cell microplate was redistributed into 200 μl of Krebs buffer and transferred to 96 well plates. Finally, fluorescence was measured (Ex 485 nm/Em 535 nm).

Nitric oxide

RAW 264.7 cells were divided into 24 well plates at 10⁶ cells/ml and 1 ml per well. In 96 well microplate, 100 μl of the cell culture supernatant and 150 μl of Griess reagent (1% sulfanilamide in 5% phosphoric acid+1% α-naphthylamide in H₂O) were mixed and reacted for 5 minutes to measure the absorbance using an ELISA microplate reader (Model: MQX200R; BioTek). Sodium nitrite (NaNO₂) was used as a standard for comparison to prepare the calibration curve.

Histamine release

After RBL 2H3 cells were divided into 10⁶ cells/ml, the sample was pretreated for 10 minutes, and melittin (0.5 μM) was treated to extricate histamine for 30 minutes. Subsequently, the supernatant and cells were separated by centrifugation. The cells were destroyed by an ultrasonic pulverizer, and distilled water was added to adjust the final volume to 2 ml. In each tube, 0.4 ml of 1 N NaOH and 0.1 ml of 1% OPT (o-phthalaldehyde, 10 mg/ml in absolute methanol) were added, mixed, and incubated at room temperature for 4 minutes or more. Subsequently, the reactions were stopped by adding 0.2 ml of 3N HCl. Fluorescence was measured by dividing 200 μl into 96 well microplate (Ex 355 nm/Em 455 nm). The amount of histamine in the supernatant was considered to be the amount of extracted histamine.

Data analysis and statistical verification

Statistical analysis was performed using SPSS Window version 21.0 (IBM Corp., Armonk, NY, USA), and the significance was tested by student’s t-test. The experiment was performed three or more times independently under the same conditions noted in mean±SD, and a statistically significant difference was observed when the p-value was less than 0.05.

Measurement of cytotoxicity

Previous studies on cell viability by the phenylpropanoid family revealed that human foreskin fibroblasts treated with 200 μM caffeic acid result in a cell survival rate similar to that of the noncaffeic acid group [18]. To investigate the effect of ferulic acid on cytotoxicity, cell viability was measured using an MTT assay. Analysis of the concentration of ferulic acid at 25, 50, and 100 μg/ml in RAW 264.7 cell revealed that ferulic acid did not exhibit cytotoxicity at all concentrations (Fig. 2). Moreover, it exhibited a strong cell viability rate of 92% at the highest concentration of 100 μg/ml. Thus, it is considered to be a safe substance when applied to the human body as a cosmetic raw material.

Figure 2. The cytotoxicity of ferulic acid. Values are presented as mean±SD from 4 separate experiments (*p<0.05, **p<0.005).

Effects of DPPH radical scavenging

Phenylpropanoid-based compounds have hydroxyl and methoxyl groups in the benzene group and are known to have various pharmacological activities, including antioxidant activity. In particular, the DPPH radical scavenging activities of caffeic acid have been proven [19]. The DPPH assay, as an in vitro experiment, is an essential test to analyze the presence of antioxidants and is based on hydrogen atom transfer reactions [20]. The DPPH radical scavenging ability at concentrations of 25, 50, and 100 μg/ml ferulic acid revealed that the DPPH radical scavenging ability increased significantly as the concentration of ferulic acid increased, thus exhibiting a strong antioxidant activity of 64% at the highest concentration of 100 μg/ml. Further, ascorbic acid, which is already known as an antioxidant, has a similar antioxidant effect (Fig. 3).

Figure 3. Anioxidant activities of ferulic acid in the DPPH radical scavenging activity assay. Values are presented as mean±SD from 4 separate experiments (*p<0.05, **p<0.005).

ROS erasing activity in RAW 264.7 cells

Intracellular hydrogen peroxide produced in cells was measured using DCF-DA fluorescent materials. Aerobic cells produce ROS as a metabolite of oxygen restoration. Among ROS, superoxide anions are the first to be produced, and when superoxide dismutase is activated, superoxide is converted to hydrogen peroxide for removal [21]. Hydrogen peroxide is converted to water and oxygen by intracellular catalase or peroxidase. However, peroxynitrite, lipid hydroxide, and intracellular hydroperoxide may be produced in the cells during this process. Silica, which is used as a stimulant, is known to produce ROS not only in macrophages but also in fibroblasts [22]. The inhibitory effect of ROS produced by silica revealed that ferulic acid inhibited ROS production in a concentration-dependent manner and exhibited an antioxidant activity of 76% at the highest concentration of 100 μg/ml (Fig. 4). These results support the antioxidant efficacy of ferulic acid in the development of treatments for neurodegenerative diseases, such as Alzheimer’s disease, accompanied by oxidative stress, as studied by Kanski et al. [23]. In addition, the results of ferulic acid, which has various therapeutic effects, such as anti-aging, anti-tumor, and anti-hypertensive effects, were similar to those of previous studies [24] that reported cell inflammation and oxidative stress inhibition on H₂O₂-induced damage in vitro in rat vascular smooth muscle cells. These results suggest that the strong antioxidant effect of ferulic acid could be applied to cosmetics instead of water-soluble vitamin C, which is difficult to apply to cosmetics.

Figure 4. Effects of ferulic acid on reactive oxygen species generation in RAW 264.7 cells. Data were expressed as % change of silica. Values are presented as mean±SD from 4 separate experiments.

Effects of nitric oxide production on RAW 264.7 cells

Nitric oxide (NO) is an intercellular messenger present in all vertebrates as a free radical that regulates blood flow, blood clots, and nerve activity. Although NO-induced physiological reactions play an important role in nonspecific host defense, such as effective vasodilation and stimulation of tumor growth by endothelium-derived relaxing factor, NO itself is unlikely to directly reduce intracellular pathogens and tumors [25-27]. NO production is closely related to its anti-inflammatory action as a medium for inflammation in macrophages [28]. After inducing NO production using lipopolysaccharide as a stimulant, NO production was observed at each concentration of ferulic acid. At the highest concentration of 100 μg/ml, NO production was found to be 74% suppressed (Fig. 5). These results are similar to that of the inhibition of NO production by ferulic acid in inflammatory human umbilical vein endothelial cells induced by TNF-α stimulation, thus suggesting the potential of ferulic acid as a cosmetic material for the amelioration of anti-inflammatory acne.

Figure 5. Effects of ferulic acid on nitric oxide generation in RAW 264.7 cells. Data were expressed as % change of LPS. Values are presented as mean±SD from 4 separate experiments (*p<0.05, **p<0.005). LPS, lipopolysaccharide.

Histamine release active

Histamine is a biological amine synthesized from histidine, which is an amino acid. It causes inflammation and allergic reactions within the tissue, thus resulting in the secretion of mucus from the nose and bronchial mucosa, contraction of bronchial smooth muscle, and itching and pain at the nerve ends. Histamine-induced allergic reactions are induced by IgE, an immune complex with halophiles and obese cells, which are considered to be the causative cells of chronic inflammatory diseases, combined with Fc-ε receptors in the cell membrane. Therefore, it is useful for measuring the allergens bioactivity as the quantification of histamine is an indicator of degranulation in these cells [29]. Herein, histamine was induced using melittin in RBL2H3 cells, which are halophilic cells. The results revealed that ferulic acid exhibited a concentration-dependent tendency to suppress histamine extraction, and 66% histamine release at the highest concentration of 100 μg/ml (Fig. 6). Thus, ferulic acid has an inhibitory effect on histamine extraction and has potential for application as a material that can improve skin allergic diseases.

Figure 6. Effects of ferulic acid on Histamine release in RBL2H3 cells. Data were expressed as % change of melittin. Values are presented as mean±SD from 4 separate experiments.

This study demonstrated the use of ferulic acid in functional cosmetics as an anti-inflammatory and antioxidant agent. Ferulic acid has various pharmacological properties, such as antioxidant and anti-inflammatory effects, and it consists of phenolic hydroxyl groups (-OH), double bonds, and carboxyl groups (-COOH). The results of this study are as follows. In a cytotoxicity test using RAW 264.7 cells, ferulic acid did not exhibit any significant cytotoxicity at any concentration of ferulic acid. Therefore, it is considered to be a safe substance for the skin within the range of concentrations used in this experiment. DPPH measurement revealed strong antioxidant effects in a concentration-dependent manner for the self-antioxidant effect of ferulic acid, and ROS produced in cells were measured using DCF-DA fluorescent materials. The inhibitory effect of ROS induced by silica was observed; evidently, ferulic acid was found to suppress ROS production in a concentration-dependent manner and exhibited an antioxidant activity of 76% at the highest concentration of 100 μg/ml. The inhibition of NO production induced by lipopolysaccharide was measured to observe the effect of ferulic acid on anti-inflammatory activities. Ferulic acid suppressed NO production in a concentration-dependent manner and exhibited 74% of anti-inflammatory properties at the highest concentration of 100 μg/ml. In addition, melittin induced histamine inhibition in a concentration-dependent manner. These results suggest that ferulic acid can be effectively used as a functional substance for antioxidant and anti-inflammatory activities in the development of cosmetic materials.

This work was supported by the Dongnam Health University research grant in 2022.