Mixture of enzyme-processed Panax ginseng and Gastrodia elata extract prevents UVB-induced decrease of procollagen type 1 and increase of MMP-1 and IL-6 in human dermal fibroblasts

ABSTRACT

According to the previously described anti-photoaging effect of the enzyme-processed Panax ginseng extract and Gastrodia elata extract, we hypothesized that the combination of the two extracts would have superior effect to protect human skin from UVB radiation. Besides, the mixture of active components isolated from herbal extracts, ginsenoside F2, and α-gastrodin was investigated on the photo-protective capability. The expression of aging-related markers including matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and procollagen type 1 was evaluated using ELISA kits. It was reported that the herbal extract at a Panax ginseng extract to Gastrodia elata extract ratio of 1:10 (w/w) and the compound mixture with equal proportion of ginsenoside F2 and α-gastrodin exhibited significant inhibition of MMP-1 and IL-6 production, and marked upregulation of procollagen type 1 formation. Thus, the combination of either the enzyme-processed herbal extracts or their active components would enhance the properties of prevention and treatment of UVB-induced skin damage.

GRAPHICAL ABSTRACT

The reverse effect of EPG on the UVB-induced damage via inhibiting MMP-1 and IL-6 production and accelerating procollagen type 1 synthesis.

KEYWORDS:

• Panax ginseng
• Gastrodia elata
• ginsenoside F2
• α-gastrodin
• ultraviolet B (UVB) irradiation

Skin is the most voluminous organ of the human body and functions as an effective biological shield, segregating the body from the external environment [1]. The skin is made up of multiple layers of cells and tissues that are connected to the underlying structures by connective tissue [2]. The outermost layer, called the epidermis, is a waterproof covering composed of different cell types, such as self-renewing keratinocytes, which produce and store keratin, melanocytes, for pigmentation, and adnexal cells, which include sebaceous glands, sweat glands, and hair and nails [2,3]. The dermis is the layer lying beneath the epidermis and is primarily and continuously made up of fibroblasts; it is responsible for the production and secretion of procollagen and elastic fibers, which provide strength and flexibility to the skin [4]. The innermost layer, the subcutaneous tissue, includes fat and connective tissues and contains larger blood vessels and nerves that are in charge of regulating body temperature [2,5].

During a lifetime, the aging process of the skin is an inevitable result of both intrinsic and extrinsic aging factors; it is characterized by wrinkle formation, reduction of elasticity, and epidermal structure damage [6,7]. Acute exposure to solar radiation, particularly the UVB component (280–320 nm), is the main factor in extrinsic skin aging, attributing to about 80% of facial aging [1,8,9]. The long-term irradiation of UVB negatively affects the extracellular matrix (ECM) of aged skin. The ECM plays an important role in the formation and function of skin tissue networks [10–12]. The components of the ECM are fibrous proteins (i.e., collagen, elastins, fibronectins, and laminins) and proteoglycans (i.e., hyaluronic acid, heparan sulfate, and keratin sulfate), which are required to provide tensile strength, hydration, and elasticity to the skin [11,13]. In response to the stimulation of UVB radiation, there are increases in expression of pro-inflammatory factor interleukins, as well as in matrix metalloproteinases (MMPs), which mediate the degradation of the different components of the ECM [14]. Further studies on the degradation system of collagen have shown that matrix metalloproteinases-1 (MMPs-1) are involved in the breakdown of procollagen type 1, which is a precursor of collagen type 1, associated with photoaging [6,14].

Panax ginseng (P. ginseng) C. a. Meyer (Korean ginseng), which belongs to the Araliaceae family has been widely known for its pharmacological effects on physical performance, vitality, and resistance to stress and aging; it exhibits immunomodulatory activity [15,16]. The bioactive compounds of P. ginseng Meyer have been isolated and shown a variety of advantageous effects, including anti-inflammatory, antioxidant, and anticancer effects. The major ginsenosides of P. ginseng are Rb1, Rb2, Rc, Rd, Re, and Rg1, accounting for 80% of the total ginsenosides [17]. The major ginsenosides can be transformed into minor ginsenosides such as F1, F2, Rg3, Rh1, Rh2 compound Y, compound Mc, and compound K; these occupy a low percentage of, or are absent from, ginseng extract [18]. However, the previous study of Hwang et al. [19] has shown that the use of β-Glycosidases from Aspergillus niger in P. ginseng extraction can enhance the yield of minor ginsenosides, particularly ginsenoside F2, by the hydrolytic process. As two glucose molecules at C-3 and one glucose molecule at C-20 of protopanaxadiol (PPD) derivatives are hydrolyzed by enzymatic reaction, there is an increase in the yield of the newly formed ginsenoside F2 [18]. Moreover, this study demonstrated that enzyme-modified Korean ginseng extract and its active component, ginsenoside F2, can protect in vitro normal human dermal fibroblasts from UVB-induced photoaging by inhibition of MMP-1 production. The same treatment on UVB-irradiated hairless mice suppressed MMP-1 secretion and preserved procollagen type 1 expression, resulting in a decrease in wrinkle formation and an increase in hydration [19].

It has been reported that Gastrodia elata, a traditional Chinese medicine rich in phenolic components, is used pharmacologically in the treatment of epilepsy, vertigo, headache, insomnia, hypertension, and dementia due to its anticonvulsant, analgesic, calming, hypnotic, nootropic, and anti-aging properties [20,21]. Its three main phenolic components, 4-HBA, α, and β-gastrodin, have shown a noticeable reduction of oxidative stress by upregulating Nrf2, as well as inhibiting IL-6 and MAPK expression, which then depresses MMP production and increases procollagen type 1. According to Hwang et al. [22], both Gastrodia elata extract and its enzyme-processed extract, as well as its individual compounds, have reduced MMPs production and improved procollagen type 1 expression. The research also pointed out that α-gastrodin is the most effective antiaging agent and α-gastrodin content is higher in CGTase-processed Gastrodia elata extract.

In this study, it is hypothesized that the mixture of enzyme-processed Panax ginseng and enzyme-processed Gastrodia elata (EPG), as well as the combination of ginsenoside F2 and α-gastrodin, can prevent UVB irradiation-induced photoaging of human skin. In order to evaluate the hypothesis, serial assays with UVB-irradiated normal human dermal fibroblasts (NHDFs) using ELISA kits were conducted to analyze the markers of skin photoaging including MMP-1, IL-6 and procollagen type 1.

Materials and methods

Materials and extraction methods

Six-year-old Panax ginseng was supplied by Dr. Byung-Goo Cho (R&D Headquarters, Korea Ginseng Corporation, Daejeon, Korea) and extracted using a patent protocol (Korea Patent, No. 1014540660000) [23]. The dried rhizomes of Gastrodia elata were purchased from The Best Herb Company (Seoul, Republic of Korea). The extraction was prepared in 50% ethanol for 3 h [22]. Ginsenoside F2 was purchased from the Ambo Laboratory (Daejeon, Korea). α-gastrodin was synthesized in our laboratory (Hwang et al., unpublished protocol).

Preparation of Panax ginseng and Gastrodia elata extract processed with enzyme

The enzyme-processed Panax ginseng extract (EP) was obtained with its patented protocol [23]. As described by Hwang et al. [19], the crude enzyme was extracted from Aspergillus niger (KACC 40280), which was isolated from the Nuruk for Korean traditional wine. Then, the active enzyme was purified by loading into an ion-exchange resin. The dried and ground Panax ginseng (0.5 kg) was incubated with the enzyme solution containing ginsenoside-β-glucosidase and extracted from 5.0 L ethanol. The extract subsequently reacted with the enzyme at 50°C to 60°C for 24 h. After reaction, the enzyme was removed by ultrafiltration (9MWCO, 10,000 Da). The solution was then filtered and concentrated. According to our previous analysis, the F2 content accounted for 45.51 µg/mg in EP (Table 1) [19].

Table 1. Ginsenoside content of Panax ginseng and enzymatic preparation of ginseng. ND: non-detectable [19].

	Content (µg/mg)
Ginsenosides	Panax ginseng extract	Enzyme-treated Panax ginseng extract (EP)
Rb1	48.61	6.14
Rb2	7.73	ND
Rc	12.89	ND
Rd	17.59	13.32
F2	ND	45.51
Rg1	128.57	84.29
Re	20.52	60.82
Rf	19.69	4.16
Rb1 + Rg1	177.18	90.43
Rb1 + Rd + F2	66.20	64.97
Total	255.60	214.24

Industrial grade cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus stearothermophilus and glucoamylase from Rhizopus nevius were purchased from Bision Corporation (Sungnam, Gyeonggi, Korea). Gastrodia elata extract was incubated with 2250 U/100 mL CGTase, 10 mM CaCl₂, and 0.1 M phosphate buffer (pH 6.0) at 50°C for 24 h. The reaction was stopped by boiling in water for 10 min. Finally, the reaction products were continued using 80 U/100 mL glucoamylase at pH 5.0 and 50°C for 8 h. The enzyme-processed Gastrodia elata extract (EG) was reported to contain 22.64 (mAU*min in peak area) of α-gastrodin in our previous study, resulting in final EG product composing of 0.42% α-gastrodin [22].

EP and EG were mixed following their weight at different ratios (1:1; 1:3; 1:10; 3:1; 10:1). Similarly, mixture of ginsenoside F2 and α-gastrodin was also tested at these ratios.

Cell culture

NHDFs were characterized from a skin biopsy from a healthy, young male donor (MCTT Bio, Inc., Seoul, Korea). Passages 5 to 10 of the NHDF cells were utilized to culture in 100-mm dishes obtained in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin–streptomycin at 37°C in a humidified incubator under 5% CO₂.

DPPH radical scavenging

The scavenging activity of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) of the samples was determined with EPG (50 and 250 µg/mL) at various ratios (1:1; 1:3; 1:10; 3:1; 10:1). Arbutin (10, 50, and 250 µg/mL) was used as the positive control. After samples were incubated with DPPH for 30 min at 37°C in the dark, the optical density was measured at 595 nm.

UVB irradiation and sample treatment

Previously, a method for UVB irradiation and sample treatment was introduced by Hwang et al. [24]. First, cells were seeded in culture dishes. When the cells reached 80% confluence, they were subjected to UVB by a Bio-Link BLX-312 machine (VilberLourmatGmbH, VilberLourmat, Marne-la-Vallée, France) cross-linker system (312 nm 6 W*6 tube, luminescent dosimeter) with 144 mJ/cm² for 40 seconds [24]. After irradiation, the cells were treated with herbal extracts (1, 10, and 100 µg/mL), or active compounds (0.1, 1, and 10 µg/mL). Control cells were obtained from the serum-starved DMEM medium without sample and UVB exposure.

ROS generation activities

After 24 h of UVB irradiation, cells were stained with 2ʹ7’-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) and incubated for 30 min at 37°C. Then, the cells were washed twice with ice-cold PBS prior harvested and analyzed using a fluorescence microplate reader (Molecular Devices FilterMax5, USA; Excitation/Emission = 485/525 nm).

MTT assay

After 72 h of treatment with sample and UVB exposure, the supernatants were transferred to new tubes and stored at −20°C for the MMP-1, IL-6, and procollagen type 1 measurements. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenultetrazolium bromide (MTT 0.1 mg/mL), incubated at 37°C for 2 h in a CO₂ incubator. After formazan crystals were completely dissolved by dimethyl sulfoxide (DMSO), they were gently shaken for 30 min before the absorbance was measured at 595 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The experiment was repeated three times for each sample and mixture.

Measurement of MMP-1, IL-6, and procollagen type 1 secretion

The secretions of MMP-1, IL-6, and procollagen type 1 in the medium were determined using commercially available ELISA kits according to the manufacturer’s instructions.

Statistical analysis

Results were analyzed using a statistical analysis system (GraphPad Prism 5). All experiments were carried out in triplicate. Data are expressed as mean ± standard deviation (SD). Statistical comparison between different treatments was determined using one-way analysis of variance followed by Duncan’s test. For statistical analysis, Student’s t-test was used to compare the individual treatments to the controls. The level of statistical significance was set at p < 0.05.

Results

Antioxidant activity

The free radical scavenging activities of the EPG at different ratios and the arbutin were determined by DPPH assay. The mixtures, as well as the arbutin, exhibited dose-dependent increases in scavenged DPPH radicals. Additionally, at equal concentration, the free radical scavenging capacity of the herbal mixture at a Panax ginseng extract to Gastrodia elata extract ratio of 1:10 (w/w) was not only the most effective one among the defined ratios but was also superior to the positive control arbutin. In particular, arbutin inhibited DPPH radiation by 28.6% at 50 µg/mL and 45.3% at 250 µg/mL, whereas the 1:10 EPG inhibited DPPH radiation by 34.8% at 50 μg/mL and 54.8% at 250 μg/mL (Figure 1(a)). Thus, the above ratio of herbal mixture was selected for use in further experiments. Besides, the previous studies reported that ginsenoside F2 accounted for 45.51 µg/mg in EP [19], whereas α-gastrodin constituted 0.42% of EG (equivalent to 4.20 µg/mg) [22]. Therefore, the compound mixture at a ginsenoside F2 to α-gastrodin ratio of 1:1 (w/w) proportionately determined as a working ratio for further assays, based on the defined EPG ratio of 1:10.

Figure 1. Antioxidant activity of EPG. Radical scavenging activity of EPG at different ratios (a). Data are presented as the mean ± SD. ** and *** indicate the significant within-group differences (** p < 0.01 and *** p < 0.001). Levels of ROS in the UVB-exposed NHDFs were measured after 24 h of treatment with herbal extracts (b) and active compounds (c) using flow cytometry with the DCFH-DA dye. The relative ROS production of cells appears in each histogram. Values shown are the mean ± SD. # and * indicate the significant differences from the non-irradiated control and UVB-irradiated control groups. ^## p < 0.01 versus non-irradiated control. * p < 0.05 versus UVB-irradiated control.

To investigate the effects of the individual herbal extracts as well as their mixture on intracellular ROS production in UVB-irradiated NHDFs, NHDFs were stained with 30 µM DCFH-DA. As a result, ROS generation was significantly elevated to 178% by UVB exposure (Figure 1(b)). However, the enzyme-processed Panax ginseng and Gastrodia elata extracts, at the concentration of 100 µg/mL, slightly suppressed ROS production of UVB-irradiated group by 14.0% and 24.7%, respectively. Remarkably, the treatment of the UVB-exposed group with EPG exhibited synergistic effect, reducing the ROS level by 43.3% at 100 µg/mL, compared with the UVB-irradiated control treatment. Similar to the herbal mixture, the combination of their active compounds at the concentration of 10 µg/mL reduced the ROS level by 32.3% in comparison with the UVB-irradiated control group (Figure 1(c)).

Cell viability and MMP-1, IL-6, and procollagen type 1 secretion from UVB-irradiated NHDFs of EP, EG, and EPG

To examine the cytotoxicity of samples, MTT assay was conducted on NHDFs treated with EP, EG, and EPG at concentrations ranging from 1 to 100 μg/mL under UVB-irradiated condition. The above treatment did not significantly affect cell viability in the UVB-irradiated cells (Figure 2(a)).

Figure 2. Effect of EP, EG, and EPG on cell viability (a) and the production of MMP-1 (b and c), IL-6 (d and e), and procollagen type 1 (f and g). Values shown are the mean ± SD. #, *, and ^a indicate the significant differences from the non-irradiated control, the UVB-irradiated control groups, and single extract-treated groups. ^# p < 0.05, ^## p < 0.01 versus non-irradiated control. * p < 0.05, ** p < 0.01 versus UVB-irradiated control, ^a p < 0.05 versus single extract-treated groups.

The impact of the individual herbal extracts and their mixture on UVB-induced MMP-1, IL-6, and procollagen type 1 expression were continuously evaluated. In the non-irradiated groups, the individual supplement of EP and EG at 100 µg/mL reduced MMP-1 and IL-6 production by 35.6% and 37.1%, and 23.6% and 34.9%, respectively. In addition, under the presence of EP and EG at 100 µg/mL in culture medium, procollagen type 1 was upregulated by 55.0% and 80.0%, respectively. Moreover, the synergetic effect of the herbal extract diminished the level of MMP-1 and IL-6 to 46.9% and 48.6%, respectively; meanwhile, the expression of procollagen type 1 was raised significantly to 111.3%, compared with the non-treated cells (Figure 2(b, d, and f)). Under UVB irradiation, the production of MMP-1 and IL-6 was increased, whereas procollagen type 1 level was suppressed, compared with the non-irradiated group. In particular, MMP-1 and IL-6 secretion were increased by 122.6% and 84.7%, respectively, and the production of procollagen type 1 dropped to 57.2% (Figure 2(c, e, and g0). Nevertheless, single herbal extracts at 100 µg/mL diminished the levels of MMP1 by 27.5% and 24.2%, meanwhile, IL-6 level was inexpressively inhibited. As regards restoration of procollagen type 1, EP and EG treatment at 100 µg/mL enhanced the protein level by 110.9% and 134.3%, respectively. Noticeably, both of MMP-1 and IL-6 production were decreased by 47.7% and 29.4%, respectively, in EPG-treated cells at the highest concentration (100 µg/mL) (Figure 2(c and e)). Moreover, in the UVB-induced group, the EPG-treated cells exhibited a sharp rise in procollagen type 1 secretion (178.1%), compared with the UVB-irradiated control (Figure 2(g)). The individual herbal treatment also showed relative effects on the UVB-induced changes in the photo-aging markers; however, the results were not as efficient as the treatment of their mixture had been (Figure 2(c, e, and g)). These results demonstrate that both independent treatment of herbal extracts and their combined extract are capable of inhibiting collagen degradation by down-regulating the MMP-1 and IL-6 expression and enhancing the procollagen type 1 secretion in UVB-irradiated NHDFs, resulting in prevention the UVB-induced photo-aging effect on skin.

The effect of different-defined ratios of herbal extract mixture on MMP-1 production was also evaluated to confirm the superiority of 1:10 EPG extract. As expected, in the group without UVB treatment, MMP-1 expression reduced by 35.1% and 46.9% at 1:10 EPG concentrations of 10 and 100 µg/mL, respectively, being better than down-regulating effect of the remained ratios (Figure 3(a)). In UVB-irradiated group, UVB radiation increased MMP-1 level by 112.6%, compared to the non-irradiated cells. However, 1:10 EPG exhibited surpassing MMP-1 inhibition, resulting in protein level reduction of 23.6% and 47.7% at 1:10 EPG concentrations of 10 and 100 µg/mL, respectively (Figure 3(b)).

Figure 3. Effect of different ratios of EPG on the protein expression of MMP-1 on non-irradiated (a) and irradiated cells (b). Values shown are the mean ± SD. # and * indicate the significant differences from the non-irradiated control and the UVB-irradiated control groups. ^## p < 0.01 versus non-irradiated control. * p < 0.05, ** p < 0.01 versus UVB-irradiated control.

Cell viability and MMP-1, IL-6, and procollagen type 1 secretion from UVB-irradiated NHDFs of active compounds

The viability of UVB-irradiated NHDFs was evaluated by MTT assay after 72 h of treatment with ginsenoside F2 and α-gastrodin. UVB exposure decreased cell viability to approximately 89% of that of the non-irradiated fibroblasts. The treatment with active compounds slightly enhanced the cell viability, compared with the irradiated control. As shown in Figure 4(a), the cell viability of the experiment group treated with ginsenoside F2, α-gastrodin, and their mixture were 96%, 97%, and 108%, respectively, at the concentration of 10 μg/mL.

Figure 4. Effect of the active compounds and their 1:1 mixture on cell viability (a) and the production of MMP-1 (b and c), IL-6 (d and e), and procollagen type 1 (f and g). Values shown are the mean ± SD. #, *, and ^a indicate the significant differences from the non-irradiated control, the UVB-irradiated control groups, and single extract-treated groups. ^## p < 0.01, ^### p < 0.001 versus non-irradiated control. * p < 0.05, ** p < 0.01, *** p < 0.001 versus UVB-irradiated control, ^a p < 0.05 versus single extract-treated groups.

To investigate the effect of the active compounds on the secretion of MMP-1, IL-6, and procollagen type 1, NHDFs were treated with ginsenoside F2, α-gastrodin, and their mixture. Under non-irradiated condition, MMP-1 and IL-6 production were not markedly affected by the treatment of each compounds; however, α-gastrodin at 10 µg/mL promoted procollagen type 1 formation by 48.8%. Continuously, the synergetic of active components at 10 µg/mL significantly inhibited MMP-1 and IL-6 expression by 38.3% and 45.5%, respectively, whereas the same treatment up-regulated procollagen type 1 formation by 80% (Figure 4(b, d, and f)). In UVB-exposed group, MMP-1 and IL-6 secretion increased by 281.1% and 84.8%, respectively, while type 1 procollagen decreased by 33.5% in the UVB-irradiated control group (Figure 4(c, e, and g)). On the other hand, the treatment with the active components inhibited the over-secretion of MMP-1 and IL-6 in a dose-dependent manner. At the concentration of 10 µg/mL, single compounds reduced MMP-1 level by 61.7% and 54.2% (ginsenoside F2 and α-gastrodin, respectively). Single active compound-treated cells also showed marked increase in procollagen type 1 by 76.6% and 125.9% under 10 µg/mL ginsenoside F2 and 10 µg/mL α-gastrodin treatment, respectively. Although each compound did not present significant effect on IL-6 production of irradiated cells, the combination of ginsenoside F2 and α-gastrodin suppressed the expression of MMP-1 and IL-6 by 82% and 35%, respectively, while type 1 procollagen production was increased by 200%, compared with the positive control group (Figure 4(c, e, and g)).

Discussion

Nowadays, chemical compounds are no longer widely used due to their toxicity and adverse effects, such as allergic reactions or irritation [25]. Instead, herbal plants have been promoted as promising materials with biological and pharmacological properties and have been used for centuries as traditional drugs. Moreover, their derived and enzymatic compounds are being developed as new products bringing about greater effects in treatments [26]. Indeed, several studies have pointed out the protective and preventive effects of enzyme-processed extracts, as well as their hydrolyzed major active compounds against UVB-induced photo-aging by increasing the collagen expression, reducing MMP production and wrinkle formation, with no cytotoxicity both in vitro [27,28], and in vivo [29]. Among them, Enzyme-processed Gastrodia elata (EG) extract was demonstrated to be more effective than the non-modified extract in treating UVB-induced photo-aging due to the higher level of α-gastrodin (accounting for 0.42%) in terms of content [22]. Also, dietary Enzyme-processed Panax ginseng (EP) containing ginsenoside F2 which was not detected on original Panax ginseng extract reversed the inhibitory effect of UVB damage to filaggrin and procollagen type 1, reduced skin dryness, and increased moisture in irradiated mice [6]. Based on the studies on anti-photoaging effect of EP and EG on NHDFs of Hwang et al., 10 and 100 µg/mL were determined as the two most effective concentrations, respectively [19,22]. Consequently, the current study demonstrated that the herbal mixture at a Panax ginseng extract to Gastrodia elata extract ratio of 1:10 (w/w) was the commensurable to the effectiveness of reported working doses of the herbs. Besides, bio-transformed ginsenoside F2 and α-gastrodin which were enriched in Panax ginseng and Gastrodia elata extracts after enzymatic reaction, supposed to be the main pharmacologically active compounds of ginseng and possess anti-aging [19], antioxidant [30], antidepressant [31], anticancer [32], and pro-immune properties [33]. Thus, we hypothesized that the combination of the two enzyme-processed herbal extracts, as well as its bio-transformed active compounds, could result in synergistic effect on the prevention of UVB-induced damage in NHDFs.

Over-exposure to Ultraviolet (UV) radiation usually leads to oxidative stress, release of inflammatory mediators, and creates gene mutations, which in turn cause skin damage [34]. ROS is generated by rapid uptake of oxygen and then can release free radicals such as superoxide anion (O₂⁻•), perhydroxyl radical (HO₂•), hydroxyl radical (▪OH), nitric oxide, and other species such as hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), hypochlorous acid (HOCl), and peroxynitrite (ONOO⁻). ROS attacks the chain reactions of proteins and fatty acids resulting in significant cellular damage [35]. Hence, ROS is believed to play a key role in the process of initiating skin photo-aging [36,37]. In order to reduce ROS-induced damage, antioxidants are supplemented in the diet to balance free radicals by scavenging activities, aside from the development of antioxidant defense systems inside the body [38]. It is hypothesized that Protopanaxadiol (PPD) and Protopanaxatriol (PPT) components of Panax ginseng enroll in the ROS-scavenging process. PPD and PPT can inhibit the action of several enzymes which are responsible for the generation of ROS. These active compounds can activate or promote antioxidants which abolish the ROS species, such as glutathione and superoxide dismutase. Besides, gastrodin components in Gastrdia elata, which is a natural phenol, are known to significantly depress the levels of •OH, O2 •⁻, NO•, or OONO⁻ by acting as electron or hydrogen atom donors and to inhibit reactive species production by deactivating its precursor. In order to boosting the ROS inhibitory effect of the mentioned active compounds in plant extracts, the strategy of this study was analyzing the effective proportion between each herbal extract and estimating the superiority of the synergetic action. In this study, ROS production was increased by UVB-induced irradiation on NHDFs; however, it was delayed and reduced by supplement of the enzyme-processed herbal extracts and their bioactive components to the medium. Cells treated with 100 µg/mL herbal mixture and 10 µg/mL compound mixture described a dramatic reduction of ROS generation. Besides, individual plant extracts or active compounds also exhibited a decrease of ROS production, but this was not significant. Therefore, the synergetic of herbal extracts as well as their bio-transformed compounds enhanced inhibitory impact on ROS formation as expected.

Collagen type 1, which is the major component of the dermal Extracellular Matrix (ECM), accounts for approximately 90% of the proteins and creates the elasticity and integrity of the skin [14,39]. A precursor molecule of collagen type 1 is synthesized by dermal fibroblasts and is called procollagen type 1, which is then converted to collagen [40]. Collagen suppression is considered to be the main cause of wrinkle formation and skin aging [41]. Photo-aging is induced by chronic UVB exposure, which can reduce collagens by increasing ROS and the expression of activator protein (AP-1) signaling [42]. AP-1 is generated by c-Jun and c-Fos family proteins and activates the transcription of Matrix-Metalloproteinase (MMP). MMPs are a family of zinc-dependent endopeptidases, playing crucial roles in various pathophysiological processes including photo-aging, wound healing, skeletal growth and remodeling, arthritis, inflammation, angiogenesis, and cancer by the breakdown of the ECM [41,43,44]. Activated MMP-3 can activate MMP-1, which is responsible for initiating the degradation of collagen type 1, inhibiting the production of procollagen type 1 by blocking the regulation of transforming growth factor (TGF-β), and eventually destroying the structural integrity of the dermis [45–47]. Additionally, UVB irradiation also activates pro-inflammatory factors, such as IL-6 and tumor necrosis factor (TNF-α) [48]. It has been reported that IL-6 participates in inhibiting TGF-β, stimulating the expression of MMP levels (1, 3, and 9), resulting in the degradation of collagens [49]. A variety of studies indicate the antioxidant activity and protective effects against UVB-induced photo-aging of botanical extracts such as marigold [50], Camellia sinensis [51], and a mixture of rosemary and grapefruit [52] by inhibiting MMP overexpression, enhancing procollagen type 1 production and elasticity, and decreasing wrinkle formation. Similar to the reported results of inhibitory impacts on MMPs and interleukins production, and procollagen up-regulation of EP and EG in several studies [22,45,53,54], the current study presented the photo-protective effects of independent treatment EP, EG, ginsenoside F2, and α-gastrodin via proper regulations of the aging-related markers; however, EPG and their active component mixture showed the surpassing outcomes of procollagen type 1 up-regulation, and MMP-1 and IL-6 down-regulation, compared to the individual treatment of each sample.

Other studies have also shown the protective effects of herbal plants and their active compounds against skin photo-aging. Antioxidants can balance the oxidative stress, prevent oxidation, and protect skin from damage. A study on a mixture of citrus and rosemary (MCR) was conducted by Pérez-Sánchez et al. [55]. In this study, HaCaT cells were treated with various doses of MCR (12.5–100 µg/mL) under UVB irradiation. In the presence of citrus, rosemary, and MCR, cells promoted significant protection in a dose-dependent manner, especially the MCR, which maintained 70% and 60% of cell viability under 800 J/m² and 1200 J/m² UVB irradiation. The treatment of MCR at 75 and 100 µg/mL also dramatically decreased the ROS synthesis by 64% and 71%, respectively, compared with irradiated control group. In the present study, ROS production was significantly inhibited by 43% with the treatment of EPG at 100 µg/mL compared with the irradiated cells. On the other hand, the study of Gęgotek et al. which research on the synergistic effect of bioactive compounds including ascorbic acid and rutin, resulting in a slight increase in cell viability in UVB-induced fibroblasts [56]. In addition, the combined action of ascorbic acid and rutin was exhibited to be more conducive than separate compound action in reducing ROS production in animal tissues [57]. Another study demonstrated that the combination of quercetin and resveratrol significantly decreased TNF-α-stimulated IL-6 secretion, whereas quercetin and resveratrol separately did not inhibit the release of this cytokine [58]. The current study also presented a similar improvement when UVB-irradiated NHDFs were treated with compound mixture instead of single components. Therefore, these results strengthened our hypothesis about the potential effects of EPG and the bio-transformed compound mixtures in the treatment of photo-aging as safety products.

n conclusion, this study demonstrated that EPG and their active compounds may inhibit the photo-aging process by the up-regulation of procollagen type 1 and the down-regulation of MMP-1 and IL-6; simultaneously limiting the production of intracellular ROS without damaging cells. The results have opened a new direction on studying bio-transformed compounds from herbal plants treated with enzymes. To gain better knowledge about anti-aging signaling pathways of EPG and their active compounds in an artificial skin model and clinical trials, follow-up experiments are required.

Contribution of authors

The experiments were designed by H.T.T.N, E.H., and T-H.Y. The experiments were performed by H.T.T.N, S.a.S, and J-E.Y. The data were analyzed by H.T.T.N, E.H., Q.T.N.N, and N.Q.D. The manuscript was edited by H.T.T.N, Q.T.N.N, and N.Q.D. The experiment was supervised by T-H.Y.

Disclosure statement

The authors have no conflicts of interest to declare.