Antiphotoaging properties of Zingiber montanum essential oil isolated by solvent-free microwave extraction against ultraviolet B-irradiated human dermal fibroblasts

Abstract

Maintaining youthful skin from photoaging with natural products, including essential oils, is a vital strategy that has piqued the interest of researchers in the pharmaceutical and cosmetic industries. This research aimed to investigate the protective properties of Zingiber montanum (J. Koenig) Link ex A. Dietr. essential oil against ultraviolet B (UVB)-induced skin damage and photoaging in normal human dermal fibroblast (HDFn) cells. The essential oil was extracted from fresh plant rhizomes using solvent-free microwave extraction. Its antiphotoaging properties in HDFn cells were investigated using reactive oxygen species (ROS)-scavenging, wound healing, matrix metalloproteinases (MMP-1, MMP-3, and MMP-9) expression, procollagen synthesis, and elastase and tyrosinase inhibitory assays. The results showed that the test oil exhibited no significant toxicity in HDFn at concentrations up to 10 mg/mL, with cell viability exceeding 90%. Following UVB irradiation at 30 mJ/cm², Z. montanum oil demonstrated time and concentration-dependent ROS radical scavenging capabilities. In a cell migration assay, the essential oil demonstrated wound-healing properties. Z. montanum oil suppressed the expression of MMPs and enhanced the synthesis of type I procollagen at a concentration of 0.1–1 mg/mL. In addition, 0.1–1 mg/mL Z. montanum oil inhibited elastase activity in a concentration-dependent manner but did not affect tyrosinase activity. From these findings, the essential oil of Z. montanum could have potential applications in developing cosmeceutical products to prevent skin photoaging.

Introduction

Skin aging is a serious medical issue that is linked to the inevitability of human aesthetics. This is a complicated process marked by progressive loss of structural integrity, elasticity, laxity, and wrinkle formation caused by intrinsic and extrinsic factors [1]. Intrinsic skin aging is associated with internal physiological factors like genetics, hormones, and cellular metabolism that occur as humans age [2]. On the other hand, extrinsic skin aging is primarily caused by chronic exposure to ultraviolet (UV) and infrared radiation, pollution, and cigarette smoking [3]. Skin aging and photoaging have caught scientists’ attention worldwide. Excessive acute UV exposure causes sunburn, acute inflammation, structural skin tissue damage, and skin tanning due to UV stimulation of melanin production. Most people experience these effects within a few days of exposure to UV light [4]. In contrast, chronic UV exposure causes degenerative changes in the skin cells, fibrous tissue, and blood vessels, accelerates the gradual loss of skin elasticity, and results in wrinkles and photoaging [5].

The molecular mechanism of skin photoaging caused by excessive UV exposure is summarized below. Excessive UV exposure promotes the formation of intracellular reactive oxygen species (ROS), which increases the activity of matrix metalloproteinases (MMPs) by regulating gene expression and proenzyme activation [1, 6, 7]. MMPs are a family of zinc endopeptidases with proteolytic activity against dermal collagen the most abundant protein in connective skin tissue and the main structural component of the extracellular matrix, elastic fibers, and glycosaminoglycans [8]. ROS mediates switching MMP (MMP-2, MMP-3, MMP-9, and MMP-13) expression via the mitogen-activated protein kinase (MAPK) signaling pathway [9, 10]. This pathway involves the increased activation of nuclear factor-κB (NF-κB) and activator protein 1 (AP-1), which in turn upregulate MMP expression, and increase skin wrinkles by inducing extracellular matrix degradation [11–13]. Targeting ROS, MMP expression, elastase activity, and collagen (I, III, and VII) synthesis are, thus, at the forefront of photoaging skincare strategies.

For perspective, the global cosmetic market was worth $380.2 billion in 2019 and is expected to reach $463.5 billion by 2027. This suggests a compound annual growth rate (CAGR) of 5.3% over the 6 years from 2021 to 2027 [14]. Because of their important biological properties, natural ingredients, including essential oils, have piqued the pharmaceutical and cosmetic industries’ interest in combating photoaging [15–17]. Zingiber montanum (J. Koenig) Link ex A. Dietr. (Zingiberaceae family), also known as cassumunar ginger, is native to Southeast Asia and has been widely cultivated for various medicinal purposes throughout tropical Asia [18–20]. In many Asian countries, including China, India, Indonesia, Laos, Malaysia, Thailand, the Philippines, and Vietnam, the dermal and oral routes of administration and application of the herbal medicinal products containing bioactive ingredients from this plant species are among the most famous traditional and modern medicinal treatments [20]. A recent systematic review and meta-analysis of the clinical effects of Z. montanum has been published. The study concluded that creams containing 14% w/w of this plant’s essential oil significantly reduced muscle pain and ankle sprain [18].

The anti-inflammatory properties of this herb are well documented. Its curcuminoids attenuated oxidative stress caused by H₂O₂ in rat thymocytes [21]. In lipopolysaccharide-induced gingival fibroblast inflammation, the ethanolic extract of Z. montanum suppressed the expression of cyclooxygenase-2 and MMP-2 by inhibiting the activation of the proinflammatory pathway, phosphorylation of ERK1/2 and JNK, and p38 MAPK [22]. Terpinen-4-ol, α-terpinene, and (E)-1-(3,4-dimethoxyphenyl)but-3-en-1-ol inhibited the activity of cyclooxygenase and lipoxygenase, two enzymes responsible for the inflammatory process, in carrageenan-induced paw edema in rats [19, 23–25]. 4-[2,4,5-trimethoxy-phenyl)but-1,3-diene and cassumunaquinone suppressed the expression of nitric oxide (NO) and interleukin-1 (IL-1) in lipopolysaccharide-induced inflammation in mouse macrophages [26]. The ethanolic, hydrophilic extract, and essential oil of Z. montanum inhibited the activity of β-hexosaminidase, a mediator released from activated mast cells involved in the allergic response with an IC₅₀ of 12.9 μg/mL [27]. The crude extract of Z. montanum and (E)-4-(3´,4´-dimethoxyphenyl)but-3-en-2-ol inhibited proMMP-9 cleavage and attenuated the PMA-induced MMP-9 gene expression and release from human lung mucoepidermoid carcinoma (NCI-H292) cells [28]. Furthermore, (E)-4-(3´,4´-dimethoxyphenyl)but-3-en-2-ol inhibited the expression of genes involved in cartilage erosion MMPs (MMP-1, 3, and 13) and IL-1β, a unique inflammatory cytokine in human synovial fibroblast cells [29].

The first cosmetic potentials of the petroleum ether and ethyl acetate extracts from this plant rhizomes containing some phenylbutenoids, namely (E)-3(3,4-dimethoxyphenyl)-2propenal, (Z)-3-(3,4-dimethoxyphenyl)-4-[(E)-2,4,5-trimethoxystyryl]cyclohex-1-ene, 1-feruloyloxy cinnamic acid, (1E,4E,6E)-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one, bisdemethoxycurcumin, and curcumin, have recently been identified as antiaging, skin whitening, and anti-inflammatory agents [30]. Apart from these lipophilic extracts, the rhizomes of this herb contain 0.57–2.10% v/w essential oil, primarily consisting of sabinene (25–45%), γ-terpinene (5–10%), α-terpinene (2–5%), terpinen-4-ol (25–45%), and (E)-1-(3,4-dimethoxyphenyl)butadiene (1–10%), and exhibit strong anti-inflammatory properties [23, 29, 31].

Our ongoing research focused on searching for natural products with antiphotoaging; an eco-friendly solvent-free microwave extraction was applied to optimize this plant’s essential oil extraction process. The findings suggested its advantage over the oil isolated using a conventional hydrodistillation method in bioeconomy and sustainable pharmaceutical development. A closer look at the literature revealed some gaps. There is limited literature regarding the antiphotoaging effects of essential oil isolated from this plant material. Thus, the present study aimed to investigate the antiphotoaging properties of Z. montanum essential oil isolated by solvent-free microwave extraction. Its molecular mechanisms related to the inhibition of wrinkle formation caused by extrinsic factors were elucidated using ultraviolet B (UVB)-irradiated normal human dermal fibroblast (HDFn) cells. This will highlight the potential mechanisms responsible for the essential oil’s antiphotoaging properties and its ability to maintain youthful skin.

Materials and methods

Chemicals

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin–EDTA solution were purchased from Gibco Invitrogen Corporation (Grand Island, NY, USA) and EnzChek^® elastase assay kit was purchased from Molecular Probes (Eugene, OR, USA). Assay kits for MMP-1, MMP-3, MMP-9, type Iα procollagen, 2´,7´-dichlorofluorescein diacetate (DCFH-DA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), l-DOPA, kojic acid, N-acetylcysteine (NAC), mushroom tyrosinase, and C₈–C₂₀ alkane homolog standard solution were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other reagents and solvents used were of the highest grade available.

Extraction of essential oil from Z. montanum rhizomes

The essential oil of Z. montanum was prepared per the method previously described [32]. Briefly, cultivated 2-year-old Z. montanum rhizomes (voucher specimen no. BCY UBU 2016031) was then sliced into small pieces, and the essential oil was extracted immediately at 700 W and 2.45 GHz for 70 min using solvent-free microwave extraction (UBU01, Microwave extraction system, Ubon Ratchathani University, Ubon Ratchathani, Thailand). The essential oils collected from a Clevenger-type apparatus were dehydrated with anhydrous sodium sulfate and kept at 4 °C for subsequent analysis.

Gas chromatography-mass spectrometry (GC–MS) analysis of essential oil

The chemical composition of the essential oil was analyzed by GC–MS using the method developed by this group of authors with some modifications [33]. The essential oil was diluted in n-hexane (1% v/v) (1 μL) (1:50 split ratio) and injected into GC–MS (Clarus 680 GC/SQ8 MS model, Perkin Elmer Inc., MA, USA). A fused silica capillary column (Elite-5MS; 30 m length × 0.32 mm diameter × 0.25 μm film thickness) was used to separate the volatile compounds in the sample. The temperature program was set by holding at 50 ºC for 5 min, then increasing linearly from 50 to 250 ºC (3 ºC/min), and finally holding at 250 ºC for 5 min. Other operating conditions included: carrier gas, helium; flow rate, 1.2 mL/min; electron ionization energy, 70 eV; injection temperature, ion source temperature, and interface temperature, 250 ºC; interface temperature, 250 ºC; mass scan range, m/z 45–500 amu. Each individual quantified oil component was identified based on mass fragmentation pattern, linear retention indices related to C₈–C₂₀ alkane homologs on an Elite-5MS column, and mass fragmentation pattern obtained from literature and the National Institute of Standards and Technology (NIST) electronic library [34]. The analysis was performed with Turbo Mass 6.1.0 software, and the proportion of each volatile compound was calculated based on the relative peak area in the total oil.

Cytotoxicity study

The cytotoxicity of Z. montanum essential oil was evaluated in vitro using a mitochondrial-based MTT cell viability assay [35]. The HDFn cells (ATCC, Manassa, VA, USA) (5 × 10³ cells/well) were seeded in a 96-well culture plate and incubated overnight at 37 °C in a CO₂ incubator. The cells were then treated with various concentrations of essential oil (0–10 mg/mL) for 24 h. Nontreated control cells were incubated in media containing dimethyl sulfoxide (DMSO) (0.1% final concentration), which was used to dissolve the oil. Following incubation, 100 μL of 0.5 mg/mL MTT solution in culture medium was added to each well, and the cells were incubated for an additional 3 h. The resulting formazan was dissolved in 100 μL of DMSO. Optical density was measured at 570 nm using a PowerWave XS2 microplate reader (BioTek, Winooski, VT, USA). Relative cell viability of treatment was expressed as a percentage of the vehicle control group.

Next, HDFn cells were cultured overnight in a 96-well culture plate to determine the protective effects of essential oil against UVB-induced damage. Following pretreatment with the essential oil at various concentrations for 3, 6, or 12 h, the cells were irradiated with 30 mJ/cm² of UVB for 2 h, and cultured in fresh serum-free DMEM. MTT assay was performed 24 h after irradiation. NAC solution (5 mM) served as the positive control.

Cell proliferation assay

The HDFn cells were seeded at a density of 5 × 10³ cells/well in 96-well plates and incubated overnight. The cells were serum-starved for 12 h and then incubated with essential oil (0–1 mg/mL) and supplemented serum-free medium. At 24, 48, and 72 h of incubation, 0.5 mg/mL MTT solution was added and the cells were incubated for another 3 h at 37 ºC. The supernatant was aspirated, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was then measured at 570 nm using a PowerWave XS2 microplate reader. All experiments were performed in triplicate, and cell proliferation was then calculated.

Intracellular ROS study

Intracellular ROS levels were determined using the DCFH-DA fluorescent staining assay [36]. HDFn cells were seeded in 96-well black plates (5 × 10³ cells/well) and incubated in a CO₂ incubator overnight at 37 ºC. The cells were then pre-treated with essential oil (0–1 mg/mL) for 3, 6, or 12 h before being exposed to 50 μM of DCFH-DA for 40 min at 37 ºC, followed by 2 h of UVB-irradiation at 30 mJ/cm². The fluorescent intensity of 2´,7´-dichlorofluorescein was detected using a PowerWave XS2 microplate reader at excitation and emission wavelengths of 485 nm and 528 nm, respectively. The fold of fluorescence intensity (ROS generation) was compared to that of control cells that had not been treated with essential oil. Positive control was performed on the cells pre-treated with an NAC solution (5 mM).

Wound healing assay

HDFn cells were grown in 24-well plates (2 × 10⁵ cells/mL) overnight for the wound healing assay [37, 38]. After the cells had reached confluence, a wounded area in the culture dish was created by scratching a straight line in the fibroblast with a sterile 10 μL pipette tip. The cells were cultured for another 24 h with essential oil (0–1 mg/mL), and the percentage of wound closure was determined by measuring the wound area at 0, 6, 12, and 24 h after the injury. The cells were observed under a converted microscope, and the wound closure area/original wound area ratio was calculated using the Image-Pro Plus 4.5 software (Media Cybernetics, Inc., MD, USA).

Determination of MMPs secretions by ELISA

HDFn cells were seeded into 6-well plates at a density of 1 × 10⁵ cells/well and cultivated for 24 h. The cells were then treated with essential oil (0–1 mg/mL) in serum-free medium for 6, 12, and 24 h before 30 mJ/cm² of UVB irradiation for 2 h. After UVB irradiation, the cell-free supernatants were collected from each well and centrifuged at 15,000 rpm for 15 min. The MMP-1, 3, and 9 levels in the culture media were assessed using Sigma MMP-1, 3, and 9 ELISA kits, according to the manufacturer’s instructions. Cells were treated with l-ascorbic acid solution (200 μg/mL), which served as a positive control.

Type Iα procollagen assay

HDFn cells were incubated in 6-well plates at a density of 1 × 10⁵ cells/well, and the cells were then treated with essential oil (0–1 mg/mL) in a serum-free medium for 48 h. After incubation, the cell-free supernatant was collected from each well, and the collagen content was determined using a type Iα assay kit (Cambridge, MA, USA) following the manufacturer’s instructions. l-ascorbic acid solution (200 μg/mL) was used as a positive control, and the results were normalized with confluent cell numbers.

Elastase inhibitory test

Elastase inhibition activity was assessed using the EnzChek^® elastase assay kit (Thermo Fisher Scientific, MA, USA), following the manufacturer’s instructions. In brief, porcine pancreatic elastase, DQ^® elastin substrate, and various concentrations of the essential oil (0–1 mg/mL) were separately dissolved in pH 8 Tris–HCl buffer. Diluted samples (50 μL) were preincubated with 0.4 U/mL porcine pancreatic elastase (100 μL) for 15 min. After that, 50 μL of 0.1 mg/mL DQ^® elastin was then added to the mixture, and the samples were incubated with light protection for 30 min. At the end of incubation, the fluorescence intensity was measured, with the excitation and emission wavelengths at 485 nm and 538 nm, respectively. Epigallocatechin gallate (EGCG) in a concentration of 250 μM was used as a positive inhibitor. The ability of essential oil to inhibit elastase activity was calculated as follows:

$$\text{Inhibition}\left(\%\right)=\left[\frac{\left(C-D\right)}{\left(A-B\right)}\right]\times 100$$

where, A denotes the absorbance of blank after incubation; B represents the absorbance of test sample before incubation; C is the absorbance of the test sample after incubation; D is absorbance without test sample before incubation.

Mushroom tyrosinase inhibitory study

Tyrosinase inhibition activity was assessed using diphenolase activity assay described in a previous study with minor modification [39]. Briefly, 40 μL of 10 mM l-DOPA was mixed with 80 μL of 0.1 M phosphate buffer pH 6.8 in a 96-well culture plate. Thereafter, 40 μL of essential oil (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 5 mg/mL) in 10% DMSO and 40 μL of 250 U/mL in PBS of mushroom tyrosinase were added to each well on the plate. The reaction mixture was incubated on ice for 10 min, and the absorbance characteristics of the reaction mixtures were measured at 475 nm using a PowerWave XS2 microplate reader at 1 min intervals for 15 min. PBS and kojic acid (50 μg/mL) were used as a negative blank and positive control, respectively. The inhibition of tyrosinase was calculated as follows:

$$\text{Inhibition}\left(\%\right)=\left[\frac{{\text{Abs}}_{\mathit{control}}-{\text{Abs}}_{\mathit{sample}}}{{\text{Abs}}_{\mathit{control}}}\right]\times 100$$

where, Abs_control and Abs_sample represent the absorbance of control and sample, respectively.

Statistical analysis

All results are presented as mean ± standard deviation (SD) for three independent experiments. Statistical analysis was performed using the SPSS version 21 software (IBM Corporation, North Castle, NY, USA). Different means were analyzed using the one-way analysis of variance (ANOVA) followed by a Tukey’s test for multiple comparisons. Differences were considered statistically significant at p < 0.05.

Results

Chemical composition of Z. montanum essential oil

The yield of essential oil isolated from Z. montanum rhizomes via solvent-free microwave extraction was 1.22 ± 0.10% v/w with a pale-yellow color. Figure 1a, b shows the total ion GC–MS chromatogram of this essential oil. A total of 29 different types of oil compounds were identified to contain 97.78 ± 0.63% of the oil. Six major components, including sabinene (32.40 ± 0.08%), terpinen-4-ol (29.85 ± 0.02%), (E)-1-(3,4-dimethoxyphenyl)butadiene (17.42 ± 0.09%), γ-terpinene (5.11 ± 0.10%), α-terpinene (2.30 ± 0.02%), and β-pinene (2.05 ± 0.10%) were discovered in the oil. Additionally, this oil contained significant amounts of various minor constituents of compounds that contributed less than the major components (Table 1). Based on three independent experiments, the proportions of monoterpene hydrocarbons, sesquiterpenes, and oxygenated compounds were calculated to be 48.01 ± 0.28%, 0.19 ± 0.02%, and 49.57 ± 0.33%, respectively. The complex combination of these constituents in Z. montanum essential oil produces a cool, earthy, herbaceous, and woody odor.

Fig. 1.

Total ion gas chromatography chromatogram showing volatile components in Z. montanum essential oil isolated by solvent-free microwave extraction: a total ion chromatogram of essential oil, and b zoomed-in version of the GC chromatogram

Table 1.

Chemical composition of the essential oil of Z. montanum rhizomes isolated by solvent-free microwave extraction

No.a	Compound	RIexpb	RIlitc	Relative peak area (%)	Identification methodd	References
1	α-Thujene	923	924	0.35 ± 0.01	RI, MS	[32]
2	α-Pinene	929	932	1.34 ± 0.02	RI, MS, Co	[32]
3	Camphene	949	946	0.10 ± 0.01	RI, MS	[32]
4	Sabinene	967	969	32.40 ± 0.08	RI, MS	[32]
5	β-Pinene	979	974	2.05 ± 0.10	RI, MS	[32]
6	β-Myrcene	990	988	0.82 ± 0.03	RI, MS	[32]
7	α-Phellandrene	1003	1002	0.05 ± 0.02	RI, MS	[32]
8	α-Terpinene	1015	1014	2.30 ± 0.02	RI, MS, Co	[32]
9	p-Cymene	1021	1020	0.95 ± 0.04	RI, MS	[32]
10	β-Phellandrene	1024	1025	1.95 ± 0.07	RI, MS	[32]
12	γ-Terpinene	1053	1054	5.11 ± 0.10	RI, MS	[32]
13	(Z)-Sabinene hydrate	1066	1065	0.44 ± 0.03	RI, MS	[32]
14	Terpinolene	1085	1086	0.59 ± 0.03	RI, MS	[32]
15	(E)-Sabinene hydrate	1097	1098	0.41 ± 0.03	RI, MS	[32]
17	(E)-p-Menth-2-en-1-ol	1036	1135	0.32 ± 0.02	RI, MS	[32]
18	Borneol	1166	1165	0.22 ± 0.01	RI, MS	[32]
19	Terpinen-4-ol	1175	1174	29.85 ± 0.02	RI, MS	[32]
21	(Z)-Piperitol	1195	1195	0.07 ± 0.06	RI, MS	[32]
22	(E)-Piperitol	1206	1207	0.30 ± 0.06	RI, MS	[32]
23	α-Terpinyl acetate	1348	1346	0.10 ± 0.01	RI, MS	[32]
24	α-Zingiberene	1492	1493	0.08 ± 0.00	RI, MS	[32]
26	β-Sesquiphellandrene	1522	1521	0.03 ± 0.02	RI, MS	[32]
27	Germacrene B	1548	1543	0.09 ± 0.04	RI, MS	[32]
28	(E)-1-(3,4-Dimethoxyphenyl)but-1-ene	1591	1592	0.45 ± 0.04	RI, MS	[32]
29	(E)-1-(3,4-Dimethoxyphenyl)butadiene	1637	1636	17.42 ± 0.09	RI, MS, Co	[32]
	Total identified compounds (%)			97.78 ± 0.63
	Essential oil yield (% v/w)			1.22 ± 0.10

^aThe missing compounds were discovered in trace amounts with < 0.01% relative peak area in the total oil. These compounds included 11) 1,8-cineole (RI_exp = 1027 and RIl_it = 1026); 16) (Z)-p-Menth-2-en-1-ol (RI_exp = 1118 and RIl_it = 1118); 20) α-terpineol (RI_exp = 1191 and RIl_it = 1190); and 25) β-bisabolene (RI_exp = 1507 and RIl_it = 1509)

^bRI_exp, retention indices determined on the Elite-5MS capillary column relative to the C₈-C₂₀ alkane homolog

^cRI_lit, literature retention indices [34, 76]

^dMS, identification based on the comparison of mass spectral data to those from computer mass libraries and the NIST 11 library; RI, literature retention indices [34, 76]; Co, co-injection with a laboratory-available authentic standard

Cytotoxicity evaluation

The MTT assay was used to assess the cytotoxicity of essential oil on HDFn cells. The test sample had no toxic effects on HDFn cells at concentrations up to 10 mg/mL, with cell viability exceeding 90%; thus, the IC₅₀ value of the essential oil could not be determined based on the data (Fig. 2a). Consequently, experiments on the protective effect of essential oil against UVB-induced skin aging used concentrations of up to 1 mg/mL. As illustrated in Fig. 2b, pretreatment of HDFn cells with oil at 0.001–1 mg/mL resulted in concentration-dependent attenuation of UVB-induced cytotoxicity. At these concentrations, the cytoprotective effects of the essential oil were less than that of 5 mM NAC, which restored cell viability (~ 80%) almost to that in untreated control cells. Despite the differences, these findings support the notion that the test oil has cytoprotective properties against UVB-induced HDFn cell death.

Fig. 2.

The cytotoxicity of Z. montanum essential oil in HDFn cells with or without UVB irradiation. a The viability of HDFn cells after 24 h of treatment with various concentrations of essential oil. b The viability of HDFn cells after 3, 6, or 12 h pre-treatment with various essential oil concentrations or 5 mM N-acetylcysteine (NAC) solution, followed by a 2 h exposure to 30 mJ/cm² UVB. Cell viability was determined by MTT assay. Data were expressed as the means ± SD of three independent experiments. Different letters above the bars represent statistically significant differences between treatments (p < 0.05)

Cell proliferation assessment

The HDFn cells were monitored with an MTT assay, which gauged the degree of the effect of essential oil on cell proliferation. It was discovered that while the oil did not cause cytotoxicity, it affected cell proliferation. Compared to untreated control cells, HDFn cells treated with essential oil 0.001–1 mg/mL increased cell proliferation dose and time-dependently, reaching a maximum of 40% at 0.1 mg/mL after 72 h (Fig. 3a).

Fig. 3.

a The effect of different concentrations of Z. montanum essential oil on the proliferation of HDFn cells was determined by MTT assay at 24, 48, and 72 h. b The protective effects of Z. montanum essential oil against the formation of reactive oxygen species (ROS). The HDFn cells were pre-treated for 3, 6, or 12 h with various essential oil concentrations or N-acetylcysteine (NAC) before being exposed to a 30 mJ/cm² for 2 h. Intracellular ROS levels were measured by the DCFH-DA assay. The data were expressed as the means ± SD of three independent experiments. Different letters above the bars denote statistically significant differences in treatments (p < 0.05)

Inhibitory effect of essential oil on intracellular ROS formation

The ROS-scavenging activity of essential oil was determined to evaluate its protective effects against oxidative damage. UVB at 30 mJ/cm² caused a 4-fold increase in ROS compared to untreated cells (p < 0.05), and the effects were concentration-dependent but not time-dependent. The ROS-scavenging effect of Z. montanum essential oil, however, was significantly lower than that of 5 mM NAC (Fig. 3b).

Scratch wound recovery

In the essential oil treatment group, cells exhibited an accelerated rate of wound closure compared with the untreated control. An in vitro scratch wound recovery assay revealed that the original wound area reduced by about 20–30% after treatment with oil at 0.01–1 mg/mL, in both dose and time-dependent manners, compared with the untreated control group. However, at 24 h, the original wound gap was not completely closed in both the essential oil-treated and untreated control HDFn cell monolayers (Fig. 4a, b). Additional evidence points to the conclusion that essential oil enhances HDFn cell migration.

Fig. 4.

Effects of Z. montanum essential oil on scratch wound healing of confluent HDFn cells: a micrographs of HDFn cells and wound closure at different treatment time intervals; and b Wound closure area/original wound area ratio. Cells cultured in 24-well plates were mechanically scratched with a sterile 10 μL pipette tip before being kept at 37 ºC for 0, 6, 12, and 24 h in the presence or absence of essential oil. Data were expressed as the means ± SD of three independent experiments. Different letters above the bars indicate statistically significant differences between treatments (p < 0.05)

Inhibitory effect against MMPs expression

Skin wrinkles are induced by natural aging and photoaging; thus, the activity level of MMPs (MMP-1, 3, and 9) on collagen degradation was determined using ELISA kits. UVB-irradiated cells had more than four times the MMP-1, MMP-3, and MMP-9 expression levels compared to nonirradiated cells. However, when the cells were treated with 0.01–1 mg/mL of essential oil for 6, 12, and 24 h, the MMP expression levels were significantly reduced time- and concentration-dependently, compared with UVB irradiated cells (p < 0.05). MMP expression was markedly decreased when the cells were pretreated for 24 h before UVB irradiation with 1 mg/mL essential oil (p < 0.05) (Fig. 5a–c).

Fig. 5.

The effect of Z. montanum essential oil on the expression of MMPs and the synthesis of type I procollagen in UVB-irradiated HDFn cells: a MMP-1 expression level; b MMP-3 expression level; c MMP-9 expression level; and d type I procollagen synthesis. The data are expressed as the means ± SD of three independent experiments. Different letters above the bars denote statistically significant differences in treatments (p < 0.05)

Enhanced type I procollagen synthesis

Procollagen synthesis is a critical factor with a direct impact on skin regeneration. HDFn cells were treated with essential oil at nontoxic concentrations to examine its effect on collagen synthesis. Essential oil of Z. montanum at 0.1 (1587.62 ± 61.66 pg/mL) and 1 mg/mL (1907.16 ± 47.54 pg/mL) significantly improved type I procollagen production in a concentration-dependent manner when compared with the untreated control (789.59 ± 99.43 pg/mL) (p < 0.05). Nonetheless, the essential oil induced significantly less type I procollagen production than 200 μg/mL l-ascorbic acid solution (2799.50 ± 122.47 pg/mL) (p < 0.05), the positive control used in the study (Fig. 5d).

Elastase inhibition of essential oil

The anti-elastase activity of essential oil was tested in vitro using DQ^® elastin substrate, with EGCG as a positive control. The essential oil of Z. montanum at the concentrations of 0.1 and 1 mg/mL significantly inhibited elastase activity by 21.53 ± 0.91% and 27.80 ± 2.11%, respectively, compared with negative control (p < 0.05). This potency was weaker than that of EGCG, which inhibited elastase activity by 77.39 ± 2.48% (Fig. 6).

Fig. 6.

Antielastase property of Z. montanum essential oil. Data were expressed as the means ± SD of three independent experiments. Different letters above the bars indicate statistically significant differences between treatments (p < 0.05)

Tyrosinase inhibition of essential oil

In the presence of molecular oxygen, tyrosinase catalyzes two distinct reactions: the hydroxylation of monophenol (monophenolase activity) and the oxidation of o-diphenol to o-quinone (diphenolase activity) [40–42]. In the current study, the effect of essential oil on mushroom diphenolase activity was investigated using l-DOPA as a substrate and kojic acid as a reference standard. There were no significant changes in absorbance (p < 0.05), indicating that the essential oil does not interact with l-DOPA or inhibit the tyrosinase activity (data not shown). By contrast, kojic acid inhibited tyrosinase strongly, with an IC₅₀ value of 47.63 ± 0.81 μg/mL.

Discussion

Although lipophilic extracts (not essential oil) of this herb have been shown to have cosmetic potential as anti-aging agents [30], research on the antiphotoaging activity of the essential oil of Z. montanum lacks in the literature. The antiphotoaging effects of Z. montanum essential oil on skin cells exposed to UVB irradiation were evaluated in this study. This is the first investigation of Z. montanum oil extracted using solvent-free microwave extraction. The selected essential oil has some advantages over the oils obtained through conventional extraction methods, as previously reported, in terms of sustainable chemistry and pharmacy, as well as bioeconomy [32]. In terms of contents of major components, solvent-free microwave extracted essential oil had a higher proportion of oxygenated compounds than hydrodistilled essential oil (data not shown). This observation agrees with Yingngam and Brantner [32], which was similar to that investigated in this study.

The process of skin damage caused by chronic UV light exposure is known as photoaging. Photoaging refers to the process of skin damage caused by chronic UV light exposure [43]. UVB (290–320 nm wavelength), which can penetrate the epithelial layer and cause damage to dermal fibroblasts, plays a vital role in skin photoaging. Dermal fibroblasts are well-known for producing an extracellular matrix, which helps to maintain skin thickness and elasticity. As a result, photodamage hastens the appearance of visible wrinkles on the skin. This complex progression inhibits the down-regulation of type I procollagen expression and collagen synthesis [44, 45]. Because there are various legal and ethical constraints on studying aging in humans, model organisms and cellular model systems are essential. The UVB-irradiated HDFn cell model was, thus, chosen for this work because its potential has been extensively described in the dermo-cosmetic field [46, 47]. Since these cells are easily grown and susceptible to numerous age-inducing stimuli, HDFn in monolayer culture is regarded as a suitable model for researching this extrinsic aging aspect. The test oil did not cause cytotoxicity when cells were treated with the sample at concentrations of up to 10 mg/mL (Fig. 2a), indicating its safety for dermal use. These findings are in line with those found in the literature, where the essential oil of Z. montanum at 14% w/w is recommended for pain management in topical market products [18].

The essential oil of Z. montanum increased the differentiation and proliferation of HDFn cells. This effect was dose and time-dependent, with a maximum increase of more than 40% compared with untreated control cells (Fig. 3a). Surprisingly, pretreatment with essential oil prevented UVB-induced cell death in HDFn (Figs. 2b and 3a). The formation of ROS was then assessed using a DCFH-DA staining assay [36]. The intracellular ROS levels increased by more than 4-fold; however, Z. montanum essential oil caused a reduction of UVB-induced ROS production (Fig. 3b). This is an intriguing discovery and suggests that when the essential oil is incorporated into topical formulations, it may have antiphotoaging properties against UVB irradiation.

UVB-induced skin aging is associated with the generation and accumulation of intracellular ROS, which triggers various signaling pathways, including those involving MAPKs. This pathway mediates the activation of the transcription factors NF-κB, AP-1, and the expression of inflammatory cytokines such as COX-2, IL-6, and MMPs [48–50]. Chronic UVB exposure induces the expression of MMPs, the main extracellular matrix enzymes involved in collagen degradation and the inhibition of procollagen synthesis. Among the several MMPs isoforms found in normal skin, only MMP-1, 3, and 9 are significantly induced in response to UV irradiation [51, 52]. MMP-1, also known as collagenase, degrades fibrillar collagens type I and III into specific fragments at a single site within the central triple helix. MMP-3 degrades type I collagen and activates MMP-1 and MMP-9, whereas MMP-9 degrades type IV collagen [8, 52].

ROS mediates MMP overexpression, thereby regulating the degradation of the connective tissue’s structural components. This is a critical step in the progression of skin damage and the formation of wrinkles [53–55]. It has been demonstrated that inhibiting MMP activity and expression prevents UVB-induced photoaging [7, 56–59]. Additionally, changes in elastin level, a major component contributing to tissue resilience and elastics, have been proposed during the photoaging process [60–62]. Although only trace amounts of elastin are degraded under normal physiological circumstances, the rate of degradation and elastin fragmentation increases with UV exposure. Age has been shown to cause elastin degradation by activating enzymes in the photoaging pathway—elastin loss results in the appearance of wrinkles on the skin [63, 64]. The essential oil of Z. montanum suppressed intracellular ROS production in HDFn cells, implying that this extract protects the skin from UVB irradiation because ROS plays a critical role in the UVB-irradiation induced photoaging process.

A variety of factors cause delays in wound healing due to aging, mainly an altered inflammatory response, suppressed collagen synthesis, increased elastin degradation, retarded angiogenesis, and delayed epithelialization [11, 65–67]. The in vitro wound scratch test was applied to evaluate the wound healing activity of Z. montanum essential oil. The radius of the wound area was used to observe cell migration and proliferation. Cell migration increased to 32.42% and 35.94% by treating with 0.1 and 1 mg/mL of essential oil, respectively, compared with the untreated control group. This phenomenon appears to be time- and concentration-dependent (Fig. 4a–b). Our experimental data explain this effect by the enhanced cell proliferation and collagen synthesis induced by Z. montanum essential oil. Besides, the potential anti-inflammatory capability of this oil could be an additional property that enhances that effect [29, 30].

Another property of Z. montanum oil investigated was its ability to inhibit UVB-induced skin wrinkles. UVB irradiation caused changes by inducing MMP (MMP-1, and MMP-3 to MMP-9) expressions and increasing elastase activity in HDFn cells. This irradiation suppressed type I collagen synthesis (Fig. 5a–d). Pretreatment of the cells with 1 mg/mL of essential oil, in contrast, significantly suppressed the expression of MMPs (p < 0.05) while increasing type I procollagen synthesis by more than two-fold compared with the untreated control cells (Fig. 5d). MMP activity analysis is currently considered a vital stage in cell migration, invasion, and tissue remodeling. Wherever possible, quantitative zymography or ELISA assays should be used to determine the active forms of MMPs. The previous studies have proven a strong correlation between zymography-based and ELISA-based measurements of MMPs activity [68–70]. Thus, in the current investigation, only commercially available ELISA kits were utilized to quantify MMPs activity, and zymography was not used to compare bioactivity. The reason could partly be explained because the ELISA assay is straightforward, quantitative, and effective. However, additional research using zymography technique is necessary to clarify the intricacies of this phenomenon. This poses an area for future work.

The exact mechanism underlying the regulation of MMPs expression by the test essential oil was not explored here. It is known that ROS mediates switching MMPs (MMP-2, MMP-3, MMP-9, and MMP-13) expression via the MAPK signaling pathway, which could be one possible mode of action in alleviating UVB-induced MMP secretion activity [9, 10]. This pathway involves enhanced NF-κB and AP-1 activation, which increases MMPs production and skin wrinkles by triggering extracellular matrix decomposition [11–13]. However, the limitation in the exact mode of action of the test essential oil should be anticipated and addressed in the future.

The functional activity of elastase was significantly reduced by the essential oil, although the inhibitory effect was weak compared to that of EGCG, which inhibited elastase activity by 77.39% (Fig. 6). Considering the correlation between antiphotoaging and the inhibition of enzymatic collagen degradation activity, MMPs, elastase enzymes, and increased type I procollagen synthesis, the current study supports our hypothesis that Z. montanum essential oil may protect against UVB-induced skin damage.

Melanin synthesis is primarily responsible for skin color and plays a crucial role in preventing sun-related injuries [71]. Many pigmentation disorders are caused by melanocyte hyperreactivity and excess melanin production [72–74]. Thus, the regulation of melanogenesis by inhibiting tyrosinase activity is a meaningful way to prevent pigmentation disorders. The above results revealed no significant changes in absorbance in the essential oil-treated groups. There was also no statistically significant difference in the rate of o-quinone formation between the essential oil-treated groups and the blank control group. This can partly be explained by the fact that the extract does not interact with l-DOPA and does not inhibit tyrosinase activity in vitro. A more detailed analysis by other researchers demonstrated that (E)-4-(3,4-dimethoxyphenyl)but-3-en-l-ol, the principal nonvolatile constituent found in the Z. montanum extract, enhanced melanogenesis via the activation on ERK and p38. This activation increased the levels and nuclear localization of upstream stimulating factor-1, increasing tyrosinase expression in B16F10 murine melanoma cells and guinea pigs [30, 75]. Nevertheless, the evidence from previous studies is limited and cannot be extrapolated to this study’s findings. This is attributed to the essential oil not containing the nonvolatile (E)-4-(3,4-dimethoxyphenyl)but-3-en-ol. As a result, the essential oil of Z. montanum cannot be used as a pigmenting agent.

Theoretically, the chemical components of essential oils influence the bioactivity benefits they produce. In this study, the oil isolated by solvent-free microwave extraction contained 29 volatile substances. The major metabolites in that plant oil were identified as sabinene, terpinen-4-ol, (E)-1-(3,4-dimethoxyphenyl)butadiene, γ-terpinene, α-terpinene, and β-pinene. Because excessive UV radiation can cause skin inflammation, the obtained results can be directly compared to previously reported findings on the anti-inflammatory properties of the essential oil isolated by steam distillation. According to Pongprayoon et al. [25], (E)-1-(3,4-dimethoxyphenyl)butadiene, terpinen-4-ol and α-terpinene significantly inhibited the formation of hind paw edema in carrageenan-induced rats, whereas sabinene and γ-terpinene did not produce the same effect. The dosage of essential oil required to inhibit inflammation by 50% (ID₅₀) was calculated to be 22 mg oil/paw. Interestingly, (E)-1-(3,4-dimethoxyphenyl)butadiene (ID₅₀ = 3 mg/paw) was twice as effective as diclofenac (ID₅₀ = 6 mg/paw) at reducing inflammation. Comparative analysis of the other biological activities of each compound in the test oil is impossible because there are literature limitations. Taken together, the above-mentioned biological properties of Z. montanum essential oil could be attributed to its volatile components.

In conclusion, these findings highlight the potential of Z. montanum essential oil to prevent UVB-induced skin aging in vitro. The molecular mechanisms involved are inhibiting ROS formation and MMPs expression, decreasing elastase activity, increasing type I procollagen synthesis, and enhancing wound healing properties. On the other hand, the test essential oil did not affect skin photoaging via melanogenesis, as demonstrated by the experimental data, which revealed that it did not involve both tyrosinase and melanin production. The outcome of various experimentation leads to the conclusion that the essential oil of Z. montanum could be a viable candidate as a new active ingredient for antiphotoaging applications. This is where the strength of our discovery lies. The development of this essential oil in dermo-cosmetic products and its in vivo antiphotoaging efficacy study remain unaddressed; thus, future study is hereby suggested.

Abbreviations

AP-1

Activator protein 1

GC–MS

Gas chromatography-mass spectrometry

HDFn

Normal human dermal fibroblast

IL

Interleukin

MAPKs

Mitogen-activated protein kinases

MMPs

Matrix metalloproteinases

NAC

N-Acetylcysteine

NF-κB

Nuclear factor-κB

ROS

Reactive oxygen species

UVB

Ultraviolet B

Z. montanum

Zingiber montanum (J. Koenig) Link ex A. Dietr

Author contributions

AN funding acquisition, conceived the idea and design of the study, carried out the experimental studies and data analysis, wrote and final revised manuscript. BY participated in the conceptualization of the design, advised for statistical analysis, supervised the research project, prepared the sample extract, performed GC–MS analysis, and collected and interpreted data. RM participated in the conceptualization of the design and supervised the research project. All authors read and approved the final manuscript.

Declarations

Conflict of interest

The authors declare no conflicts of interest.