Evaluation of protective effect of cyclodextrin glucanotransferase-treated Gastrodia elata Blume extract on ultraviolet B-induced premature skin aging

Purpose: To investigate the protective effect of Gastrodia elata Blume (G. elata, GE) and cyclodextrin glucanotransferase (CGTase) enzyme-treated G. elata extract (EGE) against premature skin aging using ultraviolet B (UVB)-exposed normal human dermal fibroblasts (NHDFs). Methods: The extract was characterized by liquid chromatography with tandem mass spectrometry (LC-MS/MS), ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC–QToF–MS) and nuclear magnetic resonance spectroscopy (NMR). The expression of matrix metalloproteinases (MMP-1,3), interleukin-6 (IL-6), transforming growth factor (TGF-β1) and procollagen type I was assayed using ELISA kits. Safety evaluation of EGE’s dietary administration and topical application was performed by in vivo acute oral toxicity and local lymph node tests. Results: Lower MMP-1 and IL-6 and higher procollagen type I and TGF-β1 levels were observed after treatment with EGE than with GE, indicating that EGE was more effective than GE in treating UVBinduced photoaging. With respect to phenolic composition, EGE had lower 4-hydroxybenzaldehyde (4HBA) level and higher α-gastrodin level than GE. In UVB-irradiated NHDFs, α-gastrodin exhibited higher anti-aging activity than 4-HBA and β-gastrodin based on the expression of MMP-1, MMP-3, and procollagen type I. The in vivo data indicate that EGE was safe at concentrations of up to 2000 mg/kg for dietary administration and 0.1 % for topical application. Conclusion: EGE protects UVB-induced photoaged human skin better than GE owing to its higher αgastrodin content. Thus, EGE may be potentially useful agent in anti-aging cosmetic products.

Extrinsic skin aging was recently reported to be a result of exposure to UVB (280-320 nm).UVB can reduce skin collagens, which are the main components of extracellular matrix (ECM).Matrix metalloproteinases (MMPs) are increased in aged human skin to cleave collagen and other ECM proteins [8].Additionally, transforming growth factor-β (TGF-β) has an important role in ECM biosynthesis [9].Pro-inflammatory factors, such as interleukins (ILs), are also elevated and the oxidant-antioxidant cellular homeostasis is disturbed upon UVB exposure [10].Thus, regulation of the expression of these factors is crucial to control extrinsic skin aging.
In the present study, it was hypothesized that the enzyme-treated ethanolic G. elata Blume extract (EGE) would enhance the synthesis of procollagen type I in exposed normal human dermal fibroblasts (NHDFs).Although phenolic compounds possess low toxicity, some plant extracts can irritate the skin by causing skin allergies.Based on this hypothesis, we investigated the anti-aging effect of EGE in vitro and its oral and topical safety in vivo.

EXPERIMENTAL Plant material and extraction methods
The dried rhizomes of  , and 0.1 M of phosphate buffer (pH 6.0) was incubated for 24 or 48 h at 50 C.The reaction was stopped by boiling in water for 10 min [11].Finally, reaction products were continued using glucoamylase (40, 80, and 120 U) at pH 5.0 and 50 C for 8 h.

Measurement of CGTase and glucoamylase activities
CGTase (or glucoamylase) and 0.3% soluble starch were dissolved at 40°C in a 20 mM acetate buffer (pH 5.5) containing 1 mM CaC1 2 for 10 min [11].One unit of enzyme activity was defined as the amount of enzyme catalyzing 10% decrease in absorbance per minute under these conditions [12].

Cell culture, UVB irradiation and sample treatment
NHDFs were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA).Cells were seeded at 2 × 10 4 cells/cm 2 density for 24 h and then exposed to UVB (144 mJ/cm 2 ) radiation as described previously [13].After UVB irradiation, cells were immediately incubated in serum-free medium with or without sample treatment.Normal cells were cultured in the same incubator conditions without UVB exposure or sample treatment.

MTT assay
To measure cell viability, 1 mg/mL MTT was added into cell culture dishes after 72 h of treatment with UVB and samples.After 2 h incubation with MTT, cell supernatants were discarded and 800 µL of DMSO (98% purity) was added to dissolve the formazan crystals.After gentle shaking for 10 min, the absorbance was measured at 570 nm h on a microplate reader (Molecular Devices FilterMax5, USA).

Measurement of secreted proteins
The concentrations of MMP-1, MMP-3, procollagen type I, IL-6, and TGF-β1 were determined in the cell culture supernatants after 72 h of sample treatment using ELISA kits according to the kit-specific instructions.

Local lymph node assay (LLNA)
The assay was conducted according to OECD Guideline 442B (OECD, 2010).Twenty-five female BALB/c mice (7-weeks-old; 17-20 g) were purchased from Central Lab.Animal Inc. (Seoul, Korea).No experiment was carried out during adaptation (first week).EGE was suspended in acetone: olive oil (4:1, v/v) and applied to the ear skin of the mice.As a positive control, 25% hexylcinnamaldehyde (HCA) in the same delivery vehicle was used.Concentrations of 0.05, 0.1, and 0.5% (v/v) of EGE extract were chosen for the 5 groups (n = 5) for the LLNA study.
Ears of each mouse were observed for erythema and scored using the OECD 442B criteria.On the 1 st , 3 rd , and 6 th day, ear thickness was measured with an electronic digital caliper (Mitutoyo Korea Corporation).The experimental protocol was conducted according to GLP and conducted from 2017 July 20 th to September 4 th at ChemOn Inc. (Gyeonggi-do, Korea) [15].

Lymphocyte proliferation
A BrdU ELISA colorimetric kit (Roche, Germany) was used to evaluate lymphocyte proliferation.The stimulation index (SI) was calculated by dividing the mean BrdU labeling index/mouse in each test substance group and the positive control group by the mean BrdU labeling index for the solvent group.The BrdU labeling index is defined by calculating absorbance (ABS) values as: (ABS 370 -ABS blank370 ) -(ABS 492 -ABS blank492 ).The test result is considered positive when SI ≥ 1.6 [15].

Statistical analysis
Tukey's test was performed to compare the difference between treatments using one-way analysis of variance.Statistical significance was set at p < 0.05 for in vitro and in vivo data.

RESULTS
α-Gastrodin content of EGE Among the 5 different concentrations of ethanol used and the 3 different duration of extraction, the 50% ethanolic extract processed after 3 h was identified as the most efficient based on its highest content of 4-HBA.The 4-HBA content of GE all extracts is shown in Table 1.Among 18 methods of enzyme modification, the 24-h treatment of CGTase (2250 U/100 mL) and glucoamylase (80 U/100 mL) gave the most active EGE, which contained 22.64 (mAU*min in peak area) of α-gastrodin, a targeted biotransformation product (Table 2, Figure 1).The biotransformed compound was identified as α-gastrodin by NMR analysis (Figure 2).Three batches were thereafter processed on a larger scale as shown in Figure 3.In 15 kg of crude GE, extraction yield was 0.920±0.030g with 0.19% of 4-HBA.Finally, EGE containing 0.42% of αgastrodin (C 13 H 18 O 7 , 285.10636 MW) was selected for the skin anti-aging tests.

Effects of GE and EGE on MMP-1, procollagen type I, IL-6, and TGF-β1 secretion from UVB-irradiated NHDFs
No toxicity was observed in the control group as well as in the experiment group treated with GE and EGE.After UVB exposure, MMP-1 and IL-6 production was significantly higher while procollagen type I and TGF-β1 expression was lower than that in the normal group.However, treatment with GE and EGE reversed these responses by fibroblasts.EGE at 10 and 100 μg/mL significantly reduced MMP-1 production by 36.17 and 58.92 % as shown in Figure 4. GE treatment at 10 and 100 μg/mL reduced MMP-1 production by 25.07 and 35.60 %.In 100 μg/mL EGE-treated cells, procollagen type I and TGF-β1 levels increased by 197.80 and 216.82 %, respectively.

Acute oral toxicity of EGE
Upon oral administration of EGE, SD rats did not exhibit any signs of toxicity in the skin, hair, eyes, mucous membranes, respiratory, circulatory, and autonomic, and central nervous systems, or abnormalities in behavior patterns.A negligible increase was observed after 15 days approximately 16 -18 g/rat for all groups (Table 3).No death was observed in any of the groups (even at a dose of 2000 mg/kg); therefore, EGE may be considered to be safe for oral administration.

In vivo skin sensitization potential of EGE
Ear thickness, erythema index, and individual animal observations indicated that only one mouse in the 0.5% EGE treatment condition had local dermal irritation.The BrdU labeling indexes of the normal and 0.05% EGE groups were not significantly different (Table 4, Table 5, Table 6, and Table 7).The SI value of the positive control was 2.54.Treatment with EGE resulted in a dose-dependent increased in lymphocyte proliferation.However, SI values of EGE-treated mice ears were all below 1.6.Based on these SI values, EGE was not considered as a dermal sensitizer in the LLNA.

DISCUSSION
It is increasingly accepted that the effects of UVB on the skin can be modeled in vitro using UVB-irradiated NHDFs.In the present study, EGE exhibited a greater anti-aging activity than GE in UVB-irradiated NHDFs.EGE regulated MMP-1 and procollagen type I production via IL-6 and TGF-β1 expression.The higher content of αgastrodin contributed to the higher activity of EGE than that of GE.Both dietary and topical application of EGE was recognized as safe at concentrations of up to 2000 mg/kg and 0.1% (w/v), respectively.4-HBA and β-gastrodin are the main phenolic constituents of GE [16].They reduce oxidative stress by upregulating Nrf2 [17,18].β-Gastrodin downregulated IL-6 and MAPKs [6].Sun et al.
and Hwang et al. [20] indicated that an antiaging candidate that promoted Nrf2/ARE signaling and reduced IL-6 and MAPKs could inhibit the expression of MMPs and increase procollagen type I. MMP-1 and procollagen type I are important markers in photoaging.Thus, we hypothesized that GE and EGE containing 4-HBA and β-gastrodin could have an effect on the secretion of procollagen type I.In this study, EGE was found to contain 4-HBA, β-gastrodin, and α-gastrodin.Both GE and EGE, as well as their individual compounds decreased the production of MMPs and increased the production of procollagen type I.Among the 3 main compounds studied, the anti-aging activity of α-gastrodin was demonstrated to be the best.EGE possibly possessed a greater anti-aging activity than GE because of its higher α-gastrodin content.

CGTase
(EC 2.4.1.19)from B. stearothermophilus is an α-amylase which is generally recognized as safe (GRAS) and is widely used in the food, pharmaceutical, chemical and cosmetic industries [21].α-Gastrodin-enriched EGE possessed high antiaging and no significant toxic activities in UVBirradiated NHDFs as well as in SD rats and BALB/c mice.The results also showed that EGE is safe for ingestion as well as topical use (at < 2000 mg/kg and <0.1 %w/v, respectively).

CONCLUSION
The results suggest that UV-induced premature skin aging may be regulated by treatment with GE and EGE.This study allows us to better appreciate biotransformed functional foods or cosmetics with tested safety.GE and EGE containing α-gastrodin, β-gastrodin, and 4-HBA show protective effects in photoaged fibroblasts in vitro as well as in rats and mice in vivo.Followup research is required to determine the antiaging or anti-wrinkle activities of EGE in artificial human skin models and in clinical trials.

Table 1 :
GE and its content of 4-HBA

Table 2 :
Large-scale extraction of EGE and its content of α-gastrodin* *The unit of peak area of α-gastrodin in EGE extracts was mAU*min

Table 3 :
Body weight of SD rats The first day of administration is designated as Day 1.All values are presented as mean ± SD (g); # Day; * Body weight gained after 15 days