Phytochemical investigation on the aerial parts of Veratrum versicolor f. viride Nakai and their biological activities

Veratrum spp. have traditionally been used in folk medicine to treat various pathologies. In this study, nine compounds, comprising one simple phenolic compound (1), three stilbenoids (2–4), and five flavonoids (5–9), were isolated from the aerial parts of Veratrum versicolor f. viride Nakai. The structures of these compounds were elucidated by spectroscopic analyses and comparison with reported data. Together, all reported compounds were isolated from V. versicolor f. viride for the first time in the study. Among them, two flavone aglycone tricetins (7 and 9) have never been isolated from the genus Veratrum or the family Melanthiaceae. The ethanol extract and isolated compounds were assessed for their inhibitory effects on elastase, tyrosinase, and melanin synthesis. Compounds 5 and 7 inhibited elastase (IC50: 292.25 ± 14.39 and 800.41 ± 5.86 μM, respectively), whereas compounds 2–5 inhibited tyrosinase with IC50 values in the range of 6.42 ~ 51.19 μM, respectively. In addition, compounds 3–6 and 8 exhibited dose-dependent inhibition (70.4% ~ 91.0%) of melanogenesis at a concentration of 100 μM.


General
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend III 700 spectrometer (Bruker BioSpin GmbH., Rheinstetten, Germany) in DMSO-d 6 and CD 3 OD at room temperature.Chemical shifts are in ppm (δ), relative to tetramethylsilane as an internal standard, and coupling constants are in hertz; 1 H, 13 C, DEPT, COSY, HSQC, and HMBC were performed using the standard pulse sequences.Electrospray ionization mass (ESI-MS) spectra were acquired on an Agilent 6130 series quadrupole LC/MS System (Agilent Technologies, Santa Clara, CA, USA).Open column chromatography was performed using Diaion HP-20 adsorbent resin (Mitsubishi Chemical Corp, Tokyo, Japan).Mediumpressure liquid chromatography (MPLC) was conducted using a CombiFlash Rf flash chromatography system (Teledyne ISCO Inc., Lincoln, NE, USA), and the separations were performed on a RediSep ® Rf C 18 column with a flow rate of 40 mL/min.Preparative high-performance liquid chromatography (HPLC) was performed on a Thermo Scientifc Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an HPG-3200BX biocompatible binary semipreparative pump and a rapid separations PDA detector (Ultimate DAD-3000) controlled by Chromeleon 7.2 software.The separations were carried out on a Kromasil 100-5-C18 column (5 μm, 21.2 × 250 mm, Nouryon Chemicals Finance B.V., Amsterdam, Netherlands).Thin layer chromatography (TLC) was performed using DC-Fertigfolien ALUGRAM SIL G/UV 254+366 (0.2 mm, Macherey-Nagel GmbH & Co. KG, Düren, Germany) plates, and spots were visualized by a 10% vanillin-sulfuric acid reagent.All chemicals and solvents were of analytical grade and were used without further purification.

Plant material
The aerial parts of V. versicolor f. viride Nakai [23] were collected from Cheorwon-gun, Gangwon-do, Republic of Korea, in July 2022.The collection area is 868 m above sea level and located at GPS 38°8′30.87″N127°26′22.95″Eand they grow naturally at the edge of the ridge.Organic matter in soil is moderate (2%∼4%) and it is black forest soil.The crown density is 80% and the quantity of light is poor.The botanical materials were authenticated by one of the authors (Prof.J.S. Oh) and a voucher specimen (G105) was deposited at the Bio-Center, Gyeonggido Business & Science Accelerator (GBSA), Suwon, Republic of Korea.

Spectral data of isolated compounds
The structures of the isolated compounds were elucidated by MS and 1D/2D NMR data analyses and compared with the corresponding data reported in the literature.

Biological assay 2.5.1. Tyrosinase inhibition assay
The reaction was carried out in a 0.1 M potassium phosphate buffer (pH 6.5) containing 1.5 mM L-tyrosine and 1250 unit/ mL mushroom tyrosinase and the reaction mixture was incubated at 37 °C for 20 min.The test samples were assayed for tyrosinase inhibition by measuring its effect on tyrosinase activity using an ELISA reader at 490 nm.Arbutin was used as a positive control.The inhibitory activity of the sample was expressed as the concentration that inhibits 50% of the enzyme activity (IC 50 ) [32].

Elastase inhibition assay
The reaction was carried out in a 50 mM Tris-HCl buffer (pH 8.5) containing 1 mg/mL N-succinyl-(Ala)3-p-nitroanilide and 0.6 U/mL PPE (porcine pancreas elastase).The test sample was added to the reaction mixture, and elastase inhibition was incubated at 25 °C for 10 min.The change in absorbance was measured at 405 nm using an ELISA reader.Ursolic acid was used as a positive control.The inhibitory activity of the sample was expressed as the concentration that inhibits 50% of the enzyme activity (IC 50 ) [33,34].2.5.3.Cell culture B16F10 mouse melanoma cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin in a 37 °C incubator with 5% CO 2 .The cells were seeded in 100-mm tissue culture dishes to be used for subsequent experiments [32].2.5.4.Measurement of relative melanin contents B16F10 cells (1 × 10 5 /well) were seeded in 6 well plates for 24 h.The cells were then incubated in the presence of 100 nM α-MSH, and treated with various concentrations (25, 50, and 100 μM) of sample for 72 h.After being washed twice with PBS, the cells were dissolved of 1 N NaOH and 10% DMSO, incubated at 60 °C for 1 h, and mixed to solubilize the melanin.Relative melanin content was determined with an ELISA reader by absorbance at 405 nm [32].

Statistical analysis
All data are presented as means ± standard deviation (SD).The results were analyzed for statistical significance using Student's t-test and one-way analysis of variance (ANOVA).Values of *p < 0.05 and **p < 0.01 were considered statistically significant.
The structure of 9 was closely related to 7 based on UV and 1 H and 13 C NMR spectroscopic data.However, 9 had one carbon and two protons less than 7.The appearance of a sharp singlet at δ H 9.42 in the 1 H NMR spectrum of 9 and the absence of the signals of a methoxy group and a methoxy carbon in the 1 H and 13 C NMR spectra of 9, respectively, suggested a hydrogen bonded phenolic hydroxyl group at C-5′.The HMBC experiment of 9 showed correlations between 5′-OH (δ H 9.42)/C-5′ (δ C 145.9), C-4′ (δ C 138.6) and C-6′ (δ C 107.5), confirming the substitution of the phenolic hydroxyl group at C-5′ (Figure 2).On the basis of this evidence, the structure of the molecule was confirmed as 4′,5′,5,7-tetrahydroxy-3′ methoxyflavone (3′-O-methyltricetin) [31].To the best of our knowledge, this is the first report of all aforementioned compounds being isolated from this plant, as well as compounds 7 and 9 from the family Melanthiaceae.The HPLC profiles of the isolated compounds are shown in Figure 3.
The tyrosinase inhibitory effects of the isolated compounds were examined, and IC 50 was calculated using a dosedependent response curve.The IC 50 values for compounds 2-5 were as follows: 51.19 ± 5.50, 6.42 ± 0.45, 19.82 ± 0.77, and 37.89 ± 1.04 μM, respectively (Table ).The IC 50 values of arbutin (tyrosinase inhibitor) as a positive control were 473.65 μM [36].With this bioassay, no tyrosinase inhibitory activity (IC 50 > 100 M) was detected for other compounds.Moreover, inhibition of elastase enzyme (porcine pancreas) activity was used to evaluate the antiaging properties of the isolated compounds, and the results are shown in the Table.It was observed that compounds 5 and 8 showed moderate antielastase activity with IC 50 values of 292.25 ± 14.39 and 800.41 ± 5.86 μM, respectively, compared to ursolic acid [37] (IC 50 : 96.88 μM), which was used as the positive control.A melanin content assay was conducted to observe the inhibitory effect of the isolated constituents on melanin production.Here B16F10 melanoma cells were stimulated by α-MSH and cotreated with compounds in three different concentrations (25,50, and 100 μM) or arbutin (400 μM).According to the results shown in Figure 4, compounds 4, 5, and 8 greatly decreased the melanin content in α-MSH-stimulated B16F10 melanoma cells at 100 μM, as compared to those of arbutin.In the range of 50 ~ 100 μM, the inhibition percentages of resveratrol (4), luteolin (5), and chrysoeriol (8) were 72.7% ~ 82.7%, 83.5% ~ 91.0%, and 72.6% ~ 76.6%, respectively, whereas the inhibition rate of 400 μM arbutin was 69.2% (Supporting Information, Table S1) [38].In conclusion, extracts and active compounds effectively inhibited elastase and mushroom tyrosinase activities and inhibited melanin synthesis by B16F10 melanoma cells.Thus, V. versicolor f. viride extracts and several of their chemical constituents can be considered useful for application in developing multitarget cosmeceuticals.

Conclusion
As part of our ongoing work in the search for biologically active constituents from Korean resource plants, an EtOH extract of V. versicolor f. viride was selected for phytochemical investigation.Nine natural compounds were isolated from the aerial parts of V. versicolor f. viride, comprising one phenolic analog (1), three stilbenoids (2-4), and five flavonoids (5)(6)(7)(8)(9), and the effectiveness of their biological potential was demonstrated.Based on enzyme and cell inhibitory assays, the  isolated compounds could exhibit good antityrosinase, antielastase properties, and melanogenesis in B16F10 melanoma cells, thereby conferring a comprehensive attenuating effect against skin aging-related factors.Hence, the results of our study suggest that V. versicolor f. viride has great potential to be used as an effective multifunctional bioactive agent for cosmeceutical formulations.However, additional in-depth studies and clinical evaluations are needed to estimate the skincare potential of extracts and active compounds.1. Apparatus and chromatographic conditions HPLC analysis was performed on a Waters alliance e2695 (Waters Co., Milford, MA, USA) system composed of a 2998 PDA detector and column heater/cooler with a passive preheater.The separation was achieved using a YMC-Triart C18 column (250 × 4.6 mm I.D., 5 μm particle size) (YMC Co., Ltd., Japan).The mobile phase consisted of water-TFA (99.95:0.05;v/v) (solvent A) and acetonitrile (solvent B).The elution was performed using the following gradient: initial 90:10 (A:B v/v); 60 min 60:40 (A:B v/v).The mobile phase was prepared daily, filtered through a 0.45-mm, WTP 0.5-mm membrane (Whatmann, Maidstone, UK), sonicated before use and delivered at a flow rate of 1.0 mL/min.The injection volume was 10 μL and the column temperature was at 25 °C.All the operations, the acquiring and analysis of data were controlled by Empower 3 Software (Waters Co., Milford, MA, USA).

Figure 1 .
Figure 1.Tyrosinase (A), elastase (B), and melanogenesis (C) inhibitory activity of extracts obtained from aerial parts of V. versicolor f. viride.The asterisks represent a significant difference between the columns.**, p < 0.01.The bars represent standard errors.

Figure 4 .
Figure 4. Effects of isolated compounds and arbutin (positive control) on melanin production in B16F10 mouse melanoma cells.The data are expressed as the mean value ± standard deviation of three independent experiments (*, p < 0.05).

Table .
In vitro tyrosinase and elastase inhibitory activity of the isolated compounds.
a All compounds were examined in a set of experiments three times.b Positive control.

Table S1 .
Inhibitory activity (melanin contents) of compounds isolated from V. versicolor f. viride on melanogenesis in B16 mouse melanoma cells.