All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), except 3,5-bis(trifluoromethyl)pyrazole (BTP2), which was obtained from Tocris (Bristol, UK). Stock solutions of capsaicin (10 mM), inositol triphosphate (InsP₃; 20 mM), BTP2 (10 mM), and 4-(3-chloro-2-pyridinyl)-N-[4- (1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC; 10 mM) were prepared in dimethyl sulfoxide (DMSO). All stock solutions were stored at −20°C.

The dried and pulverized leaves of S. plebeia (200 g) were purchased from Omniherb drug store (Seoul, Korea) and extracted twice with 70% methanol (1 l) under reflux to obtain the methanolic extract. After evaporation of the solvent, the solid extracted material was obtained (yield, 43.0 g), which was then successively partitioned with n-hexane (200 ml), dichloromethane (200 ml), ethylacetate (100 ml) and n-butanol (100 ml) to obtain the corresponding fractions with yields of 1.8, 1.5, 2.4 and 5.0 g, respectively. Dried extracts were diluted with DMSO to prepare stock solutions (30 and 100 mg/ml) of each extract.

GC-MS (Agilent GC/Pegasus 4D, Agilent Technologies, Santa Clara, CA, USA) analyses of the dichloromethane and ethylacetate fractions were performed under the following conditions: Injector split ratio, 10:1; injection volume, 1.0 µl dichloromethane solution; injector temperature, 250°C; column, Agilent J&W DM-5MS (30 m × 0.25 mm ID × 0.25 µm; Agilent Technologies); carrier gas, helium; flow rate, 1.0 ml/min; oven temperature programming, 50°C (3 min)→10°C/min→320°C (18 min). For the MS analysis, the following conditions were used: Ion source, electron impact; 230°C; analyzer, quadrupole; 150°C; mass range, 35–800 m/z (mass-to-charge ratio).

HEK293T cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator containing 20% O₂ and 10% CO₂ (as 3.7 g/l NaHCO₃ requires 10%). All media were supplemented with 10% fetal bovine serum (WelGENE, Daegu, South Korea), 100 U/ml penicillin and 100 g/ml streptomycin (Thermo Fisher Scientific, Inc.). The cells were subcultured every 2–3 days.

For the patch clamp studies, the cells were transferred in 25-cm² culture flasks (Thermo Fisher Scientific, Inc.) 1 day prior to transfection. HEK293T cells were transiently transfected with a mammalian expression vector carrying human (h)TRPV1 or hORAI1 and hSTIM1 using the Turbofect transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In all transfection studies, the cells were co-transfected with a plasmid vector coding the enhanced green fluorescent protein (pEGFP-N1) to sort out the transfected cells. To record TRPV1 currents (I_TRPV1), hTRPV1 and pEGFP-N1 were co-transfected at a ratio of 9:1. To record ORAI1 currents (I_ORAI1), hORAI1, hSTIM1 and pEGFP-N1 were triple-transfected at a ratio of 4.5:4.5:1, respectively. Human TRPV1 (hTRPV1) plasmid (pcDNA5/FRT) was generously donated by Dr Sung Joon Kim (Seoul National University, Seoul, Korea). Human ORAI1 (hOrai1) and human STIM1 (hSTIM1) were purchased from Origene Technologies (Rockville, MD, USA). hSTIM1 and hORAI1 cDNA were subcloned into pcDNA3.1 (Thermo Fisher Scientific, Inc.). Experiments were performed within 24–36 h of transfection.

Conventional whole-cell patch clamp methods were used to measure the hTRPV1 and hORAI1 currents at room temperature. Recordings were acquired using an Axopatch 700B amplifier interfaced with a Digidata 1440A (Molecular Devices, Sunnyvale, CA, USA). The recorded data were digitized at 10 kHz and low-pass filtered at 5 kHz using the pCLAMP 10.4 software (Molecular Devices). Patch pipettes were produced from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL, USA) using a horizontal Flaming Brown micropipette puller (model P-97). Pipette tips were fire-polished to a resistance of 2–3 MOhm to facilitate gigaseal formation (Narishige, East Meadow, NY, USA). The transfected cells were transferred to a perfusion chamber (Warner Instruments, Hamden, CT, USA) mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan). Bath solutions were perfused at 3 ml/min. To measure the hTRPV currents, voltage ramp protocols ranging from −100–100 mV over 100 msec were applied every 20 sec at a −10-mV holding potential. For hORAI1, ramp-like pulses from −130-70 mV over 100 msec were applied every 30 sec at a −10-mV holding potential to obtain the current-voltage association. The junction potentials were canceled prior to patch formation and pipette capacitances were compensated for electronically after gigaseal formation. All the voltage and current trace data were analyzed using the Clampfit software 10.4, Prism 6.0 (GraphPad, Inc., La Jolla, CA, USA) and Origin 8.0 (MicroCal, Northampton, MA, USA).

For the hTRPV1 whole-cell patch clamping, the external solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM ethylene glycol tetraacetic acid (EGTA), 5 mM D-glucose and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4). The internal solution contained 140 mM CsCl, 10 mM NaCl, 5 mM EGTA, 3 mM adenosine triphosphate magnesium salt and 10 mM HEPES (pH 7.2). The capsaicin-evoked activity was observed by applying capsaicin (1 µM) in the external solution after confirming the basal current. For the hORAI1 whole-cell patch clamp recording, the external solution contained 3.6 mM KCl, 10 mM CaCl₂, 1 mm MgCl₂, 5 mM D-glucose, and 10 mM HEPES (pH 7.4). The internal solution contained 130 mM Cs-glutamate, 20 mM 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetracetic acid, 1 mM MgCl₂, 3 mM MgATP, 0.002 mM sodium pyruvate and 20 mM HEPES (7.2). To activate the hORAI1 currents, 20 µM InsP₃ was added to the internal solution.

The tyrosinase inhibitory activity of S. plebeia was measured using a modified method (36). In brief, a mixture of sodium phosphate buffer (pH 6.8; 0.066 M), L-3,4-dihydroxyphenylalanine (1.5 mM) and DMSO (10 mg/ml) was added to the enzyme solution (500 U/ml), and the mixture was incubated at 30°C for 10 min. After enzyme inactivation using an ice bath, the optical density was measured using spectrophotometer (Ultraspec 2000; Amersham Pharmacia Biotech, NJ, USA) at a wavelength of 475 nm. Kojic acid (200 µg/ml) was used as a positive control.

Inhibition of porcine pancreatic elastase was estimated using a modified method (37). In brief, a mixture of Tris-HCl buffer (pH 8.0; 0.1 M), N-succinyl-Ala-Ala-Ala-p-nitroanilide (1 mM) and sample solution (plant extract or fractions, 10 mg/ml in DMSO) was added to the enzyme solution (elastase in Tris-HCl buffer, 0.1 U/ml), followed by incubation at 25°C for 20 min. After enzyme inactivation using an ice bath, the optical density was measured at a wavelength of 410 nm using an Ultraspec 200 spectrophotometer. Ursolic acid (200 µg/ml) was used as a positive control.

Values are expressed as the mean ± standard error of the mean. The N values indicate the number of separate cells used in the experiment. Comparison tests were performed using one-way analysis of variance with Bonferroni's post-hoc comparison. P<0.05 was considered to indicate a statistically significant difference. Prism 6.0 software (GraphPad, Inc.) and Origin 8.0 (MicroCal) were used for statistical analyses.

To evaluate whether TRPV1 and ORAI1 are affected by the S. plebeia leaf crude extract and fractions, whole-cell currents in HEK293T cells overexpressing TRPV1 or ORAI1 and STIM1 were measured. To evaluate the inhibitory effects of this herbal extract on the basal I_TRPV1, 1 mM capsaicin was used to activate I_TRPV1. After confirming the I_TRPV1 steady state, 10, 30 or 100 µg/ml of the extract or fractions were added to the external solution. A total of 1 µM of the selective TRPV1 inhibitor BCTC was used as a positive control. Fig. 1A shows a typical data recording for the capsaicin-induced I_TRPV1 and its inhibition by the butanolic fraction. The associated I–V curves at (a) the peak I_TRPV1 and at (b) 10, (c) 30, and (d) 100 µg/ml butanolic fraction were achieved using a ramp-like pulse protocol from −100–100 mV (Fig. 1B). Fig. 1B further shows that the application of capsaicin produced a strong outward rectifying current potential reversal (0 mV). To analyze the fraction-induced inhibition, the normalized amplitudes of the fraction-treated currents (I/I_con ×100%) were obtained at 100- and −60-mV clamp voltages. As shown in Fig. 1C, the level of I_TRPV1 inhibition by the butanolic fraction at −60 mV was 5±11, 39±10 and 84±8% at 10, 30 and 100 µg/ml, respectively, and at 100 mV, it was 4±9, 37±4 and 86±1% at 10, 30 and 100 µg/ml, respectively.

To evaluate the inhibition of I_ORAI1, 20 µM InsP₃ was added to the internal solution in order to deplete ER calcium stores. When the ER calcium concentration drops, STIM1 binds to and activates ORAI1, and thereby allows for calcium entry. After obtaining the whole-cell configuration, an inwardly rectifying current with reversal potential at 50 mV was obtained in the ORAI1/STIM1 co-transfected cells. After confirming the steady state of the I_ORAI1, 10, 30 or 100 µg/ml extract or fraction of S. plebeia leaf was added to the bath solution to analyze the S. plebeia-mediated inhibition of ORAI1. BTP2 (10 µM), which is a selective ORAI1 inhibitor, was added to the external solution to confirm the basal current. As presented in Fig. 2A, the serial application of 10, 30 and 100 µg/ml of the butanolic fraction inhibited I_ORAI1. Fig. 2B shows the I–V association curve between the (a) control and butanolic fraction treatment at (b) 10, (c) 30 and (d) 100 µg/ml. At −120 mV, the butanolic fraction inhibited I_ORAI1 by 31±5, 62±5 and 87±2% at 10, 30 and 100 µg/ml, respectively (Fig. 2C; n=6). The hexane fraction had a greater inhibitory effect on I_ORAI1 than the butanolic fraction (Fig. 3); at −120 mV, the hexane fraction inhibited I_ORAI1 by 56±2, 82±1 and 92±2% at 10, 30 and 100 µg/ml, respectively (Fig. 3C; n=7). The TRPV1 and ORAI1 inhibition rates are presented in Table I. The butanolic fraction had the strongest effect on I_TRPV1 and I_ORAI1.

To verify the skin-lightening effects of the S. plebeia leaves tyrosinase assays were performed using the extract and fractions. Tyrosinase is a key enzyme that initiates melanogenesis. Decreased tyrosinase activity corresponds with reduced melanin production. kojic acid, a tyrosinase inhibitor, was used as a positive control (80.7±0.69%). As shown in Fig. 4, the dichloromethane (32.4±0.69%) and hexane (22.6±0.96%) fractions inhibited tyrosinase activity.

Elastase regulates wrinkle formation by deteriorating and remodeling the extracellular matrix. Therefore, elastase assays can be used to evaluate the effect of S. plebeia on wrinkle formation. Ursolic acid, an elastase inhibitor, was used as a positive control (83.9±0.79%). As presented in Fig. 5, the ethylacetate and butanolic fractions inhibited elastase by 65.2±1.30 and 31.7±1.23%, respectively.