Article pubs.acs.org/jnp
Valencene from the Rhizomes of Cyperus rotundus Inhibits Skin Photoaging-Related Ion Channels and UV-Induced Melanogenesis in B16F10 Melanoma Cells Joo Hyun Nam,†,‡ Da-Yeong Nam,§ and Dong-Ung Lee*,§ †
Department of Physiology, Dongguk University College of Medicine, 123 Dongdae-ro, Gyeongju 780-714, Republic of Korea Channelopathy Research Center (CRC), Dongguk University College of Medicine, 27 Dongguk-ro, Ilsan Dong-gu, Goyang 410-773, Republic of Korea § Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Ultraviolet (UV) radiation deeply penetrates skin and causes inflammation and pigmentary changes and triggers immune responses. Furthermore, accumulating evidence suggests that calcium ion channels, such as TRPV1 and ORAI1, mediate diverse dermatological processes including melanogenesis, skin wrinkling, and inflammation. The rhizomes of Cyperus rotundus have been used to treat inflammatory diseases including dermatitis. However, their effects on UV-induced photoaging-related ion channels remain unknown. Therefore, this study was undertaken to evaluate the antagonistic effects of C. rotundus extract and their constituents on TRPV1 and ORAI1 channels. Electrophysiological analysis revealed that valencene (1) isolated from the hexane fraction potently inhibited capsaicin-induced TRPV1 and ORAI1 currents at 90 μM (69 ± 15% and 97 ± 2% at −60 and −120 mV, respectively). The inhibitory effect of 1 on cytoplasmic Ca2+ concentrations in response to ORAI1 activation (85 ± 2% at 50 μM) was also confirmed. Furthermore, 1 concentrationdependently decreased the melanin content after UVB irradiation in murine B16F10 melanoma cells by 82.66 ± 2.14% at 15 μg/ mL. These results suggest that C. rotundus rhizomes have potential therapeutic effects on UV-induced photoaging and indicate that the therapeutic and cosmetic applications of 1 are worth further investigation.
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increase the intracellular Ca2+ concentration.7−9 Therefore, controlling intracellular calcium concentration is an important strategy for preventing UV-induced skin pigmentation. The calcium release-activated calcium channel protein 1 (ORAI1) plays an important physiological role in UV-induced melanogenesis.4,10 The Gq protein-coupled receptor (including ET-1, rhodopsin, and α-MSH)-mediated activation of phospholipase C produces inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 then binds to the endoplasmic reticulum (ER) and induces stored calcium release, which causes a transient depletion in the levels of ER Ca2+.5 Furthermore, ER Ca2+ levels are modulated by stromal interaction molecules 1 and 2 (STIM1 and STIM2), which activate Ca2+ release-activated Ca2+ (CRAC) channels encoded by the ORAI gene.11 The CRAC-mediated increase in intracellular Ca2+ ([Ca2+]i) is an important step in melanogenesis, as it controls the active transport of L-phenylalanine and its turnover to L-tyrosine via calmodulin-dependent Ca2+-ATPase.12 This process provides sufficient concentrations of L-tyrosine, which is the substrate required for melanogenesis.13 With respect to wrinkle
kin is continuously exposed to a variety of environmental factors, such as sunlight, air pollutants, and mechanical stress, which accelerate the effect of aging. Although skin aging is also determined by genetic background (referred to as intrinsic aging), the influence of the external environment (extrinsic aging) is also critical.1 Ultraviolet (UV) radiation in sunlight may account for up to 80% of the visible signs of skin aging that constitute photoaging.2 These clinical signs include mottled pigmentation, deep coarse wrinkling, sagging, and laxity.3 The mechanisms of UV-induced melanogenesis and wrinkle formation are well established. A recent study reported that the photopigment rhodopsin is expressed in human epidermal melanocytes (HEMs), which are involved in UV phototransduction.4 UV exposure rapidly induces Ca2+ mobilization via the Gq protein-coupled signaling pathway and generates early Ca2+-dependent melanin synthesis. Furthermore, following exposure to UV radiation, endothelin-1 (ET-1) secreted from keratinocytes subsequently binds to the Gq proteincoupled ET receptors (ETA and ETB) of melanocytes,5,6 then initiates melanocyte proliferation and synthesis of melanin. In addition to ET-1, keratinocytes and melanocytes also increase α-melanocyte-stimulating hormone (α-MSH), which can © XXXX American Chemical Society and American Society of Pharmacognosy
Received: December 30, 2015
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DOI: 10.1021/acs.jnatprod.5b01127 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Structures of the compounds isolated from the rhizomes of Cyperus rotundus.: 1, valencene; 2, camphene; 3, nootkatone; 4, caryophyllene oxide; 5, α-cyperone.
(Figure S1C). To confirm the remaining ITRPV1, we applied 1 μM 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]1-piperazinecarboxamide (BCTC, ≥98% purity), a selective TRPV1 antagonist, to the bath solution at the end of each experiment and as a positive control (Figure S1A). To identify the constituents in the hexane fraction responsible for the observed inhibitory effects on ITRPV1 and ICRAC, we isolated five compounds from the hexane fraction (Figure 1). These compounds were screened for their inhibitory effects at a concentration of 90 μM; among them, 1 exhibited a significant inhibitory effect of 69 ± 15% at a clamp voltage of −60 mV (Table 1). Figure 2A illustrates the mean I−V relationship
formation, the transient receptor potential vanilloid 1 (TRPV1) channel is a key molecule involved in the UV-induced productions of matrix metalloproteinases (MMPs), such as collagenase (MMP-1), gelatinase B (MMP-9), stromelysin-1 (MMP-3), and metalloelastase (MMP-12), which degrade collagen, elastin, and hyaluronan.14 In a recent report, it was suggested that an increase in [Ca2+]i might regulate the expressions and activations of MMPs.15 Furthermore, UV exposure activates TRPV1 channels, which induce [Ca2+]i increases, thereby activating calcium-dependent protein kinase C (PKC) and promoting MMP-1 expression.16 Moreover, elevated extracellular calcium levels have been shown to concentration-dependently increase MMP-9 expression.17 Therefore, the discovery that the inhibition of UV-induced photoaging-related ion channels, such as TRPV1 and ORAI1, might prevent the degradation of collagen and melanogenesis offered a potential means of inhibiting cutaneous photoaging. Cyperus rotundus L. (Cyperaceae) is a perennial that grows naturally and is cultivated widely in tropical, subtropical, and temperate regions. In oriental medicine, its rhizomes have been used as sedatives and analgesics, and it has also been prescribed for gynecological disorders, such as dysmenorrhea and irregular mensturation.18 In addition, this fragrant, edible herb has been reported to exhibit a wide spectrum of activities, such as antioxidative, antiproliferative, anti-inflammatory,19−21 and sedative22 effects and smooth muscle-relaxing properties.23 C. rotundus has been reported to contain saponins,24 alkaloids,25 and more than 20 sesquiterpenes, including α-cyperone (the primary sesquiterpene constituent).26,27 Recently, we demonstrated the extract of C. rotundus, its fractions, and oleanolic acid isolated from the ethyl acetate fraction inhibit human TRPV1 (hTRPV1) channels.28 In the present study, the inhibitory effects of other constituents in the hexane fraction of C. rotundus on TRPV1 and ORAI1 ion channels were investigated and the antimelanogenesis effect of the most active compound was evaluated.
Table 1. Effects of Constituents of the Rhizomes of Cyperus rotundus on ICRACa (I/Icon) × 100 (%) ± SEM compound controlb camphene caryophyllene oxide α-cyperone nootkatone 1
TRPV1 at −60 mV (90 μM) 100 115 45 94 176 31
± ± ± ± ±
11 10 15 18 15
(3) (6) (3) (3)c (3)c
Orai1 at −120 mV (90 μM) 100 79 44 50 53 5
± ± ± ± ±
3 2 8 7 2
(3) (7)d (5)d (5)d (4)d
a
Values in parentheses are the total number (n) of experiment at each concentration. bControl means normalized value at the steady-state peak current before treatment of the compounds. Data represent remaining current compared to control, normalized to 100%, and are mean ± SEM. cp < 0.05 versus control group. dp < 0.0001 versus control group.
curve of 1 μM capsaicin-induced ITRPV1 before (solid black line) and after (dashed gray line) treatment with 1 (90 μM). A summary of the normalized inhibition rates of 1 at 90 μM is presented in Figure 2B. ORAI1 Channel Inhibition. The inhibitory effects of each fraction on the ORAI1 channel were assessed. ER Ca2+depleted conditions were induced in the whole-cell configuration by including 20 mM 1,2-bis(o-aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid (BAPTA) and 20 μM IP3 in the pipet solution. Under these conditions, spontaneous activation of inwardly rectifying currents (ICRAC) was consistently observed in HEK293 cells overexpressing wild-type ORAI1 channels. After confirming the steady state of ICRAC, C. rotundus extract or its fractions were applied to bath solutions to determine whether they inhibited ICRAC. As shown in Table S1, all fractions inhibited ICRAC, and the hexane fraction had the greatest effect. Sequential applications of 10 (2), 30 (3), and 100 (4) μg/mL demonstrated the hexane fraction inhibited ICRAC in a concentration-dependent manner (Figure S1D and E). These results showed the hexane fraction efficiently inhibited ITRPV1 and ICRAC in HEK293 cells and suggested this fraction contained chemical constituents with potential TRPV1 and ORAI1 channel inhibitory effects.
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RESULTS AND DISCUSSION TRPV1 Channel Inhibition. To determine whether the skin-photoaging-related ion channels TRPV1 and ORAI1 were inhibited by the crude extract and fractions, we performed whole-cell patch-clamp experiments using the HEK293T cell line expressing these channels. In a previous study, we evaluated the inhibition of capsaicin-induced TRPV1 activation by C. rotundus extracts. The results revealed that only the hexane and ethyl acetate fractions (100 μg/mL) inhibited ITRPV1 at 100 mV.28 The generation of [Ca2+]i signaling requires a critical influx of Ca2+ through TPRV1 channels; that is, it requires an inward rather than outward current. At −100 mV, the hexane fraction most potently inhibited ITRPV1 (Table S1, Supporting Information), whereas the ethyl acetate fraction had no effect. At 10 μg/mL the hexane fraction did not inhibit ITRPV1, but experiments conducted at 30 and 100 μg/mL demonstrated it concentration-dependently inhibited ITRPV1 B
DOI: 10.1021/acs.jnatprod.5b01127 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. Valencene (1) inhibited both inward transient receptor potential vanilloid 1 currents (ITRPV1) and inwardly rectifying calcium-releaseactivated calcium currents (ICRAC). (A) Mean I−V relationship curve of ITRPV1 of control and 90 μM 1. Numbers in parentheses represent the number of replicate experiments. (B) Effects of 1 on ITRPV1 at −60 and 100 mV. 1 blocked ITRPV1 more efficiently at negative than positive voltages. *p < 0.05 and **p < 0.01 versus control group. (C) Mean I−V relationship curve of IOrai1 for the control and after treatment with 1 (10, 30, or 90 μM). Numbers in parentheses represent the number of replicate experiments. (D) Effects of 1 on IOrai1 at −120 mV. ****p < 0.0001 versus control group.
Figure 3. Inhibition of anti-CD3 antibodies (α-CD3) induced store-operated calcium (Ca2+) entry (SOCE) by valencene (1). (A, B) Treatment of α-CD3 induced a transient increase in intracellular calcium ([Ca2+]i) that was completely reversed to basal level by 1 treatment. After confirming steady-state [Ca2+]i, serial application of 1 (10, 20, and 30 μM) gradually decreased [Ca2+]i to basal level. (C) Summary of inhibitory effects on [Ca2+]i. [Ca2+]i,steady indicates the normalized intracellular Ca2+ concentrations at steady state. Inhibition rate (y-axis) indicates the difference of [Ca2+]i levels between [Ca2+]i,steady and [Ca2+]i after 10, 30, and 50 μM 1 treatment.
at 90 μM. The application of graded concentrations (10, 30, and 90 μM) of 1 demonstrated concentration-dependent inhibition of ICRAC (Figure 2C and D). Figure 2C shows the
Next, the inhibitory effects on ICRAC of compounds isolated from the hexane fraction were investigated. All were found to be active (Table 1). Compound 1 inhibited ICRAC by 95 ± 2% C
DOI: 10.1021/acs.jnatprod.5b01127 J. Nat. Prod. XXXX, XXX, XXX−XXX
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mean I−V relationship curve of ICRAC control and tested concentrations. Specifically, 1 inhibited ICRAC by 13 ± 4%, 45 ± 8%, and 94 ± 2%, at 10, 30, and 90 μM, respectively (Figure 2D, n = 4), which suggests that it inhibited TRPV1 and ORAI1. As mentioned above, TRPV1 and ORAI1 channels are important mediators of wrinkle formation and melanogenesis, and therefore, we considered 1 may be a potentially useful therapeutic agent for the prevention of skin aging. Calcium Determination. Store-operated calcium (Ca2+) entry (SOCE) pathways modulate the increase in [Ca2+]i by ER store depletion. We used the whole-cell patch-clamp assay to determine if inhibition of ICRAC by 1 was mediated by SOCE pathways. Human T lymphocytes have a well-developed SOCE pathway. Therefore, we used a human T lymphocyte cell line (Jurkat-T) for the SOCE experiments. We reported previously29 that treatment with anti-CD3 antibodies (α-CD3, 5 μg/mL) induced a biphasic increase in [Ca2+]c (Δ[Ca2+]c,CD3) in Jurkat T cells. This increase was characterized by an initial transient increase in the Δ[Ca2+]c,CD3 due to Ca2+ release from ER stores and an increase the secondary sustained Δ[Ca2+]c,CD3 phase, which is almost solely due to SOCE. As shown in Figure 3A, treatment with 50 μM 1 almost abolished α-CD3-induced sustained Δ[Ca2+]c,CD3. Interestingly, while the whole-cell patch-clamp study showed less than 50% inhibition of ICRAC by 1 at 10 and 30 μM, fura-2 Ca2+ measurements revealed the inhibitory effect was greater than 50% at 10 μM (Figure 3B). Normalized inhibitory rates of 1 at 10, 30, and 50 μM are presented in Figure 3C. Application of 1 decreased the sustained increase in [Ca2+]c,CD3 in a concentration-dependent manner by 53 ± 3%, 75 ± 3%, and 85 ± 2% at 10, 30, and 50 μM, respectively. These results show 1 efficiently inhibited the SOCE pathway and suggest it as a suitable candidate for potential development as an anti-skin-photoaging agent. However, we were unable to determine why 1 showed more potent effects based on fura-2 Ca2+ measurements than by patch-clamp experiments. This phenomenon might be due to 1 modulating other cation channels such as transient receptor potentials (TRPs). Nevertherless, additional mechanisms and other targets of 1 should be explored. Melanin Content after UV Exposure. In order to identify the antimelanogenesis effect of 1, which potently inhibited ORAI1, the UV-induced melanin contents were measured in B16F10 cells (a murine melanoma cell line). As we mentioned earlier, the generation of intracellular Ca2+ signaling via rhodopsin and ET-1 is an important factor in UV-induced melanogenesis. Therefore, we pretreated cells with 7.5 or 15 μg/mL (36.7 or 73.4 μM) of 1 before UVB exposure and then measured the melanin levels after 48 h. Treatment with 1 (7.5 or 15 μg/mL) concentration-dependently decreased the amount of melanin produced by UVB irradiation by 94.82 ± 2.77% and 82.66 ± 2.14%, respectively, while N-[4-[3,5bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3thiadiazole-5-carboxamide (BTP2), a potent inhibitor30 of ORAI1, inhibited melanogenesis by 41.58 ± 2.05% at a concentration of 3 μg/mL (7.1 μM) (Figure 4A). The MTT assay was performed before melanin determination to verify the effects of compounds on cell viability; 1 and BTP2 at concentrations of
