Tamariscinols U–W, new dihydrobenzofuran-type norneolignans with tyrosinase inhibitory activity from Selaginella tamariscina

Phytochemistry Letters 34 (2019) 79–83

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Tamariscinols U–W, new dihydrobenzofuran-type norneolignans with tyrosinase inhibitory activity from Selaginella tamariscina

T

Xiao-Ru Hea, Liu-Yun Xub, Chen Jina, Peng-Fei Yuea, Zhi-Wang Zhoub, Xin-Li Lianga,

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a b

Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of TCM, Nanchang, 330004, China School of Pharmacy, Nanchang University, Nanchang, 330006, China

ARTICLE INFO

ABSTRACT

Keywords: Selaginella tamariscina Selaginellaceae Norneolignan Tyrosinase inhibitors

Three minor new dihydrobenzofuran-type norneolignans, tamariscinols U–W (1–3), and two known phenolics were isolated from the ethanolic extract of Selaginella tamariscina. Their structures were elucidated on the basis of extensive spectroscopic analysis. All isolates were evaluated for their inhibitory effects against the mushroom tyrosinase. Compounds 1 was the most potent compound (IC50 = 5.75 μM), which exhibited a threefold activity as that of the positive control, kojic acid.

1. Introduction Tyrosinase is a copper-containing enzyme implicated in the biosynthesis of mammalian melanin which protects the skin from photodamage by absorbing ultraviolet (UV) rays and removing reactive oxygen species (Kishore et al., 2018). Tyrosinase inhibitors, therefore, have potential therapeutic and cosmetic applications for the treatment of some dermatological disorders associated with melanin hyperpigmentation, such as age spots, melasma, and chloasma (Kim and Uyama, 2005). Additionally, tyrosinase has also been found to be responsible for the browning of some fruits and vegetables (Mayer, 1987). Its inhibitors are applicable to food preservation by inhibiting this undesirable browning process so as to improve the food lifespan. Despite the existence of a lot of tyrosinase inhibitors, only a few are marketed as safe to date (Adhikari et al., 2008; Chiari et al., 2010). Thus, there is an urgent need to search for safe and efficient new tyrosinase inhibitors in cosmetics, medicinal products, and food industries. Selaginella tamariscina (Beauv.) Spring (Selaginellaceae), a perennial herb was first recorded by “Shen Nong Ben Cao Jing” (The Divine Farmer’s Materia Medica) in 2737 BCE As a well-known Traditional Chinese Medicine (TCM), this herb has been extensively used to treat amenorrhea, dysmenorrhea, metrorrhagia, and abdominal lumps in women (Kim et al., 2012; Lee et al., 2013), and used in folk medicine as an anticancer, chronic hepatitis, anti-inflammatory, and antidiabetic agent (Yao et al., 2017). Moreover, S. tamariscina has also been reported to lower blood glucose levels and to facilitate the repair of pancreatic islet β-cells injured by alloxan (Jung et al., 2011; Millon et al., 2011; Nitin et al., 2009; Zou et al., 2005). Previous chemical investigations

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showed that the major constituents of this plant include flavonoids (Liu et al., 2009, 2010), biflavonoids (Cao et al., 2012), sterols (Gao et al., 2007), anthraquinones (Liu et al., 2018), lignans (Dat et al., 2017; Zheng et al., 2004a, b), and selaginellins (Cheng et al., 2008; Xu et al., 2011a, b; Xu et al., 2015; Zhu et al., 2019). Among them biflavonoids and selaginellins have attracted increasing attention in recent years due to their diverse biological activities, such as antifungal (Jung et al., 2007; Lee et al., 2009), anti-inflammatory (Shim et al., 2018) and vasorelaxant activity (Kang et al., 2004), together with cytotoxicity (Yang et al., 2012; Zhang et al., 2012), neuroprotection (Cao et al., 2017), and inhibition of protein tyrosine phosphatase 1B (PTP1B) (Le et al., 2017; Nguyen et al., 2015a, b), matrix metalloproteinase-1 (MMP-1) (Lee et al., 2008), and phosphodiesterase-4 (Yao et al., 2017; Woo et al., 2019). In our ongoing effort to discover novel tyrosinase inhibitors from traditional medicinal plants (Wang et al., 2018), a fraction of the ethanolic extract of S. tamariscina showed significant inhibitory activity against the mushroom tyrosinase (IC50 8.60 μg/mL), which prompted us to conduct the phytochemical investigation of this plant. As a result, six phenolics including three new dihydrobenzofuran-type norneolignans (1–3) and two known compounds (4 and 5) were isolated. Herein, we describe the structural elucidation of the new compounds as well as the tyrosinase inhibitory activities of all isolates. 2. Results and discussion The air-dried powder of S. tamariscina was extracted with 95% EtOH three times at room temperature. The EtOAc-soluble fraction of the

Corresponding author. E-mail address: [email protected] (X.-L. Liang).

https://doi.org/10.1016/j.phytol.2019.08.013 Received 29 May 2019; Received in revised form 27 July 2019; Accepted 28 August 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. Chemical constituents isolated from S. tamariscina.

Table 1 1 H (400 MHz) and

13

C (100 MHz) NMR Data for Compounds 1−3 in CD3OD (δ in ppm, J in Hz)a.

No.

1 δH

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 3-OMe 5-OMe 3′-OMe 1′′ 2′′ 3′′ 4′′ a

6.69, s

6.69, s 5.54, d, 6.4 3.51, m 3.78, dd, 10.8, 3.2 3.86, dd, 10.8, 5.2 6.85, s

6.85, s 4.29/4.28, q, 6.4 1.40, d, 6.4 3.81, s 3.81, s 3.88, s 3.21/3.20, s

2 δC 133.8 104.2 149.4 136.4 149.4 104.2 89.4 55.5 64.9 138.3/138.2 111.9/111.8 145.6/145.5 148.9/148.9 130.1/130.0 116.2/116.0 81.0/81.0 24.2/24.0 56.8 56.8 56.8 56.5/56.5

δH

3 δC

6.69, s

6.69, s 5.53, d, 6.6 3.51, m 3.79, dd, 11.2, 3.2 3.86, dd, 11.2, 5.2 6.85, s

6.85, s 4.40, q, 6.6 1.40/1.40, d, 6.6 3.83, s 3.83, s 3.88, s 3.37, q, 7.2 1.16, t, 7.2

133.5 104.2 149.4 136.4 149.4 104.2 89.3 55.5 64.9 138.9 111.8/111.7 145.5 148.8 130.0/130.0 115.9 79.2/79.1 24.4/24.3 56.8 56.8 56.8 64.8/64.8 15.6

δH 6.69, s

6.69, s 5.54, d, 6.4 3.51, m 3.79, dd, 11.2, 3.2 3.86, dd, 11.2, 5.2 6.85, s

6.85, s 4.37/4.37, q, 6.4 1.40, d, 6.4 3.82, s 3.82, s 3.88, s 3.34, t, 7.2 1.52, m 1.36, m 0.89, t, 7.2

δC 133.6 104.2 149.4 136.4 149.4 104.2 89.4 55.4 65.0 139.0 111.8 145.5 148.8 130.0 116.0 79.3/79.3 24.5/24.4 56.8 56.8 56.8 69.3/69.2 33.1 20.4 14.2

compounds 1–3 are isolated as C-7 epimers.

EtOH extract was purified using various column chromatographies to afford three new dihydrobenzofuran-type norneolignans (1–3) which were isolated as C-7 epimers together with two known compouns (Fig. 1). The known compounds were identified as (2R, 3S)-dihydro-2(3, 5-dimethoxy-4-hydroxyphenyl)-7-methoxy-5-acetyl-benzofuran (4) (Bi et al., 2004) and selariscinin D (5) (Nguyen et al., 2015a) by analysis of their spectroscopic data and comparison with literature data. Tamariscinol U (1) was obtained as a yellow gum. Its molecular formula was determined to be C21H26O7 by HR-ESI-MS at m/z 413.1577 [M + Na]+ (calcd for C21H26O7Na+, 413.1576). The 1H-NMR data (Table 1) displayed signals for two 1, 2, 3, 5-tetrasubstituded benzene rings [δH 6.69 (2H, brs) and 6.85 (2H, s)], four methoxyls [δH 3.81, 3.81, 3.88, and 3.21/3.20 (each 3H, s)], one methyl [δH 1.40 (3H, d, J =6.4 Hz)], one oxygenated methylene [δH 3.78 (1H, dd, J = 10.8, 2.8 Hz) and 3.86 (1H, dd, J = 10.8, 5.2 Hz)], one methine [3.51 (1H, m)], and two oxymethine protons [δH 5.54 (1H, d, J =6.4 Hz) and 4.29/4.28 (1H, q, J =6.4 Hz)]. The 13C-NMR (Table 1) and HSQC spectra (Figure S3, Supporting Information) showed 21 carbon signals consisting of eight sp2 quaternary carbons (including five oxygenated ones), four sp2 methines, three sp3 methines (including two oxygenated ones), one sp3 oxymethylene carbon, one methyl, and four methoxyls. The 1D-NMR data of 1 were very similar with those of tamariscinol T, a dihydrobenzofuran-type norneolignan isolated by Kim (Kim et al., 2015) from the same plant, except for the presence of one more methoxyl [3.21/3.20 (3H, s)]. This methoxy group was then assigned to connect with C-7′ by HMBC correlation of H3-1′′ (3.21/3.20, s, 3 H) to C-7′ (δC 81.0/81.0) (Fig. 2 and Figure S4, Supporting Information). Further detailed HSQC, HMBC, and 1H-1H COSY spectral analysis

completely established the planar structure of 1 as shown (Fig. 2 and Figure S3, S4 and S5, Supporting Information). The occurrence of pairs of H-7′ and H-1′′ signals in the 1H-NMR (Table 1 and Figure S1, Supporting Information) and of pairs of C-1′–8′ and C-1′′ signals in the 13C-NMR spectrum (Table 1 and Figure S2, Supporting Information) indicated 1 was a mixture of two C-7′ epimers in a ratio of about 1: 1.3 based on integration, which was unable to be separated successfully by further HPLC purification neither using a YMC-Pack ODS-A column nor using two normal-phase chiral columns (Daicel Chiralcel ODeH and ADHe). The absolute configuration of 1 was determined using NOESY and CD spectroscopy. The NOESY spectrum revealed a correlation between H-7 (δH 5.54) and H-9 (δH 3.86) that suggested a trans- relationship between H-7 and H-8 (Fig. 2 and Figure S6, Supporting Information). In the CD spectrum, the negative Cotton effect around 290 nm indicated that 1 had a 7R, 8S configuration on the basis of the reversed helicity rule of the 1Lb band CD for the 7-methoxy-2, 3-dihydrobenzo[b]furan chromophore (Antus et al., 2001) (Fig. 3). Thus, the structure of tamariscinol U (1) was established as (7R, 8S)-4, 9-dihydroxy-3, 3′, 5, 7′-tetramethoxy-9′-nor-4′, 7-epoxy5′, 8-neolignan. Tamariscinol V (2) and Tamariscinol W (3) were also obtained as yellow gum. Their 1H-NMR spectra (Table 1 and Figure S8 and S15, Supporting Information) closely resembled those of 1 and indicated they were the ethyl ether and the n-butyl ether homologues of 1, respectively, due to the presence of signals for one ethoxy group [δH 3.37 (2H, q, J = 7.2 Hz) and 1.16 (3H, t, J = 7.2 Hz)] in 2 and signals for one butoxyl unit [δH 3.34 (2H, t, J = 7.2 Hz), 1.52 (2H, m), 1.36 (2H, m), and 0.89 (3H, t, J = 7.2 Hz)] in 3. This aforementioned deduction 80

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Fig. 2. 1H-1H COSY (bold line), Key HMBC (H→C) and NOESY (H H) correlations of 1.

3. Experimental 3.1. General experimental procedures The 1D and 2D NMR were recorded on a Bruker AVANCE 400 NMR spectrometer with tetramethylsilane (TMS) as an internal reference. All HRESI-MS spectra were analyzed on a Waters UPLC-QTOF mass spectrometer equipped with ESI source (Waters, Milford, MA, USA). UV spectra were obtained on a Shimadzu UV-2550 Spectrophotometer. Silica gel (300 − 400 mesh, Qingdao Marine Chemical Plant, Qingdao, P. R. China), C18 reversed-phase silica gel (150 − 200 mesh, Merck), MCI gel (CHP20 P, 75 − 150 μM, Mitsubishi Chemical Industries Ltd., Japan), and Sephadex LH-20 gel (75 − 150 μM, GE Healthcare, USA) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant) were used for TLC. Semi-preparative HPLC was performed on an Agilent 1200 system equipped with a VWD G1314B detector and a YMC-Pack ODS-A column (250 × 10 mm, 5 μm). All solvents used were of analytical grade (Xilong Chemical Reagent Co., Ltd., Guangdong, P. R. China). Both the mushroom tyrosinase and L-DOPA were purchased from the Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Kojic acid was purchased from the Solarbio Science & Technology Co., Ltd (Beijing, P. R. China).

Fig. 3. CD spectra of compounds 1―3.

was corroborated by the 1H-1H COSY and HMBC spectra (Figure S11, S12, S18, and S19, Supporting Information) of 2 and 3 as well as their molecular formula, C22H28O7 and C24H32O7, as determined by HR-ESIMS. Further detailed 2D NMR (HSQC and HMBC) spectral analysis confirmed above assignment and established the planar structures of 2 and 3 as shown (Figure S22, Supporting Information). Moreover, both the 1D NMR spectra of 2 and those of 3 yielded two inseparable C-7′ epimers, respectively, as the occurrence of pairs of C-7′, C-8′, and C-1′′ signals in their 13C-NMR spectra (Table 1 and Figure S9 and 16, Supporting Information). In the CD spectrum, their similar Cotton effect curves with that of 1 indicated that 2 and 3 possess a 7R, 8S configuration (Fig. 3). Therefore, tamariscinol V (2) and tamariscinol W (3) were established in turn as (7R, 8S)-7′-ethoxy-4, 9-dihydroxy-3, 3′, 5trimethoxy-9′-nor-4′, 7-epoxy-5′, 8-neolignan and (7R, 8S)-7′-butoxy-4, 9-dihydroxy-3, 3′, 5-trimethoxy-9′-nor-4′, 7-epoxy-5′, 8-neolignan. Compounds 1–5 exhibited significant inhibitory activities against the mushroom tyrosinase (Table 2). Among them, compound 1 was the most potent compound (IC50 = 5.75 μM), which exhibited a threefold activity as that of the positive control, kojic acid. Compound 2 also showed a better inhibitory activity than kojic acid with an IC50 value of 11.86 μM, followed by selariscinin D (5) with a slightly weaker activity (IC50 = 23.79 μM). While another 9′-norneolignan, compound 3 exhibited a moderate activity with an IC50 value of 30.37 μM. From above results, it can be seen that the substituent group at C-7′ of these 9′norneolignans (1–3) was crucial for their inhibitory activities. Moreover, compound 4 exhibited a moderate activity with an IC50 value of 67.76 μM.

3.2. Plant material The herb of S. tamariscina was collected in the Jiangxi Province of China in August 2017, and the plant material (ST-201708) was identified by Prof. Yun Lin of the School of Pharmacy, Nanchang University. A voucher specimen has been deposited in our laboratory. 3.3. Extraction and isolation The dry powder of S. tamariscina (5.0 kg) was extracted with 95% EtOH three times at ambient temperature to yield a crude extract (240.0 g), which was suspended in water and then extracted with EtOAc. The EtOAc extract (49.6 g) was subjected to MCI gel column chromatography (CC) eluted with a CH3OH/H2O gradient (3: 7→10: 0, v/v) to afford eight fractions, Fr.1−Fr.8. Fr.2 (1.6 g) was separated over Sephadex LH-20 (EtOH) to yield four subfractions, Fr.2A−Fr.2D. Fr.2A (78.4 mg) was then separated by semi-preparative HPLC (CH3CN/H2O, 30: 70, 3.0 ml/min) to give two compounds which were further purified by Sephadex LH-20 (EtOH) to afford 1 (tR =42.3 min, 4.0 mg) and 2 (tR =25.4 min, 1.5 mg), respectively. Fr.2C (102.3 mg) was chromatographed over a silica gel column (petroleum ether/acetone, 2: 1) to yield 5 (4.1 mg). Fr.4 (3.6 g) was separated by Sephadex LH-20 (EtOH) to give subfractions Fr.4A−Fr.4 F. Fr.4C (343.7 mg) was purified by CC on silica gel (CHCl3/MeOH, 30: 1), followed by semi-preparative HPLC (CH3CN/H2O, 50: 50, 4.0 ml/min) to afford 3 (tR =13.7 min, 1.3 mg). Fr.1 (1.6 g) was subjected to CC with Sephadex LH-20 (EtOH) to give three fractions (Fr.1A−Fr.1C). Fr.1B (743.5 mg) was separated on silica gel CC (CHCl3/MeOH, 70: 1→35:1) to afford seven subfractions (Fr.1B1−Fr.1B7). Fr.1B4 (112.9 mg) was then purified by semi-preparative HPLC (CH3CN/H2O, 30: 70, 3.0 ml/min) to afford 4 (tR =15.1 min, 3.1 mg).

Table 2 Tyrosinase Inhibitory Activity of the Compounds 1–5. compound

IC50/(μM)

compound

IC50/(μM)a

1 2 3

5.75 ± 0.16 11.86 ± 0.09 30.37 ± 0.21

4 5 kojic acid

67.76 ± 0.13 23.79 ± 0.11 17.32 ± 0.24

a

Each value represents the mean ± SD of three determinations. 81

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3.4. Spectroscopic data

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Tamariscinol U (1): Yellow gum; 1H- and 13C-NMR data: see Table 1; (+) HR-ESI-MS m/z 413.1577 [M+Na]+ (calcd for C21H26O7Na, 413.1576). Tamariscinol V (2): Yellow gum; 1H- and 13C-NMR data: see Table 1; (+) HR-ESI-MS m/z 427.1715 [M+Na]+ (calcd for C22H28O7Na, 427.1733). Tamariscinol W (3): Yellow gum; 1H- and 13C-NMR data: see Table 1; (+) HR-ESI-MS m/z 455.2043 [M+Na]+ (calcd for C24H32O7Na, 455.2046) and 887.4149 [2M+Na]+ (calcd for C48H64O14Na, 887.4194). 3.5. Tyrosinase inhibitory assay The tyrosinase assay was conducted as reported by us previously (Wang et al., 2018). All testing samples were dissolved in dimethyl sulfoxide (DMSO) and used at a concentration series of 100, 50, 25, 10, 5 μg/mL (or μM for pure compounds). The tyrosinase assay was performed in 96-well microplates with 200 μl total testing solution using kojic acid, a known tyrosinase inhibitor, as positive control. The assay mixture consist 40 μL of sample solution, 40 μL L-DOPA (2 mmol/L in 0.1 M phosphate buffer pH 6.8), 40 μL of mushroom tyrosinase solution (2 U/mL in 0.1 M phosphate buffer pH 6.8), and 80 μL of 0.1 M phosphate buffer pH 6.8. After incubation at room temperature for 30 min, the assay mixture was measured the absorbance at 475 nm in triplicate with a microplate reader (Thermo Electron Corporation, CA, USA). Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21362023 and 30901855), the Natural Science Foundation of Jiangxi Province, China (No. 20142BAB215021 and 20151BAB205083), and the Project of Jiangxi Provincial Education Department (No. GJJ150843). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.08.013. References Adhikari, A., Devkota, H.P., Takano, A., Masuda, K., Nakane, T., Basnet, P., SkaikoBasnet, N., 2008. Screening of Nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition. Int. J. Cosmet. Sci. 30, 353–360. Antus, S., Kurtan, T., Juhasz, L., Kiss, L., Hollosi, M., Majer, Z.S., 2001. Chiroptical properties of 2,3-dihydrobenzo[b]furan and chromane chromophores in naturally occurring O-heterocycles. Chirality 13, 493–506. Bi, Y.F., Zheng, X.K., Feng, W.S., Shi, S.P., 2004. Isolation and structural identification of chemical constituents from Selaginella tamariscina (Beauv.) Spring. Acta Pharm. Sin. B 39, 41–45. Cao, Y., Wu, Y.P., Wen, X.Z., Weng, Y., Wang, Q., 2012. Cytotoxic constituents from Selaginella tamariscina. Nat. Prod. Res. Dev. 24, 150–154. Cao, Q., Qin, L., Huang, F., Wang, X., Yang, L., Shi, H., Wu, H., Zhang, B., Chen, Z., Wu, X., 2017. Amentoflavone protects dopaminergic neurons in MPTP-induced Parkinson’s disease model mice through PI3K/Akt and ERK signaling pathways. Toxicol. Appl. Pharmacol. 319, 80–90. Cheng, X.L., Ma, S.C., Yu, J.D., Yang, S.Y., Xiao, X.Y., Hu, J.Y., Lu, Y., Shaw, P.C., But, P.P.H., Lin, R.C., 2008. Selaginellin A and B, two novel natural pigments isolated from Selaginella tamariscina. Chem. Pharm. Bull. 56, 982–984. Chiari, M.E., Joray, M.B., Ruiz, G., Palacios, S.M., Capinella, M.C., 2010. Tyrosinase inhibitory activity of native plants from central Argentina: isolation of an active principle from Lithrea molleoides. Food Chem. 120, 10–14. Dat, L.D., Zhao, B.T., Hung, N.D., Lee, J.H., Min, B.S., Woo, M.H., 2017. Lignan derivatives from Selaginella tamariscina and their nitric oxide inhibitory effects in LPS-stimulated RAW 264.7 cells. Bioorg. Med. Chem. Lett. 27, 524–529.

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Zhu, Q.F., Shao, L.D., Wu, X.D., Liu, J.X., Zhao, Q.S., 2019. Isolation, structural assignment of isoselagintamarlin A from Selaginella tamariscina and its biomimetic synthesis. Nat. Prod. Bioprospect. 9, 69–74. Zou, C., Wang, Y., Shen, Z., 2005. 2-NpBDG as a fluorescent indicator for direct glucose uptake measurement. J. Biochem. Biophys. Methods 64, 207–215.

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