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Phytochemical investigation of Eremostachys moluccelloides Bunge (Lamiaceae)
Biochemical Systematics and Ecology 84 (2019) 17–20
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phytochemical investigation of Eremostachys moluccelloides Bunge (Lamiaceae)
T
Faxuan Qiua, Manana Khutsishvilib, George Fayvushc, Kamilla Tamanyanc, Daniel Athad, Robert P. Borrisa,∗ a
School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China National Herbarium of Georgia, Ilia State University, Tbilisi, Georgia c Institute of Botany, Armenian National Academy of Sciences, Yerevan, Armenia d New York Botanical Garden, Bronx, NY, USA b
ARTICLE INFO
ABSTRACT
Keywords: Lamiaceae Lamioideae Eremostachys moluccelloides Diterpene Chemotaxonomy
Phytochemical investigation of the aerial parts of Eremostachys moluccelloides Bunge led to the identification of a new diterpene, 2β,14-dihydroxy −11-formyl- 12-carboxy-13-des-isopropyl-13-hydroxymethyl-abieta-8,11,13triene- 16(17)- lactone (1), along with the known compounds 12, 18-dicarboxy-14-hydroxy-13-des -isopropyl13-hydroxymethyl- abieta-8,11,13-triene-16(17)-lactone (2), 5-hydroxy-3′,4′,7-trimethoxyflavone (3), 5-hydroxy-4’,7-dimethoxyflavone (4), luteolin-7-O-β-glucoside (5), verbascoside (6), luteolin 7-O-(6″-O-β-D-apiofuranosyl) -β-D-glucopyranoside (7), chlorogenic acid (8), echinacoside (9), apigenin-7-O-β-D-glucoside (10), pcoumaric acid (11), vanillic acid (12), apigenin-7-O-(6″-E-p-coumaroyl)-β-D-glucopyranoside (13), apigenin-7O-(3″,6″-E-p-dicoumaroyl)-β-glucoside (14), lamalbide (15), 6β-hydroxy-7-epi-loganin (16), phloyoside II (17) The structures were elucidated on the basis of 1D and 2D NMR spectroscopy, UV, MS and by comparison with compounds previously reported in the literature. Compounds 1–4, 8, 9, 11, 12, 14 have not been reported previously from any species within the genus Eremostachys. Compounds 1–14, 17 were obtained from this species for the first time. The chemotaxonomic significance of the isolated compounds is discussed.
1. Subject and source The genus Eremostachys Bunge (Lamiaceae) comprises about 60 species and is widely distributed through West and Central Asia (Rabe et al., 2014). The plants in this genus are perennial herbs with thick roots. Previous studies have demonstrated various biological effects from a number of Eremostachys species, such as anti-inflammatory, antinociceptive and antioxidant activities (Asnaashari et al., 2015). In this study, the aerial parts of Eremostachys moluccelloides Bunge were collected in Kotayk Province, Armenia, between Garni and Zovashen, near Kotayk (GPS: N 40.0950, E 44.6389) in May 2006. A voucher specimen documenting this collection (K. Tamanyan 26–2006) has been deposited in the herbarium of the Institute of Botany, Armenian National Academy of Sciences (ERE), and the New York Botanical Garden (NY). 2. Previous work To the best of our knowledge, there are five previous reports on the chemical constituents of E. moluccelloides. Among them, four reports
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were focused on the oils of the seeds (Panekina et al., 1978; Gusakova and Umarov, 1975a, 1975b, 1976) and one report described iridoid glucosides from the aerial parts of the plant (Çalış et al., 2007). Phytochemical investigations on other species of Eremostachys revealed the presence of essential oils and glycosides of iridoids, phenylethanoids, flavonoids, and diterpenes (Rustaiyan et al., 2011). Other studies have focused on essential oils (Nori-Shargh et al., 2007). The limited phytochemical information on Eremostachys Bunge contains little, if anything, of chemotaxonomic significance. 3. Present study 3.1. General experimental conditions All solvents used throughout these studies were HPLC grade (Concord Technologies, Tianjin, China). Thin-layer chromatography (TLC) was performed on precoated layers of Silica gel GF254 (Haiyang Chemicals Corp., Qingdao, China). Column chromatography (CC) was performed on either silica gel 40–65 μm, 60 A (Sorbent Technologies,
Corresponding author. E-mail address: [email protected] (R.P. Borris).
https://doi.org/10.1016/j.bse.2019.03.002 Received 24 January 2019; Received in revised form 6 March 2019; Accepted 16 March 2019 0305-1978/ © 2019 Elsevier Ltd. All rights reserved.
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Norcross GA, USA), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), or Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan). Flash chromatography was performed using an ISCO CombiFlash Rf + instrument (Teledyne ISCO, Lincoln NE, USA) using prepacked columns of silica gel 25–40 μm (SepaFlash, Santai Technologies, Changzhou, China). Analytical and semipreparative HPLC separations were performed on an Agilent 1260 V L quad gradient system (G1311C pump, G1329B autosampler, G1316A thermostatted column compartment and G1315D photodiode array detector, Agilent Technologies, Santa Clara CA, USA), using Hypersil Gold C18, C8 or aQ analytical column (3 μm, 2.1 × 150 mm, Thermo Scientific, Waltham MA, USA) or a Hypersil Gold C18 or aQ semipreparative column (5 μm, 10 × 250 mm). Preparative HPLC separations were performed on an Agilent 1260 V L quad gradient system (G1311C pump, and G1315D photodiode array detector) equipped with a Rheodyne 7725i manual injection valve (IDEX Health & Science, Middleboro MA, USA) and Shimadzu CTO-20 A column oven (Shimadzu Scientific Instruments, Kyoto, Japan). Preparative separations were conducted using a Hypersil Gold C18 column (5 μm, 21.2 × 250 mm). Mass spectra were determined on an Agilent Technologies 6230 TOF LC-MS or an Agilent Technologies 6420 triple quadrupole LC-MS. NMR spectra were recorded on Bruker Avance III spectrometers (Bruker Biospin Corp., Billerica MA, USA) operating at 400 or 600 MHz (1H) and 100 or 150 MHz (13C). Residual solvent resonances were used as internal reference, and chemical shifts are reported in PPM (δ). Optical rotations were determined with an Autopol-II polarimeter (Rudolf Research Analytical, Hackettstown NJ, USA). Circular dichroism studies were performed using a Bio-Logic SAS MOS-500 instrument (Bio-Logic SAS, Claix, France).
The n-BuOH fraction (14.49 g) was chromatographed on the macroporous resin, Diaion HP20 (330 g), eluted with water, 25%, 50%, 75% and 100% methanol-water (600 ml each). Fractions containing similar compounds were combined and evaporated to give fifteen fractions (Frs.1–15). Fr.14, eluted with MeOH, was subjected to a reverse-phase HPLC on a Hypersil Gold C18 column eluted with 12% MeCN:H2O to obtain compound 5 (11.5 mg), HPLC retention time 18.40 min (Hypersil Gold C18, 12% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.10, eluted with 75% methanol, was analyzed by HPLC then fractionated by preparative HPLC eluted with 12% MeCN:H2O to give 5 subfractions (A-E). Subfraction E and C were purified by preparative HPLC with 12% MeCN:H2O to yield compound 6 (37.6 mg) and compound 7 (28.4 mg), HPLC retention times of 22.56 min, 10.81 min, respectively (Hypersil Gold C18, 12% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Subfraction A was subjected to semipreparative HPLC on a Hypersil Gold aQ column eluted with 5% MeCNH2O to yield compound 8 (30.9 mg), HPLC retention time 17.78 min (Hypersil Gold aQ, 5% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Further purification of subfraction B by preparative HPLC with 10% MeCN:H2O to afford compound 9 (26.4 mg), HPLC retention time 14.07 min (Hypersil Gold C18, 10% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Compound 10 (1.1 mg) was obtained from Fr.11 by semipreparative HPLC on a Hypersil Gold C18 column eluted with 14% MeCN:H2O. HPLC retention time 7.80 min (Hypersil Gold C18, 14% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.9, eluted with 50% methanol, was separated in a similar manner to afford compound 11 (2.1 mg), HPLC retention time 12.57 min (Hypersil Gold C18, 12% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C) and compound 12 (0.5 mg), HPLC retention time 12.55 min (Hypersil Gold C18, 5% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.13, eluted with 100% methanol, was subjected to semipreparative HPLC on a Hypersil Gold C18 column eluted with 21% MeCN:H2O to afford compound 13 (10.8 mg), HPLC retention time 25.01 min (Hypersil Gold C18, 21% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Further elution with 33% MeCN:H2O afforded compound 14 (4.6 mg), HPLC retention time 19.58 min (Hypersil Gold C18, 33% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.2, eluted with 25% methanol, was purified by semipreparative HPLC on a Hypersil Gold aQ column eluting with 3% aqueous MeOH to yield compound 15 (18.1 mg) and compound 16 (9.4 mg). HPLC retention time 7.56 min and 6.16 min, respectively, (Hypersil Gold aQ, 5% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Further purification of Fr.2 by semipreparative HPLC on a Hypersil Gold aQ column with 3% MeCN:H2O afforded compound 17 (4.5 mg). HPLC retention time 6.76 min (Hypersil Gold aQ, 10% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C).
3.2. Extraction and isolation The dried and powdered aerial parts of E. moluccelloides Bunge (1 kg) were extracted with methanol (3 × 6 L) at ambient temperature and the combined methanol extracts were concentrated in vacuo to yield a tarry residue (115.9 g). The resulting residue was suspended in 90% (aqueous) methanol (250 ml) and partitioned with n-hexane (3 × 200 ml). The defatted methanol extract was freed of solvent in vacuo, dispersed in water (250 ml) and extracted successively with dichloromethane (DCM) and n-BuOH (each 3 × 200 ml) to obtain a gross separation into hexane-, dichloromethane-, butanol-, and water-soluble fractions. The dichloromethane extract (2.3 g) was subjected to flash chromatography on silica gel eluting with a step-gradient of dichloromethane and ethyl acetate mixtures (40:1, 20:1, 10:1, 4:1, 1:1) followed by linear gradient of ethyl acetate-methanol (100:0–0:100) to afford 24 fractions (Fr.1-Fr.24). Fr.18, eluted with DCM and ethyl acetate (4:1), was purified by silica gel column chromatography (DCM:MeOH = 40:1, 20:1 step gradient), chromatography over Sephadex LH-20 in MeOH followed by semi-preparative HPLC (Hypersil Gold C18, 40 °C, 28% MeCN:H2O, 4 ml/min) to obtain compound 1 (2.3 mg) and 2 (3.0 mg). HPLC retention time 7.84 and 8.98 min, respectively (Hypersil Gold C18, 35% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.11, eluted with DCM and ethyl acetate (20:1), was analyzed by HPLC (Hypersil Gold C18, 40 °C, 45% MeCN:H2O, 0.2 ml/min) and then purified by semi-preparative HPLC (Hypersil Gold C18, 40 °C, 45% MeCN:H2O, 4 ml/min) to yield compound 3 (3.2 mg). HPLC retention time 11.72 min (Hypersil Gold C18, 50% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C. Fr.8, eluted with DCM and ethyl acetate (40:1), was re-chromatographed on silica gel eluting with a linear gradient of hexane and DCM (100:0–0:100) then purified on Sephadex LH-20 eluting with chloroform-methanol (1:1) to afford compound 4 (3.3 mg). HPLC retention time 9.68 min (Hypersil Gold C18, 65% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C).
3.3. Structure elucidation Compound 1 was obtained as a yellow solid; [ ]18 D + 50.0 (c 0.1, MeOH); UV (MeOH) λmax 210, 246, and 300 nm. CD [θ]210 3.4 × 104 deg cm2/dmol, [θ]246 4.5 × 105 deg cm2/dmol, [θ]300–2.8 × 104 deg cm2/dmol. IR (microscope) νmax 3426, 1631 cm−1. Its molecular formula C20H24O5 with 9 degrees of unsaturation was determined based on a HR-ESI-MS pseudomolecular ion at m/z 367.1516 ([M+Na]+, calcd 367.1521). 1H NMR spectral data (600 MHz, CD3OD) δH 1.04 (3H, s, H3-19), 1.07 (3H, s, H3-18), 1.37 (1H, dd, J = 12.7, 2.1 Hz, H-5), 1.53 (1H, dd, J = 13.1, 3.4 Hz, H-3), 1.64 (3H, s, H3-20), 1.67 (1H, m, H-3), 1.76 (1H, m, H-6), 1.91 (1H, ddd, J = 1.7, 5.8, 7.2 Hz, H-6), 1.95 (1H, dd, J = 13.1, 6.4 Hz, H-1), 2.10 (1H, dd, J = 13.1, 7.9 Hz, H-1), 2.63 (1H, ddd, J = 18.4, 12.7, 7.2 Hz, H-7), 3.05 (1H, dd, J = 18.4, 6.1 Hz, 18
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Fig. 1. Chemical structures of isolated compounds (1–17) from E. moluccelloides Bunge.
reduced [δH 5.26, 5.29 each (1H, d, J = 15.2 Hz)]. The 13C NMR, DEPT 135° and HSQC spectra indicated a total of 20 carbon signals comprising three methyls, five methylenes, two methines, one carboxyl, one aldehyde, and eight quaternary carbons, six of which belong to an aromatic ring. These data, together with other spectroscopic characteristics, suggested that compound 1 is a tetracyclic compound related to the abietane diterpenes. The aliphatic spin systems were established on the basis of a COSY experiment and supported by HMBC. H2-1 (δH 1.95, 2.10) were coupled to H-2 (δH 3.96) as were H2-3 (δH 1.53, 1.67). The lack of coupling between H2-1 and H2-3 clearly established that the hydroxyl was located between these groups. NOESY
H-7), 3.96 (1H, m, H-2), 5.26 (1H, d, J = 15.2 Hz, H-17), 5.29 (1H, d, J = 15.2 Hz, H-17), 10.95 (1H, s, H-15). 13C-NMR spectral data (150 MHz, CD3OD) δC 19.4 (C-6), 25.8 (C-18), 27.4 (C-7), 28.8 (C-20), 32.5 (C-19), 33.7 (C-4), 42.1 (C-10), 46.1 (C-3), 48.2 (C-5), 50.2 (C-1), 67.2 (C-2), 69.6 (C-17), 125.1 (C-12), 130.3 (C-13), 131.3 (C-11), 131.9 (C-8), 152.0 (C-14), 154.0 (C-9), 172.4 (C-16), 198.8 (C-15). The 1H NMR spectrum revealed the presence of three methyl groups [δH 1.04 (3H, s, H-19), δH 1.64 (3H, s, H-20) and δH 1.07 (3H, s, H-18)], one aldehyde group [δH 10.95 (1H, s)], one oxygenated methine [δH 3.96 (1H, m)], and an oxygenated methylene which appeared as an AB quartet in which the outer member of each doublet were greatly 19
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correlations between H-2 (δH 3.96) and the methyl groups H3-20 (δH 1.64) and H3-18 (δH 1.07) confirms that the hydroxy group at C-2 is equatorial. That the remaining aliphatic protons form a single isolated spin system supports the angular arrangement of the ring system with the aromatic ring fused across C-8 and C-9 rather than a linear arrangement. In the HMBC spectrum, correlations are observed between protons of the angular methyl group H3-20 (δH 1.64) and C-1 (δC 50.2), C-5 (δC 48.2), C-9 (δC 154.0), and C-10 (δC 42.1). The aldehydic proton H-15 (δH 10.95) shows an HMBC correlation with its point of attachment, C-11 (δC 131.2) as well as C-9 (δC 154.0). H-1 (δH 1.95) also shows a correlation to C-9, further supporting this assignment. The AB quartet comprising H2-17 (δH 5.26, 5.29) displays strong HMBC correlations with C-13 (δC 130.2) and C-16 (δC 172.4), establishing the presence of a fused γ-lactone. Weaker 3-bond correlations between these protons and C-12 (δC 125.1) and C-14 (δC 152.0) but not C-11 (δC 131.2) support the orientation of the lactone shown in Fig. 1. Finally, HMBC correlations between H2-7 (δH 2.63, 3.06) and both C-9 (δC 154.0) and C-14 (δC 152.0) confirm the substitution pattern on the aromatic ring. These observations support the assignment of 1 as 2β,14dihydroxy-11-formyl-12-carboxy-13-des-isopropyl-13-hydroxymethylabieta-8,11,13-triene-16(17)-lactone. Compound 1 is analogous to (18) previously reported from Phlomis tuberosa (Yang et al., 2015), differing in the oxidation state of C-2 and C-18. The structures of the obtained known compounds were elucidated by 1D and 2D homonuclear and heteronuclear NMR spectroscopy and by comparison of their spectral data (1H NMR, 13C NMR, MS) with those reported in the literature. The 16 compounds were determined to be 12,18-dicarboxy-14-hydroxy-13-des-isopropyl −13-hydroxymethylabieta-8,11,13-triene-16(17)-lactone (2) (Yang et al., 2015), 5-hydroxy3′,4′,7-trimethoxyflavone (3) (Sipos and Kónya, 2018), 5-hydroxy-4’,7dimethoxyflavone (4) (Sipos and Kónya, 2018), luteolin-7-O-β-glucoside (5) (Refaat et al., 2015), verbascoside (6) (Lan et al., 2018), luteolin 7-O-(6″-O-β-D-apiofuranosyl)-β-D-glucopyranoside (7) (Bucar et al., 1998), chlorogenic acid (8) (Okonkwo et al., 2016), echinacoside (9) (Porter et al., 2015), apigenin-7-O-β-D-glucoside (10) (Marzouk et al., 2016), p-Coumaric acid (11) (Liao et al., 2014), vanillic acid (12) (Ahmed et al., 2014), apigenin-7-O-(6″-E-p-coumaroyl)-β-D-glucopyranoside (13) (Chang et al., 2017), apigenin-7-O-(3″,6″-E-p-dicoumaroyl)-β-glucoside (14) (Sweidan and Abu Zarga, 2016), lamalbide (15) (Zhang et al., 2009), 6β-hydroxy-7-epi-loganin (16) (Zhang et al., 2009), phloyoside II (17) (Kasai et al., 1994), (Fig. 1). It should be noted that, in our opinion, the name originally applied to 2 by Yang et al., 14-hydroxyabieta-8,11,13 -triene-17-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone is incorrect. We propose 12,18-dicarboxy-14-hydroxy −13-des-isopropyl-13-hydroxymethyl -abieta8,11,13-triene-16(17)-lactone as a more appropriate name for this compound.
the related betonicosides (e.g., betonicoside-A aglycone 19) and betolide (20) in which the lactone ring has formed by condensation of the carboxylic acid at C-12 of the aromatic ring with a hemiacetal at C-11, rather than with the hydroxymethyl group at C-13 (Miyase et al., 1996; Bankova et al., 1999; Tkachev et al., 1987). Since the original description of betolide in 1988, neither of these two, closely related, tetracyclic skeleta have been found anywhere else in the plant kingdom. Their narrow, known distribution, to date, suggests that they may become useful as chemotaxonomic markers for this subfamily, if not for the Lamiaceae as a whole, once a broader systematic search is undertaken. Declaration of interest None. Acknowledgements This study was supported, in part, by the National Basic Research Program of China (2015CB856500). The authors also gratefully acknowledge their respective institutions for financially supporting this research. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bse.2019.03.002. References Ahmed, A.F., Saad, H.E., Abd El-Karim, E.M., 2014. Molecules 19, 5940–5951. Asnaashari, S., Afshar, F.H., Ebrahimi, A., Moghadam, S.B., Delazar, A., 2015. Bioimpacts 5, 135–140. Bankova, V., Koeva-Todorovska, J., Stambolijska, T., Ignatova-Groceva, M.-D., Todorova, D., Popov, S., 1999. Z. Naturforsch. 54c, 876–880. Bucar, F., Ninov, S., Ionkova, I., Kartnig, T., Schubert-Zsilavecz, M., Asenov, I., Konuklugil, B., 1998. Phytochemistry 48, 573–575. Çalış, İ., Güvenç, A., Armağan, M., Koyuncu, M., Gotfredsen, C.H., Jensen, S.R., 2007. Helv. Chim. Acta 90, 1461–1466. Chang, Y.-X., Zhang, P., Zhang, X., Chen, J.-P., Rausch, W.D., Gula, A., Bao, B.Q., 2017. Nat. Prod. Res. 31, 1223–1227. Gusakova, S.D., Umarov, A.U., 1975a. Khim. Prir. Soedin. 11 (3), 324–328. Gusakova, S.D., Umarov, A.U., 1975b. Khim. Prir. Soedin. 11 (4), 511–512. Gusakova, S.D., Umarov, A.U., 1976. Khim. Prir. Soedin. 12 (6), 717–723. Kasai, R., Katagiri, M., Ohtani, K., Yamasaki, K., Yang, C.R., Tanaka, O., 1994. Phytochemistry 36, 967–970. Lan, Y.-H., Chi, X.-F., Zhou, G.-Y., Zhao, X.-H., 2018. Record Nat. Prod. 12 (4), 332–339. Liao, C.-R., Kuo, Y.-H., Ho, Y.-L., Wang, C.-Y., Yang, C.-S., Lin, C.-W., Chang, Y.-S., 2014. Molecules 19, 9515–9534. Marzouk, M.M., Hussein, S.R., Kassem, M.E., Kawashty, S.A., EI-Negoumy, S.I., 2016. Nat. Prod. Res. 30, 1537–1541. Miyase, T., Yamamoto, R., Ueno, A.S., 1996. Chem. Pharm. Bull. 44 (8), 1610–1613. Nori-Shargh, D., Kiaei, S.M., Deyhimi, F., 2007. Nat. Prod. Res. 21 (8), 733–735. Okonkwo, T.J., Osadebe, P.O., Proksch, P., 2016. Phytother Res. 30, 78–83. Panekina, T.V., Gusakova, S.D., Tabak, M. Ya, Umarov, A.U., 1978. Khim. Prir. Soedin. 14 (1), 44–48. Porter, E.A., Kite, G.C., Veitch, N.C., Geoghegan, I.A., Larsson, S., Simmonds, M.S.J., 2015. Phytochemistry 117, 185–193. Rabe, S.Z.T., Mahmoudi, M., Ahmadsimab, H., Rabe, S.S.Z.T., Emami, A., 2014. Food Agric. Immunol. 25 (4), 578–585. Refaat, J., Samy, M.N., Desoukey, S.Y., Ramadan, M.A., Sugimoto, S., Matsunami, K., Kamel, M.S., 2015. Med. Chem. Res. 24, 2939–2949. Rustaiyan, A., Masoudi, S., Ezzatzadeh, E., Akhlaghi, H., Aboli, J., 2011. J. Essential OilBearing J. Essent. Oil Bearing Plants 14, 84–88. Sipos, Z., Kónya, K., 2018. Synthesis 50, 1610–1620. Sweidan, N.I., Abu Zarga, M.H., 2016. Lett. Org. Chem. 13, 277–282. Tkachev, V.V., Nikonov, G.K., Atovmyan, L.O., Kobzar, A.Ya, Zinchenko, T.V., 1987. Khim. Prir. Soedin. 23 (6), 811–817. Yang, Y.B., Gu, L.H., Xiao, Y., Liu, Q., Hu, H.J., Wang, Z.T., Chen, K.X., 2015. PLoS One 10 (2). https://doi.org/10.1371/journal.pone.0116922. e0116922. Zhang, H.-J., Rothwangl, K., Mesecar, A.D., Sabahi, A., Rong, L.-J., Fong, H.H., 2009. J. Nat. Prod. 72, 2158–2162.
4. Chemotaxonomic significance The present study reports the identification of a new diterpene (1), along with one known diterpene (2), seven flavonoids (3, 4, 5, 7, 10, 13, 14), three phenolic acid (8, 11, 12), two phenylethanoids (6, 9) and three iridoid glucosides (15–17) from the aerial parts of E. moluccelloides Bunge (Fig. 1). Nine compounds (1–4, 8, 9, 11, 12, 14) have not been previously isolated from genus Eremostachys. And fifteen compounds (1–14, 17) have not been previously reported from this species. While most of these compounds are well known and widely distributed, the two diterpenes (1 and 2) are noteworthy as this is only the second report of compounds having this tetracyclic skeleton. Compounds 2 and 18 were isolated previously only from Phlomis tuberosa, which is a member of the subfamily Lamioideae. Similarly, the genera Stachys L. and Betonica L. (also in the Lamioideae) have afforded
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phytochemical investigation of Eremostachys moluccelloides Bunge (Lamiaceae)
T
Faxuan Qiua, Manana Khutsishvilib, George Fayvushc, Kamilla Tamanyanc, Daniel Athad, Robert P. Borrisa,∗ a
School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China National Herbarium of Georgia, Ilia State University, Tbilisi, Georgia c Institute of Botany, Armenian National Academy of Sciences, Yerevan, Armenia d New York Botanical Garden, Bronx, NY, USA b
ARTICLE INFO
ABSTRACT
Keywords: Lamiaceae Lamioideae Eremostachys moluccelloides Diterpene Chemotaxonomy
Phytochemical investigation of the aerial parts of Eremostachys moluccelloides Bunge led to the identification of a new diterpene, 2β,14-dihydroxy −11-formyl- 12-carboxy-13-des-isopropyl-13-hydroxymethyl-abieta-8,11,13triene- 16(17)- lactone (1), along with the known compounds 12, 18-dicarboxy-14-hydroxy-13-des -isopropyl13-hydroxymethyl- abieta-8,11,13-triene-16(17)-lactone (2), 5-hydroxy-3′,4′,7-trimethoxyflavone (3), 5-hydroxy-4’,7-dimethoxyflavone (4), luteolin-7-O-β-glucoside (5), verbascoside (6), luteolin 7-O-(6″-O-β-D-apiofuranosyl) -β-D-glucopyranoside (7), chlorogenic acid (8), echinacoside (9), apigenin-7-O-β-D-glucoside (10), pcoumaric acid (11), vanillic acid (12), apigenin-7-O-(6″-E-p-coumaroyl)-β-D-glucopyranoside (13), apigenin-7O-(3″,6″-E-p-dicoumaroyl)-β-glucoside (14), lamalbide (15), 6β-hydroxy-7-epi-loganin (16), phloyoside II (17) The structures were elucidated on the basis of 1D and 2D NMR spectroscopy, UV, MS and by comparison with compounds previously reported in the literature. Compounds 1–4, 8, 9, 11, 12, 14 have not been reported previously from any species within the genus Eremostachys. Compounds 1–14, 17 were obtained from this species for the first time. The chemotaxonomic significance of the isolated compounds is discussed.
1. Subject and source The genus Eremostachys Bunge (Lamiaceae) comprises about 60 species and is widely distributed through West and Central Asia (Rabe et al., 2014). The plants in this genus are perennial herbs with thick roots. Previous studies have demonstrated various biological effects from a number of Eremostachys species, such as anti-inflammatory, antinociceptive and antioxidant activities (Asnaashari et al., 2015). In this study, the aerial parts of Eremostachys moluccelloides Bunge were collected in Kotayk Province, Armenia, between Garni and Zovashen, near Kotayk (GPS: N 40.0950, E 44.6389) in May 2006. A voucher specimen documenting this collection (K. Tamanyan 26–2006) has been deposited in the herbarium of the Institute of Botany, Armenian National Academy of Sciences (ERE), and the New York Botanical Garden (NY). 2. Previous work To the best of our knowledge, there are five previous reports on the chemical constituents of E. moluccelloides. Among them, four reports
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were focused on the oils of the seeds (Panekina et al., 1978; Gusakova and Umarov, 1975a, 1975b, 1976) and one report described iridoid glucosides from the aerial parts of the plant (Çalış et al., 2007). Phytochemical investigations on other species of Eremostachys revealed the presence of essential oils and glycosides of iridoids, phenylethanoids, flavonoids, and diterpenes (Rustaiyan et al., 2011). Other studies have focused on essential oils (Nori-Shargh et al., 2007). The limited phytochemical information on Eremostachys Bunge contains little, if anything, of chemotaxonomic significance. 3. Present study 3.1. General experimental conditions All solvents used throughout these studies were HPLC grade (Concord Technologies, Tianjin, China). Thin-layer chromatography (TLC) was performed on precoated layers of Silica gel GF254 (Haiyang Chemicals Corp., Qingdao, China). Column chromatography (CC) was performed on either silica gel 40–65 μm, 60 A (Sorbent Technologies,
Corresponding author. E-mail address: [email protected] (R.P. Borris).
https://doi.org/10.1016/j.bse.2019.03.002 Received 24 January 2019; Received in revised form 6 March 2019; Accepted 16 March 2019 0305-1978/ © 2019 Elsevier Ltd. All rights reserved.
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Norcross GA, USA), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), or Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan). Flash chromatography was performed using an ISCO CombiFlash Rf + instrument (Teledyne ISCO, Lincoln NE, USA) using prepacked columns of silica gel 25–40 μm (SepaFlash, Santai Technologies, Changzhou, China). Analytical and semipreparative HPLC separations were performed on an Agilent 1260 V L quad gradient system (G1311C pump, G1329B autosampler, G1316A thermostatted column compartment and G1315D photodiode array detector, Agilent Technologies, Santa Clara CA, USA), using Hypersil Gold C18, C8 or aQ analytical column (3 μm, 2.1 × 150 mm, Thermo Scientific, Waltham MA, USA) or a Hypersil Gold C18 or aQ semipreparative column (5 μm, 10 × 250 mm). Preparative HPLC separations were performed on an Agilent 1260 V L quad gradient system (G1311C pump, and G1315D photodiode array detector) equipped with a Rheodyne 7725i manual injection valve (IDEX Health & Science, Middleboro MA, USA) and Shimadzu CTO-20 A column oven (Shimadzu Scientific Instruments, Kyoto, Japan). Preparative separations were conducted using a Hypersil Gold C18 column (5 μm, 21.2 × 250 mm). Mass spectra were determined on an Agilent Technologies 6230 TOF LC-MS or an Agilent Technologies 6420 triple quadrupole LC-MS. NMR spectra were recorded on Bruker Avance III spectrometers (Bruker Biospin Corp., Billerica MA, USA) operating at 400 or 600 MHz (1H) and 100 or 150 MHz (13C). Residual solvent resonances were used as internal reference, and chemical shifts are reported in PPM (δ). Optical rotations were determined with an Autopol-II polarimeter (Rudolf Research Analytical, Hackettstown NJ, USA). Circular dichroism studies were performed using a Bio-Logic SAS MOS-500 instrument (Bio-Logic SAS, Claix, France).
The n-BuOH fraction (14.49 g) was chromatographed on the macroporous resin, Diaion HP20 (330 g), eluted with water, 25%, 50%, 75% and 100% methanol-water (600 ml each). Fractions containing similar compounds were combined and evaporated to give fifteen fractions (Frs.1–15). Fr.14, eluted with MeOH, was subjected to a reverse-phase HPLC on a Hypersil Gold C18 column eluted with 12% MeCN:H2O to obtain compound 5 (11.5 mg), HPLC retention time 18.40 min (Hypersil Gold C18, 12% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.10, eluted with 75% methanol, was analyzed by HPLC then fractionated by preparative HPLC eluted with 12% MeCN:H2O to give 5 subfractions (A-E). Subfraction E and C were purified by preparative HPLC with 12% MeCN:H2O to yield compound 6 (37.6 mg) and compound 7 (28.4 mg), HPLC retention times of 22.56 min, 10.81 min, respectively (Hypersil Gold C18, 12% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Subfraction A was subjected to semipreparative HPLC on a Hypersil Gold aQ column eluted with 5% MeCNH2O to yield compound 8 (30.9 mg), HPLC retention time 17.78 min (Hypersil Gold aQ, 5% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Further purification of subfraction B by preparative HPLC with 10% MeCN:H2O to afford compound 9 (26.4 mg), HPLC retention time 14.07 min (Hypersil Gold C18, 10% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Compound 10 (1.1 mg) was obtained from Fr.11 by semipreparative HPLC on a Hypersil Gold C18 column eluted with 14% MeCN:H2O. HPLC retention time 7.80 min (Hypersil Gold C18, 14% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.9, eluted with 50% methanol, was separated in a similar manner to afford compound 11 (2.1 mg), HPLC retention time 12.57 min (Hypersil Gold C18, 12% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C) and compound 12 (0.5 mg), HPLC retention time 12.55 min (Hypersil Gold C18, 5% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.13, eluted with 100% methanol, was subjected to semipreparative HPLC on a Hypersil Gold C18 column eluted with 21% MeCN:H2O to afford compound 13 (10.8 mg), HPLC retention time 25.01 min (Hypersil Gold C18, 21% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Further elution with 33% MeCN:H2O afforded compound 14 (4.6 mg), HPLC retention time 19.58 min (Hypersil Gold C18, 33% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.2, eluted with 25% methanol, was purified by semipreparative HPLC on a Hypersil Gold aQ column eluting with 3% aqueous MeOH to yield compound 15 (18.1 mg) and compound 16 (9.4 mg). HPLC retention time 7.56 min and 6.16 min, respectively, (Hypersil Gold aQ, 5% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Further purification of Fr.2 by semipreparative HPLC on a Hypersil Gold aQ column with 3% MeCN:H2O afforded compound 17 (4.5 mg). HPLC retention time 6.76 min (Hypersil Gold aQ, 10% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C).
3.2. Extraction and isolation The dried and powdered aerial parts of E. moluccelloides Bunge (1 kg) were extracted with methanol (3 × 6 L) at ambient temperature and the combined methanol extracts were concentrated in vacuo to yield a tarry residue (115.9 g). The resulting residue was suspended in 90% (aqueous) methanol (250 ml) and partitioned with n-hexane (3 × 200 ml). The defatted methanol extract was freed of solvent in vacuo, dispersed in water (250 ml) and extracted successively with dichloromethane (DCM) and n-BuOH (each 3 × 200 ml) to obtain a gross separation into hexane-, dichloromethane-, butanol-, and water-soluble fractions. The dichloromethane extract (2.3 g) was subjected to flash chromatography on silica gel eluting with a step-gradient of dichloromethane and ethyl acetate mixtures (40:1, 20:1, 10:1, 4:1, 1:1) followed by linear gradient of ethyl acetate-methanol (100:0–0:100) to afford 24 fractions (Fr.1-Fr.24). Fr.18, eluted with DCM and ethyl acetate (4:1), was purified by silica gel column chromatography (DCM:MeOH = 40:1, 20:1 step gradient), chromatography over Sephadex LH-20 in MeOH followed by semi-preparative HPLC (Hypersil Gold C18, 40 °C, 28% MeCN:H2O, 4 ml/min) to obtain compound 1 (2.3 mg) and 2 (3.0 mg). HPLC retention time 7.84 and 8.98 min, respectively (Hypersil Gold C18, 35% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C). Fr.11, eluted with DCM and ethyl acetate (20:1), was analyzed by HPLC (Hypersil Gold C18, 40 °C, 45% MeCN:H2O, 0.2 ml/min) and then purified by semi-preparative HPLC (Hypersil Gold C18, 40 °C, 45% MeCN:H2O, 4 ml/min) to yield compound 3 (3.2 mg). HPLC retention time 11.72 min (Hypersil Gold C18, 50% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C. Fr.8, eluted with DCM and ethyl acetate (40:1), was re-chromatographed on silica gel eluting with a linear gradient of hexane and DCM (100:0–0:100) then purified on Sephadex LH-20 eluting with chloroform-methanol (1:1) to afford compound 4 (3.3 mg). HPLC retention time 9.68 min (Hypersil Gold C18, 65% MeCN:H2O containing 0.1% formic acid at 0.2 ml/min, 40 °C).
3.3. Structure elucidation Compound 1 was obtained as a yellow solid; [ ]18 D + 50.0 (c 0.1, MeOH); UV (MeOH) λmax 210, 246, and 300 nm. CD [θ]210 3.4 × 104 deg cm2/dmol, [θ]246 4.5 × 105 deg cm2/dmol, [θ]300–2.8 × 104 deg cm2/dmol. IR (microscope) νmax 3426, 1631 cm−1. Its molecular formula C20H24O5 with 9 degrees of unsaturation was determined based on a HR-ESI-MS pseudomolecular ion at m/z 367.1516 ([M+Na]+, calcd 367.1521). 1H NMR spectral data (600 MHz, CD3OD) δH 1.04 (3H, s, H3-19), 1.07 (3H, s, H3-18), 1.37 (1H, dd, J = 12.7, 2.1 Hz, H-5), 1.53 (1H, dd, J = 13.1, 3.4 Hz, H-3), 1.64 (3H, s, H3-20), 1.67 (1H, m, H-3), 1.76 (1H, m, H-6), 1.91 (1H, ddd, J = 1.7, 5.8, 7.2 Hz, H-6), 1.95 (1H, dd, J = 13.1, 6.4 Hz, H-1), 2.10 (1H, dd, J = 13.1, 7.9 Hz, H-1), 2.63 (1H, ddd, J = 18.4, 12.7, 7.2 Hz, H-7), 3.05 (1H, dd, J = 18.4, 6.1 Hz, 18
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Fig. 1. Chemical structures of isolated compounds (1–17) from E. moluccelloides Bunge.
reduced [δH 5.26, 5.29 each (1H, d, J = 15.2 Hz)]. The 13C NMR, DEPT 135° and HSQC spectra indicated a total of 20 carbon signals comprising three methyls, five methylenes, two methines, one carboxyl, one aldehyde, and eight quaternary carbons, six of which belong to an aromatic ring. These data, together with other spectroscopic characteristics, suggested that compound 1 is a tetracyclic compound related to the abietane diterpenes. The aliphatic spin systems were established on the basis of a COSY experiment and supported by HMBC. H2-1 (δH 1.95, 2.10) were coupled to H-2 (δH 3.96) as were H2-3 (δH 1.53, 1.67). The lack of coupling between H2-1 and H2-3 clearly established that the hydroxyl was located between these groups. NOESY
H-7), 3.96 (1H, m, H-2), 5.26 (1H, d, J = 15.2 Hz, H-17), 5.29 (1H, d, J = 15.2 Hz, H-17), 10.95 (1H, s, H-15). 13C-NMR spectral data (150 MHz, CD3OD) δC 19.4 (C-6), 25.8 (C-18), 27.4 (C-7), 28.8 (C-20), 32.5 (C-19), 33.7 (C-4), 42.1 (C-10), 46.1 (C-3), 48.2 (C-5), 50.2 (C-1), 67.2 (C-2), 69.6 (C-17), 125.1 (C-12), 130.3 (C-13), 131.3 (C-11), 131.9 (C-8), 152.0 (C-14), 154.0 (C-9), 172.4 (C-16), 198.8 (C-15). The 1H NMR spectrum revealed the presence of three methyl groups [δH 1.04 (3H, s, H-19), δH 1.64 (3H, s, H-20) and δH 1.07 (3H, s, H-18)], one aldehyde group [δH 10.95 (1H, s)], one oxygenated methine [δH 3.96 (1H, m)], and an oxygenated methylene which appeared as an AB quartet in which the outer member of each doublet were greatly 19
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correlations between H-2 (δH 3.96) and the methyl groups H3-20 (δH 1.64) and H3-18 (δH 1.07) confirms that the hydroxy group at C-2 is equatorial. That the remaining aliphatic protons form a single isolated spin system supports the angular arrangement of the ring system with the aromatic ring fused across C-8 and C-9 rather than a linear arrangement. In the HMBC spectrum, correlations are observed between protons of the angular methyl group H3-20 (δH 1.64) and C-1 (δC 50.2), C-5 (δC 48.2), C-9 (δC 154.0), and C-10 (δC 42.1). The aldehydic proton H-15 (δH 10.95) shows an HMBC correlation with its point of attachment, C-11 (δC 131.2) as well as C-9 (δC 154.0). H-1 (δH 1.95) also shows a correlation to C-9, further supporting this assignment. The AB quartet comprising H2-17 (δH 5.26, 5.29) displays strong HMBC correlations with C-13 (δC 130.2) and C-16 (δC 172.4), establishing the presence of a fused γ-lactone. Weaker 3-bond correlations between these protons and C-12 (δC 125.1) and C-14 (δC 152.0) but not C-11 (δC 131.2) support the orientation of the lactone shown in Fig. 1. Finally, HMBC correlations between H2-7 (δH 2.63, 3.06) and both C-9 (δC 154.0) and C-14 (δC 152.0) confirm the substitution pattern on the aromatic ring. These observations support the assignment of 1 as 2β,14dihydroxy-11-formyl-12-carboxy-13-des-isopropyl-13-hydroxymethylabieta-8,11,13-triene-16(17)-lactone. Compound 1 is analogous to (18) previously reported from Phlomis tuberosa (Yang et al., 2015), differing in the oxidation state of C-2 and C-18. The structures of the obtained known compounds were elucidated by 1D and 2D homonuclear and heteronuclear NMR spectroscopy and by comparison of their spectral data (1H NMR, 13C NMR, MS) with those reported in the literature. The 16 compounds were determined to be 12,18-dicarboxy-14-hydroxy-13-des-isopropyl −13-hydroxymethylabieta-8,11,13-triene-16(17)-lactone (2) (Yang et al., 2015), 5-hydroxy3′,4′,7-trimethoxyflavone (3) (Sipos and Kónya, 2018), 5-hydroxy-4’,7dimethoxyflavone (4) (Sipos and Kónya, 2018), luteolin-7-O-β-glucoside (5) (Refaat et al., 2015), verbascoside (6) (Lan et al., 2018), luteolin 7-O-(6″-O-β-D-apiofuranosyl)-β-D-glucopyranoside (7) (Bucar et al., 1998), chlorogenic acid (8) (Okonkwo et al., 2016), echinacoside (9) (Porter et al., 2015), apigenin-7-O-β-D-glucoside (10) (Marzouk et al., 2016), p-Coumaric acid (11) (Liao et al., 2014), vanillic acid (12) (Ahmed et al., 2014), apigenin-7-O-(6″-E-p-coumaroyl)-β-D-glucopyranoside (13) (Chang et al., 2017), apigenin-7-O-(3″,6″-E-p-dicoumaroyl)-β-glucoside (14) (Sweidan and Abu Zarga, 2016), lamalbide (15) (Zhang et al., 2009), 6β-hydroxy-7-epi-loganin (16) (Zhang et al., 2009), phloyoside II (17) (Kasai et al., 1994), (Fig. 1). It should be noted that, in our opinion, the name originally applied to 2 by Yang et al., 14-hydroxyabieta-8,11,13 -triene-17-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone is incorrect. We propose 12,18-dicarboxy-14-hydroxy −13-des-isopropyl-13-hydroxymethyl -abieta8,11,13-triene-16(17)-lactone as a more appropriate name for this compound.
the related betonicosides (e.g., betonicoside-A aglycone 19) and betolide (20) in which the lactone ring has formed by condensation of the carboxylic acid at C-12 of the aromatic ring with a hemiacetal at C-11, rather than with the hydroxymethyl group at C-13 (Miyase et al., 1996; Bankova et al., 1999; Tkachev et al., 1987). Since the original description of betolide in 1988, neither of these two, closely related, tetracyclic skeleta have been found anywhere else in the plant kingdom. Their narrow, known distribution, to date, suggests that they may become useful as chemotaxonomic markers for this subfamily, if not for the Lamiaceae as a whole, once a broader systematic search is undertaken. Declaration of interest None. Acknowledgements This study was supported, in part, by the National Basic Research Program of China (2015CB856500). The authors also gratefully acknowledge their respective institutions for financially supporting this research. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bse.2019.03.002. References Ahmed, A.F., Saad, H.E., Abd El-Karim, E.M., 2014. Molecules 19, 5940–5951. Asnaashari, S., Afshar, F.H., Ebrahimi, A., Moghadam, S.B., Delazar, A., 2015. Bioimpacts 5, 135–140. Bankova, V., Koeva-Todorovska, J., Stambolijska, T., Ignatova-Groceva, M.-D., Todorova, D., Popov, S., 1999. Z. Naturforsch. 54c, 876–880. Bucar, F., Ninov, S., Ionkova, I., Kartnig, T., Schubert-Zsilavecz, M., Asenov, I., Konuklugil, B., 1998. Phytochemistry 48, 573–575. Çalış, İ., Güvenç, A., Armağan, M., Koyuncu, M., Gotfredsen, C.H., Jensen, S.R., 2007. Helv. Chim. Acta 90, 1461–1466. Chang, Y.-X., Zhang, P., Zhang, X., Chen, J.-P., Rausch, W.D., Gula, A., Bao, B.Q., 2017. Nat. Prod. Res. 31, 1223–1227. Gusakova, S.D., Umarov, A.U., 1975a. Khim. Prir. Soedin. 11 (3), 324–328. Gusakova, S.D., Umarov, A.U., 1975b. Khim. Prir. Soedin. 11 (4), 511–512. Gusakova, S.D., Umarov, A.U., 1976. Khim. Prir. Soedin. 12 (6), 717–723. Kasai, R., Katagiri, M., Ohtani, K., Yamasaki, K., Yang, C.R., Tanaka, O., 1994. Phytochemistry 36, 967–970. Lan, Y.-H., Chi, X.-F., Zhou, G.-Y., Zhao, X.-H., 2018. Record Nat. Prod. 12 (4), 332–339. Liao, C.-R., Kuo, Y.-H., Ho, Y.-L., Wang, C.-Y., Yang, C.-S., Lin, C.-W., Chang, Y.-S., 2014. Molecules 19, 9515–9534. Marzouk, M.M., Hussein, S.R., Kassem, M.E., Kawashty, S.A., EI-Negoumy, S.I., 2016. Nat. Prod. Res. 30, 1537–1541. Miyase, T., Yamamoto, R., Ueno, A.S., 1996. Chem. Pharm. Bull. 44 (8), 1610–1613. Nori-Shargh, D., Kiaei, S.M., Deyhimi, F., 2007. Nat. Prod. Res. 21 (8), 733–735. Okonkwo, T.J., Osadebe, P.O., Proksch, P., 2016. Phytother Res. 30, 78–83. Panekina, T.V., Gusakova, S.D., Tabak, M. Ya, Umarov, A.U., 1978. Khim. Prir. Soedin. 14 (1), 44–48. Porter, E.A., Kite, G.C., Veitch, N.C., Geoghegan, I.A., Larsson, S., Simmonds, M.S.J., 2015. Phytochemistry 117, 185–193. Rabe, S.Z.T., Mahmoudi, M., Ahmadsimab, H., Rabe, S.S.Z.T., Emami, A., 2014. Food Agric. Immunol. 25 (4), 578–585. Refaat, J., Samy, M.N., Desoukey, S.Y., Ramadan, M.A., Sugimoto, S., Matsunami, K., Kamel, M.S., 2015. Med. Chem. Res. 24, 2939–2949. Rustaiyan, A., Masoudi, S., Ezzatzadeh, E., Akhlaghi, H., Aboli, J., 2011. J. Essential OilBearing J. Essent. Oil Bearing Plants 14, 84–88. Sipos, Z., Kónya, K., 2018. Synthesis 50, 1610–1620. Sweidan, N.I., Abu Zarga, M.H., 2016. Lett. Org. Chem. 13, 277–282. Tkachev, V.V., Nikonov, G.K., Atovmyan, L.O., Kobzar, A.Ya, Zinchenko, T.V., 1987. Khim. Prir. Soedin. 23 (6), 811–817. Yang, Y.B., Gu, L.H., Xiao, Y., Liu, Q., Hu, H.J., Wang, Z.T., Chen, K.X., 2015. PLoS One 10 (2). https://doi.org/10.1371/journal.pone.0116922. e0116922. Zhang, H.-J., Rothwangl, K., Mesecar, A.D., Sabahi, A., Rong, L.-J., Fong, H.H., 2009. J. Nat. Prod. 72, 2158–2162.
4. Chemotaxonomic significance The present study reports the identification of a new diterpene (1), along with one known diterpene (2), seven flavonoids (3, 4, 5, 7, 10, 13, 14), three phenolic acid (8, 11, 12), two phenylethanoids (6, 9) and three iridoid glucosides (15–17) from the aerial parts of E. moluccelloides Bunge (Fig. 1). Nine compounds (1–4, 8, 9, 11, 12, 14) have not been previously isolated from genus Eremostachys. And fifteen compounds (1–14, 17) have not been previously reported from this species. While most of these compounds are well known and widely distributed, the two diterpenes (1 and 2) are noteworthy as this is only the second report of compounds having this tetracyclic skeleton. Compounds 2 and 18 were isolated previously only from Phlomis tuberosa, which is a member of the subfamily Lamioideae. Similarly, the genera Stachys L. and Betonica L. (also in the Lamioideae) have afforded
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