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The isolation and structural elucidation of three new biflavonoid glycosides from Lomatogonium carinthiacum a
a
a
Bagenna Bao , Qinghu Wang , Xiaolan Wu & Wenquan Tai
a
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College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, PR China Published online: 27 Mar 2015.
Click for updates To cite this article: Bagenna Bao, Qinghu Wang, Xiaolan Wu & Wenquan Tai (2015) The isolation and structural elucidation of three new biflavonoid glycosides from Lomatogonium carinthiacum, Natural Product Research: Formerly Natural Product Letters, 29:14, 1358-1362, DOI: 10.1080/14786419.2015.1025399 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1025399
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Natural Product Research, 2015 Vol. 29, No. 14, 1358–1362, http://dx.doi.org/10.1080/14786419.2015.1025399
The isolation and structural elucidation of three new biflavonoid glycosides from Lomatogonium carinthiacum Bagenna Bao, Qinghu Wang1*, Xiaolan Wu and Wenquan Tai College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, PR China
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(Received 29 December 2014; final version received 25 February 2015)
Three biflavonoid glycosides, named carinoside B, C and D (1, 2 and 3, respectively), were isolated from the n-BuOH crude extract of the aerial part of Lomatogonium carinthiacum (Wulfen) Rchb (L. carinthiacum). The structures of 1, 2 and 3 were elucidated by spectroscopic methods, including UV, IR, HR-ESI-MS and extensive 1D and 2D NMR techniques. Keywords: Lomatogonium carinthiacum (Wulfen) Rchb; carinoside B; carinoside C; carinoside D; NMR; HR-ESI-MS
1. Introduction Lomatogonium carinthiacum is a member of the family Gentianaceae, genus Lomatogonium and distributed throughout Inner Mongolia, Shanxi, Xinjiang of China. It is used as a remedy for plague, influenza, typhoid, liver disease, jaundice in Mongolian medicine (Jia et al. 2010). Terpenoids and flavonoids (Massia et al. 1981; Jia and Yuan 2011) have been reported from L. carinthiacum. In our previous studies, flavonoids (Bao et al. 2014), two new monoterpene glucoside derivatives (Wang, Dai, et al. 2014), two new xanthones (Wang, Bao, et al. 2014), and a biflavonoid glycoside (Wang et al. 2015) were isolated from the CHCl3, EtOAc and n-BuOH crude extract of this plant. In the continuation of our work on the constituents of the n-BuOH crude extract from L. carinthiacum, three biflavonoid glycosides were isolated.
2. Results and discussion The ethanol extract of L. carinthiacum was suspended in water, and then partitioned with petroleum ether (PE), CHCl3, EtOAc, and n-BuOH. The n-BuOH-soluble fraction was separated
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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by chromatography and afforded three biflavonoid glycosides, named carinoside B, C and D (Figure 1). Carinoside B (1) was obtained as a yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was flavonoid glycoside. The molecular formula was determined to be C54H58O27 by HR-ESI-MS at m/z 1139.3243 [M þ H]þ. In the 1H NMR spectrum (Table S1), two characteristic resonance for H-3 of two flavones at dH 6.84 (1H, s, dC 103.7 by HSQC) and 6.86 (1H, s, dC 103.6 by HSQC), which confirmed by long-range correlations of dH 6.84 with dC 182.7 (C-4), 164.4 (C-2), 105.1 (C-10) and 121.4 (C-10 ), and 6.86 with dC 182.4 (C-4), 164.2 (C-2), 104.5 (C-10) and 121.4 (C-10 ) in the HMBC spectrum (Figure S1). Likewise two second singlet resonance in the aromatic region at dH 6.83 (1H, s, dC 91.6 by HSQC) and 6.82 (1H, s, dC 91.7 by HSQC) were assigned to H-8 (I) and H-8 (II) on the basis of long-range correlations of dH 6.83 with dC 110.1 (C-6) and 105.1 (C-10), and dH 6.82 with dC 110.0 (C-6) and 104.5 (C-10). The remaining aromatic signals at dH 7.97 (4H, d, J ¼ 7.5 Hz) and 6.94 (4H, d, J ¼ 7.5 Hz) indicated the presence of two A2B2 system. Correlations of OH-5 (dH 13.50) to C-5 (dC 160.7), C-6 (dC 110.1) and C-10 (dC 105.1), and OH5 (dH 13.49) to C-5 (dC 159.9), C-6 (dC 110.0) and C-10 (dC 104.5) were observed by HMBC experiment, and they helped us to determine the substituents on the pentasubstituted A rings. According to the above analysis, there are two 1,4-disubstituted B and B0 rings and two pentasubstituted A and A0 rings. The A and B rings were called as the partial structure I and the A0 and B0 rings as the partial structure II. It is similar to those of carinoside A (Wang et al. 2015), except for the sugar moieties. The 13C NMR spectrum of 1 (Table S1) also showed the presence
Figure 1. Structures of compounds 1 – 3.
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of 20 carbon signals except for the aglycone carbons. The presence of two L -arabinopyranoses (d (C) 103.5, 70.7, 70.8, 67.7 and 65.2, and 103.5, 70.7, 70.8, 67.6 and 65.1) was confirmed via sugar analysis (Tanaka et al. 2007). The anomeric protons appearing at d (H) 4.10 (2H, d, J ¼ 4.0 Hz) and their corresponding carbons resonating at d (C) 103.5 (C-1000 , I and II) from the HSQC experiment also suggested the presence of two a-L -arabinopyranoses. The remaining signals at d (C) 70.6, 79.3, 71.3, 80.3 and 69.6, and 70.0, 79.2, 71.2, 80.2, 69.3 belong to two L arabinofuranoses, which was confirmed by comparing their spectral data with those reported in the literature (Gorin & Mazurek 1975). In addition, the HMBC correlations from H-11 (I) to C100 (d (C) 70.6, I) and C-200 (d (C) 79.3, I), and H-11 (II) to C-100 (d (C) 70.0, II) and C-200 (d (C) 79.2, II) indicated that the two L -arabinofuranoses were attached to C-11(I and II). The HMBC correlations (Figure S1) of H-1000 (I and II) to C-600 (I and II) confirmed that the two a-L arabinopyranose were linked at C-600 (I and II), respectively. The anomeric configuration in the L -arabinofuranoses was determined as b according to the coupling constant 10.0 Hz. Compound 1 was thus identified as 3-methoxy-apigenin-C(I)-6-C(I)-11-O-C-11(II)-C-6(II)-3-methoxyapigenin-11(I),11(II)-b-L -arabinofuranosyl-(1000 →600 )-O-a-L -arabinopyranoside, and named as carinoside B. Carinoside C (2) was obtained as a yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was flavonoid glycoside. The molecular formula was determined to be C56H62O29 by HR-ESI-MS at m/z 1199.3454 [M þ H]þ. 1H and 13C NMR spectra (Table S2) of compound 2 were similar to those of compound 1, except for the sugar moieties. Namely, the two L -arabinopyranoses in compound 1 were substituted by the two D glucose d (C) 103.5, 73.8, 77.2, 70.4, 77.3 and 61.4, and 103.5, 73.7, 77.2, 70.4, 77.3, 61.4 and this was confirmed via sugar analysis (Tanaka et al. 2007). The anomeric protons appearing at d (H) 4.13 (2H, d, J ¼ 6.0 Hz), and their corresponding carbons resonating at d (C) 103.5 (C-1000 , I, II) from the HSQC experiment also suggested the presence of two b-D -glucose. The HMBC correlations (Figure S1) of H-1000 (I and II) to C-600 (I and II) confirmed that the two b-D -glucose were linked at C-600 (I and II), respectively. Compound 2 was thus identified as 3-methoxyapigenin-C-6(I)-C-11(I)-O-C-11(II)-C-6(II)-3-methoxy-apigenin-11(I),11(II)-b-L -arabinofuranosyl-(1000 →600 )-O-b-D -glucopyranoside, and named as carinoside C. Carinoside D (3) was obtained as a yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was flavonoid glycoside. The molecular formula was determined to be C44H42O21 by HR-ESI-MS at m/z 907.2295 [M þ H]þ. 1H and 13C NMR spectra (Table S3) of compound 3 were similar to those of carinoside A (Wang et al. 2015), except for B ring of the flavone aglycone. Namely, the A2B2 system in carinoside A was substituted by the ABX system in compound 3 and this was confirmed by the carbon signals at dc 121.7, 114.0, 150.4, 146.3, 116.5 and 119.5, and 121.8, 113.8, 150.2, 146.2, 116.5 and 119.5. Compound 3 was thus 7-methoxy-lutelin-C-6(I)-C-11(I)-O-C-11(II)-C-6(II)-7-methoxy-lutelin11(I),11(II)-b-L -arabinofuranoside, and named as carinoside D. 3. Experimental 3.1. General experimental procedures The UV spectra were recorded on a Shimadzu UV-2201 spectrometer (Shimadzu, Kyoto, Japan). The IR spectra were recorded in KBr discs on a Thermo Nicolet 200 double beam spectrophotometer (Shimadzu). The HR-ESI-MS spectra were measured on Bruker Daltonics MicroTOFQ (Bruker Daltonics Inc., Karlsruhe, Germany). NMR spectra were measured on a Bruker Avance III 500 NMR spectrometer (Bruker, Germany) with tetramethylsilane (TMS) as the internal reference, and chemical shifts are expressed in d (ppm). Semi-preparative HPLC was performed by using a Japanese liquid chromatograph equipped with an EZ0566 column. Column chromatography was performed by using silica gel (200 – 300 mesh, Marine Chemical Factory,
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Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Fractions were monitored by TLC (silica gel GF25410– 40 mm, Marine Chemical Factory, Qingdao, China), and spots were visualised by heating silica gel plates sprayed with 10% H2SO4 in EtOH.
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3.2. Plant material The aerial part of L. carinthiacum, used as experimental material, were collected in Xilinhaote, Inner Mongolia of China, in August 2012, and identified by Professor Buhebateer (Inner Mongolia University for Nationalities). A voucher (No. 20120810) has been deposited in the School of Traditional Mongolian Medicine of Inner Mongolia University for Nationalities. 3.3. Extraction and isolation The air-dried aerial part of L. carinthiacum (5.0 Kg) were powdered and extracted twice under reflux 95% EtOH (50 L). Evaporation of the solvent under reduced pressure delivered the 95% EtOH extract. The extract was partitioned with PE, CHCl3, EtOAc and n-BuOH. The n-BuOHsoluble fraction (30.0 g) was separated by column chromatography on silica gel and gradiently eluted with CHCl3 –CH3OH (60:1 to 5:1) to give 6 fractions (Fractions 1 – 6). Fraction 3 [340 mg, CHCl3 – MeOH (5:1) elute] was further chromatographed on Sephadex LH-20 column eluting with MeOH, and then separated by semi-preparative HPLC [The separation was performed on EZ0566 column (250 mm £ 4.6 mm, 5 mm) with CH3OH:H2O (13:87) as the mobile phase and a detection wavelength at 205 nm. The retention time of carinoside B, carinoside C and carinoside D were 17.12, 18.65, 21.35 min, respectively] yielding 1 (21 mg), 2 (30 mg), 3 (36 mg). Carinoside B (1): Yellow powder. m.p.195.2 –195.48C. ½a25 D ¼ 2 11.9 (c ¼ 0.1, MeOH). UV (MeOH): 260 (4.56), 332 (4.02). IR (KBr): 3492, 1654, 1605, 1489, 1384, 1249, 1205, 1180. 1 H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectral data see Table S1. HR-ESI-MS: m/z 1139.3243 [M þ H]þ (calculated for C54H59O27, 1139.3238). Carinoside C (2): Yellow powder. m.p.197.1– 197.48C. ½a25 D ¼ 2 12.3 (c ¼ 0.1, MeOH). UV (MeOH): 261 (4.55), 333 (3.94). IR (KBr): 3493, 1656, 1603, 1487, 1386, 1242, 1203, 1181. 1 H-NMR(500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectral data see Table S2. HR-ESI-MS: m/z 1199.3454 [M þ H]þ (calculated for C56H63O29, 1199.3450). Carinoside D (3). Yellow powder; m.p.192.1 – 193.68C; ½a25 D 2 13.3 (c ¼ 0.1, MeOH), UV (MeOH) lmax (log 1): 262 (4.64) and 364 (3.96) nm; IR (KBr) nmax (cm21): 3395, 1643, 1605, 1489, 1386, 1248, 1203, 1179 cm21; 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectral data see Table S3; HR-ESI-MS: m/z 907.2295 [M þ H]þ (calculated for 907.2291). 4. Conclusion Three new biflavonoid glycosides were isolated from the n-BuOH crude extract of the whole plant of L. carinthiacum. Supplementary material Supplementary material relating to this paper is available online. Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was supported by the National Natural Science Foundation of China [grant number 81360673].
Note 1. The authors contributed equally to this work.
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References Ba GN, Chen YL, Ba DRH, Tong YF, Ye RG, Wang QH. 2014. Two new xanthones from Lomatogonium carinthiacum. Chin J Nat Med. 12:693– 696. Gorin PAJ, Mazurek M. 1975. Further studies on the assignment of signals in 13C magnetic resonance spectra of aldoses and derived methyl glycosides. Can J Chem. 53:1212– 1219. Jia LY, Li Q, Yuan JZ. 2010. Isolation and identification of chemical constituents of whole plant of Lamatogonium rotaum (L.) Fries ex Nvm. J Shen Pharm Univ. 27:704–706. Jia LY, Yuan JZ, Sun QS. 2011. Isolation and identification of chemical constituents of whole plant of Lomatogonium rotatum (L.) Fries ex Nym (2). J Shen Pharm Univ. 28:260– 262. Massia MS, Carbonnier J, Molho D. 1981. Xanthones and C-glucosyflavones from Gebtiana corymbifera. Phytochemistry. 20:1577–1578. Tanaka T, Nakashima T, Ueda T, Tom K, Kouno I. 2007. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem Pharm Bull. 55:899–902. Wang QH, Bao BGN, Chen YL. 2014. Structure elucidation and NMR assignments of two unusual xanthones from Lomatogonium carinthiacum (Wulf) Reichb. Magn Reson Chem. 52:37–39. Wang QH, Dai NYT, Han NRCKT. 2014. The structural elucidation and antimicrobial activities of two new monoterpene glucoside derivatives from Lomatogonium carinthiacum (Wulf) Reichb. Magn Reson Chem. 52:511–514. Wang QH, Han NRCKT, Wu XL, Tai WQ, Dai NYT, Wu RJ, Wu JS, Bao BGN. 2015. A biflavonoid glycoside from Lomatogonium carinthiacum (Wulf) Reichb. Nat Prod Res. 29:77–81.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
The isolation and structural elucidation of three new biflavonoid glycosides from Lomatogonium carinthiacum a
a
a
Bagenna Bao , Qinghu Wang , Xiaolan Wu & Wenquan Tai
a
a
College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, PR China Published online: 27 Mar 2015.
Click for updates To cite this article: Bagenna Bao, Qinghu Wang, Xiaolan Wu & Wenquan Tai (2015) The isolation and structural elucidation of three new biflavonoid glycosides from Lomatogonium carinthiacum, Natural Product Research: Formerly Natural Product Letters, 29:14, 1358-1362, DOI: 10.1080/14786419.2015.1025399 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1025399
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2015 Vol. 29, No. 14, 1358–1362, http://dx.doi.org/10.1080/14786419.2015.1025399
The isolation and structural elucidation of three new biflavonoid glycosides from Lomatogonium carinthiacum Bagenna Bao, Qinghu Wang1*, Xiaolan Wu and Wenquan Tai College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, PR China
Downloaded by [New York University] at 08:10 18 June 2015
(Received 29 December 2014; final version received 25 February 2015)
Three biflavonoid glycosides, named carinoside B, C and D (1, 2 and 3, respectively), were isolated from the n-BuOH crude extract of the aerial part of Lomatogonium carinthiacum (Wulfen) Rchb (L. carinthiacum). The structures of 1, 2 and 3 were elucidated by spectroscopic methods, including UV, IR, HR-ESI-MS and extensive 1D and 2D NMR techniques. Keywords: Lomatogonium carinthiacum (Wulfen) Rchb; carinoside B; carinoside C; carinoside D; NMR; HR-ESI-MS
1. Introduction Lomatogonium carinthiacum is a member of the family Gentianaceae, genus Lomatogonium and distributed throughout Inner Mongolia, Shanxi, Xinjiang of China. It is used as a remedy for plague, influenza, typhoid, liver disease, jaundice in Mongolian medicine (Jia et al. 2010). Terpenoids and flavonoids (Massia et al. 1981; Jia and Yuan 2011) have been reported from L. carinthiacum. In our previous studies, flavonoids (Bao et al. 2014), two new monoterpene glucoside derivatives (Wang, Dai, et al. 2014), two new xanthones (Wang, Bao, et al. 2014), and a biflavonoid glycoside (Wang et al. 2015) were isolated from the CHCl3, EtOAc and n-BuOH crude extract of this plant. In the continuation of our work on the constituents of the n-BuOH crude extract from L. carinthiacum, three biflavonoid glycosides were isolated.
2. Results and discussion The ethanol extract of L. carinthiacum was suspended in water, and then partitioned with petroleum ether (PE), CHCl3, EtOAc, and n-BuOH. The n-BuOH-soluble fraction was separated
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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by chromatography and afforded three biflavonoid glycosides, named carinoside B, C and D (Figure 1). Carinoside B (1) was obtained as a yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was flavonoid glycoside. The molecular formula was determined to be C54H58O27 by HR-ESI-MS at m/z 1139.3243 [M þ H]þ. In the 1H NMR spectrum (Table S1), two characteristic resonance for H-3 of two flavones at dH 6.84 (1H, s, dC 103.7 by HSQC) and 6.86 (1H, s, dC 103.6 by HSQC), which confirmed by long-range correlations of dH 6.84 with dC 182.7 (C-4), 164.4 (C-2), 105.1 (C-10) and 121.4 (C-10 ), and 6.86 with dC 182.4 (C-4), 164.2 (C-2), 104.5 (C-10) and 121.4 (C-10 ) in the HMBC spectrum (Figure S1). Likewise two second singlet resonance in the aromatic region at dH 6.83 (1H, s, dC 91.6 by HSQC) and 6.82 (1H, s, dC 91.7 by HSQC) were assigned to H-8 (I) and H-8 (II) on the basis of long-range correlations of dH 6.83 with dC 110.1 (C-6) and 105.1 (C-10), and dH 6.82 with dC 110.0 (C-6) and 104.5 (C-10). The remaining aromatic signals at dH 7.97 (4H, d, J ¼ 7.5 Hz) and 6.94 (4H, d, J ¼ 7.5 Hz) indicated the presence of two A2B2 system. Correlations of OH-5 (dH 13.50) to C-5 (dC 160.7), C-6 (dC 110.1) and C-10 (dC 105.1), and OH5 (dH 13.49) to C-5 (dC 159.9), C-6 (dC 110.0) and C-10 (dC 104.5) were observed by HMBC experiment, and they helped us to determine the substituents on the pentasubstituted A rings. According to the above analysis, there are two 1,4-disubstituted B and B0 rings and two pentasubstituted A and A0 rings. The A and B rings were called as the partial structure I and the A0 and B0 rings as the partial structure II. It is similar to those of carinoside A (Wang et al. 2015), except for the sugar moieties. The 13C NMR spectrum of 1 (Table S1) also showed the presence
Figure 1. Structures of compounds 1 – 3.
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of 20 carbon signals except for the aglycone carbons. The presence of two L -arabinopyranoses (d (C) 103.5, 70.7, 70.8, 67.7 and 65.2, and 103.5, 70.7, 70.8, 67.6 and 65.1) was confirmed via sugar analysis (Tanaka et al. 2007). The anomeric protons appearing at d (H) 4.10 (2H, d, J ¼ 4.0 Hz) and their corresponding carbons resonating at d (C) 103.5 (C-1000 , I and II) from the HSQC experiment also suggested the presence of two a-L -arabinopyranoses. The remaining signals at d (C) 70.6, 79.3, 71.3, 80.3 and 69.6, and 70.0, 79.2, 71.2, 80.2, 69.3 belong to two L arabinofuranoses, which was confirmed by comparing their spectral data with those reported in the literature (Gorin & Mazurek 1975). In addition, the HMBC correlations from H-11 (I) to C100 (d (C) 70.6, I) and C-200 (d (C) 79.3, I), and H-11 (II) to C-100 (d (C) 70.0, II) and C-200 (d (C) 79.2, II) indicated that the two L -arabinofuranoses were attached to C-11(I and II). The HMBC correlations (Figure S1) of H-1000 (I and II) to C-600 (I and II) confirmed that the two a-L arabinopyranose were linked at C-600 (I and II), respectively. The anomeric configuration in the L -arabinofuranoses was determined as b according to the coupling constant 10.0 Hz. Compound 1 was thus identified as 3-methoxy-apigenin-C(I)-6-C(I)-11-O-C-11(II)-C-6(II)-3-methoxyapigenin-11(I),11(II)-b-L -arabinofuranosyl-(1000 →600 )-O-a-L -arabinopyranoside, and named as carinoside B. Carinoside C (2) was obtained as a yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was flavonoid glycoside. The molecular formula was determined to be C56H62O29 by HR-ESI-MS at m/z 1199.3454 [M þ H]þ. 1H and 13C NMR spectra (Table S2) of compound 2 were similar to those of compound 1, except for the sugar moieties. Namely, the two L -arabinopyranoses in compound 1 were substituted by the two D glucose d (C) 103.5, 73.8, 77.2, 70.4, 77.3 and 61.4, and 103.5, 73.7, 77.2, 70.4, 77.3, 61.4 and this was confirmed via sugar analysis (Tanaka et al. 2007). The anomeric protons appearing at d (H) 4.13 (2H, d, J ¼ 6.0 Hz), and their corresponding carbons resonating at d (C) 103.5 (C-1000 , I, II) from the HSQC experiment also suggested the presence of two b-D -glucose. The HMBC correlations (Figure S1) of H-1000 (I and II) to C-600 (I and II) confirmed that the two b-D -glucose were linked at C-600 (I and II), respectively. Compound 2 was thus identified as 3-methoxyapigenin-C-6(I)-C-11(I)-O-C-11(II)-C-6(II)-3-methoxy-apigenin-11(I),11(II)-b-L -arabinofuranosyl-(1000 →600 )-O-b-D -glucopyranoside, and named as carinoside C. Carinoside D (3) was obtained as a yellow powder. The positive reactions to the Molish and HCl-Mg tests suggested that the compound was flavonoid glycoside. The molecular formula was determined to be C44H42O21 by HR-ESI-MS at m/z 907.2295 [M þ H]þ. 1H and 13C NMR spectra (Table S3) of compound 3 were similar to those of carinoside A (Wang et al. 2015), except for B ring of the flavone aglycone. Namely, the A2B2 system in carinoside A was substituted by the ABX system in compound 3 and this was confirmed by the carbon signals at dc 121.7, 114.0, 150.4, 146.3, 116.5 and 119.5, and 121.8, 113.8, 150.2, 146.2, 116.5 and 119.5. Compound 3 was thus 7-methoxy-lutelin-C-6(I)-C-11(I)-O-C-11(II)-C-6(II)-7-methoxy-lutelin11(I),11(II)-b-L -arabinofuranoside, and named as carinoside D. 3. Experimental 3.1. General experimental procedures The UV spectra were recorded on a Shimadzu UV-2201 spectrometer (Shimadzu, Kyoto, Japan). The IR spectra were recorded in KBr discs on a Thermo Nicolet 200 double beam spectrophotometer (Shimadzu). The HR-ESI-MS spectra were measured on Bruker Daltonics MicroTOFQ (Bruker Daltonics Inc., Karlsruhe, Germany). NMR spectra were measured on a Bruker Avance III 500 NMR spectrometer (Bruker, Germany) with tetramethylsilane (TMS) as the internal reference, and chemical shifts are expressed in d (ppm). Semi-preparative HPLC was performed by using a Japanese liquid chromatograph equipped with an EZ0566 column. Column chromatography was performed by using silica gel (200 – 300 mesh, Marine Chemical Factory,
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Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Fractions were monitored by TLC (silica gel GF25410– 40 mm, Marine Chemical Factory, Qingdao, China), and spots were visualised by heating silica gel plates sprayed with 10% H2SO4 in EtOH.
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3.2. Plant material The aerial part of L. carinthiacum, used as experimental material, were collected in Xilinhaote, Inner Mongolia of China, in August 2012, and identified by Professor Buhebateer (Inner Mongolia University for Nationalities). A voucher (No. 20120810) has been deposited in the School of Traditional Mongolian Medicine of Inner Mongolia University for Nationalities. 3.3. Extraction and isolation The air-dried aerial part of L. carinthiacum (5.0 Kg) were powdered and extracted twice under reflux 95% EtOH (50 L). Evaporation of the solvent under reduced pressure delivered the 95% EtOH extract. The extract was partitioned with PE, CHCl3, EtOAc and n-BuOH. The n-BuOHsoluble fraction (30.0 g) was separated by column chromatography on silica gel and gradiently eluted with CHCl3 –CH3OH (60:1 to 5:1) to give 6 fractions (Fractions 1 – 6). Fraction 3 [340 mg, CHCl3 – MeOH (5:1) elute] was further chromatographed on Sephadex LH-20 column eluting with MeOH, and then separated by semi-preparative HPLC [The separation was performed on EZ0566 column (250 mm £ 4.6 mm, 5 mm) with CH3OH:H2O (13:87) as the mobile phase and a detection wavelength at 205 nm. The retention time of carinoside B, carinoside C and carinoside D were 17.12, 18.65, 21.35 min, respectively] yielding 1 (21 mg), 2 (30 mg), 3 (36 mg). Carinoside B (1): Yellow powder. m.p.195.2 –195.48C. ½a25 D ¼ 2 11.9 (c ¼ 0.1, MeOH). UV (MeOH): 260 (4.56), 332 (4.02). IR (KBr): 3492, 1654, 1605, 1489, 1384, 1249, 1205, 1180. 1 H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectral data see Table S1. HR-ESI-MS: m/z 1139.3243 [M þ H]þ (calculated for C54H59O27, 1139.3238). Carinoside C (2): Yellow powder. m.p.197.1– 197.48C. ½a25 D ¼ 2 12.3 (c ¼ 0.1, MeOH). UV (MeOH): 261 (4.55), 333 (3.94). IR (KBr): 3493, 1656, 1603, 1487, 1386, 1242, 1203, 1181. 1 H-NMR(500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectral data see Table S2. HR-ESI-MS: m/z 1199.3454 [M þ H]þ (calculated for C56H63O29, 1199.3450). Carinoside D (3). Yellow powder; m.p.192.1 – 193.68C; ½a25 D 2 13.3 (c ¼ 0.1, MeOH), UV (MeOH) lmax (log 1): 262 (4.64) and 364 (3.96) nm; IR (KBr) nmax (cm21): 3395, 1643, 1605, 1489, 1386, 1248, 1203, 1179 cm21; 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectral data see Table S3; HR-ESI-MS: m/z 907.2295 [M þ H]þ (calculated for 907.2291). 4. Conclusion Three new biflavonoid glycosides were isolated from the n-BuOH crude extract of the whole plant of L. carinthiacum. Supplementary material Supplementary material relating to this paper is available online. Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was supported by the National Natural Science Foundation of China [grant number 81360673].
Note 1. The authors contributed equally to this work.
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