- Email: [email protected]
Chemical constituents from Tithonia diversifolia and their chemotaxonomic significance
Biochemical Systematics and Ecology 44 (2012) 250–254
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemical constituents from Tithonia diversifolia and their chemotaxonomic significance Gui-Jun Zhao a,1, Zhong-Xin Xi a,1, Wan-Sheng Chen b, Xia Li b, Lei Sun c, Lian-Na Sun a, * a
School of Pharmacy, Second Military Medical University, Guohe Road 325#, Shanghai 200433, People’s Republic of China Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Fengyang Road 415#, Shanghai 200003, People’s Republic of China c Department of Pharmacy, Fujian University of Traditional Chinese Medicine, Hutuo Road 1#, Fuzhou 350108, People’s Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 February 2012 Accepted 3 June 2012 Available online 28 June 2012
Two new compounds 600 -O-b-D-apiofuranosyl-trichocarpin (15) and 1-heptade-4,6-diyne3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16), together with fourteen known compounds were isolated from Tithonia diversifolia. The chemotaxonomic significance of these compounds was discussed in the article. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Tithonia diversifolia Lignins Polyacetylene Alkaloids Phenolics
1. Subject and source Tithonia diversifolia (Hemsl.) A. Gray (Compositae: Heliantheae), known as Mexican sunflower, is native to Mexico and Central America (Chagas-Paula et al., 2011). Presently, T. diversifolia has been introduced into other countries for ornamental purposes or for their pharmacological action (Zhai et al., 2010). And T. diversifolia is widespread in southern parts of China. The aerial parts of T. diversifolia were collected from Mengzi, Yunnan province of China, in September 2008, and were authenticated by Prof. Wansheng Chen (Department of Pharmacy, Changzheng Hospital, Second Military Medical University). A voucher specimen (NO.TD20080819) was deposited in Department of Pharmacognosy, Second Military Medical University, Shanghai, P. R. China. 2. Previous work Many classes of secondary metabolites have been isolated from the Tithonia species, including sesquiterpenoids and diterpenoids, as well as flavonoids. Further minor classes such as phytosterols, xanthanes, coumarins, ceramides, chromones, and chromenes have been also found (Chagas-Paula et al., 2012). The most studied species of the genus Tithonia is T. diversifolia, from which more than 150 compounds have been isolated. Previous phytochemical investigations on T. diversifolia have reported the presence of sesquiterpenes, including germacrane type sesquiterpenoids, eudesmane type sesquiterpenoids, guaiane type sesquiterpenoids, cadinane type sesquiterpenoids and xanthane type sesquiterpenoids (Baruah et al., 1979, Rüngeler et al., 1998, Kuroda et al., 2007; Kuo and Chen, 1998; Gu et al., 2002; Bordoloi et al., 1996; Kuo and Lin, 1999).
* Corresponding author. Tel./fax: þ86 21 81871308. E-mail address: [email protected] (L.-N. Sun). 1 These authors contributed equally to this work. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.06.019
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
251
Moreover, chromenes (Kuo and Lin, 1999), diterpenoids, quininic acids and three flavonoids (Kuroda et al., 2007) have also been isolated. 3. Present study The air-dried aerial parts of T. diversifolia (22 kg) were powdered and extracted with 80% aqueous ethanol (Zhao et al., 2010). After removing the solvent under reduced pressure using a rotary evaporator at 60 C, the resulting gum was dried to yield 2596 g of crude extract. A portion of the crude extract (2500 g) was suspended in distilled water and successively partitioned with petroleum ether (yield: 150 g), EtOAc (yield: 130 g), n-BuOH (yield: 160 g). The EtOAc extract (120 g) was chromatographed on silica gel column with the gradient petroleum ether/EtOAc (20:1, 10:1, 5:1, 3:1, 2:1, 1:1) as eluent. The eluate of petroleum ether/EtOAc (5:1) was purified by a Sephadex LH-20 to afforded compounds 1 (46 mg) and 2 (78 mg). The n-BuOH extract (160 g) was applied on silica column eluted with gradient CH2Cl2/MeOH system (50:1, 20:1, 10:1, 5:1, 2:1, 1:1). The obtained fractions were combined on the basis of silica gel TLC and six fractions (Fr1–Fr6) were obtained. The Fr1 was subjected to silica gel column chromatography and eluted with CH2Cl2/MeOH (20:1, 15:1, 10:1) to afford compounds 3 (18 mg) and 4 (7 mg). Fr2 was retreated on silica gel column and eluated with CH2Cl2/MeOH (10:1) to yield compound 5 (11 mg). Fr3 was treated on silica gel column once more and eluted with CH2Cl2/MeOH (8:1) to yield compounds 6 (25 mg), 7 (100 mg) and 8 (10 mg). Fr4 was separated by reversed-phase silica gel (RP-18) with MeOH/H2O (1:9, 1:4, 2:3, 1:1, 1:0) and finally reapplied to a Sephadex LH-20 column using MeOH/H2O (1:1) to yield compounds 9 (32 mg), 10 (21 mg), 11 (12 mg), 12 (13 mg), 13 (28 mg) and 14 (80 mg). Compound 15 (18 mg) was isolated from Fr5 by RP-18 with MeOH/H2O (1:4), while compound 16 (7 mg) was purified from Fr6 by Sephadex LH-20 gel column with MeOH/H2O (1:1). The structures of isolated compounds (Fig. 1) were elucidated by analysis of their spectral data (including IR, NMR and HRESIMS) and by comparison with the literature. The compounds identified include a new phenolic glycoside 600 -O-b-Dapiofuranosyl-trichocarpin (15) and a new polyyne glucoside 1-heptade-4,6-diyne-3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16), together with fourteen known compounds 3-indolecarboxylic acid (1) (Feng et al., 2007), protocatechuic acid (2) (Shen et al., 2009), phloroglucinol trimethyl ether (3), 2-mercaptobenzothiazole (4), uracil (5) (Hu et al., 2008), pinoresinol (6) (Duan et al., 2002), methyl 3,5-dicaffeoyl quinate (7) (An et al., 2008), 2-hydroxy-5-acetylbenzoic acid (8), arbutin (9) (Sun et al., 2006), 3-(4-hydroxyphenyl)-3-oxopropyl-b-D-glucopyranoside (10) (Shen et al., 1999), vanilloloside (11) (Ida et al., 1994), harman-3-carboxylic acid (12) (Cardoso et al., 2004), ()-isolariciresinol-3a-O-b-D-glucopyranoside (13) (Latté et al., 2008), 3,5-dicaffeoyl quinate (14) (An et al., 2008). Compound 15 obtained as pale yellow powder, the molecular formula of it was C25H30O13 based on HR-ESIMS m/z 561.1560 [M þ Na]þ (calc 561.1579). The 1H-NMR spectrum of 15 (Table 1) displayed one set of aromatic proton signals at d 7.27 (1H, d, J ¼ 7.2 Hz, H-40 ), 7.31 (2H, dd, J ¼ 7.2, 7.2 Hz, H-30 , 50 ) and 7.47 (2H, d, J ¼ 7.2 Hz, H-20 , 60 ), as well as an aromatic ABX system d 7.66 (1H, d, J ¼ 3.0 Hz, H-6), 7.85 (1H, d, J ¼ 8.4 Hz, H-3) and 7.42 (1H, dd, J ¼ 3.0, 8.4 Hz, H-4). The 13C-NMR spectrum of compound 15 (Table 1) showed 25 signals, assigned as four oxymethylene, seven oxymethines, eight methines, one carbonyl carbon, five quaternary carbons. The chemical shifts and coupling constants of these signals, in combination with the observed HMBC and 1H-1H COSY correlations, also suggested the presence of one monosubstituted aromatic ring and one trisubstituted aromatic ring. Furthermore, a methine the HMBC correlations from H-20 and H-60 to methylene (d 66.8, C-70 ), from H-6 to carbonyl carbon (d 166.7, C-7) as well as from H-70 a (d 5.33, d, J ¼ 12.0 Hz) and H-70 b (d 5.38, d, J ¼ 12.0 Hz) to C-7 indicated the presence of a benzoic acid benzyloate moiety. Besides above signals, a glucose could be assigned at d 105.1, 75.1, 77.4. 71.4, 78.0, 68.9, d 4.10–5.33 and an apiose at 111.1, 77.7, 80.3, 74.9, 65.3, d 4.09–5.77, with HSQC, 1H-1H COSY and HMBC correlations. The HMBC correlations from H-1000 (d 5.77, d, J ¼ 2.4 Hz) to C-60 ’ (d 68.9) identified an apiofuranosyl (1/6) glucopyranosyl linkage, while the glucose moiety was located at the C-2 of the benzoyl, which was confirmed by the HMBC correlations from H-100 (d 5.33, d, J ¼ 7.2 Hz) to C-2 (d 151.2). The b-anomeric configuration for the glucose was determined from large coupling constant value of H-1000 (d 5.33, d, J ¼ 7.2 Hz), also, the b-anomeric configuration for the apiose was indicated from the anomeric signal of H-1000 (d 5.77, d, J ¼ 2.4 Hz). The glucose and apiose in compound 15 were tentatively assigned the D-configuration because of naturally occurring. Thus, the structure of compound 15 was established as 600 -O-b-Dapiofuranosyl-trichocarpin. Compound 16 was obtained as white power, whose molecular formula C23H36O9 was inferred from the positive-ion HRESIMS (m/z 479.2279 [M þ Na]þ), indicating six degrees of unsaturation. IR also gave an absorption band at 2254 cm1 suggesting it was an acetylenic derivative. The 1H NMR spectrum (Table 1) showed the presence of three olefinic proton signals at d 5.24 (ddd, J ¼ 1.2, 2.4, 10.8 Hz, H-1a), 5.48 (ddd, J ¼ 1.2, 10.8, 17.4 Hz, H-1b) and 5.94 (ddd, J ¼ 5.4, 1.8, 17.4 Hz, H-2), which were coupled to the 13C NMR resonances at d 118.2 (C-1) and 135.9 (C-2) in the HMQC spectrum, respectively. Furthermore, an oxymethine d 69.1 (C-3), which was coupled to the 1H NMR resonances at d 5.26 in the HMQC, was linked to C-2 on the basis of 1H-1H COSY (H-2/H-3). By long-range HMBC correlations from H-3 to the acetylenic carbons at C-4 (d 72.8), C-5 (d 65.2), C-6 (d 73.5) and from H2-8 (d 2.42) to C-7 (d 82.7), C-6, C-5, a conjugated diyne was deduced between C-3 and C-8 (d 16.2). The connection of C-8 to C-10 was deduced from the 1H-1H COSY correlations H2-8 with H2-9 (d 1.58 and 1.69), H2-9 with H-10 (d 3.62), coupled with the HMBC correlations of H2-8 with C-9 (d 36.8) and C-10 (d 71.0), H-9 (d 1.58) with C-8 and C10, H-10 with C-8. As similarly, the connection of C-16 to C-17 was deduced by the 1H-1H COSY correlation H-16 (d 3.58) with H-17 (d 3.47). The 1H NMR spectrum of 16 exhibited a sugar moiety with an anomeric proton at d 4.60 (d, J ¼ 7.8); The 13C NMR data of 16 (Table 1) also revealed the presence of a glucose moiety: five oxygenated methine carbons at d 101.1, 74.9, 78.0, 71.7, 78.2 and one methylene carbon at d 62.8. The location of the glucosylmoiety was concluded to be C-3 by the HMBC correlation
252
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
Fig. 1. Chemical structures of compounds 1–16.
of H-10 (d 4.60) with C-3 (d 69.1) while the anomeric configuration of the glucose was determined to be b-linkage from the coupling constant (J ¼ 7.8 Hz) of anomeric proton. In addition, the DEPT data of 16 revealed the presence of five methylenes at d 38.3, 26.7, 30.8, 26.7 and 34.4. Thus, C-10 connected to C-16 through five methylenes, which was confirmed by 1H-1H COSY correlations of H-10 with H-11 (d 1.45), H-16 with H-15 (d 1.36), along with the HMBC correlations of H-11 with C-9 (d 38.3), H17 with C-15 (d 34.4). On the basis of this examination, the structure of 16 was proposed as 1-heptade-4,6-diyne-3,10,16,17tetraol-3-O-b-D-glucopyranoside.
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
253
Table 1 1 H and 13C NM Ra spectral data of 15 and 16. Position
15b
Position
dH
dC
1
–
122.6
1
2 3 4 5 6 7 10 2 0 , 60
– 7.85 (d, J ¼ 8.4 Hz) 7.42(dd, J ¼ 3.0, 8.4 Hz) – 7.66 (d, J ¼ 3.0 Hz) – – 7.47 (d, J ¼ 7.2 Hz)
151.2 121.2 121.8 153.9 117.5 166.7 136.6 128.3
2 3 4 5 6 7 8 9
30 , 50 70
16c
dH
dC 118.2
J ¼ 7.2, 7.2 Hz) ¼ 12.0 Hz) ¼ 12.0 Hz) ¼ 7.2 Hz)
128.8 66.8
10 11
100
7.31 5.33 5.38 5.33
105.1
12
200
4.31 (dd, J ¼ 7.2, 9.0 Hz)
75.1
13
300
4.20 (dd, J ¼ 9.0, 8.4 Hz)
77.4
14
400
4.09 (dd, J ¼ 9.0, 9.0 Hz)
71.4
15
500
4.29 4.18 4.73 5.77 4.78 – 4.34 4.59 4.15 4.17
(dd, (dd, (d, J (d, J (d, J
J ¼ 9.0, 6.6 Hz) J ¼ 10.2, 6.6) ¼ 10.2 Hz) ¼ 2.4 Hz) ¼ 2.4 Hz)
78.0 68.9
16 17
(d, (d, (d, (d,
¼ ¼ ¼ ¼
111.1 77.7 80.3 74.9
10 20 30 40
5.24 (ddd, J ¼ 1.2, 2.4, 10.8) 5.48 (ddd, J ¼ 1.2, 1.8, 17.4) 5.94 (ddd, J ¼ 5.4, 10.8, 17.4) 5.26 (m) – – – – 2.42 (2H, m) 1.58 (m) 1.69 (m) 3.62 (m) 1.42 (m) 1.45 (m) 1.35 (m) 1.46 (m) 1.29 (m) 1.35 (m) 1.35 (m) 1.50 (m) 1.36 (m) 1.50 (m) 3.58 (m) 3.42 (dd, J ¼ 5.6, 10.8) 3.47 (dd, J ¼ 5.4, 10.8) 4.60 (d, J ¼ 7.8) 3.21(dd, J ¼ 7.8, 9.0) 3.39 (dd, J ¼ 9.0, 9.0) 3.30 (dd, J ¼ 7.8, 9.0)
65.3
50
3.29 (m)
78.2
60
3.66 (dd, J ¼ 6.0, 12.0) 3.88 (dd, J ¼ 1.8, 12.0)
62.8
600 10 00 20 00 30 00 40 00 50 00
(dd, (d, J (d, J (d, J
J J J J
9.0 Hz) 9.0 Hz) 11.4 Hz) 11.4 Hz)
135.9 69.1 72.8 65.2 73.5 82.7 16.2 36.8 71.0 38.3 26.7 30.8 26.7 34.4 73.2 67.4 101.1 74.9 78.0 71.7
a 1 H and 13C NMR were obtained at 600 MHz and 150 MHz, respectively. Chemical shifts were given in ppm; J values were in parenthesses and reported in Hz. b In C5D5N. c In CD3OD.
4. Chemotaxonomic significance The genus Tithonia is phylogenetically close to the genera Viguiera and Helianthus. This evolutionary relationship was first proposed based only on morphological data. Later, phytochemical studies of several taxa from these genera have been carried out, the common presence of some sesquiterpene lactones support the phylogenetic relationship among Viguiera, Helianthus, and Tithonia (Chagas-Paula et al., 2012). The present phytochemical investigation on T. diversifolia led to the isolation of two new compounds, 600 -O-b-D-apiofuranosyl-trichocarpin (15) and 1-heptade-4,6-diyne-3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16), but they do not occur in Viguiera and Helianthus. Therefore, it could be tentatively concluded that these compounds might be a useful chemotaxonomic marker of the species T. diversifolia and could be used to differentiate Tithonia from Viguiera and Helianthus. The result confirmed morphological data and also clarified ambiguous relationships among these genera. The genus Tithonia comprises 13 taxa, which are distributed in eleven species and two sections as follows: section Tithonia and section Mirasola (Morales 2000). Interesting to note is that 2-mercaptobenzothiazole (4), uracil (5), pinoresinol (6), ()-isolariciresinol-3a-O-b-D-glucopyranoside (13), 600 -O-b-D-apiofuranosyl-trichocarpin (15) and 1-heptade-4,6-diyne3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16) were isolated herein for the first time from T. diversifolia. Therefore, it could be useful to differentiated T. diversifolia from other species of Tithonia. Acknowledgements Research was supported by a grant Shanghai Science and Technology Committee (08DZ1971503) and a foundation National Natural Science Foundation of China (20872177).
254
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
References An, R.B., Sohn, D.H., Jeong, G.S., Kim, Y.C., 2008. Arch. Pharm. Res. 31, 594–597. Baruah, N.C., Sharma, R.P., Madhusudanan, K.P., Thyagarajan, G., Herz, W., Murari, R., 1979. J. Org. Chem. 44, 1831–1835. Bordoloi, M., Barua, N.C., Ghosh, A.C., 1996. Phytochemistry 41, 557–559. Cardoso, C.L., Castro-Gamboa, I., Silva, D.H., Furlan, M., Epifanio Rde, A., Pinto Ada, C., Moraes de Rezende, C., Lima, J.A., Bolzani Vda, S., 2004. J. Nat. Prod. 67, 1882–1885. Chagas-Paula, D.A., Oliveira, R.B., Silva, V.C., Gobbo-Neto, L., Gasparoto, T.H., Campanelli, A.P., Faccioli, L.H., Da Costa, F.B., 2011. J. Ethnopharmacol. 136, 355– 362. Chagas-Paula, D.A., Oliveira1, R.B., Rocha, B.A., Da Costa, F.B., 2012. Chem. Biodivers. 9, 210–235. Duan, H.Q., Takaishi, Y., Momota, H., Ohmoto, Y., Taki, T., 2002. Phytochemistry 59, 85–90. Feng, Y., Li, X.M., Wang, B.G., 2007. Chin. Tradit Herb Drugs 38, 1301–1303. Gu, J.Q., Gills, J.J., Park, E.J., Mata-Greenwood, E., Hawthorne, M.E., Axelrod, F., Chavez, P.I., Fong, H.H., Mehta, R.G., Pezzuto, J.M., kinghorn, A.D., 2002. J. Nat. Prod. 65, 532–536. Hu, W.C., Yang, N.Y., Deng, T., 2008. Cent. South Pharm. 6, 437–438. Ida, Y., Satoh, Y., Ohtsuka, M., Nagasao, M., Shoji, J., 1994. Phytochemistry 35, 209–215. Kuo, Y.H., Chen, C.H., 1998. J. Nat. Prod. 61, 827–828. Kuo, Y.H., Lin, B.Y., 1999. Chem. Pharm. Bull. 47, 428–429. Kuroda, M., Yokosuka, A., Kobayashi, R., Jitsuno, M., Kando, H., Nosaka, K., Ishii, H., Yamori, T., Mimaki, Y., 2007. Chem. Pharm. Bull. 55, 1240–1244. Latté, K.P., Kaloga, M., Schäfer, A., Kolodziej, H., 2008. Phytochemistry 69, 820–826. Morales, E., 2000. Evolution 54, 475–484. Rüngeler, P., Lyb, G., Castro, V., Mora, G., Pahl, H.L., Merfort, L., 1998. Planta Med. 64, 588–593. Shen, Y.B., Kojima, Y., Terazawa, M., 1999. J. Wood Sic 45, 332–336. Shen, X.J., Ge, R.L., Wang, J.H., 2009. J. Henan Univ. 28, 196–199. Sun, X.H., Shen, G.M., Tian, X., 2006. Xi Bei Zhi Wu Xue Bao 26, 412–415. Zhai, H.L., Zhao, G.J., Yang, G.J., Sun, H., Yi, B., Sun, L.N., Cheng, W.S., Zheng, S.Q., 2010. Chem. Nat. Comd. 46, 198–200. Zhao, G.J., Zhang, C.X., Wu, Z.J., Li, X., Yang, Y.B., Sun, L.N., Zheng, S.Q., 2010. Acad. J. Second Mil. Med. Univ. 31, 189–192.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemical constituents from Tithonia diversifolia and their chemotaxonomic significance Gui-Jun Zhao a,1, Zhong-Xin Xi a,1, Wan-Sheng Chen b, Xia Li b, Lei Sun c, Lian-Na Sun a, * a
School of Pharmacy, Second Military Medical University, Guohe Road 325#, Shanghai 200433, People’s Republic of China Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Fengyang Road 415#, Shanghai 200003, People’s Republic of China c Department of Pharmacy, Fujian University of Traditional Chinese Medicine, Hutuo Road 1#, Fuzhou 350108, People’s Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 February 2012 Accepted 3 June 2012 Available online 28 June 2012
Two new compounds 600 -O-b-D-apiofuranosyl-trichocarpin (15) and 1-heptade-4,6-diyne3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16), together with fourteen known compounds were isolated from Tithonia diversifolia. The chemotaxonomic significance of these compounds was discussed in the article. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Tithonia diversifolia Lignins Polyacetylene Alkaloids Phenolics
1. Subject and source Tithonia diversifolia (Hemsl.) A. Gray (Compositae: Heliantheae), known as Mexican sunflower, is native to Mexico and Central America (Chagas-Paula et al., 2011). Presently, T. diversifolia has been introduced into other countries for ornamental purposes or for their pharmacological action (Zhai et al., 2010). And T. diversifolia is widespread in southern parts of China. The aerial parts of T. diversifolia were collected from Mengzi, Yunnan province of China, in September 2008, and were authenticated by Prof. Wansheng Chen (Department of Pharmacy, Changzheng Hospital, Second Military Medical University). A voucher specimen (NO.TD20080819) was deposited in Department of Pharmacognosy, Second Military Medical University, Shanghai, P. R. China. 2. Previous work Many classes of secondary metabolites have been isolated from the Tithonia species, including sesquiterpenoids and diterpenoids, as well as flavonoids. Further minor classes such as phytosterols, xanthanes, coumarins, ceramides, chromones, and chromenes have been also found (Chagas-Paula et al., 2012). The most studied species of the genus Tithonia is T. diversifolia, from which more than 150 compounds have been isolated. Previous phytochemical investigations on T. diversifolia have reported the presence of sesquiterpenes, including germacrane type sesquiterpenoids, eudesmane type sesquiterpenoids, guaiane type sesquiterpenoids, cadinane type sesquiterpenoids and xanthane type sesquiterpenoids (Baruah et al., 1979, Rüngeler et al., 1998, Kuroda et al., 2007; Kuo and Chen, 1998; Gu et al., 2002; Bordoloi et al., 1996; Kuo and Lin, 1999).
* Corresponding author. Tel./fax: þ86 21 81871308. E-mail address: [email protected] (L.-N. Sun). 1 These authors contributed equally to this work. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.06.019
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
251
Moreover, chromenes (Kuo and Lin, 1999), diterpenoids, quininic acids and three flavonoids (Kuroda et al., 2007) have also been isolated. 3. Present study The air-dried aerial parts of T. diversifolia (22 kg) were powdered and extracted with 80% aqueous ethanol (Zhao et al., 2010). After removing the solvent under reduced pressure using a rotary evaporator at 60 C, the resulting gum was dried to yield 2596 g of crude extract. A portion of the crude extract (2500 g) was suspended in distilled water and successively partitioned with petroleum ether (yield: 150 g), EtOAc (yield: 130 g), n-BuOH (yield: 160 g). The EtOAc extract (120 g) was chromatographed on silica gel column with the gradient petroleum ether/EtOAc (20:1, 10:1, 5:1, 3:1, 2:1, 1:1) as eluent. The eluate of petroleum ether/EtOAc (5:1) was purified by a Sephadex LH-20 to afforded compounds 1 (46 mg) and 2 (78 mg). The n-BuOH extract (160 g) was applied on silica column eluted with gradient CH2Cl2/MeOH system (50:1, 20:1, 10:1, 5:1, 2:1, 1:1). The obtained fractions were combined on the basis of silica gel TLC and six fractions (Fr1–Fr6) were obtained. The Fr1 was subjected to silica gel column chromatography and eluted with CH2Cl2/MeOH (20:1, 15:1, 10:1) to afford compounds 3 (18 mg) and 4 (7 mg). Fr2 was retreated on silica gel column and eluated with CH2Cl2/MeOH (10:1) to yield compound 5 (11 mg). Fr3 was treated on silica gel column once more and eluted with CH2Cl2/MeOH (8:1) to yield compounds 6 (25 mg), 7 (100 mg) and 8 (10 mg). Fr4 was separated by reversed-phase silica gel (RP-18) with MeOH/H2O (1:9, 1:4, 2:3, 1:1, 1:0) and finally reapplied to a Sephadex LH-20 column using MeOH/H2O (1:1) to yield compounds 9 (32 mg), 10 (21 mg), 11 (12 mg), 12 (13 mg), 13 (28 mg) and 14 (80 mg). Compound 15 (18 mg) was isolated from Fr5 by RP-18 with MeOH/H2O (1:4), while compound 16 (7 mg) was purified from Fr6 by Sephadex LH-20 gel column with MeOH/H2O (1:1). The structures of isolated compounds (Fig. 1) were elucidated by analysis of their spectral data (including IR, NMR and HRESIMS) and by comparison with the literature. The compounds identified include a new phenolic glycoside 600 -O-b-Dapiofuranosyl-trichocarpin (15) and a new polyyne glucoside 1-heptade-4,6-diyne-3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16), together with fourteen known compounds 3-indolecarboxylic acid (1) (Feng et al., 2007), protocatechuic acid (2) (Shen et al., 2009), phloroglucinol trimethyl ether (3), 2-mercaptobenzothiazole (4), uracil (5) (Hu et al., 2008), pinoresinol (6) (Duan et al., 2002), methyl 3,5-dicaffeoyl quinate (7) (An et al., 2008), 2-hydroxy-5-acetylbenzoic acid (8), arbutin (9) (Sun et al., 2006), 3-(4-hydroxyphenyl)-3-oxopropyl-b-D-glucopyranoside (10) (Shen et al., 1999), vanilloloside (11) (Ida et al., 1994), harman-3-carboxylic acid (12) (Cardoso et al., 2004), ()-isolariciresinol-3a-O-b-D-glucopyranoside (13) (Latté et al., 2008), 3,5-dicaffeoyl quinate (14) (An et al., 2008). Compound 15 obtained as pale yellow powder, the molecular formula of it was C25H30O13 based on HR-ESIMS m/z 561.1560 [M þ Na]þ (calc 561.1579). The 1H-NMR spectrum of 15 (Table 1) displayed one set of aromatic proton signals at d 7.27 (1H, d, J ¼ 7.2 Hz, H-40 ), 7.31 (2H, dd, J ¼ 7.2, 7.2 Hz, H-30 , 50 ) and 7.47 (2H, d, J ¼ 7.2 Hz, H-20 , 60 ), as well as an aromatic ABX system d 7.66 (1H, d, J ¼ 3.0 Hz, H-6), 7.85 (1H, d, J ¼ 8.4 Hz, H-3) and 7.42 (1H, dd, J ¼ 3.0, 8.4 Hz, H-4). The 13C-NMR spectrum of compound 15 (Table 1) showed 25 signals, assigned as four oxymethylene, seven oxymethines, eight methines, one carbonyl carbon, five quaternary carbons. The chemical shifts and coupling constants of these signals, in combination with the observed HMBC and 1H-1H COSY correlations, also suggested the presence of one monosubstituted aromatic ring and one trisubstituted aromatic ring. Furthermore, a methine the HMBC correlations from H-20 and H-60 to methylene (d 66.8, C-70 ), from H-6 to carbonyl carbon (d 166.7, C-7) as well as from H-70 a (d 5.33, d, J ¼ 12.0 Hz) and H-70 b (d 5.38, d, J ¼ 12.0 Hz) to C-7 indicated the presence of a benzoic acid benzyloate moiety. Besides above signals, a glucose could be assigned at d 105.1, 75.1, 77.4. 71.4, 78.0, 68.9, d 4.10–5.33 and an apiose at 111.1, 77.7, 80.3, 74.9, 65.3, d 4.09–5.77, with HSQC, 1H-1H COSY and HMBC correlations. The HMBC correlations from H-1000 (d 5.77, d, J ¼ 2.4 Hz) to C-60 ’ (d 68.9) identified an apiofuranosyl (1/6) glucopyranosyl linkage, while the glucose moiety was located at the C-2 of the benzoyl, which was confirmed by the HMBC correlations from H-100 (d 5.33, d, J ¼ 7.2 Hz) to C-2 (d 151.2). The b-anomeric configuration for the glucose was determined from large coupling constant value of H-1000 (d 5.33, d, J ¼ 7.2 Hz), also, the b-anomeric configuration for the apiose was indicated from the anomeric signal of H-1000 (d 5.77, d, J ¼ 2.4 Hz). The glucose and apiose in compound 15 were tentatively assigned the D-configuration because of naturally occurring. Thus, the structure of compound 15 was established as 600 -O-b-Dapiofuranosyl-trichocarpin. Compound 16 was obtained as white power, whose molecular formula C23H36O9 was inferred from the positive-ion HRESIMS (m/z 479.2279 [M þ Na]þ), indicating six degrees of unsaturation. IR also gave an absorption band at 2254 cm1 suggesting it was an acetylenic derivative. The 1H NMR spectrum (Table 1) showed the presence of three olefinic proton signals at d 5.24 (ddd, J ¼ 1.2, 2.4, 10.8 Hz, H-1a), 5.48 (ddd, J ¼ 1.2, 10.8, 17.4 Hz, H-1b) and 5.94 (ddd, J ¼ 5.4, 1.8, 17.4 Hz, H-2), which were coupled to the 13C NMR resonances at d 118.2 (C-1) and 135.9 (C-2) in the HMQC spectrum, respectively. Furthermore, an oxymethine d 69.1 (C-3), which was coupled to the 1H NMR resonances at d 5.26 in the HMQC, was linked to C-2 on the basis of 1H-1H COSY (H-2/H-3). By long-range HMBC correlations from H-3 to the acetylenic carbons at C-4 (d 72.8), C-5 (d 65.2), C-6 (d 73.5) and from H2-8 (d 2.42) to C-7 (d 82.7), C-6, C-5, a conjugated diyne was deduced between C-3 and C-8 (d 16.2). The connection of C-8 to C-10 was deduced from the 1H-1H COSY correlations H2-8 with H2-9 (d 1.58 and 1.69), H2-9 with H-10 (d 3.62), coupled with the HMBC correlations of H2-8 with C-9 (d 36.8) and C-10 (d 71.0), H-9 (d 1.58) with C-8 and C10, H-10 with C-8. As similarly, the connection of C-16 to C-17 was deduced by the 1H-1H COSY correlation H-16 (d 3.58) with H-17 (d 3.47). The 1H NMR spectrum of 16 exhibited a sugar moiety with an anomeric proton at d 4.60 (d, J ¼ 7.8); The 13C NMR data of 16 (Table 1) also revealed the presence of a glucose moiety: five oxygenated methine carbons at d 101.1, 74.9, 78.0, 71.7, 78.2 and one methylene carbon at d 62.8. The location of the glucosylmoiety was concluded to be C-3 by the HMBC correlation
252
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
Fig. 1. Chemical structures of compounds 1–16.
of H-10 (d 4.60) with C-3 (d 69.1) while the anomeric configuration of the glucose was determined to be b-linkage from the coupling constant (J ¼ 7.8 Hz) of anomeric proton. In addition, the DEPT data of 16 revealed the presence of five methylenes at d 38.3, 26.7, 30.8, 26.7 and 34.4. Thus, C-10 connected to C-16 through five methylenes, which was confirmed by 1H-1H COSY correlations of H-10 with H-11 (d 1.45), H-16 with H-15 (d 1.36), along with the HMBC correlations of H-11 with C-9 (d 38.3), H17 with C-15 (d 34.4). On the basis of this examination, the structure of 16 was proposed as 1-heptade-4,6-diyne-3,10,16,17tetraol-3-O-b-D-glucopyranoside.
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
253
Table 1 1 H and 13C NM Ra spectral data of 15 and 16. Position
15b
Position
dH
dC
1
–
122.6
1
2 3 4 5 6 7 10 2 0 , 60
– 7.85 (d, J ¼ 8.4 Hz) 7.42(dd, J ¼ 3.0, 8.4 Hz) – 7.66 (d, J ¼ 3.0 Hz) – – 7.47 (d, J ¼ 7.2 Hz)
151.2 121.2 121.8 153.9 117.5 166.7 136.6 128.3
2 3 4 5 6 7 8 9
30 , 50 70
16c
dH
dC 118.2
J ¼ 7.2, 7.2 Hz) ¼ 12.0 Hz) ¼ 12.0 Hz) ¼ 7.2 Hz)
128.8 66.8
10 11
100
7.31 5.33 5.38 5.33
105.1
12
200
4.31 (dd, J ¼ 7.2, 9.0 Hz)
75.1
13
300
4.20 (dd, J ¼ 9.0, 8.4 Hz)
77.4
14
400
4.09 (dd, J ¼ 9.0, 9.0 Hz)
71.4
15
500
4.29 4.18 4.73 5.77 4.78 – 4.34 4.59 4.15 4.17
(dd, (dd, (d, J (d, J (d, J
J ¼ 9.0, 6.6 Hz) J ¼ 10.2, 6.6) ¼ 10.2 Hz) ¼ 2.4 Hz) ¼ 2.4 Hz)
78.0 68.9
16 17
(d, (d, (d, (d,
¼ ¼ ¼ ¼
111.1 77.7 80.3 74.9
10 20 30 40
5.24 (ddd, J ¼ 1.2, 2.4, 10.8) 5.48 (ddd, J ¼ 1.2, 1.8, 17.4) 5.94 (ddd, J ¼ 5.4, 10.8, 17.4) 5.26 (m) – – – – 2.42 (2H, m) 1.58 (m) 1.69 (m) 3.62 (m) 1.42 (m) 1.45 (m) 1.35 (m) 1.46 (m) 1.29 (m) 1.35 (m) 1.35 (m) 1.50 (m) 1.36 (m) 1.50 (m) 3.58 (m) 3.42 (dd, J ¼ 5.6, 10.8) 3.47 (dd, J ¼ 5.4, 10.8) 4.60 (d, J ¼ 7.8) 3.21(dd, J ¼ 7.8, 9.0) 3.39 (dd, J ¼ 9.0, 9.0) 3.30 (dd, J ¼ 7.8, 9.0)
65.3
50
3.29 (m)
78.2
60
3.66 (dd, J ¼ 6.0, 12.0) 3.88 (dd, J ¼ 1.8, 12.0)
62.8
600 10 00 20 00 30 00 40 00 50 00
(dd, (d, J (d, J (d, J
J J J J
9.0 Hz) 9.0 Hz) 11.4 Hz) 11.4 Hz)
135.9 69.1 72.8 65.2 73.5 82.7 16.2 36.8 71.0 38.3 26.7 30.8 26.7 34.4 73.2 67.4 101.1 74.9 78.0 71.7
a 1 H and 13C NMR were obtained at 600 MHz and 150 MHz, respectively. Chemical shifts were given in ppm; J values were in parenthesses and reported in Hz. b In C5D5N. c In CD3OD.
4. Chemotaxonomic significance The genus Tithonia is phylogenetically close to the genera Viguiera and Helianthus. This evolutionary relationship was first proposed based only on morphological data. Later, phytochemical studies of several taxa from these genera have been carried out, the common presence of some sesquiterpene lactones support the phylogenetic relationship among Viguiera, Helianthus, and Tithonia (Chagas-Paula et al., 2012). The present phytochemical investigation on T. diversifolia led to the isolation of two new compounds, 600 -O-b-D-apiofuranosyl-trichocarpin (15) and 1-heptade-4,6-diyne-3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16), but they do not occur in Viguiera and Helianthus. Therefore, it could be tentatively concluded that these compounds might be a useful chemotaxonomic marker of the species T. diversifolia and could be used to differentiate Tithonia from Viguiera and Helianthus. The result confirmed morphological data and also clarified ambiguous relationships among these genera. The genus Tithonia comprises 13 taxa, which are distributed in eleven species and two sections as follows: section Tithonia and section Mirasola (Morales 2000). Interesting to note is that 2-mercaptobenzothiazole (4), uracil (5), pinoresinol (6), ()-isolariciresinol-3a-O-b-D-glucopyranoside (13), 600 -O-b-D-apiofuranosyl-trichocarpin (15) and 1-heptade-4,6-diyne3,10,16,17-tetraol-3-O-b-D-glucopyranoside (16) were isolated herein for the first time from T. diversifolia. Therefore, it could be useful to differentiated T. diversifolia from other species of Tithonia. Acknowledgements Research was supported by a grant Shanghai Science and Technology Committee (08DZ1971503) and a foundation National Natural Science Foundation of China (20872177).
254
G.-J. Zhao et al. / Biochemical Systematics and Ecology 44 (2012) 250–254
References An, R.B., Sohn, D.H., Jeong, G.S., Kim, Y.C., 2008. Arch. Pharm. Res. 31, 594–597. Baruah, N.C., Sharma, R.P., Madhusudanan, K.P., Thyagarajan, G., Herz, W., Murari, R., 1979. J. Org. Chem. 44, 1831–1835. Bordoloi, M., Barua, N.C., Ghosh, A.C., 1996. Phytochemistry 41, 557–559. Cardoso, C.L., Castro-Gamboa, I., Silva, D.H., Furlan, M., Epifanio Rde, A., Pinto Ada, C., Moraes de Rezende, C., Lima, J.A., Bolzani Vda, S., 2004. J. Nat. Prod. 67, 1882–1885. Chagas-Paula, D.A., Oliveira, R.B., Silva, V.C., Gobbo-Neto, L., Gasparoto, T.H., Campanelli, A.P., Faccioli, L.H., Da Costa, F.B., 2011. J. Ethnopharmacol. 136, 355– 362. Chagas-Paula, D.A., Oliveira1, R.B., Rocha, B.A., Da Costa, F.B., 2012. Chem. Biodivers. 9, 210–235. Duan, H.Q., Takaishi, Y., Momota, H., Ohmoto, Y., Taki, T., 2002. Phytochemistry 59, 85–90. Feng, Y., Li, X.M., Wang, B.G., 2007. Chin. Tradit Herb Drugs 38, 1301–1303. Gu, J.Q., Gills, J.J., Park, E.J., Mata-Greenwood, E., Hawthorne, M.E., Axelrod, F., Chavez, P.I., Fong, H.H., Mehta, R.G., Pezzuto, J.M., kinghorn, A.D., 2002. J. Nat. Prod. 65, 532–536. Hu, W.C., Yang, N.Y., Deng, T., 2008. Cent. South Pharm. 6, 437–438. Ida, Y., Satoh, Y., Ohtsuka, M., Nagasao, M., Shoji, J., 1994. Phytochemistry 35, 209–215. Kuo, Y.H., Chen, C.H., 1998. J. Nat. Prod. 61, 827–828. Kuo, Y.H., Lin, B.Y., 1999. Chem. Pharm. Bull. 47, 428–429. Kuroda, M., Yokosuka, A., Kobayashi, R., Jitsuno, M., Kando, H., Nosaka, K., Ishii, H., Yamori, T., Mimaki, Y., 2007. Chem. Pharm. Bull. 55, 1240–1244. Latté, K.P., Kaloga, M., Schäfer, A., Kolodziej, H., 2008. Phytochemistry 69, 820–826. Morales, E., 2000. Evolution 54, 475–484. Rüngeler, P., Lyb, G., Castro, V., Mora, G., Pahl, H.L., Merfort, L., 1998. Planta Med. 64, 588–593. Shen, Y.B., Kojima, Y., Terazawa, M., 1999. J. Wood Sic 45, 332–336. Shen, X.J., Ge, R.L., Wang, J.H., 2009. J. Henan Univ. 28, 196–199. Sun, X.H., Shen, G.M., Tian, X., 2006. Xi Bei Zhi Wu Xue Bao 26, 412–415. Zhai, H.L., Zhao, G.J., Yang, G.J., Sun, H., Yi, B., Sun, L.N., Cheng, W.S., Zheng, S.Q., 2010. Chem. Nat. Comd. 46, 198–200. Zhao, G.J., Zhang, C.X., Wu, Z.J., Li, X., Yang, Y.B., Sun, L.N., Zheng, S.Q., 2010. Acad. J. Second Mil. Med. Univ. 31, 189–192.