Biochemical Systematics and Ecology 45 (2012) 148–150

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Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

Chemical constituents from gardneri ()

Pan Xu a, Zhining Xia a,*, Yexin Lin b a Chemistry and Chemical Engineering College, Chongqing University, No.174, Shapingba District, Chongqing 400030, PR b Bioengineering College, Chongqing University, No.174, Shapingba District, Chongqing 400030, PR China article info abstract

Article history: This work reports the isolation and characterization of twelve compounds, including six Received 29 February 2012 coumarins and two flavonoids from the ethyl acetate extract of the flowers of Edgeworthia Accepted 22 July 2012 gardneri. Their structures were elucidated on the basis of various spectroscopic methods Available online 19 August 2012 (UV, IR, 1H and 13C NMR) and identified as umbelliferone (1), edgeworin (2), edgeworthin (3), daphnoretin (4), rutamontine (5), edgeworoside A (6), 7-Hydroxy-40-methoxyflavone Keywords: 00 (7), kaempferol-3-O-b-D-(6 -E-p-coumaroyl)-glucopyranoside (8), pentadecanoic acid (9), b-sitosterol (10), b-adenosine (11), b-daucosterol (12). The chemotaxonomic significance of Thymelaeaceae Coumarins these compounds was summarized. Ó Flavonoids 2012 Elsevier Ltd. All rights reserved. Chemotaxonomy

1. Subject and source

The Edgeworthia (Thymelaeaceae) consists of five species, of which 4 species grow in China (Robert et al., 1988). One of the species, Edgeworthia gardneri, is widely distributed in the southwestern region of China. Flowers of E. gardneri, traditionally called “Lv Luohua”, have been used for the treatment of diabetes, hypertension, coronary heart disease and vascular inflammation (Zeng and Li, 2008). However, there is no phytochemical investigation on the flowers of this . In this paper we describe the first isolation and structure elucidation of twelve compounds from the flowers of E. gardneri and their chemotaxonomic significance. Flowers of E. gardneri were purchased from Huisheng Tang Pharmacy at Tibet in September 2009, and identified by Professor Guoyue Zhong, Chongqing Academy of Chinese Materia Medica. A voucher specimen (No.GH827) was deposited at Chongqing Academy of Chinese Materia Medica.

2. Previous work

Previous phytochemical investigations focus on the stem bark of E. gardneri and have yielded three bis-coumarins, which are edgeworin (Majumder et al., 1974), edgeworthin (Chakrabarti et al., 1986) and daphnoretin (Majumder et al., 1974). The previous report of the flowers from this plant reveals that the ethanol extract of the flowers exhibits antioxidant activity and inhibitive effect on a-glucosidase (Zeng and Li, 2008). Moreover, our research on the ethyl acetate extract of the flowers demonstrates its significant activation on peroxisome proliferator activator receptors (PPARs) and its therapeutic potential for diabetes mellitus (Zhong et al., 2011).

* Corresponding author. Tel./fax: þ86 023 65106615. E-mail addresses: [email protected] (P. Xu), [email protected] (Z. Xia), [email protected] (Y. Lin).

0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.031 P. Xu et al. / Biochemical Systematics and Ecology 45 (2012) 148–150 149

3. Present study

3.1. Extraction and isolation

The dried flowers of E. gardneri (1100 kg) were defatted three times with petroleum ether (1.0 L) under reflux (1.0 h). The defatted flowers were extracted three times with 70% ethanol (1.0 L) under reflux (1.0 h). The suspension was then filtered and concentrated in vacuo to give a viscous residue, which was dissolved in water and then partitioned with ethyl acetate. The ethyl acetate soluble fraction was evaporated to dryness below 45 C to afford dark-green semi-solid mass (30.52 g, 2.8%). The ethyl acetate soluble fraction was subjected to silica gel column chromatography and eluted with petroleum: ethyl acetate (100:0–0:100) to afford eleven fractions (Fr.1 – Fr.11). Crystals from fraction 6 and 10 were purified by recrystallization to give compound 5 (81 mg) and compound 6 (120 mg), respectively. Fr.2 was subjected to silica gel column chromatography and eluted with petroleum: ethyl acetate (90:10–70:30) to obtain compound 10 (47 mg). Fr.3 was applied to silica gel column chromatography eluted with a system of CHCl3:MeOH (90:10–20:80) to afford 6 fractions (3 a–f), sub-fraction 3d was purified by recrystallization in acetonetogivecompound9 (19 mg). Fr.5 was treated on silica gel column chromatography eluted with petroleum: ethyl acetate (90:10–20:80) to yield 5 fractions (5 a–e), sub-fraction 5b was purified by recrystallization in MeOH to give compound 4 (19 mg), sub-fraction 5e was purified by ODS column chromatography using a constant elution of EtOH:H2O (15:85) to afford compound 3 (23 mg). Fr.6 was subjected to silica gel column chromatography and eluted with CHCl3:MeOH (85:15 to 20:80) to afford 4 fractions (6 a–d), sub-fraction 6 b was purified by ODS column chromatography using an elution of EtOH:H2O(15:85–70:30) to afford compound 7 (32 mg), sub-fraction 6 d was recrystallized in MeOH to give compound 2 (38 mg). Fr.9 was purified by ODS column chromatography using an elution of EtOH:H2O(15:85–70:30) to afford compound 1 (24 mg) and 8 (35 mg). Fr.11 was purified by Sephadex LH-20 column chromatography eluting with MeOH to obtain compound 12 (20 mg) and 11 (42 mg).

3.2. Identification of purified compounds

The isolated compounds were identified by a combination of spectroscopic methods (UV, IR, 1H and 13C NMR) and comparison with the literature data. They are umbelliferone (1)(Zhang et al., 2011), edgeworin (2)(Majumder et al., 1974), edgeworthin (3)(Chakrabarti et al., 1986), daphnoretin (4)(Cordell, 1984), rutamontine (5)(Kabouche et al., 2003), edge- 0 00 woroside A (6)(Hu et al., 2009), 7-Hydroxy-4 -methoxyflavone (7)(Park et al., 2007), kaempferol-3-O-b-D-(6 -E-p-cou- maroyl)- glucopyranoside (8)(Jung et al., 1998), pentadecanoic acid (9)(Kozhamkulova et al., 2011), b-sitosterol (10)(Kitajima and Tanaka, 1993), b-adenosine (11)(Domondona et al., 2004), b-daucosterol (12)(Qu et al., 2001). 150 P. Xu et al. / Biochemical Systematics and Ecology 45 (2012) 148–150

4. Chemotaxonomic significance

Twelve compounds were isolated from flowers of E. gardneri including six coumarins (1–6) and two flavonoids (7–8). Edgeworin (2), edgeworthin (3) and daphnoretin (4) have previously been reported from E. gardneri (Majumder et al., 1974; Chakrabarti et al., 1986). However, this is the first report of edgeworoside A (6) from E. gardneri. It is also the first report of 00 kaempferol-3-O-b-D-(6 -E-p- coumaroyl)-glucopyranoside (8) from the genus Edgeworthia, and the first report of ruta- montine (5), 7-Hydroxy-40-methoxyflavone (7) from the family Thymelaeaceae. The family Thymelaeaceae, comprises of 40 genera and about 360 species (Robert et al., 1988). More than 40 coumarins and 60 flavonoids with various skeletal patterns have been reported from this family (Liao et al., 2005; Robert et al., 1988; Zhang et al., 2011). In the present study, six coumarins are isolated from E. gardneri, including one monomeric coumarin (1), four bis-coumarins (2–5) and one “trimeric” coumarin (6). Previous studies have shown that umbelliferone (1), edgeworin (2), edgeworthin (3), daphnoretin (4) and edgeworoside A (6) have also been isolated from (Baba et al., 1989; Su et al., 2009; Yan et al., 2006). The occurrence of these five compounds in both species indicates a close systematic relationship between them. Daphnoretin (4) is commonly distributed in the family Thymelaeaceae, including 15 species of , 8 species of , 2 species of Daphnopsis, 2 species of Edgeworthia, 2 species of Thymelea, 1 species of , 1 species of Dirca, 1 species of Enkleia, 1 species of Peddiea (Zhang et al., 2008). Therefore, daphnoretin has no significant chemotaxonomic importance in the family Thymelaeaceae. This is only the second time that rutamontine (5) is reported from a natural source. It was isolated previously from the family Rutaceae (Kabouche et al., 2003). As this is the first report of this compound from Edgeworthia and it is suggested further research is undertaken to evaluate whether it is of systematic importance within Edgeworthia or the family Thymelaeaceae. The coumarin edgeworoside A (6) has been isolated previously from two species in Thymelaeaceae, E. chrysantha (Baba et al., 1989) and Daphne retusa (Hu et al., 2009). This is the third report of edgeworoside A in this family. Moreover, to the best of our knowledge, edgeworoside A has not been reported from other families. So edgeworoside A might be a useful chemotaxonomic marker for the family Thymelaeaceae. 00 Kaempferol-3-O-b-D-(6 -E-p-coumaroyl)-glucopyranoside (8) occurs commonly in many medicinal belonging to 16 families which are Asteraceae, Cistaceae, Elaeagnaceae, Euphorbiaceae, Fagaceae, Lamiaceae, Malvaceae, Monimiaceae, Pinaceae, Plantanaceae, Polypodiaceae, Primulaceae, Rosaceae, Solanaceae, Thymeleaceae and Tiliaceae (Kowalska, 2004). In the family Thymeleaceae, this compound was isolated previously from Daphne genkwa (Song et al., 2010), but this is the first report of this compound in Edgeworthia genus. The data presented in this study show that the diversity of coumarins and flavonoids in Edgeworthia justify further study as systematic characters.

Acknowledgements

This work was financially supported by the Natural Science Foundation of China (21175159) and the International Cooperation Project of Ministry of Science and Technology (NO. 2010DFA32680).

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