A new anthraquinone and eight constituents from Hedyotis caudatifolia Merr. et Metcalf: isolation, purification and structural identification

Abstract Hedyotis caudatifolia Merr. et Metcalf. (HC), a folk medicine in Yao nationalities areas in China, was used to investigate the chemical constituents. Through silica gel and Sephadex LH-20 column chromatography, nine compounds were isolated and purified. By physical and chemical properties, IR, MS (EI-MS, high resolution EI-MS), 1D NMR (1H NMR, 13C NMR) and 2D NMR (HSQC, 1H–1H COSY, HMBC), their structures were identified as β-sitosterol (1), stigmasterol (2), scopolin (3), 2-hydroxy-1,7,8-trimethoxyanthracene-9,10-dione (4), oleanolic acid (5), ursolic acid (6), methyl barbinervate (7), β-daucosterol (8) and p-Hydroxybenzoic acid (9). These compounds were isolated from HC for the first time, and 4 a new anthraquinone whose biological activities are worth to be investigated in future. These compounds may contribute to the HC’s pharmacological effects on treating diseases, and may be used as candidates for control index in establishing the quality control standard of HC.

and eliminating phlegm, strengthening stomach and promoting digestion, homeostasis, defervescence, anti diarrhoea. Therefore, the whole herb of HC, a folk medicine was traditionally used to treat chronic bronchitis, asthma, pulmonary tuberculosis (with coughing and haemoptysis), children`s malnutritional stagnation, traumatic injury (acute soft-tissue trauma), bleeding in trauma, inflamed eye, children`s fever, sore throat and diarrhoea by the Yao nationalities in Jinxiu county of Guangxi province, China (Chinese Herbalism Editorial Board, State Administration of Traditional Chinese Medicine of the People's Republic of China 1999; Nanjing university of Chinese Medicine 2006).
Except the chemical compositions from volatile oil (Pan et al. 2012), other chemical constituents of HC had not been reported yet. The aim of this paper was mainly to isolate and purify constituents from HC, and identify their structures, which can lay a foundation for the future works, such as investigation on material basis of its pharmacological actions, study on quality control standard of HC, etc.
Other eight known compounds ( Figure 1) were identified as follows: By comparing spectroscopic data ( 1 H and 13 C NMR) and melting point data with the literatures, 1 was identified as β-sitosterol (Ahmad et al. 2010), and 2 was identified as stigmasterol (M = 412, C 29 H 48 O); Luo et al. 2003;Chung et al. 2005). 1 H and 13 C NMR spectral data of 3 were consistent with the literature values of scopolin (Vasconcelos et al. 1998;Alberto et al. 2011), so 3 was identified as scopolin (M = 192, C 10 H 8 O 4 ). 5 and 6 were respectively identified as oleanolic acid (M = 456, C 30 H 48 O 3 ) and ursolic acid (M = 456, C 30 H 48 O 3 ) by comparing their 1 H and 13 C NMR spectral data with the literature (Werner et al. 2003). 1 H and 13 C NMR spectral data of 7 were consistent with the literature values of Barbinervic acid (Su et al. 2003) except a oxygenated methyl (δ C 49.7) located at 17-COOH, which suggest that 7 is a methyl-esterification product (17-COOCH 3 ) of Barbinervic acid, so 7 was identified as Methyl barbinervate (M = 502, C 31 H 50 O 5 ). 1 H and 13 C NMR spectral data of 8 were consistent with the literature values of Stigmast-5-en-3-O-β-D-glucoside (Shaheen et al. 2001), so 8 was identified as β-daucosterol (M = 576, C 35 H 60 O 3 ). With the 1 H and 13 C NMR spectral data being consistent with the literature values of p-Hydroxybenzoic acid (Lu et al. 2009) or 4-benzyloxybenzoic acid (Sivakumar et al. 2010), 9 was identified as p-Hydroxybenzoic acid (M = 138, C 7 H 6 O 3 ).

Extraction procedures
Through percolation, powder of HC (30 kg) was successively extracted with ethanol-water (95:5, v/v, 240 L), ethanol-water (80:20, v/v, 240 L), ethanol-water (60:40, v/v, 240 L), ethanol-water (40:60, v/v, 240 L) and water (240 L), at room temperature. All above extracting solutions were mixed and the solvents were evaporated in vacuum at 60-80 °C, to get residues (2427.6 g, crude extract). 2138.6 g of crude extract was suspended by water, and then extracted successively with four portions each of petroleum ether (b.p. 60-90 °C), chloroform, ethyl acetate and n-butyl alcohol. After the solvents evaporated, 16.6 g of petroleum ether portion, 115.0 g of chloroform portion, 32.0 g of ethyl acetate portion, 268.0 g of n-butyl alcohol portion and 1654.7 g of water portion were obtained, respectively.

Experimental procedures to determine structures
The structures of the known compounds were identified by physical and spectroscopic data measurement (EI-MS, IR, 1 H NMR, 13 C NMR, DEPT) by comparing the data obtained with those of literature values. The new compound was identified by physical and spectroscopic data measurement {EI-MS, HREI-MS, IR, 1D NMR ( 1 H NMR, 13 C NMR, DEPT) and 2D NMR ( 1 H-1 H COSY, HSQC, HMBC)}.
Scopolin (3), with a content of 0.005% in HC ethanol-water extract, had been reported with homeostasis, anti-inflammatory, analgesia, relieving cough and eliminating phlegm activities, which may contribute to the HC's pharmacological effects about moistening lung, relieving cough and eliminating phlegm, treating chronic bronchitis, asthma, pulmonary tuberculosis (with coughing and haemoptysis), curing traumatic injury (acute soft-tissue trauma), treating bleeding in trauma, etc. The steroids or triterpenes were well known for their anti-inflammatory actions, which may contribute to the HC's pharmacological activities of curing traumatic injury (acute soft-tissue trauma), treating children's fever (defervescence), etc. The antibiotic activities of anthraquinones were reported widely, which may contribute to the HC's pharmacological actions on treating children`s fever, inflamed eye, sore throat and diarrhoea. Moreover, 4, a new anthraquinone, its biological activities is worth to be investigated further in future. In addition, above chemical constituents can be used as candidates for control index in establishing the quality control standard of HC.

Supplementary material
Supplementary material relating to this article is available online, alongside Table S1 and Figures S1-S7 (EI-MS, HR-EI-MS, IR, 1 H NMR, 13 C NMR, HSQC, COSY and HMBC spectra of the compound 4).