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Analytical Methods Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/methods Page 1 of 7 PleaseAnalytical do not adjust Methods margins 1 2 3 4 Journal Name 5 6 7 ARTICLE 8 9 10 11 Determination of Phenanthrenes and Stilbenoid in the Ethyl Acetate 12 Extract of Thunia alba (Lindl) by HPLC-DAD 13 1 1 1 1 1 1 1,* 14 He-Gui Yan † , Hui-Ran Zhao † , Jun Hu , An-Mei Lu , Xue-Mei Fu , Bao Jia , Ming-Hui Yang 15 The target of this work was to determine the amount phenenthrenes and stilbenoid in the ethyl acetate 16 extract of Thunia alba (Lindl). Compounds lusianthridin (1), coelonin (2) (two phenenthrenes) and 17 thunalbene (3) (a stilbenoid) were isolated from Thunia alba (Lindl) by column chromatography and 18 SephadexLH-20. Their structure were elucidated on the basis of spectral data (UV, MS, 1HNMR, 13 C NMR) 19 with refs. Then, three compounds were used as standards for HPLC determination. Method validation was 20 performed with linearity, limit of detection (LOD), limit of quantification (LOQ), precision, stability, 21 repeatability, recovery, selectivity and robustness. The experimental results showed the linearity of the Manuscript 22 method was in the range from 0.06 to 0.80 μg/ml with regression coefficients ranging from 0.9987 to 0.9996. 23 The limits of detection were 0.007, 0.009 and 0.013 μg/ml and the limits of quantification were 0.027, 0.022 24 and 0.033μg/ml for compounds 1, 2 and 3, respectively. The relative standard deviation(RSD) of precision, 25 stability and repeatability were in the range from 0.67% to 2.82%. The average recoveries (n=6) ranged from 26 96.81% to 102.69% and the RSD values varied from 1.59% to 1.81%. The method validation showed that the 27 established method is accurate, simple and repeatable and can be used to provide a reference for the quality 28 control of Thunia alba (Lindl). 29 30 4- 5 31 ulcer . The isolation of compounds of diverse structural types Accepted 32 1. Introduction from Thunia alba (Lindl) has been reported , including stilbenoids, 33 bibenzyls, phenanthrenes, 9, 10-dihydrophenanthrenes, 34 Chinese herbal medicine has played an indispensable role in phenanthropyrans and simple aromatic compounds 6-12. Our 35 preventing and treating human diseases for a long time, and thus chemical research on Thunia alba (Lindl) has resulted in the 1-2 36 has already attracted global attention . The Orchidaceae with over isolation of three known compounds (two phenanthrenes and a 37 700 genera and 25, 000 species is one of the largest plant families stilbenoid), lusianthridin (4, 7-dihydrox-2-methoxy-9, 10- 38 on earth, which has important ecological, economic, cultural and dihydrophenanthrene) 13, 14 (1), coelonin (2, 7-dihydrox-4-methoxy-9, 39 medicinal values. The genus Thunia comprises of approximately 7 10-dihydrophenanthrene)13, 15 (2), and thunalbene (3, 3'--dihydrox- 40 species, all of which are distributed in Sichuan, Xizang, Yunnan, 5-methoxy-stilbene)16 (3). Their structures are presented in Figure 1 . Methods 41 Bhutan, India, Sikkim, Indonesia, Malaysia, Myanmar, Nepal, 42 Thailand and Vietnam 3. T. alba , T. bensoniae and T. marshalliana are Phenanthrenes and bibenzyls are most characteristic as chemical 43 the representative species of the genus Thunia . All of these species, marker for Thunia alba (Lindl), which have been shown to have 44 only Thunia alba (Lindl) distributed in China and was used as various biological activities such as anti-inflammatory 17 , anti- 45 medical plant. Thunia alba (Lindl) was locally known as the "Rock oxidation 18 , anti-bacterial agent 19 , anti-allergy 20 , antialgal and 46 Angle " or "Jie-gu-dan " in Dali of Yunnan Province and was used for antitrypanosomal activities 21-22, vasorelaxant effects 23 and 47 the treatment of cough, pneumonia, bronchitis and duodenal anticancer 24-26 . Phenanthrenes, monbarbatain A, B, C and D had 48 exhibited free radical scavengining of the 2,2′- 49 diphenylpicrylhydrazyl (DPPH) by Ming-hui Yang reported 13 . Some 50 refs indicated that the compounds moscatilin, loddigesiinol D and Analytical 1 51 School of Pharmaceutical Sciences and Chemistry, WanHua pholidotol A, B can intensively inhibit NO activity27 . Lusianthridin 52 Road, Dali University, 671000, Dali, China was found to exert cytotoxic effects both in vitro and in vivo. The 53 significant activities on A549 human lung carcinoma (ED 50 : *Corresponding author: E-mail: [email protected]; Tel: +86 54 7.7μg/ml); SK-OV-3 human ovary adenocarcinoma (ED 50 : 9.4μg/ml) 55 872 2257409 and HL-60 human promyelocytic leukaemia (ED 50 : 9.8μ/ml) cell 56 lines were demonstrated 28 . 57 † These authors contributed equally to this work. 58 59 60 This journal is © The Royal Society of Chemistry 20xx J. Name ., 2013, 00 , 1-3 | 1 Please do not adjust margins PleaseAnalytical do not adjust Methods margins Page 2 of 7 ARTICLE Journal Name 1 2 High-performance liquid chromatographic (HPLC) was widely performed by Prof. Jiang-Miao Hu, Kunming Institute of Botany, 3 used for the analysis of chemical constitutes. HPLC-DAD Chinese Academy of Sciences. 4 chromatographic method is a suitable tool for the separation and 5 quantification of phenolic compounds in plant extracts because of 2.2 Extraction and isolation 6 its versatility, precision and relatively low cost. For medicinal plant 7 The crushed rhizomes of Thunia alba (Lindl)(10.0 Kg) were analysis, using HPLC-DAD method, the identification and 8 extracted in the cold-soaked way with 90% ethanol for 48 h each quantification of bioactive and marker compounds in complex 9 time, and it was repeated for 5 times . Then the combined ethanol matrices can be realized even structurally similar natural products 10 extract was concentrated under reduced pressure at 45 °C to afford based on their chromatographic retention data, spectral 11 29-31 a brown residue (252 g). The residue was diluted with H O and characteristics and quantitative information . In 2006, Li Yang 2 12 partitioned with petroleum ether, EtOAc and n-BuOH, successively. reported the method on simultaneous determination of phenols 13 The EtOAc fraction (60.8 g) was subjected to silica gel column (bibenzyl, phenanthrene, and fluorenone) in Dendrobium species 14 32 chromatography and eluted with a CHCl -CH OH (10:0 →0:10, v/v) by high-performance liquid chromatography . Subsequently, 3 3 15 gradient to afford 8 fractions (A-H). Fractions B and C were futher analytical methods of 9, 10-dihydrophenanthrenes in Bletilla striata 16 purified by column chromatography, eluted with chloroform- (Thunb.) Reichb.f. and Pholidota chinensis were reported 33-34 . 17 acetone (60:1 →0:1) and were subsequently subjected to Sephadex However, it was rarely reported the determination of 18 LH-20 to give compounds 1 (10.7 mg), 2 (9.6 mg) and 3 (9.5 mg). phenanthrenes in Thunia alba (Lindl). Especially, the less work on 19 The NMR data of compounds 1, 2 and 3 were given in Table 1 . analysis of compound 3 by HPLC has been previously reported. In 20 Three compounds were purified by semi-preparative HPLC and the addition, the aim of our study was to develop a time-saving and 21 purity was determined to be more than 98% by normalization of simple HPLC method with a DAD detector for the determination of Manuscript 22 the peak areas detected by HPLC analysis. 23 the three compounds, which was used for analysis of different 24 genus Thunia plant and different substitutes or false to ensure the 2.3 Wavelength selection 25 quality and the clinical efficacy. 26 Compounds 1, 2 and 3 were detected by a spectral scan between 27 200 and 400nm to obtain the optimal absorption wavelength. The 28 results of the UV spetra indicated that the maximum absorbance 29 peaks of the compounds 1, 2 and 3 were 212 and 279nm, 208 and 30 279nm, and 230 and 299nm, respectively. Thus, the 279nm peak 31 was selected as the wavelength for compounds 1 and 2, and 299nm Accepted 32 was used as the wavelength for compound 3, because its 33 absorption was much greater at higher wavelength. 34 35 2.4 Chromatographic conditions 36 37 Figure 1. Structures of compounds 1, 2 and 3 The HPLC analysis of the three compounds was conducted on an 38 Agilent LC-1200 apparatus (Palo Alto, USA) equipped with a quaternary pump, a vacuum degasser, an autosampler, a DAD 39 2. Experimental 40 detector and chromatographic data were processed by Agilent Methods 41 2.1 Reagents and materials Chem Station Software. The column used for the analysis was a 42 ZORBAX SB-Aq, with a 4.6 mm internal diameter, 250 mm length 43 Methanol (HPLC grade) was purchased from Merck (Darmstadt, and 5 μm particle size.