黄酮类酚酸类

Chinese Journal of Natural Medicines 2010, 8(3): 0202 0207doi: 10.3724/SP.J.1009.2010.00202ChineseJournal ofNaturalMedicinesAnalysis of Flavonoids and Phenolic Acids in Iristectorum by HPLC-DAD-ESI-MS nSHU Pan 1,2, HONG Jun-Li 1,2, WU Gang 1,2, YU Bo-Yang3, QIN Min-Jian 1,2*1Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009;2Key Laboratory of Modern Traditional Chinese Medicines (Ministry of Education),China Pharmaceutical University, Nanjing 210009; 3Department of Complex Prescription of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, ChinaAvailable online May 2010[ABSTRACT]AIM: To develop high performance liquid chromatography combined with photodiode-array detection and electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MS n) for the analysis and identification of flavonoids and phenolic acids in the rhizome of Iris tectorum Maxim.. METHOD: The structures of flavonoids and phenolic acids were identified by chroma-tographic retention times, UV spectra as well as ESI-MS n spectra. RESULTS: Ten isoflavones were identified as tectori-genin-7-O-ȕ-glucosyl-4'-O-ȕ-glucoside (3), tectoridin (5), iristectorin B (6), iristectorin A (7), iridin (8), genistein (11),tectorigenin (12), iristectorigenin A (14), iristectorigenin B (16), i and rigenin (17). Two flavanones, one flavonol and one flavanonol were tenta-tively identified as hesperetin (9), 5, 7, 3'-trihydroxy-6, 4'-dimethoxyflavanone (10), rhamnocitrin (13) and dihydrokaempferide (15), respectively. The three phenolic acids were tectoruside (1), androsin (2) and apocynin (4). CONCLUSION: The developed simple and rapid method is useful to rapidly identify the bioactive constituents in the rhizome of Iris tectorum. Two flavanones, hesperetin (9)and 5,7,3'-trihydroxy-6, 4'-dimethoxyflavanone (10) were identified from this species for the first time.[KEY WORDS]Iris tectorum Maxim.; HPLC-DAD-ESI-MS n; Flavonoids; Phenolic acids[CLC Number]R917 [Document code] A [Article ID] 1672-3651(2010)03-0202-061 IntroductionIris tectorum Maxim. (Iridaceae) is a perennial herbwidely distributed in China, called Yuan Wei in Chinese. It isalso known as Japanese Roof Iris in some literature, becauseit was first observed growing on roofs in Japan by the Rus-sian botanist, Carl Maximowicz (1827–1891) [1]. Its rhizomehas been used in traditional Japanese medicine as an emeticand laxative [2]. In traditional Chinese medicine, it was usedas a bitter medicine to treat disorders described as Zheng JiaJie Ju, which are similar to modern descriptions of tumors[3-4]. According to the latest edition of the Chinese Pharma-copoeia, the rhizome of I. tectorum is referred to as “ChuanShe Gan” (Rhizoma Iridis Tectori), which is used as a tradi-tional herbal medicine to treat sore throat, disperse phlegmand for heat-clearing as well as detoxifying [5]. Previous phy-[Received on] 18-Mar-2009[Research Funding] This project was supported by National NaturalScience Foundation of China (No. 30170103)[ Corresponding author] QIN Min-Jian: Prof., Tel: 86-025-********,Fax: 86-025-********, E-mail: minjianqin@Copyright © 2010, China Pharmaceutical University.Published by Elsevier B.V. All rights reserved.tochemical investigations resulted in the isolation of severalflavonoids [6-11], iridal-type triterpenoids [2, 12-14] and quinones[15]. Some isoflavones and phenolic acids were found to havehigh content in I. tectorum, and exhibit considerableanti-infective, antitussive, expectorant, antibacterial, cyto-toxic and hepatoprotective effects [3, 16-20]. Those compoundswere considered as the main active components of I. tectorum.However, in the Chinese Pharmacopoeia, only tectoridin hasbeen used as the chemical marker for the quality control ofthe rhizome of I. tectorum. Therefore, qualitative evaluationof these main components of I. tectorum is significant for thequality control of this medicinal herb.With the soft ionization source such as atmosphericpressure chemical ionization (APCI) and electrospray ioniza-tion (ESI), MS combined with chromatographic techniqueshas become a powerful approach in the identification, quanti-fication and structural confirmation of active components inmedicinal plants. Nowadays, HPLC with photodiode arraydetection–electrospray ionization multiple-stage mass spec-trometry (HPLC-DAD–ESI-MS n) has grown into one of themost powerful analytical techniques available for analyzingcomplex herbal extracts [21-23]. It can simultaneously provideUV and multiple-stage mass spectra, which can be applied toidentify known components by comparing on-line detected chromatograms and spectra with those of authentic com-pounds, and can elucidate unknown structures based on the tandem mass fragmentation pathways of known ones. Previ-ously, there were no reports on the qualitative research of the major components in the rhizome of I. tectorum by HPLC-DAD–ESI-MS n.In this study, a HPLC-DAD–ESI-MS n method was de-veloped and validated for the identification of ten known isoflavones, three phenolic acids, two flavanones, one fla-vonol and one flavanonol in the rhizome of I. tectorum.2 Experimental2.1 Instrumentation and reagentsLiquid chromatography separation was performed using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) composed of a quaternary pump, an on-line degasser, a column temperature controller and a diode array detector (DAD). A KH5200DB ultrasonic cleaning instru-ment (Jiangsu Kscsb Ultrasonic Instrument Co., Jiangsu, China) was used for extraction. HPLC grade acetonitrile (TEDIA, Fair¿eld, OH, USA) was used. HPLC grade water was obtained from a water purifying system (Milli-pore, Bedford, MA, USA); analytical grade acetic acid (Nanjing Reagent, Jiangsu, China) and HPLC grade methanol (Han-bang, Jiangsu, China) were used for sample preparation. For HPLC-DAD–ESI-MS n analysis, the LC system was coupled to ion trap mass spectrometer (Agilent Corp., Santa Clara, CA, USA) equipped with an ESI source.2.2 MaterialsI. tectorum was collected from Beijing, China, in August 2008. The plant was identified by Prof. QIN Min-jian and a voucher specimen (SP-08-0810) was deposited at the Her-barium of Medicinal Plants of China Pharmaceutical Univer-sity. Eight authentic compounds: Androsin, tectoridin, iris-tectorin A, iristectorin B, iridin, tectorigenin, iristectorigenin A and irigenin were isolated in our laboratory from I. tecto-rum. Their structures were elucidated by spectral data (MS, 1H NMR and 13C NMR). The purity of each compound was determined to be higher than 95% by HPLC. The samples of the herb and chemicals for analysis were stored in the refrig-erator at 20 q C.2.3 Sample preparationThe rhizomes of I. tectorum were air-dried and ground into powder. An aliquot (0.5 g) of the powder was weighed into a conical flask and 25 mL methanol (HPLC grade) was added. Then the mixture was ultrasonically extracted at room temperature for 40 min. The solution was centrifuged at 2 500 r·min 1, at room temperature for 10 min, the supernatant was filtered through a syringe filter (0.45 ȝm) before HPLC analysis.2.4 HPLC proceduresChromatographic separation was carried out on an Agilent Eclipse Plus TM C18 column (150 mm × 3.0 mm, 3.5 ȝm) at 40°C. Elution was performed at a flow rate of 0.8 mL·min 1. Solvents used were acetonitrile (A) and 0.05% acetic acid in water (B). All solvents were filtered through a 0.45 ȝm nylon filter and then degassed by sonication in an ultrasonic bath prior to use. Gradient was as follows: 5% B at 0 min, 12% B at 3 min, 15% B at 8 min, 20% B at 20 min, 28% B at 24 min, 35% B at 28 min, 65% B at 32 min, 65% B at 35 min, 100% B at 50 min, and the injection volume of sample solution was 5 ȝL. The chromatograms were recorded at 270 nm.2.5 ESI-MS parameterAgilent 1100 HPLC-MSD Trap SL mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray ionization source was used in both positive and negative ion modes. The mass spectrometry detector (MSD) parameters were as follows: negative and positive ionization modes, scan range from m/z 100 to 1 000, desol-vent gas temperature 350 °C, capillary voltage 3.1 kV (posi-tive mode) and 3.5 kV (negative mode). Nitrogen was used as nebulizing gas at a pressure of 40 psi and the flow rate was adjusted to 9.0 lL/min. All the operations, data acquisition and analysis were controlled by Chemstation software (Agilent Technologies, Palo Alto, CA, USA).3 Results and discussion3.1 Optimization of HPLC–DAD–ESI-MS conditions and method validationPhotodiode array detector (DAD) was used in HPLC analysis and the optimum monitor wavelength at 270 nm was selected from the full range spectra. Several binary solvent gradients were compared with respect to separation efficiency of phenolic acids and flavonoids. Modifiers such as formic acid, acetic acid and phosphoric acid were added to the mo-bile phase to enhance peak resolution. After several trials, a gradient solvent system described in the experimental section with acetic acid as modifier was developed and a total of 17 flavonoids and phenolic acids were resolved within 30 min. Since a complicated gradient of elution was used, variation in retention time may happen. The repeatability was assessed by analyzing six independent extracts prepared from the same batch of herb, respectively. The RSDs of the retention time were lower than 0.15% (Table 1).The flavonoids and phenolic acids were analyzed in both positive and negative ionization mode. According to the lit-erature, the negative ion mode should be more selective and more sensitive than the positive ion mode in crude plant phytochemical analysis [25]. Although the pseudomolecular ion signals of all the components investigated were observed in negative ion mode, some of the diagnostic Retro-Diels-Alder (RDA) ions were only observed in the positive ion mode which is helpful for the structural determi-nation of the A- and B-ring substitution patterns. As a result, the combined application of negative and positive ion mode appeared to be necessary for the structural analysis of flavon-oids by mass spectrometry.3.2 Identification of flavonoids and phenolic acids in I. tectorum by HPLC–DAD– ESI-MS nThe dominant fragmentation pathways of authentic compounds were studied. All authentic compounds exhibited [M + H]+ ions in positive ion mode and [M – H]- in negative ion mode with sufficient abundances that could be subjected to MS2 and MS3 analysis. MS2 and MS3 data were obtained by collision-induced dissociation (CID), and utilized for the structural identification of compounds with similar fragmen-tation patterns. Comparing retention times and the MS n spec-tra with those of the authentic standards, eight peaks were unambiguously identified as androsin (2), tectoridin (5), iris-tectorin B (6), iristectorin A (7), iridin (8), tectorigenin (12), iristectorigenin A (14) and irigenin (17). Nine other peaks were tentatively identified as tectoruside (1), tectori-genin-7-O-ȕ-glucosyl-4'-O-ȕ-glucoside (3), apocynin (4), hesperetin (9), 5,7,3'-trihydroxy-6,4'-dimethoxyflavanone (10), genistein (11), rhamnocitrin (13), dihydrokaempferide (15) and iristectorigenin B (16) by comparing their MS data and UV spectra with those reported in the literature [10, 23-28].The total ion currents (TIC) together with HPLC chro-matograms of the samples are shown in Fig. 1, and the chemical structures of the compounds from 1 to 17 are shown in Fig. 2From the above results, isoflavonoids were identified as the major constituents in the rhizome of I. tectorum. Four isoflavone O-glycosides and five aglycones were identified. Peak 5 (tectoridin) was taken as an example to discuss the fragmentation pathways in detail. The molecule ion at m/z 463 in positive ion mode showed MS2 fragment ion at m/z 301, due to the loss of one glucose residue. In the following MS3 experiment, the loss of a methyl radical (15 Da) from [M + H – 162]+ was the predominant fragmentation, indicat-ing an methoxyl group linked at the aglycone. Furthermore, the ion at m/z 301 successively yielded the diagnostic ions of isoflavonoids at m/z 183, with the neutral loss of 118 Da produced by RDA fragmentation [26, 29], suggesting that the methoxyl group was attached to the A-ring. Therefore, peak 5 was identified as tectoridin by comparing its retention time and mass fragmentation pattern with those of the standards. The proposed fragmentation pathway in positive ion mode is given in Fig. 3. Similar fragmentation pathways were ob-served in the spectra of other isoflavonoids.Table 1 HPLC-DAD-ESI-MS n data of flavonoids and phenolic acids identified in the rhizome of Iris tectorum Maxim.Peak No. t R/minRSD oft R/%UV Ȝmax/nm[M+H]+(m/z)Fragment ions (+)[M–H]-(m/z)Fragment ions(-) Identi¿cation1 4.5 0.11 226, 270, 304 491 329, 167 489 373, 327, 165 tectoruside2 5.3 0.08 228, 270, 304 - - 327 283, 165, 150 androsin3 6.7 0.07 212(sh*),264,336(sh) - - 623 461,299tectori-genin-7-O-ȕ-glucosyl-4'-O-ȕ-glucoside4 9.4 0.13 232, 278, 304 - - 165 150, 122 apocynin5 13.2 0.13 214 (sh), 266,334(sh) 463 301, 286, 183 461 446, 428, 299, 284 tectoridin6 14.7 0.10 230(sh),266,340(sh) 493 331, 316, 298, 183, 168491 437, 331, 329, 314, 262 iristectorin B7 16.8 0.14 230(sh),266,340(sh) 493 331, 316, 299, 183, 168491 437, 331, 329, 314 iristectorin A8 17.5 0.14 238(sh), 268 523 361, 346 521 506,488,466,442,359,344 iridin9 25.7 0.03 214(sh), 294 - - 301 286,273,259,257,244,193,181, 179, 151, 124hesperetin10 26.6 0.03 212(sh), 266 - - 331 316, 313, 301, 274, 251,193, 1815,7,3'-trihydroxy-6,4'-dimethoxyflava-none11 26.7 0.02 271, 210 - - 269 212, 167, 152, 118 genistein12 27.3 0.05 214(sh),266,340(sh) 301 286, 229, 168, 159 299 284, 240, 212 tectorigenin13 27.9 0.04 218(sh), 282, 338 - - 299 284, 271, 255, 132, 120 rhamnocitrin14 28.1 0.03 216(sh), 268, 340(sh) 331 316, 301, 298, 242,186, 134329 314, 299, 271 iristectorigenin A15 28.5 0.05 220(sh), 292 - - 301 283, 273, 139 dihydrokaempferide16 28.9 0.04 224(sh), 268 331 316, 301, 298, 287,273, 243, 195329 314, 301, 289 iristectorigenin B17 29.2 0.04 234(sh), 268 361 346, 328, 310, 301,286, 271, 183 359 344,299 irigenin* shoulder peak - not observedreferred to as 5, 7, 3'-trihydroxy-6, 4'-dimethoxyflavanone likewise. According to the literature, the structures of known flavonol and flavanonol as well as three phenolic acids were also tentatively identified. Results of all the HPLC-DAD and MS n analyses are listed in Table 1.4 ConclusionIn this study, fourteen known flavonoids and three phe-nolic acids were identified in the rhizome of I. tectorum by using HPLC-DAD-ESI-MS n in both positive and negative ion modes. Isoflavones seem to be the major constituents ac-cording to our study. Two flavanones were identified from this species for the first time.This newly established method was successfully applied to simultaneously identify the major constituents in the rhi-zome of I. tectorum. The results were consistent to other phytochemical analyses, but it’s timesaving and simple com-pared with the traditional phytochemical method [2, 6-15]. Moreover, with the high sensitivity of the mass spectrum detector (MSD), some components with trace amounts were also identified, and thus a full-scale chemical profile could be obtained. Those phenols identified in I. tectorum could be considered as chemical markers of this species which might be the major bioactive constituents of I. tectorum. Further quantitative analysis method of those components should be developed for the quality control of this medicinal herb. References[1] Klingaman G. Plant of the week: Japanese roof iris, Latin:Iris tectorum, Division of Agriculture, University of Arkan-sas, Little Rock, Arkansas, USA [EB/OL]. 2005. Availablefrom: /plantoftheweek/ articles/iris_ japanese_roof_3-4-05.htm,[2] Seki K, Tomihari T, Haga K, et al. Iristectorene B, a mono-cyclic triterpene ester from Iris tectorum [J].Phytochemistry,1994, 36(2): 433-438.[3] Fang R, Houghton PJ, Hylands PJ. 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