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Identification of the Phenolic Components of Vernonia amygdalina
and Ruslia equitiformis
C.E. Johnson 1*, Long-Ze Lin 2, J. M. Harnly 2, F. O. Oladeinde 1, 3,4,
A.M. Kinyua 1, 6, R. Michelin 5, Y. Bronner 1 1
School of Community Health and Policy, Morgan State University, Baltimore, MD, USA. 2Food Composition and Methods Laboratory, Beltsville Human Nutrition Rearch Center,
Agricultural Rearch Service, USDA, Beltsville, MD, USA. 3Dept. of Chemistry, Morgan State University, Baltimore, MD, USA 4Dept. of Pharmaceutical Chemistry, University of Ibadan, Ibadan, Nigeria. 5 Dept. of Biology, Morgan State University, Baltimore, MD, USA. 6Dept. of Physics, Morgan State University, Baltimore, MD, USA.
* Corresponding Author上海中考科目
(Received 18 October 2010; Revid 21 October -1o December; Accepted 11 December 2010)
ABSTRACT The leaves of Vernonia amygdalina (Family-Compositae) and Ruslia
equitiformis (Family-Scrophulariacae), were analyzed using LC-DAD-MS to identify 26 phenolics components. Among them, 9 caffeoylquinic acids, verbascoside, isoverbascoside and 7 glycosylated flavonoids were positively identified. This is the first report of the existence of caffeoylquinic acids in Vernonia amygdalina and most of the flavonoids in Ruslia equitiformis .
Keywords : Caffeoylquinic acids; Glycosylated flavonols; LC-DAD-ESI/MS analysis.
INTRODUCTION
Vernonia amygdalina Del. is a vegetable found in some African countries. The leaves are ud in traditional African medicine and are reputed to have hepatoprotective, antioxidant, antibacterial, cytoprotective, antimalarial, and antidiabetic activities (Masaba, et al., 2000; Izevbigie, 2003; Iwalokun, et al., 2006; Ibrahim, et al., 2009; Ong, et al., 2010). Chemical studies on the bioactive components of this plant have led to the identification of some squiterpene lactones and steroids (Jisaka, et al., 1992; Erasto, et al., 2006). Ruslia equitiformis is a medicinal plant considered to have anti-inflammatory, analgesic and membrane stabilizing properties (Awe, et al., 2004; 2009). Two phenylethanoid glycosides of this plant, rusctinol and rusliaoside, were identified as its active constituents (Awe, et al., 2004; 2007).
The total phenolic content of both plants ud in this study was previously quantified in a previous study (Johnson, et al., 2008). The aim of this study was to identify their phenolic components using a standardized profiling method bad on
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liquid chromatography with diode array and electro-spray ionization mass spectrometric detection (L
C-DAD-ESI/MS) (Lin and Harnly, 2007; 2008).
MATERIALS AND METHODS
Chemicals : Chlorogenic acid from Sigma Chemical Co. (Saint Louis, MO, USA), apigenin 7-O-glucoside, vitexin and orientin from Extrasynthe (Genay Cedex, France), verbascoside, isoverbascoside, 1,4-, 1,5-dicaffeoyquinic acids from ChromaDex Inc. (Irvine, CA), and the isolated 3-, 4-caffeoylquinic acids, 3,5-, 3,4-, 4,5, 1,4-dicaffeoylquinic acids in the USDA laboratory (Lin & Harnly, 2007) were ud as the standards. Formic acid and HPLC solvents (acetonitrile, methanol) from VWR Scientific (Seattle, WA), and prepared HPLC grade water were ud for HPLC-MS analysis.
Plant materials : The leaves of Vernonia amygdalina and the whole plants of Ruslia equitiformis were collected from southwestern Nigeria during the rainy ason and identified using descriptions in literature (Hostettmann, et al., 2001). Authentication was done in the Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria and a voucher specimen is deposited. The plant materials were carefully air-dried indoors at room temperature for three days, dried in an oven between 40-50˚C; comminuted into small powders and pasd through 20 mesh sieves prior to the extraction experiments.
Plant extracts: Dried powdered Vernonia amygdalina leaves (200 mg) and Ruslia equitiformis whole plants (1000 mg) were extracted with methanol-water (5.00 ml, 60:40, v/v) by sonication at room temperature for 60 minutes. Extracts were filtered and a 50 µl sample of each was injected onto the analytical column for analysis.
冰箱风冷和直冷有什么区别LC-DAD and ESI-MS conditions: The LC-DAD-ESI/MS instrument and operating parameters have been previously described (Lin and Harnly, 2007). Briefly, the LC-DAD-ESI/MS consisted of an Agilent 1100 HPLC coupled to a diode array detector and mass spectrometer (MSD, SL mode) (Agilent, Palo Alto, CA). A 250 x 4.6 mm i.d., 5 µm Symmetry (or SymmetryShield) C18 column (C18, 5 µm,) with a 20 x3.9 mm i.d., 5 µm ntry guard column (Symmetry, 3.9 x 20 mm) (Waters Corp., Milford, MA) was ud at flow rate of 1.0 ml/min. The column oven temperature was t at 25°C. The mobile pha consisted of a combination of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The gradient was varied linearly from 10% to 26% B (v/v) in 40 minutes, to 65% B at 70 minutes. The DAD was t at 350, 310, and 270 nm to provide real-time chromatographic traces and the UV spectra were recorded from 190-450 nm for plant component identification. Mass spectra were simultaneously acquired using electro-spray ionization in the positive and negative ionization (PI and NI) modes at low and high fragmentation voltages (70 V and 250 V) over the range of m/z 100-2000.
RESULTS
Identification of the extract of Vernonia amygdalina : HPLC chromatograms (at 350 nm) of the aqueous methanol extract of Vernonia amygdalina leaves were recorded on both a Waters Symmetry column and a SymmetryShield column (Fig. 2A and B, respectively). The structures of the caffeoylquinic acids are shown in Figure 1. The retention times (t R ), wavelength of maximum absorbance (λmax ), deprotonated molecules ([M-H]-), and major fragment ions are listed in Table 1. In Table 1, the asterisk “*” denotes compounds identified by comparison to standards or references all other compounds were reported previously in the plant. The two columns provided
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iq题paration of the 14 peaks (Fig. 2A and 2B). Neither column, by itlf, provided satisfactory paration. The UV λmax at 240, 300sh, 326 nm spectra is typical for caffeic acid, suggesting that the compounds are caffeic acid conjugates. The mass suggest that they are quinic acid conjugates. Positive identification of the common isomeric caffeoylquinic acids was achieved by comparison to purchad or isolated dicaffeoylquinic acids and monocaffeoylquinic acids (Lin and Harnly, 2008). Positively identified 1-mono-, 1, 4,5-, and 1,3,5-tri-caffeoylquinic acids in arnica flower
and burdock roots, and 3,4,5-tricaffeoylquinic acid in sweet potato leaves were previously profiled using the standardized method, and 13 naturally occurring isomeric caffeoylquinic acids in plants have been recorded and stored in the collected food phenolic component databa in the USDA laboratory (Lin and Harnly, 2008). The compounds in Table 1 were identified by direct comparison of their retention time and UV and mass spectra with tho of the standard. Thus, it was possible to identify peaks 1, 2, 3 and 4 as 1-, 3-caffeoylquinic acid, chlorogenic acid (5-caffeoylquinic acid), and 4-caffeoylquinic acid ([M-H]- at m/z 353). Peaks 5-10 were identified as 1,3-, 1,4-, 3,4-, 3,5-, 1,5- and 4,5-dicaffeoylquinic acids ([M-H]- at m/z 515). Finally, peaks 11, 12 and 13 were identified as 1,4,5-, 1,3,5- and 3,4,5-tricaffeoylquinic acids ([M-H]- at m/z 677) and peak 14 was identified as feruloyldicaffeoylquinic acid ([M-H]- at m/z 691). The detection limit for the compounds is depended upon their concentration in
the extract and the injection volume. Thus, as mentioned in the Materials and Methods ction, the detected phenolics in the leaves have concentrations equal to or larger than 0.0005 % by dry weight, i.e., 0.005 mg/g of dried leaves. The 3,4-, 1,5-, 3,5-, and 4,5-dicaffeoylquinic acids (peak 7,8,9 and 10) and 3,4,5-tricaffeoylquinic acid (peak 14) are the main phenolics of the leaves and are prent at concentrations much greater than the detection limit.
Identification of the phenylethanoid glycosides and flavonoids in whole plants of Ruslia equitiformis : HPLC chromatograms (at 350 nm) of the aqueous methanol extract of the whole plant Ruslia equitiformis were recorded on Waters Symmetry column (Fig. 2C). The retention times, wavelengths of maximum absorbance (λmax ), protonated and deprotonated molecules ([M+H]+ and [M-H]-), and major fragment ions are listed in Table 2. The structures of the compounds are shown in Fig. 1. Eighteen peaks were obrved in the chromatograms of this plant. Bad on a direct comparison of the data in Table 2 with our collected data for standards and reference compounds (Lin and Harnly, 2008), 9 of the peaks were identified as phenylethanoids, and 9 as flavonoids. Peaks 11 and 13 were identified as verbascoside and isoverbascoside and peaks 2, 5, 6, 9, 12, 15 and 18 were identified as 6, 8-diglucosylapigenin, 6-arabinosyl-8-glucosylapigenin, orientin (8-glucosylluteolin), vitexin (8-glucosylapigenin), luteolin 7-O -glucuronide, apigenin O -glucuronide and apigenin, respectively. The remaining peaks were minor components and provisionally assigned as listed in Table 2. Bad on an extraction of 1g of plant material with 5.0 ml of solvent and an injection volume of 50 µl, the detection limit for each compound was equal to 0.0001 % (i.e., 1 mg/ kg of dry plant material) by dry weight. The major component verbascoside, however, was prent at much higher concentrations.
DISCUSSION
A literature arch revealed that caffeoylquinic acids as identified in Vernonia
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amygdalina , have many biological activities. In addition to the common antioxidant, radical-scavenging, and anti-inflammatory activities of plant phenolic compounds, the caffeoylquinic acids, especially 3,4,5-tricaffeoylquinic acid, have been reported to show antimutagenicity, anti-human cancer cell, and anti-human immunodeficiency virus activities (Yoshimoto, et al., 2002; Tamura, et al., 2006; Kurata, et al., 2007). Verbascoside was previously isolated as one of the active antinociceptive components from the leaves of Ruslia equitiformis and named rustinol (Awe, et al., 2007). Verbascoside was also reported to exist in many herbs with biological activities similar to many of the common plant phenolics (Bilia, et al., 2008). Further rearch is necessary to determine biological activity of the newly identified flavanoids in Ruslia equitiformis.
CONCLUSION
With the u of this method, 10 caffeoylquinic acids were detected in the aqueous methanol extract of Vernonia amygdalina leaves and 7 phenylethanoids and 9 glycosylated flavonoids were detected
in Ruslia equitiformis . This is the first report of the existence of caffeoylquinic acids and most of the flavonoids in the plants.
Acknowledgements: This work was supported by the Agricultural Rearch Service of the U.S. Department of Agriculture and an Interagency Agreement with the Office of Dietary Supplements of the National Institutes of Health, and a grant from National Center for Complementary and Alternative Medicine (NIH-NCCAM # 1-T32-TA01058-01). A special thanks to Dr. Janet Makinde for her contributions to this study.
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