1
1
THE
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STEENIS, A NEW SOURCE OF ZEAXANTHIN
FLOWER
OF
RADERMACHERA
IGNEA
(KURZ)
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Duangnapa Sompong1 and Pichaya Trakanrungroj1*
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School of Chemistry, Institute of Science, Suranaree University of Technology,
Nakhon Ratchasima, Thailand 30000. Fax: 66-44-224-185; E-mail: pnakkiew@sut.ac.th
*Corresponding author
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Abstract
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The flower of Radermachera ignea (Kurz) Steenis or “Peep Thong”, the
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emblem of Suranaree University of Technology, was selected for the study as
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a potential new source of antioxidant. The main antioxidant component was
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isolated from the ethyl acetate extract of the flower and its chemical structure
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was determined by spectroscopic data as zeaxanthin (β,β-carotene-3,3'-diol),
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one of the two significant antioxidant carotenoids present in the marcula
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lutea of the retina of human eyes. The antioxidant study confirmed the
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activity of the isolated compound with the IC50 of 1.13 mol antioxidant/mol
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DPPH. This is the first report to identify the bioactive constituent in this
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plant.
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Keywords: Radermachera ignea (Kurz) steenis, peep thong, antioxidant,
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carotenoids
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Introduction
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Oxidative damage process has been indicated as a major cause of several
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degenerative diseases including cancer, cardiovascular disease, diabetes,
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Alzheimer’s disease, and Parkinson’s disease (Ames et al., 1993; Banerjee et al.,
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2005). Antioxidants have been found to play an important role to prevent and
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inhibit such process (Ames et al., 1993).
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In the search for a new source of antioxidants, the flower of Radermachera
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ignea (Kurz) Steenis or commonly known as “Peep Thong”, the emblem of
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Suranaree University of Technology, was chosen to be the subject of interest
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because of its bright orange color, which normally suggests the presence of
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natural substances with antioxidant activity. R. ignea (Kurz) Steenis belongs to the
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family Bignoniaceae. The tree is evergreen or partly deciduous with 6-15 m high
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and typically scattered in several areas of Southeast Asian region.
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Neither the data about the chemical constituent nor the antioxidant activity
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of this plant has been previously reported in the literature. Therefore, this is the
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first paper to describe the isolation and characterization of the bioactive
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compound from the flower of R. ignea (Kurz) Steenis.
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Materials and Methods
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Plant Materials
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The flower of R. ignea was collected directly from the trees around the
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campus of Suranaree University of Technology, Nakhon Ratchasima, Thailand.
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The plant specimen was authenticated by the Forest Herbarium, the Office of
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Forest and Plant Conservation Research, National Park, Wildlife and Plant
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Conservation Department, Bangkok, Thailand.
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General Experimental Procedure
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Commercial grade solvents were distilled prior to use. Silica gel 60 (0.063-
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0.200 M) and silica gel 60 (0.040-0.060 M) for flash column chromatography
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were from Merck. Unless otherwise stated, all analytical grade solvents were from
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Carlo Erba. DPPH (1,1-Diphenyl-2-picryl-hydrazyl) and L-ascorbic acid were
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from Fluka and (+)-catechin was from Sigma.
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NMR spectra were measured by Varian Inova 600 MHz, Bruker DRX 600
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MHz, and DRX 500 MHz. All NMR spectra were recorded in CDCl3 ( in ppm, J
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in Hz) with tetramethylsilane (TMS) as an internal standard. Mass spectra were
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measured using ICP-MS techniques by Agilent, LC-MS Series 1100. Infrared
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spectra were taken as KBr pallet on Perkin-Elmer, FT-IR Spectrophotometer
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Model Spectrum GX. UV spectra were obtained in ethanol (abs.) on CARY
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Varian-1E.
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Extraction and Isolation Procedures
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The fresh flowers were cleaned, dried in the oven at 40oC, and ground into
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small pieces before used. Dried flowers of R. ignea weighing (meaning “which
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weighed”. Or you can stick with the original “weighed” but this means “which
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was weighed”.) 463 g were extracted with 3.0 L of 90% methanol with soxhlet
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extractor for 24 h. The solvent was removed by rotary evaporation yielding
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methanol crude extract. The obtained methanol crude extract was extracted
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consecutively by partition with hexane followed by ethyl acetate to yield ethyl
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acetate extract. The subsequent chromatographic separations were performed until
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the antioxidant component was obtained as illustrated in Figure 1.
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Antioxidant Study
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The antioxidant activity was determined from the scavenging activity
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against DPPH radicals. Antioxidant TLC-screening assay (Cuendet, 1997;
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Hostettmann, 1999) was used for directing the isolation process. The radical
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scavenging activity was evaluated by spraying the developed TLC plates with 0.2
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mM methanolic solution of DPPH. The quantitative antioxidant activity analysis
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was measured as the IC50 values by using a modified version of the method
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described by Jiménez-Escrig et al. (2000) using DMSO as the solvent to confirm
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the activity of the isolated compound. (+)-Catechin and ascorbic acid were used as
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the antioxidant standards.
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Results and Discussion
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Isolation of the Main Antioxidant Component from the Flower of R. ignea
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(Kurz) Steenis
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The main antioxidant component was isolated (see Scheme 1) by series of
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chromatographic separations guided by the antioxidant TLC-screening assay. The
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ethyl acetate crude extract was isolated by flash column chromatography using
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hexane-EtOAc (1:1) as the mobile phase. The fraction containing the main
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antioxidant was concentrated and purified by crystallization in EtOAc to yield a
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reddish orange powder weighing (As above) 0.0660 g (0.0143% based on the
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weight of dried plant material).
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Characterization of the Main Antioxidant Component in the Flower of R.
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ignea (Kurz) Steenis
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From the LC/APCI-MS spectrum, the compound showed the molecular
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ion [M+H]+ peak at m/z 569.4. FT-IR spectrum showed the absorption peaks at
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3435 cm-1(O–H stretching), 2921 cm-1 (C–H stretching), 1628 cm-1 (C=C
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stretching), 1384 cm-1 (CH3 bending), 1049 cm-1 (C–O stretching), 958 cm-1 and
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688 cm-1 (C=C–H out of plane bending). In the UV spectrum, the observed λmax
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were at 450 and 477 nm, which are the characteristic absorption peaks of
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carotenoids (Sajilata et al., 2008).
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The 1H and
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C data are presented in Table 1. The molecular ion peak,
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[M+H]+ peak at m/z 569.4, corresponded with (make sure you need Past
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Continuous here) the chemical formula of C40H56O2. Twenty carbon peaks in the
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carbons was assigned according to HSQC and HMBC spectra. The two equivalent
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carbons bearing hydroxyl groups showed a peak at  65.1. The eight methyl
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groups showed the
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were correlating (make sure you need Past Continuous here) to the 1H signals at 
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1.07s, 1.07s, 1.74s, 1.97s, and 1.97s, respectively. The NMR data are consistent
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with those of zeaxanthin (β,β-carotene-3,3'-diol; Figure 2) as previously reported
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by Moss (1976) and Eisenreich (2002).
C spectrum indicated molecular symmetry. Correlation between protons and
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C NMR signals at  30.3, 28.7, 21.6, 12.8, and 12.8, which
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In addition to zeaxanthin, the isolation yielded a mixture of two steroidal
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compounds. Their structures were characterized based on the NMR and LC/APCI-
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MS data as -sitosterol and stigmasterol.
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Antioxidant Study
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The result of the antioxidant study is presented in Table 2. The testing
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confirmed the antioxidant activity of the isolated zeaxanthin from the flower of R.
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ignea (Kurz) Steenis. The isolated compound exhibited the antioxidant activity
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with IC50 1.13 mol antioxidant/mol DPPH, which was about the same level as (+)-
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catechin; IC50 1.10 mol /mol DPPH, and was much stronger than ascorbic acid;
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IC50 304.30 mol /mol DPPH under the same condition.
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The Content of Zeaxanthin in the Flower of R. ignea (Kurz) Steenis
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The main antioxidant isolated from the flower of R. ignea (Kurz) Steenis
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was identified as zeaxanthin in its free (native) form. It has been widely known
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that zeaxanthin along with lutein are the only two carotenoids presented in the
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marcula lutea of the retina of human eyes (Roberts et al., 2009). Several studies
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suggested that zexanthin may help protect against the development of aged-related
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marcula degeneration (AMD) and aged-related cataract formation (Weller and
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Breithaupt, 2003; Roberts et al., 2009). Like other carotenoids, zeaxanthin cannot
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be produced in human body; therefore, its presence must be obtained primarily
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from dietary intake. Although lutein is abundant in green leafy vegetables and
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fruits, only trace amount of zeaxanthin is present in common diet (Lam and But,
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1999, Humphries and Khachik, 2003).
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One of the known rich sources of zeaxanthin is Gou Qi Zi or goji berry or
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wolfberry (Lycium barabarum), which is commonly used in Chinese cooking and
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traditional herbal medicine for preventing and treating of visual problems. The
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amount of the total zeaxanthin in wolfberries obtained by hydrolysis of its
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dipalmitate derivative was ranging from 2.4 to 82.4 mg/100 g of the dried berries
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while the free zeaxanthin amount was reported at 1.22 mg/100 g of the dried
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berries (Lam and But, 1999; Weller and Breithaupt, 2003). Comparing to the
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flower of R. ignea (Kurz) Steenis, the zexanthin obtained was 0.0143% or 14.3
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mg/100 g of the dried plant material, all of which was in the free zeaxanthin form.
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Typically, a commercial production process of zeaxanthin supplement products
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requires a saponification step, where esterified derivatives of zeaxanthin are
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hydrolyzed back to their native form using strong bases such as KOH. The rather
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high amount of zeaxanthin of its native form in the flower of R. ignea (Kurz)
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Steenis suggested that the plant could be a promising alternative source for this
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antioxidant carotenoid. The isolation of zeaxanthin directly from the plant would
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be an interesting shortcut in obtaining the pure compound without going through
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the expensive saponification and further purification steps.
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Conclusions
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Zeaxanthin was identified as the main antioxidant in the flower of R. ignea (Kurz)
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Steenis and presumably responsible for its bright orange color. The results from
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the study suggested that this local plant could potentially be a promising new
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source of zeaxanthin. Our future research efforts will focus on developing
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effective isolation and purification methods to maximize the yield of this
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antioxidant carotenoid from various local plants of this type.
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Acknowledgments
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Partial funding support for this work was provided by Suranaree University of
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Technology. We would like to thank Prof. Dr. Roberts Bates, Department of
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Chemistry, The University of Arizona, for accommodating the NMR facility; and
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Assoc. Prof. Dr. James R. Ketudate-Cairns, School of Biochemistry, Suranaree
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University of Technology, for assistance with the LC-MS analysis.
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References
177
Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993). Oxidants, antioxidants,
178
and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA,
179
90:7915-7922.
180
181
Banerjee, A., Dasgupta, N., and De, B. (2005). In vitro study of antioxidant
activity of Syzygium cumini fruit. Food Chemistry, 90:727-733.
182
Cuendet, M., Hostettmann, K., Potterat, O., and Dyatmiko, W. (1997). Iridoid
183
glucosides with free radical scavenging properties from Fagraea blumei.
184
Helv. Chim. Acta, 80:1144-1152.
185
Eisenreich, W., Bacher, A., Berry, A., Bretzel, W., Hümbelin, M., Lopez-Ulibarri,
186
R., Mayer A.F, and Yeliseev A. (2002). Biosynthesis of zeaxanthin via
9
187
mevalonate in Paracoccus species strain PTA-3335. A product-based
188
retrobiosynthetic study. J. Org. Chem., 67:871-875.
189
Hostettmann, K. (1999). Strategy for the biological and chemical evaluation of
190
plant extracts. The International Conference on Bioversity and
191
Bioresources: Conservation and Utilization, 23-37 November 1997,
192
Phuket, Thailand.
193
Humphries, J.M. and Khachik, F. (2003). Distribution of lutein, zeaxanthin, and
194
related geometrical isomers in fruit, vegetables, wheat, and pasta products.
195
J. Agric. Food Chem., 51:1322-1327.
196
Jiménez-Escrig, A., Jiménez- Jiménez, I., Sánchez-Moreno, C., and Saura-Calixto,
197
F. (2000). Evaluation of free radical scavenging of dietary carotenoids by
198
the stable radical 2,2-diphenyl-1-picrylhydrazyl. J. Sci. Food. Agric.,
199
80:1686-1690.
200
201
202
203
204
205
Lam, K-W. and But P. (1999). The content of zeaxanthin in Gou Qi Zi, a potential
health benefit to improve visual acuity. Food Chemistry, 67:173-176.
Moss, G.P. (1976). Carbon-13 NMR spectra of carotenoids. Pure Appl. Chem.,
47:97-102.
Roberts, R.L., Green, J., and Lewis, B. (2009). Lutein and zeaxanthin in eye and
skinhealth. Clinics in Dermatology, 27:195-201.
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Sajilata M.G., Singhal R.S., and Kamat M.Y. (2008). The carotenoid pigment
207
zeaxanthin—a review. Comprehensive Reviews in Food Science and Food
208
Safety, 7:29-49.
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Weller, P. and Breithaupt, D.E. (2003) Identification and quantification of
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zeaxanthin
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spectrometry. J. Agri. Food. Chem., 51:7044-7049.
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213
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esters
in
plants
using
liquid
chromatography–mass
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Table 1. 1H and
216
13C
NMR dataa of the main antioxidant isolated from the
flower of R. ignea (Kurz) Steenis
1H
Position
1, 1’
C
2, 2’
CH2
NMR (J = Hz)
- (q)
13C
NMR
37.1
1.77dd(12, 2.4),
48.4
1.48t(12)
3, 3’
CH
4, 4’
CH2
4.00
65.1
2.39dd(16.8, 5.4)
42.6
2.05dd(16.8,10.2)
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218
a
5, 5’
C
- (q)
126.2
6, 6’
C
- (q)
137.8
7,7’
CH
6.10d(16.8)
125.6
8, 8’
CH
6.13d(15.6)
138.5
9, 9’
C
- (q)
135.7
10, 10’
CH
6.16d(10.8)
131.3
11, 11’
CH
6.65dd(13.2, 10.8)
124.9
12, 12’
CH
6.63d(15)
137.6
13, 13’
C
- (q)
136.5
14, 14’
CH
6.25brd(9)
132.6
15, 15’
CH
6.64d(15)
130.1
16, 16’
CH3
1.07s
30.3
17, 17’
CH3
1.07s
28.7
18, 18’
CH3
1.74s
21.6
19, 19’
CH3
1.97s
12.8
20, 20’
CH3
1.97s
12.8
The NMR spectra were measured in CDCl3 using TMS as the internal references.
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Table 2. Antioxidant activity study of the isolated zeaxanthin from the flower
of R. ignea (Kurz) Steenis.
Compound
IC50
(mol antioxidant/mol DPPH)
zeaxanthin (isolated compound)
1.13
(+)-Catechin
1.10
Ascorbic acid
304.30
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Dried flowers of R. ignea (Kurz) Steenis
(463 g)
1. Soxhlet extraction with 3.0 L of 90% MeOH
2. Filtered and evaporated in vacuo
methanol crude extract
(140.62 g)
hexane
(1.08 g)
Extract with hexane
residue
Extract with EtOAc
ethyl acetate crude extract
(9.63 g)
residue
Flash CC
(hexane: EtOAc 1:1)
Fraction #1
Fraction #2
crystallization (EtOAc)
zeaxanthin (reddish orange powder)
(0.0660 g; 0.014% yield)*
orange-brown viscous liquid
(0.2743 g; 0.06% yield)
crystallization (EtOAc/CH3CN)
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mixture of -sitosterol and stigmasterol
(a mixture of white crystal and colorless needle)
(0.0165 g)
Figure 1. Extraction diagram of the flower of R. ignea (Kurz) Steenis
* based on the weight of the dried flower
residue
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227
18'
16
17
H3C
2
CH3
1
CH3
7
8
3
5
10
15
13
12
14
4'
OH
5
3'
14'
15'
12'
13'
CH3
10'
11'
8'
9'
CH3
228
229
HO
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Figure 2. Chemical structure of zeaxanthin (β,β-carotene-3,3'-diol)
4
CH3
CH3
11
9
6
H3C
18
1'
6
2'
7'
H3C
16'
CH3
17'