Characterization of Chemical Components of ... - ACS Publications

Chapter 15

Characterization of Chemical Components of Ixeris denticulata 1

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Hang Chen , Shiming Li , Zhu Zhou , Naisheng Bai , and Chi-Tang Ho Downloaded by PENNSYLVANIA STATE UNIV on July 6, 2012 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch015

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Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 College of Pharmacy, Fudan University, Shanghai 200032, People's Republic of China 2

The extract of Ixeris denticulata was subjected to successive column chromatography to obtain pure compounds. Five compounds with potential bioactivities were isolated. They were identified as the flavonoids apigenin and luteolin, the sterols β-sitosterol and stigmasterol, and the chlorinated iridoid rehmaglutin D. With the exception of luteolin, the other compounds were reported from Ixeris denticulate for the first time. A systematic investigation on the structure-activity relationships among six flavonoids, namely, quercetin, kaempferol, catechin, epicatechin, apigenin and luteolin was also undertaken, with respect to antioxidant activity. The combined results of D P P H radical scavenging assay and O R A C antioxidant capacity assay indicated the importance of at least four structural elements to the antioxidant activity of these compounds.

© 2006 American Chemical Society

In Herbs: Challenges in Chemistry and Biology; Wang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Downloaded by PENNSYLVANIA STATE UNIV on July 6, 2012 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch015

196 There are about 50 species of Ixeris, a genus belonging to the family Compositae, across the world. Among these, 20 species are mainly distributed in Eastern and Western China. These plants are well known as wild vegetables in certain areas of China, and thus have been incorporated in the diet of the populations indigenous to these regions. Ixeris plants have also been widely used in Chinese and Korean as traditional folk medicine to treat many diseases. Ixeris denticulata is one of the most important species of Ixeris and is of particular interest to researchers (/). This plant was recently found to possess a wide spectrum of therapeutic properties, including antioxidant, cytotoxic, analgesic, antipyretic, anti-inflammatory, anti-tumor and anticancer activities (2,3). These findings validate the long and widespread use of Ixeris denticulata as a popular folk medicine in China. Thus, there is increasing interest in investigating the chemical components of Ixeris denticulata. M a and co-workers isolated three new sesquiterpene lactone glucoside, Ixerin X , Y and Z , as well as two known flavonoids, luteolin and luteolin-7-O-glucoside from Ixeris denticulata (4-6). Many of the biological properties of phytochemicals can be attributed, in large part, to their antioxidant activity and their ability to scavenge free radicals (7). Widespread awareness of the role of reactive oxygen species (ROS) in both the aging process and many chronic diseases has given rise to a high degree of public concern. Flavonoids have been reported to possess the ability to quench and scavenge ROS and various radicals (8). In this paper, we report the isolation and structure identification of bioactive compounds from Ixeris denticulata. In addition, the structure-activity relationships among several structure-related flavonoids are elucidated.

Materials and Methods Reagents Chloroform, ethyl acetate, methanol, hexane and acetone were purchased from Fisher Scientific (Pittsburgh, PA). 4-Hydroxy-3-methoxybenzyl-aldehyde (vanillin), l,l-diphenyl-2-picrylhydrazyl radical (DPPH), fluorescein, 2,2azobis-(2-amidinopropane) dihydrochloride (AAPH), quercetin, kaempferol, epicatechin and catechin standards were obtained from Sigma Chemical Co. (St. Louis, M O ) . 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Aldrich Chemical Co. (Milwaukee, WI). A l l chemicals were used without further purification.


197 Extraction


The fresh, whole Ixeris denticulata plants were collected by Dr. Zhu Zhou of the Pharmaceutical Institute in Shanghai, China. This material was dried, ground into powder and extracted three times with 95% ethanol for 3 weeks at room temperature. The ethanol extracts were pooled, filtered, concentrated and further extracted with aqueous hexane and ethyl acetate. The final extract was obtained upon drying of the ethyl acetate layer.

Column Chromatography The ethyl acetate extract was subjected to normal phase silica gel column chromatography. The solvent system employed to elute the compounds of interest consisted of mixtures of chloroform and methanol wherein the content of methanol increased with each successive volume (specifically, 1%, 5%, 10%, 20%, and 50%), and finally, 100% methanol. Fractions obtained from normal phase silica gel chromatography were screened for compounds of interest by thin layer chromatography (TLC) using a solvent system comprised of chloroform: methanol: water: acetic acid (500:100:1:1 (v/v)) and vanillin spray as the visualization agent. Fractions containing compounds of interest that were determined by T L C , to be impure were subjected to further purification by Sephadex LH-20 column chromatography with 95% ethanol as the mobile phase or normal phase silica gel chromatography. The above chromatographic procedures were repeatedly applied to the fractions until pure compounds were obtained.

Mass Spectrometry (MS) M S analysis was used to elucidate structure information of compounds of interest. The parameters of M S are the following. (1) Instruments: MicroMass AutoSpec HF (ΕΙ-MS) and MicroMass Platform II (ES-MS) (MicroMass Co., M A ) ; (2) Mass scan Range: 100-900 amu; (3) Scan rate: 0.4 sec (ES-MS); (4) Cone voltage: 36 Volts (ES-MS); (5) Corona voltage: 3.59 Κ Volts (ES-MS); (6) Source temperature: 250°C (ΕΙ-MS) and 150°C (ES-MS); (6) Mobile phase: 50/50 acetonitrile/water.


198 Nuclear Magnetic Resonance (NMR) !

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H N M R and C N M R were used to identify the structures of the compounds of interest. The spectra were obtained on Varian (300 M H z , 400 M H z and 500 MHz) instruments.


DP Ρ H Assay The l,l-diphenyl-2-picrylhydrazyl radical (DPPH) is a stable radical which exhibits a dark purple color in alcoholic solution and has a maximum absorbance at 517 nm. Interaction of this radical with antioxidants results in a decrease in its absorbance intensity, thus providing a basis for the measurement of the antioxidant activity of investigational compounds. A Model 301 spectrophotometer from Milton Roy (Ivyland, PA) was used to obtain absorbance measurements at 517 nm in the D P P H assay. Each test compound was added to a solution of D P P H radical in 3 mL of 95% ethanol to make a sample solution having 0.1 m M of D P P H and 1 m M of the test compounds. Samples were then shaken vigorously with a Vortex and left in the dark for 30 minutes at room temperature. The absorbance of the test samples at 517 nm was compared to that of a control lacking antioxidants (9). Each test compound (luteolin, apigenin, quercetin, kaempferol, epicatechin, catechin) was assayed in triplicate and the results were averaged. The % inhibition of the DPPH radical by each sample was calculated according to the formula of Yen and Duh (10). % Inhibition = [(A -A ) / A ] χ 100 0

t

0

(1)

ORAC Assay The oxygen radical absorbance capacity (ORAC) assay is a recently developed method that measures antioxidant scavenging activity against peroxyl radicals induced by 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) at 37°C. The underlying basis for this assay is the measurement of oxidative damage to a fluorescent probe caused by a peroxyl radical. The ability of a compound to inhibit this loss of fluorescence intensity is used as a measure of its antioxidant capacity against free radicals (11). The O R A C analysis was performed on a HITACHI F-3010 Fluorescence Spectrophotometer (Hitachi, Japan) with excitation and emission wavelengths of 493 nm and 515 nm, respectively. The fluorescence intensities of all standards and samples were monitored over the course of 60 minutes. Fluorescein working solution (8.16 χ 10" mM) was prepared daily with 75 m M phosphate buffer (pH=7.4). The 153 5



199 m M solution of A A P H was kept in an ice bath and discarded after eight hours. Trolox was used as a standard and diluted with 75 m M phosphate buffer (pH=7.4) to give a 10 μΜ Trolox working solution. Samples of pure flavonoids were prepared by dissolving in a 1:1 (v/v) mixture of acetone and water and then diluting with 75 m M phosphate buffer (pH 7.4) to a final concentration of 5 μΜ which was used for analysis. A mixture consisting of 3000 μ ί of working fluorescein solution, 500 μ ί of A A P H solution, 100 yL of 75 mM phosphate buffer (pH 7.4), and 400 μΐ, of sample/standard/blank solution was assayed by the Fluorescence Spectrophotometer. The fluorescence decay curve for each sample was generated by plotting the intensity data obtained by the instrument at each time point, and the final O R A C value of each standard and sample was calculated for comparison according to the formulas presented below: Antioxidant Capacity = A U C - AUC Relative O R A C Value = [ ( A U C - A U C b u ^ A U C ^ - AUC Molarity of Trolox/Molarity of Sample a n t i o x Î d a i U

(2)

b ! a n k

antioxidant

blank

)] x (3)

Results and Discussion Isolation of Compounds from Ixeris denticulate Upon fractionation of 120 g ethyl acetate extract of Ixeris denticulata by normal phase silica gel column chromatography, we identified three fractions of interest, via T L C , for further analysis. The first fraction of interest contained 277.6 mg of material. This fraction was subjected to further purification by successive column chromatographies to yield 4 mg of compound (1) and 13.9 mg of compound (5). Upon spraying with vanillin, compound (1) became yellow while compound (5) became red, indicating that they may be a flavonoid and a terpene, respectively. The second fraction of interest was dried and weighed 235.6 mg. It was determined by T L C to be primarily composed of a compound that became yellow upon spraying with vanillin, indicating that this compound may also be a flavonoid. 40.8 mg of compound (2) was obtained after purification by Sephadex LH-20 column chromatography. The final fraction of interest contained 1.59 g of material. Compound (3) (19.4 mg) and compound (4) (13.4 mg) were isolated from this fraction. Given a purple color was formed upon spraying with vanillin, these two compounds were identified as steroids.


200 Identification of Compounds Isolated from Ixeris denticulata A total of 5 compounds were isolated from Ixeris denticulata. The chemical structures of these compounds are shown in Figure 1, and the spectral data for each of the isolated compounds are listed and discussed below. Compound (1) was isolated as a white powder. We estimated the molecular formula for this compound from the results of ES-MS (m/z 271 [M+H] ) to be C i 5 H , o 0 The N M R data corresponding to compound (1) are as follows: Ή N M R (300 M H z in methanol-^): δ 6 . 2 1 (1H, s, H-6), 6.46 (1H, s, H-8), 6.60 (1H, s, H-3), 6.93 (2H, d,J=9 Hz, H-3',5'), 7.85 (2H, d, J =9 Hz, H-2',6' ). C N M R (300 M H z in DMSO-

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