Determination of Polyphenol Components of Korean Prostrate Spurge (Euphorbia supina) by Using Liquid Chromatography—Tandem Mass Spectrometry: Overall Contribution to Antioxidant Activity

The Korean prostrate spurge Euphorbia supina is a weed that has been used in folk medicine in Korea against a variety of diseases. Nine polyphenols were characterized for this plant by using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and the results were compared with the literature data. The individual components were validated using the calibration curves of structurally related external standards and quantified for the first time by using the validated method. Correlation coefficients (r 2) were >0.9907. The limit of detection and limit of quantification of the method were >0.028 mg/L and 0.094 mg/L, respectively. Recoveries measured at 50 mg/L and 100 mg/L were 76.1–102.8% and 85.2–98.6%, respectively. The total amount of the identified polyphenols was 3352.9 ± 2.8 mg/kg fresh plant. Quercetin and kaempferol derivatives formed 84.8% of the total polyphenols. The antioxidant activities of the flavonoids were evaluated in terms of 1,1-diphenyl-2-picrylhydrazyl and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging activity, and the reducing power showed a dose-dependent increase. Cell viability was effectively suppressed at polyphenol mixture concentrations >250 mg/L.


Introduction
The Korean prostrate spurge Euphorbia supina is a weed that belongs to the Euphorbiaceae family and is native to North 2 Journal of Analytical Methods in Chemistry The plant has been used in folk medicine in Korea against a variety of conditions such as diarrhea and suppurated swelling and as a styptic [2]. It was reported that the plant contains a number of biologically interesting organic substances, including terpenoids [3,4], tannins, and polyphenols [5][6][7].
Of all bioactive natural constituents, polyphenols have attracted a great deal of interest, because they have beneficial effects to human health. Epidemiological studies have shown that polyphenols render many biological benefits, including a reduced risk of chronic diseases [8,9] and antioxidant, antiaging, and antimicrobial properties [10]. In the plants, polyphenols function as physiologically active substances, such as attractants, feeding deterrents, materials used to communicate with the surrounding environment, and materials used as defense against biotic and abiotic stresses [11,12]. Although the pharmaceutical efficacy of E. supina could be ascribed, at least partly, to the polyphenols, few studies have been conducted to validate this [3,13].
The objective of the present study was to comprehensively characterize the polyphenol metabolomes of Korean E. supina by using high-performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) and to investigate their biological benefits, including antioxidant and hepatoprotective effects. HPLC-MS/MS is a useful technique for analyzing plant polyphenols, because it provides online structural information and characterizes unknown substances even when no reference standards are available [14].

Materials and
Chemicals. E. supina was purchased in mid-April 2012 from a market in Jinju, South Korea. The plant was authenticated by Professor Moo Ryong Huh, a plant taxonomist with the Research Institute of Agricultural Life Science, Gyeongsang National University. A voucher plant was deposited in the herbarium at this institute. The plant was washed with water, lyophilized, and stored in dark containers at −70 ∘ C until needed. All chemicals were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Gallic acid, protocatechuic acid, 7-hydroxycoumarin, quercetin 3-O-glucoside, and kaempferol, which were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA), were used as external standards after recrystallization in ethanol. The purity of all standards was confirmed to be >99% by using HPLC. All solvents and water were obtained from Duksan Pure Chemicals Co., Ltd. (Ansan, Republic of Korea).

Extraction and Purification.
The lyophilized E. supina tissue (10 g) was ground into powder and extracted in ethyl acetate (300 mL) at 80 ∘ C for 20 h. The extract was filtered through a Büchner funnel and concentrated at reduced pressure under 40 ∘ C by using a rotator evaporator. The concentrated solution was washed with n-hexane (100 mL × 3), extracted with ethyl acetate (100 mL × 3), and dried over anhydrous sodium sulfate (Na 2 SO 4 ). The solvent was removed under reduced pressure. The sticky residue was placed on top of a silica gel sorbent (3 × 1.7 cm i.d.) and eluted using a mixture of methanol:dichloromethane (1 : 5, 25 mL). The solvent was removed to give a mixture of polyphenols (0.9% of the dried plant). The mixtures were reconstituted in ethyl acetate (0.03 g/mL), filtered through 0.45 m cellulose membranes, transferred into silanized vials, and stored at −20 ∘ C until analysis.

HPLC-MS/MS
. HPLC-MS/MS experiments were conducted according to a previously reported method [15], except for the use of a solvent system consisting of 0.5% aqueous formic acid (A) and methanol (B). The gradient conditions of the mobile phase were from 10 to 30% B over 10 min, increased to 90% B over 40 min, and increased again to 98% B over 5 min.

Quantification and Validation.
All components were quantified using chromatograms obtained at 254 nm. The quantification was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision.
The individual components for which standards were not available, except for gallic acid (1) and protocatechuic acid (2), were quantified using the calibration curves of structurally related external standards. Thus, nodakenin (3) was quantified as 7-hydroxycoumarin, quercetin derivatives (4, 5, 8) as quercetin 3-O-glucoside, and kaempferol derivatives (6,7,9) as kaempferol. Plant polyphenols can be quantified using a standard curve of structurally related compounds [16]. A stock solution of each standard (10 mg/L) was prepared by dissolving the appropriate amounts in methanol and storing at −20 ∘ C. Linearity was assessed using six different concentrations, (1, 10, 50, 100, 1000, and 2000 mg/L) of each standard and by plotting the concentration of the standard against the peak area. LOD and LOQ were determined by injecting each standard solution into the HPLC until the signal-to-noise ratio for the standards reached 3 : 1 and 10 : 1, respectively. The accuracy of the methods was estimated as recovery = A/IS-C/B/IS-C, where A is the peak area obtained for the analyte spiked preextraction, B is the area obtained for the analyte spiked after extraction, and C is the area of the blank extraction. The precision of the method was represented as a relative standard deviation (RSD).

Effects of the Polyphenol Mixture of E. supina on Hep3B
Cell Viability 2.6.1. Cell Viability Assay. Hepatic cancer cells (1 × 10 4 cells per well) were plated onto 12-well plates and treated

Statistical Analysis.
All statistical analyses were performed according to a method described previously [16].

Separation and Characterization.
A mixture of polyphenols was isolated from E. supina by methanol extraction at 80 ∘ C, followed by elution in ethyl acetate over a silica gel cartridge. The polyphenols were characterized through HPLC by using a C 18 column, MS/MS in negative-and positiveion modes, and comparison with the previous literature data.
The HPLC chromatograms of the plant are shown in Figure 1.

Antioxidant Activity.
The polyphenol mixture isolated from Korean E. supina was evaluated for its antioxidant effects. Contemporary interest in polyphenols focuses on the epidemiological association between their potent antioxidant properties and a low incidence of chronic diseases. Epidemiological studies have shown that oxidative stress plays an important role in the pathogenesis of various chronic diseases, including cancer, cardiovascular disease, atherosclerosis, hypertension, diabetes, neurodegenerative disorders, rheumatoid arthritis, and aging [29][30][31]. Polyphenols can reduce oxidative stress and thus might protect and/or retard disease development [32,33]; therefore, it is necessary to evaluate the antioxidant properties of the polyphenols in medicinal herbs.  Antioxidant capacity can be evaluated by using a number of in vitro methods. Because the assay results are method dependent, a combined assay involving several methods is often used [34]. In this study, the antioxidant activity of the polyphenol mixture isolated from E. supina was determined by DPPH • and ABTS •+ scavenging and RP assay at a concentration ranging from 25 to 500 mg/L. In DPPH • scavenging tests, antioxidant activity is monitored by measuring the disappearance of purple DPPH • , which can be detected spectrophotometrically at 517 nm [16]. In the ABTS •+ scavenging assay, the added antioxidants reduce the deep blue ABTS •+ to ABTS, and the decrease in absorbance of ABTS •+ at 414 nm is monitored [16]. The RP assay can also serve as an indicator of antioxidant activity. The added antioxidants convert the iron ion (Fe 3+ ) to Fe 2+ . The increase in absorbance of the deep-green Fe 2+ solution at ∼700 nm is monitored [16]. The assay results are provided in Table 4. The antioxidant capacity assayed using three methods showed a similar tendency. Thus, the antioxidant capacity of the E. supina polyphenol mixture showed a dose-dependent increase. DPPH • or ABTS •+ scavenging activity can be represented as an EC 50 value, which is the antioxidant concentration required to bring about a 50% loss in absorbance at 517 nm for DPPH • and 414 nm for ABTS •+ as determined by linear regression analysis [16]. RP can be represented as EC 0.3 , which is the reducing activity presented by the sample concentration at 0.3 of the absorbance value at 700 nm. Low EC 50 and RP values signify high antioxidant activity [33]. The DPPH • and ABTS •+ scavenging activity of butylated hydroxytoluene (BHT) as the control were 121.85 ± 0.39 mg/L and 93.85 ± 0.43 mg/L, respectively. The EC 0.3 value of BHT was 26.71 ± 0.69 mg/L. The antioxidant capacity values represented in terms of the DPPH • and ABTS •+ scavenging activity and RP value were lower than those of BHT ( < 0.05).

Growth Inhibitory and Morphological Effects of Polyphenol
Mixture of Korean E. supina. The anticancer activity of the polyphenol mixture of Korean E. supina was evaluated for human hepatocellular carcinoma Hep3B cells by MTT assay. The assay is a colorimetric method that measures cancer cell viability, quantifying the activity of the mitochondrial enzyme that reduces the yellow MTT molecule to purple formazan [34]. The cell line was incubated with serial concentrations of the polyphenol mixture ranging from 31.25 to 500 mg/L for 24 h and then subjected to MTT assays. The results are shown in Figure 3. The cell viability was decreased at polyphenol mixtures >250 mg/L, and the IC 50 value was 500 mg/L. After treatment with the polyphenol mixtures for 24 h, the morphological changes such as loss of cell adhesion and floating cell debris were also observed, as shown in Figure 4. The MTT assay results and morphological changes show that the polyphenol mixtures effectively suppressed cell viability.

Conclusion
Nine polyphenols from the Korean E. supina were profiled using a single HPLC-MS/MS run. The antioxidant activities of the flavonoids were evaluated in terms of DPPH •