Antioxidant Glucosylated Caffeoylquinic Acid Derivatives in the

Dec 13, 2012 - Antioxidant Glucosylated Caffeoylquinic Acid Derivatives in the Invasive Tropical Soda Apple, Solanum viarum. Shi-Biao Wu†, Rachel S...
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Antioxidant Glucosylated Caffeoylquinic Acid Derivatives in the Invasive Tropical Soda Apple, Solanum viarum Shi-Biao Wu,† Rachel S. Meyer,‡,§ Bruce D. Whitaker,*,⊥ Amy Litt,§ and Edward J. Kennelly†,‡ †

Department of Biological Sciences, Lehman College, 250 Bedford Park Boulevard West, Bronx, New York 10468, United States The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, New York 10016, United States § The New York Botanical Garden, 2900 Southern Boulevard, Bronx, New York 10458, United States ⊥ Food Quality Laboratory, Building 002, Room 117, Beltsville Agricultural Research Center-West, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States ‡

S Supporting Information *

ABSTRACT: Eggplant and related Solanum species contain abundant caffeoylquinic acid (CQA) derivatives. Fruit of the invasive species Solanum viarum Dunal contain numerous complex CQA derivatives, but only a few have been identified. The structures of two new compounds isolated from methanolic extracts of S. viarum fruit by C18-HPLC-DAD were determined using 2D NMR and MS data. Both include two 5-CQA molecules joined by glucose via ester and glycosidic linkages. The structures of compounds 1 and 2 (viarumacids A and B) are, respectively, 5-caffeoyl- and 3-malonyl-5-caffeoyl-[4-(1β-[6-(5caffeoyl)quinate]glucopyranosyl)]quinic acid. The antioxidant activities determined by ABTS•+ and DPPH• assays were in the order 1 > 2 > 5-CQA.

Solanum viarum Dunal is an aggressive pan-tropical invasive weed that has recently become problematic in the southern United States.1 It is toxic to mammals, inducing neuronal lesions.2,3 However, this species has also been reported to contain unusual phenolic compounds with therapeutic potential that may be useful for the natural products industry.4 Toxic compounds reported in S. viarum include steroidal glycoalkaloids such as solasodine, which coincidentally are useful precursors for the synthesis of cortisone and other therapeutic steroids.5 Four recently identified new phenolic compounds are all 6-sinapoylglucosyl derivatives of 5-O-(E)-caffeoylquinic acid (5-CQA).4 Caffeoylquinic acids such as 5-CQA are the most potent antioxidants in the genus Solanum,6 and anticarcinogenic activity has also been demonstrated.7−9 It has become increasingly important to find industrial applications of invasive species. The problematic and uncontrollable spread of S. viarum warrants further investigation of the therapeutic and economic potential of its abundant phytochemicals. The marked invasivity of this species may in fact partly result from its unique phenolic composition, as suggested by the finding that increased phenolic levels in eggplant (S. melongena) hybrids of S. viarum correlated with greater resistance to the fruit and shoot borer, Leucinodes orbonalis.10 In a recent survey of phenolics in fruits of Asian eggplant and related species, it was noted that the HPLC profile of fruit from a Chinese accession of S. viarum (Meyer 311) included two prominent peaks not seen in the profile of fruit from S. viarum accession PI319855 studied by Ma et al.4 Herein we report the isolation of these two compounds by C18-HPLC-DAD and © 2012 American Chemical Society and American Society of Pharmacognosy

their structural elucidation using 2D NMR and MS data. Compounds 1 and 2, new complex 5-CQA derivatives, were tested for radical scavenging activity relative to Trolox and 5-CQA in ABTS•+ and DPPH• assays. Profiling of hydroxycinnamic acid (HCA) conjugates in a methanolic extract of S. viarum Meyer 311 fruit tissue by C18HPLC-DAD revealed two unknown compounds with similar UV spectra showing maxima at 324 and 296 nm. Compounds 1 and 2 eluted after 5-CQA (21.6 min) at 25.6 and 26.9 min and composed 10.2% and 11.3%, respectively, of the total HCA conjugate peak area determined at 325 nm. Isolation of the two compounds from a concentrated methanolic extract of powdered freeze-dried tissue from mature unripe fruit was achieved by solid-phase extraction to obtain an enriched fraction followed by C18-HPLC-DAD separation and peak collection. Two rounds of HPLC isolation yielded 1.83 mg of compound 1 and 1.74 mg of compound 2 at 87% and 85% purity (by HPLC-DAD, 325 nm), respectively. Compound 1 was obtained as a colorless oil. Its HRESIMS showed the protonated molecular ion [M + H]+ (Table 1) corresponding to the formula C38H44O22. The UV spectrum of 1 showed absorption maxima at 324 and 296 nm. Two sets of aromatic ABX protons were indicated by the 1H NMR signals at δ 7.04, 6.79, and 6.95 and those at δ 7.17, 7.12, and 7.10 (Table 2).4,11 The 1H NMR Received: August 14, 2012 Published: December 13, 2012 2246

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and 38.5 (C-6‴). These assignments were determined from HSQC data. Moreover, there were two oxygenated quaternary carbons at δ 76.3 (C-1″) and 76.5 (C-1‴) and two carboxy resonances at δ 175.0 (C-7″) and 177.7 (C-7‴) in the 13 C NMR spectrum (Table 2), which confirm the presence of two quinic acid moieties. In the HMBC spectra, correlations between δ 5.36 (H-5″) and 168.5 (C-9), and between δ 5.32 (H-5‴) and 168.6 (C-9′), indicated that the two caffeoyl groups are linked at C-5″ and C-5‴ of the two quinic acid moieties (Figure 1). Chemical shifts of the six carbons at δ 103.4 (C-1⁗), 74.8 (C-2⁗), 77.6 (C-3⁗), 71.9 (C-4⁗), 75.8 (C-5⁗), and 66.0 (C-6‴′) in the 13C NMR spectrum (Table 2) indicated a 1⁗,6⁗-disubstituted hexose moiety. Acid hydrolysis of 1 with 2 M HCl afforded β-D-glucose, identified by HPLC-TOFMS analysis and co-elution with an authentic β-D-glucose standard (tR 7.12 min). A 7.8 Hz coupling constant for the anomeric proton confirmed the β-anomeric configuration. Linkages of D-glucose within the molecular structure were established by an HMBC experiment. The H-6⁗ proton at δ 4.32 correlated with the carbonyl carbon signal δ 175.0 (C-7″) of one of the quinic acid moieties, and the signal of the anomeric proton of glucose (H-1⁗) at δ 4.86 showed a 1 H−13C long-range correlation with a signal of one of the caffeoyl moieties at δ 148.8 (C-4′), indicating that C-6⁗ of glucose is linked to C-7″ of one quinic acid moiety by an ester bond and that C-1⁗ of glucose is ether-linked to C-4′ of one caffeoyl group (Figure 1). The HRESIMS fragmentation analysis data for 1 verified the structure assigned by NMR analysis (Table 1). When the aperture 1 voltage of the MS detector was set at 60 V, TOF-TOF MS/MS yielded the fragments m/z 679.1874 (C31H35O17, [M + H − quinic acid]+), 691.2039 (C29H39O19, [M + H − caffeic acid]+), 517.1567 (C22H29O14, [M + H − quinic acid − caffeic acid]+), and 355.1025 (C16H19O9, [M + H − quinic acid − caffeic acid − glucosyl group]+), which provided further proof of caffeic acids, quinic acids, and a sugar moiety in the structure. Thus, compound

doublets at δ 7.53, 6.22, 7.68, and 6.38 (Table 2), along with 1 H−1H COSY cross-peaks H-7/H-8 and H-7′/H-8′ (Figure 1), indicated four trans-oriented olefinic protons ( 3J = 15.9 Hz). HMBC experiments showed the correlations H-7/C-1, /C-4, /C-6, and/C-9 and H-8/C-1 and/C-9, in addition to the correlations H-7′/C-1′, /C-4′, /C-6′, and/C-9′ and H-8′/C-1′ and/C-9′ (Figure 1). Taken together, the above NMR data indicated that two caffeoyl groups exist in the structure. The presence of two quinic acid moieties was indicated by 1 H NMR resonances of six oxymethine protons at δ 4.18, 3.73, 5.36, 4.17, 3.74, and 5.32, together with four pairs of sp3 methylene protons at δ 2.10−2.22, 2.01−2.19, 2.12−2.22, and 2.01−2.18 (Table 2). By inspection of the 13C NMR and DEPT spectra, these resonances were in agreement with six oxymethine resonances at δ 71.8 (C-3″), 73.9 (C-4″), 72.5 (C-5″), 71.7 (C-3‴), 74.0 (C-4‴), and 72.4 (C-5‴), as well as with four sp3 methylene carbons at δ 39.2 (C-2″), 38.5 (C-6″), 39.1 (C-2‴),

Table 1. HRESIMS Fragmental Analysis of Viarumacids A (1) and B (2) (Positive Mode) compound

fragmental ions (m/z)

ion formulas

mDa difference

ppm difference

calcd mass

structures of fragments

1

875.2228 853.2391 679.1874 691.2039 517.1567 499.1469 355.1025 337.0990 961.2240 939.2422 921.2309 853.2338 777.2108 679.1967 603.1555 585.1502 517.1563 499.1438 441.1133 423.0929

C38H44O22Na C38H45O22 C31H35O17 C29H39O19 C22H29O14 C22H27O13 C16H19O9 C16H17O8 C41H46O25Na C41H47O25 C41H45O24 C38H45O22 C32H41O22 C31H35O17 C25H31O17 C25H29O16 C22H29O14 C22H29O14 C19H21O12 C19H19O11

0.6 −1.1 −0.2 −4.7 1.0 1.7 −0.4 6.7 1.4 1.6 0.8 −6.4 1.9 9.3 −0.6 4.6 0.6 −1.4 −0.2 0.2

0.7 −1.3 −0.3 −6.8 1.9 3.4 −1.1 19.9 1.5 1.7 0.9 −7.5 2.4 13.7 −1.0 7.9 1.2 −2.8 −0.5 0.5

875.2222 853.2402 679.1874 691.2086 517.1557 499.1452 355.1029 337.1557 961.2226 939.2406 921.2310 853.2402 777.2089 679.1874 603.1561 585.1456 517.1557 499.1452 441.1033 423.0927

[M+Na]+ [M+H]+ [M+H−Qa]+ [M+H−Cb]+ [M+H−Q−C]+ [M+H−Q−C−H2O]+ [M+H−Q−C−Gc]+ [M+H−Q−C−G−H2O]+ [M+Na]+ [M+H]+ [M+H−H2O]+ [M+H−Pd]+ [M+H−C]+ [M+H − Q − P]+ [M+H-Q − C]+ [M+H−Q−C−H2O]+ [M+H−Q−C−P]+ [M+H-Q−C−P−H2O]+ [M+H−Q−C−G]+ [M+H−Q−C−G−H2O]+

2

a

Q: quinic acid group. bC:caffeoyl group. cG: glucosyl group. dP:propanedioic acid group. 2247

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Table 2. 1H and 13C NMR Data for Viarumacids A (1) and B (2)a 1 position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″

δH (J in Hz)

2 δC

δH (J in Hz)

Caffeoyl Group A 127.8, s 7.04, 1H, d (2.1) 115.6, d 7.04, 1H, d (2.1) 147.3, s 149.7, s 6.79, 1H, d (8.4) 116.7, d 6.80, 1H, d (8.1) 6.95, 1H, dd (8.4, 2.1) 123.2, d 6.96, 1H, dd (8.1, 2.1) 7.53, 1H, d (15.9) 146.9, d 7.47, 1H, d (15.8) 6.22, 1H, d (15.9) 115.5, d 6.23, 1H, d (15.8) 168.5, s Caffeoyl Group B 131.5, s 7.17, 1H, d (2.1) 117.9, d 7.18, 1H, d (2.1) 148.7, s 148.8, s 7.12, 1H, d (8.4) 116.2, d 7.14, 1H, d (8.3) 7.10, 1H, dd (8.4, 2.1) 122.8, d 7.12, 1H, dd (8.3, 2.1) 7.68, 1H, d (15.9) 146.3, d 7.61, 1H, d (15.8) 6.38, 1H, d (15.9) 117.9, d 6.40, 1H, d (15.8) 168.6, s Quinic Acid Group A 76.3, s 2.10−2.22, 2H, m 39.2, t 2.10−2.22, 2H, m (overlapped) (overlapped) 4.18, 1H, m 71.8, d 4.18, 1H, m 3.73, 1H, dd (9.0, 4.1) 73.9, d 3.72, 1H, dd (9.0, 4.1)

1 δC

position

127.8, 115.6, 147.5, 149.8, 116.7, 123.3, 146.8, 115.2, 168.4,

s d s s d d d d s

131.4, 117.8, 148.7, 148.8, 116.1, 122.5, 146.3, 117.8, 168.6,

s d s s d d d d s

δH (J in Hz)

2 δC

δH (J in Hz)

δC

Quinic Acid Group A 5″ 5.36, 1H, m 72.5, d 5.37, 1H, m 72.5, d 6″ 2.01−2.19, 2H, m 38.5, t 2.01−2.18, 2H, m 38.4, t (overlapped) (overlapped) 7″ 175.0, s 175.0, s Quinic Acid Group B 1‴ 76.5, s 76.3, s 2‴ 2.12−2.22, 2H, m 39.1, t 2.10−2.23, 2H, m 35.7, t (overlapped) (overlapped) 3‴ 4.17, 1H, m 71.7, d 5.30, 1H, m 73.6, d 4‴ 3.74, 1H, dd (8.8, 4.2) 74.0, d 3.94, 1H, dd (8.0, 3.8) 70.0, d 5‴ 5.32, 1H, m 72.4, d 5.40, 1H, m 72.3, d 6‴ 2.01−2.18, 2H, m 38.5, t 2.01−2.18, 2H, m 38.5, t (overlapped) (overlapped) 7‴ 177.7, s 177.4, s Glucose Moiety 1‴′ 4.86, 1H, d (7.8) 103.4, d 4.89, 1H, d (7.5) 103.2, d 2‴′ 3.52, 1H, dd (9.3, 7.2) 74.8, d 3.55, 1H, dd (8.0, 7.5) 74.8, d 3‴′ 3.46, 1H, dd (9.3, 7.8) 77.6, d 3.48, 1H, dd (8.0, 7.9) 77.4, d 4‴′ 3.39, 1H, dd (7.8, 7.7) 71.9, d 3.38, 1H, dd (7.9, 7.7) 71.7, d 5‴′ 3.74, 1H, m 75.8, d 3.75, 1H, m 75.7, d 6a⁗ 4.32, 1H, dd (11.7, 6.9) 66.0, t 4.34, 1H, dd (12.3, 7.0) 66.2, t 6b⁗ 4.50, 1H, dd (11.7, 1.8) 4.49, 1H, dd (12.3, 1.8) Propanedioic Acid Group 1a 168.0, s 2a 3.36, 2H, s 50.2, t 3a 170.5, s

76.4, s 39.0, t 71.7, d 73.7, d

a Assignments were based on 1H−1H COSY, HMQC, and HMBC experiments. Samples were in methanol-d4. 1H and 13C scans were at 300 and 75 MHz, respectively.

they share the same skeleton. However, compound 2 includes a propanedioic acid moiety linked with quinic acid, which is supported by its 13C NMR data and molecular formula. Moreover, from the TOF-TOF MS/MS data (Table 1) obtained under identical conditions, the fragment ions from 2 were similar to those from 1. However, the presence of the m/z 853.2338 (C38H45O22, [M + H − propanedioic acid]+), 679.1967 (C31H35O17, [M + H − quinic acid − propanedioic acid]+), and 517.1563 (C22H29O14, [M + H − quinic acid − caffeic acid − propanedioic acid]+) ions indicated compound 2 includes a propanedioic acid group. Additionally, the ions at m/z 777.2108 (C32H41O22, [M + H − caffeic acid]+), 603.1555 (C25H31O17, [M + H − quinic acid − caffeic acid]+), and 441.1133 (C19H21O12, [M + H − quinic acid − caffeic acid − glucosyl group]+) further confirmed that 2 has quinic acid, caffeic acid, and a glucosyl group. Linkage of the propanedioic acid moiety in the molecular structure was established via the HMBC correlation between H-3‴ (δ 3.46) and C-1a (δ 168.0), indicating that the propanedioic acid moiety is linked at C-3‴ by an ester bond (Figure 1). Therefore, compound 2 was identified as 1β,4β-dihydroxy-3β-carboxyacetoxy-5α-{[3-[4-[1β-(6-O-(5-(E)caffeoylquinic acid)-β-D-glucopyranosyl)oxy]-3-hydroxyphenyl](E)-1-oxo-2-propenyl]oxy}cyclohexanecarboxylic acid, which was named viarumacid B. Among the reported natural derivatives of 5-CQA, the 6-sinapoylglucosyl conjugates previously isolated from S. viarum fruit are structurally the most similar to viarumacids A and B. Specifically, two of the four compounds identified by Ma et al.4 are analogues of viarumacids A and B, wherein the 6-hydroxy group of the glucose moiety is ester linked to the carboxy group

Figure 1. Key 1H−1H COSY () and HMBC (→) NMR correlations used in structural elucidation of the new glucosylated caffeoylquinic acid derivatives.

1 was determined to be 1β,3β,4β-trihydroxy-5α-{[3-[4-[1β-(6-O(5-(E)-caffeoylquinic acid)-β-D-glucopyranosyl)oxy]-3-hydroxyphenyl]-(E)-1-oxo-2-propenyl]oxy}cyclohexanecarboxylic acid (IUPAC numbering), which was named viarumacid A. For compound 2, a prominent sodium adduct of the molecular ion in the HRESIMS spectrum, [M + Na]+, gave the molecular formula C41H46O25 (Table 1). The UV spectrum of 2 showed maxima at 325 and 296 nm. The 1H and 13C NMR spectra of 2 were similar to those of 1 (Table 2), indicating that 2248

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Standards and Reagents. 5-O-(E)-Caffeoylquinic acid (5-CQA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 1,1diphenyl-2-picrylhydrazyl (DPPH), and HPLC grade MeOH were purchased from Sigma-Aldrich (St. Louis, MO, USA). Diammonium 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was from TCIAce (Tokyo, Japan). Ultrapure H2O was prepared using a MilliRO 12 Plus system (Millipore Corp., Bedford, MA, USA). Plant Material. Seeds of Solanum viarum accession 311, collected by one of the authors (R.S.M.) in China, were obtained from The New York Botanical Garden DNA bank.13 Seeds were germinated indoors, and two plants were transferred to an outdoor bed at the Calder Research Center in Armonk, New York, where they were grown in the summer of 2009. On the basis of morphological features, the plants were confirmed to be S. viarum by Dr. Michael Nee, curator of Solanaceae at The New York Botanical Garden. For further confirmation, DNA was isolated and the nuclear ribosomal internal transcribed spacer was sequenced according to Meyer et al.,13 which showed Meyer 311 (GenBank ID JQ638805) to be a 100% match to S. viarum accession AY561275 used in the Solanum phylogenetic study by Levin et al. (2006).14 Two fruit from each plant of Meyer accession 311 were harvested at the mature unripe stage, i.e., full size but mottled green and white in color rather than yellow. The four fruit were pooled as a single sample, frozen in liquid N2, and stored at −80 °C. The frozen tissue was lyophilized and powdered with a mortar and pestle prior to extraction. Fruit Tissue Extraction and Isolation of Compounds. Two 1.0 g samples of the lyophilized, powdered tissue were each extracted twice with 30 mL of MeOH−H2O, 4:1, for 30 min using a magnetic stir bar in a 50 mL screw-cap tube that was sealed after flushing with N2. After centrifugation for 5 min at 1500g, the supernatant was vacuum filtered through a sintered glass funnel fitted with a glass fiber disk. The combined extracts were reduced to 20 mL by N2 evaporation at 40 °C, and two 10 mL portions were fractionated on 500 mg Strata X polymeric solid-phase extraction (SPE) tubes (Phenomenex, Torrance, CA, USA) with a step gradient of 25%, 40%, and 60% aqueous MeOH (10 mL each).11 Analysis of aliquots by C18-HPLC-DAD showed the 40% MeOH SPE fraction was enriched in compounds 1 and 2. This fraction was processed to yield two 1.2 mL samples in 10% aqueous MeOH plus 0.02% H3PO4, which were injected in 80 μL portions onto a Phenomenex Luna C18(2) column (5 μm particle size, 250 mm long, 4.6 mm i.d.) for isolation of compounds 1 and 2 using an HP 1100 Series HPLC system (Agilent Technologies) and a binary gradient consisting of 0.02% H3PO4 in H2O and MeOH as previously described by Ma et al.4 DPPH Radical (DPPH•) Scavenging Activity. DPPH• scavenging activity was quantified according to Ma et al.11 Five 1:2 serial dilutions of each sample made from a stock solution of 250 μg/mL were used to calculate the standard curve. Two technical replicates were performed. In addition to Trolox, 5-CQA was included as a reference standard. Activity was reported as Trolox equivalent antioxidant capacity (TEAC, μM Trolox/μM compound) after incubation for 30 min. ABTS Radical (ABTS•+) Scavenging Activity. ABTS•+ scavenging activity was quantified as described in Ma et al.11 Six 1:2 serial dilutions were made from a stock solution of 500 μg/mL, and these were used to calculate the standard curve. Seven technical replicates were performed. Activity was reported in TEAC values at 5 min intervals from 0 to 40 min. Viarumacid A (1): C38H44O22, colorless oil; [α]20D −45.0 (c 0.020, MeOH); IR νmax cm−1 3305, 2945, 2823, 1727, 1599, 1450, 1409, 1106, 1025, and 604; UV (MeOH), λmax (log ε) 324 (4.26), 296 (4.06); HRESIMS (negative) m/z 851.2282 ([M − H]− calcd for C38H43O22, 851.2246), (positive) see Table 1; 1H NMR (methanol-d4, 300 MHz) and 13C NMR (methanol-d4, 75 MHz) data, see Table 2. Viarumacid B (2): C38H44O22, colorless oil; [α]20D −54.2 (c 0.048, MeOH); IR νmax cm−1 3313, 2945, 2823, 1726, 1598, 1446, 1409, 1115, 1017, and 620; UV (MeOH), λmax (log ε) 325 (4.19), 296 (4.00); HRESIMS (negative) m/z 937.2253 ([M + H]+ calcd for C41H45O25, 937.2250), positive please see Table 1; 1H NMR (methanol-d4, 300 MHz) and 13C NMR (methanol-d4, 75 MHz) data, see Table 2.

of sinapic acid rather than to the carboxy group of quinic acid in 5-CQA. Concerning the latter linkage, it appears that esterification of quinic acid to glucose or other saccharides is extremely rare; a 6-quinylglucoside of the flavonol herbacetin from Ephedra alata12 is the only example we found in the natural products literature. Antioxidant activity was quantified for viarumacids A and B using the DPPH• and ABTS•+ radical scavenging assays. Because viarumacids A and B both include two 5-CQA moieties linked by glucose, 5-CQA was included in the assays as a reference compound in addition to Trolox. The TEAC values ± SD from the DPPH• assay were 1.707 ± 0.013, 2.471 ± 0.052, and 2.158 ± 0.043, respectively, for 5-CQA, viarumacid A, and viarumacid B. The ABTS•+ assay (Table 3) gave similar results to the DPPH• assay. Table 3. ABTS•+ Results Reported in Trolox Equivalent Antioxidant Capacity (TEAC)a 5-CQA

viarumacid A (1)

viarumacid B (2)

time (min)

TEAC

SD

TEAC

SD

TEAC

SD

0 5 10 15 20 25 30 35 40

1.44 1.67 1.68 1.60 1.72 1.65 1.71 1.72 1.73

±0.17 ±0.12 ±0.09 ±0.05 ±0.10 ±0.07 ±0.06 ±0.05 ±0.04

1.51 2.71 3.01 3.18 3.64 3.73 3.71 3.81 3.89

±0.26 ±0.15 ±0.22 ±0.34 ±0.17 ±0.37 ±0.16 ±0.16 ±0.16

−0.31 1.61 1.85 2.10 2.26 2.49 2.42 2.50 2.56

±0.25 ±0.21 ±0.21 ±0.18 ±0.22 ±0.28 ±0.18 ±0.19 ±0.20

a

Technical replicates: 7.

In both cases, viarumacid A was a better scavenger than viarumacid B. Activity observed for viarumacid A in the ABTS•+ assay was roughly twice that of 5-CQA, suggesting that the caffeic acid moieties contribute most or all of the activity (Table 3). The roughly 1.5-fold greater values for viarumacid A compared with B suggest that the malonyl group in B results in reduced activity. Another factor that distinguished the viarumacids from 5-CQA was the rate at which maximum activity was attained after initiation of incubation with the ABTS•+ radical: 5-CQA activity showed an immediate sharp increase and plateaued at 5 min, whereas activities of viarumacids A and B increased asymptotically, rising rapidly from 0 to 5 min and reaching a plateau by 20 to 25 min (Table 3). In summary, these two new compounds that include two 5-CQA moieties as part of their structure have antioxidant potentials exceeding those of 5-CQA and Trolox. Moreover, the protracted radical scavenging activity of the viarumacids compared with 5-CQA merits investigation into whether these new 5-CQA derivatives exert different biological activities potentially useful for the natural products industry.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on an AUTOPOL III polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) equipped with a sodium lamp (589 nm) and a 10 cm microcell. UV and IR spectra were obtained on a UV-2450 spectrometer (Shimadzu, Japan) and a Nicolet iS10 spectrometer (Thermo Scientific, Waltham, MA, USA), respectively. A Bruker Avance 300 NMR spectrometer, equipped with bbi (for 1H and 2D) and bbo (for 13C) probes, was operated at 300.1312 MHz for 1H and at 75.4753 MHz for 13C NMR experiments. HRESIMS was performed using an LCT Premier XE TOF mass spectrometer (Waters, Milford, MA, USA) equipped with an ESI interface and controlled by Masslynx V4.1 software. 2249

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Note

ASSOCIATED CONTENT

S Supporting Information *

1 H and 13C NMR, 1H−1H COSY, HSQC, HMBC, and HRESIMS spectra of compounds 1 and 2, a diagram of HRTOFMS fragmentation of compounds 1 and 2, and a C18-HPLC-DAD chromatogram of hydroxycinnamic acid conjugates in a methanolic extract of S. viarum 311 fruit tissues are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 301-504-6984. Fax: 301-504-5107. E-mail: bruce. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. G. Subramaniam, Department of Chemistry and Biochemistry, Queens College, CUNY, for acquisition of the UV spectra and optical rotation data, and Mr. S. Elhakem, Department of Chemistry, Lehman College, CUNY, for acquisition of the IR spectra. The authors also thank Mrs. M. Fredrick for her language assistance.

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REFERENCES

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