Quillajasides A and B: New Phenylpropanoid Sucrose Esters from the

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Quillajasides A and B: New Phenylpropanoid Sucrose Esters from the Inner Bark of Quillaja saponaria Molina Christiane Maier,† Jürgen Conrad,‡ Christof B. Steingass,# Uwe Beifuss,‡ Reinhold Carle,#,§ and Ralf M. Schweiggert*,# †

Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany ‡ Department of Bioorganic Chemistry, Institute of Chemistry, University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany # Department of Plant Foodstuff Technology and Analysis, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, 70599 Stuttgart, Germany § Department of Biological Sciences, King Abdulaziz University, P.O. Box 80257, Jeddah 21589, Saudi Arabia ABSTRACT: The phenolic composition of freshly prepared aqueous extracts of the inner bark of Quillaja saponaria Molina was compared to that of commercially available Quillaja extracts, which are currently used as emulsifiers in foods and cosmetics. Major phenolics in both extracts were (+)-piscidic acid and several p-coumaroyl sucrose esters. Among the latter, two new compounds were isolated and characterized: α-L-rhap-(1→4)-α-L-rhap-(1→3)-(4-O-(E)-p-coumaroyl)-α-D-glup-(1→2)-(3-O(E)-p-coumaroyl)-β-D-fruf (quillajaside A) and β-D-apif-(1→4)-α-L-rhap-(1→4)-α-L-rhap-(1→3)-(4-O-(E)-p-coumaroyl)-α-Dglup-(1→2)-(3-O-(E)-p-coumaroyl)-β-D-fruf (quillajaside B). In addition, a putative biosynthetic pathway of at least 20 structurally related p-coumaroyl sucrose esters was tentatively identified. Besides their antioxidant activity and their potential function as substrate for enzymatic browning reactions, the new compounds are highly characteristic for both the inner bark of Q. saponaria and commercial extracts derived therefrom. Consequently, they might serve as authenticity markers for the detection of Quillaja extracts in food and cosmetic formulations. KEYWORDS: Quillaja saponaria Molina, saponin, polyphenol, browning, MS/MS, NMR spectroscopy

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INTRODUCTION The inner bark of the soapbark tree (Quillaja saponaria Molina) is naturally rich in highly surface-active saponins. Aqueous extracts of the bark or wood have therefore been used as soaps by indigenous inhabitants of Chile since pre-Columbian times (quillean = “to wash” in the Mapuche language).1 Today, Quillaja saponins, mostly being olean-12-ene-type saponins such as quillaic acid, are used as adjuvants in vaccines and pharmaceutical formulations and as emulsifiers in foods, beverages, and cosmetics.2−4 Although the chemical structures of the emulsifying saponins have been elucidated in detail as summarized by van Setten and van de Werken,3 knowledge about the phenolic compounds in different commercial saponin-rich Q. saponaria extracts is scarce, despite their importance for potential browning reactions and the antioxidant potential of the extracts. In our previous paper,5 total polyphenol contents of up to 4.6% (w/w, dry matter (DM)) were determined by HPLC-PDA in several commercial extracts, and (+)-piscidic acid was shown to be the major phenolic compound (75−87% w/w of the total phenol content). Besides this uncommon phenolic acid, two previously unidentified derivatives of p-coumaric acid were the most abundant phenolic compounds. We now report the full structural elucidation of these compounds, which represented 6.2−18.8% (w/w) of the total phenolics according to our previous HPLC-PDA-MSn analyses.5 After isolation by preparative HPLC, the structures of these previously unknown compounds were elucidated by HRESI-MS, HPLC-PDA-MSn, GC-MS, and NMR spectroscopy. In © XXXX American Chemical Society

addition, several less abundant but structurally highly related compounds were detected in the inner bark as well as in commercial extracts derived therefrom, indicating that the described phenolics may be only two representatives of a class of rhamnosylated p-coumaroyl sucrose esters, which were so far only found in Q. saponaria and commercial products obtained therefrom.

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MATERIALS AND METHODS

Materials. All solvents used were at least of analytical or HPLC grade. They were purchased from VWR (Darmstadt, Germany) unless stated otherwise. L-Cysteine methyl ester, L-glucose, L-rhamnose, Lfructose, p-coumaric acid, and the silylation mixture (10% (v/v) hexamethyl disilazane/trimethyl chlorosilane (HMDS/TMCS, 2:1, v/ v) in pyridine) were obtained from Sigma-Aldrich (St. Louis, MO, USA); D-glucose, hexane, and trifluoroacetic acid were purchased from Merck (Darmstadt, Germany). D-Apiose and D-rhamnose were obtained from Carbosynth (Compton, UK). DMSO-d6 and pyridined5 were purchased from Deutero (Kastellaun, Germany). The “Andean QDP ultra organic” (batch 1718-0211-PSD-06 ORG) commercial Quillaja extract and natural inner bark (harvested in Chile in September 2010) were generously provided by Pera Ingredients (Springe-Eldagsen, Germany). A voucher specimen (no. 2015-CMQS-001) has been deposited at the chair Plant Foodstuff Technology Received: July 20, 2015 Revised: September 16, 2015 Accepted: September 16, 2015

A

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry and Analysis of the University of Hohenheim. Ultrapure water was used throughout the study (Milli-Q system, Millipore, France). HPLC-PDA-ESI/MSn Analyses. The inner bark of Q. saponaria Molina was crushed with a food processor until a fine powder was obtained. An aliquot of 3.8 g of the sample powder was combined with 20 mL of water, the pH was adjusted to pH 2 with diluted formic acid, and the mixture was stirred at room temperature for 12 h in the dark under nitrogen atmosphere. Samples were consecutively filtered through a folded filter (5−8 μm, cellulose) and a membrane filter (0.45 μm, regenerated cellulose) to obtain the crude bark extract. In contrast to the crude bark extract, the commercial Quillaja extract was dissolved in 1% (v/v) aqueous formic acid and membrane filtered (0.45 μm, regenerated cellulose). Subsequent HPLC-PDA-ESI/MSn analyses were performed as previously described by Maier et al.5 Preparative Isolation of p-Coumaroyl Sucrose Esters. Due to the availability of larger quantities, the compounds of interest were only isolated from commercial Quillaja extracts. The similarity of the isolated compounds to those found in the natural bark was verified by HPLC-PDA-MSn. Preparative isolation of phenolic constituents from Quillaja extracts was conducted as previously described by Maier et al.5 Briefly, the targeted phenolic compounds present in the abovedescribed extracts were separated with an Ultimate 3000 preparative HPLC system equipped with two solvent delivery modules, a diode array detector (DAD-3000), and an automatic fraction collector (AFC3000). The separation column was a preparative 250 × 21.1 mm i.d., 5 μm particle size, 125 Å pore size, Phenomenex Aqua C18 reversedphase column. The used eluents were water (eluent A) and methanol (eluent B), both being acidified to pH 2 with formic acid. Separation was started by isocratic elution at 20% B for 10 min, followed by a linear gradient from 20 to 40% B over 18 min. After isocratic elution at 40% B for 20 min, a linear gradient from 40 to 100% B within 2 min was applied. After 8 min of isocratic elution at 100% B, a linear gradient to 20% B was applied within 2 min, and the system was equilibrated at 20% B for 10 min. Total run time was 70 min at a constant flow rate of 8 mL/min. The injection volume was 1000 μL. The retention times of the compounds to be isolated, 14 and 15 (Table 1), were 40.2 and 41.0 min, respectively. Compounds were monitored at 280 and 310 nm. After solvent evaporation under reduced pressure (Tmax = 40 °C), the collected fractions were freezedried and stored at −20 °C until further use. Aliquots of the isolated compounds were dissolved in aqueous 4 N HCl and heated to ∼85 °C for 120 min. After subsequent neutralization with aqueous NaOH, the hydrolysate was membrane filtered (0.45 μm) and analyzed by HPLCPDA-MSn as described above. High-Resolution ESI-MS Analyses. High-resolution ESI-MS analyses of the isolated compounds 14 and 15 were performed using a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an API ion source and a HESI-II probe. The instrument was operated in negative ionization mode and was externally calibrated according to the manufacturer’s guidelines. Compounds isolated by preparative HPLC were redissolved in 100 μL of aqueous methanol (50%, v/v) and were analyzed by direct infusion at a flow rate of 5 μL/min. The MS conditions were as follows: capillary temperature, 250 °C; spray voltage, −3200 V; sheath gas and auxiliary gas were not applied. Mass spectra were acquired in the range of m/z 150−2000 at resolution of 70000 (at m/z 200). Mass spectrometric data were analyzed using Xcalibur 3.0.63 software (Thermo Fisher Scientific). High-resolution mass data for minor compounds 5, 6, 9, 11−13, and 16−29 were determined by HPLC-ESI-HR-MS. An Agilent 1200 series, set up according to the above-mentioned HPLC method,5 was coupled online to a micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in negative electrospray ionization mode. Capillary voltage was set at +4.5 kV. Nebulizing and drying gas was both nitrogen applied at 3.0 bar and 8.8 L/min. Drying gas was heated to 250 °C. Mass spectra were recorded at 20 Hz (2 GHz sampling rate) in the range of m/z 250−2500 at a resolution of 12900 (at m/z 996.8). The instrument was calibrated with sodium formate according to the manufacturer’s instructions. Chemical

formulas were generated by accurate mass measurements and DataAnalysis 3.4 software. Determination of the Absolute Configuration of the Sugar Moieties. Isolated compounds (ca. 1 mg) were heated with aqueous TFA (2 M) at 85 °C for 2 h, subsequently evaporated in vacuo to 0.2 mL (45 °C). Toluene and pyridine (both 4 × 3.0 mL) were used consecutively to remove TFA and water without evaporating to dryness to avoid the degradation of apiose. Subsequently, the sample was combined with 0.1 mL of L-cysteine methyl ester hydrochloride in pyridine (120 mg/mL) and heated at 65 °C for 1 h. After the addition of 0.2 mL of the silylation reagent, heating was continued for a further 0.5 h. Analytes were partitioned between hexane and H2O (0.3 mL each), and the hexane layer was analyzed by GC-MS equipped with a 30 m × 0.25 mm, df = 0.25 μm, HP-5ms column (temperature program: 100 °C for 1 min, raised at 15 °C/min to 200 °C, raised at 3 °C/min to 240 °C, raised at 15 °C/min to 300 °C, and held for 5 min; carrier gas, helium at 1.2 mL/min). Reference standards were subjected directly to the derivatization reaction (0.2 mL, 0.1 g/L in pyridine). In contrast, 10 μL of aqueous L-apiose was derivatized according to the protocol given above without TFA hydrolysis. Retention times (retention indices relative to n-alkanes) of the derivatives obtained were as follows: D-apiose, 14.0 min (2240); Lrhamnose, 15.7 min (2336); D-fructose, 17.3 min (2426); and Dglucose, 18.5 min (2483). The D-rhamnose, L-fructose, and L-glucose derivatives revealed retention times (indices) of 15.9 min (2347), 17.1 min (2412), and 18.8 min (2502). An authentic standard of L-apiose was unavailable. EI-MS (70 eV) base peak fragment ions were m/z 217 (apiose, rhamnose, glucose) and m/z 248 (fructose). NMR Spectroscopy. 1H and 13C NMR spectra of isolated compounds were recorded on a Varian Unity Inova 500 MHz spectrometer (Darmstadt, Germany). Compounds 14 and 15 (Figure 1) were dissolved in DMSO-d6 (plus a drop of D2O) and pyridine-d5, respectively. The 1H and 13C chemical shifts were referenced to residual solvent signals at δH/C 2.49/39.5 (DMSO-d6) and 8.71/149.8 (pyridine-d5) relative to TMS. Adiabatic 2D broadband and bandselective gHSQC and gHMBC, gDQFCOSY, ROESY, and TOCSY as well as selective 1D TOCSY spectra were recorded using CHEMPACK 4.0 pulse sequences (implemented in Varian VnmrJ 2.1B software). Coupling constants J (in hertz) were directly taken from the spectra and are not averaged.

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RESULTS AND DISCUSSION Comparison of Freshly Prepared Bark Extracts and Commercially Available Wood Extracts of Q. saponaria Molina. A total of 31 phenolic compounds were previously described to be present in commercially available Quillaja wood extracts,5 which are widely used as emulsifiers in foods (e.g., as E999 in the EU) and cosmetics. In the present study, we compared the composition of such commercial extracts to that of a freshly prepared aqueous extract of the inner bark. The HPLC-PDA-MSn-based comparison revealed a highly similar phenolic pattern (Figure 2; Table 1). In agreement with our previous work,5 (+)-piscidic acid, 1, was found to be the major phenolic, whereas several vanillic acid derivatives (2, 3), syringic acid derivatives (4, 8), and catechin derivatives (7, 10) were found at minor signal intensities. However, we previously described several unknown p-coumaric acid derivatives (5, 6, 9, 11−29), two of them (14, 15) at very high signal intensities. Notably, we were unable to identify some trace compounds in the inner bark extracts, which were previously detected in commercial extracts.5 The latter compounds were mostly free phenolic acids, such as vanillic, syringic, p-coumaric, and ferulic acid, possibly representing artifacts generated during the manufacture of the commercial extracts. Characterization of New p-Coumaroyl Sucrose Esters. After preparative isolation, compound 14 was obtained as a B

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. HPLC separation of phenolic compounds from an aqueous extract of the inner bark of Q. saponaria Molina at 280 and 310 nm.

Figure 1. Chemical structures of the new compounds quillajaside A and quillajaside B extracted from the inner bark of Q. saponaria Molina.

NMR spectrum showed a total of 17 characteristic signals of one apiosyl and two rhamnosyl moieties (Table 2; Figure 3). Each of the sugar moieties was assigned by evaluation of the gDQFCOSY, TOCSY, selective 1D TOCSY, and gHSQC spectra. Moreover, ROESY correlations (Figure 3A) and HMBC correlations of the H-4″″″ with C-1″″″, C-2″″″, and C-3″″″ as well as correlations of H-5″″″a/b with C-2″″″ and C3″″″ unambiguously established apiose as one sugar moiety in 14 (Figure 3B). The downfield-shifted rhamnosyl C-4″″′ (δ 77.81) as well as long-range HMBC and ROESY correlations between the rhamnosyl H-4″″′ and the apiosyl C-1″″″ revealed a 1,4 linkage between both sugar moieties. The observation of a doublet at δ 5.17 with a coupling constant of J 2.5 Hz of H1″″″, established the β-configuration of the apiose according to previous papers.6−10 The absence of downfield-shifted C atoms other than the anomeric C-1″″″ indicated the terminal localization of the apiosyl moiety (Figure 3). The downfieldshifted C-4″″″ (δ 80.02) of the second rhamnose along with long-range HMBC interactions of H-4″″ with C-1″″′ and H1″″′ with C4″″ as well as an 1H NMR signal of H-1″″′ at δ 4.82 (bs) suggested the α-1,4 linkage of the two rhamnosyl moieties (Figure 3). Besides rhamnose and apiose, the 13C NMR spectrum displayed 12 carbon signals of a glucose- and fructose-based disaccharide. Analysis of the homo- and heteronuclear 1D and 2D NMR spectra ascertained the disaccharide to be a trisubstituted sucrose (Figure 3B). The glucosyl C-3″ revealed a characteristic downfield shift at δ 78.49 and displayed a long-

white amorphous powder. HR-FT-ESI-MS measurements indicated a molecular formula of C47H62O27 (see Table 1). The acidic hydrolysis yielded free p-coumaric acid as its only phenolic constituent as shown by HPLC-PDA-MSn data of an authentic standard. In HPLC-ESI(−)-MS experiments, the nonhydrolyzed compound yielded a pseudomolecular ion [M − H]− at m/z 1057. Collision-induced dissociation (CID) experiments yielded a predominant fragment at [M − H − 146]− at m/z 911 (Table 1), indicating the presence of a deoxyhexose moiety. The second most abundant, nonisotopic fragment ion [M − H − 132]− at m/z 925 was possibly produced after the potential loss of the pentose moiety. The fragment ion at m/z 779 may have resulted from the consecutive loss of the above-mentioned deoxy-hexose (146 Da) and pentose (132 Da) moieties. The fragment ion at m/z 633 may represent the additional loss of a deoxy-hexose moiety (146 Da loss), that is, being a fragment species [M − H − 146 − 146 − 132]− produced by the loss of two deoxy-hexose (146 Da) and one pentose moiety (132 Da). In agreement, a fragment ion [M − H − 146 − 146]− at m/z 765 generated by the loss of two deoxy-hexose (146 Da) units was also observed (Table 1). Further fragments at lower m/z values were unavailable due to insufficient signal intensity. In agreement with the MS/MS-based proposition of one pentose and two deoxy-hexose moieties, analyses of the 13C C

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

314

314 314 314 314 314 312 311 304 313

28.6 28.8 29.0 29.4 29.7 31.0 31.1 31.3 31.7 32.3 32.6 32.9

quillajaside A

quillajaside B isomer

quillajaside B isomer quillajaside A isomer p-coumaroyl sucrose der p-coumaroyl sucrose der quillajaside A isomer tri-p-coumaroyl trirhamnosyl monoapiosyl sucrose tri-p-coumaroyl trirhamnosyl sucrose tri-p-coumaroyl trirhamnosyl monoapiosyl sucrose tri-p-coumaroyl trirhamnosyl sucrose tri-p-coumaroyl trirhamnosyl sucrose tri-p-coumaroyl dirhamnosyl sucrose tri-p-coumaroyl dirhamnosyl sucrose tri-p-coumaroyl trirhamnosyl sucrose

15

16

17 18 19 20 21 22

D

304 304 312 314

33.1 33.5 33.7 35.1

312

1217.3864 (1217.3930)

1071.3311 (1071.3351)

1071.3344 (1071.3351)

1217.3917 (1217.3930)

1217.3859 (1217.3930)

1349.4390 (1349.4353)

1217.3897 (1217.3930)

1057.3354 (1057.3406) 925.2964 (925.2983) na (low signal) na (low signal) 925.2979 (925.2983) 1349.4365 (1349.4353)

1057.3341 (1057.3406)

1057.3346 // 1057.34131 (1057.3406)a 925.2980 // 925.29932 (925.2983)a

na 633.2022 (633.2036) 779.2564 (779.2615) 1203.3957 (1203.3985)

nac na na na 487.1417 (487.1457) 779.2581 (779.2615) na na 911.3010 (911.3038)

HPLC-ESI-(−)-HR-MS meas m/z (calcd m/z) [M − H]−a

329(4). 192(4), 178(4), 168(9), 167(100), 161(8), 152(11), 143(5) 197(100), 182(12), 123(3) 341(9), 323(3), 307(53), 217(2), 179(1), 163(34), 145(100) 635(3), 634(36), 633(100), 616(5), 615(11), 599(3), 487(2), 453(2), 433(2), 341(2), 307(2) 289(100), 245(7),, 161(4) 455(6), 359(3), 197(100), 182(28), 167(5), 153(4) 779(4), 766(19), 765(100), 749(12), 747(17), 732(4), 731(27), 633(3), 585(11), 509(4)

209(5), 193(21), 179(5), 166(10), 165(100), 149(5)

[1217]: 1072(43), 1071(100), 926(13), 925(44), 907(7), 779(7), 633(1)

[1071]: 926(40), 925(100), 780(12), 779(47), 761(5), 633(2)

[1071]: 926(22), 925(100), 780(4), 779(20), 633(2), 487(0.3)

[1217]: 1072(41), 1071(100), 926(5), 925(26), 779(6), 633(1)

[1349]: 1350(7), 1349(16), 1217(2), 1204(51), 1203(100), 1072(7), 1071(15), 1058(7), 1057(20), 926(2), 925(5), 911(7), 799(1), 731(1), 633(1) [1217]: 1072(46), 1071(100), 1053(4), 926(12), 925(53), 907(7), 779(6), 761(2), 633(2)

na [633]: 615(1), 597(1), 488(19), 487(100), 469(9), 341(2), 307(5), 217(1), 179(1) [779]: 635(3), 634(34), 633(100), 616(3), 615(8), 487(2), 453(1), 433(1), 341(2), 307(1) [1203]: 1204(5), 1203(9), 1072(5), 1071(10), 1058(40), 1057(100), 925(4), 912(6), 911(17), 893(8), 779(3), 633(1) [1057]: 1057(18), 926(7), 925(18), 912(39), 911(100), 893(3), 779(9), 765(6), 745(3), 731(3), 633(1) [1057 → 633]: 487(72), 341(100), [925]: 780(28), 779(100), 761(5), 634(2), 633(17), 615(3) [925 → 633]: 487(63), 471(61), 341(100), 231(19) [1057]: 1058(7), 1057(17), 937(2), 926(4), 925(20), 912(37), 911(100), 893(4), 779(8), 765(8), 747(3), 731(3), 633(1) [1057]: 1057(18), 926(7), 925(22), 912(37), 911(100), 893(4), 779(7), 765(5), 747(3), 731(3), 633(1) [925]: 780(31), 779(100), 761(4), 634(2), 633(21), 615(5) [1057]: 1058(5), 1057(14),, 925(5), 912(38), 911(100), 893(6), 779(4), 765(7), 633(1) [1057]: 1057(14), 926(5), 925(16), 912(34), 911(100), 893(4), 779(6), 765(5), 731(3), 633(1) [925]: 780(31), 779(100), 762(2), 761(7), 634(2), 633(13), 615(3) [1349]: 1350(30), 1349(80), 1218(18), 1217(41), 1071(17), 1058(19), 1057(40), 1204(45), 1203(100), 925(6), 911(5), 895(5), 779(2), 731(1), 633(1) [1217]: 1072(41), 1071(100), 926(14), 925(50), 907(7), 779(7), 633(2)

[255]: na [461]: [359]: [487]: [779]: [451]: [491]: [911]:

HPLC-ESI-MSn expt (m/z)b

a First displayed m/z was measured by HPLC-HR-MS; second displayed m/z was measured by direct HR-MS of the isolated compound. bMost reported signals of pseudomolecular ions [M − H]− also occurred as chloride adducts [M + 35]−. cna, not available. dder, derivative.

29

28

27

26

25

24

23

14 314

280 313 313 313

21.6 22.2 24.0 27.3

10 11 12 13

275 254/293 254/292 262/292sh 313 313 280 262/293sh 315

HPLC-PDA abs max (nm)

12.3 16.7 18.4 19.6 20.7 20.9 21.0 21.2 21.6

1 2 3 4 5 6 7 8 9

ret time (min)

(+)-piscidic acid vanillic acid 4-hexoside vanillic acid 4-hexosyl-pentoside glucosyringic acid p-coumaroyl sucrose p-coumaroyl dirhamnosyl sucrose catechin hexoside syringic acid hexosyl-pentoside p-coumaroyl dirhamnosyl monoapiosyl sucrose catechin hexosyl derd p-coumaroyl rhamnosyl sucrose p-coumaroyl dirhamnosyl sucrose di-p-coumaroyl trirhamnosyl monoapiosyl sucrose quillajaside B

compd identity

no.

Table 1. Identification of Phenolic Compounds in Crude Aqueous Extracts from the Inner Bark of Quillaja saponaria Molina by HPLC-PDA-MSn

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data of Quillajaside B, 14 (β-D-apif-(1→4)-α-L-rhap-(1→4)-α-L-rhap-(1→3)(4-O-p-coumaroyl)-α-D-glup-(1→2)-(3-O-p-coumaroyl)-β-D-fruf) carbona

δC (DMSO)b

p-coumaroyl moiety at α-Dglup 1 125.25 2 130.50 3 116.34 4 160.00 5 116.34 6 130.50 7 145.70 8 114.06 9 166.09 p-coumaroyl moiety at β-Dfruf 1′ 125.48 2′ 130.73 3′ 116.24 4′ 160.18 5′ 116.24 6′ 130.73 7′ 145.73 8′ 114.37 9′ 166.02 α-D-glup 1″ 91.57 2″ 72.33 3″ 78.49 4″ 69.10 5″ 71.07 6″ 60.55

protona

δH (J in Hz)

carbona

p-coumaroyl moiety at α-D-glup

2 3

7.26 d-like (7.8) 6.76 d-like (8.3)

5 6 7 8

6.76 7.26 7.40 6.03

β-D-fruf 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

d-like (8.3) d-like (7.8) d (16.1) d (16.0)

7.54 d-like (8.1) 6.73 d-like (8.5)

5′ 6′ 7′ 8′

6.73 7.54 7.61 6.38

α-D-glup 1″ 2″ 3″ 4″ 5″ 6″a 6″b

5.26 d (3.3) 3.43 bd (10.5) 3.74 ov 4.82 bdd (9.6, 9.6) 4.00−3.94 m 3.34 ov 3.44 ov

63.5 103.9 77.81 72.83 83.50 62.02

α-L-rhap (1→3) 1″″ 101.71 2″″ 71.08 3″″ 71.15 4″″ 80.02 5″″ 67.45 6″″ 18.75 α-L-rhap (1→4) 1″″′ 101.87 2″″′ 70.99 3″″′ 71.148 4″″′ 77.81 5″″′ 67.54 6″″′ 17.7 β-D-apif (1→4) 1″″″ 109.70 2″″″ 76.67 3″″″ 79.25 4″″″ 63.53

p-coumaroyl moiety at β-D-fruf

2′ 3′

δC (DMSO)b

d-like (8.5) d-like (8.1) d (15.9) d (16.0)

5″″″

73.50

protona

δH (J in Hz)

β-D-fruf 1‴a 3.40 ov 1‴b 2‴ 3‴ 5.34 d (7.5) 4‴ 4.12 dd (7.4) 5‴ 3.78 ov, m 6‴a 3.63 dd (6.5, 11.6) 6‴b 3.59 dd (2.6, 11.3) α-L-rhap (1→3) 1″″ 4.81 bs 2″″ 3.65 bs 3″″ 3.43−3.37 m 4″″ 3.18 t-like (9.2) 5″″ 3.32 m 6″″ 0.79 d (6.0) α-L-rhap (1→4) 1″″′ 4.82 bs 2″″′ 3.63−3.64 bs 3″″′ 3.4 ov 4″″′ 3.27 ov 5″″′ 3.26 ov 6″″′ 0.86 d (6.3) β-D-apif (1→4) 1″″″ 5.17 d (2.5) 2″″″ 3.76 bd (2.5) 4″″″a 4″″″b 5″″″

3.85 d (8.5) 3.85 ov 3.36 ov

a

For numbering, see Figure 3A. bAssignments were based on 1H−1H COSY, sel 1D TOCSY, gHSQC, TOCSY, gDQFCOSY, ROESY, and gHMBC experiments.

Its acidic hydrolysis yielded free p-coumaric acid as the only phenolic constituent. Compound 15 gave a pseudomolecular ion [M − H]− at m/z 925, yielding predominant fragments [M − H − 146]− at m/z 779 and [M − H − 146 − 146]− at m/z 633 during CID experiments (Table 1). The consecutive losses of 146 Da indicated the presence of two deoxy-hexose moieties. A fragment ion [M − H − 164]− at m/z 761 was possibly caused by the loss of the above-mentioned p-coumaric acid moiety. In agreement with the proposition of two deoxy-hexose moieties, analysis of the 13C NMR spectrum showed 12 characteristic signals of 2 rhamnosyl moieties, respectively (Table 3). Comparison of the 1H and 13C NMR signals (Table 3) as well as HMBC and ROESY long-range correlations (Figure 4) revealed a highly similar structural identity of compound 14 and 15, consisting of two p-coumaroyl, one glucosyl, one fructosyl, and two rhamnosyl moieties. Glycosidic linkages, anomeric configurations, and (E)-configuration of the p-coumaroyl double bonds of compound 15 were identical to those of compound 14 as shown by the NMR data in Tables 2 and 3. Likewise, the absolute sugar configurations were identical to those described for compound 14. In summary, the structure of compound 15 was shown to be a α-L-rhap-(1→ 4)-α-L-rhap-(1→3)-(4-O-(E)-p-coumaroyl)-α-D-glup-(1→2)-

range correlation with the rhamnosyl H-1″″ in the gHMBC spectrum, indicating the rha(1 → 3)glu linkage as shown in Figure 3B. The 1H NMR signal of H-1″″ at δ 4.81 (bs) indicated the α-configuration of the anomeric rhamnose atom. The remaining 18 signals in the 13C NMR spectrum were assigned as two p-coumaroyl moieties (Table 2; Figure 3). HMBC correlations of the carboxylic C-9 of a p-coumaroyl moiety and H-4″ of the glucose revealed the position of the pcoumaroyl moiety at C-4″ as shown in Figure 3B. By analogy, a long-range correlation of the fructose H-3‴ and the second pcoumaroyl carboxyl C-9′ unveiled the presence of a further pcoumaroyl ester (Figure 3B). Both p-coumaroyl moieties were in (E)-configuration as indicated by the size of the coupling constants, J = 15.9−16.1 Hz, of the respective 1H NMR signals (H-7, H-8, H-7′, H-8′; Table 2) as previously reported for (E)and (Z)-coumaroyl moieties.11 According to our GC-MS analyses, the above-mentioned sugars were D-glucose, Dfructose, L-rhamnose, and D-apiose. Therefore, the structure of compound 14 was established to be β-D-apif-(1→4)-α-Lrhap-(1→4)-α-L-rhap-(1→3)-(4-O-(E)-p-coumaroyl)-α-D-glup(1→2)-(3-O-(E)-p-coumaroyl)-β-D-fruf. Compound 15 was obtained as a white amorphous powder. Its molecular formula was determined to be C 42H54O23 according to our HR-FT-ESI-MS measurements (Table 1). E

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 3. 1H (500 MHz) and 13C (125 MHz) NMR Data of Quillajaside A, 15 (α-L-rhap-(1→4)-α-L-rhap-(1→3)-(4-O-pcoumaroyl)-α-D-glup-(1→2)-(3-O-p-coumaroyl)-β-D-fruf) carbona

δC (pyridine)b

p-coumaroyl moiety at α-

D-glup

1 126.27 2 130.90 3 116.96 4 161.62 5 116.96 6 130.90 7 146.40 8 114.68 9 167.39 p-coumaroyl moiety at βD-fruf 1 126.44 2′ 130.97 3′ 116.83 4′ 161.74 5′ 116.83 6′ 130.97 7′ 146.40 8′ 114.74 9′ 167.46 α-D-glup 1″ 92.9 2″ 73.2 3″ 80.0 4″ 70.4 5″ 72.4 6″ 61.8

Figure 3. Important (A) ROESY and (B) HMBC long-range correlations between carbon and hydrogen atoms of quillajaside B.

β-D-fruf 1‴

(3-O-(E)-p-coumaroyl)-β-D-fruf. Due to the complexity of their systematic names, we proposed to name the smaller compound 15 quillajaside A, whereas the larger compound 14 was named quillajaside B. Tentative Identification of Further p-Coumaroyl Sucrose Esters. A tentative biosynthetic series of precursors and products of quillajasides A and B may be proposed when detected compounds with highly related UV and mass spectra are considered. The most basic detectable p-coumaroylated substance was compound 5, presumably representing a pcoumaroylated sucrose (C21H27O13) according to its UV spectrum, its [M − H]− at m/z 487.1417 (calcd value in Table 1), and several characteristic CID losses and fragment ions (Table 1). A p-coumaroyl rhamnosyl sucrose (C27H37O17, compound 11) was tentatively identified by its UV and mass spectra, particularly, its [M − H]− at m/z 633.2022 (Table 1). Because rhamnosyl- and p-coumaroyl moieties are nominally isobaric, HR-MS was crucial to distinguish the identity of compound 11 clearly from a putative di-p-coumaroyl sucrose (C30H33O15), which was not detected in the extract. Possibly representing a direct biosynthetic derivative of the quillajaside A, a p-coumaroyl dirhamnosyl sucrose (compound 12) was found according to our analytical data (Table 1). A total of three di-p-coumaroyl dirhamnosyl sucrose esters (compounds 15 (quillajaside A), 18, and 21) were identified, whereas di-p-coumaroyl (mono)-rhamnosyl sucroses were not detected. Furthermore, two di-p-coumaroyl dirhamnosyl apiosyl sucrose esters (i.e., compounds 14 (quillajaside B), 16, and 17) were detected. A rhamnosylated derivative of

2‴ 3‴ 4‴ 5‴ 6‴

65.0 105.2 79.5 73.8 84.7 62.7

α-L-rhap (1→3) 1″″ 103.2 2″″ 72.5 3″″ 76.62 4″″ 80.7 5″″ 68.5 6″″ 19.4 α-L-rhap (1→4) 1″″′ 103.15 2″″′ 72.1 3″″′ 72.4 4″″′ 73.6 5″″′ 70.1 6″″′ 18.2

δH (J in Hz)

protona

p-coumaroyl moiety at α-D-glup

2′ 3′

7.58 d-like (8.8) 7.37 d-like (8.7)

5′ 6′ 7 8

7.37 7.58 7.97 6.52

d-like (8.7) d-like (8.8) d (15.9) d (16.2)

p-coumaroyl moiety at β-D-fruf

2′ 3′

7.69 d-like (8.4) 7.25 d-like (8.6)

5′ 6′ 7′ 8′

7.69 7.25 8.09 6.73

α-D-glup 1″ 6.14 2″ 4.14 3″ 4.79 4″ 5.75 5″ 4.90 6″a 4.12 6″b 4.30 β-D-fruf 1‴a 4.30 1‴b 4.30 2‴ 3‴ 6.40 4‴ 5.30 5‴ 4.76 6‴a 4.56 6‴b 4.45 α-L-rhap (1→3) 1″″ 5.89 2″″ 4.70 3″″ 4.51 4″″ 4.27 5″″ 4.31 6″″ 1.50 α-L-rhap (1→4) 1″″′ 6.07 2″″′ 4.78 3″″′ 4.41 4″″′ 4.24 5″″′ 4.19 6″″′ 1.45

d-like (8.4) d-like (8.6) d (15.8) d (16.0)

d (3.7) dd (3.4, 9.5) dt (9.9) t (9.6) ddd (2.5, 5.1, 10.2) dd (4.9, 12.4) ov ov ov d (7.4) dd (7.3, 7.4) ddd (3.7, 5.5, 7.2) dd (6.3, 12.2) dd (3.7, 12.2) d (1.7) dd (1.7, 3.2) dd (3.2, 9.3) t-like (9.4) ov d (6.0) d (1.5) dd (1.6, 3.5) dd (3.3, 9.0) t-like (9.2) m, ov d (6.0)

a For numbering, see Figure 4A. bAssignments were based on 1H−1H COSY, sel 1D TOCSY, gHSQC, TOCSY, gDQFCOSY, ROESY, and gHMBC experiments.

quillajaside B was observed (compound 13), being tentatively identified as di-p-coumaroyl trirhamnosyl apiosyl sucrose ([M F

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*(R.M.S.) Phone: +49 711 459 22995. Fax: +49 711 459 24110. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Regina Walther from Pera Ingredients (Springe-Eldagsen, Germany) for generously donating the inner bark of Q. saponaria Molina. We also acknowlege Dr. Jens Pfannstiel (University of Hohenheim) for conducting the highresolution mass spectrometric analyses on the isolated compounds 14 and 15. We thank Joachim Trinkner (University of Stuttgart, Germany) for conducting the HPLC-ESI-HR-MS analyses.

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REFERENCES

(1) San Martín, R.; Briones, R. Industrial uses and sustainable supply of Quillaja saponaria (Rosaceae) saponins. Econ. Bot. 1999, 53, 302− 311. (2) Marciani, D. J. Triterpene saponin analogs having adjuvant and immunostimulatory activity. Patent US 5977081 A, 1999. (3) van Setten, D. C.; van de Werken, G. Molecular structures of saponins from Quillaja saponaria Molina. In Saponins Used in Traditional and Modern Medicine−Adances in Experimental Medicine and Biology; Waller, G. R., Yamasaki, K., Eds.; Springer: New York, 1996; Vol. 404, pp 185−193. (4) Oleszek, W.; Hamed, A. Saponin-based surfactants. In Surfactants from Renewable Resources; Kjellin, M., Johannson, I., Eds.; Wiley: Hoboken, NJ, USA, 2010; pp 239−249. (5) Maier, C.; Conrad, J.; Carle, R.; Weiss, J.; Schweiggert, R. M. Phenolic constituents in commercial aqueous quillaja (Quillaja saponaria Molina) wood extracts. J. Agric. Food Chem. 2015, 63, 1756−1762. (6) da Silva, B. P.; Velozo, L. S. M.; Parente, J. P. Biochanin A triglycoside from Andira inermis. Fitoterapia 2000, 71, 663−667. (7) Golovchenko, V. V.; Ovodova, R. G.; Shashkov, A. S.; Ovodov, Y. S. Structural studies of the pectic polysaccharide from duckweed Lemna minor L. Phytochemistry 2002, 60, 89−97. (8) Guo, S.; Kenne, L. Characterization of some O-acetylated saponins from Quillaja saponaria Molina. Phytochemistry 2000, 54, 615−623. (9) Ishii, T.; Yanagisawa, M. Synthesis, separation and NMR spectral analysis of methyl apiofuranosides. Carbohydr. Res. 1998, 313, 189− 192. (10) Snyder, J. R.; Serianni, A. S. DL-Apiose substituted with stable isotopes: synthesis, NMR-spectral analysis, and furanose anomerization. Carbohydr. Res. 1987, 166, 85−99. (11) Lu, Y.; Yeap Foo, L. Flavonoid and phenolic glycosides from Salvia of f icinalis. Phytochemistry 2000, 55, 263−267. (12) Zhao, W.; Huang, X.-X.; Yu, L.-H.; Liu, Q.-B.; Li, L.-Z.; Sun, Q.; Song, S.-J. Tomensides A−D, new antiproliferative phenylpropanoid sucrose esters from Prunus tomentosa leaves. Bioorg. Med. Chem. Lett. 2014, 24, 2459−2462. (13) Takasaki, M.; Konoshima, T.; Kuroki, S.; Tokuda, H.; Nishino, H. Cancer chemopreventive activity of phenylpropanoid esters of sucrose, vanicoside B and lapathoside A, from Polygonum lapathifolium. Cancer Lett. 2001, 173, 133−138. (14) Liu, T.; Yip, Y. M.; Song, L.; Feng, S.; Liu, Y.; Lai, F.; Zhang, D.; Huang, D. Inhibiting enzymatic starch digestion by the phenolic compound diboside A: a mechanistic and in silico study. Food Res. Int. 2013, 54, 595−600.

Figure 4. Important (A) ROESY and (B) HMBC long-range correlations between carbon and hydrogen atoms of quillajaside A.

− H]− at m/z 1203.3957, calcd values in Table 1). Further detected compounds (22−29) represented sucrose derivatives with three p-coumaroyl moieties, at least two rhamnosyl moieties, and eventually an apiosyl moiety (Table 1). The highest molecular weight compounds detected in the aqueous extracts were compounds 22 and 24 with an [M − H]− at m/z 1349, representing tri-p-coumaroyl trirhamnosyl monoapiosyl sucroses (Table 1). In brief summary, the aqueous extracts of the inner bark of Q. saponaria Molina were shown to contain a total of 22 phenylpropanoid sucrose esters, being linked to up to 1 apiosyl, 3 rhamnosyl, and 3 p-coumaroyl moieties. Full structural elucidation was only possible for compounds 14 and 15 (quillajasides A and B). Nevertheless, because the occurrence of these unique compounds is highly characteristic of Quillaja extracts including the food additive E999, they may represent valuable authenticity markers for the detection of Quillaja extracts in food and cosmetic formulations. According to a previous paper,12 ca. 150 phenylpropanoid sucrose esters have been identified previously, some of which exerted biological activities such as a cancer chemopreventive activity.13 Moreover, a p-coumaroyl sucrose ester from wild buckwheat (Fagopyrum esculentum Moench) was recently shown to inhibit α-amylase activity and, thus, proposed to serve as a functional food ingredient for controlling postprandial hyperglycemia by modulating starch degradation during digestion.14 Further studies on these p-coumaroyl sucrose esters, their biofunctionality in the bark of Q. saponaria, and their bioactivity in humans should be encouraged, particularly as these compounds are ingested daily by consumers worldwide. G

DOI: 10.1021/acs.jafc.5b03532 J. Agric. Food Chem. XXXX, XXX, XXX−XXX