Phenolic Constituents in Commercial Aqueous Quillaja (Quillaja

Article pubs.acs.org/JAFC

Phenolic Constituents in Commercial Aqueous Quillaja (Quillaja saponaria Molina) Wood Extracts Christiane Maier,† Jürgen Conrad,‡ Reinhold Carle,§,∥ Jochen Weiss,† and Ralf Martin 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: Phenolic compounds in aqueous, saponin-rich soapbark tree (Quillaja saponaria Molina) extracts were qualitatively and quantitatively characterized by HPLC-PDA−MSn and NMR spectroscopy. (+)-Piscidic acid represented the major constituent (75−87% (w/w) of total phenolics) in all examined extracts (n = 4), ranging from 22.1 ± 0.1 to 34.0 ± 0.2 mg/g of dry matter (DM). Derivatives of p-coumaric acid were present at concentrations from 2.2 to 9.3 mg/g of DM (8.1− 20.4% of total phenolics), whereas other phenolic constituents such as glucosyringic acid and vanillic acid derivatives accounted for less than 7% of total phenolics. Generally, all Quillaja extracts showed a highly similar but unique pattern, potentially being useful to authenticate Quillaja extracts in foods, cosmetics, and pharmaceutical formulations. Furthermore, the desired antioxidant activity as well as undesired browning reactions in the final product might also be explained by these phenolic compounds, which were identified for the first time in Q. saponaria extracts. KEYWORDS: saponin extract, emulsifier, polyphenol, piscidic acid, antioxidant, mass spectrometry, NMR spectroscopy

■

described for numerous phenolic compounds.5,6 Therefore, the objective of this work was to qualitatively and quantitatively characterize phenolic compounds in different saponin-rich Q. saponaria extracts.

INTRODUCTION Quillaja wood extracts are obtained from the Chilean soapbark tree (Quillaja saponaria Molina) by aqueous extraction. Due to their high saponin content, they exert excellent emulsifying properties.1,2 Since pre-Columbian times, these extracts were used by indigenous people from Chile as soaps, and consequently, the tree’s generic name is derived from the native Mapuche word quillean, which means “to wash”.3 To date, such aqueous saponin-rich extracts are widely used as natural emulsifiers in cosmetics, foods, and beverages,4 e.g., in root beer and ginger ale. Furthermore, they serve as adjuvants in vaccines and pharmaceutical formulations.4 In the past, the chemical structures of a variety of emulsifying saponins have been elucidated in detail. However, the extracts contain many other components, such as calcium oxalate, proteins, sugars, salts, tannins, and other polyphenols,3,4 potentially contributing to the desired product characteristics. San Martı ́n and Briones3 briefly summarized various refinement processes for the purification and concentration of the saponins from the primary aqueous extract. Using affinity chromatography or ultrafiltration, enrichment from 20% to 65−90% (w/w) (dry matter, DM) saponins and the simultaneous partial removal of undesired constituents such as phenolic compounds have been achieved. Nevertheless, most commercial Quillaja extracts still contain significant amounts of phenolic compounds, since their complete purification is a highly elaborate process. However, residual phenolic compounds contained in the extracts may affect the color and taste of the final product. At the same time, their presence may be desired for certain applications due to the antioxidant and potentially health-promoting properties © XXXX American Chemical Society

■

MATERIALS AND METHODS

Materials. All solvents used in this study were of analytical or HPLC grade and were purchased from VWR (Darmstadt, Germany), unless stated otherwise. Syringic acid was purchased from FlukaChemie (Buchs, Switzerland), while ferulic acid and p-coumaric acid were obtained from Carl Roth (Karlsruhe, Germany). Vanillic acid, (+)-catechin, L-cysteine methyl ester, L-glucose, and the silylation mixture (10% (v/v) hexamethyldisilazane−trimethylchlorosilane (HMDS/TMCS), 2/1, v/v) in pyridine were acquired from SigmaAldrich (St. Louis, MO). D-Glucose, hexane, and trifluoroacetic acid were from Merck (Darmstadt, Germany). D2O and MeOH-d4 were purchased from Deutero (Kastellaun, Germany). The following commercial Quillaja extracts were provided by Pera Ingredients (Springe-Eldagsen, Germany). According to the manufacturer, they were obtained by milling of the wood (mostly branches), water extraction, filtration through diatomaceous earth, removal of impurities by membrane filtration, and evaporative concentration.7 “Andean Q ultra” (no. 310 709 02) represented a liquid formulation with a triterpenic saponin content of 13 ± 0.5% (w/w) and 0.1% sodium benzoate as a preservative as stated by the manufacturer. According to the supplier’s specification, two batches of the spray granulated preparation “Andean QDP ultra organic” (no. 050 111-PSD-07 ORG Received: December 25, 2014 Revised: January 26, 2015 Accepted: January 27, 2015

A

DOI: 10.1021/jf506277p J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Identification of Phenolic Compounds in Commercial Aqueous Q. saponaria Molina Wood Extracts compd no.

a

retention time (min)

compd identity

HPLC−UV/Vis abs. max. (nm)

HPLC−ESI[(−) and (+)]-MS experiment (m/z) −

1 2

7.1 11.4

unknown (+)-piscidic acida

280 275

[M − H] : 271 [M − H]−: 255

3

16.3

vanillic acid 4-hexoside

253/291

[M + Na]+: 279 [M − H]−: 329

4

18.4

vanillic acid 4-hexosylpentoside

254/292

[M − H]−: 461

5

19.4

syringic acid 4-O-β-Dglucopyranoside (glucosyringic acid)a

262/292sh

[M − H]−: 359

6

20.7

catechin hexoside

280

[M − H]−: 451

7

21.0

syringic acid hexosylpentoside

263/293sh

[M − H]−: 491

8

21.5

catechin hexosyl derivative

280

[M − H]−: 739

9

21.7

p-coumaric acid derivative

281/315

[M − H]−: 1007

10

22.0


285/316

[M − H]−: 1007

11 12 13 14

22.1 24.1 25.2 25.8

vanillic acid syringic acid unknown vanillic acid derivative

261/293 275 303 261/292

[M [M [M [M

− − − −

H]−: H]−: H]−: H]−:

167 197 175 611

15 16 17 18

26.1 26.4 27.7 28.2

unknown p-coumaric acid ferulic acid unknown

262/285 298/310 295sh/325 273/275

[M [M [M [M

− − − −

H]−: H]−: H]−: H]−:

189 163 193 467

19

28.5


314

[M − H]−: 1057

20

28.7


314

[M − H]−: 925

21 22

28.9 29.6

unknown p-coumaric acid derivative

303 314

[M − H]−: 313 [M − H]−: 925

23 24 25 26 27 28 29 30 31

30.9 31.3 31.6 32.2 32.6 33.0 33.5 33.7 35.5

p-coumaric p-coumaric p-coumaric p-coumaric unknown unknown unknown p-coumaric p-coumaric

314 314 313 313 304 304 304 313 313

[M [M [M na [M [M [M na na

acid acid acid acid

derivative derivative derivative derivative

acid derivative acid derivative

− H]−: 1057 − H]−: 925 − H]−: 925 − H]−: 623 − H]−: 1217 − H]−: 1071

HPLC−ESI-MSn experiment (m/z) [271]: 253(100), 181(50), 191(49), 235(23),, 109(14) 123(12) [255]: 165(100), 193(24), 166(11), 209(10), 177(7), 149(6), 93(2) [255 → 165]: 107(100), 135(59), 58(38), 147(36), 137(29), 119(15), 93(10) [255 → 193]: 149(100), 165(30), 93(26), 99(13), 150(9), 147(8) [255 → 209]: 165(100) [279]: 189(100), 217(47), 149(42) [329]: 167(100) [329 → 167]: 152(100), 123(81), 108(21) [461]: 167(100), 152(21), 153(8), 329(8) [329 → 167]: 152(100), 123(50) [359]: 197(100), 182(12), 123(2), 167(1), 153(1) [359 → 197]: 182(100), 153(21), 138(11), 167(2), 123(1) [451]: 289(100), 245(10) [451 → 289]: 245(100), 205(40), 179(38), 125(36), 247(20), 231(18), 203(15), 161(7), 137(9), 175(5) [491]: 197(100), 182(31), 167(10), 447(7), 198(5), 359(4) [491 → 197]: 182(100), 153(17), 138(7) [739]: 587(100), 407(76), 559(39), 451(37), 567(28), 289(18), 613(17), 577(15), 533(13), 560(11), 408(8), 425(2) [1007]: 779(100), 925(83), 861(19), 633(13), 453(2) [1007 → 779]: 633(100), 259(41), 471(38) [1007]: 779(100), 925(85), 861(17), 633(9) [1007 → 779]: 633(100), 615(13), 487(1) [167]: 152(100), 123(70), 108(29) [197]: 153(100), 182(82), 138(56) [175]: 131(100) [611]: 443(100) [611 → 443]: 167(100) [189]: 171(100) [163]: 119 (100) [193]: 178 (100), 149(68), 134(24) [467]: 205(100), 163(61), 247(46), 143(36), 171(25), 449(14), 131(11), 145(7) [1057]: 911(100), 912(41), 925(23), 765(7), 893(7), 926(6), 913(6), 779(4), 633(1) [1057 → 911]: 765(100), 766(74), 337(42), 732(27), 509(18) [1057 → 633]: 341(100), 487(72) [925]: 779(100), 780(29), 633(18), 761(3) [925 → 779]: 633(100) [925 → 633]: 341(100), 487(63), 471(61), 231(19) [313]: 165(100), 221(53), 218(39), 278(38), 269(32) [925]: 779(100), 780(28), 633(23), 615(5), 634(5), 761(3), 599(3) [1057]: 911(100), 912(36), 925(8), 779(7), 765(7), 633(3) [925]: 779(100), 780(21), 633(11), 761(6) na na [623]: 315(100), 300(21), 271(15), 316(14), 255(12) [1217]: 1071(100), 1072(50), 925(17), 926(10), 779(3) [1071]: 925(100), 926(33), 779(27), 780(6), 633(3) na na

Identity confirmed by NMR spectroscopy, polarimetry (piscidic acid only), and GC−MS (glucosyringic acid only).

(batch 1) and no. 1718-0211-PSD-06 ORG (batch 2)) and one batch

60% (w/w) Quillaja saponins. In addition, 0.47 ± 0.02% sodium

of “Andean QDP ultra” (no. 310-709-02-PSD-03) contained at least

benzoate was added by the manufacturer to Andean QDP ultra. B


Article

Journal of Agricultural and Food Chemistry

Figure 1. HPLC separation of phenolic compounds in aqueous Q. saponaria wood extracts (untreated) monitored at 280 nm (A), 260 nm (B), and 310 nm (C) and in an acid-hydrolyzed extract (D) at 280 nm. of (+)-piscidic acid, the compound isolated from Quillaja extract was used. Quantitation experiments were performed in duplicate. Preparative Isolation of Major Quillaja Extract Compounds. Four fractions containing major phenolic components as identified by HPLC-PDA−MSn were isolated from the Quillaja extract Andean QDP ultra organic by preparative HPLC. The Quillaja extract was dissolved in water/methanol (90/10, v/v) containing 0.1% formic acid for the isolation of compounds 2 and 5 (Table 1 for peak assignment). For isolation of compounds 19 and 20, the extract was adjusted to pH 2 with formic acid. After membrane filtration (0.45 μm), phenolic compounds 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). Separation of compounds 2 and 5 was performed with a 150 × 21.0 mm i.d., 5 μm particle size, 175 Å pore size, Hypersil Gold silica column (Thermo Fisher Scientific, Waltham, MA). The mobile phase consisted of 0.1% (v/v) formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) using a multistep gradient as follows: 10% B isocratic (10 min), 10−40% B (5 min), 40−100% B (0.5 min), 100% B isocratic (4.5 min), 100−10% B (1 min), 10% B isocratic (9 min). The total run time was 30 min at a constant flow rate of 15 mL/min. Using this method, the retention times of compounds 2 and 5 were 8.8 and 14.2 min, respectively. Monitoring and peak detection were performed at 256 nm. Isolation of compounds 19 and 20 was performed with a preparative 250 × 21.1 mm i.d., 5 μm particle size, 125 Å pore size, Phenomenex Aqua C18 reversed-phase column. The mobile phase consisted of water (solvent A) and methanol (solvent B), which were both acidified to pH 2 with formic acid. The following gradient was used: 20% B isocratic (10 min), 20−40% B (18 min), 40% B isocratic (20 min), 40−100% B (2 min), 100% B isocratic (8 min), 100−20% B (2 min), 20% B isocratic (10 min). The total run time was 70 min at a constant flow rate of 8 mL/min. Using this method, the retention times of compounds 19 and 20 were 40.2 and 41.0 min, respectively. Monitoring and compound detection were performed at 310 nm.

Ultrapure water was used throughout the study (Milli-Q system, Millipore, France). HPLC-PDA−ESI/MSn Analyses. Dry commercial Quillaja powders and the liquid Quillaja preparation were dissolved and diluted in 1% (v/v) aqueous formic acid, respectively. All samples were membrane filtered (0.45 μm) prior to HPLC separation. HPLC-PDA analyses were performed on an 1100 series HPLC system (Agilent, Waldbronn, Germany) equipped with a 250 × 4.6 mm i.d., 5 μm particle size, 100 Å pore size, Kinetex C18 column fitted with a 4.0 × 2.0 mm i.d. guard column of the same material (Phenomenex, Torrance, Canada). The column was operated at 35 °C. The mobile phase consisted of 1% (v/ v) formic acid in water (solvent A) and 1% (v/v) formic acid in methanol (solvent B), using the following gradient program: 5% B isocratic (10 min), 5−100% B (40 min), 100% B isocratic (5 min), 100−5% B (1 min), 5% B isocratic (4 min). The total run time was 60 min at a flow rate of 1.0 mL/min. Injection volumes of 10 and 20 μL were used. Phenolic compounds were monitored at 260, 280, 310, and 370 nm, and additional UV/vis spectra were acquired in the range of 200−600 nm, employing a model G1315A photodiode array (PDA) detector (Agilent). For HPLC−ESI-MSn analyses, the HPLC system described above was serially interfaced with a Bruker (Bremen, Germany) Esquire 3000+ ion trap mass spectrometer via an electrospray ionization (ESI) source operated in both positive and negative modes. Ion mass spectra of the column eluate were recorded in the range m/z 100−1400 at a scan speed of 13 000 m/z/s. Nitrogen was used as a drying gas at a flow rate of 11.0 L/min and as a nebulizing gas at a pressure of 60.0 psi. The nebulizer temperature was set at 365 °C. Helium was used as a collision gas for collision-induced dissociation (CID) at a pressure of 4.0 × 10−6 mbar. Individual compounds were quantitated using linear calibration curves of authentic reference compounds (ferulic acid, p-coumaric acid, syringic acid, and vanillic acid). Derivatives of these phenolic acids were quantitated according to the calibration of the free compound using molecular weight correction factors. For quantitation C


Article

Journal of Agricultural and Food Chemistry Aliquots of 1000 μL of the Quillaja Andean QDP ultra organic extract were injected for each preparative HPLC run. The collected fractions were evaporated to dryness at 40 °C under reduced pressure (Tmax = 40 °C), freeze-dried, and stored at −20 °C until further use. Compound purity was checked by HPLC-PDA−MSn as described above. Acidic Hydrolysis of Extracts and Individual Compounds. The Quillaja Andean QDP ultra organic extract, p-coumaric acid, and the isolated fractions containing compounds 2, 5, 19, and 20 were dissolved in aqueous 4 N HCl, heated to ∼85 °C for 20−120 min, and subsequently neutralized with aqueous NaOH. Hydrolyzed samples were membrane filtered (0.45 μm) and analyzed with HPLC-PDA− MSn as described above. Determination of the Absolute Configuration of Compound 2 and Absolute Sugar Conformation of Compound 5. Specific rotation values for compound 2 were measured with a PerkinElmer model 341 polarimeter (PerkinElmer, Waltham, MA). Compound 5 (ca. 1 mg) was heated with aqueous 2 M TFA at 85 °C for 2 h. The reaction mixture was evaporated at 45 °C in vacuo, dissolved in pyridine (0.2 mL), and treated with L-cysteine methyl ester hydrochloride in pyridine (12 mg in 0.1 mL) at 65 °C for 1.5 h. Subsequent trimethylsilylation was carried out with the abovementioned silylation mixture (0.3 mL) at 65 °C for 1 h. The reaction mixture was partitioned between hexane and H2O (0.3 mL each), and the hexane layer was analyzed by GC−MS (column, 30 m × 0.25 mm i.d., df = 0.25 μm, HP-5 ms Agilent J&W; column temperature, 100 °C for 1 min, 15 °C/min to 200 °C, hold for 1.33 min, 2.5 °C/min to 240 °C, 15 °C/min to 300 °C, hold for 5 min; carrier gas, helium). The sugar derivative was identified as D-glucose with the retention time of 20.15 min, being identical to the respective retention time of derivatized authentic D-glucose. Under the same conditions, the Lglucose derivative revealed a retention time of 20.61 min. NMR Spectroscopy. 1H and 13C NMR spectra of isolated compounds 2 and 5 were recorded on a Varian Unity Inova 500 MHz spectrometer (Darmstadt, Germany). Compound 2 was dissolved in MeOH-d4 and compound 5 in D2O. The 1H and 13C chemical shifts were referenced to residual solvent signals at δH 3.35 and δC 49.0 for methanol as well as δH 4.70 for deuterated water relative to TMS. Adiabatic 2D broad-band and band-selective gHSQC and gHMBC as well as gDQFCOSY spectra were recorded using CHEMPACK 4.0 pulse sequences (implemented in Varian Vnmrj 2.1B software). Coupling constants, J, were directly taken from the spectra and are not averaged. Piscidic Acid, 2. 1H NMR (CD3OD, 500 MHz): δ 3.03 (1H, d, J = 13.8 Hz, 4-Ha), 3.18 (1H, d, J = 14.4 Hz, 4-Hb), 4.55 (1H, s, 2-H), 6.69 (2H, d-like, J = 8.8 Hz, 3′-H, 5′-H), 7.11 (2H, d-like, J = 8.8 Hz, 2′-H, 6′-H). 13C NMR (CD3OD, 125 MHz): δ 174.6 (C-1), 76.4 (C2), 81.4 (C-3), 42.0 (C-4), 175.9 (C-5), 128.0 (C-1′), 132.5 (C-2′, C6′), 115.7 (C-3′, C-5′), 157.2 (C-4′). NMR data of compound 2 were in agreement with previously published data for piscidic acid.8 Syringic Acid 4-O-β-D-Glucopyranoside (Glucosyringic Acid), 5. 1 H NMR (D2O, 500 MHz): δ 3.29 (1H, ddd, J = 2.3, 5.3, 9.7 Hz, 5′H), 3.42 (1H, t-like, J = 9.5 Hz, 4′-H), 3.48 (1H, t-like, 3′-H), 3.51 (1H, ov, 2′-H), 3.63 (1H, dd, J = 5.2, 12.5 Hz, 6′-Hb), 3.74 (1H, dd, J = 2.4, 12.6 Hz, 6′-Ha), 3.84 (6H, s, 8-H, 9-H, −OCH3), 5.08 (1H, d, J = 7.4 Hz, 1′-H), 7.33 (2H, s, 2-H, 6-H). 13C NMR (D2O, 125 MHz): δ 127.2 (C-1), 107.5 (C-2, C-6), 152.2 (C-3, C-5), 137.6 (C-4), 170.1 (C-7, O−CO), 56.3 (C-8,C-9, −OCH3), 102.6 (C-1′), 73.6 (C-2′), 75.6 (C-3′), 69.3 (C-4′), 76.3 (C-5′), 60.4 (C-6′). This compound was previously reported by Wolfram et al.9 and Liu et al.10

identified by comparison of their retention times, UV/vis spectra, and mass spectra to those of authentic reference substances (Figure 2). Furthermore, compounds 1 and 2 were

Figure 2. Major phenolic compounds in aqueous Q. saponaria wood extracts.

present in both untreated and hydrolyzed extracts. The minor compound 1 remained unidentified in our study. Compound 2 eluted at 11.4 min and represented the major phenolic component in all extracts, showing a UV absorption maximum at 275 nm. In negative ionization mass spectrometry, it produced a quasi-molecular ion, [M − H]−, at m/z 255, while a corresponding sodium adduct ion, [M + Na]+, at m/z 279 was produced in positive ionization mode (Table 1). The latter ion [M + Na]+ at m/z 279 produced a dominant fragment, [M + Na − 90]+, at m/z 189 after CID, possibly representing the simultaneous loss of two carboxyl groups. Fragmentation of the analogous negative quasi-molecular ion [M − H]− at m/z 255 yielded a strong signal, [M − H − 90]−, at m/z 165. The predominant MS3 fragments of this daughter ion at m/z 165 were observed at m/z 147, 135, and 107, indicating the loss of water, a CH2O group, and a C2H2O2 group, respectively. The presumably corresponding ion of the latter C2H2O2 group was detected at m/z 58 at high signal intensity (Table 1). After isolation by preparative HPLC, NMR spectroscopic data allowed the identification of compound 2 as piscidic acid. Since a rotational value of [α]25 D +41.0° (c 5.4, H2O) was determined in good agreement with previously described data for (+)-piscidic acid ([α]D24 +40.5° and [α]D23 +42.5°),11 compound 2 was unambiguously identified as (2S,3R)(+)-piscidic acid (Figure 2), which was previously found in Piscidia erythrina L. (Jamaican dogwood).12,13 Compounds 3 and 4 exhibited highly similar UV absorption spectra and identical dominant MS2 daughter ions at m/z 167, derived from parent ions at m/z 329 and 461, respectively. Moreover, MS3 spectra of these ions at m/z 167 yielded similar major fragments at m/z 152, 123, and 108, being characteristic for the elimination of a methyl moiety, decarboxylation, and a combination of both losses, respectively. In agreement with

■

RESULTS AND DISCUSSION Characterization of Phenolic Compounds. Typical chromatograms of an aqueous saponin-rich Q. saponaria extract are illustrated in parts A (280 nm), B (260 nm), and C (310 nm) of Figure 1. Acidic sample hydrolysis led to a simplified chromatogram shown in Figure 1D (280 nm). In both untreated and hydrolyzed extracts, vanillic acid (11), syringic acid (12), p-coumaric acid (16), and ferulic acid (17) were D


Article

Journal of Agricultural and Food Chemistry

Table 2. Quantification of Phenolic Compounds in Aqueous Extracts of Wood from Q. saponaria Molina (mg/g of DM) Andean QDP ultra organic compd no.

compd identity

Andean Q ultra

Andean QDP ultra

batch 1

batch 2

2 3 4 5 9 10 11 12 14 19 20

(+)-piscidic acid vanillic acid 4-hexoside vanillic acid 4-hexosylpentoside glucosyringic acid p-coumaric acid derivative p-coumaric acid derivative vanillic acid syringic acid vanillic acid derivative p-coumaric acid derivative p-coumaric acid derivative

23.60 ± 0.05 0.27 ± 0.00 0.10 ± 0.00 0.47 ± 0.00 0.24 ± 0.00 0.29 ± 0.00 tra 0.29 ± 0.00 0.17 ± 0.00 0.55 ± 0.00 1.12 ± 0.00

22.09 ± 0.12 0.23 ± 0.00 0.17 ± 0.00 0.41 ± 0.00 0.25 ± 0.00 0.37 ± 0.00 0.06 ± 0.05 0.21 ± 0.01 0.22 ± 0.01 1.30 ± 0.01 2.46 ± 0.01

33.99 ± 0.15 0.29 ± 0.01 0.45 ± 0.00 0.81 ± 0.01 0.46 ± 0.00 0.26 ± 0.00 0.05 ± 0.00 0.29 ± 0.01 0.39 ± 0.01 3.33 ± 0.01 5.21 ± 0.05

32.75 ± 0.31 0.29 ± 0.00 0.42 ± 0.01 0.78 ± 0.00 0.35 ± 0.01 0.27 ± 0.03 0.04 ± 0.00 0.26 ± 0.00 0.38 ± 0.01 2.78 ± 0.01 4.17 ± 0.01

27.10

27.78

45.52

42.49

total polyphenols (mg/g of DM) a

The abbreviation “tr” means nonquantitated traces.

that of compound 6 and a quasi-molecular ion, [M − H]−, at m/z 739. The main daughter ions obtained at m/z 587, 407, and 559 could not be specified, while the daughter ions at m/z 451 and 289 indicate a possible structural similarity to compound 6, a catechin hexoside. Therefore, compound 8 was tentatively identified as a catechin hexosyl derivative. A total of 11 different compounds (9, 10, 19, 20, 22−26, 30, 31) were assigned to p-coumaric acid derivatives. All of them showed a UV absorption spectrum highly similar to a pcoumaric acid standard, while ESI(−)-MS analyses yielded signals at uncommonly high m/z values. Compounds 20, 22, 24, and 25 exhibited a quasi-molecular ion, [M − H]−, at m/z 925, while [M − H]−, ions at m/z 1007 were observed for compounds 9 and 10 and at m/z 1057 for compounds 19 and 23. Irrespective of their parent ion, common fragment ions were observed at m/z 925, 779, and 633 for all of the abovementioned compounds. Compounds 19 and 20 were isolated by preparative HPLC and subjected to acidic hydrolysis, yielding free p-coumaric acid (Figure 2) as identified by comparing the retention times and UV and mass spectra to those of an authentic standard. In agreement, the neutral loss of 164 Da (p-coumaric acid) to yield daughter ions [M − H − 164]− at m/z 893 and 761 for compound 19 and 20, respectively, was observed during ESI(−) experiments (Table 1). Therefore, the above-mentioned compounds were assigned to yet uncharacterized p-coumaric acid derivatives (Table 1). Further investigations for their detailed characterization by NMR spectroscopy are ongoing. Compounds 28 and 29 revealed quasi-molecular ions at m/z 1217 and 1071, respectively. Their fragmentation pattern yielded daughter ions at m/z 925, 779, and 633, highly similar to those of the above-described p-coumaric acid derivatives (e.g., compounds 19 and 20). However, their UV absorption maximum was different (ca. 304 nm) as compared to that of pcoumaric acid and its derivatives (ca. 314 nm). Several other unknown compounds displaying analogous absorption spectra were found (compounds 13, 21, and 27−29). While compounds 21, 27, 28, and 29 disappeared after acidic hydrolysis, compound 13 was still retained in the hydrolyzed extract. Thus, compound 13 may represent a basic structural element of the above-mentioned compounds. In agreement, the quasi-molecular ion of compound 13 was detected at m/z 175, whereas compounds 21, 27, 28, and 29 produced signals at higher m/z 313, 623, 1217, and 1071, respectively. The quasi-

Fischer et al.14 and data obtained by an authentic vanillic acid standard, both compounds were identified as vanillic acid derivatives. Compound 3 yielded the fragment [M − H − 162]− at m/z 167 after elimination of a putative hexose moiety from the molecular ion at m/z 329. The quasi-molecular ion of compound 4 at m/z 461 exhibited a loss of 294 Da, possibly representing a hexose−pentose moiety. Their UV absorption spectrum showed high similarity to that of vanillic acid (11). While esterification of vanillic acid with hexoses was reported to cause a bathochromic shift, the absorption bands remain mostly unaltered after glycosylation of the hydroxyl group.14,15 Therefore, compounds 3 and 4 were tentatively identified as vanillic acid 4-hexoside and 4-hexosylpentoside, respectively. Compound 14 was also tentatively identified as a vanillic acid derivative, eluting at 25.8 min and exhibiting a characteristic UV absorption spectrum and a dominant CID fragment at m/z 167 (Table 1). By analogy to compounds 3 and 4, the quasi-molecular ions [M − H]− of compounds 5 (m/z 359) and 7 (m/z 491) showed identical losses of 162 and 294 Da, although both yielded a strong MS2 signal at m/z 197. MS3 spectra of the daughter ion at m/z 197 yielded characteristic fragments at m/z 182, 167, and 153, which allowed the tentative assignment of compounds 5 and 7 to syringic acid derivatives. After isolation of compound 5 by preparative HPLC, acidic hydrolysis resulted in the appearance of free syringic acid, supporting the tentative identification. In agreement, NMR spectroscopy allowed the complete elucidation of compound 5 as syringic acid 4-O-β-Dglucopyranoside (glucosyringic acid) (Figure 2). The absolute configuration of the sugar moiety of compound 5 was determined to be D-glucose by GC−MS. Since UV absorption and mass spectra indicated a high structural similarity of compounds 5 and 7, except for the above-described difference in primary mass loss of 294 Da, compound 7 was tentatively assigned to be a yet uncharacterized syringic acid hexosylpentoside. Compound 6 showed a UV absorption maximum at 280 nm and a quasi-molecular ion at m/z 451, which produced a strong signal, [M − H − 162]−, at m/z 289 due to the putative loss of a hexose moiety after CID. Since the MS3 fragmentation pattern of this daughter ion at m/z 289 was highly similar to the fragmentation pattern of an analytical (+)-catechin standard, compound 6 was tentatively identified as catechin hexoside. Compound 8 revealed a UV absorption spectrum similar to E


Article

Journal of Agricultural and Food Chemistry molecular ion of compound 13, [M − H]−, at m/z 175 produced a single detectable fragment at m/z 131, indicating the elimination of CO2 and, hence, the presence of a carboxyl function. However, we were unable to identify compound 13 and its tentative derivatives, compounds 21 and 27−29 (Table 1). Quantification of Phenolic Compounds. The identified (+)-piscidic acid represented the major phenolic compound (75−87% of all phenolic compounds) in all Quillaja extracts, ranging from 22.09 ± 0.12 to 33.99 ± 0.15 mg/g of DM (Table 2). Although quantitative reports about the natural occurrence of piscidic acid are scarce, dried Cimicifuga rhizomes were reported to contain from 0.28 to 4.47 mg of piscidic acid/g of DM previously.16 Compounds 3−5, 9−12, and 14 were minor phenolic compounds at levels below 3 mg/g of DM (7% of the total identified phenolic compounds). The p-coumaric acid derivative contents (compounds 19 and 20) ranged between 1.67 mg/g of DM (6.2%) and 8.54 mg/g of DM (18.8%) as shown in Table 2. Generally, all four Quillaja extract batches showed similar portions of their polyphenolics. However, both Andean QDP ultra organic batches revealed slightly higher total polyphenol concentrations (42.5 and 45.5 mg/g of DM, respectively) when compared to the batches of Andean QDP ultra and Andean Q ultra (27.8 and 27.1 mg/g of DM, respectively). Previously, the total phenolic content of a less purified Q. saponaria bark extract (20% sapogenins) has been assessed by the highly unspecific Folin−Ciocalteu assay to contain 72.14 mg of total phenolics/g of DM.17 Since the extracts investigated are frequently used for emulsification in commercial applications, we sought to compare the phenolic contents of Quillaja wood extracts with those of other polyphenol-rich emulsifiers. For instance, Rombouts and Thibault18,19 reported a polyphenol content of 1−2% (DM) in the alcohol-insoluble residue (AIR) obtained from sugar beet pulp, containing feruloyl groups covalently linked to the pectin molecule. Cashew tree gum exudate, representing a natural emulsifier, has been found to contain less than 0.5% (DM) phenols.20 Consequently, Quillaja extracts contain substantially higher levels of total phenolic compounds (up to 4.6%, w/w) than sugar beet pectin and cashew gum exudate. Since a number of characteristic phenolic acids were present in Quillaja wood extracts, their occurrence might be useful for the detection of Quillaja extracts in foods, cosmetics, and pharmaceutical formulations. In particular, the high content of 2.2−3.4% (w/w, DM) piscidic acid in the dried extracts is highly characteristic. Due to its monohydroxylated benzene moiety, piscidic acid is most likely susceptible to enzymatic oxidation. Monophenol oxidases (tyrosinase, EC 1.14.18.1) from plants and microorganisms efficiently catalyze the hydroxylation of monohydroxy phenolics to o-dihydroxy phenolics. The latter are rapidly oxidized to o-quinones by odiphenol:oxygen oxidoreductases (EC 1.10.3.2), which then nonenzymatically polymerize to yield commonly undesired brown or black melanins.21,22 Oxidases originating from other starting materials should be completely inactivated to prevent undesired browning during Quillaja processing. However, (+)-piscidic acid was previously shown to effectively complex Fe3+ in pigeon pea root exudates23 and, therefore, may also be capable of inactivating phenol oxidases by sequestering the copper cation cofactor of these enzymes. Besides enzymatic oxidation, autoxidation of Quillaja polyphenols might contrib-

ute to discoloration. The presence of catalysts such as excessive metal ions beyond the complexing capacity of piscidic acid as well as excessive oxygen and heat exposure might further accelerate the autoxidation of polyphenols.24,25 In addition, polyphenolic compounds with high affinity for iron ions were previously shown to exert an antimicrobial activity.26 Besides their high content in saponins, the ironsequestering piscidic acid may therefore represent a further antimicrobial and hemolytic agent of Quillaja extracts.27,28 In summary, the qualitative and quantitative characterization of phenolic compounds in different commercially available saponin-rich Q. saponaria extracts revealed a unique profile of polyphenols that might be applicable for their authentication. Furthermore, the high levels of polyphenols observed in the extracts might exert desired antioxidant properties but, at the same time, cause undesired browning reactions within the food matrix.

■

AUTHOR INFORMATION

Corresponding Author

*Phone +49 711 459 22995. Fax: +49 711 459 24110. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

■

REFERENCES

We acknowledge Pera Ingredients GmbH (Springe-Eldagsen, Germany) for generously providing Q. saponaria extract samples.

(1) Marciani, D. J., Triterpene saponin analogs having adjuvant and immunostimulatory activity. Patent US 5977081 A, 1999. (2) van Setten, D. C.; van de Werken, G. Molecular structures of saponins from Quillaja saponaria Molina. In Saponins Used in Traditional and Modern Medicine; Waller, G. R., Yamasaki, K., Eds.; Advances in Experimental Medicine and Biology, Vol. 404; Springer: New York, 1996; pp 185−193. (3) San Martín, R.; Briones, R. Industrial uses and sustainable supply of Quillaja saponaria (Rosaceae) saponins. Econ. Bot. 1999, 53, 302− 311. (4) San Martín, R.; Briones, R. Quality control of commercial quillaja (Quillaja saponaria Molina) extracts by reverse phase HPLC. Sci. Food Agric. 2000, 80, 2063−2068. (5) Kähkönen, M. P.; Hopia, A. I.; Vuorela, H. J.; Rauha, J.-P.; Pihlaja, K.; Kujala, T. S.; Heinonen, M. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 1999, 47, 3954− 3962. (6) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biol. Med. 1996, 20, 933−956. (7) Walther, R. U.; Padilla, L.; González, J.; Otero, R. Quillaja saponaria wood extract: Refined processing and forestry management guarantee sustainability and ecological benefits. Compend. Detergency Suppl. Household Pers. Care Today 2011, 20−22. (8) Takahira, M.; Kusano, A.; Shibano, M.; Kusano, G.; Miyase, T. Piscidic acid and fukiic acid esters from Cimicifuga simplex. Phytochemistry 1998, 49, 2115−2119. (9) Wolfram, K.; Schmidt, J.; Wray, V.; Milkowski, C.; Schliemann, W.; Strack, D. Profiling of phenylpropanoids in transgenic lowsinapine oilseed rape (Brassica napus). Phytochemistry 2010, 71, 1076− 1084. (10) Liu, J. X.; Di, D. L.; Shi, Y. P. Diversity of chemical constituents from Saxifraga montana H. J. Chin. Chem. Soc. 2008, 55, 863−870.

F


Article

Journal of Agricultural and Food Chemistry (11) Toshima, H.; Saito, M.; Yoshihara, T. Total syntheses of all four stereoisomers of piscidic acid via catalytic asymmetric dihydroxylation of (Z)- and (E)-trisubstituted olefins. Biosci., Biotechnol., Biochem. 1999, 63, 1934−1941. (12) Culbreth, D. M. A Manual of Materia Medica and Pharmacology; Health Research Books: Pomeroy, WA, 1996. (13) Bridge, W.; Coleman, F.; Robertson, A. Constituents of “Cortex Piscidiae erythrinae.” Part I. The structure of piscidic acid. J. Chem. Soc. 1948, 257−260. (14) Fischer, U. A.; Carle, R.; Kammerer, D. R. Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MSn. Food Chem. 2011, 127, 807−821. (15) Vanholme, R.; Storme, V.; Vanholme, B.; Sundin, L.; Christensen, J. H.; Goeminne, G.; Halpin, C.; Rohde, A.; Morreel, K.; Boerjan, W. A systems biology view of responses to lignin biosynthesis perturbations in Arabidopsis. Plant Cell 2012, 24, 3506− 3529. (16) Takahira, M.; Yanagi, M.; Kusano, A.; Shibano, M.; Baba, K.; Kusano, G.; Sakurai, N.; Nagai, M. Phenolic constituents of Cimicifuga species rhizomes. Nat. Med. 1998, 52, 330−338. (17) Nasri, S.; Luciano, G.; Vasta, V.; Aouadi, D.; Priolo, A.; Makkar, H. P. S.; Ben Salem, H. Effect of Quillaja saponaria dietary administration on colour, oxidative stability and volatile profile of muscle longissimus dorsi of Barbarine lamb. Meat Sci. 2012, 92, 582− 586. (18) Rombouts, F. M.; Thibault, J.-F. Enzymic and chemical degradation and the fine structure of pectins from sugar-beet pulp. Carbohydr. Res. 1986, 154, 189−203. (19) Rombouts, F. M.; Thibault, J.-F. Feruloylated pectic substances from sugar-beet pulp. Carbohydr. Res. 1986, 154, 177−187. (20) Marques, M. R.; Xavier-Filho, J. Enzymatic and inhibitory activities of cashew tree gum exudate. Phytochemistry 1991, 30, 1431− 1433. (21) Mayer, A. M. Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry 2006, 67, 2318−2331. (22) Martinez, M. V.; Whitaker, J. R. The biochemistry and control of enzymatic browning. Trends Food Sci. Technol. 1995, 6, 195−200. (23) Ae, N.; Arihara, J.; Okada, K.; Yoshihara, T.; Johansen, C. Phosphorus uptake by pigeon pea and its role in cropping systems of the Indian subcontinent. Science 1990, 248, 477−480. (24) Nkhili, E.; Loonis, M.; Mihai, S.; El Hajji, H.; Dangles, O. Reactivity of food phenols with iron and copper ions: Binding, dioxygen activation and oxidation mechanisms. Food Funct. 2014, 5, 1186−1202. (25) Talcott, S. T.; Howard, L. R. Phenolic autoxidation is responsible for color degradation in processed carrot puree. J. Agric. Food Chem. 1999, 47, 2109−2115. (26) Engels, C.; Schieber, A.; Gänzle, M. G. Inhibitory spectra and modes of antimicrobial action of gallotannins from mango kernels (Mangifera indica L.). Appl. Environ. Microbiol. 2011, 77, 2215−2223. (27) Hassan, S.; Byrd, J.; Cartwright, A.; Bailey, C. Hemolytic and antimicrobial activities differ among saponin-rich extracts from guar, quillaja, yucca, and soybean. Appl. Biochem. Biotechnol. 2010, 162, 1008−1017. (28) Wallace, R. J. Antimicrobial properties of plant secondary metabolites. Proc. Nutr. Soc. 2004, 63, 621−629.

G


Phenolic Constituents in Commercial Aqueous Quillaja (Quillaja

Recommend Documents