Article pubs.acs.org/JAFC
Identification and Characterization of the Phenolic Glycosides of Lagenaria siceraria Stand. (Bottle Gourd) Fruit by Liquid Chromatography−Tandem Mass Spectrometry Rakesh Jaiswal* and Nikolai Kuhnert* Chemistry, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany S Supporting Information *
ABSTRACT: Bottle gourd, Lagenaria siceraria Stand. (Cucurbitaceae), fruit is used in folk medicines and for culinary purposes in Asia. The phenolics of bottle gourd fruit were investigated qualitatively by LC−MSn. Twenty-two phenolic glycosides were detected and characterized on the basis of their unique fragmentation pattern in the negative ion mode tandem MS spectra. Twenty of them were extracted for the first time from this source, and twelve of them have not been reported previously in nature. It was also possible to distinguish between the individual classes of isobaric phenolic glycosides by tandem and highresolution mass spectrometry. In this study we also discuss the mass spectrometric fragmentation mechanism of 6(hydroxycinnamoyl)glucoses. This is the first report of the full characterization of phenolic glycosides of bottle gourd fruit by LCMS2−4. KEYWORDS: phenolic glycosides, caffeoylglucose, p-coumaroylglucose, feruloylglucose, Lagenaria siceraria Stand., LC−MSn, bottle gourd, caffeoyl glucoside−4-hydroxybenzyl alcohol, caffeoyl glucoside−3,4-dihydroxybenzyl alcohol, feruloyl glucoside−4-hydroxybenzyl alcohol, feruloyl glucoside−3,4-dihydroxybenzyl alcohol
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INTRODUCTION The phenolics comprise one of the largest and most diverse groups of phytochemicals found in plants and are essential components of the human and animal diets. Polyphenol compounds can range from simple phenolic acids (such as hydroxybenzoic acids, hydroxycinnamic acids, hydroxycinnamates, gallates, hydroxybenzoates, gallic acid, and vanillic acid) to complex natural polymers (such as tannins). The phenolics are claimed to have a series of fascinating biological activities, including antioxidant activity1 and anticancer,2,3 antimutagenic,4 antidiabetic,5 anti-inflammatory,6 and anti-HIV7 properties. These phenolics are also beneficial for wound healing8 and skin diseases,9 reduce the risks of cardiovascular diseases,10 and protect from drug toxicity11 and UV radiation. Bottle gourd (Lagenaria siceraria Stand.) fruit is used as a vegetable in Asia, especially in India, China, Japan, and Korea. The fruit is used in folk medicines and Ayurvedic medicines for the treatment of jaundice, diabetes, ulcer, piles, hypertension, skin diseases, constipation, nervous disorders, rheumatism, hair loss, and heart diseases.12,13 The fruit shows antibacterial, analgesic, anti-inflammatory, antihyperlipidemic, diuretic, antihelmintic, antistress, immunomodulatory, antihepatotoxic, and antioxidant activities.12−17 Bottle gourd fruit is a good source of nutrients such as vitamins, carbohydrates, proteins, fiber, essential amino acids, and minerals.13 It also contains flavonoids, terpenoids, essential oils, phenolic acids, and phenolic acid glycosides.17−19 Due to the presence of a large variety of phenolics and their biological activities and uses in medicine, bottle gourd is of great interest to be studied for its phenolic contents to identify suitable candidates for biological studies. © 2014 American Chemical Society
Recently, we used mass spectrometric methods for the positive identification and characterization of chlorogenic acids, hydroxycinnamoyl shikimates, methyl quinates, chlorogenic acid lactones, water addition products of chlorogenic acids, and proanthocyanidins.20−36 In the present study phenolic glycosides have been identified and characterized to their regioisomeric level by their unique fragmentation pattern in tandem mass spectra. This is the first time that LC−MSn has been used to identify and characterize phenolic glycosides of bottle gourd fruit.
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MATERIALS AND METHODS
All the chemicals (reagent and analytical grades) were purchased from Sigma-Aldrich (Bremen, Germany). The phenolics, caffeic acid, ferulic acid, p-coumaric acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, and 3,4-dihydroxybenzyl alcohol, were purchased from Sigma-Aldrich (Bremen, Germany). Fresh fruit of L. siceraria Stand. was purchased from an Indian supermarket in Bremen (Germany). Sample Preparation. Fruit (5 g) of L. siceraria Stand. was freezedried at −20 °C overnight, extracted with aqueous methanol (100 mL, 70%), homogenized with a blender, and ultrasonicated for 30 min. This extract was filtered through Whatman no. 1 filter paper. The solvents were removed by evaporation in vacuo, and the extract was stored at −20 °C until required, thawed at room temperature, dissolved in methanol (60 mg/10 mL of methanol), filtered through a membrane filter, and used directly for LC−MS. LC−MSn. The LC equipment (Agilent 1100 series, Bremen, Germany) comprised a binary pump, an autosampler with a 100 μL Received: Revised: Accepted: Published: 1261
November 29, 2013 January 21, 2014 January 22, 2014 January 22, 2014 dx.doi.org/10.1021/jf4053989 | J. Agric. Food Chem. 2014, 62, 1261−1271
Journal of Agricultural and Food Chemistry
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Figure 1. continued
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dx.doi.org/10.1021/jf4053989 | J. Agric. Food Chem. 2014, 62, 1261−1271
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Article
Figure 1. Representative structures of Lagenaria siceraria Stand. phenolic glycosides. loop, and a DAD detector with a light-pipe flow cell (recording at 254, 280, and 320 nm and scanning from 200 to 600 nm). This was interfaced with an ion-trap mass spectrometer fitted with an ESI source (Bruker Daltonics HCT Ultra, Bremen, Germany) operating in full-scan, auto-MSn mode to obtain fragment ion m/z. Tandem mass spectra were acquired in auto-MSn mode (smart fragmentation) using a ramping of the collision energy. The maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. MS operating conditions (negative mode) had been optimized using 5-Ocaffeoylquinic acid with a capillary temperature of 365 °C, a dry gas flow rate of 10 L/min, and a nebulizer pressure of 10 psi. Highresolution LC−MS was performed using the same HPLC instrument equipped with a MicrOTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) fitted with an ESI source, and internal calibration was achieved with 10 mL of 0.1 M sodium formate solution injected through a six-port valve prior to each chromatographic run. Calibration was done using the enhanced quadratic mode. HPLC Coupled to MS. Separation was achieved on a 250 × 3 mm i.d. column containing diphenyl 5 μm, with a 5 mm × 3 mm i.d. guard column (Varian, Darmstadt, Germany). Solvent A was water/formic acid (1000:0.05, v/v), and solvent B was methanol. Solvents were delivered at a total flow rate of 500 μL/min. The gradient profile was from 10% B to 70% B linearly in 70 min followed by isocratic conditions for 10 min and a return to 10% B at 90 and isocratic conditions for 10 min to re-equilibrate. Base Hydrolysis. A 10 mg sample of plant extract was dissolved in 2 mL of 0.5 M NaOH solution and the resulting solution stirred at room temperature for 2 h. The reaction mixture was neutralized by adding 1 g of acidic resin (Amberlite IR-120, H+). The reaction mixture was filtered, washed with water (2 mL), and directly used for HPLC−MSn. NMR. 1H and 13C NMR spectra were recorded on a JEOL-ECX 400 spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR at room temperature in acetone-d6 or methanol-d4 using a 5 mm probe. The chemical shifts (δ) are reported in parts per million and were referenced to the residual solvent peak. The coupling constants (J) are quoted in hertz. Synthesis of 6-O-Caffeoylglucoses 17 and 18. To a solution of 1,2-O-isopropylidene-α-D-glucofuranose (1.00 g, 4.54 mmol) and DMAP (200 mg, 1.63 mmol) in DCM (50 mL) were added pyridine (5 mL, 62 mmol) and 3,4-di-O-allylcaffeoyl chloride (1.58 g, 5.70 mmol)22 at room temperature. The reaction mixture was stirred for 24 h at room temperature and acidified (pH ≈ 3) with 2 M HCl. The layers were separated, and the aqueous phase was extracted with DCM (3 × 50 mL). The combined organic layers were dried over sodium
sulfate and filtered, and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate−petroleum ether, 30−50%) to give 6-O-(3,4-di-O-allylcaffeoyl)-1,2-O-isopropylidene-α-D-glucofuranose (80%): δH (acetone-d6) 7.62 (1H, d, J = 15.6, CArCH), 7.32 (1H, dd, J = 1.8, CArH), 7.17 (1H, dd, J = 8.7, 2.3 CArH), 6.99 (1H, d, J = 8.2, CArH), 6.41 (1H, d, J = 16.0, CArCHCH), 6.08 (2H, m, CH2CH), 5.85 (1H, d, J = 3.6, H3), 5.43 (2H, m, CHHCH), 5.23 (2H, m, CHHCH), 4.65 (4H, m, CArOCH2), 4.49 (1H, d, J = 3.7, H-2), 4.31 (2H, m, H-6a, H-6b), 4.12 (2H, m, H-4, H-5), 1.39 (3H, s, OCH3), 1.24 (3H, s, OCH3); δC (acetone-d6) 166.6 (−COOC), 150.9 (CArOCH2), 148.9 (CArOCH2), 144.5 (CArCH), 133.9 (CH2CH), 133.5 (CH2CH), 127.7 (CArCH), 122.9 (CAr), 116.9 (CH2CH), 116.5 (CHCOO), 115.7 (CAr), 113.7 (CAr), 112.6 (H3COCOCH3), 111.2 (H3COCOCH3), 105.0 (C-3), 85.3 (C-6), 80.4 (C-1), 74.4 (C-2), 69.4 (C-4), 69.2 (CArOCH2), 67.1 (CArOCH2), 66.8 (C-5), 26.4 (OCH3), 25.6 (OCH3). To a solution of 6-O-(3,4-di-O-allylcaffeoyl)-1,2-O-isopropylideneα-D-glucofuranose (1.00 g, 2.23 mmol) and p-TsOH (50 mg) in methanol−water (9:1, 50 mL) was added 10% Pd/C (350 mg) at room temperature. The reaction mixture was heated at 70 °C for 48 h, cooled to room temperature, and filtered, and methanol was removed in vacuo. The aqueous reaction mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried over sodium sulfate and filtered, and the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel (ethyl acetate−petroleum ether, 50−95%) to give 6-O-caffeoyl-1,2-Oisopropylidene-α-D-glucofuranose (62%). The resulting ester 6-Ocaffeoyl-1,2-O-isopropylidene-α-D-glucofuranose (400 mg, 1.10 mmol) was dissolved in a mixture of 20 mL of TFA and water (8:2) at room temperature and the resulting solution stirred for 1 h. The solvents were removed in vacuo to obtain the resulting esters 6-Ocaffeoylglucoses 17 and 18 in quantitative yield: δH (methanol-d3) 7.54 (1H, d, J = 16.0, CArCH), 7.02 (1H, d, J = 2.2, CArH), 6.92 (1H, dd, J = 8.2, 1.4, CArH), 6.75 (1H, d, J = 8.2, CArH), 6.25 (1H, J = 16.5, CArCHCH), 5.10 (1H, d, J = 3.7, gluc H-1), 4.45 (1H, m, gluc H6a), 4.30 (1H, m, gluc H-6b), 4.05 (1H, m, gluc H-4), 3.28−3.37 (2H, m, gluc H-2, gluc H-5); δC (methanol-d3) 167.9 (COOC), 148.3 (CAr), 145.8 (CArCH), 145.5 (CAr), 126.4 (CAr), 123.6 (CAr), 121.6 (CAr), 115.2 (CH2CH), 113.7 (CHCOO), 113.6 (CH2CH), 96.8 (βgluc C-1), 92.6 (α-gluc C-1), 76.7 (β-gluc C-3), 74.9 (β-gluc C-2), 74.1 (β-gluc C-5), 73.5 (α-gluc C-3), 72.2 (α-gluc C-2), 71.6 (α-gluc C-5), 70.4 (α-gluc C-4), 69.6 (β-gluc C-4), 63.6 (β-gluc C-6), 63.5 (α-gluc C6). 1263
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Article
RESULTS AND DISCUSSION
For all the compounds the high-resolution mass data were in good agreement with the theoretical molecular formulas, all displaying a mass error of below 5 ppm, thus confirming their elemental composition. In general, peak identities were consistent both within and between analyses. However, when the mass spectrum for a particular substance included two ions of similar mean intensities, within-analysis experimental error dictated that in some individual MS scans one would be more intense while for other scans the reverse would be true. This phenomenon was encountered primarily when the signal intensity was lower, that is, with quantitatively minor components and/or higher order spectra. For example, one of the phenolics (21) produces MS2 ions at m/z 235 and 265 which were essentially coequal in some spectra. However, in this particular case the higher mass ion was assigned consistently as the base peak. Fragment ions with intensities
