Article pubs.acs.org/JAFC
Quantitative Analysis of Phenylpropanoid Glycerol Glucosides in Different Organs of Easter Lily (Lilium longiflorum Thunb.) John P. Munafo, Jr. and Thomas J. Gianfagna* Plant Biology Department, RutgersThe State University of New Jersey, 59 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT: The Easter lily (Lilium longiflorum Thunb.) is esteemed worldwide as an attractive ornamental plant, and the flower buds and bulbs are used for both culinary and medicinal purposes in many parts of the world. L. longif lorum contains significant amounts of phenylpropanoid glycerol glucosides, a group of compounds that may contribute to plant pathogen defense, ultraviolet/high-intensity visible light (UV/high light) protection, and the purported medicinal uses of lilies. To define the natural distribution of these compounds within the plant, a liquid chromatography−mass spectrometry (LC-MS) method performed in selected ion monitoring (SIM) mode was employed for the quantitative analysis of five phenylpropanoid glycerol glucosides, namely, (2S)-1-O-caffeoyl-2-O-β-D-glucopyranosylglycerol, 1; (2R)-1-O-β-D-glucopyranosyl-2-O-p-coumaroylglycerol, 2; (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosylglycerol, 3; (2S)-1-O-caffeoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 4; and (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 5, in the different organs of L. longif lorum. The pcoumaroyl-based 3 and its acetylated derivative 5 were determined to be the most abundant of the phenylpropanoid glycerol glucosides found in Easter lily bulbs, at 776.3 ± 8.4 and 650.7 ± 32.6 μg/g dry weight, respectively. The acetylated p-coumaroyland caffeoyl-based derivatives, 5 and 4, accumulated to the highest concentration in the closed flower buds, at 4925.2 ± 512.8 and 3216.8 ± 406.4 μg/g dry weight, respectively. Compound 4, followed by 5 and 1, proved to be the most abundant in the mature flowers, occurring at 6006.2 ± 625.8, 2160.3 ± 556.5, and 1535.8 ± 174.1 μg/g dry weight, respectively. Total concentrations of the phenylpropanoid glycerol glucosides were 10−100-fold higher in the above-ground plant organs as compared to the bulbs and fleshy roots. Two of the five compounds, 1 and 2, were identified in L. longif lorum for the first time. The quantitative analysis of phenylpropanoid glycerol glucosides in the different plant organs of L. longif lorum will establish the direction of investigations aimed at defining how these compounds function in the physiology and chemical ecology of the plant and also as a starting point for determining their possible effects on human health, which has not been investigated. KEYWORDS: Lilium longiflorum Thunb., Liliaceae, Easter lily, phenylpropanoid glycerol glucosides, regalosides
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flavonoids,6 carotenoids,7 sterols,7 steroidal saponins,2,8−17 and steroidal glycoalkaloids.15,17−19 In addition, the Lilium genus contains an abundant supply of cinnamic acid derivatives, in particular, phenylpropanoid glycerol glucosides, a group of natural products, with many structural analogues, first isolated and characterized from Lilium regale E. H. Wilson and thus trivially named regalosides.20 Cinnamic acid derivatives, most notably p-coumaric, caffeic, and ferulic acids, are widely distributed in plants and naturally occur as both esters and glycosides. Regalosides are acylated glycerol glucosides. As such, they characteristically contain p-coumaric, caffeic, or ferulic acids, but less often cinnamic acid. Typically, they are esterified at the C1 or C2 carbon of a glucosylated glycerol backbone, with S configuration at the 2-position, (2S)regalosides, being the most common, and R configuration, (2R)-regalosides, less usual. Moreover, a few acetylated and methylated regalosides have been described.6,9,20−22 Regalosides have been identified in members of the Liliaceae family including L. regale,20 Lilium henryi Baker,21 Lilium pardalinum Kellogg,3 Lilium auratum Lindl.,3,23 Lilium lancifolium Thunb.,22
INTRODUCTION The Easter lily (Lilium longiflorum Thunb., family Liliaceae) is primarily grown as an ornamental plant because of its large, fragrant, white flowers. Although not well-known as a food in the United States, lily flower buds and bulbs are used as both food and medicine in many Asian countries, especially China and Japan. In Chinese cuisine the bulbs of various lily species, including L. longif lorum, are a common ingredient in soups, stirfries, and stew-like dishes such as “hot pot”. Lily bulbs and flower buds are an important part of the Asian diet and are consumed on a regular basis. Despite this fact, very little is known about their chemical composition and impact on human health. In addition to their culinary value, lily bulbs have a long history of documented medicinal use as sedatives, antiinflammatory agents, and antitussives.1−3 In fact, preparations of lily bulbs from Lilium species, referred to in China as “Baihe,” are employed in Traditional Chinese Medicine (TCM) as effective treatments for inflammation and some lung ailments.1,2 However, although the medicinal use of L. longif lorum is well documented, the bioactive phytochemicals responsible for the putative medicinal properties as well as their mechanisms of action remain elusive. Plants from the Lilium genus are an abundant source of a number of natural products. Many classes of compounds have been reported from the genus, including phenolics,4 alkaloids,5 © 2015 American Chemical Society
Received: Revised: Accepted: Published: 4836
February April 23, April 23, April 23,
16, 2015 2015 2015 2015 DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
Article
Journal of Agricultural and Food Chemistry L. longif lorum,6,22 Lilium brownii F. E. Br. ex Miellez,2,15 Lilium speciosum Thunb.,14 and Lilium mackliniae Sealy.9 Earlier studies have identified phenylpropanoid glycerol glucosides in the bulbs of L. longif lorum, specifically, (2S)-1O-p-coumaroyl-2-O-β-D-glucopyranosylglycerol, 3; (2S)-1-Ocaffeoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 4; and (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 5.22 Additionally, a 4-acetyl derivative of (2S)-1-O-pcoumaroyl-2-O-β-D-glucopyranosylglycerol has been reported from L. longif lorum flowers that exhibited mild cyclooxygenase and lipid peroxidation inhibition activities.6 Although these compounds may be involved in plant pathogen defense, ultraviolet/high-intensity visible light (UV/high light) protection, and the putative medicinal activities of lilies,1−3,17 reports on the biological activities of phenylpropanoid glycerol glucosides are scarce. Phenylpropanoid glycerol glucosides are reported to be bitter in taste,20 which may suggest a possible role in antiherbivory; however, the bitter taste thresholds of the compounds have not been quantitated nor have the antiherbivory activities of the compounds been investigated. The dual aim of our present investigation has been to develop a liquid chromatography−mass spectrometry (LC-MS) method for the quantitative analysis of five phenylpropanoid glycerol glucosides in plant tissues and to identify and quantitate the concentration of the compounds in the different plant organs of Easter lily. The results were anticipated to lay the groundwork for defining how these compounds function in the physiology and chemical ecology of the plant and also as a starting point for determining their possible effects on human health when consumed as a food, which has not been investigated.
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residue was then allowed to dry overnight in a fume hood. The dried defatted residue was extracted with ethanol and DI water (7:3, v/v; 2 × 150 mL) for 45 min at room temperature on a shaker. The extract was centrifuged (5000 rpm) for 10 min and the supernatant was saved and vacuum filtered. The supernatant was rotary evaporated (30 °C; 1.0 × 10−3 bar) and lyophilized, yielding a crude bulb extract (fraction I, 12.4 g). Next, the lyophilized crude bulb extract was dissolved in DI water (100 mL) and extracted with ethyl acetate (5 × 100 mL), and the aqueous phase, containing the highly polar compounds such as carbohydrates and steroidal glycosides, was discarded. The organic phase was rotary evaporated to dryness (30 °C; 1.0 × 10−3 bar) (fraction II), redissolved in DI water (100 mL), and sonicated for 10 min. The extract was centrifuged (5000 rpm) for 10 min, vacuum filtered, and the supernatant was collected. The aqueous phase was then extracted with 1-butanol (5 × 100 mL). The organic phase was rotary evaporated (30 °C; 1.0 × 10−3 bar) and lyophilized, yielding 1butanol extract (fraction III, 1.9 g). Semipreparative Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The 1-butanol extract (fraction III) was fractionated by semipreparative RP-HPLC on a 250 mm × 21.2 mm i.d., 10 μm, Luna C18 column (Phenomenex, Torrance, CA) to afford 1−5 (Figure 1) using an LC-6AD liquid chromatograph (Shimadzu
MATERIALS AND METHODS Figure 1. Structures of compounds 1−5 quantitated in the various L. longif lorum organs.
Plant Material. Cultivar 7-4 L. longiflorum bulbs were cultivated, harvested, lyophilized, and milled according to the methods described in Munafo et al.19 Three fully mature plants, containing all stages of the vegetative and reproductive organs, were harvested and dissected into fleshy bulb scales, fleshy roots, leaves, lower stems, upper stems, flower buds, and mature flowers. The fleshy bulb scales were 0.8−2.0 cm in width and 0.9−4.0 cm in length. The fleshy roots were approximately 2−4 mm in diameter and the leaves about 6−14 cm in length. Lower stems (the part growing below the soil surface) ranged in size from 6 to 10 cm and were white to yellow in color. Upper stems (the part growing above the soil surface) ranged in size from 19 to 31 cm and were green. The flower buds were approximately 3−6 cm in length, fully closed, and green in appearance. The mature flowers ranged between 6 and 14 cm in length, were fully open, and were white in appearance. Each of the organ types from an individual plant were pooled together. Three individual plants were used in the analysis (n = 3). After harvest, all samples were immediately frozen under liquid nitrogen, lyophilized, and stored at −80 °C prior to analysis. Chemicals. All solvents, acetonitrile, 1-butanol, dimethyl sufoxide (DMSO), ethyl acetate, ethanol, formic acid, methanol, tetrohydrofuran (THF), and pentanes were of chromatographic grade and were obtained from Thermo Fisher Scientific Inc., Fair Lawn, NJ, USA. Methanol-d4 (0.03% v/v TMS) was obtained from Sigma-Aldrich, St. Louis, MO, USA. Deionized (DI) water (18 MΩ cm) was generated using a Milli-Q-water purification system (Millipore, Bedford, MA, USA). Purification of Phenylpropanoid Glycerol Glucosides 1−5 as Analytical Standards. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs. Lyophilized lily bulbs (100 g) were milled to a fine powder after freezing in liquid nitrogen. To remove lipids, the bulb powder was extracted with pentanes (3 × 100 mL) at room temperature for 15 min each time on a shaker. The extract was centrifuged (5000 rpm) for 10 min and the solvent was decanted. The
Scientific Instruments Inc., Columbia, MD, USA) equipped with a UV/vis detector and a 2 mL injection loop. The mobile phases were mixtures of (A) 0.1% formic acid in DI water and (B) 0.1% formic acid in acetonitrile. The flow rate was set to 20 mL/min, column temperature was 23 ± 2 °C, and UV detection was recorded at λ 210 nm. The 1-butanol extract (fraction III, 200 mg) was dissolved in a mixture of mobile phase A and mobile phase B (90:10, v/v; 2 mL) and filtered through a 0.45 μm PTFE syringe filter prior to injection (injection volume = 1 mL). The butanol extract was fractionated using a linear gradient of 5−24.4% B for 35 min and then ramped to 90% B in 5 min, with a hold at 90% B for additional 5 min. The solvent mixture was reset to 5% B for 10 min before subsequent injections. Fractions containing the compounds of interest were collected (Figure 2), and the solvent was rotary evaporated (30 °C; 1.0 × 10−3 bar) and lyophilized, yielding 1 (15.2 mg), 2 (12.1 mg), 3 (39.6 mg), 4 (10.2 mg), and 5 (40.3 mg) as pale yellow amorphous powders with >98% purity as determined by LC-MS and NMR. Compound 1, (2S)-1-O-Caf feoyl-2-O-β-D-glucopyranosylglycerol (regaloside K). 1D and 2D NMR data were consistent with 1H NMR and 13 C NMR reported in the literature.9 Compound 2, (2R)-1-O-β-D-Glucopyranosyl-2-O-p-coumaroylglycerol (regaloside H). 1D and 2D NMR data were consistent with 1H NMR and 13C NMR reported in the literature.23 Compound 3, (2S)-1-O-p-Coumaroyl-2-O-β-D-glucopyranosylglycerol (regaloside D). 1D and 2D NMR data were consistent with 1H NMR and 13C NMR reported in the literature.3 Compound 4, (2S)-1-O-Caf feoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol (regaloside E). 1D and 2D NMR data were consistent with 1H NMR and 13C NMR reported in the literature.3 4837
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Journal of Agricultural and Food Chemistry
Figure 2. Semipreparative RP-HPLC chromatogram (λ 210 nm) of 1−5 purified from L. longif lorum 1-butanol extract (fraction III).
Figure 3. LC-MS chromatogram for the quantitative analysis of compounds 1−5 in a L. longif lorum bulb scale operating in selective ion monitoring (SIM) mode. Compound 5, (2S)-1-O-p-Coumaroyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol (regaloside B). 1D and 2D NMR data were consistent with 1 H NMR and 13C NMR reported in the literature.20 Nuclear Magnetic Resonance Spectroscopy (NMR). 1D 1H NMR spectra was acquired with a 400 MHz spectrometer (Bruker, Rheinstetten, Germany). 2D heteronuclear multiple-bond coherence (HMBC), heteronuclear single-quantum coherence (HSQC), and H− H correlation spectroscopy (H−H COSY) spectra were acquired on a 500 MHz spectrometer (Bruker). Samples for NMR analysis were dissolved in methanol-d4, and chemical shifts were calculated as δ values with reference to tetramethylsilane (TMS). Optimization of Sample Extraction Parameters for Quantitative Analysis. Solvent Composition and Extraction Efficiency Determination. Six neat organic solvents (methanol, ethanol, DMSO, acetone, acetonitrile, and THF) as well as different ratios of methanol and DI water (1:0, 9:1. 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:1, v/ v) were used for experiments aimed at optimization of the extraction solvent system for phenylpropanoid glycerol glucosides 1−5 from lyophilized L. longiflorum tissue. Performing successive extraction of the same plant tissue from one to three times determined extraction efficiency. Lyophilized lily bulb tissue was extracted with methanol and DI water (8:2, v/v) solvent mixture. Results from each successive extraction were collected separately and filtered through a 0.45 μm PTFE syringe filter prior to LC-MS analysis.
Quantitative Analysis of Phenylpropanoid Glycerol Glucosides 1−5 in L. longif lorum. Sample Preparation. Following removal from the freezer, lyophilized lily organ samples were thawed to ambient temperature. The samples (40 mg each) were milled to pass through a 200 mesh sieve, weighed separately, transferred into a 15 mL centrifuge tube, and covered with methanol and DI water (8:2, v/v; 7 mL each). The samples were extracted with shaking (10 min), sonicated (10 min), and filled to final volume (10 mL) with methanol and DI water (8:2, v/v). The samples were vortexed (1 min), and centrifuged (5000 rpm for 10 min), and the supernatant was collected. The pellet was re-extracted with methanol and DI water (8:2, v/v; 10 mL), shaken (10 min), sonicated (10 min), centrifuged (5000 rpm for 10 min), and the supernatants were combined (total volume ∼ 20 mL). Prior to LC-MS analysis, all samples were filtered through a 0.45 μm PTFE syringe filter. Analytical Standard Preparation. Phenylpropanoid glycerol glucosides 1−5, isolated and purified as described above, were used as analytical standards. Each compound was weighed into volumetric flasks (10 mL), dissolved with methanol and DI water (8:2, v/v; 7 mL each), and sonicated for 5 min before taken to full volume. Working solutions were prepared for calibration equations by diluting the stock solutions. External standard equations were calculated from six data points covering a concentration range of 1−300 μg/mL. To establish calibration equations, mean peak areas (n = 3) from the standard 4838
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Journal of Agricultural and Food Chemistry
Figure 4. Evaluation of six organic solvents on the extraction of compounds 1−5 from L. longif lorum bulbs. Concentrations are means of triplicates ± SD. solutions were plotted versus concentration. Standard solutions were stored at 4 °C and equilibrated to ambient temperature before use. Liquid Chromatography−Mass Spectrometry (LC-MS). LC-MS analysis of L. longif lorum extracts was performed using an Agilent 1100 series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an autoinjector, quaternary pump, column heater, diode array detector and interfaced to a 1100 series single-quadruple mass selective detector (MSD) equipped with an API-ESI ionization source. Chromatographic separations were made on a 250 mm × 4.6 mm i.d., 5 μm Prodigy C18 column (Phenomenex) operated at a flow rate of 1.0 mL/min, column temperature set to 23 ± 2 °C, and an injection volume of 10 μL. The binary mobile phase consisted of (A) 0.1% formic acid in DI water and (B) 0.1% formic acid in acetonitrile. Phenylpropanoid glycerol glucosides were separated using a linear gradient of 15−34.6% B for 28 min and then ramped to 95% B in 5 min with a hold at 95% B for 5 min. The system was reset to 15% B for 10 min before each sample injection. ChemStation software was used for data acquisition and analysis. Negative ionization mode was used to quantify the phenylpropanoid glycerol glucosides using SIM. The following ions were selected for each compound: 1, m/z 415; 2, m/z 399; 3, m/z 399; 4, m/z 457; and 5, m/z 441 (Figure 3). Ionization conditions were as follows: capillary voltage, 3.5 kV; nebulizer pressure, 35 psi; drying gas flow, 10.0 mL/min; and drying gas temperature, 350 °C. MSD signal parameters included mode, SIM; polarity, negative; fragmentor voltage, 70 V; gain, 1.0; dwell time, 144 ms; and percent relative dwell time, 25. Recovery. The standard addition method was used to calculate recovery rates.24 Lyophilized lily organs were milled to pass through a 200 mesh sieve, separately weighed (40 mg), and transferred into 15 mL centrifuge tubes containing one of three different concentrations (50, 100, and 200 μg/g) of pure compounds dissolved in methanol and DI water (8:2, v/v). To the sample tubes was added a solution of methanol and DI water (8:2, v/v; 7 mL), and the extract was shaken (10 min), sonicated (10 min), and then filled to final volume (10 mL) with the same solvent. The samples were vortexed (1 min) centrifuged (5000 rpm for 10 min), and the supernatant was recovered. For a second time, the centrifuge tube containing the pellet was filled to final volume (10 mL), extracted with shaking (10 min), sonicated (10 min), and centrifuged (5000 rpm for 10 min), and the supernatants from the two extractions were pooled (total volume ∼ 20 mL). All samples were syringe filtered (0.45 μm PTFE) and the phenylpropanoid glycerol glucosides quantified as described above. The percentage recovery for each of the phenylpropanoid glycerol glucosides 1−5 in vegetative and reproductive organs was determined by subtracting the amount found in the lily organ sample that was not spiked with additional standards
(control) from the amount of standard found in the spiked sample, with three replicate samples for each analysis. Statistical Analysis. To compare differences in phenylpropanoid glycerol glucosides content in the lily plant organs, LC-MS data were first transformed to the logarithmic function, and an analysis of variance (ANOVA) was conducted. Means were separated with Tukey’s test (α = 0.05) using JMP 10 software (SAS Institute Inc., Cary, NC, USA). Two-way hierarchal cluster analysis (HCA) and the resulting color map, visually depicting the differences in concentration of compounds 1−5 throughout the lily plant organs, were generated using JMP 10 software (SAS Institute Inc.).
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RESULTS AND DISCUSSION
Isolation and Identification of Compounds 1 and 2 from L. longif lorum Bulbs. The crude 1-butanol extract was fractionated by repeated semipreparative RP-HPLC to yield compounds 1−5. On the basis of 1D 1H and 2D (HMBC, HSQC, and H−H COSY) NMR, compounds 3, 4, and 5 were confirmed to be (2S)-1-O-p-coumaroyl-2-O-β-D-gucopyranosylglycerol, (2S)-1-O-caffeoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, and (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosyl3-O-acetylglycerol, respectively. All three of these compounds have been previously reported from L. longif lorum bulbs.22 On the basis of 1D 1H and 2D (HMBC, HSQC, and H−H COSY) NMR, compounds 1 and 2 were confirmed to be (2S)-1-Ocaffeoyl-2-O-β-D-glucopyranosylglycerol, 1, and (2R)-1-O-β-Dglucopyranosyl-2-O-p-coumaroylglycerol, 2, respectively. Compound 1 was previously reported from fresh bulb scales of L. mackliniae9 and 2 from L. auratum.23 This is the first report of 1 and 2 as natural products identified in L. longif lorum. Optimization of Quantitative Extraction Parameters. Solvent Composition Determination. The determination of an effective extraction solvent system for the quantitative analysis of 1−5 was achieved by first analyzing the resulting extracts prepared from six neat organic solvents, namely, methanol, ethanol, DMSO, acetone, acetonitrile, and THF (Figure 4). Various combinations of methanol and DI water were then evaluated with the goal of determining a solvent system that effectively extracted 1−5. On the basis of these data, the combination of methanol and DI water (8:2, v/v) was chosen 4839
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Journal of Agricultural and Food Chemistry
each of the plant organs (bulb scales, 94.7−100.8%; roots, 94.8−101.4%; leaves, 96.7−102.9%; lower stems, 93.7−99.8%; upper stems, 94.9−103.3%; flower buds, 96.3−102.0%; and mature flowers, 95.2−101.9%). For compounds 1−5 the recovery rates in all of the plant organs assayed were between 93.7 and 103.3%. The precision of the method was evaluated by analyzing the same sample multiple times (bulb; n = 6) and subsequently calculating the relative standard deviation (RSD) of 1−5 in the sample. The RSD values for 1−5 were 4.14, 3.53, 2.81, 2.73, and 3.57%, respectively. These data demonstrate satisfactory linearity, recovery rates, and RSD, which suggests that the LC-MS method operating in SIM mode is a suitable method for the quantitation of phenylpropanoid glycerol glucosides 1−5 in L. longif lorum plant organs. Quantitation of Phenylpropanoid Glycerol Glucosides 1−5 in the Different Organs of L. longif lorum. Concentrations of 1−5 were quantitatively determined in all plant organs of L. longif lorum (Table 1; Figure 6). The pcoumaroyl-based compound 3 and its acetylated derivative 5 were determined to be the most abundant of the phenylpropanoid glycerol glucosides found in Easter lily bulbs, at 776.3 ± 8.4 and 650.7 ± 32.6 μg/g dry weight (dw), respectively. As previously mentioned, lily bulbs are regularly consumed as a food, and preparations from lily bulbs, referred to in China as “Bai-he”, are employed as effective treatment for various inflammations and some lung ailments; however, the bioactive phytochemicals responsible for the putative medicinal properties remain unknown.1,2 Compounds 3 and 5 occurred at the highest concentration in the root tissues at 584.4 ± 34.3 and 345.8 ± 32.6 μg/g dw, respectively. Similar to the bulbs and roots, 3 and 5 occurred at the highest concentration in the upper stem and lower stem. Compound 3 occurred at higher concentrations in the lower stem versus the upper stem, and the concentrations of compound 5 were similar in both the lower and upper stems. The above-ground plant organs of Easter lily (upper stems, leaves, buds, and flowers) showed a different trend of phenylpropanoid glycerol glucoside concentrations from that of the below-ground plant organs (bulbs, roots, and lower stem). The above-ground organs displayed more caffeoyl-based compounds and a greater abundance of acetylation than the below-ground organs. The caffeoyl-based compounds 1 and 4 were the highest in the leaves, at 2007.4 ± 207.5 and 1786.0 ± 200.9 μg/g dw, followed by the p-coumaroyl-based compounds 3, 2, and 5, at 1074.8 ± 53.1, 1003.5 ± 36.4, and 935.1 ± 103.9 μg/g dw, respectively. The acetylated p-coumaroyl- and caffeoyl-based derivatives, 5 and 4, had the highest concentrations in the closed flower buds, at 4925.2 ± 512.8 and 3216.8
as the optimum solvent system of the solvent combinations evaluated (Figure 5).
Figure 5. Evaluation of various ratios of methanol and DI water on the extraction of compounds 1−5 from L. longiflorum bulbs. Concentrations are means of triplicates ± SD.
Extraction Efficiency. Sequential solvent extraction of the lyophilized lily bulb powder using methanol and DI water (8:2, v/v) was employed to estimate the optimal number of extraction cycles for quantitative analysis. The results indicated that between 77.6 and 89.6% of compounds 1−5 were recovered in the first extraction. An additional 9.3−17.7% was found with the second extraction and approximately 0−5.1% after the third cycle. On the basis of these observations, the combined supernatants generated from two successive extraction cycles were chosen for further quantitative analysis, resulting in an estimation of 94.4−98.9% extraction efficiency for compounds 1−5. Method Validation. To quantitate the distribution of five phenylpropanoid glycerol glucosides in L. longif lorum organs, 1−5 were purified from lily bulbs as analytical reference standards. For quantitative analysis, extracts were prepared from lyophilized plant parts and analyzed using LC-MS in SIM mode. To determine linear relationships between peak area and concentration for the quantitative method, six-point calibration plots were constructed, which demonstrated good linearity over the concentration range of 1.0−300 μg/mL for compounds 1− 5. The correlation coefficients ranged from R2 = 0.9998 to 0.9999. Accuracy of the method was assessed by calculating recovery rates for 1−5 in each of the plant organs using the standard addition method.24 Recovery rates were calculated for
Table 1. Concentrations of Compounds 1−5 in the Different Organs of L. longif lorum compound concentrationa (μg/g dw) organ bulb root leaf lower stem upper stem bud flower
1 68.4c 95.7c 2007.4a 381.3c 274.1c 762.9d 1535.8b
± ± ± ± ± ± ±
2 b
11.4 12.6 207.5 49.0 15.6 34.0 174.1
80.3bc 62.8d 1003.5b 152.8e 242.7c 636.7d 385.9d
3 ± ± ± ± ± ± ±
8.43 1.70 36.4 13.0 25.4 40.5 10.6
776.3a 584.4a 1074.8b 1281.7a 1011.3a 1771.2c 736.2c
± ± ± ± ± ± ±
4 8.4 34.3 53.1 26.5 35.2 104.3 16.4
116.7b 85.5c 1786.0a 275.8d 619.0b 3216.8b 6006.2a
± ± ± ± ± ± ±
5 29.1 12.5 200.9 14.1 34.6 406.4 625.8
650.7a 345.8b 935.1b 860.0b 1125.0a 4925.2a 2160.3b
± ± ± ± ± ± ±
32.6 17.1 103.9 47.2 105.7 512.8 556.5
Concentrations are means of triplicates ± SD, expressed on a dry weight basis (dw). bValues with the same letter in each row are not significantly different (p < 0.05). a
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DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Journal of Agricultural and Food Chemistry
Figure 6. Two-way hierarchal cluster analysis (HCA). Color map visually depicting the differential concentrations of compounds 1−5 in the different plant organs of L. longif lorum. Colored branches on the dendrogram denote clusters. Upper stem, lower stem, root, and bulb, red cluster; flower and leaf, blue cluster; bud, green cluster.
± 406.4 μg/g dw, respectively. In parts of Asia, lily flower buds, similarly to lily bulbs, are commonly consumed as snacks and in soups, stir-fries, and stew-like dishes. Easter lily flower buds are rich in acetylated phenylpropanoid glycerol glucosides 5 and 4 and differ dramatically from the phytochemical profile of the bulbs. Most abundant in the mature flowers was compound 4, followed by 5 and 1, occurring at 6006.2 ± 625.8, 2160.3 ± 556.5, and 1535.8 ± 174.1 μg/g dw, respectively. Interestingly, mature Easter lily flowers are also a plentiful source of acetylated phenylpropanoid glycerol glucosides. The p-coumaroyl-based compound 3 and its acetylated derivative compound 5 had similar distribution profiles in L. longif lorum plant parts; however, compound 5 occurred in higher concentrations than compound 3 in the flower buds and mature flowers. Compounds 3 and 5 were the highest occurring phenylpropanoid glycerol glucosides measured in the bulb scales, but their highest concentrations were determined to be in the flower buds as compared to the other plant organs. Notably, compounds 3 and 5 were both higher in the flower buds as compared to mature flowers. Compound 2 was determined to be a structural isomer of compound 3, differing only in the linkage arrangement of the glucose and p-coumaroyl moiety to the glycerol backbone. In compound 2, the acyl group is linked to the C2 carbon and the glucose to the C1 carbon, (2R)-1-O-β-D-glucopyranosyl-2-O-pcoumaroylglycerol, whereas in compound 3, the acyl group is linked to the C1 carbon and the glucose to the C2 carbon of the glycerin backbone, (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosylglycerol. An acetylated derivative of compound 2 was not identified in the present investigation. Its natural distribution differed from that of the other compounds
investigated in the study. The highest concentration of compound 2 was determined to be in the leaves as compared to the other plant organs (Table 1). The caffeyol-based compound 1 and its acetylated derivative compound 4 had similar profiles in the various organs of L. longif lorum; however, as with compounds 3 and 5, the acetylated derivative, compound 4, occurred in higher concentrations than compound 1 in the flower buds and mature flowers (Table 1). Compound 4 was the most abundant phenylpropanoid glycerol glucoside measured in the mature flowers. Upon comparison of the total phenylpropanoid glycerol glucoside concentrations in the plant organs, it is clear that the highest concentrations of these compounds are found in the above-ground portions of the plant (Figure 6). Leaves, flower buds, and flowers have 10−100 times the concentrations of phenylpropanoid glycerol glucosides compared to bulbs and roots, suggesting possible functions for these compounds unique to the above-ground environment. In general, free phenylpropanoid acids do not accumulate in plants, but are usually found as glycosides or esterified to glycerol or organic, fatty, and amino acids. In Arabidopsis, malic acid esters and glucosides of the phenylpropanoid sinapic acid accumulate in the leaves, where they provide protection from UV-B light stress and function as sunscreens.24 It is possible that phenylpropanoid glycerol glucosides may play a similar role in Easter lily, a plant native to the unshaded rocky shores of the subtropical Ryukyu Islands of Japan. The high concentrations of phenylpropanoid glycerol glucosides in the leaves and flower buds may also serve as a constitutive reserve of fungitoxic phenolic acids as in cucumber leaves, where attack by a 4841
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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(8) Shimomura, H.; Sashida, Y.; Mimaki, Y. Steroidal saponins, pardarinoside A−G from the bulbs of Lilium pardarinum. Phytochemistry 1989, 28, 3163−3170. (9) Sashida, Y.; Ori, K.; Mimaki, Y. Studies on the chemical constituents of the bulbs of Lilium mackliniae. Chem. Pharm. Bull. 1991, 39, 2362−2368. (10) Nakamura, O.; Mimaki, Y.; Nishino, H.; Sashida, Y. Steroidal saponins from the bulbs of Lilium speciosum x L. nobilissimum ’Star Gazer’ and their antitumour-promoter activity. Phytochemistry 1994, 36, 463−467. (11) Mimaki, Y.; Satou, T.; Kuroda, M.; Sashida, Y.; Hatakeyama, Y. Steroidal saponins from the bulbs of Lilium candidum. Phytochemistry 1999, 51, 567−573. (12) Mimaki, Y.; Satou, T.; Kuroda, M.; Sashida, Y.; Hatakeyama, Y. New steroidal constituents from the bulbs of Lilium candidum. Chem. Pharm. Bull. 1998, 46, 1829−1832. (13) Mimaki, Y.; Sashida, Y.; Nakamura, O.; Nikaido, T.; Ohmoto, T. Steroidal saponins from the bulbs of Lilium regale and Lilium henryi. Phytochemistry 1993, 33, 675−682. (14) Mimaki, Y.; Sashida, Y. Steroidal and phenolic constituents of Lilium speciosum. Phytochemistry 1991, 30, 937−940. (15) Mimaki, Y.; Sashida, Y. Steroidal saponins and alkaloids from the bulbs of Lilium brownii var. colchesteri. Chem. Pharm. Bull. 1990, 38, 3055−9. (16) Mimaki, Y.; Nakamura, O.; Sashida, Y.; Satomi, Y.; Nishino, A.; Nishino, H. Steroidal saponins from the bulbs of Lilium longiflorum and their antitumour-promoter activity. Phytochemistry 1994, 37, 227− 232. (17) Munafo, J. P., Jr.; Gianfagna, T. J. Chemistry and biological activity of steroidal glycosides from the Lilium genus. Nat. Prod. Rep. 2015, 32, 454−477. (18) Munafo, J. P., Jr.; Gianfagna, T. J. Antifungal activity and fungal metabolism of steroidal glycosides of Easter lily (Lilium longiflorum Thunb.) by the plant pathogenic fungus, Botrytis cinerea. J. Agric. Food Chem. 2011, 59, 5945−5954. (19) Munafo, J. P., Jr.; Ramanathan, A.; Jimenez, L. S.; Gianfagna, T. J. Isolation and structural determination of steroidal glycosides from the bulbs of Easter lily (Lilium longif lorum Thunb.). J. Agric. Food Chem. 2010, 58, 8806−8813. (20) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Iida, N. Regaloside A and B, acylated glycerol glucosides from Lilium regale. Phytochemistry 1988, 27, 451−454. (21) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Iitaka, Y. Studies on the chemical constituents of Lilium henryi Baker. Chem. Pharm. Bull. 1988, 36, 2430−2446. (22) Shimomura, H.; Sashida, Y.; Mimaki, Y. New phenolic glycerol glucosides, regaloside D, E, and F from the bulbs of Lilium species. Chem. Pharm. Bull. 1989, 43, 64−70. (23) Mimaki, Y.; Sashida, Y.; Shimomura, H. Lipid and steroidal constituents of Lilium auratum var. platyphyllum and Lilium tenuifolium. Phytochemistry 1989, 28, 3453−3458. (24) Skoog, D.; Holler, F.; Nieman, T. Introduction. In Principles of Instrumental Analysis, 5th ed.; Brooks/Cole Publishing: Belmont, CA, USA, 1997. (25) Li, J.; Ou-Lee, T.-M.; Raba, R.; Amundson, R. G.; Last, R. L. Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 1993, 5, 171−179. (26) Daayf, F.; Ongena, M.; Boulanger, R.; El Hadrami, I.; Bélanger, R. R. Induction of phenolic compounds in two cultivars of cucumber by treatment of healthy and powdery mildew-infected plants with extracts of Reynoutria sachalinensis. J. Chem. Ecol. 2000, 26, 1579− 1593. (27) Munafo, J. P., , Jr.; Gianfagna, T. J. Quantitative analysis of steroidal glycosides in different organs of Easter lily (Lilium longif lorum Thunb.) by LC-MS/MS. J. Agric. Food Chem. 2011, 59, 995−1004.
powdery mildew fungus triggers the hydrolysis of glucosides of p-coumaric acid methyl ester and other phenylpropanoid acid conjugates, significantly reducing disease symptoms.26 The unequal distribution pattern of the phenylpropanoid glycerol glucosides between above- and below-ground organs is similar to the distribution of two types of steroidal glycosides of lily bulbs. The proportion of the steroidal glycoalkaloids to furostanol saponins in light-exposed organs was greater; moreover, this ratio decreased from the above-ground organs to those growing farther below the soil surface, suggesting that these compounds may be involved in different physiological processes particular to their growing environment.27 In conclusion, this is the first report of the natural distribution of phenylpropanoid glycerol glucosides within the different organs of a plant and the first report that these compounds are present in plant organs other than the bulbs and flowers. The p-coumaroyl-based compound 3 and its acetylated derivative 5 were determined to be the most plentiful of the phenylpropanoid glycerol glucosides found in Easter lily bulbs, whereas the acetylated p-coumaroyl- and caffeoyl-based derivatives 5 and 4 were found in the highest concentrations in the flower buds. Quantitative analysis of phenylpropanoid glycerol glucosides in the component parts of L. longif lorum is a prelude to understanding their function in plant physiology, development, and pathogen defense. The results of this study will also form the basis for research on the benefits of lily bulb consumption to human health and for the development of functional foods, cosmetics, and pharmaceuticals containing lily bulb compounds.
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AUTHOR INFORMATION
Corresponding Author
*(T.J.G.) Phone: (848) 932-6369. Fax: (732) 932-6441. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are very grateful to Dr. Karsten Siems from AnalytiCon Discovery GmbH (Potsdam, Germany) for the skillful acquisition of the NMR spectra.
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REFERENCES
(1) Ori, K.; Mimaki, Y.; Mito, K.; Sashida, Y.; Nikaido, T.; Ohmoto, T.; Masuko, A. Jatropham derivatives and steroidal saponins from the bulbs of Lilium hansonii. Phytochemistry 1992, 31, 2767−2775. (2) Mimaki, Y.; Sashida, Y. Steroidal saponins from the bulbs of Lilium brownii. Phytochemistry 1990, 29, 2267−2271. (3) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Kudo, Y.; Maeda, K. New phenylpropanoid glycerol glucosides from the bulbs of Lilium species. Chem. Pharm. Bull. 1988, 36, 4841−4848. (4) Sang Tai, C.; Uemoto, S.; Shoyama, Y.; Nishioka, I. Biologically active phenolics from Lilium longif lorum. Phytochemistry 1981, 20, 2565−2568. (5) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Minegishi, Y. Jatropham glucoside from the bulbs of Lilium hansonii. Phytochemistry 1987, 26, 582−583. (6) Francis, J. A.; Rumbeiha, W.; Nair, M. G. Constituents in Easter lily flowers with medicinal activity. Life Sci. 2004, 76, 671−683. (7) Tsukida, K.; Ikeuchi, K. Epoxycarotenoids. VIII. Pollen carotenoids of Lilium longif lorum and of its cultivated hybrid. Bitamin 1965, 32, 222−226. 4842
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
Quantitative Analysis of Phenylpropanoid Glycerol Glucosides in Different Organs of Easter Lily (Lilium longiflorum Thunb.) John P. Munafo, Jr. and Thomas J. Gianfagna* Plant Biology Department, RutgersThe State University of New Jersey, 59 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT: The Easter lily (Lilium longiflorum Thunb.) is esteemed worldwide as an attractive ornamental plant, and the flower buds and bulbs are used for both culinary and medicinal purposes in many parts of the world. L. longif lorum contains significant amounts of phenylpropanoid glycerol glucosides, a group of compounds that may contribute to plant pathogen defense, ultraviolet/high-intensity visible light (UV/high light) protection, and the purported medicinal uses of lilies. To define the natural distribution of these compounds within the plant, a liquid chromatography−mass spectrometry (LC-MS) method performed in selected ion monitoring (SIM) mode was employed for the quantitative analysis of five phenylpropanoid glycerol glucosides, namely, (2S)-1-O-caffeoyl-2-O-β-D-glucopyranosylglycerol, 1; (2R)-1-O-β-D-glucopyranosyl-2-O-p-coumaroylglycerol, 2; (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosylglycerol, 3; (2S)-1-O-caffeoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 4; and (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 5, in the different organs of L. longif lorum. The pcoumaroyl-based 3 and its acetylated derivative 5 were determined to be the most abundant of the phenylpropanoid glycerol glucosides found in Easter lily bulbs, at 776.3 ± 8.4 and 650.7 ± 32.6 μg/g dry weight, respectively. The acetylated p-coumaroyland caffeoyl-based derivatives, 5 and 4, accumulated to the highest concentration in the closed flower buds, at 4925.2 ± 512.8 and 3216.8 ± 406.4 μg/g dry weight, respectively. Compound 4, followed by 5 and 1, proved to be the most abundant in the mature flowers, occurring at 6006.2 ± 625.8, 2160.3 ± 556.5, and 1535.8 ± 174.1 μg/g dry weight, respectively. Total concentrations of the phenylpropanoid glycerol glucosides were 10−100-fold higher in the above-ground plant organs as compared to the bulbs and fleshy roots. Two of the five compounds, 1 and 2, were identified in L. longif lorum for the first time. The quantitative analysis of phenylpropanoid glycerol glucosides in the different plant organs of L. longif lorum will establish the direction of investigations aimed at defining how these compounds function in the physiology and chemical ecology of the plant and also as a starting point for determining their possible effects on human health, which has not been investigated. KEYWORDS: Lilium longiflorum Thunb., Liliaceae, Easter lily, phenylpropanoid glycerol glucosides, regalosides
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flavonoids,6 carotenoids,7 sterols,7 steroidal saponins,2,8−17 and steroidal glycoalkaloids.15,17−19 In addition, the Lilium genus contains an abundant supply of cinnamic acid derivatives, in particular, phenylpropanoid glycerol glucosides, a group of natural products, with many structural analogues, first isolated and characterized from Lilium regale E. H. Wilson and thus trivially named regalosides.20 Cinnamic acid derivatives, most notably p-coumaric, caffeic, and ferulic acids, are widely distributed in plants and naturally occur as both esters and glycosides. Regalosides are acylated glycerol glucosides. As such, they characteristically contain p-coumaric, caffeic, or ferulic acids, but less often cinnamic acid. Typically, they are esterified at the C1 or C2 carbon of a glucosylated glycerol backbone, with S configuration at the 2-position, (2S)regalosides, being the most common, and R configuration, (2R)-regalosides, less usual. Moreover, a few acetylated and methylated regalosides have been described.6,9,20−22 Regalosides have been identified in members of the Liliaceae family including L. regale,20 Lilium henryi Baker,21 Lilium pardalinum Kellogg,3 Lilium auratum Lindl.,3,23 Lilium lancifolium Thunb.,22
INTRODUCTION The Easter lily (Lilium longiflorum Thunb., family Liliaceae) is primarily grown as an ornamental plant because of its large, fragrant, white flowers. Although not well-known as a food in the United States, lily flower buds and bulbs are used as both food and medicine in many Asian countries, especially China and Japan. In Chinese cuisine the bulbs of various lily species, including L. longif lorum, are a common ingredient in soups, stirfries, and stew-like dishes such as “hot pot”. Lily bulbs and flower buds are an important part of the Asian diet and are consumed on a regular basis. Despite this fact, very little is known about their chemical composition and impact on human health. In addition to their culinary value, lily bulbs have a long history of documented medicinal use as sedatives, antiinflammatory agents, and antitussives.1−3 In fact, preparations of lily bulbs from Lilium species, referred to in China as “Baihe,” are employed in Traditional Chinese Medicine (TCM) as effective treatments for inflammation and some lung ailments.1,2 However, although the medicinal use of L. longif lorum is well documented, the bioactive phytochemicals responsible for the putative medicinal properties as well as their mechanisms of action remain elusive. Plants from the Lilium genus are an abundant source of a number of natural products. Many classes of compounds have been reported from the genus, including phenolics,4 alkaloids,5 © 2015 American Chemical Society
Received: Revised: Accepted: Published: 4836
February April 23, April 23, April 23,
16, 2015 2015 2015 2015 DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Journal of Agricultural and Food Chemistry L. longif lorum,6,22 Lilium brownii F. E. Br. ex Miellez,2,15 Lilium speciosum Thunb.,14 and Lilium mackliniae Sealy.9 Earlier studies have identified phenylpropanoid glycerol glucosides in the bulbs of L. longif lorum, specifically, (2S)-1O-p-coumaroyl-2-O-β-D-glucopyranosylglycerol, 3; (2S)-1-Ocaffeoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 4; and (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, 5.22 Additionally, a 4-acetyl derivative of (2S)-1-O-pcoumaroyl-2-O-β-D-glucopyranosylglycerol has been reported from L. longif lorum flowers that exhibited mild cyclooxygenase and lipid peroxidation inhibition activities.6 Although these compounds may be involved in plant pathogen defense, ultraviolet/high-intensity visible light (UV/high light) protection, and the putative medicinal activities of lilies,1−3,17 reports on the biological activities of phenylpropanoid glycerol glucosides are scarce. Phenylpropanoid glycerol glucosides are reported to be bitter in taste,20 which may suggest a possible role in antiherbivory; however, the bitter taste thresholds of the compounds have not been quantitated nor have the antiherbivory activities of the compounds been investigated. The dual aim of our present investigation has been to develop a liquid chromatography−mass spectrometry (LC-MS) method for the quantitative analysis of five phenylpropanoid glycerol glucosides in plant tissues and to identify and quantitate the concentration of the compounds in the different plant organs of Easter lily. The results were anticipated to lay the groundwork for defining how these compounds function in the physiology and chemical ecology of the plant and also as a starting point for determining their possible effects on human health when consumed as a food, which has not been investigated.
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residue was then allowed to dry overnight in a fume hood. The dried defatted residue was extracted with ethanol and DI water (7:3, v/v; 2 × 150 mL) for 45 min at room temperature on a shaker. The extract was centrifuged (5000 rpm) for 10 min and the supernatant was saved and vacuum filtered. The supernatant was rotary evaporated (30 °C; 1.0 × 10−3 bar) and lyophilized, yielding a crude bulb extract (fraction I, 12.4 g). Next, the lyophilized crude bulb extract was dissolved in DI water (100 mL) and extracted with ethyl acetate (5 × 100 mL), and the aqueous phase, containing the highly polar compounds such as carbohydrates and steroidal glycosides, was discarded. The organic phase was rotary evaporated to dryness (30 °C; 1.0 × 10−3 bar) (fraction II), redissolved in DI water (100 mL), and sonicated for 10 min. The extract was centrifuged (5000 rpm) for 10 min, vacuum filtered, and the supernatant was collected. The aqueous phase was then extracted with 1-butanol (5 × 100 mL). The organic phase was rotary evaporated (30 °C; 1.0 × 10−3 bar) and lyophilized, yielding 1butanol extract (fraction III, 1.9 g). Semipreparative Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The 1-butanol extract (fraction III) was fractionated by semipreparative RP-HPLC on a 250 mm × 21.2 mm i.d., 10 μm, Luna C18 column (Phenomenex, Torrance, CA) to afford 1−5 (Figure 1) using an LC-6AD liquid chromatograph (Shimadzu
MATERIALS AND METHODS Figure 1. Structures of compounds 1−5 quantitated in the various L. longif lorum organs.
Plant Material. Cultivar 7-4 L. longiflorum bulbs were cultivated, harvested, lyophilized, and milled according to the methods described in Munafo et al.19 Three fully mature plants, containing all stages of the vegetative and reproductive organs, were harvested and dissected into fleshy bulb scales, fleshy roots, leaves, lower stems, upper stems, flower buds, and mature flowers. The fleshy bulb scales were 0.8−2.0 cm in width and 0.9−4.0 cm in length. The fleshy roots were approximately 2−4 mm in diameter and the leaves about 6−14 cm in length. Lower stems (the part growing below the soil surface) ranged in size from 6 to 10 cm and were white to yellow in color. Upper stems (the part growing above the soil surface) ranged in size from 19 to 31 cm and were green. The flower buds were approximately 3−6 cm in length, fully closed, and green in appearance. The mature flowers ranged between 6 and 14 cm in length, were fully open, and were white in appearance. Each of the organ types from an individual plant were pooled together. Three individual plants were used in the analysis (n = 3). After harvest, all samples were immediately frozen under liquid nitrogen, lyophilized, and stored at −80 °C prior to analysis. Chemicals. All solvents, acetonitrile, 1-butanol, dimethyl sufoxide (DMSO), ethyl acetate, ethanol, formic acid, methanol, tetrohydrofuran (THF), and pentanes were of chromatographic grade and were obtained from Thermo Fisher Scientific Inc., Fair Lawn, NJ, USA. Methanol-d4 (0.03% v/v TMS) was obtained from Sigma-Aldrich, St. Louis, MO, USA. Deionized (DI) water (18 MΩ cm) was generated using a Milli-Q-water purification system (Millipore, Bedford, MA, USA). Purification of Phenylpropanoid Glycerol Glucosides 1−5 as Analytical Standards. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs. Lyophilized lily bulbs (100 g) were milled to a fine powder after freezing in liquid nitrogen. To remove lipids, the bulb powder was extracted with pentanes (3 × 100 mL) at room temperature for 15 min each time on a shaker. The extract was centrifuged (5000 rpm) for 10 min and the solvent was decanted. The
Scientific Instruments Inc., Columbia, MD, USA) equipped with a UV/vis detector and a 2 mL injection loop. The mobile phases were mixtures of (A) 0.1% formic acid in DI water and (B) 0.1% formic acid in acetonitrile. The flow rate was set to 20 mL/min, column temperature was 23 ± 2 °C, and UV detection was recorded at λ 210 nm. The 1-butanol extract (fraction III, 200 mg) was dissolved in a mixture of mobile phase A and mobile phase B (90:10, v/v; 2 mL) and filtered through a 0.45 μm PTFE syringe filter prior to injection (injection volume = 1 mL). The butanol extract was fractionated using a linear gradient of 5−24.4% B for 35 min and then ramped to 90% B in 5 min, with a hold at 90% B for additional 5 min. The solvent mixture was reset to 5% B for 10 min before subsequent injections. Fractions containing the compounds of interest were collected (Figure 2), and the solvent was rotary evaporated (30 °C; 1.0 × 10−3 bar) and lyophilized, yielding 1 (15.2 mg), 2 (12.1 mg), 3 (39.6 mg), 4 (10.2 mg), and 5 (40.3 mg) as pale yellow amorphous powders with >98% purity as determined by LC-MS and NMR. Compound 1, (2S)-1-O-Caf feoyl-2-O-β-D-glucopyranosylglycerol (regaloside K). 1D and 2D NMR data were consistent with 1H NMR and 13 C NMR reported in the literature.9 Compound 2, (2R)-1-O-β-D-Glucopyranosyl-2-O-p-coumaroylglycerol (regaloside H). 1D and 2D NMR data were consistent with 1H NMR and 13C NMR reported in the literature.23 Compound 3, (2S)-1-O-p-Coumaroyl-2-O-β-D-glucopyranosylglycerol (regaloside D). 1D and 2D NMR data were consistent with 1H NMR and 13C NMR reported in the literature.3 Compound 4, (2S)-1-O-Caf feoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol (regaloside E). 1D and 2D NMR data were consistent with 1H NMR and 13C NMR reported in the literature.3 4837
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Figure 2. Semipreparative RP-HPLC chromatogram (λ 210 nm) of 1−5 purified from L. longif lorum 1-butanol extract (fraction III).
Figure 3. LC-MS chromatogram for the quantitative analysis of compounds 1−5 in a L. longif lorum bulb scale operating in selective ion monitoring (SIM) mode. Compound 5, (2S)-1-O-p-Coumaroyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol (regaloside B). 1D and 2D NMR data were consistent with 1 H NMR and 13C NMR reported in the literature.20 Nuclear Magnetic Resonance Spectroscopy (NMR). 1D 1H NMR spectra was acquired with a 400 MHz spectrometer (Bruker, Rheinstetten, Germany). 2D heteronuclear multiple-bond coherence (HMBC), heteronuclear single-quantum coherence (HSQC), and H− H correlation spectroscopy (H−H COSY) spectra were acquired on a 500 MHz spectrometer (Bruker). Samples for NMR analysis were dissolved in methanol-d4, and chemical shifts were calculated as δ values with reference to tetramethylsilane (TMS). Optimization of Sample Extraction Parameters for Quantitative Analysis. Solvent Composition and Extraction Efficiency Determination. Six neat organic solvents (methanol, ethanol, DMSO, acetone, acetonitrile, and THF) as well as different ratios of methanol and DI water (1:0, 9:1. 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:1, v/ v) were used for experiments aimed at optimization of the extraction solvent system for phenylpropanoid glycerol glucosides 1−5 from lyophilized L. longiflorum tissue. Performing successive extraction of the same plant tissue from one to three times determined extraction efficiency. Lyophilized lily bulb tissue was extracted with methanol and DI water (8:2, v/v) solvent mixture. Results from each successive extraction were collected separately and filtered through a 0.45 μm PTFE syringe filter prior to LC-MS analysis.
Quantitative Analysis of Phenylpropanoid Glycerol Glucosides 1−5 in L. longif lorum. Sample Preparation. Following removal from the freezer, lyophilized lily organ samples were thawed to ambient temperature. The samples (40 mg each) were milled to pass through a 200 mesh sieve, weighed separately, transferred into a 15 mL centrifuge tube, and covered with methanol and DI water (8:2, v/v; 7 mL each). The samples were extracted with shaking (10 min), sonicated (10 min), and filled to final volume (10 mL) with methanol and DI water (8:2, v/v). The samples were vortexed (1 min), and centrifuged (5000 rpm for 10 min), and the supernatant was collected. The pellet was re-extracted with methanol and DI water (8:2, v/v; 10 mL), shaken (10 min), sonicated (10 min), centrifuged (5000 rpm for 10 min), and the supernatants were combined (total volume ∼ 20 mL). Prior to LC-MS analysis, all samples were filtered through a 0.45 μm PTFE syringe filter. Analytical Standard Preparation. Phenylpropanoid glycerol glucosides 1−5, isolated and purified as described above, were used as analytical standards. Each compound was weighed into volumetric flasks (10 mL), dissolved with methanol and DI water (8:2, v/v; 7 mL each), and sonicated for 5 min before taken to full volume. Working solutions were prepared for calibration equations by diluting the stock solutions. External standard equations were calculated from six data points covering a concentration range of 1−300 μg/mL. To establish calibration equations, mean peak areas (n = 3) from the standard 4838
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Figure 4. Evaluation of six organic solvents on the extraction of compounds 1−5 from L. longif lorum bulbs. Concentrations are means of triplicates ± SD. solutions were plotted versus concentration. Standard solutions were stored at 4 °C and equilibrated to ambient temperature before use. Liquid Chromatography−Mass Spectrometry (LC-MS). LC-MS analysis of L. longif lorum extracts was performed using an Agilent 1100 series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an autoinjector, quaternary pump, column heater, diode array detector and interfaced to a 1100 series single-quadruple mass selective detector (MSD) equipped with an API-ESI ionization source. Chromatographic separations were made on a 250 mm × 4.6 mm i.d., 5 μm Prodigy C18 column (Phenomenex) operated at a flow rate of 1.0 mL/min, column temperature set to 23 ± 2 °C, and an injection volume of 10 μL. The binary mobile phase consisted of (A) 0.1% formic acid in DI water and (B) 0.1% formic acid in acetonitrile. Phenylpropanoid glycerol glucosides were separated using a linear gradient of 15−34.6% B for 28 min and then ramped to 95% B in 5 min with a hold at 95% B for 5 min. The system was reset to 15% B for 10 min before each sample injection. ChemStation software was used for data acquisition and analysis. Negative ionization mode was used to quantify the phenylpropanoid glycerol glucosides using SIM. The following ions were selected for each compound: 1, m/z 415; 2, m/z 399; 3, m/z 399; 4, m/z 457; and 5, m/z 441 (Figure 3). Ionization conditions were as follows: capillary voltage, 3.5 kV; nebulizer pressure, 35 psi; drying gas flow, 10.0 mL/min; and drying gas temperature, 350 °C. MSD signal parameters included mode, SIM; polarity, negative; fragmentor voltage, 70 V; gain, 1.0; dwell time, 144 ms; and percent relative dwell time, 25. Recovery. The standard addition method was used to calculate recovery rates.24 Lyophilized lily organs were milled to pass through a 200 mesh sieve, separately weighed (40 mg), and transferred into 15 mL centrifuge tubes containing one of three different concentrations (50, 100, and 200 μg/g) of pure compounds dissolved in methanol and DI water (8:2, v/v). To the sample tubes was added a solution of methanol and DI water (8:2, v/v; 7 mL), and the extract was shaken (10 min), sonicated (10 min), and then filled to final volume (10 mL) with the same solvent. The samples were vortexed (1 min) centrifuged (5000 rpm for 10 min), and the supernatant was recovered. For a second time, the centrifuge tube containing the pellet was filled to final volume (10 mL), extracted with shaking (10 min), sonicated (10 min), and centrifuged (5000 rpm for 10 min), and the supernatants from the two extractions were pooled (total volume ∼ 20 mL). All samples were syringe filtered (0.45 μm PTFE) and the phenylpropanoid glycerol glucosides quantified as described above. The percentage recovery for each of the phenylpropanoid glycerol glucosides 1−5 in vegetative and reproductive organs was determined by subtracting the amount found in the lily organ sample that was not spiked with additional standards
(control) from the amount of standard found in the spiked sample, with three replicate samples for each analysis. Statistical Analysis. To compare differences in phenylpropanoid glycerol glucosides content in the lily plant organs, LC-MS data were first transformed to the logarithmic function, and an analysis of variance (ANOVA) was conducted. Means were separated with Tukey’s test (α = 0.05) using JMP 10 software (SAS Institute Inc., Cary, NC, USA). Two-way hierarchal cluster analysis (HCA) and the resulting color map, visually depicting the differences in concentration of compounds 1−5 throughout the lily plant organs, were generated using JMP 10 software (SAS Institute Inc.).
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RESULTS AND DISCUSSION
Isolation and Identification of Compounds 1 and 2 from L. longif lorum Bulbs. The crude 1-butanol extract was fractionated by repeated semipreparative RP-HPLC to yield compounds 1−5. On the basis of 1D 1H and 2D (HMBC, HSQC, and H−H COSY) NMR, compounds 3, 4, and 5 were confirmed to be (2S)-1-O-p-coumaroyl-2-O-β-D-gucopyranosylglycerol, (2S)-1-O-caffeoyl-2-O-β-D-glucopyranosyl-3-O-acetylglycerol, and (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosyl3-O-acetylglycerol, respectively. All three of these compounds have been previously reported from L. longif lorum bulbs.22 On the basis of 1D 1H and 2D (HMBC, HSQC, and H−H COSY) NMR, compounds 1 and 2 were confirmed to be (2S)-1-Ocaffeoyl-2-O-β-D-glucopyranosylglycerol, 1, and (2R)-1-O-β-Dglucopyranosyl-2-O-p-coumaroylglycerol, 2, respectively. Compound 1 was previously reported from fresh bulb scales of L. mackliniae9 and 2 from L. auratum.23 This is the first report of 1 and 2 as natural products identified in L. longif lorum. Optimization of Quantitative Extraction Parameters. Solvent Composition Determination. The determination of an effective extraction solvent system for the quantitative analysis of 1−5 was achieved by first analyzing the resulting extracts prepared from six neat organic solvents, namely, methanol, ethanol, DMSO, acetone, acetonitrile, and THF (Figure 4). Various combinations of methanol and DI water were then evaluated with the goal of determining a solvent system that effectively extracted 1−5. On the basis of these data, the combination of methanol and DI water (8:2, v/v) was chosen 4839
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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each of the plant organs (bulb scales, 94.7−100.8%; roots, 94.8−101.4%; leaves, 96.7−102.9%; lower stems, 93.7−99.8%; upper stems, 94.9−103.3%; flower buds, 96.3−102.0%; and mature flowers, 95.2−101.9%). For compounds 1−5 the recovery rates in all of the plant organs assayed were between 93.7 and 103.3%. The precision of the method was evaluated by analyzing the same sample multiple times (bulb; n = 6) and subsequently calculating the relative standard deviation (RSD) of 1−5 in the sample. The RSD values for 1−5 were 4.14, 3.53, 2.81, 2.73, and 3.57%, respectively. These data demonstrate satisfactory linearity, recovery rates, and RSD, which suggests that the LC-MS method operating in SIM mode is a suitable method for the quantitation of phenylpropanoid glycerol glucosides 1−5 in L. longif lorum plant organs. Quantitation of Phenylpropanoid Glycerol Glucosides 1−5 in the Different Organs of L. longif lorum. Concentrations of 1−5 were quantitatively determined in all plant organs of L. longif lorum (Table 1; Figure 6). The pcoumaroyl-based compound 3 and its acetylated derivative 5 were determined to be the most abundant of the phenylpropanoid glycerol glucosides found in Easter lily bulbs, at 776.3 ± 8.4 and 650.7 ± 32.6 μg/g dry weight (dw), respectively. As previously mentioned, lily bulbs are regularly consumed as a food, and preparations from lily bulbs, referred to in China as “Bai-he”, are employed as effective treatment for various inflammations and some lung ailments; however, the bioactive phytochemicals responsible for the putative medicinal properties remain unknown.1,2 Compounds 3 and 5 occurred at the highest concentration in the root tissues at 584.4 ± 34.3 and 345.8 ± 32.6 μg/g dw, respectively. Similar to the bulbs and roots, 3 and 5 occurred at the highest concentration in the upper stem and lower stem. Compound 3 occurred at higher concentrations in the lower stem versus the upper stem, and the concentrations of compound 5 were similar in both the lower and upper stems. The above-ground plant organs of Easter lily (upper stems, leaves, buds, and flowers) showed a different trend of phenylpropanoid glycerol glucoside concentrations from that of the below-ground plant organs (bulbs, roots, and lower stem). The above-ground organs displayed more caffeoyl-based compounds and a greater abundance of acetylation than the below-ground organs. The caffeoyl-based compounds 1 and 4 were the highest in the leaves, at 2007.4 ± 207.5 and 1786.0 ± 200.9 μg/g dw, followed by the p-coumaroyl-based compounds 3, 2, and 5, at 1074.8 ± 53.1, 1003.5 ± 36.4, and 935.1 ± 103.9 μg/g dw, respectively. The acetylated p-coumaroyl- and caffeoyl-based derivatives, 5 and 4, had the highest concentrations in the closed flower buds, at 4925.2 ± 512.8 and 3216.8
as the optimum solvent system of the solvent combinations evaluated (Figure 5).
Figure 5. Evaluation of various ratios of methanol and DI water on the extraction of compounds 1−5 from L. longiflorum bulbs. Concentrations are means of triplicates ± SD.
Extraction Efficiency. Sequential solvent extraction of the lyophilized lily bulb powder using methanol and DI water (8:2, v/v) was employed to estimate the optimal number of extraction cycles for quantitative analysis. The results indicated that between 77.6 and 89.6% of compounds 1−5 were recovered in the first extraction. An additional 9.3−17.7% was found with the second extraction and approximately 0−5.1% after the third cycle. On the basis of these observations, the combined supernatants generated from two successive extraction cycles were chosen for further quantitative analysis, resulting in an estimation of 94.4−98.9% extraction efficiency for compounds 1−5. Method Validation. To quantitate the distribution of five phenylpropanoid glycerol glucosides in L. longif lorum organs, 1−5 were purified from lily bulbs as analytical reference standards. For quantitative analysis, extracts were prepared from lyophilized plant parts and analyzed using LC-MS in SIM mode. To determine linear relationships between peak area and concentration for the quantitative method, six-point calibration plots were constructed, which demonstrated good linearity over the concentration range of 1.0−300 μg/mL for compounds 1− 5. The correlation coefficients ranged from R2 = 0.9998 to 0.9999. Accuracy of the method was assessed by calculating recovery rates for 1−5 in each of the plant organs using the standard addition method.24 Recovery rates were calculated for
Table 1. Concentrations of Compounds 1−5 in the Different Organs of L. longif lorum compound concentrationa (μg/g dw) organ bulb root leaf lower stem upper stem bud flower
1 68.4c 95.7c 2007.4a 381.3c 274.1c 762.9d 1535.8b
± ± ± ± ± ± ±
2 b
11.4 12.6 207.5 49.0 15.6 34.0 174.1
80.3bc 62.8d 1003.5b 152.8e 242.7c 636.7d 385.9d
3 ± ± ± ± ± ± ±
8.43 1.70 36.4 13.0 25.4 40.5 10.6
776.3a 584.4a 1074.8b 1281.7a 1011.3a 1771.2c 736.2c
± ± ± ± ± ± ±
4 8.4 34.3 53.1 26.5 35.2 104.3 16.4
116.7b 85.5c 1786.0a 275.8d 619.0b 3216.8b 6006.2a
± ± ± ± ± ± ±
5 29.1 12.5 200.9 14.1 34.6 406.4 625.8
650.7a 345.8b 935.1b 860.0b 1125.0a 4925.2a 2160.3b
± ± ± ± ± ± ±
32.6 17.1 103.9 47.2 105.7 512.8 556.5
Concentrations are means of triplicates ± SD, expressed on a dry weight basis (dw). bValues with the same letter in each row are not significantly different (p < 0.05). a
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DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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Figure 6. Two-way hierarchal cluster analysis (HCA). Color map visually depicting the differential concentrations of compounds 1−5 in the different plant organs of L. longif lorum. Colored branches on the dendrogram denote clusters. Upper stem, lower stem, root, and bulb, red cluster; flower and leaf, blue cluster; bud, green cluster.
± 406.4 μg/g dw, respectively. In parts of Asia, lily flower buds, similarly to lily bulbs, are commonly consumed as snacks and in soups, stir-fries, and stew-like dishes. Easter lily flower buds are rich in acetylated phenylpropanoid glycerol glucosides 5 and 4 and differ dramatically from the phytochemical profile of the bulbs. Most abundant in the mature flowers was compound 4, followed by 5 and 1, occurring at 6006.2 ± 625.8, 2160.3 ± 556.5, and 1535.8 ± 174.1 μg/g dw, respectively. Interestingly, mature Easter lily flowers are also a plentiful source of acetylated phenylpropanoid glycerol glucosides. The p-coumaroyl-based compound 3 and its acetylated derivative compound 5 had similar distribution profiles in L. longif lorum plant parts; however, compound 5 occurred in higher concentrations than compound 3 in the flower buds and mature flowers. Compounds 3 and 5 were the highest occurring phenylpropanoid glycerol glucosides measured in the bulb scales, but their highest concentrations were determined to be in the flower buds as compared to the other plant organs. Notably, compounds 3 and 5 were both higher in the flower buds as compared to mature flowers. Compound 2 was determined to be a structural isomer of compound 3, differing only in the linkage arrangement of the glucose and p-coumaroyl moiety to the glycerol backbone. In compound 2, the acyl group is linked to the C2 carbon and the glucose to the C1 carbon, (2R)-1-O-β-D-glucopyranosyl-2-O-pcoumaroylglycerol, whereas in compound 3, the acyl group is linked to the C1 carbon and the glucose to the C2 carbon of the glycerin backbone, (2S)-1-O-p-coumaroyl-2-O-β-D-glucopyranosylglycerol. An acetylated derivative of compound 2 was not identified in the present investigation. Its natural distribution differed from that of the other compounds
investigated in the study. The highest concentration of compound 2 was determined to be in the leaves as compared to the other plant organs (Table 1). The caffeyol-based compound 1 and its acetylated derivative compound 4 had similar profiles in the various organs of L. longif lorum; however, as with compounds 3 and 5, the acetylated derivative, compound 4, occurred in higher concentrations than compound 1 in the flower buds and mature flowers (Table 1). Compound 4 was the most abundant phenylpropanoid glycerol glucoside measured in the mature flowers. Upon comparison of the total phenylpropanoid glycerol glucoside concentrations in the plant organs, it is clear that the highest concentrations of these compounds are found in the above-ground portions of the plant (Figure 6). Leaves, flower buds, and flowers have 10−100 times the concentrations of phenylpropanoid glycerol glucosides compared to bulbs and roots, suggesting possible functions for these compounds unique to the above-ground environment. In general, free phenylpropanoid acids do not accumulate in plants, but are usually found as glycosides or esterified to glycerol or organic, fatty, and amino acids. In Arabidopsis, malic acid esters and glucosides of the phenylpropanoid sinapic acid accumulate in the leaves, where they provide protection from UV-B light stress and function as sunscreens.24 It is possible that phenylpropanoid glycerol glucosides may play a similar role in Easter lily, a plant native to the unshaded rocky shores of the subtropical Ryukyu Islands of Japan. The high concentrations of phenylpropanoid glycerol glucosides in the leaves and flower buds may also serve as a constitutive reserve of fungitoxic phenolic acids as in cucumber leaves, where attack by a 4841
DOI: 10.1021/acs.jafc.5b00893 J. Agric. Food Chem. 2015, 63, 4836−4842
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(8) Shimomura, H.; Sashida, Y.; Mimaki, Y. Steroidal saponins, pardarinoside A−G from the bulbs of Lilium pardarinum. Phytochemistry 1989, 28, 3163−3170. (9) Sashida, Y.; Ori, K.; Mimaki, Y. Studies on the chemical constituents of the bulbs of Lilium mackliniae. Chem. Pharm. Bull. 1991, 39, 2362−2368. (10) Nakamura, O.; Mimaki, Y.; Nishino, H.; Sashida, Y. Steroidal saponins from the bulbs of Lilium speciosum x L. nobilissimum ’Star Gazer’ and their antitumour-promoter activity. Phytochemistry 1994, 36, 463−467. (11) Mimaki, Y.; Satou, T.; Kuroda, M.; Sashida, Y.; Hatakeyama, Y. Steroidal saponins from the bulbs of Lilium candidum. Phytochemistry 1999, 51, 567−573. (12) Mimaki, Y.; Satou, T.; Kuroda, M.; Sashida, Y.; Hatakeyama, Y. New steroidal constituents from the bulbs of Lilium candidum. Chem. Pharm. Bull. 1998, 46, 1829−1832. (13) Mimaki, Y.; Sashida, Y.; Nakamura, O.; Nikaido, T.; Ohmoto, T. Steroidal saponins from the bulbs of Lilium regale and Lilium henryi. Phytochemistry 1993, 33, 675−682. (14) Mimaki, Y.; Sashida, Y. Steroidal and phenolic constituents of Lilium speciosum. Phytochemistry 1991, 30, 937−940. (15) Mimaki, Y.; Sashida, Y. Steroidal saponins and alkaloids from the bulbs of Lilium brownii var. colchesteri. Chem. Pharm. Bull. 1990, 38, 3055−9. (16) Mimaki, Y.; Nakamura, O.; Sashida, Y.; Satomi, Y.; Nishino, A.; Nishino, H. Steroidal saponins from the bulbs of Lilium longiflorum and their antitumour-promoter activity. Phytochemistry 1994, 37, 227− 232. (17) Munafo, J. P., Jr.; Gianfagna, T. J. Chemistry and biological activity of steroidal glycosides from the Lilium genus. Nat. Prod. Rep. 2015, 32, 454−477. (18) Munafo, J. P., Jr.; Gianfagna, T. J. Antifungal activity and fungal metabolism of steroidal glycosides of Easter lily (Lilium longiflorum Thunb.) by the plant pathogenic fungus, Botrytis cinerea. J. Agric. Food Chem. 2011, 59, 5945−5954. (19) Munafo, J. P., Jr.; Ramanathan, A.; Jimenez, L. S.; Gianfagna, T. J. Isolation and structural determination of steroidal glycosides from the bulbs of Easter lily (Lilium longif lorum Thunb.). J. Agric. Food Chem. 2010, 58, 8806−8813. (20) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Iida, N. Regaloside A and B, acylated glycerol glucosides from Lilium regale. Phytochemistry 1988, 27, 451−454. (21) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Iitaka, Y. Studies on the chemical constituents of Lilium henryi Baker. Chem. Pharm. Bull. 1988, 36, 2430−2446. (22) Shimomura, H.; Sashida, Y.; Mimaki, Y. New phenolic glycerol glucosides, regaloside D, E, and F from the bulbs of Lilium species. Chem. Pharm. Bull. 1989, 43, 64−70. (23) Mimaki, Y.; Sashida, Y.; Shimomura, H. Lipid and steroidal constituents of Lilium auratum var. platyphyllum and Lilium tenuifolium. Phytochemistry 1989, 28, 3453−3458. (24) Skoog, D.; Holler, F.; Nieman, T. Introduction. In Principles of Instrumental Analysis, 5th ed.; Brooks/Cole Publishing: Belmont, CA, USA, 1997. (25) Li, J.; Ou-Lee, T.-M.; Raba, R.; Amundson, R. G.; Last, R. L. Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 1993, 5, 171−179. (26) Daayf, F.; Ongena, M.; Boulanger, R.; El Hadrami, I.; Bélanger, R. R. Induction of phenolic compounds in two cultivars of cucumber by treatment of healthy and powdery mildew-infected plants with extracts of Reynoutria sachalinensis. J. Chem. Ecol. 2000, 26, 1579− 1593. (27) Munafo, J. P., , Jr.; Gianfagna, T. J. Quantitative analysis of steroidal glycosides in different organs of Easter lily (Lilium longif lorum Thunb.) by LC-MS/MS. J. Agric. Food Chem. 2011, 59, 995−1004.
powdery mildew fungus triggers the hydrolysis of glucosides of p-coumaric acid methyl ester and other phenylpropanoid acid conjugates, significantly reducing disease symptoms.26 The unequal distribution pattern of the phenylpropanoid glycerol glucosides between above- and below-ground organs is similar to the distribution of two types of steroidal glycosides of lily bulbs. The proportion of the steroidal glycoalkaloids to furostanol saponins in light-exposed organs was greater; moreover, this ratio decreased from the above-ground organs to those growing farther below the soil surface, suggesting that these compounds may be involved in different physiological processes particular to their growing environment.27 In conclusion, this is the first report of the natural distribution of phenylpropanoid glycerol glucosides within the different organs of a plant and the first report that these compounds are present in plant organs other than the bulbs and flowers. The p-coumaroyl-based compound 3 and its acetylated derivative 5 were determined to be the most plentiful of the phenylpropanoid glycerol glucosides found in Easter lily bulbs, whereas the acetylated p-coumaroyl- and caffeoyl-based derivatives 5 and 4 were found in the highest concentrations in the flower buds. Quantitative analysis of phenylpropanoid glycerol glucosides in the component parts of L. longif lorum is a prelude to understanding their function in plant physiology, development, and pathogen defense. The results of this study will also form the basis for research on the benefits of lily bulb consumption to human health and for the development of functional foods, cosmetics, and pharmaceuticals containing lily bulb compounds.
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AUTHOR INFORMATION
Corresponding Author
*(T.J.G.) Phone: (848) 932-6369. Fax: (732) 932-6441. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are very grateful to Dr. Karsten Siems from AnalytiCon Discovery GmbH (Potsdam, Germany) for the skillful acquisition of the NMR spectra.
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REFERENCES
(1) Ori, K.; Mimaki, Y.; Mito, K.; Sashida, Y.; Nikaido, T.; Ohmoto, T.; Masuko, A. Jatropham derivatives and steroidal saponins from the bulbs of Lilium hansonii. Phytochemistry 1992, 31, 2767−2775. (2) Mimaki, Y.; Sashida, Y. Steroidal saponins from the bulbs of Lilium brownii. Phytochemistry 1990, 29, 2267−2271. (3) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Kudo, Y.; Maeda, K. New phenylpropanoid glycerol glucosides from the bulbs of Lilium species. Chem. Pharm. Bull. 1988, 36, 4841−4848. (4) Sang Tai, C.; Uemoto, S.; Shoyama, Y.; Nishioka, I. Biologically active phenolics from Lilium longif lorum. Phytochemistry 1981, 20, 2565−2568. (5) Shimomura, H.; Sashida, Y.; Mimaki, Y.; Minegishi, Y. Jatropham glucoside from the bulbs of Lilium hansonii. Phytochemistry 1987, 26, 582−583. (6) Francis, J. A.; Rumbeiha, W.; Nair, M. G. Constituents in Easter lily flowers with medicinal activity. Life Sci. 2004, 76, 671−683. (7) Tsukida, K.; Ikeuchi, K. Epoxycarotenoids. VIII. Pollen carotenoids of Lilium longif lorum and of its cultivated hybrid. Bitamin 1965, 32, 222−226. 4842
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