Major Chemical Constituents of Bamboo Shoots (Phyllostachys

Nov 2, 2015 - Bamboo shoots are a delicacy in Asia. Two novel compounds, adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate (16) and (−)-(7R...
2 downloads 9 Views 892KB Size
Download PDF
Article pubs.acs.org/JAFC

Major Chemical Constituents of Bamboo Shoots (Phyllostachys pubescens): Qualitative and Quantitative Research Jia Sun,†,∥ Zhao-Qing Ding,§,∥ Quan Gao,§,∥ Hang Xun,† Feng Tang,*,† and Er-Dong Xia† †

State Forestry Administration Key Open Laboratory, International Centre for Bamboo and Rattan, Beijing 100102, China Anhui Key Laboratory of Agricultural Products, School of Resource and Environment, Anhui Agricultural University, Hefei 230036, China

§

S Supporting Information *

ABSTRACT: Bamboo shoots are a delicacy in Asia. Two novel compounds, adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate (16) and (−)-(7R,8S)-(4-hydroxy-3-methoxyphenylglycerol 9-O-β-D-[6-O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside (20), together with 12 known nucleosides (1−12), 3 amino acids (13−15), β-carboline (17), and 2 megastigmane glycosides (18, 19) were isolated from bamboo shoots (Phyllostachys pubescens). Their structures and absolute configurations were rigorously determined by detailed spectroscopic analysis, and the composition of carbohydrates in bamboo shoots was qualitatively detected and quantitatively analyzed with ion chromatography. A simple, rapid, sensitive, and accurate HPLC-UV analysis was built for routine edible quality control of bamboo shoots, and 12 major components of bamboo shoots were quantitatively analyzed. The major chemical constituents of bamboo shoots were determined to be carbohydrates, amino acids, and nucleotides. These findings are correctives to the usual view of bamboo shoots chemical composition, and the previous research reports about the chemical composition of bamboo shoots may have taken the aromatic amino acids and nucleotides for flavonoids and phenolic acids. KEYWORDS: bamboo shoots, Phyllostachys pubescens, adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate, (−)-(7R,8S)-(4-hydroxy-3- methoxyphenylglycerol 9-O-β-D-[6-O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside, carbohydrates, affix recovery

■

phenolic acids and flavones existed in bamboo shoots. Besides, the preliminary analyses of major chemical compositions of bamboo shoots performed on ultraviolet spectrum and mass spectrometry also showed unmatched data with flavonoids and phenolic acids. Therefore, the primary chemical components of bamboo shoots remain to be determined. In the present study, 20 compounds were first isolated from bamboo shoots (P. pubescens). Two novel compounds (16 and 20), 12 nucleosides (1−12), 3 amino acids (13−15), βcarboline (17), and 2 megastigmane glycosides (18 and 19) were identified. The composition of carbohydrates in bamboo shoots was also qualitatively detected and quantitatively analyzed with ion chromatography. Moreover, a simple, rapid, sensitive, and accurate HPLC-UV analysis was built for routine edible quality control of bamboo shoots, and 12 major components of bamboo shoots were quantitatively analyzed. The results revealed the major chemical constituents of bamboo shoots to be carbohydrates, amino acids, and nucleotides, but the contents of flavonoids and phenolic acids in bamboo shoots were too low to be detected or quantitatively analyzed.

INTRODUCTION Bamboo is a type of perennial evergreen plant whose bud is called a shoot.1 Owing to its fragrance and taste, bamboo shoots are treated as a delicacy in Asia.2 As a traditional forest vegetable in China for more than 2500 years, bamboo shoots are not only delicious but also rich in nutrients and rank among the five most popular health foods in the world.3 At present, Phyllostachys pubescens is one of the highest yield bamboo shoots and is harvested in winter from December to January.1 The moisture content of fresh bamboo shoots is approximately 85%.4 There has been abundant research on bamboo shoots.3 For instance, some research has reported that bamboo shoot extract possesses antioxidant and angiotensin-converting enzyme inhibition activities.5−7 In addition, many studies have reported that the bioactivity of bamboo shoots mainly arises from flavonoids and phenolic acids,3,5,7−10 and the previous studies on the chemical compositions of bamboo shoots were based on the research results of bamboo leaves and other vegetables.5,7 However, so far, there have been few reports about exact compounds confirmed from bamboo shoots. When we compared the contents of flavonoids and phenolic acids from bamboo leaves with bamboo shoots under the same analysis conditions, high contents of flavonoids and phenolic acids were found from the leaf extract, whereas few flavonoids and phenolic acids were found in shoots. For further investigations, we selected and tested a lot of extraction and purification methods for obtaining flavonoids and phenolic acids from bamboo shoots, but the results still confirmed few © XXXX American Chemical Society

Special Issue: Phytochemicals in Food (ISPMF 2015) Received: August 24, 2015 Accepted: November 2, 2015

A

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. NMR Spectroscopic Data (Measured at 500 MHz) for Compounds 16 and 20 (in DMSO) from the Extracted Fractions of Bamboo Shoots (Phyllostachys pubescens) compound 16 no.

δC type

2 4 5 6 6-NH2 8 1′ 2′ 2′-OH 3′ 3′-OH 5′

153.4 149.9 119.7 156.7

■

140.5 88.3 72.4 70.1

compound 20

δH (J in Hz) 8.16, 1H, s

7.27, 8.38, 6.16, 4.84, 5.71, 4.72, 5.57,

2H, 1H, 1H, 1H, 1H, 1H, 1H,

brs s d, 5.5 m brs m brs

162.7

no.

δC type

1 2 3 4 5 6 7 8 9 3-OCH3 1′ 2′

134.6 111.4 147.4 145.7 115.2 119.5 72.9 74.3 71.3 55.9 103.9 74.0

δH (J in Hz) 6.88, 1H, s

6.63, 6.68, 4.46, 3.64, 3.47, 3.70, 4.19, 3.02,

1H, d, 8.0 1H, d, 8.0 1H, d, 6.0 1H, m 3.17, 2H, m 1H, s 1H, d, 8.0 1H, m

no.

δC type

3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 3″-OCH3

76.8 70.6 74.2 64.4 121.0 113.0 147.8 152.0 115.7 124.0 166.0 56.0

δH (J in Hz) 3.18, 3.18, 3.43, 4.53,

1H, 1H, 1H, 1H,

m m m dd, 12.0, 2.5; 4.17, 1H, m

7.42, 1H, s

6.89, 1H, d, 8.0 7.46, 1H, d, 8.0 3.77, 1H, s

extracted three times with 60% aqueous ethanol at room temperature (3 days each for 40 L). The filtrates were combined and concentrated under reduced pressure to remove the organic solvent. The concentrated aqueous fraction (2.5 kg) was separated on a macroporous resin column (200 × 20 cm) using a gradient of water/ethanol with five ratios (100:0, 85:15, 70:30, 50:50, 5:95) fraction. The 15% ethanol fraction (35.1 g) was applied to an ODS-A column and preparative HPLC and eluted with acetonitrile/ water(7:93) to yield compounds 1 (21 mg), 2 (10 mg), 3 (39 mg), 4 (20 mg), 6 (16 mg), 7 (33 mg), 9 (18 mg), 10 (13 mg), 11 (18 mg), 12 (29 mg), 13 (11 mg), 14 (1102 mg), and 15 (94 mg). The 30% ethanol fraction (16.5 g) was separated by ODS-A column preparative HPLC and eluted with acetonitrile/water (12:88), resulting in the isolation of compounds 5 (3 mg), 8 (9 mg), 16 (5 mg), 17 (7 mg), 18 (10 mg), and 19 (11 mg). The 50% ethanol fraction (5.8 g) was subjected to preparative HPLC with an ODS-A column and eluted with acetonitrile/water (15:85) to yield compound 20 (6 mg). Structures and absolute configurations of compounds 1−20 were determined by detailed spectroscopic analysis (UV, IR, HRESIMS, NMR, and CD). Adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate (16): white amorphous powder; [α]D +37.3° (c 0.50, methanol); HRESIMS (C10H11N5O5) m/z 282.0836 [M + H]− (calculated for 282.0838); IR νmax 3357, 3196, 2921, 2360, 2340, and 1261; UV λmax (methanol) (log ε) 258.8 nm; 1H (500 MHz) and 13C (125 MHz) NMR (DMSO-d6), see Table 1; CD (C 5 × 10−3, Mo2(OAC)4, DMSO) Δε285 nm +0.053, Δε310 nm −0.007, Δε334 nm −0.1991, Δε400 nm +0.1079. (−)-(7R,8S)-(4-Hydroxy-3-methoxyphenylglycerol 9-O-β-D[6-O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside (20): white amorphous powder; [α]D +10.1° (c 2.0, methanol); HRESIMS (C24H30O13) m/z 549.1580 [M + Na]+ (calculated for 549.1584); IR νmax 3356, 2920, 2851, 1701, 1656, 1513, 1281, and 1113; UV λmax (methanol) (log ε) 265.3 nm; 1H (500 MHz) and 13C (125 MHz) NMR (DMSO-d6), see Table 1; CD (C 2.5 × 10−3, MeOH) Δε233 nm +0.089, Δε278.5 nm −0.039. The sugar unit configuration of compound 20 after cellulose hydrolysis was analyzed by GC analysis of its trimethylsilyl L-cysteine derivative.11,12 Analysis of Carbohydrate Composition. The analysis of the composition of polysaccharides was based on the method of sulfuric acid acidolysis13 with some modification. Fresh bamboo shoots (P. pubescens) (1 g) were minced and dried, and the specimens (107 mg) were treated with 72% H2SO4 (2.5 mL) and ultrapure water (27 mL) in a brown bottle at 105 °C for 2.5 h and shaken every 30 min. Once cooled, the acidolysis solution was neutralized to pH 7.0 by 2.0 mM NaOH, and a portion of the sample (5 mL) was added to 10 mL of ultrapure water and then filtered using a membrane filter with a pore size of 0.22 mm; the filtrate (10 mL) was then tested. Detection of the sugars (i.e., glucose, xylose, fructose, galactose, and arabinose) and analyses of the carbohydrates were conducted in ultrapure water with 2.0 mM NaOH and 0.5 mM NaAc in a mobile phase at a rate of 1 mL/

MATERIALS AND METHODS

Plant Material. Fresh bamboo shoots (P. pubescens), cultivated and processed in Hangzhou city (Zhejiang province, China), were collected from a bamboo pilot field at the Research Institute of Subtropical Forestry, Chinese Academy of Forestry, China. A voucher specimen (No. 00-A3-006) was deposited in the State Forestry Administration Key Open Laboratory, International Centre for Bamboo and Rattan, Beijing, China. General Procedure. Preparative HPLC was performed using a Shimadzu LC-6AD instrument with an SPD-20A detector (Shimadzu, Kyoto, Japan) and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm, YMC, Kyoto, Japan). HPLC-PAD analysis was performed using a Waters 2695 system and a PDA detector 2996 (Waters, Milford, MA, USA) with a YMC-Pack ODS-AQ C18 column (250 × 4.6 mm, 5 μm, YMC), and the PAD detection wavelength was monitored in the range of 210−400 nm. The optical rotation was measured with a PerkinElmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). Ultraviolet (UV) spectra were obtained using a Waters 2695 HPLC with a photodiode array detector (Waters). Infrared (IR) spectra were taken with a ThermoFisher Scientific Nicolet iN10 (Thermo, Waltham, MA, USA) spectrophotometer with LN-cooled MCT. One- and two-dimensional nuclear magnetic resonance (NMR) spectra were run with Bruker 500 spectrometers (Bruker, Zurich, Switzerland) using DMSO-d6 as solvent and tetramethylsilane (TMS) as an internal standard, operating at 500 MHz for 1H and at 125 MHz for 13C. Chemical shifts are expressed in δ (ppm), and coupling constants are reported in hertz. High-resolution electrospray ionization mass spectroscopy (HRESIMS) spectra were obtained using an Agilent 6540 high-resolution time-of-flight (Q-TOF) mass spectrometer (Agilent, Santa Clara, CA, USA). Gas chromatography (GC) analysis was performed with an Agilent 6890N gas chromatograph (Agilent). Circular dichroism (CD) spectra were recorded using a JASCO J-815 CD spectrometer (JASCO, Tokyo, Japan) on methanol and dimethyl sulfoxide (DMSO) solutions. The carbohydrates were analyzed by ion chromatography using an amperometric detector (Metrohm, Herisau, Switzerland) with a Hamilton RCX-30 column (250 × 4.6 mm, 7 μm; Hamilton, Reno, NV, USA). Chemicals and Reagents. Column chromatography was performed with macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan), Rp-18 (50 μm, YMC), and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). All reagents and sugars were purchased from Beijing Chemical Works (Beijing, China), except as otherwise specified. HPLC grade methanol (MeOH) and ethanol (EtOH) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Extraction, Isolation, Purification, and Identification of Compounds from Bamboo Shoots (P. pubescens). Fresh bamboo shoots (P. pubescens) (50 kg) were minced into pulp and filtered by a 300 mesh strainer. The filtrate was squeezed out, and the residue was B

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Compounds 1−20 isolated from bamboo shoots (Phyllostachys pubescens). min; the injection volume was 2 mL, and the run time was 60 min. Five monosaccharide standard stock solutions (0.5 mg/mL) were prepared by mixing in a 10 mL volumetric flask and were diluted to six different concentrations (1, 5, 25, 50, 150, and 250 mg/L) with ultrapure water to examine the linear relationship. Optimum Extraction. Extraction was conducted by using accumulation of two optimum extraction phenolic and flavonoid conditions from bamboo shoots (P. pubescens) with minor modification,7,14 and it was further confirmed by using an orthogonal test about extraction solvent, extraction time, and extraction cycles with three levels. Fresh bamboo shoots (P. pubescens) (2 g) were minced into pulp and filtered by a 300 mesh strainer. The filtrate was squeezed out, and the residue was extracted three times with 50 mL of 95% ethanol for 24 h by magnetic stirring at room temperature; after suction filtration, the residue was again extracted with 50 mL of 60% aqueous ethanol for 3 h at 80 °C in three replicates and the second extract suction filtration. All obtained filtrates and extracts were combined and concentrated, then diluted with water to 10 mL. Authentic samples were prepared as water solutions. All sample solutions were filtered through a 0.45 μm (Millipore) filter before injection for the HPLC analysis. HPLC-UV Analysis of 12 Components. HPLC-UV analysis was conducted using a Waters 2695-2996 system. The optimal mobile phase for analysis was a binary gradient elution system consisting of solvent A (acetonitrile) and solvent B (water containing 0.5% acetic acid). The gradient was programed as 3% (solvent A) and 97% (solvent B) for 0−10 min, changed to 10% (solvent A) and 90% (solvent B) for 11 min, changed to 12% (solvent A) and 88% (solvent

B) for 17 min, and changed to 100% (solvent A) for 37 min. The column was a YMC-PACK ODS-AQ C18 column. The flow rate was 1 mL/min, and the column temperature was set at 25 °C. The injection volume was 10 μL. The UV detection wavelength was monitored at 270 nm (λmax for compounds 1, 2, 3, 7, 12, 13, and 15) and at 254 nm (λmax for compounds 4, 6, 10, 11, and 14). The HPLC-UV detection peaks were confirmed by previous isolation and identification as well as the UV absorption and retention times of the authentic samples. All experiments were performed in triplicate, and the results are expressed as mean values. Quantification of the Main Constituents. The standards were obtained from the previously isolated and identified compounds 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 14, and 15 from bamboo shoots (P. pubescens), which were prepared and diluted to a mixture of 0.2 mg/ mL for compound 1, 10, and 11 standards, 0.3 mg/mL for compound 3 and 12 standards, 0.1 mg/mL for compound 2 and 13 standards, 0.05 mg/mL for compound 4 and 6 standards, 0.5 mg/mL for compound 7 standards, 15 mg/mL for compound 14 standards, and 5 mg/mL for compound 15 standards in a volumetric flask and were diluted to five different concentrations. The series concentrations of the mixed standard solutions were prepared to assess the linear relationships, and each of the different concentrations of solution was injected using the chromatographic conditions to generate corresponding regression equations. Validation of the Analysis. The HPLC-UV analysis was validated for limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy.15 The LOD and LOQ for the main constituents were measured by duplicate injections of standard solutions based on S/N C

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry ratios of 3 and 10, respectively. The precision of the method was studied through five replicate injections of the mixed standard solutions and was expressed as RSD. The intraday precision was studied by injecting the mixed standard solutions five times on the same day (0, 2, 4, 8, and 12 h), and the interday precision was evaluated by injecting the mixed standard solutions four times on four consecutive days. Results are expressed as RSD. The accuracy was based on a standard addition recovery test of the method, which was performed with a 10 g sample of fresh bamboo shoots, to which was added three concentrations of mixed standard solutions. Three replicate spiked samples were analyzed by HPLC. Subsequently, the recovery rates of the 12 components were calculated. A blank control was also analyzed.

Figure 2. Significant HMBC and 1H−1H COSY correlations of compounds 16 and 20.

■

dimolybdenum reagent Mo2(OAC)4 method analysis and a ROESY experiment. A convenient spiral rule for the dimolybdenum reagent Mo2(OAC)4 in DMSO-induced CD data was demonstrated to be useful to determine the absolute configuration of C-2′ and C-3′ (vic-diols).29 Application of the method to 16 indicated that the absolute configuration at C-2′ was R, and C-3′ was R by the positive Cotton effect at 310 and 400 nm.30 NOESY correlations of H-1′ with H-3′ were not observed, confirming that H-1′ and H-3′ were on the other side of the molecule and indicating that the absolute configuration of H-1′ was R. Thus, the structure of 16 was elucidated to be adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate. Compound 20 was obtained as a white amorphous powder with [α]D +10.1° (c = 0.20, MeOH, 20 °C), having a molecular formula of C24H30O13, and was indicated by positive HRESIMS at m/z 549.1580 [M + Na]+, calcd for 549.1584). All spectra of compound 20 are presented in the Supporting Information, and NMR assignments are gathered in Table 1. The UV spectrum exhibited absorption maximum at 265.3 nm. The IR spectrum exhibited absorption bands for hydroxyl (3356 cm−1), methyl (2920 cm−1) methylene (2851 cm−1), carbonyl (1701 cm−1), and aromatic (1656 and 1515 cm−1) functional groups. The 1H NMR spectrum of 20 in DMSO exhibited signals attributed to a meta−ortho-trisubstituted phenyl group of three aromatic protons at δH 6.88 (1H, s), δH 6.68 (1H, d, J = 8.0), and δH 6.63 (1H, d, J = 8.0); a meta−ortho-substituted benzoyl group of three aromatic protons at δH 7.42 (1H, s), δH 7.46 (1H, d, J = 8.0), and δH 6.89 (1H, d, J = 8.0); and two aromatic methoxy groups at δH 3.77 (3H, s) and δH 3.70 (3H, s). In addition, the 1H NMR spectrum exhibited signals assignable to a β-glucopyranosyl unit, of which the anomeric proton resonated at δH 4.19 (1H, d, J = 8.0), whereas the oxymethylene protons were at δH 4.53 (1H, dd, J = 12.0, 2.5) and δH 4.53 (1H, m). The presence of a β-glucopyranosyl unit was confirmed by acid hydrolysis of 20 and produced glucose identified by a gas chromatography (GC) comparison to an authentic sugar sample. The glucose isolated from the hydrolysate produced a positive optical rotation, indicating that it was β-D-glucose.31 The 13C NMR and DEPT spectra of 20 exhibited carbon signals corresponding to the above units, one 4-hydroxy-3-methoxyphenyl, one 4-hydroxy-3-methoxybenzoyl, and one β-glucopyranosyl and also included were two oxygenbearing methines at δC 72.9 and δC 74.3 and an oxygen-bearing methylene at δC 71.3. In the HMBC spectrum, the correlations from δH 4.46 (H-7) to δC 134.6 (C-1), δC 111.4 (C-2), and δC 119.5 (C-6); δH 3.64 (H-8) to δC 134.6 (C-1) and δC 72.9 (C7); and δH 3.47, 3.17 (H-9) to δC 72.9 (C-7) revealed a 4hydroxy-3-methoxyphenylglycerol moiety, and the anomeric protons at δH 4.19 (H-1′) were correlated to δC 71.3 (C-9) (Figure 2), indicating that β-glucopyranosyl was located at C-9. Moreover, HMBC correlation of δH 4.53, 4.17 (H-6′) with δC

RESULTS AND DISCUSSION Isolation and Characterization. Repeated column chromatography over macroporous resin and preparative HPLC of the extract from fresh bamboo shoots (P. pubescens) led to the isolation of 2 novel compounds, adenine(1′R,2′R,3′R)-cyclic butanetetraol carbonate (16) and (−)-(7R,8S)-(4-hydroxy-3-methoxyphenylglycerol 9-O-β-D-[6O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside (20), in addition to 18 known compounds. The known compounds were identified as cytidine (1),16,17 2′-deoxycytidine (2),18 adenine (3),16,17 adenosine (4),19 2′-deoxyadenosine (5),16,17 uracil (6),16,17 uridine (7),16,17 2′-O-methyl-adenosine (8),20 guanine (9),16,17 guanosine (10),16,17 2′-deoxyguanosine (11),21 thymidine (12),22 L-tryptophan (13),23 L-phenylalanine (14),24,25 L-tyrosine (15),24,25 β-carboline (17),26 (−)-(6S,9R)cis-roseoside (18),27 and (−)-(6S,9R)-roseoside (19)28 (Figure 1) by comparison of their spectroscopic and physical data with those previously reported in the literature. This was the first isolation of all 20 compounds from bamboo shoots (P. pubescens). Compound 16 was obtained as a white powder, [α]D +37.3° (c = 0.50, MeOH, 20 °C), and its molecular formula was determined to be C10H11N5O5 by positive HRESIMS (m/z 282.0836 [M + H]−, calcd for 282.0838). All spectra of compound 16 are presented in the Supporting Information, and NMR assignments are gathered in Table 1. The UV spectrum exhibited absorption maximum at 258.3 nm, and the IR spectrum exhibited absorption bands for amino and hydroxyl (3357 cm−1), secondary amines (3196 cm−1), methylene (2921 cm−1), and carbonic anhydride (2360 cm−1). The 1H NMR spectra of compound 16 revealed signals attributable to an adenine moiety with two aromatic protons at δH 8.38 (s, 1H) and δH 8.16 (s, 1H) and amino group protons at δH 7.27 (brs, 2H). In addition, one anomeric proton was found at δH 6.16 (1H, d, J = 5.5), two oxymethines at δH 4.84 (1H, m) and δH 4.72 (1H, m), and one oxymethylene at δH 4.31 (1H, t, J = 1.5), 4.20 (1H, m). The 13C NMR spectrum (Table 1) exhibited 10 carbon signals in addition to the adenine group at δC 156.7, δC 153.4, δC 149.9, δC 140.5, and δC 119.7, one carbonyl carbon at δC 162.7, three oxymethines at δC 88.3, δC 72.4, and δC 70.1, and one oxymethylene at δC 85.0. In the 1 H−1H COSY experiment, the correlation of δH 6.16 (H-1′) to δH 4.84 (H-2′), δH 4.84 (H-2′) to δH 4.72 (H-3′), and δH 4.72 (H-3′) to δH 4.31, 4.20 (H-4′) combined with the HMBC correlation from δH 4.31, 4.20 (H-4′) to δC 162.7 (C-5′) confirmed the presence of a seven-membered carbonate ring unit (Figure 2). The correlations of δH 6.16 (H-1′) to δC 149.9 (C-4) and δC 140.5 (C-8) were observed in the HMBC spectrum, confirming the connection of C-1′ to N-9 (Figure 2). The relative configuration of 16 was determined by D

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 3. HPLC chromatogram of the 12 component mixed standards.

Table 2. Linear Relationships between the Peak Area and Concentrations of 12 Components Found in Bamboo Shoots (Phyllostachys pubescens)

a

chemical constituent

retention time (min)

cytidine (1) 2′-deoxycytidine (2) adenine (3) adenosine (4) uracil (6) uridine (7) guanosine (10) 2′-deoxyguanosine (11) thymidine (12) L-tryptophan (13) L-phenylalanine (14) L-tyrosine (15)

2.479 2.137 2.937 9.635 4.670 6.718 10.669 13.856 15.885 17.073 11.603 5.502

regression equationa Y Y Y Y Y Y Y Y Y Y Y Y

= = = = = = = = = = = =

37574X + 6632.2 62529X + 27807 60815X + 2837.3 41059X − 879.95 59626X − 2793.8 28093X + 2762.6 32471X + 2626.1 48481X + 2543.1 40313X + 8733.6 28807X + 3999.5 493.3X − 895 3830.8X − 5931.6

R2

linear range (mg/L)

0.9999 0.9997 0.9999 0.9999 0.9999 0.9999 0.9999 0.9999 0.9999 0.9999 0.9998 0.9995

0.5−50 0.5−50 0.1−20 0.5−50 0.2−20 0.3−20 0.5−50 0.5−50 0.5−50 0.5−50 10−300 2−200

contentb (mg/kg) 4.50 2.52 8.20 0.83 1.14 12.20 4.17 5.23 7.23 2.16 269.98 122.06

± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.03 0.03 0.01 0.02 0.12 0.02 0.08 0.06 0.02 1.17 0.91

Y = peak area and X = compound concentration. bMean ± SD (n = 3).

(Fru); and y = 45.362x + 5.9619, r2 = 0.9966 (Gal). The HPLC chromatogram of mixed monosaccharide standards and the HPLC chromatogram of bamboo shoot extract carbohydrate composition are presented in the Supporting Information. The carbohydrate composition of the bamboo shoot (P. pubescens) was Glc (2.04 ± 0.54 g/kg), Fru (9.11 ± 1.02 g/kg), and Gal (0.24 ± 0.08 g/kg). Fructose was confirmed to be the main component of the bamboo shoot carbohydrates. HPLC Quantitative Analysis of 12 Components. A typical chromatogram obtained from the mixed standards is presented in Figure 3. The linear relationships observed are presented in Table 2. The standard curves in the corresponding ranges exhibited good linearity. The chromatogram of fresh bamboo shoot extract is presented in Figure 4. The contents of the 12 components in bamboo shoots (P. pubescens) are presented in Table 2, and the extract of fresh bamboo shoots

166.0 (C-7″) demonstrated that the 4-hydroxy-3-methoxybenzoyl moiety was substituted at C-6′ (Figure 2). The Δ δC8‑C7 value in DMSO was 1.4 ppm, suggesting a 7,8-erythro configuration for 20.32 In the CD spectrum of 20, a positive Cotton effect at 233 nm indicated the 8S configuration.33 Therefore, the structure of compound 20 was determined to be (−)-(7R,8S)-(4-hydroxy-3-methoxyphenylglycerol 9-O-β-D-[6O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside. Analysis of the Carbohydrate Composition. In addition to hydrolysate, glucose, fructose, and galactose were detected by HPLC−ion chromatography; their retention times were 13.0 (Gal), 14.8 (Glc), and 20.3 (Fru) min, and six different concentrations established the calibration curve. Good linearity was established for all tested authentic samples. Subsequently, calibration curves were made accordingly to yield the following regression equations for quantificational analysis: y = 458.97x − 64.636, r2 = 0.9956 (Glc); y = 24.443x − 3.0048, r2 = 0.9965 E

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 4. HPLC chromatogram of fresh bamboo shoots (Phyllostachys pubescens).

Table 3. Limits of Detection, Accuracy, and Precision for 12 Components of Bamboo Shoots (Phyllostachys pubescens) chemical constituent cytidine (1) 2′-deoxycytidine (2) adenine (3) adenosine (4) uracil (6) uridine (7) guanosine (10) 2′-deoxyguanosine (11) thymidine (12) L-tryptophan (13) L-phenylalanine (14) L-tyrosine (15)

LOD (mg/kg)

LOQ (mg/kg)

accuracy precision (RSD%, n = 4)

intraday precision (RSD%, n = 5)

interday precision (RSD%, n = 4)

0.17 0.21 0.17 0.44 0.28 0.36 0.36 0.30

0.57 0.70 0.57 1.47 0.94 1.20 1.20 1.00

0.96 0.65 0.95 0.86 0.78 0.77 0.99 0.90

0.70 0.71 1.42 1.19 0.50 0.20 0.78 0.76

2.74 2.35 2.53 2.36 2.42 2.43 2.89 2.52

0.11 0.31 4.60 1.98

0.37 1.03 16.06 6.60

0.88 0.74 1.06 1.10

0.84 1.12 1.13 0.63

2.76 2.34 2.74 2.31

was observed to be rich in L-phenylalanine (269.98 ± 1.17 mg/ kg) and L-tyrosine (122.06 ± 0.91 mg/kg). Using the optimum conditions for the extraction of flavonoids and phenolic acids from the bamboo shoots, the detection wavelength was set at 270 and 254 nm because flavonoids and phenolic acids exhibit stronger absorption at 270 and 254 nm. However, no obvious peak was observed (Figure 4); therefore, we believe that flavonoids and phenolic acids are not primary components of bamboo shoots. Validation. The HPLC method was validated in-house for LOD, LOQ, accuracy, intraday and interday precision, and recovery. The LOD, LOQ, accuracy, and intraday and interday precision data are presented in Table 3. LOD and LOQ were in

suitable ranges for the 12 components. The values of RSD for the accuracy and intraday and interday precision studies were 70%. The method was found to be feasible for qualitative and quantitative determination of the main components in bamboo shoots. According to our results, the major chemical constituents of bamboo shoots (P. pubescens) are primary metabolites, carbohydrates, amino acids, and nucleotides. These chemical components affect the taste of fresh bamboo shoots; the high F

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 4. Recovery of 12 Components of Bamboo Shoots (Phyllostachys pubescens) 1 chemical constituent

addition quantity (mg/kg)

cytidine (1) 2′-deoxycytidine (2) adenine (3) adenosine (4) uracil (6) uridine (7) guanosine (10) 2′-deoxyguanosine (11) thymidine (12) L-tryptophan (13) L-phenylalanine (14) L-tyrosine (15)

2 addition quantity (mg/kg)

recovery (%)

32 16

99.89 ± 0.04 98.86 ± 0.09

0.04 0.09

20 10

88.20 ± 0.80 90.83 ± 1.76

0.68 1.41

12 6

89.83 ± 0.77 89.78 ± 3.24

0.47 1.87

48 8 8 80 32 32

94.41 97.25 94.63 96.88 89.74 96.14

± ± ± ± ± ±

0.42 0.88 1.62 0.56 1.68 1.24

0.38 0.94 1.58 0.56 1.88 1.2

30 5 5 50 20 20

94.12 92.73 95.53 92.98 93.07 90.10

± ± ± ± ± ±

1.50 1.44 0.98 1.42 2.18 2.00

1.12 1.42 0.81 1.15 1.91 1.57

18 3 3 36 12 12

86.74 93.67 93.78 92.68 89.72 90.31

± ± ± ± ± ±

0.92 2.80 3.13 2.35 1.09 5.16

0.5 2.08 1.91 1.55 0.72 2.9

48 16 2400

100.17 ± 2.01 88.29 ± 1.28 95.43 ± 0.80

1.98 1.43 0.92

30 10 1500

90.16 ± 0.73 87.60 ± 0.95 94.94 ± 2.04

0.6 0.84 0.96

18 6 900

91.24 ± 1.13 93.28 ± 2.37 91.18 ± 1.55

0.67 2.76 1.12

800

97.07 ± 0.57

0.57

500

90.10 ± 2.46

2.01

360

87.03 ± 2.07

1.4

recovery (%)

RSD (%)

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to the staff of the analytical group at the State Forestry Administration Key Open Laboratory in the International Centre for Bamboo and Rattan and the Institute of Materia Medica (IMM) at the Chinese Academy of Medical Sciences & Peking Union Medical College for measuring the spectroscopic data.

■

REFERENCES

(1) Jiang, Z. H. Conservation of bamboo resources and genetic improvement. Bamboo and Rattan in the World; China Forestry Publishing House: Beijing, China, 2007; pp 200−201. (2) Jin, C.; Wang, Y. Y.; Zhang, W. J.; Zhang, Y. H.; Chen, G. L.; Lin, M. S. Literature analysis and development strategy on bamboo shoot research in China. J. Zhejiang Agric. For. Univ. 2000, 17, 75−79. (3) Chongtham, N.; Bisht, M. S.; Haorongbam, S. Nutritional properties of bamboo shoots: potential and prospects for utilization as a health food. Compr. Rev. Food Sci. Food Saf. 2011, 10, 153−168. (4) Zheng, L. U.; Lin, Q. X.; Wang, H.; Lu, Z. J. Hot pump drying characteristics of bamboo shoots and the rehydrating rates of the products. J. Fujian Agric. For. Univ. 2002, 31, 117−119. (5) Liu, L. L.; Liu, L. Y.; Lu, B. Y.; Xia, D. Z.; Zhang, Y. Evaluation of antihypertensive and antihyperlipidemic effects of bamboo shoot angiotensin converting enzyme inhibitory peptide in vivo. J. Agric. Food Chem. 2012, 60, 11351−11358. (6) Park, E. J.; Jhon, D. Y. Effects of bamboo shoot consumption on lipid profiles and bowel function in healthy young women. Nutrition 2009, 25, 723−728. (7) Park, E. J.; Jhon, D. Y. The antioxidant, angiotensin converting enzyme inhibition activity, and phenolic compounds of bamboo shoot extracts. LWT−Food Sci. Technol. 2010, 43, 655−659. (8) Wang, W. W.; Yuan, Y.; Wu, J. W.; Tai, Y. L.; Hu, F. HPLC-MS analysis on chemical principles of ethanol extracts from bamboo shoots and waste materials. Acta Laser Biol. Sinica 2011, 20, 703−710. (9) Huang, X. B.; Li, J. H.; Zhang, W. H.; Peng, S. D.; Zhu, Y. T.; Lin, L. J. Nutritional components and active ingredient analysis of different

ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05167. 1 H and 13C, DEPT, HSQC, HMBC, and NOESY spectra of adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate (16) and 1H and 13C, DEPT, HSQC, and HMBC spectra of (−)-(7R,8S)-(4-hydroxy-3-methoxyphenylglycerol 9O-β-D-[6-O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside (20) in DMSO; UV, IR, HRESIMS, and CD spectra of adenine-(1′R,2′R,3′R)-cyclic butanetetraol carbonate (16) and (−)-(7R,8S)-(4-hydroxy-3-methoxyphenylglycerol 9-O-β-D-[6-O-4-hydroxy-3-methoxybenzoyl])-glucopyranoside; HPLC chromatogram of mixed monosaccharide standards and HPLC chromatogram of fresh bamboo shoot carbohydrate composition (PDF)

AUTHOR INFORMATION

Corresponding Author

*(F.T.) Phone: + 86 10 8478 9821. Fax: + 86 10 8478 9821. Email: [email protected]. Author Contributions ∥

addition quantity (mg/kg)

This work was supported by the Basic Science Research Fund Program of the International Centre for Bamboo and Rattan (ICBR) (1632016002) and the National Science and Technology Infrastructure Program (No. 2012BAD23B03).

S Supporting Information *

■

recovery (%)

RSD (%)

Funding

content of fructose makes the shoots taste sweet, L-phenylalanine makes them taste slightly bitter, and nucleotides contribute an umami taste. The previous research on flavonoids and phenolic acids in bamboo shoots may have mistaken the aromatic amino acids and nucleotides for flavonoids and phenolic acids, and the major factor causing the mistake is mixup of these compounds, which have adjacent retention time with inappropriate HPLC analysis method. The results are also consistent with theoretical analysis from the perspective of plant physiology; bamboo shoots are in the primary metabolism phase of bamboo and, thus, should not contain high levels of secondary metabolites such as flavonoids and phenolic acids. Compound 2, which is a novel adenosine that bears a rare carbonate heptatomic ring group, is speculated to be a new marker involved in a new metabolic pathway. In addition, the chemical composition analysis method for bamboo shoots established in our research may be applicable to other species of bamboo shoots.

■

3

RSD (%)

J.S., Z.-Q.D., and Q.G. contributed equally. G

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry parts of winter moso bamboo shoots. Food Sci. Technol. 2014, 39, 59− 63. (10) Wu, J. W.; Yuan, Y.; Wang, W. W.; Yuan, C. H. Antioxidant activity of crude extracts from shoots and shells of different bamboos. Acta Laser Biol. Sinica 2011, 20, 703−710. (11) Liang, D.; Hao, Z. Y.; Zhang, G. J.; Zhang, Q. J.; Chen, R. Y.; Yu, D. Q. Cytotoxic triterpenoid saponins from Lysimachia clethroides. J. Nat. Prod. 2011, 74, 2128−2136. (12) Sun, J.; Tang, F.; Yue, Y. D.; Xun, H.; Guo, X. F. Two new compounds from the dry leaves of Pleioblastus amarus (Keng) keng f . J. Asian Nat. Prod. Res. 2014, 16, 930−935. (13) Wang, L. C.; Zhang, K.; Liu, Q. D.; Liu, R.; Wu, H. Isolation and structural elucidation of novel homogenous polysaccharide from Mactra venerformis. Carbohydr. Polym. 2011, 86, 982−987. (14) Lin, S.; Lin, M. F.; W, J. Y.; Lu, Y. D.; W, Z. H. Study on the extraction and antioxidant activity of flavonoid compounds from shells of bamboo shoots. Appl. Chem. Ind. 2012, 41, 445−448. (15) Wang, S.; Tang, F.; Yue, Y. D.; Yao, X.; Wei, Q.; Yu, J. Simultaneous determination of 12 coumarins in bamboo leaves by HPLC. J. AOAC Int. 2013, 96, 942−946. (16) Pretsh, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds Tables of Spectral Data; Springer-Verlag: Berlin, Germany, 2000; pp 154−155. (17) Pretsh, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds Tables of Spectral Data; Springer-Verlag: Berlin, Germany, 2000; pp 237−239. (18) Brzezinska, J.; Gdaniec, Z.; Popenda, L.; Markiewicz, W. T. Polyaminooligonucleotide: NMR structure of duplex DNA containing a nucleoside with spermine residue, N-[4,9,13-triazatridecan-1-yl]-2′deoxycytidine. Biochim. Biophys. Acta, Gen. Subj. 2014, 1840, 1163− 1170. (19) Kinjo, J.; Masumoto, K.; Inoue, M.; Takeshita, T.; Nohara, T. A new sapogenol and other constituents in Abri Semen, the seeds of Abrus precaturius L. Chem. Pharm. Bull. 1991, 39, 116−119. (20) Zhang, Z. H.; Li, W. J.; Lin, R. C.; Dai, Z.; Li, X. F. Isolation and structure elucidation of alkaloids from Pinellia ternata. Heterocycles 2013, 87, 637−643. (21) Zhuo, R. P.; Fu, H. Z.; Zhang, L. H.; Lin, W. H. Nucleosides from Anthopleura stell. Chin. Tradit. Herb Drugs 2001, 32, 289−291. (22) Shi, X. F.; Tang, X. L.; Li, G. Q.; Wang, C. Y.; Guan, H. S. Study on chemical constitutes of Muriceides collaris from the south China. Chin J. Marine Drugs. 2009, 28, 18−21. (23) Shi, H. M.; Wen, J.; Tu, P. F. Chemical constitutes of Abrus cantoniensis. Chin. Tradit. Herb Drugs 2006, 37, 1610−1613. (24) Pretsh, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds Tables of Spectral Data; Springer-Verlag: Berlin, Germany, 2000; pp 150−151. (25) Pretsh, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds Tables of Spectral Data; Springer-Verlag: Berlin, Germany, 2000; pp 234−235. (26) Shen, L.; Jiang, S. P.; Zhu, H. J. Chemical constituents in herb of Incarvillea younghusbandii Sprague. Nat. Prod Res. Dev. 2012, 24, 1210−1213. (27) Pabst, A.; Barron, D.; Semon, E.; Schreier, P. Two diastereomeric 3-oxo-α-ionol-β-glucosides from raspberry fruit. Phytochemistry 1992, 31, 1649−1652. (28) Sueyoshi, E.; Liu, H.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Aramoto, M.; Takeda, Y. Bridelionosides A−F: megastigmane glucosides from Bridelia glauca f. balansae. Phytochemistry 2006, 67, 2483−2493. (29) Di Bari, L.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. Determination of absolute configuration of acyclic 1,2-diols with Mo2 (OAc)4. 1. Snatzke’s method revisited. J. Org. Chem. 2001, 66, 4819− 4825. (30) Snatzke, G.; Ahmad, H. The CD in situ complexation method as a tool for determination of absolute configurations of Cottonogenic derivatives. J. Am. Chem. Soc. 1993, 115, 12533−12544.

(31) Wang, F.; Jiang, Y. P.; Wang, X. L.; Wang, S. J.; Bu, P. B.; Lin, S.; Zhu, C. G.; Shi, J. G. Aromatic glycosides from the flower buds of Lonicera japonica. J. Asian Nat. Prod. Res. 2013, 15, 492−501. (32) Sun, J.; Yu, J.; Zhang, P. C.; Yue, Y. D. Enantiomeric determination of four diastereoisomeric oxyneolignans from Bambusa tuldoides Munro. Phytochem. Anal. 2015, 26, 54−60. (33) Xiong, L.; Zhu, C. G.; Li, Y. R.; Tian, Y.; Lin, S.; Yuan, S. P.; Hu, J. F.; Hou, Q.; Chen, N. H.; Yang, Y. C.; Shi, J. G. Lignans and neolignans from Sinocalamus af f inis and their absolute configurations. J. Nat. Prod. 2011, 74, 1188−1200.

H

DOI: 10.1021/acs.jafc.5b05167 J. Agric. Food Chem. XXXX, XXX, XXX−XXX