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New Screw Lactam and Two New Carbohydrate Derivatives from the Methanol Extract of Rice Bran Wei Wang, Jia Guo, Junnan Zhang, Tianxing Liu, and Zhihong Xin* Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China ABSTRACT: A new screw lactam and two new carbohydrate derivatives, oryzalactam (1), oryzasaccharide A (2), and oryzasaccharide B (3), have been isolated from the methanol extract of rice bran together with four other known compounds, including momilactone A (4), butyl β-D-xylopyranose (5), ethyl β-D-xylopyranose (6), and methyl β-D-xylopyranose (7). The structures of these compounds were determined using a combination of spectroscopic methods and chemical analysis. This work represents the first recorded example of the isolation of compounds 1, 2, 3, 5, 6, and 7 from rice bran. The antioxidant experiments revealed that compound 1 possessed strong ABTS+ (ABTS = 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)) and DPPH (DPPH = diphenyl(2,4,6-trinitrophenyl) iminoazanium) radical scavenging with IC50 values of 33.38 ± 1.58 and 40.20 ± 1.34 μM, respectively. Antimicrobial assays revealed that compound 4 showed high levels of selectivity toward Escherichia coli with a minimal inhibitory concentration value of 5 μM. KEYWORDS: rice bran, structural identification, screw lactam, oligosaccharide derivatives, antioxidant and antimicrobial activities

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INTRODUCTION

General Methods. UV spectra were recorded on a Beckman DU640 spectrophotometer (Beckman Coulter, Beijing, China). IR spectra were taken from KBr discs on a Nicolet Nexus 470 spectrophotometer (Thermo Scientific, Beijing, China). All of the 1H and 13C NMR spectra were recorded on a Bruker Avance 300, 400, and 500 spectrometer (Bruker BioSpin GmbH, Beijing, China), using tetramethylsilane (TMS) as an internal standard. The chemical shifts in the NMR spectra were recorded as δ values. Electrospray ionization mass spectrometry (ESI-MS) analyses were measured on a QTof Ultima Global GAA076 LC mass spectrometer (Waters Asia, Ltd., Singapore). Thin-layer chromatography (TLC) analysis was performed on plates precoated with silica gel GF254 (10−40 μm). Column chromatography (CC) was performed over silica gel (300−400 mesh, Qingdao Marine Chemical Factory, Qingdao, China) or Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA). Materials and Chemicals. Rice bran was purchased from Wei Gang trade market in Nanjing city (it is near the Yangtze River in east China and is one of the major rice-producing areas; the weather in this region is rainy and wet all year round and is very suitable to the growth of rice), Jiangsu Province, China. Diphenyl(2,4,6-trinitrophenyl) iminoazanium (DPPH), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), Dimethyl sulfoxide-d6 (DMSO-d6), methanol-d4, and CDCl3 were obtained from Merck (Darmstadt, Germany). All of the other chemicals and solvents (e.g., methanol, 99.5%) used in the current study were purchased as the analytical grade.

Rice (Oryza sativa (O. sativa) L.) is the most important cereal crop in the world and consumed by over half of the world population.1 Rice consists of the hull, embryo, endosperm, and bran, of which rice bran, a natural byproduct produced from the milling process of rice, is the outer layer of the rice grain, constitutes about 10% of the total weight while accounting for 60% of the nutrients, especially contains abundant healthpromoting nonnutrient components, commonly referred to as unique phytochemicals that are not present in significant quantities in fruits and vegetables,2 and is considered a major contributor to this health-beneficial potential. It is reported that rice bran may contain over 100 different phytochemicals;3 the most health-promoting groups of phytochemicals found in rice bran can be classified as γ-oryzanol,4 vitamins,5 unsaturated fatty acid,6 phytosterols,7 and phenolic compounds.8 These bioactive phytochemicals play an important role in the prevention of certain major chronic diseases, such as diabetes,9 chronic inflammation,10 cardiovascular disease,11 and certain kinds of cancer,12 as strongly supported by clinical trials and epidemiological studies.13 However, the individual phytochemicals in rice bran have not been extensively investigated as much as those in fruits and vegetables. In our ongoing efforts toward identifying new bioactive components from cereal grain, fruits, vegetables, and other natural products, we have conducted an investigation of the methanol extract from rice bran and isolated a new screw lactam and two new oligosaccharide derivatives, as well as four known compounds, and evaluated their antioxidant and antimicrobial activities. Herein, we describe the experimental details of the separation process as well as provide information pertaining to the elucidation of the structures of these compounds based on their spectroscopic properties and chemical reactivity. © XXXX American Chemical Society

MATERIALS AND METHODS

Received: August 5, 2014 Revised: October 6, 2014 Accepted: October 12, 2014

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dx.doi.org/10.1021/jf5037273 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 1. Extraction and separation process for the methanol extract of rice bran.

Extraction and Isolation. Rice bran (0.8 kg) was homogenized using a Polytron homogenizer before being extracted three times with 8 L of 80% acetone at ambient temperature for 24 h. The resulting mixture was then filtered after each extraction, and the filtrate was collected and concentrated under vacuum at 38 °C until approximately 90% of the solvent had been evaporated. The final solution was then extracted three times with equal volumes of methanol, and the combined extracts were concentrated under vacuum at 40 °C to give the crude methanol extract (7.5 g). The crude methanol extract (7.5 g) was separated into four fractions (Fr. 1-4; Figure 1) by normal-phase silica gel CC (20 g of silica gel, 300−400 mesh) using a stepwise gradient elution of petroleum ether:acetone:MeOH (100:0:0 to 0:100:0 to 0:0:100, v/v/v). Fraction 1 (2.3 g) was separated into two subfractions (Fr. 11 and Fr. 1-2) on a silica gel CC with a CHCl3: acetone eluent (200:3, v/v). Fr. 1-1 was passed through a Sephadex LH-20 column with a CHCl3:MeOH eluent (1:1, v/v) to give Fr. 1-11, which was purified over a silica gel CC using a petroleum ether:acetone eluent (20:1, v/v) to give 1 (13.2 mg). Fr. 1-2 was purified by CC over silica gel with a cyclohexane:ethyl acetate eluent (1:5, v/v) to give 2 (2.7 mg). Fraction 2 (1.9 g) was purified over a silica gel CC with a petroleum ether:acetone eluent (50:1, v/v) to yield Fr. 2-1 and Fr. 2-2. Fr. 2-1 was then purified over a silica gel CC with a CHCl3:MeOH eluent (100:1, v/v) to give 3 (3.1 mg). Fr. 2-2 was separated through a Sephadex LH-20 column with a CHCl3:MeOH eluent (1:1, v/v) to yield Fr. 2-2-1, which was further purified on a silica gel CC using a petroleum ether:acetone eluent (60:1, v/v) to yield 4 (2.0 mg). Fraction 3 (1.8 g) was also purified on a normal-phase silica gel CC using a petroleum ether:acetone eluent (10:1, v/v) to yield Fr. 3-1. Fr. 3-1 was purified over a Sephadex LH-20 column with a CHCl3:MeOH eluent (1:1, v/v) to give Fr. 3-11, which was further purified on a silica gel CC using a

CHCl3:MeOH eluent (200:1, v/v) to give 5 (2.3 mg) and 6 (1.9 mg). Fraction 4 (1.5 g) was subjected to CC over silica gel using a petroleum ether:acetone eluent (5:1, v/v) to yield Fr. 4-1. Fr. 41 was subsequently purified on a silica gel CC using a CHCl3: MeOH eluent (300:1, v/v) to give Fr. 4-1-1, which was purified by CC over silica gel, eluting with CHCl3:MeOH (30:1, v/v) to yield 7 (2.1 mg). Assay of ABTS+ and DPPH Radical Scavenging. Radical-scavenging activity against ABTS+ was performed as described in the literature.14 DPPH radical-scavenging assay was performed according to a previously reported protocol.15 Vitamin C was employed as a positive control. The antioxidant activities of the test compounds were expressed as IC50, with the IC50 being defined as the concentration of the test compounds required to inhibit the formation of radicals by 50%.16 All of the samples were analyzed in triplicate. Antimicrobial Bioassay. The antimicrobial assay was carried out on the pure compounds using the disc diffusion method described in the literature.17 The commercial antimicrobial agent, gentamicin (National Institute for the Control of Pharmaceutical and Biological products, Beijing, China), was employed as a positive control. Several test microorganisms were used, including Escherichia coli (E. coli; ATCC 25922), Staphylococcus aureus (S. aureus; ATCC 25923), Bacillus subtilis (B. subtilis; ATCC 6633), Mycobacterium smegmatis (My. smegmatis; CMCC 93321), Clostridium perf ringens (C. perf ringens; ATCC 13124), Micrococcus tetragenus (Mi. tetragenus; ATCC 35098), Candida albicans (C. albicans; CMCC 98001), and Mycobacterium phlei (My. phlei; AS 4.1180). The tested strains were incubated in Luria− Bertani (LB) agar plates for bacteria and in YPD agar plates for C. albicans at 37 °C. The minimal inhibitory concentrations (MICs) were determined. After an incubation period of 24 h, the zones of inhibition (millimeters in diameter) were recorded, and the MICs values were determined. B



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Figure 2. Key 1H−1H COSY and HMBC correlations for compounds 1, 2, 3, and 4.

Figure 3. Structures of the compounds isolated from the methanol extract of rice bran.

Statistical Analysis. All of the data were analyzed according to Duncan’s multiple comparison test (p < 0.05), using version 8.1 of the SAS software package. The resulting data were presented as mean ± SD.

HMBC correlations (Figure 2) from 2-OH to C-1, C-2, and C3, from 10-OH to C-5, C-9, and C-10, and from 6-NH to C-5, C-8, and C-10. The double bond at C-3 and C-4 was supported by COSY correlations between H-3 and H-4, as well as confirmed by the key correlation from H-4 to C-1, C-2, C-3, and C-5. The proton signal at δ 2.91 was assigned to C-8 on the basis of the results provided by the HSQC and HMBC spectra, respectively. A key correlation from H-12 to C-11was observed in the HMBC experiment, indicating that a methoxy at C-12 was connected to the carbonyl at C-11. Consequently, the structure of 1 was determined to be methyl 2,10-dihydroxy-7oxo-6-azaspiro[4.5]deca-1,3,9-triene-9-carboxylate and named Oryzalactam (Figure 3). Compound 2 was isolated as a white amorphous powder, and its molecular formula was determined to be C16H32O12 based on HRESIMS analysis of the pseudomolecular ion peak at m/z 415.1826 [M − H]− (calcd 415.1816). The UV spectrum of compound 2 revealed only one strong absorption band at 205 nm. The IR spectrum displayed strong absorption bands at 3416 and 1029 cm−1 which are characteristic of glycosidic-type structures. The 1H NMR spectrum of 2 showed the presence of

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RESULTS AND DISCUSSION Structural Elucidation of the Isolated Compounds. Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was determined to be C11H11NO5 based on HRESIMS analysis of the pseudomolecular ion peak at m/z 260.0542 [M + Na]+ (calcd 260.0535), indicating seven degrees of unsaturation. The UV spectrum of compound 1 revealed three strong absorption bands at 210, 265, and 320 nm. The IR spectrum displayed strong absorption bands at 3274, 1721, 1478, and 1204 cm−1, which were attributed to hydroxyl, amide, ester carboxyl, and olefinic functionalities. The 1H NMR spectrum showed three exchangeable protons in the downfield at δ 10.01 (s, 1H), 9.01 (s, 1H), and 6.07 (s, 1H) corresponding to two hydroxyl group linked to C-10 and C2, and an amide group at the position of 6-NH, respectively, these three functionalities were further confirmed by the key C



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two anomeric protons at δ 4.12 (d, J = 7.77 Hz, 1H) and 4.63 (d, J = 3.72 Hz, 1H), which was further supported by the chemical shift at δ 103.0 and 98.7 in the 13C NMR spectrum. Analysis of the 1H−1H COSY, HSQC, and HMBC spectra (Figure 2) of compound 2 allowed for the complete assignment of the 1H and 13C NMR spectral data. The sequence from an anomeric proton H-1 to oxymethylene proton H-6 through oxymethine protons H-2, H-3, H-4, and H-5, as well as H-7 to H-8, was confirmed by 1H−1H COSY. Similarly, the fragments from H-1′ to H-6′ and from H-7′ to H-8′ were also established. Three sets of mutual key long-range couplings from H-1 to C-7 and from H-7 to C-1, and from H-1′ to C-7′ and from H-7′ to C-1′, as well as from H-2′ to C-4 and from H-4 to C-2′, were observed in the HMBC experiments, indicating that two ethyl groups and another sugar unit in a ring-opening form were connected directly to the C-1, C-1′, and C-4 by ether linkages. The pyranosyl sugar units were identified as a β-glucopyranosyl group on the basis of its 3JH‑1,H‑2 coupling constant of 7.77 Hz. On the basis of this evidence, the structure of 2 was characterized as shown Figure 3 and trivially named oryzasaccharide A. Compound 3 was obtained as a white amorphous powder. Its molecular formula was established as C20H40O12 by negative HRESIMS [M − H]− at m/z 471.2451 (calcd 471.2444). The UV spectrum of compound 3 revealed only one strong absorption band at 205 nm. The IR spectrum displayed strong absorption bands at 3420 and 1033 cm−1 which are typical of glycosidic-type structures. The 1D NMR data (Table 3) revealed the presence of a fructofuranose, a ring-opening pyranose moiety and two butyl group fragments. The 13C NMR of 3 revealed the presence of 20 carbon signals, with six of these signals being assigned to the fructofuranose, six to the ringopening pyranose moiety, and the remaining eight to two butyl ether fragments. The chemical shifts of all of the protons and carbons belonging to these three parts were assigned using a combination of 1H−1H COSY, HSQC, and HMBC analysis (Figure 2). Several key correlations in the HMBC spectra, including correlations from H-7 to C-2, and two mutual longrange couples from H-12 to C-1′ and H-1′ to C-12, and from H-12 to C-1″ and H-1″ to C-12, were observed, indicating that two butyl groups and the ring-opening pyranose moiety were connected directly to the C-1′, C-1″, and C-2 by three ether linkages, respectively. Taken together, these data were indicative of a typical sugar ether structure with unique ringopening moiety and two butyl groups. Consequently, the structure of 3 was elucidated, as shown in Figure 3 and named oryzasaccharide B (Figure 3). Compound 4 was isolated as a colorless needle. The molecular formula of the material was determined to be C20H26O3 on the basis of its 1H and 13C NMR and ESI-MS data. ESI-MS analysis of the material revealed a peak at m/z 337.2, corresponding to [M + Na]+, indicating that the molecular weight of 4 was 314. The 1H NMR and 13C NMR spectra revealed the presence of three tertiary methyls at δ 0.94 (s, 3H), 1.58 (s, 3H), and 1.04 (s, 3H), three vinylic protons at δ 5.90 (dd, J = 10.76, 17.48 Hz, 1H), 5.01 (d, J = 17.48 Hz, 1H), and 4.98 (d, J = 10.76 Hz, 1H), one oxygenated methane at δ 4.89 (t, J = 5.12 Hz, 1H), and one trisubstituted olefinic protons at δ 5.76 (d, J = 5.00 Hz, 1H), as well as one γ-lactone carbon at δ 174.5 and one carbonyl carbon at δ 205.3 in the molecule. The structure of 4 was further confirmed by the COSY and HMBC correlations (Figure 2) and by comparing the NMR data with those of momilactone A in the literature.18

Compound 4 was consequently identified as momilactone A (Figure 3). Compound 5 was isolated as a white amorphous powder. The molecular formula was determined to be C9H18O5 on the basis of its 1H NMR, 13C NMR, and ESI-MS spectra. ESI-MS analysis gave m/z values of 205.1 and 229.1, corresponding to [M − H]− and [M + Na]+. The 1H and 13C NMR spectra of 5 were typical of a sugar ether compound. The 13C NMR spectrum revealed the presence of nine carbons corresponding to one xylopyranose unit and one butyl group, respectively. Analysis of the 1H−1H COSY, HSQC, and HMBC spectra of compound 5 allowed for the complete assignment of the 1H and 13C NMR spectral data. From the 1H−1H COSY spectrum, the molecular fragments from C-1 to C-5 and from C-1′ to C4′ could be established. Key mutual correlations from H-1 to C1′ and from H-1′ to C-1 were observed in the HMBC experiments, indicating that the xylopyranose unit and the butyl group were connected by ether linkages. The xylopyranose unit was identified as a β-xylopyranose group on the basis of its 3 JH‑1,H‑2 coupling constant of 6.90 Hz. Based on a comparison of these data with data published in the literature,19 compound 5 was identified as butyl β-D-xylopyranose (Figure 3). Compound 6 was isolated as a white amorphous powder, and its molecular formula was determined to be C7H14O5 on the basis of its 1H and 13C NMR and ESI-MS spectra. ESI-MS analysis of the material gave m/z values of 177.1 and 201.1, corresponding to [M − H]− and [M + Na]+, respectively. A careful comparison of the 1H and 13C NMR spectra of 6 with those of 5 revealed the existence of a close structural relationship between the two compounds. Compared to the spectra of 5, five ethyoxyl proton signals at δ 3.51 (m, 2H, H1′), 1.25 (t, J = 6.87 Hz, 3H, H-2′) in 6 instead of nine butyoxyl proton signals at δ 3.54 (m, 2H, H-1′), 1.61 (m, 2H, H-2′), 1.38 (m, 2H, H-3′), and 0.93 (t, J = 7.35 Hz, 3H, H-4′) in 5 were observed in the 1H NMR spectrum. As expected, two methene carbon signals at δ 31.6 (C-2′) and 19.1 (C-3′) in 5 were missed in the 13C NMR spectrum of 6. By a comparison with data available in the literature,19 compound 6 was determined to be ethyl β-D-xylopyranose (Figure 3). Compound 7 was isolated as a white amorphous powder. The molecular formula was determined to be C6H12O5 on the basis of its 1H NMR, 13C NMR, and ESI-MS spectra. ESI-MS analysis gave m/z values of 163.0 and 187.1, corresponding to [M − H]− and [M + Na]+, respectively. The 1H and 13C NMR spectra for 7 and 6 are very similar, with the exception that an ethyoxyl group in 6 was replaced by a methoxy group in 7. Based on a comparison of these data with information reported in the literature,19 compound 7 was identified as methyl β-Dxylopyranose (Figure 3). Antioxidant Activities of the Pure Compounds Isolated from Rice Bran. The antioxidant activities of the seven pure compounds were tested according to their ABTS+ and DPPH radical-scavenging assay. The results of these experiments have been shown in Figure 4 and Figure 5. Only compound 1 showed 98.57% and 77.33% of ABTS+ and DPPH radical-scavenging ability at the concentration of 100 μM, respectively (Figure 4A,B) and were comparable to that of positive control vitamin C; other compounds were inactive. Based on the data obtained, compound 1 was selected to further determine whether their ABTS+ and DPPH radicalscavenging capacity was in a dose-dependent manner , and the results are shown in Figure 5. The ABTS+ radical-scavenging activities for compound 1 and vitamin C were comparable and D



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Figure 5. ABTS+ radical-scavenging activity (A) and DPPH radicalscavenging activity (B) of compound 1 under different concentrations.

Figure 4. ABTS+ radical-scavenging activity (A), DPPH radicalscavenging activity (B) of the isolated compounds. oryzalactam (1), oryzasaccharide A (2), oryzasaccharide B (3), momilactone A (4), butyl β-D-xylopyranose (5), ethyl β-D-xylopyranose (6), and methyl βD-xylopyranose (7). Each value has been presented as the mean ± SD of three experiments. Note: a−e, results with a different letter differ significantly (p < 0.05).

NMR and ESI-MS Spectroscopic Data. Oryzalactam (1). Yellow amorphous powder. 1H NMR and 13C NMR data, see Table 1. ESI-MS m/z 260.0542 [M + Na]+. Oryzasaccharide A (2). White amorphous powder. 1H NMR and 13C NMR data, see Table 2. ESI-MS m/z 415.1826 [M − H]−. Oryzasaccharide B (3). White amorphous powder. 1H NMR and 13C NMR data, see Table 3. ESI-MS m/z 471.2451 [M − H]−. Momilactone A (4). Colorless needles. 1H NMR (400 MHz, CDCl3, TMS): δ 5.90 (dd, J = 10.76, 17.48 Hz, 1H, H-15), 5.76 (d, J = 5.00 Hz, 1H, H-7), 5.01 (d, J = 17.48 Hz, 1H, H-16b), 4.98 (d, J = 10.76 Hz, 1H, H-16a), 4.89 (t, J = 5.12 Hz, 1H, H6), 2.67 (m, 2H, H-2), 2.37 (d, J = 5.08 Hz, 1H, H-5), 2.26 (d, J = 12.04 Hz, 1H, H-14b), 2.11 (d, J = 12.04 Hz, 1H, H-14a), 1.84 (m, 1H, H-9), 1.78 (m, 2H, H-11), 1.66 (m, 2H, H-1),1.60 (m, 2H, H-12), 1.58 (s, 3H, H-18), 1.04 (s, 3H, H-20), 0.94 (s, 3H, H-17); 13C NMR: δ 31.4 (C-1), 35.0 (C-2), 205.3 (C-3), 53.7 (C-4), 46.5 (C-5), 73.1 (C-6), 114.1 (C-7), 148.9 (C-8), 50.3 (C-9), 32.6 (C-10), 24.1 (C-11), 37.4 (C-12), 40.3 (C13), 47.7 (C-14), 149.1 (C-15), 110.3 (C-16), 22.1 (C-17), 21.4 (C-18), 174.5 (C-19), 21.9 (C-20). ESI-MS m/z 337.2 [M + Na]+. Butyl β-D-xylopyranose (5). Colorless amorphous powder. 1 H NMR (500 MHz, CDCl3, TMS): δ 4.28 (d, J = 6.90 Hz, 1H, H-1), 3.98 (m, 1H, H-5a), 3.85 (m, 1H, H-4), 3.54 (m, 2H, H1′), 3.53 (m, 1H, H-3), 3.41 (m, 1H, H-2), 3.28 (m, 1H, H-5b),

increased sharply with increasing concentration in a dosedependent manner at the range of 0−80 μM, increased to maximal values of 96.70% and 97.10% ABTS+ inhibitory effect at the concentration of 80 μM, and then were kept constant, and their IC50 values were 33.38 ± 1.58 and 34.70 ± 1.19 μM, respectively. The results suggested that the ABTS+ radicalscavenging activity of compound 1 was almost identical to that of vitamin C. Similarly, as shown in Figure 4B and Figure 5B, compound 1 and vitamin C showed antioxidant activity profiles that resembled one another and a dose-dependent manner in DPPH scavenging-radical assay, and their IC50 values were 40.2 ± 1.34 and 47.59 ± 1.41 μM, respectively, indicating that the DPPH scavenging-radical activity of compound 1 was slightly weaker than that of Vitamin C. Antimicrobial Bioassay. Seven pure compounds were tested for their antimicrobial activities against eight pathogenic strains. Only compound 4 exhibited a significant inhibition zone against E. coli with a MIC value of 5 μM, indicating that compound 4 possessed high levels of selectivity toward E. coli. All of the other compounds isolated were found to be inactive. E



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Table 1. NMR Spectral Data for Compound 1 in DMSO-d6 at 400 (1H) and 100 MHz (13C) position

H (J, Hz)

HSQC

1 2 2-OH 3 4 5 6 7 8 9 10 10-OH 11 12

6.59 (d, J = 1.44 Hz, 1H)

115.5 152.8

9.01 (s, 1H) 6.77 (t, J = 1.44 Hz, 1H) 6.59 (d, J = 1.44 Hz, 1H)

112.5 110.3 73.5

6.07 (s, 1H) 2.91 (d, J = 4.32 Hz, 2H)

1

H−1H COSY

C-3, C-4, C-5 C-2, C-3, C-4 C-1, C-2, C-4, C-5 C-1, C-2, C-3, C-5

H-4

C-1, C-8, C-10 178.1 41.9 134.5 132.3

10.01 (s, 1H) 3.42 (s, 3H)

HMBC (H→C)

C-7, C-10, C-11

C-1, C-10, C-9 169.5 51.6

C-11

Table 2. NMR Spectral Data for Compound 2 in DMSO-d6 at 400 (1H) and 100 MHz (13C) H (J, Hz)

position 1 2 2-OH 3 3-OH 4 5 6 6-OH 7 8 1′ 1′-OH 2′ 3′ 3′-OH 4′ 4′-OH 5′ 5′-OH 6′ 6′-OH 7′ 8′

4.12 2.92 4.91 3.04 4.80 3.06 3.39 3.62 4.40 3.81 1.14 4.63 4.56 3.17 3.40 4.66 3.04 4.63 3.12 4.86 3.43 4.40 3.40 1.14

(d, J = 7.77 Hz, 1H) (m, 1H) (d, J = 4.83 Hz, 1H) (m, 1H) (d, J = 5.40 Hz, 1H) (m, 1H) (m, 1H) (m, 1H), 3.43 (m, 1H) (m, 1H) (m, 1H), 3.52 (m, 1H) (m, 3H) (d, J = 3.72 Hz, 1H) (d, J = 6.36 Hz, 1H) (m, 1H) (m, 1H) (d, J = 4.68 Hz, 1H) (m, 1H) (m, 1H) (m, 1H) (d, J = 5.04, 7.59 Hz, 1H) (m, 1H), 3.62 (m, 1H) (m, 1H) (m, 1H), 3.64 (m, 1H) (m, 3H)

HSQC

HMBC (H→C)

103.0 73.8

C-3, C-7 C-1, C-3 C-1, C-2, C-3

77.2 C-3, C-4 70.5 73.8 61.5 64.2 15.5 98.7 72.4 73.1

C-4, C-5 C-6 C-1 C-7 C-2′, C-7′ C-1′, C-2′ C-2′, C-3′ C-2′, C-3′, C-4′ C-3′, C-4′

1

H−1H COSY H-2 H-2 H-2 H-3 H-3 H-4 H-5 H-6 H-8 H-2′ H-1′ H-2′, H-4′ H-3′

70.9 C-3′, C-4′, C-5′ 77.2 C-4′, C-5′ 61.5 62.7 15.6

C-6′ C-1′ C-7′

H-4′ H-4′ H-5′ H-5′ H-6′ H-8′

(C-4), 66.1 (C-5), 56.4 (C-1′). ESI-MS m/z 163.0 [M − H]−, 187.1 [M + Na]+. An investigation of the methanol extract of rice bran allowed for the isolation of a new screw lactam and two new oligosaccharide derivatives, oryzalactam (1), oryzasaccharide A (2), and oryzasaccharide B (3), as well as four known compounds, including momilactone A (4), butyl β-D-xylopyranose (5), ethyl β-D-xylopyranose (6), and methyl β-Dxylopyranose (7), and the structures of all seven of these compounds were elucidated using a combination of spectroscopic methods. To the best of our knowledge, this is the first report concerning the isolation of compounds 1, 2, 3, 5, 6, and 7 from rice bran. Oryzalactam is a new screw lactam and extremely rare. To date, there are no compounds with this particular skeleton have been isolated from natural sources. This compound exhibited strong antioxidant activity against ABTS+ and DPPH radical and might represent a new potential natural antioxidant agent.

1.61 (m, 2H, H-2′), 1.38 (m, 2H, H-3′), 0.93 (t, J = 7.35 Hz, 3H, H-4′). 13C NMR: δ 102.9 (C-1), 72.9 (C-2), 76.0 (C-3), 69.6 (C-4), 64.9 (C-5), 69.7 (C-1′), 31.6 (C-2′), 19.1 (C-3′), 13.8(C-4′). ESI-MS m/z 205.1 [M − H]−, 229.1 [M + Na]+. Ethyl β-D-xylopyranose (6). Colorless amorphous powder. 1 H NMR (300 MHz, CDCl3, TMS): δ 4.28 (d, J = 6.84 Hz, 1H, H-1), 4.19 (m, 1H, H-5a), 3.94 (m, 1H, H-4), 3.51 (m, 2H, H1′), 3.50 (m, 1H, H-3), 3.38 (m, 1H, H-2), 3.26 (m, 1H, H-5b), 1.25 (t, J = 6.87 Hz, 3H, H-2′). 13C NMR: δ 102.9 (C-1), 72.9 (C-2), 76.0 (C-3), 69.6 (C-4), 65.4 (C-5), 65.3 (C-1′), 15.1 (C-2′). ESI-MS m/z 177.1 [M − H]−, 201.1 [M + Na]+. Methyl β-D-xylopyranose (7). Colorless amorphous powder. 1 H NMR (300 MHz, DMSO-d6, TMS): δ 3.99(d, J = 7.5 Hz, 1H, H-1), 3.70 (m, 1H, H-5a), 3.41 (m, 1H, H-4), 3.34 (s, 3H, 1-OCH3), 3.30 (m, 1H, H-3), 3.01 (m, 1H, H-2), 2.94 (m, 1H, H-5b). 13C NMR: δ 105.2 (C-1), 73.7 (C-2), 77.0 (C-3), 70.1 F



Article

Table 3. NMR Spectral Data for Compound 3 in Methanol-d4 at 400 (1H) and 100 MHz (13C) H (J, Hz)

position 1 2 3 4 5 6 7 8 9 10 11 12 1′ 2′ 3′ 4′ 1″ 2″ 3″ 4″

3.50 (m, 1H), 3.67 (m, 1H) 4.13 3.94 3.75 3.60 3.75 3.70 3.80 3.60 3.30 3.66 3.40 4.79 3.77 1.65 1.45 0.97 3.46 1.65 1.45 0.97

(d, J = 8.08 Hz, 1H) (t, J = 7.72 Hz, 1H) (m, 1H) (m,1H) (m, 1H) (t, J = 2.80 Hz, 1H) (d, J = 2.36 Hz, 1H) (m, 1H) (d, J = 9.64 Hz, 1H) (dd, J = 3.20, 5.76 Hz, 1H) (dd, J = 3.76, 9.68 Hz, 1H) (d, J = 3.72 Hz, 1H) (m, 2H) (m, 2H) (m, 2H) (m, 3H) (m, 2H) (m, 2H) (m, 2H) (m, 3H)

HSQC

HMBC (H→C)

60.8 103.7 77.0 75.9 81.9 63.6

C-3, C-5, C-6 C-4, C-6 C-4, C-5

61.3

C-2, C-8, C-9

1

H−1H COSY

C-2, C-3

72.2 70.4 73.7 72.2 98.6 67.4 31.3 19.0 12.8 67.4 32.0 18.9 12.9

H-4 H-5 H-5 H-8 H-9

C-7, C-8, C-10, C-11 C-8, C-12 C-10, C-11, C-1′, C-1″ C-2′, C-3′, C-12 C-1′ C-1′, C-2′, C-4′ C-2′ C-2″, C-3″, C-12 C-1″ C-1″, C-2″, C-4″ C-2″

H-11 H-11 H-2′ H-3′ H-4′ H-2″ H-3″ H-4″

potential of rice bran as natural antioxidant to the food industry, as well as enhancing our better understanding of the relationships between the individual component and human health in rice bran. Further studies should be carried out to assess the bioactivities to validate the real effects of the compounds on the prevention and therapy of some chronic human diseases in future, especially on antioxidant activities of the compounds isolated, e.g., bioaccessibility, bioavailability, and enrichment for developing a food by using these compounds, as well as possibilities of interactions with other biomolecules.

It is worth mentioning, however, the bioactivities of this compound in vitro are not necessarily equal to its actual beneficial effects on human health. It is determined by the bioaccessibility and bioavailability of the compound in vivo where it is commonly mixed with different macromolecules such as carbohydrates, lipids, and proteins to form the food matrix.20 Several factors interfere with the real effects of a compound, such as food source and chemical interactions with other phytochemicals and biomolecules present in the food matrix. Therefore, research concerning the bioaccessibility and bioavailability of a compound from rice bran are important, since a compound is only released from the food matrix by the action of digestive enzymes in the small intestine or bacterial microflora in the large intestine and then can be absorbed to exert their beneficial effects.2 Oryzasaccharides A and B and the three other known compounds are carbohydrate derivatives with a diverse side chain. It has been reported that rice bran contains about 6.5% free sugars on a dry-weight basis, more than 50% of the sugar being simple saccharides (mainly glucose and sucrose); therefore, the current research indicated that the free sugar in the rice bran might exist in more complex forms. Momilactone A obtained in this study is one of the diterpenoid phytoalexins and originally isolated from the seed husk of rice. So far, three momilactones members (momilactone A−C) have been found from rice in all.21,22 It has been reported that momilactones are accumulated in rice during vegetative growth and are released into the soil, causing growth inhibition of neighboring plants, and responding to UV irradiation and pathogenesis such as fungal invasion, suggesting that momilactones in rice also play a role in plant defense.23,24 In summary, the current study has effectively demonstrated that various compounds with antioxidant and antimicrobial activities are present in rice bran. Oryzalactam, in particular, has been identified as a new screw lactam and showed strong antioxidant activities. The results of the current study represent a useful addition to understanding the potential application and health benefits of rice bran and confirming the interesting

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +86 25 8439 5618. E-mail: [email protected]. Funding

This work was financially supported by the Fundamental Research Funds for the Central Universities (Grant KYZ201118), the Project of National Key Technology Research and Development Program for the 12th Five-year Plan (Grant 2012BAD33B10), the special funds of agroproduct quality safety risk assessment of Ministry of Agriculture of the People's Republic of China (Grant 2014FP11), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institution. Notes

The authors declare no competing financial interest.

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