Article pubs.acs.org/JAFC
Inhibitory Effects of the Leaves of Loquat (Eriobotrya japonica) on Bone Mineral Density Loss in Ovariectomized Mice and Osteoclast Differentiation Hui Tan,† Syoko Furuta,† Toshiro Nagata,† Koichiro Ohnuki,§ Taiki Akasaka,# Bungo Shirouchi,⊥ Masao Sato,⊥ Ryuichiro Kondo,† and Kuniyoshi Shimizu*,† †
Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan § Department of Biological and Environmental Chemistry, Kinki University, Kayanomori 11-6, Iizuka, Fukuoka 820-8555, Japan # Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan ⊥ Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan S Supporting Information *
ABSTRACT: The loquat, Eriobotrya japonica Lindl. (Rosaceae), is a small tree native to Japan and China that is widely cultivated for its succulent fruit. Its leaves are used as an ingredient of a tasty tea called “Biwa cha” in Japanese. The antiosteoporosis effects of the leaves of loquat in vitro and in vivo have been investigated. After 15 days of feeding normal diet or diet supplemented with 5% loquat leaves, the body weight, viscera weights, and bone mineral density (BMD) of both groups of eight ovariectomized (OVX) mice were compared. The result showed that the loss of BMD in loquat-fed mice was significantly prevented in three parts of the body, especially in the trabecular bone of the head (P < 0.05), abdomen (P < 0.01), and lumbar (P < 0.05) compared to the control group. No hypertrophy in the uterus by the loquat leaves diet was observed. The effect of the extract (447.25 g) prepared from the dried leaves of loquat (2.36 kg) was further studied on RANKL-induced osteoclast differentiation and cell viability. The extract suppressed the differentiation of osteoclasts under 50, 125, 250, and 500 μg/mL. Through bioactivity-guided fractionation, ursolic acid (1) was isolated and inhibited osteoclast differentiation under 4 and 10 μg/ mL. It was concluded that loquat leaves possess the potential to suppress ovariectomy-induced bone mineral density deterioration. KEYWORDS: tea, osteoporosis, Eriobotrya japonica, osteoclast differentiation, ursolic acid
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cause of age-related bone loss in women.5 Decreased bone density and the resulting bone dysfunction greatly affect the quality of life, because the bone is a supporting organ, hematopoietic tissue, and immune tissue.6 This imbalance of bone remodeling is caused by excess osteoclast activity. Osteoclasts are large multinucleated cells, which can resorb bone. These multinucleated cells express known markers, including tartrate-resistant acid phosphatase (TRAP),7 a glycosylated monomeric metalloenzyme expressed in mammals, that is highly expressed in osteoclasts associated with osteoblast migration to bone resorption sites. This initiates osteoblast differentiation, proliferation, and activation.8 Hormone replacement therapy (HRT) is a treatment for the prevention of bone loss and for restoring the rate of bone resorption and formation.9 However, long-term HRT may increase a patient’s risk of developing breast cancer, endometrial cancer, thromboembolic events, or vaginal
INTRODUCTION Biwa (Japanese) is a flowering plant known as loquat, Eriobotrya japonica, and belongs to the Rosaceae family. It probably had its origin in southeastern China and has been cultivated in Japan since ancient times. It is widely cultivated for its succulent fruit. Its leaves are used as the ingredient of tasty teas called as “Biwa cha” in Japanese. It has been reported that the extracts of the leaves from E. japonica exerted an antiobesity effect through regulation of plasma lipid and adiponectin levels in mice and adipogenic transcription factors PPARγ and C/ EBPα in 3T3-L1 adipocytes.1 Also, the leaves of E. japonica were reported to have a potent inhibitory effect on the inflammatory mediator via the attenuation of NF-κB translocation to the nucleus.2 E. japonica significantly delayed carcinogenesis induced by peroxynitrite (initiator) and TPA (promoter), and its potency was comparable to that of a green tea polyphenol, (−)-epigallocatechin 3-O-gallate, in the same assay.3 Osteoporosis is a growing public health problem, affecting millions of people worldwide.4 It is a major growing health problem for elderly women associated with ovarian hormone deficiency following menopause and is by far the most common © 2014 American Chemical Society
Received: Revised: Accepted: Published: 836
June 26, 2013 December 28, 2013 January 9, 2014 January 9, 2014 dx.doi.org/10.1021/jf402735u | J. Agric. Food Chem. 2014, 62, 836−841
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bleeding.10 Therefore, because of women’s reluctance to comply with HRT, safer ingredients originating from foods or beverages such as tea are needed for the treatment or prevention of menopausal symptoms, such as osteoporosis. As far as we know, the antiosteoporosis effect of the leaves of E. japonica has not yet been investigated. The objective of this study was to investigate the effect of the leaves of E. japonica on the bone mineral density (BMD) of ovariectomized mice and RANKL-induced osteoclast differentiation and to identify the active substance.
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AIN-76 diet and (2) the loquat group was fed the modified AIN-76 diet incorporating 5% leaves of loquat. The treatment continued to the 22nd day, and then the mice were sacrificed and dissected. The body weights and food intakes of all animals were measured every day. The handling and sacrificing of all animals were carried out in accordance with nationally prescribed guidelines, and ethical approval for the studies was granted by the Animal Care and Use Committee of Kyushu University (Authorization A22-234-0). X-ray CT Scan. On the 21st day of the diet regimen, each mouse underwent a computed tomography (CT) scan. BMD was measured by a computerized tomography scanner (X-ray CT) (La Theta, Aloka Co. Tokyo) at a scan pitch of 5 mm for mouse body parts. The BMD of the head, chest, abdomen, and lumbar (including the trabecular bone, cortical bone, and whole) were analyzed. Anatomy. At the end of the experiment, the mice were euthanized with pentobarbital sodium at 50 mg/kg body weight, and the liver, uterus, kidney, spleen, brain, and fat mass (fat around the kidney and fat around the uterus) were carefully removed and weighed. Cell Cultures. Cells of the murine macrophage cell line RAW 264.7 were maintained in α-MEM with 10% FBS and 1% antibiotics. For the osteoclastogenesis experiments, cells were cultured in a 96-well plate at the density of 6 × 103 cells/well, and after 24 h, the adherent cells were cocultured with an additional α-MEM medium + 10% FBS + 1% antibiotics containing cytokines (40 ng/mL RANKL and 10 ng/mL TNF-α). Samples at appropriate concentrations were dissolved into DMSO (v/v, 0.1% in each well) and added into each well. The cells were then cultured for 4 days. Assay for Osteoclastic TRAP Activity. The protocol for the assay of osteoclastic TRAP activity was as follows. After the cell culture period, the media were removed and adherent cells were fixed in 37% formaldehyde mixed with acetone and citrate solution (8:65:25, v/v/v) for 3 min and then washed with water. The cells were then stained for TRAP with the TRAP staining kit 387-A according to the manufacturer’s instructions: the cells were stained with a fast garnet GBC base solution mixed with sodium nitrite solution, naphthol AS-BI phosphoric solution, acetate solution, and tartrate solution. After 1 h of staining in the dark, the stained cells were washed with water and colored by hematoxylin solution for 2 min and then rinsed with 0.1 N NaOH. The number of TRAP-positive multinucleated cells in each well was counted. Oleic acid was used as a positive control based on reference.13 Assay for Cell Proliferation. Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RAW cells (1 × 105 cells/well) were seeded into 96-well plates for 24 h. Samples at appropriate concentrations were dissolved in DMSO (v/v, 0.1% in each well) and added into each well for another 24 h. After culturing, the cells were treated with 5 μL of MTT (final concentration = 5 mg/mL dissolved in phosphate buffer solution) for 4 h, and the precipitated dye was solubilized by isopropanol (with 0.04 N HCl). The absorbance at 570 nm was measured. Extraction and Isolation. For the extraction and isolation of bioactive principles, 5.10 kg of fresh leaves of loquat was freeze-dried and ground into powder, and the resulting 2.36 kg of the dried leaves of loquat were extracted with methanol (35 L) and evaporated. Then, 447.25 g of methanol extract was obtained (yield = 18.95%). First, a part of the methanol extract (234.22 g) was separated by silica gel column chromatography (20 cm i.d. × 50 cm, 4.49 kg of Wakogel C200) and eluted with an n-hexane/EtOAc solvent system [10:0 (11 L), 9.8:0.2 (2 L), 9.7:0.3 (2 L), 9.6:0.4 (2 L), 9.5:0.5 (2 L), 9:1 (2 L), 8.5:1.5 (11 L), 8:2 (8 L), 7:3 (5 L), 6.5:3.5 (12 L), 6:4 (5 L), 5.5:4.5 (2.5 L), 5:5 (6.5 L), 4:6 (3 L), 3:7 (3 L), 2:8 (2 L), 0:10 (2 L), and methanol 100% (13 L)] to yield 14 fractions. The remaining part of the methanol extract (213.03 g) was applied to column chromatography again (20 cm i.d. × 50 cm, 4.21 kg of Wakogel C-200) and eluted with an n-hexane/EtOAc solvent system [10:0 (10 L), 9:1 (5 L), 8.5:1.5 (5 L), 8:1.5 (5 L), 8:2 (7 L), 7.5:2.5 (6 L), 7:3 (5 L), 6.5:3.5 (5 L), 6:4 (5 L), 5.5:4.5 (5 L), 5:5 (5 L), 4:6 (5 L), 3:7 (5 L), 2:8 (5 L), 1:9 (5 L), 0:10 (14 L), and methanol 100% (10 L)] to yield 14 fractions. On the basis of the TLC results, twice-separated fractions were combined into nine fractions [Fr. 1 (17.58 g), Fr. 2 (8.37 g), Fr. 3
MATERIALS AND METHODS
Chemicals. E. japonica (Thunb.) Lindl. (Rosaceae) was obtained from Japan in 2010, and a voucher specimen (No. EJ001) has been deposited in the Laboratory of Systematic Forest and Forest Products Sciences (Kyushu University, Fukuoka, Japan). All leaves were airdried and powdered mechanically. Methanol and water extracts of the leaves of loquat were prepared with three times extraction at room temperature. Mice (C57BL/6JJc1) used for the experiment were purchased from Clea Co. (Osaka, Japan). RAW 264.7 macrophage cells were purchased from the Riken Bioresource Center Cell Bank (Tsukuba, Japan). Minimal essential medium, alpha modification (αMEM medium), was from Gibco BRL (Grand Island, NY, USA); fetal bovine serum (FBS) and antibiotics−antimycotics were obtained from Gibco and Invitrogen (Carlsbad, CA, USA), respectively. Receptor activator of NF-κB (RANKL) from Escherichia coli was purchased from PeproTech EC (London, UK), and tumor necrosis factor alpha (TNFα) was obtained from Roche Molecular Biochemicals (Mannheim, Germany). TRAP staining kit 387-A was obtained from Sigma-Aldrich (St. Louis, MO, USA). Oleic acid was obtained from Wako (Osaka, Japan). Dimethyl sulfoxide (DMSO) was also obtained from Wako. Animals and Diets. Sixteen 12-week-old female mice were bilaterally ovariectomized at 11 weeks of age. Groups of four mice were randomly housed in one animal cage. The animal room was conditioned at 23 ± 1 °C and illuminated according to a 12 h light/ dark cycle (light on at 8:00 a.m. and off at 8:00 p.m.). All animals were allowed free access to distilled water and fed ad libitum. AIN-76 diets with (loquat group) or without the leaves of loquat (control group) were used on the basis of the diet reported by the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies.11 This diet was composed on the basis of the average energy intake for Japanese 40−49-year-old women with a slight modification of protein, fat, and carbohydrate weight.12 The total calorie count in both groups’ diets was the same. The main diet ingredients were vitamins, minerals, DL-glutamin, choline bitartrate, cellulose, casein, safflower oil, starch, and sucrose (Table 1). Animal Experiment Protocol. At the beginning of the experiment, the mice were acclimated for 6 days by feeding the control diets to both groups. Then the 16 mice were randomly divided into two groups (n = 8 each), and (1) the control group was fed the modified
Table 1. Compositions of the AIN-76 Diet in the Control Group and Loquat Group percentage (%) composition
control group
loquat group
vitamins minerals DL-glutamine choline bitartrate cellulose casein safflower oil corn starch leaves of loquat powder sucrose
1.0 3.5 0.3 0.2 5.0 20.6 12.71 15.49 0 41.15
1.0 3.5 0.3 0.2 2.69 20.29 12.53 15.56 5.0 38.93 837
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Figure 1. Effect of diet with or without the leaves of loquat on the bone mineral density (BMD) of the head (A), chest (B), abdomen (C), and lumbar (D) in OVX mice. Data showed ± SD (n = 8); Student’s t test: (∗) P < 0.05; (∗∗) P < 0.01. were compared with the literature14 (see the Supporting Information, Figure S5). Statistical Analysis. All values are expressed as means ± SD. The significance of differences between means for two groups was determined by Student’s t test. Differences were considered significant at (∗)P < 0.05 and (∗∗)P < 0.01.
(4.86 g), Fr. 4 (7.55 g), Fr. 5 (12.46 g), Fr. 6 (13.34 g), Fr. 7 (14.44 g), Fr. 8 (14.03 g), and Fr. 9 (366.45 g)]. On the basis of bioactivityguided fractionation on osteoclastic TRAP activity, Fr. 4 (7.10 g) was subjected to further separation. Fraction 4 was applied to silica gel column chromatography (6 cm i.d. × 120 cm, 963 g of Wakogel C200) and eluted with an n-hexane/EtOAc solvent system [10:0 (1 L), 9:1 (1.5 L), 8:2 (1.5 L), 7:3 (1.5 L), 6.8:3.2 (1 L), 6.5:3.5 (1 L), 6.2:3.8 (1 L), 6:4 (1 L), 5:5 (1 L), 2.5:8.5 (1 L), and methanol 100% (2 L)] to give eight fractions. The amount of each fraction was shown as Fr. 4-1 (74.11 mg), Fr. 4-2 (3.79 mg), Fr. 4-3 (17.52 mg), Fr. 4-4 (74.16 mg), Fr. 4-5 (85.29 mg), Fr. 4-6 (1.18 g), Fr. 4-7 (1.04 g), and Fr. 4-8 (4.79 g). Further fractionation procedures were applied only to Fr. 4-7, due to its highest osteoclastic TRAP inhibitory activity. Fraction 4-7 (989.1 mg) was applied to a Sephadex LH 20 column (5 cm i.d. × 50 cm, Sephadex amount = 100 g) and eluted with MeOH/ CHCl3 (500 mL) to give eight fractions: Fr. 4-7-1, Fr. 4-7-2, Fr. 4-7-3, Fr. 4-7-4, Fr. 4-7-5, Fr. 4-7-6, Fr. 4-7-7, and Fr. 4-7-8. The 15 mg of fraction 4-7-6 (see the HPLC chromatogram in the Supporting Information, Figure S1) dissolved in methanol was separated by recycling preparative HPLC (LC- 9204, Japan Analytical Industry Co., Tokyo, Japan). The total injection amount was 3 mL, with the flow rate at 5 mL/min; mobile phase was methanol 95% + water 5%, and the column was an Inertsil ODS-3 (20 × 250 mm, GL Science, Toyko, Japan) with detection for absorbance at 210 nm to give compound 1 (6.27 mg) (see the Supporting Information, Figure S2). To verify the purity, the isolated compound 1 was confirmed by HPLC (see the Supporting Information, Figure S3). HPLC chromatograms for individual fractions (Fr. 1−9, Fr. 4-1, 4-3−4-8, Fr. 4-7-1−4-7-8) and methanol extract were also studied (see the Supporting Information, Figure S4). GC-MS and NMR Identification. For the gas chromatography− mass spectrometry (GC-MS), compound 1 (white solid) was treated with trimethylsilyldiazomethane (Tokyo Chemical Industry, Tokyo, Japan) in a solution of benzene (20% ethanol) for 30 min at room temperature and then subjected to a GC-MS analysis. Conditions were as follows: column temperature, 260 °C for 60 min; injection temperature, 250 °C; detector temperature, 300 °C; column, DB-5 capillary column, 0.25 mm i.d. × 30 m; helium rate, 1.4 mL/min; injection volume, 1 μL; split ratio, 5:1; m/z range, 50−600. EI/MS m/ z (relative intensity %): 470 (3), 412 (3), 262 (100), 233 (6), 203 (97), 189 (24), 133 (75), 119 (27). Then compound 1 was dissolved in DMSO-d6 and identified by 1H NMR and 13C NMR as ursolic acid (1) (optical rotation [α]24 D +31.3 (methanol, c 0.117). The above data
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RESULTS Effect of the Leaves of Loquat on Body Weight and the Weight of Each Organ. The two groups of mice showed no significant difference in body weight or viscera weights; notably, their uterine weights were the same. This outcome suggests that the leaves of loquat did not influence the actions of estrogen or its receptors; it also indicates that there is no potential side effect caused by unexpected estrogenic activity. Effect of the Leaves of Loquat on BMD of Various Body Parts. We investigated the effect of the leaves of loquat on the BMD of various body parts of mice and found that the loss of BMD in the trabecular cells of head, abdomen, and lumbar parts was inhibited after treatment with the diet containing the leaves of loquat compared to the control group. As shown in Figure 1, the BMD of the loquat group was significantly higher in the trabecular bone of the head, abdomen, and lumbar. Effect of the Extracts Prepared from the Leaves of Loquat on Osteoclast Differentiation. To determine the effect of the extracts prepared from the leaves of loquat on osteoclast differentiation, we applied water extract or methanol extract with various concentrations to RAW 264.7 cells. As shown in Figure 2, the multinucleated osteoclast-like cells were significantly inhibited by the methanol extract in a dosedependent manner (∗∗, P < 0.01). Under these concentrations, the methanol extract did not affect the cell viability of RAW 264.7 cells, suggesting that the methanol extract of the leaves of loquat inhibited osteoclast differentiation specifically without any cytotoxicity. It should be noted that the water extract showed no influence on osteoclast differentiation up to 500 μg/ mL. 838
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Figure 2. Effect of methanol extract of the leaves of loquat on osteoclast differentiation and cell viability of each concentration on RAW 264.7 cells. Oleic acid (50 μg/mL) was used as a positive control. The concentrations of methanol extract of the leaves of loquat were 12.5, 25, 50, 125, 250, and 500 μg/mL. Data showed ± SD (n = 3). Student’s t test against control: (∗) P < 0.05; (∗∗) P < 0.01.
Effect of Subfractions Prepared from Methanol Extract of the Leaves of Loquat by Chromatography on Osteoclast Differentiation. We examined the effects of the nine fractions separated from the methanol extract on osteoclast differentiation. Under the concentration of 20 μg/ mL, Fr. 4 showed significant osteoclast differentiation inhibitory activity with less cytotoxicity (Figure 3A), so it was further fractionated into eight fractions (Fr. 4-1−4-8), and their effects on osteoclast differentiation activity under 20 μg/mL were examined. As shown in Figure 3B, fraction 4-7 significantly inhibited the osteoclast differentiation; other fractions had less effect. In light of the search for bioactive principles, we applied Fr. 4-7 to further fractionation procedures and evaluated their effects on osteoclast differentiation. Among the eight fractions (Fr. 4-7-1−4-7-8), Fr. 4-7-6 showed significant inhibitory activity Figure 3C). Effect of an Isolated Compound on Osteoclast Differentiation. Fraction 4-7-6 was further separated by recycling preparative HPLC to give compound 1. Compound 1 was identified as ursolic acid (1) (Figure 4B). Ursolic acid (1) inhibited osteoclast differentiation in a dose-dependent manner shown in Figure 4A. We concluded that ursolic acid (1) should be the major principle for antiosteoclast activity of the leaves of loquat.
Figure 3. Effect of subfractions separated from methanol extract (A), Fr. 4 (B) and Fr. 4-7 (C) on osteoclast differentiation and cell viability on RAW 264.7 cells. Oleic acid (50 μg/mL) was used as a positive control. The concentration of subfractions separated from methanol extract and Fr. 4 was 20 μg/mL, and the concentration of subfractions separated from Fr. 4-7 was 10 μg/mL. Data showed means ± SD (n = 3). Student’s t test against control: (∗) P < 0.05; (∗∗) P < 0.01.
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DISCUSSION Ovariectomized animal models (OVX) have been widely used to study preventive treatments for postmenopausal osteoporosis with estrogen insufficiency.15 In OVX animals, as in postmenopausal women, bone loss induced by ovarian deficiency results mainly from trabecular bone loss.15 In the present study, the administration of the leaves of loquat had a significant preventive effect against BMD loss of trabecular bone in OVX mouse compared to the control, especially in the head, abdomen, and lumbar. However, there is no obvious effect on the weight of the uterus and other organs in ovariectomized mice, indicating that the mechanism of the preventive effect of bone loss is not related to estrogenic activity.7 The loss of bone mass and the deterioration of bone microstructure have been linked to an imbalance between bone formation and bone resorption.16 Here we found that the methanol extract of the leaves of loquat significantly inhibited the expression of multinucleated osteoclast cells, which are responsible for bone resorption, by evaluating the TRAP
activity of osteoclast cells. Dose-dependent inhibitory effects of the extract on the differentiation of the osteoclasts without any cytotoxicity were observed. The leaves of loquat exhibit significant in vivo and in vitro bone preventive activity; thus, it will be of interest to further investigate bioactive compounds. By using a bioactivity-guided fractionation judged by bioassay using RAW 264.7 cells, the effects of subfractions prepared from methanol extract on osteoclast differentiation at different concentrations were screened; the most significant bioactive fractions were tracked and further fractionated. One bioactive compound separated from bioactive fractions, ursolic acid (1), showed osteoclast differentiation inhibitory activity dose-dependently. On the basis of previous study on the leaves of loquat, pentacyclic triterpene acids make up the greatest percentage of secondary metabolites. Ursolic acid (1) accounted for >50% of the quantified triterpene fraction.17 The yield of ursolic acid (1) extracted by methanol is higher than that by water due to its 839
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Figure 4. Effect of ursolic acid (1) on osteoclast differentiation and cell viability on RAW 264.7 cells (A) and chemical structure of ursolic acid (1) (B). Oleic acid (50 μg/mL) was used as a positive control. The concentrations were 1 μg/mL (2.19 μM), 4 μg/mL (8.77 μM), and 10 μg/mL (21.9 μM). Data showed ± SD (n = 3). Student’s t test against control: (∗) P < 0.05; (∗∗) P < 0.01.
relatively low polarities.18 In our study, through quantified study by HPLC, the percentage of ursolic acid in methanol extract of loquat leaves is nearly 33%, and the percentage of ursolic acid in the dried loquat leaves is 6.25% (see the Supporting Information Figure S4 and Table 1). A number of studies have demonstrated ursolic acid (1) can indeed suppress multiple molecular targets that play a pivotal role in anticancer potential, anti-inflammatory in vitro, and in vivo human clinical trials.19−21 However, more detailed investigations are needed to completely understand its exact mode of action against different diseases. There are few reports of ursolic acid (1) in the study of osteoporosis diseases. Lee et al. observed that ursolic acid (1) has bone-forming activity in an in vivo mouse calvarial bone formation model. Also, ursolic acid (1) stimulates osteoblast differentiation and mineralization with the induction of osteoblast-specific gene expressions.22 However, no study focused on the inhibitory activity of ursolic acid (1) on osteoclast differentiation. We found that ursolic acid (1) inhibited osteoclast differentiation with an IC50 value of 3.49 μM. To the best of our knowledge, the present study is the first to report the effect of the leaves of loquat in the treatment of osteoporosis. Our results showed that the leaves of loquat in the diet could prevent BMD loss in OVX mice. Ursolic acid (1) is responsible for the osteoclast differentiation inhibitory activity. In conclusion, the leaves of loquat should be a functional ingredient of tea for the prevention and treatment of osteoporosis.
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Notes
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
S Supporting Information *
TLC, HPLC, and recycling preparative HPLC chromatogram of Fr. 4-7-6; HPLC chromatogram of compound 1, fraction (Fr. 1−9, 4-1, 4-3−4-8, 4-7-1−4-7-8) and methanol extract; quantified data for ursolic acid in fraction 4, 4-7, 5, 6, 7 and methanol extract. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*(K.S.) Phone/fax: +81 092 642 3002. E-mail: shimizu@agr. kyushu-u.ac.jp. Funding
The costs of publication were supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. 840
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Journal of Agricultural and Food Chemistry
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dx.doi.org/10.1021/jf402735u | J. Agric. Food Chem. 2014, 62, 836−841
