Monogalactosyldiacylglycerol: an abundant galactosyllipid of Cirsium brevicaule A. GRAY leaves inhibits the expression of gene encoding fatty acid synthase.
Background: The leaves of Cirsium brevicaule A. GRAY (CL) significantly decreased hepatic lipid accumulation and the expression of fatty acid synthase gene (FASN) in mice.
Purpose: We aimed to purify and identify the active compound(s) from CL and determine the inhibitory mechanism of expression of FASN.
Methods: We purified monogalactosyldiacylglycerol (MGDG) from extracts of CL (CL-MGDG) and showed that it was the active CL component through analyses of its effects on the expression of genes of human breast cancer cell line, SKBR-3.
Results: The content and fatty acid composition of CL-MGDG are distinctly different from those of other vegetable-derived MGDGs. Treatment of SKBR-3 cells with MGDG decreased the level of FASN mRNA as well as the levels of mRNA encoding other protein involved in lipogenesis. Further, MGDG treatments significantly inhibited luciferase activities of constructs containing liver X receptor response element in FASN promoter region without altering the levels of mRNA encoding transcription factors. MGDG and the FASN inhibitor C75 decreased the viabilities of SKBR-3 cells in a concentration-dependent manner. CL-MGDG more potently inhibited cell viability than a commercial MGDG preparation.
Conclusions: CL represents a good source of glycoglycerolipids with potential as functional ingredients of food.
Cirsium brevicaule A. GRAY
Fatty acid synthase
Throughout southern Japan and Taiwan, Cirsium brevicaule A. GRAY is a wild perennial herb growing in rocky gravels or forest margins along maritime coastlines (Kadota 1990). Its leaves, stems, and roots have traditionally been used as food and medicine in the Okinawa and Amami Islands of Japan. We showed that dietary C. brevicaule A. GRAY leaves (CL) significantly decreased the expression of the gene encoding fatty acid synthase (FASN) in livers and white adipose tissues of mice fed a high-fat diet (Inafuku et al. 2013). Further, we detected a significant reduction in serum free fatty acid (FA) levels in mice fed a diet containing CL. An increase of free-FA influx into circulation, which leads to enhanced free-FA uptake by multiple tissues, is associated with the pathogenesis of metabolic syndromes such as obesity, non-alcoholic fatty liver disease, and type 2 diabetes. Moreover, dietary CL decreased hepatic lipid levels in mice fed a high-fat diet (Inafuku et al. 2013). Therefore, CL has recently been drawing attention as a new functional food material.
FASN is the enzyme that catalyzes the NADPH-dependent condensation of acetyl-CoA and malonyl-CoA to produce the saturated FA palmitate. The prognostic marker OA-519, which is expressed by breast cancer cells of patients with very poor prognosis, was identified as FASN (Kuhajda et al. 1994). Breast cancer is currently the leading cause of death in women, accounting for 23% of all cancer-related deaths (Donepudi et al. 2014). The expression of FASN increases dramatically at the transcriptional and posttranscriptional levels in patients with breast cancer (Alo et al. 1996; Wang et al. 2001) as well as other cancers, and the expression of FASN is undetectable in normal tissues other than liver, adipose and breast (Menendez and Lupu 2007). Further, overexpression of FASN is associated with advanced stage, metastasis and poor prognosis of cancer (Menendez et al. 2004; Wang et al. 2001). These differences in the expression of FASN between normal and cancer tissues imply that FASN represents a promising target for anti-tumor therapy. Further, inhibition of FASN catalytic activity by agents such as cerulenin and C75 preferentially induce apoptosis of cancer cells (Pizer et al. 1998; Puig et al. 2008) and inhibit tumor growth in xenograft models (Pizer et al. 2000). The potential of FASN as an anti-tumor target is indicated by analyses of RNA interference-mediated silencing of expression of FASN (Menendez et al. 2005).
These findings inspired us to purify and identify the bioactive compound from CL to determine how it inhibits expression of FASN. Further, we evaluated the antitumor effects of the bioactive compound on the human breast cancer cell line, SKBR-3.
Materials and methods
Monogalactosyldiacylglycerol (MGDG) was purchased from Avanti Polar Lipids, Inc. (AL, USA). McCoy's 5A medium, C75, GW3965, and LG100268 were purchased from Sigma-Aldrich (MO, USA). FBS was obtained from AusGeneX PTY Ltd. (Oxenford, Australia) and inactivated at 56[degrees]C for 30 min before use.
Preparation of extracts and a bioactive compound from CL
C. brevicaule A. GRAY (Kousyunsou[R]) was harvested on Tokunoshima Island in Kagoshima Prefecture, Japan. The freeze-dried and ground powder of CL was generously provided by Tokunoshima-cho (Kagoshima, Japan). The dried-CL powder was serially extracted by incubation with 10 volumes each of hexane, chloroform, ethanol and water for 2 h at 37[degrees]C. The filtrates were evaporated or freeze-dried in vacuo, and stored at -80[degrees]C. The CL chloroform extract was applied to a Hi-Flash silica-gel column (Yamazen Corp., Osaka, Japan) and eluted with chloroform/methanol (95/5, v/v). The eluate was evaporated to dryness, dissolved in chloroform, and purified using an HPLC with a silica gel column (Cosmosil 5SL-II, Nacalai Tesque, Inc., Kyoto, Japan) with chloroform/methanol/water/formic acid (92.0/7.1/0.4/0.5, v/v). The active fractions were further evaporated, dissolved in methanol, and further purified through an HPLC reverse-phase column (Cosmosil 5C22-AR-11, Nacalai Tesque) with methanol/water/trifluoroacetic acid (91.8/8.0/0.2, v/v). All extracts and isolated fractions were evaporated, dissolved in DMSO and stored at -80[degrees]C.
Determination of the chemical structure and fatty acid composition of the bioactive compound isolated from CL
To identify the chemical structure of the purified active compound, NMR spectra were acquired using a Bruker UltraShield 400 MHz NMR spectrometer (Bruker Corporation, Billerica, MA, USA) with CDC13 as an internal standard. ESI-MS and MS/MS data were acquired using a Bruker Esquire 3000 Plus Ion Trap Mass Spectrometer.
To determine its FA composition, MGDG was methylated and FA methyl esters were analyzed using a gas chromatography system as described elsewhere (Wongtangtintharn et al. 2005).
The human breast cancer cell line SKBR-3 was purchased from the JCRB Cell Bank (Tokyo, Japan) and maintained at 37[degrees]C in a humidified atmosphere containing 5% C[O.sub.2]. The cells were grown in McCoy's 5A medium supplemented with 10% FBS and 1% penicillin-streptomycin.
Twenty-four hours after seeding the cells into a 24-well plate (1.5 x [10.sup.5] cells per well), the cells were treated with purified fractions or chemicals for 24 h. Protein was extracted and equal amounts of protein were separated using SDS-PAGE. The proteins were electrophoretically transferred onto a polyvinyiidene difluoride membrane. The blots were probed with antibodies against FASN and [beta]-actin (Cell Signaling Technology, Tokyo, Japan) as well as liver X receptor (LXR)-[alpha], LXR-[beta] and retinoid X receptor (RXR)-[alpha] (Abeam, Cambridge, UK) according to the manufacturers' protocols. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody (Biosource International, Inc., CA, USA) for 2 h at room temperature. Band intensities were quantified using ImageJ (NIH, MA, USA), and the level of each protein was normalized to that of [beta]-actin.
Analysis of mRNA levels
Twenty-four hours after seeding the cells in a 96-well plate (2 x [10.sup.4] cells per well), cells were treated with CL extracts, MGDGs, a-linolenic acid (ALA) and 5 [micro]M of the LXR agonist (GW3965) or 1 [micro]M of the RXR agonist (LG100268) for 24 h, washed with PBS, and cDNA was synthesized using a Gene Expression Cell-to-CT kit (Life Technologies, CA, USA). The TaqMan primer/probe sets for FASN (Hs01005622_ml), acetyl-CoA carboxylase [alpha] (ACACA, Hs01046047_ml), sterol regulatory element binding transcription factor 1 (SREBF1, Hs01088691_ml), stearoyl-CoA desaturase (SCD, Hs01682761_ml), nuclear receptor subfamily 1, group H, member (NR1H) 3 (LXR-[alpha], Hs00172885_ml), NR1H2 (LXR-[beta], Hs01027215_gl), RXR-[alpha] (Hs01067640_ml), and actin, beta (ACTS, Hs01060665_gl) were purchased from Applied Biosystems (CA, USA). To measure the relative abundance of target transcripts, amplifications were performed using TaqMan Fast Advanced Master Mix with the StepOne Real-Time PCR System (Applied biosystems), and the amounts of target transcripts were normalized to those of ACTB.
Construction of luciferase reporter plasmids
Fragments of the human FASN promoter were generated using nested-PCR. In brief, a PCR fragment (-1677/+128 bp) was amplified using human genomic DNA as a template (Promega Corp., WI, USA) and then used as a template for a second-round of PCR. To generate -1600, -835, -150, and -106 bp fragments, specific upstream primers were combined with the common downstream primer at +70 bp. These amplicons were sequenced and cloned into a firefly luciferase reporter vector [pGL4.23 (luc2/minP), Promega Corp.] that is digested with Kpnl and Hindlll using an In-Fusion HD Cloning Kit (Clontech Laboratories, Inc., CA, USA). The plasmids used for transfection experiments were purified from E. coli using a PureYield Plasmid Miniprep System (Promega Corp.).
Transient DNA transfection and luciferase assay
SKBR-3 cells were seeded at 4 x 104 cells per well in a 96-well plates 24 h before transfection. The cells were transiently transfected with the plasmids using the FuGENE HD transfection reagent (Promega Corp.). The pGL4.13 [luc2/SV40] vector served as the positive control, and the pGL4.73 [hRluc/SV40] vector (Promega Corp.) was cotransfected with each firefly luciferase-reporter plasmids as an internal control. Forty-eight hours after transfection, the cells were treated with MGDGs and an LXR or RXR agonist for 24 h, and then luciferase activities were determined using a DualGlo Luciferase Assay System (Promega Corp.). The firefly luciferase activities of test plasmids were normalized to renilla luciferase activities.
Celt viability assay
Cells were seeded at 2 x [10.sup.3] cells per well in a 96-well plate and allowed to attach for 24 h and then treated with serially diluted chemicals for another 24 h. After treatment, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS).
Data are expressed as the mean [+ or -] SEM. The statistical significance of the difference between two experimental groups was determined using the Student t-test. The Tukey-Kramer multiple comparison test was used to determine the significance of the differences among the means of more than three groups. Statistical significance was considered at P < 0.05.
Effects of CL extracts on expression of FASN and identification of the bioactive compound
The effects of serial extracts of CL on expression of FASN by SKBR-3 cells were tested using real-time PCR (Supplementary Fig. 1). No significant effects on FASN mRNA levels were detected in cells treated with the vehicle and hexane, ethanol, or water extracts of CL. However, FASN mRNA levels were significantly decreased in SKBR-3 cells treated with the CL chloroform extract compared with that of the vehicle.
The bioactive compound was purified from the CL chloroform extract as described in the materials and methods section. These data are in perfect accordance with literature data for MGDG (Larsen et al. 2003; Murakami et al. 1995; Wegner et al. 2000). ESIMS analysis of the compound (positive-ion mode) showed a quasi-molecular ion at m/z 797.8 [[(M+Na).sup.+]] and m/z 813.7 [[(M+K).sup.+]], ESI-MS/MS analysis of the [(M+Na).sup.+] ion showed that the fragment ion at m/z 519.4 [[(M+[Na.sup.-]278.4).sup.+]] indicated the loss of a FA residue. The FA component of CL-derived MGDG (CL-MGDG) was ALA, which represented approximately 97% of all species and differed significantly from that of MGDG (commercial MGDG) purchased from Avanti Polar Lipids, Inc. (Fig. 1 and Table 1).
Effects of MGDG on the expressions of genes involved in lipogenesis
We assessed the effects of MGDGs on the expression of FASN in SKBR-3 cells and observed that CL-MGDG caused concentration-dependent decrease in levels of FASN mRNA (Fig, 2a) and FASN (Fig. 2b). This treatment significantly inhibited the expression of SCD1 and SREBF1, which mediate lipogenesis, and tended to decrease expression of ACACA (Fig. 2c). Although similar results were obtained for cells treated with commercial MGDG, there were larger decreases in mRNA levels when cells were treated with CL-MGDG.
Effects of MGDG on FASN promoter activity and the expression of transcription factors
To gain further insight into the effect of MGDGs on expression of FASN, we determined the activities of human FASN promoter constructs in SKBR-3 cells. The four promoter constructs, hFASN (-1600/+70), hFASN (-835/+70), hFASN (-150/+70) and hFASN (-105/+70), include the liver X receptor (LXR) response element (LXRE), sterol regulatory element (SRE), stimulatory protein 1 (SP1) binding site, and the non-canonical sterol regulatory element binding protein recognition site (SREBP). Treatment with commercial MGDG or CL-MGDG significantly decreased the transcriptional activities of the FASN promoters in SBKR-3 cells transfected with hFASN (-1600/+70) or hFASN (-835/+70) but not those transfected with hFASN (-150/+70) or hFASN (-105/+70) (Fig. 3).
We assessed the effects of MGDG on the mRNA levels of liver X receptor (LXR), LXR[alpha] (NR1H3) and LXR[beta] (NR1H2), and retinoid X receptor [alpha] (RXR[alpha], NR2B1) (Fig. 4a). No significant changes were detected in LXR[alpha] mRNA levels in SKBR-3 cells treated with MGDGs. Although the mRNA levels of LXR[beta] and RXR[alpha] increased significantly in cells treated with 50 [micro]M MGDG compared with vehicle, they were unchanged when the cells were treated with other concentration of MGDGs. No significant changes were observed in the levels of LXR[alpha], LXR[beta], and RXRa (Fig. 4b).
Effects of MGDG on the expressions of genes involved in lipogenesis in the presence of the agonists of LXR or RXR
To understand the effects of MGDGs on lipid metabolism, we determined the mRNA levels of genes that mediate lipogenesis expressed by SKBR-3 cells treated with the LXR agonist (GW3965) or the RXR agonist (LG100268). Significant increases were detected in the levels of FASN, SREBF1, and SCD mRNA (Fig. 5a), which were prevented by treating cells with CL-MGDG. Although the levels of ACACA mRNA were significantly increased in cells treated with LG100268 but not with GW3965, CL-MGDG treatment significantly decreased ACACA mRNA levels in cells treated with each agonist.
There was a statistically significant enhancement but biologically to a lesser extent in FASN promoter activity in cells transfected with hFASN (-835/+70) and hFASN (-150/+70) by GW3965 treatment (Fig. 5b). These effects were significantly inhibited by treating cells with MGDG. In contrast, no significant differences were detected in luciferase activities when cells were stimulated with LG100268. However, the luciferase activity of hFASN (-835/+70), which was slightly increased in the presence of LG100268, was significantly inhibited by CL-MGDG compared with the vehicle.
Effects of MGDG and a FASN inhibitor on the viability of SKBR-3 cells
The systemic FASN inhibitor C75 decreased the viability of SKBR-3 cells as function of its concentration (Fig. 6). Treatment with MGDGs decreased cell viability more than C75. Although the inhibitory effects of these MGDGs were similar after 24 h. CL-MGDG decreased cell viability more than commercial MGDG after 48 h.
We reported that the dietary intake of CL significantly decreases FASN mRNA and serum-free FA levels in mice fed a high-fat diet (Inafuku et al. 2013). Here, we isolated CL-MGDG as the bioactive compound that inhibited the expression of FASN (Fig. 1). MGDGs are present in a wide range of plants, and plant galactosyl lipids constitute approximately 80% of the membrane lipids, of which MGDG is the most abundant one and accounts for approximately 50% of the thylakoid lipids. Spinach and parsley contain the highest amounts of MGDG among the vegetables tested (481 mg and 450 mg per 100 g dried vegetables, respectively) (Watson and Preedy 2010). Therefore, we did not expect the MGDG contents of CL to be higher than those of commercial spinach leaves and whole parsley grown in Okinawa and Chiba, respectively (Supplementary Table 1).
Here, we show that culturing SKBR-3 cells in a medium containing MGDG significantly decreased the levels of FASN, SCD, and SREBF1 mRNA (Fig. 2). MGDG treatments, compared with the vehicle, significantly decreased the promoter activity of FASN (Fig. 3). These effects were only detected in cells transfected with constructs containing LXRE. Contrary, the constructs lacking LXRE in FASN promoter showed very poor luciferase activities, suggesting that LXRE is critical for the basal promoter activity of FASN in SKBR-3 cells. LXRs are members of the nuclear receptor superfamily of ligand-activated transcription factors, which regulate the metabolism of lipids such as cholesterol and bile acids. LXR forms an obligate heterodimer with RXR and then binds to LXRE of promoters. The LXR/RXR heterodimer expression of the genes by binding to LXRE in the promoters of lipogenic-related genes such as FASN, and indirectly increases their transcription by up-regulating the expression of the gene encoding the transcription factor SREBF1 that regulates the expression of the genes involved in fatty acid synthesis, such as FASN, SCD, and ACACA (Schultz et al. 2000; Talukdar and Hillgartner 2006).
Significant decreases in the mRNA levels of genes that mediate lipogenesis and FASN promoter activities were detected in SKBR-3 cells cultured in the presence of MGDG, although the levels of expression of LXRa, LXR/3, and RXRa were unchanged (Fig. 4). It was noted that PUFA decrease SREBF1 promoter activity by inhibiting the binding of LXR to LXRE of SREBF1 promoter (Yoshikawa et al. 2002). When SKBR-3 cells were treated with MGDGs, the mRNA levels of SREBF1 were significantly decreased with no significant change in the promoter activities of constructs with SRE, although a significant reduction was shown in the construct including LXRE (Fig. 5). It is noteworthy that SREBF1 encodes a transcription factor, SREBP1 that binds to SRE. The promoter activity of the construct with SRE but not an LXRE was significantly decreased when stimulated with LXR agonist, GW3965. It is well known that ALA, one of PUFAs, inhibits the expression of lipogenic-related genes by inhibiting the interaction of LXRs with LXRE of their promoter region as described above. Indeed, similar results in mRNA levels of FASN and the promoter activities were obtained when SKBR-3 cells were treated with ALA (Supplementary Fig. 2). Therefore, these results suggest that MGDG suppresses the expression of FASN through the similar mechanisms to ALA.
FASN was identified as a prognostic marker for breast cancer cells (Kuhajda et al. 1994), because it is expressed at increased levels in breast cancer and other cancers (Alo et al. 1996; Rashid et al. 1997). These results indicate that fatty acid synthesis plays an important role in breast cancer and suggest that inhibiting FASN activity is a promising target for anticancer therapies. For example, cerulenin and C75 are commonly used as inhibitors of FASN through inhibition of the reaction of [beta]-ketoacyl synthase on FASN. Moreover, these inhibitors are used as cancer-therapeutics (reviewed in (Menendez and Lupu 2007) and (Kuhajda 2000)). As shown in Fig. 6, decrease in SKBR-3 cell viability was greater in treatment with MGDG than that with FASN inhibitor C75. In addition, MGDG was more active in SKBR-3 cells, which expressed more FASN mRNA, compared with other cancer cell lines, MCF-7 and HepG2 (Supplementary Fig. 3). These results might indicate that MGDG-induced drop in viability of cancer cells is caused by FASN downregulation.
MGDG from spinach inhibits mammalian replicative DNA polymerase activity (Kuriyama et al. 2005), tumor growth (Mizushina et al. 2012), and angiogenesis (Matsubara et al. 2005). Moreover, MGDG purified from thermophilic blue-green algae acts as an anti-inflammatory agent in human articular cartilage (Ulivi et al. 2011). Although the acyl groups of spinach-derived MGDG are mainly palmitoleic acid (C16:1) and ALA (Akasaka et al. 2013), two major FAs of MGDGs isolated from spinach leaves were palmitic acid (C16:0) and ALA (Supplementary Table 2), Further, two major FAs of parsley-derived MGDG are C17:1 and ALA. MGDG with two ALA moieties inhibits DNA polymerase activity and cancer cell growth compared with MGDG derivatives containing other FAs such as C18:0 and C18:1 (Matsui et al. 2009). Flere, we showed that ALA represents 97% of the FAs content of CL-MGDG, suggesting that [greater than or equal to] 95% of the CL-MGDG preparation comprised two ALAs.
MGDG was rapidly decomposed into FAs and its corresponding monogalactosylglycerols basically by lipase hydrolysis when exposed to pancreatic juice, suggesting that MGDG does not enter into the circulation as its intact form (Sugawara and Miyazawa 2000). However, it has been reported that dietary MGDG strongly and dose-dependently inhibits the tumor growth in mouse model (Maeda et ai. 2013), although the antitumor activity is due to its constituent FAs in part (Murray et al. 2015). Thus, these results suggest that MGDG, which escapes the digestion is absorbed partially or is re-synthesized from the digested component in the intestinal epithelial cells of mice, explaining the efficacy of dietary CL in our previous study (lnafuku et al. 2013).
Further, it is suggested that MGDG significantly decreases the cell viability by inhibiting the expression of FASN as well as DNA polymerase activity of SKBR-3 cells, explaining the large decrease in cell viability in the presence of MGDG compared with that of C75 and that in cell line expressing FASN at high levels (Supplementary Fig. 3). Moreover, the difference in FA composition between CL-MGDG and commercial MGDG may contribute to their distinct effects on the expression of genes and viability of SKBR-3 cells (Figs. 2 and 6). MGDG from spinach inhibits tumor growth in mice, suggesting that its efficacy is associated with its ability to inhibit angiogenesis and proliferation and to induce apoptosis in tumor tissue without side effects (Maeda et al. 2013). These results led us to propose that the significant decrease in the FASN expression caused by dietary intake of CL, as shown in our previous study (lnafuku et al. 2013), can be attributed in part to CL-MGDG.
In conclusion, the present study demonstrates that CL-MGDG is the bioactive compound present in CL that suppresses the FASN expression and decreases the viability of SKBR-3 cells, suggesting that CL represents a good source of glycoglycerolipids as well as the potential as a functional food ingredient.
Received 16 November 2015
Revised 19 February 2016
Accepted 25 February 2016
Abbreviations: ACACA, acetyl-CoA carboxylase or, ACTB, actin, beta; ALA, [alpha]-linolenic acid; CL Cirsium brevicaule A GRAY leaves; EI-MS, electron ionizationmass spectrometry; FA, fatty acid; FASN. fatty acid synthase; LXR, liver X receptor; LXRE, liver X receptor response element; MGDG, monogalactosyldiacylglycerol; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; RXR, retinoid X receptor; SCD, stearoyl-CoA desaturase; SP1, stimulatory protein 1 site; SRE, sterol regulatory element; SREBF, sterol regulatory element binding transcription factor; SREBP, non-canonical sterol regulatory element binding protein recognition site.
Conflict of interest
There are no conflicts of interest to declare.
We thank Tokunosima-cho of Kagoshima Prefecture, Japan, and Shojiro Fujimyama for generously providing the freeze-dried and ground powder of CL used in this study.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.02.023.
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Masashi Inafuku (a), Kensaku Takara (b), Naoyuki Taira (a,c), Ruwani N. Nugara (a,c), Yasuo Kamiyama (d), Hirosuke Oku (a), *
(a) Center of Molecular Biosciences, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213, Japan
(b) Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan
(c) United Graduate School of Agricultural Sciences. Kagoshima University, Kagoshima 890-0065, Japan
(d) Kobe Tokushukai Hospital, Hyogo 655-0017, Japan
* Corresponding author. Tel./fax: +81 98 895 8972.
E-mail address: firstname.lastname@example.org, email@example.com (H. Oku).
Table 1 Fatty acid compositions of monogalactosyldiacylglycerols (MGDGs). Fatty acid CL-MGDG (%) coMGDG C16:0 1.2 1.6 C16:1 0.2 1.5 C16:3 0.8 37.6 C18:2 0.2 2.8 C18:3 97.4 56.6 Total 100.0 100.0 CL-MGDG was isolated and purified from CL, and coMGDG was purchased from Avanti Polar Lipids, Inc.
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|Author:||Inafuku, Masashi; Takara, Kensaku; Taira, Naoyuki; Nugara, Ruwani N.; Kamiyama, Yasuo; Oku, Hirosuke|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||May 15, 2016|
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