trans-Resveratrol in Gnetum gnemon Protects against Oxidative

Jul 16, 2013 - Institute for Bee Products & Health Science, Yamada Bee Company, Inc., 194 Ichiba, Kagamino 708-0393, Japan. § Department of Aging ...
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trans-Resveratrol in Gnetum gnemon Protects against OxidativeStress-Induced Endothelial Senescence Hidetaka Ota,† Masahiro Akishita,*,† Hiroko Tani,‡ Tomoki Tatefuji,‡ Sumito Ogawa,† Katsuya Iijima,† Masato Eto,† Takuji Shirasawa,§ and Yasuyoshi Ouchi† †

Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan ‡ Institute for Bee Products & Health Science, Yamada Bee Company, Inc., 194 Ichiba, Kagamino 708-0393, Japan § Department of Aging Control Medicine, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo 113-0033, Japan ABSTRACT: Gnetum gnemon is an arboreal dioecious plant that is cultivated in Indonesia. The seeds of this species mainly contain dimeric stilbenoid compounds [gnetin C (1), gnemonoside A (2), and gnemonoside D (3)] along with trans-resveratrol (4). trans-Resveratrol has been reported to have antiaging, anticancer, and antidiabetic effects, as well as being a calorie restriction mimetic. SIRT1 exerts a protective effect against vascular senescence. In this study, the effects of these four main stilbenoid derivatives of a G. gnemon seed endosperm ethanolic extract on endothelial senescence were investigated. In streptozotocin-induced diabetic mice, administration of the G. gnemon ethanolic extract increased SIRT1 and decreased endothelial senescence. The concentration of 1 in blood plasma was 6-fold higher than 4 in these mice. Next, the in vitro effects of the four main stilbenoid derivatives of G. gnemon seeds were investigated. Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide. Endothelial senescence was inhibited by 4, which increased the expression of endothelial nitric oxide synthase and SIRT1, whereas 1−3 had no effect. These results indicated that the ethanolic extract of G. gnemon seeds inhibits endothelial senescence, suggesting that 4 plays a critical role in the prevention of endothelial senescence.

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dimer: 4.8% w/w), gnemonoside A (2) (resveratrol dimer, GCdiglucoside: 22% w/w), and gnemonoside D (3) (resveratrol dimer, GC-monoglucoside: 6.9% w/w), with smaller amounts of trans-resveratrol (4, 0.093% w/w) occurring. In Saccharomyces cerevisiae, the Sir2 (silent information regulator-2) family of genes governs budding exhaustion (cellular longevity) and replicative life span.7,8 Sir2 has been identified as an NAD+-dependent histone deacetylase and is responsible for maintaining chromatin silencing and genome stability. Mammalian sirtuin 1 (Sirt1), the closest homologue of Sir2, regulates the cell cycle, senescence, apoptosis, and metabolism, by interacting with a number of molecules such as p53. trans-Resveratrol (4) was identified as an SIRT1activating molecule in a screen for small-molecule activators of SIRT1, and the beneficial effect of 4 has been attributed to mitochondrial function.9,10 An increasing body of evidence suggests the presence of a link between endothelial senescence and vascular dysfunction, especially atherosclerosis.11 Senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis; SIRT1 has been recognized as a key regulator of vascular endothelial homeostasis, controlling angiogenesis, endothelial senescence, and dysfunction.12−14 In this study, the effects of a G. gnemon seed endosperm ethanolic extract on the inhibition of endothelial senescence in

revention of age-related diseases such as cardiovascular disease, diabetes, and Alzheimer’s disease is a primary goal of modern medicine. In lifestyle-related matters such as caloric restriction, many people pay attention not only to drugs but also to the functionality of natural products, such as polyphenols, contained in food and drinks. Gnetum gnemon L. (Gnetaceae) is one of the oldest species of trees on Earth, dating back to 200 million years, and is an arboreal dioecious plant that is cultivated widely in Southeast Asia.1 Its fruits and seeds are used widely as a dietary component in Indonesian cuisine. The fruits, which change from green to yellow, orange, and red with ripeness, are colorful and attractive, and the seeds of the ripe fruits have a hard shell. In Indonesia, popular dishes include soup prepared from G. gnemon fruits and leaves and also crackers made by crushing the seed endosperm obtained by shelling heated seeds, drying, and deep-frying. Wallace and colleagues first reported the isolation of C-glycosylflavones from G. gnemon leaves,2 and stilbenoid oligomers were isolated from dried G. gnemon roots.3 Moreover, stilbenoid derivatives are well-known components of other species in the family Gnetaceae.4 Plants in this family are used in folkloric treatments for diabetes, arthritis, and bronchitis, and their chemical constituents, stilbenoid oligomers, induce apoptosis in colon cancer cells.5 trans-Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is abundant in some red wines and is associated with the cardiovascular benefits of red wine (the French paradox).6 The seeds of G. gnemon mainly contain gnetin C (1) (resveratrol © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 12, 2012

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streptozotocin (STZ)-diabetic mice were studied. Furthermore, the activities of the four principal stilbenoid constituents (1−4) of G. gnemon seeds in the prevention of endothelial senescence have been investigated.

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RESULTS AND DISCUSSION To investigate whether the ethanolic extract of G. gnemon seed endosperm has a protective effect against vascular endothelial senescence in vivo, STZ-diabetic mice were used, since their development of endothelial senescence is well documented,15 and they are suitable for investigations in a clinical setting. STZtreated or control mice were given food containing 2% (w/w) of the powdered extract for 21 days. STZ-treated mice had elevated plasma glucose, and administration of the G. gnemon extract did not change the plasma glucose levels (Figure 1A).

Figure 2. Immunofluorescent staining for SIRT1 (green), CD-31 (red), and DAPI (blue). The bottom series shows the merged image from each column. Arrows indicate SIRT1-expressing endothelial cells. Representative samples (n = 10) are shown.

G. gnemon seeds. The amount of food ingested (g/day) was nearly identical in STZ-treated and control mice with or without the administration of the G. gnemon extract (Figure 3A). The stilbenoid concentrations in blood plasma of STZ-

Figure 1. (A) Plasma glucose in streptozotocin (STZ)-diabetic mice (*p < 0.05 as compared to control, n = 3). Number of SA-βgal-stained cells in G. gnemon seed endosperm powdered ethanolic extract (GSE)treated thoracic aortas (*p < 0.05 as compared to control, n = 3). (B) SA-βgal staining of thoracic aortas, with or without the powdered extract, at 14 days after a single intraperitoneal injection of STZ (60 mg/kg).

Figure 3. (A) Amount of food ingested (g/day) of wild-type and STZtreated mice with and without G. gnemon seed endosperm powdered ethanolic extract (GSE) (n = 5). (B) Concentrations of 1 and 4 in blood plasma of control and STZ-treated mice with this extract (*p < 0.05 as compared to 4, n.s: not significant).

The thoracic aortas of these mice were resected surgically, and the senescent phenotype was compared in mice with and without administration of the G. gnemon extract (Figure 1B). The number of senescence-associated β-galactosidase (SAβgal)-stained cells in a SA-βgal assay increased significantly in the thoracic aortas of STZ-diabetic mice but decreased in the thoracic aortas of the G. gnemon extract-administered mice (Figure 1A). Immunostaining of sections for SIRT1 showed that SIRT1 expression in aortic endothelial cells increased after administration of the extract to STZ-diabetic mice (Figure 2). Inhibition of endothelial senescence by this extract is likely attributed to the four main stilbenoid derivatives (1−4) of the

treated and control mice were measured after administration of the extract. As shown in Figure 3B, the concentration of 1 in blood plasma was about 6-fold higher than that of 4 in STZtreated mice. Next, to investigate the in vitro effects of the four main stilbenoid derivatives (1−4) of the G. gnemon seeds on the endothelial senescent phenotype, premature endothelial senescence was induced by treating human umbilical vein endothelial cells (HUVEC) with hydrogen peroxide (H2O2) B

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(100 μmol/L) for 1 h. Treatment with 4 inhibited the senescent phenotype, as judged by the SA-βgal assay and the enlarged and flattened cell morphology at 10 days (Figure 4).

Finally, as previously reported, trans-resveratrol (4) or SIRT1 promotes mitochondrial biogenesis through deacetylation and activation of PGC-1α. This is a master regulator of mitochondrial biogenesis that coactivates the nuclear respiratory factors (NRF-1 and NRF-2) inducing transcription of genes involved in mitochondrial biogenesis.16 To elucidate the molecular mechanisms of the antioxidative effects of 1−4, mitochondrial biogenesis was evaluated. Senescent endothelial cells induced by H2O2 had lower levels of MitoTracker Red fluorescence compared with untreated cells (Figure 6A). In

Figure 4. H2O2 (100 μmol/L)-induced endothelial senescence was inhibited by 4 (100 μmol/L), but not 1−3 (100 μmol/L), as judged by SA-βgal staining and morphological changes (*p < 0.05 as compared to control H2O2, n = 3).

On the other hand, HUVEC treated with compounds 1−3 did not lead to a reduced number of SA-βgal-positive cells or show specific senescent morphological changes (Figure 4). To explore the mechanism by which compounds 1−4 prevent oxidative-stress-induced endothelial senescence, the expression of SIRT1, p53, PAI-1, and eNOS was examined. Treatment with H2O2 increased the expression of PAI-1 and p53 significantly and decreased the expression of SIRT1 and eNOS (Figure 5A). Treatment with 4 returned expression

Figure 6. (A) MitoTracker Red fluorescence was evaluated in 1−4 (100 μmol/L)-treated cells at 10 days after addition of H2O2. DAPI (blue)-stained nuclei. (B) TFAM and NRF-1 quantified by real-time reverse transcription PCR. β-Actin was used as the internal control (*p < 0.05 as compared to control H2O2).

contrast, treatment with 4, but not with 1−3, partially restored the MitoTracker Red fluorescence (Figure 6A). To address whether mitochondrial transcription was increased, mRNA levels of TFAM (the principal transcription factor involved in regulating mtDNA transcription) and NRF-1 were quantified by real-time reverse transcription PCR. Concomitantly, TFAM and NRF-1 transcripts increased after treatment with 4, but not after treatment with 1−3 (Figure 6B). These results indicate that the molecular basis for the antioxidative effect of G. gnemon is attributable to increased mitochondrial biogenesis through up-regulation of SIRT1. In this study, G. gnemon inhibited oxidative-stress-induced endothelial senescence through up-regulation of SIRT1 and mitochondrial biogenesis, and resveratrol (4) played a critical role in the prevention of endothelial senescence. Resveratrol is known to be an SIRT1-activator8 and was the first compound found to mimic caloric restriction by stimulating SIRT1.7 In this study, 1−3 did not activate SIRT1, whereas 4 was able to activate SIRT1 in an assay using a fluorophore-containing peptide. Resveratrol has many targets in mammalian cells, and its ability to activate SIRT1 in vitro is dependent on the use of fluorescent substrates, calling into question its mechanism of action.17,18 Recently, some reports have suggested that the indirect activation of SIRT1 by 4 is mediated by the inhibition of phosphodiesterase or activation of AMPK.19,20 In addition, cilostazol, a phosphodiesterase 3 inhibitor, was reported to inhibit oxidative-stress-induced endothelial senescence through up-regulation of SIRT1.21 Resveratrol increases the concentration of cytosolic cAMP and subsequently activates the cAMP

Figure 5. A. SIRT1, eNOS, PAI-1, and p53 in 1-4 (100 μmol/L)treated HUVEC. B. The measurement of SIRT1 activity in 1-4 (100 μmol/L)-treated HUVEC. NAD+ (nicotinamide adenine dinucleotide) is a positive control (*p < 0.05 as compared to vehicle control, n = 3).

levels back to control levels (Figure 5A). In contrast, 1−3 did not change the expression of SIRT1 or eNOS significantly (Figure 5A). To determine whether the activation of SIRT1 was regulated by 1−4, a fluor-de-Lys SIRT1 activation assay was performed. Following such treatment, the activation of SIRT1 was markedly higher after treatment with 4 than when 1−3 were used to treat cells (Figure 5B). C

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described,3 to give 1 (69.6 mg), 2 (2.00 g), and 3 (280 mg) with more than 98% purity (determined by HPLC analysis). Preparation and HPLC analysis of a Powdered G. gnemon Seed Endosperm Ethanolic Extract. Dextrin (0.39 g) and water (5 g) were added to 6.25 g of the G. gnemon seed endosperm ethanolic extract and lyophilized to prepare a powdered extract used for biological experiments. The analysis of this extract was carried out by HPLC using a TSK gel ODS-100 V column (4.6 × 150 mm, Tosoh Co., Tokyo, Japan) with a gradient solvent system of CH3CN containing 1% CH3COOH (20−22%, 10 min linear, 22−30%, 10 min linear, 37%, 15 min hold, and 20%, 10 min hold) in 1% CH3COOH at a flow rate of 0.8 mL/min and a column temperature of 40 °C with detection at 320 nm. The powdered extract used in this study contained 3.5% w/w 1, 14% w/w 2, 4.5% w/w 3, and 0.1% w/w 4. Animal Experiments. The animal experiments were approved by the Institutional Review Board of the University of Tokyo (protocol number M-P09-056) and observed U.S. National Institutes of Health guidelines. Twelve-week-old SPF wild-type, male C57/BL6 mice (weighing approximately 25 g) were supplied by Charles River Laboratories, Inc. (Boston, MA, USA). Mouse feed was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). These mice were assigned randomly to two treatment groups (control group, n = 10; G. gnemon powdered seed extract group, n = 10). Each group was orally administered vehicle alone or food containing 2% of the powdered extract for 21 days ad libitum. Diabetic mice were made by a single intraperitoneal injection of STZ (60 mg/kg) at 7 days after administration of the extract. Tail blood glucose levels were assayed 3 days after injection using glucose test strips. The mice were killed by cervical dislocation. Aortas were removed for histological examination, after systemic perfusion with phosphate-buffered saline (PBS). Then, the aortas were washed with PBS three times, embedded in OCT medium, and cryosectioned. The proportion of SA-βgal-positive cells was analyzed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The primary antibody was purified rat anti-mouse CD-31 monoclonal antibody from Pharmingen (San Jose, CA, USA). DAPI was used to stain nuclei. Secondary antibodies (Alexa Fluor 488 donkey anti-rat IgG and Alexa Fluor 594 donkey anti-rat IgG) were purchased from Invitrogen (Carlsbad, CA, USA). Cells. Human umbilical vein endothelial cells were purchased from Cambrex (Walkersville, MD, USA). Population doubling levels (PDL) were calculated as described previously,24 and all experiments were performed at a PDL of 10−11. Senescence-Associated β-Galactosidase (SA-βgal) Staining. HUVEC were grown in 100 mm collagen-coated dishes to 80% confluence. HUVEC were pretreated with vehicle (0.05% DMSO), 1 (100 μmol/L), 2 (100 μmol/L), 3 (100 μmol/L), and 4 (100 μmol/ L) diluted in EGM-2 medium for 1 day. HUVEC were washed three times with EGM-2 and then treated for 1 h with 100 μmol/L H2O2 diluted in EGM-2. After treatment, HUVEC were trypsinized, reseeded at a density of 1 × 106 in 100 mm dishes, and cultured with EGM-2 containing these compounds for 10 days. At 10 days after treatment with H2O2, HUVEC were fixed and the proportion of SAβgal-positive cells was determined as described by Dimri et al.25 Cells were stained with hematoxylin to clarify the cellular appearance. Antibodies and Immunoblotting. Cells were lysed on ice for 1 h in buffer (50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1% NP40, 0.1% SDS, 1 mmol/L dithiothreitol, 1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/ mL leupeptin, and 10 mmol/L sodium fluoride). After blocking, the filters were incubated with the following antibodies: anti-eNOS (BD Biosciences, San Jose, CA, USA), anti-p53, anti-SIRT1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-PAI-1 (Molecular Innovations, Inc., Novi, MI, USA), and anti-β-actin (Sigma-Aldrich). After washing and incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (GE Healthcare Life Sciences, Pittsburgh, PA, USA) for 1 h, the antigen−antibody complexes were visualized using an enhanced chemiluminescence system (GE Healthcare Life Sciences).

effector protein Epac1, inducing AMPK phosphorylation via a calcium/calmodulin-dependent protein kinase II. The activation of AMPK increases NAD+ levels, thus leading to SIRT1 activation.19 More recently, strong mechanistic evidence has indicated that SIRT1 is activated directly by 4 through an allosteric mechanism.22 Therefore, 4 is an activator of SIRT1, and since G. gnemon seeds contain this compound, ingestion of this species in the diet may show beneficial effects on cardiovascular disease such as atherosclerosis. In the present investigation, the concentrations of stilbenoids 1−4 in blood plasma of STZ-treated and control mice after administration of the ethanolic extract of G. gnemon seed endosperm were measured, and the concentration of 1 was about 6-fold higher than that of 4 in STZ-treated mice (Figure 3B). STZ-treated mice that had been administered the G. gnemon extract showed reduced endothelial senescence in vivo. Furthermore, 4, but not 1−3, inhibited endothelial senescence in vitro in accordance with increased SIRT1 expression. In human plasma, the presence of 1−3 prolonged the persistence of 4 when the G. gnemon extract was administered to healthy humans (unpublished data). Therefore, 1 may prevent endothelial senescence induced by oxidative stress in vivo indirectly. Humans consume resveratrol frequently in some red wines, berries, peanuts, and other foods, but the levels are usually less than 4 mg per serving. Without knowing the effects of chronic exposure to higher levels of 4 in studies with experimental animals, the intake of resveratrol at higher doses is worthy of investigation.23 Therefore, food containing higher doses of the powdered G. gnemon seed extract (2% w/w) was tested. Thus, the average amount of food ingested by each mouse was 3.82 g/day, and the consumption of the extract was 76.4 mg/day (containing 2.7 mg of 1, 11 mg of 2, 3.4 mg of 3, and 0.1 mg of 4). No obvious side-effects were observed when mice were administered these higher stilbenoid doses. Considering that the daily consumption of G. gnemon seeds per capita in Indonesia is about 207−414 mg, the stilbenoid concentration levels tested in this study (76.4 mg/day) would be possible in daily life. In Figure 3C, the concentration of resveratrol (4) was lower in STZ-diabetic mice than for the control, although there was no significant difference in the amount of food ingested, pending a mechanistic explanation.

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EXPERIMENTAL SECTION

General Experimental Procedures. HPLC analysis was performed using an LC-20A system (Shimadzu Scientific Instruments, Kyoto, Japan). Preparative HPLC was used using a semipreparative HPLC Waters 600E multisolvent delivery system (Waters, Milford, MA, USA). LC/MS/MS were recorded on an API 4000 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) linked to an LC-20 A HPLC system (Shimadzu Scientific Instruments). Fluorescent images were acquired with a digital camera attached to a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). Plant Material. The seeds (endosperms) of G. gnemon were collected in Indonesia (Desa Bangkok, Kecamatan Gurah Kabupaten Kediri, Kediri, Jawa Timur) in July 2009 and identified by Dr. Eishin Kato. Voucher specimens (number 090716) have been deposited at Hosoda SHC Co. Ltd., Fukui, Japan. Compounds 1−3 were provided by the Institute for Bee Products & Health Science, Yamada Bee Company, Inc., Okayama, Japan. Resveratrol (4) was supplied by Sigma-Aldrich (St Louis, MO, USA). Extraction and Isolation. The dried endosperms of G. gnemon (250 g) were powdered and soaked in 55% EtOH (750 mL) at room temperature for 3 days to obtain a G. gnemon seed endosperm ethanolic extract (23.0 g). The residue was purified as previously D

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Staining Mitochondria. HUVEC were grown in chamber slides to 80% confluence. After the treatment described above, the medium was removed from the chamber slide and prewarmed (37 °C). Then, staining solution (100 nM) containing MitoTracker Red CMSRos probes was added for a 15 min incubation. After staining, the cells were washed in fresh, prewarmed growth medium. Real-Time Reverse Transcription Polymerase Chain Reaction. Total RNA from HUVEC was isolated with an ISOGEN kit (Nippon Gene Inc., Toyama, Japan). After treatment with RNase-free DNaseI for 30 min, total RNA (50 ng/μL) was reverse transcribed with random hexamers and oligo d(T) primers. The expression levels of NRF-1 and TFAM relative to β-actin were determined by means of staining with SYBR green dye and a LineGene fluorescent quantitative detection system (Bioflux Co., Tokyo, Japan), as recommended by the manufacturer. Primer quality was verified by dissociation curve analysis, the slopes of standard curves, and reactions without RT. The following primers were used; NRF-1-F: 5′-GATGGCACTGTCTCACTTATCC-3′, R: 5′-CTGATGCTTGCGTCGTCT-3′, TFAM-F: 5′-CATCTGTCTTGGCAAGTTGTCC-3′, R: 5′CCACTCCGCCCTATAAGCATC-3′, β-actin-F: 5′TGGCATGGGTCAGAAGGAT-3′, R: 5′-AAGCATTTGCGGTGGACCAT-3′. SIRT1 Activity Assay. SIRT1 activity was measured using an SIRT1 fluorescent activity assay kit (Biomol, Plymouth Meeting, PA, USA) according to the manufacturer’s instructions. Measurement of Stilbenoid Concentrations. For UPLC/MS/ MS analysis, 100 μL of mouse plasma was mixed with 300 μL of cold acetonitrile and centrifuged at 13000g for 15 min at 4 °C. The supernatants were evaporated with nitrogen gas, dissolved in 250 μL of water, and then diluted with 750 μL of citric acid/acetonitrile (1:3) solution. The solution was loaded onto a SPE column, and the eluate was evaporated with nitrogen gas. The pellet was resuspended in 200 μL of 30% aqueous acetonitrile before filtering through a 0.2 μm filter. trans-Resveratrol (4) and gnetin C (1) were separated at 40 °C using a reversed-phase column. The mobile phase consisted of water with 0.005% formic acid and acetonitrile and was pumped at a flow rate of 0.3 mL/min. The acetonitrile gradient was composed of the following steps: 5% acetonitrile (0−1 min), 5−15% acetonitrile (1−3.5 min), 15−50% acetonitrile (3.5−13.5 min), 50−100% acetonitrile (13.5− 15.5 min), linear gradient, 100% acetonitrile (15.5−17.5 min). There was a 5 min reequilibration period with the initial solvent mixture between analyses. Data Analysis. Values are shown as the means ± SEM in the text and figures. The unpaired Student t test was used for statistical analysis. Probability values less than 0.05 were considered significant.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81358008832. Fax: +81358008831. E-mail: [email protected]. Funding Notes

The authors declare the following competing financial interest(s): This study was funded by the Institute for Bee Products & Health Science, Yamada Bee Company, Inc., Okayama, Japan. The sponsor of the study had a role in measurement of stilbenoid concentrations of mice, but no role in the study design, conducting of the study, data interpretation, or preparation of this report.

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ACKNOWLEDGMENTS We thank Mr. Winarno Tohir (National Outstanding Farmer Association, Indonesia) for providing the plant material used in this investigation. E

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Journal of Natural Products

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dx.doi.org/10.1021/np300841v | J. Nat. Prod. XXXX, XXX, XXX−XXX