- Email: [email protected]
Toralactone glycoside in Cassia obtusifolia mediates hepatoprotection via an Nrf2-dependent anti-oxidative mechanism
Food Research International xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Toralactone glycoside in Cassia obtusifolia mediates hepatoprotection via an Nrf2-dependent anti-oxidative mechanism Yongtaek Seoa, Jae-Sook Songb, Young-Mi Kimb,⁎, Young Pyo Janga,c,⁎⁎ a b c
Division of Pharmacognosy, College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 02447, South Korea College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588, South Korea Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 02447, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Antioxidant enzymes Bioactivity-guided isolation Cassia seeds HepG2 cells
Cassia obtusifolia L. (Leguminosae) seeds are a well-known medicinal food in East Asia and are used to clear liver heat, sharpen vision, lubricate the intestines, and promote bowel movement. The aims of the present study were to identify the hepatoprotective components of C. obtusifolia seeds by bioactivity-guided isolation and to elucidate their mechanisms of action. Ten phenolic glycosides were isolated from the most active ethyl acetate fraction, and their chemical structures were elucidated by spectroscopic analyses. Among the isolated compounds, toralactone 9-O-gentiobioside (5) had the highest hepatoprotective efficacy against tert-butylhydroperoxide-induced cell death in HepG2 cells. Immunoblotting and real-time polymerase chain reaction analyses revealed that the hepatoprotective effects were exerted through nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent antioxidative signaling. Together, these results provide insights into the effects of this medicinal plant as well as a basis for developing hepatoprotective agents as pharmaceuticals and/or nutraceuticals.
1. Introduction The Cassia plants have been used as valuable herbal medicines and food sources in many countries in East Asia. Various studies have investigated the hepatoprotective effects of Cassia plant since antihepatotoxic naphtha-pyrone glycosides were isolated from the seeds of C. tora (Wong, Wong, Seligmann, & Wagner, 1989). Ethanol and nheptane extracts from C. occidentalis and C. fistula leaves showed serum transaminase-lowering effects in a paracetamol- and ethyl alcoholinduced rat model of liver injury (Bhakta et al., 2001; Jafri, Subhani, Javed, & Singh, 1999). The ethanol extract of C. fistula leaves also showed protective activity against diethylnitrosamine-induced hepatic injury in rats (Pradeep, Mohan, Gobianand, & Karthikeyan, 2007). In addition, an ethanol extract of Cassia seeds protected against carbon tetrachloride-induced liver injury in mice by enhancing antioxidant capacity (Xie, Guo, & Zhou, 2012). Cassia obtusifolia L. (Leguminosae) seeds are a well-known traditional herbal medicine and food in East Asia used to ‘promote liver function’, ‘improve eyesight’, and ‘promotes bowel movement’ (Yanjun,
Yuli, & Yuqing, 2001). The biological benefits of this medicinal food include neuroprotection in Parkinson's disease models (Ju et al., 2010), mitigating learning and memory impairment induced by scopolamine or transient cerebral hypoperfusion in mice(Kim et al., 2007), hepatoprotection against tacrine-induced cytotoxicity in HepG2 cells (Byun et al., 2007), antimicrobial activities (Kitanaka & Takido, 1986), larvicidal activity against mosquito species (Yang, Lim, & Lee, 2003), and in vitro inhibition of protein glycation and aldose reductase activity (Jang et al., 2007). The major active components of C. obtusifolia are anthraquinones, naphtho-pyrones, lactones, and their glycosides (Hatano et al., 1999; Wong et al., 1989; Yun-Choi, Kim, & Takido, 1990). Cassia seeds have been an effective medicinal food for the prevention of fatty liver. The ethanol extract of C. tora seeds showed hypolipidemic activity in rats with hyperlipidemia induced by a cholesterol-rich high fat diet (Awasthi et al., 2015; Patil, Saraf, & Dixit, 2004), and reduced hepatic lipids accumulation caused by high fat diet in rats by activating the 5′ adenosine monophosphateactivated protein kinase signaling pathway (Tzeng, Lu, Liou,
Abbreviations: LC-MS, liquid chromatography-mass spectrometry; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; Nrf2, nuclear factor erythroid-2-related factor 2; NQO1, NAD(P)H:quinone oxidoreductase 1; HO-1, heme oxygenase 1; t-BHP, tert-butylhydroperoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; EtOAc, ethyl acetate; ARE, antioxidant response element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase ⁎ Corresponding author. ⁎⁎ Correspondence to: Y.P. Jang, Division of Pharmacognosy, College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 02447, South Korea. E-mail addresses: [email protected] (Y.-M. Kim), [email protected] (Y.P. Jang). http://dx.doi.org/10.1016/j.foodres.2017.04.032 Received 13 January 2017; Received in revised form 11 April 2017; Accepted 29 April 2017 0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Seo, Y., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.04.032
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Chang, & Liu, 2013). Soluble fibers from C. tora seeds also showed a hypolipidemic effect in rats fed a high cholesterol diet by enhancing fecal lipid excretion (Cho, Lee, & Ha, 2007). Although various studies have investigated the hepatoprotective activity of seeds from Cassia species, to our knowledge there have been no reports on this activity in C. obtusifolia seeds. In the present study, we examined the hepatoprotective effect of the ethanol extract of C. obtusifolia seeds in HepG2 cells, and isolated and identified the active components by activity-guided fractionation. Ethanol has been known as the best solvent for extracting bioactive components from food materials because ethanol has been known as relatively safe than other organic solvents such as ethyl acetate, methylene chloride, even when it remained as residue in the extract. By using 70% of ethanol for the extraction, various range of phytochemicals from non-polar to highly polar compounds could be extracted.
2. Material and methods 2.1. Plant material The Ministry of Food and Drug Safety of South Korea (MFDS) certified C. obtusifolia L. seeds were purchased at the Kyungdong traditional herbal market in Seoul, South Korea, in August 2010 and were identified by Professor Young Pyo Jang and Professor Emeritus Chang Soo Yook of the College of Pharmacy, Kyung Hee University. A voucher specimen (KHUP-017) has been deposited at the Herbarium of College of Pharmacy, Kyung Hee University, South Korea.
2.2. Spectroscopy and chromatography Liquid chromatography-mass spectrometry (LC-MS) was performed using an Acquity H class ultra-performance LC system (Waters, Milford, MA, USA) connected to a JMS-AccuTOF mass spectrometer (JEOL, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were recorded with an Avance 500 NMR spectrometer (Bruker, Billerica, MA, USA) in dimethyl sulfoxide (DMSO) using tetramethylsilane as an internal standard. Chemical shifts are represented in parts per million (δ) and the coupling constants (J) are expressed in Hertz. Highperformance liquid chromatography (HPLC) was carried out using a Waters HPLC system consisting of a dual 515 pump, a 717 s autosampler, and a 996-photodiode array detector. Open column chromatography was carried out with a Diaion HP-20 (particle size 250–850 μm; Sigma-Aldrich, St. Louis, MO, USA), a reversed-phase silica gel (particle size 150 μm; YMC Co., Tokyo, Japan), or a Sephadex LH-20 gel (bead size 25–100 μm; Sigma-Aldrich, St. Louis, MO, USA).
Fig. 1. The chemical structure of the compounds isolated from the ethyl acetate (EtOAc) fraction of Cassia obtusifolia seeds.
2.4. Ultra performance liquid chromatography-mass spectrometry (UPLCMS) UPLC analyses were performed at 25 ± 1 °C using sample solutions filtered through 0.22 μm membrane (Whatman's syringe filter) and analysed (4 μL injected volume) using an ACQUITY UPLC™ H Class system (Waters, Milford, USA), equipped with a binary solvent delivery system and an autosampler. Separation was achieved on a Brownlee™ SPP C18 column (100 mm × 2.1 mm, 2.7 μm particle size) (Perkin Elmer Co., MA, USA); step-gradient elution was carried out using the mobile phase consisted of acetonitrile as solvent A and 0.1% formic acid in deionized water (DW) as solvent B. Separation was performed by gradient elution: 0 min 20% A, 2 min 20% A, 8 min 30% A, 15 min 100% A and 15.2 min 20% A with the flow rate of 0.4 mL/min. UV spectra of each peaks were obtained from photo diode array detector. Mass spectra were obtained from JMS-T100TD spectrometer AccuTOF® single-reflection time-of-flight mass spectrometer connected with Electrospray ionization (JEOL Ltd., Tokyo, Japan), operated with Mass Center software version 1.3.7. In positive ion mode, the need electrode was set to 4000 V; nitrogen gas was as the nebulizer and desolvating agent at flow rate of 1 and 3 L/min, respectively. The atmospheric pressure interface potential for the orifice 1, ring lens and orifice 2 for the 30 V, 10 V and 5 V, respectively. The range of analysis was set to m/z 50–1000. The temperature of desolvation chamber was
2.3. Extraction and isolation of compounds Air-dried C. obtusifolia seeds (1.2 kg) were powdered and extracted twice with 10 L of ethanol:water (7:3) at 60 °C. The extract was filtered and lyophilized, yielding 91.68 g of residue (abbreviated as CoE); 61.0 g of CoE was resuspended in 300 mL distilled water and successively partitioned with n-hexane (12.7 g), ethyl acetate (EtOAc) (11.4 g), and n-butanol (27.5 g). A portion of the EtOAc fraction (10.1 g) was separated through a Diaion HP-20 (4 × 30 cm) column packed in deionized water and eluted with a step gradient of deionized water/methanol followed by methanol, yielding 14 fractions of which four (#4, #6, #8, and #9) were subjected to semi-preparative HPLC using a YMC-Pack Pro C18 column (5 μm, 10 × 250 mm inner diameter; YMC Co., Tokyo, Japan), yielding 10 compounds (Fig. 1) (Tsuda, 2004). These were purified by HPLC with an Atlantis T3 column (3 μm, 4.6 × 150 mm, Waters, Milford, USA). Chemical structures of the isolated compounds were determined and identified by direct comparison of their spectroscopic profiles with previously reported data. 2
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Technologies, Santa Clara, CA, USA). Real-time PCR was performed with a LightCycler 480 II instrument using SYBR Green I Master solution (Roche Diagnostics, Indianapolis, IN, USA) per the manufacturer's instructions. A melting curve analysis was carried out to verify the accuracy of each amplicon. NQO1 mRNA levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following sense/antisense primer sets were used: human NQO1, 5′AGGCTGGTTTGAGCGAGT-3′/5′-ATTGAATTCGGGCGTCTGCTG-3′; and human GAPDH, 5′-GAAGGTGAAGGTCGGAGTC-3′/5′-GAAGATGGTGATGGGATTTC-3′.
set to 250 °C. 2.5. Hepatoprotection assay reagents Antibodies against the following proteins were used: nuclear factor erythroid-2-related factor 2 (Nrf2, Santa Cruz Biotechnology, Santa Cruz, CA, USA); lamin A/C (Cell Signaling Technology, Beverly, MA, USA); NAD(P)H:quinone oxidoreductase 1 (NQO1, Abcam, Cambridge, MA, USA); glutamate-cysteine ligase (GCL, Neomarker, Fremont, CA, USA); heme oxygenase 1 (HO-1, Enzo Life Sciences, Plymouth Meeting, PA, USA); and β-actin (Sigma-Aldrich). Hygromycin was purchased from Invitrogen (Carlsbad, CA, USA). Sulforaphane, tert-butylhydroperoxide (t-BHP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other reagents were purchased from SigmaAldrich. All chemicals were of analytical grade. DMSO was used as a vehicle to dissolve and dilute total extract, fractions, or isolated compounds.
2.10. Cell viability assay The cytoprotective effects of compound 5 against t-BHP-induced cell death were evaluated with the MTT colorimetric assay. HepG2 cells were seeded at a density of 5 × 104 cells per well in 48-well plates; after overnight serum starvation, the cells were treated with 500 μM t-BHP for 6 h after a 12-h pretreatment with or without compound 5. The cells were incubated with 0.5 mg/mL MTT for 1 h at 37 °C. DMSO (300 μL) was added to dissolve the formazan crystals after removal of cell media, and absorbance was measured at 570 nm using an Infinite M200 PRO microplate reader (Tecan, Salzburg, Austria). DMSO was used as a vehicle to dissolve and dilute total extract, fractions, or isolated compounds.
2.6. Cell culture Human hepatocyte-derived HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells stably transfected with pGL4.37 [luc2P/ARE/Hygro], an antioxidant response element (ARE)-driven reporter gene construct, were donated by Dr. I. J. Cho (Daegu Haany University, Gyeongsan, Korea) (Choi et al., 2016). Both cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. Hygromycin was added to the culture medium to maintain stable expression of pGL4.37 in HepG2 cells.
2.11. Statistical analysis Data are presented as means ± standard error (SE). Mean differences were assessed with student's t-test or by one-way analysis of variance with Bonferroni's multiple comparisons test. A P value < 0.05 was considered statistically significant.
2.7. Preparation of nuclear extracts and immunoblot analysis HepG2 cells were seeded in 6-well plates and grown to 70%–80% confluence, serum-starved overnight, and then exposed to compound 5 (20 μM) or sulforaphane (5 μM) for 3, 6, 12, or 24 h, respectively. Nuclear extracts were prepared and immunoblotting was carried out as previously described (Kang, Cho, Lee, & Kim, 2003; Kim et al., 2011). Briefly, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), which was blocked with 5% (w/v) skim milk in phosphate-buffered saline containing 0.25% (v/v) Tween20 for 1 h. This was followed by overnight incubation with the primary antibody at 4 °C and reaction with horseradish peroxidase-conjugated secondary antibody (Life Technologies, Grand Island, New York, USA). Proteins were detected with an enhanced chemiluminescence kit (GE Healthcare). β-Actin or lamin A/C was used as a loading control.
3. Results and discussion 3.1. CoE has hepatoprotective effects against oxidative stress-induced cell damage Oxidative stress is a key contributor to hepatocellular injury (Cichoż-Lach & Michalak, 2014). We investigated the hepatoprotective effects of CoE by assessing the viability of HepG2 cells. Exposure to 500 μM t-BHP for 6 h decreased cell viability to ~30% in HepG2 cells. Pretreatment with CoE protected hepatocytes against cell death caused by t-BHP-induced oxidative stress (Fig. 2), with a maximal effect observed with 30 μg/mL.
2.8. Luciferase assay HepG2 cells stably transfected with pGL4.37 were plated at a density of 2 × 105 cells per well in 12-well plates and allowed to grow to 70%–80% confluency. Cells were serum-starved overnight and then incubated with total extract (10 or 30 μg/mL), fractions (10 or 30 μg/ mL), or isolated compounds (20 μM) for 12 h, washed twice with icecold phosphate-buffered saline, and lysed with passive lysis buffer (Promega, Madison, WI, USA). The luciferase assay system (Promega) was used to measure luciferase activity in cell lysates per the manufacturer's protocol. 2.9. Real-time polymerase chain reaction (PCR) assay Fig. 2. Hepatoprotective effects of 70% ethanol extract of C. obtusifolia (CoE) against tbutyl hydroperoxide (t-BHP)-induced cell death. HepG2 cells were incubated with CoE for 12 h and then treated with t-BHP (500 μM, 6 h). Cell viability was evaluated with the MTT assay. Data represent means ± standard error (SE; n = 5). **P < 0.01 vs. vehicletreated control; ##P < 0.01 vs. t-BHP group. Con, control.
Total RNA was isolated from HepG2 cells using TRIzol reagent (Invitrogen) as described previously (Choi, Kim, Yang, Song, & Kim, 2015). Briefly, RNA (2 μg) was reverse-transcribed using oligo-d(T)16 primers to obtain cDNA on a SureCycler 8800 instrument (Agilent 3
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Table 1 Theoretical masses and measured mass numbers for the compounds from crude extract by LC-MS study. Compounds
Chemical formula
Measured (m/ z)
Theoretical (m/ z)
Mass difference (mmu)
1 2 3 4 5 6 7 8 9 10
C27H33O15 C25H29O12 C27H33O15 C28H33O15 C27H33O15 C23H25O12 C23H25O12 C22H23O10 C20H25O9 C27H31O14
597.18953 520.16570 597.19597 609.17485 597.19597 493.14013 493.14859 447.12912 409.14986 579.17819
597.18195 520.15808 597.18195 609.18195 597.18195 493.13460 493.13460 447.13868 409.15526 579.17138
7.58 7.62 14.02 − 7.10 14.02 5.53 13.99 9.56 5.41 6.81
crude extract with LC-MS analysis. From the UV chromatogram, several compounds were co-eluted at the same time but their identities were discriminated by corresponding accurate mass numbers (Fig. S2). The measured mass numbers and the corresponding calculated values were listed in Table 1.
Fig. 3. Effects of CoE and its sub-fractions on antioxidant response element (ARE)-driven luciferase activity in HepG2 cells. HepG2 cells stably transfected with the pGL4.37 plasmid were treated with 10–30 μg/mL total CoE or each fraction for 12 h. Relative AREdriven luciferase activity was determined in cell lysates. Data represent means ± SE (n = 6). *P < 0.05, **P < 0.01 vs. vehicle-treated control (Con).
3.2. CoE induces Nrf2 activation 3.5. Toralactone-9-O-β-D-gentiobioside from the EtOAc fraction of C. obtusifolia seeds activates Nrf2
Cellular antioxidant defense systems play an important role in the prevention of oxidative stress-induced cell death. Nrf2 is a key regulator of antioxidant and phase II detoxifying enzymes that are translocated into the nucleus upon activation and bind to the ARE sites of target genes, thereby enhancing their transcription (Ma, 2013). To evaluate the effects of CoE on Nrf2 activation, we measured ARE-driven luciferase activity in HepG2 cells stably transfected with the pGL4.37 plasmid containing four copies of ARE. Treatment with CoE (30 μg/mL) increased luciferase expression by approximately 5-fold (Fig. 3). We then compared effects of the n-hexane, EtOAc, BuOH, and H2O fractions of CoE on Nrf2 activation (Fig. 3). Nrf2/ARE-luciferase activity was strongest in cells treated with the EtOAc fraction (~ 8 and 14 folds at 10 and 30 μg/mL, respectively). Treatment with the n-hexane fraction (30 μg/mL) also increased Nrf2/ARE-luciferase activity by approximately 5.5-fold. An MTT assay revealed that no cytotoxicity was seen at the tested concentrations of EtOAc and n-hexane fractions of CoE as shown in Supplemental Fig. 1.
To identify bioactive principles from the EtOAc fraction responsible for Nrf2 activation, we assessed ARE-induced luciferase activity in HepG2 cells incubated with eight of the isolated compounds (Fig. 4). Compounds 9 and 10 were not tested owing to the negligible amounts that were available for bioactivity test. Compound 5 (toralactone-9-Oβ-D-gentiobioside) induced the greatest increase in Nrf2/ARE-luciferase activity (~ 4 fold at 20 μM). This effect was comparable to that of 5 μM sulforaphane, a naturally occurring isothiocyanate derived from cruciferous vegetables that is used to activate Nrf2/ARE signaling (Keum et al., 2006; Zhang, Talalay, Cho, & Posner, 1992). In the view of chemical structure, non-anthraquinone compounds such as xanthone (3) and naphtholactone (5) showed more potent activities than those of anthraquinones (compounds 2, 4, 7, and 8). Nuclear translocation of Nrf2 was confirmed by evaluating its nuclear levels (Fig. 5A). A time-course analysis revealed that nuclear Nrf2 levels in HepG2 cells increased within 3–6 h of treatment with compound 5 and were maintained for up to 24 h.
3.3. Identification of compounds The isolated compounds were identified through the direct comparison of their spectroscopic data with the data previously reported. The compounds were identified as rubrofusarin-6-β-gentiobioside (1, 9.7 mg) (Wong et al., 1989), chryso-obtusin-2-O-β-D-glucoside (2, 5.3 mg) (Jang et al., 2007), 8-hydroxy-3-methyl-6-methoxyxanthone1-O-β-D-gentiobioside (3, 3.0 mg) (Wang, Zuo, Ma, Wang, & Hu, 2008), physcion-8-O-β-D-gentiobioside (4, 20.0 mg) (Holzschuh, Kopp, & Kubelka, 1982), toralactone-9-O-β-D-gentiobioside (5, 32.7 mg) (Lee, Jang, Lee, Kim, & Kim, 2006), gluco-aurantioobtusin (6, 10.0 mg) (Yun-Choi et al., 1990), 6, 8-dihydroxy-1,7-dimethoxy-3methyl anthracene-9,10-dione 2-β-D-glucoside (7, 7.7 mg) (Shabana, Gonaid, Khaleel, & Yousif, 2001), physcion-8-O-β-D-glucopyranoside (8, 10.6 mg) (Lee et al., 2010), torachrysone gentiobioside (9, 1.7 mg) (Hatano et al., 1999), and chrysophanol 1-O-β-gentiobioside (10, 1.1 mg) (Ito et al., 2004) (Fig. 1). The purity of these compounds were assumed as above 95% at least evidenced in both of proton and carbon NMR spectra.
Fig. 4. Effects of compounds isolated from the ethyl acetate (EtOAc) fraction of Cassia obtusifolia seeds on antioxidant response element (ARE)-driven luciferase activity in HepG2 cells. Relative ARE-luciferase activity was measured in lysates of HepG2 cells stably transfected with the pGL4.37 plasmid and treated with each compound (20 μM) or sulforaphane (5 μM) for 12 h. Data represent means ± SE (n = 4). *P < 0.05, **P < 0.01 vs. vehicle-treated control (Con).
3.4. LC-MS study of CoE CoE sample solution (10 mg/mL) was prepared and injected on UPLC-PDA-MS system for the identification of active principles from the 4
Fig. 5. Nuclear factor erythroid-2-related factor 2 (Nrf2) activation and induction of antioxidant enzymes by compound 5 in HepG2 cells. A) Nuclear fractions prepared from HepG2 cells incubated with compound 5 (20 μM) or sulforaphane (5 μM) for the indicated times and probed with anti-Nrf2 antibodies. Signal intensity was assessed by scanning densitometry. Nuclear Nrf2 levels were normalized to those of lamin A/C. Data represent means ± SE (n = 4). B) HepG2 cells were treated with compound 5 (20 μM) for the indicated times, or exposed to sulforaphane (5 μM) for 12 h. NAD(P)H:quinone oxidoreductase 1 (NQO1) mRNA levels were determined by real-time polymerase chain reaction (PCR). Data represent means ± SE (n = 3 or 8 for compound 5 or sulforaphane, respectively). C) Lysates were obtained from cells exposed to compound 5 (20 μM) for the indicated times and probed with antibodies against NQO1, glutamate-cysteine ligase (GCL), and hemeoxygenase 1 (HO-1). *P < 0.05, **P < 0.01 vs. vehicle-treated control. Con, control; Sulfo, sulforaphane.
Y. Seo et al.
Food Research International xxx (xxxx) xxx–xxx
5
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
regulates diverse cellular antioxidant genes (Ma, 2013). Furthermore, toralactone-9-O-β-D-gentiobioside was identified as the primary component responsible for Nrf2 activation, as evidenced by increases in Nrf2/ARE-driven luciferase activity and nuclear Nrf2 levels, as well as upregulation of Nrf2 target genes. Finally, compound 5 prevented oxidative stress-induced cell damage in HepG2 cells, which may explain the hepatoprotective efficacy of C. obtusifolia seed extracts. Although non-anthraquinone structures showed more potent activities than those of anthraquinones compounds, it was not possible to speculate on the impact of xanthone or naphtholactone substitution patterns on the hepatoprotective activity of the compounds owing to a dearth of compounds available for comparison. 4. Conclusion In conclusion, the ethanol extract of Cassia obtusifolia seeds significantly protected HepG2 cells from t-BHP-induced cell death. Ten phenolic glycosides were identified from the most active EtOAc fraction by activity-guided isolation, and toralactone-9-O-β-D-gentiobioside was the most potent active principle in these seeds. The biochemical data on the active component of Cassia seeds provide insight into the effects of this medicinal plant as well as a basis for developing hepatoprotective agents as pharmaceuticals and/or nutraceuticals. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2017.04.032.
Fig. 6. Protection of HepG2 cells by compound 5 against t-butyl hydroperoxide (t-BHP)induced cell death. HepG2 cells were exposed to t-BHP (500 μM, 6 h) with or without pretreatment with compound 5 (10–20 μM) for 12 h. Cell viability was evaluated with the MTT assay. Data represent means ± SE (n = 6). **P < 0.01 vs. vehicle-treated control (Con); ##P < 0.01 vs. the t-BHP group.
3.6. Toralactone-9-O-β-D-gentiobioside induces antioxidant enzymes and protects against oxidative stress-induced cell death To determine whether nuclear translocation of Nrf2 stimulated by compound 5 induces upregulation of target genes, we measured mRNA levels of NQO1, a prototypical antioxidant enzyme, in HepG2 cells. Treatment with compound 5 increased NQO1 mRNA (Fig. 5B) as well as protein (Fig. 5C) levels. Moreover, protein levels of GCL and HO-1, which are also Nrf2-dependent antioxidant enzymes, were also upregulated in the presence of compound 5 (Fig. 5C). We next examined the ability of compound 5 to protect hepatocytes against cell death induced by t-BHP. Results of the MTT assay revealed that pretreatment with compound 5 (20 μM) attenuated t-BHP-induced damage in HepG2 cells (Fig. 6), an effect that may be attributed to induction of the Nrf2-dependent antioxidant system. t-BHP is frequently used to induce intracellular oxidative stress because it can be converted to free radical intermediates (Oh et al., 2012). Indeed, treatment with t-BHP enhances intracellular ROS production and results in cell apoptosis (Song, Kim, Choi, Oh, & Kim, 2016). Oxidative stress caused by excessive ROS can play an important role in the development and aggravation of liver diseases, such as alcoholic or drug-induced liver injury, nonalcoholic fatty liver diseases, and hepatocellular carcinoma (Bataille & Manautou, 2012; Tang, Jiang, Ponnusamy, & Diallo, 2014). Few study has been done on pharmacological efficacy of toralactone-9-O-β-D-gentiobioside except for the inhibitory activity on advanced glycation end products (AGEs) formation. Diabetes-related enzyme assays have described toralactone-9-O-β-D-gentiobioside as a potent inhibitory compound (IC50 10.7 μM, 1/45 fold as aminoguanidine, positive control) against formation of AGEs (Lee et al., 2006). Under hyperglycemic conditions, elevated blood glucose may induce formation of AGEs due to protein glycation, leading to diabetic complications caused by oxidative stress. As toralactone-9-O-β-D-gentiobioside also showed moderate inhibitory activity against protein tyrosine phosphatases 1B (PTP1B) and αglucosidase (Jung, Ali, & Choi, 2016), it was suggested as a potential candidate for the prevention of diabetic complications. The antioxidative capacity of toralactone-9-O-β-D-gentiobioside seems play a critical role in the pharmacological efficacies of both anti-diabetic and hepatoprotective activities. Previous studies have suggested that the protective effects of Cassia seed extracts in Parkinson's disease or liver injury models are related to their antioxidant properties (Ju et al., 2010; Xie et al., 2012). However, to the best of our knowledge this is the first study to demonstrate that C. obtusifolia seed extracts activate Nrf2, a transcription factor that
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author disclosure statement Young Pyo Jang and Young-Mi Kim supervised the conception and design of the study and helped in data interpretation and manuscript evaluation. Jae-Sook Song participated in the evaluation of hepatoprotective activity and gene expression data and data interpretation. Yongtaek Seo participated in the isolation of natural compounds, elucidation of compound structures, data interpretation, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. Acknowledgments We thank Editage (www.editage.co.kr) for English language editing and review of the manuscript. References Awasthi, V. K., Mahdi, F., Chander, R., Khanna, A. K., Saxena, J. K., Singh, R., ... Singh, R. K. (2015). Hypolipidemic activity of Cassia tora seeds in hyperlipidemic rats. Indian Journal of Clinical Biochemistry, 30, 78–83. Bataille, A. M., & Manautou, J. E. (2012). Nrf2: A potential target for new therapeutics in liver disease. Clinical Pharmacology & Therapeutics, 92, 340–348. Bhakta, T., Banerjee, S., Mandal, S. C., Maity, T. K., Saha, B. P., & Pal, M. (2001). Hepatoprotective activity of Cassia fistula leaf extract. Phytomedicine, 8, 220–224. Byun, E., Jeong, G.-S., An, R.-B., Li, B., Lee, D.-S., Ko, E.-K., ... Kim, Y.-C. (2007). Hepatoprotective compounds of Cassiae semen on tacrine-induced cytotoxicity in Hep G2 cells. Korean Journal of Pharmacognosy, 38, 400–402. Cho, I. J., Lee, C., & Ha, T. Y. (2007). Hypolipidemic effect of soluble fiber isolated from seeds of Cassia tora Linn. in rats fed a high-cholesterol diet. Journal of Agricultural and Food Chemistry, 55, 1592–1596. Choi, D. G., Kim, E. K., Yang, J. W., Song, J. S., & Kim, Y.-M. (2015). Nectandrin B, a lignan isolated from nutmeg, inhibits liver X receptor-alpha-induced hepatic lipogenesis through AMP-activated protein kinase activation. Pharmazie, 70, 733–739. Choi, H. Y., Lee, J.-H., Jegal, K. H., Cho, I. J., Kim, Y. W., & Kim, S. C. (2016). Oxyresveratrol abrogates oxidative stress by activating ERK-Nrf2 pathway in the liver. Chemico-Biological Interactions, 245, 110–121. Cichoż-Lach, H., & Michalak, A. (2014). Oxidative stress as a crucial factor in liver diseases. World Journal of Gastroenterology, 20, 8082–8091.
6
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Physcion-8-O-β-D-glucopyranoside enhances the commitment of mouse mesenchymal progenitors into osteoblasts and their differentiation: Possible involvement of signaling pathways to activate BMP gene expression. Journal of Cellular Biochemistry, 109(6), 1148–1157. Ma, Q. (2013). Role of nrf2 in oxidative stress and toxicity. Annual Review of Pharmacology and Toxicology, 53, 401–426. Oh, J. M., Jung, Y. S., Jeon, B. S., Yoon, B. I., Lee, K. S., Kim, B. H., ... Kim, S. K. (2012). Evaluation of hepatotoxicity and oxidative stress in rats treated with tert-butyl hydroperoxide. Food and Chemical Toxicology, 50, 1215–1221. Patil, U. K., Saraf, S., & Dixit, V. K. (2004). Hypolipidemic activity of seeds of Cassia tora Linn. Journal of Ethnopharmacology, 90, 249–252. Pradeep, K., Mohan, C. V. R., Gobianand, K., & Karthikeyan, S. (2007). Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats. Chemico-Biological Interactions, 167, 12–18. Shabana, M. M., Gonaid, M. H., Khaleel, A. E., & Yousif, M. F. (2001). Glycosides of Cassia tora L. Cultivated in Egypt. Journal of Biomedical Science, 7, 136–144. Song, J.-S., Kim, E.-K., Choi, Y.-W., Oh, W. K., & Kim, Y.-M. (2016). Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK3beta. Toxicology and Applied Pharmacology, 307, 138–149. Tang, W., Jiang, Y.-F., Ponnusamy, M., & Diallo, M. (2014). Role of Nrf2 in chronic liver disease. World Journal of Gastroenterology, 20, 13079–13087. Tsuda, Y. (2004). Isolation of natural products. University of Karachi. Tzeng, T.-F., Lu, H.-J., Liou, S.-S., Chang, C. J., & Liu, I.-M. (2013). Cassia tora (Leguminosae) seed extract alleviates high-fat diet-induced nonalcoholic fatty liver. Food and Chemical Toxicology, 51, 194–201. Wang, L. L., Zuo, J. P., Ma, L., Wang, X. C., & Hu, L. H. (2008). Two new xanthone glycosides from Ventilago leiocarpa Benth. Natural Product Communications, 3(5), 795–798. Wong, S.-M., Wong, M. M., Seligmann, O., & Wagner, H. (1989). New antihepatotoxic naphtho-pyrone glycosides from the seeds of Cassia tora. Planta Medica, 55, 276–280. Xie, Q., Guo, F.-F., & Zhou, W. (2012). Protective effects of cassia seed ethanol extract against carbon tetrachloride-induced liver injury in mice. Acta Biochimica Polonica, 59, 265–270. Yang, Y.-C., Lim, M.-Y., & Lee, H.-S. (2003). Emodin isolated from Cassia obtusifolia (Leguminosae) seed shows larvicidal activity against three mosquito species. Journal of Agricultural and Food Chemistry, 51, 7629–7631. Yanjun, H., Yuli, S., & Yuqing, Z. (2001). The advancement of the studies on the seeds of Cassia obtusifolia. Chinese Traditional and Herbal Drugs, 32, 2. Yun-Choi, H. S., Kim, J. H., & Takido, M. (1990). Potential inhibitors of platelet aggregation from plant sources, V. Anthraquinones from seeds of Cassia obtusifolia and related compounds. Journal of Natural Products, 53, 630–633. Zhang, Y., Talalay, P., Cho, C.-G., & Posner, G. H. (1992). A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation of structure. Proceedings of the National Academy of Sciences of the United States of America, 89, 2399–2403.
Hatano, T., Uebayashi, H., Ito, H., Shiota, S., Tsuchiya, T., & Yoshida, T. (1999). Phenolic constituents of Cassia seeds and antibacterial effect of some naphthalenes and anthraquinones on methicillin-resistant Staphylococcus aureus. Chemical & Pharmaceutical Bulletin, 47, 1121–1127. Holzschuh, L., Kopp, B., & Kubelka, W. (1982). Physcion-8-O-beta-D-gentiobioside, a new anthraquinone glycoside from rhubarb roots. Planta Medica, 46(3), 159–161. Ito, H., Nishida, Y., Yamazaki, M., Nakahara, K., Michalska-Hartwich, M., Furmanowa, M., Leistner, E., & Yoshida, T. (2004). Chrysophanol glycosides from callus cultures of monocotyledonous Kniphofia spp. (Asphodelaceae). Chemical and Pharmaceutical Bulletin, 52(10), 1262–1264. Jafri, M. A., Subhani, M. J., Javed, K., & Singh, S. (1999). Hepatoprotective activity of leaves of Cassia occidentalis against paracetamol and ethyl alcohol intoxication in rats. Journal of Ethnopharmacology, 66, 355–361. Jang, D. S., Lee, G. Y., Kim, Y. S., Lee, Y. M., Kim, C.-S., Yoo, J. L., & Kim, J. S. (2007). Anthraquinones from the seeds of Cassia tora with inhibitory activity on protein glycation and aldose reductase. Biological & Pharmaceutical Bulletin, 30, 2207–2210. Ju, M. S., Kim, H. G., Choi, J. G., Ryu, J. H., Hur, J., Kim, Y. J., & Oh, M. S. (2010). Cassiae semen, a seed of Cassia obtusifolia, has neuroprotective effects in Parkinson's disease models. Food and Chemical Toxicology, 48, 2037–2044. Jung, H. A., Ali, Y., & Choi, J. S. (2016). Promising inhibitory effects of anthraquinones, naphthopyrone, and naphthalene glycosides, from Cassia obtusifolia on alphaglucosidase and human protein tyrosine phosphatases 1B. Molecules, 22(1). Kang, K. W., Cho, I. J., Lee, C. H., & Kim, S. G. (2003). Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta activation in the induction of glutathione S-transferase by oltipraz. Journal of the National Cancer Institute, 95, 53–66. Keum, Y.-S., Yu, S., Chang, P. P.-J., Yuan, X., Kim, J.-H., Xu, C., ... Kong, A.-N. T. (2006). Mechanism of action of sulforaphane: Inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response elementmediated heme oxygenase-1 in human hepatoma HepG2 cells. Cancer Research, 66, 8804–8813. Kim, D. H., Yoon, B. H., Kim, Y.-W., Lee, S., Shin, B. Y., Jung, J. W., ... Ryu, J. H. (2007). The seed extract of Cassia obtusifolia ameliorates learning and memory impairments induced by scopolamine or transient cerebral hypoperfusion in mice. Journal of Pharmacological Sciences, 105, 82–93. Kim, Y. M., Lim, S.-C., Han, C. Y., Kay, H. Y., Cho, I. J., Ki, S. H., ... Kim, S. G. (2011). G(alpha)12/13 induction of CYR61 in association with arteriosclerotic intimal hyperplasia: Effect of sphingosine-1-phosphate. Arteriosclerosis, Thrombosis, and Vascular Biology, 31, 861–869. Kitanaka, S., & Takido, M. (1986). Studies on the constituents in the roots of Cassia obtusifolia L. and the antimicrobial activities of constituents of the roots and the seeds. Yakugaku Zasshi, 106, 302–306. Lee, G. Y., Jang, D. S., Lee, Y. M., Kim, J. M., & Kim, J. S. (2006). Naphthopyrone glucosides from the seeds of Cassia tora with inhibitory activity on advanced glycation end products (AGEs) formation. Archives of Pharmacal Research, 29, 587–590. Lee, S.-U., Choi, Y. H., Kim, Y. S., Park, S.-J., Kwak, H. B., Min, Y. K., ... Kim, S. H. (2010).
7
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Toralactone glycoside in Cassia obtusifolia mediates hepatoprotection via an Nrf2-dependent anti-oxidative mechanism Yongtaek Seoa, Jae-Sook Songb, Young-Mi Kimb,⁎, Young Pyo Janga,c,⁎⁎ a b c
Division of Pharmacognosy, College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 02447, South Korea College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588, South Korea Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 02447, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Antioxidant enzymes Bioactivity-guided isolation Cassia seeds HepG2 cells
Cassia obtusifolia L. (Leguminosae) seeds are a well-known medicinal food in East Asia and are used to clear liver heat, sharpen vision, lubricate the intestines, and promote bowel movement. The aims of the present study were to identify the hepatoprotective components of C. obtusifolia seeds by bioactivity-guided isolation and to elucidate their mechanisms of action. Ten phenolic glycosides were isolated from the most active ethyl acetate fraction, and their chemical structures were elucidated by spectroscopic analyses. Among the isolated compounds, toralactone 9-O-gentiobioside (5) had the highest hepatoprotective efficacy against tert-butylhydroperoxide-induced cell death in HepG2 cells. Immunoblotting and real-time polymerase chain reaction analyses revealed that the hepatoprotective effects were exerted through nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent antioxidative signaling. Together, these results provide insights into the effects of this medicinal plant as well as a basis for developing hepatoprotective agents as pharmaceuticals and/or nutraceuticals.
1. Introduction The Cassia plants have been used as valuable herbal medicines and food sources in many countries in East Asia. Various studies have investigated the hepatoprotective effects of Cassia plant since antihepatotoxic naphtha-pyrone glycosides were isolated from the seeds of C. tora (Wong, Wong, Seligmann, & Wagner, 1989). Ethanol and nheptane extracts from C. occidentalis and C. fistula leaves showed serum transaminase-lowering effects in a paracetamol- and ethyl alcoholinduced rat model of liver injury (Bhakta et al., 2001; Jafri, Subhani, Javed, & Singh, 1999). The ethanol extract of C. fistula leaves also showed protective activity against diethylnitrosamine-induced hepatic injury in rats (Pradeep, Mohan, Gobianand, & Karthikeyan, 2007). In addition, an ethanol extract of Cassia seeds protected against carbon tetrachloride-induced liver injury in mice by enhancing antioxidant capacity (Xie, Guo, & Zhou, 2012). Cassia obtusifolia L. (Leguminosae) seeds are a well-known traditional herbal medicine and food in East Asia used to ‘promote liver function’, ‘improve eyesight’, and ‘promotes bowel movement’ (Yanjun,
Yuli, & Yuqing, 2001). The biological benefits of this medicinal food include neuroprotection in Parkinson's disease models (Ju et al., 2010), mitigating learning and memory impairment induced by scopolamine or transient cerebral hypoperfusion in mice(Kim et al., 2007), hepatoprotection against tacrine-induced cytotoxicity in HepG2 cells (Byun et al., 2007), antimicrobial activities (Kitanaka & Takido, 1986), larvicidal activity against mosquito species (Yang, Lim, & Lee, 2003), and in vitro inhibition of protein glycation and aldose reductase activity (Jang et al., 2007). The major active components of C. obtusifolia are anthraquinones, naphtho-pyrones, lactones, and their glycosides (Hatano et al., 1999; Wong et al., 1989; Yun-Choi, Kim, & Takido, 1990). Cassia seeds have been an effective medicinal food for the prevention of fatty liver. The ethanol extract of C. tora seeds showed hypolipidemic activity in rats with hyperlipidemia induced by a cholesterol-rich high fat diet (Awasthi et al., 2015; Patil, Saraf, & Dixit, 2004), and reduced hepatic lipids accumulation caused by high fat diet in rats by activating the 5′ adenosine monophosphateactivated protein kinase signaling pathway (Tzeng, Lu, Liou,
Abbreviations: LC-MS, liquid chromatography-mass spectrometry; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; Nrf2, nuclear factor erythroid-2-related factor 2; NQO1, NAD(P)H:quinone oxidoreductase 1; HO-1, heme oxygenase 1; t-BHP, tert-butylhydroperoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; EtOAc, ethyl acetate; ARE, antioxidant response element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase ⁎ Corresponding author. ⁎⁎ Correspondence to: Y.P. Jang, Division of Pharmacognosy, College of Pharmacy, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 02447, South Korea. E-mail addresses: [email protected] (Y.-M. Kim), [email protected] (Y.P. Jang). http://dx.doi.org/10.1016/j.foodres.2017.04.032 Received 13 January 2017; Received in revised form 11 April 2017; Accepted 29 April 2017 0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Seo, Y., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.04.032
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Chang, & Liu, 2013). Soluble fibers from C. tora seeds also showed a hypolipidemic effect in rats fed a high cholesterol diet by enhancing fecal lipid excretion (Cho, Lee, & Ha, 2007). Although various studies have investigated the hepatoprotective activity of seeds from Cassia species, to our knowledge there have been no reports on this activity in C. obtusifolia seeds. In the present study, we examined the hepatoprotective effect of the ethanol extract of C. obtusifolia seeds in HepG2 cells, and isolated and identified the active components by activity-guided fractionation. Ethanol has been known as the best solvent for extracting bioactive components from food materials because ethanol has been known as relatively safe than other organic solvents such as ethyl acetate, methylene chloride, even when it remained as residue in the extract. By using 70% of ethanol for the extraction, various range of phytochemicals from non-polar to highly polar compounds could be extracted.
2. Material and methods 2.1. Plant material The Ministry of Food and Drug Safety of South Korea (MFDS) certified C. obtusifolia L. seeds were purchased at the Kyungdong traditional herbal market in Seoul, South Korea, in August 2010 and were identified by Professor Young Pyo Jang and Professor Emeritus Chang Soo Yook of the College of Pharmacy, Kyung Hee University. A voucher specimen (KHUP-017) has been deposited at the Herbarium of College of Pharmacy, Kyung Hee University, South Korea.
2.2. Spectroscopy and chromatography Liquid chromatography-mass spectrometry (LC-MS) was performed using an Acquity H class ultra-performance LC system (Waters, Milford, MA, USA) connected to a JMS-AccuTOF mass spectrometer (JEOL, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were recorded with an Avance 500 NMR spectrometer (Bruker, Billerica, MA, USA) in dimethyl sulfoxide (DMSO) using tetramethylsilane as an internal standard. Chemical shifts are represented in parts per million (δ) and the coupling constants (J) are expressed in Hertz. Highperformance liquid chromatography (HPLC) was carried out using a Waters HPLC system consisting of a dual 515 pump, a 717 s autosampler, and a 996-photodiode array detector. Open column chromatography was carried out with a Diaion HP-20 (particle size 250–850 μm; Sigma-Aldrich, St. Louis, MO, USA), a reversed-phase silica gel (particle size 150 μm; YMC Co., Tokyo, Japan), or a Sephadex LH-20 gel (bead size 25–100 μm; Sigma-Aldrich, St. Louis, MO, USA).
Fig. 1. The chemical structure of the compounds isolated from the ethyl acetate (EtOAc) fraction of Cassia obtusifolia seeds.
2.4. Ultra performance liquid chromatography-mass spectrometry (UPLCMS) UPLC analyses were performed at 25 ± 1 °C using sample solutions filtered through 0.22 μm membrane (Whatman's syringe filter) and analysed (4 μL injected volume) using an ACQUITY UPLC™ H Class system (Waters, Milford, USA), equipped with a binary solvent delivery system and an autosampler. Separation was achieved on a Brownlee™ SPP C18 column (100 mm × 2.1 mm, 2.7 μm particle size) (Perkin Elmer Co., MA, USA); step-gradient elution was carried out using the mobile phase consisted of acetonitrile as solvent A and 0.1% formic acid in deionized water (DW) as solvent B. Separation was performed by gradient elution: 0 min 20% A, 2 min 20% A, 8 min 30% A, 15 min 100% A and 15.2 min 20% A with the flow rate of 0.4 mL/min. UV spectra of each peaks were obtained from photo diode array detector. Mass spectra were obtained from JMS-T100TD spectrometer AccuTOF® single-reflection time-of-flight mass spectrometer connected with Electrospray ionization (JEOL Ltd., Tokyo, Japan), operated with Mass Center software version 1.3.7. In positive ion mode, the need electrode was set to 4000 V; nitrogen gas was as the nebulizer and desolvating agent at flow rate of 1 and 3 L/min, respectively. The atmospheric pressure interface potential for the orifice 1, ring lens and orifice 2 for the 30 V, 10 V and 5 V, respectively. The range of analysis was set to m/z 50–1000. The temperature of desolvation chamber was
2.3. Extraction and isolation of compounds Air-dried C. obtusifolia seeds (1.2 kg) were powdered and extracted twice with 10 L of ethanol:water (7:3) at 60 °C. The extract was filtered and lyophilized, yielding 91.68 g of residue (abbreviated as CoE); 61.0 g of CoE was resuspended in 300 mL distilled water and successively partitioned with n-hexane (12.7 g), ethyl acetate (EtOAc) (11.4 g), and n-butanol (27.5 g). A portion of the EtOAc fraction (10.1 g) was separated through a Diaion HP-20 (4 × 30 cm) column packed in deionized water and eluted with a step gradient of deionized water/methanol followed by methanol, yielding 14 fractions of which four (#4, #6, #8, and #9) were subjected to semi-preparative HPLC using a YMC-Pack Pro C18 column (5 μm, 10 × 250 mm inner diameter; YMC Co., Tokyo, Japan), yielding 10 compounds (Fig. 1) (Tsuda, 2004). These were purified by HPLC with an Atlantis T3 column (3 μm, 4.6 × 150 mm, Waters, Milford, USA). Chemical structures of the isolated compounds were determined and identified by direct comparison of their spectroscopic profiles with previously reported data. 2
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Technologies, Santa Clara, CA, USA). Real-time PCR was performed with a LightCycler 480 II instrument using SYBR Green I Master solution (Roche Diagnostics, Indianapolis, IN, USA) per the manufacturer's instructions. A melting curve analysis was carried out to verify the accuracy of each amplicon. NQO1 mRNA levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following sense/antisense primer sets were used: human NQO1, 5′AGGCTGGTTTGAGCGAGT-3′/5′-ATTGAATTCGGGCGTCTGCTG-3′; and human GAPDH, 5′-GAAGGTGAAGGTCGGAGTC-3′/5′-GAAGATGGTGATGGGATTTC-3′.
set to 250 °C. 2.5. Hepatoprotection assay reagents Antibodies against the following proteins were used: nuclear factor erythroid-2-related factor 2 (Nrf2, Santa Cruz Biotechnology, Santa Cruz, CA, USA); lamin A/C (Cell Signaling Technology, Beverly, MA, USA); NAD(P)H:quinone oxidoreductase 1 (NQO1, Abcam, Cambridge, MA, USA); glutamate-cysteine ligase (GCL, Neomarker, Fremont, CA, USA); heme oxygenase 1 (HO-1, Enzo Life Sciences, Plymouth Meeting, PA, USA); and β-actin (Sigma-Aldrich). Hygromycin was purchased from Invitrogen (Carlsbad, CA, USA). Sulforaphane, tert-butylhydroperoxide (t-BHP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other reagents were purchased from SigmaAldrich. All chemicals were of analytical grade. DMSO was used as a vehicle to dissolve and dilute total extract, fractions, or isolated compounds.
2.10. Cell viability assay The cytoprotective effects of compound 5 against t-BHP-induced cell death were evaluated with the MTT colorimetric assay. HepG2 cells were seeded at a density of 5 × 104 cells per well in 48-well plates; after overnight serum starvation, the cells were treated with 500 μM t-BHP for 6 h after a 12-h pretreatment with or without compound 5. The cells were incubated with 0.5 mg/mL MTT for 1 h at 37 °C. DMSO (300 μL) was added to dissolve the formazan crystals after removal of cell media, and absorbance was measured at 570 nm using an Infinite M200 PRO microplate reader (Tecan, Salzburg, Austria). DMSO was used as a vehicle to dissolve and dilute total extract, fractions, or isolated compounds.
2.6. Cell culture Human hepatocyte-derived HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells stably transfected with pGL4.37 [luc2P/ARE/Hygro], an antioxidant response element (ARE)-driven reporter gene construct, were donated by Dr. I. J. Cho (Daegu Haany University, Gyeongsan, Korea) (Choi et al., 2016). Both cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. Hygromycin was added to the culture medium to maintain stable expression of pGL4.37 in HepG2 cells.
2.11. Statistical analysis Data are presented as means ± standard error (SE). Mean differences were assessed with student's t-test or by one-way analysis of variance with Bonferroni's multiple comparisons test. A P value < 0.05 was considered statistically significant.
2.7. Preparation of nuclear extracts and immunoblot analysis HepG2 cells were seeded in 6-well plates and grown to 70%–80% confluence, serum-starved overnight, and then exposed to compound 5 (20 μM) or sulforaphane (5 μM) for 3, 6, 12, or 24 h, respectively. Nuclear extracts were prepared and immunoblotting was carried out as previously described (Kang, Cho, Lee, & Kim, 2003; Kim et al., 2011). Briefly, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), which was blocked with 5% (w/v) skim milk in phosphate-buffered saline containing 0.25% (v/v) Tween20 for 1 h. This was followed by overnight incubation with the primary antibody at 4 °C and reaction with horseradish peroxidase-conjugated secondary antibody (Life Technologies, Grand Island, New York, USA). Proteins were detected with an enhanced chemiluminescence kit (GE Healthcare). β-Actin or lamin A/C was used as a loading control.
3. Results and discussion 3.1. CoE has hepatoprotective effects against oxidative stress-induced cell damage Oxidative stress is a key contributor to hepatocellular injury (Cichoż-Lach & Michalak, 2014). We investigated the hepatoprotective effects of CoE by assessing the viability of HepG2 cells. Exposure to 500 μM t-BHP for 6 h decreased cell viability to ~30% in HepG2 cells. Pretreatment with CoE protected hepatocytes against cell death caused by t-BHP-induced oxidative stress (Fig. 2), with a maximal effect observed with 30 μg/mL.
2.8. Luciferase assay HepG2 cells stably transfected with pGL4.37 were plated at a density of 2 × 105 cells per well in 12-well plates and allowed to grow to 70%–80% confluency. Cells were serum-starved overnight and then incubated with total extract (10 or 30 μg/mL), fractions (10 or 30 μg/ mL), or isolated compounds (20 μM) for 12 h, washed twice with icecold phosphate-buffered saline, and lysed with passive lysis buffer (Promega, Madison, WI, USA). The luciferase assay system (Promega) was used to measure luciferase activity in cell lysates per the manufacturer's protocol. 2.9. Real-time polymerase chain reaction (PCR) assay Fig. 2. Hepatoprotective effects of 70% ethanol extract of C. obtusifolia (CoE) against tbutyl hydroperoxide (t-BHP)-induced cell death. HepG2 cells were incubated with CoE for 12 h and then treated with t-BHP (500 μM, 6 h). Cell viability was evaluated with the MTT assay. Data represent means ± standard error (SE; n = 5). **P < 0.01 vs. vehicletreated control; ##P < 0.01 vs. t-BHP group. Con, control.
Total RNA was isolated from HepG2 cells using TRIzol reagent (Invitrogen) as described previously (Choi, Kim, Yang, Song, & Kim, 2015). Briefly, RNA (2 μg) was reverse-transcribed using oligo-d(T)16 primers to obtain cDNA on a SureCycler 8800 instrument (Agilent 3
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Table 1 Theoretical masses and measured mass numbers for the compounds from crude extract by LC-MS study. Compounds
Chemical formula
Measured (m/ z)
Theoretical (m/ z)
Mass difference (mmu)
1 2 3 4 5 6 7 8 9 10
C27H33O15 C25H29O12 C27H33O15 C28H33O15 C27H33O15 C23H25O12 C23H25O12 C22H23O10 C20H25O9 C27H31O14
597.18953 520.16570 597.19597 609.17485 597.19597 493.14013 493.14859 447.12912 409.14986 579.17819
597.18195 520.15808 597.18195 609.18195 597.18195 493.13460 493.13460 447.13868 409.15526 579.17138
7.58 7.62 14.02 − 7.10 14.02 5.53 13.99 9.56 5.41 6.81
crude extract with LC-MS analysis. From the UV chromatogram, several compounds were co-eluted at the same time but their identities were discriminated by corresponding accurate mass numbers (Fig. S2). The measured mass numbers and the corresponding calculated values were listed in Table 1.
Fig. 3. Effects of CoE and its sub-fractions on antioxidant response element (ARE)-driven luciferase activity in HepG2 cells. HepG2 cells stably transfected with the pGL4.37 plasmid were treated with 10–30 μg/mL total CoE or each fraction for 12 h. Relative AREdriven luciferase activity was determined in cell lysates. Data represent means ± SE (n = 6). *P < 0.05, **P < 0.01 vs. vehicle-treated control (Con).
3.2. CoE induces Nrf2 activation 3.5. Toralactone-9-O-β-D-gentiobioside from the EtOAc fraction of C. obtusifolia seeds activates Nrf2
Cellular antioxidant defense systems play an important role in the prevention of oxidative stress-induced cell death. Nrf2 is a key regulator of antioxidant and phase II detoxifying enzymes that are translocated into the nucleus upon activation and bind to the ARE sites of target genes, thereby enhancing their transcription (Ma, 2013). To evaluate the effects of CoE on Nrf2 activation, we measured ARE-driven luciferase activity in HepG2 cells stably transfected with the pGL4.37 plasmid containing four copies of ARE. Treatment with CoE (30 μg/mL) increased luciferase expression by approximately 5-fold (Fig. 3). We then compared effects of the n-hexane, EtOAc, BuOH, and H2O fractions of CoE on Nrf2 activation (Fig. 3). Nrf2/ARE-luciferase activity was strongest in cells treated with the EtOAc fraction (~ 8 and 14 folds at 10 and 30 μg/mL, respectively). Treatment with the n-hexane fraction (30 μg/mL) also increased Nrf2/ARE-luciferase activity by approximately 5.5-fold. An MTT assay revealed that no cytotoxicity was seen at the tested concentrations of EtOAc and n-hexane fractions of CoE as shown in Supplemental Fig. 1.
To identify bioactive principles from the EtOAc fraction responsible for Nrf2 activation, we assessed ARE-induced luciferase activity in HepG2 cells incubated with eight of the isolated compounds (Fig. 4). Compounds 9 and 10 were not tested owing to the negligible amounts that were available for bioactivity test. Compound 5 (toralactone-9-Oβ-D-gentiobioside) induced the greatest increase in Nrf2/ARE-luciferase activity (~ 4 fold at 20 μM). This effect was comparable to that of 5 μM sulforaphane, a naturally occurring isothiocyanate derived from cruciferous vegetables that is used to activate Nrf2/ARE signaling (Keum et al., 2006; Zhang, Talalay, Cho, & Posner, 1992). In the view of chemical structure, non-anthraquinone compounds such as xanthone (3) and naphtholactone (5) showed more potent activities than those of anthraquinones (compounds 2, 4, 7, and 8). Nuclear translocation of Nrf2 was confirmed by evaluating its nuclear levels (Fig. 5A). A time-course analysis revealed that nuclear Nrf2 levels in HepG2 cells increased within 3–6 h of treatment with compound 5 and were maintained for up to 24 h.
3.3. Identification of compounds The isolated compounds were identified through the direct comparison of their spectroscopic data with the data previously reported. The compounds were identified as rubrofusarin-6-β-gentiobioside (1, 9.7 mg) (Wong et al., 1989), chryso-obtusin-2-O-β-D-glucoside (2, 5.3 mg) (Jang et al., 2007), 8-hydroxy-3-methyl-6-methoxyxanthone1-O-β-D-gentiobioside (3, 3.0 mg) (Wang, Zuo, Ma, Wang, & Hu, 2008), physcion-8-O-β-D-gentiobioside (4, 20.0 mg) (Holzschuh, Kopp, & Kubelka, 1982), toralactone-9-O-β-D-gentiobioside (5, 32.7 mg) (Lee, Jang, Lee, Kim, & Kim, 2006), gluco-aurantioobtusin (6, 10.0 mg) (Yun-Choi et al., 1990), 6, 8-dihydroxy-1,7-dimethoxy-3methyl anthracene-9,10-dione 2-β-D-glucoside (7, 7.7 mg) (Shabana, Gonaid, Khaleel, & Yousif, 2001), physcion-8-O-β-D-glucopyranoside (8, 10.6 mg) (Lee et al., 2010), torachrysone gentiobioside (9, 1.7 mg) (Hatano et al., 1999), and chrysophanol 1-O-β-gentiobioside (10, 1.1 mg) (Ito et al., 2004) (Fig. 1). The purity of these compounds were assumed as above 95% at least evidenced in both of proton and carbon NMR spectra.
Fig. 4. Effects of compounds isolated from the ethyl acetate (EtOAc) fraction of Cassia obtusifolia seeds on antioxidant response element (ARE)-driven luciferase activity in HepG2 cells. Relative ARE-luciferase activity was measured in lysates of HepG2 cells stably transfected with the pGL4.37 plasmid and treated with each compound (20 μM) or sulforaphane (5 μM) for 12 h. Data represent means ± SE (n = 4). *P < 0.05, **P < 0.01 vs. vehicle-treated control (Con).
3.4. LC-MS study of CoE CoE sample solution (10 mg/mL) was prepared and injected on UPLC-PDA-MS system for the identification of active principles from the 4
Fig. 5. Nuclear factor erythroid-2-related factor 2 (Nrf2) activation and induction of antioxidant enzymes by compound 5 in HepG2 cells. A) Nuclear fractions prepared from HepG2 cells incubated with compound 5 (20 μM) or sulforaphane (5 μM) for the indicated times and probed with anti-Nrf2 antibodies. Signal intensity was assessed by scanning densitometry. Nuclear Nrf2 levels were normalized to those of lamin A/C. Data represent means ± SE (n = 4). B) HepG2 cells were treated with compound 5 (20 μM) for the indicated times, or exposed to sulforaphane (5 μM) for 12 h. NAD(P)H:quinone oxidoreductase 1 (NQO1) mRNA levels were determined by real-time polymerase chain reaction (PCR). Data represent means ± SE (n = 3 or 8 for compound 5 or sulforaphane, respectively). C) Lysates were obtained from cells exposed to compound 5 (20 μM) for the indicated times and probed with antibodies against NQO1, glutamate-cysteine ligase (GCL), and hemeoxygenase 1 (HO-1). *P < 0.05, **P < 0.01 vs. vehicle-treated control. Con, control; Sulfo, sulforaphane.
Y. Seo et al.
Food Research International xxx (xxxx) xxx–xxx
5
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
regulates diverse cellular antioxidant genes (Ma, 2013). Furthermore, toralactone-9-O-β-D-gentiobioside was identified as the primary component responsible for Nrf2 activation, as evidenced by increases in Nrf2/ARE-driven luciferase activity and nuclear Nrf2 levels, as well as upregulation of Nrf2 target genes. Finally, compound 5 prevented oxidative stress-induced cell damage in HepG2 cells, which may explain the hepatoprotective efficacy of C. obtusifolia seed extracts. Although non-anthraquinone structures showed more potent activities than those of anthraquinones compounds, it was not possible to speculate on the impact of xanthone or naphtholactone substitution patterns on the hepatoprotective activity of the compounds owing to a dearth of compounds available for comparison. 4. Conclusion In conclusion, the ethanol extract of Cassia obtusifolia seeds significantly protected HepG2 cells from t-BHP-induced cell death. Ten phenolic glycosides were identified from the most active EtOAc fraction by activity-guided isolation, and toralactone-9-O-β-D-gentiobioside was the most potent active principle in these seeds. The biochemical data on the active component of Cassia seeds provide insight into the effects of this medicinal plant as well as a basis for developing hepatoprotective agents as pharmaceuticals and/or nutraceuticals. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2017.04.032.
Fig. 6. Protection of HepG2 cells by compound 5 against t-butyl hydroperoxide (t-BHP)induced cell death. HepG2 cells were exposed to t-BHP (500 μM, 6 h) with or without pretreatment with compound 5 (10–20 μM) for 12 h. Cell viability was evaluated with the MTT assay. Data represent means ± SE (n = 6). **P < 0.01 vs. vehicle-treated control (Con); ##P < 0.01 vs. the t-BHP group.
3.6. Toralactone-9-O-β-D-gentiobioside induces antioxidant enzymes and protects against oxidative stress-induced cell death To determine whether nuclear translocation of Nrf2 stimulated by compound 5 induces upregulation of target genes, we measured mRNA levels of NQO1, a prototypical antioxidant enzyme, in HepG2 cells. Treatment with compound 5 increased NQO1 mRNA (Fig. 5B) as well as protein (Fig. 5C) levels. Moreover, protein levels of GCL and HO-1, which are also Nrf2-dependent antioxidant enzymes, were also upregulated in the presence of compound 5 (Fig. 5C). We next examined the ability of compound 5 to protect hepatocytes against cell death induced by t-BHP. Results of the MTT assay revealed that pretreatment with compound 5 (20 μM) attenuated t-BHP-induced damage in HepG2 cells (Fig. 6), an effect that may be attributed to induction of the Nrf2-dependent antioxidant system. t-BHP is frequently used to induce intracellular oxidative stress because it can be converted to free radical intermediates (Oh et al., 2012). Indeed, treatment with t-BHP enhances intracellular ROS production and results in cell apoptosis (Song, Kim, Choi, Oh, & Kim, 2016). Oxidative stress caused by excessive ROS can play an important role in the development and aggravation of liver diseases, such as alcoholic or drug-induced liver injury, nonalcoholic fatty liver diseases, and hepatocellular carcinoma (Bataille & Manautou, 2012; Tang, Jiang, Ponnusamy, & Diallo, 2014). Few study has been done on pharmacological efficacy of toralactone-9-O-β-D-gentiobioside except for the inhibitory activity on advanced glycation end products (AGEs) formation. Diabetes-related enzyme assays have described toralactone-9-O-β-D-gentiobioside as a potent inhibitory compound (IC50 10.7 μM, 1/45 fold as aminoguanidine, positive control) against formation of AGEs (Lee et al., 2006). Under hyperglycemic conditions, elevated blood glucose may induce formation of AGEs due to protein glycation, leading to diabetic complications caused by oxidative stress. As toralactone-9-O-β-D-gentiobioside also showed moderate inhibitory activity against protein tyrosine phosphatases 1B (PTP1B) and αglucosidase (Jung, Ali, & Choi, 2016), it was suggested as a potential candidate for the prevention of diabetic complications. The antioxidative capacity of toralactone-9-O-β-D-gentiobioside seems play a critical role in the pharmacological efficacies of both anti-diabetic and hepatoprotective activities. Previous studies have suggested that the protective effects of Cassia seed extracts in Parkinson's disease or liver injury models are related to their antioxidant properties (Ju et al., 2010; Xie et al., 2012). However, to the best of our knowledge this is the first study to demonstrate that C. obtusifolia seed extracts activate Nrf2, a transcription factor that
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author disclosure statement Young Pyo Jang and Young-Mi Kim supervised the conception and design of the study and helped in data interpretation and manuscript evaluation. Jae-Sook Song participated in the evaluation of hepatoprotective activity and gene expression data and data interpretation. Yongtaek Seo participated in the isolation of natural compounds, elucidation of compound structures, data interpretation, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. Acknowledgments We thank Editage (www.editage.co.kr) for English language editing and review of the manuscript. References Awasthi, V. K., Mahdi, F., Chander, R., Khanna, A. K., Saxena, J. K., Singh, R., ... Singh, R. K. (2015). Hypolipidemic activity of Cassia tora seeds in hyperlipidemic rats. Indian Journal of Clinical Biochemistry, 30, 78–83. Bataille, A. M., & Manautou, J. E. (2012). Nrf2: A potential target for new therapeutics in liver disease. Clinical Pharmacology & Therapeutics, 92, 340–348. Bhakta, T., Banerjee, S., Mandal, S. C., Maity, T. K., Saha, B. P., & Pal, M. (2001). Hepatoprotective activity of Cassia fistula leaf extract. Phytomedicine, 8, 220–224. Byun, E., Jeong, G.-S., An, R.-B., Li, B., Lee, D.-S., Ko, E.-K., ... Kim, Y.-C. (2007). Hepatoprotective compounds of Cassiae semen on tacrine-induced cytotoxicity in Hep G2 cells. Korean Journal of Pharmacognosy, 38, 400–402. Cho, I. J., Lee, C., & Ha, T. Y. (2007). Hypolipidemic effect of soluble fiber isolated from seeds of Cassia tora Linn. in rats fed a high-cholesterol diet. Journal of Agricultural and Food Chemistry, 55, 1592–1596. Choi, D. G., Kim, E. K., Yang, J. W., Song, J. S., & Kim, Y.-M. (2015). Nectandrin B, a lignan isolated from nutmeg, inhibits liver X receptor-alpha-induced hepatic lipogenesis through AMP-activated protein kinase activation. Pharmazie, 70, 733–739. Choi, H. Y., Lee, J.-H., Jegal, K. H., Cho, I. J., Kim, Y. W., & Kim, S. C. (2016). Oxyresveratrol abrogates oxidative stress by activating ERK-Nrf2 pathway in the liver. Chemico-Biological Interactions, 245, 110–121. Cichoż-Lach, H., & Michalak, A. (2014). Oxidative stress as a crucial factor in liver diseases. World Journal of Gastroenterology, 20, 8082–8091.
6
Food Research International xxx (xxxx) xxx–xxx
Y. Seo et al.
Physcion-8-O-β-D-glucopyranoside enhances the commitment of mouse mesenchymal progenitors into osteoblasts and their differentiation: Possible involvement of signaling pathways to activate BMP gene expression. Journal of Cellular Biochemistry, 109(6), 1148–1157. Ma, Q. (2013). Role of nrf2 in oxidative stress and toxicity. Annual Review of Pharmacology and Toxicology, 53, 401–426. Oh, J. M., Jung, Y. S., Jeon, B. S., Yoon, B. I., Lee, K. S., Kim, B. H., ... Kim, S. K. (2012). Evaluation of hepatotoxicity and oxidative stress in rats treated with tert-butyl hydroperoxide. Food and Chemical Toxicology, 50, 1215–1221. Patil, U. K., Saraf, S., & Dixit, V. K. (2004). Hypolipidemic activity of seeds of Cassia tora Linn. Journal of Ethnopharmacology, 90, 249–252. Pradeep, K., Mohan, C. V. R., Gobianand, K., & Karthikeyan, S. (2007). Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats. Chemico-Biological Interactions, 167, 12–18. Shabana, M. M., Gonaid, M. H., Khaleel, A. E., & Yousif, M. F. (2001). Glycosides of Cassia tora L. Cultivated in Egypt. Journal of Biomedical Science, 7, 136–144. Song, J.-S., Kim, E.-K., Choi, Y.-W., Oh, W. K., & Kim, Y.-M. (2016). Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK3beta. Toxicology and Applied Pharmacology, 307, 138–149. Tang, W., Jiang, Y.-F., Ponnusamy, M., & Diallo, M. (2014). Role of Nrf2 in chronic liver disease. World Journal of Gastroenterology, 20, 13079–13087. Tsuda, Y. (2004). Isolation of natural products. University of Karachi. Tzeng, T.-F., Lu, H.-J., Liou, S.-S., Chang, C. J., & Liu, I.-M. (2013). Cassia tora (Leguminosae) seed extract alleviates high-fat diet-induced nonalcoholic fatty liver. Food and Chemical Toxicology, 51, 194–201. Wang, L. L., Zuo, J. P., Ma, L., Wang, X. C., & Hu, L. H. (2008). Two new xanthone glycosides from Ventilago leiocarpa Benth. Natural Product Communications, 3(5), 795–798. Wong, S.-M., Wong, M. M., Seligmann, O., & Wagner, H. (1989). New antihepatotoxic naphtho-pyrone glycosides from the seeds of Cassia tora. Planta Medica, 55, 276–280. Xie, Q., Guo, F.-F., & Zhou, W. (2012). Protective effects of cassia seed ethanol extract against carbon tetrachloride-induced liver injury in mice. Acta Biochimica Polonica, 59, 265–270. Yang, Y.-C., Lim, M.-Y., & Lee, H.-S. (2003). Emodin isolated from Cassia obtusifolia (Leguminosae) seed shows larvicidal activity against three mosquito species. Journal of Agricultural and Food Chemistry, 51, 7629–7631. Yanjun, H., Yuli, S., & Yuqing, Z. (2001). The advancement of the studies on the seeds of Cassia obtusifolia. Chinese Traditional and Herbal Drugs, 32, 2. Yun-Choi, H. S., Kim, J. H., & Takido, M. (1990). Potential inhibitors of platelet aggregation from plant sources, V. Anthraquinones from seeds of Cassia obtusifolia and related compounds. Journal of Natural Products, 53, 630–633. Zhang, Y., Talalay, P., Cho, C.-G., & Posner, G. H. (1992). A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation of structure. Proceedings of the National Academy of Sciences of the United States of America, 89, 2399–2403.
Hatano, T., Uebayashi, H., Ito, H., Shiota, S., Tsuchiya, T., & Yoshida, T. (1999). Phenolic constituents of Cassia seeds and antibacterial effect of some naphthalenes and anthraquinones on methicillin-resistant Staphylococcus aureus. Chemical & Pharmaceutical Bulletin, 47, 1121–1127. Holzschuh, L., Kopp, B., & Kubelka, W. (1982). Physcion-8-O-beta-D-gentiobioside, a new anthraquinone glycoside from rhubarb roots. Planta Medica, 46(3), 159–161. Ito, H., Nishida, Y., Yamazaki, M., Nakahara, K., Michalska-Hartwich, M., Furmanowa, M., Leistner, E., & Yoshida, T. (2004). Chrysophanol glycosides from callus cultures of monocotyledonous Kniphofia spp. (Asphodelaceae). Chemical and Pharmaceutical Bulletin, 52(10), 1262–1264. Jafri, M. A., Subhani, M. J., Javed, K., & Singh, S. (1999). Hepatoprotective activity of leaves of Cassia occidentalis against paracetamol and ethyl alcohol intoxication in rats. Journal of Ethnopharmacology, 66, 355–361. Jang, D. S., Lee, G. Y., Kim, Y. S., Lee, Y. M., Kim, C.-S., Yoo, J. L., & Kim, J. S. (2007). Anthraquinones from the seeds of Cassia tora with inhibitory activity on protein glycation and aldose reductase. Biological & Pharmaceutical Bulletin, 30, 2207–2210. Ju, M. S., Kim, H. G., Choi, J. G., Ryu, J. H., Hur, J., Kim, Y. J., & Oh, M. S. (2010). Cassiae semen, a seed of Cassia obtusifolia, has neuroprotective effects in Parkinson's disease models. Food and Chemical Toxicology, 48, 2037–2044. Jung, H. A., Ali, Y., & Choi, J. S. (2016). Promising inhibitory effects of anthraquinones, naphthopyrone, and naphthalene glycosides, from Cassia obtusifolia on alphaglucosidase and human protein tyrosine phosphatases 1B. Molecules, 22(1). Kang, K. W., Cho, I. J., Lee, C. H., & Kim, S. G. (2003). Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta activation in the induction of glutathione S-transferase by oltipraz. Journal of the National Cancer Institute, 95, 53–66. Keum, Y.-S., Yu, S., Chang, P. P.-J., Yuan, X., Kim, J.-H., Xu, C., ... Kong, A.-N. T. (2006). Mechanism of action of sulforaphane: Inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response elementmediated heme oxygenase-1 in human hepatoma HepG2 cells. Cancer Research, 66, 8804–8813. Kim, D. H., Yoon, B. H., Kim, Y.-W., Lee, S., Shin, B. Y., Jung, J. W., ... Ryu, J. H. (2007). The seed extract of Cassia obtusifolia ameliorates learning and memory impairments induced by scopolamine or transient cerebral hypoperfusion in mice. Journal of Pharmacological Sciences, 105, 82–93. Kim, Y. M., Lim, S.-C., Han, C. Y., Kay, H. Y., Cho, I. J., Ki, S. H., ... Kim, S. G. (2011). G(alpha)12/13 induction of CYR61 in association with arteriosclerotic intimal hyperplasia: Effect of sphingosine-1-phosphate. Arteriosclerosis, Thrombosis, and Vascular Biology, 31, 861–869. Kitanaka, S., & Takido, M. (1986). Studies on the constituents in the roots of Cassia obtusifolia L. and the antimicrobial activities of constituents of the roots and the seeds. Yakugaku Zasshi, 106, 302–306. Lee, G. Y., Jang, D. S., Lee, Y. M., Kim, J. M., & Kim, J. S. (2006). Naphthopyrone glucosides from the seeds of Cassia tora with inhibitory activity on advanced glycation end products (AGEs) formation. Archives of Pharmacal Research, 29, 587–590. Lee, S.-U., Choi, Y. H., Kim, Y. S., Park, S.-J., Kwak, H. B., Min, Y. K., ... Kim, S. H. (2010).
7