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Cite This: J. Nat. Prod. 2018, 81, 625−629
Capsicodendrin from Cinnamosma fragrans Exhibits Antiproliferative and Cytotoxic Activity in Human Leukemia Cells: Modulation by Glutathione Soumendrakrishna Karmahapatra,† Corey Kientz,† Shruthi Shetty,† Jack C. Yalowich,*,† and L. Harinantenaina Rakotondraibe*,‡ †
Division of Pharmacology and ‡Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *
ABSTRACT: Capsicodendrin (CPCD, 1), an epimeric mixture of a dimeric drimane-type sesquiterpene, is one of the major compounds present in the three endemic species of Madagascan traditional chemopreventive plants: Cinnamosma species (C. f ragrans, C. macrocarpa, and C. madagascariensis). Despite the popular use of Cinnamosma in Madagascan traditional medicine and the reported antiproliferative properties of CPCD, elucidation of its mechanism(s) of action is still to be accomplished. In the present study, CPCD at low micromolar concentrations was cytotoxic and induced apoptosis in human myeloid leukemia cells in a time- and concentration-dependent manner. The activity of CPCD in HL-60 and K562 cells was modulated by glutathione (GSH), since depletion of this intracellular thiol-based antioxidant with buthionine sulfoximine resulted in significantly (p < 0.05) greater potency in antiproliferation assays. GSH depletion also significantly potentiated the cytotoxic activity in CPCD-treated human HL-60 cells. Single-cell gel electrophoresis (Comet) assays revealed that GSH depletion in HL-60 cells enhanced the formation of DNA strand breaks in the presence of CPCD. Although CPCD does not contain an obvious Michael acceptor in its structure, 1H NMR analyses indicated that cinnamodial (2), a monomer of CPCD, was formed within a few hours when dissolved in DMSO-d6 and interacts with GSH to form a covalent bond via Michael addition at the C-7 carbon. Together the results strongly suggest that 2 is responsible for the DNA-damaging, pro-apoptotic, and cytotoxic effects of CPCD and that depletion of GSH enhances overall activity by diminishing covalent interaction between GSH and this 2-alkenal decomposition product of CPCD.
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atural products as a group are major contributors to the anticancer drug armamentarium. Nearly one-half (49%) of Food and Drug Administration (FDA) approved anticancer small molecules from the 1940s to the end of 2014 are natural products or are derived therefrom.1 Most natural products from plant, microorganism, or marine organism extracts are identified via phenotypic biological screening or are acquired from bioassay-directed isolation of bioactive compounds of plants that have been used in traditional medicines. Our systematic search for bioactive compounds from Madagascan endemic and traditional chemopreventive plants led to the isolation and identification of capsicodendrin (CPCD, 1) from Cinnamosma f ragrans as an antiproliferative compound against murine leukemia (L1210/0), human T-lymphocyte (Molt4/C8, CEM/0), and MCF-7 breast cancer cells.2−4 CPCD was also isolated from the other two plant species of the same genus Cinnamosma, C. macrocarpa and C. madagascariensis, that have been used in traditional medicine for the same purposes as C. f ragrans.2,5 Despite the observed antiproliferative activity, the mechanisms of action of CPCD have not been elucidated. Recent results focused on the antiangiogenic activity of CPCD demonstrated that it is a potent inhibitor of the VEGFR2/AKT pathway at nontoxic nanomolar concentrations and an inducer of autophagy-related angiostatic effects.6 © 2018 American Chemical Society and American Society of Pharmacognosy
To provide additional mechanistic insight, we report herein the cytotoxic activity of CPCD against human leukemia cells, associated with DNA damage, and provide evidence of the role of glutathione (GSH) in forming an adduct with cinnamodial, the monomeric decomposition product of CPCD.
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RESULTS AND DISCUSSION This work defines novel cytotoxic and apoptotic properties of 1 and suggests a biochemical mechanism modulating its activity. Recently, 1 has been reported to demonstrate antiangiogenic Special Issue: Special Issue in Honor of Susan Horwitz Received: October 25, 2017 Published: February 6, 2018 625
DOI: 10.1021/acs.jnatprod.7b00887 J. Nat. Prod. 2018, 81, 625−629
Journal of Natural Products
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Results demonstrated an interaction between these molecules with an apparent dissociation constant of 7.2 μM (Figure 4A). Michael acceptor containing compounds are known to bind covalently to GSH. Since CPCD does not contain an evident Michael acceptor site, the working hypothesis was that CPCD, in DMSO, was converted to its unsaturated dialdehyde containing monomer, cinnamodial (2), therefore allowing for covalent interaction with GSH. 1H NMR experiments of CPCD were undertaken in DMSO-d6 to identify potential chemical adduction between GSH and CPCD and/or its decomposition product(s). 1H NMR spectra of CPCD after 5 min (Figure 4Bi) and 1 h (Figure 4Bii) were recorded. The 1H NMR spectrum of the sample 1 h after preparation showed the presence of signals attributable to cinnamodial, a drimane sesquiterpene compound bearing an unsaturated aldehyde as a Michael acceptor site. The proton signals of cinnamodial were identified by comparison with a standard sample, previously isolated from Cinnamosma species.2−4 The signals of the olefinic proton at C7 (d 7.11, d, 4.1 Hz) and the two aldehydes at 9.48 and 9.69 ppm (each singlet) of cinnamodial disappeared when excess GSH was added to the 1 h solution of CPCD in DMSO-d6 and incubated a further 1 h (Figure 4Biii; see asterisks). These observations strongly support the interpretation that GSH covalently bound with cinnamodial by Michael addition at C-7 of the alkenal as well as nucleophilic attack at the C-11 aldehyde. Next, DNA damage studies (Comet assays) were performed in HL-60 cells, indicating that incubation with CPCD resulted in a concentration-dependent (0.5−5 μM) increase in DNA strand breaks (Figure 5A). Pretreatment with BSO to deplete GSH enhanced DNA damage in cells treated with CPCD with statistically significant increases at 2 and 5 μM (Figure 5A). These experiments were performed with CPCD prepared in DMSO several hours prior to experimentation. Since Figure 4B results showed time-dependent production of monomeric cinnamodial (containing a Michael acceptor site), additional DNA damage experiments were performed with CPCD incubated with HL-60 cells either immediately after preparation in DMSO or after 1.5 h in DMSO (Figure 5B). Results clearly indicate greater DNA damage after longer time in DMSO, corresponding with production of cinnamodial (Figure 4Bii), suggesting that DNA-damaging effects of CPCD are due, in part, to the 2-alkenal-containing cinnamodial. This finding is consistent with the previously published data on DNA damage in V79 lung fibroblast and Caco-2 colorectal carcinoma cells as a result of 2-alkenal exposure.7 Importantly, after incubation of HL-60 cells using solutions of CPCD (1−5 μM) known to contain cinnamodial, there was no evidence of induced reactive oxygen species (ROS), as assessed by oxidation of dichlorofluorescein (Figure 6). These results strongly suggest that the biological effects of CPCD are due, in part, to the 2-alkenalcontaining cinnamodial by mechanisms unrelated to cellular depletion of antioxidant GSH that can increase cytotoxic ROS. Rather, depletion of GSH resulted in greater potency in growth inhibition assays, enhanced DNA damage, and elevated apoptosis and cell death after treatment with CPCD, strongly implicating cinnamodial as an active component of CPCD. Moreover, it is important to note that, in addition to the Michael acceptor found in cinnamodial (C-7, C-8, C-12), there is an additional reactive aldehyde (C-11), which is not conjugated. The presence of this nonconjugated aldehyde as well as the 2-alkenal moiety in cinnamodial may be important structural features that contribute to the cytotoxic end points
and cytostatic effects at nanomolar concentrations without inducing cytotoxic effects.6 Here, the cytotoxicity of CPCD was examined via a trypan blue dye exclusion assay at 48 and 72 h, revealing time- and concentration-dependent cell killing in human acute myeloid leukemia HL-60 cells albeit at concentrations above those at which 1 produces its antiangiogenic and cytostatic effects (Figure 1A).6 CPCDinduced cell death was via apoptosis as quantified by examining nuclear morphology (Figure 1B).
Figure 1. Time- and concentration-dependent cytotoxicity and apoptosis of CPCD in HL-60 cells. HL-60 cells were incubated for 48 and 72 h with the indicated concentrations of CPCD. (A) Cytotoxicity (% trypan blue positive cells) was quantitated after counting on a hemocytometer. Results shown are the mean ± SEM from 6 to 9 experiments run on separate days. (B) Cells were stained with Hoechst 33342 and analyzed for apoptotic morphology using a Nikon Eclipse Ti fluorescent microscope. For each experimental condition, 100−200 cells were evaluated and expressed as % apoptotic cells. Results shown are the mean ± SEM from 6 to 8 experiments run on separate days.
To examine the potential role of glutathione, a major intracellular thiol-based antioxidant, on CPCD activity, HL-60 cells were pretreated with the inhibitor of GSH synthesis, buthionine sulfoximine (BSO, 50 μM, 40 h). Depletion of GSH to ∼30% of control levels (not shown) resulted in enhanced CPCD activity (Figure 2). CPCD (0.2 and 0.5 μM)-induced growth inhibition (Figure 2A), cell death (Figure 2B), and apoptosis (Figure 2C) were significantly greater in BSO-treated HL-60 cells compared to controls (p < 0.05). In addition, upon GSH depletion, lower concentrations of CPCD were required to yield 50% inhibition (i.e., a shift to the left of the concentration−response curves indicating greater potency) in both HL-60 and K562 cells (Figure 3A,B). CPCD IC50 values were 0.20 ± 0.03 and 0.09 ± 0.02 μM in the absence and presence of BSO, respectively (p < 0.05) (Figure 3C). Similar experiments in K562 cells yielded IC50 values of 0.48 ± 0.09 and 0.21 ± 0.04 μM in the absence and presence of BSO, respectively (p < 0.05) (Figure 3C). Based on the potential for GSH interaction with CPCD and its biological consequences indicated in Figure 2 and 3, binding assays between CPCD and GSH in vitro were performed. 626
DOI: 10.1021/acs.jnatprod.7b00887 J. Nat. Prod. 2018, 81, 625−629
Journal of Natural Products
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Figure 2. Effect of GSH depletion on CPCD (0.2, 0.5 μM)-induced growth inhibition, cytotoxicity, and apoptosis in HL-60 cells. HL-60 cells were incubated in the presence or absence of BSO (50 μM) for 40 h. Cells were subsequently incubated for 48 h with the indicated concentrations of CPCD for (A) growth inhibition, (B) trypan blue positivity, and (C) apoptotic morphology as described in the Experimental Section. For each experimental condition, 100−200 cells were evaluated for apoptotic morphology. Results shown are the mean ± SEM from 3 experiments runs on separate days; *p < 0.05 comparing HL-60 cells ± BSO.
Figure 3. Effect of GSH depletion on CPCD-induced growth inhibition in HL-60 and K562 cells. Cells were treated with BSO for a period of 40 h to decrease the GSH levels. (A) HL-60 cells (treated/untreated with BSO) were incubated for 72 h with various concentrations of CPCD. Cell growth was assessed by quantitation on the Coulter counter. Growth inhibition curves were plotted using SigmaPlot 13. (B) K562 cells (treated/untreated with BSO) were incubated with CPCD, and the growth inhibition was assessed after 48 h. (C) IC50 values in BSO-treated or untreated cell lines (HL-60 and K562 cells) were calculated. IC50 values were the mean ± SEM from 3 to 4 experiments runs on separate days; *p < 0.05 comparing HL-60 or K562 cells ± BSO.
mechanisms of in vitro cytotoxicity and potential in vivo anticancer activities of these natural products.
observed. Since CPCD exhibited activity in the sub-micromolar range (Figures 1−3), and its conversion to cinnamodial led to induction of DNA damage and apoptosis in the absence of increased ROS (Figures 5 and 6), we conclude that CPCD is a prodrug that has potential for development as an anticancer agent. Additional studies are under way to further characterize “activation” of CPCD to cinnamodial and to further explore
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EXPERIMENTAL SECTION
General Experimental Procedures. Glutathione (γ-L-glutamyl-Lcysteinyl-glycine) and L-buthionine-sulfoximine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Capsicodendrin (1) was isolated from C. f ragrans.2−4 Human acute leukemia (HL-60) and chronic myelogenous leukemia (K562) cell lines were obtained from the 627
DOI: 10.1021/acs.jnatprod.7b00887 J. Nat. Prod. 2018, 81, 625−629
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Figure 4. CPCD binding to GSH and 1H NMR of CPCD in the absence and presence of GSH. (A) GSH (5 μM) was combined with various concentrations of CPCD in 1× PBS buffer and incubated at 37 °C for 8 h, after which ThioGlo IV (10 μM) was added to diluted aliquots. Unreacted GSH as a result of CPCD−GSH interaction was quantified subsequent to measurement of fluorescence resulting from GSH−ThioGlo IV binding (excitation 400 nm; emission 465 nm). A ligand binding plot was performed using SigmaPlot 13. (B) 1H NMR of CPCD in DMSO-d6 was measured at different times in the presence and absence of GSH. Chemical shifts are given relative to DMSO-d6 (δ 2.50) as an internal standard. The 1H NMR of 2 mg of CPCD dissolved in 600 μL of DMSO-d6 was taken at 5 min after preparation (Bi). The presence of two aldehyde signals (δ 9.48 and 9.85), indicating the conversion of CPCD to cinnamodial (2), was observed 1 h after preparation (Bii). Asterisks denote that these two signals disappeared 1 h after addition of GSH (10 mg) (Biii). In Vitro GSH Measurement Assay. In vitro binding of CPCD to GSH was performed using thiol fluorescent probe IV (ThioGlo IV; Calbiochem) in a competitive binding assay as described.8 GSH (5 μM) was combined with various concentrations of CPCD (0.5, 1, 2, 5, 10, 20, 50, 100 μM) in 1× PBS buffer for 8 h at 37 °C. After incubation, 10 μM ThioGlo IV was added to diluted aliquots in a 96well plate, and inhibition of GSH-ThioGlo IV binding was quantified by fluorescence [excitation 400 nm; emission 465 nm). Growth Inhibition Assay. The growth inhibitory properties of CPCD were evaluated over a 72 h incubation period as described.9 HL-60 (in complete RPMI medium) and K562 (in complete DMEM medium) cells were incubated in the absence or presence of BSO (50 μM) for 40 h. Cells were then seeded at 100 000 cells/mL and incubated with CPCD (0.1, 0.2, 0.5 μM) or DMSO (1%) in a 24-well plate for 72 h at 37 °C in a cell culture incubator (5% CO2). Cells were counted on a model ZBF Coulter counter (Beckman Coulter, Danvers, MA, USA). SigmaPlot 13 was used for the preparation of growth curves and for calculation of IC50 values. Comet Assay. For the detection of DNA damage, single-cell gel electrophoresis assays (Comet assays) were performed using a Comet assay kit (Trevigen, Gaithersburg, MD, USA). Briefly, HL-60 cells (100 000 cells/mL) were incubated in the absence or presence of BSO (50 μM) for 40 h. Cells were washed and resuspended in buffer A (25 mM HEPES, 10 mM glucose, 1 mM MgCl2, 5 mM KCl, 130 mM NaCl, 5 mM NaH2PO4, pH 7.4), then treated with CPCD (0.5, 1, 2, 5 μM) or DMSO (0.2%) for 1 h at 37 °C. For indicated experiments, CPCD was dissolved in DMSO and used immediately or after 1.5 h in DMSO. After incubation, cells were washed with ice cold buffer A and resuspended at 0.28 × 106 cells/mL. The cells were then embedded in
American Type Culture Collection (Manassas, VA, USA). The cell lines were cultured in Roswell Park Memorial Institute (RPMI-1640) supplemented with 15% fetal bovine serum (FBS) (HL-60 cells) or Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (K562 cells), at 37 °C and 5% CO2. Hoechst 33342 stain was purchased from Sigma-Aldrich. SYBR Gold for Comet assays was purchased from Life Technologies (Calrlsbad, CA, USA). Other chemicals (DMSO, HEPES, sodium chloride, etc.) were purchased from Sigma-Aldrich. Trypan Blue Exclusion Assay. Exponentially growing HL-60 cells were seeded at 100 000 cell/mL and treated with DMSO solvent (0.1%) or CPCD (0.1, 0.2, 0.5 μM) for 48 and 72 h in complete RPMI medium. Cell suspensions were mixed 1:1 with trypan blue followed by hemocytometer assessment for live (trypan blue excluding) and dead (trypan blue positive) cells. Detection of Apoptosis by Hoechst Staining. HL-60 cells were seeded at 100 000 cells per mL and treated with CPCD (0.1, 0.2, 0.5 μM) or DMSO (0.1% final by volume) for 48 and 72 h in complete RPMI medium. Cells ((1−1.5) × 106) were pelleted in 1.5 mL Eppendorf tubes, the media was aspirated, and the pellets were washed with 1× PBS. Final cell pellets were then mixed with 100 μL (0.013 mg/mL) of Hoechst 33342 stain and incubated in the dark at room temperature for 20 min. Images of cells on glass slides (with coverslips) were taken using a Nikon Eclipse Ti fluorescent microscope with a 20× lens and an appropriate UV emission filter allowing blue-cyan fluorescence (at ∼460 nM). For each experimental condition, 100−200 cells were evaluated for nuclear apoptotic morphology. 628
DOI: 10.1021/acs.jnatprod.7b00887 J. Nat. Prod. 2018, 81, 625−629
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DA (2 μM) in buffer A for 30 min at 37 °C. After treatment, cells were washed and resuspended in buffer A to a final concentration of 1 × 106 cells/mL and aliquoted to 1.5 mL Eppendorf tubes (1 mL per tube for each condition). Cells were then treated with DMSO vehicle, CPCD (1, 2, 5 μM), or H2O2 (25 μM). Immediately, the cells from each condition were aliquoted (0.25 mL/well) in triplicate to a 96-well plate for recordings (emission 487 nm; excitation 515 nm) on a fluorescence plate reader (BioTek, Synergy H1). Statistical Analysis. The results are presented as the mean ± SEM, and statistical analysis was performed by Student’s t test or rank sum test. The statistical significance of differences was set at p < 0.05.
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Figure 5. Effect of GSH depletion on CPCD-induced DNA damage. Single-cell gel electrophoresis assays (Comet assays) were performed in HL-60 cells incubated in the absence or presence of BSO (50 μM) for 40 h. (A) Cells were incubated for 1 h with the indicated concentrations of various agents dissolved in DMSO. Cells were then immobilized in low-melt agarose on glass slides followed by lysis and incubation at pH 13 to allow for DNA denaturation and unwinding. Electrophoresis for 30 min at 21 V/cm was followed by staining with SYBR gold and Comet image capture on a Nikon Eclipse Ti fluorescent microscope. DNA damage was quantified as Olive tail moments using a free software program (Open Comet) from at least 100 cells for each condition. Results shown are the mean ± SEM from 5 experiments runs on separate days; p < 0.05 comparing HL-60 cells ± BSO. (B) Cells were incubated for 1 h with 5 μM CPCD, immediately after preparation (dissolved in DMSO) or after 1.5 h dissolved in DMSO. The Comet assay was performed as described in Figure 5A. Results shown are the mean ± SEM from 5 experiments runs on separate days. *p < 0.05 comparing CPCD in solution (DMSO) for 0 or 1.5 h.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00887. 1 H NMR spectra of compounds 1, 2, glutathione, and reduced GSH (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +1-614-688-5980. Fax: +1-614-292-2435. E-mail:
[email protected]. *Tel: +1-614-292-4733. Fax: +1-614-292-2435. E-mail:
[email protected]. ORCID
L. Harinantenaina Rakotondraibe: 0000-0003-2166-4992 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank The Ohio State University, College of Pharmacy, instrumentation facility for the acquisition of the NMR spectra. DEDICATION Dedicated to Dr. Susan Band Horwitz, of Albert Einstein College of Medicine, Bronx, NY, for her pioneering work on bioactive natural products.
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Figure 6. Effect of CPCD on the generation of reactive oxygen species in HL-60 cells. The increase in the DCF fluorescence induced by DMSO vehicle (control), CPCD (1−5 μM), or H2O2 (25 μM) was measured every 2 min up to 1 h and calculated as the change in fluorescence units per min. For H2O2, rates were calculated from fluorescence increases up to 6 min. Results shown are the mean ± SEM from 3 to 5 experiments run on separate days.
REFERENCES
(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661. (2) He, D.; Slebodnick, C.; Rakotondraibe, L. H. Bioorg. Med. Chem. Lett. 2017, 27, 1754−1759. (3) Harinantenaina, L.; Takaoka, S. J. Nat. Prod. 2006, 69, 1193− 1197. (4) Harinantenaina, L.; Asakawa, Y.; De Clercq, E. J. Nat. Prod. 2007, 70, 277−282. (5) Harinantenaina, L.; Matsunami, K.; Otsuka, H.; Kawahata, M.; Yamaguchi, K.; Asakawa, Y. J. Nat. Prod. 2008, 71, 123−126. (6) Pan, C. C.; Shah, N.; Kumar, S.; Wheeler, S. E.; Cinti, J.; Hoyt, D. G.; Lee, N. Y.; Beattie, C. E.; An, M.; Mythreye, K.; Rakotondraibe, L. H.; Lee, N. Y.; Lee, N. Y. Oncotarget 2017, 8, 12675−12685. (7) Glaab, V.; Collins, A. R.; Eisenbrand, G.; Janzowski, C. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2001, 497, 185−197. (8) Wu, X.; Liang, H.; O’Hara, K. A.; Yalowich, J. C.; Hasinoff, B. B. Chem. Res. Toxicol. 2008, 21, 483−493. (9) Hasinoff, B. B.; Wu, X.; Patel, D.; Kanagasabai, R.; Karmahapatra, S.; Yalowich, J. C. J. Pharmacol. Exp. Ther. 2016, 356, 397−409. (10) Vlasova, I. I.; Feng, W.-H.; Goff, J. P.; Giorgianni, A.; Do, D.; Gollin, S. M.; Lewis, D. W.; Kagan, V. E.; Yalowich, J. C. Mol. Pharmacol. 2011, 79, 479−487. (11) Olive, P. L. Methods Mol. Biol. (Totowa NJ, U. S.) 2002, 203, 179−194.
low-melt agarose, immobilized on specially treated slides (Trevigen), and lysed, followed by alkaline electrophoresis. Cells were then stained with SYBR Gold.10 Migrating DNA fragments (Comets) were visualized, and images captured on a Nikon Eclipse Ti fluorescent microscope. Olive tail moments11 of at least 100 cells per sample were analyzed using the open-source software tool OpenComet (www. cometbio.org) and ImageJ. 1 H NMR Spectroscopy. The 1H NMR spectra of CPCD and the mixture of CPCD and GSH were recorded using a Bruker AVII 400 MHz NMR. CPCD (2 mg) was dissolved in 600 μL of DMSO-d6 and poured into a 5 mm Shigemi NMR tube before 1H NMR measurements (5 min and 1 h). After 1 h, GSH (10 mg) was added to the NMR tube containing CPCD, and 1H NMR spectra were recorded (after an additional 1 h). ROS Assay. Dichlorofluorescin diacetate (DCFH-DA) dye was used for the detection of ROS induced by CPCD or H2O2 in HL-60 cells. Briefly, HL-60 cells (1 × 106 cells/mL) were loaded with DCFH629
DOI: 10.1021/acs.jnatprod.7b00887 J. Nat. Prod. 2018, 81, 625−629
