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Abstract

Mallotus japonicus has been evaluated for the treatment of dermatitis, inflammatory conditions, and cancer. Diterpenes, one of the major constituents of M. japonicus, possess various pharmacological effects. In this study, 2 known diterpenes, anomaluone (6) and 16-epiabbeokutone (7), along with other known compounds, 2-hydroxy ferulic acid (1), bergenin (2), gallocatechin (3), catechin (4), erythro,erythro-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl) ethoxy]-3,5-dimethoxyphenyl]-1,2,3- propanetriol (5), and gallincin (8), were isolated from M. japonicus. Cytotoxicity assays in blood cancer cell models demonstrated that M. japonicus compounds possess potent antiproliferative activity. In addition, treatment with compound 6 increased the number of apoptotic cells, led to cell-cycle arrest at the subG0/G1 phase, and decreased the number of cells in the S and G2/M phases. Compound 6 also displayed potent mitochondrial depolarization effects in Jurkat cells. These findings revealed that the cytotoxic effects of 6 were mediated by intracellular signaling, possibly through a mechanism involving upregulation of mitochondrial reactive oxygen species. Thus, compound 6 could be a potential multi-target therapeutic agent for leukemia.

Mallotus japonicus, which belongs to the Euphorbiaceae family, has been used for a long time as a traditional medicine for the treatment of various diseases, including gastropathy, scald, and cancer.¹ Several species of the genus Mallotus are rich in biologically active compounds, such as phloroglucinols, tannins, terpenoids, coumarins, benzopyrans, and chalcones.^2

-8 Phytochemical studies of this genus have shown that kauranes, which are produced through the mevalonate pathway and consist of a perhydrophenanthrene unit fused with a cyclopentane unit,⁹ are an important group of tetracyclic diterpenes.¹⁰ Ent-kaurane diterpene is one of 2 enantiomeric series that resulted from triterpene cyclization. Kaurane and ent-kaurane diterpenes differ in their inverted configurations of carbons C-5, C-9, and C-10.⁹ They possess many different biological activities, including plant growth regulatory, antimicrobial, antiviral, anti-inflammatory, and antitumoral activities.^11,12 The broad spectrum of biological activities of kaurane diterpenes have motivated countless studies of structural modifications of the kaurane skeleton, aiming to obtain new bioactive substances, especially those with antitumor activities.^13,14 Evasion of apoptosis is one of the most common cellular malfunctions that can facilitate the development of various cancers. Cancer cells easily evade apoptosis because of defects in the programmed cell death signaling pathway. Therefore, induction of apoptosis in cancer cells is an effective cancer prevention strategy.^15

-18 Antitumoral activities have been reported for some ent-kaurane diterpenes; oridonin induced apoptosis in Jurkat cells via activation of caspase 3,¹⁹ glaucocalyxin A induced apoptosis in HL-60 cells through mitochondrial membrane potential loss,²⁰ and ent-11α-hydroxy-16-kaurene-15-one induced apoptosis in HL-60 cells through caspase 8.²¹ Ent-Kaurane diterpenes with strong apoptosis-inducing activity are expected to have potential as anti-cancer drugs.

In this study, 2 ent-kaurane diterpenes, anomaluone (6) and 16-epiabbeokutone (7), were isolated from the roots of M. japonicus for the first time, along with other known compounds (1 - 5 and 8). Although ent-kaurane diterpenes are effective antitumor agents, the cytotoxic activity of anomaluone (6) has not been established. Here, we showed for the first time that anomaluone (6) exhibits significant cytotoxic activity against human leukemia (Jurkat) cells. To clarify some of its antileukemic mechanisms, we investigated its antiproliferation and apoptosis-inducing effects on Jurkat cells in vitro and determined that anomaluone (6) induced reactive oxygen species (ROS) production along with an increase in the sub-G1 cell population, and reduced the mitochondrial membrane potential.

The EtOAc fraction from the roots of M. japonicus was subjected to repeated column chromatography (CC), RP C18-MPLC (medium-pressure liquid chromatography), and high-performance liquid chromatography (HPLC) to yield 7n known compounds. The structures of the isolated compounds were elucidated using 1-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy (¹H, ¹³C, heteronuclear single quantum coherence, heteronuclear multiple bond correlation, correlation spectroscopy, and rotating frame overhauser effect spectroscopy [ROESY]). Comparison of the obtained spectroscopic data with those reported in the literature showed that compounds 1 to 8 were 2-hydroxy ferulic acid (1),²² bergenin (2),²³ gallocatechin (3),^24,25 catechin (4),^24,26 erythro,erythro -1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1- (hydroxymethyl) ethoxy] -3,5-dimethoxyphenyl]-1,2,3-propanetriol (5),²⁷ anomaluone (6),^4,10 16-epiabbeokutone (7),^28,29 and gallincin (8) (Figure 1(a)).³⁰

Figure 1

(a) Strnctures of compounds 1 to 8 isolated from Mallotus japonicus roots. (b) Key COSY, HMBC, and ROESY correlations for compounds 6 and 7. COSY, correlation spectroscopy; HMBC, heteronuclear multiple bond correlation; ROESY, rotating frame Overhauser effect spectroscopy.

Compound 6 (anomaluone) was obtained as white powder and its molecular formula of C₂₀H₃₀O₃ was determined by high-resolution time-of-flight mass spectrometry (HRTOFMS) (m/z 318.2190 [M⁺], calculated 318.2189). The ¹H and ¹³C NMR spectra revealed characteristic signals of 3 tertiary methyls (δ _H 0.99, 1.02, and 1.17), 7 methylenes including 1 oxygenated one (δ _C 19.0, 20.5, 26.8, 38.6, 41.1, 52.4, and 69.6), 5 methines (δ _C 40.8, 50.5, 53.3, 125.3, and 161.1), and 5 quaternary carbons (δ _C 41.1, 43.8, 44.5, 79.1, and 204.5), indicating that compound 6 is a tetracyclic diterpenoid (Table 1). Further analysis of NMR data revealed 6 to be an ent-kaurane-type diterpenoid and reported in the literature as anomaluone (please see supplemental materials S1-S3).^4,10 The configuration of compound 6 was confirmed by ROESY experiment between H-18/H-5, H-5/H-9, H-9/H-15, H-17/H-14β, H-14α/H-20, H-20/H-19,³¹ shown in Figure 1(b), and negative optical rotation $[α]_{D}^{20}$ -75.9 (c 0.32, MeOH), agreed with the majority of ent-kauranes characterized for negative optical rotation.⁹

Table 1

¹H and ¹³C Nuclear Magnetic Resonance Data for Compounds 6 and 7 (Dimethyl Sulfoxide-D ₆).

No.	6		7
No.	δ _H	δ _C	δ _H	δ _C
1	7.23 (d, 10.0)	161.1	1.36 (m)	39.2
			1.95 (overlap)
2	5.76 (d, 10.0)	125.3	2.42 (m)	34.1
3		204.5		217.2
4		44.5		46.9
5	1.55 (m)	53.3	1.41 (m)	53.9
6	1.47 (m)	20.5	1.39 (m)	21.3
7	1.42 (m)	41.1	1.38 (m)	41.0
8		43.8		43.3
9	1.26 (d, 8.8)	50.5	1.13 (d, 8.4)	55.7
10		41.4		38.5
11	1.67 (dd, 6.0, 15.2)	19.0	1.47 (dd, 6.0, 14.2)	19.3
	2.15 (m)		2.05 (m)
12	1.37 (m)	26.8	1.34 (m)	26.9
	1.81 (m)		1.74 (m)
13	1.97 (m)	40.8	1.94 (overlap)	40.8
14	1.07 (dd, 4.0, 12.0)	38.6	1.02 (overlap)	37.7
	1.78 (dd, 2.0, 12.0)		1.80 (dd, 2.0, 12.0)
15	1.30 (dd, 2.4, 14.0)	52.4	1.26 (dd, 2.4, 14.0)	52.5
	1.39 (m)		1.35 (m)
16		79.1		79.0
17	3.15 (dd, 4.0, 10.8)	69.6	3.14 (d, 10.8)	69.6
	3.25 (dd, 4.0, 10.8)		3.24 (d, 10.8)
18	1.02 (s)	28.4	0.99 (s)	27.4
19	0.99 (s)	21.6	0.93 (s)	21.1
20	1.17 (s)	21.3	1.01 (s)	17.8

Compound 7 (16-epiabbeokutone), white powder, had a molecular formula of C₂₀H₃₂O₃ based on the [M⁺] at m/z 320.2348 (calculated for, 320.2346) in the HRTOFMS. The NMR data of 7 were similar to those of 6, except for the absence of double bond at C-1 and C-2 in 7, indicating that compound 7 is also an ent-kauranoid. A careful comparison of NMR spectra for 7 suggested that it was analyzed by Liu G et al and Gustafson KR et al, as 16-epiabbeokutone (please see supplemental materials S4-S6).^28,29 This structure was confirmed by ROESY experiment (Figure 1(b)) and negative optical rotation $[α]_{D}^{20}$ -72.8 (c 0.18, MeOH). Therefore, compound 7 was determined as shown.

The antiproliferative potential of M. japonicus compounds and fractions was tested in a cell proliferation assay (CellTiter Glo) using a blood cancer-derived cell line (please see supplemental material S7). Of the isolated compounds, compound 6 showed the strongest antiproliferative activity against Jurkat cells, and this activity was dose-dependent. The antiproliferative effect increased with both the dose and treatment duration (Figure 2). Treatment with 50 µM compound 6 significantly reduced cell viability, and microscopic analysis showed that compound 6 treatment induced morphological changes compared to that of control, dimethyl sulfoxide (DMSO)-treated cells (Figure 3). These results indicated that compound 6 has potent cytotoxic activity against Jurkat cells.

Figure 2

Antiproliferative effects of Mallotus japonicus compounds 1 to 8 on Jurkat cells. Cell viability was assessed by the MTS assay at 48 and 72 hours post treatment. DMSO, dimethyl sulfoxide, ***p < 0.001 vs control group.

Figure 3

Morphological changes in Jurkat cells treated with compound 6. Cells treated with compound 6 (20 and 50 μM) show morphological changes that are characteristic of apoptosis at 48 and 72 hours post treatment. Phase contrast microscopy (400× magnification).

Compound 6-induced apoptosis was assessed using Jurkat cells. The cells were double stained with Annexin V and 7-AAD dyes to determine the percentages of early and late apoptotic cells and viable cells. The percentages of early (Annexin+, 7-AAD-) and late (Annexin+, 7-AAD+) apoptotic cells increased, while the percentage of live cells decreased at 48 hours post compound 6 treatment, confirming the apoptosis induction activity of 6 (Figure 4(a) and (b)). Compound 6-treated Jurkat cells showed an increased percentage of late apoptotic cells (11.5%) within 48 hours, indicating that apoptosis was induced by compound 6 in leukemia cells (Figure 4(b)).

Figure 4

Compound 6 induced apoptosis in Jurkat cells. (a) Jurkat cells were either treated with compound 6 or untreated for 48 hours and then apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/7AAD double staining and flow cytometry. (b) Quantification of early and late apoptosis cells. ***p < 0.001 vs control group.

To determine whether the reduction in cell viability in Jurkat cells induced by compound 6 was due to its effects on cell-cycle progression was investigated by a cell-cycle analysis (Figure 5(a) and (b)). Incubation of Jurkat cells with compound 6 resulted in an increased percentage of cells in sub-G0/G1 phase and a decreased percentage of cells in S and G2/M phases. Similar results have been reported in human leukemia cells treated with jolkinolide B, a diterpene isolated from Euphorbia fischeriana Steud.³² Our results showed that compound 6 inhibited the growth of blood cancer cells (Jurkat cells) by inducing cell-cycle arrest.

Figure 5

Compound 6 induced changes in cell-cycle progression in Jurkat cells. (a) Cell-cycle analysis of compound 6-treated cells. Cells were stained with propidium iodide for 30 minutes and analyzed by flow cytometry. (b) The percentages of cells in SubG0/G1. S. and G2/M phases from triplicate experiments were determined. ***p < 0.001 vs control group.

Next, the effects of compound 6 on mitochondrial membrane potential (ΔΨm) were evaluated by measuring tetramethylrhodamine methyl ester perchlorate (TMRM+) fluorescence intensity using flow cytometry. TMRM+ quantifies the changes in mitochondrial membrane potential in live cells. Figure 6(a) shows representative microscopic data from DMSO- and compound 6-treated Jurkat cells. Compound 6-treated cells showed significant membrane potential depolarization after 48 hours (Figure 6(b) and (c)). Thus, compound 6 induced mitochondrial depolarization in Jurkat leukemia cells. These results suggest that compound 6 is a potential chemotherapeutic agent for blood cancers.

Figure 6

Compound 6 reduced the mitochondrial membrane potential in Jurkat cells. (a) TMRM fluorescence intensity as observed by fluorescence microscopy. (b) Mitochondrial membrane potential in Jurkat cells loaded with TMRM (100 nM) as detected by flow cytometry. (c) Quantification of the mitochondrial membrane potential measured by flow cytometry. DMSO, dimethyl sulfoxide; TMRM, tetramethylrhodamine methyl ester perchlorate. ***p < 0.001 vs control group.

Cancer cells maintain ROS production by suppressing antioxidant generation.^33,34 Cancer cells exhibit a redox imbalance due to increased ROS levels compared to those in normal cells. ROS production induced by anticancer agents has been shown to be associated with promotion of apoptosis and cell-cycle arrest.^35,36 To examine how compound 6 causes Jurkat cell death, ROS levels were measured using the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. Confocal microscopy showed that compound 6 increased intracellular ROS levels in Jurkat cells after 48 hours (Figure 7(a)). ROS generation was quantified by flow cytometry, which similarly showed that ROS generation was increased by compound 6 treatment (Figure 7(b) and (c)). Collectively, these results suggest that ROS generation by compound 6 is a potential mechanism underlying ROS-dependent apoptosis in Jurkat leukemia cells.

Figure 7

Compound 6 induced changes in intracellular ROS levels in Jurkat cells. (a) ROS detection by fluorescence microscopy. (b) Measurements of ROS levels by flow cytometry with DCFDA (1 µM). (c) Quantified RO levels. DMSO, dimethyl sulfoxide; ROS, reactive oxygen species. DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate ; ROS, reactive oxygen species. ***p < 0.001 vs control group.

Experimental

General Experimental Procedures

Optical rotations were measured using a Perkin-Elmer model 343 plus polarimeter (JASCO Corp., Japan). UV spectra were recorded on a JASCO V-530 UV/VIS spectrometer (JASCO Corp., Japan). IR spectra were obtained using a JASCO FTIR 300-E spectrometer. NMR spectra were recorded by an AVANCE-400 NMR spectrometer (Bruker, Billerica, MA, USA). CC was performed using Kieselgel 60 silica gel (<0.063 mm; Merck, Germany) and octadecyl (C18) silica gel (Yamazen, Japan). Thin-layer chromatography (TLC) spots were visualized on Kieselgel 60 F254-coated normal-phase silica gel TLC plates and TLC silica gel 60 RP-18 F254 (Merck, Germany). All solvents were obtained from Daejung Chemicals & Metals Co., Ltd. (Siheung, Korea).

Plant Specimens and Reagents

Mallotus japonicus roots were collected in June 2018 from the Medicinal Plant Garden of Boseong, Jeollanam-do, Korea and were identified by H.S. Park (Medicinal Plant Garden of Boseong). A voucher specimen (CNU-0602) was deposited at the College of Pharmacy, Chonnam National University, South Korea.

Sample Extraction

The dried roots of M. japonicus (2.0 kg) were percolated with 100% MeOH at room temperature. Evaporating in vacuo yielded a residue (120 g) that was suspended in H₂O and fractionated with EtOAc and n-butanol consecutively. The EtOAc extract (30 g) was subjected to CC using silica gel and eluted with a gradient of CH₂Cl₂:MeOH (100:1-1:100, v/v), which yielded 6 fractions (E1-E6). Fraction E2 (2.1 g) was subjected to RP C18-MPLC (10% MeOH to 100% MeOH), which yielded 6 subfractions (M1-M6). Compound 1 (19.8 mg) was obtained from subfraction M1 by preparative HPLC with 30% acetonitrile. Subfraction M3 (178.8 mg) was subjected to silica gel CC (CH₂Cl₂:MeOH = 50:1, v/v to 100% MeOH), which yielded 6 subfractions (M31-M36). Subfraction M31 (78.9 mg) was further purified using preparative HPLC (CH₃CN:H₂O = 4:6), which yielded compound 7 (12.6 mg). Subfraction M32 (80.7 mg) was separated by preparative HPLC using 40% acetonitrile, which yielded compound 6 (45.7 mg). Fraction E4 (1.1 g) was subjected to RP C18-MPLC (10% MeOH to 100% MeOH), which yielded 4 subfractions (M7-M10). Subfraction M8 (140.7 mg) was separated by preparative HPLC with 30% acetonitrile, which yielded 6 subfractions (M81-M86). Subfraction M84 (7.9 mg) was purified by preparative HPLC (CH₃CN:H₂O = 5:5), which yielded compound 5 (4.1 mg). Fraction E5 (2.6 g) was subjected to RP C18-MPLC with an MeOH:H₂O gradient (1:9% to 100% MeOH), which yielded 5 subfractions (M11-M15). Subfraction M11 (96.0 mg) was purified by preparative HPLC with 12% acetonitrile, which yielded compound 3 (38.7 mg). Compound 2 (920.0 mg) was separated from subfraction M12 by purification with MeOH and was confirmed by preparative HPLC with a CH₃CN:H₂O gradient (1:9% to 100% CH₃CN). The methanol-soluble fraction of subfraction M12 was isolated by preparative HPLC (CH₃CN:H₂O = 1:9), which yielded 3 subfractions (M121-M123). Subfraction M123 (80.8 mg) was subjected to preparative HPLC with 15% acetonitrile, which yielded compounds 4 (50.6 mg) and 8 (3.5 mg).

Cell Culture

Jurkat cells were obtained from the American Type Culture Collection (ATCC). Cell-culture medium was prepared as recommended by ATCC. The cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C and subcultured (1:5) when the cell density reached 80%-90%, which was every 3 or 4 days.

Antiproliferation Assay

The antiproliferative effect of the M. japonicus compounds on the cells was measured using the CellTiter 96 AQueous One solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). Cells were seeded at a density of 5 × 10⁵ cells/well in RPMI 1640 (100 µL) and cultured for 24 hours. Then, the cells were treated with different concentrations of each compound (10, 25, and 50 µM). DMSO-treated cells were included as a control. To estimate cell viability (%) at 48 hours post treatment, the cells were incubated with 20 µL of CellTiter 96 AQueous One solution Cell Proliferation Assay reagent for 2 hours at 37°C. Then, the optical density at 490 nm was measured using a microplate reader (Synergy HTX, BIO-TEK Instruments, Inc.).

Microscopy

To detect morphological changes, compound-treated Jurkat cells were examined under a phase contrast microscope (Olympus). Photomicrographs of the cells (400× magnification) were taken at 48 and 72 hours post compound treatment and analyzed for changes in shape, size, and number.

Cytotoxicity Assay

To explore the mechanism underlying compound-induced cytotoxicity in Jurkat cells, we investigated the changes in cell-cycle progression and apoptosis using flow cytometry.

Apoptosis Analysis

Compound-treated Jurkat cells were visualized by flow cytometry using the PE Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend). Annexin V binds to phosphatidylserine (PS) when PS flips from the inner leaflet to the outer leaflet of the plasma membrane during early apoptosis. In contrast, PI stains DNA when the cell membrane is disrupted during late apoptosis. To distinguish PE fluorescence from PI fluorescence and thus discriminate between early (Annexin V+) and late (7-AAD+) apoptotic cell populations, the FL-2 and FL-3 channels were selected to measure the PE and 7-AAD fluorescence, respectively. Compound-treated and untreated (control) Jurkat cells were seeded in 6-well plates and incubated at 37°C for 48 hours. The cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in binding buffer (1×). The cells were stained with annexin V-PE and 7-AAD, according to the manufacturer’s instructions. After staining, the cells were categorized as early (Annexin V+/7-AAD-) or late (Annexin V+/7-AAD+) apoptotic cells using a flow cytometer (BD FACSVerse, BD Biosciences), and the cell percentages were determined using FlowJo software (BD Biosciences).

Tetramethylrhodamine Methyl Ester Perchlorate (TMRM) Assay

Compound-treated Jurkat cells were harvested at 48 hours post-treatment, washed with PBS, and incubated with the cell-permeable fluorescent indicator TMRM (100 nM; Thermo Fisher Scientific), which was prepared by diluting the 10 mM stock in PBS, for 30 minutes at 37°C. To assess mitochondrial membrane potential, the cells were washed, resuspended in PBS, and analyzed with a flow cytometer (BD FACSVerse) and FlowJo v10 software.

Cell-Cycle Analysis

Univariate analysis of the cellular DNA content of compound-treated cells enables the detection of cells in different cell-cycle phases (Sub-G1/G1 vs. S vs. G2/M) and the detection of apoptotic cells with fractional DNA content. Compound-treated Jurkat cells were seeded in 6-well plates and incubated for 48 hours. Then, the cells were harvested, washed with PBS, and fixed with ice-cold 70% ethanol for 3 hours at 4°C. For the cell-cycle analysis, the cells were resuspended in PBS containing RNase A (5 µg/mL) and PI (50 µg/mL) and then evaluated using a flow cytometer (BD FACSVerse,). The percentages of cells in G0/G1, S, and G2/M phases were analyzed using FlowJo software.

Measuring ROS

Intracellular ROS levels were detected using 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (H2DCFDA) (Thermo Fisher Scientific). Cells were continuously perfused with PBS buffer (37°C) and imaged using an inverted fluorescent microscope (Olympus). For this analysis, the cells were incubated with DCFDA (1 µM) for 30 minutes at room temperature, washed with cold PBS, and then resuspended in PBS supplemented with 1% fetal bovine serum. Intracellular fluorescence accumulation was analyzed using a flow cytometer (BD FACSVerse).

Statistical Analyses

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc.), and the data are presented as the mean ± SD. The results were analyzed by 1-way analysis of variance and t-test. P values less than 0.05 were considered statistically significant.

Anomaluone (6)

White powder; $[α]_{D}^{20}$ -75.9 (c 0.32, MeOH); UV (MeOH) λ _max (log ε) 232 (3.99) nm; IR (MeOH) v _max 3410, 2932, 2865, 1667, 1452, 1378, 1264, 1161, 1055, 1025, 892 cm^-1 ^{; 1}H NMR (400 MHz, DMSO-d₆ ) and ¹³C NMR (100 MHz, DMSO-d₆ ) data, see Table 1; HRTOFMS m/z 318.2190 [M⁺] (calculated for C₂₀H₃₀O₃, 318.2189).

16-epiabbeokutone (7)

White powder; $[α]_{D}^{20}$ -72.8 (c 0.18, MeOH); UV (MeOH) λ _max (log ε) 308 (3.77), 220 (3.73) nm; IR (MeOH) v _max 3404, 2935, 2866, 1702, 1455, 1341, 1057, 1028, 895 cm^-1 ^{; 1}H NMR (400 MHz, DMSO-d₆ ) and ¹³C NMR (100 MHz, DMSO-d₆ ) data, see Table 1; HRTOFMS m/z 320.2348 [M⁺] (calculated for C₂₀H₃₂O₃, 320.2346).

Supplemental Material

Supplementary Material - Supplemental material for An Antiproliferative ent-Kaurane Diterpene Isolated from the Roots of Mallotus japonicus Induced Apoptosis in Leukemic Cells

Supplemental material, Supplementary Material, for An Antiproliferative ent-Kaurane Diterpene Isolated from the Roots of Mallotus japonicus Induced Apoptosis in Leukemic Cells by Joo-Eun Lee, Nguyen Thi Thanh Thuy, Youngju Lee, Namki Cho and Hee Min Yoo in Natural Product Communications

Footnotes

Acknowledgment

The authors are grateful to the Center for Research Facilities at the Chonnam National University for their assistance in the analysis of the organic structure (FT-IR, FT-NMR, HRTOFMS).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Korea Research Institute of Standards and Science for the “Development of New Chemical Medical Measurement Standard Technology” [grant number KRISS-2019-GP2019-0009]; the National Research Foundation of Korea (NRF) [grant number NRF-2018R1C1B5083127]; and Chonnam National University [grant number 2018-3487].

ORCID ID

Namki Cho

Hee Min Yoo

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Supplementary Material

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An Antiproliferative ent -Kaurane Diterpene Isolated from the Roots of Mallotus japonicus Induced Apoptosis in Leukemic Cells

Abstract

Experimental

General Experimental Procedures

Plant Specimens and Reagents

Sample Extraction

Cell Culture

Antiproliferation Assay

Microscopy

Cytotoxicity Assay

Apoptosis Analysis

Tetramethylrhodamine Methyl Ester Perchlorate (TMRM) Assay

Cell-Cycle Analysis

Measuring ROS

Statistical Analyses

Anomaluone (6)

16-epiabbeokutone (7)

Supplemental Material

Supplementary Material - Supplemental material for An Antiproliferative ent-Kaurane Diterpene Isolated from the Roots of Mallotus japonicus Induced Apoptosis in Leukemic Cells

Footnotes

Acknowledgment

Declaration of Conflicting Interests

Funding

ORCID ID

References

Supplementary Material