Chemical Constituents of Eupatorium japonicum and Anti-Inflammatory, Cytotoxic, and Apoptotic Activities of Eupatoriopicrin on Cancer Stem Cells

Eupatorium japonicum Thunb. of the plant family Asteraceae is a popular traditional herb in Vietnam. However, its chemical constituents as well as bioactive principles have not been investigated yet. We investigated the phytochemistry of E. japonicum in Vietnam and isolated seventeen compounds (1–17) including phytosterols, terpenoids, phenolic acids, flavonoids, fatty alcohols, and fatty acids. They were structurally determined by MS and NMR analysis. Except for compounds 6 and 12, all the other compounds were identified for the first time from E. japonicum. Since many sesquiterpene lactones with α-methylene γ-lactone ring are reported as anti-inflammatory and anticancer agents, eupatoriopicrin (10), 1-hydroxy-8-(4,5-dihydroxytigloyloxy)eudesma-4(15),11(13)-dien-6,12-olide (11) were selected among the isolates for biological assays. Compound 10 was identified as the main bioactive sesquiterpene lactone of E. japonicum showing its potent anti-inflammatory and cytotoxic activity through inhibiting NO production and the growth of HepG2 and MCF-7 human cancer cell lines. For the first time, eupatoriopicrin (10) was demonstrated to strongly inhibit NTERA-2 human cancer stem cell (CSC) line in vitro. It is noticeable that the cytotoxicity of eupatoriopicrin against NTERA-2 cells is mediated by its apoptosis-inducing capability of 10 as demonstrated by the results of Hoechst 33342 staining, flow cytometry apoptosis analysis, and caspase-3 activity assays. The biological activities of the main bioactive constituents 1–7, 10, 12, and 15 supported the reported anti-inflammatory and anticancer properties of extracts from E. japonicum.


Introduction
In Vietnam, traditional herbal remedies are considered complementary or alternative medicines in the healthcare system. However, many herbal remedies are clinically used without any scientific evidence of their efficacy and safety. Since biological activities are the links between medicinal plants and their pharmacology and medicinal uses, we have investigated the chemical constituents and biological activities of common Eupatorium species used in Vietnamese traditional medicine such as E. fortunei [1] and E. triplinerve [2]. Among the compounds isolated from the genus Eupatorium, a number of sesquiterpenoids have been proven to possess different levels of cytotoxic, anti-inflammatory, antifungal, antibacterial, and insecticidal activities [3]. E. japonicum unb. belonging to the family Asteraceae has been recorded as a medicinal plant in Vietnam, Japan, and China. Its leaves are used for the treatment of nausea, vomiting, indigestion, and diarrhea [4]. In Vietnam, the species is known under the name "Son lan" or "Yen bach Nhat" (ydvn.net) and distributed in the mountainous Sapa district of the Northwestern Lao Cai province. is perennial herb grows 1-2 m tall, blooms from August to September, and fruits from September to November. e whole plant and root of "Son lan" are used by Vietnamese people to treat cough after a cold, back pain due to cold, and irregular menstruation (ydvn.net) [5]. A very few chemical studies reported the isolation of coumarin and euponin (a guaiane-type lactone) [6] as well as the identification of pyrrolizidine alkaloids by mass spectrometry (MS) [7] from the leaves of E. japonicum. Forty years later, in a recent communication, 19 compounds were reported from the flowers of E. japonicum in China including coumarin, 2-hydroxy-2,6-dimethylbenzofuran-3(2H)-one, 1-(2-hydroxy-4-methylphenyl)propan-1,2-dione, subamone, (7R * )-opposit-4(15)-ene-1β,7-diol, gentisic acid, caffeic acid, 9-hydroxythymol, protocatechuic acid, trans-Ohydroxycinnamic acid, umbelliferone, quercetin, taraxasterol, 9-acetoxythymol 3-O-tiglate, taraxasterol acetate, linoleic acid, 9-angeloyloxythymol, stigmasterol, and palmitic acid [7]. e present study describes the first investigation of the chemical constituents of the leaves of E. japonicum in Vietnam which resulted in the isolation of 17 compounds of different polarities and structural classes ranging from fatty alcohol, fatty acid, monoterpene, sesquiterpene lactone, and phytosterol to phenolic acid and flavonoid glycoside. Except for compounds 6 and 12, all of the compounds were isolated and identified for the first time from E. japonicum.
Compounds derived from nature create prominent screening libraries for drug discovery. Owing to a vast reservoir of unexploited medicinal plants, the Vietnamese medicinal plants have the potential to offer compounds of various structural and stereochemical types for the screening of lead compounds [8]. e safety of ethanol extracts prepared from E. japonicum and Foeniculum vulgare was evidenced from a recent toxicity test in mice [9]. Stem extracts from E. japonicum suppressed lipid accumulation and inhibited the expression of adipocyte markers and coumaric acid and its methyl ester were isolated from the extracts as the bioactive components [10]. Extracts from E. japonicum exerted anti-inflammatory effects through the suppression of various molecular targets such as NO production, interleukin, iNOS, COX-2, MMP-9 transcription, NF-κB activation, and TRIF-dependent signaling pathway of toll-like receptors [4,11,12]. In addition, apoptotic effects through the ROS-induced ATF4 and CHOP expression [4] and antimetastatic effects through inhibiting cell migration, invasion, and adhesion of MDA-MB-231 human breast cancer cells in dose-dependent manners were observed for the extracts [13]. e major compounds responsible for these biological activities of E. japonicum have not been revealed yet. Since many sesquiterpene lactones with α-methylene c-lactone ring are reported to regulate antiinflammatory and anticancer targets [14,15] in order to validate the biological effects of E. japonicum extracts in relation to its chemical constituents eupatoriopicrin (10), a germacranolide and 1-hydroxy-8-(4,5-dihydroxytigloyloxy) eudesma-4(15),11(13)-dien-6,12-olide (11), an eudesmanolide were selected for the study of NO-inhibitory activity and cytotoxicity against human cancer stem cells (CSCs). Being disclosed as cytotoxic, the mechanism of cell death through apoptosis induction on CSCs would be examined using Hoechst 33342 staining, flow cytometry apoptosis analysis, and caspase-3 activity assays on NTERA-2 pluripotent human embryonal carcinoma cell line.
Nitric oxide (NO) is a short-lived free radical derived from L-arginine by nitric oxide synthase (NOS) that mediates diverse functions by acting on various cells through interactions with different molecular targets. e excessive production of NO in prolonged inflammation can cause cellular and tissue damage. e adverse effect of this inflammation can be prevented by the intervention of antiinflammatory agents. Compounds able to inhibit the production of NO or other proinflammatory molecules are considered potential anti-inflammatory agents. CSCs are the cells that have the ability to self-renew, differentiate into defined progenies, initiate, and maintain tumor growth [16].
Despite being a small percentage in tumor cells, CSCs are present in most tissues including breast, brain, lung, head and neck, prostates, testis, ovary, esophagus, colon, and liver and play a role in cancer metastasis and therapeutic resistance, both of which are the major causes of cancer mortality and cancer recurrence [17].
ere is evidence that the current chemotherapy for the treatment of cancer is insufficient in eliminating CSCs from a number of cancer types. e lingering CSCs are able to form new, fully developed tumors from a small number of cells or even a single cell. erefore, recent studies target the discovery of natural drugs against CSCs involved in tumor development for different cancers [18]. Apoptosis is a highly regulated cell death process and occurs both during normal development and under certain pathological conditions. Apoptosis plays a crucial role in maintaining tissue homeostasis by the selective elimination of excessive cells. e evasion of apoptosis is an essential hallmark of cancer; therefore, killing cancer cells through the induction of apoptosis is a valuable tool for cancer treatment.
2.5. Nitric Oxide Assay. RAW 264.7 cells were seeded to a 96well plate at a concentration of 2 × 10 5 cells/well. After incubation for 24 h, the cultured medium was refreshed by DMEM without FBS. en, the cells were treated with samples at different concentrations in the presence of LPS (1 µg/mL) for 24 h. N G -Methyl-L-arginine acetate (L-NMMA) (Sigma) was used as the positive control and the cells treated with a diluted solution of DMSO (1.0%) were used as the negative control. Nitrite (NO _ 2 ), an indicator of NO production, was determined by the Griess reagent system (Promega Corporation, WI, USA). Briefly, 100 µL medium was mixed with 50 μL 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 50 μL 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride in a 96-well plate. e plate was then incubated at room temperature for 10 min. e optical density (OD) was measured at 540 nm by using a microplate reader (Biotek, Winooski, VT, USA). e concentration of nitrite in each well was calculated by using the NaNO 2 standard curve. e ability of NO inhibition of a sample was calculated by the following formula: (%) inhibition � 100%-(concentration of NO sample /concentration of NO negative control ) × 100. e value of IC 50 (the halfmaximal inhibitory concentration) was determined by using

Cell Viability MTT Assay.
e proliferation of cells treated with samples was determined by MTT assay [20]. 10 µL MTT (5 mg/mL) was added to the wells containing 5 × 10 3 RAW 264.7 cells treated with samples and then incubated at 37°C for 4 h. e purple formazan crystals formed by metabolically active cells were diluted by adding 100 µL DMSO (100%) to each well after removing the medium. e OD was measured by using a microplate reader (BioTek, Winooski, VT, USA) at 540 nm. e percentage of cell viability was determined by the following formula: (%) cell viability � (OD (sample) -OD (blank) )/(OD (negative control) -OD (blank) ) × 100%. All analyses were performed in triplicate and data were reported as mean ± SEM (standard error of the mean).

SRB Colorimetric
Assay. SRB assay was used for determining the cytotoxic activity of tested samples in a 96-well plate according to the method of Skehan et al. [21]. e test evaluates the cell density based on the measurement of the total cellular protein content stained with sulforhodamine B (SRB). Briefly, trypsinized cells were seeded in a 96-well plate and incubated with sample for 48 h. e cell-containing wells treated with diluted solution serve as the negative control. After a period of incubation time, the cells were fixed with TCA for 1 h and then stained with SRB for 30 min at 37°C. e plates were washed three times with acetic acid to remove all nonstaining dye and allowed to dry at room temperature. SRB staining protein in cells was dissolved in 10 mM unbuffered Tris base and the plate was gently shaken for 10 min at room temperature. e OD at the wavelength of 540 nm was measured by using an ELISA plate reader (BioTek, Winooski, VT, USA)). e inhibition percentage of cell growth in the presence of the treated sample is determined by the following formula: (%) inhibition � 100%-(OD (sample) -OD (day 0) )/ (OD (negative control) -OD (day 0) ) × 100. e test was triplicated to ensure accuracy. Ellipticine was used as the positive control. e IC 50 value was determined by using the TableCurve 2Dv4 computer software.

Hoechst 33342 Staining.
e morphology of cell nuclei treated with samples was determined through Hoechst 33342 staining (Allen et al. [22]). NTERA-2 cells grew stable in a 6-well plate for 24 h before being treated with samples, the reference (camptothecin) or the negative control (DMSO 1%). After 48 h of treatment, cells were fixed with formaldehyde 4% for 30 min and then washed with phosphate-buffered saline (PBS) before staining with Hoechst 33342 (0.5 µg/mL) for 10 min. e morphological change of nuclei was observed under a fluorescence microscope at 350⁄ 461 nm (excitation/ emission). e apoptotic cells have brighter nuclei or fragmented chromatin.

Flow Cytometry Apoptosis Analysis.
e flow cytometry protocol was performed using Kit Annexin V and PI/dead cell apoptosis ® (Invitrogen, ermo Fisher Scientific) according to Ngo et al. [23]. Basically, cells (1 × 10 6 cells) treated with samples were collected after 48 h incubation and washed with PBS before staining with Annexin V-FITC for 15 min. e cells were continuously stained with propidium iodide (PI) (5 μL) in Annexin V binding buffer. Total 10.000 cells for each sample were analyzed using Attune NxT flow cytometer ( ermo Fisher Scientific, USA). e unaffected cells were PI-negative and Annexin V-negative, while the apoptotic cells were Annexin V-positive, and the necrotic cells were only positive for PI.

Caspase-3 Activity Assay.
e experiment was performed using the caspase-3 colorimetric assay kit (BioVision Inc., USA). Briefly, cells were grown in a 6-well plate for 24 h before they were treated with samples and incubated in a high-humidity CO 2 incubator at 37°C for further 48 h. Cells that were treated with DMSO 10% were considered the negative control. After the treatment period, cells were detached with Trypsin-EDTA and then washed with PBS. e cell pellet was lysed with 50 µL lysis buffer from the kit on ice for 10 min. e cell lysis solution was then reacted with 10 mM DTT and 4 mM DEVD-pNA and incubated at 37°C for 2 h. Samples were read at 405 nm by using a microplate reader to determine the OD. Fold-change in caspase-3 activity of the sample was measured by comparing with the OD of the negative control [24].

Statistical Analysis.
e data were expressed as the mean of three replicate determinations ± standard deviation (SD). Statistical comparisons were made with Student's test; p values < 0.05 were considered significantly different.

Cytotoxic Activity of Compounds 10 and 11.
NTERA-2 is an embryonal carcinoma cell line, which expresses a gene profile related to pluripotency of stem cells such as OCT3/4 and SSEA-4. e cell line presents a lot of early embryo antigens that could be used to identify undifferentiated human embryonic stem cell (hESC) [46,47]. In addition, this cell line is easy to culture and grows quickly in a medium without any additional growth supplements except FBS. us, this cell line was widely used as  Evidence-Based Complementary and Alternative Medicine a model for CSC research in vitro and in discovering novel anticancer compounds against CSCs, a research area of great interest nowadays. e cytotoxicity of compounds 10 and 11 on human breast cancer cell line (MCF7), human hepatocellular carcinoma (HepG2), and pluripotent human embryonal carcinoma cell line (NTERA-2) was accessed using SRB assay. As shown in Figure 2 and Table 2, eupatoriopicrin (10) showed strong cytotoxic activity on all tested cell lines with IC 50 values of 1.22 ± 0.10 µg/mL, 0.94 ± 0.12 µg/mL, and 0.88 ± 0.05 µg/mL, respectively. Compound 11 did not show cytotoxic effects on HepG2, MCF-7, and NTERA-2 cell lines at the concentrations tested. Hitherto compound 10 expressed its cytotoxic activity against KB, HeLa, HL-60, EN19, EAT, P388, FIO 26, and L5178Y cell lines [28,48,49]. e structural requisite for the potent cytotoxic activity of 10 may be associated with its α-methylene-c-butyrolactone moiety [50]. e oncolytic activity was also demonstrated in an in vivo study  showing the tumor growth delay after administration of eupatoriopicrin [51,52]. e present study is the first report on the cytotoxicity of 10 against human CSCs (NTERA-2) as well as human cancer cell lines HepG2 and MCF-7. Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways [53]. An in vitro study by Woerdenbag et al. showed that significant GSH reduction began to occur with concentrations of eupatoriopicrin ≥1 μg/mL in FIO 26 cells [52]. Eupatoriopicrin may react with the sulfhydryl group of cellular GSH to form a Michael adduct and its cytotoxicity may increase by the action of GSH depletion. Two other distinct pathways of cell death are apoptosis and necrosis. To investigate the possibility of apoptosis in the cytotoxicity of 10, we assayed further the apoptosis-inducing activity of 10 in NTERA-2 cells.      Evidence-Based Complementary and Alternative Medicine 3.4. Apoptosis-Inducing Activity of Compound 10. One of the promising strategies for the identification of cytotoxicity of anticancer compounds is the induction of apoptosis of cancer cells. Based on the strong cytotoxicity of compound 10 on NTERA-2 CSC line, 10 was further evaluated for its apoptosis-inducing activity to better understand the possible apoptosis pathway. We examined the possible induction of apoptotic cell death by 10 using flow cytometry analysis and activation of caspase-3 in CSCs. us, NTERA-2 cancer cells were treated with 10 and stained with Hoechst 33342. Using this nuclear staining dye, apoptotic cells with condensed and fragmented nuclei were detected, while most negative control cells showed nuclei with homogeneously staining morphology ( Figure 3). e experiment exhibited that compound 10 could induce apoptosis on NTERA-2 cells in a dose-dependent manner (Table 3), resulting in the inhibition of the proliferation of NTERA-2 cells. e flow cytometric analysis confirmed the apoptosisinducing activity of compound 10. As shown in Figure 4, the number of apoptotic cells, which was positive with Annexin V, increased along with raising concentrations of 10. At the highest concentration, 4 µg/mL 10 could induce up to 44.38% of apoptotic cells, while the anticancer drug camptothecin induced 65.26% of apoptotic cells. However, compound 10 also caused necrosis at this concentration, which was 27.14%. e necrosis was reduced when cells were exposed to lower concentrations of 10 such as 1.0 and 2.0 µg/ mL; meanwhile, the apoptosis-inducing activity of 10 was maintained at 19.41 and 28.70%, respectively. e results indicated that the induction of apoptosis could be a mechanism of the cytotoxicity of 10 against NTERA-2 cancer cells.
In order to confirm the apoptosis-inducing activity of compound 10 towards CSCs, caspase-3 activity was measured. e initiator phase of apoptosis is characterized by initiator caspase (cysteine-dependent aspartate-directed protease) activation (Franco and Cidlowski). Caspase-3 is one of the key enzymes in apoptosis and is activated in both the intracellular and extracellular apoptotic pathways [54,55]. is enzyme specifically activates the caspase-activated DNAse which causes chromosomal degradation and condensation in the nucleus, which are important feature characteristics of apoptosis [56]. Compound 10 significantly induced caspase-3 production at all tested concentrations (p < 0.05) ( Figure 5). us, compound 10 induced apoptosis in NTERA-2 cancer cells by the activation of caspase-3. However, the strongest activity was observed at the concentration of 1.0 µg/mL (p < 0.01). Higher or lower concentrations also mediated caspase-3 production but the induction was not as strong as that at 1.0 µg/mL. ese results indicated that this concentration might be the optimized concentration of 10 to induce apoptosis in the NTERA-2 CSC line through the activation of caspace-3. roughout, 10 expressed the potential activity against CSCs by apoptosis and/or necrosis induction. A challenge of cancer therapy is multidrug resistant, which is closely related to CSCs [57]. Multidrug resistance of CSCs is caused by multiple reasons such as overexpression of ATP-binding cassette (ABC) transporters, high level of multidrug resistance (MDR), or detoxification proteins. e high survival capacity of CSCs under the treatment of drugs is caused by induced apoptosis resistance through changing a series of apoptosis mechanisms as downregulation p53 gene, miRNA action, or inhibiting apoptotic proteins [58][59][60]. erefore, drugs as 10 that reduce survival rates of CSCs and induce apoptosis attract considerable attention.

Conclusions
In this study, 15 compounds out of 17 isolates were isolated for the first time from E. japonicum. Belonging to the privileged group of anti-inflammatory and anticancer structures, two sesquiterpene lactones 10 and 11 were selected for biological assays. Eupatoriopicrin (10) was found to strongly inhibit NO production, MCF-7, HepG2, and NTERA-2 cancer cell lines; meanwhile, compound 11 was not active. e cytotoxicity on the NTERA-2 cancer cell line is related to significant apoptosis induction by the action of 10 in the concentration-and caspase-dependent manners.
e importance of the findings is that the study provides a chemical and biological basis for developing new approaches to explore therapeutic values of extracts or herbal medicines containing the herb E. japonicum. In addition, anti-inflammatory and anticancer botanical drugs enriched with bioactive constituents can be formulated and prepared from E. japonicum. Eupatoriopicrin (10) is also exposed in the study as a lead compound targeting inflammation or cancer diseases. Further projects on the synthesis of chemical derivatives or analogs of 10 addressing optimization of drug safety and effectiveness in combating inflammatory or cancer diseases are underway.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Minh Giang Phan carried out extraction of plant material, isolation of compounds, structure determination, and preparation of the manuscript. i Viet Huong Do, Minh Trang Vu, and Ngoc Phuc Dong contributed to the isolation of compounds. i ao Do and i Nga Nguyen evaluated biological activities and contributed to the preparation of the manuscript. All the authors have read the final manuscript and approved the submission.