Three New Dihydrophenanthrene Derivatives from Cymbidium ensifolium and Their Cytotoxicity against Cancer Cells

From the aerial parts of Cymbidium ensifolium, three new dihydrophenanthrene derivatives, namely, cymensifins A, B, and C (1–3) were isolated, together with two known compounds, cypripedin (4) and gigantol (5). Their structures were elucidated by analysis of their spectroscopic data. The anticancer potential against various types of human cancer cells, including lung, breast, and colon cancers as well as toxicity to normal dermal papilla cells were assessed via cell viability and nuclear staining assays. Despite lower cytotoxicity in lung cancer H460 cells, the higher % apoptosis and lower % cell viability were presented in breast cancer MCF7 and colon cancer CaCo2 cells treated with 50 µM cymensifin A (1) for 24 h compared with the treatment of 50 µM cisplatin, an available chemotherapeutic drug. Intriguingly, the half-maximum inhibitory concentration (IC50) of cymensifin A in dermal papilla cells at >200 µM suggested its selective anticancer activity. The obtained information supports the further development of a dihydrophenanthrene derivative from C. ensifolium as an effective chemotherapy with a high safety profile for the treatment of various cancers.


Introduction
Cymbidium spp. (Orchidaceae) comprises about fifty species widely dispersed across tropical and temperate Asia, especially in Thailand, China, and Nepal. Their beautiful flowers and structural appearance have made them more known for ornamental purposes rather than medicinal potentials. However, various parts of Cymbidium spp. are used in folkloric medicine to treat/manage many diseases. The leaves, roots, and pseudobulb of some Cymbidium spp. are employed in Nepali folkloric medicine for the treatment of boils, fever, paralysis, chronic illnesses, otitis, and as a tonic on rift bones [1]. Some species in this genus have been used as traditional Thai medicine. For example, the leaves of C. aloifolium and C. findlaysonianum have been used to treat otitis media [2]. Cymbidium plants are rich sources of phenanthrenes and bibenzyls, some of which exhibit several pharmacological activities such as cytotoxic, antimicrobial, free radical scavenging, and anti-inflammatory activities [3][4][5].
Cymbidium ensifolium (L.) Sw., known as "nang kham" or "chulan" in Thai [6], is an elegantly shaped orchid with beautiful aromatic flowers. Its flowers are white blended with yellow and red longitudinal lines along with the sepals and petals (Figure 1). It is distributed in north and northeastern Thailand [7]. The roots of this plant have been used as traditional Thai medicine to alleviate liver dysfunction and nephropathy [2,8]. In our continuing studies on bioactive compounds from orchids [9,10], we have explored the chemical constituents from the aerial parts of C. ensifolium with no previous reports on the chemical investigation. In the present study, a methanolic extract prepared from the aerial parts of this plant exhibited a significant cytotoxic effect against various cancer cells. This prompted us to investigate the plant to identify the cytotoxic compounds.
dobulb of some Cymbidium spp. are employed in Nepali folkloric medicine treatment of boils, fever, paralysis, chronic illnesses, otitis, and as a tonic on rift b Some species in this genus have been used as traditional Thai medicine. For exam leaves of C. aloifolium and C. findlaysonianum have been used to treat otitis m Cymbidium plants are rich sources of phenanthrenes and bibenzyls, some of whic several pharmacological activities such as cytotoxic, antimicrobial, free radical ing, and anti-inflammatory activities [3][4][5].
Cymbidium ensifolium (L.) Sw., known as "nang kham" or "chulan" in Thai elegantly shaped orchid with beautiful aromatic flowers. Its flowers are white with yellow and red longitudinal lines along with the sepals and petals (Figure distributed in north and northeastern Thailand [7]. The roots of this plant have be as traditional Thai medicine to alleviate liver dysfunction and nephropathy [2,8 continuing studies on bioactive compounds from orchids [9,10], we have expl chemical constituents from the aerial parts of C. ensifolium with no previous re the chemical investigation. In the present study, a methanolic extract prepared aerial parts of this plant exhibited a significant cytotoxic effect against variou cells. This prompted us to investigate the plant to identify the cytotoxic compoun

Structure Determination
The phytochemical study of a methanolic extract from the leaves of Cymbid folium resulted in the purification of three novel dihydrophenanthrene derivativ along with two known compounds cypripedin (4) [11] and gigantol (5) [12] (F The structures of the new compounds were elucidated through extensive spect data.
Compound 2, a red amorphous solid, showed a similar molecular formula with 1 as C 16   A of 2, a methoxyl group (δ 3.86) was placed at C-2 according to its NOESY interaction with H-3. On ring B, the position of the methoxy and hydroxyl groups of 2 was alternated when compared with 1. The second methyl group (δ 3.76) of 2 was located at C-8, as evidenced by the HMBC correlations of C-8 (δ 145.3) with MeO-8, H-6, H 2 -9, and HO-7. The HMBC correlations of C-7 (δ 153.1) with H-5 and HO-7 and the NOESY correlation between H-6 and HO-7 support the placement of the hydroxyl group at C-7 ( Figure 3). Based on the above spectral evidence, 2 was identified as 7-hydroxy-2,8-dimethoxy-9,10dihydrophenanthrene-1,4-dione and named cymensifin B. On ring B, the position of the methoxy and hydroxyl groups of 2 was alternated when compared with 1. The second methyl group (δ 3.76) of 2 was located at C-8, as evidenced by the HMBC correlations of C-8 (δ 145.3) with MeO-8, H-6, H2-9, and HO-7. The HMBC correlations of C-7 (δ 153.1) with H-5 and HO-7 and the NOESY correlation between H-6 and HO-7 support the placement of the hydroxyl group at C-7 ( Figure 3). Based on the above spectral evidence, 2 was identified as 7-hydroxy-2,8-dimethoxy-9,10-dihydrophenanthrene-1,4-dione and named cymensifin B.

Cytotoxic Effects against Various Cancer Cells
Because cytotoxic effects of cypripedin (4) and gigantol (5) were previously demonstrated in lung cancer cells [11,15], compounds 1-3 were evaluated for their anticancer potential against various human cancers, including lung, breast, and colon cancer cells. Cisplatin, a recommended chemotherapeutic agent [16], which at 50 µM caused the 50% reduction of viability in lung cancer cells, was selected for a positive control. The preliminary investigation via MTT assay showed that a culture with methanolic extract from C. ensifolium at 50 µg/mL for 24 h significantly diminished viability in lung cancer H460 and breast cancer MCF7 cells, but not in colon cancer CaCo 2 cells when compared with control cells which were treated with the solvent vehicle 0.5% DMSO ( Table 2). It should be noted that treatment with a lower concentration (10 µg/mL) of the methanolic extract did not obviously decrease cell viability in all cancer cells (data not shown). Among the three new dihydrophenanthrene derivatives, 24 h treatment with compound 1 (50 µM) showed the highest anticancer effect against H460, MCF7, and CaCo 2 cells, as evidenced with the lowest % cell viability assessed via an MTT assay. When compared with cisplatin (Sigma-Aldrich Chemical, St. Louis, MO, USA), the higher anticancer potency of compound 1 was indicated with the lower % viability in MCF7 and CaCo 2 cells. Additionally, Figure 4a-c illustrate the dose-response relationship of three new dihydrophenanthrene derivatives in H460, MCF7, and CaCo 2 cells, respectively. In all cancer cells, compound 1 and cisplatin demonstrated anticancer activity in a concentration dependence. When compared with cisplatin treatment at the same concentration, the significantly lower % cell viability was presented in CaCo 2 cells cultured with 50-200 µM of compound 1. All data were presented as means ± standard error of the mean (SEM) from independent experiments.  All data were presented as means ± standard error of the mean (SEM) from three independent experiments.
These were corresponded with apoptosis cell death detected via costaining with Hoechst 33342 and propidium iodide. Figure 5a-c respectively demonstrates apoptosis cells presenting with bright-blue fluorescence of condensed DNA or fragmented nuclei stained with Hoechst 33342 in H460, MCF7, and CaCo 2 cells, which were cultured with either compound 1 (50 µM) or cisplatin (50 µM) for 24 h. Notably, there were no necrosis stained with the red fluorescence of propidium iodide in all treated cells. When compared with cisplatin treatment, the culture with compound 1 significantly increased % apoptosis in breast cancer MCF7 and colon cancer CaCo 2 cells, although there was lower % apoptosis in lung cancer H460 cells (Figure 6a-c).
It is the fact that the purity of an isolated natural compound critically influences its bioactivity [17]. Composed of known anticancer compounds, cypripedin (4) and gigantol (5), as well as compound 1, a new dihydrophenanthrenequinone with a higher potency anticancer effect, the methanolic extract from C. ensifolium exhibited potent cytotoxicity against lung cancer and breast cancer cells (Table 2 and Figure 5a,b). Despite no toxicity presented in C. ensifolium extract-treated CaCo 2 cells (Table 2 and Figure 5c), the dramatic reduction of % cell viability (Figure 4c) and accumulated apoptosis indicated in CaCo 2 cells (Figures 5c and 6c) incubated with compound 1 suggested specific cytotoxicity of compound 1 against colon cancer cells.
Molecules 2022, 27, x FOR PEER REVIEW stained with the red fluorescence of propidium iodide in all treated cells. When pared with cisplatin treatment, the culture with compound 1 significantly increa apoptosis in breast cancer MCF7 and colon cancer CaCo2 cells, although there was % apoptosis in lung cancer H460 cells (Figure 6a-c).  It is the fact that the purity of an isolated natural compound critically influenc bioactivity [17]. Composed of known anticancer compounds, cypripedin (4) and gig (5), as well as compound 1, a new dihydrophenanthrenequinone with a higher pot anticancer effect, the methanolic extract from C. ensifolium exhibited potent cytotox against lung cancer and breast cancer cells (Table 2 and Figure 5a,b). Despite no tox presented in C. ensifolium extract-treated CaCo2 cells (Table 2 and Figure 5c), the dram reduction of % cell viability (Figure 4c) and accumulated apoptosis indicated in C cells (Figures 5c and 6c) incubated with compound 1 suggested specific cytotoxici compound 1 against colon cancer cells.
As the most affected normal cells, human dermal papilla cells (DPCs) are chose evaluating the safety profile of potential anticancer compounds [18,19]. Intrigui DPCs cultured with 50 µM of three new dihydrophenanthrene derivatives (1-3) e ited higher % cell viability (Table 2 and Figure 4d) and less apoptosis cells (Figure  and 6d) compared with the treatment of cisplatin. Due to the highest anticancer pote among three new dihydrophenanthrene derivatives, the half-maximum inhibitory centration (IC50) and selectivity index (S.I.) of compound 1 were calculated. Table 3 cates that when compared with cisplatin, compound 1 possessed lower IC50 valu Figure 6. The percent (%) apoptosis calculated from nuclear staining assay in (a) lung cancer H460, (b) breast cancer MCF7, (c) colon cancer CaCo 2 , and (d) dermal papilla cells (DPCs) cultured with methanolic extract and isolated compounds 1-3 from Cymbidium ensifolium for 24 h. Cisplatin, a recommended anticancer drug, was used as a positive control. The data were presented as means ± standard error of the mean (SEM) from three independent experiments. * p < 0.05 compared with control cells treated with 0.5% DMSO as a solvent vehicle, # p < 0.05 compared with the cisplatin treatment group.
As the most affected normal cells, human dermal papilla cells (DPCs) are chosen for evaluating the safety profile of potential anticancer compounds [18,19]. Intriguingly, DPCs cultured with 50 µM of three new dihydrophenanthrene derivatives (1-3) exhibited higher % cell viability (Table 2 and Figure 4d) and less apoptosis cells (Figures 5d and 6d) compared with the treatment of cisplatin. Due to the highest anticancer potential among three new dihydrophenanthrene derivatives, the half-maximum inhibitory concentration (IC 50 ) and selectivity index (S.I.) of compound 1 were calculated. Table 3 indicates that when compared with cisplatin, compound 1 possessed lower IC 50 values in MCF7 (93.04 ± 0.86 µM) and CaCo 2 cells (55.14 ± 3.08 µM), but it accounted for higher values of IC 50 in H460 (66.71 ± 6.62 µM) and DPCs cells (>200 µM). It has been reported that the selectivity index of an anticancer agent, which selectively causes toxicity to cancer cells, should be more than 1 [20]. The higher selectivity index of compound 1 was indicated in MCF7 (>2.15) and CaCo 2 cells (>3.62) compared with cisplatin treatment (MCF7: <0.59, CaCo 2 : <0.59). Although the selectivity index of compound 1 (>3.00) was lower than cisplatin (5.48 ± 0.25) in lung cancer cells, both values are more 1. These data clearly demonstrate the selective anticancer activity of compound 1 against various cancer cells.
Taken together, these results strongly suggest the potent anticancer effect against various types of cancer with the high safety profile of compound 1 isolated from the C. ensifolium extract. Nevertheless, the underlying mechanisms involved in the apoptosis inducing effect of compound 1 should be further investigated to clarify the therapeutic target.

Cell Culture
Human lung cancer H460, breast cancer MCF7, and colon cancer CaCo 2 cells were obtained from The American Type Culture Collection (ATCC, Manassas, VA, USA). Lung cancer H460 cells were cultured in the Roswell Park Memorial Institute (RPMI; Gibco, Gaithersburg, MA, USA) medium whereas human MCF7 and CaCo 2 cells were maintained in Dulbecco's modified eagle medium (DMEM; Gibco, Gaithersburg, MA, USA). Human dermal papilla cells (DPCs) purchased from Applied Biological Materials Inc. (Richmond, BC, Canada) were cultured in a Prigrow III medium (Applied Biological Materials Inc., Richmond, BC, Canada). All culture mediums were supplemented with 2 mM L-glutamine, 10% FBS (fetal bovine serum) and 100 units/mL penicillin/streptomycin. The cells were maintained in at 37 • C with 5% CO 2 until 70-80% confluence before using in further experiments.

Cell Viability Assay
After 24 h of indicated treatment, cells seeded at a density of 1 × 10 4 cells/well in a 96-well plate was further incubated with 0.4 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich Chemical, St. Louis, MO, USA) for 3 h at 37 • C and kept from light. Then, the formed formazan crystal was solubilized in a DMSO prior measurement of optical density (OD) at 570 nm by using a microplate reader (Anthros, Durham, NC, USA). The OD ratio of treated to control cells, which were incubated with 0.5% DMSO as a solvent vehicle, was calculated and presented as percent cell viability [19,21].
Furthermore, the half-maximum inhibitory concentration (IC 50 ) was calculated and used for determination of selective index in each cancer cell. The selectivity index was represented from the ratio between the IC 50 value in dermal papilla cells and the IC 50 value in cancer cells [20].

Detection of Mode of Cell Death
Apoptosis and necrosis cell death were evaluated by nuclear staining assay. The treated cells were costained with 0.02 µg/mL Hoechst 33342 (Sigma-Aldrich Chemical, St. Louis, MO, USA) and 0.01 µg/mL propidium iodide (Sigma-Aldrich Chemical, St. Louis, MO, USA) at 37 • C for 30 min. The mode of cell death was visualized under a fluorescence microscope (Olympus IX51 with DP70, Olympus, Tokyo, Japan). Apoptosis cells were characterized with a bright-blue fluorescence of Hoechst 33342 which stained fragmented DNA and condensed nuclei. Meanwhile, necrosis cells were distinguished by red fluorescence of propidium iodide. The percentage of apoptosis was represented from the ratio between the number of apoptosis cells to the total cell number [19,22].

Statistical Analysis
The statistical analysis was performed via SPSS version 22 (IBM Corp., Armonk, NY, USA) with one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Any p-value under 0.05 was considered as a statistical significance.

Conclusions
In this study, three novel dihydrophenanthrenes derivatives, cymensifins A-C (1-3), together with two known compounds, cypripedin (4) and gigantol (5), were isolated from the aerial parts of C. ensifolium. The structures of the new compounds were elucidated by spectroscopic analysis. The three new compounds from this plant were evaluated for their cytotoxicity on human lung cancer H460, breast cancer MCF7, and colon cancer CaCo 2 cells. Cymensifin A (1) showed a promising anticancer effect against various cancer cells with higher safety profiles compared with cisplatin, an available chemotherapy. To the best of our knowledge, this research is the first record on the chemical constituents and biological activity of this plant.