Identification of Cytotoxic Constituents from the Whole Plant of Isodon ternifolius

Abstract

A new oxygenated spiroketone, isodonspiroketone (1), and 4 known ones (2-5) were isolated from the whole plant of Isodon ternifolius (D.Don) Kudô. The structure of isodonspiroketone (1) was determined by nuclear magnetic resonance, mass spectroscopy, and circular dichroism spectral data. Compound 3 has not been previously isolated from I. ternifolius. Their cytotoxic activities were evaluated against A549, HepG2, and MDA-MB-231 cancer cell lines in vitro. New compound (isodonspiroketone, 1) showed moderate cytotoxic activities against A549, HepG2, and MDA-MB-231 cancer cell lines with half-maximal inhibitory concentration values of 23.84 ± 2.73, 27.77 ± 3.01, and 17.26 ± 1.61 μM, respectively; meanwhile, the others were inactive.

Keywords

Lamiaceae phenolic spiroketone cytotoxic activity cancer cell lines

Isodon ternifolius (D.Don) Kudô is a member of the Isodon genus, which belongs to Lamiaceae family. This genus consists of approximately 150 species, which are primarily distributed in tropical and subtropical Asia.¹ Previous studies examining the chemical constituents in I. ternifolius have identified the presence of diterpenoid,^2

-6 lignan, and phenylethanoid compounds.⁷ I. ternifolius has been commonly used in folk medicine for the treatment of enteritis,⁸ icterohepatitis,⁸ inflammation,^7,8 and cancer.^3,4 Despite the great medicinal value, there has been limited report on chemical constituents and cytotoxic activities against A549, HepG2, and MDA-MB-231 cancer cell lines. Therefore, this work describes the isolation and structural elucidation of isolated compounds as well as the evaluation of their cytotoxic activities against 3 cancer cell lines.

The methanol extract was partitioned with n-hexane, ethyl acetate (EtOAc), and n-butanol (BuOH) to obtain n-hexane, EtOAc, and BuOH-soluble fractions, respectively. Based on bioactivity-guided fractionation (data not shown), the n-hexane and EtOAc fractions were subjected to column chromatography on a silica gel and C18-RP silica gel column to obtain a new compound (1) and 4 known ones (2-5) (Figure 1).

Figure 1

Chemical structure of compounds (1-5) isolated from Isodon ternifolius.

Compound 1 was white amorphous powder obtained following crystallization from acetone. The molecular formula of 1 was determined as C₁₈H₁₆O₈ by the high-resolution time-of-flight mass spectroscopy (HR-TOF-MS) at m/z 359.0743 [M − H]⁻ (Supplemental Material). The ultraviolet (UV) spectrum displayed maximum absorption bands at 238, 276, and 336 nm. The infrared (IR) spectrum of 1 suggested the presence of a hydroxyl group (3400 cm⁻¹), ketone group (1702 cm⁻¹), conjugated carbonyl group (1632 cm⁻¹), and aromatic ring (1568 and 1512 cm⁻¹). The ¹H-nuclear magnetic resonance (NMR) spectrum (in acetone-d ₆) of 1 revealed the presence of an olefinic proton at δ _H 6.28 (1H, s, H-8), 3 methoxy protons [δ _H 3.86 (3H, s, 3-OCH₃), 4.23 (3H, s, 4-OCH₃), and 3.89 (3H, s, 6-COOCH₃)], and 1 proton as broad singlet at δ _H 5.65 (1H, s, 5-OH). The ¹H-NMR spectrum of 1 further showed 5 aromatic protons [δ _H 7.91 (2H, m, H-11/H-15), 7.58 (2H, m, H-12/H-14), and 7.66 (1H, m, H-13)], which indicates the presence of phenyl group (Table 1 and Supplemental Material). The ¹³C-NMR (in acetone-d ₆), distortionless enhancement by polarization transfer, and heteronuclear single-quantum coherence (HSQC) spectra of 1 exhibited 18 signals due to 2 olefinic carbons [δ _C 101.3 (C-8) and 186.6 (C-9)], 3 methoxy carbons [δ _C 59.9 (3-OCH₃), 60.8 (4-OCH₃), and 54.3 (6-COOCH₃)], and 6 sp ² carbons [δ _C 129.0 (C-10), 128.0 (C-11/C-15), 130.1 (C-12/C-14), and 134.1 (C-13)], indicating the presence of 1 benzene ring (Table 1, Supplemental Material). In addition, 2 ketone carbon signals [δ _C 185.8 (C-2) and 194.7 (C-7)], 1 carbonyl carbon at δ _C 169.9 (C-6) together with 4 other carbon atoms [δ _C 95.6 (C-1), 138.9 (C-3), 165.5 (C-4), and 80.4 (C-5)] were also detected in the ¹³C-NMR and HSQC spectra (Table 1, Supplemental Material). The ¹H- and ¹³C-NMR spectra of 1 were similar to those of limnophilaspiroketone isolated from Limnophila geoffravi,⁹ and (±)-Murrayaspiroketone isolated from Murraya paniculata,¹⁰ except that a para-hydroxyl group located at C-13 [δ _H 8.41 (1H, br s, 13-OH), δ _C 162.1 (C-13)] in limnophilaspiroketone is replaced by an aromatic proton [δ _H 7.66 (1H, m, H-13), δ _C 134.1 (C-13)] in 1 (Figure 1, Supplemental Material). This was confirmed by heteronuclear multiple bond correlations (HMBCs) of the aromatic proton at δ _H 7.66 (1H, m, H-13) to δ _C 128.0 (C-11/C15) and the cross-peak between H-12, H-14, and H-13 in the correlation spectroscopy spectrum of 1 (Figure 2, Supplemental Material). The HMBCs of H-11 and H-15 to C-8, and H-8 to C-10 as well as the correlation of H-8 to H-11 observed in the nuclear Overhauser effect spectroscopy (NOESY) spectrum additionally confirmed the existence of a phenyl group and its connection to furan moiety at C-9 (Figure 2, Supplemental Material). Furthermore, the HMBCs of the methoxy proton (3-OCH ₃) to C-3, methoxy proton (4-OCH ₃) to C-4, and the other methoxy proton (6-COOCH ₃) to C-6 were also observed indicating the location of 3 methoxy groups in 1 (Figure 2, Supplemental Material). Additionally, the correlations of the proton as a broad singlet at δ _H 5.65 (1H, s, 5-OH) to C-1, C-4, C-5, and C-6 were also observed in the HMBC spectrum, suggesting that the hydroxyl group was located at C-5 (Figure 2, Supplemental Material).

Table 1

¹H-NMR and ¹³C-NMR Spectroscopic Data for Compound.

Position	1
	δ _H (ppm, J in Hz)^a	δ _C (ppm)^b	δ _H (ppm, J in Hz)^c	δ _C (ppm)^d
1		95.6		94.7
2		185.8		184.2
3		138.9		138.4
4		165.5		163.4
5		80.4		78.9
6		169.9		169.4
7		194.7		194.5
8	6.28 (1H, s)	101.3	6.05 (1H, s)	100.8
9		186.6		186.9
10		129.0		128.0
11	7.91 (1H, m)	128.0	7.76 (1H, m)	127.4
12	7.58 (1H, m)	130.1	7.48 (1H, m)	129.1
13	7.66 (1H, m)	134.1	7.58 (1H, m)	133.6
14	7.58 (1H, m)	130.1	7.48 (1H, m)	129.1
15	7.91 (1H, m)	128.0	7.76 (1H, m)	127.4
3-OCH₃	3.86 (3H, s)	59.9	3.94 (3H, s)	59.8
4-OCH₃	4.23 (3H, s)	60.8	4.26 (3H, s)	60.7
5-OH	5.65 (1H, br s)
6-COOCH₃	3.89 (3H, s)	54.3	3.94 (3H, s)	54.6

Abbreviation: Abbreviation: NMR, nuclear magnetic resonance.

^a ¹H NMR (500 MHz in acetone-d ₆, δ values) spectroscopic data.

^b ¹³C NMR (125 MHz in acetone-d ₆, δ values) spectroscopic data.

^c ¹H NMR (500 MHz in CDCl₃, δ values) spectroscopic data.

^d ¹³C NMR (125 MHz in CDCl₃, δ values) spectroscopic data.

Figure 2

Important HMBCs and NOESY spectra of compound 1. HMBC, heteronuclear multiple bond correlation; NOESY, nuclear Overhauser effect spectroscopy.

The optical rotation value of compound 1 was found to be zero ( $[α]_{D}^{25}$ 0° [c 0.1, MeOH]) together with the lack of Cotton effects in the circular dichroism spectrum (Supplemental Material), suggesting that isodonspiroketone (1) is a racemate. In addition, there are no NOESY (measured in acetone-d ₆) correlations from OH-5 to H-11 and H-15 as well as from OH-5 to H-12 and H-14 in compound 1 (Supplemental Material). Therefore, the absolute configurations at the chiral centers of compound 1 have not been determined. Based on these findings, compound 1 was determined to be as shown and, thus, assigned to the trivial name, isodonspiroketone.

The known compounds were identified to be 1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl-3′,4′,5′-trimethoxybenzoate (2),¹¹ 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-ol-3´,4´,5´-trimethoxybenzoate (3),¹² moslosooflavone (7,8-dimethoxy-5-hydroxyflavone, 4),¹³ and techtochrysine (7-methoxy-5-hydroxyflavone, 5)¹⁴ by comparison of their NMR data with those in the literatures.

Five isolated compounds (1, 5) were tested for their cytotoxic activity against A549, HepG2, and MDA-MB-231 cancer cell lines in vitro assay by the method as described previously.^15,16 Among them, a new compound (isodonspiroketone, 1) showed moderate cytotoxic activities against A549, HepG2, and MDA-MB-231 cancer cell lines with half-maximal inhibitory concentration (IC₅₀) values of 23.84 ± 2.73, 27.77 ± 3.01, and 17.26 ± 1.61 µM, respectively; meanwhile, the others were inactive (Table 2). In this assay, ellipticine was used as a positive control. Ellipticine exhibited cytotoxic activity against A549, HepG2, and MDA-MB-231 cancer cell lines with IC₅₀ values of 1.67 ± 0.20, 2.11 ± 0.20, and 1.83 ± 0.12 µM, respectively.^15
-17

Table 2

Cytotoxic Activity of Isolated Compounds (1–5) Against Cancer Cell Lines.

Compounds	IC₅₀ (µM)
Compounds	A549	HepG2	MDA-MB-231
1	23.84 ± 2.73	27.77 ± 3.01	17.26 ± 1.61
2	>100	>100	>100
3	–^a	–	–
4	>100	>100	>100
5	>100	>100	>100
Ellipticine^b	1.67 ± 0.20	2.11 ± 0.20	1.83 ± 0.12

Abbreviation: Abbreviation: IC₅₀, half-maximal inhibitory concentration.

^aNot tested.

^bUsed as a positive control.

Experimental

General Experimental Procedures

¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) were measured on a Varian Unity Inova 500 MHz spectrometer. TOF-MS was obtained from a Varian Fourier transform mass spectrometer and MicroQ-TOF III (Bruker Daltonics, Ettlingen Germany). IR spectroscopy was fulfilled on Nicolet Impact 410 spectrometer. UV was performed in spectroscopic V-630 UV-VIS instrument. Column chromatography was carried out on silica gel (Si 60 F₂₅₄, 40-63 mesh, Merck, St Louis, MO, USA). All solvents were redistilled before use. Compounds were visualized under UV radiation (254, 365 nm) and by spraying plates with 10% H₂SO₄ followed by heating with a heat gun. Thin-layer chromatography (TLC) was carried out on silica gel F254-precoated glass plates and RP-18 F254S plates (Merck, Germany). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-ethylenediamine tetraacetic acid 0.25%, streptomycin, and penicillin were obtained from Hyclone (Logan, UT, USA).

Plant Material

The whole plant of Isodon ternifolius (D.Don) Kudô was collected in Thai Nguyen province, Vietnam, and authenticated by Assoc. Prof. Dr Tran Huy Thai, Institute of Ecology and Biological Resources, VAST. A voucher specimen (IS20171001.HN) has been stored at the Institute of Natural Products Chemistry, VAST.

Extraction and Isolation

Dried, powdered whole I. ternifolius plant (4.0 kg) was extracted using MeOH (3 × 8 L) by refluxing to produce 380 g crude extract. The crude extract was suspended in water (800 mL) and successively partitioned, using n-hexane (4 × 1.5 L), EtOAc (4 × 1.5 L), and BuOH (4 × 1.5 L). An activity-guided fractionation study resulted in the n-hexane and EtOAc extracts being chosen for further study. The n-hexane extract (48.84 g) was chromatographed on an open silica gel column chromatography (CC) and eluted with n-hexane-acetone (20:1 to 0:1), yielding 10 fractions (CMH.1-CMH.10) according to their TLC profiles. Fraction CMH.4 (4.33 g) was further subjected to silica gel CC and eluted with n-hexane-EtOAc (8:1 to 0:1) to afford 11 subfractions (CMH.4-1 to CMH.4-11). Further purification of the subfraction CMH.4-2 (250 mg), using an RP-C18 CC, eluting with acetone:water (3:1), resulted in the isolation of compounds 2 (186.2 mg) and 3 (4.0 mg). Fraction CMH.6 (4.44 g) was also subjected to silica gel CC, eluted with n-hexane-EtOAc (5:1 to 0:1), yielding 9 subfractions (CMH.6-1 to CMH.6-9). Further purification of subfraction CMH.6-2 (450 mg), using an RP-C18 column, eluted with acetone:water (2:1), resulted in the isolation of compounds 4 (316.0 mg) and 5 (11.5 mg). The EtOAc extract (54.8 g) was applied to an open silica gel CC and eluted with n-hexane−acetone (10:1 to 0:1), yielding 10 fractions (CME1-CME10). Fraction CME6 (2.2 g) was further subjected to silica gel CC, using CHCl₃−MeOH (10:1 to 0:1) as the eluent, to afford 6 subfractions (CME6-1 to CME6-6). Compound 1 (16.8 mg) was isolated by recrystallization (from acetone) from subfraction CME6-5 (150 mg).

Isodonspiroketone (1)

White amorphous powder.

$[α]_{D}^{25}$ 0° (c 0.1, MeOH).

UV λ _max (MeOH): 238, 276, 336 nm.

IR (KBr) ν _max 3384 (OH), 1702, 1632 (C = O), 1568, 1512, 1428, 1012 cm⁻¹.

¹H NMR (500 MHz, acetone-d ₆) and ¹³C NMR (125 MHz, acetone-d ₆), see Table 1.

HR-TOF-MS m/z 359.0743 [M−H]⁻ (calculated for C₁₈H₁₅O₈, 359.0756).

Cytotoxic Activity Assay

The cancer cell lines were maintained in RPMI 1640, which included L-glutamine with 10% FBS and 2% penicillin-streptomycin. Cells were cultured at 37°C in a 5% CO₂ incubator. Cytotoxic activity was measured using a modified MTT assay.^18,19 Viable cells were seeded in the growth medium (100 µL) into 96-well microtiter plates (1 × 10⁴ cells per well) and incubated at 37°C in a 5% CO₂ incubator. The test sample was dissolved in dimethyl sulfoxide (DMSO) and adjusted to final sample concentrations ranging from 5.0 to 150 µM by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to <0.1%. After standing for 24 hours, 10 µL of the test sample was added to each well. The same volume of DMSO was added to the control wells. On removing medium after 48 hours of the test sample treatment, MTT (5 mg/mL, 10 µL) was also added to each well. After 4-hour incubation, the plates were removed, and the resulting formazan crystals were dissolved in DMSO (150 µL). The optical density was measured at 570 nm. The IC₅₀ value was defined as the concentration of the sample that reduced absorbance by 50% relative to the vehicle-treated control.

Supplemental Material

Supplementary Material 1 - Supplemental material for Identification of Cytotoxic Constituents from the Whole Plant of Isodon ternifolius

Supplemental material, Supplementary Material 1, for Identification of Cytotoxic Constituents from the Whole Plant of Isodon ternifolius by Minh Quan Pham, Thi-Thuy-Huong Le, Tien-Lam Do, Thi-Hong-Minh Pham, Quoc-Long Pham, Phi-Hung Nguyen and Dao-Cuong To in Natural Product Communications

Footnotes

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 research is funded by Graduate University of Science and Technology under grant number GUST.STS.DDT2017-HH07.

ORCID iDs

Minh Quan Pham

Phi-Hung Nguyen

Dao-Cuong To

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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