Cytotoxic Homoisoflavonoids from Ophiopogon japonicus Tubers

Abstract

A phytochemical fractionation of a methanol extract of Ophiopogon japonicus tubers led to the isolation of a new homoisoflavanone, homoisopogon A (1), and three new homoisoflavanes, homoisopogon B–D (2–4). Their chemical structures were elucidated by mass, NMR, and circular dichroism (CD) spectroscopic methods. Homoisopogon A (1) exhibited potent cytotoxicity against human lung adenocarcinoma LU-1, human epidermoid carcinoma KB, and human melanoma SK-Mel-2 cancer cells with IC₅₀ values ranging from 0.51 to 0.66 µM.

Ophiopogon japonicus (L.f) KER-GAWL. (Convallariaceae) is distributed widely in Southeast Asia. In traditional Vietnamese medicine, it has been used widely as a tonic agent for the treatment of cough, fever, epistaxis, inflammation, respiratory disease, constipation, and gastrointestinal disorders.¹⁾ Previous phytochemical investigations have revealed that O. japonicus contains steroidal saponins, homoisoflavonoids, polysaccharides, phenolic acids, and sesquiterpenes.^2,3) Anti-inflammatory, antitumor, antidiabetic, anti-oxidant activities of O. japonicus have been reported.²⁾ Homoisoflavonoids belong to a special flavonoid class which their B-ring connected to C-ring by an additional CH₂ group. Homoisoflavonoids isolated from Ophigopogon have been shown to exert various biological activities including protective effects on cerebral ischemia/reperfusion (I/R) injury, antioxidant, anti-inflammation and cytotoxic activities.^4–8)

In the present study, a methanol extract of O. japonicus tubers showed strong cytotoxicity against human lung cancer cell lines. Phytochemical fractionation of the methanol extract of O. japonicus tubers led to the isolation of a homoisoflavanone (1), and three homoisoflavanes (2–4) (Fig. 1). The cytotoxic activity against human lung adenocarcinoma LU-1, human epidermoid carcinoma KB, and human melanoma SK-Mel-2 cancer cells of the isolated compounds was investigated.

Fig. 1. The Chemical Structure of Compounds 1–4

Results and Discussion

The high resolution-electrospray ionization (HR-ESI)-MS spectrum of compound 1 had a molecular ion peak at m/z 345.1329 [M+H]⁺, corresponding to the molecular formula C₁₉H₂₀O₆. Its ¹H-NMR spectrum showed the characteristic resonances of a homoisoflavanone at δ_H 11.84 (1H, s, OH-5), 4.16 (1H, dd, J=10.0, 11.0 Hz, Ha-2), 4.43 (1H, dd, J=5.0, 11.0 Hz, Hb-2), 3.06 (1H, m, H-3), 2.82 (1H, dd, J=6.0, 14.0 Hz, Ha-9), and 3.00 (1H, dd, J=5.0, 14.0 Hz, Hb-9)⁹⁾ (Table 1). Moreover, the 2′-OH and 4′-OCH₃ substitution patterns in the B-ring were deduced from the ABX type aromatic proton resonances at δ_H 6.46 (1H, d, J=2.5 Hz, H-3′), 6.41 (1H, dd, J=2.5, 8.0 Hz, H-5′), 6.94 (1H, d, J=8.0 Hz, H-6′). In the ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra, one methyl, two methoxyls, two methylenes, one aliphatic methine, four aromatic methines, eight aromatic quaternary carbons and a carbonyl group and were observed. The heteronuclear single quantum coherence (HSQC) spectrum of 1 established the connectivity between the carbons and respective protons, which confirmed the presence of twelve direct H–C correlations corresponding to twelve proton signals of one methyl, two methoxyls, two methylene, one aliphatic methine, and four aromatic methine groups. The polysubstituted A-ring structure was identified by assignment of the aromatic proton singlet at δ_H 5.98 (1H, s), which was attributed to H-8, according to its heteronuclear multiple bond connectivity (HMBC) correlations with C-4a, C-6, C-7, and C-8a. The HMBC correlations from methyl protons (δ_H 1.97) to C-5, C-6, and C-7 indicated the presence of methyl group at C-6. The ABX system of B-ring was confirmed by the HMBC correlations from H-6′ to C-2′, C-4′, from H-3′ to C-1′, C-5′, and from H-5′ to C-1′, C-3′. Furthermore, the coupling from H-9 to C-2, C-4, C-1′, C-2′, C-6′, and from H-6′ to C-9 indicated that the B-ring connected to C-ring via a methylene bridge. Additionally, the location of two methoxyl groups (δ_H 3.83 and 3.75) at C-7 and C-4′ was deduced from the HMBC correlations (Fig. 2). The absolute configuration of C-3 was determined to be R in accordance with the positive Cotton effect at 290–294 nm in the circular dichroism (CD) spectra.¹⁰⁾ Finally, the structure of compound 1 was elucidated as (3R)-2′,5-dihydroxy-4′,7-dimethoxy-6-methylhomoisoflavanone; named homoisopogon A.

Table 1. ¹H-NMR Data of Compounds 1–4 (δ in ppm, J in Hz)

Position	1	2	3	4
2	4.16, dd (10.0, 11.0)	4.06, dd (2.0, 11.0)	4.09, dd (2.5, 11.0)	4.10, m
	4.43, dd (5.0, 11.0)	3.83, dd (6.0, 11.0)	3.83, dd (6.5, 11.0)	3.75, dd (8.0, 11.0)
3	3.06, m	2.25, m	2.28, m	2.21, m
4	—	2.80, dd (5.5, 16.0)	2.86, dd (5.5, 16.0)	2.69, dd (5.5, 15.5)
		2.44, dd (6.5, 16.0)	2.50, dd (6.0, 16.0)	2.38, dd (8.0, 15.5)
5	—	6.76, s	6.92, d (8.5)	6.72, brs
6	—	—	6.47, dd (2.5, 8.5)	—
8	5.98, s	6.34, s	6.44, d (2.5)	6.26, s
9	3.00, dd (5.0, 14.0)	2.64, dd (9.0, 14.0)	2.65, dd (9.0, 14.0)	2.60, dd (7.5, 14.0)
	2.82, dd (6.0, 14.0)	2.52, dd (6.5, 14.0)	2.54, dd (6.5, 14.0)	2.51, dd (7.5, 14.0)
2′	—	—	—	6.67, d (2.5)
3′	6.46, d (2.5)	6.38, d (2.5)	6.44, d (2.5)	—
5′	6.41, dd (2.5, 8.0)	6.40, dd (2.5, 8.0)	6.45, dd (2.5, 8.0)	6.74, d (8.0)
6′	6.94, d (8.0)	6.98, d (8.0)	7.00, d (8.0)	6.62, dd (2.5, 8.0)
6-CH3	1.97, s	2.10, s	—	2.13, s
7-OCH3	3.83, s	3.74, s	3.76, s	—
4′-OCH3	3.75, s	3.73, s	3.75, s	—
–O–CH2–O–	—	—	—	5.93, s
5-OH	11.84, s	—	—	—

Fig. 2. Keys HMBC Correlations of 1–4

Table 2. ¹³C-NMR Data of Compounds 1–4

Position	1	2	3	4
2	70.0	69.2	69.3	69.7
3	45.8	33.2	33.1	34.4
4	199.5	30.2	30.4	30.2
4a	102.4	112.4	113.5	113.2
5	160.5	131.4	130.5	131.5
6	106.0	119.1	107.7	115.9
7	166.2	156.7	159.0	152.7
8	90.5	98.9	101.4	102.7
8a	161.6	152.7	154.9	153.1
9	26.3	31.0	30.9	37.7
1′	116.4	118.0	117.8	133.2
2′	155.4	155.0	154.9	109.3
3′	102.6	102.0	102.0	147.6
4′	160.0	159.3	159.4	145.9
5′	106.4	106.1	106.2	108.1
6′	131.6	131.5	131.5	121.8
6-CH3	6.85	15.3	—	14.9
7-OCH3	55.8	55.2	55.3	—
4′-OCH3	55.3	55.3	55.3	—
–O–CH2–O–	—	—	—	100.8

Compound 2 was obtained as a yellow powder with the molecular formula C₁₉H₂₂O₄, which was established from the HR-ESI-MS data (m/z 315.1602 [M+H]⁺). The ¹H-NMR spectrum showed characteristic resonances at δ_H 4.06 (1H, dd, J=2.0, 11.0 Hz) and 3.83 (1H, dd, J=6.0, 11.0 Hz) corresponding to H-2 protons, δ_H 2.25 (1H, m) corresponding to H-3, δ_H 2.80 (1H, dd, J=5.5, 16.0 Hz) and 2.44 (1H, dd, J=6.5, 16.0 Hz) corresponding to H-4 protons, and δ_H 2.64 (1H, dd, J=9.0, 14.0 Hz) and 2.52 (1H, dd, J=6.5, 14.0 Hz) corresponding to H-9 protons. The ¹H-NMR spectrum also showed signals at δ_H 6.38 (1H, d, J=2.5 Hz, H-3′), 6.40 (1H, d, J=2.5, 8.0 Hz, H-5′), and 6.98 (1H, d, J=8.0 Hz, H-6′) suggesting a 1,2,4-trisubstituted pattern for the B ring. Additionally, two aromatic singlet protons at δ_H 6.76 (1H, s, H-5) and 6.34 (1H, s, H-8) were detected, indicating the presence of a tetrasubstituted A ring. In the ¹³C and DEPT spectra, one methyl, two methoxyls, three methylenes, one aliphatic methine, five aromatic methines, and seven aromatic quaternary carbons were observed. These data suggested that 2 possesses a homoisoflavane skeleton.¹¹⁾ The HMBC correlations from the aromatic methyl group at δ_H 2.10 (3H, s) to C-5 (δ_C 131.4), C-6 (δ_C 119.1), and C-7 (δ_C 156.7), and from the methoxy signals δ_H 3.74 to C-7, and δ_H 3.73 to C-4′ (δ_C 159.3), indicated that the methyl and methoxyl groups attached to C-6, C-7, and C-4, respectively. The absolute configuration of C-3 was determined to be R based on the Cotton effect at 230 nm (negative) and 285 nm (positive) in the CD analysis.¹¹⁾ Accordingly, the structure of 2 was elucidated as (3R)-4′,7-dimethoxy-2′-hydroxy-6-methylhomoisoflavane, named homoisopogon B.

The ¹H- and ¹³C-NMR spectroscopic data of compound 3 were nearly identical to those of 2, except for the absence of the methyl group, and ABX-type protons appeared in the A-ring [6.92 (1H, d, J=8.5 Hz, H-5), 6.47 (1H, d, J=2.5, 8.5 Hz, H-6), and 6.44 (1H, d, J=2.5 Hz, H-8)]. Consistently, HR-ESI-MS revealed a molecular ion peak at m/z 301.1425 [M+H]⁺ corresponding to the molecular formula C₁₈H₂₀O₄. The absolute configuration of C-3 was determined to be R based on the CD analysis.¹¹⁾ Thus, the structure of 3 was established as (3R)-4′,7-dimethoxy-2′-hydroxyhomoisoflavane, named homoisopogon C.

Compound 4 was formulated as C₁₈H₁₈O₄ based on HR-ESI-MS (m/z 299.1271 [M+H]⁺). The ¹H- and ¹³C-NMR data for 4 were similar to those for 2 except for the presence of a methylenedioxy group [δ_H 5.93 (2H, s)] instead of two methoxy groups. Also, the HMBC correlations of H-2′/C-9, C-4′, and C-6′, and H-5′/C-1′, C-3′, and C-4′, as well as H-6′/C-9, C-2′, and C-4′, indicated the presence of a 1,3,4-trisubstituted benzene ring in 6, instead of the 1,2,4-trisubstituted benzene ring in 2. The position of the methylenedioxy group at C-3′-C-4′ was confirmed by HMBC correlations (Fig. 2). The absolute configuration of C-3 was determined to be R based on the CD analysis.¹¹⁾ Accordingly, compound 4 was elucidated as (3R)-7-hydroxy-3′,4′-methylenedioxy-6-methylhomoisoflavane, named homoisopogon D.

Compounds 1–4 were evaluated for their cytotoxic effect against LU-1, KB, and SK-Mel-2 cells. As indicated in Table 3, homoisopogon A (1) was the most potent, followed by homoisopogon B (2) and homoisopogon C (3). Interestingly, compound 1 exhibited a strong cytotoxic effect on all tested cell lines with the IC₅₀ values of 0.51–0.66 µM. The activity is comparable to that of the positive control, ellipticine (Table 3). Compound 1 was probably responsible for the cytotoxic effect observed of the MeOH extract since other isolated components showed weak or null effect. The cytotoxic effect of homoisoflavonoids has been indicated elsewhere,¹²⁾ and the structure–activity relationship has been investigated.¹³⁾ Accordingly, the 2′-hydroxy and 4′-methoxy groups seem to have a contribution to the activity. In our study, compounds 1, 2, and 3 possessing 2′-hydroxy and 4′-methoxy substituent showed positive effect on at least one cancer cell line. Compound 4 with a methylenedioxy group at C-3′-C-4′ and lack of hydroxyl group at C-2′, was inactive against all tested cells.

Table 3. Cytotoxic Effect of Compounds 1–4 (IC₅₀±S.D., µg/mL for Extract, µM for Compounds)

Samples	LU-1	KB	SK-Mel-2
MeOH extract	8.7±1.24	15.8±2.31	18.6±3.82
1	0.66±0.09	0.51±0.03	0.66±0.03
2	17.14±2.41	>30	28.29±3.52
3	27.66±3.18	>30	>30
4	>30	>30	>30
Ellipticine	0.43±0.02	0.51±0.02	0.27±0.02

In conclusion, four new homoisoflavonoids including one homoisoflavanone (1) and three homoisoflavanes (2, 3, and 4) were isolated from tubers of Ophiopogon japonicus KER-GAWLER. In a cytotoxic activity investigation, compound 1 exhibited potent effect against three cancer cells LU-1, KB, and SK-Mel-2. The results indicated that homoisoflavoinoids from O. japonicus may be a potential material for the development of anticancer agents.

Experimental

General Experimental Procedures

TLC was performed using precoated Kiesel gel 60 F254 and visualized by UV light 254 nm and 10% H₂SO₄ reagent with heat. Column chromatography was performed using Silica gel 60 (Merck, 70–230 mesh). Optical rotation was recorded a JASCO P-2000 digital polarimeter. The IR spectra were obtained from a Tensor 37 FT-IR spectrometer (Bruker, Ettlingen, Germany). CD spectra were obtained with a JASCO J-1100 spectropolarimeter. NMR experiments were carried out on a Bruker AM500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) using tetramethylsilane (TMS) as internal standard. The HR-ESI-MS were recorded on a Waters Q-TOF micromass spectrometer Waters Q-TOF micromass spectrometer. Absorbance of bioassay solutions was read on an xMark microplate spectrophotometer.

Plant Materials

The tubers of O. japonicus were collected in Feb. 2014 at Me Linh, Hanoi and identified by Prof. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens were deposited at the Department of Bioactive Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology.

Extraction and Isolation

The air-dried and powdered tubers of O. japonicus (2.4 kg) were extracted with methanol (4 L×3 times) in a sonic bath for 30 min at 40°C. The combined extracts were concentrated under a vacuum to obtain a crude residue (360 g), which was then resuspended in water (2 L), and extracted by chloroform (1 L×3 times) to obtain chloroform (8 g) and water residues. The chloroform residue was chromatographed on a silica gel column eluted with a gradient of 1–100% ethyl acetate in hexane to afford nine fractions F1–F9. Fraction F3 was fractionated on a silica gel column eluted with hexane–ethyl acetate (9 : 1, v/v) to give four fractions F3.1–F3.3. Compound 2 (125.6 mg) and 4 (46.3 mg) were isolated from F3.1 by using a reverse phase C₁₈ column eluted with acetone–water (1 : 1, v/v). The fraction F3.2 was passed through a C₁₈ column (acetone–water 3 : 2, v/v) to obtain 3 (31.5 mg). Compound 1 (40.4 mg) was purified from F3.3 by using a reverse phase C₁₈ column (acetone–water 3 : 2, v/v) and a silica gel column (hexane–ethyl acetate 7 : 1, v/v).

Homoisopogon A (1)

Yellow amorphous powder; [α]_D²⁵=−25.0 (c=0.1, MeOH); CD (c=0.29 mM, MeOH) λ_max (Δε) nm 290 (+16.8); IR ν_max (KBr): 3368, 2925, 1644, 1588, 1280 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃): see Tables 1 and 2; HR-ESI-MS: m/z 345.1329 [M+H]⁺ (Calcd for C₁₉H₂₁O₆ 345.1338).

Homoisopogon B (2)

Yellow amorphous powder; [α]_D²⁵=+18.0 (c=0.1, MeOH); CD (c=0.31 mM, MeOH) λ_max (Δε) nm 230 (−8.8), 285 (+10.4); IR ν_max (KBr): 3393, 1620, 1509, 1190 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃): see Tables 1 and 2; HR-ESI-MS: m/z 315.1602 [M+H]⁺ (Calcd for C₁₉H₂₃O₄ 315.1596).

Homoisopogon C (3)

Yellow amorphous powder; [α]_D²⁵=+23.0 (c=0.1, MeOH); CD (c=0.33 mM, MeOH) λ_max (Δε) nm 230 (−12.1), 285 (+8.4); IR ν_max (KBr): 3430, 1620, 1508, 1159 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃): see Tables 1 and 2; HR-ESI-MS: m/z 301.1425 [M+H]⁺ (Calcd for C₁₈H₂₁O₄ 301.1440).

Homoisopogon D (4)

Yellow amorphous powder; [α]_D²⁵=+12.5 (c=0.1, MeOH); CD (c=0.33 mM, MeOH) λ_max (Δε) nm 290 (+9.1); IR ν_max (KBr): 1625, 1501, 1242 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) and ¹³C-NMR (125 MHz, CDCl₃): see Tables 1 and 2; HR-ESI-MS: m/z 299.1271 [M+H]⁺ (Calcd for C₁₈H₁₉O₄ 299.1283).

Cell Culture and Cytotoxic Assay

KB (human epidermoid carcinoma), LU-1 (human lung adenocarcinoma), and SK-Mel-2 (human melanoma) cell lines, obtained from ATC C (American Type Culture Collection, Manassas, VA, U.S.A.), were maintained as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; GIBCO, Grant Island, NY, U.S.A.), sodium bicarbonate, penicillin G, and streptomycin at 37°C under a humidified atmosphere of 5% CO₂. The cytotoxicity of the isolated compounds was determined using the sulforhodamine B (SRB) method as previously reported.¹⁴⁾

Acknowledgments

This work is supported by the National Foundation for Science and Technological Development (NAFOSTED 104.01-2014.05). We thank the Institute of Chemistry (VAST) for the NMR experiments; and Prof. Dongho Lee (Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea) for HR-ESI-MS and CD measurements.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials (spectroscopic data for compounds including 1D-, 2D-NMR and CD spectra).

References

• 1) Do T. L., “Vietnamese medicinal herbs and remedies,” Medicinal Publisher, Hanoi, Vietnam, 2004.
• 2) Chen M. H., Chen X. J., Wang M., Lin L. G., Wang Y. T., J. Ethnopharmacol., 181, 193–213 (2016).
• 3) Liu S. Q., Kang L. P., Zhang J., Liu Y. X., Zhao Y., Liu B., Ma B. P., J. Asian Nat. Prod. Res., 18, 520–527 (2016).
• 4) Lin Y., Zhu D., Qi J., Qin M., Yu B., J. Pharm. Biomed. Anal., 52, 757–762 (2010).
• 5) Lin M., Sun W., Gong W., Zhou Z., Ding Y., Hou Q., PLoS ONE, 10, e0124558 (2015).
• 6) Hung T. M., Thu C. V., Dat N. T., Ryoo S. W., Lee J. H., Kim J. C., Na M., Jung H. J., Bae K., Min B. S., Bioorg. Med. Chem. Lett., 20, 2412–2416 (2010).
• 7) Duan C. L., Kang Z. Y., Lin C. R., Jiang Y., Liu J. X., Tu P. F., J. Asian Nat. Prod. Res., 11, 876–879 (2009).
• 8) Zhou C. X., Zou L., Mo J. X., Wang X. Y., Yang B., He Q. J., Gan L. S., Helv. Chim. Acta, 96, 1397–1405 (2013).
• 9) Chang J. M., Shen C. C., Huang Y. L., Chien M. Y., Ou J. C., Shieh B. J., Chen C. C., J. Nat. Prod., 65, 1731–1733 (2002).
• 10) Guo H., Zhao H., Kanno Y., Li W., Mu Y., Kuang X., Inouye Y., Koike K., Jiang H., Bai H., Bioorg. Med. Chem. Lett., 23, 3137–3139 (2013).
• 11) Awale S., Miyamoto T., Linn T. Z., Li F., Win N. N., Tezuka Y., Esumi H., Kadota S., J. Nat. Prod., 72, 1631–1636 (2009).
• 12) Lin L. G., Liu Q. Y., Ye Y., Planta Med., 80, 1053–1066 (2014).
• 13) Alali F., El-Elimat T., Albataineh H., Al-Balas Q., Al-Gharaibeh M., Falkinham J. O. III, Chen W.-L., Swanson S. M., Oberlies N. H., J. Nat. Prod., 78, 1708–1715 (2015).
• 14) Huong P. T., Diep C. N., Thanh N. V., Tu V. A., Hanh T. H., Cuong N. T., Thao N. P., Cuong N. X., Thao D. T., Thai T. H., Nam N. H., Ban N. K., Kiem P. V., Minh C. V., Nat. Prod. Commun., 9, 1255–1257 (2014).