Sage Journals: Discover world-class research

Abstract

A new benzofurane 7-methoxy-2-(3-hydroxy-4-methoxyphenyl)-benzofuran-5-carboxylate (1) and 5 known compounds, such as propacin (2), hisbiscolatone A (3), heliclactone (4), rosmarinic acid (5), and syringaldehyde (6), were isolated from methanol extract of the Helicteres hirsuta stems. Their structures were elucidated by spectroscopic analysis as Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) Furthermore, compounds 1 to 6 were evaluated for their cytotoxic activity against 4 human cancer cell lines such as Lu-1, MCF7, HepG2, and KB. The results showed that compound 2 exhibited moderate cytotoxic activity against Lu-1, MCF7, HepG2, and KB cell lines with IC₅₀ values of 43.97 ± 2.12, 46.53 ± 0.75, 52.63 ± 0.24, and 56.37 ± 1.04 µg/mL, respectively. This is the first report of compounds 2 to 6 from H. hirsuta.

Keywords

benzofuran derivative cytotoxic activity

Helicteres genus is a large genus of tropical trees and shrubs (family Sterculiaceae) with axillary flowers and fruits consisting of 5 twisted carpels.¹ Helicteres genus consisting of about 60 species distributed in tropical America and Asia. Many of them are used as folk medicines for diabetes, intestinal infection, stomach affection, empyema, uterus pain, etc.^2,3 In Vietnam, there are 9 Helicteres species, among them 6 species being used as traditional medicines.^4,5 The primary chemical constituents of this genus were triterpenoids,^6,7 cucurbitans,^8,9 lignans,¹⁰ flavonoids,^11,12 and coumarins,¹³ which possessed cytotoxic, antinociceptive, analgesic, anti-inflammatory, and antibacterial effects and hypoglycemic activities. Helicteres hirsuta is a small tree that is widely distributed in Asia such as Thailand, Indonesia, Cambodia, and Vietnam. Six lignans from the stems of Indonesian H. hirsuta, including (±)-pinoresinol, exposed potent cytotoxic effects against a small panel of cancer cell lines.¹⁰ Twelve known compounds from H. hirsuta. including 5 lupane triterpenoids exhibited weak cytotoxic activity.¹⁴ This paper describes the isolation and structural elucidation of a new benzofuran derivative 7-methoxy-2-(3-hydroxy-4-methoxyphenyl)-benzofuran-5-carboxylate (1), together with 5 known compounds such as propacin (2), hisbiscolatone A (3), heliclactone (4), rosmarinic acid (5), and syringaldehyde (6) from the methanol extract of H. hirsuta stems (Figure 1). Cytotoxic activity of these compounds against 4 cancer cell lines (KB, Lu-1, HepG2, and MCF7) was also demonstrated.

Figure 1

Structures of compounds 1 to 6.

Compound 1 was isolated as an amorphous white powder. The Infrared (IR) spectrum of 1 presented hydroxyl bands (3429 cm⁻¹), aromatic ring C-H stretch (3009, 2923 cm⁻¹), and C-O-C stretch (1018 cm⁻¹) functionalities. The molecular formula of 1 was deduced as C₁₈H₁₆O₆ by an ion peak at m/z 329.1019 [M+H]⁺ (Calc. for C₁₈H₁₇O₆: 329.1025). The ¹H-NMR spectrum of 1 showed the presence of 3 methoxyl groups at δ _H 3.90, 3.92, and 4.09; 2 olefin protons at δ _H 7.91 (1H, d, J = 1.0 Hz) and 7.11 (1H, d, J = 1.0 Hz) which are assigned to form a 1,3,5,6-tetrasubstituted benzene ring. Three olefin protons appeared at δ _H 7.09 (1H, d, J = 8.0 Hz, H-5′), 7.42 (d, J = 1.5 Hz, H-2′), and 7.43 (dd, J = 1.5, 8.0 Hz, H-6′) confirming the presence of a 1,3,4-trisubstitued benzene ring, and finally, 1 proton at 7.21 was observed. The ¹³C-NMR and DEPT spectra of 1 presented the resonances of 18 carbons including 3 methoxyl carbons at δ _C 52.3, 56.4, and 56.6; 6 methine sp ² carbons at δ _C 101.5 (C-3), 108.1 (C-6), 112.7 (C-2′), 112.8 (C-5′), 116.3 (C-4), and 117.8 (C-6′); 3 quaternary carbons at δ _C123.9 (C-1′), 127.2 (C-5), and 132.0 (C-3a); 5 oxygenated quaternary carbons at δ _C 145.9 (C-7), 147.2 (C-7a), 147.9 (C-3′), 149.6 (C-4′), and 158.5 (C-2); and 1 carbonyl carbon at δ _C 167.4. The 1D-NMR, Heteronuclear Single Quantum Coherence (HSQC) spectra, Heteronuclear Multiple Bond Correlation (HMBC) spectra, Nuclear Overhauser Effect Spectroscopy (NOESY) and High Resolution Electron Spray Ionization Mass Spectrometry (HR-ESI-MS) data identified a benzofuran ring, an ABX system benzene ring, and 3 methoxyl groups including a methyl carboxylate moiety. In detail, HMBC correlations from H-4 (δ _H 7.91)/H-6 (δ _H 7.51)/H-3 (δ _H 7.21) to C-7a (δ _C 147.2) and from H-3 (δ _H 7.21) to C-2 (δ _C 158.5)/C-7a (δ _C 147.2)/C-3a (δ _C 132.0) confirmed the presence of a benzofuran ring; HMBC correlations from protons H-12 (δ _H 4.09)/H-6 (δ _H 7.51) to C-7 (δ _C 145.9) determined the location of the methoxyl group at C-7, which were further confirmed by NOESY correlation of H-6 with methoxyl protons. Other methoxyl group was linked to carbonyl carbon which was confirmed by HMBC correlations from methoxyl proton at δ _H 3.90 to carbon at δ _C 167.4.

Additionally, HMBC correlations from protons H-4 (δ _H 7.91)/H-6 (δ _H 7.51) to carbonyl carbon (δ _C 167.4) indicated that the carboxylate group joined to the benzofuran ring at C-5. Similarly, HMBC correlations from H-3 (δ _H 7.21)/H-2′ (δ _H 7.42)/H-6′ (δ _H 7.43) to C-2 (δ _C 158.5) and C-1′ (δ _C 123.9), as well as from H-5′ (δ _H 7.09) to C-1′ (δ _C 123.9), supported the linkage between the benzofuran ring and the benzene ring at C-2 (δ _C 158.5). Finally, the HMBC correlations from protons H-2′ (δ _H 7.42) and hydroxyl proton (δ _H 7.95) to carbons C-3′ (δ _C 147.9) and C-4′ (δ _C 149.9), and from the methoxyl proton (δ _H 4.09) to C-4′ (δ _C 149.6) determined the position of the hydroxyl group at C-3′ and methoxyl group at C-4′, which was further confirmed by NOESY correlation from H-5′ (δ _H 7.09) to methoxyl proton (δ _H 3.92) (Figure 2). From the above evidence, compound 1 was characterized as 7-methoxy-2-(3-hydroxy-4-methoxyphenyl)-benzofuran-5-carboxylate, a new compound, and named as helisuta A.

Figure 2

Key HMBC and NOESY correlations of compounds 1 and 2.

The ¹H-NMR data of 2 were essentially the same as those of propacin, a coumarino-lignan found from Protium opacum in 1981.^15

-18 However, the ¹³C-NMR and 2D-NMR spectra of this compound have not been previously reported. Analysis of the ¹³C-NMR spectrum with the aid of HSQC experiment revealed the resonances of 20 carbons, including 5 sp ² methine, 2 methoxyl, 2 methine, 9 quaternary carbons (6 oxygenated), 1 carbonyl, and 1 methyl group. The carbon chemical shifts at δ _C 81.4 and 74.4 Table 1 suggested their linkages to oxygen atom. In the HMBC spectrum, the correlations from H-3 (δ _H 6.31), H-4 (δ _H 7.60) to C=O (δ _C 160.8) and C-10 (δ _C 111.6), and from H-5 (δ _H 6.51) to C-6 (δ _C 145.9), C-7 (δ _C 137.8), C-10 (δ _C 111.6), C-9 (δ _C 138.9), and C-4 (δ _C 143.7) assigned the formation of the courmarin ring. Additionally, HMBC correlations from H-9′ (δ _H 1.29) to C-8′ (δ _C 74.4), C-7′ (δ _C 81.4), and from H-2′ (δ _H 6.86), H-6′ (δ _H 6.90) to C-1′ (δ _C 127.8), CH-7′ (δ _C 81.4) established the C6-C3 lignan ring. Besides, 2 methoxyl groups were at C-6 and C-3′ which were confirmed by HMBC correlations from methoxyl protons at δ _H 3.88 and 3.92 to C-6 (δ _C 145.9) and C-3′ (δ _C 147.0), respectively (Figure 2). From the above evidence, compound 2 was established as propacin.

Table 1

NMR Data of Compounds 1 and 2 (¹H: 500 MHz and ¹³C: 125 MHz).

1			2
Pos.	δ _C	δ _H (J, Hz)	Pos.	δ _C	δ _H (J, Hz)
2	158.5	-	2	160.8	-
3	101.5	7.21 s	3	114.1	6.31 d (9.5)
3a	132.0	-	4	143.7	7.60 d (9.5)
4	116.3	7.91 d (1.0)	5	100.1	6.51 s
5	127.2	-	6	145.9	-
6	108.1	7.51 d (1.0)	7	137.8	-
7	145.9	-	8	132.3	-
7a	147.2	-	9	138.9	-
1′	123.9	-	10	111.6	-
2′	112.7	7.42 d (1.5)	1′	127.8	-
3′	147.9	-	2′	109.8	-
4′	149.6	-	3′	147.0	-
5′	112.8	7.09 d (8.0)	4′	146.6	-
6′	117.8	7.43 d (1.5, 8.0)	5′	114.7	-
COOCH₃	167.4	-	6′	121.3	-
COOCH ₃	52.3	3.90 s	7′	81.4	4.66 d (7.5)
7-OCH₃	56.6	4.09 s	8′	74.4	4.22 m
4′-OCH₃	56.4	3.92 s	9′	17.1	1.29 d (6.0)
3′-OH		7.95 s	4′-OH		5.69 s
			3′-OMe	56.1	3.92 s
			6-OMe	56.3	3.88 s

The known compounds were identified as hisbiscolatone A (3),¹⁹ heliclactone (4),²⁰ rosmarinic acid (5),²¹ and syringaldehyde (6)²² by analyses of their ESI-MS, 1D-NMR, and 2D-NMR spectral data in comparison with those reported in the literature.

Compounds 1 to 6 were evaluated for their cytotoxicity against 4 cancer cell lines, KB (mouth epidermal carcinoma cells), HepG2 (human liver hepatocellular carcinoma cells), Lu-1 (human lung adenocarcinoma cells), and MCF7 (human breast cancer cells). The cytotoxic effects of these compounds were estimated in terms of growth inhibition percentage and expressed as IC₅₀ which is the concentration of compound which reduces the absorbance of treated cells by 50% with reference to the control (untreated cells). As the results shown in Table 2, 2 displayed significant growth inhibition in the range of 94% to 99% at 128 µg/mL on 4 cancer cell lines and 3 expressed inhibition ability in the range of 70% to 72% at the same concentration.

Table 2

Inhibition Capacity of 1 to 6 Against Cell Lines at Concentration of 128 µg/mL.

Compounds	Inhibition percentage
Compounds	HepG2	KB	Lu-1	MCF7
1	35	35	25	23
2	97	99	99	94
3	70	72	70	70
4	30	35	25	25
5	32	30	30	31
6	28	32	32	26

The other remaining compounds had inhibition less than 50% at 128 µg/mL on the cell lines. As the results shown in Table 3, compound 2 exhibited moderate cytotoxic activity with IC₅₀ values in 43.97 ± 2.12, 46.53 ± 0.75, 52.63 ± 0.24, and 56.37 ± 1.04 µg/mL and compound 3 exposed less cytotoxic activity than 2 with IC₅₀ values of 90.34 ± 1.04, 94.27 ± 1.67, 94.69 ± 1.85, and 95.73 ± 0.38 µg/mL toward Lu-1, MCF7, HepG2, and KB cell lines, respectively. Compounds 1, 4, 5, and 6 displayed no cytotoxic effects.

Table 3

Cytotoxicity of Compounds 1 to 6 (IC₅₀ in µg/mL).

Compounds	HepG2	KB	Lu-1	MCF7
1	>128	>128	>128	>128
2	52.63 ± 0.24	56.37 ± 1.04	43.97 ± 2.12	46.53 ± 0.75
3	95.73 ± 0.38	94.69 ± 1.85	90.34 ± 1.04	94.27 ± 1.67
4	>128	>128	>128	>128
5	>128	>128	>128	>128
6	>128	>128	>128	>128
Ellipticine	0.41 ± 0.06	0.34 ± 0.07	0.39 ± 0.07	0.35 ± 0.08

Experimental

General Procedure

Melting points were measured in melting point meter Buchi B545. HR-ESI-MS were measured on a FT-ICR 910 MS TQFTMS-7T mass spectrometer. NMR spectra were recorded on a Bruker 500.13 MHz spectrometer, operating at 125.76 MHz for ¹³C NMR and at 500.13 MHz for ¹H-NMR. ¹H chemical shifts were referenced to CDCl₃ and CD₃OD at δ 7.27 and 3.31 ppm, respectively, while the ¹³C chemical shifts were referenced to the central peak at δ 77.0 (CDCl₃) and 49.0 (CD₃OD). For HMBC experiments, the delay (1/2J) was 70 ms, and for the NOESY experiments, the mixing time was 150 ms.

Plant Material

H elicteres hirsuta L. was collected in 2018 at Thua Thien-Hue, Vietnam and identified by Dr Tran The Bach. A specimen (Tra292018) was deposited at Institute of Ecology and Natural Resources, Vietnam Academy of Science and Technology.

Extraction and Isolation

Dried stems of H. hirsuta L. (3.2 kg) were extracted in alcohol at room temperature (1.0 L × 5) overnight. After evaporating under reduce pressure, the crude extract was obtained (106 g). This extract was dissolved in water (1.0 L) and successively extracted in hexane and EtOAc. The hexane, EtOAc, and water solutions were concentrated under diminished pressure to afford 20 g, 32 g, and 50 g dry extracts, respectively. The EtOAc extract (32 g) was subjected to a silica gel column chromatography (CC) with a gradient solvent of hexane/acetone to furnish 9 fractions. Fraction 1 was separated on a silica gel CC (gradient hexane/acetone) to yield 3 subfractions (1-3). Subfraction 2 was purified by preparative TLC (hexane/acetone: 85/15) and then purified on Sephadex LH-20 column (MeOH/CH₂Cl₂: 9/1, v/v) to give compound 1 (4 mg). Fraction 3 was separated by CC on a silica gel (gradient CH₂Cl₂/acetone) and subjected on a Sephadex LH-20 column to get compound 2 (7 mg). Fractions 5 and 6 were combined and separated on a Sephadex LH-20 column (MeOH/CH₂Cl₂: 9/1, v/v) to get compound 3 (8 mg). Fraction 8 was purified by Sephadex LH-20 column (MeOH/CH₂Cl₂: 9/1), yielding compounds 4 (6 mg) and 5 (5 mg). Fraction 9 was chromatographed on a silica gel CC (gradient hexane/acetone) to give 4 subfractions (1-4). Subfraction 3 was subjected on a Sephadex LH-20 column (MeOH/CH₂Cl₂: 9/1), yielding compound 6 (5 mg).

7-Methoxy-2-(3-Hydroxy-4-Methoxyphenyl)-Benzofuran-5-Carboxylate (1)

Colorless power.

MP: 165°C-168°C.

R _f: 0.5 (hexane-acetone, 8:2, v/v).

¹H-NMR and ¹³C-NMR (CDCl₃): see Table 1.

HR-MS-ESI: found m/z 329.1019 [M+H], calcd for C₁₈H₁₇O₆: 329.1025.

Cytotoxic Activity Assay

The 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay detects the reduction of MTT (Sigma) by mitochondrial dehydrogenase to blue formazan product, which reflects the normal function of mitochondria and hence the measurement of cytotoxicity cell and viability.^23,24 Cells were maintained in Dulbecco’s modified Eagle medium, supplemented with 10% fetal bovine serum, l-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100 µg/mL), and amphotericine B (10 µg/mL). Stock solution of test compounds were prepared in dimethylsulfoxide (DMSO) and diluted in H₂O. The cytotoxicity assays were carried out in 96-well microplates with 1 × 10⁴ viable cells/mL and incubated at 37°C in air/CO₂ (95:5) with test compounds. After 72 hours incubation, 20 µL of MTT were added per well. After 4 hours incubation, medium was removed, 100 µL of DMSO were added per well and shaken for 5 to 10 minutes. Viable cells were estimated by optical density at 540 nm with Epocher 2 (BioTek) microplates reader. The IC₅₀ value was determined as the concentration of compound that inhibits 50% cell growth compared to the control. Ellipticine was used as a reference compound.

Footnotes

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

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 financially supported by Vietnam Academy Science and Technology (Grant no. VAST 04.03/1819).

References

Merriam Webster Helicteres . USA: Merriam-Webster; [Online] Available from. http://www.merriam-webster.com/dictionary/Helicteres [Accessed on 31 st July, 2013]

Libman

Bouamanivong

Southavong

Sydara

Soejarto

. Medicinal plants: an important asset to health care in a region of central Laos. J Ethnopharmacol. 2006;106(3):303-311.doi:10.1016/j.jep.2005.11.034

Venkatesh

Laxmi

KS.

Reddy

BM.

Ramesh

. Antinociceptive activity of Helicteres isora . Fitoterapia. 2007;78(2):146-148.doi:10.1016/j.fitote.2006.09.024

. Dictionary of common plants. Hanoi, Vietnam: Hanoi Science and Technology Publishing House. 2004;2:1349-1350.

Takhtajan

. Flowering plants. Springer; 2009:269-270.

Pan

M-H.

Chen

C-M.

Lee

S-W.

Chen

Z-T

. Cytotoxic triterpenoids from the root bark ofHelicteres angustifolia . Chem Biodivers. 2008;5(4):565-574.doi:10.1002/cbdv.200890053

Gao

Y-qiao.

Liang

Y-guang.

Zhang

T-you.

Mei

Q-xi

. Study on triterpenoids from Helicteres angustifolia by high-speed countercurrent chromatography. Zhong Yao Cai. 2016;39(5):1053-1056.

Chen

Z-T.

Lee

S-W.

Chen

C-M

. Cucurbitacin B 2-sulfate and cucurbitacin glucosides from the root bark of Helicteres angustifolia . Chem Pharm Bull. 2006;54(11):1605-1607.doi:10.1248/cpb.54.1605

Bean

MF.

Antoun

Abramson

Chang

CJ.

McLaughlin

JL.

Cassady

. Cucurbitacin B and isocucurbitacin B: cytotoxic components of Helicteres isora . J Nat Prod. 1985;48(3):500.doi:10.1021/np50039a033

10.

Chin

Y-W.

Jones

WP.

Rachman

et al . Cytotoxic lignans from the stems of Helicteres hirsuta collected in Indonesia. Phytother Res. 2006;20(1):62-65.doi:10.1002/ptr.1806

11.

Kamiya

Saiki

Hama

et al . Flavonoid glucuronides from Helicteres isora . Phytochemistry. 2001;57(2):297-301.doi:10.1016/S0031-9422(01)00005-X

12.

Fernandes

Souza

Teles

et al . New sulphated flavonoids and larvicidal activity of Helicteres velutina K. Schum (Sterculiaceae). Molecules. 2018;23(11):2784-2794.doi:10.3390/molecules23112784

13.

Chen

Tang

Lou

Zhao

Wenliang

Weidong

Liguang

Weimin

. Pregnane, coumarin and lupane derivatives and cytotoxic constituents from Helicteres angustifolia . Phytochemistry. 2006;67(10):1041-1047.doi:10.1016/j.phytochem.2006.03.005

14.

Quang

DN.

Pham

CT.

LTK

et al . Cytotoxic constituents from Helicteres hirsuta collected in Vietnam. Nat Prod Res. 2018;6:1-5 [Published online: 16 Nov 2018].doi:10.1080/14786419.2018.1490907

15.

B. Zoghbi

MDG.

Roque

NF.

Gottlieb

. Propacin, a coumarinolignoid from Protium opacum . Phytochemistry. 1981;20(1):180.doi:10.1016/0031-9422(81)85248-X

16.

Hirroshi

Masaya

Kazuhiko

Kazuo

. Total synthesis of coumarinolignans, propacin and its regioisomer. Chem Pharm Bull. 1988;36:1738-1743.

17.

Waree

Bongkoch

Yodhathai

. (+)- Jatrophol, (+)-marmesin, propacin and jatrophin from the roots of Jatropha curcas (Euphorbiaceae). Science Asia. 1994;20:073-083.

18.

Almeida

EX.

Conserva

LM.

Lyra Lemos

RP.

Rosangela

Lemos

. Coumarins, coumarinolignoids and terpenes from Protium heptaphyllum . Biochem Syst Ecol. 2002;30(7):685-687.doi:10.1016/S0305-1978(01)00130-2

19.

Sood

RP.

Suri

KA.

Suri

OP.

Dhar

KL.

Atal

. Sesquiterpene lactone from Salmalia malbarica . Phytochemistry. 1982;21(8):2125-2126.doi:10.1016/0031-9422(82)83063-X

20.

Wang

MS.

Liu

WG.

. Structure elucidation of heliclactone. Acta Chim Sin. 1988;46:768-771.

21.

Elias

. Isolation and structural elucidation of rosmarinic acid by nuclear magnetic resonance spectroscopy. Am Res J Chem. 2017;2:17-23.

22.

Panyo

Matsunami

Panichayupakaranant

. Bioassay-guided isolation and evaluation of antimicrobial compounds from Ixora megalophylla against some oral pathogens. Pharm Biol. 2016;54(9):1522-1527.doi:10.3109/13880209.2015.1107106

23.

Mosmann

. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63.doi:10.1016/0022-1759(83)90303-4

24.

Scudiero

DA.

Shoemaker

RH.

Paull

KD.

Kenneth

et al . Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988;48(17):4827-4833.

A New Benzofuran Derivative From the Stems of Helicteres hirsuta

Abstract

Keywords

Experimental

General Procedure

Plant Material

Extraction and Isolation

7-Methoxy-2-(3-Hydroxy-4-Methoxyphenyl)-Benzofuran-5-Carboxylate (1)

Cytotoxic Activity Assay

Footnotes

Declaration of Conflicting Interests

Funding

References