Phytochemical Constituents from the Aerial Parts of Acanthus ebracteatus Vahl. and Their Cytotoxic Activity

Abstract

Objective

Acanthus ebracteatus Vahl. (family Acanthaceae) is distributed in the mangrove areas of northern Vietnam. The whole plant has the effect of dissolving blood stasis, reducing swelling, relieving pain, dissolving phlegm, and is often used to treat back pain, numbness, cough, and asthma. In our screening program for seeking cytotoxic active compounds, the methanol extract of this plant significantly showed activity and was selected for further study. In this paper we report the chemical constituents of A. ebracteatus and their cytotoxic activity towards two cancer cell lines, HepG2 and SK-LU1 in vitro.

Methods

The dried and powdered aerial parts of A. ebracteatus were ultrasonically extracted with methanol. This extract was suspended in water and successively partitioned with n-hexane, dichloromethane, and ethyl acetate to give n-hexane, dichloromethane, ethyl acetate, and water-soluble extracts. These extracts were fractionated and purified by column and HPLC chromatographic methods. The chemical structures of the isolates were determined by analysis of HRESIMS, and 1D and 2D NMR, and CD spectra. The isolates were screened for their in vitro cytotoxic activity by SRB assay.

Results

One new flavone glycoside (1) and ten known compounds (2-11), including five flavones (2-6), three alkaloids (7-9), and two phenolic glycosides (10 and 11), were isolated from the aerial parts of A. ebracteatus. Compounds 7–9 showed moderate cytotoxic activity against HepG2 and SK-LU1 cell lines with IC₅₀ values ranging from 28.2 to 31.1 µM. Compounds 2–6, 8, 9, and 11 are reported herein from A. ebracteatus for the first time.

Conclusion

One new (1) and five known flavonoids (2-6), together with three alkaloids (7-9) and two phenolic glycosides (10 and 11) were recorded from A. ebracteatus. Our results preliminarily revealed that alkaloids may be cytotoxic ingredients of A. ebracteatus.

Keywords

acanthaceae alkaloid flavonoid acanebranoside A cytotoxic activity

Introduction

Acanthus ebracteatus Vahl. (family Acanthaceae) is a small tree about 1–1.5 m tall, with rounded, hairless stems, opposite leaves, hairless leaf blades, and sharp serrated margins.^1,2 This plant is distributed along the beaches in Vietnam, India, China, Thailand, Malaysia, and Indonesia. A. ebracteatus flowers all year round. The whole plant is used by decoction in drinking water to treat intestinal diseases, diarrhea, and infections. Leaves are used to treat snake bites, and seeds to treat worms.^1,2 Previous studies suggested that the ethanol extract of this plant enhances the efficacy of the collagen scaffold in wound closure,³ showing potential for skin infection treatment,⁴ effects on human dermal papilla and murine macrophages.⁵ Phytochemical study of this plant led to the isolation of verbascoside,⁵ megastigmane, an aliphatic alcohol and benzoxazinoid.⁶ In our screening to seek bioactive components from Vietnamese plants, A. ebracteatus methanol extract showed cytotoxic activity on the KB cell line with an IC₅₀ value of 54.6 µg/mL and, therefore, was selected for further study. In this paper, we report the isolation and structural determination of eleven compounds, including a new flavone glycoside, and their cytotoxic activity on two cancer cell lines, HepG2 and KB.

Results and Discussion

Compound 1 (Figure 1) was obtained as a yellow powder. Its IR spectrum showed the presence of hydroxy (3390, cm⁻¹), carbonyl (1711), olefinic (1642 cm⁻¹), and ether (1043 cm⁻¹) functionalities. The molecular formula of 1 was C₃₂H₃₈O₁₈, as revealed from the HR-ESI mass spectrum (m/z 711.2131 [M + H]⁺, calcd for [C₃₂H₃₉O₁₈]⁺: 711.213; m/z 733.1925 [M + Na]⁺, calcd for [C₃₂H₃₈O₁₈Na]⁺: 733.1950; m/z 709.1989 [M-H]⁻, calcd for [C₃₂H₃₇O₁₈]⁻: 709.1974; m/z 745.1765 [M + ³⁵Cl]⁻, calcd for [C₃₂H₃₈O₁₈³⁵Cl]⁻: 745.1752; m/z 747.1749 [M + ³⁷Cl]⁻, calcd for [C₃₂H₃₈O₁₈³⁷Cl]⁻: 747.1723), indicating fourteen indices of hydrogen deficiency. The NMR spectral data (Table 1) suggested that compound 1 was a flavone glycoside containing a para-substituted aromatic ring (B ring) [δ_C/δ_H: 122.9, 129.7/7.92 (2H, d, J = 8.5 Hz), 117.1/6.95 (2H, d, J = 8.5 Hz), 163.0], two olefinic methines at C-3 and C-8 [δ_C/δ_H: 104.2/6.72 and 96.0/7.06, respectively), one glucose moiety [δ_C/δ_H: 102.7/5.17 (d, J = 8.0 Hz), 74.9/3.61, 78.0/3.53, 71.3/3.44, 78.6/3.58, and 62.5/3.74 (dd, J = 12.0, 5.0 Hz) and 3.98 (dd, J = 12.0, 2.0 Hz), one rhamnose (δ_C/δ_H: 101.9/4.96 (d, J = 1.5 Hz), 72.7/3.90 (dd, J = 3.0, 1.5 Hz), 72.0/3.66, 74.0/3.22, 69.7/3.23, and 17.5/0.68 (3H, d, J = 6.0 Hz), and a C-glycoside of an arabinofuranose moiety [δ_C/δ_H: 74.1/5.53 (d, J = 8.0 Hz), 85.1/5.14 (dd, J = 8.0, 7.0 Hz), 77.7/4.27 (dd, J = 8.5, 7.0 Hz), 83.7/4.29, and 62.5/3.80 and 3.68], similar to those of luteolin 6-C-α-L-arabinopyranosyl-7-O-β-D-glucopyranoside and other reported O- and C-glycosides.^7,8 Three clear COSY correlations of H-1ʹʹ/ H-2ʹʹ/ H-3ʹʹ/ H-4ʹʹ/ H-5ʹʹ/ H-6ʹʹ, H-1ʹʹʹ/ H-2ʹʹʹ/ H-3ʹʹʹ/ H-4ʹʹʹ/ H-5ʹʹʹ, and H-1ʹʹʹʹ/ H-2ʹʹʹʹ/ H-3ʹʹʹʹ/ H-4ʹʹʹʹ/ H-5ʹʹʹʹ/H-6ʹʹʹʹ in combination with HSQC and HMBC spectra further confirmed the above structure suggestions. In the HMBC, H-1ʹʹ correlated with C-7, H-1ʹʹʹ with C-5/C-6/C-7, and H-1ʹʹʹʹ with C-2ʹʹʹ, indicating that the glucose moiety was attached to C-7, the arabinose linked to C-6 by a C-glycoside linkage, and the rhamnose linked to C-2ʹʹʹ by an O-glycoside linkage. These locations of the sugar moieties were further evidenced by HMBC correlation from H-8 to C-6/C-7/C-8/C-9. In the NOESY spectrum, H-2ʹʹʹ correlated with H-4ʹʹʹ, while H-3ʹʹʹ correlated with H₂-5ʹʹʹ, confirming the orientations of the arabinofuranose structure, and H-1ʹʹ (δ_H 5.17) correlated with H-8 (δ_H 7.06) further evidencing the location of the glucose unit at C-7 (Figure 2). In addition, the proton coupling constants of the anomeric protons at δ_H 5.17 (J_{H−1ʹʹ/H−2ʹʹ} = 8.0 Hz), δ_H 5.53 (J_{H−1ʹʹʹ/H−2ʹʹʹ} = 8.0 Hz), and 4.96 (1.5 Hz) indicated the β-form of the glucoside and α-form of the remaining sugar moieties.^7,8 Acid hydrolysis of 1 obtained D-glucose and L-rhamnose, which were identified by TLC comparison with authentic samples and from the positive sign of the optical rotations.^9,10 From the above evidence, the chemical structure of compound 1 was determined to be 7-O-β-D-glucopyranosylapigenin 6-C-α-L-rhamnopyranosyl-(1→2)-α-L-arabinofuranoside, a new compound and named acanebranoside A (Supplemental Material Figures S1-S13).

Figure 1.

Chemical structures of 1-11.

Figure 2.

Key ¹H-¹H COSY, HMBC, and NOESY correlations of 1.

Table 1.

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

Pos.	^a,b δ _C	^a,cδ_H (mult, J in Hz)	Pos.	^a,b δ _C	^a,cδ_H (mult, J in Hz)
2	166.8	⁻	4ʹʹ	71.3	3.44 (t, 9.0)
3	104.2	6.72 (s)	5ʹʹ	78.6	3.58 (m)
4	184.2	-	6ʹʹ	62.5	3.98 (dd, 12.0, 2.0) 3.74 (dd, 12.0, 5.0)
5	161.7	-	6-C-Ara
6	112.9	-	1ʹʹʹ	74.1	5.53 (d, 8.0)
7	163.4	-	2ʹʹʹ	85.1	5.14 (dd, 7.0, 8.0)
8	96.0	7.06 (s)	3ʹʹʹ	77.7	4.27 (dd, 7.0, 8.5)
9	158.6	-	4ʹʹʹ	83.7	4.24 (m)
10	107.1	-	5ʹʹʹ	62.5	3.80 (dd, 12.0, 2.5) 3.68 dd, 12.0, 5.0)
1ʹ	122.9	-	2ʹʹʹ-O-Rha
2ʹ, 6ʹ	129.7	7.92 (d, 8.5)	1ʹʹʹʹ	101.9	4.96 (d, 1.5)
3ʹ, 5ʹ	117.1	6.95 (d, 8.5)	2ʹʹʹʹ	72.7	3.90 (dd, 1.5, 3.5)
4ʹ	163.0	-	3ʹʹʹʹʹ	72.0	3.66 (dd, 3.5, 9.0)
7-O-Glc			4ʹʹʹ	74.0	3.22 (t, 9.0)
1ʹʹ	102.7	5.17 (d, 8.0)	5ʹʹʹʹ	69.7	3.23 (m)
2ʹʹ	74.9	3.61 (dd, 8.0, 9.0)	6ʹʹʹʹ	17.5	0.68 (d, 6.0)
3ʹʹ	78.0	3.53 (t, 9.0)

Measured in CD₃OD.

150 MHz, ^c600 MHz.

All the NMR data assignments were made from ¹H NMR, ¹³C NMR, HSQC, HMBC, COSY, and NOESY spectra.

The known compounds were identified as chrysoeriol 7-O-rutinoside (2),¹¹ luteolin 7-O-rutinoside (3),¹² luteolin 7-O-glucopyranoside (4),¹³ 3,5,7,3ʹ,4ʹ-pentamethoxyflavone (5),¹⁴ 5,7,4ʹ-trimethoxyflavone (6),¹⁵ acanthaminoside (7),¹⁶ acanthiline A (8)¹⁷ 1,4-benzoxazine-2,3-dione (9),¹⁸ acteoside (10), and isoacteoside (11)¹⁹ by comparison of their physical and NMR data with those reported in the literature. Among these, to the best of our knowledge, compounds 2–6, 8, 10, and 11 are reported from A. ebracteatus for the first time.

Compounds 1–11 were evaluated for their cytotoxic effects against HepG2 and SK-LU1 cell lines (Supporting information). Our results (Table S1) indicated that alkaloids 7–9 showed moderate cytotoxic activity against the two cell lines with IC₅₀ values ranging from 28.2 to 31.1 µM. These results suggested that alkaloids may be the cytotoxic ingredients of this plant.

Materials and Methods

General experimental procedures

The used characterization equipment is the same as that described in our previous work.^20,21

Refer to Supplemental Material.

Plant materials

The aerial parts of Acanthus ebracteatus Vahl. (Acanthaceae) were collected from Xuan Thuy National Park, Nam Dinh Province, Vietnam, and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. The voucher specimen (NCCT-P172) has been deposited at IMBC, VAST.

Extraction and isolation

Refer to Supplemental Material.

7-O-β-D-Glucopyranosyl-5,4′-Dihydroxyflavone 6-C-α-L-Rhamnofranosyl-(1→2)-α-L-Arabinofuranoside (1)

A yellow powder, $[α]_{D}^{25}$ : −34.5 (c 0.03, MeOH); UV (MeOH) λ_max: 205, 250, 271, 333 nm; IR ν_max (KBr) 3390, 2923, 2853, 1711, 1642, 1518, 1464, 1043 cm⁻¹; HRESIMS m/z 711.2131 [M + H]⁺, calcd for [C₃₂H₃₉O₁₈]⁺: 711.213 (Δ = 0 ppm); m/z 733.1925 [M + Na]⁺, calcd for [C₃₂H₃₈O₁₈Na]⁺: 733.1950 (Δ = -3.4 ppm); m/z 709.1989 [M-H]⁻, calcd for [C₃₂H₃₇O₁₈]⁻: 709.1974 (Δ = + 2.1 ppm); m/z 745.1765 [M + ³⁵Cl]⁻, calcd for [C₃₂H₃₈O₁₈³⁵Cl]⁻: 745.1752 (Δ = + 1.7 ppm); 747.1749 [M + ³⁷Cl]⁻, calcd for [C₃₂H₃₈O₁₈³⁷Cl]⁻: 747.1723 (Δ = + 2.5 ppm); ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) data are shown in Table 1.

Cytotoxic assay

The cytotoxic assay is the same as that described in our previous work.^21,²²

Refer to Supplemental Material.

Acid Hydrolysis

See Supplemental Material.^9,10

Conclusions

In this study, a new flavonoid, 7-O-β-D-glucopyranosylapigenin 6-C-α-L-rhamnopyranosyl-(1→2)-α-L-arabinofuranoside (named acanebranoside A) and ten known compounds (2-11) were isolated from the aerial parts of A. ebracteatus. Except for compounds 7 and 9, the remaining compounds are reported herein for the first time from this plant. In addition, alkaloids 7–9 exhibited in vitro cytotoxic activity effects against two cell lines HepG2 and SK-LU1 with IC₅₀ values ranging from 28.2 to 31.1 µM, respectively.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231166547 - Supplemental material for Phytochemical Constituents from the Aerial Parts of Acanthus ebracteatus Vahl. and Their Cytotoxic Activity

Supplemental material, sj-docx-1-npx-10.1177_1934578X231166547 for Phytochemical Constituents from the Aerial Parts of Acanthus ebracteatus Vahl. and Their Cytotoxic Activity by Bui Thi Mai Anh, Tran Thi Thuy Nga, Hoang Thi Tuyet Lan and Nguyen Thi Mai, Phan Thi Thanh Huong, Bui Huu Tai, Nguyen Xuan Nhiem, Phan Van Kiem in Natural Product Communications

Footnotes

List of abbreviations:

Acknowledgements

The authors would like to thank Dr Nguyen The Cuong at the Institute of Ecology and Biological Resources, VAST for the plant identification.

Author Contribution

Research idea, NT Mai, PV Kiem, NX Nhiem; Isolation, cytotoxic assay, PTT Huong, BTM Anh, TTT Nga, NT Mai; Structure elucidation and writing, NT Mai, PV Kiem, BH Tai.

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

ORCID iDs

Bui Huu Tai

Nguyen Xuan Nhiem

Phan Van Kiem

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

This research has been financially supported by Ministry of Education and Training (Code B2022-GHA-562-05).”

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

References

Matos

Batista

Figueirinha

. A review of the ethnomedicinal uses, chemistry, and pharmacological properties of the genus Acanthus (Acanthaceae). J Ethnopharmacol. 2022;293:115271. doi:10.1016/j.jep.2022.115271

Chi

. Dictionary of Vietnamese medicinal plants. Medicine Publishing House; 2018, pp. 393.

Somchaichana

Bunaprasert

Patumraj

. Acanthus ebracteatus vahl. Ethanol extract enhancement of the efficacy of the collagen scaffold in wound closure: a study in a full-thickness-wound mouse model. J Biomed Biotechnol. 2012;2012:1‐8.

Sittiwet

Niamsa

Puangpronpitag

. Antimicrobial activity of Acanthus ebracteatus vahl. aqueous extract: the potential for skin infection treatment. Int J Biol Chem Sci. 2009;3(2):95‐98.

Wisuitiprot

Ingkaninan

Chakkavittumrong

. Effects of Acanthus ebracteatus vahl. extract and verbascoside on human dermal papilla and murine macrophage. Sci Rep. 2022;12:1491. doi:10.1038/s41598-022-04966-w

Kanchanapoom

Kasai

Picheansoonthon

. Megastigmane, aliphatic alcohol and benzoxazinoid glycosides from Acanthus ebracteatus. Phytochemistry. 2001;58(5):811‐817.

Wang

Chen

Zhang

, et al. Flavone C-glycosides from the leaves of Lophatherum gracile and their in vitro antiviral activity. Planta Med. 2012;78(1):46‐51.

Gluchoff-Fiasson

Jay

Viricel

. Flavone O- and C-glycosides from Setaria italica. Phytochemistry. 1989;28(9):2471‐2475.

Abe

Okabe

Yamauchi

, et al. Pregnane glycosides from Marsdenia tomentosa. Chem Pharm Bull. 1999;47(6):869‐875.

10.

Voutquenne-Nazabadioko

Gevrenova

Borie

, et al. Triterpenoid saponins from the roots of Gypsophila trichotoma wender. Phytochemistry. 2013;90:114‐127.

11.

Nakano

Kawada

Yoshida

, et al. Isolation and identification of flavonoids accumulated in proanthocyanidin-free barley. J Agri Food Chem. 2011;59(17):9581‐9587.

12.

Wang

Simon

Aviles

, et al. Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). J Agri Food Chem. 2003;51(3):601‐608.

13.

Foo

. Flavonoid and phenolic glycosides from Salvia officinalis. Phytochemistry. 2000;55(3):263‐267.

14.

Dong

Gou

Cao

, et al. Eicosenones and methylated favonols from Amomum koenigii. Phytochemistry. 1999;50(10):899‐902.

15.

Iinuma

Matsuura

Kusuda

. ¹³C-Nuclear Magnetic resonance (NMR) spectral studies on polysubstituted flavonoids. I. ¹³C-NMR spectra of flavones. Chem Pharm Bull. 1980;28(3):708‐716.

16.

Amer

Abou-Shoer

Abdel-Kader

, et al. Alkaloids and flavone acyl glycosides from Acanthus arboreus. J Braz Chem Soc. 2004;15(2):262‐266.

17.

Cai

Sun

Tang

, et al. Acanthiline A, a pyrido[1,2-a]indole alkaloid from Chinese mangrove Acanthus ilicifolius. J Asian Nat Prod Res. 2018;20(11):1088‐1092.

18.

Xia

Wang

Xia

, et al. Quinoxaline-2,3-diones: potential D-amino acid oxidase (DAAO) inhibitors. Med Chem Res. 2014;23:4977‐4989. doi:10.1007/s00044-014-1068-z

19.

Kiem

Quang

Huong

, et al. Chemical constituents of Acanthus ilicifolius L. And effect on osteoblastic MC3T3E1 cells. Arch Pharm Res. 2008;31(7):823‐829.

20.

Tai

Yen

Hoang

, et al. New dibenzocyclooctadiene lignans from Kadsura induta with their anti-inflammatory activity. RSC Adv. 2022;12:25433. doi:10.1039/d2ra05052h

21.

Hang

DTT

Trang

Dung

, et al. Guaianolide sesquiterpenes and benzoate esters from the aerial parts of Siegesbeckia orientalis L. and their xanthine oxidase inhibitory activity. Phytochemistry. 2021;190:112889. doi:10.1016/j.phytochem.2021.112889

22.

Trang

Hang

DTT

Dung

, et al. Rhabdastrenones A-D from the sponge Rhabdastrella globostellata. RSC Adv. 2022;12:10646. doi:10.1039/d2ra01674e

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

1.37 MB