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Abstract

A new biflavonoid, (2″S)-8″-C-methyl-2″,3″-dihydrorobustaflavone-4′-methyl ether (1) and 5 known robustaflavones (2-6) were isolated from the 70% EtOH extract of Selaginella braunii for the first time. The structures of 1 and the known compounds were identified by performing a comprehensive analysis of spectroscopic data including ultraviolet-visible, high-resolution electrospray ionization mass spectrometry, 1-dimensional and 2-dimensional nuclear magnetic resonance, and circular dichroism spectra. Compounds 1-6 were determined for cytotoxic activity against 3 human cancer cell lines (SMMC-7721, MCF-7, and A549). Compound 1 exerted apparent antiproliferative effects on SMMC-7721, MCF-7, and A549 cells, with IC₅₀ values of 18.6, 16.7, and 25.5 μM, respectively.

Keywords

cytotoxicity robustaflavone cancer natural product

Introduction

Numerous anticancer drugs are exploited from traditional Chinese medicine (TCM). They are hopeful candidates for drugs for cancer prevention and therapy.¹ Anticancer drugs from TCM showed strong effects and low cytotoxicity in the patients.² In the past, a large number of natural compounds strongly induce the death of cancer cells, including compounds of natural sources, synthetic and semisynthetic small molecule compounds.³ Several biological active biflavonoid connection modes with 2 flavone units have been separated from the genus Selaginella plants, which has proved that it exhibits significant anticancer biological activity among many tumor cells.^4–16 Selaginella braunii has been used as a folk medicine for antiphlogistic, detoxicating, heat-clearing, and cough-relieving treatments. The chemical composition of the 70% ethanol extract of S braunii was investigated to seek natural flavonoids with biological activity. Six biflavonoids (1-6) were isolated from S braunii (Figure 1). Chemical structures of those biflavonoids were identified by performing a comprehensive analysis of spectroscopic data including high-resolution electrospray ionization mass spectrometry (HRESIMS), ultraviolet (UV)-visible, 1-dimensional/2-dimensional nuclear magnetic resonance (NMR), and circular dichroism (CD) spectra. Compound 1 was obtained as a new flavonoid. Compounds 2-6 were separated from the whole plants of S braunii for the first time. Moreover, all isolated compounds' cytotoxicity was evaluated against 3 human cancer cell lines (SMMC-7721, MCF-7, and A549). Herein, the isolation, structural elucidation, and bioactivity evaluation of compounds 1-6 are set out in this paper.

Figure 1.

The structures of compounds 1-6 from Selaginella braunii.

Experimental

General Experimental Procedures

HRESIMS spectra were recorded with an Agilent 1290 UPLC Q-TOF MS instrument. NMR spectra were obtained on an AVANCE III 600 MHz Bruker instrument (Bruker). Silica gel (Qingdao Marine Chemical Factory) and Sephadex LH-20 (Shanghai yuanye bio-technology Co. Ltd) were used for CC. The high-performance liquid chromatography (HPLC) analyses and semipreparative were accomplished on an Agilent 1260 HPLC system with a preparative YMC Pack ODS-A (10 μm, 250 × 10 i.d mm). CD spectrum was measured on an Applied Photophysics spectrometer.

Plant Material

Dried whole S braunii Baker, collected from Yichang, Hubei Province, China in July 2021, was authenticated by Professor Ping Zhang (Hubei University of Chinese Medicine, Wuhan, China). A voucher specimen (20210701) was deposited in the Clinical College of Tradition Chinese Medicine, Hubei University of Chinese Medicine.

Extraction and Isolation

The air-dried whole S braunii plants (5.0 kg) were extracted by reflux 3 times with 70% EtOH (3 × 2 h × 40 L). The 70% ethanol extraction of S braunii was concentrated under reduced pressure, subsequently suspended in purified H₂O, and partitioned with petroleum ether, and ethyl acetate (EA). The EA fraction was preliminarily subjected to silica gel CC. Gradient eluting with CH₂Cl₂/MeOH (from 1:0.01 to 1:1) and obtained 10 fractions (Fr. A-J). Fr. D (CH₂Cl₂/MeOH, 1:0.04) was efficiently separated by Sephadex LH-20 column eluting with 65% MeOH/H₂O to obtain 7 subfractions (Fr. D1-D7). Fr. D2 was separated on a Sephadex LH-20 column, eluted with 60% MeOH/H₂O, and then purified RP-18 semipreparative HPLC and eluted with 40% ACN/H₂O (3.0 ml/min) to obtain compounds 1 (1.4 mg), 2 (3.4 mg), and 3 (3.6 mg). Fr. D5 was separated by using semipreparative HPLC eluted with 40% ACN/H₂O (3.0 ml/min) to obtain compounds 4 (4.2 mg), 5 (2.5 mg), and 6 (3.1 mg).

(2″S)-8″-C-Methyl-2″,3″-Dihydrorobustaflavone-4′-Methyl Ether (1)

Yellow amorphous powder; [α]25 D −2.0 (c 0.01, MeOH); CD (MeOH) nm λ_max (Δε): 215 (+1.8), 291 (–2.3), 332 (+0.8); UV (MeOH) λ_max: 225, 288, 335 nm; HRESIMS: m/z 569.1458 [M + H]⁺ (calcd for C₃₂H₂₅O₁₀, 569.1447); ¹H NMR and ¹³C NMR data, see Table 1.

Table 1.

¹H and ¹³C NMR Spectroscopic Data, and HMBC Correlations of Compound 1 in DMSO-d₆.

Position	δ_H (J in Hz)	δ _C	HMBC	Position	δ_H (J in Hz)	δ _C	HMBC
2		160.5		2″	5.64 (1H, dd, 12.1, 2.8)	78.9	C-3″/1‴
3	6.80 (1H, s)	103.8	C-2/4/1′	3″	3.22 (1H, dd, 16.8, 12.1) 2.83 (1H, dd, 16.8, 2.8)	42.4	C-4″/10″
4		178.8		4″		188.8
5		161.1		5″		152.6
6	6.09 (1H, d, 2.1)	95.7	C-5/7/8	6″		105.5
7		155.4		7″		162.7
8	6.57 (1H, d, 2.1)	93.6	C-7/9/10	8″		107.5
9		163.6		9″		158.8
10		102.4		10″		104.5
1′		122.9		1‴		122.6
2′	7.73 (1H, d, 2.4)	130.8	C-3″/4″/6″	2‴/6‴	7.35 (2H, d, 8.1)	128.7	C-3‴/4‴
3″		124.3		3‴/5‴	6.82 (2H, d, 8.1)	114.7	C-2‴/4‴
4″		161.2		4‴		158.9
5″	7.21 (1H, d, 8.9)	112.1	C-3″/4″/6″	4″-OCH₃	3.78 (3H, s)	56.4	C-4″
6″	8.03 (1H, dd, 8.9, 2.4)	127.7	C-1″/6″	8″-CH₃	1.98 (3H, s)	7.87	C-7″/8″/9″

Abbreviations: NMR, nuclear magnetic resonance; HMBC, heteronuclear multiple bond correlation; DMSO-d₆, dimethyl sulfide-d₆.

Cell Cytotoxicity Assay

Compounds 1-6 were tested for cell cytotoxicity against SMMC-7721, MCF-7, and A549 cells using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in vitro. Briefly, the cells (1 × 10⁵ cells/mL) were plated into 96-well microplates and were then incubated at the indicated concentrations of compounds 1-6 (1, 2, 4, 8, 16, and 100 μM). After 48 h of cultivation, 100 μL of MTT solution (1 mg/mL) was added to each well and incubated at 37 °C for another 4 h. Then, each well's optical density was measured at 490 nm using an ELISA reader. The cell growth inhibition rates and IC₅₀ values were calculated according to a previously reported method.¹⁶

Results

Identification of Compounds 1-6

Compound 1 was obtained as a yellow powder. Its molecular formula was determined to be C₃₂H₂₆O₁₀ by positive HRESIMS [M + H]⁺ at m/z 569.1458 (calcd for C₃₂H₂₇O₁₀, 569.1447). The λ_max (MeOH) at 225, 288, and 335 nm of 1 in its UV spectrum suggests that it is a flavone. The ¹³C NMR and heteronuclear single quantum coherence spectroscopic data confirmed in the presence of 32 carbons of 1, assignable to 2 carbonyl carbons, 9 linked-oxygen unsaturated carbons, 17 sp² quaternary carbons, and 2 aliphatic carbons, 1 methoxyl group carbon, and 1 methyl group carbon. The ¹H NMR spectrum of 1 showed resonances for 3 double doublets δ_H 5.64 (1H, dd, J = 12.1, 2.8 Hz, H-2″), 3.22 (1H, dd, J = 16.8, 12.1 Hz, H-3″ _ax ), 2.83 (1H, dd, J = 16.8, 2.8 Hz, H-2″ _eq ), and 1 proton singlet at δ_H 6.80 (1H, s, H-3), which are the characteristic signals for the flavone. And the ¹H NMR spectrum displayed 2 meta-coupled proton resonances at δ_H 6.57 (1H, d, J = 2.1 Hz, H-8), δ_H 6.09 (1H, d, J = 2.1 Hz, H-6), 1 proton singlet signal at δ_H 6.80 (1H, s, H-3), and 1 set of AA′BB″ spin system signal at δ_H 7.35 (2H, d, J = 8.1 Hz), δ_H 6.82 (2H, d, J = 8.1 Hz) assigned to H-2‴/H-6‴, H-3‴/H-5‴, respectively. In addition, the ¹H NMR spectrum exhibited the presence of an ABM coupling system with δ_H 8.03 (1H, dd, J = 8.9, 2.4 Hz, H-6′), 7.73 (1H, d, J = 2.4 Hz, H-2′), and 7.21 (1H, d, J = 8.9 Hz, H-5′), indicating that C-3′ was the position of linkage of 2 flavanone units. Above all, 1 was a biflavonoid with C-3′-C-6″ interflavonoid connection corresponding to the robustaflavone type based on heteronuclear multiple bond correlation (HMBC) of H-2′ (δ_H 7.73) to C-3′ (δ_C 124.3)/C-6″ (δ_C 105.5) (Figure 2). The HMBC of δ_H 3.78 (3H, s) to δ_C 161.2 (C-4′), suggested that the methoxy group substituted at C-4′. Further, a proton signal at δ_H 1.98 (3H, s) showed the key HMBC of C-7″/C-8″/C-9″, which confirmed that the methyl group is attached to C-8″. A CD experiment spectrum was used to determine the absolute configuration of C-2″ of 1, it exhibited a negative Cotton effect sign (π→π* transition) at 291 nm and a positive Cotton effect (n→π* transition) at 332 nm. Accordingly, the absolute configuration of C-2″ of 1 was confirmed as the (S)-configurations via comparison with the literature CD experiment data.^5,17 Consequently, the structure of 1 was confirmed as shown and named (2″S)-8″-C-methyl-2″,3″-dihydrorobustaflavone-4′-methyl ether. For the ¹H/¹³C NMR data, and HMBC of 1, see Table 1.

Figure 2.

¹H-¹H COSY and HMBC of compound 1.

In addition, by comparison of spectroscopic data with the literature,^4–6 the structures of 5 known compounds (2-6) were confirmed as robustaflavone (2), (2″S)-2″,3″-dihydrorobustaflavone-7-methyl ether (3), (2″S)-2″,3″-dihydrorobustaflavone (4), robustaflavone-4′-methyl ether (5), (2″S)- 2″,3″-dihydrorobustaflavone-4′,7″-dimethyl ether (6), respectively. For the ¹H NMR and ¹³C NMR data of compounds 2-6, see Table S1 and S2 in the Supplemental material.

Cytotoxicity Effects of Compounds 1-6

All the compounds (1-6) aimed to determine the cytotoxicity effects against the A549, SMMC-7721, and MCF-7 cancer cells using the MTT assay. Dimethyl sulfoxide (DMSO) was treated as a negative control group. Compounds 1-6 exerted inhibitory effects on the proliferative of the 3 human cancer cells (A549, SMMC-7721, and MCF-7), with an IC₅₀ value ranging from 13.3 to 91.2 μM (Table 2). Furthermore, as opposed to the negative control group, the cell viability of SMMC-7721, MCF-7, and A549 cells was much lower in the 1-treated-48 h group than in the negative control group with an IC₅₀ value of 18.6, 16.7, and 25.5 μM, respectively. As shown in Table 2, compound 1 possesses potential cytotoxic on A549, SMMC-7721, and MCF-7 cell lines.

Table 2.

Cytotoxicity of Compounds 1-6 Against 3 Cancer Cell Lines (IC₅₀, μM).

Compounds	1	2	3	4	5	6
MCF-7	16.7	44.2	45.6	14.1	15.7	79.6
SMMC-7721	18.6	59.3	81.2	55.7	20.1	17.9
A549	25.5	13.3	34.5	78.9	88.1	91.2

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221125056 - Supplemental material for Cytotoxic Biflavonoids from Selaginella braunii

Supplemental material, sj-docx-1-npx-10.1177_1934578X221125056 for Cytotoxic Biflavonoids from Selaginella braunii by Xue-Feng Liu, Tong-Fei Fu, Jun-Song Wu, Bi-Chao Lu, Ping Zhang and He-Jing Liu in Natural Product Communications

Footnotes

Author Contributions

XFL and TFF performed the experiments and wrote the manuscript. JSW and BCL analyzed the data. PZ and HJL revised the manuscript.

Declaration of Conflicting Interests

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

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Xue-Feng Liu

Ping Zhang

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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