Two New Bisbibenzyl Derivatives from Dendrobium findlayanum

Abstract

Objective

This study aimed to isolate and structurally characterize compounds from Dendrobium findlayanum, and to evaluate their cytotoxic activities against human tumor cells.

Methods

Compounds were purified using conventional column chromatography combined with semi-preparative liquid chromatography. Structural elucidation was carried out by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). Cytotoxicity screening was performed in vitro using the MTT assay on human tumor cell lines.

Results

Two new bisbibenzyl derivatives, dronbibisline E (1) and dronbibisline F (2), together with six known analogues (3-8), were isolated from Dendrobium findlayanum. Among them, compounds 1 and 3 exhibited significant inhibitory activity against two breast cancer cell lines, MDA-MB-231 and MCF-7.

Conclusion

Two new bisbibenzyl derivatives were obtained from Dendrobium findlayanum. Compound 1 showed IC₅₀ values of 9.25 μM against MDA-MB-231 and 23.19 μM against MCF-7, whereas compound 3 exhibited an IC₅₀ of 5.89 μM against MDA-MB-231 cells.

Keywords

Orchidaceae bisbibenzyl spectroscopic analyses cytotoxicity

Introduction

The genus Dendrobium (Orchidaceae), encompassing approximately 1100 species, represents one of the most taxonomically diverse groups within the family, exhibiting a broad paleotropical distribution across Asia, Europe, and Australia. The Chinese flora documents 74 indigenous Dendrobium species, more than 50 of which have been pharmacologically utilized in traditional Chinese medicine for the treatment of various pathological conditions including malignancies, chronic atrophic gastritis, dermatological aging processes, febrile disorders, and cardiovascular pathologies.¹ Phytochemical investigations have established that the medicinal efficacy of Dendrobium species is mediated through an array of bioactive secondary metabolites, including but not limited to alkaloids, sesquiterpenoids, phenanthrenes, bibenzyls, and polysaccharides. These constituents exhibit a broad spectrum of pharmacological activities, including immunomodulatory, neuroprotective, hepatoprotective, antitumor, antioxidant, antidiabetic, antiplatelet aggregation, hypoglycemic, and antibacterial effects.^2–5 Our current research aims to isolate and purify novel compounds from Dendrobium species (Orchidaceae) and to evaluate their antitumor activities.^6–9 In the present study, two new bisbibenzyl derivatives, designated as dronbibisline E (1) and dronbibisline F (2), along with six known analogues (3-8) (Figure 1), were isolated from the whole plants of Dendrobium findlayanum.

Materials and Methods

General Equipment and Plant Material

1D and 2D-NMR spectra were recorded on a Bruker-AVANCE III-600 instrument (Bruker, Karlsruhe, Germany) using TMS as the internal reference at room temperature. HR-ESI-MS spectra were measured using a Thermo Scientific LTQ Orbitrap XL high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA). Column chromatography (CC) was performed using silica gel (thin-layer and 200-300, 300-400 mesh from Qingdao Marine Chemical Inc., Qingdao, China). Semi-preparative HPLC was performed on an LC3000 system (Beijing Chuangxintongheng Science & Technology Co., Ltd, Beijing, China) equipped with YMC-Pack ODS-A column (10 × 250 mm, 5 μm) and a ChromCore PEP column (10 × 250 mm, 5 μm). All solvents used for CC were of at least analytical grade, sourced from Shanghai Chemical Reagents Company, Ltd, in Shanghai, China. The plant material was authenticated by Professor Faming Wu of Zunyi Medical University, and a voucher specimen (No. 20171016) has been deposited in the herbarium of the School of Pharmacy at Zunyi Medical University.

Extraction and Isolation

The air-dried and finely powdered stem of D. findlayanum (3 kg) was extracted with 92% methanol (MeOH) four times (3 h each). The combined methanol extract was concentrated under reduced pressure to obtain a crude extract (500 g), which was then suspended in water and successively partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (n-BuOH) (three times each). The EtOAc extract (89 g) and the n-BuOH extract (98 g) were combined and subjected to silica gel column chromatography, eluted with a gradient of PE-EtOAc (5:1 to 0:1, v/v) to afford five fractions Fr.1 – Fr.5. Fr.2 was purified by semi-preparative HPLC (MeOH:H₂O 70:30 to 100: 0, v/v, 20.0 mL/min) to afford twelve fractions Fr.2.1 – Fr.2.12. Fr.2.5 was further purified by semi-preparative HPLC (MeOH:H₂O 76:24, v/v, 3.2 mL/min) to yield dengibsin (5) (t_R = 36.2 min, 11.2 mg).¹⁰ Fr.2.9 was purified by semi-preparative HPLC (MeOH:H₂O 75:25, v/v, 6.0 mL/min) to yield dendrocandin F (8) (t_R = 12.6 min, 48.9 mg).¹¹ Fr.3 was subjected to silica gel column chromatography, eluted with a gradient of PE-EtOAc (10:1 to 0:1, v/v) to afford six fractions Fr.3.1 – Fr.3.6. Fr.3.2 was purified by semi-preparative HPLC (MeOH:H₂O 75:25, v/v, 20 mL/min) to yield dendroflorin (6) (t_R = 8.8 min, 10.0 mg).¹⁰ Fr.3.3 was subjected to semi-preparative HPLC (MeOH:H₂O 75:25, v/v, 20 mL/min) to afford eight fractions Fr.3.3.1 – Fr.3.3.8. Fr.3.3.4 was purified by semi-preparative HPLC (MeOH:H₂O 60:40, v/v, 6 mL/min) to yield naringenin (7) (t_R = 14.6 min, 5.2 mg).¹² Fr.3.5 was purified by semi-preparative HPLC (MeOH:H₂O 88:12, v/v, 3 mL/min) to yield dronbibisline E (1) (t_R = 16.8 min, 12.5 mg) and dendrocandin I (3) (t_R = 14.0 min, 53.7 mg).¹¹ Fr.5 was subjected to semi-preparative HPLC (MeOH:H₂O 60:40 to 100:0, v/v, 20 mL/min) to afford nine fractions Fr.5.5.1 – Fr.5.5.8. Fr.5.5.3 was purified by semi-preparative HPLC (MeOH:H₂O 82:28, v/v, 3 mL/min) to yield dronbibisline F (2) (t_R = 14.0 min, 11.5 mg). Fr.5.5.5 was repeatedly recrystallized to obtain dendrocandin B (4) (36.6 mg).¹³

Cytotoxicity Assay

Cells viability assay was carried out as previously described.^8,9 All compounds were initially screened at a uniform concentration of 20 μM against four cell lines: human thyroid carcinoma cells 8305C, human breast cancer cells MDA-MB-231, human thyroid cancer cells FTC-133, and human breast cancer cells MCF-7). Doxorubicin (DOX) was used as positive control.

Statistical Analysis

The data were statistically analyzed using GraphPad Prism 9 software. One-way ANOVA was performed to compare means among groups, with the Bonferroni t-test applied for data with homogeneous variance and Dunnett's T3 test for data with non-homogeneous variance. A P-value of less than 0.05 was considered statistically significant.

Results

Compound 1 was obtained as a colorless gum. HR-ESI-MS displayed its quasi-molecular ion peak at m/z 545.2169 [M + H]⁺ (calcd. for C₃₂H₃₃O₈⁺: 545.2172), establishing its molecular formula as C₃₂H₃₂O₈ with 17 degrees of unsaturation. The ¹H NMR spectrum (600 MHz, CD₃OD, Table 1) exhibited signals for two methylene protons [δ_H 2.75–2.78 (2H, m), 2.79–2.82 (2H, m)], four methoxy groups [δ_H 3.63 (3H, s), 3.73 (3H, s), 3.74 (3H, s), 3.74 (3H, s)], two oxygenated methine protons [δ_H 4.63 (1H, d, J = 8.0 Hz), 4.76 (1H, d, J = 8.0 Hz)], and twelve aromatic protons [δ_H 6.13 (1H, d, J = 1.6 Hz), 6.31 (1H, d, J = 1.6 Hz), 6.35 (1H, d, J = 1.6 Hz), 6.43 (1H, d, J = 1.6 Hz), 6.78 (2H, d, J = 8.5 Hz), 6.80 (2H, d, J = 8.5 Hz), 7.01 (2H, d, J = 8.5 Hz), 7.06 (2H, d, J = 8.5 Hz)], suggesting the presence of two para-substituted benzene rings. The ¹³C NMR spectrum (150 MHz, CD₃OD, Table 1) revealed two methylene carbons (δ_C 38.3, 39.2), four methoxy carbons (δ_C 55.8, 55.8, 56.7, 56.8), two oxygenated methylene carbons (δ_C 81.8, 82.1), and 24 aromatic carbons (δ_C 104.7-161.3), indicating four benzene ring units. Based on comprehensive NMR analysis, compound 1 was tentatively identified as a bibenzyl dimer derivative.¹⁴ In the HMBC spectrum (Figure 2), correlations between H-4 and C-2, C-3, C-5, C-6, C-7; H-7 and C-4, C-6, C-8, C-9; and H-8 and C-5, C-7, C-10 confirmed one bibenzyl unit as 1,2,3,5-tetrasubstituted with para-substitution. Similarly, HMBC correlations between H-7′ and C-4′, C-6′, C-8′, C-9′, as well as H-8′ and C-5′, C-7′, C-10′, established the second bibenzyl unit with the same substitution pattern. Key HMBC correlations between H-6 and C-1, C-2, C-4, along with the cross-peak of 1-OCH₃ to C-1, confirmed a methoxy group at C-1. The remaining three methoxy groups were assigned to C-12, C-3′, and C-12′ by analogous reasoning. Comparative analysis revealed that compound 1 closely resembled 3 (dendrocandin I) in NMR data, differing only slightly at C-4′, 5′, 6′, 9′, 10′, and 14′. This suggested that both compounds share the same bibenzyl-dioxane linkage but differ in connectivity.¹¹ The coupling constant (J = 8.0 Hz) between H-7′ and H-8′ indicated a trans-configuration, consistent with literature.¹⁴ Since compound 1 exhibited zero optical rotation and a flat ECD spectrum, it was deduced to be a racemic mixture. Due to limited quantity, chiral resolution was not performed. Thus, the relative configuration of compound 1 was determined, and the compound was named dronbibisline E.

Figure 1.

Structures of compounds 1‒8.

Figure 2.

Key HMBC () correlations of compounds 1 and 2.

Figure 3.

Effects of compounds 1 and 3 on cells vitality of MDA-MB-231 and MCF-7 (n=5; compared with control group, **P< 0.01).

Table 1.

NMR Data for Compounds 1 and 2 (600/150 MHz, δ in ppm, J in Hz).

	1			2
No.	δ _H ^a	δ _C ^a	δ _C ^b	δ _H ^b	δ _C ^b
1		150.0	148.8		149.1
2		133.4	131.9		129.0
3		145.8	144.4		143.7
4	6.43 (1H, d, 1.6)	110.8	109.6	6.53 (1H, d, 1.5)	109.8
5		135.8	134.6		134.9
6	6.35 (1H, d, 1.6)	106.6	105.0	6.36 (1H, d, 1.5)	105.7
7	2.75–2.78 (2H, m)	39.2	38.2	2.83 (2H, m)	37.8
8	2.79–2.82 (2H, m)	38.3	37.2	2.89 (2H, m)	38.1
9		135.5	134.1		143.4
10	7.06 (1H, d, 8.5)	130.7	129.6	6.74 (1H, br.s)	114.5
11	6.80 (1H, d, 8.5)	114.8	113.9		159.8
12		159.5	158.0	6.75 (1H, d, 7.7)	111.3
13	6.80 (1H, d, 8.5)	114.8	113.9	7.21 (1H, t, 7.7)	129.5
14	7.06 (1H, d, 8.5)	130.7	129.6	6.79 (1H, br.d, 7.7)	121.0
1′		145.4	143.8		147.5
2′		135.3	132.7		134.7
3′		149.3	146.8		147.5
4′	6.13 (1H, d, 1.6)	104.7	103.3	6.62 (1H, br.s)	102.7
5'		128.7	129.4		127.0
6'	6.31 (1H, d, 1.6)	109.8	108.7	6.62 (1H, br.s)	102.7
7'	4.63 (1H, d, 8.0)	82.1	80.8	5.20 (1H, d, 2.7)	75.5
8'	4.76 (1H, d, 8.0)	81.8	80.4	4.57 (1H, m)	77.8
9'		130.6	128.9	3.49 (1H, dd, 12.0, 3.6); 3.75 (1H, dd, 12.0, 8.7)	58.9
10’, 14'	7.01 (1H, d, 8.5)	130.3	129.0
11’, 13'	6.78 (1H, d, 8.5)	114.6	113.8
12'		161.3	159.8
1-OCH₃	3.74 (3H, s)	55.8	55.5	3.85 (3H, s)	56.2
11-OCH₃				3.79 (3H, s)	55.3
12-OCH₃	3.74 (3H, s)	55.8	55.5
1'-OCH₃				3.92 (3H, s)	56.6
3'-OCH₃	3.73 (3H, s)	56.8	56.3	3.92 (3H, s)	56.6
12'-OCH₃	3.63 (3H, s)	56.7	56.3
2'-OH				1.69 (1H, br.s)

in CD₃OD; ^b in CDCl₃.

Compound 2 was obtained as a white powder. HR-ESI-MS established its molecular formula as C₂₇H₃₀O₈ (observed m/z 481.1868 [M – H]^–, calcd. 481.1862) with 13 degrees of unsaturation. The ¹H NMR spectrum (Table 1) and HSQC data revealed: two methylene protons [δ_H 2.83, 2.89 (2H × 2, m)]; four methoxy groups [δ_H 3.79, 3.84 (each 3H, s) and 3.92 (6H, s)]; two oxygenated methine protons [δ_H 4.57 (1H, m), 5.20 (1H, d, J = 2.7 Hz)]; one oxygenated methylene [δ_H 3.49 (1H, dd, J = 12.0, 3.6 Hz), 3.75 (1H, dd, J = 12.0, 8.7 Hz)]; eight aromatic protons, including 1,3-disubstituted benzene ring protons [δ_H 7.21 (1H, t, J = 7.7 Hz), 6.75 (1H, d, J = 7.7 Hz), 6.79 (1H, br.d, J = 7.7 Hz), 6.74 (1H, br. s)], two meta-coupled protons [δ_H 6.36, 6.53 (each 1H, d, J = 1.5 Hz)], and two protons in chemically equivalent environments [δ_H 6.62 (2H, s)]. The ¹³C NMR data (Table 1) and HSQC spectrum displayed: two methylene carbons (δ_C 37.8, 38.1); four methoxy carbons (δ_C 55.3, 56.2, 56.6, 56.6); two oxygenated methine carbons (δ_C 77.8, 75.5); one oxygenated methylene carbon (δ_C 58.9); eighteen aromatic carbons (δ_C 102.7-159.8). Based on these observations, compound 2 was deduced to be a bibenzyl derivative. Further literature investigation revealed close structural similarity between compound 2 and dendronbiline C,¹⁴ except for the substitution orientation. The cis-configuration was assigned based on the coupling constant (J = 2.7 Hz) between H-7′ and H-8′,¹⁵ which was further corroborated by NOESY correlations (H-9′/H-4′, H-9′/H-6′). The CD analysis (showing a negative Cotton effect at 220-230 nm) established the absolute configuration as (7′S,8′R).^16,17 Consequently, compound 2 was named dronbibisline F.

Preliminary screening results (see Supporting Information) demonstrated that compounds 1 and 3 exhibited potent inhibitory effects against two breast cancer cell lines (MDA-MB-231 and MCF-7). Subsequent IC₅₀ determination revealed that compound 1 showed IC₅₀ values of 9.25 μM against MDA-MB-231 cells and 23.19 μM against MCF-7 cells, while compound 3 displayed an IC₅₀ of 5.89 μM against MDA-MB-231 cells (Figure 3).

Discussion

In this study, Two new bisbibenzyl derivatives, dronbibisline E and F (1 and 2), along with six known analogues (3−8), were isolated from Dendrobium findlayanum. The structures of the new compounds were elucidated through comprehensive spectroscopic analysis, incorporating 1D and 2D NMR techniques and HR-ESI-MS. All isolated compounds were evaluated for cytotoxic activity against four human tumor cell lines (8305C, MDA-MB-231, FTC-133, and MCF-7) using the MTT assay. Among them, compound 1 exhibited IC₅₀ values of 9.25 μM against MDA-MB-231 cells and 23.19 μM against MCF-7 cells, whereas compound 3 showed potent activity with an IC₅₀ of 5.89 μM against MDA-MB-231 cells.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X251407007 - Supplemental material for Two New Bisbibenzyl Derivatives from Dendrobium findlayanum

Supplemental material, sj-doc-1-npx-10.1177_1934578X251407007 for Two New Bisbibenzyl Derivatives from Dendrobium findlayanum by Ze-Yu Hou, Jun-Ru Guo and Shi-Ji Xiao in Natural Product Communications

Footnotes

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (Grant No. 32560106), the Science and Technology Department of Guizhou Province (Grant No. QKHJC-ZK[2024]331), and the Zunyi Bureau of Industry and Science & Technology (Grant No. ZSKHHZZ[2024]204).

ORCID iDs

Ze-Yu Hou

Jun-Ru Guo

Shi-Ji Xiao

Statement of Informed Consent

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

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China, (grant number 32560106), the Science and Technology Department of Guizhou Province (Grant No. QKHJC-ZK[2024]331), and the Zunyi Bureau of Industry and Science & Technology (Grant No. ZSKHHZZ[2024]204).

Declaration of Conflicting Interests

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

Statement of Human and Animal Rights

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

Supplemental Material

Supplemental material for this article is available online.

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