Bibenzyl Derivatives From Leaves of Dendrobium officinale

Abstract

Nine compounds were isolated from leaves of Dendrobium officinale, including 1 new bibenzyl derivative, denofficin (1), and 8 known structurally related compounds, dendrocandin B (2), dendrocandin U (3), 3,4-dihydroxy-5,4′-dimethoxy bibenzyl (4), moscatilin (5), 4,4′-dihydroxy-3,5-dimethoxy bibenzyl (6), (S)-3,4,α-trihydroxy-5,4′-dimethoxy bibenzyl (7), gigantol (8), densiflorol A (9). The structures of these compounds were identified by spectroscopic methods. All isolated compounds were screened for their cytotoxicity against human cervical cancer cell line HeLa cells. Of them, compounds 1-3, 5, 6, and 8 were found to have the capabilities of proliferation inhibition against HeLa cells with half-maximal inhibitory concentration values ranging from 8.0 to 92.4 μM.

Dendrobium officinale, an herb plant endemic to China, belongs to a member of Dendrobium plants (Orchidaceae) and mainly distributed in eastern and southeastern China. The dried or fresh stems of this plant have been used as the precious traditional Chinese medicine for antipyretic, eyes-benefiting, and tonic purposes for thousands of years. In the Chinese Pharmacopoeia (2010 Edition), D. officinale has been recorded officially as the original material of “Tiepishihu”. ¹ In the process of making “Tiepishihu”, the leaves of D. officinale are usually removed, which leads to a huge waste of biological resources. Although multiple phytochemical studies focusing on stems of D. officinale have displayed the occurrence of polysaccharides, essential oils, alkaloids, and bibenzyls, etc, ² the chemical constituents of its leaves still remain unknown. As a part of the comprehensive development and utilization of leaf resources of D. officinale, the phytochemical investigation was performed. Herein, we described the isolation, structural elucidation of one new bibenzyl derivative, denofficin (1), as well as 8 structurally related known compounds, dendrocandin B (2), dendrocandin U (3), 3,4-dihydroxy-5,4′-dimethoxy bibenzyl (4), moscatilin (5), 4,4′-dihydroxy-3,5-dimethoxy bibenzyl (6), (S)-3,4,α-trihydroxy-5,4′-dimethoxy bibenzyl (7), gigantol (8), densiflorol A (9). Besides, their cytotoxic potential for human cervical cancer cell line HeLa cells was evaluated.

Denofficin (1), a whitish amorphous powder, has a positive reaction with ferric chloride reagent, proving the presence of the phenolic moiety. The molecular formula was determined to be C₃₆H₃₈O₁₀ by the high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) (supplemental Figure S1) which afforded the quasi-molecular ion peak at m/z 629.2388 ([M − H]⁻, calcd. for C₃₆H₃₇O₁₀, 629.2392). The infrared spectrum (supplemental Figure S2) of 1 displayed the absorptions for hydroxyl group (3519 and 3445 cm⁻¹), methoxy group (2936 and 2852 cm⁻¹), carbonyl group (1733 cm⁻¹), and aromatic ring (1606, 1516, and 1454 cm⁻¹). The ultraviolet absorption (Figure S3) maxima at λ _max 273 nm suggested the presence of a bibenzyl skeleton. ^{3 -5} The ¹H and ¹³C NMR data (Table 1) of 1 is highly similar to those of the known bibenzyl derivative, dendrocandin B (2, Figure 1). ³ The main difference is the extra appearance of a set of signals assigned for p-hydroxylphenyl propionyl group (δ _H 7.04 [2H, d, J = 8.3 Hz], 6.70 [2H, d, J = 8.3 Hz], 2.83 [2H, m], and 2.58 [2H, m]; δ _C 132.4, 129.4, 115.4, 154.1, 29.9, 35.9 and 172.4) in 1. This indicated that 1 was the p-hydroxylphenyl propionyl acylation derivative of dendrocandin B, which was further supported by the HR-ESI-MS data. The p-hydroxylphenyl propionyl acylation took placed at the hydroxyl of C-9″, as suggested by the heteronuclear multiple bond correlation (HMBC) correlations (Figure 2 and supplemental figure S11) from H_α-9″ (δ _H 4.34)/H_β-9″(δ _H 4.02) to C-9″′ (δ _C 172.4). Interpretation of the ¹H-¹H correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and HMBC spectra (supplemental Figures S9-S11) of 1 confirmed the substitution pattern and fully assigned all ¹H and ¹³C nuclear magnetic resonance (NMR) signals. The relative configuration between C-7″ and C-8″ was assigned as trans in view of the diaxial coupling constant J _7″,8″ = 7.9 Hz. ³ As for the absolute configuration of 1, it can be determined by the biogenetic law. The compound 2 was reported previously to have S configuration both at C-7″ and C-8″, ³ and it was isolated from D. officinale where 1 was also found herein; from the biogenetic point of view, 1 and 2 would share some similar biosynthetic pathways and consequently have the same stereochemical configuration, which was further supported by the fact that they possess the same rotation value (−5.8 for 1 and −4.6 for 2). Thus, the structure of 1 was elucidated as ((2S,3S)-3-(4-hydroxy-3,5-dimethoxyphenyl)-8-methoxy-6-(4-methoxyphenethyl)-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methyl 3-(4-hydroxyphenyl)propanoate (Figure 1) and it was named denofficin.

Table 1

¹H and ¹³C Nuclear Magnetic Resonance Data of Compound 1 and Dendrocandin B (2) in Deuterated Chloroform (CDCl₃).

Position	1 a		Dendrocandin B (2) a
	δ H (m, J in Hz)	δ C	δ H (m, J in Hz)	δ C
1		134.6		134.5
2	6.49 (1 H, d, 1.6)	109.4	6.52 (1 H, d, 1.0)	109.5
3		143.9		144.1
4		130.8		131.0
5		148.5		148.4
6	6.32 (1 H, d, 1.6)	105.2	6.32 (1 H, d, 1.5)	104.8
1′		133.7		133.7
2′, 6′	7.10 (2 H, d, 8.5)	129.4	7.10 (1 H, d, 8.0)	129.4
3′, 5′	6.83 (2 H, d, 8.5)	113.8	6.83 (1 H, d, 8.0)	113.7
4′		157.9		160.1
a	2.81 (2 H, m)	37.9	2.82 (2 H, m)	38.0
a′	2.85 (2 H, m)	36.9	2.82 (2 H, m)	37.0
1″		126.9		127.3
2″, 6″	6.55 (2 H, s)	104.2	6.68 (2 H, s)	104.0
3″, 5″		147.3		147.2
4″		135.5		135.2
7″	4.77 (1H, d, 7.9)	76.6	4.96 (1 H, d, 8.5)	76.4
8″	4.20 (1 H, m)	75.5	3.98 (1 H, m, 8.0, 3.0, 3.0)	78.2
9″α	4.34 (1 H, dd, 12.3, 3.1)	63.1	3.55 (1 H, dd, 12.0, 3.0)	61.5
9″β	4.02 (1 H, dd, 12.3, 4.6)		3.90 (1 H, m)
1″′		132.4
2″′, 6″′	7.04 (2 H, d, 8.3)	129.4
3″′, 5″′	6.70 (2 H, d, 8.3)	115.4
4″′		154.1
7″′	2.83 (2 H, m)	29.9
8″′	2.58 (2 H, m)	35.9
9″′		172.4
MeO-5	3.84 (3 H, s)	56.2	3.85 (3 H, s)	56.0
MeO-4′	3.79 (3 H, s)	55.3	3.79 (3 H, s)	55.3
MeO-3″, 5″	3.87 (6 H, s)	56.4	3.92 (6 H, s)	56.4

^aBruker Avance 600 spectrometer; chemical shifts (ppm) referred to CDCl₃ (δ _H 7.26; δ _C 77.16).

Figure 1

Structures of compounds 1 and 2.

Figure 2

The key heteronuclear multiple bond correlations (H→C) of 1.

Together with the new bibenzyl derivative (1), 8 known structurally related compounds (2-9) were isolated from D. officinale. By comparison of their ¹H, ¹³C NMR and ESI-MS spectral data with those reported in literatures, the known compounds were identified as dendrocandin B (2), ³ dendrocandin U (3), ⁵ 3,4-dihydroxy-5,4′-dimethoxy bibenzyl (4), ⁶ moscatilin (5), ⁷ 4,4′-dihydroxy-3,5-dimethoxy bibenzyl (6), ⁸ (S)-3,4,α-trihydroxy-5,4′-dimethoxy bibenzyl (7), ⁹ gigantol ^8,10 densiflorol A (9). ¹¹ Among them, compounds 5 and 9 were reported here from the title plant for the first time.

The cytotoxicity of all isolated compounds against human cervical cancer cell line HeLa cells was evaluated by a colorimetric cell counting kit-8 (CCK-8) assay described previously. ¹² As shown in Table 2, compound 6 revealed the highest cell proliferation inhibition against HeLa cells among the isolated compounds, with a half-maximal inhibitory concentration (IC₅₀) value of 8.0 µM, which was comparable to that of the positive control, 5.3 µM. Compounds 1-3, 5, and 8 also exhibited mild cytotoxicity against HeLa cells with IC₅₀ values ranging from 16.8 to 92.4 µM, while compounds 4, 7, and 9 were found to be inactive. It is worth noting that the preliminary structure-activity relationship could be inferred from these activity data. For example, for the common bibenzyls, the phenolic hydroxyl group at C-4′ might be important for their cytotoxicity. This was supported by the observation that compounds 5, 6, and 8, the three common bibenzyls with a hydroxyl substitution at C-4′, are active, while those ones without substitution or bearing methoxy group at C-4′ (4, 7, and 9) displayed no activity. For those structurally complex bibenzyl derivatives with a phenylpropane unit linked to the C6–C2–C6 skeleton through a dioxane bridge, the hydroxyl group at C-4′, however, appears inessential to their cytotoxicity. This was suggested by the observation that 1 bearing methoxy group at C-4′ is over twofold more active than 3 (with hydroxyl substitution at C-4′) and also 2. Our results indicated, in part, that the cell proliferation inhibition abilities of this special class of bibenzyl derivatives might be strengthened by the p-hydroxylphenyl propionyl acylation substitution at C-9″. However, the structure–activity relationship proposed here is preliminary and activity data of more bibenzyl derivatives will be required to confirm it.

Table 2

Cytotoxicity of Bibenzyls 1-9 From D. officinale Against HeLa Cells.

Compounds	IC50 (μM)	Compounds	IC50 (μM)
1	20.2 ± 1.3	6	8.0 ± 1.5
2	91.1 ± 11.2	7	>100
3	41.5 ± 2.4	8	92.4 ± 6.4
4	>100	9	>100
5	16.8 ± 2.6	Cisplatin a	5.3 ± 0.7

IC₅₀, half-maximal inhibitory concentration.

^aPositive control.

Experimental

General Procedure

IR spectrum was executed on Shimadzu Iraffinity-1 fourier-transform infrared spectrometer with potassium bromide disc (Shimadzu Co., Kyoto, Japan). Optical rotation was determined on a JASCO P-1020 polarimeter (JASCO International Corp., Ltd, Tokyo, Japan) at room temperature. NMR spectra were recorded on a Bruker Avance 600 spectrometer (Bruker Biospin, Rheinstetten, Germany) using CDCl₃ as a solvent. HR-ESI-MS analyses were implemented on an AB SCIEX Triple TOF 5600+ mass spectrometer (AB SCIEX Co., Framingham, MA, USA). Column chromatography (CC) was performed on HP-20 (75-150 μm, Mitsubishi Chemical Co., Tokyo, Japan), octadecylsilyl (ODS) gel (75-150 μm, YMC Co., Kyoto, Japan), MCI GEL CHP20P (75-150 μm, Mitsubishi Chemical Co., Tokyo, Japan), and Sephadex LH-20 (25-100 μm, GE Healthcare Bio-Sciences, Amersham, Sweden). Precoated thin layer chromatography plates with silica gel GF₂₅₄ (10-40 μm; Yantai Jiang You silicone Development Co., Ltd., Yantai, China) were used to detect the purity of the isolates achieved by coating with 10% sulfuric acid (H₂SO₄) in ethanol (EtOH), followed by heating. Preparative high-performance liquid chromatography (PHPLC) was executed on a LC3000 liquid chromatograph (Beijing Tong Heng Innovation Technology Co., Ltd, Beijing, China) armed with an ODS column (5 µm, 250 mm × 30 mm i.d., Sepax Technologies, Inc.).

Plant Materials

The leaves of D. officinale were collected from the planting base of Yueqing Yanfeixue Shihu Co. Ltd., Yueqing city, Zhejiang province, China, in December 2014, and identified by Dr Xu Cheng, associate researcher of College of Life Sciences, Zhejiang University. The voucher specimen (TCM20140151) was deposited in the Herbarium of the Department of Pharmacognosy, Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine.

Extraction and Isolation

The air-dried and powdered leaves of D. officinale (11.0 kg) were extracted with 95% EtOH 3 times (100 L for each extraction) at room temperature. The filtrate was evaporated in vacuo to produce a residue (485.6 g), which was fractionated by a HP-20 macroporous resin column chromatography (CC) (15 × 45 cm) eluted with a gradient of EtOH/water (H₂O) (0% → 95%) to give 10 fractions (frs. H1–H10). Fr. H5 (5.8 g) was subjected to MCI CHP-20P resin CC (4 × 30 cm) eluted with a gradient of EtOH/H₂O (30% → 95%) to yield 10 subfractions (Frs. H5M1–H5M10). Fr. H5M4 (1.4 g) was further separated by Sephadex LH-20 gel CC (2 × 200 cm) eluted with 100% methanol to afford 4 subfractions (Frs. H5M4L1–H5M4L4). Fr. H5H4L3 (63.4 mg) was then purified by PHPLC (ODS, 5 µm, 2 × 25 cm) eluting with 45% methanol to obtain compound 6 (5.4 mg, t _R 25 minutes). Fr. H5M5 (0.8 g) was further fractioned by CC (3 × 25 cm) on silica gel (300 mesh) eluted with petroleum/ethyl acetate (11:1, 10:1, 9:1, 8:1, v/v) to give 7 subfractions (Frs. H5M5S1–H5M5S7). Fr. H5M5S1 (0.3 g) was purified by Sephadex LH-20 gel CC (2 × 200 cm) eluted with 100% methanol, followed by PHPLC (ODS, 5 µm, 2 × 25 cm) eluted with 30% acetonitrile to give 4 (2.6 mg, t _R 21 minutes). In a similar manner, 5 (2.3 mg, t _R 24 minutes) was obtained from Fr. H5M5S1. Fr. H6 (9.3 g) was subjected to MCI CHP-20P resin CC (4 × 30 cm) eluted with a gradient of EtOH/H₂O (40%→90%) to produce 6 subfractions (Frs. H6M1–H6M6). Fr. H6M5 (0.7 g) was separated by CC (2 × 200 cm) over Sephadex LH-20 gel eluted with 100% methanol to afford 5 subfractions (Frs. H6M5L1-H6M5L5). Fr. H6M5L4 (51.9 mg) was purified by PHPLC (ODS, 5 µm, 2 × 25 cm) eluted with 70% acetonitrile to obtain 9 (2.1 mg, t _R 28 minutes). In a similar manner, 2 (40.7 mg, t _R 31 minutes), 7 (5.1 mg, t _R 20 minutes) and 8 (3.5 mg, t _R 36 minutes) were obtained from Fr. H6M3 (2.7 g), respectively. Fr. H7 (23.6 g) was subjected to CC over MCI CHP-20P resin (4 × 30 cm) eluted with a gradient of EtOH/H₂O (20% → 90%) to yield 8 fractions (Frs. H7M1–H7M8). Fr. H7M6 (3.3 g) was separated by CC (2 × 200 cm) over Sephadex LH-20 gel eluted with 90% methanol to afford 10 subfractions (Frs. H7M6L1–H7M6L10). H7M6L6 (143.2 mg) was purified by PHPLC eluted with 50% acetonitrile to obtain 1 (5.8 mg, t _R 32 minutes) and 3 (2.2 mg, t _R 26 minutes), respectively.

Denofficin (1)

Whitish amorphous powder.

$[\alpha {]}_{\mathrm{D}}^{25}$ : −5.8 (c 0.25, MeOH).

IR (KBr): 3519, 3445, 2936, 2852, 1733, 1606, 1516, 1454, 1346, 1232, 1116 cm⁻¹.

UV (MeOH) λ_max (log ε) 273 (3.80) nm.

¹H and ¹³C NMR (CDCl₃): Table 1.

HR-ESI-MS: m/z [M − H]⁻ calcd. for C₃₆H₃₇O₁₀629.2392; found: 629.2388.

Evaluation for in Vitro Cytotoxicity Against HeLa Cells

One day after exponentially growing HeLa cells were seeded at 5 × 10³ cells/well in a 96-well plate, the culture medium was changed to the experimental medium supplemented with compounds tested at a series of concentrations. After incubation for 24 hours, 10 µL of CCK-8 was added and incubated for an additional 3 hours, and then optical density (OD) value was measured by spectrophotometer under 450 nm. Cell inhibitory rate was calculated as follows: cell inhibitory rate = (OD_control − OD_experiment) / (OD_control − OD_blank) × 100%.

Supplemental Material

Supplementary material - Supplemental material for Bibenzyl Derivatives From Leaves of Dendrobium officinale

Supplemental material, Supplementary material, for Bibenzyl Derivatives From Leaves of Dendrobium officinale by Gang Ren, Wen-Zan Deng, Yan-Fei Xie, Chun-Hua Wu, Wen-Yan Li, Chuan-Yun Xiao and Yun-Long Chen in Natural Product Communications