Megastigmane Glycosides From Phoebe tavoyana

Abstract

Two new megastigmane glycosides, phoebenoside A (1) and phoebenoside B (2) and 4 known compounds, dendranthemoside A (3), lyoniresinol (4), (+)-3-O-L-rhamnopyranoside-5-methoxyisolariciresinol (5), and (+)-lyoniresinol 3α-O-β-D-glucopyranoside (6) were isolated from the leaves of Phoebe tavoyana (Meisn.) Hook. Their chemical structures were elucidated by analyses of their spectroscopic data, and comparison with those reported in the literature.

Phoebe genus (Lauraceae family), evergreen trees and shrubs, is distributed in tropical and subtropical areas. ¹ There are about 12 Phoebe species, often used to treat skin injuries and headaches, in Vietnam. ² Phytochemistry of Phoebe genus indicated the presence of alkaloids and flavonoids. ¹ There are few reports about chemical and biological studies related to Phoebe tavoyana. ³ Herein, we report the isolation, structural elucidation of 2 new and 4 known compounds from the leaves of P. tavoyana.

The molecular formula of 1 was determined to be C₁₉H₃₂O₇ by high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) ion at m/z 407.1843 [M+Cl]⁻ (calcd. for C₁₉H₃₂O₇Cl, 407.1842). The ¹H-nuclear magnetic resonance (NMR) spectrum of 1 showed the signals of 3 tertiary methyl groups at δ _H 1.23 × 2 and 1.79, 1 secondary methyl group at δ_H 1.32 (d, J = 6.5 Hz) assigning to a megastigmane aglycone; an anomeric proton at δ _H 4.39 (d, J = 7.5 Hz) suggesting the presence of 1 sugar unit (Table 1). The ¹³C-NMR and heteronuclear single quantum correlation (HSQC) spectra of 1 revealed the signals of 19 carbons, including 1 carbonyl, 3 quaternary carbons, 6 methines, 5 methylenes, and 4 methyl carbons. In the ¹H-¹H correlation spectroscopy (COSY), cross peaks between protons H-2 (δ _H 1.82) and H-3 (δ _H 2.47), H-8 (δ _H 1.70) and H-7 (δ _H 2.32/2.54)/ H-9 (δ _H 3.94), and between H-9 and H-10 (δ _H 1.32) were observed. The heteronuclear multiple bond correlation (HMBC) from H-13 (δ _H 1.79) to C-4 (δ _C 201.5)/C-5 (δ _C 131.7)/C-6 (δ _C 168.4); from H-11/H-12 (δ _H 1.23) to C-1 (δ _C 37.6)/C-2 (δ _C 38.4)/C-6 (δ _C 168.4) suggested the positions of an oxo group at C-4 and the double bond at C-5/C-6. The HMBC correlations between H-10 (δ _H 1.32) and C-8 (δ _C 36.4)/C-9 (δ _C 77.7) suggested the oxygenated group at C-9 (Figure 1). Furthermore, acid hydrolysis of 1 revealed D-glucose. The multiplicity of glc H-1′ [δ _H 4.39 (d, J = 7.5 Hz)] showed the presence of β-d-glucopyranosyl. The HMBC between H-1′ (δ _H 4.39) and C-9 (δ _C 77.7) suggested the location of β-d-glucopyranosyl at C-9. The chemical shifts of C-9 (δ _C 77.7), C-10 (δ _C 21.8), and C-1′ (δ _C 104.1) confirmed the 9S configuration by comparing with the corresponding data of (3R,9S)-megastigman-5-en-3,9-diol 9-O-β-d-glucopyranoside [C-9 (δ _C 77.9), C-10 (δ _C 21.8), and C-1′ (δ _C 103.9)] and (3R,9R)-megastigman-5-en-3,9-diol 9-O-β-d-glucopyranoside [C-9 (δ _C 76.1), C-10 (δ _C 19.8), and glc C-1′ (δ _C 102.2)] in the same solvent CD₃OD. ⁴ Consequently, the new structure of 1 was determined to be (9S)-hydroxymegastigman-5-en-4-one 9-O-β-d-glucopyranoside and named as phoebenoside A (Figure 1).

Table 1

The ¹H- and ¹³C-NMR Data for Compounds 1 and 2 in CD₃OD.

C		1		2
	δC	δH (mult., J, Hz)	δC	δH (mult., J, Hz)
Aglycone
1	37.6	-	39.1	-
2	38.4	1.82 (t, 7.0)	38.5	1.38 (dd, 1.5, 15.0, α)2.07 (dd, 3.0, 15.0, β)
3	35.1	2.47 (t, 7.0)	68.3	4.07 (dd, 3.0, 3.0)
4	201.5	-	82.9	3.86 (dd, 3.0, 3.0)
5	131.7	-	33.2	2.36 (dq, 3.0, 7.0)
6	168.4	-	80.2	-
7	27.6	2.32 (m)/2.54 (m)	132.7	5.57 (d, 15.5)
8	36.4	1.70 (m)	135.2	5.73 (dd, 6.0, 15.5)
9	77.7	3.94 (m)	69.2	4.30 (m)
10	21.8	1.32 (d, 6.5)	24.1	1.26 (d, 6.5)
11	27.2	1.23 (s)	27.6	1.15 (s)
12	27.2	1.23 (s)	26.5	0.90 (s)
13	11.7	1.79 (s)	12.9	1.03 (d, 7.0)
O-Glc
1′	104.1	4.39 (d, 7.5)	102.7	4.29 (d, 8.0)
2′	75.4	3.20 (dd, 7.5, 9.0)	74.9	3.16 (dd, 7.5, 8.0)
3′	77.9	3.35 (t, 9.0)	78.1	3.35 (dd, 7.5, 8.5)
4′	71.7	3.34 (t, 9.0)	71.7	3.29 (dd, 7.5, 8.5)
5′	78.3	3.30 (m)	78.2	3.25 (m)
6′	62.8	3.69 (dd, 5.0, 12.0)3.88 (dd, 2.0, 12.0)	62.9	3.69 (dd, 5.5, 12.0)3.89 (d, 2.5, 12.0)

Figure 1

Chemical structures, key heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) correlations of compounds 1 and 2.

The molecular formula of 2 was established as C₁₉H₃₄O₉ based on its ion at m/z 441.1900 [M+Cl]⁻ (calcd. for [C₁₉H₃₄O₉Cl]⁻, 441.1897) in the HR-ESI-MS spectrum. The ¹H-NMR spectrum of 2 exhibited the signals of 4 methyl groups at δ _H 0.90 (s), 1.03 (d, J = 7.0 Hz), 1.15 (s), and 1.26 (d, J = 6.5 Hz), assigning to a megastigmane and an anomeric proton at δ _H 4.29 (d, J = 8.0 Hz) suggesting the presence of 1 sugar moiety. The ¹³C-NMR and HSQC spectra (Table 1) showed the signals of 19 carbons, including, 2 non-protonated carbons, 11 methines, 2 methylenes, and 4 methyl carbons. The HMBC correlations between H-13 (δ _H 1.03) and C-4 (δ _C 82.9)/C-5 (δ _C 33.2)/C-6 (δ _C 80.2); between H-11 (δ _H 1.15)/H-12 (δ _H 0.90) and C-1 (δ _C 39.1)/C-2 (δ _C 38.5)/C-6 (δ _C 80.2); between H-4 (δ _H 3.86) and C-3 (δ _C 69.2)/C-6 (δ _C 80.2)/C-13 (δ _C 12.9) as well as ¹H-¹H COSY cross peaks of H-2/H-3/H-4/H-5/H-13 suggested the positions of hydroxyl groups at C-3 and C-6; oxygenated group at C-4 (Figure 1). Acid hydrolysis of 2 revealed D-glucose (see Supporting information). In addition, the location of β-d-glucopyranosyl at C-4 was confirmed by HMBC correlations between H-1′ (δ _H 4.29) and C-4 (δ _C 82.9). Moreover, the HMBC correlations from H-10 (δ _H 1.26) to C-8 (δ _C 135.2)/C-9 (δ _C 69.2); from H-7 (δ _H 5.57) to C-6 (δ _C 80.2)/C-8 (δ _C 135.2)/C-9 (δ _C 69.2), and coupling constant of H-7 and H-8, J = 15.5 Hz) suggested the position of double bond (E geometry) at C-7/C-8 and a hydroxyl group at C-9. The relative configuration of 2 was deduced from analysis of coupling constants and the NOESY spectrum (Figure 1). The small coupling constants of H-2 and H-3 (J = 3.0 Hz); H-3 and H-4 (J = 3.0 Hz) and H-4 and H-5 (J = 3.0 Hz), suggested configurations of both hydroxyl groups at C-3 and C-4 to be axial orientation. The NOESY correlation between H-5 and H-11 indicated that H-5 and C-11 were axial (β-configuration). The hydroxyl group at C-6 was determined to be α-oriented based on the strong NOESY correlations between H-7 and H-5/H-11. Enzymatic hydrolysis of 2 gave aglycone. The aglycone of 2 was found to be identical to (3S,4S,5R,6R,9R)-3,4,6,9-tetrahydroxymegastigman-5-ene (2a), a compound reported from Cinnamomum wilsonii and confirmed by single-crystal X-ray ⁵ by comparing their spectroscopic data and optical rotation. Thus, structure of 2 was elucidated as (3S,4S,5R,6R,9R)-3,4,6,9-tetrahydroxymegastigman-5-ene 4-O-β-d-glucopyranoside and named as phoebenoside B (Figure 1).

The known compounds were elucidated as dendranthemoside A (3), ⁶ lyoniresinol (4), ⁷ (+)-3-O-l-rhamnopyranoside-5-methoxyisolariciresinol (5), ⁸ (+)-lyoniresinol 3α-O-β-d-glucopyranoside (6) ⁹ by analyses of spectroscopic data and comparison with those reported in the literature.

All the isolated compounds were evaluated against 3 human cancer cell lines, HT-29 (human colon cancer), A-549 (human lung cancer), and A-2058 (human melanoma cancer cell) using the MTT assay. However, they did not show any cytotoxic activity (IC₅₀ > 50 µM).

Experimental

General

See Supporting Information.

Plant Materials

The leaves of P. tavoyana (Meisn.) Hook. were collected at Me Linh, Vinh Phuc province, Vietnam in May, 2016 and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (NCCT-P59) was deposited at the Institute of Marine Biochemistry.

Extraction and Isolation

The dried leaves of P. tavoyana (3.2 kg) were extracted with hot MeOH (3 × 5 L) using sonicator to yield 200 g of extract. The MeOH extract was suspended in water and successively partitioned with CH₂Cl₂, ethyl acetate (EtOAc) to obtain CH₂Cl₂ (PT1), EtOAc (PT2), and water (PT3) fractions. PT3 was chromatographed on a Diaion HP-20 column eluting with water to remove sugars, then increasing concentration of methanol in water (25%, 50%, 75%, and 100%, each 1 L) to obtain 4 fractions, PT3A-PT3D, respectively. PT3A was subjected on a RP-18 column chromatography (CC) using MeOH/water (1/3, v/v) to give 5 fractions, PT3A1-PT3A5. PT3A2 was chromatographed on a Sephadex LH-20 column using MeOH/water (1/1, v/v) to give 3 fractions, PT3A2A-PT3A2C. PT3A2B was chromatographed on a silica gel CC (CH₂Cl₂/acetone/water, 1/4/0.3, v/v/v) to yield 5 (10.0 mg) and 6 (8.0 mg). PT3A4 was chromatographed on a silica gel CC eluting with CH₂Cl₂/MeOH/water (2.5/1/0.1, v/v/v) to give 2 fractions, PT3A4A-PT3A4B. PT3A4A was chromatographed on a silica gel CC eluting with EtOAc/MeOH/water (7/1/0.05, v/v/v) to yield 1 (8.8 mg). Compound 2 (20.0 mg) was obtained from PT3A4B on a silica gel CC eluting with CH₂Cl₂/acetone/water (1/4/0.3, v/v/v). PT3A5 was loaded on a silica gel CC eluting with CH₂Cl₂/MeOH/water (2.5/1/0.1, v/v/v) to give 3 fractions, PT3A5A-C. Compound 3 (10.0 mg) was obtained from PT3A5A using a RP-18 CC eluting with MeOH/water (1/3, v/v). PT3C was chromatographed on silica gel CC eluting with CH₂Cl₂/MeOH (8/1, v/v) to give 4 (50.0 mg).

Phoebenoside A (1)

White amorphous powder.

[α]_D ²⁵: –43.0 (c 0.1, MeOH).

¹H- and ¹³C-NMR (CD₃OD): Table 1.

HR-ESI-MS m/z 407.1843 (calcd. for [C₁₉H₃₂O₇Cl]⁻ 407.1842).

Phoebenoside B (2)

White amorphous powder.

[α]_D ²⁵: –55.0 (c 0.1, MeOH).

¹H- and ¹³C-NMR (CD₃OD): Table 1.

HR-ESI-MS m/z 441.1900 (calcd. for [C₁₉H₃₄O₉Cl]⁻ 441.1897).

Enzymatic Hydrolysis, Acid Hydrolysis, and Cytotoxic Assays

See Supporting Information as detailed in the Supplementary data section.