Two antiproliferative saponins of Tarenna grevei from the Madagascar dry forest [^]

Abstract

Antiproliferative bioassay-guided fractionation of the ethanol extract of the endemic Malagasy Rubiaceous plant Tarenna grevei led to the isolation of two new antiproliferative oxygenated oleanane triterpene saponins. The structures of the two active compounds were elucidated as 23-hydroxylongispinogenin 3-O-βD-glucopyranoside (1) and longispinogenin 3-O-β-D-glucopyranosyl (1→2)-β-D-glucopyranoside (3) by analyses of their spectral data including 1D- and 2D-NMR spectroscopy and chemical evidence. Compounds 1 and 3 displayed modest antiproliferative activity against the A2780 ovarian cancer cell line with IC₅₀ values of 7.6 and 4 μM, respectively.

As part of our ongoing search for potential anticancer drugs from Madagascan plants in the framework of the International Cooperative Biodiversity Group (ICBG) program, an ethanol extract of leaves and fruit of Tarenna grevei (Drake) Homolle (Rubiaceae) exhibited weak antiproliferative activity (IC₅₀ 21 μg/mL) against the A2780 ovarian cancer cell line, and this extract was selected for investigation. The genus Tarenna Gaertn. contains about 180 species distributed in the old world tropics [2]. The presence of iridoid glycosides, lignans, chalcones, sesquiterpenes, cycloartane glycosides, indole alkaloids and other phenolic compounds have been reported to be present in some species of this genus [3 11]. In Madagascar, the powdered bark and the heartwood of Tarenna madagascariensis (Baill.) Homolle, locally called “masonjoany”, has been used as a sunscreen.

Liquid-liquid partition of the ethanol extract of Tarenna grevei using hexanes, ethyl acetate and water gave an active ethyl acetate fraction (IC₅₀ 7.9 μg/mL). Successive Sephadex LH-20, octadecylsilyl silica gel and silica-gel column chromatographies afforded two bioactive oxygenated oleanane saponins (1 and 3). The isolation, the structure elucidation and the antiproliferative properties of 1 and 3 are reported herein.

Compound 1 had the molecular formula C₃₆H₆₀O₉ as determined by high resolution ESIMS (m/z 659.4098 [M+Na⁺], calculated for C₃₆H₆₀O₉Na, 659.4135). The ¹H-NMR spectroscopic data of 1 (Table 1) displayed resonances of the β-anomeric proton of a hexopyranosyl ring at δ 4.39 ppm (d, J = 7.9 Hz), signals for six quaternary methyl groups at δ 0.72, 0.90, 0.93, 1.01, 1.03 and 1.25 (each a singlet), two oxymethylene groups (δ 3.30, J = 11.7 Hz, 1H; δ 3.65, J = 11.7 Hz, 1H; and δ 3.82, J = 12 Hz, 2H) and an olefinic proton at δ 5.24 (brt, J = 2.9 Hz). Inspection of the ¹³C-NMR revealed the presence of a set of signals ascribable to a β-glucopyranosyl unit (δ 105.8, 75.6, 78.3, 71.5, 77.7 and 62.7) [12], together with 30 carbon resonances (six methyls, eleven methylenes, two of which are oxygenated; six methines, one of which is olefinic, and seven quaternary carbons as observed in the DEPT 135 spectrum, Table 2).

Table 1.

¹H-NMR data for compounds 1 and 3 (600 MHz, MeOH)

Position	1	3
1	1.63 m, 0.99 m	1.63 m, 1.03 m
2	1.75 m	1.44 m, 2.13 m
3	3.64 dd (11.7, 4.9)	3.22 dd (11.5, 5)
5	1.62 m	0.81 brd (12.6)
6	1.43 m, 1.54 m	1.62 m
7	1.43 m	1.43 m
9	1.26 overlapped	1.59 overlapped
11	1.92 m	1.92 m
12	5.24 br t (2.9)	5.26 t (3.4)
15	1.33 overlapped, 1.82 t (12.0)	1.38 overlapped, 1.82 t (12.0)
16	4.26 dd (12.0, 4.7)	4.28 dd (12.0, 4.7)
18	2.21 dd (13.8, 4.4)	2.23 dd (13.8, 4.4)
19	1.71 t (13.8), 1.06 m	1.73 t (13.4), 1.08 m
21	1.35 m, 1.73 m	1.38 m, 1.62 m
22	1.43 m, 2.13 m	1.44 m, 2.13 m
23	3.30 d (11.7), 3.65 d (11.7)	1.11 s
24	0.72 s	0.89 s
25	1.01 s	1.00 s
26	1.03 s	1.06 s
27	1.25 s	1.26 s
28	3.82 (12)	3.86 (11.8)
29	0.90 s	0.90 s
30	0.93 s	0.92 s
3-O-Glc
1′	4.39 d (7.9)	4.46 d (7.6)
2′	3.17 t (7.9)	3.58 t (7.6)
3′	3.34 m	3.38 t (7.6)
4′	3.28 overlapped	3.24 t (7.6)
5′	3.27 overlapped	3.29 overlapped
6′	3.84 dd (12, 2.9)	3.86 dd (12, 3.0)
	3.66 dd (12, 5.0)	3.65 dd (12, 5.0)
2′-O-Glc
1″		4.69 d (7.7)
2″		3.24 t (7.7)
3″		3.57 t (7.7)
4″		3.29 overlapped
5″		3.29 overlapped
6″		3.84 dd (12, 2.9)
		3.66 dd (12, 5.0)

Table 2.

¹³C-NMR data for compounds 1–4 (150 MHz)

Position	1a	2b	3a	4b	DEPT
1	39.6	38.6	40.4	39.5	CH2
2	26.2	25.5	26.4	26.3	CH2
3	83.3	82.3	91.4	89.0	CH
4	43.8	43.0	39.9	38.9	C
5	48.0	47.4	56.9	55.9	CH
6	18.7	17.9	19.3	18.4	CH2
7	34.7	32.4	34.7	33.3	CH2
8	41.3	40.0	41.1	40.1	C
9	48.1	46.9	48.2	47.2	CH
10	37.5	36.5	37.7	36.7	C
11	24.6	23.6	24.6	24.1	CH2
12	123.8	122.3	123.9	122.6	CH
13	144.3	143.6	144.2	143.8	C
14	44.7	43.7	44.6	43.9	C
15	36.5	36.3	36.6	36.6	CH2
16	67.6	66.7	67.7	66.9	CH
17	41.6	40.6	41.6	39.5	C
18	44.9	44.4	45.0	44.6	CH
19	47.7	46.9	47.8	47.2	CH2
20	31.7	30.7	31.7	30.9	C
21	33.2	33.9	33.8	33.5	CH2
22	25.9	25.9	26.0	26.3	CH2
23	64.7	64.7	28.4	28.1	CH2/CH3
24	13.4	13.1	16.8	16.5	CH3
25	16.5	16.0	15.9	15.6	CH3
26	17.3	16.8	17.4	16.9	CH3
27	27.4	26.9	27.6	27.0	CH3
28	68.9	68.9	68.8	69.8	CH2
29	33.0	33.0	33.6	33.0	CH3
30	23.9	23.9	24.2	23.0	CH3
3-O-Glc/Gal
1′	105.8	105.7	105.4	105.4	CH
2′	75.6	72.8	81.1	81.1	CH
3′	78.3	74.9	77.7	71.7	CH
4′	71.5	69.7	71.9	69.8	CH
5′	77.7	76.0	77.9	76.4	CH
6′	62.7	61.9	63.1	62.0	CH2
2′-O-Glc
1″			104.5	105.0	CH
2″			76.3	75.1	CH
3″			78.5	77.8	CH
4″			71.5	71.7	CH
5″			78.3	77.5	CH
6″			62.7	62.7	CH2

^aIn methanol-d₄,

^bIn pyridine-d₅ [13] and [14].

The ¹H- and ¹³C-NMR spectroscopic data coupled with the molecular formula suggested that 1 is an oleanane-type triterpene bearing one sugar unit at C-3 with two oxygenated methyl groups. The allocations of the double bond to C-12/C-13, the oxygenated methyl groups to C-23 and C-28, the sugar unit to C-3 and the oxygen-bearing methine to C-16 were determined as follows. The clear long-range HMBC correlation observed from the anomeric proton (δ 4.39) of the sugar unit to C-3 (δ 83.3) demonstrated the attachment of the sugar moiety to be at C-3. In addition, the presence of the trans-decalin ring system in 1 was substantiated by the presence of the proton-proton spin systems from H-3/H-2 and H-2/H-1; and H-5/H-6 and H-6/H-7; and the ³J HMBC cross-peaks from H-3 (δ3.64, dd, J= 11.7, 4.9 Hz, axial proton) to C-1, and C-5; from H-7 (δ1.43, m) to C-5 and C-9; and the NOESY correlations between H-5 and CH₃-24, and the oxymethylene CH₂-23 and the methyl group at C-10. Furthermore, the methyl groups at C-10 and C-4 were assigned by the observation of the ²J and ³J HMBC correlations from CH₃-25 (δ 1.01, s) to C-1, C-9 and C-10 and from CH₃-24 (δ 0.72, s) to C-3, C-4 and C-5. The double bond was elucidated to be at C-12/C-13 by the long range HMBC cross peaks observed from H-12 (δ 5.24, br t, J = 2.9) and C-9, C-14 and C-18 (Figure 1). Moreover, the attachments of the two hydroxymethylene groups to be at C-4 and C-17, the methyl groups at C-4, C-8, C-14 and C-20 were deduced from careful analysis of the HMBC spectral data. Inspection of the ¹³C-NMR spectral data combined with the molecular formula obtained by ESIMS demonstrated that the 1 must have an oxygen-bearing methine in its oleanane-type aglycone. In order to confirm the assignment of the sugar moiety and to locate the oxygen-bearing methine, a 2D-H2BC experiment was carried out. Correlations from H-1′ to C-2′, H-2′ to C-3′, H-3′ to C-4′, H-4′ to C-5′ and H-5′ to C-6′ of the glucopyranosyl unit, as well as from H-16 to C-15 (and vice versa) were clearly observed in the H2BC spectrum (Figure 1).

Figure 1.

Important HMBC correlations observed in 1.

Comparison of the spectroscopic data of 1 with those of similar compounds reported in the literature demonstrated that the ¹³C-NMR data of 1 are superposable with those of 23-hydroxylongispinogenin 3-O-β-D-galactopyranoside (2) (Table 2), previously isolated from Chorchorus acutangulus [13]. The only differences arise from the signal of the carbons of the sugar unit. For compound 1, the carbon chemical shifts of the β-glucopyranosyl ring (δ 105.8, 75.6, 78.3, 71.5, 77.7 and 62.7) are observed instead of those for the β-galactopyranosyl ring of 2 at δ 105.7, 72.8, 74.9, 69.7, 76.0 and 61.9. The absolute configuration of glucose was established as D by comparison of the optical rotation of 1a ([α]_D: +61 (c 0.1, H₂O) after allowing to stand overnight at room temperature with that of authentic D-glucose ([α]_D: +65 (c 0.18, H₂O) under the same conditions. The structure of 1 was thus concluded to be 23-hydroxylongispinogenin 3-O-β-D-glucopyranoside.

The molecular formula of 3 was determined to be C₄₂H₇₀O₁₃ by high resolution ESIMS (m/z 805.4741 [M+Na], calculated for C₄₂H₇₀O₁₃Na, 805.4816). The ¹H- and ¹³C-NMR spectral data of 3 were very similar with those of 1 except for the absence of the oxymethylene group at C-4 and the presence of an additional sugar unit in the molecule. In the ¹H-NMR spectrum of 3, two anomeric proton signals of two β-glucopyranosyl units at δ 4.46 ppm (d, J = 7.6 Hz, H-1′) and δ4.69 (d, J = 7.6 Hz, H-1″), and seven methyl signals (δ0.89, 0.90, 0.92, 1.00, 1.06, 1.11, 1.26, each a singlet) were observed instead of one anomeric proton and six methyl signals in 1. The assignment of the functionalities present in 3 was achieved by the interpretation of the HMBC spectral data. The second sugar present in 3 was assigned to be attached at C-2′ due to the observation of ³J HMBC correlations from the anomeric H-1″ (δ 4.69) proton to C-2′ (δ 81.1) and C-5″ (δ 78.3) and from H-2′ to C-1″ (δ 104.5) and C-4′ (δ 71.9). The ¹³C spectroscopic data of the triterpenoid skeleton of 3 were very similar to those of chorchorsin D₂ (4), a compound present in Chorchorus acutangulus [14] (Table 2). The D-configurations of the glucoses in 3 were confirmed by comparison of the TLC of the thiazolidine derivative of the sugars obtained from acid hydrolysis of 3 with those of the corresponding derivatives of D- and L-glucose [15]. Thus the structure of compound 3 was deduced to be longispinogenin 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside.

Although cycloartane saponins have been isolated from one species of the genus Tarenna [34], this is the first report on the presence of oleanane-type saponins in the genus. Compounds 1 and 3 showed moderate antiproliferative activity (IC₅₀ values of 7.6 and 4 μM, respectively) against the A2780 ovarian cancer cell line.

Experimental

General experimental procedures

Optical rotations were recorded on a JASCO P-2000 polarimeter. IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers, respectively. ¹H and ¹³C NMR spectra were recorded on a Bruker 600 spectrometer in CD₃OD with TMS as internal standard. Mass spectra were obtained on a Finnigan LTQ LC/MS. Silica-gel and silica gel C-18, both 40–63 μm, were used for open column chromatography.

Antiproliferative Bioassay

The A2780 ovarian cancer cell line assay was performed at Virginia Polytechnic Institute and State University as previously reported [16]. The A2780 cell line is a drug-sensitive ovarian cancer cell line [17].

Plant material

Leaves and fruits of Tarenna grevei (Drake) Homolle were collected in the dry forests of Orangéa, Diana region of the Antsiranana province, Madagascar, on June 11, 2005 at 12°16′43″S 049°23′19″E. The plant was a shrub 3 meters tall, with a diameter at breast height of 4 cm and yellow fruit growing on rocky terrain. Voucher specimens with collection number Razafitsalama & al. 698 have been deposited in herbaria at the Parc Botanique and Zoologique de Tsimbazaza (TAN), at the Centre National d’Application des Recherches Pharmaceutiques in Antananarivo, Madagascar (CNARP), at the Missouri Botanical Garden in St. Louis, Missouri (MO), and at the Muséum National d’Histoire Naturelle in Paris, France (P).

Extraction and Isolation

Dried leaf and fruit of T. grevei (250 g) were ground in a hammer mill, then extracted with EtOH by percolation for 24 hours at room temperature to give the crude extract MG 3249 (26.8 g), of which 9.59 g was available at Virginia Tech) for investigation. Two grams of MG 3249 were dissolved in MeOH and extracted with hexanes (3 × 300 mL) to afford 212 mg of the hexanes soluble fraction. The MeOH layer was then evaporated, suspended in H₂O (400 mL) and extracted with EtOAc (3 × 300 mL) to yield 390 mg of EtOAc soluble fraction. The EtOAc fraction was found to be active in the A2780 antiproliferative assay (IC₅₀ 7.9 μg/mL) and was divided into six fractions by size exclusion column chromatography by using Sephadex LH-20. Fractions 3 (Fr. 3, 66 mg) and 4 (Fr. 4, 130.1 mg) were active (IC₅₀ 8.4 and 6.8 μg/mL, respectively). Reversed-phase C-18 column chromatography of fraction Fr. 4 using 70% MeOH and 100% MeOH as solvent system afforded five fractions. The most active fraction (Fr. 2, 60.9 mg) was subjected to a silica gel column chromatography (solvent system CHCl₃:MeOH; 8:2) to give compounds 1 (5.3 mg) and 3 (2.5 mg).

23-Hydroxylongispinogenin 3-O-β-D-glucopyranoside

[α]_D: +27 (c 0.2, MeOH).

¹H NMR (600 MHz, CD₃OD) and ¹³C NMR (150 MHz, CD₃OD): See Tables 1 and 2.

ESIMS: m/z [M+Na⁺] calcd for C₃₆H₆₀O₉: 659.4135; found 659.4098.

Longispinogenin 3-O-β-D-glucopyranosyl (1→2)-β-D-glucopyranoside

[α]_D: +16 (c 0.12, MeOH).

¹H NMR (600 MHz, CD₃OD) and ¹³C NMR (150 MHz, CD₃OD): See Tables 1 and 2.

ESIMS: m/z [M+Na⁺] calcd for C₄₂H₇₀O₁₃: 805.4816; found 805.4741.

Acid hydrolysis of 1

Five milliliters of HCl (1N) were added to 2 mg of compound 1 and refluxed for 2 h. The reaction mixture was evaporated and dissolved in 10 mL H₂O before neutralization with Amberlite IRA 400 (Cl). The sugar fraction was purified on a C-18 SPE column eluted with H₂O to afford the carbohydrate component of 1, which was identified as D-glucose by measuring its optical rotation after allowing it to stand overnight at room temperature: [α]_D: +61 (c 0.1, H₂O).

Acid hydrolysis of 3

Compound 3 (1 mg) was dissolved in 1N HCl (5 mL) and the solution refluxed for 2 h. The reaction mixture was evaporated and dissolved in 7 mL H₂O before neutralization with Amberlite IRA 400 (Cl). The sugar fraction was purified on a C-18 SPE column eluted with H₂O.

Preparation of thiazolidine derivatives

A pyridine solution (1 mL) of the sugar fraction from the previous experiment and L-cysteine methyl ester hydrochloride (3 mg) were mixed and heated at 60 °C for 1 h as described [15] After removal of the solvent by evaporation, the residue was dissolved in water 2 ml and extracted with n-BuOH (2 ml). The organic layer after evaporation was shown to contain methyl 2-(D-gluco-pentahydroxypentyl)-thiazolidine-4(R)-carboxylate R_f: 0.42, 0.5 (C-2 epimers of thiazolidine) by TLC on silica gel, solvent system: CH₂Cl₂–MeOH–H₂O (25 : 10 : 1.5) [18].

Figure 2.

Structures of compounds 1–2.

Figure 3.

Structures of compounds 3–4.