Carbohydrate Research 402 (2015) 225–231
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Triterpenoid saponins from root bark of Zanha golungensis (Sapindaceae) Catherine Lavaud a,⇑, Charlotte Sayagh a, Franck Humbert a, Isabelle Pouny b, Clément Delaude c a
Institut de Chimie Moléculaire de Reims, CNRS UMR 7312, Université de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 02, France Epigenetic Targeting of Cancer, CNRS USR 3388, Centre de Recherche et de Développement Pierre Fabre, BP 13562, 31035 Toulouse Cedex 01, France c Institut de Chimie, Université de Liège, B36, Sart Tilman, B-4000 Liège, Belgium b
a r t i c l e
i n f o
Article history: Received 18 June 2014 Received in revised form 29 September 2014 Accepted 2 October 2014 Available online 8 October 2014 Keywords: Zanha golungensis Triterpenoid saponin Zanhic acid Zanhasaponin
a b s t r a c t The chemical investigation of the methanolic extract from root bark of Zanha golungensis Hiern led to the isolation of five new and one known triterpenoid saponins. Their structures were elucidated by full analysis of their spectroscopic data and by partial hydrolysis. These glycosides contain zanhic acid as aglycone, a rare oleanane-type triterpenoid found in species belonging to Sapindaceae, Caryophyllaceae, Asteraceae, and Fabaceae. Two new saponins are esterified saponins by 3,3-dimethylacryloyl and 3-hydroxy-2-methyl-butanoyl residues located on the sugar part. The new compounds were named zanhasaponins D–H following previous isolation of similar compounds from Zanha africana. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Zanha golungensis Hiern is a 6–25 m high tree or more rarely a 2–3 m high shrub growing all over tropical Africa. This plant has various uses such as timber for construction, chew-sticks, fodder, food, and it is one of the most common medicinal plants in whole Africa.1 New medical uses were recently reported for its leaves, bark, twigs, and roots, the latter being used against dropsy, swellings, edema, and gout.1,2 The presence of saponins and of oleanane-type triterpenes in the root bark of Z. golungensis has been previously reported.3,4 There are two species of Zanha accepted in The Plant List growing in Africa, and the second one, Zanha africana, was the object of several biological evaluations. The CH2Cl2 extract of the roots exhibited trypanocidal and antiviral activities, while the methanolic extract showed antifungal and anti-inflammatory activities.5–8 Both root bark extracts demonstrated tumoricidal activity.9 Antibacterial and fungicidal activities were reported for the methanolic bark extract.10,11 From a chemical viewpoint, three saponins were
⇑ Corresponding author. Tel.: +33 3 26913139; fax: +33 3 26913166. E-mail address: [email protected] (C. Lavaud). http://dx.doi.org/10.1016/j.carres.2014.10.001 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
isolated from the root bark of Z. africana and named zanhasaponins A–C, and they exhibited antiphospholipase A2 activity.12,13 Given the observations made on the saponins of Z. africana, it seemed interesting to complete the previous investigations and to isolate and identify new saponins from the root bark extract of this peculiar species, Z. golungensis.
2. Results and discussion The MeOH extract obtained from the root bark of Zanha golungensis was freed of salts by cationic exchange through a resin and fractionated by reverse-phase silica gel column chromatography. Nine fractions were thus obtained, which were purified by semi-prep. HPLC to yield six saponins (1–6) among them five new compounds named zanhasaponins D–H. Acid hydrolysis of the saponin extract gave five sugars identified by chiral chromatography as D-glucuronic acid, D-glucose, D-xylose, L-rhamnose, and D-fucose.
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2.1. Structure of aglycone of saponins 1–6 Zanhic acid (or 16-a-hydroxymedicagenic acid or 2b,3b,16 a-trihydroxy-12-oleanene-23,28-dioic acid) was identified as the aglycone of all saponins 1–6. The spectroscopic data were in agreement with those reported in the literature for the glycosides of zanhic acid (Table 1).12,14–17 In the 13C NMR spectra, three hydroxymethine carbons were detected at dC 70.8 ± 0.5 (C-2), 85 ± 1.5 (C-3), and 75 ± 0.3 (C-16), two carbonyl carbons at dC 181.8 ± 0.8 (C-23) and 177.9 ± 0.5 (C-28), and a pair of olefinic carbons at dC 123.7 ± 0.5 (C-12) and 145.1 ± 0.5 (C-13). The H-3 appeared as a doublet near dH 4.3 with a JH-3–H-2 coupling constant of 3.75 ± 0.25 Hz, confirming the a-equatorial position of H-2 and the a-axial position of H-3; the H-16 (dH 4.48 ± 0.1) shared with both H-15 a vicinal coupling constant of the order of 3 Hz attesting its b-equatorial position. The observation of a ROE correlation between H-3 and H-5 indicated their a-axial orientation; the acid function was located at C-23 on the basis of a ROE interaction between the b-oriented CH3-24 and CH3-25. 2.2. Structure of zanhasaponins D (1) and E (2) The molecular formula of zanhasaponin D (1) was established as C36H54O13 from the positive ion HRESIMS which exhibited a pseudo-molecular ion at m/z 717.3455 [M+Na]+ (calcd 717.3462 for C36H54O13Na). Similarly, the HRESIMS of zanhasaponin E (2) showed a pseudo-molecular ion at m/z 731.3620 [M+Na]+ (calcd 731.3619 for C37H56O13Na) in agreement with a molecular formula of C37H56O13. Consequently, these two compounds were deduced to contain a pyranosyl uronic acid linked to zanhic acid aglycone. The hypothesis that this pyranosyl uronic acid was methoxylated in compound 2 was confirmed by its NMR analysis, which showed a supplementary methyl at dC 52.9 and dH 3.8 (s). The anomeric signals of this sugar were detected at dC105 and dH 4.43 (1) or 4.45 (2) (d, J = 7.7 or 7.5 Hz) indicating an axial position; the analysis of COSY, HSQC, and HMBC experiments, together with the previous result of the acid hydrolysis of saponin extract, allowed its identification as a b-D-glucuronopyranosyl acid in 1 and 2 (Table 2). In
the HMBC spectrum of zanhasaponin E (2), H-5 of glucuronopyranosyl acid and the methoxy group share a correlation with the carbonyl carbon at dC 171.2 ppm attributed to C-6 of glucuronopyranosyl acid, and H-1 of glucuronopyranosyl acid showed a 3JH-C correlation with C-3 of zanhic acid. The structure of zanhasaponin D (1) was thus deduced to be 3-O-b-D-glucuronopyranosyl-zanhic acid, and that of zanhasaponin E (2) to be the 3-O-(6-O-methyl)b-D-glucuronopyranosyl-zanhic acid. Zanhasaponin D was previously isolated as a prosapogenin (zanhin) after partial acid hydrolysis of saponins from Zanha, but it is the first time that this compound was identified as a native saponin.4,12 The natural or artefactual character of zanhasaponin E is open to discussion since this compound might result from addition of methanol. To provide evidence of the original character of this saponin, we reacted saponin 1 with methanol under various operating conditions similar to those used in the extraction step and in the purification stage, such as in cold and then boiling methanol, in the presence of silica gel, or in the presence of HPLC eluent. After HPLC analysis of these reactions, it was found that none generated saponin 2, suggesting that it was a genuine compound. Analysis of the NMR data for saponins (3–6) showed that the 3-O-b-D-glucuronopyranosyl-zanhic acid (1) belonged to these four saponins (Tables 1 and 2). Differences with 1 arose from the presence of another glycosidic moiety linked to zanhic acid. The chemical shifts of C-28 at dC 177.4 ± 0.1 and of C-1 of second glycosidic unit at dC 95.1 ± 0.1, confirmed its esterification by this second oligosaccharidic chain. 2.3. Characterization of a common core of structure in zanhasaponins F–H (3–5) Comparison of 1H NMR spectra of zanhasaponins F–H (3–5) showed great similarities in the signals of the sugar part with three anomeric doublets at dH 4.43 ± 0.02 (assigned to b-D-glucuronic acid as in 1), 5.29 ± 0.01 (J = 1.5 Hz), and 5.41 ± 0.01 (J = 8 Hz); those protons were correlated to their respective carbons at dC 105 ± 0.5, 102 ± 0.1, and 95.1 ± 0.1 in the HSQC experiment (Table 2). These two latter sugars were identified as 6-deoxyhexoses
Table 1 NMR data of aglycone part (500 MHz for 1H and 125 MHz for No.
C, CD3OD, d in ppm, J in Hz) for zanhasaponins D–H (1–5)
1
2
3
4
5
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
acid 1.32 (m), 2.16 (br d, 14.5) 4.32 (br d, W1/2 = 9) 4.11 (d, 3.6) — 1.65 (m) 1.20 (m), 1.72 (m) 1.32 (m), 1.60 (m) — 1.70 (m) — 1.95–2.05 (m) 5.33 (br t, 3.0) — — 1.36 (m), 1.83 (dd, 14.0, 4.0) 4.49 (br t, 3.0) — 3.04 (dd, 14.0, 4.4) 1.06 (dd, 13.0, 3.5), 2.31 (t, 13.0) — 1.18 (m), 1.97 (m) 1.84 (m), 1.94 (m) — 1.45 (s) 1.32 (s) 0.84 (s) 1.41 (s) — 0.92 (s) 1.00 (s)
44.6 70.8 86.3 53.1 53.0 21.6 33.8 40.8 49.8 37.3 24.5 123.2 144.9 42.7 35.9 75.1 50.1 41.9 47.6 31.3 36.4 32.7 181.0 13.5 17.0 17.5 27.2 nd 33.3 24.7
1.32 (m), 2.14 (dd, 14.2, 1.5) 4.27 (br d, W1/2 = 9) 4.12 (br d, 3.0) — 1.62 (m) 1.20 (m), 1.65 (m) 1.32 (m), 1.60 (m) — 1.68 (m) — 1.95–2.05 (m) 5.34 (br t, 3.5) — — 1.36 (m), 1.83 (dm, 13.6) 4.49 (br s)
44.5 71.3 83.5 nd 53.1 21.5 34.0 41.0 48.7 37.4 24.9 123.3 144.9 42.5 36.0 75.3 42.1 48.0 31.4 36.5 33.0a 182.2 13.5a 17.0a 17.8a 27.3a nd 33.4 24.9a
44.7 70.8 86.0 nd 53.1 22.0 33.8 41.2 49.0 37.4 24.6 123.5 145.0 42.9 36.4 74.8 50.1 42.4 48.0 31.3 36.2 32.0 nd 13.8 17.3 17.8 27.1 177.4 33.4 25.0
1.33 (m), 2.15 (dd, 14.1, 1.6) 4.32 (m, W1/2 = 10) 4.12 (d, 3.5) — 1.65 (m) 1.18 (m), 1.68 (m) 1.43 (m), 1.58 (m) — 1.68 (m) — 1.95–2.05 (m) 5.38 (br t, 3.0) — — 1.43 (m), 1.80 (dd, 13.1, 3.0) 4.47 (br t, 3.0) — 2.99 (dd, 14.2, 4.1) 1.08 (dm, 13.5), 2.32 (t, 13.5) — 1.22 (m), 1.95 (m) 1.92 (m) — 1.42 (s) 1.32 (s) 0.85 (s) 1.41 (s) — 0.92 (s) 1.01 (s)
45.0 70.8 86.3 nd 53.5 21.7 33.8 41.2 48.5 37.4 24.7 123.4 144.6 42.9 36.2 75.0 50.2 42.4 48.0 31.0 36.5 31.4 181.5 13.6 17.4 18.0 27.2 177.5 33.4 25.1
1.31 (m), 2.18 (dd, 14.0, 2.0) 4.35 (br s, W1/2 = 10) 4.10 (d, 4.0) — 1.62 (d, 11.2) 1.18 (m), 1.68 (m) 1.43 (m), 1.58 (dd, 12.4, 8.5) — 1.68 (m) — 1.95–2.05 (m) 5.39 (br t, 3.0) — — 1.43 (m), 1.78 (dd, 13.5, 3.5) 4.47 (br s)
3.04 1.06 — 1.18 1.80 — 1.41 1.31 0.83 1.41 — 0.91 1.00
1.32 (m), 2.15 (br d, 14.5) 4.34 (br s, W1/2 = 10) 4.13 (br s, W1/2 = 4) — 1.62 (m) 1.20 (m), 1.66 (m) 1.43 (m), 1.63 (m) — 1.68 (m) — 1.98–2.05 (m) 5.38 (br t, 3.0) — — 1.43 (m), 1.78 (dd, 13.3, 3.0) 4.48 (br t, 3.0) — 2.99 (dd, 14.0, 3.2) 1.08 (m), 2.32 (br t, 13.4) — 1.22 (m), 1.95 (m) 1.82 (m), 1.95 (m) — 1.45 (s) 1.32 (s) 0.85 (s) 1.41 (s) — 0.92 (s) 1.00 (s)
44.9 70.8 86.4 53.3 53.2 21.7 33.9 41.3 49.5 37.4 24.7 123.3 144.6 42.9 36.3 74.8 50.3 42.3 48.1 31.4 36.5 31.6 181.7 13.6 17.4 18.0 27.2 177.4 33.5 25.1
(dd, 14.1, 4.0) (m), 2.31 (t, 13.5) (m), 1.97 (m) (m), 1.94 (m) (s) (s) (s) (s) (s) (s)
2.99 1.08 — 1.22 1.92 — 1.42 1.31 0.85 1.41 — 0.91 1.00
(dd, 14.2, 4.0) (m), 2.31 (t, 13.6) (m), 1.95 (m) (m) (s) (s) (s) (s) (s) (s)
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Zanhic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
13
nd = not determinated. a Detected from HSQC correlation.
227
228
Table 2 NMR data of glycosidic part (500 MHz for 1H and 125 MHz for No.
1 dH
13
C, CD3OD, d in ppm, J in Hz) for zanhasaponins D–H (1–5).
2 dC
dH
b-D-Fucopyranosyl (at C-28 of zanhic acid) 1 2 3 4 5 6
dH
105.0 74.8 77.0 73.2 76.5 171.2 52.9
4.45 3.31 3.45 3.50 3.72 —
4 dC
dH
(br d, 7.0) (nd) (nd) (nd) (nd)
104.5 74.8 77.5 73.3 nd nd
4.43 3.29 3.39 3.51 3.80 —
5.40 3.75 3.90 5.15 3.86 1.10
(d, 8.1) (dd, 9.1, 8.3) (dd, 9.3, 3.5) (d, 3.6) (q, 6.4) (d, 6.4)
95.1 75.6 74.3 75.1 71.1 16.6
5.29 3.95 3.67 3.41 3.76 1.30
(br s) (m, W1/2 = 6.0) (dd, 9.4, 3.3) (t, 9.4) (m) (d, 6.2)
102.0 72.0 72.1 73.8 70.4 18.4
5 dC
dH
dC
(d, 7.7) (dd, 9.0, 7.8) (t, 9.0) (t, 9.0) (d, 9.0)
105.0 74.9 77.5 73.8 76.2 171.5
4.42 3.30 3.40 3.48 3.79 —
(d, 7.7) (dd, 9.1, 7.8) (t, 9.1) (nd) (d, 9.0)
105.0 74.7 77.2 73.3 76.5 nd
5.42 3.77 3.91 5.15 3.87 1.08
(d, 8.0) (dd, 9.3, 8.0) (dd, 9.4, 3.6) (d, 3.6) (br q, 6.5) (d, 6.4)
95.2 76.2 74.2 74.0 71.2 16.7
5.41 3.76 3.91 5.13 3.88 1.12
(d, 8.0) (dd, 9.1, 8.1) (dd, 9.3, 3.6) (d, 3.5) (q, 6.5) (d, 6.5)
95.0 75.7 74.0 75.1 70.8 16.6
5.28 3.95 3.67 3.51 3.76 1.29
(d, 1.5) (dd, 3.2, 1.7) (dd, 9.5, 3.3) (t, 9.5) (m) (d, 6.2)
102.1 72.0 72.1 73.8 70.5 18.4
5.29 3.95 3.67 3.41 3.76 1.30
(d, 1.5) (dd, 3.3, 1.5) (dd, 9.4, 3.4) (t, 9.5) (m) (d, 6.2)
102.0 71.9 72.0 73.7 70.4 18.3
a-L-Rhamnopyranosyl (at C-2 of fucopyranose) 1 2 3 4 5 6 1 2 3 4 5 10 20 30 40 50 nd = not determinated.
Acetyl (at C-4 of fucopyranose) — 172.8 2 .20 (s) 20.8
3,3-Dimethylacryloyl (at C-4 of fucopyranose) — 167.9 5.87 (sept., 1.3) 116.8 — 159.0 2.23 (br s) 20.7 1.99 (br s) 27.6
Niloyl-(3-10 )-niloyl (at C-4 of fucopyranose) — 175.9 2.83 (qt, 6.9) 46.4 5.21 (qt, 6.4) 72.3 1.34 (d, 6.3) 18.2 1.32 (d, 7.0) 13.8 — 2.50 3.95 1.21 1.14
(qt, 7.1) (qt, 6.5) (d, 6.3) (d, 7.1)
175.1 49.2 69.9 20.3 13.8
C. Lavaud et al. / Carbohydrate Research 402 (2015) 225–231
b-D-Glucuronopyranosyl acid (at C-3 of zanhic acid) 1 4.43 (d, 7.7) 105.0 4.45 (d, 7.5) 2 3.30 (dd, 9.1, 7.8) 74.7 3.29 (br dd, 8.0, 9.1) 3 3.39 (t, 9.0) 77.2 3.39 (t, 9.2) 4 3.51 (t, 9.1) 73.0 3.53 (dd, 9.5, 9.2) 5 3.81 (d, 9.0) nd 3.88 (d, 9.7) 6 — 170.0 — OCH3 3.80 (s)
3 dC
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the with methyl group (CH3-6) as doublet near dH 1.10 (J = 6.4 Hz) and 1.30 (J = 6.2 Hz), respectively. Joint analysis of COSY and TOCSY spectra starting from anomeric protons and methyl groups, and the values of proton coupling constants revealed the presence of a b-D-fucopyranose and of an a-L-rhamnopyranose. The HMBC spectra of compounds 4 and 5 showed cross-peaks between H-1 of rhamnopyranose (dH 5.28/5.29) and C-2 of fucopyranose (dC 76.2/75.7), between H-1 of fucopyranose (dH 5.42/5.41) and C-28 of zanhic acid (dC 177.4/177.5), and as previously observed for 1 between H-1 of glucuronopyranosyl acid (dH 4.42/4.43) and C-3 of zanhic acid (dC 86.3/86.4). In the ROESY spectra of saponins 3–5, the effects observed between H-1 of rhamnopyranose and H-2 of fucopyranose, between H-18 of zanhic acid and CH3-6 of fucopyranose, and between H-1 of glucuronopyranosyl acid and H-3 of zanhic acid, confirmed the sequence of the glycosidic chains and their relative stereochemistry. The deshielding chemical shift of H-4 in fucopyranose at dH 5.14 ± 0.01 and its HMBC correlation observed with a supplementary carboxylic carbon detected between dC 167 and 176 suggested that this glycosidic position was esterified in zanhasaponins F–H. Thus, the substructure comprised in saponins 3–5 was the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)-b-D-(4-O-acyl-fucopyranosyl)]-zanhic acid, which corresponded to a C48H74O21 fragment. Although run in different solvents, the NMR data were similar to those reported in the literature for structures with similar ester saccharide chain comprised of a 4-acylated b-D-fucopyranosyl residue.18,19 2.4. Structure of zanhasaponins F–H (3–5) Zanhasaponin F (3) exhibited a molecular peak at m/z 1027.4721 [MH] in the negative mode HRESIMS (calcd 1027.4755) in agreement with a C50H76O21 molecular formula. Subtracting the common molecular skeleton, yielded a C2H2O fragment (42 units) corresponding to an acetyl group. A three proton singlet was effectively detected at dH 2.2 in the 1H NMR spectrum, and in the 13C NMR spectrum two supplementary carbons were attributed to a methyl group at dC 20.8 and a carboxyl of ester at dC 172.8. Consequently, zanhasaponin E (3) is the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)-b-D-(4-O-acetylfucopyranosyl]-zanhic acid. The HRESIMS of zanhasaponin G (4) indicated a C53H80O22 molecular formula, as deduced from the [M+Na]+ ion at m/z 1091.5033 (calcd 1091.5039). The ester moiety linked to position 4 of fucopyranose was in this case a C5H6O unit (82 amu). The signal of one olefinic proton was detected at dH 5.87 (septuplet, J = 1.3 Hz), and it had COSY correlations with two broad methyl singlets at dH 1.99 and 2.23, suggesting a dimethylallyl group. A supplementary carbonyl carbon at dC 167.9 exhibited HMBC correlations with the methyl proton signals. These signals were obviously assigned to a 3-methyl-2-butenoic acid (3,3-dimethylacrylic acid). Thus, zanhasaponin G (4) is the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)-b-D-(4-O-3,3-dimethylacryloyl-fucopyranosyl]-zanhic acid. Zanhasaponin H (5) exhibited a molecular peak at m/z 1185.5704 [MH], and by subtraction of the common molecular C48H74O21 fragment, its molecular weight was increased by 200 amu corresponding in this case to an ester group of formula C10H16O4. The characteristic NMR signals for two niloyl (2-methyl-3-hydroxybutanoyl) groups were observed on the 1H NMR spectrum of 5 with four methyl doublets at dH 1.14 (J = 8 Hz) 1.21 (J = 6.3 Hz), 1.32 (J = 8 Hz), and 1.34 (J = 6.3 Hz), correlated in COSY experiment to four protons being as quintets at dH 2.5 (J = 7.2 Hz), 3.95 (J = 6.5 Hz), 2.83 (J = 7 Hz), and 5.21 (J = 6.4 Hz), respectively.20 Two carboxyl carbons were detected at dC 175.1 and 175.9; the first exhibited an HMBC correlation with the deshielded quintet at dH 5.21, while the second had the correlation with H-4 of fucopyranose. Thus, the C10 ester
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group was composed of the sequence of two nilic acids: 2-methyl3-hydroxy-butanoyl-(1-3)-2-methyl-3-hydroxy-butanoyl. The threo configuration of nilic acid was determined from the value of vicinal coupling constant of methine protons (J >6.3 Hz), and its absolute configuration was confirmed after basic hydrolysis of zanhasaponin H (5); the positive value of optical rotation (+8, H2O) suggested a 2S,3S-configuration.21,22 The structure of zanhasaponin H (5) is the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)b-D-(4-O-[30 -S-hydroxy-20 -S-methyl-butyroyloxy]-3-S-hydroxy-2S-methyl-butyroyloxy)-fucopyranosyl]-zanhic acid. 2.5. Structure of known saponin (6) Analysis of HRESIMS and NMR data of saponin 6 allowed its identification as 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)[b-D-xylopyranosyl-(1?3)]-b-D-(4-O-acetyl-fucopyranosyl]-zanhic acid. This compound was recently isolated from Ganophyllum giganteum, a species belonging to Doratoxyleae tribe as Z. golungensis.23 3. Conclusion To our knowledge, this study is the first report on the characterization of saponins from Z. golungensis. Although this species is a widespread medicinal plant used for a large variety of pains, to the best of our knowledge, no phytochemical analyses have been previously reported. The originality of isolated saponins is the nature of the ester parts, and the structure of aglycone, the rarely distributed zanhic acid. These oleanane-type triterpenoid saponins were found in a few species belonging to genera Zanha, Filicium and Ganophyllum (Sapindaceae), Aster (Asteraceae), Medicago (Fabaceae), Physena (sole genus of Physenaceae), and Herniaria (Caryophyllaceae).12,13,15–17,23–26 Compounds isolated from Sapindaceae and Caryophyllaceae species possessed a glycosidic chain at C-28 of zanhic acid, containing as first disaccharide a-L-rhamnopyranosyl-(1?2)-b-D-fucopyranosyl moiety with the C-4 position of fucopyranose typically esterified. Esterified saponins of Z. golungensis contained isoprenyl residues which are usually located on hydroxyl groups of polyhydroxylated rings D and E of aglycones. Thus, it is the fourth time that esters as 3,3-dimethylacryloyl and 3-hydroxy-2-methyl-butanoyl residues were located on the sugar part.20,23,27 4. Experimental 4.1. General methods UV spectra were obtained using a SchimadzuÒ UV 2450 spectrophotometer. Optical rotations were measured in MeOH with a Perkin-Elmer 341 automatic polarimeter. 1H and 13C NMR spectra were recorded in CD3OD on a BrukerÒ Avance DRX-500 spectrometer (1H at 500 MHz and 13C at 125 MHz), and 2D-NMR experiments were performed using standard Bruker microprograms (XWIN NMR version 2.6 software). HR-ESI-MS was performed using a BrukerÒMicroTOF or a MicromassÒESI-Q-TOF apparatus using ESI ionization, and the samples were introduced by infusion in MeOH solution. For analysis of saponins, TLC was carried out on precoated silica gel 60F254 (MerckÒ ) with CHCl3–MeOH–H2O (60:40:7) and on reversed phase silica gel RP-18 (MerckÒ ) with MeOH–H2O (7:3 or 11:9), and spots were visualized by spraying with 50% aq H2SO4. Column chromatography (CC) was carried out on Kieselgel (63–200 lm, MerckÒ) or Lichroprep RP-18 (40-63 lm, MerckÒ). HPLC for purification of saponins was performed on a MerckÒ apparatus equipped with a AP 250 ArmenÒ pump, a Merck–KnauerÒ K-2501 UV detector, and a BuchiÒ C-660 fraction collector; column packed with MerckÒ Lichrospher RP-18 (50 200 mm, 12 lm) was
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used for preparative HPLC with binary gradient elution (solvent A: H2O; solvent B: MeOH), a flow rate of 35 mL/min, and UV detection at 205 nm. Semi-preparative HPLC of saponin fractions was performed on a DionexÒ apparatus equipped with a P580 pump, a UVD 340S diode array detector, an ASI-100 automated sample injector, a STH 585 column oven, and the Chromeleon software; prepacked column (PhenomenexÒ Luna RP-18, 15 250 mm, 5 lm) was used with a binary gradient elution (solvent A, H2O–0.025% TFA; solvent B, MeCN) and a flow rate of 8 mL/min, and the chromatogram was monitored at 205 nm. HPLC for separation of sugars was performed on a WatersÒ apparatus equipped with a 600 E pump, a WatersÒ 140 refractive index detector, an autosampler 717 Plus injector, a JascoÒ CO-965 column oven, and Empower software. Prepacked column (PhenomenexÒ Rezex ROA 21.2 250 mm) was used for semi-preparative HPLC with a H2SO4 2.5 mM solvent elution, a flow rate of 3.5 mL/min, and a column and detector temperature of 40 °C. Analytical chiral HPLC of sugars was performed on the same WatersÒ apparatus with a prepacked column (ChiraltechÒ Chiralpak IC 4.6 250 mm, 5 lM), hexane–EtOH–TFA (80:20:0.1) as solvent elution, a flow rate of 0.5 mL/min, and a column and detector temperature of 35 °C. 4.2. Plant material
gradient of MeOH–H2O as solvent elution (from 50:50 to 100:0); fr. 15 (32 mg) eluted with 60:40 was purified by semi prep. HPLC. 4.3.1. Zanhasaponin D (1) 1 [a]20 D 2° (c 0.08, MeOH); H NMR (CD3OD, 500 MHz) see Tables 1 and 2; 13C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 717.3455 [M+Na]+, calcd 717.3462 for [C36H54O13+Na]+. 4.3.2. Zanhasaponin E (2) 1 [a]20 D 2° (c 0.26, MeOH); H NMR (CD3OD, 500 MHz) see Tables 13 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 731.3620 [M+Na]+, calcd 731.3619 for [C37H56O13+Na]+. 4.3.3. Zanhasaponin F (3) 1 [a]20 D 12° (c 0.26, MeOH); H NMR (CD3OD, 500 MHz) see Tables 13 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 1027.4721 [MH], calcd 1027.4755 for [C50H76O22H]. 4.3.4. Zanhasaponin G (4) 1 [a]20 D 7° (c 0.53, MeOH); UV (MeOH) kmax 216 nm; H NMR 13 (CD3OD, 500 MHz) see Tables 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 1091.5033 [M+Na]+, calcd 1091.5039 for [C53H80O22+Na]+.
The plant was collected by one of us (C.D.) in the Forest Reserve of Luki, Democratic Republic of Congo in 1990. A voucher specimen was compared to an authentic sample (Donis 1413) from the Botanical Garden of Belgium (Brussels).
4.3.5. Zanhasaponin H (5) 1 [a]20 D 5° (c 0.96, MeOH); H NMR (CD3OD, 500 MHz) see Tables 13 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 1185.5704 [MH], calcd 1185.5693 for [C58H90O25H].
4.3. Extraction and isolation
4.4. Sugar analysis and determination of absolute configuration
The extraction of saponins from the root bark of Z. golungensis was conducted according to the protocol described in Dimbi et al., 1984; the yield of saponin extract was 38 g/kg. Root bark powder (1 kg) was macerated with 10 L of 80% aqueous MeOH and further refluxed for 3 h. After cooling, the solution was filtered, evaporated and the residue (180 g) suspended in methanol (1 L). The solution was poured into 4 L of diethyl ether and the precipitate was filtered and washed with diethyl ether. This precipitate was then dissolved in pure water (600 mL) and dialyzed against water in seamless cellulose tubing for 96 h. Freeze-drying of the tube content yielded 100 g of powdered residue, which was dissolved in 400 mL of MeOH, and purified by decolorization with activated charcoal. After elimination of absorbant by filtration, the solution was precipitated in diethyl ether for a second time. The precipitate was dried over KOH in vacuo and yielded 38 g of saponin extract. An aliquot of saponin extract (2.2 g) was passed through an ion exchange IRN 77 (H+) Amberlite resin column and then, purified by silica gel CC (80 g) using a gradient of CHCl3– MeOH–H2O (from 100:0:0 to 60:40:7) to give 123 fractions (160 mL) pooled into 24 groups according their TLC profiles. The first group (frs. 38–53) eluted with CHCl3–MeOH (80:20) was purified by semi-prep. Dionex HPLC to give saponins 1 (15 mg), 4 (2 mg), and 5 (11 mg). The following groups were passed individually through an ion exchange IRN 77 (H+) Amberlite resin column, and then combined and subjected to prep. RP-18 HPLC (solvent MeOH–H2O from 25% to 100%). Thirty-six fractions were collected among them fr. (16) gave saponin 6 (18 mg). Frs. (17), (19–20), (21–22), and (25) were purified on semi-prep. HPLC using a gradient of MeCN–H2O (from 35:65 to 55:45). Fr. (17) yielded saponin 6 (9 mg); saponins 1 (18 mg), 2 (5 mg), and 5 (5 mg) were obtained from frs. (19–20); fr. (21–22) contained saponins 1 (5 mg) and 5 (19 mg); the purification of fr. (25) yielded saponins 2 (7 mg) and 4 (12 mg). Saponin 3 (2 mg) was obtained from a purification of exchanged saponin mixture (1 g) by Lichroprep RP-18 CC using a
A part of the crude saponin mixture (600 mg) was refluxed with 0.02 N H2SO4 (37.5 mL) and 6% perchloric acid (37.5 mL) for 7 h. After filtration, the acid aq layer was neutralized with 2 M KOH and freeze-dried. The residue was solubilized in pyridine, and after filtration the soln. was evaporated. The five sugars were purified by prep. HPLC on a Rezex ROA column with H2SO4 2.5 mM as solvent: glucuronic acid (Rt 8.450), glucose (Rt 9.736), xylose (Rt 10.505), rhamnose (Rt 11.087), fucose (Rt 12.242). Each fraction was then neutralized with NaOH 50 mM and freeze-dried. The residues were solubilized in pyridine and soln. were filtrated and then evaporated. Each sugar was dissolved in hexane–EtOH–TFA (50:50:1) by ultrasonication. The solutions were analyzed by chiral HPLC with a Chiralpak IC using a mixture of hexane–EtOH–TFA (80:20:0.1) as solvent. By comparison with authentic D or L monosaccharide samples, the configurations were identified as D for glucuronic acid (Rt 19.1), glucose (Rt 19.3 and 24.5), xylose (Rt 17.3 and 18.7), and fucose (Rt 13.7 and 34.4), and L for rhamnose (Rt 12.1). 4.5. Determination of absolute configuration of nilic acid An aliquot of saponin 5 (11 mg) was dissolved in 1% K2CO3 (10 mL) and then heated at 100 °C in sand bath for 6 h. The reaction was neutralized with 10% HCl until pH 4. Then the mixture was extracted successively with CHCl3 (25 mL) and AcOEt (25 mL). The aqueous layer was freeze-dried. The residue was purified by flash-chromatography under the following conditions: a RevelerisÒ chromatograph equipped with a C18 cartridge (12 g); mobile phase: gradient of CH3OH–H2O from 0% to 100%; flow rate: 10 mL/min; UV 205 nm and DEDL detection. Fractions (32–51) were purified by semi-prep. HPLC with mobile phase: isocratic MeCN–H2O (+TFA 0.025%) 5% and flow rate of 4 mL/min. The peak with Rt 2.982 min was collected and after freeze-drying (2 mg), its optical rotation was measured ([a]20 D +8° (c 0.64, H2O)).
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Acknowledgments We thank Dr. G. Massiot for stimulating discussions and linguistic advice, and Dr. D. Harakat for MS spectra. Supplementary data Supplementary data (NMR spectra of compounds 1–6) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2014.10.001. References 1. Burkill, H. M. The Useful Plants of West Tropical Africa, 2nd ed.; Royal Botanic Gardens: Kew, Richmond, 2000; Vol. 5, p 686. 2. Bruschi, P.; Morganti, M.; Mancini, M.; Signorini, M. A. J. Ethnopharmacol. 2011, 138, 543–563. 3. Dimbi, M. Z.; Warin, R.; Delaude, C.; Huls, R. Bull. Soc. Chim. Belg. 1984, 93, 323– 328. 4. Dimbi, M. Z.; Warin, R.; Delaude, C.; Huls, R. Bull. Soc. Chim. Belg. 1987, 96, 207– 217. 5. Nibret, E.; Ashour, M. L.; Rubanza, C. D.; Wink, M. Phytother. Res. 2010, 24, 945– 947. 6. Beuscher, N.; Bodinet, C.; Neumann-Haefelin, D.; Marston, A.; Hostettmann, K. J. Ethnopharmacol. 1994, 42, 101–109. 7. Runyoro, D. K. B.; Matee, M. I. N.; Ngassapa, O. D.; Joseph, C. C.; Mbwambo, Z. H. BMC Complement. Altern. Med. 2006, 6, 11. 8. Recio, M. C.; Giner, R. M.; Manez, S.; Rios, J. L.; Marston, A.; Hostettmann, K. Phytother. Res. 1995, 9, 571–574.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Triterpenoid saponins from root bark of Zanha golungensis (Sapindaceae) Catherine Lavaud a,⇑, Charlotte Sayagh a, Franck Humbert a, Isabelle Pouny b, Clément Delaude c a
Institut de Chimie Moléculaire de Reims, CNRS UMR 7312, Université de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 02, France Epigenetic Targeting of Cancer, CNRS USR 3388, Centre de Recherche et de Développement Pierre Fabre, BP 13562, 31035 Toulouse Cedex 01, France c Institut de Chimie, Université de Liège, B36, Sart Tilman, B-4000 Liège, Belgium b
a r t i c l e
i n f o
Article history: Received 18 June 2014 Received in revised form 29 September 2014 Accepted 2 October 2014 Available online 8 October 2014 Keywords: Zanha golungensis Triterpenoid saponin Zanhic acid Zanhasaponin
a b s t r a c t The chemical investigation of the methanolic extract from root bark of Zanha golungensis Hiern led to the isolation of five new and one known triterpenoid saponins. Their structures were elucidated by full analysis of their spectroscopic data and by partial hydrolysis. These glycosides contain zanhic acid as aglycone, a rare oleanane-type triterpenoid found in species belonging to Sapindaceae, Caryophyllaceae, Asteraceae, and Fabaceae. Two new saponins are esterified saponins by 3,3-dimethylacryloyl and 3-hydroxy-2-methyl-butanoyl residues located on the sugar part. The new compounds were named zanhasaponins D–H following previous isolation of similar compounds from Zanha africana. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Zanha golungensis Hiern is a 6–25 m high tree or more rarely a 2–3 m high shrub growing all over tropical Africa. This plant has various uses such as timber for construction, chew-sticks, fodder, food, and it is one of the most common medicinal plants in whole Africa.1 New medical uses were recently reported for its leaves, bark, twigs, and roots, the latter being used against dropsy, swellings, edema, and gout.1,2 The presence of saponins and of oleanane-type triterpenes in the root bark of Z. golungensis has been previously reported.3,4 There are two species of Zanha accepted in The Plant List growing in Africa, and the second one, Zanha africana, was the object of several biological evaluations. The CH2Cl2 extract of the roots exhibited trypanocidal and antiviral activities, while the methanolic extract showed antifungal and anti-inflammatory activities.5–8 Both root bark extracts demonstrated tumoricidal activity.9 Antibacterial and fungicidal activities were reported for the methanolic bark extract.10,11 From a chemical viewpoint, three saponins were
⇑ Corresponding author. Tel.: +33 3 26913139; fax: +33 3 26913166. E-mail address: [email protected] (C. Lavaud). http://dx.doi.org/10.1016/j.carres.2014.10.001 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
isolated from the root bark of Z. africana and named zanhasaponins A–C, and they exhibited antiphospholipase A2 activity.12,13 Given the observations made on the saponins of Z. africana, it seemed interesting to complete the previous investigations and to isolate and identify new saponins from the root bark extract of this peculiar species, Z. golungensis.
2. Results and discussion The MeOH extract obtained from the root bark of Zanha golungensis was freed of salts by cationic exchange through a resin and fractionated by reverse-phase silica gel column chromatography. Nine fractions were thus obtained, which were purified by semi-prep. HPLC to yield six saponins (1–6) among them five new compounds named zanhasaponins D–H. Acid hydrolysis of the saponin extract gave five sugars identified by chiral chromatography as D-glucuronic acid, D-glucose, D-xylose, L-rhamnose, and D-fucose.
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2.1. Structure of aglycone of saponins 1–6 Zanhic acid (or 16-a-hydroxymedicagenic acid or 2b,3b,16 a-trihydroxy-12-oleanene-23,28-dioic acid) was identified as the aglycone of all saponins 1–6. The spectroscopic data were in agreement with those reported in the literature for the glycosides of zanhic acid (Table 1).12,14–17 In the 13C NMR spectra, three hydroxymethine carbons were detected at dC 70.8 ± 0.5 (C-2), 85 ± 1.5 (C-3), and 75 ± 0.3 (C-16), two carbonyl carbons at dC 181.8 ± 0.8 (C-23) and 177.9 ± 0.5 (C-28), and a pair of olefinic carbons at dC 123.7 ± 0.5 (C-12) and 145.1 ± 0.5 (C-13). The H-3 appeared as a doublet near dH 4.3 with a JH-3–H-2 coupling constant of 3.75 ± 0.25 Hz, confirming the a-equatorial position of H-2 and the a-axial position of H-3; the H-16 (dH 4.48 ± 0.1) shared with both H-15 a vicinal coupling constant of the order of 3 Hz attesting its b-equatorial position. The observation of a ROE correlation between H-3 and H-5 indicated their a-axial orientation; the acid function was located at C-23 on the basis of a ROE interaction between the b-oriented CH3-24 and CH3-25. 2.2. Structure of zanhasaponins D (1) and E (2) The molecular formula of zanhasaponin D (1) was established as C36H54O13 from the positive ion HRESIMS which exhibited a pseudo-molecular ion at m/z 717.3455 [M+Na]+ (calcd 717.3462 for C36H54O13Na). Similarly, the HRESIMS of zanhasaponin E (2) showed a pseudo-molecular ion at m/z 731.3620 [M+Na]+ (calcd 731.3619 for C37H56O13Na) in agreement with a molecular formula of C37H56O13. Consequently, these two compounds were deduced to contain a pyranosyl uronic acid linked to zanhic acid aglycone. The hypothesis that this pyranosyl uronic acid was methoxylated in compound 2 was confirmed by its NMR analysis, which showed a supplementary methyl at dC 52.9 and dH 3.8 (s). The anomeric signals of this sugar were detected at dC105 and dH 4.43 (1) or 4.45 (2) (d, J = 7.7 or 7.5 Hz) indicating an axial position; the analysis of COSY, HSQC, and HMBC experiments, together with the previous result of the acid hydrolysis of saponin extract, allowed its identification as a b-D-glucuronopyranosyl acid in 1 and 2 (Table 2). In
the HMBC spectrum of zanhasaponin E (2), H-5 of glucuronopyranosyl acid and the methoxy group share a correlation with the carbonyl carbon at dC 171.2 ppm attributed to C-6 of glucuronopyranosyl acid, and H-1 of glucuronopyranosyl acid showed a 3JH-C correlation with C-3 of zanhic acid. The structure of zanhasaponin D (1) was thus deduced to be 3-O-b-D-glucuronopyranosyl-zanhic acid, and that of zanhasaponin E (2) to be the 3-O-(6-O-methyl)b-D-glucuronopyranosyl-zanhic acid. Zanhasaponin D was previously isolated as a prosapogenin (zanhin) after partial acid hydrolysis of saponins from Zanha, but it is the first time that this compound was identified as a native saponin.4,12 The natural or artefactual character of zanhasaponin E is open to discussion since this compound might result from addition of methanol. To provide evidence of the original character of this saponin, we reacted saponin 1 with methanol under various operating conditions similar to those used in the extraction step and in the purification stage, such as in cold and then boiling methanol, in the presence of silica gel, or in the presence of HPLC eluent. After HPLC analysis of these reactions, it was found that none generated saponin 2, suggesting that it was a genuine compound. Analysis of the NMR data for saponins (3–6) showed that the 3-O-b-D-glucuronopyranosyl-zanhic acid (1) belonged to these four saponins (Tables 1 and 2). Differences with 1 arose from the presence of another glycosidic moiety linked to zanhic acid. The chemical shifts of C-28 at dC 177.4 ± 0.1 and of C-1 of second glycosidic unit at dC 95.1 ± 0.1, confirmed its esterification by this second oligosaccharidic chain. 2.3. Characterization of a common core of structure in zanhasaponins F–H (3–5) Comparison of 1H NMR spectra of zanhasaponins F–H (3–5) showed great similarities in the signals of the sugar part with three anomeric doublets at dH 4.43 ± 0.02 (assigned to b-D-glucuronic acid as in 1), 5.29 ± 0.01 (J = 1.5 Hz), and 5.41 ± 0.01 (J = 8 Hz); those protons were correlated to their respective carbons at dC 105 ± 0.5, 102 ± 0.1, and 95.1 ± 0.1 in the HSQC experiment (Table 2). These two latter sugars were identified as 6-deoxyhexoses
Table 1 NMR data of aglycone part (500 MHz for 1H and 125 MHz for No.
C, CD3OD, d in ppm, J in Hz) for zanhasaponins D–H (1–5)
1
2
3
4
5
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
acid 1.32 (m), 2.16 (br d, 14.5) 4.32 (br d, W1/2 = 9) 4.11 (d, 3.6) — 1.65 (m) 1.20 (m), 1.72 (m) 1.32 (m), 1.60 (m) — 1.70 (m) — 1.95–2.05 (m) 5.33 (br t, 3.0) — — 1.36 (m), 1.83 (dd, 14.0, 4.0) 4.49 (br t, 3.0) — 3.04 (dd, 14.0, 4.4) 1.06 (dd, 13.0, 3.5), 2.31 (t, 13.0) — 1.18 (m), 1.97 (m) 1.84 (m), 1.94 (m) — 1.45 (s) 1.32 (s) 0.84 (s) 1.41 (s) — 0.92 (s) 1.00 (s)
44.6 70.8 86.3 53.1 53.0 21.6 33.8 40.8 49.8 37.3 24.5 123.2 144.9 42.7 35.9 75.1 50.1 41.9 47.6 31.3 36.4 32.7 181.0 13.5 17.0 17.5 27.2 nd 33.3 24.7
1.32 (m), 2.14 (dd, 14.2, 1.5) 4.27 (br d, W1/2 = 9) 4.12 (br d, 3.0) — 1.62 (m) 1.20 (m), 1.65 (m) 1.32 (m), 1.60 (m) — 1.68 (m) — 1.95–2.05 (m) 5.34 (br t, 3.5) — — 1.36 (m), 1.83 (dm, 13.6) 4.49 (br s)
44.5 71.3 83.5 nd 53.1 21.5 34.0 41.0 48.7 37.4 24.9 123.3 144.9 42.5 36.0 75.3 42.1 48.0 31.4 36.5 33.0a 182.2 13.5a 17.0a 17.8a 27.3a nd 33.4 24.9a
44.7 70.8 86.0 nd 53.1 22.0 33.8 41.2 49.0 37.4 24.6 123.5 145.0 42.9 36.4 74.8 50.1 42.4 48.0 31.3 36.2 32.0 nd 13.8 17.3 17.8 27.1 177.4 33.4 25.0
1.33 (m), 2.15 (dd, 14.1, 1.6) 4.32 (m, W1/2 = 10) 4.12 (d, 3.5) — 1.65 (m) 1.18 (m), 1.68 (m) 1.43 (m), 1.58 (m) — 1.68 (m) — 1.95–2.05 (m) 5.38 (br t, 3.0) — — 1.43 (m), 1.80 (dd, 13.1, 3.0) 4.47 (br t, 3.0) — 2.99 (dd, 14.2, 4.1) 1.08 (dm, 13.5), 2.32 (t, 13.5) — 1.22 (m), 1.95 (m) 1.92 (m) — 1.42 (s) 1.32 (s) 0.85 (s) 1.41 (s) — 0.92 (s) 1.01 (s)
45.0 70.8 86.3 nd 53.5 21.7 33.8 41.2 48.5 37.4 24.7 123.4 144.6 42.9 36.2 75.0 50.2 42.4 48.0 31.0 36.5 31.4 181.5 13.6 17.4 18.0 27.2 177.5 33.4 25.1
1.31 (m), 2.18 (dd, 14.0, 2.0) 4.35 (br s, W1/2 = 10) 4.10 (d, 4.0) — 1.62 (d, 11.2) 1.18 (m), 1.68 (m) 1.43 (m), 1.58 (dd, 12.4, 8.5) — 1.68 (m) — 1.95–2.05 (m) 5.39 (br t, 3.0) — — 1.43 (m), 1.78 (dd, 13.5, 3.5) 4.47 (br s)
3.04 1.06 — 1.18 1.80 — 1.41 1.31 0.83 1.41 — 0.91 1.00
1.32 (m), 2.15 (br d, 14.5) 4.34 (br s, W1/2 = 10) 4.13 (br s, W1/2 = 4) — 1.62 (m) 1.20 (m), 1.66 (m) 1.43 (m), 1.63 (m) — 1.68 (m) — 1.98–2.05 (m) 5.38 (br t, 3.0) — — 1.43 (m), 1.78 (dd, 13.3, 3.0) 4.48 (br t, 3.0) — 2.99 (dd, 14.0, 3.2) 1.08 (m), 2.32 (br t, 13.4) — 1.22 (m), 1.95 (m) 1.82 (m), 1.95 (m) — 1.45 (s) 1.32 (s) 0.85 (s) 1.41 (s) — 0.92 (s) 1.00 (s)
44.9 70.8 86.4 53.3 53.2 21.7 33.9 41.3 49.5 37.4 24.7 123.3 144.6 42.9 36.3 74.8 50.3 42.3 48.1 31.4 36.5 31.6 181.7 13.6 17.4 18.0 27.2 177.4 33.5 25.1
(dd, 14.1, 4.0) (m), 2.31 (t, 13.5) (m), 1.97 (m) (m), 1.94 (m) (s) (s) (s) (s) (s) (s)
2.99 1.08 — 1.22 1.92 — 1.42 1.31 0.85 1.41 — 0.91 1.00
(dd, 14.2, 4.0) (m), 2.31 (t, 13.6) (m), 1.95 (m) (m) (s) (s) (s) (s) (s) (s)
C. Lavaud et al. / Carbohydrate Research 402 (2015) 225–231
Zanhic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
13
nd = not determinated. a Detected from HSQC correlation.
227
228
Table 2 NMR data of glycosidic part (500 MHz for 1H and 125 MHz for No.
1 dH
13
C, CD3OD, d in ppm, J in Hz) for zanhasaponins D–H (1–5).
2 dC
dH
b-D-Fucopyranosyl (at C-28 of zanhic acid) 1 2 3 4 5 6
dH
105.0 74.8 77.0 73.2 76.5 171.2 52.9
4.45 3.31 3.45 3.50 3.72 —
4 dC
dH
(br d, 7.0) (nd) (nd) (nd) (nd)
104.5 74.8 77.5 73.3 nd nd
4.43 3.29 3.39 3.51 3.80 —
5.40 3.75 3.90 5.15 3.86 1.10
(d, 8.1) (dd, 9.1, 8.3) (dd, 9.3, 3.5) (d, 3.6) (q, 6.4) (d, 6.4)
95.1 75.6 74.3 75.1 71.1 16.6
5.29 3.95 3.67 3.41 3.76 1.30
(br s) (m, W1/2 = 6.0) (dd, 9.4, 3.3) (t, 9.4) (m) (d, 6.2)
102.0 72.0 72.1 73.8 70.4 18.4
5 dC
dH
dC
(d, 7.7) (dd, 9.0, 7.8) (t, 9.0) (t, 9.0) (d, 9.0)
105.0 74.9 77.5 73.8 76.2 171.5
4.42 3.30 3.40 3.48 3.79 —
(d, 7.7) (dd, 9.1, 7.8) (t, 9.1) (nd) (d, 9.0)
105.0 74.7 77.2 73.3 76.5 nd
5.42 3.77 3.91 5.15 3.87 1.08
(d, 8.0) (dd, 9.3, 8.0) (dd, 9.4, 3.6) (d, 3.6) (br q, 6.5) (d, 6.4)
95.2 76.2 74.2 74.0 71.2 16.7
5.41 3.76 3.91 5.13 3.88 1.12
(d, 8.0) (dd, 9.1, 8.1) (dd, 9.3, 3.6) (d, 3.5) (q, 6.5) (d, 6.5)
95.0 75.7 74.0 75.1 70.8 16.6
5.28 3.95 3.67 3.51 3.76 1.29
(d, 1.5) (dd, 3.2, 1.7) (dd, 9.5, 3.3) (t, 9.5) (m) (d, 6.2)
102.1 72.0 72.1 73.8 70.5 18.4
5.29 3.95 3.67 3.41 3.76 1.30
(d, 1.5) (dd, 3.3, 1.5) (dd, 9.4, 3.4) (t, 9.5) (m) (d, 6.2)
102.0 71.9 72.0 73.7 70.4 18.3
a-L-Rhamnopyranosyl (at C-2 of fucopyranose) 1 2 3 4 5 6 1 2 3 4 5 10 20 30 40 50 nd = not determinated.
Acetyl (at C-4 of fucopyranose) — 172.8 2 .20 (s) 20.8
3,3-Dimethylacryloyl (at C-4 of fucopyranose) — 167.9 5.87 (sept., 1.3) 116.8 — 159.0 2.23 (br s) 20.7 1.99 (br s) 27.6
Niloyl-(3-10 )-niloyl (at C-4 of fucopyranose) — 175.9 2.83 (qt, 6.9) 46.4 5.21 (qt, 6.4) 72.3 1.34 (d, 6.3) 18.2 1.32 (d, 7.0) 13.8 — 2.50 3.95 1.21 1.14
(qt, 7.1) (qt, 6.5) (d, 6.3) (d, 7.1)
175.1 49.2 69.9 20.3 13.8
C. Lavaud et al. / Carbohydrate Research 402 (2015) 225–231
b-D-Glucuronopyranosyl acid (at C-3 of zanhic acid) 1 4.43 (d, 7.7) 105.0 4.45 (d, 7.5) 2 3.30 (dd, 9.1, 7.8) 74.7 3.29 (br dd, 8.0, 9.1) 3 3.39 (t, 9.0) 77.2 3.39 (t, 9.2) 4 3.51 (t, 9.1) 73.0 3.53 (dd, 9.5, 9.2) 5 3.81 (d, 9.0) nd 3.88 (d, 9.7) 6 — 170.0 — OCH3 3.80 (s)
3 dC
C. Lavaud et al. / Carbohydrate Research 402 (2015) 225–231
the with methyl group (CH3-6) as doublet near dH 1.10 (J = 6.4 Hz) and 1.30 (J = 6.2 Hz), respectively. Joint analysis of COSY and TOCSY spectra starting from anomeric protons and methyl groups, and the values of proton coupling constants revealed the presence of a b-D-fucopyranose and of an a-L-rhamnopyranose. The HMBC spectra of compounds 4 and 5 showed cross-peaks between H-1 of rhamnopyranose (dH 5.28/5.29) and C-2 of fucopyranose (dC 76.2/75.7), between H-1 of fucopyranose (dH 5.42/5.41) and C-28 of zanhic acid (dC 177.4/177.5), and as previously observed for 1 between H-1 of glucuronopyranosyl acid (dH 4.42/4.43) and C-3 of zanhic acid (dC 86.3/86.4). In the ROESY spectra of saponins 3–5, the effects observed between H-1 of rhamnopyranose and H-2 of fucopyranose, between H-18 of zanhic acid and CH3-6 of fucopyranose, and between H-1 of glucuronopyranosyl acid and H-3 of zanhic acid, confirmed the sequence of the glycosidic chains and their relative stereochemistry. The deshielding chemical shift of H-4 in fucopyranose at dH 5.14 ± 0.01 and its HMBC correlation observed with a supplementary carboxylic carbon detected between dC 167 and 176 suggested that this glycosidic position was esterified in zanhasaponins F–H. Thus, the substructure comprised in saponins 3–5 was the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)-b-D-(4-O-acyl-fucopyranosyl)]-zanhic acid, which corresponded to a C48H74O21 fragment. Although run in different solvents, the NMR data were similar to those reported in the literature for structures with similar ester saccharide chain comprised of a 4-acylated b-D-fucopyranosyl residue.18,19 2.4. Structure of zanhasaponins F–H (3–5) Zanhasaponin F (3) exhibited a molecular peak at m/z 1027.4721 [MH] in the negative mode HRESIMS (calcd 1027.4755) in agreement with a C50H76O21 molecular formula. Subtracting the common molecular skeleton, yielded a C2H2O fragment (42 units) corresponding to an acetyl group. A three proton singlet was effectively detected at dH 2.2 in the 1H NMR spectrum, and in the 13C NMR spectrum two supplementary carbons were attributed to a methyl group at dC 20.8 and a carboxyl of ester at dC 172.8. Consequently, zanhasaponin E (3) is the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)-b-D-(4-O-acetylfucopyranosyl]-zanhic acid. The HRESIMS of zanhasaponin G (4) indicated a C53H80O22 molecular formula, as deduced from the [M+Na]+ ion at m/z 1091.5033 (calcd 1091.5039). The ester moiety linked to position 4 of fucopyranose was in this case a C5H6O unit (82 amu). The signal of one olefinic proton was detected at dH 5.87 (septuplet, J = 1.3 Hz), and it had COSY correlations with two broad methyl singlets at dH 1.99 and 2.23, suggesting a dimethylallyl group. A supplementary carbonyl carbon at dC 167.9 exhibited HMBC correlations with the methyl proton signals. These signals were obviously assigned to a 3-methyl-2-butenoic acid (3,3-dimethylacrylic acid). Thus, zanhasaponin G (4) is the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)-b-D-(4-O-3,3-dimethylacryloyl-fucopyranosyl]-zanhic acid. Zanhasaponin H (5) exhibited a molecular peak at m/z 1185.5704 [MH], and by subtraction of the common molecular C48H74O21 fragment, its molecular weight was increased by 200 amu corresponding in this case to an ester group of formula C10H16O4. The characteristic NMR signals for two niloyl (2-methyl-3-hydroxybutanoyl) groups were observed on the 1H NMR spectrum of 5 with four methyl doublets at dH 1.14 (J = 8 Hz) 1.21 (J = 6.3 Hz), 1.32 (J = 8 Hz), and 1.34 (J = 6.3 Hz), correlated in COSY experiment to four protons being as quintets at dH 2.5 (J = 7.2 Hz), 3.95 (J = 6.5 Hz), 2.83 (J = 7 Hz), and 5.21 (J = 6.4 Hz), respectively.20 Two carboxyl carbons were detected at dC 175.1 and 175.9; the first exhibited an HMBC correlation with the deshielded quintet at dH 5.21, while the second had the correlation with H-4 of fucopyranose. Thus, the C10 ester
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group was composed of the sequence of two nilic acids: 2-methyl3-hydroxy-butanoyl-(1-3)-2-methyl-3-hydroxy-butanoyl. The threo configuration of nilic acid was determined from the value of vicinal coupling constant of methine protons (J >6.3 Hz), and its absolute configuration was confirmed after basic hydrolysis of zanhasaponin H (5); the positive value of optical rotation (+8, H2O) suggested a 2S,3S-configuration.21,22 The structure of zanhasaponin H (5) is the 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)b-D-(4-O-[30 -S-hydroxy-20 -S-methyl-butyroyloxy]-3-S-hydroxy-2S-methyl-butyroyloxy)-fucopyranosyl]-zanhic acid. 2.5. Structure of known saponin (6) Analysis of HRESIMS and NMR data of saponin 6 allowed its identification as 3-O-b-D-glucuronopyranosyl-28-O-[a-L-rhamnopyranosyl-(1?2)[b-D-xylopyranosyl-(1?3)]-b-D-(4-O-acetyl-fucopyranosyl]-zanhic acid. This compound was recently isolated from Ganophyllum giganteum, a species belonging to Doratoxyleae tribe as Z. golungensis.23 3. Conclusion To our knowledge, this study is the first report on the characterization of saponins from Z. golungensis. Although this species is a widespread medicinal plant used for a large variety of pains, to the best of our knowledge, no phytochemical analyses have been previously reported. The originality of isolated saponins is the nature of the ester parts, and the structure of aglycone, the rarely distributed zanhic acid. These oleanane-type triterpenoid saponins were found in a few species belonging to genera Zanha, Filicium and Ganophyllum (Sapindaceae), Aster (Asteraceae), Medicago (Fabaceae), Physena (sole genus of Physenaceae), and Herniaria (Caryophyllaceae).12,13,15–17,23–26 Compounds isolated from Sapindaceae and Caryophyllaceae species possessed a glycosidic chain at C-28 of zanhic acid, containing as first disaccharide a-L-rhamnopyranosyl-(1?2)-b-D-fucopyranosyl moiety with the C-4 position of fucopyranose typically esterified. Esterified saponins of Z. golungensis contained isoprenyl residues which are usually located on hydroxyl groups of polyhydroxylated rings D and E of aglycones. Thus, it is the fourth time that esters as 3,3-dimethylacryloyl and 3-hydroxy-2-methyl-butanoyl residues were located on the sugar part.20,23,27 4. Experimental 4.1. General methods UV spectra were obtained using a SchimadzuÒ UV 2450 spectrophotometer. Optical rotations were measured in MeOH with a Perkin-Elmer 341 automatic polarimeter. 1H and 13C NMR spectra were recorded in CD3OD on a BrukerÒ Avance DRX-500 spectrometer (1H at 500 MHz and 13C at 125 MHz), and 2D-NMR experiments were performed using standard Bruker microprograms (XWIN NMR version 2.6 software). HR-ESI-MS was performed using a BrukerÒMicroTOF or a MicromassÒESI-Q-TOF apparatus using ESI ionization, and the samples were introduced by infusion in MeOH solution. For analysis of saponins, TLC was carried out on precoated silica gel 60F254 (MerckÒ ) with CHCl3–MeOH–H2O (60:40:7) and on reversed phase silica gel RP-18 (MerckÒ ) with MeOH–H2O (7:3 or 11:9), and spots were visualized by spraying with 50% aq H2SO4. Column chromatography (CC) was carried out on Kieselgel (63–200 lm, MerckÒ) or Lichroprep RP-18 (40-63 lm, MerckÒ). HPLC for purification of saponins was performed on a MerckÒ apparatus equipped with a AP 250 ArmenÒ pump, a Merck–KnauerÒ K-2501 UV detector, and a BuchiÒ C-660 fraction collector; column packed with MerckÒ Lichrospher RP-18 (50 200 mm, 12 lm) was
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used for preparative HPLC with binary gradient elution (solvent A: H2O; solvent B: MeOH), a flow rate of 35 mL/min, and UV detection at 205 nm. Semi-preparative HPLC of saponin fractions was performed on a DionexÒ apparatus equipped with a P580 pump, a UVD 340S diode array detector, an ASI-100 automated sample injector, a STH 585 column oven, and the Chromeleon software; prepacked column (PhenomenexÒ Luna RP-18, 15 250 mm, 5 lm) was used with a binary gradient elution (solvent A, H2O–0.025% TFA; solvent B, MeCN) and a flow rate of 8 mL/min, and the chromatogram was monitored at 205 nm. HPLC for separation of sugars was performed on a WatersÒ apparatus equipped with a 600 E pump, a WatersÒ 140 refractive index detector, an autosampler 717 Plus injector, a JascoÒ CO-965 column oven, and Empower software. Prepacked column (PhenomenexÒ Rezex ROA 21.2 250 mm) was used for semi-preparative HPLC with a H2SO4 2.5 mM solvent elution, a flow rate of 3.5 mL/min, and a column and detector temperature of 40 °C. Analytical chiral HPLC of sugars was performed on the same WatersÒ apparatus with a prepacked column (ChiraltechÒ Chiralpak IC 4.6 250 mm, 5 lM), hexane–EtOH–TFA (80:20:0.1) as solvent elution, a flow rate of 0.5 mL/min, and a column and detector temperature of 35 °C. 4.2. Plant material
gradient of MeOH–H2O as solvent elution (from 50:50 to 100:0); fr. 15 (32 mg) eluted with 60:40 was purified by semi prep. HPLC. 4.3.1. Zanhasaponin D (1) 1 [a]20 D 2° (c 0.08, MeOH); H NMR (CD3OD, 500 MHz) see Tables 1 and 2; 13C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 717.3455 [M+Na]+, calcd 717.3462 for [C36H54O13+Na]+. 4.3.2. Zanhasaponin E (2) 1 [a]20 D 2° (c 0.26, MeOH); H NMR (CD3OD, 500 MHz) see Tables 13 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 731.3620 [M+Na]+, calcd 731.3619 for [C37H56O13+Na]+. 4.3.3. Zanhasaponin F (3) 1 [a]20 D 12° (c 0.26, MeOH); H NMR (CD3OD, 500 MHz) see Tables 13 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 1027.4721 [MH], calcd 1027.4755 for [C50H76O22H]. 4.3.4. Zanhasaponin G (4) 1 [a]20 D 7° (c 0.53, MeOH); UV (MeOH) kmax 216 nm; H NMR 13 (CD3OD, 500 MHz) see Tables 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 1091.5033 [M+Na]+, calcd 1091.5039 for [C53H80O22+Na]+.
The plant was collected by one of us (C.D.) in the Forest Reserve of Luki, Democratic Republic of Congo in 1990. A voucher specimen was compared to an authentic sample (Donis 1413) from the Botanical Garden of Belgium (Brussels).
4.3.5. Zanhasaponin H (5) 1 [a]20 D 5° (c 0.96, MeOH); H NMR (CD3OD, 500 MHz) see Tables 13 1 and 2; C NMR (CD3OD, 125 MHz) see Tables 1 and 2; ESIMS m/z 1185.5704 [MH], calcd 1185.5693 for [C58H90O25H].
4.3. Extraction and isolation
4.4. Sugar analysis and determination of absolute configuration
The extraction of saponins from the root bark of Z. golungensis was conducted according to the protocol described in Dimbi et al., 1984; the yield of saponin extract was 38 g/kg. Root bark powder (1 kg) was macerated with 10 L of 80% aqueous MeOH and further refluxed for 3 h. After cooling, the solution was filtered, evaporated and the residue (180 g) suspended in methanol (1 L). The solution was poured into 4 L of diethyl ether and the precipitate was filtered and washed with diethyl ether. This precipitate was then dissolved in pure water (600 mL) and dialyzed against water in seamless cellulose tubing for 96 h. Freeze-drying of the tube content yielded 100 g of powdered residue, which was dissolved in 400 mL of MeOH, and purified by decolorization with activated charcoal. After elimination of absorbant by filtration, the solution was precipitated in diethyl ether for a second time. The precipitate was dried over KOH in vacuo and yielded 38 g of saponin extract. An aliquot of saponin extract (2.2 g) was passed through an ion exchange IRN 77 (H+) Amberlite resin column and then, purified by silica gel CC (80 g) using a gradient of CHCl3– MeOH–H2O (from 100:0:0 to 60:40:7) to give 123 fractions (160 mL) pooled into 24 groups according their TLC profiles. The first group (frs. 38–53) eluted with CHCl3–MeOH (80:20) was purified by semi-prep. Dionex HPLC to give saponins 1 (15 mg), 4 (2 mg), and 5 (11 mg). The following groups were passed individually through an ion exchange IRN 77 (H+) Amberlite resin column, and then combined and subjected to prep. RP-18 HPLC (solvent MeOH–H2O from 25% to 100%). Thirty-six fractions were collected among them fr. (16) gave saponin 6 (18 mg). Frs. (17), (19–20), (21–22), and (25) were purified on semi-prep. HPLC using a gradient of MeCN–H2O (from 35:65 to 55:45). Fr. (17) yielded saponin 6 (9 mg); saponins 1 (18 mg), 2 (5 mg), and 5 (5 mg) were obtained from frs. (19–20); fr. (21–22) contained saponins 1 (5 mg) and 5 (19 mg); the purification of fr. (25) yielded saponins 2 (7 mg) and 4 (12 mg). Saponin 3 (2 mg) was obtained from a purification of exchanged saponin mixture (1 g) by Lichroprep RP-18 CC using a
A part of the crude saponin mixture (600 mg) was refluxed with 0.02 N H2SO4 (37.5 mL) and 6% perchloric acid (37.5 mL) for 7 h. After filtration, the acid aq layer was neutralized with 2 M KOH and freeze-dried. The residue was solubilized in pyridine, and after filtration the soln. was evaporated. The five sugars were purified by prep. HPLC on a Rezex ROA column with H2SO4 2.5 mM as solvent: glucuronic acid (Rt 8.450), glucose (Rt 9.736), xylose (Rt 10.505), rhamnose (Rt 11.087), fucose (Rt 12.242). Each fraction was then neutralized with NaOH 50 mM and freeze-dried. The residues were solubilized in pyridine and soln. were filtrated and then evaporated. Each sugar was dissolved in hexane–EtOH–TFA (50:50:1) by ultrasonication. The solutions were analyzed by chiral HPLC with a Chiralpak IC using a mixture of hexane–EtOH–TFA (80:20:0.1) as solvent. By comparison with authentic D or L monosaccharide samples, the configurations were identified as D for glucuronic acid (Rt 19.1), glucose (Rt 19.3 and 24.5), xylose (Rt 17.3 and 18.7), and fucose (Rt 13.7 and 34.4), and L for rhamnose (Rt 12.1). 4.5. Determination of absolute configuration of nilic acid An aliquot of saponin 5 (11 mg) was dissolved in 1% K2CO3 (10 mL) and then heated at 100 °C in sand bath for 6 h. The reaction was neutralized with 10% HCl until pH 4. Then the mixture was extracted successively with CHCl3 (25 mL) and AcOEt (25 mL). The aqueous layer was freeze-dried. The residue was purified by flash-chromatography under the following conditions: a RevelerisÒ chromatograph equipped with a C18 cartridge (12 g); mobile phase: gradient of CH3OH–H2O from 0% to 100%; flow rate: 10 mL/min; UV 205 nm and DEDL detection. Fractions (32–51) were purified by semi-prep. HPLC with mobile phase: isocratic MeCN–H2O (+TFA 0.025%) 5% and flow rate of 4 mL/min. The peak with Rt 2.982 min was collected and after freeze-drying (2 mg), its optical rotation was measured ([a]20 D +8° (c 0.64, H2O)).
C. Lavaud et al. / Carbohydrate Research 402 (2015) 225–231
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