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New sucrose esters from the fruits of Physalis solanaceus
Carbohydrate Research 352 (2012) 211–214
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
New sucrose esters from the fruits of Physalis solanaceus Ana-Lidia Pérez-Castorena a,⇑, Minerva Luna a, Mahinda Martínez b, Emma Maldonado a a b
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, D.F., Mexico Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Avenida de las Ciencias S/N, Col. Juriquilla 76230, Querétaro, Mexico
a r t i c l e
i n f o
Article history: Received 19 November 2011 Received in revised form 31 January 2012 Accepted 9 February 2012 Available online 18 February 2012
a b s t r a c t Three new sucrose esters (1–3) along with several known compounds were isolated from the fruits of Physalis solanaceus. The structural elucidation of the isolates was based on their spectroscopic characteristics mainly those of MS and NMR. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Solanaceae Physalis Sucrose esters Solanoses Physalin
The systematic chemical research of the genus Physalis practically started in the 60s with Physalis alkekengi var. francheti,1,2 and from that time, the aerial parts or the whole plant or the stems together with leafs have been the vegetal material more utilized for chemical studies. In these works, withasteroids (compounds derived from ergostane)3–6 have been the main metabolites reported, although labdane diterpenes,7,8 flavonoids,9,10 and sucrose esters,8,11 among other compounds, have also been isolated. From the early 90s, only a few reports on the chemical components of fruits, calyxes, and roots have been published. Nor-tropane and pyrrolidine alkaloids,12,13 withasteroids,14–16 and polysaccharides17 have been the main metabolites obtained from roots of the Physalis species. Polysaccharides,18 withasteroids,19 carotenoids,20 and sucrose esters21 have been isolated from the fruits; while physalins (highly oxidized seco-withasteroids) and flavonoids22 were obtained from the calyxes. Among the reasons that make Physalis an interesting genus is the use of some species in the traditional medicine of different countries, can be mentioned, for example: P. alkekengi var. francheti which is used for treatment of sore throat, cough, eczema, hepatitis, malaria, urinary problems and tumors in China,22,23 and Physalis philadelphica whose fruits are used to treat respiratory problems, sore throat, and cough in Mexico.8 Also, there is an economic interest in these plants because the fruits of some species are edible such as those of Physalis angulata and P. philadelphica.4,20 Additionally, various extracts and metabolites obtained from Physalis species have been shown to ⇑ Corresponding author. Tel.: +52 55 56 224412; fax: +52 55 56 162217. E-mail address: [email protected] (A.-L. Pérez-Castorena). 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2012.02.003
possess different biological activities such as antifeedant, antiinflammatory, cytotoxic, antioxidant, antimycobacterial, and immunomodulatory activities.22,24–26 As part of our systematic study on the Mexican Physalis species,4,7,8,21,27–30 we have reported the isolation of several physalins from the stems and leafs of Physalis solanaceus (Schldl.) Axelius,27 now we describe the chemical study of the fruits of this species, which resulted in the isolation of three new sucrose esters (1–3) together with the known physordinose B (4),8 25-hydroxyphysalin N (5),31 b-sitosteryl b-D-glucopyranoside, and sucrose. Compound 1, obtained as colorless oil, showed in the IR spectrum absorption bands at 3439 and 1745 cm1 attributable to hydroxyl and ester groups. The presence of a sucrose unit was deduced from the analysis of the NMR spectra (Tables 1 and 2), which showed the anomeric CH signals of the glucopyranose (dH 5.56 d, J = 3.5 Hz and dC 89.6; CH-1) and that of the anomeric carbon of the fructofuranose (d 102.2, C-20 ). The CH-1-O-C-20 glycosidic linkage between the monosaccharides was established by the correlation of the H-1 signal of the glucopyranose with the C20 signal of the fructofuranose observed in the HMBC spectrum of 1. The same spectrum let to locate the H2-10 (d 4.03 and 3.92) and H-50 (d 3.93) signals by their cross peaks with the C-20 signal. The correlations showed in the 1H–1H COSY, HSQC, and HMBC spectra allowed us to identify the signals of all the hydrogen and carbon atoms of the sucrose unit of compound 1 (Tables 1 and 2). The chemical shift of the H-2 (d 4.90), H-3 (d 5.20), H2-10 , and H-30 (d 5.25) signals indicated their gem position to ester functions.8 The presence of these ester groups was confirmed by the four 13C NMR signals for the carbonyl carbons of these functions
212
A.-L. Pérez-Castorena et al. / Carbohydrate Research 352 (2012) 211–214
H O OH
O
6
1'
O
3
CH3(CH2)10
O
O 5'
R2 = H R2 = iVal R2 = H R2 = H
4
R3 = iBu R3 = H R3 = H R3 = H
(Table 2). One of the acyl groups was identified as lauroyl by the characteristic twelve signals observed in the 13C NMR spectrum (Table 2).8 The other three acyl groups were identified as one isovaleryl32 and two isobutyryls21 on the basis to their 1H and 13C NMR signals (Tables 1 and 2). This allowed to propose the molecular formula C37H64O15 for compound 1, which was confirmed by the pseudomolecular ion peak [M+Na+] at m/z 771.4111 obtained by HRESI MS. The HMBC correlations between H-2 of the glucopyr-
Table 1 1 H NMR data (CDCl3, 500 MHz) for compounds 1–3a 2
Position
1
1 2 3 4 5 6a 6b 10 a 10 b 30 40 50 60 a 60 b
5.56 4.90 5.20 3.53 4.06 3.96 3.75 4.03 3.92 5.25 4.60 3.93 3.92 3.72
d (3.5) dd (10.5,3.5) dd (10.5,9.5) dd (10,9.5) ddd (10,6,2) dd (12,2) dd (12,6) d (12) d (12) d (8.5) dd (8.5,8.5) m m m
5.60 4.90 5.50 4.94 4.14 3.62 3.62 3.59 3.47 5.18 4.59 3.95 3.91 3.72
d (3.5) dd (10.5,3.5) dd (10.5,10) dd (10.5,10) ddd (10.5,5,3) m m d (12) d (12) d (8) dd (8.5,8) m dd (15,2.5) dd (15,1.5)
5.56 4.86 5.25 3.52 4.05 3.97 3.75 3.59 3.49 5.18 4.55 3.93 3.90 3.73
d (3.5) dd (10.5,3.5) dd (10.5,9.5) dd (9.5,9.5) ddd (10,6,2) dd (12,2) dd (12,6.5) d (12) d (12) d (8) dd (8,8) m m m
Lauroyl-O-2 200 300 400 –1100 1200
2.30 1.58 1.25 0.88
m m br s t (7.5)
2.27 1.55 1.26 0.88
m quint (7) br s t (7)
2.28 1.56 1.25 0.88
m, 2.24 m quint (7) br s t (7)
Isovaleryl-O-3 2.30 2000 3000 2.05 4000 0.95 5000 0.95
m m d (6.5) d (6.5)
2.05 2.00 0.91 0.91
m nona (7) d (7) d (7)
2.20 m 2.06 nona (7) 0.943 d (7) 0.941 d (7)
Isobutyryl-O-30 200 00 2.73 hept (7) 300 00 1.29 d (7) 00 00 4 1.26 d (7)
2.77 hept (7) 1.32 d (7) 1.29 d (7)
2.74 hept (7) 1.29 d (7) 1.25 d (7)
Isovaleryl-O-4 200 00 0 300 00 0 400 00 0 500 00 0
2.16 2.05 0.94 0.94
Isobutyryl-O-10 200 00 0 2.60 hept (7) 300 00 0 1.20 d (7) 00 00 0 4 1.19 d (7) a
Coupling constants in Hz (J).
OH
16
28
O O
O R1 = iVal R1 = iVal R1 = iVal R1 = iBu
O
14
O CH(CH3)2
1 2 3 4
HO
1
OH
3'
O
25
H
O HO
1
O
23
OR3
O
5
R2O R1 O
O
27
OH 5
anose and C-100 of the lauroyl group (d 173.1) as well as those of H-3 and C-1000 of the isovaleryl group (d 174.2), H2-10 of the fructofuranose and C-100 000 of one isobutyryl group (d 176.0), and H-30 and C-100 00 of a second isobutyryl group (d 177.3), led to establish the position of the acyl groups. Therefore, the structure of 1 was elucidated as 10 ,30 -di-O-isobutyryl-3-O-isovaleryl-2-O-lauroylsucrose and named solanose A. The IR and NMR (Tables 1 and 2) spectra of compound 2 were similar to those of 1. This similarity pointed to 2 as an esterified sucrose. The main differences observed in the 1H NMR spectra (Table 1) were the absence of the signals for the isobutyryl at-
3
m nona (7) d (7) d (7)
Table 2 13 C NMR data (CDCl3, 125 MHz) for compounds 1–3a Position
1
2
3
1 2 3 4 5 6 10 20 30 40 50 60
89.6 CH 69.3 CH 72.9 CH 70.0 CH 74.6 CH 62.3 CH2 64.0 CH2 102.2 C 78.5 CH 70.5 CH 82.3 CH 59.5 CH2
89.6 70.2 68.9 68.4 72.2 61.7 64.7 103.9 79.6 71.3 82.5 59.7
89.7 69.0 72.6 70.1 74.0 62.3 64.3 103.9 79.1 71.1 82.2 59.9
Isovaleryl-O-3 1000 2000 3000 4000 , 5000
174.2 C 43.3 CH2 25.7 CH 22.3 CH3
171.3 43.2 25.4 22.3
173.9 43.3 25.7 22.3, 22.25
Isobutyryl-O-30 100 00 200 00 300 00 400 00
177.3 C 34.0 CH 19.2 CH3 18.7 CH3
178.0 34.0 19.1 18.7
178.1 33.9 19.2 18.7
Isovaleryl-O-4 100 00 0 200 00 0 300 00 0 400 00 0 , 500 00 0 Isobutyryl-O-10 100 00 0 200 00 0 300 00 0 , 400 00 0
172.2 C 43.0 CH2 25.5 CH 22.4 CH3 176.0 C 33.8 CH 18.94, 18.91
a Lauroyl-O-2: C-100 d 173.1 (1), 172.9 (2),173.0 (3); CH2-200 d 33.9 (1–3); CH2-300 d 24.6 (1, 3), 24.5 (2); CH2-400 d 29.1 (1-3); CH2-500 to CH2-1100 d 29.4, 29.3, 29.2, 29.6, 31.9, 22.7 (1–3), CH3-1200 d 14.1 (1–3).
A.-L. Pérez-Castorena et al. / Carbohydrate Research 352 (2012) 211–214
tached to the O-10 of 1; the presence of signals attributable to a second isovaleryl group (d 2.16 m, H2-2; d 2.05 nona, H-3; d 0.94 d, H34 and H3-5); the down field shift of H-4 (d 4.94) and the up field shift of H2-10 (d 3.59 and 3.47) in relation with the same hydrogens of 1. The position of the second isovaleryl group at O-4 of compound 2 was established by the correlation between its carbonyl carbon (d 172.2) and H-4 of the glucopyranose observed in the HMBC experiment. The pseudomolecular ion peak [M+Na+] obtained by HRESI MS supported the molecular formula of compound 2, C38H66O15, which was deduced by analysis of the NMR data. Therefore, this new compound named solanose B (2) corresponds to 30 -O-isobutyryl-3,4-di-O-isovaleryl-2-O-lauroylsucrose. Compound 3 also showed in its IR and NMR (Tables 1 and 2) spectra the profile of an esterified sucrose. In the 1H NMR spectrum the chemical shifts of H-4 (d 3.52) of the glucopyranose and H2-10 (d 3.59 and 3.49) of the fructofuranose indicated that the hydroxyl groups at C-4 and C-10 were not esterified. Additionally, only the signals of three acyl groups (lauroyl, isovaleryl, and isobutyryl) were observed in the NMR spectra. These acyl groups were located at O-2, O-3, and O-30 , as is shown in the structure 3, on the basis of the correlations (C-100 /H-2, C-1000 /H-3, C-100 00 /H-30 ) observed in the HMBC spectrum. In consequence, the new compound named solanose C (3) corresponds to 30 -O-isobutyryl-3-O-isovaleryl-2-O-lauroylsucrose. The molecular formula of 3, C33H58O14, was deduced from the analysis of its NMR data, and confirmed by the pseudomolecular ion peak [M+Na+] obtained in the HRFAB MS. The NMR data of 25-hydroxyphysalin N (5), also isolated from the fruits, are described here for the first time, and since a NOE effect between H3-28 and H-27b was observed in the NOESY spectrum, an a-orientation of OH-25 was proposed. In the literature, it has been described that a treatment of physalin N with activated charcoal–MeOH–ammonium acetate produced 25-hydroxyphysalin N (5) after stirring the mixture at room temperature for 17.5 h.31 The same treatment on physalin B afforded its 25-hydroxy derivative.31,33 In the present work, fractions containing 5 were decolored with activated charcoal–Me2CO, although this process took less than 3 h, the natural occurrence of 5 is uncertain. On the other hand, b-sitosteryl glucoside and physordinose B (4) were identified by the analysis of their spectroscopic data, which were compared with those described in the literature. Also, these compounds were compared with original samples. Finally, this is the second report of sucrose esters on Physalis fruits and the fourth one about their occurrence on the genus Physalis.
213
1.2. Plant material Physalis solanaceus (Schldl.) Axelius was collected in Cerro del Azteca, state of Querétaro, México, in August 2002. A voucher specimen of the plant (M. Martínez 6366) has been deposited in the Herbarium of the Universidad Autónoma de Querétaro. 1.3. Extraction and isolation Fresh fruits (328 g) were blended and exhaustively extracted with MeOH. The MeOH extract (35.4 g) was purified by column chromatography operated with vacuum (CC1) and eluted with a gradient of MeOH in CH2Cl2 to give the fractions A (CH2Cl2–MeOH 99:1 and 98:2), B (CH2Cl2–MeOH 95:5), C (CH2Cl2–MeOH 92:8), and D (CH2Cl2–MeOH 70:30?0:100). Fraction A (2.56 g) was decolored with activated charcoal and Me2CO and fractioned by several CC (eluents hexane–Me2CO 70:30; CH2Cl2–MeOH 97:3 and 94:6) to obtain two mixtures (A1, 226 mg and A2, 200 mg). Mixture A1 was submitted to three consecutive preparative TLCs (eluents CH2Cl2–Me2CO 90:10, CH2Cl2–MeOH 97:3 and 95:5) to give 7.4 mg of 25-hydroxyphysalin N (5).31 Fraction B (1.09 g) yielded, by crystallization, 6.1 mg of b-sitosterol glucoside as amorphous solid. Its mother liquor was purified by CC (eluent hexane–Me2CO 65:35) followed by two successive preparative TLCs (eluent hexane–Me2CO 70:30) to give two mixtures (A2, 90 mg and B1, 620 mg). Fraction C (570 mg) was fractioned by two successive CC (eluents CH2Cl2–Me2CO 55:45, CH2Cl2–MeOH 97:3?90:10) to yield 14 mg of mixture B1. Fraction D (8.0 g) afforded 448 mg of sucrose. Mixture A2 (200 mg) was purified by several preparative RPTLC (eluent MeOH–H2O 70:30, 5–9 elutions) to give 5.1 mg of solanose A (1) and 14 mg of solanose B (2). Mixture B1 (400 mg) was purified similarly to mixture A to produce 10 mg of solanose C (3) and 6 mg of physordinose B (4).8 1.3.1. Solanose A (1) Colorless oil; ½a25 D +8.0 (c 0.2, CHCl3); IR (CHCl3); m 3439 (OH), 1745 (ester) cm1; 1H and 13C NMR data, see Tables 1 and 2; HRESI MS m/z: 771.4111 [M+Na+] (calcd for C37H64O15Na: 771.4137). 1.3.2. Solanose B (2) Colorless oil; ½a25 D +9.8 (c 0.32, CHCl3); IR (CHCl3); m 3425 (OH), 1748 (ester) cm1; 1H and 13C NMR data, see Tables 1 and 2; HRESI MS m/z: 785.4293 [M+Na+] (calcd for C38H66O15Na: 785.4294).
1.1. General methods
1.3.3. Solanose C (3) Colorless oil; ½a25 D +13.13 (c 0.32, CHCl3); IR (CHCl3); m 3417 (OH), 1743 (ester) cm1; 1H and 13C NMR data, see Tables 1 and 2; FABMS m/z: 701 [M+Na]+, 429, 327, 233, 183, 127, 85, 71, 57, 43; HRFAB MS m/z: 701.3730 (calcd for C33H58O14Na: 701.3724).
Melting points were determined on a Fisher Jones melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-360 digital polarimeter. IR spectra were recorded on a Nicolet Magna-IR 750 spectrophotometer. NMR spectra were obtained on a Varian Unity Plus 500 spectrometer, with TMS as internal standard. FAB-MS and HRFAB-MS were obtained on a JEOL JMS-SX102A mass spectrometer. ESI-MS and HRESI-MS spectra were performed in positive mode on an ESI Ion Trap Bruker Esquire 600 and a Bruker MicrOTOF-II spectrometers, respectively. Column chromatography (CC) was carried out under vacuum using Silica Gel 60 G (Macherey-Nagel). Flash chromatography was performed with Silica Gel 60 (230–400 mesh, Macherey-Nagel). Sephadex LH-20 (25–100 lm; Amersham Pharmacia Biotech AB) was used for size exclusion chromatography. Preparative TLC was performed on precoated Sil G-100UV254 plates (Macherey-Nagel) and on Sil RP-18W/UV254 plates of 1.0 mm thickness (Macherey-Nagel).
1.3.4. 25-Hydroxyphysalin N (5) Amorphous powder (hexane–Me2CO); 1H NMR (DMSO-d6, 500 MHz): d 6.91 (1H, ddd, J = 10, 5, 3 Hz, H-3), 6.51 (1H, s, OH25), 6.36 (1H, s, OH-13), 5.82 (1H, dd, J = 10, 2 Hz, H-2), 5.68 (1H, dd, J = 6, 2 Hz, H-6), 4.61 (1H, dd, J = 4, 2 Hz, H-22), 4.35 (1H, br dd, J = 3, 2 Hz, H-7), 4.01 (1H, d, J = 12.5 Hz, H-27a), 3.40 (1H, d, J = 12.5 Hz, H-27b), 3.30 (2H, m, H-4a and H-9), 3.13 (1H, d, J = 2.5 Hz, OH-7), 3.09 (1H, s, H-16), 2.92 (1H, br dd, J = 22, 5 Hz, H-4b), 2.33 (1H, dd, J = 14.5, 4 Hz, H-23a), 2.22–2.1 (2H, m, H-11a and H-12a), 1.93 (1H, dd, J = 12, 3 Hz, H-8), 1.78 (1H, m, H-23b), 1.76 (3H, s, H3-21), 1.43 (1H, dd, J = 14, 9 Hz, H-12b), 1.12 (3H, s, H3-28), 1.05 (3H, s, H3-19), 1.05 (1H, m, H-11b); 13C NMR (DMSO-d6, 125 MHz, assignments by DEPT and HSQC experiments): d 208.5 (C-15), 201.3 (C-1), 171.4 (C-18), 168.5 (C-26), 146.1 (C-3), 139.0 (C-5), 126.9 (C-2), 125.3 (C-6), 106.1 (C-14), 80.6 (C-17), 79.4 (C-20), 78.0 (C-13), 76.5 (C-22), 73.6 (C-25),
1. Experimental
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64.8 (C-27), 61.3 (C-4), 52.9 (C-16), 52.5 (C-10), 43.8 (C-9), 35.7 (C24), 32.2 (C-23), 27.7 (C-8), 25.2 (C-12), 24.0 (C-11), 22.0 (C-21), 18.8 (C-28), 15.6 (C-19). Acknowledgements We are indebted to Héctor Ríos, Elizabeth Huerta, Beatriz Quiroz, Isabel Chávez, Eréndira García, Carmen Márquez, Luis Velasco, and Javier Pérez from the Instituto de Química, Universidad Nacional Autónoma de México, for technical assistance. We also thank to Nieves Zavala from the Centro Conjunto de Investigación en Química Sustentable, Universidad Autónoma del Estado de México-Universidad Nacional Autónoma de México for the HRESI MS determinations. References 1. Tukalo, E. A. Chem. Abstr. 1965, 62, 10821d. 2. Kawai, M.; Taga, G.; Osaki, K.; Matsuura, T. Tetrahedron Lett. 1969, 1087–1088. 3. Ray, A. B.; Gupta, M. In Progress in Chemistry of Organic Natural Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W., Tamm, C., Eds.; Springer-Verlag: Austria, 1994; Vol. 63, pp 1–106. 4. Maldonado, E.; Pérez-Castorena, A. L.; Garcés, C.; Martínez, M. Steroids 2011, 76, 724–728. 5. Fang, S.-T.; Liu, J.-K.; Li, B. J. Asian Nat. Prod. Res. 2010, 12, 618–622. 6. Lee, S.-W.; Pan, M.-H.; Chen, C.-M.; Chen, Z.-T. Chem. Pharm. Bull. 2008, 56, 234– 236. 7. Pérez-Castorena, A.-L.; Oropeza, R. F.; Vázquez, A. R.; Martínez, M.; Maldonado, E. J. Nat. Prod. 2006, 69, 1029–1033. 8. Pérez-Castorena, A.-L.; Martínez, M.; Maldonado, E. J. Nat. Prod. 2010, 73, 1271– 1276. 9. Ismail, N.; Alam, M. Fitoterapia 2001, 72, 676–679. 10. Elliger, C. A.; Eash, J. A.; Waiss, J. A. C. Biochem. Syst. Ecol. 1992, 20, 268.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
New sucrose esters from the fruits of Physalis solanaceus Ana-Lidia Pérez-Castorena a,⇑, Minerva Luna a, Mahinda Martínez b, Emma Maldonado a a b
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, D.F., Mexico Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Avenida de las Ciencias S/N, Col. Juriquilla 76230, Querétaro, Mexico
a r t i c l e
i n f o
Article history: Received 19 November 2011 Received in revised form 31 January 2012 Accepted 9 February 2012 Available online 18 February 2012
a b s t r a c t Three new sucrose esters (1–3) along with several known compounds were isolated from the fruits of Physalis solanaceus. The structural elucidation of the isolates was based on their spectroscopic characteristics mainly those of MS and NMR. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Solanaceae Physalis Sucrose esters Solanoses Physalin
The systematic chemical research of the genus Physalis practically started in the 60s with Physalis alkekengi var. francheti,1,2 and from that time, the aerial parts or the whole plant or the stems together with leafs have been the vegetal material more utilized for chemical studies. In these works, withasteroids (compounds derived from ergostane)3–6 have been the main metabolites reported, although labdane diterpenes,7,8 flavonoids,9,10 and sucrose esters,8,11 among other compounds, have also been isolated. From the early 90s, only a few reports on the chemical components of fruits, calyxes, and roots have been published. Nor-tropane and pyrrolidine alkaloids,12,13 withasteroids,14–16 and polysaccharides17 have been the main metabolites obtained from roots of the Physalis species. Polysaccharides,18 withasteroids,19 carotenoids,20 and sucrose esters21 have been isolated from the fruits; while physalins (highly oxidized seco-withasteroids) and flavonoids22 were obtained from the calyxes. Among the reasons that make Physalis an interesting genus is the use of some species in the traditional medicine of different countries, can be mentioned, for example: P. alkekengi var. francheti which is used for treatment of sore throat, cough, eczema, hepatitis, malaria, urinary problems and tumors in China,22,23 and Physalis philadelphica whose fruits are used to treat respiratory problems, sore throat, and cough in Mexico.8 Also, there is an economic interest in these plants because the fruits of some species are edible such as those of Physalis angulata and P. philadelphica.4,20 Additionally, various extracts and metabolites obtained from Physalis species have been shown to ⇑ Corresponding author. Tel.: +52 55 56 224412; fax: +52 55 56 162217. E-mail address: [email protected] (A.-L. Pérez-Castorena). 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2012.02.003
possess different biological activities such as antifeedant, antiinflammatory, cytotoxic, antioxidant, antimycobacterial, and immunomodulatory activities.22,24–26 As part of our systematic study on the Mexican Physalis species,4,7,8,21,27–30 we have reported the isolation of several physalins from the stems and leafs of Physalis solanaceus (Schldl.) Axelius,27 now we describe the chemical study of the fruits of this species, which resulted in the isolation of three new sucrose esters (1–3) together with the known physordinose B (4),8 25-hydroxyphysalin N (5),31 b-sitosteryl b-D-glucopyranoside, and sucrose. Compound 1, obtained as colorless oil, showed in the IR spectrum absorption bands at 3439 and 1745 cm1 attributable to hydroxyl and ester groups. The presence of a sucrose unit was deduced from the analysis of the NMR spectra (Tables 1 and 2), which showed the anomeric CH signals of the glucopyranose (dH 5.56 d, J = 3.5 Hz and dC 89.6; CH-1) and that of the anomeric carbon of the fructofuranose (d 102.2, C-20 ). The CH-1-O-C-20 glycosidic linkage between the monosaccharides was established by the correlation of the H-1 signal of the glucopyranose with the C20 signal of the fructofuranose observed in the HMBC spectrum of 1. The same spectrum let to locate the H2-10 (d 4.03 and 3.92) and H-50 (d 3.93) signals by their cross peaks with the C-20 signal. The correlations showed in the 1H–1H COSY, HSQC, and HMBC spectra allowed us to identify the signals of all the hydrogen and carbon atoms of the sucrose unit of compound 1 (Tables 1 and 2). The chemical shift of the H-2 (d 4.90), H-3 (d 5.20), H2-10 , and H-30 (d 5.25) signals indicated their gem position to ester functions.8 The presence of these ester groups was confirmed by the four 13C NMR signals for the carbonyl carbons of these functions
212
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H O OH
O
6
1'
O
3
CH3(CH2)10
O
O 5'
R2 = H R2 = iVal R2 = H R2 = H
4
R3 = iBu R3 = H R3 = H R3 = H
(Table 2). One of the acyl groups was identified as lauroyl by the characteristic twelve signals observed in the 13C NMR spectrum (Table 2).8 The other three acyl groups were identified as one isovaleryl32 and two isobutyryls21 on the basis to their 1H and 13C NMR signals (Tables 1 and 2). This allowed to propose the molecular formula C37H64O15 for compound 1, which was confirmed by the pseudomolecular ion peak [M+Na+] at m/z 771.4111 obtained by HRESI MS. The HMBC correlations between H-2 of the glucopyr-
Table 1 1 H NMR data (CDCl3, 500 MHz) for compounds 1–3a 2
Position
1
1 2 3 4 5 6a 6b 10 a 10 b 30 40 50 60 a 60 b
5.56 4.90 5.20 3.53 4.06 3.96 3.75 4.03 3.92 5.25 4.60 3.93 3.92 3.72
d (3.5) dd (10.5,3.5) dd (10.5,9.5) dd (10,9.5) ddd (10,6,2) dd (12,2) dd (12,6) d (12) d (12) d (8.5) dd (8.5,8.5) m m m
5.60 4.90 5.50 4.94 4.14 3.62 3.62 3.59 3.47 5.18 4.59 3.95 3.91 3.72
d (3.5) dd (10.5,3.5) dd (10.5,10) dd (10.5,10) ddd (10.5,5,3) m m d (12) d (12) d (8) dd (8.5,8) m dd (15,2.5) dd (15,1.5)
5.56 4.86 5.25 3.52 4.05 3.97 3.75 3.59 3.49 5.18 4.55 3.93 3.90 3.73
d (3.5) dd (10.5,3.5) dd (10.5,9.5) dd (9.5,9.5) ddd (10,6,2) dd (12,2) dd (12,6.5) d (12) d (12) d (8) dd (8,8) m m m
Lauroyl-O-2 200 300 400 –1100 1200
2.30 1.58 1.25 0.88
m m br s t (7.5)
2.27 1.55 1.26 0.88
m quint (7) br s t (7)
2.28 1.56 1.25 0.88
m, 2.24 m quint (7) br s t (7)
Isovaleryl-O-3 2.30 2000 3000 2.05 4000 0.95 5000 0.95
m m d (6.5) d (6.5)
2.05 2.00 0.91 0.91
m nona (7) d (7) d (7)
2.20 m 2.06 nona (7) 0.943 d (7) 0.941 d (7)
Isobutyryl-O-30 200 00 2.73 hept (7) 300 00 1.29 d (7) 00 00 4 1.26 d (7)
2.77 hept (7) 1.32 d (7) 1.29 d (7)
2.74 hept (7) 1.29 d (7) 1.25 d (7)
Isovaleryl-O-4 200 00 0 300 00 0 400 00 0 500 00 0
2.16 2.05 0.94 0.94
Isobutyryl-O-10 200 00 0 2.60 hept (7) 300 00 0 1.20 d (7) 00 00 0 4 1.19 d (7) a
Coupling constants in Hz (J).
OH
16
28
O O
O R1 = iVal R1 = iVal R1 = iVal R1 = iBu
O
14
O CH(CH3)2
1 2 3 4
HO
1
OH
3'
O
25
H
O HO
1
O
23
OR3
O
5
R2O R1 O
O
27
OH 5
anose and C-100 of the lauroyl group (d 173.1) as well as those of H-3 and C-1000 of the isovaleryl group (d 174.2), H2-10 of the fructofuranose and C-100 000 of one isobutyryl group (d 176.0), and H-30 and C-100 00 of a second isobutyryl group (d 177.3), led to establish the position of the acyl groups. Therefore, the structure of 1 was elucidated as 10 ,30 -di-O-isobutyryl-3-O-isovaleryl-2-O-lauroylsucrose and named solanose A. The IR and NMR (Tables 1 and 2) spectra of compound 2 were similar to those of 1. This similarity pointed to 2 as an esterified sucrose. The main differences observed in the 1H NMR spectra (Table 1) were the absence of the signals for the isobutyryl at-
3
m nona (7) d (7) d (7)
Table 2 13 C NMR data (CDCl3, 125 MHz) for compounds 1–3a Position
1
2
3
1 2 3 4 5 6 10 20 30 40 50 60
89.6 CH 69.3 CH 72.9 CH 70.0 CH 74.6 CH 62.3 CH2 64.0 CH2 102.2 C 78.5 CH 70.5 CH 82.3 CH 59.5 CH2
89.6 70.2 68.9 68.4 72.2 61.7 64.7 103.9 79.6 71.3 82.5 59.7
89.7 69.0 72.6 70.1 74.0 62.3 64.3 103.9 79.1 71.1 82.2 59.9
Isovaleryl-O-3 1000 2000 3000 4000 , 5000
174.2 C 43.3 CH2 25.7 CH 22.3 CH3
171.3 43.2 25.4 22.3
173.9 43.3 25.7 22.3, 22.25
Isobutyryl-O-30 100 00 200 00 300 00 400 00
177.3 C 34.0 CH 19.2 CH3 18.7 CH3
178.0 34.0 19.1 18.7
178.1 33.9 19.2 18.7
Isovaleryl-O-4 100 00 0 200 00 0 300 00 0 400 00 0 , 500 00 0 Isobutyryl-O-10 100 00 0 200 00 0 300 00 0 , 400 00 0
172.2 C 43.0 CH2 25.5 CH 22.4 CH3 176.0 C 33.8 CH 18.94, 18.91
a Lauroyl-O-2: C-100 d 173.1 (1), 172.9 (2),173.0 (3); CH2-200 d 33.9 (1–3); CH2-300 d 24.6 (1, 3), 24.5 (2); CH2-400 d 29.1 (1-3); CH2-500 to CH2-1100 d 29.4, 29.3, 29.2, 29.6, 31.9, 22.7 (1–3), CH3-1200 d 14.1 (1–3).
A.-L. Pérez-Castorena et al. / Carbohydrate Research 352 (2012) 211–214
tached to the O-10 of 1; the presence of signals attributable to a second isovaleryl group (d 2.16 m, H2-2; d 2.05 nona, H-3; d 0.94 d, H34 and H3-5); the down field shift of H-4 (d 4.94) and the up field shift of H2-10 (d 3.59 and 3.47) in relation with the same hydrogens of 1. The position of the second isovaleryl group at O-4 of compound 2 was established by the correlation between its carbonyl carbon (d 172.2) and H-4 of the glucopyranose observed in the HMBC experiment. The pseudomolecular ion peak [M+Na+] obtained by HRESI MS supported the molecular formula of compound 2, C38H66O15, which was deduced by analysis of the NMR data. Therefore, this new compound named solanose B (2) corresponds to 30 -O-isobutyryl-3,4-di-O-isovaleryl-2-O-lauroylsucrose. Compound 3 also showed in its IR and NMR (Tables 1 and 2) spectra the profile of an esterified sucrose. In the 1H NMR spectrum the chemical shifts of H-4 (d 3.52) of the glucopyranose and H2-10 (d 3.59 and 3.49) of the fructofuranose indicated that the hydroxyl groups at C-4 and C-10 were not esterified. Additionally, only the signals of three acyl groups (lauroyl, isovaleryl, and isobutyryl) were observed in the NMR spectra. These acyl groups were located at O-2, O-3, and O-30 , as is shown in the structure 3, on the basis of the correlations (C-100 /H-2, C-1000 /H-3, C-100 00 /H-30 ) observed in the HMBC spectrum. In consequence, the new compound named solanose C (3) corresponds to 30 -O-isobutyryl-3-O-isovaleryl-2-O-lauroylsucrose. The molecular formula of 3, C33H58O14, was deduced from the analysis of its NMR data, and confirmed by the pseudomolecular ion peak [M+Na+] obtained in the HRFAB MS. The NMR data of 25-hydroxyphysalin N (5), also isolated from the fruits, are described here for the first time, and since a NOE effect between H3-28 and H-27b was observed in the NOESY spectrum, an a-orientation of OH-25 was proposed. In the literature, it has been described that a treatment of physalin N with activated charcoal–MeOH–ammonium acetate produced 25-hydroxyphysalin N (5) after stirring the mixture at room temperature for 17.5 h.31 The same treatment on physalin B afforded its 25-hydroxy derivative.31,33 In the present work, fractions containing 5 were decolored with activated charcoal–Me2CO, although this process took less than 3 h, the natural occurrence of 5 is uncertain. On the other hand, b-sitosteryl glucoside and physordinose B (4) were identified by the analysis of their spectroscopic data, which were compared with those described in the literature. Also, these compounds were compared with original samples. Finally, this is the second report of sucrose esters on Physalis fruits and the fourth one about their occurrence on the genus Physalis.
213
1.2. Plant material Physalis solanaceus (Schldl.) Axelius was collected in Cerro del Azteca, state of Querétaro, México, in August 2002. A voucher specimen of the plant (M. Martínez 6366) has been deposited in the Herbarium of the Universidad Autónoma de Querétaro. 1.3. Extraction and isolation Fresh fruits (328 g) were blended and exhaustively extracted with MeOH. The MeOH extract (35.4 g) was purified by column chromatography operated with vacuum (CC1) and eluted with a gradient of MeOH in CH2Cl2 to give the fractions A (CH2Cl2–MeOH 99:1 and 98:2), B (CH2Cl2–MeOH 95:5), C (CH2Cl2–MeOH 92:8), and D (CH2Cl2–MeOH 70:30?0:100). Fraction A (2.56 g) was decolored with activated charcoal and Me2CO and fractioned by several CC (eluents hexane–Me2CO 70:30; CH2Cl2–MeOH 97:3 and 94:6) to obtain two mixtures (A1, 226 mg and A2, 200 mg). Mixture A1 was submitted to three consecutive preparative TLCs (eluents CH2Cl2–Me2CO 90:10, CH2Cl2–MeOH 97:3 and 95:5) to give 7.4 mg of 25-hydroxyphysalin N (5).31 Fraction B (1.09 g) yielded, by crystallization, 6.1 mg of b-sitosterol glucoside as amorphous solid. Its mother liquor was purified by CC (eluent hexane–Me2CO 65:35) followed by two successive preparative TLCs (eluent hexane–Me2CO 70:30) to give two mixtures (A2, 90 mg and B1, 620 mg). Fraction C (570 mg) was fractioned by two successive CC (eluents CH2Cl2–Me2CO 55:45, CH2Cl2–MeOH 97:3?90:10) to yield 14 mg of mixture B1. Fraction D (8.0 g) afforded 448 mg of sucrose. Mixture A2 (200 mg) was purified by several preparative RPTLC (eluent MeOH–H2O 70:30, 5–9 elutions) to give 5.1 mg of solanose A (1) and 14 mg of solanose B (2). Mixture B1 (400 mg) was purified similarly to mixture A to produce 10 mg of solanose C (3) and 6 mg of physordinose B (4).8 1.3.1. Solanose A (1) Colorless oil; ½a25 D +8.0 (c 0.2, CHCl3); IR (CHCl3); m 3439 (OH), 1745 (ester) cm1; 1H and 13C NMR data, see Tables 1 and 2; HRESI MS m/z: 771.4111 [M+Na+] (calcd for C37H64O15Na: 771.4137). 1.3.2. Solanose B (2) Colorless oil; ½a25 D +9.8 (c 0.32, CHCl3); IR (CHCl3); m 3425 (OH), 1748 (ester) cm1; 1H and 13C NMR data, see Tables 1 and 2; HRESI MS m/z: 785.4293 [M+Na+] (calcd for C38H66O15Na: 785.4294).
1.1. General methods
1.3.3. Solanose C (3) Colorless oil; ½a25 D +13.13 (c 0.32, CHCl3); IR (CHCl3); m 3417 (OH), 1743 (ester) cm1; 1H and 13C NMR data, see Tables 1 and 2; FABMS m/z: 701 [M+Na]+, 429, 327, 233, 183, 127, 85, 71, 57, 43; HRFAB MS m/z: 701.3730 (calcd for C33H58O14Na: 701.3724).
Melting points were determined on a Fisher Jones melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-360 digital polarimeter. IR spectra were recorded on a Nicolet Magna-IR 750 spectrophotometer. NMR spectra were obtained on a Varian Unity Plus 500 spectrometer, with TMS as internal standard. FAB-MS and HRFAB-MS were obtained on a JEOL JMS-SX102A mass spectrometer. ESI-MS and HRESI-MS spectra were performed in positive mode on an ESI Ion Trap Bruker Esquire 600 and a Bruker MicrOTOF-II spectrometers, respectively. Column chromatography (CC) was carried out under vacuum using Silica Gel 60 G (Macherey-Nagel). Flash chromatography was performed with Silica Gel 60 (230–400 mesh, Macherey-Nagel). Sephadex LH-20 (25–100 lm; Amersham Pharmacia Biotech AB) was used for size exclusion chromatography. Preparative TLC was performed on precoated Sil G-100UV254 plates (Macherey-Nagel) and on Sil RP-18W/UV254 plates of 1.0 mm thickness (Macherey-Nagel).
1.3.4. 25-Hydroxyphysalin N (5) Amorphous powder (hexane–Me2CO); 1H NMR (DMSO-d6, 500 MHz): d 6.91 (1H, ddd, J = 10, 5, 3 Hz, H-3), 6.51 (1H, s, OH25), 6.36 (1H, s, OH-13), 5.82 (1H, dd, J = 10, 2 Hz, H-2), 5.68 (1H, dd, J = 6, 2 Hz, H-6), 4.61 (1H, dd, J = 4, 2 Hz, H-22), 4.35 (1H, br dd, J = 3, 2 Hz, H-7), 4.01 (1H, d, J = 12.5 Hz, H-27a), 3.40 (1H, d, J = 12.5 Hz, H-27b), 3.30 (2H, m, H-4a and H-9), 3.13 (1H, d, J = 2.5 Hz, OH-7), 3.09 (1H, s, H-16), 2.92 (1H, br dd, J = 22, 5 Hz, H-4b), 2.33 (1H, dd, J = 14.5, 4 Hz, H-23a), 2.22–2.1 (2H, m, H-11a and H-12a), 1.93 (1H, dd, J = 12, 3 Hz, H-8), 1.78 (1H, m, H-23b), 1.76 (3H, s, H3-21), 1.43 (1H, dd, J = 14, 9 Hz, H-12b), 1.12 (3H, s, H3-28), 1.05 (3H, s, H3-19), 1.05 (1H, m, H-11b); 13C NMR (DMSO-d6, 125 MHz, assignments by DEPT and HSQC experiments): d 208.5 (C-15), 201.3 (C-1), 171.4 (C-18), 168.5 (C-26), 146.1 (C-3), 139.0 (C-5), 126.9 (C-2), 125.3 (C-6), 106.1 (C-14), 80.6 (C-17), 79.4 (C-20), 78.0 (C-13), 76.5 (C-22), 73.6 (C-25),
1. Experimental
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64.8 (C-27), 61.3 (C-4), 52.9 (C-16), 52.5 (C-10), 43.8 (C-9), 35.7 (C24), 32.2 (C-23), 27.7 (C-8), 25.2 (C-12), 24.0 (C-11), 22.0 (C-21), 18.8 (C-28), 15.6 (C-19). Acknowledgements We are indebted to Héctor Ríos, Elizabeth Huerta, Beatriz Quiroz, Isabel Chávez, Eréndira García, Carmen Márquez, Luis Velasco, and Javier Pérez from the Instituto de Química, Universidad Nacional Autónoma de México, for technical assistance. We also thank to Nieves Zavala from the Centro Conjunto de Investigación en Química Sustentable, Universidad Autónoma del Estado de México-Universidad Nacional Autónoma de México for the HRESI MS determinations. References 1. Tukalo, E. A. Chem. Abstr. 1965, 62, 10821d. 2. Kawai, M.; Taga, G.; Osaki, K.; Matsuura, T. Tetrahedron Lett. 1969, 1087–1088. 3. Ray, A. B.; Gupta, M. In Progress in Chemistry of Organic Natural Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W., Tamm, C., Eds.; Springer-Verlag: Austria, 1994; Vol. 63, pp 1–106. 4. Maldonado, E.; Pérez-Castorena, A. L.; Garcés, C.; Martínez, M. Steroids 2011, 76, 724–728. 5. Fang, S.-T.; Liu, J.-K.; Li, B. J. Asian Nat. Prod. Res. 2010, 12, 618–622. 6. Lee, S.-W.; Pan, M.-H.; Chen, C.-M.; Chen, Z.-T. Chem. Pharm. Bull. 2008, 56, 234– 236. 7. Pérez-Castorena, A.-L.; Oropeza, R. F.; Vázquez, A. R.; Martínez, M.; Maldonado, E. J. Nat. Prod. 2006, 69, 1029–1033. 8. Pérez-Castorena, A.-L.; Martínez, M.; Maldonado, E. J. Nat. Prod. 2010, 73, 1271– 1276. 9. Ismail, N.; Alam, M. Fitoterapia 2001, 72, 676–679. 10. Elliger, C. A.; Eash, J. A.; Waiss, J. A. C. Biochem. Syst. Ecol. 1992, 20, 268.
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