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12Physalins from the calycesof Physalis alkekengi L. var. franchetii
Hai-Xue Kuang*, Zhi-Bin Wang, Bing-You Yang, Zhun-Peng Shu, Xin-Li Li and Qiu-Hong Wang
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040, People's Republic of
China
Abstract.
From the calyces of Physalis alkekengi L. var. franchetii (Solanaceae), Four new physalins14,27-seco-physalin D (1), 5a, 6p-dihydroxy physalin R (2), 3a-hydroxy-2, 3-dihydrophysalin A (3) and 14, 27-seco-physalin P (4), together with eightknown physalins were isolated. Their structures were determined mainly by spectroscopic techniques including 2D-NMR (HMBC, HSQC, NOSEY) and MS experiments.
1.Introduction
The calyces of Physalis alkekengi L.var. Franchetii (Chinese name: Jindenglong) recorded in Pharmacopoeia of the People's Republic of China has been used as the treatment for sore throat, sound dumb, phlegm-heat , cough, dysuria, stranguria, pemphigus and eczema[1]. Physalins are the major active steroidal constituents of the calyces of this plant, and their rare 13, 14-seco-16, 24-cycloergostane skeletons have been established by X-ray crystallographic analysis[2-4]. physalins derived from the genus Physalisare reported to display a wide spectrum of biological activities such as immunomodulatory^, cytotoxic[6-8], immunosuppressive[9],anti-inflammatory[10], antimicrobial activities [11, 12]. The novel structure and multiple biological activities of physalins make us to further study the calyces of P. alkekengi L.var. Franchetii. We separated four new physalins 14,27-seco-physalin D (1), 5a, 6p-di-hydroxy physalin R (2), 3a-hydroxy-2, 3-di-hydrophysalin A (3) and 14, 27-seco-physalin P (4) together with eight known compounds from the calyces of the plant. Known compounds were identified by detailed 1D- and 2D-NMR analyses,comparison of their ESI-MS and spectral data with those reported in literature [11-13] as physalins E(5), D(6), B(7), N(8), O(9), F (10), P(11), J(12). This paper describes the structural elucidation of four new physalins.
3.Results and Discussion
Compound 1 was obtained as a white amorphous powder. An UV absorption maximum at 226 nm indicated the presence of an ^-unsaturated ketone chromophore. The HR-ESI-MS of 1 showed a pseudomolecular ion [M+H]+ at m/z 529.2104. Taking into account the 28 C-atoms display in its 13C-NMR spectrum, the molecular formula was determined to be C28H32O10. The NMR spectra of 1 were similar to those of physalin D[14],suggesting that 1 was also a physalin. The 1H-NMR spectrum (in C5D5N) of 1(Table) showed the presence of four methyl singlets at SH 1.41 (H-28), Sh 1.59 (H-19), Sh 2.32 (H-21), and Sh 1.22 (H-27)(3H, d, J = 7.6 Hz) and two olefinic protons at Sh 6.61 (1H, ddd, J = 2.4, 4.8, 10.0 Hz,H-3) and Sh 6.00 (1H, dd, J = 2.4, 10.0 Hz,H-2).
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The C-NMR spectrum (in C5D5N) of 1 (Table) showed the presence of two ketone carbonyl signals at Sc 216.4 (C-15)and Sc 204.3 (C-1), two lactone carbonyl signals at Sc 174.7 (C-18) and Sc 172.7 (C-26), and two olefine carbons at Sc 142.2 (C-3) and Sc 128.9 (C-2). One additional methyl at SC 17.2 (C-27) and a characteristic C-atom at SC 103.6 (C-14) supported the nonexistence of C(14)-O-C(27) ether bridge in compound 1. The HMBC correlations (Fig.2) from the olefinic methine proton at SH 6.61 (H-3) to ketone carbon at SC 204.3 (C-1) and olefinic methine proton at SH 6.00 (H-2) to methylene carbon at Sc36.4 (C-4) and a quaternary carbon at Sc55.7 (C-10) indicated the presence of 2-ene-1-one system in compound 1.Furthermore, the HMBC correlations from an OH-substituted methine proton at Sh 4.26 (C-6) to a oxygenated quaternary carbon at Sc77.8 (C-5), to a methine carbon at SC41.7 (C-8), and to a methyl at SC 13.8 (C-19) indicated the OH groups were present at C-5 and C-6. Concerning the relative configuration of 1, The NOESY spectrum of 1 exhibited NOE correlations between SH1.59 (H-19) and SH3.20 (H-8) indicating P-orientation of H-8. But H-8 and H-6 did not exist the NOE correlations, therefore the P-orientation of 6-OH
group.Concerning the relative configuration of 1, the NOESY spectrum(in DMSO) correlations between OH-C(5) and H-C(6), so the orientations of OH-C(5) were determined to be a. On the basis of the 1H,13C, and 2D NMR (HSQC, HMBC, NOESY) data,the structure of 1 was unambiguously established as 14, 27-seco-physalin D[13] (Fig.1).
Compound 2, named 5a, 6p-di-hydroxy physalin R,was obtained as a white amorphous powder. An UV absorption maximum at 228 nm indicated the presence of an ^-unsaturated ketone chromophore. The HR-ESI-MS of 2 showed a pseudomolecular ion [M+H]+ at m/z
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545.2006. Taking into account the 28 C-atoms display in its C-NMR spectrum, the molecular formula was estalished as C28H32On . The 1H-NMR spectrum (in C5D5N) of 2 (Table) showed the presence of the characteristic signals for three methyl groups at Sh 1.58 (H-28), 1.77 (H-19) and 2.28 (H-21) and two olefinic protons at SH 6.73 (H-3) and 6.00 (H-2). Two geminally coupled signals of an oxygenated methine at Sh5.37(H-27) and 4.76 (H-27) and one methine at Sh2.83 (H-25) indicated that the presence of C(14)-O-(27) ether bridge in compound 2. The 13C-NMR spectrum (in C5D5N) of 2 (Table) showed the presence of one ketone carbonyl signal at Sc204.7 (C-1) and two lactone carbonyl signals at SC176.1(C-18) and 169.3(C-26), but no signals of a ketone carbonyl signal. These facts indicated the basic skeleton of 2 was of the physalin-R type [15]. The HMBC correlations (Fig.2) of an OH-substituted methine proton at SH 4.25 (H-6) with a oxygenated quaternary carbon at Sc 78.6 (C-5) and with a methyl at Sh1.77 (H-19) indicated the hydroxy groups were present at C-5 and C-6. Concening the relative configuration of 2, NOESY data of 1 and 2 were analogous and indicated that the hydroxy at C-5 and C-6 was determined to be a-orientation and P-orientation respectively. On the basis of the presented spectral data, compound 2 was identified as 5a,6p-di-hydroxy physalin R (Fig.1).
Compound 3, named3a-hydroxy-2, 3-di-hydrophysalin A, was obtained as a white amorphous powder. The HR-ESI-MS of 3 showed a pseudomolecular ion [M+Na]+ at m/z 567.1786, so the molecular formula was estalished as C28H32O11. The NMR spectra of 3 were similar to those of physalin A,[16] suggesting that 3 was also a physalin. The 1H-NMR spectrum (in C5D5N)(Table)of 3 showed the presence of three methyl groups at SH1.16(H-19), 1.71(H-28), and 2.02(H-21) and three olefinic protons at Sh6.94 (1H, br s,H-27), 5.89 (1H, br s,H-27), and 5.95 (1H, dd, J = 1.6, 6.0
13
Hz,H-6). The C-NMR spectrum of 3 yielded signals for 28 C-atoms, including two ketone carbonyl signals at Sc 214.4(C-15) and 210.7(C-1), two lactone carbonyl signals at Sc 173.3(C-18) and 162.7(C-26) and four olefine carbons at Sc 142.5 (C-5), 129.0 (C-6), 138.79 (C-25), 133.4 (C-27). The 13C-NMR spectrum of 3 showed an additional characteristic C-atom at Sc 102.5 (C-14) that indicated the nonexistence of C(14)-O-C(27) ether bridge in compound 3. The HMBC correlations (Fig.2) of the OH-substituted methine protons at Sh 5.26 (H-7) with two olefine carbons Sc 142.5 (C-5), 129.0(C-6), with a methylene C-atom at Sc 39.2 (C-4), and with a methine C-atom at Sc 47.5 (C-8) inferred that the OH group was present at C-7. Concerning the relative configuration of 3, the NOESY cross-peaks between H-3 and the coupling constants of H-3 and H-2 [J (2a, 3) = 3.6 and J (2P, 3) =2.8 Hz] indicated that 3-OH is a-orientation. The NOESY spectrum of 3 exhibited NOE correlations between Sh 2.48(H-8) and 1.16(H-19);Sh 2.48(H-8) and 5.26(H-7), so the orientations of H-7 was determined to be p, the 7-OH was determined to be a. On the basis of the presented spectral data, compound 3 was unambiguously established as 3a-hydroxy-2, 3-di-hydrophysalin A (Fig.1).
Compound 4, named 14, 27-seco-physalin P, was obtained as a white amorphous powder. An UV absorption maximum at 228 nm indicated the presence of an a,p-unsaturated ketone chromophore. The HR-ESI-MS of 4 showed a pseudomolecular ion [M+Na]+ at
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m/z551.1858.Taking into account the 28 C-atoms display in its C-NMR spectrum, the molecular formula was estalished as C28H32Oio. The XH-NMR (in C5D5N) (Table) spectrum of 4 showed the presence of four methyl groups at Sh 1.52 (H-28), 0.98 (H-19), 2.15 (H-21), and 1.37 (1H, d, J = 7.6 Hz,H-27) and four olefinic protons at Sh6.43 (1H, ddd, J = 2.4, 4.4, 10.6 Hz,H-3), 5.97 (1H, dd, J = 2.4, 10.6 Hz,H-2), and 6.49 (1H, dd, J = 5.2, 10.6 Hz,H-7), 5.90 (1H, d, J = 10.6 Hz,H-6). In the
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C-NMR spectrum of 4showed the presence of one ketone carbonyl signal at SC201.9 (C-1) and
three lactone carbonyl signals at Sc174.1 (C-15), 178.8 (C-18), 172.9 (C-26) were present, but no signals of a ketal group characteristic for physalin derivates. This implied that the structure of 4 had a structure different from that of common physalin derivates, namely a neophysalin skeleton, which is considered to be derived from the physalin structure by a benzilic acid rearrangement. One additional methyl at SC16.3 (C-27) and methine at SC39.5 (C-25) supported the nonexistence of C(27)-O-C(14) ether bridge in compound 4. The HMBC correlations (Fig.2) from the olefinic methine proton at SH 6.43 (H-3) to SC201.9 (C-1) and to methylene carbon at SC37.7 (C-4) indicated the presence of 2-ene-1-one system. The HMBC correlations from two olefinic methine protons at Sh 5.90 (H-6) and 6.49(H-7) to an OH-subtituted quaternary carbon at Sc71.8 (C-5) and to methine proton at Sc48.1 (C-8) inferred that the OH group was present at C-5. The stereochemistry of the OH group was determined by the CD spectrum of 4 which showed a negative cotton effect ([9]-6170) at 337nm. The 5a- and 5fi- steroids, respectively, with the 2 ene-1-one system, are known exhibit negative and positive Cotton effect due to the n-n* transition reflecting negative and positive helicity of the transoid enone moiety. Accordingly, the a-configuration was assigned to the OH group at the C-5 position[15]. Concerning the relative configuration of 4, The NOESY spectrum of 4 exhibited NOE correlations between H-19 and H-8, so the orientations of H-8 were determined to be p. On the basis of the presented spectral data, compound 4 was identified as 14, 27-seco-physalin P (Fig.1).
3.Experimental section
3.1General Experimental Procedures.
Open column chromatography (CC) was carried out using silica gel (200-300 mesh, Qingdao Marine Chemical Co, (Qingdao, China). TLC employed precoated silica gel plates (5-7 ^m, Qingdao Marine). Preparative HPLC was carried out on a Waters 600 instrument equipped with a Waters UV-2487 detector. A Waters Sunfire prep C18 ODS (19 x 250 mm i.d.) column was used for this purpose. The IR spectra were recorded as KBr pellets on a Jasco 302-A spectrometer. CD spectra were recorded on a J-600 spectropolarimeter. HR-ESI-MS were measured on a FTMS-7 instrument (Bruker Daltonics). The fH-, 13C-and 2D-(HSQC, HMBC, NOESY) NMR spectra were recorded on a Bruker AMX-400 spectrometer using standard pulse sequences. Chemical shifts are reported in ppm (5), and scalar coupling are reported in Hz. Other reagents were purchased from various commercial sources.
3.2Plant materials
The calyces of Physalis alkekengi L. var. franchetii. were collected from Shangzhi, Heilongjiang province in July 2007 and identified by Prof. ZhenYue Wang (Heilongjiang University of Chinese Medicine, Heilongjiang, China). The voucher specimen was authenticated and is deposited in the Heilongjiang University of Chinese Medicine (No.2007-07-1).
3.3Extraction and isolation
The air dried calyces of Physalis alkekengi L. var. franchetii (6kg) were extracted two times with 70% EtOH for 2h at 60°C. The solvent was evaporated to dryness, and the dry residue (1.5kg) was subjected to AB-8 macroporous adsorptive resin, eluted with water, 50% EtOH, 95% EtOH respectively to yield three fractions (Fractions 1-3). Fraction 2 (105 g) was separated by column chromatography on silica gel eluting with CH2Cl2-MeOH (10:1, 5:1, 1:1, and MeOH) to give nine fractions (Fr.1-Fr.9). Fr.1 was recrystallized from MeOH to give 11.Fr.1 was separated by column chromatography on silica gel eluting with CH2Cl2-MeOH (30:1, 15:1, 10:1, and MeOH) to give three fractions(Fr.1_1-Fr.1_3). Frx_2 was recrystallized from MeOH to give 7.Fr.1_3 was further purified by ODS column chromatography and HPLC [MeOH-H2O (60:40 v/v)] to give 10.Fr.i_3 was separated by ODS column chromatography and HPLC [MeOH-H2O (45:55 v/v)] to give8 and 9. Fr.3 was recrystallized from MeOH to give 1..Fr.3 was separated by column chromatography on silica gel eluting with CH2Cl2-MeOH (20:1, 15:1, 10:1, 8:1, 5:1, and MeOH) to yield give four fractions (Fr.3-1-Fr.3_4). Fr.3_4 was further separated by column chromatography on silica gel eluting with CH2Cl2-MeOH (15:1, 10:1, 8:1, 5:1, 1:1 and MeOH) to give two fractions (Fr.3-4-1-Fr.3-4-2).Fr.3_4_1 was further purified by ODS column chromatography and HPLC [MeOH-H2O (40:60
v/v)] to give 4.Fr.3-4-2 was separated by ODS column chromatography and HPLC [MeOH-H2O (40:60 v/v)] to give3and 2..Fr.4 was separated by column chromatography on silica gel eluting with MeOH (10:1) to give seven fractions (Fr.4-i~Fr.4-7).Fr.4-2 was separated by column chromatography on silica gel eluting with CH2Cl2-MeOH (15:1) to give three fractions (Fr.4-2-1~Fr.4-2-3).Fr.4-2-3 was separated by ODS column chromatography and HPLC [MeOH-H2O (50:50 v/v)] to give6.Fr.4-4 was further purified by ODS column chromatography and HPLC [MeOH-H2O (40:60 v/v)] to give5. 3.4. Characteristics of compounds (1-4)
3.4.1. Compound 1 (14,27-seco-physalin D) White amorphous powder (MeOH). UV [MeOH, nm, logs]: 226 (3.01). IR (film, cm-1): 3406, 3382, 2931, 2858, 1778, 1712, 1614, 1253, 1218, 1166, 1130, 1095, 1064, 1045. XH-NMR and 13C-NMR data: Table. The positive ESI-MS (m/z, %):529 (100).
3.4.2. Compound 2 (5a, 6ß-di-hydroxy physalin R) White amorphous powder (MeOH). UV [MeOH, nm, logs]: 228 (2.78). IR (film, cm-1): 3446, 3421, 2945, 2894, 1762, 1740, 1604, 1166, 1134, 1060. XH-NMR and 13C-NMR data: Table. The positive ESI-MS(m/z, %):545 (100).
3.4.3. Compound 3 (3a-hydroxy-2, 3-di-hydrophysalin A) White amorphous powder (MeOH). UV [MeOH, nm, logs]: 208 (2.90). IR (film, cm-1): 3384, 2941, 2856, 1780, 1083, 1060, 1045. 1H-NMR and 13C-NMR data: Table. The positive ESI-MS(m/z, %):567 (M+, ), 527 (100).
3.4.4. Compound 4 (14, 27-seco-physalin P) white amorphous powder (MeOH). UV [MeOH, nm, logs]: 228 (2.78). IR (film, cm-1): 3475, 2923, 2850, 1791, 1718, 1224.1, 1134, 1093, 1060. 1H-NMR and 13C-NMR data: Table . The positive ESI-MS(m/z, %): 551 (M+), 511 (100).
References
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7S. Nagafuji, H. Okabe, H. Akahane, F. Abe, Biol. Pharm. Bull. 2004;27:193.
8 C. C. Lee, P. Houghton, J. Ethnopharmacol. 2005;100:273.
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Y., Ogawa, K., Hayashi, M. Bull. Chem. Soc. Jpn. 1994;67:222-226.
12Taga, T., Miwa, Y., Machida, K., Kawai, M., Buisugan, Y. Acta Crystallogr. C 1991;47:2188-2191.
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Kazushi Shingu, Shaji Yahara, Toshihiro Nohara etal., Three new withanolides, Physalin A, B and D from Physalis angulata. Chem. Pharm. Bull. 1992;40:2088-2091. 14Makino B., Kawai M., Kito K., Yamamura H., Butsugan Y., New physalins possessing an additional carbon-carbon bond from Physalis alkekengi var. franchetii. Tetrahedron 1995;51:12529-12538.
15R. Tschesche, M. Baumgrath and P. Welzel, Tetrahedron. 1968;24:5169. 16Kawai M., Matsumoto A., Makino B., Mori H., Ogura T., Butsugan Y., Ogawa K., Hayashi M., The strueture of Physalin P, a neophysalin from Physalis alkekengi. Bull. Chem. Soc. Jpn. 1993;66:1299-1300.
Table ^H and 13C-NMR Data of compounds 1-4 (in C5D5N, 5 in ppm, J inHz)
Position 1 2 3 4
SH SC SH SC SH Sc
1 - 204.3 - 204.7 - 210.7 - 201.9
2 6.00 (dd, J = 128.9 6.00 (dd, J 128.7 2.96 (dd, J =3.6, 46.6 5.97 (dd, J =2.0, 127.7
2.4, 10.0) =2.8, 10.4) 13.6, Ha) 2.60 (dt, J =2.8, 13.6, Hp) 10.6)
3 6.61 (ddd, J = 2.4, 4.8, 10.0) 142.2 6.73 (ddd, J =2.8, 4.8, 10.4) 143.7 4.46 (t, J =1.6) 67.5 6.43 (ddd, J =2.0, 4.4, 10.6) 140.9
4 3.65 (dt, J =2.8, 19.6, Ha) 2.40 (dt, J =2.8, 19.6, Hp) 36.4 3.51 (m) 2.40 (dt, J =2.8, 19.6) 32.5 2.73 (d, J =14.8) 2.44 (d, J =14.8) 39.2 3.39-3.43 (m) 2.49-2.57 (m) 37.7
5 - 77.8 - 78.6 - 142.5 - 71.8
6 4.26 (br s) 75.1 4.25 (br s) 76.0 5.95 (dd, J =1.6, 6.0) 129.0 5.90 (d, J =10.6) 132.3
7 2.89-3.00 (m) 30.4 2.93-3.02 (m) 31.0 5.26 (d, J =6.0) 63.2 6.49 (dd, J =5.2, 10.6) 129.9
8 3.20-3.30 (m) 41.7 3.65 (dm, J =19.6) 41.7 2.48 (dd, J =2.0, 12.0) 47.5 3.01-3.07 (m) 48.1
9 4.30 (dd, J =5.0, 11.8) 32.5 4.08 (m) 42.9 3.98 (dd, J =8.8, 12.0) 30.5 2.84-2.91 (m) 31.9
10 - 55.7 - 55.8 - 58.9 - 53.9
11 2.11 (dd, J =9.2,16.0, Ha) 1.91 ( d, J =16.0, Hp) 27.0 3.07 (t, J =6.0) 49.3 3.01-3.06 (m) 2.10-2.19 (m) 31.4 2.97-3.05, 1.421.51 (2m) 25.4
12 2.19-2.28, 1.62-1.73 (2 m ) 26.0 2,35-2.42 (m) 34.1 2.25-2.43 (m) 2.11 (dd, J = 9.2, 16.0) 24.6 3.00-3.09, 2.452.52 (2m) 28.0
13 - 80.8 - 87.4 - 83.4 - 79.3
14 - 103.6 - 114.3 - 102.5 - 82.4
15 - 216.4 - 77.5 - 214.4 - 174.1
16 3.11 (s) 55.8 2.39 (s) 51.4 3.29 (s) 54.5 3.34 (s) 57.3
17 - 83.5 - 83.6 - 80.7 - 85.2
18 - 174.7 - 176.1 - 173.3 - 178.8
19 1.59 (s) 13.8 1.77 (s) 15.0 1.16 (s) 15.4 0.98 (s) 16.9
20 - 83.8 83.3 83.1 - 83.4
21 2.32 (s) 22.0 2.28 (s) 21.6 2.02 (s) 21.9 2.15 (s) 21.6
22 4.69 (d, J =3.2) 78.0 4.63 (t, J =2.8) 77.1 4.70 (t, J =2.8) 76.7 4.67 (t, J =2.4) 75.4
23 3.27-3.38 (m) 2.24-2.32(m) 30.9 2.00 (ddd, J =1.6, 10.8, 16.0) 1.91 ( dd, J =1.6, 16.0) 36.2 2.04 (d, J =2.8) 32.1 2.09 (dd, J =4.4, 15.2) 1.79 (d, J = 15.2)9 28.3
24 - 35.3 - 36.0 - 36.4 - 35.1
25 3.52 (q, J =7.6) 42.1 2.83 (d, J =4.0) 51.9 - 138.7 4.11 (q, J =7.6) 39.5
26 - 172.7 - 169.3 - 162.7 - 172.9
27 1.22 (d, J =7.6) 17.2 5.37 (d, J =10.4) 4.76 (dd, J =4.0,10.4) 61.2 6.94 (br s) 5.89 (br s) 133.3 1.37 (d, J =7.6) 16.3
28 1.41 (s) 27.0 1.58 (s) 29.6 1.71 (s) 27.6 1.52 (s) 28.0
1 5a-OH, 6P-OH 9 A5, 7a-OH
OH 2
O
'JO
O^ /O
5 5a-OH, 7a-OH
6 5a-OH, 6P-OH
7 A5
8 A5, 7a-OH
10 5p, 6p-epoxy 12 5 a, 6a-epoxy
Fig. 1. structures of compounds 1-12
3
H
1 2 Fig.2. Key HMBC (^)and 1H-1H COSY(—) correlations of compounds 1-2
