Two Novel Norwithasteroids with Unusual Six- and Seven-Membered Ether Rings in Side Chain from Flos Daturae
Chemical investigation of 50% ethanol eluate fraction of macroporous resin for the flower of Datura metel L. collected in Jiangsu province of China resulted in the isolation of two novel naturally occurring norwithasteroids, baimantuoluoline I (1) and baimantuoluoside J (2). Their structures were elucidated as 5α, 6β, 12β-trihydroxy-1-oxo-2-en-ergosta-21,24;22,29-diepoxy-26-carboxylic acid (1) and 5α, 6β, 12β, 25-tetrahydroxy-1-oxo-2-en-ergosta-21,24;22,29-diepoxy-26-carboxylic acid (2) on the basis of extensive spectroscopic analysis, including 1D, 2D-NMR, and HR-ESI-MS. According to the literatures, this study represents the first report of the norwithasteroids in the side chain with unusual six- and seven-membered ether rings instead of those with an unmodified skeleton (-lactone or -lactol side chain) and a modified skeleton (γ-lactone or γ-lactol side chain) in the family of withanolides. Meanwhile, compounds 1 and 2 were evaluated for their immunosuppressive activity against mice splenocyte proliferation in vitro.
Flos Daturae is the dry flower of Datura metel L. (Solanaceae), which widely distributed in Jiangsu, Guangdong, and Fujian province of China . It has been used in traditional Chinese medicine for the treatment of asthma, convulsions, pain, and rheumatism for centuries . It is said that it was almost one of the most important anesthetic of the ancient times. It has a very long history and is referred to in many early writings in China. For instance, according to the legend as early as 200 AD, a Chinese skilled doctor, Huatuo, once made use of “mafeisan” on patients in surgery [2, 3]. A recent study found that it has an obvious effect on the treatment of psoriasis [4, 5]. Moreover, it had been for clinical use in the first affiliated hospital of Heilongjiang University of Chinese Medicine, China [6, 7]. Our research group had done some work on its pharmacological actions for psoriasis and extraction and isolation of active constituents [5–8]. As a part of a continuing project to study the active constituents of Flos Daturae against psoriasis [3–5], we investigated 50% ethanol eluate fraction of macroporous resin for the flowers of D. metel L., which resulted in the discovery of two novel ergostane derivatives (Figure 1).
Naturally occurring withanolides are not widely distributed in the plant kingdom and isolated mainly from flowers, leaves, and seeds of Solanaceae plants [9–11]. Most of the withanolides may be subdivided into two subgroups: those with an unmodified skeleton (δ-lactone or δ-lactol side chain) and a modified skeleton (γ-lactone or γ-lactol side chain) [12, 13]. In this paper, we present the isolation and structural characterization of the two novel naturally occurring norwithasteroids on the basis of the interpretation of spectral data, including 1H-NMR, 13C-NMR, DEPT, 1H-1H-COSY, HSQC, HMBC, NOESY, and HR-ESI-MS. According to the literatures, this study represents the first report of naturally occurring norwithasteroids in the side chain with unusual six- and seven-membered ether rings formed rather than those with an unmodified skeleton (δ-lactone or δ-lactol side chain) and a modified skeleton (γ-lactone or γ-lactol side chain) in the family of withanolides. Meanwhile, compounds 1 and 2 were evaluated for their immunosuppressive activity against mice splenocyte proliferation in vitro.
2. Materials and Methods
IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer. NMR spectra were recorded on a Bruker DPX 400 NMR instrument (400 MHz for 1H NMR and 100 MHz for 13C NMR). Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as internal standard, and coupling constants are given in Hz. HR-ESI-MS were carried out using IonSpec Ultima 7.0 T FTICR. Preparative HPLC (Waters, Delta 600–2487) was performed on a Hypersil-ODS II (10 m, 20 × 300 mm, Yilite, Dalian, China).
2.2. Plant Material
The dry flowers of D. metel were collected in Nanjing city of Jiangsu province of China in September 2002, and identified by prof. Zhenyue Wang. A voucher specimen (number 2002035) is deposited at the Herbarium of Heilongjiang University of Chinese Medicine, China.
2.3. Extraction and Isolation
The dried flowers (30 kg) of D. metel L. were extracted with 70% EtOH under reflux (2 × 100 L) for 2.5 h (each), and the combined solution was filtered and evaporated under vacuum to a syrup, followed by suspension in H2O. The suspension was acidified with 0.1% HCl and then filtered and exchanged for Styrene-DVB (001 × 7). The exchanged solution was passed through AB-8 Crosslinked Polystyrene and sequentially eluted with H2O, 50% EtOH, and 95% EtOH, respectively. 50% EtOH elution was concentrated under vacuum to yield a syrup (52.0 g), and this crude residue was subjected to silica gel and eluted successively with CHCl3/MeOH (10 : 1→1 : 1) gradient to give 10 fractions (Fr. 1–10). Fr. 7 (5 g) continues silica gel chromatography eluted with CHCl3/MeOH (5 : 1→1 : 1) to afford a number of subfractions A1–A13. Compounds 1 (45 mg, min) and 2 (39 mg, min) were obtained by prep. HPLC chromatography of the sub-fraction A7 (0.5 g) with MeOH/H2O (2 : 3).
Baimantuoluoline I (1): white amorphous powder, = +12.3 (c = 0.1, MeOH). IR (KBr): = 3423, 2921, 1698, 1384, 1281, 1132, 1091, 992 cm−1. HR-ESI-MS (positive): m/z = 533.30958 (cal. for C30H45O8 533.31144, [M+H]+), 555.29130 (cal. for C30H44NaO8, 555.29339, [M+Na]+) and 571.26598 (cal. for C30H44KO8, 571.26733, [M+K]+). 1H and 13C-NMR: see Table 1.
Baimantuoluoside J (2): white amorphous powder, = +18 (c = 0.1, MeOH). IR (KBr): = 3407, 3323, 2940, 2917, 2855, 2578, 1675, 1068, 1033 cm−1. HR-ESI-MS (positive): m/z = 549.30349 (cal. for C30H45O9 549.30636, M+H]+), 571.28576 (cal. for C30H44NaO9, 571.28830, [M+Na]+) and 587.25940 (cal. for C30H44KO9, 587.26224, [M+K]+). 1H and 13C-NMR: see Table 1.
2.4. Con A-Induced Mouse Splenocyte Proliferation
Mouse splenocyte proliferation was assayed by MTT method as previously described . Splenocytes were seeded into a 96-well flat-bottom microtiter plate at 1 × 108 cells/mL in 100 μL of complete medium. Serial drug dilutions were prepared in medium immediately prior to each assay. Thereafter, 100 μL aliquots of serial dilution of each test compound were added (parallel triplicate wells were set), and then the cells were incubated in the absence or presence of Concanavalin A (Con A, final concn. 5 μg/mL) for 44 h in the humidified 5% CO2 incubator at 37°C. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) in phosphate buffered saline (PBS) at 5 mg/mL (10 μL) was added to each well, plates were incubated at 37°C for 4 h, and the formazan crystals formed were dissolved through addition of 100 μL of DMSO/well. The absorption of the samples was measured using an ELISA reader (Uniscan Titertec) at a wavelength of 570 nm. Cyclosporine was used for positive control. The immunosuppressive activity of each compound was expressed as the concentration that inhibited splenocyte proliferation to 50% (IC50) of the control value.
3. Results and Discussion
3.1. Structural Elucidation of Compound 1
Compound 1 was obtained as a white amorphous powder and showed negative results for the Molish reagent and positive results for the Liebermann-Burchard reaction, which indicated that there may be a triterpenoid or steroid aglycone. Its molecular formula was established as C30H44O8 by the positive HR-ESI-MS from [M+H]+ at m/z 533.30958 (cal.533.31144), [M+Na]+ at m/z 555.29130 (cal. 555.29339), and [M+K]+ at m/z 571.26598 (cal. 571.26733), indicating 9 degrees of unsaturation.
The 1H-NMR spectrum of 1 (Table 1) showed several characteristic signals in A-D rings of steroid skeleton. Two signals at δ 0.77 (3H, s) and 1.30 (3H, s) were attributed to Me-18 and Me-19, respectively. A Me-21 signal was missing and was replaced by two doublets at δ 3.47 (1H, br. t, J = 12.0 Hz) and 3.86 (1H, d, J = 12.0, 5.2 Hz), suggesting that C-21 was substituted by the oxygen-bearing group. Additional two methyl signals at δ1.33 (3H, s) and 1.37 (3H, d, J = 6.0 Hz) were undisputed to be assigned to the side chain. The vinylic hydrogen signals at δ 5.77 (1H, dd, J = 10.1, 2.4 Hz) and δ 6.65 (1H, ddd, J = 10.1, 5.1, 2.1 Hz) were attributed to H-2 and H-3 protons, respectively, in a steroidal Δ2-1-one system. The multiplicity of the H-2 and H-3 signals indicated that position C-4 was unsubstituted. A triplet resonating at δ 3.51 (1H, t, J = 2.2 Hz) was due to the oxygenated C-6 methine proton. The β-configuration of the hydroxyl at C-6 was established by a NOESY experiment. The NOESY spectrum showed the correlation between Me-19 and H-4β and between H-6 and H-4α, indicating that H-6 has an α-configuration. Besides, there is a double doublet at δ 3.44 (1H, dd, J = 10.8, 4.4 Hz) in 1H-NMR spectrum. Its coupling constants and splitting pattern were characteristic of 12β-hydroxywithanolide [3–5].
The 13C-NMR spectrum of 1 showed resonances for all 30 carbons (Table 1). The carbon signals from C-1 to C-21 were easily assigned according to 2D-NMR and the comparison of similar substituted patterns of withanolides from the A ring to the D ring. The characteristic downfield signals at δ 207.3 were due to ketone carbonyl, along with the characteristic doublets at δ 128.9 and 144.0 for the vinylic carbons at C-2 and C-3, respectively, in the ring A. The signals appearing at δ16.2 and 9.2 were assigned to the Me-19 and Me-18, respectively. The typical signals at δ78.3, 75.2, 79.8.0, and 65.5 were assigned to the oxygenated carbons at C-5, C-6, C-12, and C-21, respectively. The chemical shifts of the C-5 and C-6 further indicate the presence of a 5α, 6β-dihydroxyl steroid residue because their chemical shifts and multiplicity agreed with a similar substitution pattern . The downfield chemical shifts of C-21 suggested that there may be such a 21, 24 epoxy structure segment in the side chain. In that case, a tetrahydropyrane ring was proposed, including C-20 (δ 44.9), C-21 (δ65.5), C-22 (δ 68.3), C-23 (δ 40.8), and C-24 (δ 75.5). This tetrahydropyrane ring plays an important role in connection of steroid mother nucleus and the side chain. Further support this assumption was obtained that a serial of long-range correlations between H-21 [δ 3.47 (1H, br. t, J = 11.6 Hz) and 3.86 (1H, dd, J = 11.6, 5.2 Hz)] and C-17, C-20, C-22, and C-24, between H-22 [δ 3.89 (1H, dd, J = 10.4, 4.8 Hz)] and C-17, C-20, and C-24, and between H-23 [δ1.93 (1H, br. t, J = 12.8 Hz) and 1.71 (1H, dd, J = 12.8, 4.4 Hz)] and C-20, C-22, and C-24 (Figure 2).
Except for confirmed 24 carbon signals mentioned above, additional six carbon signals should be elucidated in the side chain, including δ178.2 (C), 75.8 (CH), 52.3 (CH), 33.6 (CH2), and 21.1 (CH3) assigned by DEPT spectrum. The characteristic downfield signals at δ 178.2 and 75.8 were due to free carboxyl group and oxygenated carbon signals, respectively. The signal appearing at δ21.1 was assigned to the two methyl carbon signals [δH, 1.33 (3H, s) and 1.37 (3H, d, J = 6.0 Hz)] according to its HSQC spectrum. By 1H-1H COSY spectrum, a structural segment was confirmed as the –CH [δ 2.92(1H, dd, J = 12.0, 8.8 Hz)]–CH2 [δ 2.41 and 1.83 (each, 1H, m)]–CH [δ 4.46 (1H, dt, J = 10.0, 5.8 Hz)]–O– residue (Figure 2). Taking into account the NMR spectral data and the 9 degrees of unsaturation calculated from the empirical formula of compound 1, it was suggested that it is possible to be presence of another ether ring except for an α, β-unsaturation carbonyl group, four rings of steroid skeleton, one tetrahydropyrane ring, and one carboxyl. In that case, another seven-membered-ether ring was proposed as shown in Figure 1. Further support this assumption was obtained that a serial of long-range correlations between H-25 [δ2.92 (1H, dd, J = 12.0, 8.8 Hz)] and C-24, C-26, C-27, and C-29, between H-27 [δ2.41 and 1.83 (each, 1H, m)] and C-24, C-25, C-26, and C-29, between Me-28 [δ1.33 (3H, s)] and C-23, C-24, and C-25, and between Me-30 [δ1.37 (3H, d, J = 6.0 Hz)] and C-29 and C-27. Thus, an oxepane ring moiety was determined in ring E, as shown in Figure 2. Therefore, the structure of 1 was deduced as 5α, 6β, 12β-trihydroxy-1-oxo-2-en-ergosta-21,24;22,29-diepoxy-26-carboxylic acid, which was named baimantuoluoline I.
3.2. Structural Elucidation of Compound 2
Compound 2 was obtained as a white amorphous powder and showed negative results for the Molish reagent and positive results for the Liebermann-Burchard reaction. Its molecular formula was established as C30H44O9 by the positive HR-ESI-MS from [M+H]+ at m/z 549.30349 (cal. 549.30636), [M+Na]+ at m/z 571.28576 (cal. 571.28830), and [M+K]+ at m/z 587.25940 (cal. 587.26224), indicating 9 degrees of unsaturation.
The 1H-NMR spectrum of 2 (Table 1) showed distinct resemblance to those of baimantuoluoside I. The only notable difference was the change of H-25 signal, which was missing. The 13C-NMR (DEPT) spectrum showed an additional downfield C-atom signal at δ 82.2 in 2 (Table 1), which was affirmatively assigned to the C-25, indicating that C-25 was substituted by a hydroxyl group. On the basis of previous data, the structure of 2 was identified to be 5α, 6β, 12β, 25-tetrahydroxy-1-oxo-2-en- ergosta-21,24;22,29-diepoxy-26-carboxylic acid, which was named baimantuoluoline J.
3.3. Possible Biosynthetic Pathway to Proposed Structures
The biosynthetic pathway of common withanolides in plants is derived from phytosterol, which possibly experienced a sequential oxidation, hydroxylation, and cyclization in the side chains and finally formed the basic skeleton of withanolides (Figure 3(a)) . For compounds 1 and 2, they have two more carbon atoms than the common withanolide compounds in the side chain, which is never to be seen in other phytosterol compounds. A logical biosynthetic pathway of compounds 1 and 2 is postulated as shown in Figure 3(b). The compounds 1 and 2 may be derived from the ordinary phytosterol compounds as precursors that firstly experience hydroxylation and oxidation and along with an acetate-malonate (AA-MA) pathway resulting in the side chains increase two carbon atoms and finally involve a complex process such as reduction, cyclization, and oxidation to form this kind of unusual six- and seven-membered ether rings in side chain. This type of biosynthetic pathway is rarely seen, and detailed evidences also need to be further confirmed.
3.4. Effect of Compounds on the Splenocyte Proliferation of Mouse
The colorimetric assay using MTT for cell proliferation was then carried out to evaluate Con A-stimulated mouse splenocyte in the presence of various concentrations of 1 and 2. The results of this assay showed that compounds 1 and 2 possess the obvious immunosuppressive activity with the IC50 values of 10.2 nM and 13.5 nM (Table 2), respectively. However, their activities were much lower than that of the positive medicine cyclosporine. Compounds 1 and 2 did not show cytotoxicity in the range of tested concentrations, indicating that the immunosuppressive effects observed in vitro may be not due to the toxicity of compounds.
Over the period of August 1996 to March 2010, 360 new naturally occurring withanolides were isolated largely from the flowers, leaves, and seeds of Solanaceae plants such as Withania somnifera, W. cagalans, W. aristata, Physalis pubescens, P. peruviana, P. minima, D. ferox, D. fastuosa, D. inoxia, D. metel, and D. stramoniam, [9–11]. The characteristic feature of their skeleton of withanolides can be summarized as the two subgroups: those with an unmodified skeleton (δ-lactone or δ-lactol side chain) and a modified skeleton (γ-lactone or γ-lactol side chain) , which have been shown to be associated with many biological activities including cytotoxic, anti-inflammatory, antioxidant, antiarthritic, anticholinesterase, immunoprotective, trypanocidal, antimalarial, leishmanicidal, and diuretic .
However, till now, no studies on ergostane derivatives with unusual six- and seven-membered ether rings formed in the side chain have been found in the family of Solanaceae. At the same time, these two naturally occurring norwithasteroids have been found to possess immunosuppressive activity by mice splenocyte proliferation in vitro. It is worthy of mentioning that tropane alkaloids have for a long time been considered as characteristic ingredients of Flos Daturae . This finding revealed another kind of biological constituents of unusual norwithasteroids in Flos Daturae, enlarged our understanding of Flos Daturae against psoriasis, and was possible to uncover the real material foundation of Flos Daturae against psoriasis.
|DEPT:||Distortionless enhancement by polarization transfer;|
|HSQC:||1H detected heteronuclear single quantum coherence|
|HMBC:||1H detected heteronuclear multiple bond correlation|
|NOESY:||Nuclear overhauser effect spectroscopy|
B.-Y. Yang, Y.-G. Xia contributed equally to this work.
Our work was supported by the Major State Basic Research Development Program of China (973 Program 2006CB504708), the National Natural Science Foundation of China (no. 30371736, 30672633), Special Fund Project of National Excellent Doctoral Dissertation of China (200980), Heilongjiang Excellent Young Foundation (JC201105), Chang Jiang Scholar Candidates and New Century Excellent Talents Programme for Provincial Universities in Heilongjiang, Heilongjiang Province Postdoctoral Special Funding, and Innovative Talents Funding of Heilongjiang University of Chinese Medicine.
State Pharmacopoeia Committee, Pharmacopoeia of People’s Republic of China, 2010.
Jiangsu New Medical college, Zhong Yao Da Ci Dian, vol. 2, Shanghai Science and Technology Press, Shanghai, China, 1999.
H. Kuang, B. Yang, L. Tang, Y. Xia, and D. Dou, “Baimantuoluosides A-C, three new withanolide glucosides from the flower of Datura metel L,” Helvetica Chimica Acta, vol. 92, no. 7, pp. 1315–1323, 2009.View at: Publisher Site | Google Scholar
B. Yang, Q. Wang, Y. Xia, W. Feng, and H. Kuang, “Baimantuoluolines D-F, three new withanolides from the flower of Datura metel L,” Helvetica Chimica Acta, vol. 91, no. 5, pp. 964–971, 2008.View at: Publisher Site | Google Scholar
B. Y. Yang, Y. G. Xia, Q. H. Wang, D. Q. Dou, and H. X. Kuang, “Baimantuoluosides D-G, four new withanolide glucosides from the flower of Datura metel L,” Archives of Pharmacal Research, vol. 33, no. 8, pp. 1143–1148, 2010.View at: Publisher Site | Google Scholar
Q. H. Wang, H. B. Xiao, B. Y. Yang, F. Y. Yao, and H. X. Kuang, “Studies on pharmacological actions of the effective parts for psoriasis in Flos Daturae (I),” China Journal Experimental Traditional Medical Formulae, vol. 14, no. 7, pp. 48–51, 2008 (Chinese).View at: Google Scholar
Y. X. Wang, “the report on Traditional Chinese medicine yangjinhua (Datura metel) is given priority to treat 242 patients with psoriasis,” Journal of Traditional Chinese Medicine, vol. 35, no. 2, pp. 32–33, 1985 (Chinese).View at: Google Scholar
H. X. Kuang, B. Y. Yang, Y. G. Xia, and Q. H. Wang, “Two new withanolide lactones from flos daturae,” Molecules, vol. 16, no. 7, pp. 5833–5839, 2011.View at: Publisher Site | Google Scholar
E. Glotter, I. Kirson, A. Abraham, and D. Lavie, “Constituents of Withania somnifera Dun—13. The withanolides of chemotype III,” Tetrahedron, vol. 29, no. 10, pp. 1353–1364, 1973.View at: Publisher Site | Google Scholar
Y. Pan, X. Wang, and X. Hu, “Cytotoxic withanolides from the flowers of Datura metel,” Journal of Natural Products, vol. 70, no. 7, pp. 1127–1132, 2007.View at: Publisher Site | Google Scholar
D. Benjumea, D. Martín-Herrera, S. Abdala et al., “Withanolides from Whitania aristata and their diuretic activity,” Journal of Ethnopharmacology, vol. 123, no. 2, pp. 351–355, 2009.View at: Publisher Site | Google Scholar
L. X. Chen, H. He, and F. Qiu, “Natural withanolides: an overview,” Natural Product Reports, vol. 28, no. 4, pp. 705–740, 2011.View at: Publisher Site | Google Scholar
M. H. Mirjalili, E. Moyano, M. Bonfill, R. M. Cusido, and J. Palazón, “Steroidal lactones from Withania somnifera, an ancient plant for novel medicine,” Molecules, vol. 14, no. 7, pp. 2373–2393, 2009.View at: Publisher Site | Google Scholar
H. Kuang, Y. Xia, B. Yang, Q. Wang, and Y. Wang, “Screening and comparison of the immunosuppressive activities of polysaccharides from the stems of Ephedra sinica Stapf,” Carbohydrate Polymers, vol. 83, no. 2, pp. 787–795, 2011.View at: Publisher Site | Google Scholar