Withanolides are a group of modified C28 ergostane-type steroids with a C-22, C-26 δ-lactone side chain. These compounds are structurally diverse and distributed mainly in the genera Withania, Physalis, Datura, Nicandra, Dunalia, Lycium, Tubocapsicum and Jaborosa of the family Solanaceae . Withanolides have been considered to be of great medicinal value since they display significant pharmacological activities such as anti-inflammatory , antitumor [3-4], immunosuppres-sive  and antioxidant properties . Physalis minima L., known in China as 'xiaosuanjiang', is an annual herb and widely distributed in China. It has been used in Chinese folk medicine as tonic, diuretic, laxative, applied in inflammations, enlargement of the spleen, ascites, and as a helpful remedy in ulceration of the bladder [6-7]. In our previous study, withaphysalin A and 2, 3-dihydro-withaphysalin C, two major compounds with strong anti-inflammatory activities were isolated from this plant, which made a clarification of bioac-tive material basis of P. minima for inflammation treatment . In order to find new withanolides with stronger biological activity, the remaining anti-inflammatory fractions of title plant were re-investigated. Consequently, six new withaphy-salin-type withanolides (1-6) were discovered. Herein, the isolation, structure elucidation, and bioactivities evaluation of these compounds were reported.Results and Discussion
Withaminima A (1) was isolated as amorphous solid with the molecular formula of C27H34O5 by its HR-ESI-MS (m/z 439.247 6 [M + H]+, calculated for 439.247 9) and 1D NMR data. The UV absorption maximum at 224 nm and strong IR absorption at 3435 and 1707 cm-1 indicated the presence of hydroxyl and α, β-unsaturated ketone groups . The NMR spectroscopic data (Tables 1 and 2) of 1 showed four charac-teristic methyl singlets at δH 1.20 (s, 3H), 1.38 (s, 3H), 1.90 (s, 3H) and 1.95 (s, 3H), which were assigned to H3-19, H3-21, H3-27 and H3-28 , respectively. A double doublet at δH 4.83 (dd, J = 13.2, 3.0 Hz, H-22) and a set carbon signals (δc 166.0, 122.9 and 147.5) with two methyl singlets (H3-27, H3-28) above mentioned suggested the presence of a typical α, β-unsaturated-δ-lactone of withanolide skeleton in the side chain . The vinylic proton signals at δH 5.86 (1H, dd, J = 10.0, 2.4 Hz) and δH 6.77 (1H, ddd, J = 10.0, 4.9, 2.4 Hz) indicated an α, β-unsaturated ketone in ring A as observed in many withanolides . Another olefinic proton at δH 5.60 (1H, dt, J = 4.9, 2.5 Hz) along with two olefinic carbons at δc 134.3 and 125.4 in the 13C NMR spectrum correspond to one double bond linked at C-5/6 [9-10]. In addition to these signals, the 1H NMR spectrum displayed a proton signal at δH 3.84 (1H, m) linked at C-13 (δc 59.6), in conjunction with the 13C NMR spectroscopic data showed 27 carbons, led to the conclusion that compound 1 was a C-18 norwithaphysalin-type withanolide . The chemical shifts of C-12 (δc 105.1) and C-20 (δc 84.1) in the 13C NMR spectrum and the hydroxyl group signal showed by characteristic UV spectrum indicated that 1 contained a 12, 20-γ-lactol moiety . Above deductions were confirmed by the key HMBC correlations from H-13 (δH 3.84) to C-12 (δc 105.1), C-15 (δc 21.6) and C-17 (δc 42.4), and from H3-21 (δH 1.38) to C-17 and C-20 (δc 84.1), as shown in Fig. 2. Thus, the planar structure of 1, C-18 norwithaphysalin-type withanolide featuring a rare 12, 20-γ-lactol moiety, was elucidated.
The relative stereochemistry of 1 was established on the basis of relevant correlations observed in its ROESY spectrum (Fig. 2). The ROESY cross-peaks from H-8 to H3-19 and H-9 to H-13were observed which could deduce the configuration of C-8 and C-9. The correlations from H3-21 to H-13, H-14, and H-22, from H-17 to H-13 and H-22 revealed that these protons were adopted as α configuration and the C-12, C-20 oxygen ether bridge was β-orientation [11-12]. The configuration of C-22 was determined in the ECD spectrum by its positive Cotton effect at 250 nm based on the n→π* transition of an α, β-unsaturated δ-lactone having a 22R configuration [12-13]. Accordingly, the structure of 1 was established finally and named withaminima A.
The molecular formula of compound 2 was determined as C28H34O8 by HR-ESI-MS (m/z 533.1951 [M + Cl-, calculated for 533.1948). The 1H and 13C NMR data of 2 indicated that it was a withaphysalin-type withanolide contained an α, β-unsaturated ketone in ring A, a 5-en moiety and an α, β-unsaturated-δ-lactone side chain . The obvious HMBC correlations (Fig. 3) from H3-21 (δH 1.50) to C-17 (δc 57.7), from H-12 (δH 2.32) to C-18 (δc 178.6) indicated that compound 2 contained the characteristic 18, 20-γ-lactone moiety . Additionally, a carbonyl carbon signal at δc 217.1 (C-14), along with the HMBC correlations from H-16 (δH 2.60) to C-14, and from H-17 (δH 2.78) to C-12 (δc 28.2) and C-15 (δc 69.7) revealed that 2 was a C13/14 seco-with aphysalin and C-14 was existed as ketone and a hydroxyl was located at C-13 (δc 77.4) [9, 14]. In its ROESY spectrum, the correlations of H3-19 (β-orientation) with H-8, of H3-21 (α-orientation) with H-17, and of H-15 with H-17 and H-9 suggested the trans fusing of B/C rings and the cis junction of C (D) /18, 20-γ-lactone ring . The α-orientation of 13-OH were deduced by the cis junctions of C (D) /18, 20-γ-lactone ring and the biogenetic arguments . Correlations between H-17 and H-15, H3-21 suggested the β-orientation of 15-OH. The presence of the positive Cotton effect at 250 nm in its ECD spectrum indicated the R configuration at C-22 . Thus, compound 2 was established as an (22R) C13/14 seco-with aphysalin and named withaminima B.
Compound 3 was obtained as amorphous solid with a molecular ion at m/z 547.1742 [M + Cl]- in the HR-ESI-MS, corresponding to an molecular formula of C28H32O9. The characteristic signals at δc 77.4 (C-13), δc 206.7 (C-14) and δc 178.6 (C-18) indicated that 3 possessed the same 13, 14-seco-withaphysalin skeleton as 2 . Comparison of its 1H and 13C NMR data (Table 1 and 2) with those of 2 revealed that the 5-en of 2 was replaced by 5, 6-epoxy unit in 3 (δH 3.15 (d, J = 5.5 Hz, H-6); δc 62.5, C-5; δc 60.2, C-6) . And the C-15 carbonyl moiety (δc 202.6) in 3 was deduced by the upfield shifts of the C-14 from δc 217.1 in 2 to 206.7 and the key HMBC correlations from H-16 (δH 3.56) and H-17 (δH 3.04) to C-15 (δc 202.6). The relative configuration of the 5, 6-epoxy moiety in 3 was assigned as β-orientation by the ROESY correlations between H-4α (δH 1.93) with H-6. The absolute configuration of C-22 was established as R according to its positive Cotton effect at 255 nm in the ECD spectrum . Thus, the structure of 3 was finally determined and named as withaminima C.
Withaminimas D-F (4-6) were also isolated as amorphous solid. The similar molecular formulas and 1D NMR features (Tables 1 and 2) suggested that they are typical C/D rings intact withaphysalin homologues with α, β-unsaturated ketone in ring A, 5-en moiety in ring B, and 18, 20-γ-lactone ring . The main difference between these three was the number and location of hydroxyl moieties. For compound 4, the presence of the oxygenated methylene proton signals at δH 4.38 (each 1H, d, J = 12.6 Hz) and its HMBC correlations with C-24 (δc 151.2), C-25 (δc 126.5), and δc C-26 (165.3) suggested that a hydroxymethyl group (CH2OH-28) linked at C-25. Compared the NMR data of 4 and 5, the downfield shifts of C-14 from δc 55.6 to δc 83.6 indicated the presence of a hydroxy moiety at C-14 in 5 [16-18]. The obvious HMBC correlations from two oxygenated methylene signals (δH 4.38) to C-24 (δc 151.9), C-25 (126.1), and C-26 (165.0) implied a hydroxymethyl group linked at C-25 in 5 as that of 4. For compound 6, these similar HMBC correlations were observed to C-24 (δc 151.3), C-25 (δc 121.7), and C-23 (δc 26.2), which implied the hydroxymethyl group (CH2OH-27) linked at C-24 rather than at C-25 in 5. The α-orientation of 14-OH in 5/6 were determined by the identical 1D NMR data with reported 14-hydroxywithaphysalins [2, 10]. The ECD spectra of 4-6 also showed a positive Cotton effect near 254 nm, indicating the 22R configuration of 4-6 . Therefore, their structures were determined as shown in Fig. 1 and named as withaminimas D-F, respectively.
According to our and other previous researches, the crude methanol extract and chloroform fraction of the aerial parts of P. minima showed marked anti-inflammatory in mice [6, 8]. Thus, these six new withaphysalin-type withanolides were screened to demonstrate their anti-inflammatory activities by testing inhibitory effects of NO production on lipopolysaccharide-activated RAW264.7 macrophages . MTT assay suggested that no significant cytotoxic effects (over 90% cell survival) at concentrations up to 100 μmol·L-1. NG-monomethyl L-arginine (L-NMMA) was used as a positive control and had an IC50 value of 39.20 μmol·L-1. Based on the results summarized in table 3, compounds 1-4 effectively inhibited the NO production with IC50 values range from 4-20 μmol·L-1, and 5/6 demonstrated equivalent inhibitory to positive drug. These results further proved that the withanolides in P. minima would be responsible for its anti-inflammatory effect and traditional usages .
UV spectra were recorded with a Shimadzu UV-2501 PC spectrophotometer. IR data were measured with a Bruker Tensor 27 spectrometer. Optical rotations were determined on a JASCO P-1020 polarimeter. 1D and 2D NMR spectra were performed on a Bruker Avance Ⅲ NMR spectrometer at 500 MHz (1H) and 125 MHz (13C), using TMS as the internal standard. HRESI mass spectra were recorded on an Agilent 6520B UPLC-Q-TOF mass spectrometer. ECD spectra were recorded on a JASCO 810 spectropolarimeter. The HPLC analysis was run on an Agilent 1200 instrument equipped with multiple wavelength diode array detector (DAD). Preparative HPLC was proformed on a Agilent 1100 instrument equipped with a shim-pack RP-C18 column (20 mm × 250 mm, 10 μm), the flow rate was 10.0 mL·min-1. Column chromatography (CC) was carried out using macroporous resin D-101 (pore size B 13-14 nm, 26-60 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Silica gel (100-200 mesh and 200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and ODS RP-C18 (40-63 μm, FuJi, Japan).Plant material
The plant of Physalis minima was obtained from Linyi City (Shandong Province, China) in August 2014, which was identified by Professor ZHANG Mian of the Research Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. PM-201408-LY) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.Extraction and isolation
The extraction and fraction using D-101 macroporous resin were as same as previous description . Fr. C (21 g) was subjected to silica gel with increasing polarities of CH2Cl2-MeOH solvent system (40 : 1, 20 : 1, 10 : 1, 0 : 100 V/V) to obtain four subfractions (Frs. C1-C4). Fr. C1 was applied to ODS MPLC eluted with MeOH-H2O (40 : 60 to 65 : 35, V/V) to afford four subfractions (Frs. C1A-D). Fr. C1A was applied to ODS MPLC eluted with isocratic MeOH-H2O (40 : 60, V/V) to afford three subfractions (Frs. C1A1-3). Fr. C1A1 was separated by repeated preparative HPLC (MeOH-H2O, 50 : 50, V/V) to afford 3 (17 mg). Fr. C1A2 was separated by repeated preparative HPLC (MeOH-H2O, 50 : 50, V/V) to afford 2 (8.0 mg), 5 (2.3 mg), 6 (4.3 mg). Part of Fr. C1B was chromatography over ODS with MeOH-H2O (45 : 55, V/V) to give two subfractions (Frs. C1B1-2). Fr. C1B1 was applied to preparative HPLC with MeOH-H2O (50 : 50, V/V) to yield 4 (4.7 mg). Fr. C1D was purified by silica gel to afford 1 (14.1 mg).withaminima A (1)
White amorphous solid; [α]D22 +98.3 (c 0.12, MeOH); CD Δε250 +8.45, Δε337 -4.68 (MeOH; c 0.3); UV (MeOH) λmax (log ε) 224 (4.78); IR (KBr) νmax 3435, 2925, 1707, 1666, 1382, 1127 cm-1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 2; HR-ESI-MS m/z 439.2476 [M + H]+ (Calcd. for C27H35O5, 439.2479).withaminima B (2)
White amorphous solid; [α]D22 +29.3 (c 0.09, MeOH); CD Δε250 +4.19, Δε337 -2.09 (MeOH; c 0.3); UV (MeOH) λmax (log ε) 225 (4.86); IR (KBr) νmax 3436, 2959, 2922, 1771, 1710, 1459, 1379, 1136 cm-1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 2; HR-ESI-MS m/z 533.1951 [M + Cl]- (Calcd. for C28H34ClO8, 533.1948).withaminima C (3)
White amorphous solid; [α]D22 +53.0 (c 0.14, MeOH); CD Δε255 +3.48, Δε338 -0.65 (MeOH; c 0.3); UV (MeOH) λmax (log ε) 225 (4.90); IR (KBr) νmax 3444, 2924, 1773, 1711, 1458, 1383, 1137 cm-1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 2; HR-ESI-MS m/z 547.1742 [M + Cl]- (Calcd. for C28H32ClO9, 547.174).withaminima D (4)
White amorphous solid; [α]D22 +50.5 (c 0.11, MeOH); CD Δε253 +4.24, Δε311 -1.29 (MeOH; c 0.3); UV (MeOH) λmax (log ε) 221 (4.73); IR (KBr) νmax 3439, 2923, 2868, 1755, 1709, 1664, 1453, 1384, 1110 cm-1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 2; HR-ESI-MS m/z 501.2044 [M + Cl]- (Calcd. for C28H34ClO6, 501.2049).withaminima E (5)
White amorphous solid; [α]D22 +38.0 (c 0.10, MeOH); CD Δε252 +5.74, Δε337 -3.52 (MeOH; c 0.3); UV (MeOH) λmax (log ε) 220 (4.73); IR (KBr) νmax 3430, 2923, 1752, 1701, 1385, 1127cm-1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 2; HR-ESI-MS m/z 517.2001 [M + Cl]- (Calcd. for C28H34ClO7, 517.1999).withaminima F (6)
White amorphous solid; [α]D22 +28.7 (c 0.14, MeOH); CD Δε254 +2.22, Δε327 -0.78 (MeOH; c 0.3); UV (MeOH) λmax (log ε) 222 (4.31); IR (KBr) νmax 3439, 2925, 1752, 1714, 1453, 1382, 1122 cm-1; 1H NMR (CDCl3, 500 MHz) see Table 1; 13C NMR (CDCl3, 125 MHz) see Table 2; HR-ESI-MS m/z 517.1998 [M + Cl]- (Calcd. for C28H34ClO7, 517.1999).NO production bioassay
The protocol for NO production bioassays was provided in vitro as described previously, and NG-Monomethyl-L-argi-nine (L-NMMA) was used as the positive control . All experiments were carried out in triplicate.
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