2013 Volume 61 Issue 8 Pages 873-876
Two new compounds with five known compounds have been isolated from EtOH extract of the seeds of Nigella glandulifera. On the basis of their spectroscopic and chemical evidence, the new compounds were elucidated as methoxynigeglanine (1) and 6-methoxythymol-3-O-β-D-glucopyranoside (4). Compounds 1–4 showed moderate antitubercular activity against Mycobacterium tuberculosis strain H37Rv with minimal inhibitory concentration (MIC) values of 32–250 µg/mL.
Tuberculosis (TB) remains an important public health problem worldwide, the latest estimates are that there were almost 9 million new cases in 2011 and 1.4 million TB deaths.1) The spread of drug resistant TB and emergence of multidrug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant TB poses a real threat to global TB control.2,3) Therefore, the need for new antitubercular agents is urgent, especially those from natural products. As part of our ongoing search for antitubercular natural compounds from Traditional Chinese Medicine (TCM),4–6) potent antitubercular activity was shown by the EtOH extract of the Seeds of Nigella glandulifera which are commonly eaten in many food preparations by the Uigur people in West China. The seeds of N. glandulifera are believed to have diuretic, analgesic, spasmolytic, galactagogue, bronchodilator properties, and to cure edema, urinary calculus, and bronchial asthma.7,8) Bioassay-guided investigation of the EtOH extract of the seeds of N. glandulifera led to the isolation of two new compounds (1, 4), with five known compounds (2, 3, 5, 6, 7) (Fig. 1). The isolation and structure elucidation of the compounds and an assessment of their in vitro antitubercular activity are described herein.
Of the seven compounds elucidated, the five known compounds were subsequently identified as methoxynigellidine (2),9) fuzitine (3),7) α-hederin (5),10) 3-O-β-[D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinpyranosyl]-hederagenin (6)11) and 3-O-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinpyranosyl]-28-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-hederagenin (7)12) (Fig. 1), by comparisons of their spectral data (NMR and MS) with those reported in the literature. The purity of these compounds was confirmed by TLC and HPLC (purity >95% for all compounds).
Compound 1 was obtained as an brown amorphous powder with a molecular formula of C13H18N2O2 as found by high resolution-electron ionization (HR-EI)-MS (m/z 234.1345 [M]+). The 13C-NMR experiment combined with analysis of the heteronuclear multiple quantum coherence (HMQC) spectrum permitted differentiation of the 13 resonances into four quaternary carbons, three methines, four methylenes and two methyls. The 1H-NMR spectrum of compound 1 displayed two three-proton singlets at δH 2.43 and 3.71 indicating the presence of Ar–CH3 and OCH3 groups respectively, the 13C-NMR spectrum showed resonances at δC 22.1 and 56.0 attributable to the methyl and methoxy carbons. Four methylene carbons, which had chemical shifts of δC 51.5, 46.0, 19.8 and 19.5, were assigned to C-13, C-10, C-11 and C-12, respectively, assignments were confirmed by distortionless enhancement by polarization transfer (DEPT), 1H–1H double quantum filtered-correlation spectroscopy (DQF-COSY), HMQC and heteronuclear multiple bond coherence (HMBC). The 13C-NMR resonances of indazole nucleus were confirmed by HMQC and HMBC correlations (Table 1).
| Position | δH (J in Hz) | δC | HMBC |
|---|---|---|---|
| 2 | 9.07 (1H, s) | 129.8 | C-3, 4, 8, 13 |
| 3 | 112.3 | ||
| 4 | 154.5 | ||
| 5 | 6.45 (1H, d, 2.5) | 104.6 | C-3, 4, 6, 7, 6-CH3 |
| 6 | 147.0 | ||
| 7 | 7.02 (1H, d, 2.5) | 99.3 | C-3, 5, 6, 8, 6-CH3 |
| 8 | 3.50 (1H, m) | 143.2 | |
| 10 | 4.39 (2H, m) | 46.0 | C-8, 11, 12 |
| 11 | 2.14 (2H, m) | 19.8 | C-10, 12,13 |
| 12 | 2.19 (2H, m) | 19.5 | C-10, 11, 13 |
| 13 | 4.60 (1H, m) | 51.5 | C-2, 11, 12 |
| 4-OCH3 | 3.71 (3H, s) | 56.0 | C-4 |
| 6-CH3 | 2.43 (3H, s) | 22.1 | C-5, 6, 7 |
The 13C-NMR data were compared with those of a known compound nigeglanine hydroxide,7) indicating that the 13C-NMR data of the compound 1 were very similar to those of nigeglanine hydroxide, except one more methoxy carbon in 1; correlations could be achieved between δH 3.71 (3H, s OCH3) and δC 154.5 (C-4) by analyzing HMBC spectrum experiment as shown in Table 1. These suggested the methoxyl should be linked at C-4. All above analysis showed compound 1 to be methoxynigeglanine.
Liu et al.7) have reported that nigeglanine hydroxide came from nigeglanine by heating of a water bath at 60°C overnight in the solvent (mixture of CHCl3–MeOH), it is likely that the trace HCl in the solvent (CHCl3) catalyzed the reaction. Ali et al.13) got 4-O-sulfite of nigellidine from seeds of N. sativa, they presumed that 4-O-sulfite of nigellidine may represent the true natural product leading to nigellidine via hydrolysis of the sulfate functionality during the isolation process. However we didn’t get nigeglanine and nigeglanine hydroxide from the seeds of N. glandulifera, more surprising, methoxynigeglanine could be detected in EtOH extracts of seeds by HPLC method (tR 20.3 min). In addition, we also got another methide of nigellidine (tR 14.7 min) from seeds of N. glandulifera, we therefore suppose that methide of indazole may represent a more stable state during postharvest storage.
Compound 4 was obtained as white amorphous powder. The molecular formula was established as C17H26O7 by HR-electron spray ionization (ESI)-MS analysis (365.1547 [M+Na]+), the positive ESI-MS gave a fragment at m/z 181.4 ([M+H−162]+), besides a quasi-molecular ion at m/z 343.4 ([M+H]+), indicating the potential presence of one hexose unit.
The 1H-NMR spectrum of compound 4 (Table 2) shows signals of four methyl group at δH 3.77 (3H, s, H-11), 2.13 (3H, s, H-7), 1.19 (3H, d, J=7.0 Hz, H-10), 1.18 (3H, d, J=7.0 Hz, H-9), and two aromatic protons at δH 6.96 (1H, s, H-2), 6.69 (1H, s, H-5), and a proton at δH 4.72 (1H, d, J=7.5 Hz, H-1′) indicating its β-linkage.
| Position | δH (J in Hz) | δC | HMBC |
|---|---|---|---|
| 1 | 124.5 | ||
| 2 | 6.96 (1H, s) | 119.3 | C-1, 3, 4, 6, 7 |
| 3 | 148.8 | ||
| 4 | 137.0 | ||
| 5 | 6.69 (1H, s) | 108.1 | C-1, 3, 4, 6, 8 |
| 6 | 154.8 | ||
| 7 | 2.13 (3H, s) | 15.1 | C-1, 2, 6 |
| 8 | 3.51 (1H, m) | 26.4 | C-3, 4, 5, 9, 10 |
| 9 | 1.18 (3H, d, 7.0) | 22.6 | C-4, 8, 10 |
| 10 | 1.19 (3H, d, 7.0) | 22.7 | C-4, 9, 10 |
| 11 | 3.77 (3H, s) | 55.3 | C-6 |
| Glc-1′ | 4.72 (1H, d, 7.5) | 103.3 | C-3 |
| 2′ | 3.44 (1H, t, 7.5) | 74.2 | |
| 3′ | 3.35 (1H, t, 7.5) | 77.4 | |
| 4′ | 3.37 (1H, t, 7.5) | 70.6 | |
| 5′ | 3.42 (1H, q, 7.5) | 77.1 | |
| 6′ | 3.85 (1H, dd, 12.5, 7.5) | 61.7 | |
| 3.69 (1H, dd, 12.5, 7.5) |
The 13C-NMR spectrum (Table 2) combined with HMQC spectrum exhibits the signals for 17 carbons: four methyls, and one methine, six aromatic carbons, along with 6 carbons of sugar, All 1H- and 13C-NMR signals were assigned by DEPT, 1H–1H DQF-COSY, HMQC and HMBC. HMBC and HMQC correlations (Table 2) indicated the aglycone of 4 was 6-methoxythymol.
The hexose was suggested to be a D-glucose by the comparison of data with those reported in the literature,4) and confirmed by GC analysis after the acid hydrolysis and preparation of its thiazolidine derivative.14) The HMBC correlation achieved between the anomeric proton H-1′ and C-3 suggested that C-3 of 4 should be glycosylated.
Thus, the structure of 4 was deduced as 6-methoxythymol-3-O-β-D-glucopyranoside.
The seven compounds were evaluated for their antitubercular activity against M. tuberculosis H37Rv in vitro. The clinically used antimycobacterial agents ethambutol and isoniazide were used as positive controls. As shown in Table 3, compounds 1–4 had minimal inhibitory concentration (MIC) values of 32–250 µg/mL, however compounds 5–7 lack antitubercular activity.
| Compounds | MIC (µg/mL) |
|---|---|
| 1 | 32 |
| 2 | 250 |
| 3 | 250 |
| 4 | 62.5 |
| 5 | >1000 |
| 6 | >1000 |
| 7 | >1000 |
| Ethambutola) | 6.25 |
| Isoniazidea) | 0.062 |
a) Ethambutol and Isoniazide were used as positive control.
Among the indazole analogs (1–3), 2 and 3 (with 2-aromatic substitution) exhibited lower antimycobacterial activity than 1 against M. tuberculosis H37Rv in vitro, indicated that indazole of 2-aromatic substitution may be reduced antimycobacterial activity.
HR-EI-MS was obtained on an Autospec-Ultima ETOF spectrometer, HR-ESI-MS and ESI-MS spectra were taken on a Bruker Daltonics Apex III mass spectrometer. All NMR spectra were recorded on a Bruker ARX-500 and ARX-125 MHz NMR spectrometer equipped with a CH dual 5µ probe. Samples were dissolved in 0.6 mL CD3OD, DMSO-d6 or pyridine-d5 and transferred into a 5 mm NMR tube. All chemical shifts are expressed as δ (ppm) relative to the internal standard TMS (δ=0 ppm), and scalar coupling constants are reported in Hz. 1H–1H DQF-COSY, DEPT, HMQC and HMBC spectra were recorded using conventional pulse sequences. GC-MS was performed using a Shimadzu QP5050A instrument. HPLC analyses were performed on a Waters 2695 series HPLC system together with column compartment and waters 2998 series photodiode array detector (PDA). HPLC-grade acetonitrile was purchased from Fisher Chemicals (Fair Lawn, NJ, U.S.A.). Thin-layer chromatography was performed with silica gel GF254 pre-coated plates (Qingdao Haiyang), silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., China), D101 macroporous resin (Chemical Plant of Nankai University, Tientsin, China), Sephadex LH-20 (Amersham Pharmacia Biotech) and octadecylsilyl (ODS) (35–50 µm, Alltech) were used for column chromatography. The water used in the experiment was doubly distilled in the laboratory. Other chemicals and solvents were of analytical grade.
Plant MaterialThe seeds of Nigella glandulifera FREYN et SINT. were collected from Ürümuqi in Xinjiang Uigur autonomy, China, in Octber 2010, and identified by one of the authors (Lin Zhang). A voucher specimen was deposited in the Herbarium of the College of Biomedical Engineering and Instrument Sciences, Zhejiang University, P. R. China.
Extraction and IsolationThe dried seeds materials (3.5 kg) were refluxed with petroleum ether and 50% EtOH twice respectively, the EtOH extracts were combined, then concentrated. The concentrates were submitted to D101 macroporous resin column chromatoraphy eluting successively with water, 30% EtOH and 70% EtOH. The 30% EtOH eluate (15.3 g) was chromatographed on silica gel column, eluting with a gradient of CHCl3–MeOH to give six fractions (Frs. A1–A6). Fr. A2 (368.0 mg) was purified by silica gel column [CHCl3–MeOH–H2O (80 : 20 : 2)] and Sephadex LH-20 column with MeOH–H2O (60 : 40) to give 1 (58.0 mg), Fr. A3 (1.2 g) was purified by silica gel column [CHCl3–MeOH–H2O (77 : 23 : 2)] and Sephadex LH-20 column with MeOH–H2O (60 : 40) to give 2 (77.0 mg), Fr. A5 (3.6 g) was purified by silica gel column [CHCl3–MeOH–H2O (70 : 30 : 5)] and Sephadex LH-20 column with MeOH–H2O (60 : 40) to give 3 (745.0 mg). 70% EtOH eluate (58.0 g) was chromatographed on silica gel column, eluting with a gradient of CH3Cl3–MeOH to give five fractions (Frs. B1–B5); Fr. B1 (3.8 g) was purified by repeated silica gel column [CH3Cl3–MeOH (85 : 15)] and Sephadex LH-20 column with MeOH to give 4 (228.0 mg); Fr. B3 (7.3 g) was purified by repeated silica gel column [CH3Cl3–MeOH–H2O (75 : 25 : 5)] and ODS column [MeOH–H2O (80 : 20)] to give 5 (225.0 mg) and 6 (710.0 mg); Fr. B5 (13.3 g) was purified by silica gel column [CH3Cl3–MeOH–H2O (65 : 35 : 7)] and repeated ODS [MeOH–H2O (68 : 32)] to give 7 (2.9 g).
Methoxynigeglanine (1): Brown amorphous powder. HR-EI-MS: m/z 234.1345 (Calcd for C13H18N2O2, 234.1368), EI-MS m/z(%): 234.1 [M]+ (23), 203.2 (52), 148.1 (5). 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) see Table 1.
6-Methoxythymol-3-O-β-D-glucopyranoside (4): White amorphous powder. Positive-ion HR-ESI-MS: m/z 365.1547 (Calcd for C17H26O7Na, 365.1576) [M+Na]+, positive-ion ESI-MS m/z 365.3 [M+Na]+, 343.4 [M+H]+, 181.4 [M+H−162]+; 1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD) see Table 2.
Enzymatic Hydrolysis and Determination of the Absolute Configuration of the MonosaccharideEach solution of compounds 4, 5, 6 and 7 in 0.1 M acetate buffer (pH 4.0, 1.0 mL) was treated with naringinase (Sigma Chemical Co., 3.0 mg) and then the reaction mixture was stirred at 40°C for 36 h. The reaction mixture was passed through a Sep-Pak C18 cartridge using H2O and MeOH. The H2O layer was concentrated under reduced pressure to dryness, to give a resdue of the sugar fraction. The residue was dissolved in pyridine (0.1 mL), to which 0.08 M L-cysteine methyl ester hydrochloride in pyridine (0.15 mL) was added. The mixture was kept at 60°C for 1.5 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H2O (0.3 mL each) and then the n-hexane extract was analyzed by GC-MS under the following conditions: capillary column, EQUITY™-1 (30 m×0.25 mm×0.25 µm, Supelco); column temperature, 230°C; injection temperature, 250°C; carrier N2 gas; detection in EI mode, ionization potential, 70 eV; ion-source temperature, 280°C. L-Arabinose (10.89 min) and L-rhamnose (12.15 min) in 5, 6 and 7, D-glucose (17.18 min) in 4 and 7, D-xylose (11.99 min) in 6 and 7, were confirmed by comparison of the retention times of its derivatives with those of standard D and L monosaccharide derivatives prepared in a similar way.
HPLC AnalysisCrushing and screening 5 g seeds of Nigella glandulifera, defatted with petroleum ether, were extracted three times with 200 mL 30% ethanol to give an extract, which was dissolved in 25 mL 30% ethanol. The compounds 1 and 2 were confirmed in the 30% ethanol solution.All samples were stored at −20°C until analyzing. The final solution was passed through a 0.22 µm membrane before use. An aliquot of 20 µL of each sample solution was injected into the HPLC for analysis. The HPLC analysis was carried out on a Waters Symmetry C18 column (250 mm×4.6 mm i.d., 5 µm). A binary gradient elution system, which was composed of acetonitrile as solvent A and 0.1% acetic acid in water as solvent B, was applied for analysis with the gradient elution as follows: 0–40 min, 0–10% A; 40–45 min, 10–95% A; 45–55 min, 95% A. The flow rate of mobile phase was 1 mL/min, and column temperature was maintained at 30°C. The PDA detector was set at 270 nm, and the on-line UV spectra were recorded in the range of 210–400 nm.
Antitubercular AssaysThe antitubercular activity of every tested compound was evaluated using Mycobacterium tuberculosis strain H37Rv. Middlebrook 7H10 agar was used to determine MIC values, as recommended by the proportion method.15) Briefly, every test compound was added to Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) at 50–56°C, by serial dilution, to yield a final concentration of 100 to 0.8 µg/mL. Then 10 mL of each concentration of test compound-containing medium was dispensed into plastic quadrant Petri dishes. Several colonies of the test isolate of M. tuberculosis were selected to make a suspension with Middlebrook 7H9 broth and used as the initial inoculum. The inoculum of test isolate of M. tuberculosis was prepared by diluting the initial inoculum in Middlebrook 7H9 broth until turbidity was reduced to that equivalent to the McFarland No. 1 standard. Final suspensions were prepared by adding Middlebrook 7H9 broth and preparing 10−2 dilutions of the standardized bacterial suspensions. After solidification of the Middlebrook 7H10 medium, 33 µL of the 10−2 dilution of the standardized bacterial suspensions was placed on each quadrant of the agar plates. The agar plates were then incubated at 35°C with 10% CO2 for 2 weeks. The minimal inhibitory concentration is the lowest concentration of test compound that completely inhibited the growth of the test isolate of M. tuberculosis, as detected by the unaided eye.
This work was supported by the National Science Foundation of China (Grant No. 81073155), the Zhejiang Provincial Natural Science Foundation of China (Grant No. R2100014, Y2100765), Science and Technology Project of Traditional Chinese Medicine of Zhejiang Province (Grant No. 2010ZZ011). The authors are thankful to Dr. Rui-Liang Jin (Shanghai Key Laboratory of Mycobacterium Tuberculosis, Shanghai Pulmonary Hospital) for performing the antitubercular assays.
