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Amaryllidaceae alkaloids from Crinum latifolium with cytotoxic, antimicrobial, antioxidant, and anti-inflammatory activities
Fitoterapia 130 (2018) 48–53
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Amaryllidaceae alkaloids from Crinum latifolium with cytotoxic, antimicrobial, antioxidant, and anti-inflammatory activities
T
⁎
Ming-Xin Chena,b, Jian-Min Huoa, , Jiang Hub, Zi-Ping Xua, Xue Zhanga a b
Department of Respiratory, First Affiliated Hospital of Harbin Medical University, Harbin 150001, China Institute of Characteristic Medicinal Resource of Ethnic Minorities, Qujing Normal University, Qujing 655011, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Crinum latifolium Alkaloids Cytotoxic Antimicrobial Antioxidant Anti-inflammatory activity
Four novel and potently bioactive Amaryllidaceae alkaloids, 4,8-dimethoxy-cripowellin C (1), 4,8-dimethoxycripowellin D (2), 9-methoxy-cripowellin B (3), and 4-methoxy-8-hydroxy-cripowellin B (4), together with one known alkaloid, cripowellin C (5) were isolated from the 95% EtOH extract of the bulbs of Crinum latifolium. Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR as well as spectroscopy high resolution mass spectrometry. All isolates were in vitro evaluated for their cytotoxic activity against seven lung cancer cell lines, in addition to antimicrobial activity for eight bacteria, scavenging potential using ABTS·+ and DPPH test, and anti-inflammatory activity for Cox-1 and Cox-2 which had not previously been tested for crinane-type alkaloids with the cleavage between C-1 and C-13. Consequently, alkaloids 1–5 exhibited potent cytotoxicity against all of seven tested tumor cell lines with (IC50 < 30 nM). Alkaloids 3 and 4 displayed the significant antimicrobial activity with IC50 values < 0.50 mM and antioxidant activity in the ABTS·+ and DPPH test. Additionally, Alkaloids 1–5 exhibited comparable inhibition of Cox-1 (> 64%) and Cox-2 (> 90%) with positive control.
1. Introduction Crinum L. is the only pantropical genus of the family Amaryllidaceae, which is mainly distributed in Africa, America, Australia, and southern Asia [1–3]. The genus Crinum contains around 110 accepted species with over 270 synonyms [4]. Some Extracts from Crinum species have been traditionally used especially in Africa, tropical Asia and South America as emetics, laxatives, expectorants, tonics, antipyretics, diuretics, diaphoretics, anti-asthmatics, anti-malarial, antiaging, anti-tumor, and lactagogues [5]. Previous investigations reported that alkaloids are the main chemical constituents of this genus with interesting pharmacological effects [6–8], including immunoregulant [9], immuno-stimulant [10], antibacterial, and antifungal activities [11–14], particularly the antiproliferative action [15]. Crinum latifolium grows abundantly in the upper Gangetic Plain and is also cultivated as a garden flower. Extracts of different parts of this species are used in popular medicine for treatment of a rubefacient in rheumatism and piles, and for abcess treatment to promote suppuration [16]. Previous studies on C. latifolium reported the isolation of a series of crinane-type alkaloids [1]. To find more structurally interesting substances of the genus Crinum, present phytochemical studies on C. latifolium led to the isolation of four new Amaryllidaceae alkaloids, 4,8-
⁎
dimethoxy-cripowellin C (1), 4,8-dimethoxy-cripowellin D (2), 9methoxy-cripowellin B (3), and 4-methoxy-8-hydroxy-cripowellin B (4), along with one known alkaloid, cripowellin A (5). In this paper, we described the isolation and structure elucidation of the new compounds on the basis of spectroscopic methods. Furthermore, all the triterpenoids were in vitro evaluated for their cytotoxic, antimicrobial, radical scavenging, and anti-inflammatory potential. 2. Experimental part 2.1. General Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers (Tokyo, Japan), respectively. The NMR spectra were measured in CDCl3 on a Bruker AM-600 spectrometer (Fällanden, Switzerland). Chemical shifts were reported using residual CDCl3 (δH 7.26 and δC 77.0 ppm) and CD3OD (δH 3.30 and δC 49.0 ppm) as internal standard. High resolution ESIMS spectra were obtained on a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer. Silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany),
Corresponding author. E-mail address: [email protected] (J.-M. Huo).
https://doi.org/10.1016/j.fitote.2018.08.003 Received 28 July 2018; Received in revised form 9 August 2018; Accepted 12 August 2018 Available online 13 August 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.
Fitoterapia 130 (2018) 48–53
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LiChroprep RP-18 (Merck, 40–63 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used for column chromatography (CC). HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance detector, with a Prevail (250 × 10 mm i.d.) preparative column packed with C18 (5 μm). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for analytical thin-layer chromatography analyses.
3414, 2935, 2835, 1694, 1655, 1502, 1488, 1351, 1233, 1145, 1085, 1037, 933, 731 cm−1; NMR data (CDCl3 see Table 1); ESI-MS m/z: 576 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 576.2052 ([M + Na]+, C26H35NO12Na. calc. 576.2057). 2.3.4. 4-methoxy-8-hydroxy-cripowellin B (4) White amorphous powder; [α]D21.0 = −49.10 (c 1.0 × 10−5, MeOH); UV (MeOH) λmax: 212 (3.10), 290 (2.48) nm; IR (KBr) νmax: 3413, 2934, 2837, 1693, 1653, 1503, 1490, 1353, 1232, 1147, 1086, 1038, 934, 731 cm−1; NMR data (CDCl3 see Table 1); ESI-MS m/z: 548 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 548.2102 ([M + Na]+, C25H35NO11Na. calc. 548.2108).
2.2. Plant material The bulbs of Crinum latifolium were collected in the Yongning, Guanxi province, China, in July 2017. A specimen (CL20170701), identified by one of the authors (X. Zhang), was deposited in the Herbarium of Harbin Medical University, Haerbin, China.
2.4. Cytotoxicity assay in vitro The cytotoxic activity of the isolated compounds were determined using the revised MTT method [17] against seven lung cancer cell lines (A549, ATCC, H446, H460, H292, 95-D, and SPCA-1). Doxorubicin was used as the positive control. Cancer cells (4 × 103 cells) suspended in 100 μL/well of DMEM medium containing 10% fetal calf serum were seeded onto a 96-well culture plate. After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The concentrations of the assayed compounds were 0.04, 0.2, 1.0, 5, 25, and 125 μM, respectively. The optical density (OD) of each well was detected using a microplate reader at 550 nm and for correction at 595 nm. Each determination represented the average mean of six replicates. All the IC50 results represent an average of a minimum of three experiments and were expressed as means ± standard deviation (SD).
2.3. Extraction and isolation The fresh bulbs of C. latifolium (400 kg) were chopped up to small pieces and extracted with 95% EtOH (50 L × 3) for 24 h at room temperature. After removal of EtOH under reduced pressure, the aqueous brownish syrup was suspended in H2O (l L) and then partitioned with EtOAc (1 L × 3) to afford EtOAc fraction (247 g). The EtOAc soluble extract was subjected to column chromatographied over silica gel column (12 cm × 150 cm), eluting with gradient CHCl3/MeOH (from 100:1 to 1:1, each 10 L) to afford 5 ractions (Fr.1- Fr.5). Fraction 3 (16.1 g) was divided into three subfractions (3A-3C) by MCI gel CC (8 cm × 40 cm) and eluted with MeOH/H2O (from 50% to 95%, each 4 L). Subfraction 3B (186 mg) was further separated by preparative HPLC (MeOH/H2O, from 30% to 55%, 240 nm, 220 × 25 mm, 10 μm, Merck) to afford 3 (30.1 mg, retention time: 15.3 min). Subfraction 3C (201 mg) was chromatographed on a Sephadex LH-20 column (150 cm × 10 cm, CHCl3/MeOH, 1:1) and preparative HPLC (MeOH/ H2O, from 45% to 70%, 240 nm, 220 × 25 mm, 10 μm, Merck), yielding 1 (29.9 mg, retention time: 15.9 min). Fraction 4 (9.7 g) was subjected to MCI gel (6 cm × 30 cm, MeOH/H2O, from 50% to 95%, each 2 L), silica gel (1.5 cm × 50 cm, CHCl3/MeOH, from 300:1 to 200:1; CHCl3/ Me2CO, 20:1, each 500 mL) CC to afford three subfractions (4A-4C). Further chromatography of subfraction 4A on silica gel column eluting with CHCl3/MeOH (95:5, v/v) and then purified by preparative HPLC, (MeOH/H2O, from 55% to 70%, 240 nm, 220 × 25 mm, 10 μm, Merck), gave 2 (37.1 mg) and 4 (30.4 mg). Subfraction 4C was separated on silica gel column eluting with CHCl3/MeOH (95:5, v/v) and on puried Sephadex LH-20 column (150 cm × 10 cm, CHCl3/MeOH, 1:1, v/v) to produce 5 (43.1 mg).
2.5. Antimicrobial assays in vitro A total of 8 microorganisms were assayed among which three Grampositive bacteria: Streptococcus pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis, five Gram-negative bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, Enterobacter cloacae, and Shigella dysenteriae. Antimicrobial activity was evaluated by the disc diffusion method by measuring the zone of inhibitions [18]. Standard antibiotic netilmicin was used in order to control the sensitivity of the tested bacteria respectively. The tested compounds were dissolved in MeOH. For each experiment control disc with pure solvent was used as blind control. All the paper discs had a diameter of 6 mm and were deposited on the surface of the seeded trypticase soy-agar Petri dishes. The plates were inoculated with the tested organisms to give a final cell concentration of 107 cell/ml and were incubated for 48 h at 37 °C. The experiments were repeated three times and the results (diameters in millimeters) were expressed as average values. The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method in 96well plates. All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China).
2.3.1. 4,8-dimethoxy-cripowellin C (1) White amorphous powder; [α]D21.0 = −69.66 (c 1.0 × 10−5, MeOH); UV (MeOH) λmax: 210 (3.25), 289 (2.45) nm; IR (KBr) νmax: 3434, 2893, 1693, 1653, 1504, 1490, 1355, 1235, 1123, 1105, 1081, 1040, 1005, 976, 933, 734 cm−1; NMR data (CDCl3 see Table 1); ESIMS m/z: 560 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 560.2102 ([M + Na]+, C26H35NO11Na. calc. 560.2108). 2.3.2. 4,8-dimethoxy-cripowellin D (2) White amorphous powder; [α]D21.0 = −30.01 (c 1.0 × 10−6, MeOH); UV (MeOH) λmax: 208 (3.44), 291 (2.49) nm; IR (KBr) νmax: 3414, 2933, 2836, 1694, 1652, 1505, 1489, 1355, 1234, 1145, 1087, 1039, 976, 731 cm−1; NMR data (CDCl3 see Table 1); ESI-MS m/z: 546 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 546.2309 ([M + Na]+, C26H37NO10Na. calc. 546.2315).
2.6. Microplate assay for radical scavenging activity DPPH Microplate DPPH assay was performed as described according to Luo, Zhou, Ma, and Fu [19]. Briefly, successive sample dilutions (standard stocks of different samples 5 mM) in a 96-well plate afforded DPPH solution (40 μM in methanol) in a total volume of 0.2 mL. The
2.3.3. 9-methoxy-cripowellin B (3) White amorphous powder; [α]D21.0 = −47.09 (c 1.0 × 10−5, MeOH); UV (MeOH) λmax: 213 (3.08), 289 (2.50) nm; IR (KBr) νmax: 49
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Table 1 1 H (500 MHz)
13
C NMR (125 MHz) spectroscopic data of compounds 1–4 (in CDCl3).
δH (mult., J in Hz) No.
1
1 3 6 9 11-a 11-b 14 15 16-a 16-b 18-a 18-b 19-a 19-b 4-OCH3 8-OCH3 9-OCH3 1′ 2′ 3′ 4′ 5′ 6′-a 6′-b 1″-a 1″-b 2″-a 2″-b 2′-OCH3 3′-OCH3 4′-OCH3
3.27, 6.54, – 6.71, 5.34, 4.66, 4.70, 4.13, 3.10, 2.24, 3.19, 2.31, 4.53, 2.90, 3.88, 3.85, – 4.43, 3.31, 3.51, 2.92, 3.35, 1.35, – 4.93, 4.81, 5.01, 4.86, – – 3.53,
dd (3.9, 3.2) s s d (17.6) d (17.6) d (8.2) ddd (11.8, 8.2, 3.8) dd (14.0, 11.8) dd (14.0, 3.8) m m m m s s d (8.0) dd (8.9, dd (8.9, dd (9.3, dd (9.3, d (6.0) d d d d
s
(6.1) (6.1) (5.8) (5.8)
8.0) 8.6) 8.6) 6.0)
δC 2
3
4
No.
1
2
3
4
3.28, dd (4.0, 3.1) 6.55, s – 6.73, s 5.32, d (17.4) 4.67, d (17.4) 4.67, d (7.9) 4.12, ddd (11.7, 7.9, 3.9) 3.06, dd (14.3, 11.7) 2.26, dd (14.3, 3.9) 3.19, m 2.29, m 4.54,m 2.89, m 3.89, s 3.84, s – 4.32, d (7.9) 2.95, dd (9.0, 7.9) 3.07, dd (9.0, 8.2) 2.79, dd (9.3, 8.2) 3.29, dd (9.3, 6.1) 1.32, d (6.1) – – – – – 3.44, s 3.58, s 3.53, s
3.26, dd (4.4, 3.0) 6.45, s 6.00, s – 5.12, d (17.5) 4.54, d (17.5) 4.70, d (7.6) 4.15, ddd (11.6, 7.6, 4.3) 3.09, dd (14.2, 11.6) 2.26, dd (14.2, 4.3) 3.14, m 2.26, m 4.34, m 2.76, m – – 4.00, s 4.41, d (8.0) 3.15, dd (9.0, 8.0) 2.95, dd (9.0, 8.4) 3.05, dd (9.0, 8.4) 3.31, ddd (9.0, 7.0, 3.2) 3.85 dd (11.5, 7.0) 3.64, dd (11.5, 3.2) – – – – 3.44, s 3.50, s 3.59, s
3.24, dd (4.4, 3.0) 6.50, s 6.74, s – 5.23, d (17.6) 4.47, d (17.6) 4.64, d (7.5) 4.12, ddd (11.7, 7.5, 4.4) 3.08, dd (14.4, 11.7) 2.24, dd (14.4, 4.4) 3.35, m 1.95, m 4.32, m 2.78, m 3.86, s – – 4.39, d (8.1) 3.14, dd (9.2, 8.1) 2.95, dd (9.2, 8.5) 3.04, dd (9.2, 8.5) 3.30, ddd (9.2, 7.1, 3.4) 3.85 dd (11.3, 7.1) 3.63, dd (11.3, 3.4) – – – – 3.43, s 3.50, s 3.58, s
1 2 3 4 6 8 9 10 11 13 14 15 16 17 18 19 4-OCH3 8-OCH3 9-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 2′-OCH3 3′-OCH3 4′-OCH3
55.6 128.5 114.8 149.6 – 149.3 110.1 124.8 53.3 171.0 69.6 83.8 40.1 206.4 35.6 42.3 56.0 55.9 – 100.2 79.6 83.8 82.9 71.8 17.7 91.5 92.4 – – 61.1
55.5 125.5 114.8 149.7 – 149.3 110.0 128.2 53.4 171.1 69.9 84.2 40.1 206.3 35.6 42.3 56.1 55.9 – 102.3 83.7 86.3 85.1 71.3 17.5 – – 60.5 60.8 60.6
55.6 128.5 105.8 149.1 101.3 135.3 141.1 122.0 51.4 170.3 71.0 85.5 39.9 205.8 36.5 41.7 – – 59.0 103.1 80.1 86.6 83.9 75.8 62.4 – – 60.5 60.9 60.5
55.5 128.8 114.1 146.3 – 146.3 113.2 127.6 55.0 170.8 71.0 86.3 39.6 207.0 36.9 41.8 56.1 – – 103.2 80.1 86.7 84.0 75.8 62.5 – – 60.6 61.0 60.5
2.8. Anti-inflammatory assay in vitro
absorbance value was recorded at 550 nm with a microplate reade in triplicate. Results were determined each 5 min until 60 min in order to measure kinetic behaviour of the reaction. The percentage of remaining DPPH was calculated using the following: % DPPHrem = 100 × ([DPPH]sample/[DPPH]blank). A calibrated Trolox (3.9 mM initial concentration) standard curve was also made. The percentage of remaining DPPH against the standard concentration was plotted in an exponential regression, to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (IC50).
The anti-inflammatory activity was measured according to the literature with slight modifications [17]. The reaction system was incubated at 25 °C for 5 min, by sequential addition of the buffer, heme, pure compounds, and Cox-1 or Cox-2 into the system followed by mixing with arachidonic acid and TMPD. The optical density was recorded at 590 nm after another 15 min of incubation at 25 °C. SC-560 and NS-398 were used as positive controls which gave the inhibition of Cox-1 (63.5%) and Cox-2 (97.0%) respectively (Table 4). 3. Results and Discussion
2.7. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation decolorization assay
Compound 1 was obtained as white amorphous powder. The positive HR-ESI-MS spectrum displayed a pseudomolecular ion at m/z 560.2102 [M + Na]+ (calcd for C26H35NO11Na, 560.2108) consistent with a molecular formula of C26H35NO11, corresponding to 10 degrees of unsaturation. The 1H and 13C NMR spectra of 1 showed the characteristic resonances of a crinane-type alkaloid and a glucose moiety (Table 1). The anomeric proton observed at δH 4.43 (d, J = 8.0 Hz, 1H) along with a doublet methyl at δH 1.35 (d, J = 6.0 Hz, 3H) indicated the presence of a 6-deoxypyranosyl sugar. The more unusual protons associated with the sugar moiety were found in the 1,3,5-trioxepane ring system, which is formed by part of the sugar moiety (C-2′ and C-3′) and by two isolated methylene units containing four diastereotopic protons. The methoxy group at δH 3.53/δC 61.1 (s, 3H) had a single HMBC correlation to C-4′, confirming its attachment to C-4′ of the sugar moiety. The NOESY correlations from H-1′ to H-3′ and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as [2′,3′][1,3,5]-trioxepane-4′-methoxy-β-D-quinovose. In addition to the signals of sugar moieties, the alkaloid moiety of 1 contained two OMe groups (δC 56.0 and 55.9), four CH2 groups (δC 53.3, 40.1, 35.6 and 42.3), six olefinic C-atoms (δC 128.5, 114.8, 149.6,
ABTS·+ scavenging activity was determined by the method of Gasca [20]. The radical cation was generated by the reaction between 7 mM ABTS in H2O with 2.45 mM potassium persulphate, stored at room temperature in the dark for 16 h. Before usage, the solution was diluted with phosphate buffer (pH 7.4, 0.05 M) to reach an absorbance of 0.800 ± 0.035 at 734 nm. Different concentrations of isolated compounds solution in methanol were added into 1 mL of ABTS·+ solution. The mixture was incubated in the dark at 37 °C. After 30 min of incubation, the percentage inhibition of absorbance at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). All determinations were carried out in triplicate with Trolox as reference. The capability to scavenge the ABTS·+ was calculated equation as follows: ABTS·+ scavenging effect (%) = 100 − [(ASample/ AControl) × 100]. Where in ASample is absorbance of the remaining concentration of ABTS·+ in the presence of different compounds and AControl is the initial concentration of the ABTS·+. The stock concentrations of Trolox and tested compounds are the same as reported in DPPH assay. 50
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Fig. 1. Structures of compounds 1-5.
aglycone of compound 3 showed many similarities to those of 2, indicating that they are structural analogs except for the methylenedioxy between C-4 and C-8 in 3 instead of two methoxy groups at C-4 and C-8 in 2, and for the presence one more methoxy at C-9, which was further confirmed by the HMBC correlation from the methylenedioxy (δH 6.00) to C-4 (δC 149.1) and C-8 (δC 135.3), and from the methoxy (δH 4.00) to C-9 (δC141.1), respectively. Comparison of NMR data of 6-dexoypyranosyl moieties of 3 and 2 indicated that they were almost the same with the only difference being the presence of OH-6′ in 3. In the HMBC spectrum, the correlation from the H-6′ (δH 3.85 and 3.64) to C-4′ (δC 83.9) confirmed that a hydroxy group was attached to C-6′ in 6-dexoypyranosyl moiety. The HMBC correlation of the anomeric proton at δH 4.41 to δC 85.5 (C-15) indicated the location of the sugar moiety at C-15. The relative configuration of 3 was the same as that of 2 as established by the NOESY spectrum. Consequently, compound 3 was unambiguously determined as 9-methoxy-cripowellin B (Fig. 2). Compound 4, a white amorphous powder, was assigned a molecular formula of C25H35NO11, based on the HREIMS spectrum which showed a molecular ion at m/z 548.2102 [M+ Na]+ (calcd. 548.2108). The general features of its IR and NMR spectra closely resembled those of cripowellin B which was previously isolated from the plant [20]. Comparing the 1H and 13C NMR data of 4 with those of cripowellin B, the data were almost identical. The only significant difference was that the methylenedioxy group C-4 and C-8 in cripowellin B was replaced by a methoxy group at C-4 and a hydroxyl at C-8 in 4, which was confirmed by the HMBC correlations of the proton signal of the methoxy group at δH 3.86 to the aromatic carbon signal at δC 146.3 (C-4) and the NOESY correlation of the proton signal at δH 6.50 (s, H-3) to the methoxy proton signal. NOESY correlations from H-1′ to H-15, H-3′, and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as 2′,3′,4′-methoxy-β-D-quinovose. The location of the sugar moiety at C-15 was elucidated by the HMBC correlation of the anomeric proton at δH 4.39 to δC 86.3 (C-15). The stereochemistry of 4 was expected to be identical with that of 3 on the
149.3, 110.1, and 124.8), three CH groups (δC 55.6, 69.6 and 83.8), two oxygenated quaternary carbons (δC 171.0 and 206.4) according to the NMR data, which suggested that it was similar to the aglycone of cripowellin C (4) [4]. Comparing with the NMR data of 1 and cripowellin C, the only significant difference was that the methylenedioxy substituent between C-4 and C-8 in cripowellin C was replaced by two methoxy groups in 1, which was further supported by the HMBC correlations between the two methoxy (δH 3.88 and 3.85) with C-4 (δC 149.6) and C-8 (δC 149.3) respectively and the NOESY correlation of the two methoxy groups. The HMBC correlation from the anomeric proton observed at δH 4.43 to δC 83.8 (C-15) indicated the attachment of the sugar moiety to C-15. The NOESY correlations of H-1/H-4, H-1′/ H-5, H-5/H-18, and H-18/H-19 established that the α-oriented relative configuration of hydroxyl at C-14, H-15 and the methylene bridge of C18 and C-19. Thus, 1 was named as 4,8-dimethoxy-cripowellin C and the structure was showed in Fig. 1. Compound 2 was obtained as a white amorphous powder. The HRESI-MS revealed the [M + Na]+ peak at m/z 546.2309 (calcd. for C26H37NO10Na. 546.2315), corresponding to the molecular formula C26H37NO10. The 1H- and 13C-NMR spectra of 2 repealed the presence of a crinane-type alkaloid and a glucose moiety. Comparison of the 1H NMR data of 2 with those of 1, the aglycones of the two compounds were identical with the exception of the resonances of 6-dexoypyranosyl sugar moiety. The significant difference was that the presence of two additional methoxy groups, and that the 1,3,5-trioxepane ring moiety was absent in the 6-dexoypyranosyl moiety of 2. 2D NMR spectroscopic data, including COSY, HSQC, HMBC, and NOESY, were used to determine the connectivity and configuration of the sugar moiety. Once the connectivity around the ring from H-1′ to H-6′ had been established as before from HSQC and COSY correlations, HMBC was used to determine the locations of the methoxy groups. The methoxy at δH 3.44/δC 60.5 (s, 3H), δH 3.58/δC 60.8 (s, 3H), and δH 3.53/δC 60.6 (s, 3H) each had a single HMBC correlation to their respective attachment locations at C-2′ (δC 83.7), C-3′ (δC 86.3), and C-4′ (δC 85.1) respectively. NOESY correlations from H-1′ to H-15, H-3′, and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as 2′,3′,4′-methoxy-β-D-quinovose. In the HMBC spectrum, the correlation from the anomeric proton observed at δH 4.32 (d, J = 7.9 Hz, 1H) to δC 84.2 (C-15) elucidated the attachment of the sugar moiety to C-15. On the basis of the observation of NOESY data similar to those of 1, the stereochemistry of the aglycones of 1 and 2 was expected to be the same. Accordingly, the structure of 2 was established as 4,8-dimethoxy-cripowellin D and the structure was showed in Fig. 1. Compound 3 was isolated as white amorphous powder. The HR-ESIMS measurements revealed a pseudomolecular ion at m/z 576.2052, corresponding to the formula C26H35NO12, which had one more degree of unsaturation relative to that of 2. 1H and 13C NMR spectral data revealed a crinane-type alkaloid, a 6-dexoypyranosyl sugar moiety, and a methylenedioxy bridge. The 1H and 13C NMR data (Table 1) of the
Fig. 2. Key HMBC ( 51
) correlations of compound 1 and 3.
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Table 2 The cytotoxicity of compounds 1–5 against seven human tumor cell lines (nM).
1 2 3 4 5 Doxorubicin
A549
ATCC
H446
H460
H292
95-D
SPCA-1
23.2 ± 13.7 14.8 ± 1.6 9.4 ± 5.3 9.4 ± 4.1 27.9 ± 3.2 24.7
27.1 ± 5.1 17.3 ± 0.9 8.3 ± 2.7 10.1 ± 4.4 28.4 ± 6.9 21.8
28.0 ± 21.1 15.7 ± 1.3 7.3 ± 1.1 7.1 ± 3.2 27.7 ± 4.5 33.7
25.8 ± 1.7 14.5 ± 0.7 8.6 ± 1.4 9.1 ± 3.8 29.0 ± 3.9 28.4
26.7 ± 2.6 15.3 ± 0.9 7.1 ± 0.9 7.4 ± 2.1 28.4 ± 5.3 22.3
26.3 ± 15.7 16.6 ± 1.1 9.6 ± 6.3 9.7 ± 5.4 26.5 ± 9.7 14.1
25.6 ± 10.7 15.7 ± 1.3 8.8 ± 3.6 8.6 ± 0.7 28.2 ± 8.1 13.7
Table 3 Antimicrobial and antifungal activities (zones of inhibition/MIC mM, n = 3) of compounds 1–5.
1 2 3 4 5 Netilmicin
S. pneumoniae
S. aureus
S. epidermidis
K. pneumoniae
P. aeruginosa.
H. influenzae
E. cloacae
S. dysenteriae
20/1.22 18/0.97 23/0.35 24/0.25 18/1.68 21/0.01
20/0.92 18/0.97 24/0.31 23/0.87 17/1.87 25/0.01
20/0.82 17/0.89 24/0.35 24/0.15 18/1.74 24/0.03
19/1.10 18/0.99 24/0.29 22/0.27 17/1.82 22/0.02
21/1.17 18/0.77 23/0.27 23/0.32 19/1.67 25/0.02
20/0.98 19/1.01 23/0.31 20/0.23 18/1.87 23/0.18
20/1.03 19/1.08 24/0.41 20/0.32 17/2.09 23/0.03
20/0.98 18/1.13 24/0.30 20/0.31 18/1.99 24/0.02
possessed potent cytotoxic, antimicrobial, radical scavenging, and antiinflammatory activity relatively to other crinane-type alkaloids while 3 and 4 exhibited higher bioactivities than 1, 2 and 5. These results indicated that the cleavage between C-1 and C-13 in crinane alkaloid skeleton might be essential for this type alkaloids and the hydroxyl at C6′ could significantly enhance bioactivities.
Table 4 In vitro free radical scavenging and anti–inflammatory activities of compounds 1–5. Compounds
1 2 3 4 5 Trolox SC–560 NS–398
Free radical scavenging activitya
Anti–inflammatory Activityb
DPPH IC50
ABTS·+ IC50
COX–1
COX–2
124.0 80.1 62.1 69.9 130.7 43.2
104.7 73.4 52.2 67.3 125.6 80.9
69.7 66.8 70.2 67.9 64.1
93.6 92.7 91.1 95.2 90.4
Conflict of interest The authors declare that there are no conflicts of interest. References
63.5 97.0
[1] T.T.H. Hanh, D.H. Anh, P.T.T. Huong, N.V. Thanh, N.Q. Trung, T.V. Cuong, N.T. Mai, N.T. Cuong, N.X. Cuong, N.H. Nam, C.V. Minh, Crinane, augustamine, and β-carboline alkaloids from Crinum latifolium, Phytochem. Lett. 24 (2018) 27–30. [2] A.W. Meerow, D.J. Lehmiller, J.R. Clayton, Phylogeny and biogeography of Crinum L. (Amaryllidaceae) inferred from nuclear and limited plastid non-coding DNA sequences, Bot. J. Linn. Soc. 141 (2003) 349–363. [3] D.A. Snijman, H.P. Linder, Phylogenetic relationships, seed characters, and dispersal system evolution in Amaryllideae (Amaryllidaceae), Ann. Mo. Bot. Gard. 83 (1996) 362–386. [4] C.C. Presley, P. Krai, S. Dalal, Q. Su, M. Cassera, M. Goetz, D.G.I. Kingston, New potently bioactive alkaloids from Crinum erubescens, Bioorg. Med. Chem. 24 (2016) 5418–5422. [5] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part V. Biological profiles, Int. J. Pharm. Sci. Res. 4 (2013) 1239–1252. [6] P.T. Son, T.B. Duong, P.M. Giang, N.T. Minh, W.C. Taylor, Study on alkaloids from the bulbs of Crinum latifolium L. (Amaryllidaceae), Vietnam, J. Chemother. 39 (2001) 83–88. [7] P.T. Son, T.B. Duong, P.M. Giang, N.T. Minh, H.T. Huong, L.M. Huong, Investigation of antimicrobial and cytotoxic properties of alkaloids from some species of Crinum in Vietnam, Tap. Chi. Duoc. Hoc. 43 (2003) 18–21. [8] N.T.N. Tram, Z. Kamenarska, V. Bankova, S. Popov, E. Zvetkova, E. Katzarovo, L.M. Huong, Cytotoxic activities of alkaloid fractions from Crinum latifolium L., Amaryllidaceae, Tap. Chi. Duoc. Hoc. 41 (2001) 21–23. [9] S. Ghosal, P.H. Rao, K.S. Saini, Natural occurrence of 11-O-acetylambelline and 11O-acetyl-l,2-β-epoxyambelline in Crinum latifolium: immuno-regulant alkaloids, Pharm. Res. 2 (1985) 251–252. [10] S. Ghosal, K.S. Saini, V.K. Arora, 1,2-β-Epoxyambelline, an immunostimulant alkaloid from Crinum latifolium, J. Chem. Res. (1984) 232–233 (S). [11] C. Ianello, J. Bastida, F. Bonvicini, F. Antognoni, G.A. Gentilomi, F. Poli, Chemical composition and in vitro antibacterial and antifungal activity of an alkaloid extract from Crinum angustum Steud, Nat. Prod. Res. 28 (2014) 704–710. [12] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part I- Crinum alkaloids: lycorine-type alkaloids, Int. J. Pharm. Sci. Res. 3 (2012) 1883–1890. [13] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part II. Crinum alkaloids: crinine-type alkaloids, Int. J. Pharm. Sci. Res. 3 (2012) 3091–3100. [14] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part III; Crinum alkaloids: belladine- galanthamine-, lycorenine-, tazettine-type alkaloids and other minor types, Int. J. Pharm. Sci. Res. 3
All compounds and reference drug are expressed as IC50 values in μM. b Percent inhibition (all compounds and reference drugs concentration: 100 μM). a
basis of the similarity of NOESY data. Accordingly, the structure of 4 was established as 4-hydroxy-8-methoxy-cripowellin B. The Amaryllidaceae alkaloids from Crinum genus have been reported to show strong cytotoxicity against various types of cancer cell lines [4], which were agreed well with our results. The in vitro cytotoxic activity of these compounds against seven lung cancer cell lines using the modified MTT method was summarized in Table 2. Among the tested compounds, alkaloid 2–4 exhibited potent the cytotoxic potential against all tested tumor cell lines, with IC50 values of 14.5–17.3, 7.1–9.6, and 7.4–10.1 nM, respectively. Alkaloids 1 and 5 showed high cytotoxicity against all tested tumor cell lines with IC50 values < 30 nM (Table 2). significant. The antimicrobial, radical scavenging, and anti-inflammatory activities for crinane-type alkaloids with the cleavage between C-1 and C13 has been reported for the first time. The isolated alkaloids were tested for their antimicrobial activity by measuring the zone of inhibitions and exhibited interesting and promising antimicrobial activity (Table 3). Alkaloids 3 and 4 had the highest antimicrobial potential against all assayed microorganisms with MIC value < 0.50 mM (Table 3). Antioxidant evaluation by both DPPH and ABTS assays (Table 4) showed that compounds 2–4 showed significant antiradical activity in both the tested radical of DPPH and ABTS·+. The compounds 1–5 were tested in vitro for their anti-inflammatory activity (Table 4) and displayed comparable inhibition of Cox-1 (> 64%) with positive control SC–560 and of Cox-2 (> 90%) with positive control NS–398, respectively (Table 4). Alkaloids 1–5 52
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[18] S.Y. Long, C.L. Li, J. Hu, Q.J. Zhao, D. Chen, Indole alkaloids from the aerial parts of Kopsia fruticosa and their cytotoxic, antimicrobial and antifungal activities, Fitoterapia 129 (2018) 145–149. [19] Y.H. Luo, Z.Q. Zhou, S.C. Ma, H.Z. Fu, Three new antioxidant furofuran lignans from Callicarpa nudiflora, Phytochem. Lett. 7 (2014) 194–197. [20] R. Velten, C. Erdelen, M. Gehling, A. Göhrt, D. Gondol, J. Lenz, O. Lockhoff, U. Wachendorff, D. Wendisch, Cripowellin A and B, a novel type of amaryllidaceae alkaloid from Crinum powellii, Tetrahedron Lett. 39 (1998) 1737–1740.
(2012) 3630–3638. [15] S. Berkov, S. Romani, M. Herrera, F. Viladomat, C. Codina, G. Momekov, I. Ionkova, J. Bastida, Antiproliferative alkaloids from Crinum zeylanicum, Phytother. Res. 25 (2011) 1686–1692. [16] S. Ghosal, K.S. Saini, A.W. Frahm, Alkaloids of Crinum latifolium, Phytochemistry 22 (1983) 2305–2309. [17] J. Hu, Y. Song, X. Mao, Z. Wang, Q.J. Zhao, Limonoids isolated from Toona Sinensis and their radical scavenging, anti-inflammatory and cytotoxic activities, J. Funct. Foods 20 (2016) 1–9.
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Amaryllidaceae alkaloids from Crinum latifolium with cytotoxic, antimicrobial, antioxidant, and anti-inflammatory activities
T
⁎
Ming-Xin Chena,b, Jian-Min Huoa, , Jiang Hub, Zi-Ping Xua, Xue Zhanga a b
Department of Respiratory, First Affiliated Hospital of Harbin Medical University, Harbin 150001, China Institute of Characteristic Medicinal Resource of Ethnic Minorities, Qujing Normal University, Qujing 655011, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Crinum latifolium Alkaloids Cytotoxic Antimicrobial Antioxidant Anti-inflammatory activity
Four novel and potently bioactive Amaryllidaceae alkaloids, 4,8-dimethoxy-cripowellin C (1), 4,8-dimethoxycripowellin D (2), 9-methoxy-cripowellin B (3), and 4-methoxy-8-hydroxy-cripowellin B (4), together with one known alkaloid, cripowellin C (5) were isolated from the 95% EtOH extract of the bulbs of Crinum latifolium. Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR as well as spectroscopy high resolution mass spectrometry. All isolates were in vitro evaluated for their cytotoxic activity against seven lung cancer cell lines, in addition to antimicrobial activity for eight bacteria, scavenging potential using ABTS·+ and DPPH test, and anti-inflammatory activity for Cox-1 and Cox-2 which had not previously been tested for crinane-type alkaloids with the cleavage between C-1 and C-13. Consequently, alkaloids 1–5 exhibited potent cytotoxicity against all of seven tested tumor cell lines with (IC50 < 30 nM). Alkaloids 3 and 4 displayed the significant antimicrobial activity with IC50 values < 0.50 mM and antioxidant activity in the ABTS·+ and DPPH test. Additionally, Alkaloids 1–5 exhibited comparable inhibition of Cox-1 (> 64%) and Cox-2 (> 90%) with positive control.
1. Introduction Crinum L. is the only pantropical genus of the family Amaryllidaceae, which is mainly distributed in Africa, America, Australia, and southern Asia [1–3]. The genus Crinum contains around 110 accepted species with over 270 synonyms [4]. Some Extracts from Crinum species have been traditionally used especially in Africa, tropical Asia and South America as emetics, laxatives, expectorants, tonics, antipyretics, diuretics, diaphoretics, anti-asthmatics, anti-malarial, antiaging, anti-tumor, and lactagogues [5]. Previous investigations reported that alkaloids are the main chemical constituents of this genus with interesting pharmacological effects [6–8], including immunoregulant [9], immuno-stimulant [10], antibacterial, and antifungal activities [11–14], particularly the antiproliferative action [15]. Crinum latifolium grows abundantly in the upper Gangetic Plain and is also cultivated as a garden flower. Extracts of different parts of this species are used in popular medicine for treatment of a rubefacient in rheumatism and piles, and for abcess treatment to promote suppuration [16]. Previous studies on C. latifolium reported the isolation of a series of crinane-type alkaloids [1]. To find more structurally interesting substances of the genus Crinum, present phytochemical studies on C. latifolium led to the isolation of four new Amaryllidaceae alkaloids, 4,8-
⁎
dimethoxy-cripowellin C (1), 4,8-dimethoxy-cripowellin D (2), 9methoxy-cripowellin B (3), and 4-methoxy-8-hydroxy-cripowellin B (4), along with one known alkaloid, cripowellin A (5). In this paper, we described the isolation and structure elucidation of the new compounds on the basis of spectroscopic methods. Furthermore, all the triterpenoids were in vitro evaluated for their cytotoxic, antimicrobial, radical scavenging, and anti-inflammatory potential. 2. Experimental part 2.1. General Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers (Tokyo, Japan), respectively. The NMR spectra were measured in CDCl3 on a Bruker AM-600 spectrometer (Fällanden, Switzerland). Chemical shifts were reported using residual CDCl3 (δH 7.26 and δC 77.0 ppm) and CD3OD (δH 3.30 and δC 49.0 ppm) as internal standard. High resolution ESIMS spectra were obtained on a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer. Silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany),
Corresponding author. E-mail address: [email protected] (J.-M. Huo).
https://doi.org/10.1016/j.fitote.2018.08.003 Received 28 July 2018; Received in revised form 9 August 2018; Accepted 12 August 2018 Available online 13 August 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.
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LiChroprep RP-18 (Merck, 40–63 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used for column chromatography (CC). HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance detector, with a Prevail (250 × 10 mm i.d.) preparative column packed with C18 (5 μm). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for analytical thin-layer chromatography analyses.
3414, 2935, 2835, 1694, 1655, 1502, 1488, 1351, 1233, 1145, 1085, 1037, 933, 731 cm−1; NMR data (CDCl3 see Table 1); ESI-MS m/z: 576 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 576.2052 ([M + Na]+, C26H35NO12Na. calc. 576.2057). 2.3.4. 4-methoxy-8-hydroxy-cripowellin B (4) White amorphous powder; [α]D21.0 = −49.10 (c 1.0 × 10−5, MeOH); UV (MeOH) λmax: 212 (3.10), 290 (2.48) nm; IR (KBr) νmax: 3413, 2934, 2837, 1693, 1653, 1503, 1490, 1353, 1232, 1147, 1086, 1038, 934, 731 cm−1; NMR data (CDCl3 see Table 1); ESI-MS m/z: 548 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 548.2102 ([M + Na]+, C25H35NO11Na. calc. 548.2108).
2.2. Plant material The bulbs of Crinum latifolium were collected in the Yongning, Guanxi province, China, in July 2017. A specimen (CL20170701), identified by one of the authors (X. Zhang), was deposited in the Herbarium of Harbin Medical University, Haerbin, China.
2.4. Cytotoxicity assay in vitro The cytotoxic activity of the isolated compounds were determined using the revised MTT method [17] against seven lung cancer cell lines (A549, ATCC, H446, H460, H292, 95-D, and SPCA-1). Doxorubicin was used as the positive control. Cancer cells (4 × 103 cells) suspended in 100 μL/well of DMEM medium containing 10% fetal calf serum were seeded onto a 96-well culture plate. After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The concentrations of the assayed compounds were 0.04, 0.2, 1.0, 5, 25, and 125 μM, respectively. The optical density (OD) of each well was detected using a microplate reader at 550 nm and for correction at 595 nm. Each determination represented the average mean of six replicates. All the IC50 results represent an average of a minimum of three experiments and were expressed as means ± standard deviation (SD).
2.3. Extraction and isolation The fresh bulbs of C. latifolium (400 kg) were chopped up to small pieces and extracted with 95% EtOH (50 L × 3) for 24 h at room temperature. After removal of EtOH under reduced pressure, the aqueous brownish syrup was suspended in H2O (l L) and then partitioned with EtOAc (1 L × 3) to afford EtOAc fraction (247 g). The EtOAc soluble extract was subjected to column chromatographied over silica gel column (12 cm × 150 cm), eluting with gradient CHCl3/MeOH (from 100:1 to 1:1, each 10 L) to afford 5 ractions (Fr.1- Fr.5). Fraction 3 (16.1 g) was divided into three subfractions (3A-3C) by MCI gel CC (8 cm × 40 cm) and eluted with MeOH/H2O (from 50% to 95%, each 4 L). Subfraction 3B (186 mg) was further separated by preparative HPLC (MeOH/H2O, from 30% to 55%, 240 nm, 220 × 25 mm, 10 μm, Merck) to afford 3 (30.1 mg, retention time: 15.3 min). Subfraction 3C (201 mg) was chromatographed on a Sephadex LH-20 column (150 cm × 10 cm, CHCl3/MeOH, 1:1) and preparative HPLC (MeOH/ H2O, from 45% to 70%, 240 nm, 220 × 25 mm, 10 μm, Merck), yielding 1 (29.9 mg, retention time: 15.9 min). Fraction 4 (9.7 g) was subjected to MCI gel (6 cm × 30 cm, MeOH/H2O, from 50% to 95%, each 2 L), silica gel (1.5 cm × 50 cm, CHCl3/MeOH, from 300:1 to 200:1; CHCl3/ Me2CO, 20:1, each 500 mL) CC to afford three subfractions (4A-4C). Further chromatography of subfraction 4A on silica gel column eluting with CHCl3/MeOH (95:5, v/v) and then purified by preparative HPLC, (MeOH/H2O, from 55% to 70%, 240 nm, 220 × 25 mm, 10 μm, Merck), gave 2 (37.1 mg) and 4 (30.4 mg). Subfraction 4C was separated on silica gel column eluting with CHCl3/MeOH (95:5, v/v) and on puried Sephadex LH-20 column (150 cm × 10 cm, CHCl3/MeOH, 1:1, v/v) to produce 5 (43.1 mg).
2.5. Antimicrobial assays in vitro A total of 8 microorganisms were assayed among which three Grampositive bacteria: Streptococcus pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis, five Gram-negative bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, Enterobacter cloacae, and Shigella dysenteriae. Antimicrobial activity was evaluated by the disc diffusion method by measuring the zone of inhibitions [18]. Standard antibiotic netilmicin was used in order to control the sensitivity of the tested bacteria respectively. The tested compounds were dissolved in MeOH. For each experiment control disc with pure solvent was used as blind control. All the paper discs had a diameter of 6 mm and were deposited on the surface of the seeded trypticase soy-agar Petri dishes. The plates were inoculated with the tested organisms to give a final cell concentration of 107 cell/ml and were incubated for 48 h at 37 °C. The experiments were repeated three times and the results (diameters in millimeters) were expressed as average values. The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method in 96well plates. All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China).
2.3.1. 4,8-dimethoxy-cripowellin C (1) White amorphous powder; [α]D21.0 = −69.66 (c 1.0 × 10−5, MeOH); UV (MeOH) λmax: 210 (3.25), 289 (2.45) nm; IR (KBr) νmax: 3434, 2893, 1693, 1653, 1504, 1490, 1355, 1235, 1123, 1105, 1081, 1040, 1005, 976, 933, 734 cm−1; NMR data (CDCl3 see Table 1); ESIMS m/z: 560 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 560.2102 ([M + Na]+, C26H35NO11Na. calc. 560.2108). 2.3.2. 4,8-dimethoxy-cripowellin D (2) White amorphous powder; [α]D21.0 = −30.01 (c 1.0 × 10−6, MeOH); UV (MeOH) λmax: 208 (3.44), 291 (2.49) nm; IR (KBr) νmax: 3414, 2933, 2836, 1694, 1652, 1505, 1489, 1355, 1234, 1145, 1087, 1039, 976, 731 cm−1; NMR data (CDCl3 see Table 1); ESI-MS m/z: 546 ([M + Na]+). HR-ESI-MS (pos.) m/z: calc. 546.2309 ([M + Na]+, C26H37NO10Na. calc. 546.2315).
2.6. Microplate assay for radical scavenging activity DPPH Microplate DPPH assay was performed as described according to Luo, Zhou, Ma, and Fu [19]. Briefly, successive sample dilutions (standard stocks of different samples 5 mM) in a 96-well plate afforded DPPH solution (40 μM in methanol) in a total volume of 0.2 mL. The
2.3.3. 9-methoxy-cripowellin B (3) White amorphous powder; [α]D21.0 = −47.09 (c 1.0 × 10−5, MeOH); UV (MeOH) λmax: 213 (3.08), 289 (2.50) nm; IR (KBr) νmax: 49
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Table 1 1 H (500 MHz)
13
C NMR (125 MHz) spectroscopic data of compounds 1–4 (in CDCl3).
δH (mult., J in Hz) No.
1
1 3 6 9 11-a 11-b 14 15 16-a 16-b 18-a 18-b 19-a 19-b 4-OCH3 8-OCH3 9-OCH3 1′ 2′ 3′ 4′ 5′ 6′-a 6′-b 1″-a 1″-b 2″-a 2″-b 2′-OCH3 3′-OCH3 4′-OCH3
3.27, 6.54, – 6.71, 5.34, 4.66, 4.70, 4.13, 3.10, 2.24, 3.19, 2.31, 4.53, 2.90, 3.88, 3.85, – 4.43, 3.31, 3.51, 2.92, 3.35, 1.35, – 4.93, 4.81, 5.01, 4.86, – – 3.53,
dd (3.9, 3.2) s s d (17.6) d (17.6) d (8.2) ddd (11.8, 8.2, 3.8) dd (14.0, 11.8) dd (14.0, 3.8) m m m m s s d (8.0) dd (8.9, dd (8.9, dd (9.3, dd (9.3, d (6.0) d d d d
s
(6.1) (6.1) (5.8) (5.8)
8.0) 8.6) 8.6) 6.0)
δC 2
3
4
No.
1
2
3
4
3.28, dd (4.0, 3.1) 6.55, s – 6.73, s 5.32, d (17.4) 4.67, d (17.4) 4.67, d (7.9) 4.12, ddd (11.7, 7.9, 3.9) 3.06, dd (14.3, 11.7) 2.26, dd (14.3, 3.9) 3.19, m 2.29, m 4.54,m 2.89, m 3.89, s 3.84, s – 4.32, d (7.9) 2.95, dd (9.0, 7.9) 3.07, dd (9.0, 8.2) 2.79, dd (9.3, 8.2) 3.29, dd (9.3, 6.1) 1.32, d (6.1) – – – – – 3.44, s 3.58, s 3.53, s
3.26, dd (4.4, 3.0) 6.45, s 6.00, s – 5.12, d (17.5) 4.54, d (17.5) 4.70, d (7.6) 4.15, ddd (11.6, 7.6, 4.3) 3.09, dd (14.2, 11.6) 2.26, dd (14.2, 4.3) 3.14, m 2.26, m 4.34, m 2.76, m – – 4.00, s 4.41, d (8.0) 3.15, dd (9.0, 8.0) 2.95, dd (9.0, 8.4) 3.05, dd (9.0, 8.4) 3.31, ddd (9.0, 7.0, 3.2) 3.85 dd (11.5, 7.0) 3.64, dd (11.5, 3.2) – – – – 3.44, s 3.50, s 3.59, s
3.24, dd (4.4, 3.0) 6.50, s 6.74, s – 5.23, d (17.6) 4.47, d (17.6) 4.64, d (7.5) 4.12, ddd (11.7, 7.5, 4.4) 3.08, dd (14.4, 11.7) 2.24, dd (14.4, 4.4) 3.35, m 1.95, m 4.32, m 2.78, m 3.86, s – – 4.39, d (8.1) 3.14, dd (9.2, 8.1) 2.95, dd (9.2, 8.5) 3.04, dd (9.2, 8.5) 3.30, ddd (9.2, 7.1, 3.4) 3.85 dd (11.3, 7.1) 3.63, dd (11.3, 3.4) – – – – 3.43, s 3.50, s 3.58, s
1 2 3 4 6 8 9 10 11 13 14 15 16 17 18 19 4-OCH3 8-OCH3 9-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 2′-OCH3 3′-OCH3 4′-OCH3
55.6 128.5 114.8 149.6 – 149.3 110.1 124.8 53.3 171.0 69.6 83.8 40.1 206.4 35.6 42.3 56.0 55.9 – 100.2 79.6 83.8 82.9 71.8 17.7 91.5 92.4 – – 61.1
55.5 125.5 114.8 149.7 – 149.3 110.0 128.2 53.4 171.1 69.9 84.2 40.1 206.3 35.6 42.3 56.1 55.9 – 102.3 83.7 86.3 85.1 71.3 17.5 – – 60.5 60.8 60.6
55.6 128.5 105.8 149.1 101.3 135.3 141.1 122.0 51.4 170.3 71.0 85.5 39.9 205.8 36.5 41.7 – – 59.0 103.1 80.1 86.6 83.9 75.8 62.4 – – 60.5 60.9 60.5
55.5 128.8 114.1 146.3 – 146.3 113.2 127.6 55.0 170.8 71.0 86.3 39.6 207.0 36.9 41.8 56.1 – – 103.2 80.1 86.7 84.0 75.8 62.5 – – 60.6 61.0 60.5
2.8. Anti-inflammatory assay in vitro
absorbance value was recorded at 550 nm with a microplate reade in triplicate. Results were determined each 5 min until 60 min in order to measure kinetic behaviour of the reaction. The percentage of remaining DPPH was calculated using the following: % DPPHrem = 100 × ([DPPH]sample/[DPPH]blank). A calibrated Trolox (3.9 mM initial concentration) standard curve was also made. The percentage of remaining DPPH against the standard concentration was plotted in an exponential regression, to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (IC50).
The anti-inflammatory activity was measured according to the literature with slight modifications [17]. The reaction system was incubated at 25 °C for 5 min, by sequential addition of the buffer, heme, pure compounds, and Cox-1 or Cox-2 into the system followed by mixing with arachidonic acid and TMPD. The optical density was recorded at 590 nm after another 15 min of incubation at 25 °C. SC-560 and NS-398 were used as positive controls which gave the inhibition of Cox-1 (63.5%) and Cox-2 (97.0%) respectively (Table 4). 3. Results and Discussion
2.7. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation decolorization assay
Compound 1 was obtained as white amorphous powder. The positive HR-ESI-MS spectrum displayed a pseudomolecular ion at m/z 560.2102 [M + Na]+ (calcd for C26H35NO11Na, 560.2108) consistent with a molecular formula of C26H35NO11, corresponding to 10 degrees of unsaturation. The 1H and 13C NMR spectra of 1 showed the characteristic resonances of a crinane-type alkaloid and a glucose moiety (Table 1). The anomeric proton observed at δH 4.43 (d, J = 8.0 Hz, 1H) along with a doublet methyl at δH 1.35 (d, J = 6.0 Hz, 3H) indicated the presence of a 6-deoxypyranosyl sugar. The more unusual protons associated with the sugar moiety were found in the 1,3,5-trioxepane ring system, which is formed by part of the sugar moiety (C-2′ and C-3′) and by two isolated methylene units containing four diastereotopic protons. The methoxy group at δH 3.53/δC 61.1 (s, 3H) had a single HMBC correlation to C-4′, confirming its attachment to C-4′ of the sugar moiety. The NOESY correlations from H-1′ to H-3′ and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as [2′,3′][1,3,5]-trioxepane-4′-methoxy-β-D-quinovose. In addition to the signals of sugar moieties, the alkaloid moiety of 1 contained two OMe groups (δC 56.0 and 55.9), four CH2 groups (δC 53.3, 40.1, 35.6 and 42.3), six olefinic C-atoms (δC 128.5, 114.8, 149.6,
ABTS·+ scavenging activity was determined by the method of Gasca [20]. The radical cation was generated by the reaction between 7 mM ABTS in H2O with 2.45 mM potassium persulphate, stored at room temperature in the dark for 16 h. Before usage, the solution was diluted with phosphate buffer (pH 7.4, 0.05 M) to reach an absorbance of 0.800 ± 0.035 at 734 nm. Different concentrations of isolated compounds solution in methanol were added into 1 mL of ABTS·+ solution. The mixture was incubated in the dark at 37 °C. After 30 min of incubation, the percentage inhibition of absorbance at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). All determinations were carried out in triplicate with Trolox as reference. The capability to scavenge the ABTS·+ was calculated equation as follows: ABTS·+ scavenging effect (%) = 100 − [(ASample/ AControl) × 100]. Where in ASample is absorbance of the remaining concentration of ABTS·+ in the presence of different compounds and AControl is the initial concentration of the ABTS·+. The stock concentrations of Trolox and tested compounds are the same as reported in DPPH assay. 50
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Fig. 1. Structures of compounds 1-5.
aglycone of compound 3 showed many similarities to those of 2, indicating that they are structural analogs except for the methylenedioxy between C-4 and C-8 in 3 instead of two methoxy groups at C-4 and C-8 in 2, and for the presence one more methoxy at C-9, which was further confirmed by the HMBC correlation from the methylenedioxy (δH 6.00) to C-4 (δC 149.1) and C-8 (δC 135.3), and from the methoxy (δH 4.00) to C-9 (δC141.1), respectively. Comparison of NMR data of 6-dexoypyranosyl moieties of 3 and 2 indicated that they were almost the same with the only difference being the presence of OH-6′ in 3. In the HMBC spectrum, the correlation from the H-6′ (δH 3.85 and 3.64) to C-4′ (δC 83.9) confirmed that a hydroxy group was attached to C-6′ in 6-dexoypyranosyl moiety. The HMBC correlation of the anomeric proton at δH 4.41 to δC 85.5 (C-15) indicated the location of the sugar moiety at C-15. The relative configuration of 3 was the same as that of 2 as established by the NOESY spectrum. Consequently, compound 3 was unambiguously determined as 9-methoxy-cripowellin B (Fig. 2). Compound 4, a white amorphous powder, was assigned a molecular formula of C25H35NO11, based on the HREIMS spectrum which showed a molecular ion at m/z 548.2102 [M+ Na]+ (calcd. 548.2108). The general features of its IR and NMR spectra closely resembled those of cripowellin B which was previously isolated from the plant [20]. Comparing the 1H and 13C NMR data of 4 with those of cripowellin B, the data were almost identical. The only significant difference was that the methylenedioxy group C-4 and C-8 in cripowellin B was replaced by a methoxy group at C-4 and a hydroxyl at C-8 in 4, which was confirmed by the HMBC correlations of the proton signal of the methoxy group at δH 3.86 to the aromatic carbon signal at δC 146.3 (C-4) and the NOESY correlation of the proton signal at δH 6.50 (s, H-3) to the methoxy proton signal. NOESY correlations from H-1′ to H-15, H-3′, and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as 2′,3′,4′-methoxy-β-D-quinovose. The location of the sugar moiety at C-15 was elucidated by the HMBC correlation of the anomeric proton at δH 4.39 to δC 86.3 (C-15). The stereochemistry of 4 was expected to be identical with that of 3 on the
149.3, 110.1, and 124.8), three CH groups (δC 55.6, 69.6 and 83.8), two oxygenated quaternary carbons (δC 171.0 and 206.4) according to the NMR data, which suggested that it was similar to the aglycone of cripowellin C (4) [4]. Comparing with the NMR data of 1 and cripowellin C, the only significant difference was that the methylenedioxy substituent between C-4 and C-8 in cripowellin C was replaced by two methoxy groups in 1, which was further supported by the HMBC correlations between the two methoxy (δH 3.88 and 3.85) with C-4 (δC 149.6) and C-8 (δC 149.3) respectively and the NOESY correlation of the two methoxy groups. The HMBC correlation from the anomeric proton observed at δH 4.43 to δC 83.8 (C-15) indicated the attachment of the sugar moiety to C-15. The NOESY correlations of H-1/H-4, H-1′/ H-5, H-5/H-18, and H-18/H-19 established that the α-oriented relative configuration of hydroxyl at C-14, H-15 and the methylene bridge of C18 and C-19. Thus, 1 was named as 4,8-dimethoxy-cripowellin C and the structure was showed in Fig. 1. Compound 2 was obtained as a white amorphous powder. The HRESI-MS revealed the [M + Na]+ peak at m/z 546.2309 (calcd. for C26H37NO10Na. 546.2315), corresponding to the molecular formula C26H37NO10. The 1H- and 13C-NMR spectra of 2 repealed the presence of a crinane-type alkaloid and a glucose moiety. Comparison of the 1H NMR data of 2 with those of 1, the aglycones of the two compounds were identical with the exception of the resonances of 6-dexoypyranosyl sugar moiety. The significant difference was that the presence of two additional methoxy groups, and that the 1,3,5-trioxepane ring moiety was absent in the 6-dexoypyranosyl moiety of 2. 2D NMR spectroscopic data, including COSY, HSQC, HMBC, and NOESY, were used to determine the connectivity and configuration of the sugar moiety. Once the connectivity around the ring from H-1′ to H-6′ had been established as before from HSQC and COSY correlations, HMBC was used to determine the locations of the methoxy groups. The methoxy at δH 3.44/δC 60.5 (s, 3H), δH 3.58/δC 60.8 (s, 3H), and δH 3.53/δC 60.6 (s, 3H) each had a single HMBC correlation to their respective attachment locations at C-2′ (δC 83.7), C-3′ (δC 86.3), and C-4′ (δC 85.1) respectively. NOESY correlations from H-1′ to H-15, H-3′, and H-5′, and from H-2′ to H-4′, and from H-4′ to H-6′ confirmed the stereochemistry of the sugar moiety as 2′,3′,4′-methoxy-β-D-quinovose. In the HMBC spectrum, the correlation from the anomeric proton observed at δH 4.32 (d, J = 7.9 Hz, 1H) to δC 84.2 (C-15) elucidated the attachment of the sugar moiety to C-15. On the basis of the observation of NOESY data similar to those of 1, the stereochemistry of the aglycones of 1 and 2 was expected to be the same. Accordingly, the structure of 2 was established as 4,8-dimethoxy-cripowellin D and the structure was showed in Fig. 1. Compound 3 was isolated as white amorphous powder. The HR-ESIMS measurements revealed a pseudomolecular ion at m/z 576.2052, corresponding to the formula C26H35NO12, which had one more degree of unsaturation relative to that of 2. 1H and 13C NMR spectral data revealed a crinane-type alkaloid, a 6-dexoypyranosyl sugar moiety, and a methylenedioxy bridge. The 1H and 13C NMR data (Table 1) of the
Fig. 2. Key HMBC ( 51
) correlations of compound 1 and 3.
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Table 2 The cytotoxicity of compounds 1–5 against seven human tumor cell lines (nM).
1 2 3 4 5 Doxorubicin
A549
ATCC
H446
H460
H292
95-D
SPCA-1
23.2 ± 13.7 14.8 ± 1.6 9.4 ± 5.3 9.4 ± 4.1 27.9 ± 3.2 24.7
27.1 ± 5.1 17.3 ± 0.9 8.3 ± 2.7 10.1 ± 4.4 28.4 ± 6.9 21.8
28.0 ± 21.1 15.7 ± 1.3 7.3 ± 1.1 7.1 ± 3.2 27.7 ± 4.5 33.7
25.8 ± 1.7 14.5 ± 0.7 8.6 ± 1.4 9.1 ± 3.8 29.0 ± 3.9 28.4
26.7 ± 2.6 15.3 ± 0.9 7.1 ± 0.9 7.4 ± 2.1 28.4 ± 5.3 22.3
26.3 ± 15.7 16.6 ± 1.1 9.6 ± 6.3 9.7 ± 5.4 26.5 ± 9.7 14.1
25.6 ± 10.7 15.7 ± 1.3 8.8 ± 3.6 8.6 ± 0.7 28.2 ± 8.1 13.7
Table 3 Antimicrobial and antifungal activities (zones of inhibition/MIC mM, n = 3) of compounds 1–5.
1 2 3 4 5 Netilmicin
S. pneumoniae
S. aureus
S. epidermidis
K. pneumoniae
P. aeruginosa.
H. influenzae
E. cloacae
S. dysenteriae
20/1.22 18/0.97 23/0.35 24/0.25 18/1.68 21/0.01
20/0.92 18/0.97 24/0.31 23/0.87 17/1.87 25/0.01
20/0.82 17/0.89 24/0.35 24/0.15 18/1.74 24/0.03
19/1.10 18/0.99 24/0.29 22/0.27 17/1.82 22/0.02
21/1.17 18/0.77 23/0.27 23/0.32 19/1.67 25/0.02
20/0.98 19/1.01 23/0.31 20/0.23 18/1.87 23/0.18
20/1.03 19/1.08 24/0.41 20/0.32 17/2.09 23/0.03
20/0.98 18/1.13 24/0.30 20/0.31 18/1.99 24/0.02
possessed potent cytotoxic, antimicrobial, radical scavenging, and antiinflammatory activity relatively to other crinane-type alkaloids while 3 and 4 exhibited higher bioactivities than 1, 2 and 5. These results indicated that the cleavage between C-1 and C-13 in crinane alkaloid skeleton might be essential for this type alkaloids and the hydroxyl at C6′ could significantly enhance bioactivities.
Table 4 In vitro free radical scavenging and anti–inflammatory activities of compounds 1–5. Compounds
1 2 3 4 5 Trolox SC–560 NS–398
Free radical scavenging activitya
Anti–inflammatory Activityb
DPPH IC50
ABTS·+ IC50
COX–1
COX–2
124.0 80.1 62.1 69.9 130.7 43.2
104.7 73.4 52.2 67.3 125.6 80.9
69.7 66.8 70.2 67.9 64.1
93.6 92.7 91.1 95.2 90.4
Conflict of interest The authors declare that there are no conflicts of interest. References
63.5 97.0
[1] T.T.H. Hanh, D.H. Anh, P.T.T. Huong, N.V. Thanh, N.Q. Trung, T.V. Cuong, N.T. Mai, N.T. Cuong, N.X. Cuong, N.H. Nam, C.V. Minh, Crinane, augustamine, and β-carboline alkaloids from Crinum latifolium, Phytochem. Lett. 24 (2018) 27–30. [2] A.W. Meerow, D.J. Lehmiller, J.R. Clayton, Phylogeny and biogeography of Crinum L. (Amaryllidaceae) inferred from nuclear and limited plastid non-coding DNA sequences, Bot. J. Linn. Soc. 141 (2003) 349–363. [3] D.A. Snijman, H.P. Linder, Phylogenetic relationships, seed characters, and dispersal system evolution in Amaryllideae (Amaryllidaceae), Ann. Mo. Bot. Gard. 83 (1996) 362–386. [4] C.C. Presley, P. Krai, S. Dalal, Q. Su, M. Cassera, M. Goetz, D.G.I. Kingston, New potently bioactive alkaloids from Crinum erubescens, Bioorg. Med. Chem. 24 (2016) 5418–5422. [5] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part V. Biological profiles, Int. J. Pharm. Sci. Res. 4 (2013) 1239–1252. [6] P.T. Son, T.B. Duong, P.M. Giang, N.T. Minh, W.C. Taylor, Study on alkaloids from the bulbs of Crinum latifolium L. (Amaryllidaceae), Vietnam, J. Chemother. 39 (2001) 83–88. [7] P.T. Son, T.B. Duong, P.M. Giang, N.T. Minh, H.T. Huong, L.M. Huong, Investigation of antimicrobial and cytotoxic properties of alkaloids from some species of Crinum in Vietnam, Tap. Chi. Duoc. Hoc. 43 (2003) 18–21. [8] N.T.N. Tram, Z. Kamenarska, V. Bankova, S. Popov, E. Zvetkova, E. Katzarovo, L.M. Huong, Cytotoxic activities of alkaloid fractions from Crinum latifolium L., Amaryllidaceae, Tap. Chi. Duoc. Hoc. 41 (2001) 21–23. [9] S. Ghosal, P.H. Rao, K.S. Saini, Natural occurrence of 11-O-acetylambelline and 11O-acetyl-l,2-β-epoxyambelline in Crinum latifolium: immuno-regulant alkaloids, Pharm. Res. 2 (1985) 251–252. [10] S. Ghosal, K.S. Saini, V.K. Arora, 1,2-β-Epoxyambelline, an immunostimulant alkaloid from Crinum latifolium, J. Chem. Res. (1984) 232–233 (S). [11] C. Ianello, J. Bastida, F. Bonvicini, F. Antognoni, G.A. Gentilomi, F. Poli, Chemical composition and in vitro antibacterial and antifungal activity of an alkaloid extract from Crinum angustum Steud, Nat. Prod. Res. 28 (2014) 704–710. [12] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part I- Crinum alkaloids: lycorine-type alkaloids, Int. J. Pharm. Sci. Res. 3 (2012) 1883–1890. [13] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part II. Crinum alkaloids: crinine-type alkaloids, Int. J. Pharm. Sci. Res. 3 (2012) 3091–3100. [14] J. Refaat, M.S. Kamel, M.A. Ramadan, A.A. Ali, Crinum; an endless source of bioactive principles: a review. Part III; Crinum alkaloids: belladine- galanthamine-, lycorenine-, tazettine-type alkaloids and other minor types, Int. J. Pharm. Sci. Res. 3
All compounds and reference drug are expressed as IC50 values in μM. b Percent inhibition (all compounds and reference drugs concentration: 100 μM). a
basis of the similarity of NOESY data. Accordingly, the structure of 4 was established as 4-hydroxy-8-methoxy-cripowellin B. The Amaryllidaceae alkaloids from Crinum genus have been reported to show strong cytotoxicity against various types of cancer cell lines [4], which were agreed well with our results. The in vitro cytotoxic activity of these compounds against seven lung cancer cell lines using the modified MTT method was summarized in Table 2. Among the tested compounds, alkaloid 2–4 exhibited potent the cytotoxic potential against all tested tumor cell lines, with IC50 values of 14.5–17.3, 7.1–9.6, and 7.4–10.1 nM, respectively. Alkaloids 1 and 5 showed high cytotoxicity against all tested tumor cell lines with IC50 values < 30 nM (Table 2). significant. The antimicrobial, radical scavenging, and anti-inflammatory activities for crinane-type alkaloids with the cleavage between C-1 and C13 has been reported for the first time. The isolated alkaloids were tested for their antimicrobial activity by measuring the zone of inhibitions and exhibited interesting and promising antimicrobial activity (Table 3). Alkaloids 3 and 4 had the highest antimicrobial potential against all assayed microorganisms with MIC value < 0.50 mM (Table 3). Antioxidant evaluation by both DPPH and ABTS assays (Table 4) showed that compounds 2–4 showed significant antiradical activity in both the tested radical of DPPH and ABTS·+. The compounds 1–5 were tested in vitro for their anti-inflammatory activity (Table 4) and displayed comparable inhibition of Cox-1 (> 64%) with positive control SC–560 and of Cox-2 (> 90%) with positive control NS–398, respectively (Table 4). Alkaloids 1–5 52
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(2012) 3630–3638. [15] S. Berkov, S. Romani, M. Herrera, F. Viladomat, C. Codina, G. Momekov, I. Ionkova, J. Bastida, Antiproliferative alkaloids from Crinum zeylanicum, Phytother. Res. 25 (2011) 1686–1692. [16] S. Ghosal, K.S. Saini, A.W. Frahm, Alkaloids of Crinum latifolium, Phytochemistry 22 (1983) 2305–2309. [17] J. Hu, Y. Song, X. Mao, Z. Wang, Q.J. Zhao, Limonoids isolated from Toona Sinensis and their radical scavenging, anti-inflammatory and cytotoxic activities, J. Funct. Foods 20 (2016) 1–9.
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