2 School of Biological Science and Technology, University of Jinan, Jinan 250022, China
Ipomoea nil (L.) Roth (formerly known as Pharbitis nil (L.) Choisy) is an ornamental plant widely cultivated all over the world, and its seeds, called 'Qian niu zi' in traditional Chinese medicine, have commonly been used as diuretic and insecticide or to eliminate phlegm . According to the color, 'Qian niu zi' is normally divided into two types, namely, 'Hei chou' and 'Bai chou' . Previous investigations into the chemical constituents of I. nil seeds have revealed diverse structural types including lignans [3-5], diterpenoids and their glycosides [6-8], lipid glycosides [9-10], simple phenolic constituents [5, 11], and so on [12-14]. Most of these natural products were reported to show cytotoxicity against a variety of human cancer cell lines [3-7, 9, 12]. However, the non-polar lipid constituents of I. nil seeds have seldom been reported . In our present work, we carried out an intensive chemical investigation on the EtOAc partition generated from the ethanolic extract of the seeds of I. nil, which resulted in the isolation and identification of a pair of new octadecanoid glycerides (1 and 2), two new octadecanoid ethyl esters (3 and 4), and a series of fatty acid analogues (5–16). The structures of these compounds were elucidated by comprehensive spectroscopic analyses, and the absolute configurations of 1 and 2 were determined by an in situ dimolybdenum ECD method. All the compounds (1–16) were tested for their ABTS radical scavenging and α-glucosidase inhibitory activities, while only the known 10 and 13 exhibited inhibitory effects toward α-glucosidase with IC50 of 92.73 ± 3.12 and 11.39 ± 2.18 μmol·L–1, respectively. Herein, we describe the separation, structural determination and biological evaluations of these lipid molecules.Results and Discussion
Compounds 1/2 were assigned the molecular formula of C21H38O5 by (+)-HR-ESIMS analysis at m/z 371.2792 ([M + H]+, calcd. 371.2792). The 1H NMR data (Table 1) revealed the presence of a cis-form double bond [δH 5.27 (1H, dtt, J = 10.8, 7.2, 1.5 Hz), and 5.37 (1H, dtt, J = 10.8, 7.3, 1.5 Hz)], a glycerol moiety [δH 3.58 (1H, dd, J = 11.2, 5.9 Hz), 3.68 (1H, brd, J = 11.2 Hz), 3.92 (1H, m), 4.13 (1H, dd, J = 11.6, 6.2 Hz), 4.17 (1H, dd, J = 11.6, 4.7 Hz)], a methyl [δH 0.87 (t, J = 7.0 Hz)], and 13 aliphatic methylenes [δH 1.30 (12H, m), 1.54 (2H, m), 1.60 (2H, m), 2.01 (2H, qd, J = 7.3, 1.5 Hz), 2.28 (2H, qd, J = 7.4, 1.4 Hz), 2.33 (2H, t, J = 7.5 Hz), 2.38 (2H, t, J = 7.4 Hz), 2.42 (2H, t, J = 7.4 Hz)]. The 13C NMR data (Table 1) showed signals for a ketone (δC 211.3, C-9), an ester carbonyl (δC 174.3, C-1), two olefinic (δC 127.8, 131.4), 16 methylenes (δC 21.8–65.3) and a methyl (δC 14.2) carbons. These spectral features were similar to those of (2'S)-1-O- (9-oxo-10(E), 12(E)-octadecadienoyl) glycerol isolated from the tuber-barks of Colocasia antiquorum var. esculenta , and the only difference was that compounds 1/2 only had one double bond. The positions of the double bond and the ketone were confirmed by examination of 2D NMR data (Fig. 2), with key 1H–1H COSY correlations across H2-10 to H2-14 and pivotal HMBC correlations from H3-18 to C-16, H2-14 to C-12 and C-16, and H2-7, H2-8, H2-10 and H2-11 to the C-9 ketone. The glycerol moiety was connected to C-1 to form an ester bond, based on the HMBC correlations from H2-1' to C-1 and the downfield chemical shifts for H2-1' due to esterification. Thus the planar structures of compounds 1/2 were characterized as 1-O-(9-oxo-12Z-octadecamonoyl) glycerol with only one chiral center. When measuring the optical rotation of 1/2, the near zero value alerted that the enantiomeric purity might need to be checked. Subsequent chiral HPLC analysis revealed a pair of enantiomers in a ratio of ca. 3: 2, which led to the further chiral separation of 1 ([α]D24 +3.0) and 2 ([α]D24 –3.3). The absolute configuration 2 was determined to be 2'R based on the positive Cotton effect at 311 nm (Δε +6.4) (Fig. 3) in the in situ dimolybdenum ECD experiment developed by Snatzke and Frelek [17-18], and that of 1 was thus assigned to be 2'S.
Compound 3, colorless oil, was assigned the molecular formula of C20H34O3 by (+)-HR-ESIMS analysis at m/z 345.2398 ([M + Na]+, calcd. 345.2400). The 1H and 13C NMR data of 3 (Table 1) were highly similar to those of (10E, 12Z, 15Z)-9-hydroxy-10, 12, 15-octadecatrienoic acid methyl ester , except that the methoxyl group [δH 3.66 (s); δC 51.4] in the latter was replaced by an ethoxyl moiety [δH 1.25 (3H, t, J = 7.2 Hz), 4.12 (2H, q, J = 7.2 Hz); δC 14.4, 60.3] in 3. This assignment was further supported by careful inspection of 2D NMR data (Fig. 2). Therefore, the structure of 3 was identified as (10E, 12Z, 15Z)-9-hydroxy-10, 12, 15-octadecatrienoic acid ethyl ester.
Similarly, compound 4 was assigned the molecular formula of C20H36O3 by (+)-HR-ESIMS analysis at m/z 347.2550 ([M + Na]+, calcd 347.2557). The 1H and 13C NMR data of 4 (Table 1) were highly similar to those of (10E, 12E)-9-hydroxy-10, 12-octadecadienoic acid methyl ester , except that the methoxyl group [δH 3.67 (s); δC 51.3] in the latter was replaced by an ethoxyl moiety [δH 1.25 (3H, t, J = 7.1 Hz), 4.12 (2H, q, J = 7.2 Hz); δC 14.4, 60.3] in 4. Detailed examination of 2D NMR data (Fig. 2) further confirmed this structural assignment. Thus the structure of 4 was determined as (10E, 12E)-9-hydroxy-10, 12-octadecadienoic acid ethyl ester.
Both 3 and 4 with an ethoxyl group in the structure were possibly artifacts from esterification of their respective free acid or from interesterification of their respective methyl ester, due to use of ethanol as the extracting solvent. The two compounds had only one stereocenter in their structures and showed very small [α]D values (see Experimental), which also alerted us to check their enantiomeric purities like we did to compounds 1/2. Interestingly, no separable peaks were resolved for both compounds on our three different types of chiral columns (two normal phases and one reversed phase, see Experimental).
The known compounds were identified, on the basis of detailed spectroscopic interpretation, to be 9-octadecenoic acid-2', 3'-dihydroxy propyl ester (5) , (Z)-12-octadecenic- α-glycerol monoester (6) , glycerol 1-9', 12'-octadecadienoate (7) , 1-octadecatetraenoyl glycerol (8) , 2-linoleoylglycerol (9) , 13-hydroxy-9Z, 11E-octadecadienoic acid (10) , 9-oxooctadec-cis-12-enoic acid (11) , linoleic acid (12) , rabdosia acid A (13) , oxylipin (14) , (Z)-9, 10, 11-trihydroxy-12-octadecenoic acid (15)  and (8R, 9R, 10S, 6Z)-trihydroxyoctadec-6-enoic acid (16)  (see Fig. S1, Supporting information).
All the isolates were evaluated in two bioassays, namely, ABTS radical scavenging and α-glucosidase inhibitory tests. While none of them showed ABTS radical scavenging activity (at 100μmol·L–1, ascorbic acid as positive control), compounds 10 and 13 displayed inhibitory activity against α-glucosidase with IC50 of 92.73 ± 3.12 and 11.39 ± 2.18 μmol·L–1, respectively (acarbose as positive control, IC50 167.7 ± 1.55 μmol·L–1).Experimental General experimental procedures
Optical rotations were measured on a Rudolph Ⅵ polarimeter (Rudolph Research Analytical, Hackettstown, USA) with a 10 cm length cell. NMR experiments were recorded on a Bruker Avance DRX600 spectrometer (Bruker BioSpin AG, Fallanden, Switzerland) and referenced to residual solvent peaks (CDCl3: δH 7.26, δC 77.16). HR-ESIMS spectra were obtained on an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany). ESIMS analyses were carried out on an Agilent 1260-6460 Triple Quad LC-MS instrument (Agilent Technologies Inc., Waldbronn, Germany). UV spectra were obtained on a Shimadzu UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan) with a 1 cm pathway cell. All normal HPLC separations were performed using an Agilent 1260 series LC instrument (Agilent Technologies Inc., Waldbronn, Germany) coupled with an Agilent SB-C18 column (9.4 mm × 250 mm, Agilent Technologies Inc., Santa Clara, USA) unless specified. CHIRALPAK AD-H and OD-H (both 4.6 mm × 250 mm) columns (Daicel Corporation, Tokyo, Japan) and Chiral MZ (2) RH 5u (4.6 mm × 250 mm) column (Phenomenex, Washington D.C., USA), were used for chiral HPLC analysis and separation. Column chromatography (CC) was performed on D101-macroporous absorption resin (Sinopharm Chemical Reagent Co., Ltd., Shanghai), reversed phase C18 silica gel (Merck KGaA, Darmstadt, Germany), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and silica gel (300–400 mesh; Qingdao Marine Chemical Co. Ltd., Qingdao, China). All solvents used for CC were of analytical grade (Tianjin Fuyu Fine Chemical Co. Ltd., Tianjin, China) and solvents used for HPLC were of HPLC grade (Oceanpak Alexative Chemical Ltd., Goteborg, Sweden). Pre-coated silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd., Qingdao, China) were used for TLC monitoring.Plant materials
The seeds of Ipomoea nil (L.) Roth were bought in Kunming 'Juhuayuan' herbal market and were authenticated by Prof. ZHOU Jie from University of Jinan. A voucher specimen has been deposited at School of Biological Science and Technology, University of Jinan (Accession number: npmc-024).Extraction and isolation
The air-dried powder of the seeds of I. nil (30 kg) was extracted with 95% EtOH at room temperature three times to afford a crude extract (2.8 kg). The extract was then suspended in 2.0 L water and partitioned with EtOAc (2.0 L × 3). The EtOAc soluble extract (445 g) was subjected to CC over D101-macroporous absorption resin, eluted with EtOH–H2O (30%, 50%, 80% and 90%), to afford four fractions (A, B, C and D). Fraction C (74 g) was subjected to silica gel CC, eluted with petroleum ether–ethyl acetate (50 : 1 to 1 : 2, V/V), to produce 40 subfractions (C1–C40). Fraction C12 was separated by silica gel CC (petroleum ether–chloroform, 5 : 1 to 1 : 2, V/V) to produce three subfractions (C12-1–C12-3), and then C12-1 was first fractionated by Sephadex LH-20 CC (MeOH) and further purified by HPLC (3.0 mL·min–1, 80% MeCN-H2O) to afford 12 (1.6 mg, tR = 15.0 min). Fraction C14 was separated by silica gel CC, eluted with petroleum ether–chloroform (5 : 1 to 1 : 2, V/V), to produce two subfractions (C14-1 and C14-2), and then C14-2 was first fractionated by Sephadex LH-20 CC (MeOH) and further separated by HPLC (3.0 mL·min–1, 72% MeCN–H2O, tR = 11.6, 12.5 and 15.0 min, respectively) to afford 3 (2.8 mg), 10 (5.4 mg) and 4 (2.8 mg). Fraction C20 was separated by silica gel CC, eluted with petroleum ether–acetone (10 : 1 to 1 : 2, V/V), to produce three subfractions (C20-1–C20-3), and then C20-2 was purified by HPLC (3.0 mL·min–1, 50% MeCN–H2O) to afford 5 (5.6 mg, tR = 11.5 min). Fraction C28 was subjected to Sephadex LH-20 CC (MeOH) to give one major subfraction which was further purified by HPLC (3.0 mL·min–1, 67% MeCN–H2O) to furnish 13 (15.0 mg, tR = 11.6 min). Fraction C33 was separated by silica gel CC, eluted with petroleum ether-chloroform (10 : 1 to 1 : 2, V/V), to produce three subfractions (C33-1–C33-3), and then C33-2 was first fractionated by Sephadex LH-20 CC (MeOH) and further purified by HPLC (3.0 mL·min–1, 55% MeCN-H2O, tR = 7.0, 9.0 and 9.5 min, respectively) to afford 14 (1.2 mg), 15 (3.1 mg) and 16 (2.4 mg). Fraction C37 was first subjected to Sephadex LH-20 CC (MeOH) to give two major subfractions (C37-1 and C37-2) and C37-1 was then purified by HPLC (3.0 mL·min–1, 50%–95% MeCN–H2O in 20 min, tR = 5.0, 7.0, 8.5, 16.0, 18.5 and 21.5 min, respectively) to afford 1/2 (22.5 mg), 8 (2.5 mg), 11 (1.6 mg), 9 (15.2 mg), 7 (163.8 mg) and 6 (33.3 mg). Compounds 1 (1.7 mg, tR = 11.0 min) and 2 (1.1 mg, tR = 12.0 min) were then separated from each other on an OD-H chiral column (1.0 mL·min–1, 10% isopropanol in n-hexane).Identification of new compounds Compounds 1/2
Colorless oil; [α]D24 +3.0 (c 0.17, MeOH) for 1, –3.3 (c 0.11, MeOH) for 2; (+)-ESIMS m/z 371.2 [M + H]+; (+)-HR- ESIMS m/z 371.2792 [M + H]+ (Calcd. for C21H39O5, 371.2792); 1H and 13C NMR data (CDCl3) see Table 1.Compound 3
Colorless oil; [α]D24 –1.3 (c 0.28, MeOH); UV (MeOH) lmax (log ε): 204 (3.57), 217 (3.50) nm; (+)-ESIMS m/z 345.2 [M + Na]+; (+)-HR-ESIMS m/z 345.2398 [M + Na]+ (Calcd. for C20H34O3Na, 345.2400); 1H and 13C NMR (CDCl3) data see Table 1.Compound 4
Colorless oil; [α]D24 +1.4 (c 0.28, MeOH); UV (MeOH) λmax (log ε): 224 (3.66) nm; (+)-ESIMS m/z 347.2 [M + Na]+; (+)-HR-ESIMS m/z 347.2550 [M + Na]+ (Calcd. for C20H36O3Na, 347.2557); 1H and 13C NMR data (CDCl3) see Table 1.α-Glucosidase inhibitory assay
Briefly, 0.2 U of α-glucosidase from Saccharomyes cerevisiae (Sigma-Aldrich, St. Louis, MO, USA) was diluted by 0.1 mol·L–1 phosphate buffer consisting of Na2HPO4 and NaH2PO4 (pH 6.8). The assay was conducted in a 200 μL reaction system containing 98 μL of buffer, 25 μL of diluted enzyme solution and 2 μL of DMSO (blank control) or tested samples (dissolved in DMSO). After 20 min of incubation in 96-well plates at 37 ℃, 25 μL of 0.4 mmol·L–1 PNPG (4-nitrophenyl-β-D-glucopyranoside, Aladdin, Shanghai, China) was added as substrate to start the enzymatic reaction. The plate was incubated for an additional 15 min at 37 ℃, followed by the addition of 50 μL of 0.2 mol·L–1 Na2CO3 to stop the reaction. The optical density (OD) was measured at an absorbance wavelength of 405 nm using a Microplate Reader (Tecan, Switzerland). The inhibitory effects of all the isolates were first assessed at the concentration of 100 mmol·L–1, and only compounds showing > 50% inhibition rate were further subjected for IC50 measurements. Acarbose (Aladdin, Shanghai, China) was used as a positive control with an IC50 of 167.7 ± 1.55 μmol·L–1.ABTS radical scavenging assay
The ABTS radical scavenging assay was performed as we reported previously . All the compounds were first assessed at the concentration of 100 μmol·L–1, while none of them exhibited enough activity for further IC50 measurement. Ascorbic acid was used as a positive control.Acknowledgements
We thank Prof. ZHOU Jie for the identification of the plant materials.Supporting information
Original spectroscopic data including chiral HPLC separation, HR-ESIMS and NMR (1H, 13C, 1H-1H COSY, HSQC and HMBC) spectra for compounds 1–4 were provided.
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