Limonin isolated from the seeds of Citrus limetta (Indian sweet lemon)

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1 Limonin isolated from the seeds of Citrus limetta (Indian sweet lemon) ISSN A. K. Bauri, a Sabine Foro b and Quynh Nguyen Do Nhu c * Received 18 January 2016 Accepted 27 January 2016 Edited by H. Stoeckli-Evans, University of Neuchâtel, Switzerland Keywords: crystal structure; limonin; tetracyclic terpenoid; polyoxgenated; antiproliferative; C HO hydrogen bonding. CCDC reference: Structural data: full structural data are available from iucrdata.iucr.org a Bioorganic Division, Bhabha Atomic Research Centre, Trombay, Mumbai , India, b FB Material-und Geowissenschaften, Technische Universität Darmstadt, Alarich-Weiss-Strasse 2, Darmstadt, Germany, and c Accident and Emergency Department, Franco Vietnamese Hospital, 7 Nguyen Luong Bang Street, Ho Chi Minh City, Vietnam. *Correspondence nguyendonhuquynh@yahoo.com The title molecule, C 26 H 30 O 8 [systematic name: (4aS,14bR)-12-(furan-3-yl)- 6,6,8a,12a-tetramethyldecahydro-1H,3H-oxireno[2,3-d]pyrano[4 0,3 0 :3,3a]isobenzofuro[5,4-f]isochromene-3,8,10(6H,9aH)-trione], commonly known as limonin, is an antiproliferative polyoxgenated bioactive molecule. It comprises a fused cyclic system containing a cyclohexane (D), a cyclohexanone (C), a furan (B), an epoxide and two lactone rings (A and E). In addition to these rings, a second furan ring (F) is present as a substituent. Only the cyclohexane (D) ring has a chair conformation; the other six-membered rings (A, C and E) have twist-boat conformations due to steric hinderance or bonding strain. In the crystal, molecules are linked by C HO hydrogen bonds, forming a threedimensional structure. Structure description The fruit of Citrus limetta or Indian sweet lemon, is popularly known as Mousombi in Mumbai, India. It was procured from local markets for chemical profiling of the seeds in our search for antiproliferative compounds. The roles of citrous limonoids as functional chemicals in agricultural foods have been reviewed (Berhow et al., 2000). The juices of Citrus limetta possess a pleasant aroma and have been used as a natural antioxidant and preservative in various food industries, whereas its seeds are quiet bitter in taste. The seeds have been segregated in two parts such as pericarp (outer coat/peel of the seeds) and seed kernel (inner part of the seeds). The inner part of the seeds is quiet soft and comparatively heavier than the pericarp which was extracted with methanol. The chemical analysis of this extract revealed that the major constituent is a pale-yellow oil, a glycerol ester of different long-chain fatty acid mixtures. Besides this oil, this extract is a 1of3

2 Table 1 Hydrogen-bond geometry (Å, ). D HA D H HA DA D HA C8 H8AO5 i (3) 171 C4 H4AO6 ii (3) 156 C21 H21O6 iii (3) 124 Symmetry codes: (i) x 1 2 ; y þ 3 2 ; z þ 1; (ii) x þ 1; y 1 2 ; z þ 1 2 ; (iii) x þ 1 2 ; y þ 2; z 1 2. Table 2 Experimental details. Figure 1 The molecular structure of the title compound, showing the atom labelling and displacement ellipsoids drawn at the 50% probability level rich source of some limonoids and their glycosides. Limonoids are highly oxygenated tetracyclic terpenoids. Herein we report on the extraction and crystal structure of the major constituent, the title compound limonin. A view of the molecular structure of the title compound is shown in Fig. 1. The molecule is composed of a series of fused rings. It contains a cyclohexane ring (D) with a chair conformation, a cyclohexanone and two lactone rings (D, A and E) with twist-boat conformations, a furan (B) and an epoxide ring. In addition to these rings, a second furan ring (F) witha flat conformation is present as a substituent. In the crystal, a three-dimensional structure is formed by molecules being linked by C HO hydrogen bonds (Table 1 and Fig. 2). Synthesis and crystallization The source of the title compound is a citrus fruit known as sweet lemon. The crude product has been separated by column chromatography over silica gel by gradient solvent elution. In order to purify the target molecule, it was subjected to Crystal data Chemical formula C 26 H 30 O 8 M r Crystal system, space group Orthorhombic, P Temperature (K) 299 a, b, c (Å) (1), (2), (2) V (Å 3 ) (5) Z 4 Radiation type Cu K (mm 1 ) 0.84 Crystal size (mm) Data collection Diffractometer Enraf Nonius CAD-4 No. of measured, independent and 5062, 4081, 3659 observed [I > 2(I)] reflections R int (sin /) max (Å 1 ) Refinement R[F 2 >2(F 2 )], wr(f 2 ), S 0.036, 0.110, 1.10 No. of reflections 4081 No. of parameters 308 H-atom treatment H-atom parameters constrained max, min (e Å 3 ) 0.20, 0.14 Absolute structure Flack (1983); 1742 Friedel pairs Absolute structure parameter 0.2 (2) Computer programs: CAD-4-PC Software (Enraf Nonius, 1996), REDU4 (Stoe & Cie, 1987), SHELXS97 (Sheldrick, 2008), SHELXL97 (Sheldrick, 2008), PLATON (Spek, 2009). preparative thin layer chromtography followed by crystallization. Crystals suitable for X-ray diffraction were obtained by recrystallization of the title compound from hexane ethylacetate (4:1) at room temperature by slow evaporation. Refinement Crystal data, data collection and structure refinement details are summarized in Table 2. The final refined Flack x parameter is 0.2 (2), and the Hooft y parameter is 0.02 (10), which despite using Cu K radiation does not justify the determination of the absolute structure. Figure 2 The crystal packing of the title compound, viewed along the a axis. The C HO hydrogen bonds are shown as dashed lines (see Table 1). Acknowledgements The authors thank Professor Dr Hartmut Fuess, FG Strukturforschung, FB Material-und Geowissenschaften, Technische Universität Darmstadt, Alarich-Weiss-Strasse 2, Darmstadt, for diffractometer time. 2of3 Bauri et al. C 26 H 30 O 8

3 References Berhow, M. A., Hasegawa, S. & Manners, G. D. (2000). Editors. Citrous limonoids: Functional Chemicals in Agriculture Food. ACS Symposium Series, Vol. 758, Washington, DC: Oxford Press. Enraf Nonius (1996). CAD-4-PC Software. Enraf Nonius, Delft, The Netherlands. Flack, H. D. (1983). Acta Cryst. A39, Sheldrick, G. M. (2008). Acta Cryst. A64, Spek, A. L. (2009). Acta Cryst. D65, Stoe & Cie (1987). REDU4. Stoe & Cie GmbH, Darmstadt, Germany. Bauri et al. C 26 H 30 O 8 3of3

4 full crystallographic data [ Limonin isolated from the seeds of Citrus limetta (Indian sweet lemon) A. K. Bauri, Sabine Foro and Quynh Nguyen Do Nhu (4aS,14bR)-12-(Furan-3-yl)-6,6,8a,12a-tetramethyldecahydro-1H,3H-oxireno[2,3- d]pyrano[4,3 :3,3a]isobenzofuro[5,4-f]isochromene-3,8,10(6H,9aH)-trione Crystal data C 26 H 30 O 8 M r = Orthorhombic, P Hall symbol: P 2ac 2ab a = (1) Å b = (2) Å c = (2) Å V = (5) Å 3 Z = 4 F(000) = 1000 Data collection Enraf Nonius CAD-4 diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω/2θ scans 5062 measured reflections 4081 independent reflections 3659 reflections with I > 2σ(I) Refinement Refinement on F 2 Least-squares matrix: full R[F 2 > 2σ(F 2 )] = wr(f 2 ) = S = reflections 308 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map D x = Mg m 3 Melting point: 320 K Cu Kα radiation, λ = Å Cell parameters from 25 reflections θ = µ = 0.84 mm 1 T = 299 K Prism, colourless mm R int = θ max = 67.0, θ min = 3.9 h = k = 17 1 l = standard reflections every 120 min intensity decay: 1.0% Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ 2 (F o2 ) + (0.0611P) P] where P = (F o 2 + 2F c2 )/3 (Δ/σ) max = Δρ max = 0.20 e Å 3 Δρ min = 0.14 e Å 3 Extinction correction: SHELXL97 (Sheldrick, 2008), Fc * =kfc[ xfc 2 λ 3 /sin(2θ)] -1/4 Extinction coefficient: (3) Absolute structure: Flack (1983); 1742 Friedel pairs Absolute structure parameter: 0.2 (2) data-1

5 Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wr and goodness of fit S are based on F 2, conventional R-factors R are based on F, with F set to zero for negative F 2. The threshold expression of F 2 > 2sigma(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 ) x y z U iso */U eq C (2) (14) (12) (4) C (3) (17) (13) (5) H2A * H2B * C (3) (18) (15) (6) C (3) (16) (13) (6) H4A * H4B * C (2) (16) (13) (5) H * C (3) (17) (14) (6) C (2) (15) (13) (5) H * C (3) (16) (14) (5) H8A * H8B * C (3) (16) (13) (5) C (2) (15) (12) (5) C (2) (14) (12) (4) H * C (3) (15) (12) (5) H12A * H12B * C (3) (16) (13) (5) H13A * H13B * C (2) (14) (12) (4) C (2) (15) (13) (5) C (3) (17) (14) (5) H * C (3) (17) (15) (5) C (2) (16) (13) (5) H * C (2) (17) (13) (5) C (3) (2) (14) (6) data-2

6 H * C (3) (2) (15) (6) H * C (3) (19) (14) (6) H * C (3) (2) (19) (8) H23A * H23B * H23C * C (3) (2) (15) (7) H24A * H24B * H24C * C (3) (18) (13) (5) H25A * H25B * H25C * C (3) (16) (13) (5) H26A * H26B * H26C * O (2) (13) (9) (4) O (17) (12) (10) (4) O (18) (12) (10) (4) O (2) (14) (10) (5) O (3) (18) (13) (8) O (2) (12) (11) (5) O (17) (11) (9) (4) O (2) (15) (12) (6) Atomic displacement parameters (Å 2 ) U 11 U 22 U 33 U 12 U 13 U 23 C (10) (11) (10) (9) (8) (9) C (11) (13) (12) (10) (9) (10) C (14) (13) (14) (12) (12) (11) C (12) (13) (13) (10) (10) (11) C (10) (12) (11) (9) (9) (10) C (11) (13) (14) (10) (10) (11) C (10) (11) (11) (9) (9) (9) C (11) (12) (12) (9) (10) (9) C (11) (12) (12) (9) (10) (10) C (10) (11) (11) (9) (9) (9) C (9) (10) (10) (9) (8) (9) C (11) (11) (11) (9) (9) (10) C (11) (11) (11) (9) (9) (9) C (10) (11) (11) (9) (9) (9) C (10) (11) (12) (9) (9) (9) data-3

7 C (11) (13) (12) (10) (10) (11) C (11) (13) (14) (10) (10) (12) C (10) (11) (11) (9) (9) (9) C (10) (12) (11) (10) (9) (10) C (14) (15) (12) (12) (11) (11) C (14) (16) (13) (13) (12) (12) C (15) (14) (14) (12) (12) (12) C (14) (15) (2) (12) (15) (15) C (15) (18) (15) (14) (12) (13) C (10) (14) (11) (11) (10) (11) C (11) (13) (12) (10) (10) (10) O (9) (10) (8) (9) (8) (8) O (7) (9) (10) (7) (7) (8) O (8) (10) (9) (8) (7) (8) O (11) (12) (9) (10) (8) (8) O (16) (17) (12) (15) (12) (12) O (12) (9) (11) (9) (10) (9) O (7) (9) (9) (7) (7) (8) O (11) (14) (13) (11) (10) (11) Geometric parameters (Å, º) C1 C (3) C13 H13A C1 C (3) C13 H13B C1 C (3) C14 C (3) C1 C (3) C14 C (3) C2 O (3) C14 C (3) C2 H2A C15 O (3) C2 H2B C15 C (3) C3 O (3) C16 O (3) C3 O (3) C16 C (3) C3 C (4) C16 H C4 C (3) C17 O (3) C4 H4A C17 O (3) C4 H4B C18 O (3) C5 O (3) C18 C (3) C5 H C18 H C6 O (3) C19 C (4) C6 C (4) C19 C (3) C6 C (4) C20 O (3) C6 C (3) C20 H C7 C (3) C21 C (4) C7 H C21 O (3) C8 C (3) C21 H C8 H8A C22 H C8 H8B C23 H23A C9 O (3) C23 H23B C9 C (3) C23 H23C data-4

8 C10 C (3) C24 H24A C10 C (3) C24 H24B C10 C (3) C24 H24C C11 C (3) C25 H25A C11 H C25 H25B C12 C (3) C25 H25C C12 H12A C26 H26A C12 H12B C26 H26B C13 C (3) C26 H26C C2 C1 C (18) C14 C13 H13B C2 C1 C (18) H13A C13 H13B C5 C1 C (17) C15 C14 C (18) C2 C1 C (17) C15 C14 C (17) C5 C1 C (17) C26 C14 C (17) C7 C1 C (16) C15 C14 C (18) O1 C2 C (18) C26 C14 C (18) O1 C2 H2A C18 C14 C (17) C1 C2 H2A O7 C15 C (14) O1 C2 H2B O7 C15 C (18) C1 C2 H2B C16 C15 C (19) H2A C2 H2B O7 C15 C (18) O5 C3 O (3) C16 C15 C (19) O5 C3 C (3) C14 C15 C (17) O1 C3 C (2) O7 C16 C (14) C3 C4 C (19) O7 C16 C (2) C3 C4 H4A C15 C16 C (2) C5 C4 H4A O7 C16 H C3 C4 H4B C15 C16 H C5 C4 H4B C17 C16 H H4A C4 H4B O8 C17 O (2) O2 C5 C (18) O8 C17 C (2) O2 C5 C (17) O3 C17 C (2) C4 C5 C (19) O3 C18 C (17) O2 C5 H O3 C18 C (18) C4 C5 H C19 C18 C (18) C1 C5 H O3 C18 H O2 C6 C (2) C19 C18 H O2 C6 C (2) C14 C18 H C23 C6 C (2) C20 C19 C (2) O2 C6 C (17) C20 C19 C (2) C23 C6 C (2) C22 C19 C (2) C24 C6 C (2) C19 C20 O (2) C6 C7 C (18) C19 C20 H C6 C7 C (18) O4 C20 H C8 C7 C (18) C22 C21 O (2) C6 C7 H C22 C21 H C8 C7 H O4 C21 H data-5

9 C1 C7 H C21 C22 C (3) C9 C8 C (18) C21 C22 H C9 C8 H8A C19 C22 H C7 C8 H8A C6 C23 H23A C9 C8 H8B C6 C23 H23B C7 C8 H8B H23A C23 H23B H8A C8 H8B C6 C23 H23C O6 C9 C (2) H23A C23 H23C O6 C9 C (2) H23B C23 H23C C8 C9 C (19) C6 C24 H24A C9 C10 C (18) C6 C24 H24B C9 C10 C (18) H24A C24 H24B C15 C10 C (18) C6 C24 H24C C9 C10 C (17) H24A C24 H24C C15 C10 C (17) H24B C24 H24C C25 C10 C (18) C10 C25 H25A C12 C11 C (17) C10 C25 H25B C12 C11 C (17) H25A C25 H25B C1 C11 C (16) C10 C25 H25C C12 C11 H H25A C25 H25C C1 C11 H H25B C25 H25C C10 C11 H C14 C26 H26A C11 C12 C (17) C14 C26 H26B C11 C12 H12A H26A C26 H26B C13 C12 H12A C14 C26 H26C C11 C12 H12B H26A C26 H26C C13 C12 H12B H26B C26 H26C H12A C12 H12B C3 O1 C (19) C12 C13 C (18) C5 O2 C (16) C12 C13 H13A C17 O3 C (18) C14 C13 H13A C21 O4 C (2) C12 C13 H13B C16 O7 C (14) C5 C1 C2 O (3) C26 C14 C15 C (2) C7 C1 C2 O (3) C18 C14 C15 C (3) C11 C1 C2 O (19) C13 C14 C15 C (2) O5 C3 C4 C (3) C26 C14 C15 C (2) O1 C3 C4 C (3) C18 C14 C15 C (18) C3 C4 C5 O (2) C13 C14 C15 C (2) C3 C4 C5 C (3) C9 C10 C15 O (2) C2 C1 C5 O (2) C25 C10 C15 O (3) C7 C1 C5 O (2) C11 C10 C15 O (19) C11 C1 C5 O (18) C9 C10 C15 C (3) C2 C1 C5 C (2) C25 C10 C15 C (3) C7 C1 C5 C (18) C11 C10 C15 C (19) C11 C1 C5 C (2) C9 C10 C15 C (2) O2 C6 C7 C (2) C25 C10 C15 C (19) C23 C6 C7 C (3) C11 C10 C15 C (3) data-6

10 C24 C6 C7 C (3) C14 C15 C16 O (2) O2 C6 C7 C (2) C10 C15 C16 O (2) C23 C6 C7 C (2) O7 C15 C16 C (2) C24 C6 C7 C (2) C14 C15 C16 C (3) C2 C1 C7 C (2) C10 C15 C16 C (2) C5 C1 C7 C (2) O7 C16 C17 O (3) C11 C1 C7 C (17) C15 C16 C17 O (2) C2 C1 C7 C (3) O7 C16 C17 O (3) C5 C1 C7 C (19) C15 C16 C17 O (3) C11 C1 C7 C (2) C15 C14 C18 O (2) C6 C7 C8 C (2) C26 C14 C18 O (2) C1 C7 C8 C (3) C13 C14 C18 O (17) C7 C8 C9 O (2) C15 C14 C18 C (18) C7 C8 C9 C (3) C26 C14 C18 C (2) O6 C9 C10 C (3) C13 C14 C18 C (2) C8 C9 C10 C (18) O3 C18 C19 C (2) O6 C9 C10 C (3) C14 C18 C19 C (3) C8 C9 C10 C (2) O3 C18 C19 C (3) O6 C9 C10 C (2) C14 C18 C19 C (3) C8 C9 C10 C (2) C22 C19 C20 O4 0.6 (3) C2 C1 C11 C (2) C18 C19 C20 O (2) C5 C1 C11 C (3) O4 C21 C22 C (3) C7 C1 C11 C (19) C20 C19 C22 C (3) C2 C1 C11 C (2) C18 C19 C22 C (2) C5 C1 C11 C (18) O5 C3 O1 C (3) C7 C1 C11 C (2) C4 C3 O1 C (3) C9 C10 C11 C (17) C1 C2 O1 C (3) C15 C10 C11 C (2) C4 C5 O2 C (2) C25 C10 C11 C (2) C1 C5 O2 C (2) C9 C10 C11 C (2) C23 C6 O2 C (2) C15 C10 C11 C (17) C24 C6 O2 C (2) C25 C10 C11 C (2) C7 C6 O2 C5 5.8 (2) C1 C11 C12 C (19) O8 C17 O3 C (2) C10 C11 C12 C (2) C16 C17 O3 C (3) C11 C12 C13 C (2) C19 C18 O3 C (2) C12 C13 C14 C (2) C14 C18 O3 C (3) C12 C13 C14 C (2) C22 C21 O4 C (3) C12 C13 C14 C (19) C19 C20 O4 C (3) C26 C14 C15 O (17) C17 C16 O7 C (2) C18 C14 C15 O (2) C14 C15 O7 C (2) C13 C14 C15 O (2) C10 C15 O7 C (2) Hydrogen-bond geometry (Å, º) D H A D H H A D A D H A C8 H8A O5 i (3) 171 data-7

11 C4 H4A O6 ii (3) 156 C21 H21 O6 iii (3) 124 Symmetry codes: (i) x 1/2, y+3/2, z+1; (ii) x+1, y 1/2, z+1/2; (iii) x+1/2, y+2, z 1/2. data-8