Extraction and Crystal Structure of β-Sitosterol

WU Xiao-Yi CHAO Zhi-Mao WANG Chun SUN Wen ZHANG Gui-Feng

?

Extraction and Crystal Structure ofSitosterol

WU Xiao-Yi CHAO Zhi-Mao WANG Chun SUN Wen ZHANG Gui-Feng

(,,100700)

The title compoundsitosterol (C29H50O), an active phytosterol in many medicinal and edible plants, was characterized by X-ray diffraction analysis and extensive nuclear magnetic resonance (NMR) data. It crystallizes in monoclinic system, space group21with C29H50O·1/2H2O,= 9.4226(7),= 7.4824(9),= 36.889(3) ?,= 2597.0(4) ?3,= 4,D= 1.084 g/cm3,M= 423.70,(000) = 948, and= 0.064 mm-1. The final= 0.0886 and= 0.2234 for 10157 observed reflections (> 2()). The molecular crystal structure ofsitosterol shows relative stereochemistry of24R-ethylcholest-5-en-3-ol. The molecule is composed of one steroid nucleus (3 six-membered rings and1 five-membered ring) and one sidechain of 10 carbons. There are two C29H50O molecules and one H2O molecule in a symmetrical unit, and the title compound is stacked into aspecial laminated structure through hydrogen bonds and van der Waal forces. The special laminated structure was first reported.

-sitosterol, crystal structure, NMR, X-ray diffraction, laminated structure

1 INTRODUCTION

Sitosterol, one of the most abundant and wide phytosterols, exists in some edible plants, such as, fruit of[1], seed of[2], leaf of, and rhizome of[3], and in some traditional Chinese medicines, such as fruit of[4],andseeds of,, and[5].Sitosterol can reduce the low-density lipoprotein cholesterol (LDL-C) level on hypercholesterolemic children by 20% at a dose of 6 g/day[6]and a tall oil containing 20% sitostanol and 80% phytosterols can reduce the LDL-C level on hypercholesterolemic men by 15% at a dose of 1.7 g/day[7].Sitosterol exerts antipro- liferative effect via mitochondrial pathway and membrane death receptor pathway at concentrations of 1～15 μmol/L[8]. The urinary flow parameters of the symptomatic benign prostatic hyperplasia can be significantly improved with 20 mgsitosterol three times per day[9].Sitosterol has anti-androgen and anti-inflammation activities for the inhibition on experimental mice prostatic hyperplasia at the doses of 8.0 and 16.0 mg/kg·d for 15 d[10].

The incomplete crystallographic data of mono- hydrate, hemihydrate, and microcrystallinesito- sterol were reported[11-13], and all assignments of NMR data ofsitosterol have not yet been provi- ded[1, 14, 15]. In the project of exemplary preparation of certified reference material based on the Inter- national Standardization Organization (ISO) Guide 35:2006 (GB/T 15000.3-2008), a batch ofsito- sterol crystal was extracted, its stereochemical struc- ture was confirmed by X-ray diffraction, and the qualitative and quantity determinations of some tra- ditional Chinese medicine and agricultural products containingsitosterol were carried out. Here, we report the crystal structure determination of hemi- hydrate-sitosterol by X-ray diffraction and NMR data. Meanwhile, we describe the special laminated structure of-sitosterol which may play an impor- tant role in pharmacological activity.

2 EXPERIMENTAL

2. 1 Extraction and crystallization

The fatty oil (2.25 kg) was extracted by hot petroleum ether from the dried ripe seeds (9.56 kg) ofL. (Cruciferae). The unsaponifiable matter (90.2 g) was obtained by 5% KOH-MeOH (w/v) reaction and a silica gel column chroma- tography was carried out with a gradient elution of petroleum ether and ethyl acetate.Sitosterol (8.0 g) was obtained with a purity of 75% by high per- formance liquid chromatography (HPLC) with eva- porative light scattering detector (ELSD) and dis- solved with 1600 mL of chromatographic MeOH (Merck) in a 2 L flask at a 60 ℃ water bath. The saturated solution ofsitosterol was formed and cooled to room temperature at 25 ℃ slowly. Two days later, some thick crystals were obtained. After recrystallizing twice in methanol, 3.6 g of columnar transparentsitosterol crystals were obtained, with a purity of 95% by HPLC-ELSD analysis.

2. 2 Physical and chemical properties

Colorless, soft columnar crystal, m.p: 137～139 ℃. Elemental analysis was performed on an Ele- mentarVarioEL cube V2.0.1 elemental analyzer for C29H50O·1/2H2O as C 82.27 and H 12.06% (Found C 82.24 and H 12.06%). IR spectrum was recorded on a Shimadzu FTIR-8400s spectrometer with KBr pellet. IR (KBr): (OH) 3421, (CH3) 2961, 2868, 1375, (CH2) 2935, 2850, 1465, (C-O-C) 1060, (C=CH) 800 cm-1. EI-MS were recorded on Thermo Quest Trace mass spectrometry as/(%): 414(M+, 56), 399(M-CH3, 22), 396(M-H2O, 33), 381(M- CH3-H2O, 22), 273(M-SC, 20), 255(M-SC-H2O, 35),255(M-SC-H2O, 35), 231(M-SC-42, 20), 213(M-213(M-SC-42-H2O, 35), 43(Me2CH, 100). NMR spectra (1H,13C, DEPT, COSY, HMQC, and HMBC) were measured on a Bruker AV400 instrument (400 MHz for1H) with CDCl3as a solvent and TMS as an internal reference. The detailed assignment of all1H and13C NMR is summarized in Table 1. These data are consistent with references[1, 14, 15].

Table 1. NMR Spectral Assignment of β-Sitosterol (in CDCl3)

2. 3 Crystal structure determination

The intensity data were collected on a Gemini E X-ray single-crystal diffractometer (Agilent Tech- nologies) (= 0.71073 ?) radiation at 101(2) K. The structure was solved by direct methods and re- fined by full-matrix least-squares methods on2employing the SHELXL-97 software[16, 17]. All the non-hydrogen atoms were refined anisotropically. The hydrogen atoms were located by geometrical calculations, and their positions and thermal para- meters were fixed during the structure refinement. The crystallographic data and structural refinement details are summarized in Table 2. The structure reversion of this compound was detected, and theindices of the enantiomer were similar to the com- pound. Theindices (all data) gave= 0.0998 and= 0.2316, and the finalindices (> 2()) gave= 0.0886 and= 0.2234.

3 RESULTS AND DISCUSSION

The molecular structure of one compound is shown in Fig. 1. The perspective view ofthe crystal stacking through hydrogen bonds is shown in Fig. 2. The hydrogen bonding structure of the crystal stackingis shown in Fig. 3. The 3D supramolecular laminated structure of the title compound is shown in Fig. 4. The selected bond lengths and bond angles are given in Tables 3 and 4, respectively.The de- tailed information of hydrogen bonds is given in Table 5.

Fig. 1. Molecular structure and atomicnumbering scheme of the compound

In Fig. 1,sitosterol possesses a steroid skeleton and a sidechain. There are one ethyl group at 24- position withconfiguration and one methyl group at 21-position withconfiguration in the sidechain of ten carbons. The sidechain links at 17-position of the steroid skeleton withconfiguration. The steroid skeleton is composed of one five-membered ring of D and three six-membered rings of A, B, and C with ring junctures of B/Cand C/Dconfiguration. There are one double bond in C(5)– C(6) with a bond length of 1.340(5) ?, one hy- droxyl group at the 3-position withconfiguration, and two methyl groups at 10- and 13-positions withconfiguration in the steroid nucleus.

The 2D laminated structure ofsitosterol is shown inFig. 2. Because of the distinctive la- minated structure, the2D laminated structure can be classified to a sidechain layer, steroid skeleton layer, and water layer. The sidechain layer can easily com- bine with the lipid of cell membrane, the steady and regular steroid skeleton layer can play a role in decreasing the membrane fluidity, and the water layer likes to interact with hydrophilic matter. The regular laminated structure may interpret some pharmacological mechanisms, which regulates the tumor growth by decreasing the membrane flui- dity[18], reduces the fluidity of fatty acyl group of phospholipid[19], increases the thickness of double layer coating of lipidosome[20], and maintains the stability of lipidosome[21].

Fig. 2. 2D laminated structure of the title compound viewed from a

Fig. 3. Hydrogen bonding structure of the crystal stacking

Fig. 4. 3D supramolecularlaminated structure of the title compound viewed from b

Table 2. Crystal Data and Structure Refinement for β-Sitosterol

a=1/[2(F2)+(0.1087)2+ 3.5936], where= (F2+ 2F2)/3

Table 3. Selected Bond Lengths (?)

Table 4. Selected Bond Angles (°)

Table 5. Hydrogen Bonds forβ-Sitosterol(?, °)

As shown in Fig. 3, there are five hydrogen bonds which contribute to a five-membered ring of O(1)– H(1)···O(3)–H(3B)···O(2)···H(3A)–O(3)–H(3B)···O(2)–H(2)···O(1)–H(1) in the crystal packing. The O(2) and O(1) atoms are from two title compounds and O(3) atom is from H2O. The detailed infor- mation of hydrogen bonds can be seen in Table 5.

The crystal packing is regular and tidy in Fig. 4. The formation of the 3D supramolecular laminated structure is contributed by the hydrogen bonds and van der Waal forces. The steroid skeleton plays an important role in the laminated structure. In each lamina, the steroid skeletons are arranged closely and steadily and slide difficultly. The intralamina and interlamina structures are regular and tide for the stability of steroid skeletons. The sidechain, a long chain saturated aliphatic hydrocarbon of ten carbons, can swing freely. The terminal swing of each sidechain can interact with the sidechains of the neighbouring laminaes as a whole, which coin- cide with the character of this soft crystal. The severe thermal motion of the removable side chains and the tiny thermal motion of rigid steroid ske- letons are consistent with the result of X-ray analysis that the ratio of large non-solvent Hisomax/isominis 10.0.

It is shown in Figs. 2, 3 and 4 that the unit cell contains two molecules ofC29H50O and one mole- cule of H2O.

(1) Wu, X. Y.; Chao, Z. M.; Liu, H. P.; Wang, C.; Tan, Z. G.; Sun, W. Research advance on phytosterol of Cucurbitaceae medicinal plants.2012, 24, 169-175.

(2) Hong, Q. C.; Wang, M.; Jiang, W.; Zhou Z. Identify and test the main sterols of2002, 23,103-106.

(3) Moreau, M. A.; Whitaker, B. D.; Hicks, K. B. Phytosterols, phantostanols, and their conjugates in food: structural diversity, quantitative analysis, and health-promoting uses.2002, 41, 457-500.

(4) Zhao, Y. Q.; Yuan, C. L. Studies on the chemical constituents of the fruit ofC. A. Meyer.

1993, 18, 296-297.

(5) Chao, Z. M.; Zhang, N.; Tang, C. F.; Wang, C. Thin-layer chromatography of unsaponifiable matter from 36 oily Chinese materia medica.2012, 18, 79-83.

(6) Becker, M.; Staab, D.; Bergmann, K. Treatment of severe familial hypercholesterolemia in childhood with sitosterol and sitostanol.. 1993, 122, 292-296.

(7) Jones, P. J.; Ntanio, F.; Raeini-Sarjaz, M.; Vanstone, C. A. Cholesterol-lowering efficacy of a sitostanol-containing phytosterol mixture with a prudent diet in hyperlipidemic men.1999, 69, 1144-1150.

(8) Zhang, Z. Q.; Xing, Y. J.; Hu, G. Q.; Xie, S. Q. Antiproliferative effects mechanism ofsitosterol in hepatoma HepG2 cells.2011, 36, 2145-2148.

(9) Berges, R. R.; Windeler, J.; Trampisch, H. J.; Senge, T. H. Randomised, placebo-controlled, double-blind clinical trail ofsterol in patients with benign prostatic hyperplasia.. 1995, 345, 1529-1532.

(10) Wu, G. X.; Lin, Y. X.; Ou, M. R.; Tan, D. F. An experimental study (II) on the inhibitition of prostatic hyperplasia by extract of seeds of.2003, 28, 643-646.

(11) Bernal, J. D.; Crowfoot, D.; Fankuchen, I. X-ray crystallography and the chemistry of steroids.

Part I.1940, 239, 135-182.

(12) Christiansen, L. I.; Rantaned, J. T.; Bonsdorff, A. K.; Karjalainen, M. A.; Yliruusi, J. K. A novel method of producing a microcrystallinesitosterol suspension in oil.. 2002, 15, 261-269.

(13) Kawachi, H.; Tanaka, R.; Hirano, M.; Igarashi, K.; Ooshima, H. Crystallization ofsitosterol using a water-immiscrible solvent hexane.2006, 39, 869-875.

(14) Zhang, X.; Geoffroy, P.; Miesch, M.; Julien-David, D.; Raul, F.; Aoude-Werner, D.; Marchioni, E. Gram-scale chromatographic purification ofsitosterol synthesis and characterization ofsitosterol oxides.2005, 70, 886-895.

(15) Chaturvedula, V. S. P.; Prakash, I. Isolation of stigmasterol andsitosterol from the dichloromethane extract of.2012, 1, 239-242.

(16) Sheldrick, G. M.. University of G?ttingen, Germany 1997.

(17) Sheldrick, G. M.. University of G?ttingen, Germany 1997.

(18) Awad, A. B.; Fink, C. S. Phytosterols as anticancer dietary components: evidence and mechanism of action.2000, 130, 2127-2130.

(19) Schuler, I.; Milon, A.; Nakatani, Y.; Ourisson, G.; Albrecht, A. M.; Benveniste, P.; Hartman, M. A. Differential effects of plant sterols on water permeability and on acyl chain ordering of soybean phosphatidylcholine bilayers.1991, 88, 6926-6930.

(20) Gallova, J.; Uhrikova, D.; Kucerk, N.; Teixeira, J.; Balgavy, P. Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and beta-sitosterol in unilamellar vesicles.2008, 1778, 2627-2632.

(21) He, R.; Liu, G. Q.; Li, L. Effect ofsitosterol on antioxidation and stability of liposomes.2012, 37, 57-61.

27 June 2013;

23 March 2014 (CCDC 901013)

the public welfare research special project in State Administration for Quality Supervision and Inspection and Quarantine (No. 201210209)

. Tel: 010-64014411-2869, Fax: 010-64013996, E-mail: chaozhimao@icmm.ac.cn