Ailanthus altissima (Miller) Swingle fruit - new acyl β-sitosteryl glucoside and in vitro pharmacological evaluation

Abstract β-Sitosterol-3-O-(6ʹ-O-13ʺ-octadecenoyl)-β-D-glucoside (1), a new acyl β-sitosteryl glucoside, along with three known compounds β-sitosterol-3-O-β-D-glucoside (2), β-sitosterol (3) and methyl gallate (4) have been isolated from the ethyl acetate soluble fraction of methanolic extract of Ailanthus altissima fruits. Their structures were elucidated through spectroscopic data including 2D NMR, ESI-MS, methanolysis and oxidative cleavage of double bond. Antibacterial, antifungal, cytotoxic, phytotoxic and insecticidal activities were evaluated of compound 1, crude extract and its fractions so far for the first time. Pharmacological activities results showed that n-butanol fraction was good active against Pseudomonas aeruginosa and Salmonella typhi bacteria, and moderate active against Microsporum canis fungus. Crude extract, n-butanol and aqueous fractions showed good cytotoxicity. Moreover, compound 1, extract and all fractions showed notable phytotoxicity at higher concentrations, whereas all inactive against assayed insects.


Introduction
The genus Ailanthus (Simaroubaceae) comprises about 10 species distributed in Asia, Europe and Australia (Peng & Thomas 2009). The genus Ailanthus in Pakistan is represented by two species, ABSTRACT (1), a new acyl β-sitosteryl glucoside, along with three known compounds βsitosterol-3-O-β-D-glucoside (2), β-sitosterol (3) and methyl gallate (4) have been isolated from the ethyl acetate soluble fraction of methanolic extract of Ailanthus altissima fruits. Their structures were elucidated through spectroscopic data including 2D NMR, ESI-MS, methanolysis and oxidative cleavage of double bond. Antibacterial, antifungal, cytotoxic, phytotoxic and insecticidal activities were evaluated of compound 1, crude extract and its fractions so far for the first time. Pharmacological activities results showed that n-butanol fraction was good active against Pseudomonas aeruginosa and Salmonella typhi bacteria, and moderate active against Microsporum canis fungus. Crude extract, n-butanol and aqueous fractions showed good cytotoxicity. Moreover, compound 1, extract and all fractions showed notable phytotoxicity at higher concentrations, whereas all inactive against assayed insects. one of them is Ailanthus altissima (Miller) Swingle which is deciduous tree commonly known as tree-of-heaven (Adamik & Brauns 1957). In folk medicine, A. altissima is used as remedy for proliferation, colds, gastric diseases, gonorrhoea, haemorrhoids, amoebic dysentery, anaemia, spasm, cardiac depression, astringency and epilepsy (De Feo et al. 2003;Singh et al. 2009;Kožuharova et al. 2014). Various inhibitory potentials including antioxidant, antimalarial, antiviral, insecticide, antituberculosis, anti-inflammation, cytotoxicity, anti-proliferation, antiasthmatic, phosphodiesterase, phytotoxicity, antimicrobial and cyclooxygenase (Seo et al. 2012;Albouchi et al. 2013;Kožuharova et al. 2014;Popa et al. 2015) have previously been reported for this plant species. These biological potentials were attributed to the presence of constituents such as quassinoids (Joshi et al. 2007;Yang et al. 2014), steroids (Zhao et al. 2005), phenols (El-Baky et al. 2000), coumarins (Hwang et al. 2005), alkaloids (Zhang et al. 2007, cerebrosides (Zhao et al. 2006), flavonoids, terpenoids, lipids, fatty acids, volatile oils and many others (Kožuharova et al. 2014). The chemotaxonomic and ethnopharmacological importance of the genus Ailanthus prompted us to carry out further phytochemical studies on its species A. altissima. We herein report the isolation and structural elucidation of a new acyl β-sitosteryl glucoside namely β-sitosterol- (3) and methyl gallate (4) along with antibacterial, antifungal, cytotoxicity, phytotoxicity and insecticide activities of compound 1, crude extract and its fractions so far for the first time.

Results and discussion
The methanolic extract of shed dried A. altissima fruits was suspended into water and successively partitioned into n-hexane, AcOEt, n-butanol and water soluble fractions. On AcOEt soluble fraction, a series of column chromatographic technique was applied on silica gel to afford compounds 1-4, respectively. The new compound 1, crude extract and its fractions were also pharmacologically evaluated.
Compound 1 was obtained as white amorphous solid. The high-resolution FAB-MS showed the pseudo-molecular ion [M + H] + peak at m/z 841.6911 corresponding to the molecular formula C 53 H 93 O 7 . The UV spectrum showed the absorption maxima at 205 and 243 nm. The IR absorptions showed the presence of hydroxyl (3421 cm −1 ), ester (1741 cm −1 ), olefinic (1640 cm −1 ) and O-C (1325 cm −1 ) functionalities. The EI-MS showed characteristic peaks at m/z 414 and 255 for the β-sitosterol unit.
The 13 C-NMR [broad band decoupled and distortionless enhancement by polarisation transfer] (Table S1, Figure S1) spectra showed 53 signals comprising 7 methyl, 26 methylene, 16 methine and 4 quaternary carbons. The carbon signal at δ 174.7 revealing the presence of ester moiety, whereas the four signals appeared at δ 140.3, 130.0, 128.1 and 122.1 indicating the presence of two olefinic bonds. The only oxymethine carbon was observed at δ 79.7. The methine and methylene carbons were resonated in the range of δ 21.1-38.9, and six methyl carbons showed signals at δ 19. 8, 19.4, 19.0, 18.8, 12.0 and 11.9. The hexose unit carbons were appeared in the range of δ 62.7-103.1 and the methylenes of the aliphatic chain resonated in the range of δ 23.1-34.2 with the terminal methyl carbon at δ 14.1. In the I H-NMR spectrum (Table S1, Figure S2), the signals at δ 5.36-5.34 (m, 1H) and 5.32-5.30 (m, 2H) were attributed to three olefinic protons while the oxymethine proton resonated at δ 3.49 (t, J = 7.5, 1H). The upfield region of the spectrum showed the signals of two methylenes at δ 2.79 (m, 2H) and 2.00 (m, 2H) indicating their attachments with the olefinic bond and another methylene appeared at δ 2.32 (t, J = 7.5 Hz) revealing its attachment with the carbonyl carbon. The methylene protons of the aliphatic long chain were resonated at δ 1.24-1.26 (br. s, 10 × CH 2 ) with the terminal methyl group observed at δ 0.84 (t, J = 7.5, 3H). The rest of the methine and methylene protons were observed in the range of δ 0.89-2.31, whereas the six methyl groups appeared at δ 0.96, 0.89, 0.82, 0.81, 0.79 and 0.67. The anomeric proton showed signal at δ 4.38 (d, J = 8.0 Hz) and its large coupling constant (8.0 Hz) confirming its β-configuration. All other hexose unit protons were resonated in the range of δ 3.15-4.37. The hexose unit was confirmed β-D-glucose by its sign of optical rotation ([α] D + 76.6) [Ali et al. 2012] through acid methanolysis. MS and NMR data confirm that compound 1 is an acyl β-sitosteryl glucoside. The NMR ( 1 H and 13 C) spectral data were similar to the reported data al. 2002] except the absence of signals of an olefinic bond.
The aliphatic chain length was concluded of 18 carbons by acid methanolysis. The acid methanolysis of compound 1 furnished the methyl ester of fatty acid, showed [M] + peak at m/z 296 in EI-MS and characterised as methyl octadecenoate. From the EIMS of the methyl ester, the position of the olefinic bond was suggested at C-13 by the diagnostic fragmentation peaks at m/z 237 and 213 due to the cleavage of allylic bonds by Mclafferty rearrangement. The position of the olefinic bond was further confirmed by the oxidative cleavage of 1. The GC-MS analysis of oxidative mixture gave various peaks including m/z 272 for dimethyl tridecanedioate and m/z 116 for methyl pentanoate, confirming the olefinic bond position at C-13. The cis-geometry of the olefinic bond was confirmed by the allylic methylenes chemical shifts at δ 27.2 (C-15ʺ) and δ 25.6 (C-12ʺ) [Tuntiwachwuttikul et al. 2004].

Pharmacological evaluation
Of A. altissima, the crude extract, its fractions and new compound 1 were in vitro pharmacologically evaluated for antibacterial, antifungal, cytotoxic, phytotoxic and insecticidal activities. In antibacterial activity, only n-butanol fraction showed 50 and 43% inhibitions against Pseudomonas aeruginosa and Salmonella typhi as compared to the standard imipenum, respectively (Table 1). Minimal inhibitory concentrations of n-butanol fraction against P. aeruginosa and S. typhi were 500 and 300 μg/ml, respectively, for 100% inhibition. The antifungal activity results indicated that n-butanol fraction was merely moderate active against Microsporum canis (Table 1). The cytotoxicity results exhibited that the extract, n-butanol and aqueous fractions were active with lD 50 17.97, 16.46 and 22.57, respectively (Table 2). Phytotoxicity results indicated that the extract, fractions and compound 1 showed significant activity at higher doses (Table 2). In insecticidal activity, all were inactive against selected insects.  Sifting of the literature about A. altissima shows that chloroform root extract and isolated quassinoids (Tamura et al. 2003;De Feo et al. 2005) from methanolic aerial parts extract showed the cytotoxic and antiproliferative (anti-tumour and anti-cancer) activities. Its ethanolic fruits extract was weakly active against Escherichia coli, Staphylococcus aureus, P. aeruginosa and Salmonella typhiuriun (Zhao et al. 2005). Insecticidal studies on its leaf and roots extracts have previously been conducted on insecticides (De Feo et al. 2009), different from the present insects. Antifungal, cytotoxic and phytotoxic studies so far have not been conducted on methanol fruits extract, previously. In this study, plant fruits were moderate active against assayed six bacteria and five fungi. Three previously estimated bacteria (E. coli, S. aureus and P. aeruginosa) against ethanol fruit extract, again assayed with methanol fruits extract, similar results obtained again. Fruits extract, fractions and isolated compound 1 showed the notable cytotoxic and phytotoxic potentials. Present cytotoxic potential of fruits supports the previous reported cytotoxic and antiproliferative activities of its aerial parts and roots extracts. The isolation of compound 1 (acyl β-sitosteryl glucoside) from this plant also supports its previous reported anti-proliferative potential, as acyl β-sitosteryl glucosides (ASGs) have cell anti-proliferative potentials (Shai et al. 2001;Yeol et al. 2002).

General experimental procedures
Melting points were measured on a Gallenkamp apparatus (loughborough, England) and are uncorrected. Optical rotations were measured on JASCO DIP-360 polarimeter (Jasco, Tokyo, Japan). UV spectra were recorded on Hitach UV-3200 spectrophotometer (Hitachi, Tokyo, Japan), while the IR spectra were recorded on Shimadzu FTIR-8900 spectrometer (Shimadzu, Kyoto, Japan) as KBr pellets. The 1 H-and 13 C-NMR spectra were recorded on Bruker AM-400 and AM-500 spectrometers (Bruker BioSpin, Faellanden, Switzerland) in deuterated solvents. The 2D (COSY, HMQC, HMBC) NMR spectra were recorded on the same instruments. The chemical shifts are reported in ppm (δ), relative to the tetramethylsilane (TMS) as an internal standard and the scalar couplings are reported in Hz. Mass spectra (El and HR-EI) were obtained in an electron impact mode on Finnigan MAT-112 and MAT-113 spectrometers (Finnigan, Walthan, MA, USA) and FAB (Fast atomic bombardment) mass spectra were carried out on Jeol JMS HX 110 spectrometer (Jeol, Tokyo, Japan) with glycerol as matrix, and ions are given in m/z (%). Column chromatography (CC) was performed on silica gel (70-230 mesh, E. Merck, Darmstadt, Germany). Thin-layer chromatography (TlC) was performed on pre-coated silica gel G-25-UV 254 plates (E. Merck, Darmstadt, Germany), and detection was done at 254 and 366 nm or by spraying ceric sulphate in 10% H 2 SO 4 (heating). GC-MS was performed on HP6890 gas chromatograph-mass spectrometer using a 5% diphenyl-polysiloxane/95% dimethyl-ploysiloxane HP5-MS capillary column. Column temperature ranges from 80 to 250 °C at 5 °C/min., with helium used as carrier gas at 1.4 kg/cm 2 pressure, and EI mode at 70 eV for mass.

Plant material
The shed dried plant material was purchased from market Mansehra district of Khyber Pakhtunkhwa (KPK) province of Pakistan in June 2009 and identified by Prof Dr Muqarrab Shah, Dean, Faculty of Health Sciences, Hazara University, Mansehra, and a voucher specimen has been deposited in the university herbarium (voucher No. HOH/ang/020).

Oxidative cleavage of double bond of 1
To a solution of 1 (6 mg) in acetone were added 0.04 M K 2 CO 3 (3 ml), aqueous solution of 0.02 M KMnO 4 (18 ml) and 0.09 M NaIO 4 , and allowed to progress for 18 h at 37 °C. After acidification with 5 N H 2 SO 4 , the reaction mixture was decolourised with 1 M oxalic acid and extracted with Et 2 O (3 × 25 ml). The extracts obtained with Et 2 O were dried over Na 2 SO 4 desiccator, filtered and concentrated. The resulting mixture of carboxylic acids was methylated with ethereal solution of diazomethane and analysed by GC-MS, which gave various peaks including at m/z 272 for dimethyl tridecanedioate and m/z 116 for methyl pentanoate, respectively.

Pharmacological evaluation assays
The in vitro antibacterial, antifungal, cytotoxic, phytotoxic and insecticidal activities of compound 1, crude extract and its fractions of A. altissima were carried out through modified reported protocols, which are described in supplementary material.

Conclusion
A new compound β-sitosterol-3-O-(6ʹ-O-13ʺ-octadecenoyl)-β-D-glucoside (1) was isolated from AcOEt fraction of the methanol extract of A. altissima. Pharmacological activities results determine that plant is moderate antimicrobial and cytotoxic active and good phytotoxic active. It is concluded from the present along with previous studies that this plant species may be used for cell proliferation and modest microbial infections, but need to be explored properly to develop the drug.