Chemical constituents, Antibacterial and Acetylcholine esterase inhibitory activity of Cupressus macrocarpa leaves

Abstract The chemical constituents of Cupressus macrocarpa were investigated. A new neolignan glycoside (1) in addition to nine known compounds were isolated. The acetylcholinesterase (AChE) inhibitory activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) of different fractions and isolates of C. macrocarpa were evaluated. The light petroleum fraction showed the highest activity in both assays with IC50 value of 88.79 µg/ml and 152.58 µg/ml for the AChE inhibitory activity and MRSA antibacterial activities, respectively. Weak to moderate activity were detected for the isolated compounds. Graphical Abstract


Introduction
Cupressus is one of several genera within the family Cupressaceae that has the common name cypress (Chaudhary et al. 2012). In warm, Mediterranean climates, Cupressus macrocarpa Hartw. represents one of the most abundant cypress trees.
Cypress is rich in secondary metabolites such as flavonoids, tannins, saponins, phenolics, terpenes and essential oils. Cypress trees have been widely used in folk medicine practices mainly due to its antibacterial, antifungal, antiviral, antiparasitic, antioxidant, wound healing, estrogenic, anticoagulant, anti-inflammatory and insecticidal activities (Ibrahim et al. 2009;Kuiatea et al. 2006;Loizzo et al. 2008). The aim of the current study isto explore the chemical constituents of C.macrocarpa and evaluate the anticholinesterase activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) of different C.macrocarpa extracts, fractions and isolates.
Compound (1) was obtained as colorless crystals, m.p146-147 C. Its molecular formula C 25 H 32 O 9 was deduced by 13 C-NMR spectroscopic analyses and negative ion mode ESI-MS (m/z 474.7, [M-H] -). The 1 H-NMR spectrum showed the presence of 6 aromatic proton signals assigned as two doublet of doublets [(d H 6.70, J ¼ 6.4, 1.6 Hz, H-6) and 6.96 (J ¼ 6.4, 1.2 Hz, H-2')] indicating meta and ortho couplings, two doublets [(d H 6.51, J ¼ 6.8 Hz, H-5) and 6.75 (J ¼ 6 Hz, H-3')] indicating an ortho coupling. In addition to two broad singlets at d H 6.62 and 6.95 assigned to H-2 and H-6', respectively. This suggests the presence of two tri-substituted benzene rings (1,3,4-and 1',4',5'-substitution). DEPT-135 NMR spectrum showed the presence of 13 methines, three methyl groups and four methylenes including two carbon signals resonating at d C 60.5 and 66.2 ppm assigned to the 2 hydroxymethylenes (C-9, C-9', respectively). Furthermore, a methine carbon signal appeared at d C 56.0 ppm with its attached proton at d H 3.39, which is evident from the HMQC spectrum was attributed to the C-8 benzofuran nucleus. A downfield signal at d C 87.0 with its attached proton doublet at d H 5.70 was assigned to the aryl-substituted benzofuranmethine (C-7). Based on the previous pattern and comparison with previously reported data (Guria et al. 2013), compound (1) was concluded to be dihydrobenzofuran neolignan.The pattern of benzene rings substitution was confirmed through HMBC correlations beside the established biogenetic pathway for neolignans synthesis (Teponno et al. 2016). The 1 H-NMR spectrum also showed a methyl singlet at d H 3.71 assigned for a methoxyl group, which was correlated with the aromatic quaternary carbon at d C 147.5 (C-3) in the HMBC spectrum. From COSY correlations, the diastereotopic protons at d H 3.28 and 3.52 (H-9') were coupled with the methylene multiplet at d H 1.73 (H-8'), which was further coupled with benzyl methylene protons at d H 2.51 (H-7'), suggesting the presence of an n-propanol moiety (Lina et al. 2014). The most t upfield signals at d C 31.7 and 31.4 with their respective proton signals at d H 2.51 and 1.73 were assigned to the benzylic methylene carbon (C-7') and the central methylene carbon (C-8') of the n-propanol moiety, respectively.The carbon signal at d C 136.7 (C-1') was correlated, in HMBC spectrum, with the proton signals at d H 2.51 (H-7') and 6.94 (H-6'), which indicated the n-propanolmoiety substitution at C-1'. The presence of a rhamnopyranosyl moiety was supported by two characteristic a-L-rhamnopyranosyl signals in the 1 H-NMR (a broad singlet at d H 4.54 and a methyl doublet at d H 1.23 (J ¼ 6.1 Hz) corresponding to the anomeric proton signal (H-1'') and H-6'', protons respectively, andthe 6 carbon signals at d C 100.4, 71.1, 71.2, 72.4, 68.9 and 18.3 matching the reported values for rhamnose moiety (De Bruynet al.1976). The presence of rhamnose was further confirmed through controlled acid hydrolysis of compound (1). The site of attachment of the rhamnopyranosyl unit at C-9'of the neolignanaglycone moiety was confirmed by the HMBC experiment which showed correlation between the carbon at d C 66.2 (C-9') and the anomeric proton (d H 4.54, H-1").Based on these spectral data, compound (1) was assigned as 3'-demethoxy dihydrodehydrodiconiferyl alcohol-9'-Oa-L-rhamnopyranoside. To the best of our knowledge, this is the first report for the isolation of compound (1) from a natural source.
Acetylcholinesterase (AChE) inhibitory activities of different fractions and isolates of C. macrocarpa were evaluated. The light petroleum fraction showed the most potent AChE inhibitory activity with IC 50 value of 88.79 mg/ml followed by the methylene chloride fraction (181.79 mg/ml), while the ethyl acetate and n-butanol fractions exhibited a relatively weak inhibitory activity (507.21 and 735.46 mg/ml, respectively). AChE inhibitory activity of the isolates showed that terpenoidal compounds isolated from the methylene chloride fraction exhibited a considerable activity. Compound (7) showed the most potent AChE inhibitory activity with IC 50 value of 144.31 lg/ml followed by compound (3) (195.99 lg/ml). Compounds (4) and (6) exhibited weaker AChE inhibitory activity with IC 50 values of 228.47 and 263.68 lg/ml, respectively (supplementary material, Table S1).
The antibacterial activity of the ethanolic extract, different fractions and isolated compounds (1-10) of C. macrocarpa was assessed against methicillin-resistant S. aureus (MRSA). The total ethanolic extract of C. macrocarpa leaves exhibited modest activity against MRSA with IC 50 values of 152.5. Among C. macrocarpa different fractions, the light petroleum fraction was the most effective with IC 50 values of 123.00 mg/ml against MRSA. This is mainly attributed to essential oil components as previously suggested by Salem et al. 2018. No or weak activity was detected for the isolated compounds (1-10) (supplementary material, Table S1).

Plant material
C. macrocarpa leaves were collected in 2015 from Alexandria, Egypt and the plant was identified by Dr. Salama Aldarier, Faculty of Science, Alexandria University. The plant was further authenticated by Dr. Hesham Ali, Antoniadis Research centre and a voucher specimen (CM 2015/11) was deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria Egypt.

Extraction and isolation
Powdered air-dried leaves of C. macrocarpa (7 Kg) were extracted twice at room temperature with 90% ethanol (2 X 20 L) (supplementary material, scheme S1) The combined alcoholic extract was distilled off under reduced pressure to yield a dark brown residue (800 g). The dried residue was dissolved in 70% aqueous ethanol and subjected to successive fractionation using light petroleum, methylene chloride, ethyl acetate and n-butanol. The fractions obtained were evaporated under reduced pressure to yield 90, 80, 30 and 82 g dry weight, respectively. The light petroleum fraction (10 g) was fractionated over a silica gel column (300 g, 4 cm X 120 cm). The elution was performed using methylene chloride: methanol mixtures with gradual increase in polarity (0-30%). The 2% methanol fraction yielded 60 mg colorless needles of compound (2). The methylene chloride fraction (15 g) was chromatographed over a silica gel column (430 g, 4 cm X 180 cm). The elution was performed using methylene chloride: methanol mixtures with gradual increase in polarity (0-100%) giving 65 fractions. Fractions eluted with 5-11% methanol were further subjected to column chromatography and preparative thin layer chromatography leading to the isolation of 6 compounds (1, 3-7). A part of the n-butanol fraction (16g) was fractionated over a silica gel column (480 g, 4 cm X 190 cm). The elution was performed using methylene chloride: methanol mixtures with gradual increase in polarity (5-100%).The collected fractions were combined into 15 fractions according to their TLC pattern. Subfractions eluted with 40-60% methanol were further purified using column chromatography and preparative thin layer chromatography leading to the isolation of 3 compounds (8-10).
3.3. In vitro Acetylcholinesteraase (AChE) inhibition assay using modified Ellman's method (Ellman et al. 1961) Enzyme aliquots (100 ll) and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) (100 ll) were added to 0.1 M phosphate buffer (pH 8.0, 2.8 mL). To this mixture, sample solutions of extracts, fractions and isolates (100 ll) prepared in ethanol at different concentrations were added. The reaction was initiated by adding ATChI (20 ll) followed by incubation at 37 C for 15 min. Methanol/methylene chloridewere used as negative controls while Physostigmine was used as a positive control. The change in absorption at 412 nm was monitored on Sequoia-Turner Model 340 spectrophotometer. All experiments were done in triplicates. Specific activity of AChEenzyme was calculated for each concentration and control. AChE inhibition % ¼ [1-SAT/SAC] Â 100, where SAT is specific activity of the enzyme in treatment and SAC is specific activity of the enzyme in negative control.
Methicillin-resistant S. aureus (MRSA) ATCC 33591, used in the current study, was obtained from the American Type Culture Collection (Manassas, VA). Microbial inocula were prepared by correcting the optical density (OD630) of microbe suspensions in incubation broth to afford final target inocula. Ciprofloxacin, cefotaxime, meropenem, methicillin and vancomycin (ICN Biomedicals, Ohio, USA) are included as positive controls, while DMSO was used as a negative control in each assay. MRSA was read at 630 nm using the Biotek Power wave XS plate reader (BioTek Instruments, Vermont) prior to and after incubation. Percent growth was plotted versus test concentration to afford IC 50 values.

Conclusion
A new neolignan glycoside, 3'-demethoxy-dihydrodehydrodiconiferylalcohol-9'-O-a-Lrhamnopyranoside, in addition to 9 known compounds were isolated from C. macrocarpa leaves. The AChE inhibitory activity and antibacterial activities against MRSA of C. macrocarpa fractions and isolates were assessed. The light petroleum fraction showed the most potent activities in both assays. The detected activities suggest that C. macrocarpa extracts can be considered as a promising natural AChE inhibitor and antibacterial agent against antibiotic resistant bacteria. Further studies are required to identify key minor compounds responsible for the observed biological activities.