New jacaranone glucoside from Jacaranda oxyphylla leaves

Abstract Jacaranda oxyphylla Cham. is popularly known as ‘caroba-de-São-Paulo’ and it is used in traditional medicine for microbial infections. A new phytoquinoid (α/β-glucoside-4-phenylacetate-6-(1-hydroxy-4-oxo-2,5-cyclohexadiene-1-acetate) (1) was isolated from J. oxyphylla leaves, together with three known compounds: quercetin-3-O-β-d-galactoside (2), verbascoside (3) and polystyrene (4). Their chemical structures were elucidated using spectroscopic techniques and by comparison with the related known compounds. In addition, it was found a pronounced acetylcholinesterase inhibitory activity for the quinoid 1 (100.0 ± 0.8%) and phenolic compounds 2 and 3 (99.9 ± 0.7 and 99.3 ± 0.5%, respectively), if compared to the standard eserine (92.7 ± 0.4%), that was analysed by a microplate spectrophotometer.


Introduction
The genus Jacaranda (Bignoniaceae) is well known due to the biological properties and chemical composition. Different classes of phytochemicals have been identified in Jacaranda species, and the main metabolites described are sterols, triterpenes, fatty acids, flavonoids, phenylpropanoids, phenylethanoids and quinones (Mostafa et al. 2014). For instance, a recent study with extracts from Jacaranda caroba leaves identified thirteen phenolic compounds (Ferreres et al. 2013) while four acidic triterpenes were isolated from Jacaranda puberula leaves (De Almeida et al. 2014).
In the folk medicine, Jacaranda species are used for the treatment of skin diseases, venereal infections, rheumatism and gastrointestinal disorders (Gachet & Schühly 2009). Many pharmacological screenings have been conducted to support the traditional uses. In this way, extracts from Jacaranda cuspidifolia showed to be active against Staphylococcus aureus, Proteus mirabilis and Serratia marcescens (minimum inhibitory concentration in ranging from 6.1 to 9.3 mg mL −1 ), and these antimicrobial actions were assigned to verbascoside, the major compound identified in the extracts (Arruda et al. 2011). The ethanol extract from the leaves of Jacaranda oxyphylla presented high activity against Gram-positive bacteria Bacillus cereus and S. aureus, with 96.9 and 96.6% of grow inhibition of micro-organisms, respectively (Pereira et al. 2015). These results provide important information to confirm the medicinal value of J. oxyphylla.
There are few studies on Bignoniaceae species with activity on neurodegenerative disorders such as Alzheimer's disease. For example, acetylcholinesterase inhibitory action was observed for the hexane extract from J. cuspidifolia (Arruda 2009). Moreover, extracts from aerial parts of J. oxyphylla presented a pronounced in vitro inhibition activity against acetylcholinesterase, with % of enzyme inhibition ranging from 40.3 to 84.1 (Pereira et al. 2015). These preliminary findings revealed the potential neuroprotective effect of J. oxyphylla.
Flavonoids and its derivatives have been shown a potential activity to the development of new antimicrobial agents (Cushnie & Lamb 2005). To exemplify, quercetin was assayed against Gram-positive and Gram-negative bacteria and presented selective antibacterial activity against S. aureus (Hirai et al. 2010). Phenylethanoids, such as verbascoside, presented antibacterial activity (MIC = 62.5 μM) against S. aureus and Klebsiella pneumonia (Pendota et al. 2013). The cholinesterase inhibitory activity of flavonoid derivatives was evaluated, and these compounds revealed a neuroprotective effect and quercetin exhibited a number of strong hydrogen bonds and hydrophobic interactions with several important amino acid residues of cholinesterase enzymes (Khana et al. 2009). Moreover, verbascoside revealed promissory action to treat neurodegenerative diseases (Alipieva et al. 2014).
In this work, we report the isolation of compounds 1-4 ( Figure 1) from J. oxyphylla extracts. A new jacaranone glucoside (a quinoid) and other three compounds from different classes (flavonoid, phenylethanoid and polymer) were obtained. Moreover, these compounds were evaluated for their acetycholinesterase inhibitory and antimicrobial activities.
Correlations from 2D NMR experiments (Figures S6-S8) allowed the assignment of all the carbon and hydrogen resonances in compound 1 (see Table S1). In the HMBC spectrum, an important cross-peak between the signals at δ C 168.3 and δ H 4.03 prompted assigning the phenylacetate group to C-4 position in the sugar, and the cross-peak between the signals at δ C 170.5 and δ H 4.83 permitted assigning the acetyl group to the C-6 position in the sugar moiety. Figure  S8 shows the key HMBC correlations which confirmed the structure of compound 1 as α/βglucoside-4-phenylacetate-6-(1-hydroxy-4-oxo-2,5-cyclohexadiene-1-acetate). Jacaranone glucoside analogues were previously isolated in Jacaranda mimosifolia leaves (elusyian & Olugbade 2011), but all different from the quinoid isolated here. A recent study identified three new jacaranone derivatives from aerial parts of Senecio chrysanoides that presented cytotoxic activity (Wu et al. 2015). The known compounds ( Figure 1) were identified as quercetin-3-O-β-d-galactoside (2), verbascoside (3) and polystyrene (4) by comparison with their NMR data with literature reports (Pham et al. 1991;Schlauer et al. 2004;Pereira et al. 2012). Compound 2 is also known as hyperoside and it has been isolated from Jacaranda decurrens leaves (Blatt et al. 1998), while compound 3 (1.6% of etOH extract) was previously identified from the hydroalcoholic extract of J. mimosifolia leaves (Moharram & Marzouk 2007).

Acetylcholinesterase inhibition assay
Compounds 1-3 (quinoid, flavonoid and phenylethanoid) showed high acetylcholinesterase inhibitory activity (99.3 to 100.0% of inhibition), as presented in Table 1. The presence of sugar residues and/or phenolic groups in compounds 1-3 could explain the high acetylcholinesterase inhibition, since hydroxyl moieties could be involved in hydrogen bonding with amino acid residues of acetylcholinesterase active site (Mukherjee et al. 2011). Khana et al. (2009 evaluated the molecular target in neuroprotective effect of quercetin and showed strong hydrogen bonds and hydrophobic interactions between the amino acid residues of enzymes and the flavonoid skeleton.

Antimicrobial screening
All compounds were tested against Gram-positive bacteria B. cereus and S. aureus, Gramnegative bacteria Escherichia coli and Salmonella typhimurim and the yeast Candida albicans. Compounds 1 and 2 presented a discrete antibacterial activity against S. typhimurim, with values of growth inhibition of 30.0 and 37.5%, respectively. Compounds 1-4 did not revealed promising action against the other micro-organisms. Table 1 shows the overall results of the antimicrobial assay.

General experimental procedures
The IR spectra were recorded from KBr pellets on the Shimadzu IR-408 spectrometer (Tokyo, Japan) or from ATR on the Perkinelmer Spectrum One spectrometer (Shelton, uSA). 1 H and Table 1. In vitro aceylcholinesterase (ache) and micro-organisms inhibition induced by compounds 1-4. a results are mean values of quintuplicate assays ± standard deviation (expressed as % inhibition). b eserine for aceylcholinesterase, ampicillin for bacteria and nystatin for yeast; compounds were assayed in concentration of 100 μg ml −1 ; c-l the means followed by the same letter do not differ significantly by the tukey test (p < 0.05). C NMR spectra were recorded at 400 and 100 MHz, respectively, as well as COSY, HSQC and HMBC were performed on a Bruker DRX400 Avance spectrometer (Rheinstetten, Germany). Chemical shifts (δ) were registered in ppm, and the coupling constants (J) were registered in Hz. High-resolution electrospray mass spectrometry (HR-eSIMS) spectra were acquired on Shimadzu LCMS-IT-TOF system (Tokyo, Japan) in m/z (rel. %). Column chromatography (CC) was carried out using silica gel 60 (SiO 2 , 70-230 mesh). The solvents chloroform, ethyl acetate and methanol were purchased from Vetec (São Paulo, Brazil) and used without further purification.

Plant material
The aerial parts of the plant were collected in São João da Chapada, near Diamantina city (MG, Brazil)

Extraction and isolation
Plant material was placed on paper sheets and dried at room temperature until a constant weight. Dried leaves (1.26 kg) were powdered and successively extracted with n-hexane, chloroform and ethanol by maceration, followed by filtration. The extracts were prepared at room temperature and concentrated under vacuum using the Ika rotary evaporator (Guangzhou, China) to afford crude extracts from leaves in n-hexane (13.0 g), chloroform (56.4 g) and ethanol (215.1 g).
Part of the crude CHCl 3 and etOH extracts (20 g each) were submitted to CC (CHCl 3 , etOAc and MeOH as eluents, in order of increasing polarity). Fractions of 150 mL were collected and concentrated under vacuum in a rotary evaporator. After thin layer chromatography analysis, similar fractions were pooled in groups. Successive CC purifications and recrystallisations were carried out for isolation and final purification of compounds 1-4.

Acetylcholinesterase inhibition assay
Compounds 1-4 were also screened on a quantitative assay of the inhibitory activity of acetylcholinesterase (AChe), based on ellman's method (1961).
Subsequently, eight readings were obtained (one every minute) on the BioTek eLx800 microplate spectrophotometer (Winooski, uSA) at 405 nm (control). Once read, solution of AChe (0.222 u mL −1 ) in Tris/HCl 50 mM pH 8 containing bovine serum albumin 0.1% (w V −1 ) was added (25 μL) to each well. The absorbance was measured again 10 times. The results obtained were treated in Origin ® software version 6.0 and expressed as % inhibition of mean ± standard deviation.

Antimicrobial screening
Compounds 1-4 obtained from the leaves of J. oxyphylla were subjected to antimicrobial screening by broth microdilution method with adaptations (Cueva et al. 2010). Two Grampositive bacteria (B. cereus ATCC 11778 and S. aureus ATCC 29212), two Gram-negative bacteria (E. coli ATCC 25922 and S. typhimurim ATCC 14028) and the yeast C. albicans ATCC 18804 were assayed. The microbial inocula were prepared with sterile distilled water until 74-75% of absorbance on the Biospectro SP-22 spectrophotometer (Curitiba, Brazil) at 625 nm.
Compounds were solubilised in DMSO to a concentration of 10 mg mL −1 and were diluted in brain heart infusion (BHI) broth to achieve a concentration of 100 μg mL −1 . In 96-well microplates, 100 μL of the extracts was applied and the test samples were added to 100 μL of microbial inocula previously prepared.
In parallel, it prepared a blank assay (100 μL of each sample diluted in BHI plus 100 μL of sterile distilled water), the positive control (100 μL of microbial strains plus 100 μL of BHI broth) and negative control (100 μL of sterile distilled water plus 100 μL of BHI broth). Ampicillin was tested as antibacterial standard and nystatin as antifungal standard (100 μg mL −1 ). Bioassay of each micro-organism was performed on a different microplate, and it was performed in quintuplicate.
The plates were incubated on the Quimis Q-316 oven (Diadema, Brazil) at 35 °C. After 24 and 48 h, absorbance was read on the BioTek eLx800 microplate spectrophotometer (Winooski, uSA) at 495 nm. The results of this assay were expressed as % inhibition of antimicrobial growth.

Statistical analysis
Bioassay data were analysed statistically by one-way variance (ANOVA), and the means were compared by Tukey's multiple test using the program R (version 2.10.1). It was considered statistically different than the results with p < 0.05 (ANOVA and Tukey's test).

Conclusion
This phytochemical study of J. oxyphylla leaves reported the isolation of a new jacaranone glucoside (α/β-glucoside-4-phenylacetate-6-(1-hydroxy-4-oxo-2,5-cyclohexadiene-1-acetate) and the identification of three other known compounds: quercetin-3-O-β-d-galactoside, verbascoside and polystyrene. Their antimicrobial and acetylcholinesterase inhibition activities were evaluated, and these compounds (mainly phenolic compounds and jacaranone derivative) show to be promising models to assist the development of new drugs to the treatment of Alzheimer's disease.