Phenolic profile by HPLC-ESI-MS/MS of six Brazilian Eugenia species and their potential as cholinesterase inhibitors

Abstract Eugenia genus is known for its phenolic metabolites, which may influence the progression of the Alzheimer Disease. This study aimed to evaluate the anticholinesterase effects of six Eugenia species from Brazil. Leaves and stems were submitted to maceration (methanol) and partitioned with dichloromethane and ethyl acetate (EtOAc). Samples were screened (200 μg mL−1) for the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). HPLC-ESI-MS/MS analysis allowed the identification of twenty-eight phenolic compounds. Regarding the enzymatic activity, EtOAc fraction of E. mattosii exhibited the best results. Chemical and pharmacological aspects of seasonal E. mattosii extracts were evaluated. The extract from leaves collected in the winter was the most effective for AChE, and the extract from leaves collected in the spring was the most effective for BuChE. Correlating the enzymatic results with the chemical data, it was possible to associate these effects to isoquercitrin, quercetin, catechin, epicatechin, procatecuic acid and myricitrin content.


Introduction
Alzheimer's disease (AD) is a multifactorial, progressive and incapacitating neurodegenerative disorder, characterised as the most prevalent age-related disease in the elderly. The inhibition of key enzymes related to AD, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), has been recognised as standard treatment (Saeedi et al. 2017). Besides, antioxidants may also be effective in AD therapy, impairing the disease progression (Knez et al. 2018). In this sense, phenolic compounds have already been described as ChE inhibitors, as well as powerful antioxidants (Dias et al. 2015). One genus that is widely recognised for presenting a wide variety of compounds belonging to this class is Eugenia (Vechi et al. 2018). Therefore, this study aimed to identify phenolic compounds in extracts obtained from different species of Eugenia collected in Brazil, and to evaluate their inhibitory effect on enzymes related to AD.

Results and discussion
E. brevistyla, E. handroana, E. catharinae, E. stigmatosa, E. candolleana and E. mattosii are species with wide distribution in Brazil. However, most of them had never been studied, neither chemically nor pharmacologically. Therefore, these species were selected for further chemical and pharmacological studies. For plant material information, please see Experimental Section (Table S1).
Twenty-eight phenolic compounds were identified in the extracts of Eugenia species (Table S2 and S3). Salicylic acid, caffeic acid, syringic acid, gallic acid, quercetin and isoquercetin were present in most Eugenia species. These compounds are largely described for the Eugenia genus, and have been identified in different species Tenfen et al. 2017). However, these compounds have not been quantified in previous studies.
Some studies showed the anticholinesterase activity of phenolic compounds, widely present in the Eugenia genus (Knez et al. 2018;Moneim 2015). Therefore, six Eugenia species were studied regarding their anticholinesterase activity (Table S1). Both the methanol extracts and ethyl acetate fractions (at 200 lg mL À1 ) from E. handroana, E.
stigmatosa and E. candolleana were not able to inhibit AChE activity significantly. For E. brevistyla, E. catharinae and E. mattosii, the methanol extract and the ethyl acetate fraction (at 200 lg mL À1 ) were able to inhibit AChE activity (up to 83%).
Some species of Eugenia have already been evaluated for their anti-AChE potential. Magina et al. (2012) demonstrated inhibitory activity for the extracts, fractions and isolated compounds of E. brasiliensis from 6% to 63% of inhibition at 100 mg mL À1 . The highest inhibitory activity was recorded for the non-soluble fraction (63%), while the ethanolic extract demonstrated 40% and the ethyl acetate fraction, 50% inhibition. The authors also evaluated quercetin which exhibited 35% of inhibition. On the other hand, catechin was not active. Gasca et al. (2017) evaluated the extracts of E. dysenterica and showed that the aqueous extract presented moderate inhibitory activity for AChE at the concentration of 100 mg mL À1 (66.33 ± 0.52%) and an IC 50 value of 155.20 ± 2.09 mg mL À1 . They isolated two flavonoids of this plant; quercetin and catechin, being the quercetin the most active compound (IC 50 46.59 ± 0.49 mg mL À1 for AChE). This compound was identified in all Eugenia species evaluated in this study.
Taking into account that E. mattosii showed the best results (Table S4) and the highest chemical diversity (Table S3), it was selected for further evaluation to correlate the chemical and pharmacological data (Table S5 and S6). The inhibition of BuChE was also evaluated. Comparing the seasonal pharmacological variation of E. mattosii, it can be seen that the leaves showed better results, especially the extract from winter for AChE (IC 50 ¼ 146 mg mL À1 ) and from spring (IC 50 ¼ 108 mg mL À1 ) and summer (IC 50 ¼ 133 mg mL À1 ) for BuChE activity. Comparing chemical and pharmacological data by multivariate data analysis ( Figure S1 and S2), anti-AChE activity was highly influenced by the content of quercetin, catechin, epicatechin, procatecuic acid and myricitrin. For BuChE, the compounds that were highly correlated to the enzymatic inhibition were quercetin and isoquercitrin.
In general, phenolic compounds have demonstrated anti-inflammatory potential (Nhiiem et al. 2011;Yang et al. 2011), as well as other protector activities related to neurodegeneration, such as antioxidant and lipid peroxidation inhibition (Wu et al. 2003). Also, it is well established that they can attenuate oxidative stress and neuronal apoptosis after ischaemia or reperfusion injury (Dai et al. 2018). These compounds have also exhibited metal chelating properties, which promote memory, learning, and cognitive functions and consequently, demonstrate a role in neuroprotection (Jalili-Baleh et al. 2018). As multifunctional compounds, different phenolic compounds work together and are an interesting source for the discovery of new potential therapies to treat Alzheimer's disease and other neuro dysfunctions.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was financially supported by Conselho Nacional de Desenvolvimento Cient ıfico e Tecnol ogico -CNPq (grant to VCF), Coordenação de Aperfeiçoamento de Pessoal de N ıvel