The effect of flavonols in Anacardium occidentale L. leaf extracts on skin pathogenic microorganisms

Abstract Cashew (Anacardium occidentale L.) leaf is traditionally used to treat skin infections. Although many flavonols have been identified from its leaf extract, their inhibitory effects on skin pathogens are not yet determined. The aims of this study were to determine the antimicrobial (against skin pathogenic microbes) and antioxidant activities of four flavonol glycosides from the crude extract and three flavonol aglycones from the hydrolyzed extract. The hydrolyzed extract was found to show higher activities than the crude extract. Myricetin showed the highest activity against all the tested bacteria and yeast with the lowest Minimum Inhibition Concentration (MIC) of 7.81 μg/mL on Corynebacterium minutissimum ATCC23348. Myricetin also exhibited good primary antioxidant activities with the effective concentration with 50% of activity (EC50) values ranged between 2.23 μg/mL and 6.40 μg/mL. The highest secondary antioxidant activity was indicated by myricetin-3-O-rhamnoside. Thus, myricetin can be considered as a bioactive compound of the hydrolyzed extract. Graphical Abstract


Introduction
Plants have been traditionally used to treat skin disorders caused by microorganisms (Ghuman et al. 2019). The medicinal values of plant extracts are often relied on the bioactive phytochemicals such as flavonoids. Plants mostly contain higher glycosidic form of flavonoids than the free one. Ekuadzi et al. (2014) had identified four flavonoid glycosides from the stem bark of Margaritaria discoidea, which is traditionally used to heal skin infections. Based on a comparative study on the antibacterial effects of flavonoids, three aglycones (non-hydroxylated flavone, quercetin and naringenin) were found to show a broad spectrum of activities (Rauha et al. 2000).
Anacardium occidentale L. or cashew is cultivated worldwide. The leaf paste is applied to the skin in infants for treating staphylococcal-scalded skin syndrome or also known as Ritter-von-Ritterschein disease (Burkill 1966). The leaves are also used to treat eczema, syphilis-related skin disorders, and venereal diseases (Mitra et al. 2007). Previous studies had identified flavonoids such as kaempferol-3-O-glucoside, myricetin-3-O-rhamnoside, quercetin-3-O-glucoside, and quercetin-3-O-rhamnoside (Shukri and Alan 2010;Costa et al. 2020) from the leaf extracts. Andarwulan et al. (2012) had reported the presence of kaempferol, myricetin and quercetin in the hydrolyzed extract of the leaves.
To our knowledge, most of the previous antimicrobial and antioxidant studies on the leaves of A. occidentale were only using the extracts. Although many flavonols have been identified from the extracts, their contributions to the activities are not yet determined. Therefore, the objectives of this study were to evaluate the antimicrobial and antioxidant activities of the crude and hydrolyzed extracts, and seven flavonols found in the extracts. For the antimicrobial evaluation, only skin pathogenic microorganisms were selected as there is no available report on the susceptibility of this group of microorganisms to the extracts and compounds.

Results and discussion
The extracts were found to have a broad spectrum of activities by inhibiting all the microbes excluding Malassezia furfur (Table S1A). The hydrolyzed extract was found to show higher activity than the crude extract. The hydrolyzed extract was also found to inhibit two fungi (Microsporum canis and Trichophyton rubrum) with MIC value of 500 lg/mL. The primary antioxidant activities were measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing power (FRAP), 2,2 0 -azino-bis(3-ethylbenzthiazoline-6sulphonic acid (ABTS), and b-carotene bleaching assays. The hydrolyzed extract shows greater primary antioxidant activity than the crude extract (Table S2A). The metal-chelating assay was used to measure the secondary antioxidant activity and this activity was only detected in the crude extract. Figure S1 depicts the Ultra-Performance Liquid Chromatography (UPLC) chromatograms and UV spectra of the flavonols in the crude and hydrolyzed extracts. To date there was only one bioactivity-guided fractionation study on the leaf extracts of A. occidentale by Ajileye et al. (2015), and the antibacterial activity was found to be derived from the synergistic effect of two quercetin glycosides. Table S1 shows the highest activity of myricetin against all the tested ATCC bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus luteus, Corynebacterium minutissimum and Pseudomonas aeruginosa, and yeast (Candida albicans). Many earlier comparative studies had reported the pronounced antimicrobial activities of myricetin (Xu and Lee 2001;Rashed et al. 2014). Myricetin was also found in various extracts with antimicrobial activities (Abuga et al. 2020).
Quercetin-3-O-glucoside and the hydrolyzed extract share the same MIC against P. aeruginosa. Other microorganisms were found to be more susceptible to the hydrolyzed extract than the glycosidic flavonols. Numerous studies have discovered higher antibacterial activities of the aglycones in comparison with the glycosides (Rauha et al. 2000;Rashed et al. 2014). This is due to the hydrophilic characteristic of the glycosides that might interfere them to across the cell membranes (Echeverr ıa et al. 2017).
As indicated in Table S2, all aglycones show higher DPPH radical scavenging activities than the hydrolyzed extract with the lowest EC 50 value of myricetin. The ABTS activities of most compounds were greater than the extracts. Myricetin, quercetin and quercetin-3-O-glucoside were found to show stronger FRAP activities than the hydrolyzed extract. All aglycones demonstrate higher b-carotene bleaching activities than the extracts, and kaempferol gave the best activity. The metal chelating activity can be obtained only from myricetin-3-O-rhamnoside and kaempferol-3-O-glucoside.
The antimicrobial and primary antioxidant activities are mainly influenced by the location of hydroxyl groups on the benzene rings, and the substitutions of the aglycones (Xu and Lee 2001;Santos et al. 2021). The results showed that glycosylation has reduced both activities.

Conclusions
This study highlights higher antimicrobial and antioxidant activities of the hydrolyzed extract. The hydrolysis process is crucial to remove the glycosides and the lipophilic aglycones that are produced, can be passively diffused across the cell membranes of the skin pathogenic microorganisms. Myricetin may play a major role in potentiating the activities. The results suggest that this leaf is a valuable source of bioactive flavonols that can be developed as a natural inhibitory agent for skin pathogens.