Eucalyptus botryoides’ resin and its new 2-O-galloyl-1,6-O-di-trans-p-coumaroyl-β-D-glycopyranoside compound display good antimicrobial activity

Abstract Fungal resistance to different therapeutic drugs has become a growing challenge. This crucial health problem requires new effective drug alternatives. Herein, we report the study of Eucalyptus botryoides’ resin used in folk medicine as antimicrobial. Thus, E. botryoides’ resin was extracted with aqueous-ethanol and fractionated using Sephadex chromatography, furnishing its major compounds. The crude extracts and the isolated compounds were evaluated for their in vitro antimicrobial activity against bacteria and yeasts. The crude extract displayed MIC of 25 μg/mL against S. salivarius, and for C. albicans, C. glabrata, and C. tropicalis the MIC were between 2.9 and 5.9 μg/mL. The 7-O-Methyl-aromadendrin was the most effective against C. glabrata and C. krusei (MIC = 1.6 μg/mL). 2-O-Galloyl-1,6-O-di-trans-p-coumaroyl-β-D-glycopyranoside, first time reported, showed MIC of 3.1 μg/mL against C. glabrata and C. krusei. Overall, this work gave promising results, indicating that Eucalyptus botryoides’ resin and its compounds have the potential for developing anti-yeast products. Graphical Abstract


Introduction
The Eucalyptus genus (Myrtaceae) is originally from Australia, and it bears approximately 800 species cultivated in tropical, subtropical, and temperate regions (Neiva et al. 2015). The Eucalyptus plants are widely used in energy production, essential oils extraction, wood for construction, and paper production (Carrillo et al. 2018). This plant species is also broadly applied in natural medicine, and it is traditionally used to treat respiratory diseases such as common cold, influenza, and sinus congestion (Salehi et al. 2019). It is also used for the treatment of respiratory disorders (Galan et al. 2020), wound healing (Dhakad et al. 2018), as insecticide/insect repellent (Lucia et al. 2012), nematicide (Taur et al. 2010), and in the manufacture of perfumes and soaps (Dhakad et al. 2018). There are several biological activities reported for Eucalyptus essential oil, including antimicrobial (Mekonnen et al. 2016), antioxidant (Chen et al. 2014), anti-inflammatory (Silva et al. 2003), cytotoxic (D€ oll-Boscardin et al. 2012), and antimalarial activities (Milhau et al. 1997).
Beyond its essential oils' therapeutic and commercial values, Eucalyptus species produce wood exudates, known as kino, another common source of medicinal agents. The kino, composed of a high concentration of tannins, plays a crucial role in the plant's defense against pathogenic infections caused by insects or fungi (Von Martius et al. 2012). It has been used to treat diarrhea and wounds with antimicrobial properties. In a recent study, kino's ethanolic extracts from E. citriodora and E. torelliana showed high antimicrobial activity against Gram-negative (Pseudomonas aeruginosa and Escherichia coli), Gram-positive (Staphylococcus aureus), and against Candida albicans microorganisms (Nobakht et al. 2017).
In the last decades, Candida spp. infections have become more frequent, mainly as one agent of hospital-acquired fungal infection (Cortegiani et al. 2018). Morbidity and mortality rates in critically ill patients with invasive Candida infections are high, reaching mortalities from 40% to 70% (Salci et al. 2018). Although the most common invasive fungal infections have been related to Candida albicans, other Candida species, such as C. parapsilosis, C. tropicalis, and C. glabrata have significantly increased their incidences, which could be associated with the extensive use of broad-spectrum antimicrobials and immunosuppressive drugs (Grau et al. 2016).
Additionally, fungal resistance to different therapeutic drugs has become a growing challenge for treating invasive fungal infections, currently representing a crucial health problem (Salci et al. 2018). Biofilm-associated Candida infections, for example, is one of the problems associated with anti-yeast drug resistance since most anti-yeast drugs do not penetrate the exopolymeric matrix of the biofilm (Morace et al. 2014). Because of the increased drug resistance to antifungals, it is necessary to consider the complexity of fungal cells, owing to fungi and humans' eukaryotic similarity, limiting the number of drugs available in therapy.
In this context, natural products compounds are promising sources for developing new therapeutic agents against many microorganisms (Pejin et al. 2012). Therefore, searching for new effective drugs from natural sources is an excellent alternative to treat resistant Candida strains and other fungal infections (Morace et al. 2014;Pejin et al. 2016;Sobel and Sobel 2018).

Isolation of the main compounds from eucalyptus botryoides' resin
The crude extract from E. botryoides' resin ( Figure 1S, supplementary materials) was fractionated using Sephadex chromatography, and each fractions obtained were purified by preparative HPLC-UV to furnish the pure compounds 1 (233 mg), 2 (17 mg), 3 (8 mg), and 4 (2 mg), all with relative purity above 97% by HPLC-DAD at 275 nm, as described in Sections 3.1. and 3.2.
Chemically, Eucalyptus resin is a complex mixture of lipophilic compounds. From the barks of E. botryoides, the triterpenes Oleanolic acid, Betulinic acid, Ursolic acid, and Asiatic acid were identified (Ferreira et al. 2018). The Eucalyptus leaves' essential oil is mainly composed of monoterpenes and sesquiterpenes, and their composition varies according to their geographic location. The monoterpene 1,8-Cineole (22%) is the chemical marker of Eucalyptus botryoides essential oil from Morocco, while in Italy, the same species presented a-Pinene (27%) as the chemical marker (Elaissi et al. 2011), highlighting the importance of phytochemical studies with this species of medicinal and commercial relevance.  citriodora and E. maculata and other plants, such as Populus alba. This compound was also isolated from propolis samples produced by Apis mellifera bees and geopropolis produced by Melipona subnitida bees, which shows the participation of Eucalyptus spp. species as a botanical source in propolis production. 7-O-Methyl aromadendrin displays several biological activities, including anti-inflammatory, anti-cancer, and antimicrobial apoptotic inducers (De Souza et al. 2013;Ribeiro et al. 2021).

Antimicrobial activity in vitro
The crude extract (HRE) was evaluated against eleven types of aerobic bacteria, being eight gram-positive (S. aureus, S. mutans, S.mitis, S. sanguinis, S. sobrinus, L. casei, S. salivarius, E.faecalis) and three gram-negative (E. coli, P. aeruginosa, S. choleraesuis). Tetracycline and chlorhexidine, antibacterial drugs, were used as positive controls (Table 1). The crude extract (HRE) displayed antimicrobial MIC values ranging from 100 to 400 lg/mL. Extracts should display antibacterial activity at concentrations below 100 lg/mL to be considered with potential for further studies (Ribeiro et al. 2021), and the best result was against the oral bacteria S. salivarius with MIC of 25 lg/mL ( Table 1).
The in vitro anti-yeast evaluation of the hydroalcoholic extract and its isolated compounds showed promising results ( Table 2). The crude extract of Eucalyptus resin (HRE) showed MIC values ranging from 2.9 to 46.9 lg/mL, effective against all tested yeasts. The lowest MIC's value of HRE was against C. glabrata, one of the moscommon Candida species (Table 2). The HRE and isolated compounds were also evaluated against Candida strains, and Amphotericin was a positive control ( Table 2).
The isolated compounds (1-3) exhibited good activity against C. krusei with MIC values lower than 3.1 lg/mL (1.6 < MIC < 3.1 lg/ml). Compound 1 showed the best MIC values (1.6 lg/mL) against C. glabrata. Compound 3 was the only one effective against the C. tropicalis, with MIC of 6.3 lg/mL. According to Rios and Recio (R ıos and Recio 2005) and Gibbons (Gibbons 2008), pure compounds can be considered promising when their MICs are lower than 10 lg/mL. Therefore, we suggest that these compounds have potential against opportunistic agents, such as Candida species.
Von Martius and collaborators (Von Martius et al. 2012) evaluated the antimicrobial activity of E. botryoides kino, reporting that it was not active against E. coli, P. aeruginosa, and yeasts, but it inhibited the growth of S. aureus. These results contrast with our findings since the HRE showed good activity against Candida microorganisms. The authors also found a positive correlation between total tannins content and antibacterial effect. However, tannins are often more toxic than their related monomeric phenolic structures (Von Martius et al. 2012). Elaissi and collaborators (Elaissi et al. 2011) evaluated the activity of the essential oil from leaves of E. botryoides, which showed activity against E. coli, P. aeruginosa, E. faecalis, and S. aureus.

Plant material and hydroalcoholic extraction
The Eucalyptus botryoides' resin was collected in the city of Bambu ı, state of Minas Gerais, Brazil, in June 2018 and stored at À20 C. The species E. botryoides was identified by professor Milton Groppo and a voucher specimen (no. 17670) was deposited in the herbarium of the Department of Biology, Faculty of Philosophy, Sciences, and Literature of Ribeirão Preto, University of São Paulo (SPFR). Twelve grams of E. botryoides's resin were extracted with 120 mL aqueous-ethanol (7:3) solution under stirring (120 RPM) in a mechanical shaker at 30 C for 24 h. The solution was filtrated, concentrated under reduced pressure, and lyophilized, furnishing seven grams of the hydroalcoholic resin extract (HRE).

Isolation of the main compounds from eucalyptus botryoides' resin
The HRE (seven grams) was solubilized in 20 mL of MeOH, applied to a gel filtration chromatography with Sephadex LH 20, and eluted with a step gradient of H2O/MeOH (9:1 to 1:9, v/v). Twenty-three fractions of 20 mL each were collected, and their chromatographic profiles were analyzed by thin-layer chromatography (TLC). The ones with similar profiles were combined. Then, the fractions were analyzed by HPLC-DAD using an Ascentis reverse-phase chromatographic column (5 lm, 250 Â 4.6 mm). The elution program was as follows: 5 min isocratic with 25% of B (acetonitrile) and 75% of A (94.6% water, 0.4% formic acid, 5% de methanol), followed by gradient elution with 35% B at 10 min, 38% B at 15 min, 40% B at 20 min, 45% B at 45 min, 70% B at 50 min, 80% B at 55 min, and 100% B at 60 min, at a flow rate of 1.0 mL/min and detection at 275 nm. Fractions eight, eleven and fifteen were dissolved in methanol (HPLC grade), filtered through a 0.45 lm membrane (Sartorius) and injected into preparative HPLC-UV on a Synergi Fusion reverse phase column (4 lm, 250 Â 10 mm), at a flow rate of 4.0 mL/min, detection at 275 nm, furnishing compounds 1 (fraction eight), 2-3 (fraction eleven) and 4 (fraction fifteen) (Rodrigues et al. 2020). As detailed herein and in the supplementary materials, their chemical structures were confirmed by spectroscopic ( 1 H NMR) and spectrometric (High-resolution ESI-MS) analyses.

7-O-Methyl-aromadendrin (1)
Flavonoid 1 was previously reported as a white solid in Eucalyptus citriodora (Freitas et al. 2007). The molecular weight of 302.28 g/mol (C 16 H 14 O 6 ) was established from HPLC-MS analysis. The identity of this compound was confirmed by 1 H NMR (500 MHz, CD 3 OD), 13 C NMR (125 MHz, CD 3 OD), ESI-MS analysis (Table 1S, supplementary materials), and data comparison with the reported literature (Freitas et al. 2007).
3.2.2. 1-O,2-O-Digalloyl-6-O-trans-p-coumaroyl-b-D-glycopyra noside (2) This phenolic glycoside 2 was isolated as a white solid and previously reported in Eucalyptus citriodora (Freitas et al. 2007). The molecular weight of this compound, 630.51 g/mol (C 29 H 26 O 16 ) was confirmed by 1 H NMR (500 MHz, CD 3 OD), 13 C NMR (125 MHz, CD 3 OD), and ESI-MS analysis (Table 2S, supplementary materials) and data comparison with the reported literature (Freitas et al.,. 2007).   (CLSI, 2012). The broth microdilution method, conducted in 96-well microplates, was used to determine the Minimum inhibitory concentrations (MIC). The samples were dissolved in DMSO (Merck) at 1.0 mg mL À1 . Then, the samples were diluted in RPMI broth medium. The serial dilution was at concentrations ranging from 0.195 to 400 mg mL À1 . The inoculum was adjusted at 625 nm in a spectrophotometer to give a cell concentration of 1.103 CFU ml À1 . The microplates were incubated at 37 C for 24 h. After that, 30 mL of aqueous resazurin (Sigma-Aldrich) solution at 0.02% was added to each well. Blue and red colors represented the absence and presence of microbial growth, respectively. An aliquot of the inoculum was aseptically removed from each well before resazurin addition determining the minimum bactericidal concentration (MBC). This aliquot was plated into Sabouraud agar. The plates were also incubated at 37 C for 24 h. Tetracycline and chlorhexidine were used as a positive control for bacteria and amphotericin B for yeasts.
Overall, this study provides promising results with MIC < 10 lg/mL for the isolated compounds and MIC < 100 lg/mL for the crude extract, indicating that Eucalyptus botryoides compounds have potential for the development of anti-yeast products.