Bactericidal and anti-inflammatory effects of Moquilea tomentosa Benth. flavonoid-rich leaf extract

In the context of an infection disease, it is important to eliminate the pathogen, and also minimize the immune response induced by the infectious agent, since the immune response can cause tissue damage, in certain circumstances. Thus, both aspects are related, and worthy to be investigated. Since M. tomentosa is used for intestinal disorders in traditional medicine, we decided to investigate both the anti-bacterial and anti-inflammatory properties of the plant leaf extract.

A previous study has suggested that the plant effect in intestinal tract could be due to the presence of flavonoids with antibacterial activity in the leaves [38].

Flavonoids are a very diverse class of natural products that present a wide range of biological activities such as antibacterial, antiviral, antioxidant, antitumor, antithrombotic, and immunomodulatory [23]. Flavonoid structure consists in two benzene rings, A and B, plus a heterocyclic pyrane ring that is usually glycosylated in plants [32]. In general, phenolic compounds (such as flavonoids and tannins) and saponins are mostly recovered from polar partitions, such as in EtOAc, when liquid–liquid extraction is used [10]. Several flavonoids have been described for the Licania and other Chrysobalanaceae genera, mainly myricetin, quercetin and kaempferol [8, 16].

Previous studies showed that M. tomentosa hydroalcoholic leaf extracts were active against different Gram-positive and -negative bacteria, with MIC values ranging from 32 to ≥ 512 μg/mL [38]. Ethanol extraction of M. tomentosa seeds also showed bactericidal activity [15]. In both studies, the extracts used were crude and non-partitioned, limiting the systematic investigation of the molecules involved in the bactericidal effect.

Here we observed that the EtOAc partition presented the strongest antibacterial activity, with concentrations varying from 125 to 500 μg/mL (Table 1). Interestingly, the EtOAc partition was more active against Gram-positive than Gram-negative bacteria (Table 1). According to [37], natural products that lead to MIC values lower than 500 μg/mL have strong bioactive and pharmaceutical potentials. Thus, our results confirmed the antibacterial ability of M. tomentosa, and showed that this activity was present at the EtOAc fraction. It is important to point that, Gram-positive bacteria are less relevant than Gram-negatives in the context of intestinal disorders, and although the EtOAc partition was less effective against Gram-negative, we tested only few reference strains. For better correlation of traditional medicine use of M. tomentosa and its effect in intestinal bacteria, more clinical Gram-negative isolates should be investigated.

The mass spectrometry analysis of the most active M. tomentosa EtOAc fraction showed the presence of a major compound (m/z 595), tentatively assigned as a flavonol glycoside myricetin 3-O-xylosyl-rhamnoside. The annotation of this compound was based on previously putatively annotated compounds with no chemical standard reference, based on spectral similarities following the Metabolomics Standards Initiative (MSI) recommendations [40]. The observation of the ion m/z 595 fragmentation revealed the formation of two major ionic species, m/z 316 and m/z 271, where the ion m/z 316 [M-279]− is related to the loss of the disaccharide xylosyl-rhamnoside moiety, corresponding to the aglycone myricetin [20]. The MS3 data of ion m/z 316 showed fragments m/z 271 [M-H–CO-H2O]− and m/z 179, typically found in retro Diels–Alder reactions of flavan-3-ols with a dihydroxilated-A ring, a characteristic of myricetin derivatives [34].

Myricetin can be isolated from different plant sources such as berries, vegetables and herbs, and it may occur in both free and glycosylated forms. Several myricetin derivatives have been identified in the Chrysobalanaceae family [3-8, 18] (Table S1). Bilia and co-workers also reported a glycosylated myricetin with m/z 595 identified as myricetin-3-(2’-xylosyl)-rhamnoside, in both L. carii [4] and Moquilea pyrifolia [3] (Table S1) species. Whether the M. tomentosa myricetin 3-O-xylosyl-rhamnoside compound presents similar glycosylation sites remains to be investigated.

Environmental stress conditions such as the presence of certain antibiotics, influences bacterial ability to produce biofilms that act as a physical barrier to increase bacterial tolerance to these molecules protecting bacteria from death [21, 24, 26]. For S. aureus for example, a sub-inhibitory concentration of β-lactams increased 10 times the production of biofilm [22], and amoxicillin increased bacterial biofilm and altered biofilm composition [28].

Here we showed that M. tomentosa EtOAc fraction also stimulated biofilm production by two strains of S. aureus, showing that this partition generated a stressful environment for bacteria. Besides our work, it was demonstrated that Microdesmia rigida leaf extract (2048 μg/mL) was able to modulate biofilm production by different species of Candida [18]. From the nine tested Candida spp., three exhibited an increase of biofilm production, while in three others the biofilm production was decreased. Interestingly, this extract also contained glycosylated flavons myricetin-3-O-rhamnoside (myricitrin) and myricetin-O-hexoside (Table S1), and myricitrin was the most abundant molecule of the extract [18]. It remains unclear which mechanisms are related to the ability of this extract to induce or decrease biofilm production by bacteria and yeast, and if myricetin glycosylation contributes to the molecule activity.

Several studies have shown that myricetin has diverse biological activities such as anticarcinogen, antimutagen, antioxidant, anti-inflammatory, and it is also able to reduce platelet aggregation, viral infection and replication (revised by [1, 35]).

Hou et al. [19] have shown that free myricetin reduced LPS-induced pro-inflammatory molecules such as TNF-α, IL-6, IL-1β, COX-2 and iNOS (NOS2) by RAW264.7 macrophages in vivo, and also lung inflammation induced by LPS in a mice model. This effect was associated to the suppression of NF-κB p65 and AKT in NF-κB pathway and JNK, p-ERK and p38, in MAPK signaling pathway.

An anti-inflammatory activity was also observed for the glycosylated myricetin, myricitrin present in the leaf extract from Myrica rubra. The authors showed that the myricitrin enriched extract was able to inhibit TNF-α production by RAW 264.7 cells and reduce IgE levels in DO11.10 mice model [36]. Myricitrin was also able to reduce NO production and iNOS (NOS2) expression induced by LPS [11].

Similarly, we showed that M. tomentosa-enriched myricetin 3-O-xylosyl-rhamnoside fraction was able to reduce IL-6, TNF-α and NO production by RAW 264.7 macrophages stimulated with LPS, showing that both free and glycosylated myricetin have a potential anti-inflammatory effect. The suppression mechanisms underlying the anti-inflammatory effect of M. tomentosa EtOAc fraction are still under investigation.

Other studies have highlighted the importance of glycosylation for solubility, stability and biological activity of myricertin [41]. For example, [30], showed that myricetin 3-rhamnoside and myricetin 3-(6-rhamnosylgalactoside) are more effective than free myricetin to reduce HIV infection, since glycosylation may enhance flavonoid internalization by the cells allowing myricetin to act intracellularly.

Thus, isolation, structural and pharmacological studies of different glycosylated myricetin from plant sources is crucial to better understand the biological effects of these flavonoids and to improve the efficacy of these molecules for pharmacological purposes.

Collectively, our results indicate that the EtOAc fraction isolated from M. tomentosa leaves could act reducing bacterial survival and also inhibiting inflammatory response in vitro. Thus, we demonstrate that M. tomentosa leaf extract presents potential pharmacological properties. Whether this activity is relevant in vivo, is a subject to be further investigated using animal models of experimental digestive infection.

留言 (0)

沒有登入
gif