The antibiotic potentiating activity against standard and multidrug-resistant Escherichia coli and Staphylococcus aureus bacterial strains of the natural compound 5”-O-α-L-raminopyranosyl-1’-β-D-glycopyranosyl-4’,6-dimethoxyflavone (pectolinarin) isolated from L. camara leaves was evaluated. Tests for antibacterial activity of the pure natural substance and analysis of the potentiation of antibacterial activity of pectolinarin associated with antibiotics were carried out against standard and multiresistant bacterial strains of Escherichia coli and Staphylococcus aureus by microdilution. Pectolinarin, when combined with the antibiotic gentamicin, showed synergism, potentiating growth inhibition against Gram-positive S. aureus strains. The pectolinarin flavonoid when combined with the gentamicin antibiotic potentiated its action Gram-positive S. aureus bacteria. Moreover, an antagonistic effect was observed when the pectolinarin was combined with the penicillin antibiotic against the multiresistant S. aureus 358 strain. This research suggests that pectolinarin is a compound with potential application as an antibacterial drug.

Keywords: flavonoid, Lantana camara, antibacterial activity

Flavonoids are one of the most diverse and important classes among plant chemical constituents, with over 6,000 flavonoids described.^1–3 Flavonoids present as their most significant subclasses: anthocyanins, catechins, chalcones, dihydroflavonols, flavonols, flavones, flavanones, and isoflavones.⁴ In plants, certain functions such as protection against pathogenic microorganisms, antioxidant action, allelopathic action, enzymatic inhibition, and protection against the incidence of ultraviolet rays are attributable to flavonoids.^5–8 From the flavonoid subclasses, flavones stand out given their protective action, protecting plant cells from damage caused by photooxidation,⁹ as well as protecting the body against free radical species.¹⁰ Substances extracted from plants have been arousing much scientific interest due to the diversity of the biological activities they present.^11–15 One such plant species that has been of interest to the scientific community is the L. camara from the Verbenaceae family, originating from the Americas and Africa.¹⁶ L. camara is a plant that possesses a toxin, which considering its toxicity, has been used as a repellent against Aedes mosquito larvae.¹⁷ Medicinal plants are an important source for obtaining new molecules. While several synthesis methods are used to get effective drugs to treat certain diseases, plants can produce chemical constituents that are difficult to obtain through synthesis procedures.¹⁸ In this context, obtaining new substances isolated from medicinal plants and investigating their structural and pharmacological properties is essential. In the present study, the antibiotic potentiating activity against standard and multidrug-resistant Escherichia coli and Staphylococcus aureus bacterial strains of the natural compound 5”-O-α-L-raminopyranosyl-1’-β-D-glycopyranosyl-4’,6-dimethoxyflavone (pectolinarin) (Figure 1) isolated from L. camara leaves was evaluated.

Figure 1 Molecular structure of the pectolinarin.

Plant material

Leaves from L. camara were collected in Vale do Alemão site in Pernambuquinho, Guaramiranga-Ceará (Brazil) district (S4°12’22.7” and W38°57’00.8”) in March 2016. The material was identified by Dr. Maria Iracema Bezerra Loiola at the Herbário Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil, where the voucher specimens (No. 0056696) were deposited.¹⁹

Extraction and isolation of flavonoid

Leaves (100 g) of L. camara were ground to powder and extracted with ethanol (EtOH) at room temperature. The solvent was removed under reduced pressure to give an EtOH extract. The EtOH extract (35.49 g) was fractionated coarsely on a silica gel column by elution with hexane, EtOAc and MeOH. The ethyl acetate fraction resulted in the formation of a precipitate yielded bioside flavone named 5”-O-α-L-raminopyranosyl-1’-β-D-glycopyranosyl-4’,6-dimethoxyflavone (Figure 1). The extraction, isolation and structural determination of the flavonoid used in this study were previously reporting in the literature.¹⁹

Microorganisms

The standard microorganisms used in the tests were obtained through the National Institute for Quality Control in Health (INCQS) of the Oswaldo Cruz Foundation, Ministry of Health, and the multi-resistant ones were obtained from the Federal University of Pernambuco- UFPB. The bacterial strains used were Escherichia coli (standard E. coli ATCC 10536 and multidrug-resistant E. coli 27) and Staphylococcus aureus (standard S. aureus ATCC 25923 and multiresistant S. aureus 358), with their resistance profiles being identified and reported in Table 1. All strains were maintained on Heart Infusion Agar (HIA, Difco Laboratories Ltda.). Prior to the assays, the strains were cultured for 24h at 37°C in Heart Infusion Agar (HIA, Difco Laboratories Ltda.).

Drugs

The antibiotics gentamicin (aminoglycoside), norfloxacin (fluoroquinolone), and penicillin (β-lactam) were used to evaluate the antibiotic potentiating activity of the pectolinarin flavonoid. All drugs were diluted in sterile water to concentration 5000 mg/mL. The solutions were prepared following the recommendations of the National Clinical Laboratory Standards Committee - NCCLS (NCCLS, 2003).

Antimicrobial activity

The MIC (minimum inhibitory concentration) was determined in a 96 well sterile microdilution assay²⁰ 100 μL of the inoculum suspension (10⁵ CFU/mL^-1) were removed and added to a Brain Heart Infusion broth (10% BHI), thereafter 100 μL of this solution was added to each well, followed by 100 μL of the pectolinarin compound which was added to the first well, followed by serial (1:1) dilutions up to the penultimate well of the microplate. The final concentration of the sample ranged from 512 - 8 μg/mL. The MIC was recorded as the lowest concentration capable of inhibiting growth.

Antibiotic potentiating activity of the flavonoid pectolinarin

The pectolinarin compound was evaluated as a potentiator of antibiotic action against clinically isolated microorganisms. The MIC of the antibiotics was evaluated in the presence and absence of the test solution in sterile microplates. The antibiotics were evaluated at concentrations ranging from 2500 to 2.5 µg/mL. The test solution at subinhibitory concentrations (MIC/8) was mixed with 10% BHI broth and the inoculum suspensions. Then, 100 μL of the antibiotic was added to the first well of the plate, and serial dilutions (1:1) were performed up to the penultimate well of the microplate. The plates were incubated at 37 ºC for 24 hours, and after this period, the reading was observed using resazurin.

Statistical analysis

Test results were performed in triplicates and expressed as the geometric mean. Statistical analysis was performed using a one-way ANOVA followed by Tukey's post-hoc test, performed using the GraphPad Prism 6.02 software, considering significance with p<0.05.

Antibacterial activity evaluation of the pectolinarin alone and in combination with antibiotics

The minimum inhibitory concentration (MIC) obtained for the flavone pectolinarin was ≥1024 µg/mL. Thus no differences were observed in the sensitivity of the pure natural substance against standard bacterial S. aureus and E. coli strains. However, the modulation by associating pectolinarin with gentamicin hears a change from resistant to sensitive with S. aureus 358 (Table 1).

The MIC determination of the antibiotics norfloxacin, gentamicin and penicillin in the presence and absence of the pectolinarin substance at a subinhibitory concentration (MIC/8=128 μg/mL) against multiresistant S. aureus 358 and E. coli 27 bacteria are shown in Figure 2.

Figure 2 MIC of the antibiotics norfloxacine, gentamicin, and pencilillin the presence and absence of the pectolinarin in concentration MIC/8 (128µg/mL), towards S. aureus 358 and E. coli 27. Statisically significant values with *** p<0.0001- Non significant values (ns) with p>0.05.

In the antibiotic activity test against S. aureus 358, pectolinarin was seen to potentiate the inhibition of bacterial growth when associated with gentamicin, showing significant synergism with p<0.0001. An antagonistic effect was observed with the association of the compound with penicillin with a significance of p<0.0001, while non-significant results were obtained with the antibiotic norfloxacin. When E. coli 27 was the bacterial strain tested, no significant changes in MICs were observed with the association of pectolinarin with the tested antibiotics.

Aminoglycosides present bactericidal action against aerobic Gram-negative bacteria, some staphylococci, and Mycobacterium tuberculosis.²¹ However, Gram-positive bacteria are more sensitive to antimicrobial action due to the presence of a bacterial wall that normally does not restrict the penetration of toxic molecules into the bacteria, whereas Gram-negative bacteria possess a barrier system consisting of an outer membrane with a cell wall formed by phospholipids, lipopolysaccharides and proteins making it impermeable to antibacterial agents, resulting in a greater resistance of these bacteria to antibiotics.^22,23

In vitro studies have shown that pectolinarin possesses antiviral activity against the herpes simplex HSV-1 and dengue DENV-2 virus,²⁴ anti-inflammatory and anti-allergic actions,²⁵ as well as presenting an effective hepatoprotective action in a rat model of liver damage caused by D-galactosamine.²⁶

Antimicrobial resistance has become a major public health problem affecting all countries,^27,28 where antimicrobial resistance may be a natural phenomenon developed by the pathogenic organism itself. However, the poor use of medications accelerates this process increasing resistance levels (WHO 2005).²⁹    

Treating various types of infectious diseases has become difficult, mainly due to the low efficiency of antimicrobials against today's bacteria,^30,31 which reinforces the need for the validation of new plant-derived drugs through research that will be useful in combating the spread of pathogenic microorganisms.^32–34

The pectolinarin flavonoid when combined with the gentamicin antibiotic potentiated its action Gram-positive S. aureus bacteria. Moreover, an antagonistic effect was observed when the pectolinarin was combined with the penicillin antibiotic against the multiresistant S. aureus 358 strain. This research suggests that pectolinarin is a compound with potential application as an antibacterial drug.

We thank the financial support from CNPq, CAPES, and FUNCAP. We acknowledge the Northeastern Center for the Use and Application of Nuclear Magnetic Resonance (CENAUREM) for the NMR measurements. H.S. Santos acknowledges financial support from the PQ/BPI-FUNCAP (Grant BP4-0172-00075.01.00/20), A.M.R. Teixeira acknowledges financial support from CNPq (Grant 305719/2018-1).

The authors declare no conflict of interest.

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