Structural and Microbiological Characterization of 5-Hydroxy-3,7,4′-Trimethoxyflavone: A Flavonoid Isolated from Vitex gardneriana Schauer Leaves

Abstract

Introduction:

Staphylococcus aureus represents the most common etiologic agent of purulent infections, affecting humans and animals. Escherichia coli is one of the principal causes of infectious diseases, mainly diarrheal diseases due to enterotoxin action. There are many reports indicating that these bacteria are multidrug-resistant (MDR) pathogens.

Objective:

In this study, we investigated the antimicrobial and modulatory activities of 5-hydroxy-3,7,4′-trimethoxyflavone (VG.EF.CLII) against E. coli and S. aureus strains.

Methods:

5-Hydroxy-3,7,4′-trimethoxyflavone was isolated from Vitex gardneriana Schauer leaves and structurally characterized using nuclear magnetic resonance. The antibacterial effect of VG.EF.CLII and modulation of antibiotic activity, both determined by minimum inhibitory concentration, were assessed using microtiter plates.

Results:

VG.EF.CLII showed bacterial growth inhibition at concentrations ≤512 μg/mL, and synergistic effects were observed for the modulation of two distinct antibiotic classes (the fluoroquinolone norfloxacin and the aminoglycoside gentamicin).

Conclusion:

5-Hydroxy-3,7,4′-trimethoxyflavone isolated from V. gardneriana showed promising antimicrobial activity against MDR bacterial strains S. aureus 358 and E. coli 27 when associated with the antibiotics norfloxacin and gentamicin. Therefore, this natural product can contribute to the control of bacterial resistance.

Introduction

Natural compounds isolated from medicinal plants have become a promising alternative in the fight against microbial infections, and when associated with antibiotics, they have been demonstrated to be a very attractive alternative for the challenges posed by drug-resistant bacterial infections, with the potential to decrease the development of antimicrobial resistance.^1,2 Combined therapy with antibiotics and natural products may avoid physiological and pharmacological side effects in living cells.^3–6 Thus, isolated compounds from medicinal plants are a viable alternative for the treatment of infections caused by multidrug-resistant (MDR) strains.^7,8 In this context, the use of natural substances to modulate bacterial resistance to antibiotics has been shown to be an alternative for drugs that are no longer efficient in the treatment of infectious diseases.²

The Vitex genus represents 36 species of which 16 have known pharmacological activity and are used in popular medicine as an alternative or complementary treatment for various diseases.⁹ Phytochemical studies of the genus Vitex revealed the presence of flavonoids, terpenoids, saponins, reducing sugars and phenylpropanoglycosides.⁹ Some of the specimens of the Vitex genus have been used as phytotherapeutic drugs; for instance, Vitex agnus-castus and V. negundo have been used to relieve premenstrual syndrome disorders and to treat rheumatoid arthritis, respectively.⁹

Vitex gardneriana Schauer, popularly known as “Jaramataia,” possesses in its phytochemical constitution flavonoids, iridoids, monoterpenoids, sesquiterpenoids, triterpenoids, diterpenoids, steroids, saponins, phenylpropanoglycosides, and reducing sugars, showing congruence with secondary metabolites produced by other members of the Vitex genus.¹⁰ It is worth highlighting the presence of the triterpenoids and steroids β-amirine and β-sitosterol.¹⁰ V. gardneriana is used to treat various illnesses such as flu, verminoses, abdominal pain, prostatic inflammation, bone pain, kidney problems, and spinal pain.^11,12

In this study, a structural and microbiological characterization of 5-hydroxy-3,7,4′-trimethoxyflavone isolated from V. gardneriana leaves was carried out. This natural compound was first obtained from Aniba sp., also known as Aniba canellila (Kunth) Mez.¹³ Nevertheless, this is the first time that this compound is found in the Vitex genus. The structural determination of this natural compound was performed by the nuclear magnetic resonance (NMR) technique, and a study on the modulatory and antimicrobial activity against standard and MDR bacterial strains of Escherichia coli and Staphylococcus aureus was performed.

Materials and Methods

Collection and identification of plant material

The leaves of V. gardneriana (Jaramataia, Lamiaceae Martinov) were collected in April 2014 at the experimental farm of the State University Vale do Acarau (03° 36′ 44′′ S; 40° 18′ 37′′ W), located 11 km from the city of Sobral, Ceara, Brazil, according to the methodology described by Cartaxo et al.¹⁴ The species was identified and deposited in the Francisco Jose de Abreu Matos Prisco Bezerra Herbarium of the State University Vale do Acarau (Sobral-CE, Brazil) as exsiccate #17,703.

Extraction, isolation, and identification of 5-hydroxy-3,7,4′-trimethoxyflavone

Silica gel 60 (230–400 mesh; Merck, Darmstadt, Germany) was used for analytical thin-layer chromatography (TLC). Silica gel 60 (70–230 mesh; Merck) was used for column chromatography (CC). TLC using vanillin-perchloric acid-EtOH followed by heating allowed visualization of all the compounds.¹⁵

Vitex gardneriana leaves were ground, subjected to cold methanol extraction and concentrated under reduced pressure to yield the leaf methanolic extract denoted VGFMeOH. Part of the VGFMeOH extract was subjected to liquid/liquid partition, yielding the hexane, chloroform, and AcOET fractions. A portion of the AcOET fraction was subjected to silica gel CC using hexane, AcOET, and methanol as eluents in order of increasing polarity, providing the hexane (F 10–10), hexane/AcOEt 9:1 (F 11–93), hexane/AcOEt 8:2 (F 94–310), hexane/AcOEt 6:4 (F 311–370), and hexane/AcOET 5:5 (F 371–780) fractions. The fraction 371–740, obtained with hexane/AcOET 6:4, was subjected to silica gel CC using hexane, ethyl acetate, and methanol as eluents in order of increasing polarity, yielding the hexane (F′ 1–79), hexane/AcOET 7:3 (F′ 80–243), and hexane/AcOET 3:7 (F′ 244–439) fractions. The fraction F′ 80–145, obtained with hexane/AcOET 7:3, was subjected to silica gel CC using hexane, ethyl acetate, and methanol as eluents in order of increasing polarity, providing the hexane (F′′ 1–90), hexane/AcOET 6:4 (F′′ 91–190), and hexane/AcOET 5:5 (F′′ 191–230). In the F′′ 91–140 fraction, obtained with hexane/AcOET 6:4, the presence of a greenish solid denoted VG.EF.CLII was observed, which was shown to be pure through TLC analysis.

NMR measurements

¹H and ¹³C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for ¹H and 125 MHz for ¹³C); chemical shifts are given in ppm (¹³C and ¹H).

Bacterial isolates

The bacterial strains used were E. coli (EC-ATCC 10536 and EC27) and S. aureus (SA-ATCC 25923 and SA358), which are both clinical strains isolated from surgical wounds. All strains were maintained in heart infusion agar (Difco Laboratories Ltd., Detroit, MI). Before performing the assays, the strains were cultivated for 18 h at 37°C in brain heart infusion broth (BHI; Difco Laboratories Ltd.).

Drugs and reagents

To evaluate the antibiotic modulatory activity of 5-hydroxy-3,7,4′-trimethoxyflavone (VG.EF.CLII), the antibiotics gentamicin, norfloxacin, and penicillin were used. Solutions were prepared based on the established recommendations in Clinical and Laboratory Standards Institute¹⁶ and diluted in sterile water to antibiotic concentrations of 5,000 mg/mL.

Antimicrobial and modulatory activities

The antibacterial effect of VG.EF.CLII and the modulation of antibiotic activity, both determined by minimum inhibitory concentration (MIC), were assessed using microtiter plates according to the methodology described by Coutinho et al.¹⁷

The MIC was determined in 10% BHI by the broth microdilution method, using a 100 μL inoculum of each strain in a concentration of 10⁵ colony forming unit/mL in microdilution plates with 96 wells and performing a 1:1 serial microdilution. In each well, 100 μL of the substance was added, and the final concentration ranged from 512 to 8.0 μg/mL.

Statistical analysis

The antibacterial assays were performed in triplicate, and the results are expressed as the geometric mean for the evaluation of antibacterial activity. A one-way analysis of variance statistical analysis was applied for the analysis of the modulatory antimicrobial activity of VG.EF.CLII flavonoid, with Tukey's test as a post hoc test for both analyses, which were performed using GraphPad Prism 6.02 software.

Results and Discussion

Structural determination by NMR

The compound isolated from V. gardneriana named VG.EF.CLII was isolated as a green solid. The chelated hydroxyl hydrogen at δ_H 12.70 and the carbonyl group at δ_C 178.98 were also consistent with a 5-hydroxyflavone. The aromatic region of the ¹H NMR spectrum of VG.EF.CLII displayed five hydrogen resonance signals at δ_H 8.01 (d, J = 8.6 Hz, H-2′/6′), 7.05 (d, J = 8.6 Hz, H-3′/5′), 6.39 (s) (H-6) and 6.48 (s) (H-8). These spectral data were in agreement with a 5,3,7,4′-substituted flavone. In addition, three methoxyl resonances were observed at δ_H 3.90 (MeO-7), 3.91 (MeO-3), and 3.94 (MeO-4’). The ¹³C NMR spectrum showed signals for methoxyl groups at δ_C 55.63 (MeO-4′), 55.99 (MeO-7), and 60.33 (MeO-3). The signal at δ_C 178.98 (C-4) is compatible with a conjugated carbonyl of a nucleus flavonoid. The signals at δ_C 130.36 and 114.26 correlated in the Heteronuclear Multiple-Quantum Correlation (HMQC) spectrum with the hydrogen signals δ_H 8.01 and 7.05, respectively, corroborating that the aromatic ring is para substituted. The NMR data reveal the molecular structure for VG.EF.CLII, which is the flavonoid 5-hydroxy-3,7,4′-trimethoxyflavone, with the chemical formula C₁₈H₁₆O₆. The molecular structure of this compound is shown in Fig. 1, and the NMR data are given in Table 1. These data are compatible with those reported in the literature.¹³

FIG. 1.

Molecular structure with atom numbering for the flavonoid 5-hydroxy-3,7,4′-trimethoxyflavone (VG.EF.CLII).

Table 1.

¹H and ¹³C Nuclear Magnetic Resonance Data for the Flavonoid 5-Hydroxy-3,7,4′-Trimethoxyflavone (in CDCl3), J in Hz

	HSQC		HMBC
	δ_C	δ_H	²J_CH	³J_CH
C
2	156.15			H-2′/H-6′
3	139.06			MeO-3
4	178.98
5	161.88		H-6
7	165.61		H-6; H-8	MeO-7
9	156.96		H-8
10	106.25			H-6; H-8
1′	123.01			H-3′/H-5′
4′	161.22		H-3′/H-5′	H-2′/H-6′; MeO-4’
CH
6	98.01	6.39 (s)		H-8
8	92.35	6.48 (s)		H-6
2′/6′	130.36	8.11 (d, 8.6)
3′/5′	114.26	7.05 (d, 8.6)
HO-5	—	12.69 (sl)
MeO-3	60.33	3.91 (s)
MeO-7	55.99	3.90 (s)
MeO-4′	55.63	3.94 (s)

Chemical displacements δC and δH are in ppm.

C is for non-hydrogen-bonded carbons; CH is for the carbons bonded to hydrogen atoms.

HSQC, Heteronuclear Single Quantum Correlation; HMBC, Heteronuclear Multiple Bond Correlation.

Antimicrobial activity

The 5-hydroxy-3,7,4′-trimethoxyflavone (VG.EF.CLII) compound isolated from V. gardneriana showed a MIC ≤512 μg/mL against MDR bacterial strains. This result is considered clinically relevant¹⁷ and, therefore, we can claim that the VG.EF.CLII compound presents antimicrobial activity against the microorganisms tested. This is the first report demonstrating the antibacterial effect of any compound from V. gardneriana.

Antibiotic modulation activity

Figure 2 shows the different antibiotic modulation of preparations with the VG.EF.CLII compound isolated from V. gardneriana leaves against MDR S. aureus 358 and E. coli 27 strains. A synergistic effect was observed in both MDR strains in the presence of the VG.EF.CLII compound and the antibiotic norfloxacin, where a significant MIC (p < 0.0001 vs. norfloxacin) decrease was observed. A synergistic action can also be observed in gentamicin modulation with the VG.EF.CLII compound against the S. aureus 358 and E. coli 27 strains, where a significant MIC (p < 0.0001 vs. gentamicin) decrease was also observed. However, an antagonistic effect occurred for penicillin modulation with the VG.EF.CLII compound against the S. aureus 358 strain, as seen in the graph where the MIC increased significantly (p < 0.0001 vs. penicillin). No effect was observed for the E. coli 27 strain in the presence of VG.EF.CLII compound associated with the antibiotic penicillin since there was no significant difference in MIC (p > 0.05 vs. penicillin).

FIG. 2.

MICs in the presence and absence of flavonoid 5-hydroxy-3,7,4′-trimethoxyflavone (VG.EF.CLII) isolated from Vitex gardneriana Schauer against Staphylococcus aureus and Escherichia coli strains. Statistically significant value with ****p < 0.0001. MIC, minimum inhibitory concentration.

The Staphylococcus genus is part of the microbiota of the skin and mucosa of animals and birds,¹⁸ with S. aureus, S. epidermidis, S. saprophyticus, and S. haemolyticus being the most important species causing community-acquired and nosocomial human infection. In addition, S. aureus has been the most common etiological agent of infections in different tissues and/or organs (e.g., furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis, and bacterial arthritis).^19,20

Escherichia coli is one of the main causes of infectious diseases in humans. These bacteria are known to produce enterotoxins whose properties and role in diarrheal disease have been widely investigated. E. coli not only is a very common intestinal pathogen but also is often involved in extraintestinal diseases in the intensive care unit and in surgical wound infections.^21,22

Different natural products have demonstrated antibacterial effects against both species of bacteria, alone or in association with antibiotics: plant natural products as essential oils^23,24 and phytocompounds such as gallic acid, caffeic acid, vanillin, and others.^25–28

In developing countries, such as Brazil, the risk of infections and the exposure to health threats is greater than that in developed countries, with this risk being ∼20 times greater in developing countries than in developed countries. However, the discovery of antibiotics derived from natural products has saved millions of people from bacterial infections. Therefore, the use of antibiotic modulation with an isolated substance from medicinal plants that decreases bacterial resistance has become an alternative for the research and treatment of infectious diseases.^4,7,8

Recently, we have reported the structural and microbiological characterization of labdane diterpene 15,16-epoxy-4-hydroxy-labda-13(16),14-dien-3,12-dione isolated from the stems of Croton jacobinensis Baill.²⁶ and of 2-hydroxy-3,4,6-trimethoxyacetophenone isolated from Croton anisodontus Müll.Arg.²² The results of these studies corroborate that a natural product can contribute to the control of bacterial resistance. The antibiotic modulatory activity strategy is already a clinical practice used to promote synergistic actions leading to a decrease in the dose necessary to achieve desired therapeutic antimicrobial effects, but this strategy has been used for different antibiotics rather than natural products.

Conclusion

A flavonoid isolated from the leaves of V. gardneriana was structurally characterized using NMR. The NMR data indicated that the isolated compound is 5-hydroxy-3,7,4′-trimethoxyflavone with the chemical formula C₁₈H₁₆O₆. Our microbiological tests demonstrate that this flavone, which was obtained for the first time from the Vitex genus, presents antimicrobial activity and potential antibiotic modulatory activity against the MDR bacterial strains S. aureus 358 and E. coli 27 when associated with norfloxacin and gentamicin. Therefore, this natural compound can potentiate the antibiotic activity against and contribute to the control of resistant bacteria.

Footnotes

Disclosure Statement

No competing financial interests exist.

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