Cytotoxicity and antibacterial activity of scutellarein and carajurone-enriched fraction obtained from the hydroethanolic extract of the leaves of Fridericia chica (Bonpl.) L.G. Lohmann

Abstract The hydroethanolic extract obtained from the dry leaves of Fridericia chica (HEFc) underwent several fractionations by different chromatographic techniques. The ethyl acetate and dichloromethane fraction were subjected to phytochemical analysis, resulting in the identification and isolation of scutellarein (1) and in a fraction rich in carajurone (2). They were tested for cytotoxicity in CHO-K1 and the antibacterial activity and mode of action by in vitro assays. The HEFc and scutellarein (1) presented no cytotoxicity. The results showed good antibacterial effect of HEFc against Streptococcus pyogenes and Staphylococcus aureus and moderate activity for Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella typhimurium. The fraction containing the compound carajurone (2) showed good activity against Staphylococcus aureus and Staphylococcus epidermidis and moderate activity against Streptococcus pyogenes. Scutellarein (1) showed no activity against the bacteria tested. HEFc antibacterial mode of action appeared to be associated with changes in the permeability of bacterial membranes and nucleotide leakage. Graphical Abstract


Introduction
Fridericia chica (Bonpl.) L.G. Lohmann ((synonyms: Arrabidaea chica (Humb. & Bonpl.) B. Verlt.)), Bignoniaceae family, popularly known as crajiru or pariri, is a perennial treecreeper distributed from southern Mexico to Guyana and Brazil Central (Barbosa et al. 2008). In Brazil, it was found in the Cerrado, Atlantic Forest and Amazon biomes (Medeiros et al. 2011). It is a species of economic and medicinal importance that features in its composition of dry leaves red pigments that are used by riverine communities as a craft dye in matrinxã leather dyeing, a fish of the Amazon basin and in the dying of palm fibres Astrocaryum chambira Burret (tucum) for making domestic utensils (Lorenzi and Matos 2008). Indigenous ethnic groups use the decoction of the leaves for cleaning chronic wounds and treating fungal infections and herpes, to protect the skin against sunlight and to repel insects (Barbosa et al. 2008), and also for body painting (Lorenzi and Matos 2008), a practice that refers to the identity of different ethnic groups and indigenous culture mark.
The Brazilian Ministry of Health increased the number of medicinal plants of interest that can be used in its Unified Health System (Sistema Unico de Sa ude-SUS) (Ministry of Health 2009). Among the seventy one medicinal plants of this list, F. chica also included, and thus its standardization and knowledge of the biomarkers responsible for its biological activity is part of a regulatory requirement.

Results and discussion
The HEFc was fractionated and purified by standard chromatographic methods and structure of the compounds were established by NMR spectral data 1D ( 1 H, 13 C and DEPT) and 2 D (HSQC and HMBC) and ESI-MS/MS, and comparison of their spectral data with literature. The UV spectra and fragmentation in the spectrum (Supplementary material, Figure S1) show that the hydroethanolic extract consisting mainly of flavonoids when compared with published data for this compound (Siraichi et al. 2013). It is observed in the 12.3 min retention time characterization of apigenin compound of molecular formula C 15 H 10 O 7 m/z 271, present in the HEFc. The compounds scutellarein and carajurone are present in DCM and EtOAc fractions, and retention time of 10.7 minutes to ion fragments m/z 285. The DCM fraction is observed the presence of anthocyanin derivative compounds of the retention times of 14.8 and 16.8 minutes fragments at m/z 585 and m/z 443, molecular formula C 34 H 31 O 10 and C 33 H 29 O 11 , respectively. For an isolated compound of the DCM phase, scutellarein flavonoid discussed fragments were observed for the ion m/z 285. The NMR spectrum showed a chemical shift in mono-and bi-dimensional spectra, similar to other authors (Siraichi et al. 2013), characterizing it as scutellarein confirmed in Figure S2 (Supplementary material).
Scutellarein (1) is a flavone with significant bioactivity; however, it is very difficult to obtain in nature, compared to its derivative scutellarin (Qian et al. 2012). Carajurone (2) is one of the anthocyanidins responsible for the red-brown colour of the leaves extracts and the marker substance, quite specific and useful for the characterization of fingerprints for this species of the plant (Schiozer et al. 2012). The cytotoxicity result demonstrates the viability of CHO-K1 cells treated for 72 h with different concentrations of the hydroethanolic extract of dry leaves of F. chica (HEFc) and scutellarein (1). The HEFc and scutellarein (1) were demonstrated to be non-cytotoxic with IC 50 > 200 mg/mL and IC 50 > 40 mg/mL, respectively. Doxorubicin, the positive control in this assay, was highly cytotoxic with IC 50 ¼ 0.30 ± 0.04 mg/mL (Suffness and Pezzuto 1990) (Supplementary material, Figure S3).
Extracts obtained from plants are routinely classified as antimicrobial agents on the basis of susceptibility tests to determine the minimum inhibitory concentration (MIC), and it is considered an important useful scientific method in the validation of ethnopharmacology knowledge of medicinal plants (Formagio et al. 2013). The results of the antimicrobial test are represented in Table S1 (Supplementary material). HEFc was active against strains of Gram-positive and Gram-negative bacterias and showed good activity against Streptococcus pyogenes (MIC ¼ 12.5 mg/mL), Staphylococcus aureus and Staphylococcus epidermidis, both with MIC ¼ 50 mg/mL, and MIC ¼ 100 mg/mL for both Pseudomonas aeruginosa and Salmonella typhimurium.
The AcOEt fraction showed a broad spectrum of activity with MIC of 50 mg/mL against Pseudomonas aeruginosa, Enterobacter cloacae, Enterobacter aerogenes, Staphylococcus epidermidis and Streptococcus pyogenes and MIC of 100 mg/mL against Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Shigella flexneri, Proteus mirabilis and Burkholderia cepacia. The DCM fraction was active against Staphylococcus aureus, Streptococcus pyogenes and Staphylococcus epidermidis with MIC of 100 mg/mL and for all other strains (Table S1, Supplementary material). AcOEt and DCM fractions have two chemical and biological characteristics including medium polarity and minimum cytotoxicity on test strains. These solvents would be extracted a wide range of organic compounds. They can help to extract many biological compounds (polar and non-polar) responsible for antibacterial activities (Mughal et al. 2006).
The fraction containing the anthocyanidin carajurone (2) was also active against S. pyogenes (MIC ¼ 100 mg/mL), S. aureus and S. epidermidis (both with a MIC ¼ 50 mg/ mL). To determine the minimal bactericide concentration (MBC), bacteria that were sensitive to HEFc were incubated with concentrations greater than or equal to its MIC. The HEFc and fraction containing the scutellarein (1) and carajurone (2) showed bacteriostatic action. Tannins, anthocyanins, and flavonoids detected in the HEFc may be responsible for the antinbacterial effect and perhaps exert their action either by binding to the microbial cell membrane (causing lysis), or by inactivation of the enzymes required for ion transport and metabolism (Devia et al. 2002). Due to HEFc having demonstrated a potent activity against S. pyogenes (MIC ¼ 12.5 mg/mL) and S. aureus (MIC ¼ 50.0 mg/mL) this sample and these microorganisms were selected as targets to study the antibacterial mechanism of action, using nucleotide extravasation assay. Models of bacterial targets, such as outer membrane permeability, potassium efflux and nucleotides leakage have been reported in the literature in elucidating the antimicrobial activities of compounds obtained from plants (Silva et al. 2014).
In the group containing only S. pyogenes (Supplementary material, Figure S4a) and S. aureus (Supplementary material, Figure S4b) suspended in PBS, the leakage of nucleotides was negligible throughout the time tested. The addition of HEFc to the medium containing S. pyogenes (MIC ¼ 12.5 mg/mL) caused an increase in the leakage of the nucleotide of 50.0% when compared with the control group (p < 0.001), while the amoxicillin, the standard drug, caused leakage in the order of 80.0% at the time of 4 h. However, within 12 h there was an increase in the leakage by HEFc, in the order of 88.9% when compared with the control group (p < 0.001), and it was higher than that of the amoxicillin, which continued to be 80.0%. Of note is the effect of the association of HEFc with amoxicillin, resulted in in the nucleotide leakage of 90% when compared to the control group (p < 0.001) and was 50% higher than the amoxicillin used alone. The results of the HEFc alone and in combination with amoxicillin were similar (Supplementary material, Figure S4b). However, when compared with the conventional antibiotic, the results were quite significant. That is, the addition of HEFc to the medium containing only S. aureus (MIC ¼ 50.0 mg/mL) caused an increase in leakage nucleotide by 95.0% when compared to the control (p < 0.001) in the first hour. On the other hand, amoxicillin caused extravasation in the order of 50.0%, only at the time of 4 h, increasing to 90.0% in 8 h and remained so until the final 12 h of incubation when compared with the control. HEFc and its association with amoxicillin peaked at 4 h (96.7%) (p < 0.001) and remained so until the end of the 12 h of incubation.
The nucleotide leakage test revealed that HEFc increased the permeability of the cytoplasmatic membrane and caused efflux of nucleotides from the intracellular compartment. The use of various natural compounds in association or not with synthetic antibiotics can be effective in the treatment of infections caused by pathogens of various species (Chou, 2010). Research involving flavonoids suggests that there are three mechanisms of action possible for antibacterial activity: damage to the cytoplasmatic membrane caused by perforation or extravasation, inhibition of the synthesis of nucleic acids, caused by inhibition of topoisomerase and inhibition of energy metabolism, caused by inhibition of NADH-cytochrome c reductase. There is a strong evidence that flavonoids may interfere with virulence factors of various bacteria, including enzymes, toxins and signal the receptors (Cushnie and Lamb 2011). It might be suggested then that the action is due to the combination of metabolites in HEFc. These results justify the traditional use of F. chica in the treatment of gastrointestinal disorders, cramps, diarrhea with blood and are relevant, especially when one considers that the bacteria in question have become resistant to many antibiotics, by altering their genetic makeup, and are responsible for infections causing diarrhoea, urinary tract infection and sepsis (Cant on et al. 2012).

Conclusion
In summary, the exposition of cells CHO-K1 to HEFc and fraction containing the scutellarein (1) demonstrated to be non-cytotoxic. The antibacterial activity of HEFc and fraction containing the anthocyanidin carajurone (2) have been confirmed and expanded against strains of Gram-positive and Gram-negative bacteria in these studies. However, scutellarein (1) was inactive against the tested microorganisms. Based on the results of the bacterial membrane permeability and nucleiotide lekage studies, HEFc antibacterial mode of action appears to be associated with changes in the permeability of the bacterial cell wall and the bacterial cytoplasmic membrane. These results justify the traditional use of F. chica for infectious diseases by several communities in the Amazon region and its inclusion in the list of medicinal plants of interest for use by the National Health System in Brazil.

Disclosure statement
The authors declare no conflict of interest.