Gallesia integrifolia (Spreng.) Harms: In vitro and in vivo antibacterial activities and mode of action

Gallesia integrifolia (Spreng.) Harms: In vitro and in vivo antibacterial activities and mode of action

Author’s Accepted Manuscript Gallesia integrifolia (Spreng.) Harms: in vitro and in vivo antibacterial activities and mode of action Karuppusamy Aruna...

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Author’s Accepted Manuscript Gallesia integrifolia (Spreng.) Harms: in vitro and in vivo antibacterial activities and mode of action Karuppusamy Arunachalam, Sérgio Donizeti Ascêncio, Ilsamar Mendes Soares, Raimundo Wagner Souza Aguiar, Larissa Irene da Silva, Ruberlei Godinho de Oliveira, Sikiru Olaitan Balogun, Domingos Tabajara de Oliveira Martins

PII: DOI: Reference:

www.elsevier.com/locate/jep

S0378-8741(16)30110-6 http://dx.doi.org/10.1016/j.jep.2016.03.005 JEP10012

To appear in: Journal of Ethnopharmacology Received date: 25 August 2015 Revised date: 16 February 2016 Accepted date: 1 March 2016 Cite this article as: Karuppusamy Arunachalam, Sérgio Donizeti Ascêncio, Ilsamar Mendes Soares, Raimundo Wagner Souza Aguiar, Larissa Irene da Silva, Ruberlei Godinho de Oliveira, Sikiru Olaitan Balogun and Domingos Tabajara de Oliveira Martins, Gallesia integrifolia (Spreng.) Harms: in vitro and in vivo antibacterial activities and mode of action, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Gallesia integrifolia (Spreng.) Harms: in vitro and in vivo antibacterial activities and mode of action Karuppusamy Arunachalama, Sérgio Donizeti Ascênciob, Ilsamar Mendes Soaresb, Raimundo Wagner Souza Aguiarc, Larissa Irene da Silvaa, Ruberlei Godinho de Oliveiraa, Sikiru Olaitan Baloguna, Domingos Tabajara de Oliveira Martinsa* a

Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato

Grosso (UFMT), Av. Fernando Correa da Costa, no.2367, Coxipó, Cuiabá, Mato Grosso 78060-900, Brazil. b

Research Laboratory of Natural Products, Federal University of Tocantins, Faculty of

Medicine, Palmas, Tocantins 77020-210, Brazil c

Department of Biotechnology, Federal University of Tocantins, Gurupi, Tocantins 77404-

970, Brazil. *Correspondence to: Universidade Federal de Mato Grosso, Faculdade de Medicina, Av. Fernando Corrêa da Costa, n.2367 Câmpus Universitário,78060-900 Cuiabá, Mato Grosso, Brazil. Tel.: +55 6536156231. E-mail address: [email protected] (D.T.de O. Martins)

Abstract Ethnopharmacological relevance Gallesia integrifolia (Phytolaccaceae) is commonly known as “pau-d'alho” in Brazil or “garlic plant” due to the strong scent of garlic peculiar to all parts of the plant. The bark decoction is used for the treatment of microbial infections among other diseases by different ethnic groups in Brazil, Peruvian Amazonians, Bolivia and Mosetene Indians. This study aimed to advance in the antibacterial activity and characterize the mode of action of the hydroethanolic extract of the inner stem bark of G. integrifolia (HEGi) using in vivo and in vitro experimental models.

1

Materials and methods The qualitative and quantitative phytochemical analyzes of HEGi were carried out using colorimetric and HPLC technique. The cytotoxic potential of HEGi was evaluated against CHO-K1 cells by Alamar blue assay and its acute toxicity was assessed by the Hippocratic screening test using Swiss-Webster mice. The antibacterial activity was evaluated by microdilution method against ten strains of Gram-positive and Gram-negative bacteria. The mode of action of HEGi was investigated by outer membrane permeability, nucleotide leakage and potassium efflux assays. In vivo infection model was established by using Staphylococcus aureus infection model Wistar rats. Results Qualitative phytochemical analysis of HEGi revealed the presence of saponins, alkaloids, phenolic compounds and flavonoids. Phytochemical quantification of HEGi showed that higher total phenolic (80.10 ± 0.62 mg GAE/g) and flavonoid (16.10 ± 0.03 mg RE/g) contents. HPLC fingerprint analysis revealed the presence of gallic acid, rutin, and morin. In the Alamar blue assay no cytotoxic effect of HEGi in CHO-K1 cells was observed up to 200 µg/mL, and no signs or symptoms of acute toxicity were observed in mice of both sexes at higher doses of up to 2000 mg/kg, p.o. HEGi demonstrated bacteriostatic effect against selected Gram positive and Gram negative bacterial pathogens. Its mode of action is associated, at least partly, with changes in the permeability of bacterial membranes, evidenced by the increased entry of hydrophobic antibiotic in P. aeruginosa, intense K+ efflux and nucleotides leakage in S. flexneri, S. pyogenes and S. aureus. HEGi attenuated the experimental blood borne S. aureus infection in rats at all the tested doses levels (10, 50 and 250 mg/kg). Conclusion

2

HEGi is safe at the dose tested when used acutely, and it presented broad antibacterial effect, which support its traditional use in the treatment of bacterial infections. It contains well known important phytochemicals, recognized to be active against bacterial pathogens in vitro and might be collectively responsible for the antibacterial activity of HEGi. It is bacteriostatic in nature, with membrane perturbation being one of it mode of action. HEGi represent a potential phytotherapic antibacterial agent. Keywords: Gallesia integrifolia, Antimicrobial, Cytotoxicty, Flavonoids, Polyphenols, Traditional Medicine.

1. Introduction Antibiotic resistance is a complication that continues to challenge the healthcare sector in a large part of the world in both developing and developed countries. The spread of multidrug resistant bacteria in hospital and community settings remains a widely unresolved problem and a heavy burden to health services. In spite of advances in antibiotic therapy, infectious complications remain an important cause of mortality and morbidity among hospitalized patients (Rivera, 2015). Therefore, there is greater need for alternative source that can potentially effective in the treatment of bacterial infections. Natural products have been used since ancient times and in folklore for the treatment of many diseases and illnesses. Many studies have indicated that a broad range of plant extracts that may effectively act against bacterial resistance, demonstrating their enormous therapeutic potential (Abreu et al., 2012). Towards these ends, advances in identifying new sources of natural products with antibacterial activities and expanding antibiotic arsenal are providing chemical leads for new drugs (Rivera, 2015).

3

Gallesia

integrifolia

(Spreng.)

Harms

(syn.

Crateva gorarema Vell.,

Gallesia gorazema (Vell.) Moq., Gallesia integrifolia var. ovata (O.C.Schmidt) Nowicke, Gallesia ovata O.C.Schmidt, Gallesia scorododendrum Casar., Thouinia integrifolia Spreng. and Gallesia gorazema Spreng), Phytolaccaceae, is a native and endemic plant of Brazil. It is a tree with glossy, elliptical leaves that is widely distributed in Amazon, Caatinga, Cerrado and Atlantic rain forest of Brazil where, it is commonly known as "pau-d'alho" or "garlic plant" due to the strong scent of garlic peculiar to all parts of the plant (Akisue et al., 1986; Bussmann et al., 2011). Popular medicinal uses, including in the respiratory and skin infections, of this plant have been reported in many ethnopharmacological studies from different countries of Latin America (Bussmann and Glenn, 2010; Bussmann et al., 2011; Bottazzi et al., 2013; Bussmann et al., 2014). Preparations from different parts of the plant are indicated in some Latin American countries in the treatments of infections (Duke, 2009), such as remedies in the treatment of abscesses and infections (bacterial and fungal) by different ethnic groups in the Amazon basin (Akisue et al., 1986; Balbach, 1992; Duke, 2009). In additions, its use in traditional medicine, in the form of teas made from its leaves and bark are used for treating cough and skin afflictions (Muñoz et al., 2000), infections of the throat (Carneiro et al., 2014), diarrhoea, asthma, anti-rheumatism, antispasmodic, helmintic infections, anti-hemorrhagic, as a febrifuge agent (Bourdy et al., 2000; Barbosa et al., 1997) and in inflammatory conditions (Agra et al., 2008; Bieski et al., 2015). There are reports of scientific studies on its antinociceptive, anti-inflammatory, antiviral (De Jesus Silva Júnior et al., 2013), antioxidant (Duke, 2009), antibacterial (Bussmann et al., 2010) and antifungal (Freixa et al., 1998) activities. Previous phytochemical study of G. integrifollia

methanol leaf extract indicated the presence of

alkaloids, steroids, flavonoids and sesquiterpenoids (Ordoñez et al., 2006). De Jesus Silva

4

Júnior et al. (2013) reported 28-hydroxyoctacosyl ferulate from the root extract.

The

essential oils from the fresh leaves and bark were shown to be composed of sulphurcontaining compounds such as dimethyl sulfone, methyl methane thiosulfonate and 1methylsulfonyl-2,3-dithiabutane, among others, which explain the strong garlic scent characteristic of the entire plant (Barbosa et al., 1997; Barbosa et al., 1999). Although there is evidence of preliminary antibacterial activity with ethanolic extract of G. integrifolia bark (Bussmann et al., 2011), nothing is known about the mechanisms involved in its antibacterial action. Therefore, the objectives of this study was to advance on the antibacterial activity and investigate the mode of action of the hydroethanolic extract of G. integrifolia inner stem bark (HEGi), against clinically important bacteria, using in vitro and in vivo assays. Additionally, we evaluated the acute toxicity and analysed selected secondary metabolites of HEGi.

2. Materials and methods 2.1. Animals Female Wistar rats (150–200 g) and Swiss-Webster mice of both sexes (25–30 g) from the Central Animal House of Federal University of Mato Grosso (UFMT) were used. The animals were maintained in propylene cages at 25±1ºC in a 12 h light–dark cycle and were provided with free access to standard pellet chow (Labina, Goiás, Brazil) and water. They were allowed to acclimatize to the laboratory environment for 48 h. Groups of 5-6 animals were used for the experiments. Procedures concerning animal treatments and experiments in this study were reviewed and approved by the Animal Use Ethics Committee of the UFMT (Process no.23108.082449/2015-70). 2.2. Microorganisms

5

Antibacterial activity was evaluated using microorganisms from the American Type Culture Collection (ATCC, Rockville, MD, USA). The ATCC bacterial strains used were Staphylococcus aureus (25923), Staphylococcus epidermidis (12228), Streptococcus pyogenes (19615), Enterococcus faecalis (29212), Salmonella typhimurium (14028), Pseudomonas aeruginosa (27853), Shigella flexneri (12022), Klebsiella pneumoniae (13883), Escherichia coli (25922), Helicobacter pylori (43504) and Bacillus subtilis (6633). All strains were maintained on slopes of Skim Milk (Oxoid®), stored at -20 ºC, and subcultured two days before the assays to prevent morphological and metabolic transformations.

2.3. Cell line Epithelial cells of Chinese hamster ovary (CHO-K1) from the Rio de Janeiro Cell Bank, Brazil, were used to investigate the cytotoxic effects of HEGi. The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) + Ham's Nutrient Mixtures F10 (HAM F10) supplemented with10% (v/v) fetal bovine serum (FBS) in tissue culture flasks. The cells were sub cultured in fresh medium twice a week and incubated at 37ºC in a humidified atmosphere of 95% air and 5% CO2.

2.4. Plant collection and extract preparation G. integrifolia inner stem bark material was collected from the Valley of the Juruena, Aripuanã (S 09º58.774 coordinates 'H 060º07.431'), Municipality of Mato Grosso State, Brazil, in May 10, 2011, and its botanical identity was carried out at Herbário-UFMT, Mato Grosso State, Brazil. The scientific name and the distribution of the species was confirmed with the databases of The Plant List (http://www.theplantlist.org/) and Missouri Botanical Garden (MOBOT) (available at http://www.tropicos.org), while the geographical origin status was based on Rio de Janeiro Botanical Garden database of list of species of the Brazilian

6

flora (available at http: http://floradobrasil.jbrj.gov.br/). Voucher that was collected in the flowering state was identified by comparison with the specimens at the Herbário- UFMT with a voucher number BI-34108 was deposited at the same Herbarium. The G. integrifolia species is not on the endangered species list and access to the genetic heritage components of the sample for scientific research purposes was authorized by the National Council for Scientific and Technological Development (CNPq) via the process no. 010728/2013-9. The bark material (1 kg) was collected, cleaned and dried to constant weight at 40ºC in an oven for 3 days. The dried bark materials were shredded in an electric mill with a sieve having a mesh size of 40 (model TE-625 TECNAL, Piracicaba, SP, Brazil). The dried powdered plant material was macerated with 70% ethanol (1:3 w/v) at 25 ºC for 7 days. The macerate was filtered and concentrated under reduced pressure at 40 ºC in a rotary evaporator (Fisaton 801, São Paulo, SP, Brazil) to obtain HEGi. The remaining solvent was eliminated in an oven at 40ºC, the HEGi obtained was frozen and then lyophilized (JJ-CIENTIFICA - JJ02, São Carlos, SP, Brazil). The extract yield was 1.3 % (w/w of the dried powdered whole plant). HEGi was kept protected from light and stored at 4 ºC. The extract was solubilized in 0.04% dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO, USA) as at the time of use.

2.5. Phytochemical analysis 2.5.1. Qualitative and quantitative analysis of secondary metabolites in the HEGi extract Preliminary phytochemical analysis was performed according to the methods described by Matos (2009), which rely on chemical reactions of coloration, precipitation and foam formation. The secondary metabolites were analyzed through qualitative and quantitative methods using spectrophotometer and high performance liquid chromatography (HPLC).

7

The quantification of the total phenols was performed by the Folin–Ciocalteu method described by Amorim et al. (2008) and as modified by Balogun et al. (2014), using gallic acid as a standard. The total phenolic content was expressed as mg gallic acid equivalents (GAE) per gram of extract (GAE mg/g HEGi). All experiments were performed in triplicate. The quantification of total flavonoid content was performed according to the description of Peixoto Sobrinho et al. (2008) as modified by Balogun et al. (2014) using rutin as standard and was expressed as milligrams of rutin equivalents (RE) per gram of extract (mg RE/g HEGi).

2.5.2. HPLC analysis HPLC (Shimadzu, Tokyo, Japan) was analyzed following in the method described by Balogun et al. (2014). Briefly, Shimadzu® chromatograph (LC-10 Avp series, Tokyo, Japan) equipped with a (LC-10 AD, Tokyo, Japan) pump, (DGU-14A, Tokyo, Japan) degasser, UV– vis (SPD-0A, Tokyo, Japan) detector, column oven (CTO-10A, Tokyo, Japan), manual injector Rheodyne (loop 20 μL) and CLASS (LC-10A) integrator. The separation was carried out by a gradient system, using a reverse-phase Phenomenex Luna 5 mm C18 (2) (250 x 4.6 mm2) column with direct-connect C18 Phenomenex Security Guard Cartridges (4 x 3.0 mm2) filled with similar material as the main column. Phases were mobile phase A = 2% formic acid in Milli-Qwater (model no. B 330, Sao Paulo, Brazil) and mobile phase, B = 2% formic acid in methanol and C = methanol/ Milli-Qwater/ acetic acid (1:18:1 v/v). Program gradients were 0.00-0.01 min, 100% C; 0.01-3.5 min, 100% C; 3.5-4 min, 0% C; 4-4.10 min, 0% B; 4.10-5 min, 25% B; 5-10 min, 60% B; 10-12 min, 70% B; 12-15 min, 80% B; 15-17 min, 100% B; 17-17.5 min, 100% B; 17.5-18 min, 0% B; 18-18.10 min, 0% C; 18.10-18.5 min, 100% C; 18.5-35 min, 100% C. Flow rate: was 1 m/min, temperature was 40 oC, UV detection was done at 280 nm. The compounds were identified by comparing the retention

8

times of samples and authentic standards such as gallic acid, rutin, morin, from (Sigma®, Sao Paulo, Brazil). The content of the compounds was expressed as micrograms per milligram of extract (μg/mg) by correlating the area of the analyse with the calibration curve of standards built in concentrations of 1.95-125 μg/mL using the generated equations: y = 43132x + 65219 adjusted R2 = 0.9996 (gallic acid); y = 20146x + 129656 adjusted R2 = 0.9963 (rutin); y = 82139x + 146734 adjusted R2 = 0.9977 (morin). The extract solutions and standards were prepared with methanol and filtered through a Millipore® (0.22 mM pore size, Sao Paulo, Brazil) membrane.

2.6. Cytotoxicity assay with Alamar blue This assay was performed by adopting the Alamar blue assay (Nakayama et al., 1997), with slight modifications. The CHO-k1 cells (density 2x104/well) were seeded on 96well plates in 200 μL of DMEM and treated with/without HEGi (3.125–200 μg/mL, serial dilution). Doxorubicin (0.0058–58 μg/mL, serial dilution) was used as a positive control, while some wells had the same amount of medium as the negative control. The treatments were removed after 24 or 72 h incubation and 200 μL of 10% Alamar Blue (Resazurin) was added to each well and incubated again for 5 h. The conversion of resazurin to resorufin by the cells was measured at 540 nm (oxidised state) and at 620 nm (reduced state) using microplate spectrophotometer (Multiskan EX, Thermo Scientific, Tewksbury, Massachusetts, USA). The cell viability was expressed as inhibitory concentration at 50% inhibition (IC50 ± SD). The IC50 < 4 µg/ml and < 30 μg/mL were considered cytotoxic for pure compounds and extract, respectively (Suffness and Pezzuto, 1990). 2.7. Hippocratic screening test The effect of HEGi on the general behaviour of conscious animals was evaluated in both sexes of mice as described by Malone and Robichaud (1962) as modified by Balogun et 9

al. (2014). Briefly, the animals were separated in six groups (n=6/group) and received by oral gavage (p.o) of HEGi at doses of 1000, 2000 mg/kg body weight (b.w.). One control animal per group, received the vehicle (2% Tween - 80, 10 mL/kg). Animals were observed individually in open field at 5, 10, 15, 30, 60, 120 and 240 min and once a day, for a period of 14 days, observing any clinical signs or mortality, according to established criteria Malone and Robichaud (1962). 2.8. Antibacterial assays All experiments were performed in triplicate with three independent experiments on the same day.

2.8.1. Broth microdilution method

The antibacterial activity of HEGi was evaluated by determining the minimal inhibitory concentration (MIC) according to guidelines established by Clinical and Laboratory Standards Institute (CLSI, 2003). MIC values were determined using 96- well micro plates (Global Plast-655111T, China) according to CLSI guidelines as modified by Oliveira et al. (2015). We considered inhibitory concentration at which 99.9% growth inhibition is observed. Stock solution of extract in distilled water was diluted to give serial two fold dilutions which were added to each medium, resulting in concentrations ranging from 800 to 0.39 μg/mL of the extract. Inoculates of 100 μL (final concentration104 CFU/mL) were added to Mueller-Hinton broth. Clarithromycin (0.39–6.25 μg/mL) was used as positive controls. The culture medium containing 0.04% DMSO in sterile distilled water served as the negative control. Plates were incubated for 24 h at 35ºC. The results expressed in MIC (μg/mL). MIC values were classified based on the Kuete (2010) method, as modified by Oliveira et al. (2015). Extracts with MIC<100 ≤ μg/mL, 100 ≤ MIC ≤ 625 μg/mL, MIC >

10

625 μg/mL ≤ 800 μg/mL and MIC > 800 μg/mL were classified as having high activity, moderate activity, low activity and no activity, respectively.

2.8.2. Minimum bactericidal concentration (MBC) The bactericidal activity of HEGi was determined according to the method of Mbah et al. (2012) with modifications as described by Oliveira et al. (2015). Briefly, An aliquot (10 µL) of bacterial cells from the MIC test plate was subcultured on solid Müller-Hinton agar by making streaks on the surface of the agar and incubated at 37ºC for 24 h. The concentration of the wells inoculated with MIC which showed no bacterial growth was recorded as the minimal bactericidal concentration (MBC) and those that presented bacterial growth were considered bacteriostatic. 2.9. Mode of antibacterial action of HEGi 2.9.1. Outer membrane permeability Outer membrane permeability was determined according to the method described by Hao et al. (2009), as modified by Oliveira et al. (2015). Briefly, an overnight culture (5×107 CFU/mL) was inoculated into Mueller-Hinton broth containing ½ MIC of HEGi (200 μg/mL) in combination with erythromycin or rifampicin. The media were then poured into sterilized microplates of 96 wells and incubated at 37 ºC for 24 h. P. aeruginosa growth was measured in a Microplate Reader Multiskan® (Thermo Scientific, Waltham, MA, USA) at 450 nm.

2.9.2. Intracellular K+ efflux The potassium efflux effect was performed according to the method of Hao et al. (2009) as modified by Oliveira et al. (2015). Briefly, S. flexneri, S. pyogenes, and S. aureus cells were cultured overnight at 37ºC. The cells were washed and resuspended at a concentration of 1×107 cells/mL in phosphate buffered saline (PBS) of pH 7.2. Subsequently, 1 mL of the bacterial suspensions with HEGi 2 MIC was incubated at 37ºC for different 11

times. Bacterial strains incubated with PBS alone were used as control. After centrifugation, the amounts of released K+ in the supernatants were measured with Microprocessor Flame Photometer (TKS, Model No. 1382, São Paulo, SP, Brazil). The value obtained was used as an indicator of minor damage to the cytoplasmic membrane.

2.9.3. Nucleotide leakage The experiment was performed according to Tang et al. (2008) as modified by Oliveira et al. (2015). Briefly, S. flexneri, S. pyogenes, and S. aureus cells in logarithmic growth phase were washed and resuspended in10m MPBS (pH 7.4). The bacterial strains were incubated with HEGi at ½ MIC for different periods (2, 4, 6, 8, 10 and 12 h). Bacterial strains incubated with PBS alone served as the control. The mixture was filtered through 0.2 μm to remove the bacteria cells and absorbance of the filtrate was determined in a spectrophotometer at 260 nm. Nucleotide leakage was used as an indicator of major damage to the cytoplasmic membrane.

2.10. In vivo antibacterial activity In vivo antibacterial activity of HEGi was performed according to the method of Singh et al. (2014) with major modifications, using female albino Wistar rats of 40−45 days old, 180−200 g b.w distributed in 6 groups (n=5/each). Briefly, S. aureus was grown in the Müeller-Hinton broth (MH) after reaching the log phase of growth; the suspension was centrifuged at 1000 x g for 15 min. The supernatant was discarded, and the bacterial pellet was resuspended in phosphate buffer saline (PBS) to achieve a concentration of 4 × 106 CFU/mL. All rats were inoculated intravenously (iv.) with 0.2 mL of S. aureus. After 1 h of inoculation, rats were treated with the vehicle (sterile PBS), HEGi (10, 50 and 250 mg/kg) (p.o). A sham group was injected with sterile PBS in place of the bacterial suspension.

12

Positive control group rats were treated with 60 mg/kg of amoxicillin (p.o). To perform the quantitative evaluation of the bacteria in the blood of all groups, the blood samples were taken by retro orbital puncture of rats after 1 h of treatment and the blood samples plated on blood agar plates. The plates were incubated at 37 °C in ambient air overnight. The colonies of S. aureus were counted by colony counter (Phoenix-CP-600 Araraquara, SP, Brazil). All experiments were done aseptically, while samples and contaminated materials were sterilized before being disposed.

2.11. Data analysis Results of quantitative phytochemical analyses by spectrophotometer, the in vitro and in-vivo antimicrobial activity results were expressed in terms of mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used, followed by the Student–Newman– Keuls test when statistical difference was detected among the groups. Values of p < 0.05 were considered significant. GraphPad Prism© version 5.01 for Windows (GraphPad Software, USA) was used for statistical analysis.

3. Results 3.1. Qualitative and quantitative analysis of selected phytochemical compounds in the extract The preliminary phytochemical analysis of HEGi revealed the presence of secondary metabolites such as saponins, alkaloids, phenols and flavonoids. The total phenolic and flavonoid contents of HEGi were 80.10 ± 0.62 mg GAE/g; 16.10 ± 0.03 mg RE/g, respectively.

3.2. HPLC fingerprinting

13

Chromatographic profile confirmed the presence of phenolic and flavanoid compounds detected in the preliminary screening. In a HPLC working time of 35 min a phenolic compounds matrix was detected in the intervals between 2 and 25 min. The deducted compounds concentrations were as follows: gallic acid 12.061 μg/mg, rutin 20.979 μg/mg and morin 8.877 μg/mg extract, and thus accounting for 1.2%, 2.0%, and 0.88% respectively, of HEGi (Fig. 1). The HPLC analysis confirmed the presence of gallic acid (time: 3.659 min), rutin (time: 18.450 min), morin (time: 18.984 min) respectively.

A

B

14

Fig. 1. HPLC fingerprint of the hydroethanolic extract of Gallesia integrifolia inner stem bark (HEGi) detected at 280 nm. (A) HPLC chromatogram of authentic standards of phenolic compounds mixture. (B) HPLC chromatogram of HEGi: peak 1: gallic acid; peak 2: rutin; peak 3: morin.

3.3. Acute toxicity assays 3.3.1. Hippocratic screening In the present study, administration of HEGi, orally, at the dose of 1000 mg/kg caused no significant behavioural changes in the mice. At 2000 mg/kg transient diarrhoea was observed in a male mouse, while passiveness and escape reaction, that resolved in 8 h was noticed in one animal each of the females. There was no mortality at doses up to 2000 mg/kg in both sexes.

3.3.2. Alamar blue assay Incubation of CHO-k1 cells with HEGi did not produce any cytotoxic effect in cells at 24 and 72 h (IC50 > 200 µg/mL to both times), while doxorubicin, the standard positive control drug for this assay, caused intense cytotoxicity in these cells only at 72 h, with IC50 = 0.3 ± 0.04 µg/mL

3.4. In vitro antibacterial activity 3.4.1. Broth microdilution method The MIC of HEGi against the eleven bacterial strains is presented in Table 1. HEGi showed high antibacterial activity against S. flexneri (25 µg/mL) and moderate to S. pyogenes (100 µg/mL), E. coli (200 µg/mL), S. typhimurium (200 µg/mL), P. aeruginosa (400 µg/mL), S. aureus (400 µg/mL), and K. pneumonia (400 µg/mL). Clarithromycin as expected, caused 15

high antibacterial activity, with MIC values ranging from 0.39 to 6.25 µg/mL, confirm of bacterial strains.

Table 1. Antibacterial activity of hydroethanolic extract of Gallesia integrifolia inner stem bark (HEGi) on selected bacterial strains. MIC (µg/mL) HEGi Clarithromycin

Bacterium Gram-negative Escherichia coli

200

0.39

Helicobacter pylori

>800

0.39

Klebsiella pneumonia

400

0.39

Pseudomonas aeruginosa

400

0.39

Salmonella typhimurium

200

6.25

Shigella flexneri

25

0.78

Gram-positive Bacillus subitilis

>800

0.39

Enterococcus faecalis

>800

0.39

Staphyloccus epidermidis

>800

0.39

Staphylococcus aureus

400

0.39

Streptococcus pyogenes

100

0.39

Minimum inhibitory concentration (MIC)

3.4.2. Determination of MBC MBC assay demonstrated that HEGi is bacteriostatic against the bacterial strains employed, as it failed to inhibit the selected bacterial strains growth at the MIC concentration when the bacterial cells were subcultured from the MIC plate.

3.5. Mode of antibacterial action of HEGi 3.5.1. Outer membrane permeability

16

Fig. 2 (A and B) show the effect of HEGi (½ MIC = 200 µg/mL) in combination with erythromycin (1 µg/mL) or rifampicin (10 µg/mL) on the growth of P. aeruginosa. As designed, co-incubation of HEGi with the bacterium had no measurable inhibitory effect on the bacterial growth, whereas erythromycin (0.00048 – 1.0 µg/mL) occasioned growth inhibition of P. aeruginosa, in an exponential manner, with the maximum effect at 0.5 µg/mL. On combining HEGi with erythromycin, outer membrane permeability was enhanced as evidenced by the increased sensitivity of the bacterium to erythromycin at concentrations even below the MIC of erythromycin (0.0004 µg/mL), with an increase of 89% of antibacterial activity, as compared to erythromycin alone. Moreover, rifampicin also gives rise to growth inhibition of P. aeruginosa, in an ascending order, with maximum effect at 0.62 µg/mL. On the other hand, HEGi had no appreciable outer membrane permeability effect when it was combined with rifampicin (Fig 2B)

0.55

(A) Bacterial growth parameter (OD/620 nm)

0.50 0.45 HEGi 200

0.40

Erythromycin 4.8×10-4- 1.0 HEGi 200+Erythromycin 4.8×10-4-1.0

0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.0

0.1

0.2

0.3

0.4

0.5

0.6

Drug concentration µg/mL

17

0.7

0.8

0.9

1.0

(B) Bacterial growth parameter (OD/620 nm)

EHGi 200

0.6

Rifampicin 4.8×10-4 - 2.5 EHGi 200 +Rifampicin 4.8×10-4 - 2.5

0.4

0.2

0.0

0.5

1.0

1.5

2.0

2.5

Drug concentration µg/mL

Fig. 2. Outer membrane permeabilization effect of induced by hydroethanolic extract of Gallesia integrifolia inner stem bark (HEGi) in association with antibiotics rifampicin and erythromycin. ½ MIC of HEGi + antibiotics (A and B) against Pseudomonas aeruginosa (200 µg/mL). Each value represents mean ± SD. The data represented mean values of 3 independent experiments.

3.5.2. K+ efflux induced by HEGi As shown in Fig. 3 (A - C), the baseline potassium efflux in the control was 2 ppm and this value remained constant throughout the experimental period. HEGi co-incubation (2 times MIC) with the medium containing S. flexneri, S. pyogenes and S. aureus caused intense increases in K+ efflux in a time dependent manner. Maximal effects observed at the 4th h for S. flexneri and S. aureus increased by 75% and 83.5% respectively when compared to the respective control. In the case of S. pyogenes the maximal activity occurred at the 3rd h, with 82.9% compared with control. The peak activity of the standard at 4th h with increases of 83.6%, 90.2% and 88.6% (S. flexneri, S. pyogenes and S. aureus with amoxicillin) respectively.

18

Relative amount of K+ (ppm)

(A) 15 Control HEGi Amoxicillin

10

5

0 0

1

2

3

4

Time (h)

Relative amount of K+ (ppm)

(B) 25

Control HEGi Amoxicillin

20 15 10 5 0 0

1

2 Time (h)

19

3

4

Relative amount of K+ (ppm)

(C) 25 Control HEGi

20

Amoxicillin

15 10 5 0 0

1

2

3

4

Time (h)

Fig. 3. Effect of 2 MIC of hydro ethanolic extract of Gallesia integrifolia inner stem bark (HEGi) on the amount of K+ released from Shigella flexneri (A), Streptococcus pyogenes (B). Staphylococcus aureus (C). Each value represents mean ± SD. The data represented mean values of three independent experiments.

3.5.3. Nucleotide leakage The Fig. 4 (A - C) show total nucleotide leakage from bacterial cells as function of incubation time with HEGi. As demonstrated, co-incubation of HEGi occasioned considerable nucleotide leakages in all the bacterial strains tested, right from the 2nd h of incubation with maximum effects observed at the 12th h (86.7%, 83.8% and 97%) for S. flexneri, S. pyogenes and S. aureus respectively. When compared to the respective control cultures. This effect was superior to the standard drugs polimixin with S. flexneri (84%) and amoxicillin with S. pyogenes (61.5%) and with S. aureus (93%) respectively.

20

(A) 0.20

HEGi

Absorbance 260 nm

Polymyxin Control

0.15

0.10

0.05

0.00 0

2

4

6

8

10

12

Time (h)

(B) 0.5

HEGi

Absorbance 260 nm

Amoxicillin 0.4

Control

0.3 0.2 0.1 0.0

0

2

4

6 Time (h)

21

8

10

12

(C) 0.4

HEGi

Absorbance 260 nm

Amoxicillin 0.3

Control

0.2

0.1

0.0

0

2

4

6

8

10

12

Time (h)

Fig. 4. Effect of ½ MIC of HEGi (hydroethanolic extract Gallesia integrifolia inner stem bark) on the amount of total nucleotide released from (A) Shigella flexneri (MIC = 12.5 µg/mL), (B) Streptococcus pyogenes (MIC = 50 µg/mL), (C) Staphylococcus aureus (MIC = 200 µg/mL). Each value represents mean ± SD; three independent experiments.

3.6. In vivo antibacterial activity The bacterial load in the blood sample of the vehicle group of animals was 4.36 x 102 CFU. Treatment with HEGi reduced the bacterial load in the blood of the animals at all doses (10, 50 and 250 mg/kg p.o), in a non-dose dependent manner, with maximum effect at 50 mg/kg (93.6 %, p < 0.01), when compared to the vehicle control. Amoxicillin, the standard drug used in this experiment, reduced the bacterial load by 88.1 % (p < 0.01) (Fig. 5).

22

Viable bacterial count (log 10 CFU/mL)

80

60

40

**

20

0

Sham

Vei

10

50

250

Amox 60

mg/kg

HEGi Staphylococcus aureus 4  106 CFU Fig 5. Effects of the treatment with hydroethanolic extract of G. integrifolia (HEGi) (10, 50, and 250 mg/kg), vehicle (Veh, 2% Tween 80) and amoxicillin (Amox, 60 mg/kg) on S. aureus infected rats. Each column represents Mean ± SD. of 5 animals. One way ANOVA followed by Student–Newman–Keuls test. **p< 0.01 compared with vehicle.

4. Discussion In toxicological pharmacology, the use of in vitro and in vivo models that can be used to predict adverse effects in humans exposed to chemicals, represent an important issue, because safety has been the major concern in the process of selection and development of candidate drugs (Balogun et al., 2014). Based on these premises, we commenced the toxicological studies using Alamar blue assay cytotoxicity method with CHO-K1 as the model organism. Our results showed that HEGi can be considered as non-cytotoxic (Suffness and Pezzuto, 1990). Although in vitro tests are useful as a starting point in the evaluation of potential therapeutic agent toxicity screening, the test by itself alone is not a waterproof claim of the test substance toxic effect (Oliveira et al, 2014). Hence, we proceeded to assess the acute in vivo toxicity using both male and female mice as model organisms. HEGi at up to

23

2000 mg/kg, did not cause any toxic signs, nor were any symptoms or mortality recorded in the treated mice and no toxic effect was observed throughout the 14 days study period. Therefore in accordance with the OECD guideline 423 (OECD, 2001), HEGi may be considered potentially safe. We proceeded to carry out the antibacterial activities, based on the safety profile of the extract. In this study, HEGi showed a potent and broad spectrum of activity, with greater susceptibility displayed against S. flexneri and S. pyogenes. Interestingly, the only study found in the literature evaluated the antibacterial activity of G. integrifolia, albeit in a very preliminary manner using disc diffusion method (Bussmann et al., 2011). The ethanolic extract of G. integrifolia bark was active only against S. aureus among the four bacterial strains tested (E. coli, S. aureus, Salmonella enterica and P. aeuroginosa). In the present, activity was detected against seven of the eleven clinically important Gram-positive and Gram-negative bacterial species studied, being more active against Gram-negative bacteria than the ethanol extract reported by Bussmann et al. (2011). We proceeded further to know if HEGi is bactericidal or bacteriostatic in action based on the MIC values obtained and consequently we determined the MBC. The results obtained demonstrated that HEGi is bacteriostatic in action based on the bacterial strains and respective experimental conditions. However, in vitro classification of an agent as being bactericidal or bacteriostatic is influenced by several factors, such as the type of medium, the incubation time, the bacterial load among others, and that this classification may not hold for all bacteria (Oliveira et al., 2015). For example, Pankey and Sabath (2004) stated that the distinction between bactericidal and bacteriostatic agents is more arbitrary when agents are categorized in clinical situations. This is because the supposed superiority of bactericidal agents over bacteriostatic agents is of little relevance when treating the vast majority of infections with Gram-positive bacteria, particularly in patients with

24

uncomplicated infections and non-compromised immune systems. In fact bacteriostatic agents have been effectively used for treatment of clinical conditions (e.g. endocarditis, meningitis, and osteomyelitis) that are often considered to require bactericidal activity. Many of antibiotics in use are bacteriostatic and act by disrupting or even preventing the proliferation and bacterial growth, leaving the task to the immune system to eliminate the infection. Therefore, in the present study, we elucidated probable mode of action of HEGi, using different experimental models of outer membrane and cytoplasmic membrane perturbations (potassium efflux and nucleotide leakage). The known antibacterial mechanisms of medicinal plants against microorganisms are inhibition of cell wall synthesis (Cowan, 1999; Marcucci et al., 2001), accumulation in bacterial membranes causing energy depletion (Conner, 1993), or interference in the permeability of cell membrane which as a consequence a permeability may increase, loss of cellular constituents, membrane disruption and changes in the structure and function of key cellular constituents, that may result in mutation, cell damage, and death (Bajpai et al., 2013; Akinpelu et al., 2015). Membrane potential (MP) was chosen as important aspect to illustrate the mechanism of antibacterial action as it plays an important role in bacterial physiology (Zhang et al., 2016) and bacterial outer membrane (OM) permeability assessments are fundamental to studies of the performances of antimicrobial peptides (Ohmizo et al., 2004). The Gramnegative bacteria are more resistant to antibiotics because of having a selective barrier, represented by the outer membrane of the bacterial cell wall (Vaara, 1992). Thus, hydrophobic antibiotics such as erythromycin and rifampicin in low concentrations are not able to infiltrate the intact outer membrane of Gram-negatives (Yi et al., 2010). These antibiotics do not effectively infuse the intact OM of Gram-negative bacteria but cross the

25

OM of the certain OM imperfect mutants as well as the OM damaged by chelators or polycations (Hao et al., 2009; Oliveira et al., 2015). The results of this study demonstrate that HEGi sensitizes P. aeruginosa to erythromycin, suggesting OM perturbation (Vaara and Vaara, 1983). The same could not be proven in the case of rifampicin, suggesting that the antibacterial effect of HEGi is at least, in part due to its outer membrane permeability in Gram-negative susceptible pathogen. Cellular homeostasis is central to life and in bacterial pathogens it is attained by controlling the permeability of the cytoplasmic membrane to ions and solutes. Transport of potassium ion (K+) is a critical determinant of growth and survival through its role in regulating cytoplasmic pH and cell turgor. As a result, K+ pools are tightly regulated through controlled uptake and efflux (Roosild et al., 2010). The cytoplasmic membrane may become damaged and functionally disabled when bacterial suspensions are exposed to antibacterial agents. Various vital intracellular materials including small ions such as K+ and phophates tend to leak out, and then followed by large molecules such as DNA and RNA (Xing et al., 2009). Our findings demonstrated that HEGi possesses potent bacterial membrane damaging potential, and that its antibacterial activity is due at least in part to this effect. In a similar view the positive controls amoxicillin and polymycin also demonstrated membrane damaging effects. On the basis of the results of the in vitro bacterial studies, we proceeded to screening HEGi for in vivo antibacterial activity using Staphylococcus aureus blood infection model in rats. In vivo testing without doubt is one of the recognized, if not the most important, essential links between in vitro sensitivity testing and clinical studies. In fact, several guidelines for the clinical evaluation of efficacy and toxicity of anti-infective drugs explicitly require experimental evaluation of new compounds (or novel combinations or therapeutic

26

modalities) in animals as prerequisites for clinical trials. This is because animal models of infection are the best means available as at the present, in evaluating an antibiotic efficacy and tolerability, before its administration to humans as well as finding new approaches to the treatment of infections (Zak and O’Reilly, 1991; Zak and Sande, 1999), although studies of antimicrobial activity, for the most part, have been limited basically to in vitro tests. Staphylococcus aureus is one of the major human pathogens that causes both community- and hospital-acquired infections (Sandberg et al, 2009). The infections caused by S. aureus ranges from the relatively from superficial skin infections to endocarditis, bone and joint infections and septic shock (Abreu et al., 2012; Prabhakara et al., 2013). The choice of the doses used in this experiment was based on the pilot study and the results from acute oral toxicity study. HEGi, at all the doses tested, effectively inhibits the growth of the S. aureus in blood samples of infected animals (Fig. 5). The effect of HEGi compared well with the standard drug amoxycilin employed in this assay, thus establishing the potential in vivo antibacterial activity of HEGi and further supporting its ethnomedicinal use in the treatments of bacterial infections. The exact mechanisms by which HEGi caused the inhibition of the bacterial growth in vivo are not yet clear. The phytochemical fingerprint of HEGi revealed the presence of appreciable amounts of phenolic compounds like gallic acid, rutin and morin (Fig. 1). Literature findings related that gallic acid antibacterial mechanism of action include its bacterial membrane disruption, hyper acidification of the plasma membrane via proton donation and acidification of the intracellular cytosol (Borges et al., 2013). Cushnie and Lamb (2005) reported rutin has antimicrobial activity against human pathogenic microorganisms with some mechanisms of action such as inhibition of nucleic acid synthesis, cytoplasmic membrane function and energy metabolisms. Morin was shown to inhibit DNA synthesis of the susceptible bacteria (Arima et al., 2002). The antibacterial effects of HEGi may therefore be due in part to the

27

presence of gallic acid, rutin and morin. However, other components present in the HEGi may also contribute to its antibacterial activities and it is most probable that the combined effects of the metabolites are responsible for its antibacterial activity established in this study. To the best of our knowledge this is the first study detailing the antibacterial activity and probable mode of action of HEGi.

5. Conclusions Based on the present results, hydroethanolic extract of G. integrifolia inner stem bark has broad bacteriostatic effect majorly on Gram-negative bacteria, thus confirming its popular use against bacterial infectious diseases. HEGi is safe if used for short period and its mode of action appears to be associated with changes provoked due to increased permeability in the cell membrane. HEGi may represent a potential candidate in the development of phytotherapic antibacterial agent or even may be useful in combination with existing standard antibiotics. Acknowledgments We acknowledge CAPES and CNPq for granting postdoctoral fellowship to Karuppusamy Arunachalam and research fellowship to Sikiru Olaitan Balogun, respectively. The present study was partly funded by Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT - Process no. 205978/2011), CNPq/Bionorte (Process no. 551737/20107), CPP, and INAU. We are also express sincere thanks to Ricardo Dalla Villa PhD., Larissa Sizue da Silva Taura and Danielle Cordeiro Barbosa, of Department of Chemistry, UFMT for technical assistance in the conduction of K+ efflux assay.

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