Antibacterial Activity of Tinctures from Tree leaves belonging to the Bignoniaceae family and their Synergistic Effect with Antibiotics

Context: Some species of Bignoniaceae are widely used in medicinal practice by the natives of South America. Aims: Tinctures and infusions from twelve tree species of this family were evaluated for in vitro antibacterial activity against pathogenic bacteria. The effect of interactions between the four most active extracts and conventional antibiotics was also evaluated. Methods and Material: Bioautography and disc diffusion methods were used to select the most active extracts, then agar macrodilution and broth microdilution method were used to determine the minimal inhibitory and minimal bactericidal concentration (MIC and MBC). Time-kill assay and checkerboard method were employed to determine the type of antimicrobial effect and synergism, respectively. Results: It could be determined that tinctures from Catalpa bignonioides, Handroanthus pulcherrimus, Tabebuia nodosa and Tecoma stans were able to inhibit bacterial growth. The MIC and MBC observed were between 125-1000 μg GAE/ ml and 500-1000 μg GAE/ml, respectively. The tested extracts were more effective against Gram-positive microorganisms. Time-kill experiments indicated bacteriostatic activity. Phytochemical screening showed terpenoids, phenols and flavonoids. Alkaloids were detected only in Tecoma stans. Among these combinations, the best was Tabebuia nodosa extract plus gentamicin. In most cases, MIC values were reduced 16-32 times for antibiotics, and even 8-16 times for extracts. Conclusion: These results revealed that some of the selected combinations could efficiently inhibit the growth of tested strains at lower concentrations than those required for the lonely use of the antimicrobial. These extracts would improve the efficacy of antibiotics against resistant bacteria, hence they could be used for anti-infective therapy.


INTRODUCTION
Medicinal plants represent a rich source of antimicrobial agents.These compounds can be terpenoids, glycosteroids, flavonoids and polyphenols.Most of these molecules have weak antibiotic activity; however, plants fight infections successfully.Hence, it becomes apparent that plants adopt a synergistic strategy to combat infections. 1 Bignoniaceae Juss. is a family of trees, shrubs or climbers, which is made up of about 100 genera and 800 species.Some species of this family are widely used in medicinal practice by the natives of South America. 2,3The well-known medicinally important members of this family are Tecoma, Catalpa, Handroanthus, Tabebuia and Jacaranda.][6][7] Torres et al., 2013 8 have previously analyzed the antibacterial properties of 20 climbers of this family.The aim of this work has been to evaluate the antibacterial effect of the infusions and tinctures of leaves from twelve species of trees belonging to the Bignoniaceae family against pathogenic bacteria.Besides, a study Torres Carola Analía 1,2 , Nuñez María Beatriz 1 , Isla María Inés 3,4 , Castro Marcela Paola 1,2 , Gonzalez Ana María 1,2,5 and Zampini Iris Catiana 3,4 of synergistic effects resulting from the combination of tinctures with commercial antibiotics was conducted.

Plant material
Plant material was collected from the North of Argentine.The voucher specimens were deposited and conserved in the Herbarium of the Instituto de Botánica del Nordeste (IBONE-CONICET), Corrientes, Argentina.

Extraction and preliminary phytochemical investigation
Leaves were dried at room temperature.Then, dry leaves were crushed and milled until they reached a size between 1.70 mm and 710 µm using ASTM (Association for Testing Materials of United States) sieves.Afterwards, tinctures and infusions were prepared with the obtained powder.Tincture: 20 g of powder was macerated in 80° ethanol (100 ml) for 7 days in a dark place at room temperature.Infusion: 5 g of plant powder was put in contact with boiling water (100 ml) for 20 min.Finally, all extracts were filtered through Whatman N°1 filter paper, centrifuged at 3000 rpm for 5 min and were stored at -20°C in the dark.

Phytochemical analysis
The presence of secondary metabolites was assessed according to qualitative standard methods. 9,10The total phenolic content was quantified according to Singleton et al., 1999. 11 Flavonoid content was determined according to Woisky and Salatino, 1998. 12

Antimicrobial assays
The qualitative determination of antibacterial activity was assessed by the agar disc diffusion method 13 and the bioauthography assay 14 using a spot containing 30 µg phenolic compounds of each extract.MIC values of the most active extracts were determined by serial agar macrodilution and broth microdilution method. 15The microdilution method was also used to determine MBC values.The extracts were dried and re-suspended in dimethyl sulfoxide (DMSO, Sigma Aldrich, USA).Dilutions of crude extracts in DMSO (range concentration between 62.5 and 1000 µg GAE/ml) were prepared.A growth control of each tested strain and a DMSO control were included.MIC was defined as the lowest concentration of extract at which bacterial growth was not observed after incubation.MBC was defined as the lowest extract concentration at which 99.9% of the bacteria have been killed.All experiments were carried out in duplicate.Antibacterial effect was evaluated by the time-kill assay.In this test, a standardized suspension of bacteria (5×10 5 colony-forming unit-CFU-/ ml) was added into Müeller-Hinton broth containing the extracts to give a final concentration between 500-2000 µg/ml.These mixtures were then incubated at 37°C for 12 h shaking at 200 rpm.Aliquots of 0.01ml of the diluted samples were withdrawn at time intervals (4 h) for the determination of the number of CFU/ml. 16Crude extracts were considered to be bactericidal at the lowest concentration which reduced the original inoculum by ≥3 log 10 CFU/ml (99.9% reduction in bacterial population) in 4 h.

Estimation of synergy between plant extracts and antibiotics
Synergy between extracts and selected antibiotics (ampicillin, gentamicin and oxacillin) was studied by the checkerboard assay method. 17ombinations of oxacillin and the extracts were tested only for Grampositive bacteria.The concentrations used in the combinations for each antibiotic ranged from 0.05 to 204.8 µg/ml and for each extract between 15.62 and 500 µg GAE/ml.MIC values were determined for each antibiotic and for each of these combinations to establish any interaction effect.The FIC (Fractional in-

RESULTS
Alkaloids were only detected in leaves of Tecoma stans while saponins were not detected, but all the plants showed the presence of terpenoids, phenols and flavonoids.The results of the total phenolics and flavonoids content are presented in Table 1.The qualitative screening of antimicrobial activity showed that 30 µg of phenolic compounds from infusions were not active whereas the same amount of ethanolic extracts of many plants were able to inhibit bacterial growth.The tinctures of C. bignonioides, H. pulcherrimus, Tabebuia nodosa and Tecoma stans were selected for the determination of MIC, MBC values and synergistic effect.The four extracts were active in varying degrees against Gram-positive strains.Catalpa and Tecoma inhibited the growth of Proteus and Morganella morganii, Handroanthus was active against P. mirabilis and one clinical isolate of E. cloacae, while Tabebuia had no effect against Gram-negative bacteria (Table 2).In the time-kill assay, the extracts significantly inhibited bacterial growth when compared with the growth control; however, the reduction in growth was ≤3 log 10 CFU/ml for all isolates, indicating a bacteriostatic effect (Figure 1).FIC indices for extracts and antibiotic combinations-calculated according to the checkerboard test-are shown in Tables 3 and 4. The combination of the extracts with oxacillin diminished the MIC values of antibiotic against methicillin resistant clinical isolates of Staphylococcus (F7 and F22).The MIC value of the oxacillin against strain F7 was 25.6 µg/ml and 204.8 µg/ml for strain F22 (Table 3).However, in the combinations with extracts, the antibiotic concentrations required to obtain the same effect were much lower (sometimes 16-32 times lower).Morganella morganii, K. pneumoniae and P. mirabilis were the most sensitive to combinations within Gram negative bacteria isolates (Table 4).The best combination  was T. nodosa extract and gentamicin, synergism obtained in 81.25% of bacteria tested.Only the combination of Tecoma stans extract and ampicillin had a synergistic effect for both clinical isolates of E. cloacae.No antagonism was observed for any of the combinations evaluated.

DISCUSSION
The tinctures tested had higher antibacterial activity against Gram positive bacteria than against Gram negative ones.The latter bacteria are usually less susceptible to the action of plant extracts. 19,20 H. pulcherrimus and T. stans were able to inhibit the growth of some Gram negative strains.Some studies have demonstrated that plants of the Handroanthus genus have antibacterial activity against Helicobacter pylori 21 and S. aureus. 22ost of these results referred to studies carried out on barks, while this work considered leaves as the objects of study.Though, different extracts of leaves of T. stans have been reported for antimicrobial effects on some human pathogenic bacteria, 7,19,23,24 which partially support our results.The results of synergism tests suggest the potential use of some of these plants to improve the effect of antibiotics.MIC values were reduced 16-32 times for antibiotics, in most cases, and even eight or sixteen times for extracts, depending on the tested bacterium.No antagonism was observed for any of the combinations evaluated and when indifference or additivity were observed, a substantial decrease in the MIC of the antibiotic in the combination was detected, while the MIC extract remained unchanged or decreased only 2 times.These data are also relevant.Although no synergism was observed for these combinations, they decreased the dose of the antibiotic with a consequent reduction of side effects.
The interaction of plant extracts with antibiotics is one of the novel ways to overcome the resistance mechanisms of bacteria.Several studies on the interaction between plant extracts and antibiotics indicated a synergistic interaction with antibiotics. 25,26t is remarkable that, to our knowledge, there are not data in worldwide literature about studies of synergism between these species and commercial antibiotics.Our results revealed that some of the combinations selected can effectively inhibit the growth of tested strains at lower concentrations than those required for the individual agents.

CONCLUSION
The results suggest that the extracts of the studied Bignoniaceae species possess some compounds with antimicrobial properties; besides, they could enhance the efficacy of antibiotics against resistant bacteria.Consequently, these extracts could be used as antimicrobial agents for infectious diseases therapy in humans.Chemical studies are required to determine the compounds responsible for antibacterial effect of these species in leaves; in this sense, investigations to identify the structures of active principles are being conducted in our laboratory.

Table 3 :
MIC of conventional antibiotics and FIC Index values of combinations between plant extracts and antibiotics againstGram positive bacteria MIC antibiotic (µg/ml) Concentrations of extract and antibiotic used in the combination expressed in µg/ml and FIC index of different combinations ml of extracts.FIC Index values are shown in brackets.a Synergistic effect; b Additive effect; c Indifferent effect; ND: not detected.Sa: Staphylococcus aureus ATCC 29213; Ef: Enterococcus faecalis ATCC 29212.A: Ampicillin; Cb: Catalpa bignonioides; G: Gentamicin; Hp: Handroanthus pulcherrimus; O: Oxacillin; Tn: Tabebuia nodosa; Ts: Tecoma stans.

Table 1 : Phenolic and flavonoid content in tinctures and infusions of 12 selected tree species of Bignoniaceae family Samples Tinctures Infusions Phenolic compounds (mg GAE/g DE) Flavonoids (mg QE/g DE) Phenolic compounds (mg GAE/g DE) Flavonoids (mg QE/g DE)
c Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015

Table 2 : Antibacterial activity, MIC and MBC (μg GAE/ml) values of tinctures against pathogenic bacteria
However, it is noteworthy that among the species studied in this work C. bignonioides, Pharmacognosy Journal, Vol 7, Issue 6, Nov-Dec, 2015

Table 4 : MIC of conventional antibiotics and FIC Index values of combinations between plant extracts and antibiotics against Gram negative bacteria MIC antibiotic (µg/ml) Concentrations of extract and antibiotic used in the combination expressed in µg/ml and FIC index of different combinations
# µg GAE/ml of extracts.FIC Index values are shown in brackets.a) Synergistic effect; b) Additive effect; c) Indifferent effect; ND: Not detected.Ec: Escherichia coli ATCC 35218; Pa: Pseudomonas aeruginosa ATCC 27853.A: Ampicillin; Cb: Catalpa bignonioides; G: Gentamicin; Hp: Handroanthus pulcherrimus; O: Oxacillin; Tn: Tabebuia nodosa; Ts: Tecoma stans.