In Vitro Antibacterial,Phototoxic,and Synergistic Activity of Ethanol Extracts from Costus cf. arabicus L.

Strains

Experiments to determine minimum inhibitory concentration (MIC) levels and synergistic antibiotic activity were performed with clinical isolates of E. coli (EC27), resistant to low levels of neomycin and gentamicin, as well as to tobramycin, amikacin and kanamycin, and with isolates of S. aureus 358 (SA358) resistant to several aminoglycosides. ¹⁸ Phototoxicity assays were performed using the standard wild-type strains S. aureus ATCC 25923 and E. coli ATCC 10536. ¹⁴ The strains were maintained on heart infusion agar slants (Difco, Detroit, MI, USA). Prior to the assays, the cells were grown overnight at 37°C in brain–heart infusion broth (Difco).

Plant material

Leaves and stems of C. cf. arabicus L. were collected in Crato County, Ceará State, Brazil. This plant material represents a possible new biological species, and its identification is in progress in the Botanical Garden Institute of Rio de Janeiro, Rio de Janeiro, Brazil, under the supervision of Dr. João Marcelo A. Braga. A voucher specimen of this plant was deposited in the Dárdano de Andrade Lima Herbarium, under number 1267.

Preparation of ethanol extract of leaves and stems of C. cf. arabicus

Leaves (1,450 g) and stems (965 g) from C. cf. arabicus were dried at room temperature and powdered, respectively. The material was extracted by maceration using 1 L of ethanol as the solvent at room temperature. The homogenate was allowed to stand for 72 hours at room temperature. The extracts were filtered and concentrated under vacuum in a rotary evaporator (model Q-344B, Quimis, São Paulo, Brazil) using a warm water bath (model Q-214M2, Quimis). The plant material yielded 10.1 g of ethanol extract of leaves (EELC) and 10.4 g of ethanol extract of stems (EESC). For the tests, the dry extract material was dissolved in dimethyl sulfoxide (10 mg/mL).

Phytochemical detection

Phytochemical tests to detect the presence of heterosides, saponins, tannins, flavonoids, steroids, triterpenes, cumarins, quinones, organic acids, and alkaloids were performed according to the method described by Matos. ¹⁹ The tests were based on the visual observation of a change in color or formation of precipitate after the addition of specific reagents, and the results for the extracts studied are shown in Table 1.

Drugs

Gentamicin, kanamycin, amikacin, neomycin, and 8-methoxypsoralen were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All drug solutions were prepared according to the manufacturer's instructions. Disks with norfloxacin were obtained from Laborclin (São Paulo).

Antibacterial test (MIC) and modulation of antibiotic activity

The MIC was determined in a microdilution assay using an inoculum of 100 μL of each strain, suspended in brain–heart infusion broth up to a final concentration of 10⁵ colony-forming units/mL in 96-well microtiter plates, using twofold serial dilutions. Each well received 100 μL of each extract solution. The final concentrations of the extracts varied from 512 to 8 μg/mL. MICs were recorded as the lowest concentrations required that inhibited the growth. The MIC for the antibiotics was determined in brain–heart infusion broth by the microdilution assay using suspensions of 10⁵ colony-forming units/mL and a drug concentration range of 1,024 to 1 μg/mL (twofold serial dilutions). ²⁰ The MIC was defined as the lowest concentration at which no growth was observed. For the evaluation of the extracts as modulators of resistance to the antibiotics, MIC of the antibiotics was determined in the presence or absence of EELC and EESC at subinhibitory concentrations (64 μg/mL), and the plates were incubated for 24 hours at 37°C. Each antibacterial assay for MIC determination was carried out in triplicate.

Phototoxic assays

Assays were performed according to the technique described by Lopez et al. ¹⁰ As positive controls, a disk of norfloxacin served as a standard antibiotic with photoactivated properties. 8-Methoxypsoralen (10 mg/mL) in water was used as a positive control requiring light for activation. Twenty microliters of each extract was added to blank disks. These disks were placed on the surface of the medium inoculated with bacteria by the spread plate method. To monitor for light-activated antimicrobial activities, two replicate experiments were carried out. One replicate plate was exposed to UV light (5 W/m², 320–400 nm from four Sylvania F20T12-BLB lamps [obtained from Havells Co., São Paulo], maximum emission at 350 nm) for 2 hours, whereas the other was kept in the dark. The plates were incubated at 37°C overnight. The inhibition zones were determined using a pachymeter and recorded.

Phytochemical screening

Through phytochemical detection of the extracts, it was possible to determine the presence of various classes of secondary metabolites that show a wide variety of biological activities such as antimicrobial, antioxidant, antitumor, and anti-snake venom. ^{22 –24} The antimicrobial property of tannic acid can also be utsed in food processing to increase shelf life. ²⁵ Flavonoids are synthesized by plants in response to microbial infection and are effective against a broad range of microorganisms. ²⁶

MIC evaluation and modulation of antibiotic activity

The mechanisms by which extracts can inhibit the growth of microorganisms are varied and can be due in part to the hydrophobic nature of some components. These components can interact with the lipid bilayer of the cell membrane and affect the respiratory chain and the production of energy, ²⁷ or even make the cell more permeable to antibiotics, leading to the interruption of vital cellular activity. ²⁸ Various components of extracts can enhance the permeability of the cell membrane, increasing the penetration of antibiotics. ²⁹ The interference with bacterial enzyme systems can also be a potential mechanism of action. ³⁰ These mechanisms of action can be induced by the combination of antibiotic with extract at a subinhibitory concentration applied directly to the culture medium. ^7,31

This strategy is called “herbal shotgun” or “synergistic multieffect targeting” and refers to the utilization of plants and drugs in an approach using mono- or multi-extract combinations, which can affect not only a single target but various targets, where the different therapeutic components collaborate in a synergistic–agonistic manner. This approach is possible not only with combinations of extracts; combinations between natural products or extracts and synthetic products or antibiotics are also possible. ^{32 –36}

Phototoxic activity

Many plant substances when exposed to UV or visible light exhibit phototoxicity, so that they are referred to as phototoxins or photosensitizers. ¹⁴ A large variety of plants and fungi of various families possess phototoxic substances, possibly serving as natural defense agents against insects and nematodes or against predation or herbivorous activity. ^37,38 This activity is basically due to two mechanisms: an indirect effect through the production of free radicals, or a direct effect such as through furocumarins, which interact with DNA. ³⁹ Compounds such as polyenes, thiophenes, thiarubrines, quinones, alkaloids, furochromones, and porphyrins are widely distributed in various botanical families, and various studies have demonstrated that they possess antibacterial activity after UV light activation. ^37,38 This work is the first report of a phototoxic effect for natural products from the genus Costus.