Biochemical Composition,Antimicrobial Activities,and Anti–Quorum-Sensing Activities of Ethanol and Ethyl Acetate Extracts from Hypericum connatum Lam. (Guttiferae)

Plant material

The plant material was collected in San Luis (Argentina) in October 2010 and identified by one author (M.R.F.). A voucher specimen of the plant was maintained in the Herbarium of the National University of San Luis.

Extraction

The aerial parts of the plant (5 kg) were air-dried and then extracted with methanol for 2 days at room temperature, yielding 840 g of residue. This extract was divided into the fraction soluble in ethyl acetate and the fraction soluble in ethanol, generating 235 and 350 g of residue, respectively. A 500 mg sample of the ethanolic residue was resuspended in 5 mL of H₂O and 1 mL of ethanol; 500 mg of the ethyl acetate residue was resuspended in 5 mL of H₂O, 2 mL of ethyl acetate, and 1 mL of ethanol. The two mixtures were concentrated and resuspended in dimethyl sulfoxide (DMSO) to produce the same final concentration of 100 mg/mL of the extract and then subjected to biochemical and biological analyses.

Colorimetric analysis of total phenolic compounds

The total phenolics were determined by following the methods in Singleton and Rossi ¹¹ using the Folin–Ciocalteu reagent. The absorbance was determined at room temperature at λ=760 nm using a Cary UV/Vis spectrophotometer (Varian Cary 50 MPR). The quantification was based on a standard curve generated with gallic acid; the results were expressed as μg gallic acid equivalents (GAE)/g of extract.

Polyphenol composition

An ACQUITY Ultra Performance LCTM system (Waters, Milford, MA, USA) linked to a PDA 2996 photodiode array detector (Waters) was used for ultrahigh-performance liquid chromatography (UPLC) analyses. Empower software was used to control the instruments and for data acquisition and processing. The analysis was performed at 30°C using a reverse-phase column (BEH C₁₈, 1.7 μm, 2.1 mm×100 mm; Waters). The mobile phase consisted of Solvent A (7.5 mM acetic acid) and Solvent B (acetonitrile) at a flow rate of 250 μL/min. ¹² Gradient elution was employed, starting at 5% B for 0.8 min, 5–20% B for 5.2 min, isocratic 20% B for 0.5 min, 20–30% B for 1 min, isocratic 30% B for 0.2 min, 30–50% B for 2.3 min, 50–100% B for 1 min, isocratic 100% B for 1 min, and decreasing from 100% to 5% B for 0.5 min. At the end of this sequence, the column was equilibrated under the initial conditions for 2.5 min. The pressure ranged from 6000 to 8000 psi (41.37–55.16 MPa) during the chromatographic run. The effluent was introduced to an LC detector (scanning range 210–400 nm; resolution 1.2 nm). The injection volume was 5 μL, and the peaks were monitored at 280 nm. The phenolic compounds were identified by qualitatively and quantitatively comparing the peak areas on the chromatograms of samples with those of diluted standard solutions. ^{13 –15}

Free radical-scavenging capacity

The free radical-scavenging activity was measured using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). ¹⁶ The analysis was performed in microplates by adding 7.5 μL of extract to 303 μL of a DPPH–methanol solution (153 mM). The absorbance was then spectrophotometrically measured (Varian) at λ=517 nm. The absorbance of DPPH without the antioxidant (control sample) was used as the baseline measurement. The scavenging activity was expressed as the half-maximal effective concentration (EC₅₀) of the sample, in terms of μg/mL, necessary to inhibit the DPPH radical activity by 50% after a 60-min incubation. These experiments were performed in triplicate, and the results are expressed as the mean±standard deviation (SD).

Antimicrobial assays

To screen the activity of the H. connatum extracts against microorganisms, a filter paper disc method was used. ¹⁷ The bacteria used in this study included Gram-positive Bacillus cereus (strains DSM 4313 and DSM 4384) and S. aureus DSM 25923 and Gram-negative Escherichia coli DSM 8579 and Pseudomonas aeruginosa ATCC 50071 strains. All strains were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). The strains were incubated in a nutrient broth (Oxoid) at 37°C for 18 h. The optical densities of all cultures were adjusted to match a 0.5 McFarland standard of 1×10⁸ colony-forming units (CFU)/mL. Sterile filter paper discs (5 mm) were impregnated with 10, 25, 50, and 100 μg of the extracts and placed in Petri dishes. A disc treated with DMSO alone served as the negative control. The plates were left for 30 min at room temperature under sterile conditions and then incubated at 37°C for 24 h, and the inhibition halo around the disc was measured. Tetracycline (7 μg/disc; Sigma) was used as a reference. The experiments were performed in triplicate and averaged.

AQS assay

The Chromobacterium violaceum QS-sensing system was used for this assay. QS in this wild-type strain of bacteria is known ¹⁸ to control the production of the purple pigment violacein in response to such autoinducer molecules as C6-AHLs and C4-AHLs. The disc-diffusion method was employed to detect the AQS activity of the extracts from H. connatum. In this test, bacterial growth inhibition would result in a clear halo around the disc, whereas a positive result of QS inhibition would result in a turbid halo harboring the pigmentless bacterial cells of C. violaceum DSM 30191 used as the tester strain. Cultures of C. violaceum were prepared by incubating the bacteria in a nutrient broth (Oxoid) for 16–18 h at 30°C. The cultures were adjusted to the 0.5 McFarland standard (1×10⁸ CFU/mL). The extracts from H. connatum, previously dissolved in sterile DMSO, were added at different concentrations (from 1 to 100 μg) to C. violaceum-inoculated nutrient agar plates (0.1 mL/plate). The inoculated plates were kept under a laminar flow hood for 10 min and then incubated at 30°C for 24 h.

Statistical analysis

All of the experiments were performed in triplicate. The results are expressed as the means±SD. The data were ordered in homogeneous sets, and the Student's t-test of independence was applied. ¹⁹

Polyphenol composition and antioxidant activity

In our experiments, the ethanolic and ethyl acetate extracts showed a similar amount of polyphenols (134.11 and 119.14 mg of GAE/g of extract, respectively). Evaluation of the polyphenol profile by UPLC, starting from the same amount of polyphenols in terms of milligrams of GAE, revealed the presence in both extracts of some common metabolites, such as gallic, chlorogenic, and ferulic acids and luteolin and hyperoside (Table 1). The most abundant compounds present in the two extracts were hyperoside (82.28 and 76.17 mg GAE/g of extract, respectively) and chlorogenic acid (18.92 and 14.52 mg GAE/g of extract, respectively). The ethanolic fraction contained more gallic and chlorogenic acids than the ethyl acetate fraction and contained rutin and apigenin, which were not detected in the other fraction (Table 1). In contrast, the use of ethyl acetate allowed the extraction of such metabolites as caffeic acid, (−)-epicatechin, p-coumaric acid, and naringenin. The presence of the flavonoid rutin, found in the ethanolic extract, agrees with the literature that reported this flavonoid in Hypericum brasiliense and in the flowers of H. perforatum. ⁷ However, this compound was not detected in a methanol extract of H. connatum from South Brazil. ⁷ Rutin can be easily absorbed and is found in different tissues, the plasma, and the gut, where it affects the growth of some pathogens as demonstrated by the in vitro study of Parkar et al., ²⁰ and also acts as a potent metal chelator and a free radical scavenger. In this manner, rutin significantly influences the function of various mammalian cellular systems. Like most polyphenols, rutin has a strong antioxidant activity, plays a role in inhibiting some cancers, and is also capable of reducing the extent of mitochondrial damage and reducing oxidative stress in leukocytes in patients with rheumatoid arthritis. ²¹ Flavonols, such as (–)-epicatechin, constitute an important part of the human diet and possess different biological activities, including antioxidant and anti-inflammatory properties. ²² Cinnamic acid derivatives, such as chlorogenic, caffeic, and p-coumaric acids, have been shown to be active antioxidants ²³ that are capable of inhibiting LDL oxidation in vitro. ²⁴ The two extracts in the present study demonstrated strong antioxidant activities. Despite the similar amount of polyphenols, the ethanolic fraction was twice as strong as the ethyl acetate fraction in inhibiting DPPH free radical activity (Fig. 1), with an EC₅₀ value of 3.2 with respect to 6.6 μg of extract. It is likely that such activity in both extracts could be influenced by the high amount of hyperoside and reinforced, in the ethanolic fraction, mainly by the presence of rutin, as previously described with regard to its antioxidant properties. ²⁵

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FIG. 1.

Percentage of inhibition of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) exhibited by the ethanol and ethyl acetate extracts of Hypericum connatum. The vertical bars represent the standard deviation.

Antimicrobial activity

Using the inhibition halo technique, we evaluated the antimicrobial activity of the ethanolic and ethyl acetate extracts of H. connatum against a range of Gram-positive and Gram-negative microorganisms responsible for food-borne diseases and/or for the spoilage of contaminated products. In particular, the test was performed against the following: two strains of B. cereus (DSM 4313 and DSM 4384), an aerobic spore-former commonly found in raw and processed foods that causes several cases of foodborne illnesses; an enterotoxigenic strain E. coli, capable of causing serious cases of food poisoning; S. aureus, a pyogenic bacterium that plays a significant role in invasive skin disease; and the opportunistic pathogen P. aeruginosa, which may cause pulmonary, skin, urinary, eye, and ear infections. We found that the ethyl acetate extract was more active compared to the ethanolic extract. All strains tested were sensitive to 25 μg of the ethyl acetate extract (Table 2): the ethanolic extract showed an antimicrobial effect against almost all strains only at the highest dose (100 μg) used in the test, with inhibition halos ranging from 8.7 mm (vs. B. cereus DSM 4313) to 9.3 mm (vs. S. aureus). Both extracts were effective against the toxigenic E. coli DSM 8579, with halos up to 10 mm, but their behavior was different as regard as P. aeruginosa that resulted sensitive to the action of the ethyl acetate extract, but was resistant to all doses of the ethanolic extract used in the experimentation. These results corroborated the work of Dall'Agnol et al., ⁵ who found antimicrobial activity of the methanol extract from H. connatum against S. aureus and E. coli, and confirmed previous studies reporting the different susceptibility of B. cereus to plant phenolic extracts. ¹⁷ With regard to the components responsible for the antimicrobial activity shown, different compounds of distinct natures in these extracts can be considered as acting as antimicrobial agents. This is not surprising, because a variety of active principles present in the majority of the previously studied Hypericum species have a demonstrated antimicrobial activity. ²⁶ However, the activity of the ethyl acetate extract against the tested pathogens could be ascribed to the presence of caffeic acid, which augments the action of (–)-epicatechin and p-coumaric acid. Caffeic acid, present at a concentration of 11.07 mg GAE/g in this fraction, is one of the most common polyphenols in plants and is well recognized as an antimicrobial agent. ²⁷ Caffeic acid acts as an enzyme inhibitor, possibly through a reaction with sulfhydryl groups or through more nonspecific interactions with proteins. ²⁸ p-Coumaric acid has dual mechanisms of bactericidal activity: it disrupts the bacterial cell membranes and binds to the bacterial genomic DNA to inhibit cellular functions, ultimately leading to cell death. ²⁹ (–)-Epicatechin also acts mainly to disrupt the cell membrane of several bacteria. ³⁰

AQS activity

Many studies have emphasized that various eukaryotic specimens, including plants, fungi, and even animals, produce compounds, such as polyphenols, capable of interfering with the bacterial QS system. ^3,15

In this investigation, we assayed the potential of the ethanolic and ethyl acetate extracts of H. connatum to exhibit the QS inhibitory properties. To this end, we used the C. violaceum DSM 30191 indicator strain in the disk-diffusion assay. ¹⁸ The obtained data revealed that 25 μg of the ethanol extract inhibited QS-regulated violacein pigment production in the strain without interfering with its growth; at a lower concentration, the presence of the yellow culture was undetectable, and a higher concentration inhibited the growth of the strain, with inhibition halos of 7.3 mm and 9.3 mm using 50 and 100 μg of the ethanolic extract, respectively. In contrast, neither antiquorum-sensing (AQS) nor antimicrobial activity was observed for any amount (from 10 to 100 μg) of the ethyl acetate extract (Table 3). Although the ethanolic extract exhibited a less antimicrobial activity than the ethyl acetate extract, it exhibits an AQS activity. This behavior is most likely attributable to the presence of rutin, which might synergistically reinforce the AQS activity of apigenin, whereas the presence of other flavonoids in the ethyl acetate extract, such as naringenin, is not likely to strengthen such a property. The available literature reports that flavonoids, such as naringenin, apigenin, quercetin, and rutin, have a demonstrated AQS activity. ⁹