Antimicrobial and antioxidant potential from Piper marginatum roots

Chemicals

Folin-Ciocalteu’s phenol reagent, potassium persulfate and 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma-Aldrich (Waltham, MA, USA), and 2,2- azinobis 3-ethylbenzothiozoline-6-sulfonic acid (ABTS) was supplied by Fluka Chemie (Buchs, Switzerland).

Plant

The roots of P. marginatum were collected in an Atlantic forest fragment located on the Campus of the Federal Rural University of Pernambuco (UFRPE), Recife, Pernambuco, Brazil (8°00′53.4″S 34°57′05.5″W). The botanical material was identified by Prof. Dr. Margarethe Ferreira de Sales from the Department of Biology at UFRPE. A specimen of the collected botanical material was deposited at the Vasconcelos Sobrinho Herbarium of the Department of Biology at UFRPE under number 45870.

Extraction and compounds isolation

P. marginatum roots (500 g) were dried in a circulating air oven for a period of 48 h at 50 °C and the dried material was milled to a fine powder in a Macsalab mill (400 g). The dried root was extracted by maceration with n-butanol (3 × 400 mL) at room temperature for 48 h and concentrated in a vacuum to yield crude extract (8 g). Part of the extract (4.0 g) was subjected to fractionation on a Sephadex LH-20 (Amersham Biosciences, Amersham, UK) using methanol as eluent, yielding 84 fractions. Fractions 23 to 31 were pooled (350 mg) and further fractionated with silica gel (Merck 230-400 mesh) using hexane with increasing amounts of ethyl acetate (EtOAc) as the eluent, yielding 40 fractions with isolation of compounds 1 (20.4 mg) and 2 (18.6 mg).

HPLC analyses

HPLC analyses of extracts and the pure compound were performed using a Shimadzu LC10 instrument (Tokyo, Japan) with an SPD-M20A diode array detector using a C₁₈ column (250 mm, 4.6 mm, 5 µM, (Supelco, Bellefonte, PA, USA) eluted in a gradient mode starting with CH₃OH:H₂O (3:7) for 10 min, raising to 100% of CH₃OH (HPLC grade, Merck-Brazil, Millipore, Burlington, MA, USA) in 40 min and flow rate of 1.0 mL/min. The injection volume was 20 µL of solution 1 mg/mL in methanol.

Structure

¹H and ¹³C NMR spectra of 1 and 2 compounds were acquired in CDCl₃ on Bruker DPX-300 (Bruker, BioSpin GmbH Silberstreifen 476287 Rheinstetten Germany; 300 MHz and 75 MHz, respectively) spectrometer with pulsed field gradient and signals referenced to the residual solvent signals (CDCl₃, at δ H 7.26 and δ C 77.0 ppm, 99% purity Aldrich, St. Louis, MO, USA). GC-MS analyses (60−260 °C at 3 °C/min. heating rate) were carried out in a Varian 431-GC coupled to a Varian 220-MS instrument using (Palo Alto, CA, USA) fused-silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm) coated with DB-5. MS spectra were obtained using electron impact at 70 eV with a scan interval of 0.5 s and fragments from 40 to 550 Da. The injection volume was 1.0 µL of solution 1 mg/mL in methanol. IR spectra were obtained on a Perkin Elmer Nicolet 1750 using KBr disk (Palo Alto, CA, USA). The analyses UV-Vis were carried out Agilent 8453 spectrophotometer (Santa Clara, CA, USA), in the interval from 190 to 600 nm, using 10-mm quartz cuvettes and solution (0.5 mg/mL) in methanol.

Total phenolic content

The total phenolic content of the samples was determined using the Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the method previously reported with slight modifications of gallic acid as a standard phenolic compound (Slinkard & Singleton, 1977). Appropriate amounts of each sample (500 µL; 50 µg/mL) were diluted in a volumetric flask with distilled water (3 mL). The Folin-Ciocalteu reagent (100 µL) was added and the contents of the flask were thoroughly mixed. After 3 min, Na₂CO₃ (15%, 300 µL) was added and the mixture was completed with distilled water (5 mL) and allowed to stand for 2 h in an ultrasonic bath. The absorbance was measured at 760 nm in a spectrophotometer. The total amount of phenolic compounds was determined in micrograms of gallic acid equivalents, using the equation obtained from the standard gallic acid graph.

ABTS^•+ radical scavenging assay

The method previously reported with slight modifications was adopted for ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate, 98% purity) assay (Re et al., 1999). ABTS^•+ was generated by reacting ABTS solution (7 mM) with potassium persulphate (140 mM, final concentration) for 16 h in the dark at room temperature. Then, the ABTS^•+ solution was diluted with ethanol to obtain absorbance of 0.70 (± 0.02) at 734 nm and further equilibrated at 30 °C. The samples were diluted in ethanol at a concentration of 1 mg/mL, and the ABTS^•+ solution was added to both samples to obtain concentrations of 1.0 to 100.0 µg/mL. The absorbance of the reaction mixture was measured at 734 nm after reaction at room temperature for 10 min. Trolox (97% purity) was used as positive control. The capability of scavenging the ABTS^•+ radical was calculated using the following equation: ABTS^•+ scavenging effect (%) = [(A0 − A1 / A0) ×100].

Where A0 is the initial concentration of ABTS^•+ and A1 is the absorbance of the remaining concentration of ABTS^•+ in the presence of the sample.

In vitro antimicrobial activity

The antimicrobial potential of extract and compounds was evaluated against the gram-positive bacteria Staphylococcus aureus, S. aureus MRS, Bacillus subtilis and the gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa as well as the fungi Candida albicans, Mycobacterium smegmatis and Malassezia furfur. The bacteria and fungi came from the collection of microorganisms from the Antibiotics Department of the Federal University of Pernambuco. The suspension of microorganisms was standardized by the turbidity equivalent to a 0.5 tube on the McFarland scale in distilled water, corresponding to a concentration of approximately 108 CFU/ml for bacteria and 107 CFU/ml for fungi (Ramos et al., 2022; Bezerra & Ramos, 2021).

Determination of the minimum inhibitory concentration (MIC)

MIC was performed using the microdilution technique in 96-well multiplates. The culture media used were Sabourand Agar (for fungus) and Muelle-Hinton Agar (for bacteria). Metronidazole (2.5 µg/ml) and Fluconazole (2.5 µg/ml) were used as a positive control, while ethyl alcohol was used as a negative control. Analyses were performed in triplicate and the microplates were cultured at 37 °C for 18–24 h for bacteria and 30 °C for 48–72 h for the fungus. After the culture period, the microplates were developed with the addition of 10 µL of 0.01% resazurin solution and incubated for 3 h. The MIC was defined as the lowest concentration of the sample that inhibited the growth of the microorganisms (Ramos et al., 2022; Bezerra & Ramos, 2021).

Antimicrobial activity

The crude extract from P. marginatum roots was tested against nine microorganisms and showed an inhibition zone for two bacteria and one fungus, indicating a selective antimicrobial activity for these microorganisms (Table 2).

The minimum inhibitory concentrations (MIC) of the extract for the bacteria B. subtilis, S. aureus and fungus M. smegmatis were determined with values of 250, 250 and 125 µg/mL, respectively (Table 2). MIC values ≤ 500 µg/ml are considered strong inhibitors; MIC values from 600 to 1,500 µg/ml are considered moderate inhibitors; values 1,500 µg/ml are considered weak inhibitors (Aligiannis et al., 2001). The (E,E)-N-Isobutyl-2,4-octadienamide and (E,E)-N-Isobutyl-2,4-decadienamide amides showed strong antimicrobial activity against B. subtilis, S. aureus and M. smegmatis with value of 250 µg/mL for bacteria and 125 µg/mL for fungus, indicating that the amides are responsible for activity of butanolic extract from P. marginatum roots. A previous study revealed that essential oil from leaves of P. marginatum rich in phenylpropanoids showed antimicrobial activity against bacteria B. subtilis, S. aureus and fungus M. smegmatis with MIC values of 250, 2,500, 1,250 µg/mL, respectively (Ramos et al., 2022; De Oliveira, Silva & Ramos, 2022). The leaf extract showed activity against the bacteria S. aureus with a MIC of 1,250 µg/mL (Bezerra & Ramos, 2021).

Antioxidant activity and total phenolic content

The results obtained in the determination of total phenolics were expressed in milligrams equivalent of gallic acid per gram of extract (mg GAE/g, Table 3).

The crude root extract exhibited a total phenolic content of 42.7 ± 1.10 mg GAE/g, indicating that in addition to amides, the root extract also accumulates phenolic compounds in moderate amounts. The butanolic extract from P. marginatum roots showed the highest activity with an EC₅₀ = 47.3 ± 0.80 µg/mL. ABTS^⋅+ radical scavenging activity of roots was associated with the phenolic content, considering that EC₅₀ = for the 383.0 ± 5.19 and 340.4 ± 4.81 µg/mL, respectively, considering that values approaching 500 µg/mL do not exhibit good scavenging capacity. To our knowledge, this is the first report of the antioxidant activity for the extract and their major constituents from the roots of P. marginatum. Previous studies with essential oil of P. marginatum showed significant antioxidant activity with a DPPH IC₅₀ value between 1,200 and 1,500 µg/ml while the control ascorbic acid showed a DPPH IC₅₀ value of 1,000 µg/ml (Brú & Guzman, 2016). Essential oil from the roots of P. marginatum exhibited antioxidant activity with a DPPH IC₅₀ value of 75,300 µg /L while the Ginkgo biloba used as reference exhibited a DPPH IC₅₀ value of 46.9 mg/L (Bay-Hurtado et al., 2016). Piper umbellata has been reported to be a potent antioxidant attributed to its major constituent, 4-nerolidylcatechol (Cordeiro et al., 2013). The total phenolic concentration of the extract from the leaves of P. umbellata was of 148 mg GAE/g while antioxidant activity exhibited an EC₅₀ of 120.1 µg/mL based on ABTS (Ramos et al., 2012).