Chemical composition and antioxidant and antibacterial activity of Platymiscium gracile Benth.: A species threatened by extinction

2.1 General methods

UV spectra were measured on a Jemway 6405 instrument. ¹H and ¹³C NMR spectra were recorded on a Bruker AMX 300 spectrometer. FTIR spectra were obtained in CHCl₃ on a Perkin-Elmer RXI. Mass spectra were performed on a Hewlett-Packard 6890 (Agilent Technologies) gas chromatograph with mass selective detector-quadrupole type (HP 5973 MSD). Detention of compounds was carried out by thin layer chromatography (TLC) on silica gel F₂₅₄ (0.2 mm Merck) plates.

2.2 Plant material and extraction procedure

The pruned branches of P. gracile were obtained from trees found in the municipality of Puerto Asís, department of Putumayo (Colombia). Dr. Jorge Mario Vélez at the Herbarium “Gabriel Gutiérrez Villegas” of the Universidad Nacional de Colombia-Medellín performed the botanical identification. A voucher of specimen (MEDEL#64111) was deposited. The wood was debarked, cut, and ground to a fine powder. Then, the powder (783 g) was extracted successively with n-hexane, dichloromethane, ethyl acetate, and methanol by percolation until completion. Extracts were concentrated at 40 °C under reduced pressure and used for the determination of antimicrobial and antioxidant activities, in addition to total phenolic contents.

2.3 Isolated metabolites

The n-hexane, dichloromethane, and ethyl acetate extracts were submitted to column chromatography (on silica gel) with mixtures of n-hexane and ethyl acetate of different polarity. The fractions were combined according to TLC profile and further chromatographed by size-exclusion chromatography on Sephadex LH 20® column using the mixture n-hexane, methanol, dichloromethane (2:1:1; v/v) to yield eleven compounds. Scoparone: The compound (73.4 mg) was isolated as a white crystalline solid. The spectroscopic data agree with those previously reported (Martínez et al., 2017). Homopterocarpin: It was isolated as a white solid (60.4 mg). The spectroscopic data agree with those previously reported (Martínez et al., 2017). Calycosin: This compound was isolated as a pale yellow crystalline solid (37.9 mg). The spectroscopic data agree with those previously reported (Martínez et al., 2017). Medicarpin: A pale yellow crystalline solid was obtained (12.3 mg). The spectroscopic data agree with those previously reported (Martínez-Sotres et al., 2012). Liquiritigenin: It was isolated as an amorphous yellowish solid (8.5 mg). The spectroscopic data agree with those previously reported (Li et al., 2014). Isoliquiritigenin: An amorphous yellowish solid was isolated (6.3 mg). The spectroscopic data agree with those previously reported (Li et al., 2014). Oleanolic aldehyde acetate: The compound was isolated as a white crystal from n-hexane (37.0 mg). The spectroscopic data agree with those previously reported (Anjum et al., 2007). 8-hydroxyhomopterocarpin: It was obtained as a white crystalline solid (6.1 mg). The spectroscopic data agree with those previously reported (Morimoto et al., 2006). 8-methoxyhomopterocarpin: It was isolated as yellow oil (4.1 mg). The spectroscopic data agree with those previously reported (Morimoto et al., 2006). 3, 4-dimethoxycinnamaldehyde: This compound was isolated as yellow oil (5.1 mg). The spectroscopic data agree with those previously reported (Burgi et al., 1993). 3, 4, 5-trimethoxycinnamaldehyde: This compound was isolated as yellow oil (5.3 mg). The spectroscopic data agree with those previously reported (Mohammad et al., 1985).

2.4 Total phenolic content

Total phenolic content was determined according to the Folin-Ciocalteau colorimetric method (Singleton et al., 1999), using gallic acid as a standard phenolic compound. The mean ± standard deviation (SD) of total phenolic contents of triplicate analyses were expressed as gallic acid equivalent (mg GAE)/100 g extract.

2.5 DPPH free radical scavenging activity

The hydrogen donating ability of some pure compounds and the plant extracts from P. gracile were measured by decolorization of 2,2-diphenyl-2-picryl hydrazyl (DPPH) (Preciado et al., 2016). Results were expressed by the TEAC (Trolox equivalent antioxidant capacity) values (µmol TE)/100 g of sample.

2.6 ABTS radical cation scavenging activity

The antioxidant capacity assay was performed using the ABTS radical cation decolorization method (Preciado et al., 2016). Results were expressed by the TEAC values as µmol TE/100 g of sample.

2.7 Ferric reducing antioxidant power assay (FRAP)

The reducing power of the extracts and pure compounds of P. gracile was determined according to Preciado et al. (2016). Results were expressed by the AEAC (Ascorbic acid equivalent antioxidant capacity) values (mg AAE)/100 g of sample.

2.8 Hydrophilic oxygen radical absorbance capacity assay (H-ORAC)

Peroxyl radicals was generated using [2,2′-azobis (2-amidinopropane) dihydrochloride] (AAPH). The stock solutions were 10.0 mM sodium fluorescein and 0.6 M AAPH dissolved in phosphate buffer solution (75 mM, pH 7.4). In cells, the solution consisted of fluorescein (21 µL), the pure compound (30 µL), AAPH (50 µL) and phosphate buffer solution (3 µL). Readings were performed with an excitation wavelength of 493 nm (slit of 5 nm) and emission wavelength of 515 nm (slit of 13 nm), using both an attenuator of 1% and without attenuator plate. ORAC values were calculated based on the net area under the curve (AUC) and compared to Trolox standard curve. The antioxidant capacity (ORAC) related to Trolox was estimated as: $$\text{ORAC value}=\frac{\left(\text{AUC}sample-\text{AUC}blank\right)}{\left(\text{AUC}Trolox-\text{AUC}blank\right)}f\left(\mathit{Trolox}\right)$$ where AUC_sample, AUC_blank and AUC_Trolox are the area under the curve for: pure compounds, the blank and Trolox respectively, and f is the dilution factor. Results were expressed in µmol TE/g of sample (Preciado et al., 2016).

2.9 Antibacterial activity assay

Four food-borne bacterial strains were used in this study. The strain included Bacillus cereus (ATCC 10876; Gram “+“), Staphylococcus aureus (ATCC 29223; Gram “+“), Escherichia coli (ATCC 25922; Gram “−“), and Enterobacter faecalis (ATCC 29912; Gram “+“). Antibacterial activity was evaluated using two methods: disc diffusion (Bhuyan et al., 2017) and microdilution (Costa et al., 2017).

Disc diffusion method. Filter paper discs (6 mm diameter) were prepared with 20 μL of 1, 0.1 and 0.01% solution in water (<5% ethanol) for all extracts, in addition to the pure compounds. The discs were placed in Petri dishes containing Mueller Hinton agar (MHA) inoculated with a bacteria suspension of 1.5 × 10⁸ CFU (0.5 McFarland density). After 24 h incubation at 35 °C, inhibition zones were measured (diameters). Discs impregnated with Ampicillin (Haupt Pharma Latina, Italy) and water–ethanol (<5%) were used as positive and negative controls, respectively.

Microdilution method. The minimum inhibitory concentration (MIC) of the samples was assessed using the microdilution method. A stock solution of 8000 µg/mL extract (and 2000 µg/mL pure compound) was prepared in nutritious broth. Then, serial double dilutions were prepared in the range of 8000 to 7.8 µg/mL for extracts and 2000 to 2.0 µg/mL for pure compounds. Next, 40 µL of each dilution and 140 µL of nutritious broth were deposited in the wells of a microtiter plate. As negative control, 40 µL of solvent and 140 µL of nutritious broth was used. After, 20 µL of bacterial suspension (0.5 McFarland standard) was incorporated to the wells. The microtiter plates were incubated at 35 °C during 24 h. Subsequently, 20 µL of 5 mg/mL TTC (2,3,5-triphenyl tetrazolium chloride) was aggregated to the wells and the microtiter plate incubated at 35 °C and re-examined after 30 min. Ampicillin (Haupt Pharma Latina, Italy) was used as the positive control. The MIC was considered as the lowest concentration of antimicrobial agent that prevented the microbial growth after 24 h incubation (Balouiri et al., 2016). For the determination of the MBC (minimum bactericidal concentration), 5 µL from the well that exhibited no growth (with concentrations equal or greater than the MIC) was transferred to Petri dishes and incubated at 35 °C for 24 h. The MBC corresponds to the lowest concentration that showed no visible bacterial growth after sub-culturing.

2.10 Statistical analysis

Each experiment was performed three times and the results were reported as mean ± SD. Mean values were compared with the Fisher’s least significant differences (LSD) at the 0.05 probability level.

3.1 DPPH free radical scavenging activity

The DPPH assay is the most used chemical method for determining free radical scavenging activity by antioxidant agents. A higher TEAC value implies greater protective action against free radicals. According to the results, the DPPH radical scavenging activity ranged from 20,055 to 194,919 µmol TE/100 g. Ethyl acetate extract was the most efficient free radical scavenger with the highest TEAC value of 194,919 ± 7163 µmol/100 g, followed by the methanol (74,090 ± 386 µmol TE/100 g) and dichloromethane (71,755 ± 2247 µmol TE/100 g) extract. The n-hexane extract was the least active of all samples (20,055 ± 1341 µmol TE/100 g).

3.2 ABTS radical cation scavenging activity

The ABTS assay is frequently used to determinate the antioxidant activity of plant extracts. The ABTS radical cation scavenging activity ranged from 30,081 to 901,706 µmol TE/100 g. The ABTS assay showed that maximum scavenging activity in the samples was observed in the dichloromethane extract (901,706 ± 20,393 µmol TE/100 g), being about two-fold higher than the ethyl acetate (458,989 ± 45,720 µmol TE/100 g) extract. The weakest antioxidant capacity was exhibited by the methanol and n-hexane extract. As can be seen, ABTS assay values were higher than those of DPPH assays.

3.3 Reducing power assay (FRAP)

The results showed that FRAP values were higher in dichloromethane (32,264 ± 326 µmol AAE/100 g) and ethyl acetate (13,787 ± 1183 µmol AAE/100 g) extracts as compared to methanol (1785 ± 225 µmol AAE/100 g) extraction. FRAP values were not determined for n-hexane extract, due to difficulties in solubility.

3.4 Phenolic content

Phenolic substances are recognized for imparting antioxidant activity to vegetal extracts. According to Table 1, the phenolic contents ranged from 3235 to 36,350 mg GAE/100 g extract. HEX extract of P. gracile had the highest phenolic content (36,350 ± 3049 mg GAE/100 g), followed by the EA and DCM extracts. The ME extract presented the lowest phenolic content. The results showed that the n-hexane, dichloromethane, and ethyl acetate extraction was more efficient in extracting antioxidant compounds in P. gracile as compared to methanol.

3.5 Antibacterial activity assay

3.6 Isolated metabolites

After separation and purification procedures on the ethyl acetate, dichloromethane and n-hexane extracts, eleven known compounds were obtained. From n-hexane extract, three compounds were isolated corresponding to scoparone (1) (6,7-dimethoxycoumarin), homopterocarpin (2), and oleanolic aldehyde acetate (3) (Fig. 1). In addition, five other known compounds were isolated from dichloromethane extract, and the spectra data were in agreement with calycosin (4) and medicarpin (5) 8-hydroxyhomopterocarpin (6), 8-methoxyhomopterocarpin (7), 3,4-dimethoxycinnamaldehyde (8) and 3,4,5-trimethoxycinnamaldehyde (9). Additionally, compounds liquiritigenin (10) and isoliquiritigenin (11) were obtained from ethyl acetate extract. In review of the literature, this is the first report on the isolation of compounds (3), (5), (6), (7), (8), (9), (10) and (11) from P. gracile. Surprisingly, P. gracile is a source of a wide variety of compounds that includes coumarin, chalcone, flavanone, triterpene, cinnamic aldehyde derivatives, and pterocarpans. Some of these compounds are present in high quantities (Martinez et al., 2017).

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Fig. 1

Structure of compounds isolated from P. gracile.

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3.7 Antioxidant and antimicrobial activity of isolated compounds

Table 4 presents the results of the antioxidant activity of some of the isolated compounds. Only phenolic compounds were included, with the exception of compound (6) which was not isolated in sufficient quantity to be tested. Compounds (5) and (11) were found to possess significant antioxidant properties. Compound (11) (a chalcone) displayed the strongest antioxidant activity in all assays applied. The activity of (11) was followed by that of pterocarpan (5) in the ABTS, FRAP, and ORAC assays. For these same assays, the lowest activity was found for the isoflavone (4).

Antibacterial activity of (1), (2), and (3) using the disc diffusion method are summarized in Table 5. Compounds (6, 7, 8 and 9) were excluded of assay, due to their insufficient quantities. The highest antibacterial activity against the Gram-negative bacterium was found for (3). Both (1) and (2) were ineffective. On the other hand, (2) and (3) were slightly active against B. cereus, S. aureus, and E. faecalis.

Due to the low amount of the chalcone (11), antibacterial activity was determined using the microdilution method. In addition, (1) was analyzed in the same way. Results are reported in Table 6. Under these conditions, antibacterial activity of (1) was weak, which is in according to the disc diffusion method.

From Table 6, it becomes clear that (11) was most effective in controlling the growth of Gram-positive bacteria. Compound (11) exhibited strong inhibition of the growth of S. aureus, B. cereus, and E. faecalis with MIC values of 62.5 µg/mL. It is noteworthy that the MIC value for (11) against B. cereus was lower than for the reference antibiotic Ampicillin.