Plant material

Plant material was collected from an agroecological farm at Jardín Botánico de Bogotá Jose Celestino Mutis. Carlos I. Suárez identified the collected specimen. The voucher specimen (CISB-700) was deposited in the Herbario of Jardín Botánico de Bogotá José Celestino Mutis.

Solvents and chemicals

Gallic acid (98%) was procured from Sigma-Aldrich Chemie (Steinheim, Germany). Ethanol (96%), dimethyl sulphoxide (DMSO), Folin-Ciocalteu’s phenol reagent, anhydrous sodium sulfate, and sodium carbonate were purchased from Merck (Darmstadt, Germany). Spectrophotometric data were obtained on a Thermo Genesys model spectrophotometer and 1 cm pitch quartz cells were used.

Microorganisms

The strains of Botrytis sp. (SV-02), Aspergillus sp. (SV-97), and Penicillium sp. (SV-08) from the collection of the Bank of Jardín Botánico de Bogotá José Celestino Mutis were used for bioassays. The microorganisms were isolated from high Andean and Paramo plant species (Espeletia barclayana and Quercus humboldtii) and were reactivated on Potato Dextrosa Agar (PDA) medium and incubated at 27 ºC.

Plant extracts

The essential oil (EO) and ethanolic extract were obtained from aerial parts of P. subspathulata. The EO was extracted from fresh material (990 g) by hydro-distillation for 3 h using a Clevenger-type apparatus. The EO was kept at 4°C with anhydrous sodium sulfate until further analysis (^{Elyemni, et al., 2019}). To prepare the ethanolic extract, the plant material was dried in an oven at 40°C (127.4 g) and submitted to maceration with ethanol (96%) for a week. The solvent was removed using a Heidolph rotary evaporator (^{Albarracin et al., 2017}).

Secondary metabolites of the ethanolic extract were screened qualitatively by using the methods of ^{Soni and Sosa (2013)} and ^{Sanabria-Galindo et al. (1997)}. The presence of alkaloids (Dragendorff, Wagner, Mayer, Valser, and Hagger reagents), triterpenes and steroids (Salkowki, Liebermann-Buchard and Vanillin-phosphoric acid), flavonoids (Shinoda, Roseheim and leuco-anthocyans), quinones (acid and base test), phenols and tannins (ferric chloride and jelly-salt), and saponins (foam) were analyzed.

Total phenolic content

The total phenolic content of the ethanolic extract was determined by the Folin-Ciocalteu method (^{Singleton et al., 1999}; ^{Stanojević et al., 2009}). Gallic acid was used as a standard, and the results were expressed as mg of gallic acid equivalents (GAE)/g of extract. The calibration curve was done using a dilution of gallic acid (5, 10, 25, 50,75, and 100 mg L^-1). 5 mL of Folin-Ciocalteu's reagent (dilution 1:10) was added to 1 mL of extract or standard and left to stand for 5 min. Then, 4 mL of a 7.5% sodium carbonate solution was added. The absorbance reading at 740 nm was taken 2 h later. The tests were performed in triplicate.

Characterization for essential oil

The relative density and refractive index of the EO obtained were determined by the pycnometer method at 20°C (^{ISO, 1998a}), and the refractive index was measured by Abbe refractometer (^{ISO, 1998b}). Gas chromatography-mass spectrometry (GC/MS) analysis was carried out on an Agilent Technologies 6890 gas chromatograph coupled to a mass selective detector (MSD, AT 5973N) in the electron impact mode (IE: 70 eV), operated in full scan mode with DB-5MS (60 m x 0.25 mm x 0.25 µm) and DBWAX (60 m x 0.25 mm x 0.25 µm) capillary columns. Injection was performed in Split mode (30:1), with an injection volume of 2 µL. The components were identified by the experimental determination of the linear retention indexes (LRI) and by comparing mass spectra with Adams, Wiley, and NIST databases (^{Stashenko et al., 2008}). The analyses were performed by the chromatography and mass spectrometry laboratory (LABCROMASS) of the Universidad Industrial de Santander under contract JBB-CTO-864-2021.

Antifungal activity bioassays

The antifungal activity was determined using a mycelial radial growth inhibition technique (^{Sánchez-León et al., 2015}). The assays were performed in 60 mm Petri dishes with a capacity of 5 mL of agar. 50 µL of each sample (EO, extract, or control) prepared in DMSO was added to a Petri dish, and agar PDA was added. Fungi with seven days of incubation at 27°C were used for the assays, an agar disc containing the microorganism was placed on agar with the test substance, and a control was included in each assay. Petri dishes were incubated at 27°C until the fungus of treatment reached the border of the dish. Petri dishes were checked daily until the fungus in the control covered the dish. With Botrytis the reading was done after 5 d. In the Penicillium sp. and Aspergillus sp. trials, the reading was done after 8 d, although they did not occupy the box completely. The analyses were performed in triplicate. Mycelial growth was determined by taking two measurements of the diameter (perpendicular) of the colony. The areas occupied by the colony were determined and the inhibition percentages were calculated with the following equation (1):

where C corresponds to the radial growth of the mycelium in the control, and T to the radial growth of the mycelium in the treatment. The growth of the control will correspond to 100% because this corresponds to growth without inhibition. All tests were performed in triplicate. The minimal inhibitory concentration (MIC) of EO was defined as the lowest concentration of compound that completely inhibited visible growth after 5 and 8 d of incubation. And was determined by comparison with the control.

Statistical analysis

The data from the antifungal activity assays were evaluated using the Shapiro-Wilks test to establish whether they were normally distributed. Data that showed normal distribution were analyzed by Tukey's test for analysis of variance, and those that were not normally distributed were analyzed by the Kruskal Wallis test.

Characterization of the essential oil

The EO was an oily-liquid, clear and, yellowish with a characteristic aroma of the plant and intense with fresh and cinnamon olfactory notes. The extraction yield of the EO was 0.16%, this was lower than the yield reported for fresh leaves and stems (1.0%) and roots (0.5%) (^{De Díaz et al., 1988}). The relative density determined was 1.0226 g mL^-1 at 20°C and the refractive index at 20°C (n_D) was 1.5205. Upon observation of the EO, part of it remained on the water seal, while another portion remained in the lower part of the Clevenger trap, which means the essential oil is constituted by light compounds such as terpenes, and phenylpropanoids, that are denser than water. Moreover, the refractive index determined in this study was higher than that reported by ^{De Díaz et al. (1988)} of 1.3362. These variations may be due not only to the type of extraction but also to differences in plant materials, collection periods, and time of year, among other environmental factors. This significant difference suggests differences in the chemical composition of the EOs.

Characterization of the ethanolic extract

The ethanolic extract had a dark-colored, green-brown, liquid appearance with a sweet, herbal aroma less intense than the EO, and a yield of 34.5%. The total phenol content of the ethanolic extract was 1673±16 mg GAE/100 g dry material (48.5±0.5 mg GAE/g extract). This value had not been previously reported. However, its phenol content is close to that reported for P. pellucida extracted by maceration in butanol whose value was 42.73 (^{Phongtongpasuk and Poadang, 2014}). The phytochemical analysis revealed the presence of phenolic compounds, triterpenes and steroids, alkaloids, flavonoids, quinones, and lactonic compounds. These results were consistent with the report of Peperomia pellucida (^{Oloyede et al., 2011}) and Peperomia blanda except the presence of saponins (^{Al-Madhagi et al., 2019}).

Chemical composition of essential oil

By gas chromatography analysis coupled with mass spectrometry, 95.4% of the compounds present in the EO were identified. The compounds were listed according to the elution order on the DB-5MS column (Tab. 1). The calculated retention indices and those reported in the literature are included. The highest proportion of compounds were phenylpropanes (50.3%), followed by oxygenated sesquiterpenes (27.5%) and sesquiterpenes (13.6%). The main compounds identified were Safrole (44.3%), α-Bisabolol (24.2%), Myristicin (4.7%), trans-β-Caryophyllene (3.0%), Viridiflorene (3.0%), α-Humulene (2.3%), trans-Nerolidol (1.5%), Linalool (1.1%), Methyleugenol (1.1%) and cis-Farnesene (1.0%).

According to the chemical composition of the EO, Safrole is the major compound with 44.3%, which is also reported by ^{De Díaz et al. (1988)} with 49%. Phenylpropanes are common compounds in Peperomia species, compounds such as safrole, myristicin and methyleugenol have been isolated from Peperomia pellucida, Peperomia nitida, Peperomia campylotropa, Peperomia dindygulensis, Peperomia duclouxii, Peperomia oreophylla, Peperomia sui and Peperomia tetraphylla (^{Valero Gutiérrez et al., 2016}). The α-bisabolol is the second majority compound (24.2%) in the EO of P. subspahulata, this compound has also been identified in the EO of P. galioides (^{Villegas et al., 2001}) and P. sui (^{Cheng et al., 2003}).

Antifungal activity of ethanolic extract and essential oil

The ethanolic extract and the EO of P. subspathulata were evaluated against fungi at concentrations of 10, 100, and 1,000 µg mL^-1 (Tab. 2). The data showed a normal distribution by the Shapiro-Wilks test (ethanolic extract against Aspergillus sp. and Penicillium sp.) and were analyzed for variance by Tukey's test. The results with a non-normal distribution were analyzed statistically by the Kruskal Wallis test.

The three fungi were susceptible to the treatments with the essential oil and the ethanolic extract. The best treatment with the EO was the concentration at 1,000 µg mL^-1 as it inhibited the development of Aspergillus, Botrytis, and Penicillium by 94, 100, and 77%, respectively. In contrast, the ethanolic extract showed lower inhibition percentages than EO at 1,000 µg mL^-1, particularly, against Aspergillus sp. and Botrytis sp. Aspergillus sp. was the microorganism most susceptible to the extract (71.63%). While the percentage of inhibition against Botrytis was reduced by 43%.

The antifungal activity of the EO may be mainly correlated with the main compounds, phenylpropanoids, and terpenoids. Phenylpropanoids have shown remarkable antifungal properties. Isoeugenol was active against Fusarium oxysporum and Botrytis cinerea (^{Kfoury et al., 2016}), Eugenol against Aspergillus niger and Penicillium digitatum (^{Marei and Abdelgaleil, 2018}). ^{Valente et al. (2015)} identified Myristicin as responsible for antifungal activity against Aspergillus flavus and Aspergillus ochraceus. Safrole showed activity against Aspergillus fumigatus (^{Poudel et al., 2021}). Moreover, a sesquiterpenoid -Bisabolol was active against Botrytis cinerea (^{Kamatou and Viljoen, 2010}), and Aspergillus species (^{Brandão et al., 2020}). The antifungal effect of the ethanolic extract could be due to the presence of flavonoid compounds, some authors associate these compounds with antifungal activity (^{Jin, 2019}; ^{Al Aboody et al., 2020}; ^{Di Ciaccio et al., 2020}). Therefore, it is necessary to continue with chemical studies of the extract to identify the bioactive compounds.

The MIC determined that EO against Aspergillus sp. was 1,000 µg mL^-1 and against Botrytis sp. 500 µg mL^-1. These results are very promising for the antifungal potential of this EO. Although there are no reports of other Peperomia species. However, Safrole pure was effective against Aspergillus niger and Aspergillus fumigatus with MIC of 78.1 and 39.1 µg mL^-1, respectively (^{Poudel et al., 2021}).