Spiranthera odoratissima A. St.- Hil. (Rutaceae) leaves were collected in May, when the Cerrado region, located in Goiás state (GO), undergoes its dry season. Collection was carried out in Iporá, GO (16°24'11.2"S and 51°06'41.4"W), whose altitude is 707 m (Figure 2). Spiranthera odoratissima leaves were stored in paper bags, identified and preserved. Plant material was identified by botanist Erika Amaral and deposited in the herbarium that belongs to the Instituto Federal Goiano, Campus Rio Verde, GO, exsiccate no.1039.

Figure 2.
The Cerrado region in Iporá, GO, Brazil.

Solvents used in the experiments were hexane (NEON, Suzano, SP, Brazil), ethyl alcohol (Ls Chemicals, Ribeirão Preto, SP, Brazil), methyl alcohol (Ls Chemicals, Ribeirão Preto, SP, Brazil) and ethyl acetate (Dinâmica, Indaiatuba, SP, Brazil). Deionized water was produced by a deionizer (LUCADEMA/LUCA-315).

The methodology used for preparing extracts was described by Rodrigues, Souza Filho, and Ferreira, (2009), with modifications. The material was dried by a forced air oven at 50°C up to constant mass for about 24 hours. Afterwards, the material was ground by a Willey Star FT 50 knife mill and passed through a 5-mm sieve up to homogeneous powder.

To yield crude extracts, 100 g homogeneous powder (ground leaves) was added to 1000 mL solvent (hexane, ethyl acetate, methanol, mixtures of ethanol and water (7:3) and water (100%). All extracts were stored in closed glass bottles. In this phase, extracts were kept in a dark place. After 24 hours, solutions were filtered through qualitative filter paper (80 g m-²). Resulting liquids were submitted to a rotoevaporator at 70ºC, so that solvents could evaporate and the following extracts could be obtained: hexane (7.60 g), ethyl acetate (10.3 g), methanolic (5.80 g), hydroethanolic (12.2 g) and aqueous (3.30 g) ones. At the end, all extracts had the consistency of syrup.

In preliminary phytochemical screening, classical procedures were carried out to identify the main classes of secondary metabolites. Identification of compounds found in plant extracts may be based on certain criteria, such as color, precipitation reactions and foam formation (Mariño et al., 2019).

The following classes of metabolites were analyzed: organic acids, reducing sugars, alkaloids, flavonoids, saponin compounds, coumarin compounds, cardiac glycosides, phenolics, proteins and amino acids, purines, tannins, polysaccharides, purine compounds, catechins, benzoquinones, naphthoquinones and phenanthraquinones, flavanols, flavanones, flavononols, xanthones, anthraquinones, sesquiterpene lactones and other lactones.

The methodology described by Henriques and Almeida (2013), was applied, with modifications. In a test tube, 5 mL distilled water was added to 2 mL extract. Then, 2 mL Pascová reagent was added to it. The reaction is considered positive when the reagent loses its color.

Reducing sugars were determined by the methodology proposed by Silva, Souza, Silva, Marques, and Graebner (2019), with modifications. In a test tube, 5 mL distilled water was added to 2 mL extract. Then, 2 mL Fehling’s reagent and 2 mL Fehling’s B were added to it. The tube was transferred to a vortex shaker to homogenize the solution and then kept in a water bath for 5 minutes. Positive results show brick-red precipitate.

Non-reducing sugars were also determined by the methodology proposed by Silva, Souza, Silva, Marques, and Graebner (2019), with modifications. In a test tube, 5 mL distilled water was added to 2 mL extract. Afterwards, homogenization was carried out in a vortex shaker and 1 mL concentrated hydrochloric acid (HCl) was added to the test tube.

The solution was heated in a water bath for about 5 minutes and then cooled. Neutralization was carried out by a sodium hydroxide solution (NaOH) 20% (m v^-1) and 2 mL Fehling’s reagent and 2 mL Fehling’s B were added. The solution underwent a water bath again for 5 minutes. After cooling, it was analyzed. The reaction is considered positive when it shows red precipitate.

The methodology described by Henriques and Almeida (2013), with modifications, was used for determining the group of alkaloids. In a test tube, hydrochloric acid (HCl) at 5% (m v^-1) was added to 4 mL extract. Afterwards, 1 mL solution was placed in 4 test tubes in the following order:

Test tube 1 – extract solution + hydrochloric acid at 5% (m v^-1) + 500 µL Mayer’s reagent. The reaction is considered positive when there is either white precipitate or a slightly turbid white solution.

Test tube 2 - extract solution + hydrochloric acid at 5% (m v^-1) + 500 µL Wagner’s reagent. A positive reaction shows orangish precipitate.

Test tube 3 - extract solution + hydrochloric acid at 5% (m v^-1) + 500 µL Liebermann-Bouchard’s reagent. A positive reaction shows orangish or reddish precipitate.

Test tube 4 - extract solution + hydrochloric acid at 5% (m v^-1) + 500 µL Bertrand’s reagent. A positive reaction shows white precipitate.

The Shinoda test (concentrated HCl and magnesium) was carried out in agreement with the methodology described by Henriques and Almeida (2013), with modifications. In a test tube, a little fraction of magnesium ribbon (0.5 cm in diameter) and 1 mL concentrated HCl were added to 3 mL extract. The solution exhibited total effervescence at the end of the reaction. A positive reaction shows colors that range from blackish red to red.

The method used for determining saponin compounds was described by Silva et al. (2019), with modifications. In a test tube, 10 mL deionized water was added to 5 mL extract. It was vigorously agitated by a vortex shaker for 2 minutes to form foam. The reaction is positive if foam persists in test tubes after resting for 15 minutes.

The methodology described by Henriques and Almeida (2013), with modifications, was used for determining coumarin compounds. A test tube with 3 mL extract was covered with filter paper (80 g m-²) and 1 mL aqueous solution of sodium hydroxide at 10% (m v^-1) was applied to the paper surface. The tube was heated in a water bath for 10 minutes. After having cooled at room temperature, the test tube was placed in an ultraviolet chamber and the paper was evaluated under UV light at wavelengths of 254 and 365 nm. Result is considered positive when fluorescence on the paper is either green or yellow.

The methodology described by Kloss, Albino, Souza, and Lima (2016), with modifications, was used for carrying out this assay. In a test tube, 3 mL extract was mixed with 2 mL KEEDE B reagent. The reaction is considered positive when precipitate is formed and/or its color becomes either blue or violet.

Phenolic compounds were evaluated by the methodology proposed by Henriques and Almeida (2013), with modifications. In a test tube, 5 mL distilled water was added to 3 mL extract. Then, 1 mL ferric chloride solution, FeCl₃, at 1% (m v^-1) was also added. The reaction is considered positive when colors vary between blue and red, an evidence of simple phenolics.

Proteins and amino acids were evaluated by the methodology proposed by Henriques and Almeida (2013), with modifications. In a test tube, 3 mL extract was mixed with 500 µL aqueous vanillin solution at 1%. Then, it was kept in a water bath for 10 minutes, up to ebullition. There was change in color after the solution rested. The result is positive when the solution becomes persistently violet.

Purine compounds were determined in agreement with the methodology proposed by Henriques and Almeida (2013), with modifications. In a test tube, 500 µL aqueous solution of hydrochloric acid at 32% and 500 µL aqueous solution of hydrogen peroxide 35% were added to 2 mL extract. The solution was then heated in a water bath for 10 minutes, up to total evaporation of the liquid. Reddish residue formed at the end of the water bath. Afterwards, 500 µL aqueous solution of ammonium hydroxide 6 N was added to the solution in the test tube, which was then homogenized by agitation for 1 minute. The reaction is considered positive when it becomes reddish and/or violet.

Tannins were determined by the methodology proposed by Henriques and Almeida (2013), with modifications. In a test tube, 500 µL ferric chloride solution, FeCl₃, at 10%, was added to 2 mL extract and attention was focused on changes in color. Blue precipitate shows that there are hydrolysable tannins, while the green one shows condensed tannins.

The analysis of polysaccharide compounds was carried out in agreement with Henriques and Almeida (2013), with modifications. In a test tube, 5 mL distilled water was added to 2 mL extract. The solution was manually homogenized for 30 seconds and then 500 µL Lugol’s iodine was added to it. Results are considered positive when the solution is blue.

Catechins were evaluated in agreement with the methodology described by Silva et al. (2019), with modifications. In a test tube, 2 mL vanillin solution at 1% and 1 mL concentrated hydrochloric acid at 32% (P.A. – ACS) were added to 2 mL extract. The result is positive when the solution is reddish.

Derivatives of benzoquinones, naphthoquinones and phenanthraquinones were detected by the methodology described by Barbosa et al. (2004), with modifications. In the test tube, 500 µL aqueous solution of anhydrous sodium carbonate (Na₂CO₃) at 15%, 500 µL formaldehyde solution at 4% and 500 µL 3,5-dinitrobenzoic solution at 5% were added to about 3 mL extract. The solution was then heated in a water bath for 5 minutes. The reaction is considered positive when it becomes red.

These compounds were analyzed in agreement with the methodology described by Barbosa et al. (2004), with modifications. In a test tube, a little fraction of magnesium ribbon (0.5 cm in diameter) and 1 mL concentrated HCl were added to 3 mL extract. After total effervescence of the magnesium ribbon, colors changed. The reaction is considered positive when it becomes reddish.

Anthraquinones were evaluated by the methodology proposed by Barbosa et al. (2004), with modifications. In a test tube, 2 mL aqueous solution of ammonium hydroxide at 10% was added to 3 mL extract. The solution was manually homogenized for 30 seconds. The positive reaction is pinkish, red or violet.

The group of sesquiterpene lactones and lactones was evaluated in agreement with the methodology described by Barbosa et al. (2004), with modifications. In a test tube, 1 mL alcoholic hydroxylamine hydrochloride solution at 10% and 500 µL methanolic KOH solution at 10% were added to 2 mL extract. The solution was kept in a water bath for 10 minutes and, after cooling, it was acidified by a hydrochloric acid (HCl) solution at 1 N. Then, 500 µL aqueous solution of iron chloride III (FeCl₃) was added to it. The positive reaction is violet.

Free radical scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH), azino-bis (ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) were determined by the spectrophotometric method described by Lima et al. (2019), with modifications. In the DPPH assay, different concentrations of extracts in methanol (10–100 μg mL^-1) were added to 2 mL 0.1 mM solution of DPPH that had been previously prepared and incubated in the dark for 30 min. Absorbance was recorded at 517 nm by a UV spectrophotometer. In the ABTS assay, 1980 mL diluted ABTS solution was added to 20 μL extract that had previously been diluted in ethanol. Absorbance at 734 nm was measured 6 min. after initial mixing. BHT was used as positive control. Assays were carried out in triplicate. Inhibition percentage was calculated as (I %) = (A0 − A/A0) × 100, where A0 is the absorbance of the control and A is the absorbance of samples. Finally, total antioxidant capacity of different plant extracts was evaluated by the FRAP method. In this colorimetric assay, reduction of ferric-tripyridyltriazine complex to ferrous form is expressed as antioxidant capacity. The FRAP reagent was added to different concentrations of plant extracts (10-1000 μg mL^-1). Absorbance was read at 570 nm by a microplate reader/spectrophotometer. The IC₅₀ value was calculated as the concentration of a sample required to scavenge 50% of free radicals by graphing the I % versus extract concentration.

Listeria monocytogenes (ATCC 15313) from the American Type Culture Collection (ATCC) was employed. Minimum inhibitory concentration (MIC) is defined as the lowest concentration of a sample that can inhibit bacterial growth. MIC was determined by microdilution on 96-well microplates, in triplicate. Extract samples were tested at concentrations ranging from 0.195 to 400 µg mL^-1. Positive control was penicillin (from Sigma-Aldrich, St. Louis, MO) for Gram-positive bacteria, at concentration of 5.9 µg mL^-1. Final dimethylsulfoxide (DMSO) content in the sample was 5% (v v^-1); the same concentration was employed as the negative control (without extracts). Inoculated wells containing the microorganism were only included to control bacterial growth. Non-inoculated wells (without any microorganisms) were also employed to ensure broth sterility. Inoculum cell concentration was adjusted to 5 X 10⁵ colony-forming unit (CFUs) mL^-1 based on the absorbance read at 625 nm by the spectrophotometer Nanodrop from Thermo Scientific. The complete methodology used for evaluating antilisterial activity of extracts was the one described by Fernández et al. (2018).

Results of the phytochemical screening of extracts from Spiranthera odoratissima (Rutaceae) leaves are shown in Table 1.

Table 1
Phytochemical screening of extracts from S. odoratissima (Rutaceae) leaves

Results: (+) positive and (-) negative. GR = green for condensed tannins

Table 1 shows organic acids in aqueous, methanolic, hydroethanolic, ethyl acetate and hexane extracts from S. odoratissima leaves.

Reducing sugars were identified in methanolic, hydroethanolic, ethyl acetate and hexane extracts, but not in the aqueous one. Soares, Santos, Vieira, Pimenta, and Araújo (2016), carried out phytochemical screening and reported reducing sugars in extracts from S. odoratissima leaves, while non-reducing sugars were only found in the aqueous extract. Alkaloids were not detected in any extract under study.

Flavonoids, which are known for exhibiting antimicrobial and antioxidant activities (Machado, Nagem, Peters, Fonseca, & Oliveira, 2008), were found in aqueous, methanolic, hydroethanolic and ethyl acetate extracts from S. odoratissima leaves. Soares et al. (2016), also reported this class of compounds in S. odoratissima leaves.

Saponin compounds were only found in aqueous, methanolic and hydroethanolic extracts. They have several biological activities; the ones that should be highlighted are related to increase in immune response and rupture of erythrocyte membranes, such as immunologic adjuvant and hemolytic activities (Kaiser, Pavei, & Ortega, 2010). Soares et al. (2016) identified saponin groups in aqueous extract from S. odoratissima leaves.

Coumarin compounds were qualitatively identified in aqueous and methanolic extracts. Their biological activities include antiprotozoal, antimalarial, memory stimulating, antitumoral, antifungal, anti-inflammatory and antioxidant ones (Jalhan, Singh, Saini, Sethi, & Jain, 2017). Terezan et al. (2010), reported this group of secondary compounds in crude extract from S. odoratissima leaves. Soares et al. (2016) also described this type of compound in S. odoratissima leaves.

Cardiac glycosides were found in aqueous, methanolic and hydroethanolic extracts from S. odoratissima leaves, but neither ethyl acetate nor hexane extracts were detected. These compounds are capable of increasing the rate of contraction in cardiac muscle fibers. Thus, they have been specially used for treating congestive heart failure (CHF) (Palacios, Thompson, & Gorostiaga, 2019).

Phenolic compounds were detected in aqueous, methanolic, hydroethanolic and ethyl acetate extracts from S. odoratissima leaves. They act as the main sources of antioxidant activity in fruits and plants, since they protect species from environmental aggressions and contribute to improve human health not only as agents that decrease blood sugar levels and body mass, but also as anticarcinogens (Soares, 2002). Proteins and amino acids were not identified in any extract from S. odoratissima leaves under investigation.

Regarding tannins, this class was identified in all extracts under study, except in the hexane one. Condensed tannins have quite complex structures and astringent properties, besides exhibiting antidiarrheal, antiseptic, antimicrobial and antifungal attributes. They also help to cure burns, wounds and inflammations by developing a protective layer on which healing processes occur naturally (Monteiro, Albuquerque, Araújo, & Amorim, 2005). Soares et al. (2016), also found these compounds in S. odoratissima leaves.

Polysaccharide compounds were only identified in methanolic and hydroethanolic extracts. The class of purine compounds was qualitatively identified in all extracts under investigation, except the hexane one.

Catechins were only found in aqueous, methanolic and hydroethanolic extracts. This group of secondary metabolites has a broad number of biological activities, such as antioxidant, anti-inflammatory, anticarcinogenic and chemoprotective ones. It is also considered an agent against skin aging (Isemura, 2019). Derivatives of benzoquinones, naphthoquinones and phenanthraquinones were not found in any extract from S. odoratissima leaves under investigation.

Flavonoids and their derivatives – flavonols, flavanones and xanthones – were qualitatively identified in aqueous, methanolic, hydroethanolic and ethyl acetate extracts, but not in the hexane one, from S. odoratissima leaves. Soares et al. (2016) also found this class of metabolite in S. odoratissima leaves.

Even though anthraquinones are chemically characterized by the fact that they exhibit antibacterial, antifungal and antiviral activities, they may lead to diarrhea, vomiting and nausea when they are badly employed. In addition, overdoses may provoke severe crises of acute nephritis (Malik & Muller, 2016). These compounds were found in aqueous, methanolic, hydroethanolic and ethyl acetate extracts from S. odoratissima leaves. Classes of sesquiterpene lactones and other lactones were found in aqueous, methanolic, hexane and hydroethanolic extracts, but not in the ethyl acetate one.

Antioxidant activity of plant extracts was evaluated by both DPPH, ABTS and FRAP methods, which have been commonly used for analyzing free radical scavenging capacity in several natural products (Alves, David, David, Bahia, & Aguiar, 2010).

Table 2 shows results of hexane, ethyl acetate, methanolic, hydroethanolic and aqueous extracts in terms of IC50 values (µg mL^-1). Positive control was BHT (IC50 = 18.00 µg mL^-1). The lowest antioxidant activity was exhibited by the hexane extract (IC₅₀ = 1550.80 µg mL^-1 by DPPH, IC50 = 1358.50 µg mL^-1 by ABTS and IC50 = 1450.75 µg mL^-1 by FRAP), while the highest antioxidant activity was attributed to the aqueous extract (IC50 = 3.18 µg mL^-1 by DPPH, IC50 = 3.81 µg mL^-1 by ABTS and IC50 = 3.50 µg mL^-1 by FRAP). High antioxidant activity was also identified in ethyl acetate, methanolic and hydroethanolic extracts, whose IC50 values were close to the ones of the positive control BHT (IC₅₀ = 18.00 µg mL^-1).

Silva, Costa, Santana, and Koblitz (2010), stated that the high free radical scavenging activity may be explained by the fact that phenolic compounds and flavonoids can be found at higher concentrations, by comparison with the hexane extract, which did not exhibit classes of compounds with antioxidant potential by phytochemical screening. In general, phenolic compounds prevent free radicals from acting in the body and, since they protect molecules, such as DNA, they may interrupt some carcinogenic processes. According to Youn et al. (2019), methodologies which use scavenging of radicals DPPH, ABTS and FRAP measure the activity of compounds of hydrophilic nature, i. e., compounds with high polarity. Therefore, antioxidant activities observed by this study may be directly connected with flavonoids in extracts under investigation.

Concerning anti-Listeria monocytogenes activity, ethyl acetate, methanolic, hydroethanolic and aqueous extracts exhibited high antibacterial activity, whose MIC values ranged between 12.5 and 62.5 µg mL^-1 (Table 2). However, the hexane extract was the only one that had low activity against bacteria whose MIC value was 1000 µg mL^-1 (Table 2).

Lemes et al. (2018) have recently reported that MIC values below 100 µg mL^-1, between 100 and 500 µg mL^-1 and between 500 and 1000 µg mL^-1 correspond to promising, moderate and weak activities, respectively, whereas MIC values above 1000 µg mL^-1 denote inactivity. Thus, it confirms the antibacterial potential of the most polar extracts from S. odoratissima leaves.

A large amount of antimicrobial effects of plant extracts is believed to result mainly from flavonoids in their composition (Andrade et al., 2005). Throughout the extraction process, based on the polarity of its constituents, the hexane extract kept poor in flavonoids and rich in terpenoids. As a result, the hexane extract from leaves exhibited weak or no antimicrobial activity at all.

Due to the high susceptibility of Gram-positive microorganisms, the mechanism of antimicrobial activity of the extract may result from its interaction with the peptidoglycan found in the bacterial cell wall, which characterizes a more fragile barrier than cell walls of Gram-negative bacteria (Oliveira, Oliveira, Batalini, Rosalem, & Ribeiro, 2012).

Table 2.
Antioxidant (IC50 = µg mL^-1) and anti-Listeria monocytogenes (MIC = µg mL^-1) activities of plant extracts from S. odoratissima leaves