Chemical composition, antioxidant and antibacterial activities of two Spondias species from Northeastern Brazil

Abstract

Context: The leaves of Spondias tuberosa Arr. Cam. (Anacardiaceae) and Spondias mombin L. have been traditionally used for medicinal purposes. Some studies reveal their antibacterial, antimicrobial, and antiviral properties.

Objective: Determine the chemical composition, antioxidant, and antimicrobial activities of Spondias species to justify its ethnopharmacological use.

Materials and methods: Spondias species extracts were prepared with methanol:water 80:20 and analyzed by silica gel column chromatography and reversed phase liquid chromatography (HPLC). The antioxidant activity was evaluated by scavenging the radicals 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) and measuring antimicrobial activity (agar well diffusion method, minimum inhibitory concentration and minimum bactericidal concentrations).

Results: The HPLC analysis of Spondias extracts demonstrated the occurrence of high yield of flavonoids. Found in S. mombin were quercetin (2.36 ± 0.01 mg/g) and ellagic acid (41.56 ± 0.01 mg/g) and in S. tuberosa species rutin (53.38 ± 1.71 mg/g), quercetin (24.46 ± 0.87 mg/g), and ellagic acid (169.76 ± 0.17 mg/g). The antibacterial activity of the extracts against the various bacteria strains varied from 8.8 to 20.1 mm. MIC values from 62.5 to 125 µg/mL were satisfactory when compared with other plant products. Medium DPPH scavenging activity IC₅₀ for Spondias extracts varied from 0.042 to 0.558 mg/mL and for ABTS from 0.089 to 0.465 mg/mL. DPPH scavenging activity for constituent ellagic acid IC₅₀ = 0.042 mg/mL and for quercetin IC₅₀ = 0.081 mg/mL.

Discussion and conclusion: The chemical study of Spondias leaf extracts showed the occurrence of quercetin, rutin and ellagic acid, substances with relevant antioxidant and antimicrobial activities.

Keywords::

• Phenolic compounds
• antibacterial effect
• antioxidant activity
• Spondias sp.

Introduction

The genus Spondias (Anacardiaceae) consists of about 8–12 species distributed across tropical regions in the world (Narain et al., 2004), including the species Spondias mombin L., known as cajazeira, and Spondias tuberosa Arr. Cam., known as umbuzeiro. S. mombin is native to the Amazon region in Peru, Brazil, Venezuela, Bolivia, Colombia, the three Guianas, as well as the southern Mexico, Belize, Costa Rica, and the west Indies (Adedokun et al., 2010) and S. tuberosa is a tropical plant that plays an important role in the Caatinga biome of Northeastern Brazil, since it blooms even in dry period to furnish edible fruits, largely appreciated by the population (Braga, 2001). In folk medicine, the S. mombin leaf tea is used for wound healing, demonstrating anti-inflammatory action (Villegas et al., 1997), against acute diarrhea (Gonçalves et al., 2005), in the treatment of diabetes mellitus (Fred-Jaiyesimi et al., 2009) and anemia (Morais et al., 2005). The leaves and branches of S. mombin contain ellagitannins (geraniin and galoilgeraniin) with antiviral activity against Coxsachie B2 and Herpes simplex type 1 (Corthout et al., 1991, 1992), as well as antimicrobial phenolic acids (Corthout et al., 1994). The cattle appreciate the acid taste of S. tuberosa leaves, which are popularly used as an anti-inflammatory, to combat diarrhea, dysentery and worms (Matos, 1999; Braga, 2001). The wound healing and anti-inflammatory properties of these Spondias species could be correlated with the antimicrobial and radical scavenging activity of their extracts and constituents.

The reactive oxygen species (ROS) are deleterious to the wound healing process due to the harmful effects on cells and tissues. Repair of injured tissues occurs as a sequence of events, which includes inflammation, proliferation, and migration of different cell types. Topical applications of compounds with free-radical-scavenging properties in patients have shown to significantly improve wound healing and protect tissues from oxidative damage (Umachigi et al., 2007). For example, polar extracts of several plants used for wound healing in Oman were examined for antioxidant and antimicrobial properties and the most promising were those which combine both actions (Marwah et al., 2007).

Phenolic compounds such as flavonoids, tannins, and anthocyanins are considered important antioxidants and antimicrobial agents (Nascimento et al., 2000; Elzaawely et al., 2005). In this sense, the objectives of this study were to evaluate the antioxidant and antibacterial activities of extracts from two Spondias species native to Northeastern Brazil to validate their popular use, and analyze the chemical composition to investigate possible active principles.

Material and methods

Reagents

DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS^+• [2,2-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid)], BHT, ellagic acid, quercetin and rutin were purchased from Sigma Chemical Co. Other chemicals such as dimethylsulphoxide (DMSO) and solvents were obtained from Vetec (Brazil). All solvents used for extraction were of analytical grade.

Plant material

Plant leaves were collected in May 2008 from their natural habitats in the state of Ceará (Northeastern Brazil). Voucher specimens were deposited in the Prisco Bezerra Herbarium at the Department of Biology, Federal University of Ceará (UFC), and identified by botanist Ligia Queiroz Matias, with numbers 34,826 for S. mombin and 34,887 for S. tuberosa.

Preparation of extracts

Leaves of S. mombin (3.59 kg) and S. tuberosa (3.23 kg) were macerated in methanol:water (80:20) at room temperature (25–28°C) for 7 days. The solution was filtered through a paper filter and evaporated in a rotary evaporator. The extract was concentrated in a water bath with the temperature held below 60°C. S. tuberosa and S. mombin extract yields were 1.5 and 5.0%, respectively.

Chemical analysis of main secondary metabolite classes

Chemical tests were performed using specific reagents, observing color changes or precipitate formation, following the protocols described by Matos (1997).

Phytochemical analysis of plant extracts

The methanol:water (80:20) extract was subjected to silica gel column chromatography and eluted with hexane, chloroform, and ethyl acetate in mixtures of increasing polarity. Several 10 mL fractions were collected and, after solvent evaporation, the resultant material was compared by silica gel thin layer chromatography (TLC) plates. Similar samples were joined, purified by recrystallization and analyzed by infrared (IR) and nuclear magnetic resonance (NMR). TLC also compared the main purified compounds with standard compounds. IR spectra were recorded on a PerkinElmer FTIR spectrum 100 Spectrometer and the values are expressed in cm⁻¹. NMR spectra were recorded on a Brucker Avance DRX-500 spectrometer in MeOD.

HPLC quantification of main compounds from extracts

General

HPLC analysis was performed on a Shimadzu LC-10AD pump system equipped with a Shimadzu SPD-M10A photodiode array detector with the detection wavelength set at 350 nm. A fingerprint of main compounds contained in the methanol:water (80:20) extract of S. mombin and S. tuberosa was obtained.

Sample preparation

Quantitative analysis of rutin and quercetin in the Spondias species was carried out by HPLC analysis. The concentrated extracts of S. mombin (60 mg) and S. tuberosa (30 mg) were diluted with methanol to 10 mL. The afforded solutions were filtered through a 0.45 μm syringe filter prior to HPLC use.

Chromatographic system

HPLC analysis was performed using a reversed-phase column (Hibar Lichrosopher 100RP18 4.6 mm × 25 cm − particle 5 mm) eluted at a rate of 1.25 mL/min with a solvent mixture I:II (I: aqueous solution of H₃PO₄ (pH 2.8); II: acetonitrile) starting with 20% II in I until 12 min, then installing a gradient to reach 40% II at 17 min, maintaining this concentration until 23 min, following with a gradient to reach 80% II at 25 min, with a detection wavelength set at 350 nm and 20 mL injection. A rutin standard was used to obtain calibration curves. This compound was dissolved in methanol in different concentrations (1.14, 0.57, 0.228, 0.114 and 0.0456 mg/mL). Ellagic acid (standard) is also used to obtain calibration curves and it was dissolved in methanol for obtaining different concentrations (0.46, 0.92 and 1.84 mg/mL). The follow-up extractions and HPLC analysis were accomplished with the same procedure as for rutin. The recovery was determined as follows: recovery (%) = (A − B)/C × 100% where, A is the amount of detections, B is the amount of sample without added standard, C is the added amount of the standard. The relative standard deviations (RSD) of recoveries of the ellagic acid was 2.1 (n = 5; mean = 98). The identification of rutin, ellagic acid and quercetin (Figure 1) in Spondias extracts were evaluated by HPLC-PDA, observing the retention time (Rt) and UV-VIS spectra. The standards were prepared in methanol in a concentration of 0.1 mg/mL.

Figure 1.  Chemical structures of quercetin, rutin and ellagic acid.

Determination of anti-free radical activity of Spondias extracts and constituents

Scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH): The capacity to scavenge the DPPH free radical, standard techniques, was monitored according to a method reported by Yepez et al. (2002). The diluted extract (0.1 mL) was mixed with 3.9 mL of methanol solution containing DPPH radicals (6.5 × 10⁻⁵ mol/L) for 1 h. The reduction of the DPPH radical was measured by continuously monitoring the decrease in absorption at 515 nm. The IC₅₀ (Medium effective concentration) was calculated.

Scavenging activity against 2,2-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid (ABTS^+•): The ABTS is standard technique. The ABTS^+• solution (7 mM, 5 mL) was mixed with 88 µL of potassium persulfate (140 mM). The mixture was shaken and kept in the dark at room temperature for 16 h. Then, 1 mL of this solution was added to 99 mL ethanol. The absorbance is read at 734 nm and should be approximately 0.715. Several solutions of decreasing concentrations of plant extracts were prepared and 3.0 mL of ABTS^+• solution was added to 30 µL of these solutions after 6 min, readings were taken at 734 nm (Re et al., 1999). The IC₅₀ (medium effective concentration) was calculated.

Antimicrobial activity

Microbial strains

The in vitro antimicrobial activity of S. mombin and S. tuberosa extracts was evaluated using isolation strains obtained from Walter Cantidio University Hospital, Ceará, Brazil and supplied by the Laboratory of Microbiology of the UFC: Gram negative bacteria Serratia marcescens, Serratia liquefaciens, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Morganella morganii and Enterobacter cloacae. Cultures of these bacteria were grown in brain heart infusion (BHI) broth at 37°C and maintained in Mueller-Hinton agar (MHA) at 4°C.

Antibacterial screening

Two different methods were employed for determining antibacterial activities: agar-well diffusion method (Celiktas et al., 2007) and minimum inhibitory concentrations (MIC) with protocol established by the CLSI (2009). Also was determined the minimum bactericidal concentrations (MBCs). All tests were performed in triplicate. All extracts were weighed and dissolved in dimethylsulphoxide (125 mg/mL), followed by sterilization using a 0.45 µm membrane filter. Each microorganism was taken in sterile saline at a density adjusted to a 0.5 McFarland turbidity standard [10⁸ colony-forming units (CFU)/mL].

Agar-well diffusion assay

Twenty mL nutrient agar plates were inverted and dried at 37°C for 30 min. An overnight culture of bacteria (0.5 mL) was spread over the surface of the agar plate using a sterile glass spreader. Inoculated plates were inverted and incubated at 37°C for a further 30 min. Wells (6 mm diameter) were made in the center of each agar plate into which 0.2 mL of each extract was added. The agar plates were incubated for 24 h at 37°C and the diameter (in mm) of zones of bacterial inhibition (ID) were recorded using a caliper rule. All tests were performed in triplicate.

Broth dilution assay: determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC)

The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Spondias extract against bacterial strains was measured by broth dilution method using Mueller Hinton Broth. An overnight broth culture of each strain was prepared and the final concentration in each well was adjusted to 10⁸ CFU/mL. The 96-well plates were prepared with 95 µL of Müeller-Hinton (MH), and 5 µL of the inoculum into each well. Hundred µL from Spondias extracts initially prepared at the concentration of 500 µg/mL was added into the first wells. Then, 100 µL from their serial dilutions was transferred into 10 consecutive wells. The last well containing 195 µL of nutrient without compound and 5 µL of the inoculum on each strip was used as a negative control. The final volume in each well was 200 µL. Ciprofloxacin (CIP) at a concentration range of 500–7.8 µg/mL was prepared in a nutrient medium and used as a standard drug. Contents of each well were mixed on a plate shaker for 20 min and then incubated at appropriate temperatures for 24 h. Microbial growth was determined by absorbance at 620 nm using an ELISA instrument (Ozturk & Ercisli, 2006) and confirmed by plating 5 µL samples from clear wells on nutrient agar medium to determine as minimal bactericidal concentration (MBC).

Statistical analysis

All the experiments on the antioxidant effect were calculated as means ± standard deviation (SD). The one way analysis of variance (ANOVA) test was used to determine the statistical differences followed by Tukey’s Multiple Comparison in GraphPad Prism 3.0. The significance level was set at p < 0.05.

Results and discussion

Chemical study of S. mombin and S. tuberosa leaves: S. tuberosa and S. mombin extract yields were 1.5 and 5.0%, respectively. Both extracts contain phenols, hydrolysable tannins, flavones, flavonoids, leucoanthocyanidins and saponins. These results are in accordance with Corthout et al. (1992) who demonstrated the presence of hydrolysable tannins in S. mombin. Satpathy et al. (2011) demonstrated the presence of alkaloids, tannins and saponins in Spondias pinnata K. The presence of flavonoids and tannins in Spondias species led us to investigate their chemical structures by HPLC-PDA, comparing retention time (Rt) and UV-VIS spectra. The identity of constituents was established by overlapping their peaks (retention time) and absorption spectra with those of rutin (1), ellagic acid (2) and quercetin (3) standards. Fingerprint chromatograms of S. tuberosa and S. mombin leaf extracts are shown in Figure 2. Retention times of rutin, ellagic acid and quercetin were 5.11, 6.24 and 23.46 min, respectively. The occurrence of these compounds in S. mombin and S. tuberosa species was not previously described.

Figure 2.  HPLC Chromatograms of the methanol:water extracts of S. tuberosa (A) and S. mombin (B). Compounds 1. rutin, 2. ellagic acid, 3. quercetin.

The regression equation for rutin is Y = 25,150,017.88X + 203,139.00 (R² = 1.000); limit of quantification 0.1 μg/mL; limit of detection 0.04 μg/mL; relative standard deviations (RSD) less than 2%. The regression equation for ellagic acid is: Y = 25,916,407.45X + 309,482.00 (R² = 0.9998); limit of quantification 0.1 μg/mL; limit of detection 0.04 μg/mL; relative standard deviations less than 2%. Each flavonoid peak was quantified using the rutin and ellagic acid linear regression equations. In these Spondias species, rutin, ellagic acid and quercetin were detected in different yields (Table 1). It was found in S. mombin, quercetin (2.36 ± 0.01 mg/g) and ellagic acid (41.56 ± 0.01 mg/g) and in S. tuberosa species rutin (53.38 ± 1.71 mg/g), quercetin (24.46 ± 0.87 mg/g) and ellagic acid (169.76 ± 0.17 mg/g). The differences in antioxidant and antimicrobial action of two species could be related to the concentration of active compounds in each plant.

Table 1.  Content of flavonoids (quercetin and rutin) and ellagic acid in extracts of Spondias species from Northeastern Brazil determined by HPLC.

Compounds	Cajá (S. mombin) (mg/g extract)	Umbu (S. tuberosa) (mg/g extract)
Rutin	–	53.38 ± 1.71ª
Ellagic acid	41.56 ± 0.01ª	169.76 ± 0.17^b
Quercetin	2.36 ± 0.01^b	24.46 ± 0.87^c

ANOVA and Tukey’s Multiple Comparison; superscript letters indicate the statistical significance differences at p < 0.05, comparing data in the same column.

Phenolic acids and O-glycoside flavonols were extracted from the peels of jocote fruits (Spondias purpurea L.,) harvested in Costa Rica and characterized using ultra high-performance liquid chromatography coupled with diode array and electrospray ionization mass spectrometric detection (UHPLC–DAD–ESIMSn). The analytical system allowed the separation of 21 phenolic compounds. In addition to phenolic acids, several O-glycosides of quercetin, kaempferol, kaempferide and rhamnetin were detected (Engels et al., 2011).

Antioxidant activity measured by the DPPH and ABTS^+• radical scavenging system: DPPH is used as a free radical to evaluate antioxidant activity of plant extracts, and the degree of its discoloration is attributed to the hydrogen donating ability of these products, which is indicative of their scavenging potential (Shimada et al., 1992). Table 2 shows the results of antiradical tests, where IC₅₀ is defined as the concentration required to inhibit 50% of DPPH radical present in the solution. Both Spondias extracts exhibited high DPPH scavenging potential with IC₅₀ = 0.417 ± 0.01 mg/mL for S. mombin and IC₅₀ = 0.558 ± 0.008 mg/mL for S. tuberosa. When antioxidant activity was evaluated using the radical ABTS^+• results were statistically equal, S. mombin 0.451 ± 0.029 and S. tuberosa 0.465 ± 0.029. Rufino et al. (2010) comment that it is not always a simple task to choose the most appropriate method to determine antioxidant capacity and the ABTS^+• method is generally indicated for hydrophilic compounds. The DPPH method may be employed routinely with aqueous-organic extracts containing hydrophilic and lipophilic compounds. In the evaluation of antioxidant activities of aqueous, methanol and alkaloid extracts from Mitragyna speciosa leaves, the DPPH IC₅₀ values were 0.213, 0.104 and 0.037 mg/mL, respectively (Parthasarathy et al., 2009). A study performed on other Spondias species indicate the presence of antioxidants but failed to provide information about the profile of phenolic compounds (Hazra et al., 2008). Antioxidant activity of different Spondias pinnata K. extracts, the DPPH scavenging activity expressed as IC₅₀ ranged from 0.73 to 0.59 mg/mL (Satpathy et al., 2011).

Table 2.  Antioxidant capacities measured by DPPH and ABTS free radical scavenging activity expressed as IC₅₀ (mg/mL ± SD) of methanol:water (80:20) extracts and constituents of Spondias species.

Extract/standard	DPPH scavenging EC50	ABTS scavenging EC50
S. mombin	0.417 ± 0.01^a	0.451 ± 0.029^a
S. tuberosa	0.558 ± 0.008^b	0.465 ± 0.029^a
BHT	0.165 ± 0.01^c	0.308 ± 0.01^b
Quercetin	0.081 ± 0.03^d	0.171 ± 0.01^c
Rutin	0.295 ± 0.01^e	0.413 ± 0.02^d
Ellagic acid	0.042 ± 0.05^f	0.089 ± 0.01^e

IC₅₀ = medium effective concentration.

Superscript letters indicate statistical significant differences among data in the rows at p < 0.05.

The values found of DPPH IC₅₀ for Spondias species extracts are considered mild in relation to BHT (DPPH IC₅₀ = 0.165 mg/mL). However the DPPH scavenging activity for constituent ellagic acid with IC₅₀ = 0.042 mg/mL and for quercetin with IC₅₀ = 0.081 mg/mL, show that both constituents of Spondias extracts are better antioxidants than BHT. Therefore the presence of compounds with good antioxidant activity should contribute to overall activity of extracts.

The present life style of individuals causes over production of free radicals and reactive oxygen species. Our body has a natural defense mechanism to cope with this oxidative stress but the problem arises when the free radicals outweigh the defense mechanism, damaging essential biomolecules such as proteins, DNA and lipids. Natural antioxidants play an important role in health care as they provide protection from oxidative stress and associated diseases (Lopez et al., 2007). Phenolic compounds widely distributed in medicinal plants, spices, vegetables, fruits, grains, and seeds are an important group of natural antioxidants with possible beneficial effects on human health. Turkmen et al. (2005) demonstrated that extracts from phenolic rich plants present strong antioxidant activity. Quercetin and its glycoside rutin have shown in vitro antioxidant action (Soares et al., 2005). Over the past years, both quercetin and rutin have gained tremendous interest because of their free radical scavenging activities (Teresita et al., 2001), their protective effects on DNA damage (Undeger et al., 2004), and their physiological effects such as anti-tumor (Ren et al., 2003) and anti-bacteria (Dall’Agnol et al., 2003) potential. Ellagic acid is a known polyphenol antioxidant (Hassoun et al., 1997, 2004; Hayes et al., 2010) found in numerous fruits and vegetables and has a variety of biological activities, including anticancer (Whitley et al., 2003), antiproliferative (Seeram et al., 2005) and antimutagen activities (Loarca-Piña et al., 1998). The presence of these three phenolic compounds in Spondias species extracts guarantees their medicinal properties including antioxidant and antibacterial activities.

The antibacterial activities of S. mombin and S. tuberosa extracts were assayed in vitro by agar well diffusion method against eight Gram negative bacteria. Table 3 summarizes the microbial growth inhibition of Spondias species extracts. According to results, the S. mombin extract was found to be active against three Gram negative bacteria strains and the S. tuberosa extract was found to be active against seven Gram negative bacteria strains.

Table 3.  Antibacterial activities of methanol:water (80:20) extracts of Spondias species.

Microorganisms	S. mombin			S. tuberosa
	ID	MIC	MBC	ID	MIC	MBC
Klebsiella pneumonia	–	–	R	19.1	125	R
Serratia marcescens	11.0	125	R	11.8	62.5	R
Pseudomonas aeruginosa	–	–	R	20.1	125	R
Proteus mirabilis	9.6	125	R	8.8	62.5	R
Morganella morganii	–	–	R	10.2	125	R
Serratia liquefaciens	–	–	R	11.2	62.5	R
Enterobacter cloacae	20.0	125	R	15.0	62.5	R

*Concentration unit: µg/mL; (–) No inhibition or inactive with inhibition zones inferior to 7 mm; ID, diameter of inhibition zone expressed in mm; MIC, minimum inhibitory concentrations (µg/mL); MBC, minimum bactericidal concentrations; R, inhibition at the highest concentration used (500 µg/mL); moderate activity: inhibition zones in the range of 7–13 mm; high activity: inhibition zones greater than 14 mm.

The MIC values were studied for the bacterial strains, being sensitive to the extracts in the agar diffusion method. The standard antibiotic was ciprofloxacin (CIP) which showed MIC values of 7.8 µg/mL. S. mombin extract presented MIC = 125 µg/mL for Serratia marcescens, Proteus mirabilis and Enterobacter cloacae. S. tuberosa extract presented MIC = 62.5 µg/mL against Serratia marcescens, Serratia liquefaciens, Proteus mirabilis and Enterobacter cloacae. Against Klebsiella pneumoniae, Pseudomonas aeruginosa and for Morganella morganii the MIC was 125 µg/mL (Table 3). Meot-Duros et al. (2008) tested polar and apolar fractions of Crithmum maritimum L., Eryngium maritimum L. and Cakile maritima Scop leaves and found MIC values about 100 µg/mL. Antimicrobial activity is relevant in concentrations below 100 µg/mL for extracts and 10 µg/mL for isolated compounds (Ríos & Recio, 2005). Satpathy et al. (2011) showed antimicrobial activity against Gram negative and Gram positive with MIC ranged between 1.8 and 2.9 mg/mL of Spondias pinnata extracts.

According to Natarajan et al. (2005) the MIC method is the most suitable to evaluate antibacterial activity of natural products. Although the found activities of Spondias species were lower when compared to standard antibiotic, the increasing resistance of microorganisms to antibiotics stimulates the search for new drugs. Nikaido and Vaara (1985) point out that higher resistance of Gram negative bacteria to external agents has been earlier reported, and it is attributed to the presence of lipopolysaccharides in their outer membranes, which make them inherently resistant to antibiotics, detergent, and hydrophilic dyes. The antibacterial activity of quercetin and rutin was already demonstrated (Cushnie & Lamb, 2005) and probably contributes to Spondias species extracts antimicrobial action.

Conclusion

The chemical study of leaf extracts from Spondias species shows the occurrence of quercetin, rutin, and ellagic acid. The antioxidant and antimicrobial activities of extracts and constituents were evaluated showing relevant results. The constituents’ activities can be directly correlated to wound healing and anti-inflammatory properties of the plants then this study confirms the ethnopharmacological use of two Spondias species.

Declaration of interest

The authors thank Brazilian governmental agencies CAPES and FUNCAP (Ceará State Research Funding) for financial support.