Isolation and characterization of antioxidant and antimicrobial compounds from Anacardium occidentale L. (Anacardiaceae) leaf extract

2.1 General

All analyses of samples (fractions and isolated compounds) using thin layer chromatography (TLC) were performed at room temperature using pre-coated plates (MERCK, silica gel 60 F₂₅₄ 0.2 mm). Detection of spots was by ultraviolet lamp (254 and 366 nm). Silica gel and Sephadex LH-20 were used as stationary phases for column chromatography to fractionate active fractions. Nuclear magnetic resonance (NMR) spectra data were obtained from a Brucker Spectrometer. Chemical shifts are expressed in parts per million (ppm). Mass spectrum (TOF MS ES) was obtained from a Thermofinnigan Trace GC coupled to a Polaris Q Mass Spectrometer.

2.2 Plant material

The leaves of A. occidentale were collected (November 2010) at Obafemi Awolowo University Ile-Ife, Nigeria. Mr. Ibenesibor of the Herbarium Section, Department of Botany, OAU, Ile-Ife, authenticated the plant material collected. The voucher specimen (IFE Herbarium: 16834) was deposited at the University Herbarium. The collected leaves were air dried at room temperature for two weeks and ground to powder.

2.3 Qualitative screening for the antioxidant compounds

About 10 μL of each of the four fractions (1 mg/mL) obtained were spotted on silica gel TLC plate, the chromatogram was developed in DCM/MeOH (8.5:1.5) and the plates were sprayed with 0.2% 1,1-diphenyl-2-picrylhydrazyl (DPPH•) in MeOH for detection of antioxidant compounds in each of the fractions. From the chromatogram (results not shown), it was found that ethyl acetate and butanol fractions contained more antioxidant compounds than n-hexane and dichloromethane fractions. Ethyl acetate fraction which showed the highest number of antioxidant spots was selected for fractionation.

2.4 Isolation of antioxidant compounds from ethyl acetate fraction of A. occidentale

Accelerated Column Chromatography (AGC) using silica gel (230–400 mesh) as the stationary phase was used to fractionate ethyl acetate fraction (27.0 g). The column was eluted with n-hexane 100% followed by an increasing gradient of ethyl acetate starting with 10% up to 100% ethyl acetate. This was followed with an increasing gradient of methanol from 10% in ethyl acetate up to 100% methanol. Test tube fractions (15 mL each) collected were analysed on TLC plates using Hexane/EtOAc (3:7). This afforded five fractions (A₁–E₁).

From the chromatogram, fractions B₁ and C₁ had antioxidant compounds. Fractionation of B₁ (9.8 g) on a silica gel column using hexane (100%) was followed by an increased gradient of ethyl acetate up to 100%. This was followed in turn by an increasing gradient of methanol up to 20%. Test tube fractions collected were analysed on TLC plate using Hexane/EtOAC (1:4) solvent system. Three fractions, A₂–C₂ were obtained. Open column chromatography using Sephadex LH-20 as stationary phase was used to fractionate B₂ (5.8 g). The column was eluted using Hexane/EtOAc (1:1) followed by an increasing gradient of EtAOc up to 100%. Analysis of the fractions collected on TLC plate using 100% EtOAc yielded three fractions A₃–C₃. B₃ was further purified on silica gel column using hexane (100%) followed by an increasing gradient of ethyl acetate up to 100%. This was followed in turn by an increasing gradient of methanol up to 10%. Analysis of the test tubes fractions collected on silica gel TLC plate using EtOAc:MeOH (9:1) showed that tubes 3–18 had single spots, this afforded compound 1 (19 mg).

Purification of C₂ (1.9 g) was achieved by its fractionation on silica gel column eluted with dichloromethane followed by an increasing gradient of methanol up to 25%. The fractions collected in the test tubes were analysed on TLC plate using DCM/MeOH (4:1). Test tube 14–17 showed single spots, this yielded a mixture of compounds 2 and 3.

2.4.1

2.4.1 Structure elucidation of isolated compounds

Isolated compounds were characterized using spectroscopic techniques: mass spectrometry (ESI-TOF-MS), ¹H (400 MHz) and ¹³C NMR (100 MHz), and DEPT together with 2D experiments (gCOSY, gHSQC and gHMBC).

2.4.1.1

2.4.1.1 Compound 1

Compound 1 (19 mg) was obtained as yellow powder. Its mass spectrum (TOF-MS-ES) showed the pseudo-molecular ion peak as the base peak at m/z = 537.0826, [M−H]⁻, consistent with the molecular formula C₃₀H₁₈O_10. The ¹H NMR (MeOD) of 1 showed signals between δ 7.91 and 6.38 mainly aromatic protons. The spectrum showed two separate doublets (ortho coupled related protons) at δ 7.90 (2H, d, J = 8.0 Hz, H-2′, H-6′) and 6.96 (2H, d, J = 8.0 Hz, H-3′, H-5′) for 1, 4-disubstituted phenyl ring (ring B) representing an A₂B₂ ring system, along with two separate singlet signals at 6.58 (1H, s, H-3′′) and 6.66 (1H, s, H-3). Another ortho coupled two separate doublets appeared at δ 7.54 (2H, d, J = 8.0 Hz, H-2′′′, H-6′′′) and 6.75 (2H, d, J = 8.0 Hz, H-3′′′, H-5′′′) for the second ring B. Two separate singlets also appeared at 6.70 (1H, s, H-8) and 6.38 (1H, s, H-6′′). The ¹³C NMR and DEPT spectra of this compound revealed the presence of 26 carbons. Eight of these carbons were classified as protonated from DEPT spectrum, out of which four were assigned to two units of 1,4-disubstituted phenyl and another two were characteristic of a flavone (C-3) signal. The remaining two protonated carbons were assigned to C-8 and C-6′′. The positions of attachment of the two flavone units were established by 2D NMR, Compound 1 was determined to be agathisflavone (Fig. 1). The ¹³C NMR (MeOD) – 166.4 (C-2), 104.1 (C-3), 184.4 (C-4), 161.5 (C-5), 104.9 (C-6), 164.5 (C-7), 94.8 (C-8), 159.2 (C-9), 105.4 (C-10), 123.4 (C-1′), 129.6 (C-2′), 117.0 (C-3′), 162.9 (C-4′), 117.0 (C-5′), 129.6 (C-6′), 166.2 (C-2′′), 103.6 (C-3′′), 184.1 (C-4′′), 162.6 (C-5′′), 100.0 (C-6′′), 164.2 (C-7′′), 100.4 (C-8′′), 157.1 (C-9′′), 105.7 (C-10′′), 123.5 (C-1′′′), 129.4 (C-2′′′), 117.2 (C-3′′′), 162.7 (C-4′′′), 117.2 (C-5′′′), 129.4 (C-6′′′). The spectroscopic data are in agreement with the literature (Svenningsen et al., 2006).

Close

Figure 1

Antioxidant constituents of A. occidentale leaf extract.

Export to PPT

2.4.1.2

2.4.1.2 Mixture of compounds 2 and 3

The mixture of compounds 2 and 3 (80 mg) was obtained as brown powder. The mixture components were identified by NMR spectra (mainly 2D) peak intensities. The higher intensity signals were allocated to compound 2 while the lower intensity signals were assigned to compound 3. The ratio of the two components in the mixture was also determined by comparison of the signal intensities as 2:1 for compounds 2 and 3 in the mixture. The ¹H NMR of 2 in the mixture showed aromatic signals indicating the presence of 1,3,4-trisubstituted ring B representing an ABX ring system. The spectrum peaks appeared at δ 7.62 (1H, d, J = 2.1 Hz, H-2′), 7.59 (1H, dd, J = 2.2 and 8.4 Hz, H-6′) and 6.87 (1H, d, J = 8.4 Hz, H-5′). Two separate meta coupled doublets appeared at δ 6.38 (1H, d, J = 2.1 Hz, H-8) and 6.20 (1H, d, J = 2.1 Hz, H-6) for ring A. The ¹H NMR (MeOD) of 3 represented by lower intensity signals showed a meta related doublet at δ 7.34 (1H, d, J = 2.1 Hz, H-2′), ortho and meta related doublet of doublet at δ 7.31 (1H, dd, J = 2.1 and 8.3 Hz, H-6′) and ortho related doublet at δ 6.92 (1H, d, J = 8.3 Hz, H-5′) for ring B indicating second ABX ring unit. Another two separate meta related doublets also appeared at δ 6.36 (1H, d, J = 2.1 Hz, H-8) and 6.20 (1H, d, J = 2.1 Hz, H-6) for the ring A of the second flavonoid ring. The ¹³C NMR and DEPT spectra of these compounds revealed the presence of two quercetin derivatives. There was the presence of sugar signals indicating rhamnosyl and glucosyl moieties. Anomeric proton of rhamnosyl appeared at δ 5.36 (1H, d, J = 1.56 Hz) while that of glucosyl appeared at δ 5.17 (1H, d, J = 7.2 Hz). In the ¹³C NMR (100 MHz, MeOD) spectrum, there were thirty carbon signals for the two flavonoid units in addition to the sugar signals. The anomeric carbon of the rhamnosyl appeared at δ 103.4 and C-6 at δ 17.7. Glucosyl anomeric carbon appeared at δ 104.4 and C-6 at δ 67.3. The 2D NMR (HMBC) of this compound mixture revealed that the anomeric of the rhamnosyl attached to the C-6 of the glucosyl making the sugar a disaccharide (rutinosyl). However, the HMBC spectrum further showed that there are two points of attachment for rhamnosyl indicating the presence of two units of rhamnosyl in the mixture. This is indicated by the intensities of the rhamnosyl signals in the ¹³C NMR. The point of attachment of the rutinosyl correlated to C-3 of aglycone of compound 2 at δ 135.5 while the second point of attachment of the second unit of rhamnosyl appeared at C-3 of compound 3 at δ 136.3.

Compound 2: ¹³C NMR (MeOD): 159.0 (C-2), 135.5 (C-3), 179.5 (C-4), 163.1 (C-5), 100.0 (C-6), 166.1 (C-7), 94.8 (C-8), 158.5 (C-9), 105.7 (C-10), 123.4 (C-1′), 117.3 (C-2′), 146.1 (C-3′), 150.0 (C-4′), 116.1 (C-5′), 123.0 (C-6′), 104.4(C-1′′), 73.4 (C-2′′), 77.6 (C-3′′), 72.1 (C-4′′), 75.3 (C-5′′), 67.3 (C-6′′), 103.4 (C-1′′′), 72.0 (C-2′′′), 72.1 (C-3′′′), 72.3 (C-4′′′), 71.1 (C-5′′′), 17.7 (C-6′′′).

Compound 3: ¹³C NMR (MeOD): 159.4 (C-2), 135.3 (C-3), 179.7 (C-4), 163.2 (C-5), 99.9 (C-6), 165.9 (C-7), 94.8 (C-8), 158.6 (C-9), 106.0 (C-10), 123.1 (C-1′), 117.1 (C-2′), 146.4 (C-3′), 149.8 (C-4′), 116.5 (C-5′), 123.1 (C-6′), 103.4 (C-1′′), 72.0 (C-2′′), 72.1 (C-3′′), 72.3 (C-4′′), 71.1 (C-5′′), 17.7 (C-6′′). The spectra data are in good agreement with the literature (Aderogba et al., 2006 and Aderogba et al., 2012) for compounds 2 and 3 respectively.

2.5 Antioxidant assays

2.6 Antimicrobial assay

3.1 Structure elucidation

A bioactivity directed fractionation was used to target the antioxidative constituents in the EtOAc fraction of the 80% MeOH extract of leaves of A. occidentale. Repeated column chromatography and further purification by Sephadex LH-20 yielded a bioflavonoid, agathisflavone (1), and a mixture of quercetin 3-O-rutinoside (2) and quercetin 3-O-rhamnoside (3), (ratio 2:1). The isolated compounds were characterized using spectroscopic techniques: NMR (1D and 2D) and mass spectrometry (ESI-TOF-MS).

3.2 Antioxidant activities

Quantitative antioxidant activities of the crude extract, solvent fractions and isolated compounds were determined using three complementary antioxidant assays DPPH-radical scavenging, Total Antioxidant Capacity (TAC) and Ferric Reducing Antioxidant Power (FRAP) assays. The results of assays are shown in Tables 1–3. Of all the extracts, ethyl acetate fraction was the most active in the DPPH• assay (IC₅₀ = 5.66 ± 0.16 μg/mL) and the mixture of quercetin 3-O-rutinoside (2) and quercetin 3-O-rhamnoside (3), (ratio 2:1) elicited the highest activity (IC₅₀ = 0.966 ± 0.01 μg/mL) amongst the test samples including the two standards (ascorbic acid and quercetin) used in the assay, (Table 1). Similarly, in the TAC and FRAP assays the mixture of compounds (2) and (3) ratio (2:1), exhibited a higher activity than the other test samples with values of 5332.00 ± 3.38 mg AAE/g, and 8562.00 ± 5.43 mg QE/g in the TAC assay and (15136.00 ± 7.14 mg AAE/g, and 199530.00 ± 94.12 mg QE/g) in the FRAP assay respectively(Tables 2 and 3).

The antioxidant activity reported in this study for the isolated compounds in the DPPH assay is in agreement with similar studies undertaken on the compounds. For instance Bansala et al. (2011) reported quercetin 3-O-rutinoside from Pilea microphylla with an IC₅₀ value 20.40 ± 0.3 μmol/L. In another study, Bonacorsi et al. (2011) reported quercetin 3-O-beta-d-galactopyranoside from Anacardium humile with an EC₅₀ value 9.9 μg/mL. Recently, Li et al. (2014) reported amentoflavone with IC₅₀ value 232.55 ± 45.22 μg/mL. In this study however, the difference in activity recorded for agathisflavone in the DPPH assay (IC₅₀ value 336.37 ± 32.59 μg/mL) compared to amentoflavone could be ascribed to differences in substitution pattern of the two biflavonoids. In considering the activity given by the mixture of compounds 2 and 3 in the DPPH assay the potent activity (IC₅₀ value 0.96 ± 0.01 μg/mL) observed can be adduced to the additive combination of the two compounds.

3.3 Antimicrobial activities

The compounds were screened for possible antibacterial and antifungal activities using two fungi strains as well as three Gram-positive and four Gram-negative bacteria. The Gram-positive bacteria were B. subtilis, NCIB 3610; S. aureus ATCC 29213, and Cl. sporogens NCIB 532. The Gram-negative bacteria were E. coli ATCC 25922, P. aeruginosa ATCC 27853, P. mirabilis (clinical strain) and K. pneumonia (clinical strain), while C. albicans NCYC 6 and C. pseudotropicalis (clinical strain) were the fungi. The standard antimicrobial agents used were acriflavine and streptomycin.

The plant investigated is used in ethnomedicine to treat a wide variety of conditions including infectious diseases. The crude extract of A. occidentale has little activity against the selected bacteria and no activity against the fungi at the concentrations tested. The fractions of the extract showed comparatively better activities against gram-negative bacteria than the gram positive ones with the broadest spectrum of activities demonstrated for the ethyl acetate fraction (Table 4). No inhibitions were obtained against the fungal strains investigated.

The isolated compounds from the plant were further screened against these bacteria strains to determine their contribution to the antimicrobial activities. The results obtained indicated that the identified compounds possess essentially inhibitory rather than cidal activities against these organisms. The mixture of compounds 2 and 3 (ratio 2:1), showed higher activity than compound 1 with the former having MIC as low as 0.25 mg/mL against P. aeruginosa and Cl. sporogens, 0.5 mg/mL against S. aureus and 1.0 mg/mL against E. coli, K. pneumonia and P. sporogens. The MIC of compound 1 was observed to be 1.0 mg/mL against most of the bacteria tested except Cl. sporogens where an MIC of 0.5 mg/mL was obtained. The MBC of the compounds was also greater than 2.0 mg/mL in all cases (Table 5).

Compound 1 is agathisflavone which is a biflavonoid. A number of biological activities have been reported for this group of flavonoid, for instance Cushnie and Lamb (2005) and Pegnyemb et al. (2005) reported antibacterial activity, while Njock et al. (2012) reported anti-inflammatory activity. For agathisflavone (1), activities previously reported are antineoplastic, hepatoprotective and antiviral properties (Konan et al., 2012; Njock et al., 2012). In this study, it is demonstrated that this compound also possesses inhibitory antibacterial activities against a wide spectrum of organisms.

Quercetin and its glycosides have also been reported to demonstrate antiviral, antibacterial, anti-inflammatory, anticancer and other biological activities (Yu et al., 2007; Davis et al., 2009). A mixture of quercetin 3-O-rutinoside (2) and quercetin 3-O-rhamnoside (3) isolated in this study are glycosides of quercetin with rutinosyl and rhamnosyl respectively. Quercetin 3-O-rutinoside (2) has been reported to contribute to the antibacterial and antioxidant properties of some plant species (van der Watt and Pretorius, 2001; Ibtissem et al., 2012). It can therefore be inferred that the antibacterial activities reported for the mixture of compounds 2 and compound 3 are mainly due to the synergistic effects since the two compounds have the same flavonoid nucleus.

The good activities demonstrated by the mixture of 2 and 3 against P. aeruginosa, a pathogen characterized by a low intrinsic susceptibility to most antimicrobial agents and Cl. sporogens, a sporulating organism, also with intrinsically high resistance properties is highly instructive. It is an indication that this mixture could be utilized as effective agents against pseudomonal and clostridial infections that are often reported and known to be highly recalcitrant to therapy. The specificity in activities may be employed maximally in targeted therapy. The ethyl acetate fraction of the plant extract and identified chemical isolates may therefore be a readily available source of cheap and potent antimicrobial agents to be used in the therapy of infections caused by these often multi resistant organisms.

It has been shown also that the degree of antimicrobial activities of flavonoids depends largely on the freedom of the attached phenolic groups (Alcaraz et al., 2000). Those with free phenolic groups have been reported to demonstrate better antimicrobial activity than their substituted counterparts (Alcaraz et al., 2000; Aderogba et al., 2011). This observation is also demonstrated by the results of this study as slightly higher antibacterial activities are demonstrated for mixture of 2 and 3 compared with compound 1. Further to these, the lack of inhibitory effects by the extracts and fractions of this plant against the fungal strains is not out of place because these compounds have not been reported to express significant antifungal activities.

This study has established that the extracts and isolated compounds have strong antioxidant and moderate antimicrobial activities and could be effective in the management of oxidative stress and infectious related diseases. The findings justified the use of this plant in folk medicine.

In conclusion, the use of A. occidentale L. in the management of oxidative stress and infectious related diseases prompted us to investigate the antioxidant and antimicrobial constituents of this plant extracts. Based on the qualitative antioxidant screening using DPPH TLC assay, all the four solvent fractions (hexane, dichloromethane, ethyl acetate and butanol) contained antioxidant compounds. However, ethyl acetate fraction contained the highest number of antioxidant compounds and showed the broadest antibacterial spectrum of activities. A DPPH directed fractionation of the ethyl acetate fraction afforded agathisflavone (1) and a mixture of quercetin 3-O-rutinoside (2) and quercetin 3-O-rhamnoside (3). The plant extracts and identified constituents demonstrated varying degree of activities in the three antioxidant assays. The compounds also exhibited a broad spectrum of antimicrobial activities. These findings provided a rationale for the ethnomedicinal use of the plant in traditional medicine.