Abstract: Natural antioxidants have been proven safe and potent for the treatment and prevention of several human diseases. This study aimed to investigate the chemical profiles and antioxidant activities of essential oils extracted from the leaf and stem of Parkial biglobosa (Jacq) Benth. The leaf and stem of the plant were subjected to hydro-distillation (modified Clevenger apparatus) to obtain essential oils analyzed by GC/MS method. The antioxidant activities of the oils were determined by evaluating their scavenging activities against DPPH, ABTS and nitric oxide using different concentrations (0.5, 0.2, 0.05 and 0.025 mg mL-1). Positive controls were vitamin C and rutin. The essential oils extracted from the leaf and stem contained 34 and 15 constituents accounting for 84.9 and 95.9% of the total oil, respectively. Of the 34 eluted, 33 compounds were identified in the leaf essential oil. The major components were limonene (16.0%), hexadecanoic acid (12.5%) and farnesene (10.2%). Thirteen compounds were identified in the stem oil, the major compounds were caryophyllene oxide (16.6%), β-caryophyllene alcohol (14.9%), terpinene-4-ol (12.1%) and β-caryophyllene (8.1%). The three antioxidant assays were concentration dependent with varying antioxidant potentials. The antioxidant activity of the stem and stem oils were similar to that of the standard drugs used. The present findings suggest that the essential oils obtained from the leaf and stem of P. biglobosa posses strong antioxidant potential and can be used to produce natural antioxidants as well as natural food preservatives.
INTRODUCTION
Natural antioxidant agents possess the ability to reduce free radicals generated in the body system. The generation of free radicals such as O2•-OH•, HO2• RO•, ROO•, NO• and LOO• beyond the antioxidant capacity of a biological system gives rise to oxidative stress (Murray et al., 2004). These radicals have been implicated in the pathogenesis of a variety of human diseases such as diabetes, hypertension, inflammation, cancer, AIDS, etc. (Halliwell and Gutteridge, 1989). Therefore, antioxidants have become a major area of scientific research (Mimica-Dukic et al., 2010). Many essential oils have been investigated for their free radical scavenging activities as antioxidants (Emami et al., 2010). These radical quenching activities make certain volatile compounds in essential oils profiles strong antioxidant products. Volatile compounds such as limonene of lemon essential oil (Yang et al., 2010) and β-caryophyllene of Marrubium pergrinum essential oil are known to exhibit strong antioxidant activity (Kaurinovic et al., 2010). Other cyclic monoterpene and sesquiterpene hydrocarbons with double bonds have antioxidant capacity comparable to the activity of phenols and α-tocopherol (Misharina and Samusenko, 2008). The fermented seed of Parkia biglobosa is used in the production of a local condiment in Nigeria (Odetola et al., 2006). Its stem bark, leaves and seeds are used for the treatment of many diseases including diabetes, leprosy, hypertension, diarrhea, toothaches pneumonia, ulcer, bronchitis, violent stomachaches, conjunctivitis and severe cough (Agunu et al., 2005; Alabi et al., 2005). Numerous authors have investigated nutrition values and biological activity of extracts from P. biglobosa. The ethanol extracts of both leaves and stem bark of the plant have been documented to possess strong antioxidant activities due to presence of bioactive compounds (Millogo-Kone et al., 2006). However, there is a dearth of information on the antioxidant activity of the essential oils and its components despite, the fact that essential oils have been reported to penetrate tissue 100 times faster than water and 10,000 times faster than salts (Nweze and Okafor, 2010). Essential oils have earlier been considered as natural antioxidant and proposed as potential substitute for synthetic antioxidants due to the volatile and lipophilic nature of the oils (Huang et al., 2005). Therefore, this study was conducted to investigate the chemical profiles and the antioxidant activities of the essential oils of P. biglobosa hoping to give credence to plant medicinal uses in Nigeria.
MATERIALS AND METHODS
Chemicals: Potassium persulfate (PPS), 2, 2-Dipphenyl-1-picrylhydrazyl (DPPH) and 2, 21-Azinobis-(3- ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma-Aldrich (St Louis, USA). Methanol was purchased from Fluka Chemicals (Buchs, Switzerland). All other chemicals used were analytical grade.
Plant material: The leaf and stem were gotten from their natural habitat, Ogbomosho in Oyo state, southwest of Nigeria. The plant was authenticated by Mr.T. K. Odewo of the Botany Department, University of Lagos and the plant voucher number (LUH2007) was deposited.
Essential oil isolation: The leaves and stem were air dried for 5 to 8 days. They were pulverized and each material was hydro distilled for 3 h, using a modified Clevenger type apparatus (US Pharmacopeia, 2007). The essential oils were kept in vials at 4°C until used. The yield of the oil was calculated per gram of the plant material.
Antioxidant assays: The antioxidant activities were determined by DPPH radical-scavenging test, ABTS free radical decolorization assay and nitric oxide radical scavenging activity.
DPPH assay: The DPPH test of the essential oil was carried out as described by Bruits and Bucar (2000). A solution of 0.135 Mm DPPH in methanol was prepared and 1.0 mL of this solution was mixed with 1.0 mL of the essential oil prepared in methanol containing 0.025 mL-0.5 mg of the oils and standard drugs (vitamin C and rutin). The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Methanol was used as blank and all measurements were performed in triplicates. The ability of the essential oil to scavenge DPPH radical was calculated as inhibition by the following equation:
Abscontrol is the absorbance of the DPPH radical+methanol; Abssample is the absorbance of DPPH radical+essential oil or standard.
ABTS assay: In the ABTS free radical decolorization assay, the method of Nantitanon et al., (2007) was adopted with minor changes (ABTS stock solution diluted in methanol).The pre-formed radical of ABTS was generated (Re et al., 1999) by oxidation of ABTS solution (7 mM) with 2.4 mM Potassium persulfate solution in equal amount. The mixture was allowed to react for 12 h in the dark at room temperature. 1.0 mL of the resulting solution was diluted in 60 mL of methanol to obtain an absorbance of 0.706±0.001 at 734 nm using spectrophotometer. 1.0 mL of the ABTS radical cation solution was added to series of essential oil solutions and vitamin C and rutin of different concentrations (0.025-0.5 mg mL-1), prepared by diluting with methanol. Methanol was used as blank. The absorbance, after 7 min was measured spectrophoto-metrically at 734 nm. All measurements were carried out in triplicates. The percentage inhibitions of ABTS radical by the oils were calculated using the equation as described in the DPPH assay.
Nitric oxide radical: Nitric oxide assay was carried out as described by Sreejayan and Rao (1997) was adopted. In brief, nitric oxide radicals were generated from sodium nitroprusside solution at physiological pH. Sodium nitro-prusside (1.0 mL of 10 mM) was mixed with 1.0 mL of oils in different concentrations 0.025-0.5 mg mL-1 in phosphate buffer (pH 7.4). The mixture was incubated at 25°C for 150 min. To 1.0 mL of the incubated solution, 1.0 mL of Griess’ reagent (1% sulphanilamide, 2% o-phosphoric acid and o.1% napthyl ethylene diamine dihydrochloride) was added. Absorbance was read at 546 nm and percentage inhibition of nitric oxide radical by the oil was calculated using the equation as described in DPPH assay. All measurements were run in triplicates and mean values were calculated.
Gas chromatography/Mass spectrometry (GC/MS): The GC/MS analyses of the oils were conducted on a Hewlett- Packed HP 5973 interfaced with an HP 6890 mass spectrophotometer. The following column and temperature conditions were used: initial temperature, 70°C, maximum temperature 325°C, equilibration time 3.00 min, ramp 4°C min-1, final temperature 240°C; inlet: splitless, initial temperature 220°C, pressure 8.27 psi, purge flow 30 mL min-1, purge time 0.20 min, helium gas flow rate was at 1 mL; column: capillary, 30x0.25 mm, film thickness 0.25 μm, initial flow 0.7 mL min-1, average velocity 32 cm sec-1; MS: The ion source was set at 240°C and the electron ionization at 70 eV. The scanning range was 50 to 500 amu.
Identification of compounds: The essential oil compounds were identified by matching the MS data of the compounds with that of authentic standard held in the computer (Wiley 275, New York) and by comparison of the calculated retention indices relative to C8-C22 n-alkanes injected under the same condition as the samples. The percentage composition was calculated from the sum of the peak areas of the total oil composition (Adlard and Alan, 2001).
Statistics: Data were calculated as Mean±SD. Pearson’s correction analysis (SPSS15.0 for windows, SPSS Inc) was used to test for the significance of the relationship between the concentration and percentage inhibition.
RESULTS AND DISCUSSION
Air-dried leaf and stem of P. biglobosa gave by hydro-distillation, 0.35 and 0.27% essential oils, respectively. The compounds identified from the essential oils of P. biglobosa are listed in Table 1.
| Table 1: | Chemical profile of essential oils extracted from the leaf, stem and bark of P. biglobosa |
| aCompounds listed in order of elution from HB-5 column and identified using retention index and mass spectra, RIb: Retention., indices relative to C8 - C22 n-alkanes on HB-5 column, Ui: unidentified in the sample | |
The GC/MS analysis indicated the presence of 34 compounds in the leaf oil out of which 33 were identified. Fourteen of these are monoterpeniods, (44.5% of the leaf oil), fifteen of the leaf oil compounds are sesquiterpenoids (20.7%), while the non-terpenoids components are dodecanoic acid, hexadecanol, hexadecanoic acid, tetradecanoic acid and tetracosane, representing 19.7% of the total oil composition. The major compounds of the leaf oil are limonene (16%), β-fenchyl alcohol (9.2%), β-pinene (5.7%) and linalool (4.4%), all monoterpenoids. Other notable components of the leaf oil are camphene (1.7%), α-pinene (1.6%), terpinene- 4-ol (1.4%), terpinolene (0.7%) and eugenol (0.4%). Farnesene (10.2%) was the prominent sesquiterpene in the leaf oil. Of the four unidentified compounds in the leaf oil one is sesquiterpenoid (0.8%), while others are monoterpenoids (3.3%). The stem essential oil consists of 5 monoterpenoids and 10 sesquiterpenoids compounds. The major monoterpenoids identified are terpinene-4-ol (10.8%), fenchyl alcohol (6.8%), longifolene (6.1%) and myrtenol (6.0%), while caryophyllene oxide (16.6), α-caryophyllene alcohol (14.9%) and β-caryophyllene (8.1%), were the dominant sesquiterpenoids in the stem oil.
The composition of the monoterpenoids identified in the leaf essential oil of P. biglobosa in this study was higher than that reported from Australia by Amer and Mehlhorn (2006). This is the first time to report the chemical profile of essential oil extracted from the stem as well as the antioxidant property of the plant essential oils. Previous in vitro and in vivo studies have reported limonene (Yang et al., 2010), terpinolene (Grassmann et al., 2005), linalool (Peana et al., 2006) and β-caryophyllene (Kaurinovic et al., 2010) identified in these oils to posses antioxidant property. The results obtained from ABTS and DPPH assay indicated strong antioxidant activity in the leaf and stem oils of P. biglobosa. The percentage inhibitions of these radicals were concentration dependent (Fig. 1, 2). The IC50 values of the oils for the DPPH radicals oils were 4.2±0.01 and 5.3±0.03 mg mL-1 while that of ABTS free radicals were 10.8±0.01 and 24.6±0.01 mg mL-1, respectively.
| Fig. 1: | Antioxidant effect of the essential oils extracted from P. biglobosa on DPPH radicals |
| Fig. 2: | Antioxidant effect of the essential oils extracted from P. biglobosa on ABTS free radicals |
The free radical quenching potency of P. biglobosa stem and leaf oils were more pronounced in the DPPH assay, compared with IC50 values of vitamin C (1.5±0.01) and (rutin 0.5±0.01 mg mL-1) as shown in Table 2.
The variation in the chemical profiles of the oils in both assays could have accounted for their varied antioxidant activities (Sacchetti et al., 2005). The chemical profile of the leaf oil of P. biglobosa comprises mainly of monoterpenes such as limonene (16) which has been reported as a strong antioxidant agent (Yang et al., 2010). Alpha-Zingiberene (4.5) and terpine-4-ol (1.8) in the leaf oil were aslo earlier documented as antioxidant compounds in ginger essential oil (Ahmed et al., 2008). In contrast, the stem oil contained sesquiterpenoids such as caryophyllene oxide (16.6%), caryophyllene alcohol (14.9%), β-caryophyllene (8.1%) as major compounds. Kaurinovic et al., (2010) reported that caryophyllene oxide and β-caryophyllene of M. pergrinum essential oil demonstrated powerful scavenging activity against DPPH radical. Therefore, the combination of camphene, β-caryophyllene, caryophyllene oxide and other antioxidant terpenoids in the stem oil of P. biglobosa could justify the reason why the oil demonstrated stronger antioxidant activity than the leaf oil in DPPH and nitric oxide assays (Table 2). These results agree with the findings of previous essential oil researchers, that individual terpene has antioxidant activity and that the activity of cyclic monoterpene hydrocarbon with double bonds is comparable to the activity of phenols and α-tocopherol (Yanishlieva et al., 1999; Foti and Ingold, 2003). The antioxidant potency of the oils might have been improved by other terpenoid and phenolic compounds identified even in small amount e.g., linalool (4.4%) and eugenol (0.4%), suggesting possible synergistic interaction between the constituents (Farag et al.,1989).
The stem oil of P. biglobosa strongly inhibited the nitric oxide radicals in a concentration dependent manner with IC50 value of 3.8±0.02 mg mL-1. The stem oil nitric oxide scavenging capacity was superior to the leaf oil, rutin and compares well with that of vitamin C (Fig. 3).
| Fig. 3: | Antioxidant effect of the essential oils extracted from P. biglobosa on nitric oxides radicals |
| Table 2: | Antioxidant activities of essential oils of P. biglobosa. IC50 (mg mL-1) |
Farag et al. (1989) and Misharina and Samusenko (2008) investigations have revealed that the bioactivity of essential oils is dependent not only on the major compounds but also on the chemical nature of the entire oil. Several studies have demonstrated that chronic expression of nitric oxide is associated with various inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis, ulcerative colitis (Hou et al., 1999; Hyun et al., 2004), as well as pathogenic processes leading to vascular and myocardial dysfunctions (Dessy and Feron, 2004). The results of the stem oil scavenging power indicate the plant potency of preventing inflammatory conditions.
CONCLUSION
The present study shows that the volatile oils extracted from the P. biglobosa leaf and stem have strong antioxidant potential and can be used to produce natural antioxidants as well as natural food preservatives. The presence of terpenoids and phenolic compounds identified in the essential oils may validate the medicinal use of this plant.
ACKNOWLEDGMENTS
This research was supported by University of Lagos STEP B World Bank Project in Nigeria and Govan Mbeki Research and Development Centre, University of Fort Hare, S. Africa.