Antioxidant assessment of characterised essential oils from Calophyllum inophyllum Linn using 2 , 2-diphenyl-1-picrylhydrazyl and hydrogen peroxide methods

Recently, researches on the antioxidant evaluation of volatile constituents from medicinal plants have increased geometrically as a result of gross increase in health disorders triggered by oxidative imbalance. This imbalance is caused by reactive oxygen species (ROS), which is because of the inability of antioxidants in the body to scavenge the effects of free radicals generated in the human system. Excess amount of ROS is deleterious because they can initiate biomolecular oxidative chain reactions (Bhaskara et al. 2015; Rattan 2006). When reactive radicals are generated in the body, the process disrupts all levels of cell function, resulting in oxidative stress (OS). Oxidative stress is associated with increased production of oxidising species or a significant decrease in the effectiveness of antioxidant defences. It can result in numerous diseases and disorders, such as ageing, cancers, rheumatoid arthritis and cardiovascular diseases (Saiket et al. 2010). Antioxidants are molecules that can safely react with free radicals and terminate the chain reaction before vital molecules are damaged (Ajiboye, Moronkola & Adesomoju 2017). These free radicals and ROS may oxidise nucleic acids, proteins, lipids or deoxyribonucleic acid (DNA) and can trigger several degenerative Background: Oxidative stress is a multifactorial global health disorder that disrupts all levels of cell function. Therefore, therapeutic intervention using reliable, affordable and non-toxic natural sources is crucial.


Introduction
Recently, researches on the antioxidant evaluation of volatile constituents from medicinal plants have increased geometrically as a result of gross increase in health disorders triggered by oxidative imbalance. This imbalance is caused by reactive oxygen species (ROS), which is because of the inability of antioxidants in the body to scavenge the effects of free radicals generated in the human system. Excess amount of ROS is deleterious because they can initiate biomolecular oxidative chain reactions (Bhaskara et al. 2015;Rattan 2006). When reactive radicals are generated in the body, the process disrupts all levels of cell function, resulting in oxidative stress (OS). Oxidative stress is associated with increased production of oxidising species or a significant decrease in the effectiveness of antioxidant defences. It can result in numerous diseases and disorders, such as ageing, cancers, rheumatoid arthritis and cardiovascular diseases (Saiket et al. 2010). Antioxidants are molecules that can safely react with free radicals and terminate the chain reaction before vital molecules are damaged (Ajiboye, Moronkola & Adesomoju 2017). These free radicals and ROS may oxidise nucleic acids, proteins, lipids or deoxyribonucleic acid (DNA) and can trigger several degenerative diseases, such as atherosclerosis, stroke, diabetes and cancer, in humans (Ushio-Fukai & Nakamura 2008). Antioxidants are believed to be prophylactic for the mentioned deleterious diseases. Human cells possess an inherent ROS scavenging mechanism, but this becomes inefficient and insufficient with age and under undue environmental stresses. Hence, dietary supplementation with synthetic antioxidants is necessary (Barros et al. 2011).
Plants have been utilised from time immemorial in the alternative and complementary treatment of several disease conditions, especially in developing economies like Nigeria where affordability and access to modern treatment is a major setback. Chemical constituents in medicinal plants possess several pharmacological potentials, which have been the focus of researches targeted at prospection of reliable, affordable and potent drugs (Mohammadhosseini et al. 2016;Nunes & Miguel 2017). These constituents could be found in plant extracts or essential oils (EOs) with great activity useful for several therapeutic applications (Camilo et al. 2017;Ganesan & Xu 2017;Pavunraj, Ramasubbu & Baskar 2017).
Calophyllum inophyllum Linn is the most abundant species in genus Calophyllum and is widespread in tropical areas, with a wide variety of uses ranging from traditional, medicinal and industrial applications (Dweck & Meadowst 2002). The extracted oil from the fruit of C. inophyllum Linn is used as a remedy for sciatica, shingles, neuritis, rheumatism, ulcers and skin diseases, whilst the seed oil is reported to have medicinal and healing properties. The plant's dried leaves and its decoction are widely used in curing rheumatism, skin infections, cuts and sores (Uma et al. 2012). Its leaf and stem bark extracts have shown anti-hyperglycaemic and antihyperlipidaemic activities, whilst the leaf extract was identified to inhibit OS (Varsha et al. 2016). Its fruits are effectively utilised in the treatment of dermatitis (Yu et al. 2016). The broad spectrum of biological activities exhibited by C. inophyllum may be associated with the chemical composition of its different parts (Figures 1-3). This article was therefore designed to evaluate the antioxidant properties of gas chromatography-mass spectrophotometry (GC-MS) characterised EOs from 10 parts of C. inophyllum Linn using the generally reliable 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide models.
The oils had a distinct characteristic smell. The EOs were refrigerated until the assay was carried out.

Identification of essential oils by gas chromatographymass spectrometry analyses
Gas chromatography-mass spectrometry analyses were carried out by using an Agilent 7890B-5977B GC-MS (Santa Clara, California, United States) system operating in the EI mode at 70 eV, using an HP-5MS capillary column (5% phenylmethyl polysiloxane, 30 m, 0.25 mm internal diameter (i.d.) and 0.1 μm film thickness) (Jenning and Walter Scientific, Folsom, California, United States), which was programmed with the following conditions: 60 °C for 4 minutes, then up to 4 °C/minutes to 160 °C, then 11 °C/min up to 280 °C, held for 15 min, and finally 15 °C/min up to 300 °C. The carrier gas was helium at a flow rate of 1.2 mL/min, the injector temperature was 280 °C whilst the transfer line temperature was 300 °C, the injection volume was 1 μL, the split ratio was 1:100, the run time was 57 min and the acquisition mass range was 29 atomic mass unit (amu) -400 amu. Identification of the EO components was based on their retention indices (experimentally determined using homologous series of C8-C30 alkanes) and by comparison of their mass spectral fragmentation patterns in computer matching against library linear retention index and mass spectra taken from Adams and NIST 17 [25] FFNSC2 and MAGGI libraries (Adams 2007;FFNSC2 2012;NIST 17 2017). Relative peak area percentages were obtained by peak area normalisation without using correction factors and were the mean of the three determinations with an relative standard deviation (RSD%) in all cases below 10%.

Antioxidant activity
2,2-diphenyl-1-picrylhydrazyl assay: The free radical scavenging activity of EOs from C. inophyllum Linn was determined using the stable DPPH radical (Ebrahimzadeh & Bahramian 2009;Njenga & Mugo 2020). The dark purple colour of DPPH is lost when it is reduced to non-radicals by antioxidants and decreases in its absorbance when monitored at a characteristic wavelength of 517 nm. A 0.1 mM concentration of DPPH was prepared by dissolving 3.94 mg in 100 mL of methanol. An amount of 2 mg of the EO was dissolved in 2 mL of methanol to prepare a 1.0 mg/mL concentration of the EO, which was the stock solution. This stock solution was vortexed and serially diluted with methanol to obtain sample solutions of various concentrations, ranging from 1.0 mg/mL to 0.3125 mg/mL. The six serially dilute concentrations (1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL and 0.03125 mg/mL) of EOs and standards were prepared in triplicates. Ascorbic acid (vitamin C) and BHA were used as standard positive controls. About 0.5 mL of each of these concentrations of the triplicates was added to 3 mL of pure methanol solution of DPPH (0.1 M). The absorbance of each sample concentration against methanol solution of DPPH blank was measured at 517 nm using an ultraviolet (UV) spectrophotometer. BHA, butylated hydroxyl anisole. Note: All the results are mean ± standard deviation (SD) where n = 3. The half maximal inhibitory concentration (IC 50 ) was obtained in mg/mL using non-linear regression analysis in Microsoft Excel.  All readings were taken after 30 min of reaction time at room temperature. The decrease in absorbance of DPPH on the addition of test samples to the blank was used to calculate the percentage inhibition (I %) using the following equation: where Abs (Blank) is the absorbance measurement of the blank and Abs (Eo) is the absorbance reading of EOs at 517 nm.
Hydrogen peroxide scavenging activity: The ability of EOs from C. inophyllum Linn to scavenge hydrogen peroxide was determined using the hydrogen peroxide scavenging assay at different concentrations (1.0 mg/mL -0.03125 mg/mL) (Kamalanathan et al. 2015;Njenga & Mugo 2020;Serhat et al. 2012). A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer at pH 7.4. The concentration of hydrogen peroxide was determined by absorption at 230 nm using a UVD. Essential oils and standards in distilled water were added to a hydrogen peroxide solution (0.6 mL, 40 mM). The absorbance of hydrogen peroxide was determined after 30 min against a blank solution containing phosphate buffer without hydrogen peroxide. The absorbance value of the reaction mixture was recorded at 230 nm using ascorbic acid (vitamin C) and BHA as standard. The percentage of hydrogen peroxide scavenged by the EOs and standards was calculated as follows: where Abs (Blank) is the absorbance measurement of the blank and Abs (Eo) is the absorbance of EOs at 230 nm.

Statistical analysis
The experiments were conducted three times, all determinations were performed in triplicates (n = 3) and the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed by non-linear regression analysis on Microsoft Excel. The half maximal inhibitory concentration (IC 50 ) values of 10 parts of C. inophyllum Linn were determined using non-linear regression analysis on Microsoft Excel in comparison with standards.

Ethical consideration
This article followed all ethical standards for a research without direct contact with human or animal subjects.

Results and discussion
Percentage yield of essential oils from C. inophyllum Linn Essential oils obtained from C. inophyllum Linn gave characteristic odours (herbal, floral and woody). The oils were procured in 0.219% to 0.506% yields (Table 1), with the highest yield from fruit pulp, which gave 0.560%, and the lowest yield (0.219%) from the root, which may be because of its high fibre content.

Essential oil composition of C. inophyllum Linn
The GC-MS characterisations of the leaf, leaf stalk, flower oil, pod, peel, stem wood, stem bark, root wood and root bark EOs extracted from C. inophyllum Linn showed a total of 102 compounds, which are mostly monoterpenes, sesquiterpenes and their oxygenated derivatives as shown in Table 2.
A total of 71 compounds were characterised in leaf oil, which corresponded to 54.94% of the identified peaks. This oil consists mainly of sesquiterpenes (22.18%) and nonterpenes (27.89%). The predominant compounds in leaf oil are cis-cadina-1(6), 4-diene (6.50%), hexadecanal (6.16%) and cis-calamenene (5.41). The oils contain non-ubiquitous norisoprenoids, such as α-cyclocitral, β-cyclocitral and β-ionone.    Generally, the essential oils are dominated by cymene, terpinene and limonene. Cymene, which is present in a relatively large percentage in eight of the oils from C. inophyllum Linn, has been reported as a good antioxidant, anti-inflammatory, anti-nociceptive, anxiolytic, anticancer and antimicrobial agent (DeOliveira et al. 2015), which corroborates the ethno-medicinal applications of the plant. In a recent in vivo investigation on an experimental animal model, p-cymene was found to increase the activity of antioxidant enzymes, thereby reducing the OS; the high antimicrobial potential of Carum copticum EO was also attributed to the abundance of cymene and terpinene (Hassan et al. 2016).
The high content of γ-terpinene in leaf stalk (13.06%), seed coat (6.77%) and root bark (7.75%) oils of C. inophyllum Linn is responsible for the anti-inflammatory and antioxidant effects, thus supporting the plant's anti-osteoarthritic activity. The presence of Terpinene in Hyptis species inhibited gastric lesions, reduced volume and acidity of the gastric juice and increased gastric wall mucus (Marcelo, Rafael & Lucio 2015). Limonene, which is found in an appreciable amount in stem heartwood (23.79%), stem bark (3.24%) and root bark (13.93%) EOs of C. inophyllum Linn, is known to have sedative and stimulative effects in Lippia alba (Vale et al. 2002;Viana, Vale & Matos 2000). Consumption of diets containing fruits and vegetables rich in monoterpenes, such as limonene, is known to reduce the risk of developing cancer of the colon, mammary gland, liver, pancreas and lung. Limonene, which is known to possess high anticancer properties (Chistani et al. 2007;Marostica et al. 2009), is abundant in C. inophyllum Linn: leaf stalk (25.40%), seed (25.40%) and root bark (13.93%) oils. The presence of phenylpropanoids, norisoprenoids and other non-ubiquitous compounds, such as β-alaskene, β-acoradiene and E-anethole, is a unique feature of oils from C. inophyllum Linn as shown in Table 2.

2,2-diphenyl-1-picrylhydrazyl antioxidant activity of Calophyllum inophyllum Linn
The percentage inhibition obtained for standard antioxidants (ascorbic acid and BHA) was relatively high for the concentration range used (1.0 mg/mL -0.03125 mg/mL). A maximum percentage inhibition of 92.68% and 91.67% was obtained at 1.0 mg/mL for ascorbic acid and BHA, respectively. The 10 oils (leaf, leaf stalk, flower, seed, pod, peel, stem wood, stem bark, root wood and root bark) exhibited concentration-dependent inhibition with reference to standard synthetic antioxidants used as a positive control. Percentage inhibitions of standards were in close range with pod EO, with inhibition efficiency of 78.32% at 1.0 mg/mL as indicated in Table 2. A graph of percentage DPPH inhibition versus concentration (mg/mL) of EOs was plotted from which the IC 50 values were obtained for each oil using linear regression analysis in reference to standards ( Figure 5). An inverse relationship exists between the percentage inhibition efficiency and the IC 50 values. The higher the IC 50 value, the lower the activity of the EOs and vice versa. The following IC 50 values were obtained in the determination of DPPH inhibition: (leaf, 3.89 mg/mL; leaf stalk, 4.17 mg/mL; flower, 3.92 mg/mL; seed, 3.49 mg/mL; pod, 4.68 mg/mL; peel, 3.64 mg/mL; stem wood, 3.93 mg/mL; stem bark, 3.36 mg/mL; root wood, 3.19 mg/mL; and root bark, 3.87 mg/mL) BHA, butylated hydroxyl anisole. Note: All the results are mean ± standard deviation (SD) where n = 3. The half maximal inhibitory concentration (IC 50 ) was obtained in mg/mL using non-linear regression analysis in Microsoft Excel.

Hydrogen peroxide scavenging activity of Calophyllum inophyllum Linn
Optimum percentage inhibitions of 90.61% and 89.24% were obtained for ascorbic acid and BHA at 1.0 mg/mL and decreased slightly to 45.61% and 45.24% at 0.03125 mg/mL, respectively. This trend indicates that the percentage inhibition of the standard used in this study is concentration dependent. The 10 oils (leaf, leaf stalk, flower, seed, pod, peel, stem wood, stem bark, root wood and root bark) exhibited concentration-dependent inhibition similar to standard synthetic antioxidants used. A graph of percentage inhibition versus concentration (mg/mL) of EOs was plotted from which the IC 50 values were obtained for each oil using linear regression analysis in reference to the central standard. An inverse relationship exists between the percentage inhibition efficiency and the IC 50 values (Figure 4). The higher the IC 50 value, the lower the activity of the EOs and vice versa.

Conclusion
The antioxidant activity of characterised compounds of C. inophyllum Linn was presented for the first time and extends the knowledge in the broad range of biological activities and therapeutic prospects associated with this medicinal plant. Results from both DPPH and hydrogen peroxide assays established that C. inophyllum Linn EOs possess antioxidant and radical scavenging potential. However, the antioxidant properties were found to be slightly lower compared to standard antioxidants used (ascorbic acid and BHA). Despite having lower activity compared to standards, the antioxidant activity of C. inophyllum Linn was found to be significant for potential applications in pharmaceutical industries and could act as a potential alternative to more toxic synthetic antioxidants. Because of the toxic nature of EOs, further studies on the toxicity and other biological properties of the extract are needed prior to possible applications.