Antioxidant Potentials of the Pod Extract of Caesalpinia Pulcherrima Swarta (Fabaece) and the Theoretical Evaluation of the Antioxidant Property of the Isolated Compounds

Various diseases associated with oxidative stress have necessitated the need to investigate plants for antioxidant agents. This study aims to investigate the antioxidant potential of the extract and fractions of C. pulcherrima and estimate the quantum chemical properties of polyphenolic compounds isolated from its most active antioxidant fraction. Methanolic extract and partition fractions of the pods of the plant were assayed for their antioxidant activity using four models: 1,1-diphenyl-2-dipicrylhydrazyl radical scavenging, ferric reducing antioxidant power, total antioxidant capacity and hydroxyl radical scavenging activity. Total phenolic and avonoid contents of the extract and fractions were also investigated. Furthermore, the quantum chemical properties of two polyphenolic compounds were calculated to predict the antioxidant potential. The extract exhibited good antioxidant activity with IC 50 = 45.63 µg/ml comparable to ascorbic acid of IC 50 = 37.94 µg/ml, high reducing power with value at 376.74 ± 6.78, total antioxidant capacity obtained at ascorbic acid equivalence of 383 ± 8.16 mgAAE/g and potent hydroxyl radical scavenging activity with value at 580.97 µg/ml with ascorbic acid at 30.7 µg/ml. The total phenol and avonoid contents values at 540.53 ± 3.71 and 347.87 ± 7.13 respectively. Furthermore, the results obtained from the quantum chemical calculations reveals polyphenolic compounds; 5(4-hydroxyphenyl)-3-hydroxy-2-methoxyphenol and 3(4-methanetriiol-2,6-dihydroxyphenoxy)-3’, 4’, 5’, 5, 7-pentahydroxyavonol as promising antioxidant agents. The study concluded that C. pulcherrima pods have good antioxidant activity. Also, the polyphenolic compounds are among the chemical constituents responsible for the antioxidant activity obtained for the extract and ethyl acetate fraction.


Introduction
Oxidative stress is characterised by increased production of reactive oxygen and nitrogen species (RONS) and reduced antioxidant capacity in the cardiovascular, renal and central nervous systems [1][2] These excessive amounts can lead to cellar damage and apoptosis, contributing to several acute and chronic pathological diseases such as cancer, stroke, myocardial infarction, diabetes, acute and chronic kidney disease, neurodegenerative diseases, macular degeneration and biliary diseases [3][4][5]. Several synthetic drugs are currently used as antioxidant agent. These drugs are presented with several side effects.
Medicinal plants are known to produce antioxidant compounds that have lesser side effect. These antioxidant agents include avonoids, tannis, stilbenes, coumarins, lignin, lignans and phenolic acids [6].
Caesalpinia pulcherrima (Fabaceae/Leguminosae) is a shrub rich in a variety of phytoconstituents including diterpenoids, avonoids, peltogynoids, steroids, glycosides and more [7][8][9]. In folk medicine, extracts from various parts of the plant are used as stimulant, emenagogue, arboti cient and in the treatment of fever, ulcer, asthma, tumors, and skin diseases [9][10]. Pharmacologically, the plant has been reported to possess antimicrobial, analgesic, anti-in ammatory, anthelmintic, antimalarial, antiulcer, cytotoxic, antioxidant, antiviral, anticancer, immunosuppressive, anti-diabetic, antimicrobial activities and vasorelaxing effect [7,[9][10]. This study was aimed at investigating the antioxidant capacity of the extract and fractions of C. pulcherrima pod. It further predicted the antioxidant potentials of the polyphenolic compounds previously isolated from the ethyl acetate fraction [11] by calculating their electronic properties.

2.1
Plant materials and extraction The pods of C. pulcherrima were collected at Obafemi Awolowo University campus, Ile-Ife, Osun State, Nigeria. It was identi ed and authenticated by Mr. Ademorriyo at the herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife. The voucher specimen (IFE 17513) was prepared and deposited at the same herbarium. The pods were peeled to remove the seeds and air dried for 30 days.
The air dried pods were ground to powder and 6 kg of the powdered pods was exhaustively extracted with methanol and concentrated in vacuo to obtain its methanolic extract with 9.6 % w/w yield. The collection of the plant material was according to the guidelines of World Health Organization guidelines on good agricultural and collection practices for medicinal plants [12].

2.2
Antioxidant assays of the crude extract and the fractions

Total Flavonoid Content
The total avonoid content of the extract and the fractions was determined according to the method of Zhishen [13] as described by Miliauskas [14]. To 0.2 ml of the extract/fractions, 0.4 ml of distilled water was added. This was followed by the addition of 0.1 ml of 5 % (w/v) sodium nitrate. After 5 minutes, 0.1 ml of 10 % (w/v) aluminum chloride and 0.2 ml of 0.1 M sodium hydroxide solution were added and the volume made up to 2.5 ml with distilled water. The absorbance was then measured against the blank at 500 nm.

Total Phenolic Content
The total phenolic content of the extract and the fractions was determined using the Folin-Ciocalteu's method of Singleton and Rossi, [15] as described by Gulcin [16]. To 0.1 ml of 5 mg/ml of the extract/fractions was added to 0.9 ml of distilled water. 0.2 ml of 10 % Folin reagent was added. The resulting mixture was vortexed. After 5 minutes, 1 ml of 7 % Na 2 CO 3 solution was then added to the mixture. The solution was diluted to 2.5 ml with distilled water and then incubated for 90 minutes at room temperature. The absorbance at 750 nm was then measured against the reagent blank. Standard preparation was done by preparing a stock solution of Gallic acid 100 µl/ml, aliquots of 0.2, 0.4, 0.6, 0.8 and 1 ml were taken and made up to a total volume of 1 ml.

DPPH scavenging assay
DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity of the extract/fractions was determined using the method of Blois [17] as described by Brace [18] in which 1 ml of DPPH solution (0.3 mM DPPH dissolved in methanol) was prepared. To 1 ml each of the extract/fractions and methanol, 1 ml of DPPH was added. The mixture was vortexed and allowed to react at room temperature in a dark chamber for 30 minutes. The absorbance was measured at 517 nm against DPPH control (1 ml of methanol) as a blank using UV-VIS spectrophotometer.

Total Antioxidant Capacity Assay
The total antioxidant capacity was carried out according to Prieto [19]. Typically, 0.3 ml extract/fractions were mixed with 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95 °C for 90 minutes. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer against blank after cooling to room temperature. Methanol (0.3 ml) in place of the extract/fractions was used as a blank. The antioxidant activity was expressed as ascorbic acid equivalent (mg AAE/g extract) which served as positive control.

Hydroxyl Radical Scavenging Assay
The ability of the extract to scavenge the hydroxyl radical generated by Fenton reaction was measured according to the modi ed method of Chung [21]. 1 % TBA were added and placed in boiling water bath for 10 minutes. The resultant mixture was allowed to cool to room temperature and absorbance was recorded at 532 nm in a UV-VIS spectrophotometer.

Results And Discussion
3.1 Antioxidant activity of the extract and the fractions Total phenolic content and total avonoid content of the extract of pods of C. pulcherrima were estimated. It was observed that the crude pod extract of C. pulcherrima contained high amount of total phenol and avonoids ( Table 1). The results obtained in this study are in accordance with the ndings of other researchers who have reported high phenolic and avonoid content in the pods of C. pulcherrima [23][24]. The antioxidant property of polyphenolic compounds has been reported [25][26]. A correlation between antioxidant activity and the phenolic content has been documented [24,27]. Plant extracts with high phenolic content also show high avonoid content [28] as evident in this study. Several in vitro model systems have been used for assessing the antioxidant activity of various plant extracts. This study employed DPPH-radical scavenging, Total antioxidant capacity (TAC), Ferric Reducing Antioxidant Power (FRAP) and Hydroxyl Radical Scavenging antioxidant assays to evaluate the antioxidant potential of the crude extract and the fractions of the pods of C. pulcherrima.
The total antioxidant capacity of fractions of C. pulcherrima was also evaluated. The ethyl acetate fraction gave the highest total antioxidant capacity. Total antioxidant capacity of the fractions of C. pulcherrima from the highest to the lowest was ethyl acetate, aqueous methanol, dichloromethane and n-hexane ( Table 1). The evaluation of the total antioxidant capacity (TAC) may be an appropriate tool to determine the additive antioxidant properties of plant foods [30].
Ferric reducing antioxidant power (FRAP) of C. pulcherrima fractions was evaluated. Ethyl acetate fraction possessed the strongest ferric reducing antioxidant power of the four fractions of C. pulcherrima followed by the aqueous methanol, dichloromethane and n-hexane fractions. Ferric Reducing power assay method is an electron transfer based total antioxidant assay that is based on the principle that substances, which have reduction potential, react with potassium ferricyanide (Fe 3+ ) to form potassium ferrocyanide (Fe 2+ ), which then reacts with ferric chloride to form ferric-ferrous complex that has an absorption maximum at 700 nm. The reducing ability of a compound generally depends on the presence of reductones (antioxidants), which exert its antioxidant activity by breaking the free radical chain by donating a hydrogen atom [31]. Fe (III) reduction is often used as an indicator of electron donating activity, which is an important mechanism of phenolic antioxidant action [32].
The hydroxyl radical scavenging activity of fractions of C. pulcherrima was estimated. The ethyl acetate fraction exhibited the highest hydroxyl radical scavenging properties when compared to other fractions.
The order of hydroxyl radical scavenging ability of fractions of C. pulcherrima from the highest to the lowest was n-hexane, dichloromethane, ethyl acetate and aqueous methanol ( Table 1). All fractions scavenge hydroxyl radicals in a concentration dependent manner. The scavenging of the hydroxyl radicals may be due to the presence of hydrogen donating ability of the fractions.

Molecular modeling and quantum chemical calculations
Frontier molecular orbital analysis via computational method is of great importance as it gives information on the electronic properties of compounds and further insight on the reactivity and nature of the compounds [33]. Frontier molecular orbitals analysis often deals with electronic transitions as well as reactivity of molecules. The frontier molecular orbitals refer to the LUMO, which is largely related to the electron accepting ability of a compound and the HOMO which is concerned with its electron donating capacity. The energy difference between the frontier molecular orbital also known as energy gap is used to predict the reactivity and stability of compounds. Compounds with large energy gaps exhibit low reactivity as well as high kinetic stability while compounds with low energy gaps exhibit high reactivity and low kinetic stability [34]. The energy gap DE for 3(4-methanetriiol-2,6-dihydroxyphenoxy)-3', 4', 5', 5, 7-pentahydroxy avonol and 5(4-hydroxyphenyl)-3-hydroxy-2-methoxyphenol is 3.53 ev and 5.08 ev respectively. This suggests that 3(4-methanetriiol-2,6-dihydroxyphenoxy)-3', 4', 5', 5, 7pentahydroxy avonol is likely to be more stable and less reactive than 5(4-hydroxyphenyl)-3-hydroxy-2methoxyphenol. Figure 1 (b) shows that for 3(4-methanetriiol-2,6-dihydroxyphenoxy)-3', 4', 5', 5, 7-pentahydroxy avonol, the HOMO is distributed along the aromatic system as well as the hydroxyl groups on the aromatic system. The LUMO is localized over the other ring, Figure 2 (b). The third ring neither participates in the LUMO and HOMO. For 5(4-hydroxyphenyl)-3-hydroxy-2-methoxyphenol, the LUMO, Fig.2 (a) and HOMO, Fig. 1 (a) are distributed along the pie network of both aromatic rings.
The quantum descriptors namely chemical hardness, electrophilcity, chemical potential are excellent tools in giving a vivid description of the reactivity and stability of the compounds. These descriptors were calculated using the formulas below; the results are displayed in Table 1.
Chemical potential is the measure of alteration in energy of a molecule with respect to the electron number at a xed potential. It is strongly linked to the electrophilcity index of a molecule, which is the energy stabilization of a molecule on acquiring an extra amount of electron density from its environment [35]. Strong electrophiles often possess high chemical hardness and low chemical potential while weak electrophiles possess low chemical hardness and high chemical potential. In effect it means that strong electrophile possess high electrophilcity index values while weak electrophiles possess low electrophilcity index values [34]. Compounds with electrophilcity index values above 0.8 are referred to as strong electrophiles [35]. Hence 3(4-methanetriiol-2,6-dihydroxyphenoxy)-3', 4', 5', 5, 7-pentahydroxy avonol and 5(4-hydroxyphenyl)-3-hydroxy-2-methoxyphenol are strong electrophiles.
Molecular electrostatic potential is an important molecular descriptor that is usually used in investigating the chemical reactivity of molecules. It is a three dimensional plot of electrostatic potential over electron density of molecular systems. It indicates the position, shape and size of positive, negative and neutral electrostatic potentials using a colour grading scheme [36]. It aids understanding of system physicochemical properties in relation to their molecular structure and help to identify reactive sites of nucleophilic and electrophilic attack during bonding interactions [37]. Blue regions are the more electropositive regions, as they lack electrons while the red regions are electron rich centers; hence they are likely to be probable sites for electrophilic attack. From gure 3, in both compounds low electrostatic potential regions are observed around the oxygen of the hydroxyl groups while a high electrostatic potential is found on hydrogen atoms. This suggests that the oxygen atoms are suitable for electrophilic interactions while the hydrogen atoms are suitable for nucleophilic interactions [36]. In both cases oxygen atoms are likely to act as electron donors as the electron rich regions are located on them.

Conclusion
The crude extract as well as the fractions demonstrated appreciable antioxidant property based on the antioxidant methods employed in this study. Of all the fractions tested, ethyl acetate fraction had the highest DPPH radical scavenging activity, the strongest ferric reducing power and the highest total antioxidant capacity. This could be attributed to its higher total phenolic and total avonoid content of 540.53±3.71 mgGAE/g and 347.87 ±7.13 mg/g rutin equivalent respectively. Therefore, the results of this study indicate that antioxidant property of C. pulcherrima may be one of the mechanisms by which this plant exerts its effectiveness in several free radical mediated diseases including diabetes as reported by Faloye [11]. The density functional theory studies strongly predicts that the isolated compounds are strong electrophiles. However, they containing electron rich oxygen centers which are likely to be involved in their exhibition of antioxidant activity.