Evaluation of Antioxidant and Cytotoxic Properties of Vernonia Amygdalina

Vernonia amygdalina is a shrub that grows predominantly in the tropical Africa. Leaves from this plant serve as food vegetable and culinary herb in soup [1]. Anecdotal evidences suggest the use of V. amygdalina in the treatment of feverish condition, cough, constipation, hypertension and related vascular diseases as well as diabetes. Photochemical screening of this plant leaves extracts showed the presence of Saponins, riboflavin, polyphenols, sesquiterpene and flavonoids [2]. Strong antioxidant activities involving flavonoids extracted from V. amygdalina and its saponins have been reported to elicit anti-tumoral activities in leukaemia cells [3]. In addition, peptides from V. amygdalina are known to be potent inhibitor of Mitogen Activated Protein Kinase (MAPK) which is involved in the regulation and growth of breast tumor [4].


Introduction
Vernonia amygdalina is a shrub that grows predominantly in the tropical Africa. Leaves from this plant serve as food vegetable and culinary herb in soup [1]. Anecdotal evidences suggest the use of V. amygdalina in the treatment of feverish condition, cough, constipation, hypertension and related vascular diseases as well as diabetes. Photochemical screening of this plant leaves extracts showed the presence of Saponins, riboflavin, polyphenols, sesquiterpene and flavonoids [2]. Strong antioxidant activities involving flavonoids extracted from V. amygdalina and its saponins have been reported to elicit anti-tumoral activities in leukaemia cells [3]. In addition, peptides from V. amygdalina are known to be potent inhibitor of Mitogen Activated Protein Kinase (MAPK) which is involved in the regulation and growth of breast tumor [4].
in the maintenance of health and protection of some age related degeneration disorders, such as coronary heart disease and cancer, neurodegenerative disease [5,6,7].
Although, antioxidants from natural sources are beneficial, it is pertinent to know their bio-safety. In this regard, the brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity of plant extracts; a suggested pharmacological screening method in plant extracts. It has been used for the detection of fungal toxins, plant extract toxicity. The shrimp lethality assay was proposed by Michael and co-workers in 1956, and later developed by Vanhaecker and his group in 1981. This is based on the principle, whereby the kill laboratoryculture of an invertebrate, Artemia salina L (the brine shrimp larva) following exposure to a varied concentration of plant extracts, heavy metals, cyanobacteria toxins and pesticides, is assessed for toxicity [8].
The purpose of this study is to evaluate the acute toxicity and antioxidant properties of V. amygdalina in relation to its use as a neutralcetical.

Materials and Methods
V. Amygdalina: Fresh leaves of V. amygdalina were collected from the University Village, Kogi State University, Nigeria. The plant material was identified and authenticated by taxonomist in the Department of Botany, Kogi State University, where the voucher specimen (VA-111) was deposited. Fresh leaves of V. amygdalina were air dried under room temperature until a constant weight was obtained. Thereafter, the leaves were milled to a coarse powder with the use of laboratory Mortar and Pestle. After this, 20 g of the plant powder was weighed into a volumetric flask and then extracted using 200 mls of distilled water for 72 hours. The crude extract was obtained by concentrating the water soluble extract using rotary evaporator at 450C. The working solution of extract was prepared by weighing out 0.02g of crude extract accurately and dissolved it in 20 ml of distilled water to give an effective concentration of 1mg /ml.

Radical Scavenging Activity:
In order to determine the antioxidant properties of the plant, radical scavenging activities of the leaves extract, was determined using the stable radical DPPH (2, 2-diphenyl-1 piccrlhydrazyl hydrate) according to the method of Blois (1958) as describe by Babalola and co-workers [9]. The principle is based on the reaction of DPPH, and an antioxidant compound to generate hydrogen, which is reduced (DPPH + RH → DPPH2 + R). The observed colour change from deep violet to light yellow was measured at 517 nm. To 1 ml of varied concentrations (0.5, 0.25, 0.125, 0.0625, 0.003125 mg/ ml) of the extract or standard, was added 1 ml of 0.3 mm DPPH in methanol. The mixture was vortexed, and then incubated in a dark chamber for 30 minutes. Thereafter the absorbance was read at 517 nm against a DPPH control containing only 1 ml of methanol in place of the extract. The antioxidant activity (AA) was then calculated using the formula: AA = [(Ao -Ac)/Ao] x 100, Where: Ao = absorbance without extract and Ac = absorbance with extract.

Nitric Oxide: Sodium nitroprusside generates nitric oxide in aqueous solution at
Physiological pH, which consequently interacts with oxygen to produce nitric ions. This was measured by Griess reaction [10]. Procedure: 3 ml of the reaction mixture containing sodium nitroprusside (10 mm) in phosphate buffered saline (PBS) together with the varying concentrations of the extract (0.5, 0.25, 0.125, 0.0625, 0.003125 mg/ml) were incubated in a water bath at room temperature for 150 minutes. This was followed by the removal of 1.5 ml of the reaction mixture and the addition of 1.5 ml of Griess reagent. After which, the absorbance of the chromophore formed was read using spectrophotometer at 546 nm. Percentage inhibition of nitric oxide radical by the extract was calculated using the formula: NO = [(1-E/C)] x 100, where: C= absorbance value of the fully.
Procedure: A 50µL aliquot of extract was added to 1.5 ml of FRAP reagent in a semi-micro plastic cuvette. Absorbance measurement was taken at 593 nm (A593) exactly 10 minutes after mixing using 50µL of water as the reference. Thereafter, to standardize 50 µL of the standard, iron (III) sulphate, (1mM) was added to 1.5 ml of FRAP reagent. All measurement was taken at room temperature in the absence of light.

Evaluation of Total Phenolic content
The total phenolic of V.amygdalina extract was determined using the folin ciocalten assay method of Singleton and Rossi (1965) [11]. To 0.1ml of 1mg/ml of extract /standard was added 0.9 ml of distilled water. Thereafter, 0.2 ml of folin reagent was added. This was vortex-missed. Subsequently, 1ml of 7 % Na2CO3 solution was added to the mixture after 5 minutes. The solution was followed by dilution to 2.5 ml and then incubated for 90 minutes at room temperature. The absorbance was read at 750 nm against the reagent blank. Standard preparation was carried out by preparing a stock solution of gallic acid (1mg/ml) aliquots of 0.2,0.4, 0.6,0.8 and 1ml were taken and made up to a total volume of 2 ml.
With the equation as shown below, the total phenolic content of the plants was then calculated, and expressed as mg gallic acid equivalent (GAE)/g fresh weight. The analysis was carried out in triplicates. Equation (1)

Evaluation of Total flavonoids content
Aluminium chloride colorimetric method described by Zhilen was used for the determination of the total flavonoidal content of the plant extract [12]. Water (0.4 ml) was added to 0.1 ml of extract/standard, as well as 0.1 ml of 5 % sodium nitrite. This was left for 5 minutes. Thereafter, 0.1ml of 10 % aluminium chloride and 0.2 ml of sodium hydroxide was added to the solution, and the volume was adjusted to 2.5 ml with water. The absorbance at 510 nm was measured against the blank.

Standard preparation
A stock solution of quercetin (1 mg/ml) was prepared. Aliquots of 0.2, 0.4, 0.6, 0.8, and 1 ml were taken and the volume made up to 2 ml with distilled water.
The total flavonoid content of the plant extract was then calculated as shown in the equation below and expressed as mg quercetin equivalents per gram of the plant extract. The analysis was conducted duplicates and mean value considered. X = q×V/w: Where X= total content of flavonoid compound in quercetin equivalent; q = concentration of quercetin established from the standard curve; V=volume of extract (ml); w = weight of the crude methanolic extract obtained.

Proanthocyanidin content Determination
The proanthocyanidin content of the extract was determined spectrophotometrically [13]. Extracts were diluted to provide a spectrophometric reading between 0.1 and 0.8 absorbance units.
Procedure: A 0.25 ml sample aliquot of adequately diluted extract was added to 2.25 ml of concentrated hydrochloric acid in n-butanol (10/90, v/v) in a screw top vial. The resulting solution was mixed for 10 to 15 seconds. Extracts were then heated for 90 minutes in an 85o C water bath then cooled to 15-25o C in an ice bath. The absorbance at 550 nm was measured on a UV visible spectrophotometer. A control solution of each extract was prepared to account for background absorbance due to pigments in the extracts. The control solution consisted of the diluted extract prepared in the hydrochloric acid/n-butanol solvent without heating. The absorbance of the samples at 512 nm (A512 nm) and 700 nm (A700 nm) was measured on a UV-visible spectrophotometer. The difference in absorbance (∆A) between the anthocyanin extract diluted in pH 1.0 and pH 4.5 buffers was calculated using the equation below ∆A= (A512 pH1.0-A700 nm pH1.0)-(A512 nm pH4.5-A700 nm pH 4.5) The A700 nm was employed in the calculation of ∆A to correct for any background absorbance due to turbidity on the extracts. The anthocyanin content was expressed as mg cyaninidin 3-glucoside per 100 g berries using a molar absorbance co

Brine shrimp bioassay
Brine shrimp lethality test was carried out using hatched Brine shrimp (Artemia salina L) larvae (nauplii) according to the procedure described by Meyer et al., (1982). The eggs were hatched in artificial sea water (16 g of sea salt in 50 ml of distilled water) by adding 100 mg of brine shrimp eggs to 50 ml of sea water that was partitioned into two compartments. The compartment sprinkled with the cysts was left dark, while the other compartment was supplied with bright white fluorescent light. After 24 hours of incubation, the hatched shrimps moved to the illuminated side. Ten brine shrimps larvae were then counted and transferred to each sample vial, using a Pasteur pipette and artificial sea water was added to make 10 ml. The sample vials were previously containing solution of the extract prepared by dissolving 0.2 g of the extract in 20 ml distilled water to give concentration of 1mg/ml. The varied concentrations from the stock solution were transferred to different graduated container with the aid of a micropipette. The survivors were counted after 24 hours. Three independent studies were carried out (n =3).
Statistical Analysis: The results are expressed as mean ± SEM using Graph Pad Prism Graphical-Statistical Package version 5. The difference between groups was analyzed by Student t-test followed by Dennett's test with 5 % level of significance (p < 0.05).

Antioxidants:
The extract was assayed for total content of four major types of antioxidant properties. The antioxidant constituents were: total phenol, total flavonoid, proanthocyanidins and anthocyanins. However, the percentage yield of the crude extract used for the assays is given as 10.11 ± 1.08%. The results showed the total phenolic content as 1.588± 0.04 mgGAE/g, which is considerably high compared to the standard. The total flavonoid content expressed as quercetin equivalent per gram of the plant extract showed that the test material had 0.857 ± 0.15 mg QUE/g dry weight for the crude extract (Table 1). These two indices are pointer to an increased antioxidant activity. The concentration of anthocyanin in the sample was 0.099 ± 0.08 cyanidin 3-glucoside/100 g for the crude extract, while the concentrations of proanthocyanin was 0.038 ± 0.05 cyanidin 3-glucoside/100 g for the crude extract. Tannin was also assayed, and it gave a concentration of 1.188 ± 0.04 mg/ml (Table 1).

Antiradicals:
The result of the antiradical assays carried out on the extract is shown in Table 2. Using the DPPH (2, 2-diphenyl-1-piccrlhydrazyl hydrate) assay, a well established antiradical assay, the activity was concentration dependent i.e. activity increases with increase in concentration. The extract

Evaluation of Antioxidant and Cytotoxic Properties of Vernonia Amygdalina
Copyright: © 2018 Omede A, et al.   Table  2). The extract used showed activity despite the significant difference (P < 0.05) between the test and standard.
The nitric oxide inhibition assay also showed that V.amygdalina is a potent scavenger of nitric oxide as shown by the percentage inhibition and IC50 of 3.84±1.03 mg/ml ( Table 3). The FRAP assay result showed a concentration dependent change when the FRAP values of the test fractions were determined. Results were expressed in mmolFe2+/L. The concentration of Fe2+ in the reaction mixture at 0.5 mg/ml, was given as 1.49 ± 0.18 mmolFe2+/l for the test extract (Table 4). All values are expressed as mean ± SEM (n=3). All values are expressed as mean ± SEM (n=3).

Discussion
Studies have shown that consumption of bio safe exogenous and natural antioxidant is beneficial, as regard combating diseases such as cancer, arthritis, diabetes, among others. These diseases emanates from oxidative stress mostly caused by reactive oxygen species (ROS) [14,15,16]. Moreover, synthetic antioxidant, including tert-butylhydroquinone (TBHQ), buthylatedhydroxytoluene (BHT) and propylgallate have been found to be beneficial, but toxic, as well as with attendant effects [17,18]. This is shown by comparing the bio-safe syzygium cumini fruit juice, a natural antioxidant to the toxic BHT on serum enzymes such as ALT (alanine transferase), AST (aspartate transferase), alkaline phosphatase and urea in rats [19]. For this reason, it has become imperative to continue to investigate and search for more bio-safe antioxidants that could be relevant in the fight against oxidative stress. V. amygdalina is useful in this regard [20,21,22]. Kahaliw and his group have reported on the bio safety of this plant [23]. Moreover, anecdotal evidence attests to its use in the treatment of different ailments after boiling, as well as its use in the preparation of soup. This informed the aqueous extraction carried out, as opposed to the use of organic solvents, such as methanol and ethanol. This beneficial antioxidants; Phenols, flavonoids, proanthocyanidine and anthocyanin, amongst others, contains hydroxyl groups known for scavenging free radicals [24,25,26]. Phenolic compounds as antioxidants act as free radical chain reaction terminator. This combat oxidative stress responsible for neurodegenerative and cardiovascular diseases. Phenolic compounds constitute a large group of biologically active substances, such as Quercetin, catechin, ferrulic acid, caffeic acid, gallic acid, coumaric acid, and rutin. These are naturally occurring antioxidants. Even though the scavenging activity of the extract is not as high as the control, quercetin, the results suggest the presence of phenolics provided the active DPPH scavenging activity. The flavonoid content could have also provided the scavenging activity, as the activity-driven mechanisms of flavonoid are via chelating and scavenging process. It has been established that V. amygdalina contains these antioxidant agents [27,28]. Which is similar to the results in table 4? The slight differences observed, could be due to the polarity of the solvents used. A less polar solvent like methanol and ethanol would extract more antioxidant component of the plant compared to the water used. This properties and the report of Yagi and Khiralla qualifies this plant as a neutralcetical [28].
A lot has been reported on V. amygdalina as a functional food. In order to further establish its biosafety, the result in table 5 and the work of Kaali justifies V. amydalina as an anti-malaria agent that is bio safe for all the benefits discoursed above [29]. The study of Patnaik and Bhatnagar is in agreement with this study [30]. Moreover, Thompson showed comparable results [31]. Data from alcoholic extract of V.amygdalina is statistically indistinguishable compared to this study [32] (Table 5).

Conclusion
On the basis of the data from this current research, V. amygdalina is a potent antioxidant attributable to their flavonoid and phenolic constituent that is bio safe for all the health benefits that is known for.