Evaluation of Antioxidant Activities of Celosia trigyna (Linn) Extracts African Extinction Vegetable

Celosia trigyna recognized as a known ancient vegetable unfortunately unpopular in this modern age. In this study, antioxidant activities of Celosia trigyna leaves were evaluated in crude extract (methanol) and different fractions; chloroform, hexane, methanol and ethylacetate. Antioxidant evaluation were established using total antioxidant activity (phosphomolybdeum) and antiradical scavenging activity (2,2-Diphenyl-1-Picrylhydrazyl (DPPH)) and Hydrogen Peroxide (H2O2) methods. C. trigyna leaves showed the highest values of Total phenolics content (TP) (0.162mg, Rutin equivalent per g methanol fraction) while the lowest value of TP (0.092mg ± 0.001 GAEg-1) was recorded in the chloroform fraction. Ethylacetate fraction of C. trigyna leave extract was highest (0.192 ± 0.003mg GAEg-1) in Total Antioxidant Activity. Interestingly the C. trigyna methanol fraction exhibited the highest percentage inhibition for both DPPH and H2O2 scavenging activities of 89.22% and 88.24% respectively at concentration 0.6mg/ml. All the fractions including the C. trigyna crude methanol extract showed strong scavenging activity on DPPH as the concentration increases while the scavenging activity of the C. trigyna leaves methanol extract and fractions were high at 0.6mg/ml concentration. C. trigyna is presented as an alternative natural antioxidant to synthetic antioxidants used in foods and pharmaceutical industries.


Introduction
Recently, research focus on plants has incredibly risen all over the world and quite a several plants are discovered to have medicinal advantages especially for humans [1].Developing countries around the world invest immensely on traditional herbs in the treatment of diseases.Since civilization began, Human beings within their Habitant found remedies to ailments and adopted several strategies varying climate, socio-cultural, floral, and phytogeographic characteristics.Traditionally several methods are invoked to get rid of diseases and ailments which include the use of plants as herbs for prevention and cure [2].Several medicinal plants are found around the world, this is the plant with the potency of healing; which has therapeutic effects or foundational compounds that can be synthesized for useful drugs [3].Human has depended largely on medicinal drugs in past centuries.Though a great number of research works have been done on numerous medicinal plants especially from the Amaranthus.This study is aimed at evaluating the antioxidant activities of the crude extract of Celosia trigyna L leaf (extinction vegetable) and its fractionated extracts.
The Celosia species is an edible plant belonging to the Amaranthaceae, it is ornamental with a small genus.Its name Celosia was derived from the Greek word for 'burned' which is 'Kelos', signifying it flamelike flowerhead [4].C. trigyna L is an erect annual herb of about 120(-180) cm tall; has a simple or branched stem, grooved, glabrous, or with few hairs, usually pink to silver color.The leaves are alternate, simple, without stipule petiole up to 5(- Generally, most of the Celosia species are known in the treatment of different ailments such as diarrhea, disinfectant, inflammation, bleeding nose hematological, piles, treating cold and gynecological disorders according to traditional practitioner consulted.He further disclosed that when crushed, leaves can be used in the treatment of chest trouble and stomach aches.The C. trigyna leaves have been using to conclude the several medicinal preparations using in the treatment of women's disorder diseases, including ovarian troubles and treatment of wounds.
Antioxidant activity is a very important pharmacological property.The property of the plant is responsible for the functions it performs such as anti-aging, anti-carcinogen, antimutagenicity, and so on.Superoxide, hydroxyl, and nitric oxide radicals are reactive oxygen and nitrogen which are also referred to as the most important free radicals of the body.The consequence of cellular and metabolic activities generates these free radicals.Excessive production and leakage from their site cause degeneration of the body cells and they appear to be the major contributor to aging and diseases.These free radicals also contain unpaired elections; thus, making them unstable, having an affinity for electrons from other substances to get them neutralized.This firstly stabilizes the radicals but also generates another radical in this process causing more chain reactions which can occur within a few seconds [5].Synthetic and Natural antioxidants are the two known categories.The synthetic type is made of phenolic compounds with alkyl substitution while the natural antioxidant can either be made of phenolic, nitrogen, and carotenoids or ascorbic.Those of phenolic are in the form of tocophenols, flavonoids, and phenolic acid while nitrogen compounds are alkaloids, chlorophyll derivatives, amino acids, and amines.Plant in nature contains natural antioxidants because of their exposure to ultraviolet light during the day leading to the synthesis of various free radicals.These free radicals have a built-in system that inhibits cellular damage which may lead to dying and weathering of the plant [6].
Vitamins Such as A, C, E which are vitamin Anti-oxidants are needful to be included in the diet because they are not naturally produced by the human body, folic and beta carotene are also good anti-oxidation which are part of human daily food intakes.Vitamin A (retinol and carotenes) is very useful in the improvement of the immune system, repair of wore out tissues, eye healthiness, and control human cholesterol level.Vitamin C protects the skin from damage, infections, and promotes good iron absorption.Vitamin E helps healthy blood vessels, protecting the body membrane, and improving skin conditions.In regards to the protection against free radicals, beta-carotene is the best [7].
All of these vitamins are most commonly found in oranges, colored vegetables such as spinach, carrot.With the aid of plant antioxidants, free radicals are converted into healthpromoting less reactive species which in turn prevent degenerative diseases.Due to the latest discoveries of defects from synthetic antioxidants, the biochemical function of the plants with naturally occurring antioxidants is being focused.The human body can then be protected from oxidative stress, free radicals and associated diseases through substances derived from the plants which are a good source of natural antioxidative of the most important natural antioxidants are the phenolic compounds which reduce agents and activators of an antioxidant defenseenzyme system that suppress the damages from radicals in the biological system.Thus, this study is purposed to evaluate the new potentials of the natural anti-oxidant in Celosia trigyna L., which will be contributing to the antioxidant activity of the vegetable extract for the development of new drugs and food preservatives.

Plant Collection
Trigyna L. leaves were collected at the back of the faculty of Sciences, the Polytechnic, Ibadan, North local Government Area, Oyo State, Nigeria.The geographical location of the leaves collection area is approximately het longitude 07°25¹ 36.61°N and 07°27¹ 22.7¹ N and latitude 03°52¹ and 03°53¹ 45.81°E [8].Identification authentication of C. trigyna L was done at Herbarium section of Forest Research Institute of Nigeria with FRI NO 111896.

Preparation of Crude Extract
The leaves Celosia trigyna L. were pulverized into a coarse powder.766.9g of the powder leaves were soaked in 5liters of 95% methanol for 72hours, the extract decanted and filtered.A rewash of the residues with methanol was carried out and the two filtrates collected were further filtered using Whatman filter paper (1mm).The filtrate collected was then concentrated using a rotary evaporator that was set at 40°C.This is the Crude Methanol Extract of Celosia trigyna plant leaves, which was further concentrated using a vacuum oven set at 40°C with a pressure of 700mmHg, placed in a glass bottle, and preserved for onward use.

Fractionation of Celosia trigyna L. Leaves Methanol Extract Using Vacuum Liquid Chromatography (VLC)
Fractionation of Celosia trigyna L. leaves methanol extract was achieved by modifying the method of Okwuchi (2015) [9].Celosia trigyna L. leaves methanol extract (12 g) was dissolved in 120 ml of methanol to make it easy to mix with silica gel powder.100g of silica gel was weighed and placed into the sintered glass funnel compressed under pressure from the vacuum pump and then rinsed with n-hexane, drained dried under pressure before the crude extract mixture was packed and pressed under pressure.The Crude Method Extract of C. trigyna (CMECT) was pre-adsorbed, dried, and used to prepare the extract bed.The solvents were added from the less polar to the more and drained until a very clear solvent was observed on each elution.The fractions were concentrated and the remaining solvent in the concentrates was further allowed to dry and the contents were then stored in glass containers and stored at 4°C until ready for use.The fractions obtained were a fraction of the hexane (HFCT), chloroform fraction (CFCT), ethyl acetate fraction (EFCT) and methanol fraction (MFCT).The vacuum liquid chromatography and thin-layer chromatography techniques were used for fractionation.The different fractions (HFCT, CFCT, EFCT, and MFCT) obtained from the CMECT of Celosia trigyna leaves were spotted on a pre-coated TLC plate of the different solvent mobile phase.

Total Phenolic Content Estimation
The total phenolic content of the Celosia trigyna leaves crude and fraction extracts were determined by spectrophotometric method according to Folin -Ciocalten method using gallic acid to set up the standard curve.0.25ml of the extract was mixed with 0.5ml Folin-C phenol reagent.After 5minutes, 5ml of 7% Na 2 CO 3 solution was added to the mixture, followed by the addition of 65ml distilled water.The mixture was thoroughly mixed, allowed 90minutes incubation at 25°C and thereafter the absorbance was reading at 750nm.The total phenolic content of C. trigyna leaves crude and fraction extracts were evaluated from a gallic acid standard curve expressed as GAE meaning Gallic Acid Equivalent, ml/g of extract.Blank is distilled water and the values were in triplicates [10].

Determination of Antioxidant Activities
The antioxidant activities of Celosia trigyna leave crude and fraction extracts were established using three (3) different antioxidant assays noting that one method cannot be sufficient to prove the antioxidant efficiency of plant products [11].

DPPH Radical Scavenging Activity
The radical solution was prepared by dissolving 2.5mg of DPPH in 100ml of methanol (0.025g/l) solution.Different concentrations of the C. trigyna leaves stock extract solution (1mg/ml) were added to 3.9ml of DPPH solution and the reactants incubated at 25°C room temperature for 30 minutes.
In place of an extract, the Ascorbic Acid solution (a positive control) was used as standard.The mixture was shaken and the solution was allowed to stand in the dark at room temperature for 35 minutes.Free radical scavenging activity was calculated from absorbance values at 517 nm.Blank was also measured which was distilled water.

% Inhibition =
Abs of Control -Abs of extract Abs of Control × 100

Total Antioxidant Activity
The total antioxidant activity of the Celosia trigyna leaves crude and fraction extracts were assessed spectrophotometrically by the phosphomolybdenum method [12].1m1 of C. trigyna leaves samples was mixed separately with 3ml of reagent solution (28mM Sodium Phosphate buffer, 0.6M H 2 O 4 and 4mM ammonium molybdate); the blank solution contained 4ml of reagent solution only.The mixtures were passed through incubation for 90mins at 95°C.After, the absorbance was measured at 675nm after the mixture had cooled to room temperature.Total antioxidant activity was expressed as Ascorbic acid equivalent.

Hydrogen Peroxide Scavenging Assay
The hydrogen peroxide (H 2 O 2 ) scavenging activity of C. trigyna leaves crude and fraction extracts was determined by replacement titration method with shift mollification.Aliquot of 2ml of 1mM H 2 O 2 and 1ml of various concentrations of the extracts were mixed, then added 2 drops of 3% ammonium molybdate, 7 ml of 1.8mM potassium iodide, 10ml of 0.2 MH 2 SO 4 , and 2 drops of 1% Starch indicator.The mixtures from the C. trigyna leaves samples were titrated separately with 0.5mM sodium thiosulphate until the disappearance of blue color.Percentage of scavenging of H 2 O 2 was calculated as:

Abs control − Abs of sample
Abs of Control × 100 Where Vo and V1 were the volume of sodium thiosulphate used for the titration of blank and the sample extract respectively.IC50 (mg/ml), which denotes effective concentration yielding 50% inhibition of H 2 O 2 radicals was also calculated [13].

Results and Discussion
The percentage yield of Celosia trigyna L leaves extract (methanol crude extract) was 10.65%.The total phenolic contents of the leaves of C. trigyna methanolic extract is 0.144mg ± 0.005 GAEg-1, hexane fraction (0.094±0.003GAEg-1), chloroform fraction (0.092±0.001GAEg-1), ethyl acetate fraction (0.107±0.002GAEg-1) and methanol fraction of C. trigyna is 0.162±0.003GAEg-1 as shown in table 1.This shows that naturally occurring antioxidants are primarily phenolics that are from all parts of plants especially from fruits and vegetables [14][15][16].The DPPH scavenging activities of C. trigyna L leaves methanol crude extract and its fractions were presented in table 2. The DPPH scavenging activities of the extract and fractions were compared with Ascorbic acid.Inhibition Percentage of the C. trigyna leaf extract and its fractions on DPPH Scavenging activity were present in Table 3 where methanol fraction of C. trigyna (MFCT) has the highest Percentage Inhibition on DPPH Scavenging Activity at 0.6mg/ml relative to the standard.In vitro condition, DPPH is considered as a stable free radical where it accepts an electron or hydrogen radical to become a stable diamagnetic molecule.The reduced ability of DPPH radical Scavenging was determined by the decrease in its absorbance at 517nm and by the disappearance of purple color to a new color, yellow which is induced by antioxidants.The coloration indicates the scavenging potential of the antioxidant compound in the extract.The different fraction of the enable extract at various concentrations induced a rapid decrease in the optical density.
Total Antioxidant activities can be defined as the measure of the number of free radicals scavenged.The Total Antioxidant Activity was highest (0.192 ± 0.003mg GAEg-1) in ethyl acetate fraction of C. trigyna leave extract followed by methanol fraction of C. trigyna (0.141±0.001mgGAEg-1), leaves crude methanol extract of C. trigyna (0.093 ± 0.003mg GAEg-1), chloroform fraction of C. trigyna (0.051 ± 0.012mg GAEg-1) while hexane fraction of C. trigyna (0.010 ± 0.001 mg GAEg-1) has the least total antioxidant activity (Table 4).The Hydrogen Peroxide Scavenging Activity of the C. trigyna leaves methanol extract and its fractions and Percentage Inhibition of C. trigyna leaves extract and its extract fractions on hydrogen period (H 2 O 2 ) were presented in Tables 5 and 6 respectively.The C. trigyna methanol fraction exhibited the highest percentage inhibition for H 2 O 2 scavenging activity of 88.24% at concentration 0.6 mg/ml whereas the chloroform fraction exhibited the lowest percentage inhibition for H 2 O 2 scavenging activity of 12.50% at concentration 1.0mg/ml.

Conclusion
In the study Tin Layer Chromatography (TLC) investigation of C. trigyna L leaves extracts revealed that there is high hope of exploring more antioxidant compounds for therapeutic and drug manufacturing following the trend of all the antioxidant assays and in the food industries, C. trigyna L leaves showed a high inhibitory property both in the DPPH and H 2 O 2 scavenging activities.Moreover, Total Phenolic content and Total Antioxidant determination showed that there is hope for this wild spinach to be explored deeper for use.These indications also show that the leaves of C trigyna L have beneficial health effects in humans as they help in the treatment of sores and boils costal pains, heart complaints, stomach ache, urethral disorder, and chest pains.The Celosia trigyna L leaf is not only used in treating ailment but also very well in soup making, in that the Yoruba call it "aje fo awo, meaning eat and break plate".Interestingly finding naturally occurring antioxidants has increased to replace synthetic antioxidants which are toxic and carcinogenic.The study also established that phenolic compounds contribute greatly to the antioxidant activity of C. trigyna L leaf extracts.

Recommendation
Further studies on C. trigyna L leaf extracts are recommended to be carried out to isolate and purify the active compounds useful in the pharmaceutical and food industries because the plant has shown various compounds on thin-layer chromatography (TLC) plates.The promising compounds separated on TLC plates may be responsible for C. trigyna L leaf traditional treatment of diseases including ovarian troubles, excessive menstruation, women's disorders, cancer, and so on.
8) cm long.Celosia plants occur has almost fifty (50) species in the region of tropical and subtropical, C. trigyna L in the tropical is said to be the most widespread.Vegetables such as Celosia globosa, C. isertii, and C. leptostachya are often confused with Celosia trigyna.Among plants of this species, C. trigyna though not well known in this present age is a wild type of Celosia found almost throughout tropical Africa, South Africa, and Southern Arabia.It is known as a weed called aje fo awo meaning eat and break the plate, it is a vegetable that is common among the old people in the Southern part of Nigeria.

Table 1 .
Total Phenolics Content (GAEmg/g) of the Celosia trigyna leaves extract and its fractions.

Table 2 .
DPPH scavenging assay for Celosia trigyna leaves methanol crude extract and its fractions UV Absorbance Readings (Optical Density).

Table 3 .
Inhibition Percentage of the Celosia trigyna leaves methanol crude extract and its fractions on DPPH Scavenging activity.

Table 4 .
Total Antioxidant Activity (TAA) (GAEmg/g) of the Celosia trigyna leaves methanol crude extract and its fractions.

Table 5 .
Hydrogen Peroxide Scavenging Activity of the Celosia trigyna leaves methanol extract and its fractions.

Table 6 .
Percentage Inhibition of Celosia trigyna leaves extract and its extract fractions on hydrogen period (H2O2).