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Determination of Chemical Composition, Total Phenolic Content and Antioxidant Activity of Xylanthemum Macropodum.

Byline: Samiullah, Fariha Alam, Samina Aslam, Sajida, Rasool Bakhsh Tareen, Abdullah Khan, Naqeebullah Khan, Ali Akber, Imran Ali, Abdul Kabir Khan, Muhammad Raza Khan, Muhammad Anwer Panezai, Muhammad Aslam Buzdar, Saleha Suleman Khan and Athiq-ur-Rehman

Summary: Evaluation of the phytochemistry, total phenolic content and antioxidant activity of the endemic plant of northern Balochistan Xylanthemum Macropodum of the Asteraceae family, is reported for the first time in this document. Chemical composition of Xylanthemum Macropodum was determined using well-established chemical tests and modern spectroscopic techniques. Extracts were taken from the whole plant using methanol and the extracts were tested for phytochemicals (secondary metabolites), total phenolic content (TPC) and antioxidant activity. The phytochemical (biochemical) examination of Xylanthemum Macropodum exposed the presence of alkaloids, phenols, steroids, flavonoides, tannins, terpenoids, saponins, coumarins, carbohydrates, cardiac glycosides, reducing sugars, and quinines.

TPC of crude methanolic extract (CME) of plant was determined using Folin-Ciocalteu's reagent. The TPC determined was 256mg of tannic acid Eq/g of extract. Antioxidant activities were determined spectrophotometrically using the DPPH assay and Ferric ion (Fe+3) reducing antioxidant power assay. The potency of the DPPH assay of Xylanthemum Macropodum extract was 68% for the 0.10 mg/ml concentration and the FRAP value of the extract was 3.368 mmol Fe+2/g of extract. Xylanthemum Macropodum has proved to be very rich in secondary metabolites, natural phenolics and has a very potent antioxidant activity.

Keywords: Phytochemistry, Antioxidant activity, Total phenolic content (TPC), Xylanthemum Macropodum.

Introduction

The ancient human civilization used plants as medicines. The great ancient Chinese, Indian and North African civilizations contribute written confirmation of man's insight in using plants for the treatment of a variety of diseases. A landmark in the field of medicine was made in 19th century when therapeutics made use of the idea of isolating biologically active constituents from analeptic plants. Revelation of quinine, the famous antimalarial agent, was executed by Caventou and Pelletier, French researchers who proved to be revolutionary in the field of phyto-remediation [1].

Phytochemistry is the study of natural products executed from plants. It is concerned with the scientific study of plant constituents, elaborating their natural distribution and the process of metabolism along with their biological functions [2].

The active constituents from medicinal plants are known as phytochemicals. Phytochemicals habitually contain medicaments which are responsible for plants pigments, taste, aroma and appearance. Phytochemicals are grouped into two categories i.e. primary metabolites that include carbohydrates, lipids, proteins and secondary metabolites that encompass alkaloids, flavonoids, terpenes, saponins and phenolics, which are present in different parts of the plant such as roots, fruits, flowers, or leaves [3]. Approximately half of the world's communities trust natural product as drugs due to their safety and efficacy. It was realized between 1984 -1995 that around 39.5 % of 522 innovative medications originated from plants or was their derivatives, and most of the antiseptics and anticancer remedies exhibit plant ancestry.

A study during the start of the 21st century revealed that close to 61% of all drugs in medical judgments were from plant sources. Moreover, in 2001, out of 30 maximum marketed medicines today, eight (azithromycin, pravastatin, cyclosporine, amoxicillin, clavulanic acid, simvastatin, ceftriaxone, and paclitaxel) were of natural origin [4].

Oxidation is one of the major metabolic pathways of cells. During the oxidation process a number of reactive species are formed in the form of free radicals such as negatively charged superoxide radicals (O2)-, hydrogen oxide radical (OH)., the precursors of free radicals such as hydrogen peroxide (H2O2) and alkyl peroxides (ROOR). These free radicals can attack electron rich centers in bio molecules like fats, proteins and DNA, due to this attack the cell metabolism is disturbed, resulting in the depletion of the immune system, carcinogenesis, cardiovascular diseases and inflammatory diseases [5]. Antioxidants are the molecules, which can scavenge such oxidants and inhibit tissue damage [6]. A number of medical judgments and epidemiological examinations have discovered an inverse association among the consumption of fruits, vegetables and diseases like depletion of the immune system, cancer, cardiovascular diseases and inflammatory disease [7].

Vegetables, fruits and medicinal plants are found to be the rich sources of antioxidants [8]. Thus therapeutic potential of medicinal plants are therefore related to their antioxidant potential [9, 10]. Phenolics and flavonoids are considered as the major antioxidants found in plants [11]. Radical scavenging activity was found to be directly related with the total phenolics contents of plant extract [12]. The antioxidants isolated from medicinal plants were found to be far more active than those found in fruits and vegetables [13]. Therefore, plant extracts are investigated for their antioxidant potentials and also to isolate the antioxidants that are more active and less harmful to living organisms [14]. As a result, this is of immense importance to the public, medical practitioners, dietary experts and nutrition researchers to know the antioxidant potential of different edible and medicinal plants.

A number of assays are used to study the antioxidant power of plant extracts, both in vivo and in vitro. According to the reaction mechanism these assays are divided into two broad categories: Hydrogen atom transfer assay (HAT) such as inhibition of induced LDL auto-oxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), crocin bleaching assays and electron transfer assay (ETA) that includes the TPC assay by the Folin reagent, Trolox equivalence antioxidant capacity (TEAC), ferric ion (Fe3+) reducing antioxidant power (FRAP), "total antioxidant potential" assay where Cu(II) complex captures electrons, and DPPH assay. Every individual assay measures the antioxidant activity under particular conditions applied in that system, therefore, it is not correct to oversimplify the information obtained as indicators of "total antioxidant activity".

Antioxidant activity of the extract greatly depends on the extraction methods and conditions such as temperature and solvent. In most studies, the solvent used was ethanol, methanol, ethylacetate, acetone and boiling water. It is important to notice that the herbal medicines are used at homes under conditions that are quite different from the conditions applied in research laboratories, therefore, the antioxidant potential will be quite different in both [14-16]. Xylanthemum Macropodum is included in the Asteraceae family, known as compositae or the aster, a composite family of the blossoming plant form asterales. It is not only distributed along the northwestern juniper forests of Balochistan but also frequently found in the regions of WaliTangi and Hanna Urak. Xylanthemum Macropodum is similar in structure to the rest of the Asteraceae family having yellow flowers, erect shrubs and 30-40cm tall shrublet, which are slender, monocephalous and leafy.

Extraction can be done by using various methods such as maceration, digestion, soxhlet extraction, percolation, sonication, decoction, and supercritical fluid extraction [17]. To the best of our knowledge no work is documented on the phytochemistry, antioxidants, total phenolic contents or bioactivity of this species. However, the medicinal importance was observed by the local residents as it plays a vital role in the control of diarrhea and jaundice. This study will be the first investigation to explain the phytochemistry, phenolic content and the antioxidant capacity of this plant species.

The aim of this study is the determination of the phytochemistry, the antioxidant capacity through different assays and the determination of the total phenolic content of Xylanthemum Macropodum of Asteraceae family to understand the potential of this endemic plant as a rich source of phytochemicals and antioxidants.

Experimental

Reagents and Solutions

All plastic and glassware were washed with detergent and further rinsed with ultra-high purity water. All the plastic and glassware were kept in dry and clean place. All of the reagents were of analytical grade, supplied by Merck.

Plant Material

The plant samples were collected from Hanna valley, north of Quetta, and the plant species was identified by Professor Rasool Bakhsh Tareen.

Solvent Extraction

After drying, the plant material was ground to a coarse powder. The granular portion was separated from the fine powder and was grinded once again. The powdered plant material weighed 4kg and was soaked in methanol 10L for 3 days. The mixture was filtered through filter paper (Whatman No.1) followed by re-extraction of the left over crude with 5L methanol until the color of the solvent remained unchanged. Methanol was removed using a rotary evaporator and the dry crude methanolic extract (CME) which weighed 200g was contained in an air tight bottle and stored in a cool, dark and dry place. This methanolic extract was used for phytochemical analysis, determination of antioxidant activity and total phenolic content of Xylanthemum Macropodum.

Phytochemical Screening

The freshly obtained crude methanolic extract was qualitatively tested for the phytochemicals present in Xylanthemum Macropodum. Methods from recent literature with standard procedures were used for the determination of all the phytochemicals present in the plant material [18-21].

Test for Alkaloids

For alkaloids, 0.55-0.65g of the crude methanolic extract was mixed with 8.5ml of 1% hydrochloric acid followed by warming and filtration. The filtrate was divided into three equal parts in separate test tubes and mixed with Wagner's reagent, Mayer's reagent and Dragandroff's reagent and then observed for precipitate formation and color change.

Test for Phenolics

Two procedures were followed to test the presence of phenolics:

1. The plant CME was mixed with a few drops of neutral ferric chloride solution and was observed for intense violet color change.

2. In the second test for phenolics, a small amount of plant extract was first dissolved in water and then 3% of lead acetate (3.5ml) was added, after which formation of bulky white precipitates was observed.

Tests for Flavonoids

For testing the presence of flavonoids the plant extract was first defatted by treating with petroleum ether. To the defatted residue 80% of 20.5ml ethanol was added, mixed and filtered. The filtrate was used for the following tests:

1. The filtrate was mixed with 4.5ml of 1% potassium hydroxide and was observed for color change.

2. The presence of flavonoids was also examined by adding 5.5ml of ammonia solution and concentrated sulphuric acid and observed the color change.

Tests for Saponins

For saponins 0.55g of plant extract was shaken with distilled water and was heated till it boiled. The mixture was shaken to froth appearance.

Tests for Steroids

1. For testing of the presence of steroids, equal volumes of plant extract and chloroform was mixed with some drops of concentrated sulphuric acid and was observed for ring formation.

2. 0.55g of plant extract was mixed with 2.5ml of acetic anhydride and sulphuric acid and change in color from violet to blue was observed for steroids.

Tests for Terpenoids

For terpenoids screening, the plant extract was dissolved in distilled water and then 2.5ml of chloroform and 3.5ml of sulphuric acid was added and in the in interface a reddish brown coloration was observed.

Test for Tannins

0.55g of plant extract was dissolved in 20.5ml of distilled water and was filtered. To the filtrate 0.1% of ferric chloride was added and a brownish green coloration was observed for the presence of tannins.

Test for Coumarins

To test the presence of coumarins, 1.5ml of 10% sodium hydroxide was added to the plant extract, and a color change was observed.

Tests for Glycosides and Carbohydrates

Molisch's test

10% alcoholic alpha naphthol was added to the plant extract followed by 2.5ml sulphuric acid. A bluish violet zone indicated the presence of carbohydrates and glycosides.

Fehling's Test

To perform Fehling's test for carbohydrate presence, the Fehling's solution was prepared and was labeled as A and B. From both the solutions 5.5ml was added to the plant extract and it was heated. The formation of reddish brown precipitates was observed for the presence of reducing sugars.

Detection of Cardiac Glycosides

2.5ml of glacial acetic acid was added to 0.55ml of the plant extract, with the addition of few drops of ferric chloride (5%). To this mixture 1ml of sulphuric acid was added and establishment of a brown ring indicated the presence of cardiac glycosides.

Test for Quinones

For testing the presence of quinones, 1.5ml of sulphuric acid was added to the plant extract and a red color formation was observed.

Test for Anthraquinones

2% of hydrochloric acid was prepared for testing the presence of anthraquinones and a few drops of it was added to 0.55ml of the plant extract. A red precipitate formation indicated the presence of anthraquinones.

Test for Phlobatannins

The plant extract (0.55ml) was mixed with a 10% ammonia solution and the formation of pink precipitates was observed.

Detection of Fats

Saponification Test

The plant extract was mixed with 0.55N solution of potassium hydroxide followed by adding a few drops of phenolphthalein, and was heated for 2-3 hours on a water bath. A soap formation was observed for the presence of fats.

Determination of Total Phenolic Content (TPC) of Crude Methanolic Extract

Folin-Ciocalteu's Reagent reducing capacity of methanolic extract

The TPC of Xylanthemum Macropodum was calculated using the methods developed by Barku, Chodak (2011), Chodak (2009), Wojcikowski, Yan and Naeema [12-16 and 22]. In this method TPC was determined spectrophotometrically by using Folin's reagent. Initially, the sample solution was prepared by dissolving 1.5mg of the CME in 1ml methanol.

0.5ml of sample (1.5mg/ml) was added to equal volumes of 1/10 times diluted Folin's reagent. After 7 min, 5ml of 7% sodium carbonate (Na2CO3) solution was added to the mixture and shook properly. The mixture was placed in darkness at 23degC to avoid any interaction with light for 90 minutes before measuring the UV absorbance at 750nm. The TPC was calculated from a calibration curve derived by repeating the same procedure with tannic acid solutions of concentrations ranging from 0 to 50 mg/L (Fig. 1). The total amount of phenolics in the CME was calculated from a standard curve of tannic acid. The TPC was expressed in milligrams of tannic acid equivalents (TAE) per gram of the CME. The total phenol value was obtained from the regression equation: y= bx +- a where y is UV absorbance at 750 nm and x is the amount of tannic acid in mg/L.

DPPH Radical Scavenging Capacity Assay

The DPPH radical coupling assay was calculated following the methods developed by Barku, Naeema and Yan [12, 16 and 22]. The DPPH assay involves the following procedure while determining the antioxidants in the crude methanolic extract (CME). The solutions of the CME of different concentrations 0.02, 0.04, 0.06, 0.08, and 0.1 mg/ml were prepared in methanol. Newly prepared 1ml DPPH solution (0.1 mM=3.943 mg/100ml in methanol) was mixed with 2ml of all the samples 0.02, 0.04, 0.06, 0.08, and 0.1 mg/ml. Ascorbic acid solutions of the same concentrations 0.02 to 0.1mg/ml were prepared in the same solvent and applied as positive control in the DPPH assay. After the addition of DPPH solution to the test samples, the solutions were kept in the dark place for 30 minutes. The UV-VIS absorbance was calculated at 517 nm.

To measure the absorbance of the control the plant extract was replaced by methanol i.e. 1ml DPPH (0.1mM solution + 2 ml methanol) in order to make the concentration the same as was taken in the case of test solutions. To measure the absorbance of the plant extract alone as a blank, DPPH was omitted from the reaction mixture and replaced by methanol. To calculate the DPPH radical scavenging capacity assay, the absorbance of the blank (crude plant extract alone) was subtracted from the absorbance of the reaction mixture and expressed as the percent drop off in the absorbance of the reacting species compared with that of the control. The greater radical capturing capability of the CME of Xylanthemum Macropodum is indicated by the declined absorbance of the reaction mixture.

The considerability to capture the DPPH free radical by CME was measured through the following equation: Percentage of radical capturing activity = (Abs(control)-Abs(sample))/Abs(control)x100%

Where,

Abs(control) = Absorbance of DPPH solution

Abs (control) = Absorbance of the CME and vitamin C (ascorbic acid) solutions after reacting with the

DPPH in dark for 30 minutes. The DPPH radical capturing activity of the CME compared with vitamin C (ascorbic acid) is shown in Fig. 2.

Ferric Ion (Fe+3) Reducing Antioxidant Power Assay

The ferric ion (Fe+3) reducing antioxidant power of the CME of Xylanthemum Macropodum was determined by the procedure developed by Saeed et al [22]. Different concentrations of the extract were prepared in the range of 0.02 to 0.1mg/ml in distilled water. To one milliliter of each concentration of plant extract was added phosphate buffer (2.5ml, 0.2M, pH 6.60) and 1% Potassium ferricyanide [K3Fe(CN)6] (2.5ml). The reaction mixture was incubated at 50-51degC for 25 minutes followed by the addition of 2.5ml of 10-11% trichloroacetic acid (Cl3CCOOH). The solution was centrifuged for 15 minutes at 1500 rpm. From the upper layer, 2.5ml of the supernatant was added to equal volumes of distilled water and half milliliter of 0.1% ferric chloride (FeCl3) solution. The absorbance was calculated using UV-Vis spectrophotometer at 700 nm against a blank.

To measure the absorbance of the plant extract alone as a blank, 0.1% FeCl3 was omitted from the reaction mixture and replaced by distilled water. To determine the ferric ion reducing antioxidant power assay the absorbance of the blank was subtracted from the absorbance of the reaction mixture. Ascorbic acid was used as standard antioxidant. The raise in the absorbance of the reaction mixture shows the augment in the reducing power of the CME of Xylanthemum Macropodum (Fig. 3).

Ferric (Fe+3) Reducing Antioxidant Potential (FRAP) Assay.

The ferric reduction power of CME of Xylanthemum Macropodum was measured using an advanced method of the FRAP assay [11]. This method is based on the reduction of ferric ions (Fe+3) to ferrous ions (Fe+3) present as central metal atoms in the tripyridyltriazine complex, at a low pH. The reduction is indicated by a clear color change of a colorless Ferric complex to a blue Ferrous complex. The electron donating species present in the CME of Xylanthemum Macropodum provide the electron for this reduction. The absorbance of electrons by the Ferric complex is monitored by measuring the change in UV-VIS absorbance at 593 nm. The FRAP reagent was synthesized by the addition of 10ml of 23mM acetate buffer (pH 3.65) to 1ml of 10mM tripyridyl triazine (TPTZ) in 400mM HCl followed by the addition of 1ml of 20mM FeCl3. Several concentrations of FeSO4.7H2O ranging from 0mg/L to 500mg/L were prepared to obtain a standard curve.

All solutions were freshly prepared and used. Half milliliter of the CME solution (5mg/ml) and 1ml of distilled water were added to 2 ml of FRAP reagent. The reaction mixture was incubated for half an hour at human body temperature. The UV-VIS absorbance of the samples was measured at 593 nm. The same procedure was repeated with standard solutions of FeSO4.7H2O as well to get the values for calibration curve (Fig. 4). A blank reading was taken using acetate buffer in place of a reagent. The difference between sample absorbance and the blank was calculated and further applied to calculate the FRAP value. In this experiment, the reductive potential of the plant extract tested was measured with reference to the reaction output given by a Fe+2 solutions. FRAP values were determined as mmol Fe+2/g of the CME.

Table-1: Shows the summary of the tests performed to investigate the occurrence of various phytochemicals in Xylanthemum Macropodum

S.No Phytochemicals###Tests###Observation###Results

1###Alkaloids###2ml of plant extract + few drops of Wagner's reagent###Red precipitates are formed###+

###1ml filtrate + few drops of Mayer's reagent.###white precipitates appeared###+

###1ml of filtrate + 2ml of Dragendroff's reagent###Yellow precipitate appeared###+

2###Phenols###Plant extract + few drops of neutral FeCl3solution###Intense color developed###+

###0.5g extract dissolved in distilled H2O + 3ml of 10% lead acetate###Bulky white precipitates appeared###+

###0.5ml of plant extract + equal volume of CHCl3+ with few drops of

3###Steroids###Appearance of brown ring###+

###conc. H2SO4

###0.5g extract + 2ml of acetic anhydride + 2ml of H2SO4###Color change from violet to blue or green color###+

###An interface with a reddish brown coloration

4###Terpenoids###5ml of extract of each + 2 ml CHCl3 + 3ml of H2SO4###+

###formed

5###Flavonoides###3ml of defatted filtrate + 4ml of 1% potassium hydroxide###Dark yellow color observed###+

###Yellow coloration observed, which

###5ml of dil. ammonia solution + extract + H2SO4###+

###decolourised on addition af acid.

###0.5 g extract was boiled in 20ml of distilled water, filtered + 0.1%###Brownish green or a blue black coloration

6###Tannins###+

###FeCl3###observed

###0.5g of extract was dissolved in boiling water in a test tube, was

7###Saponins###Froth formation was observed###+

###cooled and shaken vigorously

8###Coumarins###1 ml plant extract + 10% sodium hydroxide (1ml)###Formation of yellow color###+

###Molisch's test

9###Carbohydrates###Water solution of extract + 10% alcoholic alpha naphthol + 2 ml###Appearance of bluish violet zone###+

###sulphuric acid

###Fehling's test

###Reducing

###5ml Fehling's solution A and B + aqueous solution of plant extract###Reddish brown precipitates were formed###+

###sugars

###+ heat

###Cardiac###0.5ml of plant extract + 2 ml of glacial acetic acid + few drops of Formation of brown ring at the interface was

10###+

###glycosides###5% ferric chloride + 1 ml of conc.sulphuric acid###observed

11###Quinone###Plant extract + 1 ml conc. sulphuric acid###Formation of red color was observed###+

Results and Discussion

The present work aims to evaluate the phytochemical compositionof the methanolic extract of Xylanthemum Macropodumof the Asteraceae family by qualitative methods and also executing its antioxidant property by using various standard procedures.

The phytochemical analysis of this endemic plant showed very interesting results (Table-1). Natural products are not only a good source of bioactive compounds used as drugs but also help in the design of new effective synthetic drugs. The survey of the phytochemicals extracted from this plant has revealed positive tests for the presence of alkaloids, phenols, steroids, terpenoids, flavonoids, tannins, saponins, coumarins, carbohydrates, reducing sugars, cardiac glycosides and quinones.

The presence of various poly phenols (phenolics, flavonoids, flavonols) indicates its medicinal and pharmacological importance [20]. The antioxidant activity of polyphenols is well documented and plays a vital role in decreasing coronary diseases, age related disorders, brain degeneration and more importantly their anticancer activities make them vital for living organisms [23].

The total phenolic content and all the natural antioxidants present in the CME of Xylanthemum Macropodum is determined using four well established methods.

Routine and wide use of colorimetric reactions in the laboratory and UV/VIS spectrophotometers have made them vital in quantitative and qualitative determination of different compounds in plant extracts and synthetic products [20]. The low cost and easy use of these methods have made them very useful. A standard reference, however, is always needed in colorimetric methods to calculate the concentration of unknown compound in the test sample [23].

The Folin-ciocalteu reagent forms a blue complex of phosphotungsticphosphomolybdenum with the polyphenols present in the plant extract, which can absorb the photons of the visible region and can be exploited to calculate the concentration of total polyphenols spectrophotmetrically [20]. The stability of the blue complex decreases in the alkaline solution therefore, excess reagent is used, and the resulting turbidity of the solution is cleared by adding lithum salts as suggested by Folin [24]. The increase in the absorbance indicates the higher concentration of antioxidant or polyphenols present in the CME. The complex formation reveals accurate concentrations of different polyphenolic groups depending on the structure and reaction kinetics [24, 25]. Several studies have revealed the use of the Folin-ciocalteu reagent to determine the total phenolic content in the CME of plants [26-28].

In this investigation Folin-ciocalteu reagent was used to calculate the total phenolic concentration (TPC) found in the CME of Xylanthemum Macropodum. Once the complex formation was complete in 90 minutes in the dark the absorbance was measured at 750 nm. The TPC was measured from extrapolation of the calibration curve (Fig. 1) using standard solutions of tannic acid (0 to 500mg/L). The TPC was expressed as mg of tannic acid equivalents (TAE) per gram of the CME. Total phenol value was obtained from the regression equation: y= bx +- a where y is absorbance at 750 nm and x is the concentration of tannic acid expressed in mg/L. TPC is calculated from the standard curve of tannic acid. The regression equation for the standard curve of tannic acid is: y = 0.0063x + 0.0377. Where y is absorbance at 750 nm and x is the concentration of tannic acid.

For the absorbance observed for sample (0.243) the concentration of tannic acid calculated from the calibration curve was 32mg/L. This concentration is present in the diluted solution of the extract of which the volume increased from 0.5ml to 6ml under the reaction conditions. However, in the original test solution (1.5 mg/ml) the quantity of phenolics is 384mg TAE/L. Currently, the total phenolic content in one gram of extract was determined to be equal to 256 mg TAE/g of extract. These results reveal that Xylanthemum Macropodum is rich in polyphenolics.

Free radicals are generally highly unstable molecules however, they could be stabilised through resonance and conjugation. In this investigation DPPH radical was chosen to determine the concentration of antioxidants in the CME of Xylanthemum Macropodum. Due to the stability of this radical it can be used in readily available solvents (water, methanol and ethanol) [29]. The electronic transition of this free radical involves the absorbance of photons having a wavelength of 517nm. The change in the position of the absorbance band along with colour can be easily observed once the free radical scavenges a hydrogen atom from the antioxidants present in the CME. The radical capturing capacity of the CME is observed at all concentrations (Fig. 2). The free radical capturing activity of the CME is determined by comparing it to the natural antioxidant ascorbic acid.

The percentage inhibition of DPPH increases to 68% at a concentration of 0.1mg/ml of the CME solution indicating that Xylanthemum Macropodum is rich in antioxidants.

The reducing power of the plant extract is also determined by reducing ferric ions (Fe+3) to ferrous ions (Fe+2) by the presence of antioxidants in the CME as compared to ascorbic acid (Fig. 3). This reduction is readily observed by change in the absorption maximum of potassium ferricyanide [K3Fe(CN)6], since it is reduced to potassium ferrocyanide [K4Fe(CN)6] by the CME which is then converted into ferri ferrocyanide Fe4[Fe(CN)6]3 in the presence of FeCl3. The increase in the absorbance shows higher concentration of antioxidants in the CME.

An advanced version of the FRAP assay was applied to measure the antioxidant concentration in the CME. A colourless ferric complex (Fe+3tripyridyltriazine) is reduced to a bluish colored ferrous complex (Fe+2-tripyridyltriazine) by natural antioxidants present in the plant extract in a media where less protons are available. The reduction is easily observed by measuring the change in absorbance at 593 nm. The concentration of natural antioxidants found in the CME is calculated from a standard calibration curve (Fig. 4). The FRAP assay shows a concentration of 3.368mmol Fe+2/g of extract. This concentration of antioxidants in Xylanthemum Macropodum shows that it is a rich source of natural antioxidants.

The regression equation for the standard curve of FeSO4.7H2O is: y = 0.0037x + 0.0009, where y is absorbance at 579 nm and x is the concentration of FeSO4.7H2O. The absorbance of the test solution of the extract measured at 593 nm was 2.476. The concentration of FeSO4.7H2O calculated from the calibration curve was 669mg/L. This was the concentration of the test solution under the reaction conditions i.e. it is diluted from 0.5ml to 3.5ml. For the original test solution of extract (5mg/ml), the concentration of FeSO4.7H2O becomes 4683mg/L. Thus the concentration of Fe+2 in the test solution is 943.3 mg/L.

The FRAP value for our extract is 3.368mmol Fe+2/g of the extract

This investigation discovers all the phytochemicals, polyphenolics present in the Xylanthemum Macropodum. This research also finds out the concentration of antioxidants present in the CME of plant by using four well established methods. Xylanthemum Macropodum is rich in secondary metabolites and polyphenolics. This plant also contains high concentration of antioxidants. Further research will continue on the isolation and evaluation of the bioactivity of different fractions of the CME of Xylanthemum Macropodum.

Acknowledgement

We are thankful to the University of Balochistan and Sardar Bahadur Khan Woment University for Facilitating this project.

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Publication:Journal of the Chemical Society of Pakistan
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Date:Feb 28, 2017
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