Evaluation of Phenolic Content and Antioxidant Capacity of Eleusine coracana ( L . )

The aim of this study is to evaluate the content of phenolics: total seven phenolic acids and one flavonoid were separated from the species of Eleusine coracana (L.). The separated phenolics were characterized by various spectral techniques. The antioxidant capacity of all phenolics was determined by rancimat study of sunflower oil using control and standard drugs like Glipizide and Metformin. The stability period of sunflower oil was found to increase from 0.89 hr to 1.04 hr in presence of eight extracted ingredients of Eleusine coracana (L.), which was compared with result of neat sunflower oil.


Introduction
Eleusine coracana (L.) is native to East African highland, India and China and is considered as one of the richest sources of phenolics 1 and it contains flavonoides, which have high biological activity 2 .Many synthetic antioxidants are available but these compounds must be used under strict regulation due to their potential hazards 3,4 .Therefore, new interest has been developed as purifying and characterizing safe antioxidants from natural sources.The predominant flavonoides and phenolic acids, which are almost exclusively present in Eleusine coracana (L.), are linked with glycosides 5 .
There have been many attempts made to determine the contents and physiological activity of phenolic compounds present in Eleusine coracana (L.) due to the apparent relationship of phenolics in minor millet with prevention of chronic diseases.Phenolics are natural secondary metabolites and are widely used as potential components for hypertension, diabetes, heart diseases, osteoporosis, and some forms of cancer, avian flu, Endometriosis, chronic fatigue syndrome, tetanus, different types of cancers, lyme disease, chronic ear infection and even obesity are considered chronic diseases 6 .There are many factors like surrounding atmosphere, environmental conditions and soil conditions, which affect the phenolics and their antioxidant capacity of Eleusine coracana (L.).
Minor millets are rich in phytochemicals, including phytic-acid and phytate, which are believed to lower cholesterol and cancer risk 7 .Eleusine coracana (L.) is well known minor millet for its anti-nutrient constituents such as trypsin inhibitors, phytates, phenols and tannins 8 .Unfortunately, very less attention has been given to this millet for exploring its medicinal properties and therefore, the present study; as part of the continuing investigation on the antioxidant constituents of minor millet, deals with the separation and identification of the minor anti-oxidative components found in Eleusine coracana (L.) 9,10 .
Even though, some components occur only in minor concentrations in crude extracts, they could be strong antioxidants, which make them interesting for further purification and identification.Minor anti-oxidative components from Eleusine coracana (L.) were detected and separated using semi-prep-HPLC-PDA technique followed by GC-MS for their characterization 11 .The structure elucidation of the eight components and their overall antioxidant activity were reported herein.Thus, the general purpose of this work is to extend our knowledge on the chemical compositions of Eleusine coracana (L.).This would be a first step to assess the precision of this agricultural product as a source of natural antioxidants, potentially useful for the food industry to prepare functional foods or as natural antioxidant additives from Eleusine coracana (L.) 12 .

Materials and Chemicals
Eleusine coracana (L.) was collected from Millet Research Centre, Waghai, Gujarat-India with proper authentication under the supervision of Botanist.The collected samples were preserved in dark and dry place at ambient temperature with passive ventilation prior to extraction.
Silica gel 60 (Merck) with particle size 40-65 µm was used for column chromatography, while silica gel 60 F 254 pre-coated aluminum sheets (0.25 mm, Merck) were employed for TLC.HPLC analysis and separation were conducted on a quaternary gradient HPLC system containing W600e multi solvent delivery system from Waters, a degasser with Helium and 2996-photodiode array detector with EMPOWER 2 software.The system was equipped with a normal phased semi-preparative column Xterra MSC18 (7.8 × 100 mm; 5 μM) and flow rate was kept one mL.min - for all the experiments performed.HPLC solvent A was methanol (gradient grade) and solvent B was 10 mM ammonium acetate with pH adjusted to 4.14 using glacial acetic acid.The solvents used for extraction of active ingredients from Eleusine coracana (L.) (1% acidified methanol) and chromatographic separation were glass-distilled prior to use.

Extraction of phenolics from Eleusine coracana (L.)
The phenolics of Eleusine coracana (L.) were extracted successively with the help of 1 % acidified methanol solvent of HPLC grade using Accelerated Solvent Extractor (ASE) technique with the aid of ASE-300 multi channel system from Dionex, Germany.Ottawa sand was used to remove interference from moisture.The extraction conditions were optimized based on different percentage recovery of extracts in the different experimental conditions.The solvent was evaporated under reduced pressure to give the maximum percentage yield from Eleusine coracana (L.).An aliquot of 1%-acidified methanol was chromatographed by open column chromatography on silica gel, using chloroform: methanol mixtures of increasing polarity.Fractions of 500 mL in 10 mL sub-fractions in tagged vials were collected, monitored by thin-layer chromatography (TLC), to identify eight major constituents, which were subsequently given the numbers as C-1, C-2, C-3, C-4, C-5, C-6, C-7 and C-8.
An aliquot of crude fraction was chromatographed by semi-preparative HPLC.A linear gradient from 5% -50% and back to 5% of solvent B in 90 min was used to yield pure fractions from crude extracts of Eleusine coracana (L.).

Standard and sample solution preparation
Standard drugs were purchased from local market, dissolved and diluted up to one g.mL - in methanol and these solutions were centrifuge, filtered and stored for further use.The separated phenolics were dried after extraction and dissolved in methanol for obtaining 1 mg.mL -1 stock solutions of phenolics.

Purification of antioxidant
Semi prep-HPLC-PDA technique is extremely useful for the analysis of natural products containing phenolics.The chromatogram at multiple wavelengths was retrieved from the data files after analysis 13 .The HPLC retention time and the UV-VIS spectrum for any component (LC peak) are characteristic of certain compounds.The data are rapidly previewed for unique absorption regions correlating to specific compounds or functional groups 13,14 .Quaternary gradient semi-prep HPLC system with PDA detector 600e multi solvent delivery system from WATERS (EMPOWER 2) was used along with column Xterra MSC18 (7.8 × 100 MM, 5 µm).PDA detector (2996 Photodiode Array Detector) was used and the specific wavelength used for detection of phenolics is 236 nm.

Results and Discussion
Optimization of solvent and time was performed with six different solvents having different polarities to achieve maximum percentage yield of crude extract of Eleusine coracana (L.) for phenolic content and their results are summarized in Table 1.It was found that 1% acidified methanol is more suitable solvent for extracting the phenolics from Eleusine coracana (L.).To authenticate the data total eight injections of 5000 µL of eight standards were injected 16 .The average retention time (t R ) with their standard deviation and % RSD clearly suggested that all the eight components were separated under the similar experimental conditions with fine distinct peaks as described in the Table 2. Therefore, same practice was carried out for actual sample analysis as described herein 17,18 .The actual sample injection under similar conditions leads to separation of phenolics from crude extract of Eleusine coracana (L.) and a chromatogram is shown in the fig.2. Out of twelve separated components, eight were identified based on our results of standards and summarized data are depicted in the Table 3  The statistical analysis is done by using STAST software programme available in the Department of Statistics, Bhavnagar University.

Rancidity of Sunflower oil
Oils can be particularly susceptible to rancidity because their chemistry, which makes them susceptible to oxygen damage.During the process of oxidative rancidity, oxygen molecules interact with the structure of the oil and damage its natural structure in a way that can change its odour, its taste, and its safety for consumption.Rancidification is the decomposition of fats, oils and other lipids by hydrolysis or oxidation, or both.Oxidation primarily occurs with unsaturated fats by a free radicalmediated process.These chemical processes can generate highly reactive molecules in rancid foods and oils, which are responsible for producing unpleasant and noxious odours and flavors.These chemical processes may also destroy nutrients in food.Antioxidants are often added to fat-containing foods in order to retard the development of rancidity due to oxidation.Fresh 5.0 gm samples of sunflower oil were taken into vessel of rancimat and were subjected to five different temperature programmers because increased in the temperature increased the rate of oxidative rancidity in the oil.It was found that at 140 0 C sunflower oil became rancid after 1.11hr, while oxygen damage is found rapid in the presence methanol and sunflower oil became rancid within 0.89 hour under similar experimental conditions.Two standard drugs Glipizide and Metformin [22][23][24][25] were also used under similar conditions for their induction time and sustainability.It was found that presence of standard drugs increased the stability time from 0.89 hr to 1.04 hr suggests that standard drugs have good impact on rancidity of sunflower oil because both drugs able reduce the oxygen damage in the sunflower oil 10,18 .Similarly, eight components (C1-C8) were also used to improve the stability time for sustaining the oxidative rancidity.The stability time for sunflower oil was increased from 0.89 hr to 1.04 hr, which indicates that natural antioxidants extracted from Eleusine coracana (L.) have great impact in the reduction of induction time and increased the stability of oil considerably by decreasing the rate of oxygen damage or rancidity.The rate of induction time and stability period from crude extract of Eleusine coracana (L.) and standard drugs is depicted in fig. 3. The data for oxidative rancidity of sunflower oil with MeOH, standard drugs and crude extracts is summarized in the Table 4.

Conclusion
Neat sunflower oil became rancid in 1.11 hr at 140 0 C, whereas Sunflower oil became rancid in 0.89 hr in the presence of methanol, indicates rapid oxygen exchange between both molecules.Under similar conditions, the stability time was found increased in the presence of two standard drugs and stability of an oil was increased from 0.89 hr to 1.04 hr.Similarly, extract of Eleasine coracana showed good antioxidant activity when compared with standard drugs like those that Glipizide and Metformin and stability time was found increased up to 1.04 hr.It is also evident from the study that natural antioxidants are very much useful as an additive to sustain the food gradients for longer period and therefore, crude extract of Eleasine coracana should be explored by the food industries as source of natural antioxidants.

Figure 1 .
Figure 1.HPLC chromatogram of mixture of eight standards in gradient elution with one mg.mL - concentration.

Figure 3 .
Figure 3. (a) Rate of rancidity of sunflower oil in the presence of methanol; (b) Rate of rancidity of sunflower oil in the presence of methanol and standard drug Glipizide; (c) Rate of rancidity of sunflower oil in the presence of methanol and extracted ingredients of Eleusine coracana (L.).

Table 1 .
Optimization data for maximum Extraction yield in ASE.

Table 4 .
Summarized data of Rancidity of sunflower oil in the presence of media, standard drugs and Eleusine coracana (L.) extract.