Variation in bioactive composition , antioxidant attributes and fatty acids profile of Phoenix dectylifera L . fruits in relation to different extraction solvents

The present study was planned to determine the antioxidant and fatty acids profile of different varieties of Phoenix dectylifera L. The sample of Ajwa date, Ajwa paste, Moti jhari and Basrah were taken to evaluate the antioxidant potential and fatty acids of their pulp and seeds, because unsaturated fatty acids are useful for heart diseases. The extract yields from these samples ranged from 32.68 to 55.0 g/100g dry weight (DW). Maximum yield was obtained by 80% ethanol. All extracts contained appreciable amount of total flavonoid and total phenolic contents (20.6-43.5 CE mg/g DW), (110.6-490.9 GAE mg/100g DW), respectively. All date varieties exhibited good reducing power and DPPH radical scavenging activity with contribution of EC50 2.56 to 6.2 mg/mL and IC50 2.5-7.6 mg/mL of extract, respectively. Maximum amount of total flavonoids and total phenolics was obtained by 50% methanol and 100% methanol extract, respectively, whereas DPPH radical scavenging activity and reducing potential was higher for 50% ethanol and 100% ethanol extract. Extracts of ajwa date pulp and seed exhibited maximum value of total phenolic contents and extracts of ajwa paste exhibited maximum value of total flavonoid contents. Ajwa date exhibited greater DPPH scavenging activity and reducing potential. All samples contained a wide range of phenolic acids among which Gallic acid is the most diversified phenolic acid. Ajwa date contained maximum value of total phenolic acids (1005.03mg/kg). All samples contain appreciable amount of saturated and unsaturated fatty acids among which percentage of oleic acid (21%) was higher in Ajwa dates. The present results indicated that pulp and seeds of Phoenix dectylifera L. have good antioxidant potential, considerable contents of phenolics and appreciable amount of fatty acids, so can be used for prevention of heart diseases, in food industry, oil industry and pharmaceuticals as a natural source of antioxidants and preservatives.


Introduction
Role of nutritional factors on health status has long been identified.But recent clinical and epidemiological studies demonstrated the clear insight on physiochemical and chemical mechanisms of effects of bioactive compounds present in food on human health [1].Bioactive compounds are the phytochemicals which are produced naturally, being nonnutritive are synthesized by the plants as safeguards for attack by pathogenic microorganisms and external stress [2].Naturally occurring bioactive compounds such as, fatty acids, terpenoids, phenolics, coumarins, flavonoids, xanthones, curcuminoids and tannins are found in plant products such as peels, leaves, oils, seeds and fruits, several of these are proved as efficient as synthetic antioxidant in model systems [3,4].Bioactive compounds are reported to have diverse biological effects for instance antimutagenic, antioxidant, antimicrobial, anticarcinogenic and anti-inflammatory activities [5].Current studies have declared that date fruit has been shown to have tremendous antioxidant activity among the twenty-eight fruits commonly consumed in China [6].Phenolic acid content and antioxidant activity of dates demonstrated a linear relationship in some recent studies [7].Phoenix dactylifera commonly called dates are grown in over 30 countries.Nutrient-rich dates are enjoyed for their sweet, exotic and succulent flavor.In recent years, dates have found acceptance among consumers in European Countries and North America.Date fruit is also processed into a broad variety of value-added products beside fresh consumption [8,9].They serve as good source of natural antioxidants, so, considered as functional food [10,11].Pakistan being one of the top ten dates producing countries of the world and Pakistan has become the fourth largest country in the world that is exporting dates to the rest of the world.However, very little is known about fatty acids, phenolics composition, antioxidant activity and bioactive compounds of different varieties of locally available dates.So, it would be worthwhile to evaluate the fatty acid profile and antioxidant attributes of date fruit by different solvents such as chloroform, methanol and ethyl acetate.The nature of extracting solvent have a central role in the resulting extracts yields for their antioxidant activities and other important bioactive compounds of date's fruit leading to exploring their potential prospects in pharmaceuticals and food industries as natural preservatives and antioxidants.With this in mind, TPC, TFC, fatty acids and antioxidant properties along with individual phenolic acids composition of the dates from three important and widely cultivated dates were studied in the current work

Sample preparation and extraction
All samples (Ajwa date, Moti, jhari and Basrah) were cut manually into small pieces and the seeds were separated and were air dried and ground to fine powder.The ground material was stored in polythene bags.The Ajwa paste purchased from market to check the difference between commercial and Ajwa dates available in market.The ground material that passed through 80-mesh sieve was used for the extraction of antioxidant components and other bioactives compounds.All other chemicals (analytical grade) used in different experiments were purchased from Sigma-Aldrich Chemical Corporation, Germany.Extraction of all samples for antioxidant and other bioactive compounds was done by orbital shaker, using different solvents such as 100 % methanol, 100% ethanol, 80% methanol, 80% ethanol, 50% methanol and 50% ethanol.

Estimation of antioxidant activity TPC determination
Folin-Ciocalteu assay was used for determination of total phenolic contents (TPC) as described by Kalpna et al., [12].Briefly, 0.5 mL (2mg/1mL) of extract solutions were taken in test tube and 0.1 mL of Folin-Ciocalteu (0.5 N) reagent was added in each solution.Then they were incubated at room temperature for 15 min.Following that 2.5 mL of saturated solution (7% W/V) of sodium carbonate was added.The mixtures were again incubated for 30 minutes at room temperature.The absorbance was done at 700 nm using spectrometer (CECIL CE 7200).Amount of TP was calculated using Gallic acid as standard.

TFC determination
Total flavonoid contents (TFC) were calculated by following the experiment reported by Zhishen et al., [13] 1 mL of aqueous solution of extracts (10mg/mL) was placed in 10 mL volumetric flasks.Distilled water was added to make the total volume of every flask up to 5 mL.Then 0.3 mL NaNO2 (1:20) was added in each mixture.For 5 min mixtures were kept at room temperature.Following the addition of 0.3 mL AlCl3, the mixture was kept again at room temperature for 6 minutes, followed by 2 mL 1 mol litre - 1 NaOH.Total volume was made up to 10 mL by adding distilled water.The solutions were mixed again and absorbance was taken at 510 nm against blank.The results were expressed as catechin equivalent (10-500 ppm) (R 2 = 0.9919) CE mg/g of dry weight.

DPPH scavenging activity assay
The antioxidant activity of Phoenix dectylifera L. varieties was assessed by 1, 1 diphenyl-2-picrylhydrazyl radical (DPPH .) scavenging assay.The DPPH assay was performed by using the method followed by Zhuang et al., [14] According to the assay, 1 mL of extracts solutions were taken with 0.25 mg-2mg/mL concentration. 2 mL of 0.1 mM DPPH solution in methanol was added to each mixture.Methanol was added to make the solutions up to 4 mL.The extracts were kept in dark at room temperature for 30 minutes.The absorbance was measured at 517 nm.BHA was used as a positive control.IC50, the concentration of extract which scavenged 50 % of DPPH free radicals, was calculated.% scavenging activity=(A control -A extract)/ Acontrol* 100 The % scavenging activity at 50 % is IC50

Determination of reducing power
The reducing power of Phoenix dectylifera L. extracts were determined by using method following by Zhuang et al., [14] with minute alteration.According to which different extracts concentrations (2-10) mg were formed by mixing the extracts with 3.5 mL phosphate buffer (0.2 M, pH 6.6), then 2 mL of 1 % potassium ferricyanide in all test tubes was added.The mixtures were incubated for 20 minutes at room temperature.After that 2.5 mL of 10 % trichloroacetic acid was added to the mixtures.For 10 minutes mixtures were centrifuged at 3000 rpm.2.5 mL of supernatant fluid was collected and its volume made up to 5mL by adding 2.5 mL of distilled water.After that 0.5 mL of 0.1 % ferric chloride was added.The absorbance of mixtures was taken at 700 nm.

Calculation of phenolic acids profile by HPLC
Individual phenolic acids were determined by HPLC.Separation of phenolic acids was done by Varian HPLC.Acidified acetonitrile (99.5%) was used as mobile phase.By using a microsyringe, the column was injected with 20-μL sample.It was detected at 280 nm.By matching the relative retention times with standards of targeted compounds, phenolic compounds were identified.Peak area measurement gives the contents of individual compounds.

Extraction of oil for fatty acids profile
In this method the sample was dried, grind into fine particles and placed in porous cellulose thimble which was placed in an extraction chamber.About 500ml of nhexane was placed in round bottom flask.The flask was heated on heating mantle, solvent evaporates and drop into the extraction chamber containing the sample.The process was repeated thrice.After that the solvent was evaporated and mass of the remaining oil was measured.

Formation of methyl esters for fatty acids profile determination of Phoenix dectylifera L.
One drop of oil was taken in round bottom flask having 100ml capacity.Then one pellet of KOH was added in round bottom flask having one drop of oil.Then 30ml of pure methanol was added.After that a condenser was attached to this flask with proper circulation of water.Then the round bottom flask with attached condenser was placed in heating mantle for about 30 min only to condense methanol.After 30 min heating was stopped and the round bottom flask was cooled in water immediately.Whole mixture in round bottom flask was transferred into separating funnel (250-500ml), then only 10 ml of n-hexane were added in this separating funnel, shaked it gently and allowed to stand for the formation of 2 layers.Lower layer was discarded.Then small amount of distilled water was added in separating funnel, shaked it slowly and allowed it to stay for 5 min.After the formation of two layers again, lower layer was again discarded.Repeat the addition of water two times to wash all residues of methanol in mixture.Then FAMES were poured into beaker, small amount of sodium sulphate were added in it to absorb moisture then filtered it.

Fatty acids profile by GC
The dates oil methyl esters were analyzed on a SHIMADZU gas chromatograph, model 17-A, coupled with flame ionization detector (FID).Separation was made on a methyllignocerate coated polar capillary column SP-2330(30 m*0.32mm*0.20µm;Supelco, Inc, Bellefonte, PA., USA).Nitrogen was used as a carrier gas at a flow rate of 3.0 mL/min, temperature column was programmed from 180 to 220 o C at the linear rate of 5 o C/min.Initial and final temperatures was set for 2 and 10 min, respectively.Injector and detector were set at 230 and 250 o C, respectively.A sample volume of 1.0 µL was injected using split mode (split ratio of 1:75).By comparing their relative and absolute retention times to those of authentic standards, FAMEs were identified.The internal standard used was heptadecanoic acid (C17:0).

Statistical analysis
Data was expressed as mean values.Two way ANOVA procedures were used to carry out analysis of variance.Significant difference for values relative to means of solvents was determined.P > 0.05 makes difference that is not significant.Analysis was done by using SPSS software [15].

Results and discussions
The extraction yield for antioxidant components of Phoenix dectylifera L. with methanol (100%, 80% and 50%) and ethanol (100%, 80% and 50%) is shown in Table 1.The extract yield of Phoenix dectylifera L. varied over range of 32.68% to 55.04% (g/100g).Maximum yield (55.04%) was obtained with 80% ethanol while minimum (32.68%) by 50% methanol for Phoenix dectylifera L. In the present study by seeing results, the order of extraction value of different solvents was seen to be: 80% ethanol >100% ethanol >50% ethanol > 100% methanol > 80% methanol >50% methanol.The higher yield of Phoenix dectylifera L. with 80% ethanol revealed that 80% ethanol has greater effectiveness to extract antioxidant components from Phoenix dectylifera L. A significant difference (p< 0.05) has been observed in the extracted yield relative to the solvent system and Phoenix dectylifera L. used.

DPPH scavenging activity assay
It is observed from graphical representation in Figure 1 A & B that the required concentration for reducing 50% of lipid peroxidation was significant (P < 0.05).The IC50 data for different date varieties.The IC50 for the Phoenix dectylifera species peels ranged from 2.5-8.2mg/mL.The highest value was noted for 100% ethanolic extract of basrah (8.2 mg/mL) while lowest for 50% ethanolic extract of ajwa date (2.5 mg/mL).IC50 (mg/mL) values for different species peels were as: 2.5-4.25 for ajwa date, 2.9-4.3 for ajwa paste, 2.9-4.5 for moti jhari and 5.

Reducing power assay
Antioxidant activity and reducing capacity assessment are directly related to each other.So, reducing capacity may serve as significant indicator to determine the antioxidant activity.This assay is basically based on the capability of antioxidants to reduce ferric III ions to ferrous II ions.EC50 is the concentration at which 0.5 absorbance is obtained.Greater is the value of EC50, smaller is the reducing potential [23].The EC50 for different date varieties from different solvents is given in table 5.5.EC50 value ranged from 2.56 to 6.2 mg/mL of extract.The lowest value is for moti jhari (2.56 mg/mL) from 100% ethanol as solvent while the highest was for ajwa paste (6.2 mg/mL) using 50% ethanol.The EC50 values for Phoenix dectylifera are 3.86-4.9for ajwa date, 4.56-6.2for ajwa paste, 2.56-4.5 for moti jhari and 2.65-4.4 for basrah.The ability of extraction solvent towards recovery of reducing agents from Phoenix dectylifera is in the order of: 100% methanol > 80% methanol > 50% ethanol > 80% ethanol >50% methanol > 100% ethanol as shown in Figure 2 A & B While 50% ethanol extract exhibited higher reducing potential among others indicating a significant (P< 0.05) among solvents.The reducing potential for is in Phoenix dectylifera the order of ajwa paste > ajwa date > moti jhari > basrah and thus indicates significant variations among dates variety.These readings confirmed Al-Farsi et al., [12] suggested that mostly antioxidants in dates are hydrophilic.However, the majority of antioxidants in date fruit are water soluble which is the major contribution to act as antioxidant in lipids membranes system.This can be, to some extent, due to the property of flavanols which have the ability to react in both side of water and lipid layers of cell membranes [22], and thus protecting from oxidative degradation.

A B
Figure.1A and B. Graphical representation of DPPH by using different concentration of solvents

Table 1 . Extract yields (g/100 g) from different extracts of Phoenix dectylifera
Values are means ± SD (n=3) of three separate experiments.Different caps letter in subscript within the same column show significant differences of means among Phoenix dectylifera L. varieties.Different superscript letters within the same row show significant differences of means among extraction solvent.

Table 5 . Fatty acid profile of oil extract from Phoenix dectylifera. L Relative percentage of fatty acids in different date pulp and seed oils Fatty acids Type of Fatty acids Ajwa Date% Moti Jhari % Basrah%
ConclusionAll results indicated that pulp and seeds of Phoenix dectylifera L. have good antioxidant potential so can be used in food and pharmaceutical industry as a natural antioxidant and as a preservative.Maximum value of TPC was found in extract of ajwa date and TFC was in ajwa paste extract while values of DPPH and reducing power were higher in extract of ajwa date.Phenolic acids have antioxidant and medicinal properties.The most abundant phenolic acid in dates was gallic acid.So, present findings conclude that seeds from dates would serve as valuable source of fatty acids and substitute the other vegetable oils.Depending on the regional industry, present data recommend the possible application of date seed oil in cosmetics and pharmaceutics and may be the some other more applications.Our findings supported the use of dates as a potential source of valuable natural antioxidants as well as for pharmaceutical, for development of nutraceutical, in value addition and food industries.Authors' contributionsConceived and designed the experiments: T Mehmood, Performed the experiments: ZA khan & Abdul Karim, Analyzed the data: T Mehmood, M Akram, A Afzal & F Siddique, Contributed reagents/ materials/ analysis tools: MA Shaheen, Wrote the paper: T Mehmood & MA Shaheen.