In vitro inhibitory effects onα-glucosidase andα-am ylase level and antioxidant potentia lof seeds of Phoenix dactylifera L.

Shah A lam Khan,Am ira Rashid A l Kiyum i,Manal Saif A l Sheidi,Tagreed Salim A l Khusaibi,Noura Mohammed A l Shehhi,Tanveer A lamDepartmentof Pharmacy,Oman Medical College,Muscat,Sultanate of OmanOman Medicinal Plants&Marine Natural Products,University of Nizwa,Barkat Al Mouz,Nizwa,Sultanate of Oman



In vitro inhibitory effects onα-glucosidase andα-am ylase level and antioxidant potentia lof seeds of Phoenix dactylifera L.

Shah A lam Khan1*,Am ira Rashid A l Kiyum i1,Manal Saif A l Sheidi1,Tagreed Salim A l Khusaibi1,
Noura Mohammed A l Shehhi1,Tanveer A lam21Departmentof Pharmacy,Oman Medical College,Muscat,Sultanate of Oman
2Oman Medicinal Plants&Marine Natural Products,University of Nizwa,Barkat Al Mouz,Nizwa,Sultanate of Oman

Original article http://dx.doi.org/10.1016/j.apjtb.2015.11.008

ARTICLE INFO

Article history:

Received in revised form 21Sep,2nd revised form 22Oct2015

Accepted 10 Nov 2015

Availableonline 11 Jan 2016

Phoenix dactylifera L.

Antioxidant

In vitro antidiabetic activity Date pits

ABSTRACT

Ob jective:To evaluate and compare the antioxidant activity,total phenolic contents (TPCs)and in vitro antidiabetic activity of variouspitsextracts obtained from fi veOmani date cultivars.

M ethods:Sun-dried mature fruits of fi ve Omani date varieties,namely,Fardh,Naghal, Khalas,Khinazi and Khasab were purchased from the localmarket in M uscat,Oman in the month of September 2014.Four seed extracts viz.water,ethanol,methanol and acetone were prepared for each date variety and their antioxidant activitieswere investigated by 1,1-diphenyl-2-picrylhydrazyl,hydrogen peroxide scavenging method and reducing power assay method,respectively.In vitro antidiabetic activity of the date pit extracts was evaluated by measuring their inhibitory effect onα-glucosidase andαamylase level.TPCswere also quanti fi ed colorimetrically.

Resu lts:The results indicated that TPC of date seedswas solvent dependent.Acetone, ethanol and methanol were found to be signi fi cantly better solvents than water in extracting phenolic compounds from the date seeds.Pit extracts exhibited moderate to good in vitro antioxidant activity and increased reducing power.Among all date pit extracts,water extractexhibited signi fi cant in vitro antidiabetic activity in comparison to standard drug,acarbose.

Conclusions:The present study con fi rms that disposed waste of Omani dates is a rich source of dietary antioxidant because of its high TPC.The pits due to their inhibitory effectsonα-glucosidase andα-amylase level could beused asamonotherapy along w ith an appropriate diabetic diet and exercise orm ight be in conjunction w ith antidiabetic therapy tomanage and prevent progression of diabetes.

1.Introduction

Plants have played an important role in drug development[1]. They have always been a common source ofmedications,either in the form of traditionalpreparations oraspureactive principles [2].Plants known for their curative powers are used for a w ide spectrum of diseases,from the common cold and fever to paralysis and diabetes[3].

Nature has bestowed Oman w ith an enormous wealth of medicinal plants.One common plant is the date palm[Phoenix dactylifera(P.dactylifera)]w ith almost 10 m illion grow ing along the Northern Batinah coastal strip[4].The fruit of these palms“dates”are considered as the most traditional and popular food especially for breaking the Ramadan fast. Eating dates after fasting helps tomaintain blood sugar levels and is an excellent source of dietary fi ber,potassium, magnesium,and complex sugars[5].The bene fi cial effects of dates are not lim ited to the date fruit only but date seeds or pits,which are left after consum ing date fruits,considered as disposed waste are equi-important.These pits,seeds or disposed waste of date food have been used traditionally asanimal feed,a source of oil,a coffee substitute in“Arabic Qahw a”,a raw material for activated carbon and an adsorbent for dye-containing waters throughout gulf region[6].The date seeds contain a high percentage of carbohydrate (81.0%–83.1%),protein(5.17%–5.56%),oil(10.19%–12.67%),ash(1.12%–1.15%)and oleic acid(41.3%–47.7%). Date seed oil is also reported to contain various polyphenolic com pounds,namely,hydroxytyrosol,protocatechuic acid, tyrosol,gallic acid,caffeic acid,p-coumaric acid and oleuropein[7].Several reports have indicated that there may be a correlation between phenolic content and antioxidant capacity of plant extracts[8].Hence,date seeds m ight be considered as a potential source of natural edible oil, antioxidants,and pharmaceuticals[9].

Preclinical studies have shown that the date fruits aswell as seeds possess free radical scavenging,antioxidant,antimutagenic,antim icrobial,anti-in fl ammatory,gastroprotective, hepatoprotective,nephroprotective,anticancer and immune–stimulant activities[10].Crude methanol,acetone and water extracts obtained from pits of three varieties of P.dactylifera grown in Saudi Arabia were found to bemore effective than the leaves extracts of the plant against the selected Gram positive and Gram negative pathogenic bacteria[11].The study concluded that the antimicrobial activity of date pits m ight result from the selective or synergistic action of various chem icals present in date palm.It has also been reported that treatment w ith date seed extract could protect against cerebral ischem ic damages in male rats most probably due to its antioxidant properties[12].Ardekani et al.,screened seed extracts of fourteen date varieties grown in Iran for their antioxidant activities and polyphenol contents.They observed a signi fi cant correlation between the total phenolic content (TPC)and antioxidant activity[13].Seeds of fi ve varieties of date palm grown in Qassim region,Saudi Arabia,were analyzed by Ammar and Habiba for their proximate composition,TPC and antioxidant activity by using different solvents for extraction viz.water,acetone,methanol and ethanol[14].They reported that ethanol was the best solvent for extracting phenolic compounds,followed by acetone and methanol.A ll seed extracts also exhibited signi fi cant antioxidant activity determ ined as Trolox equivalent. However,results of another sim ilar study conducted on Egyptian date seeds revealed that both non polar and polar fractions of the date seeds possess potent antioxidant and estrogen-like activity[15].Many studies conducted elsewhere have shown a direct relationship between antioxidant,TPC and antidiabetic activity[16,17].

Because there have been no studies assessing the in vitro antidiabetic effect of seeds of P.dactylifera dates grown in Oman,this study was carried out to evaluate the in vitro antioxidant potential and the inhibitory effect of date pits onαglucosidase andα-amylase level as an indication of antidiabetic activity.

2.M aterials and m ethods

2.1.Drugs and chemicals

1,1-Diphenyl-2-picrylhydrazyl(DPPH),caffeic acid,αglucosidase,α-amylase and acarbose were purchased from Sigma–A ldrich(USA).Folin–Ciocalteu reagent and ascorbic acid were obtained from Merck,Germany.A ll other chemicals and solvents used in the study were of analytical grade procured locally.

2.2.Collection of plantmaterial

Mature fruits of fi ve locally grown Omani date varieties, namely,Fardh,Naghal,Khalas,Khinaiziand Khasab which are listed among top 10 date palm cultivar produceof Oman[18]and are commonly consumed,were purchased from the localmarket of Muscat,Oman in themonth of September 2014.The dates were authenticated by the botanist and sample vouchers(DS/ PH/1–5)were also deposited in the pharmacy lab of Oman M edical College for future reference.

2.3.Evaluation of physical properties and preparation of date seed extracts

Five different cultivars ofmature date fruits having uniform size,free of physical damage,injury from insects and fungal infectionswere picked up by hand and used for preparation of extract.Sampleswere washed w ith tap water and the pitswere removed manually for preparation of extract of varying polarities.

2.3.1.Evaluation of physical characteristics of date

fruits

Twenty dates from each variety were random ly selected and individually weighed using an analytical balance.The date pit was removed manually and it was re-weighed again.The average weightof date fruit,fruit pulp,date pit,fruit fl esh percentage,total number of fruits/kg and pulp/pit ratio etc.were calculated.

2.3.2.Preparation of date seed extracts

Pits obtained from the date fruit were thoroughly washed under running tap water and then dried at 45°C for 24 h.Date pits were coarsely powdered using heavy duty grinder and fi nally passed through 1 mm sieve to obtain fi ne powder of uniform particle size.Approximately 0.5 g of powdered date seeds was suspended in 10 m L of four different solvents viz. water,ethanol,methanol and acetone separately and kept overnight in a tightclosed container.Eachm ixturewasstirred brie fl y (5–10m in)at6 h interval to speed up the extraction and fi nally centrifuged to obtain clear supernatant liquid extracts of varying polarities.

2.3.3.Qualitative phytochemical screening

The freshly prepared date pit extractswere also subjected to qualitative chem ical tests to identify various classes of bioactive chem ical constituents present in the date pits using standard procedures[19].

2.4.Estimation of TPCs

Total phenolics in date pit extracts were determ ined colorimetrically by using Folin–Ciocalteu reagent as per the reported method of Ardekani et al.[13].Caffeic acid was used as a reference standard for constructing the external calibration curve in the concentration range of 4–64μg/m L.The linear regression equation from the standard plot of caffeic acid was used to calculate TPC in various extracts and TPC resultswere expressed asmg/g caffeic acid equivalent(CAE)of dry powder.

The TPCs were calculated using the follow ing linear regression equation obtained from the external calibration curve of caffeic acid:

y=0:0133x−0:0133;R2=0:9925

where y is absorbance and x is amount of caffeic acid inμg.

2.5.Determination of antioxidant activity

2.5.1.DPPH free radical-scavenging assaymethod

The antioxidantactivity of various date pitextracts(0.1m L) was evaluated by using DPPH(1 mmol/L in methanol)free radicals as per the previously reported method[20].Ascorbic acid(5–100μg/m L)was used as a positive control for comparison purpose.

2.5.2.Hydrogen peroxide(H2O2)scavenging assay method

The ability of the date seed extracts(0.1 m L)to scavenge H2O2was determ ined according to the UV spectrophotometric method of Vadnere etal.[21].Ascorbic acid(5–200μg/m L)was used as a reference material for comparison of antioxidant activity.

2.5.3.Ferric reducing power assaymethod

Total reducing power was determ ined by colorimetric method as previously described by Nizam and M ush fi q[22]. Ascorbic served as a positive control.Increased absorbance at 700 nm by the reaction m ixture of standard or extracts indicated increased reducing power i.e.antioxidant action. Percentage increase in reducing power was also calculated by the follow ing formula:

%Increase in reducing power=［（AT/AB）−1］×100

where,ABisabsorbanceofblank(containing all reagentsexcept the testmaterial)and ATis absorbance of test solution.

2.6.In vitro antidiabetic activity

2.6.1.α-Amylase inhibition

Theα-amylase(bovine pancreaticα-amylase;EC 3.2.1.1, Sigma)inhibition by the standard drug acarbose(12.5–200μg/ m L)and pit extracts(0.1 m L)was performed by using the chromogenicmethod adapted from A li et al.[23].Potato starch (0.5%,w/v)in 20mmol/L phosphate buffer(pH 6.9)was used as a substrate solution while a mixture of sodium potassium tartrate and 3,5-dinitrosalicylic acid was used as colorimetric reagent.Inhibition ofα-amylase activity by extract or standard was determined by measuring the absorbance at540 nm which was due to the reduction of 3,5-dinitrosalicylic acid to 3-am ino-5-nitrosalicylic acid.

2.6.2.Inhibition ofα-glucosidase enzyme

α-Glucosidase inhibitory activity of pitextracts(0.1m L)was measured at540 nm using a reportedmethod[24].Brie fl y,2%w/ v sucrose and acarbose(12.5–200μg/m L)served as a substrate and positive control,respectively.

2.7.Data analysis

A ll sampleswere analyzed in triplicate and the resultswere expressed asmean±SD.The obtained data were statistically analyzed by SPSS(version 19)using one way of ANOVA and signi fi cant difference between means of tested parameters was determined by using Turkey post hoc multi-comparison test.A P-value less than 0.05 was considered statistically signi fi cant.

3.Resu lts

3.1.Physical characteristics of date fruits

The physical characteristic data of fi ve commonly consumed Omanidate varietieswere presented in Table 1.It could be seen from the results that a signi fi cant difference(P＜0.05)existed between varieties for almostall the physical parameters studied. Averagenumberof fruits/kg in date varieties ranged from 125 to 239.Khalas and Fardh were found to contain the highest and lowest number of fruits/kg,respectively.Therefore,mean weights of fruit and fl esh were also the highest(8.33 g and 8.01 g)for Fardh date.Though,Khalas date had the highest number of date fruits/kg i.e.smaller in size,the lowest fruitand fl esh weights were observed for Khinazi variety(4.28 g and 3.65 g).However,there was no signi fi cant difference between the Khalas and Khinazivarieties.Average pitweights of all fi ve cultivars ranged from 0.24 to 0.92 g.Itwasexpected thatKhalas being lighter and smaller in size(239 fruits/kg)would have the lowest pit weight(0.46 g)but contrary to this Fardh had the lowest pitweight(0.24 g).Naghal had the heaviestpit followed by Khinazi(0.63 g)and Khasab(0.54 g).A signi fi cant differencewasobserved inmean pitweights of all date varieties.The percentage of fruit fl esh in date varieties ranged from 82.84%to 96.21%and Naghaldatehad the lowestedible portion(82.84%) w ith respect to other four varieties.The highest fruit fl esh was found in Fardh(96.21%).On the other hand,the percentage of disposed waste i.e.seed or pit and fl esh/pit ratio varied greatly from 3.79%to 17.16%and 4.83%to 33.36%,respectively (Table 1).

3.2.Phytochemical screening

Prelim inary phytochem ical testing showed thatpitsof alldate varieties contained bioactive macromolecules such as tannins, carbohydrates,proteins and amino acids in all date varieties, however,steroidal compound and alkaloids were found to be absent in the date pitextracts.

3.3.TPCs

The resultsofTPCof thevariousdatepitsextracted by using four differentsolventsindicated thatacetone,ethanolandmethanolwere signi fi cantly better solvents than water in extracting phenolic compounds from thedateseeds(Table2).Among threeorganic solvents, acetone appeared to be the best solvent for the extraction of maximum phenolic compounds[(1.41±0.16)–(1.58±0.06)mg/g CAE].TPC of ethanol[(1.32±0.21)–(1.51±0.01)mg/g]and methanol[(1.40±0.03)–(1.46±0.04)mg/g]solventsshowed little variationbutwerebetter than the contentofphenolic compounds in water[(0.85±0.03)–(1.29±0.04)mg/g].ForFardhand Khinazi,the order of solvents according to extraction of phenolic compoundswasobserved tobeacetone＞methanol＞ethanol＞waterwhile for Khasab,itwasin theorderofethanol＞methanol＞acetone＞water. Fardh date pits showed the highest TPC in acetone[(1.58±0.06) mg/g]and methanol[(1.46±0.04)mg/g].Further,no signi fi cant statisticaldifferencewasobserved in TPCofdatecultivarsextracted by using acetoneandmethanol(Table 2).

Table1 Physical characteristic of date fruits.

Table2 TPC asmg/g of CAE in date seed using different solvents.

3.4.In vitro antioxidant activity

The antioxidant activity of four crude extracts of date pits was investigated by ferric reducing power assaymethod and by two other commonly used radical scavengingmethods such as DPPH and H2O2.The scavenging effect of pit extracts on the DPPH and peroxide free radicalswere expressed as%inhibition and were compared w ith standard antioxidant,ascorbic acid.

Ascorbic acid showed a dose dependent scavenging activity of DPPH radical,inhibiting 93.83%of free DPPH radicals at a concentration of 100μg/m L.The IC50value of ascorbic acid was found to be 13.68μg/m L.The resultsof antioxidantactivity of date pits presented in Table 3 indicated that the%inhibition of DPPH varied w ith the polarity of solvent.A ll the solvents exhibited moderate to good antioxidant activity by DPPH method.For Fardh,Khasab and Khinazi date pits,acetone extract exhibited the highest antioxidant activity(78.25±2.38, 73.50±8.37 and 70.92±10.16,respectively)while surprisingly,it was the least active extract for Naghal variety (17.21±11.62).M ethanol extract displayed better activity than the ethanol and water extracts.In fact,antioxidant activity of aqueous extract for all date varieties except Fardh was noted to be better than the ethanol extract.In general,Fardh and Khasab pitswere observed to possess signi fi cantantioxidantactivity by DPPH scavenging method.Acetone and ethanol extract of Naghal variety appeared to be the weakest antioxidant but methanol and water extract showed prom ising activity.The highest inhibition of DPPH for each date variety in a particular solvent were observed as follows:Fardh in acetone(78.25), Khalas in water(66.27),Khinazi in acetone(70.92),Khasab in acetone(73.50)and Naghal in methanol(69.44).A signi fi cant statistical difference in activity of each extractwas observed.

No signi fi cantdifference in%inhibition of H2O2by ascorbic acid was noted at a concentration of 25μg/m L(55.76%)and 100μg/m L(58.68%)or higher concentration of 200μg/m L (59.33%).The H2O2scavenging ability of prepared date pit extracts was shown in Table 4.The antioxidant activity of all extracts ata concentration of 5mg/m L was found to bebetween 24.49 and 40.75 i.e.their activity was comparable to ascorbic acid at a concentration of 5–10μg/m L(30.90%–46.15%).In otherwords,the percent inhibition produced by ascorbic acid at concentration of 25μg/m L was much greater than the scavenging activity of alldate pitextractsata concentration of 5mg/ m L.Acetone and water extracts exhibited better inhibition as compared to alcoholic extracts.In terms of H2O2scavenging activity of Khinazi,Khasab and Naghal date pits,acetone appeared to be the best solvent while methanol and water seemed to be ideal for Fardh and Khalas,respectively.The highestantioxidantactivity was exhibited by aqueous extract ofKhalas(40.75±1.43)and lowest activity by ethanol extractof Khinazipits.

The reducing powerof the date pitextractsata concentration of 5 mg/m L was determ ined using the potassium ferricyanide reduction method and the results were given in Table 5. Ascorbic acid was used a positive control and a standard curve was plotted to show the increase in ferric reducing power at different concentration(Figure 1).A linear relationship was obtained in the concentration ranging from 10 to 100μg/mL w ith a regression coef fi cient(r2)of 0.979 4.The highest%increase in reducing power of Khinazi in acetone(81.80%)and Khasab in ethanol(80.88%)was found to be comparable to ascorbic acid(85.36%)obtained at100μg/m L.In general,itwas observed that the reducing power of organic extracts for alldate pitswerebetter than theaqueousextract.Theactivity of acetone, methanol and ethanol extracts at tested concentration was very much sim ilar to the ascorbic acid at concentration of 75μg/m L. Itwas interesting to note that reducing power of aqueousextract of Fardh pits(67.39%)was signi fi cantly different from aqueous extractof other date varieties(31.57%–41.65%).

Table4 Antioxidantactivity of date seed extracts at5mg/m L concentration by H2O2scavenging assay method.

Figure 1.Plot show ing increase in ferric reducing power of standard ascorbic acid solution at different concentration.FRP:Ferric reducing power.

Table5 Ferric reducing power of date seed extracts at5mg/m L concentration.

3.5.In vitro antidiabetic activity

In vitro antidiabetic activity of the date pitswas investigated by studying the inhibitory effects on the level ofα-amylase and α-glucosidase.Acarbosewasused asa standard drug to compare the inhibitory effects.

The standard curve of the inhibitory effects of acarbose (12.5–200μg/m L)against intestinalα-glucosidase showed the dose dependent activity(r2=0.952 9).The IC50value of acarbosewas found to be 137.64μg/m L.At concentration of 5mg/ m L,pitsextractof fi ve date varieties exhibitedmoderate to good inhibitory activity,ranging from 5.91%to 51.71%(Table 6).It was quite evident from the results that solventhad pronounced effect on the inhibitory activity ofα-glucosidase.Water extract by far exhibited superior%inhibition(34.46±2.33 to 51.71±8.20)in comparison to the organic extracts(5.91±0.65 to 42.40±6.43).Itwas observed to be the best solvent for all date varieties in exhibiting potent in vitro antidiabetic activity by α-glucosidase inhibition method followed by methanol and ethanol.Itcould be concluded that the inhibitory effectofwater extractat5mg/m L was approximately equivalent to IC50value of acarbose i.e.,137.64μg/m L.Interestingly,acetone extract exhibited the weakest activity(5.91%–24.78%)among all pit extracts w ith Fardh and Naghal show ing the highest and the lowest activity.For Khalas,Khinazi and Naghal,the order of solvents according to activity from the highest to lowestwas as follows:water＞methanol＞ethanol＞acetone;for Fardh it was,water＞acetone＞ethanol＞methanol and for Khasab it waswater＞methanol＞acetone＞ethanol.

The IC50value calculated from the standard curve of acarbose(12.5–200μg/m L)on the inhibitory effects on the level of α-amylase was found to be 95.37μg/m L.The results presentedin Table 7 indicated the percentage inhibition shown by water extract(13.71%–51.45%)at concentration of 5 mg/m L was greater than allother extracts(1.44%–26.23%).Further,itcould be seen thatwater was the best solvent for displaying antidiabetic activity by inhibition ofα-amylase.It exhibited approximately 2,2.24 and 8.21 folds greateractivity for Fardh pits than methanol,ethanol and acetone extracts,respectively.The inhibitory activities for water,methanol,ethanol and acetone were found in the range of 13.71%–51.45%,8.55%–26.23%, 4.96%–22.99%and 1.44%–6.26%,respectively.Thus,it could be inferred thatwaterwas almost 2–3 folds better than ethanol and methanol and approximately 7–10 folds better than acetone solvent.The inhibitory effects on the level ofα-amylase by water and acetone extracts of date varieties from highest to lowest was observed to be in the follow ing order: Fardh＞Khasab＞Khinazi＞Khalas＞Naghal;formethanol it was,Fardh＞Khinazi＞Khalas＞Khasab＞Naghal;for ethanol it was,Fardh＞Khalas＞Khinazi＞Khasab＞Naghal.Thus, Fardh was the most potent and Naghal was the least potent varieties in all solvent extracts(Table 7). geographical conditions and other environmental factors,etc [4].Thus,it could be hypothesized that this variation m ight also affect the chem ical composition and other physical characteristics of disposed waste or pits of dates.Therefore, the present study was undertaken to investigate the bene fi cial actions of pits of fi ve date(P.dactylifera)varieties w idely grown and consumed in Oman by assessing their physical characteristics,in vitro antioxidant activity,TPC and inhibitory effects againstα-amylaseandα-glucosidaseenzymes.

Table6 α-Glucosidase inhibitory activity of date seed extractsat5mg/m L concentration.

Table7 α-Amylase inhibitory activity of date seed extracts at5mg/m L concentration.

The selected date cultivars showed variation in the physical characteristics such as average weight of date fruit,fruit pulp, date pit,fruit fl esh percentage,total number of fruits/kg and pulp/pit ratiowhich ismainly due to genetic variations,different environmental and grow th conditions.A good quality date is generally characterized by ahigh fl esh percentand fruit/pit ratio. Though Fardh is commonly used for industrial purposesand isa moderate quality date,it showed the highest pulp and fl esh/pit ratio,evenmuch higher than the Khalas,a prem ium quality date. Sim ilarly,the other two moderate quality dates,Naghal and Khinazi had the greatest pitweights as expected.

4.Discussion

P.dactylifera or date palm is one of the oldest and most popular fruit trees in the hot arid regions of theworld,particularly in the gulf countries of the M iddle East[25].Date fruits are popular worldw ide due to their taste,health bene fi ts and nutritional values ow ing to the presence of good amount of essential nutrients such as sugars,proteins,fi bers,trace elements,etc.,thus form ing an important part of the daily diet, especially in Oman[26].A high degree of biodiversity has been reported in size,shape,color,texture,quality and chem ical composition among numerous date fruits varieties grown in different parts of the Sultanate which probably could be due to difference in cultivation time,harvesting time,

The preliminary phytochem ical screening was also done to con fi rm the presenceofvarious classesof biologically active plant metabolites in date pitextractswhich revealed thepresenceof poly phenols(tannins),carbohydrates,proteins and am ino acids. Numerous in vitro studieshavesuggested thatantioxidantpotential and other useful biological actions of medicinal plants could be attributed to their high phenolic contents[27].The phenolic compounds by virtue of their reducing properties can absorb and neutralize free radicals,quench singlet and triplet oxygen,or decompose peroxides to act as antioxidant[28].Therefore,the TPC of date pits and effect of solvents of varying polarity in extraction of TPCwas determ ined by using a colorimetric reagent Folin–Ciocalteu,which reacts non speci fi cally w ith phenolic compoundsand formsa complex to bemeasured at765 nm[29].

The TPC determ ined in different solventextracts of date pits is expressed as CAE and ranges from 0.85 to 1.58mg/g of dry date pit powder.A l Harthy et al.determ ined the TPC of four date fruits native to Oman and it ranged from 32.24 to 35.84mg/ 100 g of CAE.Therefore,it can be deduced that phenolic content of date pits is lower than the date fruits[20].It was observed that organic solvents,namely,acetone,ethanol and methanol are signi fi cantly better than water in extracting phenolic compounds from the date seeds.This difference is mainly due to their polarity and good solubility for phenolic components from plantmaterials[30].Acetone extract showed the highest content of total phenol followed by ethanol and methanol.A sim ilar study conducted on different varieties of date pits also reported that organic solvents extract phenolic compounds better than water;however,they observed ethanol to be the bestsolvent followed by acetone andmethanol[14].

A numberof studies have ascribed the antioxidantactivity of medicinalplants to thepresenceofpolyphenolic compounds.A lso natural antioxidants are known to play an important role in the prevention ofmany age-related diseasesand promotion ofhealth. Due to the complexity of theantioxidantmechanism,it isalways preferred to adopt themulti-method approach to test theantioxidant activity[31].The antioxidant activity of date seeds was therefore,evaluated by reducing power assay method and against DPPH and H2O2anion radicals by in vitro experiment. Positive control,ascorbic acid signi fi cantly scavenged the DPPH and H2O2radicals and also showed increased reducing power in a concentration dependent manner.Though all the tested extractsexhibitedmoderate to good antioxidantactivity,a signi fi cant difference(P＜0.05)in the activity of different solvents was observed by One way ANOVA.The acetone extract showed better antioxidant activity as compared to other extracts which could be due to the fact that extraction of antioxidant compounds is solventdependentor in otherwords it depends on the TPC.Therefore,this difference in antioxidant activity could be attributed to variation in their TPC[22].Several other studies have also shown a good correlation between TPC and antioxidantactivity[32].In contrary to this,water extractof Khalas and Khasab in DPPH assay method,Khalas against H2O2and Fardh in ferric reducing power assay also showed potent antioxidant activity suggesting that phenols are not the only phytochem icals responsible for antioxidant activity,and there m ight be some other water soluble non phenolic phytoconstituents which contribute to the antioxidant activity. Nevertheless,acetone seems to be the best choice for the extraction of phenolic compounds irrespective of the date variety,vis-a-vis it exhibited high antioxidant activity.Thus,it could be concluded thatdate seedscan serveasanaturalsourceof naturalantioxidant due to their phenolic content.

Diabetes mellitus is a chronic metabolic disorder affecting m illions of people worldw ide.Its prevalence is on rise globally atan alarm ing ratewhichmakes it as one of themajor grow ing health problem[33].Oral hypoglycem ic agents and insulin are themain components of antidiabetic therapy in addition to life stylemodi fi cation.One of the therapeutic strategies to control postprandial hyperglycem ia in diabetics is to retard the hydrolysis of carbohydrates by the inhibition ofα-glucosidase andα-amylase enzymes in order to slow down the intestinal absorption of glucose[34].These two enzymes,α-glucosidase andα-amylase,in the digestive tract are responsible for the hydrolysis of starch and disaccharides to glucose and breakdown of long chain carbohydrates,respectively.The inhibitors of these enzymes are considered as the potential targets in themanagement of diabetesmellitus.

Many natural products of phenolic nature have been shown to inhibit the activity ofα-glucosidase andα-amylase.Moreover,a positive relationship between the phenolic content and inhibitory activity has been reported[16,17].In the present study, aqueous extract of almost all date pits exhibited signi fi cantαglucosidase andα-amylase inhibition activity by indicating the presence of naturalα-glucosidase andα-amylase inhibitors in date pits.However,unlike a previous study,no direct correlation between the content of total phenols or antioxidant activity and inhibitory activity againstα-glucosidase andαamylase could be observed[35].Contrary to this,water extract which extracted the lowest amount of phenolic components exhibited the best inhibitory activities.Thus,it could be proposed that date pits contain some water soluble non phenolic bioactive compounds in addition to phenolic acids and fl avonoids,which have the ability to act asα-glucosidase andα-amylase inhibitors.Thus,it can be concluded that date pits m ight prove prom ising and bene fi cial in lowering the blood glucose in diabetics by inhibiting the digestion and suppressing the hydrolysis of dietary carbohydrates.Further, detailed phytochem ical investigation of date pits can provide a lead compound that can be used effectively in the prevention and managementof diabetes and its complications.

The resultsof the presentstudy con fi rmed thatdisposedwaste ofOmanidatesisa rich sourceofdietaryantioxidantbecauseof its high TPC.Acetonewas found tobeabettersolventthanalcoholor water forextraction ofphenolic compounds from the datepitsand forexhibiting antioxidantactivity.Pitsofalldatevarietiespossess signi fi cant in vitro antidiabetic activity.However,no direct correlation wasobserved in theantidiabetic activity and the antioxidantactivity.W aterextractdisplayed themaximum inhibition of α-glucosidase andα-amylase suggesting presence of some non phenolic water soluble compounds as the potential inhibitor of these enzymes.The pits due to their inhibitory effects onαglucosidaseandα-amylase level could beused asamonotherapy alongw ith anappropriatediabetic dietand exerciseorm ightbe in conjunction w ith antidiabetic therapy to manage and prevent progression of diabetes.

Con fl ict of interest statement

We declare thatwe have no con fl ict of interest.

Acknow ledgm ents

Authors are grateful to management of Oman Medical College for providing infrastructure and necessary research facilities.Wewould also like to thank the Research CouncilofOman for fi nancially supporting this research project under Faculty M entored Undergraduate Research Award Program scheme w ide grant No FRP/OMC/14/002.

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1 Sep 2015

*Corresponding author:Dr.Shah Alam Khan,Associate Professor,Department of Pharm acy,Oman M edical College,M uscat,Su ltanate of Oman.

Tel:+968 24504608,ex t.165

E-mails:shahalamkhan@yahoo.com,sakhan@omc.edu.om

Foundation Project:Supported by Faculty M entored Undergraduate Research Award Program scheme w ide grant No FRP/OMC/14/002.

Peer review under responsibility o f Hainan M edical University.The journal implements double-blind peer review practiced by specially invited international editorial boardmembers.

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