The in vitro evaluation of antioxidative activity, α-glucosidase and α-amylase enzyme inhibitory...

Diabetes & Metabolic Syndrome: Clinical Research & Reviews xxx (2013) xxx–xxx

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DSX-359; No. of Pages 8

Original Article

The in vitro evaluation of antioxidative activity, a-glucosidase anda-amylase enzyme inhibitory of natural phenolic extracts

Amar Djeridane a,*, Aicha Hamdi b, Wafa Bensania b, Khadidja Cheifa b,Imane Lakhdari b, Mohamed Yousfi a

a Laboratory of Fundamental Sciences, University Amar Telidji, PB 37 G, 03000 Laghouat, Algeriab Department of Biology, University Amar Telidji Laghouat, Algeria

A R T I C L E I N F O

Keywords:

Medicinal plants

Polyphenols

Enzymatic inhibition

a-Amylase

a-Glycosidase

Antioxidant capacity

A B S T R A C T

Phenolic extracts from the medicinal parts of six traditional Algerian herbs were tested in screening

experiments for the antioxidant, a-amylase and a-glycosidase inhibiting activities. UV-analysis of the

extracts from the plants indicated that the total phenols content was ranged between 0.48 and 3.46 mg

equivalent of gallic acid per gram of dry matter, whereas the flavonoids content expressed as rutin

equivalent per gram of dry matter was ranged between 0.18 and 2.23 mg/g.

The study of antioxidant activity by scavenging the hydroxyl radical (OH�), the nitroxide radical (NO

�)

and the stable radical cation (ABTS�+) showed a high antioxidant power. Also, these extracts illustrated a

significant reductive power of the Fe3+–TPTZ complex. Similarly, we have found that the phenolic

extracts exhibit an imperative antioxidant status compared to synthetic antioxidants.

The study of the extract effects shows that Anabasis articulata, Agatophora alopecuroide and

Heliantheum kahiricum extracts have a powerful inhibiting capacity of the a-amylase and a-glycosidase

with a Ki values less than 10 mM. Our study, for the first time, revealed the anti-diabetic potential of the

six plants and the results of this study could be helpful to develop medicinal preparations or

nutraceuticals and functional foods for diabetes.

� 2013 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Diabetes & Metabolic Syndrome: Clinical Research &Reviews

jo ur n al h o mep ag e: www .e lsev ier . c om / loc ate /d s x

1. Introduction

In recent years, much interest has been focused on biologicallyactive compounds occurring in plants and herbs for their safetyand effectiveness in prevention and/or treatment of humandiseases. Demand for natural antioxidants has increased latelyowing to the fact that synthetic antioxidants may be related todiseases such as atherosclerosis and cancer, and that their use isstill a health concern [1,2]. The mechanism by which naturalantioxidants exert their effects is not fully understood, but aconsensus has developed that antioxidants protect against damageto membranes, proteins, and DNA by scavenging free radicalsgenerated through oxidative metabolism [3,4].

The antioxidant properties of plant extracts have beenattributed to their polyphenol contents [5–7]. So plants containinghigh-level of polyphenols have a great importance as naturalantioxidants. The antioxidant activity of phenolic compounds is

* Corresponding author at: Laboratoire des Sciences fondamentales, Universite

Amar Telidji Laghouat, BP37G Laghouat, Algeria. Tel.: +213 029 92 00 66;

fax: +213 029 92 00 66.

E-mail address: [email protected] (A. Djeridane).

Please cite this article in press as: Djeridane A, et al. The in vitro evenzyme inhibitory of natural phenolic extracts. Diab Met Syndr: Cli

1871-4021/$ – see front matter � 2013 Diabetes India. Published by Elsevier Ltd. All r

http://dx.doi.org/10.1016/j.dsx.2013.10.007

mainly due to their redox properties, which can play an importantrole in adsorbing and neutralizing free radicals, quenching singletand triplet oxygen or decomposing peroxides and have healthfunctional properties that may protect humans from variousdiseases [8,9].

There is increasing evidence that individual polyphenols orclasses of polyphenols may cause other beneficial effects,independent of their antioxidant capacities, by directly influencingthe activities of key enzymes. There have been reports thatpolyphenolic fractions from plants can cause insulin-like effects inglucose utilization. Polyphenolic extracts from a number of plantswere found to be effective inhibitors of intestinal a-glucosidaseactivity [10,11] with Ki values in the same range as syntheticinhibitors (acarbose and voglibose). Already these plants com-pounds were being used therapeutically to control non-insulindependent diabetes mellitus (NIDDM) and also there are used toinhibit a-amylase activity, which could prove to be synergistic totheir potential therapeutic effect on post-meal blood glucose levels[12].

There are no previous reports of any in vitro a-glucosidase anda-amylase inhibitory activity and antioxidant activity of these sixplant extracts. Hence, in this study we try to investigate howpolyphenolic plant extracts could inhibit the a-glucosidase and a-

aluation of antioxidative activity, a-glucosidase and a-amylasen Res Rev (2013), http://dx.doi.org/10.1016/j.dsx.2013.10.007

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A. Djeridane et al. / Diabetes & Metabolic Syndrome: Clinical Research & Reviews xxx (2013) xxx–xxx2

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amylase enzymes, and scavenge free radical aggression order tofind out new potential sources of natural enzyme inhibitors andantioxidants.

2. Materials and methods

2.1. Preparation and analysis of phenolic compound

2 g of the air dried plant material is crushed and extracted for48 h with 100 ml of 80% (v/v) aqueous methanol at roomtemperature. Another successive extraction with 50 ml of thesame hydroalcoholic solution was carried out at room temperaturefor 24 h. After removal of methanol under reduced pressure in arotary evaporator at 40 8C, the remaining aqueous solution of theextraction is defatted twice with petroleum ether to remove lipids.Then, the lyophilized solution is extracted with ethyl acetate inpresence of aqueous solution with 20% ammonium sulphate, and2% of ortho-phosphoric acid solution. The ethyl acetate fraction isdried by addition of a sufficient amount of anhydrous sodiumsulphate, and then evaporated to dryness using a rotary evapora-tor. The precipitate is dried, dissolved in 5 ml of absolute methanoland kept at �10 8C.

The concentration of total phenolics in plant extracts wasestimated by the Folin–Ciocalteu procedure [13], which isconsidered as the best method for total phenolics (includedtannins) determination [14]. The total phenolic content of thesample was expressed as gallic acid equivalents (GAE), whichreflected the phenolic content as the amount of gallic acid (mg) in1 g of dry material. Whereas, the total flavonoid content in driedaerial parts of the plants was determined by a slightly modifiedversion of the method of Zhishen et al. [15] and Dewanto et al. [16].Flavonoid contents were calculated using a standard calibrationcurve, prepared from rutin.

Chromatographic analysis of phenolic extracts was performedwith a Merck Lachrom System consisting of a degasser L-7612Merck, an auto-injector L-7200 Merck Lachrom, a injection pumpsL-7100 Merck Lachrom equipped with an injection boucle of100 ml, a Chromolith Performance RP-18e chromatographiccolumn and an UV-visible L-7455 Merck photodiode array detectorcontrolled by the D7000-HSM MilleniumTM Chromatographymanager software. The UV spectra were recorded in the range220–600 nm to identify the different classes of phenolics and testtheir purity. Chromatogram and spectrum were treated withMillenium software. Reverse phase HPLC was carried out at 30 8Con a Chromolith RP-18e column (100 mm � 4.6 mm i.d.; Merck)using a gradient with a flow rate of 1 ml min�1 (Gradient I: solventA (orthophosphoric acid in H2O, pH = 2.5), solvent B (Me-OH): 0–30 min, 10–70% B; 30–31 min, 70–100% B; 31–40 min, 100% B; 40–41 min, 100–10% B; 41–50 min, 100–10% B).

2.2. Evaluation of antioxidant capacity

The antioxidant activity of extracts was measured using fourdifferent in vitro assays; including the scavenging activity of 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation,the hydroxyl radicals (OH

�) and the nitric oxide radical (NO

�)

and a test measure the capacity of ferric reduction (FRAP assay).

2.2.1. Determination of ABTS radical cation scavenging activity

In the ABTS assay, the antioxidant capacity of the samples wasmeasured by using a slightly modified method of Arnao et al. [17]based on the ability of a substance to scavenge the ABTS

�+ radical

compared with a standard antioxidant (Trolox) in a dose–responsecurve. The working solution contained 2.0 mM ABTS and 0.86 nMhorseradish peroxidase in 50 mM phosphate buffer, pH 7.0. TheABTS

�+ chromogen was generated by adding 1 mM hydrogen


peroxide in 50 mM phosphate buffer. One milliliter ABTS�+ was

then added to glass test tubes containing various dilutions of eachextract. The absorbance at 734 nm was recorded each minute afterinitial mixing. Appropriate solvent blanks were run in each assay,controls taken, and all determinations carried out at least 5 min.Fall in absorbance was calculated and plotted against functionssuch as time and concentration of the standard and samples. Astandard curve was plotted with different concentrations of Trolox.The antioxidant activity was expressed as Trolox equivalentantioxidant capacity (TEAC) according the calibration curve of theTrolox.

2.2.2. Determination of hydroxyl radical scavenging activity

The scavenging activity for hydroxyl radicals was measuredwith Fenton reaction [18]. The assay was performed by adding0.1 ml EDTA (1 mM), 0.01 ml of FeCl3(10 mM), 0.1 ml of H2O2

(10 mM), 0.36 ml of deoxyribose (10 mM), 1.0 ml of the extractdissolved in distilled water, 0.33 ml of phosphate buffer (50 mM,pH 7.4) and 0.1 ml of ascorbic acid (1 mM) in sequence. Themixture was then incubated at 37 8C for 1 h. A 1.0 ml portion of theincubated mixture was mixed with 1.0 ml of 10% TCA and 1.0 ml of0.5% TBA to develop the pink chromogen measured at 532 nm. Thehydroxyl radical scavenging activity of the extract is reported as %inhibition of deoxyribose degradation and is calculated as theefficient concentration EC50 in mg/g of dry matter plant. Thehydroxyl radical scavenging activity obtained for each plantextract was compared with that of caffeic acid and 3-tertio-butyl-4, hydroxyanisole (BHA).

2.2.3. Determination of nitric oxide radical scavenging activity

Nitric oxide scavenging activity was measured spectrophoto-metrically [19]. Sodium nitroprusside (5 mM) in phosphatebuffered saline was mixed with different concentrations of theextract and incubated at 25 8C for 30 min. After 30 min, 1.5 ml ofthe incubation solution was removed and diluted with 1.5 ml ofGriess. The absorbance of the chromophore formed duringdiazotization of the nitrite with sulphanilamide and subsequentcoupling with naphthylethylene diamine dihydrochloride wasmeasured at 546 nm. Ascorbic acid and BHA were employed aspositive controls and the antioxidant activity of extracts wasexpressed as an EC50 value defined as the concentration (in mg l) ofthe extract that inhibited the formation of ABTS

�+ radicals by 50%.

2.2.4. Determination of the ferric reducing-antioxidant power (FRAP

assay)

The FRAP was assessed according to [20]. The method is basedon the reduction of the Fe3+–TPTZ complex to the ferrous form atlow pH. This reduction is monitored by measuring the absorptionchange at 593 nm. Briefly, 1 ml of working FRAP reagent prepareddaily was mixed with 100 ml of diluted sample. The absorbance at593 nm was recorded every minute after initial mixing after during10 min. FRAP values of phenolic extracts were calculated from thecalibration curve of caffeic acid used as a standard antioxidant andexpressed as caffeic acid equivalent antioxidant capacity (CAEAC).The reducing power of our plat extracts was compared with that ofascorbic acid and BHA.

2.3. Assay of a-amylase and a-glucosidase inhibitor activity

2.3.1. Assay of a-amylase inhibitor activity

Kinetic experiments were performed at 37 8C in 20 mM sodiumphosphate buffer pH 6.9 containing 6 mM NaCl and 1 mM sodiumazide. Substrate and inhibitor were mixed together and thereaction was initiated by adding amylase. Fore concentrations ofeach starch substrate were used. The amylase concentration was0.5 mg/ml. The reducing power of liberated sugars was determined



A. Djeridane et al. / Diabetes & Metabolic Syndrome: Clinical Research & Reviews xxx (2013) xxx–xxx 3

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according to Dygert et al. [21]. A calibration curve of absorbanceand concentration of maltose was established with known amountof maltose. One unit (U) of amylase was defined as the amount ofenzyme that liberates 1 mol of reducing sugars, measured asmaltose per min under the conditions of assay. The initial velocitywas determined from the slope, as calculated by linear regression,of the linear part of the kinetic curves giving either the number ofhydrolyzed bonds or the changes in product and substrateconcentration with time. All the experiments were repeated threeto four times.

2.3.2. Assay of a-glucosidase inhibitor activity

The effects of polyphenols on yeast a-glucosidase weremeasured by determining the release of reducing sugars fromsucrose. The a-glucosidase (0.1 U in 500 ml) was incubated with1 ml of 50 mM sucrose and 400 ml of 0.05 M phosphate buffer (pH7.0) in the presence of a phenolic extract solution (100 ml) ordistilled water (100 ml) at 37 8C for 30 min. The reducing sugarsreleased were quantified by the dinitrosalicylate method [22]. Acalibration curve of glucose was established to determine theamount of liberated glucose. The initial velocity was determinedfrom the slope, as calculated by linear regression, of the linear partof the kinetic curves. All the experiments were repeated three tofour times. Inhibition kinetics were studied by including fixedconcentration of the substrate sucrose and varying the concentra-tion of the inhibitor in the assay mixture. Reciprocal initial velocityplots versus varied substrate concentration were used to determinethe mode of inhibition and the Ki values.

3. Results and discussion

3.1. Phenolic content analysis and their antioxidant activity

It has been recognized that phenolic compounds showantioxidant activity and their effects on human nutrition andhealth are considerable. Antioxidants inhibit lipid peroxidation,scavenge free radicals and chelate divalent cations [23,24].Antioxidants have been shown to reduce the risk of diabetesonset, improve glucose disposal and some of the associatedcomplications. Especially, phenolic compounds, such as catechinand its derivatives have a wide range of health promoting effects[25]. The flavonoid content of the extract in terms of rutinequivalent was ranged from 0.11 to 2.23 mg g�1 (Table 1). The totalphenol content which measured by Folin–Ciocalteu reagent interms of gallic acid equivalent was ranged from 0.85 to 3.46 mg g�1

(Table 1). Among plant extracts, extremely high total phenoliccontent was detected in Helianthemum kahiricum and Salsola

vermiculata. The lowest total phenolic levels were detected inHammada elegans and Salsola baryosma. However, the content offlavonoids (mg/g), in rutin equivalent witch varied from 0.11 to2.23, indicates that the majority of these plants have flavonoidspercentages above 50%. The amount of polyphenolic compounds indifferent plant extracts is dependent on its origin and the method

Table 1The amount of plant phenolic compounds, according to the spectrophotometric and H

Plant name Total phenolic content

(mg GAE/g dw)

Flavonoids content

(mg RE/g dw)

Agatophora alopecuroıde 1.20 1.12

Anabasis articulata 1.60 1.22

Hammada elegans 0.85 0.18

Helianthemum kahiricum 3.46 2.23

Salsola baryosma 1.00 0.60

Salsola vermiculata 2.22 1.22


of extraction. The compounds, such as flavonoids, which containhydroxyl groups, are responsible for the radical scavenging effectin the plants. According to our study, the contents of thesephytochemicals in plant extracts can explain its antioxidantactivity.

Each extract was analyzed by HPLC using a diode array detectorfor simultaneous recording of chromatographic analysis at severalwavelengths. Because there are a significant number of com-pounds in different phenolic classes, each of which has a differentabsorption maximum, it is common to quantify each class at thewavelength of its absorption maximum. According to the HPLCanalysis and the spectral characteristic, the phenolic profile of eachplant was expressed in percentage (w/w) of caffeic acid equivalentfor hydroxycinnamic, of gallic acid for hydroxybenzoic and rutinfor flavonoids (Table 1). In all the extracts, flavonoids were themain phenolic subgroup except in S. vermiculata extract, whichcontained 50% of these compounds.

An explosion of interest in studying the involvement of freeradicals in carcinogenesis has led to the use of dietary antioxidanttreatment to terminate free radical attacks, hence promotinggeneral human health. The antioxidants may act as free radicalscavengers, reducing agents, chelating agents for transition metals,quenchers of singlet oxygen molecules, and activators of anti-oxidative defense enzyme systems to suppress radical damage inbiological systems [26,27].

The ABTS�+ assay has been used to screen the relative radical-

scavenging capacities of flavonoids and phenolics, which act aselectron- or H-donating agent or plant extracts that contains thesesubstances [28]. The green cation radical ABTS

�+ has absorbance

peaks at 630, 734 and 812 nm. On interaction with antioxidantsthe radical is reduced, suppressing the absorbance of the greenradical cation in a dose-dependent way.

In the current study, the scavenging activities of ABTS�+ radical

cation exerted by our phenolic extracts as well as vitamin C andBHA were calculated from the linear response curves representingthe variation between the inhibition percentages of Trolox withtheir concentrations in mM. The antioxidant activity of the extractwas expressed as TEAC (Trolox Equivalent Antioxidant Activity).The results of TEAC obtained by different plant extracts aresummarized in Table 2.

All plant extracts showed a high beneficial effect against free-radical cation damage compared to the BHA a powerful lipophilicantioxidant. But comparatively to the hydrophilic antioxidant(vitamin C), only the H. kahiricum extract was the best inhibitor. Itseffect was almost more than three times as BHA.

TEAC values reported in the literature are variable. Our resultsrevealed that the investigated plants exhibited on average,remarkably higher antioxidant activity and contained significantlymore phenolics than other traditional medicinal herbs andcommon vegetables and fruits (dietary plants) [29,30]. However,it appears that the TEAC values largely depend on the assayconditions [31]. The most important reason for this variation is thatthe reaction of an antioxidant with ABTS

�+ usually does not reach

PLC analysis.

Dominant phenolic compounds

Flavonoids Hydroxycinamic derivatives Hydroxybenzoic derivatives

98 0 2

95 0 5

100 0 0

70 15 15

90 4 6

50 10 40



Table 2Antioxidant capacity of plant extracts as measured using the ABTS, OH

�, NO

�and FRAP assays.

Plant extract NO�

assay EC50 (mg/l) OH�

assay EC50 (mg/l) ABTS assay TEAC (mM) FRAP assay CAEAC (M)

Agatophra alopecuroıde 300 0.54 00.27 1.49

Anabasis articulata 80 0.45 02.37 4.71

Hammada elegans 16 0.37 01.65 0.93

Helianthemum kahiricum 97 1.90 03.27 2.31

Salsola baryosma 101 0.26 01.28 1.08

Salsola vermiculata 67 0.67 01.23 2.90

Vitamin C 15 – 03.39 2.47

Caffeic acid – 0.35 – –

BHA 22 0.31 01.28 3.04


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completion within the time span applied. It should therefore, berealized that in comparing TEACs from different reports, the timeinterval used should be taken into account, although contraryresults on the time dependency in the TEAC were reported inprevious studies [32,33]. In our analysis, the TEAC value wasdetermined at 5 min and, within these 5 min, a slow reaction hadalready made a substantial contribution.

It can be observed that the content of phenolics in the extractshave no significant correlation with their antiradical activitymeasured by ABTS assay (R2 = 0.27). The unclear relationshipbetween the antioxidant activity and the total phenolics may beexplained in numerous ways, in fact, the total phenolics contentdoes not incorporate all the antioxidants synergism between theantioxidants in the mixture makes the antioxidant activity not onlydependant on the concentration, but also on the structure and theinteraction between the antioxidants. This is the reason whysamples such as S. baryosma and Agatophra alopecuroıde withsimilar concentrations of total phenolics, may vary in theirantioxidant activities (TEAC assay). It is known that only flavonoidsof a certain structure and particularly hydroxyl position in themolecule determine antioxidant properties; in general theseproperties depend on the ability to donate hydrogen or electronto a free radical. Detailed examination of phenolic composition inplant extracts is required for the comprehensive assessment ofindividual compounds exhibiting antioxidant activity.

The phenolic extract of H. kahiricum aerial parts showed strongantioxidant activity by inhibiting ABTS

�+ when compared with

other plant and standards. In addition these plants found tocontain the best amount of total phenols, which play a major role incontrolling antioxidants. The results of this study show that theseplants can be used as easily accessible source of naturalantioxidants and as a possible food supplement or in pharmaceu-tical industry. However, the components responsible for theantioxidant activity of these plants are currently unclear.Therefore, it is suggested that further works should be performedon the isolation and identification of the antioxidant components.The discrepancy between results of ABTS and FRAP assay may berelated to the different mechanisms involved in each evaluation.

In the FRAP assay, the antioxidant capacity of phenoliccompounds is measured as the ability to reduce ferric tripiridyltriazing (F+3) to a ferrous form (F+2) and may be related to thepresence of iron-chelating compounds in the samples. The totalantioxidant activities of the plant extracts are expressed as caffeicacid equivalent antioxidant activity (CAEAC) based on thecalibration curve of caffeic acid. The CAEAC parameter is definedas the millimolar concentration of caffeic acid equivalent to 1 M ofpolyphenols present in our extracts and higher CAEAC valuesmeans higher reducing capacity. Table 2 summarizes the obtainedCAEAC values of extracts and standard antioxidants expressed ascaffeic acid equivalent in mol per liter.

The CAEAC values showed a higher antioxidant capacity for theAnabasis articulata extract, and a smaller activity for the otherextracts comparatively to standard antioxidant BHA. As shown in


Table 2, there were remarkable differences in total antioxidantcapacity (FRAP) between the selected medicinal plant extracts. TheFRAP values varied from 4.71 to 0.93 molar. The correlationcoefficient between total antioxidant activity and total phenoliccontents is R2 = 0.92. This result suggests that 92% of theantioxidant capacity of Algerian medicinal plants is due to thecontribution of phenolic compounds. As it was shown, the ABTSvalues of H. kahiricum were higher than those with the FRAP assayin water-soluble as well as water-insoluble extracts. This can beexplained by the water soluble extracts containing a highproportion of antioxidants with a higher activity in the ABTSmethod than in the FRAP assay

Moreover, we reported that the lipophilic antioxidant (BHA) ismore effective than the hydrophilic antioxidant (vitamin C). Thisresult helps us to suggest that the extracts showed significantantioxidant power, can contain individual polyphenols with alipophilic character or molecules with similar structures to the BHA.Also, we have found that the extracts containing the high level ofpolyphenol contents show very interesting antioxidant activities.Furthermore, we discovered that plants rich in flavonoids exhibit asignificant antioxidant status which is correlated with literature[34,35]. Furthermore, it can be concluded that the antioxidantactivity of plant extracts is not the result of these compounds butmay be also related to the presence of some individual activephenolic compounds. In conclusion, medicinal plant extracts withconsiderable antioxidant potential can be considered as a richsource of potent antioxidants. The best results were obtained for A.

articulata extract. For that reason this plant can be considered theantioxidant queen between 6 selected medicinal plants.

Hydroxyl radical is the most reactive free radical and can beformed from superoxide anion and hydrogen peroxide in thepresence of metal ions such as copper or iron. Hydroxyl radical hasthe highest 1-electron reduction potential (2310 mV) and can reactwith anything in living organisms at the second order rateconstants of 109–1010 mol l�1 s�1 [36]. It can react with lipids,polypeptides, saccharides, nucleotides and organic acids, especial-ly thiamine and guanosine and thereby cause cell damage [37].Hydroxyl radical can be generated by the reaction of hydrogenperoxide with Fe2+ or Cu2+. To test the scavenging capacity of ourphenolics toward hydroxyl radicals, we used the Fenton reagent(Fe2+ + H2O2! Fe3+ + OH� +

�OH) as a source of hydroxyl radicals.

As shown in Table 2, phenolic extracts exhibited strong scavengingcapacity toward the hydroxyl radicals generated by Fentonreaction in the test concentration range and the scavenging effectswere dependent on concentration from 0.26 to 1.90 mg/l. Highscavenging activity was obtained from the S. baryosma and H.

elegans phenolic extracts, and relatively low scavenging activitywas found from H. kahiricum extract. A good linear relationship(R2 = 0.88) was observed between hydroxyl radical scavengingexpressed as EC50 and the concentrations of phenolic plant extractsexpressed as gallic acid equivalent, indicating that the hydroxylradical scavenging activities of polyphenolic extracts of theseplants were dose-dependent.




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In addition, the antioxidant activity of our extracts wascompared to that of synthetic antioxidants. The antioxidantschosen in our study are currently used in therapy and foodindustry, but are accused of being dangerous to health andparticularly BHA [38]. The results suggest that our extracts canreplace synthetic antioxidants because they pose a potential free

radical scavenge similar to those observed for the antioxidantreference, and in particular the extract of S. baryosma that haveregistered the greatest antioxidant potential (EC50 = 0.26 ppm).The potential scavenging abilities of phenolic substances might bedue to the active hydrogen donor ability of hydroxyl substitution.Similarly, high molecular weight and the proximity of manyaromatic rings and hydroxyl groups are more important for thefree radical scavenging by specific functional groups. On the otherhand, S. baryosma and H. elegans which contained the lowestamount of phenolics showed the best antioxidant activities in thistest. We can also conclude that the antioxidant power of plantextracts may be varied depending on the content of polyphenols;flavonoid content and structure of molecules coexist in thephenolic extract.

In the nitric oxide-scavenging activity, the procedure is basedon the principle that sodium nitroprusside in aqueous solution atphysiological pH spontaneously generates nitric oxide, whichinteracts with oxygen to produce nitrite ions that can be estimatedusing Griess reagent. Phenolic extracts as a scavenger of nitricoxide competed with oxygen, leading to reduced production ofnitrite ions. The scavenging activity of phenolic extracts wascalculated after 30 min and expressed as EC50 ‘‘efficient concen-tration’’. This is defined as the concentration of the substrate thatcauses 50% loss of the NO

�activity. The EC50 values were calculated

by the linear regression method of plots of the percent ofantiradical activity against the concentration of the testedcompounds. The concentration of inhibitors has been calculatedin mg/l. The results obtained are summarized in (Table 2). The EC50

values of the extracts ranged from 16 to 300 mg/l. All the extractsshowed the same and a low activity except the H. elegans extractwith a very high potency compared on the above basis antioxidants(BHA and ascorbic acid). It can be observed that the phenoliccontent of the extracts had no significant correlation with theirantiradical activity as measured by the NO assay (R2 < 0.1).Therefore, we observed that the antioxidant activities measured bydifferent methods are correlated no significantly. There are severalreasons to explain the ambiguous relationship between theantioxidant activity and total phenolics: (1) total phenolics contentdid not include all the antioxidants; (2) the synergism among theantioxidants in the mixture made the antioxidant activity, not onlydependant on the concentration of antioxidant, but also on thestructure and interaction among the antioxidants. In addition toreactive oxygen species, nitric oxide is also a factor involved ininflammation, cancer, and other pathological conditions. Naturalextracts may have the property to counteract the effect of NO

Table 3The inhibition constant (Ki) of a-amylase and a-glucosidase activity by different plant

Plant extract Type of inhibition

a-Amylase a-G

Agatophora alopecuroıde Mixed noncompetitive Mi

Anabasis articulata Noncompetitive No

Hammada elegans Noncompetitive No

Helianthemum kahiricum Mixed noncompetitive Mi

Salsola baryosma Competitive Co

Salsola vermiculata Mixed noncompetitive Mi

Ascorbic acid Mixed noncompetitive Mi

Trolox Mixed noncompetitive Mi

Rutin Competitive No


formation and, in turn, may be of considerable interest inpreventing the ill effects of excessive NO generation in the humanbody. Furthermore, the scavenging activity may also help to arrestthe chain of reactions initiated by excess generation of NO that aredetrimental to human health.

The results suggested that the phenolic compounds of themedicinal plants contributed significantly to the overall resistanceagainst free radical aggression. Although there was no significantcorrelation found between the antioxidant activity and thephenolic contents. During the screening of six plants in this work,H. elegans was found to be the most promising one in the hydroxyland nitroxide radicals scavenging activity. This plant whichcontained a low amount of phenolics (0.85 mg/g DW) had a veryhigh level of antiradicalar activity compared to antioxidantreferences. But according to the reduction screenings, a highlysignificant efficiency was also observed for A. articulata and H.

kahiricum whose activities were nearly similar or best to thestandard antioxidants.

3.2. Effect of plant extracts on a-amylase and a-glucose activities

The effects of the phenolic extract inhibitors on porcinepancreatic a-amylase-catalyzed starch hydrolysis and a-glucosi-dase-catalyzed sucrose hydrolysis were studied. The experimentaltreatments were done under conditions of pH 6.9 and a totalconcentration of the herbal extracts of 0.5 mg/ml. The phenolicextracts produced a weak a-amylase enzyme inhibition. Themaximum inhibition was 100% at a concentration of 0.5 mg/ml.The percentage inhibition ranged from 100% to 75% at 0.5 mg/ml.The Ki values of different phenolic extracts were determined byboth Dixon and Lineweaver–Burk representations.

The phenolic extracts produced a weak a-amylase enzymeinhibition. The maximum inhibition was 100% at a concentration of0.5 mg/ml. The initial velocity v was measured at various substrateconcentrations S and in the presence of the indicated inhibitoramount I.

Our results show that herbal extracts react with porcinepancreatic amylase, inhibiting its enzymatic activity against starchas a substrate in in vitro experiments. As illustrated, the activity ofa-amylase decreased depending on the type of the extract content.But not always does the in vitro inhibitory activity relate to thecorresponding in vivo activity.

In order to determine the constant and which inhibition modelapplies, i.e. whether the inhibition is of the competitive,noncompetitive or uncompetitive type and whether it occurs inthe steady state or at rapid equilibrium, we have outlined bothDixon and Lineweaver–Burk representations. The results of thistest show that all the phenolic extracts of our plants have thecapacity to inhibit the activity of the a-amylase with Ki values ofmicromolar range (Table 3). All these extracts present anoncompetitive and mixed noncompetitive inhibition except the

extracts.

Ki (mM)

lucosidase a-Amylase a-Glucosidase

xed noncompetitive 16.00 18.00

ncompetitive 146.00 92.00



mpetitive 07.00 16.00


xed competitive 197.00 100.00

xed competitive 73.11 450.00




Fig. 1. Lineweaver–Burk representation of a-amylase inhibitory by Salsola baryosma phenolic extract.


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S. baryosma extract which present a competitive inhibition. Thistype of inhibition can explain that the polyphenolic extract of S.

baryosma have compounds bearing functional groups similar tothose of the substrate, which has moved from the active site of theenzyme. But the noncompetitive inhibition exerted by crude otherpolyphenol extracts maybe explained by the absence of glycosidicgroups in the chemical structure of phenolic compounds or thepresence of glucosidic groups but different to that of the substrate.Therefore the fixation of phenolic compounds occurs at a site otherthan the enzyme’s active site. In this study, the majority of ourextracts revealed a mixed type of inhibition, demonstrating thatthe inhibitory effect of phenolic agents is due to the high affinity ofthese compounds to bind the enzyme-substrate complex. The Ki

values of different phenolic extracts determined by both Dixon andLineweaver–Burk representations were ranged from 146 to 7 mM.We note that the ‘‘S. baryosma’’ extract show the weaker Ki whichproves that it is a good inhibitor. The Fig. 1 shows the Lineweaver–Burk representation of a-amylase inhibitory by S. baryosma

phenolic extract.For a better understanding of the factors responsible for the a-

amylase inhibition, we have revealed the correlations between theKi values, the phenolic and flavonoid contents and the antioxidantactivity parameters. Also, we have tried to correlate the anti-amylase activity to the Trolox, vitamin C and rutin. The correlationbetween Ki values and total phenolic contents of Algerian plantshad a correlation coefficient of R2 = 0.46. This result suggests thatthe inhibitory activity was not proportional to the concentration ofphenolic compounds. Also, it may be that the inhibitory potency ofthe plant extracts is not limited to the phenolic content but may bedue to the presence of some individual active phenolic compounds.Thus the inhibitory potency of an extract cannot be explained juston the basis of its phenolic content but also requires their propercharacterization. There are several reasons to explain theambiguous relationship between inhibitory potency and totalphenolics: total phenolics content did not include all the possibleinhibitors; the synergism among the inhibitors in the mixtureaccounted for the inhibition, but was not only dependant on theconcentration of individual inhibitors, but also on the structure andinteraction among them. Consequently, A. articulata and A.

alopecuroıde, with similar concentrations of total phenolics mayvary in their inhibitory activity (16 and 146 mM respectively).Comparatively to the tested synthetic compounds under the sameconditions, we can note that all plant extracts are more effective for


the a-amylase inhibition compared to these molecules except theA. articulata extract. The rutin, a glycoside flavonoid, showed themost remarkable inhibitory potency compared to Trolox andvitamin C. This anti-amylasic activity may be due to the glucosideresidue included in the flavonoid skeleton. Consequently, we caninfer that the good inhibition of our phenolic extracts is probablyowed to the presence of the glycosidic groups into the phenolicskeletons. This suggestion is as lively with the literature [22,39].Also, the results suggested that there was no significant correlationfound between the antioxidant activity and the inhibition of a-amylase activity. But during this study, we have concluded thatelectron-donating antioxidants were low inhibition while anti-oxidants proton donors have a good inhibition. Throughout thescreening of six plants in this work, S. baryosma, Agatophora

alopecuroide and H. elegans were found to be the most promising.These plants which contained the lowest amount of phenolics(0.85–1.2 mg/g DW) had a very high level of antioxidant activityand were potent inhibitors of a-amylase enzymatic compared tothe other plants extracts.

According to the a-glucosidase inhibitory assay, we can deducethat all plant phenolic extracts were found to inhibit verysignificantly, in a concentration dependent manner, the yeast a-glucosidase activity. The best inhibitors were the phenolic extractof S. baryosma and A. alopecuroide. To investigate the type ofenzyme inhibition, we assayed the a-glucosidase activity in thepresence of different concentrations of the substrate (0.02–0.2 g/l)and polyphenol extracts. The Ki value was obtained from bothDixon and Lineweaver–Burk representations. Data from theseplots indicate that the inhibition by all investigated plants wascompetitive, noncompetitive and mixed noncompetitive inhibi-tion with a Ki values ranged from 16 to 540 mM. The resultsobtained for the phenolic extract were reported in Table 3. In Fig. 2,we show the Lineweaver–Burk representation of a-glucosidaseinhibitory by S. baryosma phenolic extract.

We sought to elucidate the relationship between the structureof phenolic compounds and their enzyme inhibition effect. Wehave quantified the total phenol and flavonoid contents byspectroscopic and chromatographic methods. Our resultshighlighted that the correlation between Ki values and totalphenolic contents had a correlation coefficient of R2 = 0.65. Thisresult suggests that the inhibition activity is inversely proportionalto the contribution of phenolic compounds. Also, it can beconcluded that inhibition of enzymatic activity of plant extracts



Fig. 2. Lineweaver–Burk representation of a-glucosidase inhibitory by Salsola baryosma phenolic extract.


G Model


is not limited of the content of these compounds but may also dueto the presence of some individual active phenolic compounds.

Regarding the relationship between the antioxidant power ofphenolic extracts and their anti-glucosidic activity, we can notethat there was no significant correlation found between the anti-a-glucosidase activity and the antioxidant capacity. But with theABTS assay, there was a significant correlation (R2 = 0.74). Thisresult indicates that the extracts exhibiting significant antiradicalcation activities showed the best inhibitory activity of a-glucosidase enzyme.

Herbs that reduce postprandial hyperglycemia by suppressingthe hydrolysis of carbohydrates may be helpful in the control ofdiabetes mellitus. In the present study, both a-amylase and a-glucosidase activities were most strongly inhibited by S. baryosma

and A. alopecuroıde phenolic extracts. It can be concluded that hotwater extract of these plant aerial parts can exert inhibitory effectagainst a-amylase and a-glucosidase enzymes and had apossibility to be consumed as antidiabetic herb infusions.However, the difference in the inhibitory effect between theseplants extracts might be attributed to variety and processing ofplant extracts products.

4. Conclusion

Recent statistical data show that type 2 diabetes is a globalhealth challenge. Management of this disease will require multi-purpose strategies, including dietary chemoprevention as part ofthe solution. Results from this investigation indicated thatalthough antioxidant activity and phenolic compounds had higherin vitro antioxidant activity. In the case of a-glucosidase and a-amylase inhibitory activities, the results indicated that the S.

baryosma had the highest activity. Also, an interesting observationwas the increased a-amylase inhibitory activity in the case of A.

alopecuroıd and Adjram extracts, which can be attributed to eitherspecific phenolics or secondary metabolite.

Phytochemicals have been linked to positive health benefits[40–42] and results from this study further indicate thatenrichment with herb phytochemicals can result in enhancedhealth functional value of a-glucosidase inhibition relevant to type2 diabetes management. In this study, phenolic compounds from


medicinal plants appeared to be a promising system for potentialapplication in type 2 diabetes management with high a-glucosidase inhibitory activity, but high a-amylase inhibitoryactivity may be of concern, due to side effects of undigestedcarbohydrates. The results from this study also suggest that a-glucosidase and a-amylase inhibitory activities correlated with theherb enrichment but without strong correlation to phenoliccontent, indicating that quality and synergy of phytochemicalsmay be more relevant. Phenolics have also antioxidant potentialand this investigation provides an insight that properly optimizedphenolic phytochemical could have a role in developing lessexpensive complimentary strategies for type 2 diabetes concurrentcombined with other nutritional and pharmacological strategies.

5. Conflict of Interest

There is no conflict of interest.

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