evaluation of in vitro antioxidant and antidiabetic

Research ArticleEvaluation of In Vitro Antioxidant and Antidiabetic Activities ofAristolochia longa Extracts

Nasreddine El Omari 1 Karima Sayah 2 Saad Fettach2 Omar El Blidi1

Abdelhakim Bouyahya 3 My El Abbes Faouzi2 Rabie Kamal2 and Malika Barkiyou1

1Laboratory of Histology Embryology and Cytogenetic Faculty of Medicine and PharmacyMohammed V University of Rabat Morocco2Biopharmaceutical and Toxicological Analysis Research Team Laboratory of Pharmacology and ToxicologyFaculty of Medicine and Pharmacy Mohammed V University of Rabat Morocco3Laboratory of Human Pathology Biology Faculty of Sciences Genomic Center of Human PathologyFaculty of Medicine and Pharmacy Mohammed V University of Rabat Morocco

Correspondence should be addressed to Nasreddine El Omari nasrelomarigmailcom

Received 26 December 2018 Revised 5 February 2019 Accepted 19 March 2019 Published 2 April 2019

Academic Editor Vıctor Lopez

Copyright copy 2019 Nasreddine El Omari et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Oxidative stress plays a major role in diabetic physiopathology hence the interest of using natural antioxidants as therapeutic toolsexists The aim of this study was the evaluation of in vitro antioxidant activity and inhibitory potential of organic extracts fromAristolochia longa roots against key enzymes linked to hyperglycemia Antioxidant activity was performed using 221015840-diphenyl-1-picrylhydrazyl (DPPH) and 22-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals and ferric reducingantioxidantpower (FRAP) methods The 120572-Glucosidase and 120573-Galactosidase inhibitory activities were investigated using an in vitro modelMoreover phytochemical analysis of tested extracts was carried out The aqueous fraction of this herb exhibited the highestantioxidant activity for both DPPH and ABTS methods IC50=12540plusmn240120583gmL and IC50=6523plusmn249 120583gmL respectivelyHowever the ethyl acetate fraction possessed the strongest inhibitory effect towards 120572-Glucosidase (IC50=1112plusmn0026mgmL)Furthermore the result showed high levels of phenolic contentThe results showed that this plant could be a significant source ofmedically important natural compounds

1 Introduction

Medicinal plants are one of the main resources of therapeuticagents Indeed 80 of the worldrsquos population uses plantsin health care [1] Recently the interest in the search fornatural substances has considerably increased because thesesubstances are intended for use in foods or drugs to replacesynthetic compounds which are limited because of their sideeffects [2] There is an increasing interest in using medicinalplants and their phytoconstituents as natural sources becauseof their well-known ability to scavenge free radicals Effec-tively plants are sources of natural antioxidants compoundsthat possess various pharmacological properties with little orno side effects and protect human health frommany diseases[3ndash5] The prevention of oxidative stress related disease by

medicinal plant products is delaying the oxidation of lipidsor other molecules by inhibiting the propagation of oxidativechain reactions [2]

Among Moroccan medicinal plants Aristolochia longa(A longa) is a medicinal plant belonging to Aristolochiaceaefamily which is widely distributed in the tropical and tem-perate regions [6] Aristolochia species contain secondarymetabolites that have well-known beneficial effects [7] Alonga locally known as ldquoBarraztamrdquo is a species commonlyused in Moroccan traditional medicine Many traditionalhealers also use a small amount of its rhizome powder withhoney or salted butter for the treatment of abdominal painand upper respiratory tract infections [8ndash10]

Likewise diabetes mellitus is a major cause of mortalityand the most common metabolic disorder characterized

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 7384735 9 pageshttpsdoiorg10115520197384735

2 Evidence-Based Complementary and Alternative Medicine

by hyperglycemia due to lack of insulin production bythe pancreas or the inability of the insulin produced tocontrol blood glucose [11 12] One interesting approach isto reduce postprandial hyperglycemia by retarding glucoseuptake through the inhibition of carbohydrate-hydrolyzingenzymes such as 120572-Glucosidase and 120573-Galactosidase [13 14]In this context the aim of this study was to evaluate the invitro antioxidant activity 120572-Glucosidase and 120573-Galactosidaseinhibitory potentials of A longa root extracts

2 Materials and Methods

21 Reagents p-Nitrophenyl-120572-D-glucopyranoside 2-Ni-trophenyl-120573-D-galactopyranoside 120572-Glucosidase fromSaccharomyces cerevisiae 120573-Galactosidase from Aspergillusoryzae Acarbose Folin-Ciocalteu reagent rutin catechin221015840-diphenyl-1-picrylhydrazyl (DPPH) 22-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) andascorbic acid were purchased from Sigma-Aldrich (France)All other reagents were of analytical grade

22 Plant Material

221 Plant Collection Roots of A longa were collected inApril 2016 in the province of Al Haouz in Morocco Thecollected plantmaterialswere authenticated at theHerbariumof Botany Department of the Scientific Institute of RabatMorocco The roots of the plant were washed dried at roomtemperature from48 to 92 h grounded into powder and thenstored in glass bottles preserved from light andmoisture untiluse

222 Preparation of Plant Extracts To prepare the extractsthe technique of continuous hot extraction by a Soxhletextractor was carried out using solvents of different polaritiesBriefly 10 g of root powder was extracted successively with100mL each of ethyl acetate methanol and water untilthe extracts were colorless in the siphon tube The aqueousextract was prepared by adding 500mL of distilled water to50 g of A longa dry roots powder After 24 h of macerationunder magnetic stirring at room temperature the mixturewas centrifuged Then all extracts already prepared werefiltered through a filter paper (Whatman) and evaporated todryness by a rotary evaporator at 50∘CThe extracts obtainedwere kept at 4∘C until further uses

23 Phytochemical Analysis

231 Determination of Total Phenolic Content Total phenoliccontent of A longa extracts was assessed by Folin-Ciocalteumethod [15] as described by Spanos andWrolstad [16] Gallicacid was used as standard for the calibration curve and theresults are expressed as mg of gallic acid equivalent per g ofextract dry weight (mg GAEg edw)

232 Determination of Total Flavonoid Content The totalflavonoid content of A longa extracts was determined

according to the method described by Dewanto et al [17]using Aluminium Chloride (AlCl3) Rutin was used toperform the standard curve and the results were expressedas rutin equivalent per gram of extract dry weight (mg REgedw)

233 Determination of Proanthocyanidin Content The pro-anthocyanidin content of A longa extracts was determinedas reported by Julkunen-Tiitto [18] Catechin was used as astandard for constructing the calibration curve and the resultsare expressed as catechin equivalent per gram of extract dryweight (mg CEg edw)

24 Antioxidant Activity

241 DPPH Radical Scavenging Activity Assay Radical scav-enging activity of the extracts was measured using the stableradical DPPH such as that determined by Sayah et al[19] with some modifications In tubes 25mL of differentconcentrations of each extract were introduced and 05mL ofmethanol solution of DPPH (02mM of DPPH dissolved inmethanol) freshly prepared was added The mixture is vigor-ously vortexed and left in the dark at ambient temperature for30min Then the absorbance of the mixture was measuredat 517 nm in a spectrophotometer The antioxidant activity ofour extracts is expressed as the percentage of DPPH radicalinhibition and the IC50 was calculated for comparing theobtained results

242 ABTSRadical ScavengingAssay TheTEAC test (TroloxEquivalent Antioxidant Capacity) or discoloration test ofABTS∙+ was carried out according to the method describedby Sayah et al [19] Briefly the cationic radical (ABTS∙+) wasprepared by the reaction between 10mL of ABTS (2mM) inH2O and 100120583L of potassium persulfate (K2S2O8) (70mM)The mixture was incubated in the dark for 16 hours at roomtemperature Then the ABTS∙+ solution was diluted withmethanol to obtain an absorbance of 070 at 734 nm Then200120583L of each extract was mixed with 2mL of the dilutedABTS∙+ solution and allowed to react for 1 minute After theabsorbance of the ABTS∙+ radical is measured at 734 nm Allsamples were made in triplicate The results were representedas Trolox equivalent per gram of extract dry weight (mgTEedw)

243 Ferric ReducingAntioxidant Power (FRAP) Assay TheFRAP test is performed according to themethod described bySayah et al [19] Briefly 1mL of each extract (1mgmL) wasmixed with 25mL of the phosphate buffer solution (02MpH 66) and 25mL of the 1 potassium ferricyanide aqueoussolution After incubation at 50∘C for 20min 25mL of 10trichloroacetic acid were added to the mixture and then themixture was centrifuged at 3000 rpm for 10min At the end25mL of the supernatant was mixed with 25mL of distilledwater and 05mL of aqueous ferric chloride solution FeCl3(01 wv) The absorbance was measured at 700 nm Theresults are expressed as ascorbic acid equivalent per gram ofextract dry weight (mg AAEg edw)

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Total phenolic flavonoid and proanthocyanidin content of extracts

Extracts Polyphenolic content (mgof GAEg of extract)

Flavonoid content (mg ofREg of extract)

Proanthocyanidins content(mg of CEg of extract)

MF 2448plusmn163b 700plusmn061b 0578plusmn028a

EAF 3255plusmn043c 11686plusmn412c 7764plusmn193c

AF 2890plusmn136bc 0760plusmn011b 1079plusmn049b

AE 1313plusmn048a 0380plusmn032a 0687plusmn032ab

The values are the mean of three determinations plusmn standard errorValues in the same column not sharing a common letter (a to c) differ significantly at 119901 lt 005MFmethanolic fraction EAF ethyl acetate fraction AF aqueous fraction AE aqueous extract GAE gallic acid equivalent RE rutin equivalent CE catechinequivalent

25 In Vitro Antidiabetic Effects

251 120572-Glucosidase Inhibitory Assay The 120572-Glucosidaseinhibitory activity was monitored using the substratep-Nitrophenyl 120572-d-glucopyranoside (pNPG) which ishydrolyzed by 120572-Glucosidase to release p-Nitrophenyl (acolored agent which can be monitored at 405 nm) accordingto the method described by Marmouzi et al [20 21] Theresults are expressed as percentage inhibition while forcomparing results the concentrations of inhibitor requiredto inhibit 50 of enzyme activity (IC50) were determined

252 120573-Galactosidase Inhibitory Assay The 120573-Galactosidaseinhibitory activity was assessed according to the methodof Bouabid et al [22] using 2-Nitrophenyl 120573-D-Galac-topyranoside as substrate which is hydrolyzed by 120573-Galactosidase to release 2-nitrophenyl (a colored agentwhich can be monitored at 410 nm) Briefly a mixtureof 150120583L of the samples at different concentrations (05-5mgmL) and 100120583L of sodium phosphate buffer 01M(pH=76) containing the enzyme 120573-Galactosidase solution(01UmL) was incubated at 37∘C for 10min After preincu-bation 200 120583L of gala solution 1mM in sodium phosphatebuffer 01M (pH=76) was added The reaction mixtureswere incubated at 37∘C for 30min After incubation 1mLof 01M of Na2CO3 were added to stop the reaction andthe absorbance was recorded at 405 nm using the spec-trophotometer The 120573-Galactosidase inhibitory activity wasexpressed as percentage inhibition and calculated using thesame formula as the 120572-Glucosidase test and the IC50 valueswere determined Quercetin was used as positive control andthe experiment was carried out in triplicate

26 Statistical Analysis Results are expressed as a mean plusmnstandard error of the mean Differences between the meanswere determined by one-way analysis of variance (one-wayANOVA) A difference in the mean values of 119901 lt 005 wasconsidered to be statistically significant All analyses wereperformed with GraphPad Prism 6 Also IC50 values weredetermined using a nonlinear regression curve with the sameprogram

3 Results and Discussion

31 Total Phenolic Flavonoid and Proanthocyanidin Con-tents Total phenolic flavonoid and proanthocyanidins

contents are presented in Table 1 The phenolic contentsin ethyl acetate fraction (EAF) of A longa were found tobe 3255plusmn043mg GAEg edw which are significantly (plt005) greater than methanolic fraction and aqueous extract(2448plusmn163mg GAEg edw and 1313plusmn048mg GAEg edw)respectively There is no significant difference between EAFand aqueous fraction (AF) (2890plusmn136mg GAEg edw)Results of flavonoid and proanthocyanidin contents showthat ethyl acetate fraction resulted also in significantly (plt005) higher values of those compounds (11686plusmn412mgREg edw and 7764plusmn193 mgCEg edw respectively) followedby the aqueous fractions that were 760plusmn011mg REg edwand 1079plusmn049mg CEg edw respectively The interestingphenolic contents of this herb indicate an important healthpromoting activity Indeed Djeridane et al [23] worked onthe A longa roots and they estimated the total phenolicsand flavonoids content as 147plusmn002mg GAEg edw and081plusmn002mg REg edw respectively [23] These compoundsare secondary plant metabolites possessing a wide range ofpharmacological activities such as anticancer antiviral anti-inflammatory activities and effects on capillary fragility [24ndash26] Previous studies which have been interested in otherspecies of Aristolochia species such as A bracteolata and Aindica revealed that the methanol extract from each plantcontains high amount of phenols and flavonoids [25 26]Phenolic compounds are an important and complex group ofchemical constituents present in plants and are classic defensecompounds to protect plants from herbivores pathogenicand parasite infections [27]

32 Antioxidant Activity Recently many scientific studieshave shown that free radicals play a major role in thedevelopment of cancer heart disease aging cataracts andimmune system damage [28] These unstable free radicalscan be eliminated by antioxidants that inhibit the rate ofoxidation and protect cells from damage [28] Antioxidantdrugs are used for the prevention and treatment of oxidativestress related disease such as diabetes Alzheimerrsquos diseaseatherosclerosis stroke and cancer [29 30] However its sideeffects and high prices force many people to take herbalmedicines which have fewer side effects [31] Numerousmethods for the analysis of in vitro and in vivo antioxidantactivity have been developed but only a few fast and reliablemethods for a large number of plant extract samples exist[32ndash34] Furthermore to study the antioxidant activity of


0

20

40

60

80

100

DPP

H in

hibi

tion

()

100 200 300 400 5000Concentration (gmL)

MF

EAF

AF

AE

Figure 1 DPPH radical scavenging activity of extracts The valuesare the mean of three determinations plusmn standard error

our plant extracts the ability to scavenge the stable freeradical DPPH and the cation ABTS and their ferric reducingantioxidant power (FRAP) was evaluated

DPPH is one of the stable free radicals commerciallyavailable nitrogen centered and largely used for evaluat-ing scavenging activity of antioxidant standards and plantextracts with a characteristic absorbance at 517 nm [35]which decreases in the presence of free radical scavengersBy accepting hydrogen from a corresponding donor theDPPH solution loses the characteristic dark purple color andbecomes yellow diphenylpicryl hydrazine [36ndash39]This scav-enging activity has been widely used as a quick and reliableparameter to evaluate the general in vitro antioxidant activityof plant extracts [40 41] Recently numerous studies reportedthe antioxidant properties of medicinal plant products usingDPPH assay [42ndash44] From these assay several moleculesfrom medicinal plants were developed as antioxidant agentsFigure 1 illustrated the DPPH radical scavenging activity ofdifferent extracts of A longa at various concentrations AllA longa extracts showed scavenging effect which increaseswith the concentration of samples At 416 120583gmL concentra-tion aqueous and methanolic fraction of A longa exhibitedincreased DPPH radical scavenging activity of 7717 and7466 respectively Moreover aqueous extract showed lessactivity at all concentrations These results showed that Alonga roots contained high amount of radical scavengingcompounds with proton-donating ability Similar result wasobserved in one previous study of Benmehdi et al [45] whichshowed a dose-dependent scavenging of DPPH radicals usingA clematitis roots

The radical cation of 22-azino-bis-3-ethylbenzothia-zoline-6-sulfonic acid (ABTS) is stable in its free formThe concentration of this radical can be determined bymeasuring the absorbance at 734 nm The addition of anantioxidant to a solution of this radical leads to its reduction

MF

EAF

AF

AE

0

20

40

60

80

100

４３

bull+in

hibi

tion

()

50 100 150 2000Concentration (gmL)

Figure 2 ABTS radical scavenging activity of extracts The valuesare the mean of three determinations plusmn standard error

and a decrease in absorbance This decrease depends onthe antioxidant activity of the test compound but also onthe time and the concentration [46] The experiments werecarried out using an improved ABTS decolorization assay[47] The ability of the extracts to scavenge ABTS cation wasexpressed in Figure 2 The aqueous fraction exhibited potentABTS radical cation scavenging activity in concentrationdependent manner At 181 120583gmL concentration aqueousand methanolic fraction of A longa possessed 9089 and8258 scavenging activity on ABTS At the concentrationsinvestigated this effect may indicate the capacity of the herbto minimize oxidative damage to certain vital tissues of thebody [48]These results are in agreement with the findings ofJegadeeswari et al [49] using the same ABTS test on anotherAristolochia species

In the FRAP method antioxidants present in the samplereduced the Fe3+ferricyanide complex to the blue ferrousform [50] which can serve as a measure of the antioxi-dant capacity interpreted as the reducing power [51] Thereducing power of the extracts is represented in Figure 3A higher absorbance indicates a higher reducing power At2000120583gmL concentration and among the solvents testedaqueous fraction exhibited higher reducing activity 8073The results obtained are consistent with the studies carriedout on A longa (aerial part) [52] and A indica (aerial part)[53] and (roots) [54] which indicate that they have a reducingpower

To compare the results the IC50 are calculated as shownin Table 2 A lower value of IC50 indicates greater antioxidantactivity Indeed the aqueous fraction showed the highestDPPH radical inhibition value (IC50=12540plusmn240120583gmL)while the methanolic fraction was the most active againstthe ABTS radical cation (IC50=6158plusmn215 120583gmL) and theTrolox value for both tests was 179plusmn035120583gmL and070plusmn001120583gmL respectively Our results are in analogy to


MFEAF AF AE

0

20

40

60

80

100

mg

of A

AE

g of

extr

act

Figure 3 Ferric reducing antioxidant power of extracts The valuesare the mean of three determinations plusmn standard error

Table 2 IC50 values (120583gmL) of extracts and Trolox on DPPH andABTS inhibition activity

Products DPPH (IC50 120583gmL) ABTS (IC50 120583gmL)MF 19935plusmn125b 6158plusmn215b

EAF 22080plusmn240b 10362plusmn862bc

AF 12540plusmn240b 6523plusmn249b

AE 35460plusmn520c 14440plusmn207c

Trolox 179plusmn035a 070plusmn00a

The values are the mean of three determinations plusmn standard errorValues in the same column not sharing a common letter (a to c) differsignificantly at 119901 lt 005

those of Ramalalingam et al [53] using the DPPH assayon the aqueous extract of A indica who showed IC50value 18231plusmn031120583gmL while standard showed the valueof 3012plusmn011120583gmL [53] In another study also using theDPPHassayA longa roots had theweakest activity comparedto Trolox among all the plants tested [23] Furthermoreour study showed similar findings with this work In ABTSmethodwe share the same results froma study of five extractsof A bracteolate who found that the methanolic extract hadpotent antioxidant activity [25] However there is variabilityin the recorded activity of the different extracts this is cer-tainly related to the difference in the chemical composition ofthe extracts especially polyphenols and flavonoids detectedby phytochemical analysis in our study These constituentshave different degree of antioxidant activity against differentfree radicals [55] The effectiveness of antioxidants may bedue to the high amount of the main constituents and also tothe presence of other constituents in small quantities or tothe synergy between them Indeed previous studies reportedthe presence of chemical compounds such as Aristolactamla (ALIa) Limonen-6-ol and Maaliol [56ndash58] Phenolicsare antioxidants with redox properties the hydroxyl grouphelps them to work as reducing agents hydrogen donorsand singlet oxygen quenchers [59 60] Plant extracts thathave high amounts of phenolic compounds do not alwaystranslate into high antioxidant capacity it may be due to thepresence of different active compounds the synergistic effects

of these compounds and also to the position and extent ofhydroxylation and conjugation [61] Moreover our activitywas significantly weak compared to Trolox (p lt005) thisresult can be interpreted by the fact that the extracts have sev-eral compounds while the concentrations of those who couldperform this activity will be really low It is also noted thatthe antioxidant effects change depending on the test used Infact the antioxidant activity depends on the interactions inthe reaction media between the substrate(s) (radicals) andthe active molecule(s) that trap them Other studies haveshown that the roots of A longa possess antioxidant activity[23] Moreover there is another study that was interestedin the aerial part of this plant [52] and we found almostthe same results for the DPPH method IC50 values of fruitaqueous extract and fruit methanol extract were found to be14515plusmn078120583gmL and 18621plusmn624120583gmL respectively Thedifference between the results is due as already mentionedto the difference of the phytoconstituents also to the methodused the harvest period and the studied part (root leafstem )

33 120572-Glucosidase and 120573-Galactosidase Inhibitory ActivitiesDiabetes is characterized by high blood sugar levels whichcan lead to serious complications so the goal of treatingpatients with diabetes is to maintain near-normal levels ofglycemia control In modern medicine there is no treatmentor medication to treat diabetes without side effects which arerelated to the use of insulin and oral hypoglycemic agents[62] Medicinal plants with antidiabetic properties may bea useful source for finding safer cost-effective antidiabeticdrugs In the present research different extracts of A longaare evaluated for their antidiabetic activity Two different invitro assays were used to evaluate this activity 120572-Glucosidaseand 120573-Galactosidase uptake assay120572-Glucosidase catalyzes the final step in the digestion

of carbohydrates and is located in the brush-border surfacemembrane of intestinal cells [63] Its inhibitors can retard theuptake of dietary carbohydrates in the small intestine andreduce postprandial hyperglycemia which may be a usefulmechanism in the preparation of antidiabetic drugs [64]Thisis largely used as an effective pharmacological strategy formanaging hyperglycemia related to the early stages of type2 diabetes [65]

On the other hand 120573-Galactosidase catalyzes the hy-drolysis of 120573-Galactosides subtracts to simple carbohydratesin the intestine Subsequently the inhibition of this enzymecan lead to the intestinal hydrocarbon reduction andeventually decreases the glucose level A longa extractsshowed inhibitory effects on both enzymes tested aspresented in Table 3 The result revealed that the testedextracts inhibited 120572-Glucosidase and 120573-Galactosidaseactivity concentration dependently (05ndash5mgmL) Indeedat the concentration of 15mgmL the ethyl acetate fractionhas the highest inhibitory activity against 120572-Glucosidase(7656plusmn254) and 120573-Galactosidase (1270plusmn127) Themethanolic fraction showed a moderate inhibitionof 120572-Glucosidase (2194plusmn134) and 120573-Galactosidase(205plusmn122) However the aqueous extract inhibited only


Table 3 120572-Glucosidase and 120573-Galactosidase enzyme inhibition test results

ExtractStandard

120572-Glucosidase inhibition 120573-Galactosidase inhibition of inhibition at 15

mgmL IC50 (mgmL) of inhibition at 15mgmL IC50 (mgmL)

Ethyl acetate fraction 7656plusmn254b 1112plusmn0026b 1270plusmn127b gt5Methanolic fraction 2194plusmn134a 2378plusmn0037b 205plusmn122a gt5Aqueous fraction na gt5 na gt5Aqueous extract na gt5 220plusmn013a gt5Acarbose 9678plusmn003c 0199plusmn0014a ---- ----Quercetin ---- ---- 9262plusmn014c 0247plusmn0006na nonactive mean plusmn SD (n=3)Values in the same column not sharing a common letter (a to c) differ significantly at plt 005

the enzymatic activity of 120573-Galactosidase (220) Theseresults are consistent with those of Janani and Revathi [66]who worked on another species of Aristolochiaceae (Aindica) Their study revealed that the methanolic extract ofwhole plant showed 120572-Glucosidase inhibitory activity whichincreased with the increasing concentration

To measure the inhibitory effectiveness of each extractwe used the IC50 which represents the concentration ofan inhibitor that is required for 50 inhibition of its tar-geted enzyme The fractions of ethyl acetate and methanolshowed a strong inhibitory capacity against 120572-Glucosidasewith IC50 values of 1112plusmn0026 and 2378plusmn0037mgmLrespectively These inhibition values are greater than thatobtained byAcarbose (0199plusmn0014mgmL) used as standardantidiabetic Similar effects were observed on A indica[67] Likewise the 120572-Glucosidase inhibition values by theaqueous extract and aqueous fraction are above 5mgmLFor 120573-Galactosidase inhibitory capacity all extracts showedan IC50 value greater than 5mgmL The fact that 120572-Glucosidase and 120573-Galactosidase showed difference is dueto structural differences related to the origins of enzymes[68]

The inhibitory effects of A longa extracts against theenzyme 120572-Glucosidase demonstrate their potential abilitiesto reduce the postprandial increase of blood glucose levelsin diabetic patients and their capacities to prevent type2 diabetes Hence it is suggested that the mechanism ofantihyperglycemic may be due to the antioxidant activity ofthis herb Our finding is in accordance with earlier reportsthat showed that in animal models two of its species aerialparts of A indica [69] and the A ringens roots extracts[70] showed a reduction in elevated blood glucose levelMoreover the results are in line with a study performed on71 herb plants to test their antidiabetic effects that showedthat 36 herbs had 120572-Glucosidase inhibition including aspecies of Aristolochiaceae (Asarum heterotropoides) [71]The differences observed for the inhibitory activity of theenzyme could be explained by the changes in the percentageof inhibition relative to the phytochemical composition ofthe plant species and also by the sensitivity of enzymesConsequently as mentioned above phytochemical studieson A longa demonstrated its abilities to produce a highamount of phenolic compounds and several flavonoids

including alkaloids [57] saponins and tannins [72] Thephenolic compounds are known by their capacity to inhibitthe activities of carbohydrate-hydrolyzing enzymes becauseof their ability to bind to proteins [73] Moreover thepresence of flavonoids especially in ethyl acetate fractionmay account for the inhibitory activity observed Indeedflavonoids have been known to possess high inhibitorypotential towards 120572-Glucosidase in both in vitro and in vivostudies [74] and may prevent the malfunction of pancreaticbeta cell due to oxidative stress and can thus reduce theonset of type 2 diabetes [75] Importantly some researchershave indicated that there is a positive relationship betweentotal flavonoid and polyphenol content and the ability toinhibit 120572-Glucosidase [76]The inhibitory effect observed formethanolic fraction of A longa may be associated with thepresence of other phytoconstituents like alkaloids tanninsand saponins [76 77] These last have been responsible forsuppressing the absorption of liquid and glucose at the brushborders [78] These compounds which can also inhibit 120572-Glucosidase have fewer side effects and are less expensivecompared to synthetic pharmacotherapeutics like Acarbose[79] and they perform several other biological activities suchas antibacterial antioxidant and anticancer [80] Generallyherbal medicine is based on the therapeutic action of amixture of different compounds acting often in synergyto exert all their beneficial effects This suggests that thebiologically active compounds present in the extracts studiedmay act in a synergistic way to exercise their carbohydrate-hydrolyzing enzymes inhibition activities and antioxidanteffects The observed variations in chemical composition ofAristolochia sp from different parts of the world are notonly due to the type of specie but also different agroclimaticconditions extraction method harvest period and charac-terization techniques [81 82] and also to the selected part ofplant and polarity of extraction solvents Concerning plantextracts other studies have shown that some of them canincrease insulin secretion and insulin signaling in adiposeand skeletal muscles [83] In the present study we haveinvestigated the antidiabetic potential of A longa which isused in traditional medicine for the treatment of severaldiseases This herb was not previously investigated for its invitro antidiabetic activity The results from this work shouldbe relevant to the human body


4 Conclusion

The aqueous fraction of the A longa roots had the bestantioxidant effects against the DPPH and ABTS radicalsand a strong ferric reducing power This suggests that Alonga can be used to prevent and control the oxidativestress induced by free radicals The antidiabetic activity wasalso investigated focusing on the inhibitory effects on 120572-Glucosidase and 120573-Galactosidase Our study is the first toreport a potential mode of action of A longa and suggeststhat the effect of this plant is due to the inhibition of digestiveenzymes On the other hand the presence of flavonoidsand phenols concludes that this herb has multiple biologicalproperties Other studies must be conducted to isolate theactive ingredients of this plant identify them and study theirbioactivity

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

The authors have no conflicts of interest to declare

References

[1] S G D Oliveira F R R de Moura F F Demarco P D SNascente F A B D Pino and R G Lund ldquoAn ethnomedicinalsurvey on phytotherapy with professionals and patients frombasic care units in the brazilian unified health systemrdquo Journalof Ethnopharmacology vol 140 no 2 pp 428ndash437 2012

[2] Y S VeliogluG Mazza L Gao and B D Oomah ldquoAntioxidantactivity and total phenolics in selected fruits vegetables andgrain productsrdquo Journal of Agricultural and Food Chemistry vol46 no 10 pp 4113ndash4117 1998

[3] M J Piao K A Kang R Zhang et al ldquoAntioxidant proper-ties of 12346-penta-O-galloyl-120573-d-glucose from Elaeocarpussylvestris var ellipticusrdquo Food Chemistry vol 115 no 2 pp 412ndash418 2009

[4] V Di Matteo and E Esposito ldquoBiochemical and therapeuticeffects of antioxidants in the treatment of Alzheimers diseaseParkinsons disease and amyotrophic lateral sclerosisrdquo CurrentDrugTargets-CNSampNeurological Disorders vol 2 no 2 pp 95ndash107 2003

[5] M Gerber M-C Boutron-Ruault S Hercberg E Riboli AScalbert and M-H Siess ldquoFood and cancer state of the artabout the protective effect of fruits and vegetablesrdquo Bulletin duCancer vol 89 no 3 pp 293ndash312 2002

[6] A Shirwaikar A P Somashekar A L Udupa S L Udupa andS Somashekar ldquoWound healing studies of Aristolochia bracte-olata Lam with supportive action of antioxidant enzymesrdquoPhytomedicine vol 10 no 6-7 pp 558ndash562 2003

[7] A G Pacheco P M De Oliveira D Pilo-Veloso and A F DeCarvalho Alcantara ldquoC-NMR data of diterpenes isolated fromAristolochia speciesrdquo Molecules vol 14 no 3 pp 1245ndash12622009

[8] J Bellakhdar R Claisse J Fleurentin and C Younos ldquoReper-tory of standard herbal drugs in the Moroccan pharmacopoeardquoJournal of Ethnopharmacology vol 35 no 2 pp 123ndash143 1991

[9] A Benchaabaneand and A Abbad ldquoMedicinal Plants markedin Marrakechrdquo in Trace of the Present Marrakech 1997

[10] J Bellakhdar The Traditional Moroccan Pharmacopoeia IbisPress 1997

[11] B K Bailes ldquoDiabetes mellitus and its chronic complicationsrdquoAORN Journal vol 76 no 2 pp 265ndash282 2002

[12] D G Gardner and D M Shoback Greenspanrsquos Basic amp ClinicalEndocrinology McGraw-Hill Medical New York NY USA 9thedition 2011

[13] M R Bhandari N Jong-AnurakkunG Hong and J Kawabataldquo120572-Glucosidase and 120572-amylase inhibitory activities of Nepalesemedicinal herb Pakhanbhed (Bergenia ciliata Haw)rdquo Journal ofFood Chemistry and Nutrition vol 106 no 1 pp 247ndash252 2008

[14] W Puls U Keup H P Krause G Thomas and F HoffmeisterldquoGlucosidase inhibition A new approach to the treatmentof diabetes obesity and hyperlipoproteinaemiardquo Naturwis-senschaften vol 64 no 10 pp 536-537 1977

[15] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1999

[16] G A Spanos and R E Wrolstad ldquoInfluence of processingand storage on the phenolic composition of thompson seedlessgrape juicerdquo Journal of Agricultural and Food Chemistry vol 38no 7 pp 1565ndash1571 1990

[17] V Dewanto X Wu and R H Liu ldquoProcessed sweet corn hashigher antioxidant activityrdquo Journal of Agricultural and FoodChemistry vol 50 no 17 pp 4959ndash4964 2002

[18] R Julkunen-Tiitto ldquoPhenolic constituents in the leaves ofNorthern willows methods for the analysis of certain pheno-licsrdquo Journal of Agricultural and Food Chemistry vol 33 no 2pp 213ndash217 1985

[19] K Sayah I Marmouzi H Naceiri Mrabti Y Cherrah and ME A Faouzi ldquoAntioxidant activity and inhibitory potential ofcistus salviifolius (L) and cistus monspeliensis (L) aerial partsextracts against key enzymes linked to hyperglycemiardquo BioMedResearch International vol 2017 Article ID 2789482 7 pages2017

[20] I Marmouzi E M Karym N Saidi et al ldquoIn vitro and in vivoantioxidant and anti-hyperglycemic activities of moroccan oatcultivarsrdquo Antioxidants vol 6 no 4 2017

[21] A Berrani I Marmouzi M Kharbach et al ldquoAnabasis are-tioidesCoss ampMoq phenolic compounds exhibit in vitro hypo-glycemic antioxidant and antipathogenic propertiesrdquo Journal ofBasic and Clinical Physiology and Pharmacology 2018

[22] K Bouabid F Lamchouri H Toufik K Sayah Y Cherrah andM E Faouzi ldquoPhytochemical screening and in vitro evaluationof alpha amylase alpha glucosidase and beta galactosidaseinhibition by aqueous and organic Atractylis gummifera Lextractsrdquo Plant Science Today vol 5 no 3 pp 103ndash112 2018

[23] A Djeridane M Yousfi B Nadjemi S Maamri F Djireb andP Stocker ldquoPhenolic extracts from various Algerian plants asstrong inhibitors of porcine liver carboxylesteraserdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 21 no 6 pp719ndash726 2006

[24] E S S Abdel-Hameed S A Bazaid and M M ShohayebldquoPhytochemical Studies and Evaluation of Antioxidant Anti-cancer and Antimicrobial Properties of Conocarpus erectus LGrowing in Taif Saudi Arabiardquo European Journal of MedicinalPlants vol 2 no 2 pp 93ndash112 2012


[25] S Badami C K Jose C R K Kumar et al ldquoIn vitro antioxidantactivity of various extracts of Aristolochia bracteolata leavesrdquoOriental Pharmacy and Experimental Medicine vol 5 no 4 pp316ndash321 2005

[26] D Sivaraj S Shanmugam M Rajan et al ldquoEvaluation ofAristolochia indica L and Piper nigrum L methanol extractagainst centipede Scolopendramoristans L usingWistar albinorats and screening of bioactive compounds by high pressureliquid chromatography a polyherbal formulationrdquo Biomedicineamp Pharmacotherapy vol 97 pp 1603ndash1612 2018

[27] N JWaltonM JMayer andA Narbad ldquoMolecules of interestvanillinrdquo Phytochemistry vol 63 no 5 pp 505ndash515 2003

[28] O A Asimi N P Sahu and A K Pal ldquoAntioxidant activityand antimicrobial property of some Indian spicesrdquo InternationalJournal Scientific Research Publications vol 3 no 3 pp 1ndash82013

[29] T P A Devasagayam J C Tilak K K Boloor et al ldquoFreeradicals and antioxidants in human health current status andfuture prospectsrdquoThe Journal of the Association of Physicians ofIndia vol 52 no 794804 4 pages 2004

[30] S I M Howlader M M Rahman A B R Khalipha MM Rahman and F Ahmed ldquoAntioxidant and antidiarrhoealpotentiality of Diospyros blancoirdquo International Journal ofPharmacology vol 8 no 5 pp 403ndash409 2012

[31] C P Kala ldquoCurrent status of medicinal plants used by tra-ditional Vaidyas in Uttaranchal state of Indiardquo EthnobotanyResearch amp Applications vol 3 pp 267ndash278 2005

[32] N J Miller C Rice-Evans M J Davies V Gopinathan andA Milner ldquoA novel method for measuring antioxidant capacityand its application to monitoring the antioxidant status inpremature neonatesrdquoClinical Science vol 84 no 4 pp 407ndash4121993

[33] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWT - FoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[34] O I Aruoma and S L Cuppett Antioxidant Methodology InVivo and In VitroConceptsTheAmericanOil Chemists SocietyPress Champaign IL USA 1997

[35] S L Helfand and B Rogina ldquoGenetics of aging in the fruit flyDrosophila melanogasterrdquoAnnualReview of Genetics vol 37 no1 pp 329ndash348 2003

[36] D Huang B Ou and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005

[37] F Conforti S Sosa M Marrelli et al ldquoIn vivo anti-inflammatory and in vitro antioxidant activities of Mediter-ranean dietary plantsrdquo Journal of Ethnopharmacology vol 116no 1 pp 144ndash151 2008

[38] G Tirzitis and G Bartosz ldquoDetermination of antiradical andantioxidant activity basic principles and new insightsrdquo ActaBiochimica Polonica vol 57 no 1 pp 139ndash142 2010

[39] K Prashith M Manasa G Poornima et al ldquoAntibacterialcytotoxic and antioxidant potential of Vitex negundo varnegundoand Vitex negundo var purpurascens- A comparativestudyrdquo Science Technology and Arts Research Journal vol 2 no3 pp 59ndash68 2013

[40] F Bonina C Puglia A Tomaino et al ldquoIn-vitro antioxidantand in-vivo photoprotective effect of three lyophilized extractsof Sedum telephium L leavesrdquo Journal of Pharmacy andPharmacology vol 52 no 10 pp 1279ndash1285 2000

[41] J R Soares T C P Dinis A P Cunha and L M AlmeidaldquoAntioxidant activities of some extracts of Thymus zygisrdquo FreeRadical Research vol 26 no 5 pp 469ndash478 1997

[42] S Sarr A Fall R Gueye et al ldquoEtude de lrsquoactivite antioxydantedes extraits des feuilles de Vitex doniana (Verbenacea)rdquo Inter-national Journal of Biological and Chemical Sciences vol 9 no3 pp 1263ndash1269 2015

[43] F Amezouar W Badri M Hsaine N Bourhim and HFougrach ldquoAntioxidant and anti-inflammatory activities ofMoroccan Erica arborea Lrdquo Pathologie Biologie vol 61 no 6pp 254ndash258 2013

[44] N Ghedadba H Bousselsela L Hambaba S Benbia and YMouloud ldquoEvaluation de lrsquoactivite antioxydante et antimicrobi-enne des feuilles et des sommites fleuries deMarrubiumvulgareLEvaluation of the antioxidant and antimicrobial activitiesof the leaves and flowered tops of Marrubium vulgare LrdquoPhytotherapie vol 12 no 1 pp 15ndash24 2014

[45] H Benmehdi A Behilil F Memmou and A Amrouche ldquoFreeradical scavenging activity kinetic behaviour and phytochem-ical constituents of Aristolochia clematitis L rootsrdquo ArabianJournal of Chemistry vol 10 pp S1402ndashS1408 2017

[46] A Cano M Acosta and M B Arnao ldquoA method to measureantioxidant activity in organic media Application to lipophilicvitaminsrdquo Redox Report vol 5 no 6 pp 365ndash370 2000

[47] R Re N Pellegrini A ProteggenteA PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[48] S E Atawodi ldquoAntioxidant potential of African medicinalplantsrdquo African Journal of Biotechnology vol 4 no 2 pp 128ndash133 2005

[49] P Jegadeeswari A Nishanthini S Muthukumarasamy and VR Mohan ldquoEvaluation of antioxidant activity of AristolochiaKrysagathra (Aristolochiaceae)- An important medicinal herbrdquoInternational Journal of Pharmacy vol 4 no 1 pp 410ndash4162014

[50] I GulcinMOktay E Kirecci and O I Kufrevıoglu ldquoScreeningof antioxidant and antimicrobial activities of anise (Pimpinellaanisum L) seed extractsrdquo FoodChemistry vol 83 no 3 pp 371ndash382 2003

[51] S P Samaradivakara R Samarasekera S M Handunnetti andO V D S J Weerasena ldquoCholinesterase protease inhibitoryand antioxidant capacities of Sri Lankan medicinal plantsrdquoIndustrial Crops and Products vol 83 pp 227ndash234 2016

[52] N Merouani R Belhattab and F Sahli ldquoEvaluation of thebiological activity ofAristolochia longa l Extractsrdquo InternationalJournal of Pharmaceutical Sciences andResearch vol 8 no 5 pp1978ndash1992 2017

[53] V Subramaniyan R Saravanan D Baskaran and SRamalalingam ldquoIn vitro free radical scavenging and anticancerpotential of aristolochia indica L Against MCF-7 cell linerdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 7 no 6 pp 392ndash396 2015

[54] S H Nile A S Nile and Y-S Keum ldquoTotal phenolicsantioxidant antitumor and enzyme inhibitory activity ofIndian medicinal and aromatic plants extracted with differentextraction methodsrdquo 3 Biotech vol 7 no 1 76 pages 2017

[55] J Sun Y-F Chu X Wu and R H Liu ldquoAntioxidant andantiproliferative activities of common fruitsrdquo Journal of Agricul-tural and Food Chemistry vol 50 no 25 pp 7449ndash7454 2002

[56] J Hinou CDemetzos CHarvala andC Roussakis ldquoCytotoxicand antimicrobial principles from the roots of aristolochia


longardquo International Journal of Crude Drug Research vol 28 no2 pp 149ndash151 1990

[57] M Dhouioui A Boulila H Chaabane M S Zina and HCasabianca ldquoSeasonal changes in essential oil composition ofAristolochia longa L ssp paucinervis Batt (Aristolochiaceae)roots and its antimicrobial activityrdquo Industrial Crops and Prod-ucts vol 83 pp 301ndash306 2016

[58] M Aneb A Talbaoui A Bouyahya et al ldquoIn vitro cytotoxiceffects and antibacterial activity of moroccan medicinal plantsaristolochia longa and lavandulamultifidardquo European Journal ofMedicinal Plants vol 16 no 2 pp 1ndash13 2016

[59] M-T Chua Y-T Tung and S-T Chang ldquoAntioxidant activ-ities of ethanolic extracts from the twigs of Cinnamomumosmophloeumrdquo Bioresource Technology vol 99 no 6 pp 1918ndash1925 2008

[60] TMudoi D C Deka and R Devi ldquoIn vitro antioxidant activityof Garcinia pedunculata an indigenous fruit of North Eastern(NE) region of Indiardquo International Journal of PharmTechResearch vol 4 no 1 pp 334ndash342 2012

[61] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[62] R J Marles and N Farnsworth ldquoPlants as sources of antidia-betic agentsrdquo Economic andMedicinal Plant Research vol 6 pp149ndash187 1994

[63] W F Caspary ldquoSucrosemalabsorption inman after ingestion of120572-glucosidehydrolase inhibitorrdquo The Lancet vol 311 no 8076pp 1231ndash1233 1978

[64] M Toeller ldquo120572-glucosidase inhibitors in diabetes efficacy inNIDDM subjectsrdquo European Journal of Clinical Investigationvol 24 Suppl 3 pp 31ndash35 1994

[65] K Hanhineva R Torronen I Bondia-Pons et al ldquoImpactof dietary polyphenols on carbohydrate metabolismrdquo Interna-tional Journal of Molecular Sciences vol 11 no 4 pp 1365ndash14022010

[66] N Janani and K Revathi ldquoIn vitro evaluation of Aristolochiaindica for its anti-inflammatory antidiabetic and anticancerefficacyrdquo International Journal of Current Research in MedicalSciences vol 4 no 6 pp 23ndash30 2018

[67] S H Nile and Y S Keum ldquoAntioxidant anti-inflammatoryand enzyme inhibitory activities of 10 selected unani herbsrdquoBangladesh Journal of Pharmacology vol 12 no 2 pp 162ndash1642017

[68] S Chiba ldquoMolecular mechanism in 120572-glucosidase and glu-coamylaserdquo Bioscience Biotechnology and Biochemistry vol 61no 8 pp 1233ndash1239 1997

[69] S K Karan S KMishra D Pal andAMondal ldquoIsolation of120573-sitosterol and evaluation of antidiabetic activity of Aristolochiaindica in alloxan-induced diabetic mice with a reference to in-vitro antioxidant activityrdquo Journal of Medicinal Plants Researchvol 6 no 7 pp 1219ndash1223 2012

[70] A O Sulyman J O Akolade S A Sabiu R A Aladodo andH F Muritala ldquoAntidiabetic potentials of ethanolic extract ofAristolochia ringens (Vahl) rootsrdquo Journal of Ethnopharmacol-ogy vol 182 pp 122ndash128 2016

[71] S M Shin Y J Jeong D W Park et al ldquoScreening for anti-diabetic effects of prescribed korean traditional medicinesrdquoKorean Journal of Plant Resources vol 25 no 6 pp 670ndash6812012

[72] B Benarba and B Meddah ldquoEthnobotanical study antifungalactivity phytochemical screening and total phenolic content ofAlgerianAristolochia longardquo Journal of Intercultural Ethnophar-macology vol 3 no 4 p 150 2014

[73] S Shobana Y N Sreerama and N G Malleshi ldquoCompositionand enzyme inhibitory properties of finger millet (Eleusinecoracana L) seed coat phenolics mode of inhibition of 120572-glucosidase and pancreatic amylaserdquo Food Chemistry vol 115no 4 pp 1268ndash1273 2009

[74] S A Adefegha and G Oboh ldquoIn vitro inhibition activityof polyphenol-rich extracts from Syzygium aromaticum (L)Merr amp Perry (Clove) buds against carbohydrate hydrolyzingenzymes linked to type 2 diabetes and Fe2+-induced lipidperoxidation in rat pancreasrdquo Asian Pacific Journal of TropicalBiomedicine vol 2 no 10 pp 774ndash781 2012

[75] Y Song J E Manson J E Buring H D Sesso and S LiuldquoAssociations of dietary flavonoids with risk of type 2 diabetesand markers of insulin resistance and systemic inflammationin women a prospective study and cross-sectional analysisrdquoJournal of the American College of Nutrition vol 24 no 5 pp376ndash384 2005

[76] K M Ramkumar B Thayumanavan T Palvannan and PRajaguru ldquoInhibitory effect of Gymnema montanum leaveson 120572-glucosidase activity and 120572-amylase activity and theirrelationship withpolyphenolic contentrdquo Medicinal ChemistryResearch vol 19 no 8 pp 948ndash961 2010

[77] E V Shabrova O Tarnopolsky A P Singh J Plutzky N Vorsaand L Quadro ldquoInsights into the molecular mechanisms ofthe anti-atherogenic actions of flavonoids in normal and obesemicerdquo PLoS ONE vol 6 no 10 Article ID e24634 2011

[78] M F Mahomoodally A H Subratty A Gurib-Fakim M IChoudhary and S Nahar Khan ldquoTraditional medicinal herbsand food plants have the potential to inhibit key carbohydratehydrolyzing enzymes in vitro and reduce postprandial bloodglucose peaks in vivordquo The Scientific World Journal vol 2012Article ID 285284 9 pages 2012

[79] E A H Mohamed M J A Siddiqui L F Ang et al ldquoPotent120572-glucosidase and 120572-amylase inhibitory activities of standard-ized 50 ethanolic extracts and sinensetin from Orthosiphonstamineus Benth as anti-diabetic mechanismrdquo BMC Comple-mentary and Alternative Medicine vol 12 no 1 176 pages 2012

[80] Q He Y Lv and K Yao ldquoEffects of tea polyphenols onthe activities of 120572-amylase pepsin trypsin and lipaserdquo FoodChemistry vol 101 no 3 pp 1178ndash1182 2006

[81] L A Usman M F Zubair S A Adebayo I A OladosuN O Muhammad and J Akolade ldquoChemical compositionof leaf and fruit essential oils of Hoslundia opposite Vahlgrown in Nigeriardquo American-Eurasian Journal Agricultural andEnvironmental Science vol 8 no 1 pp 40ndash43 2010

[82] J O Akolade L A Usman O E Okereke and N O Muham-mad ldquoAntidiabetic potentials of essential oil extracted from theleaves of hoslundia opposita vahlrdquo Journal of Medicinal Foodvol 17 no 10 pp 1122ndash1128 2014

[83] D Ahmed V Kumar M Sharma and A Verma ldquoTargetguided isolation in-vitro antidiabetic antioxidant activity andmolecular docking studies of some flavonoids from AlbizziaLebbeck Benth barkrdquo BMC Complementary and AlternativeMedicine vol 14 no 1 155 pages 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


by hyperglycemia due to lack of insulin production bythe pancreas or the inability of the insulin produced tocontrol blood glucose [11 12] One interesting approach isto reduce postprandial hyperglycemia by retarding glucoseuptake through the inhibition of carbohydrate-hydrolyzingenzymes such as 120572-Glucosidase and 120573-Galactosidase [13 14]In this context the aim of this study was to evaluate the invitro antioxidant activity 120572-Glucosidase and 120573-Galactosidaseinhibitory potentials of A longa root extracts

2 Materials and Methods

21 Reagents p-Nitrophenyl-120572-D-glucopyranoside 2-Ni-trophenyl-120573-D-galactopyranoside 120572-Glucosidase fromSaccharomyces cerevisiae 120573-Galactosidase from Aspergillusoryzae Acarbose Folin-Ciocalteu reagent rutin catechin221015840-diphenyl-1-picrylhydrazyl (DPPH) 22-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) andascorbic acid were purchased from Sigma-Aldrich (France)All other reagents were of analytical grade

22 Plant Material

221 Plant Collection Roots of A longa were collected inApril 2016 in the province of Al Haouz in Morocco Thecollected plantmaterialswere authenticated at theHerbariumof Botany Department of the Scientific Institute of RabatMorocco The roots of the plant were washed dried at roomtemperature from48 to 92 h grounded into powder and thenstored in glass bottles preserved from light andmoisture untiluse

222 Preparation of Plant Extracts To prepare the extractsthe technique of continuous hot extraction by a Soxhletextractor was carried out using solvents of different polaritiesBriefly 10 g of root powder was extracted successively with100mL each of ethyl acetate methanol and water untilthe extracts were colorless in the siphon tube The aqueousextract was prepared by adding 500mL of distilled water to50 g of A longa dry roots powder After 24 h of macerationunder magnetic stirring at room temperature the mixturewas centrifuged Then all extracts already prepared werefiltered through a filter paper (Whatman) and evaporated todryness by a rotary evaporator at 50∘CThe extracts obtainedwere kept at 4∘C until further uses

23 Phytochemical Analysis

231 Determination of Total Phenolic Content Total phenoliccontent of A longa extracts was assessed by Folin-Ciocalteumethod [15] as described by Spanos andWrolstad [16] Gallicacid was used as standard for the calibration curve and theresults are expressed as mg of gallic acid equivalent per g ofextract dry weight (mg GAEg edw)

232 Determination of Total Flavonoid Content The totalflavonoid content of A longa extracts was determined

according to the method described by Dewanto et al [17]using Aluminium Chloride (AlCl3) Rutin was used toperform the standard curve and the results were expressedas rutin equivalent per gram of extract dry weight (mg REgedw)

233 Determination of Proanthocyanidin Content The pro-anthocyanidin content of A longa extracts was determinedas reported by Julkunen-Tiitto [18] Catechin was used as astandard for constructing the calibration curve and the resultsare expressed as catechin equivalent per gram of extract dryweight (mg CEg edw)

24 Antioxidant Activity

241 DPPH Radical Scavenging Activity Assay Radical scav-enging activity of the extracts was measured using the stableradical DPPH such as that determined by Sayah et al[19] with some modifications In tubes 25mL of differentconcentrations of each extract were introduced and 05mL ofmethanol solution of DPPH (02mM of DPPH dissolved inmethanol) freshly prepared was added The mixture is vigor-ously vortexed and left in the dark at ambient temperature for30min Then the absorbance of the mixture was measuredat 517 nm in a spectrophotometer The antioxidant activity ofour extracts is expressed as the percentage of DPPH radicalinhibition and the IC50 was calculated for comparing theobtained results

242 ABTSRadical ScavengingAssay TheTEAC test (TroloxEquivalent Antioxidant Capacity) or discoloration test ofABTS∙+ was carried out according to the method describedby Sayah et al [19] Briefly the cationic radical (ABTS∙+) wasprepared by the reaction between 10mL of ABTS (2mM) inH2O and 100120583L of potassium persulfate (K2S2O8) (70mM)The mixture was incubated in the dark for 16 hours at roomtemperature Then the ABTS∙+ solution was diluted withmethanol to obtain an absorbance of 070 at 734 nm Then200120583L of each extract was mixed with 2mL of the dilutedABTS∙+ solution and allowed to react for 1 minute After theabsorbance of the ABTS∙+ radical is measured at 734 nm Allsamples were made in triplicate The results were representedas Trolox equivalent per gram of extract dry weight (mgTEedw)

243 Ferric ReducingAntioxidant Power (FRAP) Assay TheFRAP test is performed according to themethod described bySayah et al [19] Briefly 1mL of each extract (1mgmL) wasmixed with 25mL of the phosphate buffer solution (02MpH 66) and 25mL of the 1 potassium ferricyanide aqueoussolution After incubation at 50∘C for 20min 25mL of 10trichloroacetic acid were added to the mixture and then themixture was centrifuged at 3000 rpm for 10min At the end25mL of the supernatant was mixed with 25mL of distilledwater and 05mL of aqueous ferric chloride solution FeCl3(01 wv) The absorbance was measured at 700 nm Theresults are expressed as ascorbic acid equivalent per gram ofextract dry weight (mg AAEg edw)




















0

20

40

60

80

100

DPP

H in

hibi

tion

()


MF

EAF

AF

AE





MF

EAF

AF

AE

0

20

40

60

80

100

４３

bull+in

hibi

tion

()







MFEAF AF AE

0

20

40

60

80

100

mg

of A

AE

g of

extr

act
















ExtractStandard









4 Conclusion


Data Availability




References



























































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






































0

20

40

60

80

100

DPP

H in

hibi

tion

()


MF

EAF

AF

AE





MF

EAF

AF

AE

0

20

40

60

80

100

４３

bull+in

hibi

tion

()







MFEAF AF AE

0

20

40

60

80

100

mg

of A

AE

g of

extr

act
















ExtractStandard









4 Conclusion


Data Availability




References



























































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















MFEAF AF AE

0

20

40

60

80

100

mg

of A

AE

g of

extr

act
















ExtractStandard









4 Conclusion


Data Availability




References



























































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















ExtractStandard









4 Conclusion


Data Availability




References



























































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















4 Conclusion


Data Availability




References



























































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















evaluation of in vitro antioxidant and antidiabetic

Documents