Hindawi Publishing Corporation International Journal of Carbohydrate Chemistry Volume 2014, Article ID 287281, 12 pages http://dx.doi.org/10.1155/2014/287281 Research Article Twenty Traditional Algerian Plants Used in Diabetes Therapy as 𝛼 Strong Inhibitors of -Amylase Activity IhcenKhacheba,AmarDjeridane,andMohamedYousfi LaboratoiredesSciencesFondamentale,Universite´AmarTelidji,Laghouat,Algeria CorrespondenceshouldbeaddressedtoMohamedYousfi;[email protected] Received1May2014;Revised7July2014;Accepted8July2014;Published5August2014 AcademicEditor:JianjunLi Copyright©2014IhcenKhachebaetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense, whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. Inthepresentwork,wehavestudiedtheinhibitoryeffectsofaqueousandalcoholicextractsofsixAlgerianmedicinalplantsknown bytheirtherapeuticvirtuesagainstdiabetes.Thetotalphenoliccompoundscontent,assayedusingFolin-Ciocalteu’sreagent,ofthe samplesrangedfrom0.183mg/gto43.088mg/gandfrom1.197mg/gto7.445mg/g,expressedasgallicacidequivalent(GAE),for the,respectively,whereasthetotalflavonoidsconcentrations,detectedusing2%ofthealuminiumchloride,rangedfrom0.41mg/g to11.613mg/gandfrom0.0097mg/gto1.591mg/g,expressedasrutinequivalents(RE),fortheaqueousandmethanolicextracts, respectively.ThemajorplantswerefoundtoinhibitenzymaticactivitiesofAspergillusoryzae-amylaseinaconcentrationdependent manner.Thevaluesoftheinhibitionconstants(𝐾𝑖)havebeendeterminedaccordingtotheDixonandLineweaver-Burkmethods. Theresultsshowedthatthe𝐾𝑖valueswerelessthan55ppmfortheallextracts.Astronginhibitionwasfoundinthephenolicextract ofSalviaofficinaliswitha𝐾𝑖of8ppm. 1.Introduction of medicament against various diseases. These properties are usually attributed to secondary metabolites that are the Diabetes mellitus (DM) is a chronic metabolic disorder subject of a lot of research in this field. This is particularly causedbyanabsoluteorrelativelackofresistancetoinsulin. the case of polyphenol plants that are widely renowned in It is characterized by hyperglycemia and accompanied by therapeuticsasanti-inflammatories,enzymeinhibitors,and various chronic vascular complications [1–3]. About 171 antioxidants,particularlyflavonoids[6,17–19]. million people worldwide have diabetes, which is likely to Plant hypoglycemic properties have been used in folk bemorethandoubleby2030andaround3.2milliondeaths medicine from very ancient time. Medicinal plants used every year are attributable to complication of diabetes, six to treat hyperglycemic are of considerable interest to eth- deathseveryminute[4,5]. nobotanical community as they are recognized to contain Onetherapeuticapproachtodecreasethehyperglycemia valuable medicinal properties in different parts of the plant is to retard and reduce the digestion and absorption of [7, 11, 20] and, because of their effectiveness, fewer side ingested carbohydrate hydrolyzing enzymes (such as 𝛼- effectsandrelativelylowcost.Tothisend,researchhasbegun amylase and/or 𝛼-glucosidase) in the digestive organs [6– to embrace traditional medicines from various cultures, as 10]. The inhibition of enzymes involved in the digestion scientistssearchforcluestodiscovernewtherapeuticdrugs ofcarbohydratescansignificantlydecreasethepostprandial [7,21]. increase of blood glucose after a mixed carbohydrate diet InthetownofLaghouatinthestepperegionofAlgeria, by delaying the process of carbohydrate hydrolysis and the list of plants that fit neatly within this framework absorption [6, 11–13]. Therefore, safer natural amylase and is exhaustive; they are used as teas, extracts, or complex glucosidaseinhibitorshavebeenreportedfromplantsources preparations,withoutknowingthemechanismoftreatment. [14–16]. In this study, we reported the screening results for Arisingfromtheirbiodiversityandtheirwealthofactive amylase inhibitory activity of 21 herbal extracts, to confirm ingredients,plantshavebeenusedfromantiquityassources theirantidiabeticactivitybytheinhibitionof𝛼-amylase. 2 InternationalJournalofCarbohydrateChemistry Table1:Thenameandtheaerialpartofthe21plants. 2.3. Extraction. Assuming that the active ingredients are polarcompounds,theextractionofthesewasmadeinitially Name Aerialpartused with distilled water. The air-dried aerial parts of each plant Ajugaiva Alltheaerialparts werefinelypowdered.Onegramofeachpowderwasheated ∘ Aloesocotrina Theresins in 20mL of distilled water at 75 C for 20 minutes. The extractwasfilteredandevaporatedtodrynessusingarotary Anthemisarvensis Theflowers evaporator. The dried residue was dissolved in 5mL of Berberisvulgaris Thebark ∘ distilledwaterandkeptat4 C. Cistus Theleavesandstems Depending on the results of the inhibition test on the Equisetumarvense Grainsandleaves enzymeactivity,wehaveselectedsixplantsfromtheplants Erythraeacentaurium Alltheaerialparts investigatedwhichare“Cistus,”“Oudneyaafricana,”“Equise- Haloxylonscoparium Theflowers tumarvense,”“Matricariapubescens,”“Salviaofficinalis,” and Helianthemumlippii Alltheaerialparts “Thapsiagarganica”whichshowedinhibitionrateabove70%. Marrubiumvulgare Alltheaerialparts Thesixselectedplantsaresubjectedtoaseriesofextractions withdistilledwaterandmethanol.1goftheplantpowderwas Matricariapubescens Alltheaerialparts ∘ heatedin20mLofdistilledwaterfor20minat75 C. Ononisangustissima Theleaves After filtration, the residues of the plant were then Oudneyaafricana Thepodsandtheleaves macerated for 72h with 10mL of absolute methanol. The Pituranthoschloranthus Thegrains filtratesobtainedfrombothextractionstepsareevaporatedto Rhamnusalaternus Theleaves drynessandtheprecipitatewasdissolvedin5mLofdistilled Rhanteriumadpressum Theflowers waterand5mLofabsolutemethanolforaqueousextractsand Salviaofficinalis Alltheaerialparts methano¨ıque extracts, respectively. The plant extracts were ∘ keptat4 C. Teucriumpolium Alltheaerialparts Thapsiagarganica Theflowers 2.4. Determination of Total Phenolics Compound. The Trigonellafoenum Thegrains amount of total phenolics in the samples was determined Zygophyllumalbum Alltheaerialparts with the Folin-Ciocalteu reagent using the method of SingletonandRoss(1965)[22].Theprocedureisasfollows: 100𝜇L of each sample was added to 500𝜇L of the aqueous Thereforewehaveproceededtotheextractionofthepolar solution of Folin-Ciocalteu reagent at 10%. After 2min of bioactivecompoundsofthe21differentmedicinalplantswith incubation at room temperature, 2mL of 2% (w/v) sodium two polar solvents, and we have tested the effect of these carbonate in water was added. Blanks were prepared by extractsontheenzymaticactivitytoconfirmtheirinhibitory replacing the reagent by water to correct for interfering activities.Thisisthefirstworkinthevalidationofsomelocal compound. After 30min of incubation in the dark at room plantsusedfrequentlyintraditionalAlgerianmedicine. temperature,theabsorbanceofallsampleswasmeasuredat 760nmusingtheShimadzu1601visiblespectrophotometer. 2.MaterialsandMethods Thegallicacidwasusedasastandardandalltheassayswere carriedoutatleastintriplicate. 2.1. Plant Material. 21 plants have been evaluated in this study. The names and the parts used in each plant are 2.5. Quantification of Flavonoids Content. The flavonoids summarizedinTable1.Theplantsselectedforthestudyofthe inhibitoryeffecton𝛼-amylaseactivityareEquisetumarvense content in the extracts was determined spectrophotomet- rically according to the method of Laimaison and Carnat (Equisetaceae),Matricariapubescens(Asteraceae),Oudneya (1991) [23], using a method based on the formation of africana(Brassicaceae),Salviaofficinalis(Lamiaceae),Thap- the complex flavonoids-aluminium, having an absorption siagarganica(Ombellifereae),andCistuswhosetypehasnot maximum at 409nm. Rutin was used for the calibration beendetermined. curve. 1mL of diluted sample was mixed with 1mL of 2% Nine of these medicinal plants were purchased from aluminumchloridemethanolicsolution.Afterincubationat differentherbalistsofLaghouatCitybecausetheyareknown roomtemperaturefor20min,theabsorbanceofthereaction bytheirtherapeuticeffectagainstdiabetesmellitus,theothers mixturewasmeasuredat409nmwithaShimadzu1601visible plants were collected from different locations around the spectrophotometerandtheflavonoidscontentisexpressedin townofLaghouatinthestepperegionofAlgeria.Thevarious mgpergrutinequivalent(RE)ofdryweightmaterial. data(localname,medicinaluses,usedpartsofplant,method ofpreparation,andadministration)werecollectedfromlocal inhabitantsandherbalistshavingknowledgeofthecurative 2.6. Assay for Fungal 𝛼-Amylase Inhibitory Activity. In the propertiesoftheseplants. firsttime,wehavetestedtheeffectofthe23aqueousextracts atthesameconcentrationonthe𝛼-amylaseactivity. 2.2. Reagents. All chemicals were purchased from Sigma Thefungal𝛼-amylaseinhibitoryactivitywasdetermined (USA), Aldrich (Milwaukee, USA), Fluka Chemie (Buchs, accordingtoaliteraturemethod[24],onitssubstratestarch Switzerland),andMerck(Germany). usingneocuproineasareagent. InternationalJournalofCarbohydrateChemistry 3 Themethodisbasedonthereducingpowerofthemaltose Table2:Totalamountofplantphenolicscompoundandflavonoids. productthatreactswithabasicsolutionofglycine-copper(A Totalphenolics Flavonoids solution)withbluecolor,developingayelloworangecolorin Nameofplant content content thepresenceofneocuproine(Bsolution). In brief, 200𝜇L of sodium phosphate buffer containing (mgGAE/gdw)a (mgRE/gdw)b 6mMNaCl(pH6,9)wasmixedwith100𝜇Lofsolublestarch Ajugaiva 1.80±0.03 0.66±0.02 (0.05%)asasubstrateand100𝜇Lofsuitablealiquotsofour Aloesocotrina 8.91±0.06 8.39±0.07 aqueous extracts, whereas 100𝜇L of the buffer was used in Anthemisarvensis 3.94±0.05 0.95±0.04 the place of the plant extract for the blank sample. After BerberisVulgaris 6.74±0.09 0.53±0.1 thoroughly mixing, both sample and blank test tubes were Cistus 43.08±0.02 2.07±0.01 ∘ preincubatedat37 Cfor10min;thenthereactionisstarted bytheadditionof100𝜇Lof𝛼-amylasefromAspergillusoryzae Equisetumarvense (i)Thegrains 2.48±0.07 0.06±0.08 (13units;oneunitofenzymeactivityisdefinedastheamount ofenzymerequiredtoreleaseonemicromoleofmaltosefrom (ii)Theleaves 1.92±0.31 0.51±0.3 starchperminuteunderassayconditions).Afterincubation Erythraeacentaurium 7.32±0.14 6.49±0.15 ∘ at37 Cfor5minthereactionwasstoppedbyadding1mLof Haloxylonscoparium 26.71±0.15 1.73±0.12 Asolutionand1mLofBsolution.Thereactionmixturewas Helianthemumlippii 1.39±0.01 1.35±0.02 ∘ incubatedat100 Cfor10min;afterthatthetubeshavebeen Marrubiumvulgare 1.36±0.07 0.21±0.06 cooledwithtapwater. Matricariapubescens 1.71±0.03 0.43±0.04 Enzymeactivitywasquantifiedbymeasuringopticalden- sity proportional to the quantity of the maltose equivalents Ononisangustissima 1.91±0.52 1.16±0.51 releasedfromstarchat450nmandtheinhibitoryactivitywas Oudneyaafricana calculatedusingthefollowingformula: (i)Thepods 9.17±0.04 3.61±0.03 (ii)Theleaves 15.86±0.02 6.54±0.03 (𝐴 −𝐴 ) Inhibitory activity(%)=[ 0 𝑠 ]×100, (1) Pituranthoschloranthus 3.62±0.46 0.91±0.36 𝐴0 Rhamnusalaternus 3.01±0.02 2.11±0.03 Rhanteriumadpressum 2.88±0.33 1.11±0.30 where𝐴0isabsorbanceofcontrolwithoutinhibitor,and𝐴𝑠 Salviaofficinalis 16.12±0.30 11.61±0.15 isabsorbanceoftestsamplewithinhibitor. Teucriumpolium 5.61±0.01 3.00±0.05 Thapsiagarganica 8.10±0.04 2.83±0.03 3.ResultsandDiscussion Trigonellafoenum 0.18±0.08 0.04±0.07 Zygophyllumalbum 2.16±0.005 1.97±0.01 3.1. Total Phenolic Content. The polar extracts of twenty plantsknownfortheirtherapeuticpropertiesagainstdiabetes aMilligramsofgallicacidequivalentpergramofdryweightofplant. intraditionalArabmedicineweretestedfortheirinhibitory bMilligramsofrutinequivalentpergramofdryweightofplant. activity towards purified fungal 𝛼-amylase. A number of spectrophotometric methods for quantification of pheno- lic compound in plant materials were developed. These inhibition[2,28–30].Therefore,thecontentofthisgroupof tests were based on different principles and were used to phenolicswasdeterminedintheextracts. determinethevariousstructuralgroupspresentinphenolic The content of phenolic compounds (mg/g) in the 23 compounds[25].Thephenoliccontentofeachplantextract aqueousextractsofthe20plantstestedvariedbetween0.18 was estimated by the Folin-Ciocalteu procedure, and the and 43.088mg/g. The highest amounts were found in the amount of polyphenols in the plants was calculated from extractof“Trigonellafoenum”whilethelowestamountswere the calibration curve of gallic acid previously realized and found in the extract of “Cistus.” The plant Helianthemum expressed in milligrams of gallic acid equivalents per gram kahiricumshowedthehighestvaluesintotalphenolsinthe of dry matter, while the quantification of flavonoids in our twodichloromethaneandethylacetatefractionsagainstthe extracts was determined by complexation with trichloride plantSalsolabaryosmawhichshowedthelowestvalue. aluminumfromthecalibrationcurveofrutinandexpressed Theamountoftotalphenoliccompoundsinallthetested in milligrams of rutin equivalent per gram of dry matter plantsislessthanthatinotherstudiesonotherplantspecies (Table2). in the region of Laghouat [31], but near to other studies of So far, as plant phenolics constitute one of the major Djeridane et al. [32]. This lowness among our plants may groups of compounds acting as enzyme inhibitor, it was be related to a poverty of our plant in polyphenols and reasonable to determine their total amount in the selected probably to favorable biotic conditions our plants grew in plant extracts. Flavonoids as one of the most diverse and whichdidnotstimulatethebiosynthesisofthesemolecules. widespread groups of natural compounds are probably the Thecontentofflavonoids(mg/g),inrutinequivalents,varied most important natural phenolics [17, 26, 27]. These com- from0.041to11.613.Thehighestamountsofflavonoidswere poundspossessabroadspectrumofchemicalandbiological found in the aqueous extract of Salvia officinalis. All the activitiesincludingradicalscavengingpropertiesandenzyme aqueousextractsshowedthepresenceofflavonoid.Theplant 4 InternationalJournalofCarbohydrateChemistry Table3:Totalamountofphenolicscompoundandflavonoidsofthe inflavonoidsmainlytheextractsofthetwoplantsEquisetum sixmethanolicextracts. arvense (the grains) and Thapsia garganica which can be explainedbythefactthatthisplantcontainsotherclassesof Teneuren Teneuren phenolicscompoundexcepttheflavonoids. Nomdelaplante phe´nolstotaux: flavono¨ıdes: mg/gMS mg/gMS Cistus 5.71±0.01 2.03±0.001 3.2.InhibitionofFungal𝛼-Amylase. Inordertoconfirmthe Equisetumarvense(thegrains) 2.45±0.005 0±0.00 antidiabetic effect of these 20 plants by the inhibition of the fungal 𝛼-amylase used in this work, we studied in vitro Matricariapubescens 3.16±0.05 1.04±0.006 the effects of our phenolic extracts on the activities of the Oudneyaafricana(thepods) 7.06±0.1 0.27±0.003 enzyme,atvaryingconcentrationsofextractsandsubstrates, Salviaofficinalis 11.42±0.004 0.02±0.10 toidentifyplantswithinhibitoryabilitiesontheenzyme. Thapsiagarganica 1.87±0.2 0±0.00 The enzymatic activities of 𝛼-amylase were titrated using starch as a substrate which releases maltose with a spectrophotometerdetectionafterreactionwithcomplexing agents. Salviaofficinalisshowedthepresenceofthehighestamounts Based on the calibration curve of maltose, we have of both phenols and flavonoids which can be explained by determined the concentrations of maltose liberated in the thefactthatthisplantwasgrowninbadweatherconditions reactionfordifferentconcentrationsofstarch.Thevaluesof causingthesynthesisoflargequantityinphenoliccompound. concentrations allowed us to plot that the enzyme shows a If we compare the values of these flavonoids contents kineticsimilartothekineticofMichaelis. to those of phenolic compounds, we see that they are all Toidentifyplantswithinhibitorycapacitieswesubjected smaller than the latter, indicating that the extracts contain our aqueous extracts to inhibition assay at the same con- otherphenoliccompoundshavingchemicalstructuresother centration of extracts. These tests showed that all aqueous thanflavonoids(phenolicacid,tannins,stilbenes,etc.).Some extracts have a significant inhibition of 𝛼-amylase enzyme plantshaveprovenrichinflavonoidssuchasAloesocotrina, with the exception of two plants “Zygophyllum album” and Erythraeacentaurium,andHelianthemumlippii. “Trigonellafoenum”(Table4). Followingtheinhibitiontest ofthetwenty plantsinves- According to the results, we note that different levels tigated in this work, we have selected six plants that have a of inhibition ranged between 8.29 and 100%. The most percentage higher than 70% for the further study of more inhibitoryactivitybycalculatingtheir𝐾𝑖.Forthesesixplants importantpercentagesofinhibitionarenotedfortheextracts of“Cistus,”thepodsof“OudneyaAfricana,”andthegrains we have repeated a similar extraction to that of the twenty of“Equisetumarvense.” plantsfollowedbymacerationinabsolutemethanol. The quantification of total phenols and flavonoids in Theinhibitionratesinalmostallofthetestedplantsare the six methano¨ıque extracts was performed by the same higher than that in other studies on other plant species in procedures described above. The results are summarized in theregionofLaghouatofthestudiesofkhachebaetal.[33]. Table3. Thishighamongregisteredforourplantsmayberelatedto The content of phenolic compounds (mg/g) in the six atypeofextraction(infusion)andthehighlypolarnatureof methanolicextractsvariedbetween2.45and11.42mg/g.The thesolventusedwhichextractsalargenumberofinhibitory plant Salvia offisinalis showed the highest values in total molecules which explains the use of these herbal plants by phenols against the plant Equisetum arvense which showed infusionintraditionalmedicine. thelowestvalue. The herbalists confirm that, by the use of “Zygophyllum Ifwecomparethecontentofphenoliccompoundsinboth album” in traditional medicine against diabetes, we can aqueousandmethanolicextractsofthesixplantsstudied,we explain the noninhibition of its aqueous extract by another note that the aqueous extracts contain a higher amount of level to reduce high blood sugar levels which is due to the totalphenolscomparedtothemethanolicextracts.Asimple inhibition of 𝛼-glucosidase or by promoting the secretion explicationcanbeconsideredbythefactthatthemethanol of insulin. Another explanation proposed is that the active extractionhasbeenprecededbyanaqueousextraction,the ingredientsresponsiblefortheinhibitionoftheenzymeare methanolwasextractedthecompoundswhicharenotextract notextractablebydistilledwater. in distilled water excepted the methanol extract of “Matri- Followingtheinhibitionratesofthe23aqueousextracts caria pubescens” which presented a total phenolic content of the investigated plants, we have chosen six plants that higher than the aqueous extract which can be explained by haveapercentagehigherthan70%suchasCistus,Equisetum thefactthattheplant“Matricariapubescens”containsother arvense(thegrain),Matricariapubescens,Oudneyaafricana phenolics compounds non-extractable by water and which (the pods), Salvia officinalis, and Thapsia garganica, for a arepassedintothemethanol.Also,thedistilledwaterhasthe new and a same extraction with distilled water followed abilitytoextractotherpolarcompoundsaspolyphenols,such by an extraction with methanol to investigate the type of as alkaloids, proteins, sugars, and other compounds which enzymeinhibitionandtodeterminetheinhibitionconstants are assayed by the Folin Cioncalteu procedure, resulting in (𝐾𝑖) for each extract. The 𝛼-amylase activities were assayed increased levels of phenolics. Through the results listed in in the presence of different concentrations of the substrate Table3,weseethattheplantsstudiedcontainapoormaterial (0.4–1.11g/L) and different concentrations of aqueous and InternationalJournalofCarbohydrateChemistry 5 Table4:Theinhibitionratesofthe23aqueousextracts. can be attributed to the presence of higher amounts of polyphenolsascomparedtoaqueousextracts.Itmeansthat Nameofplant Inhibitionrates(%) they are more efficient in cell walls and seeds degradation Ajugaiva 70.31±0.001 which have unpolar character and cause polyphenols to be Aloesocotrina 30.19±0.005 releasedfromcells.Moreusefulexplanationforthedecrease Anthemisarvensis 61.95±0.002 inactivityofaqueousextractcanbeascribedtotheenzyme BerberisVulgaris 48.17±0.1 polyphenol oxidase, which degrades polyphenols in water Cistus 100±0.0005 extracts,whereasinmethanolandethanoltheyareinactive. Moreover,waterisabettermediumfortheoccurrenceofthe Equisetumarvense (i)Thegrains 100±0.0008 microorganisms[34]. (ii)Theleaves 32.48±0.2 Inconclusion,wecansaythatnostandardextractionpro- cedurecanleadtothedegradationofphytochemicalspresent Erythraeacentaurium 25.69±0.3 inplantsandvariationsleadingtothelackofreproducibility. Haloxylonscoparium 53.61±0.01 Buteffortsshouldbemadetoproduceprocessesasconsistent Helianthemumlippii 43.18±0.02 aspossibleinquality(inthenarrowestpossiblerange)andto Marrubiumvulgare 8.29±0.5 developandfollowthebestextractionmethods. Matricariapubescens 86.90±0.004 All the plants showed different values of inhibition Ononisangustissima 19.25±0.03 constants with the majority being close to each other and Oudneyaafricana all above 20𝜇g/mL. This can be explained by the fact that (i)Thepods 100±0.004 the two solvents used for the extraction distilled water and (ii)Theleaves 51.38±0.02 methanol may have the same polar molecules with near Rhamnusalaternus 43.18±0.08 chemical structure and were able to react similarly against theenzymesand,astheinhibitionphenomenonistheresult Rhanteriumadpressum 76.75±0.003 of a synergy between several molecules, these values could Salviaofficinalis 77.81 ±0.004 be lower if the molecules responsible for inhibition in the Teucriumpolium 51.47±0.003 twosolventsofextractionweretogetherinthesamereaction Thapsiagarganica 76.75±0.01 medium. Trigonellafoenum 0±0.00 Ifwecomparethevaluesof𝐾𝑖inthisworkwiththoseof Zygophyllumalbum 0±0.00 the study of Khacheba et al. [33], we found that our values arehigher.Thismadecanconcludethatoursamplesarelow inhibteur which is the result of the type of extraction; our methanolic extracts (88–258𝜇g/mL). The 𝐾𝑖 value (74– extractsaretoopolarandhaveextracteallpolarcompounds 393𝜇g/mL and 24–587𝜇g/mL for aqueous and methanolic mayinterferewiththemechanismofinhibitionorbythefact extracts, resp.) was obtained from a Lineweaver-Burk and that the plants studied by Khacheba and their collaborator Dixonplots(Figures1,2,3,and4). presenttheexsitanceofcompoundswithdifferentstructure The 𝐾𝑖 value (24–587𝜇g/mL) was obtained from givesthemalargestinhibitoryactivity.Bycomparisonwith Lineweaver-Burk plots (Figures 1, 2, 3, and 4) which anotherstudyofShobanaetal.[8]whosevaluesrangefrom showed that the inhibition by all the investigated plants 2to10𝜇g,ourvaluesarestillinsignificant. was noncompetitive mixed, competitive, and competitive The general techniques of medicinal plant extrac- mixedwithlow𝐾𝑖 valuesoftheorderof𝜇g/mL.Theresults tion include maceration, infusion, percolation, digestion, are summarized in Table5. These two types of inhibition decoction, hot continuous extraction (Soxhlet), aqueous- can be explained. The competitive inhibition found can be alcoholicextractionbyfermentation,countercurrentextrac- explained by the fact that methanolic and aqueous extracts tion, microwave-assisted extraction, ultrasound extraction possesscompoundswithsimilarfunctionalgroupstothose (sonication), supercritical fluid extraction, and phytonic ofthesubstratestarch,whichhasmovedfromtheactivesite extraction (with hydrofluorocarbon solvents). However the oftheenzyme. quality of the extract of these techniques is influenced by 𝐾𝑖valueswithrespecttoconcentrationsofcrudeextracts several parameters, which are plant part used as starting determined representation of both Dixon and Lineweaver- material,solventusedforextraction,andextractionprocedure Burkrangesfrom74𝜇g/mLto393𝜇g/mLandfrom24𝜇g/mL [34]. We conclude that the plant source (the nature of the to 587𝜇g/mL for the aqueous and methanolic extracts, plantmaterial,itsorigin,growthstage,climaticconditionsfor respectively.Itisalsonotedthatitis“Salviaofficinalis”which growth,degreeofprocessing,moisturecontent,andparticle present a low 𝐾𝑖 for both aqueous and methanolic extracts size)[34]remainsthefirstpointinfluencingthesynthesisof whichprovesthatthisisagoodinhibitor. inhibitory molecules and their action which are influenced Thebestinhibitorswereinthemethanolicextractsforthe bytheatmosphericandclimaticconditionsunderwhichthe six plants. The best value was recorded for the methanolic plantisgrown.Thephenoliccompoundsandothersecondary fractionofSalviaofficinaliswitha𝐾𝑖 valueof24𝜇g/mLand metabolites (responsible for the pharmacological effects of methanolic fraction of Thapsia garganica was least potent theplant)representachemicalinterfacebetweentheplants with a 𝐾𝑖 value of 587𝜇g/mL. The higher activity of the andtheenvironment,andtheirsynthesisisoftenaffectedby methanolic extracts as compared to the aqueous extract environmentalfactors[35]. 6 InternationalJournalofCarbohydrateChemistry 0.1 0.08 0.08 0.06 M) 0.06 M) 𝜇 𝜇 min/ min/ 0.04 V ( 0.04 V ( 1/ 1/ 0.02 0.02 0 0 −1 0 1 2 3 4 0 1 2 3 4 1/[S] (L/g) 1/[S] (L/g) (a) (b) 0.1 0.12 0.08 0.1 M) 0.06 𝜇 0.08 n/ mi M) V ( 0.04 𝜇n/ 0.06 1/ mi V ( 0.04 0.02 1/ 0.02 0 0 0 1 2 3 4 −1 0 1 2 3 4 1/[S] (L/g) −0.02 1/[S] (L/g) (c) (d) 0.24 0.12 0.2 0.16 M) 0.08 M) 𝜇 𝜇 0.12 n/ n/ mi V (mi 0.08 1V/ ( 0.04 1/ 0.04 0 0 0 1 2 3 4 −1 0 1 2 3 4 −0.04 1/[S] (L/g) 1/[S] (L/g) I3 I1 I3 I1 I2 Control I2 Control (e) (f) Figure1:Lineweaver-Burkplotsofinhibitionof𝛼-amylaseaccordingtothetotalphenolconcentrationoftheaqueousfractionplantof (a)Cistus;(b)Equisetumarvense(thegrains);(c)Matricariapubescens;(d)Oudneyaafricana(thepods);(e)Salviaofficinalis;(f)Thapsia garganica. Activity was determined by formation of maltose at several substrate concentrations ranging from 0.2 to 0.6g/L and three concentrationsofinhibitorsI1,I2,andI3rangingfrom88to258𝜇g/mL. InternationalJournalofCarbohydrateChemistry 7 0.08 0.12 0.1 0.06 M) 0.08 𝜇 M) min/ 0.04 𝜇n/ 0.06 V ( mi 1/ V ( 0.04 0.02 1/ 0.02 0 0 −1 0 1 2 3 4 0 1 2 3 4 −0.02 1/[S] (L/g) 1/[S] (L/g) (a) (b) 0.12 0.2 0.1 0.16 0.08 0.12 M) M) 𝜇 0.06 𝜇 min/ min/ 0.08 V ( 0.04 V ( 1/ 1/ 0.04 0.02 0 0 −2 −1 0 1 2 3 4 0 1 2 3 4 −0.02 1/[S] (L/g) −0.04 1/[S] (L/g) (c) (d) 0.2 0.12 0.16 M) 𝜇 0.12 0.08 n/ mi M) 1V/ ( 0.08 𝜇min/ 0.04 0.04 V ( 1/ 0 0 −1 0 1 2 3 4 −1 0 1 2 3 4 −0.04 1/[S] (L/g) 1/[S] (L/g) −0.04 I3 I1 I2 Control I3 I1 I2 Control (e) (f) Figure2:Lineweaver-Burkplotsofinhibitionof𝛼-amylaseaccordingtothetotalphenolconcentrationofthemethanolicfractionplantof (a)Cistus;(b)Equisetumarvense(thegrains);(c)Matricariapubescens;(d)Oudneyaafricana(thepods);(e)Salviaofficinalis;(f)Thapsia garganica. Activity was determined by formation of maltose at several substrate concentrations ranging from 0.2 to 0.6g/L and three concentrationsofinhibitorsI1,I2,andI3rangingfrom88to258𝜇g/mL. 8 InternationalJournalofCarbohydrateChemistry 0.1 0.08 0.08 0.06 M) M) 𝜇 0.06 𝜇 min/ min/ 0.04 V ( 0.04 V ( 1/ 1/ 0.02 0.02 0 0 −0.2 0 0.2 0.4 0.6 −0.4 −0.2 0 0.2 0.4 0.6 [I] (g/L) [I] (g/L) (a) (b) 0.1 0.12 0.1 0.08 M) M) 0.08 0.06 𝜇 𝜇 min/ min/ 0.06 V ( 0.04 V ( 1/ 1/ 0.04 0.02 0.02 0 0 −0.5 0 0.5 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 [I] (g/L) [I] (g/L) (c) (d) 0.12 0.24 0.2 0.08 0.16 M) 𝜇 𝜇n/M) 0.12 V (min/ mi 1/ 0.04 V ( 0.08 1/ 0.04 0 0 −0.2 0 0.2 0.4 0.6 −1 −0.5 0 0.5 −0.04 [I] (g/L) [I] (g/L) S1 S3 S1 S3 S2 S4 S2 S4 (e) (f) Figure3:Dixonplotsofinhibitionof𝛼-amylaseaccordingtothetotalphenolconcentrationoftheaqueousfractionplantof(a)Cistus;(b) Equisetumarvense(thegrains);(c)Matricariapubescens;(d)Oudneyaafricana(thepods);(e)Salviaofficinalis;(f)Thapsiagarganica.Activity wasdeterminedbyformationofmaltoseatseveralsubstrateconcentrationsrangingfrom0.2to0.6g/L. InternationalJournalofCarbohydrateChemistry 9 0.08 0.12 0.1 0.06 M) 0.08 𝜇 V𝜇 (min/M) 00..0046 1V/ (min/ 00..0024 1/ 0.02 0 0 −0.4 −0.2 0 0.2 0.4 0.6 −0.4 −0.2 0 0.2 0.4 0.6 −0.02 [I] (g/L) [I] (g/L) (a) (b) 0.12 0.2 0.1 0.16 0.08 M) M) 0.12 1V𝜇/ (min/ 000...000246 1V𝜇/ (min/ 00..0048 0 0 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 −0.4 −0.2 0 0.2 0.4 0.6 −0.02 −0.04 [I] (g/L) [I] (g/L) (c) (d) 0.2 0.12 0.16 0.12 0.08 M) V𝜇 (min/ 0.08 𝜇min/M) 0.04 1/ 0.04 V ( 1/ 0 0 −0.2 0 0.2 0.4 0.6 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 −0.04 [I] (g/L) [I] (g/L) −0.04 S1 S3 S2 S4 S1 S3 S2 S4 (e) (f) Figure4:Dixonplotsofinhibitionof𝛼-amylaseaccordingtothetotalphenolconcentrationofthemethanolicfractionplantof(a)Cistus; (b)Equisetumarvense(thegrains);(c)Matricariapubescens;(d)Oudneyaafricana(thepods);(e)Salviaofficinalis;(f)Thapsiagarganica. Activitywasdeterminedbyformationofmaltoseatseveralsubstrateconcentrationsrangingfrom0.2to0.6g/L. Secondly, the successful determination of biologically compounds extracted, ease of subsequent handling of the active compounds from plant material is largely dependent extracts, toxicity of the solvent in the bioassay process, and on the type of solvent used in the extraction procedure. potentialhealthhazardoftheextractants[34]. The factors affecting the choice of solvent are quantity of Andfinallythetypeofextractionvariationsindifferent phytochemicals to be extracted, rate of extraction, diversity extraction methods that will affect quantity and secondary of different compounds extracted, diversity of inhibitory metabolitecompositionofanextractdependsupontypeof 10 InternationalJournalofCarbohydrateChemistry Table5:𝐾𝑖andinhibitortypefortotalaqueousandmethanolicextractsobtainedfor𝛼-amylase. Nameofplant Typeofinhibition 𝐾𝑖(𝜇g/mL) Aqueousextracts Cistus Inhibitioncompetitive 240±0.004 Equisetumarvense(Lesgrains) Inhibitioncompetitivemixed 210±0.001 Matricariapubescens Inhibitioncompetitivemixed 350±0.005 Oudneyaafricana(thepods) Inhibitioncompetitive 370±0.007 Salviaofficinalis Inhibitioncompetitivemixed 74±0.004 Thapsiagarganica Inhibitioncompetitive 393±0003 Methanolicextracts Cistus Inhibitionnoncompetitivemixed 220±0.002 Equisetumarvense(thegrains) Inhibitioncompetitivemixed 210±0.001 Matricariapubescens Inhibitionnoncompetitivemixed 360±0.006 Oudneyaafricana(thepods) Inhibitioncompetitivemixed 130±0.003 Salviaofficinalis Inhibitionnoncompetitivemixed 24±0.004 Thapsiagarganica Inhibitioncompetitivemixed 587±0.007 extraction,timeofextraction,temperature,natureofsolvent, diabetes mellitus may be due to the amylase-inhibiting solvent concentration, and polarity [34]. The length of the activityofthephenoliccompounds. extraction period must be also taken into consideration, Inthecurrentstudy,weinvestigatedtheeffectofaqueous which depends on solvent used, pH of the solvent, temper- and methanolic extracts from some Algerian medicinal ature, particle size of the plant tissues, and the solvent-to- plantsonfungal𝛼-amylase. sampleratio[34]. We have demonstrated, for the first time in vitro, the It may be thought that the inhibitory potency of the inhibitory effect of some Algerian plant extracts on the 𝛼- plantextractsisnotlimitedtothephenoliccontentbutmay amylase.Theresultsobtainedthroughthistestshowthatthe be due to the presence of some individual active phenolic majority of these plants have significant inhibitory effects. compounds. Thevaluesoftheconstants(𝐾𝑖)thusobtainedindicatethat Thus the inhibitory potency of an extract can not just theseplantscanbeinvestigatedinpharmacotherapeuticfor be explained on the basis of its phenolic content but also eventual treatment, including the plant “Salvia officinalis” requiresitspropercharacterization.Thereareseveralreasons whichpresentedthelowestvaluesofinhibitionconstantfor to explain the ambiguous relationship between inhibitory bothaqueousandmethanolicextracts. potencyandtotalphenolics:totalphenolicscontentdidnot The phenolic compounds present in these plants may include all the possible inhibitors; the synergism among alsoserveasleadcompoundsforthesynthesisofaseriesof the inhibitors in the mixture accounted for the inhibition; inhibitors. In several cases, the use of the plants and their however,itwasdependentnotonlyontheconcentrationof respective therapeutic prescription in popular medicine are individual inhibitors, but on the structure and interaction not easy to understand, and we can question if there is a amongthemaswell. relation between the therapeutic properties of these plants andtheirinhibitoryeffectson𝛼-amylase. 4.Conclusion Our study is the first report on potential inhibition of these plants extracts of digestive enzyme 𝛼-amylase. In Therelativeinefficacyofalpha-amylaseinhibitorsinaffecting conclusion,theresultsfromthisstudygivescientificsupport human digestion of starch has been highlighted by recent to the use of these plants in traditional medicine for the scientific and public controversy over the commercial sales treatmentofdiabetes.Thisstudywouldbehelpfultoexplain of so-called starch-blockers or slimming pills [36]. Alpha- thepharmacologicalmechanismandalsotodevelopmedic- amylase and its inhibitors are drug-design targets for the inal preparations and nutraceutical or functional foods for developmentofcompoundsfortreatmentofdiabetes,obesity, diabetesandrelatedsymptoms. andhyperlipidaemia[37]. Furtherisolationofthebioactivecompoundsresponsible Plantextractshavelongbeenusedfortheethnomedical for the inhibition of the enzyme must be done to elucidate treatment of diabetes in various systems of medicine and their molecular structure and to study their mechanism of arecurrentlyacceptedasanalternativefordiabetictherapy. actiontoconfirmtheirantidiabeticactivity.Inaddition,more Inhibitoryactivityagainstamylasebyflavonoidsandantho- experimentsmustbecarriedoutinvivotopavethewaytothe cyaninshasbeenreported[38,39].Herbsusedintraditional developmentofnewagentsforthetreatmentofdiabetesand Algerianmedicinefordiabetesmellitustreatmentsareknown itscomplications. tocontainphenoliccompounds,aswellastheflavonoids.We It is possible to expand the panel inhibition tests, using hypothesize that the health benefits of these herbs against other substrates or other types of enzymes. There are still