Research Article Twenty Traditional Algerian Plants Used in Diabetes Therapy as Strong Inhibitors of

13 Pages·2015·1.88 MB·English

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