Co esponding au ho : Sa a Aslam
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
Ca alluma-enhanced b ead: A no el app oach o comba ad anced glyca ion end
p oduc s and me abolic synd ome- ela ed enzymes
Aimal Khan 1, Im an Khan 2, Bilal Khan 2, Sa a Aslam 3, *, Shan Muzamail 1 and Sada Ra iq 4
1 G adua e Ins i u e o Nu i ion, China Medical Uni e si y, Taiwan.
2 Depa men o Human Nu i ion, The Uni e si y o Ag icul u e, Peshawa , Pakis an.
3 Depa men o Ag icul u e chemis y & Biochemis y, The Uni e si y o Ag icul u e, Peshawa , Pakis an.
4 Ins i u e o Biological Sciences, Gomal uni e si y, D.I.K, Pakis an.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 001-012
Publica ion his o y: Recei ed on 24 No embe 2024; e ised on 30 No embe 2024; accep ed on 01 Janua y 2025
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.21.1.1111
Abs ac
Whea b ead is a commonly consumed bake y i em known o i s high ene gy con en . Ne e heless, b ead p oduced
om e ined lou may lack in nu i ional quali y. To boos he nu i ional p o ile o b ead, a ious ce eals, legumes,
and medicinal he bs ha e been blended wi h whea lou . This esea ch aims o e alua e he o e all polyphenol
con en (TPs), an ioxidan po en ial, an iglyca ion cha ac e is ics, inhibi ion o enzymes linked o me abolic synd ome
(such as α-amylase, α-glucosidase, and panc ea ic lipase), and he senso y appeal o b ead enhanced wi h ca alluma
ex ac powde (CEP). Di e en p opo ions o CEP (2%, 4%, and 6%) we e subs i u ed o whea lou o c ea e
composi e b ead. The b ead was p epa ed ollowing a s anda d ecipe, d ied, and s o ed o subsequen analysis. TPs
we e quan i ied using he Folin-Ciocal eu me hod, and an ioxidan po en ial was gauged by measu ing i s abili y o
sca enge ABTS and DPPH adicals. The Bo ine Se um Albumin-Glucose (BSA-Glu) assay was used o asce ain he
impac on an iglyca ion. Addi ionally, in i o examina ions we e conduc ed o measu e he inhibi o y e ec o CEP-
en iched b ead on alpha-amylase, alpha-glucosidase, and panc ea ic lipase. To de e mine he pala abili y o he b ead
samples among consume s, a 9-poin hedonic scale was used o he e alua ion. S a is ical analysis in ol ed ANOVA
(One-way analysis o a iance) ollowed by he LSD pos - es o compa e and analyze he ou comes o b ead wi h
di e en CEP le els agains he con ol b ead. B ead con aining 2%, 4%, and 6% CEP showed signi ican ly highe
le els o TPs (147.15 mg GAE/100g) and an ioxidan capaci y (ABTS 132.81 µmol TE/100g, DPPH 172.36 µmol
TE/100g) compa ed o he con ol b ead. CEP-en iched b ead also exhibi ed signi ican ly s onge inhibi o y e ec s
(p<0.05) agains he o ma ion o ad anced glyca ion end p oduc s (20.46%-32.24%) compa ed o he con ol
(10.18%). Inhibi o y e ec s on me abolic enzymes—α-amylase (6.26%-17.24%), α-glucosidase (10.26%-22.24%), and
panc ea ic lipase (8.26%- 19.24%)—inc eased signi ican ly wi h highe le els o CEP in he b ead. Senso y analysis
indica ed ha all CEP-inco po a ed b ead samples ecei ed an accep able sco e (≥ 6), sugges ing hei pala abili y. In
conclusion, b ead en iched wi h CEP displayed signi ican ly imp o ed phy ochemical p ope ies a all inco po a ion
le els, as well as subs an ial inhibi ion o ad anced glyca ion end p oduc s and me abolic synd ome- ela ed enzymes.
Fu he esea ch should in es iga e he e ec s o maximum CEP inco po a ion in b ead.
Keywo ds: Ca alluma Ex ac Powde (CEP); Func ional B ead; An iglyca ion; Me abolic Synd ome Enzymes;
Polyphenols and An ioxidan s; Senso y E alua ion
1. In oduc ion
Me abolic synd ome, o en known as synd ome X, is a p e alen me abolic disease ha is linked o he wo ldwide
epidemic o obesi y and diabe es. The me abolic synd ome (MS), which comp ises insulin esis ance, abdominal obesi y,
dyslipidemia and hype ension, is a collec ion o condi ions a he han a single disease. The como bidi ies linked o
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me abolic synd ome include a p o h ombo ic s a e, p oin lamma o y condi ion, ep oduc i e issues and non-alcoholic
a y li e disease. Clus e ing componen s like o e -nu i ion, excess adiposi y and seden a y li es yle e lec s he
me abolic synd ome [1]. The Na ional Choles e ol Educa ion P og am's Adul T ea men Panel III ca ego ized me abolic
synd ome as a signi ican public heal h conce n in he Uni ed S a es wi h an impac on app oxima ely 34% o he
popula ion [2]. Pa ien s wi h me abolic synd ome a e ypically counselled o abs ain om ciga e e use and consume
nu i ious oods, bu changing hei o e all li es yle choices will be a mo e c ucial o educing hei chance o
de eloping me abolic synd ome [3].
Since ancien imes, people ha e used plan s o heal a a ie y o illnesses. Being na u al, plan s ha e less ad e se e ec s
and a e mo e pala able o human bodies. E en be o e he in en ion o syn he ic mode n medicine, many diseases
including yphoid, measles, chole a and o he mic obiological diseases we e ea ed using medicinal he bs. These he bs
we e u ilized by people as a pain elie e , a cu e o poison, a he apy o many so s o in lamma ion, a emedy o
snake bi es and o many o he issues. Because o he nega i e side e ec s o mode n medica ions, socie y is u ning
back o he bal he apy [4]. Medicinal plan s con ain a a ie y o compounds, including polyphenols, la onoids,
iso la onoids, e penoids, ca o enoids, phy os e ols, and glucosinola es. Because o he exis ence o hese bioac i e
subs ances, medicinal plan s can be used he apeu ically o pu poses such as an i-cance , an i-aging, an i-diabe ic,
analgesic, an i-alle gic, an i-in lamma o y and hepa op o ec i e [5,6]. Medicinal plan s ind applica ions in he
p oduc ion o mode n medica ions, bo h h ough di ec and indi ec means [7].
Ca alluma (Ca alluma imb ia a) s ands ou as a aluable medicinal plan . This edible succulen cac us belongs o he
Asclepiadaceae amily, as documen ed by [8]. I s habi a p edominan ly spans ac oss semi-a id egions in sou he n
Eu ope, India, A ica, A ghanis an, A abia, and Pakis an. In Pakis an, i goes by he name "choogan" in U du, while in
India, pa icula ly in Tamil, i is e e ed o as "ka allamu.". I is ea en in Pakis an and India in a a ie y o ways, including
as a pickle o chu ney and occasionally as a egula ege able. Wes e n Indian hun e s ha e been u ilizing ca alluma o
sa ia e hei hunge and hi s o cen u ies. In India, i is commonly known as " amine ood," as indica ed by [9]. Up o
his poin , he e ha e been no epo ed ad e se e ec s associa ed wi h he consump ion o ca alluma. Ca alluma is said
o ha e he apeu ic e ec s due o i s phy onu ien concen a ion. I con ains annins, saponins, la onoids, phenolic
compounds, p egnane glycosides and asco bic acid as well as o he an ioxidan s [10].
As he excep ional nu i ional p o ile o Ca alluma, i is u ilized o ea a a ie y o diso de s. Resea ch on Ca alluma
ex ac powde has shown ha in addi ion o o he heal h ad an ages, i also possesses an i-obesogenic, an i-diabe ic
and enal-p o ec ing quali ies. Besides i s ole in alle ia ing oxida i e s ess, he p esence o an ioxidan s, such as
phenolic compounds and la onoids in ca alluma, con ibu es o i s e ec i eness in ea ing in lamma ion and ce ain
ypes o cance , as highligh ed by [11]. Fu he mo e, a s udy in ol ing s ep ozocin-induced diabe ic a s aimed o
e alua e he an idiabe ic p ope ies o me hanolic ex ac om Ca alluma. The esul s indica ed a signi ican educ ion
in he diabe es le els o he es ed animals, as epo ed by [12]. Wis a a s we e used in a u he in es iga ion. These
a s we e ed a high- a die in o de o modi y hei lipid p o iles. These a s we e hen adminis e ed a hyd o-alcoholic
ex ac o ca alluma o he ollowing 90 days. The ou comes shown ha powde ed ca alluma ex ac may be use ul in
lowe ing lipid al e a ions [13]. I indica es ha Ca alluma has been known o ha ing an i-in lamma o y, an ioxidan ,
an i-obesogenic and an icance p ope ies and has ne e been associa ed wi h any nega i e side e ec s [14]. The
pu pose o his s udy is o assess how adding Ca alluma ex ac powde o b ead a ec s he p oduc ion o AGEs and
enzymes ac i i y linked o he me abolic synd ome, such as α-amylase, α-glycosidase, and panc ea ic lipase.
B ead is a widely consumed s aple ood in a ious o ms. I s p ima y ing edien , whea , is ecognized o i s ich ene gy
con en and nu i ional alue. Howe e , when compa ed o o he g ains and pseudoce eals, whea alls sho in e ms
o i s o e all nu i ional and unc ional quali ies. Regula ly including whi e b ead in one's daily die may inc ease he
isk o de eloping me abolic condi ions ha is ype II diabe es, hea condi ions, and high blood p essu e. I is possible
o add a ious g ains and medicinal he bs o whi e b ead and o he whea -based oods o inc ease hei nu i ional
bene i s. This s udy is ocused on inco po a ing ca alluma ex ac powde in o b ead o augmen i s nu i ional con en .
Consequen ly, his esea ch aims o e alua e he impac o b ead en iched wi h ca alluma ex ac powde on o al
polyphenolic con en , an ioxidan p ope ies, an iglyca ion e ec s, inhibi ion o α-amylase, α-glucosidase and lipase
enzymes and i s accep able senso y quali y.
2. Ma e ial and me hods
2.1. S udy Loca ion
Resea ch was conduc ed a he Uni e si y o Ag icul u e Peshawa , in he Depa men s o Human Nu i ion and Animal
Heal h.
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2.2. P ocu emen o ing edien s
The s anda d b ead ing edien s, including whea lou , sal , yeas , oil and suga , we e p ocu ed om ma ke in
Peshawa . Comme cially a ailable Ca alluma imb ia a ex ac was also u ilized in he s udy.
2.3. B ead making
To p epa e he dough o b ead making, he ollowing s eps we e ollowed. Whea lou , 100g, was placed in a clean
bowl and o he baking ing edien s including 5 g o oil and 1 g o sal we e added and mixed ho oughly. Yeas ac i a ion
was pe o med by aking 60ml o lukewa m wa e in a sepa a e clean glass. To his, 1 g o yeas and 6 g o suga we e
added and s i ed using a spoon. The glass con aining he yeas mix u e was subsequen ly placed in a d y a ea o 5
minu es o ini ia e yeas ac i a ion. Following his du a ion, he ac i a ed yeas mix u e was blended wi h he lou o
c ea e he dough. The mix u e unde wen a 10-minu e kneading p ocess and was hen le in a d y loca ion o 40
minu es o acili a e e men a ion. A e his ini ial 40-minu e pe iod, he dough was kneaded again o an addi ional 10
minu es and allowed o e men o ano he 40 minu es. Meanwhile, he baking o en was p ehea ed a 220 °C o 15
minu es. A e he second e men a ion pe iod, he dough was placed in a baking pan and he pan was pu in he
p ehea ed o en o baking. The baking p ocess was ca ied ou o 15 minu es a 220 °C. Fo he o mula ion o he
b ead, whea lou was subs i u ed a di e en le els in combina ion wi h ca alluma ex ac powde (CEP) (2% CEP,
4% CEP, 6% CEP). The emaining baking ing edien s including wa e , yeas , sal , oil and suga we e added in he
p opo ions as speci ied in he Table numbe 1.
Table 1 Fo mula ion o whea & ca alluma ex ac powde composi e b ead (/100 g lou ).
Ing edien s
Con ol
B ead
T ea men B ead
2% CEP
4% CEP
6% CEP
Whea Flou s (g)
100 g
98 g
96 g
94 g
Ca alluma ex ac powde (g)
0 g
2 g
4 g
6 g
Oil (g)
5 g
5 g
5 g
5 g
Yeas (g)
1 g
1 g
1 g
1 g
Sal (g)
1 g
1 g
1 g
1 g
Suga (g)
6 g
6 g
6 g
6 g
Wa e (ml)
60ml
60ml
60ml
60ml
CEP: ca alluma ex ac powde
2.4. D ying o B ead
To ind he o al polyphenols, enzymes inhibi ion and an ioxidan s ac i i y all he ou b eads we e d ied in a 50°C o en
o 24 hou s.
2.5. Milling o b ead
The d ied b ead was g ound in o a ine powde using a p o essional g inde .
2.6. Sample p ese a ion
The powde was ca e ully ans e ed in o p e-labeled ai igh plas ic bo les made o polye hylene. These plas ic
bo les we e subsequen ly w apped wi h aluminum oil and s o ed in a e ige a o a a empe a u e o 4°C in
p epa a ion o analysis.
2.7. Ex ac ion o samples o assess o al phenolics and an ioxidan ac i i y
Sample ex ac s o he de e mina ion o an ioxidan ac i i y, o al phenolics and inhibi ion o enzyme we e p epa ed
ollowing he p ocedu e p oposed by [15]. A o al o 1g o samples (including whea lou , Ca alluma ex ac powde in
i s aw o m, con ol b ead, 2% CEP b ead, 4% CEP b ead and 6% CEP b ead) we e accu a ely weighed and placed in o
p e-labeled Falcon ubes. Nex , 10ml o a 99% Me hanolic HCl solu ion (comp ising 99ml o me hanol and 1ml o HCl)
was added o each Falcon ube con aining he samples. The con en s we e ho oughly mixed by gen ly agi a ing he
Falcon ubes, ensu ing p ope blending. To acili a e ho ough mixing, he Falcon ubes we e placed ho izon ally in a
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es ube ack. The Falcon ubes, con aining he sample-ex ac ion solu ion, we e hen imme sed in a wa e shake ba h
o a du a ion o 1 hou a a empe a u e o 25°C, wi h con inuous shaking a 100 oscilla ions pe minu e. Following he
one-hou ex ac ion pe iod, he samples in Falcon ubes we e ans e ed o a cen i uge machine o he pu pose o
sepa a ion. Cen i uga ion was ca ied ou o 20 minu es a a speed o 3000 pm. Following cen i uga ion, he uppe
pa , known as he supe na an , was ca e ully sepa a ed om he ex ac ion ubes using a mic opipe e wi h a clean ip.
The ex ac ed supe na an was hen placed in o p e-labeled s e ilized glass ials, co e ed wi h aluminum oil o p o ec
i om ligh exposu e and inally s o ed in a e ige a o a a empe a u e o -20°C. These ials we e kep o u he
analysis.
2.8. To al Phenol de e mina ion
To al phenolic con en was examined using he colo ime ic me hod in ol ing he Folin-Ciocal eu eagen , as de eloped
by [16]. The s udy included six samples o analysis: ex ac s o whea lou , Ca alluma ex ac powde , con ol b ead,
2% CEP b ead, 4% CEP b ead and 6% CEP b ead. The p ocedu e was consis en o all samples. To p epa e a 10- old
dilu ed FC eagen solu ion, 1ml o FC eagen was mixed wi h 10ml o dis illed wa e . Fo each expe imen , 0.2ml o
he sample ex ac was placed in o s e ilized glass es ubes. Subsequen ly, 0.8ml o he dilu ed FC eagen solu ion was
added o each es ube using a mic opipe e. The es ubes we e hen placed in he da k o a pe iod o 3 minu es.
Following a 3-minu e incuba ion pe iod, 2ml o a 15% sodium ca bona e solu ion was in oduced in o each es ube.
The mix u e's inal olume was adjus ed o 5ml by inco po a ing 2ml o dis illed wa e . Subsequen ly, he esul ing
solu ion was kep in da kness a oom empe a u e o 1 hou . Following he 1-hou incuba ion, he abso bance was
measu ed in duplica e a a wa eleng h o 760nm using a spec opho ome e (Genesys 10 UV). Dis illed wa e was used
as a blank e e ence. Gallic acid in concen a ions anging om 0 o 0.5mg/ml was used as a s anda d. The esul s we e
exp essed as millig ams o Gallic acid equi alen pe sample.
2.9. Assessing an ioxidan ac i i y using ABTS assay
The ABTS (2,2'-Azino-Bis-3-E hylbenzo hiazoline-6-sul onic Acid) adical sca enging ac i i y o bo h composi e b ead
and aw sample ex ac s was de e mined using a me hod adap ed om [17] wi h sligh modi ica ions. He e's he
p ocedu e. To c ea e he ABTS adical s ock solu ion, po assium pe sul a e (K2S2O8) powde and ABTS powde we e
me iculously weighed using a op-loading balance (CY 510C, Poland) in sepa a e clean glass beake s. A o al o 33mg o
po assium pe sul a e and 9.6mg o ABTS we e used o his pu pose. These subs ances we e hen combined in dis illed
wa e o o m he ABTS s ock solu ion. In one glass beake , 33mg o po assium pe sulpha e was dissol ed in 50ml o
dis illed wa e , while in ano he glass beake , 9.6mg o ABTS powde was dissol ed in 2.5ml o dis illed wa e . Bo h
solu ions we e s i ed using a magne ic s i e . A e ho ough s i ing, 5ml o he po assium pe sulpha e solu ion was
mixed wi h 5ml o he ABTS solu ion in a conical lask a a 1:1 a io. The lask was subsequen ly co e ed wi h aluminum
oil and placed in a da k en i onmen o a du a ion o 2 hou s o induce he gene a ion o ee adicals. Following his
2-hou incuba ion pe iod, he ABTS solu ion was dilu ed wi h me hanol un il i eached a inal abso bance alling wi hin
he ange o 0.7nm o 0.8nm. This abso bance was de e mined using a spec opho ome e (Genesys 10 UV) a a
wa eleng h o 734nm. Subsequen ly, 2.85ml o he p epa ed ABTS s ock solu ion wi h an op ical densi y o 0.7nm was
in oduced in o each p e-labeled small es ube. To hese es ubes, 150ul o he sample ex ac was added. Abso bance
measu emen s o he es samples we e aken a ime in e als o 0, 10, 20, and 30 minu es. The abso bance was
measu ed a a wa eleng h o 734nm using a spec opho ome e . All measu emen s we e eco ded in iplica e.
2.10. Assessing an ioxidan ac i i y ia he DPPH assay
The DPPH (2,2-diphenyl-1-pic ylhyd azyl adical) assay, as desc ibed by [18], was u ilized o assess he an ioxidan
ac i i y o bo h he composi e b ead and aw sample ex ac s. To p epa e he DPPH solu ion, 3.943mg o DPPH powde
was accu a ely weighed using a op load balance (CY 510C, Poland). The DPPH powde was placed in a clean olume ic
conical lask, which was hen w apped wi h aluminum oil. To his lask, 100ml o me hanol was added and he con en s
we e ho oughly mixed. This mix u e esul ed in a da k pu ple-colo ed solu ion. F om he p epa ed DPPH solu ion,
2.85ml was dispensed in o s e ilized small glass es ubes. To each es ube, 150ul o he sample ex ac was added.
The ini ial abso bance o hese samples was eco ded in iplica e using a spec opho ome e a a wa eleng h o 517nm.
Following he ini ial abso bance measu emen , he es samples, con aining he DPPH solu ion, we e placed in a da k
loca ion. A e a 30-minu e incuba ion pe iod, he inal abso bance o he samples was eco ded. Con ol samples we e
c ea ed by combining 2.85ml o he DPPH solu ion wi h 150ul o me hanol, in place o he sample ex ac . A s anda d
subs ance, T olox ( anging om 0 o 1mM), was employed o e e ence pu poses, which was dissol ed in me hanol.
The an ioxidan capaci y esul s ob ained om he DPPH assay we e calcula ed as pe cen inhibi ion (%I) using he
ollowing equa ion.
% I = [(Ac- As /As)] x 100
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2.11. Ex ac ion o samples o Ad anced Glyca ion End P oduc s analysis
Sample ex ac s o he de e mina ion o ad anced glyca ion end p oduc s (AGEs) we e p epa ed om bo h b eads and
lou samples ollowing he p ocedu e p oposed by [19]. Weighed 1g o he espec i e samples, which included whea
lou , Ca alluma ex ac powde in i s aw o m, con ol b ead, 2% CEP b ead, 4% CEP b ead and 6% CEP b ead. Added
he weighed samples in o sepa a e Falcon ubes. P epa ed an 80% Me hanol solu ion by mixing 80ml o me hanol wi h
20ml o dis illed wa e . This solu ion was used o he ex ac ion p ocess. Added 5ml o he 80% Me hanol solu ion o
each Falcon ube con aining he samples. Ensu ed ha he Falcon ubes we e p ope ly adjus ed in a es ube ack
ho izon ally o acili a e ho ough mixing o he samples wi h he ex ac ion solu ion. Placed he Falcon ubes wi h he
sample-ex ac ion solu ion in o a wa e shake ba h. The ex ac ion was ca ied ou o 40 minu es a a empe a u e o
25°C wi h con inuous shaking a 100 oscilla ions pe minu e. A e he 40-minu e ex ac ion pe iod, he Falcon ubes
wi h he samples we e ans e ed o a cen i uge machine. Cen i uga ion was pe o med o 10 minu es a a speed o
5000 pm. Following cen i uga ion, he uppe pa o he supe na an was sepa a ed om he ex ac ion ube using a
mic opipe e wi h a clean ip. The sepa a ed supe na an was hen placed in o p e-labeled s e ilized glass ials. The
glass ials con aining he supe na an we e placed in an o en o 36 hou s a a empe a u e o 40°C. Du ing his ime,
he me hanol was emo ed om he samples. Following he me hanol emo al, 5ml o a 0.1M sodium phospha e bu e
wi h a pH o 7.4 was in oduced in o he glass ials. Subsequen ly, he ials we e placed in a e ige a o a -20°C o
subsequen analysis.
2.12. Assessmen o he inhibi o y impac on ad anced glyca ion end p oduc s
The p ocedu e o assessing he inhibi o y e ec o composi e b ead a di e en pe cen ages and aw lou samples on
he o ma ion o ad anced glyca ion end p oduc s (AGEs) ollowed he me hod ou lined by [20]. To c ea e a 22% Bo ine
Se um Albumin (BSA) solu ion, 10ml o 22% BSA was mixed wi h 10ml o 0.1M sodium phospha e bu e (pH 7.4) and
0.6mg o sodium azide (NaN3) in a glass ial o p e en mic obial ac i i y. The solu ion was ho oughly mixed. A blank
sample was p epa ed by combining 1ml each o BSA, D-glucose, and 0.1M sodium phospha e bu e (pH 7.4) in a cleaned
and labeled glass ial. Tes samples we e p epa ed in cleaned, labeled glass ials by combining 1ml each o BSA, D-
glucose, and he sample ex ac . A s anda d sample was p epa ed by combining 1ml each o BSA, D-glucose, and u in
solu ion in a labeled glass ial. All samples we e incuba ed in an incuba o a 55°C o 40 hou s. A e he incuba ion
pe iod, he samples we e ans e ed o a clean mic opla e wi h 12 wells in he ho izon al posi ion and 8 wells in he
e ical posi ion, and hei a angemen was eco ded. The luo escence in ensi y o each sample was measu ed using
a mic opla e eade (ELISA eade ALLshang Model-100) wi h an exci a ion wa eleng h o 330nm and an emission
wa eleng h o 410nm. Quad uplica e samples we e analyzed o each se , and he pe cen age inhibi ion o AGEs by each
b ead ex ac and u in solu ion was calcula ed using he app op ia e equa ion.
% Inhibi ion = ( luo escence o he solu ion wi h inhibi o s/ luo escence o he solu ion wi hou inhibi o s) * 100
2.13. Assessmen o α-Amylase inhibi o y ac i i y
The alpha-amylase inhibi ion assay was conduc ed ollowing he me hod ou lined by [21]. To assess he inhibi o y e ec
o whea b ead en iched wi h ca alluma ex ac powde on alpha-amylase ac i i y, a con ol solu ion was p epa ed by
combining 500ul o 0.02M sodium phospha e bu e (pH 6.9) and 50ul o alpha-amylase enzyme in a labeled glass ial,
which was hen incuba ed a 25°C o 10 minu es in an o en. A e he ini ial incuba ion, 500ul o 1% s a ch mix u e
was added o he eac ion mix u e, ollowed by an addi ional 10-minu e incuba ion a he same empe a u e. A con ol
blank, lacking he enzyme, was simila ly p epa ed by mixing 500ul o 0.02M sodium phospha e bu e and 500ul o 1%
s a ch solu ion in a p e-labeled glass ial and incuba ing i as well. Fo he sample solu ion, 500ul o he sample ex ac
and 50ul o alpha-amylase enzyme we e combined in a labeled glass ial and incuba ed unde he same condi ions.
Following he ini ial incuba ion in he sample ial, 500ul o 1% s a ch solu ion was added and he mix u e was subjec ed
o an addi ional 10-minu e incuba ion. To hal he eac ions, 1.0 ml o dini osalicylic acid (DNSA) colo eagen was
added o all ials and hey we e placed in a boiling wa e ba h a 100°C o 5-10 minu es be o e being cooled o oom
empe a u e. The spec opho ome e was used o measu e he abso bance a 540nm o each solu ion. A bu e was
used as a con ol we also eco ded sample blanks, which con ained bu e ins ead o enzyme. The o al blank sample
was sub ac ed om he inal ex ac abso bance (A540 ex ac ) o ge he inal alue. In he end he ollowing equa ion
was used o ind ou he alpha-amylase inhibi o y ac i i y.
%Inhibi ion = (A540 con ol – A540 ex ac / A540 con ol) * 100
2.14. Assessmen o α-Glucosidase inhibi o y ac i i y
The Alpha-glucosidase inhibi ion es as epo ed by [22] was used o he in es iga ion. We made solu ions, including
1% suc ose solu ion, α-glucosidase enzyme, 0.02 M sodium phospha e bu e (pH 6.9), and 96 mM 3,5-Dini osalicylic
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Acid solu ion (DNS). The ollowing me hod was used o ind ou ha whe he he Ca alluma con aining whea b ead can
a ec he alpha-glucosidase ac i i y. We made many a numbe o solu ions ha is 20ul o α-glucosidase solu ion, 20ul
o 1% suc ose solu ion, and 20ul o 0.02M sodium phospha e bu e (pH 6.9) we e mixed in a labelled ial o make he
i s solu ion, named as con ol solu ion. A 60ul o 0.02M sodium phospha e bu e (pH 6.9) o a ial, I made a con ol
blank. This blank was ee om bo h enzyme and subs a e. In ma ked ja s, we blend 20ul o sample ex ac wi h 20ul
o he enzyme α-glucosidase and 20ul o 1% suc ose solu ion o make sample solu ion. We also added 20ul o sample
ex ac and 40ul o 0.02M sodium phospha e bu e (pH 6.9), wi hou he enzyme o subs a e, o a labelled ial o make
he sample blank. Then, each sample mix u e was kep in o en and hea ed o 37 °C o 60 minu es con inuously.
Following he incuba ion pe iod, he eac ions we e e mina ed by adding 1.0 ml o dini osalicylic acid (DNSA) colo
eagen . The ials we e hen subjec ed o a boiling wa e ba h a 100°C o 5-10 minu es. A e cooling, he solu ions
we e dilu ed wi h 10ml o dis illed wa e , and hei abso bance was measu ed a 540nm using a spec opho ome e .
Reco dings we e made o sample blanks, which con ained bu e ins ead o enzyme, and o a con ol, which con ained
bu e ins ead o sample ex ac . The α-glucosidase inhibi o y ac i i y was subsequen ly calcula ed using he ele an
equa ion.
%Inhibi ion = (A540 con ol – A540 ex ac / A540 con ol) * 100
2.15. Assessmen o inhibi ion o Panc ea ic lipase ac i i y
The de e mina ion o lipase inhibi o y ac i i y was conduc ed using a spec oscopic me hod wi h adjus men s inspi ed
by he p ocedu e ou lined by [23]. Fo he lipase inhibi ion assay, he ollowing solu ions we e employed: hog panc ea ic
lipase enzyme, 4-Ni ophenyl palmi a e (pNPP), and a bu e . To p epa e he hog panc ea ic lipase enzyme solu ion,
2mg o lipase enzyme was ca e ully weighed and added o 2ml o 0.1M sodium phospha e bu e (pH 6.9), which was
subsequen ly mixed using a o ex. Fo he p epa a ion o 4-Ni ophenyl palmi a e (pNPP), 6mg o 4-Ni ophenyl
palmi a e was weighed and combined wi h 2ml o 0.01M isop opanol, ollowed by mixing using a o ex. Rega ding he
bu e solu ion, 0.55mg o A abic gum and 1.15mg o sodium deoxychola e we e weighed and blended in 1.8ml o 0.1M
sodium phospha e bu e (pH 6.9), wi h s i ing acili a ed by a magne ic s i e . To e alua e he inhibi o y e ec o
whea b ead en iched wi h ca alluma ex ac powde on he enzyme, a sample was c ea ed by adding 20ul o sample
ex ac and 20ul o hog panc ea ic lipase enzyme o a p e-labeled es ube, while a blank was p epa ed wi hou he
sample. In bo h he sample and blank, 20ul o bu e and 20ul o hog panc ea ic lipase enzyme we e added o sepa a e
p e-labeled es ubes. Subsequen ly, bo h se s o eac ion mix u es we e incuba ed a 37°C o 10 minu es in an o en.
A e incuba ion, 20ul o 4-ni ophenyl palmi a e and 1800ul o bu e we e in oduced o he solu ions o bo h he
sample and he blank. The eac ion mix u es we e once again incuba ed in he o en a 37°C o 10 minu es. The
abso bance o he solu ions was measu ed a 410nm using a spec opho ome e , and he lipase inhibi o y ac i i y was
calcula ed using he app op ia e equa ion.
2.16. Senso y E alua ion
Senso y e alua ion p ocess in ol ed 50 un ained subjec s who pa icipa ed a he Uni e si y o Ag icul u e Peshawa ,
human nu i ion depa men . Bo h males and emales pa icipa ed in he assessmen . Hedonic scale was u ilized o
conduc senso y e alua ion. Be o e he assessmen , pa icipan s we e gi en ins uc ions o abs ain om consuming
ood o be e ages o a leas 3 hou s. All ou b ead ypes, including he con ol, CEPB 2%, CEPB 4%, and CEPB 6%,
we e subjec ed o e alua ion conce ning a ibu es like ex u e, colo . Pa icipan s we e p esen ed wi h pla es ha we e
p e-labeled and coded, each con aining slices o he a ious b ead samples. Be o e as ing each piece o b ead,
pa icipan s we e ins uc ed o sip wa e . The e alua ion was conduc ed using a nine-poin hedonic scale ques ionnai e.
The codes used o labeling he b ead samples we e 013 o con ol, 012 o CEP B 2%, 011 o CEP B 4% and 010 o
CEP B 6%. All pa icipan s we e in o med abou he s udy's p o ocols and objec i es be o e engaging in he senso y
e alua ion.
2.17. S a is ical Analysis
Fo he s a is ical s udy, we used e sion 21 o he S a is ical Package o he Social Sciences (SPSS). Va ious
cha ac e is ics, including o al phenolics, an ioxidan capaci y, an i-glyca ion impac , enzyme inhibi ion (alpha-amylase,
alpha-glucosidase, and lipase), as well as senso y accep abili y we e examined. We pe o med a s a is ical examina ion
u ilizing a one-way analysis o a iance (ANOVA) and a leas signi ican di e ence (LSD) pos -hoc es o assess he
e ec o b ead in used wi h Ca alluma. All da a analysis me hods we e subjec ed o a signi icance cu o o P <0.05.
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3. Resul s and discussion
The pu pose o his s udy was o e alua e he e ec s o inco po a ing powde ed ca alluma ex ac in o whea lou
b ead on a ious pa ame e s. The s udy examined se e al c i e ia, including he measu emen o o al phenolics,
an ioxidan ac i i y ia DPPH and ABTS es s, inhibi ion o panc ea ic lipase, alpha-amylase, and alpha-glucosidase,
p e en ion o ad anced glyca ion end p oduc (AGE) o ma ion, and senso y accep abili y. The b ead samples we e
ca ego ized in o ou g oups based on he amoun s o ca alluma ex ac powde used as aw ma e ials: 100% whea
lou b ead wi h CEP le els o 2%, 4%, and 6%.
3.1. Phenolic con en analysis in aw ing edien s and b ead samples
Bo h aw ing edien s and b ead samples we e analyzed o o al polyphenols and exp essed in millig ams o gallic acid
equi alen pe 100 g ams (mg GAE/100g). Gallic acid se ed as he s anda d o his measu emen and he s anda d
cu e o Gallic acid concen a ion (mg) is illus a ed in Figu e-Ⅰ. In he analysis, i was ound ha ca alluma ex ac
powde con ained a signi ican ly highe (p < 0.05) o al phenolic con en (284.75 mg GAE/100g) compa ed o whea
lou (50.10 mg GAE/100g). Among he b ead samples, he one con aining 6% ca alluma ex ac powde (147.15 mg
GAE/100g) exhibi ed signi ican ly highe o al phenolic le els compa ed o he con ol b ead (100% WF) (36.12 mg
GAE/100g). This obse a ion sugges s ha he o al phenolic con en o composi e b ead inc eased wi h he addi ion o
ca alluma ex ac powde . Such esul s a e consis en wi h expec a ions due o he p esence o phy ochemicals in
ca alluma.
Values a e means ± SD o quad uplica e analyses. WF: whea lou , CEP: ca alluma ex ac powde , WB: whi e b ead.
Figu e 1 To al phenolic con en s o aw ma e ials and b ead samples (d y basis)
3.2. E alua ion o he An ioxidan Po en ial in Raw Ing edien s and B ead Sample
The an ioxidan p ope ies o bo h he aw cons i uen s and he composi e b eads we e assessed using he ABTS and
DPPH me hods and he esul s a e exp essed in mic omoles o T olox equi alen pe 100 g ams (µmol TE/100g) as
shown in able 2. T olox se ed as he s anda d o hese measu emen s and he s anda d cu e o T olox
concen a ion (µmol) o he ABTS and DPPH assays is p o ided in Figu e-II and Figu e-Ⅲ, espec i ely. In he ABTS
sca enging ac i i y analysis, ca alluma ex ac powde exhibi ed signi ican ly highe an ioxidan ac i i y (291.31 ± 6.41
µmol TE/100g) a e ou di e en ime in e als. Simila ly, in he DPPH sca enging ac i i y analysis, ca alluma ex ac
powde displayed signi ican ly highe an ioxidan ac i i y (356.33 ± 10.19 µmol TE/100g) in compa ison o whea lou
(86.17 ± 7.72 µmol TE/100g). Fu he mo e, he b ead samples con aining ca alluma ex ac powde exhibi ed
signi ican ly highe an ioxidan ac i i y compa ed o he con ol b ead. These indings sugges ha he inco po a ion o
ca alluma ex ac powde in o b ead signi ican ly enhances i s an ioxidan p ope ies, po en ially con ibu ing o i s
heal h-p omo ing e ec s.
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Table 2 An ioxidan ac i i y o aw ma e ials and b ead samples (d y basis) *
Sample
ABTS (µmol TE/100g)
DPPH (µmol TE/100g)
WF
44.42 ± 4.63e
86.17 ± 7.72e
CEP
291.31 ± 6.41a
356.33 ± 10.19a
WB
23.21 ± 4.16
67.51 ± 9.31
2% CEP
79.53 ± 5.39d
101.41 ± 10.35d
4% CEP
95.04 ± 4.76c
137.44 ± 9.33c
6% CEP
132.81 ± 5.14b
172.36 ± 9.41b
*Values a e means ± SD o iplica e analyses. Means in he same columns wi h di e en le e s a e signi ican ly di e en (P < 0.05, LSD es ). WF:
whea lou , CEP: ca alluma ex ac powde , WB: whi e b ead.
3.3. The Supp ession o Sample on Ad anced Glyca ion End P oduc Fo ma ion
The impac o bo h aw ma e ials and whea b ead en iched wi h ca alluma ex ac powde on he gene a ion o
ad anced glyca ion end p oduc s (AGEs) as illus a ed Figu e 2. The inhibi o y e ec o ca alluma ex ac powde
(49.43%) was no ably g ea e (p<0.05) in compa ison o whea lou (14.23%), unde sco ing he signi ican AGEs
inhibi o y po en ial o ca alluma ex ac powde . Mo eo e , b ead con aining ca alluma ex ac powde demons a ed
a signi ican ly supe io (p<0.05) abili y o inhibi AGEs p oduc ion when compa ed o he con ol b ead. In all samples,
mos p onounced inhibi ion e ec obse ed in he b ead which con ained he highes le el ca alluma ex ac , exhibi ing
inhibi ion a es anging om 20.46% o 32.24% o inco po a ion le els o 2% CEP, 4% CEP, and 6% CEP, espec i ely.
Values a e means ± SD o quad uplica e analyses. WF: whea lou , CEP: ca alluma ex ac powde , WB: whi e b ead.
Figu e 2 Inhibi o y e ec s o aw ma e ials and b ead samples on he o ma ion o AGEs in BSA-glucose model
3.4. Inhibi ion o α-Amylase Ac i i y in Raw Ing edien s and B ead Samples
The inhibi o y ac i i y agains he α-amylase enzyme exp essed as a pe cen age o bo h he aw ma e ials and a ious
b ead samples as displayed in Figu e 3. No ably, b ead con aining ca alluma ex ac powde exhibi ed a signi ican ly
highe inhibi o y e ec on α-amylase compa ed o he con ol g oup (p<0.05). Among all he b ead samples es ed, he
g ea es inhibi o y e ec was obse ed in he b ead wi h highe le els o ca alluma ex ac powde , showing inhibi ions
anging om 6.26% o 17.24% o inco po a ion le els o 2% CEP, 4% CEP, and 6% CEP, espec i ely.
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WF: whea lou , CEP: ca alluma ex ac powde , WB: whi e b ead.
Figu e 3 α- amylase inhibi o y ac i i y o aw ma e ials and b ead samples.Values a e means ± SD o quad uplica e
analyses
3.5. Inhibi ion o α-glucosidase ac i i y in bo h he aw ma e ials and b ead samples
The pe cen age o inhibi o y ac i i y agains he α-glucosidase enzyme o bo h aw ma e ials and b ead samples as
illus a es in Figu e 4. B ead en iched wi h ca alluma ex ac powde displayed a signi ican ly ele a ed inhibi o y e ec
on α-glucosidase inhibi ion (p<0.05). Among all he b ead samples examined, he highes le el o inhibi ion was
obse ed in hose con aining g ea e quan i ies o ca alluma ex ac powde , exhibi ing inhibi ions anging om
10.26% o 22.24% o inclusion le els o 2% CEP, 4% CEP, and 6% CEP, espec i ely. This inc eased inhibi o y e ec in
hese composi e b eads can be a ibu ed o hei ele a ed polyphenolic con en .
Values a e means ± SD o iplica e analyses. WF: whea lou , CEP: ca alluma ex ac powde , WB: whi e b ead.
Figu e 4 α- glucosidase inhibi o y ac i i y o aw ma e ials and b ead samples
3.6. Inhibi ion o panc ea ic lipase ac i i y in bo h he aw ma e ials and b ead samples
The inhibi o y ac i i y agains he panc ea ic lipase enzyme, exp essed as a pe cen age, o bo h aw ma e ials and
di e en b ead samples as illus a ed in Figu e 5. Signi ican ly, b ead con aining Ca alluma ex ac powde exhibi ed a
no ably s onge inhibi o y e ec on panc ea ic lipase inhibi ion (p < 0.05). All he samples o b ead which we e es ed,
mos p ominen inhibi o y e ec was obse ed in ha b ead which con ained high le els o Ca alluma ex ac , he
inhibi ion pe cen ages we e anging om 8.26% o 19.24% o inco po a ion le els o 2% CEP, 4% CEP, and 6% CEP,
espec i ely.
