Research ArticleAntidiabetic Activity of Aloe vera Leaves

Alethia Muntildeiz-Ramirez 1 Rosa M Perez2 Efren Garcia3 and Fabiola E Garcia4

1CONACYT-IPICYTCIIDZA Camino a la Presa de San Jose 2055 Col Lomas 4 Seccion CP 78216 San Luis Potosı SLPMexico2Laboratorio de Investigacion de Productos Naturales Escuela Superior de Ingenierıa Quımica e Industrias ExtractivasInstituto Politecnico Nacional Av Instituto Politecnico Nacional SN Unidad Profesional Adolfo Lopez MateosCP 07708 Ciudad de Mexico Mexico3Laboratorio de Quımica Supramolecular y Nanociencias Instituto Politecnico Nacional Acueducto SNBarrio la laguna Ticoman CP 07340 Ciudad de Mexico Mexico4Facultad de Ciencias Quımicas Universidad Autonoma de San Luis Potosı Av Dr Manuel Nava No 6-Zona UniversitariaCP 78210 San Luis Potosı SLP Mexico

Correspondence should be addressed to Alethia Muntildeiz-Ramirez alethiamunizipicytedumx

Received 19 December 2019 Revised 25 February 2020 Accepted 27 March 2020 Published 25 May 2020

Academic Editor Ronald Sherman

Copyright copy 2020AlethiaMuntildeiz-Ramirez et al+is is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

+is research evaluated the potential of using the methanol extract of Aloe vera (L) Burmf (AVM) to prevent the formation ofAGEs by means of the BSAglucose assay BSA-methylglyoxal assay arginine-methylglyoxal assay fructosamine Nε-(carbox-ymethyl) lysine (CML) thiol groups and carbonyl protein in vitro +e effect of AVMwas also evaluated with regard to inhibitingthe enzymes α-amylase α-glucosidase and pancreatic lipase For this the plant was dried ground and subsequently maceratedwith methanol Aloe vera methanol extract (AVM) significantly decreased the formation of AGEs as well as the formation offructosamine CML and carbonyl protein +e concentration of 5mgml of AVM presented the best results AVM significantlyinhibited the α-amylase and α-glucosidase enzymes As regards the content of thiol groups a significant increase was observedduring the four weeks of the experiment So we can conclude that Aloe vera methanol extract decreases the formation of AGEsand may inhibit the increase in postprandial glucose suggesting that AVM can prevent diabetes complications associatedwith AGE

1 Introduction

Protein glycation is considered one of the main causes ofcomplications of diabetes such as vasculopathy retinopa-thy nephropathy and neuropathy cataracts and chronickidney disease [1]+e glycation of proteins brings about theformation of advanced glycation end products (AGEs)which modify the structure of proteins and alter enzymaticactivity [2] AGEs are formed by the nonenzymatic reactionof the free amino group of a protein with the carbonyl of asugar or a reducing aldehyde (called the Maillard reaction)[3] resulting in the formation of Schiff bases subsequentlythrough a series of reactions and fluorescent compoundssuch as pentosidine and nonfluorescent compounds such ascarboxymethyl lysine (CML) are formed [4] Some AGEs

such as CML and pentosidine have become biomarkers forglycooxidizing damage [5] +erefore taking into accountthe pathological implications of glycation it becomes es-sential to discover inhibitors of protein glycation which mayhelp reduce andor prevent complications in diabetesmellitus [2] For centuries herbal medicines have beenwidely used to treat a wide variety of diseases To date manyof these plants are still used as a first alternative to curecertain diseases in developing countries around the worlddue to the few side effects they present it has also beenreported that around 20 of the medicines used throughoutthe world come from plants [6] In this context there is agrowing interest in discovering safe and nontoxic plantsources to find alternative or complementary medicines thathelp the treatment of various chronic diseases such as

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020 Article ID 6371201 9 pageshttpsdoiorg10115520206371201

diabetes mellitus Aloe vera (L) Burmf has been used bydifferent cultures such as the Egyptian Indian Chinese andEuropean cultures for more than 5000 years due to its ex-traordinary medicinal properties [7 8] +e genus Aloegrows in arid tropical and subtropical areas this genusincludes approximately 450 species It is a succulent plantwith no stem or a short stem and can grow to be 60minus100 cmhigh its leaves are fleshy thick triangular and spiny [8]which gives the appearance of cactus but in fact it belongs tothe lilac (Liliacea) family Its leaves have the ability to retainwater which allows the plant to survive in environmentswith long periods of drought where most of the vegetationdisappears [9] It contains more than 70 active compounds[7] including vitamins minerals enzymes polysaccharidesphenolic compounds and organic acids It has been reportedthat the polysaccharides present in the A vera gel havetherapeutic properties such as anti-inflammatory healingantibacterial antioxidant anticarcinogenic antidiabeticand antiaging properties among others [8 9] Taking intoaccount the pathological effects that glycation brings aboutit is very important to find compounds that inhibit thenonenzymatic glycation of proteins to help prevent thegeneration of secondary complications which is why thisstudyrsquos objective was to assess the capacity of the methanolextract of Aloe vera (AVM) to inhibit the protein glycationreaction by means of the BSAglucose assay

2 Materials and Methods

21 Collection of Raw Materials Aloe vera was collected inMay 2018 in the municipality of Armadillo San Luis PotosıA specimen was taken to the herbarium of the AutonomousMetropolitan University for future reference (specimennumber ARC-53578)

22 Preparation of the Extract +ree hundred grams of thewhole leaf was dried and pulverized in a mechanical millobtaining 110 g of dry weight +e powdered material wasextracted with 3 L of methanol using a Soxhlet apparatus+e extract (AVM) was filtered and concentrated by a rotaryvacuum evaporator for the complete removal of solvents

23 Inhibition Tests for the Formation of Advanced GlycationEnd Products

231 In vitro Glycation of Bovine Albumin +e glycation ofthe BSA was carried out using the method used by Gutierrezet al [10] with some modifications +e AVM extract wasdissolved in DMSO (0031ndash0500mgml) adding 10mgmlof BSA 11M glucose 01M phosphate buffer at pH 74 and02 sodium azide +e solution was incubated at 37degC forfour weeks +e formation of the glycated BSA was deter-mined at an excitation wavelength of 355 nm and emissionof 460 nm aminoguanidine was used as a positive control

232 Determination of Fructosamine +eAVM extract wasdissolved in DMSO (0031ndash0500mgml) adding 10mgmlof BSA 11M glucose 01M phosphate buffer at pH 74 and02 sodium azide +e solution was incubated at 37degC forfour weeks After four weeks of incubation the fructosamineconcentration of the Amadori product was assessed by thenitroblue tetrazolium assay (NTB) [11] 10 μl of glycated BSAwas incubated with 90 μl of 05mMNTB in 01M carbonatebuffer at a pH of 104 at 37degC After 10 to 15 minutes theabsorbance was read at 530 nm +e concentration offructosamine was calculated by comparing it with 1-deoxy-1-morpholino-fructose (1-DMF) which was used asstandard

233 BSA-Methylglyoxal Assay +is test was performed toevaluate the middle stage of protein glycation Methylglyoxalwas added (60mM 1ml) at different concentrations ofAVM (030 060 12 25 and 5mgml) and catechin (15mgml 1ml) in sodium phosphate buffer (50mM pH 74) andsodium azide (002) at 37 deg C for 2 h BSA (30mgml 1ml)was subsequently added to the mixture and incubated at 37degfor six days +e phosphate buffer (1ml) was used as anegative control without the addition of AVM extract As apositive control aminoguanidine (10mM final concentra-tion) was used After the incubation time the samples wereread in a microplate reader at 340 nm excitation and 380 nmemission [12] +e percentage of inhibition of AGEs wascalculated using

percentage inhibition 1 minusfluorescent intensity with inhibitor

fluorescent intensity without inhibitor1113888 1113889 times 100 (1)

234 Arginine-Methylglyoxal Assay +is test analyzes themain and specific source of AGEs formation Methylglyoxalwas added (60mM 1ml) at different concentrations ofAVM (030 060 12 25 and 5mgml) and catechin(15mgml 1ml) in sodium phosphate buffer (50mM pH74) and sodium azide (002) at 37degC for 2 h Subsequentlyarginine (60mM 1ml) was added to the different samplesand incubated at 37degC for six days As a negative controlphosphate buffer (1ml) was used without the addition ofAVM extract As a positive control aminoguanidine

(10mM final concentration) was used After the incubationtime the samples were read on a microplate reader at340 nm excitation and 380 nm emission [12] +e percentageof inhibition of AGEs was calculated using (1)

235 Determination of Nε-(Carboxymethyl) Lysine At theend of the fourth week of incubation the Nε-(carbox-ymethyl) lysine (CML) was determined and a major anti-genic AGE structure was determined by using the enzyme

2 Evidence-Based Complementary and Alternative Medicine

linked immunosorbant assay (ELISA) kit +e concentrationof CML was calculated by using the standard CML-BSAcurve from the assay kit

24 Determination of Protein Carbonyl Content After fourweeks of incubation the content of the carbonyl group wasdetermined in glycosylated BSA which is a marker of ox-idative protein damage using the method described byJoglekar et al [2] with some modifications 800 μl of 10mM2-4-dinitrophenylhydrazine (DNPH) in 25M HCL wasadded to 200 μl of glycated samples +e samples wereallowed to incubate in the dark for 1 h and then the proteinswere precipitated using 1ml of trichloroacetic acid (TCA) at20 (pV) leaving the samples on ice for five minutes andthey were then centrifuged at 10000 g for 10min at 4degC +esupernatant was washed three times using 500 μl of amixture of ethanolethyl acetate (1 1) and then the proteinsupernatant was dissolved in 500 μl of 6M guanidine hy-drochloride +e absorbance reading was 370 nm +ecarbonyl group concentration of the samples was calculatedusing the absorption coefficient (ε 22000Mminus1lowast cmminus1) +eresults were expressed as nmol carbonylmg protein

25Aiol Group Estimation After four weeks of incubationfree thiol groups were determined in glycated BSA using themethod described by [11] with some modifications 70 μl ofthe sample was incubated with 130 μl of 5mM 55prime-dithiobis(2-nitrobenzoic acid) (DTNB) in 01M PBS pH 74 at 25degCfor 15 minutes After the absorbance of the samples was readat 412 nm the concentration of free thiol was calculatedusing a standard curve of L-cysteine +e results wereexpressed as nmolmg of protein

26 Enzymatic Assays

261 Inhibition of α-Amylase +e inhibition of α-amylaseactivity was performed using a modified method with2-chloro-4-nitrophenyl-4-β-D-galactopyranosylmaltoside(GalG2CNP) as substrate and saliva fraction enriched withα-amylase (HSA-f ) [13] +e HSA-f was diluted 100-fold in50mmol Lminus1 2-(N-morpholino) ethanesulfonic acid (MES)buffer containing 5mmol Lminus1 CaCl2 140mmol Lminus1 po-tassium thiocyanate and 300mmol Lminus1 NaCl (pH 60)Different concentrations of AVM (030 060 12 25 and5mgml) previously diluted in 5 DMSO were incubatedwith HSA-f (1 10 v vminus1) at 37degC for 30min +e reactionwas initiated by adding 12mmol Lminus1 GalG2CNP substrateincreases in absorbance (ie CNP release) were measuredat 37degC at 405 nm +is assay was also carried out withacarbose (purity gt 95) as a positive control and 5DMSOas a negative control +e results were presented as apercentage of α-amylase inhibition (AI) calculatedaccording to the following

AI() Anc minus Asample1113872 1113873

Anctimes 100 (2)

whereAnc is the absorbance of negative control andAsample isthe absorbance of the extract incubated with HSA-f

262 Inhibition of α-Glucosidase +e inhibition of α-glu-cosidase activity was performed using a modified methodwith 4-nitrophenyl α-D-glucopyranoside (p-NPG) as sub-strate and α-glucosidase-enriched fraction from intestinalacetone powders from rats (AG-f) [13] Different concen-trations of AVM (030 060 12 25 and 5mgml) previ-ously diluted in 5 DMSO were incubated with AG-f and15mmol Lminus1 reduced glutathione (diluted in 50mmol Lminus1

phosphate buffer pH 68) for 20min at 37degC +e reactionwas started by adding 4mmol Lminus1 p-NPG (diluted in50mmol Lminus1 phosphate buffer pH 68) and absorbancevalues were measured at 405 nm for 30min at 37degC +isassay was also carried out with acarbose (puritygt 95) as apositive control and 5 DMSO as a negative control +eresults were presented as a percentage of α-glucosidaseinhibition (GsI) calculated according to the following

GsI() Anc minus Asample1113872 1113873

Anctimes 100 (3)

whereAnc is the absorbance of negative control andAsample isthe absorbance of the extract incubated with AG-f

263 Inhibition of Pancreatic Lipase +e inhibition ofpancreatic lipase activity was performed using a modifiedmethod with p-nitrophenyl palmitate (p-NPP) as substrateand porcine pancreatic lipase (PL) (type II Sigma) [14]Different concentrations of AVM (030 060 12 25 and5mgml) previously diluted in 5 DMSO were incubatedwith 10 g Lminus1 PL (diluted in 50mM Tris-HCl buffer pH 80containing 10mmol Lminus1 CaCl2 and 25mmol Lminus1 NaCl) for20min at 37degC +e reaction was started by adding08mmol Lminus1 p-NPP substrate (diluted in 10 isopropanoland 50mmol Lminus1 Tris-HCl buffer containing 10mmol Lminus1

CaCl2 25mmol Lminus1 NaCl and 05 Triton X-100) andabsorbance values were measured at 410 nm for 30min at30degC +is assay was also carried out with orlistat(puritygt 98) as a positive control and 5 DMSO as anegative control +e results were presented as a percentageof lipase inhibition (LI) calculated according to thefollowing

LI() Anc minus Asample1113872 1113873

Anctimes 100 (4)

whereAnc is the absorbance of negative control andAsample isthe absorbance of the extract incubated with PL

27 Statistical Analysis +e results were expressed as themeanplusmn standard error of the mean (SEM) (n 3) +estatistical significance of the results was evaluated by usingone-way ANOVA +e least significant difference (LSD) testwas used for mean comparisons and Plt 005 was statisti-cally significant

Evidence-Based Complementary and Alternative Medicine 3

3 Results

31 Inhibition Tests for the Formation of Advanced GlycationEnd Products

311 In vitro Glycation of Bovine Albumin In Figure 1 thefluorescence intensity is shown during the fourweeks ofincubation +e results show that fluorescence was signifi-cantly increased over the weeks in the BSAglucose systemcompared to the groups incubated with BSAglucoseAVMat different concentrations where a significant decrease influorescence was observed depending on the concentrationAfter four weeks of the experiment the BSAglucoseAVMgroup (5mgml) showed the greatest decrease in fluores-cence intensity (8564) while the AG (5mgml) showed adecrease of 8794 in comparison with the BSA glycated inthe same time interval

312 Determination of Fructosamine +e effect of AVM onfructosamine levels during the four weeks is shown in Ta-ble 1 +e results show that the concentration of fructos-amine in the glycosylated BSA increased significantly overtime By adding AVM to different concentrations fruc-tosamine levels increased at a lower rate during the fourweeks of the test In the fourth week a fructosamine value of775mM was observed when using the BSAglucoseAVMsystem (5mgml) compared to the BSAglucoseAG systemwhich showed a value of 758mM+e glycated BSA showeda value of 1196mM in the same time interval

313 BSA-Methylglyoxal Assay +e effect of AVM at dif-ferent concentrations in the BSA-methylglyoxal test isshown in Figure 2 It can be observed that the greatestinhibition was presented when using the BSAglucoseAVMsystem (5mgml) showing an inhibition of 65 comparedto the BSAglucoseAG system (5mgml) which showed aninhibition of 91

314 Arginine-Methylglyoxal Assay +e antiglycation testusing the arginine-methylglyoxal model showed that theBSAglucoseAVM system (5mgml) inhibited the genera-tion of fluorescent AGEs at 65 (Figure 3) +e BSAglu-coseAG system (5mgml) showed an inhibition percentageof 80

315 Nε-(Carboxymethyl) Lysine +e effect of AVM on theinhibition of Nε-(carboxymethyl) lysine (CML) after fourweeks of incubation is shown in Figure 4 where it can beseen that the concentration of CML in the BSAglucosesystem increased During the experiment when evaluatingthe results of the BSAAVMglucose system it was observedthat the CML values were concentration-dependentobtaining a significant 73 inhibition in the formation ofCML at a concentration of 5mgml compared to the BSAAGglucose system (5mgml) which showed a 74 inhi-bition compared to the glycated BSA

32 Determination of Protein Carbonyl Content Table 2shows the protein carbonyl content during the four weeksWhen observing the glycated BSA an increase in proteincarbonyl content can be seen during the four weeks of theexperiment While adding AVM the BSAglucose system atdifferent concentrations showed a significant decrease inprotein carbonyl content during the four weeks that theexperiment lasted obtaining the highest decrease in the BSAglucoseAVM system at a concentration of 5mgml whichshowed a value of 8288 nmolmg protein +e BSAglucoseAG system and the glycated BSA showed values of8256 nmolmg protein and 8915 nmolmg protein re-spectively in the same time interval

33 Aiol Group Estimation +e effect of AVM during thefour weeks at different concentrations is shown in Table 3 +eBSAglucose system showed a significant increase in thiolgroups throughout the experiment By adding AVM to theBSAglucose system a significant decrease in thiol-dependentgroups can be observed+e greatest decrease was obtained at aconcentration of 5mgml (8912nmolmg protein) at fourweeks of the experiment while the BSAglucoseAG andglycated BSA system showed values of 7808 and 7005nmolmg protein respectively in the same time interval

34 Enzymatic Assays

341 α-Amylase α-Glucosidase and Lipase InhibitionActivity Figure 5 shows the inhibitory effect of AVM on theactivity of the alpha-amylase enzyme+e AVM concentrationof 5mgml showed a significant inhibition of 87 compared toacarbose (5mgml) which was used as a positive control andshowed an inhibition percentage of 97+e concentrations of060 and 030mgml AVM did not show inhibitory activityRegarding the inhibition of the α-glucosidase enzyme (Fig-ure 5) the concentration of 5mgml of AVM significantlyinhibited the activity of the enzyme by 66 compared toacarbose at the same concentration which presented an 85inhibition +e concentrations of 060 and 030mgml of theAVM extract showed no inhibition of the enzyme α-glucosi-dase Regarding the inhibition of pancreatic lipase the con-centrations of 25 and 5mgml of AVM showed an inhibitoryactivity of 9 and 15 respectively (Figure 6) compared to thecontrol drug which presented an inhibition of 99

4 Discussion

AGEs are a group of compounds that are formed by thenonenzymatic reaction of reducing sugars and metabolitesrelated to proteins and amino acids to produce Schiff basesSubsequently Amadori products (such as fructosamine) areformed which degrade into dicarbonyl compounds to finallyproduce stable AGEs [1 12 15] AVM reduced fructosaminelevels which are associated with decreased AGE formation+e reduction of fructosamine therefore is a therapeuticway to delay incident vascular complications [16] To thisend the capacity of AVM to inhibit the protein glycationreaction using the BSAglucose system was evaluated It was

4 Evidence-Based Complementary and Alternative Medicine

observed that AVM can inhibit the glycation reaction (whichis related to complications in diabetes) in a way similar to theaminoguanidine (AG) used as a control Since the formation

of AGEs is favored by oxidative reactions AVM could haveinhibited them by reducing the formation of reactive oxygenspecies (ROS) or by eliminating the ROS formed in vitro by

Week 1 Week 2 Week 3 Week 4

Fluo

resc

ence

inte

nsity

BSAglucoseAVM (030mgml)BSAglucoseAVM (12mgml)BSAglucoseAVM (5mgml)

BSAglucoseAG (5mgml)

BSAglucoseBSAglucoseAVM (060mgml)BSAglucoseAVM (25mgml)

bb

a

b

b

b

b

a

cc

cc

c c

a

dd

dd

d d

aa

aa

a a

0

200

400

600

800

1000

1200

1400

1600

Figure 1 Effects of AVM extract on formation of fluorescent advanced glycation end products (AGEs) in BSA incubated with glucose Eachvalue represents the meanplusmn SE (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose atweek two cplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAglucose at week four

Table 1 Effect of AVM on fructosamine levels during four weeks of incubation using the BSAglucose system

Experimental groupsFructosamine (mM)

Week 1 Week 2 Week 3 Week 4BSAglucose 872plusmn 08 979plusmn 09 1085plusmn 16 1196plusmn 12BSAglucoseAVM (030mgml) 867plusmn 12 855plusmn 13a 863plusmn 08a 869plusmn 14aBSAglucoseAVM (060mgml) 834plusmn 08a 843plusmn 05b 851plusmn 12c 856plusmn 17dBSAglucoseAVM (12mgml) 801plusmn 11a 827plusmn 13b 835plusmn 09c 841plusmn 13dBSAglucoseAVM (25mgml) 778plusmn 12a 792plusmn 18b 814plusmn 05c 827plusmn 14dBSAglucoseAVM (5mgml) 744plusmn 15a 754plusmn 12b 771plusmn 06c 775plusmn 14dBSAglucoseAG (5mgml) 721plusmn 14a 739plusmn 15b 745plusmn 12c 758plusmn 09d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose at week twocplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAfructose at week four

Gly

catio

n in

hibi

tion

()

BSA

met

hylg

lyox

al as

say

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

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AV

M(5

mg

ml)

BSA

glu

cose

AG

(5 m

gm

l)

0

20

40

60

80

100

120

Figure 2 Glycation inhibitory activity analysis of the methanol extract from Aloe vera in BSA-methylglyoxal model Values (mean-plusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Evidence-Based Complementary and Alternative Medicine 5

the oxidation of sugars andor by the oxidative degradationof Amadori products [17] It has been reported that theadministration of the ethanolic extract of A vera gel helps

prevent excessive free radical formation through variousbiochemical pathways and also reduces the glycosylation ofenzymes [18]

BSA

glu

cose

AV

M(0

30m

gm

l)

BSA

glu

cose

AV

M(0

60m

gm

l)

BSA

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M(1

2m

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BSA

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M(2

5m

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M(5

mg

ml)

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(5m

gm

l)

0

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90

Gly

catio

n in

hibi

tion

()

Arg

inin

e-m

ethy

lgly

oxal

assa

y

Figure 3 Glycation inhibitory activity analysis of the methanol extract from Aloe vera (AVM) in arginine-methylglyoxal model Values(meanplusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Nε-

(car

boxy

met

hyl)

lysin

e(n

gm

l)

aa

BSA

glu

cose

BSA

glu

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AV

M(0

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M(5

mg

ml)

Glib

encla

mid

e(5

mg

ml)

0

1

2

3

4

5

6

Figure 4 Effect of AVM on the inhibition of Nε-(carboxymethyl) lysine (CML) after four weeks of incubation Each value represents themeanplusmn SEM (n 3) aplt 005 compared to BSAglucose

Table 2 Effect of AVM on protein carbonyl content using the BSAglucose system

Experimental groupsProtein carbonyl content (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 8772plusmn 18 8859plusmn 14a 8891plusmn 14a 8915plusmn 12aBSAglucoseAVM (030mgml) 8644plusmn 07a 8719plusmn 08b 8709plusmn 11c 8692plusmn 11dBSAglucoseAVM (060mgml) 8631plusmn 11a 8704plusmn 17b 8653plusmn 14c 8637plusmn 11dBSAglucoseAVM (12mgml) 8511plusmn 99a 8617plusmn 14b 8681plusmn 12c 8507plusmn 09dBSAglucoseAVM (25mgml) 8433plusmn 12a 8594plusmn 08b 8418plusmn 14c 8466plusmn 11dBSAglucoseAVM (5mgml) 8324plusmn 06a 8433plusmn 07b 8357plusmn 07c 8288plusmn 06dBSAglucoseAG (5mgml) 8341plusmn 17a 8378plusmn 07b 8292plusmn 03c 8256plusmn 11d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

6 Evidence-Based Complementary and Alternative Medicine

When blood sugar levels rise the accumulation offructose in different organs can occur Both fructose andproducts derived from oxidation such as methylglyoxal can

react with proteins and form AGEs [12 15] Arginine is oneof the main targets of protein glycosylation by reactivecarbonyls which react with the guanidine group of the

Table 3 Effect of AVM on thiol group using the BSAglucose system

Experimental groups+iol group (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 7708plusmn 12 7538plusmn 007a 7588plusmn 005a 7005plusmn 008aBSAglucoseAVM (030mgml) 7595plusmn 10 7773plusmn 009b 7916plusmn 007b 7952plusmn 006BSAglucoseAVM (060mgml) 7708plusmn 051 7885plusmn 012b 7931plusmn 008b 8443plusmn 004dBSAglucoseAVM (12mgml) 7629plusmn 083a 7979plusmn 009b 8046plusmn 011b 8660plusmn 009dBSAglucoseAVM (25mgml) 7641plusmn 011a 8138plusmn 007b 8368plusmn 008b 8706plusmn 004dBSAglucoseAVM (5mgml) 7668plusmn 008a 8329plusmn 005b 8561plusmn 009b 8912plusmn 006dBSAglucoseAG (5mgml) 7652plusmn 008a 7737plusmn 002b 7756plusmn 004b 7808plusmn 009d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

AVM (12 mgml) AVM (25 mgml) AVM (5 mgml)

α-Amylaseα-Glucosidase

Acarbose (5 mgml)

a

a

aa

a

a

a

a

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Inhi

bitio

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)

Figure 5 α-Amylase and α-glucosidase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standarddeviation) expressed as a percentage of enzymatic inhibition AVM methanol extract Letters indicate statistical significance(plt 005)

BSAglucoseAVM(25 mgml)

BSAglucoseAVM(5 mgml)

Orlistat (5 mgml)0

20

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Lipa

se in

hibi

tion

()

Figure 6 Lipase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standard deviation) expressed as apercentage of enzymatic inhibition AVM methanol extract

Evidence-Based Complementary and Alternative Medicine 7

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

linked immunosorbant assay (ELISA) kit +e concentrationof CML was calculated by using the standard CML-BSAcurve from the assay kit

24 Determination of Protein Carbonyl Content After fourweeks of incubation the content of the carbonyl group wasdetermined in glycosylated BSA which is a marker of ox-idative protein damage using the method described byJoglekar et al [2] with some modifications 800 μl of 10mM2-4-dinitrophenylhydrazine (DNPH) in 25M HCL wasadded to 200 μl of glycated samples +e samples wereallowed to incubate in the dark for 1 h and then the proteinswere precipitated using 1ml of trichloroacetic acid (TCA) at20 (pV) leaving the samples on ice for five minutes andthey were then centrifuged at 10000 g for 10min at 4degC +esupernatant was washed three times using 500 μl of amixture of ethanolethyl acetate (1 1) and then the proteinsupernatant was dissolved in 500 μl of 6M guanidine hy-drochloride +e absorbance reading was 370 nm +ecarbonyl group concentration of the samples was calculatedusing the absorption coefficient (ε 22000Mminus1lowast cmminus1) +eresults were expressed as nmol carbonylmg protein

25Aiol Group Estimation After four weeks of incubationfree thiol groups were determined in glycated BSA using themethod described by [11] with some modifications 70 μl ofthe sample was incubated with 130 μl of 5mM 55prime-dithiobis(2-nitrobenzoic acid) (DTNB) in 01M PBS pH 74 at 25degCfor 15 minutes After the absorbance of the samples was readat 412 nm the concentration of free thiol was calculatedusing a standard curve of L-cysteine +e results wereexpressed as nmolmg of protein

26 Enzymatic Assays

261 Inhibition of α-Amylase +e inhibition of α-amylaseactivity was performed using a modified method with2-chloro-4-nitrophenyl-4-β-D-galactopyranosylmaltoside(GalG2CNP) as substrate and saliva fraction enriched withα-amylase (HSA-f ) [13] +e HSA-f was diluted 100-fold in50mmol Lminus1 2-(N-morpholino) ethanesulfonic acid (MES)buffer containing 5mmol Lminus1 CaCl2 140mmol Lminus1 po-tassium thiocyanate and 300mmol Lminus1 NaCl (pH 60)Different concentrations of AVM (030 060 12 25 and5mgml) previously diluted in 5 DMSO were incubatedwith HSA-f (1 10 v vminus1) at 37degC for 30min +e reactionwas initiated by adding 12mmol Lminus1 GalG2CNP substrateincreases in absorbance (ie CNP release) were measuredat 37degC at 405 nm +is assay was also carried out withacarbose (purity gt 95) as a positive control and 5DMSOas a negative control +e results were presented as apercentage of α-amylase inhibition (AI) calculatedaccording to the following

AI() Anc minus Asample1113872 1113873

Anctimes 100 (2)

whereAnc is the absorbance of negative control andAsample isthe absorbance of the extract incubated with HSA-f

262 Inhibition of α-Glucosidase +e inhibition of α-glu-cosidase activity was performed using a modified methodwith 4-nitrophenyl α-D-glucopyranoside (p-NPG) as sub-strate and α-glucosidase-enriched fraction from intestinalacetone powders from rats (AG-f) [13] Different concen-trations of AVM (030 060 12 25 and 5mgml) previ-ously diluted in 5 DMSO were incubated with AG-f and15mmol Lminus1 reduced glutathione (diluted in 50mmol Lminus1

phosphate buffer pH 68) for 20min at 37degC +e reactionwas started by adding 4mmol Lminus1 p-NPG (diluted in50mmol Lminus1 phosphate buffer pH 68) and absorbancevalues were measured at 405 nm for 30min at 37degC +isassay was also carried out with acarbose (puritygt 95) as apositive control and 5 DMSO as a negative control +eresults were presented as a percentage of α-glucosidaseinhibition (GsI) calculated according to the following

GsI() Anc minus Asample1113872 1113873

Anctimes 100 (3)

whereAnc is the absorbance of negative control andAsample isthe absorbance of the extract incubated with AG-f

263 Inhibition of Pancreatic Lipase +e inhibition ofpancreatic lipase activity was performed using a modifiedmethod with p-nitrophenyl palmitate (p-NPP) as substrateand porcine pancreatic lipase (PL) (type II Sigma) [14]Different concentrations of AVM (030 060 12 25 and5mgml) previously diluted in 5 DMSO were incubatedwith 10 g Lminus1 PL (diluted in 50mM Tris-HCl buffer pH 80containing 10mmol Lminus1 CaCl2 and 25mmol Lminus1 NaCl) for20min at 37degC +e reaction was started by adding08mmol Lminus1 p-NPP substrate (diluted in 10 isopropanoland 50mmol Lminus1 Tris-HCl buffer containing 10mmol Lminus1

CaCl2 25mmol Lminus1 NaCl and 05 Triton X-100) andabsorbance values were measured at 410 nm for 30min at30degC +is assay was also carried out with orlistat(puritygt 98) as a positive control and 5 DMSO as anegative control +e results were presented as a percentageof lipase inhibition (LI) calculated according to thefollowing

LI() Anc minus Asample1113872 1113873

Anctimes 100 (4)

whereAnc is the absorbance of negative control andAsample isthe absorbance of the extract incubated with PL

27 Statistical Analysis +e results were expressed as themeanplusmn standard error of the mean (SEM) (n 3) +estatistical significance of the results was evaluated by usingone-way ANOVA +e least significant difference (LSD) testwas used for mean comparisons and Plt 005 was statisti-cally significant

Evidence-Based Complementary and Alternative Medicine 3

3 Results

31 Inhibition Tests for the Formation of Advanced GlycationEnd Products

311 In vitro Glycation of Bovine Albumin In Figure 1 thefluorescence intensity is shown during the fourweeks ofincubation +e results show that fluorescence was signifi-cantly increased over the weeks in the BSAglucose systemcompared to the groups incubated with BSAglucoseAVMat different concentrations where a significant decrease influorescence was observed depending on the concentrationAfter four weeks of the experiment the BSAglucoseAVMgroup (5mgml) showed the greatest decrease in fluores-cence intensity (8564) while the AG (5mgml) showed adecrease of 8794 in comparison with the BSA glycated inthe same time interval

312 Determination of Fructosamine +e effect of AVM onfructosamine levels during the four weeks is shown in Ta-ble 1 +e results show that the concentration of fructos-amine in the glycosylated BSA increased significantly overtime By adding AVM to different concentrations fruc-tosamine levels increased at a lower rate during the fourweeks of the test In the fourth week a fructosamine value of775mM was observed when using the BSAglucoseAVMsystem (5mgml) compared to the BSAglucoseAG systemwhich showed a value of 758mM+e glycated BSA showeda value of 1196mM in the same time interval

313 BSA-Methylglyoxal Assay +e effect of AVM at dif-ferent concentrations in the BSA-methylglyoxal test isshown in Figure 2 It can be observed that the greatestinhibition was presented when using the BSAglucoseAVMsystem (5mgml) showing an inhibition of 65 comparedto the BSAglucoseAG system (5mgml) which showed aninhibition of 91

314 Arginine-Methylglyoxal Assay +e antiglycation testusing the arginine-methylglyoxal model showed that theBSAglucoseAVM system (5mgml) inhibited the genera-tion of fluorescent AGEs at 65 (Figure 3) +e BSAglu-coseAG system (5mgml) showed an inhibition percentageof 80

315 Nε-(Carboxymethyl) Lysine +e effect of AVM on theinhibition of Nε-(carboxymethyl) lysine (CML) after fourweeks of incubation is shown in Figure 4 where it can beseen that the concentration of CML in the BSAglucosesystem increased During the experiment when evaluatingthe results of the BSAAVMglucose system it was observedthat the CML values were concentration-dependentobtaining a significant 73 inhibition in the formation ofCML at a concentration of 5mgml compared to the BSAAGglucose system (5mgml) which showed a 74 inhi-bition compared to the glycated BSA

32 Determination of Protein Carbonyl Content Table 2shows the protein carbonyl content during the four weeksWhen observing the glycated BSA an increase in proteincarbonyl content can be seen during the four weeks of theexperiment While adding AVM the BSAglucose system atdifferent concentrations showed a significant decrease inprotein carbonyl content during the four weeks that theexperiment lasted obtaining the highest decrease in the BSAglucoseAVM system at a concentration of 5mgml whichshowed a value of 8288 nmolmg protein +e BSAglucoseAG system and the glycated BSA showed values of8256 nmolmg protein and 8915 nmolmg protein re-spectively in the same time interval

33 Aiol Group Estimation +e effect of AVM during thefour weeks at different concentrations is shown in Table 3 +eBSAglucose system showed a significant increase in thiolgroups throughout the experiment By adding AVM to theBSAglucose system a significant decrease in thiol-dependentgroups can be observed+e greatest decrease was obtained at aconcentration of 5mgml (8912nmolmg protein) at fourweeks of the experiment while the BSAglucoseAG andglycated BSA system showed values of 7808 and 7005nmolmg protein respectively in the same time interval

34 Enzymatic Assays

341 α-Amylase α-Glucosidase and Lipase InhibitionActivity Figure 5 shows the inhibitory effect of AVM on theactivity of the alpha-amylase enzyme+e AVM concentrationof 5mgml showed a significant inhibition of 87 compared toacarbose (5mgml) which was used as a positive control andshowed an inhibition percentage of 97+e concentrations of060 and 030mgml AVM did not show inhibitory activityRegarding the inhibition of the α-glucosidase enzyme (Fig-ure 5) the concentration of 5mgml of AVM significantlyinhibited the activity of the enzyme by 66 compared toacarbose at the same concentration which presented an 85inhibition +e concentrations of 060 and 030mgml of theAVM extract showed no inhibition of the enzyme α-glucosi-dase Regarding the inhibition of pancreatic lipase the con-centrations of 25 and 5mgml of AVM showed an inhibitoryactivity of 9 and 15 respectively (Figure 6) compared to thecontrol drug which presented an inhibition of 99

4 Discussion

AGEs are a group of compounds that are formed by thenonenzymatic reaction of reducing sugars and metabolitesrelated to proteins and amino acids to produce Schiff basesSubsequently Amadori products (such as fructosamine) areformed which degrade into dicarbonyl compounds to finallyproduce stable AGEs [1 12 15] AVM reduced fructosaminelevels which are associated with decreased AGE formation+e reduction of fructosamine therefore is a therapeuticway to delay incident vascular complications [16] To thisend the capacity of AVM to inhibit the protein glycationreaction using the BSAglucose system was evaluated It was

4 Evidence-Based Complementary and Alternative Medicine

observed that AVM can inhibit the glycation reaction (whichis related to complications in diabetes) in a way similar to theaminoguanidine (AG) used as a control Since the formation

of AGEs is favored by oxidative reactions AVM could haveinhibited them by reducing the formation of reactive oxygenspecies (ROS) or by eliminating the ROS formed in vitro by

Week 1 Week 2 Week 3 Week 4

Fluo

resc

ence

inte

nsity

BSAglucoseAVM (030mgml)BSAglucoseAVM (12mgml)BSAglucoseAVM (5mgml)

BSAglucoseAG (5mgml)

BSAglucoseBSAglucoseAVM (060mgml)BSAglucoseAVM (25mgml)

bb

a

b

b

b

b

a

cc

cc

c c

a

dd

dd

d d

aa

aa

a a

0

200

400

600

800

1000

1200

1400

1600

Figure 1 Effects of AVM extract on formation of fluorescent advanced glycation end products (AGEs) in BSA incubated with glucose Eachvalue represents the meanplusmn SE (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose atweek two cplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAglucose at week four

Table 1 Effect of AVM on fructosamine levels during four weeks of incubation using the BSAglucose system

Experimental groupsFructosamine (mM)

Week 1 Week 2 Week 3 Week 4BSAglucose 872plusmn 08 979plusmn 09 1085plusmn 16 1196plusmn 12BSAglucoseAVM (030mgml) 867plusmn 12 855plusmn 13a 863plusmn 08a 869plusmn 14aBSAglucoseAVM (060mgml) 834plusmn 08a 843plusmn 05b 851plusmn 12c 856plusmn 17dBSAglucoseAVM (12mgml) 801plusmn 11a 827plusmn 13b 835plusmn 09c 841plusmn 13dBSAglucoseAVM (25mgml) 778plusmn 12a 792plusmn 18b 814plusmn 05c 827plusmn 14dBSAglucoseAVM (5mgml) 744plusmn 15a 754plusmn 12b 771plusmn 06c 775plusmn 14dBSAglucoseAG (5mgml) 721plusmn 14a 739plusmn 15b 745plusmn 12c 758plusmn 09d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose at week twocplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAfructose at week four

Gly

catio

n in

hibi

tion

()

BSA

met

hylg

lyox

al as

say

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5 m

gm

l)

0

20

40

60

80

100

120

Figure 2 Glycation inhibitory activity analysis of the methanol extract from Aloe vera in BSA-methylglyoxal model Values (mean-plusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Evidence-Based Complementary and Alternative Medicine 5

the oxidation of sugars andor by the oxidative degradationof Amadori products [17] It has been reported that theadministration of the ethanolic extract of A vera gel helps

prevent excessive free radical formation through variousbiochemical pathways and also reduces the glycosylation ofenzymes [18]

BSA

glu

cose

AV

M(0

30m

gm

l)

BSA

glu

cose

AV

M(0

60m

gm

l)

BSA

glu

cose

AV

M(1

2m

gm

l)

BSA

glu

cose

AV

M(2

5m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5m

gm

l)

0

10

20

30

40

50

60

70

80

90

Gly

catio

n in

hibi

tion

()

Arg

inin

e-m

ethy

lgly

oxal

assa

y

Figure 3 Glycation inhibitory activity analysis of the methanol extract from Aloe vera (AVM) in arginine-methylglyoxal model Values(meanplusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Nε-

(car

boxy

met

hyl)

lysin

e(n

gm

l)

aa

BSA

glu

cose

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

Glib

encla

mid

e(5

mg

ml)

0

1

2

3

4

5

6

Figure 4 Effect of AVM on the inhibition of Nε-(carboxymethyl) lysine (CML) after four weeks of incubation Each value represents themeanplusmn SEM (n 3) aplt 005 compared to BSAglucose

Table 2 Effect of AVM on protein carbonyl content using the BSAglucose system

Experimental groupsProtein carbonyl content (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 8772plusmn 18 8859plusmn 14a 8891plusmn 14a 8915plusmn 12aBSAglucoseAVM (030mgml) 8644plusmn 07a 8719plusmn 08b 8709plusmn 11c 8692plusmn 11dBSAglucoseAVM (060mgml) 8631plusmn 11a 8704plusmn 17b 8653plusmn 14c 8637plusmn 11dBSAglucoseAVM (12mgml) 8511plusmn 99a 8617plusmn 14b 8681plusmn 12c 8507plusmn 09dBSAglucoseAVM (25mgml) 8433plusmn 12a 8594plusmn 08b 8418plusmn 14c 8466plusmn 11dBSAglucoseAVM (5mgml) 8324plusmn 06a 8433plusmn 07b 8357plusmn 07c 8288plusmn 06dBSAglucoseAG (5mgml) 8341plusmn 17a 8378plusmn 07b 8292plusmn 03c 8256plusmn 11d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

6 Evidence-Based Complementary and Alternative Medicine

When blood sugar levels rise the accumulation offructose in different organs can occur Both fructose andproducts derived from oxidation such as methylglyoxal can

react with proteins and form AGEs [12 15] Arginine is oneof the main targets of protein glycosylation by reactivecarbonyls which react with the guanidine group of the

Table 3 Effect of AVM on thiol group using the BSAglucose system

Experimental groups+iol group (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 7708plusmn 12 7538plusmn 007a 7588plusmn 005a 7005plusmn 008aBSAglucoseAVM (030mgml) 7595plusmn 10 7773plusmn 009b 7916plusmn 007b 7952plusmn 006BSAglucoseAVM (060mgml) 7708plusmn 051 7885plusmn 012b 7931plusmn 008b 8443plusmn 004dBSAglucoseAVM (12mgml) 7629plusmn 083a 7979plusmn 009b 8046plusmn 011b 8660plusmn 009dBSAglucoseAVM (25mgml) 7641plusmn 011a 8138plusmn 007b 8368plusmn 008b 8706plusmn 004dBSAglucoseAVM (5mgml) 7668plusmn 008a 8329plusmn 005b 8561plusmn 009b 8912plusmn 006dBSAglucoseAG (5mgml) 7652plusmn 008a 7737plusmn 002b 7756plusmn 004b 7808plusmn 009d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

AVM (12 mgml) AVM (25 mgml) AVM (5 mgml)

α-Amylaseα-Glucosidase

Acarbose (5 mgml)

a

a

aa

a

a

a

a

0

20

40

60

80

100

Inhi

bitio

n (

)

Figure 5 α-Amylase and α-glucosidase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standarddeviation) expressed as a percentage of enzymatic inhibition AVM methanol extract Letters indicate statistical significance(plt 005)

BSAglucoseAVM(25 mgml)

BSAglucoseAVM(5 mgml)

Orlistat (5 mgml)0

20

40

60

80

100

Lipa

se in

hibi

tion

()

Figure 6 Lipase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standard deviation) expressed as apercentage of enzymatic inhibition AVM methanol extract

Evidence-Based Complementary and Alternative Medicine 7

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

3 Results

31 Inhibition Tests for the Formation of Advanced GlycationEnd Products

311 In vitro Glycation of Bovine Albumin In Figure 1 thefluorescence intensity is shown during the fourweeks ofincubation +e results show that fluorescence was signifi-cantly increased over the weeks in the BSAglucose systemcompared to the groups incubated with BSAglucoseAVMat different concentrations where a significant decrease influorescence was observed depending on the concentrationAfter four weeks of the experiment the BSAglucoseAVMgroup (5mgml) showed the greatest decrease in fluores-cence intensity (8564) while the AG (5mgml) showed adecrease of 8794 in comparison with the BSA glycated inthe same time interval

312 Determination of Fructosamine +e effect of AVM onfructosamine levels during the four weeks is shown in Ta-ble 1 +e results show that the concentration of fructos-amine in the glycosylated BSA increased significantly overtime By adding AVM to different concentrations fruc-tosamine levels increased at a lower rate during the fourweeks of the test In the fourth week a fructosamine value of775mM was observed when using the BSAglucoseAVMsystem (5mgml) compared to the BSAglucoseAG systemwhich showed a value of 758mM+e glycated BSA showeda value of 1196mM in the same time interval

313 BSA-Methylglyoxal Assay +e effect of AVM at dif-ferent concentrations in the BSA-methylglyoxal test isshown in Figure 2 It can be observed that the greatestinhibition was presented when using the BSAglucoseAVMsystem (5mgml) showing an inhibition of 65 comparedto the BSAglucoseAG system (5mgml) which showed aninhibition of 91

314 Arginine-Methylglyoxal Assay +e antiglycation testusing the arginine-methylglyoxal model showed that theBSAglucoseAVM system (5mgml) inhibited the genera-tion of fluorescent AGEs at 65 (Figure 3) +e BSAglu-coseAG system (5mgml) showed an inhibition percentageof 80

315 Nε-(Carboxymethyl) Lysine +e effect of AVM on theinhibition of Nε-(carboxymethyl) lysine (CML) after fourweeks of incubation is shown in Figure 4 where it can beseen that the concentration of CML in the BSAglucosesystem increased During the experiment when evaluatingthe results of the BSAAVMglucose system it was observedthat the CML values were concentration-dependentobtaining a significant 73 inhibition in the formation ofCML at a concentration of 5mgml compared to the BSAAGglucose system (5mgml) which showed a 74 inhi-bition compared to the glycated BSA

32 Determination of Protein Carbonyl Content Table 2shows the protein carbonyl content during the four weeksWhen observing the glycated BSA an increase in proteincarbonyl content can be seen during the four weeks of theexperiment While adding AVM the BSAglucose system atdifferent concentrations showed a significant decrease inprotein carbonyl content during the four weeks that theexperiment lasted obtaining the highest decrease in the BSAglucoseAVM system at a concentration of 5mgml whichshowed a value of 8288 nmolmg protein +e BSAglucoseAG system and the glycated BSA showed values of8256 nmolmg protein and 8915 nmolmg protein re-spectively in the same time interval

33 Aiol Group Estimation +e effect of AVM during thefour weeks at different concentrations is shown in Table 3 +eBSAglucose system showed a significant increase in thiolgroups throughout the experiment By adding AVM to theBSAglucose system a significant decrease in thiol-dependentgroups can be observed+e greatest decrease was obtained at aconcentration of 5mgml (8912nmolmg protein) at fourweeks of the experiment while the BSAglucoseAG andglycated BSA system showed values of 7808 and 7005nmolmg protein respectively in the same time interval

34 Enzymatic Assays

341 α-Amylase α-Glucosidase and Lipase InhibitionActivity Figure 5 shows the inhibitory effect of AVM on theactivity of the alpha-amylase enzyme+e AVM concentrationof 5mgml showed a significant inhibition of 87 compared toacarbose (5mgml) which was used as a positive control andshowed an inhibition percentage of 97+e concentrations of060 and 030mgml AVM did not show inhibitory activityRegarding the inhibition of the α-glucosidase enzyme (Fig-ure 5) the concentration of 5mgml of AVM significantlyinhibited the activity of the enzyme by 66 compared toacarbose at the same concentration which presented an 85inhibition +e concentrations of 060 and 030mgml of theAVM extract showed no inhibition of the enzyme α-glucosi-dase Regarding the inhibition of pancreatic lipase the con-centrations of 25 and 5mgml of AVM showed an inhibitoryactivity of 9 and 15 respectively (Figure 6) compared to thecontrol drug which presented an inhibition of 99

4 Discussion

AGEs are a group of compounds that are formed by thenonenzymatic reaction of reducing sugars and metabolitesrelated to proteins and amino acids to produce Schiff basesSubsequently Amadori products (such as fructosamine) areformed which degrade into dicarbonyl compounds to finallyproduce stable AGEs [1 12 15] AVM reduced fructosaminelevels which are associated with decreased AGE formation+e reduction of fructosamine therefore is a therapeuticway to delay incident vascular complications [16] To thisend the capacity of AVM to inhibit the protein glycationreaction using the BSAglucose system was evaluated It was

4 Evidence-Based Complementary and Alternative Medicine

observed that AVM can inhibit the glycation reaction (whichis related to complications in diabetes) in a way similar to theaminoguanidine (AG) used as a control Since the formation

of AGEs is favored by oxidative reactions AVM could haveinhibited them by reducing the formation of reactive oxygenspecies (ROS) or by eliminating the ROS formed in vitro by

Week 1 Week 2 Week 3 Week 4

Fluo

resc

ence

inte

nsity

BSAglucoseAVM (030mgml)BSAglucoseAVM (12mgml)BSAglucoseAVM (5mgml)

BSAglucoseAG (5mgml)

BSAglucoseBSAglucoseAVM (060mgml)BSAglucoseAVM (25mgml)

bb

a

b

b

b

b

a

cc

cc

c c

a

dd

dd

d d

aa

aa

a a

0

200

400

600

800

1000

1200

1400

1600

Figure 1 Effects of AVM extract on formation of fluorescent advanced glycation end products (AGEs) in BSA incubated with glucose Eachvalue represents the meanplusmn SE (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose atweek two cplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAglucose at week four

Table 1 Effect of AVM on fructosamine levels during four weeks of incubation using the BSAglucose system

Experimental groupsFructosamine (mM)

Week 1 Week 2 Week 3 Week 4BSAglucose 872plusmn 08 979plusmn 09 1085plusmn 16 1196plusmn 12BSAglucoseAVM (030mgml) 867plusmn 12 855plusmn 13a 863plusmn 08a 869plusmn 14aBSAglucoseAVM (060mgml) 834plusmn 08a 843plusmn 05b 851plusmn 12c 856plusmn 17dBSAglucoseAVM (12mgml) 801plusmn 11a 827plusmn 13b 835plusmn 09c 841plusmn 13dBSAglucoseAVM (25mgml) 778plusmn 12a 792plusmn 18b 814plusmn 05c 827plusmn 14dBSAglucoseAVM (5mgml) 744plusmn 15a 754plusmn 12b 771plusmn 06c 775plusmn 14dBSAglucoseAG (5mgml) 721plusmn 14a 739plusmn 15b 745plusmn 12c 758plusmn 09d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose at week twocplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAfructose at week four

Gly

catio

n in

hibi

tion

()

BSA

met

hylg

lyox

al as

say

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5 m

gm

l)

0

20

40

60

80

100

120

Figure 2 Glycation inhibitory activity analysis of the methanol extract from Aloe vera in BSA-methylglyoxal model Values (mean-plusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Evidence-Based Complementary and Alternative Medicine 5

the oxidation of sugars andor by the oxidative degradationof Amadori products [17] It has been reported that theadministration of the ethanolic extract of A vera gel helps

prevent excessive free radical formation through variousbiochemical pathways and also reduces the glycosylation ofenzymes [18]

BSA

glu

cose

AV

M(0

30m

gm

l)

BSA

glu

cose

AV

M(0

60m

gm

l)

BSA

glu

cose

AV

M(1

2m

gm

l)

BSA

glu

cose

AV

M(2

5m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5m

gm

l)

0

10

20

30

40

50

60

70

80

90

Gly

catio

n in

hibi

tion

()

Arg

inin

e-m

ethy

lgly

oxal

assa

y

Figure 3 Glycation inhibitory activity analysis of the methanol extract from Aloe vera (AVM) in arginine-methylglyoxal model Values(meanplusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Nε-

(car

boxy

met

hyl)

lysin

e(n

gm

l)

aa

BSA

glu

cose

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

Glib

encla

mid

e(5

mg

ml)

0

1

2

3

4

5

6

Figure 4 Effect of AVM on the inhibition of Nε-(carboxymethyl) lysine (CML) after four weeks of incubation Each value represents themeanplusmn SEM (n 3) aplt 005 compared to BSAglucose

Table 2 Effect of AVM on protein carbonyl content using the BSAglucose system

Experimental groupsProtein carbonyl content (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 8772plusmn 18 8859plusmn 14a 8891plusmn 14a 8915plusmn 12aBSAglucoseAVM (030mgml) 8644plusmn 07a 8719plusmn 08b 8709plusmn 11c 8692plusmn 11dBSAglucoseAVM (060mgml) 8631plusmn 11a 8704plusmn 17b 8653plusmn 14c 8637plusmn 11dBSAglucoseAVM (12mgml) 8511plusmn 99a 8617plusmn 14b 8681plusmn 12c 8507plusmn 09dBSAglucoseAVM (25mgml) 8433plusmn 12a 8594plusmn 08b 8418plusmn 14c 8466plusmn 11dBSAglucoseAVM (5mgml) 8324plusmn 06a 8433plusmn 07b 8357plusmn 07c 8288plusmn 06dBSAglucoseAG (5mgml) 8341plusmn 17a 8378plusmn 07b 8292plusmn 03c 8256plusmn 11d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

6 Evidence-Based Complementary and Alternative Medicine

When blood sugar levels rise the accumulation offructose in different organs can occur Both fructose andproducts derived from oxidation such as methylglyoxal can

react with proteins and form AGEs [12 15] Arginine is oneof the main targets of protein glycosylation by reactivecarbonyls which react with the guanidine group of the

Table 3 Effect of AVM on thiol group using the BSAglucose system

Experimental groups+iol group (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 7708plusmn 12 7538plusmn 007a 7588plusmn 005a 7005plusmn 008aBSAglucoseAVM (030mgml) 7595plusmn 10 7773plusmn 009b 7916plusmn 007b 7952plusmn 006BSAglucoseAVM (060mgml) 7708plusmn 051 7885plusmn 012b 7931plusmn 008b 8443plusmn 004dBSAglucoseAVM (12mgml) 7629plusmn 083a 7979plusmn 009b 8046plusmn 011b 8660plusmn 009dBSAglucoseAVM (25mgml) 7641plusmn 011a 8138plusmn 007b 8368plusmn 008b 8706plusmn 004dBSAglucoseAVM (5mgml) 7668plusmn 008a 8329plusmn 005b 8561plusmn 009b 8912plusmn 006dBSAglucoseAG (5mgml) 7652plusmn 008a 7737plusmn 002b 7756plusmn 004b 7808plusmn 009d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

AVM (12 mgml) AVM (25 mgml) AVM (5 mgml)

α-Amylaseα-Glucosidase

Acarbose (5 mgml)

a

a

aa

a

a

a

a

0

20

40

60

80

100

Inhi

bitio

n (

)

Figure 5 α-Amylase and α-glucosidase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standarddeviation) expressed as a percentage of enzymatic inhibition AVM methanol extract Letters indicate statistical significance(plt 005)

BSAglucoseAVM(25 mgml)

BSAglucoseAVM(5 mgml)

Orlistat (5 mgml)0

20

40

60

80

100

Lipa

se in

hibi

tion

()

Figure 6 Lipase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standard deviation) expressed as apercentage of enzymatic inhibition AVM methanol extract

Evidence-Based Complementary and Alternative Medicine 7

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

observed that AVM can inhibit the glycation reaction (whichis related to complications in diabetes) in a way similar to theaminoguanidine (AG) used as a control Since the formation

of AGEs is favored by oxidative reactions AVM could haveinhibited them by reducing the formation of reactive oxygenspecies (ROS) or by eliminating the ROS formed in vitro by

Week 1 Week 2 Week 3 Week 4

Fluo

resc

ence

inte

nsity

BSAglucoseAVM (030mgml)BSAglucoseAVM (12mgml)BSAglucoseAVM (5mgml)

BSAglucoseAG (5mgml)

BSAglucoseBSAglucoseAVM (060mgml)BSAglucoseAVM (25mgml)

bb

a

b

b

b

b

a

cc

cc

c c

a

dd

dd

d d

aa

aa

a a

0

200

400

600

800

1000

1200

1400

1600

Figure 1 Effects of AVM extract on formation of fluorescent advanced glycation end products (AGEs) in BSA incubated with glucose Eachvalue represents the meanplusmn SE (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose atweek two cplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAglucose at week four

Table 1 Effect of AVM on fructosamine levels during four weeks of incubation using the BSAglucose system

Experimental groupsFructosamine (mM)

Week 1 Week 2 Week 3 Week 4BSAglucose 872plusmn 08 979plusmn 09 1085plusmn 16 1196plusmn 12BSAglucoseAVM (030mgml) 867plusmn 12 855plusmn 13a 863plusmn 08a 869plusmn 14aBSAglucoseAVM (060mgml) 834plusmn 08a 843plusmn 05b 851plusmn 12c 856plusmn 17dBSAglucoseAVM (12mgml) 801plusmn 11a 827plusmn 13b 835plusmn 09c 841plusmn 13dBSAglucoseAVM (25mgml) 778plusmn 12a 792plusmn 18b 814plusmn 05c 827plusmn 14dBSAglucoseAVM (5mgml) 744plusmn 15a 754plusmn 12b 771plusmn 06c 775plusmn 14dBSAglucoseAG (5mgml) 721plusmn 14a 739plusmn 15b 745plusmn 12c 758plusmn 09d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAglucose at week one bplt 005 when compared to BSAglucose at week twocplt 005 when compared to BSAglucose at week three dplt 005 when compared to BSAfructose at week four

Gly

catio

n in

hibi

tion

()

BSA

met

hylg

lyox

al as

say

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5 m

gm

l)

0

20

40

60

80

100

120

Figure 2 Glycation inhibitory activity analysis of the methanol extract from Aloe vera in BSA-methylglyoxal model Values (mean-plusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Evidence-Based Complementary and Alternative Medicine 5

the oxidation of sugars andor by the oxidative degradationof Amadori products [17] It has been reported that theadministration of the ethanolic extract of A vera gel helps

prevent excessive free radical formation through variousbiochemical pathways and also reduces the glycosylation ofenzymes [18]

BSA

glu

cose

AV

M(0

30m

gm

l)

BSA

glu

cose

AV

M(0

60m

gm

l)

BSA

glu

cose

AV

M(1

2m

gm

l)

BSA

glu

cose

AV

M(2

5m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5m

gm

l)

0

10

20

30

40

50

60

70

80

90

Gly

catio

n in

hibi

tion

()

Arg

inin

e-m

ethy

lgly

oxal

assa

y

Figure 3 Glycation inhibitory activity analysis of the methanol extract from Aloe vera (AVM) in arginine-methylglyoxal model Values(meanplusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Nε-

(car

boxy

met

hyl)

lysin

e(n

gm

l)

aa

BSA

glu

cose

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

Glib

encla

mid

e(5

mg

ml)

0

1

2

3

4

5

6

Figure 4 Effect of AVM on the inhibition of Nε-(carboxymethyl) lysine (CML) after four weeks of incubation Each value represents themeanplusmn SEM (n 3) aplt 005 compared to BSAglucose

Table 2 Effect of AVM on protein carbonyl content using the BSAglucose system

Experimental groupsProtein carbonyl content (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 8772plusmn 18 8859plusmn 14a 8891plusmn 14a 8915plusmn 12aBSAglucoseAVM (030mgml) 8644plusmn 07a 8719plusmn 08b 8709plusmn 11c 8692plusmn 11dBSAglucoseAVM (060mgml) 8631plusmn 11a 8704plusmn 17b 8653plusmn 14c 8637plusmn 11dBSAglucoseAVM (12mgml) 8511plusmn 99a 8617plusmn 14b 8681plusmn 12c 8507plusmn 09dBSAglucoseAVM (25mgml) 8433plusmn 12a 8594plusmn 08b 8418plusmn 14c 8466plusmn 11dBSAglucoseAVM (5mgml) 8324plusmn 06a 8433plusmn 07b 8357plusmn 07c 8288plusmn 06dBSAglucoseAG (5mgml) 8341plusmn 17a 8378plusmn 07b 8292plusmn 03c 8256plusmn 11d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

6 Evidence-Based Complementary and Alternative Medicine

When blood sugar levels rise the accumulation offructose in different organs can occur Both fructose andproducts derived from oxidation such as methylglyoxal can

react with proteins and form AGEs [12 15] Arginine is oneof the main targets of protein glycosylation by reactivecarbonyls which react with the guanidine group of the

Table 3 Effect of AVM on thiol group using the BSAglucose system

Experimental groups+iol group (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 7708plusmn 12 7538plusmn 007a 7588plusmn 005a 7005plusmn 008aBSAglucoseAVM (030mgml) 7595plusmn 10 7773plusmn 009b 7916plusmn 007b 7952plusmn 006BSAglucoseAVM (060mgml) 7708plusmn 051 7885plusmn 012b 7931plusmn 008b 8443plusmn 004dBSAglucoseAVM (12mgml) 7629plusmn 083a 7979plusmn 009b 8046plusmn 011b 8660plusmn 009dBSAglucoseAVM (25mgml) 7641plusmn 011a 8138plusmn 007b 8368plusmn 008b 8706plusmn 004dBSAglucoseAVM (5mgml) 7668plusmn 008a 8329plusmn 005b 8561plusmn 009b 8912plusmn 006dBSAglucoseAG (5mgml) 7652plusmn 008a 7737plusmn 002b 7756plusmn 004b 7808plusmn 009d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

AVM (12 mgml) AVM (25 mgml) AVM (5 mgml)

α-Amylaseα-Glucosidase

Acarbose (5 mgml)

a

a

aa

a

a

a

a

0

20

40

60

80

100

Inhi

bitio

n (

)

Figure 5 α-Amylase and α-glucosidase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standarddeviation) expressed as a percentage of enzymatic inhibition AVM methanol extract Letters indicate statistical significance(plt 005)

BSAglucoseAVM(25 mgml)

BSAglucoseAVM(5 mgml)

Orlistat (5 mgml)0

20

40

60

80

100

Lipa

se in

hibi

tion

()

Figure 6 Lipase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standard deviation) expressed as apercentage of enzymatic inhibition AVM methanol extract

Evidence-Based Complementary and Alternative Medicine 7

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

the oxidation of sugars andor by the oxidative degradationof Amadori products [17] It has been reported that theadministration of the ethanolic extract of A vera gel helps

prevent excessive free radical formation through variousbiochemical pathways and also reduces the glycosylation ofenzymes [18]

BSA

glu

cose

AV

M(0

30m

gm

l)

BSA

glu

cose

AV

M(0

60m

gm

l)

BSA

glu

cose

AV

M(1

2m

gm

l)

BSA

glu

cose

AV

M(2

5m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

BSA

glu

cose

AG

(5m

gm

l)

0

10

20

30

40

50

60

70

80

90

Gly

catio

n in

hibi

tion

()

Arg

inin

e-m

ethy

lgly

oxal

assa

y

Figure 3 Glycation inhibitory activity analysis of the methanol extract from Aloe vera (AVM) in arginine-methylglyoxal model Values(meanplusmn standard deviation) are expressed as a percentage of glycation inhibition AVM methanol extract from Aloe vera

Nε-

(car

boxy

met

hyl)

lysin

e(n

gm

l)

aa

BSA

glu

cose

BSA

glu

cose

AV

M(0

30

mg

ml)

BSA

glu

cose

AV

M(0

60

mg

ml)

BSA

glu

cose

AV

M(1

2 m

gm

l)

BSA

glu

cose

AV

M(2

5 m

gm

l)

BSA

glu

cose

AV

M(5

mg

ml)

Glib

encla

mid

e(5

mg

ml)

0

1

2

3

4

5

6

Figure 4 Effect of AVM on the inhibition of Nε-(carboxymethyl) lysine (CML) after four weeks of incubation Each value represents themeanplusmn SEM (n 3) aplt 005 compared to BSAglucose

Table 2 Effect of AVM on protein carbonyl content using the BSAglucose system

Experimental groupsProtein carbonyl content (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 8772plusmn 18 8859plusmn 14a 8891plusmn 14a 8915plusmn 12aBSAglucoseAVM (030mgml) 8644plusmn 07a 8719plusmn 08b 8709plusmn 11c 8692plusmn 11dBSAglucoseAVM (060mgml) 8631plusmn 11a 8704plusmn 17b 8653plusmn 14c 8637plusmn 11dBSAglucoseAVM (12mgml) 8511plusmn 99a 8617plusmn 14b 8681plusmn 12c 8507plusmn 09dBSAglucoseAVM (25mgml) 8433plusmn 12a 8594plusmn 08b 8418plusmn 14c 8466plusmn 11dBSAglucoseAVM (5mgml) 8324plusmn 06a 8433plusmn 07b 8357plusmn 07c 8288plusmn 06dBSAglucoseAG (5mgml) 8341plusmn 17a 8378plusmn 07b 8292plusmn 03c 8256plusmn 11d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

6 Evidence-Based Complementary and Alternative Medicine

When blood sugar levels rise the accumulation offructose in different organs can occur Both fructose andproducts derived from oxidation such as methylglyoxal can

react with proteins and form AGEs [12 15] Arginine is oneof the main targets of protein glycosylation by reactivecarbonyls which react with the guanidine group of the

Table 3 Effect of AVM on thiol group using the BSAglucose system

Experimental groups+iol group (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 7708plusmn 12 7538plusmn 007a 7588plusmn 005a 7005plusmn 008aBSAglucoseAVM (030mgml) 7595plusmn 10 7773plusmn 009b 7916plusmn 007b 7952plusmn 006BSAglucoseAVM (060mgml) 7708plusmn 051 7885plusmn 012b 7931plusmn 008b 8443plusmn 004dBSAglucoseAVM (12mgml) 7629plusmn 083a 7979plusmn 009b 8046plusmn 011b 8660plusmn 009dBSAglucoseAVM (25mgml) 7641plusmn 011a 8138plusmn 007b 8368plusmn 008b 8706plusmn 004dBSAglucoseAVM (5mgml) 7668plusmn 008a 8329plusmn 005b 8561plusmn 009b 8912plusmn 006dBSAglucoseAG (5mgml) 7652plusmn 008a 7737plusmn 002b 7756plusmn 004b 7808plusmn 009d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

AVM (12 mgml) AVM (25 mgml) AVM (5 mgml)

α-Amylaseα-Glucosidase

Acarbose (5 mgml)

a

a

aa

a

a

a

a

0

20

40

60

80

100

Inhi

bitio

n (

)

Figure 5 α-Amylase and α-glucosidase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standarddeviation) expressed as a percentage of enzymatic inhibition AVM methanol extract Letters indicate statistical significance(plt 005)

BSAglucoseAVM(25 mgml)

BSAglucoseAVM(5 mgml)

Orlistat (5 mgml)0

20

40

60

80

100

Lipa

se in

hibi

tion

()

Figure 6 Lipase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standard deviation) expressed as apercentage of enzymatic inhibition AVM methanol extract

Evidence-Based Complementary and Alternative Medicine 7

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

When blood sugar levels rise the accumulation offructose in different organs can occur Both fructose andproducts derived from oxidation such as methylglyoxal can

react with proteins and form AGEs [12 15] Arginine is oneof the main targets of protein glycosylation by reactivecarbonyls which react with the guanidine group of the

Table 3 Effect of AVM on thiol group using the BSAglucose system

Experimental groups+iol group (nmolmg protein)

Week 1 Week 2 Week 3 Week 4BSAglucose 7708plusmn 12 7538plusmn 007a 7588plusmn 005a 7005plusmn 008aBSAglucoseAVM (030mgml) 7595plusmn 10 7773plusmn 009b 7916plusmn 007b 7952plusmn 006BSAglucoseAVM (060mgml) 7708plusmn 051 7885plusmn 012b 7931plusmn 008b 8443plusmn 004dBSAglucoseAVM (12mgml) 7629plusmn 083a 7979plusmn 009b 8046plusmn 011b 8660plusmn 009dBSAglucoseAVM (25mgml) 7641plusmn 011a 8138plusmn 007b 8368plusmn 008b 8706plusmn 004dBSAglucoseAVM (5mgml) 7668plusmn 008a 8329plusmn 005b 8561plusmn 009b 8912plusmn 006dBSAglucoseAG (5mgml) 7652plusmn 008a 7737plusmn 002b 7756plusmn 004b 7808plusmn 009d

Results are expressed as meanplusmn SEM (n 3) aplt 005 when compared to BSAfructose at week one bplt 005 when compared to BSAfructose at week twocplt 005 when compared to BSAfructose at week three dplt 005 when compared to BSAfructose at week four

AVM (12 mgml) AVM (25 mgml) AVM (5 mgml)

α-Amylaseα-Glucosidase

Acarbose (5 mgml)

a

a

aa

a

a

a

a

0

20

40

60

80

100

Inhi

bitio

n (

)

Figure 5 α-Amylase and α-glucosidase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standarddeviation) expressed as a percentage of enzymatic inhibition AVM methanol extract Letters indicate statistical significance(plt 005)

BSAglucoseAVM(25 mgml)

BSAglucoseAVM(5 mgml)

Orlistat (5 mgml)0

20

40

60

80

100

Lipa

se in

hibi

tion

()

Figure 6 Lipase inhibition activity analysis of the methanol extract from Aloe vera Values (meanplusmn standard deviation) expressed as apercentage of enzymatic inhibition AVM methanol extract

Evidence-Based Complementary and Alternative Medicine 7

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

arginine to form AGEs [19] +erefore inhibiting the gly-cation of proteins and their oxidizing products is of para-mount importance to prevent the complications of diabetes

Our results show that the Aloe vera methanol extract(AVM) presents inhibitory activity in the formation ofmethylglyoxal and arginine and this could be due to thepresence of flavonoids [8] It has been reported that fla-vonoids as well as proanthocyanidins and phenolic acidsamong others are capable of inhibiting the glycation process[20 21] since they inhibit the formation of AGEs by cap-turing precursors such as 12-dicarbonyl or interacting withglucose preventing it from joining proteins

High concentrations of postprandial glucose are a riskfactor for microvascular and macrovascular complicationsin diabetes mellitus AVM showed inhibitory activity of theenzymes α-amylase and α-glucosidase Several flavonoidshave been reported as inhibitors of these enzymes for theirability to bind to proteins [22] which may have contributedto the inhibitory effect of AVM Previous studies ofAloe veraextract indicate that Aloe can act as a hypoglycemic agentthrough the potent inhibition of pancreatic amylase activity+is action decreases the breakdown of starch and offersgood postprandial glycemic control [23]

AVM inhibited pancreatic lipase (PL) and it is knownthat insulin sensitivity is related to increased body fat [24] soinhibiting PL can be a way to inhibit fat absorption Previousstudies report that flavonoids inhibit PL since these com-pounds can inactivate lipase through nonspecific binding ofthe enzyme which could explain the inhibitory activity ofAVM

It has been reported that AG inhibits protein glycosyl-ation eliminating beta-dicarbonyls [2] However in clinicaltrials it showed adverse effects such as gastrointestinaldisorders vasculitis and abnormal liver function while indiabetic rats it showed renal tumors [25]

+erefore the discovery of inhibitors of the proteinglycation reaction to reduce the formation of AGEs providesa promising therapeutic approach to prevent and treat di-abetic complications

Carboxymethyl lysine (CML) is formed by the oxidativedegradation of Amadori products [19] and it is one of thebest characterized AGEs chemically which has been used asthe main marker of AGEs in many studies [26] Recentresearch has shown that CML can interact with the RAGEreceptor by inactivating signaling pathways which leads tothe expression of proinflammatory genes [27] CML hasbeen found in the blood vessels of the retina in people withtype 2 diabetes mellitus and it has been linked to the degreeof retinopathy [28] and therefore it is important to findcompounds that inhibit the formation of CML In thisregard AVM showed the ability to reduce the formation ofCML significantly which could be an alternative to reducethe complications of diabetes associated with AGEs

Protein carbonyl compounds are formed by the oxida-tion of amino acid residues which together with the proteinthiol groups are considered as markers of the modificationof proteins by oxidation +is modification is measured bythe formation of hydrazone 24-dinitrophenylhydrazineadducts (DNPH) [2] In this study it was observed that

AVM is effective in eliminating carbonyls bound to proteinsand increasing the concentration of thiol protein groupshelping to prevent oxidative damage in proteins

5 Conclusions

Aloe veramethanol extract effectively inhibited the glycationreaction of proteins in the BSAglucose system possibly dueto the oxidative degradation of fructosamine however it isnecessary to determine the mechanism of action by whichAVM inhibits AGEs More research is required to describethe interactions of AVM with the enzymes α-amylaseα-glucosidase and pancreatic lipase to provide a basis for thedevelopment of new natural inhibitors

Data Availability

+e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

+e authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Zeng H Ding X Hu G Zhang and D Gong ldquoGalangininhibits α-glucosidase activity and formation of non-enzy-matic glycation productsrdquo Food Chemistry vol 271 pp 70ndash79 2019

[2] M M Joglekar L N Bavkar S Sistla and A U ArvindekarldquoEffective inhibition of protein glycation by combinatorialusage of limonene and aminoguanidine through differentialand synergistic mechanismsrdquo International Journal of Bio-logical Macromolecules vol 99 pp 563ndash569 2017

[3] N J Kellow and M T Coughlan ldquoEffect of diet-derivedadvanced glycation end products on inflammationrdquoNutritionReviews vol 73 no 11 pp 737ndash759 2015

[4] A Muntildeiz E Garcia D Gonzalez and L Zuntildeiga ldquoAntioxi-dant activity and in Vitro antiglycation of the fruit of spondiaspurpureardquo Evidence-Based Complementary and AlternativeMedicine vol 2018 Article ID 5613704 7 pages 2018

[5] R G Khalifah J W Baynes and B G Hudson ldquoAmadorinsnovel post-Amadori inhibitors of advanced glycation reac-tionsrdquo Biochemical and Biophysical Research Communica-tions vol 257 no 2 pp 251ndash258 1999

[6] L Zhang Z-C Tu X Xie et al ldquoAntihyperglycemic anti-oxidant activities of two Acer palmatum cultivars andidentification of phenolics profile by UPLC-QTOF-MSMSnew natural sources of functional constituentsrdquo IndustrialCrops and Products vol 89 pp 522ndash532 2016

[7] L Langmead R J Makins and D S Rampton ldquoAnti-in-flammatory effects of aloe vera gel in human colorectalmucosa in vitrordquo Alimentary Pharmacology andAerapeuticsvol 19 no 5 pp 521ndash527 2004

[8] B Benzidia et al ldquoChemical composition and antioxidantactivity of tannins extract from green rind of Aloe vera (L)Burm Frdquo Journal of King Saud University-Science vol 31no 4 pp 1175ndash1181 2018

[9] A A Maan A Nazir M K I Khan et al ldquo+e therapeuticproperties and applications of Aloe vera a reviewrdquo Journal ofHerbal Medicine vol 12 pp 1ndash10 2018

8 Evidence-Based Complementary and Alternative Medicine

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9

[10] R M P-Gutierrez ldquoInhibitory activities of guaianolides fromthe seeds of byrsonima crassifolia against protein glycation invitrordquo Medicinal Chemistry vol 5 no 5 pp 217ndash225 2015

[11] S Adisakwattana W Sompong A Meeprom S Ngamukoteand S Yibchok-Anun ldquoCinnamic acid and its derivativesinhibit fructose-mediated protein glycationrdquo InternationalJournal of Molecular Sciences vol 13 no 2 pp 1778ndash17892012

[12] W Wang Y Yagiz T J Buran C d N Nunes and L GuldquoPhytochemicals from berries and grapes inhibited the for-mation of advanced glycation end-products by scavengingreactive carbonylsrdquo Food Research International vol 44no 9 pp 2666ndash2673 2011

[13] A B Justino M N Pereira D D Vilela et al ldquoPeel ofaraticum fruit (Annona crassiflora Mart) as a source of an-tioxidant compounds with α-amylase α-glucosidase andglycation inhibitory activitiesrdquo Bioorganic Chemistry vol 69pp 167ndash182 2016

[14] M N Pereira A B Justino M M Martins et al ldquoStepha-lagine an alkaloid with pancreatic lipase inhibitory activityisolated from the fruit peel of Annona crassiflora Martrdquo In-dustrial Crops and Products vol 97 pp 324ndash329 2017

[15] A B Justino N C Miranda R R Franco M M MartinsN M d Silva and F S Espindola ldquoAnnona muricata Linnleaf as a source of antioxidant compounds with in vitroantidiabetic and inhibitory potential against α-amylaseα-glucosidase lipase non-enzymatic glycation and lipidperoxidationrdquo Biomedicine amp Pharmacotherapy vol 100pp 83ndash92 2018

[16] N Jariyapamornkoon S Yibchok-anun and S AdisakwattanaldquoInhibition of advanced glycation end products by red grapeskin extract and its antioxidant activityrdquo BMC Complementaryand Alternative Medicine vol 13 no 1 p 171 2013

[17] A Ardestani and R Yazdanparast ldquoCyperus rotundus sup-presses AGE formation and protein oxidation in a model offructose-mediated protein glycoxidationrdquo InternationalJournal of Biological Macromolecules vol 41 no 5 pp 572ndash578 2007

[18] M H Radha and N P Laxmipriya ldquoEvaluation of biologicalproperties and clinical effectiveness of aloe vera a systematicreviewrdquo Journal of Traditional and Complementary Medicinevol 5 no 1 pp 21ndash26 2015

[19] N Ahmed ldquoAdvanced glycation endproducts-role in pa-thology of diabetic complicationsrdquo Diabetes Research andClinical Practice vol 67 no 1 pp 3ndash21 2005

[20] N T Florence M Z Benoit K Jonas et al ldquoAntidiabetic andantioxidant effects of Annona muricata (Annonaceae)aqueous extract on streptozotocin-induced diabetic ratsrdquoJournal of Ethnopharmacology vol 151 no 2 pp 784ndash7902014

[21] P C Feng L J Haynes K E Magnus J R Plimmer andH S A Sherratt ldquoPharmacological screening of some westIndian medicinal plantsrdquo Journal of Pharmacy and Phar-macology vol 14 no 1 pp 556ndash561 1962

[22] G J Mcdougall and D Stewart ldquo+e inhibitory effects ofberry polyphenols on digestive enzymesrdquo BioFactors vol 23no 4 pp 189ndash195 2005

[23] A Mourad H Abo-Youssef B Anwar and S MessihaldquoBeneficial effects of Aloe vera in treatment of diabetescomparative in vivo and in vitro studiesrdquo Bulletin of Faculty ofPharmacy vol 51 no 1 pp 7ndash11 2013

[24] A Golay and J Ybarra ldquoLink between obesity and type 2diabetesrdquo Best Practice amp Research Clinical Endocrinology ampMetabolism vol 19 no 4 pp 649ndash663 2005

[25] N Rabbani and P J +ornalley ldquoAdvanced glycation endproducts in the pathogenesis of chronic kidney diseaserdquoKidney International vol 93 no 4 pp 803ndash813 2018

[26] Y Chen S Tang Y Chen et al ldquoStructure-activity rela-tionship of procyanidins on advanced glycation end productsformation and corresponding mechanismsrdquo Food Chemistryvol 272 pp 679ndash687 2019

[27] M-P Wautier P-J Guillausseau and J-L Wautier ldquoActi-vation of the receptor for advanced glycation end productsand consequences on healthrdquo Diabetes amp Metabolic Syn-drome Clinical Research amp Reviews vol 11 no 4 pp 305ndash309 2017

[28] S-Y Goh and M E Cooper ldquo+e role of advanced glycationend products in progression and complications of diabetesrdquoAe Journal of Clinical Endocrinology amp Metabolism vol 93no 4 pp 1143ndash1152 2008

Evidence-Based Complementary and Alternative Medicine 9