The Inhibitory Effects of Pure Flavonoidson in Vitro Protein Glycosylation

Seddighe Asgary, PhDGholam Ali Naderi, PhD

Nizal Sarraf-Zadegan, MDRoshanak Vakili, MS

ABSTRACT. Non-enzymatic glycosylation of proteins is the majorcause of diabetic complications, such as cardiovascular disorders, retino-pathy, nephropathy and neuropathy. It seems that protein glycosylationcan be inhibited effectively by antioxidants. Several flavonoids, such asrutin, kaempferol, quercetin, apigenin, naringin, morin and biochanin Awere selected to determine their antioxidant effects on in vitro insulin,hemoglobin and albumin glycosylation. The optimal glucose concentra-tion and incubation time were obtained for each protein. Then, the inhi-bition percentage of protein glycosylation was measured in the presenceof three different concentrations (0.5, 5, 10 J.lglml)of each flavonoids by acolorimetric method. The results demonstrated that biochanin A, the bestinhibitor of insulin and hemoglobin glycosylation, inhibits their glyco-sylation 100% and 60%, respectively. Glycosylation of albumin was in-hibited 100% by both biochanin A and apigenin. Therefore, it seemsprobable that plants containing flavonoids may have preventive effectsin diabetic complications. (Article copies available for a fee from TheHaworth Document Delivery Service: 1-800-HAWORTH.E-mail address:<getinfo@haworthpressillc.com> Website: <http://www.HaworthPress.com>@2002by The HaworthPress, Inc.All rightsreserved.]

KEYWORDS. Flavonoids, protein glycosylation, antioxidants, albu-min, insulin, hemoglobin

Seddighe Asgary, Gholam Ali Naderi, Nizal Sarraf-Zadegan, and Roshanak Vakiliare affiliated with the Cardiovascular Research Center, Isfahan, Iran.

Address correspondence to: Seddighe Asgary, PhD, Isfahan Cardiovascular Re-search Center, P.O. Box 81465-1148, Isfahan, Iran (E-mail: isfcarvasrc@hotmail.com). ---

Journal of Herbal Pharmacotherapy, Vol. 2(2) 2002@ 2002 by The Haworth Press, Inc. All rights reserved. 47

48 JOURNAL OF HERBAL PHARMACOTHERAPY

INTRODUCTION

Diabetes mellitus,the most commonendocrinedisease is the fourthleading cause of death in the United States, with a prevalence of3-10%.1.2Highmorbidityandmortalityof diabetesare mainlydueto itsmicro- and macrovascularcomplicationswhich lead to adverseeffectson thekidneys,eyes andcardiovascularandcentralnervoussystems.3,4

Evidence from clinical experience and animal model studies indi-cates that diabetichyperglycemiamay be the majorfactorcontributingto the progressive secondary complications.4,5Non-enzymaticglyco-sylationof bodyproteinssuchashemoglobin,duetoprolongedhyper-glycemia, is an important consequence of such a pathophysiologicmechanism.3-7Such glycosylation modifies the structural and func-tional properties of a number of proteins, including membranelipo-proteins,erythrocyte membraneproteins,plateletand lensa-crystallinproteinshumanserumproteins,suchas albumin,hemoglobin,collagen,myelin,as well as insulin.6-17

Antioxidantinhibitionof proteinglycosylationis considereda possi-ble preventive mechanism.Plant polyphenolicflavonoids,widelydis-tributedin vegetablesand otherplant foods, havebeenstudiedfor theircapacity to scavengefree hydroxyland peroxylradicals.21-25These di-etary sources of antioxidantscontributeover 4,000 chemicallyuniquesubstancesfrom allparts of vascularplantsincludingfruits,vegetables,nuts, seeds, stems and flowers, as well as from tea and wine.26The in-hibitory effects of rutin, quercetin and kaempferol on hemoglobinglycosylationhave been shown, in vitroP Theaim of this studywas tomeasure the inhibitory effects of seven flavonoids, including rutin,quercetin, kaempferol,morin, apigenin, naringinand biochaninA, onglycosylationof albumin,hemoglobinand insulin,in vitro.

MATERIALS AND METHODS

Preparation of Protein Solutions

Hemoglobin

The method is the same as our previous report on antioxidant effectofflavonoids.27 Blood from normal volunteers was drawn using EDTAas an anticoagulant. The red blood cells were washed thrice with 0.14 MNaCI solution. Then one vol. of red blood cells suspension was lysed

Asgary et at. 49

with two vol. of 0.01 M phosphate buffer (pH 7.4) and 0.5 vol. CCI4.After lysing,the hemolysatewas freed from debris by centrifugation.Theupperlayerwasseparatedandhemoglobinconcentrationwasmea-suredby the Drabkinmethod.18,19

Albumin

Human albumin (Sigma 5 g) was diluted .with 0.0I M phosphatebuffer(pH7.4) to 100ml.

Insulin

Regular bovine insulin (100 IU/ml) manufactured by Lilly was pre-pared by adding to 0.5 ml of 0.0 I M phosphate buffer (pH 7.4).

Determining the Optimal Conditions for Protein Glycosylation

Glucose solutions (0,4, 10, 20, 30, 40 and 50mg/ml) were preparedwith 0.01 M phosphate buffer (pH 7.4) and gentamycin (20 mg/l 00 nil).The optimal concentration of glucose and incubation 'time were deter-

. mined separately for each protein solution. .

.. The protein solutions were incubated for periods of 24, 48, 72 and96 hours. After incubation, they were washed twice with one ml ofCCI3COOH(20%)by centrifugingat 3000\rpm\orl0 min. The sedi- :ments obtained were heated at 100°C for one hour in the hot water bathwith one ml of 0.0 I M phosphate buffer (pH 7.4) and 0.5 ml of 0.3 N ox-alic acid. Cooled down to room temperature (25°C) and washed with0.5 ml of CC 13COOH(40%) by centrifugation at 3000 rpm for 10 min.The supernatant was separated and heated at 40°C for 30 min with 0.5ml of 0.05 M thiobarbituric acid. The end product was measured at 443nm by a colorimetric method.29_ !

Preparation of Flavonoid Solutions

Stock solutions (I mg/ml) of the tlavonoids (rutin, quercetin and .

kaempferol from Merck, morin, naringin and apigenin from Sigma, andbiochanin A from Aldrich) were prepared with ethanol, and then the re-quired concentrations (10, 100and 200 ~g/ml) were prepared in DMSO.

Assay

One mlof albumin(50 mg/ml), I ml of hemoglobin(50mg/ml),and0.5 ml of regular insulin (100 IU/ml), in 0.5 ml of 0.01 M phosphate

50 JOURNAL OF HERBAL PHARMACOTHERAPY

buffer (pH 7.4) were incubatedfor 72 hours separatelyin the presenceof differentconcentrationsof the fIavonoidsand I ml of a solutioncon-tainingglucose(30 mg/mlfor insulinand albumin,20 mg/mlforhemo-globin)and gentamycin20 mg/tOOml in 0.01 M phosphatebuffer(pH7.4). Also, the control solutions containingproteins and glucosewereincubated in the absence of flavonoids.Then the glycosylationdegreein thepresenceof differentproductsandalso intheirabsenceweremea-sured by the colorimetric measurements. 18-28

RESULTS

The optimal time and glucose concentration were found for each pro-tein solution are shown in Figures 1-3.The inhibition percentage of pro-tein glycosylation was used to express the effect of the tested fIavonoids(Tables 1-3). For each concentration of each of the proteins the experi-ments were repeated thrice, and the figures reported are means of threemeasurements. The Student's t-test showed a significant difference (P <0.05) between a test sample and its respective control in all cases. Com-parison of the inhibitory potency of each flavonoid (in the maximal con-centration) for non-enzymatic glycosylation of the proteins mentionedshowed the following results:

. Rutin had its strongest inhibitory effect on insulin glycosylation(100%).Theorder of inhibitionwas insulin> albumin> hemoglo-bin.

. Kaempferolinhibitoryeffecton the productionof glycatedinsulinwas mostpronounced(93%)whencomparedto the othertwopro-teins; theorderof inhibitionwas insulin> albumin>hemoglobin.

. Quercetin had the highest inhibitory effect (85%) on albuminglycosylation;the order of inhibitionwasalbumin> insulin> he-moglobin.. Apigeninshowedthe strongestinhibitoryeffecton albuminglyco-sylation(100%), the order of inhibitionwas albumin> hemoglo-bin> insulin. .. Naringin inhibited albumin glycosylation quite appreciably (97%).The order of inhibition was albumin> insulin> hemoglobin.. Morininhibitedalbuminglycosylation,withthehighestinhibitoryeffect (at a concentrationof 10Ilg/ml)as 90%.The inhibitorypo-tency order was albumin> insulin> hemoglobin.

Asgary etat. 51

· FinaHy, biochanin A showed the strongest inhibitory effect on al-bumin and insulin glycosylation (100%); the order of inhibitorypotency was albumin, insulin> hemoglobin.

DISCUSSION

Despite insulin therapy,diabeticpatients suffer from some chronicclinicalcomplicationsdue to highbloodglucosewhichinducesnon-en-zymaticglycosylationof naturalproteinssuch as hemoglobin,lenspro-teins,biomembraneproteins,albumin,collagen and myelin.12-17,2o.

In our previousstudy, the preventing effect on hemoglobinglyco-sylationat final concentrationsof 0.5, 5 and J0 Ilg/mlwas found to beJ1%,27%and42%for rutin,3%,37%and52% forquercetin,and 10%,J2% and 15%for kaempferoJ,respectively (27)..In the present study,theantioxidants,i.e.,flavonoids,weteassayedfor theirpotentialinhibi~toryeffectson oxidativeglycosyJation. :

Since the glycosylationinhibitory potency was concentration-de-pendent,thecomparisonsweremadeat similarconcentrationsfor eachflavonoid.The potency increasesas the concentrationincreases in thecaseof aHtheflavonoidsstudied..Thewidedifferencesobservedamongthe fIavonoidsas regards the protein glycosylation inhibitorypotency

FIGURE1. The glycosylation percentage of hemoglobin in different concentra-tions of glucose and incubation times (P < 0.05)

120

100

oo

....----..10 20 30

Concentration of glucose (mgfml)

40 50

52 JOURNAL OF HERBAL PHARMACOTHERAPY

FIGURE 2. The glycosylation percentage of albumin in different concentrationsof glucose and incubation times (P < 0.05)

120

100

i!80~

tOO(540 24hr

48hr

I 72 hr,",*"96 hr

oo 10 20 30

Concentration of glucose (mgfml)

40 50

FIGURE 3. The glycosylation percentage of insulin in different concentrationsof glucose and incubation times (P < 0.05)

120

100

~ 80

.§

t 60(5 40

24hr~ 48hr

72 hr!"'*"96hr

oo 10 20 30

Concentration of glucose (mglmJ)

40 50

maybe partly due to differences in the types andsitesof the functionalgroups and the reactivity of the protein molecule.More generally, aprotein's molecular structure may partly explain the differences..Forexample, at a concentration of 0.5 ,..g/ml,rutin showedthe lowest in-hibitory effect (6%) in the case of albumin,while the effect wasquite

Asgary et at. 53

TABLE 1.The inhibition percentage of flavonoids on hemoglobin glycosylation

Concentration of tested compound (j.lg/ml)

Compounds 0.5 5 10

~~ . G ~ G

Apigenin 29 60 64

~~ ~ ~ ~Biochanin A 47 35 66

For quercetin, rutin and kaempferol see Ref. 27.

TABLE 2. The inhibition percentage of flavonoids on albumin glycosylation

Compounds

Rutin

Quercetin

Kaempferol

. Morin

Apigenin

Naringin

/1iochanin A

Concentration of tested compound (j.lg/ml)

Q5 5 10

6 55 67

55 65 85

60 70 78

72 78 90

82 99 100

72 79 97

~ ~ 100

TABLE 3. The inhibition percentage of flavonoids on insulin glycosylation

Compounds

Rutin

Quercetin

KaempferolMorin

Apigenin

NaringinBiochaninA

Concentration of tested compound (j.lg/ml)

Q5 5 10

62 83 93

45 67 79

62 81 93

67 77 82

42 47 61

64 72 72

70 96 100

54 JOURNAL OF HERBAL PHARMACOTHERAPY

appreciable in the case of the other two proteins. The fact that it wasthe glycosylationof albuminwhichwasmoststronglyinhibited(100%)by apigenin (10 J.1g/ml)could further indicate the possible role of thestructureand chemicalpropertiesof the protein.On the other hand,thestructure and antioxidant propertiesof the flavonoidsthemselvescanalso explainthe differences.It is very interestingto note that biochaninA (10 J.1g/ml)with the strongest inhibitoryeffecton all the 3 proteinsisan isoflavonewith an antioxidantfeature, whileapigenin(10 flg/ml),aflavone, exhibits a comparable inhibitory antioxidanteffect (100%)only on albuminglycosylation.The findingsof the presentstudyshowthat flavonoidsinhibit glycosylationof albumin,hemoglobinand insu-lin in vitro.It is highlyrecommendedthatstudiesbeconductedto assessa possibleinvitro inhibitoryeffect inanimalandhumanmodels,as wellas to understandthe exact mechanismsinvolved.

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..:.~\.~.!:,'

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SUBMITTED: 03/12/01REVISED: 06/22/01

ACCEPTED: 07/11/01