Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 787138 8 pageshttpdxdoiorg1011552013787138
Research ArticleScopoletin Inhibits Rat Aldose Reductase Activity andCataractogenesis in Galactose-Fed Rats
Junghyun Kim Chan-Sik Kim Yun Mi Lee Eunjin SohnKyuhyung Jo So Dam Shin and Jin Sook Kim
Korean Medicine Based Herbal Drug Development Group Herbal Medicine Research Division Korea Institute of Oriental Medicine(KIOM) 1672 Yuseongdaero Yuseong-gu Daejeon 305-811 Republic of Korea
Correspondence should be addressed to Jin Sook Kim jskimkiomrekr
Received 15 February 2013 Revised 13 August 2013 Accepted 13 August 2013
Academic Editor Ravirajsinh Jadeja
Copyright copy 2013 Junghyun Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Cataracts are a major cause of human blindness Aldose reductase (AR) is an important rate-limiting enzyme that contributes tocataract induction in diabetic patients Scopoletin is the main bioactive constituent of flower buds from Magnolia fargesii and isknown to inhibit AR activity To assess scopoletinrsquos ability to mitigate sugar cataract formation in vivo we studied its effects in arat model of dietary galactose-induced sugar cataracts Galactose-fed rats were orally dosed with scopoletin (10 or 50mgkg bodyweight) once a day for 2 weeks Administering scopoletin delayed the progression of the cataracts that were induced by dietarygalactose Scopoletin also prevented galactose-induced changes in lens morphology such as lens fiber swelling and membranerupture Scopoletinrsquos protective effect against sugar cataracts was mediated by inhibiting both AR activity and oxidative stressThese results suggest that scopoletin is a useful treatment for sugar cataracts
1 Introduction
Cataracts are the leading cause of blindness worldwideHyperglycemia and diabetes increase the risk of developingcataracts [1] Cataractogenesis under diabetic or galactosemicconditions is directly linked to the aldose-reductase- (AR-)catalyzed accumulation of sorbitol or galactitol from glucoseor galactose respectively Accumulating excess sorbitol orgalactitol initiates osmotic stress altering lens cell perme-ability and redox homeostasis as well as decreasing ATPaseactivity and crystallin synthesis [2 3]
Although cataracts can be successfully treated withsurgery it remains important to find nonsurgical treatmentsfor this condition Synthetic AR inhibitors (ARIs) have beenstudied to treat diabetic cataracts The use of traditionalmedicines which are mainly derived from plant sources hasremained critical for managing many chronic diseases [4]Consuming foods or medicinal plants containing micronu-trients with potential anti-AR activities may protect againstcataracts [5ndash9]
Scopoletin (Figure 1) is one of the major coumarin con-stituents of the flower buds ofMagnolia fargesii this plant has
been used to treat various inflammatory diseases as a compo-nent of traditional Chinesemedicines [10] Several coumarinsreportedly block angiogenesis by inhibiting endothelial cellgrowth [11 12] Of the substances found in this plant extractscopoletin was chosen for study because it possesses awide range of biological effects including anti-inflammatoryhypouricemic and antioxidant activities [13ndash15] Recentlyscopoletin was reported to regulate hyperglycemia anddiabetes [16] In our previous study scopoletin from theflower buds ofM Fargesii inhibited protein glycation aldosereductase and cataractogenesis ex vivo [17] However invivo anticataract activity and the biochemical mechanismof scopoletin have not been understood yet In this studywe investigated the effect of scopoletin on galactose-inducedcataracts and studied the biochemical mechanism of thisprotection
2 Materials and Methods
21 Animals and Experimental Design To elucidate the effectof scopoletin treatment on sugar cataracts in vivo a galactose-fed rat model was used Scopoletin was purchased from
2 Evidence-Based Complementary and Alternative Medicine
HO
H3CO
O O
Figure 1 Chemical structure of scopoletin
Sigma (St Louis MO USA) Male Sprague-Dawley (SD)rats (sim200 g) were randomized into four groups of 10 ratsGroup 1 rats received a normal diet Group 2 rats received50 galactose diet (50 ww with normal diet) Group 3 andGroup 4 rats were fed the galactose diet and treated orallywith scopoletin (10 and 50mgkg body weight resp) oncea day for 2 weeks All animal procedures were performed inaccordance with the ARVO Statement for the Use of Animalsin Ophthalmic and Vision Research and approved by theKorea Institute of Oriental Medicine Institutional AnimalCare and Use Committee
22 Analysis of Cataract Formation and Its Severity GradationAfter two weeks of treatment the rats eyes were enucleatedunder deep anesthesia after an intraperitoneal injection of10mgkg zolazepam (Zoletil Virbac Carros France) mixedwith 10mgkg xylazine hydrochloride (Rumpun BayerFrankfurt Germany) The lenses were excised from theeyeball under an optical microscope and the lensesrsquos wetweights were calculated The lenses were transferred onto24-well plates with each well each containing 2mL salinesolution andwere photographed under an opticalmicroscopewith a CCD camera The severity of the cataracts wasevaluated using the following gradation grade 0 no opacitygrade I vacuoles present at a part of the cortical equatorgrade II vacuoles present at all parts of the cortical equatorgrade III vacuoles and their confluents spreading from thecortical equator toward the center of the cortex grade IVlarge interconnected opacities covering thewhole cortexTheopaque areas of the lenses were analyzed using an imagingprogram (ImageJ NIH USA) The data are expressed as thepercentage of opaque area relative to the entire lens area
23 Analysis of Lens Fiber Degeneration The isolated lenseswere fixed in 10 neutralized formalin for 24 h and embed-ded in paraffin To analysis the lens fiber degenerationfiber cells were visualized by labeling their membranes withwheat germ agglutininThe lens sections were deparaffinizedin xylene and rehydrated The sections were reacted with25mgmL rhodamine-conjugated wheat germ agglutinin(Vector Laboratories CA USA) for 60 minutes All speci-mens were examined with a fluorescence microscope (BX51Olympus Tokyo Japan)
24 Determination of AR Activity A 10 lens homogenatewas prepared from two to three pooled lenses in a 50mMphosphate buffer (pH 74)The incubation mixture contained135 mmolL Na K-phosphate buffer (pH 70) 100mmolL
lithium sulfate 003mmolL NADPH 004mmolL dL-glyceraldehyde and 150120583L of lens homogenate in a total vol-ume of 10mLThe reaction was initiated by adding NADPHat 37∘C and stopped by adding 03mL of 05N hydrochloricacid Subsequently 1mL 6N NaOH containing 10mmolLimidazole was added and the mixture was incubated at60∘C for 10min to convert NADP into a fluorescent productThe fluorescence was measured at room temperature with aspectrofluorophotometer (ExEm=360 nm460 nm SynergyHT Bio-Tek VTUSA) Allmeasurements were performed intriplicate
25 Lens Polyol Levels The lens galactitol was measured aspreviously reported [18] Briefly each lens was homogenizedin a ground glass homogenizer and an aliquot of thehomogenate was removed for colorimetric protein quan-tification using a DC Protein Assay (Bio-Rad LaboratoriesCA USA) with bovine serum albumin (BSA) protein stan-dards Seventy-five microliters of 10 trichloroacetic acid(TCA) was added to 125 120583L centrifuged lens homogenatethe mixture was centrifuged at 12000 rpm for 5min To15 120583L protein-free supernatant we added 50 120583L 1N HCland 250120583L 25 nM NaIO
4 The mixture was incubated for
30min at 37∘C Afterward 50120583L 14N NaOH and 50120583L10 ZnSO
4were added The mixture was allowed to stand
for a few minutes after vortexing before adding 500 120583L 2Mammonium acetate containing 20mM pentanedione Themethyltoluidine (absorbance maximum 415 nm) content wasmeasured in the supernatant after incubation for 1 h at 37∘CThe polyol standards were treated in the same manner andthe galactitol in the lens homogenate samples was completelyrecovered
26 Glutathione Levels Each lens was homogenized in aground glass homogenizer and the insoluble proteins wereremoved by centrifugation at 4∘C The remaining cell super-natants were deproteinized with equal volumes of 20TCA and the glutathione (GSH) levels in the deproteinizedsupernatant were measured at 412 nm using the DTNB (5-51015840-dithiobis[2-nitrobenzoic acid]) method [19]
27 Immunofluorescence Staining Lens sections weredeparaffinized and hydrated by sequential immersions inxylene and graded alcohol solutionsThe slides were placed in10mM sodium citrate buffer (pH 60) and autoclaved at 121∘Cfor 10min Sections were then blocked with normal serumobtained from the same species with a secondary antibodydeveloped to block nonspecific staining The sectionswere first labeled with mouse anti-AR antibody (1 250Santa Cruz Biotechnology CA USA) overnight at 4∘CAfter washing the sections were labeled with fluorescein-isothiocyanate- (FITC-) conjugated goat anti-mouse IgG(1 1000 Santa Cruz Biotechnology CA USA) for 1 h atroom temperature Finally the slides were analyzed usingfluorescence microscopy (BX51 Olympus) The negativecontrols for immunostaining were run by incubating thesections with nonimmune serum instead of the primaryantibody
Evidence-Based Complementary and Alternative Medicine 3
NOR GAL SCO-10 SCO-50
(a)
Grade 0Grade 1Grade 2
Grade 3Grade 4
100
80
60
40
20
0
Inci
denc
e (
)
GAL SCO-10 SCO-50NOR
(b)
NOR GAL SCO-10 SCO-50
90
80
70
20
10
0
Lens
opa
city
()
60
50
40
30
lowast
lowast
(c)
Figure 2 Lens opacity (a) Representative images of the lenses in each group (b) Cataract grading The cataracts were assessed on a scale of0ndash4 (c) Analyses of lens opacitiesThe opacities were analyzed for each lens from the normal rats (NOR) the vehicle-treated galactose-fed rats(GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletinat concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control rats lowast119875 lt 001 versusvehicle-treated galactose-fed rats
28 Statistical Analysis The results were statistically evalu-ated using a one-way analysis of variance (ANOVA) followedby Tukeyrsquos multiple comparison test using GraphPad Prism50 software (GraphPad CA USA)
3 Results
31 Cataract Formation Analysis During the cataract anal-ysis all of the animals fed on the galactose diet developedmature cataracts after two weeks (70 were in grade III and30 were in grade IV Figures 2(a) and 2(b)) The scopoletintreatment delayed the onset of galactose-induced cataractsin a dose-dependent manner The highest dose of scopoletintreatment (50mgkgday) delayed the onset of cataracts (80were in grade I and 20 were in grade II Figures 2(a)and 2(b)) When analyzing the lensesrsquo opacification themean opaque area of the lenses was increased 8-fold in thegalactose-fed rats relative to the normal rats the opacity wassuppressed by the scopoletin treatment in a dose-dependentmanner (Figure 2(c) 119875 lt 001) Therefore scopoletin slowedthe development of galactose-induced cataracts
32 Lens Fiber Cell Degeneration In Figure 3 themembrane-labeled lens section illustrates the histological findings afterthe two weeks of study No significant alterations in thecuboidal epithelium or lens fiber were observed in thelenses of the control rats The lenses of galactose-fed ratswere swollen degenerated vacuolated and liquefied withdegenerated lens fibers Although the lenticular damage wassevere no corneal or retinal damage was observed Howeverthis histological change in the lens fibers of galactose-fed ratswas prevented in a dose-dependent manner after scopoletintreatment
33 Polyol Pathway in Lens The galactose-fed rats werekilled after two weeks some rats were progressing towardgrade 3 cataracts and extensive protection by scopoletin wasobserved AR is a key enzyme in the polyol pathway its activ-ity was significantly elevated in the galactose-fed rats TheAR activity in the lenses from the scopoletin-treated animalswas decreased (Figure 4(a)) agreeing with our observationsduring our in vitro studies [17] In addition the galactitollevels in the galactose-fed rats increased relative to the control
4 Evidence-Based Complementary and Alternative Medicine
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Figure 3 Lens fiber changes The lens sections from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fedrats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletin at concentration 50mgkg(SCO-50) were labeled with rhodamine-conjugated wheat germ agglutinin Fiber cell liquefaction swelling and membrane rupture wereobserved in galactosemic cataractous lens
NOR GAL SCO-10 SCO-50
7
6
5
4
3
2
1
0
AR
activ
ity (U
mg
lens
wei
ght)
lowast
(a)
NOR GAL SCO-10 SCO-50
75
50
25
0
Gal
actit
ol (n
mol
mg
lens
wei
ght)
lowast
(b)
Figure 4 Polyol pathway (a) Aldose reductase (AR) activity (b) galactitol levels in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control ratslowast
119875 lt 001 versus vehicle-treated galactose-fed rats
Evidence-Based Complementary and Alternative Medicine 5
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Negative control
(e)
Figure 5 Immunofluorescence stained AR Representative immunostained AR in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) AR was strongly immunoreactive in the cytoplasm of the lens epithelial cells and lenscortical fibers The negative control section was incubated with nonimmune mouse IgG and remained unstained
rats (Figure 4(b)) as expected because the polyol pathwaywasactivated However administering scopoletin resulted in lessgalactose-induced lenticular galactitol accumulation
34 AR Protein Expression in Lens In vehicle-treatedgalactose-fed rats immunoreactive straining for ARincreased in the cytoplasm of lens epithelial cells andextended into the deeper cortical fibers However thescopoletin treatment prevented AR expression in the lensepithelial cells and inhibited the extension of AR beneath theepithelial region (Figure 5)
35 GSH Levels in Lens The GSH status after treatmentindicated that the rats fed with galactose displayed lowerGSH levels in their lenses relative to the control The scopo-letin treatment given with the dietary galactose preventeddecreases in GSH levels in the lenses (Figure 6)
4 Discussion
In this study we investigated the protective effects ofscopoletin against cataractogenesis in galactose-fed rats Thescopoletin treatment delayed the progression and reduced theextent of cataract formation Currently the only treatment for
cataracts is surgery It has been estimated that a 10-year delayin the onset and progression of a cataract could reduce theneed for cataract surgery by 50 [20]
Galactosemic and diabetic cataractogenesis in experi-mental animals and humans might be primarily due to theincreased formation of polyols from the reduced aldose sug-ars produced by aldose reductase and nicotinamide adeninedinucleotide phosphate (NADPH) [21] Polyols may accumu-late in the lens fiber cells causing increased cell hydrationmembrane stretching and dysfunction Galactose-fed ratsare a popular model used to examine the role of the ARpathway in diabetic complications In addition the galactose-induced cataracts develop within a week of feeding thismodel has been used extensively to study the morphologicaland biochemical changes during cataractogenesis Galactitolis a metabolite of galactose by AR that can accumulate in thelens Because the cellular lens membranes are impermeableto galactitol hyperosmotic cell swelling occurs causing lightscattering and diminished lens transparency [22ndash24] In thisstudy scopoletin inhibited the lenticular AR activity andthe accumulation of galactitol in galactose-fed rats This ARinhibition corresponded to the anticataractogenic activity
ARIs such as sorbinil prevented sugar cataractogenesisin experimental animals [25 26] Among the ARI onlysorbinil has reached advanced clinical trials in cataract
6 Evidence-Based Complementary and Alternative Medicine
NOR GAL SCO-10 SCO-50
2
1
0
4
3
GSH
(nm
olm
g le
ns w
eigh
t)
lowast
Figure 6 Glutathione (GSH) alteration GSH was measured in thelenses from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fed rats treated with scopoletin atconcentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data areexpressed as the means plusmn SE 119899 = 10
119875 lt 001 versus normalcontrol rats lowast119875 lt 001 versus vehicle-treated galactose-fed rats
prevention programs However due to the manifestationof skin rashes the trial was discontinued [27] Althoughseveral previous studies demonstrated that ARIs inhib-ited sugar cataracts by inhibiting AR no single agent hasbeen proven clinically effective during the treatment ofsugar cataracts Many naturally occurring compounds havestrong AR inhibitory activity in vitro [28] Recently scopo-letin demonstrated effective AR inhibitory activity [17 29]Coumarins are bicyclic phenolic compounds that harbor alactone moiety this functionality might participate in ARinhibition by hydrogen bondingwith the TYR48HIS110 andTRP111 residues inAR [30] Based on these results preventingsugar cataracts with scopoletin is partially related to ARinhibition and galactitol accumulation in the lens
In our previous study scopoletin had an excellent inhib-itory activity on AR displaying an IC
50value of 432 120583gmL
[17] Liu et al reported that the peak plasma scopoletinconcentrations (Cmax) were 051 068 and 149120583gmL andreached approximately 40 minutes after administering 50100 and 250mgkg scopoletin in rabbits respectively [31]The peak plasma concentration of scopoletin was 82 120583gmLafter administering 50mgkg scopoletin in rats [32] Becausescopoletin was highly lipophilic it absorbed effectively afteroral administration and spreadwidely to different tissues [33]Based on its previously reported pharmacokinetics and our invitro results we chose 10 and 50mgkg doses of scopoletin toevaluate its anti-AR activity in ratsWe found that the effectivedose of scopoletin was 50mgkd agreeing with the in vitroresult
The enzymatic distribution of AR activity was sup-ported by ARrsquos localized immunofluorescence In humanand rat lenses AR is primarily localized in the epithelialand superficial cortical fiber cells [34] In galactose-fed rats
the enhanced immunoreactive staining of AR was observedin the epithelial cells and the cortex region This stainingdecreased progressing from the superficial region to thedeeper cortex Scopoletin inhibited the extension of ARbeneath the epithelial region Therefore the decrease in ARactivity observed in the scopoletin-treated rats was caused bythe reduced amount of AR protein
Although the prevention of sugar-induced cataracto-genesis by ARIs appears to be caused by AR inhibitionthe osmotic hypothesis might not fully explain diabeticcataracts in human subjects because even during severehyperglycemia the examined tissues including the lens didnot have sorbitol levels gt2mM [35] Antioxidants effectivelyslow sugar cataract formation Butylated hydroxytolueneis a well-known synthetic phenolic antioxidant that slowscataract formation in rat lenses cultured under high-glucoseconditions although the sorbitol and fructose levels in thelenses remains elevated [36] ConsequentlyWolff andCrabbesuggested that the ARIs protected against sugar cataracts dueto the antioxidant nature of these inhibitors [37] Scopoletinhas demonstrated benefits for oxidative injury as an antioxi-dant [38 39] In this study scopoletin preserved the lenticularGSH content Therefore one of the possible mechanisms forscopoletin during sugar cataract development may involvethe protection of the lens cell membrane from oxidativedamage
In summary this study reveals that scopoletin mayexert beneficialprotective effects during the sugar cataractdevelopment Scopoletin inhibits the AR activity polyolaccumulation and reduction of the GSH levels We suggestthat the scopoletin may be particularly useful in treatingsugar cataracts
Acknowledgments
This research was supported by a Grant (K12040) from theKorea Institute of Oriental Medicine (KIOM)
References
[1] SNUghade S P Zodpey andVAKhanolkar ldquoRisk factors forcataract a case control studyrdquo Indian Journal of Ophthalmologyvol 46 no 4 pp 221ndash227 1998
[2] HMCheng andRGGonzalez ldquoThe effect of high glucose andoxidative stress on lensmetabolism aldose reductase and senilecataractogenesisrdquoMetabolism vol 35 no 4 pp 10ndash14 1986
[3] A Y Lee and S S Chung ldquoContributions of polyol pathway tooxidative stress in diabetic cataractrdquo FASEB Journal vol 13 no1 pp 23ndash30 1999
[4] J K Grover S Yadav and V Vats ldquoMedicinal plants of Indiawith anti-diabetic potentialrdquo Journal of Ethnopharmacology vol81 no 1 pp 81ndash100 2002
[5] J Lee D S Jang N H Kim Y M Lee J Kim and J SKim ldquoGalloyl glucoses from the seeds of Cornus officinalis withinhibitory activity against protein glycation aldose reductaseand cataractogenesis ex vivordquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 443ndash446 2011
[6] D S Jang Y M Lee I H Jeong and J S Kim ldquoConstituentsof the flowers of Platycodon grandiflorum with inhibitory
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
2 Evidence-Based Complementary and Alternative Medicine
HO
H3CO
O O
Figure 1 Chemical structure of scopoletin
Sigma (St Louis MO USA) Male Sprague-Dawley (SD)rats (sim200 g) were randomized into four groups of 10 ratsGroup 1 rats received a normal diet Group 2 rats received50 galactose diet (50 ww with normal diet) Group 3 andGroup 4 rats were fed the galactose diet and treated orallywith scopoletin (10 and 50mgkg body weight resp) oncea day for 2 weeks All animal procedures were performed inaccordance with the ARVO Statement for the Use of Animalsin Ophthalmic and Vision Research and approved by theKorea Institute of Oriental Medicine Institutional AnimalCare and Use Committee
22 Analysis of Cataract Formation and Its Severity GradationAfter two weeks of treatment the rats eyes were enucleatedunder deep anesthesia after an intraperitoneal injection of10mgkg zolazepam (Zoletil Virbac Carros France) mixedwith 10mgkg xylazine hydrochloride (Rumpun BayerFrankfurt Germany) The lenses were excised from theeyeball under an optical microscope and the lensesrsquos wetweights were calculated The lenses were transferred onto24-well plates with each well each containing 2mL salinesolution andwere photographed under an opticalmicroscopewith a CCD camera The severity of the cataracts wasevaluated using the following gradation grade 0 no opacitygrade I vacuoles present at a part of the cortical equatorgrade II vacuoles present at all parts of the cortical equatorgrade III vacuoles and their confluents spreading from thecortical equator toward the center of the cortex grade IVlarge interconnected opacities covering thewhole cortexTheopaque areas of the lenses were analyzed using an imagingprogram (ImageJ NIH USA) The data are expressed as thepercentage of opaque area relative to the entire lens area
23 Analysis of Lens Fiber Degeneration The isolated lenseswere fixed in 10 neutralized formalin for 24 h and embed-ded in paraffin To analysis the lens fiber degenerationfiber cells were visualized by labeling their membranes withwheat germ agglutininThe lens sections were deparaffinizedin xylene and rehydrated The sections were reacted with25mgmL rhodamine-conjugated wheat germ agglutinin(Vector Laboratories CA USA) for 60 minutes All speci-mens were examined with a fluorescence microscope (BX51Olympus Tokyo Japan)
24 Determination of AR Activity A 10 lens homogenatewas prepared from two to three pooled lenses in a 50mMphosphate buffer (pH 74)The incubation mixture contained135 mmolL Na K-phosphate buffer (pH 70) 100mmolL
lithium sulfate 003mmolL NADPH 004mmolL dL-glyceraldehyde and 150120583L of lens homogenate in a total vol-ume of 10mLThe reaction was initiated by adding NADPHat 37∘C and stopped by adding 03mL of 05N hydrochloricacid Subsequently 1mL 6N NaOH containing 10mmolLimidazole was added and the mixture was incubated at60∘C for 10min to convert NADP into a fluorescent productThe fluorescence was measured at room temperature with aspectrofluorophotometer (ExEm=360 nm460 nm SynergyHT Bio-Tek VTUSA) Allmeasurements were performed intriplicate
25 Lens Polyol Levels The lens galactitol was measured aspreviously reported [18] Briefly each lens was homogenizedin a ground glass homogenizer and an aliquot of thehomogenate was removed for colorimetric protein quan-tification using a DC Protein Assay (Bio-Rad LaboratoriesCA USA) with bovine serum albumin (BSA) protein stan-dards Seventy-five microliters of 10 trichloroacetic acid(TCA) was added to 125 120583L centrifuged lens homogenatethe mixture was centrifuged at 12000 rpm for 5min To15 120583L protein-free supernatant we added 50 120583L 1N HCland 250120583L 25 nM NaIO
4 The mixture was incubated for
30min at 37∘C Afterward 50120583L 14N NaOH and 50120583L10 ZnSO
4were added The mixture was allowed to stand
for a few minutes after vortexing before adding 500 120583L 2Mammonium acetate containing 20mM pentanedione Themethyltoluidine (absorbance maximum 415 nm) content wasmeasured in the supernatant after incubation for 1 h at 37∘CThe polyol standards were treated in the same manner andthe galactitol in the lens homogenate samples was completelyrecovered
26 Glutathione Levels Each lens was homogenized in aground glass homogenizer and the insoluble proteins wereremoved by centrifugation at 4∘C The remaining cell super-natants were deproteinized with equal volumes of 20TCA and the glutathione (GSH) levels in the deproteinizedsupernatant were measured at 412 nm using the DTNB (5-51015840-dithiobis[2-nitrobenzoic acid]) method [19]
27 Immunofluorescence Staining Lens sections weredeparaffinized and hydrated by sequential immersions inxylene and graded alcohol solutionsThe slides were placed in10mM sodium citrate buffer (pH 60) and autoclaved at 121∘Cfor 10min Sections were then blocked with normal serumobtained from the same species with a secondary antibodydeveloped to block nonspecific staining The sectionswere first labeled with mouse anti-AR antibody (1 250Santa Cruz Biotechnology CA USA) overnight at 4∘CAfter washing the sections were labeled with fluorescein-isothiocyanate- (FITC-) conjugated goat anti-mouse IgG(1 1000 Santa Cruz Biotechnology CA USA) for 1 h atroom temperature Finally the slides were analyzed usingfluorescence microscopy (BX51 Olympus) The negativecontrols for immunostaining were run by incubating thesections with nonimmune serum instead of the primaryantibody
Evidence-Based Complementary and Alternative Medicine 3
NOR GAL SCO-10 SCO-50
(a)
Grade 0Grade 1Grade 2
Grade 3Grade 4
100
80
60
40
20
0
Inci
denc
e (
)
GAL SCO-10 SCO-50NOR
(b)
NOR GAL SCO-10 SCO-50
90
80
70
20
10
0
Lens
opa
city
()
60
50
40
30
lowast
lowast
(c)
Figure 2 Lens opacity (a) Representative images of the lenses in each group (b) Cataract grading The cataracts were assessed on a scale of0ndash4 (c) Analyses of lens opacitiesThe opacities were analyzed for each lens from the normal rats (NOR) the vehicle-treated galactose-fed rats(GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletinat concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control rats lowast119875 lt 001 versusvehicle-treated galactose-fed rats
28 Statistical Analysis The results were statistically evalu-ated using a one-way analysis of variance (ANOVA) followedby Tukeyrsquos multiple comparison test using GraphPad Prism50 software (GraphPad CA USA)
3 Results
31 Cataract Formation Analysis During the cataract anal-ysis all of the animals fed on the galactose diet developedmature cataracts after two weeks (70 were in grade III and30 were in grade IV Figures 2(a) and 2(b)) The scopoletintreatment delayed the onset of galactose-induced cataractsin a dose-dependent manner The highest dose of scopoletintreatment (50mgkgday) delayed the onset of cataracts (80were in grade I and 20 were in grade II Figures 2(a)and 2(b)) When analyzing the lensesrsquo opacification themean opaque area of the lenses was increased 8-fold in thegalactose-fed rats relative to the normal rats the opacity wassuppressed by the scopoletin treatment in a dose-dependentmanner (Figure 2(c) 119875 lt 001) Therefore scopoletin slowedthe development of galactose-induced cataracts
32 Lens Fiber Cell Degeneration In Figure 3 themembrane-labeled lens section illustrates the histological findings afterthe two weeks of study No significant alterations in thecuboidal epithelium or lens fiber were observed in thelenses of the control rats The lenses of galactose-fed ratswere swollen degenerated vacuolated and liquefied withdegenerated lens fibers Although the lenticular damage wassevere no corneal or retinal damage was observed Howeverthis histological change in the lens fibers of galactose-fed ratswas prevented in a dose-dependent manner after scopoletintreatment
33 Polyol Pathway in Lens The galactose-fed rats werekilled after two weeks some rats were progressing towardgrade 3 cataracts and extensive protection by scopoletin wasobserved AR is a key enzyme in the polyol pathway its activ-ity was significantly elevated in the galactose-fed rats TheAR activity in the lenses from the scopoletin-treated animalswas decreased (Figure 4(a)) agreeing with our observationsduring our in vitro studies [17] In addition the galactitollevels in the galactose-fed rats increased relative to the control
4 Evidence-Based Complementary and Alternative Medicine
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Figure 3 Lens fiber changes The lens sections from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fedrats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletin at concentration 50mgkg(SCO-50) were labeled with rhodamine-conjugated wheat germ agglutinin Fiber cell liquefaction swelling and membrane rupture wereobserved in galactosemic cataractous lens
NOR GAL SCO-10 SCO-50
7
6
5
4
3
2
1
0
AR
activ
ity (U
mg
lens
wei
ght)
lowast
(a)
NOR GAL SCO-10 SCO-50
75
50
25
0
Gal
actit
ol (n
mol
mg
lens
wei
ght)
lowast
(b)
Figure 4 Polyol pathway (a) Aldose reductase (AR) activity (b) galactitol levels in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control ratslowast
119875 lt 001 versus vehicle-treated galactose-fed rats
Evidence-Based Complementary and Alternative Medicine 5
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Negative control
(e)
Figure 5 Immunofluorescence stained AR Representative immunostained AR in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) AR was strongly immunoreactive in the cytoplasm of the lens epithelial cells and lenscortical fibers The negative control section was incubated with nonimmune mouse IgG and remained unstained
rats (Figure 4(b)) as expected because the polyol pathwaywasactivated However administering scopoletin resulted in lessgalactose-induced lenticular galactitol accumulation
34 AR Protein Expression in Lens In vehicle-treatedgalactose-fed rats immunoreactive straining for ARincreased in the cytoplasm of lens epithelial cells andextended into the deeper cortical fibers However thescopoletin treatment prevented AR expression in the lensepithelial cells and inhibited the extension of AR beneath theepithelial region (Figure 5)
35 GSH Levels in Lens The GSH status after treatmentindicated that the rats fed with galactose displayed lowerGSH levels in their lenses relative to the control The scopo-letin treatment given with the dietary galactose preventeddecreases in GSH levels in the lenses (Figure 6)
4 Discussion
In this study we investigated the protective effects ofscopoletin against cataractogenesis in galactose-fed rats Thescopoletin treatment delayed the progression and reduced theextent of cataract formation Currently the only treatment for
cataracts is surgery It has been estimated that a 10-year delayin the onset and progression of a cataract could reduce theneed for cataract surgery by 50 [20]
Galactosemic and diabetic cataractogenesis in experi-mental animals and humans might be primarily due to theincreased formation of polyols from the reduced aldose sug-ars produced by aldose reductase and nicotinamide adeninedinucleotide phosphate (NADPH) [21] Polyols may accumu-late in the lens fiber cells causing increased cell hydrationmembrane stretching and dysfunction Galactose-fed ratsare a popular model used to examine the role of the ARpathway in diabetic complications In addition the galactose-induced cataracts develop within a week of feeding thismodel has been used extensively to study the morphologicaland biochemical changes during cataractogenesis Galactitolis a metabolite of galactose by AR that can accumulate in thelens Because the cellular lens membranes are impermeableto galactitol hyperosmotic cell swelling occurs causing lightscattering and diminished lens transparency [22ndash24] In thisstudy scopoletin inhibited the lenticular AR activity andthe accumulation of galactitol in galactose-fed rats This ARinhibition corresponded to the anticataractogenic activity
ARIs such as sorbinil prevented sugar cataractogenesisin experimental animals [25 26] Among the ARI onlysorbinil has reached advanced clinical trials in cataract
6 Evidence-Based Complementary and Alternative Medicine
NOR GAL SCO-10 SCO-50
2
1
0
4
3
GSH
(nm
olm
g le
ns w
eigh
t)
lowast
Figure 6 Glutathione (GSH) alteration GSH was measured in thelenses from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fed rats treated with scopoletin atconcentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data areexpressed as the means plusmn SE 119899 = 10
119875 lt 001 versus normalcontrol rats lowast119875 lt 001 versus vehicle-treated galactose-fed rats
prevention programs However due to the manifestationof skin rashes the trial was discontinued [27] Althoughseveral previous studies demonstrated that ARIs inhib-ited sugar cataracts by inhibiting AR no single agent hasbeen proven clinically effective during the treatment ofsugar cataracts Many naturally occurring compounds havestrong AR inhibitory activity in vitro [28] Recently scopo-letin demonstrated effective AR inhibitory activity [17 29]Coumarins are bicyclic phenolic compounds that harbor alactone moiety this functionality might participate in ARinhibition by hydrogen bondingwith the TYR48HIS110 andTRP111 residues inAR [30] Based on these results preventingsugar cataracts with scopoletin is partially related to ARinhibition and galactitol accumulation in the lens
In our previous study scopoletin had an excellent inhib-itory activity on AR displaying an IC
50value of 432 120583gmL
[17] Liu et al reported that the peak plasma scopoletinconcentrations (Cmax) were 051 068 and 149120583gmL andreached approximately 40 minutes after administering 50100 and 250mgkg scopoletin in rabbits respectively [31]The peak plasma concentration of scopoletin was 82 120583gmLafter administering 50mgkg scopoletin in rats [32] Becausescopoletin was highly lipophilic it absorbed effectively afteroral administration and spreadwidely to different tissues [33]Based on its previously reported pharmacokinetics and our invitro results we chose 10 and 50mgkg doses of scopoletin toevaluate its anti-AR activity in ratsWe found that the effectivedose of scopoletin was 50mgkd agreeing with the in vitroresult
The enzymatic distribution of AR activity was sup-ported by ARrsquos localized immunofluorescence In humanand rat lenses AR is primarily localized in the epithelialand superficial cortical fiber cells [34] In galactose-fed rats
the enhanced immunoreactive staining of AR was observedin the epithelial cells and the cortex region This stainingdecreased progressing from the superficial region to thedeeper cortex Scopoletin inhibited the extension of ARbeneath the epithelial region Therefore the decrease in ARactivity observed in the scopoletin-treated rats was caused bythe reduced amount of AR protein
Although the prevention of sugar-induced cataracto-genesis by ARIs appears to be caused by AR inhibitionthe osmotic hypothesis might not fully explain diabeticcataracts in human subjects because even during severehyperglycemia the examined tissues including the lens didnot have sorbitol levels gt2mM [35] Antioxidants effectivelyslow sugar cataract formation Butylated hydroxytolueneis a well-known synthetic phenolic antioxidant that slowscataract formation in rat lenses cultured under high-glucoseconditions although the sorbitol and fructose levels in thelenses remains elevated [36] ConsequentlyWolff andCrabbesuggested that the ARIs protected against sugar cataracts dueto the antioxidant nature of these inhibitors [37] Scopoletinhas demonstrated benefits for oxidative injury as an antioxi-dant [38 39] In this study scopoletin preserved the lenticularGSH content Therefore one of the possible mechanisms forscopoletin during sugar cataract development may involvethe protection of the lens cell membrane from oxidativedamage
In summary this study reveals that scopoletin mayexert beneficialprotective effects during the sugar cataractdevelopment Scopoletin inhibits the AR activity polyolaccumulation and reduction of the GSH levels We suggestthat the scopoletin may be particularly useful in treatingsugar cataracts
Acknowledgments
This research was supported by a Grant (K12040) from theKorea Institute of Oriental Medicine (KIOM)
References
[1] SNUghade S P Zodpey andVAKhanolkar ldquoRisk factors forcataract a case control studyrdquo Indian Journal of Ophthalmologyvol 46 no 4 pp 221ndash227 1998
[2] HMCheng andRGGonzalez ldquoThe effect of high glucose andoxidative stress on lensmetabolism aldose reductase and senilecataractogenesisrdquoMetabolism vol 35 no 4 pp 10ndash14 1986
[3] A Y Lee and S S Chung ldquoContributions of polyol pathway tooxidative stress in diabetic cataractrdquo FASEB Journal vol 13 no1 pp 23ndash30 1999
[4] J K Grover S Yadav and V Vats ldquoMedicinal plants of Indiawith anti-diabetic potentialrdquo Journal of Ethnopharmacology vol81 no 1 pp 81ndash100 2002
[5] J Lee D S Jang N H Kim Y M Lee J Kim and J SKim ldquoGalloyl glucoses from the seeds of Cornus officinalis withinhibitory activity against protein glycation aldose reductaseand cataractogenesis ex vivordquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 443ndash446 2011
[6] D S Jang Y M Lee I H Jeong and J S Kim ldquoConstituentsof the flowers of Platycodon grandiflorum with inhibitory
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
Evidence-Based Complementary and Alternative Medicine 3
NOR GAL SCO-10 SCO-50
(a)
Grade 0Grade 1Grade 2
Grade 3Grade 4
100
80
60
40
20
0
Inci
denc
e (
)
GAL SCO-10 SCO-50NOR
(b)
NOR GAL SCO-10 SCO-50
90
80
70
20
10
0
Lens
opa
city
()
60
50
40
30
lowast
lowast
(c)
Figure 2 Lens opacity (a) Representative images of the lenses in each group (b) Cataract grading The cataracts were assessed on a scale of0ndash4 (c) Analyses of lens opacitiesThe opacities were analyzed for each lens from the normal rats (NOR) the vehicle-treated galactose-fed rats(GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletinat concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control rats lowast119875 lt 001 versusvehicle-treated galactose-fed rats
28 Statistical Analysis The results were statistically evalu-ated using a one-way analysis of variance (ANOVA) followedby Tukeyrsquos multiple comparison test using GraphPad Prism50 software (GraphPad CA USA)
3 Results
31 Cataract Formation Analysis During the cataract anal-ysis all of the animals fed on the galactose diet developedmature cataracts after two weeks (70 were in grade III and30 were in grade IV Figures 2(a) and 2(b)) The scopoletintreatment delayed the onset of galactose-induced cataractsin a dose-dependent manner The highest dose of scopoletintreatment (50mgkgday) delayed the onset of cataracts (80were in grade I and 20 were in grade II Figures 2(a)and 2(b)) When analyzing the lensesrsquo opacification themean opaque area of the lenses was increased 8-fold in thegalactose-fed rats relative to the normal rats the opacity wassuppressed by the scopoletin treatment in a dose-dependentmanner (Figure 2(c) 119875 lt 001) Therefore scopoletin slowedthe development of galactose-induced cataracts
32 Lens Fiber Cell Degeneration In Figure 3 themembrane-labeled lens section illustrates the histological findings afterthe two weeks of study No significant alterations in thecuboidal epithelium or lens fiber were observed in thelenses of the control rats The lenses of galactose-fed ratswere swollen degenerated vacuolated and liquefied withdegenerated lens fibers Although the lenticular damage wassevere no corneal or retinal damage was observed Howeverthis histological change in the lens fibers of galactose-fed ratswas prevented in a dose-dependent manner after scopoletintreatment
33 Polyol Pathway in Lens The galactose-fed rats werekilled after two weeks some rats were progressing towardgrade 3 cataracts and extensive protection by scopoletin wasobserved AR is a key enzyme in the polyol pathway its activ-ity was significantly elevated in the galactose-fed rats TheAR activity in the lenses from the scopoletin-treated animalswas decreased (Figure 4(a)) agreeing with our observationsduring our in vitro studies [17] In addition the galactitollevels in the galactose-fed rats increased relative to the control
4 Evidence-Based Complementary and Alternative Medicine
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Figure 3 Lens fiber changes The lens sections from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fedrats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletin at concentration 50mgkg(SCO-50) were labeled with rhodamine-conjugated wheat germ agglutinin Fiber cell liquefaction swelling and membrane rupture wereobserved in galactosemic cataractous lens
NOR GAL SCO-10 SCO-50
7
6
5
4
3
2
1
0
AR
activ
ity (U
mg
lens
wei
ght)
lowast
(a)
NOR GAL SCO-10 SCO-50
75
50
25
0
Gal
actit
ol (n
mol
mg
lens
wei
ght)
lowast
(b)
Figure 4 Polyol pathway (a) Aldose reductase (AR) activity (b) galactitol levels in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control ratslowast
119875 lt 001 versus vehicle-treated galactose-fed rats
Evidence-Based Complementary and Alternative Medicine 5
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Negative control
(e)
Figure 5 Immunofluorescence stained AR Representative immunostained AR in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) AR was strongly immunoreactive in the cytoplasm of the lens epithelial cells and lenscortical fibers The negative control section was incubated with nonimmune mouse IgG and remained unstained
rats (Figure 4(b)) as expected because the polyol pathwaywasactivated However administering scopoletin resulted in lessgalactose-induced lenticular galactitol accumulation
34 AR Protein Expression in Lens In vehicle-treatedgalactose-fed rats immunoreactive straining for ARincreased in the cytoplasm of lens epithelial cells andextended into the deeper cortical fibers However thescopoletin treatment prevented AR expression in the lensepithelial cells and inhibited the extension of AR beneath theepithelial region (Figure 5)
35 GSH Levels in Lens The GSH status after treatmentindicated that the rats fed with galactose displayed lowerGSH levels in their lenses relative to the control The scopo-letin treatment given with the dietary galactose preventeddecreases in GSH levels in the lenses (Figure 6)
4 Discussion
In this study we investigated the protective effects ofscopoletin against cataractogenesis in galactose-fed rats Thescopoletin treatment delayed the progression and reduced theextent of cataract formation Currently the only treatment for
cataracts is surgery It has been estimated that a 10-year delayin the onset and progression of a cataract could reduce theneed for cataract surgery by 50 [20]
Galactosemic and diabetic cataractogenesis in experi-mental animals and humans might be primarily due to theincreased formation of polyols from the reduced aldose sug-ars produced by aldose reductase and nicotinamide adeninedinucleotide phosphate (NADPH) [21] Polyols may accumu-late in the lens fiber cells causing increased cell hydrationmembrane stretching and dysfunction Galactose-fed ratsare a popular model used to examine the role of the ARpathway in diabetic complications In addition the galactose-induced cataracts develop within a week of feeding thismodel has been used extensively to study the morphologicaland biochemical changes during cataractogenesis Galactitolis a metabolite of galactose by AR that can accumulate in thelens Because the cellular lens membranes are impermeableto galactitol hyperosmotic cell swelling occurs causing lightscattering and diminished lens transparency [22ndash24] In thisstudy scopoletin inhibited the lenticular AR activity andthe accumulation of galactitol in galactose-fed rats This ARinhibition corresponded to the anticataractogenic activity
ARIs such as sorbinil prevented sugar cataractogenesisin experimental animals [25 26] Among the ARI onlysorbinil has reached advanced clinical trials in cataract
6 Evidence-Based Complementary and Alternative Medicine
NOR GAL SCO-10 SCO-50
2
1
0
4
3
GSH
(nm
olm
g le
ns w
eigh
t)
lowast
Figure 6 Glutathione (GSH) alteration GSH was measured in thelenses from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fed rats treated with scopoletin atconcentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data areexpressed as the means plusmn SE 119899 = 10
119875 lt 001 versus normalcontrol rats lowast119875 lt 001 versus vehicle-treated galactose-fed rats
prevention programs However due to the manifestationof skin rashes the trial was discontinued [27] Althoughseveral previous studies demonstrated that ARIs inhib-ited sugar cataracts by inhibiting AR no single agent hasbeen proven clinically effective during the treatment ofsugar cataracts Many naturally occurring compounds havestrong AR inhibitory activity in vitro [28] Recently scopo-letin demonstrated effective AR inhibitory activity [17 29]Coumarins are bicyclic phenolic compounds that harbor alactone moiety this functionality might participate in ARinhibition by hydrogen bondingwith the TYR48HIS110 andTRP111 residues inAR [30] Based on these results preventingsugar cataracts with scopoletin is partially related to ARinhibition and galactitol accumulation in the lens
In our previous study scopoletin had an excellent inhib-itory activity on AR displaying an IC
50value of 432 120583gmL
[17] Liu et al reported that the peak plasma scopoletinconcentrations (Cmax) were 051 068 and 149120583gmL andreached approximately 40 minutes after administering 50100 and 250mgkg scopoletin in rabbits respectively [31]The peak plasma concentration of scopoletin was 82 120583gmLafter administering 50mgkg scopoletin in rats [32] Becausescopoletin was highly lipophilic it absorbed effectively afteroral administration and spreadwidely to different tissues [33]Based on its previously reported pharmacokinetics and our invitro results we chose 10 and 50mgkg doses of scopoletin toevaluate its anti-AR activity in ratsWe found that the effectivedose of scopoletin was 50mgkd agreeing with the in vitroresult
The enzymatic distribution of AR activity was sup-ported by ARrsquos localized immunofluorescence In humanand rat lenses AR is primarily localized in the epithelialand superficial cortical fiber cells [34] In galactose-fed rats
the enhanced immunoreactive staining of AR was observedin the epithelial cells and the cortex region This stainingdecreased progressing from the superficial region to thedeeper cortex Scopoletin inhibited the extension of ARbeneath the epithelial region Therefore the decrease in ARactivity observed in the scopoletin-treated rats was caused bythe reduced amount of AR protein
Although the prevention of sugar-induced cataracto-genesis by ARIs appears to be caused by AR inhibitionthe osmotic hypothesis might not fully explain diabeticcataracts in human subjects because even during severehyperglycemia the examined tissues including the lens didnot have sorbitol levels gt2mM [35] Antioxidants effectivelyslow sugar cataract formation Butylated hydroxytolueneis a well-known synthetic phenolic antioxidant that slowscataract formation in rat lenses cultured under high-glucoseconditions although the sorbitol and fructose levels in thelenses remains elevated [36] ConsequentlyWolff andCrabbesuggested that the ARIs protected against sugar cataracts dueto the antioxidant nature of these inhibitors [37] Scopoletinhas demonstrated benefits for oxidative injury as an antioxi-dant [38 39] In this study scopoletin preserved the lenticularGSH content Therefore one of the possible mechanisms forscopoletin during sugar cataract development may involvethe protection of the lens cell membrane from oxidativedamage
In summary this study reveals that scopoletin mayexert beneficialprotective effects during the sugar cataractdevelopment Scopoletin inhibits the AR activity polyolaccumulation and reduction of the GSH levels We suggestthat the scopoletin may be particularly useful in treatingsugar cataracts
Acknowledgments
This research was supported by a Grant (K12040) from theKorea Institute of Oriental Medicine (KIOM)
References
[1] SNUghade S P Zodpey andVAKhanolkar ldquoRisk factors forcataract a case control studyrdquo Indian Journal of Ophthalmologyvol 46 no 4 pp 221ndash227 1998
[2] HMCheng andRGGonzalez ldquoThe effect of high glucose andoxidative stress on lensmetabolism aldose reductase and senilecataractogenesisrdquoMetabolism vol 35 no 4 pp 10ndash14 1986
[3] A Y Lee and S S Chung ldquoContributions of polyol pathway tooxidative stress in diabetic cataractrdquo FASEB Journal vol 13 no1 pp 23ndash30 1999
[4] J K Grover S Yadav and V Vats ldquoMedicinal plants of Indiawith anti-diabetic potentialrdquo Journal of Ethnopharmacology vol81 no 1 pp 81ndash100 2002
[5] J Lee D S Jang N H Kim Y M Lee J Kim and J SKim ldquoGalloyl glucoses from the seeds of Cornus officinalis withinhibitory activity against protein glycation aldose reductaseand cataractogenesis ex vivordquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 443ndash446 2011
[6] D S Jang Y M Lee I H Jeong and J S Kim ldquoConstituentsof the flowers of Platycodon grandiflorum with inhibitory
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
4 Evidence-Based Complementary and Alternative Medicine
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Figure 3 Lens fiber changes The lens sections from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fedrats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treated with scopoletin at concentration 50mgkg(SCO-50) were labeled with rhodamine-conjugated wheat germ agglutinin Fiber cell liquefaction swelling and membrane rupture wereobserved in galactosemic cataractous lens
NOR GAL SCO-10 SCO-50
7
6
5
4
3
2
1
0
AR
activ
ity (U
mg
lens
wei
ght)
lowast
(a)
NOR GAL SCO-10 SCO-50
75
50
25
0
Gal
actit
ol (n
mol
mg
lens
wei
ght)
lowast
(b)
Figure 4 Polyol pathway (a) Aldose reductase (AR) activity (b) galactitol levels in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data are expressed as the means plusmn SE 119899 = 10 119875 lt 001 versus normal control ratslowast
119875 lt 001 versus vehicle-treated galactose-fed rats
Evidence-Based Complementary and Alternative Medicine 5
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Negative control
(e)
Figure 5 Immunofluorescence stained AR Representative immunostained AR in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) AR was strongly immunoreactive in the cytoplasm of the lens epithelial cells and lenscortical fibers The negative control section was incubated with nonimmune mouse IgG and remained unstained
rats (Figure 4(b)) as expected because the polyol pathwaywasactivated However administering scopoletin resulted in lessgalactose-induced lenticular galactitol accumulation
34 AR Protein Expression in Lens In vehicle-treatedgalactose-fed rats immunoreactive straining for ARincreased in the cytoplasm of lens epithelial cells andextended into the deeper cortical fibers However thescopoletin treatment prevented AR expression in the lensepithelial cells and inhibited the extension of AR beneath theepithelial region (Figure 5)
35 GSH Levels in Lens The GSH status after treatmentindicated that the rats fed with galactose displayed lowerGSH levels in their lenses relative to the control The scopo-letin treatment given with the dietary galactose preventeddecreases in GSH levels in the lenses (Figure 6)
4 Discussion
In this study we investigated the protective effects ofscopoletin against cataractogenesis in galactose-fed rats Thescopoletin treatment delayed the progression and reduced theextent of cataract formation Currently the only treatment for
cataracts is surgery It has been estimated that a 10-year delayin the onset and progression of a cataract could reduce theneed for cataract surgery by 50 [20]
Galactosemic and diabetic cataractogenesis in experi-mental animals and humans might be primarily due to theincreased formation of polyols from the reduced aldose sug-ars produced by aldose reductase and nicotinamide adeninedinucleotide phosphate (NADPH) [21] Polyols may accumu-late in the lens fiber cells causing increased cell hydrationmembrane stretching and dysfunction Galactose-fed ratsare a popular model used to examine the role of the ARpathway in diabetic complications In addition the galactose-induced cataracts develop within a week of feeding thismodel has been used extensively to study the morphologicaland biochemical changes during cataractogenesis Galactitolis a metabolite of galactose by AR that can accumulate in thelens Because the cellular lens membranes are impermeableto galactitol hyperosmotic cell swelling occurs causing lightscattering and diminished lens transparency [22ndash24] In thisstudy scopoletin inhibited the lenticular AR activity andthe accumulation of galactitol in galactose-fed rats This ARinhibition corresponded to the anticataractogenic activity
ARIs such as sorbinil prevented sugar cataractogenesisin experimental animals [25 26] Among the ARI onlysorbinil has reached advanced clinical trials in cataract
6 Evidence-Based Complementary and Alternative Medicine
NOR GAL SCO-10 SCO-50
2
1
0
4
3
GSH
(nm
olm
g le
ns w
eigh
t)
lowast
Figure 6 Glutathione (GSH) alteration GSH was measured in thelenses from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fed rats treated with scopoletin atconcentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data areexpressed as the means plusmn SE 119899 = 10
119875 lt 001 versus normalcontrol rats lowast119875 lt 001 versus vehicle-treated galactose-fed rats
prevention programs However due to the manifestationof skin rashes the trial was discontinued [27] Althoughseveral previous studies demonstrated that ARIs inhib-ited sugar cataracts by inhibiting AR no single agent hasbeen proven clinically effective during the treatment ofsugar cataracts Many naturally occurring compounds havestrong AR inhibitory activity in vitro [28] Recently scopo-letin demonstrated effective AR inhibitory activity [17 29]Coumarins are bicyclic phenolic compounds that harbor alactone moiety this functionality might participate in ARinhibition by hydrogen bondingwith the TYR48HIS110 andTRP111 residues inAR [30] Based on these results preventingsugar cataracts with scopoletin is partially related to ARinhibition and galactitol accumulation in the lens
In our previous study scopoletin had an excellent inhib-itory activity on AR displaying an IC
50value of 432 120583gmL
[17] Liu et al reported that the peak plasma scopoletinconcentrations (Cmax) were 051 068 and 149120583gmL andreached approximately 40 minutes after administering 50100 and 250mgkg scopoletin in rabbits respectively [31]The peak plasma concentration of scopoletin was 82 120583gmLafter administering 50mgkg scopoletin in rats [32] Becausescopoletin was highly lipophilic it absorbed effectively afteroral administration and spreadwidely to different tissues [33]Based on its previously reported pharmacokinetics and our invitro results we chose 10 and 50mgkg doses of scopoletin toevaluate its anti-AR activity in ratsWe found that the effectivedose of scopoletin was 50mgkd agreeing with the in vitroresult
The enzymatic distribution of AR activity was sup-ported by ARrsquos localized immunofluorescence In humanand rat lenses AR is primarily localized in the epithelialand superficial cortical fiber cells [34] In galactose-fed rats
the enhanced immunoreactive staining of AR was observedin the epithelial cells and the cortex region This stainingdecreased progressing from the superficial region to thedeeper cortex Scopoletin inhibited the extension of ARbeneath the epithelial region Therefore the decrease in ARactivity observed in the scopoletin-treated rats was caused bythe reduced amount of AR protein
Although the prevention of sugar-induced cataracto-genesis by ARIs appears to be caused by AR inhibitionthe osmotic hypothesis might not fully explain diabeticcataracts in human subjects because even during severehyperglycemia the examined tissues including the lens didnot have sorbitol levels gt2mM [35] Antioxidants effectivelyslow sugar cataract formation Butylated hydroxytolueneis a well-known synthetic phenolic antioxidant that slowscataract formation in rat lenses cultured under high-glucoseconditions although the sorbitol and fructose levels in thelenses remains elevated [36] ConsequentlyWolff andCrabbesuggested that the ARIs protected against sugar cataracts dueto the antioxidant nature of these inhibitors [37] Scopoletinhas demonstrated benefits for oxidative injury as an antioxi-dant [38 39] In this study scopoletin preserved the lenticularGSH content Therefore one of the possible mechanisms forscopoletin during sugar cataract development may involvethe protection of the lens cell membrane from oxidativedamage
In summary this study reveals that scopoletin mayexert beneficialprotective effects during the sugar cataractdevelopment Scopoletin inhibits the AR activity polyolaccumulation and reduction of the GSH levels We suggestthat the scopoletin may be particularly useful in treatingsugar cataracts
Acknowledgments
This research was supported by a Grant (K12040) from theKorea Institute of Oriental Medicine (KIOM)
References
[1] SNUghade S P Zodpey andVAKhanolkar ldquoRisk factors forcataract a case control studyrdquo Indian Journal of Ophthalmologyvol 46 no 4 pp 221ndash227 1998
[2] HMCheng andRGGonzalez ldquoThe effect of high glucose andoxidative stress on lensmetabolism aldose reductase and senilecataractogenesisrdquoMetabolism vol 35 no 4 pp 10ndash14 1986
[3] A Y Lee and S S Chung ldquoContributions of polyol pathway tooxidative stress in diabetic cataractrdquo FASEB Journal vol 13 no1 pp 23ndash30 1999
[4] J K Grover S Yadav and V Vats ldquoMedicinal plants of Indiawith anti-diabetic potentialrdquo Journal of Ethnopharmacology vol81 no 1 pp 81ndash100 2002
[5] J Lee D S Jang N H Kim Y M Lee J Kim and J SKim ldquoGalloyl glucoses from the seeds of Cornus officinalis withinhibitory activity against protein glycation aldose reductaseand cataractogenesis ex vivordquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 443ndash446 2011
[6] D S Jang Y M Lee I H Jeong and J S Kim ldquoConstituentsof the flowers of Platycodon grandiflorum with inhibitory
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
Evidence-Based Complementary and Alternative Medicine 5
NOR
(a)
GAL
(b)
SCO-10
(c)
SCO-50
(d)
Negative control
(e)
Figure 5 Immunofluorescence stained AR Representative immunostained AR in lenses from the normal rats (NOR) the vehicle-treatedgalactose-fed rats (GAL) the galactose-fed rats treated with scopoletin at concentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) AR was strongly immunoreactive in the cytoplasm of the lens epithelial cells and lenscortical fibers The negative control section was incubated with nonimmune mouse IgG and remained unstained
rats (Figure 4(b)) as expected because the polyol pathwaywasactivated However administering scopoletin resulted in lessgalactose-induced lenticular galactitol accumulation
34 AR Protein Expression in Lens In vehicle-treatedgalactose-fed rats immunoreactive straining for ARincreased in the cytoplasm of lens epithelial cells andextended into the deeper cortical fibers However thescopoletin treatment prevented AR expression in the lensepithelial cells and inhibited the extension of AR beneath theepithelial region (Figure 5)
35 GSH Levels in Lens The GSH status after treatmentindicated that the rats fed with galactose displayed lowerGSH levels in their lenses relative to the control The scopo-letin treatment given with the dietary galactose preventeddecreases in GSH levels in the lenses (Figure 6)
4 Discussion
In this study we investigated the protective effects ofscopoletin against cataractogenesis in galactose-fed rats Thescopoletin treatment delayed the progression and reduced theextent of cataract formation Currently the only treatment for
cataracts is surgery It has been estimated that a 10-year delayin the onset and progression of a cataract could reduce theneed for cataract surgery by 50 [20]
Galactosemic and diabetic cataractogenesis in experi-mental animals and humans might be primarily due to theincreased formation of polyols from the reduced aldose sug-ars produced by aldose reductase and nicotinamide adeninedinucleotide phosphate (NADPH) [21] Polyols may accumu-late in the lens fiber cells causing increased cell hydrationmembrane stretching and dysfunction Galactose-fed ratsare a popular model used to examine the role of the ARpathway in diabetic complications In addition the galactose-induced cataracts develop within a week of feeding thismodel has been used extensively to study the morphologicaland biochemical changes during cataractogenesis Galactitolis a metabolite of galactose by AR that can accumulate in thelens Because the cellular lens membranes are impermeableto galactitol hyperosmotic cell swelling occurs causing lightscattering and diminished lens transparency [22ndash24] In thisstudy scopoletin inhibited the lenticular AR activity andthe accumulation of galactitol in galactose-fed rats This ARinhibition corresponded to the anticataractogenic activity
ARIs such as sorbinil prevented sugar cataractogenesisin experimental animals [25 26] Among the ARI onlysorbinil has reached advanced clinical trials in cataract
6 Evidence-Based Complementary and Alternative Medicine
NOR GAL SCO-10 SCO-50
2
1
0
4
3
GSH
(nm
olm
g le
ns w
eigh
t)
lowast
Figure 6 Glutathione (GSH) alteration GSH was measured in thelenses from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fed rats treated with scopoletin atconcentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data areexpressed as the means plusmn SE 119899 = 10
119875 lt 001 versus normalcontrol rats lowast119875 lt 001 versus vehicle-treated galactose-fed rats
prevention programs However due to the manifestationof skin rashes the trial was discontinued [27] Althoughseveral previous studies demonstrated that ARIs inhib-ited sugar cataracts by inhibiting AR no single agent hasbeen proven clinically effective during the treatment ofsugar cataracts Many naturally occurring compounds havestrong AR inhibitory activity in vitro [28] Recently scopo-letin demonstrated effective AR inhibitory activity [17 29]Coumarins are bicyclic phenolic compounds that harbor alactone moiety this functionality might participate in ARinhibition by hydrogen bondingwith the TYR48HIS110 andTRP111 residues inAR [30] Based on these results preventingsugar cataracts with scopoletin is partially related to ARinhibition and galactitol accumulation in the lens
In our previous study scopoletin had an excellent inhib-itory activity on AR displaying an IC
50value of 432 120583gmL
[17] Liu et al reported that the peak plasma scopoletinconcentrations (Cmax) were 051 068 and 149120583gmL andreached approximately 40 minutes after administering 50100 and 250mgkg scopoletin in rabbits respectively [31]The peak plasma concentration of scopoletin was 82 120583gmLafter administering 50mgkg scopoletin in rats [32] Becausescopoletin was highly lipophilic it absorbed effectively afteroral administration and spreadwidely to different tissues [33]Based on its previously reported pharmacokinetics and our invitro results we chose 10 and 50mgkg doses of scopoletin toevaluate its anti-AR activity in ratsWe found that the effectivedose of scopoletin was 50mgkd agreeing with the in vitroresult
The enzymatic distribution of AR activity was sup-ported by ARrsquos localized immunofluorescence In humanand rat lenses AR is primarily localized in the epithelialand superficial cortical fiber cells [34] In galactose-fed rats
the enhanced immunoreactive staining of AR was observedin the epithelial cells and the cortex region This stainingdecreased progressing from the superficial region to thedeeper cortex Scopoletin inhibited the extension of ARbeneath the epithelial region Therefore the decrease in ARactivity observed in the scopoletin-treated rats was caused bythe reduced amount of AR protein
Although the prevention of sugar-induced cataracto-genesis by ARIs appears to be caused by AR inhibitionthe osmotic hypothesis might not fully explain diabeticcataracts in human subjects because even during severehyperglycemia the examined tissues including the lens didnot have sorbitol levels gt2mM [35] Antioxidants effectivelyslow sugar cataract formation Butylated hydroxytolueneis a well-known synthetic phenolic antioxidant that slowscataract formation in rat lenses cultured under high-glucoseconditions although the sorbitol and fructose levels in thelenses remains elevated [36] ConsequentlyWolff andCrabbesuggested that the ARIs protected against sugar cataracts dueto the antioxidant nature of these inhibitors [37] Scopoletinhas demonstrated benefits for oxidative injury as an antioxi-dant [38 39] In this study scopoletin preserved the lenticularGSH content Therefore one of the possible mechanisms forscopoletin during sugar cataract development may involvethe protection of the lens cell membrane from oxidativedamage
In summary this study reveals that scopoletin mayexert beneficialprotective effects during the sugar cataractdevelopment Scopoletin inhibits the AR activity polyolaccumulation and reduction of the GSH levels We suggestthat the scopoletin may be particularly useful in treatingsugar cataracts
Acknowledgments
This research was supported by a Grant (K12040) from theKorea Institute of Oriental Medicine (KIOM)
References
[1] SNUghade S P Zodpey andVAKhanolkar ldquoRisk factors forcataract a case control studyrdquo Indian Journal of Ophthalmologyvol 46 no 4 pp 221ndash227 1998
[2] HMCheng andRGGonzalez ldquoThe effect of high glucose andoxidative stress on lensmetabolism aldose reductase and senilecataractogenesisrdquoMetabolism vol 35 no 4 pp 10ndash14 1986
[3] A Y Lee and S S Chung ldquoContributions of polyol pathway tooxidative stress in diabetic cataractrdquo FASEB Journal vol 13 no1 pp 23ndash30 1999
[4] J K Grover S Yadav and V Vats ldquoMedicinal plants of Indiawith anti-diabetic potentialrdquo Journal of Ethnopharmacology vol81 no 1 pp 81ndash100 2002
[5] J Lee D S Jang N H Kim Y M Lee J Kim and J SKim ldquoGalloyl glucoses from the seeds of Cornus officinalis withinhibitory activity against protein glycation aldose reductaseand cataractogenesis ex vivordquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 443ndash446 2011
[6] D S Jang Y M Lee I H Jeong and J S Kim ldquoConstituentsof the flowers of Platycodon grandiflorum with inhibitory
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
6 Evidence-Based Complementary and Alternative Medicine
NOR GAL SCO-10 SCO-50
2
1
0
4
3
GSH
(nm
olm
g le
ns w
eigh
t)
lowast
Figure 6 Glutathione (GSH) alteration GSH was measured in thelenses from the normal rats (NOR) the vehicle-treated galactose-fed rats (GAL) the galactose-fed rats treated with scopoletin atconcentration 10mgkg (SCO-10) and the galactose-fed rats treatedwith scopoletin at concentration 50mgkg (SCO-50) All data areexpressed as the means plusmn SE 119899 = 10
119875 lt 001 versus normalcontrol rats lowast119875 lt 001 versus vehicle-treated galactose-fed rats
prevention programs However due to the manifestationof skin rashes the trial was discontinued [27] Althoughseveral previous studies demonstrated that ARIs inhib-ited sugar cataracts by inhibiting AR no single agent hasbeen proven clinically effective during the treatment ofsugar cataracts Many naturally occurring compounds havestrong AR inhibitory activity in vitro [28] Recently scopo-letin demonstrated effective AR inhibitory activity [17 29]Coumarins are bicyclic phenolic compounds that harbor alactone moiety this functionality might participate in ARinhibition by hydrogen bondingwith the TYR48HIS110 andTRP111 residues inAR [30] Based on these results preventingsugar cataracts with scopoletin is partially related to ARinhibition and galactitol accumulation in the lens
In our previous study scopoletin had an excellent inhib-itory activity on AR displaying an IC
50value of 432 120583gmL
[17] Liu et al reported that the peak plasma scopoletinconcentrations (Cmax) were 051 068 and 149120583gmL andreached approximately 40 minutes after administering 50100 and 250mgkg scopoletin in rabbits respectively [31]The peak plasma concentration of scopoletin was 82 120583gmLafter administering 50mgkg scopoletin in rats [32] Becausescopoletin was highly lipophilic it absorbed effectively afteroral administration and spreadwidely to different tissues [33]Based on its previously reported pharmacokinetics and our invitro results we chose 10 and 50mgkg doses of scopoletin toevaluate its anti-AR activity in ratsWe found that the effectivedose of scopoletin was 50mgkd agreeing with the in vitroresult
The enzymatic distribution of AR activity was sup-ported by ARrsquos localized immunofluorescence In humanand rat lenses AR is primarily localized in the epithelialand superficial cortical fiber cells [34] In galactose-fed rats
the enhanced immunoreactive staining of AR was observedin the epithelial cells and the cortex region This stainingdecreased progressing from the superficial region to thedeeper cortex Scopoletin inhibited the extension of ARbeneath the epithelial region Therefore the decrease in ARactivity observed in the scopoletin-treated rats was caused bythe reduced amount of AR protein
Although the prevention of sugar-induced cataracto-genesis by ARIs appears to be caused by AR inhibitionthe osmotic hypothesis might not fully explain diabeticcataracts in human subjects because even during severehyperglycemia the examined tissues including the lens didnot have sorbitol levels gt2mM [35] Antioxidants effectivelyslow sugar cataract formation Butylated hydroxytolueneis a well-known synthetic phenolic antioxidant that slowscataract formation in rat lenses cultured under high-glucoseconditions although the sorbitol and fructose levels in thelenses remains elevated [36] ConsequentlyWolff andCrabbesuggested that the ARIs protected against sugar cataracts dueto the antioxidant nature of these inhibitors [37] Scopoletinhas demonstrated benefits for oxidative injury as an antioxi-dant [38 39] In this study scopoletin preserved the lenticularGSH content Therefore one of the possible mechanisms forscopoletin during sugar cataract development may involvethe protection of the lens cell membrane from oxidativedamage
In summary this study reveals that scopoletin mayexert beneficialprotective effects during the sugar cataractdevelopment Scopoletin inhibits the AR activity polyolaccumulation and reduction of the GSH levels We suggestthat the scopoletin may be particularly useful in treatingsugar cataracts
Acknowledgments
This research was supported by a Grant (K12040) from theKorea Institute of Oriental Medicine (KIOM)
References
[1] SNUghade S P Zodpey andVAKhanolkar ldquoRisk factors forcataract a case control studyrdquo Indian Journal of Ophthalmologyvol 46 no 4 pp 221ndash227 1998
[2] HMCheng andRGGonzalez ldquoThe effect of high glucose andoxidative stress on lensmetabolism aldose reductase and senilecataractogenesisrdquoMetabolism vol 35 no 4 pp 10ndash14 1986
[3] A Y Lee and S S Chung ldquoContributions of polyol pathway tooxidative stress in diabetic cataractrdquo FASEB Journal vol 13 no1 pp 23ndash30 1999
[4] J K Grover S Yadav and V Vats ldquoMedicinal plants of Indiawith anti-diabetic potentialrdquo Journal of Ethnopharmacology vol81 no 1 pp 81ndash100 2002
[5] J Lee D S Jang N H Kim Y M Lee J Kim and J SKim ldquoGalloyl glucoses from the seeds of Cornus officinalis withinhibitory activity against protein glycation aldose reductaseand cataractogenesis ex vivordquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 443ndash446 2011
[6] D S Jang Y M Lee I H Jeong and J S Kim ldquoConstituentsof the flowers of Platycodon grandiflorum with inhibitory
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
Evidence-Based Complementary and Alternative Medicine 7
activity on advanced glycation end products and rat lens aldosereductase in vitrordquo Archives of Pharmacal Research vol 33 no6 pp 875ndash880 2010
[7] A Kato Y Higuchi H Goto et al ldquoInhibitory effects of Zin-giber officinale roscoe derived components on aldose reductaseactivity in vitro and in vivordquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6640ndash6644 2006
[8] H Matsuda H Cai M Kuro H Tosa and M Inuma ldquoStudyon anti-cataract drugs from natural sources II Effects ofBuddlejae Flos on in vitro aldose reductase activityrdquo Biologicaland Pharmaceutical Bulletin vol 18 no 3 pp 463ndash466 1995
[9] M Kubo H Matsuda K Tokuoka Y Kobayashi S Ma and TTanaka ldquoStudies of anti-cataract drugs from natural sources IEffects of a methanolic extract and the alkaloidal componentsfrom Corydalis tuber on in vitro aldose reductase activityrdquoBiological and Pharmaceutical Bulletin vol 17 no 3 pp 458ndash459 1994
[10] MMiyazawa H Kasahara andH Kameoka ldquoPhenolic lignansfrom flower buds of Magnolia fargesiirdquo Phytochemistry vol 31no 10 pp 3666ndash3668 1992
[11] N-H Nam Y Kim Y-J You D-H Hong H-M Kim andB-Z Ahn ldquoPreliminary structure-antiangiogenic activity rela-tionships of 4-senecioyloxymethyl-67-dimethoxycoumarinrdquoBioorganic and Medicinal Chemistry Letters vol 12 no 17 pp2345ndash2348 2002
[12] S Lee K Sivakumar W Shin F Xie and Q Wang ldquoSynthesisand anti-angiogenesis activity of coumarin derivativesrdquo Bioor-ganic and Medicinal Chemistry Letters vol 16 no 17 pp 4596ndash4599 2006
[13] P-D Moon B-H Lee H-J Jeong et al ldquoUse of scopoletinto inhibit the production of inflammatory cytokines throughinhibition of the I120581BNF-120581B signal cascade in the human mastcell line HMC-1rdquo European Journal of Pharmacology vol 555no 2-3 pp 218ndash225 2007
[14] Z Ding Y Dai and Z Wang ldquoHypouricemic action of scopo-letin arising from xanthine oxidase inhibition and uricosuricactivityrdquo Planta Medica vol 71 no 2 pp 183ndash185 2005
[15] C-Y Shaw C-H Chen C-C Hsu C-C Chen and Y-C Tsai ldquoAntioxidant properties of scopoletin isolated fromSinomonium acutumrdquo Phytotherapy Research vol 17 no 7 pp823ndash825 2003
[16] S Panda and A Kar ldquoEvaluation of the antithyroid antioxida-tive and antihyperglycemic activity of scopoletin from Aeglemarmelos leaves in hyperthyroid ratsrdquo Phytotherapy Researchvol 20 no 12 pp 1103ndash1105 2006
[17] J Lee N H Kim J W Nam et al ldquoScopoletin from the flowerbuds of Magnolia fargesii inhibits protein glycation aldosereductase and cataractogenesis ex Vivordquo Archives of PharmacalResearch vol 33 no 9 pp 1317ndash1323 2010
[18] A C Woollard Z A Bascal G R Armstrong and S P WolffldquoAbnormal redox status without increased lipid peroxidation insugar cataractrdquo Diabetes vol 39 no 11 pp 1347ndash1352 1990
[19] M F Lou and J E Dickerson Jr ldquoProtein-thiol mixed disulfidesin human lensrdquo Experimental Eye Research vol 55 no 6 pp889ndash896 1992
[20] L B Ellwein and C Kupfer ldquoStrategic issues in preventingcataract blindness in developing countriesrdquoBulletin of theWorldHealth Organization vol 73 no 5 pp 681ndash690 1995
[21] J H Kinoshita ldquoMechanisms initiating cataract formationProctor lecturerdquo Investigative Ophthalmology vol 13 no 10 pp713ndash724 1974
[22] M F Lou J E Dickerson Jr R Garadi and B M York JrldquoGlutathione depletion in the lens of galactosemic and diabeticratsrdquoExperimental Eye Research vol 46 no 4 pp 517ndash530 1988
[23] V N Reddy D Schwass B Chakrapani and C P LimldquoBiochemical changes associated with the development andreversal of galactose cataractsrdquo Experimental Eye Research vol23 no 5 pp 483ndash493 1976
[24] I Miwa M Kanbara H Wakazono and J Okuda ldquoAnalysisof sorbitol galactitol and myo-inositol in lens and sciaticnerve by high-performance liquid chromatographyrdquo AnalyticalBiochemistry vol 173 no 1 pp 39ndash44 1988
[25] D Dvornik N Simard Duquesne and M Krami ldquoPolyolaccumulation in galactosemic and diabetic rats control by analdose reductase inhibitorrdquo Science vol 182 no 4117 pp 1146ndash1148 1973
[26] S Lightman ldquoDoes aldose reductase have a role in the develop-ment of the ocular complications of diabetesrdquo Eye vol 7 no 2pp 238ndash241 1993
[27] S K Gupta V K Selvan S S Agrawal and R SaxenaldquoAdvances in pharmacological strategies for the prevention ofcataract developmentrdquo Indian Journal of Ophthalmology vol 57no 3 pp 175ndash183 2009
[28] D R Tomlinson E J Stevens and L T Diemel ldquoAldosereductase inhibitors and their potential for the treatment ofdiabetic complicationsrdquoTrends in Pharmacological Sciences vol15 no 8 pp 293ndash297 1994
[29] H A Jung M D N Islam Y S Kwon et al ldquoExtraction andidentification of three major aldose reductase inhibitors fromArtemisia montanardquo Food and Chemical Toxicology vol 49 no2 pp 376ndash384 2011
[30] ZWang B Ling R Zhang and Y Liu ldquoDocking andmoleculardynamics study on the inhibitory activity of coumarins onaldose reductaserdquo Journal of Physical Chemistry B vol 112 no32 pp 10033ndash10040 2008
[31] S L Liu M T Hsieh and C H Liu ldquoPlasma scopoletinlevels after a single dose oral administration in rabbitsrdquo ChinesePharmaceutical Journal vol 52 no 4 pp 203ndash210 2000
[32] Y Xia Y Dai Q Wang and H Liang ldquoDetermination ofscopoletin in rat plasma by high performance liquid chromato-graphic method with UV detection and its application to apharmacokinetic studyrdquo Journal of Chromatography B vol 857no 2 pp 332ndash336 2007
[33] R J Yin X F Xiao Y Y Xu et al ldquoResearch information andreview on the leaves of Diospyros kaki L II Pharmacokineticsof major active compounds of Diospyros kaki Lrdquo Asian Journalof Pharmacogynamics and Pharmacokinetics vol 10 no 4 pp271ndash285 2010
[34] M J Lizak E F Secchi J W Lee et al ldquo3-FG as substratefor investigating flux through the polyol pathway in dog lensby 19F-NMR spectroscopyrdquo Investigative Ophthalmology andVisual Science vol 39 no 13 pp 2688ndash2695 1998
[35] D J Heaf and D J Galton ldquoSorbitol and other polyols in lensadipose tissue and urine in diabetes mellitusrdquo Clinica ChimicaActa vol 63 no 1 pp 41ndash47 1975
[36] S K Srivastava and N H Ansari ldquoPrevention of sugar-inducedcataractogenesis in rats by butylated hydroxytoluenerdquoDiabetesvol 37 no 11 pp 1505ndash1508 1988
[37] S P Wolff and M J C Crabbe ldquoLow apparent aldose reductaseactivity produced by monosaccharide autoxidationrdquo Biochemi-cal Journal vol 226 no 3 pp 625ndash630 1985
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
8 Evidence-Based Complementary and Alternative Medicine
[38] P M Abreu S Matthew T Gonzalez et al ldquoIsolation andidentification of antioxidants from Pedilanthus tithymaloidesrdquoJournal of Natural Medicines vol 62 no 1 pp 67ndash70 2008
[39] C Shaw C Chen C Hsu C Chen and Y Tsai ldquoAntioxidantproperties of scopoletin isolated from Sinomonium acutumrdquoPhytotherapy Research vol 17 no 7 pp 823ndash825 2003
![Index [researchonline.jcu.edu.au] · aldose reductase inhibitors 157 aldosterone antagonists 147, 57 ALLHAT ... aromatase inhibitors. use in polycystic ovarian syndrome 100 arterial](https://static.documents.pub/doc/80x56/5fd9b9529bcf935e482a7b5a/index-aldose-reductase-inhibitors-157-aldosterone-antagonists-147-57-allhat.jpg)
