Research Article Metabolic Effects of Mulberry Leaves:...
Transcript of Research Article Metabolic Effects of Mulberry Leaves:...
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 948627 10 pageshttpdxdoiorg1011552013948627
Research ArticleMetabolic Effects of Mulberry Leaves Exploring PotentialBenefits in Type 2 Diabetes and Hyperuricemia
A Hunyadi1 E Liktor-Busa1 Aacute Maacuterki2 A Martins34 N Jedlinszki1
T J Hsieh5 M Baacutethori1 J Hohmann1 and I Zupkoacute2
1 Institute of Pharmacognosy Faculty of Pharmacy University of Szeged Eotvos u 6 Szeged 6720 Hungary2Department of Pharmacodynamics and Biopharmacy Faculty of Pharmacy University of Szeged Eotvos u 6 Szeged 6720 Hungary3 Department of Medical Microbiology and Immunobiology Faculty of Medicine University of Szeged Dom ter 13Szeged 6720 Hungary
4Unidade de Parasitologia e Microbiologia Medica Instituto de Higiene e Medicina TropicalUniversidade Nova de Lisboa Rua da Junqueira 100 1349-008 Lisboa Portugal
5 Department of GenomeMedicine College ofMedicine KaohsiungMedical University Shih Chuan 1st Rd 100 Kaohsiung 807 Taiwan
Correspondence should be addressed to A Hunyadi hunyadiapharmu-szegedhu and I Zupko zupkopharmu-szegedhu
Received 24 July 2013 Accepted 18 October 2013
Academic Editor C S Cho
Copyright copy 2013 A Hunyadi 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
The leaves ofMorus alba L have a long history in Traditional ChineseMedicine and also became valued by the ethnopharmacologyof many other culturesThe worldwide known antidiabetic use of the drug has been suggested to arise from a complex combinationeffect of various constituents Moreover the drug is also a potential antihyperuricemic agent Considering that type 2 diabetesand hyperuricemia are vice-versa in each otherrsquos important risk factors the use of mulberry originated phytotherapeutics mightprovide an excellent option for the prevention andor treatment of both conditions Here we report a series of relevant in vitroand in vivo studies on the bioactivity of an extract of mulberry leaves and its fractions obtained by a stepwise gradient on silicagel In vivo antihyperglycemic and antihyperuricemic activity plasma antioxidant status as well as in vitro glucose consumptionby adipocytes in the presence or absence of insulin xanthine oxidase inhibition free radical scavenging activity and inhibitionof lipid peroxidation were tested Known bioactive constituents of M alba (chlorogenic acid rutin isoquercitrin and loliolide)were identified and quantified from the HPLC-DAD fingerprint chromatograms Iminosugar contents were investigated byMSMS 1-deoxynojirimycinwas quantified and amounts of 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin and fagominewereadditionally estimated
1 Introduction
Chronically elevated uric acid concentration in the serumhasimpact on human health at various levels it frequently leadsto the development of gout [1] and it is a relevant risk factorof several further chronic diseases For example it representsa significant risk for various cardiovascular and cerebrovas-cular diseases [2ndash4] and a tight connection between hyper-uricemia and diabetes has recently been revealed apparentlythese two diseases mutually increase each otherrsquos incidence[5] Hyperinsulinemia in type 2 diabetes can significantlyincrease reabsorption of uric acid in the proximal tubules
while its overproduction due to an increased activity ofxanthine-oxidase usually also takes place [5 6] On the otherhand high uric acid levels might predict the developmentof metabolic syndrome consequentially leading to diabetes[7 8] and can also increase severity of the already developeddisease by leading to a higher incidence of certain diabeticcomplications [9] The mechanism by which these metabolicstates are interconnected seems yet to be clarified althoughthere is evidence that hyperuricemia metabolic syndromeand type II diabetes share the same causal origin in whichinsulin resistance would play a key role [5] Anyhow devel-opment of novel therapeutic agents targeting both diabetes
2 Evidence-Based Complementary and Alternative Medicine
and hyperuricemia appears to be a highly relevant strategy forovercoming difficulties attributed to the current therapeuticapproaches Appropriately chosen phytotherapeutics [10 11]representing complex bioactivity profiles might serve asexcellent tools to fulfill this objective either as monotherapyor in combination with already existing approaches
Mulberry leaves are probably best known by their rolein the silk production but medicinal use of this drug alsodates back at least two thousand years it is alreadymentionedin the ldquoDivine Farmerrsquos Materia Medicardquo (pinyin ShennongBencao Jıng) written during the reign of the Han dynastyIn Traditional Chinese Medicine leaves of M alba possesssweet slightly bitter and slightly cold properties and theirprimary uses are described as ldquoto expel wind and heatfrom the lungs as well as to clear the liver and the eyesrdquo[12] Anti-diabetic use of mulberry leaves had also beenpopular moreover this indication became part of the localtraditional medicine wherever the tree has been naturalized[13ndash16] Based on this a large number of herbal preparations(including many food supplements) are worldwide availablefor diabetes treatment and easily accessible to everyone evenvia online shopping This activity of mulberry leaves hasbeen verified by a number of studies including several animalexperiments [14ndash16] and a few human trials as well [17 18]but to our knowledge the active constituents and their role inthe activity still remain to be fully described Nevertheless acomplex cocktail of various bioactive constituents is thoughtto be responsible for this activity [19] among which the roleof iminosugars [18] and certain phenolics mainly chlorogenicacid and rutin [16] might be the most significant
Furthermore several traditional Chinese preparationsutilize the branch of Morus alba for the treatment of goutarthritis and rheumatism [20] Various constituents of thedrugwere found to have significant antihyperuricemic poten-tial including mulberroside A a stilbene glycoside [21] anda number of flavonoids primarily morin [22 23]
Based on the above our objectives were to explore thepotential of Morus alba leaves as dual-target phytotherapeu-tics to prevent and treat both diabetes and hyperuricemiaand to investigate whether a simple chromatographic frac-tionation can lead to the enrichment of the main bioactiveconstituents valuable for both therapeutic targets of interest
2 Materials and Methods
21 Plant Material Chemicals and Reagents The leaves ofMorus alba were collected near Asotthalom (nearby SzegedHungary) in May 2007 and botanically identified by AHunyadi A voucher specimen (MA052007) was deposited inthe Institute of Pharmacognosy University of Szeged SzegedHungary All chemicals if otherwise not specified werepurchased from Sigma-Aldrich (Budapest Hungary) Rutin(2) and isoquercitrin (3) were purchased from ChromaDex(Irvine CA USA) and Extrasynthese (Genay France)respectively Loliolide (4) was previously isolated from thedry leaves ofMorus alba [19] and 1-Deoxynojirimicin (1-DN)was purchased fromWako Pure Chemical Industries (OsakaJapan) HPLC grade methanol was obtained from Fischer
Scientific ultrapure water was obtained by using a MilliporeDirect-Q UV3 equipment
22 Extraction and Chromatographic Fractionation 25 kgof the dried and ground plant material was extracted bypercolation with 30 L of 70 aqueous methanol and thesolvent was evaporated under vacuum at 50∘C to obtain67536 g dry extract (EX) 170 g of the drymaterial was furtherprocessed it was dissolved in 1000mL of water and extractedwith 10 times 500mL of n-butanol After solvent evaporationdry residue of the aqueous (FR-W) and organic phase (FR-B) was 784 and 8809 g respectively The butanol phase wasadsorbed onto triple amount (276 g) of silica (Kieselgel 60Merck Darmstadt Germany) and administered on the topof a previously prepared silica column of 1840 g A stepwisegradient of CH
2Cl2 CH2Cl2 EtOH (95 5 9 1 8 2 7 3
6 4 and 1 1) and EtOH was used and one single fractionper solvent was collected After the first fraction of 18 L eachfollowing was of 10 L volume After solvent evaporation dryresidues of the fractions were 1372 976 448 819 1052 993550 and 1087 g respectively Based on high similarities intheir TLC fingerprints the last three fractions were joinedhence finally six fractions referred to as FR1-6 were used forthe experiments discussed here
23 HPLC-DAD Analysis of Fractions FR1-6 Diode-arraydetected high performance liquid chromatography (HPLC-DAD) was performed on a gradient system of two JascoPU2080 pumps connected to a Jasco MD-2010 Plus diode-array detector (DAD) and equipped with a Jasco AS-2055Plus autosampler (Jasco Co Tokyo Japan) by usinga Zorbax Eclipse XDB-C8 (5 120583m 46 times 150mm) analyticalcolumn Samples were dissolved in 30 of aqueous MeOH at2mgmL and 30 120583L of each was injected Gradient elutionwas performed from 30 to 100 of aqueous MeOH in 13minutes kept at 100 for four more minutes and returned to30 at 171min Chromatograms were recorded for 24minand DAD data was collected from 200 to 650 nm Baselineswere corrected by subtracting the chromatogram of a single30 120583L injection of 30 MeOH
24 Quantitative Analysis of Compounds 1ndash4 Single-pointquantitative analysis was performed for the previously iden-tified major constituents in their respective fractions suchas chlorogenic acid (1) rutin (2) and isoquercitrin (3) inEX FR-B and FR5-6 and loliolide (4) in FR2 Calibrationcurves were taken by analysing dilutions of a 1mgmL stocksolution of the corresponding standard at concentrations of 105 04 03 02 01 and 005mgmL 1198772 values were 0997009990 09904 and 09978 for the calibration lines of 1ndash4respectively In case of FR5 and FR6 peaks of 2 and 3 werepartially overlapping deconvolution was performed by usingGaussian approximations with the Fityk 097 software (FreeSoftware Foundation Poland) and quantities were calculatedbased on the peak areas revealed this way
25 Determination of Iminosugars Iminosugar content wasqualitatively investigated by thin-layer chromatography
Evidence-Based Complementary and Alternative Medicine 3
(TLC) in each fraction by using DC-Alufolien Kieselgel60F254
plates (Merck Darmstadt Germany) and CH2Cl2
MeOH NH3(3 6 2 vvv) as solvent system For quan-
titative analysis an API 2000 MSMS spectrometer was usedwith a Shimadzu autosampler and an electrospray ionization(ESI) interface set in positive mode 1-DN 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin (Gal-DN) and fagom-ine contents were investigated in FR-W FR-B and FR4-6Injected sampleswerewashed into the spectrometerwith 50aqueous MeOH at a flow rate of 200 120583Lmin temperatureof the ion source was 300∘C Multiple reaction monitoring(MRM) was used with transitions of mz 164rarr 69 for 1-DNmz 326rarr 164 for GAL-DN andmz 148rarr 86 for fagomineaccording to literature data [24] Parameter optimizationand data acquisition and evaluation were performed byusing the Analyst 151 software Calibration line of 1-DN(1198772 = 10000) was obtained by means of six measurementpoints in triplicates
26 In Vitro Assay on Xanthine Oxidase (EC 11732)Inhibition The activity of the enzyme was calculated fromthe increase of uric acid concentration determined bymicroplate-based kinetic photometry [25] Briefly theabsorption of uric acid generated from xanthine (50120583M atstart-up) was followed at 120582 = 290 nm for 125 s (SpectrostarNano BMG Labtech Ortenberg Germany) The activityof xanthine oxidase was described as the slope of theabsorbance versus time curve and allopurinol was used aspositive control Stock solutions prepared with dimethyl-sulfoxide were used for the in vitro assays and any substantialeffect of the solvent was excluded All in vitro assays werecarried out in duplicates
27 Determination of Free Radical Scavenging Activity byDPPH Assay In Vitro The activity for scavenging 11-diphenyl-2-picrylhydrazyl (DPPH) radicals was tested foreach fraction as described earlier [26] Briefly differentamounts of the samples were added to 01mM DPPH dis-solved in ethanol The mixture was shaken and allowed tostand for 30min and then the absorbance of the solutionwas measured at 120582 = 517 nm trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid) a water-soluble ana-log of vitamin E was used as positive control
28 Determination of Lipid Peroxidation Inhibitory ActivityThe antioxidant properties of the fractions were additionallymeasured by means of inhibition of the autooxidation ofunsaturated fatty acids present in animal brain tissue [27]A lipid-rich fraction was prepared from the brains of maleSprague-Dawley rats (Charles River Laboratories BudapestHungary body mass 250ndash300 g) by homogenization andcentrifugation The fatty acids in such a fraction get spon-taneously oxidized during an incubation of 1 h at 37∘C andthis oxidation can be inhibited by antioxidants The oxidizedproducts were determined by colorimetry at 532 nm afterreaction with thiobarbituric acid All in vitro experimentswere carried out in duplicates and statistically evaluatedSigmoid curves were fitted to the results of both antioxidant
assays and IC50
values were calculated by using GraphPadPrism 4 (GraphPad Software San Diego CA USA)
29 Testing the Effect on the Glucose Consumption of Adipo-cytes Effect of each fractionwas tested on the in vitro glucoseconsumption of adipocytes as published before at concentra-tions of 200120583gmL [19] Briefly 3T3-L1 preadipocytes (5times105cells BCRC no 60159 Bioresource Collection and ResearchCenter Taiwan) were seeded and cultured in 10 CS DMEMcontaining 55mM D-glucose The cells were induced todifferentiate and at day 3 samples dissolved in DMSO wereadded to the cells at 200120583gmL either in presence or absenceof 032 120583M insulin 24 h changes in the glucose contents of thetesting media were recorded and compared to those beforethe addition of the samples
210 Animal Studies Animals were treated in accordancewith the European Communities Council Directives(86609ECC) and the Hungarian Act for the Protectionof Animals in Research (XXVIIItv32sect) Experimentsinvolving animal subjects were carried out with the approvalof the Hungarian Ethical Committee for Animal Research(registration no IV01758-22008) Male Sprague-Dawleyrats (Charles River Laboratories Budapest Hungary bodymass 180ndash200 g 8 animals in each group) were housed intemperature (20ndash23∘C) humidity (40ndash60) and light (12 hof light 12 h of dark) regulated rooms with tap water androdent food (Bioplan Isaszeg Hungary) intake availablead libitum The tested extracts were orally administered in025 methylcellulose containing 2 Cremophor EL usinga dosing volume of 5mLkg Three doses of all extracts (3060 and 120mgkg) were selected for daily treatment for 3consecutive days and each treatment was performed after16 hours of fasting One-way analysis of variance (ANOVA)with Dunnettrsquos multiple comparison test by GraphPad Prism4 was used for statistical evaluation of all in vivo experiments
211 Determination of Antihyperuricemic Antihyperglycemicand Antioxidant Properties of the Fractions In Vivo In orderto minimize the number of animals antihyperuricemicantihyperglycemic and antioxidant properties effects weredetermined from the same groups of rats Experimentalhyperuricemiamodel induced by uricase inhibitor potassiumoxonate was utilized to assess the antihyperuricemic proper-ties of the tested fractions [28] 250mgkg potassium oxonatewas suspended in 025 methylcellulose and administeredintraperitoneally at the time of the third oral treatment50mgkg of allopurinol a clinically used antigout drug wasorally administered as a positive control One hour laterrats were orally treated with 25 gkg starch suspension ina dosing volume of 5mLkg in order to characterize theantihyperglycemic effects of the fractions [25] After anotherone hour venous blood samples were obtained from the tailand the plasma glucose concentrations were determined bya commercially available kit (Reanal Budapest Hungary)based on the glucose oxidase-peroxidasemethod [29] Imme-diately after blood sampling rats were anesthetized in 4isoflurane and additional blood samples were collected by
4 Evidence-Based Complementary and Alternative Medicine
cardiac puncture for determination of uric acid concentrationand total antioxidant capacity Since inhalational anestheticsare reported to substantially elevate the blood glucose levelseparate blood samplings were needed for determination ofall the planned parameters [30] Serum samples were pre-pared by centrifugation and stored at ndash70∘Cuntil the analysesAll determinations by colorimetric uric acid (BioAassay Sys-tems Hayward CA USA) and antioxidant (Sigma-AldrichBudapest Hungary) assay kits were performed in duplicatesaccording to themanufacturersrsquo suggestions Separate groupsorally treated with glibenclamide and trolox (10mgkg forboth) were included as reference for the antihyperglycemicand antioxidant assays respectively
3 Results and Discussion
31 Chemical Composition of the Fractions Obtained Thefractions obtained by solvent-solvent extraction and a roughchromatographic separation of the organic phase on silicarepresented fundamentally different chemical compositions(Figure 1) As expected highly water soluble hydrophiliccompounds (including iminosugars of the plant see below)remained in the water phase (Figure 1(b)) while most ofthe still polar chlorogenic acid (1) could already be detectedin the organic phase (Figure 1(c)) along with vast major-ity of the supposedly ldquodrug-likerdquo secondary metabolites ofmulberry leaves These would be the compounds with thehighest probability for therapeutic value mainly the too lowlog 119875 value typically results in poor penetration throughmembranes (iminosugars a constituent group of possibleexception to this are discussed below) As seen from theDAD fingerprints FR1 (Figure 1(d)) and FR2 (Figure 1(e))mostly contained compounds with lower wavelengths of UVabsorbance maxima probably including several terpenoidsandor phenylpropanes [19] The dominant peak of FR2was detected and quantified as loliolide (4) (033) amonoterpene lactone we have recently reported fromM albaleaves [19] Of all fractions FR3 (Figure 1(f)) contained thesmallest amount of UV absorbing material its main con-stituents remained unidentified Based on its UV spectrumand retention time chief constituent of FR4 (Figure 1(g)) issuggested to be a flavone aglycone (342ndash350 expressed inequivalents of isoquercitrin (3) or rutin (2)) while 3 (242)was also present in this fraction FR5 (Figure 1(h)) containedthe majority of 3 (979) along with a smaller amountof 2 (377) while the previously mentioned unidentifiedflavonoid could still be detected FR6 (Figure 1(i)) the mostpolar fraction obtained from the column chromatographycontained 1 (899) 2 (1041) and 3 (564) as major UVactive constituents
Both the extract and the fractions contained very smallamounts of iminosugars By means of MSMS 1-DN con-tents were found as low as 02694permil(EX) 03007permil (FR-W)00742permil (FR-B) sim0016permil (FR4 around detection limit)00558permil (FR5) and 02770permil(FR6) On the other handslope of the calibration lines of GAL-DN and fagomine isaround 13 and 6 times higher respectively as compared tothat of 1-DN based on a recent publication using the sameMRM transitions with ESI-MSMS [24] This allowed only
a rough estimation on the quantity of these compounds byusing our calibration obtained for 1-DN Amounts of GAL-DN were about 002permil (EX) 003permil (FR-W) 0003permil (FR-B) and 0009permil(FR6) and trace amounts of this com-pound were detected in FR4 and FR5 Fagomine contentscould be estimated as around 003permil (EX) 004permil (FR-W)001permil (FR-B) 001permil (FR5) and 003permil (FR6) with a traceamount of this compound also present in FR4 Structures ofthe compounds identified from the fractions are shown inFigure 2
32 Inhibition of Xanthin Oxidase In Vitro All fractions weretested at the final concentration of 5120583gmL The enzymeactivity determined in the solvent-treated condition wasconsidered 100 and all other conditions were comparedto that control None of the prepared fractions exerted anysubstantial action on the enzyme xanthine oxidase while asexpected the reference compound allopurinol resulted in anearly complete (gt90) inhibition of the enzyme activity at5 120583gmL and its IC
50value was determined as 103 120583gmL
Based on these results XO inhibition does not seem to be animportant mechanism for neither the antihyperuricemic northe antioxidant activity of mulberry preparations
33 In Vitro Antioxidant Properties Two different bioassayswere utilized to investigate the in vitro antioxidant propertiesof the fractions DPPH assay and lipid peroxidation (LOX)assay Even though the results obtained by these twomethodsare frequently parallel certain agents can exhibit substantialdifferences in these assays [31] Generally a compoundeffective in the DPPH assay can be considered as a freeradical scavenger functioning in an organic solvent againsta chemically pure molecule (ie the DPPH radical) LOXassay on the other hand is performed in a more complex exvivo biological system containing lipidswith unsaturated fattyacids a substance active in this assay may protect these lipidsfrom the spontaneous oxidation in aqueous conditions
DPPH assay was performed with the concentration rangeof 0001ndash015mgmL to characterize the free radical scav-enging capacity of the prepared fractions Fractions FR1and FR2 exhibited no substantial activities in the utilizedconcentrations Fractions FR3 and FR4 were moderatelyactive while FR5 and FR-W showed the highest potenciesFR6 containing significant amounts of phenolic compounds(compounds 1ndash3) was equipotent with the reference agenttrolox In agreement with these lipid peroxidation assayshowed that the antioxidant capacities of fractions FR3 andFR6 were close to that of trolox while FR5 and FR-W wereslightly less active Fractions FR2 andFR4 exhibitedmoderateactivities and similarly to the case of the DPPH assay FR1was inactive Results of both assays are summarized inTable 1
34 Effect on the In Vitro Glucose Consumption of AdipocytesAlthough some minor changes could be observed in thisbioassay no statistically significant activities were foundThis was somewhat surprising since a lipophilic fractionobtained by a simple solvent-solvent distribution from thehot water extract of the same plant collection was previously
Evidence-Based Complementary and Alternative Medicine 5
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6 Evidence-Based Complementary and Alternative Medicine
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(i)
Figure 1 HPLC-DAD fingerprints of the samples and the correspondingmaximum absorbance chromatograms from 120582 = 200 to 450 nm (a)Crude extract (EX) (b) FR-W (c) FR-B and (d)ndash(i) FR1-FR6 respectivelyMarked peaks represent chlorogenic acid (1) rutin (2) isoquercitrin(3) loliolide (4) and an unidentified flavone derivative (f)
found to exert strong effect in this test [19] The most likelyexplanation is the low relative amount of the constituentsresponsible for this effect based on which we can alsoconclude that increasing the glucose consumption of adiposetissue has little importance in the complex anti-diabeticactivity of less processed phytotherapeutics originated frommulberry leaves
35 Determination of the Antihyperuricemic Effects of theFractions In Vivo The serum uric acid concentration in ratsis considerably low because it is metabolized into allantoinby the enzyme uricase Therefore uric acid accumulationwas induced by a single administration of K-oxonate forthe in vivo investigation of the antihyperuricemic properties
of the fractions Results are shown in Figure 3 Allopurinol(50mgkg intraperitoneally) used as positive control sub-stantially and significantly decreased the accumulation ofuric acid The lowest dose (30mgkg) of FR1 the highestdose (120mgkg) of FR2 and 60mgkg of FR5 exertedantihyperuricemic actions comparable to that of allopurinolUnexpectedly treatment with FR-W (60 and 120mgkg)resulted in an elevation of serum uric acid levels No cleardose-response relationships were detected for some of thefractions
The renal excretion of uric acid is a complex and species-dependent procedure involving its glomerular filtrationtubular secretion and tubular reabsorption Since theseprocesses can independently be modulated with exogenous
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
and hyperuricemia appears to be a highly relevant strategy forovercoming difficulties attributed to the current therapeuticapproaches Appropriately chosen phytotherapeutics [10 11]representing complex bioactivity profiles might serve asexcellent tools to fulfill this objective either as monotherapyor in combination with already existing approaches
Mulberry leaves are probably best known by their rolein the silk production but medicinal use of this drug alsodates back at least two thousand years it is alreadymentionedin the ldquoDivine Farmerrsquos Materia Medicardquo (pinyin ShennongBencao Jıng) written during the reign of the Han dynastyIn Traditional Chinese Medicine leaves of M alba possesssweet slightly bitter and slightly cold properties and theirprimary uses are described as ldquoto expel wind and heatfrom the lungs as well as to clear the liver and the eyesrdquo[12] Anti-diabetic use of mulberry leaves had also beenpopular moreover this indication became part of the localtraditional medicine wherever the tree has been naturalized[13ndash16] Based on this a large number of herbal preparations(including many food supplements) are worldwide availablefor diabetes treatment and easily accessible to everyone evenvia online shopping This activity of mulberry leaves hasbeen verified by a number of studies including several animalexperiments [14ndash16] and a few human trials as well [17 18]but to our knowledge the active constituents and their role inthe activity still remain to be fully described Nevertheless acomplex cocktail of various bioactive constituents is thoughtto be responsible for this activity [19] among which the roleof iminosugars [18] and certain phenolics mainly chlorogenicacid and rutin [16] might be the most significant
Furthermore several traditional Chinese preparationsutilize the branch of Morus alba for the treatment of goutarthritis and rheumatism [20] Various constituents of thedrugwere found to have significant antihyperuricemic poten-tial including mulberroside A a stilbene glycoside [21] anda number of flavonoids primarily morin [22 23]
Based on the above our objectives were to explore thepotential of Morus alba leaves as dual-target phytotherapeu-tics to prevent and treat both diabetes and hyperuricemiaand to investigate whether a simple chromatographic frac-tionation can lead to the enrichment of the main bioactiveconstituents valuable for both therapeutic targets of interest
2 Materials and Methods
21 Plant Material Chemicals and Reagents The leaves ofMorus alba were collected near Asotthalom (nearby SzegedHungary) in May 2007 and botanically identified by AHunyadi A voucher specimen (MA052007) was deposited inthe Institute of Pharmacognosy University of Szeged SzegedHungary All chemicals if otherwise not specified werepurchased from Sigma-Aldrich (Budapest Hungary) Rutin(2) and isoquercitrin (3) were purchased from ChromaDex(Irvine CA USA) and Extrasynthese (Genay France)respectively Loliolide (4) was previously isolated from thedry leaves ofMorus alba [19] and 1-Deoxynojirimicin (1-DN)was purchased fromWako Pure Chemical Industries (OsakaJapan) HPLC grade methanol was obtained from Fischer
Scientific ultrapure water was obtained by using a MilliporeDirect-Q UV3 equipment
22 Extraction and Chromatographic Fractionation 25 kgof the dried and ground plant material was extracted bypercolation with 30 L of 70 aqueous methanol and thesolvent was evaporated under vacuum at 50∘C to obtain67536 g dry extract (EX) 170 g of the drymaterial was furtherprocessed it was dissolved in 1000mL of water and extractedwith 10 times 500mL of n-butanol After solvent evaporationdry residue of the aqueous (FR-W) and organic phase (FR-B) was 784 and 8809 g respectively The butanol phase wasadsorbed onto triple amount (276 g) of silica (Kieselgel 60Merck Darmstadt Germany) and administered on the topof a previously prepared silica column of 1840 g A stepwisegradient of CH
2Cl2 CH2Cl2 EtOH (95 5 9 1 8 2 7 3
6 4 and 1 1) and EtOH was used and one single fractionper solvent was collected After the first fraction of 18 L eachfollowing was of 10 L volume After solvent evaporation dryresidues of the fractions were 1372 976 448 819 1052 993550 and 1087 g respectively Based on high similarities intheir TLC fingerprints the last three fractions were joinedhence finally six fractions referred to as FR1-6 were used forthe experiments discussed here
23 HPLC-DAD Analysis of Fractions FR1-6 Diode-arraydetected high performance liquid chromatography (HPLC-DAD) was performed on a gradient system of two JascoPU2080 pumps connected to a Jasco MD-2010 Plus diode-array detector (DAD) and equipped with a Jasco AS-2055Plus autosampler (Jasco Co Tokyo Japan) by usinga Zorbax Eclipse XDB-C8 (5 120583m 46 times 150mm) analyticalcolumn Samples were dissolved in 30 of aqueous MeOH at2mgmL and 30 120583L of each was injected Gradient elutionwas performed from 30 to 100 of aqueous MeOH in 13minutes kept at 100 for four more minutes and returned to30 at 171min Chromatograms were recorded for 24minand DAD data was collected from 200 to 650 nm Baselineswere corrected by subtracting the chromatogram of a single30 120583L injection of 30 MeOH
24 Quantitative Analysis of Compounds 1ndash4 Single-pointquantitative analysis was performed for the previously iden-tified major constituents in their respective fractions suchas chlorogenic acid (1) rutin (2) and isoquercitrin (3) inEX FR-B and FR5-6 and loliolide (4) in FR2 Calibrationcurves were taken by analysing dilutions of a 1mgmL stocksolution of the corresponding standard at concentrations of 105 04 03 02 01 and 005mgmL 1198772 values were 0997009990 09904 and 09978 for the calibration lines of 1ndash4respectively In case of FR5 and FR6 peaks of 2 and 3 werepartially overlapping deconvolution was performed by usingGaussian approximations with the Fityk 097 software (FreeSoftware Foundation Poland) and quantities were calculatedbased on the peak areas revealed this way
25 Determination of Iminosugars Iminosugar content wasqualitatively investigated by thin-layer chromatography
Evidence-Based Complementary and Alternative Medicine 3
(TLC) in each fraction by using DC-Alufolien Kieselgel60F254
plates (Merck Darmstadt Germany) and CH2Cl2
MeOH NH3(3 6 2 vvv) as solvent system For quan-
titative analysis an API 2000 MSMS spectrometer was usedwith a Shimadzu autosampler and an electrospray ionization(ESI) interface set in positive mode 1-DN 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin (Gal-DN) and fagom-ine contents were investigated in FR-W FR-B and FR4-6Injected sampleswerewashed into the spectrometerwith 50aqueous MeOH at a flow rate of 200 120583Lmin temperatureof the ion source was 300∘C Multiple reaction monitoring(MRM) was used with transitions of mz 164rarr 69 for 1-DNmz 326rarr 164 for GAL-DN andmz 148rarr 86 for fagomineaccording to literature data [24] Parameter optimizationand data acquisition and evaluation were performed byusing the Analyst 151 software Calibration line of 1-DN(1198772 = 10000) was obtained by means of six measurementpoints in triplicates
26 In Vitro Assay on Xanthine Oxidase (EC 11732)Inhibition The activity of the enzyme was calculated fromthe increase of uric acid concentration determined bymicroplate-based kinetic photometry [25] Briefly theabsorption of uric acid generated from xanthine (50120583M atstart-up) was followed at 120582 = 290 nm for 125 s (SpectrostarNano BMG Labtech Ortenberg Germany) The activityof xanthine oxidase was described as the slope of theabsorbance versus time curve and allopurinol was used aspositive control Stock solutions prepared with dimethyl-sulfoxide were used for the in vitro assays and any substantialeffect of the solvent was excluded All in vitro assays werecarried out in duplicates
27 Determination of Free Radical Scavenging Activity byDPPH Assay In Vitro The activity for scavenging 11-diphenyl-2-picrylhydrazyl (DPPH) radicals was tested foreach fraction as described earlier [26] Briefly differentamounts of the samples were added to 01mM DPPH dis-solved in ethanol The mixture was shaken and allowed tostand for 30min and then the absorbance of the solutionwas measured at 120582 = 517 nm trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid) a water-soluble ana-log of vitamin E was used as positive control
28 Determination of Lipid Peroxidation Inhibitory ActivityThe antioxidant properties of the fractions were additionallymeasured by means of inhibition of the autooxidation ofunsaturated fatty acids present in animal brain tissue [27]A lipid-rich fraction was prepared from the brains of maleSprague-Dawley rats (Charles River Laboratories BudapestHungary body mass 250ndash300 g) by homogenization andcentrifugation The fatty acids in such a fraction get spon-taneously oxidized during an incubation of 1 h at 37∘C andthis oxidation can be inhibited by antioxidants The oxidizedproducts were determined by colorimetry at 532 nm afterreaction with thiobarbituric acid All in vitro experimentswere carried out in duplicates and statistically evaluatedSigmoid curves were fitted to the results of both antioxidant
assays and IC50
values were calculated by using GraphPadPrism 4 (GraphPad Software San Diego CA USA)
29 Testing the Effect on the Glucose Consumption of Adipo-cytes Effect of each fractionwas tested on the in vitro glucoseconsumption of adipocytes as published before at concentra-tions of 200120583gmL [19] Briefly 3T3-L1 preadipocytes (5times105cells BCRC no 60159 Bioresource Collection and ResearchCenter Taiwan) were seeded and cultured in 10 CS DMEMcontaining 55mM D-glucose The cells were induced todifferentiate and at day 3 samples dissolved in DMSO wereadded to the cells at 200120583gmL either in presence or absenceof 032 120583M insulin 24 h changes in the glucose contents of thetesting media were recorded and compared to those beforethe addition of the samples
210 Animal Studies Animals were treated in accordancewith the European Communities Council Directives(86609ECC) and the Hungarian Act for the Protectionof Animals in Research (XXVIIItv32sect) Experimentsinvolving animal subjects were carried out with the approvalof the Hungarian Ethical Committee for Animal Research(registration no IV01758-22008) Male Sprague-Dawleyrats (Charles River Laboratories Budapest Hungary bodymass 180ndash200 g 8 animals in each group) were housed intemperature (20ndash23∘C) humidity (40ndash60) and light (12 hof light 12 h of dark) regulated rooms with tap water androdent food (Bioplan Isaszeg Hungary) intake availablead libitum The tested extracts were orally administered in025 methylcellulose containing 2 Cremophor EL usinga dosing volume of 5mLkg Three doses of all extracts (3060 and 120mgkg) were selected for daily treatment for 3consecutive days and each treatment was performed after16 hours of fasting One-way analysis of variance (ANOVA)with Dunnettrsquos multiple comparison test by GraphPad Prism4 was used for statistical evaluation of all in vivo experiments
211 Determination of Antihyperuricemic Antihyperglycemicand Antioxidant Properties of the Fractions In Vivo In orderto minimize the number of animals antihyperuricemicantihyperglycemic and antioxidant properties effects weredetermined from the same groups of rats Experimentalhyperuricemiamodel induced by uricase inhibitor potassiumoxonate was utilized to assess the antihyperuricemic proper-ties of the tested fractions [28] 250mgkg potassium oxonatewas suspended in 025 methylcellulose and administeredintraperitoneally at the time of the third oral treatment50mgkg of allopurinol a clinically used antigout drug wasorally administered as a positive control One hour laterrats were orally treated with 25 gkg starch suspension ina dosing volume of 5mLkg in order to characterize theantihyperglycemic effects of the fractions [25] After anotherone hour venous blood samples were obtained from the tailand the plasma glucose concentrations were determined bya commercially available kit (Reanal Budapest Hungary)based on the glucose oxidase-peroxidasemethod [29] Imme-diately after blood sampling rats were anesthetized in 4isoflurane and additional blood samples were collected by
4 Evidence-Based Complementary and Alternative Medicine
cardiac puncture for determination of uric acid concentrationand total antioxidant capacity Since inhalational anestheticsare reported to substantially elevate the blood glucose levelseparate blood samplings were needed for determination ofall the planned parameters [30] Serum samples were pre-pared by centrifugation and stored at ndash70∘Cuntil the analysesAll determinations by colorimetric uric acid (BioAassay Sys-tems Hayward CA USA) and antioxidant (Sigma-AldrichBudapest Hungary) assay kits were performed in duplicatesaccording to themanufacturersrsquo suggestions Separate groupsorally treated with glibenclamide and trolox (10mgkg forboth) were included as reference for the antihyperglycemicand antioxidant assays respectively
3 Results and Discussion
31 Chemical Composition of the Fractions Obtained Thefractions obtained by solvent-solvent extraction and a roughchromatographic separation of the organic phase on silicarepresented fundamentally different chemical compositions(Figure 1) As expected highly water soluble hydrophiliccompounds (including iminosugars of the plant see below)remained in the water phase (Figure 1(b)) while most ofthe still polar chlorogenic acid (1) could already be detectedin the organic phase (Figure 1(c)) along with vast major-ity of the supposedly ldquodrug-likerdquo secondary metabolites ofmulberry leaves These would be the compounds with thehighest probability for therapeutic value mainly the too lowlog 119875 value typically results in poor penetration throughmembranes (iminosugars a constituent group of possibleexception to this are discussed below) As seen from theDAD fingerprints FR1 (Figure 1(d)) and FR2 (Figure 1(e))mostly contained compounds with lower wavelengths of UVabsorbance maxima probably including several terpenoidsandor phenylpropanes [19] The dominant peak of FR2was detected and quantified as loliolide (4) (033) amonoterpene lactone we have recently reported fromM albaleaves [19] Of all fractions FR3 (Figure 1(f)) contained thesmallest amount of UV absorbing material its main con-stituents remained unidentified Based on its UV spectrumand retention time chief constituent of FR4 (Figure 1(g)) issuggested to be a flavone aglycone (342ndash350 expressed inequivalents of isoquercitrin (3) or rutin (2)) while 3 (242)was also present in this fraction FR5 (Figure 1(h)) containedthe majority of 3 (979) along with a smaller amountof 2 (377) while the previously mentioned unidentifiedflavonoid could still be detected FR6 (Figure 1(i)) the mostpolar fraction obtained from the column chromatographycontained 1 (899) 2 (1041) and 3 (564) as major UVactive constituents
Both the extract and the fractions contained very smallamounts of iminosugars By means of MSMS 1-DN con-tents were found as low as 02694permil(EX) 03007permil (FR-W)00742permil (FR-B) sim0016permil (FR4 around detection limit)00558permil (FR5) and 02770permil(FR6) On the other handslope of the calibration lines of GAL-DN and fagomine isaround 13 and 6 times higher respectively as compared tothat of 1-DN based on a recent publication using the sameMRM transitions with ESI-MSMS [24] This allowed only
a rough estimation on the quantity of these compounds byusing our calibration obtained for 1-DN Amounts of GAL-DN were about 002permil (EX) 003permil (FR-W) 0003permil (FR-B) and 0009permil(FR6) and trace amounts of this com-pound were detected in FR4 and FR5 Fagomine contentscould be estimated as around 003permil (EX) 004permil (FR-W)001permil (FR-B) 001permil (FR5) and 003permil (FR6) with a traceamount of this compound also present in FR4 Structures ofthe compounds identified from the fractions are shown inFigure 2
32 Inhibition of Xanthin Oxidase In Vitro All fractions weretested at the final concentration of 5120583gmL The enzymeactivity determined in the solvent-treated condition wasconsidered 100 and all other conditions were comparedto that control None of the prepared fractions exerted anysubstantial action on the enzyme xanthine oxidase while asexpected the reference compound allopurinol resulted in anearly complete (gt90) inhibition of the enzyme activity at5 120583gmL and its IC
50value was determined as 103 120583gmL
Based on these results XO inhibition does not seem to be animportant mechanism for neither the antihyperuricemic northe antioxidant activity of mulberry preparations
33 In Vitro Antioxidant Properties Two different bioassayswere utilized to investigate the in vitro antioxidant propertiesof the fractions DPPH assay and lipid peroxidation (LOX)assay Even though the results obtained by these twomethodsare frequently parallel certain agents can exhibit substantialdifferences in these assays [31] Generally a compoundeffective in the DPPH assay can be considered as a freeradical scavenger functioning in an organic solvent againsta chemically pure molecule (ie the DPPH radical) LOXassay on the other hand is performed in a more complex exvivo biological system containing lipidswith unsaturated fattyacids a substance active in this assay may protect these lipidsfrom the spontaneous oxidation in aqueous conditions
DPPH assay was performed with the concentration rangeof 0001ndash015mgmL to characterize the free radical scav-enging capacity of the prepared fractions Fractions FR1and FR2 exhibited no substantial activities in the utilizedconcentrations Fractions FR3 and FR4 were moderatelyactive while FR5 and FR-W showed the highest potenciesFR6 containing significant amounts of phenolic compounds(compounds 1ndash3) was equipotent with the reference agenttrolox In agreement with these lipid peroxidation assayshowed that the antioxidant capacities of fractions FR3 andFR6 were close to that of trolox while FR5 and FR-W wereslightly less active Fractions FR2 andFR4 exhibitedmoderateactivities and similarly to the case of the DPPH assay FR1was inactive Results of both assays are summarized inTable 1
34 Effect on the In Vitro Glucose Consumption of AdipocytesAlthough some minor changes could be observed in thisbioassay no statistically significant activities were foundThis was somewhat surprising since a lipophilic fractionobtained by a simple solvent-solvent distribution from thehot water extract of the same plant collection was previously
Evidence-Based Complementary and Alternative Medicine 5
600000
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)586e + 004 121e + 005 184e + 005 246e + 005 309e + 005 371e + 005 434e + 005 496e + 005
120 160 200 240
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
60
60
100
100
140
140
180
180
220
220
20
20
Inte
nsity
(120583AU
)
1
2
3
(a)
00 40 80
Retention time (min)120 160 200 24060
60
100
100
140
140
180
180
220
220
20
20
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
(b)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
400000
350000
300000
250000
200000
150000
100000
50000
0
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
23
(c)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
minus10000
105e + 004 218e + 004 330e + 004 443e + 004 555e + 004 668e + 004 780e + 004 893e + 004
Inte
nsity
(120583AU
)
(d)
70000
60000
50000
40000
30000
20000
10000
0
minus10000
703e + 003 145e + 004 220e + 004 295e + 004 370e + 004 445e + 004 520e + 004 595e + 004
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
4
(e)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
527e + 003 109e + 004 165e + 004 221e + 004 278e + 004 334e + 004 390e + 004 446e + 004
Inte
nsity
(120583AU
)
(f)
Figure 1 Continued
6 Evidence-Based Complementary and Alternative Medicine
140000
120000
100000
80000
60000
40000
20000
0
164e + 004 339e + 004 514e + 004 689e + 004 864e + 004 104e + 005 121e + 005 139e + 005
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
f
(g)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
2
3
f
(h)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
12
3
(i)
Figure 1 HPLC-DAD fingerprints of the samples and the correspondingmaximum absorbance chromatograms from 120582 = 200 to 450 nm (a)Crude extract (EX) (b) FR-W (c) FR-B and (d)ndash(i) FR1-FR6 respectivelyMarked peaks represent chlorogenic acid (1) rutin (2) isoquercitrin(3) loliolide (4) and an unidentified flavone derivative (f)
found to exert strong effect in this test [19] The most likelyexplanation is the low relative amount of the constituentsresponsible for this effect based on which we can alsoconclude that increasing the glucose consumption of adiposetissue has little importance in the complex anti-diabeticactivity of less processed phytotherapeutics originated frommulberry leaves
35 Determination of the Antihyperuricemic Effects of theFractions In Vivo The serum uric acid concentration in ratsis considerably low because it is metabolized into allantoinby the enzyme uricase Therefore uric acid accumulationwas induced by a single administration of K-oxonate forthe in vivo investigation of the antihyperuricemic properties
of the fractions Results are shown in Figure 3 Allopurinol(50mgkg intraperitoneally) used as positive control sub-stantially and significantly decreased the accumulation ofuric acid The lowest dose (30mgkg) of FR1 the highestdose (120mgkg) of FR2 and 60mgkg of FR5 exertedantihyperuricemic actions comparable to that of allopurinolUnexpectedly treatment with FR-W (60 and 120mgkg)resulted in an elevation of serum uric acid levels No cleardose-response relationships were detected for some of thefractions
The renal excretion of uric acid is a complex and species-dependent procedure involving its glomerular filtrationtubular secretion and tubular reabsorption Since theseprocesses can independently be modulated with exogenous
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
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OncologyJournal of
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Evidence-Based Complementary and Alternative Medicine
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Evidence-Based Complementary and Alternative Medicine 3
(TLC) in each fraction by using DC-Alufolien Kieselgel60F254
plates (Merck Darmstadt Germany) and CH2Cl2
MeOH NH3(3 6 2 vvv) as solvent system For quan-
titative analysis an API 2000 MSMS spectrometer was usedwith a Shimadzu autosampler and an electrospray ionization(ESI) interface set in positive mode 1-DN 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin (Gal-DN) and fagom-ine contents were investigated in FR-W FR-B and FR4-6Injected sampleswerewashed into the spectrometerwith 50aqueous MeOH at a flow rate of 200 120583Lmin temperatureof the ion source was 300∘C Multiple reaction monitoring(MRM) was used with transitions of mz 164rarr 69 for 1-DNmz 326rarr 164 for GAL-DN andmz 148rarr 86 for fagomineaccording to literature data [24] Parameter optimizationand data acquisition and evaluation were performed byusing the Analyst 151 software Calibration line of 1-DN(1198772 = 10000) was obtained by means of six measurementpoints in triplicates
26 In Vitro Assay on Xanthine Oxidase (EC 11732)Inhibition The activity of the enzyme was calculated fromthe increase of uric acid concentration determined bymicroplate-based kinetic photometry [25] Briefly theabsorption of uric acid generated from xanthine (50120583M atstart-up) was followed at 120582 = 290 nm for 125 s (SpectrostarNano BMG Labtech Ortenberg Germany) The activityof xanthine oxidase was described as the slope of theabsorbance versus time curve and allopurinol was used aspositive control Stock solutions prepared with dimethyl-sulfoxide were used for the in vitro assays and any substantialeffect of the solvent was excluded All in vitro assays werecarried out in duplicates
27 Determination of Free Radical Scavenging Activity byDPPH Assay In Vitro The activity for scavenging 11-diphenyl-2-picrylhydrazyl (DPPH) radicals was tested foreach fraction as described earlier [26] Briefly differentamounts of the samples were added to 01mM DPPH dis-solved in ethanol The mixture was shaken and allowed tostand for 30min and then the absorbance of the solutionwas measured at 120582 = 517 nm trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid) a water-soluble ana-log of vitamin E was used as positive control
28 Determination of Lipid Peroxidation Inhibitory ActivityThe antioxidant properties of the fractions were additionallymeasured by means of inhibition of the autooxidation ofunsaturated fatty acids present in animal brain tissue [27]A lipid-rich fraction was prepared from the brains of maleSprague-Dawley rats (Charles River Laboratories BudapestHungary body mass 250ndash300 g) by homogenization andcentrifugation The fatty acids in such a fraction get spon-taneously oxidized during an incubation of 1 h at 37∘C andthis oxidation can be inhibited by antioxidants The oxidizedproducts were determined by colorimetry at 532 nm afterreaction with thiobarbituric acid All in vitro experimentswere carried out in duplicates and statistically evaluatedSigmoid curves were fitted to the results of both antioxidant
assays and IC50
values were calculated by using GraphPadPrism 4 (GraphPad Software San Diego CA USA)
29 Testing the Effect on the Glucose Consumption of Adipo-cytes Effect of each fractionwas tested on the in vitro glucoseconsumption of adipocytes as published before at concentra-tions of 200120583gmL [19] Briefly 3T3-L1 preadipocytes (5times105cells BCRC no 60159 Bioresource Collection and ResearchCenter Taiwan) were seeded and cultured in 10 CS DMEMcontaining 55mM D-glucose The cells were induced todifferentiate and at day 3 samples dissolved in DMSO wereadded to the cells at 200120583gmL either in presence or absenceof 032 120583M insulin 24 h changes in the glucose contents of thetesting media were recorded and compared to those beforethe addition of the samples
210 Animal Studies Animals were treated in accordancewith the European Communities Council Directives(86609ECC) and the Hungarian Act for the Protectionof Animals in Research (XXVIIItv32sect) Experimentsinvolving animal subjects were carried out with the approvalof the Hungarian Ethical Committee for Animal Research(registration no IV01758-22008) Male Sprague-Dawleyrats (Charles River Laboratories Budapest Hungary bodymass 180ndash200 g 8 animals in each group) were housed intemperature (20ndash23∘C) humidity (40ndash60) and light (12 hof light 12 h of dark) regulated rooms with tap water androdent food (Bioplan Isaszeg Hungary) intake availablead libitum The tested extracts were orally administered in025 methylcellulose containing 2 Cremophor EL usinga dosing volume of 5mLkg Three doses of all extracts (3060 and 120mgkg) were selected for daily treatment for 3consecutive days and each treatment was performed after16 hours of fasting One-way analysis of variance (ANOVA)with Dunnettrsquos multiple comparison test by GraphPad Prism4 was used for statistical evaluation of all in vivo experiments
211 Determination of Antihyperuricemic Antihyperglycemicand Antioxidant Properties of the Fractions In Vivo In orderto minimize the number of animals antihyperuricemicantihyperglycemic and antioxidant properties effects weredetermined from the same groups of rats Experimentalhyperuricemiamodel induced by uricase inhibitor potassiumoxonate was utilized to assess the antihyperuricemic proper-ties of the tested fractions [28] 250mgkg potassium oxonatewas suspended in 025 methylcellulose and administeredintraperitoneally at the time of the third oral treatment50mgkg of allopurinol a clinically used antigout drug wasorally administered as a positive control One hour laterrats were orally treated with 25 gkg starch suspension ina dosing volume of 5mLkg in order to characterize theantihyperglycemic effects of the fractions [25] After anotherone hour venous blood samples were obtained from the tailand the plasma glucose concentrations were determined bya commercially available kit (Reanal Budapest Hungary)based on the glucose oxidase-peroxidasemethod [29] Imme-diately after blood sampling rats were anesthetized in 4isoflurane and additional blood samples were collected by
4 Evidence-Based Complementary and Alternative Medicine
cardiac puncture for determination of uric acid concentrationand total antioxidant capacity Since inhalational anestheticsare reported to substantially elevate the blood glucose levelseparate blood samplings were needed for determination ofall the planned parameters [30] Serum samples were pre-pared by centrifugation and stored at ndash70∘Cuntil the analysesAll determinations by colorimetric uric acid (BioAassay Sys-tems Hayward CA USA) and antioxidant (Sigma-AldrichBudapest Hungary) assay kits were performed in duplicatesaccording to themanufacturersrsquo suggestions Separate groupsorally treated with glibenclamide and trolox (10mgkg forboth) were included as reference for the antihyperglycemicand antioxidant assays respectively
3 Results and Discussion
31 Chemical Composition of the Fractions Obtained Thefractions obtained by solvent-solvent extraction and a roughchromatographic separation of the organic phase on silicarepresented fundamentally different chemical compositions(Figure 1) As expected highly water soluble hydrophiliccompounds (including iminosugars of the plant see below)remained in the water phase (Figure 1(b)) while most ofthe still polar chlorogenic acid (1) could already be detectedin the organic phase (Figure 1(c)) along with vast major-ity of the supposedly ldquodrug-likerdquo secondary metabolites ofmulberry leaves These would be the compounds with thehighest probability for therapeutic value mainly the too lowlog 119875 value typically results in poor penetration throughmembranes (iminosugars a constituent group of possibleexception to this are discussed below) As seen from theDAD fingerprints FR1 (Figure 1(d)) and FR2 (Figure 1(e))mostly contained compounds with lower wavelengths of UVabsorbance maxima probably including several terpenoidsandor phenylpropanes [19] The dominant peak of FR2was detected and quantified as loliolide (4) (033) amonoterpene lactone we have recently reported fromM albaleaves [19] Of all fractions FR3 (Figure 1(f)) contained thesmallest amount of UV absorbing material its main con-stituents remained unidentified Based on its UV spectrumand retention time chief constituent of FR4 (Figure 1(g)) issuggested to be a flavone aglycone (342ndash350 expressed inequivalents of isoquercitrin (3) or rutin (2)) while 3 (242)was also present in this fraction FR5 (Figure 1(h)) containedthe majority of 3 (979) along with a smaller amountof 2 (377) while the previously mentioned unidentifiedflavonoid could still be detected FR6 (Figure 1(i)) the mostpolar fraction obtained from the column chromatographycontained 1 (899) 2 (1041) and 3 (564) as major UVactive constituents
Both the extract and the fractions contained very smallamounts of iminosugars By means of MSMS 1-DN con-tents were found as low as 02694permil(EX) 03007permil (FR-W)00742permil (FR-B) sim0016permil (FR4 around detection limit)00558permil (FR5) and 02770permil(FR6) On the other handslope of the calibration lines of GAL-DN and fagomine isaround 13 and 6 times higher respectively as compared tothat of 1-DN based on a recent publication using the sameMRM transitions with ESI-MSMS [24] This allowed only
a rough estimation on the quantity of these compounds byusing our calibration obtained for 1-DN Amounts of GAL-DN were about 002permil (EX) 003permil (FR-W) 0003permil (FR-B) and 0009permil(FR6) and trace amounts of this com-pound were detected in FR4 and FR5 Fagomine contentscould be estimated as around 003permil (EX) 004permil (FR-W)001permil (FR-B) 001permil (FR5) and 003permil (FR6) with a traceamount of this compound also present in FR4 Structures ofthe compounds identified from the fractions are shown inFigure 2
32 Inhibition of Xanthin Oxidase In Vitro All fractions weretested at the final concentration of 5120583gmL The enzymeactivity determined in the solvent-treated condition wasconsidered 100 and all other conditions were comparedto that control None of the prepared fractions exerted anysubstantial action on the enzyme xanthine oxidase while asexpected the reference compound allopurinol resulted in anearly complete (gt90) inhibition of the enzyme activity at5 120583gmL and its IC
50value was determined as 103 120583gmL
Based on these results XO inhibition does not seem to be animportant mechanism for neither the antihyperuricemic northe antioxidant activity of mulberry preparations
33 In Vitro Antioxidant Properties Two different bioassayswere utilized to investigate the in vitro antioxidant propertiesof the fractions DPPH assay and lipid peroxidation (LOX)assay Even though the results obtained by these twomethodsare frequently parallel certain agents can exhibit substantialdifferences in these assays [31] Generally a compoundeffective in the DPPH assay can be considered as a freeradical scavenger functioning in an organic solvent againsta chemically pure molecule (ie the DPPH radical) LOXassay on the other hand is performed in a more complex exvivo biological system containing lipidswith unsaturated fattyacids a substance active in this assay may protect these lipidsfrom the spontaneous oxidation in aqueous conditions
DPPH assay was performed with the concentration rangeof 0001ndash015mgmL to characterize the free radical scav-enging capacity of the prepared fractions Fractions FR1and FR2 exhibited no substantial activities in the utilizedconcentrations Fractions FR3 and FR4 were moderatelyactive while FR5 and FR-W showed the highest potenciesFR6 containing significant amounts of phenolic compounds(compounds 1ndash3) was equipotent with the reference agenttrolox In agreement with these lipid peroxidation assayshowed that the antioxidant capacities of fractions FR3 andFR6 were close to that of trolox while FR5 and FR-W wereslightly less active Fractions FR2 andFR4 exhibitedmoderateactivities and similarly to the case of the DPPH assay FR1was inactive Results of both assays are summarized inTable 1
34 Effect on the In Vitro Glucose Consumption of AdipocytesAlthough some minor changes could be observed in thisbioassay no statistically significant activities were foundThis was somewhat surprising since a lipophilic fractionobtained by a simple solvent-solvent distribution from thehot water extract of the same plant collection was previously
Evidence-Based Complementary and Alternative Medicine 5
600000
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)586e + 004 121e + 005 184e + 005 246e + 005 309e + 005 371e + 005 434e + 005 496e + 005
120 160 200 240
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
60
60
100
100
140
140
180
180
220
220
20
20
Inte
nsity
(120583AU
)
1
2
3
(a)
00 40 80
Retention time (min)120 160 200 24060
60
100
100
140
140
180
180
220
220
20
20
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
(b)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
400000
350000
300000
250000
200000
150000
100000
50000
0
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
23
(c)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
minus10000
105e + 004 218e + 004 330e + 004 443e + 004 555e + 004 668e + 004 780e + 004 893e + 004
Inte
nsity
(120583AU
)
(d)
70000
60000
50000
40000
30000
20000
10000
0
minus10000
703e + 003 145e + 004 220e + 004 295e + 004 370e + 004 445e + 004 520e + 004 595e + 004
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
4
(e)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
527e + 003 109e + 004 165e + 004 221e + 004 278e + 004 334e + 004 390e + 004 446e + 004
Inte
nsity
(120583AU
)
(f)
Figure 1 Continued
6 Evidence-Based Complementary and Alternative Medicine
140000
120000
100000
80000
60000
40000
20000
0
164e + 004 339e + 004 514e + 004 689e + 004 864e + 004 104e + 005 121e + 005 139e + 005
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
f
(g)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
2
3
f
(h)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
12
3
(i)
Figure 1 HPLC-DAD fingerprints of the samples and the correspondingmaximum absorbance chromatograms from 120582 = 200 to 450 nm (a)Crude extract (EX) (b) FR-W (c) FR-B and (d)ndash(i) FR1-FR6 respectivelyMarked peaks represent chlorogenic acid (1) rutin (2) isoquercitrin(3) loliolide (4) and an unidentified flavone derivative (f)
found to exert strong effect in this test [19] The most likelyexplanation is the low relative amount of the constituentsresponsible for this effect based on which we can alsoconclude that increasing the glucose consumption of adiposetissue has little importance in the complex anti-diabeticactivity of less processed phytotherapeutics originated frommulberry leaves
35 Determination of the Antihyperuricemic Effects of theFractions In Vivo The serum uric acid concentration in ratsis considerably low because it is metabolized into allantoinby the enzyme uricase Therefore uric acid accumulationwas induced by a single administration of K-oxonate forthe in vivo investigation of the antihyperuricemic properties
of the fractions Results are shown in Figure 3 Allopurinol(50mgkg intraperitoneally) used as positive control sub-stantially and significantly decreased the accumulation ofuric acid The lowest dose (30mgkg) of FR1 the highestdose (120mgkg) of FR2 and 60mgkg of FR5 exertedantihyperuricemic actions comparable to that of allopurinolUnexpectedly treatment with FR-W (60 and 120mgkg)resulted in an elevation of serum uric acid levels No cleardose-response relationships were detected for some of thefractions
The renal excretion of uric acid is a complex and species-dependent procedure involving its glomerular filtrationtubular secretion and tubular reabsorption Since theseprocesses can independently be modulated with exogenous
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
cardiac puncture for determination of uric acid concentrationand total antioxidant capacity Since inhalational anestheticsare reported to substantially elevate the blood glucose levelseparate blood samplings were needed for determination ofall the planned parameters [30] Serum samples were pre-pared by centrifugation and stored at ndash70∘Cuntil the analysesAll determinations by colorimetric uric acid (BioAassay Sys-tems Hayward CA USA) and antioxidant (Sigma-AldrichBudapest Hungary) assay kits were performed in duplicatesaccording to themanufacturersrsquo suggestions Separate groupsorally treated with glibenclamide and trolox (10mgkg forboth) were included as reference for the antihyperglycemicand antioxidant assays respectively
3 Results and Discussion
31 Chemical Composition of the Fractions Obtained Thefractions obtained by solvent-solvent extraction and a roughchromatographic separation of the organic phase on silicarepresented fundamentally different chemical compositions(Figure 1) As expected highly water soluble hydrophiliccompounds (including iminosugars of the plant see below)remained in the water phase (Figure 1(b)) while most ofthe still polar chlorogenic acid (1) could already be detectedin the organic phase (Figure 1(c)) along with vast major-ity of the supposedly ldquodrug-likerdquo secondary metabolites ofmulberry leaves These would be the compounds with thehighest probability for therapeutic value mainly the too lowlog 119875 value typically results in poor penetration throughmembranes (iminosugars a constituent group of possibleexception to this are discussed below) As seen from theDAD fingerprints FR1 (Figure 1(d)) and FR2 (Figure 1(e))mostly contained compounds with lower wavelengths of UVabsorbance maxima probably including several terpenoidsandor phenylpropanes [19] The dominant peak of FR2was detected and quantified as loliolide (4) (033) amonoterpene lactone we have recently reported fromM albaleaves [19] Of all fractions FR3 (Figure 1(f)) contained thesmallest amount of UV absorbing material its main con-stituents remained unidentified Based on its UV spectrumand retention time chief constituent of FR4 (Figure 1(g)) issuggested to be a flavone aglycone (342ndash350 expressed inequivalents of isoquercitrin (3) or rutin (2)) while 3 (242)was also present in this fraction FR5 (Figure 1(h)) containedthe majority of 3 (979) along with a smaller amountof 2 (377) while the previously mentioned unidentifiedflavonoid could still be detected FR6 (Figure 1(i)) the mostpolar fraction obtained from the column chromatographycontained 1 (899) 2 (1041) and 3 (564) as major UVactive constituents
Both the extract and the fractions contained very smallamounts of iminosugars By means of MSMS 1-DN con-tents were found as low as 02694permil(EX) 03007permil (FR-W)00742permil (FR-B) sim0016permil (FR4 around detection limit)00558permil (FR5) and 02770permil(FR6) On the other handslope of the calibration lines of GAL-DN and fagomine isaround 13 and 6 times higher respectively as compared tothat of 1-DN based on a recent publication using the sameMRM transitions with ESI-MSMS [24] This allowed only
a rough estimation on the quantity of these compounds byusing our calibration obtained for 1-DN Amounts of GAL-DN were about 002permil (EX) 003permil (FR-W) 0003permil (FR-B) and 0009permil(FR6) and trace amounts of this com-pound were detected in FR4 and FR5 Fagomine contentscould be estimated as around 003permil (EX) 004permil (FR-W)001permil (FR-B) 001permil (FR5) and 003permil (FR6) with a traceamount of this compound also present in FR4 Structures ofthe compounds identified from the fractions are shown inFigure 2
32 Inhibition of Xanthin Oxidase In Vitro All fractions weretested at the final concentration of 5120583gmL The enzymeactivity determined in the solvent-treated condition wasconsidered 100 and all other conditions were comparedto that control None of the prepared fractions exerted anysubstantial action on the enzyme xanthine oxidase while asexpected the reference compound allopurinol resulted in anearly complete (gt90) inhibition of the enzyme activity at5 120583gmL and its IC
50value was determined as 103 120583gmL
Based on these results XO inhibition does not seem to be animportant mechanism for neither the antihyperuricemic northe antioxidant activity of mulberry preparations
33 In Vitro Antioxidant Properties Two different bioassayswere utilized to investigate the in vitro antioxidant propertiesof the fractions DPPH assay and lipid peroxidation (LOX)assay Even though the results obtained by these twomethodsare frequently parallel certain agents can exhibit substantialdifferences in these assays [31] Generally a compoundeffective in the DPPH assay can be considered as a freeradical scavenger functioning in an organic solvent againsta chemically pure molecule (ie the DPPH radical) LOXassay on the other hand is performed in a more complex exvivo biological system containing lipidswith unsaturated fattyacids a substance active in this assay may protect these lipidsfrom the spontaneous oxidation in aqueous conditions
DPPH assay was performed with the concentration rangeof 0001ndash015mgmL to characterize the free radical scav-enging capacity of the prepared fractions Fractions FR1and FR2 exhibited no substantial activities in the utilizedconcentrations Fractions FR3 and FR4 were moderatelyactive while FR5 and FR-W showed the highest potenciesFR6 containing significant amounts of phenolic compounds(compounds 1ndash3) was equipotent with the reference agenttrolox In agreement with these lipid peroxidation assayshowed that the antioxidant capacities of fractions FR3 andFR6 were close to that of trolox while FR5 and FR-W wereslightly less active Fractions FR2 andFR4 exhibitedmoderateactivities and similarly to the case of the DPPH assay FR1was inactive Results of both assays are summarized inTable 1
34 Effect on the In Vitro Glucose Consumption of AdipocytesAlthough some minor changes could be observed in thisbioassay no statistically significant activities were foundThis was somewhat surprising since a lipophilic fractionobtained by a simple solvent-solvent distribution from thehot water extract of the same plant collection was previously
Evidence-Based Complementary and Alternative Medicine 5
600000
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)586e + 004 121e + 005 184e + 005 246e + 005 309e + 005 371e + 005 434e + 005 496e + 005
120 160 200 240
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
60
60
100
100
140
140
180
180
220
220
20
20
Inte
nsity
(120583AU
)
1
2
3
(a)
00 40 80
Retention time (min)120 160 200 24060
60
100
100
140
140
180
180
220
220
20
20
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
(b)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
400000
350000
300000
250000
200000
150000
100000
50000
0
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
23
(c)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
minus10000
105e + 004 218e + 004 330e + 004 443e + 004 555e + 004 668e + 004 780e + 004 893e + 004
Inte
nsity
(120583AU
)
(d)
70000
60000
50000
40000
30000
20000
10000
0
minus10000
703e + 003 145e + 004 220e + 004 295e + 004 370e + 004 445e + 004 520e + 004 595e + 004
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
4
(e)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
527e + 003 109e + 004 165e + 004 221e + 004 278e + 004 334e + 004 390e + 004 446e + 004
Inte
nsity
(120583AU
)
(f)
Figure 1 Continued
6 Evidence-Based Complementary and Alternative Medicine
140000
120000
100000
80000
60000
40000
20000
0
164e + 004 339e + 004 514e + 004 689e + 004 864e + 004 104e + 005 121e + 005 139e + 005
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
f
(g)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
2
3
f
(h)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
12
3
(i)
Figure 1 HPLC-DAD fingerprints of the samples and the correspondingmaximum absorbance chromatograms from 120582 = 200 to 450 nm (a)Crude extract (EX) (b) FR-W (c) FR-B and (d)ndash(i) FR1-FR6 respectivelyMarked peaks represent chlorogenic acid (1) rutin (2) isoquercitrin(3) loliolide (4) and an unidentified flavone derivative (f)
found to exert strong effect in this test [19] The most likelyexplanation is the low relative amount of the constituentsresponsible for this effect based on which we can alsoconclude that increasing the glucose consumption of adiposetissue has little importance in the complex anti-diabeticactivity of less processed phytotherapeutics originated frommulberry leaves
35 Determination of the Antihyperuricemic Effects of theFractions In Vivo The serum uric acid concentration in ratsis considerably low because it is metabolized into allantoinby the enzyme uricase Therefore uric acid accumulationwas induced by a single administration of K-oxonate forthe in vivo investigation of the antihyperuricemic properties
of the fractions Results are shown in Figure 3 Allopurinol(50mgkg intraperitoneally) used as positive control sub-stantially and significantly decreased the accumulation ofuric acid The lowest dose (30mgkg) of FR1 the highestdose (120mgkg) of FR2 and 60mgkg of FR5 exertedantihyperuricemic actions comparable to that of allopurinolUnexpectedly treatment with FR-W (60 and 120mgkg)resulted in an elevation of serum uric acid levels No cleardose-response relationships were detected for some of thefractions
The renal excretion of uric acid is a complex and species-dependent procedure involving its glomerular filtrationtubular secretion and tubular reabsorption Since theseprocesses can independently be modulated with exogenous
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
600000
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)586e + 004 121e + 005 184e + 005 246e + 005 309e + 005 371e + 005 434e + 005 496e + 005
120 160 200 240
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
60
60
100
100
140
140
180
180
220
220
20
20
Inte
nsity
(120583AU
)
1
2
3
(a)
00 40 80
Retention time (min)120 160 200 24060
60
100
100
140
140
180
180
220
220
20
20
500000
400000
300000
200000
100000
0
00 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
(b)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
400000
350000
300000
250000
200000
150000
100000
50000
0
469e + 004 969e + 004 147e + 005 197e + 005 247e + 005 297e + 005 347e + 005 397e + 005
Inte
nsity
(120583AU
)
1
23
(c)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
minus10000
105e + 004 218e + 004 330e + 004 443e + 004 555e + 004 668e + 004 780e + 004 893e + 004
Inte
nsity
(120583AU
)
(d)
70000
60000
50000
40000
30000
20000
10000
0
minus10000
703e + 003 145e + 004 220e + 004 295e + 004 370e + 004 445e + 004 520e + 004 595e + 004
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
4
(e)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
527e + 003 109e + 004 165e + 004 221e + 004 278e + 004 334e + 004 390e + 004 446e + 004
Inte
nsity
(120583AU
)
(f)
Figure 1 Continued
6 Evidence-Based Complementary and Alternative Medicine
140000
120000
100000
80000
60000
40000
20000
0
164e + 004 339e + 004 514e + 004 689e + 004 864e + 004 104e + 005 121e + 005 139e + 005
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
f
(g)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
2
3
f
(h)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
12
3
(i)
Figure 1 HPLC-DAD fingerprints of the samples and the correspondingmaximum absorbance chromatograms from 120582 = 200 to 450 nm (a)Crude extract (EX) (b) FR-W (c) FR-B and (d)ndash(i) FR1-FR6 respectivelyMarked peaks represent chlorogenic acid (1) rutin (2) isoquercitrin(3) loliolide (4) and an unidentified flavone derivative (f)
found to exert strong effect in this test [19] The most likelyexplanation is the low relative amount of the constituentsresponsible for this effect based on which we can alsoconclude that increasing the glucose consumption of adiposetissue has little importance in the complex anti-diabeticactivity of less processed phytotherapeutics originated frommulberry leaves
35 Determination of the Antihyperuricemic Effects of theFractions In Vivo The serum uric acid concentration in ratsis considerably low because it is metabolized into allantoinby the enzyme uricase Therefore uric acid accumulationwas induced by a single administration of K-oxonate forthe in vivo investigation of the antihyperuricemic properties
of the fractions Results are shown in Figure 3 Allopurinol(50mgkg intraperitoneally) used as positive control sub-stantially and significantly decreased the accumulation ofuric acid The lowest dose (30mgkg) of FR1 the highestdose (120mgkg) of FR2 and 60mgkg of FR5 exertedantihyperuricemic actions comparable to that of allopurinolUnexpectedly treatment with FR-W (60 and 120mgkg)resulted in an elevation of serum uric acid levels No cleardose-response relationships were detected for some of thefractions
The renal excretion of uric acid is a complex and species-dependent procedure involving its glomerular filtrationtubular secretion and tubular reabsorption Since theseprocesses can independently be modulated with exogenous
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
140000
120000
100000
80000
60000
40000
20000
0
164e + 004 339e + 004 514e + 004 689e + 004 864e + 004 104e + 005 121e + 005 139e + 005
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
Inte
nsity
(120583AU
)
f
(g)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
2
3
f
(h)
00 40 80
Retention time (min)120 160 200 24060 100 140 180 22020
60 100 140 180 2202000 40 80
Retention time (min)120 160 200 240
200
300
400
Wav
eleng
th (n
m)
600000
500000
400000
300000
200000
100000
0
703e + 004 145e + 005 220e + 005 295e + 005 370e + 005 445e + 005 520e + 005 595e + 005
Inte
nsity
(120583AU
)
12
3
(i)
Figure 1 HPLC-DAD fingerprints of the samples and the correspondingmaximum absorbance chromatograms from 120582 = 200 to 450 nm (a)Crude extract (EX) (b) FR-W (c) FR-B and (d)ndash(i) FR1-FR6 respectivelyMarked peaks represent chlorogenic acid (1) rutin (2) isoquercitrin(3) loliolide (4) and an unidentified flavone derivative (f)
found to exert strong effect in this test [19] The most likelyexplanation is the low relative amount of the constituentsresponsible for this effect based on which we can alsoconclude that increasing the glucose consumption of adiposetissue has little importance in the complex anti-diabeticactivity of less processed phytotherapeutics originated frommulberry leaves
35 Determination of the Antihyperuricemic Effects of theFractions In Vivo The serum uric acid concentration in ratsis considerably low because it is metabolized into allantoinby the enzyme uricase Therefore uric acid accumulationwas induced by a single administration of K-oxonate forthe in vivo investigation of the antihyperuricemic properties
of the fractions Results are shown in Figure 3 Allopurinol(50mgkg intraperitoneally) used as positive control sub-stantially and significantly decreased the accumulation ofuric acid The lowest dose (30mgkg) of FR1 the highestdose (120mgkg) of FR2 and 60mgkg of FR5 exertedantihyperuricemic actions comparable to that of allopurinolUnexpectedly treatment with FR-W (60 and 120mgkg)resulted in an elevation of serum uric acid levels No cleardose-response relationships were detected for some of thefractions
The renal excretion of uric acid is a complex and species-dependent procedure involving its glomerular filtrationtubular secretion and tubular reabsorption Since theseprocesses can independently be modulated with exogenous
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
HO
HO
HO
COOH
S
S S SS
S
S
R R
RRR R
RR
RR R
RR
R
R
R O
O
O O
O
OO
OO
HO
HOHO
HO
Glu
NH
NH
NH
H
1-DN Gal-DN
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH OH
OH
OH
Fagomine
1 2 R = 120572-L-rhamnopyranosyloxy3 R = H
4
Figure 2 Chemical structures of the compounds identified in the fractions chlorogenic acid (1) rutin (2) isoquercitrin (3) loliolide (4)1-deoxynojirimycin (1-DN) 2-O-alpha-D-galactopyranosyl-1-deoxynojirimycin (Gal-DN) and fagomine Glu 120573-D-glucopyranosyloxy
9
8
7
6
5
4
3
2
1
0
Basa
l
Con
trol
Allo
purin
ol
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowastlowast
lowastlowast
lowastlowast
lowastlowast lowastlowast
Seru
m u
ric ac
id (m
gdL
)plusmnSE
M
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 3 In vivo antihyperuricemic activity of the fractions obtained Hyperuricemia was induced by a single administration of K-oxonate50mgkg of allopurinol was used as positive control and samples were tested at 30 60 and 120mgkg lowast and lowastlowast 119875 lt 005 and 001respectively as compared to the negative control by means of one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8
substances the overall action of a drug is frequently biphasicAspirin at dosages of gt3 gday promotes uricosuria by inhibi-tion of the reabsorption while lower dosages (1-2 gday) maycause uric acid retention presumably by interfering with thetubular secretion [32] This together with the chemical com-plexity of the fractions could provide a simple explanation forthe unclear dose-response relationships observed in certaincases several interactions between various constituents of thefractions might take place in which the active componentswould influence these renal functions differently
36 Determination of the Serum Antioxidant Capacity Theantioxidant capacities of the serum samples of the treatedanimals were determined by means of a photometric assay in
which 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) is converted into a chromogen radical cation(ABTS∙+) The generation of radical cation is suppressed byantioxidants and can be detected as a decrease of color inten-sity results of this assay are shown in Figure 4 Treatmentwith trolox (10mgkg) resulted in a substantial increase inthe antioxidant capacity (expressed as trolox equivalents) butmost of the tested fractions failed to induce significant changein this serum parameter The higher doses of FR6 (60 and120mgkg) exhibited similar antioxidant effects to that oftrolox which effect can most likely be attributed to the highchlorogenic acid (1) rutin (2) and isoquercitrin (3) content ofthis fraction On the other hand 30mgkg of FR3 decreasedthe oxidative status of the serum indicating a prooxidantpotential for certain constituents
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
FR130
mg
kg
FR160
mg
kg
FR1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
Con
trol
lowast lowast lowast06
05
04
03
02
01
00
minus01
minus02
minus03
minus04
minus05
Trol
ox
Trol
ox eq
uiva
lent
(mM
)plusmnSE
M
lowastlowastFR
330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 4 Effects on the serum antioxidant capacity in vivo lowast119875 lt 005 as compared to the negative control by one-way ANOVA followed byDunnettrsquos multiple comparison test 119899 = 8
Con
trol
FR130
mg
kg
FR160
mg
kgFR
1120
mg
kg
FR230
mg
kg
FR260
mg
kg
FR2120
mg
kg
lowast
lowastlowast
lowastlowastlowastlowastlowastlowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowastlowast
Plas
ma g
luco
se (m
M)plusmn
SEM
Fasti
ngG
liben
clam
ide
9
8
7
6
5
4
3
2
1
0
FR330
mg
kg
FR360
mg
kg
FR3120
mg
kg
FR430
mg
kg
FR460
mg
kg
FR4120
mg
kg
FR530
mg
kg
FR560
mg
kg
FR5120
mg
kg
FR630
mg
kg
FR660
mg
kg
FR6120
mg
kg
FRW
30
mg
kg
FRW
60
mg
kg
FRW
120
mg
kg
Figure 5 Effects on the postprandial hyperglycemia of normal rats after starch loadinglowast andlowastlowast119875 lt 005 and 001 respectively as comparedto the negative control by one-way ANOVA followed by Dunnettrsquos multiple comparison test 119899 = 8 The fasting blood glucose level wasdetermined in preliminary experiments and its dataset was not included in the statistical evaluation
37 Determination of the Postprandial Plasma Glucose LevelsTreatment with the positive control glibenclamide (10mgkg)resulted in a substantial and significant decrease of thepostprandial plasma glucose levels obtained in the starch loadmodel FR1 was inactive up to 120mgkg while FR3 exhibitedantihyperglycemic action at all applied doses Fractions FR2FR-W and FR5 were effective at 120 60 and 120mgkgrespectively Fractions FR4 and FR6 exhibited some anti-hyperglycemic properties but no clear dose-response rela-tionships were observed results are shown in Figure 5 Itis worthy to note however that the present in vivo model
investigated the effect of a single-dose administration onthe postprandial hyperglycemia of normal rats after starchloading therefore these results can hardly be compared withthose we obtained previously for a longer treatment in type 2diabetic rats with ad libitum access to standard food [16]
4 Conclusions
The investigated extract ofM alba showedmultiple beneficialbioactivities in view of both targeted chronic metabolicdiseases It can also be concluded based on the different
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9
Table 1 In vitro antioxidant activities as calculated from the DPPHassay and from the inhibition of the spontaneous lipid peroxidation
Sample DPPH Lipid peroxidationEC50 (120583gmL) IC50 (120583gmL)
FR-W 722 710FR1 mdasha mdasha
FR2 mdasha 1380FR3 1860 473FR4 2450 1140FR5 550 777FR6 217 558Trolox 218 425Trolox positive control in both casesaExhibited no substantial activity in the applied concentration range
effects exerted by the fractions that separate compoundgroups are responsible for the individual actions which havea significant potential for a positive combined effect
FR1 FR2 and FR5 are of interest in view of the anti-hyperuricemic activity as potential uricosuric agents FR2ndash6were all found valuable for their antihyperglycemic activitiesbut FR3 was the strongest exerting significant effects at alladministered doses Somewhat unsurprisingly FR6 contain-ing most of the phenolic constituents was found to be thestrongest antioxidant both in vitro and in vivo which makesthis fraction particularly useful against the high oxidativestress present in both diabetes and chronic hyperuricemia
Furthermore considering our original objectives frac-tions of relatively low value could also be revealed Althoughthe water phase of the first solvent-solvent extraction (FR-W) could effectively scavenge DPPH radicals in vitro andshowed at least a weak antihyperglycemic activity in vivo(possibly due to the low iminosugar content of our sample)this fraction was also found to significantly increase theplasma uric acid levels hence it is potentially unwanted ina well-designed mulberry preparation FR1 the less polarfraction that was eluted with dichloromethane from the silicacolumn was found inactive in most bioactivity tests exceptfor its non-dose-dependent antihyperuricemic activity at 30mgkg in vivo This might be of interest for further researchbut due to the nearly 20 amount of this fraction by weightin the dry butanolic phase removing this polarity range ofconstituents could as well be considered for increasing theoverall therapeutic benefits of a phytotherapeutic product
Acknowledgments
Thisworkwas supported by theHungarianNational ResearchFund (OTKA PD75383) and it was performed within theframework of a bilateral mobility Grant from the NationalScience Council Taiwan and the Hungarian Academy ofSciences (102-2911-I-037-501 and SNK-792013) The authorsacknowledge financial support from the Pick Szeged ZrtSzeged Hungary Grants from the EuropeanUnion cofundedby the European Social Fund (TAMOP-422B-101-2010-0012 and TAMOP-422A-111KONV-2012-0035) the Grant
from the National Science Council of Taiwan (NSC 101-2314-B-037-033) and support of the Fundacao para a Cienciae a Tecnologia Portugal (PEsT-OESAUUI00742011) AMartins acknowledges theGrant SFRHBPD811182011 FCTPortugal and Professor Leonard Amaral for scientific dis-cussion The authors wish to express their special thanks toIbolya Heverne Herke for her contribution to the lab work
References
[1] E W Campion R J Glynn and L O DeLabry ldquoAsymptomatichyperuricemia risks and consequences in the normative agingstudyrdquoTheAmerican Journal of Medicine vol 82 no 3 pp 421ndash426 1987
[2] E Krishnan B J Pandya L Chung and O Dabbous ldquoHyper-uricemia and the risk for subclinical coronary atherosclerosisdata from a prospective observational cohort studyrdquo ArthritisResearch ampTherapy vol 13 p R66 2011
[3] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009
[4] P Pacher A Nivorozhkin and C Szabo ldquoTherapeutic effects ofxanthine oxidase inhibitors renaissance half a century after thediscovery of allopurinolrdquo Pharmacological Reviews vol 58 no1 pp 87ndash114 2006
[5] L Changgui H Ming-Chia and C Shun-Jen ldquoMetabolicsyndrome diabetes and hyperuricemiardquo Current Opinion inRheumatology vol 25 no 2 pp 210ndash216 2013
[6] M A Suriyajothi R Sangeetha and R Venkateswari ldquoActivityof Xanthine oxidase in diabetics its correlation with agingrdquoPharmacologyonline vol 2 pp 128ndash133 2011
[7] S Ryu J Song B-Y Choi et al ldquoIncidence and risk factors formetabolic syndrome in Korean male workers ages 30 to 39rdquoAnnals of Epidemiology vol 17 no 4 pp 245ndash252 2007
[8] X Sui T S Church R A Meriwether F Lobelo and S NBlair ldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008
[9] H Ito M Abe M Mifune et al ldquoHyperuricemia is inde-pendently associated with coronary heart disease and renaldysfunction in patientswith type 2 diabetesmellitusrdquoPloSONEvol 6 no 11 Article ID e27817 2011
[10] C L T Chang Y Lin A P Bartolome Y C Chen S C Chiuand W C Yang ldquoHerbal therapies for type 2 diabetes mellituschemistry biology and potential application of selected plantsand compoundsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 378657 33 pages 2013
[11] Z Wang J Wang and P Chan ldquoTreating type 2 diabetesmellitus with traditional Chinese and Indian medicinal herbsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 343594 17 pages 2013
[12] D Bensky and A Gamble Chinese Herbal Medicine MateriaMedica Eastland Press Seattle Wash USA 1986
[13] J Anjaria M Parabia G Bhatt and R KhamarNature Heals AGlossary of Selected Indigenous Medicinal Plants of India SristiInnovations Ahmedabad India 2002
[14] A N B Singab H A El-Beshbishy M Yonekawa T Nomuraand T Fukai ldquoHypoglycemic effect of Egyptian Morus albaroot bark extract effect on diabetes and lipid peroxidation of
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Evidence-Based Complementary and Alternative Medicine
streptozotocin-induced diabetic ratsrdquo Journal of Ethnopharma-cology vol 100 no 3 pp 333ndash338 2005
[15] I Lemus R Garcıa E Delvillar and G Knop ldquoHypoglycaemicactivity of four plants used in Chilean popular medicinerdquoPhytotherapy Research vol 13 no 2 pp 91ndash94 1999
[16] A Hunyadi A Martins T J Hsieh A Seres and I ZupkoldquoChlorogenic acid and rutin play amajor role in the in vivo anti-diabetic Activity of Morus alba leaf extract on type II diabeticratsrdquo PloS ONE vol 7 no 11 Article ID e50619 2012
[17] M Mudra N Ercan-Fang L Zhong J Furne and M LevittldquoInfluence of mulberry leaf extract on the blood glucose andbreath hydrogen response to ingestion of 75 g sucrose by type 2diabetic and control subjectsrdquo Diabetes Care vol 30 no 5 pp1272ndash1274 2007
[18] T Kimura K Nakagawa H Kubota et al ldquoFood-grade mul-berry powder enriched with 1-deoxynojirimycin suppresses theelevation of postprandial blood glucose in humansrdquo Journal ofAgricultural and Food Chemistry vol 55 no 14 pp 5869ndash58742007
[19] A Hunyadi K Veres B Danko et al ldquoIn vitro anti-diabeticactivity and chemical characterization of an apolar fraction ofMorus alba leaf water extractrdquo Phytotherapy Research vol 27pp 847ndash851 2013
[20] Y W Shia C P Wang X Wang et al ldquoUricosuric and neph-roprotective properties of Ramulus Mori ethanol extract inhyperuricemic micerdquo Journal of Ethnopharmacology vol 143no 3 pp 896ndash904 2012
[21] C-P Wang Y Wang X Wang et al ldquoMulberroside A pos-sesses potent uricosuric and nephroprotective effects in hyper-uricemic micerdquo Planta Medica vol 77 no 8 pp 786ndash794 2011
[22] Z Yu P FWing andCHKCheng ldquoThedual actions ofmorin(3572101584041015840-pentahydroxyflavone) as a hypouricemic agent uri-cosuric effect and xanthine oxidase inhibitory activityrdquo Journalof Pharmacology and Experimental Therapeutics vol 316 no 1pp 169ndash175 2006
[23] Z Yu P F Wing and C H K Cheng ldquoMorin (3572101584041015840-pentahydroxyflavone) exhibits potent inhibitory actionson urate transport by the human urate anion transporter(hURAT1) expressed in human embryonic kidney cellsrdquo DrugMetabolism and Disposition vol 35 no 6 pp 981ndash986 2007
[24] K Nakagawa K Ogawa O Higuchi T Kimura T MiyazawaandMHori ldquoDetermination of iminosugars inmulberry leavesand silkworms using hydrophilic interaction chromatography-tandem mass spectrometryrdquo Analytical Biochemistry vol 404no 2 pp 217ndash222 2010
[25] H G Vogel Ed Drug Discovery and Evaluation Pharmacolog-ical Assays Springer Berlin Germany 2002
[26] Z Hajdu J Hohmann P Forgo et al ldquoDiterpenoids andflavonoids from the fruits of Vitex agnus-castus and antioxidantactivity of the fruit extracts and their constituentsrdquo Phytother-apy Research vol 21 no 4 pp 391ndash394 2007
[27] I Zupko J Hohmann D Redei G Falkay G Janicsak andI Mathe ldquoAntioxidant activity of leaves of Salvia species inenzyme-dependent and enzyme-independent systems of lipidperoxidation and their phenolic constituentsrdquo Planta Medicavol 67 no 4 pp 366ndash368 2001
[28] I Fridovich ldquoThe competitive inhibition of uricase by oxonateand by related derivatives of s-triazinesrdquoThe Journal of biologi-cal chemistry vol 240 pp 2491ndash2494 1965
[29] P Trinder ldquoDetermination of glucose in blood using oxidasewith an alternative oxygen acceptorrdquo Annals of Clinical Bio-chemistry vol 6 pp 24ndash27 1969
[30] C J Zuurbier F J Hoek J Van Dijk et al ldquoPerioperative hyper-insulinaemic normoglycaemic clamp causes hypolipidaemiaafter coronary artery surgeryrdquoBritish Journal of Anaesthesia vol100 no 4 pp 442ndash450 2008
[31] R Minorics T Szekeres G Krupitza et al ldquoAntiproliferativeeffects of some novel synthetic solanidine analogs on HL-60human leukemia cells in vitrordquo Steroids vol 76 no 1-2 pp 156ndash162 2011
[32] D Caspi E Lubart E Graff B Habot M Yaron and R SegalldquoThe effect of mini-dose aspirin on renal function and uric acidhandling in elderly patientsrdquo Arthritis and Rheumatism vol 43pp 103ndash108 2000
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom