researcharticle smallanthus sonchifolius) leaf extract...

Research ArticleYacon (Smallanthus sonchifolius) Leaf ExtractAttenuates Hyperglycemia and Skeletal Muscle Oxidative Stressand Inflammation in Diabetic Rats

Klinsmann Carolo dos Santos12 Bianca Guerra Bueno1 Luana Ferreira Pereira1

Fabiane Valentini Francisqueti1 Mariana Gobbo Braz1 Lahis Fernandes Bincoleto1

Lilian Xavier da Silva3 Ana Luacutecia A Ferreira1 Ana Claacuteudia de Melo Stevanato Nakamune3

C-Y Oliver Chen2 Jeffrey B Blumberg2 and Camila Renata Correcirca1

1Sao Paulo State University (UNESP) Medical School Botucatu SP Brazil2Antioxidants Research Laboratory Jean Mayer USDA Human Nutrition Research Center on Aging Tufts UniversityBoston MA USA3Sao Paulo State University (UNESP) Dentistry School Aracatuba SP Brazil

Correspondence should be addressed to Camila Renata Correa correacamila9gmailcom

Received 15 March 2017 Revised 6 June 2017 Accepted 12 June 2017 Published 20 July 2017

Academic Editor Luigi Milella

Copyright copy 2017 Klinsmann Carolo dos Santos et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The effects of hydroethanolic extract of Yacon leaves (HEYL) on antioxidant glycemic and inflammatory biomarkers were tested indiabetic rats Outcome parameters included glucose insulin interleukin-6 (IL-6) and hydrophilic antioxidant capacity (HAC) inserum and IL-6 HAC malondialdehyde (MDA) superoxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GPx)in soleusThe rats (10group) were divided as follows C controls C + Y HEYL treated DM diabetic controls andDM+Y diabeticrats treated with HEYL Diabetes mellitus was induced by administration of streptozotocin C + Y and DM + Y groups received100mgkg HEYL daily via gavage for 30 d Hyperglycemia was improved in the DM + Y versus DM group Insulin was reduced inDM versus C group DM rats had higher IL-6 and MDA and lower HAC in the soleus muscle HEYL treatment decreased IL-6 andMDA and increased HAC in DM rats DM + Y rats had the highest CAT activity versus the other groups GPx was higher in C +Y and DM + Y versus their respective controls The apparent benefit of HEYL may be mediated via improving glucoregulation andameliorating oxidative stress and inflammation particularly in diabetic rats

1 Introduction

Type 1 diabetes mellitus (T1DM) occurs by autoimmune-mediated destruction of pancreatic 120573-cells leading to insulindeficiency and loss of glycemic control The hyperglycemiathat occurs in diabetes increases the production of reactiveoxygen species (ROS) and weakens antioxidant defenseresulting in enhanced oxidative stress [1] ROS mediateseveral biochemical and molecular pathways that can exacer-bate oxidative stress [2] such as activating the transcriptionfactor nuclear factor kappa B (NF-120581B) which increases thetranscription of inflammatory cytokines and chemokines [3]

promoting inflammation Moreover uncontrolled ROS gen-eration could also attack the cellular proteins lipids andnucleic acids leading to cellular dysfunction including loss ofenergy metabolism alteration on cell signaling and cell cyclecontrol mutations and inflammation In addition it playsa role in several pathological processes in skeletal muscle[4] These reactive species are important signaling moleculesnecessary for muscle function and for adaptive response tostress [5] However overproduction of ROS and decreaseof the antioxidant defense have negative impact on musclefunction as impairedmuscle growth and strength and alteredmetabolic capacity [6]

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 6418048 9 pageshttpsdoiorg10115520176418048

2 Evidence-Based Complementary and Alternative Medicine

Medicinal plants are widely used as alternative therapeu-tics for the prevention or treatment of diseases Recently greatattention has been paid to the use of natural compoundsdue to their nutritional and pharmacological characteristics[7]

Yacon (Smallanthus sonchifolius [Poepp amp Endl] HRobinson Asteraceae) is a native Andean plant cultivated forits tubers which are commonly used as a food in SouthAmer-ica Some studies have reported the presence of large amountsof phenolic compounds in extracts from Yacon leaves andtubers mainly chlorogenic protocatechuic ferulic ros-marinic gallic gentisic and caffeic acids and their derivatives[8] Evidence has also emerged about the antioxidant acti-vity [9] protective effects on oxidative damage and glucosemetabolism in rat hepatocytes and insulin-like effects ofYacon leaf extracts [10 11]

Antioxidant compounds have long been known to dimin-ish inflammatory and oxidative stress responses In additionantioxidants scavenge ROS and increase the capacity of theantioxidant defense enzyme system [2] Therefore antioxi-dants can help diminish oxidative damage and inflammationand slow or prevent the progression of diabetic complica-tions

Natural products are the groundwork of preventing andcuring several diseases Moreover ethnopharmacologicalknowledge is one attractive way to enhance the probability ofsuccess in new drug-finding efforts Regarding diabetic com-plications plants that can be effectively used based on theirtherapeutic applications for example for diabetes areworthyof special attention and studies are needed regarding theirside effects the ability to maintain normal levels of glycemiaand their possible control on oxidative stress and inflamma-tion Considering the complications of type 1 diabetes andprevious data on Yaconrsquos activities the present study wasundertaken to elucidate the antioxidant anti-inflammatoryand antihyperglycemic activity of hydroethanolic extractfrom S sonchifolius leaves (HEYL) in the serum and skeletalmuscle of STZ-induced diabetic rats

2 Materials and Methods

21 Plant Material and Extract Preparation The leaves ofS sonchifolius were collected in June (2014) by KlinsmannCarolo dos Santos in Curitiba PR Brazil The specimen wasprovided by Dr Atila FranciscoMogor from the Departmentof Plant Science and Crop Protection Federal Universityof Parana Curitiba Parana Brazil and identified by DrLin Chau Ming from Sao Paulo State University (UNESP)School of Agriculture Botucatu SP Brazil and the voucherspecimen was deposited to the Herbarium at the Sao PauloState University (UNESP) Institute of Biosciences BotucatuSP Brazil under the register 32752 for future referenceThe leaves from S sonchifolius were dried for seven daysat 50∘C powdered (3 120583m) and subjected to percolationat room temperature using a mixture of ethanol H2O(7 3 vv) with a flux of 20mLminkg The solvents wereevaporated to dryness under a low pressure (45∘C) usingrotary evaporator in vacuum system to afford the crudeHEYL

22 Characterization of Phenolics For the characterization ofphenolics 10mg HEYL was reconstituted in 1mL methanolfollowed by acidic hydrolysis with 1mL of 24M HCl at80∘C for 2 h in the dark After the incubation the solutionwas filtered through a 045120583m nylon membrane (Milli-pore Corp Bedford MA) and then injected onto a HPLCsystem equipped with a Zorbax SB-C18 column (46 times250mm 35 120583m) and a CoulArray 5600A electrochemicaldetector (ESA Inc Chelmsford MA) Phenolic acids andflavonoidswere quantified according to themethod of Li et al(2009) [12] The limits of quantitation for phenolic acid andflavonoids were 1 ng on column The linearity of calibrationcurves of authentic standards with concentrations rangingfrom 001 to 2 ngml was at least ge0991 The identificationof each compound was based on a comparison of the reten-tion time and electrochemical response of the authenticatedstandards The results are expressed in 120583g100mg HEYL

23 Dose-Response Profile of HEYL Treatment To establisha dose-response profile for the antihyperglycemic activityof Yacon leaves we used varying doses of HEYL (25 50and 100mgkg body weightday constituted in 1mL of 09saline) to identify the lowest dose that could elicit an optimalantihyperglycemic effect FifteenmaleWistar rats 60 d of agewere maintained in an environmentally controlled room (22plusmn 3∘C 12-hour lightdark cycle and relative humidity of 60 plusmn5) and were fed with a standard rat pellet diet (Purina LtdCampinas SP Brazil) and water ad libitumThe animals wererandomly assigned to one of three groups HEYL 25 HEYL50 and HEYL 100 Diabetes mellitus was induced by one ipadministration of streptozotocin (STZ 40mgbody weight)and the animals received HEYL for gavage for 2 weeks afterthe establishment of diabetic condition We found that after2 weeks of the treatment the highest dose showed the mostpotent hyperglycemic effect in STZ model of diabetes Thusthe subsequent experimentswithHEYLwere carried outwiththe dose of 100mgkg administered orally

24 Animals and Experimental Groups Forty male Wistarrats 60 d of age were maintained in an environmentally con-trolled room (22 plusmn 3∘C 12-hour lightdark cycle and relativehumidity of 60 plusmn 5) and were fed with a standard rat pelletdiet (Purina Ltd Campinas SP Brazil) and water ad libitumThe experimental protocol was approved by the Ethics Com-mittee on theUse ofAnimals (CEUA) at theBotucatuMedicalSchool Sao Paulo State University (UNESP) under number1082-2014 (approved in April 24 2014) The animals wererandomly assigned to one of four groups (n = 10) C (controlgroup) normal rats C + Y normal rats receiving HEYL DMdiabetic rats and DM+Y diabetics rats receiving HEYLDia-betes mellituswas induced by ip administration of streptozo-tocin for one time (STZ 40mgbody weight) Blood glucosewas measured 48 h and 7 days after the STZ administrationThe animals with blood glucose greater than 250mgdL wereconsidered diabetic The animals received HEYL (100mgkgbody weightday constituted in 1mL of 09 saline) for gav-age for 30 days after the 7th day of established diabetic condi-tion Control animals were given the same volume of salineThe animals were fasted overnight and killed by decapitation

Evidence-Based Complementary and Alternative Medicine 3

after anaesthesia with ketamine (50mgkg) and xylazine(05mgkg) by intraperitoneal injection and all efforts weremade to minimize suffering Blood was collected in tubesand then centrifuged at 3500 rpmtimesg The serum and soleusmuscle were collected and stored at minus80∘C until analysis

25 Preparation of the Soleus Muscle for Analysis Soleusmuscle was weighed (100mg) and homogenized in 10mLcold PBS (pH 74) using ULTRA-TURRAX T25 basic IKAWerke StaufenGermany After centrifugation at 800timesg at4∘C for 10min the supernatant was collected for mal-ondialdehyde (MDA) and IL-6 determinations For theantioxidant enzymes determination 100mg soleus musclewas homogenized (1 10 vv) in KH2PO4 (10mmolL)KCl(120mmolL) pH 74 and centrifuged at 2000timesg for 20min

26 Biochemical Measurements in Serum and Soleus MuscleAn enzymatic colorimetric kit was used to measure serumglucose (Bioclin Belo Horizonte Minas Gerais Brazil)Insulin (Immuno-Biological Laboratories Inc) and IL-6(RampD Systems Inc) were measured by an immunoassayusing a microplate reader (Spectra Max 190 MolecularDevices)

27 Pancreatic Beta-Cell Function Pancreatic beta-cell func-tion was determined using the index of homeostasismodel assessment (HOMA) [13] using the following for-mula HOMA-BETA (Homeostasis Model Assessment Beta-Cell Function) = 20 times Fasting Insulin (120583UmL)FastingGlucose (mM) minus 35

28 Malondialdehyde (MDA) Analysis in Soleus Muscle A100 120583L aliquot of soleus muscle homogenate was used forMDA analysis Briefly we added 700 120583L of 1 orthophos-phoric acid and 200120583L of thiobarbituric acid (42mM) tothe sample and then boiled it for 60min in a water baththe sample was cooled on ice immediately after that Twohundred 120583L was transferred to a 2mL tube containing200120583L sodium hydroxide-methanol (1 12 vv) The samplewas vortex-mixed for 10 s and centrifuged for 3min at13000 timesg The supernatant (200120583L) was transferred to a300 120583L glass vial and 50 120583L injected onto the column TheHPLC was a Shimadzu LC-10AD system (Kyoto Japan)equipped with a C18 Luna column (5 120583m 150 times 460mmPhenomenex Inc Torrance CA USA) a Shimadzu RF-535fluorescence detector (excitation 525 nm emission 551 nm)and 05mLmin flowof phosphate buffer (KH2PO4 1mM pH68) [14] MDA was quantified by area determination of thepeaks in the chromatograms relative to a standard curve ofknown concentrations

29 Measurement the Hydrophilic Antioxidant Capacity(HAC) in Serum and Soleus Muscle The hydrophilic antiox-idant capacity was determined fluorometrically as describedby Beretta et al (2006) [15] using a VICTOR X2 reader(Perkin Elmer Boston MA) The antioxidant activity wasquantitated by comparing the area under the curve relating tothe oxidation kinetics of the suspension phosphatidylcholine

(PC) which was used as reference biological matrixThe per-oxyl radical 2101584021015840-azobis-(2-amidinopropane) dihydrochlo-ride (AAPH) was used as an initiator of the reaction Theresults represent the percent inhibition (44 difluoro-5-(4-phenyl 1-3 butadiene)-4-bora-34-diaza-s-indacene) (BOD-IPY) 581591 plasma with respect to the control sample ofBODIPY 581591 PC liposome All analyses were performedin triplicate The results are reported as percentage of protec-tion

210 Antioxidant Enzymes Activity Evaluation in Soleus Mus-cle Superoxide dismutase activity was measured based onthe inhibition of a superoxide radical reactionwith pyrogalloland the absorbance values were measured at 420 nm [16]The values are expressed as units per milligram of proteinCatalase activity was evaluated by following the decrease inthe levels of hydrogen peroxide The absorbance values weremeasured at 240 nm [17] The activity is expressed as pmoleof H2O2 reducedminmg protein Glutathione peroxidaseactivity was measured by following 120573-nicotinamide adeninedinucleotide phosphate (NADPH) oxidation at 340 nm asdescribed by Flohe and Gunzler (1984) [18] the results wereexpressed as 120583mol hydroperoxide reducedminmg proteinProtein was quantified based on Lowryrsquos method [19] usingbovine serum albumin as the standard

211 Statistical Analysis Results are expressed as mean andstandard error of the mean (SEM) and significance wascalculated by two-way ANOVA followed by Holm-Sidakmethod The software used was SigmaStat version 35 forWindows (Systat Software Inc San Jose CA USA) Differ-ences were considered significant at 119875 lt 005

3 Results

31 Glycemia Insulin and HOMA-BETA STZ-induced dia-betic rats (DM and DM + Y) showed 325- and 308-foldrespectively higher blood glucose levels than the controlgroups (Figure 1(a)) in the beginning of the experiment (7 dafter administration of STZ) After treatment with HEYL theDM + Y animals showed reduction of glycemia to valuessimilar to the controls (Figure 1(b)) The insulin was lowerin DMwhen compared with the control groups (Figure 1(c))The same was found for HOMA-BETA DM presented thelowest values when compared to C and DM + Y whereasthe treated DM group presented increase of HOMA-BETA(Figure 1(d))

32 Characterization of Phenolics Ten phenolics in thehydroethanolic extract of Yacon were quantified using aHPLC-ECD method The active principles with their con-centrations retention time (RT) and peak area (120583C) arepresented in Table 1

33 Antioxidant Enzymes and Lipoperoxidation Marker inSoleus Muscle The treatment with Yacon leaves in DM + Ydecreased MDA (Figure 3(d)) in soleus muscle when com-pared to DM which presented the highest values for thisvariable DM + Y group presented the highest catalase


C C + Y DM DM + Y

500

400

300

200

100

0

P lt 0001

P lt 0001

Initi

al g

lyce

mia

(mg

dL)

7da

ys a

er S

TZ

(a)C C + Y DM DM + Y

400

300

200

100

0

P lt 0001 P lt 0001

Fina

l gly

cem

ia (m

gdL

)30＞

of tr

eatm

ent

(b)

C C + Y DM DM + Y

50

40

30

20

10

0

P = 0003

Insu

lin (p

mol

L)

30＞

of tr

eatm

ent

(c)C C + Y DM DM + Y

P lt 0001

P lt 0001P lt 0001

80

60

40

20

0

P = 0043

HO

MA-

BETA

30＞

of tr

eatm

ent

(d)

Figure 1 (a) Initial glycemia (7 d after STZ administration) (b) final glycemia (30 d of treatment) (c) insulin (30 d of treatment) (d) HOMA-BETA (30 d of treatment) of the different experimental groups C (control group) normal rats C + Y normal rats receiving HEYL DMdiabetic rats and DM + Y diabetics rats receiving HEYL The results are expressed as the mean plusmn SEM

Table 1 Phenolic content of hydroethanolic extract of Yacon leaves

RT (min) Name Peak area (120583C) Concentration1529 Protocatechuic acid 698 10112583 Gentisic acid 878 7643313 Chlorogenic acid 230 8173597 Vanillic acid 308 1343765 Caffeic acid 6610 27564225 Epicatechin 220 5114667 p-Coumaric acid 1950 169814978 Ferulic acid 705 13215043 Sinapic acid 641 4687535 Quercetin 1280 39900RT retention time Concentration is expressed in 120583g100mg of HEYL


C C + Y DM DM + Y

P = 0015P = 002015

10

5

0

Glu

tath

ione

per

oxid

ase

(m

olm

g pr

otei

nm

in)


(pm

olm

g pr

otei

nm

in)

Cata

lase

P = 0029

P = 00067

6

5

4

3

2

1

0

(b)

C C + Y DM DM + Y

150

120

90

60

30

0

Supe

roxi

de d

ismut

ase

(Um

g pr

otei

nm

in)


(m

olm

g of

tiss

ue)

MD

A

P lt 0001 P lt 000103

02

01

00

(d)

Figure 2 (a) Catalase (CAT) activity (b) superoxide dismutase (SOD) activity (c) glutathione peroxidase (GPx) activity (d) MDAconcentration in soleus of the different experimental groups C (control group) normal rats C + Y normal rats receivingHEYL DM diabeticrats and DM + Y diabetics rats receiving HEYL The results are expressed as the mean plusmn SEM

activity among the groups (Figure 3(b)) whereas the GPx(Figure 3(a)) was higher in C + Y and DM + Y No significantdifference was found for SOD (Figure 3(c)) among thegroups

34 Hydrophilic Antioxidant Capacity (HAC) and IL-6 inSerum and Soleus Muscle There were no significant dif-ferences for plasma IL-6 (Figure 2(c)) Plasma HAC washigher in C + Y when compared to C group although whenevaluated in soleus muscle a 22-fold increase of IL-6 with a291 decrease ofHACwas observed inDMgroup comparedto C group (Figure 2) HEYL promoted decrease of IL-6and increase of HAC in DM + Y group when compared tountreated group

4 Discussion

Streptozotocin (STZ) is a widely used chemical for theinduction of experimental diabetes [20] Type 1 diabetes canbe induced in rodents by a single STZ injection [21] Allthese STZ-induced diabetic animal models have been usefulin elucidating the mechanisms of diabetic pathogenesis and

in screening natural products and pharmacological agentsthat are potentially capable of lowering blood glucose levels[22] and attenuating the oxidative stress and inflammationUnder our experimental conditions Wistar rats treated witha single dose of 40mg STZkg bw underwent a markedhyperglycemia (395ndash416mgdL)

The administration of HEYL (100mgkgd) in diabeticanimals reduced serum glucose These results are in agree-ment with Aybar et al (2001) [23] and Genta et al (2010)[24] studies in which different extracts preparations anddoses of Yacon administered orally reduced glycemia inSTZ-induced diabetic rats although Raga et al (2010) [25]demonstrated that a dose of 100mgkg bw of Yacon teapresents more potential activity on glycemic controlThe rateof blood glucose reduction in the present study occurred insynergy with the normally functioning pancreatic cells TheDM group without treatment has the lowest concentrationsof insulin in plasma The HEYL promoted a slight increaseof insulin concentrations (Figure 1(c)) even without signif-icant difference when compared to untreated DM groupand HOMA-BETA (Figure 1(d)) suggesting regeneration offunctional 120573-cells


C C + Y DM DM + Y

P = 0001

HAC

seru

m(

of p

rote

ctio

nm

g pr

otei

n)90

80

70

60

50

40

30

20

10

0


P = 0003

P = 0004

HAC

sole

us(

of p

rote

ctio

nm

g pr

otei

n)

120

100

80

60

40

20

0

(b)

C C + Y DM DM + Y

IL-6

seru

m (p

gm

L)

03

02

01

00


P lt 0001 P lt 0001

02

01

00

IL-6

sole

us(p

g10

0Ａ

of ti

ssue

)

(d)

Figure 3 (a) Serum hydrophilic antioxidant capacity (HAC) (b) soleus HAC (c) serum interleukin-6 (IL-6) (d) soleus IL-6 of the differentexperimental groups C (control group) normal rats C + Y normal rats receiving HEYL DM diabetic rats and DM + Y diabetics ratsreceiving HEYL The results are expressed as the mean plusmn SEM

It is well known that hyperglycemia is the major cause ofdiabetic complications Oxidative stress is one of the potentialmechanisms by which hyperglycemia can result in diabeticcomplications [26] Improvement of glycemic control thatachieves near-normoglycemia can decrease the developmentand progression of its complications [25] Regeneration orprotection of pancreatic cells that were partially destroyedby STZ with increase of insulin concentrations in plasmaand probably increase in the peripheral utilization of glucosecould be factors that can explain the significant decrease offasting blood glucose in the present study [27] Additionallysome phytochemicals such as flavonoids and polyphenolshave been found to be effective due to some other extrapan-creatic mechanisms [28] Further studies are in progress toestablish the precise mechanism involved in the antihyper-glycemic effect of HEYL

It has been shown that the solvent used in the prepara-tion of plant extracts can affect positively or negatively thebiologically active principles of these plants [8] Baroni etal (2008) [29] showed that the hydroethanolic extract ofYacon leaves was the best extraction to promote reductionof glycemia in diabetic and nondiabetic animals Also thepolyphenolics in Yacon leaves may regulate the free radical

activity of STZ diabetes induction [10] and the pathogen-esis of diabetes [30] Plants rich in phenolic compoundshave potential hypoglycemic effects [31 32] Ferulic acid p-coumaric acid caffeic acid chlorogenic acid protocatechuicacid and quercetin were the highest compounds found inthe extract Jung et al (2011) [33] found that low doses ofonion peel hydroethanolic extract ameliorate hyperglycemiaand insulin resistance in high-fat dietSTZ-induced diabeticrats in 8 weeks of treatment Additionally Pereira Braga etal (2013) [34] described that isolated quercetin promotesglucose regulation and decrease of lipid peroxidation indiabetic animals Caffeic and chlorogenic acids are known fortheir antioxidant and free radical scavenging properties [35]Recently caffeic acid in particular has been associated withreduced blood glucose [36] Baroni et al (2016) [37] showedthat the phytochemical analysis of the hydroethanolic extractof Yacon identified the presence of phenolic compounds suchas caffeic acid ferulic acid gallic acid and chlorogenic acidcorroborating with the present study The phytochemicalprofile may explain the antioxidant and antihyperglycemicactivities noted in our study

It is known that the pathogenesis of DM and its compli-cations are associated with the overproduction of ROS and


depletion of the endogenous antioxidant system leading tooxidative stress [38] It is also known that skeletal muscle is aprimary tissue in the response to metabolic alteration induc-ing physiopathological stimulus Several signaling pathwaysin striated muscle can be activated by an increase in ROSproduction [39] HEYL increased the activity of catalase andGPx (Figure 3) in the soleus muscle is likely attributed toimprovement in glucose oxidation or direct modulation ofantioxidant enzymes Some reports suggest that oxidativestress is a key player to diabetic complications which maybe associated with alterations in the metabolism [40 41]In addition it has been reported that STZ induces severeoxidative stress in diabetic animals caused by the peroxida-tion of polyunsaturated fatty acids leading to the formationof MDA as by-products of lipid peroxidation [42] Excessivelipid peroxidation can readily attack the polyunsaturatedfatty acids of the lipid membrane which in turn can disruptthe structure of biological membranes and produce toxicmetabolites such asmalondialdehyde [43]MDA is often usedas a marker of oxidative damage [44 45] In summary excessROS overwhelm antioxidant defenses leading to oxidativestress

No significant alterations were found in the plasma ofdiabetic animals for IL-6 (Figure 2(c)) when compared withcontrols Although we did not observe changes in oxidativestress and inflammation markers when they were systemi-cally evaluated after Yacon treatment the leaves efficientlyreduced metabolic markers such as hyperglycemia andoxidativeinflammation stress in soleus muscle In additionwe observed that HAC decreased while MDA and IL-6increased in the soleus of diabetic animals (Figure 3) showingthe oxidative stress and inflammation in this disorder

The antioxidant activities of various vegetables fruitsand plants are mainly attributed to their content of phenoliccompounds [46] The radical scavenging activity of polyphe-nols depends on the molecular structure and the substitutionpattern of the hydroxyl groups the availability of phenolichydrogens and the possibility of stabilization of the resultingphenoxyl radicals via hydrogen donation or by expandedelectron delocalization [47]This radical scavenging ability ofextracts could be related to the nature of phenolics thus con-tributing to their election transferhydrogen donating system

In the present study for the first time the significantincrease of the antioxidant status (HAC) and endogenousantioxidant activities (GPx and CAT) and decrease ofmarkers of lipid peroxidation (MDA) and proinflammatorycytokine (IL-6) in soleus muscle in diabetic rats treated withHEYL suggest the antioxidant and anti-inflammatory activityof Yacon extract in this tissue These results indicate thatYacon leaves have significant effects on scavenging free radi-cals promoting decrease of oxidative stress under diabeticconditions

Although the HEYL promoted several benefits on STZ-induced diabetic model especially those regarding the glu-cose homeostasis and antioxidant activities this study haslimitations The precise mechanisms by which HEYL pro-moted antihyperglycemic activity and increase of insulinconcentrations have not been evaluated even if they arehypothetically attributed to regenerationpreservation of

pancreatic beta-cells But further studies are in progress toinvestigate the precise mechanismpathways involved

STZ administration induces hyperglycemia and increasesMDA and IL-6 in soleus muscle toxic intermediates in thedevelopment of oxidative stress and inflammation in dia-betes Moreover experimental diabetes decreases the capac-ity of antioxidant defenses in soleus muscle In summarythese results demonstrate hyperglycemia-induced oxidativestress in skeletal muscle of diabetic rats In conclusion thehydroethanolic extract from S sonchifolius leaves (HEYL)protects against hyperglycemia oxidative stress and inflam-mation in skeletal muscle and also promotes increase ofserum insulin concentrations in STZ-induced diabetic modelin rats These findings provide information that can guidefuture studies aimed at finding therapeutic alternatives fordiabetic complications

Conflicts of Interest

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

Authorsrsquo Contributions

All authors participated in the design interpretation of thestudies analysis of the data and review of the manuscriptKlinsmann Carolo dos Santos and Camila Renata Correaelaborated the experimental design Klinsmann Carolo dosSantos Bianca Guerra Bueno Lahis Fernandes BincoletoFabiane Valentini Francisqueti Lilian Xavier da Silva AnaClaudia de Melo Stevanato Nakamune Lahis FernandesBincoleto and Mariana Gobbo Braz conducted the experi-ments Klinsmann Carolo dos Santos Ana Claudia de MeloStevanatoNakamune C-Y Oliver Chen Jeffrey B Blumbergand Camila Renata Correa analyzed the data KlinsmannCarolo dos Santos Lahis Fernandes Bincoleto MarianaGobbo Braz C-Y Oliver Chen Jeffrey B Blumberg andCamila Renata Correa revised the manuscript KlinsmannCarolo dos Santos C-Y Oliver Chen Jeffrey B Blumbergand Camila Renata Correa wrote the manuscript

Acknowledgments

The authors acknowledge the Coordenacao de Aperfeicoa-mento de Pessoal de Nıvel Superior (CAPES) for researchgrant (PGCI 02211) and scholar stipend (BEX 942114-9)and Fundacao de Amparo a Pesquisa do Estado de Sao PauloBrazil for financial support (FAPESP Proc 201510626-0)and scholar stipend (FAPESP Proc 201417364-8) They alsothank Paulo Cesar Georgete and Corina Tomasetti for theirtechnical support and Dr Atila Francisco Mogor and Dr LinChau Ming for providing the plant material

References

[1] P-T Yeh H-W Huang C-M Yang W-S Yang and C-HYang ldquoAstaxanthin Inhibits Expression of Retinal OxidativeStress and Inflammatory Mediators in Streptozotocin-InducedDiabetic Ratsrdquo PLoS One vol 11 no 1 Article ID e01464382016


[2] K Carolo Dos Santos C Pereira Braga P Octavio Barbanera FRodrigues Ferreira Seiva A Fernandes Jr and A A HenriqueFernandes ldquoCardiac energy metabolism and oxidative stressbiomarkers in diabetic rat treated with resveratrolrdquo PLoS ONEvol 9 no 7 Article ID e102775 2014

[3] T S Kern ldquoContributions of Inflammatory Processes to theDevelopment of the Early Stages of Diabetic RetinopathyrdquoExperimental Diabetes Research vol 2007 Article ID 951032007

[4] V Rani G Deep R K Singh K Palle and U C S YadavldquoOxidative stress and metabolic disorders pathogenesis andtherapeutic strategiesrdquo Life Sciences vol 148 no 11 pp 183ndash1932016

[5] E Barbieri and P Sestili ldquoReactive Oxygen Species in SkeletalMuscle Signalingrdquo Journal of Signal Transduction pp 1ndash17 2012

[6] S K Coleman I A Rebalka DD M Souza and T J HawkeldquoSkeletal muscle as a therapeutic target for delaying type 1diabetic complicationsrdquo World Journal of Diabetes vol 6 pp1323ndash1336 2015

[7] D Russo N Malafronte D Frescura et al ldquoAntioxidantactivities and quali-quantitative analysis of different Smallan-thus sonchifolius [(Poepp and Endl) H Robinson] landraceextractsrdquoNatural Product Research vol 29 no 17 pp 1673ndash16772014

[8] B Simonovska I Vovk S Andrensek K Valentova and J Ulri-chova ldquoInvestigation of phenolic acids in yacon (Smallanthussonchifolius) leaves and tubersrdquo Journal of Chromatography Avol 1016 no 1 pp 89ndash98 2003

[9] K Valentova F Sersen and J Ulrichova ldquoRadical scavengingand anti-lipoperoxidative activities of Smallanthus sonchifoliusleaf extractsrdquo Journal of Agricultural and Food Chemistry vol53 no 14 pp 5577ndash5582 2005

[10] K Valentova L Cvak A Muck J Ulrichova and V SimanekldquoAntioxidant activity of extracts from the leaves of Smallanthussonchifoliusrdquo European Journal of Nutrition vol 42 no 1 pp61ndash66 2003

[11] K Valentova A Moncion I DeWaziers and J Ulrichova ldquoTheeffect of Smallanthus sonchifolius leaf extracts on rat hepaticmetabolismrdquoCell Biology and Toxicology vol 20 no 2 pp 109ndash120 2004

[12] L Li G Aldini M Carini et al ldquoCharacterisation extractionefficiency stability and antioxidant activity of phytonutrients inAngelica keiskeirdquo Food Chemistry vol 115 no 1 pp 227ndash2322009

[13] D R Matthews J P Hosker A S Rudenski B A Naylor DF Treacher and R C Turner ldquoHomeostasis model assessmentinsulin resistance and 120573-cell function from fasting plasmaglucose and insulin concentrations in manrdquo Diabetologia vol28 no 7 pp 412ndash419 1985

[14] D T Pierine M E L Navarro I O Minatel et al ldquoLycopenesupplementation reduces TNF-120572 via RAGE in the kidney ofobese ratsrdquoNutrition and Diabetes vol 4 no 11 article no e1422014

[15] G Beretta G Aldini R M Facino R M Russell N I Krinskyand K-J Yeum ldquoTotal antioxidant performance a validatedfluorescence assay for themeasurement of plasma oxidizabilityrdquoAnalytical Biochemistry vol 354 no 2 pp 290ndash298 2006

[16] S L Marklund ldquoProduct of extracellular-superoxide dismutasecatalysisrdquo FEBS Letters vol 184 no 2 pp 237ndash239 1985

[17] H Aebi ldquo[13] Catalase in vitrordquo Methods in Enzymology vol105 pp 121ndash126 1984

[18] L Flohe and W A Gunzler ldquoAssays of glutathione peroxidaserdquoMethods Enzymol vol 105 pp 114ndash120 1984

[19] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 pp 265ndash275 1951

[20] S Lenzen ldquoThe mechanisms of alloxan- and streptozotocin-induced diabetesrdquoDiabetologia vol 51 no 2 pp 216ndash226 2008

[21] D Yin J Tao D D Lee et al ldquoRecovery of islet 120573-cell functionin streptozotocin-induced diabetic mice an indirect role for thespleenrdquo Diabetes vol 55 no 12 pp 3256ndash3263 2006

[22] S Kumar N Vasudeva and S Sharma ldquoGC-MS analysisand screening of antidiabetic antioxidant and hypolipidemicpotential of Cinnamomum tamala oil in streptozotocin induceddiabetesmellitus in ratsrdquoCardiovascular Diabetology vol 11 no95 2012

[23] M J Aybar A N Sanchez Riera A Grau and S S SanchezldquoHypoglycemic effect of the water extract of Smallantus sonchi-folius (yacon) leaves in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 74 no 2 pp 125ndash132 2001

[24] S B Genta W M Cabrera M I Mercado A Grau C ACatalan and S S Sanchez ldquoHypoglycemic activity of leaforganic extracts from Smallanthus sonchifolius Constituents ofthe most active fractionsrdquo Chemico-Biological Interactions vol185 no 2 pp 143ndash152 2010

[25] D D Raga A B Alimboyoguen R S Del Fierro and CY Ragasa ldquoHypoglycaemic effects of tea extracts and ent-kaurenoic acid from Smallanthus sonchifoliusrdquoNatural ProductResearch vol 24 no 18 pp 1771ndash1782 2010

[26] M Brownlee ldquoThe pathobiology of diabetic complications aunifying mechanismrdquo Diabetes vol 54 no 6 pp 1615ndash16252005

[27] W J Arion W K Canfield F C Ramos et al ldquoChlorogenicacid analogue S 3483 A potent competitive inhibitor of thehepatic and renal glucose-6-phosphatase systemsrdquo Archives ofBiochemistry and Biophysics vol 351 no 2 pp 279ndash285 1998

[28] D K Patel S K Prasad R Kumar and S HemalathaldquoAn overview on antidiabetic medicinal plants having insulinmimetic propertyrdquoAsian Pacific Journal of Tropical Biomedicinevol 2 pp 320ndash330 2012

[29] S Baroni F Suzuki-Kemmelmeier S M Caparroz-Assef R KN Cuman and C A Bersani-Amado ldquoEffect of crude extractsof leaves of Smallanthus sonchifolius (yacon) on glycemiain diabetic ratsrdquo Revista Brasileira de Ciencias Farmaceuti-casBrazilian Journal of Pharmaceutical Sciences vol 44 no 3pp 521ndash530 2008

[30] M M Gupta and S Chari ldquoLipid peroxidation and antioxidantstatus in patients with diabetic retinopathyrdquo Indian Journal ofPhysiology and Pharmacology vol 49 pp 187ndash192 2005

[31] M Jung M Park H C Lee Y Kan E S Kang and S K KimldquoAntidiabetic agents from medicinal plantsrdquo Current MedicinalChemistry vol 13 no 10 pp 1203ndash1218 2006

[32] Y H Zhang J Y Cai H L Ruan H F Pi and J Z WuldquoAntihyperglycemic activity of kinsenoside a high yielding con-stituent from Anoectochilus roxburghii in streptozotocin dia-betic ratsrdquo Journal of Ethnopharmacology vol 114 no 2 pp 141ndash145 2007

[33] J Y Jung Y Lim M S Moon J Y Kim and O Kwon ldquoOnionpeel extracts ameliorate hyperglycemia and insulin resistance inhigh fat dietstreptozotocin-induced diabetic ratsrdquo Nutrition ampMetabolism vol 8 no 18 2011


[34] C Pereira Braga A C Momentti F Barbosa Peixoto et alldquoInfluence of treatment with quercetin on lipid parameters andoxidative stress of pregnant diabetic ratsrdquo Canadian Journal ofPhysiology and Pharmacology vol 91 no 2 pp 171ndash177 2013

[35] M Nardini F Natella V Gentili M D Felice and C ScaccinildquoEffect of caffeic acid dietary supplementation on the anti-oxidant defense system in rat an in vivo studyrdquo Archives of Bio-chemistry and Biophysics vol 342 no 1 pp 157ndash160 1997

[36] F-L Hsu Y-C Chen and J-T Cheng ldquoCaffeic acid as activeprinciple from the fruit of Xanthium strumarium to lowerplasma glucose in diabetic ratsrdquo Planta Medica vol 66 no 3pp 228ndash230 2000

[37] S Baroni B A da Rocha J Oliveira de Melo J F Comar SM Caparroz-Assef and C A Bersani-Amado ldquoHydroethanolicextract of Smallanthus sonchifolius leaves improves hyper-glycemia of streptozotocin induced neonatal diabetic ratsrdquoAsian Pacific Journal of Tropical Medicine vol 9 no 5 pp 432ndash436 2016

[38] A C Maritim R A Sanders and J B Watkins III ldquoDiabetesoxidative stress and antioxidants a reviewrdquo Journal of Biochem-ical and Molecular Toxicology vol 17 no 1 pp 24ndash38 2003

[39] K Sharma ldquoMitochondrial hormesis and diabetic complica-tionsrdquo Diabetes vol 64 no 3 pp 663ndash672 2015

[40] G D Lopaschuk J R Ussher C D L Folmes J S Jaswal andW C STANLEY ldquoMyocardial Fatty Acid Metabolism in Healthand Disease Physiolrdquo Rev vol 90 pp 207ndash258 2010

[41] G Vassort and B Turan ldquoProtective role of antioxidants indiabetes-induced cardiac dysfunctionrdquo Cardiovascular Toxicol-ogy vol 10 no 2 pp 73ndash86 2010

[42] T Mahesh and V P Menon ldquoQuercetin Allievates OxidativeStress in Streptozotocin-induced Diabetic Ratsrdquo Phyther Resvol 18 pp 123ndash127 2004

[43] H J He G Y Wang Y Gao W H Ling Z W Yu and T R JinldquoCurcumin attenuates Nrf2 signaling defect oxidative stress inmuscle and glucose intolerance in high fat diet-fedmicerdquoWorldJournal of Diabetes vol 3 no 5 pp 94ndash104 2012

[44] R Mittal S Sharma S Chhibber and K Harjai ldquoEvaluation ofinterleukin-10 production in Pseudomonas aeruginosa inducedacute pyelonephritisrdquo Journal of Infection and Public Health vol2 no 3 pp 136ndash140 2009

[45] A Arya C Yeng Looi S Chuen Cheah M Rais Mustafa andM Ali Mohd ldquoAnti-diabetic effects ofCentratherum anthelmin-ticum seeds methanolic fraction on pancreatic cells 120573-TC6 andits alleviating role in type 2 diabetic ratsrdquo Journal of Ethno-pharmacology vol 144 no 1 pp 22ndash32 2012

[46] C-Y O Chen A Kamil and J B Blumberg ldquoPhytochemicalcomposition and antioxidant capacity ofwholewheat productsrdquoInt J Food Sci Nutr vol 66 pp 63ndash70 2015

[47] V Benkovic N Kopjar A Horvat Knezevic et al ldquoEvaluationof radioprotective effects of propolis and quercetin on humanwhite blood cells in vitrordquo Biological and Pharmaceutical Bul-letin vol 31 no 9 pp 1778ndash1785 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Medicinal plants are widely used as alternative therapeu-tics for the prevention or treatment of diseases Recently greatattention has been paid to the use of natural compoundsdue to their nutritional and pharmacological characteristics[7]

Yacon (Smallanthus sonchifolius [Poepp amp Endl] HRobinson Asteraceae) is a native Andean plant cultivated forits tubers which are commonly used as a food in SouthAmer-ica Some studies have reported the presence of large amountsof phenolic compounds in extracts from Yacon leaves andtubers mainly chlorogenic protocatechuic ferulic ros-marinic gallic gentisic and caffeic acids and their derivatives[8] Evidence has also emerged about the antioxidant acti-vity [9] protective effects on oxidative damage and glucosemetabolism in rat hepatocytes and insulin-like effects ofYacon leaf extracts [10 11]

Antioxidant compounds have long been known to dimin-ish inflammatory and oxidative stress responses In additionantioxidants scavenge ROS and increase the capacity of theantioxidant defense enzyme system [2] Therefore antioxi-dants can help diminish oxidative damage and inflammationand slow or prevent the progression of diabetic complica-tions

Natural products are the groundwork of preventing andcuring several diseases Moreover ethnopharmacologicalknowledge is one attractive way to enhance the probability ofsuccess in new drug-finding efforts Regarding diabetic com-plications plants that can be effectively used based on theirtherapeutic applications for example for diabetes areworthyof special attention and studies are needed regarding theirside effects the ability to maintain normal levels of glycemiaand their possible control on oxidative stress and inflamma-tion Considering the complications of type 1 diabetes andprevious data on Yaconrsquos activities the present study wasundertaken to elucidate the antioxidant anti-inflammatoryand antihyperglycemic activity of hydroethanolic extractfrom S sonchifolius leaves (HEYL) in the serum and skeletalmuscle of STZ-induced diabetic rats

2 Materials and Methods

21 Plant Material and Extract Preparation The leaves ofS sonchifolius were collected in June (2014) by KlinsmannCarolo dos Santos in Curitiba PR Brazil The specimen wasprovided by Dr Atila FranciscoMogor from the Departmentof Plant Science and Crop Protection Federal Universityof Parana Curitiba Parana Brazil and identified by DrLin Chau Ming from Sao Paulo State University (UNESP)School of Agriculture Botucatu SP Brazil and the voucherspecimen was deposited to the Herbarium at the Sao PauloState University (UNESP) Institute of Biosciences BotucatuSP Brazil under the register 32752 for future referenceThe leaves from S sonchifolius were dried for seven daysat 50∘C powdered (3 120583m) and subjected to percolationat room temperature using a mixture of ethanol H2O(7 3 vv) with a flux of 20mLminkg The solvents wereevaporated to dryness under a low pressure (45∘C) usingrotary evaporator in vacuum system to afford the crudeHEYL

22 Characterization of Phenolics For the characterization ofphenolics 10mg HEYL was reconstituted in 1mL methanolfollowed by acidic hydrolysis with 1mL of 24M HCl at80∘C for 2 h in the dark After the incubation the solutionwas filtered through a 045120583m nylon membrane (Milli-pore Corp Bedford MA) and then injected onto a HPLCsystem equipped with a Zorbax SB-C18 column (46 times250mm 35 120583m) and a CoulArray 5600A electrochemicaldetector (ESA Inc Chelmsford MA) Phenolic acids andflavonoidswere quantified according to themethod of Li et al(2009) [12] The limits of quantitation for phenolic acid andflavonoids were 1 ng on column The linearity of calibrationcurves of authentic standards with concentrations rangingfrom 001 to 2 ngml was at least ge0991 The identificationof each compound was based on a comparison of the reten-tion time and electrochemical response of the authenticatedstandards The results are expressed in 120583g100mg HEYL

23 Dose-Response Profile of HEYL Treatment To establisha dose-response profile for the antihyperglycemic activityof Yacon leaves we used varying doses of HEYL (25 50and 100mgkg body weightday constituted in 1mL of 09saline) to identify the lowest dose that could elicit an optimalantihyperglycemic effect FifteenmaleWistar rats 60 d of agewere maintained in an environmentally controlled room (22plusmn 3∘C 12-hour lightdark cycle and relative humidity of 60 plusmn5) and were fed with a standard rat pellet diet (Purina LtdCampinas SP Brazil) and water ad libitumThe animals wererandomly assigned to one of three groups HEYL 25 HEYL50 and HEYL 100 Diabetes mellitus was induced by one ipadministration of streptozotocin (STZ 40mgbody weight)and the animals received HEYL for gavage for 2 weeks afterthe establishment of diabetic condition We found that after2 weeks of the treatment the highest dose showed the mostpotent hyperglycemic effect in STZ model of diabetes Thusthe subsequent experimentswithHEYLwere carried outwiththe dose of 100mgkg administered orally

24 Animals and Experimental Groups Forty male Wistarrats 60 d of age were maintained in an environmentally con-trolled room (22 plusmn 3∘C 12-hour lightdark cycle and relativehumidity of 60 plusmn 5) and were fed with a standard rat pelletdiet (Purina Ltd Campinas SP Brazil) and water ad libitumThe experimental protocol was approved by the Ethics Com-mittee on theUse ofAnimals (CEUA) at theBotucatuMedicalSchool Sao Paulo State University (UNESP) under number1082-2014 (approved in April 24 2014) The animals wererandomly assigned to one of four groups (n = 10) C (controlgroup) normal rats C + Y normal rats receiving HEYL DMdiabetic rats and DM+Y diabetics rats receiving HEYLDia-betes mellituswas induced by ip administration of streptozo-tocin for one time (STZ 40mgbody weight) Blood glucosewas measured 48 h and 7 days after the STZ administrationThe animals with blood glucose greater than 250mgdL wereconsidered diabetic The animals received HEYL (100mgkgbody weightday constituted in 1mL of 09 saline) for gav-age for 30 days after the 7th day of established diabetic condi-tion Control animals were given the same volume of salineThe animals were fasted overnight and killed by decapitation











3 Results





C C + Y DM DM + Y

500

400

300

200

100

0

P lt 0001

P lt 0001

Initi

al g

lyce

mia

(mg

dL)

7da

ys a

er S

TZ


400

300

200

100

0

P lt 0001 P lt 0001

Fina

l gly

cem

ia (m

gdL

)30＞

of tr

eatm

ent

(b)

C C + Y DM DM + Y

50

40

30

20

10

0

P = 0003

Insu

lin (p

mol

L)

30＞

of tr

eatm

ent


P lt 0001

P lt 0001P lt 0001

80

60

40

20

0

P = 0043

HO

MA-

BETA

30＞

of tr

eatm

ent

(d)





C C + Y DM DM + Y

P = 0015P = 002015

10

5

0

Glu

tath

ione

per

oxid

ase

(m

olm

g pr

otei

nm

in)


(pm

olm

g pr

otei

nm

in)

Cata

lase

P = 0029

P = 00067

6

5

4

3

2

1

0

(b)

C C + Y DM DM + Y

150

120

90

60

30

0

Supe

roxi

de d

ismut

ase

(Um

g pr

otei

nm

in)


(m

olm

g of

tiss

ue)

MD

A

P lt 0001 P lt 000103

02

01

00

(d)




4 Discussion





C C + Y DM DM + Y

P = 0001

HAC

seru

m(

of p

rote

ctio

nm

g pr

otei

n)90

80

70

60

50

40

30

20

10

0


P = 0003

P = 0004

HAC

sole

us(

of p

rote

ctio

nm

g pr

otei

n)

120

100

80

60

40

20

0

(b)

C C + Y DM DM + Y

IL-6

seru

m (p

gm

L)

03

02

01

00


P lt 0001 P lt 0001

02

01

00

IL-6

sole

us(p

g10

0Ａ

of ti

ssue

)

(d)


















Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results





C C + Y DM DM + Y

500

400

300

200

100

0

P lt 0001

P lt 0001

Initi

al g

lyce

mia

(mg

dL)

7da

ys a

er S

TZ


400

300

200

100

0

P lt 0001 P lt 0001

Fina

l gly

cem

ia (m

gdL

)30＞

of tr

eatm

ent

(b)

C C + Y DM DM + Y

50

40

30

20

10

0

P = 0003

Insu

lin (p

mol

L)

30＞

of tr

eatm

ent


P lt 0001

P lt 0001P lt 0001

80

60

40

20

0

P = 0043

HO

MA-

BETA

30＞

of tr

eatm

ent

(d)





C C + Y DM DM + Y

P = 0015P = 002015

10

5

0

Glu

tath

ione

per

oxid

ase

(m

olm

g pr

otei

nm

in)


(pm

olm

g pr

otei

nm

in)

Cata

lase

P = 0029

P = 00067

6

5

4

3

2

1

0

(b)

C C + Y DM DM + Y

150

120

90

60

30

0

Supe

roxi

de d

ismut

ase

(Um

g pr

otei

nm

in)


(m

olm

g of

tiss

ue)

MD

A

P lt 0001 P lt 000103

02

01

00

(d)




4 Discussion





C C + Y DM DM + Y

P = 0001

HAC

seru

m(

of p

rote

ctio

nm

g pr

otei

n)90

80

70

60

50

40

30

20

10

0


P = 0003

P = 0004

HAC

sole

us(

of p

rote

ctio

nm

g pr

otei

n)

120

100

80

60

40

20

0

(b)

C C + Y DM DM + Y

IL-6

seru

m (p

gm

L)

03

02

01

00


P lt 0001 P lt 0001

02

01

00

IL-6

sole

us(p

g10

0Ａ

of ti

ssue

)

(d)


















Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C C + Y DM DM + Y

500

400

300

200

100

0

P lt 0001

P lt 0001

Initi

al g

lyce

mia

(mg

dL)

7da

ys a

er S

TZ


400

300

200

100

0

P lt 0001 P lt 0001

Fina

l gly

cem

ia (m

gdL

)30＞

of tr

eatm

ent

(b)

C C + Y DM DM + Y

50

40

30

20

10

0

P = 0003

Insu

lin (p

mol

L)

30＞

of tr

eatm

ent


P lt 0001

P lt 0001P lt 0001

80

60

40

20

0

P = 0043

HO

MA-

BETA

30＞

of tr

eatm

ent

(d)





C C + Y DM DM + Y

P = 0015P = 002015

10

5

0

Glu

tath

ione

per

oxid

ase

(m

olm

g pr

otei

nm

in)


(pm

olm

g pr

otei

nm

in)

Cata

lase

P = 0029

P = 00067

6

5

4

3

2

1

0

(b)

C C + Y DM DM + Y

150

120

90

60

30

0

Supe

roxi

de d

ismut

ase

(Um

g pr

otei

nm

in)


(m

olm

g of

tiss

ue)

MD

A

P lt 0001 P lt 000103

02

01

00

(d)




4 Discussion





C C + Y DM DM + Y

P = 0001

HAC

seru

m(

of p

rote

ctio

nm

g pr

otei

n)90

80

70

60

50

40

30

20

10

0


P = 0003

P = 0004

HAC

sole

us(

of p

rote

ctio

nm

g pr

otei

n)

120

100

80

60

40

20

0

(b)

C C + Y DM DM + Y

IL-6

seru

m (p

gm

L)

03

02

01

00


P lt 0001 P lt 0001

02

01

00

IL-6

sole

us(p

g10

0Ａ

of ti

ssue

)

(d)


















Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C C + Y DM DM + Y

P = 0015P = 002015

10

5

0

Glu

tath

ione

per

oxid

ase

(m

olm

g pr

otei

nm

in)


(pm

olm

g pr

otei

nm

in)

Cata

lase

P = 0029

P = 00067

6

5

4

3

2

1

0

(b)

C C + Y DM DM + Y

150

120

90

60

30

0

Supe

roxi

de d

ismut

ase

(Um

g pr

otei

nm

in)


(m

olm

g of

tiss

ue)

MD

A

P lt 0001 P lt 000103

02

01

00

(d)




4 Discussion





C C + Y DM DM + Y

P = 0001

HAC

seru

m(

of p

rote

ctio

nm

g pr

otei

n)90

80

70

60

50

40

30

20

10

0


P = 0003

P = 0004

HAC

sole

us(

of p

rote

ctio

nm

g pr

otei

n)

120

100

80

60

40

20

0

(b)

C C + Y DM DM + Y

IL-6

seru

m (p

gm

L)

03

02

01

00


P lt 0001 P lt 0001

02

01

00

IL-6

sole

us(p

g10

0Ａ

of ti

ssue

)

(d)


















Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C C + Y DM DM + Y

P = 0001

HAC

seru

m(

of p

rote

ctio

nm

g pr

otei

n)90

80

70

60

50

40

30

20

10

0


P = 0003

P = 0004

HAC

sole

us(

of p

rote

ctio

nm

g pr

otei

n)

120

100

80

60

40

20

0

(b)

C C + Y DM DM + Y

IL-6

seru

m (p

gm

L)

03

02

01

00


P lt 0001 P lt 0001

02

01

00

IL-6

sole

us(p

g10

0Ａ

of ti

ssue

)

(d)


















Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















researcharticle smallanthus sonchifolius) leaf extract...

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