Research ArticleAdipogenic Activity of Wild Populations ofRhododendron groenlandicum a Medicinal Shrub fromthe James Bay Cree Traditional Pharmacopeia

Michel Rapinski12 Lina Musallam13 John Thor Arnason14

Pierre Haddad13 and Alain Cuerrier12

1Canadian Institutes of Health Research Team in Aboriginal Antidiabetic Medicines Universite de Montreal MontrealQC Canada H3C 3J72Institut de Recherche en Biologie Vegetale Jardin Botanique de Montreal Universite de Montreal 4101 Sherbrooke EstMontreal QC Canada H1X 2B23Natural Health Products and Metabolic Diseases Laboratory Department of Pharmacology Universite de MontrealMontreal QC Canada H3C 3J74Centre for Research in Biotechnology and Biopharmaceuticals Department of Biology University of Ottawa OttawaON Canada K1N 6N5

Correspondence should be addressed to Pierre Haddad pierrehaddadumontrealcaand Alain Cuerrier alaincuerrierumontrealca

Received 20 March 2015 Accepted 25 May 2015

Academic Editor Nunziatina De Tommasi

Copyright copy 2015 Michel Rapinski et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The traditional medicinal plant Labrador tea (Rhododendron groenlandicum (Oeder) Kron amp Judd Ericaceae) present in thepharmacopoeia of the Cree of Eeyou Istchee has shown glitazone-like activity in the 3T3-L1 adipogenesis bioassayThis activity hasbeen attributed to phenolic compounds which have been shown to vary in this plant as a function of insolation parameters Thegoal of this study was to determine if these changes in phenolic content were pharmacologically significant Leaves were harvestedin 2006 throughout the James Bay region of Northern Quebec and ethanol extracts were tested in vitro using the 3T3-L1 murinecell line adipogenesis bioassay This traditional medicinal plant was found active in the assay However there was no detectablespatial pattern in the accumulation of intracellular triglycerides suggesting that such patterns previously observed in the phenolicprofile of Labrador tea were not pharmacologically significant Nonetheless a reduction in the adipogenic activity was observedand associated with higher concentrations of quercetin for which selected environmental variables did not appropriately explainits variation

1 Introduction

In a previous study on the phytochemistry of the NorthAmerican medicinal plant Rhododendron groenlandicum(Oeder) Kron amp Judd (Ericaceae) Labrador tea we foundthe concentration of biologically active compounds to varyin Northern Quebecrsquos Hudson and James Bay region [1]Labrador tea is a common species in Canadarsquos boreal forestMore importantly it is a popularmedicinal plant found in thetraditional pharmacopoeia of indigenous populations fromthe Algonquian Salish Wakashan Tsimshian and Eskimo-Aleut linguistic families [2ndash9]

In ethnobotanical studies conducted in six communitiesof the Cree Nation of Eeyou Istchee (CEI) we found Rgroenlandicum to be the top-ranked plant species used for thetreatment of symptoms associated with type 2 diabetes (T2D)[10ndash13]The inherent cultural relevance of this species to CEItraditional medicine (CTM) warrants further investigationinto its antidiabetic potential

The CIHR Team on Antidiabetic Aboriginal Medicines(CIHR-TAAM) formed through collaborative work betweenCEI communities the Cree Board of Health and SocialServices of James Bay (CBHSSJB) and Canadian academicresearchers has screened many of the multiple plants

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 492458 7 pageshttpdxdoiorg1011552015492458

2 Evidence-Based Complementary and Alternative Medicine

present in the CEI pharmacopoeia [14ndash17] Of these Rgroenlandicum was shown to possess in vitro glitazone-likeactivity comparable to rosiglitazone in an adipogenic assaymeasuring the lipid accumulation in differentiating 3T3-L1preadipocytes [14]

The antidiabetic drug rosiglitazone induces an increasein the sensitivity to insulin acting as a PPAR120574 receptoragonist [18] the expression of this transcription factor isparticularly implicated in the differentiation of adipocytes[19ndash22] and in insulin sensitivity [18] Hence it plays acritical role in the pathogenesis of T2DThe action of PPAR120574results in an improvement in the absorption of fatty acidsin differentiated adipocytes which store them as triglycerides(TG) [18] Adipocytes therefore provide storage for fattysubstances that would otherwise accumulate in tissues suchas skeletalmuscle and liver thereby contributing tometabolicdisorders such as insulin resistance [22]

The pharmacological activity of R groenlandicumhas been attributed to phenolic compounds [14 16 23]Bioassay-guided fractionation using adipogenesis of 3T3-L1murine cells confirmed that specific phenolics are the mostactive compounds [24] In developing culturally appropriateapproaches to treating T2D in the CEI communities thevariation of these compounds in R groenlandicum hasimportant implications in ensuring the quality control oftraditional medicinal plants or to develop standardizednatural health products (NHPs)

In this study we assessed possible variations in theantidiabetic potential of R groenlandicum Our objectivewas to determine if the phytochemical variations observedin the speciesrsquo phenolic profile are biologically significantWe evaluated the in vitro pharmacological activity of crudeextracts from various localities using the adipogenesis bioas-say and hypothesized that high concentrations of phenoliccompounds would result in a stronger adipogenic activity

2 Materials and Methods

21 Sampling Extraction and Phytochemical Analysis Thesampling extraction and analytical methods for phyto-chemical identification and quantification are thoroughlydescribed in Rapinski et al [1] which reports on thephytochemistry of R groenlandicum A subsample selectedrandomly of previously reported samples was used in this invitro study

Briefly mature leaves were sampled during the summerof 2006 around the communities of Mistissini NemaskaEastmain Wemindji and Whapmagoostui thus coveringmuch of the northsouth gradient in Eeyou Istchee Fiveaccessions each containing leaves from multiple individualplants were collected within a 50 km radius around eachcommunity and selected for this study Samples were air-dried and preserved in paper bags at room temperature

Samples were milled through a Wiley Mill at 40 meshand extracted overnight in 25mLg of 80 EtOH by orbitalshaking at room temperature at 250 RPM The pelletwas extracted overnight in 15mL 80 EtOH An aliquot(1mL) of the pooled supernatants (adjusted to 500mL ina volumetric flask) was prepared for High Performance

Liquid Chromatography coupled with Diode Array Detector(HPLC-DAD) The leftover crude extracts were dried usinga speedVac the trace water was removed by lyophilizationusing SuperModulo freeze dryer and the dehydrated extractswere stored at minus80∘C

Finally 10120583L of each extract aliquot was injected throughan autosampler and detected by DAD at 290 nm bandwidth4 reference off The separations were performed on a LunaC18 column (250 times 46mm 5 120583M particle size) Peak identi-fication was undertaken by cochromatographic comparisonof the spectral data adopted in our in-house metabolomicsspectral library [23] A standard curve was constructed byinjection of serially diluted marker compounds in methanolThe quantification was based on peak area The quantitationof putatively identified quercetin-glycosides was achievedbased on calibration curve of quercetin-3-galactoside Eachsample was analyzed in triplicate and averaged to account forinstrumental variation

22 Cell Culture 3T3-L1 murine preadipocyte cells weregrown to confluence in 24-well plates in DMEMproliferationmedium containing 10 FBS Media were changed every 2days At 24 h after confluence (day 0) cells were inducedto differentiate with a short-term differentiation medium ofDMEM supplemented with 10 FBS 1 120583M DMX 250120583MIBMX and 500 nM insulin After 48 h the media werereplaced with DMEM containing 10 FBS and 500 nMinsulin for long-termdifferentiation Cells were differentiatedfor a total of 5 days with media change every 2 daysRhododendron groenlandicum crude extracts (75120583gmL) androsiglitazone (10 120583M positive control) were dissolved inDMSO and added to the cells as of day 0 of differentiationThe final concentration of DMSO was kept at 01 through-out the differentiation period

23 Adipogenesis We measured intracellular TG content atday 5 of differentiation using the AdipoRed reagent accord-ing to the manufacturerrsquos instructions Methods have beenpreviously described in Spoor et al [14] and Harbilas et al[16] In short wells containing adipocytes were washed twicewith phosphate-buffered saline (PBS) before 1mL of PBScontaining 30 120583L of AdipoRed reagent was added to each welland incubated for 15minutes at room temperature AdipoRedbecomes fluorescent when partitioned in a hydrophobiccompartment namely intracellular triglycerides (TG) Thefluorescence of each well was measured with a WallacVictor2 fluorimeter (Perkin-Elmer Saint-Laurent QC) at anexcitation wavelength of 485 nm and an emission wavelengthof 572 nm The results were reported as percentage of thevehicle control 01 DMSO

24 Cell Lines Chemicals Biochemicals and StandardsFor the identification and quantification of phenolic mark-ers (+)-catechin (1) chlorogenic acid (2) (minus)-epicatechin(3) p-coumaric acid (4) rutin (5) quercetin-3-galactoside(6) quercetin-3-glucoside (7) quercetin-3-rhamnoside (12)myricetin (13) and quercetin (14) were purchased fromSigma-Aldrich (Oakville Ontario Canada) and Extrasyn-these (Genay France) HPLC grade water acetonitrile and

Evidence-Based Complementary and Alternative Medicine 3

formic acid (99 purity) were purchased from Sigma-Aldrich

For cell culture and adipogenesis preadipocyte 3T3-L1cell line was purchased from the American Type CultureCollection (ATCC Manassas VA) Dexamethasone (DMX)bovine pancreatic insulin 3-isobutyl-1-methylxanthine (IBMX)and Dimethyl sulfoxide (DMSO) were purchased fromSigma-Aldrich (Oakville ON) Rosiglitazone was obtainedfrom Alexis Biochemicals (Hornby ON)

DulbeccorsquosModified EagleMedium (DMEM) fetal bovineserum (FBS) and bovine calf serum (NCS) were fromWisentInc (Saint-Bruno QC) AdipoRed reagent was purchasedfrom Cambrex Bio Science Walkersville Inc (WalkersvilleMD)

25 Environmental Data Annual estimates of bioclimaticvariables namely annual temperature range correspondingto our sampling year and long-term estimates for insolationvariables were provided by the Canadian Forest Services ofNatural Resources Canada [25] Long-term estimates werederived frommultidecademeteorological data collected from1971 to 2000 [26 27]

26 Statistical Analysis To reduce interassay variation TGcontent was normalized relative to each assayrsquos vehicle con-trol 01 DMSO set at 100 Rhododendron groenlandicumand rosiglitazone always induced significant increases inactivity as verified by the fact that the 95 confidenceinterval of the mean activity (quadruplicate determinations)did not include the 100 adipogenic activity reference (119901 lt005) Differences between communities were analyzed byone-way analysis of variance The relationships betweenTG content and compounds were analyzed by multipleand simple linear regressions To represent the adipogenicactivity of R groenlandicum and the quantified compoundsprincipal components analysis (PCA) was performed on thematrix of these compounds using the correlation matrixIndividual samples were scored onto the PCA axes andrepresentedwith the vectors for each compounds TG contentwas subsequently projected as a supplementary variableonto the principal components in order to interpret thedimensions of variability In doing so the calculation ofdistances between each of the samples and the constructionprincipal components depends only on their phytochemicalprofile Using the transition formulae described by Le et al[28] the coordinates for TG content are calculated usingthe original eigenvalues Finally we partitioned the variationin the adipogenic activity of R groenlandicum between thetwo sets of variables compounds and environmental factorsThis was done using a partial-redundancy analysis (partial-RDA) approach [29 30] All analyses were performed usingR statistical language [31] Results are reported asmeans plusmn SDand statistical significance is set at 120572 = 005

3 Results and Discussion

The phytochemistry of the same R groenlandicum accessionshas already been described and discussed in greater length in

500

400

300

200

100

0

DM

SO (01

)

Rosig

litaz

one

R gr

oenl

andi

cum

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

lowast

lowast

Figure 1 Effect of R groenlandicum crude leaf extracts fromNorthern Quebec on lipid accumulation Intracellular triglyceridecontent was measured by AdipoRed fluorescence in live 3T3-L1murine adipocytes incubated with plant extracts for 5 days afterdifferentiation Means plusmn SD (119899 = 4 for rosiglitazone 119899 = 100for R groenlandicum) are normalized to the vehicle control (01DMSO) Asterisk (lowast) indicates significant differences with respectto the DMSO control at 120572 = 005

Rapinski et al [1] Here we present the results of a subsampleof 2006 accessions in the adipogenesis bioassay

The glitazone-like activity ofR groenlandicum to increasethe accumulation of intracellular TG in 3T3-L1 adipocyteswas measured at day 5 of differentiation Extracts increasedadipogenesis with an average content of TG of 1590 thatof DMSO (Figure 1) and a 95 confidence interval of 1388ndash1791 of DMSOThe adipogenic activity of R groenlandicumwas roughly half of the positive control rosiglitazone This islower than what has previously been reported for this speciesSpoor et al [14] reported the stimulation of adipogenesis tobe comparable to rosiglitazone while later determinationsmeasured an activity representing two-thirds that of theantidiabetic drug [24] With few exceptions (Figure 2) ourresults nonetheless confirm the adipogenic potential of thisspecies It is important to consider the fact that previousdeterminations of activity were carried out using extractsprepared from large quantities of source material (largenumber of individual plants) collected a few years prior to thematerial used in the present studies Hence interindividualvariations were absent and different climatic conditionsmay have prevailed This can explain at least in part thedifferences in adipogenic potential observed between thestudies

There were no statistically distinct spatial patternsdetected in the pharmacological activity of R groenlandicumNone of the communities sampled possessed accessionswhich significantly increased intracellular TG more than theothers (119901 = 0348 Figure 3) We have previously foundthat biologically active phenolics were greater in collectionsmade around the communities of Nemaska Eastmain andWemindji [1] The adipogenic activity of R groenlandicum

4 Evidence-Based Complementary and Alternative Medicine

10

8

6

4

2

0

Num

ber o

f sam

ples

41

ndash100

101

ndash140

141

ndash180

181

ndash220

Intracellular triglyceride content( of DMSO control)

221

ndash260

Figure 2 Frequency distribution of the adipogenic activity from 25samples of R groenlandicum leaves collected throughout NorthernQuebec Intracellular triglyceride content was measured by Adi-poRed fluorescence in live 3T3-L1 murine adipocytes incubatedwith plant extracts for 5 days after differentiation Triglyceridecontent was normalized to the vehicle control (01 DMSO)Samples with content levels below 100 (in white) were consideredinhibitory and decreased lipid accumulation

followed a similar trend as can be observed in Figure 3although statistical significance of a polynomial relationshipwas not achieved (119901 = 0170) possibly due to high variabilityThis suggests that variations in the phytochemical profilesobserved in Rapinski et al [1] may be pharmacologicallyrelevant but further studies will be necessary to confirm thispoint

Indeed we found that quantified compounds explainedconsiderable variability obtained in this speciesrsquo pharmaco-logical activity (119901 = 00279 1198772adj = 0491) The distributionof R groenlandicum samples based on their phytochemicalprofile was reconstructed into a reduced three-dimensionalspace which represented 6992 of the samplesrsquo variationover three statistically constructed principal components oraxes (Figure 4) Each principal component from the first tothe third respectively explained 3599 (120582

1= 396) 2297

(1205822= 253) and 1096 (120582

3= 120) of the variation The

direction and proximity of arrows for some major markerssuggest that these are highly correlated (Figure 4) Whenprojecting intracellular TG onto this plot it did not appearto be well correlated with the bulk of these markers many ofwhich were found near a 90∘ angle from it thus indicatingweek or null correlations One of the only markers for whicha significant relationshipwith TG appears to exist is quercetin(Figures 4(b) and 4(c)) This suggests that out of all thecompounds assessed variations in the adipogenic activityof R groenlandicum are most vulnerable to changes in thecontent of quercetin found in the crude extracts Figure 5further illustrates the linear correlation (119901 = 00458 1198772 =0162) whereby the adipogenic activity of R groenlandicumdecreases as the concentration of quercetin in the sampleincreases This is consistent with observations from ourown group [24] where pure quercetin was found to inhibit

Mist

issin

i

Nem

aska

Eastm

ain

Wem

indj

i

Wha

pmag

oostu

i

Community

250

200

150

100

50

0

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

Figure 3 Effect of R groenlandicum crude leaf extracts preparedfrom accessions collected around five communities in NorthernQuebec on lipid accumulation Intracellular triglyceride contentwas measured by AdipoRed fluorescence in live 3T3-L1 murineadipocytes incubated with plant extracts for 5 days after differentia-tionMeans plusmn SD (119899 = 5) are normalized to the vehicle control (01DMSO)Therewere no significant differences between communities(119901 = 0348)

adipogenesis in a dose-dependent manner The activity ofquercetin is well studied and has also been consistently shownby others to be a potent inhibitor of adipocyte differentiationand adipogenesis [18 32ndash34]

Our results suggest that while the geographical locationdoes not appear to have a statistically significant impact onthe adipogenic activity of crude extracts of localized R groen-landicum samples variations in active compounds do explaina significant proportion of variability in pharmacologicalactivity We have shown that annual temperature rangesand insolation parameters such as solar radiation couldsignificantly explain some of the variation in the speciesrsquophenolic compounds [1] Although we did not find in thisstudy that these environmental variables could significantlyexplain the variation in TG content (119901 = 0150 1198772adj = 0162)we found nonetheless that they explained an importantproportion of the variation in the phytochemical profilesof R groenlandicum which could significantly explain TGcontent (nontestable see Table 1 Figure 6) Indeed variationpartitioning of TG content with both phytochemical andenvironmental variables indicates that while the relationshipwith compounds was statistically significant (119901 = 002791198772

adj = 0491) their unique contribution to explaining TGcontent no longer was when the contribution of environ-mental variables albeit small was taken into account andremoved (119901 = 00619 1198772adj = 0424)

This confirms the caveat that environmental variablesplay an underlying role in affecting the content of biologicallyactive compounds Conversely quercetin the only significant

Evidence-Based Complementary and Alternative Medicine 5

PC1 (3599)

PC3

(10

96

)

PC2 (22

97)

QcRa

TG content

QcGu

QcG4QcGa

QcG3CatQcG2ECat

ChlA

Qc

minus2

minus1

0

1

2

3

minus4 minus2 0 2 4

minus4minus2

02

4

minus3

QcG1

Figure 4 Principal component analysis biplot of 11 phenoliccompounds in R groenlandicum leaves Solid lines represent relativeloadings of these variables on axes 1 2 and 3 TG content (boldarrow) was selected as a supplementary variable and plotted ontoprincipal components generated from the phytochemical markersScores for individual samples are represented by symbols forthe communities of Mistissini (998771) Nemaska (e) Eastmain (◼)Wemindji (X) and Whapmagoostui (998787) Abbreviations representcompounds as follows (+)-catechin Cat chlorogenic acid ChlA(minus)-epicatechin ECat quercetin-3-galactoside QcGa quercetin-3-glucoside QcGu quercetin-glycoside 1 QcG1 quercetin-glycoside2 QcG2 quercetin-glycoside 3 QcG3 quercetin-glycoside 4 QcG4quercetin-3-rhamnoside Qc-Ra and quercetin Qc

250

200

150

100

50

0004 006 008 010 012 014 016

Quercetin concentration (120583M)

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

R2 = 0162

Figure 5 Intracellular triglycerides content of 3T3-L1 murineadipocytes exposed to 75 120583gmL of R groenlandicum leaf extractscollected from various locations Quercetin concentrations in crudeextract were significantly and negatively associated with the speciesrsquoadipogenic activity (119901 = 00458) Triglyceride contents are normal-ized to the vehicle control (01 DMSO)

Table 1 Variation partitioning of the adipogenic activity ofR groen-landicum leaf extracts explained by the content in biologically activecompounds (compounds) and the effect of bioclimatic variables(environment) Fraction [119886] corresponds to the unique contributionof compounds once the environment has been taken into accountwhereas fraction [119888] represents the reverse Fraction [119887] representsthe shared portion or overlap between the effect of compounds andenvironment The variation (1198772adj) of each fraction is represented inFigure 6 Asterisk (lowast) indicates significant fractions at 120572 = 005

Fractions 1198772

adj p[119886 + 119887] = compounds 0491 00279lowast

[119887 + 119888] = environment 0109 0150[119886 + 119887 + 119888] = compounds +environment (full model) 0534 00437lowast

[119886] = compounds|environment 0424 00619[119887] = shared 00668 Not testable[119888] = environment|compounds 00424 0302[119889] = unexplained (residuals) 0466 Not testable

100

80

60

40

20

0

Varia

tion

()

a

bc

d

Fraction

Figure 6 Variation partitioning of the adipogenic activity ofR groenlandicum leaf extracts explained by the content in bio-logically active compounds and the effect of bioclimatic vari-ables 119886 = compounds|environment 119887 = shared component 119888 =environment|compounds and119889=unexplained (see Table 1 formoredetails) The full model fraction [119886 + 119887 + 119888] as well as the modelincluding compounds only fraction [119886 + 119887] was significant at 120572 =005

compound related to changes in the adipogenic activity of Rgroenlandicum was not found to be strongly associated withenvironmental variables [1] This may hence explain why theportion of variation those environmental variables contributeto the model and more specifically to the content of biologi-cally active compounds which best explains the variation inTG content is considerably small (Table 1 Figure 6)

On the other hand our results provide support for thehypothesis that synergistic interactions may occur between

6 Evidence-Based Complementary and Alternative Medicine

compounds For instance the inhibitory action of Hibiscussabdariffa (Malvaceae) was greater than the sum of its partswhen polyphenols had been fractionated isolated and testedindividually [35] More importantly in bioassay-guided frac-tionation experiments the activity of crude Labrador teaextracts was higher than that of each active compound testedindividually [24] Finally quercetin and resveratrol togetherdecreased lipid accumulation considerably more than each ofthese used separately at the same dose [34]

Many of the compounds quantified in this paperhave shown adipogenic activity in some form or anotherQuercetin-3-glucoside has been found toxic to adipocytesat relatively low concentration (50 120583M) but was not foundto affect adipogenic activity [33] Quercetin-3-rhamnosidehas been found inactive at low concentrations yet inhibitedadipogenesis at high concentrations and chlorogenic acid hasalso been found to inhibit intracellular triglycerides accumu-lation [33] Content variations of some of these particularly(+)-catechin and (minus)-epicatechin have been explained byenvironmental variables [1] Although the individual effect ofthese compounds was not detected in our study it does notundermine the role they may play when found in a cocktailof substances

4 Conclusion

We have previously shown that latitude acted as a markerfor the impact of environmental variables on phytochemicalconcentrations [1] Therefore a trend could possibly existbetween abiotic factors and concentration of targeted sec-ondary metabolites but a larger sample size might be neededto detect it There may also be other environmental climaticand even biotic factors that were not taken into accountwhich explain the changes in quercetin content These maybetter explain the ecophysiological processes affecting theantidiabetic potential of R groenlandicum Increase in theadipogenic potential of this traditional medicine was asso-ciated primarily with lower concentrations of quercetin butthe cause for its variation will require further investigationNonetheless our results do not provide enough evidence tojustify the idea that specific accessions of Labrador tea mayhave reproducibly better adipogenic potential than othersalong a latitudinal gradient Conversely our study impliesthat random harvesting of R groenlandicum in the Eeyouchterritory of NorthernQuebec should not have amajor impacton the quality of traditional preparations orNHPsmade fromthis plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by the Canadian Institutes ofHealthResearch (CIHR) Team Grant (CTP-79855) to Pierre S Had-dad J T Arnason and Alain Cuerrier and discovery grant toJ T Arnason as well as funding from theNatural Sciences and

Engineering Research Council (NSERC) Canadarsquos NorthernInternship program and Network Environments for Abo-riginal Health Research (NEAHR) to M Rapinski Specialthanks are due to the Elders of the Eeyou Istchee CreeNationsof Mistissini Nemaska Waskaganish Eastmain Wemindjiand Whapmagoostui for sharing their traditional knowledgeand allowing us to collect medicinal plants from their landswith the purpose of bridging indigenous knowledge andcontemporary scienceThe authors also thank the Cree Boardof Health and Social Services of James Bay for their constantsupport as well as A Leger N Roy A Downing Y TendlandB Walsh-Roussel C H Ta D Vallerand N Shang and MOuchfoun for helping out with field and lab work Specialrecognition is due to Jonathan Ferrier who also providedcomments ideas and support Finally thanks are due to SDaigle and P Legendre for statistical advice

References

[1] M Rapinski R Liu A Saleem J T Arnason and A CuerrierldquoEnvironmental trends in the variation of biologically activephenolic compounds in Labrador tea Rhododendron groen-landicum from northern Quebec Canadardquo Botany vol 92 no11 pp 783ndash794 2014

[2] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of Eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[3] R A Zieba Healing and Healers among the Northern CreeUniversity of Manitoba 1990

[4] E V Siegfried Ethnobotany of the Northern Cree of WabascaDesmarais University of Calgary 1994

[5] R J Marles C Clavelle L Monteleone et al Aboriginal PlantUse in Canadas Northwest Boreal Forest Natural ResourcesCanada Edmonton Canada 2008

[6] A Cuerrier and Elders of Kangiqsujuaq The Botanical Knowl-edge of the Inuit of Kangiqsujuaq Avataq Cultural InstituteInukjuak Canada 2011

[7] A Cuerrier and Elders of Kangiqsualujjuaq The BotanicalKnowledge of the Inuit of Kangiqsualujjuaq Nunavik AvataqCultural Institute Inukjuak Canada 2011

[8] A Cuerrier Elders of Umiujaq and Elders of KuujjuarapikTheBotanical Knowledge of the Inuit of Umiujaq and KuujjuarapikNunavik Avataq Cultural Institute Inukjuak Canada 2011

[9] A Cuerrier and L Hermanutz Our Plants Our Land Plantsof Nain and Torngat Mountains Basecamp and Research Station(Nunatsiavut) Institut de Recherche en Biologie VegetaleMontreal Canada Memorial University of Newfoundland StJohnrsquos Canada 2012

[10] M-H Fraser Ethnobotanical Investigation of Plants Used forthe Treatment of type 2 Diabetes by Two Cree Communities inQuebec Quantitative Comparisons and Antioxidant EvaluationMcGill University 2006

[11] C Leduc J Coonishish P Haddad and A Cuerrier ldquoPlantsused by the Cree Nation of Eeyou Istchee (Quebec Canada)for the treatment of diabetes a novel approach in quantitativeethnobotanyrdquo Journal of Ethnopharmacology vol 105 no 1-2pp 55ndash63 2006

[12] A Downing Inter and Intra-Specific Differences in MedicinalPlant Use for the Treatment of Type II Diabetes Symptoms by theCree Elders of Eeyou Istchee (QC) Universite de Montreal 2010

Evidence-Based Complementary and Alternative Medicine 7

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

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Hindawi Publishing Corporationhttpwwwhindawicom 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

present in the CEI pharmacopoeia [14ndash17] Of these Rgroenlandicum was shown to possess in vitro glitazone-likeactivity comparable to rosiglitazone in an adipogenic assaymeasuring the lipid accumulation in differentiating 3T3-L1preadipocytes [14]

The antidiabetic drug rosiglitazone induces an increasein the sensitivity to insulin acting as a PPAR120574 receptoragonist [18] the expression of this transcription factor isparticularly implicated in the differentiation of adipocytes[19ndash22] and in insulin sensitivity [18] Hence it plays acritical role in the pathogenesis of T2DThe action of PPAR120574results in an improvement in the absorption of fatty acidsin differentiated adipocytes which store them as triglycerides(TG) [18] Adipocytes therefore provide storage for fattysubstances that would otherwise accumulate in tissues suchas skeletalmuscle and liver thereby contributing tometabolicdisorders such as insulin resistance [22]

The pharmacological activity of R groenlandicumhas been attributed to phenolic compounds [14 16 23]Bioassay-guided fractionation using adipogenesis of 3T3-L1murine cells confirmed that specific phenolics are the mostactive compounds [24] In developing culturally appropriateapproaches to treating T2D in the CEI communities thevariation of these compounds in R groenlandicum hasimportant implications in ensuring the quality control oftraditional medicinal plants or to develop standardizednatural health products (NHPs)

In this study we assessed possible variations in theantidiabetic potential of R groenlandicum Our objectivewas to determine if the phytochemical variations observedin the speciesrsquo phenolic profile are biologically significantWe evaluated the in vitro pharmacological activity of crudeextracts from various localities using the adipogenesis bioas-say and hypothesized that high concentrations of phenoliccompounds would result in a stronger adipogenic activity

2 Materials and Methods

21 Sampling Extraction and Phytochemical Analysis Thesampling extraction and analytical methods for phyto-chemical identification and quantification are thoroughlydescribed in Rapinski et al [1] which reports on thephytochemistry of R groenlandicum A subsample selectedrandomly of previously reported samples was used in this invitro study

Briefly mature leaves were sampled during the summerof 2006 around the communities of Mistissini NemaskaEastmain Wemindji and Whapmagoostui thus coveringmuch of the northsouth gradient in Eeyou Istchee Fiveaccessions each containing leaves from multiple individualplants were collected within a 50 km radius around eachcommunity and selected for this study Samples were air-dried and preserved in paper bags at room temperature

Samples were milled through a Wiley Mill at 40 meshand extracted overnight in 25mLg of 80 EtOH by orbitalshaking at room temperature at 250 RPM The pelletwas extracted overnight in 15mL 80 EtOH An aliquot(1mL) of the pooled supernatants (adjusted to 500mL ina volumetric flask) was prepared for High Performance

Liquid Chromatography coupled with Diode Array Detector(HPLC-DAD) The leftover crude extracts were dried usinga speedVac the trace water was removed by lyophilizationusing SuperModulo freeze dryer and the dehydrated extractswere stored at minus80∘C

Finally 10120583L of each extract aliquot was injected throughan autosampler and detected by DAD at 290 nm bandwidth4 reference off The separations were performed on a LunaC18 column (250 times 46mm 5 120583M particle size) Peak identi-fication was undertaken by cochromatographic comparisonof the spectral data adopted in our in-house metabolomicsspectral library [23] A standard curve was constructed byinjection of serially diluted marker compounds in methanolThe quantification was based on peak area The quantitationof putatively identified quercetin-glycosides was achievedbased on calibration curve of quercetin-3-galactoside Eachsample was analyzed in triplicate and averaged to account forinstrumental variation

22 Cell Culture 3T3-L1 murine preadipocyte cells weregrown to confluence in 24-well plates in DMEMproliferationmedium containing 10 FBS Media were changed every 2days At 24 h after confluence (day 0) cells were inducedto differentiate with a short-term differentiation medium ofDMEM supplemented with 10 FBS 1 120583M DMX 250120583MIBMX and 500 nM insulin After 48 h the media werereplaced with DMEM containing 10 FBS and 500 nMinsulin for long-termdifferentiation Cells were differentiatedfor a total of 5 days with media change every 2 daysRhododendron groenlandicum crude extracts (75120583gmL) androsiglitazone (10 120583M positive control) were dissolved inDMSO and added to the cells as of day 0 of differentiationThe final concentration of DMSO was kept at 01 through-out the differentiation period

23 Adipogenesis We measured intracellular TG content atday 5 of differentiation using the AdipoRed reagent accord-ing to the manufacturerrsquos instructions Methods have beenpreviously described in Spoor et al [14] and Harbilas et al[16] In short wells containing adipocytes were washed twicewith phosphate-buffered saline (PBS) before 1mL of PBScontaining 30 120583L of AdipoRed reagent was added to each welland incubated for 15minutes at room temperature AdipoRedbecomes fluorescent when partitioned in a hydrophobiccompartment namely intracellular triglycerides (TG) Thefluorescence of each well was measured with a WallacVictor2 fluorimeter (Perkin-Elmer Saint-Laurent QC) at anexcitation wavelength of 485 nm and an emission wavelengthof 572 nm The results were reported as percentage of thevehicle control 01 DMSO

24 Cell Lines Chemicals Biochemicals and StandardsFor the identification and quantification of phenolic mark-ers (+)-catechin (1) chlorogenic acid (2) (minus)-epicatechin(3) p-coumaric acid (4) rutin (5) quercetin-3-galactoside(6) quercetin-3-glucoside (7) quercetin-3-rhamnoside (12)myricetin (13) and quercetin (14) were purchased fromSigma-Aldrich (Oakville Ontario Canada) and Extrasyn-these (Genay France) HPLC grade water acetonitrile and

Evidence-Based Complementary and Alternative Medicine 3

formic acid (99 purity) were purchased from Sigma-Aldrich

For cell culture and adipogenesis preadipocyte 3T3-L1cell line was purchased from the American Type CultureCollection (ATCC Manassas VA) Dexamethasone (DMX)bovine pancreatic insulin 3-isobutyl-1-methylxanthine (IBMX)and Dimethyl sulfoxide (DMSO) were purchased fromSigma-Aldrich (Oakville ON) Rosiglitazone was obtainedfrom Alexis Biochemicals (Hornby ON)

DulbeccorsquosModified EagleMedium (DMEM) fetal bovineserum (FBS) and bovine calf serum (NCS) were fromWisentInc (Saint-Bruno QC) AdipoRed reagent was purchasedfrom Cambrex Bio Science Walkersville Inc (WalkersvilleMD)

25 Environmental Data Annual estimates of bioclimaticvariables namely annual temperature range correspondingto our sampling year and long-term estimates for insolationvariables were provided by the Canadian Forest Services ofNatural Resources Canada [25] Long-term estimates werederived frommultidecademeteorological data collected from1971 to 2000 [26 27]

26 Statistical Analysis To reduce interassay variation TGcontent was normalized relative to each assayrsquos vehicle con-trol 01 DMSO set at 100 Rhododendron groenlandicumand rosiglitazone always induced significant increases inactivity as verified by the fact that the 95 confidenceinterval of the mean activity (quadruplicate determinations)did not include the 100 adipogenic activity reference (119901 lt005) Differences between communities were analyzed byone-way analysis of variance The relationships betweenTG content and compounds were analyzed by multipleand simple linear regressions To represent the adipogenicactivity of R groenlandicum and the quantified compoundsprincipal components analysis (PCA) was performed on thematrix of these compounds using the correlation matrixIndividual samples were scored onto the PCA axes andrepresentedwith the vectors for each compounds TG contentwas subsequently projected as a supplementary variableonto the principal components in order to interpret thedimensions of variability In doing so the calculation ofdistances between each of the samples and the constructionprincipal components depends only on their phytochemicalprofile Using the transition formulae described by Le et al[28] the coordinates for TG content are calculated usingthe original eigenvalues Finally we partitioned the variationin the adipogenic activity of R groenlandicum between thetwo sets of variables compounds and environmental factorsThis was done using a partial-redundancy analysis (partial-RDA) approach [29 30] All analyses were performed usingR statistical language [31] Results are reported asmeans plusmn SDand statistical significance is set at 120572 = 005

3 Results and Discussion

The phytochemistry of the same R groenlandicum accessionshas already been described and discussed in greater length in

500

400

300

200

100

0

DM

SO (01

)

Rosig

litaz

one

R gr

oenl

andi

cum

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

lowast

lowast

Figure 1 Effect of R groenlandicum crude leaf extracts fromNorthern Quebec on lipid accumulation Intracellular triglyceridecontent was measured by AdipoRed fluorescence in live 3T3-L1murine adipocytes incubated with plant extracts for 5 days afterdifferentiation Means plusmn SD (119899 = 4 for rosiglitazone 119899 = 100for R groenlandicum) are normalized to the vehicle control (01DMSO) Asterisk (lowast) indicates significant differences with respectto the DMSO control at 120572 = 005

Rapinski et al [1] Here we present the results of a subsampleof 2006 accessions in the adipogenesis bioassay

The glitazone-like activity ofR groenlandicum to increasethe accumulation of intracellular TG in 3T3-L1 adipocyteswas measured at day 5 of differentiation Extracts increasedadipogenesis with an average content of TG of 1590 thatof DMSO (Figure 1) and a 95 confidence interval of 1388ndash1791 of DMSOThe adipogenic activity of R groenlandicumwas roughly half of the positive control rosiglitazone This islower than what has previously been reported for this speciesSpoor et al [14] reported the stimulation of adipogenesis tobe comparable to rosiglitazone while later determinationsmeasured an activity representing two-thirds that of theantidiabetic drug [24] With few exceptions (Figure 2) ourresults nonetheless confirm the adipogenic potential of thisspecies It is important to consider the fact that previousdeterminations of activity were carried out using extractsprepared from large quantities of source material (largenumber of individual plants) collected a few years prior to thematerial used in the present studies Hence interindividualvariations were absent and different climatic conditionsmay have prevailed This can explain at least in part thedifferences in adipogenic potential observed between thestudies

There were no statistically distinct spatial patternsdetected in the pharmacological activity of R groenlandicumNone of the communities sampled possessed accessionswhich significantly increased intracellular TG more than theothers (119901 = 0348 Figure 3) We have previously foundthat biologically active phenolics were greater in collectionsmade around the communities of Nemaska Eastmain andWemindji [1] The adipogenic activity of R groenlandicum

4 Evidence-Based Complementary and Alternative Medicine

10

8

6

4

2

0

Num

ber o

f sam

ples

41

ndash100

101

ndash140

141

ndash180

181

ndash220

Intracellular triglyceride content( of DMSO control)

221

ndash260

Figure 2 Frequency distribution of the adipogenic activity from 25samples of R groenlandicum leaves collected throughout NorthernQuebec Intracellular triglyceride content was measured by Adi-poRed fluorescence in live 3T3-L1 murine adipocytes incubatedwith plant extracts for 5 days after differentiation Triglyceridecontent was normalized to the vehicle control (01 DMSO)Samples with content levels below 100 (in white) were consideredinhibitory and decreased lipid accumulation

followed a similar trend as can be observed in Figure 3although statistical significance of a polynomial relationshipwas not achieved (119901 = 0170) possibly due to high variabilityThis suggests that variations in the phytochemical profilesobserved in Rapinski et al [1] may be pharmacologicallyrelevant but further studies will be necessary to confirm thispoint

Indeed we found that quantified compounds explainedconsiderable variability obtained in this speciesrsquo pharmaco-logical activity (119901 = 00279 1198772adj = 0491) The distributionof R groenlandicum samples based on their phytochemicalprofile was reconstructed into a reduced three-dimensionalspace which represented 6992 of the samplesrsquo variationover three statistically constructed principal components oraxes (Figure 4) Each principal component from the first tothe third respectively explained 3599 (120582

1= 396) 2297

(1205822= 253) and 1096 (120582

3= 120) of the variation The

direction and proximity of arrows for some major markerssuggest that these are highly correlated (Figure 4) Whenprojecting intracellular TG onto this plot it did not appearto be well correlated with the bulk of these markers many ofwhich were found near a 90∘ angle from it thus indicatingweek or null correlations One of the only markers for whicha significant relationshipwith TG appears to exist is quercetin(Figures 4(b) and 4(c)) This suggests that out of all thecompounds assessed variations in the adipogenic activityof R groenlandicum are most vulnerable to changes in thecontent of quercetin found in the crude extracts Figure 5further illustrates the linear correlation (119901 = 00458 1198772 =0162) whereby the adipogenic activity of R groenlandicumdecreases as the concentration of quercetin in the sampleincreases This is consistent with observations from ourown group [24] where pure quercetin was found to inhibit

Mist

issin

i

Nem

aska

Eastm

ain

Wem

indj

i

Wha

pmag

oostu

i

Community

250

200

150

100

50

0

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

Figure 3 Effect of R groenlandicum crude leaf extracts preparedfrom accessions collected around five communities in NorthernQuebec on lipid accumulation Intracellular triglyceride contentwas measured by AdipoRed fluorescence in live 3T3-L1 murineadipocytes incubated with plant extracts for 5 days after differentia-tionMeans plusmn SD (119899 = 5) are normalized to the vehicle control (01DMSO)Therewere no significant differences between communities(119901 = 0348)

adipogenesis in a dose-dependent manner The activity ofquercetin is well studied and has also been consistently shownby others to be a potent inhibitor of adipocyte differentiationand adipogenesis [18 32ndash34]

Our results suggest that while the geographical locationdoes not appear to have a statistically significant impact onthe adipogenic activity of crude extracts of localized R groen-landicum samples variations in active compounds do explaina significant proportion of variability in pharmacologicalactivity We have shown that annual temperature rangesand insolation parameters such as solar radiation couldsignificantly explain some of the variation in the speciesrsquophenolic compounds [1] Although we did not find in thisstudy that these environmental variables could significantlyexplain the variation in TG content (119901 = 0150 1198772adj = 0162)we found nonetheless that they explained an importantproportion of the variation in the phytochemical profilesof R groenlandicum which could significantly explain TGcontent (nontestable see Table 1 Figure 6) Indeed variationpartitioning of TG content with both phytochemical andenvironmental variables indicates that while the relationshipwith compounds was statistically significant (119901 = 002791198772

adj = 0491) their unique contribution to explaining TGcontent no longer was when the contribution of environ-mental variables albeit small was taken into account andremoved (119901 = 00619 1198772adj = 0424)

This confirms the caveat that environmental variablesplay an underlying role in affecting the content of biologicallyactive compounds Conversely quercetin the only significant

Evidence-Based Complementary and Alternative Medicine 5

PC1 (3599)

PC3

(10

96

)

PC2 (22

97)

QcRa

TG content

QcGu

QcG4QcGa

QcG3CatQcG2ECat

ChlA

Qc

minus2

minus1

0

1

2

3

minus4 minus2 0 2 4

minus4minus2

02

4

minus3

QcG1

Figure 4 Principal component analysis biplot of 11 phenoliccompounds in R groenlandicum leaves Solid lines represent relativeloadings of these variables on axes 1 2 and 3 TG content (boldarrow) was selected as a supplementary variable and plotted ontoprincipal components generated from the phytochemical markersScores for individual samples are represented by symbols forthe communities of Mistissini (998771) Nemaska (e) Eastmain (◼)Wemindji (X) and Whapmagoostui (998787) Abbreviations representcompounds as follows (+)-catechin Cat chlorogenic acid ChlA(minus)-epicatechin ECat quercetin-3-galactoside QcGa quercetin-3-glucoside QcGu quercetin-glycoside 1 QcG1 quercetin-glycoside2 QcG2 quercetin-glycoside 3 QcG3 quercetin-glycoside 4 QcG4quercetin-3-rhamnoside Qc-Ra and quercetin Qc

250

200

150

100

50

0004 006 008 010 012 014 016

Quercetin concentration (120583M)

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

R2 = 0162

Figure 5 Intracellular triglycerides content of 3T3-L1 murineadipocytes exposed to 75 120583gmL of R groenlandicum leaf extractscollected from various locations Quercetin concentrations in crudeextract were significantly and negatively associated with the speciesrsquoadipogenic activity (119901 = 00458) Triglyceride contents are normal-ized to the vehicle control (01 DMSO)

Table 1 Variation partitioning of the adipogenic activity ofR groen-landicum leaf extracts explained by the content in biologically activecompounds (compounds) and the effect of bioclimatic variables(environment) Fraction [119886] corresponds to the unique contributionof compounds once the environment has been taken into accountwhereas fraction [119888] represents the reverse Fraction [119887] representsthe shared portion or overlap between the effect of compounds andenvironment The variation (1198772adj) of each fraction is represented inFigure 6 Asterisk (lowast) indicates significant fractions at 120572 = 005

Fractions 1198772

adj p[119886 + 119887] = compounds 0491 00279lowast

[119887 + 119888] = environment 0109 0150[119886 + 119887 + 119888] = compounds +environment (full model) 0534 00437lowast

[119886] = compounds|environment 0424 00619[119887] = shared 00668 Not testable[119888] = environment|compounds 00424 0302[119889] = unexplained (residuals) 0466 Not testable

100

80

60

40

20

0

Varia

tion

()

a

bc

d

Fraction

Figure 6 Variation partitioning of the adipogenic activity ofR groenlandicum leaf extracts explained by the content in bio-logically active compounds and the effect of bioclimatic vari-ables 119886 = compounds|environment 119887 = shared component 119888 =environment|compounds and119889=unexplained (see Table 1 formoredetails) The full model fraction [119886 + 119887 + 119888] as well as the modelincluding compounds only fraction [119886 + 119887] was significant at 120572 =005

compound related to changes in the adipogenic activity of Rgroenlandicum was not found to be strongly associated withenvironmental variables [1] This may hence explain why theportion of variation those environmental variables contributeto the model and more specifically to the content of biologi-cally active compounds which best explains the variation inTG content is considerably small (Table 1 Figure 6)

On the other hand our results provide support for thehypothesis that synergistic interactions may occur between

6 Evidence-Based Complementary and Alternative Medicine

compounds For instance the inhibitory action of Hibiscussabdariffa (Malvaceae) was greater than the sum of its partswhen polyphenols had been fractionated isolated and testedindividually [35] More importantly in bioassay-guided frac-tionation experiments the activity of crude Labrador teaextracts was higher than that of each active compound testedindividually [24] Finally quercetin and resveratrol togetherdecreased lipid accumulation considerably more than each ofthese used separately at the same dose [34]

Many of the compounds quantified in this paperhave shown adipogenic activity in some form or anotherQuercetin-3-glucoside has been found toxic to adipocytesat relatively low concentration (50 120583M) but was not foundto affect adipogenic activity [33] Quercetin-3-rhamnosidehas been found inactive at low concentrations yet inhibitedadipogenesis at high concentrations and chlorogenic acid hasalso been found to inhibit intracellular triglycerides accumu-lation [33] Content variations of some of these particularly(+)-catechin and (minus)-epicatechin have been explained byenvironmental variables [1] Although the individual effect ofthese compounds was not detected in our study it does notundermine the role they may play when found in a cocktailof substances

4 Conclusion

We have previously shown that latitude acted as a markerfor the impact of environmental variables on phytochemicalconcentrations [1] Therefore a trend could possibly existbetween abiotic factors and concentration of targeted sec-ondary metabolites but a larger sample size might be neededto detect it There may also be other environmental climaticand even biotic factors that were not taken into accountwhich explain the changes in quercetin content These maybetter explain the ecophysiological processes affecting theantidiabetic potential of R groenlandicum Increase in theadipogenic potential of this traditional medicine was asso-ciated primarily with lower concentrations of quercetin butthe cause for its variation will require further investigationNonetheless our results do not provide enough evidence tojustify the idea that specific accessions of Labrador tea mayhave reproducibly better adipogenic potential than othersalong a latitudinal gradient Conversely our study impliesthat random harvesting of R groenlandicum in the Eeyouchterritory of NorthernQuebec should not have amajor impacton the quality of traditional preparations orNHPsmade fromthis plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by the Canadian Institutes ofHealthResearch (CIHR) Team Grant (CTP-79855) to Pierre S Had-dad J T Arnason and Alain Cuerrier and discovery grant toJ T Arnason as well as funding from theNatural Sciences and

Engineering Research Council (NSERC) Canadarsquos NorthernInternship program and Network Environments for Abo-riginal Health Research (NEAHR) to M Rapinski Specialthanks are due to the Elders of the Eeyou Istchee CreeNationsof Mistissini Nemaska Waskaganish Eastmain Wemindjiand Whapmagoostui for sharing their traditional knowledgeand allowing us to collect medicinal plants from their landswith the purpose of bridging indigenous knowledge andcontemporary scienceThe authors also thank the Cree Boardof Health and Social Services of James Bay for their constantsupport as well as A Leger N Roy A Downing Y TendlandB Walsh-Roussel C H Ta D Vallerand N Shang and MOuchfoun for helping out with field and lab work Specialrecognition is due to Jonathan Ferrier who also providedcomments ideas and support Finally thanks are due to SDaigle and P Legendre for statistical advice

References

[1] M Rapinski R Liu A Saleem J T Arnason and A CuerrierldquoEnvironmental trends in the variation of biologically activephenolic compounds in Labrador tea Rhododendron groen-landicum from northern Quebec Canadardquo Botany vol 92 no11 pp 783ndash794 2014

[2] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of Eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[3] R A Zieba Healing and Healers among the Northern CreeUniversity of Manitoba 1990

[4] E V Siegfried Ethnobotany of the Northern Cree of WabascaDesmarais University of Calgary 1994

[5] R J Marles C Clavelle L Monteleone et al Aboriginal PlantUse in Canadas Northwest Boreal Forest Natural ResourcesCanada Edmonton Canada 2008

[6] A Cuerrier and Elders of Kangiqsujuaq The Botanical Knowl-edge of the Inuit of Kangiqsujuaq Avataq Cultural InstituteInukjuak Canada 2011

[7] A Cuerrier and Elders of Kangiqsualujjuaq The BotanicalKnowledge of the Inuit of Kangiqsualujjuaq Nunavik AvataqCultural Institute Inukjuak Canada 2011

[8] A Cuerrier Elders of Umiujaq and Elders of KuujjuarapikTheBotanical Knowledge of the Inuit of Umiujaq and KuujjuarapikNunavik Avataq Cultural Institute Inukjuak Canada 2011

[9] A Cuerrier and L Hermanutz Our Plants Our Land Plantsof Nain and Torngat Mountains Basecamp and Research Station(Nunatsiavut) Institut de Recherche en Biologie VegetaleMontreal Canada Memorial University of Newfoundland StJohnrsquos Canada 2012

[10] M-H Fraser Ethnobotanical Investigation of Plants Used forthe Treatment of type 2 Diabetes by Two Cree Communities inQuebec Quantitative Comparisons and Antioxidant EvaluationMcGill University 2006

[11] C Leduc J Coonishish P Haddad and A Cuerrier ldquoPlantsused by the Cree Nation of Eeyou Istchee (Quebec Canada)for the treatment of diabetes a novel approach in quantitativeethnobotanyrdquo Journal of Ethnopharmacology vol 105 no 1-2pp 55ndash63 2006

[12] A Downing Inter and Intra-Specific Differences in MedicinalPlant Use for the Treatment of Type II Diabetes Symptoms by theCree Elders of Eeyou Istchee (QC) Universite de Montreal 2010

Evidence-Based Complementary and Alternative Medicine 7

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

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 3

formic acid (99 purity) were purchased from Sigma-Aldrich

For cell culture and adipogenesis preadipocyte 3T3-L1cell line was purchased from the American Type CultureCollection (ATCC Manassas VA) Dexamethasone (DMX)bovine pancreatic insulin 3-isobutyl-1-methylxanthine (IBMX)and Dimethyl sulfoxide (DMSO) were purchased fromSigma-Aldrich (Oakville ON) Rosiglitazone was obtainedfrom Alexis Biochemicals (Hornby ON)

DulbeccorsquosModified EagleMedium (DMEM) fetal bovineserum (FBS) and bovine calf serum (NCS) were fromWisentInc (Saint-Bruno QC) AdipoRed reagent was purchasedfrom Cambrex Bio Science Walkersville Inc (WalkersvilleMD)

25 Environmental Data Annual estimates of bioclimaticvariables namely annual temperature range correspondingto our sampling year and long-term estimates for insolationvariables were provided by the Canadian Forest Services ofNatural Resources Canada [25] Long-term estimates werederived frommultidecademeteorological data collected from1971 to 2000 [26 27]

26 Statistical Analysis To reduce interassay variation TGcontent was normalized relative to each assayrsquos vehicle con-trol 01 DMSO set at 100 Rhododendron groenlandicumand rosiglitazone always induced significant increases inactivity as verified by the fact that the 95 confidenceinterval of the mean activity (quadruplicate determinations)did not include the 100 adipogenic activity reference (119901 lt005) Differences between communities were analyzed byone-way analysis of variance The relationships betweenTG content and compounds were analyzed by multipleand simple linear regressions To represent the adipogenicactivity of R groenlandicum and the quantified compoundsprincipal components analysis (PCA) was performed on thematrix of these compounds using the correlation matrixIndividual samples were scored onto the PCA axes andrepresentedwith the vectors for each compounds TG contentwas subsequently projected as a supplementary variableonto the principal components in order to interpret thedimensions of variability In doing so the calculation ofdistances between each of the samples and the constructionprincipal components depends only on their phytochemicalprofile Using the transition formulae described by Le et al[28] the coordinates for TG content are calculated usingthe original eigenvalues Finally we partitioned the variationin the adipogenic activity of R groenlandicum between thetwo sets of variables compounds and environmental factorsThis was done using a partial-redundancy analysis (partial-RDA) approach [29 30] All analyses were performed usingR statistical language [31] Results are reported asmeans plusmn SDand statistical significance is set at 120572 = 005

3 Results and Discussion

The phytochemistry of the same R groenlandicum accessionshas already been described and discussed in greater length in

500

400

300

200

100

0

DM

SO (01

)

Rosig

litaz

one

R gr

oenl

andi

cum

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

lowast

lowast

Figure 1 Effect of R groenlandicum crude leaf extracts fromNorthern Quebec on lipid accumulation Intracellular triglyceridecontent was measured by AdipoRed fluorescence in live 3T3-L1murine adipocytes incubated with plant extracts for 5 days afterdifferentiation Means plusmn SD (119899 = 4 for rosiglitazone 119899 = 100for R groenlandicum) are normalized to the vehicle control (01DMSO) Asterisk (lowast) indicates significant differences with respectto the DMSO control at 120572 = 005

Rapinski et al [1] Here we present the results of a subsampleof 2006 accessions in the adipogenesis bioassay

The glitazone-like activity ofR groenlandicum to increasethe accumulation of intracellular TG in 3T3-L1 adipocyteswas measured at day 5 of differentiation Extracts increasedadipogenesis with an average content of TG of 1590 thatof DMSO (Figure 1) and a 95 confidence interval of 1388ndash1791 of DMSOThe adipogenic activity of R groenlandicumwas roughly half of the positive control rosiglitazone This islower than what has previously been reported for this speciesSpoor et al [14] reported the stimulation of adipogenesis tobe comparable to rosiglitazone while later determinationsmeasured an activity representing two-thirds that of theantidiabetic drug [24] With few exceptions (Figure 2) ourresults nonetheless confirm the adipogenic potential of thisspecies It is important to consider the fact that previousdeterminations of activity were carried out using extractsprepared from large quantities of source material (largenumber of individual plants) collected a few years prior to thematerial used in the present studies Hence interindividualvariations were absent and different climatic conditionsmay have prevailed This can explain at least in part thedifferences in adipogenic potential observed between thestudies

There were no statistically distinct spatial patternsdetected in the pharmacological activity of R groenlandicumNone of the communities sampled possessed accessionswhich significantly increased intracellular TG more than theothers (119901 = 0348 Figure 3) We have previously foundthat biologically active phenolics were greater in collectionsmade around the communities of Nemaska Eastmain andWemindji [1] The adipogenic activity of R groenlandicum

4 Evidence-Based Complementary and Alternative Medicine

10

8

6

4

2

0

Num

ber o

f sam

ples

41

ndash100

101

ndash140

141

ndash180

181

ndash220

Intracellular triglyceride content( of DMSO control)

221

ndash260

Figure 2 Frequency distribution of the adipogenic activity from 25samples of R groenlandicum leaves collected throughout NorthernQuebec Intracellular triglyceride content was measured by Adi-poRed fluorescence in live 3T3-L1 murine adipocytes incubatedwith plant extracts for 5 days after differentiation Triglyceridecontent was normalized to the vehicle control (01 DMSO)Samples with content levels below 100 (in white) were consideredinhibitory and decreased lipid accumulation

followed a similar trend as can be observed in Figure 3although statistical significance of a polynomial relationshipwas not achieved (119901 = 0170) possibly due to high variabilityThis suggests that variations in the phytochemical profilesobserved in Rapinski et al [1] may be pharmacologicallyrelevant but further studies will be necessary to confirm thispoint

Indeed we found that quantified compounds explainedconsiderable variability obtained in this speciesrsquo pharmaco-logical activity (119901 = 00279 1198772adj = 0491) The distributionof R groenlandicum samples based on their phytochemicalprofile was reconstructed into a reduced three-dimensionalspace which represented 6992 of the samplesrsquo variationover three statistically constructed principal components oraxes (Figure 4) Each principal component from the first tothe third respectively explained 3599 (120582

1= 396) 2297

(1205822= 253) and 1096 (120582

3= 120) of the variation The

direction and proximity of arrows for some major markerssuggest that these are highly correlated (Figure 4) Whenprojecting intracellular TG onto this plot it did not appearto be well correlated with the bulk of these markers many ofwhich were found near a 90∘ angle from it thus indicatingweek or null correlations One of the only markers for whicha significant relationshipwith TG appears to exist is quercetin(Figures 4(b) and 4(c)) This suggests that out of all thecompounds assessed variations in the adipogenic activityof R groenlandicum are most vulnerable to changes in thecontent of quercetin found in the crude extracts Figure 5further illustrates the linear correlation (119901 = 00458 1198772 =0162) whereby the adipogenic activity of R groenlandicumdecreases as the concentration of quercetin in the sampleincreases This is consistent with observations from ourown group [24] where pure quercetin was found to inhibit

Mist

issin

i

Nem

aska

Eastm

ain

Wem

indj

i

Wha

pmag

oostu

i

Community

250

200

150

100

50

0

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

Figure 3 Effect of R groenlandicum crude leaf extracts preparedfrom accessions collected around five communities in NorthernQuebec on lipid accumulation Intracellular triglyceride contentwas measured by AdipoRed fluorescence in live 3T3-L1 murineadipocytes incubated with plant extracts for 5 days after differentia-tionMeans plusmn SD (119899 = 5) are normalized to the vehicle control (01DMSO)Therewere no significant differences between communities(119901 = 0348)

adipogenesis in a dose-dependent manner The activity ofquercetin is well studied and has also been consistently shownby others to be a potent inhibitor of adipocyte differentiationand adipogenesis [18 32ndash34]

Our results suggest that while the geographical locationdoes not appear to have a statistically significant impact onthe adipogenic activity of crude extracts of localized R groen-landicum samples variations in active compounds do explaina significant proportion of variability in pharmacologicalactivity We have shown that annual temperature rangesand insolation parameters such as solar radiation couldsignificantly explain some of the variation in the speciesrsquophenolic compounds [1] Although we did not find in thisstudy that these environmental variables could significantlyexplain the variation in TG content (119901 = 0150 1198772adj = 0162)we found nonetheless that they explained an importantproportion of the variation in the phytochemical profilesof R groenlandicum which could significantly explain TGcontent (nontestable see Table 1 Figure 6) Indeed variationpartitioning of TG content with both phytochemical andenvironmental variables indicates that while the relationshipwith compounds was statistically significant (119901 = 002791198772

adj = 0491) their unique contribution to explaining TGcontent no longer was when the contribution of environ-mental variables albeit small was taken into account andremoved (119901 = 00619 1198772adj = 0424)

This confirms the caveat that environmental variablesplay an underlying role in affecting the content of biologicallyactive compounds Conversely quercetin the only significant

Evidence-Based Complementary and Alternative Medicine 5

PC1 (3599)

PC3

(10

96

)

PC2 (22

97)

QcRa

TG content

QcGu

QcG4QcGa

QcG3CatQcG2ECat

ChlA

Qc

minus2

minus1

0

1

2

3

minus4 minus2 0 2 4

minus4minus2

02

4

minus3

QcG1

Figure 4 Principal component analysis biplot of 11 phenoliccompounds in R groenlandicum leaves Solid lines represent relativeloadings of these variables on axes 1 2 and 3 TG content (boldarrow) was selected as a supplementary variable and plotted ontoprincipal components generated from the phytochemical markersScores for individual samples are represented by symbols forthe communities of Mistissini (998771) Nemaska (e) Eastmain (◼)Wemindji (X) and Whapmagoostui (998787) Abbreviations representcompounds as follows (+)-catechin Cat chlorogenic acid ChlA(minus)-epicatechin ECat quercetin-3-galactoside QcGa quercetin-3-glucoside QcGu quercetin-glycoside 1 QcG1 quercetin-glycoside2 QcG2 quercetin-glycoside 3 QcG3 quercetin-glycoside 4 QcG4quercetin-3-rhamnoside Qc-Ra and quercetin Qc

250

200

150

100

50

0004 006 008 010 012 014 016

Quercetin concentration (120583M)

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

R2 = 0162

Figure 5 Intracellular triglycerides content of 3T3-L1 murineadipocytes exposed to 75 120583gmL of R groenlandicum leaf extractscollected from various locations Quercetin concentrations in crudeextract were significantly and negatively associated with the speciesrsquoadipogenic activity (119901 = 00458) Triglyceride contents are normal-ized to the vehicle control (01 DMSO)

Table 1 Variation partitioning of the adipogenic activity ofR groen-landicum leaf extracts explained by the content in biologically activecompounds (compounds) and the effect of bioclimatic variables(environment) Fraction [119886] corresponds to the unique contributionof compounds once the environment has been taken into accountwhereas fraction [119888] represents the reverse Fraction [119887] representsthe shared portion or overlap between the effect of compounds andenvironment The variation (1198772adj) of each fraction is represented inFigure 6 Asterisk (lowast) indicates significant fractions at 120572 = 005

Fractions 1198772

adj p[119886 + 119887] = compounds 0491 00279lowast

[119887 + 119888] = environment 0109 0150[119886 + 119887 + 119888] = compounds +environment (full model) 0534 00437lowast

[119886] = compounds|environment 0424 00619[119887] = shared 00668 Not testable[119888] = environment|compounds 00424 0302[119889] = unexplained (residuals) 0466 Not testable

100

80

60

40

20

0

Varia

tion

()

a

bc

d

Fraction

Figure 6 Variation partitioning of the adipogenic activity ofR groenlandicum leaf extracts explained by the content in bio-logically active compounds and the effect of bioclimatic vari-ables 119886 = compounds|environment 119887 = shared component 119888 =environment|compounds and119889=unexplained (see Table 1 formoredetails) The full model fraction [119886 + 119887 + 119888] as well as the modelincluding compounds only fraction [119886 + 119887] was significant at 120572 =005

compound related to changes in the adipogenic activity of Rgroenlandicum was not found to be strongly associated withenvironmental variables [1] This may hence explain why theportion of variation those environmental variables contributeto the model and more specifically to the content of biologi-cally active compounds which best explains the variation inTG content is considerably small (Table 1 Figure 6)

On the other hand our results provide support for thehypothesis that synergistic interactions may occur between

6 Evidence-Based Complementary and Alternative Medicine

compounds For instance the inhibitory action of Hibiscussabdariffa (Malvaceae) was greater than the sum of its partswhen polyphenols had been fractionated isolated and testedindividually [35] More importantly in bioassay-guided frac-tionation experiments the activity of crude Labrador teaextracts was higher than that of each active compound testedindividually [24] Finally quercetin and resveratrol togetherdecreased lipid accumulation considerably more than each ofthese used separately at the same dose [34]

Many of the compounds quantified in this paperhave shown adipogenic activity in some form or anotherQuercetin-3-glucoside has been found toxic to adipocytesat relatively low concentration (50 120583M) but was not foundto affect adipogenic activity [33] Quercetin-3-rhamnosidehas been found inactive at low concentrations yet inhibitedadipogenesis at high concentrations and chlorogenic acid hasalso been found to inhibit intracellular triglycerides accumu-lation [33] Content variations of some of these particularly(+)-catechin and (minus)-epicatechin have been explained byenvironmental variables [1] Although the individual effect ofthese compounds was not detected in our study it does notundermine the role they may play when found in a cocktailof substances

4 Conclusion

We have previously shown that latitude acted as a markerfor the impact of environmental variables on phytochemicalconcentrations [1] Therefore a trend could possibly existbetween abiotic factors and concentration of targeted sec-ondary metabolites but a larger sample size might be neededto detect it There may also be other environmental climaticand even biotic factors that were not taken into accountwhich explain the changes in quercetin content These maybetter explain the ecophysiological processes affecting theantidiabetic potential of R groenlandicum Increase in theadipogenic potential of this traditional medicine was asso-ciated primarily with lower concentrations of quercetin butthe cause for its variation will require further investigationNonetheless our results do not provide enough evidence tojustify the idea that specific accessions of Labrador tea mayhave reproducibly better adipogenic potential than othersalong a latitudinal gradient Conversely our study impliesthat random harvesting of R groenlandicum in the Eeyouchterritory of NorthernQuebec should not have amajor impacton the quality of traditional preparations orNHPsmade fromthis plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by the Canadian Institutes ofHealthResearch (CIHR) Team Grant (CTP-79855) to Pierre S Had-dad J T Arnason and Alain Cuerrier and discovery grant toJ T Arnason as well as funding from theNatural Sciences and

Engineering Research Council (NSERC) Canadarsquos NorthernInternship program and Network Environments for Abo-riginal Health Research (NEAHR) to M Rapinski Specialthanks are due to the Elders of the Eeyou Istchee CreeNationsof Mistissini Nemaska Waskaganish Eastmain Wemindjiand Whapmagoostui for sharing their traditional knowledgeand allowing us to collect medicinal plants from their landswith the purpose of bridging indigenous knowledge andcontemporary scienceThe authors also thank the Cree Boardof Health and Social Services of James Bay for their constantsupport as well as A Leger N Roy A Downing Y TendlandB Walsh-Roussel C H Ta D Vallerand N Shang and MOuchfoun for helping out with field and lab work Specialrecognition is due to Jonathan Ferrier who also providedcomments ideas and support Finally thanks are due to SDaigle and P Legendre for statistical advice

References

[1] M Rapinski R Liu A Saleem J T Arnason and A CuerrierldquoEnvironmental trends in the variation of biologically activephenolic compounds in Labrador tea Rhododendron groen-landicum from northern Quebec Canadardquo Botany vol 92 no11 pp 783ndash794 2014

[2] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of Eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[3] R A Zieba Healing and Healers among the Northern CreeUniversity of Manitoba 1990

[4] E V Siegfried Ethnobotany of the Northern Cree of WabascaDesmarais University of Calgary 1994

[5] R J Marles C Clavelle L Monteleone et al Aboriginal PlantUse in Canadas Northwest Boreal Forest Natural ResourcesCanada Edmonton Canada 2008

[6] A Cuerrier and Elders of Kangiqsujuaq The Botanical Knowl-edge of the Inuit of Kangiqsujuaq Avataq Cultural InstituteInukjuak Canada 2011

[7] A Cuerrier and Elders of Kangiqsualujjuaq The BotanicalKnowledge of the Inuit of Kangiqsualujjuaq Nunavik AvataqCultural Institute Inukjuak Canada 2011

[8] A Cuerrier Elders of Umiujaq and Elders of KuujjuarapikTheBotanical Knowledge of the Inuit of Umiujaq and KuujjuarapikNunavik Avataq Cultural Institute Inukjuak Canada 2011

[9] A Cuerrier and L Hermanutz Our Plants Our Land Plantsof Nain and Torngat Mountains Basecamp and Research Station(Nunatsiavut) Institut de Recherche en Biologie VegetaleMontreal Canada Memorial University of Newfoundland StJohnrsquos Canada 2012

[10] M-H Fraser Ethnobotanical Investigation of Plants Used forthe Treatment of type 2 Diabetes by Two Cree Communities inQuebec Quantitative Comparisons and Antioxidant EvaluationMcGill University 2006

[11] C Leduc J Coonishish P Haddad and A Cuerrier ldquoPlantsused by the Cree Nation of Eeyou Istchee (Quebec Canada)for the treatment of diabetes a novel approach in quantitativeethnobotanyrdquo Journal of Ethnopharmacology vol 105 no 1-2pp 55ndash63 2006

[12] A Downing Inter and Intra-Specific Differences in MedicinalPlant Use for the Treatment of Type II Diabetes Symptoms by theCree Elders of Eeyou Istchee (QC) Universite de Montreal 2010

Evidence-Based Complementary and Alternative Medicine 7

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

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

4 Evidence-Based Complementary and Alternative Medicine

10

8

6

4

2

0

Num

ber o

f sam

ples

41

ndash100

101

ndash140

141

ndash180

181

ndash220

Intracellular triglyceride content( of DMSO control)

221

ndash260

Figure 2 Frequency distribution of the adipogenic activity from 25samples of R groenlandicum leaves collected throughout NorthernQuebec Intracellular triglyceride content was measured by Adi-poRed fluorescence in live 3T3-L1 murine adipocytes incubatedwith plant extracts for 5 days after differentiation Triglyceridecontent was normalized to the vehicle control (01 DMSO)Samples with content levels below 100 (in white) were consideredinhibitory and decreased lipid accumulation

followed a similar trend as can be observed in Figure 3although statistical significance of a polynomial relationshipwas not achieved (119901 = 0170) possibly due to high variabilityThis suggests that variations in the phytochemical profilesobserved in Rapinski et al [1] may be pharmacologicallyrelevant but further studies will be necessary to confirm thispoint

Indeed we found that quantified compounds explainedconsiderable variability obtained in this speciesrsquo pharmaco-logical activity (119901 = 00279 1198772adj = 0491) The distributionof R groenlandicum samples based on their phytochemicalprofile was reconstructed into a reduced three-dimensionalspace which represented 6992 of the samplesrsquo variationover three statistically constructed principal components oraxes (Figure 4) Each principal component from the first tothe third respectively explained 3599 (120582

1= 396) 2297

(1205822= 253) and 1096 (120582

3= 120) of the variation The

direction and proximity of arrows for some major markerssuggest that these are highly correlated (Figure 4) Whenprojecting intracellular TG onto this plot it did not appearto be well correlated with the bulk of these markers many ofwhich were found near a 90∘ angle from it thus indicatingweek or null correlations One of the only markers for whicha significant relationshipwith TG appears to exist is quercetin(Figures 4(b) and 4(c)) This suggests that out of all thecompounds assessed variations in the adipogenic activityof R groenlandicum are most vulnerable to changes in thecontent of quercetin found in the crude extracts Figure 5further illustrates the linear correlation (119901 = 00458 1198772 =0162) whereby the adipogenic activity of R groenlandicumdecreases as the concentration of quercetin in the sampleincreases This is consistent with observations from ourown group [24] where pure quercetin was found to inhibit

Mist

issin

i

Nem

aska

Eastm

ain

Wem

indj

i

Wha

pmag

oostu

i

Community

250

200

150

100

50

0

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

Figure 3 Effect of R groenlandicum crude leaf extracts preparedfrom accessions collected around five communities in NorthernQuebec on lipid accumulation Intracellular triglyceride contentwas measured by AdipoRed fluorescence in live 3T3-L1 murineadipocytes incubated with plant extracts for 5 days after differentia-tionMeans plusmn SD (119899 = 5) are normalized to the vehicle control (01DMSO)Therewere no significant differences between communities(119901 = 0348)

adipogenesis in a dose-dependent manner The activity ofquercetin is well studied and has also been consistently shownby others to be a potent inhibitor of adipocyte differentiationand adipogenesis [18 32ndash34]

Our results suggest that while the geographical locationdoes not appear to have a statistically significant impact onthe adipogenic activity of crude extracts of localized R groen-landicum samples variations in active compounds do explaina significant proportion of variability in pharmacologicalactivity We have shown that annual temperature rangesand insolation parameters such as solar radiation couldsignificantly explain some of the variation in the speciesrsquophenolic compounds [1] Although we did not find in thisstudy that these environmental variables could significantlyexplain the variation in TG content (119901 = 0150 1198772adj = 0162)we found nonetheless that they explained an importantproportion of the variation in the phytochemical profilesof R groenlandicum which could significantly explain TGcontent (nontestable see Table 1 Figure 6) Indeed variationpartitioning of TG content with both phytochemical andenvironmental variables indicates that while the relationshipwith compounds was statistically significant (119901 = 002791198772

adj = 0491) their unique contribution to explaining TGcontent no longer was when the contribution of environ-mental variables albeit small was taken into account andremoved (119901 = 00619 1198772adj = 0424)

This confirms the caveat that environmental variablesplay an underlying role in affecting the content of biologicallyactive compounds Conversely quercetin the only significant

Evidence-Based Complementary and Alternative Medicine 5

PC1 (3599)

PC3

(10

96

)

PC2 (22

97)

QcRa

TG content

QcGu

QcG4QcGa

QcG3CatQcG2ECat

ChlA

Qc

minus2

minus1

0

1

2

3

minus4 minus2 0 2 4

minus4minus2

02

4

minus3

QcG1

Figure 4 Principal component analysis biplot of 11 phenoliccompounds in R groenlandicum leaves Solid lines represent relativeloadings of these variables on axes 1 2 and 3 TG content (boldarrow) was selected as a supplementary variable and plotted ontoprincipal components generated from the phytochemical markersScores for individual samples are represented by symbols forthe communities of Mistissini (998771) Nemaska (e) Eastmain (◼)Wemindji (X) and Whapmagoostui (998787) Abbreviations representcompounds as follows (+)-catechin Cat chlorogenic acid ChlA(minus)-epicatechin ECat quercetin-3-galactoside QcGa quercetin-3-glucoside QcGu quercetin-glycoside 1 QcG1 quercetin-glycoside2 QcG2 quercetin-glycoside 3 QcG3 quercetin-glycoside 4 QcG4quercetin-3-rhamnoside Qc-Ra and quercetin Qc

250

200

150

100

50

0004 006 008 010 012 014 016

Quercetin concentration (120583M)

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

R2 = 0162

Figure 5 Intracellular triglycerides content of 3T3-L1 murineadipocytes exposed to 75 120583gmL of R groenlandicum leaf extractscollected from various locations Quercetin concentrations in crudeextract were significantly and negatively associated with the speciesrsquoadipogenic activity (119901 = 00458) Triglyceride contents are normal-ized to the vehicle control (01 DMSO)

Table 1 Variation partitioning of the adipogenic activity ofR groen-landicum leaf extracts explained by the content in biologically activecompounds (compounds) and the effect of bioclimatic variables(environment) Fraction [119886] corresponds to the unique contributionof compounds once the environment has been taken into accountwhereas fraction [119888] represents the reverse Fraction [119887] representsthe shared portion or overlap between the effect of compounds andenvironment The variation (1198772adj) of each fraction is represented inFigure 6 Asterisk (lowast) indicates significant fractions at 120572 = 005

Fractions 1198772

adj p[119886 + 119887] = compounds 0491 00279lowast

[119887 + 119888] = environment 0109 0150[119886 + 119887 + 119888] = compounds +environment (full model) 0534 00437lowast

[119886] = compounds|environment 0424 00619[119887] = shared 00668 Not testable[119888] = environment|compounds 00424 0302[119889] = unexplained (residuals) 0466 Not testable

100

80

60

40

20

0

Varia

tion

()

a

bc

d

Fraction

Figure 6 Variation partitioning of the adipogenic activity ofR groenlandicum leaf extracts explained by the content in bio-logically active compounds and the effect of bioclimatic vari-ables 119886 = compounds|environment 119887 = shared component 119888 =environment|compounds and119889=unexplained (see Table 1 formoredetails) The full model fraction [119886 + 119887 + 119888] as well as the modelincluding compounds only fraction [119886 + 119887] was significant at 120572 =005

compound related to changes in the adipogenic activity of Rgroenlandicum was not found to be strongly associated withenvironmental variables [1] This may hence explain why theportion of variation those environmental variables contributeto the model and more specifically to the content of biologi-cally active compounds which best explains the variation inTG content is considerably small (Table 1 Figure 6)

On the other hand our results provide support for thehypothesis that synergistic interactions may occur between

6 Evidence-Based Complementary and Alternative Medicine

compounds For instance the inhibitory action of Hibiscussabdariffa (Malvaceae) was greater than the sum of its partswhen polyphenols had been fractionated isolated and testedindividually [35] More importantly in bioassay-guided frac-tionation experiments the activity of crude Labrador teaextracts was higher than that of each active compound testedindividually [24] Finally quercetin and resveratrol togetherdecreased lipid accumulation considerably more than each ofthese used separately at the same dose [34]

Many of the compounds quantified in this paperhave shown adipogenic activity in some form or anotherQuercetin-3-glucoside has been found toxic to adipocytesat relatively low concentration (50 120583M) but was not foundto affect adipogenic activity [33] Quercetin-3-rhamnosidehas been found inactive at low concentrations yet inhibitedadipogenesis at high concentrations and chlorogenic acid hasalso been found to inhibit intracellular triglycerides accumu-lation [33] Content variations of some of these particularly(+)-catechin and (minus)-epicatechin have been explained byenvironmental variables [1] Although the individual effect ofthese compounds was not detected in our study it does notundermine the role they may play when found in a cocktailof substances

4 Conclusion

We have previously shown that latitude acted as a markerfor the impact of environmental variables on phytochemicalconcentrations [1] Therefore a trend could possibly existbetween abiotic factors and concentration of targeted sec-ondary metabolites but a larger sample size might be neededto detect it There may also be other environmental climaticand even biotic factors that were not taken into accountwhich explain the changes in quercetin content These maybetter explain the ecophysiological processes affecting theantidiabetic potential of R groenlandicum Increase in theadipogenic potential of this traditional medicine was asso-ciated primarily with lower concentrations of quercetin butthe cause for its variation will require further investigationNonetheless our results do not provide enough evidence tojustify the idea that specific accessions of Labrador tea mayhave reproducibly better adipogenic potential than othersalong a latitudinal gradient Conversely our study impliesthat random harvesting of R groenlandicum in the Eeyouchterritory of NorthernQuebec should not have amajor impacton the quality of traditional preparations orNHPsmade fromthis plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by the Canadian Institutes ofHealthResearch (CIHR) Team Grant (CTP-79855) to Pierre S Had-dad J T Arnason and Alain Cuerrier and discovery grant toJ T Arnason as well as funding from theNatural Sciences and

Engineering Research Council (NSERC) Canadarsquos NorthernInternship program and Network Environments for Abo-riginal Health Research (NEAHR) to M Rapinski Specialthanks are due to the Elders of the Eeyou Istchee CreeNationsof Mistissini Nemaska Waskaganish Eastmain Wemindjiand Whapmagoostui for sharing their traditional knowledgeand allowing us to collect medicinal plants from their landswith the purpose of bridging indigenous knowledge andcontemporary scienceThe authors also thank the Cree Boardof Health and Social Services of James Bay for their constantsupport as well as A Leger N Roy A Downing Y TendlandB Walsh-Roussel C H Ta D Vallerand N Shang and MOuchfoun for helping out with field and lab work Specialrecognition is due to Jonathan Ferrier who also providedcomments ideas and support Finally thanks are due to SDaigle and P Legendre for statistical advice

References

[1] M Rapinski R Liu A Saleem J T Arnason and A CuerrierldquoEnvironmental trends in the variation of biologically activephenolic compounds in Labrador tea Rhododendron groen-landicum from northern Quebec Canadardquo Botany vol 92 no11 pp 783ndash794 2014

[2] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of Eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[3] R A Zieba Healing and Healers among the Northern CreeUniversity of Manitoba 1990

[4] E V Siegfried Ethnobotany of the Northern Cree of WabascaDesmarais University of Calgary 1994

[5] R J Marles C Clavelle L Monteleone et al Aboriginal PlantUse in Canadas Northwest Boreal Forest Natural ResourcesCanada Edmonton Canada 2008

[6] A Cuerrier and Elders of Kangiqsujuaq The Botanical Knowl-edge of the Inuit of Kangiqsujuaq Avataq Cultural InstituteInukjuak Canada 2011

[7] A Cuerrier and Elders of Kangiqsualujjuaq The BotanicalKnowledge of the Inuit of Kangiqsualujjuaq Nunavik AvataqCultural Institute Inukjuak Canada 2011

[8] A Cuerrier Elders of Umiujaq and Elders of KuujjuarapikTheBotanical Knowledge of the Inuit of Umiujaq and KuujjuarapikNunavik Avataq Cultural Institute Inukjuak Canada 2011

[9] A Cuerrier and L Hermanutz Our Plants Our Land Plantsof Nain and Torngat Mountains Basecamp and Research Station(Nunatsiavut) Institut de Recherche en Biologie VegetaleMontreal Canada Memorial University of Newfoundland StJohnrsquos Canada 2012

[10] M-H Fraser Ethnobotanical Investigation of Plants Used forthe Treatment of type 2 Diabetes by Two Cree Communities inQuebec Quantitative Comparisons and Antioxidant EvaluationMcGill University 2006

[11] C Leduc J Coonishish P Haddad and A Cuerrier ldquoPlantsused by the Cree Nation of Eeyou Istchee (Quebec Canada)for the treatment of diabetes a novel approach in quantitativeethnobotanyrdquo Journal of Ethnopharmacology vol 105 no 1-2pp 55ndash63 2006

[12] A Downing Inter and Intra-Specific Differences in MedicinalPlant Use for the Treatment of Type II Diabetes Symptoms by theCree Elders of Eeyou Istchee (QC) Universite de Montreal 2010

Evidence-Based Complementary and Alternative Medicine 7

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

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 5

PC1 (3599)

PC3

(10

96

)

PC2 (22

97)

QcRa

TG content

QcGu

QcG4QcGa

QcG3CatQcG2ECat

ChlA

Qc

minus2

minus1

0

1

2

3

minus4 minus2 0 2 4

minus4minus2

02

4

minus3

QcG1

Figure 4 Principal component analysis biplot of 11 phenoliccompounds in R groenlandicum leaves Solid lines represent relativeloadings of these variables on axes 1 2 and 3 TG content (boldarrow) was selected as a supplementary variable and plotted ontoprincipal components generated from the phytochemical markersScores for individual samples are represented by symbols forthe communities of Mistissini (998771) Nemaska (e) Eastmain (◼)Wemindji (X) and Whapmagoostui (998787) Abbreviations representcompounds as follows (+)-catechin Cat chlorogenic acid ChlA(minus)-epicatechin ECat quercetin-3-galactoside QcGa quercetin-3-glucoside QcGu quercetin-glycoside 1 QcG1 quercetin-glycoside2 QcG2 quercetin-glycoside 3 QcG3 quercetin-glycoside 4 QcG4quercetin-3-rhamnoside Qc-Ra and quercetin Qc

250

200

150

100

50

0004 006 008 010 012 014 016

Quercetin concentration (120583M)

Intr

acel

lula

r trig

lyce

ride c

onte

nt(

of D

MSO

cont

rol)

R2 = 0162

Figure 5 Intracellular triglycerides content of 3T3-L1 murineadipocytes exposed to 75 120583gmL of R groenlandicum leaf extractscollected from various locations Quercetin concentrations in crudeextract were significantly and negatively associated with the speciesrsquoadipogenic activity (119901 = 00458) Triglyceride contents are normal-ized to the vehicle control (01 DMSO)

Table 1 Variation partitioning of the adipogenic activity ofR groen-landicum leaf extracts explained by the content in biologically activecompounds (compounds) and the effect of bioclimatic variables(environment) Fraction [119886] corresponds to the unique contributionof compounds once the environment has been taken into accountwhereas fraction [119888] represents the reverse Fraction [119887] representsthe shared portion or overlap between the effect of compounds andenvironment The variation (1198772adj) of each fraction is represented inFigure 6 Asterisk (lowast) indicates significant fractions at 120572 = 005

Fractions 1198772

adj p[119886 + 119887] = compounds 0491 00279lowast

[119887 + 119888] = environment 0109 0150[119886 + 119887 + 119888] = compounds +environment (full model) 0534 00437lowast

[119886] = compounds|environment 0424 00619[119887] = shared 00668 Not testable[119888] = environment|compounds 00424 0302[119889] = unexplained (residuals) 0466 Not testable

100

80

60

40

20

0

Varia

tion

()

a

bc

d

Fraction

Figure 6 Variation partitioning of the adipogenic activity ofR groenlandicum leaf extracts explained by the content in bio-logically active compounds and the effect of bioclimatic vari-ables 119886 = compounds|environment 119887 = shared component 119888 =environment|compounds and119889=unexplained (see Table 1 formoredetails) The full model fraction [119886 + 119887 + 119888] as well as the modelincluding compounds only fraction [119886 + 119887] was significant at 120572 =005

compound related to changes in the adipogenic activity of Rgroenlandicum was not found to be strongly associated withenvironmental variables [1] This may hence explain why theportion of variation those environmental variables contributeto the model and more specifically to the content of biologi-cally active compounds which best explains the variation inTG content is considerably small (Table 1 Figure 6)

On the other hand our results provide support for thehypothesis that synergistic interactions may occur between

6 Evidence-Based Complementary and Alternative Medicine

compounds For instance the inhibitory action of Hibiscussabdariffa (Malvaceae) was greater than the sum of its partswhen polyphenols had been fractionated isolated and testedindividually [35] More importantly in bioassay-guided frac-tionation experiments the activity of crude Labrador teaextracts was higher than that of each active compound testedindividually [24] Finally quercetin and resveratrol togetherdecreased lipid accumulation considerably more than each ofthese used separately at the same dose [34]

Many of the compounds quantified in this paperhave shown adipogenic activity in some form or anotherQuercetin-3-glucoside has been found toxic to adipocytesat relatively low concentration (50 120583M) but was not foundto affect adipogenic activity [33] Quercetin-3-rhamnosidehas been found inactive at low concentrations yet inhibitedadipogenesis at high concentrations and chlorogenic acid hasalso been found to inhibit intracellular triglycerides accumu-lation [33] Content variations of some of these particularly(+)-catechin and (minus)-epicatechin have been explained byenvironmental variables [1] Although the individual effect ofthese compounds was not detected in our study it does notundermine the role they may play when found in a cocktailof substances

4 Conclusion

We have previously shown that latitude acted as a markerfor the impact of environmental variables on phytochemicalconcentrations [1] Therefore a trend could possibly existbetween abiotic factors and concentration of targeted sec-ondary metabolites but a larger sample size might be neededto detect it There may also be other environmental climaticand even biotic factors that were not taken into accountwhich explain the changes in quercetin content These maybetter explain the ecophysiological processes affecting theantidiabetic potential of R groenlandicum Increase in theadipogenic potential of this traditional medicine was asso-ciated primarily with lower concentrations of quercetin butthe cause for its variation will require further investigationNonetheless our results do not provide enough evidence tojustify the idea that specific accessions of Labrador tea mayhave reproducibly better adipogenic potential than othersalong a latitudinal gradient Conversely our study impliesthat random harvesting of R groenlandicum in the Eeyouchterritory of NorthernQuebec should not have amajor impacton the quality of traditional preparations orNHPsmade fromthis plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by the Canadian Institutes ofHealthResearch (CIHR) Team Grant (CTP-79855) to Pierre S Had-dad J T Arnason and Alain Cuerrier and discovery grant toJ T Arnason as well as funding from theNatural Sciences and

Engineering Research Council (NSERC) Canadarsquos NorthernInternship program and Network Environments for Abo-riginal Health Research (NEAHR) to M Rapinski Specialthanks are due to the Elders of the Eeyou Istchee CreeNationsof Mistissini Nemaska Waskaganish Eastmain Wemindjiand Whapmagoostui for sharing their traditional knowledgeand allowing us to collect medicinal plants from their landswith the purpose of bridging indigenous knowledge andcontemporary scienceThe authors also thank the Cree Boardof Health and Social Services of James Bay for their constantsupport as well as A Leger N Roy A Downing Y TendlandB Walsh-Roussel C H Ta D Vallerand N Shang and MOuchfoun for helping out with field and lab work Specialrecognition is due to Jonathan Ferrier who also providedcomments ideas and support Finally thanks are due to SDaigle and P Legendre for statistical advice

References

[1] M Rapinski R Liu A Saleem J T Arnason and A CuerrierldquoEnvironmental trends in the variation of biologically activephenolic compounds in Labrador tea Rhododendron groen-landicum from northern Quebec Canadardquo Botany vol 92 no11 pp 783ndash794 2014

[2] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of Eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[3] R A Zieba Healing and Healers among the Northern CreeUniversity of Manitoba 1990

[4] E V Siegfried Ethnobotany of the Northern Cree of WabascaDesmarais University of Calgary 1994

[5] R J Marles C Clavelle L Monteleone et al Aboriginal PlantUse in Canadas Northwest Boreal Forest Natural ResourcesCanada Edmonton Canada 2008

[6] A Cuerrier and Elders of Kangiqsujuaq The Botanical Knowl-edge of the Inuit of Kangiqsujuaq Avataq Cultural InstituteInukjuak Canada 2011

[7] A Cuerrier and Elders of Kangiqsualujjuaq The BotanicalKnowledge of the Inuit of Kangiqsualujjuaq Nunavik AvataqCultural Institute Inukjuak Canada 2011

[8] A Cuerrier Elders of Umiujaq and Elders of KuujjuarapikTheBotanical Knowledge of the Inuit of Umiujaq and KuujjuarapikNunavik Avataq Cultural Institute Inukjuak Canada 2011

[9] A Cuerrier and L Hermanutz Our Plants Our Land Plantsof Nain and Torngat Mountains Basecamp and Research Station(Nunatsiavut) Institut de Recherche en Biologie VegetaleMontreal Canada Memorial University of Newfoundland StJohnrsquos Canada 2012

[10] M-H Fraser Ethnobotanical Investigation of Plants Used forthe Treatment of type 2 Diabetes by Two Cree Communities inQuebec Quantitative Comparisons and Antioxidant EvaluationMcGill University 2006

[11] C Leduc J Coonishish P Haddad and A Cuerrier ldquoPlantsused by the Cree Nation of Eeyou Istchee (Quebec Canada)for the treatment of diabetes a novel approach in quantitativeethnobotanyrdquo Journal of Ethnopharmacology vol 105 no 1-2pp 55ndash63 2006

[12] A Downing Inter and Intra-Specific Differences in MedicinalPlant Use for the Treatment of Type II Diabetes Symptoms by theCree Elders of Eeyou Istchee (QC) Universite de Montreal 2010

Evidence-Based Complementary and Alternative Medicine 7

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

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

compounds For instance the inhibitory action of Hibiscussabdariffa (Malvaceae) was greater than the sum of its partswhen polyphenols had been fractionated isolated and testedindividually [35] More importantly in bioassay-guided frac-tionation experiments the activity of crude Labrador teaextracts was higher than that of each active compound testedindividually [24] Finally quercetin and resveratrol togetherdecreased lipid accumulation considerably more than each ofthese used separately at the same dose [34]

Many of the compounds quantified in this paperhave shown adipogenic activity in some form or anotherQuercetin-3-glucoside has been found toxic to adipocytesat relatively low concentration (50 120583M) but was not foundto affect adipogenic activity [33] Quercetin-3-rhamnosidehas been found inactive at low concentrations yet inhibitedadipogenesis at high concentrations and chlorogenic acid hasalso been found to inhibit intracellular triglycerides accumu-lation [33] Content variations of some of these particularly(+)-catechin and (minus)-epicatechin have been explained byenvironmental variables [1] Although the individual effect ofthese compounds was not detected in our study it does notundermine the role they may play when found in a cocktailof substances

4 Conclusion

We have previously shown that latitude acted as a markerfor the impact of environmental variables on phytochemicalconcentrations [1] Therefore a trend could possibly existbetween abiotic factors and concentration of targeted sec-ondary metabolites but a larger sample size might be neededto detect it There may also be other environmental climaticand even biotic factors that were not taken into accountwhich explain the changes in quercetin content These maybetter explain the ecophysiological processes affecting theantidiabetic potential of R groenlandicum Increase in theadipogenic potential of this traditional medicine was asso-ciated primarily with lower concentrations of quercetin butthe cause for its variation will require further investigationNonetheless our results do not provide enough evidence tojustify the idea that specific accessions of Labrador tea mayhave reproducibly better adipogenic potential than othersalong a latitudinal gradient Conversely our study impliesthat random harvesting of R groenlandicum in the Eeyouchterritory of NorthernQuebec should not have amajor impacton the quality of traditional preparations orNHPsmade fromthis plant

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by the Canadian Institutes ofHealthResearch (CIHR) Team Grant (CTP-79855) to Pierre S Had-dad J T Arnason and Alain Cuerrier and discovery grant toJ T Arnason as well as funding from theNatural Sciences and

Engineering Research Council (NSERC) Canadarsquos NorthernInternship program and Network Environments for Abo-riginal Health Research (NEAHR) to M Rapinski Specialthanks are due to the Elders of the Eeyou Istchee CreeNationsof Mistissini Nemaska Waskaganish Eastmain Wemindjiand Whapmagoostui for sharing their traditional knowledgeand allowing us to collect medicinal plants from their landswith the purpose of bridging indigenous knowledge andcontemporary scienceThe authors also thank the Cree Boardof Health and Social Services of James Bay for their constantsupport as well as A Leger N Roy A Downing Y TendlandB Walsh-Roussel C H Ta D Vallerand N Shang and MOuchfoun for helping out with field and lab work Specialrecognition is due to Jonathan Ferrier who also providedcomments ideas and support Finally thanks are due to SDaigle and P Legendre for statistical advice

References

[1] M Rapinski R Liu A Saleem J T Arnason and A CuerrierldquoEnvironmental trends in the variation of biologically activephenolic compounds in Labrador tea Rhododendron groen-landicum from northern Quebec Canadardquo Botany vol 92 no11 pp 783ndash794 2014

[2] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of Eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[3] R A Zieba Healing and Healers among the Northern CreeUniversity of Manitoba 1990

[4] E V Siegfried Ethnobotany of the Northern Cree of WabascaDesmarais University of Calgary 1994

[5] R J Marles C Clavelle L Monteleone et al Aboriginal PlantUse in Canadas Northwest Boreal Forest Natural ResourcesCanada Edmonton Canada 2008

[6] A Cuerrier and Elders of Kangiqsujuaq The Botanical Knowl-edge of the Inuit of Kangiqsujuaq Avataq Cultural InstituteInukjuak Canada 2011

[7] A Cuerrier and Elders of Kangiqsualujjuaq The BotanicalKnowledge of the Inuit of Kangiqsualujjuaq Nunavik AvataqCultural Institute Inukjuak Canada 2011

[8] A Cuerrier Elders of Umiujaq and Elders of KuujjuarapikTheBotanical Knowledge of the Inuit of Umiujaq and KuujjuarapikNunavik Avataq Cultural Institute Inukjuak Canada 2011

[9] A Cuerrier and L Hermanutz Our Plants Our Land Plantsof Nain and Torngat Mountains Basecamp and Research Station(Nunatsiavut) Institut de Recherche en Biologie VegetaleMontreal Canada Memorial University of Newfoundland StJohnrsquos Canada 2012

[10] M-H Fraser Ethnobotanical Investigation of Plants Used forthe Treatment of type 2 Diabetes by Two Cree Communities inQuebec Quantitative Comparisons and Antioxidant EvaluationMcGill University 2006

[11] C Leduc J Coonishish P Haddad and A Cuerrier ldquoPlantsused by the Cree Nation of Eeyou Istchee (Quebec Canada)for the treatment of diabetes a novel approach in quantitativeethnobotanyrdquo Journal of Ethnopharmacology vol 105 no 1-2pp 55ndash63 2006

[12] A Downing Inter and Intra-Specific Differences in MedicinalPlant Use for the Treatment of Type II Diabetes Symptoms by theCree Elders of Eeyou Istchee (QC) Universite de Montreal 2010

Evidence-Based Complementary and Alternative Medicine 7

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

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

[13] M Rapinski Ethnobotanique de la Nation crie dEeyou Istchee etvariation geographique des plantes medicinales antidiabetiquesUniversite de Montreal 2012

[14] D C A Spoor L C Martineau C Leduc et al ldquoSelectedplant species from theCree pharmacopoeia of northernQuebecpossess anti-diabetic potentialrdquo Canadian Journal of Physiologyand Pharmacology vol 84 no 8-9 pp 847ndash858 2006

[15] M-H Fraser A Cuerrier P S Haddad J T Arnason P LOwen and T Johns ldquoMedicinal plants of Cree communities(Quebec Canada) antioxidant activity of plants used to treattype 2 diabetes symptomsrdquo Canadian Journal of Physiology andPharmacology vol 85 no 11 pp 1200ndash1214 2007

[16] D Harbilas L C Martineau C S Harris et al ldquoEvaluation ofthe antidiabetic potential of selected medicinal plant extractsfrom the Canadian boreal forest used to treat symptoms ofdiabetes part IIrdquo Canadian Journal of Physiology and Pharma-cology vol 87 no 6 pp 479ndash492 2009

[17] C S Harris L-P Beaulieu M-H Fraser et al ldquoInhibition ofadvanced glycation end product formation by medicinal plantextracts correlates with phenolic metabolites and antioxidantactivityrdquo Planta Medica vol 77 no 2 pp 196ndash204 2011

[18] X-K Fang J Gao and D-N Zhu ldquoKaempferol and quercetinisolated from Euonymus alatus improve glucose uptake of 3T3-L1 cells without adipogenesis activityrdquo Life Sciences vol 82 no11-12 pp 615ndash622 2008

[19] P A Grimaldi ldquoThe roles of PPARs in adipocyte differentia-tionrdquo Progress in Lipid Research vol 40 no 4 pp 269ndash281 2001

[20] J BHansen andK Kristiansen ldquoRegulatory circuits controllingwhite versus brown adipocyte differentiationrdquoThe BiochemicalJournal vol 398 no 2 pp 153ndash168 2006

[21] O A MacDougald and S Mandrup ldquoAdipogenesis forces thattip the scalesrdquo Trends in Endocrinology amp Metabolism vol 13no 1 pp 5ndash11 2002

[22] E D Rosen and O A MacDougald ldquoAdipocyte differentiationfrom the inside outrdquoNature ReviewsMolecular Cell Biology vol7 no 12 pp 885ndash896 2006

[23] A Saleem C S Harris M Asim et al ldquoA RP-HPLC-DAD-APCIMSD method for the characterisation of medicinal Eri-caceae used by the Eeyou Istchee Cree First Nationsrdquo Phyto-chemical Analysis vol 21 no 4 pp 328ndash339 2010

[24] M Ouchfoun Validation des effets antidiabetiques de Rhodo-dendron groenlandicum une plante medicinale des Cri de laBaie James dans le modele in vitro et in vivo elucidationdes mecanismes daction et identification des composes actifsUniversite de Montreal 2011

[25] LAASGLFCCFS et al SelectedModeledClimateData for PointLocations LAAS Sault Ste Marie Canada 2014

[26] D W McKenney J H Pedlar K Lawrence K Campbell andM F Hutchinson ldquoBeyond traditional hardiness zones usingclimate envelopes to map plant range limitsrdquo BioScience vol 57no 11 pp 929ndash937 2007

[27] D McKenney P Papadopol K Lawrence et al ldquoCustomizedspatial climate models for Canadardquo 2007

[28] S Le J Josse and F Husson ldquoFactoMineR an R package formultivariate analysisrdquo Journal of Statistical Software vol 25 no1 pp 1ndash18 2008

[29] D Borcard P Legendre and P Drapeau ldquoPartialling out thespatial component of ecological variationrdquo Ecology vol 73 no3 pp 1045ndash1055 1992

[30] AMeot P Legendre andD Borcard ldquoPartialling out the spatialcomponent of ecological variation questions and propositions

in the linear modelling frameworkrdquo Environmental and Ecolog-ical Statistics vol 5 no 1 pp 1ndash27 1998

[31] R Core Team R A Language and Environment for StatisticalComputing 2014 httpwwwr-projectorg

[32] K Iwashtta K Yamaki and T Tsushida ldquoEffect of flavonoidson the differentiation of 3T3-L1 adipocytesrdquo Food Science andTechnology Research vol 7 no 2 pp 154ndash160 2001

[33] C-L Hsu and G-C Yen ldquoEffects of flavonoids and phenolicacids on the inhibition of adipogenesis in 3T3-L1 adipocytesrdquoJournal of Agricultural and Food Chemistry vol 55 no 21 pp8404ndash8410 2007

[34] J-Y Yang M A Della-Fera S Rayalam et al ldquoEnhancedinhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetinrdquoLife Sciences vol 82 no 19-20 pp 1032ndash1039 2008

[35] M Herranz-Lopez S Fernandez-Arroyo A Perez-Sanchez etal ldquoSynergism of plant-derived polyphenols in adipogenesisperspectives and implicationsrdquo Phytomedicine vol 19 no 3-4pp 253ndash261 2012

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