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Phytomedicine 21 (2014) 109– 117

Contents lists available at ScienceDirect

Phytomedicine

j ourna l h o mepage: www.elsev ier .de /phymed

ffects of fermented rooibos (Aspalathus linearis) on adipocyteifferentiation

icheline Sandersona,∗, Sithandiwe E. Mazibukoa, Elizabeth Joubertb,c, Dalene de Beerb,abia Johnsona, Carmen Pheiffera, Johan Louwa, Christo J.F. Mullera

Diabetes Discovery Platform, South African Medical Research Council, P.O. Box 19070, Tygerberg 7505, South AfricaPost-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South AfricaDepartment of Food Science, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch 7602, South Africa

r t i c l e i n f o

rticle history:eceived 6 June 2013eceived in revised form 19 July 2013ccepted 9 August 2013

eywords:ooibos (Aspalathus linearis)T3-L1 adipocytesdipogenesisdipocyte metabolismlucose uptake

a b s t r a c t

Rooibos (Aspalathus linearis) contains a rich complement of polyphenols, including flavonoids, consideredto be largely responsible for its health promoting effects, including combatting obesity. The purpose ofthis study was to examine the effect of fermented rooibos hot water soluble solids on in vitro adipocytedifferentiation by using differentiating 3T3-L1 adipocytes. Hot water soluble solids were obtained whenpreparing an infusion of fermented rooibos at “cup-of-tea” strength. The major phenolic compounds(>5 mg/g) were isoorientin, orientin, quercetin-3-O-robinobioside and enolic phenylpyruvic acid-2-O-�-d-glucoside. Treatment of 3T3-L1 adipocytes with 10 �g/ml and 100 �g/ml of the rooibos soluble solidsinhibited intracellular lipid accumulation by 22% (p < 0.01) and 15% (p < 0.05), respectively. Inhibitionof adipogenesis was accompanied by decreased messenger RNA (mRNA) expression of PPAR�, PPAR�,SREBF1 and FASN. Western blot analysis exhibited decreased PPAR�, SREBF1 and AMPK protein expres-

MPKeptin

sion. Impeded glycerol release into the culture medium was observed after rooibos treatment. None ofthe concentrations of rooibos hot water soluble solids was cytotoxic, in terms of ATP content. Interest-ingly, the higher concentration of hot water soluble solids increased ATP concentrations which wereassociated with increased basal glucose uptake. Decreased leptin secretion was observed after rooibostreatment. Our data show that hot water soluble solids from fermented rooibos inhibit adipogenesis andaffect adipocyte metabolism, suggesting its potential in preventing obesity.

ntroduction

Obesity is a chronic lifestyle-related disease that has become global epidemic. Several serious and life-threatening co-orbidities such as type 2 diabetes (T2D), hypertension, coronary

eart disease, chronic inflammation and some cancers often accom-any obesity, especially visceral obesity (Guh et al., 2009). A needxists for the development of safe and effective agents against obe-ity and its underlying disorders.

Obesity or the excessive formation of white adipose tissue masss caused when caloric intake persistently exceeds expenditure.dipogenesis, the process whereby adipose tissue mass is formed,

nvolves proliferation of preadipocytes that undergo differentiationo form mature, lipid-accumulating adipocytes. Adipocyte differ-

ntiation is highly regulated involving the coordinated expressionf a number of proteins (Gregoire, 2001; Siersbæk et al., 2012).eroxisome proliferator-activated receptor gamma (PPAR�) is

∗ Corresponding author. Tel.: +27 219380479; fax: +27 219380456.E-mail address: micheline.sanderson@mrc.ac.za (M. Sanderson).

944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.phymed.2013.08.011

© 2013 Elsevier GmbH. All rights reserved.

regarded as the ‘master regulator’ of adipocyte differentiation(Rosen and MacDougald, 2006). Together with other transcrip-tion factors such as sterol regulatory binding factor (SREBF1) andperoxisome proliferator-activated receptor alpha (PPAR�), PPAR�controls the expression of proteins required for adipogenesis andlipid metabolism (Gregoire, 2001; Siersbæk et al., 2012).

Anti-obesity drugs currently available are plagued with inef-ficacy and side-effects ranging from mild to potentially harmful(Gooda Sahib et al., 2012; Rodgers et al., 2012). Consequently, theuse of natural products to combat obesity is rapidly increasing.Research to identify targets from natural and alternative sourcesfor development into clinical treatments against obesity is gain-ing momentum (Gooda Sahib et al., 2012). Various preparations ofCamelia sinensis teas, for example green and oolong teas, have tra-ditionally been used to treat several metabolic disorders includingobesity (Sajilata et al., 2008; Chacko et al., 2010). Likewise the SouthAfrican herbal tea, rooibos, has anti-oxidant, anti-mutagenic and

anti-cancer effects (as reviewed in Joubert et al., 2008). Rooibos hasalso demonstrated ameliorative effects against several metabolicconditions such as hyperglycemia, hyperlipidemia, oxidative stressand cardiovascular disease (Joubert et al., 2008; Beltrán-Debón

110 M. Sanderson et al. / Phytomedicine 21 (2014) 109– 117

Table 1Taqman gene expression assays used in the study.

Taqman gene expression assay Assay number†

Sterol regulatory element bindingtranscription factor 1 (SREBF1)

Mm00550338 m1

Fatty acid synthase (FASN) Mm00662319 m1Peroxisome proliferator activatedreceptor alpha (PPAR�)

Mm00440939 m1

Peroxisome proliferator activatedreceptor alpha (PPAR�)

Mm01184322 m1

�-Actin 4352341EGlyceraldehyde-3-phosphate 4352339E

e2a2

orKttp2ltfe

spoha

M

M

c((cB(GvEOprHCSLMtbrsaU

Fig. 1. HPLC chromatograms of the fermented rooibos HWSS. The phenolic com-pounds identified by comparison of data from reference standards at 288 nm (A) and350 nm (B) are 1: PPAG; 2: Isoorientin; 3: Orientin; 4: Aspalathin; 5: Quercetin-3-O-robinobioside; 6: Vitexin; 7: Hyperoside; 8: Rutin; 9: Isovitexin; 10: Isoquercitrin;11: Nothofagin.

Table 2The phenolic composition of the hot water fermented rooibos infusion.

Type Compound Soluble solids(mg/g)

Infusion (mg/l)

Phenylpropenoid PPAGb 6.30 9.54Dihydrochalcone Aspalathin 3.67 5.56

Nothofagin 0.64 0.97

Flavone Isoorientin 14.44 21.88Orientin 8.62 13.06Vitexin 1.91 2.89Isovitexin 2.29 3.47

Flavonol Quercetin-3-O-robinobioside

10.24 15.51

Hyperoside 2.91 4.41Rutin 2.31 3.50Isoquercitrin 1.38 2.09

HWSSa 1515.00

a HWSS, hot water soluble solids in fermented rooibos, extracted during 5 min

dehydrogenase

† Assay number indicates the Applied Biosystems product number.

t al., 2011; Marnewick et al., 2011; Pantsi et al., 2011; Muller et al.,012; Son et al., 2012). Recently, the anti-obesity potential of anqueous rooibos extract has been reported (Beltrán-Debón et al.,011).

Several in vitro and in vivo studies have demonstrated the anti-besity action of individual phenolic compounds also present inooibos (Ahn et al., 2008; Choi et al., 2006; Gosmann et al., 2012;im et al., 2010; Lee et al., 2010; Yang et al., 2008). It is proposed that

hese compounds act by inhibiting adipogenesis-related transcrip-ion factors and proteins such as PPAR�, CCAAT/enhancer-bindingrotein alpha (C/EBP�) and SREBF1 (Ahn et al., 2008; Choi et al.,006; Gosmann et al., 2012; Kim et al., 2010). Enzymes involved in

ipid metabolism and energy homeostasis including fatty acid syn-hase (FASN) and AMP-activated protein kinase (AMPK) were alsoound to be regulated by these compounds (Ahn et al., 2008; Leet al., 2010).

The aim of this study was to investigate the effect of hot wateroluble solids (HWSS) extracted from fermented rooibos whenreparing an infusion at a “cup-of-tea” strength on adipocyte devel-pment. We utilized differentiating 3T3-L1 adipocytes to studyow adipogenesis and related gene and protein expression wereffected by rooibos soluble solids.

aterials and methods

aterials and reagents used

3T3-L1 mouse embryonic fibroblasts (CL-173TM) were pur-hased from the American Type Culture Collection® (ATCC)Manassas, VA, USA). Dulbecco’s modified Eagle’s mediumDMEM), Dulbecco’s phosphate buffered saline (DPBS), peni-illin/streptomycin and trypsin/versene were bought from Lonzaiowhitaker® (Walkersville, MD, USA). Newborn calf serumNBCS) and fetal bovine serum (FBS) were obtained fromibco®, InvitrogenTM (EU Approved, origin: South America). Iso-itexin, hyperoside and orientin standards were obtained fromxtrasynthese (Genay, France). Vitexin, isoorientin and luteolin-7--glucoside were bought from Roth (Karlsruhe, Germany). Enolichenylpyruvic acid-2-O-�-d-glucoside (PPAG) was isolated fromooibos (purity > 95% by HPLC and LC–MS) and supplied by the Post-arvest & Wine Technology Division of the Agricultural Researchouncil of South Africa (ARC Infruitec-Nietvoorbij, Stellenbosch,outh Africa). Aspalathin and nothofagin (purity > 95% by HPLC andC–MS) were supplied by the PROMEC Unit of the South Africanedical Research Council (Cape Town, South Africa). The lep-

in (EZML-82K) and adiponectin (EZMADP-60K) ELISA kits wereought from Millipore Corporation (Billerica, MA, USA). All other

eagents including ferulic acid, rutin, isoquercitrin, dexametha-one, 3-isobutyl-1-methylxanthine (IBMX), insulin and phenol red-nd glucose-free DMEM were procured from Sigma (St. Louis, MO,SA), unless otherwise stated.

infusion at “cup-of-tea” strength.b PPAG, enolic phenylpyruvic acid-2-O-�-d-glucoside.

Preparation and analyses of freeze-dried, fermented rooibosinfusion

Unpasteurized, fermented rooibos plant material, comprisingleaves and thin stems, was obtained from a commercial processingfacility (Rooibos Ltd., Clanwilliam, South Africa). The plant mate-rial was refined by sifting and steam-pasteurization as previouslydescribed (Joubert et al., 2012). Preparation of an infusion at “cup-of-tea” strength entailed adding 600 ml boiling deionised water to7.5 g of rooibos and infusing for 5 min. The infusion, filtered con-secutively through a tea strainer and Whatman No. 4 filter paper,was cooled to room temperature, frozen at −20 ◦C and freeze-dried.

Aliquots of the filtrate were analyzed gravimetrically for the hotwater soluble solids (HWSS) content of 20 ml aliquots (Joubert et al.,2012).

M. Sanderson et al. / Phytomedicine 21 (2014) 109– 117 111

Fig. 2. Reduction of intracellular triglyceride content in differentiating 3T3-L1 adipocytes by fermented rooibos HWSS (freeze-dried infusion). Undifferentiated 3T3-L1preadipocytes controls cultured without rooibos (A) and differentiated adipocytes in the absence (B) and presence (C) of 10 �g/ml rooibos HWSS are illustrated. Quantificationof Oil-Red-O staining intensity in differentiating 3T3-L1 adipocytes was measured after 9 days of chronic exposure to 0.5, 10 and 100 �g/ml of rooibos HWSS (D). Resultsf eateda rystaS

dbd(se

C

aNc4f

C

rw(m0w

or the Oil-Red-O staining shown are of a representative experiment which was reps measured using crystal violet staining is expressed relative to the control (E). Cignificance is depicted as * where p ≤ 0.05 and ** where p ≤ 0.01.

For quantification of the major phenolic compounds the freeze-ried extract was reconstituted in purified water and analyzedy high-performance liquid chromatography with diode-arrayetection (HPLC–DAD) as described by in detail by Joubert et al.2012). All compounds were quantified using authentic referencetandards, except quercetin-3-O-robinobioside (expressed as rutinquivalents) as no standard was available.

ell culture and differentiation

3T3-L1 preadipocytes were cultured, maintained and differenti-ted according to recommendations of the ATCC (Technical Bulletino. 9; 2011) until day 5. On day 5, the cell culture medium washanged to DMEM without phenol red and supplemented with.5 g/l d-glucose, 3.7 g/l NaHCO3, 1 �g/ml insulin and 0.1% (w/v)atty acid-free bovine serum albumin until day 8.

ell treatments

For cell culture, a concentrated solution of the freeze-driedooibos HWSS (100 mg/ml) was prepared in tissue culture-gradeater, where after a 1 mg/ml stock was made in culture medium

adipocyte differentiation medium or adipocyte maintenanceedium). Rooibos working solutions of 100 �g/ml, 10 �g/ml and

.5 �g/ml were prepared by diluting the 1 mg/ml stock solutionith culture medium.

two more times in quadruplicate with similar trends. The percentage cell densityl violet results shown represent the means of 3 independent experiments ± SEM.

3T3-L1 preadipocytes were differentiated in the presence andabsence of the rooibos HWSS. Cells were chronically exposed torooibos for 9 days (day 8), with medium supplemented with therooibos HWSS being changed daily. All experiments were termi-nated on day 8, thus 9 days after the induction of differentiation.

Intracellular lipid content determination

Oil-Red-O staining was adapted from Suzuki et al. (2011). Tocompensate for cell density, the 3T3-L1 adipocytes were alsostained with 0.01% crystal violet by modifying the method of Gillieset al. (1986). Lipid content (Oil-Red-O measured at 490 nm) wasnormalized to cell density (crystal violet measured at 570 nm).Percentage lipid content was determined using the following cal-culation:

(A490 nm/A570 nm)sample(A490 nm/A570 nm)control

× 100

Free glycerol determination

The amount of free glycerol in the collected cell culture medium

was ascertained using the fluorescent glycerol release assay kitfrom Biovision Inc. (Milpitas, CA, USA) according to the manu-facturer’s protocol. Free glycerol content was normalized to celldensity (crystal violet measured at 570 nm).

112 M. Sanderson et al. / Phytomedicine 21 (2014) 109– 117

Fig. 3. Chronic exposure to fermented rooibos HWSS leads to pronounced reduction in adipocyte-related mRNA expression. Differentiating 3T3-L1 adipocytes were growni olateds SN (Da

M

at

A

k

n the presence and absence of 100 �g/ml of rooibos HWSS until day 8. Total RNA ishown are the relative mRNA expression of PPAR� (A), PPAR� (B), SREBF1 (C) and FAs * where p ≤ 0.05 and ** where p ≤ 0.01 and n = 3.

easurement of glucose uptake

Glucose uptake was measured with the fluorescent glucosessay kit from Biovision Inc. according to the manufacturer’s pro-ocol.

TP content determination

ATP content was determined using the ATP fluorometric assayit from Biovision Inc. according to the manufacturer’s guidelines.

from cells that were not treated with rooibos HWSS was used as the control. Data), normalized to two references genes (�-actin and GAPDH). Significance is depicted

RNA isolation and gene expression analysis

3T3-L1 cells were cultured in 6-well CellBIND® plates. Threewells per treatment were pooled and total RNA was extractedusing 1 ml of Qiazol (Qiagen, Hilden, Germany). Thereafter, RNAwas purified with the RNeasy Mini Kit (Qiagen) according to themanufacturer’s instructions. Residual genomic DNA was removed

with the Turbo DNase kit (Ambion Inc., Austin, TX, USA) and com-plimentary DNA (cDNA) prepared using the High Capacity ReverseTranscription kit (Applied Biosystems, Foster City, CA, USA). Quan-titative real-time PCR was performed as follows: 5 �l Taqman

M. Sanderson et al. / Phytomedicine 21 (2014) 109– 117 113

Fig. 4. Chronic exposure to fermented rooibos HWSS diminished adipocyte-related protein expression. 3T3-L1 adipocytes were grown in the presence and absence of1 ion ofa

(gwRt9n

00 �g/ml of rooibos for 9 days (day 8). Data shown are the relative protein expressre representative of three independent experiments.

Applied Biosystems) gene expression master mix, 0.5 �l of Taqmanene expression assay (Table 1), 1 �l cDNA equivalent to 12.5 ng andater to a final volume of 10 �l. PCR reactions were run on a 7500

eal Time PCR system (Applied Biosystems) using cycling condi-ions: 50 ◦C for 2 min and 95 ◦C for 10 min, followed by 40 cycles of5 ◦C for 15 s and 60 ◦C for 1 min. Messenger RNA expression wasormalized to �-actin and GAPDH.

PPAR� (A), PPAR� (B) and SREBF1 (C), normalized to �-actin. Western blots shown

Protein extraction and western blot analysis

Proteins were extracted according to the method described by

Mazibuko et al. (2013). For western blot analysis, forty microgramof the extracted proteins was separated using electrophoresis andtransferred to PVDF membranes (Amersham Pharmacia Biotech,Aylesbury, UK). Membranes were blocked with 5% (w/v) fat-free

114 M. Sanderson et al. / Phytomed

Fig. 5. Glycerol release in differentiating 3T3-L1 adipocytes is reduced by treat-mRc

mP((pauU

E

m

S

sASocDa

R

P

tm3Fofa5p(ao

A substantial decrease of 70% (p ≤ 0.001) was observed in leptin

ent with fermented rooibos HWSS. Data are depicted as percentage of the control.esults are shown as mean ± SEM for 3 independent experiments with four repli-ates each. Significance is depicted as ** where p ≤ 0.01.

ilk powder and incubated overnight at 4 ◦C with anti-rabbitPAR� (1:1500) (Cell Signaling, Beverly, MA, USA), PPAR� (1:1500)Cell Signaling), SREBF1 (1:8000) (Sigma) or anti-�-actin (1:1000)Santa Cruz, Dallas, TX, USA) antibodies. After membrane washing,roteins were incubated with the relevant HRP conjugated 2◦ IgGntibody (Santa Cruz). Immuno-responsive proteins were detectedsing chemiluminescence (KPL laboratories, Gaithersburg, MD,SA). �-Actin was used as the reference control.

nzyme-linked immunosorbent assay (ELISA)

The leptin and adiponectin ELISA assays were done according toanufacturer’s instructions.

tatistical analysis

Statistical analysis of data was done using GraphPad Prism 5oftware (GraphPad Software Inc., La Jolla, CA, USA). One-wayNOVA test with a Dunnett’s post-hoc test was carried out. Atudent’s t-test was used to ascertain any significant differencesbtained in the analysis of the relative expression of mRNA trans-ripts. P-value ≤ 0.05 was considered as statistically significant.ata shown are from three independent experiments, where n ≤ 3nd are presented as mean ± SEM.

esults

henolic composition of fermented rooibos infusion

The constituents of the rooibos infusion are representative ofhe complement of polyphenols found in a cup of commercial fer-

ented rooibos tea. HPLC–DAD chromatograms at 288 nm and50 nm, depicting the major phenolic compounds, are shown inig. 1A and B, respectively. All compounds were present at 1.4 mg/gf the HWSS (freeze-dried infusion) or higher (Table 2), excepterulic acid and luteolin-7-O-glucoside that were not presentt detectable levels. The compounds quantified comprised ca..47% of the HWSS (1520 mg/l infusion). The main phenolic com-

ounds were isoorientin (14.4 mg/g), quercetin-3-O-robinobioside10.2 mg/g), orientin (8.6 mg/g) and PPAG (6.30 mg/g), whereasspalathin was present at 3.7 mg/g. The remaining compoundsccurred at less than 3 mg/g of the HWSS.

icine 21 (2014) 109– 117

Fermented rooibos reduces lipid accumulation in differentiating3T3-L1 adipocytes

Microscopic analysis of Oil-Red-O stained differentiating 3T3-L1adipocytes, cultured in the absence (Fig. 2A) and presence (Fig. 2Band C) of rooibos, revealed the accrual of intracellular lipid droplets.Chronic treatment with the rooibos HWSS appeared to inhibit lipidaccumulation (Fig. 2C). Quantification of the extracted Oil-Red-Ostain showed that the lipid content was significantly reduced by∼22% (p ≤ 0.01) when treated at 10 �g/ml and ∼15% (p ≤ 0.05) at100 �g/ml rooibos HWSS as compared to the adipogenesis control(Fig. 2D), indicating a reduction in intracellular lipid accumulation.Rooibos treatment caused a modest increase in crystal violet inten-sity, reaching significance at a concentration of 100 �g/ml (p ≤ 0.05)(Fig. 2E). This suggests increased cell density due to continued cellproliferation.

Fermented rooibos suppresses adipocyte-related gene and proteinexpression concomitantly in differentiating 3T3-L1 adipocytes

Differentiation of 3T3-L1 cells with 100 �g/ml of the rooi-bos HWSS decreased the mRNA abundance of PPAR� (Fig. 3A)and PPAR� (Fig. 3B) by 85% (0.2 ± 0.06 fold; p ≤ 0.01) and 84%(0.2 ± 0.03 fold; p ≤ 0.01), respectively. The mRNA expression ofSREBF1 (p ≤ 0.05) (Fig. 3C) and FASN (p ≤ 0.01) (Fig. 3D) were corre-spondingly decreased by 35% (0.5 ± 0.06 fold) and 56% (0.6 ± 0.02fold) following treatment compared to non-treated adipogenesiscontrols.

Rooibos treatment had no effect on PPAR� protein levels(Fig. 4A). Chronic exposure to 100 �g/ml rooibos HWSS reducedthe protein levels of PPAR� (Fig. 4B) and SREBF1 (Fig. 4C) by 37%and 68%, respectively, albeit not significant when compared to theadipogenesis control.

Glycerol release in differentiating 3T3-L1 adipocytes is suppressedby fermented rooibos

The glycerol content in the cell supernatant was decreased by22% (p ≤ 0.01) in 3T3-L1 adipocytes differentiated with 100 �g/mlof the rooibos HWSS compared to control cells (Fig. 5). No changein glycerol secretion was observed at the other concentrationstested.

Fermented rooibos enhances cellular metabolism indifferentiating 3T3-L1 adipocytes

Basal glucose uptake was improved in the presence of the rooi-bos HWSS (Fig. 6A), with treatment at 10 �g/ml resulting in asignificant increase of 48.5% (p ≤ 0.05) relative to the adipogenesiscontrol. Chronic administration of the rooibos HWSS elevated theATP content in differentiating 3T3-L1 adipocytes by 19%, 45% and188% in a dose related manner, although only the 100 �g/ml rooibostreatment was significant (p ≤ 0.01) (Fig. 6B). At this same concen-tration of rooibos HWSS, phosphorylation of AMPK was decreasedrelative to the control (Fig. 6C).

Fermented rooibos affects adipokine secretion in differentiating3T3-L1 adipocytes

secretion by differentiating 3T3-L1 adipocytes after chronic treat-ment with rooibos HWSS at a concentration of 100 �g/ml (Fig. 7A).Adiponectin secretion remained unaffected by these treatmentconditions (Fig. 7B).

M. Sanderson et al. / Phytomedicine 21 (2014) 109– 117 115

Fig. 6. Fermented rooibos HWSS affects cellular metabolism in differentiating 3T3-L1 adipocytes. Glucose uptake (A) and ATP (B) content were measured. Results areexpressed as a percentage of the relevant control (set at 100%) and shown as mean ± SEM for 3 independent experiments, done in quadruplicate. The extent of AMPKp 0 �g/mb alize

D

btcstrea(

hosphorylation after chronic exposure of differentiating 3T3-L1 adipocytes to 10lot shown is representative of three independent experiments and data were norm

iscussion

The phenolic composition of the South African herbal tea, rooi-os, differs qualitatively and quantitatively from that of C. sinensiseas, in particular the absence of flavan-3-ols such as epigallocate-hin gallate, whilst containing dihydrochalcones. In this and othertudies, rooibos, its infusions and extracts were shown to con-ain aspalathin, orientin, isoorientin, quercetin-3-O-robinobioside,

utin and PPAG, amongst others (Rabe et al., 1994; Beltrán-Debónt al., 2011; Joubert et al., 2012; Muller et al., 2012). The anti-obesityction of a few of these individual rooibos compounds are reportedChoi et al., 2006; Ahn et al., 2008; Yang et al., 2008 Kim et al.,

l of rooibos HWSS was determined using Western blot analysis (C). The westernd to �-actin. Significance is depicted as * where p ≤ 0.05 and ** where p ≤ 0.01.

2010; Lee et al., 2010; Gosmann et al., 2012), however, synergismbetween these compounds could possibly enhance the anti-obesityaction of rooibos extracts (Ulrich-Merzenich et al., 2010). In thisstudy we investigated the effect of HWSS, extracted from fer-mented rooibos during the preparation of an infusion, on adipocytedifferentiation. Others have previously reported anti-obesity prop-erties using an aqueous rooibos extract (Beltrán-Debón et al., 2011).These authors established the hypolipidemic effect of rooibos in

LDLr−/− mice fed a high fat diet, but this effect was stringentlydependent on diet type. In this present study, we chose to usean infusion since rooibos is commonly consumed in this form(Joubert and de Beer, 2011).

116 M. Sanderson et al. / Phytomedicine 21 (2014) 109– 117

F onic tr ollectc one in

idDds(taoaBaIaTtesebe

mTuc

wpesaa2t

stace

ig. 7. Adipokine secretion in differentiating 3T3-L1 adipocytes is affected by chrooibos HWSS for the duration of the differentiation period. Culture medium was control (set at 100%) and given as mean ± SEM of three independent experiments, d

We showed that the rooibos HWSS inhibited adipogenesisn 3T3-L1 cells. Inhibition of adipogenesis was accompanied byecreased mRNA expression of PPAR�, PPAR�, SREBF1 and FASN.ecreased protein expression of PPAR� and SREBF1 was alsoemonstrated. PPAR�, PPAR� and SREBF1 control the expres-ion of proteins required for adipogenesis and lipid metabolismGregoire, 2001; Siersbæk et al., 2012). Our results suggest thathe decreased lipid accumulation observed was due to inhibition ofdipocyte differentiation, indicated by the continued proliferationf preadipocytes, decreased expression of key adipogenic proteinsnd the lipogenic protein, FASN as well as reduced glycerol release.eltrán-Debón et al. (2011) described the anti-lipogenic effect of anqueous rooibos extract in fully differentiated 3T3-L1 adipocytes.n contrast to our results, these authors were unable to demonstraten anti-adipogenic effect in differentiating 3T3-L1 preadipocytes.his discrepancy could be attributed to compositional variations ofhe extracts used and differences in the experimental proceduresmployed. Our findings are in accordance with several other in vitrotudies using differentiating 3T3-L1 adipocytes and polyphenolicxtracts derived from sources such as grape skin (Jeong et al., 2011),ilberry (Suzuki et al., 2011), mate (Ilex paraguariensis) (Gosmannt al., 2012) and various other plant sources (Roh and Jung, 2012).

We found that rooibos treatment increased glucose uptake anditochondrial activity in differentiating 3T3-L1 adipocytes as well.

he increased ATP levels, possibly as a result of increased glucoseptake in combination with decreased lipogenesis and lipolysisould be directly related to the decrease in AMPK activation.

Leptin, a protein associated with obesity (Maffei et al., 1995),as decreased in 3T3-L1 adipocytes treated with rooibos in theresent study. Our results are similar to findings for other plantxtracts from Irvingia gabonensis (Oben et al., 2008) and Blumea bal-amifera (Kubota et al., 2009). These plant extracts were reported tolso upregulate the mRNA and protein expression of adiponectin,

protein involved in glucose and lipid metabolism (Kubota et al.,009; Oben et al., 2008). However, in our case, adiponectin secre-ion remained unchanged.

Taken together, our results indicate that chronic rooibos con-umption may have the potential to inhibit adipogenesis and

hereby reduce adipocyte tissue mass expansion and also influencedipocyte metabolism in vivo. However, further work is needed tolarify the precise molecular mechanism through which rooibosxerts its anti-adipogenic effects. For example, the effect of rooibos

reatment with fermented rooibos HWSS. Cells were exposed to 100 �g/ml of theed on day 8 for the respective ELISA assays. Results are expressed as percentage of

quadruplicate. Significance is depicted as *** where p ≤ 0.001.

on enzymes involved in fatty acid metabolism such as acetyl-CoA carboxylase, hormone sensitive lipase and adipose triglyceridelipase could be investigated. Additional investigations into themolecular mechanism(s) through which rooibos alleviates obesity-associated conditions such as hyperglycemia is also justified.

Acknowledgments

This work was supported by the South African Rooibos Council,the Agricultural Research Council and the South African MedicalResearch Council.

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