Mulberry anthocyanins suppresses body weight gain in high-fat diet fed C57BL mice

Dietary supplementation with purified mulberry (Morus australis Poir)anthocyanins suppresses body weight gain in high-fat diet fed C57BL/6miceTao Wu a ,b ,Xueming Qi a ,Yan Liu a ,Jun Guo a ,Ruiyu Zhu a ,Wei Chen a ,Xiaodong Zheng a ,b ,⇑,Ting Yu a ,⇑a College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou,Zhejiang 310058,China bFuli Institute of Food Science,Zhejiang University,Hangzhou,Zhejiang 310058,Chinaa r t i c l e i n f o Article history:Received 21November 2012Received in revised form 29January 2013Accepted 13March 2013Available online 21March 2013Keywords:Mulberry Anthocyanin High-fat dietBody weight gain Obesitya b s t r a c tWe present our experiment about adding anthocyanins to the daily food of mice.Three kinds of antho-cyanins (cyanidin-3-glucoside,cyanidin-3-rutinoside and pelargonidin-3-glucoside)purified from Chi-nese mulberry (Morus australis Poir)were evaluated for suppressing body weight gain of the male C57BL/6mice fed with high-fat diet (HFD).The results from a 12-week experiment show that consump-tion of purified mulberry anthocyanins (MACN)of 40or 200mg/kg can significantly inhibit body weight gain,reduce the resistance to insulin,lower the size of adipocytes,attenuate lipid accumulation and decrease the leptin secretion.Thus,dietary supplementation with MACN can protect against body weight gain of the diet-induced obese mice.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionObesity has become one of the main public health problems in recent decades because it could cause an increased risk of chronic diseases,such as type II diabetes and coronary heart disease (Ha-slam &James,2005;Weyer,Foley,Bogardus,Tataranni,&Pratley,2000).It is a complex metabolic disorder induced by an imbalance between calorie intake and metabolic expenditure,which is ex-pressed as an increase in adipocyte number (hyperplasia)and size (hypertrophy)(Arner &Spalding,2010;Hirsch &Batchelor,1976;Suzuki et al.,2011).Currently available therapeutic approaches for treating obesity have a number of limitations,such as adverse effects and high rates of secondary failure (Derosa &Maffioli,2012;Mayer,Hocht,Puyo,&Taira,2009),so investigating potential natural products to prevent obesity has increasingly attracted the interest of the academic community (Yun,2010).In recent years,many natural pigments from vegetables and fruits have attracted scientist’s interest because of their non-toxic-ity and beneficial health effects (Hsu &Yen,2007;Kim &Park,2011;Pandey &Rizvi,2009).As a natural pigment,anthocyanin is one of the major sub-group of flavonoids responsible for the red,blue and purple colour of many plant tissues (Castaeda-Ovando et al.,2009;He &Giusti,2010).It also possesses antineoplastic,anti-inflammatory and lipoxygenase inhibition effects (Kim &Park,2011).What is more,some anthocyanins have been documented in the prevention of diet-induced obesity (Prior,2010;Zafra et al.,2007),such as anthocyanidins from purple corn (Zea mays L.)(Tsu-da,Horio,Uchida,Aoki,&Osawa,2003),black soybean (Glycine max L.Merr.)(Kwon et al.,2007),blood orange (Citrussinensis L.Osbeck)(Titta et al.,2010),cornelian cherries (Cornus mas )(Jayaprakasam,Olson,Schutzki,Tai,&Nair,2006),blueberries (Vaccinium angustifo-lium )(Prior et al.,2009,2010),strawberries (Fragaria ananassa )(Prior et al.,2008,2009)and blackberries (Rubus sp.)(Kaume,Gil-bert,Brownmiller,Howard,&Devareddy,2012).These have been shown to be useful to ameliorate obesity in vivo .However,all these studies,with the exception of blueberries and strawberries,have used anthocyanins rich extracts.So increasing knowledge about how purified anthocyanins exert influence on the preventing obes-ity is necessary and worthwhile.Chinese mulberry (Morus australis Poir)is traditionally used in medicines because of its pharmacological effects,for example,anti-fever diuretics,liver protection,blood pressure reduction and cardiovascular diseases amelioration (Huang,Chang,Wu,Huang,&Wang,2011;Priya,2012).Recent research has suggested that mulberry extracts exhibit an anti-obesity effect (Peng,Liu,Chuang,Chyau,&Huang,2011).Although mulberry is rich in anthocyanins,it is still unknown whether its specific anthocyanins0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.03.046⇑Corresponding authors.Address:College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou,Zhejiang 310058,China.Tel.:+8657186098861;fax:+8657186971139(X.Zheng),tel.:+8657188982398(T.Yu).E-mail addresses:xdzheng@ (X.Zheng),tingyu@ (T.Yu).possess pharmacological effects on anti-obesity.In this study,we purified anthocyanins from mulberry and investigated their effect on the development of obesity.2.Materials and methods2.1.MaterialsCyanidin-3-glucoside,cyanidin-3-rutinoside and pelargonidin-3-glucoside were obtained from Polyphenols Laboratories(Sand-nes,Norway).All the other chemicals were reagent grade.2.2.Methods of purifying anthocyanins from mulberry(MACN)Fresh mulberry was purchased from a local fruit market in Hangzhou.4kg of mulberry were weighed out and extracted three times with methanol/formic acid(9:1,v/v).The combined extract was subjected to vacuum evaporation to remove the solvent and subsequently loaded onto an equilibrated Amberlite XAD-7col-umn.The column was saturated with1%formic acid,and subse-quently the binding anthocyanins were eluted with1%formic acid in methanol.The methanol eluent was collected and subjected to vacuum evaporation again.Once evaporated,the concentrate was extracted with ethyl acetate until there was no colour change in the organic layer.The aqueous layer was lyophilized and stored atÀ80°C until using.The purified anthocyanins were identified by using the Chromatograph HPLC/ESI/MS/MS(from Agilent1290 Infinity LC)coupled to a6400series triple quadrupole mass spec-trometer.The concentrations of each anthocyanin were deter-mined by using UltiMate3000series HPLC.2.3.Animals and dietsThe male C57BL/6mice with4weeks of age were purchased from the Branch of National Breeder Center of Rodents(Shanghai, China).After purchase the mice were housed at23±3°C,and sub-jected to a12h light/dark cycle.The mice had free access to both food and water,and were handled according to the guidelines of the Instituted Animal Care and Use Committee of Zhejiang Univer-sity for the care and use of laboratory animal(Zju2012-2-01-011Y).After7-day adaptation,all the experimental mice(n=48)were randomly divided into four groups according to the daily food in-take of:(1)the control group fed with low-fat diet(LFD,10%calo-ries from fat);(2)the control group fed with high-fat diet(HFD, 45%calories from fat);(3)the test group fed with HFD added with MACN of40mg every kilogramme food;(4)the test group fed with HFD added with MACN of200mg of every kilogramme food.The low-fat diet(LFD)was to contain3.85kcal in each gramme of food,of which there were20%kcal from protein,70%kcal from carbohydrates and10%kcal from fat.The high-fat diet(HFD)was to contain4.73kcal in each gramme of food,of which there were 20%kcal from protein(from Mediscience Ltd.,Yangzhou,Chi-na).The diets were replaced once every2days to prevent oxidisa-tion of the fats in diets.Body weight and the food intake quantity were measured once weekly.2.4.Biochemical analyses of serumSerum ALT,AST,glucose,triglycerides and total cholesterol lev-els were analysed by enzymatic methods using commercially available kits(Elabscience).Serum insulin and leptin levels were measured by immunoassay using a corresponding rat/mouse ELISA kit according to the manufacturer’s protocols.The homeostatic model assessment for insulin resistance(HOMA-IR)was calculated from insulin and glucose values by using Matthew’s formula(Mat-thews et al.,1985).2.5.Histology analysis of liver and epididymal white adiposesThe liver and epididymal white adipose werefixed in a buffer solution of10%formalin.Liver samples were stained with Oil Red O and hematoxylin to examine their cellular architecture and lipid accumulation.Thefixed epididymal white adipose tissues were stained with haematoxylin and eosin(H&E)to analyse its adi-pocyte’s size.2.6.Hepatic lipid analysisThe samples of mice liver were homogenised in phosphate-buf-fered saline and the total lipids were estimated by Folch’s method (Folch,Lees,&Sloane Stanley,1957).The liver triglycerides and to-tal cholesterol were quantified with the same enzymatic method as used for serum analysis.2.7.Statistical analysisStatistical analyses for all sample groups were performed by the SPSS statistical software.A mean and its standard error for each group were calculated.Significant differences between these means were determined by post hoc Duncan’s multiple range tests. Probability values lower than0.05were accepted as significant.3.Results3.1.Characterization of purified mulberry anthocyanin(MACN)The Chromatograph HPLC/ESI/MS/MS was used to determine anthocyanin compositions of MACN.Table1suggests there are three kinds of anthocyanins in MACN,which are cyanidin-3-gluco-side,cyanidin-3-rutinoside and pelargonidin-3-glucoside.Then the identified anthocyanins were characterised by HPLC.As shown in Fig.1,the retention times of cyanidin-3-glucoside,cyanidin-3-ruti-noside and pelargonidin-3-glucoside are15.9,17.5and20.6min, respectively.In addition,the concentration of each anthocyanin was measured(see Table1).The results indicate that the main anthocyanins of mulberry are cyanidin-3-glucoside and cyanidin-3-rutinoside,which is in agreement with the previous studies (Dugo,Mondello,Errante,Zappia,&Dugo,2001;Huang et al., 2011;Liu,Lee,Shih,Chyau,&Wang,2008;Liu et al.,2009;Peng et al.,2011).3.2.Influence of diet MACN on food intake and body weightIn order to determine whether MACN affects body weight of the C57BL/6mice,MACN(at40or200mg/kg)was administered daily for12weeks.There were no abnormal clinical signs throughout the12-week experiment.After12weeks,the mice fed with HFD induced greater weight gain in comparison with the control LFD mice.Weight gain was44.7%higher for the HFD group as com-pared to the LFD group(see Fig.2A).Furthermore,administration of MACN at40mg/kg suppressed the body weight gain for theTable1Mass spectrometry data and the content of purified mulberry anthocyanins(MACN).Peak Anthocyanins composition[M+]m/z MS2m/z Concentration(l g/mg)1Cyanidin-3-glucoside449287548.75±8.76 2Cyanidin-3-rutinoside595448/287371.69±3.35 3Pelargonidin-3-glucoside43327125.04±3.22Values are expressed as mean±SEM.T.Wu et al./Food Chemistry141(2013)482–487483HFD-fed mice by11.8%,but the higher level of MACN produced a 21.4%decrease instead(see Fig.2A).Food intake for the LFD group was3.4g/day throughout the experiment,which was higher than three HFD group,whereas the caloric intake(13.1kcal/day)was lower(see Fig.2B).We no-ticed that there were no significant differences about daily food in-take and caloric intake between two HFD groups with MACN and the group without MACN.These data shows MACN does not affect the mouse’s food intake and caloric intake(see Fig.3),but it can significantly decrease a HFD-induced body weight gain(see Fig.2B).There are no significant differences in the weights of heart,liver and kidney of the mice in these four groups(not shown here). However,as expressed in a percentage of body weight,heart,liver and kidney are smaller for the HFD-fed mice than the LFD-fed mice (see Table2).The weight of epididymal fat is much higher for the HFD-fed mice compared to the control LFD-fed mice,but in the administration of MACN its weight will be not increased so much.3.3.Influence of diet MACN on serum parametersSerum glucose,triglyceride and total cholesterol levels were significantly increased for the HFD-fed mice relative to the control LFD-fed mice(see Table2).Although MACN decreased HFD-in-duced serum cholesterol level,it was still higher than the control LFD-fed pared to the effect on serum cholesterol,MACN affected serum glucose significantly.Furthermore,we found an interesting phenomenon that MACN increased serum triglyceride, which is contrary to the previous experiment by Prior et al.(2010).The HFD-fed mice showed dramatic elevation of serum param-eters of liver injury(ALT and AST)compared to the LFD-fed mice (see Table2).Intake of MACN did not alter AST level,but effectively attenuated ALT level for the HFD-fedmice.B CC57BL/J6mice.LFD with MACNÇ)or control, high-fat intake for C57BL/6(),(),()().(), (),()().Values expressed as mean means marked superscript others.141(2013)482–4873.4.Influence of diet MACN on adipose and liver morphology The effects of MACN on lipid accumulation in liver were deter-mined by Oil Red O staining.Mice fed with HFD stained for intense lipid accumulation in the liver (see Fig.3A).In contrast,MACN alle-viated the lipid accumulation,even higher level of MACN stained similar to the control LFD mice.Furthermore,morphological anal-ysis indicated that the lipid droplet size was the largest in the HFD-fed mice,followed successively by the HFD with MACN at 40mg/kg,the HFD with MACN at 200mg/kg and the LFD-fed mice (see Fig.3A).These suggested that MACN were responsible for suppres-sion of liver lipid accumulation.Fig.3B exhibits the histology of epididymal white adipose tissue of mice by HE staining.The mice fed with HFD show hypertrophy of the adipocytes in the adipose tissue.MACN whether adminis-trated at low or high levels,lowered the size of adipocytes in the adipose tissue.3.5.Influence of diet MACN on hepatic lipidsFig.4A shows that hepatic content of total lipid,triacylglycerol and cholesterol are elevated for all mice fed with HFD relative to the control LFD,but it is obvious that MACN can reduce lipid,triac-ylglycerol and cholesterol of liver.3.6.Influence of diet MACN on insulin and leptinThe insulin and leptin levels in mice serum were examined (Fig.4B).The results revealed that insulin and leptin secretions into serum increased by comparison with the LFD-fed mice.Although MACN administrated at low and high levels were unable to alter the insulin secretion,the HOMA-IR values (Fig.4B)and leptin secretion were significantly reduced.ABLFD HFD+MACN 40 mg/kgLFD HFD HFD+MACN 40 mg/kg HFD+MACN 200 mg/kgHFD5 µm 5 µm 5 µm5 µm100 µm100 µm100 µm100 µmHFD+MACN 200 mg/kgTable 2Tissue weight and serum parameters for the male C57BL/6mice fed with LFD,HFD and HFD plus MACN (40or 200mg/kg).ItemLFDHFDHFD +MACN 40mg/kg HFD +MACN 200mg/kg Tissue weight Heart weight (%)0.52±0.03c 0.37±0.02a 0.45±0.04b 0.43±0.05b Liver weight (%) 4.00±0.07b 2.85±0.04a 2.85±0.12a 4.03±0.14b Kidney weight (%)1.30±0.05c 0.95±0.12a 1.12±0.09b 1.22±0.06c Epididymal weight (%) 1.99±0.01d 5.97±0.16a 4.71±0.35b 3.77±0.31c Serum ALT (U/L)29.75±1.43b 37.25±1.03a 25.55±2.42c 25.75±1.17c AST (U/L)92.55±3.32b 130.75±4.32a125.55±5.24a129.55±3.78aGlucose (mmol/L)5.84±0.21a 7.29±0.33a 3.75±0.12b 3.70±0.11b Triglyceride (mmol/ml)0.78±0.03c 1.57±0.07a 1.90±0.0.07b 1.84±0.02b Cholesterol (mmol/ml)2.38±0.09d4.52±0.13a4.00±0.14b3.56±0.13cValues are expressed as mean ±SEM.ALT,aspartate aminotransferase;AST,alanine aminotransferase.The means marked with superscript letters are significantly different relative to others.T.Wu et al./Food Chemistry 141(2013)482–4874854.DiscussionAnthocyanins are important plant pigments,which have been considered to be associated with a reduced risk of developing chronic diseases(He&Giusti,2010;Kim&Park,2011;Prior et al.,2010;Zafra et al.,2007).In this study we focused on the ef-fects of purified mulberry anthocyanins on the development of obesity.The purified mulberry anthocyanins(MACN)containing cyanidin-3-glucoside,cyanidin-3-rutinoside and pelargonidin-3-glucoside have been used in this experiment.The predominant compositions of MACN are cyanidin-3-glucoside and cyanidin-3-rutinoside(see Table1),which is consistent with previous studies (Dugo et al.,2001;Huang et al.,2011;Liu et al.,2008,2009;Peng et al.,2011).As expected,the present investigation has confirmed that HFD can induce a great gain of body weight,an increase of serum and liver lipids,as well as an elevation of insulin and leptin levels.If purified,MACN can be administrated into HFD,body weight gain could be attenuated,serum cholesterol and leptin levels brought down,and hepatic lipids(total lipid,triacylglycerol and choles-terol)decreased.Noticeably,all of the effects exerted by MACN did not cause abnormal clinical signs throughout our12-week experiment.Some researchers have shown consumption of iso-lated strawberry anthocyanins(primarily elargonidin-3-glucoside) and purified blueberry anthocyanins(mainly composed of malvi-din-3-glucoside and delphinidin-3-glucoside)had lowered body weight gain and body fat.Furthermore,blueberry anthocyanins, composed of two major anthocyanins(malvidin-3-glucoside and malvidin-3-galactoside)decreased serum leptin levels and im-proved b cell function(Prior et al.,2009,2010).In the present study,the predominant anthocyanins of MACN are cyanidin-3-glu-coside and cyanidin-3-rutinoside,and both of these also exhibit the similar functions.Based upon the data available,cyanidin-3-glucoside and cyanidin-3-rutinoside may contribute to suppress body weight gain,but the specific impact of anthocyanin on the development of obesity is still unknown and further investigation is needed.The control mice fed with LFD consumed more food than the mice fed with HFD.However,intake of MACN did not affect the food intake(see Fig.2B).There were no significant differences in daily food intake among three HFD-fed groups.Daily calorie intake was not different between the HFD-fed mice and the HFD with MACN(at40or200mg/kg,respectively)fed mice(see Fig.2B). These data suggest that a decrease in weight gain induced by MACN,is irrelevant with decreasing food or calorie intake,and decreasing food efficiency may account for this(Jayaprakasam et al.,2006;Min et al.,2012).The C57BL/6mice are susceptible to diet induced obesity,which displayed as massive liver lipid accumulation(see Fig.3A).Feeding mice HFD increases liver lipids,triglycerides and cholesterol signif-icantly(see Fig.4A),but MACN dramatically countered this.The ac-tion of MACN to suppress hepatic cholesterol and triglycerides in HFD mice,should result in a reduction of cholesterol and triglycer-ides in body tissues,but its effect on serum triglyceride contrasts to this.It that may be the consumption of MACN might regulate lipid metabolism by affecting hepatic lipid oxidation lipogenesis(Jayap-rakasam et al.,2006).The increase of serum insulin level and HOMA-IR values indi-cate that insulin resistance appeared in the HFD fed mice(Prior et al.,2010;Weyer et al.,2000).In the present study,insulin resis-tance(assessed by HOMA-IR)slightly appeared in the HFD-fed mice,and the intake of MACN reduced resistance to insulin (Fig.4B).Leptin is a product of the obese gene,secreted in white adipose tissue,which plays an important role in regulating energy homeo-stasis and lipid metabolism.Most obese animals exhibit high ser-Fig.Hepatic contents of total lipid,triacylglycerol and cholesterol.(B)Serum insulin levels,leptin levels and HOMA-IR in mice.LFD(),HFD(), ()().The means marked with superscript letters are significantly different relative to others.um leptin concentrations(Haslam&James,2005).In agreement with previous studies(Prior et al.,2009,2010),the leptin level of HFD-fed mice has been elevated(Fig.4B).In addition,MACN treat-ments have slowed down the development of obesity,and a similar trend present in the leptin levels.In the current investigation,feed-ing mice with HFD could increase weight of their epididymal fat and induced hypertrophy adipocytes(Fig.3B).The intake of MACN could reduce weight of their epididymal fat and lower the size of adipocytes.This phenomenon may suggest that MACN directly af-fect both the number and the size of adipocytes in adipose tissue, which is likely associated with production of leptin(Prior,2010).In summary,consumption of MACN suppresses body weight gain of the high-fat fed C57BL/6mice.Intake of MACN at40mg/ kg inhibits body weight gain by11.8%,and the high level of MACN produces a21.4%decrease.Further,supplementation of MACN can improve impaired hepatic function,reduce the resistance to insu-lin,lower the size of adipocytes in the adipose tissue and signifi-cantly decrease the leptin secretion.AcknowledgementsThis work was supported by National Key Technology R&D Pro-gram of China(2012BAD33B08),National Natural Science Founda-tion of China(No.31000775),Zhejiang Innovation Program for Graduates(YK2011008)and Special Fund for Agro-scientific Re-search in the Public Interest(200903044).ReferencesArner,P.,&Spalding,K.L.(2010).Fat cell turnover in humans.Biochemical and Biophysical Research Communications,396,101–104.Castaeda-Ovando,A.,Pacheco-Hernández,M.L.,Elena,Ma,Páez-Hernández,M.E., RodráguezGalán-Vidal,J.A.,&Galán-Vidal,C.A.(2009).Chemical studies of anthocyanins:A review.Food Chemistry,113,859–871.Dugo,P.,Mondello,L.,Errante,G.,Zappia,G.,&Dugo,G.L.(2001).Identification of anthocyanins in berries by narrow-bore high performance liquid chromatography with electrospray ionization detection.Journal of Agricultural and Food Chemistry,49(8),3987–3992.Derosa,G.,&Maffioli,P.(2012).Anti-obesity drugs:A review about their effects and their safety.Expert Opinion on Drug Safety,11(3),459–471.Folch,L.,Lees,M.,&Sloane Stanley,G.H.(1957).A simple method for the isolation and purification of total lipids from animal tissues.The Journal of Biological Chemistry,226,497–509.Haslam,D.W.,&James,W.P.S.(2005)ncet,366,1197–1209.He,J.,&Giusti,M.M.(2010).Anthocyanins:Natural colorants with health-promoting properties.Annual Review of Food Science and Technology,1,163–187. 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