International Journal of Biological Macromolecules 62 (2013) 254–256
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International Journal of Biological
Macromolecules
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /i j b i o m a
c
Hypolipidemic activity of the polysaccharides from Enteromorpha prolifera
Zhaolin Teng a ,Li Qian b ,∗,Yan Zhou b
a Department of Neurosurgery,The Second People’s Hospital of Lianyungang,41East Hailian Road,Xinpu 222006,China
b
Laboratory Microbiology Laboratory,The Second People’s Hospital of Lianyungang,41East Hailian Road,Xinpu 222006,China
a r t i c l e
i n f o
Article history:
Received 18August 2013Accepted 15September 2013
Available online 20 September 2013
Keywords:
Enteromorpha prolifera Hypolipidaemic activity Polysaccharides
a b s t r a c t
Enteromorpha prolifera is an edible alga that is used as a traditional herb to treat many diseases.This study prepared E.prolifera polysaccharides (EPPs)and determined their hypolipidaemic activity in rats.
Forty-eight male Sprague-Dawley rats were divided into three groups and fed with diets containing either high fat (HF),EPPs or normal fat (NF)for 6weeks.Compared with the HF group,the EPP group showed decreased body weight gain,plasma triacylglycerol (TG),total cholesterol (TC),plasma low-density lipoprotein cholesterol level,liver TG,liver TC and liver weight.The faecal fat,cholesterol and plasma high-density lipoprotein cholesterol of the EPP group were higher than those of the NF and HF groups.Results suggested that EPPs had a high hypolipidaemic activity and could be a suitable alternative hypolipidaemic source for humans.
© 2013 Elsevier B.V. All rights reserved.
1.Introduction
Enteromorpha prolifera is a common green alga that is widely distributed in seashore.Since ancient times,E.prolifera has been used not only as a food source but also as a traditional herb to treat many diseases,such as heat dissipation and hydropic diseases,as documented in Chinese materia medica [1].
E.prolifera polysaccharides (EPPs)are the main active ingre-dients present in algal cells that can invigorate immunity as well as anti-bacterial,anti-viral,antioxidant,anti-tumour,anti-inflammatory and mo
isturising activities [1–9].Tang et al.[1]reported that EPPs effectively decrease plasma triacylglycerol (TG),total plasma cholesterol (TC),plasma low-density lipopro-tein cholesterol (LDL-C)and plasma high-density lipoprotein cholesterol (HDL-C).However,the effects of EPPs on other lipid parameters,including body weight gain (WG),liver TC and liver TG,remain unclear.
This study aims to extract EPPs and characterise their hypolipi-daemic activity,including body WG,food intake (FI),food efficiency ratio (FER),faecal cholesterol,faecal TC,plasma TC,plasma TG,plasma LDL-C,plasma HDL-C,liver weight,liver TC and liver TG.Results of this study enhance the current understanding on the hypolipidaemic activity of EPPs.
∗Corresponding author.Tel.:+8651885776016;fax:+8651885775003.E-mail address:qianli008@yahoo (L.Qian).
2.Materials and methods
2.1.Materials
E.prolifera was collected in the coast of Lianyungang in China.Male Sprague-Dawley rats were purchased from the Experimen-tal Animal Center of Nanjing University.TG,TC,HDL-C,hepatic lipase an
d lipoprotein lipase kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu,China).All chemicals were reagent grade.
2.2.Preparation of EPPs
Dried samples of E.prolifera were powdered and extracted with organic solvents in a Soxhlet apparatus (light petroleum,acetone and methanol).The materials were soaked in distilled water to yield a suspension at a concentration of 1%(v/w).The reactor was maintained in a thermostatic water bath at 90◦C for 4h.The sus-pensions were filtered through a Whatman GF/A filter paper and concentrated to ∼15%(w/v).Proteins were removed by the Sevag method,precipitated with 3volumes of absolute ethanol,filtered again through a Whatman GF/A filter paper and then freeze dried.
2.3.Characterisation of EPPs
The phenol–sulphuric acid colorimetric method was carried out to determine the total sugar content using glucose as the standard [10].The protein content was measured according to Bradford’s method using bovine serum albumin as the standard
0141-8130/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.ijbiomac.2013.09.010
Z.Teng et al./International Journal of Biological Macromolecules62 (2013) 254–256255
[11].The sulphate group was determined according to the methods described by Dodgson and Price[12].The uronic acid content was examined by3-hydroxydiphenyl assay using glucuronic acid as the standard[13].
HPGFC determined the molecular weight(Mw)of EPP on an Ultrahydrogel Size Exclusion Column,which can detect Mw in the range of103–106.NaNO3(0.1N)was used as an eluent at a flow rate of0.9mL/min in the size exclusion chromatography stud-ies.A high-sensitive refractive index detector(Model ERC-7515A; ERC Inc.,Japan)was used.Calibration of the detector was carried out with known concentrations of Dextran standards(Sigma).A 20␮L aliquot was injected to the column afterfiltration through a 0.45␮m milliporefilter at ambient temperature;the procedure was repeated thrice.The Multi-channel Chromatography Data Station (Version144A,1993–1997Ampersand Ltd.)software was used.
2.4.Test animals
Male Sprague-Dawley rats weighing107±8g were used in the experiment.The rats were individually housed in metabolic cages fitted with glass separators for urine and faeces collection and kept in an ai
r-conditioned room at22±1◦C on a12h light–dark cycle. All animal protocols were approved by the institutional animal care and use committee of The Second People’s Hospital of Liyungang (Jiangsu,China).Rats were allowed free access to food and water.
Forty-eight rats were fed ad libitum with a commercial diet and water for5d.The rats were randomly divided into three groups (16rats per group):normal-fat group(NF),received basic diet in which the composition conformed to AIN76(provided by Shanghai SLAC Laboratory Animal Co.Ltd.,Shanghai,China);high-fat(HF) control group(HF),received HF diet containing10%(w/w)lard, 15%(w/w)egg yolk powder,1%(w/w)cholesterol and commercial diet to100%(w/w);and EPP group,received95%(w/w)HF diet plus 5%(w/w)EPPs.Powdered commercial basal diet was blended with other ingredients and test materials,and then pelleted.
2.5.Experimental design
The rats were deprived of food overnight,and blood sample was withdrawn to measure the initial plasma lipid levels prior to the experiments.FI was monitored daily.Body masses were initially determined and thereafter on alternate days,and the WG of the rats during the experimental period was recorded.At the sixth week, the faeces were collected in three continuous days and then dried at9
0◦C for2h to determine the fat and cholesterol contents.All rats were given diethyl ether after fasting for18h.Blood was collected, and plasma was obtained by centrifugation at3000×g for10min. Plasma samples were stored in a−20◦C freezer until use for further analysis[14].
2.6.Serum,liver lipids and lipase activities of rats
Serum TG,TC and HDL-C levels were measured with commer-cial assay kits.The LDL content in serum was calculated using the Friedewald equation:
LDL-C(mmol/L)=TC-HDL-C-TG/2.2[15].Liver lipid was extracted following the method of Folch et al.[16],and liver TG and TC levels were measured with commercial assay kits.
2.7.Faecal fat and cholesterol
The faeces were dried,pulverized and then weighed.Fae-cal fat content was determined gravimetrically using the Soxhlet method,with diethyl ester as the extraction solvent[17].Faecal lipid wasfirst extracted from100mg of dried faeces with2mL of chloroform–methanol(2:1,v/v)according to the method described Table1
Body weight gain of rats(WG),food intake(FI),food efficiency ratio(FER)of rats fed on high fat(HF),Enter
omorpha prolifera polysaccharides(EPP),and normal fat(NF) diets.
Parameters Dietary group
HF EPP NF
WG(g)43.4±1.71a35.2±1.65c39.0±1.76b FI146.5±6.2a133.5±5.8b142.7±6.2a FER25.64±1.17a26.36±1.12b27.32±1.21b
Values are expressed as mean±SD(n=16).Means with different superscripts within a row indicate significant differences(p<0.05).
by Folch et al.[16]to determine the level of faecal cholesterol. The lower phase was dried under nitrogen gas.Faecal cholesterol was extracted as previously described[18].The cholesterol con-tent was enzymatically determined with a commercial assay kit. The measurements were conducted in triplicate.
2.8.Statistical analysis
All data are presented as mean±S.D.ANOVA was used to com-pare the means of two rat groups.Statis
tical significance at the 95%and99%probability levels were set at p<0.05and p<0.01, respectively.
3.Results and discussion
3.1.Characterisation of EPPs
EPPs were water soluble gray powder.The total sugar,protein, uronic acid and sulphate contents of EPPs were54.6%,10.1%,12.4% and17.9%,respectively.The Mw of EPPs was134.07kDa,indicat-ing that the average degree of EPP polymerisation was∼800–900. This average value is attributed to the composition of the EPPs, principally consisting of rhamnose,xylose,mannose,galactose and glucose[1].
3.2.Body WG,FI and FER
Table1shows the WG and FI of the rats.The WG of the HF group was sharply increased compared with that of the NF control group,indicating that the HF diet used in this study significantly increased the body weight of the rats.Although no significant dif-ferences in FI were observed among the three groups,the EPP group showed lower FER(Table1).This result suggests that the difference in rat growth among these groups was not due to FI but due to the different nutrient efficiencies caused by EPP addi
tion.Therefore, the results indicate that the EPPs were effective in inhibiting the increase in body weights of the rats
3.3.Faecal fat and cholesterol excretion of rats
Table2shows the measured lipid contents of the dried faeces collected in the last3d.The faecal fat and cholesterol contents in Table2
Fecal fat and cholesterol excretion of rats fed on high fat(HF),Enteromorpha prolifera polysaccharides(EPP),and normal fat(NF)diets.
Parameters Dietary group
HF EPP NF
Fecal fat10.7±0.51b13.1±0.62a7.9±0.33c Fecal cholesterol18.6±0.72b21.8±0.91a11.3±0.50c
Values are expressed as mean±SD(n=16).Means with different superscripts within a row indicate significant differences(p<0.05).
256Z.Teng et al./International Journal of Biological Macromolecules62 (2013) 254–256
Table3
Plasma lipid concentrations of rats fed on high fat(HF),Enteromorpha prolifera polysaccharides(EPP),and normal fat(NF)diets.
Parameters Dietary group
HF EPP NF
TC(mmol/L)  3.08±0.12a  1.14±0.05c  1.52±0.06b TG(mmol/L)  3.09±0.14a  1.28±0.05c  1.89±0.08b HDL-C(mmol/L)0.59±0.14c0.77±0.11a0.66±0.12b LDL-C(mmol/L)0.69±0.03a0.48±0.02c0.59±0.02b
Values are expressed as mean±SD(n=16).Means with different superscripts within a row indicate significant differences(p<0.05).
Table4
The liver weight,liver TC and TG of rats fed on high fat(HF),Enteromorpha prolifera polysaccharides(EPP),and normal fat(NF)diets after six weeks of diets. Parameters Dietary group
HF EPP NF
Liver weight(g)  2.34±0.08a  1.79±0.06c  1.07±0.04b TC(mmol/L)13.49±0.57a10.37±0.43b8.21±0.37c TG(mmol/L)32.05±1.05a22.12±0.96b12.07±0.58c
Values are expressed as mean±SD(n=16).Means with different superscripts within a row indicate significant differences(p<0.05).
the EPP group were higher than those in the other groups(p<0.05), indicating that the EPPs had considerable fat-and cholesterol-binding capacities.Similarly,Zhang et al.[19]reported that the faecal lipids of the high-Mw chitosan group were higher than those of the HF and NF groups.Whether the EPPs have a mechanism of decreasing faecal fat and cholesterol contents similar to that of chitosan remains unknown.characterise
3.4.Plasma lipid concentrations of rats
Table3lists the plasma lipid values in the three rat groups.After 6weeks of feeding HF diets,the plasma TC,TG and LDL-C levels of the HF group decreased but the HDL-C increased compared with those of the NF control(p<0.05).EPP addition decreased the plasma TC,TG and LDL-C levels and increased the HDL-C level.These results are consistent with those of a previous report[1].
3.5.Liver lipids of rats
Table4lists the values of liver weight and liver lipid of rats.The liver weight,liver TG and liver TC levels of the HF group were higher than those of the NF control.The liver weight,liver TC and liver TG levels were decreased in the EPP group compared with those in the HF group.This result indicates that the EPPs were effective in decreasing the accumulation of lipids in the liver.Thesefindings are consistent with the results of WG and blood lipids,all proving the good hypolipidaemic activities of the EPPs.
4.Conclusions
The present study demonstrated the hypolipidaemic activity of EPPs in rats.The results indicate that EPPs can inhibit the increase in body weight,decrease the plasma LDL-C,TG and TC levels and liver weight and increase the HDL-C level of rats fed with HF diets.Given its better and safer hypolipidaemic profile,EPPs deserve further studies to identify their possible mechanism of action.
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