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Water-soluble-polysaccharides-extracted-from-Pterocladia-capillacea-and-Dictyopteris-membranacea-and

Carbohydrate Polymers 113(2014)62–66

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Carbohydrate

Polymers

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 /c a r b p o

Water soluble polysaccharides extracted from Pterocladia capillacea and Dictyopteris membranacea and their biological activities

A.H.Abou Zeid a ,?,E.A.Aboutabl b ,A.A.Sleem c ,H.M.El-Ra?e a ,?

Pharmacognosy Department,National Research Centre,Dokki,Cairo 12311,Egypt

Pharmacognosy Department,Faculty of Pharmacy,Kasr Al-Aini,Cairo University,11562,Egypt c

Pharmacology Department,National Research Centre,Dokki,Cairo 12311,Egypt

a r t i c l e

i n f o

Article history:

Received 6February 2014

Received in revised form 2May 2014Accepted 2June 2014

Available online 7July 2014

Keywords:

Pterocladia capillacea

Dictyopteris membranacea Polysaccharides Antimicrobial Antitumor

Anticoagulant activities

a b s t r a c t

Cold and hot water extracts (CWE,HWE)of both the red alga Pterocladia capillacea (P.capillacea )and the brown alga Dictyopteris membranacea (D.membranacea )were studied for their polysaccharide contents.In both (CWE)and (HWE)extracts.Relatively higher yields were obtained in case of P.capillacea pillacea (2.87and 6.46%,respectively).The polysaccharide contents of the CWE hydrolyzate of both studied algae analyzed by HPLC were found to be enriched with glucuronic acid,arabinose and glucose,whereas,HWE hydrolyzate were found to be rich in glucuronic acid and fructose.The polysaccharide contents of the CWE and HWE extracts of (D.membranacea )showed appreciable antimicrobial activity in addition to a moderate antitumor activity against HELA (Cervix carcinoma cell line)at IC50=9.83?g/dl,respectively.Whereas the polysaccharide contents of the CWE and HWE extracts of (P.capillacea )exhibited a promising anticoagulant activity.

1.Introduction

Recently,macroalgae have been used as a novel food with potential nutritional bene?ts in industry and medicine for various purposes (Agili &Mohamed,2012;Khair &El-Shafay,2013).Fur-thermore,macroalgae have shown to provide a rich source of nat-ural bioactive compounds with antiviral,antifungal,antibacterial,antioxidant,anti-in?ammatory,hypercholesterolemic,hypolipi-demic and antineoplasteic properties (El-Baroty et al.,2007).Thus,there is a growing interest in the area of research on the positive effect of macroalgae on human health and other bene?ts.

In particular,marine algae contain large amounts of polysac-charides,which are polymers of simple sugars (monosaccharides)linked together by glycosidic bonds,and they have numerous commercial applications in products such as stabilizers,thicken-ers,emulsi?ers,food,feed,beverages etc.(Bixler &Porse,2010;Chandini,Ganesan,&Bhaskar,2008;Zhou,Hu,Wu,Pan,&Sun,2008).Over the last decade,bioactive polysaccharides isolated from brown and red algae have attracted much attention in the ?elds of pharmacology and biochemistry (Agili &Mohamed,2012;Huang,Zhou,&Zhang,2006;Tiago et al.,2012).Functional polysaccha-rides such as fucans,alginic acid and carrageenans derivatives

?Corresponding authors.Tel.:+2/02/26328886.

E-mail address:abouzeida@https://www.wendangku.net/doc/3818197984.html, (A.H.Abou Zeid).

produced by brown and red algae are known to exhibit different biological properties including anticoagulant,anti-in?ammatory,antiviral,antitumoral activities and tissue engineering and drug delivery approaches (Sahera &Fatimah 2013;Silva et al.,2012).Algal polysaccharides have been described to possess anticoagu-lant activity similar to heparin.Various polysaccharides have been extracted from species of Dictyotales and Fucales (Phaeophyta)(Medeiros et al.,2008).The proposed mechanisms of action of these compounds are predominantly related to the “in vitro ”inhibition of factors Xa and IIa mediated by antithrombin and heparin cofactor II (Li,Lu,Wei,&Zhao,2008).Algal substances are also said to possess bacteriostatic and bactericidal activity and have been extensively studied by several researchers (Freile-Pelegrin &Morales,2004;Salvador,Gomez-Garreta,Lavelli,&Ribera,2007).

A role of polysaccharides from algae as antineoplastic agents has also been suggested.Several investigations have reported that polysaccharides have antiproliferative activity in cancer cell lines in vitro ,as well as inhibitive activity in tumors growing in mice (Queiroz et al.,2006).Moreover,these polymers have been reported to induce apoptosis in several cancer lines and stimulate immune system cells to induce tumor cell death (Maruyama,Tamauchi,Hashimoto,&Nakano,2003).In addition,they have antimetastatic activity blocking the interactions between cancer cells and the basement membrane (Rocha et al.,2005).On the basis of these considerations,the purpose of the present study to study polysac-charide contents of the two algal species and to evaluate their

A.H.Abou Zeid et al./Carbohydrate Polymers113(2014)62–6663

anticoagulant,antimicrobial and antitumor activity against cancer cell lines

2.Experimental

2.1.Materials

2.1.1.Algal material

The red alga P.capillacae and the brown alga Dictyopteris membranacea were collected from along the coast of Abo-Qire, Alexandria,Cairo,Egypt,in February,March and May,respectively in two successive years2003and2004.The algal samples were brought to the laboratory in an ice box,cleaned thoroughly with fresh water,the epiphytes were removed.The cleaned algae were dried in shade at room temperature and well grinned.

2.1.2.Material for antimicrobial study

2.1.2.1.Tested microorganisms.Four pure strains of bacteria were tested viz.,Gram-positive bacteria;Bacillus cereus and Staphy-lococcus aureus,Gram-negative bacteria;Escherechia coli and Pseudomonas?uoresens,yeast strain;Saccharomyces cervesia,fungal strains;Fusarium oxysporium and Aspergillus niger were obtained from the Microbial Chemistry Department,National Research Cen-tre,Egypt.

2.1.2.2.Nutrient media.Nutrient media used were Lauri-Bertani medium[tryptone10g,yeast extract5g,NaCl10g,15g agar and water to1l,the pH was adjusted to7.5with sodium hydrox-ide(Moniantis,Fritssh,&Sambrook,1980),Yeast extract peptone medium[dextrose20g,peptone20g,yeast extract10g,water to 1l](Dillon,Nasm,&Nestmann,1985)and potato dextrose agar medium[peeled and sliced potato200g,d-glucose20g,agar15g, water to1l].These media were used for bacteria,yeast and fungi, respectively.The media were sterilized by autoclaving for20min at121?C.

2.1.

3.Material for in vitro antitumor activity

The used cancer cell lines were U251(Brain tumor cell line), MCF7(Breast adino carcinoma cell line),HELA(Cervix carcinoma cell line),HEPG2(Liver carcinoma cell line),H460(Lung carcinoma cell line),HCT116(Colon carcinoma cell line).They were obtained from the American Type Culture Collection,University Boulevard, Manassas,USA.

2.1.4.Experimental animals for anticoagulant activity

Anticoagulant activity was conducted on male albino rats (120–160g).Animals were obtained from the animal house colony, N.R.C.,Cairo,Egypt.They were housed six animals/cage and fed on standard laboratory diet.Thromboplastine with calcium Kit (Biomerieux Company)was used.

2.2.Methods

2.2.1.Extraction of water soluble polysaccharides

Five grams of the dry algal powder were extracted in distilled water(D.W)at room temperature.(CWE)of each alga was?l-tered and concentrated under reduced pressure at temperature not exceeding40?C and precipitated with ethanol(4volume,v/v) the precipitated polysaccharides were?ltered,washed twice with absolute ethanol and dried at40?C to obtain the crude cold water-soluble polysaccharides.The algal residue after cold water was extracted with(D.W)at80?C for3h.From the?ltrate hot water sol-uble polysaccharides were obtained following the same procedure used for cold water soluble polysaccharides.2.2.2.Acid hydrolysis of both cold and hot water soluble polysaccharides

The precipitated polysaccharides(0.1g)were added to10ml 1N H2SO4and heated in a boiling water bath for5h to hydrolyze the polysaccharides.Barium carbonate was added then centrifuged and the precipitate was washed twice by water,then the solu-tion was evaporated under reduced pressure at temperature not exceed exceeding40?C.The residue,in each case was extracted with2ml of hot pyridine.The pyridine extract was evaporated to dryness under reduced pressure and kept for chromatographic analysis.

2.2.

3.Paper chromatographic investigation

Part of the CWE and HWE hydrolyzates as well as authentic sugars were dissolved in10%isopropanol/water then spot-ted on Whatman No.1paper sheets.The chromatograms were developed adopting the descending technique for18h using S1[(n-butanol–acetic acid–water(4:1:5,v/v/v)]and S2 [isopropanol–water(4:1,v/v)].The spots were visualized by spray-ing with aniline phthalate reagent and heating in an oven for5min at110?C.

2.2.4.HPLC chromatographic investigation

Ten mg of the CWE and HWE hydrolyzates as well as authentic reference were homogenized with acetonitrile/water (76/24,v/v).The extract was?ltered through a through a What-man No.1?lter paper and micro?lter(0.45?m),partitioned three times with ethyl acetate and injected in HPLC appara-tus model HP1050equipped with UV detector on APS column (4.6mm×200mm),the mobile phase was the same used in the extraction and the UV detector was adjusted at192nm,?ow rate 2ml/min.

2.2.5.Antimicrobial activity

The antimicrobial test was carried out according to the antibiotic assay disk method(Gnanamanickam&Mans?eld,1981).Nutrient agar media for the bacteria,fungi,and yeast were prepared and sterilized,then distributed in sterile Petri dishes of12cm diameter. Each suspension of the tested organism was,separately,inoculated onto the surface of a number of Petri dishes.Each antibiotic assay disk(6mm diameter)was loaded with100?g/disk)of the tested extracts.The air-dried discs were?rmly applied to the surface of the inoculated agar plates.Ampicillin as antibacterial(100?g/disk) and Clotrimazole(100?g/disk)as antifungal were used as refer-ence drugs.This assay was replicated three times.All these steps were carried out under aseptic conditions,bacterial plates incu-bated at37?C for24h,while those containing yeast and fungi were incubated at28?C for48–72h,diameter of the inhibition zones were recorded for each replicate and the average diameters were calculated.

2.2.6.Antitumor activity

Antitumor activity against the human tumor cell lines previ-ously mentioned was done Skehan et al.(1977).Cells were plated in96multi-well plate(104cells/well)for24h before treatment with the extracts to allow attachment of the cell to the wall of the plate.Different concentrations of the extracts(0,1,2.5,5 and10?g/dl)were prepared and added to the cell monolayer.Trip-licate wells were prepared for each individual dose.Monolayer cells were incubated with each extract for48h at37?C and in atmosphere of5%CO2.After48h,cells were?xed,washed and stained with sulphorhodamine B(SRB)stain.Excess stain was washed with acetic acid and attached stain was recovered with tri-EDTA buffer.Color intensity was measured in an ELIAS reader and the relation between surviving fraction and the algal extracts

64 A.H.Abou Zeid et al./Carbohydrate Polymers113(2014)62–66

Table1

Yields of the cold and hot water-soluble polysaccharides obtained from P.capillacea and D.membranacea.

Extract%(g/100g)

P.capillacea D.membranacea

Cold water extract 2.87 2.14

Hot water extract 6.46 3.89 concentration is plotted to get the survival curve of each tumor cell line.

2.2.7.Anticoagulant activity

The anticoagulant activities were evaluated using the whole blood clotting time and prothrombin time(6-h and24-h),blood samples were obtained from the retro-orbital venous plexus of each animal using0.11mol/litter trisodium citrate as anticoagu-lant for prothrombin time test.Thromboplastine with calcium kit (Biomerieux Company)was used.The treated groups of animals received the compounds orally(100mg/kg body weight),the blood samples were taken at six and twenty four hours after administra-tion and the clotting and prothrombine time were recorded(Young, Kisker,&Doty,1978).

3.Results and discussion

Several methods can be used to extract polysaccharides from marine algae.However,the yield can vary depending on the tech-nique and the algae used(Rodrigo,José,Márjory,Paulo,&Norma, 2011;Rodrigues et al.,2011).Our results revealed that the total yield of the water-soluble polysaccharide extracted from the red alga Pterocladia capillacea and the brown alga D.membranacea at room temperature(25?C)were2.87,2.14%and at80?C were6.46, 3.89,respectively(Table1).Paper chromatography and HPLC anal-ysis of the polysaccharide hydrolyzates of cold and hot water of P. capillacea illustrated in Tables2and3,showed mainly the pres-ence of six sugars of the cold hydrolyzate constituting80.87%and 59.10%,respectively.The major sugars were galactose(25.36%), glucose(20.59%),arabinose(12.61%)and fructose(9.17%),while galactos(17.81%),glucose(16.99%),fructose(9.38%)and mannose (7.37%)were the major sugars of the hot hydrolyzate.Mean-while the HPLC analysis of cold and hot water hydrolyzates of D. membranacea revealed the identi?cation of seven and nine sug-ars constituting87.29%and67.87%,respectively.The major sugars of the cold hydrolyzate were glacturonic acid(32.53%),glucuronic acid(30.54%)and fructose(7.56%),while glacturonic acid(27.32%), glucuronic acid(22.02%)and mannose(6.01%)were the major sugars of the hot hydrolyzate.The chemical and structural char-acteristics of the marine algal polysaccharides are considered a Table3

HPLC analysis of the soluble polysaccharide hydrolyzate of P.capillacea and D. membranacea.

Authentic sugars R t Relative%

P.capillacea D.membranacea

Cold Hot Cold Hot

Fucose1:08– 5.32 1.73 Rhamnose1:41–––Maltose1:76–––Lactose1:80–––Sorbose1:93–––Galactose2:2125.3617.81– 1.87 Galacturonic acid2:40––32.5327.32 Ribose2:62–––Xylose2:89––0.61 Sorbitol3:19 5.92 2.02––Arabinose3:3912.61 6.53 3.79 2.71 Fructose3:889.179.387.56 4.07 Mannose4:297.227.37 5.13 6.01 Glucose4:4620.5916.99 2.42 1.53 Glucuronic acid5:50––30.5422.02 Total identi?ed sugar–80.8759.1087.2967.87

prerequisite for understanding their biological activity(Errea& Matulewicz,1996;Rodrigo et al.,2011;Zhang et al.,2008).

Natural compounds produced by red and brown marine algae specially their polysaccharides,are able to prevent QS-regulated virulence factor production(quorum sensing systems which are involved in the adaptation of populations of bacteria to environ-mental cues.)in several human pathogens,including the bacteria associated with cholera and cystic?brosis-associated lung infec-tions(Aoun,Said,&Farhat,2010;Diane,Rice,&Kjelleberg,2006; Laio,Lin,Shich,Jeng,&Huang,2003).This re?ects the necessity of testing the antimicrobial activity of the algal extracts under investi-gation.Results depicted in Table4revealed that cold and hot water extracts of P.capillacea showed wide range of antimicrobial activity against(G?ve and G+ve)bacteria and fungi.Besides their higher antifungal activity(125%activity)than clotrimazol(100%activ-ity)against Fusarium oxysporium.For D.membranacea as shown in Table4,both cold and hot water extracts has no effect on G+ve bacteria and fungi but have a good effect on G?ve bacteria.Our results were in agreement with the results obtained by Salvador et al.(2007).

They reported that the extracts that were prepared from fresh and lyophilized samples of algae showed differences in their antimicrobial activities depending on the season that samples were collected and the type of samples which the extracts have been pre-pared.Also Taskin,Caki,Ozturk,and Taskin(2010)proved that the difference observed in the antimicrobial activities are contributed

Table2

Paper chromatographic investigation of polysaccharide hydrolyzates of P.capillacea and D.membranacea.

Spot no.R f value P.capillacea D.membranacea Color of spot

S1S2I II I II

10.590.69????Yellowish brown Rhamnose

20.540.63????Brown Ribose

30.380.56??++Brown Fucose

40.320.55???+Reddish brown Xylose

50.270.52++++Brown Arabinose

60.250.48++++Yellowish brown Fructose

70.180.46++++Brown Glucose

80.160.45++?+Brown Galactose

90.160.38??++Pale brown Glucuronic acid 100.200.30??++Pale brown Galacturonic acid 110.290.61++++Brown Mannose

S1,n-butanol–acetic acid–water(4:1:5,v/v/v);S2,isopropanol–water(4:1);I,Hydrolyzate of cold water extract;II,hydrolyzate of hot water extract.

A.H.Abou Zeid et al./Carbohydrate Polymers113(2014)62–6665 Table4

Anti-microbial activity of cold and hot water extracts of P.capillacea and D.membranacea.

Micro-organism Diameter of inhibition zone in mm(%activity)

I II III IV A B

Bacillus cereus(G+)6(33.3)6(33.3)––18(100)–Staphylococcus aureus(G+)12.5(56.8)9.5(43.2)––22(100)–Streptococcus pyogen(G+)15(39.5)14.5(38.1)––38(100)–Escherichia coli(G?)––2(20)11(110)20(100)–Pseudomonas?ouresens(G?)10(40)9.5(38)18(72)–25(100)–Candida albicans––7.2(25.7)7(25)–20(100) Saccharomyces cerevisea–––––28(100) Asperagillus niger–––––22(100) Macrophomina phaseli––8.2(41)8.4(42)–25(100) Fusarium oxysporium25(125)––––20(100) I,CWE of P.capillacea;II,HWE of P.capillacea;III,CWE of D.membranacea;IV,HWE of D.membranacea;A,Ampicillin;B,Clotrimazol.

Table5

The percentage of surviving fraction of different cell lines against different concentrations of the cold and hot water extracts of P.capillacea.

Conc.in(?g/dl)%of surviving fraction

Cold water extract Hot water extract

I II III IV V VI I II III IV V VI

0.099.5099.90100.2100.010010099.5099.90100.2100.0100100

1.0094.0088.5090.5099.40100.1098.0097.0086.90101.496.10104.1010

2.3

2.5089.2086.9085.0096.5098.5092.3085.0086.4098.6094.00100.1084.40

5.0074.4079.7084.4087.4092.3081.2074.4084.9095.2087.409

6.8084.10 10.0059.107

7.7081.0086.0085.5081.2059.1081.3084.4085.808

8.3080.90 I,(HELA)cell line;II,(MCF7)cell line;III,(HCT116)cell line;VI,(U251)cell line;V,(H460)cell line;VI,(HEPG2)cell line.

Table6

The percentage of surviving fraction of different cell lines against different concentrations of the cold and hot water extracts of D.memmbranacea.

Conc.in(?g/dl)%of surviving fraction

Cold water extract Hot water extract?

I II III IV V VI I II III IV V VI

0.099.5099.90100.2100.010010099.5099.90100.2100.0100100

1.0061.2086.9097.308

2.1096.2070.106

3.9086.9095.9089.1089.5096.70

2.505

3.3073.6092.5069.7091.1063.3059.1073.609

4.6081.2086.1077.60

5.0051.5073.6088.4068.508

6.6062.3054.9073.6074.806

7.6082.7074.80 10.0049.7073.6065.3062.7079.9059.4053.6073.6071.4064.3080.9069.80 I,(HELA)cell line;II,(MCF7)cell line;III,(HCT116)cell line;VI,(U251)cell line;V,(H460)cell line;VI,(HEPG2)cell line.

Table7

Anticoagulant activity of cold and hot water extracts of P.capillacea and D.membranace.

Extract no.Anticoagulant activity

6-h24-h

Clotting time(s)Prothrombin time(s)Clotting time(s)Prothrombin time(s)

I91.3±3.4*36.1±1.4*78.2±2.3*35.6±2.9*

II82.4±2.9*47.6±1.8*63.1±3.7*48.1±2.5*

III67.9±2.3*28.6±1.3*51.2±1.524.4±0.4*

IV54.6±2.119.8±0.9*56.2±1.7*21.2±0.3* Control46.2±2.712.8±0.347.3±2.913.5±1.2

I,CWE of P.capillacea;II,HWE of P.capillacea;III,CWE of D.membranacea;IV,HWE of D.membranacea.

*Signi?cantly different from control at P<0.01.

by several factors,mainly infar-speci?c variabilities in the produc-tion of secondary metabolites.

Antitumor activity of algal polysaccharides has been reported in recent years(Costa et al.,2010;Queiroz et al.,2006;Yubin,Chong, Tao,&Chenfeng,2007).These reports were in agreement with the results obtained in this study,where,both the cold and hot water polysaccharides extracted from D.membranacea represented an effective antitumor activity against HELA(Cervix carcinoma cell line)at IC50=9.83?g(Table6).In contrast,P.capillacea showed insigni?cant antitumor activity upon application on the same cell line(Table5).

Table7shows the anticoagulant activity of polysaccha-rides extracted from the two algae,in the current work.As is evident,the polysaccharides extracted from P.capillacea by the cold and hot method of extraction exhibited the highest anticoagulant activity as compared with those of D.membr-nacae.This may be due to the differences in the type and ratio of these polysaccharides in the two examined algae (Hayakawa et al.,2000;Shanmugam&Mody,2000).This outcome is coincident with that reported by other authors (Mao et al.,2008;Mour?o,2004;Mour?o,2008;Zhang et al., 2008).

66 A.H.Abou Zeid et al./Carbohydrate Polymers113(2014)62–66

4.Conclusion

The water soluble extracts(cold and hot)of the red alga P.capil-lacae and the brown alga D.membranacea exhibited appreciable antimicrobial activity against Gram-positive,Gram-negative bacte-ria and fungi in comparison with ampicillin and clotrimazol as the standard antibiotics.In addition,these algal extracts possessed noticeable anticoagulant and antitumor activitry.From the present study it is evident that both the cold and hot extracts of the stud-ded algae could be utilized as a good natural source of antimicrobial, anticoagulant and antitumor against HELA cell line.However,the nature of the speci?c bioactive components responsible for the observed biological activities from these two algae remains to be evaluated for pharmaceutical and industrial applications. References

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