Heike Helmholz1, Antje Wichels2, Gunnar Gerdts2

1. GKSS Research Centre, Institute for Coastal Research/Physical and Chemical Analysis, Max-Planck-St. 1, 21502 Geesthacht, Germany

2. Marine Chemistry and Natural Products, Biologische Anstalt Helgoland at the Alfred Wegener Institute, Kurpromenade, 27483 Helgoland, Germany

Screening for bioactive Glycolipids in Sponges from

the Orkney Islands

Material and Methods

Results and Discussion

Introduction

Sample collection and taxonomical analysis:

Sponge species (Demospongiae and Calcarea), were collected from subtidal waters off the Orkney and Shetland Islands (Great Britain) during

a cruise with RV Heincke in June 2001 and from the Island of Helgoland. Sampling was performed by SCUBA diving and dredging. All samples

were deep frozen and stored at –20°C until extract preparation. Sponges were identified on the basis of spicule and tissue preparations on

board the RV Heincke and at the Zoologisch Museum, Universiteit van Amsterdam, The Netherlands.

Extraction:

The dry biomass was crushed and grumbeld to destroy cell

walls mechanically. The pulverized material was homogenized

three times with chloroform (CHCl3) : methanol (MeOH) (2:1)

and the obtained extracts were dried and stored at –18°C

until further use.

Thin layer chromatography (TLC) analysis:

Eluent: CHCl3: MeOH: water (70:30:4); Detection: orcinol-H2SO4. The

samples were analyzed with a Gengenius2 system; software GeneTools

(Syngene, Cambridge, UK). Quantitative analysis was performed 5 times

compared to octylmaltoside (OM) as standard glycolipid (range 0,1-5µg

OM/spot; linearity - Pearson factor 0,9991)

High-Performance Liquid Affinity Chromatography:

To analyse the sugar content, the extracts were solubilized (harsh stirring and

ultrasound) in MeOH : water (1:1) and filtered (0,2µm). A HPLC (Merck

LaChrom L7000, Darmstadt, FRG) was used. For lectin-affinity

chromatography two different lectins: Ricinus communis Agglutinin (RCA) and

Concanavalin A (ConA) were immobilized as ligands.

Separation conditions:

RCA-polymer [3] adsorption 0,01M phosphatbuffer (pH7,2) and gradient

elution 0,1M lactose in phosphatbuffer;

ConA-polymer (Sigma-Aldrich, Munich, FRG) adsorption 0,1M

acetatbuffer (pH6) and elution 0,1M methylmannose in acetatbuffer.

Figure 3: Pachymatisma johnstonia

GlycoproteinGlycolipids

Sugar side chain

The marine environment provides a huge variety of metabolites used as potential biopharmaceuticals.Especially sponges are known as a prolific resource for new bioactive substances. Sponges (Porifera) aremulticellular aquatic animals, which do not possess organs and true tissues. Compared to species fromtropical zones, sponges from the Northern Atlantic are not well characterized concerning theirpharmacological active metabolites, e.g. glycolipids. Glycolipids are amphiphilic molecules, involved in manyphysiological processes and show a proven pharmacological activity [1, 2]. As components of cellmembranes (fig. 1) they arrange the characteristic cell surface and modify interaction, communication anddifferentiation processes.

The yield of the CHCl3:MeOH extracts varies between 1,0 and 21,4 % (figure 2).

The TLC profile analysis of the extracts shows different glycolipid contents and patterns (fig. 5 and 6). Only

traces of glycolipids could be found in Leucosolenia complicata, Sycon ciliatum, Isodyctia palmata, Phakealia

ventilabrum, Stelligera stuposa and Axinella polypoides. Same elution conditions were applied to obtain

comparable results for the different species. Separation performance has to be optimized for purification

processes.

Samples from Pachymatisma johnstonia and Cliona celata (fig. 3 and 4) were selected to demonstrate the

further processing. Both extracts contain yellow and brown pigments beside different glycolipids. A

quantitative analysis of the glycolipid content shows that 1% of the total dry biomass of P. johnstonia could be

the desired compound whereas there are only 0,007% glycolipid in C. celata (able 1). These results are

estimated compared to OM as standard material because a difference in the red-violet colour of orcinol

detection could cause a change in quantitative analysis and the contaminating pigments influence the

separation although their concentration can be decreased by filtration. One can see from figure 5 and 6 that

there are more glycolipids than mentioned in table 1 but the concentration is below the limit of determination.

Orkney Islands

Rousay Sound

Waukmill Bay

Westray

Saviskaill Bay

Here 19 species collected in the area off the Scottish Orkney Islands and one species from the IslandHelgoland (North Sea) were investigated according their glycolipid contents.

Figure 1: Model of a cell membrane

Figure 4: Cliona celata

50µg

Figure 5: TLC extract profile of P. johnstonia

500µg

Figure 6: TLC extract profile of C. celata

1,10

1,83

2,35

%B

0 2 4 6 8 10 12 14 16 18

Retention Time (min)

0,00

0,02

0,04

0,06

0,08

0,10

Absorbance (AU)

0

20

40

60

80

100

Solvent (%)

2,64

23,29

0 5 10 15 20 25 30

Retention Time (min)

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

Absorbance (AU)

Figure 8: HPLAC of C. celata extract (A) ConA – adsorbent(B) RCA - adsorbent

A B

Species Biomass

[g]Extract

[mg]Extract

[%]Peak

(Fig.5 & 6)Glycolipid

[mg]Glycolipid

[%]

P. johnstonia 5,14 638,8 12,43

446,31

(± 2,02)1,11

5 1,12 (± 0,31)

0,03

C. celata 6,68 858,4 12,85 6 0,49 (± 0,17)

0,007

Table 1: Extract yield and glycolipid content of two selected sponges

High-Performance Liquid Affinity Chromatography:

Lectin affinity chromatography is based on biospecific interaction between the carbohydrate

recognizing ligand and the complementary glycoconjugate. RCA binds galactose/lactose and ConA

mannose/glucose sugar side chains. The first peaks at the beginning of all chromatograms (fig. 7

and 8) indicate that there are contaminants that do not bind to the lectins. Only the crude extract of

P. johnstonia seem to contain both lactose and mannose/glucose bearing glycolipids indicated by

the small peak at 6,91 min for ConA and 9,37 min for RCA, respectively. This delay can be due to a

weak biospecific interaction between the lectins and the glycolipids.

These results can be used for the development of a selective, structure based purification strategie

for the desired glycolipids. A purification should be performed before detecting the biological

activity of the screened glycolipids.

2,64

3,22

23,26

0 5 10 15 20 25 30

Retention Time (min)

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

Absorbance (AU)

9,37

B

Figure 7: HPLAC of P. johnstonia extract (A) ConA - adsorbent (B) RCA - adsorbent

1,04

1,79

2,43

6,91

%B

0 2 4 6 8 10 12 14 16 18

Retention Time (min)

0,00

0,05

0,10

0,15

0,20

0,25

Absorbance (AU)

0

20

40

60

80

100

Solvent (%)

A

Literature

1. Fattorusso, E., and Mangoni, A., (1997). Marine Glycolipide. Progress in the Chemistry of Organic Natural Products 72, 215-3012. Costantino, V., Fattorusso, E., Mangoni, A., Di Rosa, M., and Ianaro, A. (1999). Glycolipids from sponges. VII: Simplexides, novel immunsupressive glycolipids from the caribbean sponge Plakortis simplex. Bioorganic and medicinal Chemistry Letters 9,2-276.3. Cartellieri, S., Helmholz, H., and Niemeyer B. (2001). Preparation and Evaluation of Ricinus communis Agglutinin Affinity Adsorbents Using Polymeric Supports. Analytical Biochemistry 295, 66-75

Figure 2: Extract yield; % of dry biomass

0

5

10

15

20

25

Pa

chym

ati

sma

joh

nst

on

ia

Ha

liclo

na

ro

sea

Esp

eri

op

sis

fuco

rum

Le

uco

sole

nia

com

plic

ata

Syc

on

cili

atu

m

My

xil

lain

cru

sta

ns

Clio

na

ce

lata

Ha

liclo

na

cin

ere

a

Iso

dyc

tia

pa

lma

ta

Ph

ake

alia

ven

tila

bru

m

Ste

llig

era

stu

po

sa

Po

eci

llats

ra

com

pre

ssa

Myc

ale

ro

tari

s

Su

be

ritis

su

be

ria

Te

tilla

ze

tlan

dic

a

Axin

ella

infu

nd

ibu

lifo

rmis

Po

lym

ast

ia

ma

mill

ari

s

Axin

ella

po

lyp

oid

es

Su

be

riti

s

carn

osu

s

Ha

loch

on

dri

a

pa

nic

ea

Ext

ract

yie

ld [

%]