Aquous extracts of 24 species of green seaweeds, 13 of Caulerpa Lamour and 11 of Ulva Linn, were collected from the coastal areas near Karachi, Pakistan and tested for haemagglutination against human erythrocytes of blood groups A,B,AB and O and compared. All the species of Caulerpa and seven of Ulva exhibited a positive activity, the former genus (a member of Bryopsidophyceae) appeared to possess more phycohaemagglutinins than the latter (a member of Ulvophyceae). The haemagglutinic activity proved to be quite helpful in the distinction of various species of both the genera

Amjad, M.T.

Shameel, M.

Aquatic Commons

Pakistan Journal ofMarine Sciences, Vol.2(2), 113-117, 1993 
COMPARATIVE HAEMAGGLUTINIC ACTIVITY IN THE 
SPECIES OF CAULERPA AND ULVA (CHLOROPHYTA) 
OF KARACHI COAST 
M. T. Amjad and Mustafa Sham eel 
Department of Botany, University of Karachi, Karachi-75270, Pakistan 
ABSTRACT: Aquous extracts of24 species of green seaweeds, I3 ofCaulerpa Lamour and II of Ulva Linn., were 
collected from the coastal areas near Karachi, Pakistan and tested for haemagglutination against human erythrocytes 
of blood groups A,B, AB and 0 and compared. All the species of Caule1pa and seven of Ulva exhibited a positive 
activity, the former genus (a member ofBryopsidophyceae) appeared to possess more phycohaemagglutinins than 
the latter (a member ofUlvophyceae). The haemagglutinic activity proved to be quite helpful in the distinction of 
various species of both the genera. 
KEY WORDS: Phycohaemagglutinins -lectins- Caule1pa spp.- Ulva spp.- Chlorophyta- Karachi coast. 
INTRODUCTION 
The haemagglutinic activity of plants was known since the last century, when it 
was observed that an extract of Ricinus communis L. agglutinated erythrocytes of 
different mammals. The immunological, nutritional, chemical and other aspects of 
phycohaemagglutinins and lectins of higher plants have been thoroughly investigated 
(Lis and Sharon, 1973; Bird, 1977; Jeffe, 1977, 1983; Oppenheimer, 1977; Sharon 
and Lis, 1977; Cheeke and Shull, 1985), but there are only a few reports on the 
preliminary screening of haemagglutinic activity of seaweeds (Boyd et a/., 1966; 
Blunden eta/., 1975; Fabregas eta/., 1984, 1985, 1986; Naqvi eta/., 1992). In this 
communication an attempt has been made to compare the haemagglutinic activity of 
various species of Caulerpa Lamour. and Ulva Linn., growing in the coastal waters 
of Karachi, with the intention to obtain some information about specific 
differentiations. 
MATERIALS AND METHODS 
The seaweeds were collected from the rocky ledges and sandy bays near Goth Haji 
Ali (French Beach), Buleji, Kara.chi, Pakistan during September 1991 to May 1992. 
The healthy specimens of each species were selected, thoroughly washed to remove 
epiphytes, epizoons and attached debris, dried in shade and minced. The chopped and 
ground material was soaked in distilled water in a percolater for 1 month under sterile 
conditions. It was then filtered and the extracts were concentrated under reduced 
pressure below 30°C using a thin film rotary evaporator. The concentrated extract was 
lyophilized in Eyela Fr~eze Dryer model FD-1 (Tokyo Rikakikai), thereafter 
completely dried and powdered. About 5 mg of the powdered extract was dissolved 
in 10 ml of distilled water and serial double dilutions of the extracts (1 :2, 1:4, 1:8, 
1:16, 1:32 and 1:64) were prepared. 
114 Pakistan Journal of Marine Sciences, Vol.2(2), 1993 
The phosphate buffer (pH 7) was prepared by mixing 35 ml of stock solution of 
NaH2P04.2H20 (26 g/l) and 65 ml of stock solution of anhydrous Na2HP04 (18 g/1). 
Samples of Rh+ human blood were obtained from Diagnostic Pathological Labs. 
(Al-Tashkhees), Block C-1, Doctors Plaza, Taj Complex, M.A. Jinnah Road, Karachi, 
and erythrocytes (RBCs) were collected by simple centrifugation. A 2%, RBC 
suspension was prepared in phosphate buffer. Haemagglutinic activity was 
investigated serially in six dilutions of each seaweed extract against all the four blood 
groups (A, B, AB and 0). For this purpose 1 ml of each dilution was added to 1 ml of 
2% suspension of erythrocytes in a test tube of 5 ml capacity followed by incubation 
in a water bath at 25°C. Controls were also run similarly in distilled water instead of 
seaweed extract. A rough granular deposition in the tube due to agglutination of 
erythrocytes showed a positive activity,_ while the smooth button formation due to 
settling of erythrocytes at the bottom indicated a negative reaction. 
RESULTS AND DISCUSSION 
Twenty-four species of Caulerpa and Ulva, collected from the coastal areas of 
Karachi, were tested for their haemagglutinic activity, out of which 20 showed 
positive results (Table I). No doubt, some of these species exhibited 
haemagglutination against only one type of blood group or under a few dilutions only, 
but as a whole 83% of the investigated species of green seaweeds were found to be 
bioactive. Naqvi et al. (1992) observed only 52% positive activity in the species of 
green seaweeds of Karachi seashore. However, 80% of the brown seaweeds 
investigated from the north western coast of Spain (Fabrigas et al., 1986) exhibited 
the haemagglutinic activity. 
The eleven investigated species of Ulva showed specific variations in their 
haemagglutinic activity, four of them were completely inactive, three partially active 
against one or two blood groups and four completely active. Naqvi et al. (1992) 
investigated nine of these species from Karachi coast and observed a negative 
response to agglutination in all of them under all the dilutions. However, Blunden et 
a!. (197 5) observed a positive activity in U. lactuca, and a similar activity was noticed 
in other species of Ulva by Boyd eta/. (1966). The different results obtained by Naqvi 
et al. (1992) are probably due to their extraction technique, as they obtained ethanolic 
extract of seaweeds, during which some of the phycohaemagglutinins were lost, 
denatured or changed their structure. It is, therefore, advisable to obtain aquous 
extracts of seaweeds for agglutination studies, as was done by previous workers (Boyd 
et al., 1966; Blunden et al., 1975). However, no species of Ulva showed a positive 
activity against all the blood groups at the least dilution (1 :2). 
Ulva anandii exhibited positive agglutination against all the four blood groups, 
while U. bifrons showed a complete negative reaction in all the dilutions. This 
indicated that they are completely different species. They were previously included 
under a single specific epithet i.e. U. indica (Anand, 1940), but have recently been 
separated form one another (Amjad and Shameel, 1993). Ulva reticulata showed a 
positive haemagglutinic activity against all the blood groups and U. saifullahii 
Table 1: Haemagglutinic activity ( +) of six dilutions of the aquous extracts of different species of Caulerpa and 
Ulva against human erythrocytes (1=1:2, 2=1:4, 3=1:8, 4=1:16, 5=1:32 and 6=1:64 dilutions). 
Seaweeds species 
Caulerpa chemnitzia (Esper) Lamour. 
C. faridii Nizam. 
C. fastigiata Mont. 
C. lentillifora J. Ag. 
C. manorensis Nizam. 
C. peltata Lamour. 
C. qureshii Nizam. 
C. racemosa (Forssk) J. Ag. 
C. scalpelliformis (R. Brown) C. Ag. 
C. sertularioides (Gmel.) Howe 
C. taxifolia (Vahl) C. Ag. 
C. veravalensis Thivy et Chauhan 
C. vickersiae BQ'fg. 
Ulva anandii Amjad et Shameel 
U. bifrons Ardre 
U. fasciata Delile 
U. fenestrata Post. et Rupr. 
U. gran dis Saif. et Nizam. 
U. lactuca Lim. 
U. reticulata Forssk. 
U. rigida C. Ag. 
U. sorensenii Chapm. 
U. saifullahii Amjad et Shameel 
U. taeniata (Setch) Setch et Gardn. 
Blood group A 
123456 
++++++ 
++++++ 
--++++ 
++++++ 
++++++ 
++++++ 
--++++ 
++++++ 
++++++ 
-+++++ 
-+++++ 
++++++ 
--++++ 
- +++++ 
--++++ 
-+++++ 
Blood Group B 
1 2 3 4 5 6 
++++++ 
++++++ 
++++++ 
++++++ 
++++++ 
--++++ 
----++ 
++.++++ 
++++++ 
- +++++ 
-+++++ 
++++ 
++++ 
-+++++ 
--++++ 
- +++ 
- - + + 
-+++++ 
Blood group AB 
1 2 3 4 5 6 
++++++ 
++++++ 
-- ++++ 
++++++ 
++++++ 
-- ++++ 
-- ++++ 
++++++ 
++++++ 
- +++++ 
- +++++ 
++++ 
++++ 
- +++++ 
++++ 
+++ 
- - + + 
- +++ ++ 
Blood group 0 
1 2 3 4 5 6 
++++++ 
++++++ 
-- ++++ 
-+++ 
- - + + 
++++++ 
++++++ 
- +++++ 
- +++++ 
-- ++++ 
++++++ 
++++ 
- + + 
- +++++ 
++++ 
-+++ 
- +++++ 
> 
.a. 
~ p.. 
[ 
(/) 
r (p 
...... 
:r:: 
~ 
s 
~ 
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s· 
....... 
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en 
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VI 
116 Pakistan Journal ofMarine Sciences, Vol.2(2), 1993 
Similarly, U. fasciata exhibited strong haemagglutinic activity against all the blood 
groups and U. fasciata did not, showing specific differences between the two. 
Previously, they were considered as conspecific (Saifullah and Nazimuddin, 1977), 
but the Karachi specimens have recently been separated as distinct species (Amjad 
and Shameel, 1993) and the current study further supports this separation. 
The thirteen investigated species of Caulerpa also showed specific variations 
regarding their haemagglutinic activity (Table I), three of them were completely active 
in all the dilutions against all the blood groups, seven were partially active in certain 
dilutions against all the four blood groups, two were active againstthree blood groups, 
and one was active against only one blood group. The specie's of Caulerpa, which is 
a member of the class Bryopsidophyceae, exhibited much more agglutinic activity than 
those of Ulva, which belongs to the class Ulvophyceae. This difference in activity 
might be due to their different taxonomic positions. 
Caulerpa manorensis, C. qureshii and C. veranalensis exhibited remarkable 
differences in their haemagglutinic activity, indicating that they are distinct species. 
Coppejans and Meinesz (1988) recorded C. manorensis from Paupa New Guinea, 
discussed its similarities with C. qureshii and C. veranalensis, and considered the last 
two as conspecific. Recently, Shameel and Shaukat (1992) have made a taxometric 
study of these species and from hierarchical agglomerative clustering of their 
operational taxonomic units (OTUs) showed three distinct groups at 85% similarity 
level, indicating that they are separate species. The fatty acid composition of these 
species has further revealed that they are independent and widely different species 
(Shameel and Khan, 1991). Similarly, C. chemnitzia, C. pelata and C. racemosa also 
differ in their haemagglutinic activity from one another, indicating that they are 
distinct species. They were considered as conspecific by some phycologists, e.g. Silva 
et al. (1987). 
The haemagglutinins, which cause the clumping of RBCs, are proteinaceous 
secondary metabolites (Fabregas et al., 1984). They produce adverse effects like 
reduced growth, diarrhoea, decreased nutrient absorption and increased incidence of 
bacterial infections in human body (Jeffe, 1977). They bind themselves with the cells 
in the intestinal wall and cause a nospecific interference with the nutrient absorption 
(Jeffe, 1983). The haemagglutinins also impair the immune system, thus enhancing 
the susceptibility for bacterial infections (Cheeke and Shull, 1985). Some of the 
phytohaemagglutinins have .been found useful in blood banking (Bird, 1977), 
therefore, it appears worthwhile to investigate chemically the phycohaemagglutinins 
isolated from seaweeds. 
REFERENCES 
Amjad, M.T. and M. Shameel. 1993. Two new species and two new reports of Ulva L. (Ulvophyceae) from the 
coast of Karachi, Pakistan. Pakistan Journal of Marine Sciences 2: 5-17. · 
Anand, P.L. 1940. Marine Algae from Karachi. I. Cholorophyceae. Punjab University Botanical Publication., 
Lahore, Pakistan. Pp. 1-52. 
Bird, G.W.G. 1977. Lectins in blood banking: A brief review. Biotesting Bulletin 2: 2-12. 
Blunden, G., D.J. Rogers and W.F. Farnham. 1975. Survey of British seaweeds for hemagglutinins. Lloydia 38: 
162-168. 
Amjad and Shameel: Haemagglutinic activity in seaweeds 117 
Boyd, W.C., L.R. Almodovar and L.G. Boyd. 1966. Agglutinins in marine algae for human erythrocytes. 
Transfusion (Philadelphia) 6: 82-85. 
Cheeke, P.R. and L.R. Shull. 1985. Natural Toxicants in Feeds and Poisonous Plants. A vi. Publication Co. Inc., 
Connecticut, USA. Pp. 240-244. 
Coppejans E. and A. Meinesz. 1988. Marine algae of Paupa New Guinea (Mandang Prov.) I. Caulerpaceae 
(Chlorophyta-Caulerpales). Blumea 33: 181-196. 
Fabregas, J.A., A. Munoz, J. Liovo and J. Abalde. 1984. Agglutinins in marine red algae. IRCS Medical Sciences 
12: 298-299. 
Fabregas, J., J. Liovo and A. Munoz. 1985. Hemagglutinins in red seaweeds. Botanica Marina 28: 517- 520. 
Fabregas, J., A. Munoz and J. Liovo. 1986. Hemagglutinins in brown seaweeds. Journal of Experimental Biology 
and Ecology 97: 213-219. . 
JefTe, W.G. 1977. Immunology of plant lectins. In: Immunological Aspects of Foods (Ed. N. Catsimpooles). John 
Wiley & Sons, New York. Pp. 1-170. 
J effe, W. G. 19 83. Nutritional significance oflectins. In: Handbook ofN aturally Occurring Food Toxicants (Ed. M. 
Rechcigl Jr.). CRC Press Inn., Boca Raton, Florida. USA Pp. 31-38. 
Lis, H. and N. Sharon. 1973. The biochemistry of plant lectins (phytohemagglutinins). Annual Review of 
Biochemistry 42: 541-545. 
Naqvi, S.B.S., D. Sheikh, K. Usmanghani, M. Shameel and R. Sheikh. 1992. Screening of marine algae ofKarachi 
for haemagglutinin activity. Pakistan Journal of Pharmaceutical Sciences 5: 129-138. 
Oppenheimer, S.B. 1977. Interaction of lectins with embryonic cell surface. Current Topics of Developmental 
Biology 11: 3-5. 
Saifullah, S.M. and M. Nizamuddin. 1977. Studies of the marine algae from Pakistan: Ulvales. Botanica Marina 
20: 521-535. 
Shameel, M. and R. Khan. 1991. Fatty acid composition of nine green seaweeds. Botanica Marina 34: 501- 504. 
Shameel, M. and S.S. Shaukat. 1992. A taxometric study of three related species of Caulerpa (Bryopsidophyceae) 
from northern Arabian Sea. Marine Research 1: 1-7. 
Sharon, N. and H. Lis. 1977. Lectins: Cell agglutiniating and sugar specific proteins. Science 177: 949-950. 
Silva, P.C., E.G. Menez and R.L. Moe. 1987. Catalog of the benthic marine algae of the Philippines. Smithsonian 
Contributions to the Marine Sciences 27: 1-179. 


Comparative haemagglutinic activity in the species of Caulerpa and Ulva (Chlorophyta) of Karachi coast

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Comparative haemagglutinic activity in the species of Caulerpa and Ulva (Chlorophyta) of Karachi coast

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