Abstract: The isolated heparin and heparin-like Glycosaminoglycans (GAGs) from the cuttlefish Euprymna berryi was studied for the antimicrobial activity. The bacterial strains such as (Bacillus subtilis, Pseudomonas aeruginosa, Escheriachia coli, Staphylococcus aureus, Shigella flexineri) and fungal strains such as Aspergillus fumigatus Fusarium sp., Cryptococcus neoformans, Microsporium sp. and Candida albicans present study. various concentration (25, 50, 75 and 100%) used in this study. The heparin and heparin-like (GAGs) crude and purified sample showed activity against all the pathogenic bacterial strains. Whereas the antifungal activity in crude and purified sample was no activity against Microsporium sp. Fusarium sp. (crude) and Microsporium sp. (purified) respectively in all the concentration. The other all the fungal strains having activity in all the concentration. The activity of heparin and heparin-like GAGs extract was found to be higher in 100% concentration than the other concentration. In general the increasing concentration showed increasing activity of the extract. The heparin and heparin-like extract showing good antimicrobial activity are under going further analysis to identify the active constituents.
INTRODUCTION
Marine organisms are a rich source of structurally novel and biologically active metabolites. So far chemically unique compounds of marine origin with different biological activity have been isolated and a number of them are under investigation and/or are being developed as new pharmaceuticals (Da Rocha et al., 2001; Faulkner, 2000a, b; Schwartsmann et al., 2001).
Antibiotics are one of our most important weapons in fighting bacterial infections and have greatly benefited the health-related quality of human life since their introduction. However, over the past few decades these health benefits are under threat as many commonly used antibiotics have become less and less effective against certain illnesses not only because many of them produce toxic reactions but also due to emergence of drug resistant bacteria. It is essential to investigate newer drugs with lesser resistance. Systematic studies among various pharmacological compounds have revealed that any drug may have the possibility of possessing diverse functions and thus may have useful activity in completely different spheres of medicine (Sarkar et al., 2003). Therefore development of new technologies in search of novel bioactive compounds from marine sources will bring unique challenges and opportunities from seafood industry (Kim and Mendis, 2006). In the present an attempt has been made to study the antimicrobial activity of heparin and heparin-like GAGs of crude and purified samples isolated from the cuttlefish E. berryi was studied.
MATERIALS AND METHODS
Antibacterial and Antifungal Activity
The animals were collected from the Mudasalodai landing centre, east coast
of India, Tamil Nadu. The study was conducted in our laboratory situated at
CAS in Marine biology, parangipettai. The crude and purified heparin and heparin-like
GAGs was extracted from E. berryi using the method of (Holick et al.,
1985) and were used to study their antibacterial and antifungal activities.
Preparation of Stock Solution
One milligram of each crude and purified heparin and heparin-like GAGs was
dissolved in 2 mL of distilled water. From this 0.25, 0.50, 0.75 and 1.0 mL
was taken and made up to 1.0 mL by adding distilled water to prepare various
concentrations containing 125, 250, 375 and 500 μg of crude and purified
sample corresponding to 25, 50, 75 and 100%, respectively.
Antibacterial Activity
Antibacterial activity was determined against Bacillus subtilis,
Escheriachia coli, Pseudomonas aeruginosa, Staphylococcus aureus
and Shigella flexineri (obtained from Rajah Muthaiah Medical College,
Annamalai University, Annamalai Nagar) using the paper disc assay method (El
Masry et al., 2000). Whatman No. 1 filter paper disc of 6 mm diameter
was sterilized by autoclaving for 15 min at 15 lbs (121°C).
Nutrient broth was prepared and sterilized in an autoclave at 15 lbs for 15 min. All the five bacterial strains were inoculated into the nutrient broth and incubated at 28±2°C for 24 h.
Nutrient agar medium was also prepared and transferred aseptically into sterile petridishes. The solidified nutrient agar medium in petridishes was incubated with the bacterial cells of 24 h old in the nutrient broth using the sterile cotton swab. The sterile discs were saturated with different concentrations of crude and purified samples (100, 75, 50 and 25%). The control with respective solvent (distilled water) was also prepared. The saturated disc was placed on the medium suitably spaced apart and the plates were incubated at 37°C for 24 h. The zone of growth inhibition, if any, was measured after the incubation period. Each extract was tested thrice for confirming the effect.
Antifungal Activity
Antifungal activity was tested against Aspergillus fumigatus, Fusarium
sp., Cryptococcus neofromans, Microsporium sp. and Candida
albicans (obtained from Rajah Muthaiah Medical College, Annamalai University,
Annamalai Nagar) using the paper disc assay method as previously described in
the antibacterial assay using the same stock solution for the four different
concentrations.
The potato-dextrose broth was prepared and sterilized in an autoclave at 15 lbs. All the five fungal strains were inoculated into the potato-dextrose broth and incubated at room temperature for three days.
Potato-dextrose agar medium was also prepared and transferred aseptically into sterile petridishes. The solidified medium in the petriplates was incubated with the fungal cells of three days old in the potato-dextrose broth using sterile cotton swab.
The saturated discs (in different concentrations of crude and purified samples) were placed on the medium suitably spaced apart and the plates were incubated at 37°C for 24 h. The control with respective solvent (Distilled water) was also prepared. The zone of growth inhibition, if any, was measured after the incubation period. Each extract was tested thrice for confirming the effect.
RESULTS
Antibacterial Activity
The crude and purified sample of E. berryi showed activity against
all pathogenic bacterial strains. The activity was higher in 100% concentration
and lower in 25% concentration but activity was absent in control (Table
1, 2).
In 100% concentration, the highest inhibition zone was observed against Shigella sp. (5 mm) in crude sample; whereas in purified sample higher inhibition zone was observed against Bacillus sp. and Staphylococcus aureus (5 mm). The lowest activity, in terms of inhibition zone, was observed against S. aureus in crude sample (4 mm). But at the same time, 4.5 mm inhibition zone was recorded against P. aeruginosa, E. coli and Shigella sp. in purified sample and B. subtilis and E. coli in crude sample.
In 75% concentration, heparin and heparin-like GAGs showed maximum activity (4.5 mm inhibition zone) against B. subtilis and S. aureus in purified sample but in crude sample 4 mm inhibition zone was recorded against B. subtilis, P. aeruginosa, E. coli and Shigella sp. The lowest activity with 3.5 mm inhibition zone and moderate activity with 4 mm inhibition zone were observed against E. coli and P. aeruginosa respectively in the purified sample.
In 50% concentration, the maximum activity (4 mm inhibition zone) was recorded against P. aeruginosa in crude sample of heparin and heparin-like GAGs, but in purified, only 3.5 mm inhibition zone was observed against P. aeruginosa and Shigella sp. Minimum activity (3 mm inhibition zone) was showed against B. subtilis, E. coli and S. aureus in purified sample and 3.5 mm against B. subtilis, E. coli and Shigella sp. in crude sample.
At 25% concentration, the crude and purified samples showed more or less similar activity in all strains with the inhibition zone of 1.5 to 2.5 mm. The maximum of 2.5 mm inhibition zone was seen against S. aureus and Shigella sp. in crude sample and B. subtilis in purified sample. The moderate activity with 2 mm inhibition zone was recorded against B. subtilis and P. aeruginosa in crude sample and P. aeruginosa, E. coli, S. aureus and Shigella sp. in purified sample. The minimum activity (1.5 mm inhibition zone) was showed against E. coli in crude sample.
| Table 1: | Antibacterial activity of various concentrations of the crude heparin and heparin-like glycosaminoglycans from the whole body tissue of E. berryi |
| Table 2: | Antibacterial activity of various concentrations of the purified heparin and heparin-like glycosaminoglycans from the whole body tissue of E. berryi |
Antifungal Activity
The activity against the crude and purified heparin and heparin-like GAG
sample was showed only by three and four fungal strains, respectively. In crude
and purified samples there was no activity against Microsporium sp. And
Fusarium sp. and Microsporium sp., respectively in all the concentrations
tested (100, 75, 50 and 25%) (Table 3, 4).
At 100% concentration, the maximum inhibition zone of 5.5 mm was observed against C. albicans and A. fumigatus in crude and purified sample, respectively. The minimum of 3 mm inhibition zone was observed against C. albicans in purified sample. In purified sample 5 and 3.5 mm inhibition zone was observed against C. neofromans and Fusarium sp., respectively. At the same time 3.5 mm inhibition zone was observed against A. fumigatus and C. neofromans in crude sample.
In 75% concentration, highest activity (with 4.5 mm inhibition zone) was noted against C. albicans and A. fumigatus in crude and purified sample, respectively. Whereas 4 and 3 mm against C. neofromans and Fusarium sp., respectively in purified sample and 2.5 mm against A. fumigatus and C. albicans in crude and purified sample respectively was noticed. The lowest activity with only 2 mm inhibition zone was observed against C. neofromans in crude sample.
In 50% concentration, 3.5 mm inhibition zone was observed against A. fumigatus in purified sample, 3 mm against C. albicans in crude sample. In purified sample 2.5 and 2.0 mm inhibition zone was seen against C. neofromans and C. albicans and Fusarium sp. but in crude sample the lowest activity with 1.5 and 1 mm of inhibition zone was noted against A. fumifatus and C. neofromans, respectively.
At 25% concentration, the activity (in terms of inhibition zone) was observed between 0.5 and 1.5 mm. 1.5 mm inhibition zone was recorded against A. fumigatus in purified sample, 1 mm inhibition zone was noted against C. albicans and C. neofromans, Microsporium sp. and Fusarium sp. in crude and purified sample, respectively. The lowest activity with only 0.5 mm inhibition zone was recorded against A. fumigatus and C. neofromans in crude sample.
| Table 3: | Antifungal activity of various concentrations of the crude heparin and heparin-like glycosaminoglycans from the whole body tissue of E. berryi |
| Herpirn like glycosaminoglycans form the whole body tissue of E. berry | |
| Table 4: | Antifungal activity of various concentrations of the purified heparin and heparin-like glycosaminoglycans from the whole body tissue of E. berryi |
DISCUSSION
Antimicrobial Activity of Heparin and Heparin-Like GAGs
Multicellular organisms express a blend of antimicrobial peptides. The various
pathogenic bacteria are responsible for release of glycosaminoglycans from epithelia
and connective tissues (Andersson et al., 2004). Heparin inhibits the
growth of microorganisms gram-positive organisms are relatively susceptible
and to this effect, gram-negative organisms are relatively resistant (Rosett
and Hodges, 1980). The EDTA extract (polysaccharide) of D. sibogae gladius
recorded 10 mm inhibition zone against E. coli and K. pneumoniae,
9 mm inhibition zone against S. aureus and 7 mm against S. typhii.
Whereas the EDTA extract of L. duvauceli extract showed only low activity
i.e., 5 mm against P. aeroginosa, 4 mm against S. typhii and E.
coli. At the same time, the gladius extract of both the species showed no
activity against V. cholerae. The polysaccharide extract from the gladius
of D. sibogae recorded potent antibacterial activity against all the
bacterial strain mentioned above and at the same time the polysaccharides of
the L. duvauceli gladius extract recorded only low activity. The polysaccharide
extract from the gladius of L. duvauceli showed antifungal activity against
A. fumigatus, A. flavus and Rhizopus sp; whereas
gladius extract of D. sibogae recorded the antifungal activity against
A. fumigatus and Rhizopus sp. only. Whereas both the species
showed no activity at all against Candida sp. (Barwin Vino, 2003). The
antibacterial activity was predominant among cuttlebone extracts (using EDTA)
of the cuttlefishes such as S. aculeata and S. brevimana against
almost all the 9 pathogenic bacterial strains tested viz., B. subtilis,
E. coli, K. pneumoniae, S. aureus, V. parahaemolyticus,
V. cholerae, S. typhii, P. aeroginosa and Shigella
sp. The activity was recorded in almost all the concentrations except in control.
The antifungal activity of cuttlebone extract of S. aculeata and S.
brevimana against four fungal strains such as A. fumigatus, A.
flavus, Candida sp. and Rhizopus sp. showed the maximum activity
of 100% and activity was found to be in an increasing order from the lower to
higher concentration. On comparison the activity was higher in the cuttlebone
extract of S. aculeate than S. brevimana (Mahalakshmi, 2003).
In the present study, antibacterial activity of crude and purified heparin and
heparin-like GAGs of E. berryi was studied in terms of the inhibitory
zone produced by various bacterial and fungal strains and the diameter of inhibitory
zone reported by the microorganisms was as follows: E. coli -4.5 mm
(100% crude and purified), P. aeruginosa -5 mm (100% crude) and 4.5 mm
(100% purified), S. aureus -4 and 5 mm (100% crude and purified) and
B. substilis -4.5 mm (100%) and 5 mm (100% crude and purified) and antifungal
activity in C. albicans -5.5 and 3 mm (100% crude and purified), respectively
in which inhibition zone is higher than the above studies.
Likewise in the study of Sarkar (2003), on the antimicrobial activity of eight species of microorganisms, it was reported that the gram-positive Cocci (S. aureus, S. epidermidis and C. albicans) were more susceptible to heparin than were the gram-negative rods (Citrobacter sp., K. pneumoniae, E. aerogenes and Enterobacter cloacae) and an intermediate susceptibility of heparin was showed by the gram-negative rods E. coli and P. aeroginosa.
The activity of heparin and heparin-like GAGs extracts was found to be high in 100% concentration than the other concentrations. In general the increasing concentration showed increasing activity of the extracts. Therefore, it could be concluded that the antibacterial activity depends on the concentration. In the present investigation, the extract (both crude and purified) of heparin and heparin-like GAGs from E. berryi showed the antibacterial activity against all the five human pathogenic bacterial strains (B. substilis, E. coli, P. aeruginosa, S. aureus and S. flexineri) in increasing order in all the four concentrations (25, 50, 75 and 100%) tested. Likewise the antifungal activity was also studied in four different concentrations (25, 50, 75 and 100%) of the extracted heparin and heparin-like GAGs against five strains of fungi such as A. fumigatus, Fusarium sp., C. neofromans, Microsporium sp. and C. albicans. Though all the above fungi reported varying activities by showing difference in the inhibition zone, the crude sample showed no activity in Fusarium sp. and Microsporium sp. and also the purified sample, against Microsporium sp. The result of the present provides larger information to the researchers involved in the filed of pharmacology and the scientists looking for useful drugs or drugs basic principles for the antibiotics and antifungal drugs against the human pathogens.
The uniqueness of the present study is that use of heparin and heparin-like extract obtained from cuttlefish, E. berryi is an efficient and cheap source of marine organism with remarkable antimicrobial activity. Detailed analysis at molecular level should be conducted to identify the active constituents that are responsible for this activity.
ACKNOWLEDGMENTS
The authors are thankful to the Director, CAS in Marine Biology and authorities of Annamalai University for providing all facilities and ICMR for financial assistance.