1. Caulerpa
sertularioides
Crude Extract
Ethyl acetate
partition
Chloroform
partition
1
2
3
4
1’
2’
3’
4’
5’
1’’
2’’
3’’
4’’
5’’
6’’
7’’
6’
5
6
7
8
9
30%
methanol/water
partition
Water partition
Growth Inhibition of Vibrio sp.
Growth Promotion of Vibrio sp.
Bioassay-guided Fractionation of the Green Feather Alga
Caulerpa sertularioides from the Florida Keys
Tomasz Jurga, Stanley Budzynski and Melany P. Puglisi
Chicago State University, College of Pharmacy, 9501 S. King Dr., Chicago, IL 60628
Abstract
Algae are a rich source of biologically active metabolites some of which are chemical defenses against
herbivores, competitiors and microorganisms. In this investigation, the active crude extract from the
green feather alga Caulerpa sertularioides was separated using bio-assayed guided fractionation against the
marine bacteria Vibrio sp., Firmicutes sp., and Thalassospira sp. isolated from the surface of conspecific
algae. Bacterial cultures were incubated on a shaker table in A1 liquid media. Standard assays with
fractions were conducted in a 96-well plates format. The crude extract was partitioned between
chloroform and 30% methanol/water. Both fractions were separated by repeated Si gel medium pressure
liquid chromatography (MPLC). Initially, extracts from C. sertularioides specifically inhibited the growth of
Vibrio sp. MPLC fractions from the chloroform and methanol/water fractions were analyzed by proton
nuclear magnetic resonance (1H NMR) The methanol/water extract yielded 9 mg of a compound that
promotes the growth of Vibrio sp., a microorganism isolated from the surface of Caulerpa spp. We
suggest that there is a potential for a positive relationship between the alga and the microorganism. 1H
NMR analysis suggests that the metabolite has an aromatic region, an aldehyde, and a methoxy group.
13C, DEPT and mass spectrometry experiments are in progress to determine the structure..
Introduction
Algae are rich sources of secondary metabolites (Puglisi et al., 2014 and references cited within).
Green algae from the genus Caulerpa are widely distributed in tropical waters (Nagappan and
Vairappan, 2013). Members of this genus are invasive in the Mediterranean Sea, Costa Rica (Lowe et
al., 2004, Fernández and Cortés, 2005). Secondary metabolites from Caulerpa spp. have been shown to
deter feeding by generalist herbivores and promote growth of the host plants. For example, Caulerpin
plays an important role in plant growth promotion and has also been found to exhibit moderate
antibacterial activity against some marine bacteria (Vairappan, 2004). Caulerpin and its analog
caulerpicin have been shown to be detrimental to the reproductive success of fish and mosquitoes
(Anitha et al., 2013; Nielsen PG, 1982). The sesquiterpene, caulerpenyne is a fish deterrent (Bansemir
et al., 2004). In broad studies of algal extracts, extracts from C. cuppresoides were active against the
micro-alga Schizochytrium aggregatum and the marine bacterium Pseudoalteromonas bacteriolytica, a known
algal pathogen. C. sertularioides was shown to inhibit the water mold Halophytophthora spinosa as well as P.
bacteriolytica (Puglisi et al, 2006; Engel et al, 2006).
In our previous study, extracts from C. mexicana and Udotea looensis showed antimicrobial activity
against Vibrio sp. and Bacillus sp. The purpose of this study was to isolate the active metabolites from
Caulerpa sertularioides using bioassay-guided fractionation against Vibrio sp., Firmicutes sp., and
Thalassospira sp.
Methods
• C. sertularioides was collected in the Florida Keys in July, 2014 by snorkeling.
• Samples were immersed for 48 hours in a 1:1 solvent mixture of ethyl acetate and methanol and then partitioned
between distilled water and ethyl acetate.
• The ethyl acetate partition was further partitioned using chloroform and a 3:7 mixture of methanol and distilled water.
• Bacteria were grown in 25 mL of liquid A1 media, placed on a shaker table, and regrown every 72 hours.
• Bacterial cultures were diluted to 1:10,000.
• Chloroform and Methanol/Water partition was subjected to bio-assay guided fractionation using Vibrio sp., Firmicutes sp.,
and Thalassospira sp. in a 96-well plate.
• Plates were incubated on a shaker table for 48 hours and read on a BioTek microplate reader
• The chloroform extract was further separated into fractions using Si gel medium-pressure liquid chromatograph (MPLC)
using bioassay-guided isolation.
• A fraction with a single compound was to be identified via proton nuclear magnetic resonance (H-NMR) spectroscopy.
Results Discussion
• Bioassay-guided fractionation yielded a metabolite (fraction 2”) that promotes the
growth of an environmental strain of Vibrio spp.
• This strain was isolated from the surface of conspecific Caulerpa spp. in the same
habitat.
• We suggest that there may be a positive relationship between the alga and the
microorganism. Algae like Delisea ocellata have been shown to produce compounds
that regulate the growth of bacteria that live in the marine environments.
Depending on the conditions that the alga is exposed to, it can produce
compounds that either promote or inhibit the growth of these marine bacteria
(Puglisi MP, 2014).
• Initially the crude extracts from C. sertularioides inhibited the growth of Vibrio sp.,
while at the same time it had very little growth effect on the other strains of
bacteria. After one step of separation on the MPLC, the first four fractions showed
significant growth promotion.
Conclusion
• The chloroform and methanol/water partitions of Caulerpa sertularioides yielded
fractions that inhibited and promoted the growth of Vibrio sp.
• 1H-NMR analysis of the growth promoting fraction suggests that there is an
aromatic molecule in the extract responsible for the observed activity.
• The structure will be determined using standard NMR and MS experiments.
• Once the structure is complete, the IC50 of the compound will be determined
and it will be submitted for testing against human pathogens.
Acknowledgements
We thank Drs. Jaclyn Sievers, Heta Mewada and Albert Nelson and the staff of the
Keys Marine Lab for assistance with collecting the samples in the Florida Keys.
We thank Daniel Pietryla for assistance in the laboratory. We also than Dr. Robert
LeSeur for assistance with the NMR and the College of Pharmacy for supporting
our research.
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Caulerpin and caulerpicin
Stigmast-5,24-dien-3-ol,
fucosterol
Caulerpenyne
Caulerchlorin
Caulerpals A and B
Figure 7. H-NMR is significant of a benzene ring and hydroxyl groups present within the compound.
Compound has been found to promote growth of Vibrio sp.
68.36%
200.30%
143.43%
115.57%
222.01%
97.05%
88.02%
100%
0.00%
50.00%
100.00%
150.00%
200.00%
250.00%
1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 7’’ Methanol
Caulerpa sertularioides chloroform fractions obtained from previous MPLC of Fraction 5'
Growth of Vibrio sp. in the Final Fractionation Step of Extract from C.
Sertularioides as compared to Methanol Control
Graph 4. Growth of bacterium Vibrio sp. grown in
response to extract and compared as a percentage of
growth to methanol as a control. The fractions were
isolated using MPLC from the fifth C. sertularioides
chloroform extract fraction, which in turn came from the
first four previous fractions. In this case, there was a
combination of growth effects of the bacterium.