Antibiotic Properties of Marine Algae

IIL Cyinopolia barbat a

ΝΟΕΜί G. MARTlNEZ NADAL, CARMEN M. CASILLAS CHAPEL, LUZ V. RODRiGUEZ,
JOS£ R. RODRfGUEZ PERAZZA and LUIS TORRES VERA
Received: 9. 3, 196$

Two antibiotic substances, sarganin and chonalgin, reported by ΜΑΚΤΪΝΕΖ NADAL,

et al (1964) and described s being related, although not identical, have been found
to comprise, principally, of two different but chemically similar components, designated
s sarganin A and sarganin B, which together with impurities constitute the sarganin
complex (MARTINEZ NADAL, et al [1965]).
Sarganin complex is a broad-spectrum antibiotic substance, isolated originally from
Sargassum natans by ΜΑΚΤΪΝΕΖ NADAL in 1958.
The present study was carried out to determine the possibility that this complex or
part of its activity might be present in the marine alga Cymopolia barbata. This alga was
screened in its natural habitat by BURKHOLDER (1960) who reported limited antibiotic
activity.
Ecology
Cymopolia barbata (Linnens) belongs to the Chlorophyceae which contain Chlorophyll
and small quantities of xanthophyll and carotenoid pigments. This alga, like all
Dasydadaceae, assimilates calcium carbonate and is characterized by possessing calcified
thalli. The Cymopolia barbata is a Caribbean alga found abundantly in P. R. in the regions
of Vega Alta and Camuy, It is found in live waters in depths of z—8 feet. It lives in
rocky Substrates, far away from rivers although it is prolific in dirty waters. It is found
submerged since it does not resist light exposure. It is abundant in the spring and
summer.
Methods
The alga used in our studies was dried (4.2—4*9% moisture) and in some cases it
was kept frozen until future use.
Similar methods used for the extraction of sarganin complex from Sargassum natans
were followed in our experiments (MARTINEZ NADAL, et al [1964]). These methods
involved Soxhlet extraction, liquid-liquid extraction, maceration and warring blendor
extraction, Anhydrous ether (peroxide free), ethanol, benzene, Chloroform, acetone
and distilled water were used s solvents. Extracts were concentrated in vacuo and
their antimicrobial spectra was determined.
Figure I, which f ollows, shows a schematic view of the different extractions and
their components. Figure 2, shows schematic extraction of Cymopolia barbata with
ethyl ether (peroxide free).
Results
Grude extracts obtained by the different extractions were contaminated with colouring
and other f oreign matter. As in the case of Sargassum natans and Chondria littoralis^ the
most active extracts were those obtained with ethyl ether (see figures i and 2).

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22 .· Botanica Marina, Vol. IX, Fase. 1/2

Figure i

Cymopolia barbata

YT - ι·. Λ ** * nnt

Extraction with Extraction with

. ethyl ether ethyl alcohol Extrac
, Extraction with H with w
pccroleum cther (20-30°Ο) Positive
| antibiosis * |
Ether Carbohydratc
Po si ti vc
^^ ·* · ΎΪ L. i antibiosi 5 ι No apparent Ethci
dcoh l Positive antibiosis extra
steroids °
1 antibiosis · positivc
Crystals 1
Positive ndi in Small - Water antibiosis ^ " |
"~ antibiosis potassium antibiosis .Separation «»«αοη ^
anion uni- extra
Alcohol dentified No apparent
andbiosis Positive
white Water p antibiosis
unidenufied extraction Mixrure
crystals Clear Dark ?
Positive fraction . fraction
antibiosis 1 · os
t> l^To sppnrcn 1 i^7c .
antibiosis Distiilacion Distillation
growth in
^ _.„^_

Residue Distillate
- „» positive no antibiosis
^Ρ antibiosis - Paper Aromatography
Paper chromatography ^9 *C with butanol
Butanol 1 6c/o - water NoRf determincd
No Rfdetermined · Positiv-e. ' due to coincidence
„„β- antibic)5iS with solvent riscs
33°C
. ' positive Positive
antibiosis antibiosis
No car-
bohy- 1
d rat es
W E er * Positive
carbohydratcs
biosis solublc so) uble
ction Drop shows cest
untreated fraction fra
Strips untre a t cd
Antibiosis Strips
No anti- undetcrmined .
biosis No antibiosis
Strips
pH *= /

3 i ? '

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 Martin« Nadal et aL, Antibiotic Propertics of Marine Algae — IIL Cymopolia b rbata 23

Figutc z

Extraction with ethyl ether

Sulphur Po*itive
present Ethyl antibioi
Infrared aJcohol
absorption Separation of
Cryitali
dark + clear
m.p,83-85°C tf*f***f*nm
' 1 ImUfriWItf
m, p, I29-135°C 1
Rcactton ' l
with NaOH Crystali SmaU
wich amibiosii
potasiium Clear fraction Dark fraction
Separation Water
t Mixturc

Basic part Acid part

No amibi iEU Positive
antibiosis

Slight Marked
antibiosis andbiosii
Distillation Oistillation

Infrared
—^ absorption ·» -. -
at 2 * 7" 3 · 4 u
Paper oiromatography

No apparenc
Butanol I6°/9-water
now dctcrmined
1
[ absorpuon ^_^^ t
in dilute 33°Cfr 59 Cfr, Disullaie
no
Solutions positive positive antibiosis
antibiosis antibiosis
rt *, Residuc
Residuc
Trap
Absorption at positive
infrarcd radiation antibiosi*
at μ Po5
Watcr Ether ^ve
soluble, soluble, anubiosy
no small Paper diro-
- antibiosis antibiosis ' matography
with butanol
No carbohydrates,
no antibiosis
on any stnp [
1
Drop on Strip
shows anti-
Distil ation
biosis if
of ck ar part untrcatcd

Carbohydrates,
9
no antibiosis
.61-63°C £.1 £,a f*
6j-o" krf £Q 71°Γ*
Ρ7-/Λ L·. TJ^f*
/* l— resi uc
" on treaced
pH «4- 5 τ- j j^ τv 4 c nT-T T
positive ^p
posidve antibiosis
antibiosis

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24 Botanica Marina, Vol. IX, Fase. 1/2

Extraction with ethyl ether

Grude ether extracts had the same appearance o£ previous extracts obtained from
other algae; they were acid in nature (pH 4.5—5.0) and had the characteristic strong
smell of sarganin complex. When concentrated in vacuo, they separated into a semisolid
greenish black-mass and a volatile oily portion o£ a strong irritating smell. Both parts
of the extracts separately and united inhibited total growth of pathogenic bacteria, The
inhibitory activity was specific f or the volatile oily portion and the inhibition obtained
from the solid mass was due to contamination.
Ethyl extractions which were the ones that contained the major portion of active
drug (see figure 2) were purified by vacuum distillation, Antimicrobial spectra was
determined and compared withJoiown antibiotic controls in the form of sensitivity
disks (see figure 3).
Outstanding activity was obtained against bacteria and fuiigi. These crude and purified
extracts gave positive for sulfur and nitrogen; negative test with FeQ3, Millon, Fehling,
ninhydrin and isotin, Its molecular structure was studied by inf rared absorption patterns

Figure 3
Antibiotic and antifungal spectra of crude extract of Cymopdia barbate
and known antibiotic controls in the form of sensitivity disks (5—30) mcg

Antibiotics Test Organisms

Crude extract Control anti- Bacilltis Myco- Escherichia. Proieus Pstudomonas Staphylo-
of Cymepeliü biotic sensitiv- subtilts bacterium co/i vttlgaris aeruginosa coccus
(25—3o)mcg. ity disks smegmatis aureus
(5—30) mcg. mm. mm* mm. mm. mm* mm.

Aureomycin 28.0 65-0 22.0 32.0 17.0 36.0

30 mcg/
Erythromycin 30.0 25.0 40.0
15 mcg.
Chloromycetin 23.0 16.0 2J.O 28.0 . 32.0
30 mcg.
Cymopolia 140 140 T4O 140 95 ?o ·
crude extract (total (total (total (total
30 mcg. plate) plate plate) plate)

.J&tndida Kheckera Schi%osac~ Sacchar- Ptniättium Torulopsis

albitans apicitlata cbaromycts ömyces cbrysogenunt utilis gillus
pombe cerwisiae niger
Mycostatin 22
(control)
IOP mcg.
Cymopolia 70 140 140 T
4° 75
crude extract (total (total (total (total (total
30 mcg. plate) plate) plate) plate) plate)

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26 Botanica Marina, Vol. IX, Fase. 1/2

(see figure 4). Inf rared analysis indicated, äs in sarganin complex, the presence of a
phenolic hydrogen bonding OH (2.7—3,3 ) and probably an acid anhydride group
*(5·5 ·
Sarganin A und B were separated from crude extracts of this alga by descending
paper chromatography and located by bioautography on trays of agar seeded with
Bacillus subtilis.
Acknowledgements
The work was carried out at the Research Center Laboratories, University of Puerto
Rico at Mayaguez. The students Jos6 R, Rodriguez Perazza and Luis Torres Vera
were aided by an Undergraduate Research Academic Program, National Science
Foundation Grant E 2/3/43—3440. All alga used in our experiments was identified by
Dr. Manuel Diaz-Piferrer, Algologist.
v

Summary
A study was made of the antibiotic properties of the marine alga Cymopolia barbata. Two activities
present in sarganin complex, broad-spectrum antibiotic isolated from Sargassum natans and Chondria
littoralisy were also spotted in purified extracts of Cymopotia barbata. It is interesting to note that this
antimicrobial substance has been found in three marine algae, which belong to different families and
species.
Zusammenfassung
Die antibiotischen Eigenschaften der marinen Alge Cymopolia barbata wurden einer Untersuchung
unterzogen- Zwei aktive Substanzen, die im Sarganin-Komplex, einem Breitspektrum-Antibiotikum
aus Sargassum natans und Chondria lUtoralis vorhanden sind, wurden ebenfalls in gereinigten Extrakten
aus Cymopolia barbata festgestellt* Es ist bemerkenswert, daß diese antimikrobielle Substanz in drei
marinen Algen gefunden wurde, die 2u unterschiedlichen Familien und Species gehören.
Literature
Burkholder, et al. Botanica Marina, Vol. II, Fase. 1/2 (1960)*
Martinen Nadal, G. Noemi Research Department. Information Bulletin 1958—59. University of
Puerto Rico.
Martinez Nadal, G. Noemi, Lux V, Rodriguex and Carmen Casillas. 1963- Sarganin and
Chonalgin, New Antibiotic Substances from Marine Algae from P. R. Antimicrobial Agents and
Chemotherapy, 1964. Pgs. 6,8—72,
Martinez Nadal, G. Noemi, Luz V. Rodriguex and Carmen Casillas. 1964. Isolation and
Characterization of Sarganin Complex-A New Broad-Spectrum Antibiotic Isolated from Marine
Algae. Antimicrobial Agents and Chemotherapy: 131—134 (1965)

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