Post on 05-Dec-2020
96
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INTRODUCTION
Marine organisms have formed a valuable source of new bioactive
compounds, many of which are being used in the treatment of many diseases
and serve as compounds of interest both in their natural form and as a
template for synthetic modification (Supriya and Yogesh, 2010). The biological
and chemical diversity of the marine environment represents unlimited
resource of new active substances in the field of the development of bioactive
natural products (Aneiros and Garateix, 2004). Antimicrobial peptides are
generally effective components for developing innate immunity and they are
ubiquitous in both plant and animal kingdom (Boman, 2003; Ganz, 2004).
The competitive marine environment enables the organisms to produce
potent compounds of ecological relevance (Aneiros and Garateix, 2004). The
marine compounds possess varied functions of interest to humans like
anthelminthic, anti-bacterial, anticoagulant, anti-diabetic, anti-fungal, anti-
inflammatory, anti-malarial, anti-platelet, anti-protozoal, anti-tuberculosis and
anti-viral (Mayer and Hamann, 2005). More than 20,000 natural products have
been discovered from marine organisms since 1960s (Gu-Ping et al., 2011).
The main source of bioactive metabolites are invertebrates like
sponges, jelly fish, sea anemones, corals, bryozoans, molluscs, echinoderms,
97
tunicates and crustaceans (Bhakuni and Rawat, 2005). The marine secondary
metabolites possess unique chemical novelty and could be categorised into
steroids, terpenoids, isoprenoids, nonisoprenoids, quinones, brominated
compounds, nitrogen heterocyclics and nitrogen sulphur heterocyclics. Based
on the chemical structure of the compounds isolated during 1985 to 2008, Gu-
Ping et al. (2011) reported that these natural products could be divided into
seven classes: terpenoids, steroids (including steroidal saponins), alkaloids,
ethers (including ketals), phenols (including quinones), strigolactones, and
peptides. Terpenes are the dominant among the isolated marine compounds
followed by alkaloids (Gu-Ping et al., 2011). Spongouridin and
spongothymidin are the first marine compounds isolated from the sponge
Cryptotethya crypta (Bergmann and Feeney, 1951) followed by
prostaglandins from the Caribbean gorgonian Plexaura homomalla
(Weinheimer and Spraggins, 1969).
Bryozoans are a rich and excellent source of novel and biologically
active secondary metabolites (Faulkner, 2001). Though over 8000 species
were known from the Phylum Ectoprocta (Bryozoa), the number of natural
compounds isolated from this group compared to other invertebrates is
limited. It represented only 1% of natural products reported (Blunt et al.,
2003). The most well known bryozoan compounds are the bryostatins (Pettit,
1991), flustramines (Carle and Christophersen, 1980), securamines and
securines (Rahbaek et al., 1996), tambjamines (Carte and Faulkner, 1983),
amathamides (Blackman and Matthews, 1985), amathaspiramides (Morris
and Prinsep, 1999), convolutamydines (Kamano et al., 1995) and
98
alternatamides (Lee et al., 1997). More than 15 tryptophan-derived alkaloids
have been isolated from Flustra (Holst et al., 1994a). The brominated pyrrolo
indole deformylflustrabromine was isolated from the bryozoan Flustra foliacea
from North Sea (Nicola et al., 2002). New cytotoxic oxygenated sterols were
isolated from the marine bryozoan Cryptosula pallasiana (Tian et al., 2011).
In the present study, the active metabolite from the crude diethyl ether
extract of bryozoan Zoobotryon verticillatum was isolated through bioassay
guided fractionation through chromatographic techniques and an attempt was
made to elucidate the structure of the active compound with the help of
Fourier Transform Infrared spectrometry (FTIR), Carbon and Proton Nuclear
Magnetic resonance (NMR) and Mass spectrometry (MS).
MATERIALS AND METHODS
The collection and extraction of bryozoan Zoobotryon verticillatum with
diethyl ether was carried out as described in Chapter 4. The human bacterial
pathogenic strains and the antibacterial assay detailed in Chapter 4 were
used for bioassay guided fractionation.
Partitioning
The crude diethyl ether extract was partitioned to assess the polarity
and to localize the active component (Riguera, 1997; Wright, 1998). The
concentrated crude extract was partitioned between ethyl acetate and water
and then, this water phase was subsequently partitioned against n-butanol
(Wright, 1998; Slattery et al., 1995). Then, the 3 phases (Ethyl acetate,
99
butanol and water) were collected separately; evaporated, concentrated and
antibacterial assay was carried out against 10 human bacterial pathogens in
triplicates using 100 µg/disc concentrations.
Thin Layer Chromatography (TLC)
The crude diethyl ether extract was also eluted using Thin Layer
Chromatography plates (pre-coated silica gel plates E–Merck, Germany, Art
5554 Kiesel 60F254 with 0.2 mm thickness) (Gibbons and Alexander, 1998)
with different solvent combinations to evolve solvent elution scheme for
column fractionation.
Column fractionation
The active crude diethyl ether extract was partially purified using
column chromatography by following the method of Wright (1998). The crude
extract was fractionated using normal phase Silica gel (200-400 mesh, LOBA
Chemical) column chromatography, employing a step gradient of increasing
polarity from hexane to diethyl ether to methanol (100%H; 80%H:20%DEE;
60%H:40%DEE; 40%H:60%DEE; 20%H:80%DEE; 100%DEE;
80%DEE:20%M; 60%DEE:40%M; 40%DEE:60%M; 20%DEE:80%M;
100%M). The eleven fractions eluted were collected individually, concentrated
through evaporation and the antibacterial potentialities of these fractions were
determined against human bacterial pathogens using 50 µg/disc
concentrations in triplicates. The obtained fractions were spotted on the TLC
plates to check the purity of the eluted fractions. The fractions with similar Rf
values were combined and eluted again in column chromatography.
100
The primary column pooled fractions of 20%H:80%DE, 100%DEE,
80%DEE:20%M were further fractionated using normal phase Silica gel (200-
400 mesh, LOBA Chemical) column chromatography, employing a step
gradient of increasing polarity from diethyl ether to Dichloromethane
(90%DEE:10%DCM, 80%DEE:20%DCM, 70%DEE:30%DCM,
60%DEE:40%DCM, 50%DEE:50%DCM, 40%DEE:60%DCM,
30%DEE:70%DCM, 20%DEE:80%DCM, 10%DEE:90%DCM, 100%DCM).
The ten eluted fractions were collected separately, concentrated through
evaporation and assayed for antibacterial activity using 25 µg/disc
concentrations. The obtained active fractions were spotted in TLC plates to
check the purity of the active fraction.
HPLC, IR, NMR and MS
The active column fraction 90% DEE: 10% DCM which showed single
band in TLC was subjected to Reverse phase Semi preparative HPLC (HPLC
Shimadzu Class-Vp) to further assess the purity and to collect the pure
fractions. HPLC was carried out at Indian Institute of Crop Processing
Technology, Thanjavur. Infrared spectrum (for functional group analysis) of
the active column fraction (90% DEE: 10% DCM) was recorded using FTIR
instrument. Infrared spectrum of the active compound was obtained with
Shimadzu spectrometer at Sankara Lingam Bhuvaneswari collage of
Pharmacy, Sivakasi. 1H NMR, 13C (Nuclear Magnetic Resonance (NMR)
spectra were recorded on a Brucker Advance III 500 MHz instrument at Indian
Institute of Technology, Chennai. Molecular spectrometry was used to predict
101
molecular weight of potential bioactive compound. Molecular spectrometry
was analyzed in Finnigan MAT 8230 instrument at Indian Institute of
Technology, Chennai. An attempt was made with the available data to
elucidate the molecular formula and structural characteristics of the active
compound.
RESULTS
Partitioning
The bioassay guided partitioning of the crude diethyl ether extract
indicated wide spectrum activity in ethyl acetate phase and the zone of
inhibition ranged from 6 to 10 mm (Table 6.1) The butanol phase showed
antibacterial activity against 80% of the tested bacterial pathogens with an
inhibition zone range of 1 to 7 mm. The water phase showed very low
antibacterial activity (against 20% of bacterial pathogens). The results
indicated the non polar nature of the active fraction.
Column fractionation
Among 11 primary column fractions, the fraction 6 exhibited broad
spectrum of antibacterial activity against human pathogens (Table 6.2). The
40%H:60%DEE, 20%H:80%DEE, 100%DEE, 80%DEE:20%M,
60%DEE:40%M and 20%DEE:80%M column fractions displayed wide
spectrum activity against human pathogenic bacteria followed by 60%H: 40%
DEE fraction against 90%, 40%DEE: 60%M fraction against 80%, 80%H: 20%
DEE fraction against 40% and 100%M fraction against 30% of bacterial
pathogens. No activity was observed in 100% Hexane fraction. Comparatively
102
three fractions (20%H: 80%DEE, 100%DEE and 80%DEE: 20%H) showed
prominent level of antibacterial activity. The Thin Layer Chromatography of
these three potentially active fractions exhibited same Rf value and mixture of
some other compounds was noticed. So, these three fractions were pooled
and further fractionated with secondary column fractionation.
In the secondary column fractionation, out of ten fractions, only two
fractions exhibited broad range of antibacterial activity against ten human
pathogenic bacteria at 25 µg/disc (Table 6.3). Among the two, the 90%DEE:
10% DCM fraction (Fraction ZV2-1) exhibited prominent level of antibacterial
activity with 3 to 7 mm of inhibition zone and hence, this fraction was selected
for further characterization and anticancer work. The purity of potentially
active 90%DEE: 10% DCM fraction (Fraction ZV2-1) was confirmed by Thin
Layer Chromatography with the appearance of a single band.
HPLC, IR, NMR and MS
The HPLC of the Fraction ZV2-1 exhibited only two peaks, one
prominent peak with 93.68% (RT 11.21) area percentage Peak and another
one minor peak with 6.32% (RT 15.492) area percentage peak (Fig 6.1). The
infrared spectra indicated the presence of various functional groups including
O-H, CH, C=O (Fig. 6.2). Infrared 3379 cm-1 spectrum indicated Hydroxyl
group (O-H); 2924 cm-1, 2856 cm-1peak pointed out the presence of CH group
and 1734cm-1 indicated C=O functional group. The 1H NMR and 13C NMR
spectrum are given in Figs. 6.3 and 6.4. The molecular ion peak for the potent
secondary column fraction ZV2-1 was observed at m/z 423 and quadrant
103
peak reflected the presence of Br3 group (Quadrants = 423, 425, 427, 429).
The loss of –N (CH3)2 is clearly seen at m/z 378 fragments (Fig. 6.5). The IR,
NMR and MS data indicated that the potential active column fraction ZV2-1
may be an alkaloid, 2,5,6-tribromo-N-methylgramine. The molecular weight of
the compound was 422 (DI) (M+).
Characteristic factor Result
Molecular Weight (MW) 422
Predicted molecular formula
C12H13Br3N2
Predicted molecular structure
2,5,6-tribromo-N-methylgramine
The possible structure of the potential bioactive compound alkaloid
2,5,6-tribromo-N-methylgramine may be
Br
Br N
N(CH3)2
CH3
Br
DISCUSSION
Bryozoans are a rich source of bioactive metabolites, particularly
alkaloids, and production of these compounds must have a defensive role
against predators (Prinsep et al., 2004). Though bryozoans produce
secondary metabolites for a variety of uses, including antifouling and
104
antipredation (Lopanik et al., 2004: Al-Ogily and Knight-Jones, 1977), the
bioactive compounds recorded from bryozoans were less when compared to
other marine invertebrates. And, most of the identified products from
bryozoans are alkaloids (Blunt et al., 2004).
The activity in ethyl acetate phase in partitioning showed that the active
ingredient may be of non-polar compound. The subsequent primary column
fractionation indicated activity in diethyl ether combination and higher activity
was noticed in 100% diethyl ether fraction. The activity decreased with
decreasing diethyl ether content. In the secondary column fractionation also,
the activity was localized in higher diethyl ether fractions. The polarity index of
the diethyl ether (2.8) indicated that the active component may be of medium-
polar in nature. However, the possibility of presence of multiple compounds
was there as activities, though varied, were observed in some other fractions
as well from the two column fractionations. The secondary column
fractionation revealed higher activity in the first fraction of 90%DEE: 10%DCM
(Fraction ZV2-1) and the HPLC study of which revealed one major metabolite
and another minor metabolite. The minor metabolite could not be removed in
secondary column fractionation process and a single band was viewed in
TLC.
In the present study, the potential active compound (Fraction ZV2-1)
was identified to be an alkaloid, 2,5,6-tribromo-N-methylgramine. Many
previous works reported the isolation of alkaloids from marine invertebrates,
especially bryozoans like isoquinoline alkaloid from marine bryozoan Biflustra
105
perfragilis (Blackman et al., 1993), galogenated alkaloid volutamides from
Atlantic bryozoan Amathia convoluta (Antonio et al., 1996), alkaloid 2,4,6-
tribromo-3-methoxyphenethylamine from Floridian marine bryozoan Amathia
convolute (Hirofumi et al., 2000), alkaloid securamines A-G from bryozoan
Securiflustra securifrons (Rahbaek et al., 1996; Rahbaek and Christophersen,
1997) and three mono-brominated enamide analogs of natural alkaloids from
Tasmanian marine bryozoan Amathia wilson (Moises et al., 2011). Similar
compound has been isolated from the same bryozoan species (Sato and
Fenical, 1983). Also, Konya et al. (1994) isolated 2,5,6-tribromo-1-
methylgramine from Zoobotryon pellucidum.
The present observation of the marine bryozoan compound exhibiting
promising antibacterial and anticancer activities coincided with that of
antifouling, antibiotic and antiviral and potent muscle relaxant activities of
mixtures of extracts from the bryozoan family Flustridae including
Securiflustra securifrons (Christophersen, 1985), antibacterial activity of
bromoalkaloids from bryozoan Flustra foliacea (Maurice et al.,1986),
cytotoxic, antibacterial, antifungal and antiviral activities of β-carboline alkaloid
from bryozoan Cribricellina cribreria (Prinsep et al., 1991) and antimicrobial
activity of indole alkaloids from the bryozoan Flustra foliaceae (Holst et
al.,1994a), antitumor and antibacterial activities of Pterocellins A and B from
the New Zealand marine bryozoan Pterocella vesiculosa (Yao et al., 2003)
and Amathaspiramide A from the bryozoan Amathia wilsoni (Prinsep et al.,
2004). Also, antibacterial activity against several gram positive bacteria
including Staphylococcus aureus, Bacillus subtilis and Enterococcus faecium
106
by alternamide, a bryozoan compound (Lee et al., 1997) and antimicrobial
activity against Escherichia coli, Enterobacter cloacae, Serratia marcescens
and Pseudomonas aeruginosa by Flustramine D from the bryozoan Flustra
foliacea (Laycock et al.,1986) coincided with the present observation of
activity of Zoobotryon verticillatum compound against Staphylococcus aureus
and B. subtilis.
107
Table 6.1: Antibacterial activity of partitioned diethyl ether crude
extract of Zoobotryon verticillatum
Human pathogenic bacteria
Partitioned extracts (100 µg/disc)
Ethyl acetate
Butanol Water
Zone of inhibition in mm±SD
Staphylococcus aureus 8±0.58 7±0.58 -
Micrococcus luteus 10±1.53 5±0.58 -
Streptococcus pyogenes 7±0.58 1±0 3±0
Bacillus subtilis 10±0.58 - -
Enterobacter faecalis 6±0.58 - -
Klebsiella pneumoniae 7±0 2±0.58 1±0
Escherichia coli 8±0.58 2±0 -
Shigella sonnei 8±0.58 1±0 -
Salmonella typhimurium 6±0.58 3±0.58 -
Vibrio cholerae 7±0.58 2±0 -
(- no activity
10
8
Tab
le 6
.2:
An
tib
ac
teri
al acti
vit
y o
f p
rim
ary
co
lum
n p
uri
fied
fra
cti
on
s d
ieth
yl eth
er
extr
act
of
Zo
ob
otr
yo
n v
ert
icilla
tum
(H
-He
xan
e,
DE
E-D
ieth
yl eth
er,
M-M
eth
ano
l; -
no
act
ivity)
Hu
ma
n b
ac
teri
al
pa
tho
gen
s
Pri
mary
Co
lum
n f
rac
tio
ns
(5
0 µ
g/d
isc)
100%H
80%H: 20%DEE
60%H: 40%DEE
40%H: 60%DEE
20%H: 80%DEE
100%DEE
80%DEE: 20%M
60%DEE: 40%M
40%DEE: 60%M
20%DEE: 80%M
100%M
Zo
ne o
f in
hib
itio
n (
mm
±S
D)
Sta
phylo
co
ccu
s a
ure
us
- -
- 2±0
.58
5
±0
8±0
.58
7
±0
4
±0
1±0
3
±0
.58
-
Mic
rococcus lute
us
- -
2±0
9±0
.58
1
0±1
14
±1
1
2±0
.58
7±0
.58
2
±0.5
8
3±0
-
Str
ep
tococcus p
yog
ene
s
- -
1±0
4±0
.58
4
±0
13
±0.5
8
9±0.5
8
5±0
3±0
1
±0
-
Ba
cill
us
su
btil
is
- 1±0
3
±0
7±0
.58
8
±0
.58
14
±0.5
8
11±1
7±0
.58
2
±0.5
8
4±0
.58
1
±0.5
8
En
tero
ba
cte
r fa
ecalis
-
1±0.5
8
2±0
.58
5±0
.58
6
±0
8
±0
6
±0.5
8
6±0
3±0
2
±0
-
Kle
bsie
lla p
neu
mo
nia
e
- -
1±0
.58
5±0
.58
4
±0
.58
10
±0.5
8
8±0.5
8
6±0
.58
3
±0.5
8
4±0
-
Esche
richia
coli
- 1±0.5
8
3±0
.58
4±0
.58
5
±0
.58
10
±0.5
8
7±0.5
8
4±0
.58
2±0
3
±0
.58
1
±0.5
8
Sh
ige
lla s
onn
ei
- -
1±0
.58
3±0
5
±0
.58
9
±0
5
±0.5
8
2±0
-
1±0
-
Sa
lmo
nella
typ
him
uri
um
-
1±0.5
8
2±0
2±0
.58
4
±0
.58
8±0
.58
6
±0
5
±0
-
3±0
1
±0.5
8
Vib
rio
cho
lera
e
- -
2±0
.58
3±0
.58
3
±0
7
±0
7
±0.5
8
4±0
.58
2
±0.5
8
3±0
.58
-
10
9
Ta
ble
6.3
: A
nti
bacte
rial ac
tivit
y o
f s
ec
on
dary
co
lum
n p
uri
fie
d f
racti
on
s o
f Z
oo
bo
try
on
ve
rtic
illa
tum
Hu
ma
n b
ac
teri
al
path
og
en
s
Se
co
nd
ary
co
lum
n f
rac
tio
ns
(2
5 µ
g/d
isc)
90%DEE:10% DCM
80%DEE:20% DCM
70%DEE:30% DCM
60%DEE:40% DCM
50%DEE:50% DCM
40%DEE:60% DCM
30%DEE:70% DCM
20%DEE:80% DCM
10%DEE:90% DCM
100% DCM
Zo
ne o
f in
hib
itio
n i
n (
mm
±S
D)
Sta
ph
ylo
co
ccu
s a
ure
us
7±0
3
±0
3
±0
.58
2
±0
.58
2
±0
-
- 2±0
.58
-
-
Mic
roco
ccu
s lu
teus
4±0
.58
2
±0
2
±0
.58
1
±0
1
±0
2
±0
-
- -
-
Str
epto
co
ccu
s p
yo
ge
nes
4±0
1
±0
-
- -
- -
- -
-
Bacill
us s
ub
tilis
6
±0
.58
2
±0
.58
1
±0
1
±0
1
±0
.58
-
- 1
±0
-
-
Ente
rob
acte
r fa
ecalis
5
±0
2
±0
1
±0
.58
1
±0
.58
1
±0
-
- -
- -
Kle
bsie
lla p
ne
um
on
iae
7±0
.58
4
±0
.58
2
±0
.58
2
±0
2
±0
.58
2
±0
.58
2
±0
1
±0
-
-
Esch
eri
chia
coli
3±0
.58
1
±0
1
±0
1
±0
.58
-
- 2
±0
.58
1
±0
-
-
Shig
ella
so
nne
i 3
±0
2
±0
1
±0
-
- -
- -
- -
Salm
on
ella
typ
him
uri
um
4
±0
2
±0
2
±0
.58
2
±0
2
±0
.58
-
- -
- -
Vib
rio
ch
ole
rae
5±0
.58
3
±0
.58
2
±0
-
- 1
±0
-
- 1
±0
-
(DE
E-D
ieth
yl eth
er,
DC
M-D
ich
loro
meth
an
e;
- n
o a
ctivity)
110
Fig. 6.1: High Performance Liquid Chromatography of fraction ZV2-1 of Zoobotryon verticillatum
111
Fig. 6.2: FTIR Spectrum of fraction ZV2-1 of Zoobotryon verticillatum
112
Fig. 6.3: Carbon NMR of fraction ZV2-1 of Zoobotryon verticillatum
113
Fig. 6.4: Proton NMR of fraction ZV2-1 of Zoobotryon verticillatum
114
Fig. 6.5: Mass spectrometry of fraction ZV2-1 of Zoobotryon verticillatum