96

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BBRRYYOOZZOOAANN ZZoooobboottrryyoonn vveerrttiicciillllaattuumm

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