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Anti-inflammatory and immunomodulatory activity of isolated

compounds from marine sponge Cliona lobata

Conference Paper · February 2015

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Anti-inflammatory and immunomodulatory
activity of isolated compounds from marine
sponge Cliona lobata

AMAN K DUBE

1 13-02-2015
NCRAAS
Department of Applied Zoology, Mangalore University
Guide: Dr Maushmi S Kumar
 Introduction – Marine Sponges Likely
2
Paved the Way For All Life on Earth
 They are animals belonging to the phyllum Porifera, having porous bodies

 They are known to posses various pharmacological activities such as

immunomodulatory, anti-inflammatory, antibacterial and antiviral.
3 Objectives of the work

 Extraction of active agents from marine sponge Cliona lobata and isolation
of compound by column chromatography
 To characterize the isolated compounds using UV, FT-IR and LC MS.
 To study the anti-inflammatory and immunomodulatory activity of Cliona
lobata

Halichondria glabrata Spirastrella pachyspira Cliona lobata

4 Characterization of Cliona lobata
 The sponge was characterized by observing the nature of the
spicules which are made of silica and serve as sponge skeleton
 The composition, size, and shape of spicules is one of the largest
determining factors in sponge taxonomy
Spicule Length Width Length/ Width
No. (µm) (µm) ratio
1 65.2 2.5
26.08
2 56.6 2.3
24.60
3 66.7 3.2
20.84
4 70.6 2.9
24.34
5 57.7 2.7
21.37
6 68.3 3.1
22.03
7 59.8 2.7
22.14
8 68.9 3.4
Spicule of Cliona lobata observed 20.26
9 66.4 3.2
under 10x magnification 20.75
10 40.3 1.9
21.21
 Preparation of Sponge Extract
5
& TLC Optimization
 500 g of C. lobata sponge was macerated with 6L of methanol
 The extract was evaporated to dryness using at rota evaporator at 55° C
at 500 mm Hg pressure
 Extract obtained = 36. 45 g

Distance Rf • Mobile phase:

Ethyl acetate: Butanol:
Spot (cm) Value Chloroform: Ethanol in
ratio 6:2:1:1
Solvent front 4.8 • Visualization reagent:
Spot 1 4.5 0.93 p- Anisladehyde
• Detection of steroids:
Spot 2 3.1 0.74 Steroids turn violet in
Spot 3 2.6 0.60 colour after heating at
105° C for 5 minutes
Spot 4 1.4 0.15
Spot 5 0.9 0.09
Spot 6 0.4 0.06
6
Isolation of compounds using
column chromatography
 Glass column (18×300 mm cap) was filled with a slurry of silica gel
(230-400 mesh)
 2g extract dissolved in ethyl acetate was loaded on the column and
fractions of 1ml were collected in eppendorf
 This was followed by elution with butanol, chloroform and ethanol,
till the complete elution of compounds
 Four compounds were identified by TLC
Compound Rf Colour Weight after Solvent in
value drying which
(mg) obtained
1 0.92 Brown 1569 Ethyl acetate
2 0.76 Green 116. Ethanol
3 0.60 Green 213 Butanol

4 1.54 Orange 120 chloroform

 7
Characterization of Isolated compounds
by Infrared Spectroscopy
Compound 1 Compound 2

Peaks Functional Groups Peaks Functional Groups

3696.89 O-H stretching (Alcohols & 2846.68 Aldehydes C-H
Phenols) 2917.76 Hydrocarbons C-H stretching
2357.32 C≡C Stretching (Alkynes)
1637.44 C=O stretching
1390.18 O-C=O stretching
8
Characterization of Isolated compounds
by Infrared Spectroscopy

Compound 3 Compound 4

Peaks Functional Groups Peaks Functional Groups

2846.45 C-H stretching (Aldehydes) 2351.71 C≡N Stretching
2917.29 C- H stretching 1280.66 O-C=O stretching
(Hydrocarbons)
9 Anti inflammatory activity of
Isolated compounds
The in vitro anti- inflammatory activity of the methanolic extract
of Cliona lobata was evaluated usnig the following test :

 Inhibition of albumin denaturation

 Membrane stabilization using rat RBCs

The in vivo anti-inflammatory activity of the methanolic extract

of Cliona lobata was evaluated by studying

 Carrageenan induced rat paw edema

10 In vitro - Inhibition of albumin
denaturation
 The reaction mixture consisting of bovine serum albumin at a
concentration of 1mM in phosphate buffered saline (pH 6.4) was incubated
with varying concentration of methanolic extract of marine sponge (10-
100 μg/ mL ) and isolated compounds (25, 50 & 100 μg/ mL ) at 37°C for
15 min in a BOD incubator
 The reaction was terminated by heating at 70°C for min. The tubes were
allowed to cool. Absorbance was read at 660 nm using a Perkin Elmer UV
Spectrophotometer
 Diclofenac sodium at concentrations ranging from 10- 100 μg/ mL, served
as the standard keeping the same incubation conditions
 Similar volume of distilled water instead of the drug served as the control.
 The percent inhibition of protein denaturation was calculated from the
following formula:
% Inhibition = 100 ˣ [ Vt/ Vc - 1]
Where, Vt - Absorbance of test, Vc - Absorbance of control
 11 Inhibition of albumin denaturation

120

albumin denaturation
Methanolic extract of 100
Diclofenac sodium marine sponge R² = 0.977

% Inhibition of
80
60
Absor % Absor 40
Concentra bance Concentra bance 20
tion at 660 Inhibiti tion at 660 % 0
0.0 50.0 100.0 150.0
(µg/ml) nm on (µg/ml) nm Inhibition
10.000 0.201 0.500 10.000 0.198 -1.000 Concentration of Diclofenac sodium
(µg/ml)
20.000 0.225 12.500 20.000 0.200 0.000
% Inhibition correlation with concentration of drug
30.000 0.276 38.000 30.000 0.276 38.000

% Inhibition of albumin
120
IC50 40.000 0.298 49.000 40.000 0.299 49.500 100 R² = 0.9572

denaturation
50.000 0.309 54.500 50.000 0.315 57.500 80
60
60.000 0.328 64.000 60.000 0.328 64.000 40
70.000 0.354 77.000 70.000 0.354 77.000 20
0
80.000 0.367 83.500 80.000 0.399 99.500 0.0 50.0 100.0 150.0 200.0
Concentration of Methanolic extract of
90.000 0.397 98.500 90.000 0.435 117.500
marine sponge (µg/ml)
100.000 0.409 104.500 100.000 0.508 154.000
12 In vitro - Membrane stabilization activity
 The membrane stabilization activity was studied using a 10 % rat RBCs
suspension
 Methanolic extract of marine sponge ranging from concentration of 10-100
μg/mL or isolated compounds (25, 50 & 100 μg/ mL ) were added to the
suspension of RBCs and heated to 56°C for 30 minutes in a water bath
 The reaction mixture was cooled under running tap water and centrifuged at
5000 rpm for 5 minutes. The supernatant was collected and absorbance was
read at 540 nm.
 Diclofenac sodium at concentrations ranging from 10- 100 μg/ mL, served
as the standard keeping the same incubation conditions.
 Similar volume of distilled water instead of the drug served as the control.
 The percent inhibition of heat induced haemolysis was calculated from the
following formula:
% Inhibition = 100 ˣ [ (Vc - Vt)/ Vc ]
Where, Vt - Absorbance of test, Vc - Absorbance of control
13 Membrane stabilization activity
Methanolic extract of
Diclofenac sodium marine sponge

Absor %
Concentra bance Concentra Absorb %
tion at 560 Inhibit tion ance at Inhibit
(µg/ml) nm ion (µg/ml) 560 nm ion

10.000 0.623 32.503 10.000 0.648 29.794

20.000 0.597 35.320 20.000 0.630 31.744

30.000 0.548 40.628 30.000 0.528 42.795 % Inhibition correlation with concentration of drug
40.000 0.501 45.720 40.000 0.507 45.070

50.000 0.417 54.821 50.000 0.425 53.954

60.000 0.351 61.972 60.000 0.368 60.130

70.000 0.287 68.906 70.000 0.312 66.197

80.000 0.223 75.840 80.000 0.248 73.131

90.000 0.198 78.548 90.000 0.212 77.031

100.000 0.125 86.457 100.000 0.201 78.223

 In vivo - Carrageenan induced rat
14
paw edema
 Rat paw edema was measured using a Plethysmometer after 1,2,3,4 and 5
hours of challenge.
Animal treatment:
 Group I: Negative control- 0.1 ml saline sub-plantar
 Group II: Positive control- 0.1 ml 0.5 % carrageenan sub-plantar
 Group III: Standard:- 0.1 ml 0.5 % carrageenan sub-plantar + Diclofenac (25
mg/kg p.o. )
 Group IV: MECL- 0.1 ml 0.5 % carrageenan sub-plantar + methanolic extract
of Cliona lobata (200 mg/kg p.o. )
 In vivo - Immunomodulatory
15
activity

On the 7th day, blood was collected from the retro-orbital plexuses and
collected in eppendorf tubes without any anticoagulant. The serum was
collected and stored at -80°C for further use. The foot thickness was
again measured on this day.
 Immunomodulatory activity – Delayed
16
type hypersensitivity
 Immunomodulatory activity-Humoral type
17
antibody titre
18 Conclusion & Future scope

 Four compounds were separated and these compounds showed the

presence of steroid moiety.
 Thus Cliona lobata possess both anti-inflammatory and
immunomodulatory activity.
 In the future, marine ecosystems could represent an increasingly
important source of medical treatments, nutritional supplements,
pesticides, cosmetics, and other commercial products.
 Drugs from the ocean are without question one of the most promising
new directions of marine science today such as eribulin which is an
anti cancer drug from sponge
 19 References
 Newman D., Cragg G. Marine natural products and related compounds in
clinical and advanced preclinical trials. J. Nat. Prod. 2004; 67:1216–1238
 Wu M.L., Ho Y.C., Lin C.Y., Yet S.F. Heme oxygenase-1 in inflammation and
cardiovascular disease. Am. J. Cardiovasc. Dis. 2011; 1:150–158.
 Arya V. and Gupta V.K. A review on marine immunomodulatory. Int. J. of
Pharm. & Life Sci.2011; 2(5): 751-758.
 Alejandro M.S. Mayera, Mark T. Hamannb. Marine pharmacology in 2001–
2002: Marine compounds with anthelmintic, antibacterial, anticoagulant,
antidiabetic, antifungal, anti-inflammatory, antimalarial, antiplatelet,
antiprotozoal, antituberculosis, and antiviral activities; affecting the
cardiovascular, immune and nervous systems and other miscellaneous
mechanisms of action. Comparative Biochemistry and Physiology. 2005, 140:
265 – 286.
 Burkhard Haefner. Drugs from the deep: marine natural products as drug
candidates. DDT. 2003, 8: 536- 544 .
 20

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