SUPPLEMENTARY MATERIAL
α-glucosidase inhibitory activity of marine sponges collected in the Mauritius Waters
Avin Ramanjoolooa†, Thierry Cresteilb†, Cindy Lebrassea†, Girish Beedesseea, Preeti Oogaraha,
Rob W.M. van Soestc and Daniel E.P. Mariea*
a
Mauritius Oceanography Institute (MOI), France Centre, Victoria Avenue, Quatre-Bornes,
Mauritius
b
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Centre de Recherche de Gif,
Gif sur Yvette, France
c
Netherlands Centre for Biodiversity Naturalis, Leiden, The Netherlands
†: Contributed equally to this work.
*Corresponding author Tel + 230 4274434, fax +230 4274433
Email: depmarie@moi.intnet.mu
Abstract
This report describes the use of α-glucosidase to evaluate the anti-diabetic potential
of extracts from marine sponges collected in the Mauritius Waters. Initial screening
at 1.0 mg/mL of 141 extracts obtained from 47 sponge species revealed 10 extracts
with inhibitory activity greater than 85 %. Seven of the ten extracts were further
tested at 0.1 mg/mL and 0.01 mg/mL and only the methanol extract of two sponges
namely Acanthostylotella sp (ASSM) and Echinodictyum pykei (EPM) showed
inhibition activity greater than 60 % at 0.1 mg/mL with an IC50 value of 0.16 ± 0.02
mg/mL and 0.04 ± 0.01 mg/mL respectively while being inactive at 0.01 mg/mL.
Keywords: α-glucosidase; postprandial hyperglycemia; Mauritius; marine sponges
Experimental
Chemicals
Hexane, ethyl acetate, butanol and methanol were from SFDC Ltd. α-D glucopyranoside was
from Carbosynt. Saccharomyces cerevisiae α-glucosidase was from Sigma.
Sponge collection
The sponge specimens were collected through SCUBA diving at a depth varying from 5 to 40 m
around Mauritius. Samples were photographed in situ for better species characterization and
identification. Voucher samples of each species were deposited with the Zoological Museum of
University of Amsterdam, Netherlands, and were identified with the help of Dr. Rob Van Soest.
The physical appearance and the voucher number of the sponge species examined in this study
are described in table S4.
Preparation of extracts
Freshly collected marine sponges were set free of any debris, cut into small pieces, weight, and
freeze-dried. Two batches of specimens were used. The first batch of dried sponges (cf. Table
S1; Entries 1-20, with mass ranging between 49-400 g) was macerated with MeOH/CH2Cl2 (1:
1) for 24 hour. After maceration, the solution was decanted, filtered and evaporated to dryness on
a rotatory vacuum evaporator set at a maximum temperature of 40°. This process was repeated
several times until solution appeared almost colourless. This constituted the crude extract, which
was dissolved in distilled water and subsequently partitioned with hexane followed by ethyl
acetate (AcOEt), and ended with butanol (BuOH) to afford non-polar, semi-polar, and polar
extracts, respectively. The second batch (cf. in Table S1; Entries 21-47, with mass ranging
between 0.2-136 g) of dried/wet sponges was macerated and partitioned in the same way as
described for the first batch except the final water extract was evaporated first and then dissolved
in methanol to afford the polar extract (MeOH extract) instead of doing partitioning with
butanol. Inorganic salts present in both BuOH and MeOH extracts were removed by redissolving the extract in MeOH, followed by filtration. Thus, three extracts namely hexane, ethyl
acetate and butanol/methanol were obtained from each sponge and were subsequently used for
investigating α-glucosidase activity.
Biological evaluation
α-Glucosidase activity was assayed with 8 mM solution of 4-methylumbelliferyl α-Dglucopyranoside and Saccharomyces cerevisiae α-glucosidase in 100 mM NaHPO4 buffer, pH
6.8 at 30 °C in a 384 well microplate format based on the methods by Ouairy et al., (2013). 4Methylumbelliferyl-α-D-glucopyranoside is a substrate for α-glucosidase and becomes
fluorogenic by cleavage of the free 4-methylumbelliferyl moiety. Acarbose (10 mM final) was
used as reference inhibitor. Fluorescence was monitored (excitation 365 nm, emission 445 nm)
over a 20 min period. For the initial screening, compounds were added at a concentration of 1
mg/mL. Potent extracts were further screened at 0.1 mg and 0.01 mg/mL in duplicate.
Preliminary chemical screening of bioactive extracts
The potent extracts were analyzed for their chemical composition based on methods by Sarker et
al., (2006) and Houghton & Raman., (1998). The extracts were dissolved in methanol and were
placed on thin layer chromatographic (TLC) plates. The plates were eluted with the solvent
system of dichloromethane and methanol of ratio 7: 3 (v/v). After elution, the plates were dried
and sprayed with each of the locating agents:
(i) Anisaldehyde reagent; p-anisaldehyde (0.5 g) was mixed with glacial acetic acid,
sulphuric acid and methanol in the ratio (5:10:85);
(ii) Ammonium molybdate solution; ammonium molybdate (VI) (5.0 g) was dissolved in
concentrated sulphuric acid (50 mL);
(iii) Phosphomolybdic acid solution; phosphomolybdic acid (5.0 g) was dissolved in 96%
ethanol (100 ml).
(iv) Dragendorff reagent; Solution A: bismuth (III) nitrate (0.42) was dissolved in glacial
acetic acid (5 mL) and distilled water (20 mL). Solution B: potassium iodide (8.0 g) was
dissolved in distilled water (20 mL). Equal parts of solution A and B were mixed to make a
stock solution, which was stored in an amber-colored bottle for longer period time. The
spraying solution constituted stock solution (1 mL), glacial acetic acid (2 mL) and distilled
water (10 mL);
(v) Ninhydrin solution; ninhydrin (0.5 g) was dissolved in acetone (100 mL);
(vi) Aluminium chloride solution; aluminium chloride (1.0 g) was dissolved in 95 % ethanol
(100 mL) and
(vii) 2, 4-dinitrophenyl hydrazine solution; 2, 4-dinitrophenyl hydrazine (0.2 g) was
dissolved in 2 N HCl (50 mL)
Phosphomolybdic acid was used to detect terpenes (blue color), anisaldehyde detects terpenoids
(purple, blue or red colors) and some other compounds, such as lignans, sugars and flavonoids,
ammonium molybdate detects diterpenes (blue color), dragendroff detects alkaloids (dark orange
to red coloration), ninhydrin detects amino acids/amines (red colors), aluminium chloride detects
flavonoids (yellow fluorescence in UV at 366 nm), 2, 4-dinitrophenyl hydrazine detects
aldehydes and ketones with a yellow to red coloration.
Statistical analyses
The enzyme activity of the seven extracts was conducted in duplicate, and results are expressed
as mean ± standard deviation (SD).
Table S1: Inhibitory activity (%) of sponge extracts on the enzyme α-glucosidase at 1.0 mg/mL.
Entry
Sponge specimens
Extracts
Hexane
EtOAc
BuOH/MeOH
1
Petrosia tuberosa
16.1
39.8
15.0
2
Dragmacidon coccineum
27.0
34.5
9.5
3
Dragmacidon durrissima
64.7
30.4
7.9
4
Jaspis diastra
43.1
60.4
47.1
5
Plakortis nigra
9.5
17.0
5.3
6
Petrosia mauritiana
9.7
19.0
17.3
7
Dysidea aff. cinerea
30.6
39.6
26.8
8
Iotrochota purpurea
80.6
86.4
60.7
9
Rhabdastrella globostellata
18.9
85.6
35.9
10
Biemna tubulosa
-6.9
23.9
-9.5
11
Acanthella pulcherrima
9.9
41.7
-5.0
12
Axinella donnani
48.8
79.0
97.9
13
Acanthella cavernosa
23.6
10.9
6.4
14
Pericharax heteroraphis
8.3
67.3
33.6
15
Acanthostylotella sp
70.0
94.7
97.4
16
Liosina paradoxa
51.3
58.8
47.5
17
Haliclona sp.
32.8
40.1
19.7
18
Dactylospongia sp.
86.8
87.0
21.1
19
Spheciospongia sp.
12.7
27.4
7.1
20
Stylissa sp.
19.9
38.8
12.9
21
Cinachyrella sp.
69.2
75.0
-2.1
22
Pachychalina sp.
-3.5
-0.9
-3.3
23
Echinodictyumpykei
71.6
52.3
99.7
24
Pseudosuberites sp.
6.5
12.3
-1.9
25
Spirastrella sp.
23.1
26.6
20.0
26
Mycale tenuispiculata
52.7
53.2
51.3
27
Suberites sp.
25.7
26.8
22.9
28
Smenospongia sp.
30.5
38.6
18.5
29
Spheciospongia vagabunda
17.1
25.6
19.4
30
Myrmekioderma granulatum
82.0
61.0
17.7
31
Agelas marmarica
15.9
57.3
58.4
32
Psammoclema sp.
10.5
11.0
6.4
33
Amphimedon sp.
24.0
11.5
19.1
34
Biemna trirhaphis
5.1
9.6
-2.8
35
Phyllospongia papyracea
7.8
28.8
1.2
36
Paratetilla sp.
12.9
31.6
-8.1
37
Neopetrosia exigua
42.8
49.7
43.4
38
Hyrtios sp
48.5
52.4
23.3
39
Gelliodes incrustans
30.2
85.2
26.7
40
Acantella sp.
23.3
49.7
-13.8
41
Cinachyrella australiensis
11.0
79.7
-0.1
42
Biemna tubulata
-4.0
39.7
-9.4
43
Epipolasis suluensis
43.5
67.5
-0.3
44
Stylissa carteri
13.3
35.4
52.1
45
Amphimedon navalis pulitzer
16.5
78.2
-23.2
46
Neopetrosia tuberosa
55.9
88.3
49.9
47
Callyspongia sp
41.8
16.0
17.5
EtOAc: Ethyl acetate ;BuOH: Butanol; MeOH: methanol
Acarbose (10 mM) showed inhibitory activity of 78 %.
Table S2: Enzyme activity of seven extracts tested at 1.0, 0.1 and 0.01 mg/mL
Sponge specimens
Extracts
% Enzyme activity
Concentration (mg/mL)
EtOAc
1
Axinella donnani
2
Acanthostylotella sp
BuOH/MeOH
1.0
0.1
0.01
-
ADB
1.2 ± 0.4
60.1 ± 2.2
90.0 ± 0.6
ASSE
-
15.7 ± 3.8
79.4 ± 3.0
72.8 ± 11.5
ASSM
1.3 ± 2.1
37.1 ± 5.3
86.9 ± 2.6
3
Echinodictyum pykei
-
EPM
- 2.9 ± 0.3
21.8 ± 0.9
72.0 ± 0.1
4
Iotrochota purpurea
ICPE
-
20.3 ± 0.4
93.0 ± 3.8
107.5 ± 0.3
5
Neopetrosia tuberosa
NETE
-
22.2 ± 3.8
79.4 ± 1.6
123.2 ± 4.7
6
Rhabdastrella globostellata
RGE
-
12.6 ± 0.5
55.7 ± 1.7
92.2 ± 1.3
EtOAc: Ethyl acetate ;BuOH: Butanol; MeOH: methanol
Table S3: Preliminary chemical screening of theseven extracts
Spray reagent
Compounds
Observation
test
Anisaldehyde
Phosphomolydic
Extracts
RGE
ADB
ASSM
ASSE
EPM
ICPE
NETE
Terpenoids
Purple, blue or red
+
+
+
+
+
+
+
Terpenes
Blue spots on a
+
+
+
+
+
+
+
acid
yellow
background
Ammonium
Diterpenes
Blue color
+
+
+
+
+
+
+
Alkaloids
Dark orange to
-
+
+
+
+
-
+
-
+
+
+
+
+
-
-
+
+
+
-
+
+
-
-
-
-
-
+
-
molybdate
Dragendorff
red coloration
Ninhydrin
Aluminium
Amino acids/
Purple/red color
amines
and yellow
Flavonoids
Yellow
chloride
fluorescence in
UV light (360nm)
2, 4-dinitrophenyl
Aldehydes/
Yellow to red
hydrazine
Ketones
coloration
+: presence, -: absence
Table S4: Physical appearance and voucher specimen number of sponge species examined in this study
Petrosia tuberosa
Voucher
specimen number
ZAMPOR18315
Family
Petrosiidae
2
Dragmacidon coccineum
ZAMPOR22201
Axinellidae
3
Dragmacidon durrissima
ZAMPOR18314
Axinellidae
4
Jaspis diastra
ZAMPOR21796
Coppatiidae
5
Plakortis nigra
6
Petrosia mauritiana
7
Dysidea aff. cinerea
ZAMPOR18309
Dysideidae
8
Iotrochota purpurea
ZAMPOR22177
Mycalidae
9
Rhabdastrella globostellata
ZMAPOR21718
Geodiidae
10
Biemna tubulosa
ZAMPOR18313
Desmacellidae
11
Acanthella pulcherrima
ZAMPOR22189
Axinellidae
12
Axinella donnani
ZAMPOR20940
Axinellidae
13
Acanthella cavernosa
ZAMPOR18311
Axinellidae
14
Pericharax heteroraphis
ZAMPOR21792
Leucettidae
15
Acanthostylotella sp
ZAMPOR19134
Raspailiidae
Entry
1
Sponge specimens
ZAMPOR18310
ZAMPOR18307
Plakinidae
Petrosiidae
Physical appearance
Pale brown; large, compact structure without
defined shape, visible oscules.
Bright orange; thin sheet-like coating of the
substrate with rough surface
Bright orange; thick sheet-like coating of the
substrate with rough surface
Orange; very soft texture with prominent oscules
Black; very soft texture with small occules on the
smooth surface
Green; distinct tubes with oscules at the apex
with rough surface
Dark green; soft and flexible texture with many
flattened lobes projected from base
Black; thin, string like sponge with spiny surface
Yellow; massive, bitter gourd shape with
prominent oscules
Yellowish brown; large, compact specimen
without a definite shape with smooth surface.
Reddish orange; soft texture with large oscules,
thickly encrusting
Bright orange; hard texture firmly attached to
substrate with very small oscules
Orange; massive and spherical with soft texture
Greenish yellow; solid free-standing sponge with
a big opening at one end
Greenish yellow; soft, thickly encrusting with
randomly distributed oscules
16
Liosina paradoxa
ZAMPOR21719
Dictyonellidae
Silvery grey; body is fleshy, resilient and tough
17
Haliclona sp.
ZAMPOR22192
Chalinidae
18
Dactylospongia sp.
ZAMPOR22191
Thorectidae
Light brown; multiple clumps of hollow
cylinders, smooth and delicate.
Grey; thick, fleshly encrusting sponge
19
Spheciospongia sp.
ZAMPOR22199
Spirastrellidae
Dark brown; medium size smooth sponge
20
Stylissa sp.
ZAMPOR22185
Dictyonellidae
ZAMPOR22176
Tetillidae
ZAMPOR22184
Niphatidae
Red; soft thickly encrusting with prominent
oscules raise on short stalks
Reddish brown; small, spherical and hard texture
with no visible oscules
Reddish white, large size smooth sponge
ZAMPOR22180
Raspailiidae
ZAMPOR22181
Suberitidae
ZAMPOR22175
Spirastrellidae
ZAMPOR22182
Mycalidae
ZAMPOR22179
Suberitidae
ZAMPOR22199
Thorectidae
ZAMPOR22174
Spirastrellidae
ZAMPOR22020
Halichondriidae
ZAMPOR22204
Agelasidae
21
22
23
24
25
26
27
28
29
30
31
32
33
Cinachyrella sp
Pachychalina sp
Echinodictyum pykei
Pseudosuberites sp
Spirastrella sp
Mycale tenuispiculata
Suberites sp
Smenospongia sp
Spheciospongia vagabunda
Myrmekioderma granulatum
Agelas marmarica
Psammoclema sp
Amphimedon sp
ZAMPOR22200
ZAMPOR22195
Dark purple; rough and spiky projection with no
visible oscules
Dark brown; large and spherical with soft texture
Brown; hard texture without definite shape with
no visible oscules
Brown; body tinge with reddish surface; large
with no visible oscules
Brown; tinge with blue surface; large and hard
texture with no visible oscules
Yellow green; large with visible oscules
Milky green; smooth sponge with large
prominent oscules
Pale brown; smooth with no visible oscules
Orange; massive and smooth with prominent
oscules
Phoriospongiidae Dark brown; small and soft with no visible
oscules
Niphatidae
Creamy body with reddish surface; large and hard
texture with visible oscules
34
Biemna trirhaphis
ZAMPOR22194
Desmacellidae
Greenish-brown; soft with no visible oscules
35
Phyllospongia papyracea*
ZAMPOR22197
Spongiidae
Brown; flat and large with no visible oscules
36
Paratetilla sp
ZAMPOR21806
Tetillidae
Dark brown; hard texture with no visible oscules
ZAMPOR21776
Petrosiidae
ZAMPOR21785
Thorectidae
39
Gelliodes incrustans
ZAMPOR21804
Chalinidae
Dark brown; smooth and velvety texture with
small visible oscules
Brown and purple; rough surface and irregular
shape
Purple; very soft texture with no visible oscules
40
Acantella sp
ZAMPOR21794
Axinellidae
Bright orange; spiky shape and no visible oscules
ZAMPOR21790
Tetillidae
ZAMPOR21781
Desmacellidae
ZAMPOR21800
Halichondriidae
ZAMPOR22188
Dictyonellidae
37
38
Neopetrosia exigua
Hyrtios sp
45
Amphimedon navalis pulitzer
ZAMPOR18323
Niphatidae
Creamy brown; smooth with a spherical shape
with no visible oscules
Dark brown; large and soft texture with visible
oscules
Creamy brown; large and hard texture with no
visible oscules
Reddish brown; hard texture with no visible
oscules
Sky blue; smooth with visible oscules
46
Neopetrosia tuberosa
ZAMPOR22178
Petrosiidae
Dark brown; large with visible oscules
47
Callyspongia sp
ZAMPOR21734
Callyspongiidae
Light brown; tube sponge with visible oscules
41
42
43
44
Cinachyrella australiensis
Biemna tubulata
Epipolasis suluensis
Stylissa carteri
*Collected from St Brandon
Physical appearance for entries 1-20 has been reproduced and edited from Beedessee et al. 2010
References
Beedessee G, Ramanjooloo A, Aubert G, Eloy L, Surnam-Boodhun R, Van Soest RWM, Cresteil
T, Marie DEP. 2012. Cytotoxic activities of hexane, ethyl acetate and butanol extracts of
marine sponges from Mauritian Waters on human cancer cell lines. Environmental Toxicol.
Pharmacol. 34(2):397-408.
Houghton PJ, Raman A. 1998. Laboratory handbook for the fractionation of natural extracts.
Springer: Chapman & Hall; p. 103-105.
Sarker SD, Latif Z, Gray A. 2006. An introduction to natural products isolation. In Natural
Products Isolation (2nd edn). New Jersey: p. 88-89.
Ouairy C, Cresteil T, Delpech B, Crich D. 2013. Synthesis and evaluation of 3-deoxy and 3deoxy-3-fluoro derivatives of gluco- and manno-configured tetrahydropyridoimidazole
glycosidase inhibitors. Carbohydr. Res. 377:35-43.