Bourdy, Geneviève

Debitus, Cécile

Lavayre, J.

Paillard, J.J.

Païs, M.

Quirion, J.C.

Vassas, A.

Horizon / Pleins textes

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Naturally Occurring Somatostatin and Vasoactive 
Intestinal Peptide Inhibitors. Isolation of halo ids  
from Two Marine Sponges I 
A. Vassas'*6, G. Bourdy', J. J. Paillard3, J. Lauayre3, M. Pai's4, J. C. Quirion4, and C. Debitus' 
Laboratoire de Botanique, Phytochimie et Mycologie, Faculte de Pharmacie, F-34060 Montpellier, France 
ORSTOM, CP 9214, La Paz, Bolivia 
Rhône-Poulenc Rorer S. A., CRVA, BP14, F-94403 Vitry/Seine Cedex, France 
CNRS, Institut de chimie des substances naturelles, F-91198 Gif sur Yvette Cedex, France 
ORSTOM, BP A5, Nouméa, Nouvelle-Calédonie 
Address for correspondence 
Ex : w 4 s  1 :t I cote: B-X ZGZO? Received: May 15,1995; Revisionaccepted: August 5,1995 
L B 
Abstract 
The vasoactive intestinal peptide (VIP) 
and somatostatin (somatotropin release inhibiting factor, 
SRIF) are important neurotransmitters in a number of 
basic physiological events. Their disturbances have 
been reported in many diseases such as cystic fibrosis, 
impotent man (VIP), Alzheimer's disease, and some 
tumours (SRIF). Xestospongine B (11, sceptrine (2), and 
ageliferine (3), three alkaloids isolated from Xesto- 
Spongia sp. and Agelas nouaecaledoniae are reported 
as somatostatin and VIP inhibitors. The natural prod- 
ucts 1,2, and 3 exhibited a high affinity for somatostatin 
(IC5, = 12  pM, 0.27 pM, and 2.2 pM, respectively), 2 and 
3 showed an affinity for VIP (19.8 pM and 19.2 pM, re- 
spectively). Due to the interaction between non-peptidic 
compounds and somatostatin/VIP receptors, these three 
alkaloids could be promising agents in the research on 
natural non-peptidic compounds for therapeutical in- 
terventions. 
Key words 
Porifera, Nepheliospongida, Axinellida, 
ageliferine, sceptrine, xestospongine B, somatostatin re- 
ceptor, vasoactive intestinal peptide receptor. 
I 
I Abbreviations 
VIP: vasoactive intestinal peptide 
SRIF: somatotropin release inhibiting factor 
CNS: central nervous system 
PNS: peripheral nervous system 
I3H1-QNB: specific ligand of mAChR 
mAChR: muscarinic acetylcholine receptors 
Introduction 
The vasoactive intestinal peptide (VIP) is an 
important neurotransmitter for basic physiological events: 
Planta Med. 62 (1996) 28-30 
O Georg Thieme Verlag Stuttgart. New York 
it may regulate the cortical blood flow, the energy metabo- 
lism and the neuronal activity in the central nervous sys- 
tem (CNS), and has a modulator role in pituitary hormone 
secretion. VIP has also a wide distribution in the peripheral 
nervous system (PNS): the main actions are stimulation of 
secretions (oesophagus, stomach, airway smooth muscle, 
salivary, water and bicarbonate), control of release of 
glucagon and insulin, relaxation of vascular and non- 
vascular smooth muscle, and vasodilatation of the genital 
system. The deficiency in VIP was reported in Hirsch- 
sprung's disease, achalasia of oesophagus, cystic fibrosis, 
and in impotent man (1). A VIP antagonist inhibited pro- 
liferation in nan-small cell lung cancer (2). As far as we 
know, a high a f f i t y  between the VIP receptor and a non- 
peptidic compound has not been described previously. 
On the other hand, the somatostatin neuro- 
transmitter (somatotropin release inhibiting factor, SRIF) 
displayed in some tumour processes (pituitary, pancreas, 
brain, and breast tumours) (3). Somatostatin presumably 
plays a role in Alzheimer's disease on account of its low 
level found as the most consistent biochemical abnormality 
(4,5). Recently, the peptidic compounds, octreotide and its 
analogs, were reported as ligands of somatostatin receptor 
67). 
Because of the well-known number of bio- 
logically active compounds isolated from marine organisms 
with high affinities for cellular receptors (81, more than 300 
marine invertebrates from New Caledonia were screened 
on 20 receptors using binding methods (9). Nine percent of 
organisms showed a high affinity to some receptors. 
Xestospongia sp. and Agelas novaecaledoniae were then 
investigated for VIP and somatostatin receptors by a radio- 
receptor binding assay. This is the first time that non- 
peptidic compounds from these marine organisms are re- 
ported as VIP and somatostatin inhibitors. 
Materials and Methods 
Bioactiue samples study 
Animal material: The sponge Xestospongia sp. 
(order Nepheliospongida; family Nepheliospongiidae) was COI- 
lected at Baie de Prony, New Caledonia at a depth of 10m, and 
Agelas nouaecaledoniae (order Axinellida; family Agelasidae) was 
dredged on the Norfolk ridge at  200 m deep. They were identified 
Planta Med. 62 (1996) 29 - Naturally Occurring Somatostatin and Vasoactive Intestinal Peptide Inhibitors 
by Pr. Claude Levi of Museum National d'Histoire Naturelle (Paris). 
Voucher specimens (R602 and R1413, respectively) are available 
from Zoothèque at ORSTOM, Nouméa. 
Purifications: The bioactive compounds were 
isolated by bioassay-guided chromatography. 
. 1. Xestospongia sp.: Xestospongine B (1) was 
extracted according to the method described by Quirion et al. (10). 
In short, the freeze-dried sponge (1OOg) was extracted at room 
temperature with EtOAc (2 x 500 ml, 24 h). The organic phase was 
then extracted with dilute HC1 (pH 3). The aqueous phase was 
basified with aqueous NH40H to pH 10 and extracted with EtOAc 
to give the alkaloid fraction (3.8g). The ethyl acetate layer was 
purified by a silica gel (2 x 40cm) flash chromatography 
(hexane/EtzO/MeOH/NH,OH, 20 : 70 : 10 : 0.5) affording com- 
pound 1 (64mg) and other alkaloids. 
2. Agelas novaecaledoniae: The freeze-dried 
sponge (1OOg) was extracted with 80% EtOH (3 x 500ml) 
according to Pak (personal communication). The solvent was 
evaporated to dryness. The residue (11 g) was dissolved in water 
(600ml), filtered, and passed through a column of Amberlite 
XAD-7 (250 ml) (2 ml/min). The column was washed with water 
and then eluted with MeOH/HzO (1 : 1). The methanol-water frac- 
tion (2.35 g) was subjected to a medium pressure silica gel (Merck: 
7736) column chromatography eluted with EtOAc/2-butanone/ 
formic acid/H20 (5 : 3 : 0.5 : 0.5) (11) affording slightly impure 
sceptrine formate (1.20 g) and ageliferine formate (0.20g), which 
were purified using flash chromatography on a C18 column 
(Waters 5.5- 10.5 cm). Pure formate of sceptrine (2; 0.45 g) and 
ageliferine (3; 0.12 g) were eluted with 250ml of H20/MeOH (3 : 2). 
2 -formate salt: FAB-MS: m/z = 621 (MH+); IR, 
UV, 'H- and I3C-NMR were identical to the published spectra for 
the acetate (12,13) except for signals at 6 = 8.26 (lH, s) in the 'H- 
NMR (DMSO-d,) and 6= 172.29 in theI3C-NMR (H20) for HC0,H. A 
solution of the formate salt (0.12 g) in MeOH was passed through a 
column of IRA-410 resin in OH- form yielding the free base, which 
was transformed into the hydrochloride form using HCl, and then 
chromatographed on a C18 column (1 x 10 cm) H20/MeOH (3 : 2) 
yielding the pure hydrochloride salt (0.80 g) which was crystallized 
from water, mp 117-130°C dec), [do: -22" (c 1, MeOH); Lit. (1): 
(c 4.33, MeOH). 
' 
mp 115-125 "c (dec),  ID: - 7.4"(C 1.2, MeOH); Lit. (3): [alo: - 1,s' 
3 -formate salt: FAB-MS: m/z = 621 (MH'); IR, 
W, 'H- and I3C-NMR were identical to the published spectra (13, 
14) except for signals at 6 = 8.50 (lH, s) in the 'H-NMR (DMSO-d,) 
and 6 = 170.89 in the I3C-NMR (H20) for HC02H. The formate salt 
was transformed into the hydrochloride salt as described for 2, 
[&: + 7" (C 0.7, MeOH); Lit. (2): [alo + 15.5" (C 0.11, MeOH); Lit. (3): 
[ClIo: + 4.1" ( C  0.146, MeOH). 
Screening 
Extraction (screened extracts): The freeze-dried 
powder of the two marine sponges (100 g) was extracted with dis- 
tilled water (4 O C ,  dark room); the extract was then centrifuged and 
the freeze-dried supernatant gave extract A. 
The residue was extracted with (3 x 500ml) 
80% EtOH at room temperature and the pooled extract was con- 
centrated in vacuo. The residue was diluted with water (lOOOml, 
24h) and partitioned with CHzClz (3 x 1OOOml) .  The aqueous 
layer was freeze-dried, and the powder dissolved with absolute 
EtOH to obtain extract B. The dichloromethane fraction gave ex- 
tract C. 
Extracts A and B were further desalted and pro- 
teins eliminated through successive RP-8 (Merck: 9324) and silica 
gel 60H columns (Merck: 7736). 
Binding bioassays 
Membranes were obtained from the brain cortex 
of Sprague-Dawley male rats, homogenized in tris-HC1 buffer - 
50nM HC1 (pH 7.5), and concentrated by ultracentrifugation 
(70,000 x g for 10min). The average concentration in protein was 
8- 14  mg/ml. 
Somatostatin binding: Somatostatin binding 
receptor assay used ['25Z]-somatostatin (0.02 nM k 0.002 nM) as 
the labelling ligand. The equilibrium binding experiment was done 
at 25 O C  with 0.05 mg/ml membrabe preparation in HEPES-KOH 
buffer 20nM (pH 7 3 ,  containing 5mM MgClZ, 0.6% BSA, 
0.25 mg/ml bacitracin, and 1 mg/ml aprotinin. Aliquots (0.6 ml) of 
this preparation were incubated for 60 min. Non-specific binding 
was determined using 100 nM somatostatin. Binding values of 1, 
2, and 3 (5 pg/ml) were determined as the deplacement of the spe- 
cific binding of the labelling ligand. The binding was terminated by 
filtration through Whatman GF/B glass fibre filters with a cell 
harvester, followed by three washes with 3 ml of 20 nM HEPES- 
KOH buffer (pH 7.5). The radioactivity retained on the filters was 
determined directly with a gamma scintillation counter. 
Vasoactive intestinal peptide (WP) binding: The 
VIP receptor assay used ['"SII-VIP (0.02nM ? 0.002nM) as the 
labelling ligand. The equilibrium binding experiment was done at 
37 "C with 0.05 mg/ml membrane preparation in 10 nM PIPES 
buffer (pH 7.4) containing 2.5 mM MgC12, 0.1 M mercaptoethanol 
and 0.2 mg/ml bacitracin. Aliquots (0.6 ml) of this preparation 
were incubated for 120min. Non-specific binding was determined 
using 100 nM VIP. The binding of 2 and 3 was determined as the 
deplacement of labelling VIP from the specific binding. The 
binding was terminated by filtration through Whatman GF/B glass 
fibre filters with a cell harvester, followed by three washes with 
4 ml of 10 nM PIPES buffer (pH 7.5). The radioactivity retained on 
the filters was determined directly with a gamma scintillation 
counter. 
1 Xestospongine B 2 sceptrine 
3 Agel i fe r ine  
A. Vassas et al. 
4 _.' 
30 hanta Med. 62 (1996) - 
Table 1 Somatostatin and VIP inhibition from different extracts and Dure ComDounds. 
Results and Discussion 
Xestospongia sp.: The solvent bioassay- 
guided partition showed that the alkaloid fraction of the 
Xestospongia alcohol extract was active (Table 1). Five al- 
kaloids were previously isolated and identified from this 
sponge, four of them belonging to the xestospongine series 
[araguspongine F, xestospongine B (11, xestospongine D, 
demethylxestospongine BI and xestoamine. These alka- 
loids (base) were assayed on the somatostatin receptor, 
and 1 exhibited a good activity with an IC50 = 1 2  pM (Ki = 
1 3.4 pM). 
This compound, previously isolated from 
the Australian marine sponge X. exigua, has been reported 
to be a vasodilatative compound, inducing relaxation of 
blood vessels in uivo (13, and to demonstrate also general 
cytotoxic activity (10). This is the fEst time, as far as we 
know, that this compound has been reported to be an in- 
hibitor of somatostatin receptors. 
Agelas nouaecaledoniae: The compounds 
2 and 3 (hydrochloride salts), showed a strong activity on 
the somatostatin receptor and a medium activity on the VIP 
receptor (Table 1). 
Somatostatin receptor: 2: IC50 = 0.27pM 
(Ki = 0.30 pM), 3: IC50 = 2.21 pM (Ki = 2.47 PMI. 
Vasoactive intestinal peptide receptor: 2: 
TC50 - 19.2pM (Ki = 61.8pMh 3: IC50 = 19.8pM (& = 
63.8pM). 
The compounds 2 and 3 have already been 
isolated previously in other Agelas species (13, 14): 3 iso- 
lated from an Okaniwan Agelas sp. was found to be a 
potent actomyosin ATPase'jactivator (14). This compound 
also inhibits growth of Bacillus subtilis, and is active 
against Escherichia coli, Herpes simplex virus type 1 and 
Vesicular stomatitis virus; 3 has a moderate barnacle 
settlement inhibition, showing some potential antifouling 
activity (13). 
The compound 2 ísolated from A. conifera 
was found to be an ichtyotoxic principle (16) and, isolated 
as a dichloride, was reported as the major antimicrobial 
constituent of Agelas spectrum (121, as an antagonist of 
serotonergic receptor (17), and as'a competitive inhibitor 
of r3H1-QNB binding to rat brain mAChR (18). The activity 
of 2 was 143 times more potent on the somatostatin re- 
ceptor and 2 times more potent on the VIP receptor than 
the activity displayed on the I3H1-QNB binding assay. 
The non-peptidic compounds 2 and 3 ex- 
hibited a high affinity for somatostatin and VIP receptors 
and 1 for the somatostatin receptor. This information 
makes it possible to generate a new class of drugs used for 
therapeutical intervention in VIP and SRIF systems. 
Acknowledgements 
We thank the laboratory of Biology and 
Screening from Rhône-Poulenc Rorer (France) for the binding 
tests and Dr. S. Rapior for her assistance (Laboratory of Botany 
and Phytochemistry, Montpellier). This work was supported by the 
ORSTOM-CNRS program called SMIB (Substances Marines 
d'Interi3 Biologique) Nouméa, New Caledonia. 
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Naturally occuring somatostatin and vasoactive intestinal peptide inhibitors : isolation of alkaloids from two marine sponges

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