398
Effects of Skeletonema costa turn Extracts on the Central Nervous System R. Villar"2, M. R. Laguna', f.M. Calleja'. and!. Cadavid' Departamento de FarmacologIa. Farmacia y TecnologIa Farmacéutica. Facultad de Farmacia. Universidad de Santiago de Compostela, Galicia. Spain 2 Address for correspondence
Abstract We report the effects of aqueous extracts of the microalga Skeletonema costaturn Greve (Cleve) (Bacillariophyceae) on spontaneous motor activity, rec-
there is little information available on this species. The relevant data may be summarized as follows: — their chemical composition shows fatty acids (2. 3) and amino acids (4), — a compound identified as f-1,3-n-glucan was isolated (5)'
their biological activity shows an antibacterial action whose active part seems to be a fatty acid (2, 6).
tal temperature, motor coordination, amphetamine-
—
induced hypermotility, exploratory behaviour, muscle relaxation, catalepsy, conditioned avoidance responses. oxotremorin-induced cholinergic syndrome, and pentylenetetrazole-induced convulsions. The S. costatum extract at dosages of 310 and 620mg/kg works like an antidopaminergic drug with anticholinergic properties,
The regilar use of S. costatuin in animal nutrition, especially in aquiculture, and the results of assays of acute toxicity (the presence of toxicity may indicate a potential biological activity) led us to select S.
that does not induce catalepsy and with a notable
costatum, along with other microalgal species, for a
muscle relaxing activity.
pharmacological study with the intention of elucidating the effects of an aqueous extract of the microalga on the central nervous system.
Key words
Skeletonema costatum, CNS activity, antidopaminergic drug, muscle relaxant activity, anticholinergic properties.
Materials and Methods Material Microalga cultures were supplied by the Xunta de Galicia's Marine Culture Centre at Ribadeo (Lugo, Spain).
Introduction The use of the sea as a source of food dates back to antiquity, but much more recent is the study of its potential as a source of biologically active products. Plant
and animal marine life forms have been studied with a
view to obtaining such products. In particular, in pharmacognosy, the centre of interest is biologically active substances with therapeutic potential. Our interest along
Culture conditions and preparation of algal extracts (I) S. costatum was cultured in seawater that, 24 h before addition of the algae, had been passed through 0.45 pm filters, sterilized at 120°C for 30mm, and enriched with ErdSchreiber medium. The unialgal culture was maintained at 18 0.1 °C under 2636 lm/m3 constant fluorescent illumination and with aeration by a current of air containing 1% of CO2.
S. costatum was recovered from culture by
this line centres on the use of marine microalgae as the
centrifuging for 40 mm at 2000g. The wet cell pellet was weighed,
materia prima for biologically active substances.
ruptured by sonication with a Branson B12 Sonifier. The ruptured cell suspension was centrifuged for 40 mm at 2000 g, and the su-
The first results of a systematic study of the
effects of the marine microalgae Phaeodactylum tncornutum and Dunaliella tertiolecta upon the central nervous system have already been reported (1). Such was the interest of the results that it was decided to extend the study to other microalgal species from which promising re-
suits were also to be expected. One such species is the diatom Skeletonema costatum Greve (Cieve). In keeping with the lack of studies on microaigae, and those concerning their pharmacological properties in particular,
and the cells were resuspended in double-distilled water and pernatant was recentrifuged for 20 mm at 20000 g before being
passed through 5, 1.2. and 0.45pm Millipore filters and lyophilized. Immediately prior to the performance of assays, the iyophilized hydrosoluble extract was dissolved in 0.9% NaCl.
Acute toxicity The possible acute toxicity of the S. costatum extract was tested using CD-I mice of either sex weighing 30 3g. Extract solutions were injected in the caudal vein by continuous perfusion at a rate of 25p1/min; perfusion was stopped upon the
Downloaded by: Universite Laval. Copyrighted material.
Received: October 10. 1991
Planta Med. 58(1992) 399
Effects of Skeletonerna costatum Extracts on the Central Nervous System death of the animal or after injection of 1500 M'. whichever was the
the same mice served as controls. The mice were placed on the
sooner. Controls were injected with l500l of isotonic saline
Rota-rod 30mm later and the time they stayed there was
solution. Each concentration was assayed on three mice. The first animal deaths appeared after injection of a mean volume of 920 il of extract at a concentration of 200 mg/mi.
observed; they were considered to have passed or failed the test according to whether they did or did not keep on the rod for 180 s.
Assay of effects on amphetamine-induced
Activity in the central nervous system The activity of the S. costa turn aqueous extract on the CNS was evaluated by performing assays of its effects on
spontaneous motor activity, rectal temperature, motor coordination (Rota-rod performance), amphetamine-induced
behaviour test (holeboard hypermotility, exploratory performance), muscle relaxation (traction test performance), catalepsy, conditioned avoidance responses (CAR). oxotremorininduced cholinergic symptoms, and pentylenetetrazole-induced convulsions.
hypermotility The effects of the extract on amphetamineinduced hypermotility were assayed as described by Knoll (9). For control and each extract dosage. 5 mg/kg of dextroamphetamine
sulphate was administered intraperitoneally to 5-mouse groups 30 mm after administration of extract. Motility was recorded in an activity cage every 10 mm from amphetamine administration until 60 mm after.
Assay of effects on exploratory behaviour:
Male CD-I mice weighing 25 2 g were used except for the test of conditioned avoidance responses when male Sprague-Dawley rats weighing 250 50g were employed. Animals were kept in a soundless room thermostatted at 22 I °C under a light regimen comprising 12 h of artificial light(8 a.m.— 8 p.m.) and 12 h darkness. To avoid any influence of circadian
holeboard test
Exploratory activity has been measured by the "holeboard" apparatus (10) which consists of a black board with 16 holes. 3cm in diameter, symmetrically distributed in 4 rows. A photobeam system placed under the box provided an automated measure of the number of head-dips. Measurement of exploratory activity started 30 mm after administration of the extract to 5mouse groups and was counted in 5mm periods over one hour.
rhythms, all assays were performed at the same time of the day.
Drugs and dosage
The following drugs and dosages were used: oxotremorin sesquifumarate (Sigma) 0.5 mg/kg; pentylenetetrazole (Sigma) 100mg/kg; dextroamphetamine sulphate
(Sigma) 5mg/kg; haloperidol (Sintex Latino S.A.) 0.1 and 0.5 mg/kg; S. costatum extract 155, 310, and 620 mg/kg. All drugs were dissolved in isotonic saline solution (0.9% NaCI) immediately before use and were
Assay of effects on muscular relaxation:
traction test
Following the method of Julou-Courvoisier (11), later described by Boissier (12). mice were suspended by their hind legs from a taut metal wire and the time taken to get hold of the wire with their front legs recorded; they were considered to have passed or failed the test according to whether this did or did not occur within 5 sec. Failure was considered to be synonymous with muscle relaxation. Experiments were performed 30, 60. and 90mm after the administration of extract.
administered by intraperitoneal injection of 0.1 mI/lOg (body weight) of the resulting solutions. When two or more drugs were administered, different injection sites in the abdomen were used. Control mice received, under the same conditions, isotonic saline solution. When it was of help, the drug haloperidol was used as a comparison. The haloperidol dosage was 0.5 mg/kg, apart from the C.A.R. test in which 0.1 mg/kg was used.
Assay of effects on spontaneous motor
activity The locomotor activity of the animals was re-
gistered with an activity cage. This cage contains an electromagnetic field that is sensitive to any motion within it. Movement of the animal causes an alteration in the energy of the field that is recorded as a locomotion count (7). Mice, in groups of 5, were placed on the cage five minutes after receiving the extract as an i.p. injection and the total number of steps in 10-mm periods over one hour was recorded.
Assay of effects on rectal temperature Rectal temperature was measured with a thermoelectric probe linked to a digital thermometer and carefully inserted 2cm into the rectum of the animals placed in individual containers. The temperature was recorded 30 mm prior to (T30) and just before (T0) injection of the algal extract and at 30, 60, 90, and 120 mm after administration.
Assay of effects on motor coordination:
rota-rod test The method of Boissier (8) was used. For each extract dosage, 4-mouse groups were administered extract and
Assay of cataleptogenic effects:
catalepsy test The method of Caillard (13) was used. The animals were placed in an uncomfortable condition (stretched between two horizontal metal wires spaced apart at different levels). Catalepsy was assumed, and the test failed, for animals remaining 20 s or more in this position. Experiments were performed 30, 60, and 90 mm after administration of the microalgae extract.
Assay of effects on conditioned avoidance responses (C.A.R.) Rats were trained in a Shuttle-box with two compartments that can be electrified separately (Shuttle-box LI 916 and shock module LI 2706). One session consisted of 20 trials
repeated at variable intervals. Each trial consisted of 5 sec of visual and auditory stimuli in one compartment; if the rat did not cross into the other compartment in this time, a I mA electric shock was applied to the grid for 3 sec. Twenty seconds later the same sequence was repeated. If the rat crossed during the light phase, no electric shock was applied (avoidance response). On the
other hand, an escape response occurred whenever the rat jumped onto the other compartment during shock. Each animal underwent 7 sessions: the first 30 mm before administration of extract, the second immediately before (T = 0) and the remainder 30,
60, 120, 180. and 240mm after administration. Only those animals which in earlier assays had shown avoidance responses in at least 80% of trials were used.
Downloaded by: Universite Laval. Copyrighted material.
Animals
400 PlantaMed. 58(7992) Assay of anticholinergic effects: oxotremorin test Oxotremorin was administered by i.p. injection 30 mm after administration of extract. Tremor was quantified visually 30, 60, 90. and 120 mm after administration of oxotremorin using the scale of Kulkarni (14): 0 = no tremor. 1 = slight tremor upon being handled. 2 = slight or intermittent spontaneous tremor, 3 = continuous intense spontaneous tremor.
R. Villar eta!.
tract, though hypothermia was still observed up to 2 h after administration (Fig. 4).
As a follow up to these assays, an extract dosage of 310mg/kg was used in the curiosity, traction, CAR., and catalepsy tests. This dosage was the minimum in the range used in this study showing potential CNS depressant activity in the tests above.
Salivation and diarrhoea were quantified visually for respectively 30 and 60 mm after oxotremorin administration using a slightly modified form of the scale ofOgren (15): 0
1400
o Control • S.costatum 155 mg/k
no salivation/diarrhoea. I = slight salivation/diarrhoea. 2 = moderate salivation/diarrhoea. 3 = heavy salivation/diarrhoea.
administration, and 30, 60, 90. and 120 mm after oxotremorin administration.
Assay of anticonvulsant effects:
pentylenetetrazole test
S.costatum 310 mg/k
'U 4) 'U
1000
' S.costatum 620 mg/k • {aIo.eridoI 0.5 mg/kg
0
800
U 4)
U,
V (I)
l)escribed originally by Everett and Richards in 1944. mice were administered with extract 30mm before the i.p. injection of 100mg/kg ofpentylenetetrazole(16). The time before onset of clonic convulsions, the time before onset of tonic convulsion and percentage mortality were observed.
600
400 200
0 0
10
20
Statistical analysis For all assays in which the data were not frequencies, differences with respect to controls were evaluated using the Mann-Whitney U-Lest, otherwise the Chi-square test (x)
was employed. Discrepancies with p < 0.05 were considered statistically significant (17).
Results
30
40
50
60
70
Time (mm.) Fig. 1 Effect of three dosages of S. cost atum on the spontaneous motor activity. Activity was recorded every 10 mm for 60 mm. Extract was
administered at t = 0. Each point represents the mean of 10 (control) and 5 (treated) groups of animals, vertical bars represent the standard error of the mean, and differences with respect to the control group were evaluated using the Mann-Whitney U-test ('p
Effects of Skeletonema costa turn Extracts on the Central Nervous System R. Villar"2, M. R. Laguna', f.M. Calleja'. and!. Cadavid' Departamento de FarmacologIa. Farmacia y TecnologIa Farmacéutica. Facultad de Farmacia. Universidad de Santiago de Compostela, Galicia. Spain 2 Address for correspondence
Abstract We report the effects of aqueous extracts of the microalga Skeletonema costaturn Greve (Cleve) (Bacillariophyceae) on spontaneous motor activity, rec-
there is little information available on this species. The relevant data may be summarized as follows: — their chemical composition shows fatty acids (2. 3) and amino acids (4), — a compound identified as f-1,3-n-glucan was isolated (5)'
their biological activity shows an antibacterial action whose active part seems to be a fatty acid (2, 6).
tal temperature, motor coordination, amphetamine-
—
induced hypermotility, exploratory behaviour, muscle relaxation, catalepsy, conditioned avoidance responses. oxotremorin-induced cholinergic syndrome, and pentylenetetrazole-induced convulsions. The S. costatum extract at dosages of 310 and 620mg/kg works like an antidopaminergic drug with anticholinergic properties,
The regilar use of S. costatuin in animal nutrition, especially in aquiculture, and the results of assays of acute toxicity (the presence of toxicity may indicate a potential biological activity) led us to select S.
that does not induce catalepsy and with a notable
costatum, along with other microalgal species, for a
muscle relaxing activity.
pharmacological study with the intention of elucidating the effects of an aqueous extract of the microalga on the central nervous system.
Key words
Skeletonema costatum, CNS activity, antidopaminergic drug, muscle relaxant activity, anticholinergic properties.
Materials and Methods Material Microalga cultures were supplied by the Xunta de Galicia's Marine Culture Centre at Ribadeo (Lugo, Spain).
Introduction The use of the sea as a source of food dates back to antiquity, but much more recent is the study of its potential as a source of biologically active products. Plant
and animal marine life forms have been studied with a
view to obtaining such products. In particular, in pharmacognosy, the centre of interest is biologically active substances with therapeutic potential. Our interest along
Culture conditions and preparation of algal extracts (I) S. costatum was cultured in seawater that, 24 h before addition of the algae, had been passed through 0.45 pm filters, sterilized at 120°C for 30mm, and enriched with ErdSchreiber medium. The unialgal culture was maintained at 18 0.1 °C under 2636 lm/m3 constant fluorescent illumination and with aeration by a current of air containing 1% of CO2.
S. costatum was recovered from culture by
this line centres on the use of marine microalgae as the
centrifuging for 40 mm at 2000g. The wet cell pellet was weighed,
materia prima for biologically active substances.
ruptured by sonication with a Branson B12 Sonifier. The ruptured cell suspension was centrifuged for 40 mm at 2000 g, and the su-
The first results of a systematic study of the
effects of the marine microalgae Phaeodactylum tncornutum and Dunaliella tertiolecta upon the central nervous system have already been reported (1). Such was the interest of the results that it was decided to extend the study to other microalgal species from which promising re-
suits were also to be expected. One such species is the diatom Skeletonema costatum Greve (Cieve). In keeping with the lack of studies on microaigae, and those concerning their pharmacological properties in particular,
and the cells were resuspended in double-distilled water and pernatant was recentrifuged for 20 mm at 20000 g before being
passed through 5, 1.2. and 0.45pm Millipore filters and lyophilized. Immediately prior to the performance of assays, the iyophilized hydrosoluble extract was dissolved in 0.9% NaCl.
Acute toxicity The possible acute toxicity of the S. costatum extract was tested using CD-I mice of either sex weighing 30 3g. Extract solutions were injected in the caudal vein by continuous perfusion at a rate of 25p1/min; perfusion was stopped upon the
Downloaded by: Universite Laval. Copyrighted material.
Received: October 10. 1991
Planta Med. 58(1992) 399
Effects of Skeletonerna costatum Extracts on the Central Nervous System death of the animal or after injection of 1500 M'. whichever was the
the same mice served as controls. The mice were placed on the
sooner. Controls were injected with l500l of isotonic saline
Rota-rod 30mm later and the time they stayed there was
solution. Each concentration was assayed on three mice. The first animal deaths appeared after injection of a mean volume of 920 il of extract at a concentration of 200 mg/mi.
observed; they were considered to have passed or failed the test according to whether they did or did not keep on the rod for 180 s.
Assay of effects on amphetamine-induced
Activity in the central nervous system The activity of the S. costa turn aqueous extract on the CNS was evaluated by performing assays of its effects on
spontaneous motor activity, rectal temperature, motor coordination (Rota-rod performance), amphetamine-induced
behaviour test (holeboard hypermotility, exploratory performance), muscle relaxation (traction test performance), catalepsy, conditioned avoidance responses (CAR). oxotremorininduced cholinergic symptoms, and pentylenetetrazole-induced convulsions.
hypermotility The effects of the extract on amphetamineinduced hypermotility were assayed as described by Knoll (9). For control and each extract dosage. 5 mg/kg of dextroamphetamine
sulphate was administered intraperitoneally to 5-mouse groups 30 mm after administration of extract. Motility was recorded in an activity cage every 10 mm from amphetamine administration until 60 mm after.
Assay of effects on exploratory behaviour:
Male CD-I mice weighing 25 2 g were used except for the test of conditioned avoidance responses when male Sprague-Dawley rats weighing 250 50g were employed. Animals were kept in a soundless room thermostatted at 22 I °C under a light regimen comprising 12 h of artificial light(8 a.m.— 8 p.m.) and 12 h darkness. To avoid any influence of circadian
holeboard test
Exploratory activity has been measured by the "holeboard" apparatus (10) which consists of a black board with 16 holes. 3cm in diameter, symmetrically distributed in 4 rows. A photobeam system placed under the box provided an automated measure of the number of head-dips. Measurement of exploratory activity started 30 mm after administration of the extract to 5mouse groups and was counted in 5mm periods over one hour.
rhythms, all assays were performed at the same time of the day.
Drugs and dosage
The following drugs and dosages were used: oxotremorin sesquifumarate (Sigma) 0.5 mg/kg; pentylenetetrazole (Sigma) 100mg/kg; dextroamphetamine sulphate
(Sigma) 5mg/kg; haloperidol (Sintex Latino S.A.) 0.1 and 0.5 mg/kg; S. costatum extract 155, 310, and 620 mg/kg. All drugs were dissolved in isotonic saline solution (0.9% NaCI) immediately before use and were
Assay of effects on muscular relaxation:
traction test
Following the method of Julou-Courvoisier (11), later described by Boissier (12). mice were suspended by their hind legs from a taut metal wire and the time taken to get hold of the wire with their front legs recorded; they were considered to have passed or failed the test according to whether this did or did not occur within 5 sec. Failure was considered to be synonymous with muscle relaxation. Experiments were performed 30, 60. and 90mm after the administration of extract.
administered by intraperitoneal injection of 0.1 mI/lOg (body weight) of the resulting solutions. When two or more drugs were administered, different injection sites in the abdomen were used. Control mice received, under the same conditions, isotonic saline solution. When it was of help, the drug haloperidol was used as a comparison. The haloperidol dosage was 0.5 mg/kg, apart from the C.A.R. test in which 0.1 mg/kg was used.
Assay of effects on spontaneous motor
activity The locomotor activity of the animals was re-
gistered with an activity cage. This cage contains an electromagnetic field that is sensitive to any motion within it. Movement of the animal causes an alteration in the energy of the field that is recorded as a locomotion count (7). Mice, in groups of 5, were placed on the cage five minutes after receiving the extract as an i.p. injection and the total number of steps in 10-mm periods over one hour was recorded.
Assay of effects on rectal temperature Rectal temperature was measured with a thermoelectric probe linked to a digital thermometer and carefully inserted 2cm into the rectum of the animals placed in individual containers. The temperature was recorded 30 mm prior to (T30) and just before (T0) injection of the algal extract and at 30, 60, 90, and 120 mm after administration.
Assay of effects on motor coordination:
rota-rod test The method of Boissier (8) was used. For each extract dosage, 4-mouse groups were administered extract and
Assay of cataleptogenic effects:
catalepsy test The method of Caillard (13) was used. The animals were placed in an uncomfortable condition (stretched between two horizontal metal wires spaced apart at different levels). Catalepsy was assumed, and the test failed, for animals remaining 20 s or more in this position. Experiments were performed 30, 60, and 90 mm after administration of the microalgae extract.
Assay of effects on conditioned avoidance responses (C.A.R.) Rats were trained in a Shuttle-box with two compartments that can be electrified separately (Shuttle-box LI 916 and shock module LI 2706). One session consisted of 20 trials
repeated at variable intervals. Each trial consisted of 5 sec of visual and auditory stimuli in one compartment; if the rat did not cross into the other compartment in this time, a I mA electric shock was applied to the grid for 3 sec. Twenty seconds later the same sequence was repeated. If the rat crossed during the light phase, no electric shock was applied (avoidance response). On the
other hand, an escape response occurred whenever the rat jumped onto the other compartment during shock. Each animal underwent 7 sessions: the first 30 mm before administration of extract, the second immediately before (T = 0) and the remainder 30,
60, 120, 180. and 240mm after administration. Only those animals which in earlier assays had shown avoidance responses in at least 80% of trials were used.
Downloaded by: Universite Laval. Copyrighted material.
Animals
400 PlantaMed. 58(7992) Assay of anticholinergic effects: oxotremorin test Oxotremorin was administered by i.p. injection 30 mm after administration of extract. Tremor was quantified visually 30, 60, 90. and 120 mm after administration of oxotremorin using the scale of Kulkarni (14): 0 = no tremor. 1 = slight tremor upon being handled. 2 = slight or intermittent spontaneous tremor, 3 = continuous intense spontaneous tremor.
R. Villar eta!.
tract, though hypothermia was still observed up to 2 h after administration (Fig. 4).
As a follow up to these assays, an extract dosage of 310mg/kg was used in the curiosity, traction, CAR., and catalepsy tests. This dosage was the minimum in the range used in this study showing potential CNS depressant activity in the tests above.
Salivation and diarrhoea were quantified visually for respectively 30 and 60 mm after oxotremorin administration using a slightly modified form of the scale ofOgren (15): 0
1400
o Control • S.costatum 155 mg/k
no salivation/diarrhoea. I = slight salivation/diarrhoea. 2 = moderate salivation/diarrhoea. 3 = heavy salivation/diarrhoea.
administration, and 30, 60, 90. and 120 mm after oxotremorin administration.
Assay of anticonvulsant effects:
pentylenetetrazole test
S.costatum 310 mg/k
'U 4) 'U
1000
' S.costatum 620 mg/k • {aIo.eridoI 0.5 mg/kg
0
800
U 4)
U,
V (I)
l)escribed originally by Everett and Richards in 1944. mice were administered with extract 30mm before the i.p. injection of 100mg/kg ofpentylenetetrazole(16). The time before onset of clonic convulsions, the time before onset of tonic convulsion and percentage mortality were observed.
600
400 200
0 0
10
20
Statistical analysis For all assays in which the data were not frequencies, differences with respect to controls were evaluated using the Mann-Whitney U-Lest, otherwise the Chi-square test (x)
was employed. Discrepancies with p < 0.05 were considered statistically significant (17).
Results
30
40
50
60
70
Time (mm.) Fig. 1 Effect of three dosages of S. cost atum on the spontaneous motor activity. Activity was recorded every 10 mm for 60 mm. Extract was
administered at t = 0. Each point represents the mean of 10 (control) and 5 (treated) groups of animals, vertical bars represent the standard error of the mean, and differences with respect to the control group were evaluated using the Mann-Whitney U-test ('p
