Download (A CNS ACTIVE SUBSTANCE) ISOLATED FROM

Folia Medica Indonesiana Vol. 44 No. 2 April – June 2008 :
PHARMACODYNAMIC EFFECTS OF LIGUSTROSIDE GLYCOSIDE (A CNS ACTIVE
SUBSTANCE) ISOLATED FROM FRAXINUS GRAFFITHII Clarke AGAINST
CHEMICALLY INDUCED SEIZURES ON MICE
Achmad Basori
Department of Pharmacology
Airlangga University School of Medicine
ABSTRACT
A study was conducted to prove whether ligustroside (A CNS Active substance) isolated from bark of Fraxinus
Graffithii Clarke has antiseizure effects against chemically induced seizure. antiseizure test indicated that ligustroside
increased seizure treshold on epileptic animals injected by CD90 metrazol i.p. (ED50 anti Met : 278.54 mg/kg BW),
CD90 bicuculline (ED50 anti Bicuc : 424.57 mg/kg BW), and CD90 picrotoxin (ED50 anti Picro : 466.53 mg/kg BW).
These experiments also reported that ligustroside decreased metrazol lethality test on animals. The capability of
ligustroside to increase seizure threshold against anti metrazol test , anti bicuculline test, and anti picrotoxin test,
meant that ligustroside may have antiseizure effects by increasing GABA- facilitated inhibition in CNS through
interaction with benzodiazepine receptor of GABA-BZD-BARB/PICRO-CL receptor complexes. It is strongly supposed
that clinically ligustroside would be active against absence seizures and myoclonic seizures. Further research will be
needed to explore interaction between ligustroside and GABA-BZD-BARB/PICRO-CL receptor complex.
Keyword: ligustroside, chemically induced seizure, antiseizure effect
Correspondence: Achmad Basori, Department of Pharmacology, Airlangga University School of Medicine,
Jl Prof Dr Moestopo 4-7, Surabaya 60131, phone 62-31-5020251 ext. 174
INTRODUCTION
Fraxinus griffithii Clarke (trees: 10 – 20 m) usually
grows in forest margins, near villages, by rivers; 100–
2000 m in several regions in Bangladesh, India, Ryukyu
Islands, Myanmar, Philippines, Vietnam and Indonesia.
In Java island, Fraxinus griffithii Clarke is known as
Pohon Tiken (Kabupaten Lumajang), Bedali Gombong
(Kabupaten Probolinggo), and Pohon Orang Aring
(Perkebunan Pancur Angkrek, Prajekan) (Heyne 1987).
Extract of the bark and the leaves of Fraxinus griffithii
Clarke (so called: ekstrak Tiken) have been used as an
adulterant of illegal opium in certain areas in Indonesia
(Boerlage & Kooders 1987; Heyne 1987; Sutarjadi &
Zaini 1973; Wehmer 1931). Phytochemical studies
found that Tiken extract contains saponin, tannine, and
glycosides, and no alkaloids (Sutarjadi & Zaini 1973).
Survey in 1975 reported that the workers who prepared
this extract felt sedation and sometimes went to sleep
(Ahaditomo 1975). Explorative study in mice showed
that Tiken extract caused little sedation and reduced
motor activity (Husein 1975). Furthermore, Basori et al
(1998) found that Tiken extract 1500 mg/kg BW up to
2500 mg/kg BW given orally to animals caused little
sedation, reduced locomotor activity, did not cause
coordination disturbances and muscle paralysis, and did
not have hypnogenic effects. All those findings strongly
suggested that Fraxinus griffithii Clarke may contain a
CNS (Central Nervous System) active substance.
In 1980, Sutarjadi modified the method of Inouye
(1975) to isolate ligustroside glycoside from the bark of
Fraxinus griffithii Clarke. For further research and
development of ligustroside to be lead compound of
CNS depressant drug, a large amount of ligustroside and
high quality is needed. Basori (1997) modified the
method of Sutarjadi to isolate ligustroside by doing pre
extraction process with dichlormethane. This
modification proposed to remove all non glycoside
compounds from the solution and expected to give a
more pure ligustroside.
The previous pharmacodynamic screening showed that
ligustroside at doses of 100 mg/kg, 200 mg/kg , and 400
mg/kg given intraperitoneally to mice caused little
sedation, reduced locomotor activity, did not cause
motor coordination disturbances and muscle paralysis,
and did not have hypnogenic effect. These results
suggested that ligustroside may have CNS activity as
CNS depressant (Basori 1999). However, the precise
pharmacodynamic profile of its CNS depressant has not
been studied in detail yet. Further research is needed to
explore pharmacodynamic profile of its CNS depressant
effect.
Pharmacodynamic Effects of Ligustroside Glycoside … (Achmad Basori)
A very detailed pharmacodynamic study was conducted
by Basori (2000), to explore the profile of CNS
depressant effects of ligustroside. Those studies
concluded that ligustroside has pharmacodynamic
activity as CNS depressant, did not have neuromuscular
blocking properties. The primary site of action of its
CNS depressant is in the brain, and the mechanism of
action may involve the enhancement of GABAfacilitated inhibition in the brain neurons.
Pharmacodynamic study with electrical stimulation
indicated that ligustroside has anti seizure effect and to
prevent seizure spreads on animals. Pharmacologically,
ligustroside was very potential to be developed as
antiseizure drug candidate (Basori 2004). Further
research will be needed to investigate the other effects
of ligustroside against chemically induced seizure. The
present research aimed to identify whether ligustroside
isolated from bark of Fraxinus Graffithii Clarke has an
effect to increase seizure thresholds against some
chemically induced seizures on mice.
MATERIALS AND METHODS
Material used for the present work was dried bark of
Fraxinus griffiti Clarke (Pohon Orang Aring). Wet bark
was collected from plants used as shade trees at a
Pancur Angkrek coffee plantation of PTP XXVI located
near Prajekan, East Java. The bark was cut into pieces
and dried at room temperature, avoiding direct sunlight,
for 1 month. This was done in order to keep active
substances from degradation by direct sunlight. Later
on, the dried bark was milled with a milling machine
into smaller particles, sieved into pulves, and stored in
tightly sealed plastic bottles. ligustroside was isolated,
identified and purified according to the previous method
(Basori 1999). Male BULB/c mice (25–30 g, age: 2
months) used in all experiments were purchased from
Pusvetma, Surabaya. Animals were acclimatized for at
least one week before starting the experiments. They
were kept in a soundless room with normal room
temperature. During the experiments the animals were
not allowed to drink or eat. Finally, the animals were
given a code for experiments.
The following drugs and dosages were used:
ligustroside 100, 200, 300, 400 and 500 mg/kg. This
drug was dissolved in solutio Petit (10% absolute
alcohol; 20% Propylene glycol : 80% water pro
injection) and administered to animals by i.p. injection.
Control animals (positive and negative control) were
dosed with Solutio Petit and water pro injection. The
previous study found that solutio Petit did not have
antiseizure effects (Basori 2000).
To find the effect of ligustroside against metrazole
induced seizure, the animals were randomly divided into
4 groups (each group consisted of 20 mice). Each group
of animals were given ligustroside 100 mg/kg BW, 200
mg/kg BW, 300 mg/kg BW, and 400 mg/kg BW i.p.
Animals were placed in individual transparent plastic
cages. Forty minutes after ligustroside administration,
all animals were injected with metrazole 66.75 mg/kg
BW i.p (Convulsive Dose 90). The capability of a
substance to protect animals against clonic seizure
regarded as anti–metrazole activity (anti Met effect)
(Krall 1978; Leppik 1993, 1994; Swinyard 1972;
Swinyard et al. 1985; White et al. 1995). In this
experiment, the dose – responses curves were estimated
by testing four doses and twenty animals per dose. The
calculation of ED50 anti metrazol (a dose that protects
50% of the animals against metrazol induced clonic
seizure), and the statistical analyses were performed
according to Probit method by using SPSS 9.
To find the effects of ligustroside against bicuculline
induced seizure, the animals were randomly divided into
3 groups (each group consists of 20 mice). Each group
of animals was given with ligustroside 200 mg/kg BW,
400 mg/kg BW, and 500 mg/kg BW i.p. Animals were
placed in individual transparent plastic cages. Forty five
minutes after given with ligustroside, all animals were
injected with bicuculline 3.18 mg/kg BW i.p
(Convulsive Dose 90). The capability of a substance to
protect animals against clonic seizures regarded as anti
– bicuculline test (Leppik 1993, 1994; White et al.
1995). In this experiment, the dose–responses curves
were estimated by testing three doses and twenty
animals per dose. The calculation of ED50 anti
bicuculline and the statistical analyses were performed
according to Probit method by using SPSS 9.
To find the effects of ligustroside against picrotoxin
induced seizures, the animals were randomly divided
into 3 groups (each group consisted of 20 mice). Each
group of animals was given with ligustroside 200 mg/kg
BW, 400 mg/kg BW, and 500 mg/kg BW i.p. Animals
were placed in individual transparent plastic cages.
Forty five minutes after given with ligustroside, all
animals were injected with picrotoxin 3.20 mg/kg BW
i.p (Convulsive Dose 90). The capability of a substance
to protect animals against clonic seizures regarded as
anti – picrotoxine test (Swinyard et al. 1985; Leppik
1993, 1994; White et al. 1995). In this experiment, the
dose–responses curves were estimated by testing three
doses and twenty animals per dose. The calculation of
ED50 anti picrotoxine and the statistical analyses were
performed according to Probit method by using SPSS 9.
To find the effects of ligustroside against strychnine
nitrate induced seizures, the animals were randomly
Folia Medica Indonesiana Vol. 44 No. 2 April – June 2008 :
divided into one group (consisted of 20 mice). One
group of animals was given with ligustroside 500 mg/kg
BW i.p. Animals were placed in individual transparent
plastic cages. Forty five minutes after given
ligustroside, all animals were injected with Strycnine
Nitrate 0.88 mg/kg BW i.p. (Convulsive Dose 90). The
capability of a substance to protect animals against
maximal tonic extensor regarded as anti–Strychnine
Nitrate test (Leppik 1993, 1994; Swinyard et al. 1985;
White et al. 1995). Finally, to find the effects of
ligustroside against INH induced seizures, the animals
were randomly divided into one group (consisted of 20
mice). One group of animals was given with
ligustroside 500 mg/kg BW i.p. Animals were placed in
individual transparent plastic cages. Forty five minutes
after given ligustroside, all animals were injected with
INH 136.28 mg/kg BW i.p (Convulsive Dose 90). The
capability of a substance to protect animals against
clonic seizures regarded as anti–INH test (Leppik 1993,
1994; Swinyard et al. 1985; White et al. 1995).
RESULTS
The specific objective of these pharmacodynamic
studies was to identify antiseizure effect of ligustroside
against chemically induced seizure. This experiment
used many models of chemically induced seizures by
giving many epileptogens to animal. After being given
with epileptogen, animal experiences seizure. Every
type of seizures i.e. : clonic , clonic–tonic, or tonic
extensor seizures, depends on every underlying cellular
seizure mechanism (Table 1). In this research, it was
proved that ligustroside had anti metrazol, anti
bicuculline, and anti picrotoxin test. Those results can
be seen in Table 2, Table 3, and Table 4. Table 2 also
showed that ligustroside depressed mortality rate of
animals on metrazol lethality test. While up to the dose
of 500 mg/kg BW i.p. both animal groups given CD90
Strychnine Nitrate and INH showed no seizure
response. These results can be seen in Table 5 and 6.
While Safety Ratio (SR) analysis (SR = TD3/ED 97)
showed that SR of anti metrazole = 1.73, anti picrotoxin
= 1.16, and anti bicuculline = 1.18 (Table 7).
Table 1. Cellular mechanism of various chemically induced seizures on mice
No
Chemical epileptogen
Cellular mechanism of
seizures
Type of seizures
1 Bicuculline
Treshold clonic seizures
GABA-A receptor Antagonist
2 Metrazole
Clonic-tonic seizures
GABA-A receptor Antagonist
3 Strychnine Nitrate
Tonic extensor seizures
Glycine receptor Antagonist
4 INH
Treshold clonic seizures
GABA-transaminase inhibitor
5 Picrotoxin
Treshold clonic seizures
Picrotoxin receptor Antagonist
From: Steve White et al (1995); McDonald & Meldrum (1995); Brazil & Pedley (1995)
Table 2. Effects of ligustroside against seizure threshold
on mice after given with CD90 metrazol
intraperitoneally
Ligustrosid Dose
(mg/kg BW, i.p)
100
200
300
400
No. of seizure
mice / no. of
total mice
20 / 20
16 / 20
10 / 20
2 / 20
Antiseizure
treshold
(%)
0
20
50
90
No. of dead
mice / no. of
total mice
14 / 20
8 / 20
2 / 20
0 / 20
Mortality rate
(%)
70
40
10
0
Table 3. Effects of ligustroside against seizure threshold
on mice after given with CD90 bicuculline
intraperitoneally
Ligustroside Dose
(mg/kg BW, ip)
No. of seizure mice /
no. of total mice
200
400
500
20 / 20
10 / 20
6 / 20
Antiseizure
treshold
(%)
0
50
70
Table 4. Effects of ligustroside against seizure threshold
on mice after given with CD90 picrotoxin
intraperitoneally
Ligustrosid Dose
(mg/kg BW, ip)
No. of seizure mice /
no. of total mice
200
400
500
20 / 20
14 / 20
6 / 20
Antiseizure
treshold
(%)
0
30
70
Table 5. Effects of ligustroside against seizure threshold
on mice after given with CD90 Strychnine
Nitrate intraperitoneally
Ligustrosid Dose
mg/kg BW, ip)
No. of seizure mice /
no. of total mice
500
20 / 20
Antiseizure
treshold
(%)
0
Pharmacodynamic Effects of Ligustroside Glycoside … (Achmad Basori)
Table 6. Effects of ligustroside against seizure threshold
on
mice
after
given
CD90
INH
intraperitoneally
Ligustrosid Dose
mg/kg BW, ip)
No. of seizure mice /
no. of total mice
500
20 / 20
Antiseizure
treshold
(%)
0
Table 7. ED50 antiseizure effects ligustroside, safety
ratio, and Protective Index on experimental
epileptic mice after being given with various
chemical epileptogen intraperitoneally
Epileptogen
Bicuculline
Metrazol
Picrotoxin
Strychnine Nitrate
INH
)* No effects
ED50
(mg/kg BW i.p)
S.R
(TD3/ED 97)
424.57
290.88
466.53
--- )*
--- )*
1.18
1.73
1.16
-
DISCUSSION
Based on those results as shown in Table 2, 3, 4, 5, and
6, it was proved that antiseizure mechanism of
ligustroside was to enhance GABA-ergic inhibitory
neuron in CNS (GABA-facilitated inhibition). As we
know that GABA-ergic inhibitory neuron can be
enhanced by several mechanism of actions, namely:
GABA release, inhibition of GABA destruction by
GABA-T enzyme, agonist effect at benzodiazepine
receptor, agonist effect at Barbiturate receptor, agonist
effect at GABA receptor, antagonist effect at picrotoxin
receptor, and antagonist effect at Cl ion channel (Brazil
& Pedley, Nestler et al. 1995; McDonald and Meldrum,
1995; Nestler et al. 2001; White et al. 1995). Further
research was needed to determine anti seizure
mechanism of ligustroside action.
This safety ratio (TD3/ED97) is an indicator of the most
effective anti seizure (ED97) with the less toxic effect
of substance. Based on SR values (Table 7), it was seen
that anti seizure potency of anti metrazol was more
potent than anti bicuculline and anti picrotoxin. This
means that anti metrazol effects could be sensitive
indicator of antiseizure effect of ligustroside against
chemically induced seizure. The previous research
showed that there was strong linear correlation between
Diazepam affinity and benzodiazepine derivation
against anti metrazol effect to benzodiazepine receptor
(Baestrup and Squires 1977; Okada 1977 1978; Speth et
al. 1979). Marangos et al (1979) proved that metrazol
displaced competitively binding between Diazepam and
benzodiazepine receptor. IC20 (20% Inhibition
Concentration)
and
IC50
(50%
Inhibition
Concentration) values of metrazol to displace
competitive binding between Diazepam with
benzodiazepine receptor was in the range of in vivo
level in causing seizure on animal. Both research
indicated that anti metrazol effect was an indicator of
interaction between a substance with benzodiazepine
receptor at GABA-benzodiazepine-Barbiturate-Picro
supramolecular complex. Furthermore, if these findings
was to be correlated with anti metrazol effect and anti
metrazol lethality test of ligustroside (Table 1), hence it
was strongly suspected that ligustroside might bind to
benzodiazepine receptor in CNS. Further research is
needed to explain the molecular interaction beetwen
ligustroside and GABA-benzodiazepine-BarbituratePicro supramolecular complex.
CONCLUSION
Pharmacodynamic study with chemical epileptogen
proved that ligustroside has an effect to increase seizure
threshold of chemically induced seizure on animals.
This effect indicated that ligustroside enhanced GABA–
ergic inhibitory system in CNS. Clinically, it is strongly
concluded that ligustroside would be very effective
against absence seizures and myoclonic seizures.
Further research will be needed to investigate deeply
further antiseizure mechanism of ligustroside.
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