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TABLE 1: ANTIFUNGAL ACTIVITY OF SOME PLANT EXTRACTS AGAINST 25 ISOLATES OF CANDIDA SPECIES
Candida species
C.
C.
C.
C.
C.
C.
albicans (15)a
glabrata (4)
tropicalis (2)
krusei (2)
parapsilosis (1)
guilliermondii (1)
S. jambolanum
C. siamea
500 250 100 10
500 250 100 10
(mg/ml)
3+ 2+
3+ 2+
3+ 2+
3+ 2+
3+ 2+
3+ 2+
(mg/ml)
3+ 2+
3+ 2+
3+ 2+
3+ 2+
3+ 2+
3+ 2+
4+
4+
4+
4+
4+
4+
-
4+
4+
4+
4+
4+
4+
O. wodier
-
500 250 100
2+
2+
2+
2+
2+
2+
(mg/ml)
-
S. wightii
C. scalpelliformis
10
500 250 100 10
500 250 100 10
-
(mg/ml)
3+
2+
3+
2+
3+
2+
3+
2+
3+
2+
3+
2+
4+
4+
4+
4+
4+
4+
-
4+
4+
4+
4+
4+
4+
(mg/ml)
3+ 2+
3+ 2+
3+ 2+
3+ 2+
3+ 2+
3+ 2+
-
a
indicates number of isolates; - indicates no activity; 1+ indicates zone of inhibition in average of 7 to 10 mm; 2+ indicates zone of inhibition in average of 11 to
14 mm; 3+ indicates zone of inhibition in average of 15 to 18 mm; 4+ indicates zone of inhibition in average of 19 to 22 mm.
Candida is a difÞcult organism to establish standards
of therapeutic activity. The problem is compounded
for plant extracts in which variation may be expected
between samples depending on genotype, area of
cultivation, time of harvesting, processing methods,
dilution etc. The extracts in our study are crude
and that may be the reason for the antifungal
activity of these extracts up to 100 mg/ml for the
S. jambolanum, C. siamea and C. scalpelliformis.
Probably, a more refined preparation would have
antifungal activity at a lower concentration. Further
experimental and clinical trials should be carried out
and if these antifungals are effective they could be
used as therapeutic agents.
REFERENCES
1.
2.
3.
4.
Kwon-Chung KJ, Bennet JE. Medical Mycology: Candidiasis,
Philadelphia: Lea and Febiger; 1992. p. 280-336.
McFadden R. Comparison of the inhibitory effects of various plant
extracts on the growth of Candida albicans in vitro. Eur J Herbal Med
1995;1:26-31.
Daliya JS, Rimmer RS. Phytoalexin accumulation in tissue of
Brassica napus inoculated with Leptosphaeria maculans. Phytochemistry
1988;27:3105-7.
Darvill AG, Albersheim P. Phytoalexins and their elicitors: A
defense against microbial infection in plants. Ann Rev Plant Physiol
1984;35:243-75.
5. Pesando D, Caram B. Screening of Marine Algae from the French
Mediterranean coast for antimicrobial and antifungal activity. Bot Mar
1984;381-6.
6. Milne LJR, Fungi. In: Collee JG, Fraser AG, Marmion BP, Simmons A,
editors. Mackie and MacCartney. Practical Medical Microbiology. 14th
ed. New York: Churchill Livingstone; 1996. p. 695-720.
7. Vanden Berghe DA, Vlietinck AJ, Van Hoof L. Plant products as
potential antiviral agents. Bull Inst Pasteur 1986;84:101-47.
8. Kronvall G, Karlsson I. Fluconazole and Voriconazle Multidisk testing
of Candida species for disk test calibration and MIC Estimation. J Clin
Microbial 2001;39:1422-8.
9. Klein RS, Harris CA, Small CB, Moll B, Lesser M, Friedland GH.
Oral candidasis in high risk patients as the initial manifestation of the
acquired immunodeÞciency syndrome. N Engl J Med 1984;311:354-8.
10. Barr CE, Torosian JP. Oral Manifestations in patients with AIDS or
AIDS- related complex. Lancet 1986;2:288.
11. Bennet JE. Harrison’s principles of internal medicine In: Brwunwald,
E, Fauci AS, Kasper DS. Hauser SL, Lango DL, Jameson JL. editors.
Candidiasis. 15th ed. Vol. 1, New Delhi: McGraw–Hill; 2001. p.
1176-8.
12. Perumalsamy R, Ignacimuthu S, Sen A. Screening of 34 Indian
Medicinal Plants for Antibacterial Properties. J Ethnopharmacol
1988;62:173-81.
Accepted 10 December 2008
Revised 26 May 2008
Received 30 May 2006
Indian J. Pharm. Sci., 2008, 70 (6): 801-803
Evaluation of CNS Depressant Activity of Different Plant
parts of Nyctanthes arbortristis Linn.
SANJITA DAS*, D. SASMAL AND S. P. BASU
Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi-835 215, India
Das, et al.: CNS depressant activity of Nyctanthes arbortristis Linn.
*For correspondence
E-mail: [email protected]
November - December 2008
Indian Journal of Pharmaceutical Sciences
803
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The present study was carried out with the water-soluble portion of the ethanol extracts of flowers, barks, seeds
and leaves of Nyctanthes arbortristis Linn. to confirm their CNS depressant activity. The ethanol extracts of the
plant parts were obtained by soxhlet extraction. After performing the gross behavioral study, the CNS depressant
activity was evaluated by observing the prolongation of sleeping time induced by pentobarbital sodium in mice.
Attempts have been made to explore the possible mechanism behind this activity by determining their effect on
brain monoamine neurotransmitters like dopamine and serotonin. The gross behavioral study showed that ethanol
extracts of the leaves, flowers and seeds possess significant CNS depressant activity. The leaves, flowers, seeds and
barks (600 mg/kg) showed significant and dose-dependent prolongation of onset and duration of sleep and so found
to cause decrease dopamine and increase serotonin level. From which it can be concluded that the CNS depressant
activity of the ethanol extracts of seeds, leaves and flowers may be due to the decrease in dopamine and increase
in serotonin level.
Key words: Nyctanthes arbortristis L., behavioral study, CNS depressant activity, histamine, serotonin
Nyctanthes arbortristis Linn. (Family Oleaceae),
commonly known as Harsingar or Night Jasmine,
is a common wild hardy large shrub or small tree1,2.
Nyctanthes arbortristis L. is used by the rural people
of Orissa in India to cure various ailments along
with its use in Ayurveda, Sidha and Unani systems
of medicines. It’s claimed traditional uses have been
proved on scientiÞc basis using in vitro and in vivo
experiments3-7. It has been established that its leaves
possess hypnotic and tranquillizing activities3,4 and its
ßowers possess sedative activity5. The present study is
aimed at the CNS depressant activity of the ethanol
extracts of different parts (ßowers, barks, seeds and
leaves) of Nyctanthes arbortristis L. along with an
attempt explore the responsible mechanism.
The ßowers, barks, seeds and leaves of Nyctanthes
arbortristis L. were collected from the garden of BIT,
Mesra, Ranchi and forests of Orissa. The herbarium
of the plant (CNH/I-I (20)/2005-Tech-II/254) was
authenticated as Nyctanthes arbortristis L. from
Botanical Survey of India, Kolkata. After drying
properly, the leaves, barks and seeds were powdered
coarsely and then were extracted successively with
petroleum ether, chloroform and ethanol (90%) 6,7,
whereas its fresh flowers with ethanol (50%)7. The
ethanol extracts were evaporated to dryness, having
yield values 14%, 12.5%, 26.5% and 13% w/w,
respectively. The water-soluble portions of the extracts
were subjected to the pharmacological screening.
Adult male swiss mice weighing between 20-30
g, obtained from the animal house of BIT, Mesra,
Ranchi, were used for this investigation. The
Institutional Animals Ethics Committee (Registration
No. 62/02/ac/CPCSEA) approved the experiments.
Up and down or staircase method was followed for
the estimation of acute toxicity of the water-soluble
804
portion of the ethanol extracts of different parts of
Nyctanthes arbortristis L. The dose was increased
from 400 mg/kg to 2.0 g/kg, through intraperitoneal
route of administration8,9.
The gross behavioral study was performed 30
min after the administration of the extracts to get
maximum information about the effect of the extracts
on the central nervous system of mice9. The control
group of animals was only treated with pentobarbital
sodium (45 mg/kg). Each extracts and the reference
compound were injected 30 min before pentobarbital
sodium administration. The time taken for the loss
of righting reßex was noted in all cases. The onset
of sleep was recorded by noting the time of loss
of righting reflex of mice and duration of sleep by
noting time difference between loss of righting reßex
and recovery time10.
The monoamine neurotransmitters in brain were
estimated following the method described by
Shellenderger et al.11 The animals were sacriÞced half
an hour after the administration of the extracts by
cervical dislocation. After chilling the head of mice
in chilling mixture of ice and CaCl2, their brains were
taken out and weighed11,12.
For the estimation of dopamine, brains were
homogenized with dry n-butanol at 0 o . Clear
supernatant solutions were extracted with 0.1 M
phosphate buffer. To the phosphate buffer extract 4%
EDTA, Iodine solution, alkaline sulÞte and 5N acetic
acid were added and then heated. After cooling, the
intensities of fluorescence were determined by the
help of photoßuorometer11,12.
For the estimation of serotonin, brains were
homogenized with 0.1N HCl at 0 o and then
Indian Journal of Pharmaceutical Sciences
November - December 2008
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centrifuged. To the clear supernatant solutions,
10% zinc sulfate and 1N NaOH were added and
then centrifuged. To a quartz cuvette containing
2N HCl the supernatant fluid was added and its
intensity of the ßuorescence was determined by the
photofluorometer 11,12. The results were expressed
in mean±standard error of mean. The results were
subjected to statistical analysis, using ANOVA to
determine the signiÞcance of the tested activity and
p<0.001 were considered to be signiÞcant10.
Toxicity studies revealed that the maximum tolerable
dose for the water-soluble portion of the ethanol
extracts was more than 2.0 g/kg body weights.
TABLE 1: PROLONGATION OF PENTOBARBITALINDUCED SLEEPING TIME BY THE ETHANOL EXTRACTS
OF DIFFERENT PARTS OF NYCTANTHES ARBORTRISTIS
LINN.
Treatment
Pentobarbitone
Chlorpromazine
NAF
NAB
NAS
NAL
Dose
(mg/kg)
45
3
200
400
600
200
400
600
200
400
600
200
400
600
Onset of Sleep
(min)
7.75±0.63
4.75±0.48*
5.75±0.25
5.50±0.29
5.00±0.205*
7.75±0.79
7.25±0.855
8.75±0.48
6.00±0.41
4.25±0.48*
4.25±0.25*
3.75±0.25*
3.75±0.48*
3.50±0.29*
Duration of Sleep
(min)
56.25±1.04
128.25±1.89*
81.00±1.87*
89.50±2.06*
101.75±2.135*
70.25±2.625
75.25±1.03*
84.75±1.88*
83.50±1.89*
84.25±2.17*
100.5±3.07*
86.75±2.84*
89.00±1.58*
106.25±0.95*
Values are expressed in Mean±SEM, n = 6, *p<0.001. NAF, NAB, NAS and NAL
represent the ethanol extracts of the ßowers, barks, seeds and leaves of
Nyctanthes arbortristis respectively.
From which, doses of 200, 400 and 600 mg/kg were
selected for the evaluation of CNS depressant activity
of the extracts. The gross behavioral study on mice
of the extracts showed significant CNS depressant
activity and some muscle relaxant activity. The leaves,
ßowers and seeds showed signiÞcant (p<0.001) and
dose-related prolongation of the onset and duration
of sleep, which are well comparable to that of the
standard drug chlorpromazine (Table 1). It has
been observed that the leaves possess highest CNS
depressant activity.
The changes in dopamine and 5-hydroxytryptamine
(5-HT) levels, expressed as percentage increase
or decrease from their respective control values,
are presented in Table 2. The ethanol extracts
of the leaves, seeds and flowers of Nyctanthes
arbortristis L. were found to cause decrease in
dopamine and increase in 5-HT level significantly
in a dose-dependent manner. Its barks were found
to cause these changes only at its higher dose of
600 mg/kg. Decrease in dopamine level may lead
to the development of catalepsy and depression in
the brain 13. This can be well correlated with the
antipsychotic activity of the extracts.
Many antipsychotic agents also have some affinity
for 5-HT receptors and drugs, which increase brain
5-HT and usually increase sleep14. This is evident in
case of the extracts treatment where the 5-HT level
is increased. This can be well correlated with the
potentiation of pentobarbitone-induced sleeping time
in mice by the extracts, which could be accounted to
TABLE 2: EFFECT OF THE WATER-SOLUBLE FRACTION OF THE ETHANOL EXTRACTS OF NYCTANTHES
ARBORTRISTIS ON MONOAMINE NEUROTRANSMITTERS IN THE BRAIN OF MICE
Treatment
Dose (mg/kg)
Control
(Water for injection)
Chlorpromazine
NAF
NAB
NAS
NAL
—
3
200
400
600
200
400
600
200
400
600
200
400
600
Dopamine
Serotonin
Level in
brain tissue
Percentage
decrease
Level in
brain tissue
Percentage
decrease
0.433±0.016
0.205±0.01*
0.328±0.01*
0.283±0.021*
0.243±0.071*
0.413±0.035
0.400±0.016
0.33±0.014*
0.358±0.005*
0.295±0.018*
0.25±0.008*
0.313±0.001*
0.273±0.065*
0.233±0.015*
—
56.66
24.25
34.25
44.0
4.62
7.62
23.79
17.44
31.87
42.26
27.83
37.07
46.19
0.343±0.01
0.693±0.053
0.423±0.013*
0.488±0.009*
0.635±0.01*
0.345±0.017
0.328±0.009
0.44±0.014*
0.400±0.0.016
0.528±0.015*
0.588±0.008*
0.448±0.005*
0.525±0.018*
0.638±0.012*
—
102.04
23.32
42.27
85.13
0.58
4.37
28.28
16.62
53.94
71.43
30.47
53.06
86.01
Values are expressed in Mean±SEM; *p<0.001. NAF, NAB, NAS and NAL represent the water-soluble fraction of the ethanol extracts of the ßowers, barks, seeds and
leaves of Nyctanthes arbortristis, respectively.
November - December 2008
Indian Journal of Pharmaceutical Sciences
805
www.ijpsonline.com
the increase in the concentration of 5-HT in the brain.
The overall study conÞrms that the CNS depressant
action and the potentiation of pentobarbitone-induced
sleeping time by the ethanol extracts of flowers,
seeds, leaves and barks (600 mg/kg) of Nyctanthes
arbortristis L. might be due to the decrease in
dopamine and increase in the 5-hydroxytryptamine
level in brain.
5.
6.
7.
8.
9.
10.
ACKNOWLEDGEMENTS
Authors would like to express sincere thanks to Dr
S. K. Dutta and the faculty of SIPS, Jharpokharia,
Orissa. Faculty of BIT, Mesra, Ranchi are also
thankfully acknowledged.
11.
12.
REFERENCES
13.
1.
14.
2.
3.
4.
Chopra RN, Nayar SL, Chopra JC. Glossary of Indian Medicinal
Plants. New Delhi: Publication and Information Directorate, CSIR;
1992.
Kirtikar KR, Basu BD. Indian Medicinal Plants, Vol.7. New Delhi: Sri
Satguru Publications; 2000.
Saxena RS, Gupta B, Lata S. Tranquillizing, antihistaminic
and purgative activity of Nyctanthes arbortristis leaf extract. J
Ethnopharmacol 2002;81:321-5.
Deshmukh VK, Juvekar AR. Anti-stress, antianxiety and nootropic
activity of Nyctanthes arbortristis leaves. Planta Medica 2006. p. 72.
Ratnasooriya WD, Jayakody WD, Hettiarachchi ADI, Dharmasiri MG.
Sedative effects of hot ßower infusions of Nyctanthes arbortristis on
rats. Pharm Bio 2005;43:140-6.
Patel UM, Patel KM, Patel MP, Bhavasar GC. Anti-inflammatory
activity of Nyctanthes arbortristis. Indian J Nat Prod 1998;14:9-11.
Chitravansi VC, Singh AP, Ghosal S, Krishnaprasad BN, Srivastava V
Tandon JS. Therapeutic action of Nyctanthes arbortristis against Caecal
Amoebiasis of rat. Int J Pharmacog 1992;30:71-5.
LitchÞeld JT, Jr, Wilcoxon F. A simpliÞed method of evaluating doseeffect experiments. J Pharmacol Expt Ther 1949;96:99-113.
Gupta M, Sasmal D. Behavioral Pharmacology of Ocratoxin-A in mice,
Nerv System, Pharmacol, Psychol Psychiatry. IRCS Medical Science
1979;7:454.
Ghosh MN. Fundamentals of Experimental Pharmacology, 3rd ed.
Kolkata: Hilton and Company; 2005.
Shellenderger MK, Gordon JH. A rapid, simplified procedure
for simultaneous assay of norepinephrine, dopamine and
5-hydroxytryptamine from discrete brain areas. Analytical Biochem
1971;39:356-72.
Gupta M, Sasmal D, DuttaGupta S, Bagchi GK, Changes of
Epinephrine, Norepinephrine, Dopamine in mice brain and liver and
5-Hydroxytryptamine in mice brain following treatment with Ochratoxin
A and Citrinin. Indian J Pharmacol 1984;16:102-6.
Keele CA, Neil E, Norman J. Samson Wright’s Applied Physiology,
13th ed. New York: Oxford University Press, Oxford; 2005.
Tripathi KD. Essentials of Medicinal Pharmacology, 5th ed. New Delhi:
Jaypee Brothers, Medical Publishers (p) Ltd.; 2003.
Accepted 10 December 2008
Revised 29 May 2008
Received 17 January 2006
Indian J. Pharm. Sci., 2008, 70 (6): 803-806
Effect of Withania somnifera on Sleep-Wake Cycle in
Sleep-Disturbed Rats: Possible GABAergic Mechanism
A. KUMAR* AND H. KALONIA
University Institute of Pharmaceutical Science, Panjab University, Chandigarh-160 014, India
Kumar, et al.: Withania somnifera and possible GABAergic mechanism
Sleep deprivation disrupts significantly sleep pattern and cause poor quality of sleep. The aim the present study was
to explore role of Withania somniferra root extract in sleep-disturbed rats. Male wistar rats (n=5-6/group) were sleep
deprived for 24 h using grid suspended over water method. Withania somniferra extract (100 mg/kg) was administered
intraperitoneally (i.p.) 30 min before actual recording (EEG and EMG) recording and electrophysiological recordings
are further classified as- sleep latency, slow wave sleep, paradoxical sleep, total sleep, wakefulness. One day (24 h)
sleep deprivation delayed latency sleep, reduced duration of slow wave sleep, rapid eye movement sleep, total sleep
time and increased total waking as compared to animals placed on saw dust (P<0.05). Pretreatment with Withania
somniferra extract (100 mg/kg) and diazepam (0.5 mg/kg) significantly improved electrophysiological parameters,
which was further reversed by picrotoxin (2 mg/kg) and potentiated by muscimol (0.05 mg/kg). Flumazenil (2
mg/kg) did not produce any significant effect on the sleep parameters of Withania somnifera root extract. Present
*For correspondence
E-mail: [email protected]
806
Indian Journal of Pharmaceutical Sciences
November - December 2008