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RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 72
ANTIDEPRESSANT ACTIVITY AND CYTOTOXIC EFFECTS
OF LANNEA COROMANDELICA. LEAF EXTRACT
1MD. Mamunur Rashid*
Department of Pharmacy, Stamford University Bangladesh, Dhaka, BANGLADESH
Corresponding Author
MD. Mamunur Rashid
Stamford University Bangladesh
51, Siddeswari Road,
Dhaka-1217, BANGLADESH
E-mail: [email protected]
Phone: +8801723533704
International Journal of Innovative
Pharmaceutical Sciences and Research www.ijipsr.com
Abstract
The current study describes that ethanolic extract of Lannea coromandelica (Family:
Anacardiaceae, Local name: jiga) leaf was subjected to evaluate its Phytochemical screening,
Brine shrimp lethality bioassay and CNS depressant activity. The leaves containes alkaloids,
flavonoids, tannins and saponins ete. The evaluation of brine shrimp lethality bioassay on Artemia
salina suggestes moderate cytotoxicity in which extract of leaves and Vincristine sulfate
produced lethality (LD50) at 0.15 µg/ml and 0.05 µg/ml concentrations respectively. The CNS
depressant activity of lannea coromandelica was observed using Hole Cross and Open Field
method on mice model at doses 25mg/kg, 50 mg/kg and 100mg/kg body weight while positive
control test was performed using Diazepam (1mg/kg) as standard drug. The test groups including
both Hole Cross and Open field test shows significant neuropharmacological activity as compared
with the result of standard drug.
Keywords: Lannea coromandelica, Brine Shrimp Lethality Bioassay, CNS depressant activity,
LD50, Cytotoxicity, Phytochemical screening.
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 73
INTRODUCTION
A medium –sized deciduous tree named Lannea coromandelica is located in tropical Asia, mostly
distributed in India and Africa. It belongs to the family Anacardiaceae. Apparently it looks like
ashy-grey, bark thick and leaves crowded at the end of branches. It was commonly called Indian
ash tree. It has several Bengali names such as Jiga, jika, Jeol, Bhadi, Kamila, Kaliengla etc. It has
been used by tribal people as medicinal plant for various diseases from ancient time to recent
time. Its bark used as astringent and stomachic, used as a lotion in impetigenous, eruption, leprous
and skin eruption heart disease, dysentery and mouth sores. Boiled leaves are applied as a
fomentation for local swelling and pain. The main chemical constituents of leafs are flavonols like
quercetin, iso-quercetin, kaempferol, glycosides etc and polyphenols like tannins, gallicacid, also
contain leucocyanidin, leucodelphinidin. Flowing gum composed of D-galactose and L-arabinose.
Bark contains phlobatannins, β-sitosterol, physcion, physcionanthranol B, dl-epicatechin. The
objective of this study was to examine the neuropharmacological effects and cytotoxic activity of
ethanolic extract of Lannea coromandelica leaves on the central nervous system of mice and
brine shrimp nauplii respectively. From ancient time to present day, the most widely used group
of pharmacologic agents are CNS depressants that were discovered by primitive people where
the agents has a proof, acting on Central nervous system. The effects of drugs on the central
nervous system with reference to the neurotransmitters for specific circuits, attenuation should be
developed to general organizational principles of neurons. The view that synapses represent drug-
modifiable control points within neuronal networks. It requires explicit delineation of the sites at
which given neurotransmitters may operate and the degree of specificity with which such sight
that may be affected. The specialized cancer agency of the WHO named The International
Agency for Research on Cancer (IARC) describes the latest world cancer statistics including
Global cancer burden rises to 14.1 million new cases and 8.2 million cancer deaths in 2012 where
markedly increases breast cancer. So new search for anti-cancer therapy is an important issue.
Among many recent advances in cancer chemotherapy, phytochemicals play an important role in
cancer chemotherapeutic drugs. As a search for new anti-cancer drugs, the brine shrimp lethality
bioassay is efficient, rapid and inexpensive tests that require only a least amount of sample. The
technique is easily performed and utilizes small amount of test materials which has been
successively employed for in-vivo lethality bioassay-guide fractionation of active cytotoxic and
antitumor agents [1-14].
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 74
MATERIALS AND METHODS
Lannea coromandelica leaves were collected from Bangladesh Nation Herbarium Institute,
Mirpur, Dhaka. They delivered the leaves after ensuring that the leaves are taken from Lannea
coromandelica tree which belongs to the family Anacardiaceae. The time of leaves collection was
October 2013. They also provide an accession number after proper identification. Look at a
glance in the following table.
Table 1: Local Name, Botanical Name & Accession Number of the Plant
Local Name Botanical Name & Family Accession Number
Jiga Lannea coromandelica
Anacardiaceae 39192
The leaves of Lannea coromandelica were first free from undesirable or waste materials. They
were dried on moderate heat emitted from the sun. After Proper drying the leaves were grinded by
Blender Machine (NOWAKE, JAPAN).Coarse powers were obtained after crushing of leaves.
The material which were used in this experiment is arranged in their category.
Table 2: Apparatus used in the Experiment
Serial No. Apparatus Name Source
1 Vortex Mixture China
2 LC Oven LAB-LINE USA
3 Syringe Opso saline Ltd.Bangladesh
4 Feeding Needde Local Made
5 Digital weighing Balance Denver Instrument Company USA
6 Cotton India
7 Blender NOWAKE,JAPAN
8 Aluminium Foil DIAMOND ,USA
9 Filter Paper 11 cm DIA,USA
10 Marker Pen RED LEAF,JAPAN
11 Thermostatic Water Bath Shanghai,China
Many chemicals were used as solvent which are listed in the followingTable
Table 3: List of Chemicals Used for the experiment
Serial
No. Name of Solvent & Chemicals Source
1 Ethanol Marc,Germany
2 Distilled Water Laboratory Prepared
3 Diazepam and Vincristin Sulfate Pharmacy shop
4 Di-methyl sulfoxide(DMSO) Marc,Germany
Different types of glassware were used during experimentation, these are listed bellow:
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 75
Table 4: List of glassware used
Serial No. Apparatus Name Source
1 Volumetric Flask Changdu, Chaina
2 Test tube Changdu, Chaina
3 Beaker Changdu, Chaina
4 Funnel Changdu, Chaina
5 Measuring Cylinder Changdu, Chaina
6 Glass Rod Changdu, Chaina
The special type of subject has been used for this experiment. Details are given bellow:
Table 5: Detail information of the subject
Serial No Name of the Animal Source
1 Swiss albino mouse
Weight 20-26 gm
Pharmacology Laboratory, Jahangirnagar
University, Savar, Dhaka.
2 Artema salina leach(brine
shrimp eggs)
Shomudra Bilash pet shop Katabon,
Dhaka.
The mice were given special type of Chocolate food which was supplied by the ICDDR’B
Table 6: Type of food used for the mice
Open Field Method
In Neuropharmacological study, Open Field method is one of the most important method for
determining neuropharmacologiacl activity of investigated rodents specially mice through
inspection of behavioral characteristics after application of the respective agents.
The mice were divided into three groups named vehicle group, control group and test group. Each
group containing five mice and it is remarked the number of fields crossed by each group during
experiment. The observation was done at a various time intervals like 0, 30, 60, 90 and 120
minutes after administration of drug.
Hole- Cross Method
The most consistent behavioral change is a hyperemotional response to novel environmental
stimuli. In this test the mice were categorized into three groups named vehicle group, control
group and test group and each group containing five mice. During experiment the number of
Name of the Animal Feed Source
Mice pellets (Chocolate Food) ICDDR’B, Animal House, Mohakhali, Dhaka,
Bangladesh.
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 76
chamber crossed was noted to make a assessable data. The reading was taken at different times
interval like 0, 30, 60, 90 and 120 minutes for a specified time
Brine Shrimp Lethality Bioassay
Brine shrimp eggs were hatched in stimulated sea water to get nauplii. Test samples were
prepared by dissolving in DMSO solvent and make a solution of different concentrations which
was required in this investigation.
Hatching of brine shrimp eggs were observed through visual inspection for 48 hours. After
hatching ten nauplii were taken in a test tube containing 5ml of stimulated sea water. Then added
test samples of different concentrations and observed for 24 hour to point out the toxic effect of
leave extract that made the test samples on shrimp nauplii.
Median lethal dose, LD50 was calculated to determine the toxic effect of leave extract.
Preparation of test solutions with samples of experimental plant
At first 4 mg of leaf extract of lannea coromandelica were taken and dissolved in 100µl DMSO in
a test tube to get stock solutions. Then 50µl of solution was taken in another test tube containing 5
ml sea water and 10 nauplii. Thus the concentration of first solution was 400µg/ml. Then a series
of solution of varying concentration were prepared by serial dilution method. By this way
solutions of different concentrations were obtained. These are given bellow:
400µg/ml
200µg/ml
100µg/ml
50µg/ml
25µg/ml
12.5µg/ml
6.25µg/ml
3.125µg/ml
1.5625µg/ml
0.78125µg/ml
Preparation of control group
In order to validate the test method and make sure that the result obtained from the experiment is
only due to the cytotoxic activity of the investigated leaf extract solution, usually two types of
control groups are used.
I. Positive control
II. Negative control
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 77
Preparation of positive control group
Positive control group uses Vincristine sulphate as the standard drug in cytotoxicity study. Test
groups result is compared with the result obtained from Vincristine sulphate as cytotoxic agent to
make a assessment of cytotoxicity of the test sample. Measured amount of Vincristine sulphate is
dissolved in DMSO to get an initial concentration of 20µg/ml from which serial dilutions are
made using DMSO to get -
10µg/ml
5µg/ml
2.5µg/ml
1.25µg/ml
0.625µg/ml
0.3125µg/ml
0.15625µg/ml
0.078125µg/ml
0.0390µg/ml
Preparation of negative control group
30 µl of DMSO was added to each of three premarked test tube containing 5 ml of stimulated sea
water and 10 shrimp nauplii to use as negative control group. If the brine shrimps in these vials
show a rapid mortality rate, then the test is considered invalid as the nauplii died due to some
reason other than the cytotoxicity of the compounds.
Counting of nauplii
After 24 hours later, the test tube were inspected using a magnifying glass and the number of
survived nauplii in each test tube was counted. From those data, the percentage of the brine
shrimp nauplii was calculated for each concentration.
Fig.1: Movement of Brine Shrimp Nauplii.
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 78
RESULTS AND DISCUSSION
The investigated study was done to evaluate the Neuropharmacologic action on mice model and
Cytotoxic effects on brine shrimp nauplii. The important way to evaluate whether a drug acting
on central nervous system (CNS) or not, is to observe the locomotor activity of the animal. The
activity is a measure of the level of excitability of the CNS and this decrease may be closely
related to sedation resulting from depression of the central nervous system. Leaf extract of
Lannea coromandelica decreased the locomotor activity as shown by the results of the Open Field
and Hole Cross Tests with Graphical representation. The locomotor activity lowering effect was
noted at the 2nd observation (30 min) and continued up to 5th
observation period (120 min). Both
hole cross and open field tests showed that the depression action of the extracts was evident from
the 2nd
observation period in the test animals at the doses of 25mg/kg b.w, 50mg/kg b.w and 75
mg/kg b.w. Maximum effect was observed from 3rd
(60 min) to 5th
(120 min) observation period
as comparable to the reference drug Diazepam at a dose of 1mg/kg body weight.
Table 7: Primary Data Table (Open Field Test for leaf extract)
Group Time Number of field crossed
Mean SD SEM M1 M2 M3 M4 M5
Control
(DMSO)
0 min 70 51 44 54 9 45.6 20.18 9.02
30 min 38 24 32 45 24 32.6 8.13 3.64
60 min 41 7 30 16 17 22.2 11.92 5.33
90 min 14 25 18 43 12 22.4 11.21 5.01
120 min 19 18 3 0 11 10.2 7.67 3.43
Positive
Control
(Diazepam
1mg/kg)
0 min 110 139 132 44 86 102.2 34.5 15.43
30 min 35 26 61 3 38 32.6 18.74 8.38
60 min 74 30 42 37 15 39.6 19.45 8.69
90 min 9 31 17 19 13 17.8 7.44 3.33
120 min 100 99 13 14 9 47 42.89 19.18
Group-i
(Extract
25mg/kg)
0 min 48 39 37 6 54 36.8 16.58 7.41
30 min 21 7 24 0 26 15.6 10.24 4.58
60 min 7 15 52 0 21 19 17.96 8.03
90 min 0 9 2 3 20 6.8 7.24 3.24
120 min 3 15 13 6 4 8.2 4.87 2.18
Group-ii
(Extract
50mg/kg)
0 min 72 91 101 55 80 79.8 15.81 7.07
30 min 36 65 50 36 0 37.4 21.55 9.64
60 min 15 60 51 12 1 27.8 23.26 10.4
90 min 3 5 23 5 0 7.2 8.10 3.62
120 min 3 3 33 0 0 7.8 12.67 5.66
Group-iii
(Extract
100mg/kg)
0 min 67 91 57 59 15 57.2 24.58 10.9
30 min 51 33 0 60 71 43 24.86 11.1
60 min 26 11 25 24 24 22 5.54 2.48
90 min 7 34 14 16 0 14.2 11.39 5.09
120 min 3 25 2 9 18 11.4 8.86 3.96
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 79
Table 8: Effect of leaf extract of Lannea coromandelica (Open field test)
Group
Route of
Adminis-
tratiion
Dose
(mg/kg) Observation
0 min 30 min 60 min 90 min 120 min
Control Oral -- 45.6 ±
9.02
32.6 ±
3.64
22.2 ±
5.33
22.4 ±
5.01
10.2 ±
3.43
Positive
Control IP 1
102.2 ±
15.4
32.6 ±
8.38
39.6 ±
8.69
17.8 ±
3.33
47 ±
19.18
Group-i Oral 25 36.8 ±
7.41
15.6 ±
4.58
19 ±
8.03
6.8 ±
3.24
8.2 ±
2.18
Group-ii Oral 50 79.8 ±
7.07
37.4 ±
9.64
27.8 ±
10.4
7.2 ±
3.62
7.8 ±
5.66
Group-iii Oral 100 57.2 ±
10.9
43 ±
11.1
22 ±
2.48
14.2 ±
5.09
11.4 ±
3.96
Values are expressed as Mean ± SEM (n=5)
IP= Intra-peritoneal
Graphical Representation of Leaf Extract for open field method
The graphical representation of Open Field Test data are shown in Fig 1 & Fig 2
Fig. 2: Bar diagram of Open Field Test Fig. 3: Line diagram of Open Field Test
result for leaf extract result for leaf extract
Table 9: Primary Data Table (Hole Cross Test for leaf extract)
Group Time
Number of Chamber Crossed
Mean SD SEM
M1 M2 M3 M4 M5
Control
(DMSO)
0 min 16 14 7 13 18 13.6 3.72 1.66
30 min 7 6 1 4 0 3.6 2.72 1.22
60 min 5 4 1 2 1 2.6 1.62 0.72
90 min 7 2 0 8 2 3.8 3.17 1.42
45.6
102.2
36.8
79.8
57.2
32.6 32.6
15.6
37.443
22.2
39.6
1927.8
2222.4 17.86.8 7.2
14.210.2
47
8.2 7.8 11.4
control Positive Group-i Group-ii Group-iii
Re
spo
nse
Response by test groups
0 min 30 min 60min 90 min 120 min
0
20
40
60
80
100
120
0 min 30 min 60min 90 min 120 min
Re
sp
on
se
Time Interval
Response by test groups
Control
Positive
Group-i
Group-ii
Group-iii
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 80
120 min 0 0 0 3 0 0.6 1.20 0.54
Positive
Control
(Diazepam
test 1mg/kg)
0 min 16 22 10 23 15 17.2 4.79 2.14
30 min 14 7 4 13 7 9 3.84 1.72
60 min 6 8 0 6 11 6.2 3.60 1.61
90 min 1 12 4 5 5 5.4 3.61 1.61
120 min 1 5 14 2 8 6 4.60 2.06
Group-i
(Extract
25mg/kg)
0 min 11 2 11 18 11 10.6 5.08 2.27
30 min 6 12 0 5 5 5.6 3.82 1.71
60 min 5 11 0 4 4 4.8 3.54 1.58
90 min 4 10 3 0 2 3.8 3.37 1.51
120 min 0 6 3 2 4 3 2.00 0.89
Group-ii
(Extract
50mg/kg)
0 min 12 10 0 11 3 7.2 4.79 2.14
30 min 7 2 0 6 1 3.2 2.78 1.24
60 min 8 5 3 4 1 4.2 2.31 1.03
90 min 5 5 7 4 5 5.2 0.97 0.43
120 min 2 3 3 4 3 3 0.63 0.28
Group-iii
(Extract
100mg/kg)
0 min 2 9 4 5 6 5.2 2.31 1.03
30 min 2 1 0 0 2 1 0.89 0.40
60 min 3 1 0 0 5 1.8 1.93 0.86
90 min 4 3 0 0 1 1.6 1.62 0.72
120 min 4 1 0 1 7 2.6 2.57 1.15
Table 10: Effect of leaf extract of Lannea coromandelica (Hole cross test)
Group Route of Adminis-
tration
Dose
(mg/kg) Observation
0 min 30 min 60 min 90 min 120 min
Control Oral -- 13.6 ±
1.66
3.6 ±
1.22
2.6 ±
0.72
3.8 ±
1.42
0.6 ±
0.54
Positive
Control IP 1
17.2 ±
2.14
9 ±
1.72
6.2 ±
1.61
5.4 ±
1.61
6 ±
2.06
Group-i Oral 25 10.6 ±
2.27
5.6 ±
1.71
4.8 ±
1.58
3.8 ±
1.51
3 ±
0.89
Group-
ii Oral 50
7.2 ±
2.14
3.2 ±
1.24
4.2 ±
1.03
5.2 ±
0.43
3 ±
0.28
Group-
iii Oral 100
5.2 ±
1.03
1 ±
0.40
1.8 ±
0.86
1.6 ±
0.72
2.6 ±
1.15
Values are expressed as Mean ± SEM (n=5)
IP= Intra-peritoneal
Graphical Representation of leaf extract for Hole Cross Method
The graphical representation of Open Field Test data are shown in fig.3 & fig. 4
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 81
Fig. 4: Bar diagram of Hole Cross Test Fig .5: line diagram of Hole Cross Test
result for leaf extract result for leaf extract
The cytotoxicity of the ethanolic, leaf extract of Lannea coromandelica to brine shrimp were
determined and summarized in the following table.
Table 11: Effect of ethanolic extract of leaf and vincristine sulphate on brine shrimp nauplii
Test Solution Conc.(µg/ml) Log conc. % of Mortality LC50 (µg/ml)
Ethanolic
extract of leaf
400 2.602 100
0.15
200 2.31 100
100 2 100
50 1.698 100
25 1.396 90
12.5 1.097 90
6.25 0.795 80
3.125 0.494 80
1.562 0.193 70
0.781 -0.107 60
Vincristine
Sulphate
20 1.301 100
0.05
10 1.000 100
5 0.698 90
2.5 0.397 90
1.25 0.096 80
0.625 -0.204 70
0.3125 -0.505 60
0.15625 -0.806 50
0.078125 -1.107 40
0.0390 -1.408 30
The above table gives the results of the brine shrimp lethality after 24 hour exposure to all the
samples and the positive control, Vincristine sulfate. The positive control gives remarkable
mortality rate to brine shrimp as compared with negative control group.
13.6
17.2
10.6
7.25.2
3.6
9
5.6
3.21
2.6
6.24.8 4.2
1.83.8
5.43.8
5.2
1.60.6
6
3 3 2.6
Control Positive Group-i Group-ii Group-iii
Re
spo
nse
Response by test groups
0 min 30 min 60 min 90 min 120 min
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 82
Fig. 6: Effect of ethanolic extract on Fig. 7: Effect of Vincristine Sulfate on
Brine Shrimp nauplii Brine Shrimp nauplii
The lethal concentration LC50 of the test samples after 24 hr. was obtained by a plot of
percentage of the shrimps killed against the logarithm of the sample concentration and the best- fit
line was obtained from the curve data by means of regression analysis. The analyzed data is given
in the following table.
Table 12: Regression analysis data for Vincristine sulfate and ethanolic extract of leaf
Sample LC50 Regression Equation R2
Vincristine
Sulphate 0.15 Y=27.186x + 72.463 0.97
Ethanolic extract of
leaf 0.05 Y=14.675x + 68.692 0.89
It is evident that all the test samples were into some extent lethal to brine shrimp nauplii.
However, the result obtained from test samples shows intense lethality rate. Maximum mortality
occurred at
400µg/ml
200µg/ml
100µg/ml
50µg/ml Concentrations where the least mortality occurred at 0.781µm/mg.
It is also noted that mortality increased gradually with the increase in concentration of the test
samples.
CONCLUSION
Finally, it concluded that the ethanolic leaf extract of Lannea coromandelica possesses
significant CNS-depressant and Cytotoxic activity as compared with the standard drugs. The
results of phytochemical screening were revealed the presence of alkaloids, flavonoids,
y = 14.672x + 68.692
R² = 0.8916
0
20
40
60
80
100
120
-0.5 0 0.5 1 1.5 2 2.5 3
% o
f M
orta
lity
LogC
Effect of ethanolic extract on brine shrimp nauplii
y = 27.186x + 72.463
R² = 0.9705
0
20
40
60
80
100
120
-2 -1.5 -1 -0.5 0 0.5 1 1.5
% o
f M
ort
ali
ty
LogC
Effect of Vincristine sulfate on Brine shrimp nauplii
RESEARCH ARTICLE Rashid et.al / IJIPSR / 3 (1), 2015, 72-84
Department of Pharmacognosy ISSN (online) 2347-2154
Available online: www.ijipsr.com January Issue 83
carbohydrates, saponins, tannins etc. In Neuropharmacological study, both the hole cross and
open field tests were performed which shows significant CNS depressing activity in dose
dependant manner of the leaf extract. Significant cytotoxic activity was obtained from the result
of brine shrimp lethality bioassay.
ACKNOWLEDGEMENT
All Praised to Almighty God , Here I am Md. Mamunur Rashid would like to thank my honorable
teacher & supervisor Ummey Jannatun Nahar, Exe-Lecturer , Dept. of Pharmacy, Stamford
University Bangladesh, besides I like to thanks my honorable and beloved Parents whose were
inspired me to do the projects and also thank my dear classmates.
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