International Journal of Innovative Pharmaceutical ... · PDF fileDepartment of Pharmacognosy...

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.

mailto:[email protected]

tel:%2B8801723533704




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].




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:




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.




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




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.




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




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


Control

Positive

Group-i

Group-ii

Group-iii




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


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




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






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




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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