Pharmacological Evaluation and Phytochemical characterization of Aegiceras corniculatum (Linn.

) Blanco (Family-Myrsinaceae) Bark

Presented by Vaskor Bala Roll: 021113 Pharmacy Discipline

Abstract
Phytochemical analysis of the extract of this plant indicated the presence of alkaloids, steroid, flavonoids, tannin, saponins and absence of gums and reducing sugar type of compounds. The general toxicity was investigated on brine shrimp (Artemia salina) & showed the most prominent cytotoxic activity (LC50 = 5.5 g/ml and LC90 =9.3 g/ml). At a dose of 250 & 500 mg/kg exhibited significant (p<0.001) inhibition of writhing reflex (59.8% and 74.52% respectively) with a dose dependant manner which were compared to the standard drug Diclofenac sodium at the dose of 25 mg/kg of body weight. When tested for its antidiarrhoeal effects on castor oil induced diarrhoea in mice, it increased mean latent period and decreased the frequency of defecation significantly at the oral dose of 250 and 500 mg/kg body weight (P<0.01) comparable to the standard drug Loperamide at the dose of 50 mg/kg of body weight.

Plant Preview
Botanical Name: Aegiceras corniculatum (Linn.) Blanco
Family: Myrsinaceae Botanical features Small tree or shrub, up to 4 m high, spreading duffused branched, glabrous, bark almost smooth, reddish brown or dark grey Tap roots are well developed, much branched, horizontally spreading. Fruit capsule, 2.0 cm - 9.0 cm long, completely curved with pointed apex. Regional name Bengali Kholshi Bengali Kholisa English Kajla DACB acc. no. 31381

Fig: Stem of A. corniculatum

Traditional Use: Its fruits and seeds are used as fish-poison; barks and leaves are used in the treatment of asthma, diabetes, rheumatism, etc. (Balasooriya et al; 1982).

Literature Review
Phytochemical Literature Review:

The following chemicals are isolated form A. corniulatum 2-methoxy-3nonylresorcinol, 3,7-dihydroxy-2,5-diundecylnaphthoquinone, 2,7-dihydroxy-8methoxy-3,6-diundecyldibenzofuran-1,4-dione, 2,8-dihydroxy-7-methoxy-3,9diundecyldibenzofuran-1,4-dione, and 10-hydroxy-4-O-methyl-2,11diundecylgomphilactone (Xu et al; 2004).
Wahidullah et al; 2002, reveals oleanane triterpenoid oligoglycoside (corniculatonin) and Gomez et al; 1989, 5-O-methylembelin form A. corniculatum

A. Corniculatum contains a saponin in 1.5% in the fruits and 0.5% in the leaves and barks. Hydrolysis yielded sapogenin named kanjalagin (C30H52O3) (Kincl et al;1956).
Pharmacological Literature Review: A. corniculatum afforded 5-O-methylembelin, which was toxic to fish (Tilapia nilotica) and retarded the growth of the fungus Pythium ultimum (Venkateswara, K. et al: 1961).

Phytochemical Study
Method:
Trease et al; (1983) and Myers (1982), Method was adopted for phytochemical anlalysis.

Preparation of bark extract:

The extract of the bark of the plant was prepared by using 80% ethanol as solvent . The yield of the extract was 15.66% (47gm form 300 gm).

Result
Extract Steroids Alkaloids Tannins Gums
Flavonoids

Saponins

Reducing suger
-

Ethannol

+ Presence - No reaction

Brine shrimp lethality assay

Method:
The Meyer et al; (1982) method was adopted for the study of the toxicity of A. corniculatum bark. Materials: 100mg of dried ethanolic extract of A. corniculatum was taken in 10 ml volumetric flask and volume was adjusted by DMSO and water (6:4). The concentration of this solution was 10 g/l.

Table: Result of Brine Shrimp lethality bioassay

Conc. Test Test Test (g/ml sample 1 2 ) 1 2 Ethanoli c extract 4 6 8 10 10 8 7 4 2 0 9 8 6 5 3 0 Average Alive shrimp 9.5 8 6.5 4.5 2.5 0 % mortality 5 20 35 55 75 100 5.5 9.3 LC50 (g/ml) LC90 (g/ml)

Brine shrimp lethality assay

Fig: Effect of A. corniculatum extract on brine shrimp
100 90 80 70

% Motality

60 50 40 30 20 10 0 0 1 2 3 4 5 6
(g/ml)

10

Conctration

Analgesic Activity Study

Method:
Whittle, (1982) method that is acetic acid induced writhing method was followed for the analgesic study of A. corniculatum bark.

Study Design:
Experimental animals were randomly selected and divided into four groups denoted as group-I, group-II, group-III, group- IV consisting of 5 mice in each group. Each group received a particular treatment i.e. control, positive control and the two doses of the extract.

Table: Statistical evaluation of the Analgesic Activity

Animal group Control Diclofenac (25mg/kg) Extract (250 mg/kg) Extract (500 mg/kg) Mean writhing SE 341.48 13.61.37* 21.81.12* 18.81.36* % Protection 60 35.88 44.71

SD- Standard deviation, SE- Standard error ,* = P Value<0.001

Analgesic Activity Study

Fig: Effect of crude extract of bark of A. corniculatum on acetic acid induced writhing effect of mice.
% Writhing Inhivition vs Treatment
70 60

% Writhing Inhibtion

60 50
40 30 20 10 0 Positive Control Extract 250 mg/kg Extract 500 mg/kg

44.71 35.88 Positive Control

Extract 250 mg/kg

Extract 500 mg/kg

Treatment

Antidiarrhoeal Activity Study

Method:
The castor oil induced antidiarrhoeal method was adopted form Galvez et al; (1993). The animals were all screened initially by giving 0.3 ml of castor oil and only those showing diarrhoea were selected for the final experiment. The animals were divided into control, positive control and test groups containing five mice in each group.

Table: Effect of A. corniculatum on the latent period of castor oil induced Diarrhoeal episode in mice
Group I Control (1% tween 80) II Positive Control (Loperamide) III Ethanol extract IV Ethanol extract Dose (oral) 10 ml/kg 50 mg/kg 250 mg/k) Mean Latent Period SE 0.684 0.84 1.52 0.257 0.758 0.075 t-test (P-value) 3.09 (p<0.01) 1.21 (p<0.3) 1.42 (p<0.2)

500 mg/kg

0.944 0.093

Table: Effect of Aegiceras corniculatum on castor oil induced

diarrhoea in mice at the doses of 250 and 500 mg/kg-body weight
Groups Period of study (hr)
1 2 3 4 1

Mean no. of stools

2.6 4.8 3.6 3.4 0.2 0.8 1.8 1.2 2.0 3.7 2.3 2.5 1.6 3.0 1.8 2.0

S.E.
0.51 0.67 0.68 0.87 0.20 0.37 0.37 0.20 0.41 0.52 0.61 0.32 0.51 0.32 0.45 0.63

t-test (p value)
----4.38b 5.23a 2.32c 2.47c 0.91f 1.3f 1.43d 1.00f 1.39d 2.42c 2.21e 1.30f

I Control

II (Positive Control) III Extract of Aegiceras corniculatum (250mg/kg) IV Extract of Aegiceras corniculatum (500mg/kg)

2 3 4 1 2 3 4 1 2 3 4

Values of t-test. (n=5); fp<0.3, ep<0.1, dp<0.2, cp< 0.05, bp< 0.001, ap< 0.01 vs. control

Mean latent period vs Treatment

1.8 1.6 Mean Latent Period 1.4 1.2 1 0.8 0.6 0.4 0.2 Positive Control (1.520.257) Extract 250 mg/kg (0.7580.075) Extract 500 mg/kg (0.9440.093)
Control Positive Control (0.6840.084) (1.520.257) Extract 250 Extract 500 mg/kg mg/kg (0.7580.075) (0.9440.093)

Control (0.6840.084)

Fig: Effect of crude extract of bark of A. corniculatum on castor oil induced diarrhea of mice. Each bar represents mean latent period (4hr).

Treatment

Mean no of stool

Fig: Effect of Loperamide and A. corniculatum on castor oil induced diarrhea in mice through the observation period (4hr)

Mean no. of stool vs study hr(4hr)

6 5 4 3 2 1 0 1 0.2 2 3 Study Hours 4 2.6 2 1.6 0.8 4.8

3.7
3

3.6 2.3 1.8 1.8

3.4 2.5 2 1.2

Control Positive Control Extract 250 mg/kg Extract 500 mg/kg