Antimicrobial and Anti-Inflammatory Activity of Roccella belangeriana

Akash P Dahake^*, Rita C Chakma, Chirantan Chakma and Deshbandhu Joshi

Pharmacy College, Rampura, Kakanpur. Godhara. Dist. Panchmahal. Gujarat. India.

ABSTRACT

The investigation was carried out to study the antibacterial and anti-inflammatory of the Roccella belangeriana (Lichens) in carrageenan induced rat paw oedema im albino rats. Antimicrobial activity of the acetone and aqueous extracts of Roccella belangeriana were determined against various pathogenic bacteria. The extracts were tested against various bacteria like Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli by disk diffusion method. Minimum Inhibitory Concentration (MIC) values of both extracts were determined. It is concluded that acetone extract exhibited significant antimicrobial activity. Acetone extarct of Roccella belangeriana was given at a dose of 100, 200 and 300 mg/kg of body weight (p.o) and standard drug, Diclofenac sodium at a dose of 100 mg/kg. The paw oedema volume was determined for 3 hours at every one hour interwal. The extract exhibited dose dependent activity at all three dose levels. This Study lends sceintific support for it’s use in folk medicine.

KEYWORDS: Roccella belangeriana, Carrageenan, Antimicrobial, Anti-inflammatory

INTRODUCTION:

Medicinal plants have a special place in pharmaceutical research. Natural products are permanent challenge with respect to total synthesis and stimulate the development of new reagents and reactions¹.

Roccella belangeriana is a type of fungi belonging to family Rocellaceae, commonly known as Indian lichen. Lichens have wide range of uses and are important as food for animals in the Arctic regions. The nutritive values of ice-land moss, reindeer moss and potatoes have reported the fitness of lichens for human consumption².

Lichens were also found to be useful as sources of b-carotene; the precursor of vitamin A from the Indian lichen Roccella belangeriana is highly significant in this connection³.

MATERIAL AND METHODS:

Chemicals used:

The following chemicals were used in study: Dimethyl formamide (Merck), Ampicillin , Diclofenac sodium (Cadilla pharmaceuticals).

Plant material and extraction:

Lichen specimens were collected from Godhra, Panchamahal, Gujarat and were authenticated as Roccella belangeriana by National Botanical Research Institute (NBRI), Lucknow.

Preparation of the extract:

Collected lichens were cleaned, dried at room temperature and powdered. About 100 gm of the powdered material was placed in reflux condenser and was subjected to extraction by hot percolation method using one with water and another acetone. Then the extraction was carried out for not more than 8 hours.

The crude extracts was then transferred into a silica glass tray and placed in a desiccator containing fused calcium chloride. This crude extract was then washed several times with ether.

Antibacterial Screening:

Test organisms used:

Gram positive organisms: Staphylococcus aureus, Bacillus subtilis.

Gram negative organisms: Escherichia coli, Pseudomonas aeruginosa.

Antibacterial Screening:

Antibacterial screening of acetone and aqueous extracts of Roccella belangeriana was performed against 4 pathogenic bacteria (2-gram positive and 2-gram negative) by the standard disc diffusion method^4-6.

Each Petri dish was inoculated with one of the bacterial cultures suitably diluted to contain above 10⁶cells/ml by spreading 0.1 ml suspension of the organism with a sterile cotton swab. In each plate cups of 6 mm diameter were made at equal distances using sterile cork borer. One cup was filled with 0.1 ml of standard drug, another with 0.1 ml of DMF, and others were filled with 0.1 ml of samples in sterile DMF. Ampicillin was used as an antibacterial standard. The Petri dishes were incubated at 37˚C for 48 hours. The diameter of zone of inhibition in mm was recorded after incubation. The experiment was performed in triplicates and average diameter of zone of inhibition was obtained.

Determination of MIC:

The extracts that showed antibacterial activity were subjected to minimum inhibitory concentration (MIC) assayed by serial two fold dilution method⁷. A positive control and negative control were also prepared to confirm the nutritive and sterility properties of the prepared medium respectively. All the tubes were incubated at 37ºC for 24 hours. MIC was interpreted as the lowest concentration of the sample, which showed clear fluid without development of turbidity.

Acute toxicity and selection of doses:

The acute toxicity studies were carried out in adult female albino rats weighing about 150-200 g, by up and down method as per OECD 425 guidelines⁸. Overnight fasted animals received test drug at a dose of 2000 mg/kg body weight orally. Then the animals were observed continuously once in half an hour for next 4 hrs and then after 24 hrs for general behavioral, neurological, autonomic profiles and to find out mortality. The extract was found safe up to a dose of 2000mg/kg body weight.

Anti-inflammatory activity:

The acute hind paw oedema was produced by injecting 0.1 ml of carrageenan (prepared as 1% suspension in sterile normal saline) locally into the plantar aponeurosis of the right hind paw of rats^{9, 10}. Acetone extract was administered to three different groups i.e. 100mg/kg, 200mg/kg and 300mg/kg of the body weight respectively. The other two groups served as negative and positive controls and received vehicle, normal saline (1 ml/kg, p.o.) and standard drug, diclofenac sodium (100 mg/kg, p.o.), respectively. Extracts were administered 1 h prior to the injection of carrageenan. The linear paw circumference was measured at hourly interval for 3 h¹¹. Anti-inflammatory activity was measured as the percentage reduction in oedema level when drug was present, relative to control as shown in Table 2. Increase in the paw oedema volume was considered as the difference between 1 and 3 h. Percent inhibition of oedema volume between treated and control groups was calculated as follows:

% Inhibition = {1-V_t/V_c} X100

Where V_t and V_c are the relative changes in the oedema of the test and control respectively.

Experimental design:

The animals were divided into five groups of six animals in each group:

Group I: - Normal healthy control; received normal saline.

Group II: -Acetone Extract (100 mg/kg body weight, orally)

Group III: -Acetone Extract (200 mg/kg body weight, orally)

Group IV:-Acetone Extract (300 mg/kg body weight, orally)

Group V: - Control (Diclofenac Sodium 100 mg/kg, orally)

Statistical analysis:

All data were expressed as mean ± SEM and one-way ANOVA was applied to determine the significance of the difference between the control groups and rat treated with the test compounds.

RESULTS AND DISCUSSION:

The antibacterial activity of the different extracts of Roccella belangeriana showed significant variations as shown in Table 1. Among the two extracts tested, acetone extract had greater antibacterial potential, followed by aqueous extract.

Antibacterial potency of the different extracts of Roccella belangeriana against the tested bacterial strains were expressed in MIC as presented in Table 2. The MIC values against these bacteria, and fungal strains ranged from 15.62 to 62.5µg/ml.

Carrageenan induced rat paw oedema is used widely as a working model of inflammation in the search for new anti-inflammatory drug¹². The anti inflammatory activity of the acetone extract of Roccella belangeriana was evaluated by carrageenan induced rat paw oedema method and the result is shown in Table 2. The extract was tested at three different dose levels. The results showed that the acetone extract with a dose of 300 mg/kg showed highest anti-inflammatory activity among all the dose levels on carrageenan induced rat paw oedema at third hour. It’s activity was almost equal as compared to the reference drug diclofenac sodium.

CONCLUSION:

In our present study, a wide range of human pathogenic microorganisms were examined belonging to Gram-positive and Gram-negative class. The results indicate that Roccella belangeriana extracts have broad inhibitory activities to pathogenic microorganisms and promising to act as potential antibacterial agent from natural sources.

The development of oedema in the paw of the rat after the injection of carrageenan is due to release of histamine, serotonin and prostaglandin like substances¹³. Significantly high anti-inflammatory activity of acetone extract Roccella belangeriana may be due to inhibition of the mediators of inflammation such as histamine, serotonin and prostaglandin.

Table 1: Antimicrobial activity and MIC values of extracts of Roccella belangeriana

Microorganisms	Zone of Inhibition (mm)			MIC (µg/ml)
Microorganisms	Acetone Extract	Aqueous Extract	Ampicillin	Acetone Extract	Aqueous Extract
S.aureus	21	12	24	15.62	31.25
B.subtilis	18	11	26	15.62	62.50
P.aeruginosa	17	13	21	15.62	31.25
E.coli	22	14	25	15.62	15.62

Table 2: Anti-inflammatory activity of acetone extract of Roccella belangeriana

Group

Treatment

Increase in paw volume (ml)

1 h

2 h

3 h

4 h

Control vehicle

0.43±0.09

0.73±0.01

0.87±0.31

0.77±0.41

Acetone extract

(100mg/kg)

0.39±0.23

0.23

0.33±0.11

0.28*

0.26±0.10

0.73*

0.22±0.15

0.59*

III

Acetone extract

(200 mg/kg)

0.30±0.16

0.45

0.26±0.09

0.57*

0.23±0.14

0.33*

0.19±0.09

0.52*

Acetone extract

(300 mg/kg)

0.25±0.07

0.37

0.22±02

0.65*

0.18±0.07

0.47*

0.14±0.08

0.52*

Diclofenac sodium

(100mg/kg)

0.20±0.03

0.47

0.18±0.02

0.63*

0.15±0.09

0.19*

0.12±0.01

0.26*

There is now preliminary scientific validation for the use of Roccella belangeriana for antibacterial activity. The present result also indicates the efficacy of acetone extract Roccella belangeriana as an efficient therapeutic agent in acute anti-inflammatory conditions. Further laboratory and clinical studies of this plant is required in order to understand better antibacterial and anti-inflammatory principles which will allow the scientific community to recommend their use as an accessible alternative to synthetic drugs.

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