In vitro antimicrobial screening of mangrove plant Avicennia officinalis

In vitro antimicrobial screening of mangrove plant Avicennia officinalis

Varaprasad Bobbarala, Varahalarao Vadlapudi And K. Chendrashekar Naidu

Department of Botany, Andhra University, Visakhapatnam (India).

(Received: January 28, 2009; Accepted: March 08, 2009)

ABSTRACT

In India Avicennia officinalis is commonly used for herbal preparations in the treatment of small pox sores, scabies, as a contraceptive, boils and tumors. This has, therefore, led to the investigation of the antimicrobial activities of methanolic extract of A. officinalis. Eighteen different bacterial and fungal belonging to clinical and plant pathogenic microorganisms were used. The results show that A. officinalis extracts exhibited antimicrobial activities at a concentration of 20 mg/mL. Antibacterial activity of mature leaves and bark extracts of A. officinalis was evaluated using soxhelt extraction method. Hexane, chloroform and methanol were used as solvents in order to get the plant extracts. The antibacterial activity was screened by using agar well diffusion technique against human and plant pathogenic bacteria and fungi. The length of inhibition zone was measured in millimeters from the edge of the well to the edge of the inhibition zone. Mature leaf extracts of A. officinalis in methanol exhibited promising antimicrobial activity than other solvent extracts. Phytochemical screening revealed that mature leaf of A. officinalis contained alkaloids, steroids, triterpenoids and flavonoids.

Key words: Antimicrobial activity, Avicennia officinalis.

Methodology for Antimicrobial Studies

Plant material:

The plant materials of different plant species were collected in and around the Visakhapatnam district and shade dried. After complete dryness they are chopped into small pieces and are coarsely powdered in a wiley mill.

Preparation of plant extracts:

The extraction method employed here is a known amount of coarsely powdered plant materials were successively extracted with organic solvents like hexane, chloroform, methanol and water basing on order of polarity using soxhlet apparatus. The different extracts obtained were subsequently concentrated under reduced pressure to get their corresponding residues. The extracts were screened for anti-microbial activity using the method described under the section.

Organisms and Media:

The organisms used were purchased from Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh. The strains are maintained and tested on Nutrient Agar (NA) for bacteria and Potato Dextrose Agar (PDA) for fungi.

Antimicrobial assays:

The crude extracts of the different plant parts of different species were subjected to antimicrobial assay using the cup plate method of Murray et al (1995) modified by Olurinola (1996). 20 ml of nutrient agar was dispensed into sterile universal bottles these were then inoculated with 0.2ml of cultures mixed gently and pouredinto sterile petri dishes. After setting a number 3-cup borer (6mm) diameter was properly sterilized by flaming and used to make five uniform cups/ wells in each petri dish. A drop of molten nutrientagar was used to seal the base of each cup.The cups/wells were filled with 50µl of the different extracts of 100mg/ml, 200mg/ml, 300mg/ml, 400mg/ml and 500mg/ml, and allowed diffusing for 45 minutes. The solvents used for reconstituting the extracts were similarly analyzed. The plates were incubated at 37°c for bacteria and 25°c for fungal organisms. The standard antibiotic (Streptomycin)and anti-fungal (Bavistin) drugs were used at different concentrations to get MIC (Minimum inhibitory concentrations). The zones of inhibition were measured with Antibiotic zone scale in mm and the experiment was carried out in triplicates.

Antioxidant Activity using (FRAP) Method

Determination of total antioxidant activity (FRAP assay) for plant extract by modified method of Benzie and Strain.

1. The stock solutions included 300mM acetate buffer (3.1g C2H3NaO2·3H2O and 16 ml C2H4O2), pH 3.6, 10mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40mM HCl, and 20mM FeCl3 6H2O solution.
2. The fresh working solution was prepared by mixing 25ml acetate buffer, 2.5ml TPTZ, and 2.5ml FeCl3 6H2O.
3. The temperature of the solution was raised to 37°C before use.
4. Samples (150μL) were allowed to react with 2850μl of the FRAP solution for 30 min in the dark condition. Readings of the colored product (ferrous tripyridyltriazine complex) were taken at 593 nm.
5. The standard curve was linear between 200 and 1000μM FeSO4.

Results are expressed in μM

Antioxidant Activity by DPPH radical scavenging assay

The effect of the Antioxidant potential of samples by using DPPH radical scavenging activity  by using the method according to Liyana-Pathiranan and Shahidi [29].

1. A solution of 0.135mM DPPH in methanol was prepared and 1.0 ml of this solution was mixed with 1.0 ml of extract in methanol containing 1 mg of the sample.
2. The reaction mixture left in the dark at room temperature for 30min.
3. The absorbance of the mixture was measured spectrophotometrically at 517nm.
4. BHT was used as reference.
5. The ability to scavenge DPPH radical was calculated by the following equation:
6. DPPH radical scavenging activity (%) = [(Abscontrol - Abssample)]/ (Abscontrol)] x 100

o    where Abscontrol is the absorbance of DPPH radical + methanol;

o    Abssample is the absorbance of DPPH radical + sample extract /standard.

Total Phenolics Content estimation according to Folin-Ciocalteu

An aliquot of the extract was mixed with 5ml Folin-Ciocalteu reagent (previously diluted with water at 1:10 v/v) and 4ml (75 g/l) of sodium carbonate.

The tubes were vortexed for 15 sec and allowed to stand for 30 min at 40 degree Celsius for color development.

Absorbency was then measured at 765nm using UV-VS spectrophotometer.

Samples evaluated at a final concentration of 0.1mg/ml.

Total phenolic content were expressed as mg/g Gallic acid equivalents using the following equation based on the calibration curve: y = 0.1216x, R2 = 0.9365,

           

o    Where x was the absorbance and y was the Gallic acid equivalent (mg/g). 

Nitric Oxide Scavenging Activity

Nitric Oxide radical inhibition estimated by the use of Griess Illosvoy reaction (Garrat, 1964). Nitric oxide generated from sodium nitroprusside and measured by the Griess reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitric ions that can be estimated by use of Griess reagent. Scavenger of nitric oxide competes with oxygen leading to reduced production of nitric oxide. Sodium nitroprusside (5 mM) in phosphate–buffered saline (PBS) was mixed with 3.0ml of different concentrations (10-100μg/ml) of the drugs dissolved in the suitable solvent systems and incubated at 25⁰C for 150 min.

The samples from the above were reacted with Griess reagent (1% sulphanilamide, 2%H3PO4 and 0.1% napthylethylenediamine dihydrochloride).

The absorbency of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546nm

Griess Reagent:

Physical Appearance: Colorless to lightly pink liquid

Description: Reagent for the one step determination of nitrite (NO2-) in biological media.

Form: 1 part of 0.1% naphthyl ethylenediamine dihydrochloride in distilled water plus 1 part 1% sulfanilamide (or sulfanilic acid) in 5% concentrate H3PO4.

Prolong Storage: Keep cool and dry at +4^oC

Principle: This assay relies on a diazotization reaction that was originally described by Griess in 1879. Modifications have been made to the original reaction through the years. This procedure is based on the chemical reaction shown below, which uses sulfanilamide and naphthylethylenediamine dihydrochloride (NED) under acidic conditions. Sulfanilamide and NED compete for nitrite in the Griess reaction. This reagent detects NO₂^- in a variety of biological and experimental liquid samples such as plasma, serum, urine and tissue culture medium. The limit of detection is 2.5 uM (125 pmol) nitrite (in distilled, deionized water); however, the sensitivity will vary depending upon the sample used. Individual researchers must determine the limits for their individual experiments and referred to the absorbance at standard solutions of potassium nitrite, treated in the same way with Griess reagent.

The percentage scavenging of nitric oxide of Plant extract and standard compounds was calculated using the following formula:

NO Scavenged (%) = (A cont - A test)/A cont × 100

Where A cont is the absorbance of the control reaction and

 A test is the absorbance in the presence of the sample 

MTT Assay for Cytotoxicity Studies

Cell culture
Human cell lines used in this study were procured and all cells were grown in Minimal essential medium (MEM, GIBCO) supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 5% fetal bovine serum (FBS) (growth medium) at 37°C in 5% CO₂ incubator.

  

MTT assay
The MTT assay developed by Mosmann¹ was modified and used to determine the inhibitory effects of test compounds on cell growth In Vitro. 

The trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue culture plate at a density of 5x10³ cells/well in growth medium and cultured at 37°C in 5% CO₂ to adhere. 

After 48hr incubation, the supernatant was discarded and cells were pretreated with growth medium and were subsequently mixed with different concentrations of test compounds (12.5, 25, 50, 100 and 200 µg/ml) in triplicates to achieve a final volume of 100µl and then cultured for 48 hr. 

The compound was prepared as 1.0 mg/ml concentration stock solutions in PBS. 

Culture medium and solvent are used as controls. 

Each well then added 20µl of fresh MTT (0.5mg/ml in PBS) followed by incubation for 4hr at 37°C. 

The supernatant growth medium was removed from the wells and replaced with 200 µl of DMSO to solubilize the colored formazan product. 

After 30 min incubation, the absorbance (OD) of the culture plate was read at wavelength of 492nm on an ELISA reader.


References:
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 1983, 55.