Antimicrobial activity of Leaves of Artemisia vulgaris L

Objective: To screen the antimicrobial activity of different extracts of leaves of Artemisia vulgaris. Materials and Methods: To detect the in vitro antibacterial activity, 10 bacterial strains were selected. These bacteria are both gram +ve and gram -ve. Leaves were extracted with a petroleum ether, chloroform, ethyl acetate, ethanol and aqueous . In the present work the antibacterial activity was done by cup plate method. The antibacterial activity was expressed as zone diameter in millimeters. Different extracts from leaves of the plant was compared with standards like benzyl penicillin for gram +ve bacteria and streptomycin for gram –ve bacteria using DMF as control. The readymade media for inoculum and culture was obtained from Himedia labs. For antifungal activity four fungal organisms were selected and Griseofulvin was used as standard. Results: Herbal extracts prepared from the leaves of the plant were screened against bacteria and fungal organisms at the concentration range between 50 μg and 300 μg/0.1ml. The results of antimicrobial activity revealed that the extract exhibited activity against both gram +ve, gram –ve and fungal organisms. Conclusion: The present investigation reveals that the aqueous, chloroform and ethyl acetate extracts and in some cases petroleum ether extract showed significant antimicrobial activity when compared with standard.


Introduction
 This has been the rational for the development of new antimicrobial drugs and the search for novel molecules has been extended to herbal drugs that offer better protection.
 Plants and other natural substances have been used as the rich source of medicine.

Contd..
All ancient civilizations have documented medicinal uses of plant in their own ethnobotanical texts.
Most of the remedies were taken from plants and proved to be useful.
Twenty known flavonoids were isolated from Artemisia vulgaris a plant used as an emmenagogue in traditional medicine However, the literature review revealed that Artemisia vulgaris has not been studied for the antimicrobial activity.
Hence, in the present study, the leaves of Artemisia vulgaris have been selected for phytochemical investigation, in-vitro antibacterial and antifungal activity.
Exploring the traditional medicines with proper chemical and pharmacological profiles.
To conduct systematic Pharmacognostic investigation of leaves of Artemisia vulgaris Linn.

Introduction To Plant
Artemisia vulgaris is an aromatic perennial shrub belonging to family Asteraceae.Synonyms:

Review of Literature
Hernandez H and et.al Aqueous extract of Artemisia vulgaris showed a 89.8% growth inhibition of the Plasmodium falciparum in In-vitro culture Abdual Ghani AS and et.al The effect of aqueous extract of leaves and stems of Artemisia vulgaris were studied on picrotoxin induced seizures in mice. A. vulgaris delayed the onset of seizures and decreased the mortality rate Gilani AH and et.al The effect of a crude extract of the aerial parts of Artemisia vulgaris was investigated against D-galactosamine and lipopolyssacharide induce hepatitis in mice.Pretreatment of mice with different doses of extract (150-600mg/kg) significantly reduced the toxin induced and showing hepatoprotective activity Contd..
54 volatile constituents was fractionated from solid liquid extraction with pentane dichlomethane.

Dung NX et.al.
Essential oil of the leaves of Artemisia vulgaris fourty six components have been identified of which the major ones were found to be caryophyllene (24.1%) and -cubebene(12%).

Extraction
The air-dried leaves of Artemisia vulgaris Linn.were reduced to fine powder (40 size mesh) 100 gm of powder was subjected to successive hot continuous extraction (soxhlet) with petroleum ether, chloroform, ethyl acetate and ethanol.
Another batch of powdered drug was macerated with chloroform-water I.P.
After the effective extraction, solvent were concentrated using rotary flash evaporator

Antibacterial Activity By Cup-plate Method
In the present work to know the antibacterial activity cup-plate method is employed.
The antibacterial activity is expressed as zone diameter in millimeters, which is measured with a divider.Different extracts of leaves of the plant was compared with standards and DiMethyl Formamide (DMF) as control for antimicrobial activity.

Antifungal Activity
In the present study antifungal extract is diffused from the cup through an agar layer in a petri dish or plate to an extent such that the growth of added fungus is restricted entirely in circular area or zone around the cavity containing the solution of an antifungal substances.
The antifungal activity is expressed as zone diameter in millimeters, which is measured with a divider. Preparation of standard solutions  Standard benzyl penicillin injection IP 1,00,000 units. As per IP 1mg of benzyl penicillin=1500-1750 IU.

Preparation of inoculum for antibacterial and antifungal activity
About 28 gm of prepared medium was taken in 1000 ml distilled water and boiled to dissolve completely.The microorganisms were streaked under aseptic conditions, and the slants were incubated at 37±1ºC for 24 hrs.
These 24 hrs cultures were used for preparation of inoculum.The suspension of the microorganisms was prepared in 10 ml of sterile water and 0.5 ml of this suspension was added to 100 ml of the Nutrient agar medium (Himedia labs)

Preparation of cultural medium
27 gm of nutrient agar (Himedia) readymade medium was dissolved in freshly prepared distilled water (in 1000 ml) by gentle heating.

Preparation of agar plate
The sterilized medium was cooled at 40ºC and 0.5 ml of inoculum per 100 ml of medium was added to the conical flask.This was shaken gently to avoid the formation of air bubbles and then transferred into Petri dishes so as to obtain 6 mm thickness of medium.The medium in the plate was allowed to solidify at room temperature.

Contd…
Experimental procedure for antibacterial and antifungal activity The sterile borer was used to prepare 4 cups of 8 mm diameter in the medium of each Petri dish.An accurately measured 0.1 ml solution of each concentration of solution of extracts and standard samples were added to the cups in the medium with the help of micropipette.
[ All the plates were kept at room temperature for effecting diffusion of drug extracts and standards later they were incubated at 37±1ºC for 24 hrs.The presence of definite zones around the cup of any size indicated antibacterial and antifungal activity.
The control was run simultaneously to assess the activity of DMF, which was used as vehicle for extract and fractions.
The diameter of the zone of inhibition was measured and recorded.

Antibacterial activity shown by extracts
For the gram +ve organisms like Bacillus subtilis, Bacillus cerius, Staphylococcus aureus the chloroform and ethyl acetate extracts showed significant anti bacterial activity at 50 µg/0.1ml,when compared with standard.
For Salmonella typhi, the aqueous and ethyl acetate extracts showed minimum inhibitory concentration (MIC) at 100 µg/0.1ml.
For the gram -ve organisms like Pseudomonas aerogenosa, the aqueous and ethyl acetate extracts showed significant antibacterial activity at 50 µg/0.1ml,For Escherichia coli, the aqueous and chloroform has MIC at 50 µg/0.1ml,for Klebsiella pneumonae, The ethyl acetate and chloroform extracts has MIC at 300 µg/0.1ml,For Vibrio cholerae, the aqueous and chloroform has MIC at 300 µg/0.1ml,For Proteus mirabilis, the aqueous and chloroform has MIC at 300 µg/0.1ml,while for Serratia marsupium it was 50 µg/0.1ml,when compared with standard.

Antifungal activity shown by extracts
For fungal organisms like Candida albicans, Rhizopus japonicum the MIC was 150 µg/0.1ml with aqueous and petroleum ether extract, For Aspergillus fumigatus the ethyl acetate and aqueous extracts has MIC 100 µg/0.1ml,For Candida tropicallis the ethyl acetate and petroleum ether extracts has MIC at 150 µg/0.1ml.
Antibacterial activity of Artemisia vulgaris Linn leaves extract on following Bacteria selected for the study.Bacillus subtilis (+) Bacillus cerius Essential oil from Artemisia vulgaris plant reported have shown insect repellent, nematocidal and insect attractant activities has been enumerated.Milhau G et.al Essential oil of Artemisia vulgaris were showing In-vitro antimalarial activity on Plasmodium falciparum.Lee SJ Twenty known flavonoids were isolated from Artemisia vulgaris a plant used as an emmenagogue in traditional medicine.Herrera CL and et.alUsing modified screening method of Hall et.al for antifertility activity in female mice extracts and/or juice of Artemisia vulgaris gave promising results 50% or more reduction in fertility Collection Of Plant Material The leaves of Artemisia vulgaris were collected from local areas of Belgaum, Karnataka and authenticated by Dr.P.S.N.Rao, Joint Director, at Botanical Survey of India (BSI), Govt. of India, Ministry of Environment and Forests, Pune, India.
Different concentration of extracts equivalent to 50 g, 100 g, 150 g, 200 g and 300 g/o.1ml by using DMF were prepared.
Macroscopic Character of Artemisia vulgaris Linn.Leaf.