ANTIMICROBIAL ACTIVITY OF ARTEMISIA VULGARIS L. LEAF AND CALLUS EXTRACTS

2cm margin on all sides for the whole manuscript Bold face, Title case, Font size 14 for the manuscript Title ANTIMICROBIAL ACTIVITY OF ARTEMISIA VULGARIS L. LEAF AND CALLUS EXTRACTS H.K.R.PRASAD. SARIPALLI* 1, TEWODROS AREGAI 2 AND ASHOK SARMA 3 Asterisk (*) for the corresponding author Superscripts for indicating affiliations of authors and co authors Normal face, upper case and font size 12 for authors and co authors 1 Department of Biotechnology, College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia. 2 Department of Biochemistry, International Medical School, Management and Science University, Malaysia. 3 Surgical Oncology, Vanderbilt University School of Medicine, Nashville, USA. Superscripts for indicating affiliations of authors and co authors Normal face, Lower case and font size 12 for affiliations of authors and co authors Asterisk (*) for the corresponding author * Corresponding author email drshkrp@yahoo.com Telephone +91 9876755651 0812 211232 Fax 0812 323433 1

ABSTRACT: Bold, upper case and font size 14 pt for all main Subheadings Abstract should be not more than 300 words and single spaced spacing with 12 pt font size and Times new roman The in vitro antimicrobial activity of Artemesia vulgaris (L.) leaf and callus extracts were studied against selected pathogenic bacteria and fungi Bacillus megaterium (ATCC 23564), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25922), Enterobacter faecalis (ATCC 35550), Proteus vulgaris (ATCC 638), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) and three fungal species Asperigillus niger (NCIM 596), Aspergillus fumigatus (NCIM 291) and Candida albicans (NCIM 670) following Agar Diffusion method. The leaves were extracted with different organic solvents such as acetone, benzene, chloroform, methanol, hexane, and petroleum ether. Among the six solvents used, chloroform extract of leaf of the plant was more effective against pathogenic bacteria and fungi, with the minimum inhibitory concentration (MIC) ranging between 0.25mg/ml to 8mg/ml KEYWORDS: 4-6 key words Artemesia vulgaris (L.), leaf and callus extracts, MIC, antimicrobial activity. INTRODUCTION: Manuscript (except Abstract) should be double spaced spacing. It should be with 12 pt font size and Times new roman The use of plants as medicines is as old as human civilization itself. Many of the existing medicinal systems such as Ayurveda, Unani, Homeopathy, Naturopathy, Siddha and other alternative medicinal systems, have been utilizing plants as effective medicines to cure many harmful diseases. India is the largest producer of medicinal herbs and is appropriately called the Botanical Garden of the World 1. The genus Artemisia, which belongs to the Asteraceae family and comprises several species, is widespread in References to the literature cited for temperate areas the (South manuscript Europe, North should Africa, be numbered North America and Asia). Among these in order of appearance in the species, Artemisia vulgaris L. (Mugwort) is a vivace herb, 40-80 cm high and wild manuscript and cited in the text with growing. Its leaves superscript and flowers numbers were used in folk medicine. Artemisia vulgaris s oil is 2

used for its insecticidal and antimicrobial properties. All parts of the plant are antihelminthic, antiseptic, antispasmodic, carminative, cholagogue, diaphoretic, digestive, emmenagogue, expectorant, nervine, purgative, stimulant, slightly tonic and used in the treatment of women's complaints 2,3,4,5,6,7. The leaves are also said to be appetizers, diuretic, haemostatic and stomachic 8,9,10. An infusion of the leaves and flowering tops is used in the treatment of nervous and spasmodic affections, sterility, functional bleeding of the uterus, dysmenorrhoea, asthma and diseases of the brain 9. The leaves have an antibacterial action, inhibiting the growth of Staphylococcus aureus, Bacillus typhi, Bacillus dysenteriae, Streptococci, Escherichia coli, Bacillus subtilis and Pseudomonas 9. The principal aim of the work was to study the antimicrobial activity of Artemisia vulgaris L. callus extracts in different solvents such as chloroform and acetone against seven species of bacteria and three species of fungi. In the experimental study different solvent fractions of the both extracts of callus cultures of the plant have been investigated at varying concentrations. MATERIALS AND METHODS: Plant Material The Artemisia vulgaris (L.) was collected from Gorantla village, Guntur district of Andhra Pradesh, India. The voucher specimen was deposited at Department of Botany, St.Ann s College, Guntur, for future reference. Leaf Extract Preparation Fresh leaf material were washed thoroughly under running tap water, shade dried and used for extraction. 4 week-old-calli derived from the leaf were collected and dried in an oven at 50±1 C for 60-72 h. Both the dried leaf and calli were homogenized to a fine powder and stored in airtight bottles. 25 g of leaf/calli powder were extracted with 150 ml 3

of solvent (acetone, benzene, chloroform, methanol, hexane and petroleum ether) for 24 h by using Soxhlet apparatus. The extract was dried in a flash evaporator for 30 min and the left over powder was considered 100%. Different concentrations; 0.25, 0.5, 1, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 80 and 100 mg/ml were prepared by redissolving the extracted powder in the same solvent which was used in the extraction. Test Organisms Tests were performed on seven species of selected bacteria such as Bacillus megaterium (ATCC 23564), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25922), Enterobacter faecalis (ATCC 35550), Proteus vulgaris (ATCC 638), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) and three fungal species Asperigillus niger (NCIM 596), Aspergillus fumigatus (NCIM 291) and Candida albicans (NCIM 670). All the above test bacterial species were maintained on Nutrient Agar medium. 36 hour-old bacterial culture was inoculated into Nutrient broth and incubated on a rotary shaker at 35 ± 2 C at 100 rpm. After 36 hours of incubation, the bacterial suspension was centrifuged at 10000 rpm for 15 min. The pellet was resuspended in sterile distilled water and the concentration was adjusted to 1 10 8 cfu/ml using UV Visible Spectrophotometer. By reading the OD of the solution to 0.45Å (610nm) it was used for further studies. Fungal colonies were harvested from 9-10days old cultures, which were maintained on Potato Dextrose Agar. The spores were suspended in sterile distilled water and the spore suspension was adjusted to 1 10 8 spores/ml (NCCLS). Antimicrobial Assay 4

Different concentrations of solvent extracts of the calli were tested for antimicrobial activity by using Antibiotic Sensitivity test. Microbial suspension was evenly mixed with sterile agar medium and poured into the sterile petri plates. After allowing the media to solidify at room temperature, wells of 6mm diameter were bored in the agar with sterile cork borer. Each concentration was checked for antimicrobial activity by introducing equal amounts of the sample (40ul) into the wells. The method was repeated in five plates. Plates were allowed to stand at room temperature for 1 hour to enable extract diffusion into agar media and then incubated at 37 C for 24-48 hours. The zone of growth inhibition around the wells was measured and area of inhibition zone was calculated. Simultaneously the activity of seven standard antibiotics i.e.streptomycin(10μg/ml),gentamycin(10μg/ml),choloromphenicol(30μg/ml),vancomy cin(10μg/ml), Rifampicin (5μg/ml), Kanamycin (10μg/ml), Nystatin(10μg/ml) were also tested against the seven species of bacteria and three species of fungi under similar conditions so as to compare the degree of inhibition by the calli extracts. Agar wells fed with corresponding solvents served as control. Minimum Inhibitory Concentration (MIC) which was determined as the lowest concentration of callus extracts inhibiting the growth of organisms was determined based on the readings. RESULTS AND DISCUSSION: Phytomedicines are effective in treating most of the infectious diseases mainly skin infections. Most of the secondary metabolites, serve as a plant defence mechanism against microorganisms, insects and herbivores 12. The ethno botanical screening tests of 5

Callus extract Artemisia vulgaris Leaf Extract The table heading may have smaller bold leaf and calli extracts of Artemisia font vulgaris titles (L.) but in should different not solvents be less against than both 10 bacteria pt in size (12 pt is preferable). and fungi using antibiotic sensitivity test are depicted in Table 1. Table 1 Minimum inhibitory concentration (MIC) of Artemesia vulgaris (L.) for antimicrobial activity. Source Solvents MIC (mg/ml) A B C D E F G H I J Acetone 2.00 2.00 2.00 2.00 1.00 2.00 2.00 1.00 1.00 4.00 Benzene - - * - - * - - - 10.00 Chloroform 3.00 4.00 4.00 4.00 2.00 1.00 4.00 4.00 4.00 2.00 Methanol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 4.00 Hexane - - - * * * - * * - Petroleum ether * * * - - - - - - * Acetone 1.00 2.00 4.00 2.00-4.00 2.00 2.00 2.00 6.00 Benzene * - * - - * - * - * Chloroform * - 4.00 4.00 4.00 * 6.00 4.00 4.00 6.00 Methanol 10.00 30.00 2.00 4.00 2.00 2.00 4.00 - - - Hexane - - - - * - - * * * Petroleum ether * * * * * * - - - - Standard 6.00 10.00 6.00 4.00 4.00 4.00 6.00 4.00 4.00 6.00 Values are the average of five determinations, -;Not active * ; Shows poor inhibition (A) Bacillus megaterium (B) B. subtilis (C) Escherichia coli (D) Enterobacter faecalis (E) Proteus vulgaris (F) Pseudomonas aeruginosa (G) Staphylococcus aureus (H) Aspergillus niger (I) Aspergillus fumigatus (J) Candida albicans Standard : Streptomycin (10 µg/ml) for E. coli; gentamicin (10 µg/ml) for P. aeruginosa and P. vulgaris; chloramphenicol (30 µg/ml) for S. aureus; vancomycin (10 µg/ml) for B. subtilis; rifampicin (5 µg/ml) for E. faecalis; kanamycin (10 µg/ml) for B. megaterium; nystatin (10 µg/ml) for Aspergillus niger, A. fumigatus and Candida albicans. The extracts are found to be more effective against bacteria rather than fungi. Benzene, hexane and petroleum ether extracts of leaf and callus were found to be ineffective or showed poor inhibition of bacterial and fungal growth. The MIC values of the solvent extracts of Artemitia vulgaris L. were graphically represented in the Graph 1. The leaf extract of acetone showed MIC of 2.0 mg/ml against all tested bacteria except P. vulgaris (1.0 mg/ml) and 1.0 mg/ml against the tested fungi except C. albicans (4.0 6

mg/ml). Chloroform leaf extracts showed MIC of 4.0 mg/ml against B. subtilis, E. coli, The graph heading may have smaller bold font E. faecalis, A. niger and A. fumigatus, titles where but should as 3.0 mg/ml not be against less than B. megaterium, 10 pt in size (12 pt is referable). The graph heading MUST 2.0mg/ml for P. vulgaris, C. albicans be and in 1.0 the mg/ml text against form P. and aeruginosa. should not form part of the image. Graph 1. MIC of different solvent extracts of Artemitia vulgaris L. Methanol leaf extract showed MIC of 2.0 mg/ml against all the tested organisms except C. albicans (4.0 mg/ml).the MIC of Choloroform callus extracts was 4.0 mg/ml aginst E. coli, B. subtilis, P. vulgaris, A. niger and A. fumigatus, 6.0 mg/ml against S. aureus and C. albicans. It showed poor inhibitory action against B. megaterium and P. aeruginosa and was not active on B. subtilis. For methanol callus extracts, the MIC was 2.0 mg/ml for E. coli, P. vulgaris, P. aeruginosa, 4.0 mg/ml aginst B. megaterium, S. aureus, 10.0 mg/ml for B. megaterium and 30.0 mg/ml for B. subtilis and not active on any fungal species ( Figure 1). 7

Photographs must be clear and sharp and should follow the following guidelines 300dpi or higher sized to fit journal page, JPEG, GIF, TIFF and PDF formats are preferred Figure 1 Antimicrobial activity of solvent extracts of Artemitia vulgaris L. The figure heading may have smaller bold font titles but should not be less than 10 pt in size (12 pt is preferable). The figure heading MUST be in the text form and should not form CONCLUSION: part of the image. The results suggest that leaf extracts are more effective than callus extracts against the tested organisms. The MIC values suggest the medicinal properties of the experimental plant, which are found better than standard values. Further work is needed to isolate the active principle from the plant extracts and to carry out pharmaceutical studies. 8

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