E Subudhi, S Mohanty, S Mohanty, A Kuanar, M Panda
antimicrobial assay, essential oils, medicinal plants, patogens, plant extracts
E Subudhi, S Mohanty, S Mohanty, A Kuanar, M Panda. In Vitro Antimicrobial Study of Plant Essential Oils and Extracts. The Internet Journal of Microbiology. 2009 Volume 8 Number 2.
Present investigation provides comprehensive and quantifiable information on
Extracts of plant origin have been known to possess many therapeutic properties since thousands of years 1. Use of plant parts as folklore medicine have been trailed by traditional healers since time immemorial though scientific basis of their antimicrobial actions, specificity in performance against microbial strains, role of active ingredients or major constituents of their products, application dosage range and toxic effects have only been understood in recent times after plant essential oils and extracts have been subjected to various types of analytical studies with the advent of sophisticated instruments as well as development of newer methodologies. With time these oils and extracts have been understood to encompass the attributes accounted not only for their fragrance and flavor 2 but also for their antimicrobial nature for treating plant animal and human diseases 3-6 and food preservative properties 7. Consequently attempts have been taken by different workers on
Materials & Methods
Fresh leaves of
Test Organisms and their maintenance
Six bacterial and four fungal pure cultures (
Extraction of Essential oil
Essential oils from fresh, clean, weighed leaves of tulsi, lemongrass, curry leaves and rhizome of turmeric extracted by hydro steam distillation using Clevenger’s apparatus were collected and stored in sterile vials.
Successive solvent extraction
Successive solvent extraction was performed for
Muller Hilton agar (MHA) and Sabraud dextrose agar (SDA) were used for cultivating microorganisms and Dimethyl formamide (DMF) solvent was used for dilution of essential oil. All these chemicals were obtained from Hi Media laboratories private limited, Mumbai, India.
For evaluation of inhibition zone diameter (IZD), inoculum from 10-1 dilution of 24 hours incubated sub-cultures for bacteria and 4-8 days incubated sub-cultures of fungi were prepared from their freshly grown cultures of 10 different microorganisms. 0.5 ml. (equivalent to 106 CFU/ml of fungal spore or bacterial cell) of such diluted cultures was used and adjustment of the inoculums size was first done by making necessary dilutions after plate counting is done for these microorganisms so as to ensure the concentration of these organisms to contain approximately 1X 106 CFU/ml in 0.5ml of inoculums.
Microbial assay study
IZD was determined by disc diffusion method (24) by placing 5mm diameter Whatman’s No.1 filter paper disc dipped in essential oil onto microorganism inoculated solidified media. Anti microbial property of standard antibiotic discs impregnated each with 10μg of either streptomycin or cotrimazole used against bacteria and fungi respectively, were evaluated against all the extracts. The antibiotic discs were obtained from Hi Media laboratories private limited, Mumbai, India. Three such replicates were incubated at 27ºC for 48-72 hours for fungi and 37ºC for 24-48 hours for bacteria to evaluate the zone diameter in mm. The average of three measurements was recorded. For all above experiments one positive and one negative control were run parallel. Dimethyl formamide solvent used to dilute essential oil and six chemical solvents used for successive extraction were examined for having any antimicrobial property.
Antibacterial index (AbI) and antifungal index (AfI) for each extract type were calculated as the average value of zone of inhibition against a group of gram +ve and gram -ve bacterial and fungal test strains respectively and the average of Antibacterial index (AbI) and antifungal index (AfI) gives total Activity index (AI) of a particular cultivar. Susceptibility index (SI) denotes the range of susceptibility of any microorganism against a range of extract(s) of a plant that is calculated from the average of antimicrobial activity (IZD) obtained against an individual microbial isolate.
Results and discussion
It is clear from figure 1 that all extract types respond almost equally towards gram +ve and gram –ve bacteria except lemongrass essential oil and acetone extract of
Lower susceptibility index (SI<11mm) exhibited by chemical extracts of
Stevia also exhibited lower susceptibility index (SI<10mm) but it is clearly depicted in figure-4 that among the bacterial pathogens selected for this study, highest susceptibility (SI9.27mm) was exhibited by
As shown in figure 2 the highest anti fungal index (AfI-15) for stevia was found in case of petroleum ether extract irrespective of 4 fungal species used, namely
In figure 3 the anti microbial index for essential oil of
Highest range of susceptibility index (SI) was exhibited by almost all microbial isolates exposed to essential oil of 10 lemongrass cultivars establishing lemongrass proving to be the best antimicrobial potential among the plant extracts tested. Highest value of all indices (AfI, AbI & AI) also supports the above statement, which is clearly evident in all the figures (fig 1,2,3,4).
However, there lies a lot of variation in its activity with respect to cultivar types and microorganisms as well as certain range of specificity is also exhibited towards few isolates. The highest growth inhibiting potential was exhibited by the essential oil from cultivars CF4 and CF5 against test organisms
The present study has demonstrated that volatile essential oil extract of
Essential oil of majority of plants exposed to four food spoiling and plant disease causing fungal pathogens
The above screening results enumerates the existing potential of plant chemical extracts and essential oils to be used as suitable candidate as medicine, pharmaceuticals and food preservatives of plant origin for treating plant and animal disease causing pathogens and food spoiling microorganisms so that a safe alternate to existing chemical at low cost may be identified. However, for whatever purpose it may be used, issue of safety and toxicity needs to be addressed following recommended
Authors are grateful to Dr. S. C. Si, Dean, Center of Biotechnology and Dr M. Nayak, President, Siksha ‘O’ Anusandhan University, Bhubaneswar for providing the necessary facilities to carry out above work.