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Screening of Antimicrobial Potential and Brine Shrimp Lethality Bioassay of the Whole Plant Extract of Spilanthes Paniculata Wall. Ex Dc

Rokeya Siddiqui
Department of Microbiology, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh

Md. Masud Alam
Department of Microbiology, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh

Mohammad Ruhul Amin
Department of Microbiology, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh

A. F. M. Shahid-Ud-Daula
Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh

M. M. Hossain
Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh

Abstract/ Executive Summary:

Bangladesh possesses a rich flora of medicinal plant. Out of the estimated 5000 species of phanerogams and pteridophytes growing in this country, more than a thousand are regarded as having medicinal properties. The aim of this study was to evaluate the in vitro antimicrobial activity and brine shrimp lethality of the whole plant extracts of Spilanthes paniculata, a plant belonging to the family Asteraceae. The crude extract, n-Hexane, chloroform and ethyl acetate soluble fraction of crude extract showed significant to moderate antimicrobial activity against three Gram positive and three Gram negative microorganisms. The zones of inhibition produced by the crude extract (methanolic extract), n-hexane, choloform and ethyl acetate soluble fractions were found to be 14.89 mm-19.40 mm, 14 mm-19.40 mm, 10.66 mm-13.50 mm and 9.50 mm-13.26 mm, consecutively at a concentration of 30µg/disc. N-Hexane, chloroform and ethyl acetate soluble fractions of methanolic extract of Spilanthes paniculata were screened for antitumor properties using brine shrimp lethality bioassay. From the results of the brine shrimp lethality bioassay, it can be well predicted that n-hexane, chloroform and ethyl acetate soluble fractions of methanolic extract possess mild cytotoxicity on shrimp naupalii. The median lethal concentration (LC₅₀, the concentration at which 50% mortality of brine shrimp nauplii occurred) of n-hexane, chloroform and ethyl acetate were 48.978 mg/ml, 92.61 mg/ml and 216.770 mg/ml, respectively, comparison with positive control vincristine sulphate with 0.839 mg/ml.

Keywords: Spilanthes paniculata; Antimicrobial activity; Brine shrimp lethality; Cytotoxicity, LC50

Location: Department of Microbiology, Noakhali Science and Technology University, Bangladesh (

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Shrimp

Objectives:

1. The aim of the present work was to evaluate the antimicrobial potential and cytotoxicity to support the pharmacological effects and phytochemical investigation of the plant.

Methodology:

Bacterial strain. Six gram positive and gram negative bacterial strains were obtained from the stock culture of the Department of Microbiology, Noakhali Science and Technology University, Bangladesh. These strains were grown in nutrient broth (Oxoid, USA) for 18 hour at 37⁰C and maintained on agar slant at 4⁰C. Plant material. The plant Spilanthes paniculata was collected from the Noakhali district, Bangladesh. The plant sample was identified by the experts of Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh and the identification number is 35400. The collected whole plant parts were washed with water for several time, separated from undesirable materials and then air-dried under shade to protect from sunlight for one week. The dried plant parts were ground into a fine powder with the help of a hammer mill and fine powder was stored in cool and dry place in an airtight container until analysis commenced. Preparation of extract. Whole plant extracts were made by cold extraction method. 400 gm of powdered material was soaked in 1300 ml of 90% methanol. The container with its contents was sealed and kept for 7 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then it was filtered through whatman filter paper (Cat. No. 1440125, Whatman Incorporation, UK) and the filtrate thus obtained was concentrated by using traditional spontaneous natural vaporization method at room temperature. Then, 5 gm of methanolic extract was dissolved in 100 ml of 90% methanol. This is the mother solution, which was partitioned off successively by three solvents of different polarity such as n-hexane, chloroform and ethyl acetate. Antibacterial Screening. The total viable bacterial count was carried out by the spread plate technique. The sample (0.1ml) of each dilution was taken onto each sterile petridish and evenly spread on the solid nutrient medium and incubated at 37 °C for 24 hours. The plates were screened for the presence of discrete colonies after incubation period and the actual numbers of bacteria were estimated as colony forming unit in per ml (cfu/ml). Then the results per dilution were recorded. Quantitative analysis for the presence or absence of specific microorganisms was conducted by plating on selective media. Total coliform count (TCC) was done using MacConkey agar medium. All the viable counts were the average of at least three independent experiments. Bacterial isolates were then identified according to the Bergey’s manual of determinative bacteriology. Tested bacterial sample from the stock culture were transferred to nutrient agar slants medium. The inoculated slants were then incubated at 37⁰C for 18-24 hours and then the fresh culture was transferred to the test tube containing nutrient broth to make a uniform suspension of organisms. The bacterial suspension was immediately poured onto nutrient agar plate to give a uniform layer of bacteria. Excess bacterial suspension was taken out with a sterile Pasteur pipette. Impregnated test sample, positive and negative control discs were placed aseptically on the freshly seeded solidified agar plate using sterile forceps. The spatial arrangement of discs were such that the discs were not closer than 20 mm to the edge of the plate and far enough apart to prevent the overlapping the zone of inhibition. The plates were kept at refrigeration temperature for 3-4 hour for better absorption, during this time microorganisms will not grow but absorption of extract would take place. Finally the plates were incubated upside down at 37⁰C for 12-18 hours.

Source of Information: Stamford Journal of Microbiology, December 2013. Vol. 3, Issue 1, p. 1-5; ISSN: 2074-5346

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present study indicates that the extracts of Spilanthes paniculata (methanolic, n-hexane, chloroform and ethyl acetate) have profound antimicrobial and mild cytotoxic effect. From the previous studies and our current investigation, it may be concluded that the flavonoids and tannins are responsible for aforementioned activity. This novel finding will aid us to conduct bioactivity guided isolation and characterization of leading compounds in due course.

Knowledge Management: Journal