S. S. Roy
Department of Applied Chemistry and Chemical, Islamic University, Kushtia-7003, Bangladesh
M. A. Rahman
Department of Applied Chemistry and Chemical, Islamic University, Kushtia-7003, Bangladesh
M. M. Rahman
Department of Biotechnology and Genetic Engineering, Faculty of Biological Sciences, Islamic University, Kushtia-7003, Bangladesh
Antibacterial, Phytochemical, Antibiotics, Gram-positive, Gram-negative, Catharanthus roseus
Kushtia Sadar and campus area of Islamic University, Bangladesh.
Development of Host and Medicinal Plants
The fresh and mature flowers of C. roseus were collected from various locations in Kushtia Sadar and the campus area of Islamic University, Bangladesh. The flowers were washed carefully 2-3 times with running tap water and finally washed with sterile water for removing dust and surface microbes. Then the flowers were dried about 20-30 days under the shading place for collecting flowers extracts. The extraction of samples was prepared by soaking 5 g of dried flower powder in 50 mL of each solvent such as ethanol, ethyl acetate, chloroform, and petroleum ether and kept them overnight in a rotary shaker. The solution was left for 72 hours at room temperature and then it was filtered with the help of sterilized Whatsman No.1 (5mm diameter) filter paper. The filtrate solution samples were concentrated using a water bath and stored at 4°C for further use. In order to phytochemical analysis, we prepared three kinds of reagent solution such as Mayer’s reagent: it is used for the detection of alkaloids. Solution (A) was made by dissolving 0.68g mercuric chloride in 30 mL of distilled water. Solution (B) was made by dissolving 2.5 g of potassium iodide in 10 mL of distilled water. Solution A & B were mixed and the volume was adjusted to 100 mL with distilled water. 1% Sodium hydroxide (NaOH) solution: 1g pellets of NaOH was weighted and dissolved in 100mL distilled water. It was used in detecting the presence of quinine. Mercuric chloride solution (HgCl2): the amount of 7.4 g of mercuric chloride was dissolved in 100 mL of distilled water at about 20 °C temperature. This solution was used for the protein test. all the tested bacteria. The zones of inhibition around the wells were measured accurately using a metric ruler to the nearest millimetre. Different qualitative chemical tests can be performed for establishing a profile of methanol and aqueous extract for its chemical composition. The following tests were performed on extracts to detect various phytoconstituents present in them. Detection of Alkaloids: Previously isolated 1 mL extraction from C. roseus flowers was added to few drop of Iodine solution which formed yellow colour precipitate indicates the presence of alkaloids (Kabesh et al., 2015). Detection of Protein: Similarly 1 mL of extraction of C. roseus flowers was added a few drop of mercuric chloride and the formation of yellow colour indicates the presence of protein (Kabesh et al., 2015). Detection of Steroid: The one (1) mL of extraction of C. roseus flowers mixed with 1 mL of chloroform and concentrated with sulphuric acid (H2SO4) sidewise. A red colour presence at the lower chloroform layer indicates presence of steroids (Kabesh et al., 2015). Detection of Flavonoids (Alkaline reagent test): The extraction amount (2mL) of C. roseus flowers was treated with few drops of 20% sodium hydroxide (NaOH) solution and the mixtures solution was created acute yellow colour which disappeared on the addition of 2 drops dilute hydrochloric acid (HCL) indicates the presence of flavonoids (Gul et al., 2017). Detection of Phenols: (Ferric chloride test): The amount (2 mL) of C. roseus flowers extracts was treated with 0.5 mL of aqueous 5% ferric chloride (FeCl3) and observed for formation of deep blue or black colouration, which confirms the presence of phenols (Hema et al., 2012). Detection of Saponins (Foam test): Distilled water (6 mL) was added in 2 mL of C. roseus flowers extract. The mixture was shaken thoroughly in graduated cylinders for 15 minutes and observed for the formation of constant foam to confirm the presence of saponins (Dubey, 2014). Detection of Terpenoids (Salkowki’s test): Chloroform (2 mL) was added to 2 mL of extract and added a few drops of concentrated H2SO4. The mixture was shaken well. A reddish brown precipitate produced immediately indicates the presence of terpenoids (Amin et al., 2013). Detection of Quinines: To 1 mL of extract added 1 mL of 1% NaOH and mixed well. Appearance of blue green or red indicates presence of Quinines (Kabesh et al., 2015). Inoculums preparation- Each bacterial strain was sub-cultured overnight at 35 °C in Nutrient agar slants. The bacterial growth was harvested using 5 ml of sterile saline water, its absorbance was adjusted at 580 µm and diluted to attain viable cell count of 107 CFU/ml using spectrophotometer. Antibacterial analysis of plant extract- In this study, ethanol, ethyl acetate, petroleum ether and chloroform flower extracts of C. roseus were tested for their antimicrobial effects against four different (one Gram- positive and three Gram-negative) MDR food-borne pathogenic bacteria. The studied microorganisms included MDR clinical strains of Gram-negative bacteria (Escherichia coli BTGE-K_8, Aeromonas caviae BTGE-K_1, Citrobacter freundii BTGE-K_4) and and Gram-positive bacteria (Staphylococcus hominis BTGE-K_11) were collected from Microbiology laboratory, Department of Biotechnology and Genetic Engineering, Faculty of Biological Sciences, Islamic University, Kushtia-7003, Bangladesh. A antibacterial assay was performed by agar well diffusion method (Chaman, Sharma, and Reshi 2013). Petri dishes were prepared by pouring 20 ml of Nutrient Agar medium and allowed to solidify. Plates were solidified and 100 µL of bacterial culture was poured and uniformly spread and the inoculum was allowed to dry for 5 minutes. Agar well of 5 mm in diameter were prepared with the help of a sterilized stainless cork borer. The wells were labelled appropriately and to each well were loaded with 500µg/50µl, 250µg/60 µl and 125µg/70 µl plant extract and corresponding solvent (50µl) of the extract using a micro-pipette. Standard reference antibiotic Doxycycline (30 µg/disc) was used as controls for the tested against bacteria. The plates were incubated at 37°C for 24 hours. After incubation for 24 hours, the zones of inhibition around the wells were measured accurately using a metric ruler to the nearest millimetre. Measurement of minimum inhibitory concentrations (MIC’s) of the C. roseus L. flower plants extract- The minimum inhibitory concentrations (MIC) is defined as the lowest concentration of plant extract that inhibit bacterial growth within a certain time (24 h) incubation (Mostafa et al. 2018). It was necessary to prepare a different concentration of plant extracts. The serial dilution technique of plant extracts was as follows: mother stock was prepared by dissolving 5 mg dry extract into 10 mL of the corresponding solvent. Thus the concentration of mother stock became 500 µg/mL. Then the autoclaved eppendorf conical tubes (PolyLab, India) were taken for serial dilution. From mother stock, 5 mL solution was taken into first eppendorf tube and same amount (5 mL) of corresponding solvent was added to make the final volume 10 mL and final concentration became 250 µg/mL. Then, 5 mL aliquot was transferred from second into third eppendorf tube and the same amount of the corresponding solvent was added to make the final volume 10 mL and the final concentration became 125 µg/mL. This process was repeated for several times and made solution of 62.50, 31.25, 15.62, 7.813, 3.906 and 1.953 µg/mL concentrations and the final one was only respective solvent. We made four wells on each petri dish with borer. Among four wells, three were filled with different concentration of plant extract and one was filled with only corresponding solvent extraction. The extract concentrations of 500, 250, and 125 µg/mL were added in each well and the amount of volume of 50, 60 and 70 µl, respectively. The corresponding solvent of the extract was added to the well which volume was 50 µl. Finally, one antibiotic disk of Doxycycline (30 µg/disc) was applied in the middle position of the cultured medium of each petri dish. A similar procedure was followed for each solvent extract of C. roseus. Each experiment was completed in triplicate and antimicrobial activity of different extracts were evaluated by measuring the diameter of zones of inhibition in mm against all tested bacteria. Statistical analyses were performed One- way analysis of variance (ANOVA) using IBM SPSS Statistics version 21. Post Hoc (Duncan’s multiple range test, DMRT) Tests were done by SPSS and differences were considered as significant at the level of p < 0.05. All the data are expressed as means ± standard deviation (n = 3).
Journal of Agriculture, Food and Environment, Vol 1 No 4 December 2020 Pages 87-93
Journal