Inhibition of dehydrogenase activity in S. typhimurium by ethanolic and methanolic extracts of Carica papaya and Ocimum gratissimum

Dehydrogenase and inhibitions of dehydrogenase activities in Salmonella typhimurium by ethanolic and methanolic leaf extracts of Ocimum gratissimum and Carica papaya were investigated. Dehydrogenase activity assay was carried out using 2, 3, 5-triphenyl tetrazolium chloride (TTC) as the electron acceptor. Pure culture of S. typhimurium was exposed to varied concentrations of ethanolic and methanolic extracts of Ocimum gratissimum and Carica papaya [0-4000 μg/ml]. The ethanolic and methanolic extracts exhibited a concentration dependent response against the tested organism. Results obtained revealed that the ethanolic extracts showed a higher bactericidal effect on the test organism than the methanolic extracts at the threshold and total inhibitory concentrations. The IC50 were 45.349 and 15.697; IC100 were 43.732 and 35.526 for ethanolic extracts of Carica papaya and Ocimum gratissimum respectively while the IC50 were 7.108 and 13.696; IC100 were 40.815 and 31.104 for methanolic extracts of Carica papaya and Ocimum gratissimum respectively. This in-vitro study further revealed that the leaf of Carica papaya was more potent on Salmonella typhimurium than the leaf of Ocimum gratissimum. The findings from this study seem to provide the in-vitro evidence that justifies Carica papaya and Ocimum gratissimum as good candidate medicinal plants for the potential treatment of Salmonella typhimurium infections


INTRODUCTION
Ocimum gratissimum and Carica papaya are valuable multi-purpose medicinal plants which belong to the family Lamiaceae and Caricaceae respectively and are distributed in tropical and warm regions. They are commonly used in the treatment of various diseases such as upper respiratory tract infections, diarrhea, headache, fever, ophthalmic and skin diseases and pneumonia (Gopi et al., 2006). Extracts of the plants contain antimicrobial, antibacterial, antifungal (Lemos et al., 2005), antimalarial (Ezekwesili et al., 2004) and antiprotozoal (Holetz et al., 2003) activities. The active compounds present as volatile oil from the leaves consist mainly of thymol (32-65%) and eugenol (Adeola et al., 2014). They also contain xanthones, terpenes and lactones together with cardiac glycosides, saponins, tannins and alkaloids (Akujobi et al., 2004).
Measurement of microbial enzyme activity has been used in the assessment of ecotoxicological impacts of environmental substrates. In this regard, dehydrogenase activity has been widely used. The dehydrogenase assay is an effective primary test for assessing the potential toxicity of metals to soil microbial activities (Anyanwu et al. 2016;Nwuche and Ugoji, 2008;Nwachukwu et al. 2011), toxicity of metals to planktonic (Nweke et al., 2006) and heterotrophic (Nweke et al., 2007) bacteria from tropical river sediments. Toxicity of plant extracts to pathogenic bacteria has been assessed using the dehydrogenase assay (Nwaogu et al., 2007;Nwaogu et al., 2008;Alisi et al., 2008).
Salmonella typhimurium has posed a problem in causing regular infections in hospitals and public health centers and has become a persistent pathogen in the environment able to easily survive and proliferate to cause serious infections in animals and humans thereby posing a major risk to public health (Martelli and Davies, 2012). Human infections with S. typhimurium originate mainly from livestock products such as meats, eggs and other products when consumed raw or undercooked as well as environmental contaminations from household pets or contaminated birds (De Knegt et al., 2015).
Thus this study carried out in Anthony van Leeuwenhoek Research Laboratory, Nekede in Imo State, Nigeria isolated Salmonella typhimurium from feces of different categories of livestock using selective/differential media and determined the effects of ethanolic and methanolic extracts of O. gratissimum and C. papaya on the dehydrogenase activity of the recovered Salmonella typhimurium. Several research questions were put forward to help us understand the efficacy of O. gratissimum and C. papaya against Salmonella typhimurium in order to validate or disprove the claim of herbalists who use the leaf extracts of these plants as cheaper antimicrobial herbal remedies against the expensive conventional antibiotics that are currently in use for these ailments.
The study will make for more economic and optimal use of O. gratissimum and C. papaya in alternative medicine. This is because the outcome of this study will contribute to current knowledge on the variations in the effects of ethanolic and methanolic extracts of O. gratissimum and C. papaya on the dehydrogenase activity of Salmonella typhimurium. This will be potentially useful to the relevant public health authorities, abattoir workers, livestock farmers, for the prevention, control and management of health problems caused by Salmonella typhimurium.

Collection and Identification of Ocimum gratissimum and Carica papaya
Fresh leaves of O. gratissimum and C. papaya were collected from Ihiagwa and the forests in the Federal University of Technology Owerri, in Owerri West Local Government Area of Imo state. The plants were identified by a plant taxonomist, Dr. S. E. Okeke in the Department of Plant Science and Biotechnology, Imo State University, Owerri, Nigeria. The freshly collected leaves were air-dried completely. The air-dried leaves were macerated and ground into powdery form using washed, air-dried and oven sterilized electric blender to avoid microbial contamination and stored in a clean airtight container until further use.

Preparation of the test bacterial isolate
Pure culture of Salmonella typhimurium was recovered by direct plating on selective/enrichment media of samples obtained from one-year surveillance in an integrated pig, poultry and cattle production farms. The recovered isolate of Salmonella typhimurium is pale on deoxycholate citrate agar (DCA). Microscopic identification and biochemical confirmation tests were performed to re-identify and confirm the identity of the test organism. Biochemically identified and confirmed Salmonella typhimurium was then grown to mid exponential phase (20 minutes) in nutrient broth on a rotary incubator (150 rpm) at room temperature (28 ± 2°C). The cells were harvested by centrifugation at 6000rpm for 8 min. Harvested cells were washed three times in deionized distilled water and re-suspended in water. The re-suspended cells were adjusted in a spectrophotometer to an optical density comparable to 0.5 McFarland turbidity standards that is equal to 1.5 x 10 8 (One hundred and fifty million colony forming units/ml (CFU/ml) of bacterial suspension. The standardized cell suspension was used as the inoculum in the dehydrogenase activity assay as described by Alisi et al. (2008).

Extraction of Plant Materials
A 20g portion of O. gratissimum and C. papaya powder was weighed into 100ml of ethanol and methanol and kept in a conical flask for 72hrs. Soluble extracts from filtration (filtrate) in a Whatman number 42-filter paper was concentrated under vacuum and air-dried. A 0.4g portion of dried extracts was dissolved in 40mls of DMSO for further extraction in a conical flask. The extracts were then stored in a freezer at 4°C.

Dehydrogenase Assay
The dehydrogenase assay method as described by Alisi et al. (2008) was adopted for the study. The dehydrogenase activity (DHA) was determined using 2, 3, 5-triphenyltetrazolium chloride (TTC) as the artificial electron acceptor, which was reduced to the red colored triphenylformazan (TPF). The assay was done in 4 ml volumes of nutrient broth-glucose-TTC medium supplemented with varying concentrations (0-4000 μg/ml) of the ethanolic and methanolic leaf extracts in separate screwcapped test tubes. About 0.3 ml volume of the standardized bacterial suspension was inoculated into triplicate glass tubes containing 0.4 ml of phosphate-buffered (pH 6.8) nutrient broth-glucose medium supplemented with varying concentrations of the extract solution 0, 50, 100, 200, 400, 800, 1600, 3200 and 4000 μg/ml in different test tubes. The different test tubes were incubated in a rotary incubator (150 rpm) at room temperature (28±2°C) for 30 min. Thereafter, 0.1 ml of 0.1% (w/v) TTC in deionized water was added to each tube to obtain final extract concentrations of 0, 50, 100, 200, 400, 800, 1600, 3200 and 4000 μg/ml in different test tubes. The control consisted of Salmonella typhimurium and the media without ethanolic or methanolic extracts of C. papaya or O. gratissimum. The reaction mixtures were further incubated at room temperature (28 ± 2°C) for 16 hours. The triphenylformazan produced was extracted in 4 ml of amyl alcohol and the absorbance determined using spectrophotometer at 500 nm. The amount of formazan produced was determined from a standard dose-response curve [0-4000 μg/ml TPF (Sigma) in amyl alcohol]. Dehydrogenase activity was expressed as mg of triphenylformazan (TPF) formed per mg dry weight of cell biomass per hour. Inhibition of dehydrogenase activity in the test organism by ethanolic and methanolic C. papaya and O. gratissimum extracts was calculated relative to the control. The percentage inhibition for the test organism was linearized against the concentrations of the extracts using gamma parameter (r-) [r-= % inhibition/ (100-% inhibition)] (Alisi et al., 2008). The toxicity threshold concentrations (IC50) were determined from the linear regression plots. The total inhibitory concentrations (IC100) were extrapolated from the plot of the inhibition data.

Statistical analysis
Data was analyzed using a two-way analysis of variance (ANOVA) and values for P<0.05 were considered statistically significant.

RESULTS AND DISCUSSION
The results of this study on the effects of different concentrations of ethanolic and methanolic extracts of C. papaya and O. gratissimum on Salmonella typhimurium with respect to the dehydrogenase activity and its inhibition are shown in Figures 1, 3, 5 and 7. Dehydrogenase activities observed in the control samples (0 μg/ml i.e. no plant extract) of ethanolic and methanolic extracts of C. papaya and O. gratissimum indicated that the test organism was able to reduce TTC to the red formazan (Figures 1, 3, 5 and 7). The organism's dehydrogenase activity decreased with increase in concentration of ethanolic and methanolic extracts of C. papaya and O. gratissimum (0 -4000 μg/ml) (Figures 1, 3, 5  and 7). The ethanolic extracts of C. papaya and O. gratissimum seem to have a higher rate of inhibition of dehydrogenase activity than the methanolic extracts of O. gratissimum and C. papaya. The evidence is seen from the threshold and total inhibitory concentrations data in Figures 1 and 3 In conclusion, results obtained from this in vitro study show that the ethanolic and methanolic extracts of O. gratissimum and C. papaya inhibited the dehydrogenase activity of S. typhimurium. The inhibitory action may be due to the presence of different phytochemicals contained in these plants. The result of this in vitro study indicated that extracts of O. gratissimum and C. papaya were significantly effective against the tested organism and may serve as a cheaper antimicrobial herbal remedy in the management of salmonellosis.