INHIBITION KINETICS OF E. COLI BY METHANOLIC ROOT EXTRACT OF ALSTONIA SCHOLARIS
HTML Full TextINHIBITION KINETICS OF E. COLI BY METHANOLIC ROOT EXTRACT OF ALSTONIA SCHOLARIS
Pratyush Kumar, C. S. Misra, Joel James, Lipin Dev M. S., Arun Kumar Thaliyil Veettil and Thankamani V*
School of Bio Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India
ABSTRACT
The potential of methanol extract as a potent antimicrobial agent has been observed to be an effective means to combat disease causing pathogenic organisms. Growth pattern of Escherichia coli cells showed that the growth of cells decreased with an increase in concentration of extract. The specific relationship between the specific growth rate and the substrate concentration was thus be obtained by fitting these data into Monod kinetic model. The non competitive and uncompetitive inhibition kinetic models were also tested in order to determine the inhibitory effects of the methanol extract of roots on Escherichia coli. Longest lag phase of 8 hrs was obtained at nutrient broth concentration of 2 g/L. Kinetic studies showed that with an increase in concentration of extract (10-30 g/L) the specific growth rate and inhibition constant decreased but saturation constant increased. Therefore the biokinetic constant estimated by these models have laid the possibility of obtaining suitable antibiotic from Alstonia scholaris.
Keywords:
Alstonia scholaris, Antimicrobial, Biokinetic, |
Monod
INTRODUCTION: Whole plants or their parts have been used extensively since time immemorial in treating various disorders and have been alternative to the modern chemical based drugs because of potent side effects. The family Apocynaceae to which Alstonia scholaris belongs have more than 250 genera and 2000 different species of tropical plants like Rauwolfia serpentine, Alstonia scholaris, Alstonia venenata and Vinca rosea1. Most of these plants have been used against various diseases of skin, heart diseases, liver diseases, dysentery tumors and hypertension.
Alstonia scholaris is a tropical evergreen tree most commonly found in south Asia. So far almost all parts of plants have found to show some therapeutic activity. Leaves, stem bark, roots and inflorescence have shown antimicrobial properties against most common disease causing organisms 1, 2. The various alkaloids (Echitamine, Ditamine) found in bark have been used in the treatment of diarrohea and malaria 3, 4, 5. The bark extract is also a useful remedy for treating asthma, lung cancer, hypertension, and pneumonia while the leaf extract is used to treat fever6. It is effective against boils and ulcers and can be cured by applying milky latex or young leave as poultice over affected area.
The latex obtained from the various plant parts is useful in treating ailments like ulcers, sores, tumours, and rheumatoid pain 7. Roots however have been reported to have very rare medicinal use in enlarged liver and pain but no therapeutic intervention is available so far.
The existing data on the antimicrobial activities can be evidenced by the pathogen-extract interaction. It is well recognized by cell biologists that loss of cell viability may be greatly attributed to the large increase in membrane permeability because of the destruction of plasma membrane in the presence of high concentrations of extracts 8. Therefore the present study was undertaken to establish the effect of inhibitory substance like methanolic root extract on pathogen using Monod kinetic and the toxic effects of the inhibitor concentration (Methanolic concentration) could be studied using inhibition models viz. Non Competitive and Un Competitive 9, 10, 11.
MATERIAL AND METHODS
Collection and processing of plant materials: The fresh plant parts of Alstonia scholaris were collected and powdered which was then subjected to solvent extraction using methanol as a solvent in a soxhlet apparatus. The phytochemical investigation and antimicrobial screening of the various plant extracts were done. The plant extract with highest antimicrobial activity was chosen to proceed with the kinetic modelling of pathogen inhibition.
Toxicity Studies:
Microorganism & Growth Media: In the present study, E. coli culture was maintained in nutrient agar media and was stored at 4.±1oC. The culture was inoculated in nutrient broth with concentrations varying from 2g/L to 10 g/L and methanol root extracts with concentrations varying from 10 g/L to 30 g/L respectively. The cultures were kept under shaking conditions at 37 °C. The biomass (g/L) was harvested at different time intervals and was compared with respect to the control conditions (0 g/L extract).
Kinetic Approach: The relationship between the specific growth rate and substrate concentration in absence of inhibitory substance can be described by the Monod equation as given below.
……………………………………………………………. (1)
Where S is the substrate concentration (g l-1), µ and µm are the specific growth rate and the maximum specific growth rate of microorganism (h-1), respectively and Ks is the saturation constant (gl-1), Eq. (1) can be linearized in double-reciprocal form:
……………………………………………………. (2)
Ks and µm values can be determined from the plot of 1/ µ versus 1/So (assuming S= So at the beginning of exponential growth) yields a linear line with a slope of Ks/ µm and y-axis intercept of 1/ µm. In presence of inhibitory (toxic) substances in nutrient media, microbial growth becomes inhibited, and specific growth rate depends on inhibitor concentration12. Inhibitions models viz. Non competitive and Uncompetitive are classified according to the effects of toxic compounds on the specific growth rate and saturation constant Ks and the rate expressions of the models are expressed as follows:
Non-competitive inhibition:
…………………………………………… (3)
Or;
……………………………………………………………. (4)
Where;
………………………………………………….. (5)
Where; Co and KI are the methanol root extract concentration (g/L) and inhibition constants of extract (g/L), respectively. Eq. (3) can be linearized as similar to Eq. (2):
……………………………………………. (6)
The µm,app values can be determined from the linear plot of 1/ µ versus 1/S at different initial extract concentrations and then the value KI can be calculated from Eq. (5).
Non-competitive inhibition model describing the extract inhibition was selected for assessing the dynamic behaviour of E.coli cells 13, 14.
Uncompetitive inhibition:
.................................... (7)
This equation can be linearized as follows;
................................................... (8)
Where;
………………………………………………….. (9)
RESULT AND DISCUSSION:
Toxicity Studies: Influence of concentration of methanol extracts of root on growth pattern of Escherichia coli was studied. The growth pattern of Escherichia coli as a function of initial methanol root extract concentration was carried out by varying the extract concentration (0, 10, 20, 30g/L) in 2, 4, 6, 8, 10g/L nutrient broth with optimized pH values. The growth curves are shown in Fig. 1-5 respectively. A decrease in biomass with an increase in extract concentration was seen at all concentrations of nutrient broth.
Minimum growth was noted in case of 30 g/L extract concentration at all concentrations of nutrient broth as compared to that of 10 g/L and 20 g/L extract concentration. Longest lag phase of 8 hrs at 30 g/L extract concentration and 2 g/L nutrient broth concentration was noted. Shortest lag phase of 3 hrs at 6,8 and 10 g/L of nutrient broth concentrations and 0 g/L of extract concentration was noted. The decrease in biomass at increasing concentrations of extract was caused due to toxicity of extract on Escherichia coli 15.
Inhibition Models: In absence of Methanol root extract concentration, the values of µm and KS for Escherichia coli was determined as 0.136 h-1 and 0.484 g/L. respectively from Monod equation by linear regression method. As the Monod expression for growth kinetics did not represent the inhibitory effects of the extract, Non competitive inhibition model and Uncompetitive model were tested to characterize the methanol root extract inhibition kinetics of Escherichia coli. The µmapp values could be determined from the intercept of linear plot of 1/ µ versus 1/S at different extract concentrations (Fig. 6).The values of inhibition constant KI was also estimated from found µmapp and known extract concentration values.
FIG. 6: LINEWEAVER-BURK PLOT FOR METHANOL EXTRACT OF ROOT OF ALSTONIA SCHOLARIS
Table 1 and 2 showed the values of Non competitive and Uncompetitive model parameters. The values of inhibition constant indicate the tolerance limit of Escherichia coli species. The inhibition constant values were obtained as 184.29, 180.35, 176.59g/L for 10, 20, 30g/L of extract concentration in case of both Non competitive and Uncompetitive inhibition models which confirmed the inhibitory effect of extract on growth of Escherichia coli. As parameters of both the models (Non competitive and Uncompetitive) showed similar response in terms of µmapp and KI and almost similar response in case of KS, Non competitive inhibition model was selected 12.
TABLE 1: NON COMPETITIVE INHIBITION MODEL PARAMETERS
Concentration (g/L) | µmapp (hr)-1 | KS (g/L) | KI (g/L) |
10 | 0.129 | 0.539 | 184.29 |
20 | 0.122 | 0.634 | 180.35 |
30 | 0.116 | 0.729 | 176.69 |
TABLE 2: UNCOMPETITIVE INHIBITION MODEL PARAMETERS
Concentration (g/L) | µmapp (hr)-1 | KS (g/L) | KI (g/L) |
10 | 0.129 | 0.562 | 184.29 |
20 | 0.122 | 0.674 | 180.35 |
30 | 0.116 | 0.745 | 176.69 |
CONCLUSION: Growth profile of Escherichia coli cells was obtained in presence of extract ranging from 10 to 30 g/L concentration. The cultures were prepared in nutrient broth ranging from 2 g/L to 10 g/L. The growth was found to decrease with an increase in extract concentration. Longest lag phase of 8 hrs was obtained at 2 g/L nutrient broth concentration and 30 g/L Methanol extract concentration.
Monod Kinetics was used to calculate maximum specific growth and saturation constant values of 0.136 h-1 and 0.484 g/L. Two inhibition models namely Non competitive and Uncompetitive models were used to confirm the toxic effects of Methanol root extracts on Escherichia coli. The toxicity parameters were calculated from Lineweaver Burk plot. A decrease in specific growth rate and inhibition constant values and an increase in saturation constant values confirmed the toxic effects on E. coli cells.
ACKNOWLEDGEMENT: The authors would like to thank VIT University for providing necessary facilities and support to carry out this research work.
REFERENCES:
- Thankamani V, James J, Veettil AKT and Sagadevan LDM: Phytochemical screening and anti microbial activity of Alstonia scholaris flowers (L) R.BR. Fam: Apocynaceae. International Journal of Pharmaceutical Research and Development 2011; 3(3): 172-178.
- Misra CS, Pratyush K, Sagadevan LDM, James J, Veettil AKT and Thankamani V: A comparative study on phytochemical screening and antibacterial activity of roots of Alstonia scholaris with the roots, leaves and stem bark. International Journal of Research in Phytochemistry and pharmacology 2011; 1(2):77-82.
- Baliga MS, Jagetia GC, Ulloor JN, Baliga MP, Venkatesh P, Reddy R, Rao KVNM, Baliga BS, Devi S, Raju SK, Veeresh V , Reddy TK, Bairy LK: The evaluation of the acute toxicity and long term safety of hydroalcoholic extract of Sapthaparna (Alstonia scholaris)in mice and rats. Toxicology Letters 2004; 151: 317-326.
- Jagetia GC, Baliga MS, Venkatesh P: Effect of Sapthaparna (Alstonia scholaris Linn) in modulating the benzo(a)pyrene-induced forestomach carcinogenesis in mice. Toxicological Letters 2003; 144: 183-193.
- Singh MP and Panda H: Medicinal Herbs with Their Formulations. Daya Publishing house, Delhi, 2005: 88-90.
- Channa S, Dar A, Ahmed S, Rahman A: Evaluation of Alstonia scholaris leaves for broncho-vasodilatory activity. Journal of Ethnopharmacology 2005; 97:469-476.
- Daniel M: Medicinal plants: Chemistry and Properties. Science Publisher, USA, 2006: 24-25.
- Flores MV, Voget CE, Ertola RJJ: Permeabilization of yeast cells (Kluyveromyces lactis) with organic solvents. Enzyme Microb. Technol.1994; 16: 340-346.
- Charumathi D and Das N: Bioaccumulation of Synthetic Dyes by Candida tropicalis growing in Sugarcane Bagasse Extract Medium. Advances in Biological Research 2010; 4 (4): 233-240.
- Kleckal GM and Maier WJ: Kinetics of Microbial Growth on Pentachlorophenol. Applied and Environmental Microbiology 1985; 49(1): 46-53.
- Agarry SE, Solomon B O and Layokun SK: Kinetics of batch microbial degradation of phenols by indigenous binary mixed culture of Pseudomonas aeruginosa and Pseudomonas fluorescence. African Journal of Biotechnology 2008; 7 (14): 2417-2423.
- Das D, Charumathi D, Das N: Combined effects of sugarcane bagasse extract and synthetic dyes on the growth and bioaccumulation properties of Pichia fermentans MTCC 189. Journal of Hazardous Materials 2010; 183: 497–505.
- Shuler ML and Kargi F: In: Bioprocess engineering. Prentice Hall, New Jersey, 1992: 170–191.
- Aksu Z, Donmez G: Combined effects of molasses sucrose and reactive dye on the growth and dye bioaccumulation properties of Candida tropicalis. Process Biochemistry 2005; 40: 2443–2454.
- Dey S and Mukherjee S: Performance and kinetic evaluation of phenol biodegradation by mixed microbial culture in a batch reactor. International Journal of Water Resources and Environmental Engineering 2010; 2(3): 40-49.
Article Information
23
2884-2888
526
916
English
Ijpsr
Pratyush Kumar, C. S. Misra, Joel James, Lipin Dev M. S., Arun Kumar Thaliyil Veettil and Thankamani V*
School of Bio Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India
08 July, 2011
05 August, 2011
17 October, 2011
http://dx.doi.org/10.13040/IJPSR.0975-8232.2(11).2884-88
01 November, 2011