In vitro and in vivo Antibacterial Effects of Leaf Extracts of Ocimum sanctum and Argemone mexicana 
P. Varshney1 
,
S. K. Dash1,2 
,
A. K. Bhatia1,3
,
S. K. Dash1,2 ,
A. K. Bhatia1,3
1. Department of Microbiology and Immunology, College of Veterinary Science and Animal Husbandry, DUVASU, Mathura- 281001, UP, India
2. High Security Animal Disease Laboratory (HSADL), IVRI, Bhopal-462021 MP, India
3. Department of Biotechnology, GLA Institute of Technology and Management NH-2, Mathura-Delhi Highway, P.O. Chaumuhan, Mathura-281406 UP, India
Author Correspondence author
Medicinal Plant Research, 2013, Vol. 3, No. 9 doi: 10.5376/mpr.2013.03.0009
Received: 25 Jul., 2013 Accepted: 29 Jul., 2013 Published: 31 Jul., 2013
2. High Security Animal Disease Laboratory (HSADL), IVRI, Bhopal-462021 MP, India
3. Department of Biotechnology, GLA Institute of Technology and Management NH-2, Mathura-Delhi Highway, P.O. Chaumuhan, Mathura-281406 UP, India
Author Correspondence author
Medicinal Plant Research, 2013, Vol. 3, No. 9 doi: 10.5376/mpr.2013.03.0009
Received: 25 Jul., 2013 Accepted: 29 Jul., 2013 Published: 31 Jul., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Varshney et al., 2013, In vitro and in vivo Antibacterial Effects of Leaf Extracts of Ocimum sanctum and Argemone mexicana, Medicinal Plant Research, Vol.3, No.9 63-69 (doi: 10.5376/mpr.2013.03.0009)
Abstract
The present study was undertaken to explore the in vitro antibacterial effects of four different leaves extracts of Ocimum sanctum (OS) and Argemone mexicana (AM) against Salmonella enterica serovar Typhimurium and Escherichia coli O26. Besides in vivo antibacterial effects of the hot aqueous extracts (HAE) of OS and AM against the above said enteric pathogens was evaluated in chicken model. In vitro antibacterial activity was determined by disc diffusion and it was concentration dependent. Disc containing 20 mg concentration of all the extracts of OS and AM showed maximum inhibitory effect on the growth of S. enterica serovar Typhimurium and E. coli O26. Among all the extracts methanolic extracts of both the plants had stronger antibacterial activity. On prolonged incubation bacterial colonies reappeared within the zone of inhibition indicating bacteriostatic effect than bactericidal activity. 250 mg/kg body weight oral dose of OS and AM was found ideal and nontoxic in chickens and experimental chickens were fed this dose for 21 days for determination of in vivo antibacterial effect. On 22nd day respective groups of chickens were challenged orally with ID50 dose of Salmonella enterica serovar Typhimurium and Escherichia coli. 83% chickens of OS fed groups and 66% chickens of AM fed groups were protected from challenge of S. enterica serovar Typhimurium and E. coli O26. OS provided better clearance of both the pathogens from blood as compared to AM.
Keywords
Antibacterial; Argemone mexicana; Ocimum sanctum; Leaf extract; S. enterica serovar Typhimurium; E. coli O26
Poultry reared under intensive conditions become more prone to bacterial and viral diseases. Indiscriminate use of antibiotics in feed leads to development of drug resistant microbes. Microbes have the genetic ability to transfer and acquire resistance leading to multi drug resistant strains which further worsen the situation. Due to high production of meat and eggs there is lowering of immunity in birds thus easily becoming susceptible to infectious diseases. High input cost is required to overcome this which may have adverse effect on future growth of poultry industry (Tamara et al., 2009). To keep up the pace of growth in poultry sector and to make it contribute to support rural livelihood, there is a need to remove the constraints being faced by the industry. One of the alternative corrective measures may be the proper use of medicinal plants with scientific validation. Medicinal plants could act as a potential source of new antimicrobial agent (Ahmad et al., 1998). Phytotherapy for infectious agents are used due to their easy availability, fewer side effects and reduced toxicity (Lee et al., 2007).
Among these plants O. sanctum (OS) occupies significant place in the indigenous system of medicine of many Asian, African and South American countries. Earlier various workers have reported the antibacterial properties of OS (Ahmad et al., 1998; Ahmad and Beg, 2001; Mishra and Mishra, 2011). Phytochemical analysis revealed antibacterial properties are due to glycosides, phenols and tannins (Ahmad and Beg, 2001).
Besides these well-established medicinal herbs, there are some weeds like A. mexicana (AM) known for their toxicity. There are few reports stating antibacterial activity of AM is limited to seeds and leaves (Kempraj and Bhat, 2010).
Keeping these preliminary things in mind the present study was focused on to find the in vitro and in vivo antibacterial activity of different leaves extracts of OS and AM against two common poultry pathogens such as Salmonella enterica serovar Typhimurium and Escherichia coli O26.
1 Results
1.1 In vitro antibacterial effects of different extracts of OS leaves by disc diffusion method
Antibacterial activity of OS was examined against S. enterica serovar Typhimurium and E. coli O26. Four concentrations (2 mg/disc, 5 mg/disc, 10 mg/disc and 20 mg/disc) of four different preparations (cold aqueous, hot aqueous, methanolic and hydromethanolic) of OS leaves extract were employed. All the experiment was performed in triplicates and the mean value are given (Table 1; Table 2). Antibacterial activity was concentration dependent. 2 mg concentration/disc of all the extractsexhibited insignificant antibacterial effect against both bacterial isolates, whereas disc containing 20 mg concentration of all the extractsshowed greatest inhibitory effect on the growth of S. enterica serovar Typhimurium and E. coli O26. Among all extracts, methanolic leaves extract exhibited greatest antibacterial effect showing 20 mm and 18 mm zone of inhibition against S. enterica serovar Typhimurium and E. coli O26 respectively. All the extracts were relatively less effective against E. coli O26 (Figure 1). It was also noted that within the zone of inhibition induced by 10 mg concentration/disc, resistant bacterial colonies were present. It was further noted that on prolonged incubation zone of inhibition was narrowed down.
Antibacterial activity of OS was examined against S. enterica serovar Typhimurium and E. coli O26. Four concentrations (2 mg/disc, 5 mg/disc, 10 mg/disc and 20 mg/disc) of four different preparations (cold aqueous, hot aqueous, methanolic and hydromethanolic) of OS leaves extract were employed. All the experiment was performed in triplicates and the mean value are given (Table 1; Table 2). Antibacterial activity was concentration dependent. 2 mg concentration/disc of all the extractsexhibited insignificant antibacterial effect against both bacterial isolates, whereas disc containing 20 mg concentration of all the extractsshowed greatest inhibitory effect on the growth of S. enterica serovar Typhimurium and E. coli O26. Among all extracts, methanolic leaves extract exhibited greatest antibacterial effect showing 20 mm and 18 mm zone of inhibition against S. enterica serovar Typhimurium and E. coli O26 respectively. All the extracts were relatively less effective against E. coli O26 (Figure 1). It was also noted that within the zone of inhibition induced by 10 mg concentration/disc, resistant bacterial colonies were present. It was further noted that on prolonged incubation zone of inhibition was narrowed down.
.png){kind=small}
Table 1 Effects of cold and hot aqueous extract of OS leaves against S. enterica serovar Typhimurium and E. coli O26
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Table 2 Effects of methanolic and hydromethanolic extracts of OS leaves against S. enterica serovar Typhimurium and E. coli O26
.png)
Figure1 In vitro Antibacterial effects of methanolic extract of O. sanctum leaves against two bacterial species
1.2 In vitro antibacterial effects of different extracts of AM leaves by disc diffusion method
Antibacterial activity of AM was also investigated against above said two bacterial isolates viz S. enterica serovar Typhimurium and E. coli O26. Four concentrations (2 mg/disc, 5mg/disc, 10mg/disc and 20 mg/disc) of four different extracts (cold aqueous, hot aqueous, methanolic and hydromethanolic) of AM leaves were tested in triplicates and the mean value are presented (Table 3; Table 4). 2 mg concentration disc of cold and hot aqueous extract had no effect against any of the two bacterial isolates. 5mg /disc produced very narrow zone of inhibition which persisted only for 24~36 hrs and then there was emergence of colonies of S. enterica serovar Typhimurium and E. coli O26 within earlier zone of inhibition. 20 mg concentration disc showed greatest zone of inhibition for these two bacterial isolates. The present study also revealed that methanolic extract was effective against S. enterica serovar Typhimuriumand E.coli O26 (Figure2).
.png){kind=small}
Table 3 Effects of cold and hot aqueous extracts of AM leaves against S. enterica serovar Typhimurium and E. coli O26
.png){kind=small}
Table 4 Effects of methanolic and hydromethanolic extracts of AM leaves against S. enterica serovar Typhimurium and E. coli O26
.png)
Figure2 In vitro Antibacterial effects of methanolic extract of A. mexicana leaves against two bacterial species
1.3 In vivo effects of hot aqueous extract (HAE) of OS and AM leaves againstS. enterica serovar Typhimuriumand E. coli O26 infection
1.3.1 Determination of ID50 dose ofS. enterica serovar Typhimurium and Escherichia coli O26 in chickens
1.68×109 CFU/ml and 6.75×109 CFU/ml were recorded ID50 dose of S. enterica serovar Typhimurium andE. coli O26 respectively. The chicken showing clinical signs such as fever, dullness, depression, diarrhea, ruffled feathers, and refusal of feeding and death, considered as infected. Respective bacteria were isolated and confirmed from infected and dead birds.
1.3.2. Effects oral feeding of HAE of OS and AM in chickens experimentally infected with ID50 dose ofS. enterica serovar Typhimurium and E. coli O26
83% chickens of OS fed groups and 66% chickens of AM fed groups were protected from challenge of S. enterica serovar Typhimurium andE. coli O26 ID50 dose as they showed no signs of illness (Table 5).
.png){kind=small} Table 5 In vivo antibacterial effects of HAE of OS and AM leaves against S. enterica serovar Typhimurium and Escherichia coli O26infection in chickens |
OS provided better clearance of both S. enterica serovar Typhimurium and E. coli O26 in comparison to control groups. While clearance of S. enterica serovar Typhimurium and E. coli O26 in AM fed chickens were less than that of OS fed chickens but higher than control group (Table 6; Figure 3; Figure 4).
.png){kind=small} Table 6 Determination of bacterial load in blood of OS and AM fed chickens |
.png) Figure 3 Line diagram showing the antibacterial effect of HAE of OS and AM leaves against S. enterica serovar Typhimurium in blood of fed and unfed chickens |
.png) Figure 4 Line diagram showing the antibacterial effect of HAE of OS and AM leaves against E. coli O26 in blood of fed and unfed chickens |
2 Disscussion
2.1 In the present investigation antibacterial property of OS and AM leaves against two enteric pathogens, S. enterica serovar Typhimurium and Escherichia coli O26 was evaluated using disc diffusion procedure (Bauer et al., 1966). Different extracts (Hot aqueous, cold aqueous, methanolic and hydromethanolic) of OS and AM were used for the study at the concentration of 2 mg, 5 mg, 10 mg and 20 mg per disc. 20 mg concentration/disc of all the extracts showed greatest zone of inhibition against these two bacterial isolates (Table 1~Table 4; Figure 1~Figure 2).Antibacterial activity appeared to be dose dependent. Methanolic extract of both the plants showed greatest antibacterial activity in comparison to all other extracts. Our results are similar to Goyal and Kaushik (2011) and Mishra et al (2011). Besides Chattopadhyay et al (2009) reported antibacterial potential of aqueous and ethanolic extract of OS against E. coli and other bacteria and found varying degree of strain specific inhibitory action. They also found higher antibacterial potential of ethanolic extract than the aqueous. In addition to this Mohamedet al (2010) found the antibacterial effect of OS against E. coli and other microbes.Besides Joshi et al (2010) reported that ethanolic extract of OS was effective against S. typhi. According to Mandal et al (2012) ethanolic leaf extract of OS in combination with chloramphenicol and trimethoprim has synergistic activity for S. enterica serovar Typhi. Similar to our findings Kempraj and Bhat (2010) have concluded that methanol extract of AM leaves had inhibitory action against E. coli O and aqueous extract had relatively broad spectrum growth inhibitory activity. In contrast to our finding Geeta et al (2001) reported that aqueous extract of OS showed wider zone of inhibition compared to alcoholic extract against E. coli and other bacteria tested. However all the extracts exhibited good antibacterial property against S. enterica serovar Typhimurium as compared to E. coli O26. Joshi et al (2010) have stated that ethanolic extract of OS were ineffective against E. coli. On prolonged incubation bacterial colonies reappeared within the zone of inhibition indicating bacteriostatic effect than bactericidal activity.
2.2 In vivo antibacterial activity of HAE of OS and AM was also investigated by giving ID50 dose of 1.68 X 109 CFU/mLof S. enterica serovar Typhimurium and 6.75×109 CFU/ml of E. coli O26 orally (Table 5).OS and AM fed chickens found more protected and there was no development of clinical signs as compared to control group. Bacterial load of S. enterica serovar Typhimurium and E. coli O26 (CFU/mL) in blood of fed and unfed (Control) chickens challenged with either of these two bacteria was conducted at 24 hrs, 48 hrs and 72 hrs post challenge. Colonies of S. enterica serovar Typhim- urium in the blood of OS and AM fed chickens was less in comparison to unfed chicken at all the studied intervals (Table 6; Figure 3). Similar results were observed when challenged with E. coli O26 (Table 6; Figure 4). This indicated a better clearance of bacteria from the blood of OS and AM fed chickens.
The present findings conclude that OS and AM leaves have antibacterial activity against S. enterica serovar Typhimurium and E. coli O26.
3 Material and methods
3.1 Preparation of hot aqueous, methanolic, hydromethanolic extracts by Soxhlet apparatus
Ocimum sanctum (OS) and Argemone mexicana (AM) plants were identified and characterized by Depar- tment of Botany, BSA College, Mathura. Leaves of OS and AM plants were collected from the University campus and adjoining areas of Mathura. Fresh leaves dried under shade were used for preparation of hot aqueous (HAE), cold aqueous (CAE), methanolic (ME), hydro methanolic (HME) extracts. For in vitro antibacterial properties all the preparations were used. For in vivo study only hot aqueous extract of OS and AM leaves were used.
50 gm of dried powder of OS/AM leaves was placed in a porous cellulose thimble which was then placed in an extraction chamber, above a collection flask containing the 750 mL solvent (TDW/Methanol/ Hydromethanol). The flask was heated and the solvent was allowed to evaporate. Temperature was adjusted according to boiling temperature of the respective solvents (TDW/Methanol/Hydromethanol). The extraction process was continued for 15~20 cycles and the flask containing the solvent and extract was removed. The solvent in the flask was then evaporated and remaining material was measured.
3.2 Preparation of cold aqueous extract from dried leaves of OS and AM
150 gm of dried leaves was powdered and steeped in 750 mL of triple distilled water at room temperature for four days. 2~4 drops of chloroform were added to prevent the fungal growth. The contents were shaken three times a day and after four days suspension was filtered through muslin cloth and then Whatman No1 filter paper followed by membrane filtration using 0.45 μm membrane filter and water content present in filtered solution was allowed to evaporate in concentrator and finally lyophilized. Extract obtained was then weighed and stored at 4 ºC for further use.
3.3 Bacterial isolates
Two isolates S. enterica serovar Typhimurium and E. coli O26obtained from the repository of Department of Microbiology and Immunology, DUVASU, Mathura, were used for the study of in vitro and in vivo antibacterial activities of extracts of OS and AM leaves.
3.4 Experimental birds
Standard pathogen free one day old chicks (Av. Wt 30~35 gm) were purchased from Uday hatchery, Mathura and reared at poultry farm, DUVASU, Mathura. All the birds were housed and fed under standard conditions. Precautions were taken to prevent the environmental contamination. In each experi- mental group, individual bird identification was made by using wing tag. Seven day old chicks were used for the experiments. All these experiments were approved by “Institutional Animal Ethics Committee” (IAEC), and research was conducted under their guidelines. 6 birds per experimental group/control group were used.
3.5 Determination of nontoxic dose of hot aqueous extract (HAE) of OS and AM
The non-toxic dose was determined by oral feeding of three doses 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight of HAE of OS and AM leaves. For in vivo study oral feeding of 250 mg/kg HAE of OS and AM leaves was done as it was ideal (Varshney et al., 2013).
3.6 Preparation of herbal discs containing different concentration of OS and AM leaves extracts:
Discs of 6 mm diameter prepared from Whatman No-1 filter paper was sterilized by hot air oven at 160ºC for 60 min. and then dipped in solution of different concentrations of OS and AM extracts of leaves. Discs were then allowed to dry and used as herbal discs. Discs containing 2 mg, 5 mg, 10 mg and 20 mg disc of cold aqueous, hot aqueous, methanolic and hydromethanolic extracts of OS and AM were used to study the antimicrobial activity against S. enterica serovar Typhimurium and E. coli O26.
3.7 Determination of in vitro antibacterial activity of different extracts
Disc diffusion method was followed (Bauer et al., 1966). 0.5 ml of respective bacterial culture, containing about 3×104 CFU per ml was uniformly spreaded over the plate. Discs containing different concentrations (2 mg, 5 mg, 10 mg and 20 mg) of different OS and AM extracts were planted 8~10 cm apart on inoculated nutrient agar plates. The plates were then incubated at 37ºC for 48 hours. The antibacterial activity of extracts marked by diameter of the zone of inhibition of bacterial growth around the disc was noted. The zone of inhibition was measured in mm. Each experiment was repeated three times.
3.8 In vivo effects of HAE of OS and AM leaves against S. enterica serovar Typhimurium and E. coli O26 infection
3.8.1 Determination of nontoxic of HAE of OS and AM leaves by oral feeding to chickens
Hot aqueous extracts (HAE) of O. sanctum and A. mexicana leaves were given orally to chickens. There were three groups for OS as well as for AM for doses, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight, fed orally for 21 days and one control group which was fed placebo (Triple distilled water) in place of HAE for determination of the safe and nontoxic dose. Out of the above 3 doses 250 mg/kg of OS and AM were ideal (Varshney et al., 2013). This dose was used for study of in vivo antibacterial activity.
3.8.2 Determination of ID50 dose of S. enterica serovar Typhimurium and E. coli O26 in chickens
Smooth colonies of pure culture of S. enterica serovar Typhimuriumand E. coli O26 from Luria Bertani (LB) agar plates were inoculated in LB broth. Inoculated LB broth was incubated overnight at 37ºC and then centrifuged at 3000 rpm for 10 min. Supernatant was discarded and cell pellet was washed thrice with PBS and finally resuspended in PBS to make 9×109 cells/ml of S. enterica serovar Typhimurium and E. coli O26 suspension using McFarland’s nephelometer. Then two fold dilutions were made and 1ml of first four dilutions was inoculated orally to respective group of chickens comprising 6 chickens in each. After inoculation all the birds were monitored twice daily for 10 days for clinical signs such as fever, dullness, depression, diarrhoea, ruffled feathers, and refusal of feeding and death. Respective bacteria was isolated and confirmed from the infected and dead chickens. ID50 was calculated by the method described by Reed and Muench (1938).
3.8.3 Effects of HAE of OS/AM leaves in chickens experimentally infected with ID50 dose of S. enterica serovar Typhimurium and E. coli O26
In vivo effects of HAE of OS and AM leaves against ID50 of S. enterica serovar Typhimurium and E. coli O26 was determined by clinical signs and Bacterial load in blood. 6 groups (GI to GVI) having 6 chickens in each were taken. Chickens of GII and GIII were fed orally with 250 mg/kg body wt. HAE of OS for 21 days. Chickens of GV and GVI were fed orally with 250 mg/kg body wt. HAE of AM leaves for 21 days respectively, while GI and GIV were unfed groups and kept as control. On 22nd day chickens of GI, GII and GIII were challenged with 1.68×109 CFU/ml (ID50 dose) of S. enterica serovar Typhimurium and chickens of GIV, GV and GVI were challenged with 6.75×109 CFU/Ml (ID50 dose) E. coli O26 by oral route and observed twice daily up to 10 days for development of clinical signs of infection.
3.8.4 Determination of bacterial load in blood
Three chickens from each group were bled on day 23rd, 24th and 25th. Tenfold serial dilution (10-1 to 10-6) of collected blood was made. 0.5 mL of blood of 10-5 and10-6 dilutions were plated separately on LB agar plates using 3 plates per dilution. The inoculum was spread on plates with the help of sterile spreader and incubated at 37ºC. Colonies (CFU) were counted after 24 hrs and the mean was recorded.
Authors’ contribution
All the authors contributed equally for this study. All the authors read and approved the final version of the manuscript.
Acknowledgements
Authors are thankful to the University authorities for providing necessary facilities to carry out this research work.
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