IJCRR - 14(7), April, 2022
Date of Publication: 05-Apr-2022
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In-Vivo Screening of Citrus Maxima Oil Against, Escherichia Coli Infection in Rabbits
Author: Nisar Ahmad, Muhammad Shahab Khan, Ashfaq Ahmad, Muhammad Bilal, Farooq Akbar Khan
Abstract:Introduction: Escherichia coli is a Gram-negative facultatively anaerobic bacteria that cause serious damage in animals and humans by causing high morbidity and mortality. The increasing prevalence of antibiotic-resistant microorganisms has made it necessary to replace alternative sources of antibiotic products. There is a persistent need to recognize new and novel antimicrobial agents that would help in alleviating the problems of emerging resistant bacterial pathogens. Plant-derived natural products represent an attractive source of antimicrobial agents since they are natural and affordable, especially in rural societies in poor developing countries Objectives: The objective of the current research was to explore the antibacterial capabilities of the Citrus maxima oil against Escherichia coli infection in rabbits. The effect of oil on rabbits' hematological parameters was also assessed. Methods: Rabbits were distributed into six groups, 1st group served as a negative control, 2nd group as a positive control, infected with E. coliat the dose rate of 2.0 x 1010 CFU without medication, group 3rd, 4thand 5th were infected with E. coli and supplemented with oil at different doses and 6th group was infected with E. coli and treated with Moxifloxacin HCL (standard drug) at the dose rate of 7mg/kg. The blood samples of tested animals were collected for the analysis of haematological parameters on different days of the experiments. Result: The results showed a significant increase in WBCs, MCV, HCT and neutrophils during infection. RBCs, MCH, MCHC, haemoglobin, lymphocytes and platelets count decreased during infection. The current study provides a scientific contextual for the significant use of Citrus maxima oil for the treatment of various pathological diseases. Conclusion: The obtained result gives an insight into the antibacterial capabilities of the Citrus maxima oil against Escherichia coli infection in rabbits. Citrus maxima oil exhibited variable, but remarkable antibacterial potential. These oils could be used as a source of antimicrobial agents in pharmaceutical industries.
Keywords: Antimicrobial, bacterial pathogens, Citrus maxima oil, Escherichia coli, Hematological parameters, Medicinal plant
The increasing bacterial conflict to antibiotics has developed a growing APPREHENSION GLOBALLY.1 With the appearance and growth of microorganisms such as gram-negative (E.coli) bacteria which can cause serious infections.2 There are a number of drugs available to treat gram-positive bacteria, but less in number to treat gram-negative bacteria.3 In addition, high cost and adverse effects are commonly related to widespread synthetic antibiotics are a chief burning global issue in considering infectious diseases.4 Infectious diseases pose serious problems to health and they are the main cause of morbidity and mortality worldwide.5 The increasing prevalence of antibiotic-resistant microorganisms, has made it necessary to replace alternative sources of antibiotic products.6 Plant derived natural products represent an attractive source of antimicrobial agents since they are natural and affordable, especially in rural societies in poor developing countries.7
The literature survey revealed that the peel of Citrus maxima fruits is extremely thought to be a universal remedy within the flavoring drugs with various spectra of pharmacologic activity.8 Citrus maxima is the most extensively studied medicinal plant in recent literature. Citrus maxima is an edible fruit, its flesh is juicy, soft in texture and wealthy in nutrients and is endemic to tropical part of Asia.9E.coli is the most commonly found bacterium in the human intestinal tract. Under normal conditions, its presence is conducive to digestive processes. But when present in excess or in virulent form it causes diseases.10 E.coli, contaminate food and water supplies.11
Plants play an important role in human health because they produce a wide array of bioactive molecules which have medicinal values.12 Despite the efforts in producing the number of new antibiotics in the last three decades, resistance to these drugs by microorganisms has increased.13 The in-vitro anti-bacterial activities of three citrus Plants extracts, Citrus macrocarpa, Citrus Aurantium and Citrus maxima against S. aureus but not against E.coli.14 The oil of this fruit has been reported to possess some nutritive and medicinal potentials.15
Material and methods
The main objective of the present study was to investigate the antibacterial activity of Citrus maxima oil against E. coli infection in rabbits.A total of 30 adult Rabbits of both genderswas used in the current study as experimental animals.
Grouping of animals
Rabbits having same weight were kept in the same group, thirty rabbits of both gender, which were randomly divided into six equal groups, 1stgroup (negative control), 2ndgroup infected with Escherichia coli orally at the dose rate of 2x1010 CFU (served as positive control) which did not receive any drug or essential oil as a treatment, 3rd group, infected with E. coli at the dose rate of 2x1010 CFU and was treated with Citrus maxima oil at a dose rate of 1ml, 4th group, infected with E. coli at the dose rate of 2x1010 CFU and was fed with Citrus maxima oil at a dose rate of 1.5ml, 5th group, infected with E.coli at the dose rate of 2x1010 CFU and fed with Citrus maxima oil at a dose rate of 2ml, 6th group was injected with E.coli at the dose rate of 2x1010 CFU and was fed with standard drug, (Moxifloxacin HCL).
Chemicals and apparatus
Citrus maxima oil and Moxifloxacin HCL were purchased from the local market of QissaKhwani Bazar, Peshawar, Pakistan. The equipment used in the current study were a Haematological analyzer and a Weight scale.
Initiation of medication
All groups of rabbits received a freshly cultured sample of E. coli (2x1010 CFU) orally except the control group. After administration of the bacteria, rabbits were checked for feed intake and other clinical signs. The responses of the rabbits to E. Coli bacteria were identified by the clinical signs like temperature, diarrhea, weight loss and reduced feed intake.
Medication of infected rabbits
After development of clinical signs, group 3rd, 4th and 5th were treated with Citrus maxima oil at a dose rate of 1ml, 1.5ml and 2ml, while group 6th was treated with standard drug, Moxifloxacin HCl. Phenobarbital sodium was used to anesthetize the rabbits for the collection of blood samples. Blood samples (about 3ml) were collected from all rabbits, at day zero, day three, day six and day nine of the experimental work for the analysis of different haematological parameters.
The data obtained from the study were analysed statistically, using the analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to determine the differences between treatments. The mean and standard deviation (SD) were sorted out of each parameter, using, Graph pad prism software.
In the present study, rabbits were divided into different groups. 1st group was kept as a negative control, neither infected nor medicated, 2nd group served as a positive control which was infected, but not treated 3rd, 4th and 5th groups were treated with Citrus maxima oil at a dose rate of 1ml, 1.5ml, and 2mland group 6th was treated with standard drug (Moxifloxacin).
Blood samples was collected on day zero and the results of all the groups have been shown in: (Table 1).
1stGroup (negative control)
About 3ml blood was collected from the animals. The TRBCs of negative control was 5. 73±0. 028x106/µl and hemoglobin value was 9. 52±0. 035%. The MCH, MCHC and MCV values were 21.49±0.134 Pg, 33.0±0.848 gm/dl and 50.7±0.070pg. The WBCs count was in the range of 5.72±0.035 x 103 /µl while lymphocytes count was 27±0.494%. The neutrophils, platelets count and HCT value were also in the normal reference range (60.5±0.707%, 530.5±0.035 x 103 /µl and 37.5±0.17%).
2nd group (positive control)
The same amount of blood was also collected and were analysed for different parameters. The values of the RBCs and haemoglobin were 5.08±0.033x106/µl and 9.81±0.13%. Lymphocytes count was 28.3±0.31%. MCH and MCHC level were 19.7±0.41Pg and 30.5±0.16gm/dl. The MCV, WBCs and Neutrophils count were in the range of 50.6±0.12pg, 5.5±0.045x 103 /µland 62.6±0.25%. Platelets level was 528±0.11x 103 /µl and HCT value were 36.8±0.10%.
3rd Group three (low dose)
Before infection the RBCs and hemoglobin value were in the range of 5.70±0.17x 106 /µl and 9.45±0.057%. The MCH value was 21.45±0.14pg and MCHCs value was 33.03±0.081gm/dl while MCV was in 50.76±0.072pg range. WBCs, lymphocytes and neutrophil count were 5.71±0.036x 103 /µl, 27.05±0.26% and 62.53±0.19%. HCT value was 37.46±0.92%, platelets count was 531.52±0.27x 103 /µl while RDWC level was 15.56±0.095%.
4thGroup (medium dose)
RBC and haemoglobin of group 4th before infection were 5.73±0.23x 106 /µl and 9.52±0.27%. The concentration of MCH was 21.48±0.33pg while MCHC was 32.95±0.12gm/dl. The level of MCV and WBCs were in the range of 50.74±0.12pg and 5.71±0.076x 103 /µl. Lymphocytes, neutrophils and platelets count were 28.96±0.17%, 61.47±0.44% and 525.50±0.14x 103 /µl while HCT and RDWC values were in the range of 37.50±0.14%, and 15.60±0.26%.
5thGroup (high dose)
The RBC count was 5.76±0.074x 106 /µl while hemoglobin value was 9.75±0.38%. The values of MCH, MCHC and MCV were 21.51±0.25pg, 32.95±0.25gm/dl and 50.69±0.16pg. WBCs count was 5.71±0.036x 103 /µl and lymphocytes count was 28.08±0.28% while the neutrophil count was 60.44±0.36%. Platelets, HCT and RDWC level were 530.54±0.087x 103 /µl, 37.53±0.15% and 15.57±0.21%.
6thGroup (standard drug)
The RBCs and haemoglobin values were 5.78±0.77x 106 /µl and 9.81±0.084%. The concentration of MCH was 21.56±0.036pg and MCHC was 33.02±0.17gm/dl. MCV, WBCs and lymphocytes count were 50.72±0.34pg, 5.73±0.045x 103 /µl and 27.03±0.13% while neutrophil count was 61.52±0.27%. The platelets count was 524.53±0.053x 103 /µl, HCT value was 37.48±0.17%.
The rabbits were infected with pathogenic E. coli at the dose rate 2x1010 CFU, except group 1st (control group). After causing infection, whole blood was collected from all the infected groups for analysis of haematological parameters (Table 2).
2nd group (positive control)
The RBCs and haemoglobin count during infection were 3.47±0.042x 106 /µl and 6.82±0.042%. Neutrophils and Platelets count were 66.4±0.042% and 435±0.042x103/µl. The MCH value were20.53±0.042pg and the MCHC were 28.15±0.070gm/dl. The lymphocyte, WBCs and MCV count were 23.6±0.035%, 8.22±0.035x 103 /µl and 59.53±0.028pg. The HCT value was 41.62±0.028%. The level of RDWC was 15.42±0.10% during infection.
3rd Group (low dose)
This group was also infected with E. coli, RBC count was 3.44±0.028x 106 /µl and haemoglobin value was 6.74±0.035%. The MCH and MCHC values were 20.50±0.035pg and 28.17±0.035gm/dl. The MCV, WBCS and lymphocytes count were 66.56±0.622pg, 8.17±0.014x 103 /µl and 25.62±0.042% while HCT value was 41.64±0.056%. The level of RDWC was 15.45±0.24%, the neutrophils and platelets count were 65.41±0.414 % 491.04±0.21x 103 /µl.
4th Group (medium dose)
RBCs and haemoglobin count were 3.48±0.21x 106 /µl and 6.85±0.14%. The MCH value were 20.49±0.14pg and MCHC were 28.18±0.28 gm/dl. MCV, lymphocytes, WBCs count were 57.52±0.11pg, 24.64±0.49 %, 7.4±0.07x 103 /µl and HCT value was 42.66±0.056 %. Platelets and neutrophils count were 54.43.8±0.4, % 444.03±0.07x 103 /µl. The RDWC level was 15.45±0.24% during infection.
5th Group (high dose)
RBCs count was 3.5±0.014x 106 /µl while haemoglobin value 6.90±0.14%.
The values of MCH and MCHC were 20.52±0.14pg and 28.20±0.28 gm/dl. MCV, WBCs and lymphocytes count were 56.55±0.11pg, 8.33±0.070 x 103 /µl and 23.65±0.049%. Platelets and neutrophils count were 467.05±0.28x 103 /µl and 67.41±0.035%. The HCT value was 41.65±0.29 %.
6th Group (standard drug)
In this group the RBCs and haemoglobin values were 3.51±0.014x 106 /µl and 6.98±0.070%. MCH value was 20.54±0.14pg and MCHC was 28.22±0.28gm/dl. The lymphocyte and MCV count were 21.66±0.084% and 55.2±0.11pg. During infection. Platelets and neutrophils count were 429.06±0.28x 103 /µl and 54.44±0.141%.
Total Red Blood Cells (TRBCs)
At day six, the TRBCs count in negative control group was 5.73±0.028x 106 /µl, the positive control group was 4.54±0.028x 106 /µl. In groups, that were infected with E.coli and treated with C.maxima oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the TRBCs counts were 6.26±0.028x 106 /µl, 5.40±0.042x 106 /µl and 4.04±0.028x 106 /µl. The group which was infected with E.coli and treated with standard drug (Moxifloxacin HCl), the RBCs count was 5.83±0.035x 106 /µl. A significant difference (P<0.05) was found in the mean TRBCs counts of control group with the rest of groups (Table 3).
The haemoglobin value in control group and positive control group at day six were 9.52±0.035% and 8.85±0.035%. In groups, that were infected with E.coli and treated with C.maxima oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the haemoglobin values were 10.12±0.028%, 9.0±0.042% and 8.28±0.035%. The group that was infected with E.coliand treated with standard drugs (Moxifloxacin), the haemoglobin value was 9.6±0.035%. A significant difference (P<0.05) was found in the mean Hb value of control group with the rest of groups.
Mean Corpuscular Haemoglobin (MCH)
At day 6th, the MCH value of negative control was 21.1±0.134 pg, the positive control value group was 17.8±0.042pg. In groups, that were infected with E.coli and treated with C.maxima oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the MCH value were 18.51±0.028pg, 19.49±0.042pg and 19.7±0.035pg. That group which was infected with E.coli and treated with standard drug (Moxifloxacin HCl), the MCH value was 19.7±0.035pg. A significant difference (P<0.05) was found in the mean MCH value of control group with the rest of groups.
Mean Corpuscular Haemoglobin Concentration (MCHC)
The MCHC value in control group was 33.0±0.848gm/dl, the positive control group was 27.5±0.042gm/dl. In groups, that were infected with E.coli and treated with Citrus maxima oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the MCHC values were 30.7±0.028gm/dl, 30.9±0.035gm/dl and 30.20±035 gm/dl. The group which was infected with E.coli and treated with standard drug (Moxifloxacin), the MCHC value was 30.9±0.035 gm/dl. A significant difference (P<0.05) was found in the mean MCHC value of control group with the rest of groups.
Mean Corpuscular Volume (MCV)
In control group at day 6th, the MCV value was 50.7±0.070pg, the positive control group was 66.7±0.035. In groups, that were infected with E.coli and treated with olive oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the MCV values were 57.5±0.064pg, 56±0.035pg and 58.4±0.035pg. That group which was infected with E.coli and treated with standard drug (Moxifloxacin), the MCV value was 56.5±0.042 pg. A significant difference (P<0.05) was found in the mean MCV value of control group with the rest of groups.
White Blood Cells (WBCs)
The WBCs count in control group was 5.72±0.035x 103 /µl, the positive control group was 8.36±0.042x 103 /µl. In groups, that were infected with E. coli and treated with Citrus maxima oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the RBCs counts were 7.14±0.028x 103 /µl, 7.05±0.042x 103 /µl and 7.11±0.035x103 /µl. The group which was infected with E.coli and treated with standard drug (Moxifloxacin HCl), the WBCs count was 7.03±0.028x 103 /µl. A significant difference (P<0.05) was found in the mean WBCs counts of control group with the rest of groups.
At day 6th, the lymphocytes count in control group was 28.80±0.494%, the level of positive control group was 21.8±0.042%. In groups, that were infected with E.coli and treated with C.maxima oil at the dose rate of 1ml/kg, 1.5ml/kg and 2ml/kg, the lymphocytes counts were 26.6±0.028 %, 25.3±0.035% and 26.4±0.042%. That group which was infected with E.coli and treated with standard drugs (Moxifloxacin HCl), the lymphocytes count was 24.2±0.035%. A significant difference (P<0.05) was found in the mean lymphocytes counts of control group with the rest of groups.
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