IJCRR

INTRODUCTION

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, 1^stgroup (negative control), 2^ndgroup 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, 4^th 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 3^rd, 4^th and 5^th were treated with Citrus maxima oil at a dose rate of 1ml, 1.5ml and 2ml, while group 6^th 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.

Statistical analysis

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.

RESULTS

In the present study, rabbits were divided into different groups. 1^st group was kept as a negative control, neither infected nor medicated, 2^nd group served as a positive control which was infected, but not treated 3^rd, 4^th and 5^th groups were treated with Citrus maxima oil at a dose rate of 1ml, 1.5ml, and 2mland group 6^th was treated with standard drug (Moxifloxacin).

Pre infection

Blood samples was collected on day zero and the results of all the groups have been shown in: (Table 1).

1^stGroup (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%.

4^thGroup (medium dose)

RBC and haemoglobin of group 4^th 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%.

5^thGroup (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%.

6^thGroup (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%.

During Infection

The rabbits were infected with pathogenic E. coli at the dose rate 2x10¹⁰ CFU, except group 1^st (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.

4^th 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.

5^th 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 %.

6^th 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%.

At day 6^th, 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.