The present study was proposed to investigate the toxicological and prophylactic potential of ethanolic extracts of
From prehistoric times, herbs are being used as medicine for treatment of different illnesses [
Both of these plants are used alone by herbal practitioners; however, their prophylactic potential has not been worked out so far. On the other hand, data available on the toxicological assessment of
Flowers of
Healthy male albino mice
Extracts of
Mice were observed regularly for behavioral changes such as abdominal constriction, hyperactivity, sedation, grooming, and mortality. Initial body weight of all groups of mice was recorded once before the beginning of the first oral administration, and final body weight of all groups was recorded at the time of termination of experiment.
At days seven and fourteen after treatment, 5 mice from each group were randomly selected for bleeding. The animals were anesthetized with chloroform and the blood samples were collected from the heart in vacutainers without anticoagulant. The blood samples were allowed to clot for 15 minutes at room temperature. Serum was separated by centrifuging the blood at 3000 rpm for 10 minutes and stored at −20°C till further analysis. Small pieces of liver and kidney were collected with the help of sharp scissors and forceps. Tissues were preserved in 10% formalin and processed for histological examination. Tissue samples of liver and kidney were stained and thin sections were prepared according to Marco et al. (1992) [
Animals were observed daily for signs of toxicity including anxiety, cannibalism, depression, and weight loss, while the serum samples were subjected for evaluation of alkaline phosphate, alanine transaminase (ALT), bilirubin, uric acid, urea, and creatinine using autoanalyzer.
Pilot study was conducted to define the infective dose of the pathogen. For that purpose, animals (15) were divided into three groups, A, B, and C. Five ml of autoclaved nutrient broth was inoculated with
Animals housing has been described in the previous section (Toxicity). Twenty-five
Mice of each group were tagged. Body weight of each mouse was taken regularly till end of experiment. Mice were observed regularly for behavioral changes such as sluggishness, circumflex backbone, sunken eyes, ruffled fur, abdominal constriction, and mortality.
Following 7 days of infection, five mice from each group were sacrificed. The animals were anesthetized with chloroform and the blood samples were collected from the heart in vacutainers with anticoagulant. The blood samples were kept in refrigerator till further analysis. Samples were sent to BioMed clinical laboratories (Lahore, Pakistan) where they were processed manually for the hematological analysis. The following hematological parameters were selected: total RBC (red blood cell), hemoglobin, HCT (hematocrit), MCV (mean corpuscular volume), MCHC (mean corpuscular hemoglobin concentration), platelets, WBC (white blood cell), neutrophils, lymphocytes, and monocytes.
With the help of sterile scissors and forceps, impressions of liver, kidney, spleen, heart, and blood of each animal were taken on LB-agar plates and incubated at 37°C for 24 hours. Small pieces of the liver, kidney, and spleen of the animals were taken into eppendorf containing 1 ml of distilled, autoclaved water. The organs were triturates in water with the help of sharp objects. From each eppendorf, 100
Data was analyzed using one-way ANOVA followed by DMRT in SPSS software (version 16.0). Data are presented as mean ± SE and differences at
The initial and final body weights of both treated and nontreated mice were comparable (Table
Effects of mixture of extracts on the body weight (g) of
Groups | Treatment (mg/kg) | Initial weight | Final weight | Weight change |
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Control | 0 | 29.6 ± 1.25 | 31.20 ± 1.76 | 1.6 ± 1.72 |
Group 1 | 500 | 31.82 ± 2.13 | 33.05 ± 2.12 | 1.23 ± 2.02 |
Group 2 | 1000 | 30.03 ± 2.77 | 31.45 ± 1.98 | 1.42 ± 2.14 |
Group 3 | 1500 | 32.05 ± 2.97 | 33.29 ± 2.10 | 1.24 ± 1.95 |
Comparison among different treatment groups was performed using one-way ANOVA. No significant differences in body weight of different treatment groups could be observed. Data are presented as mean ± SEM.
The acute and chronic effects of the mixture of extracts of
Effects of extracts on biochemical parameters.
Parameters | Groups | |
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Control |
Treated |
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Mortality | 0 | 0 |
Morbidity | 0 | 0 |
Alanine transaminase (U/L) | 83.3 ± 1.32 | 29.67 ± 0.33 |
Alkaline phosphatase (U/L) | 426 ± 49.7 | 333.0 ± 57.501 |
Bilirubin (mg/dl) | 0.438 ± 0.19 | 0.433 ± 0.066 |
Blood urea (mg/dl) | 40.3 ± 3.73 | 35.00 ± 3.152 |
Creatinine (mg/dl) | 0.64 ± 0.18 | 0.500 ± 0.0577 |
Serum uric acid (mg/dl) | 4.24 ± 0.73 | 2.533 ± 0.392 |
Data are presented as mean ± SEM. Data were analyzed using independent sample
Histopathological effects of mixture of ethanolic extracts of
Comparison of photomicrographs of liver and kidney sections of animals exposed at 1500 mg/kg of mixture of extracts and control. (a) Control kidney section showed normal nucleus in lymphocytes and vacuoles are small in size. (b) Treated kidney showed slight abnormality in kidney, vacuole, and deformed nucleus can be observed. (c) Control liver section exhibited normal hepatocyte nuclei and intracellular spaces. (d) Treated liver revealed abnormalities in hepatocyte nucleus and large vacuoles indicated by arrows.
Effects of different doses on mice.
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Group B |
Group C | |
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Mortality |
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Change in behavior |
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Eye swelling |
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Discharge from eye |
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Shape of neck (circumflex backbone) |
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Sedation |
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Movement by forelimbs |
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Hyperactivity |
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Blood |
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Liver |
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Lung |
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Kidney |
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Spleen |
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Heart |
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Animals became slightly sluggish after two days of infection as compared to negative control. One animal of group three (G3) died while another animal of this group suffered from circumflex back bone. Minor variations in the body weights of the mice of infected groups (G1, G3, G4, and G5) were observed
Effects of ethanolic extracts of
Groups | Treatment | Average weights | ||||
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Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | ||
Group 1 | BI | 30.4 ± 0.65 | 32 ± 0.58 | 32 ± 0.4 | 28.8 ± 0.52 | 32.6 ± 0.72 |
Group 2 | NT | 31.6 ± 0.57 | 35 ± 0.28 | 35 ± 0.28 | 35 ± 0.37 | 34.6 ± 0.20 |
Group 3 | BI + R | 30 ± 0.45 | 29 ± 0.67 | 29 ± 0.85 | 30 ± 0.41 | 30 ± 0.55 |
Group 4 | BI + N | 30.8 ± 0.5 | 33.4 ± 0.64 | 33.4 ± 0.58 | 29.4 ± 0.58 | 32.6 ± 0.46 |
Group 5 | BI + N + R | 27.8 ± 0.52 | 32 ± 0.28 | 29.4 ± 0.33 | 30.4 ± 0.33 | 32.6 ± 0.5 |
NT: no treatment, BI: bacterial infection, N + R:
Impressions of the organs (liver, kidney, spleen, heart, and blood) of each animal were taken on tryptic soya agar plates and incubated at 37°C for 24 hours. There were too numerous colonies on the plates indicating that infection had been reached to organs. Pathogen was reisolated from the selected organs but the number of colonies was few. In group G3 (treated with
Reisolation of
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Positive control |
Negative control |
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Morbidity | 5/5 | 0/5 | 4/5 | 2/5 | 1/5 |
Mortality | 0/5 | 0/5 | 1/5 | 0/5 | 0/5 |
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Liver | 5/5 | 0/5 | 2/4 | 1/5 | 1/5 |
Lung | 5/5 | 0/5 |
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1/5 | 0/5 |
Kidney | 5/5 | 0/5 | 0/4 | 1/5 | 0/5 |
Heart | 5/5 | 0/5 |
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1/5 | 1/5 |
Spleen | 5/5 | 0/5 | 0/4 | 1/5 | 0/5 |
Two hematological parameters, neutrophils and lymphocytes counts, were found sensitive to
Effects of extracts separately and in combination on haematological parameters.
Parameters | Groups |
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Positive control | Negative control |
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RBCs (×10/ |
7.76 ± 0.30 | 7.77 ± 0.34 | 8.07 ± 0.26 | 6.65 ± 1.13 | 8.28 ± 0.28 | 0.338 |
Hemoglobin (g/dl) | 12.02 ± 0.55 | 11.6 ± 0.72 | 12.4 ± 0.77 | 10.35 ± 1.69 | 13.2 ± 0.31 | 0.420 |
PCV (%) | 39.65 ± 1.68 | 40.16 ± 3.53 | 42.3 ± 4.01 | 33.27 ± 5.17 | 43.8 ± 1.81 | 0.259 |
MCV (fL) | 53.0 ± 1.15 | 51.7 ± 2.62 | 57.8 ± 0.66 | 50.42 ± 1.31 | 54.6 ± 2.22 | 0.090 |
MCH (%) | 16.85 ± 0.85 | 15.08 ± 0.38 | 15.4 ± 0.44 | 15.72 ± 0.56 | 17.1 ± 1.15 | 0.149 |
MCHC (%) | 30.04 ± 0.67 | 29.28 ± 0.79 | 29.4 ± 0.97 | 30.97 ± 0.44 | 28.9 ± 1.16 | 0.503 |
Platelets (×10/ |
238 ± 22.53 |
247 ± 81.47 |
435 ± 19.15 |
473 ± 136.64 |
597 ± 30.96 |
0.013 |
WBCs (×10/ |
6.02 ± 0.43 | 6.32 ± 1.37 | 3.96 ± 0.09 | 5.45 ± 1.25 | 3.8 ± 0.65 | 0.142 |
Neutrophils (%) | 27.50 ± 2.5 |
12.60 ± 1.07 |
14.6 ± 0.70 |
13.25 ± 2.68 |
15.4 ± 3.53 |
0.001 |
Lymphocytes (%) | 62.5 ± 4.78 |
78.6 ± 5.03 |
83 ± 1.22 |
75 ± 4.56 |
79 ± 5.15 |
0.039 |
Monocytes (%) | 5.25 ± 4.09 | 5 ± 2.7 | 3.6 ± 1.31 | 4.0 ± 2.4 | 4.3 ± 2.02 | 0.230 |
Eosinophils (%) | 5.0 ± 1.5 | 5.0 ± 1.0 | 4.1 ± 0.70 | 3.75 ± 0.47 | 5.5 ± 0.55 | 0.195 |
Data are presented as mean ± SEM; different alphabets (in a row) indicate significant variation among groups at
Emergence of resistance in pathogenic bacteria is being reported from all parts of the world, which has accelerated research in the field of new therapeutic agents from different resources including medicinal plants. A number of plant species have been exploited for medicinal purposes with variable success. Medicinal plants and their extracts are generally considered safe and effective. However,
During the screening of drugs, determination of LD50 is a first step for the evaluation and assessment of toxicity of a substance. This is the preliminary screening step that has to be performed with all compounds for initial evaluation of toxic manifestation. Data obtained from acute toxicity experiments may serve for dose determination in animal studies [
None of the doses had adverse effects on the behavioral response of subjects except 1500 mg/kg up to 15 days of continued treatment. There was no morbidity or mortality nor effect on body weights of mice at 1000 mg/kg dose of both extracts. These findings are inconsistence with Bose et al. (2012) [
Alanine amino transaminase acts as a catalyst in the formation of oxaloacetic and pyruvic acid by transferring
A number of reports are available on the inhibitory activity of medicinal plants and different phytochemicals against pathogenic and nonpathogenic organisms through
The extract of
The potential of extracts in reducing infection was also monitored while recording physical signs of disease including change in behavior, eye secretions, sunken eyes, circumflex backbone, hyperactivity, sedation, and mortality in infected mice. High count of neutrophils and low count of lymphocyte or vice versa are indicators of different diseases and disorders. They act as primary defenders against bacterial infection physiological stress. The important components of immune system are lymphocytes, which are type of white blood cells. The difference between lymphocytes and neutrophils in adult animals is quite different: human blood is neutrophil rich (50–70% neutrophils, 30–50% lymphocytes), whereas mouse blood has a strong preponderance of lymphocytes (75–90% lymphocytes, 10–25% neutrophils) [
The DLC of G3 and G5 revealed a reduction in number of the neutrophils to lymphocyte ratio as compared to G4. The neutrophil to lymphocyte ratio was high in G1 as compared to G2, which indicated the presence of infection at tissue level. An increase in one type of white blood cells can cause a decrease in other types of white blood cells. For example, in bacterial infection, neutrophils count increases and lymphocytes count decreases. Similar results have been demonstrated by Dolma et al. (2014) [
In culmination, the hematological analysis of
These findings were further strengthened by recording reisolation of pathogen from tissue on LB-agar plates. The results showed that infection spreads throughout the body in positive control group. However, the numbers of the colonies reisolated from the treated groups (G3 and G5) were less than those from G1 (positive control). This further indicates that the plants play some role in reducing infection. G4
Platelets are type of cells that recognize damaged blood vessels and help in binging of these vessels. They are not directly associated with the bacterial infections [
The
This work is part of M.Phil. and M.S. thesis of authors Asma Kalsoom and Iqra Akbar, respectively.
The authors declare that they have no conflicts of interest.
The authors are grateful to University of the Punjab, Lahore, for funding this study.