Abstract
Background and objectives: To evaluate the safe dose range of Clerodendrum viscosum (C. viscosum) and Leucas indica (L. indica) ethanolic leaf extracts of acute and chronic oral toxicity study in Swiss Albino mice. Materials and methods: The Organization for Economic Co-operation and Development guideline was used for the toxicity studies. C. viscosum and L. indica plant extract were administered orally in a single dose of 2000 mg/kg, and general behavior, adverse effects, and mortality were studied for 72 h. For the chronic toxicity study, both plant extracts were administered orally to a separate set of animals at 300 mg/kg doses for 90 days. Animals body weight was taken out, blood and gastric juice were collected for biochemical parameters, and vital organs were collected for histopathological studies after sacrificing test and control group animals. Results: Both in acute and chronic toxicity assay, there was no significant alteration in body weight, physical signs, symptoms, hematological, biochemical parameters, and body organ weights compared to the normal group. The liver, kidney, and stomach histology did not show any drug-induced lesion. Conclusions: The result indicates that the oral administration of C. viscosum and L. indica ethanolic plant extract did not cause any toxicological effects. Hence it could be regarded as a safe natural product for therapeutic use.
Keywords: Toxicity, Swiss Albino mice, safety study, Clerodendrum viscosum, Leucas indica, ethanolic plant extract, toxicology
Introduction
Herbal medicine is one of the elements of complementary and alternative medicine, its increasing gaining favor around the world. According to popular belief, herbal remedies are “natural” and thus intrinsically safe. However, if used incorrectly, their effects can be extremely powerful and perhaps fatal, and their use as a substitute for traditional treatments may be ineffectual. Hepatotoxicity of primary ingredients, contamination of preparations by heavy metals or bacteria, unpleasant reactions owing to age, and genetic and concurrent disease characteristics of the user have all been linked to hazardous consequences [1,2].
Clerodendrum viscosum (C. viscosum) is a well know medicinal plant in Ayurveda where extracts from different plant parts have been used to treat other illnesses such as skin diseases, cancer, blood disorders, etc [1]. Despite of that very few compounds have been isolated from C. viscosum and there are limited toxicological studies. Similarly, Leucas indica (L. indica), another indigenous plant mentioned in Indian system of medicine used as analgesic, anti-inflammatory, antipyretic, antiparasitic and sedative drug [3-5]. Although this plant has tremendous potential against human diseases practically, there are no reports of its acute and or chronic toxicity screenings.
Although pharmacological effects of C. viscosum and L. indica plant extract are useful, a comprehensive awareness of its toxicity potential is still lacking.
Therefore, the current study is aimed to evaluate the acute toxicity of aqueous ethanolic extract of C. viscosum and L. indica leaves in the animal model.
Materials and methods
Plant material
The whole plants of C. viscosum and L. indica were collected in the month of June to August 2010 from the Manjanady village (13.0036° N, 75.0654° E) located in Mangaluru region of Karnataka, India. Professor Krishna Kumar G, Department of Applied Botany, Mangalore University, authenticated the plants (voucher 6/10/2010).
Extraction
Fresh leaves were separated from the whole plant and were cleaned, dried in the shade, grinded mechanically, and a fine powder was prepared. One kilogram of air-dried powdered was loaded in Soxhlet extractor in different phases and extracted using 90% ethanol as solvent for 36 h. The extract was air dried and concentrated (40-50°C) using a rotary evaporator. The dried extracted material was weighed, and the percentage yield was calculated using the formula; % Extraction = [(m1-m2)/m1] × 100, where m1 served as sample mass (gms) prior to extraction and m2 as the post-extraction sample mass (gms). The extract was suspended in 1% gum acacia and was used in these experimental studies.
Laboratory animals
The study was approved by the Institutional Animal Ethical Committee (IAEC), Yenepoya (Deemed to be University), Mangaluru, India (Ref. No.06/05/2010). The animal experiments were carried as per ‘Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, New Delhi, India.
After one week of acclimatization in the animal house, young, healthy non-pregnant female Swiss-albino mice weighing 25-30 gm were used in this study. The animals were housed in polypropylene cages with sterile paddy husk as beddings and maintained with relative humidity 55±5%, room temperature 25±2°C, and 12/12 h light/dark cycle. Standard rodent diet and purified water were fed to the animals [6,7].
Acute oral toxicity assay
For all the experiments, animals were fasted for food for 12 h (overnight) and allowed free access to water. The next day, they were randomly allocated into three groups with 6 animals in each and administered oral treatment as follows. On day 1, Group I received 1% gum acacia (3 ml/kg); Group II received an ethanolic extract of leaves of C. viscosum (2000 mg/kg); and Group III received an ethanolic extract of leaves of L. indica (2000 mg/kg). Prior to dose administration, the body weight of each animal was measured. Food was provided after one hour of dosing. All the animals were observed twice daily for the next 13 days, making a total of 14 days.
Preclinical observation
Animals were weighed, and their body weights were recorded regularly. The monitoring of general physical conditions such as observation of fur, eyes, nose, abdomen, and external genitals, including any occurrence of secretions and also autonomic nervous system activity (e.g., lacrimation, piloerection, respiratory pattern, and response to handling) was observed in each animal to determine the onset of peripheral observable toxic reactions throughout the experimental period [8].
Biochemical analysis
On 14th day, animals were sacrificed by high-dose chloroform anesthesia, and blood samples were collected by cardiac puncture for liver and renal function tests. The blood sample was collected in the plain tube (red stopper), centrifuged at 3000 rpm for 10 mins to obtain serum, and stored at -20°C until assay. After collecting the blood, the stomach was opened out and gastric juice was collected, and its volume was measured. Gastric function tests were done measurement gastric content and determination of gastric pH, total acidity, and free acidity. All the vital organs were separated for histopathological studies.
Serum samples were subjected for liver function tests which included estimation of total bilirubin, direct bilirubin, indirect bilirubin, total proteins, albumin, globulin, serum glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), gamma-glutamyltransferase gamma-glutamyltranspeptidase (Gama GT) and renal function test which included estimation of blood urea and serum creatinine which were measured by biochemical assay kits.
To determine total acidity, an aliquoted of 1 mL gastric juice was diluted with 1 mL distilled water and pipette out in a 50 ml conical flask. After adding two drops of phenolphthalein indicator, the mixture was titrated with NaOH (0.01 N) until a permanent pink color was established. The volume of NaOH utilized in titration was noted, and the total acidity was calculated using the formula: n × 0.01 × 36.45 × 1000 (meq/l); where n is the volume of NaOH consumed. The free acidity test was done using the same procedure and formula except for the Topfer’s reagent as an indicator.
The liver, kidney, and stomach’s relative organ weight (ROW) was calculated as follows: ROW = (absolute organ weight/mice body weight) × 100%. The harvested organs were immediately fixed with 10% formalin; after being embedded in paraffin wax, thin sections of 5 mm were made and stained with hematoxylin and eosin. The slides were observed under a light microscope, and tissue images were captured.
Chronic oral toxicity assay
The animals were grouped into three containing three animals in each, and drugs were administered orally. Group I served as the control, received the vehicle (1% Gum acacia) only by gavage (3 ml/kg of body weight); Group II and Group III received an ethanolic extract of leaves of C. viscosum and L. indica at a dose of 300 mg/kg body weight. All the groups received once-daily administration of the drugs 90 days. All the evaluation was carried out in the same manner as done for acute toxicity study.
Statistical analysis
Experimental data were presented as mean ± Standard error of the mean (S.E.M.), and one-way ANOVA followed by Dunnett’s multiple comparison tests was done. The p-value less than 0.05 compared to the control was considered statistically significant.
Results
The Ethanolic Extract of Leaves of C. viscosum was a green sticky mass and L. indica was a light brown mass. The % yield obtained from C. viscosum and L. indica were 12.5% w/w and 15.5% w/w with respect to dried leaves.
Behavioral pattern and body weight
The general physical condition of the animals suggested no harmful effect of C. viscosum and L. indica ethanolic extract at the dose of 200 mg/kg throughout the experimental period. The limit test with a dose of 2000 mg/kg body weights of C. viscosum and L. indica group leaf extract did not show any mortality.
Organ to body weight index
All the animal’s vital organs did not show any sign of lesion. Organ to body weight index (Table 1) also did not have any significant variation (P>0.05) among extract-treated and vehicle-treated groups.
Table 1.
Effects of EELCV and EELLI on the organ to body weight indices in mice
| Organs | Control (1% Gum acacia) 3 ml/kg | C. viscosum 2000 mg/kg | L. indica 2000 mg/kg |
|---|---|---|---|
| Liver | 7.81±0.14 | 8.03±0.12 | 7.96±0.12 |
| Kidney | 1.49±0.05 | 1.45±0.02 | 1.39±0.02 |
| Stomach | 1.34±0.01 | 1.35±0.01 | 1.35±0.01 |
Values are presented as mean ± S.E.M., N = 6; EELCV, ethanolic extract of the leaves of Clerodendrum viscosum; EELLI, ethanolic extract of the leaves of Leucas indica; organ-to-body weight index was calculated by formula (organ weight × 100)/body weight. The observations are mean ± S.E.M. P>0.05 Not Significant in extract treated and vehicle-treated groups (ANOVA followed by Dunnett’s multiple comparison test).
Biochemical analysis
The ethanolic extract of C. viscosum and L. indica had no significant difference in all the gastric function tests for measuring gastric content and determination of gastric pH, total acidity and free acidity when compared with the vehicle control group (Table 2).
Table 2.
Effect of EELCV and EELLI on gastric function test recorded during acute toxicity studies in female Swiss Albino Mice
| Tests | Control (1% Gum acacia) 3 ml/kg | C. viscosum 2000 mg/kg | L. indica 2000 mg/kg |
|---|---|---|---|
| Gastric content (ml/100 gm) | 4.45±0.13 | 3.85±0.30 | 4.15±0.06 |
| pH of the Gastric content | 3.81±0.07 | 4.22±0.16 | 4.09±0.11 |
| Total acidity of the Gastric content (eq/L) | 5753.89±190.97 | 5420.63±57.75 | 5316±78.51 |
| Free acidity of the Gastric content (meq/L) | 4677.75±113.20 | 4361.85±183.86 | 4355.77±77.46 |
Values are presented as mean ± S.E.M., N = 6; EELCV, ethanolic extract of the leaves of Clerodendrum viscosum; EELLI, ethanolic extract of the leaves of Leucas indica; S.E.M.: Standard error of the mean.
The liver function tests were normal in both control and C. viscosum and L. indica treated animals as summarized in Table 3. The alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) were significant (P<0.01 and <0.05) decreased in acute toxicity potential of hematological profile. No significant changes were observed in serum urea and serum creatinine levels of control and treated groups (Table 4).
Table 3.
Hematological examination related to liver function test recorded in acute toxicity studies of EELCV and EELLI at the dose of 2000 mg/kg in female Swiss Albino Mice
| Test | Control (Mean ± S.E.M.) | C. viscosum (Mean ± S.E.M.) | L. indica (Mean ± S.E.M.) |
|---|---|---|---|
| Total Bilirubin (mgs/dl) | 0.40±0.03 | 0.45±0.02 | 0.44±0.01 |
| Direct Bilirubin (mgs/dl) | 0.16±0.00 | 0.16±0.00 | 0.20±0.01 |
| Indirect Bilirubin (mgs/dl) | 0.24±0.01 | 0.23±0.01 | 0.25±0.03 |
| SGOT (IU/L) | 52.33±1.33 | 50.83±1.42 | 54.16±1.07 |
| SGPT (IU/L) | 65.33±3.30 | 73.16±1.70 | 71.33±1.43 |
| alkaline phosphatase (IU/L) | 151.6±3.39 | 76.82±2.56** | 79.33±1.33** |
| Total Proteins (gms/dl) | 7.28±0.25 | 7.26±0.27 | 7.73±0.09 |
| Albumin (gms/dl) | 4.03±0.14 | 3.65±0.19 | 3.68±0.12 |
| Globulin (gms/dl) | 3.88±0.16 | 3.83±0.15 | 3.95±0.11 |
| Albumin to globulin ratio | 0.80±0.03 | 0.78±0.01 | 0.81±0.01 |
| Gamma-glutamyl transferase (U/L) | 22.66±1.05 | 20±0.81* | 20.33±1.40* |
P<0.05 when compared with the vehicle control group;
P<0.01 when compared with the vehicle control group.
The observations are mean ± S.E.M. P>0.05-Not Significant, P<0.01-Highly significant as compared to control (ANOVA followed by Dunnett’s multiple comparison tests) EELCV-Ethanolic Extract of the leaves of Clerodendrum viscosum, EELLI-Ethanolic Extract of the leaves of Leucas indica, LFT-Liver Function Test, SGOT-serum glutamic oxaloacetic transaminase, SGPT-serum glutamic pyruvic transaminase, A.L.P.-Alkaline Phosphatase, Gama GT-Gamma-glutamyltransferase or gamma-glutamyltranspeptidase, mg/dL-milligrams per deciliters, IU/L-International unit per liters, gms/dl-grams per deciliters, U/L-unit per liters. S.E.M.: Standard error of the mean.
Table 4.
Hematological examination related to renal function tests recorded in acute toxicity studies of EELCV and EELLI at the dose of 2000 mg/kg in female Swiss Albino Mice
| Test | Vehicle control group | Acute Toxicity Group | |
|---|---|---|---|
|
|
|
||
| Control (Mean ± S.E.M.) | C. viscosum (Mean ± S.E.M.) | L. indica (Mean ± S.E.M.) | |
| Serum Urea (mg/dl) | 44.33±1.54 | 43.66±1.40 | 43.66±1.78 |
| Serum Creatinine (mg/dl) | 1.21±0.09 | 1.05±0.10 | 1.3±0.19 |
Values are presented as mean ± S.E.M., N = 6; EELCV = ethanolic extract of the leaves of Clerodendrum viscosum; EELLI = ethanolic extract of the leaves of Leucas indica; organ-to-body weight index = (organ weight × 100)/body weight.
Acute toxicity studies of reveals that there were no abnormal findings in histopathological examination in liver, kidney, and stomach of C. viscosum and L. indica ethanolic extract treated animals in comparison to control animals (Figure 1).
Figure 1.
Histopathology of control and ethanol extract treated groups at a limit dose (2000 mg/kg body weight). Histology of liver, kidney, and stomach in vehicle control (1% Gum acacia 3 ml/kg) mice (A, D and G); ethanolic extract of leaves of Clerodendrum viscosum animals treated mice (B, E and H); and ethanolic extract of leaves of Leucas indica treated mice (C, F and I).
Chronic oral toxicity assay
Similarly, in chronic toxicity studies, the general condition of the animals suggested no harmful effect of C. viscosum and L. indica ethanolic extract at the dose of 300 mg/kg when administered for 90 days. No changes were seen in the body weight and organ weights (liver, kidney, and stomach) when compared between the groups (Table 5). Biochemical and histopathological examination reveals no abnormality with respect to the liver, renal, and gastric function test in acute toxicity studies (Table 6). The present chronic toxicity study reveals that C. viscosum and L. indica did not produce any toxicity signs related to biochemical parameters like (total bilirubin, direct bilirubin, indirect bilirubin, SGOT, SGPT, ALP, total proteins, albumin, globulin, A/G Ratio, and gamma GT) (Tables 7, 8) and also in histopathology studies of liver, kidney, and stomach (Figure 2).
Table 5.
Effects of chronic EELCV and EELLI treatment on body weight and the organ to body weight indices in mice
| Control (1% Gum acacia) 3 ml/kg | C. viscosum 300 mg/kg | L. indica 300 mg/kg | ||
|---|---|---|---|---|
| Body weight | Day 0 | 183.83±5.13 | 184.26±4.56 | 175.18±3.94 |
| Day 90 | 186.2±3.13 | 181.2±2.71 | 177.4±1.62 | |
| Organs weight | Liver | 8.6±0.29 | 8.73±0.21 | 8.46±0.32 |
| Kidney | 1.43±0.11 | 1.4±0.10 | 1.4±0.05 | |
| Stomach | 1.38±0.00 | 1.4±0.01 | 1.39±0.00 | |
Values are presented as mean ± S.E.M., N = 3; organ-to-body weight index was calculated by formula (organ weight × 100)/body weight. S.E.M.: Standard error of the mean. P>0.05 Not Significant in extract treated and vehicle-treated groups (ANOVA followed by Dunnett’s multiple comparison test).
Table 6.
Effect of EELCV and EELLI on gastric function test recorded during chronic toxicity studies in female Swiss Albino Mice
| Tests | Control (1% Gum acacia) 3 ml/kg | C. viscosum 2000 mg/kg | L. indica 2000 mg/kg |
|---|---|---|---|
| Gastric content (ml/100 gm) | 5.01±0.32 | 4.21±0.19 | 4.16±0.25 |
| pH of the Gastric content | 3.81±0.07 | 4.22±0.16 | 4.09±0.11 |
| Total acidity of the Gastric content (meq/L) | 5528.50±370.24 | 5440.12±139.28 | 5303.47±127.23 |
| Free acidity of the Gastric content (meq/L) | 4391.22±130.66 | 4342.87±138.14 | 4276.8±141.07 |
Table 7.
Hematological examination related to liver function test recorded in chronic toxicity studies of EELCV and EELLI at the dose of 300 mg/kg in female Swiss Albino Mice
| Test | Control (Mean ± S.E.M.) | C. viscosum (Mean ± S.E.M.) | L. indica (Mean ± S.E.M.) |
|---|---|---|---|
| Total Bilirubin (mgs/dl) | 0.37±0.01 | 0.38±0.01 | 0.39±0.02 |
| Direct Bilirubin (mgs/dl) | 0.16±0.006 | 0.17±0.008 | 0.16±0.008 |
| Indirect Bilirubin (mgs/dl) | 0.20±0.01 | 0.21±0.01 | 0.23±0.02 |
| SGOT (IU/L) | 205.83±5.6 | 201.83±5.06 | 201±7.09 |
| SGPT (IU/L) | 69±4.03 | 66.33±2.92 | 66±3.06 |
| Alkaline phosphatase (IU/L) | 212.66±4.63 | 163.16±2.08* | 167.5±10.33* |
| Total Proteins (gms/dl) | 7.21±0.12 | 7.06±0.14 | 7.16±0.13 |
| Albumin (gms/dl) | 3.33±0.07 | 3.23±0.07 | 3.26±0.08 |
| Globulin (gms/dl) | 3.91±0.10 | 3.83±0.13 | 3.9±0.12 |
| Albumin to globulin ratio | 0.85±0.02 | 0.84±0.03 | 0.84±0.03 |
| Gamma-glutamyl transferase (U/L) | 22.66±1.25 | 22±1.00 | 20.16±1.22 |
P<0.05 when compared with the vehicle control group.
The observations are mean ± S.E.M. P>0.05-Not Significant as compared to control (ANOVA followed by Dunnett’s multiple comparison tests), LFT-Liver Function Test, SGOT-serum glutamic oxaloacetic transaminase, SGPT-serum glutamic pyruvic transaminase, A.L.P.-Alkaline Phosphatase, Gama GT-Gamma-glutamyltransferase or gamma-glutamyltranspeptidase, mg/dL-milligrams per deciliters, IU/L-International unit per liters, gms/dl-grams per deciliters, U/L-unit per liters. S.E.M.: Standard error of the mean.
Table 8.
Hematological examination related to renal function tests recorded in chronic toxicity studies of EELCV and EELLI at the dose of 2000 mg/kg in female Swiss Albino Mice
| Test | Vehicle control group | Chronic toxicity group | |
|---|---|---|---|
|
|
|
||
| Control (3 ml/kg) Mean ± S.E.M. | C. viscosum (300 mg/kg) Mean ± S.E.M. | L. indica (300 mg/kg) Mean ± S.E.M. | |
| Serum Urea (mg/dl) | 42.14±1.49 | 40±1.34 | 41.5±1.76 |
| Serum Creatinine (mg/dl) | 1.06±0.04 | 0.96±0.04 | 0.91±0.04 |
Values are presented as mean ± S.E.M., N = 6; EELCV = ethanolic extract of the leaves of Clerodendrum viscosum; EELLI = ethanolic extract of the leaves of Leucas indica; organ-to-body weight index = (organ weight × 100)/body weight.
Figure 2.
Histopathology of control and ethanol extract treated groups at continuous dose (300 mg/kg body weight) for 28 days. Histology of liver (A-C), kidney (D-F) and stomach (G-I) in vehicle control (1% Gum acacia 3 ml/kg) mice (A, D and G); ethanolic extract of leaves of Clerodendrum viscosum animals treated mice (B, E and H); and ethanolic extract of leaves of Leucas indica treated mice (C, F and I).
Discussion
The safety and integrity of therapeutic drugs are critical to the pharmaceutical industry, and patient care [9]. Herbal drugs generally are considered safe in contrast to the synthetic drugs that are regarded as unsafe to humans and environment [10]. Though there is huge data available on chemical and biological characterization of plant products, proven scientific studies on the toxicity and adverse effect of these plant products are needed. Both C. viscosum and L. indica plants have valuable pharmacological effects; the comprehensive awareness about its toxicity potential has not been studied till date. Therefore, the present research evaluated the acute and chronic effects of an ethanol leaf extract of C. viscosum and L. indica plant. The animal toxicity was done on mice instead of rats. It is because toxicity data collected from mice were mainly predicted with the toxic effects in human beings [11].
Indian traditional medicinal systems like Ayurveda, Siddha, and Unani have been used to treat a wide range of diseases since ancient times. Despite of advancement of modern medicine, herbal drugs contributed significantly to the health care system [10,12]. These drugs are gaining popularity as WHO encourages the use of traditional systems of medicine if possible, implant them into the mainstream of health care services [13]. Indian Council of Medical Research has made substantial research investments in medicinal plants for better drugs and remedies for various diseases [14]. Herbal medicines have played an important role in managing various chronic diseases, attracting clinical researchers to use them as alternatives to allopathic pharmaceutical drugs [15].
India is an ancient civilization with a rich repository of medicinal plants. Ayurveda’s traditional medicinal system developed in India and has been practiced for centuries [16]. Modern drugs have limited availability and usually associated side effects, therefore, researcher encourages usage of traditional medicine [17]. Increasing population, excessive treatment costs and the emergence of drug-resistant to synthetic medicines have increased the popularity of alternative medicines, contributing to countries economic value [18-20].
The leaves of C. viscosum belong to the family Verbenaceae/Lamiaceae/Labiatae and L. India belongs to family Lamiaceae/Labiatae possess anti-inflammatory and analgesic possibly due to the presence of phytosterols, triterpenes, alkaloids, glycosides, saponins and most probably due to the presence of phenolic compounds and tannins as shown by a phytochemical screening of C. viscosum and L. indica in this study [1,21]. These purified compounds and standardized plant extracts provide unlimited opportunities for new therapeutics drugs [6].
Early identification of the potential for substances to cause hepatotoxicity and nephrotoxicity is vital for human wellbeing risk appraisal [22]. The liver is frequently getting affected with chemical-induced injuries and a few factors, including drug effects effectively add to the liver’s vulnerability. Since most xenobiotics enter the body through oral route, passes through the gastrointestinal tract, and then carry to the liver, this organ is the most commonly exposed to their assault [23-25]. The subsequent explanation is that the biotransformation of synthetic substances in the body occurs in the liver [26]. The majority of time, biotransformation prompts the development of a particle that is no more or, at any rate, less naturally dynamic, more polar, and water-dissolvable, subsequently more effectively discharged from the body; but at times, the metabolic movement of the liver produces metabolites as well. Without a doubt, the biotransformation of synthetic substances into reactive metabolites has a critical occasion for nephrotoxicity. The nephrotoxic metabolites might be created locally by the activity of P450s in the kidney or they can be delivered in the liver or in different organs and transported into the kidney through foundational dissemination [27]. The high renal bloodstream and the weighty centralizations of excretory items, getting to the re-assimilation of water from the rounded liquid, are further significant elements in the kidney’s weakness to xenobiotics [28].
Acute toxicity and chronic toxicity studies were dose as per Diener et al. and Stallard et al., which served as indicators described in preclinical observation [28,29]. Most commonly, herbal drugs are administered orally, and hence gastric acid profile was evaluated in this study as both C. viscosum and L. indica have been used as analgesic, antipyretic and anti-inflammatory agents and commonly used conventional preparations related NSAIDs carry the risk of increasing gastric acidity which limits its use in a susceptible individual. Orally administered drugs usually undergo metabolism in the liver and are excreted in urine via kidney; hence liver function and renal function test was employed in this study to know major organ toxicity.
Biochemical, hematological, and histopathology examinations did not show significant changes in control and treated animals, suggesting that a dose level of 2000 mg/kg (of C. viscosum, and L. indica) is safer. The pharmaceutical drug or compound with oral LD50 more than 1000 mg/kg bw and the oral dose of 1000 mg/kg could be considered low toxic and safe [30,31]. Toxic outcomes of drugs can be visualized directly by observing the vital body organs [32]. Further, the test with chronic done of 300 mg/kg bw for 28 days of oral toxicity study also did not have any clinical sign of toxicity, and no mortality was recorded in test mice. These results indicating that the repeated dose of C. viscosum and L. indica ethanol leaf extract have no adverse effect.
Conclusions
In this study, acute toxicity studies (2000 mg/kg) were administered on a single day and observed for 14 days. Chronic toxicity studies (300 mg/kg when administered for 90 days) of Clerodendrum viscosum and Leucas indica ethanolic leaf extracts did not showed any unusual changes during preclinical observation. Physical, biochemical, and histopathological examination also reveals no abnormality when compared between the groups. Hence, study supports the use of Clerodendrum viscosum and Leucas indica ethanolic leaf extracts as an alternative system of medicine for the biological and pharmacological screening of the same.
Acknowledgements
The authors would like to thank the A. J. Institute of Medical Sciences and Research Centre for providing facilities and fund for this project.
Disclosure of conflict of interest
None.
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