EVALUATION OF ACUTE AND SUB-ACUTE TOXICITY OF ETHANOLIC EXTRACT OF SEED KERNELS OF CAESALPINIA CRISTA (LINN.) IN ALBINO MICE

EVALUATION OF ACUTE AND SUB-ACUTE TOXICITY OF ETHANOLIC EXTRACT OF SEED KERNELS OF CAESALPINIA CRISTA (LINN.) IN ALBINO MICE

S. N. Kshirsagar*¹, D.M. Sakarkar ¹ and S.S. Deshpande ²

Department of Pharmacology, Sudhakarrao Naik Institute of Pharmacy ¹, Pusad- 445 204, Maharashtra India

Department of Pharmacology, Dr. P. D. Medical College ², Amaravati, Maharashtra, India

ABSTRACT

Acute and sub-acute toxicity of ethanolic extract of Caesalpinia crista (Linn.) was evaluated in Albino mice. The acute toxicity studies were conducted as per the OECD guidelines 420, here the limit test dose of 2000mg/kg used. Observations were made and recorded after treatment at 2 hrs, 4 hrs, 8 hrs and then for seven days regularly for respiration rate, heart rate, and behavioural signs like apathy, reduced locomotor activity as well as licking. Three groups of 6 mice were made for the sub-acute toxicity study. Group I (control) received distilled water, group II 200 mg/kg and group III 400 mg/kg received ethanolic extracts every 24 hr orally for 28 days. No toxic effects of the Ethanolic extract were observed on body and organ weights between the control and the treated group after 28 days of treatment. No significant variation was found in the Hematological analysis and clinical blood chemistry. No mortality was found during study.

Hematological analysis

INTRODUCTION: Plants used in traditional medicine contain wide range of ingredients that can be used to treat chronic as well as infectious diseases. A vast knowledge of how to use the plants against different illness may be expected to have accumulated in areas where the use of plants is still of great importance ¹. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The important constitutent of these plants are alkaloids, Flavonoids,tannins and phenolic compounds ².

Caesalpinia crista (Linn.) synonym: Caesalpinia bonduc (L.) Roxb.), Caesalpinia bonducella belongs to family Caesalpiniaceae and is commonly known as kat-takaranja in Hindi and sagargota in Marathi. It is prickly shrub found throughout the hotter regions of India and srilanka ³. The seeds of plant are almost globular in shape, grey, hard with a smooth shiny surface ⁴. The seeds of the plant contain bonducin, proteins, saponin, starch, sucrose, an enzyme, two phytosterols namely sitosterol and hepatsane, fattyacids such as palmitic acid, stearic acid, lognoceric, oleic, linolenic acid. The seed kernals of the plant contain furanoditerpenes- ά-caesalpin, ß-caesalpin, γ-caesalpin, δ-caesalpin, ε-caesalpin,and F-caesalpin ⁵. The leaves of Caesalpinia crista (Lin) are tradionally used for tumors, inflammation and liver disorders.

In India, various parts of this plant has been used in various therapeutic uses like adaptogenic ⁶,  antimicrobial ⁷, antiproliferatve ⁸, antidiabetic ⁹, anti-filarial ¹⁰, contractility on uterus ¹¹, hepatprotective ¹², antitumor and antioxidant activities ¹³. Because of their various pharmacological activities, animal toxicity studies are required to identifyl adverse effects of Caesalpinia crista (Linn.). The present work was undertaken to study the acute and sub- acute toxicity of ethanolic extract of seed kernels of Caesalpinia crista (Linn.)

MATERIALS AND METHODS:

Plant Material: The Caesalpinia crista (Linn.) seeds were collected from the local market Yogesh Pharmacy Nanded Maharastra. It was authenticated by Dr. Mr. Krishna G. Kadaskar, Department of Botany P. N. College, Pusad (Maharastra).

Preparation of Extract: The seeds kernels of Caesalpinia Crista (Linn.) were shade dried at room temperature. Then the shade dried seeds kernels were powdered to get a coarse powder. 100g of coarse powder was defatted with petroleum ether and extracted exhaustively with 95% ethanol at Temperature 60oC, in a soxhlet extractor. The extract was concentrated in a rotary flash evaporator residue was dried in desiccator over sodium sulfite. This procedure was repeated for 5-6 times to receive sufficient quantity of ethanolic extract ¹⁴.

Phytochemical Investigation: Ethanolic extract of Caesalpinia crista (Linn.) seeds kernels were subjected to further preliminary qualitative phytochemical investigation ¹⁵.

Toxicology Study:

Experimental Animals: Healthy adult Albino mice of either sex weighing 24-32 gm acclimatized for 14 days. The animals were housed under standard conditions and room temperature (25±2^oC). During the acclimatization period of 14 days, animals were observed for general condition every day and weighed on the next day of arrival and on the last day of acclimatization. The animals were fed with balanced pellet and water adlibitum. The experimental protocol of toxicological studies was reviewed and approved by the Institutional Animal Ethical Committee of Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Acute Toxicity Study: The acute toxicity studies were conducted as per the OECD guidelines 420 (OECD 2001) where the limit test dose of 2000 mg/kg used ¹⁶. Observations were made at 2, 4, 8 hrs for seven days for body weight, treatment related changes like respiration rate and heart rate and behavioral signs like apathy, reduced locomotor behavior, licking activity.

Sub Acute-Toxicity Study: Healthy adult Albino mice of either sex weighing 28-32 gm were divided in to 3 groups of 6 animals each and were housed under standard conditions and room temperature (25±2^oC). The control animals (Group-I) received 0.5ml of vehicle alone and the other two groups (Group-II &III) received Caesalpinia crista (Linn.) seeds kernels ethanolic extract for 28 days at doses of 200, 400 mg/Kg body weight respectively. Toxic manifestations such as body weight, mortality, food and water intake was monitored. After 28 days, all surviving animals were fasted overnight and anesthetized with ether. The blood samples were collected in heparinized tubes for determining hematological parameters and the serum was used for clinical blood chemistry. Animals were sacrificed after blood collection and the internal organs were removed and preserved in 10 % formalin solution for histological examination.

Biochemical Estimations: Blood collected in heparinized tubes were then centrifuged at 3000 rpm for 10 minute. The serum separated was analyzed for various parameters such as serum bilirubin, Serum glutamic oxaloacetic Transaminase, Serum Glutamic pyruvic Transaminase, Serum alkaline phosphatase, Serum total proteins, serum total albumin, serum total globulin, serum cholesterol, electrolytes like Na, K, Cl, Ca, P, Blood Urea Nitrogen and creatinine by autoanalyzer.

Haematological Assay: Blood sample collected in the heparinized tubes were used to estimate hemoglobin, W.B.C, R.B.C, Platelet count, Reticulocyte count, Mean corpuscular volumn, mean corpuscular hemoglobin concentration, Percent of Neutrophils, Eosinophils, Lymphocytes and Monocytes, Packed cell volumn and mean corpuscular hemoglobin ^{17, 18}.

Histopathological Study: Histopathological investigation of the organs was done. The organs were fixed in 10% formalin for 24 hrs and washed in running water for 24 hrs. Samples were processed for weighing ¹⁹.

Statistical Analysis: The values are expressed as mean ± Standard deviation (S.D.). Results were analyzed statistically using one way Anova. The significant difference between the groups are considered at P<0.05 level.

RESULTS:

Acute Toxicity Study: The acute toxicity study was conducted as per the OECD guidelines 420, where the limit test dose of 2000mg/Kg used. No test substance related mortality was observed at 2000mg/Kg and throughout the observation period there were no significant changes in behavior (i.e., apathy, hyperactivity, hypoactivity) in any of the mice. The results of acute toxicity were illustrated in Table 1 and 2. No changes were found in body weight and changes in respiration rate and heart rate.

TABLE 1: EFFECT OF ORAL ADMINISTRATION OF ETHANOLIC EXTRACT OF CAESALPINIA CRISTA (LINN. ) SEEDS KERNELS ON BODY WEIGHT (G) AND ORGANS WEIGHT (G) OF MICE

Values are expressed as mean ± S.D of 6 mice in each group.

TABLE 2: OBSERVATIONS OF ACUTE TOXICITY STUDY (TREATMENT RELATED CHANGES)

+ Significant changes, - no change noticed, C- Control, N- normal

Sub Acute Toxicity Study: Ethanol extract of Caesalpinia crista (Linn.) seeds kernels at dose of 200,400 mg/kg orally for every 24 h for 28 days did not produce any mortality in tested animals. No sign of toxicity was observed during the experimental period. All haematological parameters such as hemoglobin, W.B.C, R.B.C, Platelet count, Reticulocyte count, Mean corpuscular volume, mean corpuscular hemoglobin concentration, percent of neutrophils,  eosinophils, lymphocytes and monocytes, packed cell volume and mean corpuscular hemoglobin and biochemical parameters such as serum bilirubin, serum glutamic xaloacetic transaminase, serum glutamic pyruvic transaminase, serum alkaline phosphatase, serum total proteins, serum  total albumin, serum total globulin and serum cholesterol and serum triglyceride were found within the normal range.

The results of hematological and biochemical parameters are showed in table 3, 4, 5.

 TABLE 3: HEMATOLOGICAL PARAMETERS AFTER 28 DAYS ORAL TREATMENT WITH ETHANOLIC EXTRACT OF CAESALPINIA CRISTA (LINN.) SEEDS KERNELS

Values are expressed as mean ± S.D of 6 mice in each group. Comparisons were made between Group-I with Group-II and Group-III. P <0.05 was considered significant. The * symbol also represent the statistical significance at P <0.05

TABLE 4: EFFECT OF TREATMENT WITH ETHANOL EXTRACT OF CAESALPINIA CRISTA (LINN.) SEEDS KERNELS ON BIOCHEMICAL PARAMETERS

TABLE 5: EFFECT OF TREATMENT WITH ETHANOL EXTRACT OF CAESALPINIA CRISTA (LINN.) SEEDS KERNELS ON BIOCHEMICAL PARAMETERS

Values are expressed as mean ± S.D of 6 mice in each group. Comparisons were made between Group-I with Group-II and Group-III. P <0.05 was considered significant. The * symbol also represent the statistical significance at P <0.05

DISCUSSION: In acute toxicity study, there was no any mortality observed up to the maximum dose level of 2000mg/kg body weight of the extract administered orally, which the single high dose is recommended by OECD guidelines 423 for testing acute toxicity. No changes were found in body weight, respiration rate, heart rate and behavioural changes. Thus, our test suggested that ethanolic extract of Caesalpinia crista (Linn.) seeds kernels does not cause any acute toxicity. The changes in body weight have been used as an indicator of adverse effects of Caesalpinia crista (Linn.) and chemicals.

In subacute toxicity study mice treated with 200,400 mg/kg doses of ethanolic extract of Caesalpinia crista (Linn.) seeds kernels had a progressive increase in body weight. The increase in weight was not significantly different from that of the control. The progressive increase in body weight at dose of 200,400 mg/kg of mice during 28 days of administration of ethanolic extract of Caesalpinia crista (Linn.) seeds kernels may indicate the improvement in the nutritional state of the animal. The growth response effect could be as a result of increased food and water intake.

The haematological status after 28 days of oral administration of ethanolic extract of Caesalpinia crista (Lin) seeds kernels was also assessed. Table 3 shows the effect of Caesalpinia crista (Linn.) seeds kernels on hematological parameters. The red blood cell was found to be significantly increased (P<0.05 ) in Group –III and white blood cell count were found to be significantly increased in Group-II and III Caesalpinia crista (Linn.) seeds kernels treated animals and slight changes in mean corpuscular hemoglobin also. With the exception of a transient increase in RBC and WBC count there were no significant alterations in the hematological parameters. Increase in WBC may indicate the impact of Caesalpinia crista (Linn.) seeds kernels in boosting the immune system of treated groups.

However, slight changes in RBC and WBC did not show any dose responsiveness. All the other parameters in all treated group remained normal without any significant difference. Transaminases (GOT and GPT) and ALPs are good indices of liver and kidney damage respectively. Results of serum biochemistry were showed in Table 4. There were no deleterious changes found in the level of transaminases and ALPs in serum of treated groups with control animals. Serum albumin and globulin level of treated groups showed significant changes when compared to control group. All other biochemical parameters were remained normal without any significant difference.

Furthermore, gross examination of internal organs like Liver, Lung, Heart, spleen and kidney were also found to be normal. No abnormalities were detected in treated animals when compared to the control. In conclusion this study presents strong evidence of nontoxic effect of ethanolic extract of Caesalpinia crista (Linn.) seeds kernels. These results showed that the use of extract of Caesalpinia crista (Linn.) seeds kernels is safe and explained the extensive utilization of the plant in traditional medicine.

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