Acute Oral Toxicity Evaluation of Ethyl Acetate Fraction of Sesbania Grandiflora Linn. Roots as Per OECD 425 TG on Albino Mice

Introduction

Herbal medicinal plants are been used since centuries to treat different ailments. The herbal medicines usage has been increased worldwide due to increasing population and, in a desire, to reduce the side effects or inefficiency of modern drugs. In the earlier days before development of synthetic and semi synthetic drugs many herbals were used as a medicine to treat various ailments and still today it is in practice. These herbal medicines are used not only for the development of new medicines but also it is used as a traditional or complementary medicine. Unlike the synthetic drugs the safety of the herbal drugs as not been scientifically confirmed but many of them believe it as a safe only because it is originated from the nature and the safety assessment of herbal drugs is very rarely done before their consumption as they are considered as inherently safe. The researcher focuses more on the efficacy and safety of the natural herbal remedies. As the one of the popular quote by the father of toxicology “all substances are poisonous there is none which is not a poison’’. It is the right dose which differentiates the remedy and the poison. Whenever chemical substances (drug) administered into the body various interactions occur between the drug and cells, it produces different responses depending on the dose administered. The responses may be desired, beneficial and it may cause hazardous toxic responses. In the drug screening process LC50 is determined as an initial step to asses and evaluate toxic characteristics of the drug.^1-6

Sesbania grandiflora Linn. belongs to the family Fabaceae was been widely employed to treat various conditions. The roots of SSesbania grandiflora was been used to treat inflammation, epilepsy, tonic and ophthalmic in ethno medicinal practices⁷ . The root rubbed into a paste with water is applied to painful or rheumatic swelling. However, the Sesbania grandiflora Linn. roots are been scientifically reported for anticancer, antioxidant, anthelmintic property, antiurolithiatic, hepatoprotective, anxiolytic, anticonvulsive, wound healing, antiulcer, antibacterial activity.^8-13 To justify traditional use, the study was conducted to evaluate the acute oral toxicity potential of ethyl acetate fractions of Sesbania grandiflora roots according to OECD TG 425 in female albino mice.

Materials and Methods

Collection of plants

Sesbania grandiflora linn. roots were collected during first week of august from the Tumakuru rural area, Karnataka, India. The plant specimen was identified and authenticated by Mr.Chidhandha ,Professor, Sree Siddaganga Women’s College, Tumakuru, Karnataka.

Preparation of plant extract

1.2 kg shade dried and powdered roots of Sesbania grandiflora were extracted using 90% ethanol (2 × 4.5 L) at a room temperature for seven days. The extract was concentrated to 100ml using to avolume of 100 mL under reduced pressure using a rotary evaporator (Buchi R -300) at a bath temperature of 40 °C, and then partitioned with hexane to afford a hexane-soluble fraction (Fr. A, 1.5 g). The aqueous ethanol soluble fraction was suspended in water and partitioned with chloroform to afford chloroform soluble fraction (Fr. B, 1.2 g). The aqueous ethanol soluble fraction was re-suspended in water and re-partitioned with ethyl acetate to afford ethyl acetate soluble fraction (Fr. C, 15 g), was designed as EFSGR (Hasan et al., 2012) and used for the present study.

Approval from Animal ethics committee

Approval was received from the institutional animal ethics committee (IAEC) of Sree Siddaganga College of Pharmacy (SSCPT/IAEC. Clear.184/2018–19 Date 09/03/2018) before performing acute oral toxicity studies.

Acute oral toxicity study¹⁴

The acute oral toxicity study was conducted according to Organization for Economic Co – operative and Development (OECD) test guidelines 425 (up and down procedure). Prior to treatment nulliparous and non – pregnant female albino mice of 6 – 8 weeks weighing 30 to 32 g were marked and randomly assigned to two groups. (n = 5). Animals were kept understandard conditioned for 5 days with free access to water adlibitium, at 24±2 °C and 55 – 65% relative humidity with 12 hours light and dark cycle in a standard animal house facility. Acute oral toxicity test was performed by administering single dose of Sesbania grandiflora ethyl acetate fraction of strength 2000mg/kg, whereas as controlled group received distilled water. Prior to the dosing mice were fasted for 5 – 6 hours with free access to water ad libitium and food was provided after one hour. The animals were closely observed for 30 min, then for 4 and 12 hours. The same procedure was followed for vehicle (normal control) treated groups containing 5 mice. Both the groups were observed for 4 hours and then at different time intervals for 14 days. At different time intervals the weights of the animals were documented and survived animals were observed for the onset of toxic reaction. At the end of the study, blood samples were collected by cardiac puncture under isoflurane anaesthesia and serum was used for biochemical estimation. Further, the remaining blood samples were stored in EDTA container tubes for hematological estimation. After sacrificing the mice, vital organs such as heart, kidney, liver and spleen were excised and weighed. For histopathological examinations these organs were preserved in 10% formalin solution.

Haematological analysis¹⁵

The complete blood count parameters (CBC) containing haemoglobin (Hb), total RBC, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), white blood cells (WBC), neutrophils (N), lymphocytes (L), monocytes (M), eosinophil (E), mean corpuscular haemoglobin (MCH), haematocrit test (HCT) were determined using auto analyser.

Biochemical analysis¹⁶

Biochemical parameters were analysed by using ERBA diagnostic kits. Serum creatinine and urea were assessed toexamine kidney function. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate, total bilirubin, direct bilirubin, indirect bilirubin, total protein, albumin, globulin was estimated to study liver function and serum cholesterol,triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL) were estimated to evaluate lipid profiles.

Histopathological study¹⁷

The isolated organs like heart, kidney, liver, lungs and spleen (vital organs) of the sacrificed mice in 1:10ratio of formalinand buffer solution is processed and embedded in paraffin wax. 5mm paraffin sections were stained with haematoxylin and eosin (H&E). The slides were observed under the light microscope and magnified images of the tissue structure were captured for further study.

Statistical analysis

The experimental data were expressed I Mean ± SEM and the statistical analysis was performed by one – way ANOVA followed by Tukey’s multiple comparison test. 

Results

1Acute oral toxicity study

Single oral dose administration of ethyl acetate fraction of Sesbania grandiflora roots at a dose of 2000mg/Kg did not produced any mortality or adverse effects during 14 days of study period. Further treatment of EFSGR did not reveal any abnormal clinical signs and mortality.

Body weight and behavioural changes

Body weights of both normal and EFSGR treated animals did not show any significant alteration during the entire study period (as shown in table 1). Behavioural observations for the EFSGR treated group showed mild itching for initial 30 minutes and increased sleeping timeat 4th hour as summarized in table 2.

Organ to body weight index

The organ weights after 14 days in the normal and EFSGR treated animals did not exhibit any statistically significant differences in the organ to body weight index (as shown in Table 3).

Haematological analysis

Haematological test is performed to predict physical and chemical changes I.e. toxicity of the blood which occurs as the drug substance of both synthetic and natural source is administered. No toxicologically significant differences were found in haematological examination between normal control and EFSGR treated mice. The data of haematologicalparameter was tabulated in Table 4.

Biochemical analysis

The various biochemical parameters post dose treatment showed no significant changes in the liver function test, renal function test and Lipid profile (Table 5).

Histopathology study

The light microscopic examination of the vital organs kidney, liver, spleen and heart of mice treated with EFSGR (2000mg/kg) exhibited no change in the treated group as compared to the control group shown in figure1. 

Discussion ^18-24

There the current study is designed to assess and focus the acute oral toxicity of ethyl acetate fraction of Sesbaniagrandfloralinn. roots (EFSGR) in albino mice as per the OECD 425 TG. This acute toxicity study using oral dose administration of single dose EFSGR(2000 mg/kg) did not revealed any signs of mortality or toxicity throughout the study period while some mild behavioural changes like increase in the sleep time and itching were noticed during initial period. The increased sleeping tendency of mice treated with EFSGR might be due to calming effect on CNS. Further based on this, EFSGR may be a good candidate to evaluate anticonvulsant activity. According to the globally harmonized system of chemical classification the EFSGR can be put into group 5 i.e. LD₅₀ greater than 2000mg/kg which is class of lower toxicity and EFSGR on acute exposure is considered safe.

No significant changes in the body weight were observed during the course of the study, food and water intake was also normal. It indicates the normal metabolism process of fats, carbohydrates and proteins within the body as these plays a major role in physiological function.

Relative organ weight is an important index of the physiological and pathological status of the animal. Kidney, heart, lungs and spleen are the vital organs of the body and are the major targeted area of any toxicity. Finally when the animals were sacrificed at the end of the study. When the animals were sacrificed there were no lesions found on macroscopic examination of vital organs in comparison with vehicle control group. No significant variations were found in organ to body weight index of mice in treatment group when compared with normal control group.

The perspective markers of physiological changes in retort to any environmental or toxic substance in animals are haematological parameters. Haematological profile of EFSRG treated group suggest that there were no significant variations in the heamatogical parameters like white blood cell count, haemoglobin, red blood cell count, MCV, MCHC, platelet and haematocrit value compared to control groups indicating that EFSGR had no effect on the blood cells of the treated group.

Sensitive biomarkers of possible tissue damage and liver damage are ALT and AST.ALT abundantly exists in the cytoplasm of hepatocytes and any liver damage can cause the elevation of ALT. AST is an enzyme present extracellular in tissues like liver, skeletal muscles, kidneys, pancreas, erythrocytes and heart. Cellular damage or change in membrane permeability causes release of AST from these cells in response. Biochemical analysis of EFSGR treated mice did not show any significant differences in these biochemical markers. Elevated levels of triglycerides, cholesterol bilirubin and reduced albumin are the significant indicators in hepatic failure. Further EFSGR did not show any significant alteration of cholesterol, HDL, LDL and triglycerides compared to the normal control mice. This indicates that no significant alteration in normal homeostasis of lipid. Histopathologicalassessments of kidney and liver tissues supports for overall biochemical estimations.

Conclusion

After exposing the mice to single doses of 2000mg/kg of EFSGR neither showed mortality nor adverse effects in their behaviour. No damages on hematopoietic and biochemical system were observed. Histopathological changes did not occur in the liver, kidney and the spleen of mice. These results of acute oral toxicity studies in mice provide supportive evidence regarding the safety of Sesbania grandiflora roots.