Safety studies of Siddha Medicine Karunjeeraga chooranam in acute and subacute toxicity wistar rat models

PG Scholar, Department of PothuMaruthuvam, GSMC,Palayamkottai, Tamilnadu, India

² Professor and Head, Department of PothuMaruthuvam, GSMC,Palayamkottai, Tamilnadu, India

³ Lecturer, Department of PothuMaruthuvam, GSMC,Palayamkottai, Tamilnadu, India

Corresponding Author

Arivoli.D, PG Scholar, Department of Pothu Maruthuvam, GSMC, Palayamkottai, Tamil Nadu. 

Background:

Herbal formulations are frequently recommended in day to day clinical practice for therapeutic purposes by many Siddha practitioners. However, the safety of Siddha herbal drugs remains a challenge whencommunicatinginto the scientific community because of the high variability of phyto-chemical components involved.

Objective:

To investigate the acute and subacute oral toxicity of Karu njeeraga chooranam in adult female wistar rat models.

Materials and Methods:

In acute oral toxicity study, Karumjeeragachooranam were administered orally initially at the dose of 50 mg/kg/body weight which was increased up to 2000mg/kg/body weight and animals were observed for toxic symptoms till 14 days as per the OECD-423 guidelines.

For subacute toxicity study, the Karun jeeragachooranam were administered for 20 days,as per the OECD guidelines-423with the doses of 50 mg, 100 mg, 200 mg and 400 mg/kg/ body weight for different groups. At the end of 20th day, the animals were sacrificed and toxicity parameters were assessed. Biochemical analysis on blood samples and histopathological evaluation of different organswere also performed to assess any toxicity.

Results:

In acute toxicity study, no mortality wasfound at a dose of 2000 mg/kg .So the dose is taken as oral LD50 of Karun jeeragachooranam. The administration of drugat varied dosesupto 400mg/kg/body weightfor20 days did not produce any significant change in haematological and biochemical parameters of wistar rats as compared to normal control group. No pathological changes were observed in histology of various organs of treated rats ascompared to normal control animals.

Conclusion:

The Karunjeeragachooranam wasfound to besafe when administered to adult wistar rat modelsfor longer duration and in maximal LD50 doses.

Keywords;, Karumjeeragachooranam, Nigella sativa , Siddha Medicine,Wistar rats

Introduction

The trial drug Karunjeeragachooranam(4), primarily composed of seed Karunjeeragam(Nigella sativa), which be­longs to the Ranunculaceae family. It is an annual herb with many pharmacological properties. The seeds of Karunjeeragam are used in the treatment of variousdiseases like bronchitis, diarrhea, rheumatism, asthma and skindisorders. Itcontains many active components, such as thymo­quinone (TQ),(1,2) alkaloids (nigellicines and nigelledine), saponins (alpha-hederin), flavonoids, proteins, fatty acids,thymohydroquinone, dithymoquinone, p-cymene, carvacrol, 4-terpineol, tanethol, sesquiterpene, longifolene, α-pineneand thymoletc that yields positive effects in the treatment of patients with different diseases&has been extensively studied for its biologicalactivities and shown to possess wide spectrum of activitiessuch as diuretic,hypolipidemic, antihypertensive, bronchodilator,gastroprotective, hepatoprotective, hypoglycemic, anticancer and Immuno-modulatory, analgesic, antimicrobial, anti-inflammatory, spasmolytic, renal protective andantioxidant properties(3).

Materials and methods

Maintenance of TestAnimals

The study was carried out in the Department of Pharmacologywith the approval of the InstitutionalAnimalEthicsCommittee,IAEC/D.ARIVOLI /TNMGRMU/MD(S)/321611001/KMCP/23/2018 KM College, Tamilnadu. Adult femaleWistar albino rats (150–200 g) were used in the study. Animals werehoused under standard laboratory conditions at 25 ± 2°C, andhumidity levels were in the range of 30 - 70% in groups withfree access to food and water ad libitum. They were acclimatizedto the laboratory conditions for a period of 2 days before the study(5).

Drug for the Toxicity study

The raw drug was authenticated by Botanists of Government Siddha Medical college,Palayamkottai, Tirunelveli.Then it was subjected to purification. The proposed drug Karunjeeragachooranamwas prepared as per Gunapaadam – Mooligaivaguppu text book (page no:464) and followed the Standard Operating Procedure for the preparatory process.

Acute toxicity study

Acute oral toxicity test was performed as per the OECD 423 guidelines (OECD, 2001). All the animals were randomly distributed into threedifferent treatment groups. The female Wistar rats (n = 3) were weighed, and the dose was calculated in reference to the body weight. The rat models were fasted overnight and provided only with water, after which the Karunjeeragachooranam(4,6)is administered by gastric intubations to the relevant group of animals orally at the dose of 50 mg.kg ^-1 body weight in Tween-80. The animals are then observed for 14 days and maintained with normal food. If the mortality rate of 2 or 3 animals in 14 days is recorded and the dose is considered to be toxic dose. But when mortality of one animal is observed, then the same dose is repeated again for confirmation. However, if mortality is not observed, the procedure is repeated for further higher doses such as 300 and 2,000 mg.kg ^-1 body weight. Toxic symptoms are observed for 72 hrs including behavioral changes, locomotion, convulsions and mortality(7).

Observations include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behavior pattern. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma(8).Body weight, food and water intake were recorded at two-day intervals. Surviving animals were fasted overnight, weighed and euthanizedon the 15^th day using anesthetic ether. All test animals were subjected to gross necropsy.

Sub-acute test for Karu n jeeraga chooranam

This experiment evaluates the toxicity potential of Karunjeeragachooranam for more than 14 days . Male and Female Wistar rats weighing 180 ± 10 g were used for this study(9). The animals were divided into five groups of six animals each. The dose of the drug administered was calculated on the basis of body weight of the animal. The animals in Group I were administered with a single daily dose of 0.5 ml of Tween 80orally for 20 days. The animals in Group II were administered with 50 mg.kg^-1b.w. of the Karunjeeragachooranamorally once in a dayfor 20 days. The animals in Group III were administered with 100 mg.kg^-1b.w. of the Karun jeeragachooranamorally once daily for 20 days. The animals in Group IV and V were administered once daily with 200 and 400 mg.kg ^-1b.w. of the Karumjeeragachooranamrespectively for 20 days orally (Pieme,et al 2006, Joshi, et al 2007, Mythilypriya, et al., 2007) .

The animals were weighed during every five days, from the initial day of the treatment, to record the weight variation. At the end of the treatment, blood samples were collected from eyes by puncturing retro orbital plexus after mild anesthesia for biochemical analysis. The collected blood sample was centrifuged within 5 min of collection at 4000 g for 10 min to obtain plasma. Itis then analyzed for total cholesterol, total triglyceride, HDL-cholesterol levels,LDL-cholesterol,plasma glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea(10).

Results

Acute toxicity study with Karu n jeeragachooranam

In acute toxicity, there was no mortality or morbidity observed in animals through the 14-days period following single oral administration at all selected dose levels of the Karun jeeragachooranam(Table-1). The animals did not show any changes in the general appearance during the observation period.

Table.1. Acute toxicity studyof Karunjeeragachooranamon experimental mice. The acute toxicityof Karunjeeragachooranamon experimental rats were tested using OECD-423 guidelines, where ST- sign of toxicity; NB- normal behaviour; D- died; S- survive. Values are expressed as number of animals (n=3).

Effect of Karu n jeeragachooranam in subacute Toxicity

The Karunjeeragachooranam was also evaluated forsubacute toxicity .The effect of Karu njeeragachooranam on the body weight changes shows that, there was significant increase (p<0.05) in body weight in all the animals observed. The results are shown in Table.2.

Table.2.The effects of Karunjeeragachooranamon body weight changes in rats. The values are expressed as mean ± S.E.M. n=6. The results of group I were compared with other groups such as II, III, IV, and V. The statistical analysis was carried out using one way ANOVA method, where **P<0.01 *P<0.05.

Effect of Karu n jeeragachooranam on kidney, heart, liver and brain in rats

The effects of Karun jeeragachooranamon kidney, heart, liver and brainof the rats were observed.From the study it was clear that, significant (p<0.01) changes in the weights of various organs of the animals occurred with higher doses of the extract (400 mg.kg^-1bwt), but macroscopic examinations did not show any changes in colour of the organs of the treated animals whencompared with the control. The results are shown in Table.3.

Table.3.The effects of Karunjeeragachooranamonkidney, heart, liver and brainof the rats. The values are expressed as mean ± S.E.M. n=6. The results of group I were compared with other groups such as II, III, IV, and V. The statistical analysis was carried out using one way ANOVA method, where **P<0.01.

Effect of Karu n jeeragachooranam on biochemical profiles of rats

The effect of Karun jeeragachooranamon various biochemical parameters of the experimental animal ‘rats’ were tested.From the study it was evident that, there was significant decrease (p<0.05) in the plasma glucose level in treated rats especially at higher dose (400 mg.kg^-1) whencompared with control rats. The control rats were administered only with 5 ml of normal saline. Significant decrease (p<0.05) in the plasma total cholesterol (TC), triglyceride (TG) and LDL-cholesterol levels were observed. But a significant increase (p<0.05) in HDL-cholesterol levels were observed in all the treated animals compared with the control animals. AST, ALT and ALP levels were also normal in the Karu njeeragachooranamtreated animals. From the results of biochemical study, there was no evidence of severe toxicity associated with the administration of higher concentration of Karu njeeragachooranam.The results are shown in Table.4.

Table.4.The effect of Karun jeeragachooranamon biochemical parameters such as glucose, cholesterol, triglyceride, HDL and LDL. The values are expressed as mean ± S.E.M. n=6. The results of group I were compared with other groups such as II, III, IV, and V. The statistical analysis was carried out using one way ANOVA method, where **P<0.01 *P<0.05

Table.5.The effects of Karun jeeragachooranamon biochemical parameters such as AST, ALT, ALP, TP and Albumin in rats. The values are expressed as mean ± S.E.M. n=6. The results of group I were compared with other groups such as II, III, IV, and V. The statistical analysis was carried out using one way ANOVA method, where **P<0.01 *P<0.05.

Effect of Karu n jeeragachooranam on haematological parameters in rats

The effects of Karun jeeragachooranamwere observed for its effect onhaematological parameterson the experimental rats. A significant increase (p<0.01) were observed in the haemoglobin contents and RBC count in the group treated with 400 mg.kg^-1 body weight of Karu njeeragachooranam. There was no significant change in the calcium level in all the treated animals compared to the control.

Table.6.The effect of Karun jeeragachooranamon haematological parameters such as HB, Calcium, RBC and WBC in rats. The values are expressed as mean ± S.E.M. n=6. The results of group I were compared with other groups such as II, III, IV and V. The statistical analysis was carried out using one way ANOVA method, where *P<0.05.

Discussion

The acute toxicity study of Karun jeeragachooranamwas carried out as per OECD-423 guidelines,no mortality was observed in both the animals of control group as well as animals treated with a maximum dose of 2000 mg.kg^-1. Hence, 1/10 ^th of 2000 mg.kg^-1 i.e. 200 mg.kg^-1 of dose was selected as a minimum dose for Chronictoxicity study. The results of subacute toxicity study shows that there was no significant change in animal behaviour due to the absence of toxicity.

There was a slight decrease in plasma glucose level when higher doses ofKarunjeeragachooranam(400 mg.kg ^-1) was administered in the treated rats. Interestingly, significant increase in the levels of hemoglobin was found in treatment with Karunjeeragachooranamwith a higher dose of 400 mg.kg^-1. The possible reason could be that one of the constituents Karun jeeragachooranam may increase absorption of iron. The overall results suggest that Karun jeeragachooranam is non toxic to the haaematopoietic and leucopoietic system.

Conclusion

These Siddha herbal drug Karun jeeragachooranamare considered safe as no adverseeffect on biochemical and hematological parameters andhistopathology of heart, kidney, liver, brain, ovaries, and testiswas observed even after administering these drugs for a long period.

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