Acute and sub-acute Toxicity study of Aqueous extracts of Canscora heteroclita (L) Gilg in Rodents

Background: Canscora heteroclita (C. heteroclita) being used in the Ayurvedic system of medicine in India for the treatment of various diseases. No systematic toxicity study for this plant was described. Objective: The present study was undertaken to assess the safe use of this plant in traditional practice. Materials and Methods: The acute oral toxicity study of aqueous extract of Canscora heteroclita (AECH) was carried out as per the OECD guidelines 423 in mice and the sub-acute toxicity was carried out at a dose of 200 mg/kg and 400 mg/kg as per OECD 407 guidelines in male and female rats. Results: Mice administered upto 2000 mg/kg as a single dose orally not caused any signs of toxicity or mortality in mice. In sub-acute toxicity study in rats, AECH at two different daily doses of 200 and 400 mg/kg for 28 days did not cause any significant change including the hematological and biochemical parameters. Histopathological examinations showed normal architecture suggesting no morphological disturbances. Conclusion: No deaths or any signs of toxicity was observed after oral administration in acute toxicity study upto a dose of 2000 mg/kg of AECH in mice and upto a dose of 400 mg/kg of AECH in sub acute toxicity study in rats.


INTRODUCTION
Canscora heteroclita (L.) Gilg ( Figure 1) (Gentianaceae) synonymically called as Canscora sessiliflora and Gentiana heteroclita is a gregarious moderately to profusely branched herb. C. heteroclita are common and abundantly available in south India and it is distributed widely in upper Gangetic plains, Gujarat, Madura hills, Rajasthan and Srilanka, 1,2 where ethno botanical studies and traditional practice revealed their use for treating fever, inflammation, tumor and for liver disorders. 3,4 In India, traditional medical practioners widely using this Canscora species for various ailments and as a substitute for shankhpushpi particularly, for the treatment of liver disorders. Decoction of the entire plant was given for cough and fever. Entire plant powder along with thadupusti legyam was used by traditional practitioners as nerve tonic powder. There is no scientific evidence or reports available in the literature for the oral acute and sub-acute toxicity study for C. heteroclita. Hence to ascertain and establish the safety for its use in traditional practice, acute and sub-acute toxicity studies of the aqueous extract of C. heteroclita was carried out as per OCED guidelines.

Plant materials
C. heteroclita (L) Gilg was collected during January 2014 and the plants were authenticated (BSI/SRC/5/23/2014-15/Tech/872) by Dr. M. Palanisamy, Scientist C, Botanical survey of India, Coimbatore, India. Voucher specimen of the plant was deposited at the Department of Pharmacognosy, KMCH College of Pharmacy, Coimbatore for future reference.

Preparation of extracts
Entire plant of C. heteroclita was cleaned with distilled water to remove earthy matter and residual materials. It was then shade dried at room temperature (32 ± 2°C) for 10 days, pulverized to coarse powder, passed through a #40 mesh sieve. Then decoction was prepared by boiling 100 g of the ground entire plant in 500 mL of distilled water for about 30 min. Filtered and concentrated separately under reduced pressure (IKA Rotary evaporator; Model No RN 10 digital V, ILMAC Germany) at 40°C, yielded 19.6 % w/w of aqueous extract of Canscora heteroclita (AECH).

Animals
Female Albino mice (7-8 weeks old, 20-30 g) and Wistar strains of either sex rats (10-12 weeks old, 125-175 g) were obtained from the animal house of Kovai medical center research and educational trust, Coimbatore, Tamilnadu. The animals were kept in polypropylene cages at a temperature of 25 ± 2°C, RH (50 ± 5%), and 12 h light and dark cycles. They were fed with standard laboratory animal diet and water ad libitum. Animals were acclimatized to laboratory conditions before the test. Experiments were designed and conducted in accordance with ethical norms approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and Institutional Animal Ethical Committee (IAEC) (KMCRET/AICTE/01/2014-15, dt.31/01/2015).

Acute toxicity study
Acute toxicity test was performed as per OECD guideline 423 5 for testing of chemicals (2001). Healthy young adult albino nulliparous, non-pregnant female mice weighing about 20-30 g were administered with 5, 50, 300 and 2000 mg/kg of AECH in distilled water as a single dose (1 ml) orally using oral feeding needle. Animals were observed individually for first 30 min, then for the first 24 h, with special attention given during the first 4 h, and daily thereafter, for a total of 14 days to observe toxicity signs like changes in skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems and for behavioural pattern. On 15 th day animals were anaesthetized, blood was collected from the mice for haematological and biochemical analysis. Mice were then sacrificed, dissected and the organs lungs, liver, spleen and kidney were carefully collected, weighed, processed and observed under photomicroscope for histopathological examination.

Sub-acute toxicity study
The sub-acute toxicity assessment of AECH was performed as per OECD Guideline 407. 6 Wistar strain of rats (125-175 g) was divided into six groups, each consisting of six male and six female rats. Male and female Control groups received only distilled water. Two other groups of each male and female rats were administered with AECH in distilled water orally using rat oral feeding needles, daily for 28 days at a dose of 200 mg/kg and 400 mg/kg respectively. During the treatment period the body weight of animals were monitored on 0, 7 th , 14 th , 21 st and 28 th day. Food consumption and water intake for all the groups were observed from day 1 to 28 days. On 29 th day, the overnight fasted rats were anaesthetized with diethyl ether inhalation in a jar containing cotton soaked with diethyl ether. Then blood samples were withdrawn from retro-orbital sinus and the collected blood samples were evaluated for hematological parameters viz. red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), platelet count, packed cell volume (PCV), differential count, Mean Platelet Volume (MPV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) and Red Blood Cell Distribution Width (RDW). A portion of the blood samples were centrifuged at 10000 rpm for 10 min and the separated serum was analyzed for biochemical parameters viz. Cholesterol, triglycerides, VLDL levels SGOT, SGPT, ALP, total bilirubin, total protein, albumin, globulin, urea, uric acid and creatinine. Biochemical investigations were carried out in an auto analyzer (Photometer 5010 V5+, Robert Riely, Berlin) using Piramal healthcare limited reagent kit. The animals were sacrificed by cervical dislocation and the organs, heart, liver, spleen, kidneys, stomach, testes and ovary were isolated, weighed, processed and observed under photomicroscope for histopathological examination.

Statistical analysis
The results are expressed as the mean ± SEM. The significance of the difference was evaluated by one-way ANOVA followed by Dunnett's test. Data were considered statistically significant if p<0.05.

Acute Toxicity Study
Single oral administration of AECH at a dose of 5, 50, 300 and 2000 mg/kg as per OECD guideline 423 for 14 days did not produce any mortality in tested animals (Table 1). No observable sign of toxicity was detected during the experimental period.

Sub-acute toxicity study
Daily oral administration of AECH at a dose of 200 mg/kg and 400 mg/kg elicited no change in skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic, central nervous systems and behaviour pattern of the rodents. Absence of tremors, convulsions, salivation, diarrhea, and sleep also noted ( Table 2). Thus in sub-acute toxicity study, no sign of toxicity viz. jerk, convulsion and mortality was observed for both the male and female rats treated with 200 and 400 mg/kg dose of AECH.

Parameters observed during acute and sub-acute toxicity studies
Body weight Gain in body weight of mice and rats was observed in acute and sub-acute toxicity study respectively for control group and AECH administered animals and the recorded weights are displayed in Table 3 and 4.

Food and distilled water consumption
Food and distilled water consumption of mice (acute toxicity) and rats (sub-acute toxicity) were continuously monitored, where there is no change in consumption was observed for both the control group and treated groups ( Figure 2 to Figure 5).

Hematological analysis
The results of hematological investigations (Table 5 and 6) conducted on day 15 th day for acute toxicity study and on 29 th day for sub-acute toxicity study revealed no significant changes in the values of RBC, WBC, Hb, platelet count, PCV, differential count, MPV, MCV, MCH, MCHC and RDW of treated groups when compared with the respective control mice and rats respectively.

Biochemical Investigations
Biochemical investigations were performed in order to review any toxic effects produced after administration of AECH on liver and kidney. There was no significant alteration in cholesterol, triglycerides and VLDL levels in treated groups of sub-acute toxicity study when compared with control group of rats (Table 7). No significant change observed in (serum glutamic oxaloacetic transaminase) SGOT, (Serum glutamic pyruvic transaminase) SGPT, (alkaline phosphatase) ALP and total bilirubin content of treated group animals when compared with control group animals (Table 8 and 9). There was no significant alteration observed in creatinine, urea and uric acid levels of treated group animals when compared with control group animals (Table 10 and 11).

Organ weight
No abnormal change in the relative weight of internal organs of mice and rats was observed when compared to control group as shown in Table 12 and 13.

Histopathological investigation Acute toxicity study in mice Lungs
Normal alveoli with normal bronchioles and normal alveoli with congested blood vessels were observed in histopathological investigation of lungs of control group of mice in 10 x and 40 x magnifications respectively. Mice treated with 2000 mg/kg of AECH showed normal alveoli with bronchioles in both the magnifications ( Figure 6).

Liver
Normal lobular architecture and normal portal tract was observed in histopathological section of liver in control group mice in 10 x and 40 x magnifications respectively. AECH treated mice at a dose of 2000 mg/kg showed normal lobular architecture, central veins and sinusoids. No toxic signs like inflammation, fatty change or fibrosis were observed ( Figure 7).

Spleen
Histological spleen section showed red pulp congestion in control group mice and in AECH treated mice (2000 mg/kg). Variation in white pulp, pencillar artery and the red pulp was not observed ( Figure 8).

Kidney
Normal cortex, medulla and normal glomeruli were observed from the histological sections of kidney from control and AECH treated mice (2000 mg/kg). Section also showed normal interstitium and inflammation or necrosis was not observed (Figure 9).

Sub-acute toxicity study in rats Heart
Histological sections of the heart of control group of rats and AECH treated male and female rats at a dose of 200 and 400 mg/kg showed normal myocytes (Figure 10 and 11).                  Lungs Normal alveoli and normal bronchioles were observed in control group of rats and AECH treated male and female group of rats at a dose of 200 and 400 mg/kg (Figure 12 and 13).

Liver
Histopathological section of liver in control group and AECH treated to male and female groups at a dose of 200 and 400 mg/kg showed normal lobular architecture. The portal tracts, hepatocytes, central veins, sinusoids are found to be normal. No evidence of toxic signs observed as there is no inflammation, fatty change or fibrosis (Figure 14 and 15).

Stomach
Section from the stomach of control group and AECH treated to male and female group of rats at a dose of 200 and 400 mg/kg showed normal mucosa, muscle layer and stomach glands (Figure 16 and 17).

Spleen
Section from the spleen of control group and AECH treated to male and female groups at a dose of 200 and 400 mg/kg showed normal spleen Indicates normal; − Indicates no effect.
Indicates normal; − Indicates no effect.   with red pulp and germinal center formation. No abnormality was observed in histological sections of spleen (Figure 18 and 19).

Kidney
Sections from kidney of control group and AECH treated to male and female groups at a dose of 200 and 400 mg/kg showed normal cortex and medulla. The cortex showed normal glomeruli. The interstitium and distal convoluted tubules are found to be normal. No inflammation or tubular necrosis was observed (Figure 20 and 21).

Testes
Sections from kidney of control group and AECH treated to male groups at a dose of 200 and 400 mg/kg showed normal testes and spermatozoa ( Figure 22).

Ovary
Sections from kidney of control group and AECH treated to female groups at a dose of 200 and 400 mg/kg showed normal ovarian follicles ( Figure 23).

DISCUSSION
Safety of aqueous extract of whole plant of C. heteroclita by determining its potential toxicity after acute and sub-acute administration in mice and rats as per OECD guidelines 423 and 407 respectively as there were no earlier reports on the safety assessment of aqueous extract of C. heteroclita.
In acute toxicity test, mortality was not observed in mice with the maximum dose of 2000 mg/kg throughout the 14 days of study. In sub-acute toxicity test also no mortality was observed in male and female rats with the maximum dose of 400 mg/kg throughout 28 days of study. Significant changes were not found in breathing, sense of touch/sound, central nervous systems, behaviour pattern and locomotor activity. Convulsion, tremor, excessive salivation, diarrhea, sedation and edema were not observed. Body weight and internal organ weight changes is sensitive and indicative marker for the first sign of toxicity when exposed to toxic substances. 7 All animals were found to be active with increase in body weight both in acute and sub-acute toxicity study. Normal food and water consumption of animals clearly support the gain in body of animals throughout the study period. Hematopoietic system is the most sensitive target for toxic substances and it is the important index of physiological and pathological status 8,9 and hence hematological investigation was carried out. No abnormality was observed in hematopoietic function indices for AECH treated groups compared with control groups indicating the extract is safe. Lipid profile assessment was performed for the animals treated with AECH to verify any changes observed in serum levels of triglycerides, cholesterol and VLDL. 10 No significant variation in cholesterol, triglycerides and VLDL levels in AECH treated groups when compared with control groups. SGOT, SGPT, ALP and bilirubin levels of the experimental animals were monitored as they are the specific markers for liver damage or injury. 11 Data provided as mean ± SEM (n=6); a p>0.05 treated groups vs control.  Data provided as mean ± SEM (n=6); a p>0.05 treated groups vs control.   Data provided as mean ± SEM (n=6); a p>0.05 treated groups vs control. No significant changes in SGOT, SGPT, ALP and bilirubin level was observed in all the treated group animals. Thus animals treated with AECH were found to be non-hepatotoxic.
To assess whether treatment of AECH to rats causes any damage to kidney, kidney function test was performed. GLDH-UV Kinetic method, Jaffe method and Uricase POD method was adopted to evaluate the important markers of renal dysfunction viz. urea, uric acid and creatinine levels respectively. 12 AECH was found to be safe and nontoxic to kidney as there is no significant change observed in creatinine, urea and uric acid levels of AECH treated group of animals both in acute and sub-acute toxicity when compared with control group animals. When compared with control groups, no variation in relative organ weight was observed for AECH treated groups both in acute and sub-acute toxicity study. The above mentioned biochemical investigations were in correlation with the histopathological studies. Section of the heart of the control and AECH treated animals showed no abnormality as shown in Figures 10 and 11. Alveoli and bronchioles were found to be normal both in control and treated group confirming that AECH did not cause any toxicity to lungs of the animals. Inflammation or cirrhosis or necrosis was not observed in the histological study of liver in mice and rats tested with AECH at a dose of 400 mg/kg. Normal cortex, medulla and normal glomeruli were observed indicating that AECH did not cause any damage to kidney. Cross section of stomach under low and high power magnification showed normal mucosal surface, normal stomach glands and normal propria indicated that AECH not caused any toxicity to rats. Histological sections of testes clearly implicated that no testicular toxicity was observed to the treated male group of rats for 28 days in sub-acute study. Observation of normal cervix, mucosa and follicles from histological sections of ovary confirmed that AECH was non toxic to ovary of female rats.

CONCLUSION
Based on our results, we conclude that AECH were found to be safe up to a dose of 2000 mg/kg. Hematological, biochemical and histopathological investigations clearly demonstrates that single oral administration upto 2000 mg/kg in acute toxicity study and daily oral administration of the AECH for 28 days upto 400 mg/kg in sub-acute toxicity study caused no significant adverse changes in the organs like heart, lungs, liver, spleen and kidney. Abnormalities were also not observed in the organs stomach, testes and ovary of the animals tested at a dose of 200 and 400 mg/kg respectively. The study provided significant data on the sub-acute toxicity profile of AECH which may be valuable in the clinical study of medicinal herb C. heteroclita (L) Gilg. The aqueous extract of C. heteroclita (L) Gilg thus may be used as one of the ingredient in traditional formulation or any other pharmaceutical formulations. The study on the interaction of this plant with other herbs in multi herbal formulation may be useful for assessment of efficacy of the formulations.