Toxicological Studies of Two Compounds Isolated from Loranthus globosus Roxb

MATERIALS AND METHODS

The experimental works were done in the department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh after the collection of plant materials.

Collection of experimental rats: For the purpose of subacute toxicity studies, 12 Long Evan’s male rats of about five weeks old weighing 120-140 g were collected from the Animal Branch of International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B).

Maintenance of the rats: The rats were kept in properly numbered iron cages individually in a hygienic animal house with an optimum temperature (25-30°C) and were given standard laboratory diet (Hawk et al., 1954). The animals were maintained in this way for 15 days before drug administration and continued up to the end of the experiment.

Grouping of the rats: Individual weight of the rats was taken and they were grouped into three. The rats of group B (4 rats, average weight 56 g) and C (4 rats, average weight 58 g) were used for experiment while those of group A were used as control (4 rats, average weight 51 g).

Administration of the sample: The compounds 1 and 2 were dissolved separately in distilled water using tween-20 as co-solvent, so that 0.3 mL contained 300 μg of the compound. The rats in group A, B and C were injected intraperitoneally with vehicle (300 μL isotonic solution), compound 1 and 2, respectively at a dose of 300 μg/day/rat for 14 consecutive days. On the 15th day blood was collected from external jugular vein under mild ether anaesthesia for the estimation of haematological and biochemical parameters. Then all the rats were sacrificed and liver, kidney, heart and lung were removed for histopathological study.

Gross general observation: During the whole experiment period their behaviour, CNS excitation and depression, reflexes, muscular weakness, salivation, diarrhoea and food intake were observed. The body weight of each rat of groups A, B and C were measured before drug administration, after completion of the treatment and prior to sacrificing the rats.

Investigation of haematological profiles: The haematological parameters like Total Count (TC) of RBC and WBC, Differential Count (DC) of WBC, platelet count and haemoglobin percentage were performed just before drug intake and also on 14th day pf drug administration. For TC, DC and platelet count blood was drawn from the tail veins of the experimental and the control rats of either groups and blood smears were made on glass slides followed by staining with Leisman reagent. Blood was also drawn from each rat with the help of capillary tube for estimating the haemoglobin percentage by Van Kampen-Ziftra’s method.

Biochemical parameters of blood: To determine biochemical parameters such as SGOT (Serum Glutamate Oxaloacetate Transaminase), SGPT (Serum Glutamate Pyruvate Transaminase), SALP (Serum Alkaline Phosphatase), serum bilirubin, creatinine and urea blood samples were collected from each rat of either group from their throat vein while they were sacrificed after 14 days of drug administration. The samples were analysed using the usual procedures reported by King and Armstrong (1934), Reitman and Frankel (1957), Fawcett and Scott (1960) and Coulombe and Favreau (1963).

Histopathological investigation: For histopathological investigation, the liver, kidney, lung and heart of all experimental and control rats were isolated after sacrificing them at the end of 14 days of drug administration. These tissues were separately sliced in pieces, fixed in 10% formaline for 3 days, processed, stained, mounted on glass slides and observed under power microscope.

RESULTS AND DISCUSSION

Table 1 showed that the rats of group A, B and C being treated with vehicle, compound 1 and 2, respectively exhibited no signs of tremor, convulsions and reflex abnormalities. The body weights of all the rats in each group were increased after treatment. Moreover, no muscular numbness of the hind and fore legs, salivation or diarrhoea was observed. The food intake per day was also found normal. So, from the results, it is decided that the drug has no effect on normal growth.

Table 2-4 showed haematological profiles that were studied on experimental rats in compared to control rats and 14 days of treatment. Each time the value of the parameters in each rat was changed slightly. However, the parameters remained within normal range.

Table 1:	Effect of the compounds 1 and 2 on body weight of rats
tc indicates calculated value; ts indicates t-value at 5% level of significance; M1 and M2, sample mean value; SD1 and SD2, standard deviation; n, number of rats; NS, not significant; +, increase; -, decrease

Table 2:	Haematological profiles of group A (rat treated with vehicle)

Table 3:	Haematological profile of group B (rat treated with compound 1)

Table 4:	Haematological profile of group-C (Rat treated with Compound 2)

Table 5:	Effect of compound 1 on biochemical parameters of rat’s blood after i.p. administration of 300 μg/rat/day for 14 consecutive days
NS’ indicates not significance

Table 6:	Effect of Compound 2 on biochemical parameters of rat’s blood after i.p. administration of 300 μg/rat/day for 14 consecutive days
NS = Not Significance

Table 5 and 6 showed biochemical parameters on control group and treatment groups of rats. The parameters remained within the normal range. This indicates that the compounds 3,4-dimethoxycinnamyl alcohol (1) and 3,4,5-trimethoxycinnamyl alcohol (2) have no adverse effect on liver and kidney function.

After the 14th day of drug treatment, the animals of both control and experimental groups were sacrificed and the organs such as liver, kidney, heart and lung were isolated and histopathological experiments were done. No abnormality was observed between the control and the drug treated rats when the tissue slides were examined under microscope. This indicates that both compounds have no effect on cellular structure i.e., the compound does not cause degeneration of cells of these organs (Table 7).

As a part of our continuous research for novel plant metabolite, we isolated the active metabolites 3,4-dimethoxycinnamyl alcohol and 3,4,5-trimethoxycinnamyl alcohol. The present study is the continuation of this toxicological study.

Table 7:	Effect of compounds 1 and 2 on histopathology of rat’s kidney, heart, liver and lung tissue after i.p., administration of 300 μL/rat/day for 14 consecutive days
NAD = No abnormality detected

The results of present study demonstrate that the compounds possess no adverse effect on Long Evan’s rats at a dose of 300 μg/rat/day. Thus the findings of this investigation and previous investigation would give valuable support to make clinical trial of the isolated compounds to get a more potent bioactive agent.

ACKNOWLEDGMENT

The authors are pleased to acknowledge Dr. Anwar Habib, Assistant Professor, Department of Pharmacology, Rajshahi Medical College, Rajshahi, Bangladesh for his cooperation and help in studying subacute toxicity study of the compounds.