Rat in vivo experiments to demonstrate the effects of Cleistanthin B

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

INTRODUCTION:
Poisoning is one of the preferred modes of committing suicide by both males and females in India. The mean age for committing suicide is around 40 years in males and 34 years in females (Tanuj Kanchan, Ritesh G. Menezes). Plant poisons are one of the common poisons, because they are easily available. One such plant is Cleistanthus collinus, which is a poisonous shrub belonging to the family Euphorbiaceae. It is found in many parts of India. It is popularly known as Oduvanthalai in Tamil.
Mortality associated with this poisoning is 28% (Kurien T et al., 1987). The mechanism of action of this poison is unknown and the treatment is symptomatic. Case reports published showed hypokalemia, metabolic acidosis, alkaline urine, respiratory failure, hypotension and cardiac arrhythmias as the major clinical findings in patients who had consumed this poison. (Eswarappa et al., 2003). Cleistanthus collinus injected intrapetineally in rats has shown to induce type I DRTA and type II respiratory failure in rats (Maneksh et al., 2010).
Cleitanthus collinus has many fractions of which the toxic ones are Cleistanthins A and B, Diphyllin and Collinusin. Cleistanthins A and B are the glycosides of Diphyllin and they have been shown to exhibit cytotoxicity on several cancer cell lines (Paulo et al., 2007). It was shown that Cleistanthin A causes DNA strand breaks induces apoptosis in cultured cells, whereas Cleistanthin B causes G1 arrest and induces apoptosis in mammalian cells (Paulo et al., 2007).
This study deals with the in vivo rat experiments with Cleistanthin B, one of the toxic fractions of Cleistanthus collinus. The aim of the study was to find out the molecular mechanism of action of this fraction. This study was started with the hypothesis that Type I DRTA and type II respiratory failure are two isolated events caused by two different fractions. Hence we started working on the isolated fractions of Cleistanthus collinus. Previous studies in our department on cleistanthin A, another toxin fraction of Cleistanthus collinus, showed respiratory acidosis and sudden respiratory arrest, DRTA was not evident in those animals (Anita M.D Thesis 2009).
AIM OF THE STUDY:
The aim of the experiment was to study the clinical picture in rats following the administration of Cleistanthin B enriched fraction.
OBJECTIVES:
To study the mechanism of action of Cleistanthin B the toxin enriched fraction was injected intraperitoneally into wistar rats and the following parameters were studied.
1. Arterial blood gases,
2. Respiration and ECG,
3. Blood pressure,
4. Urine Ph.
Two sets of experiments were done with two types of Cleistanthin B enriched fractions. Since the first Cleistanthin B fraction resolved into five fluorescent fractions, a another set of Cleistanthin B, a fairly pure compound, which was obtained using a different extraction
procedure was also studied. The vehicle used for the second set of cleistanthin B experiments was Ethanol.
Here in this book I would refer to the two sets of experiments as:
The first set of Cleistanthin B &
The second set of Cleistanthin B experiments.
MATERIALS AND METHODS:
The study has two arms, the test arm and the control arm. The rats were anaesthetized with ketamine intraperitoneally and the carotid artery was cannulated on one side. In one of the test animals the femoral artery was cannulated. The first blood sample was taken for arterial blood gas measurements. If the initial parameters were within certain ranges as mentioned below in the inclusion criteria, the animals were injected either the Cleistanthin B enriched fraction or the control solution, which was ethanol for the second set of Cleistanthin B experiments and acetone for the first set of Cleistanthin B experiments. Both the toxin and the control solution were injected intraperitoneally. The dosage used for the first set of experiments was 5-7 mg/100gm body weight. The dosage used for the second set of Cleistanthin B experiments was 2-7 mg/100gm body weight.
Blood sample was taken at regular intervals for arterial blood gas measurements. ECG, respiration and BP were monitored continuously with appropriate transducers. Urine samples for pH measurements were taken at frequent intervals in these animals. Dextrose normal saline 0.5 ml was given through the intraperitoneal route after every blood sample was taken or after every hour. The animals were monitored continuously till death or for a period of 7-8 hours after which they were sacrificed.
Inclusion and Exclusion criteria:
All animals did not have good initial blood gas values. The reasons could be respiratory infections in the rats acidosis because of the procedure per se. Hence, only rats with the following initial blood gas values were included in the study. Arterial blood pH : 7.25-7.35, Arterial PCO2 : 35-55mmHg, Arterial PO2: 75-120mmHg
Other animals were excluded from the study.
RESULTS:
Mortality with Cleistanthin B at a dose of 5-7 mg/100gm body weight is 78% in test animals, whereas in control animals it is only 29%. Of the two control animals one of animals died of suspected air embolism, the animal died immediately after the blood was injected back into the carotid. Of the animals which survived in the test group, in one animal during injection of the toxin there was some resistance in the syringe and half of the quantity spilled out, hence the animal did not receive the toxin at a dose of 7 mg/100 gm.
CONCLUSIONS:
Cleistanthin B is toxic to rats at dose of 2-7 mg/100gm body weight. The cause of death is more likely a respiratory event, the cause could be a central mechanism or a neuromuscular paralysis. Considering the more purer fraction it can be said that metabolic acidosis is seen in the rats. Acidosis per se is not the cause of death in these animals, since the control animals have also shown an equal amount of acidosis. The DRTA picture which was seen with the whole aqueous extract is not seen with Cleistanthin B, which is one of the toxic fractions of cleistanthus collinus.