The effect of pretreatment with plant extract, nicotine and caffeine on sleeping time induced by pentobarbitone in mice
Abstract
Objective: To determine the effects of plant extract, nicotine and caffeine on the activities of the liver metabolizing-enzyme induced by pentobarbitone. Materials and Method: Seven groups of mice were pretreated with high doses of sample extracts (0.4 mg/g body weight sample extract, but nicotine at 0.1 mg/g body weight) and one control group was pretreated with saline. On day 5, pentobarbitone (0.005 ml of 8 mg/ml) was administered and the sleeping time was determined. The test was repeated but at low doses (0.1 mg/g body weight sample extract, but nicotine at 0.05mg/g body weight). Results: At high doses, bitter gourd, 'tempeh', nicotine, caffeine, nicotine+bitter gourd, nicotine+'tempeh' and nicotine+caffeine induced the activities of liver metabolizing enzyme significantly compared to control. At low doses, bitter gourd, nicotine, caffeine, nicotine+bitter gourd, nicotine+'tempeh' and nicotine+caffeine induced the enzyme but 'tempeh' did not. Conclusion: The findings suggest that bitter gourd, nicotine and caffeine act as enzyme inducers, but 'tempeh' only demonstrate this ability at high dose.
Source of support: University of Malaya SuXCeS laboratory
Introduction
Cytochrome P450 is the collective name for a distinct group of protoheme containing proteins that show a Soret absorption band at around 450 nm (448 to 452 nm) in the CO-difference spectrum of dithionite-reduced sample. 450 represent a large group of hemethiolate enzymes that exhibit remarkably diverse activities for the metabolism of numerous endogenous and exogenous chemicals [1]. Some plant extracts such as leaves of Helietta apiculata [2] and grape fruit juices [3] have been documented to have certain effects on the activity of cytochrome P450.
Cytochrome P450 is responsible for the metabolism of a large proportion of drugs [1]. Whenever two or more drugs are administered concurrently, the possibility of drug interaction exist [4]. Drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extrahepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others as inhibitors [5].
In this study, the effect of bitter gourd (Momordica charantia), ‘tempeh', nicotine and caffeine on liver metabolizing enzymes were assessed. Bitter gourd is a well known tropical vegetable in South East Asia. ‘Tempeh' also known as soy cake is made from fermented soy beans and is widely consumed in daily diet in Malaysia and Indonesia. Nicotine is a major substance found in tobacco while caffeine is found in coffee, soft drinks and other beverages. The effects of these chosen sample extracts on the activity of liver metabolizing enzyme cytochrome P450, were determined by measuring the sleeping time induced by pentobarbitone. Pentobarbitone is known to be metabolized by cytochrome P450 [6]. This study also investigated the effects of different doses of these sample extracts in affecting activity of the liver metabolizing enzymes. This was done as the literature has reported that metabolic drug interaction may depend on the magnitude of the change in the concentration of active species (parent drug and/or active metabolites) at the site of pharmacological action and the therapeutic index of the drug. The smaller the difference between toxic and effective concentration, the greater the likelihood that the drug interaction will have serious clinical consequences [7].
Materials and methods
Materials
The study was carried out on mice (2 batches of 7 groups (10 mice per group)). The mice were maintained under standard laboratory conditions of food and water before the start of the experiment. The handling of mice was conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology, which was adopted by the Society of Toxicology in 1989. Bitter gourd (Momordica charantia) and ‘tempeh' were obtained from the local market, nicotine was extracted from tobacco sheets and caffeine was commercially obtained from Sigma-aldrich.
Methods
1. Sample extracts
1.1 Extracting samples
Dried weights of sample extracts were separately extracted with methanol (b.p 64-66 C) for 48 hours using Soxhlet extracting apparatus. The extracts were then concentrated using rotary evaporator under reduced pressure to give crude extract. The crude extracts were then kept in vials and in a cool, dry and dark place.
2. Animals and dietary treatment
2.1. Pretreatment of mice with sample extracts at high dose:
Mice (8 groups of 10 mice per group) were divided into two groups:
Group A (7 groups of 10 mice per group): Each group of mice were orally administered high dose of sample extract ( 0.4 mg/g body weight extract except for nicotine which was at 0.1mg/g body weight; bitter gourd, ‘tempeh', nicotine, caffeine, nicotine + bitter gourd, nicotine + ‘tempeh', nicotine + caffeine ) for 4 consecutive days prior to the sleeping time assessment.
Group B (1 group of 10 mice): The group of mice was orally administered normal saline (0.4 mg/g body weight) for 4 consecutive days prior to the sleeping time assessment.
On the fifth day, pentobarbitone (0.005 ml of 8mg/ml) was injected intraperitoneally into mice from each group. The sleeping time induced by pentobarbitone for mice in each group are listed in Table 1.
2.2 Pretreatment of mice with sample extracts at low doses.
Mice (8 groups of 10 mice per group) were divided into two groups:
Group C (7 groups of 10 mice each): Each group of mice were orally administered low doses of sample extracts (0.1 mg/g body weight of sample extracts and 0.05 mg/g body weight of nicotine; bitter gourd, ‘tempeh', nicotine, caffeine, nicotine + bitter gourd, nicotine + ‘tempeh', nicotine + caffeine) for 4 consecutive days before the sleeping time assessment.
Group D (1 group of 10 mice): The group of mice was orally administered normal saline (0.1 mg/g body weight) for 4 consecutive days prior to the sleeping time assessment.
On the fifth day, pentobarbitone (0.005 ml of 8mg/ml) was injected intraperitoneally into mice from each group. The sleeping time induced by pentorbarbitone for mice in each group are listed in Table 2.
Statistical analysis
Group results are expressed as mean ± SEM. A One Way ANOVA followed by post hoc Bonferroni test was applied for multiple comparisons between different groups. P<0.005 was taken as statistically significant.
Results
At high doses (0.4 mg/g body weight of sample extracts and 0.1 mg/g body weight of nicotine) all sample extracts demonstrated an observable effect on sleeping time. Bitter gourd, ‘tempeh', nicotine, caffeine, nicotine + bitter gourd, nicotine+ ‘tempeh' and nicotine+caffeine reduced the sleeping time induced by pentobarbitone. Post Hoc Bonferroni test showed that these effects were statistically significant (p< 0.05) compared to controls (Table 1). At low doses (0.1 mg/g body weight of sample extracts and 0.05 mg/g body weight of nicotine), all sample extracts reduced sleeping time, but tempeh did not (Table 2).
Table 1: Sleeping time (mins) of mice fed with high dose sample extracts
* [(0.4mg/g body weight of each sample extract) (0.1mg/g body weight of nicotine)]
n=10 mice in each group, df = 7, 72.
*significant at p<0.05 as compared to control
Table 2: Sleeping time (mins) of mice fed with low dose sample extracts
*[(0.1mg mg/g body weight of sample extract) (0.05 mg/g body weight of nicotine)]
n=10 mice in each group, df = 7, 72.
*significant at p<0.05 as compared to control
Discussion
Determining sleeping time induced by pentobarbitone in rodents is a common reported method for evaluating in vivo induction of drug metabolism [6]. From this in vivo study, after pretreatment with various plants, nicotine and caffeine extracts, we demonstrated that at high doses, bitter gourd, ‘tempeh', nicotine and caffeine significantly reduced sleeping time in mice whether used singly or in combination. This reduction in sleeping time suggests inductive effect towards the activity of liver metabolizing-enzyme cytochrome P450. The length of sleeping time induced by pentobarbitone is proportional to the concentration of pentobarbitone in plasma. Thus, short sleeping time indicated enzyme induction. However at low dose, ‘tempeh' per se showed no significant effect on the length of sleeping time induced by pentobarbitone.
The result of the present study showed that at high doses, all the extracts act as liver enzyme inducers. This effect was enhanced when combined with nicotine (nicotine+ bitter gourd, nicotine+ tempeh and nicotine+ caffeine), as evident from the further reduction in sleeping time. Our results are in agreement with the literature. A previous study in rats found that bitter gourd induced rat liver enzymes [8]. Other studies reported that cigarette smoking induced liver enzymes [9, 10]. Caffeine has also been reported to induce liver enzymes [11]. Further studies are however needed to reveal the exact mechanism of action responsible for the enhanced activity of these extracts when combined. This study also demonstrated inductive effect with low dose extracts of bitter gourd, nicotine and caffeine except for ‘tempeh'. Thus far, the literature has reported conflicting results pertaining to the effect of soya on liver enzymes [12, 13]. We recommend further work to study the variable effect of tempeh (soya) on liver metabolizing enzymes at different doses.
Acknowledgements
The author would like to thank University of Malaya, Department of Pharmacology SuXCeS laboratory.