Abstract: In the present study an attempt was made to study the synergetic effect of squalene and simvastatin on fecal cholesterol excretion. The rats were fed with 2% squalene and/or 20 mg simvastatin for 45 days. At the end of the experimental period the feces were collected and analyzed for total lipid and total cholesterol content. It was found that the dietary supplementation of squalene at 2% level significantly (p<0.001) increased the fecal cholesterol excretion in experimental rats. The combination of squalene and simvastatin was found to be more efficient than squalene alone. Thus the results of the present study indicate that a combination of squalene and simvastatin can be efficient in cholesterol lowering than the drugs alone. It suggests that squalene a prime HMG CoA reductase inhibitor can also be used as a prudent dietary addition for individuals using similar cholesterol lowering drugs.
INTRODUCTION
Squalene, a key intermediate in cholesterol biosynthesis, is present in shark liver oil in higher quantities (Thankappan, 2003). It also present at lower concentration in olive oil (0.2-0.7%) and a variety of other food stuffs (Ryan et al., 2007). It is the principal hydrocarbon of human surface lipids amounting up to 11% of total surface fat, where it protects the skin from harmful Ultra Violet (UV) radiation (Ohsawa et al., 1984). It is used as an effective chemopreventive agent against a variety of cancers, skin disorders and liver diseases (Gregory and Kelly, 1999). Recently, the preventive effect of squalene on myocardial infarction is established by Farvin et al. (2004).
Squalene stimulates acyl-coenzyme A cholesterol acyltransferase (ACAT, EC 2.3.1.26) activity but at the same time it is a feed back inhibitor of the enzyme hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) which has been the target of a class of cholesterol-lowering drugs (Strandberg et al., 1989). Although, oral administration of squalene appears to decrease hepatic and serum levels of sterols consistently, its impact on cholesterol metabolism is less clear. Qureshi et al. (1996) have reported that squalene administration to one-day-old chicks lowers the serum cholesterol levels. Reports of Miettinen and Vanhenen (1994) indicated that dietary supplementation of squalene for 9 weeks increases the serum total, VLDL, IDL and LDL cholesterol concentrations. In human, supplementation with squalene (900 mg day-1 for 7-30 days) is found to raise serum squalene levels (17-fold) without any significant changes in triglycerides and cholesterol contents (Strandberg et al., 1990). The compensatory mechanisms to prevent the increase of serum cholesterol during moderate long-term squalene consumption are reduced HMG-CoA reductase activity and increased fecal elimination of cholesterol (Standberg et al., 1990). Earlier an attempt has been made by Chan et al. (1996) to compare pravastatin (10 mg), a hyperlipidemic drug and squalene (860 mg) either alone or in combination for the treatment of hypercholesterolemia. They found that pravastatin is more effective than squalene in reducing total cholesterol, LDL cholesterol and triglyceride and in increasing levels of HDL cholesterol in plasma. But the combination of both pravastatin and squalene is more effective than pravastatin or squalene alone. However, there is no report on the effect of statins, on the fecal cholesterol excretion. So, a study has been undertaken to evaluate the comparative effect of squalene and simvastatin, a known cholesterol-lowering drug in the fecal excretion of cholesterol.
MATERIALS AND METHODS
Drugs and Chemicals
Simvastatin was obtained from Ranbaxy laboratories India Ltd., Gurgaon,
India. Squalene (Specific gravity: 0.853; Refractive index: 1.493; Saponification
value: 30; Iodine value: 344; Boiling point: 240-245°C) was prepared from the
shark liver oil of Centrophorus sp., caught in the Andaman waters (Farvin
et al., 2004). All the other chemicals used were of analytical grade.
Animals
Male Wistar strain albino rats, weighing 120-150 g were selected for the
study. The animals were housed individually in polypropylene cages under hygienic
and standard environmental conditions (28±2°C, humidity 60-70%, 12
h light/dark cycle). The animals were fed with a standard diet (M/s Sai Feeds,
Bangalore, India) and water ad libitum. The experiment was carried out
as per the guidelines of Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), New Delhi, India and approved by the Institutional
Animal Ethics Committee (IAEC).
Experimental Protocol
Seven days after acclimatization, the animals were divided into four groups
of 6 rats each. Group I animals were fed on standard diet with added coconut
oil at 2% level for 45 days, group II animals were fed on standard diet with
added squalene at 2% level for 45 days, group III animals were fed on standard
diet with added simvastatin at 20 mg for 45 days and group IV animals were fed
on standard diet with added 2% squalene and 20 mg simvastatin for the same period.
Feces were collected in last 10 days of the experimental period. The feces were
dried and powdered.
Biochemical Analysis
The total lipid was extracted from the feces samples by using the method
of Folch et al. (1957). In brief: 5 g of the
powdered feces sample was subjected to lipid extraction twice by using 25 volume
of chloroform-methanol mixture (2:1). The lipid extracts were transferred to
a separating funnel and added 0.2 volume of water and left overnight. Next day
the lipid extracts along with the chloroform were drained through filter paper
containing anhydrous sodium sulphate and was collected in round bottom flask.
The chloroform was evaporated off by using a flash evaporator. The lipid in
the round bottom flask was made up to 10 mL by using chloroform. From this 1.0
mL was taken into a pre-weighed vial and allowed to dry in warm temperature
to constant weight and total lipid content were calculated from the difference
in weight.
Cholesterol content was estimated by the method of Parekh and Jung (1970). In brief: 0.1 mL of the lipid sample was taken into a 25 mL glass stoppered tube and evaporated off the chloroform. Mixed well with 5 mL of freshly prepared alcoholic KOH solution and incubated in a water bath at 37°C for 55 min. After cooling to room temperature, 10 mL of petroleum ether was added and inverted the tubes to mix the contents. To this 5.0 mL of distilled water was added and the tubes were shaken vigorously for 1 min. An aliquot (0.5-2 mL) of the supernatant was taken into test tubes and petroleum ether was evaporated under nitrogen. To each of the samples 3.0 mL of glacial acetic acid and 0.1 mL distilled water was added and mixed thoroughly. To this added 2 mL of the FeCl3-H2SO4 reagent (2.0 mL of 10% FeCl3 in acetic acid was diluted to 200 mL with concentrated H2SO4) through the sides of the test tubes to form a brown ring at the interface. Bottom of the tubes were tapped well to effect mixing and a light colour appeared which changed to an immense purple colour, was measured in a Shimadzu-UV spectrophotometer at 560 nm. A blank without sample and a standard with different concentration of cholesterol was also run in the same manner to obtain a calibration curve. The cholesterol content was expressed as mg g-1 of sample.
Statistical Analysis
Mean values of each parameter tested were calculated and the results were
expressed as mean±SD of 6 animals. One way Analysis of Variance (ANOVA)
was carried out and the statistical comparisons among the groups were performed
with Tukey’s test using a statistical package program (SPSS 10.0 for windows).
A p-value<0.05 was considered as statistically significant.
RESULTS AND DISCUSSION
The present study examined the combined effect of both dietary squalene and cholesterol lowering drug simvastatin on the fecal excretion of cholesterol. Figure 1a and b shows the total lipids and cholesterol content in the feces of the normal and experimental groups of rats. After 45 days of feeding, significant (p<0.001) elevation was observed in the fecal excretion of lipids and cholesterol in Group II squalene administered rats compared to Group I control rats. Squalene feeding has been reported to increase fecal excretion of cholesterol, its non-polar derivatives and bile acids (Tilvis and Miettinen, 1983). Present study also strongly suggests that a considerable amount of the administered squalene is absorbed from the intestine and a part of it was metabolized to bile acids via cholesterol and excreted into feces. There was no significant (p<0.001) change in the excretion of lipids and cholesterol in Group III simvastatin treated animals compared to group I control rats (Fig. 1a, b). This indicates that simvastatin, lower serum cholesterol in a mechanism other than cholesterol excretion. Squalene was much potent than simvastatin in enhancing the excretion of cholesterol. One of the reasons for the antilipidemic property of the squalene might be due to the increased elimination of the lipids and cholesterol through feces. Although, cholesterol synthesis increased, fecal elimination was also up regulated resulting in no significant difference on serum cholesterol concentrations (Strandberg et al., 1989).
| Fig. 1: | (a) Level of total lipid content (mg g-1) in the feces of control and experimental groups of rats and (b) level of total cholesterol content (mg g-1) in the feces of control and experimental groups of rats. Group I animals were fed on standard diet with added coconut oil at 2% level for 45 days, group II animals were fed on standard diet with added 2% level squalene for 45 days, group III animals were fed on standard diet with added simvastatin at 20 mg for 45days and group IV animals were fed on standard diet with added squalene (2%) and simvastatin (20 mg) for the same period. Results are Mean±SD for 6 animals. Samples followed by the same letter are not significantly different in Tukey’s test using 0.05 level of significance |
Groups IV rats that obtained the combined treatment of both squalene and simvastain showed a significant increase in the excretion of cholesterol compared to both group I control rats as well as group II and group III treated rats (Fig. 1a, b), which indicates that the combination of both simvastatin and squalene is much more effective in eliminating cholesterol in feces than squalene or simvastatin alone. Even though squalene is a feed back inhibitor of HMG CoA reductase, it stimulates acylcoenzyme A: cholesterol acyltransferase (ACAT) (Standberg et al., 1989). Simvastatin has been reported to alter the activity of intestinal ACAT in rabbits leading to reduced cholesterol absorption (Owens et al., 1991). The combined effect of inhibition of ACAT by simvastatin and higher fecal cholesterol excretion by squalene might be the reason for the increased cholesterol excretion when they are used in combination.
Thus the results of the present study suggest that squalene a prime HMG CoA reductase inhibitor can also be used as a prudent dietary addition for individuals using similar cholesterol lowering drugs. However, further studies are needed for the precise evaluation of dietary squalene on cholesterol metabolism in humans as the pathway of hepatic bile acid synthesis in rats and mice are different from humans.
ACKNOWLEDGMENT
The financial aid from the Indian Council of Agricultural Research is greatly acknowledged.