Effect of ascofuranone on serum lipids of rats fed a cholesterol rich diet.

Ascofuranone, a fungal metabolite, significantly reduced serum lipid levels, when orally administered to male Wistar rats fed a cholesterol rich diet. The treatment also resulted in a marked reduction of hepatic and cardiac cholesterol contents without affecting the body weight gain. The serum albumin/globulin ratio increased significantly in the treated rats. This increase is presumably due to the decrease of beta-lipoprotein. The mode of action differentiates from clofibrate in so far as the former effectively prevents hepatic and cardiac cholesterol deposits in the cholesterol feeding rats.

Abnormal lipid metabolism plays a major role in the development of atherosclerosis and coronary heart disease in man (1-3). Ascofuranone (Af, Fig 1), a metabolite of a plant pathogenic fungus, Ascochyta r•iciae Libert, was isolated in our screening for hy polipidemic agents (4)(5)(6). Oral administration of Af resulted in significant reduction of serum lipid levels in Wistar strain rats fed a normal diet. The purpose of the present study was to establish the hypolipidemic property of Af in the rat on an atherogenic regimen.

MATERIALS AND METHODS
Thirty male Wistar rats, weighing 260 30 g, were divided equally into three groups.
The rats were housed in an air-conditioned room at 24'C and 40°,;, relative humidity under 12 hr photocycle. A hyperlipidemia inducing diet had been prepared according to Fuji wara et al. (7), but it was hardly touched by the rats so that commercial diet was admixed, as shown in 'l able I . The diet and water were given ad libiiwn. Af (20 mg/rat/day) and a positive control agent, clofibrate (30 mg/rat/day) were orally given as gum arabic sus pensions once a day for 10 consecutive days. The treated groups as well as the untreated T HOSOKA HA ET AL.
control were sacrificed under chloroform 6 hr after the final administration, since for each agent the lowest peak of serum lipid levels is attained 6-10 hr after administration. The blood was obtained by a cardiac puncture using a plastic syringe and after centrifugation in a plastic tube, serum lipids were estimated as reported in a previous paper (6). The organs were carefully removed, weighed and homogenized with phosphate buffer saline (pH 7.0) followed by extraction with a mixture of chloroform-methanol (2: 1) (8). Total cholesterol was estimated by Zurkowski's method (9) with slight modification. Tissue phospholipid, triglyceride and free fatty acid were determined as reported previously (6).

RESULTS
The positive control agent, clofibrate, can be regarded as an effective and safe drug for lowering elevated serum lipid in humans. It is remarkably free from side effects, since slight gastrointestinal disorders and transient rise of serum GOT and GPT have rarely been reported as side effects (10). Thorp and Waring (11) reported that clofibrate lowered serum In the present study, no interference with growth of the clofibrate treated rats occurred at this dose level. Af treated rats also showed the same body weight gain as the untreated control and no sign of abnormality was noted at the time of post mortem examination ( Table 2).
As shown in Table 2, male Wistar strain rats are resistant to an atherogenic regimen, since elevation of the serum lipid levels was not so high as expected even in the untreated control. The positive control agent failed to affect the serum cholesterol level (s-TC), although the serum triglyceride (s-TG) and free fatty acid (s-FFA) significantly decreased.
Grafnetter (12) observed that clofibrate was ineffective in lowering serum cholesterol in rats fed a cholesterol rich diet. In contrast with the positive control agent, Af significantly reduced s-TC together with a considerable reduction in s-TG and s-FFA.
Af slightly reduced serum total protein concentration but had little effect on serum albumin and transaminase concentrations. Serum albumin/globulin ratio rose significantly in both treated groups. This rise is attributable to the decrease of f3-lipoprotein included in globulin fraction. Whether or not Af affects a certain specific lipoprotein fraction has to be clarified.
As pointed out in a previous paper (6), clofibrate induced enlargement of liver; liver/ body weight ratio rose from 4.55,, of the untreated control to 5.47',/' of the clofibrate group in this study (Table 3). Af treatment also increased the ratio, although the change was smaller. Heart weights were unaffected by either agent. Details regarding the mechanism of action will be reported elsewhere.
Although elevation of serum lipid levels was not so high as expected, it has been con firmed in the present study that Af alters lipid metabolism of the rat fed on an atherogenic regimen without affecting body weight gain. These alterations are represented by hypo lipidemic activity and inhibition of cholesterol deposit in liver and heart. In this respect, it is very likely that the mode of action on lipid metabolism differs from that of clofibrate.
It has yet to be proven that inhibition of lipid deposit in the heart and liver decrease the rate of such deposits in the arterial wall thus preventing both occurrence and progression of atherosclerosis.
In addition to the effect on lipid metabolism, At' in small doses, shows potent hypotensive activity in Okamoto strain spontaneously hypertensive rats (personal communication). Therefore, Af is a potential candidate for a clinical agent against atherosclerosis in view of its activity and safety. Experiments are now in pro gress to determine whether or not Af therapy alteis lipid deposits in the aorta intima thereby protecting experimental animals from atherosclerosis.