Cholesterol and bile acid balance in Macaca fascicularis. Effects of alfalfa saponins.

We determine the effects of alfalfa top saponins on cholesterol and bile acid balance in eight cynomolgus macaques (Macaca fascicularis). The monkeys ate semipurified food containing cholesterol with or without added saponins. The saponins decreased cholesterolemia without changing the levels of high density lipoprotein-cholesterol; hence, they reduced the total cholesterol/high density lipoprotein-cholesterol ratio. Furthermore, they decreased intestinal absorption of cholesterol, increased fecal excretion of endogenous and exogenous neutral steroids and bile acids, and decreased the percent distribution of fecal deoxycholic and lithocholic acids. The fecal excretion of fat was also slightly increased, but steatorrhea did not occur. We saw no signs of toxicity in the monkeys after 6 or 8 wk of saponin ingestion. The data suggest that alfalfa top saponins may be of use in the treatment of patients with hypercholesterolemia, but long-term studies on possible toxicity are needed before this therapy can be recommended for humans.

Comparative effects of alfalfa saponins and alfalfa fiber on cholesterol absorption in rats.

Intestinal absorption of cholesterol was measured in control rats fed semipurified diets and in rats fed alfalfa meal, in which saponins had been previously extracted, or this extracted material plus alfalfa saponins. A dose of 2 mg radioactive cholesterol was administered intragastrically, and fecal excretion of labeled neutral steroids measured. Absorption of cholesterol was about 76% in control animals, and about 47% in alfalfa-red rats. Extraction of saponins from alfalfa eliminated the cholesterol absorption-lowering effect, while addition of 0.26% alfalfa saponins to the extracted alfalfa restored its activity. The results demonstrate that alfalfa saponins are responsible for the effect of alfalfa meal in reducing cholesterol absorption, and that alfalfa fiber is not involved in this activity.

Effect of alfalfa saponins on intestinal cholesterol absorption in rats.

Five to 20 mg of saponins obtained from alfalfa tops or roots were introduced intragastrically in rats also receiving oral and intravenous ring-labeled cholesterol. The saponins were tested before and after partial acid hydrolysis. Absorption of cholesterol was determined by estimation of fecal sterols and by a dual isotope technique involving assay of plasma radioactivity. Alfalfa top saponins (nonhydrolyzed) reduced absorption of cholesterol. Acid hydrolysis of alfalfa top or root saponins enhanced their ability to inhibit cholesterol absorption.

Alfalfa saponins and alfalfa seeds. Dietary effects in cholesterol-fed rabbits.

Since alfalfa meal prevents hypercholesterolemia and atherosclerosis in rabbits and alfalfa saponins prevent the expected rise in cholesterolemia induced by dietary cholesterol in monkeys, the experiments being reported here were performed to determine whether alfalfa saponins affect atherogenesis in rabbits. In addition, the effects of alfalfa seeds were studied. Cholesterol-feb rabbits were randomly assigned to 3 groups: (a) control animals (N = 18); (b) animals maintained on a diet containing 1.0 to 1.2% alfalfa saponins (N = 18); and (c) animals maintained on a diet containing 40% alfalfa seeds (N = 17). Results after a 4-month observation period demonstrated that alfalfa saponins and alfalfa seeds reduce hypercholesterolemia, aortic sudanophilia, and the concentration of cholesterol in aortic intima-plus-media and in the liver, but do not induce changes in the hematocrit.

Minimizing the saponin content of alfalfa sprouts and leaf protein concentrates.

Biologically active saponins were found to be concentrated in the white and green alfalfa leaf protein fractions at levels higher than in the original alfalfa. Coagulation and washing of the leaf protein at pH 8.5 resulted in a fourfold decrease in saponin compared to the protein coagulated at pH 6.0 and washed at pH 4.5. The press cakes from high and low saponin alfalfa retained 65 and 87% of the saponin. Protein fractions prepared from a low saponin alfalfa contained saponin levels less than 0.07%, compared to a saponin level of 1.33% in leaf protein prepared form a high saponin alfalfa. The saponin contents of three varieties of alfalfa sprouts ranged from 1.55 to 7.27% depending upon the maturity of the sprouts. The saponin content rapidly increased after sprouting and reached a maximum after eight days' growth. Both starch and total sugar decreased with the age of the sprouts while fiber increased from 8 to 18.7%.

Forage plant estrogens.

Forage plant estrogens occur at high levels in certain forage species. Such levels are responsible for reproductive and other effects in animals. Current progress regarding the chemical, metabolic, and biological effects, as well as occurrence of the several naturally occurring forage estrogens, is described. Forage estrogens occur as natural plant responses to foliar and other plant diseases. Their potency in animals is approximately 1/1000 to 1/10,000 that of the principal animal hormones such as estradiol. Coumestans and isoflavones comprise the two principal classes of forage estrogens, with coumestrol and genistein being the most potent and predominant of each class. Only very low levels of the coumestans occurred in alfalfa leaf protein concentrates prepared at alkaline pH.

Microbiological assay for saponin in alfalfa products.

A bioassay is described for determining medicagenin-type saponin in dried alfalfa, leaf protein concentrates, and alfalfa sprouts. Samples are extracted by refluxing 2 1/2 hr with 50% ethanol, ethanol is evaporated, and aliquots of an aqueous solution are added to potato dextrose agar (PDA) and assayed for saponin by using the fungus Trichoderma viride. The growth of the fungus on PDA is compared with a standard saponin, and saponin levels are calculated by means of a slope ratio analysis.