Alfalfa saponins and alfalfa seeds

433

Atherosclerosis, 37 (1980) 433-438 0 Elsevier/North-Holland Scientific

Publishers,

Ltd.

ALFALFA SAPONINS AND ALFALFA SEEDS Dietary Effects in Cholesterol-fed Rabbits * M.R. MALINOW, KOHLER

P. MCLAUGHLIN,

C. STAFFORD,

A.L. LIVINGSTON

and G.O.

Oregon Regional Primate Research Center, Beaverton, OR; University of Oregon Health Sciences Center, Portland, OR; and United States Department of Agriculture, Western Regional Research Center, Berkeley, CA (U.S.A.) (Received 19 February, 1980) (Revised, received 23 June, 1980) (Accepted 25 June, 1980)

Summary 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-fed 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. Key words:

Aortic intima-plus-media -Atherosclerosis

- Hematocrit - Liver

* The work described in this article, Publication No. 1099 of the Oregon Regional Primate Research Center, was supported with grants RR-00163 and HL-16687 from the National Institutes of Health and by funds from the Pacific Power and Light Company. Presented at the XIIIth Hugh Lofland Conference on Arterial Wall Metabolism, Albany, NY, 13-15 May, 1980. To whom CorrespondenCe should be addressed: MB. Malinow, M.D. Oregon Regional Primate Research Center, 505 N.W. 185th Avenue. Beaverton, OR 97006. U.S.A.

434

Introduction Alfalfa meal (sun-cured alfalfa hay) prevents hypercholesterolemia and atherosclerosis in rabbits [l-3]. Moreover, alfalfa meal substituted isocalorically in diets containing butter and cholesterol reduces hypercholesterolemia, normalizes abnormal distribution of plasma lipoproteins, and induces regression of atherosclerosis in Mczcaca fasciculuris [4]. The effects of alfalfa meal may be at least partially mediated through alfalfa saponins [5] since these glycosides decrease intestinal absorption of cholesterol in rats [6] and prevent the expected increase in cholesterolemia induced by dietary cholesterol in monkeys [7,8]. We studied the effects of alfalfa saponins in cholesterol-fed rabbits. In addition, we studied the effects of alfalfa seeds, which we had previously shown reduce cholesterolemia in humans and rats [9] ; in these animals, they also decrease absorption of cholesterol and increase fecal excretion of bile acids [9-111. Materials and Methods Fifty-three male albino New Zealand rabbits (Oryctolagus cunicuhs), weighing around 2 kg each and housed in a temperatureand light-controlled room, were offered food and water ad libitum. The animals were randomly assigned to 3 groups: (a) control (N = 18); (b) diet including alfalfa saponins (N = 18), which were added at the 1% level for 2 months and continued thereafter at the 1.2% level; and (c) diet including alfalfa seed substituted isocalorically at the 40% level (N = 17) (see diet compositions in Table 1). Plasma cholestrol levels

TABLE 1 COMPOSITION

OF DIETS ON WHICH RABBITS WERE MAINTAINED (g/loo

oats Wheat Soya meal MoIass8s corn oil Vitamins (0wP) a Salts (Hegsted IV) Vitamin D-3 (2000 IU/ml) AIfaIfa-top saponin AIfalfa seeds Cholesterol Cholesterol (m$/lOO cal) Protein (% of cal) Fat (96 of cal) CHO (% of cal)

g)

grow a

group b

group c

26 24 40 6 2.5 2 1.4 0.2 ml -

24 24 40 6 2.6 2 1.4 0.2 ml 1.0: 1.2 -

6 40 -

0.1 27 19.9 21.2 52.9

0.1 27 20.0 27.1 52.8

6.5 5 2 1.4 0.2 ml 40 0.1 27 21.7 27.1 51.2

a VaIues/kg of diet: Vitamin A acetate 12.500 U; a-tOcOph8rOl. 0.1 g; menadione. 0.04 g; ascorbic acid, 0.5 8; inositol. 1.0 g: choline chloride. 5.0 8: niacin 0.049 g; riboflavin, 0.01 g; thiamine, 0.01 g; pyridoxine, 0.01 g; calcium pantothenate, 0.03 g; biotin, 0.2 mg; folk acid, 1.0 mg; and vitamin B12, 0.02 mg.

435

were determined after 0, 1,and 2 months on the diets and at the end of the observation period with a modified version of the FeCI, method of Rude1 and Morris [ 121; blood hematocrit values were determined after 1,2, and 4 months on the diets. The animals were weighed monthly; at the end of 4 months, they were killed after Nembutal (Parke Davis and Co., Seattle, WA) had been injected intravenously. The thorax and abdomen were opened, and the organs were inspected for gross pathologic charges. The aorta was removed and split longitudinally into halves. One half was kept frozen for subsequent analysis. The other was fixed in 10% formalin and stained with Sudan IV; the extent of intimal sudanophilia was estimated and graded as follows: grade 1, no sudanophilia; grade 2, less than 10% of the surface involved; grade 3, less than 20%; grade 4, less than 50%; and grade 5, more than 50%. The intima-plus-media of the frozen aortic segment was thawed, separated from the remaining wall, dried, and weighed. It was hydrolyzed in 33% KOH for 1 h in a boiling water bath; after the addition of 2 ml of ethanol, the lipids were extracted into 10 ml of petroleum ether, the ether was washed with 5 ml of water, and the cholesterol content was determined in an aliquot of the organic solvent as indicated above. Similar determinations were performed in liver tissue. Alfalfa saponins were extracted from alfalfa hay by a modified version of the method of Walter et al. [13]; the biological activity was enhanced by partial hydrolysis [6]. For the sake of brevity the partially hydrolyzed alfalfa hay saponins are hereafter called alfalfa saponins. Alfalfa seeds were obtained from the Aloha Feed and Garden Center, Beaverton, OR, and ground with a stone wheel food grinder (Magic Mill, Salt Lake City, UT). The composition of alfalfa seeds is 88.3% dry matter, 4.4% ash, 8.1% crude fiber, 10.6% lipid, 32% nitrogen-free extract, and 33.2% protein (N = 6.25); the caloric value for a monogastric omnivorous mammal is 340 cal/ 100 g [ 141 and the saponin content is 0.3% [ 151. Results (Table 2) An epidemic of acute hemorrhagic enterocolitis (organisms not identified) reduced the number of rabbits to 10, 14, and 9 in groups a, b, and c, respectively. The deaths occurred mostly in the 2nd and 3rd months of observation, and they were distributed similarly in the 3 groups. Treatment with Sulkamytin-S@ (sulfamethazine-neomycin sulfate, Norden Labs, Inc., Lincoln, NE) in the drinking water at the 0.34% level was instituted during month 3. Because poor intake and wasting could influence the results, the affected animals were discarded. The surviving animals had no symptoms of enterocolitis. The following results were thus based on the surviving animals. Initial weights and weight increases throughout the observation period were similar in all groups of animals. Cholesterolemia increased to 438 + 69 mg/dl at the end of the observation period in the control animals, but only to 244 + 45 mg/dl and to 166 f 41 mg/dl in rabbits receiving alfalfa saponins and alfalfa seeds, respectively. However, the levels of cholesterolemia remained above the initial values observed in the animals while they were on cholesterol-free diets. The hematocrits were similar in all groups of rabbits, and only the 4-month values are shown in Table 2. The extent of aortic sudanophilia and the concentrations of cholesterol in

436 TABLE 2 VARIABLES

STUDIED IN MALE NEW ZEALAND

RABBITS

ON DIETARY

REGIMENTS

FOR FOUR

MONTHS (MEAN f SE) * Diets e

No. of animals

Body weight (kg) Initial

Plasma cholesterol

Hematocrit

(me/d0

(%o)

Final Initial

Final

Control

10

2.12 * 0.03

3.66 + 0.23

79 f 6

433 f 69

42 f 0.9

Alfalfa saponins added

14

2.15 f 0.05

3.14 f 0.16

66 f 6

244 _c45 b

40 * 1.0

9

2.14 ? 0.04

3.33 * 0.19

66 f 5

166 f 41 d

39 f 1.0 b

40% alfalfa seeds substituted

* The P (Student’s t-test) values were: b <0.05; e See Table 1.

c cO.02;

and d
the aortic intima-plus-media and in the liver were less in animals alfalfa saponins or alfalfa seeds

receiving

Discussion We have demonstrated that alfalfa saponins and alfalfa seeds reduce hypercholesterolemia, aortic sudanophilia, and the accumulation of cholesterol in aortic intima-plus-media and in the liver of cholesterol-fed rabbits. These observations extend the results of previous studies conducted with alfalfa meal in rabbits [l-3] and monkeys [ 41. Moreover, these studies, as well as those conducted in monkeys [ 41, may be relevant to conditions of moderate cholesterol intake since we have used levels of dietary cholesterol comparable to those ingested by humans in the United States [ 161. In addition, the effects of alfalfa saponins on plasma cholesterol levels in rabbits are similar to those in monkeys [7] and suggest that alfalfa saponins are involved in the hypocholesterolemic effects observed after ingestion of alfalfa seeds [ 111 and alfalfa meal [ 51. We previously showed that partial acid hydrolysis enhances the biological activity of alfalfa saponin [6]. A similar process may reduce the biological activity of other saponins [17]. It remains to be determined how removal of outer sugars of alfalfa saponin molecules affects cholesterol metabolism. Moreover, the mechanism through which alfalfa saponins prevent hypercholesterolemia and organ cholesterol deposition in rabbits and monkeys may be similar to the mechanism through which saponins from other plant sources [18-201 prevent these phenomena in chicken, i.e., the formation of an insoluble saponin : cholesterol addition product in the gastrointestinal tract [21]. This hypothesis, confirmed in vitro [ 221, may explain the decrease in intestinal absorption of cholesterol and the increase in fecal steroids observed in mice [23] and monkeys [ 81 after the intake of alfalfa saponins. However, saponins precipitate micellar cholesterol in vitro in excess of a 1 : 1 stoichiometric relationship 1171, an indication that disruption of cholesterol absorption may also be effected through perturbation of intestinal micelles or through modification of

437

Aorta sudanophilie

grade (l-6)

Cholesterol content Aorta trrlmr) dry

wt

2.9 f 0.38

29 f 82

1.6 f 0.31 c

11 f

1.2 zt 0.15 d

6 f

Liver We/me) wet wt 141 f 19.6

3.9b

80 f 14.9 c

0.3 c

66 i 11.6 d

other parameters of intestinal absorption [ 171. Additionally, alfalfa saponins [ES], as well as other components of alfalfa such as dietary fiber [24], may modify cholesterol,metabolism through increased fecal excretion of bile acids. The effects of alfalfa seeds on plasma cholesterol and on tissue cholesterol deposition seem even more pronounced than those of alfalfa saponins. It Seem8 unlikely that these effects can be attributed solely to the saponin content of the seeds since the saponin concentration in the seed diet is one order of magnitude less than in the saponin diet. The mechanism of these diverse effects is unknown, but the following factors may be involved: (1) heterogeneity in the chemical composition of saponins from hay and seeds with concomitant differences in cholesterol affinity; (2) effects of other components in alfalfa seeds; (3) greater effectiveness of saponins bound to dietary fiber than of extracted saponins. Further research is needed to clarify this mechanism. The hypocholesterolemic effects of alfalfa seed8 in rabbits are similar to those in human8 [9,10]. We did not observe anemia in the animals that ingested alfalfa seed8 or alfalfa saponins, although anemia ha8 been induced in rabbits by repeated, large intravenous doses of a saponin of unspecified origin [ 251 (probably from a source other than alfalfa). Transitory hemolytic anemia and a positive Coombs test result have been observed in a person who ingested 80-160 g of alfalfa seeds per day intermittently for 6 months [ 91. Anemia and occasionally positive Coombs test results have been observed in cynomolgus macaques fed alfalfa seeds, but anemia has not been observed in monkeys ingesting saponins obtained from alfalfa meal (unpublished data). These diverse result8 suggest that the mechanisms responsible for anemia after the ingestion of alfalfa seeds in primates are not operative in rabbits and also that saponins obtained from alfalfa meal are probably not involved in the toxic .effects of alfalfa seeds observed in human8 and monkeys. Although the substance in alfalfa seeds that is responsible for inducing anemia in primate8 remains to be identified, alfalfa saponins seem to hold promise for the reduction of hypercholesterolemia and the deposition of cholesterol in certain organs.

438

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