The divergence between the effects related with antiestrogenic activity, such as reducing the incidence of breast cancer and the effects related with estrogenic action demands an explanation. A weak bond to a estrogen receptors and a bond with approximately 80% of estradiol's affinity to the b receptors have been shown (3). This could account for the dual estrogenic-antiestrogenic action, which could be defined as a SERM-type (selective estrogen receptor modulator) activity.

Acetylcholine produces vasorelaxation through NO (4). However, if the endothelium is disrupted, it causes vasoconstriction (5). Acetylcholine's vasodilative action is therefore an indicator of the vascular endothelium's functional integrity and this endothelium is disrupted during menopause (6) and atherosclerosis (5).

In light of this data, we decided to perform two experiments to observe the effects of a commercial extract, Phytosoya^® (marketed by Arkochim), on cardiovascular and bone parameters. This extract contains genistein
1-3%, daidzein 4-6%, glycitein 2-4%, with a total iso-flavone content of 10%. It is an alcoholic extract from the hypocotyls of Glycine max LL (see product information sheet).

MATERIALS AND METHODS

Two experimental models were studied. The first consisted of female rats castrated when they were 2 months old and then kept on a low-calorie diet containing 0.15% Ca. The animals were divided into 4 study groups: A: diet control; B: diet plus 62 mg/kg/day of Phytosoya; C: diet plus 312 mg/kg/day of Phytosoya;
D: diet plus 200 mcg/week of estradiol valerianate; and E: control fed with standard diet.

After 8 weeks, the animals were killed by decapitation. The following were removed: blood, uterus, liver, heart, tibias and right femur and the third and fourth cervical vertebrae. A densitometry study was performed using the Norland XR-26 densitometer.

In the other experimental model, a total of 26 albino New Zealand rabbits weighing 2.5 kg were used. The rabbits were ovariectomized under ketamine and Xylazine anesthesia, leaving intact both uterine horns.

The rabbits were fed on a high-cholesterol diet containing 1% cholesterol and 2% saturated fat for 10 weeks. The groups were: A: diet only; B: diet plus estrogen (200 mg of estradiol valerianate/week); C: diet plus Phytosoya 62.5 mg/kg/day; and D: control group with standard diet. Two animals were killed each day by Dolethal injection. Blood was removed to assay triglyceride, LDL and HDL cholesterol. The aorta was removed to perform a vascular reactivity study on the aorta rings, analyzing the vasodilative response to acetylcholine, and the vasoconstrictive response to phenylephrine, angiotensin, and acetylcholine with l-NAME. A histological study was also performed, including analysis of atheromatous plaque.

RESULTS AND DISCUSSION

In ovariectomized rats, a low-calorie diet gives rise to a decrease in bone mass, as shown by reduced bone mineral density (BMD) measured by densitometry. Both estrogen and Phytosoya therapy significantly increased the BMD, with the largest increases being found in the estrogen-treated rats. However, these animals also show a decrease in the length of the tibia, caused by estrogen's effect on growing cartilage. However, this effect is not detected in the rats treated with Phytosoya. It can therefore be stated that Phytosoya has a beneficial effect in preventing osteoporosis induced by a low-calorie diet.

In the high cholesterol diet model, the effects are very clear. The cholesterol levels found in the animals receiving this diet were: 2100 ± 93 mg/100 ml compared with 64 ± 19 mg/100 ml in the rabbits treated with the standard diet. Neither estrogen nor Phytosoya was able to induce significant changes in the plasma lipid levels. The vascular effect was very visible. The peak vasodilatation in normal conditions in the control rabbits was equivalent to 86% of the precontraction to epinephrine. However, in the hypercholesterolemic rabbits, this peak vasodilatation was reduced to 28%. Estrogen therapy restored vasodilatation to 82% and also induced a marked weight increase of the uterus (1.4 g vs. 0.9 g). In the rabbits treated with Phytosoya, the vasodilatation function attained 61% with no effects on the weight of the uterus. In the rings pretreated with l-NAME, which blocks NO synthesis and in which acetylcholine therapy induces contraction instead of vasorelaxation, the contraction was stronger in the hypercholesterolemic rabbits (20 ± 4.5%) compared with the controls (8.6 ± 4%). Treatment with estrogen (13 ± 2%) and Phytosoya (15 ± 6%) also reduced the contraction.

However, there were no visible effects on the response to phenylephrine or angiotensin in either the diet or treatment groups.

In regards to the progression of atheromatous plaque in the aortic arch, 100% of the animals in the high-cholesterol group had plaque covering more than 50% of the vessel surface, while only 16% of the estrogen group and 71% of the isoflavone group had plaque. The reduction was significant in both cases. In the animals fed with a standard diet, 33% had surface plaque.

Our data confirm Phytosoya's beneficial effect on endothelium-dependent vascular reactivity. In our case, we assume that the model is so aggressive that it does not allow any decreases in plasma lipid values. However, endothelial protection exists even in the presence of very high lipid concentrations. This is also shown by the decrease in atheromatous plaque. All of this data confirms Phytosoya's beneficial effect on vascular function.

REFERENCES

1. Adlercreutz, H. Phytoestroges. State of the Art.Environ, Fox and P. Harmacol 1999, 7: 201-7.

2. Mazur, W. Adlercreutz, H. Overview of naturally occurring endocrine-active substances in the human diet in relation to human health. Nutrition 2000, 6: 654-8.

3. Kuiper, G.G., Lemmen, J.G., Carlsson, B. et al. Interaction of estrogenic chemicals and phytoestrogens with estroge receptor b. Endocrinology 1998, 139: 4252-63.

4. Hodgson, J.M., Marshall, J.J. Direct vasoconstriction and endothelium dependent vasodilation: Mechanisms of acetylcholine effects on coronary flow and arterial diameter in patients with nonstenotic coronary arteries. Circulation 1989,79: 1043-51.

5. Gordon, J.B., Ganz, P., Nabel, E.G. Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise. J Clin Invest 1989, 83: 1946-5.

6. Reis, S.E., Gloth, S.T., Blumenthal, R.S. et al. Ethynyl estradiol acutely attenuates abnormal vasomotor responses to acetylcholine in postmenopausal women. Circulation 1994, 89: 52-60.

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Methods and Findings in Experimental and Clinical Pharmacology Vol. 24, Suppl. A, 2002, pp. 53-54
ISSN 0379-0355 Copyright 2002 Prous Science, S.A. CCC: 0379-0355/2002 http://www.prous.com