MATERIALS AND METHODS

Samples: To be eligible for this study, Those women years post menopause 5.47±3.4 years who were non-smokers and free from diseases, not on any type of hormonal treatment during the previous 12 months and not currently using lipid-lowering drugs, antidiabetic medications, soybean-derived products, or herbal supplements. Other inclusion criteria were an intact uterus, Follicle-Stimulating Hormone (FSH) levels in blood serum exceeding 25 μL, estradiol levels less than 100 pg m^-1 and presence of hot flashes. Women with a history of uncontrolled hypertension, stroke or transient ischemic attack, cancer diagnosed less than 5 years ago, or previous myocardial infarction were excluded from the study. The study protocol was approved by the Scientific Advisory Committee and Ethical Committee of University. The length of the study was three months. All persons gave informed consent for their participation in the study after reading the protocol of this experiment and receiving information about soymilk consumption.

This study, was carried out on 37 postmenopausal women, age, 52.2±4.6 years were randomly assigned to soymilk consumption (n = 15), (exercise+soymilk) one hour walking per day+soymilk (n = 12) and control (n = 10) which were provided daily for 3 months. Soymilk was provides by Maxsoy, Co, Tehran Iran, which contained, 12.5 g of soy protein with Genistein, 13 mg and Daidzein, 4.13 mg day^-1. The information on demographic characteristics including age, ethnicity and education level were collected at the start of the study.

Menopause symptoms: Women were also queried about menopausal symptoms covered by Kuppermanet et al. (1959). This scale can be used to assess changes in different symptoms, before and after menopause treatment. Height and weight were measured with subjects wearing lightweight clothing and no shoes, body mass index (calculated as kg m^-2) was used as an estimate of obesity. The Kupperman index is a numerical conversion index and covers 11 menopausal symptoms. Each symptom on the Kupperman index was rated on a scale from 0 to 4 for no, slight, moderate, severe and very severe complaints. The score of hot flashes was based on number of complaints per day: slight (1-2), moderate (2-3), severe (5-6) and very severe more than 6.

Total antioxidant assay: After fasting for 12 h, blood samples were obtained at baseline and 3 months by venipuncture . Total Antioxidant Status (TAS) of serum was measured using the Ferric Reducing Ability of Plasma (FRAP) assay (Benzie and Strain, 1996). The FRAP assay,which depends upon the reduction of ferric tripyridyltriazine (Fe (III)-TPTZ) complex to the ferrous tripyridyltriazine (Fe(II)-TPTZ) by a reductant at low pH. (Fe(II)-TPTZ) has an intensive blue color and can be monitored at 593 nm. products of this reaction. FRAP level was calculated by plotting a standard curve of absorbance against μmol L^-1 concentration of Fe (II) standard solution. The samples were analyzed for total antioxidant concentrations using an Cecil, CE, 2501 model, 2000, series spectrophotometer.

Lipid profiles: Serum total cholesterol and triglyceride levels were measured with enzymatic techniques by using the Hitachi 704 auto-analyzer, assay by using the Randox, Co, kit, UK and Chem-enzyme, Co, Iran.

Statistical analysis: Statistical analysis Frequency counts, descriptive statistics, Pearsons correlations, stepwise multiple linear regression and repeated measure were performed using the Statistical Package for the Social Sciences (Windows version 11 SPSS). The result obtained from experiment were analyzed by inferential statistic in terms of t-test and Analysis of Variance ( ANOVA) in which post-hoc comparisons were made using the Benferonnis test. In light of multiple comparisons statistical significance will assigned at p<0.05 for all analysis.

RESULTS

Analysis of 37 subjects enrolled, revealed that the mean age of respondents was 52.2±4.6 years, years of post menopause 5.47±3.4 years and the mean height was 157.4±7.2 cm. Comparison of weight, BMI did not reveal any significant changes during different stage of study.

Menopausal symptoms: Some of menopausal symptoms such, hot flashes, nervousness, vaginal symptoms and sexual symptoms were significantly changed during different stages of the study (Fig. 1). During the treatment period, the menopausal symptoms of participants using soymilk and exercise were significantly (p<0.05) lower than soymilk treatment group. Soymilk supplementation decreased (p<0.05) of hot flashes by 72%, nervousness by 54%, vaginal symptoms by 70% and sexual symptoms by 62.5% after soymilk consumption. Exercise and soymilk treatment decreased significantly (p<0.05) hot flashes, nervousness, vagina land sexual symptoms by 83, 30, 50 and 45%, respectively.

Total antioxidant status: The total antioxidant level in control, soymilk and exercise+soymilk groups increasing of level, 642.88±66.9, 1379.11±87.4 and 1288.75±98 μmol L^-1, respectively (Fig. 2).

Fig. 1:	Percentage changed in menopausal symptoms (Kupperman Index %) in soymilk consumption and soymilk consumption+exercise in menopausal women compared with control group. *Significant level was set at below 5% (p<0.05)

Fig. 2:	Effect of soymilk and exercise on FRAP values in menopause women before and after treatment. Data are mean±SEM of (n = 10-15) samples obtained from each group. *Significant level was set at below 5% (p<0.05) compared with control group

Fig. 3:	Effect of soymilk and exercise on total triglycerides values in menopause women before and after treatment. Data are mean±SEM of (n = 10-15) samples obtained from each group

Lipid profiles: Total cholesterol mean (mg dL^-1) decreased in soymilk consumption and exercise+soymilk compared with the baseline. However, the mean of total cholesterol and triglycerides levels in two groups decreased compared with the baseline after 3 months but the results were not significant (Fig. 3, 4).

Fig. 4:	Effect of soymilk and exercise on total cholesterol values in menopause women before and after treatment. Data are mean±SEM of (n = 10-15) samples obtained from each group

DISCUSSION

This study demonstrated that 12.5 g of soy protein with Genistein, 13 mg and Daidzein, 4.13 mg day^-1 was effective in alleviating vasomotor symptoms, such as hot flashes, consistent with previous study results (Albertazzi et al., 1998). Additionally, present results showed a decrease of other subjective symptoms, which was not reported by other studies (Upmalis et al., 2000; Glazier and Bowman, 2001). In this study, consumption of Genistein, 13 mg and Daidzein, 4.13 mg day^-1 reduces hot flashes, but its real mechanism of action is not known. One possible explanation for isoflavone effect on menopausal symptoms is through its action on the estrogen receptor, which is capable of binding several structurally diverse compounds such as natural estrogens and isoflavones (Crawford et al., 1996). Another explanation is that isoflavones act through their antioxidant effects. Genistein is an inhibitor of tyrosine protein kinases, which is seen to play a role in vascular endothelial activity (Wei et al., 1995). Finally, the 17β-estradiol enhancement of the isoflavone-group patients suggests that isoflavone supplementation increases estrogen levels. It may have an indirect effect due to isoflavones acting on sex hormone-binding globulin. Isoflavones were observed to have an antioxidant activity in vitro and in vivo and augmented the activities of antioxidant enzymes in rats (Ji, 1993). It can be hypothesized that isoflavone supplementation can increase total antioxidant defence systems and reduce the impact of ROS. Soy protein with isoflavones has been reported by Anderson et al. (1995) to lower total cholesterol and to inhibit oxidizability of LDL (Tikkanen et al., 1998). Although our data showed a similar protective effect of isoflavone, such as a decrease in total cholesterol, the mechanisms have not been clearly defined. These effects may be attributed to weak estrogenic effects of isoflavones, which also possess antioxidant properties. Menopausal women who exercise regularly appear to have a better quality of life than women who don`t (Scambia et al., 2000). Additionally, our results showed significant (p<0.05) increase of total antioxidant level in control < soymilk consumption < exercise+soymilk groups. These results demonstrate that consumption of soymilk for 3 months was more effective than soymilk+exercise and could reduce some of menopausal symptoms in postmenopausal women. This could be due to the duration of exercise and amount of soymilk consumption.

CONCLUSION

These results demonstrate that consumption of soymilk with isoflavones for 3 months reduces some of menopausal symptoms and improves quality of life in postmenopausal women. So as to help guide women and clinicians in finding lifestyle changes to prevent symptoms, thus reducing or providing alternatives to use of medications. In conclusion in our future studies, we plan to determine the Total Energy Expenditure (TEE) and to include one sport group without soymilk .

ACKNOWLEDGMENTS

The author would like to thanks to, Women Research Center, Alzahra University, for the use of the laboratory facilities and wish to extend her thanks of authorities of the Max soy- Co, Tehran-Iran involved in this study.