Combined Nutraceutical Approach to Postmenopausal Syndrome and Vascular Remodeling Biomarkers

Abstract

Objective:

Menopause is associated with broad, though generally not massive changes in a large number of metabolic parameters. The aim of our study was to evaluate the efficacy and tolerability of a combined nutraceutical approach on a large number of cardiometabolic risk markers and menopause symptoms in otherwise healthy, mildly dyslipidemic postmenopausal women.

Design/setting:

Forty women were enrolled in the context of a controlled, randomized, prospective study with parallel groups at the Lipid Clinic of the University of Bologna.

Intervention:

The women were randomized to treatment with a nutraceuticals containing soy isoflavones (60 mg) and berberine (500 mg) or a placebo at a dosage of one tablet daily between meals for 12 weeks.

Results:

All patients completed the study without significant side effects. Anthropometric measures, blood pressure, HOMA index, and basal homocysteinemia significantly improved in isoflavones-berberine–treated group when compared to the baseline, but not when compared to the placebo-treated patients. Compared to placebo-treated patients, those treated with isoflavones-berberine experienced a significant improvement in plasma lipid and metalloproteinase serum levels, as well as the main menopausal symptoms.

Conclusion:

The short-term consumption of a nutraceutical containing isoflavones and berberine was well-tolerated and improved menopausal symptoms, plasma lipid level, and serum levels of matrix metalloproteinases in a cohort of mildly dyslipidemic postmenopausal women when compared with a neutral control.

Introduction

L oss of ovarian function and subsequent deficiency of endogenous estrogens are suggested to enhance cardiovascular disease risk and increase the related death rate after menopause.¹ In fact, menopause compounds many traditional cardiovascular disease risk factors, including changes in body fat distribution from a gynoid to an android pattern, reduced glucose tolerance, abnormal plasma lipids, increased blood pressure, increased sympathetic tone, endothelial dysfunction, and vascular inflammation.² However, recent evidence supports the hypothesis that this observation could be more related to the increasing mean age of women and to their incident classical risk factors (i.e increased blood pressure and cholesterol, diabetes, etc.) than to a primitive role of menopause itself in inducing cardiovascular disease.³ In any case, cardiovascular disease is the leading cause of mortality and morbidity in women after the age of 50 years in most developed countries, and the majority of its risk factors are known and potentially reversible,⁴ even if not always at a level to require a pharmacological treatment. In particular, hormonal replacement therapy, which could reverse some of these risk factors, has some relative and absolute contraindications that strongly reduce its use in current clinical practice in women with mild symptoms.⁵

In this context, a possibly safe and efficacious alternative is the use of isoflavones, which can significantly mitigate menopause symptoms.⁶ They have also a positive effect on arterial stiffness⁷ and a small but significant antihypercholesterolemic effect.⁸ To reinforce this approach, adding other nutraceuticals, such as berberine, to isoflavones could improve different metabolic risk factors. Berberine is a natural alkaloid that can improve lipid profile, glucose metabolism, and endothelial health.⁹ In particular, the lipid-lowering effect of berberine appears to be mainly due to the stabilization of hepatic low density lipoprotein (LDL)-cholesterol receptor (LDL-R) by an extracellular signal-regulated kinase (ERK)-dependent pathway and an increase in transcriptional activity of LDL-R promoter via the c-Jun N terminal kinase (JNK) pathway.^10,11 Moreover, in addition to LDL-R up-regulation, berberine activates AMP kinase (AMPK), while blocking the mitogen activated protein kinase (MAPK)/ERK pathway, resulting in inhibition of lipid synthesis.¹²

Thus the aim of our study was to evaluate the efficacy and tolerability of a combined nutraceutical approach on a large number of cardiometabolic risk markers and menopause symptoms in otherwise healthy mildly dyslipidemic postmenopausal women.

Materials and Methods

This was a controlled, single-blinded, randomized, prospective study with parallel groups. Forty otherwise healthy menopausal women who met the following inclusion criteria were consecutively enrolled: at least one typical menopausal symptom along with LDL cholesterol (LDL-C) between 130 and 190 mg/dL and/or serum triglycerides between 150 and 400 mg/dL, following a structured low-glucose, low-calorie, low-fat diet according to their clinical condition.

Exclusion criteria were diagnosis of severe primary dyslipidemia or secondary to causes other than menopause, previous history of breast cancer, and use of hormone replacement therapy or lipid-lowering drugs.

The principles of the Declaration of Helsinki were applied, with subjects being provided with an informed consent form and clearly informed of their right to withdraw from the study without giving any reason. The study was approved by the local institutional review board.

The women were randomized to a treatment with either EL (Estromineral Lipid, Rottapharm | Madaus SpA, Monza, Italy) proprietary combination of nutraceuticals containing soy isoflavones 60 mg+Lactobacillus sporogenes 1×10⁹ spores+berberine 500 mg or placebo (PL) at a dosage of one tablet daily between meals for 12 weeks. The tablets were similar in shape and size for both groups.

Menopause symptoms (subjective severity score evaluated by a visual graduated scale with values from 0 to 4), blood pressure, waist circumference and weight, and standard metabolic parameters were measured at baseline and after 4 and 12 weeks. Insulin-resistance evaluation and other nonconventional cardiovascular disease serum biomarkers were measured only at the baseline and after 12 weeks.

The systolic (first phase) and diastolic (fifth phase) blood pressure readings were measured to the nearest even digit using a standard mercury manometer (Erkameter 3000, ERKA, Bad Tolz, Germany), with a large cuff size if the arm circumference was greater than 33 cm. Three readings were recorded on the right arm of the seated participant following a minimum of 10 minutes of rest in a quiet room, and the average of the second and third readings was defined as the subject's blood pressure. Measurements were always taken by the same investigator in the morning before daily drug intake (i.e., ∼24 hours after dosing).

All plasma parameters were determined after a 12-hour overnight fast. Venous blood was taken between 8:00 and 9:00 am and samples were kept on ice prior to spinning. Plasma obtained by addition of Na₂-EDTA, 1 mg/mL, and centrifuged at 3000×g for 15 minutes at 4°C was used. Immediately after centrifugation, plasma samples were frozen and stored at −80°C for no more than 3 months. Plasma glucose was collected in NaF tubes and assayed using the glucose-oxidase method (GOD/PAP, Roche Diagnostics, Mannheim, Germany) with intra- and interassay coefficients of variation (CVs) <2%¹³. Plasma insulin was assayed with a Phadiaseph Insulin RIA (Pharmacia, Uppsala, Sweden), using a second antibody to separate the free and antibody-bound ¹²⁵I-insulin (intra- and interassay CVs 4.6% and 7.3%, respectively).¹⁴ Total cholesterol (TC) and triglyceride (TG) levels were determined using a fully enzymatic technique on a clinical chemistry analyzer (HITACHI 737; Hitachi, Tokyo, Japan).^15,16 Intra- and interassay CVs were 1.0 and 2.1 for TC determination, and 0.9 and 2.4 for TG determination, respectively. High-density lipoprotein cholesterol (HDL-C) level was measured after precipitation of plasma apo B-containing lipoproteins with phosphotungstic acid, and intra- and interassay CVs were 1.0 and 1.9, respectively; LDL-C level was calculated using the Friedewald formula.^17,18 HOMA index was calculated using the following formula: fasting plasma insulin (μU/mL)×fasting plasma glucose (mmol/L)/22.5, as described by Matthews and coworkers.¹⁹

Homocysteine was measured by a modified procedure of Araki and Sako²⁰ with high pressure liquid chromatography and fluorescence detection. The intra-assay variation of the method was 2.5%.

C-reactive protein was measured with a validated high-sensitivity assay¹⁷ on BN^™ II nephelometer (Dade Behring Inc., Newark, DE). The intra- and interassay CVs were <4% and <2%, respectively, with a detection limit of 0.20 mg/L.

Matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 levels were determined by a two-site enzyme-linked immunosorbent assay method using commercial reagents (Amersham Biosciences, Uppsala, Sweden). The intra- and interassay CVs for measuring MMP-2 levels were 5.4%, and 8.3%, respectively. The intra- and interassay CVs to evaluate MMP-9 levels were 4.9%, and 8.6%. The intra- and interassay CVs for measuring TIMP-1 levels were 9.3%, and 13.1%, respectively, while those for measuring TIMP-2 levels were 5.4%, and 5.9%, respectively.²¹

All data were statistically analyzed with the aid of SPSS^® 11.0 for Windows^® (IBM Corp. Armonk, New York). A full descriptive analysis of available parameters was carried out. Statistical analysis of baseline homogeneity and clinical efficacy was carried out by means of the chi-squared test, and nonparametric analysis of variance for parametric data and Mann-Whitney test for nonparametric data. A p level of less than 0.05 was considered significant.

Results

All patients completed the study without significant side effects. Only one patient randomized to placebo experienced one episode of nephrolithiasis, which was judged to be unrelated the treatment. The main characteristics of the subjects are reported in Tables 1 and 2; none of the studied variables were significantly different between groups.

Table 1.

Baseline Levels of the Basic Clinical and Laboratory Variables in the Two Groups of Treatment

Variable ^a	Treatment	Mean	SD	SEM
Age (years)	Placebo	51.75	4.84	1.20
	Isoflavones-berberine	52.85	4.34	0.97
Years from menopause	Placebo	0.95	1.12	0.38
	Isoflavones-berberine	1.02	1.15	0.41
BMI (kg/m²)	Placebo	22.86	2.23	0.49
	Isoflavones-berberine	23.57	1.67	0.37
SBP (mm Hg)	Placebo	134.20	5.65	1.26
	Isoflavones-berberine	136.20	4.85	1.08
DBP (mm Hg)	Placebo	87.10	5.29	1.18
	Isoflavones-berberine	88.60	3.25	0.72
HR (bpm)	Placebo	71.75	7.99	1.78
	Isoflavones-berberine	72.05	5.32	1.19
TC (mg/dL)	Placebo	237.35	21.04	4.70
	Isoflavones-berberine	237.90	21.71	4.85
HDL-C (mg/dL)	Placebo	44.15	5.75	1.28
	Isoflavones-berberine	46.50	4.52	1.01
LDL-C (mg/dL)	Placebo	150.40	29.57	6.61
	Isoflavones-berberine	141.75	24.95	5.58
Non-HDL-C (mg/dL)	Placebo	193.70	20.40	4.56
	Isoflavones-berberine	191.40	21.71	4.85
TG (mg/dL)	Placebo	216.80	76.18	17.03
	Isoflavones-berberine	255.40	83.79	18.73
FPG (mg/dL)	Placebo	91.60	10.40	2.32
	Isoflavones-berberine	94.25	9.80	2.19
FPI (μU/mL)	Placebo	11.61	3.78	0.84
	Isoflavones-berberine	11.41	4.02	0.90
HOMA index	Placebo	3.33	1.32	0.29
	Isoflavones-berberine	3.25	1.36	0.30
GOT (mg/dL)	Placebo	21.75	6.82	1.52
	Isoflavones-berberine	25.52	7.25	1.62
GPT (mg/dL)	Placebo	22.00	6.33	1.41
	Isoflavones-berberine	22.97	5.45	1.21
CPK (U/mL)	Placebo	94.75	24.29	5.43
	Isoflavones-berberine	101.35	35.35	7.90

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides; FPG, fasting plasma glucose; FPI, fasting plasma insulin; GOT, glutamic oxalacetate transaminase; GPT, glutamic pyruvate transaminase; CPK, creatinine phosphokinase.

Table 2.

Baseline Levels of the Emerging Cardiovascular Risk Factors and Vascular Remodeling Biomarkers in the Two Groups of Treatment

Variable ^a	Treatment	Mean	SD	SEM
HCYS (μmol/L)	Placebo	12.26	3.04	0.68
	Isoflavones-berberine	12.53	3.07	0.68
FBG (mg/dL)	Placebo	363.05	55.58	12.43
	Isoflavones-berberine	363.45	53.14	11.88
hsCRP (mg/dL)	Placebo	2.14	0.34	0.07
	Isoflavones-berberine	1.92	0.26	0.05
MMP-2 (ng/mL)	Placebo	1153.53	93.17	20.83
	Isoflavones-berberine	1183.01	60.62	13.55
MMP-9 (ng/mL)	Placebo	144.61	50.14	11.21
	Isoflavones-berberine	149.30	53.75	12.02
TIMP-1 (ng/mL)	Placebo	295.68	56.73	12.68
	Isoflavones-berberine	304.21	55.96	12.51
TIMP-2 (ng/mL)	Placebo	98.25	5.46	1.22
	Isoflavones-berberine	99.11	3.10	0.69

HCYS, homocysteinemia; FBG, fibrinogen; hsCRP, high sensitivity C-reactive protein; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of the metalloproteinase.

The two groups also did not differ at the baseline with regard to the severity of the analyzed menopause symptoms: flushing (p=0.560), sweating (p=0.252), palpitation (p=0.793), libido loss (p=0.311), and vaginal dryness (p=0.212).

No significant change was observed in either treatment group during the study with regard to heart rate, fibrinogen, high-sensitivity C-reactive protein, glutamic oxalacetate transaminase, glutamic pyruvate transaminase, and creatinine phosphokinase.

Both systolic (−1.3±0.8 mm Hg, 19 df, p<0.001) and diastolic (−9.8±1.2 mm Hg, 19 df, p=0.006) blood pressure improved slightly, but significantly in the isoflavones-berberine–treated group when compared to the baseline, but not when compared to the placebo-treated patients.

Anthropometric parameters such as body weight, waist circumference, and body mass index significantly improved only in the isoflavones-berberine–treated patients compared to the baseline, but not when compared to the placebo group (Table 3). Plasma lipid levels significantly improved versus baseline only in the isoflavones-berberine–treated patients (Table 3), and TC, LDL-C, TG, and non–HDL-C also significantly improved when compared with the placebo-treated group. In particular, in a comparison of the observed changes versus placebo, patients in the isoflavones-berberine group experienced a net mean decrease in LDL-C of 15.75±4.99 mg/dL (p=0.005) and in TG of 61.85±8.78 mg/dL (p<0.001).

Table 3.

Baseline and Post-Treatment Anthropometric Parameters and Serum Lipid Levels in Isoflavones-Berberine- and Placebo-Treated Patients (Values Reported as Mean±SD)

	Isoflavones-berberine		Placebo
	Baseline	Post-treatment	Baseline	Post-treatment
Body weight (kg)	62.35±5.64	61.80±5.48^a	61.40±4.21	60.75±4.32
Waist circumference (cm)	88.45±4.40	87.15±4.17^a	86.00±7.08	85.90±6.95
BMI (kg/m²)	23.57±1.67	23.36±1.53^a	22.86±2.23	22.63±2.35
SBP (mm Hg)	136.20±4.85	135.90±4.72^b	134.20±5.65	133.70±4.30
DBP (mm Hg)	88.60±3.25	87.90±2.14^b	87.10±5.29	87.5±4.75
TC (mg/dL)	237.90±21.71	207.60±18.82^b,c	237.35±21.04	236.15±21.33
HDL-C (mg/dL)	46.50±4.52	47.80±4.27^b	44.15±5.75	44.40±5.17
LDL-C (mg/dL)	141.75±24.95	126.30±26.89^b,c	150.40±29.57	149.70±30.12
Non-HDL-C (mg/dL)	191.40±21.71	159.75±18.84^b,c	193.70±20.40	193.35±20.18
TG (mg/dL)	255.40±83.79	189.55±53.89^b,c	216.80±76.18	212.80±72.23

p<0.05 vs. baseline.

p<0.01 vs. baseline.

p<0.01 vs. placebo.

With regard to parameters related to glucose metabolism, only HOMA-index was significantly reduced by isoflavones-berberine treatment (3.24±1.36 vs. 2.14±0.44, p=0.002), but not when compared to the placebo-treated group.

Homocysteine was significantly reduced by isoflavones-berberine treatment (12.54±3.1 vs. 9.66±1.68, p=0.004), but not when compared to the placebo-treated group, as well.

Treatment with isoflavones-berberine was also associated with a significant reduction in MMP-2 and MMP-9, both vs. baseline and vs. placebo (Table 4). In particular, comparing the observed changes vs. placebo, patients in the isoflavones-berberine group experienced a net mean decrease in MMP-2 of 201.83±88.21 mg/dL (p=0.028) and in TG of 45.79±19.16 mg/dL (p=0.022).

Table 4.

Baseline and Post-Treatment Matrix Metalloproteinases and Their Tissue Inhibitors (TIMP) Serum Level in Isoflavones-Berberine- and Placebo-Treated Patients (Values Reported as Mean±SD)

	Isoflavones-berberine		Placebo
	Baseline	Post-treatment	Baseline	Post-treatment
MMP-2 (ng/mL)	1433.03±60.62	1080.68±313.34^b,c	1402.53±93.17	1352.01±279.83
MMP-9 (ng/mL)	149.30±53.75	96.18±16.70^a,c	144.61±50.14	137.29±16.17
TIMP-1 (ng/mL)	304.21±55.96	297.18±59.94	295.68±56.73	246.80±60.40^a
TIMP-2 (ng/mL)	99.11±3.10	98.43±6.11	98.25±5.46	95.03±6.09

p<0.05 vs. baseline.

p<0.01 vs. baseline.

p<0.01 vs. placebo.

Median (95% CI) scores related to the main investigated menopause-related symptoms before and after the treatment in placebo and isoflavones-berberine randomized patients are reported in Table 5. When compared to the baseline, all considered symptoms beyond palpitation significantly improved in isoflavones-berberine–treated patients, only. In contrast, palpitation improved in both groups.

Table 5.

Median (95% CI) of Scores Related to the Main Investigated Menopause-Related Symptoms Before and After the Treatment in Placebo and Isoflavones-Berberine Randomized Patients

	Placebo		Isoflavones-berberine
	Baseline	After treatment	Baseline	After treatment
Flushing	1 (0.65–1.58)	1 (0.57–1.43)	1 (0.80–1.90)	0.5 (0.08–0.62)^b,c
Sweating	1 (0.45–1.65)	1 (0.39–1.01)	1 (0.66–1.44)	0.5 (0.03–0.57)^b,c
Palpitation	1 (0.63–1.27)	0.5 (0.27–0.83)^a	1 (0.63–1.47)	0.5 (0.38–0.92)^b
Libido loss	1 (0.46–1.44)	1 (0.50–1.30)	1 (0.75–1.65)	1 (0.53–1.27)^a,c
Vaginal dryness	0.5 (0.24–1.06)	0.5 (0.21–1.06)	1 (0.56–1.54)	0.5 (0.32–1.08)^a

p<0.05 vs. baseline.

p<0.01 vs. baseline.

p<0.01 vs. placebo.

Discussion

Menopause is associated with a broad, although generally not massive change in a large number of metabolic parameters.²² Even if good behavioral education could prevent some of these changes,²³ some would require care.

In our randomized, controlled study carried out on dyslipidemic postmenopausal women, we observed that the use of a combined nutraceutical containing isoflavones and berberine significantly improve a large number of subjective symptoms and cardiometabolic markers when compared to a control product. The effect observed on menopausal symptoms was larger than that expected from literature data²⁴; however, they were consistent with what has been reported for isoflavones taken with lactobacilli that improve their bioavailability.²⁵

Oral hormone replacement therapy (but not transdermal estradiol) increases the plasma level of MMP-9.^26,27 In contrast, both isoflavones²⁸ and berberine²⁹ inhibit MMP expression in vitro. To the best of our knowledge, this is the first study showing that isoflavones and berberine are also associated with a decrease in the serum level of MMP-2 and MMP-9, which are strongly associated with nearly all the main cardiovascular risk factors and serve as strong markers of atherosclerosis.³⁰

Our study has some relevant limitations. First of all, it was not a double-blind study, but rather a single-blinded, controlled, randomized study. This was due both to technical difficulties in masking the slight, but clear bitter aftertaste of berberine and because of the known positive effect of isoflavones on menopausal symptoms that would have easily identified which product contained these components. However, a priori, the enrolled patients did not know which product they were taking. We were not able to clearly distinguish from this design what type of vascular effect was due to isoflavones or to berberine, though we supposed it could be additive. Furthermore, MMP and TIMP levels could also have been related to changes in bone turn-over,³¹ which was not evaluated in our study. Finally, TIMP dosage had large CVs, which reduced the reliability of the dosage itself. However, this is a common observation in different studies on this parameter.^21,32 However, the results of this study can be considered scientifically reliable according to the aim of the study, with a random controlled design, and by the results being in agreement with the available literature data.

In conclusion, in the setting of a single-blinded, controlled, randomized trial the short-term consumption of a nutraceutical containing isoflavones and berberine was well-tolerated and improved menopausal symptoms, the plasma lipid level, and the level of MMPs in a small cohort of mildly dyslipidemic postmenopausal women in comparison with a neutral control. A statement on cardiovascular disease risk decrease associated with this approach needs longer study duration on larger patient cohorts and more extensive cardiovascular evaluations.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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