The effect of artichoke leaf extract supplementation on lipid and CETP response in metabolic syndrome with respect to Taq 1B CETP polymorphism: A randomized placebo-controlled clinical trial

European Journal of Integrative Medicine 17 (2018) 112–118

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Research paper

The effect of artichoke leaf extract supplementation on lipid and CETP response in metabolic syndrome with respect to Taq 1B CETP polymorphism: A randomized placebo-controlled clinical trial Khatereh Rezazadeha,c, Farzin Rezazadehb, Mehranghiz Ebrahimi-Mameghanic,

T

⁎

a

Talented Students Center, Student Research Committee, School of Nutrition & Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran School of Medicine, Urmia University of Medical Sciences, Urmia, Iran c Nutrition Research Center, School of Nutrition & Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran b

A R T I C L E I N F O

A B S T R A C T

Keywords: Metabolic syndrome Artichoke leaf extract Cholesterol ester transfer protein Taq IB polymorphism Lipid profile Randomised controlled trial

Introduction: The potentially favorable effects of artichoke leaf extract (ALE) have been shown on lipid profile; however, results are inconsistent. Taq IB polymorphism in cholesteryl ester transfer protein (CETP) gene may modulate the response to intervention. This study was aimed to examine the effects of ALE supplementation on serum lipid profile and CETP levels regarding CETP Taq IB polymorphism in patients with metabolic syndrome (MetS). Methods: In this double-blind placebo-controlled clinical trial, 80 patients with MetS were randomized to receive ALE (1800 mg per day as four tablets) or matching placebo for 12 weeks. Serum levels of lipid profile and CETP, as well as physical activity levels were assessed before and after the intervention. Physical activity levels were measured using short form of the International Physical Activity Questionnaire (IPAQ-SF). Moreover, patients were genotyped for CETP Taq IB polymorphism. Results: Mean age and BMI of the patients was 38.91 ± 6.90 years and 34.32 ± 4.28 kg/m2, respectively. Twenty-eight percent of the patients were male. ALE supplementation decreased serum triglyceride (TG) level compared to placebo over 12 weeks (−10% vs. −2%, p = 0.01). There was no interaction between CETP Taq IB genotype and response to ALE supplementation. The subgroup analysis showed that in men carriers of Taq IBB1B1, LDL-C level significantly decreased in ALE group compared to the placebo group (-15% vs. 9%, p = 0.004). Conclusions: ALE supplementation decreased TG levels without intervention-genotype interaction in patients with MetS. However, men with Taq IB-B1B1 genotype indicated a reduction of LDL-C in response to ALE.

1. Introduction Artichoke (Cynara Scolymus L.) is a member of Asteraceae family and its leaves are extensively used both as a food and medicine [1]. Artichoke Leaf Extract (ALE) has been traditionally used in the treatment of hepato-biliary diseases and dyspepsia. Previous studies have revealed various pharmacological activity of ALE including hepatoprotective [2], antimicrobial [3], antiatherogenic [4], antioxidant [2,5], hypoglycemic [6,7] and anticancer [8] effects. The hypolipidemic properties of ALE and its compounds has also been recently noticed [7,9–11], which are mediated through choleretic effects [12] and the inhibition of cholesterol biosynthesis [13]. The main phytochemical compounds of ALE appears to be caffeoylquinic acids (e.g.

chlorogenic acid, cynarin), caffeic acid, sesquiterpene lactones and flavonoids (luteolin, luteolin 7-o-glucoside) [1,14]. There is evidence indicating that ALE is well-tolerated without any serious side effects [7,10,15] and mutagenic or genotoxic effects [16]. Metabolic syndrome (MetS) is defined as the clustering of abdominal obesity, hypertension, hyperglycemia, and dyslipidemia (lowered high-density lipoprotein cholesterol (HDL-C) and raised triglycerides (TG) levels) [17], which can increase the risk of developing cardiovascular diseases (CVD) [18]. The rising prevalence of MetS is recognized as a major public health concern over the world [17]. Therefore, improving lipid profile could decrease CVD risk in the afflicted patients. The cholesteryl ester transfer protein (CETP) is a hydrophobic

Abbreviations: ALE, artichoke leaf extract; CETP, cholesteryl ester transfer protein; DBP, diastolic blood pressure; FBS, fasting blood sugar; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; MetS, metabolic syndrome; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride; WC, waist circumference ⁎ Corresponding author at: Nutrition Research Center, School of Nutrition & Food Sciences, Tabriz University of Medical Sciences, Attar Neyshaboori Av Golghasht St Tabriz, Iran. E-mail address: [email protected] (M. Ebrahimi-Mameghani). https://doi.org/10.1016/j.eujim.2017.12.008 Received 30 October 2017; Received in revised form 11 December 2017; Accepted 20 December 2017 1876-3820/ © 2017 Elsevier GmbH. All rights reserved.

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Fig. 1. Flow diagram of the study.

effects of ALE on lipid profile and CETP concentration in patients with MetS, considering the modulation of the response by Taq IB polymorphism.

glycoprotein with a key role in lipid metabolism via mediating the exchange of cholesteryl ester (CE) and TG between cholesterol-rich lipoproteins and TG-rich lipoproteins [19]. In a study, a significant association between the increase of CETP mass and MetS was found in men [20]. Most of the published literature suggests that Taq-IB (rs708272), a common polymorphism in intron 1 of the CETP gene, is related to the risk of CVD and response to lipid lowering therapy, however the latter has not been confirmed yet [21]. The carriers of the B2 allele have shown an increase in HDL-C levels and a decrease in TG levels, CETP activity, and MetS risk [22,23]. The promising effects of ALE on lipid metabolism [7,9–11], and the possibility of the modulation of the response to ALE by individuals' genetic differences [24] have been already recognized. However, to best of our knowledge, the efficacy of ALE on CETP concentration and the variability in response, considering Taq IB polymorphism is yet to be studied. Therefore, the present study was aimed to examine the

2. Materials and methods 2.1. Subjects This study was conducted on patients with MetS (n = 80) in Khoy, Iran. The diagnosis of MetS was based on the international criteria, i.e. having at least three out of the five criteria, as follows: TG ≥ 150 mg/ dl, fasting blood sugar (FBS) ≥ 100 mg/dl, HDL-C < 40 mg/dl for men and < 50 mg/dl for women, systolic blood pressure (SBP) ≥ 130 and/ or diastolic blood pressure (DBP) ≥ 85 mg/dl, and waist circumference (WC) ≥ 95 cm in both sexes [25]. The cut-off point for WC was determined based on a report of the Iranian National Committee of 113

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Table 2 Comparison of lipid profile, CETP levels and physical activity level between ALE and placebo groups before and after the study. Variable TG (mg/dl) Before After p‡ TC (mg/dl) Before After p‡ LDL-C (mg/dl) Before After p‡ HDL-C (mg/dl) Before After p‡ LDL-C/HDL-C Before After p‡ CETP (μg/ml)α Before After p‡ LDL-C/HDL-C Before After p‡

Fig. 2. Polymerase chain reaction-based restriction fragment length polymorphism analysis of the CETP TaqIB: 50-bp DNA ladder.

Table 1 Baseline anthropometric, biochemical and genetic characteristics of the study participants Variables

ALE group (n = 33)

Placebo group (n = 35)

p†

Age (year) Sex [n (%)] Male Female Weight (kg) Height (cm) BMI (kg/m2) WC (cm) SBP (mmHg) DBP (mmHg) Total energy intake (kcal) Total protein intake (g) Total carbohydrate intake (g) Total fat intake (g) CETP Taq 1B [n (%)] B1B1 B1B2 B2B2

38.7 (7.6)

39.1 (6.2)

0.806 0.509

8 (24.2%) 25 (75.8%) 92.6 (12.2) 161.9 (8.7) 35.3 (4.3) 109.5 (6.5) 130.7 (19.2) 83.8 (8.1) 2031.8 (638.1) 67.0 (24.5) 343.7 (122.4) 46.5 (17.6)

11 (31.4%) 24 (68.6%) 88.6 (16.0) 162.6 (9.5) 33.3 (4.0) 104.5 (18.9) 125.1 (12.9) 79.6 (7.6) 2046.7 (679.2) 68.9 (22.5) 335.1 (126.7) 50.8 (17.6)

9 (27.3) 19 (57.6) 5 (15.2)

17 (48.6) 14 (40.0) 4 (11.4)

0.198 0.758 0.051 0.256 0.168 0.030 0.934 0.776 0.800 0.368 0.195

ALE group (n = 33)

Placebo group (n = 35)

p

204.91 (93.43) 168.55 (67.23) .009

184.53 (76.00) 175.26 (88.16) .421

.010†

198.93 (37.99) 187.30 (35.29) .014

196.50 (32.75) 183.21 (35.65) .037

.459†

109.70 (28.29) 102.44 (27.29) .029

111.21 (24.27) 101.76 (22.09) .001

.605†

41.70 (8.74) 43.34 (9.06) .227

41.35 (8.15) 42.17 (8.52) .507

.430†

2.67(0.68) 2.34 (0.48) .001

2.72 (0.54) 2.45 (0.47) .001

.523†

5.46 (3.11) 4.40 (2.47) .035

4.77 (2.40) 4.23 (2.28) .138

.149†

1.19 (0.97, 1.57) 1.20 (1.02, 1.59) .239

1.20 (1.01, 1.56) 1.21 (1.02, 1.57) .246

.752†

Physical activity level [n (%)] Before Low 11 (33.3) Moderate 14 (42.4) High 8 (24.2) After Low 6 (18.2) Moderate 16 (48.5) High 11 (33.3) p£ .334

10 (28.6) 16 (45.7) 9 (25.7)

.913#

5 (14.3) 14 (40.0) 16 (45.7) .129

.062#

TG: triglyceride; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL: high density lipoprotein; CETP: cholesteryl ester transfer protein; MD: mean difference; CI = confidence interval. Values are mean (SD), unless otherwise indicated. P-values of statistical significance (p < .05) are presented in bold. α Values are geometric logarithmic mean (minimum, maximum). † Analysis of covariance (adjusted for baseline values, change of weight, physical activity, dietary intake of energy and fat). £ Mc-Nemar test. # Chi-square test. ‡ Paired t-test.

BMI: body mass index; WC: waist circumference; SBP: systolic Blood Pressure; DBP: diastolic Blood Pressure; CETP Taq 1B: cholesteryl ester transfer protein gene Taq IB polymorphism. Values are mean (SD), unless otherwise indicated. † Independent t-test for numeric variables and Pearson Chi-Square test for categorical variables.

Obesity [26]. The exclusion criteria were as follows: self-reported clinically diagnosed cardiovascular diseases, diabetes mellitus, hypo- or hyperthyroidism, cancer, liver dysfunction, renal failure, biliary tract obstruction and gallstones, recent surgery, Sprue and Crohn's disease, inflammatory disease, taking fat-lowering, anti-hypertensive or corticosteroids medications, as well as consuming antioxidant supplements such as fish oil, vitamin C, vitamin E, carotenoids, lycopene and selenium, three months prior to the study, following weight loss program, having allergy to artichoke family plants and their products, and smoking. Regarding female subjects, menopause, lactation, and pregnancy were also considered as exclusion criteria. The study was approved by the Ethics Committee of Tabriz University of Medical Sciences (reference number: 93120), and written informed consent was obtained from each subject. The trial was registered on the Iranian Registry of Clinical Trials (http://www.irct.ir/, IRCT201409033320N9).

the intervention and placebo groups using permuted block randomization procedure by Random Allocation Software (RAS). Every permuted block was consisted of four persons, matched for age and sex. The allocation was conducted by a statistician who was not involved in the clinical procedures of the study. Patients were recruited from outpatients referred to Sina clinic in Khoy, Iran through clinicians or publicly printed advertisements. All patients were screened over the phone based on the inclusion criteria, and then selected on the first visit. The eligible subjects were assigned to receive 1800 mg/day of ALE or matching placebo as four tablets (one tablet before breakfast, one before dinner, and two before lunch) for 12 weeks. This dose of ALE was chosen on the basis of prescribed doses in previous clinical trials [7,9,10,15] and the recommended daily dose of dried leaves of artichoke by the German Commission E monograph [27]. ALE and placebo tablets were provided in similar opaque plastic bottles, given to the subjects at two time points: at baseline and after 6 weeks. The participants were requested to return the remaining tablets at each visit; counting these tablets allowed us to evaluate the

2.2. Study design This randomized, double-blind, placebo-controlled clinical trial was carried out in a parallel group design. The patients were randomized to 114

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2.3. Preparation of ALE and placebo tablets

Table 3 The effect of ALE versus placebo on lipid profile and CETP levels regarding CETP Taq IB polymorphism. B1B1 (n = 26) ALE (n = 9)

TG (mg/dl) Before 235.89 (113.77) After 193.44 (84.04) .094 P‡ TC (mg/dl) Before 227.57 (47.14) After 212.43 (52.14) P‡ .201 LDL-C (mg/dl) Before 127.80 (38.97) After 120.83 (47.98) P‡ .385 HDL-C (mg/dl) Before 39.30 (6.88) After

41.05 (12.09) ‡ .634 P LDL-C/HDL-C Before 3.27 (0.67) After 2.81 (0.58) P‡ .167 TG/HDL-C Before 6.62 (2.99) After 6.11 (3.24) P‡ .579 CETP (μg/ml)α Before 1.20 (0.05) After 1.19 (0.09) P‡ .447

Fresh globe artichoke (Cynara cardunculus var. scolymus L.) leaves were collected from the Iran’s Medicinal Plants Cultivation Company in Qazvin, Iran (Voucher number 93H026-191). All leaves were identified and authenticated by a pharmacognosist, Prof. J. Afshar. The leaves of artichoke were dried and pulverized. The extract of leaves was obtained by adding 600 ml of ethanol/water (70:30) to 100 g of leaf powder at 40 °C for 12 h. The hydroalcoholic extract was concentrated under vacuum and then tablets were made. The ALE and placebo tablets were prepared by Dineh Iran. Co (Qazvin-Iran) and were identical regarding their color and size. Each tablet of ALE contained 450 mg of a hydroalcoholic extract of artichoke leaf, containing at least 4–5% chlorogenic acid. The standardization was carried out using the spectrophotometer, based on the protocol of Iranian Herbal pharmacopoeia [28]. Briefly, samples were chopped and dissolved in 250 ml of ethanol/water (50:50). The solution was heated over a boiling water bath for 30 min, centrifuged at 4000 rpm for 10 min and then filtered by a 0.2 μm syringe filter. The filtered solution (1 ml) was mixed with 2 ml of 0.5 M HCL, 2 ml of Arnow's nitrate-molybdate reagent, 2 ml of 1 M NaOH and 3 ml of water. The absorbance of samples and standards was determined at 505 ± 2 nm.

B2allel (n = 42) Placebo (n = 17)

192.35 (93.37) 161.94 (92.41) .059 188.94 (35.58) 177.94 (33.56) .105 101.26 (22.40) 95.82 (19.40) .187 39.68 (10.00) 39.00 (6.20)

P†

P†

ALE (n = 24)

Placebo (n = 18)

.964

193.29 (84.42) 159.21 (59.15) .045

176.71 (55.37) 188.59 (84.34) .466

.072

.760

190.22 (30.92) 179.65 (25.27) .045

209.60 (25.74) 184.93 (36.92) .030

.677

.917

104.76 (23.41) 97.42 (16.93) .052

121.15 (22.50) 107.70 (23.64) .000

.895

43.13 (5.36)

0.593

42.43 (9.24) 44.03 (8.14) .270

.693

45.56 (9.52)

2.4. Anthropometry and blood pressure assessments .898

Anthropometric indices and blood pressure were assessed at baseline and the end of the study by the same person. Height was measured without shoes using wall-mounting measuring tape (Soehnle 5002.01, Hamburg, Germany) to the nearest 1 mm. Body weight was measured with light clothing with no shoes on, using a digital scale (Omron BF511, kyoto, Japan) to the nearest 100 g. The following formula was used for the calculation of Body mass index (BMI): weight (kg)/height (m2). WC was measured in standing position at the midpoint between the iliac crest and the lowest rib with a precision of 1 mm. Blood pressure (BP) measurement was done in a seated position, after a 5-min rest, twice with a 5-min interval, using an automated digital sphygmomanometer (Microlife A100-30, Berneck, Switzerland), and the mean of the two measurements was reported.

.179

2.62 (0.53) 2.51 (0.50) .328

0.816

2.51 (0.60) 2.21 (0.37) .004

2.82 (0.56) 2.38 (0.45) .000

.875

5.30 (2.93) 4.09 (2.21) .028

.167

5.11 (3.12) 3.88 (1.99) .043

4.19 (1.56) 4.38 (2.42) .677

.170

1.23 (0.17) 1.24 (0.17) .367

.238

1.18 (0.14) 1.20 (1.14) .077

1.16 (0.11) 1.18 (0.10) .447

.520

TG: triglyceride; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL: high density lipoprotein; CETP: cholesteryl ester transfer protein. Data are Mean (SD). P-values of statistical significance (p < .05) are presented in bold. ‡ Paired t-test. α Analysis are based on geometric logarithmic data. † Analysis of covariance (adjusted for baseline values).

2.5. Dietary intake and physical activity assessments Dietary intake and physical activity levels were assessed at baseline and after 12 weeks. Dietary data were collected using a 3-day 24-h food recall (one weekend and two working days).The average of three-day recalls were analyzed for energy and nutrients intakes using Nutritionist IV Software modified for Iranian foods (First Databank Inc., Hearst Corp., San Bruno, CA, USA). Short form of the International Physical Activity Questionnaire (IPAQ-SF) was used to estimate physical activity levels [29]. Based on the scoring method of IPAQ-SF, the participants were classified as “high”, “moderate,” and “low” active [30].

compliance. The participants were asked to maintain their usual physical activity and food intake throughout the study and were advised to inform the researchers for any adverse effects of the supplements. The primary outcomes were changes in lipid profile and CETP level and the secondary outcomes were the modulation of response to ALE by CETP Taq IB polymorphism.

Table 4 Comparison of changes of lipid profile and CETP levels regarding CETP Taq IB polymorphism based on sex between ALE and placebo groups before and after the study. variables

TG (mg/dl) TC (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) LDL-C/HDL-C TG/HDL-C CETP (μg/ml)

Men (n = 19)

Women (n = 49)

B1B1 (n = 8)

B2allel (n = 11)

B1B1 (n = 17)

B2allel (n = 32)

94.29 (−29.76–218.35), 0.112 −18.48 (−41.67–4.70), 0.099 −20.81 (−37.03–−4.60), 0.020 −5.19 (−10.59–0.21), 0.057 −0.14 (−1.01–0.72), 0.696 3.74 (−0.13–7.62), 0.056 −0.08(−0.20–0.02), 0.104

−95.34 (−214.28–23.59), 0.097 −5.52 (−32.55–21.50), 0.635 4.07 (−20.44–28.59), 0.358 3.02 (−3.79–9.84), 0.320 −0.08 (−0.62–0.45), 0.707 −2.63 (−6.16–0.88), 0.117 0.032 (−0.13–0.17), 0.742

−30.99 (−83.02–21.02), 0.222 12.69 (−38.01–63.39), 0.593 26.15 (−9.11–61.41), 0.128 7.42 (−1.22–16.07), 0.085 0.02 (−0.90–0.94), 0.962 −0.31 (−1.64–1.02), 0.617 0.00 (−0.05–0.05), 0.952

−27.42 (−73.82–18.99), 0.237 4.21 (−22.83–31.27), 0.751 −2.69 (−13.99–8.60), 0.628 −2.65 (−8.07–2.77), 0.324 0.00 (−0.22–0.24), 0.935 −0.37 (−1.78–1.03), 0.591 0.01 (−0.02–0.05), 0.499

TG: triglyceride; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL: high density lipoprotein; CETP: cholesteryl ester transfer protein. Values are mean difference (95% confidence interval), p-value. P-values were computed using analysis of covariance (adjusted for baseline values).

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proportion of females completed the study in both groups (M/F: supplemented 8/25 vs placebo 11/24; p = .509). Table 2 represents lipid profile, CETP concentrations and physical activity level of both groups during the study, irrespective to their genotypes. ALE supplementation significantly reduced TG level compared with placebo (percent change: −10% vs. −2%, p = .010), while there was no significant differences in changes of TC, LDL-C, LDL-C/ HDL-C, TC/LDL-C, TAG/HDL-C and CETP level between groups, after adjustment for the confounders (i.e. baseline values, changes of weight, physical activity, dietary intake of energy and fat) (p > .05). Moreover, the within-group analysis indicated significant decreases in serum levels of TC (percent change: −5% in ALE group and −7% in placebo group), LDL-C (percent change: −5% in ALE group and −7% in placebo group), LDL-C/HDL-C (percent change: −10% in ALE group and −9% in placebo group) and TC/HDL-C (percent change: −8% in ALE group and −9% in placebo group) in both groups, along with a significant decrease in serum TG level (percent change: −10%), and TAG/ HDL-C (percent change: −12%) in ALE group. Physical activity level was not different between the two groups throughout the study. Genotyping was carried out for all patients. The distribution of CETP Taq IB SNP was similar in two groups (p > .05, Table 1) and genders (data not shown). The interaction between CETP Taq IB genotypes and the patient response to ALE supplementation are summarized in Table 3. The baseline values of serum levels of TC, LDL-C/HDLC, and TC/HDL-C in B1B1 carriers and LDL-C in B2 allele carriers were different between the two groups; therefore, baseline values were considered as confounders. The B2 allele carriers revealed significant reduction in serum concentrations of TAG, TC, LDL-C/HDL-C, TC/HDLC, and TAG/HDL-C in ALE group and TC, LDL-C, LDL-C/HDL-C, and TC/HDL-C in placebo group and B1B1 genotype exhibited significant reduction in TAG/HDL-C in placebo group. Based on analysis of covariance with adjustment for baseline values, ALE supplementation indicated similar changes in lipid profile and CETP level among Taq IB genotype compared to placebo. Patients were categorized by sex, to explore possible gender differences in lipid profile response to ALE. Subgroup analysis showed a statistically significant reduction in LDL-C concentration in men with B1B1 genotype in response to ALE supplemetation (percent change: −15% vs 9%, p = .004) (Table 4).

2.6. Biochemical assays Biochemical biomarkers was measured before and after the intervention. Five milliliters of fasting blood samples were centrifuged at a 3000 rpm for 10 min and then the sera were separated. The concentrations of fasting lipid profile (total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C, and TG) were measured enzymatically using an automatic biochemical Hitachi 717 analyzer (Hitachi, Boehringer Mannheim, Japan) by Pars-Azemoon kits (Tehran, Iran) on the day of blood sampling. LDL-C level was calculated using Friedewald formula as follows: LDL-C = TC-(HDL-C + TAG/5) [31]. Serum CETP concentration was assayed using human ELISA kit (Eastbiopharm, Hangzhou, china). 2.7. Genotyping Blood samples were stored at −80 °C in tubes containing anticoagulant ethylenediaminetetraacetic acid (EDTA). Genomic DNA was extracted using the conventional phenol chloroform extraction method. The polymerase-chain-reaction–based method of screening for the TaqIB polymorphism in intron 1 of the CETP gene (rs708272) was carried out as previously described in detail [32]. The final product was digested with TaqI enzyme (Thermo Scientific, Vilnius, Lithuania), for 2 h at 65 °C, and then digested product was separated by electrophoreses on 1.5% agarose gel. The resulting fragments 174 and 361 bp were referred to as B1B1 homozygotes, 535, 174 and 361 bp as B1B2 hetrozygotes and 535 bp as B2B2 homozygotes (Fig. 2). 2.8. Statistical analyses The sample size was calculated based on the changes in serum LDLC level reported by Rondanelli et al. [7]. Considering a confidence level of 95% and power of 90% and anticipating a possible dropout rate of 30%, 40 patients with MetS aged 20 to 50 years were selected for each intervention arm. All data analyses were carried out on the intention-totreat principle using SPSS software (ver. 16; SPSS Inc., IL, Chicago, USA). The Kolmogorov-Smirnov goodness-of-fit was performed for assessing the data distribution. Since CETP concentration was not symmetrically distributed, logarithm transformation was done, and data were presented as logarithmic mean (minimum, maximum). For inter and intra-comparison of quantitative variables, independent sample ttest and paired t-test were used, respectively. The chi-square or McNemar test was employed for assessing the between and within-group differences of qualitative data, respectively. Moreover, changes in lipid profile and CETP concentration in response to intervention were analyzed using analysis of covariance (ANCOVA) with adjustment for confounders (baseline values and changes of weight, physical activity, energy, and fat intake) to avoid potential bias. In addition, the interaction between the CETP Taq IB polymorphism and intervention outcomes were analyzed using ANCOVA test. Results were expressed as mean (standard deviation), and mean difference (95% confidence interval), unless otherwise stated. P < .05 was as threshold value of statistical significance.

4. Discussion In the present study, daily consumption of 1800 mg ALE for 12 weeks among patients with MetS significantly reduced TG level by 10% compared to 2% in the placebo group. Indeed, serum TC and LDL-C levels were significantly decreased in both groups, as well as LDL-C/ HDL-C and TC/HDL-C, though the between-group differences were not significant. Although hypotriglyceridemic and hypocholestrolemeic effects of ALE have been demonstrated in animal studies on rats, mice and hamsters [33–35], human interventional studies have reported different effects on lipid parameters. A recent Cochrane review [11] has confirmed the potential role of ALE in decreasing cholesterol levels, despite inconclusive results. Englisch et al. [9] conducted a study on 143 patients with TC of > 280 mg/dl with 1800 mg aqueous extract of artichoke for 6 weeks. They reported that serum levels of TC and LDL-C reduced, whereas TG levels did not change. Also, a study by Bundy et al. [10] on 75 healthy adults with plasma cholesterol in the range 232–309 mg/dl with 1280 mg of ALE for 12 weeks showed reduction of TC without significant differences between groups for LDL-C, HDL-C or TAG levels. Nonetheless, supplementation with an extract from Phaseolus vulgaris (Pv) and Cynara scolymus (Cs) on 39 overweight subjects for 2 months failed to improve serum HDL-C, LDL-C or TG status [15]. In addition, Rnodanelli et al. [7] in another study assessed the efficacy of 600 mg/d extract from Cs on glucose pattern in 55 overweight subjects with impaired fasting glycaemia (IFG) for 8 weeks. They found a significant decrease of glycometabolic parameters, TC, LDL-C and TC/

3. Results Fig. 1 shows the flowchart of the trial. Of the total 443 subjects, enrolled in the study, 256 had the first line criteria for MetS screening. The study was conducted from November 2014 to May 2015. After biochemical and anthropometric assessments, 80 patients were eligible for inclusion in the study and were divided randomly into intervention and placebo groups. Over 12 weeks, 68 patients completed the study (ALE group = 33; placebo group = 35). All dropouts during the study were irrelevant to the adverse effects of ALE or placebo (Fig. 1). Table 1 demonstrates the baseline characteristics of patients. The placebo and ALE groups were similar at the beginning of the study. An equal 116

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level was also assessed as a related factor to MetS [20]. However, the present research had the following limitations: (i): the effective constituents of ALE were not measured by high-performance liquid chromatography (HPLC) due to funding constraints; (ii) the number of given tablets to patients were high. Therefore, future clinical trials should measure CETP activity and other SNPs of CETP such as A-629C, which influence lipid profile.

HDL-C. Therefore, differences in the composition and dosage of ALE, baseline lipid profile, intervention period, dietary intake, and genotype may result no effect or moderate improvement on lipid profile. In the present study, the mild impact of ALE toward decreasing cholesterol level and absence of a significant decrease after intervention, compared to placebo may be due to normal baseline levels of cholesterol in most of the patients (77% of patients < 230 mg/dl), while previous animal studies or clinical trials have generally been carried out on hypercholesterolaemic subjects. Moreover, TG levels in 72% of our patients were higher than 150 mg/dl, which could partly clarify the modest favorable effect of ALE on reduction of TG level. The possible mechanisms for the effect of ALE on reduction of cholesterol appears to be attributed to the partial inhibition of cholesterol biosynthesis and choleretic effect [13,35]. In addition, hypotriglyceridemic effect of ALE could be mediated by action of the certain active components in the artichoke as sesquiterpens (cynaropicrin, aguerin B, and grosheimin) and sesquiterpene glycosides (cynarascolosides A, B, and C) through oxygen functional groups and exomethylene moiety [33]. Our results showed that ALE could not affect CETP level after 12 weeks. This study seems to be the first clinical trial to examine the effect of ALE on the CETP level. To date, limited clinical trials were available to investigate the effects of such intervention on variation of CETP levels. Van Venrooij et al. [36] found that atorvastatin 10 mg (A10) and 80 mg (A80) in 217 patients with diabetes for 30 weeks significantly reduced CETP mass dose-dependently by 18% (A10) and 29% (A80). This conflict of results may be due to differences in type of intervention (the primary cause), duration of intervention, CETP level assay procedure and background disease. In the current study, the allele frequency of CETP Taq IB was 0.375 for B2 allele, which is similar to the frequency reported in Iranian healthy individuals [37]. However, it is lower than the frequencies found in Egyptian patients with MetS [38], primary combined hyperlipidemia Iranian patients [39] and healthy White and East Asian population [22]. The other finding of our study was the similar changes of serum lipid profiles in response to ALE supplement between Taq IB genotype, while men in B1B1 genotype experienced significant reduction of LDL-C levels by ALE supplement versus placebo. Several clinical trials have explored the interaction between CETP Taq IB genotype and intervention, but the results are contradictory. Based on the study by Venrooij et al. [36], CETP Taq IB modulated the effect of atorvastatin on the increase of HDL-C and decrease of TG, and CETP mass. Estévez-González et al. [40] evaluated the modification effects of skim milk or oliveoil-enriched skim milk on lipid profile by CETP Taq IB polymorphism in 32 children with mild hypercholesterolemia for 6 weeks. The increase of HDL-C following intake of skim milk enriched with olive oil was more intense in B1B1 genotype. In a randomized controlled cross-over trial, Gammon et al. [41] investigated the effects of consumption of two green kiwifruit/d on plasma lipid response and modulation by four HDL-C-related SNPs on eighty-five hypercholesterolaemic men in 12 weeks. Carriers of B1B1 allele showed significantly lower TAG/HDL-C during kiwifruit intervention period compared to control intervention. Accordingly, our results regarding men are in accordance with these mentioned studies that presented improvement of some lipid parameters in B1B1 genotype in response to the intervention. In addition, more atherogenic feature of B1B1 genotype was previously demonstrated [22], especially in men [42]. However, a meta-analysis on 3 randomized, placebo-controlled, pravastatin trial indicated no interaction between CETP Taq IB genotype and response to pravastatin treatment [21]. These contradictory results could be caused by differences between study samples (number, ethnicity, and background disease) or interventions (type and duration). To the best of our knowledge, the present study is the first clinical trial to evaluate a possible interaction between Taq IB genotype and variations in plasma lipid responses to the ALE supplementation. CETP

5. Conclusion The ALE supplementation could improve TG level in patients with MetS. Although from the nutrigenetics prospective, changes of serum lipid profile were similar among CETP Taq IB genotype, men carriers of Taq IB-B1B1 had reduction of LDL-C level by ALE supplementation. Our results somewhat favor the genotype × intervention interaction, but needs to be proven in a larger sample size with longer intervention duration by consideration of sex differences. Acknowledgements The authors thank all subjects who have contributed in this study. We greatly acknowledge Dineh Iran. Co. for collaboration in preparation of Artichoke Leaf Extract and placebo tablets. We are grateful of Nutrition Research Center, Biochemistry department, Danesh laboratory, Khoy, and Immunology department for their assistance in biochemical and genetic analysis. This study was supported by a Grant from the Research Vice Chancellor, Tabriz University of Medical Sciences, Tabriz, Iran (grant number: 5/97/4653). The results of this article are derived from the Ph.D. thesis of khatereh Rezazadeh (NO, D/ 41). Conflict of interest The authors declare that there are no conflicts of interest. References [1] M.B. Salem, H. Affes, K. Ksouda, R. Dhouibi, Z. Sahnoun, S. Hammami, et al., Pharmacological studies of artichoke leaf extract and their health benefits, Plant Foods Hum. Nutr. 70 (4) (2015) 441–453. [2] R. Gebhardt, M. Fausel, Antioxidant and hepatoprotective effects of artichoke extracts and constituents in cultured rat hepatocytes, Toxicol. In Vitro 11 (5) (1997) 669–672. [3] X. Zhu, H. Zhang, R. Lo, Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and their antimicrobial activities, J. Agric. Food Chem. 52 (24) (2004) 7272–7278. [4] N. Xia, A. Pautz, U. Wollscheid, G. Reifenberg, U. Förstermann, H. Li, Artichoke, cynarin and cyanidin downregulate the expression of inducible nitric oxide synthase in human coronary smooth muscle cells, Molecules 19 (3) (2014) 3654–3668. [5] K. Rezazadeh, S. Aliashrafi, M. Asghari-Jafarabadi, M. Ebrahimi-Mameghani, Antioxidant response to artichoke leaf extract supplementation in metabolic syndrome: a double-blind placebo-controlled randomized clinical trial, Clin. Nutr. (2017), http://dx.doi.org/10.1016/j.clnu.2017.03.017 (in press). [6] N. Fantini, G. Colombo, A. Giori, A. Riva, P. Morazzoni, E. Bombardelli, et al., Evidence of glycemia-lowering effect by a Cynara scolymus L. extract in normal and obese rats, Phytother. Res. 25 (3) (2011) 463–466. [7] M. Rondanelli, A. Opizzi, M. Faliva, P. Sala, S. Perna, A. Riva, et al., Metabolic management in overweight subjects with naive impaired fasting glycaemia by means of a highly standardized extract from cynara scolymus: a double-blind, placebo-controlled, randomized clinical trial, Phytother. Res. 28 (1) (2014) 33–41. [8] S. Nadova, E. Miadokova, P. Mucaji, D. Grancai, L. Cipak, Growth inhibitory effect of ethyl acetate-soluble fraction of Cynara cardunculus L. in leukemia cells involves cell cycle arrest, cytochrome c release and activation of caspases, Phytother. Res. 22 (2) (2008) 165–168. [9] W. Englisch, C. Beckers, M. Unkauf, M. Ruepp, V. Zinserling, Efficacy of artichoke dry extract in patients with hyperlipoproteinemia, Arzneimittelforschung 50 (03) (2000) 260–265. [10] R. Bundy, A.F. Walker, R.W. Middleton, C. Wallis, H.C. Simpson, Artichoke leaf extract (Cynara scolymus) reduces plasma cholesterol in otherwise healthy hypercholesterolemic adults: a randomized, double blind placebo controlled trial, Phytomedicine 15 (9) (2008) 668–675. [11] B. Wider, M.H. Pittler, J. Thompson-Coon, E. Ernst, Artichoke leaf extract for treating hypercholesterolaemia, Cochrane Database Syst. Rev. (3) (2013), http:// dx.doi.org/10.1002/14651858.CD003335.pub3. [12] R. Gebhardt, Anticholestatic activity of flavonoids from artichoke (Cynara scolymus L.) and of their metabolites, Med. Sci. Monit. 7 (2001) 316–320. [13] R. Gebhardt, Inhibition of cholesterol biosynthesis in HepG2 cells by artichoke

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