Effects of dried licorice extract with low-calorie diet on lipid profile and atherogenic indices in overweight and obese subjects: A randomized controlled clinical trial

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ScienceDirect European Journal of Integrative Medicine 7 (2015) 287–293

Original article

Effects of dried licorice extract with low-calorie diet on lipid profile and atherogenic indices in overweight and obese subjects: A randomized controlled clinical trial Elham Mirtaheri a , Nazli Namazi b , Mohammad Alizadeh a,∗ , Nafiseh Sargheini c , Saadat Karimi a a

b

Nutrition Research Center, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Student Research Committee, Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Experimental Nutritional Medicine, Nutritional Sciences Institute, Potsdam, Germany Received 24 October 2014; received in revised form 9 March 2015; accepted 10 March 2015

Abstract Introduction: Licorice root is one of the most frequently used medicinal herbs. There is contradictory evidence about effects of the licorice on lipid profile. The aim of the present study was to compare effects of licorice extract concurrent with low-calorie diet with low calorie diet alone on the lipid profile and atherogenic indices in overweight and obese subjects. Methods: In this double blind randomized controlled clinical trial, 64 overweight and obese subjects, aged 30–60 years old, were recruited from March to September 2013. They were randomly divided into intervention (n = 32) and control (n = 32) groups. Participants and research staff were masked to the treatment allocation by randomization. Both the groups received 1.5 g/day of dried licorice extract or placebo, respectively, concurrent with weight loss diet for 8 weeks. Lipid profile, weight and dietary intake were measured at baseline and at the end of the study. Body mass index and atherogenic indices were calculated. Results: Fifty-eight participants completed the trial. At baseline, there were no significant differences for lipid profile except for low density lipoprotein-cholesterol (LDL-c) level between the groups. After the intervention, total cholesterol (TC), LDL-c levels, TC/high-density lipoprotein (HDL-c), LDL-c/HDL-c ratios and log of TG/HDL-c were significantly decreased (p < 0.01 for all variables), but no changes were observed in TG and HDL-c levels. Conclusions: It seems that supplementation with licorice extract used concurrently with a low calorie diet can efficiently improve the lipid profile in overweight and obese subjects. © 2015 Elsevier GmbH. All rights reserved. Keywords: Licorice; Lipid profile; Atherogenic status; Obesity; Weight loss

Introduction Cardiovascular disease (CVD) is an important public health concern in both developed and developing countries. A major risk factor for developing the CVD is dyslipidemia [1]. In the last decades, an increase in dyslipidemia has been observed following rising the prevalence of overweight and obesity [2]. In 2008, the World Health Organization (WHO) reported that more than 1.4 billion adults were overweight. Of these, more than 500

∗

Corresponding author. E-mail address: [email protected] (M. Alizadeh).

http://dx.doi.org/10.1016/j.eujim.2015.03.006 1876-3820/© 2015 Elsevier GmbH. All rights reserved.

million subjects were obese [3]. Weight management interventions are basically aimed at reducing dietary energy intake in combination with physical activity, life style modifications and recently pharmacotherapy [4]. Given difficulties in adherence to dietary recommendations for weight maintenance, obese and overweight subjects often turn to drugs and supplementations. More recently, there has been a greater focus on medicinal plants to ensure weight reduction [5]. Previous studies provided evidence on anti-obesity properties of many edible plants such as green tea, pepper, Camellia synensis [6] and Glycyrrhiza glabra [7]. The G. glabra root (Licorice) is one of frequently used medicinal herbs in the traditional medicine. G. glabra root has been suggested to possess many

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therapeutic and medicinal characteristics including antiviral activity, relief of peptic ulcer diseases and antioxidant/antiinflammatory effects [8]. These effects are partly attributed to glycyrrhizin, glabrol, glabridin, flavenoid fractions and volatile components of the herb [8]. Some research has suggested that licorice root could be effective in reducing abdominal fat deposition and improving lipid profile. Fuhrman et al. [9] suggested that licorice root extract consumption reduced total cholesterol (TC) and low-density lipoprotein (LDL) in moderately hypercholesterolemic patients. Also, Tominaga et al. [10] reported that licorice flavenoid oil (LFO) reduced LDL-levels after 8 weeks in overweight subjects. Whilst, Bell et al. [11] trial did not find any effect of LFO on lipid profile in overweight/obese subjects and athlete men after 8 weeks. In summary the above reports, the effects of licorice root on the lipid profile remains contradictory and it is difficult to make a conclusion about the effects of licorice on dyslipidemia. In addition, it seems that no study has been conducted to compare the concurrent effect of supplementation with Licorice and lowcalorie diet to low-calorie diet alone on lipid profile and weight loss. Therefore, the aim of the present study was to elucidate the effects of dried extract licorice along with a low calorie diet on lipid profiles in overweight and obese subjects.

and principal investigators remained masked to the treatment assignments until data collections were completed. Besides, both researchers and the participants remained blind for randomization and allocation until final data analyses. Further, all the participants were stratified for sex, age and BMI. Intervention All the participants received a low calorie diet. A dietitian designed an individualized diet to reduce energy intake by 500 kcal from the total energy requirement. Resting energy expenditure was calculated using Mifflin equation [12]. Prescribed diet contained 55% carbohydrate, 15% protein and 30% fat. Intervention and placebo groups took 1.5 g/day (divided into three times a day 30 min before each meal) of dried licorice extract and placebo (corn starch), respectively, for 8 consecutive weeks. Follow up visits were arranged in 20-days intervals. Supplements were distributed among the volunteers based on the allocation code after the randomization. In order to minimize subjects’ withdrawal and as an advocacy approach all the participants received serial weekly phone calls. Throughout the trial, the subjects were asked to maintain their usual physical activity levels. The patients also had the option to quit the study anytime during the intervention. Finally, adherence to the study protocol was assessed by number of returned capsules.

Materials and methods Preparation of licorice extract and placebo Participants and study design A randomized double-blind clinical trial was conducted on 64 overweight and obese volunteers (27 men, 37 women). The sample size was calculated based on the fat mass variables in Tominaga et al’s study and with α-value 0.05, power 90% and considering 20% loss to follow up, subjects were recruited by dietitian referral from March to September 2013 at Sheikhoraees clinic affiliated to Tabriz University of Medical Sciences, Tabriz, Iran. Inclusion criteria were as follows: age 30–60 years old and body mass index (BMI) > 25 kg/m2 . Exclusion criteria were CVD, liver, thyroid and kidney disorders, diabetes mellitus, smoking, receiving any anti-obesity medications, vitamin–mineral supplements or herbal drugs, pregnancy and lactation. At the beginning of the trial, general characteristics including age, medication history, obesity history and dietary habits were collected. The trial was approved by the Ethics Committee of Tabriz University of Medical Sciences and a written informed consent was obtained from each patient. The trial was registered on the Iranian Registry of Clinical Trials (www.irct.ir/, IRCT2013062811288N3). Randomization and allocation Eligible participants were randomly assigned into intervention (n = 32) or placebo groups (n = 32). Randomization facilitated by random number tables with a permuted block size of two. To ensure double blinding, the allocation was performed by an investigator with no clinical involvement in the study

The licorice extract was prepared by Darook pharmacological company (Esfahan, Iran) with registered number of 1228104305. Briefly, it was a dried hydroalcoholic extract of licorice root (ethanol 70:water 30% v/v) containing lowered glycyrrhizin (<0.01%). Glycyrrhizin was separated from the extract using ultrafiltration. The ultrafiltration device included a membrane which was able to selectively separate the glycyrrhizic acid from other components. The yield of the extraction was 10% (10 g extract/100 g powdered licorice root). Corn starch as placebo and licorice extract was filled in capsules in same size and color. Both licorice extract and placebo capsules were provided in the same opaque pockets. They were coded by the company and the researchers and the participants were masked for type of intervention until the data were analyzed. Assessments Assessments were included anthropometric indices, dietary intake, physical activity level and biochemical measurements at baseline and at the end of study. Weight was measured with minimum clothing without shoes to the nearest 0.1 kg. Height was measured without shoes using the SECA stadiometer to the nearest 0.1 cm by a trained staff. Body mass index (BMI) was calculated by dividing the weight in kilogram to square with height in meter. Based on WHO classification, subjects with BMI of 25–29.9, 30–34.9 and 35–39.9 kg/m2 were considered overweight, grade I obese and grade II obese, respectively. 24-h dietary record questionnaires (2 work days and 1 weekend) were filled out by patients at the

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baseline and the end of study for dietary assessment. Physical activity level was determined by International Physical activity Questionnaire (IPAQ) with face to face interview [13] at the baseline and after 8-week intervention. At baseline and at the end of trial, after an overnight fasting, 5 ml venous blood was collected. The serum samples were separated from whole blood by centrifugation at 2000 rpm for 10 min at room temperature. Lipid profile was measured on the day of sampling. The levels of serum TC, high-density lipoprotein (HDL-c) and triglyceride (TG) were measured by enzymatic colorimetric methods with a commercially available kit (Pars Azmone, Tehran, Iran) on an automatic analyzer (Abbott, model Alcyon 300, USA). For all lipid parameters, inter- and intra assay coefficient of variations (CVs) were less than 5%. Serum LDL-c was calculated by Friedewald equation [14]. Finally, atherogenic indices (log TG/HDL-c), LDL-c/HDL-c, TC/HDL-C) ratio were calculated.

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Statistical analysis Data were analyzed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). The results were expressed as mean ± SD. The normality of the distribution of data was evaluated by the one-sample Kolmogorov–Smirnov test. Paired t tests (for baseline measurements) and analysis of covariance were used to compare normal quantitative variables after intervention. Mann–Whitney U test was used for comparison of non-parametric variables. Analysis of covariance (ANCOVA) adjusting for dietary intake, weight changes and baseline values were used to identify any differences between the two groups after intervention. For calculating the percentage of mean changes of markers, at the beginning and at the end of the study, mean changes of markers from baseline were calculated in each group by [(8 weeks values-baseline values)/baseline values)] × 100. p < 0.05 was considered significant.

Fig. 1. Study flow chart. Table 1 Baseline characteristics of the study participants. Variable

Intervention group (n = 29)

Placebo group (n = 29)

p-Valuec

Age (years) Family history of obesity (%) Weight loss diet history (%) Taking anti-obesity medication history (%)

36.0 ± 11.97 83.3 50 36.1

33.6 ± 4.8 82.4 40 32.4

0.23a 0.91b 0.47b 0.74b

a b c

Independent t-test. K square. p < 0.05 considered significant.

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Table 2 Anthropometric indices in two groups at the baseline and at the end of the study. Variable Weight (kg)

Period Initial End p-Valueb

Height (cm) BMI (kg/m2 )

a b c d

Initial End p-Valueb

Intervention (n = 29) 87.6 ± 15.5a

Placebo group (n = 29) 81.9 ± 11.0 79.2 ± 8.6 <0.01

85.5 ± 16.1 <0.01

p-Value 0.08c 0.08c

161.9 ± 8.3

158.4 ± 5.8

0.04c

33.6 ± 4.8 32.8 ± 4.8 0.3

32.7 ± 3.7 32.3 ± 3.5 0.6

0.4d 0.2d

Mean ± S.D. Paired t-test; p < 0.05 considered significant. Independent t-test; p < 0.05 considered significant. ANCOVA (adjusted for height).

Results As shown in Fig. 1, of the 64 participants, 58 subjects completed the study (intervention group, n = 29; control group n = 29). Six participants (n = 3 in the Licorice group and n = 3 in the placebo group) were excluded due to compliance failure to the study procedure (n = 5) and who did not available (n = 1). Table 1 shows the baseline characteristics. No significant differences in baseline characteristics were observed between the two groups. Participants did not take any herbal or chemical medicine at the baseline and throughout the trial. They did not report any side effects for licorice supplementation.

At the baseline, no significant differences were observed in body weight (p > 0.05). After adjusting for height, BMI did not indicate any significant differences within the two groups (Table 2). At the end of the study reduction in body weight was significant in both groups as compared to the baseline (p < 0.05), but it was not significant between the two groups (p > 0.05). At the beginning of the study, no significant differences were observed in dietary intake between two groups. In both groups, energy and macronutrient intake decreased at the end of the study (p < 0.05). Cholesterol and monounsaturated fatty acid (MUFA) intake decreased significantly in the intervention group as compared to the control group (Table 3). Comparison of the baseline physical activity levels between the two groups did not indicate

Table 3 Comparison of dietary intake in the study group at baseline and at the end of the study. Variable

Period

Intervention (n = 29)

Placebo group (n = 29)

p-Valuec

Energy (kcal/day)

Initial End p-Valueb

2370.5 ± 385.6a 1431.5 ± 295.2 <0.01

2519.2 ± 47.9 1381.7 ± 233.1 <0.01

0.19 0.45

Carbohydrate (g/day) (% of Energy)

Initial End p-Valuec

Protein (g/day) (% of Energy)

Initial End p-Valuec

Fat (g/day) (% of Energy)

Initial End p-Valuec

Saturated fatty acid (g/day)

Initial End p-Valuec

14.5 ± 7.3 9.7 ± 3.75 <0.01

15.3 ± 5.9 10.5 ± 4.9 <0.01

0.64 0.63

Mono unsaturated fatty acid (g/day)

Initial End p-Valuec

13.9 ± 5 10.7 ± 4.76 0.01

13.3 ± 3.7 11.2 ± 5.9 0.2

0.57 0.74

Poly unsaturated fatty acid (g/day)

Initial End p-Valuec

15.2 ± 5.4 18.9 ± 23.8 0.3

16.1 ± 8.30 15.1 ± 12.7 0.6

0.4 0.2

a b c

Mean ± S.D. Paired t-test; p < 0.05 considered significant. Independent t-test; p < 0.05 considered significant.

313.9 ± 79.7 (52.9) 203.4 ± 48 (56.8) <0.01 95.3 ± 36.1 (16) 42.7 ± 13.3 (11.93) <0.01 112.3 ± 51 (31.1) 47.1 ± 19.7 (31.2) 0.3

322.3 ± 63.04 (51.1) 199.8 ± 45. 4 (56.2) <0.01

0.65 0.75

92.8 ± 23.5 (14.7) 41.1 ± 8.86 (11.89) <0.01

0.76 0.56

100.3 ± 37.3 (34.2) 42.9 ± 13.6 (31.9) 0.6

0.31

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Table 4 Comparison of lipid profile and atherogenic indices in the study group at the baseline and at the end of the trial. p-Valueb

Variables

Period

Intervention (n = 29)

Placebo group (n = 29)

TG (mg/dl)

Initial End p-Valuea

152.4 ± 54.1 131.5 ± 49.9 0.08

131.4 ± 69.0 140.2 ± 62.0 0.7

0.2 0.5

TC (mg/dl)

Initial End p-Value

178.3 ± 28.8 168.8 ± 29.6 0.02

193.6 ± 40.2 191.0 ± 28.4 0.2

0.1 0.01

HDL-c (mg/dl)

Initial End p-Value

38.8 ± 6.69 43.1 ± 8.6 <0.01

40.3 ± 7.6 42.3 ± 8.2 0.1

0.4 0.7

LDL-c (mg/dl)

Initial End p-Value

108.7 ± 31.0 99.8 ± 32.3 0.05

128.4 ± 34.7 124.1 ± 25.0 0.2

0.04 <0.01c

VLDL (mg/dl)

Initial End p-Value

32.6 ± 14.5 24.8 ± 10.5 <0.01

27 ± 9.7 28 ± 10.5 <0.01

0.14

TC/HDL-c

Initial End p-Value

4.5 ± 1.1 3.8 ± 1 <0.01

5.3 ± 0.8 5 ± 0.9 0.1

<0.01

LDL-c/HDL-c

Initial End p-Value

2.7 ± 1 2.3 ± 1 <0.01

3.6 ± 0.6 3.2 ± 0.8 0.1

<0.01c

Log TG/HDL-c

Initial End p-Value

0.5 ± 0.2 0.4 ± 0.1 <0.01

0.5 ± 0.2 0.5 ± 0.2 0.9

0.5 0.03

a b c

Paired t-test. ANCOVA (adjusted for dietary intake, weight changes and baseline values). ANCOVA (adjusted for LDL-C at the baseline); p < 0.05 considered significant.

any significant differences between the baseline and at the end of the trial. At the baseline, there were no significant differences for lipid profile except for LDL-c levels. After 8-weeks of the intervention, TC and LDL-c levels decreased in licorice group as compared to the placebo group (p < 0.01; ANCOVA adjusted for dietary intake, weight changes, baseline LDL-c level and baseline value). However, reductions in TG (33.6 mg/dL; −31.70%) and HDL-c levels (4.3 mg/dL; 12.12%) were not significant in the intervention group as compared to the control group (Table 4). At the baseline, differences between TC/HDL-c and LDL-c/HDL-c ratios between the two groups were significant (p < 0.01 and p = 0.03, respectively). After adjusting for the baseline values, both the ratios decreased significantly in the intervention group as compared to the control group (pvalue < 0.01). Also, comparing of logs of TG/HDL-c between the two groups did not reveal any significant differences at the beginning of the trial, but at the end of the study it decreased significantly (p = 0.03). Discussion In the present study, eight weeks supplementation of dried licorice root extract used concurrently with a weight loss diet decreased TC, LDL-c and atherogenic indices in both overweight and obese subjects. There are limited and contradictory

studies regarding the effects of licorice on body weight and lipid profile (Table 5). Some studies indicated suppressive effects of licorice on weight gain but others reported that licorice did not have any effective role on body weight. In line with our study, Bell et al. [11] reported that GlavonoidTM , “a commercial licorice extract”, did not change body weight in overweight, obese subjects and athletes. Also Hajiaghamohammadi et al. [15] could not find any effect of aqueous licorice extract on weight and BMI of patients with non-alcoholic fatty liver disease. In contrast, different group of studies indicated a suppressive effect of licorice in weight gain. Tominaga et al. [10] stated that supplementation with LFO could impede increasing rate of weight gain with not decreasing effect on body weight. In a study by Nakagawa et al. [16], obese diabetic mice, fed with a diet containing LFO, showed a suppression of weight gain. Further, Aoki et al. [17] showed a significantly slower weight gain and abdominal adipose tissue accumulation after addition of LFO to diet of obese mice. Also, Malik et al. [7] administrated that ethanolic extract of licorice prevented weight gain in rats. Differences in the dietary intake, dietary habits, physical activity level, dose and type of licorice, duration of intervention, age and BMI range and ethnicity might have resulted in the contrary results. On the other hand, previous studies did not compare the effects of licorice concurrent with weight loss diet and participants did not change their dietary intake and dietary habits throughout the trial.

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Table 5 Summary table of previous studies. Author/year

Sample

Type of licorice/dose

Duration of intervention

Result

Bell et al. (2011) [11]

Study 1: overweight or grade I–II obese men Study 2: athletic men

LFOa

8 weeks

Hajiaghamohammadi et al. (2012) [15] Tominaga et al. (2009) [16]

Non-alcoholic fatty liver disease patients Overweight subjects

Aqueous licorice root extract (2 g) LFO (300, 600, 900 mg)

2 months

No changes in lipid profile and weight in both studies Less fat gain and elevation in blood lipids in study 2 No changes in weight and BMI

Aoki et al. (2007) [17]

Obese mice

LFO (0.5%, 1%, or 2%)

8 weeks

Malik et al. (2011) [7]

Male wistar rats

8 weeks

Fuhrman et al. (2002) [9]

Hypercholesterolemic patients Albino mice

Freeze dried powder of ethanolic extract (100, 400 mg/day) Licorice root extract (0.1 g/d) Licorice root extract (1 ml of 0.2, 0.7 and 1 mg ml/day)

1 months

Saleem et al. (2011) [19]

a

8 weeks

1 month

↓Total body fat mass in all groups 900 mg/day: ↓Visceral fat, body weight, BMI, ↓LDL-c 1 and 2%: ↓Weight of abdominal white adipose tissues ↓Body weight gain 2%: ↓Adipocytes size ↓ Fatty degenerative state of the hepatocytes Pancreatic lipase inhibitor effects

↓ LDL-c ↓ TG level ↓TC, TG, LDL-C, VLDL in all groups ↑HDL-c in all groups 0.2 mg/dL was the best anti-lipidemic dosage

LFO, licorice flavonoid oil.

As same for weight loss, previous studies indicated discrepancy results about the effects of licorice on lipid profile. Fuhrman et al. [9] indicated that licorice extract reduced TC and LDL-c levels, respectively in moderately hypercholesterolemic patients. In Malik et al. [7] study, dried ethanolic extract of licorice led to a reduced TC and TG levels in a dose dependent manner. Based on Honda et al. [18] trial, diet containing LFO decreased TG and VLDL-c levels in obese rats. Saleem et al. [19] demonstrated that ethanolic extract of licorice root decreased TC, TG, LDL-c, VLDL-c and increased HDL-c levels. Also, doses of 400 and 800 mg/kg/day of licorice extract in a study by Shalaby et al. [20] decreased TC and TG levels with no changes in LDL, HDL and VLDL-c in male rats. On the contrary with the current study, Bell et al. [11] reported that LFO had no effects on lipid profile in overweight/obese subjects and athlete men after 8 weeks. Differences in the dietary intake, basal lipid profile status, dose and type of licorice and duration of interventions might resulted in the contrary results. Mechanisms for the effect of licorice on lipid profile have not yet been clarified. Losing weight and reduction in fat mass can participate in ameliorating lipid profile. Liquiritigenin is one of the flavanone components in the licorice. It can activate peroxisome proliferator-activated receptor-␥ (PPAR-␥) which may act through nuclear erythroid 2-related factor 2 (Nfr2) pathway. Also, Glabridin, a PPAR-␣ agonist, can increase resting metabolic rate [21,22]. It was reported that rising in white adipose tissue was suppressed in obese mice fed with a high fat diet containing licorice extract. Reduction in fatty

acid synthesis and rising fatty acid oxidation are two main characteristics of licorice root [10]. However, in the present study, supplementation of licorice extract concurrently with low calorie diet did not decrease body weight as compared to losing weight alone. Therefore, other possible mechanisms including effects of licorice on insulin resistance, liver function [13], oxidative [23–25] and anti-inflammatory status [26], gene expression or suppuration [27–29] might have triggered lipid profile improvement. The present study had some limitations. Firstly, duration of the intervention was short and pure effect of licorice (licorice supplementation without weight loss diet) was not evaluated. Secondly, it would have been better if we could measure serum levels of Glabridin, oxidized LDL-c, apo-lipoproteins and antioxidant enzymes to better clarify underlying mechanisms involved in regulation of serum lipid by the licorice. Thirdly, 24-h dietary recall was used for evaluating dietary intake which can be attenuated by memorial status and fidelity of the study subjects [30–32].

Conclusion Supplementation with dried extract of licorice root concurrently with low calorie diet can improve lipid profile. It seems that licorice extract can be used as an auxiliary therapy in overweight and obese subjects with dyslipidemia.

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Conflict of interest Authors declared no conflict of interest.

[16]

Acknowledgments

[17]

We are grateful to all the participants for their cooperation. The authors also would like to thank Nutrition Research Center, Tabriz University of Medical Sciences (Grant No. 9271), Tabriz, Iran for financial support.

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