**Long-Chain Polyunsaturated Fatty Acids Are Associated with Blood Pressure and Hypertension over 10-Years in Black South African Adults Undergoing Nutritional Transition**


Received: 16 August 2019; Accepted: 2 September 2019; Published: 6 September 2019

**Abstract:** Nutritional transition in Africa is linked with increased blood pressure (BP). We examined 10-year fatty acid status and longitudinal associations between individual long-chain polyunsaturated fatty acids (PUFA), BP and status of hypertension (≥140/90 mmHg and/or medication use) in black South Africans. We included 300 adults (>30 years) participating in the Prospective Urban Rural Epidemiology study, and analysed data from three consecutive examinations (2005, 2010 and 2015 study years). Fatty acids in plasma phospholipids were analysed by gas chromatography-mass spectrometry. We applied sequential linear mixed models for continuous outcomes and generalized mixed models for the hypertension outcome, in the complete sample and separately in urban and rural subjects. Mean baseline systolic/diastolic BP was 137/89 mmHg. Ten-year hypertension status increased among rural (48.6% to 68.6%, *p* = 0.001) and tended to decrease among urban subjects (67.5% to 61.9%, *p* = 0.253). Regardless of urbanisation, *n*-6 PUFA increased and eicosapentaenoic acid (EPA, C20:5 *n*-3) decreased over the 10-years. Subjects in the highest tertile of arachidonic acid (C20:4 *n*-6) had 3.81 mmHg lower systolic (95% confidence interval (CI): −7.07, −0.54) and 3.82 mmHg lower diastolic BP (DBP) (95% CI: −5.70, −1.95) compared to the reference tertile, irrespective of lifestyle and clinical confounders. Similarly, osbond acid (C22:5 *n*-6) was inversely associated with DBP. Over the 10-years, subjects in the highest EPA tertile presented with +2.92 and +1.94 mmHg higher SBP and DBP, respectively, and with 1.46 higher odds of being hypertensive. In black South African adults, individual plasma *n*-6 PUFA were inversely associated with BP, whereas EPA was adversely associated with hypertension, supporting implementation of dietary fat quality in national cardiovascular primary prevention strategies.

**Keywords:** black Africans; blood pressure; hypertension; long-chain polyunsaturated fatty acids; longitudinal study; nutritional transition; PUFA

#### **1. Introduction**

Urbanisation in sub-Saharan Africa followed by increased consumption of energy-dense food [1], is linked with an increase in cardiovascular disease, obesity and diabetes [2], and the highest

prevalence of mean blood pressure (BP) since 1980 [3]. Among black South African adults (>30 years) participating in a large-scale Prospective Urban Rural Epidemiology (PURE) study, a five-year increase in hypertension rate has been reported [4]. Nutritional interventions remain a cost-effective approach in suppressing the hypertension burden in the population. Baseline data from the PURE study indicate low total fat and omega-3 (*n*-3) intake in black South Africans [5]. In the same study, dietary *n*-3 long-chain polyunsaturated fatty acids (PUFA) were associated with serum lipids. Dietary eicosapentaenoic acid (EPA; C20:5 *n*-3) was associated with dyslipidemia and docosahexaenoic acid (DHA; C22:6 *n*-3) with favourable lipid status in the population [5]. These results indicate a unique metabolic profile in black South Africans related to fat catabolism and a specific role of individual fatty acids in cardiometabolic function.

Long-chain *n*-3 PUFA from marine foods demonstrate BP-lowering effects [6]. Clinical studies show that long-chain *n*-3 PUFA consumption diminishes the risk of cardiac death, potentially through regulation of triglycerides, heart rate and BP [7]. Measurement of the intake remains a challenge, since questionnaires are imprecise in differentiating intake of individual long-chain fatty acids. Self-reported information from dietary questionnaires is further limited by recall bias and participants' non-compliance to fat-intake related questions [8]. Fatty acids in plasma phospholipids are however reliable biomarkers reflecting fat intake of the preceding 6-8 weeks [9]. Dietary fatty acids are endogenously catalysed by desaturase-5 and desaturase-6 enzymes encoded by FADS1 and FADS2 genes, respectively. The conversion results in the formation of long-chain PUFA with diverse physiological functions. Plasma fatty acids are of raising importance as prognostic biomarkers of cardiovascular disease [10]. Data from the Women's Health Initiative study show inverse association between *n*-3 group and coronary heart disease risk in post-menopausal women [11]. A recent review underlined the importance of individual circulating fatty acids with regards to total and cause-specific mortality, type 2 diabetes mellitus and cardiometabolic indices [10]. Plasma fatty acids have been associated with BP [12–14], and observational data suggest protective associations of individual circulating *n*-3 long-chain PUFA with BP [15,16]. In middle-aged and elderly Chinese community dwellers, serum patterns presenting with high DHA levels were inversely associated with BP [12] and hypertension status [17]. Recent data from the PURE study showed association between plasma phospholipid fatty acid patterns and obesity and metabolic syndrome in black South African adults [18], however the link with vascular function has not yet been examined in the population.

Therefore, the objective of this longitudinal study was to evaluate the relationship between fat intake and BP in black South Africans. We measured and reported fatty acids in plasma phospholipids over 10 years, in a sample of black South Africans participating in the PURE and residing in rapidly urbanizing areas. To address the study objective, we examined the associations between individual long-chain *n*-3 and *n*-6 PUFA with BP and hypertension status over the 10-years. We also evaluated the 10-year associations separately in subjects residing in urban and rural areas.

#### **2. Materials and Methods**

#### *2.1. Study Design and Selection of Study Sample*

This study formed part of the South African cohort of the PURE study, an international study investigating health implications linked with urbanisation in low-, middle- and high-income countries [19]. The cohort included 2010 (1260 women and 750 men at baseline) randomly selected black adults (>30 years), from urban and rural areas of the North West Province, without use of chronic medication and/or any self-reported acute illness. Permission for the study was obtained from the provincial Department of Health. Trained fieldworkers fluent in both English and Setswana conveyed all information. All subjects voluntarily gave written informed consent for the participation in 2005, continuous consent throughout the study and again in 2010 and 2015. The study protocol adhered to the 1983 Declaration of Helsinki and was approved by the Health Research Ethics Committee of the Faculty of Health Sciences at the North-West University (Potchefstroom campus). Privacy and confidentiality were ensured during the data-gathering process, data and sample storage and management.

For the purposes of this study and analysis we applied a repeated-measures design and included data and assessments from 2005 (baseline), 2010 and 2015 (follow-up). A sub-cohort of 711 subjects were randomly selected at 2010 (Supplementary Materials Figure S1) and fatty acid analysed accordingly. Due to the loss to follow-up in the 2015 study year, we ended up with 300 complete sets of samples across the 3 study years, inclusive of fatty acid profiles and vascular outcomes. The 300 corresponding subjects were thus included in the longitudinal analysis.

#### *2.2. Clinical and Biological Measurements*

Fasting blood samples were collected from the antecubital vein with a sterile winged infusion set and were with minimal stasis. The samples were collected by a registered nurse and stored at −80 ◦C. In rural areas the samples were stored at −18 ◦C up to 5 days; afterwards transported to the laboratory facility and stored at −80 ◦C until analysis. Plasma phospholipid fatty acid composition was analysed as described previously [20]. Briefly, lipids were extracted with chloroform: methanol (2:1 v/v) from thawed ethylenediaminetetraacetic acid-plasma samples according to the modified Folch method [21]. The phospholipid fatty acid fractions were subsequently isolated by thin layer chromatography, further transmethylated to fatty acid methyl esters, and analysed by quadrupole gas chromatography electron ionization mass spectrometry using an Agilent Technologies 7890 A GC system [20]. Levels of each individual fatty acid were expressed as a percentage of the total phospholipid fatty acid pool in plasma. To examine longitudinal associations with vascular function, we used data for the long-chain *n*-3 fatty acids: EPA, DHA and docosapentaenoic acid (C22:5 *n*-3); and *n*-6 fatty acids: dihomo-γ-linolenic acid (DGLA, C20:4 *n*-6), arachidonic acid (AA, C20:4 *n*-6), adrenic acid (C22:4 *n*-6), and docosapentaenoic acid (osbond acid, C22:5 *n*-6).

Brachial BP was measured in duplicate in a sitting position by using a validated OMRON device (Omron Healthcare, Kyoto, Japan) after subjects rested for 10 min, as reported elsewhere [4]. To be categorized as hypertensive, the participants had to exceed either SBP (140) or DBP (90) or both thresholds (or had to use antihypertensive medication) [22]. The PURE-standardized demographic, socio-economic and lifestyle questionnaires were interviewer administered [19]. Education was confirmed if any formal education was present. Quantitative FFQ and the physical activity index questionnaire previously developed and validated for South Africans were used [23,24]. The FFQ was conducted in the morning on the study visit day. Study participants were provided with a list of food items (food or drinks) and were asked how often they had consumed specific foods or drinks on average in the preceding year. Assessment of height, weight, waist circumference, serum lipids and other biochemical measurements were described previously [4,5].

#### *2.3. Statistical Analyses*

Statistical analysis was performed using SAS 9.4 (SAS Institute Inc, Cary, NC, USA). Continuous variables were checked for the distribution by visual inspection of histogram and skewness. Normal, non-normal and categorical data are presented as mean ± SD, median [25th, 75th percentile] and percentages, respectively. Baseline between-subject differences across urbanisation areas were tested by independent *t*-test and Mann Whitney test, for normal and non-normal data, respectively. Between-subject differences across the three study years were analysed by general linear model. Pearson's correlation coefficients were computed to evaluate the relationship between plasma long-chain phospholipid PUFA, *n*-3 intake (cumulative intake of dietary α-linolenic acid (ALA, C18:3 *n*-3), EPA and DHA) and marine fatty acid intake (cumulative intake of EPA and DHA).

We evaluated 10-year associations between individual *n*-3 and *n*-6 long-chain PUFA (exposures) and the outcomes, by inclusion of data for the three study points. We applied linear mixed models for continuous outcomes (SBP and DBP) and generalized mixed models for the outcome of hypertension, with individual exposure fatty acids included as tertiles of the plasma phospholipid content. Sequential

regression-based models were applied: Model 1 with fatty acid predictors controlled for age, gender and level of urbanisation (urban or rural); Model 2 further controlled for lifestyle confounders, including level of education (no education or any kind of formal education), self-reported use of tobacco (current, former or never used), use of hypertension medication (yes or no), body mass index, physical activity index and dietary intake of alcohol (g per day). The urbanisation status was treated as random factor, and repeated measures design was accounted for by use of adequate syntax within the procedures. We performed a prespecified subgroup analysis, stratified by urbanisation status (rural and urban areas). The level of significance was set at 0.05 (2-tailed).

#### *2.4. Sensitivity Analyses*

We further tested whether multivariable-adjusted associations were independent on dietary and fat intake in three consecutive steps: adjusting for total energy intake, following total fat and carbohydrate intake, and lastly ratio of monounsaturated to saturated fat intake, and soluble fiber intake, latter known to be protective towards vascular health [25,26]. We also tested whether our associations survived upon adjustment for potential effect mediators linked with BP, including total cholesterol, triglycerides, fasting glucose, and γ-glutamyl transferase reported to be associated with hypertension in this population [4].

#### **3. Results**

#### *3.1. Baseline Characteristics of the 300 Rural and Urban Black South Africans*

The study sample included 300 black South Africans (mean age = 53.12 ± 9.83), out of which 91 were men, 140 rural residents (46.7%), and 42.3% overweight subjects, mostly women (53.6% and 16.5% women and men who are overweight, respectively, *p* < 0.0001). In total, 19.7% and 23% subjects had elevated total cholesterol and triglycerides, respectively. Only 2.3% of participants were either diagnosed with type 2 diabetes or actively taking anti-diabetic medications.

Urban subjects presented with higher hypertension prevalence and had higher SBP and DBP, then rural residents. The intake of total energy, total carbohydrates, total fat, and specific fat groups were higher among urban subjects (Table 1).


**Table 1.** Baseline characteristics of the 300 rural and urban black South Africans.


**Table 1.** *Cont*.

HDL-c, High-density lipoprotein cholesterol; LDL-c, Low-density lipoprotein cholesterol; GGT, γ-glutamyl transferase; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; *n*-3, Intake of EPA, DHA and plant-originated α-linolenic acid. Data are presented as mean (SD), median [25th, 75th] or percentage for categorical variables. <sup>1</sup> Significance values calculated by use of independent *t*-test or Mann-Whitney test.

#### *3.2. Ten-Year Changes in Blood Pressure and Status of Hypertension*

Within all 300 subjects, we observed a non-significant increase in hypertension rate (58.7%, 61.3% and 65% in 2005, 2010 and 2015, respectively; *p* = 0.210). There was a significant increase in the hypertension rate in rural residents (48.6%, 51.4% and 68.6% in 2005, 2010 and 2015, respectively; *p* = 0.001), and a non-significant decrease within urban dwellers (67.5%, 70% and 61.9% in 2005, 2010 and 2015, respectively; *p* = 0.253). At baseline and in 2010 there were significantly more hypertensive subjects in urban areas, with no differences in 2015. SBP and DBP significantly decreased across the 10-years in urban areas (Supplementary Materials Table S1).

#### *3.3. Ten-Year Changes in Long-Chain Plasma Phospholipid Fatty Acids*

There was a significant decrease in γ-linolenic acid. Long-chain *n*-6 PUFA (DGLA, AA, adrenic and osbond acid) increased and long-chain *n*-3 (EPA and DHA) decreased across the 10 years (Table 2). Ten-year fatty acid status across urbanisation areas is presented in Supplementary Materials Table S2. Regardless of urbanisation level, we observed increases in DGLA, AA and osbond acid. In urbans only, adrenic acid increased and EPA and DHA decreased over the 10-years. In rural subjects, docosapentaenoic acid and DHA content increased and EPA tended to decrease.


**Table 2.** Plasma phospholipid fatty acid status across 10 years in 300 black South Africans.

EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid. Age and urbanization factor-adjusted data presented as mean (SD) or median [25th, 75th]. <sup>1</sup> Probability trends associated with changes over 10-years calculated by general linear model adjusted for age and urbanization factor.

#### *3.4. Relationship Between Dietary Intake of N-3 Fatty Acids and Long-Chain Fatty Acids in Plasma Phospholipids*

A HeatMap of Pearson correlations among baseline intake of *n*-3 fatty acids and long-chain plasma phospholipid fatty acids is presented in Figure 1.

**Figure 1.** Baseline correlations between intake of *n*-3 fats and long-chain plasma fatty acids in 300 black South Africans: HeatMap of Pearson coefficients. DGLA, dihomo-γ-linoleic acid; AA, arachidonic acid; EPA, Eicosapentaenoic acid; DPA\_n3, docosapentaenoic acid; DHA, Docosahexaenoic acid; Intake\_EPADHA, Cumulative intake of preformed EPA and DHA; Intake\_Omega3, Cumulative intake of EPA, DHA and plant-originated essential α-linolenic acid; <.0001, denotes statistical threshold (*p*) of < 0.0001 associated with correlation pair.

Cumulative *n*-3 intake was correlated with status of EPA and DHA, and negatively correlated with *n*-6 long-chain PUFA: AA (*r* = −0.113, *p* = 0.052), adrenic acid (*r* = −0.280, *p* = < 0.0001) and osbond acid (*r* = −0.198, *p* = 0.001) (Supplementary Materials Table S3). Cumulative intake of preformed EPA and DHA did not correlate with status of any long-chain *n*-3 PUFA, yet negatively correlated with long-chain *n*-6 PUFA: AA (*r* = −0.136, *p* = 0.018), adrenic acid (*r* = −0.126, *p* = 0.030) and osbond acid (*r* = −0.180, *p* = 0.002).

Fatty acids in plasma phospholipids within either the *n*-3 or *n*-6 group were correlated among each other. Adrenic acid inversely correlated with EPA and DHA. Osbond acid inversely correlated with EPA, while the inverse relationship with DPA and DHA did not reach statistical significance (Supplementary Materials Table S3).

*3.5. Ten-Year Associations between Long-Chain Plasma Phospholipid Fatty Acids with Blood Pressure and Hypertension in Black South African Adults*

#### 3.5.1. Associations between *N*-3 Long-Chain Fatty Acids and Blood Pressure

Subjects in the highest tertile of plasma EPA content over the 10-years had 1.94 mmHg higher DBP in comparison with subjects in the lowest (multivariable β for T3 vs. T1: 1.94 (95% CI: 0.01, 3.87)), regardless of age, gender, BMI, educational background, intake of alcohol, smoking status, level of physical activity and use of hypertension medication (Table 3).

Urban subjects within the highest EPA content were with higher SBP (Supplementary Materials Table S4). DHA was inversely associated with DBP in rural dwellers (multivariable β for T3 vs. T1: −3.91 (95% CI: −7.04, −0.78).


**Table 3.** Ten-year associations between plasma phospholipid long-chain fatty acids and blood pressure in 300 black South Africans.

EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; T1, T2, T3, Increasing tertiles of plasma phospholipid fatty acid content. <sup>1</sup> Model 1 adjusted for age, gender and urbanization factor. <sup>2</sup> Model 2 further adjusted for level of education, use of tobacco, use of hypertension medication, body mass index, physical activity index and dietary intake of alcohol (g). <sup>3</sup> Probability values associated with β estimating absolute change in blood pressure (in mmHg) with regards to 10-year change in a fatty acid level.

#### 3.5.2. Associations between *N*-6 Long-Chain Fatty Acids and Blood Pressure

Across the 10 years AA was inversely associated with SBP and DBP (Table 3). Subjects in the highest tertile were with 3.81 and 3.82 mmHg lower SBP and DBP, respectively, in comparison with subjects within the reference tertile (multivariable β for T3 vs. T1: −3.81 (95% CI: −7.07, −0.54) for SBP and −3.82 (95% CI: −5.70, −1.95) for DBP). Osbond acid was inversely associated with DBP (Table 3). Subjects in the highest tertile had 3.71 mmHg lower DBP in comparison with reference tertile (multivariable β for T3 vs. T1: −3.71 (95% CI: −5.73, −1.70)).

The inverse associations remained significant in urban residents for both AA and osbond acid, and osbond acid was also inversely associated with SBP (Supplementary Materials Table S4). In urban dwellers DGLA was inversely associated with DBP.

#### 3.5.3. Associations between Long-Chain Plasma Fatty Acids and Status of Hypertension

Plasma phospholipid fatty acids were not associated with 10-years status of hypertension in the 300 black South African adults, except for EPA (Figure 2). Subjects in the highest tertile of EPA content were with 1.46 higher odds of being hypertensive across the 10-years, in comparison with those in the reference tertile (multivariable OR for T3 vs. T1: 1.46 (95% CI: 1.03, 2.08)) (Supplementary Materials Table S5). Adverse relationship of DGLA was lost upon controlling for potential confounders.

EPA remained adversely associated with 10-year hypertension status only in rural subjects within the highest tertile of the content. Furthermore, DGLA and osbond acid were adversely associated upon controlling for confounders known to influence the status. No associations were seen among urban dwellers.

**Figure 2.** Multivariable odds ratio of being hypertensive across 10-years depending on the individual fatty acid content in plasma phospholipids in 300 black South Africans. AA, Arachidonic acid; DGLA, Dihomo-γ-linolenic acid, EPA, Eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, Docosahexaenoic acid; T1, T2, T3, Increasing tertiles of each plasma phospholipid fatty acid content.

#### 3.5.4. Sensitivity Analyses

Observed 10-year associations with BP and hypertension status remained consistent upon sensitivity analyses evaluating contribution of dietary intake affecting fat metabolism, and serum biomarkers.

#### **4. Discussion**

Our study showed that in black middle-aged and elderly South Africans living in rapidly urbanizing areas, individual long-chain plasma phospholipid PUFA were associated with BP across 10 years. The *n*-6 fatty acids were protectively associated with office SBP and DBP, while subjects with the highest EPA content presented with higher DBP. The relationships were independent of age, gender, BMI, educational background, intake of alcohol, smoking status, level of physical activity, use of hypertension medication, total energy and intake of fat, and glucolipid biomarkers. Observed relationships between individual PUFA and vascular health confer the role of dietary fat quality in tailoring population-specific nutritional policies in black South Africans.

In our study EPA was adversely associated with 10-years status of hypertension. Previous studies suggest favourable associations of EPA intake with vascular function [7,27,28] and cardiovascular events [29]. In a prospective study among 1477 adult community dwellers, subjects in the highest quartile of erythrocyte EPA content had significantly lower SBP and DBP across 3 years [30]. However, the latter study included fatty acid biomarkers measured at single time-point, while our study considered time-dependent variations in the PUFA content by inclusion of the data from 3 consecutive examinations across the 10-years. Herein observed adverse EPA associations might be attributed to the aging of participants, an epidemiological context associated with increase in BP. EPA is a precursor of prostaglandins with limited vasodilatory properties and its physiological function might be outweighed by the natural course of aging. Further on, associations reflecting absolute changes with incremental EPA increase were relatively small. Subjects in the highest EPA tertile presented with only +2.92 and +1.94 mmHg higher SBP and DBP over the 10-years, respectively, in a multivariable-adjusted model. Of importance, baseline mean SBP/DBP was already higher (137/89 mmHg) and is with expected increasing trend over time due to aging, altogether potentially contributing to the observed 1.46 higher odds of being hypertensive with incremental EPA increase.

Our results should be interpreted in context of a population free of acute or chronic illnesses and residing in rapidly urbanizing areas. We showed raising hypertension prevalence across the 10-years, significant in rural areas. In urban dwellers we observed a non-significant decrease in hypertension rate, partly due to 10-years decline in both SBP and DBP of approximately 7 mmHg. Notably, 19.3% rural and 17.5% urban dwellers used hypertension medication at baseline. The number dramatically increased across the 10-years resulting in 35.7% and 33.1% of the respective subjects on medication in 2015, partly because study participants diagnosed with baseline high BP were instructed to their local clinics. Compliance with therapeutic protocols might be more prominent among urbans with readily available healthcare, resulting in a stabilization of hypertension prevalence across the 10-years. In our study, long-chain PUFA were not associated with status of hypertension across the 10-years, except for EPA being adversely related. Increased medication use might have masked the associations, due to the interaction with lipid metabolism [31]. The large-scale Atherosclerosis Risk in Communities study previously showed protective associations between total PUFA cholesterol ester content and 6-years prevalent and incident hypertension, with individual EPA and AA exhibiting adverse associations [32]. Overall, our results remain inconclusive on the association between fatty acids and hypertension status in the black South Africans, and larger cohorts should confirm the relationship.

The metabolic context of our results is of consideration. Within the sample of black South Africans, we found unusually high levels of long-chain PUFA in plasma phospholipids. Previously reported levels of serum AA were higher in African Americans with diabetes or metabolic syndrome, in comparison with their counterparts of European ancestry [33]. Still the levels were substantially lower (9.8 ± 1.9%) [33], in comparison with our study (mean range across the 10 years: 13.57–18.13%). In a larger population of Chinese subjects of similar age group as our participants, percentage of AA in total serum content was 6.02 ± 1.61 [12]. Also, in our study 10-years mean plasma phospholipid content of DHA exceeded 3.5%, which is above 2.5–3.4% previously reported in healthy populations [12,34–36]. Higher levels of long-chain AA and DHA observed herein might result from marked desaturase-6 activity. Observed DHA content is of special importance as only up to 1% of dietary ALA is endogenously converted to DHA [37] and our population had substantially low *n*-3 intake at baseline (year of 2005) [5] (median of 33 to 61 mg EPA +52 to 109 mg DHA below recommendations by FAO [38]). According to 2004 International Society for the Study of Fatty Acids and Lipids expert opinion, recommended combined EPA + DHA intake in general population should be at least 500 mg daily, conferring substantially low intake of the fatty acids in our subjects. A low fat, high carbohydrate

diet is reported in other urbanizing populations [39] and is associated with augmented fatty acid synthesis [40]. We thus speculate that restricted intake of *n*-3 rich food in the black South Africans might be a conditional metabolic factor enhancing desaturase activity towards physiologically active long-chain plasma products, including AA and DHA. Notably, in our study baseline intake of marine PUFA was not correlated with its plasma phospholipid status. Previous results in 1834 Chinese community dwellers demonstrated strong correlation among erythrocyte long-chain *n*-3 content and their dietary counterparts [30]. However, when we evaluated *n*-3 intake as sum of preformed EPA, DHA and plant-originated ALA we observed a direct correlation with status of EPA (*r* = 0.138, *p* = 0.017) and DHA (*r* = 0.218, *p* = 0.000). The latter suggests that in our subjects, dietary ALA is pronouncedly converted towards plasma long-chain products by activity of desaturase enzymes. Previous reports indicate specific FADS genetic make-up in populations of African descent. Results from the Diabetes Heart Study showed that 81% of African Americans are carriers of FADS rs174537 variant [33], associated with AA, eicosadienoic acid and EPA levels [41]. We suggest that historically low intake of *n*-3 PUFA in the population of black South Africans is coupled with genetically-regulated higher metabolic conversion towards AA and DHA.

We showed inverse associations of AA with BP across the 10-years. Observational studies found plasma AA to be protectively associated with coronary heart disease [42,43] and type 2 diabetes risk [44]. AA is a precursor of eicosanoids with pro-inflammatory properties and vasomodulatory function [45]. AA is also a precursor of epoxydes with anti-vasodilatory function, mediated by soluble epoxyde hydrolase [46]. A favourable balance between *n*-3 and *n*-6 intake potentiates production of vasodilatory eiocosanoids from AA and decreases BP [45,46]. Herein observed protective relationship of AA might be due to metabolic adaptation conditional to a historically low *n*-3 long-chain PUFA intake. The associations of AA were prominent within urban dwellers, potentially due to the interaction with micronutrient intake, such as magnesium known to influence desaturase-6 function [47]. In a previous cross-sectional study of 2447 middle-aged and older Chinese community dwellers, AA exhibited neutral associations with BP, but study subjects in the highest tertile of serum DHA had significantly lower SBP and DBP in comparison with those in the lowest [12]. Although there was an inverse trend, DHA was not significantly associated with BP, potentially due to limited size of our study sample. It is possible that in our population with inherently low *n*-3 PUFA intake, extensive conversion to DHA underpins its incorporation in phospholipid cellular bilayers for non-vascular beneficial effects. Prospective analysis among 381 healthy, middle-aged and elderly subjects participating in the Kuopio Ischemic Heart Disease Risk Factor study also failed to demonstrate associations between individual long-chain *n*-3 serum PUFA and BP over 10 years [48].

We observed protective associations of osbond acid with BP. Dietary contribution to osbond acid status is negligible and its physiological role is due to metabolic conversion. To our knowledge, no previous study reported associations of osbond acid with clinical outcomes. In our study, the 10-year increase in *n*-6 AA and osbond acid were related to clinically relevant 3–4 mmHg lower BP for subjects within the highest tertile of the PUFA content. The protective associations might reflect pronounced utilization of the *n*-6 long-chain products for physiological function in this population with restricted *n*-3 intake. The suggestion to increase *n*-6 intake however remains a controversial approach [49–51] and previous studies suggest neutral effects from increased *n*-6 intake to BP lowering [52,53]. As intake of essential *n*-6 linoleic acid (C18:3 *n*-6) and *n*-3 ALA are highly correlated since both are abundant in plant oils, observed inverse associations might reflect beneficial implications of higher intake of dietary ALA itself and its metabolic products [46,54].

Finally, our results should be placed in the context of a population under urbanisation coupled with transitions in nutritional habits. The protective 10-year associations of AA and osbond acid remained significant in urban dwellers only. In urban subjects only we observed decrease in EPA and DHA in plasma phospholipids, possibly be due to westernised dietary patterns characterized by cooking oils rich in linoleic acid and *n*-6 PUFA [55] and poor intake of *n*-3 sources (such as whole grains, vegetables and marine food). The finding on EPA and DHA decrease thus supports existing

policies on increasing *n*-3 intake in this population undergoing urbanisation [56]. In rural subjects only we observed an increase in DHA, which was also associated with lower DBP. It is less plausible that the increase was due to pronounced intake of DHA from marine food, rather a consequence of enhanced conversion towards long-chain *n*-3 products within rural subjects with significantly lower *n*-3 intake.

Lack of consistent association between plasma *n*-3 PUFA and BP in our study is partly in line with recent findings from ASCEND trial conducted in 16,000 diabetic middle-aged and older subjects [57]. The authors demonstrated no beneficial effects of daily consumption of *n*-3 fish oil capsules (460 mg EPA + 380 mg DHA) in comparison with placebo olive oil, and regarding incidence of serious vascular events upon 7.4 years follow-up [57]. On the other hand, REDUCE-IT showed that among 8000 patients with elevated triglycerides and stable LDL-cholesterol, receiving 2 g of highly purified EPA ethyl ester twice daily was associated with significantly lower risk of composite cardiovascular event, in comparison with placebo and despite the use of statins [58]. Based upon our results and considering the low *n*-3 PUFA intake [5] we may not discard the role of dietary *n*-3 PUFA and particularly EPA in strategies towards BP optimisation in Africa, and future intervention studies with increasing *n*-3 intake should elucidate the relationship.

The strength of our study lays in a repeated-measures design, evaluating time-dependent changes in BP and hypertension related to fat intake and metabolism. Furthermore, urbanisation-specific analyses and inclusion of a panel of demographic and clinical confounders provide robustness to the obtained relationships. We reported dietary and fat intake profiles across urbanisation categories in line with previously reported baseline dietary intake for the complete cohort (*n* 1950) [5] implying generalizability of our results to the population of black South Africans. Herein reported plasma fatty acid profiles are comparable to recent report within larger sample (*n* 711) [18] of the same cohort of black South Africans participating PURE, outweighing potential concern on the limited sample size of 300 subjects. Of note, 10-year attrition rate might have blurred some of the associations. However, we applied longitudinal analysis accounting for time-dependent variation of outcomes and exposures, providing additional reliability to the observed associations. We followed no changes in usage of medication or any other lifestyle confounder, potentially limiting our results. Although we accounted for an array of structured lifestyle, demographic and clinical confounders, the residual confounding cannot be ruled out.

In conclusion, our data advocate for a link between fat intake, blood pressure and urbanisation in a population of black South Africans with historically low omega-3 intake. Ten-year hypertension prevalence increased in the 300 subjects and only in urban residents did we observe a tendency towards 10-year optimization of hypertension status. Regardless of urbanisation areas there was an increase in individual plasma *n*-6 PUFA over 10 years, but only in urbans there was a decrease in EPA and DHA status, supporting policies on *n*-3 dietary reinforcement. The individual *n*-6 PUFA were inversely associated with blood pressure, prominently within urban dwellers. Taken together the results imply a protective mechanism linked with fat metabolism and vascular health in black South African population undergoing rapid nutritional transition. Indicated population-specific metabotype in black South Africans is possibly linked with genetic background and further research on FADS1 and FADS2 variants, desaturase activity and association with vascular function is warranted in the population.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/8/9/394/s1, Figure S1: Flow-diagram explaining selection of the 300 black South Africans for the longitudinal analysis of associations between fatty acids and blood pressure over 10 years, Table S1: Blood pressure across 10 years in black South Africans, Table S2: Plasma phospholipids fatty acid status across 10-years in rural (*n* 140) and urban (*n* 160) black South Africans, Table S3: Baseline correlations between dietary intake of *n*-3 fatty acids and long-chain fatty acids in plasma phospholipids in 300 black South Africans: Pearson correlation matrix, Table S4: Ten-year associations between plasma phospholipid long-chain fatty acids and blood pressure in rural and urban black South Africans, Table S5: Ten-year associations between plasma phospholipid long-chain fatty acids and hypertension status in black South Africans.

**Author Contributions:** Conceptualization, M.M.Z., A.E.S. and C.M.S.; Methodology, M.M.Z., A.E.S., C.R., J.B. and C.M.S.; Software, M.M.Z. and C.R.; Formal analysis, M.M.Z.; Investigation, M.M.Z., A.E.S., J.B., I.M.K. and

C.M.S.; Resources, A.E.S. and C.M.S.; Data curation, M.M.Z. and C.R.; Writing—original draft preparation, M.M.Z.; Writing—review and editing, M.M.Z., A.E.S., J.B. and C.M.S.; Visualization, M.M.Z.; Supervision, A.E.S. and C.M.S.; Project administration, I.M.K., A.E.S. and C.M.S.; Funding acquisition, A.E.S. and C.M.S.

**Funding:** This research was funded by SANPAD (South Africa Netherlands Research Programme on Alternatives in Development), South African National Research Foundation [NRF GUN numbers 2069139 and FA2006040700010], North-West University, Potchefstroom, South Africa, SASA [South African Sugar Association, Project 228], Roche Diagnostics South Africa, and the Population Health Research Institute, ON, Canada and the South African Medical Research Council. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors, and therefore, the NRF does not accept any liability in this regard.

**Acknowledgments:** We would like to acknowledge the PURE South Africa team: A Kruger, PURE-SA research team, field workers and office staff in the Africa Unit for Transdisciplinary Health Research (AUTHeR), Faculty of Health Sciences, North-West University, Potchefstroom, South Africa. The PURE International: S Yusuf and the PURE project office staff at the Population Health Research Institute, Hamilton Health Sciences and McMaster University. ON, Canada.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Bioactive Candy: E**ff**ects of Licorice on the Cardiovascular System**

**Mikkel R. Deutch <sup>1</sup> , Daniela Grimm 1,2,3, Markus Wehland <sup>2</sup> , Manfred Infanger <sup>2</sup> and Marcus Krüger 2,\***


Received: 24 September 2019; Accepted: 9 October 2019; Published: 14 October 2019

**Abstract:** Licorice, today chiefly utilized as a flavoring additive in tea, tobacco and candy, is one of the oldest used herbs for medicinal purposes and consists of up to 300 active compounds. The main active constituent of licorice is the prodrug glycyrrhizin, which is successively converted to 3β-monoglucuronyl-18β-glycyrrhetinic acid (3MGA) and 18β-glycyrrhetinic acid (GA) in the intestines. Despite many reported health benefits, 3MGA and GA inhibit the 11-β-hydrogenase type II enzyme (11β-HSD2) oxidizing cortisol to cortisone. Through activation of mineralocorticoid receptors, high cortisol levels induce a mild form of apparent mineralocorticoid excess in the kidney and increase systemic vascular resistance. Continuous inhibition of 11β-HSD2 related to excess licorice consumption will create a state of hypernatremia, hypokalemia and increased fluid volume, which can cause serious life-threatening complications especially in patients already suffering from cardiovascular diseases. Two recent meta-analyses of 18 and 26 studies investigating the correlation between licorice intake and blood pressure revealed statistically significant increases both in systolic (5.45 mmHg) and in diastolic blood pressure (3.19/1.74 mmHg). This review summarizes and evaluates current literature about the acute and chronic effects of licorice ingestion on the cardiovascular system with special focus on blood pressure. Starting from the molecular actions of licorice (metabolites) inside the cells, it describes how licorice intake is affecting the human body and shows the boundaries between the health benefits of licorice and possible harmful effects.

**Keywords:** licorice; glycyrrhizin; glycyrrhetinic acid; glabridin; 11-β-dehydrogenase isozyme 2; hyperaldosteronism; hypokalemia; hypertension

#### **1. The Sweet "Father of Herbal Medicine"**

Licorice is the root of the legume *Glycyrrhiza glabra* (Figure 1a) that grows in varieties in warm areas like the Middle East, Asia and Southern Europe. It is one of the oldest used herbs in ancient medicine and referred to as "the father of herbal medicine" [1]. Licorice, from which a sweet flavor can be extracted, has been used in herbal and traditional medicine in both Eastern and Western cultures dating back to beyond 4000 BC [2]. The early Egyptians and Assyrians are known to have cultivated the 'sweet root' that was later imported to China, where it has been used for centuries under the name '*Gan Cao*' [3]. It has also been described by ancient Greeks, including Hippocrates and Theophrastus, as well as by Romans [2,4]. Today, the Scandinavian countries seem to have the most consumers of licorice; however, licorice intake is also a popular strategy to quench thirst during Ramadan (based on its historical utilization in the desert or on battlefields, where travelers and soldiers drank licorice extracts to combat thirst sensation on long marches). Although the main active compound glycyrrhizin is considered to be 50-times sweeter than sucrose [5], licorice is rarely used for sweetening purposes alone due to its associated flavor and the brownish color that would be imparted to non-acidic foods [2,6]. Since the 18th century, the primary use comprises mainly licorice extracts (in pharmacy called *Succus liquiritae*) as a flavoring additive in tea, tobacco, candy (Figure 1b) and other sweets, but the licorice root itself (*Liquiritae radix*) is still used as a dietary supplement in some parts of the world [7]. Among people preferring alternative or complementary medicine, historical uses for licorice were revived and are still practiced today [8–10]. Ramadan (based on its historical utilization in the desert or on battlefields, where travelers and soldiers drank licorice extracts to combat thirst sensation on long marches). Although the main active compound glycyrrhizin is considered to be 50-times sweeter than sucrose [5], licorice is rarely used for sweetening purposes alone due to its associated flavor and the brownish color that would be imparted to non-acidic foods [2,6]. Since the 18th century, the primary use comprises mainly licorice extracts (in pharmacy called *Succus liquiritae*) as a flavoring additive in tea, tobacco, candy (Figure 1b) and other sweets, but the licorice root itself (*Liquiritae radix*) is still used as a dietary supplement in some parts of the world [7]. Among people preferring alternative or complementary medicine, historical uses for licorice were revived and are still practiced today [8–10].

**Figure 1.** (**a**) Inflorescence of *Glycyrrhiza glabra* L.; (**b**) licorice-containing candies; (**c**) chemical structure of the prodrug glycyrrhizin (C42H62O16), the main active compound of licorice. The molecule consists of two molecules of glucuronic acid (left) that are linked to 18β-glycyrrhetinic acid; (**d**) chemical structure of glabridin (C20H20O4), a further bioactive licorice compound. Colors indicate molecule structures used in following schematics. **Figure 1.** (**a**) Inflorescence of *Glycyrrhiza glabra* L.; (**b**) licorice-containing candies; (**c**) chemical structure of the prodrug glycyrrhizin (C42H62O16), the main active compound of licorice. The molecule consists of two molecules of glucuronic acid (left) that are linked to 18β-glycyrrhetinic acid; (**d**) chemical structure of glabridin (C20H20O<sup>4</sup> ), a further bioactive licorice compound. Colors indicate molecule structures used in following schematics.

On the one hand, the health benefits ascribed to licorice are numerous: for centuries it has been used in herbal and folk medicine to treat multiple diseases such as gastrointestinal symptoms and respiratory diseases [10]. The broad spectrum of activities known today comprises immunostimulatory and anti-ulcer effects [11–13], anti-viral and anti-microbial effects [14,15], hepatoprotective [16,17], anti-carcinogenic [18] and several other positive effects that contribute to the protection of the nervous, respiratory, endocrine and cardiovascular system [9]. Licorice is also effective against gastrointestinal problems by repairing the inner layer of the stomach and cleansing the respiratory system by increasing the production of mucus. Furthermore, other extracts of the licorice root have been tested in the treatment of gastritis induced by *Helicobacter pylori* and showed promising results [19,20]. The polyphenolic flavonoid glabridin possesses hypoglycemic effects by modulating glucose and lipid metabolism [21,22], similar to effects attributed to green tea extract [23]. On the one hand, the health benefits ascribed to licorice are numerous: for centuries it has been used in herbal and folk medicine to treat multiple diseases such as gastrointestinal symptoms and respiratory diseases [10]. The broad spectrum of activities known today comprises immunostimulatory and anti-ulcer effects [11–13], anti-viral and anti-microbial effects [14,15], hepatoprotective [16,17], anti-carcinogenic [18] and several other positive effects that contribute to the protection of the nervous, respiratory, endocrine and cardiovascular system [9]. Licorice is also effective against gastrointestinal problems by repairing the inner layer of the stomach and cleansing the respiratory system by increasing the production of mucus. Furthermore, other extracts of the licorice root have been tested in the treatment of gastritis induced by *Helicobacter pylori* and showed promising results [19,20]. The polyphenolic flavonoid glabridin possesses hypoglycemic effects by modulating glucose and lipid metabolism [21,22], similar to effects attributed to green tea extract [23]. On the other hand, it is

On the other hand, it is well-known that consuming excessive quantities of licorice can impact upon

well-known that consuming excessive quantities of licorice can impact upon cardiometabolic health by elevating blood pressure (BP), and thus, may be a cause of hypertension and other cardiovascular complications [24–30].

Hypertension is one of the major concerns for our healthcare system and was the leading contributor to premature death in 2015 [31]. Due to the higher arterial BP, it has been proven to be a major risk factor of cardiovascular diseases (CVD). The global prevalence of hypertension was estimated to be about 1.13 billion. Generally, hypertension is the cut-off BP value, where the benefits of treatment outweigh the associated risks. According to the European Society of Cardiology (ESC) "hypertension" is defined as a systolic BP ≥ 140 mmHg and a diastolic BP ≥ 90 mmHg [32]. Hypertension is divided into primary and secondary forms. It is a multifactorial disease, where the contribution of different factors is variable and with a small effect size. Most of the patients have no clear etiology, and they are classified as having primary hypertension. According to Charles et al. [33], about 5–10% of hypertensive patients have secondary hypertension, a result of a different disease affecting the cardiovascular system, such as renal diseases, primary hyperaldosteronism or obstructive sleep apnea.

Licorice and other drugs affecting the hormonal regulation of the water and electrolyte balance could be causing primary hypertension. To examine the actual cause of hypertension, some tests are needed. This would include measurements of plasma aldosterone and plasma renin. Aside from licorice, plenty of additional risk factors increase the possibility of developing hypertension [32].

In this review, we summarized and evaluated current literature about the effects of licorice ingestion on the cardiovascular system with special focus on BP. The literature was primarily identified using online databases. The search was completed on 24/9/2019. The primary registers included PubMed, Embase and ClinicalTrials.gov. Keywords that were used in the search included both "licorice" and "liquorice". Both variations were used to ensure a more complete search, since "licorice" is widely used in American literature whereas "liquorice" is common in British literature. In PubMed, the search for "liquorice" alone gave 4347 results, while "liquorice and hypertension" narrowed it down to 364 results. "Liquorice and cardiovascular disease" gave 379 results; "*Glycyrrhiza* and hypertension" resulted in 255 hits. We thoroughly collected information about the molecular and physiological mechanisms of licorice in order to explore the effects and prevalence of licorice intake in general. This way, we want to show the boundaries between its health benefits and possible harmful effects.

#### **2. Pharmacological E**ff**ects of Licorice**

#### *2.1. Licorice Digestion and Chemistry of Metabolites*

Licorice consists of up to 300 active compounds comprising phenolic acids, flavonoids, flavans, chalcones, isoflavans (including glabridin, the main compound found in the hydrophobic fraction of licorice extract) and isoflavonoids [10]. A species-dependent content of 3 to 5% the triterpenoid saponin glycyrrhizin (Figure 1c) accounts for the sweet taste of licorice root and is the main active constituent of licorice [6,34]. Although the presence of glycyrrhizin in licorice has been known for over 200 years, detailed chemical investigations have not been conducted until the mid of the 20th century [35]. In the licorice root, tribasic glycyrrhizin naturally occurs in form of its calcium and potassium salts. After oral ingestion, glycyrrhizin (which itself possesses only poor oral bioavailability) is successively hydrolyzed to 3β-monoglucuronyl-18β-glycyrrhetinic acid (3MGA) and the aglycone 18β-glycyrrhetinic acid (GA; also known as enoxolone) by intestinal bacteria possessing specialized β-glucuronidases [36,37]. GA is often considered as the active metabolite of licorice [38–40], but its pharmacokinetics seem to be more complex. After rapid absorption from the gut, 3MGA and GA circulate in the bloodstream. From there, they are transported to the liver by carrier molecules, where they are metabolized (Figure 2). In humans, hepatic processing is not yet clearly defined, but it is apparent that each metabolite can undergo further conjugation or reduction followed by biliary excretion [6]. The products are likely re-metabolized by the gut microbiome and thereby subjected to enterohepatic recycling requiring several days for complete elimination [41].

the bloodstream [43,44].

gastrointestinal bacteria as well [6].

hepatic glucuronidation. They also found that the intestine contributes to glabridin glucuronidation to a much lesser extent. After the intestinal absorption process involving P-glycoprotein, glabridin

**Figure 2.** Suggested glycyrrhizin metabolism. Dependent on the gut microbiome glycyrrhizin is stepwise hydrolyzed to 3β-monoglucuronyl-18β-glycyrrhetinic acid (3MGA) and 18β-glycyrrhetinic acid (GA; blue structure) in the intestines. Both 3MGA and GA were absorbed from the gut and transported systemically in the bloodstream. In the liver, they undergo hepatic biotransformation before products were excreted via bile. The flavonoid glabridin (yellow structure) is also absorbed from the gut and circulates in the blood in its aglycone form. The hepatic metabolization of glabridin is not shown here. Green hexagons: glucuronic acid. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0). **Figure 2.** Suggested glycyrrhizin metabolism. Dependent on the gut microbiome glycyrrhizin is stepwise hydrolyzed to 3β-monoglucuronyl-18β-glycyrrhetinic acid (3MGA) and 18β-glycyrrhetinic acid (GA; blue structure) in the intestines. Both 3MGA and GA were absorbed from the gut and transported systemically in the bloodstream. In the liver, they undergo hepatic biotransformation before products were excreted via bile. The flavonoid glabridin (yellow structure) is also absorbed from the gut and circulates in the blood in its aglycone form. The hepatic metabolization of glabridin is not shown here. Green hexagons: glucuronic acid. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0).

The digestion of licorice is still not completely understood. Interestingly, the bioavailability of glycyrrhizin is reduced when consumed as licorice [45], suggesting that some components of the licorice root may interact with glycyrrhizin during intestinal absorption, reducing its oral bioavailability [46]. Some recent animal studies on rats indicated that there might be further metabolites of GA as causal candidates for the described pharmacological effects [47,48]. In addition, it should be mentioned that the enterohepatic circulation of GA has not yet been studied in humans. However, similar steps can be expected, because GA metabolites can be hydrolyzed by human

The further bioactive constituent, glabridin (Figure 1d), has shown low oral bioavailability in rats. Microsomal studies by Cao et al. [42] demonstrated that glabridin is mainly metabolized by hepatic glucuronidation. They also found that the intestine contributes to glabridin glucuronidation to a much lesser extent. After the intestinal absorption process involving P-glycoprotein, glabridin appears in the human plasma and in the liver as the free (aglycone) form that also circulates within the bloodstream [43,44].

The digestion of licorice is still not completely understood. Interestingly, the bioavailability of glycyrrhizin is reduced when consumed as licorice [45], suggesting that some components of the licorice root may interact with glycyrrhizin during intestinal absorption, reducing its oral bioavailability [46]. Some recent animal studies on rats indicated that there might be further metabolites of GA as causal candidates for the described pharmacological effects [47,48]. In addition, it should be mentioned that the enterohepatic circulation of GA has not yet been studied in humans. However, similar steps can be expected, because GA metabolites can be hydrolyzed by human gastrointestinal bacteria as well [6].

#### *2.2. Pharmacodynamics of Licorice Constituents and Metabolites*

Licorice intake induces physiological effects similar to aldosterone and corticosteroids. Resembling steroid-like structures, both 3MGA and GA are able to bind to the mineralocorticoid receptor (MR) in the distal tubules of the kidney (direct effect), although competitive binding assays revealed that the affinities of MR for licorice metabolites were up to 10,000 times weaker than those for adrenocortical hormones [49]. In a normal physiological state, MR is activated by aldosterone to increase sodium and water resorption into the blood and potassium excretion into the urine mediating sodium and water homeostasis within the kidneys. However, it is unclear how the direct effects of 3MGA and GA on MR contribute to the effect of licorice. Although there is some evidence of this direct effect in vitro [50], the relative affinity for MR compared to aldosterone as well as low serum levels of GA after licorice consumption, which did not reach the concentrations necessary to affect aldosterone or cortisol binding to MRs in humans, question that theory [51]. In addition, hyper-mineralocorticosteroid effects were not observed in patients or animals with severe adrenal insufficiency [52]. It is much more likely that metabolites of glycyrrhizin promote a change in cortisol metabolism [53]. Cortisol acts as an agonist for aldosterone to activate MR with equal affinity but circulates in 100–1000-times higher plasma concentrations than that of aldosterone. In adult tissues, the type II isozyme of 11β-hydroxysteroid dehydrogenase (11β-HSD2) is expressed in the distal nephron of the kidney [54], in smooth muscle cells and endothelial cells of the vascular wall [55], in the heart [56] and in the brain [57], where it serves to protect the MR from being overly activated by cortisol [53,58]. 11β-HSD2 converts 'active' cortisol to the 'inactive' cortisone which has a very low affinity for MR. Monder et al. [59] described a strong inhibitory effect of GA for 11β-HSD2 using rat kidney homogenates for in vitro analysis. In addition, oral glycyrrhizin administration inhibited renal 11β-HSD2 activity in rats in a dose-dependent manner [59,60]. Kato et al. [61] suggested that 3MGA, not GA, is the mainly causative agent of licorice-induced pseudohyperaldosteronism. In the kidneys, 11β-HSD2 inhibition by 3MGA or GA (K<sup>i</sup> : 5–10 nM) results in a significant increase of active cortisol concentration in the renal tissue leading to a syndrome of apparent mineralocorticoid excess (Figure 3a) [52,62]. In the vascular wall, it increases arterial tone enhancing contractile responses to pressor hormones and reducing endothelial nitric oxide production [57,63]. Further animal studies reported a markedly inhibitory effect of GA on hepatic ring A-reduction of aldosterone by two other hepatic enzymes (5β-reductase and 3β-hydroxysteroid dehydrogenase), increasing the circulating aldosterone levels [64].

*Foods* **2019**, *8*, x FOR PEER REVIEW 6 of 20

**Figure 3.** (**a**) Correlation between licorice intake, the renin-angiotensin-aldosterone-system and licorice-induced adverse effects on the cardiovascular system. (**b**) Detailed pharmacodynamics of 3βmonoglucuronyl-18β-glycyrrhetinic acid (3MGA) and 18β-glycyrrhetinic acid (GA; blue structure) in the kidney. In addition to a possible direct binding to the mineralocorticoid receptor (MR), 3MGA and GA have inhibiting effects on 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and 5βreductase. 11β-HSD2 converts cortisol to cortisone; 5β-reductase is involved in the degradation of aldosterone in the liver. Inhibition of both enzymes contributes to apparent mineralocorticoid excess. The insert shows the localization of the processes within the Henle loop. ACE: angiotensin converting enzyme, ENaC: epithelial sodium channel, ET-1: endothelin 1, HRE: hormone response element, **Figure 3.** (**a**) Correlation between licorice intake, the renin-angiotensin-aldosterone-system and licorice-induced adverse effects on the cardiovascular system. (**b**) Detailed pharmacodynamics of 3β-monoglucuronyl-18β-glycyrrhetinic acid (3MGA) and 18β-glycyrrhetinic acid (GA; blue structure) in the kidney. In addition to a possible direct binding to the mineralocorticoid receptor (MR), 3MGA and GA have inhibiting effects on 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and 5β-reductase. 11β-HSD2 converts cortisol to cortisone; 5β-reductase is involved in the degradation of aldosterone in the liver. Inhibition of both enzymes contributes to apparent mineralocorticoid excess. The insert shows the localization of the processes within the Henle loop. ACE: angiotensin converting enzyme, ENaC: epithelial sodium channel, ET-1: endothelin 1, HRE: hormone response element, NAD(H): nicotinamide adenine dinucleotide, NO: nitric oxide, ROMK: renal outer medullary potassium channel. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0).

A vasorelaxant effect of glabridin was described in rat mesenteric arteries, which was associated with the opening of potassium channels and a concomitant rise in tissue cyclic guanosine monophosphate levels [65].

Taken together, intake of licorice induces a mild form of apparent mineralocorticoid excess causing MRs to be activated by both cortisol and aldosterone via inhibition of enzymes necessary for their catabolism (Figure 3). The direct effects of 3MGA and GA on MRs seem to be only negligible in physiological conditions. In the kidney, MR activation leads to transcription of epithelial sodium channel (ENaC), Na+/K<sup>+</sup> ATPase and mitochondrial enzymes, which accelerate adenosine triphosphate (ATP)-production (Figure 3b). The final consequences comprise elevated BP, sodium and water retention, decreased plasma potassium (hypokalemia) and caused a suppression of plasma renin and aldosterone levels [66]. In vascular smooth muscle cells, MR activation may further cause vascular stiffening by remodeling of the vascular wall [67]. Furthermore, direct effects of MR activation were described for the rat heart [68].

#### *2.3. Licorice-Induced Hypertension*

Licorice mediates its effect on BP primarily via the inhibition of renal 11β-HSD2 by 3MGA and GA (Figure 3a). Water and sodium retention in the kidney increase the blood volume and elevate BP [5]. The body countermeasures with a refractory lowering of the renin secretion in the kidneys, followed by decreased aldosterone production in the adrenal cortex via angiotensin II. However, the increasing level of cortisol (together with unrestricted activation of MR by cortisol) causes pseudohyperaldosteronism. This in turn results in further increasing blood volume and preload of the heart, thereby raising the mean arterial pressure. Furthermore, GA mediates the development of hypertension via decreased bioavailability of NO and activation of the vascular endothelin (ET-1) system (Figure 3a) which was accompanied by impaired endothelium-dependent relaxation in rats [69]. Activation of the endothelin system was also observed in human hypertension [70], and there is some evidence that increased ET-1 may be related to hypertensive end-organ damage and remodeling [71]. Interestingly, an infusion of GA into the rat brain elevated BP without affecting renal sodium and water resorption [72]. This finding indicated a central hypertensinogenic effect of licorice and suggested a more complex regulation of licorice-induced hypertension beyond the inhibition of 11β-HSD2.

Since a correlation between licorice ingestion and BP looks undeniable, further evaluation of quantities is necessary. Leskinen et al. [28] found that a daily intake of 290–370 mg licorice elevated both systolic and diastolic BP after two weeks. Furthermore, an increase of the extracellular fluid volume (hypervolemia) and amplified pressure wave reflection from the peripheral circulation was reported. Hautaniemi et al. [73] demonstrated that in addition to extracellular volume expansion, licorice increased stiffness of large arteries and systemic vascular resistance. A linear dose-response relationship between licorice intake and elevated BP was first proposed by Sigurjónsdóttir et al. [27], who found that a daily ingestion of 75 mg GA (~50 g of licorice) was sufficient to cause a significant increase in systolic BP within a period of two weeks. Similar correlations were later reported by a meta-analysis: analyzing the data of 18 studies (337 patients), systolic and diastolic BP seem to rise dose-dependently suggesting a public recommendation of avoiding excessive licorice consumption [74]. Based on the results of a 12-week experiment with 39 healthy female volunteers, van Gelderen et al. [75] proposed a no-effect level of 2 mg/kg GA per day (equal to 6 g licorice for a person with a body weight of 60 kg).

Two questions remain: 1. Is there any evidence that licorice will increase BP in patients dealing with hypotension? 2. Can general practitioners advocate the complementation of a normal diet with an intake of black licorice or other products containing GA in hypotensive patients? In 1994, it was reported that a 63-year old type 2 diabetic patient was treated for postural hypotension using licorice (3 g of GA/day) as treatment [76]. The patient's BP increased from 90/60 mmHg to 130/80 mmHg in an upright position in 7 days of therapy. Thus, there might be some indications that licorice has its place in clinical therapy, but this must be further investigated in a double-blind, randomized, place-controlled trial to avoid bias.

The case reports of licorice-induced hypertension found in the literature range from mild and reversible forms to severe resistant hypertension requiring hospitalization. In consequence of the elevated BP some patients developed hypertensive encephalopathy or cerebrovascular accidents [77–79]. Acute heart failure, pulmonary edema [80–82] or generalized edema [83–85] can be caused by the sodium retaining effect of licorice (Figure 3a). Interestingly, the occurrence of edema associated with hypertension seems to be characteristic for the 'licorice syndrome'. This is in contrast to true mineralocorticoid excess, where edema is typically absent as a result of the "sodium escape" phenomenon [86,87]. An observed increase in plasma concentration of atrial natriuretic peptide (ANP) after long-term consumption of licorice may be considered a physiological, albeit ineffective, response to prevent fluid retention and development of hypertension [88].

The effects of licorice on aldosterone secretion differ between the genders independently of the BP levels; women seem to be more susceptible to licorice intake [89,90]. A possible explanation for this gender difference are many other hormonal (estrogenic and antiandrogenic) effects exhibited by licorice in addition to its activity via MR. At least the alterations of the calcium metabolism that were observed in healthy women in response to licorice are probably influenced by several further components of the root such as glabridin, which shows estrogen-like activity [89].

There is very rare and controversial information about the correlation between licorice and the development of pulmonary hypertension. A possible contribution of licorice to pulmonary hypertension was suggested by Ruszymah et al. [91] after they had observed an increase in right atrial pressure and thickening of the pulmonary vessels of rats after GA administration. On the other hand, Yang et al. [92] described the attenuation of pulmonary hypertension progression and pulmonary vascular remodeling by glycyrrhizin in a monocrotaline-induced pulmonary hypertension rat model. Here, further studies are needed.

#### 2.3.1. Meta-analyses of Human Trials

In 2017, Penninkilampi et al. [74] reviewed the association between licorice intake, hypertension and hypokalemia. In a broad-based meta-analysis, they confirmed a significant increase in both systolic (5.45 mmHg; 95% confidence interval (CI) 3.51–7.39) and diastolic BP (3.19 mmHg; 95% CI 0.10–6.29) after chronic intake of products containing GA. Since physiological effects are not directly induced by licorice but rather by GA, the GA consumption was calculated for most of the studies. A GA content of 0.2% was approximated for black licorice [74] although the concentration of GA can obviously vary from product to product. Thus, the mean intake of 377.9 mg GA is equal to 189 g of licorice [74] and accounts for the described increase in systolic and diastolic BP. A further meta- and trial sequential analysis by Luis et al. [87] (26 trials, 985 patients) confirmed the significant increase in diastolic BP (1.74 mmHg; 95% CI 0.83–2.62) associated with the hypernatremia caused by licorice consumption. As mentioned by Penninkilampi et al. [74], most of the trials included in their meta-analysis were performed with volunteers. Selection bias in using volunteers and not random participants might be limiting results. The authors found that patients had higher increases in BP after a long intake of GA. They stratified the data in <4 weeks and ≥4 weeks and got elevations of 7.83 mmHg (95% CI 3.69–11.98) and 4.44 mmHg (95% CI 3.20–5.68), respectively. This confirmed the dose-response relationship and a positive correlation between GA dose and changes in both systolic and diastolic BP [74]. The significant increase of 5.45 mmHg might not cause adverse effects in a healthy

individual. However, combined with hypokalemia, it can lead to problems in individuals dealing with uncontrolled hypertension [74]. There have been case reports of patients with hypertensive crises where high licorice-intake in combination with hypertension caused hospitalization [93]. Compared with the modest results found in the meta-analysis on the available literature, the number of case reports with serious events or death after chronic licorice ingestion appears excessive [74]. A history of high licorice consumption alone is mostly sufficient to induce a toxic state. The degree of hypokalemia can be severe to cause a lethal arrhythmia [5].

#### 2.3.2. Treatment

In most cases, hypertension and hypervolemia induced by licorice is reversible once intake is stopped. If treatment of licorice-induced hypertension should be necessary, patients will usually be treated as normal hypertensive patients with antihypertensive therapy [94]. Different biochemical analyses will indicate a state of hyperaldosteronism by displaying low plasma potassium and lower levels of plasma renin and aldosterone. Antihypertensive therapy that targets the MR, such as spironolactone, seems to be the primary choice [69]. In rats, it was shown that blocking MR normalized BP [69]. Spironolactone works as a competitive aldosterone antagonist reducing the number of ENaC and Na+/K <sup>+</sup>-ATPase in reverse to aldosterone and cortisol. However, spironolactone treatment is only suggested for an acute hypertensive crisis. Lifestyle interventions should be advised against chronic hypertension caused by high ingestion of licorice and GA-containing products. Depending on the severity, either less ingestion of licorice or a complete stop will be necessary. The ESC guidelines state that grade 2 or 3 hypertension have to be treated with antihypertensive therapy [32]. This accounts for a clinically measured systolic BP ≥ 160 mmHg and/or a diastolic BP ≥ 100 mmHg. Since the effects on electrolyte-levels are delayed, it is furthermore important to stabilize electrolytes, with specific focus of on potassium. When licorice-induced hypertension is treated, it should be kept in mind that it can take up to six months to reverse the mineralocorticoid-like effects of licorice due to its long half-life and the duration required to normalize the renin-angiotensin-aldosterone-system [95].

Indeed, the ESC guidelines for treating hypertension mention that the intake of licorice could influence BP. They address that the medical history should include use of licorice [32]. However, there are no further comments on how licorice-induced hypertension should be treated. An intervention study aimed to investigate whether hypertensive patients were more sensitive to the inhibition of 11β-HSD2 than normotensive patients [96] and found that after 4 weeks of licorice consumption, the mean increase in systolic BP was 3.5 mmHg in healthy individuals and 15.3 mmHg in hypertensive subjects. The mean rise in diastolic BP confirmed this with an increase of 3.6 in mmHg in normotensive and 9.3 mmHg in hypertensive patients. The *p*-value showed significant differences in both systolic (*p* = 0.004) and diastolic BP (*p* = 0.03) [96]. Thus, the authors concluded that subjects with essential hypertension are more sensitive to the licorice-induced inhibition of 11β-HSD2 than normotensive subjects. This finding supports the suggestion that licorice might have stronger adverse effects in patients suffering from hypertension.

However, the available data on this topic is limited and of modest quality and only one clinical trial can be found (Table 1). Further double-blind randomized placebo-controlled studies would be necessary to determine the clinical effects of licorice intake in both healthy and non-healthy individuals.



11β-HSD2: 11-β-hydrogenase type II enzyme; ANP: atrial natriuretic peptide; BP: blood pressure; CYP3A: cytochrome P450 3A4; DBP: diastolic blood pressure; GA: 18β-glycyrrhetinic acid; SBP: systolic blood pressure; VSMC: vascular smooth muscle cell.

#### *2.4. Cardiovascular E*ff*ects of Licorice*

Licorice traditionally has been prescribed for treatment of cardiovascular disorders, but its effects are not just benign. From the cardiovascular complication described in the literature, cardiac arrhythmias are the most serious side effect caused by licorice intake due to severe hypokalemia (Figure 3a) [105]. The depletion of the body's potassium stores can cause a prolongation of the QT interval, which is closely connected with ventricular arrhythmias and tachycardia [106]. As a consequence, several patients experienced a cardiac arrest with a subsequent recovery [107–109]. Konik et al. [110] described a case of coronary artery spasm induced by licorice. The vasospastic effect of licorice was attributed to changes in endothelin and nitric oxide systems. Recently, a Polish clinical study found a correlation of arterial stiffness parameters with estimated cardiovascular risks in humans [111]. Transient visual loss, migraines and posterior reversible encephalopathy syndrome has also been demonstrated in a few cases. It is assumed that GA inhibits angiogenesis due to inhibition of reactive oxygen species generation [112]. Sobieszczyk et al. [102] found an additional attenuated vascular smooth muscle vasodilatory function without BP changes in healthy humans after 11β-HSD2 inhibition through GA. They proposed that in states of 11β-HSD2 inactivation,

non-aldosterone-mediated activation of vascular MRs may contribute to vascular dysfunction and possibly to CVDs.

In rats, cardioprotective effects of licorice and its metabolites were observed, which are mostly related to their antioxidant properties. Thirty days of licorice intake improved cardiac function and preserved histology of cardiomyocytes either by augmentation of endogenous antioxidants or by reduction in oxidative stress. Thus, licorice may delay the progression of ischemic heart disease [113]. Ohja et al. [114] further described a cardioprotective effect against oxidative stress in myocardial ischemia-reperfusion injury after ingestion of *Glycyrrhiza glabra*. Another animal study indicated that GA protects against isoproterenol-induced oxidative stress in rat myocardium decreasing lipid hydroperoxides and isoprostanes and increasing superoxide dismutase and glutathione levels [115].

Some studies suggested that the flavonoid glabridin may also have beneficial effects on the cardiovascular system. The effects described comprise inhibition of low density lipoprotein oxidation and atherogenesis [116], a possible inhibition of NADPH oxidase or an increase in the expression of antioxidant enzymes observed in macrophages [117]. Glabridin also stimulates DNA synthesis in human endothelial cells and demonstrated a bi-phasic proliferative effect on human vascular smooth muscle cells. The combination of an inhibition of smooth muscle cell proliferation and an induction of endothelial cell proliferation may be beneficial for the prevention of atherosclerosis [118,119]. Most recently, Huang et al. [120] reported that glabridin is able to prevent doxycyclin-induced cardiotoxicity in mice through the prevention of gut microbiota dysbiosis. Nevertheless, it remains unclear to which extent these effects contribute to the putative therapeutic actions of licorice.

#### *2.5. Interaction of Licorice with Prescribed Drugs*

Licorice can interfere with cardiac medications, e.g., with drugs used in the treatment of hypertension such as angiotensin converting enzyme (ACE)-inhibitors [121]. Some licorice compounds including glabridin and GA can interact with other drugs and the human liver microsomal cytochromes P450 and P450 3A4 (CYP3A4) systems [122–125]. Animal [126,127] and human studies [103] showed that glycyrrhizin has an inductive effect on CYP3A including CYP3A4 and the effect on CYP3A4 may be related to an activation of human pregnane X receptor (hPXR) [103,128]. Other studies described that CYP3A4 was inactivated by licorice extract and glabridin in a time- and concentration-dependent manner [124]. CYP3A4 is involved in the metabolism of xenobiotics [122], roughly half the drugs that are in use today, suggesting that the influence of licorice on CYP3A4 activity needs to be further investigated.

Heck et al. [129] described a toxic effect potentiation of warfarin, a cardiac drug that requires strict dosage adjustment, due to the inhibition of the hepatic microsomal enzymes by licorice.

Matsumoto et al. [130] investigated the effects of licorice on ABC-transporters. Using an in vivo ATPase assay, they demonstrated that licorice root and GA can inhibit P-glycoprotein. A two-phase randomized crossover trial by Yan et al. [104] revealed at least no induction effect on the P-glycoprotein expression after continuous glycyrrhizin administration (225 mg/day) for 6 days. The authors proposed that further research was needed to study the direct inhibition effect of glycyrrhizin on P-glycoprotein. For the pharmaceutical use, it is important to know and consider the interaction between licorice and drugs metabolized by CYP3A4 and P-glycoprotein.

Licorice decreases the bioavailability of cyclosporine and is thus contraindicated [120] in conjunction with this drug [128]. The intake of licorice should be done with caution, when using antihypertensive drugs. ACE-inhibitors, e.g., captopril, inhibit the angiotensin converting enzyme, limiting levels of angiotensin and aldosterone. It was shown that ACE-inhibitors enhance the effects of 11β-HSD2 which may contribute to the natriuretic effect [131]. Hypokalemia is one of the most serious adverse effects of licorice intake and should be completely avoided with loop-diuretics and thiazides since it can lead to serious hypokalemia and hospitalization [132].

The combination of medicine containing licorice and digitalis can cause toxicity, especially in elderly patients. There has been one reported case of digoxin toxicity due to licorice-induced hypokalemia [133].

#### *2.6. Contraindications and E*ff*ects of Licorice Overconsumption*

Licorice and its derivatives are affirmed as 'Generally Recognized as Safe' (GRAS) for use in foods by the United States Food and Drug Administration (21 CFR 184.1408). Nevertheless, tolerable upper limits of licorice intake have been provided by several institutions: the European Scientific committee of Food recommends that the daily ingestion should not exceed 100 mg of glycyrrhizin (60–70 g of licorice) [134]; the Dutch Nutrition Information Bureau advised against a daily consumption above 200 mg of glycyrrhizin (150 g of licorice) [2]. Since most consumers are not aware of possible health hazards, and there are currently no precise declaration data of glycyrrhizin on food, it is difficult to control licorice intake. Furthermore, it has to be investigated if sporadic intake carries the same risks compared to the daily consumption that is analyzed in most studies. Licorice is found in diet gum, cough mixtures, tea and herbal medicine. Having a mixed intake of these products will accumulate the quantity of GA in vivo, and therefore, increase the risk of symptoms.

In general, people aged over 40, patients with history of cardiac disease or more susceptible to cardiac arrhythmias should avoid excess licorice intake in order to obviate arrhythmias or cardiac arrest caused by licorice-induced hypokalemia. One study investigated patients treated with traditional Japanese medicine containing licorice [135]. They discovered that 24.2% of the patients treated with this medicine developed hypokalemia 34 days after administration. Hypokalemia is a serious state that increases the risk of arrhythmia and is associated with an up to 10-time increase in all-cause mortality [74]. The meta-analysis by Penninkilampi et al. [74] summarized other side effects including rhabdomyolysis, paralysis, hypertensive encephalopathy and cardiac arrest. That is why patients who are on medicines lowering potassium levels (such as thiazide or loop diuretics) should also minimize their licorice intake. The same applies for patients suffering from diarrhea or alcoholism, which can worsen hypokalemia. Licorice can be dangerous in patients treated with antihypertensive drugs such as ACE-inhibitors and diuretics. Due to the salt-retaining effect of 3MGA and GA, people suffering from congestive heart failure or resistant hypertension should completely abstain from products containing licorice. This is also advisable for patients taking digoxin or warfarin to avoid the risk of toxicity. Since 3MGA and GA are known to inhibit 11β-HSD2, licorice ingestion during pregnancy should be avoided. GA consumption impaired the development of the respiratory systems in rats because the conversion of cortisone into cortisol plays an important role in lung maturation [136].

#### **3. Conclusions**

In recent years, the mechanisms of action of licorice and its active components have become understood in more detail. The use of licorice in herbal medicine is obviously a result of some positive effects. Hence, it has become one of the most used herbs in traditional Chinese medicine and is still used in China to treat gastric symptoms and respiratory diseases today [10]. Numerous studies have reported about effects of the different compounds found in the licorice root. Glabridin has been proven to be a potent antioxidant with hypoglycemic effects [21]. Referring to studies, glycyrrhizin possesses a wide range of pharmacological effects described as antiulcer and anti-inflammatory [11–13], antiviral [14,15], anticariogenic [137,138] and antispasmodic [139,140].

The utilization of some licorice compounds in a clinical setting is still under investigation. This applies also for artificial GA derivatives such as carbenoxolone [141] or acetoxolone [142]. Glycyrrhizin was identified as an attractive drug candidate for cancer therapy after demonstrating an apoptotic effect on tumor cells [143]. Today, researchers are intensely investigating the applicability of licorice in treatment of breast and prostate cancer. The antitumor activity has attracted the attention of many scientists, since cancer is still one of the leading causes of death in humans around the globe [10].

Nevertheless, due to some safety considerations associated with chronic high-dose intake, licorice should still be consumed with caution. With the elucidation of licorice constituents and the discovery that 3MGA and GA affect the renin-angiotensin-aldosterone-system, pseudohyperaldosteronism is the obvious adverse effect; however, other side effects such as hypertension, hypokalemia and hypernatremia have also been proven. If left untreated, they can cause arrhythmia and, in a worst-case scenario, cardiac arrest. Omar et al. [30,40] have described in detail why licorice should be handled more as medicine than as a candy and that excess licorice consumption can cause serious life-threatening complications, especially in individuals already dealing with high BP and patients under treatment with anti-hypertensive drugs. Adverse effects of high-dose licorice intake have been attributed to glycyrrhizin, 3MGA and GA. Since the final toxicology report has been published in 2007, therapeutic doses of licorice are generally recommended as safe in humans [144]. Especially Scandinavian countries have a higher intake of licorice, and in addition, their licorice has a higher GA concentration [121]. This suggests a need of public focus on the negative effects of licorice on cardiovascular health. However, this is also needed in China, where licorice is widely used in medical practice; here, the knowledge of licorice's interaction with prescription medicines is quite important to avoid possible iatrogenic accidents.

**Author Contributions:** Conceptualization, M.R.D., D.G. and M.K.; methodology, M.R.D.; validation, D.G., M.W. and M.K.; investigation, M.R.D.; resources, M.I.; data curation, D.G. and M.K.; writing—original draft preparation, M.K., M.R.D. and D.G.; writing—review and editing, M.K., D.G. and M.W.; visualization, M.K.; supervision, D.G.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank Walter Welß (Botanical Garden of Erlangen) for kindly providing photos of *Glycyrrhiza glabra*.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


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