Licorice Research 1
APPLIED NUTRITIONAL INVESTIGATION
Antiatherosderotic Effects of Licorice Extract
Supplementation on Hypercholesterolemic
Patients: Increased Resistance of LDL to
Atherogenic Modifications, Reduced Plasma Lipid
Levels, and Decreased Systolic Blood Pressure
Bianca Fuhrman. DSc. Nina Volkova. MSc. Marielle Kaplan. DSc. Dita Presser. MSc.
Judith Attias. MSc. Touv Havek. MD, and Michael Aviram. DSc From the Lipid Research Labor arory, Techmon Faculty of Medicine, The Rappapon Family Institute for Research in the Medical Sciences and Rambam Medical Center,
Haifa, Israel
OBJECTIVE: We previously demonstrated the beneficial effects of diet*•• fiavonoids derived from the ethandic extract of licorice root Masut atherosclerotic lesion development in association with inhibitioo of low-density lipoprotein (LDL) oxidation in atherosclerotic mice Administration of licorice extract to normolipidemic subjects also inhibited LDL oxidaion In the present study, we extended our investigation to analyze the ant13thero genic effects of licorice-root extraa consumption in modcrady hyperchdester- olcmic patents
METHODS: Supplementation of licence root extraa (0.1 g d) to patients for 1 mo was followed by an addition^ 1 mo of placebo consumption
RESULTS: Licorice consumption 1) reduced paients‘ plasma susceptibility to oxidation (by 19°«); 2) increased resistance of pl3sma LDL 3ganst three major aherogemc modifications oxidaion (by 55°»). aggregation (by 2S*•). and retention, estimated as diondroitm sulfate binding ability (by 2 5*•); 3)reduced plasma cholesterol levels (bv 5%). which was due to a 9*« reduction in plasma LDL cholesterol levels, and 4) reduced (by 14״») plasma triacylglycerol levels After the 1 mo of placebo consumption, these parameters reversed towsd baseline levels Licorice extraa supplementation also !educed systolic Hood pressure by 10?o which was sustained during the placebo consumption
CONCLUSIONS: Dietary consumption of licorice-root extraa by hypercholesterolemic paients may aa as a moderate hypocbolestcrolemic nutrient and a potea amoxidant agent and. hence against cardiovascular disease Suwon 2002:18:268-273. tSsevier Science Inc. 2002
KEY WORDS: liccrice. tdavonoids lipid peroxidation low-density lipoprotein blood pressure, atheroscle- rosis. hypercholesterolemia
INTRODUCTION
Coronary artery disease develops as a result of risk faaots such as mac3sed plasma low-density lipoprotein (LDL) level and hyper- tension or LDL atherogenic modifications such as retention, oxi- dan on. and aggregation During early atherogenesis. circulating LDLs !made the arterial wall, where it binds to octiacellular m31nx pracoglycans. a process known as ־LDL retention". • LDL retention in the arterial wall nuv be a prerequisite to lipoprotein oxidative modification because retention of LDL to arterial pro- teoglycans increases its susceptibility to oxidation : The process of LDL oxidation appears to occur within the artery wall, and all m*ot arterv wall cells including endothelial cells, smooth muscle cells 3nd monocyte-derived macrophages. can oxidize LDL. * Ex- tensive oxidation of LDL also leads (0 its aggregation. * and oxi- dized and aggregated LDL have been found in atherosclerotic lesions 4 These LDL modifications ate constdaed atherogenic
Conesponderce to Michael A'tian. DSc. The LipidReseaichLatorscory. Rambam Medea! Center Kat'a iaael e-mail aviramga tectaonacil
Nutnnon 18268 -22002 .3ל
CElaeviet Science Inc.. 2002. Printed in the Voted Stares AO ndttstesaved.
because dies׳ contribute to macrophage cholesterol accumulation and foam cell fcemawn the hallmark of early aheroselcTosis High-density lipoprotein (HDL), in contrast is associated with aniatherogenc activity, and HDL levels are inversely related to the risk of developing atherosclerosis Paaoxonase (PON1) is an enzyme physically assoaated in serum with HDL and has been shown to protect LDL and HDL against oxidation 4
Consumption of pclyphenolic flavenoids in the dia was in- versdv assoctaed with morbidity and mortality from coronary heart disease ' Polyphenolic flavonads may prevent coronary at• toy disease by reducing pl3sma cholesterol !reels and their ability to inhibit LDL oxidation s־:: The aitioxidaii activity of fla• vonoids is related to their chemical structure :: We previously demonstrated the beneficial effects of flavonoids from red wine. >*■** pomegranate juice. '•* ginger extraa!9 and olive oil •' against LDL oxidation Further, flaxxmoids were shown to presave PON'l activity1* and increase its activity in mice '■* and humans;5 after consumption
Licorice root derived from the plan Ghcy rrluzogiobro is used widely in .Asia as a sweetener or a spice Licorice root contains flavonoids from the fl3van and chicone subclasses, which have lipophilic char aa eristics and antioxidative properties 18 .Among
0899-900' 02 $22.00 PII S0899-900*(01)00*53-J
Nutrition Volume 18. Number 3. 2002
Antiatherosderotic Effects of Licorice Extract 269
several flavonoids that were isolated and purified from licorice- root extract the isoflavaa glabridin constituted the major fla- vonoid :c We previously demonstrated that 81 vitro licorice extract and purified glabndin protea LDL from oxidation induced by copper ions or free radical-generating systems 21 Mechanstic studies have show-n that liconce-derived glabridin binds to the LDL particle and protects it from oxidation because of its capacity to scavenge &ee radicals :J׳~Structural studies have found that the antioxidant effect of glabridin resides mainly in the 2'-hydroxvl group of the isoflavan ring B צ Glabndin accumulates in macro- phages and iriubits cell-mediated oxidation of LDL due to the inhibition of maaophage nicotinamide adenine diaideotide phos- phate oxidase activity. 2* In vivo studies have shown that supple- mentation of licorice extract or glabridin to atherosclerotic mice deficient in apolipoprotetn E (E °) reduces the susceptibility of their LDL 10 oxidation and sigrificantlv reduces the developmea of aortic atherosclerotic lesions 2■•2^ In another study, licorice extract was administered to healthy normobpidemic subjects for 2 wk, consumption of 100 mg d of licorice root extraa increased their LDL resistance against oxidation 22
In the present study we extended our investigation on the antiatherogenic proparies of licorice-root extract in modaately hypacholesterolcmic patients We found that liconce-root extraa consumption by mildly hypercholestaolemic patients increases the resistance of thar LDL against major atherogenic modifica- tions and moderately reduces their plasma lipid levels and systolic blood pressures
METHODS AND MATERIALS
Materials
Licorice cthanolic extraa free of glycyrrhizinic arid was provided by Fatdizas & Chemicals Ltd (Haifa Israel) Powdered roots of commercial Glyc^rrHza glabra were extraaed in ethanol to ob- tain, after solver* evaporation. 3 brown solid extract The powder was encapsulated in a soft-gel capsule (RP Schera. Miami. FL. USA) Placebo capsules without licorice contained imertre gelat- inous mataial normally included in soft-gel capsules Na ; cthylene-diamineteiraacetic add (EDTA) was purchased from Sigma (St, Louis. MO. USA) 2,2 -Azobis 2-amiduu propane hydrochloride (AAPH) was purchased from Wako Chemical In- dustries Ltd (Osaka. Japs)
Patients
Twelve hypercholestaolemic patients (45 to 55 y old. plasma choleaaol level of 220 to 260 mg dL, and LDL cholesterol level of 120 to 170 mg dL) were seleaed for this study. .All patieas were non-smokers, with body weights of 78 : 9 kg (mean r standard deviation!, none exhibited evidence of liver, kidney, endocrine, or heart disease, and none had ever been treated with hypolipidanic drugs All patieas were first administered licorice extract (0.1 g d) in soft-gel capsules for 1 mo followed by addi- tional month of placebo supplementation To eliminate possible analytical drift or any other potentially confaundsig remits, the p an aits and the laboratory technicians did not know- which capsule (die licorice or the placebo) was consumed for the first month because both capsule types appeared the same Therefore, this study was considaed blind for all participants The subjects' mean (: standard deviation) both־ mass mdex (kg m *) was 25 (:1.7) and did not chance significantly during the study. .All patients coitinued their habitual diets dunng the study The consumption of the licorice extraa or placebo did not alter other food intakes or dies preferences, and patients did not report anv digestive or other disturbances 3!ood samples were drawn after 12 h of fasting, before study eary. 1 mo after licorice administration, and 1 mo after placebo supplementation
The study was approved by the Helsinki Committee of the Rambam Medical Ceaer. Israel Ministry of Health (no 1175).
Blood Pressure Measurement
3100d pressure was measured by a sphygmomanometer with ran- dom zero values The same apparatus was used for the entire study The measurements were made always on patients left arms; under similar coalitions The patients were seated, and each arm was placed on a table approximately at the level of the heart Blood pressure was measured after patients had relaxed, at two time points for each measuremetx. before and 30 min after blood withdrawal. The measurements did not differ significant, and the mean value was used
Serum Paraoxonase Activity
Serum PON1 activity was measured as ai•.festerase with phenyl- acetate as the substrate. Initial raes of hydrolysis were determined spectrophotometricaDy at 270 nm Hie assay mixture included 10 mM phenyl3cctate and 0.9 mM CaCl; in20 mM Tns HCl. pH 8.0. Xon-enzymatic hydrolysis of phenylacetate was subtracted from the total rate of hydrolysis The E;'0(cxtio:uon coefficient) for the reaction w as 1,310 Mem One unit of anlesterase activity is equal to 1 jiM ׳ min ־ •mL ־ of hydrolyzed phenylacetate
Plasma Lipid Peroxidation
Plasma was incubated with or without 100 mML of the free radical generator AAPH for 2 h at 37=C Plasma lipid peroxidation was determined by measuring the generaed amount of thiobarbi- turic acid-reactive substances (TBARS) 25
LDL Isolation
LDL w as separated from p!3sma by discontinuous density-gradient ultracentnfugaion29 and dialyzed against saline with EDTA (1 mML). Before the oxidation study. LDL was diluted in phosphate-buffered saline (PBS) to 1 g of protein L and dialyzed overnight against P3S at 4:C to remove the EDTA LDL protein concentration was determined with the Folin phenol reagent "
LDL Oxidation
LDL (100 mg of protein L) was incubated with 5 pM L of CuSO., for 3 h at 37°C The foemaiion of corrugated dienes was coatn- uously monitored by measuring die increase in absorbance at 234 an 2י Incubations were carried out in the spectrophotometer cu- vette (Uhraspec 3000; Pharmacia. LKB. Biochrom Ltd. Cam• budge. UK). The initial background of the samples ranged be- tween 0 1 and 0 2 optical density (OD) as recorded at 234 nm After initial absorbance was recorded, the spectrophotometer was set to zero against blank, aid the mcrcasc m absorbance of the sample that consisted of LDL and CuSO * was recorded every 10 min during LDL oxidation The lag time reqiared for thetratiarion of lipoprotein oxidation was calculated from the oxidation curve
LDL Aggregation
LDL (100 mg of protein L) was mixed by vortex a a fixed strength, and the absorbance at 680 an was moatored every 10 s against a blank solution Results after 60 mm of vortexing are reported.
Xutrtrton Volume IS, Number 3, 2002
LDL Retention (Chondroitin Sulfate Binding Ability>
LDL retention was estimated as the ability of LDL to bind to chondroitin sulfate (CS) LDL (200 mg of lipoprotein proteinL) W3S incubated with CS (100 mg L) for 30 nun at room tempera¬ture The lipoprotein was precipitated with a commercial HDL cholesterol reagent (phosphotungstic acid and MgCl Sigma) thx precipitated all the LDL in the sample* followed by a 10-tnin cennfuganon a 20<X)g After discarding the supernal am the LDL in the precipitate was dissolved in 0.1 N" NaOH and analyzed for its glvcosamnoglycan (GAG) cotient using the 1,9- dimethylm«h\1ene blue (DMMB) specoophotometnc assay for sulfated GAG's 19 Briefly. 2 5 mL of ice-cold DMMB working solution (46 pM L of DMMB. 40 mM L of glycine, 40 mM L of N'aCl in 5#o ethanol, adjusted to pH 3 0) was added to 500 *iL of the dissolved precipitate The absorbance at 525 nm was immedi¬ately measured CS was used as the standard and included within each series of assays Similar ptepiahon of LDL. with ro CS added, was used in parallel as a cortrol GAG cotient in the conrol was subtracted from the GAG content in LDL preparations that were incubated with CS
Slat is tie t
Student’s paired r test (one tail) compared the two arrays of data and analysis of variance was used when more than two groups were compared For statistical analysis of parameters without Gaussian distributions such as triacylghcerols and LDL oxidiz¬ability. the values were transformed to logarithm values to create a Gaussian distribution Results are given as mean : standard enor of the mean
RESULTS
After 1 mo of liconce-root etharolic extract consumpnon blood chemistry analyses showed no signficant changes in makers for liver, kidney, and heart functions as measured by serum blood urea mtrogen. creatinine, alamne aminotransferase, aspatae ami ro¬ll ansf erase. bilirubin and creaitne phosphokmase liable I) Pla¬cebo treaemea for 1 mo did not significantly affect those markers (Table I) In addition, serum elearohtes, including kalrum and natrium and serum alkaline phosphaase were not sigxnficandv affected by Sconce consumption or placebo (Table I) However, a small (7%) but significant (P < 0.01) reduction m serum glucose and a 10°o reduction in serum xnvlase concenraions were ob¬served after licorice consumption (Table I) After the 1 -mo placebo treatment serum glucose and amvlase levels returned to baseline values (Table I)
We next analyzed the effect of licorice-extract consumption on serum lipid profiles in the hvpercholesterolemic patio* s (Table D) Serum cholesterol levels were mmmally (5°«) but significant (P < 0 01) reduced after licorice consumption as a result of a 9% reduction in LDL cholesterol Those effects were rot sustained beyond the treatment period, after 1 mo of placebo treatment, serum and LDL cholesterol levels returned to baseline levels (Table 11) Setum HDL cholesterol levels did not change during licorice consumption
Serum triacylglycetol concentrations decreased by 13% after licorice consumption as reflected by a significant (P < 0 01) reduction of 14% in swum VLDL levels (Table U) After the placebo treatment serum triacvlgivcerol levels returned toward basekne values (Table II)
Blood pressure measuremeas showed a agraficant (P < 0 01) 10*.reduction in systolic blood pressure, with no si nunc aa effect on die diastolic blood pressure (Fig 1) After the 1-mo placebo treatment, the decrease in systolic Hood pressure remaned com¬pared with baseline levels (Fig 1).
Analysis of the effect of licorice consumption on oxidative
TABLE I
BIOCHEMICAL PARAMETERS OF HVPERCHOLESTEROLEMIC PATIENTS BEFORE AND .AFTER 1 MO OF LICORICE EXTRACT SUPPLEMENTATION .AND .AFTER .AN .ADDITIONAL MONTH OF PLACEBO SUPPLEMENTATION*
Time after liconce exuaet uppInner-ratoon
1 s» after liconce 1 mo after
Sttun cotxfrtiitiwi Baseline consunpton placebo
Glucose (mg dL) 90 = 3 0 83 : 1 0» 88 : 1.0
Blood i»ea ntzoaen 14 : 0.7 13:05 14: 06
(«g<t)
Creator*!* (tn: dL) 09 r 0 05 09:0 04 09 :0 04
Kalrum (mEqL) 4:0.1 5:01 5 =0.1
Natrnxn (mEqL) U0 = 10 140: 1 0 140 : 10
Cdciun (mg dL) 9:01 9 : 0 3 10 : 02
Amylase (UL) 82 : s o *4 : $ 0» SI : *0
.Aspartate amino 22 : 10 23 : 1.0 24 : i.o
uarsfeiase (U L)
.Alamne ammo 23 : 3 0 22 : 2.0 23 : 2.0
toareferase (U L)
Alkaline phosphatase 69 : 5.0 -<
w
II
o
O 75 : 5 0
(UL)
Cream* phophokinase 86 : *0 91 : 130 93 : 12.0
(UL)
Silirubm. totd (ms dL) 0.3 : 0.1 0.5 : 01 0 6 : 0 1
* ?.eaits are expeeurd as mean : stand*d etior et the mean (each assay was performed in duplicates)
T P < 0.001. serais baseline a studs- ertry
stress, a major risk factor for cardiovascular diseases, was per¬formed m serum samples and LDL & actions isolated bora the pacer*s’ serum before and after licorice consumption and after placebo consixnpcon
Serum PON'l. measured as aryl esterase activity, was not af¬fected by licorice consumpnon (215 r 25 aid 203 – 23 L' mL of a \ lest erase before and after licorice consumption, respectively).
TABLE II
EFFECT OF LICORICE EXTRACT SUPPLEMENTATION ON-
HYPE RCHOLESTEROLEM1C PATIENTS ON THEIR PLASMA
LIPIDS .AND LIPOPROTEINS*
Time after liconce extact supplement*!on
Strutt concentration (madL) Baseline 1 mo after liconce coruunpaon 1 mo after placebo
Cholesterol 244 : S 232 : 9r 252: 7
LDL chdestaol 156 : S 142 :9» 158 : S
HDL cholesetol 54:2 54:5 55 : 5
VLDL choleserol 36 : 5 31 : 4t 36 : 5
Triacylelcerals 174 : 23 155 = IS 184 : 23
•Resits ire expressed as mean = stand* d error of the mean (each assay •* as performed in duplicates)
r P < 0.01 after ! mo of liconce consumption versus baseline at study dlry
HDL. httvdenaty bpoprocem. LDL. low-denary lipoprotein VLDL. very-low -detorty bpoprotan
\x\
FIG 1. The effect of licorice extract supplementation on on systolic (.A) and diastolic (S) blood pie SHIM of hypercholeaerolemic patients Blood pressure was measured before licorice consumption 1 mo after liconce consumption and after an additional 1 mo of placebo consumption Results are expressed as mean — standard error of the mean *P < 0.01, versus before licorice consumption
Plasma after 1 mo of licorice extract consumption showed a significant (P < 0.01) 19% decreased susceptibility to the free radical generaor AAPH-induced lipid peroxidation measured as TBARS formation, in comparison with plasma obtained before licorice extract supplementation (37 r 1 mM before versus 30 r 1 mML after licorice consumption TBARS L of plasma). This effect was not sustained after the 1 mo of placebo-treatment and the AAPH-induced serum lipid peroxidation returned toward base¬line values (34 r 2 mM of TBARS L of plasma).
We next analyzed the effect of licorice extract supplementation on the resistance of hypercholesterolemtc patients’ LDL to three major atherogenic modifications LDL oxidation LDL aggrega¬tion. and CS binding ability. The susceptibility of LDL from hypercholesterolemic patients after consumption of licorice extract for 1 mo to copper ion-induced oxidation was reduced, as shown by the prolonged lag time of 55% required lor the initiation of LDL oxidation in compatson with the lag time of LDL isolated ftom plasma derived before licorice extract consumption (Fig 1A). This effect was partly sustained after 1 mo of placebo supplemen¬tation because the LDL derived after that period was still less susceptible to copper ion-induced lipid peroxidation as demon¬strated by an lS°o increment in the lag time, in comparison with the baseline lag time (before licorice administration Fig. 2A).
The atherogenicity of LDL has been attributed to its oxidative modification and its aggregation LDL oxidation lead to its subsequent aggregation. *-30 and werecendy reported that polyphe¬nols can reduce LDL aggregation in vitro and ex vivo. 1011 .-After analyzing the susceptibility to aggregation of LDL isolated from
FIG. 2. The effect of licorice extract supplementation on the susceptibility of LDL in hyperchoJesterolonic patients to adieroeenc modifications oxidation (A), aggregation (B). and binding ability (C). LDL was isolated from hypercholeaerolemic patienis before, 1 mo after licorice extract supplementation, and an additional 1 mo after placebo supplementation (A) LDL (100 mgofproteinl) was incubated with 5 jnnol L of CuS04for 3hat2J:C Theformaoon of conjugated dieneswaskineticallymontored at 23-i m and the lag time was measured (B) The extent of LDL aggiezatronindixedbyvortexingwas kineticaUymontotedat 6S0nm. and results after 60 s of vortexing are shown (C) LDL (200 mg of protein!) was incubated with CS (100 mgL) for 30 min at 3':C. LDL was then precipitated and the LDL-assoctated dycosan mod yean content in the precipitate was measured. Resdisare expressed as mean r standard error of the mean *? < 0.01 versus baseline at study entry. CS. chandroitin sulfate LDL, low-density lipoprotein
hypercholesterolemic patients who had consumed licorice extract for 1 mo. we found a significant (P < 0 01) reduction of 2$% in LDL aggregation (Fig. 2B) .After 1 mo of placebo consumption LDL aggregation rates returned toward baseline x-alues
Retention of LDL. which is an early step tn atherogenesis, was measured by analysis of LDL binding to the proteoglycan CS
After licorice consumption, LDL CS binding ability decreased significantly (P < 0.01) by 25®o and that effect was partly sus¬tained during the 1 mo of placebo consumption (Fig. 2C).
DISCUSSION
Consumption of licorice-root extract was shown to inhibit oxida¬tive modification of LDL in healthy human volunteers and accelerated development of atherosclerotic lesions in E 5 mice.-1 The present study expanded those findings to hypercholesterolemic patients and showed that consumption of licorice extract increases the resistance of LDL to atherogenic modifications, including CS binding ability, oxidation, and aggregatioa and moderately re-duced their plasma lipids levels and systolic blood pressures. The lipid peroxidation hypothesis of atherosclerosis is supported by the antiatherosclerotic effects of some rnrritional antioxidants, as demonstrated in humans and E c mice A3-'-10 Licorice extract ex¬hibited antioxidative characteristics against LDL oxidation tn nor¬ma! healthy subjects 21 We previously showed that LDL from hvpercholesterolemic patieres is highly-susceptible to oxidation 31 In the present study, licorice extract consumption by hvpercholes¬terolemic patietts significantlv reduced the increased susceptibility of their LDL to oxidation, further demonstrating its remarkable antioxidative capacity ex vivo. The mechanisms responsible for licorice protection of LDL against oxidation lie in its previously shown capacity to bind to LDL. scavenge free radicals, and protect other anioxidaus associated with LDL. the carotenoids, from oxidation.-1-23 However, licorice consumption did not affect wo other protectors of LDL from oxidation in serum, plasma HDL cholesterol level and serum PON1 activity. Thus, our results suggest that the protection of LDL against oxidation by licorice consumption is not mediated by an increase in HDL or POX1 activity but by a direct effect on LDL itself. These results are in contrast to what we previously found with dietary supplementation of other polyphenolic antioxidants rich nutrients K15JS such as red wine and pomegranate juice, which increased serum PON1 activity
Atherosclerosis is a multifaaorial disease, and other factors besides lipid peroxidation can accelerate atherogenesis independent or in association with bpid peroxidation Increased retention and aggregation of LDL in the arterial wall and LDL oxidation are key events in the acceleration of atherogenesis The present study showed for the first time that licorice extract consumption by hvpercholesterolemic patients reduces the susceptibility of their LDL to ex vivo CS binding ability, oxidation, and aggregation
We used vortexing to assess the susceptibility of LDL to aggregation This method was described previously as a physical procedure that leads to the exposure of the lipoprotan surface hydrophobic domains, leading to surface-induced aggregation of LDL.32 Because LDL is sensitive to surface denaturation, tins method although quite aggressive, might represent changes that apparenth' occur during the retention of LDL in the arterial wall. There are several lines of evidence that LDL aggregation occurs in the arterial wall5-33 Further, we recently demonstrated that macrophage-released proteoglycans are involved in macrophage stimulation of LDL aggregation.34 Because LDL and macrophages are retained in the atherosclerotic lesion on the arterial wall and macrophages release proteoglycans to their surroundings under atherogenic conditions, the macrophage-mediated aggregation of LDL may play a physiologic role in LDL modification in vivo. Further, because aggregated LDL can induce macrophage choles¬terol accumulation and foam cell formation 50 it is considered as a risk factor for atherosclerosis. The inhibitory effect of licorice on LDL aggregation can be attributed to posable binding of licorice extract constituents such as its polyphenols to dte LDL particle, and such irreractions between the lipoprotein hydrophobic do¬mains and the licorice polyphenols can affect interactions between lipoproteins and their subsequent aggregation These resubs are in agreement with those of our previous study, which demonstrated a reduced susceptibility to aggregation of LDL derived from athero¬sclerotic Ec mice after dietary consumption of red wine or its major polyphenols catechin or quercetin. 14
The reductions in the suscepdbrhty of LDL to oxidation, its CS binding ability, and blood pressure after licorice consumption were partly sustainfd during the month of placebo consumption. These effects could be related to the persistance of some licorice-derived components in plasma and its association with newly formed LDL, thus further protecting it from modification for an extended period over the supplementation period. However, the susceptibility to aggregation was not sustained over the supplementation period, suggesting that additional mechanisms beyond die interrelations of oxidation and aggregation are involved in licorice-mediated irhi- bition of LDL aggregation.
Administration of licorice extract to hvpercholesterolemic pa¬tients significantly reduced LDL oxidaiion and aggregation and moderately reduced their plasma and LDL cholesterol levels This hypocholesterolemic effect of licorice extract is in agreement with a previous report showing that plant foods possess cholesterol- suppressive capacities, in agreement with our previous report that the antioxidant carotenoids 0-carotene and lycopene in tomatoes act as hvpocholesterolemic 3gents secondarv to their inhibitory- effect on cellular cholesterol biosynthesis. 33
In addition to the inhibitory effect of licorice consumption on LDL quality (i.e.. susceptibility to oxidation) and quantity (serum concentration), licorice extract consumption induced a moderate reduction in patients' systolic blood pressures. The licorice ethan- olic extract used in our study is completely free of glycirhiamc arid, a known hypertensive agent. The hypotensive effect of di¬etary antioxidants has been reported in hypertensive patients. 35 Because reactive oxygen species contribute to endothdium- depmdent contraction and increase vascular resistance, antioxi¬dants might restore endothelial function and. hence, decrease blood pressure.
We conclude that consumption of licorice extract may prove beneficial m its ability to attenuate the accelerated development of atherosclerosis in hvpercholesterolemic patients Licorice-root ex¬tract consumption was associated with reduced atherogenic mod- ificaions of LDL, including reduced oxidation, reduced CS bind¬ing ability (retention) and aggregation reduced plasma lipid levels, and reduced systolic blood pressure
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