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Hypolipidemic and antioxidant effect of polyphenol-rich extracts from Moroccan thyme varieties
e-SPEN Journal 7 (2012) e119ee124
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Original article
Hypolipidemic and antioxidant effect of polyphenol-rich extracts from Moroccan thyme varieties Mhamed Ramchoun a, Hicham Harnafi b, Chakib Alem c, Berthold Büchele d, Thomas Simmet d, Mustapha Rouis e, Fouad Atmani a, Souliman Amrani a, * a
Department of Biology, Faculty of Sciences, University Mohamed I, Bd Mohamed VI BP 717, 60 000 Oujda, Morocco Laboratory Recovery and Safety of Food Products, Faculty of Sciences & Techniques, Beni-Mellal, Morocco Department of Biology, Faculty of Sciences & Techniques, University Moulay Ismail, 52000 Errachidia, Morocco d Ulm University, Institute of Pharmacology of Natural Products & Clinical Pharmacology, Helmholtzstr. 20, D-89081 Ulm, Germany e UR-04, Vieillissement, Stress and Inflammation, University Pierre et Marie Curie, Bât. A, 5ème étage. 7 Quai Saint-Bernard, 75252 Paris Cedex 05, France b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 2 February 2011 Accepted 24 February 2012
Background & aims: In the present study, the polyphenol-rich extracts of four endemic medicinal plants (Thymus satureioides, Thymus zygis L., Thymus atlanticus and Thymus vulgaris) widely used in the Errachidia area (south east of Morocco) were analyzed for their hypocholesterolemic, hypotriglyceridemic, and antioxidant activities. Methods: Hyperlipidemia was induced in rats by intraperitoneal injection of Triton WR-1339 at a dose of 200 mg/kg body weight. The animals were divided into seven groups: the normolipidemic control group (NCG), the hyperlipidemic control group (HCG), the T. atlanticus-treated group (TA), the T. zygis-treated group (TZ), the T. satureioides-treated group (TS), the T. vulgaris-treated group (TV) and the Fenofibratetreated group (FF). The hyperlipidemic groups were treated with herbal extracts at a dose of 0.2 g/100 g body weight and with fenofibrate at a dose of 6.5 mg/100 g body weight. The antioxidant activity of the polyphenol-rich extracts was assessed by using the Ferric Reducing Antioxidant Power assay (FRAP), the Radical Scavenging Activity method (RSA) and the inhibition of 2,20 -azobis (2-amidinopropane) hydrochloride (AAPH) that induces oxidative erythrocyte hemolysis. Results: After 24 h of treatment with polyphenol-rich extract from T. atlanticus, plasma total cholesterol, triglycerides and LDL-cholesterol decreased by 82.8% (P < 0.001), 96.4% (P < 0.001) and 82.2% (P < 0.001), respectively. The results demonstrate that the four aqueous thyme extracts possess antioxidant activity as evidenced by ferric reducing/antioxidant activity (equivalent to 50.79 2.02 mmol Trolox/g of extract) and radical scavenging activity (IC50: 0.44 0.02 mg/mL of extract). Conclusion: Our findings suggest that polyphenol-rich thyme extracts might be considered for therapeutic use to treat atherosclerosis. Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
Keywords: Atherosclerosis Hyperlipidemia Antioxidant Thyme Polyphenol Erythrocyte hemolysis Rats
1. Introduction Cardiovascular disease is the most frequent cause of death in industrialized countries and hyperlipidemia represents a major risk factor for the premature development of atherosclerosis and its vascular complications.1 In addition, it is well established that elevated plasma low-density lipoproteins (LDL) is a risk factor for atherosclerosis and that oxidatively modified LDL (Ox-LDL) exerts
* Corresponding author. Tel.: þ212 36 500601/02; fax: þ212 36 50060103. E-mail addresses: [email protected], [email protected] (S. Amrani).
a variety of proatherogenic effects.2 Despite, the availability of highly effective treatments, morbidity and mortality from atherosclerotic vascular diseases remain substantial. Therefore, novel therapies to improve cardiovascular outcomes are needed. Thus, natural compounds might represent an alternative therapeutic approach. Indeed, several epidemiological and experimental studies have shown that intake of a number of polyphenols from medicinal plants, which exert protective effects against oxidative stress, is inversely correlated with atherosclerosis development and cardiovascular events.3 In Morocco, as in many developing countries, most hyperlipidemic patients use medicinal plants as traditional medicine to treat hyperlipidemia and atherosclerosis. Therefore, there is a strong interest to search for
2212-8263/$36.00 Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.clnme.2012.02.005
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natural hypolipemic and antioxidant substances derived from medicinal plants. Thus, a large number of plants including Garcinia cambogia,4 Zingiber officinale5 and Emblica officinalis6 were used to lower plasma lipid levels. Moreover, various species of Thymus, aromatic plants of the Mediterranean flora commonly used as spices and traditional remedies, have been reported to possess various biological effects including antioxidant and antiinflammatory activities.7 Although, the anti-oxidative and antimicrobial properties of thyme were intensively studied.8,9 Few investigations have concerned their hypolipidimic effects. In this study, we assessed the hypolipemic and antioxidant effects of polyphenol-rich extracts obtained from endemic Moroccan thyme varieties (Lamiaceae) in Triton WR-1339-induced hyperlipidaemic rats. These plants were collected in the Errachidia area (south east of Morocco).
2. Materials and methods 2.1. Plant material Thyme varieties were collected in AprileMay 2007 in the Errachidia region, Morocco. The plants were identified by Dr. Ibn Tatou and voucher specimens were deposited at the herbarium of the Scientific Institute, University Mohammed V, Rabat, Morocco. T. satureioides Cosson (No: RAB 77497), T. zygis L. subsp. gracilis (Boiss.) R. Morales (No: RAB 77494), Thymus cf. atlanticus (Ball) Roussine (No: RAB 77496), Thymus vulgaris were cultivated in the botanic garden of the Faculty of Sciences and Techniques, Errachidia, Morocco.
2.2. High performance liquid chromatography (HPLC) analysis Gradient HPLC analysis of the thyme extracts was carried out on a Reprosil PUR C18 column equipped with a photodiode array detector. The stationary phase was a C18 analytical column (250 mm 3 mm) with a particle size of 5 mm thermostated at 28 C. Extract (100 ml) was separated at 28 C. The flow rate was 0.5 mL min1 and the absorbance changes were monitored at 215, 250 and 280 nm. The mobile phases for chromatographic analysis were: (A) methanol/water (20/80) þ 0.2% glacial acetic acid and (B) methanol/water (80/20) þ 0.2% glacial acetic acid: 100% (A) and 0% (B) at 0 min, 50% (A) and 50% (B) during 10 min, 17% (A) and 83% (B) during 20 min, which was changed to 100% (A) and 0% (B) in 5 min (35 min, total time). The retention time of standards and the corresponding UV spectra were used for identification of the compounds in thyme extracts.
2.3. Biochemical analysis of polyphenol-rich thyme extracts 2.3.1. Chemicals The following reagents were obtained from Sigma Chemical Co: FolineCiocalteu, caffeic acid, rutin, DPPH (1,1-diphenyl-2-picrylhydrazil), Trolox, TPTZ (Tripyridyl triazine), AAPH (2,20 -azobis (2amidinopropane) hydrochloride), Triton WR-1339 (Tyloxapol). 2.3.2. Preparation of thyme extracts for biochemical and antioxidant analysis Air-dried aerial parts of plants were powdered (25 g) and extracted with 600 mL of double distilled water in a Soxhlet extractor for 2 h. The yields of extraction were 10.00, 7.60, 7.60 and 14.80% for T. satureioides (TS), T. zygis L. (TZ), T. atlanticus (TA) and T. vulgaris (TV), respectively. The extracts were concentrated and dissolved in double distilled water.
2.3.3. Determination of total polyphenols content The polyphenol contents in the water extracts were determined according to the FolineCiocalteu colorimetric method10; caffeic acid was used to generate a calibration curve. 2.3.4. Determination of flavonoid contents The flavonoid contents of the extracts were determined spectrophotometrically by using a method based on the formation of a flavonoidealuminum complex, having its maximum absorption at 430 nm11; rutin was used to generate the calibration curve. The flavonoid contents were expressed in mg per g of rutin equivalents.
2.4. Hypolipidemic study of polyphenol-rich thyme extracts 2.4.1. Preparation of thyme extract for hypolipidaemic treatment The dried aerial parts of the herbs were boiled 30 min in distilled water, filtered and the obtained solution was concentrated in a rotatory evaporator under vacuum at 65 C. 2.4.2. Animals and treatment Adult female Wistar rats weighing 170e200 g were bred in the animal facility of the Biology Department (Faculty of Sciences, Oujda, Morocco) in accordance with international guidelines.12 They were housed in a controlled room with a 12 h lightedark cycle, at room temperature of 22 2 C and kept on standard chow diet (Société SONABETAIL, Oujda, Morocco). 2.4.3. Experimental animal protocol Overnight fasted rats were divided into seven groups each consisting of six rats (Table 1). The first group represents the normolipidemic control (NCG), which received both water by gavage instead of plant extracts and saline solution instead of Triton WR-1339 by intraperitoneal administration. The second group represents the hyperlipidemic control group (HCG). These animals were treated with Triton WR-1339 by intraperitoneal injection at a dose of 200 mg/kg (body weight) in physiological saline solution and received distilled water by gavage. In the treatment groups, the animals were also treated with intraperitoneal injection of Triton that was followed by gavage of aqueous extracts from T. zygis (TZ), T. atlanticus (TA), Thymus saturoides (TS), and T. vulgaris (TV) (0.2 g/100 g body weight), the last group received intraperitoneal injection of Triton plus fenofibrate used as standard hypolipidemic drug (6.5 mg/100 g body weight) by gavage. In the following 24 h, the animals had access only to tap water. After 24 h of treatment, the animals were anaesthetized with diethyl ether and blood was taken from their tail vein by using heparinized capillaries. The blood samples were immediately centrifuged for 10 min at 2500 rpm and lipid plasma samples were immediately analyzed.
Table 1 Table explaining experimental animal protocol groups and study design. Groups
Treatments
NCG HCG HCG HCG HCG HCG HCG
Distilled water Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg
þ þ þ þ þ
TA TZ TS TV FF
BW) BW) BW) BW) BW) BW)
þ þ þ þ þ þ
Distilled water AE of TA (0.2 g/100 g BW) AE of TZ (0.2 g/100 g BW) AE of TS (0.2 g/100 g BW) AE of TV (0.2 g/100 g BW) FF (6.5 mg/100 g BW)
NCG: Normolipidemic Control Group, HCG: Hyperlipidemic Control Group, TA: Thymus atlanticus, TZ: Thymus zygis, TS: Thymus satureioides, TV: Thymus vulgaris, FF: Fenofibrate, AE: Aqueous extract, BW: Body weight.
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2.4.4. Biochemical analysis of plasma Triglyceride, total cholesterol and HDL-cholesterol analysis were performed using commercial diagnostic reagents (Biosud Diagnostici S.r.l. Italy) by means of colorimetric assays. LDL-cholesterol was calculated by the Friedewald formula13: LDL-cholesterol ¼ total cholesterol e (HDL-cholesterol þ TG/5). In our experimental study, the TG levels are higher than 400 mg/ dl representing a limitation to the Friedewald equation. We applied this formula for all groups and we noted a pharmacological effect in these condition. All values are calculated with the same method.14
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3. Results 3.1. HPLC analysis of thyme polyphenol-rich extracts According to the retention time of calibration standards, thyme extracts presented a chemical profile composed of six phenolic compounds, including caffeic and rosmarinic acids as well as quercetin, hesperetin, luteolin-7-glycoside and apigenin-7glycoside. The analysis of the typical HPLC chromatogram (Fig. 1) depicted that rosmarinic acid is the major phenolic acid and quercetin is the major flavonoid especially in T. atlanticus. Rosmarinic acid was expected to represent the single most antioxidative constituent (Table 2).
2.5. Antioxidant study of polyphenol-rich thyme extracts 3.2. Induction of hyperlipidemia with Triton WR-1339 2.5.1. RSA assay (DPPH Radical Scavenging Activity) DPPH (1,1-diphenyl-2-picryl-hydrazil) is an unstable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The model of scavenging the stable DPPH radical has been widely used for a relatively rapid evaluation of antioxidant activities compared to other methods.15 The reduction capability of the DPPH radical was determined by its absorbance decrease at 517 nm, as induced by natural antioxidants. This was visualized as a discoloration from purple to yellow. An aqueous solution of the sample was added to DPPH ethanolic solution (150 mM) according to the Barros method.16 After mixing gently and leaving it for 30 min at room temperature, the absorbance was read at 517 nm. Trolox as DPPH-scavenging compound was used as positive control. The results were expressed as inhibitory concentration 50 (IC50) and measurements were done in triplicate. The capability to scavenge the DPPH radical was calculated using the following equation16:
Plasma total cholesterol and triglyceride levels of all groups were measured after 24 h as shown in Fig. 2 and Table 3. In comparison to the normal control group (NCG), Triton WR-1339 caused a marked increase of plasma total cholesterol and triglyceride levels (HCG group). In fact, 24 h after the Triton administration, the plasma total cholesterol was elevated by 391.8% (P < 0.001) in HCG (281.89 17.80 mg/dl) vs NCG (57.31 1.13 mg/ dl). Triglycerides levels are also elevated by 789.4% (P < 0.001) in HCG (517.58 19.08 mg/dl) vs NCG (58.19 1.71 mg/dl). HDL and LDL-cholesterol concentrations are shown in Fig. 3 and Table 3. The HDL-cholesterol concentration was significantly decreased by 74.0% in HCG (8.16 2.10 mg/dl) vs the control NCG (31.44 1.62 mg/dl) group. In contrast, LDL-cholesterol levels were more than 11-fold higher in the HCG (170.29 14.04 mg/dl) than in the normal control group NCG (14.23 1.14 mg/dl) (P < 0.001).
DPPH-scavenging effect (%) ¼ 100 (Acontrol Asample)/Acontrol
3.3. Effects of aqueous thyme extracts and fenofibrate on the plasma lipid profiles in rats
2.5.2. FRAP assay (Ferric Reducing Antioxidant Power) The method is based on the reduction of the Fe3þ tripyridyl triazine (TPTZ) complex to the ferrous form at low pH.17 The antioxidant activity of the thyme extracts was measured by monitoring the change in absorption at 593 nm.
After 24 h of treatment, administration of aqueous T. atlanticus extract to rats that received Triton injection significantly decreased both plasma total cholesterol and triglycerides by 82.8% and 96.4%, respectively (P < 0.001), when compared to HCG (Fig. 2, Table 3). LDL-cholesterol was significantly lowered by 82.2% (P < 0.001) but the levels of HDL remained statistically unchanged (Fig. 3, Table 3). However, the aqueous extracts obtained from T. zygis exerted no significant effect on plasma TC, TG and HDL-cholesterol, but the
2.5.3. Inhibition of the AAPH-induced oxidative erythrocyte hemolysis The antioxidant activity of the thyme extracts was measured as inhibition of the AAPH-induced oxidative erythrocyte hemolysis according to the procedure established by Prost with minor modifications.18 Blood was obtained from a rabbit and diluted with heparinized 10 mM phosphate-buffered saline (PBS) solution at pH 7.4. To induce free radical chain oxidation in the erythrocytes, aqueous peroxyl radicals were generated by thermal decomposition of AAPH (2,20 -azobis (2-amidinopropane) hydrochloride) in oxygen. The erythrocyte suspension was mixed with 1 mL of PBScontaining thyme extracts. AAPH in PBS was then added to the mixture. The reaction mixture was gently shaken during incubation at 37 C; the absorbance was read at 540 nm every 5 min. The protection of the erythrocytes by the extracts was deduced from the time required for half-hemolysis (50% reduction of A540 nm) compared to control values (PBS, pH 7.4). 2.6. Statistical analysis The data obtained were analyzed by ANOVA and the Student’s ttest. P values less than 0.05 were considered statistically significant. Results are expressed as means SEM.
Fig. 1. Comparison of the HPLC profiles from the tested Thymus extracts as detected at 280 nm. (A) Standards: (1) caffeic acid, (2) luteolin-7-glycoside, (3) rosmarinic acid, (4) daidzein, (5) quercetin, (6) hesperetin, (7) apigenin, (8) thymol, (9) carvacrol. Extracts: (B) Thymus zygis e TZ, (C) Thymus atlanticus e TA, (D) Thymus satureoides e TS, (E) Thymus vulgaris e TV.
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Table 2 Identified compounds and their retention times. Plants
Identified compound
Retention time (min)
Thymus atlanticus
Caffeic acid Rosmarinic acid Quercetin Caffeic acid Rosmarinic acid NI Luteolin-7-glycoside Rosmarinic acid Hesperetin Rosmarinic acid
11.60 17.00 21.20 11.60 17.00 13.70 15.96 17.00 22.00 17.00
Thymus zygis Thymus satureioides
Thymus vulgaris NI: No identified compound.
LDL-cholesterol was significantly elevated (þ37.7%, P < 0.01). T. satureioides shows elevated HDL-cholesterol (þ125.1%, P < 0.01) compared with HCG (Fig. 3, Table 3). T. vulgaris shows elevated concentration of TG (þ20.7%, P < 0.01), but it exerted no significant effect on plasma TC, HDL-cholesterol and LDL-cholesterol. In comparison to HCG rats, in fenofibrate-treated rats (FF) we observed a significant decrease of both plasma total cholesterol and triglycerides by 20.4%, P < 0.01 and 83.7%, P < 0.001, respectively (Fig. 2). Whereas fenofibrate increased the HDL-cholesterol by 137.1%, P < 0.001 (Fig. 3). LDL-cholesterol was not significantly changed (Fig. 3, Table 3).
3.4. Biochemical analysis and antioxidant activities of thyme extract 3.4.1. Polyphenol and flavonoid contents The total polyphenol and flavonoid contents of the four thyme varieties are shown in Table 4. The total polyphenol contents were between 356.00 9.79 (TV) and 481.10 23.42 mg eq caffeic acid/g of extract (TA). The flavonoid contents were between 144.91 2.83 and 197.93 3.18 mg eq rutin/g of extract. 3.4.2. Antioxidant activities The antioxidant activities of the four thyme varieties are shown in Table 4. The IC50 values from Moroccan thyme varieties were between 0.44 0.02 and 0.70 0.02 mg/mL of extract. The FRAP
Fig. 2. Effects of thyme extracts and fenofibrate on total plasma cholesterol and triglycerides after 24 h of hyperlipideamia induced by Triton WR-1339 in rats. Values are mean SEM from six animals. NCG: normolipidemic control group; HCG: hyperlipidemic control group; TA: Thymus atlanticus-treated group; TZ: Thymus zygis-treated group; TS: Thymus satureoides-treated group; TV: Thymus vulgaris-treated group; FF: fenofibrate-treated group; TC: total cholesterol; TG: triglycerides. Hyperlipidemic group as compared to control group. TA, TZ, TS, TV and FF are compared to HCG. **P < 0.001; *P < 0.01; NS: not significant.
Fig. 3. Effects of thyme extracts and fenofibrate on plasma HDL and LDL-cholesterol after 24 h of hyperlipidemia induced by Triton WR-1339 in rats. Values are mean SEM from six animals. NCG: normolipidemic control group; HCG: hyperlipidemic control group; TA: Thymus atlanticus-treated group; TZ: Thymus zygis-treated group; TS: Thymus satureoides-treated group; TV: Thymus vulgaris-treated group; FF: fenofibrate-treated group; HDL-C: HDL-cholesterol; LDL-C: LDL-cholesterol. Hyperlipidemic group as compared to control group. TA, TZ, TS, TV and FF are compared to HCG. **P < 0.001; *P < 0.01; NS: not significant.
assay shows that the antioxidant activity was between 38.19 0.18 and 50.79 2.02 mmol Trolox/g of extract. 3.4.3. Antihemolysis activity The inhibition of the AAPH-induced oxidative erythrocytes hemolysis by the polyphenol-rich extracts from the four varieties is shown in Table 5. Addition of AAPH induced a significant decrease in the hemolysis half-time from 73.33 2.88 min to 40.00 0.01 min (45%, P < 0.001). Application of the polyphenolrich extract from the four varieties to the erythrocytes suspension with AAPH induced an increase of the hemolysis half-time by 550% (P < 0.001), 533% (P < 0.001), 470% (P < 0.001) and 305% (P < 0.001) from T. satureioides, T. vulgaris, T. atlanticus, and T. zygis, respectively. Trolox was used as standard antioxidant showed an increase of the hemolysis half-time by 525% (P < 0.001). 4. Discussion The Triton WR-1339, a non-ionic detergent has been widely used to block the clearance of triglyceride-rich lipoproteins by inhibiting the lipoprotein lipase and to induce an acute hyperlipidemia in experimental animals.19 This model was used for a number of different objectives including studies of lipid metabolism and screening for natural and chemical hypolipidemic drugs.20,21 Hence, many plants such as Vaccinium myrtillus and Phyllanthus niruri have been investigated for their acute hypolipidemic activity in Triton WR-1339-induced hyperlipidemic animals.20 The highest levels of plasma triglycerides and total cholesterol were reached 20 h after Triton administration.20 In our study, this model yielded a similar pattern of lipid profile changes at 24 h after Triton injection and demonstrating its feasibility to assess the acute hypolipemic activity of aqueous thyme extracts. Our results clearly showed that aqueous T. atlanticus extract at a dose of 0.2 g/100 g body weight significantly lowered both plasma triglycerides and cholesterol levels after 24 h of treatment. The reduction of plasma total cholesterol was associated with a decrease of the LDL fraction, which is a major, potentially modifiable risk factor of cardiovascular diseases and the target of many hypocholesterolemic therapies. This finding led as to suggest that the cholesterol-lowering activity of these extract might be due to the enhancement of LDL-C catabolism through its hepatic receptors
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Table 3 Changes of plasma lipid parameters in control and treated rats.
NCG HCG TA TZ TS TV FF
TC (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
57.31 1.13 281.89 17.8**(þ391.8%) 48.28 3.35** (82.8%) 336.18 19.19ns 257.13 19.17ns 267.61 15.28ns 224.21 37.93* (20.4%)
58.19 1.71 517.58 19.08**(þ789.4%) 18.21 5.72** (96.4%) 452.36 37.81ns 345.26 95.05ns 624.72 13.94* (þ20.7%) 85.4 9.45** (83.7%)
31.44 1.62 8.16 2.1** (74.0%) 14.23 2.16ns 10.72 1.61ns 18.37 2.67*(þ125.1%) 14.14 3.76ns 19.35 0.01**(þ137.1%)
14.23 1.14 170.29 14.04**(þ109.6%) 30.19 3.46** (82.2%) 234.97 18.94* (þ37.7%) 169.7 37.19ns 128.52 19.12ns 183.87 38.7ns
NCG: normolipidemic control group; HCG: hyperlipidemic control group; TA: Thymus atlanticus-treated group; TZ: Thymus zygis-treated group; TS: Thymus satureioidestreated group; TV: Thymus vulgaris-treated group; FF: fenofibrate-treated group TC: total cholesterol; TG: triglycerides; HDL-C: HDL-cholesterol; LDL-C: LDL-cholesterol Hyperlipidemic group as compared to control group. TA, TZ, TS, TV and FF are compared to HCG. **P < 0.001; *P < 0.01; ns: not significant.
for the final elimination as secretory bile acids as demonstrated by Khanna et al.22 It has also recently been reported that triglycerides played a key role in the regulation of lipoprotein interactions maintaining normal lipid metabolism.23 Moreover, higher plasma TG levels have been attributed mainly to an increased population of small, dense LDL deposits, which are very atherogenic24 and enhanced cholesteryl ester mass transfer from apolipoprotein Bcontaining lipoproteins (VLDL and LDL).25 TGs have also been proposed to be the major determinant of cholesterol esterification, its transfer and HDL remodeling in human plasma. Aqueous extract from T. atlanticus significantly suppressed the elevated blood concentrations of TGs. This result suggested that this extract might be able to restore, at least partially, the catabolism of triglycerides. As hypothesized by others using different plants, this hypotriglyceridemic effect could be due to the increased stimulation of the lipolytic activity of plasma lipoprotein lipase (LPL).26 In this study, fenofibrate was used as a known reference hypolipemic drug. We found that the drug decreased plasma lipid 24 h after treatment suggesting that the lipid lowering activity of the drug was comparable to the tested extract. Fenofibrate exerted an elevated rise of HDL-cholesterol. This is in agreement with the mechanism by which fibrates act on lipid metabolism.27 Indeed, the triglycerides decreasing effect of fibrates was very important and often coupled with mild effect on LDL-C especially by both stimulation of the gene expression of lipoprotein lipase leading to enhanced catabolism of VLDL, synthesis of fatty acids and reduced VLDL secretion.26 On the other hand, it is important to note that the hypolipidemic effect of extract relatively comparable to that exerted by fenofibrate. Thus, we suggested that the hypolipidemic effect exerted by T. atlanticus extract might be explained by the same mechanisms of fenofibrate action. In this field, the possible pathways that can be implicated in the observed effect are hypothesized as catabolism of LDLcholesterol and its secretion in the form of bile acids. On the other hand, the suppression of the oxidative modification of plasma lipid and lipoprotein by antioxidants constituted one of the major targets of many antiatherogenic agents and the preferable strategy to prevent the crises of cardiovascular diseases.3 Many experimental investigations have demonstrated that a number of
secondary metabolites such as polyphenol compounds extracted from medicinal and aromatic plants possessed a high antioxidant potential due to their hydroxyl groups and protected more efficiently against some free radical-related diseases.28 In this work, we demonstrated that the four varieties of thyme originating from the Errachidia area are rich in total polyphenol and flavonoid with rosmarinic acid being the major polyphenol compound. Treatment with the polyphenol-rich thyme extracts dramatically increased erythrocyte hemolysis half-time. Hemolysis was provoked by the free radicals generated by the decomposition of the water-soluble azo compound (2,20 -azobis (2-amidinopropane) hydrochloride, AAPH) that attack erythrocyte membranes and induce lipid peroxidation leading to hemolysis. However, the increase in erythrocyte hemolysis halftime upon treatment with the thyme extracts would be due to two possible reasons: the structure of the polyphenols and flavonoid contents in the thymes which renders them potent antioxidants capable of neutralizing/scavenging free radicals that attack the erythrocyte membrane supported by the RSA and FRAP assays and/or that flavonoids might be incorporated into the erythrocyte membrane, thereby increasing its stability against hemolysis.3,29 In fact, a highly positive relationship has been established between total phenols and antioxidant activity in many plant species.30 Our results clearly demonstrated that the bioactive compound(s) contained in this plant have a polar character since they are soluble in water. This finding was in agreement with previous reports showing that polar plant extracts possessed cholesterol-suppressive capacities and the ability to attenuate the accelerated development of atherosclerosis in hypercholesterolemic animal models.31 In fact, polyphenols constitute a heterogeneous polar group of ubiquitous plant phytochemicals that exhibited a variety of pharmacological activities including anti-atherogenesis effects. Our results strongly suggested that the hypolipidemic activity of T. atlanticus plant could be attributed to the presence of polyphenolic compounds. Besides, HPLC profile of thyme polyphenol-rich extracts showed that the thyme contained rosmarinic acid as major polyphenol compound which can be probably implicated in the observed pharmacological activities.
Table 4 Polyphenol contents and antioxidant activity of the polyphenol-rich extracts (PRE) from the four thyme varieties. TA Pp (mg eq caffeic acid/gPRE) Fv (mg eq rutin/gPRE) RSA (IC50 (mg/ml PRE) FRAP (mmol trolox/gPRE)
481.1 144.91 0.44 50.31
TZ
23.42 2.83 0.02** 1.65
443.29 197.93 0.54 38.19
TS
5.82 3.18 0.02* 0.18
456.73 172.79 0.48 50.79
TV
6.94 2.12 0.01* 2.02
356.00 186.93 0.70 48.03
Trolox
9.79 2.51 0.02*** 0.01
e e 0.51 0.01 44.33 7.55
Pp: polyphenols, Fv: flavonoid, PRE: polyphenol-rich extract, FRAP: Ferric Reducing Antioxidant Power, RSA: Radical Scavenging Activity, TA: Thymus atlanticus, TZ: Thymus zygis. TS: Thymus satureioides, TV: Thymus vulgaris. RSA (***P < 0.001 TV vs Trolox, **P < 0.01 TA vs Trolox, *P < 0.05, TS and TZ vs Trolox).
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Table 5 Antihemolytic activity of aqueous thyme extracts. Hemolysis half-time (min) Control AAPH sample AAPH þ Thymus AAPH þ Thymus AAPH þ Thymus AAPH þ Thymus AAPH þ Trolox
atlanticus zygis L satureioides vulgaris
73.33 40.00 228.33 162.00 260.00 253.33 250.00
2.88 0.01* 11.54* 35.38* 17.32* 5.77* 13.26*
% Deviation 45% þ470% þ305% þ550% þ533% þ525%
AAPH: 2,20 -azobis (2-amidinopropane) hydrochloride. *P < 0.001, AAPH sample vs control; Thymus atlanticus, Thymus zygis, Thymus satureioides, Thymus vulgaris and Trolox vs AAPH sample.
5. Conclusion We concluded that the T. atlanticus polyphenol-rich extract exhibits a potent hypolipidemic capacity. In addition, the extracts of the four thyme varieties possess considerable antioxidant activities. These medicinal plants might be used as alternative intervention in prevention and treatment of atherosclerosis and related cardiovascular diseases. Author contributions MR, FA and HH contributed to the conception and design of the study. CA, SA and BB analyzed and interpreted the data. TS and MR drafted the manuscript. All authors participated in revising the manuscript and the final approval of the submitted version. Conflict of interest The authors have nothing to declare. Acknowledgments The authors wish to express their gratitude to Dr. Ibn Tatou for plant material identification. Abbreviations
LPO FRAP RSA HPLC AAPH NCG HCG TG LPL VLDL FF Ox-LDL DPPH TPTZ PBS
lipid peroxidation Ferric Reducing Antioxidant Power Radical Scavenging Activity high performance liquid chromatography 2,20 -azobis (2-amidinopropane) hydrochloride normolipidemic control group hyperlipidemic control group triglycerides lipoprotein lipase very low-density lipoprotein fenofibrate oxidatively modified LDL 1,1-diphenyl-2-picryl-hydrazil Tripyridyl triazine phosphate-buffered saline.
References 1. Castelli WP, Garisson RJ, Wilson PWF, Abbott RD, Kalousdian S, Kannel WB. Incidence of coronary heart disease and lipoprotein cholesterol levels: the Framaingham study. Journal of the American Medical Association 1986;256:2835e8. 2. Chisolm GM, Steinberg D. The oxidation modification hypothesis of atherogenesis: an overview. Free Radical Biology and Medicine 2000;28:1815e26.
3. Sesso HD, Gaziano JM, Liu S, Buring JE. Flavonoid intake and the risk of cardiovascular disease in women. American Journal of Clinical Nutrition 2003;77:1400e8. 4. Koshy AS, Vijayalakshmi NR. Impact of certain flavonoids on lipid profilespotential action of Garcinia cambogia flavonoids. Phytotherapy Research 2001;15:395e400. 5. Bhandari U, Sharma JN, Zafar R. The protective action of ethanolic ginger (Zingiber officinale) extract in cholesterol fed rabbits. Journal of Ethnopharmacology 1998;61:167e71. 6. Mathur R, Sharma A, Dixit VP, Varma M. Hypolipidemic effect of fruit juice of Emblica officinalis in cholesterol-fed rabbits. Journal of Ethnopharmacology 1996;50:61e8. 7. Chizzola R, Michitsch H, Franz C. Antioxidative properties of Thymus vulgaris leaves: comparison of different extracts and essential oil chemotypes. Journal of Agricultural and Food Chemistry 2008;56(16):6897e904. _ ska A. 8. Amarowicz R, Zegarska Z, Rafa1owski R, Pegg RB, Karama c M, Kosin Antioxidant activity and free radical-scavenging capacity of ethanolic extracts of thyme, oregano, and marjoram. European Journal of Lipid Science and Technology 2009;11:1111e7. 9. Sarica S, Ciftci A, Demir E, Kilinc K, Yildirim Y. Use of an antibiotic growth promoter and two herbal natural feed additives with and without exogenous enzymes in wheat based broiler diets. South African Journal of Animal Sciences 2005;35(1):61e72. 10. Aquino R, Morelli S, Lauro MR, Abdo S, Saija A, Tomaino A. Phenolic constituents and antioxidant activity of an extract of Anthurium versicolor leaves. Journal of Natural Products 2001;64:1019e23. 11. Jay M, Gonnet JF, Wollenweber E, Voirin B. Sur l’analyse qualitative des aglycones flavoniques dans une optique chimiotaxonomique. Phytochemistry 1975;14:1605e12. 12. Working Party of the European Commission. Recommendation for euthanasia of experimental animals. Part 1. Laboratory Animals 1996;30:293e316. 13. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of preparative ultracentrifuge. Clinical Chemistry 1972;18:499e502. 14. Nauck M, Warnick GR, Rifai N. Methods for measurement of LDL-cholesterol: a critical assessment of direct measurement by homogeneous assays versus calculation. Clinical Chemistry February 2002;48:236e54. 15. Yokozawa T, Chen CP, Dong E, Tanaka T, Nonaka GI, Nishioka I. Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2 picrylhydrazyl radical. Biochemical Pharmacology 1998;56:213e22. 16. Barros L, Baptista P, Ferreira ICFR. Effect of Lactarius piperatus fruiting body maturity stage on antioxidant activity measured by several biochemical assays. Food and Chemical Toxicology 2007;45:1731e7. 17. Benzie IFF, Strain JJ. Ferric reducing ability of plasma (FRAP) as a measure of antioxidant power. The FRAP assay. Analytical Biochemistry 1999;239:70e6. 18. Prost M. Brevet Français 1989, No 8900999. 19. Kellner A, Correll JW, Ladd AT. Sustained hyperlipemia induced in rabbits by means of intravenously injected surface-active agents. Journal of Experimental Medicine 1951;93:373e84. 20. Khanna AK, Rizvi F, Chander R. Lipid lowering activity of Phyllanthus niruri in hyperlipemic rats. Journal of Ethnopharmacology 2002;82:19e22. 21. Da Rocha JT, Sperança A, Nogueira CW, Zeni G. Hypolipidaemic activity of orally administered diphenyl diselenide in triton WR-1339-induced hyperlipidaemia in mice. Journal of Pharmacy and Pharmacology 2009;61:1673e9. 22. Khanna AK, Chander R, Chandan S, Srivastava AK, Kapoor NK. Hypolipidemic activity of Achyranthus aspera Linn in normal and triton induced hyperlipemic rats. Indian Journal of Experimental Biology 1992;30:128e30. 23. West KM, Ahuja MMS, Bennett PH, Czyzyk A, Mateo de Acosta O, Fuller JH, et al. The role of circulating glucose and triglyceride concentrations and their interactions with other “risk factors” as determinants of arterial disease in nine diabetic population samples from the WHO multinational study. Diabetes Care 1983;6(4):361e9. 24. Austin MA, Hokanson JE, Brunzell JD. Characterization of low density lipoprotein subclasses: methodologic approaches and clinical relevance. Current Opinion in Lipidology 1994;5:395e403. 25. Guerin M, Le Goff W, Lassel TS, Van Tol A, Steiner G, Chapman MJ. Proatherogenic role of elevated CE transfer from HDL to VLDL1 and dense LDL in type 2 diabetes. Arteriosclerosis Thrombosis and Vascular Biology 2001;21:282e9. 26. Shattat G, Al-Qirim T, Sweidan K, Shahwan M, El-Huneidi W, Al-Hiari Y. The hypolipidemic activity of novel benzofuran-2-carboxamide derivatives in Triton WR-1339-induced hyperlipidemic rats: a comparison with bezafibrate. Journal of Enzyme Inhibition and Medicinal Chemistry 2010;25(6):751e5. 27. Xie W, Wang W, Su H, Xing D, Cai G, Du L. Hypolipidemic mechanisms of Ananas comosus L. leaves in mice: different from fibrates but similar to statins. Journal of Pharmacological Sciences 2007;103:267e74. 28. Vaya J, Mahmood S, Goldblum A, Aviram M, Volkova N, Shaalan A, et al. Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy. Phytochemistry 2003;62:89e99. 29. De Freitas MV, Netto RM, Da Costa Huss JC, De Souza TMT, Costa JO, Firmino CB, et al. Influence of aqueous crude extracts of medicinal plants on the osmotic stability of human erythrocytes. Toxicology in Vitro 2008;22:219e24. 30. Velioglu YS, Mazza G, Gao L, Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. Journal of Agricultural and Food Chemistry 1998;46:4113e7. 31. Girija K, Lakshman K. Anti-hyperlipidemic activity of methanol extracts of three plants of Amaranthus in Triton-WR 1339 induced hyperlipidemic rats. Asian Pacific Journal of Tropical Biomedicine 2011:62e5.
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Original article
Hypolipidemic and antioxidant effect of polyphenol-rich extracts from Moroccan thyme varieties Mhamed Ramchoun a, Hicham Harnafi b, Chakib Alem c, Berthold Büchele d, Thomas Simmet d, Mustapha Rouis e, Fouad Atmani a, Souliman Amrani a, * a
Department of Biology, Faculty of Sciences, University Mohamed I, Bd Mohamed VI BP 717, 60 000 Oujda, Morocco Laboratory Recovery and Safety of Food Products, Faculty of Sciences & Techniques, Beni-Mellal, Morocco Department of Biology, Faculty of Sciences & Techniques, University Moulay Ismail, 52000 Errachidia, Morocco d Ulm University, Institute of Pharmacology of Natural Products & Clinical Pharmacology, Helmholtzstr. 20, D-89081 Ulm, Germany e UR-04, Vieillissement, Stress and Inflammation, University Pierre et Marie Curie, Bât. A, 5ème étage. 7 Quai Saint-Bernard, 75252 Paris Cedex 05, France b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 2 February 2011 Accepted 24 February 2012
Background & aims: In the present study, the polyphenol-rich extracts of four endemic medicinal plants (Thymus satureioides, Thymus zygis L., Thymus atlanticus and Thymus vulgaris) widely used in the Errachidia area (south east of Morocco) were analyzed for their hypocholesterolemic, hypotriglyceridemic, and antioxidant activities. Methods: Hyperlipidemia was induced in rats by intraperitoneal injection of Triton WR-1339 at a dose of 200 mg/kg body weight. The animals were divided into seven groups: the normolipidemic control group (NCG), the hyperlipidemic control group (HCG), the T. atlanticus-treated group (TA), the T. zygis-treated group (TZ), the T. satureioides-treated group (TS), the T. vulgaris-treated group (TV) and the Fenofibratetreated group (FF). The hyperlipidemic groups were treated with herbal extracts at a dose of 0.2 g/100 g body weight and with fenofibrate at a dose of 6.5 mg/100 g body weight. The antioxidant activity of the polyphenol-rich extracts was assessed by using the Ferric Reducing Antioxidant Power assay (FRAP), the Radical Scavenging Activity method (RSA) and the inhibition of 2,20 -azobis (2-amidinopropane) hydrochloride (AAPH) that induces oxidative erythrocyte hemolysis. Results: After 24 h of treatment with polyphenol-rich extract from T. atlanticus, plasma total cholesterol, triglycerides and LDL-cholesterol decreased by 82.8% (P < 0.001), 96.4% (P < 0.001) and 82.2% (P < 0.001), respectively. The results demonstrate that the four aqueous thyme extracts possess antioxidant activity as evidenced by ferric reducing/antioxidant activity (equivalent to 50.79 2.02 mmol Trolox/g of extract) and radical scavenging activity (IC50: 0.44 0.02 mg/mL of extract). Conclusion: Our findings suggest that polyphenol-rich thyme extracts might be considered for therapeutic use to treat atherosclerosis. Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
Keywords: Atherosclerosis Hyperlipidemia Antioxidant Thyme Polyphenol Erythrocyte hemolysis Rats
1. Introduction Cardiovascular disease is the most frequent cause of death in industrialized countries and hyperlipidemia represents a major risk factor for the premature development of atherosclerosis and its vascular complications.1 In addition, it is well established that elevated plasma low-density lipoproteins (LDL) is a risk factor for atherosclerosis and that oxidatively modified LDL (Ox-LDL) exerts
* Corresponding author. Tel.: þ212 36 500601/02; fax: þ212 36 50060103. E-mail addresses: [email protected], [email protected] (S. Amrani).
a variety of proatherogenic effects.2 Despite, the availability of highly effective treatments, morbidity and mortality from atherosclerotic vascular diseases remain substantial. Therefore, novel therapies to improve cardiovascular outcomes are needed. Thus, natural compounds might represent an alternative therapeutic approach. Indeed, several epidemiological and experimental studies have shown that intake of a number of polyphenols from medicinal plants, which exert protective effects against oxidative stress, is inversely correlated with atherosclerosis development and cardiovascular events.3 In Morocco, as in many developing countries, most hyperlipidemic patients use medicinal plants as traditional medicine to treat hyperlipidemia and atherosclerosis. Therefore, there is a strong interest to search for
2212-8263/$36.00 Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.clnme.2012.02.005
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natural hypolipemic and antioxidant substances derived from medicinal plants. Thus, a large number of plants including Garcinia cambogia,4 Zingiber officinale5 and Emblica officinalis6 were used to lower plasma lipid levels. Moreover, various species of Thymus, aromatic plants of the Mediterranean flora commonly used as spices and traditional remedies, have been reported to possess various biological effects including antioxidant and antiinflammatory activities.7 Although, the anti-oxidative and antimicrobial properties of thyme were intensively studied.8,9 Few investigations have concerned their hypolipidimic effects. In this study, we assessed the hypolipemic and antioxidant effects of polyphenol-rich extracts obtained from endemic Moroccan thyme varieties (Lamiaceae) in Triton WR-1339-induced hyperlipidaemic rats. These plants were collected in the Errachidia area (south east of Morocco).
2. Materials and methods 2.1. Plant material Thyme varieties were collected in AprileMay 2007 in the Errachidia region, Morocco. The plants were identified by Dr. Ibn Tatou and voucher specimens were deposited at the herbarium of the Scientific Institute, University Mohammed V, Rabat, Morocco. T. satureioides Cosson (No: RAB 77497), T. zygis L. subsp. gracilis (Boiss.) R. Morales (No: RAB 77494), Thymus cf. atlanticus (Ball) Roussine (No: RAB 77496), Thymus vulgaris were cultivated in the botanic garden of the Faculty of Sciences and Techniques, Errachidia, Morocco.
2.2. High performance liquid chromatography (HPLC) analysis Gradient HPLC analysis of the thyme extracts was carried out on a Reprosil PUR C18 column equipped with a photodiode array detector. The stationary phase was a C18 analytical column (250 mm 3 mm) with a particle size of 5 mm thermostated at 28 C. Extract (100 ml) was separated at 28 C. The flow rate was 0.5 mL min1 and the absorbance changes were monitored at 215, 250 and 280 nm. The mobile phases for chromatographic analysis were: (A) methanol/water (20/80) þ 0.2% glacial acetic acid and (B) methanol/water (80/20) þ 0.2% glacial acetic acid: 100% (A) and 0% (B) at 0 min, 50% (A) and 50% (B) during 10 min, 17% (A) and 83% (B) during 20 min, which was changed to 100% (A) and 0% (B) in 5 min (35 min, total time). The retention time of standards and the corresponding UV spectra were used for identification of the compounds in thyme extracts.
2.3. Biochemical analysis of polyphenol-rich thyme extracts 2.3.1. Chemicals The following reagents were obtained from Sigma Chemical Co: FolineCiocalteu, caffeic acid, rutin, DPPH (1,1-diphenyl-2-picrylhydrazil), Trolox, TPTZ (Tripyridyl triazine), AAPH (2,20 -azobis (2amidinopropane) hydrochloride), Triton WR-1339 (Tyloxapol). 2.3.2. Preparation of thyme extracts for biochemical and antioxidant analysis Air-dried aerial parts of plants were powdered (25 g) and extracted with 600 mL of double distilled water in a Soxhlet extractor for 2 h. The yields of extraction were 10.00, 7.60, 7.60 and 14.80% for T. satureioides (TS), T. zygis L. (TZ), T. atlanticus (TA) and T. vulgaris (TV), respectively. The extracts were concentrated and dissolved in double distilled water.
2.3.3. Determination of total polyphenols content The polyphenol contents in the water extracts were determined according to the FolineCiocalteu colorimetric method10; caffeic acid was used to generate a calibration curve. 2.3.4. Determination of flavonoid contents The flavonoid contents of the extracts were determined spectrophotometrically by using a method based on the formation of a flavonoidealuminum complex, having its maximum absorption at 430 nm11; rutin was used to generate the calibration curve. The flavonoid contents were expressed in mg per g of rutin equivalents.
2.4. Hypolipidemic study of polyphenol-rich thyme extracts 2.4.1. Preparation of thyme extract for hypolipidaemic treatment The dried aerial parts of the herbs were boiled 30 min in distilled water, filtered and the obtained solution was concentrated in a rotatory evaporator under vacuum at 65 C. 2.4.2. Animals and treatment Adult female Wistar rats weighing 170e200 g were bred in the animal facility of the Biology Department (Faculty of Sciences, Oujda, Morocco) in accordance with international guidelines.12 They were housed in a controlled room with a 12 h lightedark cycle, at room temperature of 22 2 C and kept on standard chow diet (Société SONABETAIL, Oujda, Morocco). 2.4.3. Experimental animal protocol Overnight fasted rats were divided into seven groups each consisting of six rats (Table 1). The first group represents the normolipidemic control (NCG), which received both water by gavage instead of plant extracts and saline solution instead of Triton WR-1339 by intraperitoneal administration. The second group represents the hyperlipidemic control group (HCG). These animals were treated with Triton WR-1339 by intraperitoneal injection at a dose of 200 mg/kg (body weight) in physiological saline solution and received distilled water by gavage. In the treatment groups, the animals were also treated with intraperitoneal injection of Triton that was followed by gavage of aqueous extracts from T. zygis (TZ), T. atlanticus (TA), Thymus saturoides (TS), and T. vulgaris (TV) (0.2 g/100 g body weight), the last group received intraperitoneal injection of Triton plus fenofibrate used as standard hypolipidemic drug (6.5 mg/100 g body weight) by gavage. In the following 24 h, the animals had access only to tap water. After 24 h of treatment, the animals were anaesthetized with diethyl ether and blood was taken from their tail vein by using heparinized capillaries. The blood samples were immediately centrifuged for 10 min at 2500 rpm and lipid plasma samples were immediately analyzed.
Table 1 Table explaining experimental animal protocol groups and study design. Groups
Treatments
NCG HCG HCG HCG HCG HCG HCG
Distilled water Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg Triton (200 mg/kg
þ þ þ þ þ
TA TZ TS TV FF
BW) BW) BW) BW) BW) BW)
þ þ þ þ þ þ
Distilled water AE of TA (0.2 g/100 g BW) AE of TZ (0.2 g/100 g BW) AE of TS (0.2 g/100 g BW) AE of TV (0.2 g/100 g BW) FF (6.5 mg/100 g BW)
NCG: Normolipidemic Control Group, HCG: Hyperlipidemic Control Group, TA: Thymus atlanticus, TZ: Thymus zygis, TS: Thymus satureioides, TV: Thymus vulgaris, FF: Fenofibrate, AE: Aqueous extract, BW: Body weight.
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2.4.4. Biochemical analysis of plasma Triglyceride, total cholesterol and HDL-cholesterol analysis were performed using commercial diagnostic reagents (Biosud Diagnostici S.r.l. Italy) by means of colorimetric assays. LDL-cholesterol was calculated by the Friedewald formula13: LDL-cholesterol ¼ total cholesterol e (HDL-cholesterol þ TG/5). In our experimental study, the TG levels are higher than 400 mg/ dl representing a limitation to the Friedewald equation. We applied this formula for all groups and we noted a pharmacological effect in these condition. All values are calculated with the same method.14
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3. Results 3.1. HPLC analysis of thyme polyphenol-rich extracts According to the retention time of calibration standards, thyme extracts presented a chemical profile composed of six phenolic compounds, including caffeic and rosmarinic acids as well as quercetin, hesperetin, luteolin-7-glycoside and apigenin-7glycoside. The analysis of the typical HPLC chromatogram (Fig. 1) depicted that rosmarinic acid is the major phenolic acid and quercetin is the major flavonoid especially in T. atlanticus. Rosmarinic acid was expected to represent the single most antioxidative constituent (Table 2).
2.5. Antioxidant study of polyphenol-rich thyme extracts 3.2. Induction of hyperlipidemia with Triton WR-1339 2.5.1. RSA assay (DPPH Radical Scavenging Activity) DPPH (1,1-diphenyl-2-picryl-hydrazil) is an unstable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The model of scavenging the stable DPPH radical has been widely used for a relatively rapid evaluation of antioxidant activities compared to other methods.15 The reduction capability of the DPPH radical was determined by its absorbance decrease at 517 nm, as induced by natural antioxidants. This was visualized as a discoloration from purple to yellow. An aqueous solution of the sample was added to DPPH ethanolic solution (150 mM) according to the Barros method.16 After mixing gently and leaving it for 30 min at room temperature, the absorbance was read at 517 nm. Trolox as DPPH-scavenging compound was used as positive control. The results were expressed as inhibitory concentration 50 (IC50) and measurements were done in triplicate. The capability to scavenge the DPPH radical was calculated using the following equation16:
Plasma total cholesterol and triglyceride levels of all groups were measured after 24 h as shown in Fig. 2 and Table 3. In comparison to the normal control group (NCG), Triton WR-1339 caused a marked increase of plasma total cholesterol and triglyceride levels (HCG group). In fact, 24 h after the Triton administration, the plasma total cholesterol was elevated by 391.8% (P < 0.001) in HCG (281.89 17.80 mg/dl) vs NCG (57.31 1.13 mg/ dl). Triglycerides levels are also elevated by 789.4% (P < 0.001) in HCG (517.58 19.08 mg/dl) vs NCG (58.19 1.71 mg/dl). HDL and LDL-cholesterol concentrations are shown in Fig. 3 and Table 3. The HDL-cholesterol concentration was significantly decreased by 74.0% in HCG (8.16 2.10 mg/dl) vs the control NCG (31.44 1.62 mg/dl) group. In contrast, LDL-cholesterol levels were more than 11-fold higher in the HCG (170.29 14.04 mg/dl) than in the normal control group NCG (14.23 1.14 mg/dl) (P < 0.001).
DPPH-scavenging effect (%) ¼ 100 (Acontrol Asample)/Acontrol
3.3. Effects of aqueous thyme extracts and fenofibrate on the plasma lipid profiles in rats
2.5.2. FRAP assay (Ferric Reducing Antioxidant Power) The method is based on the reduction of the Fe3þ tripyridyl triazine (TPTZ) complex to the ferrous form at low pH.17 The antioxidant activity of the thyme extracts was measured by monitoring the change in absorption at 593 nm.
After 24 h of treatment, administration of aqueous T. atlanticus extract to rats that received Triton injection significantly decreased both plasma total cholesterol and triglycerides by 82.8% and 96.4%, respectively (P < 0.001), when compared to HCG (Fig. 2, Table 3). LDL-cholesterol was significantly lowered by 82.2% (P < 0.001) but the levels of HDL remained statistically unchanged (Fig. 3, Table 3). However, the aqueous extracts obtained from T. zygis exerted no significant effect on plasma TC, TG and HDL-cholesterol, but the
2.5.3. Inhibition of the AAPH-induced oxidative erythrocyte hemolysis The antioxidant activity of the thyme extracts was measured as inhibition of the AAPH-induced oxidative erythrocyte hemolysis according to the procedure established by Prost with minor modifications.18 Blood was obtained from a rabbit and diluted with heparinized 10 mM phosphate-buffered saline (PBS) solution at pH 7.4. To induce free radical chain oxidation in the erythrocytes, aqueous peroxyl radicals were generated by thermal decomposition of AAPH (2,20 -azobis (2-amidinopropane) hydrochloride) in oxygen. The erythrocyte suspension was mixed with 1 mL of PBScontaining thyme extracts. AAPH in PBS was then added to the mixture. The reaction mixture was gently shaken during incubation at 37 C; the absorbance was read at 540 nm every 5 min. The protection of the erythrocytes by the extracts was deduced from the time required for half-hemolysis (50% reduction of A540 nm) compared to control values (PBS, pH 7.4). 2.6. Statistical analysis The data obtained were analyzed by ANOVA and the Student’s ttest. P values less than 0.05 were considered statistically significant. Results are expressed as means SEM.
Fig. 1. Comparison of the HPLC profiles from the tested Thymus extracts as detected at 280 nm. (A) Standards: (1) caffeic acid, (2) luteolin-7-glycoside, (3) rosmarinic acid, (4) daidzein, (5) quercetin, (6) hesperetin, (7) apigenin, (8) thymol, (9) carvacrol. Extracts: (B) Thymus zygis e TZ, (C) Thymus atlanticus e TA, (D) Thymus satureoides e TS, (E) Thymus vulgaris e TV.
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Table 2 Identified compounds and their retention times. Plants
Identified compound
Retention time (min)
Thymus atlanticus
Caffeic acid Rosmarinic acid Quercetin Caffeic acid Rosmarinic acid NI Luteolin-7-glycoside Rosmarinic acid Hesperetin Rosmarinic acid
11.60 17.00 21.20 11.60 17.00 13.70 15.96 17.00 22.00 17.00
Thymus zygis Thymus satureioides
Thymus vulgaris NI: No identified compound.
LDL-cholesterol was significantly elevated (þ37.7%, P < 0.01). T. satureioides shows elevated HDL-cholesterol (þ125.1%, P < 0.01) compared with HCG (Fig. 3, Table 3). T. vulgaris shows elevated concentration of TG (þ20.7%, P < 0.01), but it exerted no significant effect on plasma TC, HDL-cholesterol and LDL-cholesterol. In comparison to HCG rats, in fenofibrate-treated rats (FF) we observed a significant decrease of both plasma total cholesterol and triglycerides by 20.4%, P < 0.01 and 83.7%, P < 0.001, respectively (Fig. 2). Whereas fenofibrate increased the HDL-cholesterol by 137.1%, P < 0.001 (Fig. 3). LDL-cholesterol was not significantly changed (Fig. 3, Table 3).
3.4. Biochemical analysis and antioxidant activities of thyme extract 3.4.1. Polyphenol and flavonoid contents The total polyphenol and flavonoid contents of the four thyme varieties are shown in Table 4. The total polyphenol contents were between 356.00 9.79 (TV) and 481.10 23.42 mg eq caffeic acid/g of extract (TA). The flavonoid contents were between 144.91 2.83 and 197.93 3.18 mg eq rutin/g of extract. 3.4.2. Antioxidant activities The antioxidant activities of the four thyme varieties are shown in Table 4. The IC50 values from Moroccan thyme varieties were between 0.44 0.02 and 0.70 0.02 mg/mL of extract. The FRAP
Fig. 2. Effects of thyme extracts and fenofibrate on total plasma cholesterol and triglycerides after 24 h of hyperlipideamia induced by Triton WR-1339 in rats. Values are mean SEM from six animals. NCG: normolipidemic control group; HCG: hyperlipidemic control group; TA: Thymus atlanticus-treated group; TZ: Thymus zygis-treated group; TS: Thymus satureoides-treated group; TV: Thymus vulgaris-treated group; FF: fenofibrate-treated group; TC: total cholesterol; TG: triglycerides. Hyperlipidemic group as compared to control group. TA, TZ, TS, TV and FF are compared to HCG. **P < 0.001; *P < 0.01; NS: not significant.
Fig. 3. Effects of thyme extracts and fenofibrate on plasma HDL and LDL-cholesterol after 24 h of hyperlipidemia induced by Triton WR-1339 in rats. Values are mean SEM from six animals. NCG: normolipidemic control group; HCG: hyperlipidemic control group; TA: Thymus atlanticus-treated group; TZ: Thymus zygis-treated group; TS: Thymus satureoides-treated group; TV: Thymus vulgaris-treated group; FF: fenofibrate-treated group; HDL-C: HDL-cholesterol; LDL-C: LDL-cholesterol. Hyperlipidemic group as compared to control group. TA, TZ, TS, TV and FF are compared to HCG. **P < 0.001; *P < 0.01; NS: not significant.
assay shows that the antioxidant activity was between 38.19 0.18 and 50.79 2.02 mmol Trolox/g of extract. 3.4.3. Antihemolysis activity The inhibition of the AAPH-induced oxidative erythrocytes hemolysis by the polyphenol-rich extracts from the four varieties is shown in Table 5. Addition of AAPH induced a significant decrease in the hemolysis half-time from 73.33 2.88 min to 40.00 0.01 min (45%, P < 0.001). Application of the polyphenolrich extract from the four varieties to the erythrocytes suspension with AAPH induced an increase of the hemolysis half-time by 550% (P < 0.001), 533% (P < 0.001), 470% (P < 0.001) and 305% (P < 0.001) from T. satureioides, T. vulgaris, T. atlanticus, and T. zygis, respectively. Trolox was used as standard antioxidant showed an increase of the hemolysis half-time by 525% (P < 0.001). 4. Discussion The Triton WR-1339, a non-ionic detergent has been widely used to block the clearance of triglyceride-rich lipoproteins by inhibiting the lipoprotein lipase and to induce an acute hyperlipidemia in experimental animals.19 This model was used for a number of different objectives including studies of lipid metabolism and screening for natural and chemical hypolipidemic drugs.20,21 Hence, many plants such as Vaccinium myrtillus and Phyllanthus niruri have been investigated for their acute hypolipidemic activity in Triton WR-1339-induced hyperlipidemic animals.20 The highest levels of plasma triglycerides and total cholesterol were reached 20 h after Triton administration.20 In our study, this model yielded a similar pattern of lipid profile changes at 24 h after Triton injection and demonstrating its feasibility to assess the acute hypolipemic activity of aqueous thyme extracts. Our results clearly showed that aqueous T. atlanticus extract at a dose of 0.2 g/100 g body weight significantly lowered both plasma triglycerides and cholesterol levels after 24 h of treatment. The reduction of plasma total cholesterol was associated with a decrease of the LDL fraction, which is a major, potentially modifiable risk factor of cardiovascular diseases and the target of many hypocholesterolemic therapies. This finding led as to suggest that the cholesterol-lowering activity of these extract might be due to the enhancement of LDL-C catabolism through its hepatic receptors
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Table 3 Changes of plasma lipid parameters in control and treated rats.
NCG HCG TA TZ TS TV FF
TC (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
57.31 1.13 281.89 17.8**(þ391.8%) 48.28 3.35** (82.8%) 336.18 19.19ns 257.13 19.17ns 267.61 15.28ns 224.21 37.93* (20.4%)
58.19 1.71 517.58 19.08**(þ789.4%) 18.21 5.72** (96.4%) 452.36 37.81ns 345.26 95.05ns 624.72 13.94* (þ20.7%) 85.4 9.45** (83.7%)
31.44 1.62 8.16 2.1** (74.0%) 14.23 2.16ns 10.72 1.61ns 18.37 2.67*(þ125.1%) 14.14 3.76ns 19.35 0.01**(þ137.1%)
14.23 1.14 170.29 14.04**(þ109.6%) 30.19 3.46** (82.2%) 234.97 18.94* (þ37.7%) 169.7 37.19ns 128.52 19.12ns 183.87 38.7ns
NCG: normolipidemic control group; HCG: hyperlipidemic control group; TA: Thymus atlanticus-treated group; TZ: Thymus zygis-treated group; TS: Thymus satureioidestreated group; TV: Thymus vulgaris-treated group; FF: fenofibrate-treated group TC: total cholesterol; TG: triglycerides; HDL-C: HDL-cholesterol; LDL-C: LDL-cholesterol Hyperlipidemic group as compared to control group. TA, TZ, TS, TV and FF are compared to HCG. **P < 0.001; *P < 0.01; ns: not significant.
for the final elimination as secretory bile acids as demonstrated by Khanna et al.22 It has also recently been reported that triglycerides played a key role in the regulation of lipoprotein interactions maintaining normal lipid metabolism.23 Moreover, higher plasma TG levels have been attributed mainly to an increased population of small, dense LDL deposits, which are very atherogenic24 and enhanced cholesteryl ester mass transfer from apolipoprotein Bcontaining lipoproteins (VLDL and LDL).25 TGs have also been proposed to be the major determinant of cholesterol esterification, its transfer and HDL remodeling in human plasma. Aqueous extract from T. atlanticus significantly suppressed the elevated blood concentrations of TGs. This result suggested that this extract might be able to restore, at least partially, the catabolism of triglycerides. As hypothesized by others using different plants, this hypotriglyceridemic effect could be due to the increased stimulation of the lipolytic activity of plasma lipoprotein lipase (LPL).26 In this study, fenofibrate was used as a known reference hypolipemic drug. We found that the drug decreased plasma lipid 24 h after treatment suggesting that the lipid lowering activity of the drug was comparable to the tested extract. Fenofibrate exerted an elevated rise of HDL-cholesterol. This is in agreement with the mechanism by which fibrates act on lipid metabolism.27 Indeed, the triglycerides decreasing effect of fibrates was very important and often coupled with mild effect on LDL-C especially by both stimulation of the gene expression of lipoprotein lipase leading to enhanced catabolism of VLDL, synthesis of fatty acids and reduced VLDL secretion.26 On the other hand, it is important to note that the hypolipidemic effect of extract relatively comparable to that exerted by fenofibrate. Thus, we suggested that the hypolipidemic effect exerted by T. atlanticus extract might be explained by the same mechanisms of fenofibrate action. In this field, the possible pathways that can be implicated in the observed effect are hypothesized as catabolism of LDLcholesterol and its secretion in the form of bile acids. On the other hand, the suppression of the oxidative modification of plasma lipid and lipoprotein by antioxidants constituted one of the major targets of many antiatherogenic agents and the preferable strategy to prevent the crises of cardiovascular diseases.3 Many experimental investigations have demonstrated that a number of
secondary metabolites such as polyphenol compounds extracted from medicinal and aromatic plants possessed a high antioxidant potential due to their hydroxyl groups and protected more efficiently against some free radical-related diseases.28 In this work, we demonstrated that the four varieties of thyme originating from the Errachidia area are rich in total polyphenol and flavonoid with rosmarinic acid being the major polyphenol compound. Treatment with the polyphenol-rich thyme extracts dramatically increased erythrocyte hemolysis half-time. Hemolysis was provoked by the free radicals generated by the decomposition of the water-soluble azo compound (2,20 -azobis (2-amidinopropane) hydrochloride, AAPH) that attack erythrocyte membranes and induce lipid peroxidation leading to hemolysis. However, the increase in erythrocyte hemolysis halftime upon treatment with the thyme extracts would be due to two possible reasons: the structure of the polyphenols and flavonoid contents in the thymes which renders them potent antioxidants capable of neutralizing/scavenging free radicals that attack the erythrocyte membrane supported by the RSA and FRAP assays and/or that flavonoids might be incorporated into the erythrocyte membrane, thereby increasing its stability against hemolysis.3,29 In fact, a highly positive relationship has been established between total phenols and antioxidant activity in many plant species.30 Our results clearly demonstrated that the bioactive compound(s) contained in this plant have a polar character since they are soluble in water. This finding was in agreement with previous reports showing that polar plant extracts possessed cholesterol-suppressive capacities and the ability to attenuate the accelerated development of atherosclerosis in hypercholesterolemic animal models.31 In fact, polyphenols constitute a heterogeneous polar group of ubiquitous plant phytochemicals that exhibited a variety of pharmacological activities including anti-atherogenesis effects. Our results strongly suggested that the hypolipidemic activity of T. atlanticus plant could be attributed to the presence of polyphenolic compounds. Besides, HPLC profile of thyme polyphenol-rich extracts showed that the thyme contained rosmarinic acid as major polyphenol compound which can be probably implicated in the observed pharmacological activities.
Table 4 Polyphenol contents and antioxidant activity of the polyphenol-rich extracts (PRE) from the four thyme varieties. TA Pp (mg eq caffeic acid/gPRE) Fv (mg eq rutin/gPRE) RSA (IC50 (mg/ml PRE) FRAP (mmol trolox/gPRE)
481.1 144.91 0.44 50.31
TZ
23.42 2.83 0.02** 1.65
443.29 197.93 0.54 38.19
TS
5.82 3.18 0.02* 0.18
456.73 172.79 0.48 50.79
TV
6.94 2.12 0.01* 2.02
356.00 186.93 0.70 48.03
Trolox
9.79 2.51 0.02*** 0.01
e e 0.51 0.01 44.33 7.55
Pp: polyphenols, Fv: flavonoid, PRE: polyphenol-rich extract, FRAP: Ferric Reducing Antioxidant Power, RSA: Radical Scavenging Activity, TA: Thymus atlanticus, TZ: Thymus zygis. TS: Thymus satureioides, TV: Thymus vulgaris. RSA (***P < 0.001 TV vs Trolox, **P < 0.01 TA vs Trolox, *P < 0.05, TS and TZ vs Trolox).
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Table 5 Antihemolytic activity of aqueous thyme extracts. Hemolysis half-time (min) Control AAPH sample AAPH þ Thymus AAPH þ Thymus AAPH þ Thymus AAPH þ Thymus AAPH þ Trolox
atlanticus zygis L satureioides vulgaris
73.33 40.00 228.33 162.00 260.00 253.33 250.00
2.88 0.01* 11.54* 35.38* 17.32* 5.77* 13.26*
% Deviation 45% þ470% þ305% þ550% þ533% þ525%
AAPH: 2,20 -azobis (2-amidinopropane) hydrochloride. *P < 0.001, AAPH sample vs control; Thymus atlanticus, Thymus zygis, Thymus satureioides, Thymus vulgaris and Trolox vs AAPH sample.
5. Conclusion We concluded that the T. atlanticus polyphenol-rich extract exhibits a potent hypolipidemic capacity. In addition, the extracts of the four thyme varieties possess considerable antioxidant activities. These medicinal plants might be used as alternative intervention in prevention and treatment of atherosclerosis and related cardiovascular diseases. Author contributions MR, FA and HH contributed to the conception and design of the study. CA, SA and BB analyzed and interpreted the data. TS and MR drafted the manuscript. All authors participated in revising the manuscript and the final approval of the submitted version. Conflict of interest The authors have nothing to declare. Acknowledgments The authors wish to express their gratitude to Dr. Ibn Tatou for plant material identification. Abbreviations
LPO FRAP RSA HPLC AAPH NCG HCG TG LPL VLDL FF Ox-LDL DPPH TPTZ PBS
lipid peroxidation Ferric Reducing Antioxidant Power Radical Scavenging Activity high performance liquid chromatography 2,20 -azobis (2-amidinopropane) hydrochloride normolipidemic control group hyperlipidemic control group triglycerides lipoprotein lipase very low-density lipoprotein fenofibrate oxidatively modified LDL 1,1-diphenyl-2-picryl-hydrazil Tripyridyl triazine phosphate-buffered saline.
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