Effects of Glycyrrhiza glabra, Mentha piperita and their Blend Teas Infusion on Serum Lipids of Wistar Rats

The aim of this study was to demonstrate the effects of Glycyrrhiza glabra (Licorice), Mentha piperita (Peppermint) and their blend teas on serum lipids of Wistar rats. The animals were divided into control group (CG) and nine treated groups: P1, P3, P5 treated with peppermint tea; L1, L3, L5 treated with Licorice tea and P+L1, P+L3, P+L5 treated with blended peppermint and licorice tea of 10 mg/kg.BW/ml, 30 mg/kg.BW/ml and 50 mg/kg.BW/ml concentrations respectively. The teas were administered orally once in a day for 30 days. P3, P5; L3, L5 and P+L3, P+L5 animals showed significant decrease in triglycerides and total cholesterol and slight increase in HDL-c levels. The levels of LDL-c decreased in the treated groups compared to control group. The results suggested that supplementation with peppermint; licorice and their blend tea extracts can reduce the serum concentrations of cholesterol, triglycerides and LDL.


Introduction
A wide variety of plants are commonly used as remedies for the treatment of many diseases. The use of these plants to control blood lipid levels in plasma is widespread and many studies have shown that some plants do have beneficial effects [19,27].
Low HDL-c (high density lipoprotein-cholesterol) and hypercholesterolemia levels are very common today and are often associated with endothelium dysfunction and inflammation, which are often accompanied by atherosclerosis. High levels of total cholesterol, LDL-c (low density lipoprotein cholesterol) and triglyceride associated with lower HDL-c levels can also induce higher risks of cardiovascular diseases [24,25]. The combination of all these factors can trigger the Metabolic Syndrome [14,9]. Endothelial dysfunction and cardiovascular diseases can be prevented by controlling serum lipids [22].
The lack of information about the possible toxic effects of many plants, allied to the high cost of allopathic This indicates the pressing need for new studies to medicines, encourages the use of the former in the treatment of many diseases. This indicates the pressing need for new studies to clarify the real effects of medicinal plants and to provide scientific evidence establishing their usefulness.
Licorice (Glycyrrhiza glabra) is one of the oriental herbal medicines that have been most frequently prescribed for the treatment of various diseases [23] and its extracts were found to have a wide range of biological activities, including antimicrobial, antiatherosclerotic, antihepatitis, antinephritic, and cardiovascular protective activities [7,8].
Likewise, Mentha piperita is one of the ten most widely used plants in several countries [6] and is commonly used in the treatment of loss of appetite, common cold, bronchitis, fever, nausea, vomiting [2], spasmodic responses [15], and antimicrobial and antioxidant activities [16,18].
Due to the important role of high plasma lipids in the development of insulin resistance, atherogenesis and cardiovascular disease, and the widespread use of M. piperita and Glycyrrhiza glabra as complementary and alternative medicinal intervention, this study aimed to investigate the effects of teas prepared from fresh peppermint and licorice processed teas (which is how this parts of the world where peppermint and licorice plants are not available, uses this plant) on Wistar rats plasma lipids.

Animals
Healthy adult male albino rats of wistar strain weighing between 130 to 180 g were obtained from the animal house of the School of Agriculture, Federal University of Technology, Akure for the study. They were kept in rat cages at room temperature (27 ± 2°C) and humidity (55 ± 5%) and a 12 h cycle of light and dark. They were given free access to rat pellet and water ad labitum. The experiment was performed in accordance with the National Institute of Health guidelines of care and use of laboratory animals. The rats were allowed to acclimatize for a week before the experiment.

Extract preparation and administration
Licorice and Peppermint teas were bought from the Tradomedical Centre, Ibadan, Oyo state, Nigeria. The tea extracts were prepared using hot water infusion. The mixture was filtered using No 1 Whatman filter paper and the filtrate kept prior analysis. The rats were weighed daily and the calculated volumes of the extracts in milliliters (mg/Kg. BW/ml) were administered orally for 30 days.

Phytochemical investigation of the extracts
The different chemical constituents present in aqueous extracts were subjected to the tests by Kokate [32] and Trease & Evans [31].

Total Flavonoid content
The total flavonoid content of the extracts was determined using a slightly modified method reported by Chung et al [5]. Briefly, 0.5mL of enzyme digested sample was mixed with 0.5mL methanol, 50μl of 10% AlCl 3 , 50μl of 1mol L -1 potassium acetate and 1.4mL water, and allowed to incubate at room temperature for 30 min. Thereafter, the absorbance of each reaction mixture was subsequently measured at 415 nm. The total flavonoid was calculated using quercetin as standard by making use of a seven point standard curve (0, 20, 40, 60, 80,100 μg/mL). The total flavonoids content of samples was determined in triplicates and the results were expressed as mg quercetin equivalent per gram of the sample.

Total Phenolic content
The total phenolic content of the samples extract was determined by the Folin-Ciocalteu assay as described by Chanda et al [4]. 500μl of Folin reagent was added and mixed with a solution containing 100μL of the extract and 2mL of distilled water. 1.5mL of 7.5% sodium carbonate was then added to the solution and the volume was made up to 10mL with distilled water. The mixture was left to stand for 2 h after addition of the sodium carbonate. The absorbance of the mixture was measured at 760 nm using a Lambda EZ150 spectrophotometer (Perkin Elmer, USA). The standard used was tannic acid and the results were expressed as mg tannic acid equivalents per gram of the sample.

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Vol. 5

Biochemical analysis
At the end of the experiment, blood was collected from each rat by cardiac puncture method. Blood samples were centrifuged (at 2000 g for 10 min); serum was obtained for the measurement of cholesterol, triglycerides, HDL by spectrophotometer using a commercial kit package (Randox Laboratories Limited). We used standard commercial kits for analysis as recommended by the manufacturer of these kits. LDL and VLDL-cholesterol were calculated following the method by Johnson et al [10], while the atherogenic index was calculated by using the method described by Muruganandan et al [17]. LDL = TC -(HDL + Triglyceride/5) VLDL = TC-HDL-LDL Atherogenic index = (TC-HDL)/HDL

Statistical analysis
Results are expressed as mean±SEM (standard error mean) and subjected to one-way analysis of variance (ANOVA) followed by Dunnett's test and values with p<0.05 were considered to be statistically different.

Results and Discussion
Phytochemical investigation was performed and the following compounds were identified in the teas extracts as shown in Table 1

Discussion
Plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators, it was reasonable to determine their total amount in the selected plant extracts [28]. Phenolics and polyphenolics (polymeric phenolics) can provide relief from certain physical ailments and degenerative diseases in humans, including the reduction of cardiovascular disease and certain cancers [1,30].
Flavonoids present in food of plant origin are also potential antioxidants [29]. Peppermint, Licorice and their blend are good source of flavonoid ( Figure 1).

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Vol. 5 In this study, peppermint, licorice and their blend extracts did not have a significant impact on the serum content of HDL. The peppermint supplements had no significant effect on the serum content of HDL in broiler chicks [21]. The absence of concurrence among these studies may be a result of the levels at which the extract was administered. In addition, other variables, such as differences in background of the selected animals, the types of animals or genera and age may have affected the efficacy of extract usage, and therefore it was difficult to directly assess different studies that used extracts.
In this study, administering the peppermint licorice and their blend extracts at high levels decreased the serum content of LDL, triglycerides, and cholesterol in Wistar rats. It seemed that peppermint, licorice and their blend teas had anti-lipidemic benefits, but it cannot show this benefit at low levels. In relation to our findings, Johari et al. showed that peppermint extract decreased the serum contents of LDL, cholesterol, and triglycerides in Wistar rats compared with the control group [11]. The decrease of total cholesterol, LDL cholesterol, and total triglycerides of the treatment group might have been related to the involvement of the proposed components in the antioxidant and excess cholesterol protection mechanisms.
High temperature can induce the synthesis of free radicals, which can damage cell membranes by lipid peroxidation of polyunsaturated fatty acids in the cell membrane, thereby destroying the integrity of the membrane [14]. It seems antioxidants (peppermint, licorice and their

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blend extracts) decrease lipid peroxidation in the plasma and tissues. The idea was confirmed by Young et al. who showed that herbs and spices, along with vitamins C and E (anti-oxidant vitamins) were even more effective at preventing lipid peroxidation in the tissues of birds [26]. There was a positive correlation between the total phenol concentration of herbal plants and human low-density lipoprotein oxidation in vitro [20]. I believe that the decrease in the serum content of cholesterol may be a result of inhibition of HMG-COA reductive activity, which is a key regulatory enzyme in cholesterol synthesis.

Conclusion
Peppermint, licorice and their blend tea extracts treatment did not improve the serum content of HDL. The results also suggested that supplementation with peppermint, licorice and their blend tea extracts can reduce the serum concentrations of cholesterol, triglycerides, LDL and VLDL and atherogenic index. This study showed that the individual of the samples extract is able to reduce the serum lipids, thereby helping to protect the rats against the harmful aftermath of lipoperoxidation and potentially ensuring antioxidant potential.