Obesity prevention in mice through the use of lotus (nelumbo nucifera) leaf tea

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2012, Vol. 57, No. 8, pp. 148-156 This paper is available online at OBESITY PREVENTION IN MICE THROUGH THE USE OF LOTUS (Nelumbo nucifera) LEAF TEA Le Thi Tuyet, Nguyen Thi Hong Hanh and Vuong Thi Huyen Faculty of Biology, Hanoi National University of Education Abstract. The aim of this study was to investigate the use of lotus leaf tea in preventing obesity. Forty eight mice were divided into three experimental groups: the control group (NM), which consisted of normal mice, was given a basic diet; an experimental group was given a high fat diet (OM) and an experimental group (POM) was given a high fat diet along with 15 mL of lotus leaf tea/kg body weight which they were to drink each day. The mice were weighed weekly, blood lipids were measured in the 6th and 9th weeks and histological samples of the liver and arterial tissues were taken in the 9th week. The results were statistically significant. The animals’ weight and level of adverse blood lipids: triglycerides (TG), total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) of the POM sample were much lower than the OM sample and were equivalent the NM sample. The liver and arterial tissues of mice in the POM sample did not display elevated levels of TG, TC or LDL-C. This shows a positive association between the ingestion of 15 mL of lotus leaf tea/kg/day and obesity prevention in mice. Keywords: Obesity, lotus, Nelumbo nucifera, mice. 1. Introduction Obesity is simply fatness to a degree higher than overweight which may have an adverse impact on a person’s physical and mental health. Obesity has been shown to increase the risk of developing many different health problems, including type II diabetes, heart disease, stroke, high blood pressure (hypertension), high cholesterol (hypercholesterolemia), certain cancers, sleep apnea, osteoarthritis, gallbladder disease and gallstones, fatty liver disease, gastroesophageal reflux disease, gout and psychological and emotional illness [1, 4]. For many centuries, herbal remedies have been used to treat obesity. Herbs contain a wide variety of active phytochemicals such as flavanoids, terpenoids, lignans, Received September 26, 2011. Accepted February 14, 2012. Biology Subject Classification: 616 398. Contact Le Thi Tuyet, e-mail address: lttuyet@gmail.com 148 Obesity prevention in mice through the use of lotus (Nelumbo nucifera) leaf tea polyphenols, saponins, plant sterols and carotenoids. Significant interest is emerging in herbs that possess hypolipidemic, antiplatelet, anti-tumour and immune system stimulating properties [3, 4] and their association with the prevention of obesity [2]. In Vietnam a wide variety of useful herbs grow naturally. While research investigating obesity in Vietnam is limited, in 2007, a study looked into the use of mulberry leaves to treat obesity of Nguyen Quang Trung in 2010, research was done by Do Ngoc Lien on grapefruit and Clausena lasium (Lour.) Skeels [6]. In Vietnam, lotus plants are widely grown and some parts of the lotus plant have been used in traditional folk medicine. This herb is easy to find, inexpensive, non-toxic and is appreciated by local residents [4]. However, thus far in Vietnam, few in the population know that lotus leaf can be used to prevent and treat obesity [6]. The present study was designed to evaluate the anti-obesity effects of lotus leaf in mice. 2. Content 2.1. Time and place of study This study was carried out from November 2010 to July 2011 at the Faculty of Biology, Hanoi National University of Education. 2.2. Material and methodology 2.2.1. Material * Animal Fifty eight (58) 4-week old white male mice (Mus musculus) weighing 16 - 18 g were obtained from the National Institute of Hygiene and Epidemiology. * Plant materials The lotus (Nelumbo nucifera) leaf of the Nelumbonaceae family that was used in this study was collected in Gia Lam, Hanoi. 2.2.2. Methodology * Experimental design The National Institute of Hygiene and Epidemiology provided food for the mice in this experiment. The mice were kept in clean and dry cages 30 × 50 × 20 cm in size, 4 mice to a cage at 25 - 300C with 45 - 55% relative humidity. The mice were divided into 3 groups of 16 mice per group: (1) The NM group was given a basic diet (65 g food/kg body weight (BW)/day) (2) The OM group was given a high fat diet (basic diet + 13 g rich lipid food/kg BW/day) (3) The POM group was given a high fat diet and were to drink 15 mL of lotus leaf tea/kg body weight per day (1 mL lotus leaf tea was made from 24 mg lotus leaf powder, which makes 360 mg lotus powder/kg bw/day). The dose of 15 mL lotus leaf tea/kg BW/day was chosen after referring to the studies of Yuka Ono et al. [9], Taoying Zhou et 149 Le Thi Tuyet, Nguyen Thi Hong Hanh and Vuong Thi Huyen al. [8]. According to that study, an alcohol-basedNelumbo nucifera leaf extract (NNE) had an inhibitory activity on a-amylase and lipase, and these inhibitory activities disappeared due to the elimination of phenolic compounds from NNE. Extracts of N. nucifera leaves (NNE) inhibited PL with an IC50 value of 0.46 mg/mL. The inhibitory activity was attributed to the phenolic constituents of the leaves. NNE caused a concentration dependent increase in glycerol release due to the break down of oil droplet in adipocytes. This result means that NNE upregulated lipolysis in adipocytes [9]. According to the study of Taoying Zhou et al., [8], flavonoids from Nelumbo nucifera Gaertn were orally administered once a day, after 3 days of alloxan induction, at 50 and 200 mg/kg for 28 days, and the results showed that fasting blood glucose, serum total cholesterol and total glycerid levels were significantly decreased, whereas the serum HDL-C level increased. These effects may be due to low level activity of cholesterol biosynthesis enzymes and/or a low level of lipolysis which is under the control of insulin [8]. Over the 9 week period, mice were weighed weekly. In the 6th weeks and the 9th weeks, the index of blood lipids was measured and the template of liver and arterial tissues was measured in the 9th week. * Lotus leaf tea To make the lotus leaf tea, leaves were dried at 450C. The dried lotus leaves were then pulverized to produce a coarse powder and stored in a cool, dry place. A tea was made placing 600 mg of lotus leaf powder in a paper filter bag and steeped in 25 mL of boiling water for 5 to 7 minutes. * Blood lipid index analysis The blood lipid index was measured using an Olympus AU 400 automatic analyzer (Made in Japan). * Acute toxicity evaluation in mice Lotus leaf tea was tested for acute toxicity on the sample mice. This was carried out by a single oral administration of lotus leaf at doses of 5000 mg, 6000 mg, 7000 mg and 8000 mg/kg BW/day (in 25 mL water), given to different groups of mice (10 mice in each group). The mortality rate was noted and general behavior was observed for one hour, four hours, and intermittently for the next six hours and again at 24, 48 and 72 hours [8]. The parameters observed included gross behavioral changes, grooming, alertness, sedation, loss of righting reflex, tremors and convulsions [5]. * The histological study Mouse livers and arteries were dissected and then fixed using a 10% formalin solution and processed routinely. Sections 5µ thick were cut and stained using Hematoxylin and Eosin (H&E) for histological examination. * Statistical analysis Collected data was expressed as mean± S.D (Standard Deviation). Also, statistical significance was calculated using the one-way analysis of Microsoft Excel. 150 Obesity prevention in mice through the use of lotus (Nelumbo nucifera) leaf tea 2.3. Results 2.3.1. Acute toxicity studies Mice were given lotus leaf tea at doses increasing from 5000 to 8000 mg/kg BW. The results are shown in Table 1. Table 1. Acute toxicity test results Dose (mg/kg BW) Total number of mice Number of dead mice after 72 hrs Mortality (%) 5000 10 0 0 6000 10 0 0 7000 10 0 0 8000 10 0 0 The acute oral toxicity study of lotus leaves showed no mortality up to 8000 mg/kg. Even at this dose, no adverse signs of toxicity were exhibited. The purpose of this toxicity test was to get some indication of conspicuous behavioral changes and death. No adverse effects were exhibited in the limited toxicity evaluation of the male mice tested. The physiological status of mice appeared to be normal during the 72 h that they were monitored. The operation one the mice showed that the organ does not change when compared with that of the control mice. According to the study of J. Shetty Akhila et al., [5], this result demonstrated that concentrated oral doses of lotus leaf tea is not toxic to mice under experimental conditions. 2.3.2. Facilitating the creation of obesity in mice *Mice body weight After keeping the mice for 6 weeks, 100% of the mice in the OM group (16 mice) were obese. The average weight of the mice in the NM and OM group are shown in Table 2. Table 2 shows that after 6 weeks, the weight of the mice in the NM group increased 19.4 g (125.32%) while in the OM group weight increased 39.63 g (255.18%). Thus, the weight of mice in the OM group increased on average than NM lot by 19.35 g or more than 1.58 time. According to Yuka Ono et al., [9] and Srinivasan et al., [7], the increased weight of the mice in the OM group indicates that they were obese. Table 2. The average weight of mice in the NM and the OM group within 6 weeks Time Weight of mice (g) (X± SD) NM %OM The first week 15.48 ± 0.63 15.53 ± 0.52 The 6th week 34.88 ± 1.14 55.16 ± 0.93 The weight increase within 6 weeks (g) 19.40 39.63 The percent of weight increase within 6 weeks (%) 125.32 255.18 151 Le Thi Tuyet, Nguyen Thi Hong Hanh and Vuong Thi Huyen * Blood lipid indexes The blood lipid indexes that were identified included triglycerides (TG), total cholesterol (TC), high density lipoprotein - cholesterol (HDL - C) and low density lipoprotein - cholesterol (LDL - C). The results are shown in Table 3. Table 3. Blood lipid indexes in the NM and OM group Blood lipid index Level (mmol/l) (X± SD) Compare XOM with XNM NM OM Content (mmol/l) Rate (%) TG 0.73 ± 0.04 1.94 ± 0.10 ↑1.21 ↑165.75 TC 2.97 ± 0.17 5.57 ± 0.12 ↑2.6 ↑87.54 HDL – C 1.93 ± 0.04 0.90 ± 0.03 ↓1.03 ↓53.36 LDL – C 0.68 ± 0.03 1.95 ± 0.04 ↑1.27 ↑186.76 Table 3 shows that, within 6 weeks, the levels of TG, TC and LDL-C in the OM group were significantly greater than in the NM group (p < 0.05): 165.75%, 87.54% and 186.76%, respectively. However, the HDL-C levels in the OM group were lower than in the NM group by 53.37%. This result is congruent with the results of the studies by Yuka Ono et al., [9]. * Histological results After 9 weeks, the histology of the liver and arteries of 16 mice of the NM group and 16 mice of the OM group were studied, and they are shown in Figure 1 and Figure 2. The results were that 100% of the histopathological examinations of the liver (16/16) and the artery (16/16) of mice in the OM group display the presence of adipose tissue. Figure 1 shows that the livers of mice in the OM group display the presence of adipose tissue inside and outside the cells and the size of the liver cells in the OM group are larger than those of the NM sample. Figure 2 shows that the arterial histological examination of mice in the OM sample also shows the presence adipose tissue. Figure 1. Histopathological studies of liver: the NM group (A, B) and the OM group (C, D) (1. Liver cells; 2. Adipose) 152 Obesity prevention in mice through the use of lotus (Nelumbo nucifera) leaf tea Figure 2. Histopathological studies of artery: the NM group (E, F) and the OM group (G, H, I, J) (1. Tunica; 2. Adipose) The results obtained above confirm that the model chosen to experimentally induce obesity in mice was successful. 2.3.3. Obesity prevention in mice through the use of lotus leaves tea * Mice body weight After 9 weeks, the average weight of 16 mice in the POM sample was significantly lower than in the OM sample and there was the potential that it could reach the average weight in the NM sample (Table 4). Table 4. The average weight of mice in the NM group, the OM group and the POM group in the 6th and 9th week Time Average weight (g) (X± SD) Comparison NM POM OM POM-NM POM-OM The 6th week 34.88±1.14 37.54±1.22 55.16±0.93 ↑2.66 (7.63%) ↓17.62 (31.94%) The 9th week 37.68±1.29 40.07±1.61 60.99±1.37 ↑2.39 (6.34%) ↓20.92 (34.30%) After 6 weeks, the weight of mice in the POM sample was lower than those in the OM group by 17.62 g (31.94% or 1.47 times), and higher than those in the NM group by 2.66 g (7.63%). Over the next 3 weeks, the weight of mice in the POM group became 20.92 g (34.3%) lower than mice in the OM group and 2.39 g (6.34%) higher than mice in the NM group. According to the studies of Yuka Ono et al., [9] and Taoying Zhou et al., [8] this result demonstrates that lotus leaf tea is an useful method for inhibiting weight gain in mice that are on a high-fat diet. 153 Le Thi Tuyet, Nguyen Thi Hong Hanh and Vuong Thi Huyen * Testing of blood lipid index The blood lipid index in the 6th and 9th week are shown in Table 5. Table 5. The index of blood lipids of the NM group, the OM group and the POM group in the 6th and 9th week Week The blood lipid index Level (mmol/l) (X± SD) Comparison NM POM OM POM-NM POM-OM The 6th week TG 0.73±0.04 1.07±0.06 1.94±0.10 ↑0.34 (46.58%) ↓0.87 (44.85%) TC 2.97±0.17 3.36±0.12 5.57±0.12 ↑0.39 (13.13%) ↓2.21 (39.68%) HDL-C 1.93±0.04 1.72±0.04 0.90±0.03 ↓0.21 (10.88%) ↑0.82 (91.11%) LDL-C 0.68±0.03 0.94±0.05 1.95±0.04 ↑0.26 (38.24%) ↓1.01 (51.79%) The 9th week TG 0.74±0.07 1.14±0.02 2.10±0.23 ↑0.40 (54.05%) ↓0.96 (45.71%) TC 2.99±0.15 3.38±0.04 5.85±0.02 ↑0.39 (13.04%) ↓2.47 (42.22%) HDL-C 1.93±0.07 1.62±0.13 0.83±0.06 ↓0.31 (16.06%) ↑0.79 (95.18%) LDL-C 0.69±0.03 0.79±0.13 2.20±0.06 ↑0.10 (14.49%) ↓1.41 (64.09%) Table 5 shows that in the 6th week, the levels of TG, TC and LDL-C of mice in the POM group were lower than those of mice in the OM group with the corresponding levels of 44.85%, 39.68% and 51.79%, which were higher than those of the NM sample (with corresponding levels of 46.58%, 13.13% and 38.24%). The HDL-C index of mice in the POM group was higher than that of the OM group by 91.11% and lower than that of the NM group by 10.88% In the 9th week, the levels of TG, TC and LDL-C of mice in the POM group were still lower than those of the OM sample with corresponding values of 45.71%, 42.22% and 64.04%, and these were higher than those of the NM sample (with corresponding values of 54.05%, 13.04% and 14.49%). The HDL-C level of mice in the POM group was still greater than that of mice in the OM sample by 95.18% and lower than mice in the NM sample by 16.06%. According to Yuka Ono et al., [9] and Taoying Zhou et al., [8], this result demonstrated that lotus leaf tea has been shown to effectively limit the increase of adverse blood lipids (TG, TC, LDL-C) in mice that are fed a fatty diet. * Histological results After 9 weeks, a histological examination of the liver and arteries of every mouse in the NM, OM and POM group (16 mice per group) was done with the results shown in Figure 3 and Figure 4. The results were that 100% of the histopathological examinations of the liver and artery (16/16) of the POM group show no indication of unusual characteristics. 154 Obesity prevention in mice through the use of lotus (Nelumbo nucifera) leaf tea Figure 3. Histopathological studies of the liver: the NM group (A, B), the POM group (C, D) and the OM group (E, F) Figure 4. Histopathological studies of artery: the NM group (E, F) and the OM group (G, H, I,J) (1. Tunica; 2. Adipose) Figure 3 and Figure 4 display no unusual characteristics in the liver and arterial tissues of the POM group in the week 9th. The arterial diameter was still wide, howerver the size of liver cells was slighty greater than those of the NM group. No adipose tissue was seen in these samples. 3. Conclusion Lotus leaf tea is not toxic to mice. Lotus leaf tea administered in oral doses of 15 mL/kg body weight/day (1 mL tea is extracted from 24 mg the lotus leaf powder) helped mice that were consuming a high-fat diet prevent the onset of obesity. Specifically, the TG, TC and LDL-C levels of mice in the POM sample were significantly lower than those of mice in the OM group and nearly equivalent with those in the NM sample while the 155 Le Thi Tuyet, Nguyen Thi Hong Hanh and Vuong Thi Huyen levels of HDL-C were significantly higher when compared with that of the OM sample. 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