Volume 17 Supplement 1
Antioxidant and hepatic protective effects of lotus root hot water extract with taurine supplementation in rats fed a high fat diet
- Huan Du†1,
- Xu Zhao†1,
- Jeong-Soon You†1,
- Ji-Yeon Park†1,
- Sung-Hoon Kim†2 and
- Kyung-Ja Chang1Email author
© Chang et al; licensee BioMed Central Ltd. 2010
Published: 24 August 2010
Nelumbo nucifera, known as sacred lotus, is a well-known medicinal plant and this lotus root is commonly used as food compared to different parts of this plant. This study was conducted to investigate the antioxidant and hepatic protective effects of lotus root hot water extract with taurine supplementation in high fat diet-induced obese rats.
Thirty-two male Sprague-Dawley rats (4-week-old) were randomly divided into four groups (n=8) for 6 weeks (normal diet, N group; high fat diet, HF group; high fat diet + lotus root hot water extract, HFR group; high fat diet + lotus root hot water extract + taurine, HFRT group). Lotus root hot water extract was orally administrated (400mg/kg/day) to HFR and HFRT groups and the same amount of distilled water was orally administered to N and HF groups. Taurine was supplemented by dissolving in feed water (3% w/v).
The activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in serum were lower in HFR and HFRT groups compared to HF group. Thiobarbituric acid reactive substance contents in all groups fed a high fat diet were higher compared to N group. The activities of hepatic antioxidant enzymes were higher in HFR and HFRT groups compared to HF group.
These results suggest that lotus root hot water extract with taurine supplementation shows antioxidant and hepatic protective effects in high fat diet-induced obese rats.
High fat diet leads to an increase in oxidative stress levels  which is related to many human diseases such as cancer, ischemia, failures in immunity and endocrine functions . It is also believed that oxidative stress is induced by reactive oxygen species (ROS) as its primary factor in aerobic organisms. ROS formed during normal metabolic processes can easily initiate the peroxidation of membrane lipids, leading to the accumulation of lipid peroxides [3, 4]. In addition, high fat diets may induce accumulation in liver (non alcoholic fatty liver disease) .
Plants are widely used to delay the oxidation process as antioxidants and free radical scavengers. Lotus root was considered to possess a strong astringent herb in Tradition Chinese Medicine which helps to treat all manners of bleeding and haematemesis . It has been reported that lotus root has the activities of hypoglycemic, antifungal, antiinflammatory, antipyretic and antianxiety properties . A previous study showed that various extracts of lotus rhizome exhibited higher antioxidant activity. Taurine, a free amino acid, plays various physiological roles such as antiinflammation, neuroprotector, antidiabetes, immune regulation, etc. . It has been reported that taurine possesses antioxidant properties [10, 11], which is contributed by scavenging ROS  and reducing TBARS, and possesses the activity of hepatic protection .
Because lotus root has high antioxidant activity, it seems that lotus root possesses activity of hepatic protection. However, there are few papers about the effect of lotus root on hepatic protection. Therefore, this study was conducted to evaluate antioxidant and hepatic protective effects of lotus root hot water extract with taurine supplementation in rats fed a high fat diet.
Animals and diet
Composition of experimental diets (g/100g diet)
High Fat Diet
Carbohydrate (kcal / 100g)
Protein (kcal / 100g)
Fat (kcal /100g)
Total calories (kcal / 100g)
Preparation of lotus root hot water extract
A dried block of lotus root was purchased from Seonwon Temple (Ganghwa-gun, Incheon, Korea). Previous studies recommend that functional components should be extracted at 80~90℃ [16, 17]. In this study, lotus samples were extracted by water at 90℃ with a solid-liquid ratio of 2.5g/100ml for 2 hours. After vacuum filtration, the extract was introduced into a rotary evaporator (Büchi Rotavapor, Büchi Laboratoriums Teknik, Flawil, Switzerland). The concentrated liquid was dried by using a freeze dryer (Iilshin, Seoul, Korea). The brown powder was obtained with 30.3% extraction yield and then it was stored at -20℃ until application.
Sampling and tissue preparation for biochemical analysis
The animals were fasted for 12 hours before sacrifice. Blood was collected from the heart, and serum was obtained by centrifugation at 3000rpm for 20 minutes. The epididymal fat (E-fat) and retroperitoneal fat(R-fat) were weighed. Some liver tissue was removed from the rats for histological photograph. The procedure for preparation of cytosol and mitochondria was partially modified from the method reported by Lee . Approximately 5 grams of minced liver tissue was mixed with 10ml cold potassium phosphate buffer (154mM KCl, 50mM Tris-HCl, 1mM EDTA buffer, pH 7.4) in a Potter homogenizer (Sartorius, Goettingen, Germany) with gap from 0.095mm to 0.115mm. The homogenate was centrifuged for 10 min at 1000g to remove the precipitate, 1ml of above supernatant solution was stored for analyzing TBARS, and the remaining supernatant solution was centrifuged for 15min at 12,000g to remove the cell debris by using a centrifuge (Beckman J25-I, Beckman, USA). The precipitate was mitochondria, which was sonicated for 30s with a dismembrator (Fisher 300, Fisher Scientific, USA); the cytosol was obtained by centrifuging the supernatant solution for 60 min at 100,000g to remove the microsome by using an ultracentrifuge (T2080, Kontron Instruments, USA). All procedures were carried out at 0~4℃. After processing, all of the samples were immediately frozen in liquid nitrogen, and then stored at -70℃ until application (Operon, Korea).
GOT and GPT activities
Activities of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were analyzed by using UV rate method . After thawing for 30 minutes at room temperature, both were analyzed using an automatic analyzer (BPC BioSed srl, Rome, Italy). All of the results were expressed as IU/l serum.
TBARS contents and SOD, GSH-Px and catalase activities
Serum and hepatic lipid peroxide contents were analyzed using the method of thiobarbituric acid (TBA) described by Buege and Aust . The standard curve was prepared by 1,1,3,3-tetraethoxypropane from 0 to 50µmol/tube (R-square = 0.9999).
Cytosolic SOD activity was analyzed based on epinephrine autoxidation using Misra’s method . The SOD activity was expressed as inhibiting the oxidation of epinephrine by 50% is equal to 1 unit.
GSH-Px activity was analyzed using the method described by Tappel . Cumene hydroperoxide was used as the peroxide substrate in this method. The decrease in absorbance of NADPH was measured at 340nm by spectrometer (HP8453, Hewlett Packard, USA). One unit defined as 1 nmol of NADPH was converted to NADP+ per minute at 37℃ and pH 7.6. GSH-Px activity was calculated using NADPH molar extinction coefficient of 0.00622µM-1cm-1, and the results were expressed as IU per milligram protein.
Catalase activity was analyzed based on hydrogen peroxide to release oxygen and water under the catalytic influence of catalase . One unit was defined as the amount of enzyme which decomposed 1 nmol of H2O2 per minute at 25 and pH 7.0. Catalase activity is calculated using H2O2 molar extinction coefficient of 43.6M-1cm-1 which expressed as IU per milligram protein.
Hepatic morphology was analyzed based on the paraffin method using a light microscope. Fresh tissues were fixed immediately in Bouin’s solution for 6-12 hours and then fixed tissue was washed under running water. After being dehydrated through different grades of alcohol, the tissues were embedded in paraffin block at 60°C. Eight µm sections were cut and mounted on glass slides coated with an egg albumin, and then the paraffin was removed with xylem and alcohol. The glass slides were stained with hematoxylin and eosin. After being dehydrated and cleared by alcohol and xylem, the glass slides were mounted in Canada Balsam. Photomicrographs were taken with a Zeiss Axiolab light microscope equipped with a Nikon Microflex HFX microscope camera.
In vitro assay DPPH radical scavenging activity
l,l-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity was analyzed using the method described by Nanjo, Goto et al. . Sixty µl methanol solutions of sample with various concentrations were added to 60µl DPPH in methanol solution. After mixing vigorously for 10 sec, the solution was then transferred into a 100 μl Teflon capillary tube, and the scavenging activity of each enzymatic extract on DPPH radical was measured using an ESR spectrometer. A spin adduct was measured on a JES-FA ESR spectrometer (JEOL LTD., Tokyo, Japan) exactly 2 min later. Measurement conditions: central field, 3475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 5 mW; gain, 6.3×105 and temperature, 298 K.
Alkyl radical scavenging activity
Alkyl radical scavenging activity was analyzed using the method described by Hiramoto et al. . Alkyl radicals were generated by 2, 2'-azobis (2-amidinopropane) hydrochloride (AAPH). The phosphate buffered saline (pH 7.4) reaction mixtures containing 10 mM AAPH, 10 mM α-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) and indicated concentrations of samples, which were incubated in a water bath for 30 minutes at 37°C and then transferred to a capillary tube. The spin adduct was recorded on a JES-FA ESR spectrometer (JEOL LTD., Tokyo, Japan). Measurement conditions: central field, 3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 1 mW; gain, 6.3 × 105; and temperature, 298 K.
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was analyzed using the method described by Rosen and Rauckman . Hydroxyl radicals were generated by iron-catalyzed Haber–Weiss reaction (Fenton driven Haber–Weiss reaction) and the generated hydroxyl radicals rapidly reacted with nitrone spin trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). The resultant DMPO-OH adduct was detectable with an ESR spectrometer. In brief, 0.2ml sample with various concentrations was mixed with 0.2ml DMPO (0.3M), 0.2ml FeSO4 (10mM) and 0.2 ml H2O2 (10mM) in a phosphate buffer solution (pH 7.2), and then introduced into 100 µl Teflon capillary tube. After 2.5 minutes, an ESR spectrum was recorded using a JES-FA ESR spectrometer (JEOL LTD., Tokyo, Japan). Measurement conditions: central field, 3475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 1 mW; gain, 6.3×105 and temperature, 298 K.
Data were analyzed for significant difference by one-way analysis of variance followed by Duncan’s multiple range tests at a p<0.05. The 50% inhibition concentration (IC50) was calculated using probit regression analysis. All analyses were performed using SPSS 17.0 program.
Results and discussion
Body weight and adipose tissue weight
Effect of lotus root hot water extract with taurine on body and adipose tissue weights
GOT and GPT activities
Effect of lotus root hot water extract with taurine on serum GOT and GPT activities
TBARS contents and SOD, GSH-Px and catalase activities
Effect of lotus root hot water extract with taurine on TBARS and antioxidant enzymes
Hepatic TBARS (nmol/mg protein)
Serum TBARS (µmol/L serum)
SOD (IU/mg protein)
GSH-Px (IU/mg protein)
Catalase (IU/mg protein)
SOD and GSH-Px activities were significantly higher in HFR group compared to HF group. Catalase activity was significantly higher in HFR and HFRT groups compared to HF group.
Free radical scavenging activity
IC50 of DPPH, hydroxyl, alkyl radical scavenging in lotus root hot water extract and taurine
In conclusion, lotus root hot water extract alone or combined with taurine supplementation has antioxidant activity both in vivo and in vitro. Also lotus root hot water extract with taurine supplementation shows hepatic protective effects in high fat diet-induced obese rats
glutamate oxaloacetate transaminase
glutamate pyruvate transaminase
thiobarbituric acid reactive substances
result larger than 4mg/ml
lotus hot water extract
electron spin resonance
This article has been published as part of Journal of Biomedical Science Volume 17 Supplement 1, 2010: Proceedings of the 17th International Meeting of Taurine. The full contents of the supplement are available online at http://www.jbiomedsci.com/supplements/17/S1.
We thank the Dong-A Pharmaceutical Co. which donated taurine.
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