In Vivo Toxicity and the Antioxidant Activity of the Hot Water Extract of Glechoma Hederacea

Glechoma hederacea belongs to the Labiatae family and has many biological effects. Our previously in vitro studies, hot water extract of G. hederacea (HWG) possessed antioxidant and anti-inammatory activities. Also, the Ames test indicated that HWG had no mutagenicity. However, the in vivo toxicity and antioxidant capacity have not been clearly demonstrated. Thus, this study was aimed to evaluate the antioxidant properties and the safety level of HWG by using animal models.

The Council of Europe lists G. hederacea as a natural source of food avoring [5]. In China, the decoction of G. hederacea is used for ear and toothache. The most common current usage of G. hederacea is in the form of tea by infusing ground leaves in boiling water. G. hederacea tea is believed to be rich in iodine, vitamin C, and iron and is generally recognized as a good source of vitamins [10]. Our previous in-vitro study showed that hot water extract of G. hederacea (HWG) possessed antioxidant and anti-in ammatory characteristics owing to the presence of polyphenolic compounds [11][12]. The antioxidant activities of HWG were signi cantly higher than those of vitamin C and trolox in terms of superoxide anion radical-scavenging activity and Fe 2+ -chelating ability. Results of Ames test indicated that HWG had marked inhibition effects against the mutagenicities of the diagnostic mutagens with the metabolic activator system (S9 mix). In this study, we further used Sprague-Dawley rats as a testing model to con rm the antioxidant potential of HWG.
To apply HWG as an anti-mutagen or antioxidant agent, we have to ascertain its safety level. However, no clinical trials have been performed on G. hederacea [5], and the safety of G. hederacea has not been reported yet. In the present study, safety of HWG was assessed with regarding of its genotoxicity and acute and subchronic toxicity.

Chemicals
Cyclophosphamide (Product NO. C0768) were purchased from Sigma-Aldrich (MO, USA.). All other chemicals were reagent grade or purer.
Preparation of water extract of G. hederacea G. hederacea was planted in pots and the plants were harvested when the leaves grew up to 4-5 cm in diameter, which took about 2-3 weeks of growth during spring time (temperature around 20-25°C). The G. hederacea extracts were prepared in accordance with our previously reported procedures [11][12]. Brie y, fresh plants were extracted with distilled water at 100°C for 3 h using a heating mantle (NEW LAB MN-30000, Sunray Science Co., Ltd., Taipei, Taiwan). The decoctions were ltered, and then dried by a vacuum freeze-dryer. The extracts were sealed in plastic bottles and stored at -70°C until use.
The micronucleus assay was conducted according to Krishna and Hayashi [13] and Organization for Economic Cooperation and Development (OECD) [14]. Five mice were allocated randomly to each group. The mice were given HWG at a limited dose of 1.25, 2.50 or 5.00 g/kg bw by oral gavage at 09:00-11:00 am daily. The administered volume was 0.2 ml. The CP group, as the positive control, was intraperitoneal injected with 0.04 g/kg bw of cyclophosphamide. After dosing, the animals were examined for mortality and clinical signs. The animals were anesthetized using CO 2 , and 100 µL of orbital peripheral blood was withdrawn at 48 and 72 h. Slides were prepared for staining with 0.1% acridine orange hemi. The reticulocytes (RETs), which stained orange, and the micronuclei (Mn) in the RETs, which stained yellow-green, on each slide were counted under a orescence microscope (Eclipse 50i, Nikon, Japan) with blue excitation (450∼490 nm) and a barrier lter (520 nm). In total, 1000 RETs per animal were analyzed for the existence of Mn. The ratio of RETs to normochromatic erythrocytes (NCEs) was determined based on 1000 NCEs. The Mn-to-NCE ratio was recorded while counting 1000 RETs per animal and the Mn-RETs/1000 RETs (‰) was calculated.

Acute And Subacute Toxicity Tests In Rats
Animals and diets Weaned ( ve week old) SD male rats (body weight: 140-170 g) and female (body weight: 120-150 g) rats purchased from the Biolasco Taiwan Co., Ltd. (I-Lan, Taiwan) were used. The rats were acclimatized for 1 week prior to starting the experiment. Each rat was caged in a stainless steel cage (cage size: D47.3 × W25.5 × 21.5 cm) individually under controlled environmental conditions (22 ± 2℃, 65 ± 5% relative humidity, 07:00-19:00 h lighting period). The rats were allowed free access to commercial basic diets (Fu-So pellet chow Taichung, Taiwan) and water. The food intake was recorded every day, and the rats were weighed weekly. All the animals received humane care according to the guidelines of the Guidebook for the Care and Use of Laboratory Animals [15]. The study protocol was approved by the animal research ethics committee at Providence University, Taichung, Taiwan (Approval No: 20111215-A03).

Acute Oral Toxicity Study
A single dose of the test substance (1.00 and 5.00 g/kg bw) was gavaged to 10 male and 10 female rats. The administered volume was 1.0 ml. The water ad libitum was used as the vehicle for the acute and subacute studies. The animals were observed carefully for any signs of morbidity and mortality immediately after dosing, at 4 h and at 24 h and intervals, and twice daily for 14 days. The animals were sacri ced under CO 2 after 14 days following an overnight fast, and a thorough necropsy was performed on all the animals.

Subacute Oral Toxicity Study
Ten rats were allocated with body weight-based randomization to four groups, including control (water) and HWG treated groups (0.25, 0.50 and 1.00 g/kg bw) for each sex. Each group consisted of 10 males and 10 females, and the rats were gavaged daily with HWG (1.0 mL) at 9:00-11:00 am for 28 days. The limited dose level of HWG at 1.00 g/kg bw/day was selected for the high dose group, and 0.25 g/kg bw/day was considered the lowdose group. Clinical signs and mortality were observed twice daily. The food intake of the rat was recorded every day. The rats were weighed weekly.
At the end of the 28-day treatment period, the animals fasted overnight and were sacri ced under CO 2 . A 1-mL whole blood sample was taken from the abdominal aorta in an EDTA-containing tube (K3 EDTA syringes, product NO. 367835, Vacutainer, NJ, USA.) for the complete blood count (CBC) assay, and serum was obtained from 5 mL of whole blood in Vacutainer tubes (Product NO. 367955, Insepack, Japan) by centrifugation (Kubota 2010, Japan) at 775 xg for 10 min at 4℃.

Autopsy And Histology
All the animals, including those in the erythrocyte micronucleus assay and the acute / subacute toxicity tests, were subjected to necropsy, and the organ weights, histopathological examinations and biochemical analyses were evaluated. The morphologies of all the glands were examined visually, and all the organs were observed macroscopically. Selected vital organs (including the heart, liver, spleen, and kidney) were excised, blotted and weighted. Vital organs included brain, heart, liver and kidneys were examined grossly and weighed. Relative organ weight (%) is calculated as organ weight (g) / nal body weight (g) x 100. The tissues were xed in a 10% buffered formaldehyde solution and embedded in para n. The para n wax was cut into 2 µm sections, stained with hematoxylin and eosin, and examined under light microscopy (Opticphot-2, Nikon, Tokyo, Japan) for the histological examinations.

Antioxidant Activity In Rats
Tissue sampling and preparation After 28 days of feeding, food was withheld overnight and the rats were sacri ced by decapitation the next morning. The livers, heart, and brains in male rates were subjected for assessment of the antioxidant status. One portion of this tissue was immediately homogenized (0.3 g/mL) in ice-cold 0.05 mole/L phosphate buffer (pH 7.4) using a Potter-Elvehjem-type homogenizer with a Te on pestle and then centrifuged at 12,000 × g, 4 o C for 10 min (Himac CF 16 RX, Hettich, Tokyo, Japan). The supernatant was used for determining the activities of antioxidant-related enzymes including CAT, SOD, and GPx, and lipid peroxidation and the concentrations of ascorbic acid (vitamin C), vitamin E, GSH and MDA were assessed as well.

Lipid Peroxidation Measurement
The extent of tissue lipid peroxidation was determined by malonaldehyde-thiobarbituric acid (MDA-TBA) adduct according to the method described by Tatum et al [16]. MDA-TBA adduct standard was prepared by reacting 1,1,3,3-tetraethoxy propane (TEP) with TBA under acid condition.

Antioxidants And Plasma Of Total Antioxidant Status (tas) Measurement
The serum sample was used to determine TAS status. The TAS was measured by the colorimetric technique as described by Miller et al [17], using a commercialized kit (Randox Laboratoratories Ltd, UK), in which 2,2'-azinobis-(3-ethyl benzothiazoline-6-sulphonic acid (ABTS) is incubated with metmyoglobin and H 2 O 2 to produce radical cations. The absorbance of 734 nm was monitored, and this absorbance was proportional inversely to the antioxidant capacity of the tested substance. The data of TAS was expressed as a trolox equivalent.
The levels of vitamin C and E in the tissues were measured by using HPLC with electrochemical detection according to the method of Mitton and Trevithick [18]. Reduced glutathione (GSH) in tissue homogenates were analyzed by HPLC with uorimetric detection as previously described [19].
Determination of the catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities CAT activity was determined spectrophotometricaly using H 2 O 2 as the substrate [20]. The rate of H 2 O 2 dismutated to H 2 O and O 2 was proportional to the CAT activity. The decrease in H 2 O 2 amount was monitored at 240 nm at every 15 s interval over 1 min period. One unit of CAT activity was de ned as 1 mM H 2 O 2 remaining / min. Speci c activity of the enzyme was expressed as unit / mg protein.
The activity of SOD in tissue homogenate was assayed by the inhibition of autoxidation of pyrogallol as described by Marklund and Marklund [21]. Tissue supernatant was mixed with equal volume of 1% triton x-100 on ice for 30 min. After centrifuged at 9300 × g for 5 min (4°C), the supernatant was collected for SOD activity analysis. A nal 3.017 mL volume of the reaction systems contained 10 µL sample, 3 mL of 50 mM sodium phosphate buffer containing 0.1 mM ethylenediamine tetraacetic acid (EDTA, pH 8.0) and 0.7 µL 50 mM pyrogallol, and the absorbance was recorded every 15 sec for 5 min at 420 nm. One unit of SOD activity was de ned as the amount of enzyme required for producing half maximal inhibition of autoxidation.
GPx activity in the supernatant was measured as described by Paglia and Valentine [22]. Brie y, the assay mixture consisted of 0.62 mL of 250 mM phosphate buffer (pH 7.

Statistical analysis
Data were expressed as the mean ± standard deviation in each group. Mean values were compared by student's t test or analysis of variance (ANOVA) [24] using the SPSS 10.0 software (Spss Inc., Chicago, IL, USA). A signi cance level of 5% was adopted for all comparisons.

Effect of HWG on Mouse Micronuclei
In the micronucleus tests, the mice were gavage with HWG up to 5.00 g/kg bw. No clinical or body weight changes were noted in the tested mice (data not shown). Cyclophosphamide (CP), a diagnostic mutagen, caused an increase in the quantity of micronucleated reticulocytes (Mn-RET) and a decrease in the number of reticulocytes (RET) indicated that CP not only caused the damage of nucleus, but also inhibited or retarded, at least, the maturation of erythrocyte ( Table 1). The frequency of micronucleus ratios (Mn-RET/1000 RET) in the male mice for the negative control (without any treatment) was 0.80 ± 0.83 and 0.60 ± 0.55 at 48 h and 72 h for male rats, respectively, and 0.50 ± 1.00 and 0.80 ± 0.84 at 48 h and 72 h for female rats, respectively. The Mn-RET/1000 RET of All the HWG-treated animals were within the normal range of 0.60 ∼ 1.00%. Compared with negative control (normal mice), the RET/1000 RBC of the treated mice showed no difference indicating that HWG did not affect maturation of erythrocytes. Values were means ± S.D. of ve mice in each group. * Signi cantly different with the negative control group in the same sex at p < 0.05.

Effect of HWG on acute and subacute oral toxicity in rats Effects Of Heg On The Growth Of Sd Rats
The body weight gain, food e ciency ratio and relative organ weights of the rats in treatment and control groups were not signi cantly different (p > 0.05, data not shown) in this study, no matter of in acute toxicity or subacute oral toxicity studies.

Effects of HWG on acute oral toxicity study in SD rats
At a single dose of 1.0 or 5.0 g/kg bw of HWG, all the treated rats appeared normal during the observation period, and all the animals survived during the experimental period. A thorough autopsy of the treated animals revealed no treatment-related macroscopic changes. The relative organ weight (data not shown) and hematological parameters (data not shown) showed no signi cant changes in either sex in all the groups. Compared with the control group, low dose HWG-treated male rats showed lower glucose concentration in their serum ( Table 2), but still within normal range. The serum biochemical parameters and hematological parameters revealed no treatment-related alterations at the end of the study. None of the histological examinations showed signi cant ndings in the control or HWG groups (data not show). There were no signi cant clinical signs or death of the rats attributed to treatment. The acute oral LD 50 of HWG was greater than 5.0 g/kg bw in the rats.   Values were means ± S.D. (n = 10) in each group.

Effects of HWG on subacute oral toxicity study in rats
Compared with the control group, the administration of HWG at a dose of 0.25, 0.50 or 1.00 g/kg bw for 28 days did not cause any alterations in the body weight or food intake of the experimental rats (data not shown). The macroscopic appearance of all the organs observed was normal. The relative organ weight (data not shown) and hematological parameters (data not shown) of the treated animals showed no signi cant changes in both sexes of all the groups.
The serum biochemical parameters revealed no treatment-related alterations at the end of the study (Table 3). Because all the glands and organs were normal in their morphologies examined visually or macroscopically, only the heart, liver, spleen, and kidney were subjected to histopathology. The histological examination of the tissues showed no marked differences between the control and treated groups with respect to any of the organs examined (data not shown).   Values were means ± S.D. (n = 10) in each group.
There was no signi cant different among control and treatment groups (p > 0.05).

Effects Of Hwg On Tbars And Antioxidant Level
Tissue lipid peroxidation is associated with oxidant stress and MDA is a major secondary product of lipid hydroperoxides. In this study, MDA was measured as MDA-thiobarbituric acid reactive substances (TBARS) adducts using TEP as the standard. Therefore, the concentration of TBARS may re ect the degree of oxidative stress in animals and is usually used for assessing the degree of lipid peroxidation [25]. The levels of TBARS in the brain, heart and liver are presented in Table 4. Compared to the control group, HWG treatment, in all tested concentrations, did not affect signi cantly the TBARS level of brain, heart or liver in male SD rats (p > 0.05). Again, there was no signi cant difference (p > 0.05) in plasma TAS between the control and HWG-treated groups (Table 5). Values were means ± S.D. (n = 10) in each group.
There was no signi cant different among control and treatment groups (p > 0.05).
Oral administration of high dose (1.00 g HWG / kg bw) of HWG might increase the vitamin E and C levels signi cantly (p < 0.05) in brain and heart, respectively. The levels of GSH in either brain, heart or liver tissues, no matter 0.25 g HWG / kg bw, 0.50 g HWG / kg bw or 1.00 g HWG / kg bw, were not affected signi cantly by HWG treatments (p > 0.05).

Effects Of Hwg On Activities Of Antioxidant Enzymes
The effects of HWG on activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in brain, heart and liver of SD rat are shown in Table 6. Compared with the control, oral administration of HWG could signi cantly (p < 0.05) increased the SOD activity of heart tissues, however, SOD activities of the brains or livers were not affected by HWG (p > 0.05). For the catalase or GPx activity, there was no difference was observed between the HWG group and the control (p > 0.05). Values were means ± S.D. (n = 10) in each group.

Discussion
In our previous study, the mutagenicity of HWG was assessed by Ames test [11]. Results of Ames test indicated that HWG had no mutagenicity toward all tester strains including Salmonella typhimurium TA97, TA98, TA100, TA102, and TA1535 with / without the metabolic activator (S9 mix). In the present study, the safety level of HWG was further evaluated with animal models including micronucleus test for mutagenicity property, 14 days-single dose and 28 days-repeated dose studies for measuring acute and subacute toxicity. The micronucleus experimental results showed that HWG not only caused no damage to erythrocyte but also did not in uent the maturation of erythrocyte.
Animal feeding experiments are commonly used to evaluate the toxic characteristics of speci c substances [26]. In the present study of the toxicity of HWG to SD rats, HWG was administered at a single dose of 5.0 g/kg bw for acute toxicity or it was given at 1.0 g/kg bw for 28 days for subacute toxicity. The treatment-related changes showed no signi cant difference in the SD rats. Histopathology has historically been the most consistent criterion to establish the no observed adverse effect level (NOAEL) [27]. Again, the results of the histopathological examination in the acute and subacute toxicity studies revealed that HWG did not cause signi cantly toxicological effects in the rats, indicating that there was no adverse effect of HWG in rats [26][27][28]. In vivo studies indicated that a single dose of 5 g/Kg bw in a mouse (equal to 3.5 g/kg bw in rat) or a single dose of 5.0 g/kg bw in the rat or repeated doses of 1.0 g/kg bw (equal to 0.16 g in human adult) for 28 days in rats did not meet the toxicity criteria in the mouse erythrocyte micronucleus assay or the rat acute and subacute toxicity assays.
In vitro study showed that HWG possessed antioxidant characteristics and the antioxidant activities of HWG were signi cantly higher than those of vitamin C and trolox in superoxide anion radical-scavenging activity and Fe 2+ -chelating ability [11]. Therefore, animal model was applied to substantiate the antioxidant properties of HWG. Many researches showed that phytochemicals could decline the TBARS level of liver in tested animals [29]. In the present study, HWG did not show the protection effect on the tissue lipid peroxidation. Antioxidants such as vitamin C, vitamin E and GSH will provide greater protection to the organs tissues against oxidant damage. Therefore, we evaluated the effect of the supplement of the HWG on the levels of vitamin C, vitamin E and GSH in the brain, heart and liver tissues. The HWG did not improve considerably the concentration of antioxidant substances, in where, HWG only increased vitamin E and C levels in brain and heart, respectively. It is also worth to mention that HWG is not considered as a vitamin C or E enriched plant item (approximate 70-80 mg vitamin C/g). In this case, an explanation was needed to address the issue of how the HWG could increase the ascorbic acid or vitamin E level in the brain or heart tissues. Our previous data [11] showed that the HWG exhibited antioxidant activities in vitro. Moreover, HPLC analysis results showed that rosmarinic acid, chlorogenic acid, caffeic acid, rutin, genistin, and ferulic acid were the most abundant phytochemicals in HWG and possess potent antioxidant and anti-in ammatory properties [12]. Also, Belščak-Cvitanović et al [6] extracted phenolic compounds of G. hederacea with boiling water and found that the concentrations of caffeic acid, cholrogenic acid and rosmarinic acid were 249.84 ± 15.09, 1300.22 ± 58.36 and 3236.56 ± 45.78 ug/g dry weight extract, respectively. It is possible that instead of the supplement of ascorbic acid or vitamin E in the brain or heart, the phenolic compounds of HWG might perform direct free radical scavenging activity and preserve more ascorbic acid or vitamin E in the rat with the treatment of the extract. Oral administration of HWG increased the concentration of vitamin E in brains and vitamin C in hearts and the activity of SOD in hearts; on the other hand, the concentration of GSH and the activities of GPx and CAT were maintained and did not induce lipid peroxidation.

Conclusions
HWG had no genotoxicity, and did not induce acute or subacute toxicity in SD rat. The level of no observed adverse effect level (NOAEL) of HWG rats was 1 g/kg bw for subacute toxicity study. HWG possessed antioxidant potential and reduced oxidative stress by improving the antioxidant system in animal.