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Keywords:

  • acute and subacute toxicity;
  • fermentation;
  • mutagenicity;
  • rat;
  • Toona sinensis Roemor leaves

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

ABSTRACT:  The purpose of this study was to evaluate the mutagenicity and safety of water extract of fermented Toona sinensis Roemor leaves (WFTS). The WFTS was prepared by fermenting Toona sinensis Roemor leaves anaerobically for 14 d, and then extracting with boiling water. The mutagenic effects of WFTS were investigated using Ames test. No mutagenicity was found toward all tester strains (Salmonella typhimurium TA98, TA100, TA102, TA1535). In the acute oral toxicity study, a single limit dose of 2.5 or 5 g/kg body weight (bw) WFTS was given to male Sprague-Dawley (SD) rats, then the rats were observed for 14 d. No acute lethal effect at a maximal dose of 5 g/kg bw WFTS was observed in rats. In the subacute study, the male rats were administered daily by gavage at a dose of 0.5 or 1 g/kg bw/d of WFTS for 28 d. The results indicated that no significant toxic effect was found in the parameters of body and organ weight, as well as hematological, biochemical, urinary, and pathological parameters between control and the WFTS-treated rats. The level of no observed adverse effect level (NOAEL) of WFTS in male rats was 1 g/kg bw for subacute toxicity study.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Toona sinensis Roem (TS) is a domestic plant containing various nutrient compounds with a distinct flavor. The tender leaf and stem have also been used for the treatment of carminative, enteritis, dysentery, and itch in the oriental medicine (Edmonds and Staniforth 1998). Water extract of Toona sinensis (WTS) possess high antioxidant activity and many biological functions, and also no remarked toxic effect was reported (Wang 2000; Chang and others 2002; Hsu and others 2003; Yang and others 2003; Poon and others 2005; Liao and others 2006, 2007).

Many researches demonstrated that fermentation processing may enhance antioxidant properties of food materials. For example, theaflavins and thearubigin are produced during fermentation processing and provide major antioxidant capacity for black tea (Luczaj and Skrzydlewska 2005), and soybean fermentation products, such as miso, natto, and tempeh, obtained antioxidants by liberation of isoflavones in soybean (Murakami and others 1984; Esaki and others 1994; Berghofer and others 1998; Sheih and others 2000). Lin (2007) assessed the antioxidant activities of water extract of fermented TS (WFTS). In Lin's (2007) study, WFTS were prepared by fermenting TS leaves anaerobically for 14 d, and then extracting with boiling water. The free radical scavenging, superoxide anion radical scavenging, and Fe2+-chelating activities of the WFTS at a concentration of 0.16 mg/mL, reached 85%, 77%, and 88%, respectively. The results of animal study showed that WFTS increased activities of glutathione peroxidase (GPx), glutathione reductase (GR), and catalase in rat's liver tissues. Prior to applying WFTS as antioxidant agents, the assessments of their safety are essential.

Margiantoro (2003) found that probiotic bacteria including Lactobacillus (total 16 strains), Lactococcus (total 3 strains), and Streptococcus (2 strains) dominate among the flora in nature fermented TS leaves. Margiantoro (2003) also reported that the major phenolic compounds in fresh TS leaves was rutin and during fermentation processes (up to 4 wk) the concentration of rutin was decreased but concentration of quercetin glucoside increased with the fermentation processing. According to Margiantoro's (2003) study, the compositions of WFST were different from WTS; therefore the safety of WFST needed to be questioned even though our previous study demonstrated that WTS had no remarked toxicity in tested in mice (Liao and others 2007). In this study, Salmonella revertant assay (Ames test) was applied to assess the mutagenic properties of the WFST, and the Sprague-Dawely (SD) rats were served as animal models for acute and subacute toxicity tests.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Preparation of the fermented leaves water extract

For the fermented leaves, the TS leaves were capped tightly in a tin canister for 14 d to be fermented by natural microorganisms. The water extract was prepared by extracting the cut fermented leaves with water (1: 4 w/v) at 100 °C for 30 min. The supernatant was strained and then concentrated using an evaporator at −70 °C (Rotary vacuum evaporator, EYELA Type N-1, Japan) under reduced pressure. A yield of 6% was obtained for the water extract of fermented TS leaves. The water extract of fermented TS leaves were designated as WFTS and stored at −86 °C until use.

Mutagenicity and toxicity tests

Bacteria strains The Salmonella typhimurium tester strains, including TA98 (hisD3052/rfa/▵uvrB/pKM101), TA100 (hisG46/rfa/▵uvrB/pKM101), TA102 (hisG428/rfa/pKM101, pAQ1), and TA1535 (hisG46/rfa/▵uvrB), were purchased from Culture Collection and Research Center, Food Industry Research and Development Institution, Taiwan.

Preparation of rat liver microsomal enzymes and cofactors (S-9 mix) S-9 mix was prepared followed the method of Maron and Ames (1983). S-9 mix was obtained as a metabolic activation system consisting of the postmitochondrial fraction of livers from rats (MA BioService, Rockville, Md., U.S.A.). Before each test, each vial of S-9 mix was reconstituted to 2.1 mL immediately and S-9 mix was prepared fresh by adjusting S-9 mix to final concentration of 10% with dilution buffer containing 0.004 M phosphate buffer (pH 7.4), 0.008 M MgCl2, 0.033 M KCl, 0.004 M NADP, and 0.05 M G-6-P. All chemicals used for S-9 mix were purchases from Sigma Co. (St. Louis, Mo., U.S.A.). The S-9 mix was prepared fresh for each assay, and it was discarded if duration exceeded 4 h.

Bactericidal toxicity test Samples (1 to 5 mg/plate) were prepared with 0.1 mL of fresh culture of the tester strain (approximately 108 cells/mL), 0.1 mL of the WFTS, 0.2 mL of phosphate buffer (0.2 M, pH 7.4), and 0.5 mL of S-9 mix (metabolic activator) or instead with phosphate buffer. The mixture was diluted sequentially with phosphate buffer and 1 mL of diluted solution was mixed with 12 mL of nutrient agar. After incubation at 37 °C for 48 h, the number of colonies was counted. A bactericidal toxicity effect was confirmed if the standard plate count of tested compound was lower than that of control (without adding tested compound).

Bacterial reverse mutation assay (Ames test) Mutagenicity was assayed by the standard Ames test (standard plate incorporation assay) (Maron and Ames 1983; Mortelmans and Zeiger 2000). A mixture containing 0.1 mL of the WFTS (1 to 5 mg/plate), 0.5 mL of S9 mix or phosphate buffer, 0.2 mL of 0.5 mM histidine/biotin, and 0.1 mL of fresh culture of the tester strain (Salmonella typhimurium TA98, TA100, TA102, TA1535, approximately 108 cells/mL) was added to a tube containing 2 mL of Top agar (contained 0.75% agar and 0.5% NaCl). The tube was then gently vortexed and poured onto glucose minimal agar plate (MA plate) that contained 1.5% agar, 0.02% MgSO4.7H2O, 0.2% citric acid, 1% K2HPO4, 0.35% NaHNH4PO4.4H2O, and 2% glucose. The compound was tested with and without S9 mix and triplicate plates are poured for each dose of mutagen. Diagnostic mutagens including sodium azide (Az-Na), 4-nitroquinoline-N-oxide (4-NQO), and 2-anthramine (2-AA) were served as positive control chemicals. After incubation at 37 °C for 48 h, the number of revertant colonies was scored. A compound was considered a mutagen if there was a 2-fold increase in the number of revertants compared with spontaneous revertants (negative control) or a dose-related increase in the number of revertant in 1 or more strains.

Acute and subacute toxicity tests in rats

Animals and diets Five week-old SD male rats purchased from the Natl. Lab. Animal Center, Taipei, Taiwan, and acclimatized for 1 wk prior to the commencement of the experiment. Two rats were housed per cage under controlled environmental conditions (25 ± 2 °C, 65%± 5% relative humidity, 0700 to 1900 h lighting period). The rats were allowed free access to the commercial basic diets (Fu-So pellet chow, Taichung, Taiwan) and water. The food intake of the rats in each cage was recorded everyday and the value was divided by the number of the rats in a cage to represent the mean food intake for the rats in that cage. The rats were weighed weekly. All animals received humane care according to the guideline of Guidebook for the Care and Use of Laboratory Animals (Yu and others 2004). The study protocol was approved by the animal research ethics committee at Providence Univ., Taichung, Taiwan.

Acute oral toxicity study Acute oral toxicity study of WFTS was conducted mainly based on the test guidelines of U.S. Environmental Protection Agency (EPA 1998) and, where possible, with Organization for Economic Cooperation and Development (OECD) test guidelines (OECD 2001) with modification. The no-observed-adverse-effect-level (NOAEL) is an important part of the nonclinical risk assessment. The alteration of body weight of animal after treatment is 1 of important indication that is without adverse effect (Dorato and Engelhardt 2005). According to results reported by Hasumura and others (2004), diet supplemented with rutin, 1 of the major decomposed phenolic compounds in Toona sinensis Roemor leaf through anaerobic fermentation, for 13 wk in rats showed body weight gain decreases in male rats. Male rat was considered to be a sensitive target in the WFTS toxicity study; for this, male rat was used for the acute and subacute toxicity study of WFTS. Briefly, a limited dose of 2.5 or 5 g/kg bw, of WFTS was administered by single gavage to 8 male rats in each group (1 mL/rat/time). The double-distilled water (DDW) was used as the vehicle in acute study. The animals were observed carefully for sign of morbidity and mortality at 4- and 24-h intervals immediately after dosing and twice daily for the subsequent 14 d. The animals were sacrificed by blood exhaustion via abdominal aorta under ether anesthesia after 14 d following an overnight fast, and then an autopsy was carried out on all animals.

Subacute toxicity study Subacute oral toxicity study of WFTS was performed according with the test guidelines of U.S. Environmental Protection Agency (EPA 2000) and, where possible, with OECD test guidelines (OECD 1995) with modification. The limited dose of WFTS at 1 g/kg bw/d was selected as high dose group and 0.5 g/kg bw/d was considered as low-dose group. Twenty-four male rats were randomized to 3 groups of 8 male rats each, including control (DDW) and 2 WFTS treated groups (0.5 and 1 g/kg bw), and daily gavaged with WFTS for 28 d, respectively. Two rats were housed per cage. Clinical signs and mortality were observed twice daily. At the end of the 28-d period, the animals were fasted overnight and sacrificed under anesthesia. One milliliter of whole blood sample was taken from abdominal aorta by EDTA-containing tubes (K3 EDTA syringes, Vacutainer, N.J., U.S.A.) for the complete blood count (CBC) assay, and serum was obtained from 5 mL of whole blood in the Vacutainer tubes (Franklin Lakes, N.J., U.S.A.) by centrifugation (Kubota 2010, Japan) at 775 ×g for 10 min at 4 °C.

Hematology examination Complete blood count (CBC) was examined by automated hematology analyzer (Sysmex K-4500, Toa Medical Electronics Co., Ltd., Kobe, Japan), including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet (PLT) counts at a commercial analytical service center (Lian-Ming Co., Taichung, Taiwan).

Biochemical examinations The serum sample was used to determine the alkaline phosphatase (ALP), total cholesterol, triglyceride, glucose, aspartate aminotransferase (AST), alinine aminotransferase (ALT), albumin, total bilirubin, glucose, blood urine nitrogen (BUN), and creatinine by enzymatic methods using an automatic analyzer (Synchron CX-7 systems, Beckman Coulter, Fullerton, Calif., U.S.A.) at a commercial analytical service center (Lian-Ming Co., Taichung, Taiwan).

Autopsy and histology All animals included in acute oral and subacute tests were subjected to a complete necropsy, and then the organ weights, histopathological examinations, and biochemical analysis were evaluated. All organs were observed macroscopically and selected vital organs (including brain, heart, liver, spleen, pancreas, and kidneys) were excised, blotted, and weighed. Tissues were fixed in 10% buffered formaldehyde solution and embedded in paraffin. The paraffin wax were cut into 2 μm sections, stained with hematoxylin, and eosin, and then histologically examined under the light microscopy (Opticphot-2, Nikon, Tokyo, Japan) for histological examination.

Statistical analysis

Data were examined for equal variance and normal distribution prior to statistical analysis. Mean values were compared by Student's t-test or analysis of variance (ANOVA) (Steel and Torrie 1980) using the SPSS 10.0 software (SPSS, Inc., Chicago, Ill., U.S.A.). A significance level of 5% was adopted for all comparisons.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Mutagenicity assay

It is recommended that a preliminary toxic dose range experiment be performed to determine an appropriate dose range for the mutagenicity assay. Within the tested dose range (1 to 5 mg/plate), the extract did not show toxicity toward the tester strains (Salmonella typhimurium TA98, TA100, TA102, TA1535) with or without the presence of S9 mix (data not shown). Since there was no toxicity effect on the tester strains, the same dose range of the WFTS was applied in the mutagenicity assay (Ames test).

In Ames test (Table 1), the revertant numbers induced by extracts (1 to 5 mg/plate) for all tester strains were close to that of the negative control (spontaneous revertant, without extract) and much lower than that of positive controls (with diagnostic mutagens). The result of Ames test demonstrated the WFTS had no mutagenicity effect under the tested dose range.

Table 1—.  Mutagenicity of water extract of fermented Toona sinensis toward Salmonella typhimurium TA98, TA100, TA102, and TA1535.
Colony revertant parameterRevertant colonies (CFU/plate)a
TA98TA100TA102TA1535
−S9bS9−S9S9−S9S9−S9S9
  1. WFTS: Water extract of fermented Toona sinensis.

  2. aValues were means ± SD of triplicates.

  3. bS9 is a metabolic activation system consisting of the postmitochondrial fraction of the livers of rats.

  4. cNegative control: without extract was treated with aseptic water, spontaneous revertants/plate.

  5. dPositive control: TA98 and TA100 with S9: 2-AA (50 μg/mL), 2-AA (20 μg/mL), respectively, without S9.: 4-NQO (1 μg/mL), TA102 with S9.: 2-AA (200 μg/mL), without S9.: 4-NQO (20 μg/mL), TA1535 with S9.: AZ-Na (50 μg/mL), without S9.: AZ-Na (5 μg/mL).

Controlc60 ± 388 ± 4 49 ± 232 ± 7284 ± 14198 ± 165 ± 24 ± 3
Positive controld489 ± 21936 ± 231285 ± 15748 ± 581031 ± 81 724 ± 78164 ± 23 307 ± 8  
Treatment
 WFTS 5 mg/plate80 ± 7107 ± 10 46 ± 363 ± 6331 ± 10333 ± 277 ± 28 ± 3
 WFTS 4 mg/plate69 ± 696 ± 20 54 ± 11 66 ± 10355 ± 15307 ± 536 ± 26 ± 1
 WFTS 3 mg/plate71 ± 289 ± 12 65 ± 1174 ± 4355 ± 24291 ± 9 5 ± 56 ± 2
 WFTS 2 mg/plate69 ± 581 ± 1476 ± 553 ± 3301 ± 16291 ± 9 5 ± 04 ± 4
 WFTS 1 mg/plate 64 ± 1669 ± 1472 ± 9 51 ± 11253 ± 25229 ± 9 4 ± 22 ± 1

Acute and subacute oral toxicity study in rats

At a single dose of 2.5 or 5 g/kg bw, of WFTS all the treated rats appeared normal during the observation period, and all the animals survived during the experimental period. Before and after the test period of acute toxicity study, no significant difference was observed in body weight change, food progressive intakes, and water progressive intake between 3 males groups (Figure 1). A thorough autopsy of the treated animals revealed no treatment-related macroscopic changes. Serum biochemical parameters revealed no treatment-related alteration at the end of the study (data not shown). Histological examination also showed no significant findings in control and WFTS groups (data not shown). There was no significant clinical sign or death of rats that was attributed to treatment. Thus, the acute oral LD50 of WFTS was greater than 5 g/kg bw in rats.

image

Figure 1—. Changes of body weight, food, and water intake of male Sprague-Dawley rats after a single dose of water extract of fermented Toona sinensis (WFTS). (A) Body weight, (B) cumulative food intake, and (C) cumulative water intake. Data points were means ± SD (n= 8).

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Figure 2 shows the body weight change, food progressive intakes, and water progressive intake of rats in subacute toxicity study. When compared with the control group, administration of WFTS at a dose of 0.5 or 1 g/kg bw for 28 d did not cause any divergence in the body weight, or food and water intakes of the male experimental rats. The organ weight and hematological parameters of treated animals did not vary significantly from control groups (Table 2 and 3). It was noted that the WFTS group had significantly higher serum albumin level than the control rats (P < 0.05) (Table 4). Except the WFTS groups had higher nitrate contents in urine, administration of WFTS did not affect urinary parameters (data not shown). The gross examination of all organs was normal and the histological examination showed no difference between the control and the WFTS-treated group among all organs examined.

image

Figure 2—. Changes of body weight, food, and water intake of male Sprague-Dawley rats treated with water extract of fermented Toona sinensis (WFTS) by daily gavage for 28 d. (A) Body weight, (B) cumulative food intake, and (C) cumulative water intake. Data points were means ± SD (n= 8).

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Table 2—.  Organ weight changes of Sprague-Dawley male rats treated with water extract of fermented Toona sinensis by daily gavage for 28 d.
Organ parameterBrain (g)Heart (g)Liver (g)Kidney (g)aPancreas (g)Spleen (g)
  1. WFTS = water extract of fermented Toona sinensis.

  2. Data are expressed as the mean ± SD (n= 8).

  3. aBoth right and left kidneys were weighed.

Control2.00 ± 0.111.24 ± 0.1410.22 ± 1.242.65 ± 0.270.68 ± 0.1 0.90 ± 0.15
Treatment
 WFTS 0.5 g/kg bw2.08 ± 0.081.24 ± 0.1010.32 ± 0.912.71 ± 0.180.74 ± 0.110.97 ± 0.32
 WFTS 1 g/kg bw2.00 ± 0.081.34 ± 0.1210.47 ± 0.882.56 ± 0.220.79 ± 0.131.13 ± 0.21
Table 3—.  Changes in hematological parameters in Sprague-Dawley male rats treated with water extract of fermented Toona sinensis by daily gavage for 28 d.
Hematological parameteraWBC (103/μL)RBC (106/μL)HGB (g/dL)HCT (%)MCV (fl)MCH (pg)MCHC (g/dL)PLT (103/μL)
  1. WFTS: water extract of fermented Toona sinensis.

  2. Data are expressed as the mean ± SD (n= 8). All data did not show significant difference between treatments and control (P > 0.05).

  3. aWBC: white blood count; RBC = red blood cell; HGB = hemoglobin; HCT = hematocrit; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; PLT = platelet.

Control 9.93 ± 1.267.36 ± 0.1315.65 ± 0.2543.39 ± 0.6858.95 ± 0.4621.29 ± 0.3336.06 ± 0.39897.5 ± 46.59
Treatment
 WFTS 0.5 g/kg bw11.14 ± 1.007.46 ± 0.1015.82 ± 0.1443.58 ± 0.5258.33 ± 0.5421.22 ± 0.3336.35 ± 0.46989.0 ± 60.87
 WFTS 1 g/kg bw13.96 ± 1.637.36 ± 0.1115.10 ± 0.2642.56 ± 0.62 57.8 ± 0.5421.06 ± 0.2036.43 ± 0.20993.8 ± 40.44
Table 4—.  Serum biochemistry changes in Sprague-Dawley male rats treated with extract of fermented Toona sinensis by daily gavage for 28 d.
Biochemistry parameteraALP (U/L)AST (U/L)ALT (U/L)Albumin (g/dL)Total protein (g/dL)Total bilirubin (mg/dL)Cholesterol (mg/dL)Triglyceride (mg/dL)BUN (mg/dL)Creatinine (mg/dL)Glucose (mg/dL)
  1. WFTS = Water extract of fermented Toona sinensis.

  2. Data are expressed as the mean ± SD (n= 8).

  3. ASignificant difference between the control and treated groups at P < 0.05.

  4. aALP = alkaline phosphatase; AST = aspartate aminotransferase; ALT = alinine aminotransferase; BUN = blood urea nitrogen.

Control150.87 ± 8.8874.12 ± 2.3926.62 ± 1.023.12 ± 0.125.16 ± 0.090.38 ± 0.0331.62 ± 1.9442.87 ± 6.0511.37 ± 0.330.41 ± 0.01148.62 ± 9.84
Treatment
 WFTS 0.5 g/kg bw138.55 ± 7.6366.44 ± 2.4528.66 ± 0.943.24 ± 0.04A5.26 ± 0.08 0.23 ± 0.03A33.88 ± 2.7350.33 ± 6.0911.33 ± 0.6 0.37 ± 0.01148.33 ± 3.84
 WFTS 1 g/kg bw142.70 ± 9.9669.50 ± 3.1227.30 ± 1.063.30 ± 0.02A5.36 ± 0.060.33 ± 0.0428.60 ± 2.3633.80 ± 2.82  12.5 ± 0.370.40 ± 0.01148.30 ± 6.40

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

According to the results of Ames test, no reproducible and dose-depending increase in revertants to prototrophy were obtained with any of the tested TA strains exposed to WFTS either in the presence or absence of S9 mix. Therefore, WFTS should not be considered as a mutagenicity-inducing agent based on the results of the Ames assay. In the present study of toxicity of WFTS to SD rats, either administered WFTS at a single dose of 5 g/kg bw for acute toxicity or subacute administration of 1 g/kg body weight for 28 d. In the subacute toxicity study, the WFTS groups had significantly higher level of serum albumin than the control rats (3.12 ± 0.12 g/dL). Even the WFTS group showed increased serum albumin (3.24 ± 0.04 and 3.30 ± 0.02 g/dL) after the treatment of repeated dose of 0.5 or 1 g/kg bw of WFTS, however, the value of serum albumin was still within the normal range in SD rats (3.75 ± 0.37 g/dL) based on literature data at the age of 10 wk in SD rats (Liang and others 1999). The WFTS groups had a slight higher nitrate contents in urinary parameters; however, this change is considered within normal physiological conditions. No other clinical toxicity was observed based on the results of serum biochemical parameters and pathological examinations.

In our previous research, the safety of water extracts of Toona sinensis Roemor leaf (TSL-1) was evaluated (Liao and others 2007). No mutagenicity was found in the tested range of 1 to 5 mg TSL-1/plate using the Ames test. Animal feeding study using ICR mice showed that there was no acute or subacute toxicity through oral administration of a single dose of 5 g/kg bw or 1 g/kg bw/d. The phytochemical components of TSL-1 has been investigated and several compounds were isolated and identified, including gallic acid, methyl gallic acid, gallic acid 3-O-β-D-(6′-O-galloyl)-glucopyranoside, 4-O-β-D-(6′-O-galloyl)-glucopyranoside, methyl gallate, ethyl gallate, kaempferol, kaempferol-3-O-β-glucoside, quercetin, quercitrin, quercetin-3-O-β-glucoside, rutin, and catechin (Hsieh and others 2004; Chia 2005). The major compound in TSL-1 is gallic acid with the content of 10% to 13% and the rutin content (0.16%) in TSL-1 is much less than gallic acid (Chia 2005). Lin (2007) reported that total phenolic content of WFTS (0.27 ± 0.02 mg gallic acid equivalent/mg) was lower than that of TSL-1 (0.34 ± 0.02 mg gallic acid equivalent/mg). Rutin is the major decomposed phenolic compounds in Toona sinensis Roemor leaf through anaerobic fermentation, and 32.4% of rutin content was decreased after fermentation (Margiantoro 2003). According to results reported by Hasumura and others (2004), supplementation of rutin up to 5% of diet for 13 wk in Wistar rats showed no mortality or abnormal clinical sign, but the body weight gain was reduced in male rats at 5% rutin supplementation in diet. The total rutin content of WFTS used for oral administration in this study is expected to be much lower than the research of Hasumura and others (2004), and this may be contributed in part to no difference in body weight gain observed in our study.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

This study demonstrated that the WFTS (water extract of fermented Toona sinensis Roemor) was present to lack genotoxicity under the Ames assay. The results also showed no acute lethal effect at a maximal tested dose of 5 g/kg bw in rats. Histopathological examination in subacute toxicity study revealed that the WFTS did not cause any significant toxic effect in rats. The subacute toxicity study also confirmed the safety of the WFTS after the repeated oral administration of 1 g/kg bw for 28 consecutive days. The WFTS is considered to be safe in general in male rats at the limited dose level and this result indicates that no significant adverse effect of WFTS in rats (OECD 1995; EPA 2000; Dorato and Engelhardt 2005).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

We are grateful to Dr. Hseng-Kuang Hsu in Dept. of Physiology, School of Technology for Medical Sciences at Kaohsiung Medical Univ. for providing the fermented TS extract. This study was supported by the Dept. of Health, Executive Yuan, Taiwan, R.O.C. (DOH93-TD-1007).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
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