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Acute and subchronic oral toxicities of Pu-erh black tea extract in Sprague–Dawley rats
Journal of Ethnopharmacology 134 (2011) 156–164
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Acute and subchronic oral toxicities of Pu-erh black tea extract in Sprague–Dawley rats Di Wang a,b , Kunlong Xu c , Ying Zhong a,b , Xiao Luo a,b , Rong Xiao d , Yan Hou e , Wei Bao a,b , Wei Yang a,b , Hong Yan b , Ping Yao a,b , Liegang Liu a,b,∗ a Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, PR China b MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, PR China c College of Animal Science and Technology, Yunnan Agriculture University, Kunming, Yunnan 650201, PR China d College of Food Science, Yunnan Agriculture University, Kunming, Yunnan 650201, PR China e Certificate Assessment Center of Yunnan Pu-erh Tea, Yunnan Agriculture University, Kunming, Yunnan 650201, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2010 Received in revised form 22 September 2010 Accepted 28 November 2010 Available online 4 December 2010 Keywords: Pu-erh black tea extracts Acute toxicity Subchronic toxicity LD50 NOAEL
a b s t r a c t Ethnopharmacological relevance: Pu-erh black tea, which is obtained by first parching crude green tea leaves and then undergoes secondary fermentation with microorganisms, has been believed to be beneficial beverages for health for nearly 2000 years in China, Japan and Taiwan area. But its potential toxicity when administered at a high dose as concentrated extracts has not been completely investigated. The aim of the study: The present study was aimed at evaluating potential toxicity of Pu-erh black tea extracts (BTE) from acute and sub-chronic administration to male and female Sprague–Dawley (SD) rats. Materials and methods: A single BTE dose of 10,000 mg/kg of body weight was administered by oral gavage for acute toxicity in SD rats. Four groups (10 males and 10 females per group) of dose levels of 1250, 2500, and 5000 mg/kg/day of the test article, as well as controls (distilled water) were tested as the subchronic toxicity study. Results: No deaths and signs of toxicity occurred during the 14 days of the study. There were no test article related mortalities, body weight gain, feed consumption, clinical observation, organ weight changes, gross finding, clinical or histopathological alterations during the 91-day administration. Conclusions: The LD50 of BTE can be defined as more than 10,000 mg/kg, and a dose of 5000 mg/kg/day was identified as the no-observed-adverse-effect-level (NOAEL) in this study. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Tea is the most widely consumed as a daily beverage in the world, drunk in the UK for 350 years and in Asia for more than 4000 years. Data on over 7000 adults from the UK National Diet and Nutrition Survey (NDNS) (Henderson et al., 2002), indicate that 77% of people drink tea, with a mean consumption of 2.3 mugs (540 mL) per day. It is believed to have medicinal efficacy in the prevention and treatment of many diseases, and so longevity is often associated with the habit of drinking tea (Crespy and Williamson, 2004; Kao et al., 2006; Nagao et al., 2007). Usually, on the basis of the processing procedures, tea can generally be divided into three types:
∗ Corresponding author at: Department of Nutrition and Food Hygiene, Tongji Medical College, Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, PR China. Tel.: +86 27 83692711; fax: +86 27 83650522. E-mail address: [email protected] (L. Liu). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.068
nonfermented (green tea), semifermented (oolong tea), and fully fermented (black tea and Pu-erh tea) (Lin et al., 1998). Pu-erh tea, originally produced in the Yunnan province of China for about 1700 years, was recorded by Compendium of Materia Medica that Pu-erh tea can expel wind-evil, clear away heat and aid in lose weight. Pu-erh tea is obtained by first parching crude green tea leaves (Camellia sinensis var. assamica (L.) O. Kuntze; Theaceae) and then undergoes secondary fermentation with microorganisms such as Aspergillus sp. (postfermented) (Jeng et al., 2007a), resulting in a unique type of tea. During the fermentation process, catechins are oxidized into quinone by polyphenol oxidase and then condensed to form bisflavanol, theaflavin, thearubigen, and other high molecular components (Yang and Koo, 1997), otherwise, the Puerh black tea is relatively rich in natural statins (Jeng et al., 2007b). These are regarded as the biologically important active components of Pu-erh black tea and that provide health benefits such as antioxidation, hypocholesterolemia, and anti-obesity (Duh et al., 2004; Fujita and Yamagami, 2008a,b). Therefore, Pu-erh black tea, with
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its old tradition, long history and special favor, is widely consumed by the world. Though the health benefits of tea are accepted by the people all over the word, issues of safety should not be neglected. Based on available data, high dose of green tea and/or associated chemical compounds intake could alter hepatic and thyroid function adversely (Schmidt et al., 2005; Bonkovsky, 2006; Bun et al., 2006; Chandra and De, 2010). Specially, for black tea, which is consumed by nearly 80% of the word people, its potential toxicities have not been got enough attentions until now. Relevant epidemiological and clinical studies published between 1990 and 2004 reported that excess black tea consumption would be harmful. This is associated to caffeine content, which has an adverse effect on fluid balance, cognitive, function, bone health, dental health and iron status. (Gardner et al., 2007). In vitro study prove that black tea polyphenol extracts (Babich et al., 2005), a black tea theaflavin mixture (Babich et al., 2006) and specific theaflavin monomers (Schuck et al., 2008) exhibited both antioxidant and prooxidant properties. Relevant results show that these compounds could lead to morphological changes characteristic of apoptosis both in cancer and normal cells. The possible mechanism could attribute to the generation of excessive reactive oxygen species (ROS) in cell. However, these injuries are rarely observed in vivo with black tea administration. Above all, although the Pu-erh black tea becomes more popular in the worldwide, fewer data about its potential toxicity can be available when administered at a high dose as concentrated extracts or products. In this experiment, commercial Pu-erh black tea samples were selected from Yunnan high land of China, and the toxicity of its extract was evaluated with dietary administration to male and female Sprague–Dawley (SD) rats for acute 14 days and subchronic 91 days as parts of a safety assessment according to the internationally acceptable guidelines for Pu-erh tea consumption.
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2.2. Experimental animals A total of 60 male and 60 female 5-wk-old SD rats were purchased from Sino-British Sippr/BK (Shanghai, China) and used after 1-wk acclimatization. Individual body weights were recorded and detailed physical examinations were performed twice during the acclimation period to ensure the use of healthy animals. Each animal was housed in a cage suspended wiremesh and given free access to commercial laboratory feed and tap water during the nonexposure periods. Animals were feed in a separate room with a barrier system controlled for the light–dark cycle (12–12 h, lights on 7:00–19:00), ventilation (air-exchange rate of 18 times per hour), temperature (23 ± 2 ◦ C) and relative humidity (55 ± 5%) during the study. The cages and the chip bedding were exchanged twice a week. The study was performed in accordance with the guide for the care and use of laboratory animals, prepared by National Institute of Health, USA (Guide for the Care and Use of Laboratory Animals, 1996). 2.3. Single dose acute oral toxicity of BTE in rats The single dose acute oral toxicity study was evaluated following the recommendations by OECD/OCDE (2001). After overnight fasting (8–10 h), groups of 10 male and 10 female SD rats were administered a single dose of 10,000 mg/kg of body weight BTE by oral gavage. The purified water was administered to the control group including the same number of rats with BTE treated group. Feeding was restarted 4 h after dosing. All animals were observed for clinical signs including mortality and moribundity, immediately after dosing and at 1, 2, 4, 8 and 12 h, then twice daily until d 14. Abnormal findings were recorded with the time of onset and disappearance. Body weights and food consumption were measured on d 0, 1, 3, 5, 7, 10 and 14. On the 14th day, all animals were sacrificed and all organs and tissues were observed macroscopically. The abnormal organs were placed in 10% neutral buffered formalin and observed by pathological examination.
2. Materials and methods 2.4. Subchronic 91-day oral toxicity study of BTE 2.1. Tea extracts preparation The commercial Pu-erh black tea samples were collected from Yunnan Highland of China. Voucher specimens of tea (Camellia sinensis var. assamica (L.) O. Kuntze; Theaceae) were collected from agro-forests and deposited at the Kunming Institute of Botany, Chinese Academy of Sciences. The detail processing of Pu-erh black tea was introduced in previous study (Hou et al., 2009). In brief, the crude green tea leaves cultivated in the Yunnan Highlands of China was used as the raw material. Leaves were collected and heated, dried at <60 ◦ C, and molded to make unfermented Pu-erh tea. To make fermented Pu-erh tea, the unfermented Pu-erh tea was dampened and fermented with a pure culture of Aspergillus niger for 50 d at controlled temperature (40–60 ◦ C) and humidity (70–85%). Fermented Pu-erh tea was then dried at <60 ◦ C and packed. The tea sample (100 g) was cut into small pieces, and soaked in boiled distilled water for three times (2 L, 1.5 L and 1.5 L of each bulk, respectively; 20 min, 15 min and 15 min of each time, respectively). After separating from leaves by filtration, the whole extracts were evaporated by a rotary evaporator at 65 ◦ C (Yarong Biochemical Instrument, Shanghai, China) to 100 mL. The final beverages were sterilized at 121 ◦ C for 20 min and kept at 4 ◦ C in a closed container and were designated as 1 g/mL (tea/water) Pu-erh black tea extract (BTE) for later experiments. The contents of biochemical ingredients including total polyphenols, total flavonoid, free amino acid, theaflavins, thearubigins and theabrownins in Pu-erh black tea were analyzed in our prior study (Wang et al., 2010).
2.4.1. Experimental design The subchronic 91-day oral toxicity study was evaluated following the recommendations by OECD/OCDE (1998). Groups of ten males and ten females received doses of 0, 1250, 2500 or 5000 mg/kg bw of BTE at daily gavage of 1 ml/100 g bw for 91 consecutive days. Observations were made twice daily for mortality and changes in general appearance or behavior. The body weights were recorded every week, the individual dose was adjusted for the body weight to maintain the target dose level for all rats. In addition, detailed clinical examination and measurement of food consumption were preformed weekly. The animals were sacrificed by exsanguinations from the abdominal aorta at the d 92 following fasting for 12–16 h. A sample of blood (approximately 20 L) was treated with EDTA-2K to analyze hematological indexes. Serum from blood samples collected in separator tubes was stored at −20 ◦ C. During necropsy, the following organs were dissected: liver, spleen, thymus, heart, lungs, stomach, ovaries, uterus, kidney, adrenals, trachea/thyroid gland, brain, pituitary gland, pancreas, perirenal fat, testes, and epididymis. All organs were visually inspected and weighed directly after dissection to reduce mechanical damage. Defined samples of the liver, brain, pancreas, stomach, kidney, adrenals, testes and ovaries were placed in 10% neutral buffered formalin for pathological examination. 2.4.2. Hematology and blood chemistry One blood sample (approximately 20 L) was treated with EDTA-2K for white blood cells (WBC), red blood cells (RBC),
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hemoglobin (Hb), platelets (PLT), percent of lymphocytes (LY), percent of monocytes (MO), percent of granulocyte (GR), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean platelet volume (MPV), Platelet hematocrit (PCT), platelet distribution width (PDW) and red blood cell distribution width (RDW) using a hematology analyzer MEK-6318K (Nihon Kohden Co., Ltd.). Serum from blood samples collected in separator tubes was measured using a BS-200 automatic biochemistry analyzer (Mindary Co., Ltd.) including aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, creatinine (Cr), total cholesterol (TC), triglyceride (TG), total protein (TP), albumin (Alb) and glucose (Glu). Ionized calcium (iCa) and total calcium (TCa) using the 7020 automatic biochemistry analyzer (Hitachi Co., Ltd.), and for sodium (Na), potassium (K), chloride (Cl) and pH value analyses. 2.4.3. Urinalysis The 24-h pooled urine of each animal was collected by metabolism cage and volume was recorded at week 13. The fresh urine was examined for specific gravity, pH, protein, glucose, acetone body, bilirubin and urobilinogen by an UA-66 urine analyzer (Mindary Co., Ltd.). 2.4.4. Histopathological examination Samples of the liver, brain, pancreas, stomach, kidney, adrenals, testes and ovaries in every group were placed in 10% neutral buffered formalin, sectioned and stained with hematoxylin and eosin (H&E). 2.5. Statistical analysis Variance in data for body and organ weights as well as the results of hematology, and serum biochemistry was checked for homogeneity by Bartlett’s procedure. When the data were homogeneous, one-way analysis of variance for homogeneity (ANOVA) was used. In the heterogeneous cases, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the Dunnett’s multiple tests were employed for comparisons between control and treated groups. All statistical analyses were performed using SPSS 12.0 statistical software (SPSS Inc., Chicago, IL) and a difference was considered significant when P < 0.05. 3. Results 3.1. Single dose acute oral toxicity study of BTE in SD rats No deaths occurred during the 14 d of the study. There were no significant differences in body weights and food consumption of either sex between the BTE administrated group and the controls (data not shown). A short time of sluggish appeared in some BTE treated rats after oral gavage in the first hour, but the rats return to life later. Otherwise, there were no abnormal findings at other clinical signs and autopsy in the experimental or control group in either sex. Based on these, the LD50 value of BTE was greater than 10,000 mg/kg for both sexes. 3.2. Subchronic 91-day oral toxicity study of BTE in SD rats 3.2.1. Clinical signs and mortality There was no mortality attributed to any effect of BTE during the 91-day administration. One female (8th wk) given BTE with dose of 2500 mg/kg/day and two females with 1250 mg/kg/day (8th and 9th wk, respectively), and one male rat with 5000 mg/kg/day
(9th wk) died during the 13-wk oral study could be due to inadvertent gavage accidents. The body weights and food consumption of these rats were not obviously decreased as compared to other rats in the same group before death. Clinical observations in these animals prior to death consisted of intermittent convulsions, gasping, hypoactivity, labored respiration, and prostration. Anatomical results showed that no abnormal organic damages were observed in the dead rats. Moreover, there were no treatmentrelated changes at autopsy in the BTE group or control group in either sex.
3.2.2. Body weights and food consumption Mean body weights for male and female rats at 1250, 2500 and 5000 mg/kg/day were compared to control values (Fig. 1). Female SD rats in all BTE groups had significantly lower mean body weights (254.6 g, 257.7 g and 253.8 g in 5000, 2500 and 1250 mg/kg/day group, respectively) as compared to the control group (281.3 g). The mean body weights of male rats in 5000 mg/kg/day and 2500 mg/kg/day BTE groups were significantly lower than the control group (332.6 g vs 438.3 g and 392.3 g vs 438.3 g, respectively) at the thirteenth week (P < 0.01 or P < 0.05). During the thirteen-wk study, there were no differences in mean food consumption at any other observation points in any group. The data about mean daily food consumption in BTE treated groups and control group is shown in Fig. 2.
3.2.3. Organ weights The results for relative organ weights were shown in Tables 1a and 1b. In females, the relative organ weights of heart and liver in 5000 mg/kg/day group were significantly increased as compared to controls, while the relative pancreas weight was significantly lower than controls. In addition, significant decrease of relative spleen weight was found in the 2500 mg/kg/day group. In males, the relative organ weights of brain, stomach, liver and testes were significantly increased in 5000 mg/kg/day group. The perirenal fat, which was an important visceral fat in body, was also removed and weighed in present study. A dose-dependent decrease in relative perirenal fat was observed in 5000 and 2500 mg/kg/day groups both in females and males.
3.2.4. Hematology and blood chemistry In hematology parameters, statistically significant decrease and increase were observed in RBC and MCHC with dose of 5000 mg/kg/day in female rats, respectively (Table 2a). There were no significant differences between the treatment and control groups in males (Table 2b). In blood serum biochemistry of females (Table 3a), a dose-dependent decrease in TG was observed in 5000 and 2500 mg/kg/day group. Statistically significantly, the TC level in 5000 mg/kg/day was decreased as compared with control group. A slight increase was observed in high dose group, but the difference was not significant. For males (Table 3b), TC and TG were significantly decreased in 5000 mg/kg/day. Statistically significant increase of ALT in females administrated 5000 mg/kg/day was observed. Beyond that, there were no treatment-related or statistically significant effects in metallic ion and pH value.
3.2.5. Urinalysis Statistically significant decrease in 24-h urine volume in males administered 5000 mg/kg/day was observed as compared with controls (data not shown). Thus, the changes observed in urinalysis were considered to be unrelated to the treatment and no adverse effects were found at the same test doses in females and had no other clinical pathology correlation.
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159
Female 350.0
Body weights (g)
BTE 5000 mg/kg/day
300.0
BTE 2500 mg/kg/day BTE 1250 mg/kg/day Control 0 mg/kg/day
250.0 200.0 150.0 100.0 50.0
week0 week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Male 500.0
Body weights (g)
450.0
BTE 5000 mg/kg/day BTE 2500 mg/kg/day
400.0
BTE 1250 mg/kg/day
350.0
Control 0 mg/kg/day
300.0 250.0 200.0 150.0 100.0 50.0 week0 week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Fig. 1. Effects of oral administration of Pu-erh black tea extract for 13 weeks on mean body weights. Body weight was calculated and data are expressed as mean ± SEM. BTE = Pu-erh black tea extract.
Table 1a Effects of oral administration of Pu-erh black tea extract for 13 weeks on relative organ weight for female SD rats. Organs
Dose (mg/kg/day) 5000 (n = 10) a
Body weights Brainb Cerebellumb Brainstemb Pituitaryb Heartb Thyroids/parathyroidsb Thymusb Liverb Spleenb Lungb Pancreasb Stomachb Kidneysb Adrenalsb Uterusb Ovaryb Perirenal fatb
254.6 4.8 0.1 0.9 0.1 4.1 0.1 1.4 32.8 2.4 7.3 2.2 6.6 6.8 0.3 2.0 0.6 2.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
**
4.8 0.3 0.0 0.1 0.0 0.2* 0.0 0.1 1.7* 0.1 0.5 0.2* 0.1 0.2 0.0 0.3 0.0 0.2*
2500 (n = 9) 257.7 4.7 0.1 0.9 0.1 3.4 0.1 1.4 26.7 2.2 6.5 2.5 5.7 6.3 0.3 1.7 0.5 3.7
Mean organ-to-body weight ratio was calculated and data are expressed as mean ± SEM. a Unit: g. b Unit: ‰ body weights. * Statistically significant compared to controls (P < 0.05). ** Statistically significant compared to controls (P < 0.01).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1250 (n = 8) **
6.8 0.2 0.0 0.1 0.0 0.1 0.0 0.2 0.9 0.1* 0.4 0.2 0.2 0.1 0.0 0.3 0.0 0.4*
253.8 4.8 0.1 0.9 0.1 3.6 0.1 1.9 28.4 2.6 6.5 2.4 6.2 6.8 0.3 2.2 0.5 5.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0 (n = 10) **
10.7 0.1 0.0 0.1 0.0* 0.1 0.0 0.1 0.7 0.3 0.4 0.2 0.3 0.3 0.0 0.2 0.0 2.0
281.3 4.3 0.1 1.1 0.1 3.6 0.1 1.5 26.6 2.5 5.4 2.5 5.5 6.1 0.3 2.5 0.5 6.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.5 0.2 0.0 0.1 0.0 0.1 0.0 0.1 0.8 0.2 0.3 0.2 0.4 0.5 0.0 0.5 0.0 1.0
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Female Food Consumption (g/day)
40.0
BTE 5000 mg/kg/day BTE 2500 mg/kg/day BTE 1250 mg/kg/day Control 0 mg/kg/da y
30.0
20.0
10.0
0.0
week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Male
Food Consumption (g/day)
40.0 BTE 5000 mg/kg/day BTE 2500 mg/kg/day
35.0
BTE 1250 mg/kg/day Control 0 mg/kg/day
30.0 25.0 20.0 15.0 10.0
week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Fig. 2. Mean food consumption for SD rats during a 13-week oral (gavage) toxicity study with Pu-erh tea extract. Food consumption was calculated and data are expressed as mean ± SEM. BTE = Pu-erh black tea extract.
Table 1b Effects of oral administration of Pu-erh black tea extract for 13 weeks on relative organ weight for male SD rats. Organs
Dose (mg/kg/day) 5000 (n = 9)
Body weightsa Brainb Cerebellumb Brainstemb Pituitaryb Heartb Thyroids/parathyroidsb Thymusb Liverb Spleenb Lungb Pancreasb Stomachb Kidneysb Adrenalsb Testesb Epididymidesb Perirenal fatb
337.7 3.7 0.5 0.8 0.5 3.7 0.2 0.9 34.4 1.7 7.0 2.3 5.8 7.1 0.2 7.7 1.8 4.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
11.2** 0.2* 0.1 0.1 0.1 0.1 0.1 0.1 2.6* 0.1 1.1 0.3 0.4* 0.3 0.0 0.3* 0.2 1.1**
2500 (n = 10) 392.3 3.2 0.6 0.8 0.6 3.4 0.4 1.1 26.5 1.8 5.6 1.8 4.7 6.6 0.3 7.1 1.9 5.4
Mean organ-to-body weight ratio was calculated and data are expressed as mean ± SEM. a Unit: g. b Unit: ‰ body weights. * Statistically significant compared to controls (P < 0.05). ** Statistically significant compared to controls (P < 0.01).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
14.9* 0.1 0.1 0.1 0.1 0.2 0.0 0.1 1.3 0.1 0.3 0.1 0.1 0.2 0.1 0.3 0.2 0.9**
1250 (n = 10) 421.4 3.1 0.6 0.6 0.6 3.3 0.5 1.0 28.1 1.8 5.2 2.2 4.3 6.7 0.2 6.6 1.9 8.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
11.5 0.1 0.1 0.1 0.1 0.2 0.1 0.1 1.7 0.1 0.6 0.2 0.1 0.2 0.0 0.3 0.2 0.5
0 (n = 10) 438.3 3.1 0.6 0.6 0.6 3.3 0.5 1.1 25.2 1.8 5.2 2.0 4.5 6.7 0.2 6.6 2.1 10.8
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
12.4 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.3 0.1 0.2 0.0 0.2 0.3 1.4
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Table 2a Hematological data for female rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 10)
WBC (thous/L) RBC (millions/L) HGB (g/dL) PLT (thous/L) LY (%) MO (%) GR (%) HCT (%) MCH (pg) MCHC (g/dL) MCV (fl) MPV (fl) PCT (%) PDW (fl) RDW (fl)
16.06 8.27 15.42 359.50 63.32 5.68 21.00 43.58 17.12 37.06 51.62 9.91 0.29 12.50 13.31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.93 0.54* 1.44 39.71 10.43 1.68 9.30 3.26 0.92 1.26* 1.90 0.78 0.133 0.59 0.75
2500 (n = 9) 16.52 9.27 16.65 396.50 70.33 4.37 26.17 46.47 17.97 35.85 50.11 9.55 0.37 12.16 12.93
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.21 0.90 1.66 80.13 6.87 1.35 6.59 4.56 0.81 1.37 0.82 0.64 0.09 0.54 1.04
1250 (n = 8) 15.14 9.01 16.36 356.56 61.77 5.59 32.64 45.58 18.14 35.88 50.61 9.50 0.34 12.21 12.77
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.08 0.93 1.71 53.32 11.89 1.17 11.70 4.30 0.99 11.83 1.49 0.71 0.07 0.93 0.59
0 (n = 10) 13.17 8.84 15.68 339.50 70.44 5.78 23.78 44.32 17.74 35.39 50.14 9.70 0.32 12.22 12.69
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.77 0.41 0.91 46.71 12.71 1.43 12.38 2.32 0.60 13.3 1.34 0.67 0.05 0.86 0.60
Values are presented as mean ± SEM. * Significantly different from the controls at levels of P < 0.05.
Table 2b Hematological data for male rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 9)
WBC (thous/L) RBC (millions/L) HGB (g/dL) PLT (thous/L) LY (%) MO (%) GR (%) HCT (%) MCH (pg) MCHC (g/dL) MCV (fl) MPV (fl) PCT (%) PDW (fl) RDW (fl)
16.20 8.37 15.43 329.30 63.79 6.51 25.98 42.77 18.43 36.12 51.06 9.50 0.31 12.42 13.44
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2500 (n = 10)
7.23 0.47 0.92 57.37 9.92 3.11 13.06 2.94 0.78 1.35 1.69 0.40 0.05 0.90 1.10
16.59 8.45 15.88 373.30 63.00 6.17 27.55 43.40 18.77 36.53 51.38 9.44 0.35 12.15 13.28
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.90 0.69 1.57 39.42 12.77 3.31 15.69 3.38 0.84 1.04* 1.41 0.44 0.05 0.71 0.98
1250 (n = 10) 15.96 8.39 15.52 340.90 72.79 6.26 20.95 43.07 18.55 36.13 51.28 9.45 0.32 11.99 13.28
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.63 1.00 1.58 35.33 9.74 2.22 8.52 5.33 0.61 1.16 1.08 0.25 0.04 0.78 1.06
0 (n = 10) 13.22 7.98 14.45 342.10 70.90 5.34 23.76 41.13 18.14 35.15 51.60 9.36 0.32 12.32 13.36
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.72 0.85 1.54 66.11 10.28 1.37 9.63 4.40 0.72 1.08 1.46 0.33 0.06 0.79 1.20
Values are presented as mean ± SEM. * Significantly different from the controls at levels of P < 0.05.
Table 3a Serum biochemical data for female rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 10)
TC (mg/dL) TG (mmol/L) Glu (mg/dL) TP (mg/L) Alb (mg/L) ALT (U/L) AST (U/L) Cre (mmol/L) Urea (mmol/L) K (mmol/L) Na (mmol/L) Cl (mmol/L) iCa (mmol/L) TCa (mmol/L) pH
69.87 0.30 98.74 80.13 35.79 51.56 141.15 43.04 6.07 7.94 144.16 108.43 1.26 2.45 7.66
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2500 (n = 9) *
2.38 0.03** 9.67 1.79 2.26 4.27 4.62 2.42 0.33 0.17 0.83 0.75 0.04 0.08 0.01
Values are presented as mean ± SEM. * Statistically significant compared to controls (P < 0.05). ** Statistically significant compared to controls (P < 0.01).
80.24 0.33 97.40 77.50 39.34 48.50 122.53 42.00 5.52 7.83 142.18 107.50 1.27 2.48 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.90 0.01* 9.31 1.50 1.87 2.68 8.71 1.32 0.28 0.20 0.49 0.52 0.02 0.04 0.01
1250 (n = 8) 88.71 0.38 109.42 87.47 37.92 41.96 129.80 42.47 5.51 7.47 143.57 108.51 1.29 2.54 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.71 0.01 14.61 2.54 0.71 3.44 8.27 1.84 0.51 0.29 1.32 1.05 0.02 0.05 0.01
0 (n = 10) 89.46 0.40 103.58 83.98 38.74 42.88 142.26 43.20 5.64 7.44 143.65 108.27 1.31 2.55 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.98 0.01 10.82 2.28 1.04 2.62 9.23 2.49 0.27 0.29 0.88 0.88 0.02 0.04 0.01
162
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Table 3b Serum biochemical data for male rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 9)
TC (mg/dL) TG (mmol/L) Glu (mg/dL) TP (mg/L) Alb (mg/L) ALT (U/L) AST (U/L) Cre (mmol/L) Urea (mmol/L) K (mmol/L) Na (mmol/L) Cl (mmol/L) iCa (mmol/L) TCa (mmol/L) pH
76.32 0.31 103.42 70.13 35.79 49.70 124.09 42.01 5.95 7.99 142.77 105.60 1.34 2.62 7.65
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.67* 0.02* 7.68 2.16 2.26 2.17* 7.56 1.80 0.26 0.14 0.68 0.95 0.04 0.08 0.01
2500 (n = 10) 83.41 0.33 110.53 80.32 39.34 44.36 101.89 42.23 5.84 8.14 143.61 107.70 1.30 2.53 7.66
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.27 0.05 7.43 1.75 1.87 1.94 8.52 2.34 0.21 0.35 0.46 0.55 0.02 0.05 0.01
1250 (n = 10) 80.18 0.44 109.34 80.58 37.92 46.06 116.41 42.39 5.66 8.11 145.34 108.97 1.32 2.57 7.65
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.32 0.05 9.87 1.88 0.71 1.02 2.72 2.46 0.21 0.39 0.63 0.62 0.02 0.03 0.01
0 (n = 10) 86.74 0.46 115.72 76.19 38.74 39.70 106.27 41.47 4.81 8.12 144.39 108.88 1.30 2.54 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.32 0.05 13.20 1.25 1.02 1.61 5.99 1.68 0.20 0.26 0.32 0.42 0.03 0.06 0.01
Values are presented as mean ± SEM. * Statistically significant compared to controls (P < 0.05).
3.2.6. Gross necropsy and histopathology There were no macroscopic observations considered to be treatment related. No gross abnormalities attributed to the test article were noted for any of the euthanized animals necropsied at the conclusion of the 92-day observation period. Histopathological changes including minimal bile-duct hyperplasia, vacuolation, cellullar swelling, spotty necrosis and inflammation were observed in liver. For kidney, incidence of inflammation, epithelium swelling appeared. Thus these changes were not considered to be treatment related in this study, because these microscopic changes were commonly observed in untreated laboratory animals and were of comparable incidence and severity between test article-treated and control animals. 4. Discussion Pu-erh black tea, which is planted in Yunnan High Land of P.R. China, has been consumed widely by people living in southern China, Taiwan Area and Japan. As a unique type of tea, the chemical contents in Pu-erh black tea are different to other teas. Our prior study found that the catechins are easily oxidized by polyphenol oxidase, and further polymerizations lead to theaflavins (0.17%), thearubigins (5.88%), and theabrownins (9.73%), the contents of other smaller molecular-weight compounds such as amino acids were also lower than green tea during the special fermentation (Wang et al., 2010). The present study demonstrated the comprehensive safety profile of BTE in acute and subchronic oral toxicity study in SD rats. Exposure of SD rats in single dose of BTE did not produce any treatment-related effect. A number of changes including body and organ weights, parameters in hematology, serum biochemistry and histopathology were observed after subchronic exposure of BTE, but the changes were not toxicity-related. All rats at each dosage group continued to gain weight throughout the course of 91-day toxicological study. But the body weight gains in all BTE administration groups were significantly lower than controls except the 1250 mg/kg/day BTE male group. Additionally, a dose-dependent decrease in relative perirenal fat weights was observed in 5000 and 2500 mg/kg/day BTE administrated groups with both sexes. However, the food consumption results did not show significant differences in weekly food intake between BTE treatment groups and controls. The results in our study were similar to previous studies which reported that a growth depression is induced when Pu-erh black tea was given in excess to growing animals. In Jen-kun Lin’s study (Kuo et al., 2005), the body weights of rats placed on a basal diet had decreased from 5 wk to 30 wk
after given with 4% green tea, 1.5% and 4% Pu-erh tea. Michael and colleague (Fallon et al., 2008) found that fecal fat was significantly increased to approximately 15 percent in rats and body weights slightly increased receiving 3.0- and 6.0- black and green tea and mulberry mixture extracts. However, the mechanisms leading to the depression of body weight gain induced by Pu-erh black tea remain largely unknown. In vitro studies showed that black tea rich in theaflavin, which can inhibit the activity of digest enzymes, such as pancreatic lipase and salivary amylase (Zhang and Kashket, 1998; Nakai et al., 2005). Although the same conclusion has not been verified in vivo, promoting fat metabolism and inhibiting excess fatty deposits could be taken as presumptive evidence of anti-obesity effect of Pu-erh black tea. No untoward toxicity-related changes were observed for hematology parameters in male and female rats. The RBC level was significantly decreased and MCHC was increased in 5000 mg/kg/day female rats as compared to controls, but the changes were regarded as toxicologically irrelevant because the adverse results did not appear in both sexes and/or were without dose-relation. Significant increase in ALT in 5000 mg/kg/day BTE males and relative liver weights in males and females were presumably at least partly associated with hepatic changes. Though slight hepatotoxicity was observed in some studies about green tea extracts (Bonkovsky, 2006; Bun et al., 2006), separated chemicals i.e. polyphenols (Schmidt et al., 2005; Kapetanovic et al., 2009) and mixture containing black tea composition (Fallon et al., 2008), few vivo studies showed the similar hepatotoxicity in whatever single constituent in black tea or black tea extracts related experiments. As to Pu-erh black tea, A. niger was claimed to be the dominant microorganism in the Pu-erh tea manufacturing process and known to produce the mycotoxins i.e. fumonisins B2 and B4 (Noonim et al., 2009; Mogensen et al., 2010). Certain evidences show that exposure tomycotoxins in humans and animals can results in liver failure and death (Yoshida et al., 2001). However, neither production of tomycotoxins of the 47 isolates of Pu-erh tea and black tea were observed in Mogensen’s study (Mogensen et al., 2009). Therefore, increase of ALT and relative liver weight in the 5000 mg/kg/day group in males but without histopathological lesions and other injury-related evidences in the liver cannot be seen as a toxicity-related effect of BTE in present study. Other abnormal changes, like decrease in TC in 5000 mg/kg/day BTE treated group, TG in 5000 and 2500 mg/kg/day males and 5000 mg/kg/day females, were associated with the biological functions but toxicities. Previous studies had also proved that Pu-erh fermented tea cannot only significantly decrease the serum cholesterol levels in a hyperlipidemia rat model (Hou et al.,
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2009) but also slightly decrease the TC level during the 30-wk oral feeding in normal diet rats (Kuo et al., 2005). Relative liver weights were significantly increased in 5000 mg/kg/day BTE treated groups of both sexes. However, there was no microscopic correlate to the increase in mean liver weights in males and females at this dose. The other absolute and/or relative weights changes were observed in some organs, such as heart and pancreas in females with 5000 mg/kg/day BTE or brain, stomach and testes in males with 5000 mg/kg/day, they were not considered toxic-related since they were only observed in one sex, and did not exhibit a dose–response relationship. No treatment related microscopic changes were observed in this study. All microscopic changes observed in the liver and kidney were randomly distributed between test article-treated and control animals, and the incidences were within the range of normal background lesions. In conclusion, the high single dose administration of BTE (10,000 mg/kg of body weight) did not show acute oral toxicity in SD rats in present study. Based on common classification of the relative toxicity of chemicals, BTE probably belongs to the slightly toxic (LD50 : 5–15 g/kg) (Lu and Kacew, 2002). In the subchronic 13-wk oral toxicity test, no obvious toxic changes which due to administration of BTE were observed in any parameters. Therefore, the NOAEL in this subchronic toxicity study was 5000 mg/kg/day for both sexes in SD rats. In accordance with recommendations and considering an average consumption of five cups (2.5 g/cup) per person per day, the percentage of the average daily intake of Puerh black tea was about 178.6 mg/kg/day for adult (70 kg of body weight). The NOAEL level of BTE in present study is 28 times higher than the average intake by human, the present study suggested that high dose (5000 mg/kg/day) of BTE intake would be well-tolerated for long-term use as a dietary supplement whatever by animals or human. Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgements We appreciate the contribution of all the members participating in this study. We also wish to thank Dr. Yajun Du (Office of Health Assessment and Epidemiology, Department of Public Health, Los Angeles Country, USA). This work was supported by the National Key Technology R&D Program of the People’s Republic of China (2007BAD58B05). References Babich, H., Gold, T., Gold, R., 2005. Mediation of the in vitro cytotoxicity of green and black tea polyphenols by cobalt chloride. Toxicology Letters 155, 195–205. Babich, H., Pinsky, S.M., Muskin, E.T., Zuckerbraun, H.L., 2006. In vitro cytotoxicity of a theaflavin mixture from black tea to malignant, immortalized, and normal cells from the human oral cavity. Toxicology In Vitro 20, 677–688. Bonkovsky, H.L., 2006. Hepatotoxicity associated with supplements containing Chinese green tea (Camellia sinensis). Annals of Internal Medicine 144, 68–71. Bun, S.S., Bun, H., Guedon, D., Rosier, C., Ollivier, E., 2006. Effect of green tea extracts on liver functions in Wistar rats. Food and Chemical Toxicology 44, 1108–1113. Chandra, A.K., De, N., 2010. Goitrogenic/antithyroidal potential of green tea extract in relation to catechin in rats. Food and Chemical Toxicology. Crespy, V., Williamson, G., 2004. A review of the health effects of green tea catechins in in vivo animal models. The Journal of Nutrition 134, 3431S–3440S. Duh, P.D., Yen, G.C., Yen, W.J., Wang, B.S., Chang, L.W., 2004. Effects of Pu-erh tea on oxidative damage and nitric oxide scavenging. Journal of Agricultural and Food Chemistry 52, 8169–8176. Fallon, E., Zhong, L., Furne, J.K., Levitt, M., 2008. A mixture of extracts of black and green teas and mulberry leaf did not reduce weight gain in rats fed a high-fat diet. Alternative Medicine Review 13, 43–49. Fujita, H., Yamagami, T., 2008a. Efficacy and safety of Chinese black tea (Pu-Ehr) extract in healthy and hypercholesterolemic subjects. Annals of Nutrition and Metabolism 53, 33–42.
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Fujita, H., Yamagami, T., 2008b. Extract of black tea (pu-ehr) inhibits postprandial rise in serum cholesterol in mice, and with long term use reduces serum cholesterol and low density lipoprotein levels and renal fat weight in rats. Phytotherapy Research 22, 1275–1281. Gardner, E.J., Ruxton, C.H., Leeds, A.R., 2007. Black tea – helpful or harmful? A review of the evidence. European Journal of Clinical Nutrition 61, 3–18. 1996. Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources. Commission on Life Sciences. National Research Council. National Academy Press, Washington, DC. Henderson, L., Gregory, J., Swan, G., 2002. National Diet and Nutrition. Survey: Adults Aged 19–64 years. FSA, London. Hou, Y., Shao, W., Xiao, R., Xu, K., Ma, Z., Johnstone, B.H., Du, Y., 2009. Pu-erh tea aqueous extracts lower atherosclerotic risk factors in a rat hyperlipidemia model. Experimental Gerontology 44, 434–439. Jeng, K.C., Chen, C.S., Fang, Y.P., Hou, R.C., Chen, Y.S., 2007a. Effect of microbial fermentation on content of statin, GABA, and polyphenols in Pu-erh tea. Journal of Agricultural and Food Chemistry 55, 8787–8792. Jeng, K.C., Chen, C.S., Fang, Y.P., Hou, R.C.W., Chen, Y.S., 2007b. Effect of microbial fermentation on content of statin, GABA, and polyphenols in Pu-erh tea. Journal of Agricultural and Food Chemistry 55, 8787–8792. Kao, Y.H., Chang, H.H., Lee, M.J., Chen, C.L., 2006. Tea, obesity, and diabetes. Molecular Nutrition and Food Research 50, 188–210. Kapetanovic, I.M., Crowell, J.A., Krishnaraj, R., Zakharov, A., Lindeblad, M., Lyubimov, A., 2009. Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs. Toxicology 260, 28–36. Kuo, K.L., Weng, M.S., Chiang, C.T., Tsai, Y.J., Lin-Shiau, S.Y., Lin, J.K., 2005. Comparative studies on the hypolipidemic and growth suppressive effects of oolong, black, Pu-erh, and green tea leaves in rats. Journal of Agricultural and Food Chemistry 53, 480–489. Lin, J.K., Lin, C.L., Liang, Y.C., Lin-Shiau, S.Y., Juans, I.M., 1998. Survey of catechins, gallic acid, and methylxanthines in green, oolong, Pu-erh, and black teas. Journal of Agricultural and Food Chemistry 46, 3635–3642. Lu, F.C., Kacew, S., 2002. Acute, short-term and long-term toxicity studies. In: Basic Toxicology: Fund amentals, Target Organs, and Risk Assessment. Mogensen, J.M., Frisvad, J.C., Thrane, U., Nielsen, K.F., 2010. Production of fumonisin B2 and B4 by Aspergillus niger on grapes and raisins. Journal of Agricultural and Food Chemistry 58, 954–958. Mogensen, J.M., Varga, J., Thrane, U., Frisvad, J.C., 2009. Aspergillus acidus from Puerh tea and black tea does not produce ochratoxin A and fumonisin B2. International Journal of Food Microbiology 132, 141–144. Nagao, T., Hase, T., Tokimitsu, I., 2007. A green tea extract high in catechins reduces body fat and cardiovascular risks in humans. Obesity (Silver Spring) 15, 1473–1483. Nakai, M., Fukui, Y., Asami, S., Toyoda-Ono, Y., Iwashita, T., Shibata, H., Mitsunaga, T., Hashimoto, F., Kiso, Y., 2005. Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro. Journal of Agricultural and Food Chemistry 53, 4593–4598. Noonim, P., Mahakarnchanakul, W., Nielsen, K.F., Frisvad, J.C., Samson, R.A., 2009. Fumonisin B2 production by Aspergillus niger in Thai coffee beans. Food Additives and Contaminants. Part A, Chemistry, Analysis, Control, Exposure and Risk Assessment, vol. 26, pp. 94–100. OECD, 1998. Guideline for Testing of Chemicals. 408 Subchronic Oral Toxicity – Rodent: 90 Day Study. OECD, 2001. Guideline for Testing of Chemicals. 423 Acute Oral Toxicity – Acute Toxic Class Method. Schmidt, M., Schmitz, H.J., Baumgart, A., Guedon, D., Netsch, M.I., Kreuter, M.H., Schmidlin, C.B., Schrenk, D., 2005. Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture. Food and Chemical Toxicology 43, 307–314. Schuck, A.G., Ausubel, M.B., Zuckerbraun, H.L., Babich, H., 2008. Theaflavin-3,3 digallate, a component of black tea: an inducer of oxidative stress and apoptosis. Toxicology In Vitro 22, 598–609. Wang, D., Xiao, R., Hu, X., Xu, K., Hou, Y., Zhong, Y., Meng, J., Fan, B., Liu, L., 2010. Comparative safety evaluation of Chinese Pu-erh green tea extract and Pu-erh black tea extract in Wistar rats. Journal of Agricultural and Food Chemistry 58, 1350–1358. Yang, T.T., Koo, M.W., 1997. Hypocholesterolemic effects of Chinese tea. Pharmacological Research 35, 505–512. Yoshida, T., Takeda, M., Tsutsumi, T., Nagata, S., Yoshida, F., Maita, K., Harada, T., Ueno, Y., 2001. Tumor necrosis factor-␣ expression and Kupffer cell activation in hepatotoxicity caused by microcystin-LR in mice. Journal of Toxicologic Pathology 14, 259. Zhang, J., Kashket, S., 1998. Inhibition of salivary amylase by black and green teas and their effects on the intraoral hydrolysis of starch. Caries Research 32, 233–238.
Glossary ALT: alanine aminotransferase Alb: albumin AST: aspartate aminotransferase Cl: chloride Cr: creatinine Glu: glucose GA: gallic acid
164 HCT: hematocrit H&E: hematoxylin and eosin Hb: hemoglobin iCa: ionized calcium NDNS: National Diet and Nutrition Survey NOAEL: no-observed-adverse-effect-level ANOVA: one-way analysis of variance PLT: platelets PDW: platelet distribution width PCT: platelet hematocrit GR: percent of granulocyte LY: percent of lymphocytes MO: percent of monocytes
D. Wang et al. / Journal of Ethnopharmacology 134 (2011) 156–164 K: potassium BTE: Pu-erh black tea extract ROS: reactive oxygen species RDW: red blood cell distribution width RBC: red blood cells Na: sodium SD: Sprague–Dawley TC: total cholesterol TG: triglyceride TP: total protein TCa: total calcium Urea: urea nitrogen WBC: white blood cells
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Acute and subchronic oral toxicities of Pu-erh black tea extract in Sprague–Dawley rats Di Wang a,b , Kunlong Xu c , Ying Zhong a,b , Xiao Luo a,b , Rong Xiao d , Yan Hou e , Wei Bao a,b , Wei Yang a,b , Hong Yan b , Ping Yao a,b , Liegang Liu a,b,∗ a Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, PR China b MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, PR China c College of Animal Science and Technology, Yunnan Agriculture University, Kunming, Yunnan 650201, PR China d College of Food Science, Yunnan Agriculture University, Kunming, Yunnan 650201, PR China e Certificate Assessment Center of Yunnan Pu-erh Tea, Yunnan Agriculture University, Kunming, Yunnan 650201, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2010 Received in revised form 22 September 2010 Accepted 28 November 2010 Available online 4 December 2010 Keywords: Pu-erh black tea extracts Acute toxicity Subchronic toxicity LD50 NOAEL
a b s t r a c t Ethnopharmacological relevance: Pu-erh black tea, which is obtained by first parching crude green tea leaves and then undergoes secondary fermentation with microorganisms, has been believed to be beneficial beverages for health for nearly 2000 years in China, Japan and Taiwan area. But its potential toxicity when administered at a high dose as concentrated extracts has not been completely investigated. The aim of the study: The present study was aimed at evaluating potential toxicity of Pu-erh black tea extracts (BTE) from acute and sub-chronic administration to male and female Sprague–Dawley (SD) rats. Materials and methods: A single BTE dose of 10,000 mg/kg of body weight was administered by oral gavage for acute toxicity in SD rats. Four groups (10 males and 10 females per group) of dose levels of 1250, 2500, and 5000 mg/kg/day of the test article, as well as controls (distilled water) were tested as the subchronic toxicity study. Results: No deaths and signs of toxicity occurred during the 14 days of the study. There were no test article related mortalities, body weight gain, feed consumption, clinical observation, organ weight changes, gross finding, clinical or histopathological alterations during the 91-day administration. Conclusions: The LD50 of BTE can be defined as more than 10,000 mg/kg, and a dose of 5000 mg/kg/day was identified as the no-observed-adverse-effect-level (NOAEL) in this study. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Tea is the most widely consumed as a daily beverage in the world, drunk in the UK for 350 years and in Asia for more than 4000 years. Data on over 7000 adults from the UK National Diet and Nutrition Survey (NDNS) (Henderson et al., 2002), indicate that 77% of people drink tea, with a mean consumption of 2.3 mugs (540 mL) per day. It is believed to have medicinal efficacy in the prevention and treatment of many diseases, and so longevity is often associated with the habit of drinking tea (Crespy and Williamson, 2004; Kao et al., 2006; Nagao et al., 2007). Usually, on the basis of the processing procedures, tea can generally be divided into three types:
∗ Corresponding author at: Department of Nutrition and Food Hygiene, Tongji Medical College, Huazhong University of Science & Technology, 13 Hangkong Road, Wuhan 430030, PR China. Tel.: +86 27 83692711; fax: +86 27 83650522. E-mail address: [email protected] (L. Liu). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.068
nonfermented (green tea), semifermented (oolong tea), and fully fermented (black tea and Pu-erh tea) (Lin et al., 1998). Pu-erh tea, originally produced in the Yunnan province of China for about 1700 years, was recorded by Compendium of Materia Medica that Pu-erh tea can expel wind-evil, clear away heat and aid in lose weight. Pu-erh tea is obtained by first parching crude green tea leaves (Camellia sinensis var. assamica (L.) O. Kuntze; Theaceae) and then undergoes secondary fermentation with microorganisms such as Aspergillus sp. (postfermented) (Jeng et al., 2007a), resulting in a unique type of tea. During the fermentation process, catechins are oxidized into quinone by polyphenol oxidase and then condensed to form bisflavanol, theaflavin, thearubigen, and other high molecular components (Yang and Koo, 1997), otherwise, the Puerh black tea is relatively rich in natural statins (Jeng et al., 2007b). These are regarded as the biologically important active components of Pu-erh black tea and that provide health benefits such as antioxidation, hypocholesterolemia, and anti-obesity (Duh et al., 2004; Fujita and Yamagami, 2008a,b). Therefore, Pu-erh black tea, with
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its old tradition, long history and special favor, is widely consumed by the world. Though the health benefits of tea are accepted by the people all over the word, issues of safety should not be neglected. Based on available data, high dose of green tea and/or associated chemical compounds intake could alter hepatic and thyroid function adversely (Schmidt et al., 2005; Bonkovsky, 2006; Bun et al., 2006; Chandra and De, 2010). Specially, for black tea, which is consumed by nearly 80% of the word people, its potential toxicities have not been got enough attentions until now. Relevant epidemiological and clinical studies published between 1990 and 2004 reported that excess black tea consumption would be harmful. This is associated to caffeine content, which has an adverse effect on fluid balance, cognitive, function, bone health, dental health and iron status. (Gardner et al., 2007). In vitro study prove that black tea polyphenol extracts (Babich et al., 2005), a black tea theaflavin mixture (Babich et al., 2006) and specific theaflavin monomers (Schuck et al., 2008) exhibited both antioxidant and prooxidant properties. Relevant results show that these compounds could lead to morphological changes characteristic of apoptosis both in cancer and normal cells. The possible mechanism could attribute to the generation of excessive reactive oxygen species (ROS) in cell. However, these injuries are rarely observed in vivo with black tea administration. Above all, although the Pu-erh black tea becomes more popular in the worldwide, fewer data about its potential toxicity can be available when administered at a high dose as concentrated extracts or products. In this experiment, commercial Pu-erh black tea samples were selected from Yunnan high land of China, and the toxicity of its extract was evaluated with dietary administration to male and female Sprague–Dawley (SD) rats for acute 14 days and subchronic 91 days as parts of a safety assessment according to the internationally acceptable guidelines for Pu-erh tea consumption.
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2.2. Experimental animals A total of 60 male and 60 female 5-wk-old SD rats were purchased from Sino-British Sippr/BK (Shanghai, China) and used after 1-wk acclimatization. Individual body weights were recorded and detailed physical examinations were performed twice during the acclimation period to ensure the use of healthy animals. Each animal was housed in a cage suspended wiremesh and given free access to commercial laboratory feed and tap water during the nonexposure periods. Animals were feed in a separate room with a barrier system controlled for the light–dark cycle (12–12 h, lights on 7:00–19:00), ventilation (air-exchange rate of 18 times per hour), temperature (23 ± 2 ◦ C) and relative humidity (55 ± 5%) during the study. The cages and the chip bedding were exchanged twice a week. The study was performed in accordance with the guide for the care and use of laboratory animals, prepared by National Institute of Health, USA (Guide for the Care and Use of Laboratory Animals, 1996). 2.3. Single dose acute oral toxicity of BTE in rats The single dose acute oral toxicity study was evaluated following the recommendations by OECD/OCDE (2001). After overnight fasting (8–10 h), groups of 10 male and 10 female SD rats were administered a single dose of 10,000 mg/kg of body weight BTE by oral gavage. The purified water was administered to the control group including the same number of rats with BTE treated group. Feeding was restarted 4 h after dosing. All animals were observed for clinical signs including mortality and moribundity, immediately after dosing and at 1, 2, 4, 8 and 12 h, then twice daily until d 14. Abnormal findings were recorded with the time of onset and disappearance. Body weights and food consumption were measured on d 0, 1, 3, 5, 7, 10 and 14. On the 14th day, all animals were sacrificed and all organs and tissues were observed macroscopically. The abnormal organs were placed in 10% neutral buffered formalin and observed by pathological examination.
2. Materials and methods 2.4. Subchronic 91-day oral toxicity study of BTE 2.1. Tea extracts preparation The commercial Pu-erh black tea samples were collected from Yunnan Highland of China. Voucher specimens of tea (Camellia sinensis var. assamica (L.) O. Kuntze; Theaceae) were collected from agro-forests and deposited at the Kunming Institute of Botany, Chinese Academy of Sciences. The detail processing of Pu-erh black tea was introduced in previous study (Hou et al., 2009). In brief, the crude green tea leaves cultivated in the Yunnan Highlands of China was used as the raw material. Leaves were collected and heated, dried at <60 ◦ C, and molded to make unfermented Pu-erh tea. To make fermented Pu-erh tea, the unfermented Pu-erh tea was dampened and fermented with a pure culture of Aspergillus niger for 50 d at controlled temperature (40–60 ◦ C) and humidity (70–85%). Fermented Pu-erh tea was then dried at <60 ◦ C and packed. The tea sample (100 g) was cut into small pieces, and soaked in boiled distilled water for three times (2 L, 1.5 L and 1.5 L of each bulk, respectively; 20 min, 15 min and 15 min of each time, respectively). After separating from leaves by filtration, the whole extracts were evaporated by a rotary evaporator at 65 ◦ C (Yarong Biochemical Instrument, Shanghai, China) to 100 mL. The final beverages were sterilized at 121 ◦ C for 20 min and kept at 4 ◦ C in a closed container and were designated as 1 g/mL (tea/water) Pu-erh black tea extract (BTE) for later experiments. The contents of biochemical ingredients including total polyphenols, total flavonoid, free amino acid, theaflavins, thearubigins and theabrownins in Pu-erh black tea were analyzed in our prior study (Wang et al., 2010).
2.4.1. Experimental design The subchronic 91-day oral toxicity study was evaluated following the recommendations by OECD/OCDE (1998). Groups of ten males and ten females received doses of 0, 1250, 2500 or 5000 mg/kg bw of BTE at daily gavage of 1 ml/100 g bw for 91 consecutive days. Observations were made twice daily for mortality and changes in general appearance or behavior. The body weights were recorded every week, the individual dose was adjusted for the body weight to maintain the target dose level for all rats. In addition, detailed clinical examination and measurement of food consumption were preformed weekly. The animals were sacrificed by exsanguinations from the abdominal aorta at the d 92 following fasting for 12–16 h. A sample of blood (approximately 20 L) was treated with EDTA-2K to analyze hematological indexes. Serum from blood samples collected in separator tubes was stored at −20 ◦ C. During necropsy, the following organs were dissected: liver, spleen, thymus, heart, lungs, stomach, ovaries, uterus, kidney, adrenals, trachea/thyroid gland, brain, pituitary gland, pancreas, perirenal fat, testes, and epididymis. All organs were visually inspected and weighed directly after dissection to reduce mechanical damage. Defined samples of the liver, brain, pancreas, stomach, kidney, adrenals, testes and ovaries were placed in 10% neutral buffered formalin for pathological examination. 2.4.2. Hematology and blood chemistry One blood sample (approximately 20 L) was treated with EDTA-2K for white blood cells (WBC), red blood cells (RBC),
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hemoglobin (Hb), platelets (PLT), percent of lymphocytes (LY), percent of monocytes (MO), percent of granulocyte (GR), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean platelet volume (MPV), Platelet hematocrit (PCT), platelet distribution width (PDW) and red blood cell distribution width (RDW) using a hematology analyzer MEK-6318K (Nihon Kohden Co., Ltd.). Serum from blood samples collected in separator tubes was measured using a BS-200 automatic biochemistry analyzer (Mindary Co., Ltd.) including aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, creatinine (Cr), total cholesterol (TC), triglyceride (TG), total protein (TP), albumin (Alb) and glucose (Glu). Ionized calcium (iCa) and total calcium (TCa) using the 7020 automatic biochemistry analyzer (Hitachi Co., Ltd.), and for sodium (Na), potassium (K), chloride (Cl) and pH value analyses. 2.4.3. Urinalysis The 24-h pooled urine of each animal was collected by metabolism cage and volume was recorded at week 13. The fresh urine was examined for specific gravity, pH, protein, glucose, acetone body, bilirubin and urobilinogen by an UA-66 urine analyzer (Mindary Co., Ltd.). 2.4.4. Histopathological examination Samples of the liver, brain, pancreas, stomach, kidney, adrenals, testes and ovaries in every group were placed in 10% neutral buffered formalin, sectioned and stained with hematoxylin and eosin (H&E). 2.5. Statistical analysis Variance in data for body and organ weights as well as the results of hematology, and serum biochemistry was checked for homogeneity by Bartlett’s procedure. When the data were homogeneous, one-way analysis of variance for homogeneity (ANOVA) was used. In the heterogeneous cases, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the Dunnett’s multiple tests were employed for comparisons between control and treated groups. All statistical analyses were performed using SPSS 12.0 statistical software (SPSS Inc., Chicago, IL) and a difference was considered significant when P < 0.05. 3. Results 3.1. Single dose acute oral toxicity study of BTE in SD rats No deaths occurred during the 14 d of the study. There were no significant differences in body weights and food consumption of either sex between the BTE administrated group and the controls (data not shown). A short time of sluggish appeared in some BTE treated rats after oral gavage in the first hour, but the rats return to life later. Otherwise, there were no abnormal findings at other clinical signs and autopsy in the experimental or control group in either sex. Based on these, the LD50 value of BTE was greater than 10,000 mg/kg for both sexes. 3.2. Subchronic 91-day oral toxicity study of BTE in SD rats 3.2.1. Clinical signs and mortality There was no mortality attributed to any effect of BTE during the 91-day administration. One female (8th wk) given BTE with dose of 2500 mg/kg/day and two females with 1250 mg/kg/day (8th and 9th wk, respectively), and one male rat with 5000 mg/kg/day
(9th wk) died during the 13-wk oral study could be due to inadvertent gavage accidents. The body weights and food consumption of these rats were not obviously decreased as compared to other rats in the same group before death. Clinical observations in these animals prior to death consisted of intermittent convulsions, gasping, hypoactivity, labored respiration, and prostration. Anatomical results showed that no abnormal organic damages were observed in the dead rats. Moreover, there were no treatmentrelated changes at autopsy in the BTE group or control group in either sex.
3.2.2. Body weights and food consumption Mean body weights for male and female rats at 1250, 2500 and 5000 mg/kg/day were compared to control values (Fig. 1). Female SD rats in all BTE groups had significantly lower mean body weights (254.6 g, 257.7 g and 253.8 g in 5000, 2500 and 1250 mg/kg/day group, respectively) as compared to the control group (281.3 g). The mean body weights of male rats in 5000 mg/kg/day and 2500 mg/kg/day BTE groups were significantly lower than the control group (332.6 g vs 438.3 g and 392.3 g vs 438.3 g, respectively) at the thirteenth week (P < 0.01 or P < 0.05). During the thirteen-wk study, there were no differences in mean food consumption at any other observation points in any group. The data about mean daily food consumption in BTE treated groups and control group is shown in Fig. 2.
3.2.3. Organ weights The results for relative organ weights were shown in Tables 1a and 1b. In females, the relative organ weights of heart and liver in 5000 mg/kg/day group were significantly increased as compared to controls, while the relative pancreas weight was significantly lower than controls. In addition, significant decrease of relative spleen weight was found in the 2500 mg/kg/day group. In males, the relative organ weights of brain, stomach, liver and testes were significantly increased in 5000 mg/kg/day group. The perirenal fat, which was an important visceral fat in body, was also removed and weighed in present study. A dose-dependent decrease in relative perirenal fat was observed in 5000 and 2500 mg/kg/day groups both in females and males.
3.2.4. Hematology and blood chemistry In hematology parameters, statistically significant decrease and increase were observed in RBC and MCHC with dose of 5000 mg/kg/day in female rats, respectively (Table 2a). There were no significant differences between the treatment and control groups in males (Table 2b). In blood serum biochemistry of females (Table 3a), a dose-dependent decrease in TG was observed in 5000 and 2500 mg/kg/day group. Statistically significantly, the TC level in 5000 mg/kg/day was decreased as compared with control group. A slight increase was observed in high dose group, but the difference was not significant. For males (Table 3b), TC and TG were significantly decreased in 5000 mg/kg/day. Statistically significant increase of ALT in females administrated 5000 mg/kg/day was observed. Beyond that, there were no treatment-related or statistically significant effects in metallic ion and pH value.
3.2.5. Urinalysis Statistically significant decrease in 24-h urine volume in males administered 5000 mg/kg/day was observed as compared with controls (data not shown). Thus, the changes observed in urinalysis were considered to be unrelated to the treatment and no adverse effects were found at the same test doses in females and had no other clinical pathology correlation.
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159
Female 350.0
Body weights (g)
BTE 5000 mg/kg/day
300.0
BTE 2500 mg/kg/day BTE 1250 mg/kg/day Control 0 mg/kg/day
250.0 200.0 150.0 100.0 50.0
week0 week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Male 500.0
Body weights (g)
450.0
BTE 5000 mg/kg/day BTE 2500 mg/kg/day
400.0
BTE 1250 mg/kg/day
350.0
Control 0 mg/kg/day
300.0 250.0 200.0 150.0 100.0 50.0 week0 week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Fig. 1. Effects of oral administration of Pu-erh black tea extract for 13 weeks on mean body weights. Body weight was calculated and data are expressed as mean ± SEM. BTE = Pu-erh black tea extract.
Table 1a Effects of oral administration of Pu-erh black tea extract for 13 weeks on relative organ weight for female SD rats. Organs
Dose (mg/kg/day) 5000 (n = 10) a
Body weights Brainb Cerebellumb Brainstemb Pituitaryb Heartb Thyroids/parathyroidsb Thymusb Liverb Spleenb Lungb Pancreasb Stomachb Kidneysb Adrenalsb Uterusb Ovaryb Perirenal fatb
254.6 4.8 0.1 0.9 0.1 4.1 0.1 1.4 32.8 2.4 7.3 2.2 6.6 6.8 0.3 2.0 0.6 2.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
**
4.8 0.3 0.0 0.1 0.0 0.2* 0.0 0.1 1.7* 0.1 0.5 0.2* 0.1 0.2 0.0 0.3 0.0 0.2*
2500 (n = 9) 257.7 4.7 0.1 0.9 0.1 3.4 0.1 1.4 26.7 2.2 6.5 2.5 5.7 6.3 0.3 1.7 0.5 3.7
Mean organ-to-body weight ratio was calculated and data are expressed as mean ± SEM. a Unit: g. b Unit: ‰ body weights. * Statistically significant compared to controls (P < 0.05). ** Statistically significant compared to controls (P < 0.01).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1250 (n = 8) **
6.8 0.2 0.0 0.1 0.0 0.1 0.0 0.2 0.9 0.1* 0.4 0.2 0.2 0.1 0.0 0.3 0.0 0.4*
253.8 4.8 0.1 0.9 0.1 3.6 0.1 1.9 28.4 2.6 6.5 2.4 6.2 6.8 0.3 2.2 0.5 5.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0 (n = 10) **
10.7 0.1 0.0 0.1 0.0* 0.1 0.0 0.1 0.7 0.3 0.4 0.2 0.3 0.3 0.0 0.2 0.0 2.0
281.3 4.3 0.1 1.1 0.1 3.6 0.1 1.5 26.6 2.5 5.4 2.5 5.5 6.1 0.3 2.5 0.5 6.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.5 0.2 0.0 0.1 0.0 0.1 0.0 0.1 0.8 0.2 0.3 0.2 0.4 0.5 0.0 0.5 0.0 1.0
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Female Food Consumption (g/day)
40.0
BTE 5000 mg/kg/day BTE 2500 mg/kg/day BTE 1250 mg/kg/day Control 0 mg/kg/da y
30.0
20.0
10.0
0.0
week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Male
Food Consumption (g/day)
40.0 BTE 5000 mg/kg/day BTE 2500 mg/kg/day
35.0
BTE 1250 mg/kg/day Control 0 mg/kg/day
30.0 25.0 20.0 15.0 10.0
week1 week2 week3 week4 week5 week6 week7 week8 week9 week10week11week12week13
Fig. 2. Mean food consumption for SD rats during a 13-week oral (gavage) toxicity study with Pu-erh tea extract. Food consumption was calculated and data are expressed as mean ± SEM. BTE = Pu-erh black tea extract.
Table 1b Effects of oral administration of Pu-erh black tea extract for 13 weeks on relative organ weight for male SD rats. Organs
Dose (mg/kg/day) 5000 (n = 9)
Body weightsa Brainb Cerebellumb Brainstemb Pituitaryb Heartb Thyroids/parathyroidsb Thymusb Liverb Spleenb Lungb Pancreasb Stomachb Kidneysb Adrenalsb Testesb Epididymidesb Perirenal fatb
337.7 3.7 0.5 0.8 0.5 3.7 0.2 0.9 34.4 1.7 7.0 2.3 5.8 7.1 0.2 7.7 1.8 4.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
11.2** 0.2* 0.1 0.1 0.1 0.1 0.1 0.1 2.6* 0.1 1.1 0.3 0.4* 0.3 0.0 0.3* 0.2 1.1**
2500 (n = 10) 392.3 3.2 0.6 0.8 0.6 3.4 0.4 1.1 26.5 1.8 5.6 1.8 4.7 6.6 0.3 7.1 1.9 5.4
Mean organ-to-body weight ratio was calculated and data are expressed as mean ± SEM. a Unit: g. b Unit: ‰ body weights. * Statistically significant compared to controls (P < 0.05). ** Statistically significant compared to controls (P < 0.01).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
14.9* 0.1 0.1 0.1 0.1 0.2 0.0 0.1 1.3 0.1 0.3 0.1 0.1 0.2 0.1 0.3 0.2 0.9**
1250 (n = 10) 421.4 3.1 0.6 0.6 0.6 3.3 0.5 1.0 28.1 1.8 5.2 2.2 4.3 6.7 0.2 6.6 1.9 8.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
11.5 0.1 0.1 0.1 0.1 0.2 0.1 0.1 1.7 0.1 0.6 0.2 0.1 0.2 0.0 0.3 0.2 0.5
0 (n = 10) 438.3 3.1 0.6 0.6 0.6 3.3 0.5 1.1 25.2 1.8 5.2 2.0 4.5 6.7 0.2 6.6 2.1 10.8
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
12.4 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.3 0.1 0.2 0.0 0.2 0.3 1.4
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Table 2a Hematological data for female rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 10)
WBC (thous/L) RBC (millions/L) HGB (g/dL) PLT (thous/L) LY (%) MO (%) GR (%) HCT (%) MCH (pg) MCHC (g/dL) MCV (fl) MPV (fl) PCT (%) PDW (fl) RDW (fl)
16.06 8.27 15.42 359.50 63.32 5.68 21.00 43.58 17.12 37.06 51.62 9.91 0.29 12.50 13.31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.93 0.54* 1.44 39.71 10.43 1.68 9.30 3.26 0.92 1.26* 1.90 0.78 0.133 0.59 0.75
2500 (n = 9) 16.52 9.27 16.65 396.50 70.33 4.37 26.17 46.47 17.97 35.85 50.11 9.55 0.37 12.16 12.93
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.21 0.90 1.66 80.13 6.87 1.35 6.59 4.56 0.81 1.37 0.82 0.64 0.09 0.54 1.04
1250 (n = 8) 15.14 9.01 16.36 356.56 61.77 5.59 32.64 45.58 18.14 35.88 50.61 9.50 0.34 12.21 12.77
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.08 0.93 1.71 53.32 11.89 1.17 11.70 4.30 0.99 11.83 1.49 0.71 0.07 0.93 0.59
0 (n = 10) 13.17 8.84 15.68 339.50 70.44 5.78 23.78 44.32 17.74 35.39 50.14 9.70 0.32 12.22 12.69
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.77 0.41 0.91 46.71 12.71 1.43 12.38 2.32 0.60 13.3 1.34 0.67 0.05 0.86 0.60
Values are presented as mean ± SEM. * Significantly different from the controls at levels of P < 0.05.
Table 2b Hematological data for male rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 9)
WBC (thous/L) RBC (millions/L) HGB (g/dL) PLT (thous/L) LY (%) MO (%) GR (%) HCT (%) MCH (pg) MCHC (g/dL) MCV (fl) MPV (fl) PCT (%) PDW (fl) RDW (fl)
16.20 8.37 15.43 329.30 63.79 6.51 25.98 42.77 18.43 36.12 51.06 9.50 0.31 12.42 13.44
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2500 (n = 10)
7.23 0.47 0.92 57.37 9.92 3.11 13.06 2.94 0.78 1.35 1.69 0.40 0.05 0.90 1.10
16.59 8.45 15.88 373.30 63.00 6.17 27.55 43.40 18.77 36.53 51.38 9.44 0.35 12.15 13.28
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.90 0.69 1.57 39.42 12.77 3.31 15.69 3.38 0.84 1.04* 1.41 0.44 0.05 0.71 0.98
1250 (n = 10) 15.96 8.39 15.52 340.90 72.79 6.26 20.95 43.07 18.55 36.13 51.28 9.45 0.32 11.99 13.28
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.63 1.00 1.58 35.33 9.74 2.22 8.52 5.33 0.61 1.16 1.08 0.25 0.04 0.78 1.06
0 (n = 10) 13.22 7.98 14.45 342.10 70.90 5.34 23.76 41.13 18.14 35.15 51.60 9.36 0.32 12.32 13.36
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.72 0.85 1.54 66.11 10.28 1.37 9.63 4.40 0.72 1.08 1.46 0.33 0.06 0.79 1.20
Values are presented as mean ± SEM. * Significantly different from the controls at levels of P < 0.05.
Table 3a Serum biochemical data for female rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 10)
TC (mg/dL) TG (mmol/L) Glu (mg/dL) TP (mg/L) Alb (mg/L) ALT (U/L) AST (U/L) Cre (mmol/L) Urea (mmol/L) K (mmol/L) Na (mmol/L) Cl (mmol/L) iCa (mmol/L) TCa (mmol/L) pH
69.87 0.30 98.74 80.13 35.79 51.56 141.15 43.04 6.07 7.94 144.16 108.43 1.26 2.45 7.66
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2500 (n = 9) *
2.38 0.03** 9.67 1.79 2.26 4.27 4.62 2.42 0.33 0.17 0.83 0.75 0.04 0.08 0.01
Values are presented as mean ± SEM. * Statistically significant compared to controls (P < 0.05). ** Statistically significant compared to controls (P < 0.01).
80.24 0.33 97.40 77.50 39.34 48.50 122.53 42.00 5.52 7.83 142.18 107.50 1.27 2.48 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.90 0.01* 9.31 1.50 1.87 2.68 8.71 1.32 0.28 0.20 0.49 0.52 0.02 0.04 0.01
1250 (n = 8) 88.71 0.38 109.42 87.47 37.92 41.96 129.80 42.47 5.51 7.47 143.57 108.51 1.29 2.54 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.71 0.01 14.61 2.54 0.71 3.44 8.27 1.84 0.51 0.29 1.32 1.05 0.02 0.05 0.01
0 (n = 10) 89.46 0.40 103.58 83.98 38.74 42.88 142.26 43.20 5.64 7.44 143.65 108.27 1.31 2.55 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.98 0.01 10.82 2.28 1.04 2.62 9.23 2.49 0.27 0.29 0.88 0.88 0.02 0.04 0.01
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Table 3b Serum biochemical data for male rats administered with Pu-erh black tea extract for 13 weeks. Parameter
Dose (mg/kg/day) 5000 (n = 9)
TC (mg/dL) TG (mmol/L) Glu (mg/dL) TP (mg/L) Alb (mg/L) ALT (U/L) AST (U/L) Cre (mmol/L) Urea (mmol/L) K (mmol/L) Na (mmol/L) Cl (mmol/L) iCa (mmol/L) TCa (mmol/L) pH
76.32 0.31 103.42 70.13 35.79 49.70 124.09 42.01 5.95 7.99 142.77 105.60 1.34 2.62 7.65
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.67* 0.02* 7.68 2.16 2.26 2.17* 7.56 1.80 0.26 0.14 0.68 0.95 0.04 0.08 0.01
2500 (n = 10) 83.41 0.33 110.53 80.32 39.34 44.36 101.89 42.23 5.84 8.14 143.61 107.70 1.30 2.53 7.66
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.27 0.05 7.43 1.75 1.87 1.94 8.52 2.34 0.21 0.35 0.46 0.55 0.02 0.05 0.01
1250 (n = 10) 80.18 0.44 109.34 80.58 37.92 46.06 116.41 42.39 5.66 8.11 145.34 108.97 1.32 2.57 7.65
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.32 0.05 9.87 1.88 0.71 1.02 2.72 2.46 0.21 0.39 0.63 0.62 0.02 0.03 0.01
0 (n = 10) 86.74 0.46 115.72 76.19 38.74 39.70 106.27 41.47 4.81 8.12 144.39 108.88 1.30 2.54 7.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.32 0.05 13.20 1.25 1.02 1.61 5.99 1.68 0.20 0.26 0.32 0.42 0.03 0.06 0.01
Values are presented as mean ± SEM. * Statistically significant compared to controls (P < 0.05).
3.2.6. Gross necropsy and histopathology There were no macroscopic observations considered to be treatment related. No gross abnormalities attributed to the test article were noted for any of the euthanized animals necropsied at the conclusion of the 92-day observation period. Histopathological changes including minimal bile-duct hyperplasia, vacuolation, cellullar swelling, spotty necrosis and inflammation were observed in liver. For kidney, incidence of inflammation, epithelium swelling appeared. Thus these changes were not considered to be treatment related in this study, because these microscopic changes were commonly observed in untreated laboratory animals and were of comparable incidence and severity between test article-treated and control animals. 4. Discussion Pu-erh black tea, which is planted in Yunnan High Land of P.R. China, has been consumed widely by people living in southern China, Taiwan Area and Japan. As a unique type of tea, the chemical contents in Pu-erh black tea are different to other teas. Our prior study found that the catechins are easily oxidized by polyphenol oxidase, and further polymerizations lead to theaflavins (0.17%), thearubigins (5.88%), and theabrownins (9.73%), the contents of other smaller molecular-weight compounds such as amino acids were also lower than green tea during the special fermentation (Wang et al., 2010). The present study demonstrated the comprehensive safety profile of BTE in acute and subchronic oral toxicity study in SD rats. Exposure of SD rats in single dose of BTE did not produce any treatment-related effect. A number of changes including body and organ weights, parameters in hematology, serum biochemistry and histopathology were observed after subchronic exposure of BTE, but the changes were not toxicity-related. All rats at each dosage group continued to gain weight throughout the course of 91-day toxicological study. But the body weight gains in all BTE administration groups were significantly lower than controls except the 1250 mg/kg/day BTE male group. Additionally, a dose-dependent decrease in relative perirenal fat weights was observed in 5000 and 2500 mg/kg/day BTE administrated groups with both sexes. However, the food consumption results did not show significant differences in weekly food intake between BTE treatment groups and controls. The results in our study were similar to previous studies which reported that a growth depression is induced when Pu-erh black tea was given in excess to growing animals. In Jen-kun Lin’s study (Kuo et al., 2005), the body weights of rats placed on a basal diet had decreased from 5 wk to 30 wk
after given with 4% green tea, 1.5% and 4% Pu-erh tea. Michael and colleague (Fallon et al., 2008) found that fecal fat was significantly increased to approximately 15 percent in rats and body weights slightly increased receiving 3.0- and 6.0- black and green tea and mulberry mixture extracts. However, the mechanisms leading to the depression of body weight gain induced by Pu-erh black tea remain largely unknown. In vitro studies showed that black tea rich in theaflavin, which can inhibit the activity of digest enzymes, such as pancreatic lipase and salivary amylase (Zhang and Kashket, 1998; Nakai et al., 2005). Although the same conclusion has not been verified in vivo, promoting fat metabolism and inhibiting excess fatty deposits could be taken as presumptive evidence of anti-obesity effect of Pu-erh black tea. No untoward toxicity-related changes were observed for hematology parameters in male and female rats. The RBC level was significantly decreased and MCHC was increased in 5000 mg/kg/day female rats as compared to controls, but the changes were regarded as toxicologically irrelevant because the adverse results did not appear in both sexes and/or were without dose-relation. Significant increase in ALT in 5000 mg/kg/day BTE males and relative liver weights in males and females were presumably at least partly associated with hepatic changes. Though slight hepatotoxicity was observed in some studies about green tea extracts (Bonkovsky, 2006; Bun et al., 2006), separated chemicals i.e. polyphenols (Schmidt et al., 2005; Kapetanovic et al., 2009) and mixture containing black tea composition (Fallon et al., 2008), few vivo studies showed the similar hepatotoxicity in whatever single constituent in black tea or black tea extracts related experiments. As to Pu-erh black tea, A. niger was claimed to be the dominant microorganism in the Pu-erh tea manufacturing process and known to produce the mycotoxins i.e. fumonisins B2 and B4 (Noonim et al., 2009; Mogensen et al., 2010). Certain evidences show that exposure tomycotoxins in humans and animals can results in liver failure and death (Yoshida et al., 2001). However, neither production of tomycotoxins of the 47 isolates of Pu-erh tea and black tea were observed in Mogensen’s study (Mogensen et al., 2009). Therefore, increase of ALT and relative liver weight in the 5000 mg/kg/day group in males but without histopathological lesions and other injury-related evidences in the liver cannot be seen as a toxicity-related effect of BTE in present study. Other abnormal changes, like decrease in TC in 5000 mg/kg/day BTE treated group, TG in 5000 and 2500 mg/kg/day males and 5000 mg/kg/day females, were associated with the biological functions but toxicities. Previous studies had also proved that Pu-erh fermented tea cannot only significantly decrease the serum cholesterol levels in a hyperlipidemia rat model (Hou et al.,
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2009) but also slightly decrease the TC level during the 30-wk oral feeding in normal diet rats (Kuo et al., 2005). Relative liver weights were significantly increased in 5000 mg/kg/day BTE treated groups of both sexes. However, there was no microscopic correlate to the increase in mean liver weights in males and females at this dose. The other absolute and/or relative weights changes were observed in some organs, such as heart and pancreas in females with 5000 mg/kg/day BTE or brain, stomach and testes in males with 5000 mg/kg/day, they were not considered toxic-related since they were only observed in one sex, and did not exhibit a dose–response relationship. No treatment related microscopic changes were observed in this study. All microscopic changes observed in the liver and kidney were randomly distributed between test article-treated and control animals, and the incidences were within the range of normal background lesions. In conclusion, the high single dose administration of BTE (10,000 mg/kg of body weight) did not show acute oral toxicity in SD rats in present study. Based on common classification of the relative toxicity of chemicals, BTE probably belongs to the slightly toxic (LD50 : 5–15 g/kg) (Lu and Kacew, 2002). In the subchronic 13-wk oral toxicity test, no obvious toxic changes which due to administration of BTE were observed in any parameters. Therefore, the NOAEL in this subchronic toxicity study was 5000 mg/kg/day for both sexes in SD rats. In accordance with recommendations and considering an average consumption of five cups (2.5 g/cup) per person per day, the percentage of the average daily intake of Puerh black tea was about 178.6 mg/kg/day for adult (70 kg of body weight). The NOAEL level of BTE in present study is 28 times higher than the average intake by human, the present study suggested that high dose (5000 mg/kg/day) of BTE intake would be well-tolerated for long-term use as a dietary supplement whatever by animals or human. Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgements We appreciate the contribution of all the members participating in this study. We also wish to thank Dr. Yajun Du (Office of Health Assessment and Epidemiology, Department of Public Health, Los Angeles Country, USA). This work was supported by the National Key Technology R&D Program of the People’s Republic of China (2007BAD58B05). References Babich, H., Gold, T., Gold, R., 2005. Mediation of the in vitro cytotoxicity of green and black tea polyphenols by cobalt chloride. Toxicology Letters 155, 195–205. Babich, H., Pinsky, S.M., Muskin, E.T., Zuckerbraun, H.L., 2006. In vitro cytotoxicity of a theaflavin mixture from black tea to malignant, immortalized, and normal cells from the human oral cavity. Toxicology In Vitro 20, 677–688. Bonkovsky, H.L., 2006. Hepatotoxicity associated with supplements containing Chinese green tea (Camellia sinensis). Annals of Internal Medicine 144, 68–71. Bun, S.S., Bun, H., Guedon, D., Rosier, C., Ollivier, E., 2006. Effect of green tea extracts on liver functions in Wistar rats. Food and Chemical Toxicology 44, 1108–1113. Chandra, A.K., De, N., 2010. Goitrogenic/antithyroidal potential of green tea extract in relation to catechin in rats. Food and Chemical Toxicology. Crespy, V., Williamson, G., 2004. A review of the health effects of green tea catechins in in vivo animal models. The Journal of Nutrition 134, 3431S–3440S. Duh, P.D., Yen, G.C., Yen, W.J., Wang, B.S., Chang, L.W., 2004. Effects of Pu-erh tea on oxidative damage and nitric oxide scavenging. Journal of Agricultural and Food Chemistry 52, 8169–8176. Fallon, E., Zhong, L., Furne, J.K., Levitt, M., 2008. A mixture of extracts of black and green teas and mulberry leaf did not reduce weight gain in rats fed a high-fat diet. Alternative Medicine Review 13, 43–49. Fujita, H., Yamagami, T., 2008a. Efficacy and safety of Chinese black tea (Pu-Ehr) extract in healthy and hypercholesterolemic subjects. Annals of Nutrition and Metabolism 53, 33–42.
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Glossary ALT: alanine aminotransferase Alb: albumin AST: aspartate aminotransferase Cl: chloride Cr: creatinine Glu: glucose GA: gallic acid
164 HCT: hematocrit H&E: hematoxylin and eosin Hb: hemoglobin iCa: ionized calcium NDNS: National Diet and Nutrition Survey NOAEL: no-observed-adverse-effect-level ANOVA: one-way analysis of variance PLT: platelets PDW: platelet distribution width PCT: platelet hematocrit GR: percent of granulocyte LY: percent of lymphocytes MO: percent of monocytes
D. Wang et al. / Journal of Ethnopharmacology 134 (2011) 156–164 K: potassium BTE: Pu-erh black tea extract ROS: reactive oxygen species RDW: red blood cell distribution width RBC: red blood cells Na: sodium SD: Sprague–Dawley TC: total cholesterol TG: triglyceride TP: total protein TCa: total calcium Urea: urea nitrogen WBC: white blood cells