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Safety assessment of Cordyceps guangdongensis
Food and Chemical Toxicology 48 (2010) 3080–3084
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Safety assessment of Cordyceps guangdongensis Wen-juan Yan a,b, Tai-Hui Li b,⇑, Qun-ying Lin b, Bin Song b, Zi-de Jiang a,⇑⇑ a b
South China Agricultural University, Guangzhou 510642, China Guangdong Institute of Microbiology, Guangzhou 510070, China
a r t i c l e
i n f o
Article history: Received 29 December 2009 Accepted 3 August 2010
Keywords: Cordyceps guangdongensis Toxicological assessments
a b s t r a c t Cordyceps guangdongensis as a kind of fungus, has been discovered and cultivated successfully in recent years. However, its safety assessments have not been studied. In this report, a serial of tests for toxicological safety assessments were depicted in details. These tests included bacterial reverse mutation (Ames) study, bone marrow cell micronucleus test in mice, sperm aberration test in mice, teratogenicaction test in rats, acute toxicity test and 13-week oral toxicity study in rats. After a profound analysis of these tests, it clearly demonstrated that C. guangdongensis did not have any mutagenic, clastogenic nor genotoxic effects; the oral LD50 of the biomass in rats was greater than 15 g/kg body weight; the no-observed adverse-effect-levels (NOAEL) was 5.33 g/kg body weight according to the 13-week oral toxicity analysis. Therefore, a conclusion can be drawn that C. guangdongensis is considered safe for long term consumption. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cordyceps (Fr.) Link is a wide spread genus which usually grows on insects or subterranean fungi. Modern scientific studies have shown that certain species of Cordyceps possess a wide spectrum of biological activities, including antioxidation (Yamaguchi et al., 2000; Li et al., 2001), antitumor (Ohmori et al., 1986; Chen et al., 1997; Shin et al., 2003), immuno-potentiation (Xu et al., 1992; Shin et al., 2003), antiinflammation (Kim et al., 2003) and stimulation of testosterone biosynthesis (Huang et al., 2001). And they also have been used to cure many diseases, such as hyposexuality and hyperlipidemia (Kuo et al., 2006; Chen et al., 2006; Wang and Shiao, 2000). One of the most famous species is Cordyceps sinensis (Berk.) Sacc., which has been used as a healthy food supplement and traditional Chinese herb for thousands of years. Additionally, some of
other species of Cordyceps also have highly potential value for utilization, as well as some commercial products of them which are available in the market nowadays such as Didanosine from Cordyceps militaris. (Russell and Paterson, 2008). Cordyceps guangdongensis T.H. Li, Q.Y. Lin & B.Song, which discovered in South China recently (Lin et al., 2008), has been successfully cultivated. It has many similar or same ingredients to those of C. sinensis, including high contents of cordycepic acid and cordycepin. To explore the possibilities of using this fungus further, we have performed a number of tests related to safety assessments, including bacterial reverse mutation (Ames) study, bone marrow cell micronucleus test, acute toxicity test, sperm aberration test, teratogenicaction test and 13-week oral toxicity study and demonstrated that C. guangdongensis can be safely used as a food supplement.
2. Materials and methods 2.1. Study design Abbreviations: A/G, albumin to globulin ratio; Alb, albumin; ALT, alanine transaminase; AST, aspartate transaminase; BA, basophils; bw, bodyweight; BUN, blood urea nitrogen; CFU, colony forming unit; CP, cyclophosphamide; CRE, creatinine; EOS, eosinophils; GLU, glucose; HGB, hemoglobin; LD50, lethal dose; LY, lymphocytes; MCT, mercury cadmium telluride; MO, monocytes; NOAEL, noobserved adverse-effect-level; NIH, National Institutes of Health; PCB, polychlorobiphenyl; PLT, platelet count; RBC, red blood cells; SD, standard deviation; SPF, specific pathogen free; T-CHO, total cholesterol; TG, Triglycerides; UV, ultraviolet; TP, total protein; WBC, white blood cells. ⇑ Corresponding author. Tel./fax: +86 20 87688102. ⇑⇑ Corresponding author. Tel./fax: +86 20 38604779. E-mail addresses: [email protected] (T.-H. Li), [email protected] (Z.-d. Jiang). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.08.001
The study was conducted according to the guidelines of ‘‘Health Food Products Testing and Evaluation of Technical Specifications” (2003 Version) issued by Ministry of Health of the People’s Republic of China : the bacterial reverse mutation (Ames) test was conducted with PCB induced rat liver S9; the micronucleus assay, acute oral toxicity study and sperm aberration test were performed in mice; the teratogenicaction test and 13-week repeated-dose toxicity study were performed in rats.
2.2. Test substance The test substance, cultivated fruitbody of C. guangdongensis (CG), was crushed and provided by Guangdong Institute of Microbiology. The doses varied slightly from batch to batch and would be reported for each study performed.
W.-j. Yan et al. / Food and Chemical Toxicology 48 (2010) 3080–3084 2.3. Bacterial reverse mutation (Ames) study Four strains of Salmonella typhimurium (TA97, TA98, TA100, TA102) and the PCB induced rat liver S9 were offered by Guangdong Center of Disease Control and Prevention. The study was performed both with and without the S9 activation system. And doses used in this study were: 8, 40, 200, 1000 and 5000 lg/plate. Experiments were performed in triplicate. Positive controls in the experiment without S9 activation included three parts: fenaminosulf for strain TA97 and TA98, NaN2 for strains TA100, as well as mitomycin C for strains TA102. Positive controls in the experiments with S9 activation were composed of 2-FA for strains TA97, TA98 and TA100, 1,8-dihydroxyanthraquinone for strains TA102. All strains were tested for spontaneous revertant colonies using distilled water as a negative control. Frozen stock cultures were inoculated and grown overnight at 37 ± 1 °C. In brief, sterile culture tubes were filled with 0.1 ml of test strains, 0.1 ml of test article culture or control, as well as 0.5 ml of S9/cofactor mix. First, the mixture was spread onto minimal glucose plates and incubated at 37 ± 1 °C for 48 h. Second, the number of revertant colonies per plate was determined by hand counting. If the outcome of this experiment was three times over the background average number of revertant colonies on a plate (two times for negative control), or if there was a dose-related increase in colonies, the test was considered as positive. On the contrary if the output did not reach to the criteria, the test article was considered as non-mutagenic. 2.4. Micronucleus assay in mice The mice of strain NIH, which were both male and female with the age of 7– 8 weeks, were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China). At the beginning of the study, the body weights of the mice were 27.3 ± 2.7 g. The mice were randomly assigned to five groups, 10 per group, which could get the drinking water ad libitum. During this experiments, water for injection was utilized as the negative control, while 50 mg/kg bw CP served as the positive control. The test article was administered by oral gavage at a dose of 0.5 ml/10 g bw, except for the positive and negative controls which were administered only once by intraperitoneal injection on the last test article administration day. Doses of the test article, included 2.5, 5 and 10 g/kg bw, were given for 30 h. Blood samples were obtained by tail trimming approximately 48 h after the last administration. Samples were smeared on acridine orange-coated slides, and 1000 reticulocytes were scored under a fluorescence microscope for the presence of micronuclei and the slides were blind coded. The proportion of reticulocytes to total erythrocytes was an indicator to evaluate bone marrow toxicity. Percentages were determined by flow cytometry and were based on analysis of 200 erythrocytes. 2.5. Acute oral toxicity study in mice NIH mice were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China), and their body weights were 19.7 ± 2.3 g at the beginning of the experiments. The mice were assigned randomly to two groups of 10, which could get the tap water ad libitum. Cordyceps guangdongensis was administered to mice at doses of 15.0, 10.0, 4.64, 2.15 and 1.00 g/kg BW. The test article was administered by oral gavage at a dose of 0.35 ml/10 g bw three times at 4 h intervals after 16 h of non-food obtained. During the experiments, the animals were monitored daily for 7 days and were euthanized at the end of the tests for macroscopic examinations. 2.6. Sperm aberration test in mice NIH male mice were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China), and their body weights were 29.5 ± 5.3 g at the beginning of the study. The mice were randomly assigned to five groups, five per group. According to the results of acute oral toxicity obtained above, doses of the test article were designed and included 2.5, 5 and 10 g/kg bw. Distilled water served as the negative control, while 50 mg/kg bw CP served as the positive control. The test article was administered by oral gavage at a dose of 0.5 ml/10 g bw once a day for 5 days. Both sides of the epididymis were obtained by tail trimming approximately 35 days after the first administration. Samples were smeared on acridine orange-coated slides, and 1000 sperms were examined under a microscope for their number and type. The incidence of sperm malformation was calculated and analyzed by wilcoson rank and testing methods. 2.7. Teratogenicaction test in rats SD rats were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China). The pregnant rats were given the test article once a day between days 7 and 16 after mating, and their body weights were 228.3 ± 22.7 g at the beginning of the study. Distilled water was used as the negative control, doses of CG were 1.25, 2.50 and 5 g/kg bw. At the end of the treatments,
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the average numbers of living embryo, dead fetus and absorbed fetus were counted and compared with the negative control. Visual, bone and organ examination of rat embryo were performed. 2.8. Thirteen-weeks repeated-dose toxicity study in rats Eighty-four-week-old SD rats (40 male and 40 female) were purchased from The Laboratory Animal Center of Southern Medical University (Guangzhou, China), and used in this study after a 3-day acclimation period. The experiments were controlled in the conditions as follows: the room was maintained on a 12-h light–dark cycle, at the temperature of 22.0 ± 1 °C with a relative humidity of 60–80%. Each group was consisted of 20 rats (10 male and 10 female), whose bodyweights were 74.3 ± 4.5 g for male, and 72.4 ± 4.3 g for female. All rats were assigned averagely to four groups, and one of them was the control group. C. guangdongensis was administered orally by incorporation in the feed at a concentration of 3.33%, 6.67% and 10%. The casein was incorporated at a concentration of 22% into the diet. Compared to the animals in control group, the low, middle and high dose group animals received C. guangdongensis at doses of 2.67, 5.33 and 8.00 g/kg bw/day in their diet. Weekly adjustments were made for body weight changes. Clinical observations were made once daily following treatment at approximately the same time each day. All animals were checked for general conditions once a week. Body weights were measured at the initiation of the experiments, and were measured every other week. At the scheduled termination, final body weights were recorded after an overnight fast (approximately 16 h). Throughout the experiments, food was weighed weekly, and the average food consumption per animal was calculated twice at weekly intervals. At the end of the treatment period, blood samples for hematology and clinical chemistry were taken at necropsy from all surviving animals via the abdominal aorta under anesthesia after an overnight fast. The following hematological parameters were measured on samples collected by EDTA-2K, which was used as an anticoagulant: red blood cell count (RBC), white blood cell count (WBC), hemoglobin (HGB), platelet count (PLT). Blood cell morphology, including eosinophils (EOS), basophils (BA), monocytes (MO), lymphocytes (LY) count, was also assessed by microscopic examination. The following clinical chemistry measurements were made on sera obtained by centrifugation of the collected blood samples: aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), total cholesterol (T-CHO), triglycerides (TG), total protein (TP), albumin (ALB), albumin/globulin ratio (A/G). All surviving animals were sacrificed by exsanguination at the end of the treatment. During that period, gross pathological examination was performed on all organs, including organ weight and appearance. The organs of each rat were weighed, furthermore, the ratio of the organs to body weight were determined: including heart, liver, spleen, kidneys, intestine, stomach, prostate (ventral). In the end, a full histopathological examination was performed on hematoxylin–eosin stained tissue sections of the organs and tissues listed above for the control and high the dose group. In this study, the significance of differences in each parameter between the control group and treated groups were analyzed. All parameters were assessed by SPSS11.0 software. The statistical significance of differences among groups was set at P < 0.05 in both studies.
3. Results 3.1. Bacterial reverse mutation (Ames) study There were no revertants exceeding three times over the background average either with or without the S9 metabolic activation system. In addition, no dose-dependent increasing in revertants was observed. 3.2. Micronucleus assay in mice There were no significant differences in body weights between the treated groups and the control group, and no signs of toxicity were noted in clinical observations following administration of the test article at doses of 2.5, 5 and 10 g/kg bw/day. None of the treated groups were positive for statistically significant induction of micronuclei in reticulocytes, and the average ratio of the reticulocyte to total erythrocytes in the negative control group was 0.73%, while in the treated groups were 0.72%, 0.72% and 0.72% in the 2.5, 5 and 10 g/kg bw/day groups respectively, which showed that there was no significant decrease between the treated group and the negative control. On the contrary, the positive control group caused a 0.68% decrease in the ratio.
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3.3. Acute oral toxicity study in mice The results of acute oral toxicity experiments indicated that C. guangdongensis on the per-oral LD50 in mice were all >15.0 g/ kg bw, and no classification or labeling for toxicity is required for C. guangdongensis. In addition, each doses of C. guangdongensis produced no treatment-related signs in any of the animals. For instance, no death in the treated groups was observed as compared with the control group in the end of the treatment. 3.4. Sperm aberration test in mice There were no significant differences in aberration ratio of sperm between the treated groups and the control group. 3.5. Teratogenicaction test in rats There were no abnormal of physiological signs, such as appearance, behavior, urine and fur in the clinical observations for these animals. There were no significant differences in bodyweight of pregnant rats, the average number of living embryo, dead fetus and absorbed fetus between the treated groups and the negative control group. The average bodyweight, length of body and tail of living embryo of treated groups were normal by comparing with control group. There were no abnormal of physiological signs, skeleton and internal organs in the clinical observations for living embryos. 3.6. Thirteen-weeks repeated-dose toxicity study in rats During the observation of 13-week, there was no death nor abnormal of physiological signs, such as weight, appearance, behavior, urine and fur, in the clinical observations for these animals. The mean bodyweight of male in the 8 g/kg group was below that of the controls during the experiments, but was not considered related to the test substance. There were no statistically significant changes in body weight observed in other groups. A summary of total body weights can be found in Table 1. There was a decrease tread toward average weekly food consumption in the three groups of the male: 8 g/kg group at weeks 1, 3, 6, 7, 11 and 13; 5.33 g/kg group at weeks 7, 11 and 2.67 g/ kg group at week 13. A tendency for decrease in food consumption was observed in the female 8 g/kg group at weeks 1, 6, 11 and 12. These changes were thought to be related to the test substance be-
cause they were continuous throughout the treatment period. A summary of food consumption can be found in Table 2. Hematology data are shown in Table 3. No statistically significant differences were observed from the results of the hematology and clinical chemistry parameters tested in the middle stage. Gross pathological evaluation at the end of the study revealed an obvious decrease in platelet counts of high doses for male rats and in monocytes counts of low doses for female rats, however, the fluctuation was in the normal reference range. There were no statistically significant changes in other parameters, which were observed in all treated groups at the end of the study. There were no treatment-related biologically significant adverse effects of C. guangdongensis on clinical chemistry parameters in male and female rats (Table 4). However, some statistically significant differences were noted when the control and treatment groups were compared. For example, albumin (ALB) increased significantly in the male of 8 g/kg group and blood urea nitrogen (BUN) was also significantly elevated in the female of 8 g/kg groups, but it fluctuated in the range of reference value of detection. In a word, there was no abnormal of terminal clinical chemistry. The data of organ and organ to body weight ratio are shown in Table 5. No treatment-related changes of biological significance in organ weights were noted in male and female rats following administration of C. guangdongensis. The ratio of the organ weight-to-body-weight was normal for all tissues except for the heart and the testicle. There were no treatment-related macroscopic and histopathological findings at the scheduled necropsy following administration of C. guangdongensis to rats. One case of minor liver hepatic fatty changes occurred in high dose treated males and females. One case of liver cell necrosis with inflammatory cell infiltration occurred in high dose and control treated males. One case of renal interstitial inflammatory cell infiltration appeared in high dose treated males. One case of cystic dilatation of the small glands occurred in control treated males. There were no other microscopic findings suggestive of treatment-related effects in histopathological examination. All changes, including those already mentioned, were considered to be spontaneous and/or incidental in nature and unrelated to the treatment. 4. Conclusion and discussion Cordyceps has a long history of usage as a traditional Chinese medicine. More and more new products of Cordyceps are commercially available as dietary ingredients in both dietary supplements and functional foods. Safety assessments of many Cordyceps,
Table 1 Summary of body weight data in the rat subchronic 13-week oral toxicity study. Week of study
0 1 2 3 4 5 6 7 8 9 10 11 12 13 *
P < 0.05.
Males
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
74.4 ± 4.6 144.0 ± 6.8 212.5 ± 12.3 257.0 ± 14.5 306.7 ± 18.1 339.8 ± 21.8 378.7 ± 29.0 396.2 ± 28.5 433.6 ± 31.1 454.2 ± 33.8 474.8 ± 40.9 497.2 ± 46.4 508.3 ± 46.4 534.0 ± 52.9
74.1 ± 4.8 143.7 ± 5.7 212.2 ± 9.0 249.0 ± 11.8 305.0 ± 16.8 343.3 ± 20.9 378.5 ± 22.9 403.8 ± 20.3 435.3 ± 27.0 456.9 ± 25.1 475.3 ± 31.8 499.9 ± 31.4 516.8 ± 31.9 539.8 ± 37.6
74.1 ± 4.8 139.5 ± 7.9 209.0 ± 12.6 250.0 ± 14.8 299.0 ± 15.0 333.3 ± 19.0 359.6 ± 18.9 385.3 ± 15.8 412.3 ± 18.2 425.4 ± 21.5 452.3 ± 21.7 469.9 ± 22.5 490.5 ± 24.7 509.1 ± 26.8
74.6 ± 4.4 130.4 ± 7.5* 196.5 ± 27.1 225.7 ± 12.5* 276.5 ± 17.5* 305.8 ± 24.9* 332.6 ± 27.1* 353.2 ± 29.0* 379.3 ± 30.3* 395.7 ± 31.8* 416.4 ± 35.5* 431.7 ± 38.7* 447.8 ± 42.9* 457.4 ± 30.6*
72.4 ± 4.3 130.1 ± 6.6 167.3 ± 13.4 187.2 ± 14.7 211.3 ± 17.6 224.3 ± 16.5 237.0 ± 21.1 246.3 ± 22.6 262.9 ± 21.7 267.6 ± 22.1 276.1 ± 25.0 281.1 ± 23.3 288.6 ± 25.5 290.8 ± 25.2
72.5 ± 4.6 130.3 ± 6.5 168.6 ± 8.8 189.4 ± 10.5 211.9 ± 12.6 226.2 ± 14.3 239.3 ± 12.6 219.4 ± 15.9 263.7 ± 16.5 269.3 ± 16.8 277.6 ± 18.4 288.4 ± 19.3 296.0 ± 18.6 301.0 ± 17.1
72.1 ± 4.3 128.7 ± 6.2 163.6 ± 8.5 183.1 ± 4.7 206.4 ± 9.4 223.4 ± 13.9 234.4 ± 13.9 244.8 ± 18.1 259.1 ± 20.6 265.7 ± 22.2 272.9 ± 22.4 282.2 ± 21.2 288.0 ± 22.0 292.2 ± 22.8
72.7 ± 4.7 121.6 ± 8.3 159.4 ± 10.8 177.4 ± 10.7 199.4 ± 12.6 215.0 ± 12.9 223.6 ± 12.5 230.2 ± 12.9 241.9 ± 12.9 250.0 ± 13.1 258.7 ± 14.2 263.8 ± 13.2 269.3 ± 14.6 275.8 ± 13.9
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W.-j. Yan et al. / Food and Chemical Toxicology 48 (2010) 3080–3084 Table 2 Food consumption values in 13-week repeated-dose toxicity study (n = 10). Week of study
1 2 3 4 5 6 7 8 9 10 11 12 13
Males
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
139.5 ± 7.6 232.0 ± 13.7 220.8 ± 15.2 203.0 ± 15.4 232.8 ± 14.5 237.5 ± 18.4 226.7 ± 18.6 235.1 ± 19.7 226.3 ± 15.7 217.9 ± 20.1 225.2 ± 23.3 209.3 ± 21.9 215.6 ± 23.5
137.0 ± 4.7 223.5 ± 8.9 208.1 ± 10.6 210.2 ± 12.4 234.5 ± 14.4 236.5 ± 12.5 198.4 ± 10.2* 227.2 ± 16.3 247.2 ± 15.0 208.4 ± 12.3 220.8 ± 14.5 223.3 ± 16.8 182.3 ± 15.6*
130.7 ± 10.6 221.6 ± 14.4 205.8 ± 13.5 191.6 ± 11.5 221.3 ± 14.8 221.1 ± 12.4 191.0 ± 10.5* 234.8 ± 10.6 215.5 ± 12.1 220.7 ± 10.5 202.4 ± 9.5* 216.4 ± 13.4 212.3 ± 13.7
120.2 ± 8.7* 220.0 ± 29.0 197.5 ± 14.4* 197.5 ± 13.9 223.9 ± 19.8 205.3 ± 14.9* 193.5 ± 16.4* 220.6 ± 19.2 241.3 ± 21.2 207.5 ± 15.4 196.4 ± 17.5* 204.0 ± 21.6 188.2 ± 14.6*
115.8 ± 7.7 158.7 ± 12.2 134.0 ± 11.8 138.4 ± 12.7 166.4 ± 14.6 131.2 ± 11.4 135.4 ± 13.3 153.5 ± 13.6 145.3 ± 14.3 122.6 ± 11.4 134.6 ± 11.7 137.4 ± 12.9 121.9 ± 11.0
113.4 ± 6.6 159.3 ± 6.3 134.8 ± 9.7 138.1 ± 11.7 159.3 ± 10.3 126.7 ± 7.3 132.5 ± 10.7 154.6 ± 13.3 145.4 ± 8.9 128.7 ± 9.4 130.6 ± 9.7 139.0 ± 12.3 121.9 ± 10.0
115.3 ± 6.8 154.9 ± 9.0 129.6 ± 6.9 138.6 ± 5.5 157.1 ± 8.8 125.6 ± 5.8 133.3 ± 10.1 151.3 ± 10.3 146.8 ± 11.5 131.5 ± 9.4 126.9 ± 10.8 141.4 ± 8.8 133.1 ± 7.5
104.6 ± 9.4* 155.7 ± 9.5 123.1 ± 10.5 135.5 ± 12.1 167.0 ± 11.2 120.1 ± 7.1* 124.1 ± 9.7 144.0 ± 11.4 140.3 ± 9.1 119.7 ± 7.3 115.4 ± 9.6* 121.5 ± 10.2* 129.5 ± 9.5
The values are means ± SD. P < 0.05.
*
Table 3 Effect of Cordyceps guangdongensis on hematological parameters in male and female rats. Parameter
Units
Males
RBC HGB WBC PLT Lymphocytes Neutrophils Monocytes Eosinophils Basophils
1012/L g/L 109/L 109/L % % % % %
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
7.96 ± 0.32 141.1 ± 6.9 12.1 ± 2.5 1079.4 ± 84.4 79.0 ± 3.9 14.3 ± 2.6 4.8 ± 1.9 1.58 ± 0.53 0.37 ± 0.28
7.65 ± 0.14 137.8 ± 4.0 10.7 ± 1.7 1141.4 ± 92.9 74.7 ± 7.7 18.7 ± 7.5 4.7 ± 1.8 1.57 ± 0.42 0.28 ± 0.14
7.70 ± 0.35 136.1 ± 4.1 10.3 ± 1.6 1089.6 ± 98.0 75.0 ± 6.7 18.8 ± 7.4 4.2 ± 1.9 1.61 ± 0.57 0.36 ± 0.30
7.75 ± 0.41 140.0 ± 4.6 10.1 ± 2.3 913.6 ± 151.3* 77.3 ± 2.5 15.5 ± 2.4 5.3 ± 1.8 1.46 ± 0.27 0.40 ± 0.23
7.43 ± 0.40 136.5 ± 5.1 6.9 ± 2.1 889.6 ± 146.5 83.3 ± 4.1 10.7 ± 3.1 3.8 ± 1.9 1.83 ± 0.53 0.27 ± 0.24
7.35 ± 0.42 134.9 ± 5.7 6.7 ± 3.5 872.6 ± 204.5 81.4 ± 3.7 10.1 ± 3.5 5.9 ± 1.1* 2.26 ± 0.60 0.44 ± 0.27
7.28 ± 0.37 35.9 ± 5.3 8.2 ± 2.8 1004.1 ± 158.3 81.2 ± 4.2 11.5 ± 3.6 5.2 ± 1.5 1.81 ± 0.30 0.31 ± 0.23
7.22 ± 0.32 133.0 ± 4.2 7.7 ± 2.8 1035.2 ± 179.2 79.9 ± 6.8 13.3 ± 6.4 4.5 ± 1.1 1.94 ± 0.69 0.33 ± 0.42
HGB = hemoglobin; PLT = platelet count; RBC = red blood cells; WBC = white blood cells. The values are means ± SD; n = 10. P < 0.05.
*
Table 4 Effect of Cordyceps guangdongensis on clinical chemistry parameters in male and female rats. Parameter
ALT AST TP Albumin AG ratio Creatinine BUN Glucose TG Total Cholesterol
Units
U/L U/L g/L g/L
lmol/L mmol/L mmol/L mmol/L mmol/L
Males
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
24.7 ± 3.1 91.4 ± 16.3 62.9 ± 2.5 30.2 ± 1.2 0.92 ± 0.04 59.1 ± 2.6 5.76 ± 1.01 6.03 ± 0.50 1.45 ± 0.71 1.58 ± 0.23
26.4 ± 3.0 99.7 ± 18.7 62.7 ± 2.1 30.9 ± 1.3 0.97 ± 0.04 57.6 ± 4.2 5.53 ± 0.71 5.87 ± 0.37 1.42 ± 0.93 1.73 ± 0.33
30.5 ± 9.2 107.1 ± 32.8 62.8 ± 2.6 30.2 ± 0.7 0.93 ± 0.07 60.1 ± 3.5 5.96 ± 0.82 6.29 ± 0.68 0.98 ± 0.23 1.66 ± 0.27
24.2 ± 7.1 90.8 ± 21.0 64.3 ± 2.1 31.8 ± 1.1* 0.98 ± 0.04 57.5 ± 2.1 6.14 ± 0.85 6.58 ± 0.44 0.70 ± 0.30 1.44 ± 0.22
28.5 ± 13.3 79.5 ± 21.3 65.7 ± 5.2 34.1 ± 2.9 1.08 ± 0.05 63.7 ± 4.5 6.86 ± 0.74 5.95 ± 0.48 1.21 ± 0.62 2.16 ± 0.41
22.9 ± 4.5 78.3 ± 19.1 68.2 ± 5.2 35.5 ± 3.0 1.08 ± 0.05 65.9 ± 4.0 7.51 ± 1.17 6.18 ± 0.48 1.98 ± 1.51 2.29 ± 0.37
20.3 ± 3.0 78.3 ± 13.6 64.6 ± 3.4 33.3 ± 2.4 1.06 ± 0.06 62.6 ± 3.5 7.01 ± 0.98 6.25 ± 0.55 1.20 ± 0.66 2.09 ± 0.20
32.1 ± 23.0 87.7 ± 25.6 66.9 ± 3.2 34.6 ± 2.0 1.07 ± 0.05 62.0 ± 4.6 8.20 ± 1.05* 6.36 ± 0.59 0.92 ± 0.40 1.97 ± 0.38
The values are means ± SD. n = 10. ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; AG, albumin globulin; BUN, urea nitrogen; TG, triglycerides; TP, total protein. * P < 0.05.
including C. sinensis, Paecilomyces gunnii Z.Q. Liang, C. militaris and cultivated Cordyceps brasiliensis Henn have been studied (Chen et al., 2009; He et al., 2009; Gong et al., 2003; Zhou et al., 1995). Most of the tested species are non-toxic except C. brasiliensis that has teratogenesis effect on rats with the relationship of dose–response (Zhou et al., 1995). As a new species to be exploited, it is important to test its safety properties. The focus of this paper is to present a comprehensive toxicologic assessment to support the safety for long term consumption of C. guangdongensis. As parts of a pre-clinical safety evaluation program, several tests have been performed. Furthermore, the results of acute oral toxicity study were found to be similar to the results of Chen et al. (2009) and Cha et al. (2008), which
reported that C. sinensis (Berk.) Sacc. and its mycelium on the peroral LD50 in mice and rats were all >20 g/kg bw and they were non-toxic. In the bacterial reverse mutation (Ames) study, it showed that the C. guangdongensis had no mutagenic effect on any strain used in this test. The micronucleus assays indicated that C. guangdongensis did not cause signs of toxicity in the bone marrow of the mice in the range of the doses tested. As these values were not statistically significant, therefore, they did not demonstrate any signs of toxicity with administration of C. guangdongensis in the mouse peripheral blood micronucleus assay. No treated mortality, morbidity or clinical symptoms resulted from an acute oral toxicity study using five doses of 15.0, 10.0, 4.64, 2.15 and 1.00 g/kg bw. In the 13-week oral toxicity study, no toxicologically
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Table 5 Effect of Cordyceps guangdongensis on group mean organ weights adjusted to overall necropsy body weight in male and female rats. Organ (g) Organ to bodyweight (%)
Heart Heart to bodyweight Liver Liver to bodyweight Spleen Spleen to bodyweight Kidneys Kidneys to bodyweight Testicle Testicle to bodyweight
Male
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
1.21 ± 0.22 0.23 ± 0.04 13.09 ± 2.09 2.46 ± 0.37 1.08 ± 0.26 0.20 ± 0.05 3.19 ± 0.38 0.60 ± 0.06 3.51 ± 0.43 0.66 ± 0.06
1.38 ± 0.18 0.26 ± 0.03 13.63 ± 2.09 2.53 ± 0.34 1.11 ± 0.26 0.20 ± 0.05 3.27 ± 0.38 0.61 ± 0.08 3.79 ± 0.40 0.71 ± 0.09
1.30 ± 0.08 0.26 ± 0.02 12.35 ± 1.05 2.43 ± 0.20 0.95 ± 0.15 0.19 ± 0.03 2.90 ± 0.32 0.57 ± 0.07 3.59 ± 0.32 0.71 ± 0.05
1.25 ± 0.21 0.27 ± 0.03* 11.24 ± 1.61 2.45 ± 0.23 0.90 ± 0.10 0.20 ± 0.03 2.90 ± 0.36 0.63 ± 0.06 3.60 ± 0.37 0.79 ± 0.10*
0.87 ± 0.08 0.30 ± 0.02 7.42 ± 0.99 2.55 ± 0.22 0.64 ± 0.11 0.22 ± 0.03 1.81 ± 0.31 0.62 ± 0.09
0.88 ± 0.16 0.29 ± 0.06 7.77 ± 0.81 2.58 ± 0.22 0.71 ± 0.11 0.24 ± 0.04 1.82 ± 0.21 False ± 0.06
0.85 ± 0.19 0.29 ± 0.06 7.39 ± 0.78 2.53 ± 0.14 0.75 ± 0.21 0.25 ± 0.06 1.66 ± 0.30 0.57 ± 0.09
0.76 ± 0.15 0.28 ± 0.05 6.85 ± 0.78 2.48 ± 0.24 0.67 ± 0.17 0.24 ± 0.06 1.71 ± 0.16 0.62 ± 0.06
The values are means ± SD; n = 10. * P < 0.05.
significant differences were observed between the treated groups and the controlled group for food consumption, hematological and clinical chemistry evaluations. C. guangdongensis caused neither treatment-related macroscopic or microscopic signs nor changes in the organ weights of the male and female rats after the 13-week treatment. Some slight changes in organ and body weights might not be a consequence of toxicity. However, if great deviation occurred, it might be necessary for further investigation. Our experiments did not display any severe deviation in the organ and body weights and thus excluded the toxicity of C. guangdongensis. Based on the results of this study, a no-observed-effect-level (NOVEL) was concluded to be 5.33 g/kg bw for rats. According to the results of sperm aberration and teratogenicaction test, there were no toxic effect on female parent, embryo and no distort influence on body and sperm about C. guangdongensis. The significant changes of ratio of the testicle to body weight in 13-week repeated-dose toxicity study in rats revealed that C. guangdongensis could improve sexual function of males and could be possibly used as an indigenous medicine for the treatment of male sexual disorders. Although the reasons for the increased male genitals have not been investigated, the interesting results provide a unique chance for developing a new market of C. guangdongensis. Based upon the above scientific tests, C. guangdongensis is considered to be safe for long term human consumption as some other species of Cordyceps. Moreover, it would have a broad application prospects in exploiting new healthy products. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors thank Dr. Burton B. Yang from the University of Toronto, who has provided some constructive suggestions during the course of the writing. The authors also thank Guangdong Key Laboratory of Microbial Culture Collection and Application and Guangdong Center for Disease Control and Prevention for valuable assistance and constant support in the process of the experiments. This study was supported by the Projects of the Breakthrough Project in Key Fields of Guangdong and Hongkong (No. 2007168604) Science and Technology Planning Project of Guangdong Province, China (No. 2009B080702029) and Nature Science Foundation of Guangdong Province (No. E05202480).
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Safety assessment of Cordyceps guangdongensis Wen-juan Yan a,b, Tai-Hui Li b,⇑, Qun-ying Lin b, Bin Song b, Zi-de Jiang a,⇑⇑ a b
South China Agricultural University, Guangzhou 510642, China Guangdong Institute of Microbiology, Guangzhou 510070, China
a r t i c l e
i n f o
Article history: Received 29 December 2009 Accepted 3 August 2010
Keywords: Cordyceps guangdongensis Toxicological assessments
a b s t r a c t Cordyceps guangdongensis as a kind of fungus, has been discovered and cultivated successfully in recent years. However, its safety assessments have not been studied. In this report, a serial of tests for toxicological safety assessments were depicted in details. These tests included bacterial reverse mutation (Ames) study, bone marrow cell micronucleus test in mice, sperm aberration test in mice, teratogenicaction test in rats, acute toxicity test and 13-week oral toxicity study in rats. After a profound analysis of these tests, it clearly demonstrated that C. guangdongensis did not have any mutagenic, clastogenic nor genotoxic effects; the oral LD50 of the biomass in rats was greater than 15 g/kg body weight; the no-observed adverse-effect-levels (NOAEL) was 5.33 g/kg body weight according to the 13-week oral toxicity analysis. Therefore, a conclusion can be drawn that C. guangdongensis is considered safe for long term consumption. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cordyceps (Fr.) Link is a wide spread genus which usually grows on insects or subterranean fungi. Modern scientific studies have shown that certain species of Cordyceps possess a wide spectrum of biological activities, including antioxidation (Yamaguchi et al., 2000; Li et al., 2001), antitumor (Ohmori et al., 1986; Chen et al., 1997; Shin et al., 2003), immuno-potentiation (Xu et al., 1992; Shin et al., 2003), antiinflammation (Kim et al., 2003) and stimulation of testosterone biosynthesis (Huang et al., 2001). And they also have been used to cure many diseases, such as hyposexuality and hyperlipidemia (Kuo et al., 2006; Chen et al., 2006; Wang and Shiao, 2000). One of the most famous species is Cordyceps sinensis (Berk.) Sacc., which has been used as a healthy food supplement and traditional Chinese herb for thousands of years. Additionally, some of
other species of Cordyceps also have highly potential value for utilization, as well as some commercial products of them which are available in the market nowadays such as Didanosine from Cordyceps militaris. (Russell and Paterson, 2008). Cordyceps guangdongensis T.H. Li, Q.Y. Lin & B.Song, which discovered in South China recently (Lin et al., 2008), has been successfully cultivated. It has many similar or same ingredients to those of C. sinensis, including high contents of cordycepic acid and cordycepin. To explore the possibilities of using this fungus further, we have performed a number of tests related to safety assessments, including bacterial reverse mutation (Ames) study, bone marrow cell micronucleus test, acute toxicity test, sperm aberration test, teratogenicaction test and 13-week oral toxicity study and demonstrated that C. guangdongensis can be safely used as a food supplement.
2. Materials and methods 2.1. Study design Abbreviations: A/G, albumin to globulin ratio; Alb, albumin; ALT, alanine transaminase; AST, aspartate transaminase; BA, basophils; bw, bodyweight; BUN, blood urea nitrogen; CFU, colony forming unit; CP, cyclophosphamide; CRE, creatinine; EOS, eosinophils; GLU, glucose; HGB, hemoglobin; LD50, lethal dose; LY, lymphocytes; MCT, mercury cadmium telluride; MO, monocytes; NOAEL, noobserved adverse-effect-level; NIH, National Institutes of Health; PCB, polychlorobiphenyl; PLT, platelet count; RBC, red blood cells; SD, standard deviation; SPF, specific pathogen free; T-CHO, total cholesterol; TG, Triglycerides; UV, ultraviolet; TP, total protein; WBC, white blood cells. ⇑ Corresponding author. Tel./fax: +86 20 87688102. ⇑⇑ Corresponding author. Tel./fax: +86 20 38604779. E-mail addresses: [email protected] (T.-H. Li), [email protected] (Z.-d. Jiang). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.08.001
The study was conducted according to the guidelines of ‘‘Health Food Products Testing and Evaluation of Technical Specifications” (2003 Version) issued by Ministry of Health of the People’s Republic of China : the bacterial reverse mutation (Ames) test was conducted with PCB induced rat liver S9; the micronucleus assay, acute oral toxicity study and sperm aberration test were performed in mice; the teratogenicaction test and 13-week repeated-dose toxicity study were performed in rats.
2.2. Test substance The test substance, cultivated fruitbody of C. guangdongensis (CG), was crushed and provided by Guangdong Institute of Microbiology. The doses varied slightly from batch to batch and would be reported for each study performed.
W.-j. Yan et al. / Food and Chemical Toxicology 48 (2010) 3080–3084 2.3. Bacterial reverse mutation (Ames) study Four strains of Salmonella typhimurium (TA97, TA98, TA100, TA102) and the PCB induced rat liver S9 were offered by Guangdong Center of Disease Control and Prevention. The study was performed both with and without the S9 activation system. And doses used in this study were: 8, 40, 200, 1000 and 5000 lg/plate. Experiments were performed in triplicate. Positive controls in the experiment without S9 activation included three parts: fenaminosulf for strain TA97 and TA98, NaN2 for strains TA100, as well as mitomycin C for strains TA102. Positive controls in the experiments with S9 activation were composed of 2-FA for strains TA97, TA98 and TA100, 1,8-dihydroxyanthraquinone for strains TA102. All strains were tested for spontaneous revertant colonies using distilled water as a negative control. Frozen stock cultures were inoculated and grown overnight at 37 ± 1 °C. In brief, sterile culture tubes were filled with 0.1 ml of test strains, 0.1 ml of test article culture or control, as well as 0.5 ml of S9/cofactor mix. First, the mixture was spread onto minimal glucose plates and incubated at 37 ± 1 °C for 48 h. Second, the number of revertant colonies per plate was determined by hand counting. If the outcome of this experiment was three times over the background average number of revertant colonies on a plate (two times for negative control), or if there was a dose-related increase in colonies, the test was considered as positive. On the contrary if the output did not reach to the criteria, the test article was considered as non-mutagenic. 2.4. Micronucleus assay in mice The mice of strain NIH, which were both male and female with the age of 7– 8 weeks, were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China). At the beginning of the study, the body weights of the mice were 27.3 ± 2.7 g. The mice were randomly assigned to five groups, 10 per group, which could get the drinking water ad libitum. During this experiments, water for injection was utilized as the negative control, while 50 mg/kg bw CP served as the positive control. The test article was administered by oral gavage at a dose of 0.5 ml/10 g bw, except for the positive and negative controls which were administered only once by intraperitoneal injection on the last test article administration day. Doses of the test article, included 2.5, 5 and 10 g/kg bw, were given for 30 h. Blood samples were obtained by tail trimming approximately 48 h after the last administration. Samples were smeared on acridine orange-coated slides, and 1000 reticulocytes were scored under a fluorescence microscope for the presence of micronuclei and the slides were blind coded. The proportion of reticulocytes to total erythrocytes was an indicator to evaluate bone marrow toxicity. Percentages were determined by flow cytometry and were based on analysis of 200 erythrocytes. 2.5. Acute oral toxicity study in mice NIH mice were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China), and their body weights were 19.7 ± 2.3 g at the beginning of the experiments. The mice were assigned randomly to two groups of 10, which could get the tap water ad libitum. Cordyceps guangdongensis was administered to mice at doses of 15.0, 10.0, 4.64, 2.15 and 1.00 g/kg BW. The test article was administered by oral gavage at a dose of 0.35 ml/10 g bw three times at 4 h intervals after 16 h of non-food obtained. During the experiments, the animals were monitored daily for 7 days and were euthanized at the end of the tests for macroscopic examinations. 2.6. Sperm aberration test in mice NIH male mice were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China), and their body weights were 29.5 ± 5.3 g at the beginning of the study. The mice were randomly assigned to five groups, five per group. According to the results of acute oral toxicity obtained above, doses of the test article were designed and included 2.5, 5 and 10 g/kg bw. Distilled water served as the negative control, while 50 mg/kg bw CP served as the positive control. The test article was administered by oral gavage at a dose of 0.5 ml/10 g bw once a day for 5 days. Both sides of the epididymis were obtained by tail trimming approximately 35 days after the first administration. Samples were smeared on acridine orange-coated slides, and 1000 sperms were examined under a microscope for their number and type. The incidence of sperm malformation was calculated and analyzed by wilcoson rank and testing methods. 2.7. Teratogenicaction test in rats SD rats were obtained from The Laboratory Animal Center of Southern Medical University (Guangzhou, China). The pregnant rats were given the test article once a day between days 7 and 16 after mating, and their body weights were 228.3 ± 22.7 g at the beginning of the study. Distilled water was used as the negative control, doses of CG were 1.25, 2.50 and 5 g/kg bw. At the end of the treatments,
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the average numbers of living embryo, dead fetus and absorbed fetus were counted and compared with the negative control. Visual, bone and organ examination of rat embryo were performed. 2.8. Thirteen-weeks repeated-dose toxicity study in rats Eighty-four-week-old SD rats (40 male and 40 female) were purchased from The Laboratory Animal Center of Southern Medical University (Guangzhou, China), and used in this study after a 3-day acclimation period. The experiments were controlled in the conditions as follows: the room was maintained on a 12-h light–dark cycle, at the temperature of 22.0 ± 1 °C with a relative humidity of 60–80%. Each group was consisted of 20 rats (10 male and 10 female), whose bodyweights were 74.3 ± 4.5 g for male, and 72.4 ± 4.3 g for female. All rats were assigned averagely to four groups, and one of them was the control group. C. guangdongensis was administered orally by incorporation in the feed at a concentration of 3.33%, 6.67% and 10%. The casein was incorporated at a concentration of 22% into the diet. Compared to the animals in control group, the low, middle and high dose group animals received C. guangdongensis at doses of 2.67, 5.33 and 8.00 g/kg bw/day in their diet. Weekly adjustments were made for body weight changes. Clinical observations were made once daily following treatment at approximately the same time each day. All animals were checked for general conditions once a week. Body weights were measured at the initiation of the experiments, and were measured every other week. At the scheduled termination, final body weights were recorded after an overnight fast (approximately 16 h). Throughout the experiments, food was weighed weekly, and the average food consumption per animal was calculated twice at weekly intervals. At the end of the treatment period, blood samples for hematology and clinical chemistry were taken at necropsy from all surviving animals via the abdominal aorta under anesthesia after an overnight fast. The following hematological parameters were measured on samples collected by EDTA-2K, which was used as an anticoagulant: red blood cell count (RBC), white blood cell count (WBC), hemoglobin (HGB), platelet count (PLT). Blood cell morphology, including eosinophils (EOS), basophils (BA), monocytes (MO), lymphocytes (LY) count, was also assessed by microscopic examination. The following clinical chemistry measurements were made on sera obtained by centrifugation of the collected blood samples: aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), total cholesterol (T-CHO), triglycerides (TG), total protein (TP), albumin (ALB), albumin/globulin ratio (A/G). All surviving animals were sacrificed by exsanguination at the end of the treatment. During that period, gross pathological examination was performed on all organs, including organ weight and appearance. The organs of each rat were weighed, furthermore, the ratio of the organs to body weight were determined: including heart, liver, spleen, kidneys, intestine, stomach, prostate (ventral). In the end, a full histopathological examination was performed on hematoxylin–eosin stained tissue sections of the organs and tissues listed above for the control and high the dose group. In this study, the significance of differences in each parameter between the control group and treated groups were analyzed. All parameters were assessed by SPSS11.0 software. The statistical significance of differences among groups was set at P < 0.05 in both studies.
3. Results 3.1. Bacterial reverse mutation (Ames) study There were no revertants exceeding three times over the background average either with or without the S9 metabolic activation system. In addition, no dose-dependent increasing in revertants was observed. 3.2. Micronucleus assay in mice There were no significant differences in body weights between the treated groups and the control group, and no signs of toxicity were noted in clinical observations following administration of the test article at doses of 2.5, 5 and 10 g/kg bw/day. None of the treated groups were positive for statistically significant induction of micronuclei in reticulocytes, and the average ratio of the reticulocyte to total erythrocytes in the negative control group was 0.73%, while in the treated groups were 0.72%, 0.72% and 0.72% in the 2.5, 5 and 10 g/kg bw/day groups respectively, which showed that there was no significant decrease between the treated group and the negative control. On the contrary, the positive control group caused a 0.68% decrease in the ratio.
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3.3. Acute oral toxicity study in mice The results of acute oral toxicity experiments indicated that C. guangdongensis on the per-oral LD50 in mice were all >15.0 g/ kg bw, and no classification or labeling for toxicity is required for C. guangdongensis. In addition, each doses of C. guangdongensis produced no treatment-related signs in any of the animals. For instance, no death in the treated groups was observed as compared with the control group in the end of the treatment. 3.4. Sperm aberration test in mice There were no significant differences in aberration ratio of sperm between the treated groups and the control group. 3.5. Teratogenicaction test in rats There were no abnormal of physiological signs, such as appearance, behavior, urine and fur in the clinical observations for these animals. There were no significant differences in bodyweight of pregnant rats, the average number of living embryo, dead fetus and absorbed fetus between the treated groups and the negative control group. The average bodyweight, length of body and tail of living embryo of treated groups were normal by comparing with control group. There were no abnormal of physiological signs, skeleton and internal organs in the clinical observations for living embryos. 3.6. Thirteen-weeks repeated-dose toxicity study in rats During the observation of 13-week, there was no death nor abnormal of physiological signs, such as weight, appearance, behavior, urine and fur, in the clinical observations for these animals. The mean bodyweight of male in the 8 g/kg group was below that of the controls during the experiments, but was not considered related to the test substance. There were no statistically significant changes in body weight observed in other groups. A summary of total body weights can be found in Table 1. There was a decrease tread toward average weekly food consumption in the three groups of the male: 8 g/kg group at weeks 1, 3, 6, 7, 11 and 13; 5.33 g/kg group at weeks 7, 11 and 2.67 g/ kg group at week 13. A tendency for decrease in food consumption was observed in the female 8 g/kg group at weeks 1, 6, 11 and 12. These changes were thought to be related to the test substance be-
cause they were continuous throughout the treatment period. A summary of food consumption can be found in Table 2. Hematology data are shown in Table 3. No statistically significant differences were observed from the results of the hematology and clinical chemistry parameters tested in the middle stage. Gross pathological evaluation at the end of the study revealed an obvious decrease in platelet counts of high doses for male rats and in monocytes counts of low doses for female rats, however, the fluctuation was in the normal reference range. There were no statistically significant changes in other parameters, which were observed in all treated groups at the end of the study. There were no treatment-related biologically significant adverse effects of C. guangdongensis on clinical chemistry parameters in male and female rats (Table 4). However, some statistically significant differences were noted when the control and treatment groups were compared. For example, albumin (ALB) increased significantly in the male of 8 g/kg group and blood urea nitrogen (BUN) was also significantly elevated in the female of 8 g/kg groups, but it fluctuated in the range of reference value of detection. In a word, there was no abnormal of terminal clinical chemistry. The data of organ and organ to body weight ratio are shown in Table 5. No treatment-related changes of biological significance in organ weights were noted in male and female rats following administration of C. guangdongensis. The ratio of the organ weight-to-body-weight was normal for all tissues except for the heart and the testicle. There were no treatment-related macroscopic and histopathological findings at the scheduled necropsy following administration of C. guangdongensis to rats. One case of minor liver hepatic fatty changes occurred in high dose treated males and females. One case of liver cell necrosis with inflammatory cell infiltration occurred in high dose and control treated males. One case of renal interstitial inflammatory cell infiltration appeared in high dose treated males. One case of cystic dilatation of the small glands occurred in control treated males. There were no other microscopic findings suggestive of treatment-related effects in histopathological examination. All changes, including those already mentioned, were considered to be spontaneous and/or incidental in nature and unrelated to the treatment. 4. Conclusion and discussion Cordyceps has a long history of usage as a traditional Chinese medicine. More and more new products of Cordyceps are commercially available as dietary ingredients in both dietary supplements and functional foods. Safety assessments of many Cordyceps,
Table 1 Summary of body weight data in the rat subchronic 13-week oral toxicity study. Week of study
0 1 2 3 4 5 6 7 8 9 10 11 12 13 *
P < 0.05.
Males
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
74.4 ± 4.6 144.0 ± 6.8 212.5 ± 12.3 257.0 ± 14.5 306.7 ± 18.1 339.8 ± 21.8 378.7 ± 29.0 396.2 ± 28.5 433.6 ± 31.1 454.2 ± 33.8 474.8 ± 40.9 497.2 ± 46.4 508.3 ± 46.4 534.0 ± 52.9
74.1 ± 4.8 143.7 ± 5.7 212.2 ± 9.0 249.0 ± 11.8 305.0 ± 16.8 343.3 ± 20.9 378.5 ± 22.9 403.8 ± 20.3 435.3 ± 27.0 456.9 ± 25.1 475.3 ± 31.8 499.9 ± 31.4 516.8 ± 31.9 539.8 ± 37.6
74.1 ± 4.8 139.5 ± 7.9 209.0 ± 12.6 250.0 ± 14.8 299.0 ± 15.0 333.3 ± 19.0 359.6 ± 18.9 385.3 ± 15.8 412.3 ± 18.2 425.4 ± 21.5 452.3 ± 21.7 469.9 ± 22.5 490.5 ± 24.7 509.1 ± 26.8
74.6 ± 4.4 130.4 ± 7.5* 196.5 ± 27.1 225.7 ± 12.5* 276.5 ± 17.5* 305.8 ± 24.9* 332.6 ± 27.1* 353.2 ± 29.0* 379.3 ± 30.3* 395.7 ± 31.8* 416.4 ± 35.5* 431.7 ± 38.7* 447.8 ± 42.9* 457.4 ± 30.6*
72.4 ± 4.3 130.1 ± 6.6 167.3 ± 13.4 187.2 ± 14.7 211.3 ± 17.6 224.3 ± 16.5 237.0 ± 21.1 246.3 ± 22.6 262.9 ± 21.7 267.6 ± 22.1 276.1 ± 25.0 281.1 ± 23.3 288.6 ± 25.5 290.8 ± 25.2
72.5 ± 4.6 130.3 ± 6.5 168.6 ± 8.8 189.4 ± 10.5 211.9 ± 12.6 226.2 ± 14.3 239.3 ± 12.6 219.4 ± 15.9 263.7 ± 16.5 269.3 ± 16.8 277.6 ± 18.4 288.4 ± 19.3 296.0 ± 18.6 301.0 ± 17.1
72.1 ± 4.3 128.7 ± 6.2 163.6 ± 8.5 183.1 ± 4.7 206.4 ± 9.4 223.4 ± 13.9 234.4 ± 13.9 244.8 ± 18.1 259.1 ± 20.6 265.7 ± 22.2 272.9 ± 22.4 282.2 ± 21.2 288.0 ± 22.0 292.2 ± 22.8
72.7 ± 4.7 121.6 ± 8.3 159.4 ± 10.8 177.4 ± 10.7 199.4 ± 12.6 215.0 ± 12.9 223.6 ± 12.5 230.2 ± 12.9 241.9 ± 12.9 250.0 ± 13.1 258.7 ± 14.2 263.8 ± 13.2 269.3 ± 14.6 275.8 ± 13.9
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W.-j. Yan et al. / Food and Chemical Toxicology 48 (2010) 3080–3084 Table 2 Food consumption values in 13-week repeated-dose toxicity study (n = 10). Week of study
1 2 3 4 5 6 7 8 9 10 11 12 13
Males
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
139.5 ± 7.6 232.0 ± 13.7 220.8 ± 15.2 203.0 ± 15.4 232.8 ± 14.5 237.5 ± 18.4 226.7 ± 18.6 235.1 ± 19.7 226.3 ± 15.7 217.9 ± 20.1 225.2 ± 23.3 209.3 ± 21.9 215.6 ± 23.5
137.0 ± 4.7 223.5 ± 8.9 208.1 ± 10.6 210.2 ± 12.4 234.5 ± 14.4 236.5 ± 12.5 198.4 ± 10.2* 227.2 ± 16.3 247.2 ± 15.0 208.4 ± 12.3 220.8 ± 14.5 223.3 ± 16.8 182.3 ± 15.6*
130.7 ± 10.6 221.6 ± 14.4 205.8 ± 13.5 191.6 ± 11.5 221.3 ± 14.8 221.1 ± 12.4 191.0 ± 10.5* 234.8 ± 10.6 215.5 ± 12.1 220.7 ± 10.5 202.4 ± 9.5* 216.4 ± 13.4 212.3 ± 13.7
120.2 ± 8.7* 220.0 ± 29.0 197.5 ± 14.4* 197.5 ± 13.9 223.9 ± 19.8 205.3 ± 14.9* 193.5 ± 16.4* 220.6 ± 19.2 241.3 ± 21.2 207.5 ± 15.4 196.4 ± 17.5* 204.0 ± 21.6 188.2 ± 14.6*
115.8 ± 7.7 158.7 ± 12.2 134.0 ± 11.8 138.4 ± 12.7 166.4 ± 14.6 131.2 ± 11.4 135.4 ± 13.3 153.5 ± 13.6 145.3 ± 14.3 122.6 ± 11.4 134.6 ± 11.7 137.4 ± 12.9 121.9 ± 11.0
113.4 ± 6.6 159.3 ± 6.3 134.8 ± 9.7 138.1 ± 11.7 159.3 ± 10.3 126.7 ± 7.3 132.5 ± 10.7 154.6 ± 13.3 145.4 ± 8.9 128.7 ± 9.4 130.6 ± 9.7 139.0 ± 12.3 121.9 ± 10.0
115.3 ± 6.8 154.9 ± 9.0 129.6 ± 6.9 138.6 ± 5.5 157.1 ± 8.8 125.6 ± 5.8 133.3 ± 10.1 151.3 ± 10.3 146.8 ± 11.5 131.5 ± 9.4 126.9 ± 10.8 141.4 ± 8.8 133.1 ± 7.5
104.6 ± 9.4* 155.7 ± 9.5 123.1 ± 10.5 135.5 ± 12.1 167.0 ± 11.2 120.1 ± 7.1* 124.1 ± 9.7 144.0 ± 11.4 140.3 ± 9.1 119.7 ± 7.3 115.4 ± 9.6* 121.5 ± 10.2* 129.5 ± 9.5
The values are means ± SD. P < 0.05.
*
Table 3 Effect of Cordyceps guangdongensis on hematological parameters in male and female rats. Parameter
Units
Males
RBC HGB WBC PLT Lymphocytes Neutrophils Monocytes Eosinophils Basophils
1012/L g/L 109/L 109/L % % % % %
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
7.96 ± 0.32 141.1 ± 6.9 12.1 ± 2.5 1079.4 ± 84.4 79.0 ± 3.9 14.3 ± 2.6 4.8 ± 1.9 1.58 ± 0.53 0.37 ± 0.28
7.65 ± 0.14 137.8 ± 4.0 10.7 ± 1.7 1141.4 ± 92.9 74.7 ± 7.7 18.7 ± 7.5 4.7 ± 1.8 1.57 ± 0.42 0.28 ± 0.14
7.70 ± 0.35 136.1 ± 4.1 10.3 ± 1.6 1089.6 ± 98.0 75.0 ± 6.7 18.8 ± 7.4 4.2 ± 1.9 1.61 ± 0.57 0.36 ± 0.30
7.75 ± 0.41 140.0 ± 4.6 10.1 ± 2.3 913.6 ± 151.3* 77.3 ± 2.5 15.5 ± 2.4 5.3 ± 1.8 1.46 ± 0.27 0.40 ± 0.23
7.43 ± 0.40 136.5 ± 5.1 6.9 ± 2.1 889.6 ± 146.5 83.3 ± 4.1 10.7 ± 3.1 3.8 ± 1.9 1.83 ± 0.53 0.27 ± 0.24
7.35 ± 0.42 134.9 ± 5.7 6.7 ± 3.5 872.6 ± 204.5 81.4 ± 3.7 10.1 ± 3.5 5.9 ± 1.1* 2.26 ± 0.60 0.44 ± 0.27
7.28 ± 0.37 35.9 ± 5.3 8.2 ± 2.8 1004.1 ± 158.3 81.2 ± 4.2 11.5 ± 3.6 5.2 ± 1.5 1.81 ± 0.30 0.31 ± 0.23
7.22 ± 0.32 133.0 ± 4.2 7.7 ± 2.8 1035.2 ± 179.2 79.9 ± 6.8 13.3 ± 6.4 4.5 ± 1.1 1.94 ± 0.69 0.33 ± 0.42
HGB = hemoglobin; PLT = platelet count; RBC = red blood cells; WBC = white blood cells. The values are means ± SD; n = 10. P < 0.05.
*
Table 4 Effect of Cordyceps guangdongensis on clinical chemistry parameters in male and female rats. Parameter
ALT AST TP Albumin AG ratio Creatinine BUN Glucose TG Total Cholesterol
Units
U/L U/L g/L g/L
lmol/L mmol/L mmol/L mmol/L mmol/L
Males
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
24.7 ± 3.1 91.4 ± 16.3 62.9 ± 2.5 30.2 ± 1.2 0.92 ± 0.04 59.1 ± 2.6 5.76 ± 1.01 6.03 ± 0.50 1.45 ± 0.71 1.58 ± 0.23
26.4 ± 3.0 99.7 ± 18.7 62.7 ± 2.1 30.9 ± 1.3 0.97 ± 0.04 57.6 ± 4.2 5.53 ± 0.71 5.87 ± 0.37 1.42 ± 0.93 1.73 ± 0.33
30.5 ± 9.2 107.1 ± 32.8 62.8 ± 2.6 30.2 ± 0.7 0.93 ± 0.07 60.1 ± 3.5 5.96 ± 0.82 6.29 ± 0.68 0.98 ± 0.23 1.66 ± 0.27
24.2 ± 7.1 90.8 ± 21.0 64.3 ± 2.1 31.8 ± 1.1* 0.98 ± 0.04 57.5 ± 2.1 6.14 ± 0.85 6.58 ± 0.44 0.70 ± 0.30 1.44 ± 0.22
28.5 ± 13.3 79.5 ± 21.3 65.7 ± 5.2 34.1 ± 2.9 1.08 ± 0.05 63.7 ± 4.5 6.86 ± 0.74 5.95 ± 0.48 1.21 ± 0.62 2.16 ± 0.41
22.9 ± 4.5 78.3 ± 19.1 68.2 ± 5.2 35.5 ± 3.0 1.08 ± 0.05 65.9 ± 4.0 7.51 ± 1.17 6.18 ± 0.48 1.98 ± 1.51 2.29 ± 0.37
20.3 ± 3.0 78.3 ± 13.6 64.6 ± 3.4 33.3 ± 2.4 1.06 ± 0.06 62.6 ± 3.5 7.01 ± 0.98 6.25 ± 0.55 1.20 ± 0.66 2.09 ± 0.20
32.1 ± 23.0 87.7 ± 25.6 66.9 ± 3.2 34.6 ± 2.0 1.07 ± 0.05 62.0 ± 4.6 8.20 ± 1.05* 6.36 ± 0.59 0.92 ± 0.40 1.97 ± 0.38
The values are means ± SD. n = 10. ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; AG, albumin globulin; BUN, urea nitrogen; TG, triglycerides; TP, total protein. * P < 0.05.
including C. sinensis, Paecilomyces gunnii Z.Q. Liang, C. militaris and cultivated Cordyceps brasiliensis Henn have been studied (Chen et al., 2009; He et al., 2009; Gong et al., 2003; Zhou et al., 1995). Most of the tested species are non-toxic except C. brasiliensis that has teratogenesis effect on rats with the relationship of dose–response (Zhou et al., 1995). As a new species to be exploited, it is important to test its safety properties. The focus of this paper is to present a comprehensive toxicologic assessment to support the safety for long term consumption of C. guangdongensis. As parts of a pre-clinical safety evaluation program, several tests have been performed. Furthermore, the results of acute oral toxicity study were found to be similar to the results of Chen et al. (2009) and Cha et al. (2008), which
reported that C. sinensis (Berk.) Sacc. and its mycelium on the peroral LD50 in mice and rats were all >20 g/kg bw and they were non-toxic. In the bacterial reverse mutation (Ames) study, it showed that the C. guangdongensis had no mutagenic effect on any strain used in this test. The micronucleus assays indicated that C. guangdongensis did not cause signs of toxicity in the bone marrow of the mice in the range of the doses tested. As these values were not statistically significant, therefore, they did not demonstrate any signs of toxicity with administration of C. guangdongensis in the mouse peripheral blood micronucleus assay. No treated mortality, morbidity or clinical symptoms resulted from an acute oral toxicity study using five doses of 15.0, 10.0, 4.64, 2.15 and 1.00 g/kg bw. In the 13-week oral toxicity study, no toxicologically
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Table 5 Effect of Cordyceps guangdongensis on group mean organ weights adjusted to overall necropsy body weight in male and female rats. Organ (g) Organ to bodyweight (%)
Heart Heart to bodyweight Liver Liver to bodyweight Spleen Spleen to bodyweight Kidneys Kidneys to bodyweight Testicle Testicle to bodyweight
Male
Females
0
2.67
5.33
8.00
0
2.67
5.33
8.00
1.21 ± 0.22 0.23 ± 0.04 13.09 ± 2.09 2.46 ± 0.37 1.08 ± 0.26 0.20 ± 0.05 3.19 ± 0.38 0.60 ± 0.06 3.51 ± 0.43 0.66 ± 0.06
1.38 ± 0.18 0.26 ± 0.03 13.63 ± 2.09 2.53 ± 0.34 1.11 ± 0.26 0.20 ± 0.05 3.27 ± 0.38 0.61 ± 0.08 3.79 ± 0.40 0.71 ± 0.09
1.30 ± 0.08 0.26 ± 0.02 12.35 ± 1.05 2.43 ± 0.20 0.95 ± 0.15 0.19 ± 0.03 2.90 ± 0.32 0.57 ± 0.07 3.59 ± 0.32 0.71 ± 0.05
1.25 ± 0.21 0.27 ± 0.03* 11.24 ± 1.61 2.45 ± 0.23 0.90 ± 0.10 0.20 ± 0.03 2.90 ± 0.36 0.63 ± 0.06 3.60 ± 0.37 0.79 ± 0.10*
0.87 ± 0.08 0.30 ± 0.02 7.42 ± 0.99 2.55 ± 0.22 0.64 ± 0.11 0.22 ± 0.03 1.81 ± 0.31 0.62 ± 0.09
0.88 ± 0.16 0.29 ± 0.06 7.77 ± 0.81 2.58 ± 0.22 0.71 ± 0.11 0.24 ± 0.04 1.82 ± 0.21 False ± 0.06
0.85 ± 0.19 0.29 ± 0.06 7.39 ± 0.78 2.53 ± 0.14 0.75 ± 0.21 0.25 ± 0.06 1.66 ± 0.30 0.57 ± 0.09
0.76 ± 0.15 0.28 ± 0.05 6.85 ± 0.78 2.48 ± 0.24 0.67 ± 0.17 0.24 ± 0.06 1.71 ± 0.16 0.62 ± 0.06
The values are means ± SD; n = 10. * P < 0.05.
significant differences were observed between the treated groups and the controlled group for food consumption, hematological and clinical chemistry evaluations. C. guangdongensis caused neither treatment-related macroscopic or microscopic signs nor changes in the organ weights of the male and female rats after the 13-week treatment. Some slight changes in organ and body weights might not be a consequence of toxicity. However, if great deviation occurred, it might be necessary for further investigation. Our experiments did not display any severe deviation in the organ and body weights and thus excluded the toxicity of C. guangdongensis. Based on the results of this study, a no-observed-effect-level (NOVEL) was concluded to be 5.33 g/kg bw for rats. According to the results of sperm aberration and teratogenicaction test, there were no toxic effect on female parent, embryo and no distort influence on body and sperm about C. guangdongensis. The significant changes of ratio of the testicle to body weight in 13-week repeated-dose toxicity study in rats revealed that C. guangdongensis could improve sexual function of males and could be possibly used as an indigenous medicine for the treatment of male sexual disorders. Although the reasons for the increased male genitals have not been investigated, the interesting results provide a unique chance for developing a new market of C. guangdongensis. Based upon the above scientific tests, C. guangdongensis is considered to be safe for long term human consumption as some other species of Cordyceps. Moreover, it would have a broad application prospects in exploiting new healthy products. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors thank Dr. Burton B. Yang from the University of Toronto, who has provided some constructive suggestions during the course of the writing. The authors also thank Guangdong Key Laboratory of Microbial Culture Collection and Application and Guangdong Center for Disease Control and Prevention for valuable assistance and constant support in the process of the experiments. This study was supported by the Projects of the Breakthrough Project in Key Fields of Guangdong and Hongkong (No. 2007168604) Science and Technology Planning Project of Guangdong Province, China (No. 2009B080702029) and Nature Science Foundation of Guangdong Province (No. E05202480).
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