本实验试验物质为葡萄王生技股份有限公司之樟芝纯化物Antrosterol (AST)及樟芝发酵菌丝体冻干品(AC-1) (AST含量3.0 mg/g)、富含活性成分Antrosterol之樟芝发酵菌丝体冻干品(AC-2) (AST含量8.8 mg/g),经单次口服投予大鼠后,评估可能产生之急性毒性反应,系参考卫生福利部食品药物管理署:健康食品安全性评估方法(1999)。试验共设置7组,分别为逆渗透水对照组、橄榄油对照组、试验组I (8 mg/kg bw AST)、试验组II (16 mg/kg bw AST)、试验组III (24 mg/kg bw AST)、试验组IV (2 g/kg bw AC-1)、试验组V (2 g/kg bw AC-2),以上试验组为AST建议剂量依序之17、34、52、17及42倍,每组各使用5只雌性Sprague-Dawley (SD)品系大鼠,以口服管喂方式单次投予试验物质或对照物质,并进行7天临床观察。试验结束后,牺牲大鼠并采集血液、肝脏与肾脏进行血清生化分析及病理学检查。临床症状部分,各组别所有大鼠均无出现异常之症状。动物体重部分,血清生化结果显示,对照组与其对应试验组均无显着差异。试验终结进行解剖肉眼检查及组织病理学检查,所有动物皆未发现任何与试验物质处理相关之病变。综合以上实验结果,试验物质AC-1、AC-2及AST在本试验条件下,大鼠均无发现任何与试验物质相关之急性毒性反应,AC-1及AC-2其LD50皆大于2 g/kgbw,AST则大于24 mg/kg bw,此结果将作为人体使用此产品之安全性参考。 This study was conducted to a single limit dose of acute toxicity assessment in SD rats according to the safety assessment guideline of Health Food announced by Ministry of Health and Welfare of Taiwan. The test substances in this experiment are Antrosterol (AST), Antrodia cinnamomea ( A. cinnamomea ) mycelium (AC-1) (AST content 3.0 mg/g) and Antrosterol-Enriched A. cinnamomea mycelium (AC-2) (AST content 8.8 mg/g) is provided by the Biotechnology Center of Grape King Inc. SD rats were divided into seven groups and were fed with RO water, olive oil, AST 8 (Group I), 16 (Group II), 24 (Group III) mg/kg bw, 2 g/kg bw A. cinnamomea powder (AST content 3.0 mg/g) (Group IV) and 2 g/kg bw Antrosterol-Enriched A. cinnamomea powder (AST content 8.8 mg/g) (Group V). The above test group is 17, 34, 52, 17 and 42 times the recommended dose of AST. Each group has 5 experiment animals. During the study period, no abnormality occurred in clinical signs and body weight. There was no significant difference in biochemistry parameters between the treatment and control group. Necropsy and histopathological examination showed no treatment-related change. The results of this study provide evidences for the use of A. cinnamomea fermentation product as a safe agent in functional food: The LD50 of A. cinnamomea powder, its Antrosterol, A. cinnamomea powder and Antrosterol-Enriched A. cinnamomea powder than 24 mg/kg bw, 2 g/kg bw and 2 g/kg bw in SD rats respectively.
本实验试验物质为葡萄王生技股份有限公司之樟芝纯化物Antrosterol (AST)及樟芝发酵菌丝体冻干品(AC-1) (AST含量3.0 mg/g)、富含活性成分Antrosterol之樟芝发酵菌丝体冻干品(AC-2) (AST含量8.8 mg/g),经单次口服投予大鼠后,评估可能产生之急性毒性反应,系参考卫生福利部食品药物管理署:健康食品安全性评估方法(1999)。试验共设置7组,分别为逆渗透水对照组、橄榄油对照组、试验组I (8 mg/kg bw AST)、试验组II (16 mg/kg bw AST)、试验组III (24 mg/kg bw AST)、试验组IV (2 g/kg bw AC-1)、试验组V (2 g/kg bw AC-2),以上试验组为AST建议剂量依序之17、34、52、17及42倍,每组各使用5只雌性Sprague-Dawley (SD)品系大鼠,以口服管喂方式单次投予试验物质或对照物质,并进行7天临床观察。试验结束后,牺牲大鼠并采集血液、肝脏与肾脏进行血清生化分析及病理学检查。临床症状部分,各组别所有大鼠均无出现异常之症状。动物体重部分,血清生化结果显示,对照组与其对应试验组均无显着差异。试验终结进行解剖肉眼检查及组织病理学检查,所有动物皆未发现任何与试验物质处理相关之病变。综合以上实验结果,试验物质AC-1、AC-2及AST在本试验条件下,大鼠均无发现任何与试验物质相关之急性毒性反应,AC-1及AC-2其LD50皆大于2 g/kgbw,AST则大于24 mg/kg bw,此结果将作为人体使用此产品之安全性参考。
樟芝菌丝体,Antrosterol,急性毒性
Hsin-Tung Chu1, Ting-Wei Lin1, Wen-Cherng Tsai2, Yu-Hsuan Kuo3, Chang Zhao4, Chin-Chu Chen1,5,6,7*
1Grape King Bio Ltd., Taoyuan Taiwan
2CRO Laboratory, Super Laboratory Co. Ltd., New Taipei Taiwan
3Department of Cosmetic Science, Chang Gung University of Science and Technology, Taoyuan Taiwan
4Shanghai Grape King Enterprise Co., Ltd., Shanghai
5Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, Taipei Taiwan
6Institute of Food Science and Technology, National Taiwan University, Taipei Taiwan
7Bioscience Technology, Chung Yuan Christian University, Taoyuan Taiwan
Received: Dec. 4th, 2020; accepted: Feb. 16th, 2021; published: Feb. 24th, 2021
This study was conducted to a single limit dose of acute toxicity assessment in SD rats according to the safety assessment guideline of Health Food announced by Ministry of Health and Welfare of Taiwan. The test substances in this experiment are Antrosterol (AST), Antrodia cinnamomea (A. cinnamomea) mycelium (AC-1) (AST content 3.0 mg/g) and Antrosterol-Enriched A. cinnamomea mycelium (AC-2) (AST content 8.8 mg/g) is provided by the Biotechnology Center of Grape King Inc. SD rats were divided into seven groups and were fed with RO water, olive oil, AST 8 (Group I), 16 (Group II), 24 (Group III) mg/kg bw, 2 g/kg bw A. cinnamomea powder (AST content 3.0 mg/g) (Group IV) and 2 g/kg bw Antrosterol-Enriched A. cinnamomea powder (AST content 8.8 mg/g) (Group V). The above test group is 17, 34, 52, 17 and 42 times the recommended dose of AST. Each group has 5 experiment animals. During the study period, no abnormality occurred in clinical signs and body weight. There was no significant difference in biochemistry parameters between the treatment and control group. Necropsy and histopathological examination showed no treatment-related change. The results of this study provide evidences for the use of A. cinnamomea fermentation product as a safe agent in functional food: The LD50 of A. cinnamomea powder, its Antrosterol, A. cinnamomea powder and Antrosterol-Enriched A. cinnamomea powder than 24 mg/kg bw, 2 g/kg bw and 2 g/kg bw in SD rats respectively.
Keywords:Antrodia cinnamomea, Antrosterol, Acute Toxicity
Copyright © 2021 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
http://creativecommons.org/licenses/by/4.0/
樟芝(Antrodia cinnamomea)是台湾特有的真菌之一,被认为具有调节免疫、保护肝脏的功能 [
图1. Antrosterol之化学结构
樟芝(A. cinnamomea) (BCRC 35398)菌种购自台湾新竹市食品工业发展研究所生物资源保存及研究中心。在生物安全柜中,将PDA平板上长好的樟芝菌丝,取0.5 cm大小正方的菌块,接种入2 L三角瓶中(内含1 L液体培养基),于25℃、100 rpm 中震荡培养2周,后接种入500公升级发酵槽,在90 rpm的搅拌,0.5 vvm的通气量、25℃中培养7天;经冷冻干燥、磨粉后得樟芝菌丝体发酵全液冻干粉(简称AC)。液体培养基组成:1.0%葡萄糖、0.5%黄豆粉、0.5%蛋白胨、0.3% (NH4)2SO4、0.1% MgSO4∙7H2O。AC再利用HPLC进行AST定量 [
樟芝液态发酵菌丝体粉末利用乙醇萃,萃取液进行减压浓缩,得到棕色萃取物,将其加水中(1升),然后用1升乙酸乙酯分配分离共3次。乙酸乙酯层萃取物(96 g)在硅胶上进行色谱分离,正己烷和乙酸乙酯作为洗脱剂。用10%的乙酸乙酯洗脱,然后用乙醇再结晶得到AST (5.1 g)。
8周龄Sprague-Dawley (SD)雌性大鼠,购自乐斯科生物科技(BioLASCO Taiwan Co., Ltd. Taipei, Taiwan);饲养条件为22℃ ± 2℃、55% ± 15%相对湿度、12小时光照/黑暗交替、饲料MFG (Oriental Yeast Co., Ltd., Tokyo, Japan)。本试验所执行之试验程序均经委托机构之动物试验管理委员会审阅。描述于试验报告中之操作程序均以避免并降低试验动物之不适、紧迫、疼痛之方式进行。本试验之实验动物管理委员会核可编号(IACUC NO.)为109-1ae。
本试验共设置7组,每组5只大鼠,试验组I、II、III投予试验物质AST,剂量分别为8 mg/kg bw、16 mg/kg bw及24 mg/kg bw,分别为人体AST建议口服剂量(人体4.5 mg/60 kg bw之17倍、34倍及52倍。试验组IV、V分别投予试验物质AC-1 (内含AST 3.0 mg/g)、AC-2 (内含AST 8.8 mg/g),剂量皆为2 g/kg bw,分别为人体AST建议口服剂量之17倍及42倍。试验组之试验物质皆为粉状,试验前试验组I、II、III之试验物质以橄榄油分别配制成0.8 mg/mL、1.6 mg/mL、2.4 mg/mL,试验组II、III之试验物质以逆渗透水配制成200 mg/mL。动物经禁食16小时,以管喂口服投予试验物质或对照物质,投予一次,投予体积为10 mL/kg bw。试验前记录体重。投药4小时后给予食物,并在经过投予试验或对照物质的第30分钟、4小时观察一次,之后每日观察一次至投药后第7天。试验结束时(第8天),将所有存活大鼠以吸入二氧化碳的方式牺牲并进行解剖及肉眼病变检查。
实验数据以平均值(mean)及标准偏差(standard deviation, SD)表示。试验数据利用SPSS统计软件中单因子变异数分析(One-Way ANOVA)后,以Duncan’s multiple range test进行各组别间数据之差异性分析,并以p值小于0.05作为显着差异水平。
体重为试验前及牺牲时(第8天)之体重,试验组与对照组相比皆无统计显着差异(p > 0.05) (表1)。详细记录大鼠的毒性症状、发生时间、症状持续时间以及复原状态。对照组及各试验组之大鼠,于试验期间皆无异常临床症状。试验期间无大鼠之死亡情形。
组别 | 体重(g) | ||||||
---|---|---|---|---|---|---|---|
逆渗透水 对照组 | 橄榄油 对照组 | 试验组I | 试验组II | 试验组III | 试验组IV | 试验组V | |
试验前 | 197.4 ± 7.3 | 201.0 ± 4.1 | 205.0 ± 4.7 | 205.4 ± 9.6 | 207.5 ± 6.3 | 202.6 ± 9.5 | 202.7 ± 5.2 |
第八天 | 208.7 ± 6.6 | 214.2 ± 6.4 | 223.1 ± 6.6 | 219.8 ± 15.6 | 222.4 ± 8.9 | 217.6 ± 11.4 | 217.2 ± 11.6 |
表1. 试验期间大鼠体重
Data expressed as mean ± S.D., n = 5. *Significant different from control group (p < 0.05).
牺牲后采血,将血液收集于不含抗凝剂之采血管,并离心分离血清,分析血中尿素氮(blood urea nitrogen, BUN)、天门冬氨酸转氨酶(aspartate aminotransferase, AST)及丙胺酸转氨酶(alanine aminotransferase, ALT)。结果显示,试验组与对照组相比无统计显着差异(p > 0.05) (表2)。
试验结束后,肉眼检查大鼠外观、口腔、颅腔及胸、腹腔内各组织及器官。采集各组之肾脏及肝脏组织,并将组织以10%中性福尔马林溶液进行固定及保存,所有存活大鼠牺牲后进行解剖及肉眼病变检查,结果显示并无任何肉眼病变(图2)。对照组及试验组之肾脏及肝脏无明显组织病理变化(图3)。非特异性组织病理判读结果为肝脏混合性炎症细胞浸润,散发,极微(图3(A));肝细胞质空泡,大泡性,极微(图3(B));肾脏慢性进展性肾病,极微(图3(C));肾脏单核炎症细胞浸润,间质,极微(图3(D))。以上病变严重性均为极微,此病变于各组组间之病变发生率无显着差异。
组别 | 血清生化分析 | ||||||
---|---|---|---|---|---|---|---|
逆渗透水 对照组 | 橄榄油 对照组 | 试验组I | 试验组II | 试验组III | 试验组IV | 试验组V | |
BUN (mg/dL) | 21.8 ± 3.2 | 23.2 ± 4.1 | 28.5 ± 5.8 | 24.0 ± 3.9 | 21.7 ± 3.4 | 22.4 ± 3.8 | 25.7 ± 2.6 |
AST (U/L) | 96.8 ± 16.0 | 85.2 ± 6.1 | 82.4 ± 7.6 | 90.4 ± 11.2 | 82.8 ± 7.5 | 91.4 ± 12.1 | 88.0 ±11.9 |
ALT (U/L) | 48.4 ± 8.7 | 41.6 ± 12.9 | 51.6 ± 4.7 | 48.4 ± 9.3 | 48.4 ± 9.3 | 45.4 ± 8.3 | 38.2 ± 3.4 |
表2. 试验结束后血清生化分析
Data expressed as mean ± S.D., n = 5. *Significant different from control group (p < 0.05).
图2. 各组大鼠肉眼病变检查剖图。A:逆渗透水对照组;B:橄榄油对照组;C:试验组I;D:试验组II;E:试验组III;F:试验组IV;G:试验组V
图3. 非特异性组织病理学变化。A:肝脏散发混合性炎症细胞浸润(100×);B:肝脏肝细胞质空泡(100×);C:肾脏单核炎症细胞浸润(100×);D:肾脏囊泡(100×)
试验后,各组平均正常增重,试验组体重与对照组相比无统计显着差异,于临床观察及病理判读无相关病变,故判定本试验物质对于试验动物体重无不良影响。试验期间各组大鼠均无异常临床症状与死亡等毒性症状,试验结束后之病理解剖及肉眼病理学检查发现并无任何异常病变,故判定本试验物质不会对试验动物造成组织肉眼病变。血清生化结果显示,试验组与对照组相比无统计显着差异。组织病理学检查试验动物肝脏及肾脏,观察之病变属于此物种常见非特异性病变,且对照组及试验组间之病变发生率及严重程度亦无显着差异,应为非特异性病变,与试验物质无关。
试验结果显示,投予试验物质后,各组试验大鼠均存活至试验结束,于动物临床观察、肉眼病变观察无明显异常。各组大鼠试验期间,各组平均正常增重,试验组体重与对照组相比无显着差异。血清生化结果中试验组与对照组相比无统计显着差异。于临床观察及组织病理判读无试验物质相关病变,故判定本试验物质对于试验动物体重及血清生化数值无不良影响。综合以上实验结果,试验物质AC-1、AC-2及AST在本试验条件下,大鼠均无发现任何与试验物质相关之急性毒性反应,AC-1及AC-2其LD50皆大于2 g/kg bw,AST则大于24 mg/kg bw,此结果可作为毒性试验时剂量参考范围之选择及人体使用此产品之安全性参考。
朱心彤,林定威,蔡文城,郭郁萱,赵 敞,陈劲初. 樟芝发酵菌丝体及活性成分Antrosterol纯化物之大鼠急性毒性分析 An Acute Toxicity Study of Antrosterol and Antrosterol-Enriched Antrodia cinnamomea Myceliumin SD Rats[J]. 食品与营养科学, 2021, 10(01): 63-69. https://doi.org/10.12677/HJFNS.2021.101009