HJFNS Hans Journal of Food and Nutrition Science 2166-613X Scientific Research Publishing 10.12677/HJFNS.2024.131012 HJFNS-80834 HJFNS20240100000_10492958.pdf 医药卫生 香杉芝液态发酵菌丝体粉末及其萃取物之口服急性毒性分析 Acute Oral Toxicity Test of Antrodia salmonea Powder Derived from Submerged Fermented Mycelia 朱 心彤 2 1 林 定威 2 1 陈 劲初 2 3 1 葡萄王生技股份有限公司，台湾 桃园 葡萄王生技股份有限公司，台湾 桃园；实践大学食品营养与保健生技学系，台湾 台北；中原大学生物科技学系，台湾 桃园 null 31 01 2024 13 01 93 101 © Copyright 2014 by authors and Scientific Research Publishing Inc. 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

香杉芝(Antrodia salmonea)为台湾特有种真菌，经液态发酵培养可大量生产香杉芝菌丝体发酵液粉末，目前投入研究指出具有抗癌、抗发炎和保护肝脏机能等良好功效，极适合开发成为健康食品之素材。本研究为首次针对香杉芝液态发酵菌丝体粉末及其萃取物进行单一剂量口服急性毒性试验，以香杉芝液态发酵菌丝体粉末(AS)和其水萃取物(AS-W)及酒精萃取物(AS-E)，经由口服投予Institute of Cancer Research (ICR)小鼠单一极限剂量，AS-E为3 ml/kg bw外，AS及AS-W皆为9 mg/kg bw，观察2周；以及经由口服投予Sprague-Dawley (SD)大鼠单一极限剂量皆为2000 mg/kg，观察1周，以测试可能产生的毒性变化。结果显示不论在ICR小鼠或是SD大鼠的试验中，在分别投予AS、AS-W及AS-E或对照溶液后，全部动物均存活，并在试验期间皆无发现任何临床症状，而各试验物质处理组在试验期间之体重生长情形均正常及无不良反应，于血清生化分析亦未发现相关异常。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。综观以上香杉芝液态发酵菌丝体粉末及其萃取物的研究试验结果，在喂食ICR小鼠之口服急性毒性的2周试验期间，其AS-E的LD50为大于3 ml/kg bw，AS及AS-W则皆大于9 g/kg bw；在SD大鼠之口服急性毒性剂量投予至2000 mg/kg，在1周试验期间未发现不良反应，此结果将作为人体使用此产品之安全性参考。 Antrodia salmonea (AS) is a type of fungus that is found only in Taiwan. By using liquid fermentation culture, large quantities of AS mycelium fermentation liquid powder can be produced. Current research suggests that it has beneficial effects in fighting cancer, reducing inflammation, and protecting the liver. It is highly suitable for use as an ingredient in healthy food products. This study is the first to conduct a test on the oral toxicity of the liquid fermented mycelium powder and its extract from AS. The alcohol extract (AS-E) was orally administered to Crl:CD1 (ICR) mice at a maximum single dose of 3 ml/kg bw, while mycelium powder (AS) and AS-W were both adminis-tered at a maximum single dose of 9 mg/kg bw. The mice were observed for 2 weeks. In addition, the extract was orally administered to Sprague-Dawley (SD) rats at a maximum single dose of 2000 mg/kg, and these rats were observed for 1 week to check for any potential toxic changes. The re-sults showed that no abnormalities were found in the ICR mice or SD rats during the tests. All ani-mals survived after being given AS, AS-W, AS-E, or the control solution, and no clinical symptoms were observed. The weight gain of each test substance treatment group was normal, with no ad-verse reactions. Furthermore, the serum biochemical analysis did not reveal any relevant abnor-malities. Macroscopic anatomical examination and histopathological examination were conducted after the experiment, and no impairment related to the handling of the test substances was found in any of the animals. Based on the above research and test results on the liquid fermentation myce-lium powder and its extract, a 2-week oral acute toxicity test was conducted on ICR mice, and the LD50 of AS-E was found to be greater than 3 ml/kg body weight. The LD50 of AS and AS-W were both found to be greater than 9 g/kg body weight. The acute oral toxicity dose in SD rats was 2000 mg/kg, and no adverse reactions were observed during the 1-week test. These results can serve as a safety reference for future human consumption.

 香杉芝，液态发酵，口服急性毒性, Antrodia salmonea Submerged Fermentation Acute Oral Toxicity Test 摘要

香杉芝(Antrodia salmonea)为台湾特有种真菌，经液态发酵培养可大量生产香杉芝菌丝体发酵液粉末，目前投入研究指出具有抗癌、抗发炎和保护肝脏机能等良好功效，极适合开发成为健康食品之素材。本研究为首次针对香杉芝液态发酵菌丝体粉末及其萃取物进行单一剂量口服急性毒性试验，以香杉芝液态发酵菌丝体粉末(AS)和其水萃取物(AS-W)及酒精萃取物(AS-E)，经由口服投予Institute of Cancer Research (ICR)小鼠单一极限剂量，AS-E为3 ml/kg bw外，AS及AS-W皆为9 mg/kg bw，观察2周；以及经由口服投予Sprague-Dawley (SD)大鼠单一极限剂量皆为2000 mg/kg，观察1周，以测试可能产生的毒性变化。结果显示不论在ICR小鼠或是SD大鼠的试验中，在分别投予AS、AS-W及AS-E或对照溶液后，全部动物均存活，并在试验期间皆无发现任何临床症状，而各试验物质处理组在试验期间之体重生长情形均正常及无不良反应，于血清生化分析亦未发现相关异常。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。综观以上香杉芝液态发酵菌丝体粉末及其萃取物的研究试验结果，在喂食ICR小鼠之口服急性毒性的2周试验期间，其AS-E的LD50为大于3 ml/kg bw，AS及AS-W则皆大于9 g/kg bw；在SD大鼠之口服急性毒性剂量投予至2000 mg/kg，在1周试验期间未发现不良反应，此结果将作为人体使用此产品之安全性参考。

 关键词

香杉芝，液态发酵，口服急性毒性

 Acute Oral Toxicity Test of Antrodia salmonea Powder Derived from Submerged Fermented Mycelia<sup> </sup>

Hsin-Tung Chu1, Ting-Wei Lin1, Chin-Chu Chen1,2,3

1Grape King Bio. Ltd., Taoyuan Taiwan

2Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, Taipei Taiwan

3Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan Taiwan

Received: Dec. 5th, 2023; accepted: Feb. 9th, 2024; published: Feb. 19th, 2024

 ABSTRACT

Antrodia salmonea (AS) is a type of fungus that is found only in Taiwan. By using liquid fermentation culture, large quantities of AS mycelium fermentation liquid powder can be produced. Current research suggests that it has beneficial effects in fighting cancer, reducing inflammation, and protecting the liver. It is highly suitable for use as an ingredient in healthy food products. This study is the first to conduct a test on the oral toxicity of the liquid fermented mycelium powder and its extract from AS. The alcohol extract (AS-E) was orally administered to Crl:CD1 (ICR) mice at a maximum single dose of 3 ml/kg bw, while mycelium powder (AS) and AS-W were both administered at a maximum single dose of 9 mg/kg bw. The mice were observed for 2 weeks. In addition, the extract was orally administered to Sprague-Dawley (SD) rats at a maximum single dose of 2000 mg/kg, and these rats were observed for 1 week to check for any potential toxic changes. The results showed that no abnormalities were found in the ICR mice or SD rats during the tests. All animals survived after being given AS, AS-W, AS-E, or the control solution, and no clinical symptoms were observed. The weight gain of each test substance treatment group was normal, with no adverse reactions. Furthermore, the serum biochemical analysis did not reveal any relevant abnormalities. Macroscopic anatomical examination and histopathological examination were conducted after the experiment, and no impairment related to the handling of the test substances was found in any of the animals. Based on the above research and test results on the liquid fermentation mycelium powder and its extract, a 2-week oral acute toxicity test was conducted on ICR mice, and the LD50of AS-E was found to be greater than 3 ml/kg body weight. The LD50of AS and AS-W were both found to be greater than 9 g/kg body weight. The acute oral toxicity dose in SD rats was 2000 mg/kg, and no adverse reactions were observed during the 1-week test. These results can serve as a safety reference for future human consumption.

Keywords:Antrodia salmonea, Submerged Fermentation, Acute Oral Toxicity Test

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 1. 引言

香杉芝(Antrodia salmonea)为台湾特有真菌，寄生于台湾特有的香杉树(Cunninghamia konishii)枯树干中，香杉芝与樟芝同属多孔菌科(Polyporaceae)，薄孔菌属(Antrodia)，两者外型极为相似 [ 1 ] ，由于樟芝价格昂贵，早期有许多不肖商人以掺入型态相似但价格相对较便宜的香杉芝，来进行贩卖以获取暴利，但因食用者日渐众多，口碑上也不遑多让，再加上其生长速度缓慢，目前尚无法以人工经济培养，只能靠野外采集方式获得 [ 2 ] 。香杉芝的特性与樟芝相似，而樟芝液态发酵菌丝体早已成功开发，樟芝生理活性特性，及药理有关功效成分也研究的相当透彻 [ 3 ] ，因此，同属的香杉芝应也具有成为开发潜力的新兴菇菌类 [ 4 ] [ 5 ] ，液态发酵的香杉芝菌丝体中发现许多的马来酸衍生物 [ 6 ] ，其中的潜力活性成分Antrodin C，具有护肝 [ 6 ] 、对抗癌细胞(乳癌、肺癌及大肠癌) [ 7 ] [ 8 ] [ 9 ] [ 10 ] 、可预防或治疗糖尿病相关的心血管疾病 [ 11 ] 、阿兹海默症 [ 12 ] 等活性，但在安全性的研究甚微。本试验目的为初步进行香杉芝的安全性试验，依据行政院卫生福利部「健康食品安全性评估方法」 [ 13 ] [ 14 ] 及行政院卫生署药品非临床试验安全性规范 [ 15 ] [ 16 ] [ 17 ] ，以香杉芝液态发酵菌丝体粉末及其萃取物，经单一极限剂量喂食小鼠及大鼠后，分析评估口服急性毒性的影响。

 2. 材料与方法 2.1. 液态发酵菌丝体发酵条件 [<xref ref-type="bibr" rid="hanspub.80834-ref18">18</xref>] 及萃取物制备方法

香杉芝(A. salmonea) (BCRC 36937)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。在无菌操作台中，将PDA平板上长好的香杉芝菌丝体，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，液体培养基组成为1.0%葡萄糖、0.5%黄豆粉、0.5%蛋白胨、0.3% (NH4)2SO4、0.1% MgSO4∙7H2O)，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天后，可得到香杉芝菌丝体液态发酵液；经冷冻干燥、磨粉后可得到香杉芝液态发酵菌丝体粉末(AS)。取香杉芝液态发酵菌丝体粉末加入10倍量的纯水，加热至100℃萃取1小时，离心取上清液，经冷冻干燥后可得香杉芝液态发酵菌丝体水萃取物(AS-W)粉状物。取香杉芝液态发酵菌丝体粉末加入10倍量的酒精，以超音波震荡萃取1小时，离心取上清液，以减压浓缩抽干后之液状物即为香杉芝液态发酵菌丝体酒精萃取物(AS-E)。

 2.2. 试验动物 2.2.1. ICR小鼠组

5~6周龄，雌雄性各半共40只，购自乐斯科生物科技股份有限公司。投予试验物质之前，进行动物称重及临床观察。挑选健康且体重相近之动物，进行随机分组。每组10只雌雄性各半，共4组。经食品工业发展研究所实验动物照护及使用委员会审核通过之动物实验审查字号「动管字第105-01号」。

 2.2.2. SD大鼠组

7~8周龄，雄性20只，购自乐斯科生物科技股份有限公司。投予试验物质之前，进行动物称重及临床观察。挑选健康且体重相近之动物，依体重大小，进行随机分组。每组5只雄鼠，共5组。实验动物管理及使用委员会核准之计划书编号「CTPS-15-004」。

 2.3. 试验设计 2.3.1. 分组与剂量

按照表1进行试验动物分组与试验物质剂量设计。

 2.3.2. 试验物质投与

依ICR小鼠组及SD大鼠组之体重计算所需之喂食试验物质数量，AS及AS-W为粉末样品，经注射用水配制成悬浮液后，分两次喂食以达剂量；AS-E为液状物样品，于ICR小鼠组试验时直接以设计剂量口服喂食，SD大鼠组试验则以玉米油制成悬浮液后直接喂食。动物于投药前一天禁食至少16小时，按照动物投药当天之体重计算投予之体积。

Experimental design of oral acute toxicity test for AS, AS-W and AS-
试验动物 试验物质 剂量 动物数量(只)
ICR小鼠 Control (water) 10 ml/kg 雄5/雌5
AS 9 g/kg 雄5/雌5
AS-W 9 g/kg 雄5/雌5
AS-E 3 ml/kg 雄5/雌5
SD大鼠 Control (water) 15 ml/kg 雄5
Control (oil) 10 ml/kg 雄5
AS 2000 mg/kg 雄5
AS-W 2000 mg/kg 雄5
AS-E 2000 mg/kg 雄5

表1. 香杉芝液态发酵菌丝体粉末及其萃取物口服急性毒性试验之试验设计

1. Control (water)：对照组(注射用水)；2. Control (oil)：对照组(玉米油)；3. AS：香杉芝液态发酵菌丝体粉末；4. AS-W：香杉芝液态发酵菌丝体水萃取物；5. AS-E：香杉芝液态发酵菌丝体酒精萃取物。

 2.4. 试验观察及纪录 2.4.1. 临床观察

试验观察期间，每日观察及记录试验动物之临床症状，包括动物中毒症状、异常症状的发生、复原及死亡时间等；ICR小鼠观察期为2周，SD大鼠观察期为1周。

 2.4.2. 动物秤重

于试验第一天投药前及试验结束前(ICR小鼠组2周，SD大鼠组1周)进行秤重。

 2.4.3. 血清生化分析

所有试验动物于试验结束前一天禁食至少16小时，于试验结束当天，进行二氧化碳安乐死，将血液收集在不含任何抗凝剂的集血管中，离心并分离血清后，以血清生化自动分析仪(Vitros® 350, US)测定丙氨酸转氨酶(alanine aminotransferase, ALT)、天门冬氨酸转氨酶(aspartate aminotransferase, AST)、尿素氮(blood urea nitrogen, BUN)等三项生化值。

 2.4.4. 解剖与肉眼检查及组织病理切片

试验结束当天，所有动物于采血、放血后进行解剖，以肉眼检查外观及所有组织器官，并拍照记录。肝脏、肾脏及脾脏以10%中性福马林缓冲液固定保存。将所有固定之肝脏、肾脏及脾脏进行组织修切、蜡块制作、切片、染色(Hematoxylin and Eosin stain)，再进行病理镜检观察。

 2.5. 统计分析

试验数据以平均值(Mean) ± 平均数标准误差(SEM)来表示，以Dunnett t-test与对照组比较，当p < 0.05 表示具有显著性差異。

 3. 结果

本研究旨在评估以液态发酵生产所得到的香杉芝液态发酵菌丝体粉末及其萃取物，经单一极限剂量投予喂食ICR小鼠及SD大鼠后，分析对其口服急性毒性的影响。参考卫生福利部公告的「健康食品安全性评估方法」及药品非临床试验安全性规范，利用管喂方式喂食香杉芝液态发酵菌丝体粉末及其萃取物，藉由对动物临床观察，体重变化，血清生化分析，解剖与肉眼检查及组织病理切片等，评估对小鼠及大鼠的口服急性毒性影响。

3.1. 物临床观察与死亡率

在喂食所有试验物质及对照溶液后，试验期间各组试验动物均存活、活动状况正常，且无观察发现到任何临床异常症状。

 3.2. 体重变化

试验动物之平均体重增长百分率总结于表2。各试验物质处理组在试验期间之体重与对照组无显著差异(p > 0.05)。

Body weight of rats at the beginning and the end of AS, AS-W and AS-
试验动物 试验物质 体重增长百分率
ICR小鼠 Control (water) 13.4 ± 1.6/9.5 ± 1.6
AS 14.2 ± 1.0/10.4 ± 2.6
AS-W 14.0 ± 1.4/6.5 ± 1.8
AS-E 16.4 ± 0.6/10.3 ± 3.3
SD大鼠 Control (water) 47.8 ± 4.3
Control (oil) 41.8 ± 7.9
AS 43.8 ± 7.5
AS-W 41.8 ± 6.8
AS-E 39.4 ± 7.2

表2. 香杉芝液态发酵菌丝体粉末及其萃取物试验期间动物体重增长百分率变化

平均体重以平均值 ± 标准偏差表示(n = 5)，雄、雌分别计算。

 3.3. 血清生化分析

各项血清生化分析结果总结于表3。各试验物质处理组与对照组动物于所有血清生化分析项目的结果皆无显著差异(p > 0.05)。

Serum biochemical analysis values during the test of AS, AS-W and AS-
试验动物 试验物质 ALT (U/L) AST (U/L) BUN (mg/dL)
ICR小鼠 Control (water) 37.4 ± 2.8/45.2 ± 1.9 76.0 ± 7.6/75.4 ± 3.6 29.6 ± 1.3/22.8 ± 2.0
AS 31.4 ±3.6/34.2 ± 2.2 81.4 ± 7.9/98.4 ± 19.0 22.2 ± 1.0/24.0 ± 2.3
AS-W 31.0 ± 2.2/36.2 ± 3.7 78.0 ± 7.2/87.8 ± 14.7 21.8 ± 1.1/2.64 ± 1.5
AS-E 38.6 ± 2.4/32.6 ± 2.5 67.6 ± 1.2/69.8 ± 18.1 24.2 ± 1.2/18.8 ± 0.8
SD大鼠 Control (water) 60.6 ± 13.1 111.8 ± 24.0 22.18 ± 3.56
Control (oil) 149.2 ± 185.9 285.2 ± 381.4 22.96 ± 4.19
AS 68.4 ± 18.8 155.2 ± 62.9 21.40 ± 4.11
AS-W 68.0 ± 24.4 121.6 ± 35.3 22.38 ± 2.96
AS-E 60.4 ± 9.8 127.8 ± 44.1 21.14 3.48

表3. 香杉芝液态发酵菌丝体粉末及其萃取物试验期间之血清生化分析值

1. ALT：丙氨酸转氨酶(alanine aminotransferase)、AST：天门冬氨酸转氨酶(aspartate aminotransferase)、BUN：尿素氮(blood urea nitrogen)；2. 血清生化分析值以平均值 ± 平均數之标准误差表示(n = 5)，雄、雌分别计算。

 3.4. 肉眼病变解剖检查

肉眼检查结果图片总结于图1及图2。试验终结进行解剖与肉眼检查，所有动物皆未发现与试验物质处理相关之病变。各试验物质之处理组与对照组比较，ICR小鼠组与SD大鼠组之肝脏、肾脏及脾脏皆无明显与试验物质处理相关之病变。

图1. ICR小鼠组喂食香杉芝液态发酵菌丝体粉末及其萃取物后解剖肉眼检查结果

图2. SD大鼠组喂食香杉芝液态发酵菌丝体粉末及其萃取物后解剖肉眼检查结果

 3.5. 织病理评估

组织病理评估结果图片总结于图3及图4。ICR小鼠组经由组织病理切片结果显示，各试验物质之处理组之肾脏、肝脏及脾脏均无明显与试验物质有关之组织病理变化，如图3。部分鼠只出现弥漫性糖原浸润，与未禁食完全有关，与试验物质无关。其他如肝脏轻微局部或多发局部坏死等病变，于对照组与各处理组病变程度及发生率并无相关性，为非特异性病变，与试验物质无关。SD大鼠组经由组织病理切片结果显示，各试验物质之处理组之肾脏及肝脏均无与试验物质有关之病变，于SD大鼠组肝肾观察到的病变属于此物种常见之非特异性病变，如图4，病变严重性均为极微，且对照组及各试验组间之病变发生率亦无显著差异。

图3. ICR小鼠组喂食香杉芝液态发酵菌丝体粉末及其萃取物后病理组织结果

图4. SD大鼠组喂食香杉芝液态发酵菌丝体粉末及其萃取物后病理组织结果

 4. 结论

本研究为首次针对香杉芝液态发酵菌丝体粉末及其萃取物进行单一剂量口服急性毒性试验，依据行政院卫生福利部「健康食品安全性评估方法」及药品非临床试验安全性规范，分别经由口服投予ICR小鼠及SD大鼠单一极限剂量，以测试可能产生之毒性变化。结果显示不论ICR小鼠组或是SD大鼠组试验中，在分别投予AS、AS-W及AS-E或对照溶液后，全部的动物均存活，并在试验期间皆无发现任何临床症状，而各试验物质处理组在试验期间之体重生长情形均无不良反应；于血清生化分析亦未发现相关异常。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。

综观以上香杉芝液态发酵菌丝体粉末及其萃取物的研究试验结果，喂食ICR小鼠的口服急性毒性的2周试验期间，其AS-E的LD50为大于3 ml/kg bw，AS及AS-W则皆大于9 g/kg bw；在SD大鼠的口服急性毒性剂量投予至2000 mg/kg，在1周试验的期间内皆未发现不良反应，此结果将可作为人体使用此素材的安全性参考依据。

 文章引用

朱心彤,林定威,陈劲初. 香杉芝液态发酵菌丝体粉末及其萃取物之口服急性毒性分析 Acute Oral Toxicity Test of Antrodia salmonea Powder Derived from Submerged Fermented Mycelia[J]. 食品与营养科学, 2024, 13(01): 93-101. https://doi.org/10.12677/HJFNS.2024.131012

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