北蟲草與蜜環菌複方萃取物之安全性評估

Abstract

北蟲草 (Cordyceps militaris, CM) 是著名的傳統中藥材之一。由於北蟲草為冬蟲夏草的近緣種，且許多研究顯示，其功能性成分大致與冬蟲夏草相近，故逐漸取代之。現有文獻指出，北蟲草具有抗腫瘤、抗憂鬱、神經保護作用及調節免疫等功效。蜜環菌 (Armillaria mellea, AM) 具有抗暈眩、神經保護作用、免疫調節及抗腫瘤等功效，是一種與天麻 (Gastrodia elata Blume) 共生的食藥用真菌。因其具有和天麻相似的功效，故逐漸受到重視，具開發成保健食品之潛力。由於目前尚缺乏相關安全性研究證明北蟲草與蜜環菌之複方萃取物 (CM-AM) 不具毒性反應，故本研究依照衛福部食藥署公告的健康食品安全性評估方法規定，進行第二類之基因毒性及28天亞急性毒性試驗。藉由以上試驗分析此複方萃取物是否會對生物體造成亞急毒性及基因毒性傷害，以作為日後食用安全性之參考依據。由Ames test 的實驗結果顯示，CM-AM在0.3-5.0 mg/plate 之劑量下對Salmonella typhimurium TA98、TA100、TA102、TA1535及TA1537 菌株之回復突變菌數，與負控制組相比均無顯著性差異。而體外哺乳動物染色體異常分析試驗結果顯示，CM-AM在0.3-5.0 mg/ml 等劑量處理下，並未造成中國倉鼠卵巢細胞 (Chinese hamster ovary cells, CHO-K1) 產生染色體變異增加的情形。在微核試驗中的結果顯示各試驗劑量下之網狀紅血球比例及微核發生率均和負控制組相比無顯著性差異。此外，在28天亞急性毒性試驗中，SD大鼠在管餵北蟲草與蜜環菌複方萃取物後，並無明顯臨床症狀以及死亡情形發生，且在血液學檢查、血清生化分析及組織病理切片分析均無明顯與試驗物質有關的不良反應及病變情形。綜合上述結果，推知 CM-AM並不會導致基因毒性及亞急毒性產生，故本試驗之無毒性顯示劑量 (no-observed-adverse-effect level, NOAEL) 為大於高劑量組 (252 mg/kg bw/day 北蟲草及504 mg/kg bw/day 蜜環菌) 複方濃度。

Cordyceps militaris (CM) is one of the most famous traditional Chinese medicinal mushrooms. Cordyceps militaris has been increasingly viewed as a substitute for Cordyceps sinensis because of not only the relative species but also their similar active components. Currently, many studies have shown that CM has a variety of effects such as antitumor, antidepressant-like, neuroprotective and immunomodulatory effects. Armillaria mellea (AM) has many pharmacological effects such as anti-dizziness, brain protection, antitumor and immunomodulatory effects. AM is an edible and medicinal mushroom, and it grows in a symbiotic relationship with Gastrodia elata Blume. As AM has shown similar efficacy to Gastrodia elata, it has gradually drawn attention and has the potential to be developed into functional foods. So far, it still lacks of relevant safety evaluation studies to prove that the CM and AM extract formula (CM-AM) has no toxic effect. Therefore, the objective of this study is to perform genotoxicity and 28-day subacute toxicity test, which are requirements of the second category according to the regulations of Health Food Safety Assessment announced by the Ministry of Health and Welfare in Taiwan. These tests can analyze whether the CM-AM could cause subacute toxicity or genotoxic damage to organisms, and the results could be as a reference for the safety of this formula in the future. The result of Ames test showed that the treatment with CM-AM at 0.3-5.0 mg/plate did not significantly increase in revertant colonies of Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 strains. The result of in vitro mammalian chromosome aberration test showed that the treatment with CM-AM at 0.3-5.0 mg/ml did not increase in chromosome aberrations on Chinese hamster ovary cells (CHO-K1). The result in the micronucleus tests indicated that there was no statistically significant difference in the proportion of reticulocyte and the incidence of micronuclei between the CM-AM treated groups and the negative control group. Moreover, in the 28-day subacute toxicity test, there were no obvious clinical symptoms and death occurred in the SD rats fed with CM-AM, and showed no significant adverse reactions and lesions associated with the test substance in hematology, serum biochemistry, and histopathological examination. Based on above results, it could conclude that the CM-AM did not cause genotoxicity and subacute toxicity. Therefore, the no-observed-adverse-effect level (NOAEL) was higher than the high dose group which is composed of 252 mg/kg bw/day Cordyceps militaris and 504 mg/kg bw/day Armillaria mellea.