高眼压是造成青光眼重要的原因之一,若长期眼内压力过高压迫视神经,视神经会受损、萎缩,影响视力,严重情形可能造成失明。蝉花(Cordyceps cicadae )是使用历史悠久的中药材,具有护肾、调节血糖与护眼等生理活性,不过尚未有针对降低眼压的文献报导。因此本研究利用液态发酵方式培养蝉花菌丝体,以大白兔动物模式评估食用安全性后,再评估其降眼压的能力。安全性部分,以口服投予蝉花发酵菌丝体低剂量2.5 mg/kg b.w.及高剂量25 mg/kg b.w.,连续喂食10天后,采血分析以及进行组织病理切片评估。结果显示,在血液学分析、血清生化分析以及肝、肾病理组织切片中,没有观察到与试验物质相关的病变。在降眼压测试中,单次口服投予不同发酵天数的蝉花发酵菌丝体单一剂量,在喂食后不同时间点量测其眼内压。结果显示,不同发酵天数的蝉花发酵菌丝体在喂食后第1小时及第3小时皆可降低兔子眼内压。综合上述,蝉花发酵菌丝体在兔子口服安全性试验中不具短期连续喂食毒性,且对降眼压有正面的效果。 High intraocular pressure is one of the main risk factors for glaucoma. Chronic intraocular pressure may result in compression of the optic nerve with a consequence of optic nerve damage and atrophy, which can cause problems with vision and blindness in severe cases. Cordyceps cicadae is a traditional Chinese medicine that has a long history of use. It has important physiological roles in renal protection, blood sugar regulation, and eye health care. However, there is limited evidence regarding the effect of Cordyceps cicadae on intraocular pressure reduction. Therefore, this study aims to use Cordyceps cicadae mycelia using liquid fermentation and evaluate its safety and ability to lower intraocular pressure in the animal model of white rabbits. For safety assessment, 2.5 (low dose) and 25 (high-dose) mg/kg b.w. of Cordyceps cicadae mycelia were orally administered. After continuous feeding for 10 days, blood and tissues were collected for analysis. Results showed that in hematological, serum biochemical, and liver and kidney histological analyses, no observable lesions related to the test substance were found. For efficacy assessment, a single dose of Cordyceps cicadae mycelia from different fermentation days was orally administered, and the intraocular pressures of the rabbits were measured at different time points after feeding. All rabbits showed reduced intraocular pressure reduction in the first and third hour after feeding Cordyceps cicadae mycelia from different fermentation days. Taken together, these findings suggest that short-term oral supplementation of Cordyceps cicadae mycelia shows no toxicity in rabbits and has a beneficial effect on intraocular pressure-lowering efficacy.
高眼压是造成青光眼重要的原因之一,若长期眼内压力过高压迫视神经,视神经会受损、萎缩,影响视力,严重情形可能造成失明。蝉花(Cordyceps cicadae)是使用历史悠久的中药材,具有护肾、调节血糖与护眼等生理活性,不过尚未有针对降低眼压的文献报导。因此本研究利用液态发酵方式培养蝉花菌丝体,以大白兔动物模式评估食用安全性后,再评估其降眼压的能力。安全性部分,以口服投予蝉花发酵菌丝体低剂量2.5 mg/kg b.w.及高剂量25 mg/kg b.w.,连续喂食10天后,采血分析以及进行组织病理切片评估。结果显示,在血液学分析、血清生化分析以及肝、肾病理组织切片中,没有观察到与试验物质相关的病变。在降眼压测试中,单次口服投予不同发酵天数的蝉花发酵菌丝体单一剂量,在喂食后不同时间点量测其眼内压。结果显示,不同发酵天数的蝉花发酵菌丝体在喂食后第1小时及第3小时皆可降低兔子眼内压。综合上述,蝉花发酵菌丝体在兔子口服安全性试验中不具短期连续喂食毒性,且对降眼压有正面的效果。
蝉花,液态发酵,安全性,降眼压,兔眼内压模式
Jui-Hsia Hsu1, Chin-Chu Chen1,2,3,4*
1Grape King Bio Ltd., Taoyuan Taiwan
2Institute of Food Science and Technology, National Taiwan University, Taipei Taiwan
3Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, Taipei Taiwan
4Bioscience Technology, Chung Yuan Christian University, Taoyuan Taiwan
Received: Dec. 9th, 2020; accepted: Feb. 13th, 2021; published: Feb. 23rd, 2021
High intraocular pressure is one of the main risk factors for glaucoma. Chronic intraocular pressure may result in compression of the optic nerve with a consequence of optic nerve damage and atrophy, which can cause problems with vision and blindness in severe cases. Cordyceps cicadae is a traditional Chinese medicine that has a long history of use. It has important physiological roles in renal protection, blood sugar regulation, and eye health care. However, there is limited evidence regarding the effect of Cordyceps cicadae on intraocular pressure reduction. Therefore, this study aims to use Cordyceps cicadae mycelia using liquid fermentation and evaluate its safety and ability to lower intraocular pressure in the animal model of white rabbits. For safety assessment, 2.5 (low dose) and 25 (high-dose) mg/kg b.w. of Cordyceps cicadae mycelia were orally administered. After continuous feeding for 10 days, blood and tissues were collected for analysis. Results showed that in hematological, serum biochemical, and liver and kidney histological analyses, no observable lesions related to the test substance were found. For efficacy assessment, a single dose of Cordyceps cicadae mycelia from different fermentation days was orally administered, and the intraocular pressures of the rabbits were measured at different time points after feeding. All rabbits showed reduced intraocular pressure reduction in the first and third hour after feeding Cordyceps cicadae mycelia from different fermentation days. Taken together, these findings suggest that short-term oral supplementation of Cordyceps cicadae mycelia shows no toxicity in rabbits and has a beneficial effect on intraocular pressure-lowering efficacy.
Keywords:Cordyceps cicadae, Liquid Fermentation, Safety, Intraocular Pressure-Lowering, White Rabbit Animal Model
Copyright © 2021 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
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长期高眼压为青光眼的主要病因之一,眼睛房水的分泌速度过快或房水排出受到阻塞,即会造成眼内压(Intraocular pressure, IOP)异常升高,长期高眼压会压迫视神经致使其受损、萎缩,进而影响视力,为目前导致失明及视觉损伤的主要原因之一 [
蝉花(Cordyceps cicadae)为一种虫生真菌,是使用历史悠久的中药材,古籍记载其味甘、无毒,晒干后可入药,有散风热、镇惊、明目、退翳障、透疹之功效。现代药理学实验也表明,天然蝉花或其人工培养物具有调节免疫力、护肾、抗肿瘤、降血糖、镇静、镇痛解热以及抗疲劳 [
然而,目前却还没有蝉花与降眼压相关的研究报导,更无以蝉花发酵菌丝体单方作为研究样品。此外,蝉花液态发酵菌丝体的口服安全性试验已有动物90天慢性毒性试验 [
蝉花菌种取自食品工业研究所生物资源保存及研究中心(新竹,台湾),编号BCRC MU30106。取马铃薯葡萄糖洋菜平板培养基上之菌丝接种于内含1.2 L培养基(2%葡萄糖、1%酵母萃出物、1%黄豆粉、pH 6.0)的2 L烧瓶中,以转速120 rpm、温度25℃的条件下培养三天,至发酵液呈亮紫色后,再将培养出之菌丝体发酵液接种至内含400 L相同培养基的500 L发酵槽中,在温度25℃条件下培养三天,之后再接种至含16吨相同培养基的20吨发酵槽,于温度25℃下培养七天后,加热至100℃,收取其菌丝体发酵液,于55℃下减压浓缩,经冷冻干燥、磨粉,制成粉末状样品,保存于4℃备用。
本试验之实验动物皆通过管理委员会(IACUC)申请核可。本试验使用之动物为成年、8~12周龄、雌性之新西兰大白兔(New Zealand White (NZW) Rabbit),安全性试验之动物购自台湾彰化惠钧繁殖场,降眼压试验之动物购自台湾苗栗家畜卫生试验所。
实验动物以耳朵标记试验编号以区分实验动物个体,并于饲育笼上标示笼号、品系、周龄、动物试验编号、试验组别、入室日期及试验期间。饲养区之光照时间自动控制为12小时亮、12小时暗,室温23℃ ± 2℃、相对湿度40%~70%。动物可自由取得充分之食物及饮水。实验动物经检疫、驯化1周后开始进行实验。实验开始前,依据实验动物体重随机分组,使各组平均体重及体重分布趋势相近。
兔子分为空白组、对照组、低剂量组(2.5 mg/kg b.w.)及高剂量组(25 mg/kg b.w.)等4组,每组5只动物。低剂量组及高剂量组每日喂食蝉花发酵菌丝体,连续喂食10天后,采集血液做血液学分袭击血液生化分析,并牺牲取其肝脏与肾脏组织制作切片,进行组织病理判读。
依据安全性试验结果,取单一剂量进行单次口服喂食。实验动物分为2组,依组别分别以口服投予培养3天(D3)及7天(D7)之蝉花发酵菌丝体1次,投予剂量25 mg/kg b.w.,并于投予试验物质前1小时(0 hr)、后1小时(1 hr)及后3小时(3 hr)分别测量右眼之眼内压。投予样品前1小时之右眼眼内压分别作为各时间点右眼眼内压之基准量,并分别比较投药后之眼内压(IOP)、眼内压变化量(Delta IOP)、眼内压变化量百分率(percentage of Delta IOP)之表现。
眼内压变化量(Delta IOP):为各时间点相较于0 hr之眼内压变化量
Δ IOP = IOPtimepoint − IOP 0 hr
Delta IOP比例变化:为各时间点之ΔIOP相较于0 hr之IOP变化量比例
Percentageof Δ IOP = Δ IOP / IOP 0 hr
试验数据以平均值(Mean) ± 标准偏差(standard error of mean, S.D.)来表示。动物之血液学分析及血清生化值分析等数据,均利用SPSS统计软件中单因子变异数分析(One-Way ANOVA)及Duncan’s multiple range test分析各组别间数据之差异性,并以p值小于0.05表示具统计上显著差异。
试验数据以平均值(Mean) ± 标准偏差(standard error of mean, SEM)来表示。采用Paired Sample t-test与Student’s t-test比较是否具差异性,以p值小于0.05表示两试验组之间具有统计上显著差异。
血液学分析结果如表1。在所有血液学分析项目中,蝉花发酵菌丝体低剂量组与高剂量组,与空白组以及对照组间相比,均无统计差异(p > 0.05)。血清生化分析结果如表2。高剂量组的肌酸酐(creatinine)与对照组相比较低(p < 0.05),其余血清生化生析项目于低剂量组及高剂量组中,均与空白组及对照组间无统计差异。组织病理学检查结果如表3。空白组、对照组、低剂量组及高剂量组,肝脏及肾脏均无明显与试验物质有关之组织病理变化。
Item | 空白组 | 对照组 | 低剂量组 | 高剂量组 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Naïve | Vehicle | Low-dose 2.5 mg/kg b.w | High-dose 25 mg/kg b.w. | |||||||||
WBC (103/μL) | 12.03 | ± | 1.24 | 12.69 | ± | 1.89 | 12.20 | ± | 3.24 | 12.02 | ± | 2.23 |
RBC (106/μL) | 9.48 | ± | 0.47 | 9.16 | ± | 0.25 | 9.63 | ± | 0.34 | 9.66 | ± | 0.65 |
Hemoglobin (g/dL) | 16.52 | ± | 0.50 | 16.02 | ± | 0.43 | 16.44 | ± | 0.36 | 16.38 | ± | 0.79 |
Hematocrit (%) | 49.42 | ± | 1.84 | 48.08 | ± | 1.69 | 49.34 | ± | 0.62 | 48.68 | ± | 1.89 |
MCV (fL) | 52.24 | ± | 2.68 | 52.56 | ± | 2.90 | 51.27 | ± | 1.32 | 50.52 | ± | 2.39 |
MCH (pg) | 17.44 | ± | 0.63 | 17.50 | ± | 0.59 | 17.10 | ± | 0.69 | 17.00 | ± | 0.51 |
MCHC (g/dL) | 33.44 | ± | 0.70 | 33.34 | ± | 0.89 | 33.32 | ± | 0.76 | 33.62 | ± | 0.74 |
Platelet (103/μL) | 960.0 | ± | 104.7 | 969.2 | ± | 97.1 | 955.8 | ± | 150.7 | 992.8 | ± | 167.8 |
Neutrophil (%) | 20.66 | ± | 6.60 | 22.60 | ± | 8.54 | 20.50 | ± | 9.04 | 23.50 | ± | 5.73 |
Lymphocyte (%) | 73.16 | ± | 6.35 | 69.84 | ± | 9.58 | 66.86 | ± | 21.69 | 69.80 | ± | 6.61 |
Monocyte (%) | 4.80 | ± | 0.46 | 5.88 | ± | 1.27 | 5.08 | ± | 1.27 | 5.22 | ± | 0.92 |
Eosinophil (%) | 1.26 | ± | 0.55 | 1.54 | ± | 0.49 | 1.40 | ± | 0.46 | 1.36 | ± | 0.78 |
Basophil (%) | 0.12 | ± | 0.04 | 0.14 | ± | 0.05 | 0.16 | ± | 0.09 | 0.12 | ± | 0.04 |
表1. 血液学分析
所有数据均以平均值 ± 标准偏差表示,n = 5。Data expressed as mean ± standard deviation (n = 5). WBC, white blood cell. RBC, red blood cell. MCV, mean corpuscular volume. MCH, mean corpuscular hemoglobin. MCHC, mean corpuscular hemoglobin concentration.
Item | 空白组 | 对照组 | 低剂量组 | 高剂量组 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Naïve | Vehicle | Low-dose 2.5 mg/kg b.w | High-dose 25 mg/kg b.w. | |||||||||
Glucose (mg/dL) | 396.2 | ± | 101.4 | 407.4 | ± | 90.1 | 392.4 | ± | 43.7 | 342.0 | ± | 74.9 |
BUN (mg/dL) | 19.1 | ± | 2.2 | 19.2 | ± | 3.3 | 18.8 | ± | 1.4 | 19.4 | ± | 2.6 |
Creatinine (mg/dL) | 1.18 | ± | 0.11 | 1.33 | ± | 0.19 | 1.18 | ± | 0.08 | 1.09 | ± | 0.11* |
AST (U/L) | 62.4 | ± | 98.2 | 23.4 | ± | 6.1 | 19.6 | ± | 4.3 | 19.4 | ± | 3.5 |
ALT (U/L) | 68.6 | ± | 36.3 | 57.2 | ± | 15.6 | 60.2 | ± | 13.6 | 48.6 | ± | 11.2 |
Total protein (g/dL) | 6.1 | ± | 0.2 | 6.2 | ± | 0.3 | 5.9 | ± | 0.3 | 5.9 | ± | 0.4 |
Albumin (g/dL) | 4.06 | ± | 0.17 | 4.02 | ± | 0.15 | 3.94 | ± | 0.23 | 4.04 | ± | 0.26 |
ALP (U/L) | 125.8 | ± | 12.1 | 116.6 | ± | 23.7 | 181.4 | ± | 82.8 | 147.6 | ± | 27.6 |
Cholesterol (mg/dL) | 71.0 | ± | 15.9 | 85.0 | ± | 23.4 | 65.0 | ± | 21.8 | 68.8 | ± | 19.8 |
Triglyceride (mg/dL) | 63.2 | ± | 8.2 | 117.8 | ± | 96.2 | 59.2 | ± | 17.4 | 68.6 | ± | 13.0 |
Calcium (mg/dL) | 14.6 | ± | 0.3 | 14.7 | ± | 0.6 | 14.6 | ± | 0.4 | 14.7 | ± | 1.1 |
Phosphorus (mg/dL) | 8.3 | ± | 0.9 | 8.5 | ± | 0.5 | 8.2 | ± | 0.3 | 8.4 | ± | 0.9 |
Sodium (meq/L) | 142.8 | ± | 2.3 | 142.4 | ± | 2.7 | 143.2 | ± | 0.8 | 143.0 | ± | 4.4 |
表2. 血清生化分析
所有数据均以平均值 ± 标准偏差表示,n = 5。*与对照组比较具显著性差异(p < 0.05)。Data expressed as mean ± standard deviation (n = 5). BUN, blood urea nitrogen. AST, aspartate transaminase. ALT, alanine transaminase. ALP, alkaline phosphatase. *Significantly different from the control group (P < 0.05).
Organ | Lesions | 空白组 | 对照组 | 低剂量组 | 高剂量组 |
---|---|---|---|---|---|
Naïve | Vehicle | Low-dose 2.5 mg/kg b.w | High-dose 25 mg/kg b.w. | ||
Liver | Mixed type inflammatory cell infiltration, minimal, random | 1/5 | 5/5 | 2/5 | 2/5 |
Kidney | Mononuclear cell infiltration, minimal, interstitial | - | 2/5 | - | 1/5 |
表3. 肝脏及肾脏病理检查
兔子喂食样品D3及D7后第1及第3小时皆可观察到眼压下降(如图1),喂食蝉花发酵菌丝体D3后第0、1、3小时,兔子眼压分别为12.60 ± 0.28、12.07 ± 0.34、11.98 ± 0.27,在第1及第3小时的眼压变化量为−0.53 ± 0.15、−0.62 ± 0.20,其下降百分比分别为−4.25% ± 1.28%、−4.89% ± 1.59% (表4)。喂食蝉花发酵菌丝体D7后第0、1、3小时,兔子眼压分别为12.22 ± 0.09、11.44 ± 0.33、11.73 ± 0.34,在第1及第3小时的眼压变化量为−0.78 ± 0.24、−0.49 ± 0.33,其下降百分比分别为−6.42% ± 1.98%、−3.99% ± 2.72% (表4)。
蝉花发酵菌丝体已完成多项动物安全性试验,试验结果显示蝉花发酵菌丝体不具有基因毒性,在大、小鼠与猪只的口服急毒性、大鼠90天慢性毒性以及大鼠致畸胎毒性的试验中,皆没有食用安全上的疑虑 [
图1. 兔子口服蝉花发酵菌丝体样品,第0、1、3小时的(A)眼压值及(B)眼压变化量。D3:发酵3天之蝉花发酵菌丝体;D7:发酵7天之蝉花发酵菌丝体
Percentage of delta IOP (%) | ||||||
---|---|---|---|---|---|---|
Group | 1 hr | 3 hr | ||||
D3 | −4.25 | ± | 1.28 | −4.89 | ± | 1.59 |
D7 | −6.42 | ± | 1.98 | −3.99 | ± | 2.72 |
表4. 兔子眼压下降百分比
在血清生化值分析中,高剂量组的肌酸酐与对照组相比偏低,但仍于历史参考范围值内 [
在降眼压评估试验中,以安全性试验剂量,作为单次口服喂食评估蝉花发酵菌丝体短期降眼压的剂量参考。试验结果显示,喂食蝉花发酵菌丝体虽会造成眼压下降,但降幅缓,不会因喂食而在短时间内骤降。喂食发酵3天的蝉花发酵菌丝体,在第3小时后,动物眼内压降幅趋缓,此外,喂食发酵7天的蝉花发酵菌丝体,在第3小时后,可观察到动物眼内压缓慢回升,原因可能为因本研究模式非诱导高眼压模式,眼压的回升除了可能为动物本身自我调节能力之外,也可能是其中降眼压的成分易在短时间内被代谢掉,或者是蝉花发酵菌丝体具有调节并维持正常眼压的能力,不致使眼内压过度降低。
以液态发酵方式培养真菌,会因不同发酵条件而影响菌丝体中的成分,不同发酵期的菌丝体亦可能产生不同的有效成分 [
正常兔眼内压动物模式是一种广泛被使用在研究降眼内压开发治疗青光眼药物的动物模式 [
安全性方面,口服蝉花发酵菌丝体2.5 mg/kg b.w及25 mg/kg b.w,动物的血液学分析、血清生化分析以及肝、肾组织病理检查皆无观察到与试验物质相关之病变,可作为日后人体食用蝉花发酵菌丝体之安全性参考依据。降眼压方面,蝉花发酵菌丝体以正常兔眼内压动物模式试验观察到良好的降眼压效果,大白兔服用蝉花发酵菌丝体1小时后即有降眼压现象,且不论发酵时间3天或7天的样品,皆可帮助短效期降眼压,日后可进一步探究对长期降眼压的效果,作为开发护眼产品之参考依据。
徐瑞霞,陈劲初. 蝉花发酵菌丝体口服安全性及其降眼压效果评估 Intraocular Pressure-Lowering Efficacy and Safety of Fermented Cordyceps cicadae Mycelia[J]. 食品与营养科学, 2021, 10(01): 33-39. https://doi.org/10.12677/HJFNS.2021.101005