Anti-aging Effect and Mechanism of Ethanol Extract of Clinacanthus nutans on Caenorhabditis elegans
-
摘要: 目的：以秀丽隐杆线虫为模式生物，探究忧遁草乙醇提取物（Clinacanthus nutans ethanol extract，CNEE）对线虫的抗衰老作用及作用机制。方法：通过寿命、产卵、运动等实验检测忧遁草乙醇提取物对线虫抗衰老的作用；通过急性氧化应激、抗氧化酶活力测定、活性氧（ROS）测定、实时荧光定量PCR等实验初步探究忧遁草乙醇提取物的抗氧化能力及延缓衰老的作用机制。结果：忧遁草乙醇提取物在100 μg/mL时能延长N2野生型线虫寿命（P<0.001），对其运动能力也有促进作用（P<0.001），同时对产卵能力没有毒性影响（P>0.05）；该提取物还可以提高线虫在急性氧化应激条件下的寿命（P<0.05）及过氧化氢酶的活力（P<0.01），并有效降低体内ROS的堆积情况（P<0.05），上调daf-16（P<0.01）及下游ctl-1（P<0.001）基因的表达水平。但在daf-16突变株线虫中，没有观察到正常条件或急性氧化应激条件下延寿的作用，也没有提高过氧化氢酶的活力、降低ROS水平、提高daf-16和ctl-1表达水平的情况（P>0.05）。结论：忧遁草乙醇提取物可以提高秀丽隐杆线虫抗衰老的能力，其作用机制可能与daf-16的氧化应激调控有关。Abstract: Objective: To assess the anti-aging effect and mechanism underlying the effects of ethanol extract from Clinacanthus nutans (CNEE) using Caenorhabditis elegans as the model organism. Methods: The anti-aging effects of ethanol extract from Clinacanthus nutans on nematodes were detected by experiments to assay longevity, oviposition, and exercise. The antioxidant capacity of the ethanolic extracts from Clinacanthus nutans and their mechanisms of action in delaying aging were investigated by assessing the acute oxidative stress, determining the antioxidant enzyme activity, and determining the reactive oxygen species (ROS) levels along with real-time fluorescence quantitative PCR experiments. Results: The ethanol extract from Clinacanthus nutans at 100 μg/mL could prolong the life span of N2 wild type nematode (P<0.001), promote the ability to exercise (P<0.001), and exerted no toxic effects on oviposition (P>0.05). The extract also increased nematode longevity (P<0.05) and catalase activity (P<0.01) under acute oxidative stress conditions, and effectively reduced the accumulation of ROS in vivo (P<0.05) along with upregulating the levels of daf-16 (P<0.01) and downstream ctl-1 (P<0.001) gene expressions. However, in daf-16 mutant nematodes, effects on life extension under normal conditions or acute oxidative stress were not observed, nor was there an increase in catalase activity, a decrease in ROS levels, or an increase in daf-16 and ctl-1 levels (P>0.05). Conclusion: The ethanol extract from Clinacanthus nutans enhanced the ability of nematodes to resist aging and the underlying mechanism of action may be related to the regulation of oxidative stress by daf-16.
-
Key words:
- Clinacanthus nutans ethanol extract /
- Caenorhabditis elegans /
- anti-aging /
- antioxidant /
- daf-16
-
图 2 氧化应激条件下CNEE对线虫寿命的影响
Figure 2. Effect of ethanol extract of Clinacanthus nutans on the life span of nematodes under oxidative stress
图 4 CNEE对CAT活力的影响
Figure 4. Effect of ethanol extract of Clinacanthus nutans on CAT activities
图 5 忧遁草乙醇提取物对线虫基因转录水平的影响
Figure 5. Effect of ethanol extract of Clinacanthus nutans on gene transcription level of nematodes
表 1 秀丽隐杆线虫qRT-qPCR引物序列
Table 1. qRT-PCR primer sequence of Caenorhabditis elegans
基因 上游引物（5'-3'） 下游引物（5'-3'） act-1 GCTGGACGTGATCTTACTGATTACC GTAGCAGAGCTTCTCCTTGATGTC daf-16 CGGGTGCCTATGGAAACT TTGGAAGAGCCGATGAAG ctl-1 CGGATACCGTACTCGTGATGAT CCAAACAGCCACCCAAATCA 
下载: 导出CSV
表 2 CNEE对线虫平均寿命的影响
Table 2. Effect of ethanol extract of Clinacanthus nutans on the average life span of nematodes
下载: 导出CSV
表 3 CNEE对线虫生殖能力和运动能力的影响
Table 3. Effects of ethanol extract of Clinacanthus nutans on the reproductive and athletic ability of nematodes
组别 总产卵量（个） 身体弯曲频率（次/20 s） 头部摆动频率（次/min） BC 201.57±72.40 3.88±2.45 5.96±4.15 HD 221.43±61.51ns 4.03±2.09ns 8.82±4.42***
下载: 导出CSV
表 4 氧化应激条件下CNEE对线虫平均寿命的影响
Table 4. Effect of ethanol extract of Clinacanthus nutans on the average life span of nematodes under oxidative stress
线虫类型 组别 平均寿命（h） N2 BC 3.028±0.120 HD 3.322±0.129* daf-16 BC 2.483±0.116 HD 2.494±0.122ns
下载: 导出CSV
-
[1] 刘赫男. 富氢水的制备及丁酸梭菌延长秀丽线虫寿命的机制研究[D]. 合肥: 安徽医科大学, 2020LIU H N. Preparation of hydrogen-rich water and the mechanism of Clostridium butyricum to prolong the life span of Caenorhabditis elegans[D]. Hefei: Anhui Medical University, 2020. [2] 黄小兵. 抗衰老化合物筛选及阿司匹林抗衰老活性评价[D]. 昆明: 中科院昆明植物研究所, 2016HUANG X B. Screening of anti-aging compounds and evaluation of anti-aging activity of aspirin[D]. Kunming: Kunming Institute of Botany, Chinese Academy of Sciences, 2016. [3] GUO X Y, LI Q Q, SHI J, et al. Perfluorooctane sulfonate exposure causes gonadal developmental toxicity in Caenorhabditis elegans through ROS-induced DNA damage[J]. Chemosphere,2016,155:115−126. doi: 10.1016/j.chemosphere.2016.04.046 [4] SHAY J W. Role of telomeres and telomerase in aging and cancer[J]. Cancer Discovery,2016,6(6):584−593. doi: 10.1158/2159-8290.CD-16-0062 [5] 段佳辉. 忧遁草叶提取物解酒、保肝及抗氧化功能的研究[D]. 无锡: 江南大学, 2019DUAN J H. Study on the anti-drinking, hepatoprotective and antioxidant functions of the leaf extracts of Clinacanthus nutans[D]. Wuxi: Jiangnan University, 2019. [6] 王瑶, 钟希文, 张文霞. 鳄嘴花的化学成分及药理作用研究进展[J]. 中国药房,2016,27(1):108−111. [WANG Y, ZHONG X W, ZHANG W X. Advances in the chemical composition and pharmacological effects of Clinacanthus nutans[J]. Chinese Pharmacy,2016,27(1):108−111. [7] 魏瑞. 忧遁草中三萜类物质的提取纯化及功能评价[D]. 天津: 天津科技大学, 2020WEI R. Extraction, purification and functional evaluation of triterpenoids from Clinacanthus nutans[D]. Tianjin: Tianjin University of Science and Technology, 2020. [8] 段佳辉, 徐德平, 钱志娟, 等. 忧遁草抗氧化功效与成分的研究[J]. 食品科技,2019,44(3):205−210. [DUAN J H, XU D P, QIAN Z J, et al. Study on the antioxidant efficacy and composition of Clinacanthus nutans[J]. Food Science and Technology,2019,44(3):205−210. [9] 黄壮, 李静, 杜鸿志, 等. 三七醇提取物对秀丽隐杆线虫的降脂作用[J]. 世界科学技术-中医药现代化,2020,22(5):1648−1653. [HUANG Z, LI J, DU H Z, et al. Lipid lowering effect of Panax notoginseng alcohol extract on Caenorhabditis elegans[J]. World Science and Technology-Modernization of Traditional Chinese Medicine,2020,22(5):1648−1653. [10] 刘静. 金钗石斛总生物碱抗秀丽隐杆线虫衰老作用及其机制研究[D]. 遵义: 遵义医科大学, 2019LIU J. Study on the anti aging effect and mechanism of total alkaloids of Dendrobium nobile on Caenorhabditis elegans[D]. Zunyi: Zunyi Medical University, 2019. [11] HERNDON L A, WOLKOW C A, DRISCOLL M, et al. Effects of ageing on the basic biology and anatomy of C. elegans[M]. Switzerland in Ageing Lessons from C. elegans, 2017: 9−39. [12] TISSENBAUM H A. Using C. elegans for aging research[J]. Invertebrate RE Production & Development,2015,59(1):59−63. [13] 普元柱. 基于蛋白质组学研究秀丽线虫生殖系统调控衰老的机制[D]. 北京: 中国科学院大学, 2017PU Y Z. Proteomics based study on the mechanism of the reproductive system of Caenorhabditis elegans regulating aging [D]. Beijing: University of Chinese Academy of Sciences, 2017. [14] 张宗敏. 金钗石斛多糖对秀丽隐杆线虫衰老的影响[D]. 遵义: 遵义医学院, 2018ZHANG Z M. Effects of Dendrobium nobile polysaccharides on the senescence of C. elegans[D]. Zunyi: Zunyi Medical College, 2018. [15] 刘春红, 汤燚聪, 高瑜培, 等. 鹿茸乙醇提取物对秀丽隐杆线虫抗衰老的作用[J]. 食品工业科技,2021,42(7):354−359. [LIU C H, TANG Y C, GAO Y P, et al. Anti aging effect of ethanol extract of cartialgenous on C. elegans[J]. Science and Technology of Food Industry,2021,42(7):354−359. [16] LOPEZ-GARCIA G, CILLA A, BARBERA R, et al. Effect of plant sterol and galactooligosaccharides enriched beverages on oxidative stress and longevity in Caenorhabditis elegans[J]. Journal of Functional Foods,2020,65:1−8. [17] MARIA D N H, JOSE M R M, ADELAIDA E M, et al. Strawberry (Fragaria×ananassa cv. Romina) methanolic extract attenuates Alzheimer's beta amyloid production and oxidative stress by SKN-1/NRF and DAF-16/FOXO mediated mechanisms in C. elegans[J]. Food Chemistry,2022,372:1−12. [18] 于群. 忧遁草活性成分的提取及其功能评价[D]. 海口: 海南大学, 2017YU Q. Extraction and functional evaluation of the active ingredients of Clinacanthus nutans[D]. Haikou: Hainan University, 2017. [19] 杨帆, 肖曼, 陈柏岑, 等. 益智仁提取物延缓秀丽隐杆线虫衰老的研究[J]. 中华中医药学刊,2022,40(2):43−46, 265. [YANG F, XIAO M, CHEN B C, et al. Study on the effect of Alpinia oxyphylla extract on delaying the senescence of C. elegans[J]. Chinese Journal of Traditional Chinese Medicine,2022,40(2):43−46, 265. [20] 金司仪. 三七粉延缓秀丽隐杆线虫衰老的作用机制及其稳定性研究[D]. 武汉: 湖北中医药大学, 2019JIN S Y. Study on the mechanism and stability of Panax notoginseng powder in delaying the senescence of C. elegans[D]. Wuhan: Hubei University of Traditional Chinese Medicine, 2019. [21] 黄少杰, 陈宏著, 钟淳菲, 等. 铁皮石斛叶多糖对秀丽隐杆线虫体内抗衰老作用[J]. 食品科学,2022,43(21):203−208. [HUANG S J, CHEN H, ZHONG C F, et al. Anti aging effect of polysaccharides from Dendrobium nobile on C. elegans in vivo[J]. Food Science,2022,43(21):203−208. doi: 10.7506/spkx1002-6630-20211202-031 [22] 付敏, 张旭光, 杨柳, 等. 虾青素调控ROS介导的自噬延长秀丽隐杆线虫寿命作用[J]. 食品工业科技,2022,43(4):373−378. [FU M, ZHANG X G, YANG L, et al. Astaxanthin regulates ROS mediated autophagy to prolong the life span of C. elegans[J]. Science and Technology of Food Industry,2022,43(4):373−378. [23] 李煜, 敬海明, 李国君. 秀丽隐杆线虫神经毒理学研究进展[J]. 中国公共卫生,2013,29(4):622−624. [LI Y, JING H M, LI G J. Research progress in neurotoxicology of C. elegans[J]. China Public Health,2013,29(4):622−624. doi: 10.11847/zgggws2013-29-04-58 [24] 周栋. 内分泌干扰物双酚A对秀丽隐杆线虫的生态毒性效应及其作用机制研究[D]. 上海: 华东理工大学, 2016ZHOU D. Study on the ecotoxicity and mechanism of endocrine disruptor bisphenol A on C. elegans[D]. Shanghai: East China University of Technology, 2016. [25] 王力, 肖嵋方, 陈弘培, 等. 牡蛎多肽组分OE-I抗氧化活性及其对秀丽隐杆线虫抗衰老作用[J]. 食品科学,2022,43(3):152−160. [WANG L, XIAO M F, CHEN H P, et al. Antioxidant activity of oyster polypeptide OE-I and its anti-aging effect on C. elegans[J]. Food Science,2022,43(3):152−160. [26] 王锐凡, 雷欣安, 成高丽, 等. 鳄嘴花总黄酮对高糖诱导血管内皮细胞损伤的保护作用及机制研究[J]. 中国循证心血管医学杂志,2019,11(9):1077−1080. [WANG R F, LEI X A, CHENG G L, et al. Study on the protective effect and mechanism of total flavones of Clinacanthus nutans on vascular endothelial cell injury induced by high glucose[J]. Chinese Journal of Evidence Based Cardiovascular Medicine,2019,11(9):1077−1080. [27] TYAGI N, GAMBHIR K, KUMAR S, et al. Interplay of reactive oxygen species (ROS) and tissue engineering: A review on clinical aspects of ROS-responsive biomaterials[J]. Journal of Materials Science,2021,56:16790−16823. doi: 10.1007/s10853-021-06338-7 [28] LIU Y L, ZHOU Z Y, YIN L F, et al. Tangeretin promotes lifespan associated with insulin/ insulin-like growth factor-1 signaling pathway and heat resistance in Caenorhabditis elegans[J]. BioFactors (Oxford, England),2021,48(2):442−453. [29] LIN C, XIAO J, XI Y, et al. Rosmarinic acid improved antioxidant properties and healthspan via the IIS and MAPK pathways in Caenorhabditis elegans[J]. BioFactors (Oxford, England),2019,45(5):774−787. doi: 10.1002/biof.1536 [30] 宋兵兵. 蓝莓和苹果皮提取物联合抗衰老活性及作用机制研究[D]. 广州: 华南理工大学, 2020SONG B B. Study on the combined anti-aging activity and mechanism of blueberry and apple peel extracts[D]. Guangzhou: South China University of Technology, 2020. [31] 蔡洁. 米糠活性肽通过daf-16和skn-1调节线虫寿命与应激能力的研究[D]. 长沙: 中南林业科技大学, 2021CAI J. Studies on the regulation of rice bran active peptides on C. elegans longevity and stress capacity through daf-16 and skn-1[D]. Changsha: Central South Forestry University, 2021. [32] WAN Q L, FU X D, DAI W Y, et al. Uric acid induces stress resistance and extends the life span through activating the stress response factor DAF-16/FOXO and SKN-1/NRF2[J]. Aging,2020,12(3):2840−2856. doi: 10.18632/aging.102781 [33] 米象男. 3,4-二羟基苯甲酸甲酯延长秀丽线虫寿命的机制研究[D]. 广州: 暨南大学, 2018MI X N. Study on the mechanism of methyl 3,4-dihydroxybenzoate prolonging the life span of C. elegans[D]. Guangzhou: Jinan University, 2018. -
点击查看大图
图(5) / 表(4)
计量
- 文章访问数: 132
- HTML全文浏览量: 56
- PDF下载量: 14
- 被引次数: 0
