Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
INTEQUI-CONICET, Área Química Orgánica, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco 917, 5700 San Luis, Argentina.
Photodiagnosis Photodyn Ther. 2019 Jun;26:420-429. doi: 10.1016/j.pdpdt.2019.05.003. Epub 2019 May 10.
Porophyllum obscurum (Spreng) DC (Asteraceae) hexanic extract (PoHex) from aerial parts has demonstrated antifungal activity under UVA irradiation against Candida spp. isolates from patients with oropharyngeal candidiasis and four thiophenes were isolated as responsible of the activity. In the present work, we studied the photomechanisms whereby PoHex and their thiophenes produce photoinactivation of C. albicans. Reactive Oxygen Species generation by PoHex and thiophenes was evaluated: the production of superoxide anion, employing the NBT reduction assay; hydrogen peroxide, through the formation of a red quinoneimine; and singlet oxygen by using the 1,3-DPBF bleaching method. The action of ROS in fungal cells was investigated by evaluating binding of photosensitizer, leakage, apoptosis and stress sensibility that were performed by following M27-A3 guidelines, in parallel under "light" and "darkness" conditions. Results showed that the photosensitive antifungal activity of PoHex required oxygen and both type I (production of superoxide anion and hydrogen peroxide) and type II (production of singlet oxygen) reactions were involved. In addition, we found that ROS generated by PoHex did not cause release of cytoplasmic components due to membrane damage nor apoptosis of C. albicans. Treatment with PoHex and UVA increased cells sensitivities to osmotic stressors; did not reduce resistance to additional oxidative stress and possibly affected the structure of the cell wall. In addition, 2,2':5'2″terthiophene, the most active PS present in PoHex and the only one that generate single oxygen, at Minimal Fungicide Concentration, did not cause leakage nor apoptosis and did not increase sensitivities to osmotic and oxidative stressors. Results demonstrated that Photodynamic Inactivation employing PoHex under UVA does represent an alternative for topical antifungal therapy for oropharyngeal candidiasis.
块菌属植物 obscurum（Spreng）DC（菊科）的全草正己烷提取物（PoHex）在 UVA 照射下对口腔念珠菌病患者的念珠菌分离株具有抗真菌活性，并且分离出了四种噻吩作为活性物质。在本工作中，我们研究了 PoHex 及其噻吩产生白念珠菌光灭活的光机理。通过 NBT 还原试验评估了 PoHex 和噻吩产生的活性氧种类（超氧阴离子）的生成；通过形成红色醌亚胺来评估过氧化氢的产生；并通过 1,3-DPBF 漂白法评估了单线态氧的产生。通过评估光敏剂的结合、渗漏、细胞凋亡和应激敏感性，在“光照”和“黑暗”条件下平行进行，研究了 ROS 在真菌细胞中的作用，这些实验均遵循 M27-A3 指南进行。结果表明，PoHex 的光敏抗真菌活性需要氧气，并且涉及到 I 型（产生超氧阴离子和过氧化氢）和 II 型（产生单线态氧）反应。此外，我们发现 PoHex 产生的 ROS 不会因细胞膜损伤而导致细胞质成分释放或白念珠菌凋亡。PoHex 和 UVA 处理增加了细胞对渗透胁迫的敏感性；不会降低对额外氧化应激的抵抗力，并且可能影响细胞壁的结构。此外，在最低抑菌浓度下，PoHex 中最活跃的 PS 2,2':5'2″-三联噻吩和唯一能产生单线态氧的 PS，既不会引起渗漏也不会引起凋亡，并且不会增加对渗透和氧化应激源的敏感性。结果表明，在 UVA 下采用 PoHex 的光动力失活确实代表了口腔念珠菌病局部抗真菌治疗的一种替代方法。
