Ahmad Anas, Prakash Ravi, Khan Mohd Shahnawaz, Altwaijry Nojood, Asghar Muhammad Nadeem, Raza Syed Shadab, Khan Rehan
Chemical Biology Unit, Institute of Nano Science and Technology, Knowledge City, Sector 81, Mohali, Punjab 140306, India.
Laboratory for Stem Cell & Restorative Neurology, Department of Biotechnology, Era's Lucknow Medical College Hospital, Sarfarazganj, Lucknow, Uttar Pradesh 226003, India.
ACS Omega. 2022 Sep 19;7(38):34476-34484. doi: 10.1021/acsomega.2c04148. eCollection 2022 Sep 27.
Naringenin, one of the flavonoid components, is majorly found in and obtained from grapefruits and oranges. Naringenin also acts as a potent antioxidant, which possesses hypolipidemic as well as anti-inflammatory potential. Naringenin reduces the expressions of several inflammatory mediators, viz., NF-κB, cycloxygenase-2, and other cytokine mediators. In spite of having various biological effects, the clinical application of naringenin is restricted due to its very poor aqueous solubility. In the present study, the high-energy ball milling method was employed for the preparation of naringenin nanoparticles without using any chemical with an aim to enhance the anti-oxidant potential of naringenin. The milled naringenin nanoparticles were characterized for their physicochemical properties using scanning electron microscopy (SEM) and X-ray diffraction. Additionally, the effects of milling time and temperature were further assessed on the solubility of crude and milled naringenin samples. The antioxidant potential of milled naringenin was evaluated with various assays such as DHE, DCFDA, and cleaved caspase-3 using SH-SY5Y human neuroblastoma cells. The nanoparticle size of naringenin after milling was confirmed using SEM analysis. Crystalline peaks for milled and crude samples of naringenin also established that both the naringenin forms were in the crystalline form. The solubility of naringenin was enhanced depending on the milling time and temperature. Moreover, crude and milled naringenin were found to be cytocompatible up to doses of 120 μM each for the duration of 24 and 48 h. It was also observed that milled naringenin at the doses of 1, 2, and 5 μM significantly reduced the levels of reactive oxygen species (ROS) generated by HO and exhibited superior ROS scavenging effects as compared to those of crude or un-milled forms of naringenin. Furthermore, milled naringenin at the doses of 1 and 2 μM inhibited HO-induced cell death, as shown by immunofluorescence staining of cleaved caspase-3 and Annexin-V PI flow cytometry analysis. Conclusively, it could be suggested that the size reduction of naringenin using high-energy ball milling techniques substantially enhanced the antioxidant potential as compared to naïve or crude naringenin, which may be attributed to its enhanced solubility due to reduced size.
柚皮素是黄酮类成分之一，主要存在于葡萄柚和橙子中，并可从这些水果中获取。柚皮素还是一种强效抗氧化剂，具有降血脂和抗炎潜力。柚皮素可降低多种炎症介质的表达，即核因子κB、环氧化酶-2和其他细胞因子介质。尽管柚皮素具有多种生物学效应，但其临床应用因水溶性极差而受到限制。在本研究中，采用高能球磨法制备柚皮素纳米颗粒，不使用任何化学物质，旨在提高柚皮素的抗氧化潜力。使用扫描电子显微镜（SEM）和X射线衍射对研磨后的柚皮素纳米颗粒的物理化学性质进行表征。此外，进一步评估研磨时间和温度对粗制和研磨后的柚皮素样品溶解度的影响。使用SH-SY5Y人神经母细胞瘤细胞，通过多种测定方法，如二氢乙啶（DHE）、2′,7′-二氯二氢荧光素二乙酸酯（DCFDA）和裂解的半胱天冬酶-3来评估研磨后的柚皮素的抗氧化潜力。通过SEM分析确认了研磨后柚皮素的纳米颗粒大小。柚皮素研磨样品和粗制样品的结晶峰也表明，两种柚皮素形式均为结晶形式。柚皮素的溶解度随研磨时间和温度的增加而提高。此外，发现粗制和研磨后的柚皮素在剂量高达120μM、持续时间为24小时和48小时的情况下具有细胞相容性。还观察到，与粗制或未研磨形式的柚皮素相比，剂量为1、2和5μM的研磨后的柚皮素显著降低了由过氧化氢（HO）产生的活性氧（ROS）水平，并表现出优异的ROS清除效果。此外，如通过裂解的半胱天冬酶-3免疫荧光染色和膜联蛋白-V碘化丙啶（Annexin-V PI）流式细胞术分析所示，剂量为1和2μM的研磨后的柚皮素可抑制HO诱导的细胞死亡。总之，可以认为，与未处理的或粗制的柚皮素相比，使用高能球磨技术减小柚皮素的尺寸可显著提高其抗氧化潜力，这可能归因于其尺寸减小导致溶解度增加。
