Das Suryasnata, Ray Asit, Nasim Noohi, Nayak Sanghamitra, Mohanty Sujata
Centre for Biotechnology, School of Pharmaceutical Sciences, Siksha 'O' Anusandhan (Deemed to be University), Bhubaneswar, Odisha 751003 India.
PG Department of Biotechnology, Rama Devi Women's University, Bhubaneswar, Odisha 751022 India.
3 Biotech. 2019 Jan;9(1):37. doi: 10.1007/s13205-018-1565-8. Epub 2019 Jan 5.
The objective of the study is to analyze the effect of different extracting methods on the polyphenolic content and antioxidant activities in leaves. In the present research, leaf extract was prepared by sonication, Soxhlet and maceration methods using acetone (100%, v/v). The efficiency of the extraction methods was estimated by quantifying the total phenolic content (TPC) by the Folin-Ciocalteu method and total flavonoid content (TFC) by AlCl colorometric methods, and antioxidant power of the various extracts was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assay. Thin-layer chromatography (TLC) bioautography was carried out to identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. DPPH free radical scavenging capacity of the different extracts from strongest to weakest was as follows: ascorbic acid (4.27 µg/mL) > sonication (5.35 µg/mL) >, maceration (5.53 µg/mL)>, soxhlet extraction (5.83 µg/mL). Same trend was also observed for the ABTS radical scavenging capacity. Similarly, findings of this study also showed that sonication extract possessed highest phenolic and flavonoid contents followed by maceration and Soxhlet extraction. In addition, important bioactive phenolic constituents which contribute largely towards antioxidant potential such as eugenol and eugenol acetate were quantified using HPTLC (high-performance thin-layer chromatography) method. The average percent recovery of eugenol and eugenol acetate was found to be 97.28% and 98.04%, respectively. The LOD (limit of detection) and LOQ (limit of quantification) for eugenol were 5 and 15 ng/spot, whereas that of eugenol acetate were 10 and 30 ng/spot. The HPTLC densitometric determination also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in sonication followed by maceration and Soxhlet extraction. The developed HPTLC chromatogram profile may be used as a reference for the standardization of leaf extracts.
本研究的目的是分析不同提取方法对叶片中多酚含量和抗氧化活性的影响。在本研究中，使用丙酮（100%，v/v）通过超声处理、索氏提取和浸渍法制备叶提取物。通过福林-西奥尔特法测定总酚含量（TPC）和通过氯化铝比色法测定总黄酮含量（TFC）来评估提取方法的效率，并通过DPPH（2,2-二苯基-1-苦基肼）和ABTS 2,2'-联氮-双-（3-乙基苯并噻唑啉-6-磺酸）自由基清除试验测定各种提取物的抗氧化能力。进行薄层色谱（TLC）生物自显影以鉴定抗氧化剂，并通过新开发的高效薄层色谱（HPTLC）方法测定其含量。不同提取物的DPPH自由基清除能力从最强到最弱依次为：抗坏血酸（4.27 µg/mL）＞超声处理（5.35 µg/mL）＞浸渍（5.53 µg/mL）＞索氏提取（5.83 µg/mL）。ABTS自由基清除能力也观察到相同趋势。同样，本研究结果还表明，超声提取物的酚类和黄酮类含量最高，其次是浸渍和索氏提取。此外，使用HPTLC（高效薄层色谱）方法对在抗氧化潜力方面起主要作用的重要生物活性酚类成分如丁香酚和乙酸丁香酯进行了定量。丁香酚和乙酸丁香酯的平均回收率分别为97.28%和98.04%。丁香酚的检测限（LOD）和定量限（LOQ）分别为5和15 ng/斑点，而乙酸丁香酯的检测限和定量限分别为10和30 ng/斑点。HPTLC密度测定法也通过揭示超声提取物中鉴定出的抗氧化剂含量高于浸渍和索氏提取物，支持了抗氧化试验的结果。所开发的HPTLC色谱图可作为叶提取物标准化的参考。
