Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan, 304022, India.
Laboratory of Phytopathology and Microbial Biotechnology, Department of Botany, Mohanlal Sukhadia University, Udaipur, 313001, Rajasthan, India.
Plant Physiol Biochem. 2023 Aug;201:107843. doi: 10.1016/j.plaphy.2023.107843. Epub 2023 Jun 15.
The present study is aimed to isolate terpenoids from Gymnosporia senegalensis through analytical and preparative thin-layer chromatography (TLC) and to determine their antioxidant activity using the 2, 2-diphenyl-1- picrylhydrazyl (DPPH) assay and to find out the presence of β-carotene through high-performance thin-layer chromatography (HPTLC). The validation included linearity, limit of detection (LOD), limit of quantification (LOQ), specificity, precision, recovery, and robustness. All the isolated compounds from TLC exhibited significant antioxidant activity. Among all, isolated compounds from leaf showed highest IC values. The highest total terpenoid content (TTC) was found 51.6 ± 0.06 in stem, then 49.02 ± 0.01 in bark, and 46.27 ± 0.01 in leaf. DPPH results indicated that leaf-isolated compound 1 (LIC1) showed the highest IC at 7.55 ± 0.02 and stem-isolated compound 2 (SIC2) showed the lowest IC at 0.616 ± 0.01 among all the isolated compounds of G. senegalensis. HPTLC separation was carried out on aluminium plates pre-coated with silica gel 60 F254 as the stationary phase and n-hexane: ethyl acetate (6:4, v/v) as the mobile phase. Quantification was achieved based on a densitometric analysis of β-carotene in the concentration range of 100-500 ng/band at 254 nm. For the calibration plots, linear regression produced r = 0.96450 and Rf = 0.27. The LOD and LOQ were 10.15 and 30.76 ng/mL for HPTLC and relative standard deviation were 137.26 ± 2.03 and 160.43 ± 2.95 (intra-day) and 127.88 ± 2.14 and 157.27 ± 1.90 (inter-day) for 200 and 400 ng/band, respectively. The present study shows the presence of various types of terpenoids through TLC whereas the HPTLC results indicated that the developed methods were accurate and precise. It also shows that the approach is appropriate for its intended use in routine quality control testing of commercially available tablet formulations and drug assay to assist both industries and researchers in making important decisions at a reasonable cost. Moreover, due to the use of a safer and more environmentally friendly mobile phase in comparison to the toxic mobile phases used in recent analytical techniques to estimate β-carotene, this methodology is also secure and sustainable.
本研究旨在通过分析和制备薄层色谱（TLC）从非洲皂荚中分离萜类化合物，并使用 2,2-二苯基-1-苦基肼（DPPH）测定其抗氧化活性，并用高效薄层色谱（HPTLC）法检测β-胡萝卜素的存在。验证包括线性、检测限（LOD）、定量限（LOQ）、特异性、精密度、回收率和稳健性。所有从 TLC 中分离出来的化合物均表现出显著的抗氧化活性。其中，叶部分离的化合物 1（LIC1）的 IC 值最高。在茎中发现最高的总萜类化合物含量（TTC）为 51.6±0.06，然后在树皮中为 49.02±0.01，在叶中为 46.27±0.01。DPPH 结果表明，叶分离化合物 1（LIC1）在所有分离的非洲皂荚化合物中显示出最高的 IC 值，为 7.55±0.02，而茎分离化合物 2（SIC2）则显示出最低的 IC 值，为 0.616±0.01。HPTLC 分离在硅胶 60 F254 预涂覆的铝板上进行，作为固定相，正己烷：乙酸乙酯（6：4，v/v）作为流动相。基于 254nm 处β-胡萝卜素浓度范围为 100-500ng/条带的分光光度分析进行定量。对于校准曲线，线性回归产生 r=0.96450 和 Rf=0.27。HPTLC 的 LOD 和 LOQ 分别为 10.15 和 30.76ng/mL，相对标准偏差分别为 137.26±2.03 和 160.43±2.95（日内）和 127.88±2.14 和 157.27±1.90（日间）对于 200 和 400ng/条带，分别。本研究通过 TLC 显示了各种类型的萜类化合物的存在，而 HPTLC 结果表明所开发的方法准确、精密。它还表明，该方法适用于其在商业上可获得的片剂制剂的常规质量控制测试和药物测定中的用途，以协助行业和研究人员以合理的成本做出重要决策。此外，与最近用于估计β-胡萝卜素的分析技术中使用的有毒流动相相比，由于使用了更安全、更环保的流动相，因此该方法也更安全、更可持续。
