Development and validation of a high-performance thin-layer chromatography‒densitometric method and mass spectroscopy profiling for the determination of bioactive phytosterol from Manilkara zapota L. P. Royen leaves and correlating its antioxidant and antiinflammatory potential

IF 1.1 4区化学 Q4 CHEMISTRY, ANALYTICAL Jpc-journal of Planar Chromatography-modern Tlc Pub Date : 2024-01-13 DOI:10.1007/s00764-023-00280-x

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Abstract

A simple, reliable high-performance thin-layer chromatography (HPTLC)‒densitometry method was developed and validated for the quantification of β-sitosterol, a significant bioactive phytosterol, in Manilkara zapota L. P. Royen leaves. This method is combined with mass spectroscopy (MS) for relevant structural identification, responsible for their antioxidant and antiinflammatory therapeutic potential. The pet ether (PE), chloroform (CH), ethyl acetate (EA), and hydro-ethanolic (HE) leaf extracts were prepared using the cold maceration technique. These extracts were screened for preliminary qualitative tests, and based on the screening results, secondary metabolites such as flavonoids, phenols, steroids, terpenoids, and tannins were further quantified. Antioxidant potential was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), ferric reducing antioxidant power (FRAP), and nitric oxide (NO) free radical scavenging assays. antiinflammatory activity was evaluated through protein denaturation and human red blood cell (HRBC) membrane stability methods. Additionally, an HPTLC method was developed and validated for β-sitosterol quantification. Among all the extracts, the ethyl acetate leaf extract exhibited the highest steroidal content [50% inhibitory concentration (IC₅₀) 2.92 ± 0.21 mg β-sitosterol equivalent (BSE)/g dry extract (DE)], flavonoid content [IC₅₀ 22.9 ± 0.25 mg quercetin equivalent (QE)/g DE], and phenolic content [IC₅₀ 103.8 ± 0.23 mg gallic acid equivalent (GAE)/g DE]. The ethyl acetate extract also demonstrated the highest efficacy against DPPH (IC₅₀ 16.35 ± 1.49), ABTS (IC₅₀ 17.52 ± 2.36), FRAP [41.32 ± 0.02 mg ascorbic acid (AA)/g crude extract], NO (IC₅₀ 17.13 ± 2.06), protein denaturation (IC₅₀ 31.75 ± 2.1), and HRBC membrane stability (IC₅₀ 24.8 ± 2.44). Correlation analysis further supported these findings. Furthermore, following the International Council for Harmonisation guidelines, an HPTLC method was developed, and 434.4 ng of β-sitosterol was quantified, with the highest content found in the ethyl acetate extract. Therefore, M. zapota leaf extract exhibits beneficial antioxidant and antiinflammatory effects due to its richness in phytosterols. This extract has the potential to be further explored as a therapeutic agent against inflammatory diseases.

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开发和验证测定 Manilkara zapota L. P. Royen 叶中生物活性植物甾醇的高效薄层色谱-密度测定法和质谱分析法，并将其与抗氧化和抗炎潜力联系起来

摘要开发并验证了一种简便、可靠的高效薄层色谱-密度测定法，用于定量检测 Manilkara zapota L. P. Royen 叶子中具有重要生物活性的植物甾醇 β-谷甾醇。该方法与质谱（MS）相结合，用于相关的结构鉴定，以确定其抗氧化和抗炎治疗潜力。采用冷浸渍技术制备了乙醚（PE）、氯仿（CH）、乙酸乙酯（EA）和水乙醇（HE）叶提取物。对这些提取物进行了初步的定性检测，并根据检测结果对黄酮类、酚类、甾体类、萜类和单宁酸等次生代谢物进行了进一步的定量分析。抗氧化潜力采用 1,1-二苯基-2-苦基肼（DPPH）、2,2-偶氮-双（3-乙基苯并噻唑啉）-6-磺酸（ABTS）、铁还原抗氧化力（FRAP）和一氧化氮（NO）自由基清除法进行评估。此外，还开发并验证了一种 HPTLC 方法，用于定量检测 β-谷甾醇。在所有提取物中，乙酸乙酯叶提取物的甾体含量[50%抑制浓度（IC50）2.92 ± 0.21 mg β-谷甾醇当量（BSE）/g 干提取物（DE）]、黄酮含量[IC50 22.9 ± 0.25 mg 槲皮素当量（QE）/g DE]和酚含量[IC50 103.8 ± 0.23 mg 没食子酸当量（GAE）/g DE]最高。乙酸乙酯提取物对 DPPH（IC50 16.35 ± 1.49）、ABTS（IC50 17.52 ± 2.36）、FRAP [41.32 ± 0.02 毫克抗坏血酸（AA）/克粗提取物]、NO（IC50 17.13 ± 2.06）、蛋白质变性（IC50 31.75 ± 2.1）和 HRBC 膜稳定性（IC50 24.8 ± 2.44）也表现出最高的功效。相关性分析进一步证实了这些发现。此外，根据国际协调理事会的指导方针，开发了一种 HPTLC 方法，定量分析了 434.4 ng β-谷甾醇，其中乙酸乙酯提取物中的含量最高。因此，M. zapota 叶提取物因富含植物甾醇而具有有益的抗氧化和抗炎作用。这种萃取物有可能被进一步开发为治疗炎症疾病的药物。

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来源期刊

Jpc-journal of Planar Chromatography-modern Tlc 化学-分析化学

CiteScore

2.20

自引率

18.80%

发文量

审稿时长

>12 weeks

期刊介绍： JPC - Journal of Planar Chromatography - Modern TLC is an international journal devoted exclusively to the publication of research papers on analytical and preparative planar chromatography. The journal covers all fields of planar chromatography, on all kinds of stationary phase (paper, layer, gel) and with various modes of migration of the mobile phase (capillary action or forced flow).