Optimization of Protein Extraction from Hypericum perforatum Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Abstract

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.

Chemical characterization of the essential oil compositions from Iranian populations of Hypericum perforatum L.

“…Hypericum species have been used in the traditional medicine for centuries and many of them have great economic importance as natural sources of active compounds (Hosni et al, 2013). Among them, H. perforatum, commonly known as St. John's Wort, is traditionally used as a medicinal species for the treatment of mild to moderate depression as well as anxiety and insomnia (Karppinen et al, 2010;Barnes et al, 2001;Greeson et al, 2001;Anonymous, 1997). H.…”

“…perforatum is an herbaceous perennial species and contains different biologically active components including naphthodianthrones, phloroglucinols, tannins, xanthones, flavonoids, phenolic acids and essential oil (Karppinen et al, 2010;Greeson et al, 2001). In particular, the latter were defined by European Pharmacopeia (Council of Europe, 1997) as odorous products, usually of complex composition, obtained from a botanically defined plant raw materials by steam distillation, dry distillation, or a suitable mechanical process without heating.…”

Evaluation of Protein Extraction Methods for Proteomic Analysis of Non-Model Recalcitrant Plant Tissues

“…Similarly, interfering substances in samples from Hypericum perforatum organs obtained after acetone and TCA/acetone precipitation produced low-quality SDS-PAGE results. 23 We performed IEF with 300 µg of the proteins, followed by 2-DE (Figures 3-5). Differences were observed in both the number and resolution of protein spots.…”

“…24 It seems that for particularly recalcitrant tissues like the ones tested here, acetone and TCA/acetone precipitation do not sufficiently remove nucleic acids, carbohydrates and polyphenols, which cause precipitation, bad focusing and protein streaking. 20,22,23 Another issue with the TCA/acetone and acetone precipitation methods is the low yield of extracted proteins: to produce enough proteins from M. gracilis and S. tectorum for 2-DE, these methods would require up to 4 times more of the starting material than the phenol method (data not shown). Using much more starting material also increases the amount of contaminants in the samples.…”

Evolution, Three-Dimensional Model and Localization of Truncated Hemoglobin PttTrHb of Hybrid Aspen

“…Soluble proteins from leaves and stem were extracted according to Karppinen et al [39] from 100 mg of fresh powder with a 50 mM sodium borate buffer pH 9 containing 12% (w/v) polyvinylpolypyrrolidone (PVPP), 10 mM dithiothreitol (DTT) and 50 mM ascorbic acid. Proteins were quantified spectrophotometrically by the Bradford method [40] using bovine serum albumin (BSA) as a standard.…”