Phytochemical Profiling of An 80% Ethanolic Extract of Hypericum Perforatum L. Aerial Parts By Preliminary Screening and LC-MS

Abstract

Background: Hypericum perforatum L. (St. John’s wort, Hypericaceae) is a widely used medicinal plant whose biological activity is attributed to a complex mixture of flavonoids, phenolic acids, biflavonoids, naphthodianthrones and phloroglucinols. The chemical composition of an extract depends on plant part, solvent and extraction method, so each preparation needs its own characterization.

Objective: To prepare an 80% ethanolic extract of H. perforatum aerial parts and characterize its phytochemical composition by preliminary qualitative screening and liquid chromatography–mass spectrometry (LC-MS).

Methods: Shade-dried, powdered aerial parts collected in November 2025 were extracted by Soxhlet extraction with 80% ethanol for 48 h. Percentage yield was calculated on a dry-powder weight basis. The extract was screened with standard chemical tests for alkaloids, flavonoids, phenolics and tannins, anthraquinones, triterpenoids, sterols, saponins and glycosides, and was analyzed by LC-MS in electrospray negative (ES⁻) and positive (ES⁺) modes.

Results: The extractive yield was 11.45% w/w. Screening indicated flavonoids, phenolic compounds and tannins, anthraquinones, triterpenoids and phytosterols; saponins were not detected, and alkaloids gave a positive result with only one of three reagent groups. LC-MS showed about 20 peaks in ES⁻ and about 17 in ES⁺ mode. Eight compounds were assigned by retention time and molecular ions: protocatechuic acid, chlorogenic acid, isoquercitrin, rutin, hyperoside, kaempferol-3-O-glucoside, quercetin and amentoflavone. Together these accounted for 95.2% of the total chromatographic peak area. Kaempferol-3-O-glucoside (37.30%), hyperoside (32.24%) and quercetin (14.83%) were the dominant peaks.

Conclusion: The 80% ethanolic extract is a flavonoid-dominated preparation, with flavonoids and the biflavonoid amentoflavone representing 93.2% of the peak area. These data provide a chemical fingerprint for the extract and a basis for standardization and further study.