Phytochemical Screening of Anti-inflammatory Compounds of the Stinging Nettle (Urtica Spp.)

Abstract

The stinging nettle is an alimurgic plant known for its anti-inflammatory ethnomedical importance. However, there are no studies that have attempted to identify the specific anti-inflammatory compounds within the extracts of the stinging nettle leaves as well as their mechanism of action. Therefore, the objective of this study was to screen for bioactive compounds that target the cyclo-oxygenase pathway. The plant samples were identified by DNA (Deoxyribo Nucleic Acid) barcoding using ribulose bisphosphate carboxylase (rbcL) and Internal Transcribed Spacer (ITS) markers. The resulting consensus sequence was blasted to idebtify the sample species. Aqueous and methanol: dicholoromethane (1:1) plant extracts were prepared and analysed for their total phenolic and flavonoid content. In vivo carrageenan model was used to determine the anti-inflammatory capacity of these extracts. Further investigations in the form of Liquid Chromatography – Mass Spectroscopy (LC-MS) and Raman spectroscopy analysis were used for the identification of compounds. In addition to identification, the LC-MS performed the quantification of polyphenols. Molecular comparisons were done between indomethacin, a non-selective inhibitor, and the identified compounds prior to docking them with receptor Protein Databank cyclooxygenases-2 (PDB: 4COX). DNA barcoding identified Urtica sp. as the species used for this study. The total phenolic content of the aqueous and methanol: dichloromethane extracts were at 3.75 mg Gallic acid equivalent (GAE) /g dry sample and 6.26 mg GAE/g dry sample while total flavonoid content were at 0.3872 mg quercetin/g dry sample and 1.76 mg quercetin/g dry sample, respectively. An in vivo carrageenan model of inflammation based on aqueous extract administered at 750 mg/kg alleviated the carrageenan induced inflammation at 22.35% maximum inhibition rate. Raman spectroscopy identified phenolic acids (hydroxy-cinnamic acids) and flavonoids (flavan-3-ol). On the other hand, the LC-MS analysis yielded a wider range of phenolic acids specifically hydroxy-cinnamic and hydroxy-benzoic acids and flavonoids (flavonols and flavan-3-ols). Based on LC-MS, molecules belonging to the flavonol group were found to be abundant with concentrations ranging to 0.16 – 1.47 μg/g. The flavonoid compounds exhibited binding energy -6.9 to -8.6 kcal/mol while phenolic acids, -3.8 to -8.3 kcal/mol. Quercetin (-8.6 kcal/mol) scored the most favorable anti-inflammatory flavonoid compound and chlorogenic acid (-8.3 kcal/mol) for phenolic acids. These results validated the anti-inflammatory capacity of the aqueous extract of the leaves by acting as cyclooxygenases 2 inhibitors. Extensive research should be focused on isolation and purification of compounds from crude extracts and testing for their anti-inflammatory activity at both in vivo and in vitro basis.