Effects of Tissue Freezing, Storage Time and Temperature, and Extraction Conditions On Marrubiin Content of the Medicinal Plant, Marrubium Vulgare

Marrubium vulgare, white horehound, is a medicinal plant that exhibits antispasmodic, analgesic, and anti-inflammatory activities, and is used in the treatment of gastrointestinal and respiratory disorders. One of the active compounds is the furanic labdane diterpene marrubiin. Marrubiin is proposed to be formed from premarrubiin during extraction. The objective of this study was to evaluate the effect of extraction and storage conditions on the concentration of marrubiin.  Specifically, extractions of fresh versus frozen leaf tissue, storage time and temperature, and extraction solvent were compared. Fresh or frozen leaf tissue was extracted in acetone. The effect of leaf and extract storage time at -80 ºC was assessed by assaying marrubiin content over time for up to 4 weeks.  In addition, the effect of acetone or ethanol extraction solvent on marrubiin content was compared using fresh leaf tissue.  Further studies evaluated the effect of heating extracts prior to analysis because this is proposed to enhance the conversion of premarrubiin to marrubiin. Tissue was extracted for 24 hours at room temperature, dried under air flow, resuspended in 80% methanol, and subjected to C-18 column chromatography. Marrubiin concentration was determined with RP-HPLC with pure marrubiin as a standard.  Storage of both leaf tissue and extracts at -80 ºC had a marked effect on marrubiin concentration.  Marrubiin concentration decreased 24% over 4 weeks and was associated with a 2 fold increase in a compound whose retention time correlated to previous reports for premarrubiin. Heating of extracts to 30 ºC for 3 days increased the marrubiin concentration by 76% with levels of premarrubiin dropping to undetectable levels.  These concentrations remained stable upon further storage at either room temperature or -80 ºC.  Marrubiin and premarrubiin concentrations differed with the type of solvent used during extraction. However, heating of extracts eliminated the differences producing high and stable levels of marrubiin for up to a 4 week period.