Optimization of carvacrol, rosmarinic, oleanolic and ursolic acid extraction from oregano herbs (Origanum onites L., Origanum vulgare spp. hirtum and Origanum vulgare L.)

The aim of our study was to increase the extraction efficiency of carvacrol, rosmarinic, oleanolic and ursolic acid from the different species of oregano herbs (Origanum onites L., Origanum vulgare spp. hirtum and Origanum vulgare L.). Various extraction methods (ultrasound-assisted, heat-reflux, continuous stirring, maceration, percolation) and extraction conditions (different solvent, material:solvent ratio, extraction temperature, extraction time) were used, and the active substances were determined by HPLC. The lowest content of carvacrol, rosmarinic, oleanolic and ursolic acid was obtained by percolation. During heat-reflux extraction, the content of active substances depended on the solvent used: ethanol/non-aqueous solvent (glycerol or propylene glycol) mixture was more effective compared with ethanol alone. The results showed that for each species of oregano the most optimal extraction method should be selected to maximize the content of biologically active substances in the extracts.


Introduction
The genus Origanum (Lamiaceae) is rich in phenolic compounds with powerful antibacterial and antifungal properties (Govaris et al. 2010). Oregano essential oils including carvacrol (CA) have been shown to possess antioxidant, antibacterial, antifungal, diaphoretic, carminative, q 2015 Taylor & Francis antispasmodic and analgetic activities (De Falco et al. 2013). Antiviral, antibacterial, antiinflammatory, antioxidant activities of oregano (Petersen & Simmonds 2003;Radusienė et al. 2008) are associated with its constituents rosmarinic acid (RA), ursolic acid (UA) and oleanolic acid (OA). The impact of extraction methods and different oregano species on the extraction yields of RA, UA, OA and CA has not been determined yet to our knowledge. Therefore, we evaluated them in this study using three species of oregano herb: Origanum onites L., Origanum vulgare spp. hirtum and Origanum vulgare L.

Results and discussion
The highest amount of RA from O. vulgare spp. hirtum was obtained by heat-reflux extraction. This method was by 1.2^0.02 times more effective (Supplementary Table 1) than the traditional ultrasound-assisted extraction (UAE) (Jäger et al. 2009). The extracted maximal RA amount from O. vulgare L. by heat-reflux and from O. onites L. by continuous stirring was respectively by 3.85^0.03 and 2.2^0.01 times lower ( p , 0.05) compared with O. vulgare spp. hirtum species. Radusienė et al. (2008) showed that RA content in O. vulgare L. extract prepared by 72 h maceration with 96% of ethanol as solvent ranged within 0.99 -9.65 mg/g dry weight: 1.11-7.42 mg/g from leaves and 0.53 -0.77 mg/g from stems. This content was 4-9 times lower compared with the RA content identified in our extracts from O. vulgare L. herb.
UA and OA are pentacyclic triterpenoids of similar chemical structure (Zhou et al. 2007 Table 1). The latter method was also efficient for CA isolation from O. vulgare L., however, in these conditions the CA amount depended on the oregano species: it was by 3.48^0.02 and 1.1^0.05 times lower ( p , 0.05) in O. vulgare L. compared with O. onite L. and O. vulgare spp. hirtum, respectively. Başer (2008) identified carvacrol as the main compound responsible for oregano biological activities. Therefore, it could be essential to optimize the maximal CA extraction yield.
The present study revealed that different types of extraction methods had a big influence on the extraction yield of compounds. The effects of extraction parameters, i.e., extraction time, temperature, agitation speed and solvent to solid ratio were very important for the diffusion of biologically active compounds from the raw material to the extracts. Heat-reflux extraction and continuous stirring extraction gave significantly higher values, while percolation, maceration and UAE extraction yielded lower values. Based on the extraction time, heat-reflux extraction was the fastest extraction method. Based on the extraction efficiency, it was similar to continuous stirring extraction. In addition, the disadvantages of the extraction by percolation were low extraction efficiency and long extraction time.
Further, we tested the influence of non-aqueous solvent on the amounts of OA, UA, RA and CA extracted from oregano herbs. Zacchigna et al. (2014) determined that polyethylene glycol and UA conjugates could increase UA content in plant-derived extracts. Our results (Supplementary Table S2) showed that ethanol/propylene glycol (10 -30%) solvent mixture as well as ethanol/glycerol (1 -20%) solvent mixture increased only the RA and CA extraction yield by 1.4^0.3 and 1.2^0.1 times ( p , 0.05) compared with the control extract. Ethanol/ glycerol (10%) solvent mixture increased CA extraction yield by 1.1^0.08 times ( p , 0.05) compared with ethanol/propylene glycol solvent mixture (Supplementary Figure S1). A binary solvent system is more efficient compared with a mono-solvent system in the extraction of phenolic compounds in regard to their relative polarity (Dent et al. 2013). Thus, the observed effects could be related to the polarity of the investigated compounds (RA . CA . UA . OA).

Conclusions
CA, RA, OA and UA extraction from oregano herbs depended not only on the oregano species (O. vulgare L., O. onites L. ir O. vulgare spp. hirtum), but also on the extraction method used for the study. The lowest amounts of CA, RA, OA and UA were extracted by percolation. Ethanol/ non-aqueous solvent (glycerol or propylene glycol) mixture instead of ethanol alone resulted in significantly higher extraction yields of CA, RA and UA during heat-reflux extraction from O. onites L., therefore, it could be important for increasing the content of biologically active substances in the herbal preparations.

Supplementary material
Experimental materials relating to this article are available online.

Disclosure statement
No potential conflict of interest was reported by the authors.