Comparison of chemical composition of the essential oil from Marrubium vulgare L. and M. incanum Desr. during the second year of cultivation

Horehound herb (Marrubium vulgare L.) is harvested from plantations in Poland. In our country, there are also favorable conditions for M. incanum Desr. growing. The aim of the study was to compare the chemical composition of essential oils from M. vulgare L. and M. incanum Desr. in the second year of cultivation. The study revealed the presence of 31 compounds in the essential oil from M. vulgare L. and 24 compounds in the essential oil from M. incanum Desr. Chromatographic analysis revealed that the main compounds in the essential oil of M. vulgare L. from a two-year plantation were as follows: E-caryophyllene (34.51–36.78%), germacrene D (22.45–27.18%), bicyclogermacrene (9.54–11.12%), δ-amorphene (6.15–8.18%), and carvacrol (4.71–6.64%), whereas the following compounds prevailed in the oil from M. incanum Desr.: germacrene D (28.75–32.14%), E-caryophyllene (23.18–29.57%), α-cadinol (13.59–20.84%), and carvacrol (2.08–7.47%).

Marrubium vulgare L. herb contains about 0.1% of essential oil [4].The oil has relaxant, expectorant [2], and antioxidant [6] properties.Antimicrobial properties were found in the essential oil both from M. vulgare L. [7] and M. incanum Desr.[8].M. incanum Desr. is recommended for soothing stomach and heart diseases [9].Marrubium vulgare L. and M. incanum Desr.raw material is recommended to drink as a panacea, especially as an agent accelerating the digestion processes [10].Marrubium vulgare L. herb is sometimes replaced with M. incanum Desr.herb [11]; therefore, the aim of the study was to compare the chemical composition of essential oils from M. vulgare L. and M. incanum Desr. in the second year of cultivation.

Plant material
The study was carried out in 2010-2011 in the experimental section of the Department of Vegetable and Medicinal Plants, University of Life Sciences in Lublin (51°14' N 22°34' E).The plantation was established from seedlings produced in a greenhouse.Seeds of M. vulgare L. and M. incanum Desr.came from the Botanical Garden of the Maria Curie-Skłodowska University in Lublin.Plants were grown at 30 × 40 cm spacing.The experiment was conducted using two-year-old horehound plants.The herb of M. vulgare L. and M. incanum Desr.was harvested at the flowering stage (July).According to Wolski et al. [2], essential oil is extensively excreted just during this developmental stage.Plants were cut at a height of 8 cm, since the lower part of the stem was woody.Herbage was then dried in a drying chamber at 30°C for four days.

Oil isolation
Essential oils from M. vulgare L. and M. incanum Desr.were obtained from dry herb in a glass Clevenger-type distillation apparatus.Woody stems were discarded before the distillation process.Essential oils were distilled during the steam process according to the "Polish Pharmacopoeia" VIII [12].The distillation time was three hours.40 g of herb and 400 ml of distilled water were used for distillation.The distillation process was carried out in three replicates.
The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library [13] and with data available in the literature [14].The identity of the compounds was confirmed by their retention indices, taken from the literature [14].

Results
The two-year study showed that the essential oil content in the dry herb of M. vulgare L. was on average 0.06% and in the dry herb of M. incanum Desr.-0.04% (Tab.1).
The study revealed the presence of 31 compounds in the essential oil from M. vulgare L. and 24 compounds in the essential oil from M. incanum Desr.during the second year of growing.Sesquiterpene hydrocarbons dominated in the essential oil from M. vulgare L. In addition to the dominant sesquiterpene hydrocarbons in the essential oil from M. incanum Desr., also oxygenated sesquiterpenes accounted for a significant portion of oil (Tab.2, Tab.3).
The study revealed that bicyclogermacrene was also a dominant constituent in the essential oil of M. vulgare L., whereas in the oil of M. incanum Desr. the content of this compound was low.The content of δ-amorphene in the essential oil from M. vulgare L. was much the same, which was many times greater than that from M. incanum Desr.

Discussion
The content of essential oil from M. vulgare L. herb collected in the second year of cultivation was 0.06-0.07%.A similar level of oil content was reported by Zawiślak [15] for annual herbal plants containing 0.06% of essential oil.The content of essential oils in the herb of M. incanum Desr. in the second year of cultivation ranged between 0.03% and 0.05%, which was the same as in the herb harvested during the first year of cultivation, as determined by Zawiślak [5].
Studies on essential oil in other plants of the genus Marrubium sp. have also revealed its low content.The herb of M. peregrinum L. contained 0.07% of essential oil, that of M. parviflorum Fisch & C.A. Mey -0.08% [16], while in M. cuneatum Russell -up to 0.15% [17].
According to Zawiślak [5], the number of identified compounds in the essential oils from M. vulgare L. and M. incanum Desr. in the first year of cultivation remained at a similar level and it was 34 and 20 compounds, respectively.Asadipour et al. [18] indicated the presence of 43 compounds, among which 20 were identified.However, Abadi and Hassani [19] identified 50 different compounds in the essential oil from M. vulgare L.
Sesquiterpene hydrocarbons were dominant in the essential oil from M. vulgare L., which is consistent with the research findings of many authors [7,20].Sesquiterpene hydrocarbons were also dominant in M. thessalum [21].In the report of Zawiślak [5], the essential oil from annual M. incanum Desr.was also abundant in these compounds.
The content of δ-amorphene in the essential oil from M. vulgare L. was 6.15-8.18%and from M. incanum Desr.0.05-1.32% in the second year of cultivation.Zawiślak [5] reported more than twice higher content of δ-amorphene in the essential oil of M. vulgare L. than from M. incanum Desr.harvested during the first year of cultivation.
A study performed by Salama et al. [20] revealed that carvacrol was also the dominant component in the essential oil of M. vulgare L. with its content of 4.35%.This study showed a higher content of carvacrol in the oil of M. vulgare L.

Conclusions
The essential oils from M. vulgare L. and M. incanum Desr.were characterized by a high content of sesquiterpenes, which are responsible for the biological activity of horehound herb.E-caryophyllene and germacrene D are the dominant components of the essential oil of M. vulgare L. and they were also determined as the main compounds of the essential oil from M. incanum Desr.during the second year of cultivation.

Essential oil content in dry herb (%)
Tab. 1 Percentage of essential oil in the herb of Marrubium vulgare L. and M. incanum Desr.during the second year of cultivation.© The Author(s) 2015 Published by Polish Botanical Society Acta Agrobot 68(1):59-62 Zawiślak / Chemical composition of the essential oil from Marrubium Percentage composition of essential oil of Marrubium vulgare L. during the second year of cultivation.Percentage composition of essential oil of Marrubium incanum Desr.during the second year of cultivation.