Obtaining and characterization of Achillea millefolium L. extracts

Transcription

1 Available online at Journal of Agroalimentary Processes and Technologies 214, 2(2), Journal of Agroalimentary Processes and Technologies Obtaining and characterization of Achillea millefolium L. extracts Corina Iuliana Costescu 1, Bogdan Petru Rădoi 1, Nicoleta Gabriela Hădărugă 1, Alexandra Teodora Gruia 2, Adrian Riviş 1, Dorel Pârvu 1, Ioan David 1, Daniel Ioan Hădărugă 3* 1 Department of Food Science, Banat's University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania -Timişoara, Calea Aradului 119, 3645 Timişoara, Romania 2 Regional Centre for Immunology and Transplant, County Clinical Emergency Hospital Timişoara, Iosif Bulbuca Blvd. 1, 3736-Timişoara, Romania 3 Department of Applied Chemistry, Organic and Natural Compounds Engineering, Polytechnic University of Timişoara, Carol Telbisz 6, 31-Timişoara, Romania Received: 9 May 214; Accepted: 11 June 214. Abstract The paper presents the obtaining and characterization of some yarrow (Achillea millefolium L.) extracts, from various parts of plant. The yarrow essential oils were obtained by hydrodistillation method and, in order to characterize them, they were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Essential oil extraction yields were in the range of.5-1.2%, the main compounds being cyclic monoterpenes (such as α- and β-pinene, β-phellandrene) and sesquiterpenes (caryophyllene, β-cubebene, and camazulene), as well as some related epoxides (eucalyptol, bisabolol-oxides). The highest concentration in yarrow flower extract was found for β-pinene and camazulene (17% and 13%, respectively), while for leaf extract the main compound was α-bisabolol. Camazulene was in very high concentration in yarrow root and steam extracts (34% and 46%, respectively). Keywords: yarrow, Achillea millefolium L., essential oils, gas chromatography-mass spectrometry, β- phellandrene, β-pinene, camazulene, α-bisabolol 1. Introduction Yarrow (Achillea millefolium L.) is an ancient medicinal herbaceous plant belonging to the Compositae family and it is used by many people in the form of teas, having stomachic and antidiarrheic properties [1-3]. It can be also used externally in the treatment of wounds. It is a perennial plant and its leaves are bipennated and arranged spirally on the stems. White flowers are arranged in a calatidiu type inflorescence [3]. Aerial parts of the plant, harvested at flowering and then dried, contain.1-.4% volatile oil, and the inflorescence contains up to.5% [3-7]. From some areas of Romania were collected inflorescence samples which, dried in natural conditions, in air, had a.8% content of volatile oil, rich in camazulene. Current production output is.2% volatile oil, obtained from properly dried raw material. In the leaves of six species of yarrow: A. millefolium, A. filipendulina, A. tenuifolia, A. santolina, A. biebersteinii and A. eriophora were identified more than ninety compounds [4-1]. Achillea millefolium L. essential oil principally contains mono- and sesquiterpenoids. The most Corresponding author: dan_hadaruga@yahoo.com

2 concentrated from the first class were pinenes, sabinene, phellandrene, eucalyptol (1,8-cineol), terpinen-4-ol and α-terpineol, while from the second class the most important compounds were β-caryophyllene, bornyl acetate, eudesmol, camazulene, and α-bisabolol [4-7]. The paper presents the obtaining of essential oils from different parts of yarrow (Achillea millefolium L.) from different parts of wild plant grown in the North-West of Romania, by using hydrodistillation coupled with liquid-liquid extraction method and gas chromatography-mass spectrometry, respectively. 2. Materials and methods Materials used for obtaining the essential oils were different anatomical parts of Achillea millefolium L., yarrow (flowers, leaves, roots, and stems), collected in spring-summer of 27 from North- West area of Romania (Apuseni Mountains and Western Plain of Romania Salonta area). Standard solution of alkanes C 8 C 2, used for Kovats index determination, was obtained from Fluka Chemie AG. Hexane was used for liquidliquid extraction and appropriately dilutions and was of GC grade (Merck & Co., Inc.). Obtaining of the yarrow essential oil. The raw and well-grounded plant material was added to a 2 ml extraction flask together with 12 ml of water. A double mantled distillation condenser was attached to the extraction flask as well as a dropping funnel for completing the water from the distillation flask (cohobation). The hydrodistillate containing the yarrow essential oil was passed through the 15 ml GC grade hexane (liquid-liquid extraction) in a collecting 25 ml pear-bottomed flask. The hydrodistillation was performed by using a thermo-controlled heating bath and the extraction process was realized up to three hours. The hexane extract was then dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The amount of essential oil is measured and the yield was calculated as percent of the ratio between essential oil and raw plant masses. Extraction conditions of the essential oils and the results obtained are presented in Table 1. GC-MS analysis of the yarrow essential oils. Analysis of the composition of yarrow essential oils was performed with a Hewlett Packard HP 689 Series gas chromatograph coupled with a Hewlett Packard 5973 mass selective detector (GC-MS). A HP-5 MS capillary column (3 m length,.25 mm i.d.,.25 µm film thickness) was used for the GC system. The temperature program was set up from 5 C to 25 C with 6 C/min, both the injector and detector temperatures were 28 C and He was used as carrier gas. The injection volume was 2 µl. Ionization energy EI of 7eV was used for mass spectroscopy detector, with a source temperature of 15 C, scan range 5-3 amu, scan rate 1 s -1. Compounds separated by GC were identified by matching the experimental mass spectra with those from the NIST/EPA/NIH Mass Spectral Library 2. (22). 3. Results and discussions The separations of essential oils from different yarrow plant parts were achieved with good yields, especially for root and stem (.9% and 1.16%, respectively), while the flower and leaf essential oils were obtained with yields of.5-.7% (Table 1). In order to evaluate de composition of the yarrow essential oil extracted from various plant parts and further to establish the quality and pharmaceutical utility of these products, the gas chromatography coupled with mass spectrometry method was used. The appropriately diluted essential oils were injected in the GC-MS system and the separated compounds were identified by MS spectra (comparison with the NIST database, ver. 22) and/or by using the Kovats index (KI) of the compounds already identified in our earlier studies [9,1]. The KI values were obtained by using a mixture of linear alkane compounds (C 8 -C 2 ; from octane to eicosane), which have KI values corresponding to the product of the number of carbons with 1 (from 8 to 2 in the presented case). The correlation of these KIs with the values of retention times (RT) obtained from the GC-MS analysis of this alkane mixture in the same conditions such as for the samples, allows to calculate the KI values for every compound separated at a specific retention time value.

3 The GC-MS analysis of yarrow flowers essential oil (figure 1), reveals that the main compounds identified by MS and/or KI were monoterpenoids (especially cyclic monoterpenes and the corresponding oxygenated ones) and sesquiterpenoids (hydrocarbons and alcohols), including some degradation compounds (such as epoxides) (see figure 2 for the experimental and NIST database spectra). The most concentrated compounds in yarrow flowers essential oil were β-phellandrene and β-pinene from cyclic monoterpene class (12% and 17%, respectively), and camazulene from the cyclic sesquiterpenes (13%, Table 2). Other compounds identified in relatively high concentrations (2-7%) were α-pinene, eucalyptol, α-terpineol, β- caryophyllene, and β-cubeben); moreover, oxidized compounds such as caryophyllene oxide and bisabolol oxides were identified (Table 2). Table 1. Extraction conditions and results obtained for obtaining the yarrow essential oil samples N o Code Plant part used Mass of plant (g) V hexane (ml) V water (ml) Mass of E.O. * (ml) 1 Am_Fw A. millefolium L. (flowers) Am_Lf A. millefolium L. (leaves) Am_Rt A. millefolium L. (roots) Am_St A. millefolium L. (stems) * E.O. essential oil TIC: CS-S-FL.D\ data.ms Time--> Figure 1. The gas chromatogram from the GC-MS analysis of the yarrow flowers essential oil 144

4 Abundanc e Scan 2518 ( min): CS-S-FL.D\ data.ms Scan 7 (6.942 min): CS-S-FL.D\ data.ms β-phellandrene (mainlib) á-phellandrene 15 1 Scan 767 (7.95 min): CS-S-FL.D\ data.ms β-pinen (replib) á-pinene 2 Scan 98 (8.346 min): CS-S-FL.D\ data.ms Eucaliptol (1,8-cineol) (replib) Eucalyptol 18 O β-caryophyllene (mainlib) Caryophyllene

5 Abundanc e Scan 2735 ( min): CS-S-FL.D\ data.ms Sc an 3315 (22.64 min): CS-S-FR.D\ data.ms Scan 348 ( min): CS-S-FR.D\ data.ms β-cubeben (mainlib) 1H-Cyclopenta[1,3]cyclopropa[1,2]benzene, octahydro-7-methyl 9 Scan 3572 ( min): CS-S-FL.D\ data.ms Camazulene (mainlib) Azulene, 7-ethyl-1,4-dimethyl OH O α-bisabolol oxide B (mainlib) 2-Furanmethanol, tetrahydro-à,à,5-trimethyl-5-(4-methyl-3-cyclohexen-1-yl)-, [2S-[2à,5á(R*)]] OH 79 5 α-bisabolol (mainlib) à-bisabolol

6 Scan 3615 ( min): CS-S-FR.D\ data.ms OH O Bisabolol oxide A (mainlib) 2H-Pyran-3-ol, tetrahydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen Figure 2. Experimental (left) and NIST database (right) MS spectra for the main compounds identified in yarrow essential oils Table 2. Relative concentrations of the main components identified in the raw Achillea millefolium L. essential oils (Am_Fw: flowers; Am_Lf: leaf; Am_Rt: root; Am_St: steam) N o KI (a) RT (b) (min) MS Identification Area% (Am_Fw) Area% (Am_Lf) Area% (Am_Rt) Hexanal Hexenal, (E) Hexanol Area% (Am_St) α-pinen β-phellandrene β-pinen Eucalyptol (1,8-cineol) Ocimen Borneol α-terpineol Tuienyl acetate β-caryophyllene β-farnesen Humulen Caryophyllene oxide β-cubeben Elixen Caryophyllene oxide Ledol α-bisabolol oxide B α-bisabolol Camazulene Bisabolol oxide A Other minor compounds (a) Kovats index (determined with linear alkane standard solution); (b) Retention time

7 The distribution of volatile compounds in yarrow leaves essential oil (figure 3) is relatively similar with the flowers essential oil, the highest difference being in the case of α-bisabolol (16% relative concentration) and the corresponding oxides (bisabolol oxide A at a concentration of 2.8% and bisabolol oxide B with a relative concentration of 7.3%, Table 2). The other main terpenoids were generally identified in lower but important concentrations: β-phellandrene 8%, β- pinene 1%, eucalyptol 7.7%, β-caryophyllene 2.3%, β-cubeben 1.2%, caryophyllene oxide 2.7%, and camazulene 9.7% (Table 2) Time--> TIC: CS-S-TU.D\ data.ms Figure 4. The gas chromatogram from the GC-MS analysis of the yarrow roots essential oil 7 TIC: CS-S-RA.D\ data.ms TIC: CS-S-FR.D\ data.ms Time--> Figure 3. The gas chromatogram from the GC-MS analysis of the yarrow leaves essential oil The GC-MS analyses of yarrow root and stem essential oils (Figures 4 and 5) revealed that the composition differ in comparison with the corresponding flower and leaf essential oils one. Thus, very high concentration of camazulene was determined in these oils, this being 3-4 folds higher than in the other cases (33.8% in root essential oil and 45.8% in stem essential oil, Table 2). The other main terpenoids were β-phellandrene at concentrations of 2.6% and 3.2%, β-pinene 4.2% and.1%, β-caryophyllene 5% and 7.7%, β- cubeben 3.6% and 5.6%, caryophyllene oxide 4.4% and 2.4% for root and stem essential oils, respectively (Table 2). α-bisabolol and α-bisabolol oxide B were identified in higher concentrations only in yarrow root essential oil (1.3% and 3.5%, respectively. These compounds were in concentrations of only.8% for α-bisabolol and.7% for its oxide B for the case of yarrow stem essential oil (Table 2). Time--> Figure 5. The gas chromatogram from the GC-MS analysis of the yarrow steams essential oil 4.Conclusion The studies regarding the composition and distribution of volatile compounds with pharmaceutical properties in yarrow (Achillea millefolium L.) essential oils obtained from different plant parts by using the combined extraction method of hydrodistillation/liquid-liquid extraction reveal the following: (1) the highest concentration of essential oil was obtained in root and stem, probably due to a lower water content of these raw materials used; (2) the most important volatile compounds in yarrow essential oils were mono- and bicyclic monoterpenes and bicyclic sesquiterpenes and their oxides; (3) yarrow flower and leaf essential oil have the highest concentration of β-phellandrene and β-pinene from terpenoid class and camazulene from the sesquiterpenoid class; (4) the most important compound in yarrow root and stem essential oils was camazulene, at three-four times more concentrated than in the flower and leaf essential oil. 148

8 Compliance with Ethics Requirements: Authors declare that they respect the journal s ethics requirements. Authors declare that they have no conflict of interest and all procedures involving human and/or animal subjects (if exists) respect the specific regulations and standards. References 1. Dias, M.I.; Barros, L.; Dueñas, M.; Pereira, E.; Carvalho, A.M.; Alves, R.C.; Oliveira, M.B.P.P.; Santos-Buelga, C.; Ferreira, I.C.F.R., Chemical composition of wild and commercial Achillea millefolium L. and bioactivity of the methanolic extract, infusion and decoction, Food Chemistry 213, 1, Kocevar, N.; Glavac, I.; Injac, R.; Kreft, S., Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium, Journal of Pharmaceutical and Biomedical Analysis 28, 46, *** Achillea millefolium, Accessed: May 15, Tuberoso, C.I.G.; Kowalczyk, A., Chemical Composition of the Essential Oils of Achillea millefolium L. Isolated by Different Distillation Methods, Journal of Essential Oil Research 29, 21, Rahimmalek, M.; Tabatabaei, B.E.S.; Etemadi, N.; Goli, S.A.H.; Arzani, A.; Zeinali, H., Essential oil variation among and within six Achillea species transferred from different ecological regions in Iran to the field conditions, Industrial Crops and Products 29, 29, Bocevska, M.; Sovová, H., Supercritical CO2 extraction of essential oil from yarrow, Journal of Supercritical Fluids 27, 4, Chalchat, J.-C.; Gorunovic, M.S.; Petrovic, S.D., Aromatic Plants of Yugoslavia. I. Chemical Composition of Oils of Achillea millefolium L. ssp. pannonica (Scheele) Hayak, A. crithmifolia W. et K., A. serbica Nym. and A. tanacetifolia All., Journal of Essential Oil Research 1999, 11, *** Achillea, Accessed: May 15, Hădărugă, N.G.; Hădărugă, D.I.; Lupea, A.X.; Păunescu, V.; Tatu, C., Bioactive Nanoparticles (7). Essential Oil from Apiaceae and Pinaceae Family Plants/β-Cyclodextrin Supramolecular Systems, Revista de Chimie 25, 56, Costescu, C.I.; Hădărugă, N.G.; Hădărugă, D.I.; Riviş, A.; Ardelean, A.; Lupea, A.X., Bionanomaterials: Synthesis, physico-chemical and multivariate analysis of the Dicotyledonatae and Pinatae essential oil/β-cyclodextrin nanoparticles, Revista de Chimie 28, 59,