Available online at http://journal-of-agroalimentary.ro Journal of Agroalimentary Processes and Technologies 214, 2(2), 142-149 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: e-mail: dan_hadaruga@yahoo.com
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.
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).7 15 12.5 2 Am_Lf A. millefolium L. (leaves) 99.9 15 12.7 3 Am_Rt A. millefolium L. (roots) 99.9 15 12.9 4 Am_St A. millefolium L. (stems) 13.5 15 12 1.2 * E.O. essential oil TIC: CS-S-FL.D\ data.ms 6 6 6 5 5 5 5 5 4 4 4 4 3 3 3 3 3 2 2 2 2 1 1 1 Time--> 4. 6. 8.1.12.14.16.18.2.22.24.26.28.3.32. Figure 1. The gas chromatogram from the GC-MS analysis of the yarrow flowers essential oil 144
Abundanc e 19 17 15 13 11 9 7 5 3 1 Scan 2518 (17.383 min): CS-S-FL.D\ data.ms 133 79 15 12 175 189 24 3 4 5 6 7 8 9 1111213159221 13 Scan 7 (6.942 min): CS-S-FL.D\ data.ms 1 1 9 7 5 5 3 36 77 121 53 15 63 46 58 82 87 98 11115 128 136 3 4 5 6 7 8 9 1 11 12 13 14 β-phellandrene 77 136 53 65 121 17 3 4 5 6 7 8 9 1 11 12 13 14 15 (mainlib) á-phellandrene 15 1 Scan 767 (7.95 min): CS-S-FL.D\ data.ms 1 13 1 9 7 5 5 3 36 46 79 53 17 121 63 58 74 87 12 115 128 136 3 4 5 6 7 8 9 1 11 12 13 14 β-pinen 79 53 121 136 17 3 4 5 6 7 8 9 1 11 12 13 14 15 (replib) á-pinene 2 Scan 98 (8.346 min): CS-S-FL.D\ data.ms 18 1 2 154 65 99 119 37 49 87 132 139 3 4 5 6 7 8 9 1 11 12 13 14 15 16 5 Eucaliptol (1,8-cineol) 84 96 58 31 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 (replib) Eucalyptol 18 O 139 154 1 133 79 15 5 β-caryophyllene 3 5 7 9 11 13 15 17 19 21 (mainlib) Caryophyllene 12 189 175 24 145
3 3 3 3 2 2 2 2 Abundanc e 19 17 15 13 11 9 7 5 3 1 3 3 3 3 3 2 2 2 2 67 Scan 2735 (18.658 min): CS-S-FL.D\ data.ms 91 15 119 175 189 238 3 4 5 6 7 8 9 1111213159221222324 59 Sc an 3315 (22.64 min): CS-S-FR.D\ data.ms 3 4 5 6 7 8 91111213159221222324 85 95 15 179 Scan 348 (22.611 min): CS-S-FR.D\ data.ms 19 119 175 189 25 24 22 238 222233 3 4 5 6 7 8 9 1111213159221222324 1 5 133 24 β-cubeben 91 15 12 133 24 3 5 7 9 11 13 15 17 19 21 (mainlib) 1H-Cyclopenta[1,3]cyclopropa[1,2]benzene, octahydro-7-methyl 9 Scan 3572 (23.575 min): CS-S-FL.D\ data.ms 1 184 1 1 184 7 5 5 3 39 77 63 92 51 12 115 128 1 153 194 25 22 3 4 5 6 7 8 9 111121315922122 Camazulene 39 51 63 77 115 128 1 92 12 3 5 7 9 11 13 15 17 19 (mainlib) Azulene, 7-ethyl-1,4-dimethyl- 153 1 OH 5 59 85 15 121 179 O 115 152 188 α-bisabolol oxide B 187 22 22 238 3 6 9 12 15 18 21 24 (mainlib) 2-Furanmethanol, tetrahydro-à,à,5-trimethyl-5-(4-methyl-3-cyclohexen-1-yl)-, [2S-[2à,5á(R*)]]- 1 19 OH 79 5 α-bisabolol 189 3 5 7 9 11 13 15 17 19 21 (mainlib) à-bisabolol 24 146
Scan 3615 (23.827 min): CS-S-FR.D\ data.ms 1 13 11 OH 9 7 5 3 17 59 5 59 85 17 O 1 159 18 116 1 189 25 22 238 3 4 5 6 7 8 91111213159221222324 Bisabolol oxide A 1 18 22 22 238 3 6 9 12 15 18 21 24 (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) 1 847 3.64 Hexanal -.2.11-2 96 4.49 2-Hexenal, (E)- -.4 - - 3 92 4. 1-Hexanol -.1 - - Area% (Am_St) 4 994 6.8 α-pinen 3.4 1.2.4.67 5 135 6.95 β-phellandrene 12.1 8. 2.56 3.18 6 142 7.11 β-pinen 17.2 9.9 4.2.11 7 196 8.34 Eucalyptol (1,8-cineol) 6.8 7.7.64.2 8 115 8.57 Ocimen.4.18.8-9 1242 11.75 Borneol 1.5 2.3.64.45 1 1264 12.26 α-terpineol 2.1 3.3.36.66 11 1338 13.91 Tuienyl acetate 1.3..32.87 12 153 17.37 β-caryophyllene 6. 2.3 5.4 7.67 13 1527 17.88 β-farnesen -.6.42.11 14 154 18.13 Humulen -.3.63 1.2 15 1545 18.24 Caryophyllene oxide -.24.22.19 16 1566 18.65 β-cubeben 4.2 1.15 3.59 5.6 17 15 18.95 Elixen -.52..51 18 1672 2.74 Caryophyllene oxide 2.1 2.7 4. 2.42 19 1685 2.98 Ledol 2.5 1.8 3.62 3.24 2 1745 22.7 α-bisabolol oxide B - 7.3 3.48. 21 1775 22.63 α-bisabolol.68 16. 1.27.76 22 1827 23. Camazulene 12.9 9.7 33.82 45.79 23 18 23.84 Bisabolol oxide A.8 2.8.97 - Other minor compounds 26.74 21.39 23.49 25.86 (a) Kovats index (determined with linear alkane standard solution); (b) Retention time
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). 5 4 4 4 4 3 3 3 3 3 2 2 2 2 1 1 1 Time--> TIC: CS-S-TU.D\ data.ms 4. 6. 8.1.12.14.16.18.2.22.24.26.28.3.32. Figure 4. The gas chromatogram from the GC-MS analysis of the yarrow roots essential oil 7 TIC: CS-S-RA.D\ data.ms 4 3 3 3 3 3 2 2 2 2 1 1 1 TIC: CS-S-FR.D\ data.ms 65 5 45 35 3 25 15 5 Time--> 4. 6. 8.1.12.14.16.18.2.22.24.26.28.3.32. 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--> 4. 6. 8.1.12.14.16.18.2.22.24.26.28.3.32. 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
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