Artemisia annua L.: Essential oil and acetone extract composition and antioxidant capacity

Industrial Crops and Products 45 (2013) 170– 181 Contents lists available at SciVerse ScienceDirect Industrial Crops and Products journa l h o me pag e: www.elsevier.com/locate/indcrop Artemisia annua L.: Essential oil and acetone extract composition and antioxidant capacity Sandra C. Gouveia∗, Paula C. Castilho Centro de Química da Madeira, CCCEE, Universidade da Madeira, Campus Universitário da Penteada, piso 0, 9000-390 Funchal, Portugal a r t i c l e i n f o Article history: Received 26 May 2012 Received in revised form 21 September 2012 Accepted 17 December 2012 Keywords: Artemisia annua Caffeoylquinic acids Phenolic Essential oil Antioxidant a b s t r a c t Aerial parts of Artemisia annua growth in three different locations of Madeira Archipelago were studied. The essential oil composition was established by GC-MS and the main components were monoand sesquiterpenes; artemisia ketone was not detected. The presence of phenolic compounds in the acetone extracts was investigated by HPLC-DAD-ESI/MSn and a diversified phenolic profile of 40 hydrocinnamic acid derivatives and glycosylated flavonoids was found. A few compounds were reported for the first time in Artemisia annua. The antioxidant capacity of essential oils and extracts were measured by three different in vitro assays. For the essential oils, a very good antioxidant response was found and the extracts also showed a good antioxidant capacity, in particular as antiradical scavengers. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The genus Artemisia (family of Asteraceae) includes more than 300 species, mainly small herbs and shrubs (Yoon et al., 2011). There are several reports (Carvalho et al., 2011) describing Artemisia plants as dietary foods and as traditional herbal medicines against inflammatory diseases, infections by fungi, bacteria and viruses, gastric ulcer, cancer among others. One of the most studied Artemisia plants is Artemisia annua L., commonly known as “qinghao” or “annual wormwood”. It is a plant used for many centuries in Chinese folk medicine for the treatment of malaria and fever and several bioactive metabolites have been reported, the most important of them being artemisinin, an endoperoxide sesquiterpene lactone (Castilho et al., 2008). Artemisia annua leaves have a high content of essential oil (EO) containing cineole, ␣-pinene, camphene, borneol, camphor, germacrene-D and artemisia ketone (Bora and Sharma, 2011). The essential oil of Artemisia annua is referenced as having antifungal and antimicrobial activity (Juteau et al., 2002; Verma et al., 2011). In addition to these, other biological properties are associated to more polar extracts of Artemisia annua such ∗Corresponding author at: Kemihuset, Linnaeus väg 10, KB6A14, Umeå universitet, SE-901 87 Umeå, Sweden. Tel.: +46 907865182. E-mail address: [email protected] (S.C. Gouveia). as antimalarial, antibacterial, anti-inflammatory, antitumor and antiulcerogenic (Bhakuni et al., 2001; ´ Cavar et al., 2012). The antioxidant capacity referred in the literature is associated to the high flavonoid content and diversified types of compounds (Brisibe et al., 2009; Ferreira et al., 2010). Also, in vitro antimalarial and anticancer activity of artemisinin and its derivatives is enhanced by the presence certain flavonoids (Ferreira et al., 2010). The phenolic composition of Artemisia annua has been described using HPLC-MS/MS techniques by (Lai et al., 2007), (Han et al., 2008) and (Carbonara et al., 2012); Carvalho and co-workers (Carvalho et al., 2011) reported the HPLC-DAD quantification of several phenolic compounds. The main phenolic compounds reported were flavonoids and hydroxycinnamates. Based on literature survey, the chemical composition and biological properties of Artemisia annua can extensively differ according to their geographical origin and how the plant material is processed. The interest in the potential applications of this plant is still increasing and in the present study we analysed Artemisia annua plants grown in Madeira Archipelago, previously reported as having ca. 1% of artemisin (Castilho et al., 2008). The main goals were to establish the phenolic composition of crude acetone and methanol extracts by high-performance liquid chromatography with diode array detection coupled with mass spectrometry (HPLC-DAD-ESI/MSn); the EO composition by GCMS and the antioxidant capacity of the EO and the extracts were evaluated by three different assays (DPPH, ABTS and FRAP). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.12.022 S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 171 2. Materials and methods 2.1. Chemicals and standards The following reagents were purchased from Merck (Darmstadt, Germany): disodium phosphate dodecahydrated (99%), potassium persulfate (99%), ferrous sulfate heptahydrate (99%), glacial acetic acid (100%), sodium carbonate (p.a.) and sodium chloride (99.5%). 2,2-diphenyl-1-picrylhydrazyl (DPPH) (>95%), Trolox (≥99.8%, HPLC), 2,2azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (≥99%, HPLC), 2,4,6-Tri(2-Pyridyl)-s-triazine (TPTZ) (≥99.0%, TLC) and Folin-Ciocalteu’s phenol reagent were purchased from Fluka (Lisbon, Portugal). Potassium chloride (>99.5%), gallic acid (99%, HPLC), potassium acetate (p.a.), rutin (≥98%, HPLC) and ferric chloride hexahydrate (97–100%) were purchased from Panreac (Barcelona, Spain); potassium dihydrogen phosphate (99.5%), aluminium chloride (98%) and sodium acetate trihydrate (pure) were purchased from Riedel-de Haën (Hanover, Germany). All solvents used for plant extraction were AR grade, purchased from Fisher (Lisbon, Portugal). HPLC-MS grade acetonitrile (99.9%, LabScan, Gliwice, Poland) and ultra-pure water (Milli-Q Waters purification system, EUA) were used for HPLC analysis. Standards prepared in ethanol (100 ␮g/mL): apigenin (>99%), luteolin (>99%), quercetin (>99%), p-coumaric acid (>99%) and caffeic acid (>99%) from Extrasynthese (Lyon, France), kaempferol (>99%) and 5-O-caffeoylquinic acid (99%) from Acros Organics (Geel, Belgium). 1,3-O-dicaffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5O-dicaffeoylquinic acid and 3,4,5-O-tricaffeoylquinic acid (>98% by HPLC for all) were obtained from Chengdo Biopurify Phytochemicals, Ltd China (Sichuan, China). 2.2. Plant material and sample preparation Samples of Artemisia annua were obtained from seeds of artemisinin rich hybrids (CPQBA × POP) kindly offered by UNICAMP, Brazil and cultivated in three different locations of Madeira Archipelago (Preces, Jardim Botânico and Porto Santo). Before flowering, the whole plants were cut and dried. Plants grown in Madeira Botanical Garden (Jardim Botânico) experimental field reached about 3.5 m high. These plants were divided into two lots and one of them was dried under direct sunlight in order to maximize artemisinin production (Castilho et al., 2008). Leaves from the top third of plants were processed separately from those of the rest of the plant. Vouchers were deposited in the Madeira Botanical Garden Herbarium collection. The dried plant material was ground to fine powder in a mechanic grinder. Portions of this powdered plant were separately extracted by solid liquid extraction, using two solvents (acetone and methanol): The plant material (50 g) was macerated with 500 mL of solvent, at room temperature, for 24 h. In all cases, the solutions were filtered and concentrated under reduced pressure in a rotary evaporator (40 ◦C) and kept in the dark at −20 ◦C until tested. Essential oils were obtained by hydrodistillation in a Clevenger type apparatus. 2.3. Essential oil analysis by GC-MS analysis Qualitative and quantitative composition of essential oils was performed by GC and GC-MS analysis, using a HP 5889 series II and a Varian Saturn 3 ion trap system, respectively in both cases, columns DB-5 (30 m × 0.15 mm × 0.25 ␮m; J. W. Scientific) were used; helium N60 was used as carrier gas. The analysis conditions were: initial temperature 40 ◦C for 14 min, gradient of 1.5 ◦C/min up to 250 ◦C, isotherm for 10 min. Injector (and transfer line) temperature was 270 ◦C. 1 ␮L was injected in splitless mode. GC-MS analysis was performed by ESI in a mass range 24 to 400 m/z with a delay time of 2 min. Identification of components was performed by comparing the mass spectra of those of the available NIST database (GC-MS) and by their relative retention index (Kovacs Index, KI) towards a mixture of alkanes C4to C26, injected in the same experimental conditions (GC-FID). 2.4. HPLC-DAD-ESI/MSnanalysis 2.4.1. Liquid chromatography Stock solutions with concentrations (m/v) of 5 mg/mL were prepared by dissolving each dried extract in initial HPLC mobile phase (Acetonitrile/Water (20/80, v/v)). These solutions were filtered through 0.45 mm Nylon micropore membranes prior to use and 10 ␮L were injected for HPLC-DAD-ESI/MSnanalysis. Three independent assays were performed for each sample. The HPLC separation was carried out on a Dionex ultimate 3000 series instrument (California, EUA) coupled to a binary pump, a diode-array detector (DAD), an autosampler and a column compartment. Samples were separated on a Phenomenex Gemini C18 column (5 ␮m, 250 mm × 3.0 mm i.d.; Phenomenex) with a sample injection volume of 10 ␮L. The mobile phase was composed of acetonitrile (A) and water/formic acid (100/0.1, v/v) (B). A gradient program was used as follows: 20% A (0 min), 25% A (10 min), 25% A (20 min), 50% A (40 min), 100% A (42–47 min), 20% A (49–55 min). The mobile phase flow rate was 0.4 mL/min; the chromatogram was recorded at 280 nm and 350 nm and spectral data for all peaks were accumulated in the range of 190–400 nm. Column temperature was controlled at 30 ◦C. 2.4.2. Mass spectrometry For HPLC-ESI/MSnanalysis, a model 6000 ion trap mass spectrometer (Bruker Esquire, Bremen, Germany) fitted with an ESI source operating in the negative mode was used. Data acquisition and processing were performed using Esquire control software. Negative ion mass spectra of the column eluate were recorded in the range m/z 100–1000 at a scan speed of 13,000 Da/s. High purity nitrogen (N2) was used both as drying gas at a flow of 10.0 mL/min and as a nebulizing gas at a pressure of 50 psi. The nebulizer temperature was set at 365 ◦C and a potential of +400 V was used on the capillary. Ultra-high-purity helium (He) was used as collision gas at a pressure of 1 × 10−5mbar and the collision energy was set at 40 V. The acquisition of MSndata was made in auto MSnmode, with an isolation width of 4.0 m/z. For MSnanalysis, the mass spectrometer was scanned from 10 to 1000 m/z with fragmentation amplitude of 1.0 V (MSnup to MS4) and two precursor ions. 2.5. Total phenolic content (TPC) The total phenolic content the extracts was determined following the Folin-Ciocalteu method (Zheng and Wang, 2001) with some modifications (Gouveia and Castilho). Each sample aliquot of 50 ␮L (10 mg/mL, w/v) was mixed with 1.25 mL of Folin-Ciocalteu reagent (diluted 1:10 fold) and 1 mL of 7.5% sodium carbonate solution. The mixture was incubated for 30 min at room temperature and then absorbance was measured at  = 765 nm. The final results were expressed as milligrams of gallic acid equivalents per 100 g of dried plant (mg GAE/100 g). 172 S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 2.6. DPPH radical scavenging activity For the DPPH assay, 100 ␮L of the sample solutions (10 mg/mL) were added to 3.5 mL of a 0.06 mM methanol DPPH radical solution (Gouveia and Castilho, 2012a, b). The decrease in absorbance at  = 516 nm was measured during 30 min. The DPPH radical scavenging effect of the extracts was expressed as ␮mol Trolox equivalent per 100 gram of dried plant (␮mol eq. Trolox/100 g dried plant) for plant extracts and as ␮mol Trolox equivalent per mL of essential oil (␮mol eq. Trolox/mL EO) for essential oils. 2.7. ABTS•+radical scavenging activity The ABTS•+radical working solution was prepared by mixing 50 mL of 2 mM ABTS•+solution with 200 ␮L of 70 mM potassium persulfate solution (Gouveia and Castilho, 2012a,b). This mixture was kept in the dark for 16 h at room temperature. For each analysis, the ABTS•+solution was diluted with pH 7.4 phosphate buffered saline (PBS) solution to an initial absorbance of 0.700 ± 0.021 at 734 nm. This solution was freshly prepared for each analysis. For the assessment of the radical scavenging activity, an aliquot of 100 ␮L (10 mg/mL, w/v) was added to 1.8 mL of ABTS•+working solution and the absorbance decrease, at a  = 734 nm, was recorded during 6 min. Results were expressed as ␮mol Trolox equivalent per 100 g of dried plant (␮mol eq. Trolox/100 g dried plant) for plant extracts and as ␮mol Trolox equivalent per mL of essential oil (␮mol eq. Trolox/mL EO) for essential oils. 2.8. Ferric reducing activity (FRAP assay) The ferric reducing ability of the extracts was measured based on the FRAP assay (Benzie and Strain, 1996). FRAP reagent was prepared daily by mixing 2.5 mL of solution ferric trichloride hexahydrate (20 mM), 2.5 mL of solution TPTZ (10 mM in 40 mM of hydrochloric acid) and 25 mL of acetate buffer 0.3 M (pH 3.6) and incubating at 37 ◦C. For each analysis, 30 ␮L of methanolic solution (1 mg/mL, w/v) were added to 180 ␮L of distilled water and 1.8 mL of FRAP solution. The increase of absorbance was recorded at  = 593 nm in 15 s intervals, during 30 min at 37 ◦C. The FRAP results were expressed as mmol Iron(II) sulfate heptahydrate per mg of dried plant (mmol Fe(II)/mg) for plant extracts and as mmol Iron(II) sulfate heptahydrate per mL of essential oil (mmol Fe(II)/mL EO) for essential oils. 3. Results and discussion 3.1. Essential oil composition The chemical composition of essential oils obtained from dried leaves is presented in Table 1. Similar results have been published by Magalhães et al. (2004), who produced this hybrid and cultivated it in Brazil. Comparing these sets of results it was clear that the composition of essential oil was not substantially affected by climate, location or cultivating condition but it is intrinsic to the plant. Even drying in direct sunshine or shade did not affect much the essential oil composition, with a little loss of the more volatile components being observed for the plant dried under sunlight. The CPQBA × POP hybrid has no thujone derivatives or artemisia ketone, harmful compounds normally present in wild-type Artemisia annua L. (´ Cavar et al., 2012; Reale et al., 2011). The only possible troublesome, in terms of safety, component could be camphor (ca. 40%). The pair 1,8-cineole/camphor as the two major components in essential oils has been found not only in Artemisia annua (´ Cavar et al., 2012; Holm et al., 1997; M R, 2009) but in several other Artemisia subspecies (Kordali et al., 2005; Lopes-Lutz et al., 2008; Shang et al., 2012). 3.2. Phenolic composition by HPLC-DAD-ESI/MSn Comparison between the HPLC-DAD-ESI/MSnscreening of methanolic and acetone extracts showed that, for the methanolic extract, the number of peaks detected was lower and with reduced chromatographic resolution leading to a poor fragmentation of the detected compounds. This can be associated to matrix effects and high molecular weight compounds common in more polar solvents extractions. Several authors (Kallithraka et al., 1995; Korekar et al., 2011) have evaluated methanol versus acetone as extraction solvents for phenolic compounds with clear advantage for acetone. Our choice for this solvent resulted from our previous work where it was the best to extract artemisinin. The acetone extracts from plants cultivated in three different locations were very similar between them, thus we only present the chromatogram and fragmentation data for one of them (Preces). The HPLC-DAD-ESI/MSnbase peak chromatogram profile for acetone extract is shown in Fig. 1. The identification and characterization of the detected compounds was made by comparison of the HPLC retention, UV and mass spectra with those of standard compounds. Since only a limited number of standard compounds was available, structures of unknown compounds were established largely based on their tandem MSnfragmentation behaviour. Among the detected compounds, we found typical hydroxycinnamic acid UV absorptions (max 230–240, 300 sh. 320–340 nm) and flavonoids characteristic UV absorptions: flavonols derivatives exhibited two maximum absorptions at 250–270 nm and 320–360 nm, derived from the aglycone A and B rings, respectively. Peaks corresponding to flavones conjugates showed three absorptions at 210–230 nm, 250–280 nm and 330–350 nm (Mabry et al., 1970). In our study, 38 compounds were tentatively identified and some of them are reported for the first time for Artemisia annua. Fig. 2 represents the chemical structures of main classes of compounds detected in Artemisia annua. Table 2 shows the experimental analytical data: retention time (tR), wavelength of maximum absorbance (max), deprotonated molecular ions [M–H]−, and most important MSnfragment ions for each peak. Usually, the base peak in the MS1spectrum was assigned as the deprotonated molecular ion [M–H]−. When isomers were observed, their identification was performed based on literature data. 3.2.1. Identification of flavonoids The occurrence of flavonoids, in particular those conjugated with one or more sugar moieties, in Artemisia species has been previously reported (Ferreira et al., 2010, Gouveia and Castilho, Han et al., 2008; Lai et al., 2007). In HPLC-ESI/MSnscreening of Artemisia annua, mainly apigenin and quercetin conjugates have been reported. In the present work, in addition to these two types of aglycones, luteolin, isorhamnetin and kaempferol derivatives compounds were also identified. The tandem MS/MS experiments of the flavonoids resulted on the deprotonated molecular ion ([M–H]−) and the deprotonated aglycone ion (Y− 0) as a result of the loss of the sugar unit. Most of the detected flavonoids were in their glycosylated form and/or esterified with acyl groups. But two aglycones were detected on its free form – luteolin and apigenin. 3.2.1.1. Free aglycones (36 and 40). Compound 36 (tR= 27.7 min) was identified as luteolin by comparison with a reference standard (data not shown). In the MSnexperiments the [M–H]−ion at m/z 285 gave several typical luteolin fragment ions at m/z 199, 217 ([MH-C3O2]−), 175 ([M-H-C3O2-C2H2O]−) and 241 ([M-H-CO2]−). Compound 40 (tR= 33.9 min) exhibited a [M–H]−ion at m/z 269 and the its MSnfragmentation presented main fragment ions at S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 173 Table 1 Composition of the essential oil (EO) from leaves of Artemisia annuagrowth in Preces, Porto Santo and JardimBotânico (dried under direct sunlight and in the shade). KI ref*KI Compound Preces Porto Santo JardimBotânico Shade JardimBotânico Sunshine 937 937 ␣-pinene 3.58 1.55 3.19 2.79 953 954 camphene 0.28 0.01 0.23 0.31 990 902 ␤-pinene 0.60 0.27 1.35 0.88 1018 1017.0 ␣-terpinene 0.68 0.68 0.77 0.78 1026 1027.0 p-cymene 3.32 1.23 2.10 3.31 1030 1031.0 limonene 0.23 0.05 0.39 0.24 1030 1035.0 1,8-cineole 11.6 6.54 9.87 7.92 1059 1061.0 ␥-terpinene 1.27 1.22 1.35 1.28 1141 1141.0 trans-pinocarveol 0.51 0.88 0.86 0.64 1143 1149.0 camphor 42.8 45.5 42.0 44.33 1165 1173.0 borneol 2.50 8.96 2.58 4.74 1177 1180.0 terpinen-4-ol 2.80 2.59 2.45 2.51 1194 1196.0 myrtenol 1.23 1.21 1.00 1.08 1217 1222.0 trans-carveol 1.77 1.76 1.79 1.55 1242 1247.0 l-carvone 1.03 0.52 0.30 0.78 1345 1346.0 ␣-cubebene 0.10 0.10 0.08 0.13 1351 1353.0 eugenol 1.13 0.66 1.23 1.09 1376 1373.0 ␣-copaene 1.75 0.17 0.82 1.15 1389 1388.0 ␤-cubebene 1.93 1.31 1.02 1.66 1394 1392.0 cis-jasmone 0.24 0.72 0.24 0.48 1428 1417.0 ␤-caryophyllene 7.77 10.21 6.44 8.65 1444 1452.0 ␣-humulene 0.46 0.94 6.70 0.94 1480 1479.0 germacrene-D 7.15 8.41 8.61 7.77 1485 1486.0 ␤-selinene 1.93 3.09 2.78 3.04 1494 1493.0 bicyclogermacrene 3.29 1.4 1.86 1.95 *See http://www.pherobase.com/database/kovats/kovats-index.php. m/z 225 ([M-.H-CO2]−), 151 (1,3A−) and 149 (1,4B−+ 2H). Comparing these results to those obtained for a standard solution of apigenin, 40 was identified as free apigenin. Two types of flavonoid glycosilation were observed: Oglycosilation and C-glycosilation. The identification of these two groups was based on the tandem MSnfragmentation experiments. For C-glycosylated compounds the sugar unit is linked to the aglycone with a carbon-carbon bond that is resistant to rupture. The representative MSnfragments for C-glycosylated flavonoids are related to the sugar part and occurs at m/z 0,2 X−[M-H-120]−,0,3 X−[M-H-90]−and 0,2 X−[M-H-60]−(Cuyckens and Claeys, 2004). The non-observation of deprotonated aglycone ion, Y− 0, makes identification process harder. For O-glycosylated compounds the sugar moieties are lost as neutral fragments and the flavonoid structure is more easily identified due to aglycone bonds cleavages. 3.2.1.2. C-glycosides (7, 8, 9, 12 and 13). For these compounds UV spectra showed maximum absorptions bands at  = 225, 272 and 314 nm identical to characteristics absorptions bands of flavones (Mabry et al., 1970). However, the tandem MSnexperiments revealed a different pattern to that known for O-glycosylated flavones. Compound 7 (tR= 5.4 min) and 8 (tR= 5.8 min) showed a [MH]−ions at m/z 563. The MS2fragmentation revealed a behaviour typical of the asymmetrical di-C-glycosides with fragment ions at [M-H-210]−, [M-H-90]−and [M-H-60]−. The neutral loss of 60 Da (0,3X−) indicates the presence of a pentose moiety. Compound 8 has been previously described by our group in Artemisia argentea (Gouveia and Castilho, 2011a,b) and was characterized as apigenin-6-C-hexoside-8-C-pentoside. Compound 7 similar to 8 had MS3fragment ions at m/z 353 [0,2Xi0,2Xj]−and 383 [0,2Xi0,3Xj]−suggesting apigenin as the aglycone (Vukics et al., 2008). However, contrarily to compound 8, 7 did not present an intense [M-H-60]−ion at m/z 503 (2.9% of base peak). Based on these evidences and knowing that 6-C-pentoside-8-C-hexoside elutes before the isomer 6-C-hexoside-8-C-pentoside, compound 7 was identified as apigenin-6-C-pentoside-8-C-hexoside. Fig. 1. HPLC-DAD-ESI/MSnanalysis of the acetone extracts of Artemisia annua total aerial parts – HPLC-MS negative ion ESI/MSnbase peak chromatogram (BPC). 174 S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 Table 2 Characterization of phenolic components of the acetone extract from Artemisia annua by HPLC-DAD-ESI/MSn. No. tR(min) Identification max (nm) [M-H]−(m/z) HPLC-DAD-ESI/MSnm/z (% base peak) 1 2.8 Caffeic acid hexoside derivative 275, 304 473 MS2[473]: 342 (14.4), 341 (100), 179 (13.8), 161 (13.8), 131 (29.2) MS3[473 → 341]: 179 (100), 161 (43.7), 143 (10.8), 119 (44.2), 101 (11.3) MS4[473 → 341 → 179]: 119 (92.0), 113 (54.0), 101 (63.2), 89 (100) 2 3.1 Quinic acid - 191 MS2[191]: 173 (74.9), 127 (100), 111 (47.0), 109 (69.7), 85 (70.7) MS3[191 → 127]: 163 (30.8), 109 (69.7), 99 (100) 3 4.0 Kaempferol-3,7-di-Ohexoside - 609 MS2[609]: 449 (18.2), 448 (11.6), 447 (100), 285 (34.9) MS3[609 → 447]: 327 (7.6), 284 (73.0), 285 (100), 255 (62.3) MS4[609 → 447 → 284]: 256 (31.1), 255 (100), 151 (34.2) 4 4.4 3-O-Caffeoylquinic acid 241, 300, 324 353 MS2[353]: 191 (100), 179 (51.4), 135 (15.9) MS3[353 → 191]: 173 (100), 171 (16.9), 127 (56.8), 111 (30.2), 109 (39.2), 85 (37.4) MS4[353 → 191 → 127]: 109 (100), 99 (90.9), 85 (12.9) 5*5.0 5-O-Caffeoylquinic acid 242, 300, 325 353 MS2[707]: 353 (100) MS2[353]: 191 (100) MS3[707 → 353]: 191 (100) MS3[353 → 191]: 173 (57.1), 127 (100), 109 (55.9), 85 (97.7) MS4[707 → 353 → 191]: 173 (100), 127 (63.7), 135 (21.8), 111 (17.4) MS4[353 → 191 → 127]: 109 (100) 6 5.2 4-O-Caffeoylquinic acid 240, 300, 323 353 MS2[353]: 191 (18.4), 179 (58.3), 173 (100), 135 (8) MS3[353 → 173]: 111 (30.8), 93 (100) 7 5.4 Apigenin-6-Cpentoside-8-Chexoside. 224, 273, 314 563 MS2[563]: 503 (2.9), 473 (98.0), 443 (78.2), 383 (83.5), 353 (100) MS3[563 → 353]: 326 (13.4), 325 (100), 297 (78.2) MS4[563 → 352 → 325]: 298 (5.1), 297 (100) 8 5.9 Apigenin-6-Chexoside-8-Cpentoside. 224, 273, 314 563 MS2[563]: 503 (59.3), 473 (68.6), 443 (100), 383 (63.0), 353 (63.2) MS3[563 → 443]: 383 (50.0), 354 (12.0), 353 (100) MS4[563 → 443 → 353]: 326 (35.1), 325 (100), 297 (56.2) 9 6.5 Apigenin-6-Crhamnoside-8-Chexoside 224, 272, 313 577 MS2[577]: 559 (21.9), 503 (2.8), 487 (28.3), 473 (40.0), 457 (53.1), 353 (100) MS3[577 → 353]: 326 (18.3), 325 (100), 297 (71.2) MS4[577 → 353 → 325]: 297 (100) 10 6.9 3-O-Feruloylquinic acid 326 367 MS2[367]: 193 (100), 191 (1.5), 173 (3.7) MS3[367 → 193]: 149 (31.9), 134 (100), 109 (10.3) 11 7.4 5-O-Feruloylquinic acid 327 367 MS2[367]: 191 (100) MS3[367 → 191]: 179 (100), 134 (53.0), 111 (68.6), 127 (55.5), 109 (12.7), 85 (93.0) 12 7.9 Apigenin-6-Chexoside-8-Crhamnoside 224, 272, 313 577 MS2[577]: 487 (41.2), 473 (2.7), 457 (100), 353 (36.9) MS3[577 → 457]: 383 (18.5), 354 (33.0), 353 (100) MS4[577 → 457 → 353]: 326 (55.5), 325 (100), 298 (22.0) 13 8.3 Apigenin-8-C-hexoside 224, 272, 314 431 MS2[431]: 341 (23.1), 311 (100) MS3[431 → 311]: 284 (38.1), 283 (100) MS4[431 → 311 → 283]: 283 (100), 224 (77.3), 163 (23.7) 14 8.8 Quercetin-Odihexoside – 625 MS2[625]: 302 (17.1), 301 (100), 300 (19.1) MS3[625 → 301]: 271 (22.9), 212 (10.8), 179 (82.9), 151 (100) MS4[625 → 301 → 151]: 169 (100), 107 (20.2) 15 9.1 Luteolin-7-O-hexoside – 447 MS2[447]: 286 (14.0), 285 (100) MS3 [447 → 285]: 285 (100), 241 (24.9), 176 (19.0), 175 (80.1), 149 (117.5) 16 9.3 Mearnsetin-Ohexoside 257, 300, 342 493 MS2[493]: 332 (19.0), 331 (100), 330 (10.3), 316 (11.7) MS3[493 → 331]: 317 (12.8), 316 (100), 315 (16.3) MS4[493 → 331 → 316]: 287 (49.6), 271 (46.1), 229 (38.2), 166 (100) 17*9.5 Quercetin-3-Oglucoside 258, 353 463 MS2[463]: 302 (17.1), 301 (100), 300 (19.1) MS3[463 → 301]: 271 (22.9), 212 (10.8), 179 (82.9), 151 (100) MS4[463 → 301 → 151]: 169 (100), 107 (20.2) 18 9.8 Isorhamnetin-Ohexoside 256, 270, 344 477 MS2[477]: 316 (17.1), 315 (100), 300 (29.3) S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 175 Table 2 (Continued) No. tR(min) Identification max (nm) [M-H]−(m/z) HPLC-DAD-ESI/MSnm/z (% base peak) MS3[477 → 315]: 301 (11.4), 300 (100) MS4[477 → 315 → 300]: 284 (62.1), 271 (67.5), 245 (63.1), 229 (100), 213 (61.9) 19*11.8 3,4-O-Dicaffeoylquinic acid 246, 299, 325 515 MS2[515]: 354 (17.5), 353 (100), 335 (12.3), 299 (12.2), 173 (21.8) MS3[515 → 353]: 191 (24.8), 179 (60.9), 173 (100), 135 (14.8) MS4[515 → 353 → 173]: 155 (20.8), 111 (100), 93 (61.1) 20*12.5 1,5-O-Dicaffeoylquinic acid 243, 300, 328 515 MS2[515]: 353 (100) MS3[515 → 353]: 191 (100) MS4[515 → 353 → 191]: 173 (43.8), 127 (100), 109 (22.1) 21*12.9 3,5-O-Dicaffeoylquinic acid 242, 300, 328 515 MS2[515]: 354 (10.7), 353 (100) MS3[515 → 353]: 191 (100), 179 (53.9) MS4[515 → 353 → 191]: 173 (100), 127 (90.9), 93 (34.5) 22 13.1 Dicaffeoylquinic acid isomer 240, 326 515 MS2[515]: 354 (15.8), 353 (100) MS3[515 → 353]: 191 (100), 179 (52.1), 135 (15.2) MS4[515 → 353 → 191]: 85 (100), 173 (76.6), 127 (66.8), 109 (76.6) 23*14.2 4,5-O-dicaffeoylquinic acid 243, 300, 327 515 MS2[515]: 354 (14.5), 353 (100) MS3[515 → 353]: 191 (31.5), 179 (53.9), 173 (100), MS4[515 → 353 → 179]: 155 (33.6), 111 (57.0), 93 (100) 24 14.6 Luteolin-7-O-pentoside 417 MS2[417]: 285 (100), 284 (22.4) MS3[417 → 285]: 257 (5.1), 243 (72.1), 241 (54.1), 217 (93.1), 199 (98.2), 175 (100), 151 (39.2) 25 15.3 Dihydroxydimethoxyl-Ohexoside flavone 491 MS2[491]: 371 (12.1), 330 (12.5), 329 (100), 314 (10.3) MS3[491 → 329]: 315 (21.1), 314 (100) MS4[491 → 329 → 314]: 300 (22.8), 299 (100) 26 16.8 3-p-O-Coumaroyl-5-Ocaffeoylquinic acid 499 MS2[499]: 337 (100), 221 (11.4), 179 (), 163 (14.2) MS3[499 → 337]: 179 (28.2), 163 (100), 135 (15.8) MS4[499 → 337 → 163]: 119 (100) 27 18.1 Unknown 423 MS2[423]: 262 (10.6), 261 (100), 173 (100) MS3[423 → 261]: 175 (11.6), 173 (100) MS4[423 → 261 → 173]: 93 (100) 28 18.9 1-O-Caffeoyl-5-Oferuloylquinic acid 328 529 MS2[529]: 368 (12.9), 367 (100), 353 (30.0), 191 (8.5) MS3[529 → 367]: 191 (100) MS4[529 → 367 → 191]: 173 (69.0), 127 (100), 109 (52.9) 29 20.2 1 or 5-O-caffeoyl-4-Oferuloylquinic acid – 529 MS2[529]: 368 (42.4), 367 (100), 173 (23.3) MS3[529 → 367]: 193 (81.5), 191 (28.5), 173 (100) MS4[529 → 367 → 173]: 137 (21.5), 111 (14.6), 93 (100) 30 20.6 3-O-feruloyl-5-Ocaffeoylquinic acid 529 MS2[529]: 368 (2.8), 367 (100), 353 (18.4), 193 (21.4) MS3[529 → 367]: 194 (8.4), 193 (100), 191 (42.2), 135 (2.4) 31 22.2 3,4-O-diferuloylquinic acid 543 MS2[543]: 367 (19.5), 349 (100), 193 (3.8), 173 (12.4) MS3[543 → 349]: 287 (38.6), 193 (28.4), 175 (100), 155 (34.2) 32 23.2 3,5-O-diferuloylquinic acid 543 MS2[543]: 367 (100), 349 (38.2), 191 (18.28) MS3[543 → 367]: 193 (100), 173 (2.4) 33 23.8 Kaempferol-3-Ocaffeoylhexoside – 609 MS2[609]: 447 (20.1), 323 (17.1), 286 (11.5), 284 (100), 285 (98.2), 179 (29.4) MS3[609 → 285]: 213 (55.2), 151 (89.4), 107 (100) 34 26.5 Quercetin-Ocaffeoylhexoside 253, 330 625 MS2[625]: 463 (57.1), 445 (24.2), 323 (14.3), 301 (100), 300 (5.1) MS3[625 → 301]: 273 (4.1), 271 (5.9), 255 (19.6), 176 S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 Table 2 (Continued) No. tR(min) Identification max (nm) [M-H]−(m/z) HPLC-DAD-ESI/MSnm/z (% base peak) 179 (45.9), 151 (100), 107 (7.0) MS4[625 → 301 → 151]: 107 (100) 35 27.2 Unknown 579 MS2[579]: 418 (24.7), 417 (100) MS3[579 → 417]: 243 (100), 179 (26.5) MS4[579 → 417 → 243]: 225 (16.7), 199 (100), 183 (31.0) 36*27.7 Luteolin – 285 MS2[285]: 243 (38.2), 241 (92.1), 217 (12.5), 199 (69.5), 175 (100), 151 (40.2) MS3[285 → 175]: 147 (100) 37 28.5 Caffeoylcoumaroyltartaric acid 232, 300, 311 457 MS2[457]: 296 (10.5), 295 (100), 173 (12.9) MS3[457 → 295]: 163 (100), 121 (36.2) MS4[457 → 295 → 163]: 111 (38.8), 93 (100) 38*29.2 3,4,5-OTricaffeoylquinic acid 677 MS2[457]: 296 (10.5), 295 (100), 173 (12.9) MS3[457 → 295]: 163 (100), 121 (36.2) MS4[457 → 295 → 163]: 111 (38.8), 93 (100) 39 30.4 Eriodictyol-7-Ohexoside 247, 329 449 MS2[449]: 287 (100), 173 (14.3) MS3[449 → 287]: 173 (100) MS4[449 → 287 → 173]: 111 (100), 83 (73.6) 40*33.9 Apigenin 260, 331 269 MS2[269]: 227 (33.4), 225 (100), 201 (59.2), 151 (32.6), 149 (80.3) MS3[269 → 225]: 198 (23.1), 183 (66.9), 181 (100) (–) Their UV spectra have not been properly observed due to low intensity. *Comparison with reference standards. Fig. 2. Chemical structures of phenolic compounds characterized. S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 177 Compounds 9 (tR= 6.5 min) and 12 (tR= 7.9 min) displayed a [MH]−ion at m/z 577. MSnexperiments of these two peaks did not revealed the fragment corresponding to the neutral loss of 60 Da and the presence of a fragment ion at m/z 353 indicates that the sugar groups must be a hexoside or a methylpentose. Compound 9 presented MS2ions at m/z 559 (21.9% of base peak) [M-H-H2O]−and m/z 503 [M-H-74]−suggests that the rhamnoside group should be linked at 6-C-position. Thus, 9 was identified as apigenin-6-C-rhamnoside-8-C-hexoside. Compound 12 has been reported before and was identified as apigenin-6-C-hexoside-8-C-rhamnoside. Compound 13 (tR= 8.3 min) showed a [M-H]−ion at m/z 431. The MS2spectrum presented main fragment ions at m/z 311 [MH-120]− 0,2X−(base peak) and 341 0,3X−(23.1% of base peak). The loss of water molecules is indicative of C-6 isomers. This type of cleavage was not observed for compound 13 and based on literature reports (Gouveia S. and Castilho) this compound was identified as apigenin-8-C-hexoside. 3.2.1.3. O-glycosides (3, 14, 15, 16, 17, 18, 24, 25, 33 and 34). The fragments resulted from the of O-glycosilated flavonoids were labelled as proposed by Cuyckens and Claeys (2004). The i,jA−and i,jB−labels correspond to ions containing intact Aand B-rings, respectively, and i and j specify the C-ring bonds that have been broken. Compound 3 (tR= 4.0 min) displayed a [M-H]−ion at m/z 609. Its analysis by MS2fragmentation resulted in a neutral loss of 162 Da forming a fragment ion at m/z 447 and an intense fragment at m/z 285 (34.9% of base peak). MS3spectrum displayed a fragment ion at m/z 285 (loss of 162 Da), as base peak, and its subsequent fragmentation gave the typical fragments of kaempferol at m/z 255 [Y0−-CH2OH]− and 151 (1,3A−) (comparison made with a standard solution of kaempferol). The absence of a fragment ion at m/z 323 excluded the hypothesis of the two 162 Da residues being caffeoylhexosides and were characterized as being two hexosides residues. Based on the previous reports (Gouveia and Castilho, 2010) and according to the rules described by Ablajan and co-workers (Ablajan et al., 2006) 3 was identified as kaempferol-3,7-Odihexoside. Compound 33 (tR= 23.8 min) also displayed a [M-H]−ion at m/z 609. However, MSnfragmentation was quite different of that found for compound 4. The MS2spectra showed the radical aglycone ion [Y0-H]−at m/z 284, as base peak, and also an intense fragment at m/z 285 (98.2% of base peak). The loss of a 324 Da residue was attributed to a combined loss of two 162 Da groups and was confirmed by the presence of a fragment ion at m/z 447 (20.1% of base peak) formed by the loss of 162 Da. The fragment ion at m/z 323 (17.1% of base peak) assigned as [caffeoylhexoside-H]−point out to a hexoside group esterified with a caffeoyl group rather than two hexosides moieties. The aglycone was identified as being kaempferol based on the MSnfragments and comparison with a kaempferol reference solution. The favoured glycosilation positions for flavonols, such as kaempferol are 3-OH and 7-OH. When the aglycone radical is more abundant than the deprotonated aglycone ion, indicates an aglycone substituted at position 3-OH (Cuyckens and Claeys, 2005). For compound 33, the MS2spectrum base peak was the aglycone radical ion, at m/z 285, and therefore it was identified as being kaempferol-3-O-caffeoylhexoside. Compound 17 (tR= 9.5 min) exhibited a [M-H]−ion at m/z 463 which easily lost a 162 Da moiety, in MS2fragmentation, resulting in a fragment ion at m/z 301. Further fragmentation of this ion gave the representative fragments of quercetin at m/z 151 (1,2 A−-CO), 179 ([1,2A−-H]−) and 271 [M-H-CH2O]−. Therefore, compound 17 was unequivocally identified as quercetin-3-O-glucoside by comparison with a reference standard solution. Compounds 14 (tR= 8.8 min) and 34 (tR= 26.5 min) gave the same [M-H]−ion at m/z 625 and their MSnfragmentation was similar. In the MS2spectra, the same base peak at m/z 301 was observed (loss of 324 Da). The occurrence of a MS1ion at m/z 463 (loss of 162 Da) suggests that the residue of 324 Da is composed of two units of 162 Da linked. However, the nature of these two groups appears to be distinct for each compound. For 34, a MS1 ion at m/z 323 (14.3% of base peak) was observed indicating a caffeoylhexoside group which is in good agreement with the long retention time of this compound. The lower retention time of 14 and the absence of a fragment ion at m/z 323 indicate a dihexoside residue. Fragmentation of the deprotonated aglycone ion, Y− 0at m/z 301 allowed to identify common fragment ions of quercetin, as described for compound 17. Since the Y− 0ion is the MS2base peak, the substitution groups must be linked to only one OH group of aglycone structure. The aglycone radical ion was not observed, so the 3-OH position is excluded but no other fragments were found to support in which position the substitution occurs. Thus, 14 was tentatively identified as quercetin-O-dihexoside and 34 was as quercetin-Ocaffeoylhexoside. Compound 15 (tR= 9.1 min) displayed a [M-H]−in at m/z 447 and its MS2fragmentation revealed the loss of 162 Da forming a fragment ion at m/z 285, Y− 0. This compound was already described for Artemisia argentea (Gouveia and Castilho, 2011a, b) and identified as luteolin-7-O-hexoside. Another luteolin derivative was detected at a retention time of 14.6 min (compound 24). It gave a [M-H]−ion at m/z 417 easily lost a neutral group of 132 Da (pentoside) rising the deprotonated aglycone ion at m/z 285. Fragmentation of the ion at m/z 285 gave the characteristics fragments of luteolin at m/z 175 ([M-H-C3O2C2H2O]−), 217 ([M-H-C3O2]−) and 241 ([M-H-CO2]−) (Fig. 3). The favoured substitution position for flavones such as luteolin is the 7OH position (Cuyckens and Claeys, 2004). Consequently, compound 24 was characterized as luteolin 7-O-pentoside. To our knowledge it is the first time that this compound is reported for Artemisia species. Compound 16 (tR= 9.3 min) showed a [M-H]−ion at m/z 493. Its MSnfragmentation resulted in the aglycone ion at m/z 331 due to the loss of 162 Da. The MS3radical ion at m/z 316 is similar to the fragmentation behaviour described for mearnsetin-O-hexoside, also detected in Artemisia argentea and Artemisia annua (Gouveia and Castilho, 2011a,b; Han et al., 2008). Compound 18 (tR= 9.8 min) displayed a [M-H]−ion at m/z 477 and was characterized as isorhamnetin-O-hexoside by comparison with literature data (Gouveia and Castilho, 2009). This compound was not reported before for Artemisia annua but was detected in Artemisia species (Gouveia and Castilho, 2011a,b). Compound 25 (tR= 15.3 min) showed a [M-H]−ion at m/z 491. A loss of 162 Da was observed in the MS2fragmentation, forming a fragment ion at m/z 329. In the further MSnfragmentations, two losses of 15 Da each were observed and associated to two methoxyl groups. So, 25 was characterized as dihydroxy-dimethoxyl-O-hexoside flavones. This compound was also reported for Artemisia argentea (Gouveia and Castilho, 2011a,b) but it was not reported for Artemisia annua before. One compound (39) from the group of flavanones is reported for Artemisia annua for the first time. It was found at a retention time of 30.4 min and identified as a eriodictyol-7-O-hexoside. The [MH]−ion appeared at m/z 449 and the hexoside residue was easily expelled in the MS2fragmentation resulting in the deprotonated aglycone ion, Y− 0, at m/z 287. 178 S.C. Gouveia, P.C. Castilho / Industrial Crops and Products 45 (2013) 170– 181 Fig. 3. Proposed fragmentation pathway for compound 24–luteolin-O-pentoside. 3.2.2. Identification of hydroxycinnamic acids (2, 4, 5, 6, 10, 11, 19, 20, 21, 22, 23, 26, 28, 29, 30, 31, 32 and 38) Compound 2 (tR= 3.1 min) displayed a [M-H]−ion at m/z 191 and was identified as quinic acid (Gouveia and Castilho, 2011a,b). Despite that this compound was found as a relative intense component of the Artemisia annua extract, it was not reported before for this plant. A total of 19 quinic acid derivatives were detected and identified in this study, most of them quinic acid esterified with acyl groups. The identification of each compound was made based on the main fragment ions obtained in the MSnexperiments. The hierarchical key for the identification by LC-MSnof quinic acid derivatives proposed by Clifford et al. (Clifford et al., 2003, 2005) was used to identify this class of compounds. Mono-, diand tricaffeoylquinic acids were identified by comparison of the retention time, MSnfragmentation behaviour and UV spectra with those of standard compounds. It was the case of compounds 5 (tR= 4.4 min) as 5-O-caffeoylquinic acid; 19 (tR= 11.8 min) as 3,4-O-dicaffeoylquinic acid; 20 (tR= 12.5 min) as 1,5-Odicaffeoylquinic acid; 21 (tR= 12.9 min) as 3,5-O-dicaffeoylquinic acid; 23 (tR= 14.2 min) as 4,5-O-dicaffeoylquinic acid and 38 (tR= 29.2 min) as 3,4,5-O-tricaffeoylquinic acid. Additionally, two monocaffeoylquinic acids, compounds 4 and 6, were identified based on MSnfragmentation of the [M-H]−ion at m/z 353. Compound 4 (tR= 4.4 min) gave a MS2ion at m/z 191 (base peak) and an intense fragment ion at m/z 179 (51.4% of base peak) which is representative of a caffeoyl group linked to the 3-OH position of quinic acid. Thus, 4 was identified as 3-O-caffeoyquinic acid. Compound 6 (tR= 5.2 min) showed a MS2ion at m/z 173 (base peak) pointing to a quinic acid esterified at position 4-OH (Fig. 4). Therefore, 6 was classified as 4-O-caffeoylquinic acid. Remarkably, this compound gave a higher intensity than the isomer 5-O-CQA commonly found in other Asteraceae plants. Compound 22 (tR= 13.1 min) gave a [M-H]−ion at m/z 515 and further MSnfragmentation gave the characteristic fragment ions of dicaffeoylquinic acid isomers at m/z 191, 173 and 353. This should be a cis isomer of a dicaffeoylquinic acid with no substitution position 4-OH (Jaiswal et al., 2011; Ma et al., 2008). The unequivocally identification of this compound can only be established by NRM studies or comparison with a reference standard since even UV-irradiation studies would not clarify if it is a mono-cis or a di-cis compound. Compounds 10 (tR= 6.9 min) and 11 (tR= 7.4 min) exhibited the same [M-H]−ion at m/z 367 but a different MSnfragmentation behaviour. For 10, the MS2spectrum showed a fragment ion at m/z 193 (base peak). Based on the rules reported by Clifford et al. (Clifford et al., 2003) this compound was identified as 3-Oferuloylquinic acid. Compound 11 gave, as base peak of the MS2 spectrum, a fragment ion at m/z 191 [quinic acid-H]−. This pattern is consistent to that reported for 5-O-feruloylquinic acid (Clifford et al., 2005). Compound 26 (tR= 16.8 min) displayed a [M-H]−ion at m/z 499. The MS2spectrum showed a fragment ion at m/z 337, as base peak, indicating the loss of 162 Da and suggesting a coumaroylquinic acid derivative. MS3fragmentation of the ion at m/z 337 resulted in a fragment ion at m/z 163 and a MS4ion at m/z 119, as base peaks, pointing to a 3-p-O-coumaroylquinic acid structure, according to literature reports (Clifford et al., 2003). The absence of a strong fragmentation at m/z 173 suggests a 3,5-O-p-coumaroyl-caffeoylquinic acid. Since the caffeoyl group is the first to be lost it should be linked to the 5-OH position. Taking into account these data, 26 was identified as 3-p-O-coumaroyl-5-O-caffeoylquinic acid. To our knowledge, quinic acids esterified with coumaroyl groups have not been reported before in phenolic screenings for Artemisia annua. Three compounds with [M-H]−ions at m/z 529 were observed (compound 28 (tR= 18.9 min), 29 (tR= 20.2 min) and 30 (tR= 20.7 min)). In the MS2fragmentation all compounds gave MS2 base peaks at m/z 367 [feruloylquinic acid-H]−, due to the loss of a caffeoyl residue, and a secondary fragment ion at m/z 353 (ca. 20% of base peak). The distinction of these three isomers was based on the MS3fragmentation. Compound 28 gave a MS3ion at m/z 191, as base peak, which is consistent with a 5-O-feruloylquinic acid structure. According to the fragmentation data, a fragment ion at m/z 179 was not observed, thus the caffeoyl group must be linked to the 1-OH position of quinic acid. So, compound 28 was identified as 1-O-caffeoyl-5-Oferuloylquinic acid. Compound 29 (tR= 20.2 min) exhibited a MS3base peak at m/z 173 which indicates a 4-O-feruloylquinic acid structure. The exact