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Keywords:

  • LC–MS/MS;
  • iridoids;
  • phenylethanoid glycosides;
  • Globularia alypum;
  • Plantaginaceae (Globulariaceae)

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Acknowledgements
  7. References

Introduction

Globularia alypum L., belonging to the Globulariaceae family, is a perennial wild shrub found throughout the Mediterranean area, Europe, and Africa. This plant is widely used to treat many diseases, but no previous work on the phytochemical composition of the Algerian G. alypum species has yet been reported.

Objective

To investigate the phytoconstituents of the methanolic extract of G. alypum using an LC-ESI-QTOF-MS method.

Methods

Ground air-dried leaves of G. alypum were macerated with methanol at room temperature for 24 h. The supernatant was filtered and concentrated to dryness under reduced pressure in a rotary evaporator, and extracts were recovered with methanol and filtered. Afterwards, the G. alypum extract was injected into the LC-ESI-QTOF-MS system.

Results

The combined LC–MS/MS led to the tentative characterisation of 63 phytochemicals. In this work, a large number of compounds have been characterised in the leaf-extract analysis of this plant. Among others, 24 iridoids and secoiridoids were found, of which nine compounds have not previously been recorded in G. alypum. Also, nine unusual phenylethanoid glycosides were characterised for the first time in this species.

Conclusion

The method used has proved to be a valued tool for the characterisation of a wide range of compounds from G. alypum leaves. This work constitutes a detailed investigation of the chemical composition of G. alypum leaves, which are widely used in different traditional systems of medicine. Copyright © 2014 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Acknowledgements
  7. References

Globularia alypum L., of the family Plantaginaceae (formerly Globulariaceae), is a wild perennial shrub found throughout the Mediterranean area, Europe and northeastern Africa (Elbetieha et al., 2000; Es-Safi et al., 2005). In the Mediterranean region, there are two Globularia species: G. eriocephala (Pomel), which is endemic, and G. alypum L. (Boutiti et al., 2008), which is the subject of this study.

Known locally as ‘taselgha’, G. alypum is one of the most prominent plants in the Algerian folk medicine. Its leaves have been used traditionally as a hypoglycaemic, laxative, diuretic, cholagogue, stomachic, tonic, purgative and sudorific agent, and even as an aphrodisiac (Calis et al., 2002a; Jouad et al., 2002; Es-Safi et al., 2005). Moreover, this plant has been used to treat haemorrhoids and cardiovascular diseases. More recently, it has been found that the extracts from the whole plant reduced histamine and serotonin contraction in vitro and were active against lymphocytic leukaemia P-388 and neoplastic cell culture (Fehri et al., 1996; Bello et al., 2002; Es-Safi et al., 2006). It was also found to have anti-viral activity against polio (Soltan and Zaki, 2009). Concentrated decoctions of young branches and leaves are used in the treatment of boils and intermittent fever. Leaves are used to treat rheumatism and arthritis, and they have been found to have anti-tumour effects as well as phytotoxic potential (Fehri et al., 1996; Elbetieha et al., 2000).

The main biological activities described in G. alypum can be attributed to the different bioactive compounds previously reported in this plant, such as phenolic compounds and iridoid glycosides (Es-Safi et al., 2005; Taskova et al., 2006). The wide use of this plant to treat many diseases in addition to the fact that no phytochemical study has been reported on the Algerian G. alypum strain prompted us to investigate the chemical composition of this plant matrix.

Reversed-phase high-performance liquid chromatography (RP-HPLC) combined with mass spectrometry (MS) detection is one of the most important techniques used to analyse phenolic compounds. Recently, chromatographic performance has been improved by using columns packed with small particles (smaller than 2 µm) and by operating at a pressure of up to 600 bar, thus offering high resolution (Verardo et al., 2010). The on-line coupling of HPLC–MS using electrospray ionisation (ESI) as an interface yields a powerful analytical platform because of its highly efficient resolution and enables the characterisation of a wide range of polar compounds. Electrospray ionisation is one of the most versatile ionisation techniques, as well as being preferred for detecting polar compounds separated by liquid chromatography. The advantages of MS detection include the ability to determine the molecular weight and to gain structural information. Quadrupole time-of-flight (QTOF) MS can provide excellent mass accuracy over a wide dynamic range, allowing measurements of the isotopic pattern, and may be used in MS/MS experiments to provide the elemental composition of the parent and fragment ions. Therefore, QTOF/MS is a powerful detection system for identifying target compounds in highly complex matrices (Gómez-Romero et al., 2011).

Searching for new sources from natural plants or resources is of practical interest, as pharmaceutical drugs applied to several diseases are often too expensive and the exploration of traditional remedies constitutes the first resort for a deprived population. In this context, the present work uses the LC–ESI/QTOF/MS method to characterise the constituents of G. alypum, one of the most widely utilised herbal remedies in Algeria.

Experimental

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Acknowledgements
  7. References

Chemicals

All chemicals were of analytical reagent grade and used as received. Sodium hydroxide was from Fluka (Buchs, Switzerland), and acetic acid from Merck (Darmstadt, Germany). The organic solvents, methanol, acetonitrile and 2-propanol were from Sigma Aldrich (St Louis, MO, USA). Distilled water with a resistance of 18.2 mΩ was deionised by using a Milli-Q system (Millipore, Bedford, MA, USA). Filtering the sample prior to injection into the HPLC system utilized Millex filters (0.20 µm pore size; Millipore).

Sample preparation and extraction

The leaves of G. alypum were harvested in April 2009, in remote areas near the suburbs of Souk Elbatel, 2 km from Seddouk (city of Bajaia, Algeria). The sample was identified at the Botany Laboratory (University of Bajaia). Voucher specimen (D-PH-2013-37-7) was deposited at the Herbarium of the Natural History Museum of Aix-en-Provence, France. Fresh leaves were air-dried in shade at room temperature. After drying, the plant material was ground to a fine powder (diameter < 250 µm) using an electric mill (IKAR A11 basic, Staufen, Germany) and 4 g of this powder was exhaustively extracted by maceration with 50 mL of methanol, at room temperature for 24 h.

In all cases the solutions were filtered and concentrated to dryness under reduced pressure in a rotary evaporator (40°C). Stock solutions with concentrations of 1 mg/mL were prepared and filtered through 0.20-µm micropore membranes before analysis.

Liquid chromatography–mass spectrometry

Analyses were performed using an Agilent 1200 Series Rapid Resolution LC system (Agilent Technologies, Palo Alto, CA, USA), including a standard autosampler and a diode array detector (DAD). The LC column used was a Zorbax Eclipse Plus C18 (150 × 4.6 mm, 1.8 µm).

Separation was performed using as mobile phases aqueous acetic acid 0.5% (v/v) (A) and acetonitrile (B). A gradient programme was used as follows: from 5 to 15% B (0–5 min), from 15 to 30% B (5–25 min), from 30 to 95% B (25–35 min), from 95 to 5% B (35–40 min) and to hold 5% B (40–45 min). The flow rate was established at 0.5 mL/min and column temperature was controlled at 25°C. The LC system was coupled to a microTOF-Q II mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with an ESI interface. The effluent from the RP-HPLC column was split using a T-type phase separator before being introduced into the mass spectrometer (split ratio 1:2). The MS instrument was operated in the negative ion mode with spectra acquired over a mass range from 50 to 1000 m/z. The optimum values of the ESI/MS parameters were: capillary voltage, +4.5/kV; drying gas temperature, 190°C; drying gas flow, 9.0 L/min; and nebulising gas pressure, 2 bars.

During the analyses, external mass-spectrometer calibration was performed using a Cole Palmer syringe pump (Vernon Hills, IL, USA) directly connected to the interface, passing a solution of sodium acetate 5 mm. This external calibration provided accurate mass values for a complete run without the need for a dual sprayer set-up for internal mass calibration.

The accurate mass data of the molecular ions were processed through the software DataAnalysis 4.0 (Bruker Daltoniks), which provided a list of possible elemental formulae by using the Smart Formula Editor. The Editor lists and rates possible molecular formulae consistent with the accurate mass measurement and the true isotopic pattern (TIP). If the given mass accuracy leads to multiple possible formulae, the TIP adds a second dimension to the analyses, using the masses and intensities of each isotope to make a sophisticated comparison of the theoretical with the measured isotope pattern (mSigma value). The smaller the sigma value and the error, the better the fit, and therefore for routine screening an error of 5 ppm and a threshold sigma value of 0.05 are generally considered appropriate (Abu-Reidah et al., 2013).

Results and discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Acknowledgements
  7. References

The base peak chromatogram (BPC) that resulted for the G. alypum extract is depicted in Fig. 1, where the peaks are numbered according to their elution order. A large number of metabolites present in G. alypum were identified by interpretation of their MS and MS/MS spectra found by QTOF/MS combined with the data provided in the literature. The MS data of the identified compounds, including experimental and calculated m/z for the molecular formulae provided, error, mSigma value and the main fragments shown by MS/MS, as well as the proposed compound for each peak, are summarised in Table 1. The analysis of the methanolic extract by LC–ESI/QTOF/MS revealed that iridoids, secoiridoids and phenylethanoid glycosides were the major classes of secondary metabolites in G. alypum.

image

Figure 1. Base peak chromatogram (BPC) of Globularia alypum by LC–ESI/QTOF/MS in the negative ion mode. Peak labelling represents the compounds identified.

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Table 1. Proposed compounds detected in Globularia alypum extract obtained by HPLC–ESI/QTOF/MS
GroupPeaktR (min)m/z experimentalMolecular formulam/z calculatedError (ppm)mSigma valueMS/MS fragments (% relative abundance)Proposed compound
Iridoids and secoiridoids47.78361.114C15H 21O10361.11400.16.7127.0394 (46), 151.0386 (86), 169.0502 (100)Catalpol
 58.35391.1253C16H 23O11391.1246−1.813.6123.0445 (75), 149.0587 (28), 167.0703 (100), 211.0621 (37), 229.0704 (68)Shanzhiside
 610.61373.115C16 H 21O10373.1140−2.78.8123.0451 (63), 149.0597 (45), 167.0731 (32), 193.0519 (100)Gardoside/Geniposidic acid
 811.81373.1134C16 H 21O10373.11401.625.8123.0450 (100), 149.0603 (74), 167.0733 (37), 211.0609 (59)Gardoside/Geniposidic acid
 1013.00375.1292C16H 23O10375.12971.215.4125.0591 (55), 151.0797 (30), 169.0851 (26), 213.0775 (100)Mussaenosidic acid
 1213.28375.1314C16H23O10375.1397−4.72.9113.0239 (12), 151.0761 (26), 169.0879 (59), 213.0774 (100), 214.0824 (12)(epi)Loganic acid
 1313.99373.1166C16H21O10373.1140−7.018123.0444 (55), 149.0620 (100), 167.0717 (44), 193.0501 (31)Gardoside/Geniposidic acid
 1615.12445.1341C19H25O12445.13512.328.7151.0406 (11), 161.0231 (18), 179.0344 (2)Asperulosidic acid methyl ester
 1815.74489.1589C21H29O13489.16144.917.7145.0303 (42), 163.0405 (31), 205.0515 (23), 265.0715 (42)Acetylbarlerin (isomer 1)
 2016.23489.1607C21H29O13489.16141.317.2145.0306 (21), 163.0415 (24), 205.0521 (6), 265.0723 (9), 325.0995 (5)Acetylbarlerin (isomer 2)
 2116.61525.159C24H29O13525.16144.429.1161.0243 (15), 179.0393 (5), 283.0935 (2), 301.1112 (1), 345.0950 (1)6-O-Veratroylcatalpol
 2817.47523.1448C24H27O13523.14571.725.0161.0248 (52), 179.0367 (37)Verminoside
 3017.93355.1028C16H19O9355.10351.19.7149.0610 (21), 175.0401 (69), 191.0723 (35), 193.0504 (100), 235.0606 (75), 265.0724 (20), 295.0836 (67)Gentiopicroside
 3319.42371.0978C16H19O10371.09841.66.4113.0241 (22), 121.0321 (91), 249.0638 (100)Deacetylasperuloside
 3419.54507.1498C24H 27O12507.15081.931.5145.0297 (82), 163.0400 (100)Specioside
 3820.38415.1239C18H23O11415.12461.87.0113.0233 (11), 149.0604 (100), 163.0757 (27), 175.0407 (22), 191.0702 (44)Alpinoside
 4120.95509.1648C24H29O12509.16643.37.0147.0453 (100)Globularimin/Globularinin
 4221.36537.1584C25H29O13537.16145.618.3161.0222 (8), 493.1686 (10)Minecoside
 4622.27519.1505C25H27O12519.15080.517.5145.0297 (40), 163.0412 (32), 307.0822 (68)Coumaroylgeniposidic acid
5123.49551.1774C26H 31O13551.1770−0.715.9147.0454 (100), 163.0409 (8), 181.0513 (14)Decumbeside D (isomer1)
5525.09491.1566C24H 27O11491.1559−1.46.9161.0237 (12), 175.0410 (75), 315.1090 (21)Globularin/Globularicisin
5625.28551.1791C26H31O13551.1770−3.721.4147.0454 (100), 163.0409 (8), 181.0513 (14)Decumbeside D (isomer 2)
5725.98503.1550C25H27O11503.15591.718.9123.0450 (32), 147.0444 (57), 161.0541 (40), 189.0546 (34), 193.0517 (100)Serratoside A
5927.20527.1323C30H23O9527.13484.7157.5Globularioside/Baldaccioside
Phenylethanoid glycosides1113.10461.166C20H29O12461.16641.17.5113.0255 (15), 135.0446 (14), 397.1152 (19)Decaffeoylacteoside
1414.46505.1541C21H29O14505.15634.318.9161.0239 (7), 179.0349 (2), 341.0901 (1)Hebitol II
2417.06519.1721C22H31O14519.1719−0.116.2175.0391 (17), 193.0512 (25), 235.0617 (11)Globularitol
4020.81477.1379C23H25O11477.14024.927.9161.0245 (33), 179.0350 (7)Calceolarioside A (isomer 1)
4421.87623.2001C29H35O15623.1981−3.124.6161.0247 (9), 461.1673 (4)Verbascoside
4722.38477.1399C23H25O11477.14020.83.3161.0245 (64), 315.1104 (11)Calceolarioside B
5023.14623.1993C29H35O15623.1981−1.98.5161.0243 (7), 461.1636 (3)Isoverbascoside
5224.19461.1451C23H25O10461.14530.417.4145.0292 (70), 163.0397 (71), 297.0977 (40)Neosyringalide (isomer 1)
5324.54461.1467C23H25O10461.1553−3.116.6145.0297 (100), 315.1084 (19)Neosyringalide (isomer 2)
5826.95651.2287C31H39O15651.22941.128.2175.0403 (50), 193.0496 (5), 475.1828 (6)Martynoside
6028.59785.2258C34H41O21785.2246−14.310.7Rossicaside A
6130.60769.2341C38H41O17769.23491.113.7Galypumoside A
6230.90799.2423C39H43O18799.245547.9161.0209 (2), 623.1925 (1)Galypumoside B
6335.06753.235C38H41O16753.24006.621.5161.0282 (3)(−)-6’-O-(E)-Cinnamoylverbascoside
Flavonoids2517.05401.1442C18H25O10401.14532.720.1269.1024 (100), 161.0464 (75), 113.0243 (25), 101.0230 (22)Icariside F2
3118.62625.1418C27H29O17625.1410−1.213.9301.0358 (2), 461.1429 (1)Hydroxyluteolin 7-O-laminaribioside
3218.89305.0692C15H13O7305.0667−8.419.496.9592 (54), 225.1137 (100)Gallocatechin/Epigallocatechin
3519.91611.1597C27H31O16611.16182.13.4151.0001 (2), 475.1085 (8)Eriodictiol-O-disaccharide
3619.98463.0871C21H19O12463.08822.34.6301.0366 (53)Quercetin glucoside
3720.23609.1452C27H29O16609.14611.610.3285.0394 (2)Luteolin disaccharide (isomer 1)
3920.38609.1444C27H29O16609.14612.823.2285.0398 (31)Luteolin disaccharide (isomer 2)
4522.12447.0924C21H19O11447.09331.912.9151.0037 (5), 285.0414 (49)Cynaroside
4822.52533.1661C26H29O12533.16640.613.6161.0233 (11), 179.0363 (8), 323.0777 (100)Amurensin
4922.74477.1051C22H21O12477.1038−2.63.5299.0211 (5), 315.0509 (34), 462.0801 (3)Nepitrin
5424.89517.1717C26H29O11517.1715−0.411.3145.0299 (27), 209.0828 (22), 307.0826 (100)Phellamurin
Other polar compounds12.46181.0721C6H13O6181.0718−2.07.8101.0241 (100), 113.0266 (36), 163.0611 (69)Mannitol
22.73191.0564C7H11O6191.0561−1.33.4Quinic acid
32.83341.1091C12H21O11341.1089−0.613.6101.0223 (77), 113.0251 (100), 179.0558 (99)Sucrose
710.86315.0724C13H15O9315.0722−0.727.4108.0224 (39), 152.0134 (100), 153.0219 (53), 232.9773 (22)Gentisoyl hexoside
912.08315.1085C14H19O8315.10850.220.3101.0267 (34), 113.0257 (32), 119.0376 (15), 135.0439 (100), 153.0564 (16)Cornoside
1514.66341.0872C15H17O9341.08781.710.6135.0449 (23), 161.0234 (56), 179.0361 (100), 221.0461 (76), 251.0578 (28), 281.0680 (86)Caffeoylhexose (isomer 1)
1715.48341.0875C15H17O9341.08780.87.2135.0460 (23), 161.0250 (48), 179.0346 (99), 221.0470 (71), 251.0579 (32), 281.0675 (100)Caffeoylhexose (isomer 2)
1915.84163.0413C9H7O3163.0401−7.89.4p-Coumaric acid
2216.83325.0928C15H17O8325.09290.210119.0510 (24), 145.0295 (100), 161.0602 (31), 163.0405 (70), 205.0517 (79), 235.0633 (20), 265.0744 (79)p-Coumaroylhexose (isomer 1)
2316.96429.1395C19H25O11429.14021.712.1145.0303 (72) ,163.0417 (35)Neohancoside C
2617.37385.1136C17H21O10385.11401.126.7101.0239 (2), 113.0246 (3), 145.0317 (2), 207.1043 (19)Sinapic acid O-hexoside
2717.45387.165C18H27O9387.16612.64.3145.0284 (6) , 207.1021 (20)Tuberonic acid hexoside
2917.70325.0927C15H17O8325.09290.67.6119.0493 (36), 145.0301 (73), 161.0620 (30), 163.0403 (86), 205.0510 (100), 235.0616 (19), 265.0734 (85)p-Coumaroylhexose (isomer 2)
4321.46569.1892C26H33O14569.1876−2.830147.0454 (100), 199.0625 (4), 361.1140 (17), 509.1674 (7)Columbianin

Iridoids and secoiridoids

Several iridoids and secoiridoids were tentatively identified in G. alypum leaves. Peaks 4 (m/z 361), 10 (m/z 375) and 12 (m/z 375) were tentatively identified as catalpol, mussaenosidic acid and (epi)loganic acid, respectively. These compounds were characterised by the common fragments at m/z 169 and 151 corresponding to [(M − H) − 162 − CH2O] and [(M − H) − 162 − CH2O − H2O], respectively.

Compounds 16 (m/z 445), 33 (m/z 371) and 38 (m/z 415) were tentatively proposed as asperulosidic acid methyl ester, deacetylasperuloside and alpinoside, respectively, according to their MS data and information reported in the literature (Calis et al., 2001; Ren et al., 2007; Hong et al., 2010).

Compound 30 with m/z 355 was tentatively identified as gentiopicroside (Fig. 2d). Its MS/MS data showed the fragment ions at m/z 193 and 175, corresponding to the loss of the hexose moiety and its subsequent dehydration. This constitutes the first report of this secoiridoid in G. alypum.

image

Figure 2. Chemical structures of several proposed compounds in Globularia alypum leaves.

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Compounds 6, 8 and 13 showed the molecular ion at m/z 373. In view of the molecular formula provided for their accurate masses and the data given in the literature (Quirantes-Piné et al., 2009; Hong et al., 2010), they were identified as gardoside and geniposidic acid, although it was not possible to distinguish between them because they showed the same fragmentation pattern. The MS/MS spectra of these compounds showed fragments at m/z 211, 167, 149 and 123 corresponding to [(M − H) − 162] (211) and the loss of CO2 (167) from the main fragment, as well as the simultaneous elimination of water and CO2 (149). Another fragment was found at m/z 123, corresponding to [(M − H) − 162 − 88], which was shown by the loss of a 3-oxopropanic acid molecule. Gardoside and geniposidic acid have been identified in Globularia species previously (Calis et al., 2001; Taskova et al., 2006) but for the first time in G. alypum.

Peaks 28 (m/z 523) and 42 (m/z 537) were tentatively identified as verminoside (Fig. 2f) and minecoside (Fig. 2i), respectively. It was found that fragments obtained from the first compound at m/z 179 and 161 corresponded to the caffeic acid moiety and its dehydration product, respectively. Meanwhile, the second compound gave fragments at m/z 493, resulting from decarboxylation, and 161 representing the deoxyhexose. Minecoside and verminoside had never been reported in G. alypum before.

Furthermore, specioside (peak 34) was tentatively characterised by comparing its MS data with those reported in the literature (Hong et al., 2010). The MS/MS analysis of this compound (Fig. 3a) yielded the fragment ions at m/z 163 and 145, corresponding to [(M − H) − 162 − 182 Da] and [(M − H) − 162 − 182 − H2O], respectively.

image

Figure 3. The MS/MS fragmentation pattern of (a) specioside, (b) shanzhiside, (c) decumboside D and (d) serratoside.

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Compound 41 was tentatively characterised as globularimin or globularinin, and compound 55 as globularin or globularicisin (Calis et al., 2002a,2002b; Kirmizibekmez et al., 2004), although it was not possible to assign one specific compound because the data provided by MS was unable to distinguish between stereoisomers. Globularimin and globularinin are two highly oxygenated iridoid glucosides from G. alypum (Sudo et al., 1998; Kirmizibekmez et al., 2008).

Peak 59 gave a molecular ion at m/z 527 that was tentatively identified as globularioside or baldaccioside (Es-Safi et al., 2006; Kirmizibekmez et al., 2009). Peak 5 was identified as shanzhiside. The MS/MS spectrum of this compound showed the fragment ions at m/z 229, 211, 167, 149 and 123 corresponding to the successive losses of hexose (229) and water (211) as well as subsequent decarboxylation (167) and dehydration (149) from the fragment ion at m/z 211. Another fragment was found at m/z 123, corresponding to [M − H − hexose − 88], which was obtained by the loss of the 3-oxopropanic acid molecule and a subsequent dehydration. The fragmentation pathway of shanzhiside is shown in Fig. 3b. No available study has reported this iridoid hexoside in G. alypum. In addition to shanzhiside, peaks 18 and 20 were assigned as acetylbarlerin isomers. These compounds present a structure based on the shanzhiside methyl ester skeleton (Fig. 2g).

Peak 21 (m/z 525) was tentatively identified as 6-O-veratroylcatalpol (Saracoglu et al., 2011). This compound showed the fragment ions at m/z 345, 301, 283, 179 and 161. The fragment ion at m/z 345 can be attributed to the aglycone resulting from the loss of hexose, which further formed the fragments at m/z 301 and 283 by a neutral loss of CO2 and subsequent dehydration.

Peaks 51 and 56 were tentatively characterised as decumbeside D isomers. These compounds with the molecular ion at m/z 551 presented MS/MS ions at m/z 181, 163 and 147 (Fig. 3c). The fragment ions at m/z 163 and 147 were referred to the coumaroyl moiety and the formation of an aldehyde from this later, respectively.

Peak 57 was tentatively suggested to be serratoside A. The fragmentation of this iridoid hexoside led to several fragments, among which the fragment ion at m/z 193 was formed by the cleavage of a deoxyhexose and a cinnamoyl unit (m/z 148), while the fragment ions at m/z 147 and 161 could be attributed to cinnamic acid and dehydrated glucose, respectively (Fig. 3d).

Peak 46 presented a molecular ion at m/z 519. The ESI/QTOF analysis showed MS/MS fragments at m/z 307, 163 and 145. The fragment ion at m/z 307 resulted from the loss of the coumaroylhexose moiety. In addition, the m/z ions at 163 and 145 corresponded to the coumaroyl moiety after breakage of the ester bond with the sugar, as well as after an additional loss of an H2O molecule, respectively. According to these data, compound 46 was tentatively characterised as coumaroylgeniposidic acid.

Phenylethanoid glycosides

Phenylethanoid glycosides represent another important group of metabolites characterised in this study, many of which were previously described in G. alypum or in the family Globulariaceae.

Compounds 40 and 47 presented the molecular ion at m/z 477 (C23H25O11). The MS/MS spectra of both compounds led to the identification of calceolarioside A and calceolarioside B, respectively (Fig. 2b). These compounds are another example of phenyl ethyl glycosides extracted from the genus Globularia (Taskova et al., 2006; Kirmizibekmez et al., 2008). The molecular formula C34H41O21 was assigned to compound 60, although it was not possible to establish a suitable MS/MS spectrum due to its low intensity. This compound was tentatively identified as rossicaside A, on the basis of previous reports for Globularia species (Calis et al., 1999; Kirmizibekmez et al., 2009).

Compounds 61 and 62 were characterised as galypumoside A and galypumoside B, respectively, according to their MS data and the data reported in the literature (Kirmizibekmez et al., 2008). These compounds are characterised here for the first time in G. alypum.

Compound 44 gave the molecular ion at m/z 623 with molecular formula C29H35O15. This compound represents the second main peak in the chromatogram of the G. alypum extract, according to its MS data; it has been tentatively proposed to be verbascoside (Fig. 2h). Also, another verbascoside isomer was found in the extract (compound 50) that corresponded to isoverbascoside (Es-Safi et al., 2007a).

Besides these known compounds, two new phenylethanoid glycosides were found in G. alypum for the first time. Accepted data recorded from MS and MS/MS spectra enabled the tentative identification of peak 11 as decaffeoylacteoside and peak 63 as (−)-6-O-(E)-cinnamoylverbascoside.

Compound 24 with the pseudo-molecular ion at m/z 519 and the molecular formula C22H31O14 was tentatively identified as globularitol (Fig. 2j). This compound has been previously isolated as a new sugar ester from the methanolic extract of the underground parts of G. orientalis, but it has never been reported in G. alypum.

Compound 58 gave a [M − H] ion at m/z 651 with the molecular formula C31H39O15. In its MS/MS spectrum, a fragment ion was detected at m/z 475, due to the loss of 176 Da, which represented the feruloyl moiety. Other fragment ions were detected at m/z 193 and 175, corresponding to ferulic acid and further water loss, respectively. Thus, martynoside was the proposed structure for this compound (Fig. 2a), for the first time in G. alypum.

Peak 14 (m/z 505) was assigned to hebitol II (Fig. 2c). The fragmentation of this compound yielded the fragment ions at m/z 341, 179 and 161. The first fragment ion was extracted after the neutral loss of C6H12O5 corresponding to the glucityl group, while fragments ions at m/z 179 and 161 were attributed to the caffeoyl and hexose moieties, respectively.

Compounds 52 and 53 were found to be neosyringalide isomers (Fig. 2e). Their MS/MS spectrum gave fragments corresponding to the coumaroyl moiety at m/z 145 (Es-Safi et al., 2007a).

Flavonoids

In the present work, five flavone glycosides previously described in Globularia were detected. Based on the MS/MS data and on the bibliography (Es-Safi et al., 2005; Kirmizibekmez et al., 2008), peak 31 was tentatively suggested to be hydroxyluteolin-7-laminaribioside, while peaks 37 and 39 were tentatively attributed to luteolin disaccharide isomers. In addition, peaks 45 and 49 were determined as cynaroside (Ben et al., 1982) and nepitrin (Kirmizibekmez et al., 2003), respectively. Compound 35 yielded a [M − H] ion at m/z 611 and its fragmentation resulted in the aglycone ion at m/z 151, probably due to the neutral loss (324 Da) from the fragment ion at m/z 475, suggesting the presence of two hexose residues. Therefore, based on these data, compound 35 was tentatively characterised as eriodictiol O-disaccharide.

Compound 25 with the molecular formula C18H26O10 yielded the fragments at m/z 269, 161, 113 and 101. Where the product ion (m/z 269) corresponded to the loss of the [Ph − CH2 − hexose] group, while the fragments at m/z 161, 113 and 101 matched the dehydration of hexose (161) and its fragment ions at m/z 113 [hexose − 2H2O − CH2O] and m/z 101 [hexose − H2O − 2CH2O]. This compound was tentatively characterised as icariside F2, and was found in the extract of G. alypum for the first time.

Peak 32 had the molecular formula C15H13O7 and showed a fragment ion at m/z 225 corresponding to C11H13O5, which resulted from the breaking of the C-ring of gallocatechin or epigallocatechin, but it was not possible to distinguish between these isomers.

Three flavonol glycosides were found in the extract of G. alypum leaves. Peak 36 was identified as quercetin glucoside (Kirmizibekmez et al., 2009). The characterisation of this compound was based on MS data and the neutral loss of the glucose moiety, which gives rise to the fragment ion at (m/z 301) corresponding to quercetin.

Peak 48 at m/z 533 showed a MS/MS spectrum with the fragments ions at m/z 323, 179 and 161, corresponding to the loss of [M − H − hex − 2(CH3)], hexose and its dehydration, respectively. Accordingly, compound 48 was tentatively considered to be amurensin.

Peak 54 gave a molecular ion at m/z 517. In MS/MS analysis, this compound yielded the daughter fragments at m/z 307 and 145. The first fragment resulted from the neutral loss of glucose and two methyl groups from the main ion, while the latter fragment ion at m/z 145 corresponded to the loss of C9H5O2 (cleavage 1,3A) from the fragment at m/z 307. This detected fragmentation pattern is consistent with that proposed by Fernández-Arroyo et al. (2010). Therefore, compound 54 was tentatively identified as phellamurin.

Other polar compounds

In addition to the above-mentioned flavonoid derivatives identified in this work, six hydroxycinnamic acids and derivatives were characterised. According to the fragmentation profile of these compounds, peaks 15 and 17 with molecular ions at m/z 341 were tentatively characterised as caffeoylhexose isomers, while peaks 22 and 29 at m/z 325 were proposed as p-coumaroylhexoside isomers. Peak 19 was assigned to p-coumaric acid, while peak 26 was attributed to sinapic acid-O-hexoside. Most of these compounds were characterised by common losses such as the loss of the hexose and caffeoyl moieties in caffeoylhexose isomers and coumaroyl unit in isomers of coumaroylhexose. Peak 26 presented a fragment at m/z 207 found after the loss of hexose and an oxygen. These hydroxycinnamic acid glycosides were found in G. alypum for the first time.

Other polar compounds were identified in this extract by using the applied LC–ESI/QTOF/MS method. Compound 2 showed a molecular formula C7H11O6. This compound was identified as quinic acid on the basis of the main literature reports on quinic acid (Gómez-Romero et al., 2010; Gouveia and Castilho, 2010).

Peak 7 with the molecular ion at m/z 315.0724 has been tentatively assigned to gentisoyl glycoside. The MS/MS spectrum displayed the fragment ions at m/z 153 and 108 corresponding to aglycone and its decarboxylation product, respectively.

The MS/MS spectrum of compound 27 demonstrated the molecular ion at m/z 387 and MS/MS fragment ions at m/z 207 and 145, which were consistent with the loss of hexose moiety followed by successive dehydration and decarboxylation, respectively. In accordance with these data, peak 27 was proposed as tuberonic acid hexoside.

Compound 9 with the molecular formula C14H19O8, showed the daughter ions at m/z 153 and m/z 135 corresponding to the loss of hexose and dehydration, respectively. Thus, compound 9 was tentatively characterised as cornoside.

Compound 23 with a molecular ion at m/z 429 gave the fragment ions at m/z 163 and 145 corresponding to [M − H − xylose − CH2] and its dehydration product, respectively. This compound was tentatively proposed as neohancoside C. This phenol glycoside is reported for the first time in this plant. The MS2 analysis of compound 43 showed the pseudo-molecular ion at m/z 569 and the fragment ions at m/z 361, 199 and 147. The daughter ion at m/z 361 resulted from the loss of hexose and two methyl groups, whereas the fragment at m/z 199 arose after the loss of a deoxyhexose group from the fragment ion at m/z 361. Moreover, the fragment that appeared at m/z 147 was the result of the loss of (CH2) from the deoxyhexose group. Accordingly, compound 45 was tentatively assigned as columbianin.

Summary

The LC–ESI/QTOF/MS-based metabolite-profiling approach enabled the tentative identification of 63 metabolites in a G. alypum extract on the basis of their MS and MS/MS spectra in negative ion mode together with the relevant data from the literature. The method applied combined the advantages of a small-particle-size C18-column (1.8 µm), as such the high resolution made it possible to separate several isomers, with the high selectivity, sensitivity, mass accuracy and measurements of the isotopic pattern associated with QTOF/MS for both parent and fragment ions. The analyses of the leaf extract revealed a larger number of compounds, most being iridoids and phenylethanoid glycosides substituted with acyl groups. Therefore, the described HPLC–ESI/QTOF/MS method has proven to be a valuable tool for simultaneous characterisation of a wide range of bioactive compounds from G. alypum leaves. Furthermore, the data compiled may encourage further use of this plant matrix as a folk and alternative medicine in human therapy.

Acknowledgements

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Acknowledgements
  7. References

We wish to thank the Algerian Ministry of High Education and Scientific Research for sponsoring this work. This work was also supported by the project AGL2011-29857-C03-02 (Spanish Ministry of Science and Innovation), as well as P09-CTS-4564, P10-FQM-6563 and P11-CTS-7625 (Andalusian Regional Government Council of Innovation and Science), and A1/041035/11 (Spanish Agency for International Development Cooperation). The authors are grateful to the Spanish Ministry of Science and Innovation for a grant FPU.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Acknowledgements
  7. References