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Analysis of procyanidins in pine bark with reversed-phase and normal-phase high-performance liquid chromatography–electrospray ionization mass spectrometry
Analytica Chimica Acta 522 (2004) 105–112 www.elsevier.com/locate/aca
Analysis of procyanidins in pine bark with reversed-phase and normal-phase high-performance liquid chromatography–electrospray ionization mass spectrometry Maarit Karonen*, Jyrki Loponen, Vladimir Ossipov, Kalevi Pihlaja Laboratory of Environmental Chemistry, Department of Chemistry, University of Turku, FIN 20014, Turku, Finland Received 16 March 2004; received in revised form 18 June 2004; accepted 18 June 2004 Available online 19 July 2004
Abstract Three procyanidin fractions, one with shorter oligomers, one with longer oligomers and one with polymers, were obtained from Pinus sylvestris L. bark extract. Procyanidins were analyzed using reversed-phase and normal-phase high-performance liquid chromatography methods coupled with electrospray ionization mass spectrometric detection in the negative ion mode. On the grounds of mass spectral data, pine bark was found to contain procyanidins from monomers through decamers and higher polymers. # 2004 Elsevier B.V. All rights reserved. Keywords: Procyanidins; Liquid chromatography; Pine bark
1. Introduction Plant phenolics have been increasingly of interest due to their suggested advantageous health effects (maintenance of health and protection from diseases such as cancer and coronary heart disease) and possibility to use them as natural food additives, since they influence the quality and stability of foods by acting as flavourants, colourants and antioxidants. In a study where the antioxidant activities of phenolic extracts from edible and nonedible Finnish plant materials were examined by autoxidation of methyl linoleate, pine bark was ranked among the most potent plant sources for natural phenolic antioxidants [1]. In another study where antimicrobial activity of extracts prepared from Finnish plant materials against selected microbes was conducted, pine phloem extract was again one of the most active extracts [2]. In addition, pine bark extract has been used as a folk medicine since ancient times [3] and it is still used therapeutically as dietary supplement in Europe. Previously several phenolic compounds such as * Corresponding author. Tel.: +358 2 333 6830; fax: +358 2 333 6700. E-mail address: [email protected] (M. Karonen). 0003-2670/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.06.041
phenolic acid glycosides, stilbene glycosides, lignans and flavonoids have been isolated from inner Scots pine bark [4]. Scots pine bark has also been found to be rich in procyanidins [4,5]. Proanthocyanidins are oligomers and polymers of flavan3-ol monomer units most frequently linked either C4 ! C6 or C4 ! C8 (B-type proanthocyanidins). A-type proanthocyanidins possess the second interflavanoid bond resulting in oxidative coupling between C2 ! O7 positions. The most common classes are procyanidins consisting of catechin, epicatechin and/or their gallic acid esters, and prodelphinidins consisting of gallocatechin, epigallocatechin and/or their galloylated derivatives. Proanthocyanidins in bark are largely procyanidins and, to a more limited extent, prodelphinidins; Scots pine bark contains no prodelphinidins [5]. Proanthocyanidins have attracted increasing attention in the fields of nutrition, health, and medicine mainly due to their potent antioxidant capacity, e.g. it has been observed that procyanidin content in cocoa samples closely correlates to their oxygen radical absorbance capacity (ORAC) values [6]. Proanthocyanidins are known to have various physiological effects like antioxidant, antimicrobial, anti-allergy and anti-hypertensive activities [7].
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Taking into account the interesting properties of pine bark extract that can be due to procyanidins and the fact that distribution and structural characteristic of pine bark procyanidins were unknown, it was necessary to study further pine bark procyanidins. In this study, reversed-phase and normal-phase high-performance liquid chromatographic (HPLC) methods with UV-diode array detection (DAD) and electrospray ionisation–mass spectrometry (ESI–MS) were applied to detect and characterize procyanidins in pine bark.
detector and Analyst Software 1.1 data system. In HPLC– ESI–MS the ionization technique was an ionspray (pneumatically assisted electrospray). The mass spectrometer was operated in negative ion mode: spray needle voltage, 4200 V; heated nitrogen gas temperature, 310 8C (for normal-phase method) and 320 8C (for reversed-phase method); orifice plate voltage, 35 V; ring voltage, 220 V; nebulizer gas (purified air) was set at 10; curtain gas (N2) at 12. Masses were scanned from 100 to 2800 amu in steps of 0.3 amu. Split ratio was 7:3 prior to introduction into the ionization chamber.
2. Experimental
2.4. Reversed-phase HPLC
2.1. Materials
Reversed-phase separations were performed using a Merck Superspher 100 RP-18 column (75 mm 4 mm i.d., 4 mm). The binary mobile phase consisted of (A) water and formic acid (99:1, v/v) and (B) acetonitrile. A linear gradient elution was performed: 0 min: 100% A; 2 min: 100% A; 19 min: 91% A, 9% B; 35 min: 70% A, 30% B; 43 min: 30% A, 70% B. The flow rate was 1 ml/min, detection wavelength 280 nm and injection volume 20 ml.
Scots pine (Pinus sylvestris L.) phloem and bark material were collected, air dried and ground into small pieces by Ravintorengas Oy. Acetone (analytical grade) was purchased from Riedel-de Hae¨ n (Seelze, Germany), formic acid 98–100% from Fischer Scientific (Loughborough, UK), acetonitrile (HPLC-grade) from Lab-Scan (Dublin, Ireland), ethanol (99.5%, v/v) from Primalco (Rajama¨ ki, Finland), methanol (HPLC-grade) and glacial acetic acid from J.T. Baker (Denventer, Holland), and dichloromethane from Mallinckrodt (Denventer, Holland). Water was obtained using the Elgastat UHQ-PS purification system (Elga, Kaarst, Germany). (+)-Catechin and ()-epicatechin were from Sigma (St. Louis, MO, USA). 2.2. Sample preparations Pine bark powder (100 g) was extracted with 70% aqueous acetone (4 ml 1000 ml 12 h). Acetone was evaporated under reduced pressure, and the solid residue was lyophilized. The lyophilized crude extract (21.3 g) was dissolved in water to a concentration of 100 mg/ml, and 50 ml of extract was fractionated on Sephadex LH-20 using a modification of the method used earlier [4] by elution with H2O (fraction I), and stepwise gradient of aqueous ethanol (10–95%, fractions II–X) and aqueous acetone (30–70%, fractions XI–XIII). The procyanidin compositions of obtained fractions were screened with HPLC and fractions containing procyanidins were combined as follows: fr A (IV–VI; 370.0 mg), fr B (VII–XI; 379.4 mg) and fr C (XII, XIII; 231.9 mg), dissolved in water to a final concentration of 20 mg/ml, and filtered through 0.45 mm PTFE filters. 2.3. Instrumentation HPLC analyses were performed on two systems: MerckHitachi HPLC-DAD system consisted of L-7100 pump, L-7200 autosampler, L-7455 diode array detector, and D-7000 interface, and Perkin-Elmer HPLC–ESI–MS system consisted of SCIEX API 365 LC/MS/MS mass spectrometer connected to a Series 200 HPLC system with UV–vis
2.5. Normal-phase HPLC Normal-phase separations were performed using a Merck LiChrospher Si 60 column (250 mm 4 mm i.d., 5 mm). The method developed earlier [6,8] was used with minor modifications. The binary mobile phase consisted of (A) dichloromethane, methanol, water and acetic acid (82:14:2:2, v/v) and (B) methanol, water and acetic acid (96:2:2, v/v). A linear gradient elution was performed: 0 min: 100% A; 30 min: 82% A, 18% B; 45 min: 69% A, 31% B; 50 min: 12% A, 88% B; 60 min: 12% A, 88% B. The flow rate was 1 ml/min, detection wavelength 280 nm and injection volume 5 ml.
3. Results and discussion Procyanidins were isolated from pine bark crude extract by column chromatography with Sephadex LH-20 into three fractions (A–C) which were studied using reversed-phase and normal-phase HPLC with DAD and ESI–MS. Procyanidins can be detected UV spectrophotometrically without difficulty but the identification of individual procyanidins is impossible because of the similar shapes and maxima of UV spectra. Therefore, in HPLC–MS analyses, UV and mass data were collected within the same run allowing the comparison of the UV chromatogram with the total ion chromatogram (TIC). Mass data was obtained using electrospray ionization technique which is easy to use and soft usually giving just molecular ion peaks or molecular ion peaks with few fragments. In addition, electrospray is also capable to produce multiply charged ions, and the sensitivity is high. Procyanidins were detected with the negative ion mode due
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Fig. 1. Reversed-phase HPLC-UV trace (280 nm) of procyanidin fractions from pine bark: (A) fraction A, mainly from monomers to tetramers; (B) fraction B, mainly from dimers to decamers; (C) fraction C, mainly polymers. The labels 1–3 on the peaks indicate the degree of polymerization of procyanidins in the peaks and the label ‘‘tax’’ to taxifolin.
to their weakly acidic nature. Earlier, it was shown that positive mode is successful in detecting procyanidin oligomers through pentamers, but it seemed to be evident from the decreased ionization efficiency of higher oligomers that pentamers would be the limit of detection [9]. Reversed-phase and normal-phase HPLC traces of obtained fractions at 280 nm are presented in Figs. 1 and 2, respectively. In the chromatograms, the degree of polymerization of each peak due to pine bark procyanidins was
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Fig. 2. Normal-phase HPLC-UV trace (280 nm) of procyanidin fractions from pine bark: (A) fraction A, mainly from monomers to tetramers; (B) fraction B, mainly from dimers to decamers; (C) fraction C, mainly polymers. The labels 1–10 on the peaks indicate the degree of polymerization of procyanidins in the peaks and the label ‘‘tax’’ to taxifolin.
determined based on mass spectral data consisting of characteristic deprotonated molecules and fragment ions. Degrees of polymerization (DP), deprotonated molecules [M H], [2M H], [M 2H]2, [M 3H]3 and fragment ions of pine bark procyanidins are listed in Table 1. A comparison between the extracted ion chromatograms of the doubly charged oligomeric procyanidins from pentamer up to decamer from fraction B and the HPLC-UV trace of fraction B is shown in Fig. 3
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Table 1 Degrees of polymerization (DP), and [M H], [2M H], [M 2H]2, [M 3H]3 and fragment ions of pine bark procyanidins DP
[M H]
[2M H]
1 2 3 4 5 6 7 8 9 10
289.0 577.3 865.3 1153.6 1441.6 1729.9 2017.0
579.1 1155.7 1731.4
[M 2H]2
[M 3H]3
Fragment ions (m/z) 245.2 286.9, 289.0, 407.2, 425.2, 451.0 286.6, 289.0, 575.5, 577.0, 713.2, 739.3 286.9, 289.0, 575.2, 577.0, 864.7
720.7 864.7 1009.0 1152.4 1296.7 1440.7
To get the general view of pine bark procyanidins, a calculated mass spectrum of all procyanidins in fraction B accumulated from ca. 640 scans recorded by negative ion NP–HPLC–ESI–MS is presented in Fig. 4. A series of [M H] ions separated by 288 Da can be observed from m/z 289.0 to 2017.0 corresponding to B-type procyanidins from monomer to heptamer. Confirming the foreknowledge of pine bark proanthocyanidins, prodelphinidins were not detected. Two procyanidin monomers were detected (Fig. 1A) and verified using standard compounds to be (+)-catechin and ()-epicatechin, respectively. The mass spectrum of the monomer (Fig. 5A) in fraction A recorded by negative ion NP–HPLC–ESI–MS exhibited [M H] ion at m/z 289.0, [2M H] ion at m/z 579.1 and also a fragment ion at m/z 245.2 consistent with the spectrum reported in [10]. The mass spectrum of the dimer (Fig. 5B) in fraction A recorded by negative ion NP–HPLC–ESI–MS exhibited [M H] ion at m/z 577.3 and [2M H] at m/z 1155.7. Fragmentation of procyanidin dimer consisting of the extension unit and the terminal unit can happen through retroDiels-Alder (RDA) or heterocyclic ring fission (HRF) mechanisms or through quinone methide (QM) cleavage of the interflavonoid bond [11,12]. The fragmentation of the heterocyclic ring can take place on the extension unit or on the terminal unit. The fragment ion of the dimer at m/z 451.0 indicates HRF, which is also known as the elimination of the phloroglucinol molecule. The RDA fragmentation of the dimer produced the ion at m/z 425.2. RDA has been found to be the most important fragmentation for structure elucidation of dimers [11]. Fragmentation on the extension unit has been considered to be energetically more favorable since it produces fragment ions with larger p–p hyperconjugated system than RDA on the terminal unit [13]. The sequential water elimination from m/z 425.2 produced the ion at m/z 407.2. In some cases [11], m/z 407.2 has been detected in significant amounts, even greater than m/z 425.2 but in this study m/z 425.2 was more abundant. The QM cleavage of the interflavonoid bond produced fragment ions at m/z 286.9 and 289.0. Four isomeric dimers were detected with reversedphase HPLC, two of them eluting at the same peak at the retention time of 15 min. (Fig. 1A). All dimers had the same
864.7 960.1
molecular mass and similar fragments thus making the identification of individual dimers unfeasible. The identification or structural characterization, e.g. the position of the interflavanoid linkage, of any oligomeric procyanidins cannot be conducted with ESI–MS, therefore, further analysis is needed in order to identify individual procyanidins. However, a variety of difficulties in isolating and characterizing pure higher oligomers exist. The mass spectrum of the procyanidin trimer (Fig. 5C) in fraction A recorded by negative ion NP–HPLC–ESI–MS exhibited [M H] ion at m/z 865.3 and [2M H] ion at m/z 1731.4. The fragmentation of the trimer seems to be similar to that of dimers. The HRF fragmentation produced an ion at m/z 739.3. The fragment ion at m/z 713.2 indicates the RDA. Contrary to dimers the product of HRF was more abundant than that of RDA. The QM cleavage of the interflavonoid bond produced principally the ions at m/z 286.6 and 577.0 indicating that the cleavage takes place in the upper interflavanoid bond; regardless of that the ions at m/z 289.0 and 575.5 were observed implying that the cleavage can also happen in lower interflavanoid bond. A weak signal at m/z 425.2 was detected suggesting that further fragmentation of m/z 577.0 occurs. The main ion for tetrameric procyanidins (Fig. 5D) was [M H] at m/z 1153.6 whilst neither [2M H] ion nor ions indicating HRF or RDA were detectable. The fragment ions from QM cleavage were observed at m/z 286.9, 289.0, 575.2, 577.0 and 864.7. Mass spectral data (Table 1, Fig. 5) indicate that welldefined molecular ions [M H] and fragment ions were obtained for the shorter procyanidins. According to the literature with increasing degree of polymerization, higher procyanidins are prone to form multiple charges [9,14]. The charge state of the multiply charged species can be defined by the m/z difference between isotopic peaks: for a [M H] ion the distance between the isotopic peaks is one mass unit, for [M 2H]2 ion 0.5 and for [M 3H]3 ion 0.3 [14,15]. For the procyanidin pentamer (Fig. 5E) the most abundant ion is [M H]– at m/z 1441.6 but in addition to the fragment ions the doubly charged ion [M 2H]2 at m/z 720.7 can be seen. The enlarged signal of [M H] ion (Fig. 5E) shows the m/z difference between the isotopic peaks. The distance between the isotopic peaks of [M H] ion is
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Fig. 3. (A) Normal-phase HPLC-UV trace (280 nm) of procyanidin fraction B in comparison to extracted ion chromatograms of procyanidin pentamer (B), hexamer (C), heptamer (D), octamer (E), nonamer (F), and decamer (G).
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Fig. 4. The calculated mass spectrum of pine bark procyanidins accumulated from ca. 640 scans recorded by negative ion NP–HPLC–ESI–MS.
varying from 0.9 to 1.2 because in the MS conditions used the masses were scanned in steps of 0.3 amu. In the case of hexamer (Fig. 5F), the doubly charged [M 2H]2 ion at m/ z 864.7 is the most abundant but the [M H] ion at m/z 1729.9 is also visible. The enlarged signal of [M 2H]2 ion (Fig. 5F) shows that the distance between the isotopic peaks is 0.6. For the heptamer, [M 2H]2 ion at m/z 1009.0 is dominant and [M H] ion at m/z 2017.0 is weak. The molecular ion of octamer was no longer detectable. The doubly charged ions were observed for octamer, nonamer and decamer at m/z 1152.4, 1296.7 and 1440.7, respectively. In addition, triply charged [M 3H]3 ions were observed for nonamer and decamer at m/z 864.7 and 960.1. On average, the ionization efficiency decreases with the increasing degree of polymerization. In UV trace (Fig. 2), a strong peak of higher polymers exist but in the corresponding place in TIC a negative peak is observed. For the moment, the amount of polymers and their isomers can be so high that it causes a decline in the ionization efficiency and inhibits the removal of counter ions. No mass spectral data was obtained for procyanidin polymers. They were identified on the grounds of their retention times and UV spectra characteristic for procyanidins. Since procyanidins are eluted according to the increasing degree of polymerization in normalphase HPLC (Fig. 2B), the degree of polymerization for higher polymers must be >10. In a few earlier studies conducted concerning proanthocyanidins in pine, the mean degrees of polymerization (mDP) by thiolytic degradation for P. sylvestris bark procyanidins and for the pycnogenol, a food supplement extract from maritime pine tree bark, have been found to be 5.6 [5] and 7.4 [12], respectively. Earlier, it has been stated that the electrospray is superior to other ionization techniques because of its capability to produce multiply charged ions corresponding to molecular masses of higher proanthocyanidin polymers [15]. Recently, it was shown that the polymeric procyanidins fragment readily to single charged fragments instead of forming multiply charged ions in the negative ESI mode [13]. Among
other things, this was based on the fact that in the spectrum of the polymeric procyanidin standard obtained by the time of flight mass spectrometry, intervals between isotopic peaks were 1 Da indicating that the major ions were single instead of multiply charged [13]. They also suggested that the polymers tend to cleave into numerous small fragments rather than a few large ones, and some of the larger fragments will cleave further into smaller ions through a cascade process [13]. Confirming their observation, in our studies, the fragment ions at m/z 287, 289, 575, 577, 865 were frequently detected in the background for the procyanidins higher than tetramer. Using reversed-phase HPLC, the extremely good separation of individual procyanidins from monomers to trimers is achieved (Fig. 1A) noting that the order of elution is not related to the degree of polymerization. Reversed-phase method is not able to separate oligomers higher than trimers probably due to the higher number of isomers interfering with the separation. A broad and unresolved hump eluting between 20 and 35 min. was detected (Fig. 1B and C). Hence normal-phase HPLC is better suited for analysis of procyanidin oligomers higher than trimers. Using normal-phase HPLC, procyanidin monomers through decamers are separated by the degree of polymerization and the separation of higher oligomers is preferable (Fig. 2). On the other hand, by comparing the HPLC traces of fraction A from reversedphase (Fig. 1A) and normal-phase (Fig. 2A) HPLC it is obvious that the separation of individual procyanidins from monomers to trimers achieved with reversed-phase is lost in normal-phase analysis. Presently in normal phase conditions in HPLC–ESI–MS analysis the limiting degree of polymerization for the separation seems to be 10. Nevertheless by enhancing the normal-phase and ESI conditions this limit can be surely overcome. Hayasaka et al. [15] have already investigated the highly polymerized proanthocyanidins (the degree of polymerization up to 28) from grape seeds using direct injection ESI–MS. Generally, fraction A contained shorter procyanidin oligomers (Fig. 2A), mainly from monomers to tetramers, fraction B procyanidin oligomers from dimers to decamers (Fig. 2B) and fraction C procyanidin polymers (Fig. 2C). Chromatography on Sephadex LH-20 was efficient enough to purify procyanidins from other phenolic compounds except for taxifolin, which is structurally so similar to procyanidin monomer (additional oxo group in C3) that it is co-eluting at the same time with the shorter procyanidin oligomers. Using Sephadex LH-20, it is possible to roughly fractionate the procyanidins by their degree of polymerisation whereas exact classification is inaccessible. A small peak due to procyanidin polymers is evident for fraction A in normal-phase HPLC (Fig. 2A) even if polymers are not detectable in fraction A in reversed-phase HPLC (Fig. 1A). In conclusion, the reversed-phase method was developed and the normal-phase method optimized in order to study the procyanidins present in pine bark. Using these rapid HPLC methods, pine bark was found to contain an entire series of
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Fig. 5. The mass spectra of different pine bark procyanidin oligomers recorded by negative ion NP–HPLC–ESI–MS.
procyanidins from monomers through decamers and longer polymers.
Acknowledgements We are grateful to Jaana Liimatainen for her technical assistance. This work was supported by the Academy of Finland, grants nos. 71081 and 201073.
References [1] M.P. Ka¨ hko¨ nen, A.I. Hopia, H.J. Vuorela, J.-P. Rauha, K. Pihlaja, T.S. Kujala, M. Heinonen, J. Agric. Food Chem 47 (1999) 3954. [2] J.-P. Rauha, S. Remes, M. Heinonen, A. Hopia, M. Ka¨ hko¨ nen, T. Kujala, K. Pihlaja, H. Vuorela, P. Vuorela, Int. J. Food Microbiol. 56 (2000) 3. [3] G. Drehsen, in: L. Parker, M. Hiramatsu, T. Yoshikawa (Eds.), Antioxidant Food Supplements in Human Health, Academic Press, San Diego, 1999, p. 311.
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[4] H. Pan, L.N. Lundgren, Phytochemistry 42 (1996) 1185. [5] S. Matthews, I. Mila, A. Scalbert, D.M.X. Donnelly, Phytochemistry 45 (1997) 405. [6] G.E. Adamson, S.A. Lazarus, A.E. Mitchell, R.L. Prior, G. Cao, P.H. Jacobs, B.G. Kremers, J.F. Hammerstone, R.B. Rucker, K.A. Ritter, H.H. Schmitz, J. Agric. Food Chem 47 (1999) 4184. [7] C. Santos-Buelga, A. Scalbert, J. Sci. Food. Agric 80 (2000) 1094. [8] J. Rigaud, M.T. Escribano-Bailon, C. Prieur, J.-M. Souquet, V. Cheynier, J. Chromatogr. A 654 (1993) 255. [9] J.F. Hammerstone, S.A. Lazarus, A.E. Mitchell, R. Bucker, H. Schmitz, J. Agric. Food Chem. 47 (1999) 490.
[10] E. Gariboldi, D. Mascetti, G. Galli, P. Caballion, E. Bosisio, Pharma. Res. 15 (1998) 936. [11] W. Friedrich, A. Eberhardt, R. Galensa, Eur. Food Res. Technol. 211 (2000) 56. [12] L. Gu, M.A. Kelm, J.F. Hammerstone, G. Beecher, J. Holden, D. Haytowitz, R.L. Prior, J. Agric. Food Chem. 51 (2003) 7513. [13] L. Gu, M.A. Kelm, J.F. Hammerstone, Z. Zhang, G. Beecher, J. Holden, D. Haytowitz, R.L. Prior, J. Mass Spectrom. 38 (2003) 1272. [14] S. Guyot, T. Doco, J.-M. Souquet, M. Moutounet, J.-F. Drilleau, Phytochemistry 44 (1997) 351. [15] Y. Hayasaka, E.J. Waters, V. Cheynier, M.J. Herderich, S. Vidal, Rapid Commun. Mass Spectrom. 17 (2003) 9.
Analysis of procyanidins in pine bark with reversed-phase and normal-phase high-performance liquid chromatography–electrospray ionization mass spectrometry Maarit Karonen*, Jyrki Loponen, Vladimir Ossipov, Kalevi Pihlaja Laboratory of Environmental Chemistry, Department of Chemistry, University of Turku, FIN 20014, Turku, Finland Received 16 March 2004; received in revised form 18 June 2004; accepted 18 June 2004 Available online 19 July 2004
Abstract Three procyanidin fractions, one with shorter oligomers, one with longer oligomers and one with polymers, were obtained from Pinus sylvestris L. bark extract. Procyanidins were analyzed using reversed-phase and normal-phase high-performance liquid chromatography methods coupled with electrospray ionization mass spectrometric detection in the negative ion mode. On the grounds of mass spectral data, pine bark was found to contain procyanidins from monomers through decamers and higher polymers. # 2004 Elsevier B.V. All rights reserved. Keywords: Procyanidins; Liquid chromatography; Pine bark
1. Introduction Plant phenolics have been increasingly of interest due to their suggested advantageous health effects (maintenance of health and protection from diseases such as cancer and coronary heart disease) and possibility to use them as natural food additives, since they influence the quality and stability of foods by acting as flavourants, colourants and antioxidants. In a study where the antioxidant activities of phenolic extracts from edible and nonedible Finnish plant materials were examined by autoxidation of methyl linoleate, pine bark was ranked among the most potent plant sources for natural phenolic antioxidants [1]. In another study where antimicrobial activity of extracts prepared from Finnish plant materials against selected microbes was conducted, pine phloem extract was again one of the most active extracts [2]. In addition, pine bark extract has been used as a folk medicine since ancient times [3] and it is still used therapeutically as dietary supplement in Europe. Previously several phenolic compounds such as * Corresponding author. Tel.: +358 2 333 6830; fax: +358 2 333 6700. E-mail address: [email protected] (M. Karonen). 0003-2670/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.06.041
phenolic acid glycosides, stilbene glycosides, lignans and flavonoids have been isolated from inner Scots pine bark [4]. Scots pine bark has also been found to be rich in procyanidins [4,5]. Proanthocyanidins are oligomers and polymers of flavan3-ol monomer units most frequently linked either C4 ! C6 or C4 ! C8 (B-type proanthocyanidins). A-type proanthocyanidins possess the second interflavanoid bond resulting in oxidative coupling between C2 ! O7 positions. The most common classes are procyanidins consisting of catechin, epicatechin and/or their gallic acid esters, and prodelphinidins consisting of gallocatechin, epigallocatechin and/or their galloylated derivatives. Proanthocyanidins in bark are largely procyanidins and, to a more limited extent, prodelphinidins; Scots pine bark contains no prodelphinidins [5]. Proanthocyanidins have attracted increasing attention in the fields of nutrition, health, and medicine mainly due to their potent antioxidant capacity, e.g. it has been observed that procyanidin content in cocoa samples closely correlates to their oxygen radical absorbance capacity (ORAC) values [6]. Proanthocyanidins are known to have various physiological effects like antioxidant, antimicrobial, anti-allergy and anti-hypertensive activities [7].
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Taking into account the interesting properties of pine bark extract that can be due to procyanidins and the fact that distribution and structural characteristic of pine bark procyanidins were unknown, it was necessary to study further pine bark procyanidins. In this study, reversed-phase and normal-phase high-performance liquid chromatographic (HPLC) methods with UV-diode array detection (DAD) and electrospray ionisation–mass spectrometry (ESI–MS) were applied to detect and characterize procyanidins in pine bark.
detector and Analyst Software 1.1 data system. In HPLC– ESI–MS the ionization technique was an ionspray (pneumatically assisted electrospray). The mass spectrometer was operated in negative ion mode: spray needle voltage, 4200 V; heated nitrogen gas temperature, 310 8C (for normal-phase method) and 320 8C (for reversed-phase method); orifice plate voltage, 35 V; ring voltage, 220 V; nebulizer gas (purified air) was set at 10; curtain gas (N2) at 12. Masses were scanned from 100 to 2800 amu in steps of 0.3 amu. Split ratio was 7:3 prior to introduction into the ionization chamber.
2. Experimental
2.4. Reversed-phase HPLC
2.1. Materials
Reversed-phase separations were performed using a Merck Superspher 100 RP-18 column (75 mm 4 mm i.d., 4 mm). The binary mobile phase consisted of (A) water and formic acid (99:1, v/v) and (B) acetonitrile. A linear gradient elution was performed: 0 min: 100% A; 2 min: 100% A; 19 min: 91% A, 9% B; 35 min: 70% A, 30% B; 43 min: 30% A, 70% B. The flow rate was 1 ml/min, detection wavelength 280 nm and injection volume 20 ml.
Scots pine (Pinus sylvestris L.) phloem and bark material were collected, air dried and ground into small pieces by Ravintorengas Oy. Acetone (analytical grade) was purchased from Riedel-de Hae¨ n (Seelze, Germany), formic acid 98–100% from Fischer Scientific (Loughborough, UK), acetonitrile (HPLC-grade) from Lab-Scan (Dublin, Ireland), ethanol (99.5%, v/v) from Primalco (Rajama¨ ki, Finland), methanol (HPLC-grade) and glacial acetic acid from J.T. Baker (Denventer, Holland), and dichloromethane from Mallinckrodt (Denventer, Holland). Water was obtained using the Elgastat UHQ-PS purification system (Elga, Kaarst, Germany). (+)-Catechin and ()-epicatechin were from Sigma (St. Louis, MO, USA). 2.2. Sample preparations Pine bark powder (100 g) was extracted with 70% aqueous acetone (4 ml 1000 ml 12 h). Acetone was evaporated under reduced pressure, and the solid residue was lyophilized. The lyophilized crude extract (21.3 g) was dissolved in water to a concentration of 100 mg/ml, and 50 ml of extract was fractionated on Sephadex LH-20 using a modification of the method used earlier [4] by elution with H2O (fraction I), and stepwise gradient of aqueous ethanol (10–95%, fractions II–X) and aqueous acetone (30–70%, fractions XI–XIII). The procyanidin compositions of obtained fractions were screened with HPLC and fractions containing procyanidins were combined as follows: fr A (IV–VI; 370.0 mg), fr B (VII–XI; 379.4 mg) and fr C (XII, XIII; 231.9 mg), dissolved in water to a final concentration of 20 mg/ml, and filtered through 0.45 mm PTFE filters. 2.3. Instrumentation HPLC analyses were performed on two systems: MerckHitachi HPLC-DAD system consisted of L-7100 pump, L-7200 autosampler, L-7455 diode array detector, and D-7000 interface, and Perkin-Elmer HPLC–ESI–MS system consisted of SCIEX API 365 LC/MS/MS mass spectrometer connected to a Series 200 HPLC system with UV–vis
2.5. Normal-phase HPLC Normal-phase separations were performed using a Merck LiChrospher Si 60 column (250 mm 4 mm i.d., 5 mm). The method developed earlier [6,8] was used with minor modifications. The binary mobile phase consisted of (A) dichloromethane, methanol, water and acetic acid (82:14:2:2, v/v) and (B) methanol, water and acetic acid (96:2:2, v/v). A linear gradient elution was performed: 0 min: 100% A; 30 min: 82% A, 18% B; 45 min: 69% A, 31% B; 50 min: 12% A, 88% B; 60 min: 12% A, 88% B. The flow rate was 1 ml/min, detection wavelength 280 nm and injection volume 5 ml.
3. Results and discussion Procyanidins were isolated from pine bark crude extract by column chromatography with Sephadex LH-20 into three fractions (A–C) which were studied using reversed-phase and normal-phase HPLC with DAD and ESI–MS. Procyanidins can be detected UV spectrophotometrically without difficulty but the identification of individual procyanidins is impossible because of the similar shapes and maxima of UV spectra. Therefore, in HPLC–MS analyses, UV and mass data were collected within the same run allowing the comparison of the UV chromatogram with the total ion chromatogram (TIC). Mass data was obtained using electrospray ionization technique which is easy to use and soft usually giving just molecular ion peaks or molecular ion peaks with few fragments. In addition, electrospray is also capable to produce multiply charged ions, and the sensitivity is high. Procyanidins were detected with the negative ion mode due
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Fig. 1. Reversed-phase HPLC-UV trace (280 nm) of procyanidin fractions from pine bark: (A) fraction A, mainly from monomers to tetramers; (B) fraction B, mainly from dimers to decamers; (C) fraction C, mainly polymers. The labels 1–3 on the peaks indicate the degree of polymerization of procyanidins in the peaks and the label ‘‘tax’’ to taxifolin.
to their weakly acidic nature. Earlier, it was shown that positive mode is successful in detecting procyanidin oligomers through pentamers, but it seemed to be evident from the decreased ionization efficiency of higher oligomers that pentamers would be the limit of detection [9]. Reversed-phase and normal-phase HPLC traces of obtained fractions at 280 nm are presented in Figs. 1 and 2, respectively. In the chromatograms, the degree of polymerization of each peak due to pine bark procyanidins was
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Fig. 2. Normal-phase HPLC-UV trace (280 nm) of procyanidin fractions from pine bark: (A) fraction A, mainly from monomers to tetramers; (B) fraction B, mainly from dimers to decamers; (C) fraction C, mainly polymers. The labels 1–10 on the peaks indicate the degree of polymerization of procyanidins in the peaks and the label ‘‘tax’’ to taxifolin.
determined based on mass spectral data consisting of characteristic deprotonated molecules and fragment ions. Degrees of polymerization (DP), deprotonated molecules [M H], [2M H], [M 2H]2, [M 3H]3 and fragment ions of pine bark procyanidins are listed in Table 1. A comparison between the extracted ion chromatograms of the doubly charged oligomeric procyanidins from pentamer up to decamer from fraction B and the HPLC-UV trace of fraction B is shown in Fig. 3
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Table 1 Degrees of polymerization (DP), and [M H], [2M H], [M 2H]2, [M 3H]3 and fragment ions of pine bark procyanidins DP
[M H]
[2M H]
1 2 3 4 5 6 7 8 9 10
289.0 577.3 865.3 1153.6 1441.6 1729.9 2017.0
579.1 1155.7 1731.4
[M 2H]2
[M 3H]3
Fragment ions (m/z) 245.2 286.9, 289.0, 407.2, 425.2, 451.0 286.6, 289.0, 575.5, 577.0, 713.2, 739.3 286.9, 289.0, 575.2, 577.0, 864.7
720.7 864.7 1009.0 1152.4 1296.7 1440.7
To get the general view of pine bark procyanidins, a calculated mass spectrum of all procyanidins in fraction B accumulated from ca. 640 scans recorded by negative ion NP–HPLC–ESI–MS is presented in Fig. 4. A series of [M H] ions separated by 288 Da can be observed from m/z 289.0 to 2017.0 corresponding to B-type procyanidins from monomer to heptamer. Confirming the foreknowledge of pine bark proanthocyanidins, prodelphinidins were not detected. Two procyanidin monomers were detected (Fig. 1A) and verified using standard compounds to be (+)-catechin and ()-epicatechin, respectively. The mass spectrum of the monomer (Fig. 5A) in fraction A recorded by negative ion NP–HPLC–ESI–MS exhibited [M H] ion at m/z 289.0, [2M H] ion at m/z 579.1 and also a fragment ion at m/z 245.2 consistent with the spectrum reported in [10]. The mass spectrum of the dimer (Fig. 5B) in fraction A recorded by negative ion NP–HPLC–ESI–MS exhibited [M H] ion at m/z 577.3 and [2M H] at m/z 1155.7. Fragmentation of procyanidin dimer consisting of the extension unit and the terminal unit can happen through retroDiels-Alder (RDA) or heterocyclic ring fission (HRF) mechanisms or through quinone methide (QM) cleavage of the interflavonoid bond [11,12]. The fragmentation of the heterocyclic ring can take place on the extension unit or on the terminal unit. The fragment ion of the dimer at m/z 451.0 indicates HRF, which is also known as the elimination of the phloroglucinol molecule. The RDA fragmentation of the dimer produced the ion at m/z 425.2. RDA has been found to be the most important fragmentation for structure elucidation of dimers [11]. Fragmentation on the extension unit has been considered to be energetically more favorable since it produces fragment ions with larger p–p hyperconjugated system than RDA on the terminal unit [13]. The sequential water elimination from m/z 425.2 produced the ion at m/z 407.2. In some cases [11], m/z 407.2 has been detected in significant amounts, even greater than m/z 425.2 but in this study m/z 425.2 was more abundant. The QM cleavage of the interflavonoid bond produced fragment ions at m/z 286.9 and 289.0. Four isomeric dimers were detected with reversedphase HPLC, two of them eluting at the same peak at the retention time of 15 min. (Fig. 1A). All dimers had the same
864.7 960.1
molecular mass and similar fragments thus making the identification of individual dimers unfeasible. The identification or structural characterization, e.g. the position of the interflavanoid linkage, of any oligomeric procyanidins cannot be conducted with ESI–MS, therefore, further analysis is needed in order to identify individual procyanidins. However, a variety of difficulties in isolating and characterizing pure higher oligomers exist. The mass spectrum of the procyanidin trimer (Fig. 5C) in fraction A recorded by negative ion NP–HPLC–ESI–MS exhibited [M H] ion at m/z 865.3 and [2M H] ion at m/z 1731.4. The fragmentation of the trimer seems to be similar to that of dimers. The HRF fragmentation produced an ion at m/z 739.3. The fragment ion at m/z 713.2 indicates the RDA. Contrary to dimers the product of HRF was more abundant than that of RDA. The QM cleavage of the interflavonoid bond produced principally the ions at m/z 286.6 and 577.0 indicating that the cleavage takes place in the upper interflavanoid bond; regardless of that the ions at m/z 289.0 and 575.5 were observed implying that the cleavage can also happen in lower interflavanoid bond. A weak signal at m/z 425.2 was detected suggesting that further fragmentation of m/z 577.0 occurs. The main ion for tetrameric procyanidins (Fig. 5D) was [M H] at m/z 1153.6 whilst neither [2M H] ion nor ions indicating HRF or RDA were detectable. The fragment ions from QM cleavage were observed at m/z 286.9, 289.0, 575.2, 577.0 and 864.7. Mass spectral data (Table 1, Fig. 5) indicate that welldefined molecular ions [M H] and fragment ions were obtained for the shorter procyanidins. According to the literature with increasing degree of polymerization, higher procyanidins are prone to form multiple charges [9,14]. The charge state of the multiply charged species can be defined by the m/z difference between isotopic peaks: for a [M H] ion the distance between the isotopic peaks is one mass unit, for [M 2H]2 ion 0.5 and for [M 3H]3 ion 0.3 [14,15]. For the procyanidin pentamer (Fig. 5E) the most abundant ion is [M H]– at m/z 1441.6 but in addition to the fragment ions the doubly charged ion [M 2H]2 at m/z 720.7 can be seen. The enlarged signal of [M H] ion (Fig. 5E) shows the m/z difference between the isotopic peaks. The distance between the isotopic peaks of [M H] ion is
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Fig. 3. (A) Normal-phase HPLC-UV trace (280 nm) of procyanidin fraction B in comparison to extracted ion chromatograms of procyanidin pentamer (B), hexamer (C), heptamer (D), octamer (E), nonamer (F), and decamer (G).
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Fig. 4. The calculated mass spectrum of pine bark procyanidins accumulated from ca. 640 scans recorded by negative ion NP–HPLC–ESI–MS.
varying from 0.9 to 1.2 because in the MS conditions used the masses were scanned in steps of 0.3 amu. In the case of hexamer (Fig. 5F), the doubly charged [M 2H]2 ion at m/ z 864.7 is the most abundant but the [M H] ion at m/z 1729.9 is also visible. The enlarged signal of [M 2H]2 ion (Fig. 5F) shows that the distance between the isotopic peaks is 0.6. For the heptamer, [M 2H]2 ion at m/z 1009.0 is dominant and [M H] ion at m/z 2017.0 is weak. The molecular ion of octamer was no longer detectable. The doubly charged ions were observed for octamer, nonamer and decamer at m/z 1152.4, 1296.7 and 1440.7, respectively. In addition, triply charged [M 3H]3 ions were observed for nonamer and decamer at m/z 864.7 and 960.1. On average, the ionization efficiency decreases with the increasing degree of polymerization. In UV trace (Fig. 2), a strong peak of higher polymers exist but in the corresponding place in TIC a negative peak is observed. For the moment, the amount of polymers and their isomers can be so high that it causes a decline in the ionization efficiency and inhibits the removal of counter ions. No mass spectral data was obtained for procyanidin polymers. They were identified on the grounds of their retention times and UV spectra characteristic for procyanidins. Since procyanidins are eluted according to the increasing degree of polymerization in normalphase HPLC (Fig. 2B), the degree of polymerization for higher polymers must be >10. In a few earlier studies conducted concerning proanthocyanidins in pine, the mean degrees of polymerization (mDP) by thiolytic degradation for P. sylvestris bark procyanidins and for the pycnogenol, a food supplement extract from maritime pine tree bark, have been found to be 5.6 [5] and 7.4 [12], respectively. Earlier, it has been stated that the electrospray is superior to other ionization techniques because of its capability to produce multiply charged ions corresponding to molecular masses of higher proanthocyanidin polymers [15]. Recently, it was shown that the polymeric procyanidins fragment readily to single charged fragments instead of forming multiply charged ions in the negative ESI mode [13]. Among
other things, this was based on the fact that in the spectrum of the polymeric procyanidin standard obtained by the time of flight mass spectrometry, intervals between isotopic peaks were 1 Da indicating that the major ions were single instead of multiply charged [13]. They also suggested that the polymers tend to cleave into numerous small fragments rather than a few large ones, and some of the larger fragments will cleave further into smaller ions through a cascade process [13]. Confirming their observation, in our studies, the fragment ions at m/z 287, 289, 575, 577, 865 were frequently detected in the background for the procyanidins higher than tetramer. Using reversed-phase HPLC, the extremely good separation of individual procyanidins from monomers to trimers is achieved (Fig. 1A) noting that the order of elution is not related to the degree of polymerization. Reversed-phase method is not able to separate oligomers higher than trimers probably due to the higher number of isomers interfering with the separation. A broad and unresolved hump eluting between 20 and 35 min. was detected (Fig. 1B and C). Hence normal-phase HPLC is better suited for analysis of procyanidin oligomers higher than trimers. Using normal-phase HPLC, procyanidin monomers through decamers are separated by the degree of polymerization and the separation of higher oligomers is preferable (Fig. 2). On the other hand, by comparing the HPLC traces of fraction A from reversedphase (Fig. 1A) and normal-phase (Fig. 2A) HPLC it is obvious that the separation of individual procyanidins from monomers to trimers achieved with reversed-phase is lost in normal-phase analysis. Presently in normal phase conditions in HPLC–ESI–MS analysis the limiting degree of polymerization for the separation seems to be 10. Nevertheless by enhancing the normal-phase and ESI conditions this limit can be surely overcome. Hayasaka et al. [15] have already investigated the highly polymerized proanthocyanidins (the degree of polymerization up to 28) from grape seeds using direct injection ESI–MS. Generally, fraction A contained shorter procyanidin oligomers (Fig. 2A), mainly from monomers to tetramers, fraction B procyanidin oligomers from dimers to decamers (Fig. 2B) and fraction C procyanidin polymers (Fig. 2C). Chromatography on Sephadex LH-20 was efficient enough to purify procyanidins from other phenolic compounds except for taxifolin, which is structurally so similar to procyanidin monomer (additional oxo group in C3) that it is co-eluting at the same time with the shorter procyanidin oligomers. Using Sephadex LH-20, it is possible to roughly fractionate the procyanidins by their degree of polymerisation whereas exact classification is inaccessible. A small peak due to procyanidin polymers is evident for fraction A in normal-phase HPLC (Fig. 2A) even if polymers are not detectable in fraction A in reversed-phase HPLC (Fig. 1A). In conclusion, the reversed-phase method was developed and the normal-phase method optimized in order to study the procyanidins present in pine bark. Using these rapid HPLC methods, pine bark was found to contain an entire series of
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Fig. 5. The mass spectra of different pine bark procyanidin oligomers recorded by negative ion NP–HPLC–ESI–MS.
procyanidins from monomers through decamers and longer polymers.
Acknowledgements We are grateful to Jaana Liimatainen for her technical assistance. This work was supported by the Academy of Finland, grants nos. 71081 and 201073.
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