Potential Health Benefits of Citrus - American Chemical Society

Chapter 5

Downloaded by FUDAN UNIV on December 29, 2016 | http://pubs.acs.org Publication Date: June 9, 2006 | doi: 10.1021/bk-2006-0936.ch005

Powerful Analytical Tools for Citrus Characterization: LC/MS and LC/NMR Characterization of Polymethoxylated Flavones Claus O. Schmidt*, Gerhard E. Krammer, Berthold Weber, Detlef Stõckigt, Stefan Brennecke, Günter Kindel, and Heinz-Jürgen Bertram Symrise GmbH and Company KG, Mühlenfeldstrasse 1, D-37603 Holzminden, Germany *Corresponding author: telephone: +49-5531-90-2629; fax: +49-5531-90-49629; email: [email protected]

Citrus fruits are well known for their unique flavor and their health benefits. Volatile compounds such as terpenes are formed as secondary metabolites in the plant. These volatile compounds mainly contribute to the flavor and scent of the plant. However, other non-volatile metabolites are formed via biosynthesis in citrus. These non-volatile compounds contribute to certain health benefits like anticarcinogenic, antitumor, antiviral or antimicrobial activities. Our goal is to establish efficient analytical technologies to identify and characterize the volatile and non-volatile compounds from citrus plants. For a fast and reliable screening of volatile compounds in citrus flavors, solid phase micro-extraction (SPME) and solid phase dynamic extraction (SPDE) are well known techniques for flavor analysis. Residues obtained from molecular distillation of cold pressed peel oils of oranges and clementines were analyzed using LC/MS and LC/NMR to characterize non-volatile compounds.

© 2006 American Chemical Society

Patil et al.; Potential Health Benefits of Citrus ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

71


Introduction Oils of citrus fruits obtained by molecular distillation of cold pressed oils are of high importance in the flavoring of food. After pressing the citrus fruits and addition of water, the oil layer separates out from the juice phase. The oil is filtered, centrifuged and then chilled, the latter treatment resulting in precipitation of waxes. This cold pressed oil is subjected to molecular distillation thereby obtaining an almost colorless oil with a sharp sensory profile. Recently the non-volatiles of lemon peel oils were analyzed using LC/NMR (1). During our work on citrus we extended these studies to the residue obtained from Clementine peel oils. These residues were expected to be enriched in polymethoxyflavones which could be confirmed by LC/MS experiments. Further LC/NMR measurements were necessary to actually confirm the structures of the different individual components. The chromatograms obtained from LC/MS and LC/NMR experiments (detection at 210 nm) are very similar since the same stationary phase was used. In Figure 1 the chromatogram and the total ion current (TIC) from the LC/MS experiment obtained by atmospheric chemical ionization in the positive mode are shown. The retention time interval between 17 and 30 min is occupied by different polymethoxyflavones. Compounds 1 to 6 are indicated as tetra-, penta-, hexa- and heptamethoxy substituted flavones as based on the detected masses of the molecular species, with m/z = 343, 373, 403 and 433, respectively. Compound 7 is a monohydroxylated hexamethoxyflavones with MW = 418. The two-dimensional plot from the LC/NMR experiment in the on-flow mode (Figure 2) clearly shows the characteristic signal pattern for polymethoxylated flavones due to the aromatic protons between 7 and 9 ppm and also for the methoxy groups around 3.7 to 4.1 ppm. This experiment was repeated in the loop collection mode for a better signal to noise ratio and therefore better identification of the constituents. The substitution pattern of the phenyl residues of the different flavones can easily be deduced. The parasubstituted phenyl moieties of compounds 4 and 6 are deduced from two doublets at about 7.92 and 7.06 ppm and a coupling constant of about 9.0 Hz. For both compounds the typical singlet signal of the proton attached to C-3 is observed at about 6.62 ppm. Bearing in mind the remaining four methoxy groups, compound 6 can be unambiguously identified as tangeretin. The identity of compound 4 with three methoxy groups at positions C-5, C-6 and C-7 is deduced as a result of a detailed comparison with recently isolated tetramethylscutellarein (2).



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70-

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Time (min)

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Figure 1. Chromatogram (detection at 210 nm) and TIC originatingfrom LC/MS experiment.

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Table I. Structures of detected polymethoxylated flavones

1

MW

Name

R.

R2

R3

R4

Rs

R

5,6,7,3\4'-

H

OCH3

OCH3

OCH3

H

OCH3

OCH3

372

OCH3

OCH3

OCH3

H

OCH3

OCH3

402

H

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

402

H

OCH3

OCH3

OCH3

H

H

OCH3

342

OCH3

OCH

3

OCH3

OCH

3

OCH3

OCH3

OCH3

432

H

OCH3

OCH3

OCH

3

OCH3

H

OCH3

372

OCH3

OH

OCH3

OCH3

OCH3

OCH3

OCH3

418

6

R

7

Pentamethoxyflavone (sinensetin)

2

3,5,6,7,3',4'-

OCH

3

Hexamethoxyflavone

3

5,6,7,8,3',4'Hexamethoxyflavone (nobiletin)

4

5,6,7,4'Tetramethoxyflavone (Tetramethylscutellarein)

5

t

3,5,6,7,8,3',4Heptamethoxyflavone

6

5,6,7,8,4' Pentamethoxyflavone (tangeretin)

7

5-Hydroxy3,6,7,8,3',4'Hexamethoxyflavone



75

8.0

7.0

6.0

F2/BRm

4.0

Figure 2. 2D plot of the LC/NMR on-flow experiment.



76 The phenyl residues of flavones 1, 2, 3 and 5 are in fact 1,3,4-substituted isomers as concluded from the double doublet at about 7.6 (1,3) or 7.7 ppm (2,5), another doublet around 7.5 (1,3) or 7.6 ppm (2,5) and a further doublet at 7.1 ppm with coupling constants of 8.6 and 2.1 Hz. The lack of substitution at C3 for 1 and 3 is evident as a result of the presence of the signal at 6.6 ppm for the proton. Compound 3 is therefore unambiguously identified as nobiletin (Figure 3) due to the remaining four methoxy groups. Comparison of the chemical shifts of 1 with those of tetramethylscutellarein 4 and especially with respect to the protons at the chromen-4-one system, reveals almost identical shifts leading to the assignment of sinensitin for 1. For compound 5, all protons of the chromen4-one system are substituted by methoxy groups, thus confirming the structure as the trisubstituted phenyl-containing 3,5,6,7,8,3',4'heptamethoxyflavone. The LC/MS experiment indicates compound 2 as hexamethoxyflavone. The absence of the signal in the 'H-NMR spectrum at about 6.6 ppm for the proton at C-3 due to substitution by a methoxy group, raises the question about the substitution pattern on the aromatic ring of the chromen-4-one moiety by the residual methoxy groups. Both 3,5,6,7,3',4'- and 3,5,7,8,3\4'-hexamethoxyflavone have recently been reported from orange peel (3). The experimental NMR data of the former fit better with the published data (4, 5). N M R data in CDC1 of compound 2 isolated by preparative HPLC are identical to those from literature for 3,5,6,7,3',4 -hexamethoxyflavone (5). Compound 7 is indicated as hydroxy-hexamethoxyflavone, which is confirmed by the LC/NMR experiments. The 1,2,4-trisubstituted pattern is reflected by the three signals of the aromatic protons as described above. Since the chemical shifts of these protons are almost identical to those of compounds 2 and 5, three of the six methoxy groups should be attached to C-3, C-3' and C-4\ The exact position of the hydroxyl group could not be determined by the LC/NMR measurements and preparative isolation of the component was performed by HPLC. By comparison of the experimental and published *HNMR data (6), compound 7 is identified as being 5-hydroxy-3,6,7,8,3',4'hexamethoxyflavone. By comparing the intensities of the UV absorptions and the mass spectrometric total ion current resulting from the less polar constituents, UV absorption appears to be a more favorable detection method for those compounds. Using LC/NMR, compound 8 (44.5 min) can be readily identified as a sinensal isomer and 9 (48.8 min) as limonene. 3

,

Experimental Plant material Residues from the molecular distillation of cold pressed peel oil of Clementines (Citrus reticulata Blanco var. Clementine) were analyzed.


Patil et al.; Potential Health Benefits of Citrus ACS Symposium Series; American Chemical Society: Washington, DC, 2006. -T

1

1

r—|

r-

-i

1

1

1

r-

Figure 3. H-LC/NMR-spectrum of nobilitin 3 with solvent suppression around 4.5 (HDO) and 2 ppm (acetonitrile).

1

o

nobilitin (3)

OMe

_jJULi

OMe

OMe


78 HPLC/MS analyses LC/MS experiments were performed on a system consisting of a ThermoQuest LCQ mass spectrometer with an APCI interface and a Hewlett Packard HP 1100 HPLC system. APCI experiments were carried out in the positive mode. Nitrogen was used as sheath gas. Gradients with H 0 (containing 0.01% formic acid) and acetonitrile were run with a flow rate of 0.2 ml/min and a Varian C18 column (125 x 2 mm, particle size 3 |im) was used for chromatographic separations. 2


HPLC/NMR analyses For LC/NMR measurements D 0 (99,9%) was purchased from Deutero GmbH, Kastellaun, Germany, and acetonitrile (HPLC/NMR grade) was bought from Riedel de Haen, Seelze, Germany. LC/NMR experiments were performed on the INOVA system using a ^ { ^ C / ^ N J P F G triple resonance indirect detection microflow LC/NMR probe (IFC probe) with a detection volume of 60 nL at 20 °C. The HPLC system consisted of a ternary Varian ProStar 230 pump, a Varian ProStar 330 Photodiode Array Detector (210 nm) and a Varian ProStar 510 column oven (50 °C). Chromatographic separation was carried out on an OmniSpher CI8 column (250 x 4.6 mm, particle size 5 jum) running gradients with acetonitrile and D 0 (containing 0.01% TFA v/v). Solvent suppression was achieved by performing a WET pulse sequence (7). During gradient elution, shapes of selective pulses were automatically calculated on theflybased on a scout scan recorded before each increment. Chemical shifts were referenced to acetonitrile (1.93 ppm). NMR spectra of isolated compounds were also recorded on a Varian INOVA (400 MHz) spectrometer. 2

Unity

2

Un,ty

Conclusions In the past, polymethoxylated flavones were normally identified by comparing MS and UV spectra from LC/MS experiments with isolated reference materials, which is quite time-consuming, or with the aid of published data, which implies a certain tentativeness. HPLC/NMR represents a powerful tool for the identification of constituents without any prior isolation being necessary. The method is somewhat limited when dealing with molecules with only few protons. However, with increasing reported NMR data in the given solvent system D 0/acetonitrile, identification will become more easy and reliable. In addition, components which possess a strong UV absorption but low mass spectrometric ionization efficiency or vice versa can be readily characterized by LC/NMR. 2


79

Acknowledgement The author are indebted to their colleagues Dr. Ingo Reiss suppling the Clementine residues obtained from molecular distillation of the essential oils and Michael Roloff for the preparative HPLC work.


References 1. Sommer, H.; Bertram, H.-J., Krammer, G.; Kindel, G.; Kühnle, T.; Reinders, G.; Reiss, I.; Schmidt, C . O . ; Schreiber, K.; Stumpe, W.; Werkhoff, P.; Perfumer&Flavorist 2003, 28, 18-34. 2. Weber, B.; Hartmann, B. Unpublished results. 3. Malterud, K.E.; Rydland, K.M. J. Agric. Food Chem. 2000, 48, 5576-5580. 4. Joseph-Nathan, P.; Abramo-Bruno, D; Torres, M . A. Phytochemistry 1981, 20, 313-318. 5. Machida, K.; Osawa, K. Chem. Pharm. Bull. 1989, 37(4), 1092-1094. 6. Roitman, J. N.; James, L. F. Phytochemistry 1985, 24(4), 835-848. 7. Smallcombe, S.H.; Patt, S.L.; Keifer, P.A. J. Magn. Reson. Series A 1995, 117, 295-303.


Potential Health Benefits of Citrus - American Chemical Society

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