Nutraceutical Beverages - American Chemical Society

Chapter 29

Tetrahydro-β-carboline Bioactive Alkaloids in Beverages and Foods

Downloaded by COLUMBIA UNIV on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch029

Tomas Herraiz Spanish Council for Scientific Research, Instituto de Fermentaciones Industrials, Juan de la Cierva, 3, 28006, Madrid, Spain

Tetrahydro-β-carboline (THβCs) bioactive alkaloids occur in nutritional beverages and foods. Four of these compounds were identified by GC-MS and HPLC-MS in fruit juices and beverages: 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 1methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 1methyl-1,2,3,4-tetrahydro-β-carboline and 6-hydroxy-1methyl-1,2,3,4-tetrahydro-β-carboline. They were present in concentrations of up to several mg per liter in fruit juices and fermented alcoholic beverages. The formation of these alkaloids in beverages is via a nonenzymatic Pictet-Spengler condensation from indoleamines and aldehydes depending on the pH and temperature. Sulfur dioxide (SO ) binds carbonyls, thus preventing the formation of THβCs alkaloids. The relatively abundant THβC-3-carboxylic acid are oxidized under heating, storage and oxidants such as nitrite, hydrogen peroxide and Fenton reagent to give the fully aromatic βcarbolines (βCs), norharman and harman. Beverages and foods containing THβCs and βCs are a significant source of these active compounds occurring in biological tissues and fluids. 2

© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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406 Tetrahydro-P-carbolines (THpCs) and β-carbolines (PCs) are naturally occurring indole alkaloids with a common tricyclic pyrido (3,4-b)indole ring structure. THpCs and PCs exhibit a broad range of pharmacological and biological activity (1-3). Biological interest in THpCs and pCs has grown from reports suggesting their occurrence under physiological conditions in biological tissues and fluids (2,4-8). These alkaloids have attracted the attention of neurochemists who have speculated on their putative role in the central nervous system where they might function as mild neuromodulators. They inhibit monoamine oxidase, monoamine uptake, and bind to benzodiazepine-GABA receptor (2,3,7-10). Simultaneously, THpCs and pCs have been studied in relation with alcoholism where they might play a role in the etiology or addiction, or in pathological states (7,8,11-14). P-carbolines are also interesting from a toxicological point of view because they could act as co-mutagens or precursors of mutagens (75-/7), or be bioactivated to give endogenous neurotoxins (18-20). Taken together, a full delineation of the biological activity and possible toxicity of THPCs and pCs is still desirable and needed. In this regard, we have recently found that these compounds exhibit noticeable antioxidant activity (21). Since tetrahydro-P-carboline and P-carboline alkaloids may exhibit biological actions, their availability during food consumption is of interest. These compounds may be formed under mild conditions in foods by a nonenzymatic Pictet-Spengler reaction (22). As a result they occur in commercial beverages and foods (23-32). A scheme illustrating the formation of THpC and PC heterocycles in foods and biological systems from indoleamine precursors is shown in Figure 1. Therefore, the diet might surely contribute to the ultimate presence of these alkaloids in the human biological tissues and fluids. The present paper focuses on the occurrence of tetrahydro-P-carbolines in commercial beverages such as fruit juices and fermented alcoholic beverages. We have identified several THpCs by GC-MS and HPLC-MS in many of those products and subsequently studied their occurrence by SPE-HPLC-fluorescence. In addition, the factors affecting the levels of THPCs produced by a PictetSpengler reaction during food production, processing and storage were studied, along with the possible formation of the fully aromatic p-carbolines (PCs) from THpCs as shown in Figure 1.

Experimental Reference Compounds and Samples l-Methyl-l,2,3,4-tetrahydro-P-carboline-3-carboxylic acid (MTCA) was prepared from L-tryptophan and acetaldehyde through Pictet-Spengler

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


407

Figure L Tetrahydrorf-carbolines (ΓΗβϋ) producedfromindoleamine precursors (tryptophan, serotonin and tryptamine) and their oxidation to give the fully aromatic β-carbolines (βΟ).



408

condensation (33), and also purchased from Sigma (Saint Louis MO). In the same manner, 1-ethyl-l,2,3,4-tetrahydro-p-carboline-3-carboxylic acid (ETCA) (used as internal standard), and l,2,3,4-tetrahydro-P-carboline-3-carboxylic acid (THCA) were prepared from L-tryptophan and propionaldehyde or formaldehyde, respectively. The compounds l,2,3,4-Tetrahydro-P-carboline-l,3dicarboxylic acid (THCA-COOH), and 1-methyl-1,2,3,4-tetrahydro-P-carboline1,3-dicarboxylic acid (MTCA-COOH) were synthesized from L-tryptophan, and glyoxylic acid or pyruvic acid, respectively. 6-Hydroxy-1 -methyl-1,2,3,4tetrahydro-P-carboline and 1 -methyl-1,2,3,4-tetrahydro-p-carboline were obtained from the corresponding indoleamines and acetaldehyde through PictetSpengler reaction (33). Confirmation of the structures of the synthesized compounds was by NMR, MS, GC-MS, and RP-HPLC-MS (33, 34). The derivatives of Af-methoxycarbonyl-tetrahydro-P-carboline-S-carboxylic acid methyl ester were obtained with methyl chloroformate in the presence of pyridine and methanol, or methyl chloroformate and diazomethane (24). Samples containing the standards or food-isolated tetrahydro-P-carboline-3carbobylic acid (THPC-3-COOH) were derivatized to obtain the Nmethoxycarbonyl esters and subsequently analyzed by GC-MS. Commercial samples of nutritional beverages, fermented alcoholic beverages and foods, both from local and imported origin, were purchased from local supermarkets and subsequently used for the isolation and analysis of carboline alkaloids.

Analysis of THpC-3-COOHs from Nutritional Beverages and Foods THPC-3-COOHS were isolated by using SCX-solid phase extraction (25, 27, 31). Liquid samples such as fruit juices (20 mL) were added with 1 mg/mL semicarbazide (Sigma), and centrifuged (5100g, 0-5°C) for 10-15 min. Solid samples were homogenized in 0.6 M HC10 using an ultraturrax and subsequently centrifuged. An aliquot of supernatant (5.5 mL) was spiked with 0.5 mL of ETCA solution (5 mg/L) used as internal standard (IS), acidified if needed with drops of 1M HC1, and slowly passed through benzenesulfonic acid SCX columns (Bond Elut, 500mg/3mL size, Varian, Harbor City, CA) using a vacuum manifold. Washing of SCX-columns was carried out with 0.1M HC1, methanol, phosphate buffer and THpC-3-COOHs eluted with phosphate buffermethanol [25]. The analysis of THpC-3-COOHs was carried out using RP-HPLC and fluorescence detection (23). A 150 mm χ 3.9 mm, 5 μπι, Nova-pak C18 column (Waters, Milford, MA) was used for separation. Chromatographic conditions were as follows: 50 mM ammonium phosphate buffer (pH 3) (buffer A) and 20% of A in acetonitrile (buffer B). Gradient programmed from 0 (100% A) to 32% Β 4


409


in 8 min and then 90% Β at 18 min. The flow rate was 1 mL/min, the column temperature was 40°C and the injection volume 20 μΐ.. Fluorescent detection was set at 270 nm for excitation and 343 nm for emission. Calibration curves from standard solutions of THpC-3-COOH were used for quantitation. Confirmation of the identity of THpC-3-COOHs was established by HPLC-MS and GC-MS. In addition, fluorescence spectra of HPLC peaks were compared with those of reference compounds. For this, eluting peaks corresponding to THpC-3-COOHs were trapped into the flow cell of the fluorescence detector by stopping the solvent pump, and excitation and emission spectra monitored (55).

GC-MS and HPLC-MS Analysis THpC-3-COOHs were isolated from beverages and foods by solid phase extraction with SCX (benzenesulfonic silica) and C i cartridges, and injected into GC-MS following derivatization (24). GC-MS analysis was performed onto a 20 m χ 0.25mm methyl silicone capillary column by using a HP G1800A GCD system (GC-MS) working under electron ionization (EI). Oven temperature, 160°C (2 min), 4°C/min to 245°C (10 min); helium flow rate, 0.6 mL/min; injector temperature, 260°C; transfer line, 280°C, and ionization mode, 70 ev scanningfromm/z 10 to 425. Chemical identification of THPCs isolated by SCX was also accomplished by HPLC-MS on a 3.9 χ 150 mm, 5 μηι, Nova-pak CI8 column, by using an HPLC-MSD series 1100 (Hewlett Packard) (electrospray-positive ion mode). Eluents: A: formic acid (0.5%); B: 0.5% formic acid in acetonitrile; 0-30% Β in 30 min. Flow 0.5 ml/min. Cone voltage 50 V. Mass range 50-700 amu. 8

Formation of THpCs in Model Solutions and Beverages. The formation of THpCs by a nonenzymatic Pictet-Spengler condensation was investigated from model reactions containing indoleamines (tryptophan 104 mg/L or tryptamine 80 mg/L) and aldehydes (formaldehyde, 50 mg/L or acetaldehyde, 48 mg/L) at different pH values (pH 1 of HC1 and NaCl, and 50 mM phosphate buffer pH values: 3, 5, 7 and 9), temperatures (25, 37, 60 and 80 °C) and levels of sulfur dioxide (0, 87, 208, 417 and 833 mg/L added as Na S20 ). Additionally, beverages were spiked with exogenous formaldehyde or acetaldehyde and further determining the increase of THpC-3-COOHs (23, 25). For that, test tubes containing 10 mL of orange juice were separately added with acetaldehyde at zero (control), 20, 50 and 100 mg/L, or formaldehyde at zero (control), 20, 50 and 100 mg/L and kept for 48 h at 30 °C. 2

5


410 Oxidation of Tetrahydro-P-carbolines to Afford β-CarboIines (pCs) Standard solutions of tetrahydro-P-carbolines (THpCs) were prepared in 100 mM phosphate buffer pH 4 as follows: 50 μΜ THCA, 50 μΜ MTCA, 50 μΜ 1,2,3,4-tetrahydro-P-carboline, 50 μΜ l-methyl-l,2,3,4-tetrahydro-pcarboline, 50 μΜ MTCA-COOH and 50 μΜ THCA-COOH. Subsequently, the model solutions were heated at 60 and 80 °C for 1 and 3 h, or treated separately with a variety of oxidants such as H 0 (2 mM), NaN0 (100 μΜ), H 0 (50 μΜ) + FeS0 (50 μΜ), FeCl (1 mM). Then, the concentration of the aromatic pcarbolines, norharman and harman, that appeared into the model solution was determined by RP-HPLC-fluorescence. Chromatographic analysis was accomplished as for THpC-3-COOHs by changing the fluorescence detection at 300 nm for excitation and 433 nm for emission (35-57). 2


4

2

2

2

2

3

Results and Discussion Chemical Identification of THpCs in Beverages and Foods In order to accomplish the chemical identification of tetrahydro-P-carboline (THpC) alkaloids in foods by GC-MS, the electron ionization (El)-mass fragmentation pattern of THpC alkaloids and their precursors tryptamine and tryptophan, both as free and derivatized species, including W-trifluoroacetyl and JV-methoxycarbonyl derivatives, were studied (55). Fragmentation of THpCs was dominated by retro Diels-Alder rearrangement (-73 amu) which afforded the fragment m/z 143 as base peak for tetrahydro-p-carbolines, and the ion m/z 157 for 1-methyl-tetrahydro-p-carbolines (Figure 2). In addition, THpCs provided abundant molecular ions. The loss of substituents at C - l position of the tetrahydro-pyrido ring and groups at the N-pyrido and 3-carboxylates (COOCH or COOCH CH ) were also characteristic of these compounds (55). Volatile THpC-W-methoxycarbonyl methyl ester derivatives amenable for GC-MS analysis were obtained by reacting the THpCs with methyl chloroformate in the presence of pyridine and methanol, or alternatively methyl chloroformate and diazomethane as shown in Figure 2 (24,33). Taken advantage of this derivatization method, two THpC-3-COOHs (MTCA and THCA) and 1-methyltetrahydro-p-carboline were positively identified in beverages and foods by GCMS. THpC alkaloids were found in many beverages and foodstuffs such as wine, beer, wine vinegar, fruit juices, apple cider, soy and Tabasco sauces, blue cheese, yogurt and toasted bread (Table I). Figure 3 shows a GC-MS chromatogram corresponding to THpC-3-COOHs in an orange juice. 1,3Disubstituted tetrahydro-P-carbolines such as 1-methyl-1,2,3,4-tetrahydro-Pcarboline-3-carboxylic acid (MTCA) occurred as two diastereoisomers of 3

2

3


411

m/2143

m/zl57^

%.

0

H

MS

MS

Μ · 230 M -73 (157) M - C H (215) +

W- (216)

+

M -73(143) ! ^ - N ' THCA


+

V!

s

a) CICOOCH3,

Pyridine Methanol

H

N H

? MTCA C H

+

3

b) CICOOCH3, CH2N2

m/2157

MS

.0CH3

Μ · (302) M -59 (243) M - C H (287) +

1^(288) M -59 (229) +

of

OCH

OCH3

3

"CH tH

+

+

3 3

3

GC-MS

3 100

Τ

ο ιο

CO ^2

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S

τ00 00 CM

100

ιοιο^ϊτκ

i 200

m/z

100

ilu 200

000 CMCO

m/z

Figure 2. Electron Ionization massfragmentationpattern of tetrahydro-βcarbolines (ΤΗβΟ) and their N-methoxycarbonyl methyl ester derivatives used for GC-MS identification in beverages andfoods.



412

m/z 143 2000

OCH

3

m/z 288

,

1000 0

12

14

8000

20

18

16

Nutraceutical Beverages - American Chemical Society

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