Characterization of potentially mutagenic products from the nitrosation of piperine

Cancer Letters, 64 (1992) 235-239 Elsevier Scientific Publishers Ireland Ltd.

235

Characterization of potentially mutagenic products from the nitrosation of Ipiperine Narmada Bio-organic

R. Shenoy

and Ahmed

Division, Bhabha Atomic Research

S.U. Choughuley Centre,

Trombay,

Bombay

400085

(India]

(Received 10 March 1992) (Revision received 24 April 1992) (Accepted 27 April 1992)

Summary

Introduction

Pipe&e is the main pungent principle of pepper, a spice consumed by people all over the world. It is the trans-trans isomer of Ipiperoylpiperidine and contains the methylene dioxy moiety. It is known to give unidentified mutagenic products on reaction with nitrite. The nitrosation reaction of piperine is of concern as endogenous nitrosation could take place in the human stomach from ingested precursors, piperine and nitrite. Nitrites can be

Piperine is the major pungent principle of pepper, a spice consumed by people all over the world. It is the trans-trans isomer of lpiperoylpiperidine and contains the methylene dioxy moiety. Nitrosation of piperine is interesting as unidentified mutagenic products have been obtained from this reaction [1,2]. This is of concern as human beings are exposed to substantial amounts of nitrites either directly or indirectly. Nitrites are used for the curing of foodstuffs and can be readily produced by bacterial reduction of nitrates present in vegetables, some food supplies and adversely stored food [3 - 51. Nitrate as present in vegetables, water and other food supplies is essentially non-toxic. However it can be reduced to nitrite by microbial conversion in the saliva, adverse storage conditions of food, achlorhydric stomach and in infected urinary bladder [6]. Nitrite is also known to be endogenously formed from arginine. As the physiological conditions prevalent in the human stomach are ideal for nitrosation reactions to occur from ingested precursors, we thought it important to investigate the nitrosation of piperine. In our present study we have nitrosated piperine using nitrous acid in

ingested directly by consuming cured foods or indirectly as nitrates, which could be converted to nitrites under appropriate conditions. We have nitrosated pipeline using aqueous nitrous acid and have isolated and identified some Nnitroso and C-nitro compounds. Their isoiation, characterization and potential mutagenicity has been discus.sed.

Keywords: piperine; nitrosation; derivatives; potentially mutagenic

nitro

Correspondence to: Narmada R. Shenoy, Beckman Institute of the City of Hope, Division of Immunology, Duarte Rd., Duarte, California, USA 91010-0269.

Research 1450 E.

0304-3835/92/$05,00 Printed and Published

0 1992 El sevier Scientific Publishers in Ireland

Ireland Ltd.

237

h ‘I

NPA

I ’

MNAP

”

h

I

‘I

CHO DNP

c=c_c_c_c_; ; L

NPIP

L)

Scheme 1. The mechanism of nitrosation of piperine. The compounds identified from the nitrosation of piperine are piperonal (PA), 6-nitropiperonal (NPA) ,3,4-methylenedioxy cinnamaldehyde (MDCA), l-5[( 1,3-benzodioxoC6-nitro5-yl)-1-oxo-2,4,E,Egentadienyl]piperidine (MNAP), 1-5[(1,3-benzodioxol-5-yl)-l-oxo-2-nitro-2,4,E,E,-pentadienyl]piperdine (MNOP), 1-5[(1,3-benzodioxol-6-nitro-5-yl)-l-oxo-2-ni~o-2,4,E,E-pentadienyl]piperidine (DNP) and Nnitrosopiperidine (NPIP) .

reagent Eu(fod)s the olefinic protons shifted as follows: 8.8 ppm (dd., &proton), 8.53 ppm (d, S-proton), 7.8 ppm (d, o-proton) and 8.2 ppm (dd, y-proton). The compound has an absorbtion at 1320 and 1530 cm -’ (nitro), 1640 cm -l (carbonyl), 3010 cm -’ and 980 cm - ’ (trans double bond) in the IR spectra. From these spectroscopic data it was deduced that the compound was a nitro derivative of piperine. The position of the nitro group was established to be in the 6 ’ position of the aromatic ring as the other two aromatic pro-

tons gave singlets in the PMR spectrum. The compound (MNAP) was assigned the structure 1-5[(1,3-benzodioxol-6-nitro-5-yl)-l-oxo2,4,E,E_pentadienyI] piperidine (MNAP). Compound MNOP showed a molecular ion peak at m/z 330 and fragment peaks at m/z 300 (M+-NO) and m/z 285 (M +-NOs). The PMR spectrum in CDCls showed a twoproton singlet at 6.1 ppm (methylenedioxy), 1.7 ppm two six-proton multiplets at (piperidine 3,4,5 protons split magnetically due to addition of nitro group), two four-

proton multiplet at 3.29 ppm-3.63 ppm (piperidine 2,6 protons split magnetically due to nitro addition), a one-proton doublet of the aromatic proton at 6.83 ppm (C-5’ aromatic proton), a one proton-double doublet of the aromatic proton at 7.02 ppm (C-6 ’ aromatic proton) and a one-proton doublet of the aromatic proton at 6.69 ppm (C-2’ aromatic proton), a one-proton doublet at 7.79 ppm (polefinic proton), a one-proton double doublet at 6.62 ppm (y-olefinic proton) and a oneproton doublet at 7.11 ppm (b-olefinic proton). On adding the chemical shift reagent Eu(fod)s the olefinic protons shifted as follows: 8.22 ppm (d, P-proton), 7.26 ppm (d, b-proton) and 7.65 ppm (dd, y-proton). The compound has an absorbtion at 1320 and 1530 cm-’ (nitro), 1630 cm-’ (carbonyl), 3010 cm1 and 980 cm-l (trans double bond ) in the IR spectra. This data indicated the compound to be a mono nitro derivative of piperine with the nitro substitution at the olefinic at position. The compound (MNOP) was assigned the structure l-5[(1,3-benzodioxol- 5-yl)-l-oxo-2-nitro-2,4,E,E-pentadienyl] piperidine. Compound DNP showed a molecular ion peak at r+n/z 375 and fragment peaks at m/z 330 (M -NO& and m/z 285 (M -NOz & NO). The PMR spectrum in CDCls showed a two-proton singlet at 6.18 ppm (methylenedioxy), two six-proton multiplets at 1.64 ppm (piperidine 3,4,5 protons split magnetically due to addition of nitro group), two fourproton multiplet at 3.3 ppm-3.52 ppm (piperidine 2,6 protons split magnetically due to nitro addition), two singlets of the aromatic protons at 7.55 ppm (C-5’ proton) and 7.03 ppm (C-2 ’ proton), a one-proton doublet at 7.78 ppm @I-olefinic proton), a one-proton double doublet at 6.64 ppm (y-olefinic proton) and a one-proton doublet at 7.55 ppm (Aolefinic proton). On adding the chemical shift reagent Eu(fod)3 the olefinic protons shifted as follows: 8.66 ppm (d, P-proton), 8.0 ppm (d, b-proton) and 8.45 ppm (dd, y-proton). The compound has an absorbtion at 1320 and 1530 cm -’ (nitro), 1640 cm -’ (carbonyl),

3010 cm-’ and 980 cm -’ (trans double bond) in the IR spectra. From these spectroscopic data it was deduced that the compound was a dinitro derivative of piperine with one of the nitro groups substituted at the CYolefinic position and the other at the 6 ’ position of the aromatic ring. The compound (DNP) was assigned the structure l-5[(1,3benzodioxol-6-nitro-5-yl)-l-oxo-2-nitro-2,4,E,Epentadienyl] piperidine. The elemental analysis of the compounds MNAP, MNOP and DNP matched within 0.3% of the required value. The yields of the compounds identified from the nitrosation of piperine are PA: 14 mg (0.093 mm), NPA: 29 mg (0.148 mm), MDCA: 11 mg (0.063 mm), MNAP: 18 mg (0.055 mm), MNOP: 51 mg (0.154 mm), DNP: 24 mg (0.064 mm) and NPIP: 31 mg (0.271 mm). The mechanism of nitrosation of piperine has been outlined in scheme 1. Nitrous acid behaves as both a nitrosating reagent as well as an oxidising agent in this reaction. Piperine undergoes oxidative cleavage at the amide bond and at the olefinic double bonds to form piperonal and 3,4-methylene dioxycinnamaldehyde. Both these compounds probably undergo nitrosation followed by oxidation to form 6-nitropiperonal. Although the nitro derivative of MDCA has not been isolated it might very well be formed and may be present in small amounts. Cleavage of piperine at the amide bond and subsequent N-nitrosation of the cleaved piperidine molecule results in the formation of the carcinogenic N-nitrosopiperidine. The three nitro derivatives formed from uncleaved piperine are MNAP, MNOP and DNP. Two of the these three, MNAP and MNOP are formed by C-nitrosation of the aromatic ring at the 6 ’ position and across the olefinic bond at the a position, followed by oxidation forming mono-nitro derivatives of piperine . The dinitro derivative of piperine DNP is formed by C-nitrosation followed by oxidation at two positions, the aromatic 6 ’ position and the olefinic (I!position. The dinitro derivative could also result from nitrosation followed by oxidation of the mono-nitro

239

derivatives, MNOP and MNAP at the aromatic 6 ’ position and the olefinic (Yposition respectively .

The products that have been identified from the piperine nitrosation reaction mixture by earlier researchers are NPIP and NPA. lVnitrosopiperidine is a known carcinogen and NPA has been proven to be a mutagen to Salmonella typhimutium [2,7]. Earlier experiments have shown that the mutagenicity of the nitrosated reaction mixture of Piperine exceeds that can be attributed to NPIP and NPA alone [2,7]. Researchers have suggested that the Cnitro group at the conjugated double bond or aromatic C-nitro groups are essential structural features for mutagenicity of dienoic carbonyl compounds [1,2,8]. Piperonal and methylenedioxycinnamaldehyde are not known to be mutagenic. All the three nitro derivatives MNOP, MNAP and. DNP exhibit the structural features considered necessary for mutagenicity and could be the hitherto unidentified mutagens. Initial studies on the mutagenicity of these three nitro derivatives show interesting results and will be presented in a following publication. A number of other food constituents viz., ascorbic acid, rr-tocopherols, vegetable juices, plant phenolics, flavonoids, etc., are known to modify the mutagenic potential of preformed or endogenously Itormed mutagens [9, lo]. There are several naturally occuring compounds which render protective action against mutagens. Furthermore the amount of nitrite and piperine used in our experiments exceeds the amount consumed in normal diets. Along with the protective factors present in the diet, formation of the nitro derivatives of piperine

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