Chemical constituents of peppers (Piper spp.) and application to food preservation: naturally occurring antioxidative compounds.

In a structure analysis of the compounds of the genus Piper (Family Piperaceae), we identified five phenolic amides from Piper nigrum, seven compounds from P. retrofractum, and two compounds from P. baccatum. All the phenolic amides possess significant antioxidant activities that are more effective than the naturally occurring antioxidant, alpha-tocopherol. One amide, feruperine, has antioxidant activity as high as the synthetic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Naturally occurring antioxidants, therefore, may surpass BHA and BHT in their ability to inactivate mutagens in food.


Introduction
Much research has involved finding ways to prevent or to delay deterioration in foods. Recently, our research (1,2) has focused on developing safe and effective compounds from natural sources (especially from edible plants) that will prolong the storage life of food.
We studied the genus Piper of the Family Piperaceae, to identify the structure of the compounds in its species that give organoleptic (3)(4)(5), medicinal (6)(7)(8)(9), and insecticidal (10)(11)(12)(13) properties. Many species, among the 700 in this genus, are used not only as spices but also as folk medicines (14). This paper reveals the structure of the compounds and their resulting properties for Piper nigrum L., P. retrofractum Vahl., and P. baccatum Blume.

Structural Determination of Constituents
Powdered dry fruits of white pepper (P. nigrum) were extracted with methylene chloride and fractionated as shown in Figure 1. The fractions determined were neutral (83.80%), weakly acidic (1.72%), strongly *Department of Food and Nutrition, Faculty of Science of Living, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi-ku, Osaka 558, Japan. acidic (0.55%), and basic (0.22%). We found piperine [1] (a pungent principle) and more than 40 constituents isolated from P. nigrum to be neutral compounds. The weakly acidic fraction showed significant anitoxidative activity, and we subjected it to column chromatography on silica gel, using a solution of CH2Cl2 and MeOH as an eluant. The major compound [2a], purified upon recrystallization from hot chloroform (mp 144.0-144.5°C), produced colorless needles.
Mass spectral and elemental analyses of [2a] indicated the molecular formula C18H19NO4.
Three absorption bands at 3500, 3360, and 3240 cm1 in the infrared (IR) spectrum showed the presence of hydroxy or amino groups. The absorptions at 1645, 1615, and 980 cm-represented a trans-conjugated amide carbonyl group.
The Xmax at 295 and 321 nm in the ultraviolet (UV) spectrum indicated the presence of feruloyl moiety in the molecule, as shown in the spectrum of ferulic acid (Xmax 294 and 320 nm). This was also confirmed by the base peak at m/z 177 in the mass spectral (MS) data.
Further evidence for the structural characteristics was obtained from nuclear magnetic resonance (NMR) spectroscopy. One methoxy group was observed at 8 3.88, and a typical set of two doublets at 8 5.47 and 8 7.47 (both J = 16 Hz) was assigned to trans-ax and trans-,B-protons conjugated to the carbonyl group, respectively. A triplet (2H) centered at 8   a multiplet (2H) at 8 3.2 to 3.7 were attributed to the partial structure of Ar-CH2-CH2-R and RCH2-CH2-NH-R, respectively. Seven aromatic protons and an NH were observed in the range 8 6.65 to 7.2. To confirm the structure, we prepared two derivatives. The acetylated compound [2b] showed the acetate absorption at 1760 cm-1 and the disappearance of two hydroxyl groups. The methyl protons of acetyl groups at 8 2.30 (6H) in the NMR spectrum and the molecular ion at m/z 397 in the MS were good evidence for the existence of two hydroxyl groups in the structure of [2a]. Using the spin decoupling technique in the NMR analysis, irradiation of a triplet of [2b] at 8 2.84 caused an apparent quartet at 3.58 to collapse to a doublet, with a coupling constant of J = 7 Hz; whereas irradiation on the signal at 8 3.58 caused a triplet at 8 2.84 to collapse to a singlet and a triplet at 8 6.00 (NH, J = 7 Hz) to collapse to a singlet. These data supported the partial structure of this amide, -CONH-CH2-CH2Ar.
Methylation of [2a] with methyl iodide and sodium methoxide resulted in the addition of two methyl groups and a new molecular ion [2c] at m/z 341, a base peak at m/z 191, and a major fragment at m/z 134 (175%), indicating the feruloyl moiety and the tyramine moiety, respectively (Fig. 2). All ofthese data indicate the structure of the main amide [2a] to be N-feruloyl tyramine.
The second major component, coumaperine, yielded a molecular formula of C16H19N02, a base peak at m/z 173, and a strong fragment at m/z 84, suggesting acyl moiety and piperidine moiety, respectively. The IR spectrum revealed a hydroxyl group at 3200 cm-', an amide carbonyl at 1630 cm-1, and conjugated olefinic groups at 1605 cm-'. The conjugated olefinic system was also revealed in the NMR and by Xmax at 241, 315, and 338 nm in the UV spectrum, close to that of piperine [1].
Acetylation of [3a] produced a monoacetyl derivative [3b], which showed an acetate absorption at 1755 cm-1 in the IR spectrum and acetyl methyl protons at X 2.25 as a 3H singlet in the NMR spectrum.
The structures of all five compounds obtained from the weakly acidic fraction of P. nigrum were confirmed by synthesis (15,16) (see Fig. 3). We examined another Piper species, the Javanese long pepper, P. retrofractum Vahl. (17). The fruits of this species are used as spice for pickling and in medicine for digestive and intestinal disorders. Extractions and purification were performed as previously described (see Fig. 1). Six compounds from the neutral fraction and piperic acid [7a] from a strongly acidic fraction were  isolated. One of the neutral compounds recrystallized from hexane and produced needles [8] (mp 116°C). We determined the molecular formula to be C24H33NO3. The IR absorption bands indicated a conjugated amide carbonyl at 1655, piperonyl moiety at 1255, 1040, and 970 cm-1, which was also supported by a two-proton singlet at 8 5.92 in the NMR spectrum. A doublet (6H) at 8 0.91 and a triplet (2H) at 8 3. 16 suggested the presence of isobutylamino moiety in the structure of [8].
Another species, P. baccatum Blume (known as "rinu" in Indonesia), contained abundant glyceride. Fatty acid composition was determined by gas chromatography following hydrolysis and methylation with BF3-MeOH. Palmitic acid (26.9%), linoleic acid (26.1%), oleic acid (9.2%), stearic acid (5.6%), and other acids were identified. The ratio of constituents ofP. baccatum differed from those of the two preceding species: the I volatile component was as high as 40%, and the piperine component was only a trace. We isolated two compounds E from the neutral fraction of the nonvolatile component; I one was a lignan recrystallized from methanol (mp 1280C). Mass spectral analysis showed a molecular ion peak at m/z 356, a dehydrated ion at m/z 338, a base peak at m/z 203 (M+ -18 -135), and a methylenedioxytropyrium ion at -135. The NMR and IR spectra also supported the structure of hydroxy furan substi-   tuted with two methylenedioxy benzyl groups. We identified this compound as (-)-cubebin [12] (20,21). On the basis of its spectral data we determined the other compound (mp 85.0-85.5°C) to be a 2,3-seco-7, 11-dehydrogermacranolide sesquiterpene lactone.

Antioxidative Activity
Today, to depress and delay the formation of peroxide and rancidity of oils and fats, antioxidants are widely used in processed foods. The most commonly used antioxidants are synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (Ferric Thiocyanate Method; conc. 0.02%) (BHT), and a natural antioxidant, a-tocopherol. However, the use of BHA and BHT as food additives is restricted in several countries because of undesirable effects these additives produce on the enzyme systems of liver and lung (22,23). The antioxidative ability of atocopherol is less active. Therefore, finding and preparing antioxidants from natural foodstuffs is a logical alternative.
We measured antioxidative activity on the compounds isolated from pepper. Figures 5 and 6 show the antioxidative activity of piperine and five amides from P. nigrum, measured by the ferric thiocyanate method and thiobarbituric acid method, respectively. In these experiments, linoleic acid was used for the oxidation substrate. Piperine [1] showed no activity. All phenolic amides [2][3][4][5][6] showed significant activity, more effective than cx-tocopherol at the same concentration (0.01%). The activity of compound [5] was as high as that of synthetic antioxidants, BHA and BHT, at the same level. Piperic acid [7a] and some of the neutral components isolated from P. retrofractum and P. baccatum showed slight antioxidative activity, but they were less active than a-tocopherol (Fig. 7).

Antimicrobial Activity
It is known that certain pepper spices possess antifungal and antibacterial activities. Antibacterial activities of compounds [2][3][4][5][6] from P. nigrum and [7a], [7b], and [8] from P. retrofractrum were measured against eight microorganisms, including Staphylococcus aureus and Streptococcus faecalis, to determine minimum inhibition concentrations. The results of these studies revealed that the antibacterial activity of these compounds was low.
Mutagenesis in food is an obvious hazard. Continued study of the relationship between mutagens and naturally occurring antioxidants isolated from peppers and other spices is important if methods are to be developed to prevent the formation of mutagens or to inactivate them.

Experimental
Melting points, measured with a Yanagimoto micro melting-point apparatus, were left uncorrected. The UV absorption spectra were determined on Hitachi 323 and 220 spectrophotometers, and IR spectra were recorded by a Jasco IR-S. The 1H-NMR spectra were determined on a Hitachi R-600S instrument tetramethylsilane (TMS) being used as an internal standard. The MS data were obtained on a Shimadzu GCMS-7000. Column chromatography was performed using Merck silica gel-60 (70-230 mesh) and Pharmacia Sephadex LH-20; thin layer chromatography (TLC) was completed using silica gel GF-254.

Antioxidative Assay
Antioxidative activity of each sample to protect linoleic acid was measured by the ferric thiocyanate method and thiobarbituric acid method (TBA) (2). The sample solution, prepared according to our method at a concentration of 0.01% or 0.02%, was kept in a dark oven at 40°C.