PHYTOCHEMICAL ANALYSIS, VOL. 4, 120-123 (1993)

Quantification of Benzaldehyde and its Precursors in Montmorency Cherry (Prunus cerasus L.) Kernels

Amitabh Chandra and Muraleedharan G. Nair* Department of Horticulture, Bioactive Natural Products Laboratory, Michigan State University, East Lansing, Michigan 48824. USA

Kernels from Michigan-grown tart cherry Prunus cerusus L. cv. Montmorency were frozen at -40 "C, lyophilized, crushed and extracted sequentially with hexane and methanol. Amygdalin, mandelonitrile and benzaldehyde were present in the kernel extracts and were quantified by high performance liquid chromatogra- phy. Amygdalin was found to be the major component, while free mandelonitrile is detected for the first time in the tart cherry pit kernels.

Keywords: Prunus cerasus; amygdalin; mandelonitrile; benzaldehyde; HPLC quantification

INTRODUCTION

Benzaldehyde, one of the constituents of almond fla- vour, is probably the most important flavour ingredient in many food products and beverages (Broderick, 1975). It is obtained from various natural sources such as bitter almonds (Prunus amygdalus L.), apricots (P. armeniaca L.), and peaches (P . persica L.) and has developed over the years as a flavour component in candies, carbonated beverages and desserts. Among all cherries, tart cherries (P. cerasus L.) have been reported to yield the maximum amount of benzalde- hyde (Broderick, 1975). Owing to an increasing demand, high prices and limited supplies of natural benzaldehyde, synthetic benzaldehyde is being used as a substitute for artificially flavouring of some food products (Butzenlechner et a[., 1989). Therefore, pro- duction of benzaldehyde from Montmorency cherry pits is highly desirable.

Michigan's tart cherry production is about 200 mil- lion pounds per year with half of it grown and pro- cessed around Traverse City. The cherry pits now constitute a waste disposal problem. An economically viable procedure for the isolation and purification of benzaldehyde will not only help defray the costs of final disposal but also provide additional income to cherry growers (Lin et al . , 1990). The economic feasibility of benzaldehyde production from cherry pit kernels depends on its efficient recovery during isolation and purification.

Amygdalin (I) , generally in the R form (Cairns et al., 1978), is one of the major constituents of cherry kernel extract and is reported to undergo enzymatic hydrolysis (Haisman and Knight, 1967; Poulton, 1988) to produce benzaldehyde (2) and HCN via mandelonitrile (3) (Fig. 1). Broderick (1975) reported that the free ben- zaldehyde produced in the kernels is partitioned into the pulp adjoining the pit. Benzaldehyde has been detected along with other volatile components contri-

* Author to whom correspondence should be addressed

0958-0344/93/030 120-04 $07.00 0 1993 by John Wiley & Sons, Ltd

buting to the cherry essence and flavour (Stinson et al., 1969; Schmid and Grosch, 1986). Therefore, overall benzaldehyde content in cherry pits is not only related to the amygdalin and mandelonitrile content in the pits but also to their storage and handling. This paper describes the extraction and quantification of benzalde- hyde and its precursors from Montmorency cherry kernels.

EXPERIMENTAL

General. High performance liquid chromatographic (HPLC) analyses were carried out on a Capcell pak (Dychrom, Sunnyvale, CA) C-18 column (250 mm x 10 mm, 5 p). The mobile phase CH3CN:H20 (70:30v/v) was used under isoc- ratic conditions at a flow-rate of 1.5 mL/min. The samples were analysed at 210nm (aufs 0.002) using a Waters 490 variable wavelength UV-visible detector (Waters Assoc., Milford, MA). Amygdalin (Sigma Chemical Co., St. Louis, MO), mandelonitrile and henzaldehyde (Aldrich Chemical Co., Milwaukee, WI) were used as standards.

Extraction of Montmorency cherry pit kernels. Cherry pit kernels were frozen at -40°C and lyophilized. The lyophi- lized kernels (13.71 g) were then crushed using a mortar and pestle and sequentially extracted in a Soxhlet apparatus with hexane (150mL, 6h) and methanol (150mL, 12h). The extracts were combined and evaporated in uucuo to afford residue A (4.06 g), as the total kernel extract.

Extraction of benzaldehyde from pit oil. The kernels from the same batch as used for solvent extraction were pressed (Anderson International, Cleveland, OH) to afford an oil. The oil (476.30g, 500mL) was extracted with MeOH (3 X 600 mL) using a separatory funnel, and the combined MeOH extracts were evaporated in U ~ C U O to yield a yellow oily product with a strong smell of benzaldehyde (15.10g). This oil was purified by vacuum liquid chromatography (222 g silica gel) and nine fractions were collected following elution with hexane, 500 mL (fraction l), hexane, 300 mL (fraction

Received I0 September 1992 Accepred 6 December I992

BENZALDEHYDE ANALYSES IN CHERRY KERNELS 121

CH,OH w-&:s CH,OH t H20 p-glucosidaseC HO @H HO @ :s HO

OH OH OH HO OH

Am ygdalin (1 1

Prunasin

@H CH20H

HO OH Benzaldehyde (R)-Mandelonitrile

(2) (3) Figure 1. Enzymatic conversion of amygdalin to benzaldehyde.

2), hexane, 300 mL (fraction 3), hexane:acetone ( 5 : 1 ) . 320 mL (fraction 4), hexane:acetone ( 5 : I ) , 160 mL (fraction S ) , hexane:acetone ( 5 : l ) , 220 mL (fraction 6), hexane:ace- tone ( 5 : l), 200 mL (fraction 7), hexane:acetone (3: I ) , 350 mL (fraction 8) and hexane:acetone (2: I ) , 450 mL (frac- tion 9). These fractions were concentrated separately in uucuo and a thin layer chromatographic anaylis (hexane: ace- tone, 4: 1) indicated that fractions 3 to 7 contained benzalde- hyde and these were combined to yield a yellow oily residue. This oil was partially decolourized with activated charcoal (5.0 g) in hexane (250 mL) to afford a pale yellow oil (14.40 g, 3.02 %).

Quantification. Calibration curves were established for 1, 2 and 3 using appropriate standard solutions containing 0.25- 1Opg for 1 and 2 and 0.62-2.50pg for 3, respectively. Standard solutions were freshly prepared in CH,CN : H,O (70:30, v/v) prior to the analysis: 3 was stable in this solvent for at least 8 h and was not converted into 2 during that time. Concentrations of amygdalin, benzaldehyde and mandeloni- trile in the kernel extract were determined by the correlation of their HPLC peak areas to their calibration curves, respecti- vely.

The whole kernel extract, A (76.70mg), was dissolved in CH,CN:H20 (70:30) (1.0mL) and passed through a C'-18 Sep-pak column (Waters Assoc.) preactivated with CH,CN. The resulting solution was diluted with CHICN (10 mL) and an aliquot (30 vL) was analysed by HPLC to yield 1 (5.70 pg). 2 (4.10 pg) and 3 ( 1 .58 pg).

RESULTS AND DISCUSSION

Amygdalin, one of the major components present in the kernels of almonds, peaches, prunes, and cherries undergoes enzymatic conversion (Poulton, 1988) to produce benzaldehyde via mandelonitrile as shown in Fig. 1. The total benzaldehyde content in the pit (Hais- man and Knight, 1967) is proportional to the amygdalin content in the kernel. Therefore, it is essential to know the total amygdalin in the kernel in order to estimate

the amount of free and bound benzaldehyde in the pits. Cherry flavour components are commonly determined by gas chromatographic analysis (Stinson et ul., 1969; Schmid and Grosch, 1986). Recently Okuyama et ul. (1989) purified amygdalin by HPLC using a gradient of CH,CN : H20 from 95 : 5 to 65 : 35 (v/v) for 60 min on a hydroxyapatite column at a flow-rate of 1.0 mL/min. Cairns et al. (1978) have developed a method by using reversed phase HPLC with an isocratic system contain- ing 4% CH3CN:H20 (1.6mL/min) to detect an epi- meric mixture of amygdalin, the components of which were separated using the solvent system of 0.2% CH,CN:H,O and reducing the flow-rate to 1.2mL/ min. These workers have also reported that the natural amygdalin present in the kernels of the members of the family Rosaceae is in the R form. We have developed an isocratic system for the HPLC analysis of a mixture containing amygdalin, mandelonitrile and benzalde- hyde from the kernel extract of Montmorency cherries.

The hydrolysing enzymes for amygdalin present in the kernels were inactivated by freezing ( - 40°C) and lyophilization prior to their solvent extraction (Nahr- stedt, 1981). The hexane extract of the kernels con- tained benzaldehyde in addition to the oil as the major constitutent. Therefore, hexane and methanol extracts of the kernels were combined to obtain the extract A prior to the quantification of 1,2 and 3. This combined extract was dissolved in CH,CN:H20 (70:30) and was passed through a C-18 cartridge, thereby rendering the reversed phase HPLC analysis more efficient (Parli- ment, 1981; Minguez-Mosquera et al., 1992). The quantification of 1, 2 and 3 in the extract was achieved using the standard curves. The injection volumes of these samples for their HPLC analysis were adjusted so that all the three compounds would absorb in a similar manner under the HPLC conditions studied. Peaks for amygdalin (8.83 min), benzaldehyde (12.50 min) and mandelonitrile (10.81 min) were confirmed by compar- ing their retention times with the standards (Fig. 2) and by their 'H NMR spectra, which were found to be identical to their standards.

Free benzaldehyde was found to be present in an

122 A. CHANDRA AND M. G. NAIR

1

2

I I I I I I

2.5 5.0 7.5 10.0 12.5 15.0 Time (min)

Figure 2. HPLC chromatogram of the cherry pit kernel extract; (1) amygdalin, (2) benzaldehyde and (3) mandelonitrile.

amount of 0.53% in the extract A, whilst amygdalin and mandelonitrile were found in yields of 0.73% and 0.20%, respectively. In the kernels the figures were benzaldehyde 0.16%, amygdalin 0.22% and mandelo- nitrile 0.06%. Using these percentages and converting 1 and 3 to their molar equivalents of 2, the available benzaldehyde content in the cherry pit kernel is calcu- lated to be 0.26% (dry weight). In another experiment, the kernels from the same batch were pressed to eva- luate the recovery of benzaldehyde in the expelled oil, since the oil obtained by the solvent extraction of the kernels had the characteristic smell of benzaldehyde.

this is the first evidence for the presence of free mande- lonitrile in cherry kernels. It is produced as an interme- diate during the conversion of 1 to 2 in the pits during the maturity of cherry fruits while they are on the tree. The amount of benzaldehyde reported in the kernels may be slightly less than that actually present since the volatilization and oxidation of benzaldehyde during the extraction conditions is hard to control under regular laboratory conditions.

Acknowledgements The 'H NMR spectrum of the MeOH extract of the expelled oil indicated that the benzaldehyde content

higher than in the Oil Obtained from the solvent extraction of the kernels. TO our knowledge

This research is a contribution from the Michigan State University Agricultural Experiment Station. The authors are thankful to Mr. C . R. Johnston of CMI for supplying cherry pit kernels, pressed cakes and oil samples.

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