Journal of Horticultural Science & Biotechnology (2010) 85 (5) 410–414
Cyanogenic D-amygdalin contents of the kernels of cultivated almonds and wild Amygdalus webbii Spach. By G. FERRARA1, P. MAGGIO2 and M. D. R. PIZZIGALLO2* 1 Dipartimento di Scienze delle Produzioni Vegetali, University of Bari ‘Aldo Moro’, Via Amendola 165/A, 70126 Bari, Italy 2 Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, University of Bari ‘Aldo Moro’, Via Amendola 165/A, 70126 Bari, Italy (e-mail:
[email protected]) (Accepted 13 May 2010) SUMMARY D-amygdalin is a toxic compound found in the kernels of some bitter almond cultivars. This compound is toxic because of its potential to release poisonous hydrogen cyanide. The D-amygdalin contents of the kernels of 18 commercial almond cultivars (Prunus dulcis Mill. = Amygdalus communis L.) and three wild genotypes (Amygdalus webbii Spach.) were determined by HPLC. In initial tests, two extraction procedures [100% (v/v) methanol or 4% (w/v) citric acid)], two different kernel cutting sizes (powdered or roughly-cut pieces), and two shaking techniques (mechanical shaking or sonication) were assessed. The results obtained showed that the method of extraction can have a strong effect on the extent of recovery of the potentially toxic compound, which varied by a factor of approx. 20-fold across the different extraction techniques. The greatest recovery of D-amygdalin from wild almond kernels was achieved with mechanical shaking of roughly-cut kernels in 100% (v/v) methanol, and this procedure was applied for all subsequent analyses of the D-amygdalin contents of all genotypes. The highest amounts of D-amygdalin were found in “bitter” cultivars and wild genotypes (716 – 23,025 mg kg–1), with lower values in “sweet” cultivars (0 – 158 mg kg–1). High levels of variability were observed both among the 18 almond cultivars and the A. webbii genotypes tested.
mygdalin (D-mandelonitrile--D-gentiobioside), known as D-amygdalin (Figure 1) or laetrile, is a naturally-occurring diglucoside anti-feedant found not only in many species of the family Rosaceae, in particular in members of the Prunoideae and Maloideae (Nahrstedt et al., 1990), but also in members of the Leguminosae, Gramineae, Araceae, Compositae, and Euphorbiaceae (Conn, 1980). D-amygdalin can be found in green plant tissues, but it is especially common in the seeds or kernels of almond (Frehner et al., 1990; Sanchez-Perez et al., 2008), apricot (Femenia et al., 1995; Gómez et al., 1998; Haque and Bradbury, 2002), peach (Haque and Bradbury, 2002), black cherry (Poulton and Li, 1994), apple (Haque and Bradbury, 2002), plum (Haque and Bradbury, 2002; Poulton and Li, 1994), and in flax seeds and lima beans (Frehner et al., 1990). This diglucoside is toxic because of its potential to release hydrogen cyanide (HCN). Nevertheless, it has been reported that D-amygdalin can be used for medical purposes because can selectively kill cancer cells at tumour sites without systemic toxicity (Koo et al., 2005). D-amygdalin can be epimerised in boiling water, namely transformed into its epimer neo-amygdalin (L-mandelonitrile--D-gentiobioside; Figure 1; Hwang et al., 2002). Neo-amygdalin has no anti-tumour action. In almond (Prunus dulcis Mill. = Amygdalus communis L.), the bitter taste of the kernels of some cultivars is due to the presence of D-amygdalin. The precursor of D-amygdalin is the monoglucoside prunasin (D-mandelonitrile--D-glucoside; Figure 1). Prunasin is stored in the cotyledons via the apoplast (Selmar et al.,
A
*Author for correspondence.
1988), but D-amygdalin and prunasin have also been detected in the leaves of some Prunus species (Santamour, 1998) and in some almond cultivars (Sanchez-Perez et al., 2008). However, approx. 60 d after flowering, the ratio of D-amygdalin:prunasin increases in the developing fruit; and, 100 d after flowering, Damygdalin constitutes ≥ 90% of the cyanogenic glucosides in the kernel (Frehner et al., 1990). Like other diglucosides, D-amygdalin is a secondary product used to protect wild plants against herbivores. There are only a few reports on the D-amygdalin contents of kernels (Barbera et al., 1988; Siami et al., 2002), oil (Dore et al., 2004), nectar, or pollen (LondonShafir et al., 2003) of wild and domesticated almonds. In bitter almonds, prunasin synthesised in the tegument is transported in the cotyledon via the symplast and converted into D-amygdalin in the kernels (SanchezPerez et al., 2008). However, in sweet almond, the formation of D-amygdalin is prevented because prunasin is degraded by -glucosidase in the inner epidermis of the tegument (Sanchez-Perez et al., 2008).
FIG. 1 Structures of the cyanogenic -glucosides: D-amygdalin (A), neoamygdalin (B), and prunasin (C), where Gen = gentiobiose and Glc = glucose.
G. FERRARA, P. MAGGIO and M. D. R. PIZZIGALLO D-amygdalin levels are usually determined by HPLC in the reverse phase, after extraction with aqueous or organic solvents, although other analytical methods such as GC can be used (Berenguer-Navarro et al., 2002; Sanchez-Perez et al., 2008). The main drawbacks of all these methods are their low efficiency and the length of time needed to complete the analysis. Some differences in the efficiency of extraction of D-amygdalin have been observed depending on the size of the kernel particles used (e.g., powdered, whole kernels, or cut pieces; Koo et al., 2005). A large proportion of the D-amygdalin can be converted into its epimer, neo-amygdalin, during aqueous extraction, but this can be overcome by lowering the pH of the extraction solution (Hwang et al., 2002). We used a slightly different method to increase the efficiency of extraction of D-amygdalin and to prevent its epimerisation, in order to improve the discrimination of D-amygdalin contents among 21 genotypes of almond. The objective of the present work was to determine Damygdalin contents in the kernels of 18 Italian, Spanish, American, and Australian almond cultivars and in three wild-almond genotypes of Amygdalus webbii Spach. In particular, we established the optimum extraction conditions by which to classify the different genotypes by preventing the epimerisation of D-amygdalin.
MATERIALS AND METHODS Plant material Almond (Prunus dulcis Mill. = Amygdalus communis L.) fruit were collected from 18 Italian, American, Australian, and Spanish cultivars and from three genotypes of Amygdalus webbii Spach. from Apulia (southern Italy) and Spain, and grown at the ‘P. Martucci’ Experimental Station, Valenzano, Bari. Mature fruit (2 kg tree–1) were collected from three trees of each cultivar, and the hulls and shells were removed. Kernels were peeled using mildly acidic (pH 5.9) cold water in order to avoid epimerisation of the D-amygdalin (Koo et al., 2005; Hwang et al., 2002). Chemicals 8-Amino-2-naphthalene-sulphonic acid (8,2 ANS) was used as an internal standard. D-amygdalin (99% pure; used without further purification), methanol (reagent grade) and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich (Milan, Italy). All other chemicals were of analytical reagent grade. Sample preparation D-amygdalin extraction was conducted using two different solvents [100% (v/v) methanol or 4% (w/v) citric acid] and two different extraction techniques (mechanical shaking or sonication). Moreover, the kernels were powdered, or each was cut roughly into five-to-six pieces. Briefly, in the case of solvent extraction, 10 g of whole kernels were mixed in a Waring Blender with 1.0 ml 4% (w/v) citric acid and the mixture was frozen and lyophilised. Subsequently, 2.0 g aliquots of this kernel powder were added to 100 ml 100% (v/v) methanol or to 100 ml 4% (w/v) citric acid, and shaken for 24 h at room temperature using an end-over-end shaker (Model 708;
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ASAL, Milan, Italy). Each suspension was centrifuged at 1,620 g for 10 min at 25°C and the supernatant was filtered through a 0.2 µm regenerated cellulose filter. A 10 ml aliquot of each solution was then evaporated to dryness (Rotary Evaporator 4001; Heidolph, Schwabach, Germany) and the residue was dissolved in 10 ml of 4% citric acid (w/v) or 100% (w/v) methanol prior to HPLC. The same procedures as reported above were applied to 2.0 g of roughly-cut pieces of kernel after drying them at 35°C for 6 h. All extractions were conducted in triplicate. Sonication was also used for the extraction of Damygdalin (Vibracell Model VCX 500; Sonics, Newtown, CT, USA). Briefly, the samples were prepared by adding 100 ml 100% (v/v) methanol or 100 ml 4% (w/v) citric acid to 2.0 g of kernels (powdered or cut pieces) and sonicated for between 5 – 10 min at 1,300 – 2,100 J in order to evaluate the best extraction conditions. After sonication, the samples were centrifuged (1,620 g for 10 min at 4°C) and filtered as above. All extractions were conducted in triplicate. All extraction procedures were first applied to kernels of the three A. webbii genotypes and to two sweet and two bitter almond cultivars in order to verify the optimum conditions for analysis. Recovery of Damygdalin was determined in all samples by adding a standard amount of the 99% pure compound to the kernels and measuring the increase in the chromatographic peak with respect to that obtained from the almond kernel sample alone and to that obtained using only the standard solution of Damygdalin at the same concentration (the same amount). Analytical methods D-amygdalin was analysed using a Model 410 HPLC pump (Perkin Elmer Italia, Monza, Italy) with a Supercosil LC-18 reverse phase column (15 cm 3.9 mm id) and a Diode Array Detector (Perkin Elmer Series 200) set at 220 nm. The mobile phase, used for isocratic elution, consisted of 90:10 (v/v) water and acetonitrile and at a flow rate of 1.0 ml min–1. A calibration plot, conducted using 8,2-ANS as an internal standard, showed a linear response from 5 – 500 mg kg–1 Damygdalin and a detection limit (i.e., signal-noise ratio, S/N = 3) of 0.5 mg kg–1 D-amygdalin. Statistical analysis Data from the different extraction methods were analysed by three-way ANOVA (2 solvents 2 extraction techniques 2 kernel piece sizes). Variance assumptions were verified by Levene’s test and normal distributions by Lillefors’ test after the D-amygdalin contents of all genotypes were square root-transformed for homogeneity of variance. Genotype values only were analysed by one-way analysis of variance (P ≤ 0.01) and mean values separated using the Ryan-Einot-GabrielWelsch (REGWQ) test.
RESULTS AND DISCUSSION The amount of D-amygdalin extracted from each sample of kernels varied with the solvent used [100% (v/v) methanol or 4% (w/v) citric acid], the method of extraction (shaking or sonication), and the sample size
Amygdalin contents of various almond kernels
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FIG. 2 Chromatograms of a roughly-cut sample of almond kernels extracted by sonication at 2,100 J (Panel A) or at 1,300 J (Panel B). Both samples were extracted for 5 min at 25°C using 100% (v/v) methanol.
(powder or roughly-cut pieces). The recovery rates in all samples ranged from 5 – 95%, reaching its highest value in roughly-cut pieces of kernel extracted with 100% (v/v) methanol. The most important factor influencing the yield of D-amygdalin was the solvent, with a higher efficiency of extraction using methanol (Table I), a wellknown solvent for cyanogenic compounds (Frehner et al., 1990). The recovery of D-amygdalin was also always higher after mechanical shaking than after sonication (Table I). In the case of sonication, the best analytical conditions were 5 min at 1,300 J and 25°C (Figure 2). The amounts of D-amygdalin extracted using methanol from the roughly-cut pieces of kernel were higher than those from powder (Table I). Similar results were achieved by Koo et al. (2005) when extracting D-amygdalin from seeds of P. armeniaca var. ansu, with methanol or acidified water. These TABLE I Effect of the method of extraction on the amount of D-amygdalin –1 (mg kg ) recovered from wild almond kernels Shaking Solvent Methanol [100% (v/v)] Citric acid [4% (w/v)] †
Sonication
Roughly-cut Roughly-cut pieces Powder pieces Powder 454 a† 171 d
336 b 120 d
265 c 38 e
258 c 25 e
Mean values followed by a different lower-case letter are significantly different at P ≤ 0.01. Data are the means of three replicates per treatment.
authors concluded that the efficiency of extraction increased as the size of the kernel pieces increased. This result was probably a consequence of almond emulsin, an enzyme that hydrolyses D-amygdalin (-glucosidase activity) that is located on the seed surface. Emulsin was possibly present in larger amounts in lyophilised powder samples than in roughly-cut pieces as a consequence of the higher surface area of the powder (Koo et al., 2005). In general, the increased release of hydrolytic enzymes (e.g., -glucosidase) from cells as a consequence of crushing to make the crude powder, caused increased hydrolysis of D-amygdalin with the production of benzaldehyde and hydrogen cyanide (Conn, 1980). This was particularly evident in the case of sweet almond cultivars (Sanchez-Perez et al., 2008). A higher hydrolytic activity was probably also caused by sonication, because of the high energy applied which disrupted cell walls, thus releasing -glucosidase from the apoplast. In fact, the chromatographic peak of D-amygdalin decreased as the sonication energy increased (Figure 2). On the basis of the results obtained in our preliminary experiments, mechanical shaking of roughly-cut kernels, using 100% (v/v) methanol, was adopted as the standard extraction technique. D-amygdalin was not detected in seven out of the 21 cultivated and wild almond genotypes examined (Table II). The remaining 14 genotypes were grouped on the basis of their flavour and origin. Bitter almond cultivars had higher concentrations of D-amygdalin, ranging from 15,514 mg kg–1 (‘Andria’) to 23,025 mg kg–1 (‘Cicerchia’). There was also a variation among A. webbii genotypes from Apulia and Spain, with values ranging from 716 – 23,702 mg kg–1. These results confirm the high Damygdalin contents in bitter almond cultivars from TABLE II Concentrations of D-amygdalin in the kernels of 18 cultivated and 3 wild almond Almond type Bitter Group mean Bitter Group mean Sweet
Group mean Sweet Group mean Sweet Group mean Sweet Group mean Pooled mean †
Cultivar D-amygdalin content name/genotype (mg kg–1 DW) ‘Cicerchia’ ‘Padula di Terlizzi’ ‘Andria’ A. webbii F1 A. webbii B2 A. webbii A4 ‘Genco’ ‘Filippo Ceo’ ‘Tuono’ ‘Fragiulio Grande’ ‘Cristomorto’ ‘Rachele Grande’ ‘Marcona’ ‘Desmajo Largueta’ ‘Glorieta’ ‘Texas’ ‘Ne Plus Ultra’ ‘Nonpareil’ ‘Baxendale’ ‘Chellaston’ ‘J. Prolific’
23,025 b† 21,655 c 15,514 d 20,065 23,702 a 992 e 716 f 8,470 135 h 91 i 54 k 158 g 0l 0l 73 82 i 52 k 0l 45 68 j 0l 0l 23 50 k 0l 0l 17 4,109
Source Italy (Apulia) Italy (Apulia) Italy (Apulia) Italy (Apulia) Italy (Apulia) Spain Italy (Apulia) Italy (Apulia) Italy (Apulia) Italy (Apulia) Italy (Apulia) Italy (Apulia) Spain Spain Spain USA USA USA Australia Australia Australia
Mean values followed by a different lower-case letter are significantly different at P ≤ 0.01. Data are the means of three replicates per genotype.
G. FERRARA, P. MAGGIO and M. D. R. PIZZIGALLO Apulia, and the large variation in wild species. However, these two groups presented very different average values (approx. 20,065 and approx. 8,470 mg kg–1, respectively). Sweet almond cultivars had much lower D-amygdalin contents, ranging from only 50 mg kg–1 (‘Baxendale’) to 158 mg kg–1 (‘Fragiulio Grande’). The amounts of D-amygdalin detected in the sweet almond cultivars here were much lower than the particularly high values reported for the same cultivars by Barbera et al. (1988), who did not describe the method used. Our data are more in accord with data collected for wild Iranian almonds (Siami et al., 2002) and some crosses used for plant improvement (Berenguer-Navarro et al., 2002; Dicenta et al., 2002). A recent study on some Sardinian almond genotypes (Dore et al., 2004) reported D-amygdalin concentrations of 60 – 70 mg kg–1 in the extracted oil, close to our values for sweet almond cultivars (Table II). In recent work conducted in Israel (London-Shafir et al., 2003), high concentrations of Damygdalin were detected in pollen (approx. 2,000 mg kg–1), in nectar (approx. 5 – 7 mg kg–1), and in honey (approx. 3 mg kg–1) of ‘Ne Plus Ultra’, ‘Mem-Dalet’, and ‘Um-El-Phahem’ almond cultivars. In the case of A. webbii, D-amygdalin contents were much higher than the values reported for wild Iranian almond species (approx. 100 mg kg–1; Siami et al., 2002). Moreover, the amount of D-amygdalin found in both the bitter cultivars and in A. webbii were generally lower than those reported for bitter apricot kernels (50,000 – 60,000 mg kg–1) in Spain (Femenia et al., 1995; Gómez et al., 1998). Similar, high D-amygdalin contents in both bitter almond cultivars and in A. webbii suggest a possible relationship between the two species in Apulia. It has
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been reported that “kernel taste” is controlled by a single dominant/recessive gene, with sweet cultivars being heterozygous Ss or homozygous SS, and bitter cultivars being recessive ss (Dicenta and Garcia, 1993; Dicenta et al., 2002; 2007). Some authors have suggested that sweetness is due to a mutation in the original bitter almond (Dicenta et al., 2007). Sometimes, normally sweet cultivars that are heterozygous for this trait may be slightly bitter, possibly due to an alteration in the single dominant/recessive gene system (Dicenta et al., 2002). Some authors have also suggested that the genetic control of bitterness/sweetness in almond is more complex than originally proposed (Dicenta et al., 2007) and that the gene(s) determining bitterness remain to be identified (Sanchez-Perez et al., 2010). The high contents of D-amygdalin in bitter almond genotypes should be considered a danger to human health. Attention should therefore be paid to the consumption of these kernels. The limits for cyanide contents in products such as mineral water is 0.07 mg l–1 (FAO/WHO, 1997) and in cassava flour is 10 mg kg–1 (FAO/WHO, 1995). Ingested cyanide salts have a minimum lethal dose of 2 – 4 mg kg–1 body weight (Fermenia et al., 1995). Based on this lethal dose, an average person weighing 80 kg should ingest no more than 250 – 450 kernels of the bitter genotypes with highest contents of the D-amygdalin (‘Cicerchia’; Table II) to avoid being poisoned. Bitter almond kernels are generally used in small amounts in baked goods, cookies, confectioneries, and candies, and for the extraction of almond oil. Limited possible application as an anti-tumour product has also been suggested (Koo et al., 2005), but this needs to be clinically tested.
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Amygdalin contents of various almond kernels
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