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Canavanine content in sword beans (Canavalia gladiata): Analysis and effect of processing
Food and Chemical Toxicology 45 (2007) 797–803 www.elsevier.com/locate/foodchemtox
Canavanine content in sword beans (Canavalia gladiata): Analysis and effect of processing S. Ekanayake a
a,b,*
, K. Skog b, N.-G. Asp
b
Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka b Department of Food Technology, Engineering and Nutrition, Division of Applied Nutrition and Food Chemistry, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received 8 September 2005; accepted 30 October 2006
Abstract The amino acid canavanine is a potentially toxic constituent of leguminous seeds. The aim of the present study was to determine the ability of different processing methods to reduce canavanine in sword beans (Canavalia gladiata). For this purpose a method for the detection and quantification of canavanine was developed using reversed-phase high-performance liquid chromatography of the dabsylated derivatives. The recovery of canavanine using this method was 88–91%. Optimum extraction of canavanine from raw and processed beans was obtained by addition of hot water prior to overnight soaking. The results obtained with this method agree well with previously published values for raw seeds. The method is sensitive, specific and can successfully be applied to the detection of canavanine in legumes. Overnight soaking and boiling in excess water followed by decanting gave the most pronounced reduction in canavanine content (around 50%), followed by boiling and decanting excess water (34%). Roasting as used in this study and autoclaving were less effective in reducing the canavanine content. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Canavanine; Processing; Dabsyl derivative; HPLC; Sword beans
1. Introduction Sword beans (Canavalia gladiata) are one of many underutilized but exceptionally productive, large-seeded tropical legumes. The average yield under optimal agronomic management conditions can reach 720–1500 kg/ha (Bressani et al., 1987). Immature pods are consumed as a green vegetable in Sri Lanka and boiled seeds are widely consumed by tribal and village people in Southern India (Siddhuraju and Becker, 2001). In Indonesia seeds are consumed after cooking or fermenting, or are used in curries and as a substitute for mashed potatoes. Roasted, ground beans are used as a coffee substitute in Guatemala (Bressani et al., 1987). Dry * Corresponding author. Address: Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka. Tel.: +94 112 778943; fax: +94 112 852604. E-mail address: [email protected] (S. Ekanayake).
0278-6915/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.10.030
seeds were used in ancient times as a food or forage crop by natives of the south-western United States, Mexico and Central American countries (Sauer and Kaplan, 1969). Despite its nutritional potential in terms of protein content and quality (Eknayake et al., 1999; Ekanayake et al., 2003) sword beans are not commonly used as a food or cultivated like other legumes in Sri Lanka. The reason is partly the presence of antinutritional factors such as haemagglutinins (concanavalin A), protease inhibitors (Laurena et al., 1994), hydrocyanic acid (Kay, 1979), tannins, phytates and canavanine (Siddhuraju and Becker, 2001). Canavanine [2-amino-4-(guanidinoxy) butyric acid], a structural analogue of arginine, is a non-protein amino acid naturally occurring in legumes. The molecular structure of canavanine is shown in Fig. 1. The main function of canavanine is believed to be sequestering nitrogen for embryo growth since the nitrogen content is high, 31.8% (Rosenthal, 1970). A taxonomic survey has led to the
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Fig. 1. Molecular structure of canavanine.
discovery of 1500 species and 246 genera of legumes containing canavanine as a free amino acid (Turner and Harbone, 1967). These legumes may store prodigious amounts of canavanine (Rosenthal and Nkomo, 2000); in some species the canavanine content ranges from 10% to 13% of the seed dry weight and constitutes more than 95% of the free amino acid nitrogen (Rosenthal, 1977a). Canavanine is an arginine antagonist, able to manifest antimetabolic effects in viruses, bacteria and fungi, as well as in plants and animals by being incorporated in the cell nucleus and other proteins and interfering with DNA and RNA synthesis. Thus most regulatory and catalytic reactions of arginine metabolism, arginine uptake, formation of structural components and other cellular processes are mimicked (Rosenthal, 2001). In addition, induce formation of non-functional proteins in a variety of organisms (inhibit proteolytic processing of several prosecretory proteins), thus affecting plasma protein secretion (Michelangeli and Vargas, 1994). In macrophages and polymorphonuclear leukocytes, canavanine is able to prevent L-arginine derived synthesis of nitric oxide (Hrabak et al., 1994). Animal experiments have shown a number of deleterious effects following consumption of legumes containing canavanine, e.g. decreased feed intake and growth, a significant reduction in plasma concentration of basic amino acids (Herzberg et al., 1971; Rueda et al., 2003), and a reduction in plasma arginine, lysine and histidine. Biological manifestations in the adult rat included markedly elevated concentrations of ornithine in serum and urine when canavanine was administered at 2 g/kg body weight; urinary excretion of lysine + histidine and arginine was also enhanced (Thomas and Rosenthal, 1987a,b). Prolonged consumption of alfalfa seeds by humans has been associated with pancytopenia, anaemia, leuckopenia and the development of antinuclear antibodies, attributed to canavanine in the seeds (Montanaro and Bardana, 1991). The extent to which dietary canavanine exerts its antinutritional effect is not fully established. However, the antagonistic activity is observed only at low arginine concentrations (Swaffar et al., 1994). In contrast, animal studies suggest that canavanine has a marked potential as an anti-cancer agent (Thomas et al., 1986), and in combination with radiation may have clinical potential in the treatment of pancreatic cancer (Bence et al., 2002). Animal studies have also revealed that canavanine could be important in attenuation of diabetic retinopathy mainly by induction of molecular chaperone proteins (Mihaly et al., 1998).
Hydrolysis of canavanine forms canaline [2-amino-4(aminoxy) butyric acid], a structural analogue of ornithine (Thompson, 1980). Canaline is a vitamin B6 antagonist acting as a powerful inhibitor of pyridoxal-phosphate-dependent enzymes and may inhibit cell growth in plants (Swaffar and Ang, 1999). Thus, to increase the utilization of sword beans, i.e. to use sword beans as a food ingredient, it would be desirable to reduce the canavanine content. Complete elimination of canavanine in jack beans by soaking in alkaline solution followed by autoclaving (D’Mello and Walker, 1991) or 85–95% reduction by roasting at medium or high temperatures with a long exposure time (Melcion et al., 1998) has been reported. However, no reports are available on the effects of various household processing methods on canavanine content in sword beans. Spectrophotometric methods have been used for the analysis of canavanine, (Cacho et al., 1989; Rosenthal, 1977b) but these methods are time consuming and the stability of the reagent pentacyanoaminoferrate (PCAF) is limited to 48 h once prepared (Cacho et al., 1989). An HPLC method with pre-column derivatisation of canavanine with dansyl chloride and fluorescence detection has been successfully applied for the analysis of canavanine in plant tissues (Oropeza et al., 1988). However, the instability of the derivatives causes a rapid decline in fluorescence (Bianchi and Mazza, 1995). An amino acid analyser may be used for identification and quantification of canavanine (Ekanayake, unpublished data). However, long analysis time and the lack of instruments at most labs are serious drawbacks. Dabsyl chloride has been used as a pre-column derivatisation reagent for HPLC determination of biogenic amines with detection in the visible wavelength region (Lin and Chang, 1975; Lin and Lai, 1982; Bianchi and Mazza, 1995; Bartig and Klink, 1992; Krause et al., 1995; Romero et al., 2003). The dabsyl chloride method has not been reported for canavanine analysis, and we were interested in using this method for several reasons: (i) the stability of the derivatives at room temperature, (ii) detection in the visible region and (iii) high specificity and sensitivity, at pico-mole levels (Watanabe et al., 1992). In an animal study differently processed sword beans were fed to rats and it was speculated that canavanine could have been partially responsible for the low biological value that was observed for some diets (Ekanayake et al., 2003). The aim of the present study was therefore, to quantify canavanine in sword beans subjected to different household processing methods using the dabsyl chloride derivatisation method. 2. Materials and methods 2.1. Chemicals Solvents and chemicals were of HPLC or analytical grade. Water was passed through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Canavanine and caproic acid (internal standard) were
S. Ekanayake et al. / Food and Chemical Toxicology 45 (2007) 797–803 obtained from Sigma–Aldrich Sweden AB (Stockholm, Sweden). Acetonitrile was obtained from Merck, (KGaA, Germany) and Dabsyl chloride reagent was obtained from ICN Biomedical Inc., (OH, USA).
2.2. Samples Sword beans (C. gladiata) cultivated in Kandy, Sri Lanka, harvested in 2003 at full maturity were used for the experiments. The seeds were removed from the mature pods, air-dried, washed, oven-dried at 50 °C overnight (12 h) and decorticated. A portion of the cotyledons was made into flour (particle size <0.75 mm or 40–60 mesh: Cyclotec 1093, Tecator, Sweden) and another portion into grits (1–2 mm). The flour samples and the grits were stored in a desiccator at room temperature (20 °C) until processing and analysis.
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all samples were mixed with cold water (n = 3) or hot water (n = 3) and left at 20 °C for 14 h prior to extraction. Addition of hot water and soaking increased the yield of canavanine from raw flour but not of the other samples. In another preliminary experiment, extraction using alkaline conditions was performed, but this did not influence the yield. In the extraction method finally used 0.5–1.0 g of thoroughly homogenized flour samples was dispersed in 9 mL hot (95 °C) water in a 25 mL centrifuge vial, covered with parafilm and left overnight (14 h; 20 °C). Then, 1 mL 1 M HCl with the internal standard (590 mmol/L) was added and the samples were subjected to ultrasonic treatment (30 min) and centrifugation (1000g for 20 min). Part of the supernatant (1 mL) was mixed with 1 mL trichloroacetic acid (40% v/v), kept on ice for 10 min and centrifuged (2000g for 30 min). An aliquot (100 lL) of the deproteinised supernatant was completely dried under a flow of nitrogen.
2.3. Processing 2.6. Derivatisation with dabsyl chloride The samples (flour and grits) were processed in different ways as indicated in Table 1. The same processing methods were used as in a previous animal study (Ekanayake et al., 2003) with the addition of a soaked and drained grits sample. In Sample B grits were mixed with water which was completely absorbed (2 g grits to 3 mL water) and in Sample C grits were mixed with water in excess (1 g to 4 mL). Samples B and C were left at 20 °C for 15 h. The excess water in Sample C was drained off and for Sample D part of the grits from Sample C was boiled in excess water (1 g to 6 mL water) for 20 min. In Sample E grits were boiled in water (1 g to 7 mL water) for 20 min without the soaking step. In Sample F grits (1 g to 2 mL water) were autoclaved for 20 min at 121 °C at 1.5 atm. Samples B–F were then dried at 50 °C in a continuous air flow for 3.5 to 6 h. In Sample G, grits (250 g) and in Sample H, flour (250 g) were roasted at 150 °C for 30 min in closed tin containers (400 mL) attached to a rotating bar in an electric oven. The processed grits were made into flour as described above before analysis.
2.4. Basic analysis 2.4.1. Moisture Triplicates of all processed samples (2.0 g) were heated in an oven at 105 °C in pre-dried, cooled and pre-weighed crucibles, until a constant weight was attained (AOAC, 1984) and the moisture content calculated.
Derivatisation was performed according to the method of Krause et al. (1995) with minor modifications. The dried extracts or canavanine standards were dissolved in reaction buffer (0.15 mol/L NaHCO3, pH 8.6) and mixed with dabsyl chloride reagent (40 mg/10 mL acetone). The samples were incubated in a water bath at 70 °C for 20 min with intermediate mixing at 1, 5 and 12 min and kept on ice for 5–8 min. Dilution buffer (a mixture of acetonitrile, ethanol and HPLC buffer A) was added to each sample and mixed thoroughly. The samples and standards were then filtered through Millipore HV filters (0.45 lm) and injected into the HPLC or stored at 20 °C until analysis.
2.7. Effect of incubation time and temperature on derivatisation yield Preliminary experiments were carried out to study the influence of incubation time and temperature on the derivatisation yield. The same procedure as above was followed but the canavanine samples were incubated for 20 and 60 min at 20 and 70 °C. The highest yield was obtained when the reaction was carried out at 70 °C for 20 min, and these conditions were subsequently used in the study.
2.8. Extraction recovery and derivatisation yield 2.5. Extraction of canavanine from processed sword bean samples Preliminary experiments to determine the canavanine content according to Krause et al. (1995) indicated that the soaked samples had a higher canavanine content than the raw sword beans, and it was speculated that soaking increased the extractability of canavanine. Therefore,
Samples A and E (0.5 g), were mixed with internal standard (0.2 mL) and hot water (7.8 mL) in 25 mL tubes. The samples were spiked by mixing with canavanine standards (1 mL, 19 mg/mL or 1 mL 8.8 mg/mL) and the unspiked samples were mixed with 1 mL hot water. The samples were left at room temperature overnight. The volume was made upto
Table 1 Processing conditions and content of canavanine (expressed as % on dry matter basis) in sword bean samples (n = 5) Sample
Treatment
Grits/flour
A B C
Raw Soaked Soaked and drained Soaked, boiled and drained Boiled and drained Autoclaved Roasted Roasted
D E F G H
Soaking time (h)
Heating temperature (°C)
Heating time (min)
Drying time at 50 °C (h)
Canavanine (%)
Flour Grits Grits
15 15
– – –
– – –
– 3.5 4
4.1 ± 0.13c 4.0 ± 0.41c 3.7 ± 0.16d,c
Grits
15
100
20
6
2.1 ± 0.23f
Grits
–
100
20
6
2.7 ± 0.15b
Grits Grits Flour
– – –
121 150 150
20 30 30
6 – –
3.1 ± 0.10a 3.1 ± 0.14a,b 2.8 ± 0.38b
Values in columns with different superscripts differ significantly (p < 0.05). d Significantly different from roasted grits.
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10 mL by adding 1 M HCl. Extraction and derivatisation were performed as described above.
2.9. HPLC analysis An HPLC (Varian 9100) was coupled to an auto-sampler (Varian 9000) and a UV/VIS detector (Company X 206PHD). Varian Star software was used for system control, data collection and peak integration. The volume of the injection loop was 100 lL. The chromatographic conditions were adopted from Krause et al. (1995) and further optimised. The column (LiChrospher 100, RP-C18, 5 lm, length 25 cm, i.d. 4.6 mm, Merck KGaA) equipped with a guard cartridge was maintained at 50 °C (Eurotherm Thermostat). Mobile phase A consisted of NaH2PO4 (9 mmol), 4% dimethyl formamide and 0.2% triethylamine, pH adjusted to 6.55 with phosphoric acid. Mobile phase B consisted of aqueous acetonitrile (80% v/v). For best resolution with a short elution time, a gradient system was used starting at 40% B for 15 min and then increased to 58% B from 15 to 17 min, followed by an increase to 100% B from 17 to 42 min. The flow rate was 1.0 mL/min, the injection volume was 20 lL and the effluent was monitored at 436 nm. Identification and quantification were achieved by comparing retention times and peak areas with those from a standard sample run under the same conditions.
2.10. Quality parameters Raw and boiled samples (A and E) were each extracted and derivatised in triplicate on the same day for determination of within day variation and in duplicate for between day variation on three consecutive days. To check the linearity, eight standard solutions of canavanine, in the concentration range 0.025–50 lmol/mL, were derivatised on three consecutive days and injected twice on each day. For estimation of the detection limit, duplicate samples of standard solutions of canavanine (0.25–5.0 nmol/mL) were derivatised on two consecutive days.
Fig. 2. HPLC chromatogram (436 nm) of a standard canavanine sample. The peaks corresponding to excess hydrolysed dabsyl chloride (A) and canavanine are indicated by arrows (B and C).
Fig. 3. The peaks corresponding to canavanine and the internal standard are indicated by arrows. The peak at 26 min is large and well resolved. From the chromatogram it was apparent that the peaks corresponding to other amino acids were either absent or very small in comparison to the canavanine peaks. This indicates that canavanine is present at a higher quantity than other amino acids. Within day variation and day to day variation were assayed using Samples A and E and the results are presented in Table 2. The extraction efficiency in the raw
2.11. Statistical analysis Statistical evaluation was carried out using the MINITAB statistical software package. Significance was evaluated with a general linear model using Tukey’s multiple comparison tests.
3. Results 3.1. Canavanine analysis The chromatographic conditions used by Krause et al. (1995) were initially used to run standard canavanine samples and then further optimised. A chromatogram obtained from the analysis of standard canavanine is shown in Fig. 2. The broad peak eluting at 5 min corresponds to hydrolysed excess dabsyl chloride reagent. The standard canavanine samples gave rise to two peaks at retention times of 16 and 26 min, indicated by arrows in Fig. 2. The ratio between the peak areas was similar in the concentration range used. The standard curve was based on the peak eluting at 26 min due to the better resolution of this peak. A linear relationship (R2 = 0.9941) between peak area and canavanine concentration was observed over a range of 1–1000 nmol. The detection limit of canavanine was estimated to be between 100 and 150 pmol, from on a signal-to-noise ratio of more than 3. A chromatogram obtained from the analysis of canavanine in Sample E (soaked and boiled) is shown in
Fig. 3. HPLC chromatogram (436 nm) of a soaked and boiled sword bean sample. The peaks corresponding to the internal standard (B) and canavanine (A and C) are indicated by arrows.
Table 2 Data for the analysis of within day and between day variations (n = 3) Sample
A E
Within day variation
Between day variation
Mean canavanine content (mg/100 g)
CV
Mean canavanine content (mg/100 g)
CV
4141 2833
3.6 2.4
4137 2621
3.7 4.7
CV = coefficient of variation.
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and boiled samples following spiking indicated good recovery amounting to 91% and 88%, respectively. 3.2. Effect of processing The effects of processing on the canavanine content in sword beans are summarised in Table 1. Data are calculated on dry matter basis. The raw flour contained 4.1% canavanine. Soaking and draining did not significantly decrease the amount of canavanine, in contrast to heat-treated samples in which the decrease in canavanine was significant. The most significant decrease in canavanine, around 50%, was obtained in Sample D. Boiling without soaking (Sample E) decreased the canavanine content by 34%. Autoclaving (Sample F) decreased the canavanine content to a smaller extent, 24%. Roasting decreased the canavanine content of both grits and flour by 24–32% (Samples G and H). 4. Discussion Use of sword beans in food is desirable due to its high nutritional value (Eknayake et al., 1999). However, the presence of canavanine with its potential antinutritional effects calls for further studies on the possibility of reducing the canavanine content in sword beans. 4.1. Method development To investigate the effects of different cooking or processing methods on the canavanine content, it is necessary to have access to a reliable analytical method. The extraction procedure (Krause et al., 1995) was modified to include the addition of hot water and soaking, since results of preliminary investigations showed that soaked sword bean grits had a higher concentration of canavanine than raw flour. This modification of the method increased the extractability of canavanine from raw sword bean flour. Canavanine contains two primary amino groups which can undergo dabsylation to give rise to two analytes and hence two peaks in the HPLC chromatogram. The ratio between the areas of the two peaks was constant at the concentrations used in the present study. The peak at 26 min could easily be integrated from very low to high canavanine concentrations and had a better resolution than the peak that eluted at 16 min. Based on these observations we decided to use the second peak for construction of the standard curve and quantification. Several steps are needed for the analysis, and there is a risk that part of the sample will be lost especially during the extraction and derivatisation steps. Mean recoveries for raw and boiled samples were between 88% and 91%, indicating in general good recovery. The average coefficients of variation (Table 2) of within day variation and between day variation for raw flour and boiled grits were below 5%, which can be regarded as acceptable. The results show that the modified method of Krause et al. (1995); using extraction, dabsylation and HPLC sep-
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aration with visible detection, can be reliably applied to detect canavanine in plant materials at low concentrations with good recovery and precision. Several samples can be extracted and dabsylated in parallel and thus the number of samples that can be analysed per day is high. 4.2. Effect of processing on canavanine content The canavanine content of raw sword beans was 4.1% (Table 1). This value correlates well with values reported in the literature. Rosenthal and Nkomo (2000) found that the concentration of canavanine was 3–4%, while Siddhuraju and Becker (2001) reported 2.6% canavanine in raw sword beans. Despite the solubility of canavanine in water (D’Mello and Walker, 1991), our results show that soaking where all water was absorbed (Sample B) and soaking in excess water followed by draining (Sample C) were not effective methods of removing canavanine. This is in agreement with results from a study where soaking and irradiation of other legumes, e.g. Sesbania spp., did not effectively decrease the canavanine content (Siddhuraju et al., 2002). The present study shows that in order to achieve a significant reduction of canavanine, a heating step is necessary. Soaking and boiling of grits significantly decreased the canavanine content (by about 50%). When samples were boiled without soaking (Sample E), the decrease was less pronounced. The most probable explanation is that soaking for several hours facilitates cell rupture and leaching, and thus the removal of canavanine with the cooking water. Earlier animal study has shown that soaking and boiling results in improved protein quality, probably due to a reduction of antinutritional factors including canavanine (Ekanayake et al., 2003). However, further boiling of grits in water led to the formation of a gelatinized mass, which hindered the decanting process and thus removal of solubilised canavanine. Therefore, the use of intense wet heat treatment is limited so as to preserve the structure of the grits. Such grits could be used in food after drying, i.e. as grits or flour. Autoclaving (pressure cooking) of grits significantly decreased the canavanine content by 24%. This indicates that, unlike protease inhibitors and concanavalin A in legumes, which can be completely eliminated by autoclaving (Agbede and Aletor, 2005), canavanine resists complete degradation by heating under pressure. Roasting sword bean flour (Sample H) was more effective in decreasing canavanine than roasting grits (Sample G), and the smaller particle size of the flour could be the reason behind this. This is in accordance with results from another study where the decrease in canavanine content was 35% in jack beans roasted at 150 °C for 27 min. However, a 95% decrease was obtained after roasting at 164 °C for 26 min (Leon et al., 1998). The decrease could be due to the participation of canavanine in Maillard reaction and also oxidation at high temperature used for roasting. To further explore the possibility of increasing the use of sword beans in foods, greater canavanine reduction by
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roasting at increased temperature (Melcion et al., 1998), perhaps at the cost of the biological value, may be considered, and also removal of antinutritional factors using KHCO3 (D’Mello and Walker, 1991) and urea, (Udedibie et al., 1994). Acid extraction of heat treated beans could also remove a higher amount of canavanine from the seeds and is another possibility for further studies.
0.4–0.5 g. For a 60 kg person this would correspond to less than 0.01 g canavanine/kg body weight, which can be compared with the 0.7–1.5 g canavanine/kg body weight calculated from the results in animal studies discussed. Application of a safety factor of 100 which is normally used in ADI estimates, suggests that soaked and boiled sword beans may be safely used as an ingredient in food.
4.3. Toxicological aspects of canavanine
5. Conclusions
The diets of humans are complex mixtures of compounds, some of which can be harmful and some of which can have protective effects. Studies have shown that canavanine in legumes can have harmful as well as beneficial effects. Inhibition of arginine uptake in isolated brush border membranes of chicks has been reported at a canavanine concentration of 0.5 mmol (Rueda et al., 2003). Development of a condition that mimics systemic lupus-like disease in primates ingesting canavanine-containing alfalfa seeds has been attributed to canavanine (Montanaro and Bardana, 1991). On the other hand, a diet containing 1% canavanine was shown to increase longevity in mice fed a highprotein diet (23.4%) (Brown et al., 2000). With a moderate protein diet (15.7%) the life span on a 1% canavanine diet was the same as the control group (Brown, 2005). When soaked sword bean grits were fed to rats, a low biological value of the protein was obtained and it was speculated that canavanine could have been partially responsible for this (Ekanayake et al., 2003). Our results on the content of canavanine in soaked sword beans support this speculation. The highest weight gain, 72%, compared with the controls was observed for rats fed soaked and boiled sword bean grits, i.e. those with the most pronounced reduction in canavanine. The intake of canavanine corresponded to 0.7 g canavanine/kg body weight/ day. This suggests that a daily intake of canavanine at this level does not cause short-term adverse effects in rats. In the studies on life-long supply of diets containing 1% canavanine in mice showing prolonged or unaltered life span (Brown et al., 2000; Brown, 2005), the canavanine dose can be estimated to be 1.5 g/kg body weight/day (assuming a daily feed intake corresponding to 15% of the body weight). When poultry were fed diets containing jack beans roasted at 164 °C for 26 min, the weight gain was 89% of the controls (canavanine intake 0.2 g/day), while those fed jack beans roasted at 150 °C, showed only a 35% weight gain (canavanine intake 0.38 g/day) (Leon et al., 1998). The lower weight gain was attributed to reduced feed intake (40%), but as jack beans were not the only source of protein, the canavanine content could be an important factor explaining the decreased protein quality. The dietary level of canavanine that can be considered safe in humans cannot be determined without further studies. An ordinary dish may contain around 25 g beans, and thus if a daily intake of 20–25 g soaked and boiled sword beans is assumed, the intake of canavanine would be
In conclusion, the analytical HPLC method using precolumn dabsyl derivatisation and visible detection is sensitive, specific and can successfully be applied for the detection and quantification of canavanine in legumes, and presumably other plant materials. The different household processing methods involving heat treatment caused a significant reduction in canavanine, thus indicating the importance of heating. The maximum reduction, around 50%, was obtained by decorticating the seeds, making grits, soaking and boiling them for 20 min and decanting the cooking water. Recent animal studies, however, do not confirm acute or chronic toxicity of canavanine, at levels consumed when sword beans are incorporated in the diets indicating that the canavanine content in sword beans prepared as above would not prevent them from being used in food. However, more data are needed to determine safe levels of canavanine intake for humans. Conflict of interest statements for all authors None. Acknowledgements The authors thank Mia Malmquist for technical support and Dr. Roberto Romero for valuable advice on analytical issues. The financial support of the International Science Program (IPICS), Uppsala University, Sweden for Grant SRI: 07 is gratefully acknowledged. References Agbede, J.O., Aletor, V.A., 2005. Studies of the chemical composition and protein quality evaluation of differently processed Canavalia ensiformis and Mucuna pruriens seed flours. Journal of Food Composition and Analysis 18, 89–103. Association of Official Analytical Chemists (AOAC), 1984. Official methods of analysis (14th ed.) 14. 009, Virginia, DC USA. Bartig, D., Klink, F., 1992. Determination of the unusual amino acid hypusine at the picomole level by derivatization with 4-dimethylaminoazobenzene-4 0 -sulfonyl chloride and reversed phase high performance or medium pressure liquid chromatography. Journal of Chromatography 606, 43–48. Bence, A.K., Worthen, D.R., Adams, V.R., Crooks, P.A., 2002. The anitproliferative and immunotoxic effects of L-canavanine and L-canaline. Anti Cancer Drugs 13, 313–320. Bianchi, V., Mazza, L., 1995. Rapid reverse-phase high-performance liquid chromatographic method with double derivatization for the assay of urinary hydroxyproline. Journal of Chromatography 665, 295–302.
S. Ekanayake et al. / Food and Chemical Toxicology 45 (2007) 797–803 Bressani, R., Gomez, B.R., Garcia, A., Eliaz, L.G., 1987. Chemical composition, amino acid content and protein quality of Canavalia seeds. Journal of Science Food and Agriculture 40, 17–23. Brown, D., 2005. Canavanine induced longevity in mice may require diets with greater than 15.7% protein. Nutrition and Metabolism 2, 7. Brown, D.L., Naiki, M., Gershwin, M.E., 2000. Does L-canavanine ingestion induce murine SLE? Paradoxical effects on survival of BALB/c mice. Journal of Nutritional Immunology 5, 17–27. Cacho, J., Angeles, G., Ignacio, F., 1989. Selective spectrophotometric determination of canavanine. Analyst 114, 965–968. D’Mello, J.P.F., Walker, A.G., 1991. Detoxification of jack beans (Canavalia ensiformis): Studies with young chicks. Animal Feed Science and Technology 33, 117–127. Ekanayake, S., Nair, M.B., Asp, N.-G., Jansz, E.R., 2003. Effect of processing on protein nutritional quality of Canavalia gladiata. Nahrung/Food 47, 256–260. Eknayake, S., Jansz, E.R., Nair, B.M., 1999. Proximate composition, mineral and amino acid composition of Canavalia gladiata. Food Chemistry 66, 115–119. Herzberg, G., Sheerin, H., Lerner, J., 1971. Cationic amino acid transport in chicken small intestine. Comparative Biochemistry and Physiology 40A, 229–247. Hrabak, A., Bajor, T., Temesi, A., 1994. Comparison of substrate and inhibitor specificity of arginase and nitric oxide (NO) synthase for arginine analogues and related compounds in murine and rat macrophages. Biochemical and Biophysical Research Communications 198, 206–212. Kay, E.D., 1979. Food Legumes. TIP Crop and Product Digest No. 3. Tropical Products Institute, London, XVI + 435 pp. Krause, I., Bockhardt, A., Neckermann, H., Henle, T., Klastermeyer, H., 1995. Simultaneous determination of amino acid and biogenic amines by reversed phase high performance liquid chromatography of the dabsyl derivatives. Journal of Chromatography 715, 67–79. Laurena, A.C., Revilleza, M.J.R., Mendoza, E.M.T., 1994. Polyphenols, phytate, cyanogenic glycosides and trypsin inhibitor activity of several Philippine indigenous food legumes. Journal of Food Composition and Analysis 7, 194–202. Leon, A., Vargas, R.E., Michelangeli, C., Melcion, J.P., Picard, M., 1998. Detoxification of jack bean (Canavalia ensiformis) with pilot scale roasting. II. Nutritional value for poultry. Animal Feed Science and Technology 73, 231–242. Lin, J., Chang, J., 1975. Chromophoric labelling of amino acids with 4-dimethylaminoazobenzene-4 0 -sulfonyl chloride. Analytical Chemistry 47, 1634–1638. Lin, J., Lai, C., 1982. Chromophoric determination of putrescine, spermidine and spermine with dabsyl chloride by HPLC and TLC. Journal of Chromatography 227, 369–377. Melcion, J.P., Kodaira, M., Leon, A., Michelangeli, C., Vargas, R.E., Picard, M., 1998. Detoxification of the jack bean (Canavalia ensiformis L.) with pilot scale roasting. I. Technological conditions and analytical data. Animal Feed Science and Technology 73, 217–230. Michelangeli, C., Vargas, R.E., 1994. L-canavanine influences feed intake, plasma basic amino acid concentrations and kidney arginase activity in chicks. Journal of Nutrition 124, 1081–1087. Mihaly, K., Toth, S., Szlavik, L., Toth, A., Csermely, P., 1998. Attenuation of diabetic retinopathy by the molecular chaperone inducer amino acid analogue canavanine in streptozytocin diabetic rats. Cellular and Molecular Life Sciences 54, 1154–1160. Montanaro, A., Bardana, E.J., 1991. Dietary amino acid-induced systemic lupus erythematosus. Rheumatic Disease Clinics of North America 17, 323–332.
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Oropeza, C., Alpizar, L., Victo, M., Vargas, L., Quiroz, J., Scorer, K.N., 1988. Determination of L-canavanine and L-canaline in plant tissues by high performance liquid chromatography. Journal of Chromatography 456, 405–409. Romero, R., Bagur, M.G., Vinas, M.S., Gazques, D., 2003. The influence of the brewing process on the formation of biogenic amines in beers. Analytical and Bioanalytical Chemistry 376, 162–167. Rosenthal, G.A., 1970. Investigation of canavanine biochemistry in the jack bean plant, Canavalia ensiformis (L.) DC.I. Canavanine utilization in the developing plant. Plant Physiology 46, 273–276. Rosenthal, G.A., 1977a. Nitrogen allocation for L-canavanine synthesis and its relation to chemical defense of the seed. Biochemical Systematics and Ecology 5, 19–20. Rosenthal, G.A., 1977b. Preparation and colorimetric analysis of L-canavanine. Analytical Biochemistry 77, 147–151. Rosenthal, G.A., 2001. L-canavanine: A higher plant insecticidal allelochemical. Amino Acids 21, 319–330. Rosenthal, G.A., Nkomo, P., 2000. The natural abundanbce of L-canavanine, an active anticancer agent in alfalfa, Medicago sativa (L.). Pharmaceutical Biology 38, 1–6. Rueda, E., Michelangeli, C., Gonzaez-Mujica, F., 2003. L-canavanine inhibits L-arginine uptake by broiler chicken intestinal brush border membrane vesicles. British Poultry Science 44, 620–625. Sauer, J., Kaplan, L., 1969. Canavalia beans in American prehistory. American Antiquity 34, 417–424. Siddhuraju, P., Becker, K., 2001. Species/variety differences in biochemical composition and nutritional values of Indian tribal legumes of genus Canavalia. Nahrung/Food 45, 224–233. Siddhuraju, P., Osoniyi, O., Makkar, H.P.S., Becker, K., 2002. Effects of soaking and ionising radiation on various antinutritional factors of seeds from different species of an unconventional legume, Sesbania and a common legume, green gram (Vigna radiata). Food Chemistry 79, 273–281. Swaffar, D.S., Ang, C.Y., 1999. Growth inhibitory effect of L-canavanine against MIA paca-2 pancreatic cancer cells is not due to conversion of its toxic metabolite canaline. Anti Cancer Drugs 10, 113–118. Swaffar, D.S., Ang, C.Y., Desai, P.B., Rosenthal, G.A., 1994. Inhibition of growth of human pancreatic cancer cells by arginine antimetabolite L-canavanine. Cancer Research 54, 6045–6048. Thomas, D.A., Rosenthal, G.A., 1987a. Toxicity and pharmacokinetics of the nonprotein amino acid L-canavanine in the rat. Toxicology and Applied Pharmacology 91, 395–405. Thomas, D.A., Rosenthal, G.A., 1987b. Metabolism of L-[guanidinooxy14] canavanine in the rat. Toxicology and Applied Pharmacology 91, 406–414. Thomas, D.A., Rosenthal, G.A., Gold, D.V., Dickey, K., 1986. Growth inhibition of a rat colon tumor by L-canavanine. Cancer Research 46, 2898–2903. Thompson, J.F., 1980. The Biochemistry of Plants. A Comprehensive Treatise. Academic Press, New York. Turner, Harbone, J.B., 1967. Distribution of canavanine in the plant kingdom. Phytochemistry 6, 863–866. Udedibie, A.B.I., Esonu, B.O., Obaji, C.N., Durunna, C.S., 1994. Dry urea treatment prior to toasting as a method of improving the nutritive value of jackbeans (C. ensiformis) for broilers. Animal Feed Science and Technology 48, 335–345. Watanabe, A., Semba, J., Kurumaji, S., Kumashiro, S., Toru, M., 1992. Measurement of Glutamate, aspartate and glycine and its potential precursors in human brain using high-performance liquid chromatography by pre-column derivatization with diethylaminobenzene sulphonyl chloride. Journal of Chromatography 583, 241–245.
Canavanine content in sword beans (Canavalia gladiata): Analysis and effect of processing S. Ekanayake a
a,b,*
, K. Skog b, N.-G. Asp
b
Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka b Department of Food Technology, Engineering and Nutrition, Division of Applied Nutrition and Food Chemistry, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received 8 September 2005; accepted 30 October 2006
Abstract The amino acid canavanine is a potentially toxic constituent of leguminous seeds. The aim of the present study was to determine the ability of different processing methods to reduce canavanine in sword beans (Canavalia gladiata). For this purpose a method for the detection and quantification of canavanine was developed using reversed-phase high-performance liquid chromatography of the dabsylated derivatives. The recovery of canavanine using this method was 88–91%. Optimum extraction of canavanine from raw and processed beans was obtained by addition of hot water prior to overnight soaking. The results obtained with this method agree well with previously published values for raw seeds. The method is sensitive, specific and can successfully be applied to the detection of canavanine in legumes. Overnight soaking and boiling in excess water followed by decanting gave the most pronounced reduction in canavanine content (around 50%), followed by boiling and decanting excess water (34%). Roasting as used in this study and autoclaving were less effective in reducing the canavanine content. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Canavanine; Processing; Dabsyl derivative; HPLC; Sword beans
1. Introduction Sword beans (Canavalia gladiata) are one of many underutilized but exceptionally productive, large-seeded tropical legumes. The average yield under optimal agronomic management conditions can reach 720–1500 kg/ha (Bressani et al., 1987). Immature pods are consumed as a green vegetable in Sri Lanka and boiled seeds are widely consumed by tribal and village people in Southern India (Siddhuraju and Becker, 2001). In Indonesia seeds are consumed after cooking or fermenting, or are used in curries and as a substitute for mashed potatoes. Roasted, ground beans are used as a coffee substitute in Guatemala (Bressani et al., 1987). Dry * Corresponding author. Address: Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka. Tel.: +94 112 778943; fax: +94 112 852604. E-mail address: [email protected] (S. Ekanayake).
0278-6915/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.10.030
seeds were used in ancient times as a food or forage crop by natives of the south-western United States, Mexico and Central American countries (Sauer and Kaplan, 1969). Despite its nutritional potential in terms of protein content and quality (Eknayake et al., 1999; Ekanayake et al., 2003) sword beans are not commonly used as a food or cultivated like other legumes in Sri Lanka. The reason is partly the presence of antinutritional factors such as haemagglutinins (concanavalin A), protease inhibitors (Laurena et al., 1994), hydrocyanic acid (Kay, 1979), tannins, phytates and canavanine (Siddhuraju and Becker, 2001). Canavanine [2-amino-4-(guanidinoxy) butyric acid], a structural analogue of arginine, is a non-protein amino acid naturally occurring in legumes. The molecular structure of canavanine is shown in Fig. 1. The main function of canavanine is believed to be sequestering nitrogen for embryo growth since the nitrogen content is high, 31.8% (Rosenthal, 1970). A taxonomic survey has led to the
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Fig. 1. Molecular structure of canavanine.
discovery of 1500 species and 246 genera of legumes containing canavanine as a free amino acid (Turner and Harbone, 1967). These legumes may store prodigious amounts of canavanine (Rosenthal and Nkomo, 2000); in some species the canavanine content ranges from 10% to 13% of the seed dry weight and constitutes more than 95% of the free amino acid nitrogen (Rosenthal, 1977a). Canavanine is an arginine antagonist, able to manifest antimetabolic effects in viruses, bacteria and fungi, as well as in plants and animals by being incorporated in the cell nucleus and other proteins and interfering with DNA and RNA synthesis. Thus most regulatory and catalytic reactions of arginine metabolism, arginine uptake, formation of structural components and other cellular processes are mimicked (Rosenthal, 2001). In addition, induce formation of non-functional proteins in a variety of organisms (inhibit proteolytic processing of several prosecretory proteins), thus affecting plasma protein secretion (Michelangeli and Vargas, 1994). In macrophages and polymorphonuclear leukocytes, canavanine is able to prevent L-arginine derived synthesis of nitric oxide (Hrabak et al., 1994). Animal experiments have shown a number of deleterious effects following consumption of legumes containing canavanine, e.g. decreased feed intake and growth, a significant reduction in plasma concentration of basic amino acids (Herzberg et al., 1971; Rueda et al., 2003), and a reduction in plasma arginine, lysine and histidine. Biological manifestations in the adult rat included markedly elevated concentrations of ornithine in serum and urine when canavanine was administered at 2 g/kg body weight; urinary excretion of lysine + histidine and arginine was also enhanced (Thomas and Rosenthal, 1987a,b). Prolonged consumption of alfalfa seeds by humans has been associated with pancytopenia, anaemia, leuckopenia and the development of antinuclear antibodies, attributed to canavanine in the seeds (Montanaro and Bardana, 1991). The extent to which dietary canavanine exerts its antinutritional effect is not fully established. However, the antagonistic activity is observed only at low arginine concentrations (Swaffar et al., 1994). In contrast, animal studies suggest that canavanine has a marked potential as an anti-cancer agent (Thomas et al., 1986), and in combination with radiation may have clinical potential in the treatment of pancreatic cancer (Bence et al., 2002). Animal studies have also revealed that canavanine could be important in attenuation of diabetic retinopathy mainly by induction of molecular chaperone proteins (Mihaly et al., 1998).
Hydrolysis of canavanine forms canaline [2-amino-4(aminoxy) butyric acid], a structural analogue of ornithine (Thompson, 1980). Canaline is a vitamin B6 antagonist acting as a powerful inhibitor of pyridoxal-phosphate-dependent enzymes and may inhibit cell growth in plants (Swaffar and Ang, 1999). Thus, to increase the utilization of sword beans, i.e. to use sword beans as a food ingredient, it would be desirable to reduce the canavanine content. Complete elimination of canavanine in jack beans by soaking in alkaline solution followed by autoclaving (D’Mello and Walker, 1991) or 85–95% reduction by roasting at medium or high temperatures with a long exposure time (Melcion et al., 1998) has been reported. However, no reports are available on the effects of various household processing methods on canavanine content in sword beans. Spectrophotometric methods have been used for the analysis of canavanine, (Cacho et al., 1989; Rosenthal, 1977b) but these methods are time consuming and the stability of the reagent pentacyanoaminoferrate (PCAF) is limited to 48 h once prepared (Cacho et al., 1989). An HPLC method with pre-column derivatisation of canavanine with dansyl chloride and fluorescence detection has been successfully applied for the analysis of canavanine in plant tissues (Oropeza et al., 1988). However, the instability of the derivatives causes a rapid decline in fluorescence (Bianchi and Mazza, 1995). An amino acid analyser may be used for identification and quantification of canavanine (Ekanayake, unpublished data). However, long analysis time and the lack of instruments at most labs are serious drawbacks. Dabsyl chloride has been used as a pre-column derivatisation reagent for HPLC determination of biogenic amines with detection in the visible wavelength region (Lin and Chang, 1975; Lin and Lai, 1982; Bianchi and Mazza, 1995; Bartig and Klink, 1992; Krause et al., 1995; Romero et al., 2003). The dabsyl chloride method has not been reported for canavanine analysis, and we were interested in using this method for several reasons: (i) the stability of the derivatives at room temperature, (ii) detection in the visible region and (iii) high specificity and sensitivity, at pico-mole levels (Watanabe et al., 1992). In an animal study differently processed sword beans were fed to rats and it was speculated that canavanine could have been partially responsible for the low biological value that was observed for some diets (Ekanayake et al., 2003). The aim of the present study was therefore, to quantify canavanine in sword beans subjected to different household processing methods using the dabsyl chloride derivatisation method. 2. Materials and methods 2.1. Chemicals Solvents and chemicals were of HPLC or analytical grade. Water was passed through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Canavanine and caproic acid (internal standard) were
S. Ekanayake et al. / Food and Chemical Toxicology 45 (2007) 797–803 obtained from Sigma–Aldrich Sweden AB (Stockholm, Sweden). Acetonitrile was obtained from Merck, (KGaA, Germany) and Dabsyl chloride reagent was obtained from ICN Biomedical Inc., (OH, USA).
2.2. Samples Sword beans (C. gladiata) cultivated in Kandy, Sri Lanka, harvested in 2003 at full maturity were used for the experiments. The seeds were removed from the mature pods, air-dried, washed, oven-dried at 50 °C overnight (12 h) and decorticated. A portion of the cotyledons was made into flour (particle size <0.75 mm or 40–60 mesh: Cyclotec 1093, Tecator, Sweden) and another portion into grits (1–2 mm). The flour samples and the grits were stored in a desiccator at room temperature (20 °C) until processing and analysis.
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all samples were mixed with cold water (n = 3) or hot water (n = 3) and left at 20 °C for 14 h prior to extraction. Addition of hot water and soaking increased the yield of canavanine from raw flour but not of the other samples. In another preliminary experiment, extraction using alkaline conditions was performed, but this did not influence the yield. In the extraction method finally used 0.5–1.0 g of thoroughly homogenized flour samples was dispersed in 9 mL hot (95 °C) water in a 25 mL centrifuge vial, covered with parafilm and left overnight (14 h; 20 °C). Then, 1 mL 1 M HCl with the internal standard (590 mmol/L) was added and the samples were subjected to ultrasonic treatment (30 min) and centrifugation (1000g for 20 min). Part of the supernatant (1 mL) was mixed with 1 mL trichloroacetic acid (40% v/v), kept on ice for 10 min and centrifuged (2000g for 30 min). An aliquot (100 lL) of the deproteinised supernatant was completely dried under a flow of nitrogen.
2.3. Processing 2.6. Derivatisation with dabsyl chloride The samples (flour and grits) were processed in different ways as indicated in Table 1. The same processing methods were used as in a previous animal study (Ekanayake et al., 2003) with the addition of a soaked and drained grits sample. In Sample B grits were mixed with water which was completely absorbed (2 g grits to 3 mL water) and in Sample C grits were mixed with water in excess (1 g to 4 mL). Samples B and C were left at 20 °C for 15 h. The excess water in Sample C was drained off and for Sample D part of the grits from Sample C was boiled in excess water (1 g to 6 mL water) for 20 min. In Sample E grits were boiled in water (1 g to 7 mL water) for 20 min without the soaking step. In Sample F grits (1 g to 2 mL water) were autoclaved for 20 min at 121 °C at 1.5 atm. Samples B–F were then dried at 50 °C in a continuous air flow for 3.5 to 6 h. In Sample G, grits (250 g) and in Sample H, flour (250 g) were roasted at 150 °C for 30 min in closed tin containers (400 mL) attached to a rotating bar in an electric oven. The processed grits were made into flour as described above before analysis.
2.4. Basic analysis 2.4.1. Moisture Triplicates of all processed samples (2.0 g) were heated in an oven at 105 °C in pre-dried, cooled and pre-weighed crucibles, until a constant weight was attained (AOAC, 1984) and the moisture content calculated.
Derivatisation was performed according to the method of Krause et al. (1995) with minor modifications. The dried extracts or canavanine standards were dissolved in reaction buffer (0.15 mol/L NaHCO3, pH 8.6) and mixed with dabsyl chloride reagent (40 mg/10 mL acetone). The samples were incubated in a water bath at 70 °C for 20 min with intermediate mixing at 1, 5 and 12 min and kept on ice for 5–8 min. Dilution buffer (a mixture of acetonitrile, ethanol and HPLC buffer A) was added to each sample and mixed thoroughly. The samples and standards were then filtered through Millipore HV filters (0.45 lm) and injected into the HPLC or stored at 20 °C until analysis.
2.7. Effect of incubation time and temperature on derivatisation yield Preliminary experiments were carried out to study the influence of incubation time and temperature on the derivatisation yield. The same procedure as above was followed but the canavanine samples were incubated for 20 and 60 min at 20 and 70 °C. The highest yield was obtained when the reaction was carried out at 70 °C for 20 min, and these conditions were subsequently used in the study.
2.8. Extraction recovery and derivatisation yield 2.5. Extraction of canavanine from processed sword bean samples Preliminary experiments to determine the canavanine content according to Krause et al. (1995) indicated that the soaked samples had a higher canavanine content than the raw sword beans, and it was speculated that soaking increased the extractability of canavanine. Therefore,
Samples A and E (0.5 g), were mixed with internal standard (0.2 mL) and hot water (7.8 mL) in 25 mL tubes. The samples were spiked by mixing with canavanine standards (1 mL, 19 mg/mL or 1 mL 8.8 mg/mL) and the unspiked samples were mixed with 1 mL hot water. The samples were left at room temperature overnight. The volume was made upto
Table 1 Processing conditions and content of canavanine (expressed as % on dry matter basis) in sword bean samples (n = 5) Sample
Treatment
Grits/flour
A B C
Raw Soaked Soaked and drained Soaked, boiled and drained Boiled and drained Autoclaved Roasted Roasted
D E F G H
Soaking time (h)
Heating temperature (°C)
Heating time (min)
Drying time at 50 °C (h)
Canavanine (%)
Flour Grits Grits
15 15
– – –
– – –
– 3.5 4
4.1 ± 0.13c 4.0 ± 0.41c 3.7 ± 0.16d,c
Grits
15
100
20
6
2.1 ± 0.23f
Grits
–
100
20
6
2.7 ± 0.15b
Grits Grits Flour
– – –
121 150 150
20 30 30
6 – –
3.1 ± 0.10a 3.1 ± 0.14a,b 2.8 ± 0.38b
Values in columns with different superscripts differ significantly (p < 0.05). d Significantly different from roasted grits.
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10 mL by adding 1 M HCl. Extraction and derivatisation were performed as described above.
2.9. HPLC analysis An HPLC (Varian 9100) was coupled to an auto-sampler (Varian 9000) and a UV/VIS detector (Company X 206PHD). Varian Star software was used for system control, data collection and peak integration. The volume of the injection loop was 100 lL. The chromatographic conditions were adopted from Krause et al. (1995) and further optimised. The column (LiChrospher 100, RP-C18, 5 lm, length 25 cm, i.d. 4.6 mm, Merck KGaA) equipped with a guard cartridge was maintained at 50 °C (Eurotherm Thermostat). Mobile phase A consisted of NaH2PO4 (9 mmol), 4% dimethyl formamide and 0.2% triethylamine, pH adjusted to 6.55 with phosphoric acid. Mobile phase B consisted of aqueous acetonitrile (80% v/v). For best resolution with a short elution time, a gradient system was used starting at 40% B for 15 min and then increased to 58% B from 15 to 17 min, followed by an increase to 100% B from 17 to 42 min. The flow rate was 1.0 mL/min, the injection volume was 20 lL and the effluent was monitored at 436 nm. Identification and quantification were achieved by comparing retention times and peak areas with those from a standard sample run under the same conditions.
2.10. Quality parameters Raw and boiled samples (A and E) were each extracted and derivatised in triplicate on the same day for determination of within day variation and in duplicate for between day variation on three consecutive days. To check the linearity, eight standard solutions of canavanine, in the concentration range 0.025–50 lmol/mL, were derivatised on three consecutive days and injected twice on each day. For estimation of the detection limit, duplicate samples of standard solutions of canavanine (0.25–5.0 nmol/mL) were derivatised on two consecutive days.
Fig. 2. HPLC chromatogram (436 nm) of a standard canavanine sample. The peaks corresponding to excess hydrolysed dabsyl chloride (A) and canavanine are indicated by arrows (B and C).
Fig. 3. The peaks corresponding to canavanine and the internal standard are indicated by arrows. The peak at 26 min is large and well resolved. From the chromatogram it was apparent that the peaks corresponding to other amino acids were either absent or very small in comparison to the canavanine peaks. This indicates that canavanine is present at a higher quantity than other amino acids. Within day variation and day to day variation were assayed using Samples A and E and the results are presented in Table 2. The extraction efficiency in the raw
2.11. Statistical analysis Statistical evaluation was carried out using the MINITAB statistical software package. Significance was evaluated with a general linear model using Tukey’s multiple comparison tests.
3. Results 3.1. Canavanine analysis The chromatographic conditions used by Krause et al. (1995) were initially used to run standard canavanine samples and then further optimised. A chromatogram obtained from the analysis of standard canavanine is shown in Fig. 2. The broad peak eluting at 5 min corresponds to hydrolysed excess dabsyl chloride reagent. The standard canavanine samples gave rise to two peaks at retention times of 16 and 26 min, indicated by arrows in Fig. 2. The ratio between the peak areas was similar in the concentration range used. The standard curve was based on the peak eluting at 26 min due to the better resolution of this peak. A linear relationship (R2 = 0.9941) between peak area and canavanine concentration was observed over a range of 1–1000 nmol. The detection limit of canavanine was estimated to be between 100 and 150 pmol, from on a signal-to-noise ratio of more than 3. A chromatogram obtained from the analysis of canavanine in Sample E (soaked and boiled) is shown in
Fig. 3. HPLC chromatogram (436 nm) of a soaked and boiled sword bean sample. The peaks corresponding to the internal standard (B) and canavanine (A and C) are indicated by arrows.
Table 2 Data for the analysis of within day and between day variations (n = 3) Sample
A E
Within day variation
Between day variation
Mean canavanine content (mg/100 g)
CV
Mean canavanine content (mg/100 g)
CV
4141 2833
3.6 2.4
4137 2621
3.7 4.7
CV = coefficient of variation.
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and boiled samples following spiking indicated good recovery amounting to 91% and 88%, respectively. 3.2. Effect of processing The effects of processing on the canavanine content in sword beans are summarised in Table 1. Data are calculated on dry matter basis. The raw flour contained 4.1% canavanine. Soaking and draining did not significantly decrease the amount of canavanine, in contrast to heat-treated samples in which the decrease in canavanine was significant. The most significant decrease in canavanine, around 50%, was obtained in Sample D. Boiling without soaking (Sample E) decreased the canavanine content by 34%. Autoclaving (Sample F) decreased the canavanine content to a smaller extent, 24%. Roasting decreased the canavanine content of both grits and flour by 24–32% (Samples G and H). 4. Discussion Use of sword beans in food is desirable due to its high nutritional value (Eknayake et al., 1999). However, the presence of canavanine with its potential antinutritional effects calls for further studies on the possibility of reducing the canavanine content in sword beans. 4.1. Method development To investigate the effects of different cooking or processing methods on the canavanine content, it is necessary to have access to a reliable analytical method. The extraction procedure (Krause et al., 1995) was modified to include the addition of hot water and soaking, since results of preliminary investigations showed that soaked sword bean grits had a higher concentration of canavanine than raw flour. This modification of the method increased the extractability of canavanine from raw sword bean flour. Canavanine contains two primary amino groups which can undergo dabsylation to give rise to two analytes and hence two peaks in the HPLC chromatogram. The ratio between the areas of the two peaks was constant at the concentrations used in the present study. The peak at 26 min could easily be integrated from very low to high canavanine concentrations and had a better resolution than the peak that eluted at 16 min. Based on these observations we decided to use the second peak for construction of the standard curve and quantification. Several steps are needed for the analysis, and there is a risk that part of the sample will be lost especially during the extraction and derivatisation steps. Mean recoveries for raw and boiled samples were between 88% and 91%, indicating in general good recovery. The average coefficients of variation (Table 2) of within day variation and between day variation for raw flour and boiled grits were below 5%, which can be regarded as acceptable. The results show that the modified method of Krause et al. (1995); using extraction, dabsylation and HPLC sep-
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aration with visible detection, can be reliably applied to detect canavanine in plant materials at low concentrations with good recovery and precision. Several samples can be extracted and dabsylated in parallel and thus the number of samples that can be analysed per day is high. 4.2. Effect of processing on canavanine content The canavanine content of raw sword beans was 4.1% (Table 1). This value correlates well with values reported in the literature. Rosenthal and Nkomo (2000) found that the concentration of canavanine was 3–4%, while Siddhuraju and Becker (2001) reported 2.6% canavanine in raw sword beans. Despite the solubility of canavanine in water (D’Mello and Walker, 1991), our results show that soaking where all water was absorbed (Sample B) and soaking in excess water followed by draining (Sample C) were not effective methods of removing canavanine. This is in agreement with results from a study where soaking and irradiation of other legumes, e.g. Sesbania spp., did not effectively decrease the canavanine content (Siddhuraju et al., 2002). The present study shows that in order to achieve a significant reduction of canavanine, a heating step is necessary. Soaking and boiling of grits significantly decreased the canavanine content (by about 50%). When samples were boiled without soaking (Sample E), the decrease was less pronounced. The most probable explanation is that soaking for several hours facilitates cell rupture and leaching, and thus the removal of canavanine with the cooking water. Earlier animal study has shown that soaking and boiling results in improved protein quality, probably due to a reduction of antinutritional factors including canavanine (Ekanayake et al., 2003). However, further boiling of grits in water led to the formation of a gelatinized mass, which hindered the decanting process and thus removal of solubilised canavanine. Therefore, the use of intense wet heat treatment is limited so as to preserve the structure of the grits. Such grits could be used in food after drying, i.e. as grits or flour. Autoclaving (pressure cooking) of grits significantly decreased the canavanine content by 24%. This indicates that, unlike protease inhibitors and concanavalin A in legumes, which can be completely eliminated by autoclaving (Agbede and Aletor, 2005), canavanine resists complete degradation by heating under pressure. Roasting sword bean flour (Sample H) was more effective in decreasing canavanine than roasting grits (Sample G), and the smaller particle size of the flour could be the reason behind this. This is in accordance with results from another study where the decrease in canavanine content was 35% in jack beans roasted at 150 °C for 27 min. However, a 95% decrease was obtained after roasting at 164 °C for 26 min (Leon et al., 1998). The decrease could be due to the participation of canavanine in Maillard reaction and also oxidation at high temperature used for roasting. To further explore the possibility of increasing the use of sword beans in foods, greater canavanine reduction by
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roasting at increased temperature (Melcion et al., 1998), perhaps at the cost of the biological value, may be considered, and also removal of antinutritional factors using KHCO3 (D’Mello and Walker, 1991) and urea, (Udedibie et al., 1994). Acid extraction of heat treated beans could also remove a higher amount of canavanine from the seeds and is another possibility for further studies.
0.4–0.5 g. For a 60 kg person this would correspond to less than 0.01 g canavanine/kg body weight, which can be compared with the 0.7–1.5 g canavanine/kg body weight calculated from the results in animal studies discussed. Application of a safety factor of 100 which is normally used in ADI estimates, suggests that soaked and boiled sword beans may be safely used as an ingredient in food.
4.3. Toxicological aspects of canavanine
5. Conclusions
The diets of humans are complex mixtures of compounds, some of which can be harmful and some of which can have protective effects. Studies have shown that canavanine in legumes can have harmful as well as beneficial effects. Inhibition of arginine uptake in isolated brush border membranes of chicks has been reported at a canavanine concentration of 0.5 mmol (Rueda et al., 2003). Development of a condition that mimics systemic lupus-like disease in primates ingesting canavanine-containing alfalfa seeds has been attributed to canavanine (Montanaro and Bardana, 1991). On the other hand, a diet containing 1% canavanine was shown to increase longevity in mice fed a highprotein diet (23.4%) (Brown et al., 2000). With a moderate protein diet (15.7%) the life span on a 1% canavanine diet was the same as the control group (Brown, 2005). When soaked sword bean grits were fed to rats, a low biological value of the protein was obtained and it was speculated that canavanine could have been partially responsible for this (Ekanayake et al., 2003). Our results on the content of canavanine in soaked sword beans support this speculation. The highest weight gain, 72%, compared with the controls was observed for rats fed soaked and boiled sword bean grits, i.e. those with the most pronounced reduction in canavanine. The intake of canavanine corresponded to 0.7 g canavanine/kg body weight/ day. This suggests that a daily intake of canavanine at this level does not cause short-term adverse effects in rats. In the studies on life-long supply of diets containing 1% canavanine in mice showing prolonged or unaltered life span (Brown et al., 2000; Brown, 2005), the canavanine dose can be estimated to be 1.5 g/kg body weight/day (assuming a daily feed intake corresponding to 15% of the body weight). When poultry were fed diets containing jack beans roasted at 164 °C for 26 min, the weight gain was 89% of the controls (canavanine intake 0.2 g/day), while those fed jack beans roasted at 150 °C, showed only a 35% weight gain (canavanine intake 0.38 g/day) (Leon et al., 1998). The lower weight gain was attributed to reduced feed intake (40%), but as jack beans were not the only source of protein, the canavanine content could be an important factor explaining the decreased protein quality. The dietary level of canavanine that can be considered safe in humans cannot be determined without further studies. An ordinary dish may contain around 25 g beans, and thus if a daily intake of 20–25 g soaked and boiled sword beans is assumed, the intake of canavanine would be
In conclusion, the analytical HPLC method using precolumn dabsyl derivatisation and visible detection is sensitive, specific and can successfully be applied for the detection and quantification of canavanine in legumes, and presumably other plant materials. The different household processing methods involving heat treatment caused a significant reduction in canavanine, thus indicating the importance of heating. The maximum reduction, around 50%, was obtained by decorticating the seeds, making grits, soaking and boiling them for 20 min and decanting the cooking water. Recent animal studies, however, do not confirm acute or chronic toxicity of canavanine, at levels consumed when sword beans are incorporated in the diets indicating that the canavanine content in sword beans prepared as above would not prevent them from being used in food. However, more data are needed to determine safe levels of canavanine intake for humans. Conflict of interest statements for all authors None. Acknowledgements The authors thank Mia Malmquist for technical support and Dr. Roberto Romero for valuable advice on analytical issues. The financial support of the International Science Program (IPICS), Uppsala University, Sweden for Grant SRI: 07 is gratefully acknowledged. References Agbede, J.O., Aletor, V.A., 2005. Studies of the chemical composition and protein quality evaluation of differently processed Canavalia ensiformis and Mucuna pruriens seed flours. Journal of Food Composition and Analysis 18, 89–103. Association of Official Analytical Chemists (AOAC), 1984. Official methods of analysis (14th ed.) 14. 009, Virginia, DC USA. Bartig, D., Klink, F., 1992. Determination of the unusual amino acid hypusine at the picomole level by derivatization with 4-dimethylaminoazobenzene-4 0 -sulfonyl chloride and reversed phase high performance or medium pressure liquid chromatography. Journal of Chromatography 606, 43–48. Bence, A.K., Worthen, D.R., Adams, V.R., Crooks, P.A., 2002. The anitproliferative and immunotoxic effects of L-canavanine and L-canaline. Anti Cancer Drugs 13, 313–320. Bianchi, V., Mazza, L., 1995. Rapid reverse-phase high-performance liquid chromatographic method with double derivatization for the assay of urinary hydroxyproline. Journal of Chromatography 665, 295–302.
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