Food Chemistry 128 (2011) 1094–1099

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Catechin and epicatechin in testa and their association with bioactive compoundsin kernels of cashew nut (Anacardium occidentale L.)

Jennifer Trox a, Vellingiri Vadivel a, Walter Vetter b, Wolfgang Stuetz a, Dietmar R. Kammerer c,Reinhold Carle c, Veronika Scherbaum a, Ute Gola a, Donatus Nohr a, Hans Konrad Biesalski a,⇑a Institute for Biological Chemistry and Nutrition, University of Hohenheim, Germanyb Institute of Food Chemistry, University of Hohenheim, Germanyc Institute for Food Science and Biotechnology, Chair of Plant Foodstuff Technology, University of Hohenheim, Germany

a r t i c l e i n f o

Article history:Received 13 August 2010Received in revised form 7 March 2011Accepted 7 April 2011Available online 16 April 2011

Keywords:Anacardium occidentale L.Cashew nut testaCatechinEpicatechinCarotenoidsTocopherolsThiamineFatty acids

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.04.018

⇑ Corresponding author. Tel.: +49 711 459 24112; fE-mail address: Biesal@uni-hohenheim.de (H.K. Bi

a b s t r a c t

Catechins in testa and bioactive compounds in testa-free and testa-containing kernels of cashew nuts wereanalysed. The cashew nut testa contained (+)-catechin and (�)-epicatechin with concentrations of 5.70 and4.46 g per kg DM, respectively. Testa-containing kernels revealed significantly higher levels of b-carotene(218 vs. 89.6 lg/kg DM), lutein (525 vs. 292 lg/kg DM), and a-tocopherol (10.1 vs. 2.4 mg/kg DM), similaramounts of zeaxanthin (7.0 vs. 7.1 lg/kg DM), c-tocopherol (10.6 vs. 10.1 mg/kg DM), stearic acid (41 vs.43 g/kg DM), oleic acid (214 vs. 219 g/kg DM) and linoleic acid (69 vs. 62 g/kg DM), but a lower concentra-tion of thiamine (3.0 vs. 10.7 mg/kg DM) in comparison to testa-free samples. The testa-containing kernelsprovide high amounts of catechins and higher concentrations of b-carotene, lutein and a-tocopherol thando testa-free cashew nut kernels. This could have potential health benefits for consumers.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Cashew nut (Anacardium occidentale L.) kernels are regarded asa nutritious food product, worldwide. The kernels of cashew nutsare externally covered with a thin and reddish-brown-colouredskin, known as testa. The testa constitutes about 1–3% of the totalweight of cashew nuts and is found to provide a rich source ofhydrolysable tannins with polymeric proanthocyanidins as majorpolyphenols (Mathew & Parpia, 1970). Ethanolic extract of cashewnut testa exhibited a significant level of antioxidant activity, whichwas attributed to its phenolic composition (Kamath & Rajini, 2007).Unfortunately, cashew nuts for human consumption are beingmarketed without the testa. Usually after shelling, the testa is re-moved from the kernels, in order to avoid bitter/astringent tasteand also to achieve a higher price in the market.

During conventional industrial shelling, the testa is exposed to avery high processing temperature (75–200 �C) and ultimatelyeliminated from the kernels. As an alternative, Nair (2005) pat-ented (WO/2005/039322) a novel method to produce cashew nutswith intact testa. The ‘‘Flores’’ hand-cracking method has recentlybeen introduced, to retain the testa with kernels, by PT. Profil Mitra

ll rights reserved.

ax: +49 711 459 23822.esalski).

Abadi (PT. PMA), Tangerang, Indonesia. In this process, the cashewnut kernels are separated from the shell by employing a speciallydesigned hand-cracking machine. Subsequently, the cashews aredried for 3 h at a mild temperature (45 �C). Under these conditions,the testa is not damaged and remains intact. Furthermore, we havealready reported the maximal retention of various bioactive com-pounds in cashew nut kernels using the ‘‘Flores’’ hand-crackingprocess (Trox et al., 2010).

Even though the nutritional value and health benefits of cashewnut kernels were demonstrated by various research studies, thenutritional importance of testa has been ignored. Currently, thereis no information available on the phenolic composition of testaor its contribution to the nutritional quality of cashew kernels.Hence, the present study was conducted in order to characterisethe polyphenolic compounds in cashew nut testa, and also todetermine the bioactive compounds in testa-containing and tes-ta-free cashew nut kernels.

2. Materials and methods

2.1. Sample collection

The cashew nut samples were collected from agricultural farmslocated at four different villages (Rowa, Ile Padung, Ilenmedo and

J. Trox et al. / Food Chemistry 128 (2011) 1094–1099 1095

Kringa) in central and eastern Flores Island, Indonesia, located at asea level of 10–45 m. To ensure the comparability between pro-cessed samples, all the cashew nuts were harvested in October2006, and also the same type of drying and storage conditions wereapplied. To reduce water content from approximately 25% to 8–9%the raw material was sun-dried for 2–3 days. It was stored in bagsunder adequate ventilation. The collected samples were processedas described below.

2.2. ‘‘Flores’’ hand-cracking

The hand-cracking of cashew nuts was conducted on the basisof a detailed description of production (Rudolf Heering, PresidentDirector, PT, PMA, Tangerang, Indonesia). After manual removalof small samples, acceptable cashew nuts, with a mean weight of6.42 g and a water content of 6.07% (±0.4%), were passed into thebreakers. It is inevitable that a minimum amount of cashew nutshell liquid (CNSL) emerges from the mesocarp of the shell as a re-sult of the cracking process. Thus, it is important to protect the tes-ta of the cashew nut kernels from exiting the CNSL. The outersurface of the cashew nut kernel was not damaged during thecracking process, so that the cashew nuts were gained without vio-lating the testa. Further, the cashew nut kernel, which is protectedby testa, was not in contact with the CNSL. The samples werecracked and dried for 3 h at a temperature of 45 �C in a heatingblock. Then the samples were frozen at �80 �C and freeze-driedfor 48 h.

2.3. Sample preparation

The processed samples were divided into two batches (eachcontaining 50 g). In one batch the testa was removed manuallyfrom the kernels whereas, in the other batch, the cashew nut ker-nels were stored without removing the testa. The manually re-moved testa was used for the determination of phenoliccompounds, while the testa-containing and testa-free cashew nutkernels were subjected to the analysis for bioactive compounds.Testa-containing and testa-free cashew nut kernels were homoge-nised by using a mortar and pestle, freeze-dried for 24 h and storedat 9 �C prior to further use. Degradation of tocopherols was pre-vented by working under a yellow light.

2.4. Polyphenolic compounds in cashew nut testa

In a preliminary experiment, approximately 70 mg of powderedtesta were mixed with 5 ml of methanol–water mixture (40:60 v/v)and 50 ll of formic acid. This solution was centrifuged for 3 min at2500 g and the supernatant was diluted (1:100, v/v) for subsequentmeasurements by both HPLC with photodiode array (PDA) andmass spectrometric (LC–MS) detection. For HPLC/PDA analysis,20 ll were injected into a Varian HPLC (Pro Star 210) equipped witha Shimadzu PDA (PD-M20A), applying the following chromato-graphic conditions: Reprosil-Pur 120 C18 AQ column (5 lm,250 � 4.6 mm, Trentec, Gerlingen, Germany) at 40 �C, and solventA of aqueous formic acid (5% v/v) and solvent B containing formicacid, distiled water and acetonitrile (5:10:85, v/v/v). The gradientprogramme was as follows: 5% B to 26% B (12.38 min), 26% B to100% B (17.22 min), 100% B isocratic (10 min) with a flow-rate of1 ml/min and a total run time of 40 min. Spectra were recordedfrom 200 to 700 nm. Testa extracts were analysed by LC–MSaccording to the method of Stintzing et al. (2004). The separationof phenolic compounds was performed in an Agilent HPLC series1100 (Agilent, Waldbronn, Germany), under the conditions de-scribed above, applying the following gradient: 5% B to 15% B(6 min), 15% B isocratic (4 min), 15% B to 26% B (6.38 min), 26% Bto 100% B (17.62 min), 100% B isocratic (10 min).

This HPLC system was connected, in series, with a Bruker iontrap mass spectrometer (Model: Esquire 3000+, Bremen, Germany)fitted with an APcI source. The detector was operated in negativeionisation mode (range: m/z 50–1200). Nitrogen was used as thedry gas at a flow rate of 5.0 l/min and a pressure of 65.0 psi. Thenebuliser temperature was set at 350 �C, and the vaporiser temper-ature at 400 �C. Using helium as the collision gas (4.1 � 10�6 mbar),collision-induced dissociation spectra were obtained with a frag-mentation amplitude of 1.0 V.

After peak assignment by LC–MS, the testa samples wereprepared as follows: 10 mg of powdered testa were vigorouslymixed with 1 ml of methanol in a 1.5 ml Eppendorf tube, andcentrifuged (11400 g for 5 min), and supernatants were finally di-luted with water (1:25, v/v) prior to HPLC analysis. Aliquots of20 ll were injected into a Varian Pro Star 210 and the phenolicswere separated with the same column and solvents as describedabove, using the following gradient programme: 5% B to 15% B(1 min), 15% B isocratic (4 min), 15% B to 26% B (6.38 min), 26%B to 100% B (17.62 min), 100% B isocratic (10 min). (�)-Epicate-chin and (+)-catechin were identified at 278 nm, using a Watersdetector (Model: 2487). (+)-Catechin and (�)-epicatechin werequantified by using an external calibration, with respective stan-dards diluted in methanol–water (50:50, v/v).

2.5. Carotenoids and tocopherols in cashew nut kernels

The b-carotene, lutein and zeaxanthin, as well as the a- and c-tocopherol contents, in testa-containing and testa-free cashewnut kernels were analysed by RP-HPLC after extraction and sapon-ification as follows: 2 ml of ethanol, containing pyrogallol (2.5%)and b-apo-80-carotenal-methyloxime and 1 ml of 50% KOH solu-tion, were added to 100 mg of the sample in a screw-capped glasstube. The internal standard b-apo-80-carotenal-methyloxime wassynthesised as described by Sommerburg, Zang, and Van Kuijk(1997). For saponification, the suspension was covered by argonand closed with screw caps, and then the mixture was stirred for4 h in a water bath at 38 �C. After the addition of 2 ml of saline solu-tion (15%), fat-soluble substances were extracted twice with l ml ofhexane. The combined hexane phases were washed (15% salinesolution), evaporated using nitrogen gas and finally re-dissolvedin 200 ll of ethanol–water (1:3, v/v) prior to analysis on a VarianHPLC (Prostar-210) equipped with UV–VIS and fluorescence detec-tors (Waters-2487, Waters-474), using the following chromato-graphic conditions: Spherisorb ODS-2 analytical column (3 lm,250 � 4.6 mm, Trentec, Germany) at 40 �C and a mobile phaseconsisting of acetonitrile (82%), dioxane (15%) and methanol (3%,containing 100 mM ammonium acetate and 0.1% triethylamine)in a re-circulation mode with a flow rate of 1.6 ml/min. The carote-noids were detected at 450 nm, while a- and c-tocopherols weremeasured by fluorescence with an excitation/emission wavelengthof 298 and 328 nm, respectively.

2.6. Thiamine in cashew nut kernels

The determination of thiamine content of cashew nut sampleswas performed by pre-column derivatization, RP-HPLC and fluores-cence detection according to the method of Gerrits, Eidhof,Brunnekreeft, and Hessels (1997) method with modifications. Inbrief, 100 mg of the sample were mixed with 7.5 ml of 0.1 M HClsolution and stirred for l h at 30 �C in the dark. Subsequently, an ali-quot of 1.5 ml of this mixture was centrifuged at 5000 g and theclear supernatant was derivatised as follows: 500 ll of the clearsolution were mixed with 100 ll of freshly prepared oxidation re-agent (12.1 mM K3[Fe(CN)6] in 3.35 M NaOH solution). The ‘‘thio-chrome reaction’’ was stopped by the addition of 20 ll of 6 Morthophosphoric acid, and 20 ll of the aliquot were analysed on a

1096 J. Trox et al. / Food Chemistry 128 (2011) 1094–1099

Merck-Hitachi HPLC (LaChrom) equipped with a column oven (setat 40 �C), fluorescence detector (L-7480) and Clarity chromato-graphic station (DA-C50, DataApex Ltd., Praha). The separationwas achieved on a 5 lm analytical column (Grom-Sil 120 ODS-4HE, 125 � 4 mm, Grom, Rottenburg-Hailfingen, Germany), using amobile phase consisting of methanol (27.5% v/v) and phosphatebuffer (pH 7.0) at a flow rate of 0.8 ml/min. Thiamine was detectedby excitation/emission set at 367/435 nm.

2.7. Fatty acids in cashew nut kernels

Determination of fatty acids (stearic acid, oleic acid and linoleicacid) of cashew nut samples was carried out by following themethod of Thurnhofer, Lehnert, and Vetter (2008). Sample aliquots(500 mg) were extracted by means of an ASE 200 system (Dionex,Idstein, Germany), using 22 ml extraction cells. For this purpose,22 ml extraction cells were used, which were filled with isolute-HM-N. The cells were extracted twice with the azeotropic mixtureof cyclohexane and ethyl acetate (46:54, v/v) (Weichbrodt, Vetter,& Luckas, 2000). The two combined extracts were concentrated,using a rotary evaporator with vacuum controller at 60 �C and pre-cisely adjusted to a definite volume. The lipid content was deter-mined gravimetrically (Trox et al., 2010).

To determine fatty acids, the extracted oil was first transesteri-fied. The first internal standard (10,11-dichloroundecanoic acid),produced according to the method of Thurnhofer et al. (2008),was pipetted into the mixture and 500 ll of 0.5 M methanolicKOH solution were added for saponification at 80 �C. After a reac-tion time of 5 min, the mixture was cooled in an ice bath. The sub-sequent methylation was started by adding l ml of boron-trifluoridein methanol and kept for 5 min at 80 �C. After cooling, 2 ml of hex-ane and 2 ml of a saturated NaCl solution were added and shakenwell. The organic phase (180 ll) was mixed with 20 ll of the secondinternal standard, oleic acid ethyl-ester, which was preparedaccording to the method of Thurnhofer et al. (2008). Gas chroma-tography, in combination with electron ionisation mass spectrom-etry (GC–EI/MS) analyses, was performed with an HP 5890/5971system (Agilent, Waldbronn, Germany). Helium (99.999% purity)was used as the carrier gas with a flow rate of 1 ml/min. A CP-Sil88 column (50 m � 0.25 mm ID � 0.20 lm film thickness) (Chrom-pack, Middelburg, The Netherlands) was installed in the GC oven.An injection volume of 1 ll was used at a temperature of 250 �Cand analysed for 38.81 min. In the selected ion monitoring (SIM)mode, nine fragment ions were recorded and six of them were usedfor quantification: (1) m/z 74 and (2) m/z 87 for the methyl esters ofsaturated and monounsaturated fatty acids, (3) m/z 81 and (4) m/z79 for the methyl esters of polyunsaturated fatty acids, (5) m/z 88and (6) m/z 101 for the ethyl esters of saturated and monounsatu-rated fatty acids.

Fig. 1. Catechin and epicatechin contents in cashew nut testa.

2.8. Statistical analysis

The statistical analysis was performed using SPSS for WINDOWS(SPSS Inc., Chicago, IL, Version 11.0). 10 separate determinationswere carried out for catechins, carotenoids, tocopherols and thia-mine and seven separate determinations were carried out for fattyacids in testa-free and testa-containing cashew kernels; valueswithin each group were found to be normally distributed using Kol-mogorov–Smirnov-test and were described by their means (SD).Comparison of means between testa-free and testa-containingcashew kernels was determined by independent Student’s t-test;two-tailed p values <0.05 were considered statistically significant.

3. Results and discussion

3.1. Polyphenolic compounds in cashew nut testa

The RP-HPLC analysis of phenolic extract of cashew nut testa re-vealed two prominent peaks with absorption maxima at 278 nm(data not shown). Based on the comparison of mass spectrometricdata of these peaks with an earlier report by Stintzing et al. (2004),the phenolic compounds were identified as (+)-catechin and (�)-epicatechin and confirmed with authentic reference compounds.To our knowledge, this is the first report to confirm the phenolicprofile of cashew nut testa by employing RP-HPLC and LC–MStechniques.

Remarkable amounts of (+)-catechin and (�)-epicatechin werequantified with averages of 5.70 and 4.46 g per kg DM, respec-tively, in the cashew nut testa (Fig. 1). In an earlier report, it hasbeen postulated that the catechins are mainly localised in the cash-ew nut testa and not in the kernels (Mathew & Parpia, 1970), whichis in accordance with the results of the present research work. Theantioxidant and free radical-scavenging potential of cashew nutextracts, demonstrated by Sajilatha and Singhal (2006), might beattributed to the catechins found in testa, as deciphered by thepresent investigation. Further, the antioxidant-rich extract fromcashew nut skin was found to have a protective effect against orga-nophosphorus insecticide toxicity in rats (Kamath, Joshi, & Rajini,2008).

It is important to mention that the polyphenolic content ofcashew nut testa was higher than those of green tea and dark choc-olate (Khokhar & Magnusdottir, 2002; Natsume et al., 2000). Incomparison, the contents of (+)-catechin and (�)-epicatechin incashew nut testa were �20 and �5-fold higher, respectively, thanthose of the polyphenolic compounds reported in dark chocolate(Natsume et al., 2000). The high levels of (+)-catechin and (�)-epi-catechin confer high nutritional quality to the cashew nut.

The results of the analysis of different varieties of green tea, froma previous research work, showed average concentrations of0.43 mg of (+)-catechin and 6.66 mg of (�)-epicatechin per grammeDM (Khokhar & Magnusdottir, 2002). The levels of phenolic

J. Trox et al. / Food Chemistry 128 (2011) 1094–1099 1097

compounds per cup of green tea were calculated on the basis of 2 g ofgreen tea in 200 ml of water, with extraction yields of 95% for (+)-catechin and 85% for (�)-epicatechin (extraction conditions:5 min, 80 �C) (Khokhar & Magnusdottir, 2002). Based on data fromKhokhar and Magnusdottir (2002), an average cup of green tea con-tains 0.82 and 11.3 mg of (+)-catechin and (�)-epicatechin, respec-tively. From the results of the present study, one single piece ofcashew nut, along with testa (’2 g), contains 11.4 and 8.92 mg of(+)-catechin and (�)-epicatechin, respectively. Hence, the (+)-cate-chin content of one cashew nut kernel with testa is roughly equiva-lent to the amount of (+)-catechin contained in 13 cups of green tea.Further, the level of (�)-epicatechin in one cashew nut containingtesta is equivalent to 75% of the (�)-epicatechin amounts of an aver-age cup of green tea.

Cabrera, Artacho, and Gimenez (2006) reviewed the properties ofgreen tea polyphenols (mainly catechins), revealing high antioxi-dant, anti-mutagenic, anti-diabetic, anti-inflammatory, anti-bacte-rial and anti-viral activities. Further, the results of some humanstudies also indicate that the consumption of green tea reducesthe risk of cardiovascular diseases and some types of cancer (Cabreraet al., 2006). However, it is important to note that the total amount ofpolyphenolics in green tea is higher than that of cashew nut testa, asgreen tea does not only contain (+)-catechin and (�)-epicatechin,but also high quantities of gallic acid esters, (�)-epicatechin gallate,(�)-epigallocatechin and (�)-epigallocatechin gallate. These mayalso be responsible for the above-mentioned health protectiveeffects of green tea.

In addition, the effect of the drying process at 45 �C, for 3 h in theFlores hand-cracking method, on the level of polyphenolic com-pounds of cashew nut testa has not yet been examined. A significantreduction of polyphenolic content was noticed in millet as a resultof increasing the temperature from 65 to 75 �C, (Nwanguma & Eze,1996). Nonetheless, no significant change of the level of polyphe-nols or their antioxidant activity in grape pomace from red grapeswas observed at a drying temperature of 60 �C (Larrauri, Ruperez,& Saura-Calixto, 1997). Consequently, it could be concluded thatdrying at the mild temperature of 45 �C should not affect the poly-phenolic compounds of cashew nut testa significantly.

3.2. Carotenoids in cashew nut kernels

The analysis of carotenoids exhibited significantly (p < 0.001)higher contents of b-carotene and lutein in testa-containing ker-nels (2.4- and 1.8-fold, respectively) than in testa-free samples (Ta-ble 1). These significant differences in the carotenoids are mostlikely a result of occurrence of high amounts of these compoundsin the testa. Additional analyses in testa samples showed approxi-mately 2.5- to 3-fold higher levels of b-carotene and lutein, respec-tively, in the testa than in kernel (data not shown). In contrast, thecontent of zeaxanthin was virtually the same (7.1 vs. 7.0 lg/kg

Table 1Carotenoids, tocopherols and thiamine in testa-free vs. testa-containing kernels ofcashew nut1,2.

Bioactive compounds Cashew nut samples

Testa-free kernels3 Testa-containing kernels

b-Carotene (lg/kg DM) 89.6a ± 3.9 218b ± 11.8Lutein (lg/kg DM) 292a ± 26.7 525b ± 45.2Zeaxanthin (lg/kg DM) 7.1a ± 2.3 7.0a ± 2.2a-Tocopherol (mg/kg DM) 2.4a ± 0.4 10.1b ± 0.7c-Tocopherol (mg/kg DM) 10.1a ± 0.6 10.6a ± 0.6Thiamine (mg/kg DM) 10.7a ± 1.0 3.0b ± 0.5

1 Values are means ± standard deviation of 10 separate determinations (n = 10).2 Values in the same row with different superscripts are significantly different

(p < 0.001).3 Data taken from our previous report (Trox et al., 2010).

DM) in testa-free and testa-containing cashew nut kernels. Thepresence of such high amounts of these antioxidants in the testais of great importance for the development of cashew nuts duringgermination, by protecting them from insects, microbial infectionand sun-light, through the hard CNSL-containing shell. Followingthe breakdown of shell, the protective function of antioxidantscontained in the testa is activated (Chung, Wong, Wei, Huang, &Lin, 1998).

In addition to the importance as provitamin A, b-carotene has aprotective function against both UV-light and oxidative stress,through deactivation of singlet oxygen and by inhibition of lipidperoxidation (Biesalski et al., 2009). Functions of carotenoids areconsiderable as a precursor of vitamin A, which is required foradaptive immunity and plays a significant role in the developmentof both T-helper cells and B cells (Stephensen, 2001). Furthermore,carotenoids are also known to play a role in the prevention of can-cer and atherosclerosis (Krinsky & Johnson, 2005).

3.3. Tocopherols in cashew nut kernels

The levels of a- and c-tocopherols in testa-containing cashewnut kernels were found to be 10.1 and 10.6 mg/kg DM, respectively(Table 1). The testa-containing samples exhibited significantly(p < 0.001) higher level of a-tocopherol (4.2-fold) but similar levelsof c-tocopherol in relation to testa-free kernels. The explanation isthe presence of a much higher concentration of a-tocopherol (23-fold) in the testa than in kernels (data not shown).

Tocopherols are reported to constitute an essential part of bio-logical membranes. It is well known that tocopherols exhibit a pro-tective role against lipid peroxidation of membrane lipids,lipoproteins and depot fats (Biesalski et al., 2009) and thereforeprevent atherosclerosis. Vitamin E is also able to induce apoptosisin tumour cells and modulate oncogenes and this is probably thereason for the low occurrence of cancer as a result of vitamin E-richdiets, which was demonstrated in a variety of epidemiologicalstudies (Dutta & Dutta, 2003).

3.4. Thiamine in cashew nut kernels

Various enzymes of intermediary metabolism, including pyru-vate dehydrogenase, a-ketoglutarate dehydrogenase and transke-tolase, require thiamine pyrophosphate as an essential cofactor.The testa-free cashew nut kernels were found to contain consider-ably high amounts of thiamine (10.7 mg/kg DM) when comparedto testa-containing samples (3.0 mg/kg DM) (Table 1).

In testa-containing cashew nut samples, the thiamine contentwas significantly reduced, by more than 70% with respect to testa-free kernels, which is most likely due to the anti-thiamine activityof phenolic compounds in testa, because phenolic compounds, suchas catechin, epicatechin and tannins, were reported to exhibit astrong anti-thiamine activity (Hilker & Somogyi, 2006). Further, inthe present investigation, the testa-containing cashew nuts werehomogenised and stored for about 2 months prior to analysis. Dur-ing this time thiamine seems to be partially degraded, by splittingthe thiazole ring of the thiamine, or accelerating the oxidation ofdisulphide, due to the polyphenolic compounds of testa. The anti-thiamine activity of phenolic compounds is closely related to thenumber and position of OH groups on the benzene ring. Substanceswith two vicinal OH groups were reported to exhibit high anti-thia-mine activity (Somogyi & Bonicke, 1969) and, interestingly, both(�)-epicatechin and (+)-catechin show such characteristic features.Hence, it is recognised that the reduction of the thiamine level wasmost likely due to the interaction with polyphenolic compounds ofcashew nut testa.

The thiamine concentrations of commercially available testa-free and testa-containing cashew nuts are probably not much

1098 J. Trox et al. / Food Chemistry 128 (2011) 1094–1099

different, as shown in the present study, since they are stored for alonger period and also offered as whole kernels and not as a homog-enised powder. The anti-thiamine factors contained in the testamay have less association in whole kernels whereas, in pulverisedform, there are more chances for close contact with polyphenoliccompounds, which might be responsible for the reduction of thia-mine content. However, reduction of bioavailability of thiamine,due to the presence of high concentrations of (+)-catechin and(�)-epicatechin, still remains unclear upon consumption of wholecashew nuts with testa. Nonetheless, it has been shown that theconsumption of tea containing high amounts of catechin polyphe-nols causes a severe thiamine deficiency (Kositawattanakul, Tos-ukhowong, Vimokesant, & Panijpan, 1977).

3.5. Fatty acids in cashew nut kernels

We observed no significant difference between the fatty acidpatterns of the testa-containing and testa-free samples (Table 2).While the levels of oleic acid and stearic acid were comparable, lin-oleic acid was, on average, 10% higher in testa-containing cashewnut kernels than in testa-free samples. Since, the testa contributes�7% to the sample, the high concentration of linoleic acid in thetesta-containing kernels was attributed to testa. Subsequent anal-yses of (separated) testa and testa-free kernels confirmed thisassumption. The relative contribution of linoleic acid to the fattyacids in the separated testa was higher than those of stearic acidand oleic acid. For instance, linoleic acid accounted more than60% of oleic acid in the testa while the level in the testa-free nutwas <30%. Especially, linoleic acid is sensitive to oxidation, whichmeans that it must be protected from autoxidation. This wouldalso explain why the cashews with testa have higher levels of otherbioactive compounds, although they were processed similarly totesta-free cashew nuts for 3 h at 45 �C. The samples were also pro-tected against very high temperature, since on heating cytotoxicproducts, such as trans-isomers, may be formed (Martin, Milinsk,Visentainer, Matsushita, & De-Sauza, 2007).

The stearic acid level (41 g/kg DM) of testa-containing sampleswas found to be higher than those in a previous report on cashewnut (3.43 g/100 g), chestnut (0.02 g/100 g), peanut (1.30 g/100 g),hazelnut (0.94 g/100 g), coconut (1.10 g/100 g), macadamia(1.47 g/100 g), almond (0.55 g/100 g), pecan (1.51 g/100 g), pista-chios (0.98 g/100 g) and walnut (1.37 g/100 g) (Souci, Fachmann,& Kraut, 2008). The oleic acid content (214 g/ kg DM) was higherthan those of chestnut (0.98 g/100 g), coconut (2.10 g/100 g), Brazilnut (18.5 g/100 g) and walnut (11.4 g/100 g), whereas the linoleicacid level of testa-containing kernels (69 g/ kg DM) was higherthan those of coconut (0.68 g/100 g) and macadamia (1.74 g/100 g) (Souci, Fachmann, & Kraut, 2008). Linoleic acid was assessedas precursor of arachidonic acid and eicosapentaenoic acid in theeicosanoid metabolism (Tapiero, Ba, Couvreur, & Tew, 2002). It isalso a component of phospholipids, which are responsible for thestructure and function of biological membranes. In recent years,studies have demonstrated that dietary polyunsaturated fattyacids have protective effects on metabolic syndrome diseases, such

Table 2Fatty acids in testa-free vs. testa-containing kernels of cashew nut.1,2

Bioactive compounds (g/kgDM)

Cashew nut samples

Testa-freekernels

Testa-containingkernels

Stearic acid 42.8a ± 3.6 40.9a ± 6.3Oleic acid 219a ± 18.5 214a ± 33.2Linoleic acid 62.3a ± 5.1 68.6a ± 10.0

1 Values are means ± standard deviation of seven separate determinations (n = 7).2 Values in the same row with similar superscripts are not significantly different

(p < 0.001).

as Type 2 diabetes, hyper-lipidemia or cardiovascular disease.Equally promising results are now available regarding their posi-tive effects on inflammatory diseases, cancer and osteoporosis(Benatti, Peluso, Nicolai, & Menotti, 2004).

4. Conclusion

In the present study, the phenolic compounds in methanolic ex-tract of cashew nut testa were clearly identified as (+)-catechin and(�)-epicatechin by RP-HPLC and LC–MS. Further, it was demon-strated that the levels of (+)-catechin and (�)-epicatechin in thetesta of cashew nuts are higher than those reported for green teaand chocolate. The testa-containing cashew nuts possess signifi-cantly higher amounts of carotenoids and tocopherols, but a lowerlevel of thiamine when compared to testa-free kernels. Such higherlevels of carotenoids and tocopherols of cashew nut kernels werecontributed by the testa and the low thiamine content was likelydue to the diminishing effect of catechins in testa. The presenceof such potentially bioactive compounds in the testa-containingcashew nut kernels could be of interest for the food and nutraceu-tical industries, where it can be employed as an economic source ofnatural antioxidants. While the cashew nut testa is heat-treatedand removed during various conventional processes, it is possibleto retain the testa by the newly developed ‘‘Flores’’ hand-crackingmethod. Removal of cashew nut testa may significantly decreasethe uptake of essential bioactive compounds. Hence, the processingof cashew nuts should be re-considered to avoid the loss of cashewnut testa and its appreciable levels of catechins. In this context, therecently developed ‘‘Flores’’ hand-cracking process could be rec-ommended, on the industrial scale, in order to produce cashewnut kernels along with testa at a mild temperature.

Acknowledgements

Authors are deeply grateful to Mr. Jochen Wolf, ManagingDirector, Flores Farm GmbH, Germany, Rudolf Heering, PresidentDirector, PT. PMA, Tangerang, Indonesia and Dr. Frank-MichaelLange, Terra Fusca, Germany, for their immense help in providingthe cashew nut samples and continuous support. One of theauthors (VV) is thankful to the Alexander von Humboldt (AvH)Foundation, Bonn, Germany for the award of a post doctoral re-search fellowship.

References

Benatti, P., Peluso, G., Nicolai, R., & Menotti, C. (2004). Poly-unsaturated fatty acids:Biochemical, nutritional and epigenetic properties. Journal of the AmericanCollege of Nutrition, 23, 281–302.

Biesalski, H. K., Dragsted, L. O., Elmadfa, I., Grossklaus, R., Müller, M., Schrenk, D.,et al. (2009). Bioactive compounds: Safety and efficacy. Nutrition, 25,1206–1211.

Cabrera, C., Artacho, R., & Gimenez, R. (2006). Beneficial effects of green tea – Areview. Journal of the American College of Nutrition, 25, 79–99.

Chung, K. T., Wong, T. Y., Wei, C. I., Huang, Y. W., & Lin, Y. (1998). Tannins andhuman health: A review. Critical Reviews in Food Science and Nutrition, 38,421–464.

Dutta, A., & Dutta, S. K. (2003). Vitamin E and its role in prevention ofatherosclerosis and carcinogenesis: A review. Journal of the American Collegeof Nutrition, 22, 258–268.

Gerrits, J., Eidhof, H., Brunnekreeft, J. W. I., & Hessels, J. (1997). Determination ofthiamine and thiamine phosphates in whole blood by reverse-phase liquidchromatography with precolumn derivatization. Methods in Enzymology, 279,74–82.

Hilker, D. M., & Somogyi, J. C. (2006). Anti-thiamines of plant origin: Their chemicalnature and mode of action. Annals of the New York Academy of Sciences, 378,137–145.

Kamath, V., & Rajini, P. S. (2007). The efficiency of cashew-nut (A. occidentale L.) skinextract as a free radical scavenger. Food Chemistry, 103, 428–433.

Kamath, V., Joshi, A. K. R., & Rajini, P. S. (2008). Dimethoate induced biochemicalperturbations in rat pancreas and its attenuation by cashew nut skin extract.Pesticide Biochemistry and Physiology, 90, 58–65.

J. Trox et al. / Food Chemistry 128 (2011) 1094–1099 1099

Khokhar, S., & Magnusdottir, S. G. M. (2002). Total phenol, catechin and caffeinecontents of teas commonly consumed in the United Kingdom. Journal ofAgricultural and Food Chemistry, 50, 565–570.

Kositawattanakul, T., Tosukhowong, P., Vimokesant, S. L., & Panijpan, B. (1977).Chemical interactions between thiamine and tannic acid. II. Separation ofproducts. American Journal of Clinical Nutrition, 30, 1686–1691.

Krinsky, N. I., & Johnson, E. J. (2005). Carotenoid actions and their relation to healthand disease. Molecular Aspects of Medicine, 26, 459–516.

Larrauri, J. A., Ruperez, P., & Saura-Calixto, F. (1997). Effect of drying temperature onthe stability of polyphenols and antioxidant activity of red grape pomace peels.Journal of Agricultural and Food Chemistry, 45, 1390–1393.

Martin, C. A., Milinsk, M. C., Visentainer, J. V., Matsushita, M., & De-Sauza, N. E.(2007). Trans fatty acid forming processes in foods: A review. Anais da AcademiaBrasileira de Ciências, 79, 343–350.

Mathew, A. G., & Parpia, H. A. B. (1970). Polyphenols of cashew nut kernel testa.Journal of Food Science, 35, 140–143.

Nair, H. K. (2005). Edible testa-on (skin-on) cashew nuts and methods for preparingsame. International patent publication number WO/2005/039322 dt.06.05.2005, IPC: A23L1/211 (2006.01), A23L1/36 (2006.01).

Natsume, M., Osakabe, N., Yamagishi, M., Takizawa, T., Nakamura, T., Miyatake, H.,et al. (2000). Analysis of polyphenols in cacao liquor, cocoa and chocolate bynormal-phase and reversed-phase HPLC. Bioscience, Biotechnology andBiochemistry, 64, 2581–2587.

Nwanguma, B. C., & Eze, O. M. (1996). Changes in the concentration of thepolyphenolic constituents of sorghum during malting and mashing. Journal ofthe Science of Food and Agriculture, 70, 162–166.

Sajilata, M. G., & Singhal, R. S. (2006). Effect of irradiation and storage on theantioxidative activity of cashew nuts. Radiation Physics and Chemistry, 75,297–300.

Sommerburg, O., Zang, L., & Van Kuijk, J. G. M. (1997). Simultaneous detection ofcarotenoids and vitamin E in human plasma. Journal of Chromatography B, 695,209–215.

Somogyi, I. C., & Bonicke, R. (1969). Connection between chemical structure andanti-thiamine activity of various phenolic derivatives. International Journal ofVitamin Research, 39, 65–73.

Souci, S. W., Fachmann, W., & Kraut, H. (2008). Food composition and nutrition tables(7th Revised ed.). USA: Taylor of Francis, CRC Press, pp. 1171–1196.

Stephensen, C. B. (2001). Vitamin A, infection and immune function. Annual Reviewof Nutrition, 21, 167–192.

Stintzing, F. C., Kammerer, D., Schieber, A., Adama, H., Nacoulma, O. G., & Carle, R.(2004). Betacyanins and phenolic compounds from Amaranthus spinosus L. AndBoerhavia erecta L. Zeitschrift für Naturforschung, 59, 1–8.

Tapiero, H., Ba, G. N., Couvreur, P., & Tew, K. D. (2002). Polyunsaturated fatty acids(PUFA) and eicosanoids in human health and pathologies. Biomedicine andPharmacotherapy, 56, 215–222.

Thurnhofer, S., Lehnert, K., & Vetter, W. (2008). Exclusive quantification ofmethyl branched fatty acids and minor 18:1-isomers in foodstuff by GC/MSin the SIM mode using 10, 11-dichloroundecanoic acid and fatty acid ethylesters as internal standards. European Food Research and Technology, 226,975–983.

Trox, J., Vadivel, V., Vetter, W., Stuetz, W., Scherbaum, V., Gola, U., et al. (2010).Bioactive compounds in cashew nut (Anacardium occidentale) kernels: Effect ofdifferent shelling methods. Journal of Agricultural and Food Chemistry, 58,5341–5346.

Weichbrodt, M., Vetter, W., & Luckas, B. (2000). Micro-wave-assisted extraction andaccelerated solvent extraction with ethyl acetate-cyclohexane beforedetermination of organochlorines in fish tissue by gas chromatography withelectron-capture detection. Journal of AOAC International, 83, 1334–1343.