861 c -sitosterol β te of , Animal and a reduction of the 4,5 ´ on (UNDAC), College of Farming titute of Pharmaceutical Sciences, TH Zurich, Institu 2010 Society of Chemical Industry e c Ihklas A Khan, c [email protected] The leaves of maca are usually left on the field after 6 Ivan Manrique ember 2009 Published online in Wiley Interscience: 9 February 2010 ich is mainly known for its fertility-enhancing properties d Universidad Nacional Agraria – La Molina (UNALM), La Molina, Lima, University ofUniversity, Mississippi, MS 38677, USA Research Ins Universidad Nacional Daniel AlcidesSciences, School Carri of Agronomy, Cerro de Pasco, Peru Centro Internacional de la Papa (CIP), La Molina, Lima, Peru Correspondence to: Michael Kreuzer,and E Agroecosystem Sciences, Universitaetstrasse 2,E-mail: 8092 michael.kreuze Zurich, Switzerland. ETH Zurich, Institute of Plant, Animal and Agroecosystem Sciences,Switzerland 8092 Zurich, Bharathi Avula, ∗ c a e on, as colour associates with variations in concentrations of d b nging from white to black. Each colour has different biological in their biological effectiveness. For instance,spermatogenesis beneficial appear effects to on be more pronounced withcompared black maca to reddish and yellow maca, ventral prostate size inreddish rats maca. has been reported as a response to in macaene, macamides and than were leaves, b landindoleglucosinolateconcentrations.Effectsonmetabolite pocotyls were rich in glucosinolates, macaene and macamides, ns of major secondary metabolites in hypocotyls and leaves of ificant for most secondary metabolites; exceptions were Walpers; and thus 3 ´ on), a traditional Chac Lepidium meyenii ∗ a Dante D Ponce Aguirre, Diego A Diaz Grados, a ; ; macamide; macaene; phytosterol a because of its potential fertility-related 1 Walpers) 2 -sitosterol and total phenols. : 861–869 www.soci.org β 90 Lepidium peruvianum ´ ement, 2010; Lepidium meyenii The compounds of interest in maca, primarily macaene

2 -sitosterol, as well as a range of glucosinolates (GL). These β

2010 Society of Chemical Industry ´ Maca is predominantly cultivated for its hypocotyls and . eline Cl c Andean crop and a member ofas particularly the promising family, is viewed and campesterol. The lead-coloured, yellow and violet maca hy respectively.Previouscultivationaffectedmacaene,campestero concentrations in the leaves were minor. Hypocotyls were richer and were poorer in distinct bioactive metabolites. Leavessecondary may metabolites be as interesting the hypocotyls for but animal in nutrition clearly lower purposes concentrations. as they contain essentially the same Keywords: CONCLUSION: Colour type has to be considered in maca producti The hypocotyls, whichbe are found in the acoloured edible through variety yellow, part of pink, violet different ofThe and colours lead-coloured differently the to ranging coloured black. from plant, hypocotylsdifferences cream- seem in can concentrations also of to secondary metabolites correlate with J Sci Food Agric INTRODUCTION ’ secondary metabolites are gaining in interest owing topresumed their health benefits in humanplants rich nutrition. in Among these the metabolites, maca many ( sometimes called BACKGROUND: Maca is an Andean crop of the Brassicaceae family wh maca in a controlled plantingprevious cultivation experiment of in the the field Peruvian were examined. at 4130 mRESULTS: above In the sea hypocotyls, level. the colour The type effects effect was of sign colour type and of Abstract Andrea C Mayer, and Michael Kreuzer in hypocotyls and leaves of maca ( C cultivation on secondary metabolites meyenii following consumption. The hypocotyls display various colourseffects. ra The aim of this study was to analyse the concentratio (www.interscience.wiley.com) DOI 10.1002/jsfa.3896 Influence of colour type and previous Research Article Received: 24 September 2009 Revised: 21 December 2009 Accepted: 22 Dec secondary metabolites aloneexplain or the in exhibitionconsumption certain of of maca. combinations biological may effects attributed to the and macamides, are specific to thisother bioactive plant, compounds but such maca as also phytosterols like contains and campesterol effects. . et al : 861–869 ´ ement 90 C at the Centro 2010; 2–3yearsago ◦ Cultivated with maca J Sci Food Agric it was assumed that this parent 8 ) 4.5 4.8 4.6 4.4 2.71.32.1 5.5 4.4 1.6 3.3 0.7 1.0 5.8 3.7 1.8 0.13 0.10 0.18 0.09 1 16.0 24.0 6.0 9.0 13.7 27.6 51.9 60.8 − Never cultivated 144 120 176 125 ) )64574634 ) 30.2 33.6 33.1 24.1 1 1 1 − − − 12% humidity). In December, the plantlets were ± ) 0.74 0.62 0.70 0.56 ) 0.04 0.06 0.04 0.14 1 32 plots and required 3200 individual plantlets. The 1 ) 0.22 0.32 0.26 0.30 − 1 − Soil characteristics + ) − ) = 1 H 1 − C; 80% 4 − + + ◦ + 2 + + 2 × 1 3 + 4 Silt (g g K Na Al (g kg Clay (g g Sand (g g Mg Ca capacity (meq kg (dS m ± Available P (mg kg Available K (mg kg Organic matter Table 1. Time in relation to experimentTexture Before After Before After Base saturation (%) 14 33 32 58 Exchangeable ions (meq kg Cation exchange pHElectrical conductivity 5.0 4.4 5.0 5.0 × programcarriedoutoverseveralyears.Atfirsthypocotylsofyellow, pink, violet and lead hadon been the selected from basis different of landand their races is colour. partially As cleistogamous, maca reproduces autogamously material was already quitetransplanted homozygous. to These separated hypocotyls plough were furrowsstrictly to autogamous produce way. seeds This ingenerations procedure a (S1 was and S2) followed in order over toThe further two increase homozygosity. seeds used infrom the one present plant plantingfertilized per seeds. experiment colour The originated of seedsfor were this 24 cleaned second h with generation in water,on of and a Petri stored auto- dishes gibberellic and acidConviron, allowed Winnipeg, solution. to Canada) germinate They in for were a 1 week then germinator (G30, at placed 18 Internacional de la Papa (CIP) in Lima.transplanted The into germinated jiffy seeds pellets were (diameterLtd, 42 Shippagan, cm; Canada) Jiffy and Products grown (NB) for(19 1 month in a greenhouse transported to the experimental sites and planted into the soil. Experimental design Terrains were divided into 16 plots 1 mper apart, giving four maca replicates colour(with and terrain. 50 cm Each spacingper plot row between consisted (20 cm of the spacing2 four rows) between rows plants). containing Overall this 25 resulted plants in plots were randomlyThe arranged harvest for took colour placegrowth type in on July stage, of the i.e., theflowering terrains. when had second begun. year At some that at time, leavesby the yield was accustomed typical had judged maca to turned workers. be normal Nevertheless,of yellow the the hypocotyl and four yield cultivated plots was insufficient to of allow further analysis. pink All undamaged maca in the terrain never previously 3 8,9 www.soci.org C Cl suggesting S,longitude 2010 Society of Chemical Industry  c The traditional 13 4). The analysed  3,12,13 7 = 92 are recommended. ◦ n 8,9,11 and quite long fallow periods 9 ´ an(latitude10 ın) in Peru. In 1998, the demand for maca resulting in new cultivated areas in other 1 Alternatively, in response to the new demand, it 9 is that the sensory quality of maca (form, flavour or even 8–10 years and more, W, altitude 4130 m a.s.l.), department of Pasco, Peru, 9  10 75  05 ◦ In the present study, an extensive planting experiment was Maca seeds of four different colour types (yellow, collection Maca grows best at altitudes between 3500 and 4500 m above of 4 years, cultivation area is situated on a plateau at 4200Chinchaycocha m (Lake a.s.l. Jun´ around Lake increased considerably, carried out inhypocotyl the colour Peruvian type Andes andeffect previous to on form the test level of of the cultivation majorhypocotyls secondary have hypothesis metabolites and an found that in leaves. maca these Additionally, two factors it of influence was interact. examined whether soil characteristics of the two terrains are described in Table 1. number CR95S2-22-4; pink,lead-coloured, RO95S2-306-8; PLS2-284-20) violet, were obtained MOS2-142-1; fromcollection the of one germplasm of the authorsde (DD Ponce Pasco, Aguirre, Pasco, UNDAC, Cerro Peru). This collection had been established in a was chosen as it iscultivation. situated in Two an area terrains traditionallybeen used were for cultivated maca selected, before onecultivated with which with any had crop macaperiod. never and 2 The years one distance previously, between thatensured the followed had that two been by terrains the was microclimates aterrains 40 and were fallow m, soils tilled which first were withfenced similar. in a The November. plough two Fields and were thenfollowing not with common fertilized a and practice. hoe, not and on Soil irrigated, the samples two were experimentaland collected terrains: once twice again before directly tillingTwenty in after subsamples, October each harvest comprising the in soiltaken without July roots, (after were of removalan the of approximately following the 3–20 year. across cm thin each depth, grass-containing terrain. following The layer)composite a subsamples sample from per were zigzag terrain and homogenized gradient date to (total one www.interscience.wiley.com/jsfa Fields and seeds Asanexperimentalsite,Alpacay MATERIAL AND METHODS For this reason, maca can be found repeatedlysoils, planted sometimes in the in same rotation withwith different fertilization. crops and occasionally harvest of the hypocotyls.fed However, to dried livestock leaves and are guinea-pigs.similar sometimes If beneficial leaves biological can effects be tomaca shown leaves those to may seen have prove for to hypocotyls, livestock. be a valuable supplementary feed for sea level (a.s.l.). Whencontains cultivated at significant lower amounts altitudes it of no macamides. longer The producers’ experience,Combelles, as reported by Humala-Tasso and before and aftermaca cooking, is absence of producedyet cavities) on been is scientifically newly higher confirmed;different cultivated producers when have however, been terrains. observed maca to vary This significantly batchesconcentrations in the of from has its characteristic not constituents, 76 high-altitude regions such as Cusco and the Lake Titicaca area. that at least part of the variation is due to environmental factors. may appear in soils in which itThese had not differences been previously in cultivated. maca soil quality, features as its mayexploitation cultivation of result is the in generally soil resources, perceived differences as in a great

862 863 . . - 1 1 < m m − − µ -( n 6 mm; . gmL 4 µ × 6 mm; 4 . 4 × -octadecadienoic E -octadecadienamide ,8 . The isocratic elution E E 1 ,8 − E Sample amounts of 500 mg -sitosterolobtainedforsignal- . The solutions were injected DM were considered as trace g β -sitosterol, Sigma, St Louis, MO, 1 22 β − × were established. Accuracy was www.interscience.wiley.com/jsfa 1 − mol g trifluoroacetic acid) (C) and acetonitrile µ -benzyl-5-oxo-6 C. The mobile phase consisted of high- gmL 1 1 n ◦ -octodecenamide (‘macamide 3’) and . µ − Z 0 -Benzyl palmitamide (later called ‘macamide www.soci.org < N DM were considered as well. 1 − Candagainof9.Pressureofnitrogenasthenebulizer ◦ trifluoroacetic acid) (C) at a flow rate of 1.0 mL min Data were analysed with Waters Empower software 1 mol g − with minor modification in the composition of the µ C. The mobile phase consisted of distilled water (E) and 1 ◦ . 23,24 12 -benzyl-9-oxo-12 0 . n m particle size; Phenomenex, Torrance, CA, USA) maintained For the analysis of selected phytosterols, the same HPLC pump Concentrations of macaenes, macamides and phytosterols < µ (‘macamide 2’) were measured at 280 nm.major Macamides representatives 1–4 of are the thisin group, concentration, while are the not otherschemical specific are structures minor to of macaare the or shown are in major Fig. unstable. macamides 1.retention The The times and peaks by of were UV spectra. assigned macaene compounds Macaene by and comparisons were macamide of standard isolatedProduct at Research. Their the identitychromatographic National and (thin-layer Centre purity chromatography, were forby HPLC) confirmed Natural the by methods, nuclear analysis magnetic of resonance, high-resolutionspectrometry) the electrospray and through mass spectral comparisondata. with data published spectral (infrared, 1D and 2D acid (‘macaene’) and methoxybenzyl)-hexadecanamide (‘macamide 4’) were measured at a wavelength of 210 nm, while 5-oxo-6 system was used, but(SEDERE, Alfortille, equipped France) and with a Luna a C8 column Sedex (150 75 ELS detector gluconasturtiin standard eliminated this peakHowever, as it gluconasturtiin. was not possibleglucoaubrietin or to glucolimnanthin. definitely These assign GL with peaksbeing concentrations to consistently either Data were again analysed with Waters Empower software. The gradient program was to0.07 change C/0.93 linearly D from 0.5 within C/0.55 D 55 min min. to exclusively This with was acetonitrileas and followed 1/1 re-equilibration for by with 15 washing min. C/D 1’), for 5.0% of residualthe standard predefined limits deviation of and acceptability.quantificationforcampesteroland The therefore limits of well detection and within to-noise ratios of 3 and 10, respectively, were 3.0 and 10 was 0.1 E/0.9with F methanol followed in and the 20 column min. was15 then min A equilibrated with for 0.1 5 E/0.9 min F. Thewassetto40 probe washout temperature of period the ELS exclusively detector purity water (2.5 ml L were sonicated incentrifuged for 2.5 15 mL min at of 2900 methanol for 20 min and then (Milford, MA, USA). For calculatingat total macamides, those found (2.5 ml L gas was 3.5 bar. TestChromadex, Santa rungs Ana, of CA, USA; authentic standardsUSA) (campesterol, confirmed that peaksdid of not interest interfere were withthe well range other separated of 10–1000 and peaks. Linear calibration curves in mobile phase and sample size. were determined following theet al analytical protocols of Ganzera and were not statistically evaluated for treatment effects. However, they were still included in the calculation of total GL. methanol (F) at a flow rate of 1.0 mL min 5 at 30 in triplicate into thebut same equipped HPLC with pumpWaters) a system and as photodiode a usedparticle array Synergi for detector size; GL, Max (model Phenomenex,was RP 996; maintained Torrance, column at (150 CA, 40 USA). Temperature m, µ 14,15 C, as also C18, 5 ◦ 2010 Society of Chemical Industry  60 Lepidium meyenii c < Nbyanautomatic × 25 . v/v). Step B included a 1 − ¨ as,Sweden).ForNDFanalysis, using the Fibertec System M . Base saturation, describing the which were mixed before the This procedure includes internal + ¨ 16 ogan 17 . -amylase (termamyl 120L, type S, 17 α and H et al andglucolimnanthin(3-methoxybenzyl + ;Fluka,Buchs,Switzerland),aninternalGL 3 1 : 861–869 18,19 − 90 2010; co-eluted off the column. The results obtained for the -BC367) was analysed by the same method to allow peak 150 mm column, Waters). Elution was carried out for 1 min  × 20,21 Glucosinolates (GLs) were extracted and analysed in duplicate Gross nutrient composition was also determined based on 6 . Secondary metabolites in hypocotyls and leaves of 4 with purified water (step A) before switchingphase to the second (B: mobile acetonitrile in water, 0.2 L subsequent reversed-phase HPLC analysis (Waters Alliance2695, Model Milford, MA,229 USA). nm with A a Lichrosorb variable standard column UV (SunFire detector was used at Laboratory analyses All analyses weresamples carried were analysed out using established in standard duplicate. methods: The individual soil practised by maca distributors. The samples werea then milled 20-mesh with (0.85 mm) filter.the The same leaves distribution plant were and kept thenfilter under also when a ground dry. with roof a at 20-mesh GL) J Sci Food Agric sedimentation in water for texture,matter, Walkley and Olsen Black for for organic availableavailable P, K, ammonium a acetateammonium 1 : at acetate 1 at pH water pH : 7.0 soil 7.0KCl for for for mixture exchangeable exchangeable Al for cations and conductivity with and pH, and potentially marketable hypocotyls asper well plot as all were intact collected leaves placed in for a later special instrument analysis.the developed The for ‘Fitotoldo’, maca hypocotyls drying, for were called situated 3 close weeks to the in experimental site.of the This equipment a plant consisted of blacksoil. a impermeable The hypocotyls maca plastic were distributor placedtransparent sheet plastic on cover supported, which the tent-like, by sheet two was poles andends at both covered laid allowing by the on a airremoved the to and then replaced circulate. in the Each evening to morning,in ensure that temperature the variation was cover low was betweenwere day transported and to night. Finally, Lima,placed samples where in they were an coarsely industrial milled oven and for final drying at standards and determinationsamples of were added response to centrifugation factors. tubes togetherof Briefly, with sinigrin (20 0.2 mmol 0.3 mL L g identification. Gluconasturtiin (2-phenylethyl GL),(4-methoxybenzylGL) glucoaubrietin standard. The extracts were transformedGL into according eluates of to desulfo- ISO 9167-1, linear gradient starting with20 zero min, B, returning reaching exclusively to5 min B zero after eluting B(ERM with after A 5 min, for followed equilibration. by Standard another canola Novozymes A/S, Bagsvaerd, Denmark) but not withData sodium sulfite. on NDF and ADF were corrected for ash. by high-performance liquidthe chromatography ISO (HPLC) based certifiedseed, with on method small modifications. (ISO 9167-1) developed for canola (Tecator, 1021 Cold Extractor, H samples were treated with exchangeable base fraction of thecalculated. cation exchange capacity, was standard protocols. Briefly, dry matter (DM) and ashautomatically were analysed with a TGA-500USA). Crude (Leco protein (CP) was Corporation, measured as 6 St Joseph, MI, analyser (LECO C/N-Analyzer Type FP-2000,Joseph, Leco MI, Corporation, St USA),Neutral operated (NDF) according and acid toaccording detergent the to fibre Dumas (ADF) Van method. were Soest determined . - E O ,8 05; . et al E × × 0 c < P : 861–869 P OT ∗ ´ ement 90 × × × 01; . Overall 0 2010; ∗ were excluded, as < ∗∗∗ ∗∗∗ ∗∗∗ -benzyl-5-oxo-6 O P n × ∗∗ , respectively (Table 2). D 1 ∗ ∗∗∗ CT O C × × − × 001; . C d 0 2.6 < b J Sci Food Agric and P D TSEM ∗∗∗ × across × C P terrains -values for colour type, terrain and organ. P × 18 a . P ijkl , cultivated with maca 2–3 years ago) and colour type ε 16 . + dry matter) in maca + ijk 1 ε jk − -benzyl palmitamide (macamide 1); (b) + O n ij × T 05). Significance of effects: . T 0 × + C < Colour ik + P O j they were almost never significant. Aspinkmacawasonlyavailableforoneterrain,leavingpinkmacadata mentioned above, data on only for evaluation withcolour Model type 1. means Multiple weremethod. comparisons The tables among performed give least and square (LS) means, adjustedthe standard LS errors of means with (SEM) and Tukey’s colour type, terrain and their interaction.pliedtoevaluatetheeffectofallthreefactorsandtheirinteractions. The third model was ap- After testing all interactions, RESULTS After drying,tions of approximately hypocotyls 910 and 850 g and kg leaves had DM concentra- T × , never cultivated; + C 845843 – 851935 860 845 866 857 – 853 804276 –226 268 273230 – 216 231 246 225 225 12.1 3.1 2.6 i − C + TY P V L CCTC k + b O × ijkl ∗∗ µ organ ε + www.soci.org C Cl = j T + = across ijk jk y + P terrains . O i 2010 Society of Chemical Industry O ij ∗ ∗ ∗ 5 C O ε c × O + + a ∗ i T × ∗∗ P -octadecadienoic acid (macaene). µ C E + = ,8 + E ik µ ijkl ijk O y ε 1 = 1 O O Hypocotyls Leaves × ij + red) on the concentrations of nutrients (mg g y terrain effect, C N H N H ij T + = k × Colour O residual error. Model 1 was used T O C = + + j ε j 9794a – 100a 95a 93 88b 90 T ij T HO 953 – 949 955 909b 908b 908b 919a 956 952186 951 956173 – 192116 190 167 194 197 – 119 106 876 849 909 197 850 192 220 – 198 210 203 180 4.9 114 119 116 109 279 269 281 275 909 – 916 907 (a) (b) (c) ε + + 25 i + TY P V LCCTC i − + + − + − + − + − i C C C + + + µ µ µ = = = leaf) effect and ij ijk ijkl colour type effect, y y y Effects of plant organ (O, hypocotyl/leaf), type of terrain (T: e vs. = 1 (approaching significance). – , missing data. Chemical structure of the three major maca-specific compounds found: (a) . 0 C < -values for each variable within organ applying Model 2: -values for each row applying Model 1: -values for each variable applying Model 3: P Model 3: Model 2: Model 1: P SEM, standard errors of the mean. In original substance. P P Total phenolic compounds were analysed according to the Organic matter Crude protein Neutral detergent fibre d e a b c LS means within organ carrying no common letter are significantly different ( Dry matter Table 2. Acid detergent fibre × (C: Y, yellow; P, pink; V, violet; L, lead-colou Folin–Ciocalteu method. to test colour type effects within terrain, Model 2 served to analyse www.interscience.wiley.com/jsfa (hypocotyl where Statistical analysis The SAS procedure MIXEDused (SAS for Institute, analysis of Inc., variance version applying the 9.1.3) following was models: Figure 1. octadecadienamide (macamide 2); and (c) 5-oxo-6

864

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.201 .401 .400 .50.04 0.05 0.04 0.04 0.14 0.14 0.13 0.12 +

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www.soci.org 0.022 0.13 0.17 – 0.12 0.46 0.38 – 0.35 Glucoalyssin × × −

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.602 .20.18 0.32 0.22 0.16 0.66 0.51 0.69 0.53 × +

0.024 0.19 0.22 – 0.19 .1–06 0.64 0.60 – 0.51 GL alkylthioalkyl Tot.

× × −

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.4 .2 .4b0.05a 0.04ab 0.02b 0.04b .000 .00.00 0.00 0.01 0.00 +

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.0 .8 .9 0.15a 0.09b 0.08b 0.10b .100 .20.02 0.02 0.01 0.01 +

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−

∗∗ ∗ ∗ ∗ ∗∗∗ ∗

.617 .815 .903 .00.26 0.30 0.33 0.39 1.57 1.38 1.70 1.76 +

0.968 0.22 0.29 – 0.37 1.69 1.09 – 1.35 Glucosinalbin × × ×× ×× −

∗∗∗

.036 .033 .007 .60.60 0.76 0.77 0.70 3.35 2.70 3.61 3.20 +

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∗∗∗

14 77 83 88 .136 .43.23 3.14 3.63 3.71 48.84 28.30 37.78 31.43 +

.6–38 2.66 3.85 – 4.56 2.280 Glucotropaeolin 36b–2.1b50.13a 29.51ab – 23.60b

× − 2010 Society of Chemical Industry

∗∗∗ ∗∗∗∗∗ ∗∗∗ ∗∗

Lepidium meyenii c 63 30 23 37 .047 .04.08 4.20 4.72 4.80 53.77 32.39 43.09 36.39 +

.8–49 3.34 4.97 – 5.78 2.442 oa rmtcGL aromatic Total 76b–3.9b55.06a 33.39ab – 27.65b

× −

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72 41 32 47 .650 .14.45 4.71 5.08 5.16 54.78 33.22 44.10 37.23 +

oa GL Total 84b–3.0b56.00a 34.30ab – 28.42b .6–53 3.70 5.39 – 6.16 2.457

− ×

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YPVLCCTC V P TY YPVLCCTC L V P TY TSEM O OT C O CT

× × × × d

Colour P Colour P Overall P terrains terrains

b a a b c

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: 861–869

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fet fpatogn(,hpctlla) yeo eri (T: terrain of type hypocotyl/leaf), (O, organ plant of Effects 3. Table ee cultivated; never , utvtdwt aa23yasao n oortp C Y (C: type colour and ago) years 2–3 maca with cultivated , elw ,pn;V ilt ,la-oord nthe on lead-coloured) L, violet; V, pink; P, yellow; , − + 2010; Secondary metabolites in hypocotyls and leaves of J Sci Food Agric . 3 et al : 861–869 Therefore No explicit ´ ement 90 which differ 29 27 Yellow colour Flavonoids are or intermediate 2010; 4 26,30 26 27 A yellow pigmented where different maca 3 26 except for yellow maca whereas reddish and blue 8 28 J Sci Food Agric The (from the group of the 27 -sitosterol concentrations confirmed earlier results, β except for macamide 2. The lack of response in campes- 3 The characteristic colour pigments are present only in the Overall,colourtypeeffectswerequitefrequentinthehypocotyls flavonoids) probably do notground maca represent hypocotyls. These a compounds are significantcompletely obviously almost part degraded of the even by simple air-drying. terol and they are unlikelydifferences candidates in to the contribute biological to effects the described. colour type colours are typically determined by anthocyanins. hypocotyls were clearly richer than thecentrations yellow hypocotyls of for total con- GL, aromaticGL. GL, To indole GL the andspecifically authors’ alkylthioalkyl analysed for knowledge GL, lead-coloured which isther maca why this confirmation. was finding In requires never a fur- preliminary study, thin outer layer of the hypocotyls, but were rarely found in the leaves ofgenerating these plants. accessions This means of that certainassociated colour with generating types clear metabolic was differences in synthesis probablyprocesses also of underground biomass.varietyofdistinctlydifferentmacahypocotylcoloursdemonstrates The general availability ofthat a colour has been a selection criterion forAs farmers and the breeders. only knownto germplasm provide collection, maca UNDAC, ofwithout Peru, knowing different is the underlying colour able differences in typesbe genotype. for It that could planting, the though genessome responsible secondary for metabolites traits arewhen of they associated would colour be to situated and on a the those same certainpublications chromosome. for However, degree sometimes doamong colours, not especially not evenmaca between and black between differentiate reddish-types and of maca lead-coloured which accurately areor either violet. pinkish where there wasinvestigated here) only and three a other colour difference types. between black maca (not from pathways resulting in(macaene/macamides),phytosterolsandsulfur-containing polyunsaturated GL.The fattyrepeated acids/amides reports of majormetabolite colour concentrations type indicate differencesclear in that, breeding goal, secondary despite colour is the notmapping might just lack help a to casual of characterize attribute the and a underlying gene mechanisms. Effect of the terrain As anticipated fromdid the not proximity differ of much the in two their fields, characteristics. the The soils most obvious products of biosynthesis. the end product of biosynthetic pathways, relationship was observedand between the theviolet increasing metabolites and analysed intensity black. of pigmentation from pink to part typically lacks anthocynanins, colour types had beena phenotypically low selected GL from concentration producers, and of violet maca yellow was maca noted, comparedyellow different hypocotyls from to were the richest pink in present macaene study. (red) interrain The the never-cultivated and in total phenolicas across compounds both in terrains. both The terrains violetin hypocotyls as were macamides well generally 1 high findings, and 2. This is consistent with the preliminary where this colourothers is are also plainattributed white present to different pigments inside. such in as carotenoids, The anthocyanins theand colours inner investigated some part, can while be of all their intermediate products. can be associated with carotenoid pigments www.soci.org C Cl -sitosterol β 2010 Society of Chemical Industry c organ interactions were × 1; especially in the never cultivated terrain), but . 0 < P The level of the phytosterol campesterol in the hypocotyls was Colour effects were significant for macaene and macamides, The total, and most individual, GLs were much higher in www.interscience.wiley.com/jsfa DISCUSSION Effect of colour type There were certain colour type differencesthe in nutrient hypocotyls contents originating of fromThis four affected corresponding fibre accessions. especially.trations Colour of type secondary differences metabolites in were concen- larger. The lead-coloured found, mainly due to the response to colour onlyAcross in colours the hypocotyls. and terrains, macaenehigher and in total macamides concentration were compared by to factors leaves, respectively. of Onlyfound 9 to macamides be and 1 above the and 24 detection 2 limitrepresentatives for in were followed concentration. the hypocotyls These trends two found for total macamides. higher in plants grownnotaffectedbyeithercolourtypeororgan.Incontrast, on the never-cultivated terrainshowed but a higher were concentration in the leaves than in thebut hypocotyls did not respond towas the significant other for factors. total Thewith phenolic colour effect a compounds again in trend the tormaca. hypocotyls, the Total highest phenolic concentrations concentrations occurringcolour found effects in were in less yellow the pronounced, were leaves,than about where those threefold of higher the hypocotyls. the colour trends were lessrepresentatives clear of the than total for of 13 the GL found hypocotyls. includedglucoiberin, glucobrassicin, Further progoitrin, epigoitrin, gluconapoleiferin, gluconapin and glucobrassicanapin, butamounts. these were only present in trace but mostly onlycultivated for the terrain hypocotyls wheretended (Table 4). pink to In maca have thewhereas was previously the available, in highest hypocotyls hypocotyls macaene the were levels terrain significantly (not richertwo never in significant), colours macaene cultivated (significant thathypocotyls also were the before poor across other in the total bothviolet macamide terrains). concentration, yellow whereas maca Theconcentration yellow hypocotyls as were wellother (but rich. colours). not Macaene concentration Pink significantly inontheterrainpreviouslycultivatedwithmaca.Theorganeffectwas different hypocotyls maca was from higher the hadvery pronounced, and additional a colour high Although there were some significanttypes in differences terms of among nutrient colour composition ofwas the hypocotyls, small variation in magnitude.composition Colour of type had theeither no leaves plant effect organ. and on The hypocotyls nutrient therebut were richer was poorer in no organic in matter, interactions terrain CP occurring among effect and the factors in especially analysed were in minor. fibre thanconcentration the in leaves. thefactor The hypocotyls of 6 thanindividual for in GL total (often the GL). significantsignificance leaves for Colour for the type the (Table hypocotyls, 3; leaves). effects approaching particularly The were high concentration in frequent of lead-coloured many in the hypocotyls, GLs other was while colours. it Significant variedthe terrain for hypocotyls effects and in were the found indole GLterrain only group. previously cultivated Pink for with hypocotyls maca showed on a the tendency tohigher have GL concentrations than yellow and violetIn maca hypocotyls. the leaves, the highestmaca GL ( concentration was found in yellow

866 867 in O 05; 19 . ∗ × 0 c < P OT 33 P ∗ × × 01; . Overall 0 ∗∗∗ ). This means that < P 36 dry matter) and total ∗∗ 1 while that of the leaves − ∗ ∗∗ ∗∗∗ ∗ ∗∗∗ CTOC ∗∗∗ ∗∗∗∗∗∗ ∗∗∗ ∗∗∗∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 32 001; d . 0 mol g b µ < 0.062 0.213 0.087 to positive P TSEM 35 across www.interscience.wiley.com/jsfa × ∗∗∗ × × P terrains -sitosterol, the only metabolite found in and were even higher than those in fresh β a ∗ × P www.soci.org 19 . as the harvest happened before flowering. The proposed that the GLs synthesized in the leaves . 34 34 ijkl , cultivated with maca 2–3 years ago) and colour type et al ε . + ijk + ε jk + O ij × T 05). Significance of effects: . T 0 × Colour + C < The organ difference in the concentration of GL could be the Currently hypocotyls are almost exclusively consumed by ik + P O j hypocotyls investigated in that study. Consistentconcentration with in this, the the dried GL leaves inthan the that present found study was by higher the same group of previous researchers follow the principle of assimilate translocation and are transported via the phloem from sourcewould to be sink. the In hypocotyls the and present notand the case, Andreasson, seeds the as sinks suggested by Chen quite a large intakes of leavesfrom will any be potential required fertility-enhancing to effects in be livestock. able to profit result of GL translocation from the leaf toand other Andreasson plant organs. Chen concentration of total phenols found inparable to the that hypocotyls found was in a com- range of fruits, dried hypocotyls showed areported higher by GL Li concentration than those was higher; however, the latter was still rather lowto when concentrations compared found in many tropical fodder shrubs. higher concentration in thevariable (ranging leaves from than negative in the hypocotyls, are humans, while leaves are sometimesas fed feed to cattle supplement, or with guinea-pigs biological the effects. idea However, of these benefitingwhat effects from is are known the first about same deduced thethe from consumption secondary of metabolites the that hypocotyls,for are where the presumed biological effects to are be present inEffects responsible a more on concentrated fertility form. by fresh leaves. Differences between plantthose caused organs by were colour type larger in than the hypocotyls. T × , never cultivated; + C i − C + 0.09 –0.06 0.08 –0.15a 0.07 – tr. 0.08b tr. 0.07b 0.320 0.66 – 0.800.07 0.53 0.080.02 0.07 0.02 0.07 0.09 0.020.13 0.10 0.05 0.10 – 0.12 0.09 0.12 0.016 16.19 15.38 15.70 14.73 16.84 – 15.27 15.61 0.298 k + The TY P V L CCTC O µ b 31 × × × × + . = j T ijk ∗ across y + ace; i.e. detected under limit of quantification. . i 2010 Society of Chemical Industry P oncentrations of macaene, macamides and phytosterols ( ij terrains C ε ∗∗ Lepidium meyenii c ∗∗∗ + + ) and bases, as a i -sitosterol in the ∗∗∗ ∗ ∗ ∗∗∗ µ × × × ∗∗ ∗∗∗ ∗∗ ∗∗ )inmaca P C + β 1 2 = − -sitosterol and total + β µ ijkl y = ij The terrain that had been y Hypocotyls Leaves Alchornea cordifolifolia 8–11 Colour : 861–869 6.69a – 4.86b 4.93ab 0.13 0.145.655.85 0.17 – 5.72 0.10 4.61 5.21 4.89 4.91 0.45 0.34 0.41 0.43 1.30b 2.15ab 2.28a 1.78ab 1.79b 2.77ab 3.21a 2.18ab 7.21 8.240.47b0.49b 6.83 – 0.62ab 0.92a 6.03 0.40b 0.79a 0.65ab 0.83 0.86 0.88 0.71 1.40 –1.87 1.80 – 1.94 0.14 2.60 – 2.59 0.08 0.07 0.07 0.010.23 0.06 – 0.05 0.14 0.01 0.11 0.12 0.43 0.06 0.14 – 0.52 0.44 0.025 90 TY P V L CCTC − + − + + + + − + − − − + − 2010; Effects of plant organ (O, hypocotyl/leaf), type of terrain (T: 1 (approaching significance). – , missing data; tr, tr . 0 < -values for each variable within organ applying Model 2: -values for each row applying Model 1: -values for each variable applying Model 3: P P SEM, standard errors of the mean. -Sitosterol P P phenolic compounds (mg gallic acid equivalent g b c d LS means within organ carrying no common letter area significantly different ( Macaene Table 4. Total phenols Macamide 1 Macamide 2 Total macamides Campesterol β × (C: Y, yellow; P, pink; V, violet; L, lead-coloured) on the c Secondary metabolites in hypocotyls and leaves of well as ato higher the base higher saturation. claycultivation. This concentration In was and general, not most previous theoccasionally probably cultivation effects result due with on of the maca previous compositionleaves. had of Macaene only and maca campesterol hypocotyls concentrations and especiallyrespectively were higher and lower when grownpreviously on with the maca. terrain Further studies planted are neededfindings to confirm and these to clarifyconcentration why of soil these properties compounds.support the would The recommendation for influence present a long results the fallowto period do repeated subsequent not cultivation with maca. phenols were found inprevious the finding of leaves. a Thisleaves higher is than concentration in in of the agreement storage organ with of a Effect of the plant organ The two plant organs investigated – hypocotylssignificantly and different leaves in – were concentrations ofsecondary all nutrients metabolites and analysed, most finding for except leaves was for expected, ascompared they campesterol. are to typically This the more fibrous hypocotyls, whichin serve reserve as storage . organshypocotyls rich Across were all richermacamides, colours in while higher concentrations and concentrations of of terrains, GL, the macaene and recently planted with maca even yielded hypocotylsconcentration with a of higher thehealth characteristic also metabolite does macaene. notterrain previously seem Plant cultivated with to maca. have been compromised on the J Sci Food Agric difference was thata the higher soil sum cultivated of with cations maca (mostly before due had to Ca . . ) (ISO ,ed. Book et al ´ andez , Walp.) JAgric J Hered Kluwer, ´ ement C, L. Walp.), in :988–991 : 861–869 Andrologia Rapssamen, 50 ´ ement 90 Walp.) ,TROPENTAG Phytomedicine ¨ ottingen, p. 177 :255–262 (2001). Lepidium Meyenii Lepidium meyenii :347–349 (1994). 2010; 55 HPLC-Verfahren J Agric Food Chem 49 Anthocyanins as Food ). :105–110 (2002). (maca) on reproductive )onbenignprostatic CompetitionforResources Chemical Methods ız Nutritiva de los Andes Lepidium meyenii :208–212 (1981). Maca (Lepidium meyenii 59 1 :463–469 (2005). Physical and Mineralogical Teil 1: Lepidium meyenii Capsicum annuum Econ Bot :368–368 (2008). Chem Pharm Bull 16 ´ (maca) by reversed phase high a I, Variation in chemical Lepidium meyenii 74 erica, Madison, WI (1996). Food Chem (maca) collected in Peru by LC-UV .Part3: . :3583–3597 (1991). J Sci Food Agric ´ erales, in .Part1: turnino P and Schettino O, Chemical La Maca Ra´ 74 J Ethnobiol ´ en ´ ´ on) seeds, sprouts, mature plants and os CF, Glucosinolate contents in maca Lepidium meyenii Phytochemistry Lepidium meyenii Lepidium meyenii Planta Med Chac ´ ardenas R, Maca ( Phytochem Anal No. 5. Soil Science Society of America, Madison, JDairySci Lepidium meyenii ‘maca’. ´ eristiques g ´ etudes ethnobiologiques, Paris, pp. 22–44 (2007). Lepidium meyenii Lepidium meyenii :5621–5625 (2002). ˜ 50 nu and the maca. QuantificationofTanninsinTreeandShrubFoliage. . Universidad Nacional Mayor de San Marcos, Lima, BookSeries , ed. by Humala-Tasso K and Combelles PO. Pitunilla, Methods of Soil Analysis , , ed. by Markakis P. Academic Press, New York, pp. 69–92 Methods of Soil Analysis 5. Soil Science Society of Am , Separation and determination of macaene, macamides and ´ :690–693 (2005). :105–111 (2008). :460–464 (2007). :151–158 (2007). on de Popovici G, Geologia, geofisica y area de origin, in ´ os CF and Aliaga C Lepidium peruvianum Walpers) caract Andean Roots andby Tubers: Hermann Ahipa, M and Arrcacha, HellerInstitute, J. Maca Rome, International pp. and Plant 173–197 Genetic Yacon (1997). Resources Walpers) Institut andin d’ Dordrecht (2003). composition of the hypocotyl of maca ( cultivatedinCzechRepublicandinPeru,in in a Changing World: New Drive2008, for Book Rural of Development Abstracts, ed. by Tielkes(2008). E. Cuvillier, G MillenarianPeruvianFoodPlant,withHighlyNutritionalandMedicinal Properties pp. 27–44 (2001). 53 alkamides from maca ( 99 Colours (1982). Stommel JR, Effects of anthocyaninon foliage and and carotenoid immature combinations fruit color of Cusimamani E and Viehmannov Ministerio de Agricultura, Cerro(2003). de Pasco, Pasco, Peru, pp. 32–69 standardization of Lepidium meyenii with three varietiesparameters of and DNA quantification in adult male rats. hyperplasia induced by14 testosterone enanthate. 39 effect of red maca ( performance liquid chromatography. (2002). Analysis of macamidesby HPLC-UV-MS/MS. in samples of maca ( Methods of the tuber constituents of maca ( Food Chem et al phytosterols of and LC-ELSD methods. WI (1986). Series neutral detergent fiber, and nonstarchto animal in nutrition. relation Bestimmung des Glucosinolatgehaltes. 9167 : 1992), pp. 1–17 (1995). composition of ( several derived commercial products. ´ ordova Herrera HE, Cultivo, in 9 Humala-Tasso K and Combelles PO, La maca ( 8Quir 5 Gasco M, Aguilar J and Gonzales GF, Effect of chronic treatment 7 Melnikovova I, Havlik J, Halamova K, Koucek P, Fern 6 Gasco M, Villegas L, Yucra S, Rubio J and Gonzales GF, Dose–response 10 Chac 25 Makkar HPS, 26 Grisebach H, Biosynthesis of anthocyanins, in 11 C 24 Zhao J, Muhammad I, Dunbar DC, Mustafa J and Khan IA, New 27 Lightbourn GJ, Griesbach RJ, Novotny JA, Clevidence BA, Rao DD and 12 Ganzera M, Zhao J, Muhammad I and Khan IA, Chemical profiling and 22 Avula B, Wang Y-H, Zhao J, Ponce Aguirre D, Manrique I, Cl 23 Muhammad I, Zhao JP, Dunbar DC and Khan IA, Constituents of 14 Klute A, 13 McCollom MM, Villinski JR, McPhail KL, Craker LE and Gafner S, 17 International Organization for Standardization (ISO), 21 Piacente S, Carbone V, Plaza A, Zampelli A and Pizza C, Investigation 15 Sparks DJ, 16 Van Soest PJ, Robertson JB and Lewis BA, Methods for dietary fiber, 18 Johns T, The19 a Li G, Ammermann U and Quir 20 Dini A, Magliuolo G, Rastrelli L, Sa Our 37 www.soci.org C Cl maca improving 2010 Society of Chemical Industry of for c :783–792 (2007). 40 transition Utilization of Diversity in term treatments with three Food Res Int Walp), in (maca) on spermatogenesis in rats. The Transition of Maca from Neglect [Online]. Agricultural Biodiversity and prominencenbsplessons :448–454 (2006). ion/publication/the 103 market , Influence of colour type on the concentration of to , TROPENTAG 2007, Book of Abstracts, ed. by Tielkes E. Lepidium meyenii Lepidium meyenii et al ¨ ottingen, p. 33 (2007). neglect strategies.html [7 January 2010]. to MarketLivelihood Prominence. Papers 1. BiodiversityAvailable: International, Rome, Italy (2009). publications/publicat http://www.bioversityinternational.org/publications/ from use Kreuzer M, multi-pharmacological functions. potential fertility enhancing secondary metabolitesplant of the maca Andean ( Effect of short-term and long- Land Use Systems:Human Needs Sustainable andCuvillier, Organic G Approaches to Meet ecotypes of J Ethnopharmacol ´ ement C, Locher NM, Ponce Aguirre D, Khan IA, Manrique I, 1 Hermann M and Bernet T, 2 Wang Y, Wang, Y, McNeil B and Harvey LM, Maca: an Andean crop with 4 Gonzales C, Rubio J, Gasco M, Nieto J, Yucra S and Gonzales GF, 3Cl results suggest thatat this least should total beimportant glucosinolates as extended and the by active selectedand principle determining health phytosterols. in has not maca This yet been thatmetabolite(s) firmly is facilitates established. that Depending fertility on eventually the dodifferent colour turn types out are likely to to be be preferred. most bioactive, www.interscience.wiley.com/jsfa REFERENCES ACKNOWLEDGEMENTS The authors are grateful to Jianping Zhao for isolatingand the macaene macamide standardMuna compounds. Mergani We thank andanalyses. Carmen Irene We Kunz, Zbinden acknowledge the forFrossard consulting (ETH performing Zurich, activity Switzerland). part of Emmanuel of the CONCLUSIONS The present study demonstrated thatinvestigated the were sensitive secondary to metabolites colour typea and plant organ, clear giving advantagehypocotyls over in the air-dried terms leaves. Thetype of associations have with concentration colour to to besuggest confirmed the that in it air-dried otherof could accessions, the be but associations the interestingfarmers of results tend to to colour cultivate investigate and more than thea metabolites one colour certain nature in type on standardization full theeven fields, of detail. for export the As has product tobe be for to achieved. larger A initiate good markets a startingaccessions point strategic or used would breeding in programme theinclude based analyses present upon of study. the batches First for attempts macaene to and macamides. do this Interactions among factors of influence Significant interactions were most frequentand between colour plant type organ. Thetended major not to reason follow the for colourExceptions this type differences was were of that the yellow hypoctyls. theconcentration maca leaves of hypocotyls that totalopposite had in GL the the and corresponding lowest importance leaves. of macamides, previous Due cultivation with whereas to maca, interactions thecolour this among type generally and terrain was low were absent and thosewere of organ rare. and This terrain indicates thatmuch colour when maca type is cultivated effects on would different terrains. not Furtherare studies differ needed to showtypes whether of interactions soil to be type expressed. and climate cause these

868 869 gas ,Effectof in vitro -sitosterol in et al β :493–499 (2008). -sitosterol on the American ¨ anen M and Mustonen A-M, on sexual behaviour in mice :149–153 (1991). β 71 ¨ 35 att :743–758 (2001). ¨ a 39 gas production and stoichiometric www.interscience.wiley.com/jsfa :341–352 (2002). in vitro Chemosphere 139 ). Lepidium meyenii :598–602 (2000). 55 J Ethnopharmacol www.soci.org ¨ onen I, Ikonen, K, M JAgricSci ¨ ol Plant Physiol Biochem Urology Neovison vison and rats. Evaluation of reproductive safetymink of ( a lipid extract from male albino rats. production. transport. relationship between short chain fatty acid and phenolic compounds, 37 Zheng BL, He K, Kim CH, Rogers L, Shao RY, Huang ZY, 36 Nieminen P, P 35 Malini T and Vanithakumari G, Antifertility effects of 34 Chen S and Andreasson E, Update on glucosinolate metabolism and 33 Getachew G, Makkar HPS and Becker K, Tropical browses: contents of ¨ ull. Arg. J Food Sci Arabidopsis 2010 Society of Chemical Industry Lepidium meyenii c Nutt.). :4026–4037 (2004). 52 , Marabashi W and Iwaschina T, (Schumach. & Thonn.) M :80–87 (2008). 77 Amelanchier alnifolia :25–29 (2008). :263–270 (2002). J Agric Food Chem 7 115 : 861–869 90 Alchornea cordifolia 2010; J Japan Soc Hortic Sci Trends Plant Sci :122–126 (2004). era. Leclercq J, Anti-inflammatory compoundsbark from of leaves and root Anthocyanins of gladiolus cultivars and their contributioncolors. to flower affect phytochemical contents and free radical scavenging activities of Saskatoon69 berries ( J Ethnopharmacol Prior RL, Lipophilic and hydrophilicin antioxidant the capacities United of States. foods 30 Wittstock U and Halkier BA, Glucosinolate research in31 the Mavar-Manga H,Haddad M,Pieters L,BAccelin C,Penge AandQuetin- 29 Kwok BHL, Hu C, Durance T and Kitts DD, Dehydration techniques 28 Takemura T, Takatsu Y, Kasumi M 32 Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE and Secondary metabolites in hypocotyls and leaves of J Sci Food Agric