Original Papers

Metabolomic Differentiation of Maca

(Lepidium meyenii) Accessions Cultivated
under Different Conditions Using NMR
and Chemometric Analysis
Authors

Jianping Zhao 1, Bharathi Avula 1, Michael Chan 2, 3, Cline Clment 4, Michael Kreuzer 4, Ikhlas A. Khan 1, 2

Affiliations

2
3
4

Key words
" maca
l
" Lepidium meyenii
l
" Brassicaceae
l
" metabolite
l
" NMR
l
" chemometrics
l

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi,
University, MS, USA
Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS, USA
British Columbia Institute of Technology, Burnaby, B. C., Canada
ETH Zurich, Institute of Agricultural Sciences, Zurich, Switzerland

Abstract
!

To gain insights on the effects of color type, cultivation history, and growing site on the composition alterations of maca (Lepidium meyenii
Walpers) hypocotyls, NMR profiling combined
with chemometric analysis was applied to investigate the metabolite variability in different maca
accessions. Maca hypocotyls with different colors
(yellow, pink, violet, and lead-colored) cultivated
at different geographic sites and different areas
were examined for differences in metabolite expression. Differentiations of the maca accessions
grown under the different cultivation conditions
were determined by principle component analyses (PCAs) which were performed on the datasets
derived from their 1H NMR spectra. A total of 16

Introduction
!
received
revised
accepted

January 14, 2011

May 25, 2011
July 6, 2011

Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1280117
Published online August 19,
2011
Planta Med 2012; 78: 90101
Georg Thieme Verlag KG
Stuttgart New York
ISSN 00320943
Correspondence
Prof. Dr. Ikhlas A. Khan
National Center for
Natural Products Research
Research Institute of
Pharmaceutical Sciences
University of Mississippi
University, MS 38677
USA
Phone: + 1 66 29 15 78 21
Fax: + 1 66 29 15 79 89
ikhan@olemiss.edu

Maca, Lepidium meyenii Walpers (Brassicaceae), is

a perennial herbaceous plant grown on the high
Andean plateaus in Peru and Bolivia. It has been
one of the main staple crops for the indigenous
peoples of the area and is now an important economic crop [1, 2]. The cultivation history of maca
can be dated back to at least 2000 years [3]. Maca
plants have the unique ability to grow best at high
altitudes (3500 to 4500 m a. s. l.) under intense
sunlight, high wind, and fluctuating temperatures
[4, 5]. Maca is traditionally used as food. The tuberous root of maca, the hypocotyl, is generally
consumed fresh or dried, and possesses a tangy
taste and an aroma similar to that of butterscotch.
According to folk belief, maca can enhance male
sexual drive and female fertility in humans [4]. In
recent years, maca products have gained popularity throughout the world as dietary supplements
used for aphrodisiac purposes and to increase
stamina. The plant is also reputed to regulate hormonal secretion, stimulate metabolism, and im-

Zhao J et al. Metabolomic Differentiation of Planta Med 2012; 78: 90101

metabolites were identified by NMR analysis, and

the changes in metabolite levels in relation to the
color types and growing conditions of maca hypocotyls were evaluated using univariate statistical
analysis. In addition, the changes of the correlation pattern among the metabolites identified in
the maca accessions planted at the two different
sites were examined. The results from both multivariate and univariate analysis indicated that the
planting site was the major determining factor
with regards to metabolite variations in maca
hypocotyls, while the color of maca accession
seems to be of minor importance in this respect.
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica

prove memory [6]. Due to its wide spectrum of

putative qualities, maca is sometimes called Peruvian ginseng. Although many chemical and biological studies have been conducted on maca during the past three decades, the principal bioactive
components of the plant have not yet been identified.
Due to the increased interest in maca, its plantation area has drastically expanded in Peru since
the late 1990s. Cultivation of maca has extended
from the area around Lake Chinchaycocha to other high altitude areas in Peru such as Cusco and
Lake Titicaca [3, 5], as well as high altitude areas
outside of Peru. Usually, maca is preferentially
produced on sandy soils compared to heavy soils.
It has been reported that its sensory quality is improved when it is cultivated on an area that has
previously been cultivated with the plant [7, 8].
Maca hypocotyls have different colors including
yellow, pink, violet, and lead-colored. Although
farmers often use a mix of colors in order to reduce the risk of failure in harvest, distinction of
colors in marketing could make sense, as Gon-

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90

Original Papers

Materials and Methods

!

Plant materials
Maca hypocotyl samples were collected from a field experiment
conducted in 2006 in Peru (see details in [7]). Briefly, the experiment took place at two sites in Peru: Patal (1220 899 S, 7507
86 W, altitude 4086 m a. s. l.) and Alpacayn (1092129 S, 76
05748 W, altitude 4130 m a. s. l.). At each location, two nearby
experimental areas were selected; one (never cultivated) which
had never been cultivated before with any agricultural crop and
one (previously cultivated) which had been cultivated with maca
2 to 3 years earlier, followed by a fallow period. To be consistent
with local cultivation practices, the fields were not fertilized, nor
irrigated. Maca germplasm breeders were selected for color and
thus were able to offer accessions with distinct colors. The
present material was obtained from, and identified by, the owner
of the germplasm collection, Prof. Dante D. Ponce Aguirre, Universidad Nacional Daniel Alcides Carrin, Cerro de Pasco, Pasco,
Peru. The colors selected represent four of the more important
ones, but there are many others. Seedlings of accessions with yellow, pink, violet, and lead-colored hypocotyls were planted on
the experimental sites. Each area was divided into 16 plots, resulting in four replicates for each maca color type. After drying
from the day after harvest, the hypocotyls were sliced and lyophilized for later analysis. Due to limitations in harvest, only 55 samples from the plots (28 from Patal and 27 from Alpacayn) were
" Table 1, including their
available for the present study (see l
voucher numbers). Voucher specimens of all samples were de-

posited at the National Center for Natural Products Research

(NCNPR), University of Mississippi, Mississippi, USA.

Chemicals and reagents

All chemical reagents used in the present study were of analytical
grade. Deuterated methanol (CD3OD, 99.8 % D) was purchased
from Cambridge Isotope Laboratories, and sodium 3-trimethylsilyl [2,2,3,3-2H4] propanoate (TSP) was obtained from Sigma-Aldrich, Inc.

Sample preparation
Powdered and homogenized hypocotyl samples (200 mg) were
sonicated in 3 mL of methanol for 15 min followed by centrifugation for 15 min at 4500 rpm. The supernatant was transferred to a
15-mL vial. The extraction procedure was repeated thrice and the
respective supernatants combined. The extract was dried by removing the solvent under vacuum with a Thermo Savant SpeedVac system (Thermo Scientific). Parallel triplicate experiments
were conducted for each sample. The dried extract was dissolved
in 0.4 mL of CD3OD containing 0.05 % (w/v) TSP, then transferred
into a NMR tube for NMR measurement.

NMR measurements
NMR spectra were measured on an Avance DRX400 NMR spectrometer (Bruker BioSpin) equipped with a 3-mm BBO probe
(SN# 9606-3133) operating at a proton NMR frequency of
400.13 MHz at a temperature of 298 K with Bruker Topspin software (v. 1.3). A pulse program with 65 dB power level of selective
irradiation for water presaturation was used to suppress the residual water signal. For each sample, 16 transients were collected
into 64 K data points. The spectra were recorded with the following parameters: spectral width = 4845 Hz, relaxation delay = 5 s,
pulse width = 11.00 s (90). Free induction delays (FIDs) were
Fourier transformed with 0.3 Hz line broadening. The spectra
were manually phased and baseline corrected using Mnova software (v. 5.2, Mestrelab Research S. L.). The 1H NMR spectra were
calibrated to the signal of TSP at chemical shift = 0.00 ppm. The
13
C chemical shifts reported were calibrated to the CD3OD solvent
signal at 49.15 ppm. 2D NMR spectra, including 1H-1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY),
2D J-resolved spectra, 1H-13C heteronuclear single quantum coherence (HSQC), and 1H-13C heteronuclear multiple bonds coherence (HMBC) spectra, were recorded on selected samples. The
measurements of 2D spectra were performed using the standard
pulse sequences of the Bruker Topspin (ref. Tab. 1S in the Supporting Information). The optimized coupling constants for HSQC
and HMBC were 145 and 8 Hz, respectively.

Data analysis
1
H NMR spectra were bucketed with equal bin width of 0.02 ppm
over a region of 0.5 to 8.5 ppm after phase and baseline corrections. The spectral regions from 4.75 to 5.05 ppm and 3.27 to
3.37 ppm were excluded to eliminate the effects of water suppression and residual methanol signal. All binning data were normalized to the total spectral area and converted to ASCII format.
The data sets were then imported and subjected to multivariate
statistical analysis using SIMCAP+ software (v. 12.0, Umetrics).
Principal component analysis (PCA) was carried out to look for
compositional similarities and explore the overall metabolite variability in the accessions of maca. PCA is essentially a descriptive
method used to visualize samples present in an N dimensional
space of a starting set of variables into a smaller number of di-

Zhao J et al. Metabolomic Differentiation of

Planta Med 2012; 78: 90101

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zales et al. [4, 9, 10] observed that hypocotyls of different colors

demonstrated different biological properties. Until recently [7,
11], there have been no detailed studies reported on the difference of metabolites for maca hypocotyls with different colors.
The metabolites reported in maca plants include macaenes, macamides, glucosinolates, alkaloids, sterols, amino acids, organic
acids, and fatty acids. Significant variations in the concentrations
of these metabolites have been observed in maca plants obtained
from different producers [7, 12, 13]. It has been suggested that
the cultivation conditions and color types might be factors that
affect the expression of metabolites in maca [7, 11].
A systematic study through the analyses of the secondary metabolites in maca to investigate the influences of site, cultivation history, and hypocotyl color on their variation was initially performed by Clement et al. [7]. In the present study, we applied
NMR spectroscopy in combination with chemometric analysis as
an approach to investigate the metabolite variability in different
maca accessions. NMR-based metabolite profiling technique is
one of the most powerful tools in the rapidly growing field of metabolomics. In comparison with other techniques (i.e., LC, LCMS,
NIR, etc.), the NMR approach can offer holistic views on the
chemical composition of plant extracts without bias [14]. In addition, NMR spectra can provide a wealth of accurate qualitative
and quantitative information regarding the components of a mixture [15]. The intention of the present study was to gain insights
on the relation of maca quality and cultivation condition by using
the NMR technique and to provide information that should be
helpful for maca plantation practices. In addition, the study
aimed to demonstrate that NMR spectroscopy combined with
multivariate and univariate statistical analyses can be a potent
tool for investigating metabolite variations in plants cultivated
under different conditions.

91

Original Papers

Table 1 List of samples.

Site

Area

Color

Serial number

Sample number

Voucher

Patal

Never cultivated

Yellow

I
II
III
IV
I
II
III
IV
I
II
III
IV
I
II
III
IV
I
II
III
I
II
I
II
III
IV
I
II
III
I
II
III
IV
I
II
III
IV
I
II
I
II
III
IV
I
II
III
I
II
III
IV
I
II
III
I
II
III

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55

#3363
#3362
#3360
#3361
#3341
#3337
#3358
#3356
#3340
#3344
#3351
#3352
#3355
#3357
#3346
#3354
#3353
#3364
#3359
#3342
#3343
#3350
#3337
#3338
#3349
#3348
#3347
#3345
#3437
#3421
#3433
#3442
#3449
#3423
#3451
#3430
#3445
#3444
#3448
#3427
#3419
#3450
#3420
#3425
#3422
#3424
#3439
#3434
#3447
#3441
#3438
#3432
#3440
#3435
#3436

Violet

Lead

Pink

Previously cultivated

Yellow

Violet
Lead

Pink

Alpacayn

Never cultivated

Yellow

Violet

Lead
Pink

Previously cultivated

Yellow

Violet

Lead

Pink

mensions, called principal components (PCs), which represent

sources of successively maximized variance of data. By PCA, the
dataset derived from NMR spectra was transformed to a new coordinate system such that the significant variances by any projection of the data could be visualized on the new coordinates (PCs).
The main advantage of this approach was that the NMR spectra

Zhao J et al. Metabolomic Differentiation of Planta Med 2012; 78: 90101

were treated as fingerprints, all metabolites and interactions associated with the changes of metabolite levels were taken into account, giving an overall view of the data. Furthermore, a total of
16 metabolites were identified based on the NMR spectral analy" Table 2), and their distributions in the
ses in the present study (l
maca accessions were examined by using univariate statistical

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92

Original Papers

Group

Adenine

Alanine

8.19 (s)
8.23 (s)
1.50 (d, 7.2 Hz)
3.70

Choline

Fatty acids

5
6

Formic acid
Fumaric acid

Glutamine

Glucotropaeolin

Macamides

1=CH*
4=CH
-CH3*
-CH
COOH
NCH3*
-CH2
=CH
=CCH2
CH2
CH2*
CH2
-CH3
H-COOH*
CH=CH*
COOH
-CH
-CH2
-CH2*
CONH2
2,6 Ar-H
3,5 Ar-H
4 Ar-H
1 Ar-C
1 CH (glu)*
Ph-CH2 ()
Ph-CH2 ()
C=N
-CH2*
Ar-H
1Ar = C
-COCH2
-CON
-CH
-CH2*
COOH
COOH

155.6
153.6
17.5
51.9
175.9
54.9
69.1
129.2
26.6
28.4
26.1
29.3
14.5
168.8
136.7
173.1
55.7
28.0
31.6
178.2
129.4
130.0
128.3
137.4
83.0
39.7
39.7
161.1
44.1
128.6
140.2
36.0
176.3
70.2
42.3
42.3
177.6
180.4
47.2
30.7
25.2
62.7
174.6
93.8
73.4
74.6
75.8
79.5
62.3
64.1
105.6
74.5
71.4
83.7
63.6
103.2
142.7
90.7
18.4
19.3
30.8
61.7
12.4
40.8
24.7
34.7
179.5

10

Malic acid

11

Proline

12

Sucrose

13

Uridine

14

Valine

15
16

Phytosterols
-Aminobutyric acid
(GABA)

-CH
-CH2*
-CH2
-CH2
COOH
1 CH*
2 CH
3 CH
4 CH
5 CH
6 CH2
1 CH2
2 C
3 CH
4 CH
5 CH
6 CH2
1=CH
2=CH
1 CH*
-CH3
-CH3*
-CH
-CH
CH3*
-CH2*
-CH2
-CH2
COOH

3.23 (s)
3.52 (t)
5.35
2.80
2.06
1.62
1.35
0.91
8.50 (s)
6.68 (s)
3.69 (m)
2.14 (m)
2.49 (m)
7.42 (dd, 1.5 and 7.6 Hz)
7.36 (dt, 1.5 and 7.6 Hz)
7.28 (dt, 1.5 and 7.6 Hz)
4.54 (d, 8.8 Hz)
4.25 (d, 16.0 Hz)
4.09 (d, 16.0 Hz)

4.37 (s)
7.28 (d, 8.0 Hz)
2.25 (t, 7.0 Hz)

4.31 (dd, 3.0 and 7.6 Hz)

2.57 (dd, 7.6 and 15.6 Hz)
2.79 (dd, 3.0 and 15.6 Hz)

4.08 (dd, 4.8 and 4.8 Hz)

2.33 (m), 2.13 (m)
1.99 (m)
3.30 (m), 3.42 (m)
5.43 (d, 3.6 Hz)
3.49 (dd, 3.6 and 10.2 Hz)
3.78 (dd, 10.2 and 9.8 Hz)
4.04 (m)
4.14 (m)
3.76, 3.82
3.67 (m)
3.77 (d, 7.4 Hz)
3.41 (m)
3.80 (m)
3.78 (m)
5.76 (d, 8.2 Hz)
8.02 (d, 8.2 Hz)
5.93 (d, 4.6 Hz)
1.05 (d, 7.0 Hz)
1.09 (d, 7.0 Hz)
2.29 (m)
3.52
0.73 (s)
3.01 (t, 7.2 Hz)
1.93 (m)
2.39 (t, 7.2 Hz)

Table 2 Peak assignment for

NMR spectra of maca extracts.

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Compound
1

93

H and 13C chemical shifts (ppm) are reported with respect to TSP signal ( = 0.00 ppm) and MeOD solvent signal ( = 49.15 ppm),

respectively. * Signal for integration

Zhao J et al. Metabolomic Differentiation of

Planta Med 2012; 78: 90101

Original Papers

methods. Using Mnova software, the concentration levels of 16

identified metabolites were obtained based on the integration of
the characteristic peak (free from the interference of other sig" Table 2) in the 1H NMR spectrum and expressed as relnals, see l
ative intensity against the peak area of the internal standard TSP
at 0.00 ppm. The content value for each metabolite was determined as relative intensity using the following equation:
Cx = Ix / Istd Nstd/Nx
where, Ix and Istd are the intensities (area integration) of appropriate signals of the identified metabolite and the internal standard (TSP), respectively. Nx and Nstd are the number of protons
contributing to the resonances.
By comparing the values of individual metabolites from different
sample populations, general observations on the effects that different variables have on metabolite levels can be made without
requiring the determination of the exact quantity of each individual metabolite in each sample, because the experimental conditions for all of the tested samples were identical. For each sample
all replicates were pooled to give a mean value for each individual
metabolite.
Using the values obtained from 1H NMR spectra, Student t-tests
were performed to compare the relative levels of the individual
metabolites measured in the maca samples from the two separate sites (Patal and Alpacayn) as well as the samples obtained
from the two areas (previously cultivated and never cultivated)
within these sites. Kruskall-Wallis tests were used to compare
the relative metabolite levels between the different colored samples within each of the four cultivation areas. The KruskalWallis
test is a nonparametric multiple comparison test that uses
ranked data. Sets showing a significant result were subsequently
analyzed with the Mann-Whitney U test, a nonparametric method to compare two sample sets, adjusted with the Bonferroni correction. The Bonferroni correction uses a corrected significance
level of /n where is equal to the desired significance level
(0.05) and n is equal to the number of comparisons that are required in the analysis. Pearson correlation analysis was performed to test pairwise linear correlations for the identified metabolites in the maca accessions obtained from the two sites. The
correlation coefficient (r) was calculated for each pair of metabolites, and a t-test was conducted to test the statistical significance
of the correlation coefficient. A significance level of p < 0.001 was
required to reject the null hypothesis of uncorrelation between
the two variables. Correspondingly, an absolute value of r larger
than 0.6 was considered to indicate a statistically significant relationship between pairs of metabolites. The statistical analyses
were carried out in Microsoft Excel 2007 (Microsoft Corp.).

Supporting information
The parameters for the 13C and 2D NMR experiments and overview lists of the PCA for all the accessions of maca from the two
growing sites, for the accessions from the Patal the Alpacayn
sites separately, as well as for the four individual growing sites
are available as Supporting Information.

Results and Discussions

!
" Fig. 1 shows a typical 1H NMR spectrum of maca extract. The
l
signals in the 1H NMR spectra of maca extracts were assigned to
individual metabolites on the basis of thorough analyses of the

Zhao J et al. Metabolomic Differentiation of Planta Med 2012; 78: 90101

2D NMR spectra and spiking experiments. The results are shown

" Table 2. The signals of sucrose in the 1H and 13C NMR spectra
in l
could be easily identified because they were relatively strong in
comparison with the other metabolites. Maca contains a high
amount of sucrose, and sucrose crystals were obtained from
maca in our previous study. The 1H and 13C NMR chemical shift
data of sucrose in the extract were in agreement with those of
the sucrose standard reference. Maca contains rich amino acids
[6, 16]. The presence of proline in the extracts and the identification of its signals in the NMR spectra were achieved by 1D and
2D NMR analyses. The protons on the proline ring showed the
characteristic spin system correlations in the TOCSY spectrum.
The assignment was further confirmed by the observations of
the HMBC correlations of the -methine proton ( 4.08 ppm)
and the -methylene protons ( 2.33 and 2.13 ppm) to the carboxylic carbon ( 174.6 ppm) of proline.
In the maca extract, the presence of alanine, glutamine, valine,
and -aminobutyric acid (GABA) was evidenced by the characteristic signals in the 1H and 13C NMR spectra, as well as the correlation cross-peaks in the 2D spectra. The doublet peak at 1.50 ppm
with the coupling constant of 7.2 Hz was attributed to the terminal methyl group of alanine. It exhibited the 1H-1H correlation in
the COSY spectrum to the signal at 3.70 ppm corresponding to
the -methine group (C 51.9 ppm) of alanine. The assignment of
alanine was verified by the evidence that the methyl protons (
1.57 ppm) showed 1H-13C correlations to the -methine carbon
and the carboxylic carbon (C 175.9 ppm) in the HMBC spectrum.
The resonances of glutamine, valine, and GABA in the 1H NMR
spectrum of the extract were assigned also. Their 1H and 13C
chemical shifts of , , and groups, as well as the 13C chemical
shifts of their carboxylic carbons, were determined, as shown in
" Table 2.
l
Macamides are a unique class of alkamide compounds isolated
from maca. Seven macamides have been isolated and reported
in our previous study [17, 18]. The assignment of signals of macamides in the 1H NMR spectrum of maca extract was achieved by
the analyses of the HSQC and HMBC spectra and by the spiking
experiments. N-benzyl is a characteristic structural group for
macamides. The signal at 4.37 ppm, which showed HMBC correlations to the aromatic carbons at 128.6 and 140.2 ppm and
the acyl carbon at 176.3 ppm, was attributed to the methylene
protons of the N-benzyl group. All macamides exhibited the same
chemical shift at 4.37 ppm for the resonance of their characteristic N-benzyl group in the 1H NMR spectrum of maca extract.
Therefore, the integration of the peak at 4.37 ppm could be used
for the quantitative analysis of the total macamides contained in
the extract.
The resonance peaks of fatty acids in the 1H NMR spectrum of
maca extract were identified according to the characteristic
broad signals at 0.90, 1.35, and 1.62 ppm and the correlations
shown in the COSY and TOCSY spectra. The assignment was confirmed by the HSQC and HMBC experiments and by literature data [19]. The characteristic singlet at 6.68 ppm, showing the
HSQC correlation to the olefinic carbon at 136.7 ppm and the
HMBC correlation to the carboxylic carbon at 173.1 ppm, was
assigned to fumaric acid. Formic acid was identified also. It gave
the singlet at 8.50 ppm in the 1H NMR spectrum, and the peak
showed correlation to the carboxylic carbon at 170.4 ppm in the
HSQC spectrum. Malic acid was detected in the maca tuber [20].
In the present study, the presence of malic acid in the maca extract was evidenced by the resonances at 2.57 and 2.79 ppm
corresponding to the methylene group of malic acid. The COSY

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94

Fig. 1

A representative 1H NMR spectrum of maca extract.

spectrum revealed the 1H-1H correlation between the protons of

the methylene group and the hydroxylated methine group corresponding to the resonance at 4.31 ppm. The identification of
malic acid was further confirmed by the observation from the
HMBC experiment, in which the 1H-13C correlations of the methylene protons to the two carboxylic carbons at 177.6 and
180.4 ppm were clearly observed.
Two singlets with chemical shifts at 8.19 and 8.23 ppm in the
downfield region of 1H spectrum were attributed to the two
methine protons on the aromatic ring of adenine. The direct HC
couplings of these methine protons to the aromatic carbons (at
155.7 and 153.6 ppm, respectively), revealed in the HSQC spectrum, provided additional evidence for the assignment. The resonances of uridine could be clearly identified. The doublets at
8.02 and 5.76 ppm that correlated with each other with a coupling constant of 8.0 Hz in the COSY spectrum were assigned to
the signals from protons on the aromatic ring of uridine. Moreover, the HSQC spectrum showed that they correlated to the carbons at 142.7 and 103.2 ppm, respectively. The carbon at 90.7,
showing HSQC correlation to the proton at 5.93 ppm, was assigned as the anomeric carbon of a ribose. The assignment of uridine was further confirmed by the HMBC analysis, in which the
doublets at 8.02 presented 1H-13C correlations to the carbons at
165.1, 151.3, 103.2, and 90.7 ppm, and the doublet at 5.76
showed correlations to the carbons at 165.1 and 142.7 ppm. In
addition, the anomeric proton at 5.93 correlated to the carbons

at 142.7 and 151.3 ppm. The presence of uridine in maca was

previously reported [20].
Glucotropaeolin was identified in the maca extract based on the
1D and 2D NMR spectral analyses. The two methylene protons of
the benzyl moiety of glucotropaeolin showed the characteristic
doublets at 4.25 and 4.09 ppm. They correlated to each other
with a coupling constant of 16.0 Hz in the COSY spectrum, and
both were attached to the carbon with chemical shift at
39.7 ppm as shown by the HSQC spectrum. The HSQC spectrum
also showed that the anomeric proton ( 4.54) of the glucose
moiety of glucotropaeolin attached directly to the carbon at
83.0 ppm which was upfield shifted because of the CS bond.
The assignment of glucotropaeolin was further confirmed by the
evidence that the protons both on the methylene carbon of the
benzyl moiety and the proton on the anomeric carbon of the glucose moiety all showed correlations in the HMBC spectrum to the
same carbon (C=N) corresponding to the characteristic signal at
161.1 ppm.
The singlet at 3.23 ppm in the 1H NMR spectrum was tentatively
identified as coming from the methyl groups of choline. The assignment was supported by the facts that the singlet showed the
HSQC correlation to the methyl carbon at 54.9 and the HMBC
correlations to the carbons at 54.9 and 69.1. To the best of
our knowledge, this is the first time this compound has been reported as a component in maca. The signal at 0.73 ppm in the
upfield range of the 1H NMR spectrum of maca extract, which
showed clear correlations to the carbons at 41.3, 43.5, 57.5,
Zhao J et al. Metabolomic Differentiation of

Planta Med 2012; 78: 90101

95

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Original Papers

Original Papers

Fig. 2 PCA scores plot (A) and loadings plot (B) for
all the accessions of maca from the two growing
sites: the Patal site (square) and the Alpacayn site
(triangle).

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96

and 58.3 ppm in the HMBC spectrum, was provisionally assigned

to the methyl group of phytosterols. The assignment was further
confirmed by the spiking experiment with -sitosterol.
Serving as fingerprints, the 1H NMR spectra of the maca extracts
reflected the variability of metabolites in the different maca accessions. To describe the sample variations and to detect the metabolites responsible for the differentiations of samples, PCA was
performed on the dataset derived from the 1H NMR spectra of
maca accessions. PCA is an unsupervised clustering method that
requires no prior knowledge of the dataset. It is a useful data visualization method that facilitates the grouping of samples according to the difference of the metabolites in the detected sam" Fig. 2 A shows the PCA scores plot for all the maca accesples. l
Zhao J et al. Metabolomic Differentiation of Planta Med 2012; 78: 90101

sions with PC1 and PC3. The first three components of the PCA
model accounted for 49.5 %, 15.0 %, and 10.8 % of the variables, respectively (ref. Tab. 2S in the Supporting Information). As dem" Fig. 2 A, the maca accessions from the Patal site
onstrated in l
could be clearly distinguished from those from the Alpacayn
site, thus indicating the metabolites varied significantly between
the sample groups from the two different growing sites. Therefore, the growing site (comprising soil type, microclimate, occurrence of flooding, etc.) was an important factor affecting the metabolite expressions in maca. Actually, plants from the Patal site
suffered from environmental stress whereas the plants from the
Alpacayn site did not [7]. From the loadings plot corresponding
" Fig. 2 B) the metabolites
to the PC1 and PC3 of the PCA model (l

Original Papers

97

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Fig. 3 PCA scores plots for the maca accessions

from the Patal site (A) and the Alpacayn site (B).
Previously cultivated area (square); never cultivated
area (triangle).

responsible for the separation of the groups were identified. The

resonances of sucrose (3.76, 3.64, 3.46, and 4.04 ppm), glucotropaeolin (7.39, 7.33, 4.51, 4.08 ppm), fatty acids (1.26, 1.30,
5.37 ppm), and glutamine (2.46, 3.72 ppm) made the greatest
contributions to the separation of the groups in the scores plot.
PCAs were further conducted to visualize and to mine the information on the metabolite differences of the maca accessions from
the two different areas (previously cultivated and never cultivated) for each growing site, in an effort to eliminate the influ" Fig. 3 shows the PCA scores plots for
ence of geographic factor. l
the maca accessions from the Patal site (A) and the Alpacayn
site (B), respectively. For both growing sites, the differentiation
between maca planted on the area previously cultivated with

maca and that planted in the never cultivated area could be observed in the scores plot of PC1 and PC2. The results indicated
therefore that cultivation history also influenced the metabolite
variability of maca. In addition, for both sites, the PCA loadings
plots revealed that the resonances corresponding to sucrose and
glucotroaeolin were mostly responsible for the separation of the
maca accession groups harvested from the different growing
areas.
Maca hypocotyls can be classified into different color types according to skin color, and breeders offer accessions with distinct
colors. We assumed that the differences in bioactivity among different color types reported [4, 9, 10] could be directly related to
the variability of metabolites in maca. The NMR data set of maca

Zhao J et al. Metabolomic Differentiation of

Planta Med 2012; 78: 90101

Original Papers

Fig. 4 PCA scores plots for the maca accessions from the four growing
areas: A never cultivated area at the Patal site; B previously cultivated area
at the Patal site; C never cultivated area at the Alpacayn site; D previously

accessions from each of the four single area (previously cultivated

or never cultivated) located at Patal or Alpacayn was separately
subjected to PCA to exclude the interferences of the cultivation" Fig. 4, no PCA scores plot for
based factors. Still, as shown in l
maca accessions with different color types (yellow, pink, violet,
and lead-colored) showed distinct differentiations based on color
type. The results suggested that the metabolite differences
caused by color type were less than those caused by cultivation
factors.
" Table 2, a total of 16 metabolites were identified in
As shown in l
maca extracts by NMR spectral analyses. An examination of the
distribution of these metabolites would provide detailed information on the effects of the different factors investigated. Thus,
univariate statistical methods were conducted to analyze the variation of content level of individual metabolites in different maca
samples. Significant differences were observed in all of the metabolites except proline, valine, and phytosterols, when comparing the samples between the two growing sites (Patal and Alpa" Table 3). For the majority of metabolites examined, the
cayn; l
levels were found to be significantly higher in the samples grown

Zhao J et al. Metabolomic Differentiation of Planta Med 2012; 78: 90101

cultivated area at the Alpacayn site. Color-types of maca hypocotyl: yellow

(square); pink (inverted triangle); violet (triangle); lead-colored (diamond).

at the Alpacayn site with glucotropaeolin and glutamine being

the only exceptions. Sucrose, glucotropaeolin, and glutamine
had the large absolute differences between samples from the
two sites. For the samples from the Alpacayn site, the levels of
adenine, fumaric acid, uridine, and macamides were more than
two times higher than those grown in the Patal site; whereas
the levels of glucotropaeolin and glutamine were more than two
times higher in the samples from the Patal site than those from
the Alpacayn site. Since the contents of macamides are considered to be related to the quality of maca products [12], the results
suggested that soil and climatic conditions are important factors
to be taken into consideration for producing high-quality maca.
With respect to metabolite levels in maca accessions obtained
from the two different areas (previously cultivated or never culti" Table 3),
vated) at the two sites, some differences were observed (l
but these metabolite differences were smaller in magnitude and
number than those between the two sites. Sucrose, glucotropaeolin, fumaric acid, and valine were the only metabolites that
showed a significant difference in content level when comparing
between the two areas for both sites, whereas adenine, proline,

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98

Original Papers

99

Table 3 Comparison of the metabolite levels a,b in maca accessions from the different planting sites. The p value indicates the significance level obtained from a
Students t-test. Results with a p value < 0.05 were considered significantly different.
Patal site

Alpacayn site p Value

Patal site
Previously

Alpacayn site
Never cultivated p Value

cultivated
Formic acid
Adenine
Fumaric acid
Uridine
Sucrose
Glucotropaeolin
Macamides
Choline
GABA
Malic acid
Glutamine
Proline
Fatty acids
Alanine
Valine
Phytosterols
a

11.88
2.52
19.17
18.90
8 527.32
1 007.55
145.76
235.04
400.19
392.13
1 065.06
3 278.48
1 281.33
250.59
317.43
58.35

17.55
27.90
86.13
71.73
10 343.88
522.36
327.42
214.88
528.84
474.21
470.03
3 152.48
1 445.63
336.30
285.45
61.86

< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.029
< 0.001
< 0.001
< 0.001
0.387
< 0.001
< 0.001
0.623
0.054

11.70
2.70
20.84
17.91
7 489.44
878.67
148.37
240.33
443.25
383.90
1 280.52
3 222.86
1 306.89
263.52
363.45
61.98

Previously

Never cultivated

p Value

cultivated
12.06
2.34
17.01
20.34
9 911.07
1 179.36
142.34
227.99
342.77
403.07
777.83
3 352.64
1 247.22
233.37
256.08
53.46

0.824
0.509
0.029
0.225
< 0.001
< 0.001
0.779
0.339
< 0.001
0.547
< 0.001
0.554
0.211
0.123
< 0.001
< 0.001

15.84
25.47
77.67
64.80
9 286.38
450.99
354.92
199.02
516.96
430.70
421.07
3 087.54
1 418.81
334.47
259.95
61.05

19.44
30.69
95.22
79.20
11 482.65
599.31
297.86
231.96
541.67
521.06
522.77
3 222.45
1 474.56
338.25
312.93
62.70

0.037
0.056
0.015
0.008
< 0.001
< 0.001
0.022
0.005
0.311
0.007
0.116
0.508
0.264
0.829
0.001
0.466

The metabolite content is expressed as the relative integration ratio of the characteristic peak to the peak of the internal standard (TSP) at 0.00 ppm (the number of protons

contributing to the peak is taken into consideration). b The data of metabolite content is as the mean of the samples

Table 4 Results of comparison of metabolite levels in maca accessions with different color type of the hypocotyl. Kruskall-Wallis analysis was performed and sets
with a p value < 0.05 were considered significant. Only sets considered significant were compared using a Mann-Whitney U test with the Bonferroni correction.
Patal site
Compound

Area

Formic acid

Adenine
Fumaric acid
Uridine
Sucrose
Glucotropaeolin
Macamides
Choline
GABA
Malic acid
Glutamine
Proline
Fatty acids
Alanine
Valine
Phytosterols

Alpacayn site
Yellow

18.00b
13.95
3.42
2.79
20.48
17.55
16.20
25.65
6 137.37b
10 004.13
843.39
1 203.39
167.45
170.19
254.72
235.74
433.80
309.83
426.69
414.63
1 032.57a
659.43
2 850.57
3 512.88
1 368.95
1 360.22
361.11a
228.78
350.91
225.93
67.17
54.03

Violet
10.08a
7.92
2.25
1.35
20.75
12.06
15.93
16.11
7 164.27a
8 767.62
760.05
1 070.19
137.66
88.02
239.88
211.14
474.35
339.71
362.48
351.72
1 330.92a
825.53
3 212.10
2 984.09
1 317.20
1 131.39
240.24a
227.28
415.29
303.06
63.45
50.58

Lead
7.38a
12.33
2.25
2.16
19.53
17.37
20.52
18.99
8 632.80a
10 732.41
1 039.68
1 280.52
124.47
132.71
233.92
226.72
412.56
352.04
326.30
389.88
1 729.17b
981.14
3 540.69
3 365.96
1 211.76
1 222.02
202.14b
228.87
342.96
282.27
56.94
52.74

Pink

p Value

Area

11.25a
12.69
2.97
2.61
22.55
19.26
18.90
19.53
8 023.50a
9 485.28
871.56
1 093.14
163.85
163.53
232.81
233.14
452.25
365.45
420.17
443.34
1 029.38a
593.37
3 288.11
3 420.36
1 329.57
1 245.15
250.56a
248.07
344.67
220.05
60.48
55.80

0.014
0.311
0.721
0.685
0.446
0.396
0.143
0.332
0.028
0.394
0.052
0.378
0.235
0.449
0.750
0.841
0.149
0.444
0.136
0.514
0.015
0.260
0.420
0.750
0.226
0.288
0.010
0.789
0.248
0.236
0.198
0.540

Yellow

Violet

14.85
16.47
26.37
19.62
70.65
64.89
70.83
56.25
8 856.45
11 042.37
415.26
535.86
321.62
246.69
184.88
198.91
446.45
487.85
362.61a
387.77
329.22
430.56
2 688.71a
2 838.65
1 386.05
1 354.41
309.93
321.39
239.52
270.57
58.53
57.51

12.96
19.80
20.70
30.69
66.60
102.38
56.88
81.90
9 213.57
11 743.20
474.66
619.02
323.06
302.67
186.77
234.99
510.35
538.29
395.78a
514.58
407.97
418.91
2 830.77a
3 009.38
1 318.01
1 456.88
309.75
326.79
254.52
318.99
57.66
62.94

Lead

Pink

12.78
21.06
19.26
20.97
26.37
28.89
36.18
32.40
75.24
97.02
99.27
99.50
64.53
66.69
90.81
78.66
8 505.36 10 179.63
12 067.29 10 757.16
411.84
482.67
600.12
607.86
331.88
440.55
314.55
307.26
188.93
230.44
243.26
237.66
547.16
579.06
580.46
544.28
441.72a
528.17b
578.21
563.67
563.31
454.82
780.71
499.41
3 051.14a 3 761.33b
3 554.91
3 500.96
1 387.53
1 567.94
1 516.68
1 542.02
332.07
384.93
355.44
351.45
266.97
282.24
354.24
289.74
57.45
68.79
61.62
66.90

p Value
0.782
0.661
0.208
0.103
0.061
0.191
0.750
0.094
0.208
0.056
0.438
0.532
0.065
0.251
0.117
0.079
0.059
0.430
0.045
0.063
0.068
0.102
0.045
0.153
0.107
0.191
0.189
0.451
0.202
0.187
0.081
0.102

Superscripts a and b are used to indicate significant differences (p < 0.05) among the samples with different colors. Symbols + and indicate either the area that had previously
never been cultivated () or had been cultivated 23 years ago (+) with maca

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Planta Med 2012; 78: 90101

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Metabolite

Original Papers

fatty acids, and alanine were the ones that did not show a significant difference for any of the sites, and the remaining 8 metabolites showed a significant difference for one of the two sites only.
Sucrose and glucotropaeolin were the metabolites with the largest absolute differences when comparing the samples from between the two sites and between the two areas for each site. Interestingly, at the Patal site, both fumaric acid and valine had
higher levels for the samples from the previously cultivated area
than those from the never cultivated area. At the Alpacayn site,
inverse relationships were observed. Therefore, factors other
than cultivation history seemed to have an influence here.
According to the results of Kruskall-Wallis analysis, metabolite
levels did not appear to be greatly associated with the color of
" Table 4). Some color types in the certain
maca hypocotyls (l
growing areas appeared to have different quantities for some metabolites, such as formic acid, sucrose, malic acid, glutamine, pro" Table 4), than other color types. Still, the
line, and valanine (see l
differences appeared isolated and there was no apparent trend in
such differences. For instance, although the yellow maca had significantly higher formic acid levels than the other colored maca
plants in the never cultivated Patal area, there were no corresponding significant differences in any of the other three areas
for this metabolite. The number of samples used for the statistical
analysis was low for several of the colors investigated, and these
low sample numbers could have prevented the detection of significant differences between the populations. Several of the comparisons did approach significance (see those with p < 0.05 in
" Table 4). Still, apart from the missing general trend, variations
l
among values were high. Therefore, color type might have only a
minor effect on the levels of the 16 metabolites detected in the
plant.
The impact of environmental fluctuations to metabolite variability can be reflected in changes of relationships among metabolites. Correlations between the levels of various metabolites are
the result of transcriptional and/or biochemical processes occurring in cells and under the influence of external stresses [21].
" Fig. 5 shows the correlation matrices separately for the Alpal
cayn and Patal sites as built from the Pearsons correlations
among the 16 metabolites detected in the maca samples and facilitated by using scaled colors. The correlation coefficients
ranged from 0.65 to 0.94, with |r| < 0.6 or |r| > 0.6 being significant at p < 0.001. The correlations among the metabolites were
generally positive for the Alpacayn site, but many negative correlations were observed for the Patal site, and overall the correlations calculated for the Patal site were weak. For the Alpacayn site, choline, fatty acids, phytosterols, proline, and malic acid showed overall strong correlations with most other metabolites. Additionally, changes in correlation pattern (from positive
to negative, or vice versa) were observed for glutamine, glucotropaeolin, sucrose, and valine (correlated to most other metabolites) when comparing between the two sites; whereas such
changes were minimal for adenine, fatty acids, and macamides.
The correlation between valine and sucrose and that between valine and glucotropaeolin changed dramatically from high positive
(r = 0.75 and 0.59, respectively, Alpacayn site) to high negative
(r = 0.65 and 0.57, respectively, Patal site). Similar significant
changes were also observed for the correlation between phytosterols and sucrose and that between phytosterols and glucotropaeolin. Interestingly, the correlation between sucrose and glucotropaeolin, two key metabolites contributing to the differentiation of maca accessions in the PCA models, remained at a high
positive level for both sites (r = 0.69 for Alpacayn, r = 0.81 for Pa-

Zhao J et al. Metabolomic Differentiation of Planta Med 2012; 78: 90101

Fig. 5 Pearsons correlation matrices of 16 metabolites of the maca accessions from the two sites: Alpacayn (A) and Patal (B). The color scale is
relative to the Pearsons correlation coefficients. Statistically significant
correlations (p < 0.001) are considered for coefficients |r| > 0.6. The num" Table 2): 1. adenine,
bers are used for the metabolites (same as given in l
2. alanine, 3. choline, 4. fatty acids, 5. formic acid, 6. fumaric acid, 7. glutamine, 8. glucotropaeolin, 9. macamides, 10. malic acid, 11. proline, 12.
sucrose, 13. uridine, 14. valine, 15. phytosterols, 16. aminobutyric acid
(GABA).

tal). Overall, the number of correlation pairs of metabolites with

high coefficient (|r| > 0.6) was larger for the Alpacayn site than
for the Patal site. It is still not clear how or why the metabolite
correlations changed in such a way for the different growing sites
with their different environmental conditions. More studies are
required to understand in detail these relationships among metabolites and with respect to their response to environmental or
genetic variation.
In conclusion, the NMR methodological approach, combined
with multi- and univariate statistical analyses, can be used as an
effective tool for investigating metabolite variation in maca accessions and evaluating the metabolite changes in association
with the different growing conditions for maca cultivation. The

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100

information provided by this study will be helpful to our understanding of the variation of chemical composition of maca products, and to gain insights on the effects of color type, cultivation
history, and growing site on the metabolite alterations for maca
cultivation or plantation. Both ways of statistical analysis suggest
that the focus in improving maca metabolite concentration first
should be put on planting site, followed by cultivation history,
whereas color type might be of lesser importance here.

Acknowledgements
!

This research is supported in part by Science Based Authentication of Dietary Supplements and Botanical Dietary Supplement
Research funded by the Food and Drug Administration grant
numbers 5U01FD002071-09 and 1U01FD003871-01, and the
United States Department of Agriculture, Agricultural Research
Service, Specific Cooperative Agreement No. 58-6408-2-0009.
We thank Dr. Jon Parcher (Department of Chemistry & Biochemistry, University of Mississippi) for helpful conversations.

Conflict of Interest
!

All authors declare that there are no conflicts of interests in relation with this manuscript.

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