Phytochemistry 58 (2001) 1209–1212

www.elsevier.com/locate/phytochem

Betacyanins from vine cactus Hylocereus polyrhizus

S•awomir Wybranieca,*, Itzhak Platznerb, Shimona Gereshc, Hugo E. Gottliebd,
Marcela Haimberge, Michael Mogilnitzkie, Yosef Mizrahic
a
Department of Chemical Engineering and Technology, Cracow University of Technology, Warszawska 24, Cracow 31-155, Poland
b
Ben Gurion University of the Negev from NRCN, POB 9001, 84190 Beer-Sheva, Israel
c
The Institutes for Applied Research, Ben Gurion University of the Negev, POB 653, 84105 Beer-Sheva, Israel
d
Chemistry Department, Bar Ilan University, 52900 Ramat Gan, Israel
e
Research and Development Division, Makteshim Ltd., 84100 Beer-Sheva, Israel

Received 13 June 2001; received in revised form 18 July 2001

Abstract
The presence of betacyanin pigments and their isoforms has been detected in the fruit of Hylocereus polyrhizus, a vine cactus
native to South America. Along with the known betanin and phyllocactin (60 -O-malonylbetanin), a new betacyanin was structurally
elucidated as betanidin 5-O-[60 -O-(300 -hydroxy-300 -methyl-glutaryl)-b-d-glucopyranoside] (proposed trivial name hylocerenin) by
means of electrospray MS/MS, HPLC, and NMR techniques. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Vine cactus; Hylocereus polyrhizus; Cactaceae; Betacyanins; Betalains; Betanin; Phyllocactin

1. Introduction
Hylocereus polyrhizus [(F. A. C. Weber) Britton and
Rose] and its related species belong to the vine cacti
from the subfamily Cactoideae of the tribe Cacteae
(Raveh et al., 1993). This tribe contains many species
with edible fruits mostly known as pitaya, or pitahaya.
The common name of these fruits is pitaya since they
contain scales on the fruit skin and hence the name
pitaya ‘‘the scaly fruit’’ (Canto, 2000).
In the last two decades efforts have been made to
develop the cultivation of pitayas, vine species of the
genus Hylocereus. Some of them contained pulp of red
and/or purple colors of various hues (Mizrahi et al. 1997;
Mizrahi and Nerd, 1999; Canto, 2000). In Israel, some of
these cacti are already produced commercially, among
them Hylocereus polyrhizus with glowing deep red-pur-
ple fruit flesh (Fig. 1) (Mizrahi and Nerd, 1999). The
pulp of H. polyrhizus is already used in Israel for the
production of red-violet colored ice cream. They have
also the potential to be used in low temperature dairy
drinks and in light drinks with or without other fruit
* Corresponding author. Tel.: +48-12-628-2707; fax: +48-12-633-
3374.
E-mail address: swybran@chemia.pk.edu.pl (S. Wybraniec).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00336-3
juices. In cacti the most important fruit pigments are the
betacyanins and betaxanthins (Gibson and Nobel,
1986), the betalains.
The pigments in these newly domesticated species are
unknown, therefore, their structure elucidation is
reported here.
2. Results and discussion
The presence of the betacyanins (1–3) and their 15R-
isoforms (10 –30 ) has been detected by HPLC of fruit
pulp extracts from H. polyrhizus by their characteristic
spectral properties (Fig. 2). From the ratio of the
absorbances at 540 and 320 nm (1:0.14) the presence of
hydroxycinnamoyl residues as acylating moieties in 1–3
can be excluded (Heuer et al., 1994).
Compounds 1/10 were assigned as betanin and iso-
betanin, by UV-vis spectroscopy and co-chromatography
with authentic betanin from red beet. The assignment was
confirmed by ESI–MS/MS giving the expected proto-
nated molecular ion [M+H]+ (m/z 551) and the pro-
minent daughter ion at m/z 389 [betanidin+H]+.
Subsequent NMR analysis confirmed undoubtedly the
aglycones (betanidin, isobetanidin) in the compounds 1
and 10 , respectively. The only significant differences in
1210 S. Wybraniec et al. / Phytochemistry 58 (2001) 1209–1212
Fig. 1. Fruits of Hylocereus polyrhizus.
chemical shifts are a small (0.03 ppm) shielding of H-15.
The suggestion is that the pairs of compounds are 11Z
or E isomers. The small chemical shift difference between
H-4 and H-7 of 0.12 ppm is characteristic of a substitu-
tion of the hydroxyl group at C-5 (Heuer et al., 1992,
1994). The signals of the 1H NMR were comparable to
those published recently (Strack et al., 1993).
Compounds 2 and 20 were identified as already known
phyllocactin and isophyllocactin, respectively (Fig. 3)
(Minale et al., 1966). The MS and NMR data of phyllo-
cactin has been recently discussed (Kobayashi et al., 2000).
The protonated parent ion ([M+H]+) for compound 2
was found at m/z 637 [550 (betanin)+86 (malonyl)+H]+
and the daughter ions at m/z 551 [betanin+H]+ and 389
[betanidin+H]+. Additional ions at m/z 619 and 593
were assigned to losses of H2O and CO2 (Table 1). The
1D and 2D 1H NMR data of 2 and 20 were virtually the
same as published recently (Kobayashi et al., 2000). The
presence of the aglycone, glucose and malonyl moieties
was confirmed and the small chemical shift of 0.14 ppm
Fig. 2. HPLC pattern of betacyanins from fruit pulp of Hylocereus
polyrhizus. For peak identification see Table 1. Peak numbers refer to
Fig. 3.
Fig. 3. Chemical structures of analyzed betacyanins in Hylocereus
polyrhizus. Compound numbers refer to Fig. 2 and Table 1.
between H-4 and H-7 indicated the substitution at the
hydroxyl group at C-5 of betanidin. Additionally the
AB quartet of H-200 A/H-200 B at =3.46 ppm suggested a
malonate moiety. The low field chemical shift of H-60 A/
H-60 B (4.59 and 4.35) provides definitive evidence that
the malonate moiety is bound to C-60 .
The protonated parent ion ([M+H]+) for compound
3 was found at m/z 695 (Table 1). Characteristic ions at
m/z 551 and 389 indicated that compound 3 is a deri-
vative of betanin and betanidin. The mass difference (m/z
695–551) of 144 suggested the presence of aliphatic acyl
moiety connected to the glucose of 1. The additional
ions found after fragmentation of [M+H]+ for 3 and 30
at m/z 677, 651 and 633 were assigned to losses of H2O
and CO2. Compounds 3 and 30 had 1H NMR spectra
which were virtually identical to those of 2 and 20 ,
respectively, except of the absence of the malonate pro-
tons H2-200 at =3.46 ppm. Instead, an extra singlet of
H3-300 (methyl group) at =1.57 ppm and two new
methylene groups at =2.89 ppm (singlet of H-400 ) and
2.71 ppm (AB quartet of H-200 A/H-200 B) were seen.
These data are in accordance with the MS data and are
indicative for the presence of a 3-hydroxy-3-methyl-
glutaryl moiety (HMG) at C-60 of betanin.
Hence, the combination of ESI–MS and 1H NMR data
identified 3 as betanidin 5-O-[60 -O-(300 -hydroxy-300 -methyl-
glutaryl)-b-d-glucopyranoside], a new betacyanin pigment
(Fig. 3). A similar compound, amaranthin acylated with 3-
hydroxy-3-methyl-glutaric acid (iresinin-I) has been iso-
lated from Iresine herbstii (Amaranthaceae) (Minale et al.,
1966). Upon b-glucuronidase treatment iresinin-I yielded
glucuronic acid and degradation products (Minale et al.,
1966) containing most probably 60 -HMG-betanin, an
 S. Wybraniec et al. / Phytochemistry 58 (2001) 1209–1212 1211

Table 1
Retention time, HPLC–PDA and ESI–MS/MS data of betacyanins from fruit pulp of Hylocereus polyrhizus

Compounda Rt (min) Relative conc. (%) HPLC–PDA (lmax, nm) Characteristic ions from ESI–MS/MS

[M+H]+ Other characteristic ions Betacyanin

1 8.1 25.3 537 551 389 Betanin

10 11.1 7.8 537 551 389 Isobetanin
2 18.2 35.7 539 637 619; 593; 551; 389 Phyllocactin
20 24.3 7.4 538 637 619; 593; 551; 389 Isophyllocactin
3 22.6 19.8 541 695 677; 651; 633; 551; 389 Hylocereninb
30 29.8 4.0 540 695 677; 651; 633; 551; 389 Isohylocereninc
a
Compound numbers correspond to peak numbers in Fig. 2.
b
New compound: betanidin 5-O-[60 -O-(300 -hydroxy-300 -methyl-glutaryl)-b-d-glucopyranoside].
c
New compound: isobetanidin 5-O-[60 -O-(300 -hydroxy-300 -methyl-glutaryl)-b-d-glucopyranoside].

artificially formed compound, which is now shown to be 3.2. Pigment extraction

an endogenously occurring pigment in H. polyrhizus.
The 15S-forms are eluted in HPLC (ODS-type col-
umns with acidic eluents) earlier than the 15R-forms
(the isoforms) (Bokern et al., 1991; Schliemann et al.,
1996). Therefore, peaks 1, 2 and 3 can be attributed to
betanidin based forms, whereas peaks 10 , 20 and 30 to
isobetanidin based forms.
The contents of all pigments in purified extracts
before HPLC fractionation ranged between 30 and 80 mg
betanin equivalents/100 g of fresh fruit pulp, taking E538
(1%)=1120 (Forni et al., 1992) for spectrophotometric
calculations of betacyanins concentration in purified
extracts. Fruit pulp of H. polyrhizus is characterized by the
glowing purple-red colour, which can be attributed to the
presence of 3 in the fruit. Assuming that the E538(1%) of
the all pigments are similar, the relative concentrations of
H. polyrhizus pigments were calculated from the chroma-
togram (Fig. 2) and showed in Table 1.
In summary, the betacyanins of fruits of H. polyrhizus
were identified as the known betanin, phyllocactin and
their iso-forms. Additionally, a new betacyanin was
identified as betanidin 5-O-[60 -O-(300 -hydroxy-300 -methyl-
glutaryl)-b-d-glucopyranoside] and its isoform. The
formation of the 15R-isoforms (10 –30 ) may occur during
fruit maturation, but they could be also artefacts of
isolation.
3. Experimental
3.1. Plant material
Seven-year-old plants of Hylocereus polyrhizus grown
on trellis system under greenhouse conditions in Beer-
Sheva, Israel, were used (Mizrahi and Nerd, 1999). All
flowers were hand-crossed pollinated with other com-
patible clones and tagged (Weiss et al., 1994). Fruits
were harvested for analysis when reaching full ripening
stage, 35 days after pollination for H. polyrhizus (Nerd
et al., 1999). The edible pulp was taken for analysis.
One hundred grams of the peeled fruit (of watery
consistency) were shaken with 200 ml 80% aq. EtOH
for few minutes, followed by fast filtration and cen-
trifugation at 18 000 rpm and 4  C for 20 min. After
careful decantation, the extract was concentrated in
vacuo at 25  C to 3–4 ml and stored in a dark vessel at
18  C. The roots from red beet (Beta vulgaris) was
processed by the same procedure.
3.3. Extract purification
The concentrated betacyanin extracts were purified
prior to prep. HPLC by column chromatography (401.2
cm, i.d.) on Sephadex G-25 (Sigma Steinheim, Germany)
according to Adams and von Elbe (1977) and subse-
quently on LH-20-100 (Sigma Steinheim, Germany)
(Schliemann et al., 1996). The betacyanin fractions were
lyophilized.
3.4. HPLC
Preparative HPLC was carried out with a Waters
Delta Prep 4000 prep. chromatography system (Waters,
Milford MA, USA) equipped with a LiChroCART1 250-
10, LiChrospher1 60 RP-select B, (10 mm) column pro-
tected by a guard column. The separation was performed
isocratically using a mixture of 90% solvent A (0.5%
aq. TFA) with 10% solvent B (acetonitrile) for 35 min, at
a flow rate of 3.1 ml min1 (injection volume: 1 ml; detec-
tion at 538 nm). 1: Rt 14–17 min; 2: Rt 25–29 min; 3: Rt 41–
45 min. To remove the solvent, the pooled fractions were
concentrated in vacuo and subsequently lyophilised. The
yields were: 2—3.3 mg; 3—2.1 mg. After checking the
purity of the fractions by analytical HPLC the com-
pounds were dissolved in appropriate solvents prior to
analysis by UV/vis, electrospray MS/MS or 1H NMR.
Analytical HPLC was carried out on LC-10AT-VP
chromatograph with photodiode-array SPD-M10A VP
detector (Shimadzu Corp., Kyoto, Japan) equipped
1212 S. Wybraniec et al. / Phytochemistry 58 (2001) 1209–1212
with a LiChroCART1 250-4, LiChrospher1 60 RP-
select B, (5 mm) column. The elution system was the
same as for the prep. HPLC at a flow rate of 0.5 ml/min
(injection volume: 10 ml; detection at 538 nm).
3.5. UV/vis
UV/vis absorption spectra in the range of 200–800 nm
were recorded with a Jasco U-530 UV/vis spectro-
photometer (JASCO Corp., Tokyo, Japan). On-line
spectra in the range of 200–700 nm were obtained by
HPLC-photodiode array detection.
3.6. Electrospray MS/MS and NMR
Electrospray mass spectra were obtained with LCQ
Deca FINNIGAN-MAT mass spectrometer (subsidiary
of ThermoQuest Corp., USA), operating in the positive
ion, full scan MS/MS mode. The spectra were taken in
the presence of minute quantities of formic acid pro-
moting [M+H]+ ion production (electrospray voltage
4.5 kV, heated capillary temperature 210  C; sheath gas:
nitrogen; injection: 80 ml min1).
Compound 1 (betanin), (positive ion mode) (m/z, rel.
int.): 551 [M+H]+ (100). Compound 2 (phyllocactin),
(positive ion mode) (m/z, rel. int.): 637 [M+H]+ (100);
daughter ion scan mode of m/z 637: 619 [M+H–H2O]+
(20); 593 [M+H–CO2]+ (100); 551 [betanin+H]+ (65);
389 [betanidin+H]+ (18). Compound 3 (hylocerenin),
(positive ion mode) (m/z, rel. int.): 695 [M+H]+ (100);
daughter ion scan mode of m/z 695: 677 [M+H–H2O]+
(5); 651 [M+H–CO2]+ (3); 633 [M+H–CO2–H2O]+
(7); 551 [betanin+H]+ (3); 389 [betanidin+H]+ (100).
1D and 2D 1H (COSY) NMR spectra were recorded
at 300 K on a Bruker DMX-600 instrument locked to
the major deuterium resonance of the solvent, CD3OD
containing traces of DCl. Chemical shifts are given in
ppm relative to TMS and coupling constants in Hz.
Compound 3 {betanidin 5-O-[60 -O-(300 -hydroxy-300 -
methyl-glutaryl)-b-d-glucopyranoside]} 1H NMR (600.1
MHz, CD3OD/DCl) =8.69 (1H, d, J1112=12.3 Hz,
H-11), 7.34 (1H, s, H-7), 7.22 (1H, s, H-4), 6.46 (1H, s,
H-18), 6.24 (1H, d, H-12), 5.47 (1H, dd, J23A=9.9 Hz,
H-2), 4.87 (1H, d, J10 20 =7.8 Hz, H-10 ), 4.64 (1H, dd,
J1514A=5.3 Hz, J1514B=7.3 Hz, H-15), 4.56 (1H, dd,
J60 A60 B=12.1 Hz, H-60 A), 4.37 (1H, dd, H-60 B), 3.74
(1H, m, J50 60 A=2.1 Hz, J50 60 B=6.5 Hz, H-50 ), 3.73
(1H, dd, J3A3B=16.4 Hz, H-3A), 3.72 (1H, dd,
J14A14B=17.4 Hz, H-14A), 3.57 (1H, m, H-20 ), 3.53
(1H, m, H-30 ), 3.50 (1H, dd, H-3B), 3.43 (1H, m, H-40 ),
3.31 (1H, dd, H-14B), 2.89 (2H, s, H-400 ), 2.71 (2H, ABq,
H-200 A, H-200 B), 1.57 (3H, s, H-300 ).
Acknowledgements
The authors wish to express their thanks to Dr. Y.
Gilboa for his dedicated assistance in the HPLC work.
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