Identification and Quantification of Zeaxanthin Esters in Plants using Liquid Chromatography-Mass Spectrometry

Article abstract of DOI:10.1021/jf034803s

It has been suggested that lutein and zeaxanthin may decrease the risk for age-related macular degeneration. Surprisingly, oleoresins rich in zeaxanthin are not yet available on the market. Several authors have reported enhanced stability of esterified xanthophylls, so plants containing zeaxanthin esters were investigated to establish valuable sources for the production of durable oleoresins. Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [LC-(APCI)MS] was used to unequivocally identify zeaxanthin esters of a standard mixture and in several plant extracts. Zeaxanthin esters were quantified on the basis of their respective molecular masses using zeaxanthin for calibration; total zeaxanthin was determined after saponification of aliquots of the extracts. Thus, dried wolfberries (Lycium barbarum), Chinese lanterns (Physalis alkekengi), orange pepper (Capsicum annuum), and sea buckthorn (Hippophae rhamnoides) proved to be valuable zeaxanthin ester sources. The present LC-MS method allows for an even more detailed analysis of zeaxanthin esters than reported previously.

Full text of DOI:10.1021/jf034803s

7044 J. Agric. Food Chem. 2003, 51, 7044−7049
Identification and Quantification of Zeaxanthin Esters in Plants
Using Liquid Chromatography−Mass Spectrometry
PHILIPP WELLER AND DIETMAR E. BREITHAUPT*
Universita¨t Hohenheim, Institut fu¨r Lebensmittelchemie, Garbenstrasse 28,
D-70599 Stuttgart, Germany
It has been suggested that lutein and zeaxanthin may decrease the risk for age-related macular
degeneration. Surprisingly, oleoresins rich in zeaxanthin are not yet available on the market. Several
authors have reported enhanced stability of esterified xanthophylls, so plants containing zeaxanthin
esters were investigated to establish valuable sources for the production of durable oleoresins. Liquid
chromatography-atmospheric pressure chemical ionization mass spectrometry [LC-(APCI)MS] was
used to unequivocally identify zeaxanthin esters of a standard mixture and in several plant extracts.
Zeaxanthin esters were quantified on the basis of their respective molecular masses using zeaxanthin
for calibration; total zeaxanthin was determined after saponification of aliquots of the extracts. Thus,
dried wolfberries (Lycium barbarum), Chinese lanterns (Physalis alkekengi), orange pepper (Capsicum
annuum), and sea buckthorn (Hippophae rhamnoides) proved to be valuable zeaxanthin ester sources.
The present LC-MS method allows for an even more detailed analysis of zeaxanthin esters than
reported previously.
KEYWORDS: Zeaxanthin esters; LC-(APCI)MS; zeaxanthin oleoresin
INTRODUCTION
esterified zeaxanthin may be more bioavailable than the free
form, as indicated by a recent study with rodents (8). It is known
from human studies with lutein that ingestion of esterified lutein
together with a high dietary fat content enhances bioavailability
(9). On the other hand, the deposition rate in the egg yolk of
hens was higher after the feeding of free xanthophylls (e.g., ref
10). However, the fact that the expensive saponification step,
which is prone to high losses, can be omitted emphasizes the
advantages of the use of zeaxanthin ester-rich plants for potential
oleoresin production.
Zeaxanthin (â,â-carotene-3,3′-diol) and lutein (â,ꢀ-carotene-
3,3′-diol) are widely distributed in plants. Today it is accepted
that a high intake of both xanthophylls protects against age-
related macular degeneration (AMD) and age-related cataract
formation (1, 2), proving both xanthophylls to be ophthalmo-
protective. The serum concentration of lutein and zeaxanthin
may be influenced by dietary intake (3). This was confirmed
by studies of Bernstein et al. (4), in which metabolites of
zeaxanthin and lutein were isolated from human retina tissue.
Zeaxanthin concentrations of several plants were specified
by different authors (e.g., refs 11-13) and can be found in
official databases (e.g., ref 14). Humphries and Khachik (15)
reported the total concentration of all-trans- and several cis-
isomers of zeaxanthin in fruit after saponification of the extracts.
Knowledge of the native zeaxanthin ester pattern is compara-
tively scarce. Basic work has been done by Wingerath and co-
workers (16), who studied zeaxanthin esters in tangerine juice
using MALDI mass spectrometry. Besides â-cryptoxanthin
esters, zeaxanthin monopalmitate as well as five zeaxanthin
diesters (dicaprate, laurate/myristate, dimyristate, myristate/
palmitate, and dipalmitate) were identified. However, the total
amount of zeaxanthin in tangerine juice concentrate was rather
low (1.3 mg/100 g). In the following, the current knowledge of
zeaxanthin esters in plants under investigation is summarized.
As carotenoids are intensely colored, they are widely used
as food colorants. Isolated as raw oleoresin by solvent extraction,
they are available in large amounts while being relatively low-
priced. With regard to the potential health benefits, zeaxanthin
could serve as a multifunctional food additive. Surprisingly, no
zeaxanthin oleoresin can be obtained commercially on an
industrial scale. Thus, several plants containing high amounts
of total zeaxanthin were investigated for their potential use as
sources for the production of oleoresins. Because carotenoid
esters are known to be more stable than free carotenoids (5),
research was focused on plants containing native zeaxanthin
esters. Evidence for an enhanced stability of xanthophyll esters
in a lipoxygenase assay has been presented by Biacs et al. (6).
Lam and But (7) postulated that the natural occurrence of
esterified zeaxanthin contributes to the stability of the medicinal
effect of wolfberries. In addition to these technological aspects,
Red Pepper (Capsicum annuum L.). Capsanthin esters
occurring in red pepper have been studied extensively (e.g., refs
17-19). Less information is available concerning native zea-
xanthin esters, occurring as minor constituents. Biacs and Daood
* Author to whom correspondence should be addressed [telephone (49)
711-4594094; fax (49) 711-4594096; e-mail breithau@uni-hohenheim.de].
10.1021/jf034803s CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/10/2003

Zeaxanthin Esters in Plants
J. Agric. Food Chem., Vol. 51, No. 24, 2003 7045
(20) reported the occurrence of two zeaxanthin diesters but did
not identify them. In a recent study, we used liquid chroma-
tography-atmospheric pressure chemical ionization mass spec-
trometry [LC-(APCI)MS] to identify carotenoid esters in paprika
and found zeaxanthin diesters (laurate/myristate, dimyristate,
myristate/palmitate, and dipalmitate) in fruits from local markets
(19). However, individual zeaxanthin esters were not quantified.
Orange Pepper and Orange Thai Chilies (Capsicum
annuum L.). Sommerburg et al. (11) investigated the carotenoid
composition of several foods and reported that the highest molar
percentage of zeaxanthin is found in orange pepper, followed
by egg yolk, corn, and orange juice. However, the zeaxanthin
ester pattern was not investigated. Davies et al. (21) described
the carotenoid pattern of some color varieties of ornamental
pepper after saponification. They found zeaxanthin to be one
of the parent xanthophylls of orange and red fruits. However,
the detailed zeaxanthin ester pattern is still unknown.
Wolfberries (Lycium barbarum). Wolfberries (Gou Qi Zi,
Fructus lycii) are commonly used as traditional Chinese food
and herbal medicine for the therapy of a number of disorders,
including visual problems (7). The red color of the berries is
mainly caused by zeaxanthin dipalmitate, the characteristic
carotenoid in Lycium varieties (22, 23). Direct analyses of native
extracts of Lycium had been performed by Li et al. (24).
Wolfberries have been used as a dietary source of zeaxanthin
in several feeding studies (8, 25). Recently, physiological effects
of zeaxanthin dipalmitate from Lycium chinense were studied
with rats as model organisms (26). L. chinense was further
described by Khachik in a patent as a plant source for zeaxanthin
from saponified extracts (27). However, total carotenoid esters
have not been characterized using LC-(APCI)MS yet.
Sea Buckthorn Berries (Hippophae rhamnoides L.). Sea
buckthorn fruits are rich in lipids, carotenoids, vitamin C, and
several other micronutrients. Plenty of research has been done
on the different constituents. As an example, Pintea et al. (28)
studied intensively the lipid fraction of carotenolipoproteins.
Novruzov (29) investigated the carotenoids of varieties grown
in Azerbaidjan and found R- and â,â-carotene, lycopene, and
zeaxanthin. One of the main native carotenoids has been
described as zeaxanthin dipalmitate (30, 31). Further zeaxanthin
esters were not given in the literature. Although processes for
the production of food dyes from sea buckthorn are published
(31, 32), the oleoresin is not available on the market. In the
present study, two different varieties originating from Germany
and Romania were investigated.
highly concentrated lutein oleoresins comprising 4-5% zea-
xanthin (37). As far as we know, the carotenoid ester pattern
of zucchini blossoms has not been studied yet. Thus, we
investigated extracts of zucchini blossoms as one example of
an intensely yellow flowering plant with potential use for
zeaxanthin oleoresin production.
Maoka et al. (38) described the occurrence of free zeaxanthin
in plant seeds, and we have additionally investigated an
assortment of seeds [ginkgo (Ginkgo biloba), bittersweet (Celas-
trus orbiculatus), squash (Cucurbita moschata; butternut bush,
calabaza), and corn (Zea mays)].
MATERIALS AND METHODS
Chemicals. Light petroleum (boiling fraction 40-60 °C), methanol,
ethyl acetate, and acetone were purchased from Merck (Darmstadt,
Germany); tert-butyl methyl ether, n-hexane, pyridine (99.8%, over
molecular sieve), lauroyl chloride, myristoyl chloride, palmitoyl
chloride, and stearoyl chloride (purity of all acyl chlorides ) 99%)
were from Sigma-Aldrich (Taufkirchen, Germany); and silica gel
(0.063-0.2 mm) was from J. T. Baker (Griesheim, Germany). All
solvents were distilled before use. High-purity water was prepared with
a Milli-Q 185 Plus water purification system (Millipore, Eschborn,
Germany). Zeaxanthin as reference substance was generously provided
by Roche Vitamins (Basel, Switzerland).
Plant Samples. Fresh red and orange peppers (Ca. annuum L.),
orange Thai chili (Ca. annuum L.), zucchini blossoms (Cu. pepo L.),
persimmon (D. kaki L.), and kernels of fresh sweet corn (Z. mays L.),
which were purchased attached to the cob, were obtained from retail
shops. Heat-dried wolfberries (L. barbarum L.) originating from China
were kindly provided by Rich Nature Nutraceutical Labs, Lynnwood,
WA (http://www.richnature.com). Fresh Chinese lanterns (P. alkekengi)
together with their red ripe husks (not dried) were obtained from florist
R. Mergenthaler, Stuttgart, Germany. Freshly deep frozen sea buckthorn
berries (H. rhamnoides L.) from Germany and Romania were kindly
provided by Bayernwald Fru¨chteverwertung GmbH, Hengersberg,
Germany. Fresh seeds from ginkgo (G. biloba L.), a squash variety
(butternut bush; Cu. moschata), and bittersweet (Ce. orbiculatus Thunb.)
were obtained from B&T World Seeds, Olonzac, France (http://www.
b-and-t-world-seeds.com).
Zeaxanthin Ester Synthesis. Zeaxanthin was isolated from orange
peppers (Ca. annuum L.). Fruits were cut into small pieces, portions
were homogenized with an Ultra Turrax T 25 (Janke & Kunkel, Staufen,
Germany), and the homogenate was extracted immediately with
methanol/ethyl acetate/light petroleum (1:1:1 v/v/v) until the solid
residue was colorless. Combined extracts were saponified with metha-
nolic KOH (30%, w/v) in diethyl ether overnight. Zeaxanthin was
isolated using open column chromatography on silica gel. For elution,
a mixture of light petroleum/acetone 94:6 (v/v) was applied. The
concentration of the resulting solution was calculated on the basis of
an ꢀ value of 144.5 × 103 [L/(mol‚cm)] in ethanol at 450 nm (39) and
a molecular mass of 568.9 g/mol. Subsequently, zeaxanthin mono- and
diesters were synthesized as described earlier (40). In brief, zeaxanthin
(0.5-1 mg) was dissolved in pyridine (2 mL), and the respective acyl
chloride (400 µL) was added dropwise over a period of 3 h. The reaction
solution was transferred into a separatory funnel, washed with water
twice, dried over anhydrous sodium sulfate, and evaporated to dryness.
The residue was redissolved in ethanol (5 mL) and kept at -20 °C. To
separate all-trans-monoesters from diesters as well as from cis-isomers,
semipreparative HPLC on C30 material was employed using isocratic
solvent mixtures consisting of methanol and TBME. For laurate and
myristate esters, a mixture of 60:40 (v/v) was used; for palmitate and
stearate esters, 45:55 (v/v) was applied. Directly after isolation, the
combined fractions of multiple separations were evaporated to dryness
under dim light, redissolved in ethanol, and stored at -20 °C.
Preparation of Samples. Extraction of Carotenoids. Portions were
cut into small pieces and homogenized for 10-15 s. Samples of Chinese
lanterns (fruits, 3 g; husks, 0.5 g), orange and red peppers (5 g each),
orange Thai chili (2 g), sea buckthorn (10 g), wolfberry (2 g),
persimmon (5 g), kernels of sweet corn (10 g), and zucchini blossoms
Chinese Lanterns (Physalis alkekengi). In contrast to the
fruit of Physalis peruViana L. (cape gooseberry, goldenberry),
P. alkekengi is mainly used as an ornamental plant. The bitter
components present in their green parts may irritate the human
intestinal tract; therefore, the berries are normally not consumed.
The carotenoid composition of the fresh red cups was previously
studied by Booth (33), who found zeaxanthin dipalmitate as
the main component.
Persimmon (Diospyros kaki). Daood et al. (34) investigated
the carotenoids, sugars, and organic acids from kaki. Because
zeaxanthin diesters were not separated by chromatography,
individual peak assignment could not be achieved. However,
the area percentage corresponding to zeaxanthin diesters amounted
to 16%, proving the high zeaxanthin concentration. Detailed
data concerning zeaxanthin diesters are not available.
Zucchini Blossoms (Cucurbita pepo L.). The occurrence of
zeaxanthin in flower petals has been known for a long time
(35, 36). The best documented example is marigold (Tagetes
erecta L.), which is commercially used for the production of

7046 J. Agric. Food Chem., Vol. 51, No. 24, 2003
Weller and Breithaupt
Figure 1. Structures of zeaxanthin (Z) monoesters (A) [Z laurate (1; n ) 10), Z myristate (2; n ) 12), Z palmitate (3; n ) 14), Z stearate (4; n ) 16)]
and homogeneous zeaxanthin diesters (B) [Z dilaurate (5; n ) 10), Z dimyristate (6; n ) 12), Z dipalmitate (7; n ) 14), Z distearate (8; n ) 16)].
(10 g) were extracted three times with a mixture of methanol/ethyl
acetate/light petroleum (1:1:1 v/v/v; 50 mL each). Seeds from ginkgo,
butternut bush, and bittersweet were ground and extracted likewise (2
g each). The supernatants were collected and filtered, dried with
anhydrous sodium sulfate, and evaporated to dryness. The residues were
dissolved in TBME/methanol (1:1, v/v; 10 mL) and subjected to HPLC/
DAD or LC-(APCI)MS analyses.
Saponification. For saponification, an aliquot of the crude extract
(5-10 mL) was evaporated to dryness under reduced pressure and
redissolved in diethyl ether (100 mL). After the addition of methanolic
KOH (30%, w/v; 5 mL), the flask was stored overnight. The reaction
mixture was transferred into a separatory funnel, washed with water
three times, dried with anhydrous sodium sulfate, evaporated to dryness
under reduced pressure, and subjected to HPLC analysis (final volume
Figure 2. HPLC chromatogram (DAD, 450 nm) of a mixture of zeaxanthin
) 5-10 mL, according to the volume used for saponification).
mono- and diesters. Peak assignment (Z ) zeaxanthin): Z laurate (1), Z
myristate (2), Z palmitate (3), Z stearate (4), Z dilaurate (5), Z dimyristate
(6), Z dipalmitate (7), and Z distearate (8).
High-Performance Liquid Chromatography (HPLC) and LC-
(APCI)MS. Apparatus and Conditions. The HPLC consists of an HP
1100 modular system (Hewlett-Packard GmbH, Waldbronn, Germany)
with a diode array detector (DAD, 450 nm). For separation, a 250 ×
4.6 mm i.d., 5 µm, YMC analytical column (YMC Europe, Schermbeck,
Germany) with C30-reversed phase material including a 10 × 4.0 mm
i.d. precolumn was used and kept at 35 °C. LC-(APCI)MS was
performed on an HP 1100 modular HPLC system, coupled to a
Micromass (Manchester, U.K.) VG platform II quadrupole mass
spectrometer. The MS parameters and the mobile phases have been
detailed (40). Mass spectra of zeaxanthin esters were acquired with a
scan range of m/z 400-1200. Data were processed with MassLynx
3.2 software.
Calibration. Calibration of zeaxanthin was performed in the range
of 0.3-15 mg/L using zeaxanthin obtained from orange pepper (see
above). A calibration curve was created by plotting the peak area (DAD,
450 nm) versus the concentration. For the calculation of zeaxanthin
esters, the same graph was applied, specifying the zeaxanthin concen-
tration in micromoles per liter. Concentrations of zeaxanthin esters were
calculated according to the respective molecular masses.
Detection Limit of Zeaxanthin Diesters [LC-(APCI)MS]. For estimat-
ing the detection limit of zeaxanthin diesters, zeaxanthin dipalmitate
was employed as a model compound. On the basis of the intensity of
the fragment ion at m/z 1045.9 ([M + H]+), a signal-to-noise ratio of
3:1, and an injection volume of 20 µL, the detection limit was estimated
to be 0.4 µg/mL.
Figure 3. Mass spectrum (APCI, positive mode) of zeaxanthin dipalmitate
(corresponding to 7, Figure 2) as an example of the fragmentation pattern
of a homogeneous zeaxanthin diester.
removed by semipreparative HPLC. For unequivocal identifica-
tion, the standard mixture and all extracts obtained from plants
were analyzed using LC-(APCI)MS in the positive mode. In
each case, the quasimolecular ion ([M + H]+) was clearly
detectable. The fragmentation pathway was dominated by the
loss of one ([M + H - FA]+) or two ([M + H - 2FA]+) fatty
acids. In the case of zeaxanthin monoesters, this led to the
formation of m/z 551.4, whereas diesters formed m/z 533.4, the
“backbone” of zeaxanthin. An exemplary mass spectrum
obtained from zeaxanthin dipalmitate (7) is given in Figure 3,
and the data set used for identification of 1-8 is presented in
Table 1. The described fragmentation pathway not only allows
for the identification of homogeneous esters but also allows for
the assignment of mixed esters, which likewise occur in the
analyzed plant extracts. Thus, all carotenoid esters described
RESULTS AND DISCUSSION
Method Performance. To evaluate the applicability of the
HPLC method for separation of zeaxanthin esters, eight zea-
xanthin monoesters and homogeneous diesters (C12, C14, C16,
and C18; 1-8) were synthesized and separated on a C30
column. Figure 1 illustrates the structures of these compounds.
Figure 2 shows the resulting chromatogram and demonstrates
the applicability of the method for the analysis of zeaxanthin
esters. Multiple analyses and the addition of synthesized standard
compounds proved the stability of xanthophyll esters during
extraction. Small peaks correspond to cis-isomers, incompletely

Zeaxanthin Esters in Plants
J. Agric. Food Chem., Vol. 51, No. 24, 2003 7047
lanterns berries and husks were identical. Figure 4B shows a
chromatogram obtained from a native husk extract. As in
wolfberry extracts, zeaxanthin dipalmitate (7) was the main
xanthophyll. Persimmon contained only minute amounts of total
zeaxanthin (Z/C ) 5%). As discussed earlier (13), the typical
xanthophyll is â-cryptoxanthin. Extracts of zucchini blossoms
showed the most complex xanthophyll pattern of all plants under
investigation; zeaxanthin esters formed only minor components
(Z/C ) 19%). In fresh kernels of sweet corn as well as in seeds
from ginkgo, zeaxanthin was present in the free form, yet no
zeaxanthin esters were detected. However, zeaxanthin concen-
trations were low in both cases (kernels of sweet corn, 0.14
mg/100 g; ginkgo, 0.35 mg/100 g). Neither in bittersweet nor
in butternut bush seeds, a variety of squash, was zeaxanthin
present.
Table 1. LC-(APCI)MS Data of Synthesized Zeaxanthin Mono- and
Diesters (FA ) Fatty Acid; Z ) Zeaxanthin)
m/z a
compound
[M + H]+
[M + H − FA]+
[M + H − 2FA]+
C12 − Z(1)
C14 − Z(2)
C16 − Z(3)
C18 − Z(4)
C12/C12 − Z(5)
C14/C14 − Z(6)
C16/C16 − Z(7)
C18/C18 − Z(8)
751.6 (100%)
779.6 (100%)
807.7 (100%)
835.7 (100%)
933.8 (100%)
989.8 (100%)
1045.9 (100%)
1101.9 (97%)
551.4 (43%)
551.4 (43%)
551.4 (45%)
551.4 (51%)
733.6 (92%)
761.6 (81%)
789.7 (61%)
817.7 (100%)
533.4 (36%)
533.4 (21%)
533.4 (13%)
533.4 (35%)
a The fragment ions (exact masses) and signal intensities (in parentheses) are
given. The assignment of 1−8 corresponds to the peak numbering in Figure 2.
Occurrence of Antheraxanthin. In samples with high
concentrations of zeaxanthin esters (e.g., wolfberry), acylated
antheraxanthin was found as a minor component. Antheraxan-
thin esters were identified on the basis of the UV-vis spectrum,
with maxima at 421/443/473 nm (39) and the corresponding
ester masses. With the exception of orange and red pepper
extracts, antheraxanthin dipalmitate (15) (m/z 1061.9 [M + H]+;
m/z 1043.9 [M + H - H2O]+; m/z 805.6 [M + H - FA]+; m/z
549.4 [M + H - 2FA]+) was unequivocally identified in all
samples. As an example, the occurrence of 15 in an extract of
Chinese lantern husks is shown in Figure 4B. Free antherax-
anthin (9) was found in small amounts only in orange pepper
extracts (Figure 4A). The occurrence of antheraxanthin in
zeaxanthin-rich plants can be explained by the precursor function
of zeaxanthin in the enzymatic pathway of the xanthophyll
biosynthesis (41). It is noteworthy that the second epoxidation
step, which leads to symmetric violaxanthin, obviously does
not occur in the plants under investigation, as violaxanthin was
not present in saponified extracts.
in this study were identified via their molecular ions and the
resulting fragment ions by LC-(APCI)MS.
Zeaxanthin Esters in Selected Plants. Table 2 summarizes
the zeaxanthin ester patterns as well as the concentration of free
(native) and total (after saponification) zeaxanthin of all plants
investigated in this study. Additionally, the area percentages of
zeaxanthin compared to total carotenoids, calculated from
representative chromatograms of saponified samples, are given
[Z/C (%)]. Although ꢀ values of carotenoids and UV-vis
maxima are different, this value allows for estimation of the
proportion of zeaxanthin compared to total carotenoids present
in the respective extract.
Zeaxanthin esters other than those listed in Table 2 were not
detected. In the case of monoesters, no derivatives comprising
fatty acids with chain lengths shorter than C12 or longer than
C18 were found. In none of the investigated plants were
unsaturated fatty acids detectable in the zeaxanthin ester fraction.
With the exception of zeaxanthin dilaurate (5), which was found
only in orange Thai chili, and zeaxanthin distearate (8), which
was present only in zucchini blossoms, other homogeneous
diesters (6, 7) were widely distributed. With the occurrence of
mixed carotenoid esters, only fatty acids differing in two
methylene groups were present (e.g., 12 and 13, Table 2). This
is in accordance with previous results of our group (13). With
the exception of persimmon extracts, all samples contained
zeaxanthin dipalmitate (7), although in varying concentrations.
Thus, the predominant fatty acid used in the biosynthesis of
zeaxanthin esters is palmitic acid.
The predominant xanthophyll of orange pepper was free
zeaxanthin (11, Figure 4A), accompanied by small amounts of
free lutein (10) and antheraxanthin (3,3′-dihydroxy-5,6-epoxy-
â,â-carotene; 9). Zeaxanthin mono- and diesters formed minor
compounds. To our knowledge, this is the first presentation of
the complete xanthophyll pattern of commonly consumed orange
pepper. In contrast to orange pepper, orange Thai chilies and
red pepper comprised only minute amounts of free zeaxanthin,
whereas the major part occurred esterified (see Table 2). The
xanthophyll ester pattern of Thai chilies was extremely complex.
We did not focus in this research on identification of the
remaining xanthophyll esters. The xanthophyll pattern of
wolfberries differed significantly from those of all other plants
under investigation. In accordance with previous results (7),
zeaxanthin dipalmitate (7) was by far the predominant carotenoid
(Z/C ) 89%, Table 2). Furthermore, minor quantities of
â-cryptoxanthin palmitate were present (data not shown). Two
different varieties of sea buckthorn, originating from Germany
and Romania, were examined. Only the Romanian variety
additionally contained a mixed zeaxanthin ester (13) in minor
amounts (Table 2). The zeaxanthin ester patterns of Chinese
Concentration of Total Zeaxanthin. To determine the total
amount of zeaxanthin and to evaluate a potential commercial
use as zeaxanthin oleoresin sources, aliquots of all sample
extracts were saponified using methanolic KOH. The total
zeaxanthin concentrations ranged from 0.04 (persimmon) to 82.4
(wolfberry) mg/100 g (Table 2). The lower concentrations
obtained in some cases after saponification (e.g., red pepper)
may actually be a result of small losses or degradation reactions
during this workup step or may be a consequence of the presence
of cis-isomers of zeaxanthin esters, which were not taken into
account. The high concentration of zeaxanthin in wolfberries
is a result of the drying process, as the loss of water amounts
to 86% (personal communication, Rich Nature Nutraceutical
Labs). The original zeaxanthin concentration of fresh berries
therefore was ∼12 mg/100 g. Surprisingly, the zeaxanthin
concentration of the dried berries was ∼40 times that previously
reported (average ) 2.4 mg/100 g) (7). This huge discrepancy
may be explained by a special drying process or by the variety
used in this study.
To estimate the commercial use of plants as zeaxanthin
source, the total lutein concentration of marigold flower petals
is used for comparison. Piccaglia and Grandi (42) investigated
10 samples obtained from T. erecta and T. patula petals and
found a mean lutein concentration of 170.6 mg/100 g (range )
16.8-569.9 mg/100 g). Neither of the plants under investigation
showed a zeaxanthin concentration as high as that of marigold
species. However, if an oleoresin composed of zeaxanthin as
the main carotenoid is required, wolfberries (Z/C ) 89%),
Chinese lanterns (fruit, Z/C ) 69%; husk, 58%), orange pepper
(Z/C ) 44%), and sea buckthorn (Z/C ) 31-39%) may be of

7048 J. Agric. Food Chem., Vol. 51, No. 24, 2003
Weller and Breithaupt
Table 2. Concentrations of Zeaxanthin Ester as Well as Free and Total Zeaxanthin (Milligrams per 100 g) (after Saponification) in Different Plants (Z
) Zeaxanthin)a
sea buckthorn
(Germany)
sea buckthorn
(Romania)
compound
orange pepper
red pepper
orange Thai chili
C12− Z(1)
C14− Z(2)
C16− Z(3)
0.25 ± 0.04
0.69 ± 0.05
0.28 ± 0.03
6.43 ± 0.72
C12/C12 − Z(5)
C14/C14 − Z(6)
C16/C16 − Z(7)
C12/C14 − Z(12)
C14/C16 − Z(13)
free Z (11)
0.28 ± 0.03
1.68 ± 0.14
0.31 ± 0.02
1.32 ± 0.10
1.00 ± 0.07
0.04 ± 0.01
2.57 ± 0.24
16
0.38 ± 0.03
0.18 ± 0.02
0.24 ± 0.03
0.35 ± 0.03
1.86 ± 0.25
3.03 ± 0.23
44
5.42 ± 0.49
1.67 ± 0.11
5.69 ± 0.06
4.02 ± 0.03
4.50 ± 0.39
1.26 ± 0.14
16.75 ± 2.04
15
0.68 ± 0.03
0.26 ± 0.01
2.34 ± 0.01
31
0.25 ± 0.03
3.34 ± 0.06
39
total Z
b
Z/C (%)
Chinese lantern
(fruit)
Chinese lantern
(husk)
compound
wolfberryc
zucchini blossom
persimmon
C16− Z(3)
1.24 ± 0.13
7.81 ± 0.31
10.07 ± 2.06
98.28 ± 14.90
C14/C14 − Z(6)
C16/C16 − Z(7)
C 18/C18 − Z(8)
C12/C14 − Z(12)
C14/C16 − Z(13)
C16/C18 − Z
free Z (11)
0.66 ± 0.07
0.92 ± 0.12
0.15 ± 0.01
0.81 ± 0.06
0.84 ± 0.09
1.02 ± 0.13
0.46 ± 0.07
3.27 ± 0.42
19
0.05 ± 0.01
160.95 ± 0.44
1.02 ± 0.02
3.91 ± 0.59
0.007 ± 0.004
0.12 ± 0.01
6.39 ± 0.21
69
0.48 ± 0.14
54.89 ± 7.23
58
1.22 ± 0.19
82.4 ± 2.19
89
0.001 ± 0.001
0.044 ± 0.003
5
total Z
b
Z/C (%)
a Quantitative amounts (n ) 3) were calculated on the basis of the respective molecular mass. Numbers of the compounds correspond to Figures 2 and 4. b Z/C [%]
) area zeaxanthin × 100/area total carotenoids (detected at 450 nm, DAD). c Dried berries.
this study has to be replaced by an appropriate solvent (e.g.,
n-hexane) according to law. Further on, extraction yields have
to be optimized. As consumers are sensitive to food additives
in general, products with a “natural dyed” label may attract
interest. Thus, it is anticipated that the commercial use of
zeaxanthin ester oleoresins obtained from several plants men-
tioned in this study will soon be realized.
ACKNOWLEDGMENT
We thank Prof. Dr. Wolfgang Schwack, University of Hohen-
heim, for the excellent working conditions at the Institute of
Food Chemistry.
Figure 4. HPLC chromatograms (DAD, 450 nm) of an extract of orange
pepper (Ca. annuum L.) (A) and Chinese lantern husks (P. alkekengi)
(B). Peak assignment (Z ) zeaxanthin): Z laurate (1), Z myristate (2), Z
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Received for review July 21, 2003. Revised manuscript received
September 9, 2003. Accepted September 9, 2003. This work was
supported by Grant BR2173/1-1 of the Deutsche Forschungsgemein-
schaft (DFG).
JF034803S