Tocotrienol and tocopherol contents of annatto seed accessions

Abstract

The composition of Vitamin E in plants is affected by species, variety, maturity, growing conditions like weather, growing season, intensity of sunlight and soil type, as well as time and manner of harvesting. Screening of 15 accessions of annatto (Bixa orellana L) showed that total tocotrienols in the seeds ranged from 369.60 to 4422.93 pg/g. Delta-Tocotrienol is the major tocotrienol, followed by gamma-Tocotrienol. Alpha-Tocotrienol was not detectable in 7 of the accessions. For 11 annatto accessions, the Vitamin E content in dry seeds was higher than in the fresh seeds while in other 4 accessions, the Vitamin E content was higher in fresh seeds compared with the dry seeds. Almost all the Vitamin E in annatto seeds was made up of tocotrienols with a range in composition of 88.28 to 99.94%. Delta-Tocotrienol was the highest, ranging from 53.67 to 93.51% of the total Vitamin E. By comparison, gamma-Tocotrienol made up 1.09 to 37.31%, while alpha-Tocotrienol made up 0 to 16.72% of the total Vitamin E content in the seeds. The content of Vitamin E in annatto fruit skin (29.72 pg/g) was much lower than the Vitamin E content in the seed. Annatto contains high levels of tocotrienols that are increasingly being associated with multiple health benefits. Several studies have shown that high levels of alpha-Tocopherol depress the bioavailability of tocotrienols and inhibit tocotrienols in their chemo-preventive activity against degenerative diseases. This study shows that the seeds of the annatto plant are a rich source of tocotrienols which are virtually free of tocopherols.

Full text

JournalofScienceandTechnologyintheTropics 15
JournalofScienceandTechnologyintheTropics(2014)10:15-25
Tocotrienol and tocopherol contents of annatto
seed accessions
S. T. Yong1*, H. K. Wong1, M. Mardhati1 and S. L. Tan2
1Strategic Livestock Research Centre, Malaysian Agricultural Research and
Development Institute (MARDI), GPO Box 12301, 50774 Kuala Lumpur, Malaysia
2Academy of Sciences Malaysia, Menara MATRADE, West Wing,
20th Floor, Jalan Khidmat Usaha, 50480 Kuala Lumpur, Malaysia
(*E-mail: yongsuting@mardi.gov.my)
Received 8-3-2014; accepted 26-03-2014
Abstract The composition of vitamin E in plants is affected by species, variety,
maturity,growing conditions like weather, growing season, intensity of sunlight and
soiltype,as wellas timeand mannerofharvesting. Screeningof 15accessions of
annatto (Bixa orellana L.) showed that total tocotrienols in the seeds ranged from
369.60to4422.93μg/g.Delta-tocotrienolisthemajortocotrienol,followedbygamma-
tocotrienol.Alpha-tocotrienolwasnotdetectablein7oftheaccessions.For11annatto
accessions, the vitamin E content in dry seeds was higher than in the fresh seeds
whileinother4accessions,thevitaminEcontentwashigherinfreshseedscompared
with the dry seeds. Almost all the vitamin E in annatto seeds was made up of
tocotrienolswitharangeincompositionof88.28to99.94%.Delta-tocotrienolwasthe
highest,rangingfrom53.67to93.51%ofthetotalvitaminE.Bycomparison,gamma-
tocotrienolmadeup1.09to37.31%,whilealpha-tocotrienolmadeup0to16.72%of
thetotalvitaminEcontentintheseeds.ThecontentofvitaminEinannattofruitskin
(29.72μg/g)wasmuchlowerthanthevitaminEcontentintheseed.Annattocontains
highlevels oftocotrienolsthat areincreasinglybeing associatedwithmultiplehealth
benets.Severalstudieshaveshownthathighlevelsofalpha-tocopheroldepressthe
bioavailabilityoftocotrienolsandinhibittocotrienolsintheirchemo-preventiveactivity
againstdegenerativediseases.Thisstudyshowsthattheseedsoftheannattoplant
arearichsourceoftocotrienolswhicharevirtuallyfreeoftocopherols.
Keywords annatto –seed–tocotrienol–tocopherol–reversed-phaseHPLC
INTRODUCTION
Vitamin E is a collective term for eight naturally occurring compounds: four
tocopherols, namely, alpha (α), beta (β), gamma (γ) and delta (δ), and four
tocotrienols (α, β, γ and δ), that can exhibit the biological activities of α-
tocopherol [1]. Both structures are similar except that the tocotrienol structure has
double bonds on the isoprenoid units. This group of compounds are potent, lipid-
soluble, chain-breaking antioxidants that prevent the propagation of free radical
reactions [1]. Tocopherols are synthesized by most plants and are particularly
abundant in seeds. Most plant-derived foods, especially fruits and vegetables in

JournalofScienceandTechnologyintheTropics
16
the US diet, contain low to moderate levels of vitamin E while the α- and
γ-tocotrienols were at levels usually less than 0.1 mg/100 g [2]. Due to the
abundance of plant-derived foods in our diets, these foods can provide a
signicant and consistent source of vitamin E [3]. The amount of vitamin E in
fruits and vegetables is affected by species, variety, maturity, growing conditions
(weather, growing season, intensity of sunlight, and soil type) and time and
manner of harvesting [4]. Sattler et al. [5] have reported that in plants the
tocopherols are essential for seed longevity and for preventing lipid peroxidation
during germination. Dicotyledoneous plants (e.g., soybean, groundnut)
typically contain tocopherols, predominantly as γ-tocopherol, and secondarily
as δ-tocopherol and α-tocopherol [6]. Monocotyledoneous plants (e.g., oil palm
and rice) typically contain tocotrienols, predominantly as γ-tocotrienol, and
secondarily as δ-tocotrienol and α-tocotrienol [6]. β-tocopherol and β-tocotrienol
are insignicant in abundance in plants, and have negligible or unknown activity
[7]. Dicotyledoneous plants that contain tocopherols may have less (~5%)
tocotrienols, and monocotyledoneous plants that contain tocotrienols may have
more (~30%) tocopherols.
Tocopherol-free tocotrienols [7] are rare and have been reported in plants
like Bixa orellana L., commonly known as annatto. Tocotrienols have been
associated with multiple health benets [7-10], and it has been reported that
the unsaturated carbon ‘tail’ provides tocotrienols with greater anti-oxidative
activity as well as unique functions in lowering cholesterol, providing neuro-
protection and anti-tumorigenesis (anti-cancer) activity, and are thus considered
functionally superior to tocopherols. In human nutrition, vitamin E requirements
increase when intakes of polyunsaturated fatty acids are increased [11]. Several
methods to perform tocopherol and tocotrienol analysis using different detectors
have been reviewed and published [12]. Normal-phase high performance liquid
chromatography (HPLC) methods with a silica column as well as reversed-phase
HPLC methods with a C18 column are commonly used. This paper describes
the screening of annatto accessions and some vegetable oils for tocopherols and
tocotrienols composition using HPLC with a reverse phase C18 silica column.
MATERIALS AND METHODS
Standards of pure tocotrienol isomers (α), (β), (γ) and (δ) from Davos Life
Science Pte. Ltd. (Singapore) were weighed and dissolved with methanol to
provide a 0.04 g/ml stock solution. Standards of tocopherol isomers (α), (β),
(γ) and (δ) were purchased from Sigma Aldrich (St. Louis, MO) and stock
solutions (0.04 g/mL) were made as described above. Working standard

JournalofScienceandTechnologyintheTropics 17
solutions with concentration of 10 ug/ml were prepared by diluting the stock
solutions with methanol. Standards were ltered with a 0.20 µM PVDF syringe
lter prior to injection into the HPLC. The HPLC consisted of a binary pump,
a photodiode array detector, a reverse phase C18 silica column and an auto
injector. Column temperature was set at 40°C. Different ratios of methanol:
water (v/v) as the mobile phase were studied in order to determine the ratio
which gave the best separation of the different component peaks of tocopherols
and tocotrienols. The mobile phases that were evaluated to separate the vitamin
E isomers were the following methanol:water ratios – 99:1, 98:2, 97:3, 96:4
and 95:5. The wavelength was set at 290 nm absorption and the column
was equilibrated with the mobile phase at a ow rate of 1.0 mL/min until the
baseline was stable for analysis to commence.
Sample preparation
Seeds of dry and fresh fruits from 15 accessions of annatto were collected for
determination of vitamin E prole. In this paper, dry fruit refer to fruit naturally
dried on the plant and dehisced, while fresh fruit refer to those which have fully
developed colour but not dehisced. Vegetable oils (palm, soybean, olive, corn,
ricebran and sunower) were purchased from retail outlets in Petaling Jaya for
determination of their vitamin E prole.
Extraction of vitamin E
The samples were ground in a dry mill kitchen blender to ne particles before
vitamin E extraction. Tocopherols and tocotrienols are fat-soluble and readily
dissolve in organic solvents. For vegetable oils, the vitamin E was extracted
using the cold extraction method [13] with slight modications. A
chloroform:methanol (1:1) mixture (50 mL) was added to an accurately
weighed test sample (approximately 1.0 g for vegetable oils) in a 100-mL beaker,
covered and left overnight. The chloroform:methanol containing lipid was then
ltered into a round ask and evaporated to dryness using a Buchi Rotavapor
R-205 set at 40oC. Twenty-ve millilitres of methanol were then added to
dissolve the extracted vitamin E, and the samples were ltered with a 0.2-μM
PVDF syringe lter prior to injection into the HPLC. For the annatto seeds,
an accurately weighed test sample (approximately 1.0 g) was extracted by
the method of Xu [14] which includes a saponication and heating step to
weaken sample matrices to allow the solvent to fully access all tocopherols and
tocotrienols in the sample. Each sample was analyzed in duplicate and if the
results were different by more than 10%, the analysis was repeated. The nal
results were expressed in μg/g.

JournalofScienceandTechnologyintheTropics
18
RESULTS AND DISCUSSION
Quantication of tocopherols and tocotrienols
Evaluation of the different mobile phases showed that the α- and δ-tocopherols
and tocotrienols were adequately separated by the methanol:water ratio of
95:5 at a ow rate of 1.0 mL/min. However the reversed-phase HPLC method
[15] could not completely separate the isomers of β- and γ-tocopherols and
tocotrienols, and these isomers were reported together as a single value. In
reversed-phase HPLC on a C18 column, separation is based on the structure of
the side chain and the number of methyl substituents. It is thus difcult to
completely separate β- and γ-tocotrienols by reversed-phase HPLC, because
both have a similar side-chain structure and number of methyl substituents on
the chromanol ring [14]. As reported by Tan [7], the contents of β-tocopherol
and β-tocotrienol are insignicant in plants, so in this paper the isomers of β-
and γ-tocopherols and tocotrienols are reported as γ-tocopherols and tocotrienols.
The tocopherols and tocotrienols were identied according to the retention
times of the standard solutions. Quantication of the respective vitamin E
isomers was by using linear regression from the calibration curve of the respective
isomers.
The vitamin E elution proles of annatto seeds and vegetable oils are shown
in the chromatograms in Figures 1, 2, 3, 4 and 5. The elution time for all the
vitamin E isomers was less than 12.0 min. The rst isomer to elute was δ-
tocotrienol at about 6.77 min while the last isomer to elute was α-tocopherol at
11.96 min.
Figure 1. Chromatogram of tocopherol and tocotrienol isomers in annatto seeds.
a Tocotrienol = α-tocotrienol; b/g Tocotrienol = β/γ-tocotrienol; d Tocotrienol =
δ-tocotrienol; a Tocopherol = α-tocopherol; b/g Tocopherol = β/γ-tocopherol; d
Tocopherol = δ-tocopherol.

JournalofScienceandTechnologyintheTropics 19
Figure 2. Chromatogram of tocopherol and tocotrienol isomers in an annatto seed
accession showing signicant levels of α-tocotrienol.
Figure 3. Chromatogram of tocopherol and tocotrienol isomers in palm oil.
Vitamin E content in dry seeds was higher than fresh seeds in 11 of the
annatto accessions, while in the remaining 4 accessions, the vitamin E content
was higher in the fresh seeds. There is a big variation in total vitamin E content
among the accessions ranging from a low 377.87 to a high 4426.47 μg/g
(Table 1). Within the vitamin E family, annatto has very high levels of
tocotrienols ranging from 143.94 to 4422.93 μg/g. By comparison, Frega et
al. [16] using GC-MS reported that dry annatto seeds contain 1400-1470 μg/g
tocotrienols, almost all it being δ-tocotrienol. In this study, for most of the
annatto accessions, δ-tocotrienol was the main tocotrienol followed by

JournalofScienceandTechnologyintheTropics
20
Figure 4. Chromatogram of tocopherol and tocotrienol isomers in soyabean oil.
Figure 5. Chromatogram of tocopherol and tocotrienol isomers in corn oil.
γ-tocotrienol, and the presence of these major isomers has been reported by
several researchers [7, 16-18].
Almost all the vitamin E in annatto seeds was made up of tocotrienols with
a range of 88.28 to 100% (Table 2). Delta-tocotrienol was the highest, ranging
from 54.17 to 95.53% of the total vitamin E content. By comparison, γ-tocotrienol
made up 1.09 to 37.31% while α-tocotrienol made up 0 to 16.72% of the total
vitamin E content (Table 2). The content of vitamin E in annatto fruit skin (29.72
μg/g) was much lower than the vitamin E content in the seed (Table 1), and for
the skin, tocotrienols made up 99.23% (Table 2) of the vitamin E. The presence
of signicant levels α-tocotrienol in this study (Fig. 2) is unique as it has not
been reported by other researchers [7, 16-18].

Journal of Science and Technology in the Tropics 21
Table 1. Tocotrienols and tocopherols content (μg/g) in the seeds of the top 5 annatto
accessions having the highest total tocotrienol content.
Accession αT αT3 βγT βγT3 δT δT3 T T3 Total
Vit E
Dry seed 1 12.12 14.06 19.25 582.56 2.3 3427.70 33.67 4024.33 4058.01
Dry seed 6 6.51 77.33 15.80 937.36 6.84 3087.32 29.15 4102.01 4160.31
Fresh seed 7 11.77 108.87 44.42 802.37 4.34 2561.41 60.53 3472.65 3533.18
Dry seed 9 ND 58.35 3.54 1005.49 ND 3359.09 3.54 4422.93 4426.47
Dry seed 15 10.41 ND 1.66 1543.80 2.15 2835.56 14.22 4379.36 4393.58
Mean values for
15 seed accessions
(fresh and dry)
(n=30)
23.22 57.22 17.20 406.57 10.86 1409.24 51.29 1873.03 1928.76
Range for 15 seed
accessions
(fresh and dry)
(n=30)
ND-
294.63 ND-
157.23 1.45-
72.84 7.75-
1005.49 ND-
101.05 284.37-
3427.70 1.45-
368.81 369.60-
4422.93 377.87-
4426.47
Annatto fruit skin
(n=3) ND ND ND 12.87 0.23 16.62 0.23 29.49 29.72
T = Tocopherol; T3 = Tocotrienol; ND = non detectable
Table 2. Tocotrienols and tocopherols content as % of total vitamin E in the seeds of
the top 5 annatto accessions.
Accession αT αT3 βγT βγT3 δT δT3 T T3
Dry seed 1 0.30 0.35 0.47 14.36 0.06 84.47 0.83 99.17
Dry seed 6 0.16 1.86 0.38 22.53 0.16 74.21 0.70 98.60
Fresh seed 7 0.33 3.08 1.26 22.71 0.12 72.50 1.71 98.29
Dry seed 9 ND 1.32 0.08 22.72 ND 75.89 0.08 99.92
Dry seed 15 0.27 ND 0.04 35.14 0.05 64.54 0.32 99.68
Mean data for 15
seed accessions
(fresh and dry)
(n=30)
0.93 4.30 1.33 19.43 0.74 73.07 3.00 96.79
Range for 15 seed
accessions
(fresh and dry)
(n=30)
ND-
9.37 ND-
16.72 0.06-
7.02 1.09-
37.31 ND-
7.84 53.67-
93.51 0.06-
11.72 88.28-
99.94
Annatto fruit skin
(n=3) ND ND ND 43.31 0.77 55.92 0.77 99.23
T = Tocopherol; T3 = Tocotrienol; ND = non-detectable

JournalofScienceandTechnologyintheTropics
22
Vegetable oils are a major source of vitamin E in the human diet, and the
content of vitamin E in vegetable oils is shown in Table 3. Palm oil has the highest
content of vitamin E, while olive oil has the lowest content and sunower oil has
the highest tocopherol content. Palm oil also has the highest tocotrienol content
followed by rice bran oil (Table 3). Tocotrienol content was non-detectable in
olive oil and was very low in sunower and soybean oils.
Gamma-tocotrienol is the main tocotrienol in palm oil and rice bran oil,
followed by α-tocotrienol. The percentage of tocotrienols and tocopherols as
proportions of total vitamin E (Table 4) show that tocotrienols were the major
vitamin E isomers in palm oil (74.38%) and rice bran oil (64.25%) and were
only a very minor portion of the vitamin E in sunower, soybean and corn oils.
Vitamin E is one of the most important antioxidants in animals and plants.
It helps to protect unsaturated lipids in eukaryotic cells against free radicals that
damage DNA and cause age-related pigmentation and skin ageing, cataract,
neuritic plates in Alzheimer’s disease, arteriosclerosis, stroke, heart attack and
tumours [7-10]. Reviews in the literature [7, 19, 20] also show that high levels
of α-tocopherol depress or attenuate the bioavailability of tocotrienols and
interfere with the activity of tocotrienols in chemo-prevention against degenerative
diseases. Paradoxically, a meta-analysis of human randomized controlled trials
showed that natural but not synthetic α-tocopherol supplementation signicantly
increases all-cause mortality [19]. The annatto plant has a very high tocotrienol
content, and indeed some accessions are virtually tocopherol-free. The reported
tocotrienols in the literature contain about 90% δ-tocotrienol and 10% γ-
Table 3.Tocotrienols and tocopherols content (ug/g) in selected vegetable oils.
Sample αT αT3 βγT βγT3 δT δT3 T T3 Total
Vit E
Palm oil
(n=3) 154.21 101.73 3.77 307.36 4.46 62.67 162.44 471.76 634.2
Soya bean oil
(n=3) 27.44 3.64 79.62 ND 30.55 ND 137.61 3.64 141.25
Olive oil
(n=2) 75.16 ND 2.44 ND ND ND 77.60 ND 77.60
Rice bran oil
(n=2) 75.31 41.35 30.98 148.46 0.38 1.90 106.69 191.71 298.40
Sunower oil
(n=2) 345.09 ND 5.57 ND ND 0.61 350.66 0.61 351.27
Corn oil
(n=2) 39.08 8.41 57.95 9.56 2.51 ND 99.54 17.97 117.52
T = Tocopherol; T3 = Tocotrienol; ND = non-detectable

Journal of Science and Technology in the Tropics 23
Table 4.Tocotrienols and tocopherols content as % of total vitamin E in selected
vegetable oils.
Sample aT aT3 βγT βγT3 δT δT3 T T3
Palm oil (n=3) 24.32 16.04 0.59 48.46 0.70 9.88 25.61 74.38
Soya bean oil (n=3) 19.43 2.58 56.37 ND 21.63 ND 97.42 2.58
Olive oil (n=2) 96.86 ND 3.14 ND ND ND 100 ND
Rice bran oil (n=2) 25.24 13.86 10.39 49.75 0.13 0.64 35.75 64.25
Sunower oil (n=2) 98.24 ND 1.58 ND ND 0.17 99.82 0.17
Corn oil (n=2) 33.25 7.16 49.31 8.14 2.13 ND 84.70 15.29
T = Tocopherol; T3 = Tocotrienol; ND = non-detectable
tocotrienol [7, 17, 18]. Researchers have reported that the order of potency
among the isomers for cancer inhibition is δ-tocotrienol > γ-tocotrienol >
α-tocotrienol where α-tocopherol is inactive and δ-tocopherol is weakly
active. Delta-tocotrienol has been shown to inhibit the growth and survival of
pancreatic cancer cells in vitro and in vivo [24, 25], and clinical trials
sponsored by the US National Cancer Institute using pure δ-tocotrienol to treat
pancreatic cancer patients is in progress at the Moftt Cancer Center. In
November 2011, the Malaysian government [26] announced funding for six
clinical trials to be conducted by medical experts in U.S., Singapore and
Malaysia to determine whether tocotrienols are able to prevent the recurrence of
stroke, breast cancer tumour progression and colorectal cancer, and to prolong
the survival of patients with prostate cancer. Two other projects also seek to
investigate the effect of tocotrienols on diabetes mellitus, and ADHD (attention
decit hyperactive disorder).
This study detected signicant levels of α-tocotrienol in some annatto
accessions (up to 157.23 μg/g), a level higher than the content in palm oil. Several
researchers [10, 21-23] have reported that α-tocotrienol has unique functions
and was shown to protect rodents and canine brain from stroke damage, and
they concluded that this isomer of tocotrienol is a natural neuroprotective
agent. Besides oil palm, the seeds of the annatto plant are very rich sources of
tocotrienols which are virtually tocopherol-free. Tan [7], Gee [19, 20] and Trias
and Tan [27] have suggested that α-tocopherol should be eliminated, or at least
reduced, in order to enhance the potency and bioavailability of the tocotrienol-
rich fraction (TRF) for chemo-prevention. They also proposed that for vitamin
E health supplements to be effective, it is desirable to enhance the δ-tocotrienol

JournalofScienceandTechnologyintheTropics
24
and γ-tocotrienol contents in TRF as these forms have higher potencies for
chemo-prevention of cancers and cardiovascular diseases.
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