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To cite this Article Liu, Donghong, Shi, John, Posada, Luidy Rodrguez, Kakuda, Yukio and Xue, Sophia Jun(2008)'Separating
1525-6103
8755-9129
LFRI
Food
Reviews International
International, Vol. 24, No. 4, July 2008: pp. 132
Separating
Liu
et al. Tocotrienols from Palm Oil by Molecular Distillation
Introduction
Palm oil is extracted from the fleshy orange-red mesocarp of the fruit of the oil palm
(Elaeis guineensis). This plant, indigenous to West Africa, has spread to the tropical and
subtropical zones of the world, particularly Malaysia and Indonesia.(1) Crude palm oil
Address correspondence to Dr. John Shi, Guelph Food Research Center, Agriculture and
Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada. E-mail: shij@agr.gc.ca
376
377
consists mainly of glycerides and small quantities of non-glyceride components. The nonglyceride components include free fatty acids (FFA), trace metals, moisture, impurities,
and minor components.(2) Crude palm oil contains approximately 1% minor components,
which include carotenoids, vitamin E (tocopherols and tocotrienols), sterols, phospholipids, glycolipids, terpenic, and aliphatic hydrocarbons.(3) The carotenoids, tocopherols and
tocotrienols are the most important of these minor components. They contribute to the
stability and nutritional properties of palm oil.(4)
Carotenoids impart the characteristic orange-red color to palm oil. Oil from the tenera variety, which is widely planted in Malaysia, has a carotenoid content of about 500700 mg/L.(3)
Alpha and -carotenes are the major components present (ca. 36 and 54%, respectively),
and the rest are -carotene, lycopene, and xanthophylls. Carotenes, particularly -carotene,
are converted into vitamin A in vivo. Unfortunately, most of the carotenoids are degraded
during the refining, bleaching and deodorization processes, which traditionally produce
the light colored oils preferred by most consumers.(1,5)
Palm oil is potentially one of the best sources of vitamin E. The vitamin E content in
palm oil is unique in that it is composed of tocotrienols rather than tocopherols.(6) Palm oil
normally contains 6001,000 mg/L, of which 43% is -tocotrienol, 24% is -tocotrienol,
11% is -tocotrienol, and 21% is -tocopherol.(7) Tocopherols and tocotrienols are potent
natural antioxidants that play an important role in the stabilization of oils and fats.(6,8)
They extend the induction period and delay the time when oxidation produces off-flavors
and/or odors.(1)
Palm tocotrienols may also have beneficial health impacts as they have been reported
to lower plasma cholesterol by inhibiting the activity of HMG-CoA reductase, which
regulates cholesterol synthesis in the liver.(9) Tocotrienols may also play an important role
in suppressing the progression of certain types of cancer particularly breast cancer.(7)
The molecular distillation process for recovering tocopherols using soya oil deodorizer distillate is in commercial production.(8) During steam deodorization and distillation
of FFA approximately 1557% of the tocotrienols and tocopherols are removed from the
palm oil. Most of the tocopherols and tocotrienols are not lost but accumulate in the palm
fatty acid distillates (PFAD). The levels of tocopherols and tocotrienols in the distillates
may be as high as 0.71.0%, and therefore, this by-product represents a potential source
for the recovery of these valuable compounds.(5) In addition to tocopherols and
tocotrienols, other valuable minor components are concentrated in the PFAD such as sterols and terpenic hydrocarbons (e.g., squalene). Their value lies in their health promoting
functionality in the food and pharmaceutical industries.
378
Liu et al.
OH
R1
CH3 H
2
R2
R3
OH
4
R4
Tocopherol structure
CH3
CH3
2
R3
R1
R2
Tocopherol/tocotrienol
CH3 H
R4
3
Tocotrienol structure
R1
R2
R3
R4
CH3
CH3
H
H
CH3
H
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
, , , or depending on the methyl substitutions on the phenolic ring.(12) The only difference in the structure of tocotrienols and tocopherols is in the phytyl tail; tocotrienols contain
three isolated double bonds in their phytyl tail, while tocopherols have a saturated tail.(10)
The tocopherols possess three chiral centers at position 2 of the chromanol head and
at positions 4 and 8 of the phytyl tail, making a total of eight stereoisomeric forms possible.
All naturally occurring tocopherols have the RRR configuration (2D, 4D, 8D, d-tocopherols,
or (+)-tocopherols). Synthetic -tocopherol (all-rac--tocopherol) is a racemic mixture of
equal parts of each stereoisomer. On the other hand, the tocotrienols only have one chiral
center at position 2; therefore, they only have two stereoisomers. However, since there are
two double bonds at positions 3 and 7, four cis / trans geometrical isomers are possible.
Therefore, a total of 8 isomers could be present for each tocotrienol. Only the 2R, 3-trans,
and 7-trans isomers exist in nature.(13)
Two new tocotrienols have been identified from rice bran by HPLC using a normal
phase silica column.(14) These new tocotrienols have been designated desmethyl tocotrienol
(d-P21-T3) and didesmethyl tocotrienol (d-P25-T3). Using the same scheme as in Fig. 1, R1,
R2, R3, and R4 must be substituted by H, H, H, and CH3, respectively, to get the molecular
structure of d-P21-T3, and by H, H, H, and H to get the molecular structure of d-P25-T3. Evidence suggests that these tocotrienols, along with the other tocotrienols, might have therapeutic value in the prevention and treatment of cardiovascular disease and breast cancer.(14)
Antioxidant Activity of Tocotrienols and Tocopherols
The biological activity of tocopherols is generally believed to be due to their antioxidant
activity against lipid peroxidation in biological membranes. These compounds are able to
scavenge the chain-propagating peroxyl radicals. The antioxidant function per se is
379
(a)
(b)
located in the chromanol head, where the phenolic hydroxy group donates an H-atom to
quench lipid radicals. The phytyl tail has no effect on the chemical reactivity of tocopherols but is responsible for the very strong lipophilic properties of these compounds,
which determines their proper positioning in the biomembranes.(15) For tocotrienols, their
antioxidant reaction mechanisms are expected to be similar to those of tocopherols. It is
not known, either, if the unsaturation of the phytyl tail makes any difference on the activity of the tocotrienols relative to the corresponding tocopherols.(10)
The antioxidant activity of 1 mg of all-rac--tocopheryl acetate equals the activity
of 0.67 mg of d--tocopherol.(8,16) Table 2 shows the biological activities of some
tocopherols and tocotrienols relative to natural d--tocopherol as assayed by the rat
resorption-gestation test.(10) d--tocopherol has the highest antioxidant activity, while
d--tocotrienol manifest only about 30% of this activity. However, there is evidence
that suggests that d--tocotrienol has higher antioxidant activity than d--tocopherol.
For example, d--tocotrienol possesses 4060 times greater antioxidant activity against
(Fe2+ + ascorbate)- and (Fe2+ + NADPH)-induced lipid peroxidation in rat liver
microsomal membranes and 6.5 times better protection of chytochrome P-450 than d-tocopherol.(15) Another study showed that the tocotrienol rich-fraction from palm oil
(TRF) has the ability to inhibit oxidative damage induced by ascorbate-Fe2+ and light
380
Liu et al.
Figure 3. HPLC chromatogram of analysis of tocopherols and tocotrienols in palm oil. Peak a-T,
-tocopherol; a-T3, -tocotrienol; g-T3, -tocotrienol; d-T3, -tocotrienol.
Table 1
Tocotrienols and tocopherols levels (mg/L) in common dietary oils(11)
Dietary oil
Palm
Rice bran
Wheat germ
Coconut
Palm-kernel
Cocoa butter
Corn
Cottonseed
Peanut
Olive
Safflower
Soybean
Sunflower
Total
-Tocopherol -Tocotrienol -Tocotrienol -Tocotrienol tocotrienols
152
324
133
5
12
11
112
389
130
51
387
101
487
205
236
26
5
21
2
439
349
94
19
738
586
26
25
21
2
0
0
0
0
0
0
0
381
Table 2
Biological activity of some tocopherols and tocotrienols(10)
Name / configuration
d--Tocopheryl acetate (2R,4R,8R)
2R,4R,8S-d--tocopheryl acetate
2R,4S,8S-d--tocopheryl acetate
2R,4S,8R-d--tocopheryl acetate
1--Tocopheryl acetate (2S,4R,8R)
2S,4R,8S-d--tocopheryl acetate
2S,4S,8R-d--tocopheryl acetate
2S,4S,8S-d--tocopheryl acetate
d--Tocopherol
d--Tocopherol
d--Tocopherol
d--Tocopherol
d--Tocotrienol
d--Tocotrienol
d--Tocotrienol
d--Tocotrienol
photosensitization in rat liver microsomes. Among the constituents of TRF, d-tocotrienol was the most effective followed by its - and -isomers. The protective ability of TRF, against both protein oxidation and lipid peroxidation, was significantly
higher than that of d--tocopherol.(17)
Role of Tocotrienols in the Prevention of Cardiovascular Heart Disease
Cell culture and animal studies have shown that tocotrienols, in contrast to tocopherols, possess hypercholesterolemic activity.(11) Studies indicate that tocotrienols inhibit the activity of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate- limiting enzyme
in cholesterol synthesis.(9) d-P25-T3 was found to be the most potent in inhibiting HMG-CoA
reductase activity in chickens.(14) Tocopherols, on the other hand, have no effect on plasma
concentrations of total cholesterol, low-density lipoproteins (LDL), high density lipoproteins
(HDL) and very-low density lipoproteins (VLDL), and triacylglycerols.(18)
A tocotrienol rich fraction from palm oil containing d-P21-T3 and d-P25-T3 (TRF25)
reduced total serum cholesterol, LDL, apolipoprotein B, platelet factor 4, thromboxane B2,
glucose, and triglycerides in swine expressing hereditary hypercholesterolemia.(19) The
effect of tocotrienols on cholesterol-lowering might be related to HMG-CoA activity.(20)
Human studies used a TRF or TRF25 oil capsule that was administered to hypercholesterolemic individuals. The TRF mixture contained d--tocopherol and d--, d--, and
d--tocotrienol; the TRF25 contained the TRF mixture plus d-P21-T3 and d-P25-T3. Participants received 100200 mg per day for 410 weeks. Results showed a significant
decrease in total plasma cholesterol, LDL cholesterol, and apolipoprotein B levels.(21,22) In
addition, a synergistic effect between TRF25 and lovastina HMG-CoA reductase
inhibitorwas found on the reduction of cholesterol.(23)
The most effective supplements provided 1520% d--tocopherol and 60% d-- and tocotrienol, while the least effective supplements consisted of < 30% d--tocopherol and 45%
382
Liu et al.
d-- and -tocotrienol. It was postulated that the d--tocopherol attenuated the action of tocotrienols on HMG-CoA reductase activity. It was also suggested that high doses of tocotrienols
were not beneficial since a conversion of tocotrienols to tocopherols was likely to occur.(22)
Studies have shown that dietary supplementation with tocotrienols result in carotid
atherosclerosis regression. One study found a significant reduction in the atherosclerotic
lesion size of apoE mice fed d-P25-T3 or rice bran.(23) In another study, a positive effect of
TRF on the inhibition of LDL oxidation and endothelial cell lipid peroxidation in human
umbilical vein endothelial cells was found.(24) Enhanced endothelial cell activation and/or
dysfunction are associated with accelerated development of atherosclerosis.(11)
Tocotrienols have been shown to improve endothelial function by at least four mechanisms: a) decreased susceptibility of LDL oxidation; b) decreased monocyte-endothelial
cell adhesion; c) suppression of vascular smooth muscle cell proliferation; and d) suppression of platelet activation and aggregation.
Role of Tocotrienols in the Prevention of Breast Cancer
Tocotrienols from TRF have been shown to inhibit the proliferation of human breast cancer cell lines. The inhibition was found to be independent of the estrogen receptor status of
the cell lines.(7) Similarly various tocotrienols have shown concentration-dependent suppression of B16 melanoma cells proliferation.(14) In general, tocotrienols display potent
antiproliferative and apoptotic activity against breast cancer cells at doses that have no
adverse effects on cell growth or viability in vitro. Tocotrienols have also shown greater
anticancer activity than tocopherols in a variety of in vitro experimental models.(11) These
results suggest that dietary supplementation of tocotrienols might lower the risk of breast
cancer in women.
383
384
Liu et al.
1. forming a mixture with the deodorizer sludge and a solution of urea dissolved in methanol;
2. heating the mixture to form a urea complex of the fatty acids and glycerides. The reaction is controlled so as not to go above 64C;
3. cooling the mixture to precipitate the urea complex from the mother liquor containing
the tocopherols and tocotrienols; and
4. separating the mother liquor from the precipitate.
A process similar to urea complexation was developed that treated the PFAD with
calcium hydroxide in the presence of a solvent medium containing water and an inert
water miscible organic solvent to form calcium salts of the naturally occurring free fatty
acids.(32) These salts were precipitated and removed. After removal of the calcium salts,
the material in the liquid phase contained the natural tocopherols and tocotrienols in a far
more concentrated form than that present in the starting material.(32)
When PFAD is used directly in the molecular distillation process, it would not be
efficient to obtain a product with high tocotrienol and tocopherol concentration, as was the
result from the process using deodorizer distillate of soya oil.(8) Conventionally preparing
high-purity concentrate of tocotrienols and tocopherols normally involves a series of physical and chemical treatment steps. The process requires a pretreatment to transform heavier
components into components of small molecular weights. Because deodorized distillate is a
complex mixture and the properties of its components are very similar, it is difficult to
recover high-quality concentrates of tocotrienols and tocopherols with good yield. As seen
in the above examples, most of the processes developed for recovering tocopherols and
tocotrienols from PFAD are difficult to perform due to their complexity, inefficiency or cost.
385
Distillation at moderate vacuum is characterized by the use of conventional distillation equipment. Its lowest pressure limit is of the order of 1 torr, i.e., one mm-Hg.
Unobstructed-path distillation is defined as distillation in which the path between the
evaporator and the condenser is not blocked, in other words when there is a free transfer of
molecules.(37) When the distance of transfer is comparable with the mean free path of the
vapor molecules, the distillation is known as molecular distillation.
Mean free path is defined as the average distance a molecule will travel in the vapor
phase without colliding with another vapor molecule.(37) This implies that, in molecular
distillation, the vapor molecules can reach the condenser without intermolecular collisions. A dynamic equilibrium can not, therefore, be established between the vapor and the
liquid. As well, concepts like theoretical plates, stages, and other common distillation terminology do not apply. There is, however, no certainty that an individual molecule that
has evaporated will be able to travel any distance without a collision.
Molecular distillation occurs at low temperatures and, therefore, reduces the problem
of thermal decomposition. High vacuum also eliminates oxidation that might otherwise
occur in the presence of air. Unobstructed path and molecular distillation are often classified together as short-path or high vacuum distillation. They employ the same kind of
equipment; the difference is in the dimensions and operating conditions. Unobstructed
path distillation is carried out at pressures as low as 102 torr, while in molecular distillation pressures of 103 torr, i.e., a mtorr, are used. Another useful distinction between the
methods of vacuum distillation can be made with reference to the way in which the vapor
phase is formed: a) ebullition, b) evaporative distillation, or c) molecular distillation.
Ebullition is accompanied by the formation of bubbles when the saturated vapor pressure exceeds the pressure of the surrounding gas. The only limit to the rate of evaporation
in this case is the rate at which heat can be transferred to the liquid. Evaporative distillation, on the other hand, occurs when the pressure of the surrounding gas is higher than the
vapor pressure of the liquid at a given temperature, so that bubbles are not formed. The
rate of evaporation is controlled by the temperature of the liquid and the conditions above
the liquid surface.
In molecular distillation, which can also be called molecular evaporative distillation,
the rate of evaporation is controlled by the rate at which the molecules escape from the
free surface of the liquid and condense on the condenser. Theoretically, as mentioned
before, the condenser should be in the immediate vicinity of the evaporating surface. In
practice, however, distances of 510 cm are not uncommon, despite the fact that more
intermolecular collisions are likely to occur.
Rate of Evaporation Under Molecular Distillation
The theoretical evaporation rate in molecular distillation is defined by the LangmuirKnudsen Equation, which is derived from kinetic theory considerations(38):
.
GT = PO
M
,
2RT
(1)
386
Liu et al.
When an appreciable number of collisions can occur in the vapor space, some of the
molecules will return to the liquid. This leads to a decrease in the number of molecules
that reach the condensation surface. This quantity divided by the time and area of evaporation gives the actual rate of evaporation G. The Langmuir-Knudsen Equation, therefore,
provides a limiting value for the rate of molecular distillation.(39,40) The ratio of the actual
to the theoretical rate of evaporation is defined as the evaporation coefficient f:
.
f =
G
.
(2)
GT
The coefficient f, which represents the fraction of vaporized molecules that reach the
condenser, can be calculated as follows(41):
f = F + (1 F ) (2e N e 2 N ),
(3)
where
F=
AC
h
and N =
,
AC + AE
kb
where, AC is the condensation surface area in m2; AE is the evaporation surface area in m2;
h is the distance between the evaporator and condenser in m; k is a factor that has to be
determined experimentally; and is the mean free path at equilibrium conditions in m.(41)
The following empirical equation is used to calculate k for a short-path, falling-film
evaporator(42):
(4)
Relative Volatility
In a mixture of two components, the relative volatility , which is also called the separation factor, represents the ratio of the observed volatility of component 1, i.e., the more
volatile, to that of component 2. It can be expressed as Eq. (5)(41):
a=
y1 x2
,
x1 y2
(5)
where x and y are the mol fractions in the liquid and vapor phases, respectively. Further
consideration shows that, if a mixture of two components obeys Raoults law, their relative volatility is equal to P1/P2, i.e., the ratio of their equilibrium vapor pressures in the
pure state at the prevailing temperature. The ratio y/x is the distribution coefficient (when
equilibrium conditions are present it is the equilibrium distribution constant). At high rates
of evaporation under high vacuum, i.e., molecular distillation, the relative volatility
becomes(43):
a=
P1O
M2
,
M1
P2O
387
(6)
LnP O =
A
+ B,
T
(7)
14886
11365
11841
19131
19710
11365
34.230
30.663
30.819
35.450
35.646
28.393
388
Liu et al.
products by molecular distillation has gained wide application, including products derived
from refined vegetable oils. For example, deodorizer distillate of vegetable oils; palm oil
for obtaining tocotrienols and tocopherols; oil of rice for the oryzanol recovery;
monoglycerides concentration; carotenoids recovery from palm oil; heavy petroleum
characterization, and herbicides.(8,29,47,48,49,50) Molecular distillation is often used to separate or purify high-boiling materials that decompose, oxidize, or polymerize at elevated
temperatures.(51) This process can be considered for industrial uses if both distillate and
residue are high value-added products.(52) Some specific uses of molecular distillation are
listed below.
1. Production of biologically active substances such as vitamins, sterols and antioxidants
from natural oils and fats. Examples include extraction of vitamin A from fish liver and
whale oil and carotenoid recovery from esterified palm oil.(24)
2. Separation of mono and diglycerides in partially saponified fats.(52)
3. The refining of crude animal and vegetable oils. For instance, a Malaysian company
produces Carotino, an edible red palm oil, using a process that involves a pretreatment
of crude palm oil (i.e., degumming with phosphoric acid and treatment with bleaching
earth) followed by deacidification and deodorization using molecular distillation.(4)
Recently a new process of molecular distillation was developed for recovery of tocotrienols and tocopherols from rapeseed by combination of acid-catalysed methyl esterification and crystallization followed by fractional distillation of derived products.(53,54)
Conclusions
Molecular distillation has shown great potential in the separation, purification and concentration of natural products such as tocotrienols and tocopherols as well as other minor
health-promoting components from palm fatty acid distillates (PFAD). Because it uses
very low levels of temperatureunder high vacuum and short operating time for separation, while using no solventsit avoids problems of toxicity. The effects of feed-flow rate
and temperature of distillation on extraction of minor components from PFAD are related
to the yield, purity, and rate of evaporation in terms of concentrations, distribution coefficients, and relative volatilities. The molecular distillation process can remove maximum
amount of free fatty acids to increase the concentration of tocotrienols and tocopherols
without adding extra components to the system. The percentage of free fatty acids in the
distillation steam of the molecular still is larger at low feed-flow rates and low evaporator
temperatures, thus avoiding thermal decomposition.
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