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GEOMETRICAL ISOMERS OF THE MAJOR.pdf

GEOMETRICAL ISOMERS OF THE MAJOR.pdf

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23
GEOMETRICAL ISOMERS OF THE MAJOR PROVITAMIN A PALM CAROTENES,
α
- and
β
-CAROTENES IN THE MESOCARP OILOF FRESH AND STERILIZED PALM FRUITS, CRUDE PALM OIL AND PALM CAROTENE-BASED PRODUCTS: RED PALM OLEIN AND CAROTENE CONCENTRATES
*
Malaysian Palm Oil Board,P.O. Box 10620, 50720 Kuala Lumpur, Malaysia.
**
Universiti Putra Malaysia,Department of Chemistry, Faculty of Science and Environment,43400 UPM Serdang, Selangor, Malaysia.
+
Forest Research Institute of Malaysia, Kepong,52109 Kuala Lumpur, Malaysia.
GEOMETRICAL ISOMERS OF THE MAJORPROVITAMIN A PALM CAROTENES,
ααααα
- and
βββββ
-CAROTENES IN THE MESOCARP OIL OFFRESH AND STERILIZED PALM FRUITS,CRUDE PALM OIL AND PALM CAROTENE-BASED PRODUCTS: RED PALM OLEIN ANDCAROTENE CONCENTRATES
BONNIE TAY YEN PING*; CHOO YUEN MAY*;GWENDOLINE EE CHENG LIAN** and GOH SWEE HOCK
+
ABSTRACT
Polymeric C 
30
 reversed-phased (RP) liquid chromatography (LC) columns were used to identify the geometricalisomers of the major carotenes from the mesocarp oil of fresh (MOFPF) and sterilized palm fruits (MOSPF),crude palm oil (CPO) and palm carotene-based products (red palm olein [RPOo] and carotene concentrates).In MOFPF, the isomers of
α 
-carotene were all
trans
 , 13-
cis
 , 13'-
cis
 and 9-
cis
 and the isomers of
β 
-caroteneall
trans
 and 13-
cis
. MOSPF had similar geometrical isomers plus two unidentified
cis
 
α 
- and
β 
-carotenes.CPO had the
 
 geometrical isomers of MOSPF plus 9-
cis
-
β 
-carotene. The isomer profiles of carotenoid extracts from RPOo and carotene concentrates (CC1 and CC2) had similar patterns to that of CPO. The percentagecompositions of total
cis
-isomers of
α 
-and
β 
-carotene in MOFPF, MOSPF, CPO, RPOo, CC1 and CC2 were18.2%, 44.9%, 43.5%, 45.4%, 55.9%, and 38.8%, respectively.
Keywords
: geometrical isomers, mesocarp oil of palm fruits,crude palm oil, red palm olein, carotene concentrates.
NTRODUCTION
Trans
 
α
- and
β
-carotenes are the major carotenes inCPO and other palm carotene-based products (Tayand Choo, 2000; Ooi
et al.,
1994). The analyses ofpalm carotenoids are based on reverse-phase LCwith C18 as the stationary phase (Yap
et al.,
 1991;Tay and Choo, 2001) as the column is able to separatemost of the major carotenoids on their structuraldifferences. However, it is not efficient for separatingthe geometrical isomers of asymmetrical carotenes(which have the
cis
 bonds at the same carbon numberbut at the opposite end of their molecules). Thegeometrical isomers may well have differentphysiological properties but this aspect has yet tobe explored thoroughly. The provitamin A activitiesof the c
is
-isomers of
α
- and
β
-carotenes and
β
-cryptoxanthin are only 50% or less than those oftheir corresponding all
trans
 forms (Zechmeister,1962; Sweeny and Marsh, 1973). Therefore, theproper
 
determination of provitamin A activity for
 
 Journal of Oil Palm Research Vol. 13 No. 2, December 2002, p. 23-32
 
24
 JOURNAL OF OIL PALM RESEARCH 13 (2)
any biological sample should take into account the
cis
 form. Lessin
et al
. (1997) were among the first toquantify the geometrical isomers of provitamin Acarotenoids for fresh, processed fruits andvegetables.Studies (Ben-Amotz
et al
., 1989; Mokady
et al
.,1990; Levin
et al
., 1994a; Levin and Mokady, 1994b)have shown that 9-
cis
β
-carotene was preferentiallyabsorbed and stored when a mixture of all
trans
 andthe
cis
-isomer was administered to rats and chicks.Recent human trials (Stahl
et al
., 1992; 1993; Tamai
et al.,
 1993) showed that when the diet wassupplemented with an oily mixture of all
trans
β
-carotene and its 9-
cis-
isomer, only a negligibleamount of 9-
cis
was found in the blood butconsiderable amounts of 9-
cis
 and all the
trans
 forms(Stahl
et al.,
 1993) were present in the tissues. Thesestudies suggest that the absorption, metabolism anddistribution of
β
-carotene isomers may be differentin animals and humans. The antioxidant activity can be different for thegeometrical isomers. The 9-
cis-
β
-carotene has ahigher potency than the all
trans
 forms (Jimenezand Pick, 1993; Levin
et al
., 1994a; Levin andMokady, 1994b). An
in vivo
 animal study showedthat the geometrical isomer mixture in theunicellular alga,
Dunaliella bardawil
,
 
protected thecentral nervous system against oxygen toxicity.But the effect was not observed with the all
trans
or oxidized natural
β
-carotenes (Bitterman
et al.,
1994).Details on the geometrical isomers of the majorcarotenes in palm oil and its products are stilllacking. The C
30
 stationary phase column givesoutstanding shape selectivity towards thegeometrical asymmetrical isomers in pure systems(Emenhiser
et al.,
 1995; 1996a), and also in biologicalextracts (Emenhiser
et al.,
 1996b). This study, usingthe C
30
 column, was therefore undertaken to identifythe geometrical isomers of
α
- and
β
-carotenes inpalm oil of various origins
 ,
such as MOFPF, MOSPF,CPO and palm carotene-based products like RPOoand carotene concentrates.
MATERIALS AND METHODSMaterials
Fresh ripe and sterilized palm fruits werecollected from the Malaysian Palm Oil Board(MPOB) Experimental Palm Oil Mill in Labu. CPOwas obtained from two local mills. RPOo waspurchased from the local supermarket. The caroteneconcentrates
 
were
 
obtained from the experimentalplant of MPOB
 
(CC1) and Carotech Sdn. Bhd. (CC2).Hexane and ethanol used for saponification were ofanalytical grade from Merck, Darmstadt, Germany.The HPLC solvents, methanol and methyl
tert
-butylether (MTBE), were of HPLC grade from Merck.Butylated hydrotoluene (BHT) was from SigmaAldrich Chemical Company, Steinheim, Germany.
Methods
 Extraction of oil from fresh and sterilized palm fruit.
A day after the fresh palm fruits were received,their mesocarp was cut away from the nuts andground in a food processor. The ground mesocarp(1 kg) was weighed into a beaker and soaked in 4litres hexane at room temperature for a day to extractthe oil. BHT 0.01 % (w/v) was added
 
to theextracting solvent to minimize degradation of thecarotenes. For the sterilized fruit, the softenedmesocarp was peeled off and 1 kg weighed into abeaker and soaked in 4 litres hexane at roomtemperature for one day. The oil samples wereobtained after the
 
hexane was removed
in vacuo
.
 Extraction of carotenoids from mesocarp oil of  fresh sterilized fruits and CPO
.
The
 
oil sampleskept in bottle containers were first homogenizedby immersing in a
 
water bath at 35
o
C - 40
o
C. About3 g of the oil were dissolved in 30 ml ethanol andsaponified with 5 ml 50% (w/v) potasium hydroxide(KOH)
in vacuo
 at 35
o
C - 40
o
C for 40 min. Thesaponified samples were then extracted with 50 mlportions of hexane until the supernatant becamecolourless. To minimize oxidation of the carotene,the saponification mixture and extracting solventwere added with 0.01% BHT (w/v).
 
The combinedhexane extracts were washed five times with 50 mlportions of distilled water and dried over sodiumsulphate. The carotene extracts were obtainedafter removing the hexane
in vacuo
. Since lightpromotes isomerization, the whole procedure wascarried out under dim diffused light. Thesaponification reaction flask was wrapped inaluminium foil and the entire extraction carried outwithin 2 hr. The dried carotene extracts were storedat -20
o
C under a headspace of nitrogen and usedwithin 72 hr. Prior to injection into the HPLC-DAD,the extracts were dissolved in 2-5 ml 89:11methanol:MTBE mixture.
 Instrumentation.
Separation was done using twoanalytical
 
scales
 
(25 cm x 0.46 cm I.D.), DV1 andDV2, of the
 
Develosil ODS-UG 5
µ
m polymeric C
30
column protected by precolumns with the samestationary phase. The HPLC system consisted of aHP 1100 series quaternary solvent delivery systemwith a HP 1100 series on-line degasser and wasconnected to a HP 1100 series diode array detector(DAD) covering the spectral range 190 - 800 nm.The data were stored and processed by HPChemstation.
 HPLC analysis and peak identification
.
The HPLCseparation was done isocratically using a mobile
 
25
GEOMETRICAL ISOMERS OF THE MAJOR PROVITAMIN A PALM CAROTENES,
α
- and
β
-CAROTENES IN THE MESOCARP OILOF FRESH AND STERILIZED PALM FRUITS, CRUDE PALM OIL AND PALM CAROTENE-BASED PRODUCTS: RED PALM OLEIN AND CAROTENE CONCENTRATES
phase of 89:11 (v/v) methanol:MTBE at a flow rateof 1 ml min
-1
. The chromatographic peaks weretentatively identified by comparison with a previousseparation on a polymeric C
30
 column (Emenhiser
et al.,
1996b) and by comparing their accumulatedabsorption visible light spectra (
Figures 1, 2
 and
Table 1
) with the absorption maxima from previousstudies (Emenhiser
et al.,
 1995; 1996a).
RESULTS AND DISCUSSIONMesocarp Oil from Fresh Palm Fruit (MOFPF)
Figure 3
 shows a typical separation of carotenoidextract from CPO performed on a C18 reverse phasecolumn. The two major peaks between the retentiontimes of 50 min and 65 min were assigned as all
Figure 2. Electronic absorption spectra of
β 
-carotene isomers in MOFPF, MOSPF, CPO, RPOo and carotene concentrates,CC1 and CC2: a) an unidentified
cis
-
β 
-carotene, b) 13-
cis
-
β 
-carotene, c) 9-
cis
-
β 
-carotene, and d)
trans
β 
-carotene.Figure 1. Electronic absorption spectra of
α 
-carotene isomers in MOFPF, MOSPF, CPO, RPOo and carotene concentrates,CC1 and CC2: a) 13-
cis
-
α 
-carotene, b) an unidentified
cis
-
α 
-carotene, c)
trans
 
α 
-carotene, and d) 9-
cis
-
α 
-carotene.
mAU4003002001000250 300 350 400 450 500Wavelength (nm)
   A   b  s  o  r   b  a  n  c  e
(b)(c)(a)(d)mAU8007006005000250 300 350 400 450 500Wavelength (nm)
   A   b  s  o  r   b  a  n  c  e
(b)(c)(a)(d)400300200100

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