In toxic Plants and Other Natural Toxicants(Eds. T.Garland and A.C.Barr)
CAB International,198 Madison Avenue, New York, USA.p.97-100
Analysis of Gossypol in Whole Cottonseed and Rumen Fluid by HPLC
Ismartoyo 1 and T. Acamovic2
Introduction
Gossypol is a polyphenolic compound that can be toxic ti animals, microbes and cells
(Ismartoyo and Acamovic, 1994; Ismartoyo, 1997). The accurate and specific quantitation of
gossypol is therefore important.
Whole cottonsed (WCS) contains gossypol at about 4-20g/kg glanded cottonseed
(Botsoglou, 1992; Acamovic, 1994). During processing, the aldehyde groups of gossypol react
with free amino groups of proteins, forming “bound gossypol” (Jones, 1979; Calhoun et al.,
1991; Risco et al., 1997). Gossypol react with other constituents to form condensation
products that are soluble in aqueous acetone and represent “soluble bound gossypol.” Some
gossypol may be oxidized and/or degraded to various unidentified products. The remaining
gossypol and gossypol-related compounds soluble in equous acetone that have an aldehyde
moiety represent “unbound gossypol” (Botsoglou and Kufidis, 1990; Botslogou, 1992).
Determination methods for gossypol in WCS and cottonseed meal have often been
non-specific and lacking in sensitivity. In this study, an HPLC method was developed for
specific determination of unbound and acetone-soluble bound gossypol in WCS and unbound
gossypol in the liquor. Themethod was based on that by Botsoglou (1992).
Materials and Methods
The solvents were HPLC-grade methanol, acetone, chloroform, acetonitrile, hydrochloric acid
and phosphoric acid. Aqueous acetone-ascorbic acid solution was prepared by diluting 2.5 g
ascorbic acid in 150ml distilled water (H2O), which was then mixed with 350ml acetone.
Ascorbic acid solution was prepared by dissolving 1.5g ascorbic acid in 500ml distilled H2O.
Mobile phase A was prepared as follows; 1ml phosphoric acid (Sigma Ltd., Poole,
Dorset) was added to 25ml double distilled H2O in avolumetric flask and made to 1L with
double distilled H2O. Mobile phase B was prepared by adding 1 ml phosphoric acid to 1L
HPLC-grade methanol in a volumetric flask. Both solutions were filtered through a 0.45 µm
membrane filter to remove particulate material.
A stock solution of gossypol was prepared by dissolving 6mg gossypol in 25ml
acetonitrile. Working gossypol solutions of 0.5-8µg/ml were prepared.
Sample preparation and extraction
A laboratory mill ( 1mm screen) was used to grind WCS. The ground WCS sample (2g) was
weighed accurately, transferred to a 250ml glass stoppered Erlenmeyer flask, and acetoneascorbic acid solution (100ml) was added. The flask was stoppered and shaken for 1hr. The
extract was filtered through a Whatman No. 40 filter paper and three separate 25 ml aliquots
transfreed into flask for the determination of unbound and acetone-soluble bound gossypol
(USB), unbound gossypol (U), and unbound and acetone-soluble lipophilic form of bound
gossypol (USL).
1
2
Departement of Animal Science, Hasanuddin University, Ujungpandang 90245 Indonesia;
Departmenet of Biochemistry and Nutrition, SAC, Ayr KA6 5 HW
In toxic Plants and Other Natural Toxicants(Eds. T.Garland and A.C.Barr)
CAB International,198 Madison Avenue, New York, USA.p.97-100
For determination of USB, the WCS extract (25ml) wa transferred into a 50ml conical
flask and 0.05ml concentrated HCl added. The flask was stoppered and heated in a water bath
for 1 hr at 65C. the flask was removed and cooled to room temperature, then the extract was
transferred into a separating funnel. Chloroform (50ml), 100 ml ascorbic acid solution and 1ml
concentrated HCl were added to the funnel, which was shaken for 3 min and allowed to stand
for 5 min. The lower organic layer was filtered through anhydrous Na2SO4 in a Whatman No.
40 filter paper into a 100ml flask. The Na2SO4 was rinsed with chloroform and the filtrate
evaporated to dryness by rotary evaporation at 30C. The residue was dissolved in 25ml
acetonitrile for HPLC analysis. The WCS extract (25ml) for U was transferred directly into a
separating funnel and then processed as in USB, but heating with HCl was omitted.
For USL determination, the WCS extract (25ml) was trabsfrred to a separating funnel
(250 ml) and processed as in U, but instead of reconstitution in acetonitrile, the residue was
dissolved in 25ml acetone-ascorbic acid solution and then processed as in USB.
Chromatography
Analysis was performed on a varian HPLC system consisting of a 9010 pump with a 9065
polybhromator diode array detector (DAD). Data was handled using a Desk Pro 486
workstation with Varian Star software Version 4.0. The chromatographic column used was a
stainless steel Spherisorb ODS2 (S5 ODS2, 250x4.6 mm; phaseSeparation, Ltd. Deeside
Industrial Park, Clwyd, Wales). The DAD was set to collect data from 190-365nm. The
mobile phase flow rate was 1.5ml/min. The temperature was maintained at 30C using a
column heater, and injection of both sample and working standard solutions (25µl) was
achieved using a 9100R autosampler. Values of gossypol were determinated by relation of the
standards.
The calculated values from USB, U and USL allowed furher calculation of a “soluble
bound gossypol” (lipophilic and hydrophilic from of bound gossypol). The acetone-soluble
lipophilic form of bound gossypol (SLB) was determined by subtracting U from USL, or
SLB=USL-U. The acetone-soluble hydrophilic bound gossypol (SHB) was calculated by
substracting USL from USB, or SHB=USB-USL.
Gossypol determination in rumen fluid
Selected frozen rumen samples from sheep fed basal diet grass hay (1kg/d) supplemented with
500 g WCS/d were thawed at room temperature to 20Covernight and cenctrifuged at 2,000xg
for 10min. The supernatant was transferred to HPLC vials for the determination of free
gossypol. No gossypol peaks were found in any rumen liquor samples from sheep that
consumed cottonseed except the gossypol spiked controls. This suggests that the concentration
of any free gossypol in the rumen was very low (less than 0.5µg/ml).
Analysis of standard gossypol solutions by HPLC
A single pure peak of gossypol was obtained for the standard as determined by spectral
comparison and statistical analyses of the spectra across the peaks. Gossypol was eluted at
about 6min (5.90.015min).
In toxic Plants and Other Natural Toxicants(Eds. T.Garland and A.C.Barr)
CAB International,198 Madison Avenue, New York, USA.p.97-100
The regression line of the standard gossypol solutions againts peak area at 234nm was linier
r2=0.98). thus quantitation of gossypol to a concentration as low as 0.5µg/ml was attainable.
Detection at 287nm gave a linier response and a more stable baseline, but was less sensitive by
a factor of about three. The average peak purity parameter was 245.730.54nm.
Conclusions
Gossypol analusis by HPLC was rapid, repproducible, specific and sensitive for the gossypol
in WCS with the limit of detection of 0.5µg/ml, which is similar to that of Botslogou (1992).
The absence of, or very low, free gossypol concentration in the rumen samples indicated that a
large proportion of free gossypol may have been bound to protein and possibly to other
components in the rumen such aslipids (Reyes et al., 1984) and iron salts (Waldroup, 1981;
Risco et al., 1997). Jones, (1985) reported that during processing, free gossypol is bound to
cottonseed protein, resulting in bound gossypol and unavailable amino acids; lysine is
considered to be the primary amino acid to which free gossypol is bound (Baliga and Lyman,
1957; Martin, 1990). It has been suggested that not all free gossypol is bound in the rumen and
that some may be released during digestion (Calhoun et al.,1991). The incident needs further
detailed investigation, particularly the mechanism of how the free gossypol binds and its
possible release during digestion.
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