IV. RESULTS AND DISCUSSION
A. Sugarcane Wax Composition
The qualitative separation of most of the major classes of aliphatic wax components could be achieved by silica gel of thin layer chromatography
Figure 7. Compositions of the waxy materials from sugarcane cultivars were all similar. They were composed of sterol, alcohol, acid,
triacylglycerol, methyl ester, aldehyde and sterol ester. Thin layer chromatography had been widely used for qualitative analysis in waxy
materials Hwang et al. 2002; Adhikari et al. 2006; Webster et al. 2006.
Figure 7 Thin layer chromatography of waxes of several sugarcane cultivars. S: Standard.
S NiF 8 Ni 22 Ni 15
Plant sterol Policosanol
Acid Triacylglycerol
Sterol ester
Methyl ester aldehyde
Wax components of sugarcane on TLC plate could finally be visualized by cupric sulfate charring. Each component of waxy materials
was well charred and separated, except for sterol ester which split into two smear bands. These two separated bands were identified by Adhikari et al.
2006 as wax ester and sterol ester. Besides this, methyl ester and aldehyde were appeared as a single spot, while acid and policosanol as closed dense
spots. Separation of cane wax components by high performance liquid
chromatography HPLC were further quantified with evaporative light-scattering detector ELSD. Five peaks in HPLC chromatogram were
identified as groups of aldehyde, sterol ester, triacylglycerol, acid, alcohol, and sterol Figure 8. Wax component contents in analyzed samples were
determined based on the relation between peak area and calibration curve of each standards. An example of standard curve of triacylglycerol is shown in
Figure 9, it has a linear equation of y = 1E+07x – 52429. These standards Figure 10 had different concentrations covering the levels of components in
samples, i.e. 0.05–0.50 mgml. This result confirmed the same result obtained by TLC. Every peak
represented one group of wax components, except for peak 1 which was a mixture of aldehyde and sterol ester. However, Hwang et al. 2002 and
Adhikari et al. 2006 identified this peak as mixture of aldehyde, sterol ester and also wax ester. A very weak respond of hydrocarbon compound
somewhat was detected after 3 minutes retention, but it was not enough for making a single peak.
Figure 8 HPLC chromatogram of sugarcane rind of Ni 15 cultivar. The chromatogram was obtained by HPLC-ELSD method.
Peak 1 : Aldehyde, sterol ester Peak 4 : Alcohol
Peak 2 : Triacylglycerol Peak 5 : Sterol
Peak 3 : Acid
Figure 9 Standard curve of triacylglycerol.
y = 1E+07x - 524294 R² = 0.9987
1,000,000 2,000,000
3,000,000 4,000,000
5,000,000 6,000,000
0.00 0.10
0.20 0.30
0.40 0.50
0.60
P ea
k A
rea
Concentration mgml
0.0 5.0
10.0 15.0
20.0 25.0
30.0 0.2
0.4 0.6
0.8 1.0
0.0 1
2 3 4
5
Time min V
Figure 10 HLPC chromatograms of wax composition standards. a aldehyde, b sterol ester, c triacylglycerol, d acid, e alcohol, f sterol. Standards
concentrations were 0.5 mgml a-d and 0.4 mgml e-f. The chromatograms were obtained by HPLC-ELSD method.
a b
c d
e f
Wax components in sugarcane rind were composed of mixture of aldehydes and sterol esters 55.1–60.4, alcohols 31.8–39.8,
triacylglycerols 2.3–4.6, acids 2.0–2.7, and sterols 0.5–0.9, respectively Table 5, Figure 8. Significant differences p 0.05 were
observed in all wax composition of Okinawan sugarcane cultivars. The highest content of wax component was found in sugarcane rind of Ni 22
cultivar with about 1 g of mixture of aldehydes and sterol esters per 100 g sample wet weight basis. Followed with mixture of aldehydes and sterol
esters of NiF 8 and Ni 15 cultivars, i.e. 751.0 and 594.6 mg100 g. The alcohol group, policosanols, of Ni 22 cultivar sample was 462.9 mg100 g;
while sterol group was less abundant and its response became too small for making a detectable peak.
Table 5 Wax compositions of rind part of Okinawan sugarcane cultivars, obtained by HPLC-ELSD.
Cultivar Aldehyde,
sterol ester Triacylgycerol
Acid Alcohol
Sterol ww
NiF 8 55.1 ± 1.1
3.7 ± 0.4
b
2.7 ± 0.1
b
39.8 ± 4.0
a
0.5 ± 0.03
a
Ni 15
b
60.5 ± 3.0 4.6 ± 0.6
a
2.4 ± 0.4
a
31.8 ± 2.1
ab
0.9 ± 0.2
b
Ni 22
a
58.5 ± 1.8 2.3 ± 0.2
ab
2.0 ± 0.2
c
37.3 ± 1.9
b
n.d.
ab
mg100 g
‡
c
NiF 8 751.0 ± 29.4
101.7 ± 10.7
q
62.0 ± 1.6
p
367.5 ± 8.1
p
33.3 ± 0.01
q
Ni 15
p
594.6 ± 32.6 80.2 ± 6.0
r
39.0 ± 1.4
q
216.4 ± 14.7
q
25.3 ± 0.2
r
Ni 22
q
1027.1 ± 66.5 97.0 ± 6.9
p
63.2 ± 3.1
p
462.9 ± 7.6
p
n.d.
p r
Calculated as aldehyde group.
‡
Not detected.
a
Data are means ±S.D. n = 3. Means in the same column with the same letter are not significantly different p 0.05.
Similar separation method and stationary phase of wax component of plant waxes were applied in grain sorghum Hwang et al. 2002, perilla and
sesame seeds Adhikari et al. 2006, potato leaves Szafranek Synak 2006, and calanoid copepod Calanus finmarchicus Webster et al. 2006. The
amounts and compositions of these waxes were markedly different depended on their genetics, bio-functions, plant growth conditions, and environments.
Another HPLC technique of wax components separation was using alumina as stationary phase Nordback Lundberg 1999.
Plant waxes are a complex heterogeneous mixture of very long chain C20–C34 fatty acids and their derivatives. During wax biosynthesis very
long chain fatty acids are further modified to aldehydes, alkanes, ketones, and so on. Grain sorghum and carnauba waxes compositions were well
investigated by Hwang et al. 2002. Grain shorghum wax was composed of 46.3 ww fatty aldehydes, 7.5 fatty acids, 41.0 fatty alcohols, 0.7
hydrocarbons, 1.4 wax esters and sterol esters, and 0.9 triacylglycerols. Carnauba wax contained of 34.3 wax esters, 5.1 fatty acids, undetermined
amount of fatty alcohols, and 3.0 triacylglycerols, determined by HPLC. Major components of the waxy materials from Korean and Chinese
perilla seeds were alcohols 25.5 and 34.8, hydrocarbons 18.8 and 10.5, wax esters, steryl esters and aldehydes 53.0 and 49.8, acids 1.7 and
2.1, and triacylglycerols 1.0 and 2.9, based on HPLC data were reported by Adhikari et al. 2006. The principal components of leaf
cuticular waxes from potato varieties were very long chain n-alkanes, 2-methylalkanes and 3-methylalkanes 3.1–4.6 µg cm
-2
, primary alcohols 0.3–0.7 µg cm
-2
, fatty acids 0.3–0.6 µg cm
-2
, and wax esters 0.1–0.4 µg
cm
-2
In general, sugarcane stalk was composed of sugars 12–16, water 70–74, pith fiber 7, rind fiber 7, and epidermis 0.1. Wax would
abundantly found in rind part as epicuticular wax. Figure 11 shows the comparison of policosanol and long chain aldehyde contents as wax
components in sugarcane stalk. Rind and pith samples were separated by CSS Cane Separation System. This study discovered that policosanol and
long chain aldehyde compositions of sugarcane parts varied significantly. Policosanol and aldehyde compounds in the rind part of sugarcane were
found much abundant than in pith. , analyzed by GC-FID Szafranek Synak 2006. Methyl ketones,
sterols, β-amyrin, benzoic acid esters and fatty acid methyl, ethyl, isopropyl
and phenylethyl esters were found in potato waxes. A new group of cuticular wax constituents consisting of free 2-alkanols with odd and even
numbers of carbon atoms ranging from C25 to C30 was also identified.
Figure 11 Policosanol and long chain aldehyde contents in sugarcane Ni 15
cultivar, obtained by GC-FID analysis. Means in the same group with the same letter are not significantly
different p 0.05.
20 40
60 80
100
Rind Pith
Whole stalk
A m
ou n
t m
g 100 g
Policosanol Aldehyde
a p
c r
b q
The whole stalk of sugarcane Ni 15 cultivar contained 35 mg policosanols and 24 mg aldehydes per 100 g of wet weight basis. The
policosanol and aldehyde contents in pith part were negligible about 1 mg100 g, however, policosanols and adehydes of rind part was found in
high concentration, i.e. 80 mg100 g. This result is associated with surface wax present in rind of cane. The surface waxes protect plants from water
lost and environmental stress Koch et al. 2006; Dominguez Herdia 1996. Separation with CSS would make loosing an amount of epicuticular waxes in
rind, while either it separated to epidermis wax chamber or attached in rolls and blades of CSS. In this way the hand peeled rind samples were also
investigated. Rutherford and Staden 1996 reported a suggestion of sugarcane
surface wax component towards resistance to borer Eldana saccharina Walker. In their investigation, a high ratio of alcohol to aldehyde and
shorter chain length appeared to be associated with cane resistance. Furthermore, Purcell et al. 2005 described epicuticular wax as a potential
genetic marker and predictor of desirable plant traits. According to that a comprehensive study of wax compositional analysis and eco-geochemistry of
Okinawan sugarcane cultivars is needed.
B. GC Chromatogram and Mass Spectrum of Policosanols and Long Chain
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