INTRODUCTION
Among the ancillary resources obtained from rubber (Hevea brasiliensis)
plantations (i.e., wood and seeds), seed has the greatest potential use. However,
rubber seeds are not currently much in use, so they are abundant and wasted
(Achinewhu and Akpapunam, 1985).
The seeds of the rubber tree have been found to be rich in oil. Rubber seed
oil is a yellow, semi-drying oil (Aigbodion and Bakare,
2005). At the present time, the production of Rubber Seed Oil (RSO) is increasing
greatly in both quantity and quality in Malaysia because of its important role
in different industrial processes. The oil does not contain any unusual fatty
acids and it is a rich source of essential fatty acids C18:2 and
C18:3 that make up 52% of its total fatty acid composition (Ghandhi
et al., 1990).
Polyunsaturated fatty acids (PUFAs) consist of omega-6 (linoleic acid, γ-linolenic
acid) and omega-3 (α-linolenic acid, eicosapentanoic acid, docosahexaenoic).
The importance of PUFAs were highlighted when these fatty acids proved to be
essential to human health to reduce cholesterol in the body and risk of heart
attack and stroke. However, due to the inability of the human body to synthesize
these fatty acids, nutritional PUFAs are obtained from marine life, plant sources
and vegetable oils (Yong and Jumat, 2006).
However, many studies of rubber seeds have indicated that the use of RSO for
nutritional purposes faces various vital challenges, one of which is the presence
of toxins in RSO. It is well known that some concentration of poisons will always
be found in the seeds of all types of plants, including the seeds of the rubber
plant. Rubber seeds known to contain linamarin (Duke and Ducellier,
1993).
A linamarin is a cyanogenic glucoside. The hydrolysis or cyanogenesis of linamarin
by the endogenous enzyme linamarase (β-glucosidase) results in the formation
of glucose and acetonecyanohydrin, which later decomposes into hydrogen cyanide
(HCN) and acetone (Idibie et al., 2007; Sornyotha
et al., 2006). Linamarin has been demonstrated to protect the plant
from herbivores, both animals and generalized insect feeders (Siritunga
and Sayre, 2004).
In this study, analysis of RSO for the detection of linamarin using rats and
shrimps tests plays a vital role in the determination of the value of Malaysian
RSO (MRSO) for inclusion in a healthy diet for animals and human beings. Hence,
the evaluation of the levels of the toxic compound linamarin from MRSO must
be done before MRSO can be approved for consumption by both animals and human
beings.
MATERIALS AND METHODS
Seed Material and Oil Extraction
Rubber seeds were collected from Malaysia’s Rubber Research Institute,
Sungai Buloh, Selangor, Malaysia in May 2009. The seeds were shelled and dried
in the oven at 105°C for 30 min and then milled using a grinder. The seeds
were then kept in the refrigerator. The MRSO was extracted from the 500 g rubber
seeds by soxhlet extractor using hexane and a mixture of chloroform+methanol
(1:1) as solvents at 60°C for 6 h.
Physicochemical Characteristics
The physicochemical properties of MRSO such as color, free fatty acid content
(FFA%), acid value, saponification value, iodine value and unsaponifiable matter
content were determined according to the guidelines of the American Oil Chemical
Society (AOCS).
Gas Chromatography (GC)
The fatty acid composition of RSO was determined using its fatty acid methyl
esters. The GC analysis was performed on a Shimadzu GC equipped with flame ionization
detector and capillary column (30 mx0.25 mmx0.25 μm films). The detector
temperature was programmed for 280°C with flow rate of 0.3 mL min-1.
The injector temperature was set at 250°C. Nitrogen was used as the carrier
gas at a flow rate of 20 mL min-1.
Rats Toxicological Test
Nine male white rats (Rumah Haiwan Laboratories, Universiti Kebangsan Malaysia)
weighing between 248.1-248.8 g were used for toxicity study as in Nwokolo
et al. (1988). The male white rats were individually housed in stainless
steel cages in a room with controlled temperature (30-35°C) and lighting
(alternating 12 h periods of light and darkness). The male white rats were fed
a pelleted commercial laboratory feed for 3 months. The mortality, color and
the behavior of the male white rats were recorded daily, but the food consumption
was recorded every two weeks. The food consumption, food efficiency, body waist
measurement and body length measurement were also determined.
Three experiments were conducted to determine the toxicological response of
rats fed rubber seed oil. In experiment 1, 3 rats were fed a rubber seed oil
that had been extracted with hexane. In experiment 2, 3 rats were fed a rubber
seed oil that had been extracted with chloroform+methanol. In experiment 3,
3 rats were fed a normal food were used as blank control. The rubber seed oil
to be fed to rats was stored at 4°C. Toxicological evaluation of the rubber
seed oil extracted with these two solvents was carried out by an acute oral
toxicity limit test to assess the acute toxicity potential of each oil.
Shrimps Test
Sample Extraction
The samples of linamarin were extracted from two different MRSO (100 g)
using water as a solvent in a separating funnel. After gentle shaking, the mixture
was left a few minutes to allow emergence of two phases: oil phase and water
phase. The oil phase was removed and the water phase was kept for further analysis.
HCN Hydrolysis
To conserve cyanide, the samples were supplemented with 4 mL 10 M NaOH.
The sample was distilled without further pretreatment. HCN was recovered in
the presence of 10 mL zinc acetate buffer (pH 4.5). The remaining cyanide was
subsequently recovered by distillation after addition of 5 mL of MgCl2
and 5 mL sulfamic acid plus 5 mL 50% H2SO4 later added
to obtain pH 1-2 for converting to HCN during distillation (ASTM,
1998). After 3 min, 45 mL 50% H2SO4 was added and
the solution boiled under reflux for 90 min (Bjarnholt et
al., 2008; Dzombak et al., 2006). After
distillation, the samples were put in a drying vacuum to evaporate the solvents.
The released HCN was determined using the shrimp acute toxicity method.
Shrimp Acute Toxicity Method
Shrimp used in this study were obtained from a local breeder and transported
immediately to the laboratory within 20 min. In the laboratory, a total of 450
shrimps were kept in an 80 L glass aquarium containing filtered and dechlorinated
tap water (pH 6.2-6.4, dissolved oxygen concentration 7.3-8.1 mg L-1,
conductivity 64-68 μS cm-1 and ammonia <0.5 mg L-1).
The shrimp aquarium was equipped with a water-cycling device and the water was
continuously aerated for one week to remove chlorine before the shrimp were
introduced. Shrimps were acclimated for 14 days (26-27°C with 12 h light:
12 h darkness) and fed daily. Care was taken to keep the mortality rate less
than 5% for the whole acclimatization period.
The acute toxicity test was performed according to the OECD (1993) and APHA
(1998) recommendations. Laboratory static tests were conducted to determine
the median lethal concentration (LC50). Ten shrimps of similar size
were placed in the test chambers. Shrimp were exposed for 96 h to one of the
different concentrations of 5, 10, 50, 75 and 100% of the samples which were
extracted from MRSO and rubber seed. The test chambers were aerated throughout
the test period. Physiochemical parameters of the water in the chambers such
as pH, conductivity, dissolved oxygen and temperature were measured for each
solution. The tests were repeated three times for both the control and each
test solution. During the experiment, dead shrimps were removed and mortality
of the shrimps exposed to various concentrations of samples was recorded after
6, 12, 18, 24, 48, 72 and 96 h. The LC50 was calculated based on
Finneys Probit Analysis Method (US EPA, 1999). The palm
oil was used as blank control.
Statical Analysis
Data collected were subjected to analysis of variance while the Significant
of difference between means were determined by Duncan’s Multiple Range
Test (DMRT), where (p<0.05) was considered for significant difference. Each
value was determined by at least three replicates. Results were given as Mean±SD
(SAS, 1996).
RESULTS AND DISCUSSION
The physicochemical properties of MRSO which was extracted by using different
solvents such as hexane (MRSOh) and chloroform+methanol (MRSOch+mth)
were determined are given in Table 1. The present FFA% (7.55±0.02
and 8.76±0.03, respectively) and acid value (15.03±0.04 and 17.43±0.06,
respectively) show that the MRSO has a high FFA% since it had not been neutralized.
MRSOh presents as a pale yellow oil (33.98±0.08), but lighter
(higher L* value) than MRSOch+mth (30.91±0.6) because of the
high FFA% in RSOch+mth. The MRSO shows high iodine value (135.79±0.33
and 134.44±0.31, respectively) compared with the iodine value of palm
oil (52) (Onyeike and Acheru, 2002) due to the high
content of unsaturated fatty acids such as oleic acid (22.95±0.15 and
25.31±0.13%, respectively) are shown in Table 2.
The saponification values of MRSOh and MRSOch+mth (182.12±0.27
and 183.32±0.29, respectively) are with average saponification numbers
in the range of 175-250 (Gunstone et al., 1994).
The value of unsaponifiable matter of MRSO is 1.83±0.01% and 2.19±0.03,
respectively (Table 1). This value is in agreement with the
value for RSO reported in by Gandhi et al. (1990).
The fatty acid composition of the RSO is shown in Table 2.
The Fatty Acids (FA) of MRSOh and MRSOch+mth consist of
saturated FA 19.12±0.28 and 21.64±0.21%, respectively and unsaturated
FA 79.45±0.31 and 77.40±0.26%, respectively. The saturated FA
consist mainly of palmitic acid 8.56±0.07 and 9.10±0.06%, respectively
and stearic acid 10.56±0.02 and 12.63±0.01%, respectively and
the unsaturated FA consist mainly of oleic acid 22.95±0.15% and 25.31±0.135,
respectively, linoleic acid 37.28±0.10 and 36.31±0.09%, respectively
and linolenic acid 19.22±0.21 and 15.78±0.18, respectively (Table
2). The FA composition of RSO can be used as indicator of the type of each
fatty acid (Aigbodion and Bakare, 2005). The physicochemical
properties and FA composition don’t show any significant (p<0.05) difference
between MRSOh and MRSOch+mth.
| Table 1: |
Physicochemical properties of MRSO |
 |
| MRSOh: Extracted using hexane as solvent; MRSOch+mth:
Extracted using a mixture of chloroform and methanol |
| Table 2: |
Fatty acids composition of MRSO |
 |
| MRSOh: Extracted using hexane as solvent; MRSOch+mth:
Extracted using a mixture of chloroform and methanol |
Toxicological evaluation of the MRSO was carried out in white male rats by
performing an acute toxicity limit test to assess its acute toxicity potential
in a 3-month feeding study. Two different types of rubber seed oil were extracted
by using hexane (MRSOh) and chloroform+methanol (MRSOch+mth)
as solvents under the same extracting condition. A total of 9 rats, 3 for each
experimental condition were used. Table 3 shows the mortality,
color and the behavior of the experimental rats. No acute toxic potential was
observed with MRSO extracted with both types of solvents. The rats displayed
no behavioral changes and there was no mortality in any of the groups during
the 3-month feeding study. The color of the male white rats didn’t appear
to change during the feeding study.
Neither MRSOh nor MRSOch+mth had any adverse effect on
food consumption. A similar increase in the average daily gain of rats fed MRSOh
and MRSOch+mth was also observed; differences between the 3 groups
of male white rats was not statistically (p<0.05) significant. The 3 groups
of rats showed no significant (p<0.05) differences in body weight gain, food
efficiency, body waist measurement and body length measurement. The growth rates
of the 3 groups of rats is shown in Table 4 and Fig.
1. These results would indicate that MRSOh and MRSOch+mth
had no toxic or antipalatability effects agreement with the value for RSO reported
in by Gandhi et al. (1990) and Nwokolo
et al. (1988).
Table 5 shows the relation between the samples of the linamarin
that was extracted from the MRSO which was extracted using different solvents
such as hexane (MRSOh) and chloroform+methanol (MRSOch+mth)
in the same extracting condition, concentration and the mortality rate of the
shrimps.
| Table 3: |
Mortality, color, and behavior of the male white rats |
 |
| MRSOh: Extracted using hexane as solvent; MRSOch+mth:
Extracted using a mixture of chloroform and methanol |
| Table 4: |
Food consumption, body weight gain, food efficiency, body
waist measurement and body length measurement |
 |
| *Condition was assessed by visual appearance |
| Table 5: |
Mortality (%) of shrimps at various concentrations of samples
of the linamarin extracted from MRSOh, MRSOchl+mth
and palm oil |
 |
|
| Fig. 1: |
Body weight gain of rats fed MRSO and blank control |
| Table 6: |
Estimated LC values and confidence limits of toxicity on shrimps
of samples of linamarin extracted from the MRSOh, MRSOchl+mth
and palm oil |
 |
|
Palm oil was used as blank control for comparison with MRSO. The estimated
LC values and their confidence limits that resulted from the acute toxicity
testing on freshwater shrimps using samples of the linamarin extracted from
the MRSOh, MRSOchl+mth and palm oil are listed in (Table
6). Based on Finneys Probit Analysis Method (using EPA software program),
the mean LC50 value of samples of the linamarin extracted from the
MRSOh, MRSOchl+mth and palm oil using shrimps was found
to be 211.70, 139.40 and 139.40%, respectively.
In this study, shrimp has, for the first time, been used as test organism for
acute toxicity of linamarin in MRSO. The results showed that samples of the
linamarin extract from the MRSOh and MRSOchl+mth have
no toxic effects on shrimps (LC50 211.70 and 139.40%), indicating
that no hazardous linamarin was found in MRSO. The results for MRSO were compared
with those for palm oil. The palm oil did not show any toxic effect as indicated
by its LC50 (139.40%). These results would indicate that MRSOh
and MRSOch+mth had no acute toxicity toward shrimp, a result which
supported the results obtained with the other method used (rats toxicological
test).
CONCLUSION
The current study has shown that, from the nutritional and toxicological aspects,
MRSO could be considered for edible use. These initial results indicate that
the use of MRSO as an edible oil will not be restricted by toxic or antipalatability
factors.
ACKNOWLEDGMENTS
We would like to thank UKM and the Ministry of Science, Technology and Innovation
(MOSTI) for research grant No. UKM-GUP-NBT-08-27-113, UKM-OUP-NBT-28-145/2009
and we also would like to thank the Malaysian Rubber Research Institute for
supplying the rubber seed samples.
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