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Fatty acid composition and biological activities of seed oil from rubber (Hevea
brasiliensis) cultivar RRIM 600

Article  in  International Journal of Applied Research in Natural Products · March 2013

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International Journal of Applied Research in Natural Products
Vol. 6 (2), pp. 1-7.
Directory of Open Access Journals
©2008-2013. IJARNP-HS Publication

Original Research

Fatty acid composition and biological activities of seed

oil from rubber (Hevea brasiliensis) cultivar RRIM 600
Kittigowittana K1*; Wongsakul S2; Krisdaphong P1; Jimtaisong A1; Saewan N1
1
School of Cosmetic Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
2
School of Agri-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand

Summary. The oils from seeds of Hevea brasiliensis (Muëll. Arg.) cultivar RRIM 600 cultivated in Thailand (from two different sources,
Chiang Rai and Surin provinces), were subjected to the evaluation of fatty acid composition, antioxidant activities, antimicrobial activities
and cytotoxicity. The seed oils were extracted using n-hexane as a solvent and the major fatty acids were oleic and linoleic acids. The seed
oils from two different sources similarly exhibited high capability in inhibiting scavenging DPPH radicals (95%, 87% inhibition, from
Chiang Rai and Surin provinces respectively), reducing power (1.588±0.016, 1.832±0.009 mg of AAE/mL). However, moderate lipid
peroxidation inhibition activity of these two seed oils were observed (24%, 28% inhibition). The cytotoxicity effect of oil was determined on
human dermal fibroblast. It showed that the H. brasiliensis seed oil was not cytotoxic to human skin at >1000 μg/mL. Based on these results,
it was suggested that the H. brasiliensis seed oil may be considered as a potential antioxidant candidate for topical cosmetic applications.
Industrial relevance. Natural origin raw materials have gained increasing attention for cosmetics because of their effectiveness and safety
as compared to the synthetics. H. brasiliensis seed oil from this research has shown itself as a highly promising natural raw material source
for cosmetic industry. It composed of skin health benefit fatty acids and has been found to exhibit high capability in inhibiting scavenging
DPPH radicals. Moreover, from the cytotoxicity result, it indicated that the H. brasiliensis seed oil can safely be applied to human skin.
Keywords. Hevea brasiliensis; seed oil; fatty acids; biological activities

INTRODUCTION
Hevea brasiliensis or para rubber tree belongs to the family Euphorbiaceae which is the most economically important
member of the genus Hevea. It is native to South America and cultivated as an industrial crop since its introduction to South
East Asia (Indonesia, Malaysia and Thailand). In Thailand, rubber tree was first introduced in Trang province in 1899 and
has now become one of the most economically important crop. The rubber seed collection increases every year according to
larger quantity of rubber tree plantation but little or no attention has been given to seed production and its utilization. Two
products are obtained from rubber seeds which are the oil and the cake. The oil content in dried kernel varies from 35 to
45%.

Figure 1. A. Hevea brasiliensis (Muëll. Arg.) cultivar RRIM 600 (Rubber Research Institute of Thailand, Department of Agriculture) and B.
the seeds which were used to prepare the extracts.
Rubber seed oil is a semi-dried substance that is a rich source of fatty acids such as oleic acid, linoleic acid, linolenic acid,
palmitic acid, and stearic acid (about 52% of total fatty acid composition) (Ghandhi et al., 1990). Studies have shown that
rubber seed oil has many areas of potential applications including possible uses for lubricant (Njoku and Ononogbu, 1995),
___________________
*Corresponding Author.
 kkittigowittana@hotmail.com
 +66894301717
Available online http.//www.ijarnp.org
 Kittigowittana et. al.

printing ink (Reethamma et al., 2005), factice (Vijayagopalan, 1971; Fernando, 1971), biodiesel (Perera and Dunn, 1990;
Ikwuagwu et al., 2000), paints and coatings (Aigbodion et al., 2003).To date, there are a few studies that have been
conducted on the chemical composition of rubber seed oil but no studies has particularly focused to those properties relevant
to cosmetic industrial uses, such as antioxidant activity, antimicrobial activity, anti-inflammatory activity, and the
composition of beneficial fatty acids for human skin health. In this study, the fatty acid composition of extracted seed oil
using GC analysis and antioxidant activities including DPPH radical scavenging, reducing power and lipid peroxidation
inhibition activities were evaluated for further introducing the rubber seed oil as a natural raw material to cosmetic industry.
Moreover, in order to determine whether the H. brasiliensis seed oil can safely be applied to human skin, the cytotoxicity to
human dermal fibroblast cell lines was also carried out.

MATERIALS AND METHODS

Chemicals and reagents. All the chemicals used are analytical grade. Ethyl acetate, n-hexane, ethanol, methanol, and
trichloroacetic acid were purchased from Merck; ascorbic acid and butylated hydroxytoluene (BHT) were purchased from
Finechem; linoleic acid was purchased from Fluka; 2,2’-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma.
feric chloride (FeCl3), potassium ferricyanide (K3[Fe(CN)6]), ammonium thiocyanate (NH4SCN), and ferrous chloride
tetrahydrate (FeCl2·4H2O) were purchased from Fisher scientific; sodium dihydrogenphosphate (NaH2PO4·2H2O) and
disodium hydrogen phosphate (Na2HPO4•12H2O) were purchased from Prolabo.
Plant material. The seeds of rubber (Hevea brasiliensis) cultivar RRIM 600 used in this study were obtained from Rubber
Research Institute of Thailand plantations in Chiang Rai and Surin provinces, Thailand.
Extraction. Fresh rubber seeds were decorticated manually to free the kernel from the shell. Three hundred grams of the
kernel each were milled using blender. Each of these milled kernels was extracted with n-hexane. The extraction was carried
out at room temperature for 24 h. The extract was dried over Na2SO4 and evaporated in vacuo using rotary evaporator. The
oil was then collected, weighed and stored in a sealed container at 4C for further analysis.
Physiochemical characteristics of oil. Density of the oil was determined by using pycnometer and its specific gravity was
calculated by following formula
Specific gravity = density of oil/density of water

The acid value, iodine value sponification value and unsaponificable matter were determined by following the standard
method as described in US Pharmacopeia and National formulary (USP 24/NF 19, 2000).
Analysis of fatty acid composition. The fatty acid composition of H. brasiliensis seed oil was determined by converting all
fatty acids to the corresponding fatty acid methylesters (FAME) followed by GC analysis (Wongsakul et al., 2003). FAMEs
were prepared by methylation of 10 L oil with 0.5 % methanolic NaOH (500 L), followed by incubated for 20 min at
60°C. The methylesters were extracted with n-hexane (500 L) for 1 min. The n-hexane layer was washed with 200 L
distilled water and dried over anhydrous sodium sulfate. The GC analysis for the FAME samples were analyzed on the
Agilent (6890 series, USA) gas chromatograph equipped with a DB-5ms column (0.25 µm x 30 m). Helium was used as
carrier gas. Samples were injected an oven temperature of 70°C then oven was heated at 30°C/min to 170°C, at 10°C/min to
195°C and at 10°C/min to 215°C.
Determination of DPPH scavenging activity. DPPH radical scavenging activity was determined using the method
originally developed by Blois (Blois, 1958; Lai et al., 2009) with some modifications. A portion (0.1 mL) of the oil solution
(1.0 mg/mL EtOAc) in a test tube was well mixed with 3.9 mL of methanol and 0.1 mL of α,α-diphenyl-β-picrylhydrazyl
(DPPH) solution (1.0 mM in methanol). The mixture was kept at ambient temperature for 30 min prior to measurement of
the absorbance at 517 nm. Ascorbic acid was used as reference. All measurements were done in triplicate. The DPPH radical
scavenging activity was derived following equation

DPPH radical scavenging activity (%) = [(Abs control – Abs sample)/ Abs control] x 100

Determination of ferrous reducing power activity. Reducing power of oils were determined according to the ferrous
reducing power method of Takashi (Takashi et al., 2009) with some modifications and expressed as ascorbic acid
equivalents (AAE). Each 250 μL of sample or ascorbic acid (a positive control), 250 μL of 0.1 M phosphate buffer (pH 7.2)
and 500 μL of 1% potassium ferricyanide were added to test tube. After incubation at 37ºC for 60 min, 250 μL of 10%
trichloroacetic acid and 750 μL of DI water were added. Then, the absorbance was measured at 700 nm (Abs1). After 250 μL
of 0.1% ferric chloride was added to the mixture and the absorbance was measured again (Abs2). Ferrous reducing power
was calculated by following equation

Reducing Power = [Abs2 – Abs1] sample – [Abs2 – Abs1] control

Determination of lipid peroxidation inhibition activity. Inhibition of lipid peroxidation was determined according to the
thiobarbituric acid-reactive substances (TBARS) method (Miguel, 2010) with some modifications. The essential oils or
extracts 100 µl was mixed with linoleic acid 900 µL, then incubated the mixture at 100 C for 20 min. 1 mL of buffer
solution (pH 3.5) and 1 mL of the mixture of 20 mM of thiobabituric acid in 10% trichloroacetic acid were added in the
resulting mixture, and then incubated at 100 C for 30 min to obtain the pink color solution. The absorbance was measured

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 Biological activities of rubber seed oil

at 532 nm using a microplate reader. All data were run in triplicate and compared with BHT and ascorbic acid references.
The percentage inhibition of lipid peroxidation was calculated by following equation

Inhibition of lipid peroxidation (%) = [(Abs control – Abs sample)/ Abs control] x 100

Cytotoxicity assay in human dermal fibroblast cell lines. This assay was a modified version of conventional direct and
indirect contact tests conformed to the published standard methods (BS-EN30993-5 and ISO10993-5). The MTT assay
(Plumb et al., 1989) is a tetrazolium-dye based colorimetric microtitration assay. Metabolism competent cells are able to
metabolize the tetrazolium (yellow) to formazan (blue); this color change is measured spectrophotometrically with a plate
reader. It is assumed cells that are metabolically deficient will not survive, thus the MTT assay is also an indirect
measurement of cell viability. The cells were seeded in a 96-well plate at a density of 3,000 cells/well, and incubated for 48
h. The sample at various concentrations were added to the cells and incubated for 24 h. The test samples were removed from
the cell cultures and the cells were reincubated for a further 24 h. in fresh medium and then tested with MTT assay.
50 μL of MTT in PBS at 5 mg/mL was added to the medium in each well and the cells were incubated for 4 h. Medium
and MTT were then aspirated from the wells, and formazan solubilized with 200 μL of DMSO and 25 μL of Sorensen’s
Glycine buffer, pH 10.5. The optical density was read with a microplate reader (Molecular Devices) at a wavelength of 570
nm. The average of 4 wells was used to determine the mean of each point. The experiments were done 3 times to get the
values and standard deviation. The data were analyzed with the SoftMax Program (Molecular Devices) to determine the IC50
for each toxin sample. A dose-response curve was derived from 8 concentrations in the test range using 4 wells per
concentration. Results of toxic compounds are expressed as the concentration of sample required to kill 50% (IC50) of the
cells compared to controls.
Determination of antifungal activity (Candida albicans). Antifungal activity against Candida albicans was tested
according to Resazurin microplate assay (Brien et al., 2000). Candida albicans (ATCC 90028) is grown on a potato
dextrose agar (PDA) plate at 30oC for 3 days. Three to five single colonies are then cultured in a shaking flask containing
RPMI 1640 until cell density reaches 5x105 CFU/mL.
The yeast cell suspension is added to the 384-well plate; each well containing 45 μL of cell suspension and 5 μL of test
sample. The plates are then incubated at 37oC for 4 h. Thereafter, 10 μL of 62.5 μg/mL resazurin solution is added to each
well and incubated at 37°C for 30 min. Fluorescent intensity is measured at the excitation wavelength of 530 nm and the
emission wavelength of 590 nm using SpectraMax M5 microplate reader (Molecular Devices, USA). Fluorescence signal
from wells containing only medium is used for background subtraction. The percentage of inhibition is determined from the
fluorescence units of treated cells (FUT) and untreated cells (FUC) using the following equation:

% Inhibition = [1- (FUT / FUC)] × 100

The 50% inhibitory concentration (IC50) is determined from doseresponse curve, using 6 concentrations of 2-fold serially
dilution. Amphotericin B and 0.5% DMSO are used as a positive and a negative control, respectively.

Determination of antibacterial activity (Bacillus cereus). Antibacterial activity against Bacillus cereus was examined by
Resazurin microplate assay and performed according to the guidelines of National Committee for Clinical Laboratory
Standards (2006; 2007) (Satyajit et al., 2007). The gram positive strain; Bacillus cereus ATCC 11778 (TISTR 687), was
obtained from American Type Culture Collection (ATCC). The bacteria was streaked on tryptic soy agar (TSA) and
incubated at 37ºC overnight. A single colony was inoculated in 5 mL of tryptic soy broth (TSB) and incubated in 37oC, 200
rpm for 30 min (OD600 ~ 0.1). The activated B. cereus culture was then diluted 200 folds in 20 mL TSB and incubated at
37ºC, 200 rpm for 3 h (OD600 ~ 0.4-0.5). As an assay condition in 384-well format, the mixture of 7.5 μL of sample, 25 μL
of 0.25 mM resazurin and 15,000 bacterial cells were cultured in Mueller-Hinton broth (MHB) at the final volume of 75 μL
per well. The plates were incubated at 37oC for 2 h. The fluorescent intensity was measured at an excitation of 530 nm and
an emission of 590 nm with SpectraMax M5 microplate reader (Molecular device, USA). Fluorescence from Blank is used
for background subtraction. The percentage of growth inhibition is calculated from the mean of fluorescent unit of treated
cells (FUT) and untreated cells (FUC) as defined by the equation:

% Inhibition = [1- (FUT) / (FUC)] x100.

Minimum Inhibitory concentration (MIC) is determined with the cut off at 90 % inhibition. Vancomycin and 0.5 %
DMSO are used as a positive and a negative control, respectively.

Statistical analysis. Data were expressed as means ± standard deviations (SD) of three replicate determinations. One way
analysis of variance (ANOVA) and Duncan’s New Multiple-range test were used to determine the differences among the
means. P values < 0.05 were regarded as significant.

3
 Kittigowittana et. al.

RESULTS AND DISCUSSION

Physicochemical properties and fatty acid composition of seed oils. 37% yield of pale yellow oil obtained from the hexane
extraction of seeds of H. brasiliensis occurs consistently liquid at 28±2oC. No significant different in the specific gravity
values of two seed oils (0.941±0.01, 0.928±0.03 from Chiang Rai and Surin, respectively). The chemical characteristics of
cultivar RRIM 600 H. brasiliensis seed oils from two different sources was evaluated by acid value, iodine value,
saponification value and unsaponifiable matter which showed small variation between each values of two oils, depending on
geographical origin. These values of H. brasiliensis seed oil from Chiang Rai were found to be 39.53±0.08, 133.89±0.29,
190.10±0.33 and 0.95±0.01 respectively and those of oil from Surin were found as 27.88±0.05, 136.11±0.34, 183.91±0.24
and 1.24±0.01 respectively (Table 1). The saponification values obtained from two H. brasiliensis seed oils agrees with
values for most vegetable oils ranging from 188-253 mgKOH/g (Oluba et al., 2008; Atasie and Akinhanmi, 2009). This
indicates that the H. brasiliensis seed oil has high potential for soap and shampoos producing propose (Ku and Mun, 2007).
Analysis of fatty acid composition. The seed oils of H. brasiliensis were subjected to GC analysis in order to determine the
fatty acid composition (Figure 2). Palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2),
arachidonic (C20:0) acids were detected in both rubber seed oils as summarized in Table 2. The study showed that Chiang
Rai rubber seed oil contained significant amounts of linoleic acid (42.13%), and oleic acid (38.96%). Whereas the Surin
rubber seed oil contained 40.81% of linoleic acid and 42.08% of oleic acid. It showed that no significant differences in the
amounts of fatty acids from two different sources. In addition, linoleic acid and oleic acid were the predominating fatty acids
in the seed oils and they have currently become increasing popular in cosmetic industry due to their beneficial properties on
the skin. Research points to linoleic acid’s antioxidant, anti-inflammatory, acne reductive and moisture retentive properties
when applied topically on the skin. Since the rubber seeds are by products, development of cosmetic application of rubber
seed oil may improve the value of this plant and enhance the agricultural economy.

Table 1. Physicochemical characteristics of H. brasiliensis seed oils.

The H. brasiliensis seed oils

Analysis
Chiang Rai Surin
Specific gravity 0.941±0.01 0.928±0.03
Acid value (mgKOH/g) 39.53±0.08 27.88±0.05
Iodine value (mgI2/g) 133.89±0.29 136.11±0.34
Saponification value (mgKOH/g) 190.10±0.33 183.91±0.24
Unsaponifiable matter (%) 0.95±0.01 1.24±0.01

Figure 2. GC chromatogram of fatty acid composition in rubber seed oils (A: Chiang Rai seed oil; B: Surin seed oil)

Antioxidant activity. Figure 3 demonstrates that, at the concentration examined (1.0 mg/mL), the seed oils from Chiang
Rai (CSO) and Surin (SSO) displayed similar inhibition capability on DPPH radical scavenging to ascorbic acid standard
(95% and 87% inhibition, respectively). Significant differences found in each seed oils were P < 0.01 and P < 0.02. They
have been found to show reducing power ability significantly (P < 0.03 and P < 0.01), 1.588±0.016, 1.832±0.009 mg of

4
 Biological activities of rubber seed oil

AAE/mL respectively. Whereas moderate lipid peroxidation inhibition activity of these two seed oils were observed
significantly (P < 0.005 and P < 0.01) (24% and 28% inhibition, respectively). The antioxidant activities of the seed oils
may be attributed to fatty acid components. Moreover, these activities may have been partly contributed by some
constituents other than fatty acids e.g. phenolic compounds. It would be suggested to further study in chemical composition
of this rubber seed oil in order to use as a natural raw material for cosmetic applications.
Cytotoxicity assay in human dermal fibroblast cell lines. The survival of the cells cultured with samples at various
concentrations and IC50 were investigated. The indication of toxicity has been evaluated in two ranges: at % survival >50%,
it will be evaluated as no toxicity and at % survival ≤50%, it will be evaluated as toxicity with IC50. In Table 3, the
cytotoxicity results showed that the CSO and SSO were not cytotoxic to human dermal fibroblast cell lines over the test
concentration range of 1000-7.8 μg/mL. This indicated that the H. brasiliensis seed oil can safely be applied to human skin.

Table 2. Fatty acid composition of H. brasiliensis seed oils.

The H. brasiliensis seed oils

Fatty acids (%)
Chiang Rai Surin
Palmitic acid, C16:0 8.78±0.05 8.94±0.05
Palmitoleic acid, C16:1 0.23±0.02 0.25±0.02
Stearic acid, C18:0 6.17±0.16 5.69±0.11
Oleic acid, C18:1 38.96±0.36 42.08±0.10
Linoleic acid, C18:2 42.13±0.06 40.81±0.29
Linolenic acid, C18:3 2.38±0.00 ND*
Arachidonic acid, C20:0 0.66±0.02 0.97±0.13
ΣSaturated fatty acid 15.61 15.60
ΣUnsaturated fatty acid 83.70 82.57
*ND = not detectable.

Figure 3. Antioxidant activities of rubber seed oils (A: DPPH radical scavenging activity; B: reducing power; C: inhibition on lipid
peroxidation, CSO: Chiang Rai seed oil; SSO: Surin seed oil) Values are expressed as means of three replicated ± standard deviations.

5
 Kittigowittana et. al.

Table 3. Cytotoxicity of rubber seed oils (CSO: Chiang Rai seed oil; SSO: Surin seed oil) to human dermal fibroblast cell lines

Sample Concentration (μg/mL) % survival SD IC50 (μg/mL)

CSO 1000 94 5 >1000

500 97 4
250 99 5
125 91 11
62.5 98 8
31.25 100 4
15.6 97 5
7.8 97 10
SSO >1000
1000 94 10

500 93 4

250 95 10

125 93 5

62.5 91 4

31.25 99 8

15.6 96 9

7.8 95 8

CONCLUSION
To the best of our knowledge, this is the first study providing data on antioxidant activities and cytotoxicity of the H.
brasiliensis seed oil. The fatty acid composition of this seed oil was also described in detailed. The oil obtained from the
seed of H. brasiliensis cultivar RRIM 600 is quite interesting as a potential antioxidant candidate for topical cosmetic
applications.

ACKNOWLEDGEMENTS
The authors are grateful to the scientific and technological instruments center, Mae Fah Luang University for providing us
GC/MS facility.

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