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Plant proteins, minerals and trace elements of Eurycoma longifolia (Tongkat

Ali)

Article  in  Natural Product Research · April 2012

DOI: 10.1080/14786419.2012.676552 · Source: PubMed

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Plant proteins, minerals and trace

elements of Eurycoma longifolia
(Tongkat Ali)
a a a
Lee Suan Chua , Nurulaini Abdul-Rahman , Bustanur Rosidi &
a
Chew Tin Lee
a
Metabolites Profiling Laboratory, Institute of Bioproduct
Development, Universiti Teknologi Malaysia, 81310 UTM Skudai,
Johor, Malaysia

Available online: 03 Apr 2012

To cite this article: Lee Suan Chua, Nurulaini Abdul-Rahman, Bustanur Rosidi & Chew Tin Lee
(2012): Plant proteins, minerals and trace elements of Eurycoma longifolia (Tongkat Ali), Natural
Product Research: Formerly Natural Product Letters, DOI:10.1080/14786419.2012.676552

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 Natural Product Research
2012, 1–5, iFirst

Plant proteins, minerals and trace elements of Eurycoma longifolia

(Tongkat Ali)
Lee Suan Chua*, Nurulaini Abdul-Rahman, Bustanur Rosidi and Chew Tin Lee

Metabolites Profiling Laboratory, Institute of Bioproduct Development, Universiti Teknologi

Malaysia, 81310 UTM Skudai, Johor, Malaysia
(Received 2 November 2011; final version received 9 February 2012)
Downloaded by [Universiti Teknologi Malaysia] at 00:28 03 April 2012

A water extraction method has been used to extract plant proteins from the roots
of Eurycoma longifolia harvested from Perak and Pahang, Malaysia. On the basis
of the spectroscopic Bradford assay, Tongkat Ali Perak and Pahang contained
0.3868 and 0.9573 mg mL 1 of crude protein, respectively. The crude proteins
were separated by one dimensional 15% sodium dodecyl sulphate polyacrylamide
gel electrophoresis into two (49.8 and 5.5 kD) and four (49.8, 24.7, 21.1 and
5.5 kD) protein spots for Tongkat Ali Perak and Pahang, respectively. Isoleucine
was present in the highest concentration significantly. Both plant samples showed
differences in the mineral and trace element profiles, but the minerals calcium,
magnesium and potassium were present in the highest concentration. The highly
concerned toxic metals such as arsenic and lead were not detected.
Keywords: Eurycoma longifolia; SDS-PAGE; plant protein; amino acids; metals

1. Introduction
Eurycoma longifolia, also known as ‘Tongkat Ali’, belongs to a family of Simaroubaceae,
which has been widely used by the Malay community from South East Asian countries as a
herbal folk medicine for centuries. Numerous studies have reported its diverse biological
activities on antimalarial (Kuo, Damu, Lee, & Wu, 2004), antiulcer (Tada et al., 1991),
antipyretic (Chan, Lee, & Yuen, 1995), antitumour (Jiwajinda et al., 2002), antiparasitic
(Jiwajinda et al., 2002) and cytotoxic to cancer cells (Morita, Kishi, Takeya, Itokawa, &
Tanaka, 1990), which were closely associated with the bioactive compounds such as
quassinoids, canthin-6-one alkaloids, -carboline alkaloids, tirucallane-type triterpenes
and biphenylneolignans.
Besides small molecular weight of metabolites, peptides and proteins might be the plant
bioactive constituents that contribute to certain pharmacological properties. Asiah,
Nurhanan, and Mohd Ilham (2007) reported the detection of bioactive peptide (4.3 kD) as
an aphrodisiac marker in a few Malaysian plants, including E. longifolia. The proteins (7.5
and 6.0 kD) identified by Farouk, Mohd Nawi, and Hassan (2008) have been reported
having antibacterial activity against human pathogenic bacteria. Indeed, antimicrobial
peptides are ribosomally synthesised as natural antibiotics by nearly all living organisms,
from bacteria, animals to plants. It is believed that the plant still contains many more
proteins or peptides to be explored, particularly for pharmacological application.

*Corresponding author. Email: lschua@ibd.utm.my

ISSN 1478–6419 print/ISSN 1478–6427 online

ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2012.676552
http://www.tandfonline.com
 2 L.S. Chua et al.

Furthermore, the number of plant peptides discovered as signal molecules is very limited
compared with animal-based signalling peptides. Many plant peptides are likely to be
phytohormones in regulating intercellular responses for plant growth and development,
including plant defense mechanism in response to wound signal transduction by pests
(Lindsey, Casson, & Chilley, 2002).
In addition to plant proteins, the mineral and trace elemental composition is of
particular interest in natural product research. Many essential metals are involved in
cellular biochemical and physiological processes as cofactors of enzymes that needed in
numerous biological activities in response to biotic and abiotic stress for plant growth and
defense system (Wu, Susnea, Chen, Przybyski, & Becker, 2011). On the contrary, an
excessive amount of metals might disturb plant metabolism. Therefore, the elemental
profile of E. longifolia is important to provide fundamental understanding of their
Downloaded by [Universiti Teknologi Malaysia] at 00:28 03 April 2012

interactions with the proteome, especially in the response regulation of producing metal
chelate proteins for metal tolerance, accumulation and adaptive mechanism either at
transcriptional or posttranscriptional levels (Wu et al., 2011).
The elemental profile could explain the environmental pollution around the area where
the plant located. This profile could also be used to determine the geographical origin of
the plant species. The amount of minerals and trace elements up-taken by the plants is very
much dependent on the plant need and their availability. Besides mineral nutrients, the
quantity of toxic metals such as lead, arsenic and chromium need to be determined for
safety concern. This is because mostly people consume the decoction drink of
E. longifolia’s roots, which is believed where most minerals and trace elements are stored.

2. Results and discussion

2.1. Crude protein extraction
The extraction method varies dependent on the type of targeted protein and research
objective. To our knowledge, no single extraction protocol can capture full proteome.
Each protocol is developed based on the type of plant tissue and the desired research
objectives. The ideal protocol must be highly reproducible with the minimal artifactual
protein degradation for both gel and liquid chromatography-based MS analysis (Kota &
Goshe, 2011).
In this study, a simple water extraction method in the absence of extraction additives
was used to extract highly water soluble proteins. It is the easiest extraction protocol,
especially for water soluble and nonthermal labile proteins. The use of water as extractor
at ambient temperature could get rid of plant pigments, phenolics and lipids, which might
contaminate protein extract and create difficulties in the consequent protein analysis. This
protocol was able to produce superior protein yield and good resolution of protein spots in
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
The total protein content was estimated by the Bradford assay based on the
absorbance of protein-CBB complexes at 595 nm. This spectrophotometric dye-binding
assay is widely accepted for protein quantification in biological samples, mainly because of
its sensitivity and feasibility (Schmidt, Steier, & Otto, 2009). From the assay, it was found
that 0.3868 (15.5% (w/w) of extract) and 0.9573 mg mL 1 (39.3% (w/w) of extract) of
highly water soluble proteins were extracted from Tongkat Ali Perak and Pahang,
respectively. The crude protein content of Tongkat Ali Pahang was about twice higher
than in Tongkat Ali Perak. Overall, the high protein content exhibited in the Bradford
assay was very encouraging. This is because the extraction method is likely to be a
promising technique to extract a relatively low abundance of plant proteins.
 Natural Product Research 3

2.2. Protein separation by gel electrophoresis

In the subsequent analysis, the crude proteins were separated by SDS-PAGE based on
protein molecular weight. It was found that the running buffer containing Tris-glycine
gave the best results, in terms of protein band separation, spot focusing and resolution.
The contrast between the protein bands and the background was also considerably better
than the running buffer containing Tris-tricine. Even though the running buffer containing
Tris-tricine could give sharper protein bands, particularly the molecular markers, it could
not isolate low-molecular-weight proteins very well. This can be observed by the nonlinear
curve of the log plot of protein mass versus relative mobility of protein bands as presented
in Figure S1.
The loading volume of extracts (10 and 20 mL) onto the gel also statistically significant
affected the results of protein mass calculated from the log plot with the variance,
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0.1 kD. On the other hand, the loading volume did not improve the resolution of protein
spots. Figure 1 reveals that two and four protein spots were separated from the aqueous
extracts of Tongkat Ali Perak and Pahang, respectively. Both extracts contained 5.5 and
48.9 kD spots as prominent proteins. In addition to that, Tongkat Ali Pahang, which also
gave the higher total protein content contained additional two types of proteins with the
mass, 21.1 and 24.7 kD in the extract.
The smearing effect of the protein bands on the gel was most probably because of the
low purity of protein extract, in addition to the presence of proteases and oxidative
enzymes. The other coextraction of water soluble and nonproteinaceous constituents such
as polysaccharides, lipids, organic acids and phenolics, might also cause interference to
protein migration electrophoretically. This effect was more significant for low mass
proteins. The low mass proteins have to migrant longer distance and face higher resistance
during migration.
Even though trichloroacetic acid (TCA) in acetone has been proven as an efficient
precipitating and preconcentrating agent for plant protein purification in many studies
(Carpentier et al., 2005; Maserti et al., 2007), the addition of TCA in acetone in the
extracts did not improve plant protein separation in gel electrophoresis significantly
(Figure 1).

Figure 1. Crude protein separation on 15% polyacryalmide gels for Tongkat Ali Perak (PRK) and
Tongkat Ali Pahang (PHG) using molecular marker mixture, bovine serum albumin (BSA) and
lysozyme (Lys) as control in Tris-glycine (a) and Tris-tricine (b) running buffer solution.
 4 L.S. Chua et al.

2.3. Amino acid composition

Besides gel electrophoresis, the protein extracts were analysed for the amino acids content
using high performance liquid chromatography combined with photodiode array detection
at 254 nm. The chromatogram of the amino acid profile for both extracts compared with
17 types of hydrolysate standard amino acids (100 pmol mL 1) is presented in Figure S2. It
is interesting to note that both extracts have different amino acid profiles qualitatively
(Figure S2) and quantitatively (Table S1). Table S1 shows that aspartic acid, glutamic acid,
histidine, proline and cysteine were under the detection limit in both extracts. Isoleucine
was present in the highest concentration, followed by glycine. However, lysine and
arginine were only found in Tongkat Ali Pahang, whereas alanine was only found in
Tongkat Ali Perak.
Based on the formulation of Zarkadas et al. (2007), there were only 1.56% (w/w)
(15.649 mg mg 1 crude protein Tongkat Ali Perak) and 0.57% (w/w) (5.686 mg mg 1 crude
Downloaded by [Universiti Teknologi Malaysia] at 00:28 03 April 2012

protein Tongkat Ali Pahang) of total amino acids content detected from liquid
chromatographic method, compared with the crude protein content that determined
from Bradford assay.
Specificity was determined by comparing retention times obtained in the standard
amino acid mixture with those obtained from the samples. The minimal difference between
retention times of standard and hydrolysed sample peaks were less than 1.7%, except
aspartic acid (7.1%). The relatively low deviation in retention time allows the identifi-
cation of amino acid peaks with high confident level.
The linearity was studied in the range of 0 to 100 pmol mL 1 of standard amino acids.
All the 17 amino acids showed a good linearity in that range with the square of correlation
coefficient (R2) greater than 0.912, except phenylalanine (0.629).

2.4. Minerals and trace elements analysis

Based on the results in Table S2, the major elements (41 g kg 1) were nutrient minerals,
namely Ca, Mg, Na, Mn, Fe, Zn and K. There were also minerals present in minor
quantity (51 g kg 1) such as Cr, Ni, Cu and Co. Apart from minerals, the contaminant
metals such as Ba, Al, Li, Rb, Li, In, Sr, Ga and U were also detected in significant
amount. The amount of the contaminant metals is actually very much dependent on the
bioavailability of these metals in the soils, as well as the ability of the plant to store and
accumulate metals in conjunction with plant metabolism. Therefore, the elemental profile
of the plant can also be used as environmental indicator to determine contamination level.
Although aluminium presents naturally in the environment, it is released due to
anthropogenic activities such as mining and industrial uses. European Food Safety
Authority highlights that the safety level of aluminium from dietary intake is 0.2 to
1.5 mg kg 1 body weight per week in a 60 kg adult (EFSA, 2008). However, it is impossible
to leach the total amount of the metals easily, if the Tongkat Ali drink is prepared by
decoction as traditional practice. It is noteworthy that the highly toxic elements such as As
and Pb were not detected in both plant samples.

3. Conclusion
Plant proteins were extracted from the roots of E. longifolia without any extraction
additives at room temperature. The crude protein content was approximately 15.5 and
39.3% (w/w) of the plant extract from Tongkat Ali Perak and Pahang, respectively. There
were two and four protein spots detected from Tongkat Ali Perak and Pahang,
respectively, in 15% polyacrylamide gel. Both plant extracts contained 5.5 and 49.8 kD
 Natural Product Research 5

proteins, but Tongkat Ali Pahang has relatively low amount of additional protein bands,
21.1 and 24.7 kD. Therefore, they have different amino acid profiles, but both extracts
have isoleucine and glycine as the highest concentration of amino acids. The difference was
also observed in the metallic element composition of the plant samples. Zinc and
magnesium were present as the highest amount of minerals in Tongkat Ali Perak, whereas
potassium, calcium and magnesium were present in Tongkat Ali Pahang.

Supplementary material
Experimental details relating to this article are available online, alongside Figures S1 and
S2 and Tables S1 and S2.

Acknowledgements
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We thank Mohamad Subri Abdul Rahman for running the ICP-MS. Sincere thanks to Tang Boon
Seng for his valuable inputs in preparing the manuscript. We also take this opportunity to express
our gratitude to Prof. Dr. Atta-ur-Rahman for his great contribution in natural product studies.

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