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J. Sci. Dev. 2011, 9 (Eng.Iss. 1): 84 - 90 HANOI UNIVERSITY OF AGRICULTURE
EFFECT OF
MUCUNA PRURIENS
ON CATECHOLAMINE BIOSYNTHETIC ENZYME GENE
EXPRESSION IN RAT BRAIN
Ảnh hưởng của Mucuna pruriens đến sự biểu hiện gen tổng hợp catecholamine
trên mô não chuột
Nguyen Thi Hiep
1
, Truong Thi Thanh Mai
2
, Bui Thai Hang
1
, Kim Sung Jun
3
1
College of Food Industry, Da Nang City, Vietnam
2
College of Education – University of Danang, Da Nang City, Vietnam
3
College of Science, Chosun University, Gwang ju, Korea
Corresponding author email:
Received date: 13.04.2011 Accepted date: 05.05.2011
TÓM TẮT
Để xác định ảnh hưởng của Mucuna pruriens (MP) đến sự biểu hiện của một số gen tổng hợp
catecholamine, nghiên cứu này xác định sự thay đổi của hàm lượng mRNA và protein của tyrosine
hydroxylase (TH) và aromatic l- amino acid decarboxylase (AADC). TH là enzyme thứ nhất và AADC là
enzyme thứ hai trong chu trình tổng hợp catecholamine. Hàm lượng mRNA của TH và AADC được
xác định trong mô não của chuột bằng kỹ thuật RT- PCR. Hàm lượng protein được xác định bằng kỹ
thuật Western blot. Chuột được nghiên cứu với liều lượng MP khác nhau (0, 100, 200, 300 và 400
mg/kg) lần lượt trong thời gian 2 giờ và 4 giờ. Kết quả chỉ ra rằng MP làm tăng cả hàm lượng mRNA

và protein của TH và AADC ở liều lượng 400 mg/kg trong 2 giờ và ở liều lượng 200 mg/kg trong 4 giờ.
Kết quả này cho biết, MP có ảnh hưởng tích cực đến sự biểu hiện của TH và AADC gen và mức độ
ảnh hưởng đó phụ thuộc vào liều lượng cũng như thời gian sử dụng MP.
Từ khóa: AADC, biểu hiện gen, catecholamines, TH.
SUMMARY
To determine the effect of Mucuna pruriens (MP) on the catecholamine biosynthetic enzyme
encoding genes, we determined changes in mRNA and protein levels of tyrosine hydroxylase (TH) and
aromatic l- amino acid decarboxylase (AADC), the first rate – limiting and the second enzyme in the
catecholamine biosynthetic pathway. TH and AADC mRNA levels were examined in rat brain tissue by
RT- PCR. Protein levels were determined by Western blot analysis. Rats were treated with different
doses of MP (0, 100, 200, 300 and 400 mg/kg) for 2 and 4 hrs The results showed that the MP
significantly increased the levels of TH and AADC mRNA and protein at a dose of 400 mg/kg for 2 hrs
and at a dose of 200 mg/kg for 4hrs, respectively. These results indicated that MP has significant
effects on the expression of TH and AADC genes and the level of expression depends on the
treatment dose and duration of administration.
Key words: AADC, catecholamines, gene expression, TH.
1. INTRODUCTION
Parkinson’s disease (PD) is the most common
neurodegenerative movement disorder and affects
over six million people all over the world (Licker et
al., 2009). This disease is a degenerative disease of
the nervous system due to the progressive loss of
nigrostriatal dopaminergic neurons and decrease in
striatal dopamine. Dopamine is produced by
dopaminergic neuronal cells and is one of three
main neurotransmitters (dopamine, norepinephrine
and epinephrine) called "catecholamines".
Tyrosine hydroxylase (TH) is the first and
rate-limiting enzyme in the biosynthesis of
catecholamines, which is neurotransmitter involved

in a variety of important physiological functions
(Milsted et al., 2004). The regulation of TH protein
level and activity represents a central means for
controlling catecholamine synthesis (Kumer et al.,
1996). Changes in TH expression generally reflect
altered activity of dopaminergic neurons in brain.
84
Effect of Mucuna pruriens on catecholamine biosynthetic enzyme gene expression in rat brain
The aromatic l- amino acid decarboxylase
(AADC) is the second enzyme in the catecholamine
biosynthesis. AADC is a non-specific enzyme
mainly implicated in the synthesis of dopamine and
serotonin through the decarboxylation of a substrates
(L-DOPA), 5-hydroxytryptophan in neuronal and
non-neuronal cells (Yuichi Okazaki and Yoshikazu
Shizuri, 2001). Unlike other enzymes which are
involved in catecholamine biosynthetic pathway,
AADC is still generally considered not to be limiting
in regulating of catecholamine biosynthesis.
Nevertheless, some recent studies concerning the
regulation of AADC in vivo and in vitro through
phosphorylation indicate that this enzyme may play
the role in regulation of catecholamine biosynthesis
(Waymire and Haycock, 2002). The immobilization
stress stimulation induced also increases in TH and
AADC activity (Kubovcakova et al., 2004;
Micutkova et al., 2003).
Seeds of Mucuna pruriens (MP) have been
described as an useful therapeutic agent in various
diseases of the human nervous and reproductive

system including PD in the ancient Indian medical
system (Manyam et al., 2004). MP contains L-dopa
(5%), 5-indole compounds (tryptamine and 5-
hydroxytriptamine), and four tetrahy-
droisoquinolines alkaloidals (Eustace et al, 2006;
Misra and Wagner, 2004). In addition, MP consists
of co-enzyme Q-10 and nicotinamide adenine
dinucleotide (NADH), which have neuroprotective
activities (Manyam et al., 2004).
Although many researches have focused on
the therapeutic effects of natural plant extracts,
however, until now, there are few researches on
genetic analysis of catecholamine biosynthetic
enzyme genes induced by MP. Therefore, in this
study, we analyzed the effects of MP on the gene
expression of TH and AADC, the first rate –
limiting and the second enzyme in the
catecholamine biosynthetic pathway,
2. MATERIALS AND METHODS
2.1. MP sample preparation
The fresh seeds of MP (from Gwang-ju city,
South of Korea) were collected by removing peel
and then ground to a paste by grinder. This powder
was kept in deep freezer -70
0
C and freezing-dry. A
total of 100g of the dry powder was completely
dissolved in 100ml of saline before use.
2.2. Animal experiments
Male Sprague-Dawlay rats, 6 ~ 8 weeks of age

and weights from 250~300g were used in all
experiments. Animals were housed four per cage and
under controlled environmental conditions (12 hrs
light / dark cycle and room temperature 25 ± 2
o
C.
Food and water were supplied all the time. Rats were
treated by MP powder dissolved in saline with dose
of 100, 200, 300 and 400 mg/kg each for 2 and 4 hrs
(each group = 5 rats). The control groups received
the same volume of saline instead of MP.
2.3. Total RNA isolation and relative quantification
of mRNA levels by Reverse transcriptase
polymerase chain reaction (RT – PCR)
Total RNA was isolated from frozen brain
tissues by using the Trizol
TM
reagent (Invitrogen
Co. USA). Reverse transcription was performed
from 4 μg of total RNA samples using oligo (dT)
primer and Moloney murine leukemia virus
ribonuclease (M-MLV) (BioNEER co. Korea)
according to the manufacturer’s instructions.
Quality of cDNA was verified by PCR
amplification of β-actin. The cDNA was stored at -
20
o
C for further using.
The determining of catecholamine
synthesizing enzymes, TH, AADC as well as

housekeeper β-actin gene expression was carried
out by RT-PCR. Specific primers, annealing
temperatures, number of cycles and size of each
fragment for TH, AADC and β-actin mRNA are
shown in table 1. PCR products were analyzed on
1.2% agarose gel in 0.5 X TBE (Tris-Boric acid-
EDTA) buffers containing EtBr. Intensity of
individual bands was evaluated by Gel Quant
software (DNR Bio-Imaging Systems Ltd.).
Table 1. Oligonucleotide sequence of primers used in PCR for amplification
Genes Sequences of primers
Temp.of
annealing
No. of
cycles
Size of
fragment
Gene
Reference
TH
5’ GCTGTCACGTCCCCAAGGTT 3’
3’ TCAGACACCCGACGCACAGA 5’
64
0
C/45’’ 35 380bp M10244
AADC
5’ CTTCAGATGGCAACTACTCC 3’
3’ CTTCGGTTAGGTCAGTTCTC 5’
60
0

C/45’’ 35 345 bp U31884
β-actin
5’ CCTCTATGCCAACACAGT 3’
3’ AGCCACCAATCCACACAG 5’
56
0
C/45’’ 30 155 bp BC063166
85
Nguyen Thi Hiep, Truong Thi Thanh Mai, Bui Thai Hang, Kim Sung Jun
Table 2. Antibodies used for western blot assay
Antibodies Species Dilution Source
Primary antibodies
Anti-TH (0MA-04051)
Anti-AADC (ab3905)
Actin

Mouse (monoclonal IgG1)
Rabbit polyclonal
Mouse (monoclonal IgG1)

1:2000
1:1000
1:2000

Affinity Bio Reagents
Abcam plc
Biomeda crop.
Secondary antibodies
HRP conjugated(Sc-2054)
HRP conjugated(Sc-2055)

Goat-anti-mouse IgG1
Goat-anti-rabbit IgG1

1:1000
1:1000
Santa Cruz Biotechnology
Santa Cruz Biotechnology

2.4. Total protein isolation and western blot
analysis
Total protein was separated in the organic
phase during the preparation of total RNA and
subsequently precipitated with isopropanol, and
then it was washed three times with 0.3 M
guanidine hydrochloride. After washing with 70%
ethanol, protein was dried and dissolved in 1%
SDS. Protein concentration was determined by
using a Bicinchoninic acid (BCA) protein assay kit
(Cabres Co. USA). Bovine serum albumin (BSA)
(Sigma-Aldrich Co. USA) was used as the
standard.
Total 10 μg of protein isolated from brain
tissue was separated by sodium dodecyl sulphate-
polyarylamide gel (SDS-PAGE; 5% stacking gel
and 10.5% separating gel) and then transfered to a
polyvinylidene fluoride (PVDF) membrane. Non-
specific binding sites were blocked by immersing
the membranes in 3% BSA in Tris- buffered saline
Tween (TBST) for overnight at 4

o
C on a shaker.
Levels of the TH and AADC immunoreactive
protein were determined using TH and AADC
primary antibody (Table 2). After membranes
washed several times with washing buffer TBS-T,
the membranes were incubated with primary anti-
TH and AADC antibody for 2hrs at 4
o
C, followed
by incubation with secondary antibodies (Table 2)
for 2hrs at room temperature. The same amount of
protein was loaded and reacted with anti-mouse
actin antibody (Table 2) which was used for
normalization of protein loading. All primary
antibodies and secondary antibodies were diluted in
TBS-T solution.
To reveal the reaction bands, the membrane
was reacted with WEST-ZOL (plus) Western blot
detection system (Intron Biotechnology, Inc.) and
exposed to X-ray film (BioMax MS-1, Eastman
Kodak). A digital image system was used to
determine the density of the bands (Gel Quant,
DNR Bio-Imaging Systems Ltd.).
2.5. Statistical analysis
Data were presented as mean ± S.E.M. Results
were evaluated by Student’s test and by one- way
analysis of variance (ANOVA). A value of P ≤ 0.05
was considered statistically significant.
3. RESULTS

3.1. Effects of MP on TH and AADC mRNA levels
expression in rat brain tissue
To determine the effects of MP on the
catecholamine biosynthetic enzyme genes, we
determined changes in mRNA level of TH and
AADC, the first rate-limiting and the second
enzyme in this pathway. TH and AADC mRNA
levels were examined by RT-PCR. Rats were
treated with different doses of MP (0, 100, 200,
300, and 400 mg/kg) each for 2 and 4 hrs. As
shown in Fig.1 and Fig. 2, TH and AADC mRNA
levels increased in dose-dependent manner.
However, the AADC mRNA level was less than
TH mRNA level. In particular, at 2 hrs TH mRNA
increased by 2.2 ± 3 folds (P<0.001), while AADC
mRNA level rose to 1.86 and 2 folds with doses
300 mg/kg, 400 mg/kg, respectively. At 4hrs, TH
mRNA level increased sharply and was higher than
AADC mRNA level, the highest expression (TH
mRNA 7.4 times (P<0.0001) and AADC mRNA
1.3 times (P<0.05)) was observed with dose 200
mg/kg. The expression of TH and AADC mRNA
levels depend on MP doses and duration of
administration.
86
Effect of Mucuna pruriens on catecholamine biosynthetic enzyme gene expression in rat brain
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin

380 pb
345 pb
150 pb
(㎎/㎏)
Con 100 200 300 400
Relative gene expression
0.0
0.5
1.0
1.5
2.0
2.5
TH
AADC
**
**
**
**
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb
345 pb
150 pb
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb

345 pb
150 pb
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb
345 pb
150 pb
(㎎/㎏)
Con 100 200 300 400
Relative gene expression
0.0
0.5
1.0
1.5
2.0
2.5
TH
AADC
**
**
**
**
(㎎/㎏)
Con 100 200 300 400
Relative gene expression
0.0
0.5
1.0

1.5
2.0
2.5
TH
AADC
(㎎/㎏)
Con 100 200 300 400
Relative gene expression
0.0
0.5
1.0
1.5
2.0
2.5
TH
AADC
**
**
**
**

Relative gene expression
Figure. 1. The effects of MP on the TH and AADC mRNA levels in rat brain tissue at after 2hrs
treatment. Values (n=5/group) are presented as mean ± S.E and compared by one-way ANOVA
and Tukey’s test: *P<0.05 and ** P< 0.001 versus control
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb

345 pb
150 pb
*
(㎎/㎏)
Con 100 200 300 400
Relative gene expression
0
2
4
6
8
TH
AADC
**
**
*
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb
345 pb
150 pb
Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb
345 pb
150 pb

Con 100 200 300 400 (mg/kg)
TH
AADC
β-actin
380 pb
345 pb
150 pb
*
)
(㎎/㎏
Con 100 200 300 400
Relative gene expression
0
2
4
6
8
TH
AADC
**
**
*
*
)
(㎎/㎏
Con 100 200 300 400
Relative gene expression
0
2
4

6
8
TH
AADC
(㎎/㎏)
TH
AADC
**
**
*
0
2
4
6
8
Relative gene expression
Relative gene expression
Con 100 200 300 400

Figure. 2. The effects of MP on the TH and AADC mRNA levels in rat brain tissue at after 4hrs
treatment. Values (n=5/group) are presented as mean ± S.E and compared by one- way ANOVA
and Tukey’s test: *P<0.05 and ** P< 0.001 versus control
Con

(mg/kg)

87
Nguyen Thi Hiep, Truong Thi Thanh Mai, Bui Thai Hang, Kim Sung Jun
3.2. Effect of MP on TH and AADC protein level
expression in rat brain tissue

The protein levels of TH and AADC were
determined by western blot analysis at 2 and 4 hrs
after treatment. All different doses of MP induced
TH and AADC protein levels at 2hrs. TH protein
levels increased significantly at 1.34, 5.62, 6.92
and 9.74 folds with the doses of 100, 200, 300, 400
mg/kg, respectively (Fig. 3), AADC protein levels
increased highly significantly at a dose of 100 mg/kg
by 2.53 times. But only doses of 200 mg/kg and 300
mg/kg have significantly upregulated the TH protein
for 4hrs, while doses of 100 mg/kg and 400 mg/kg
showed the down regulation (Fig. 4). AADC protein
levels did not change at different doses of MP in the
4hrs treatment. The above results indicate that TH
and AADC protein levels were significantly up
regulated with the treatment of MP.
(㎎/㎏)
Con 100 200 300 400
Relative protein levels
0
2
4
6
8
10
TH
AADC
*
**
**

**
(㎎/㎏)
Con 100 200 300 400
Relative protein levels
0
2
4
6
8
10
TH
AADC
*
**
**
**
(㎎/㎏)
Con 100 200 300 400
Relative protein levels
0
2
4
6
8
10
TH
AADC
(㎎/㎏)
TH
AADC

*
**
**
**
0
2
4
6
8
10
Relative protein levels
Relative protein levels

Con 100 200 300 400
Figure. 3. The effects of MP on the TH and AADC protein levels in rat brain tissue at after 2hrs
treatment. Values (n=5/group) are presented as mean ± S.E and compared by one- way ANOVA
and Tukey’s test: *P<0.05 and ** P< 0.001 versus control
(㎎/㎏)
Con 100 200 300 400
Relative protein levels
0
1
2
3
TH
AADC
*
**
**
(㎎/㎏)

Con 100 200 300 400
Relative protein levels
0
1
2
3
TH
AADC
*
**
**
(㎎/㎏)
Con 100 200 300 400
Relative protein levels
0
1
2
3
TH
AADC
(㎎/㎏)
*
**
**
TH
AADC
Con 100 200 300 400
Relative protein levels
0
1

2
3
Relative protein levels

Figure. 4. The effects of MP on the TH and AADC protein levels in rat brain tissue at after 4hrs
treatment. Values (n=5/group) are presented as mean ± S.E and compared by one- way ANOVA
and Tukey’s test: *P<0.05 and ** P< 0.001 versus control
88
Effect of Mucuna pruriens on catecholamine biosynthetic enzyme gene expression in rat brain
4. DISCUSSION
The seed powder of MP has been used in
traditional Ayurvedic India medicine for disease
therapy including PD. All compounds of MP seed
have been identified by several studies (Eustace et
al., 2006; Misra 2004). These researches showed
that MP has high contents of crude protein, amino
acids, total phenols, tannins and, especially, L-
dopa. In additional, MP contains co-enzyme Q-10
and nicotinamide adenine dinucleotide (NADH),
which have neuroprotective activities (Manyam et
al., 2004). MP treatment is known to increase the
dopamine content in the rat brain and MP exhibited
twice the antiparkinsonian activity compared with
synthetic levodopa (Manyam et al., 2004).
Furthermore, synthetic levodopa causes drug –
induced dyskinesias in majority of patients with
PD. MP, however, is reputed to provide anti-
parkinsonian benefits without inducing dyskinesias
(Christopher et al., 2010).
The regulation of TH and AADC expression

has attracted much attention in the field of
neurology. Indeed, the biological function of TH
and AADC is very important for the dopamine
biosynthesis as well as for brain function under
physiological and pathological conditions. The
study of Duan et al., (2005) found that the
expression of TH and AADC genes could fulfill the
function of dopamine synthesis. The present study
was carried out to evaluate the effects of MP on the
catecholamine biosynthetic enzyme genes,
particularly TH and AADC. The results showed
that the levels of TH mRNA and protein increased
significantly at different doses of MP at both 2hrs
and 4hrs treatment. Similarly, the AADC mRNA
and protein levels rose slightly at different doses of
MP at 2hrs treatment. In contrast, the level of
AADC protein did not change at different doses of
MP at 4hrs after treatment. The levels of TH
mRNA and protein are always higher than those of
AADC at different doses of MP during the
treatment period. These results indicate that MP has
a complex and different effect on TH and AADC
gene expression in the rat brain tissue.
5. CONCLUSIONS
The present study shows that both
catecholamine biosynthetic enzymes encoding
genes TH and AADC were up – regulated by the
treatment of MP. The expression of TH and AADC
genes depends on MP doses and duration of
administration. Thus, MP may provide a platform

for future drug discoveries and novel therapy
strategies for PD.
Acknowledgements
This work was support by a reseach grant
provied by Chosun University, 2007.
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