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

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

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

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)

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

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

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

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

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
*
**
**

*
**
**
**
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
*
**
**
(㎎/㎏)

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

for future drug discoveries and novel therapy
strategies for PD.
Acknowledgements
This work was support by a reseach grant
provied by Chosun University, 2007.
REFERENCES
Christopher A. Lieu, R. Kunselman Allen,
V.Manyam Bala, Venkiteswaran Kala, and
Subramanian Thyagarajan. (2010). A water
extract of Mucuna pruriens provides long – term
amelioration of parkinsonism with reduced risk
for dyskinesias. Parkinsonism and related
disorders. 16: 458 – 465.
Duan Chun- Li, Yue Su, Chun –Li Zhao, Qun –
Yuan Xu and Hui Yang.(2005). The assays of
activities and function of TH, AADC and GCH1
and their potential use in ex vivo gene therapy of
PD. Brain Research Protocols. 16: 37 – 43.
Eustace A lyayi, Holger Kluth and Markus
Rodehutscord.(2006). Chemical composition,
antinutritional constituents, precaecal crude
protein and amino acid digestibility in three
unconventional tropical legumes in broilers. J
Sci Food Agric. 86: 2166 – 2171.
Katzenschlarger R, Evans A, Manson A, P N
Patsalos, N Ratnaraj, H Watt, L Timmermann, R
Van der Geissen, A J Lees.(2004). Mucuna
pruriens in Parkinson’s disease: a double blind
chinical and pharmacological study. J Neuol
Neurosurg Psychiatry.75: 1672 -1677.

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