Effects of epigallocatechin gallate on tissue protection and functional recovery after contusive spinal cord injury in rats

Abstract

Recent studies revealed the neuroprotective effects of epigallocatechin gallate (EGCG) on a
variety of neural injury .The purpose of this study was to determine the effects of EGCG on
the tissue protection and behavioral improvement after spinal cord injury (SCI). Rats were
randomly divided into four groups of 18 rats each as follows: sham-operated group, trauma
group, and EGCG treatment groups (50 mg/kg, i.p., immediately and 1 hour after SCI). Spinal
cord samples were taken 24 hours after injury and studied for determination of
malodialdehyde (MDA) levels, immunohistochemistry of Bax and Bcl-2, and TUNEL
reaction. Behavioral testing was performed weekly up to 6 weeks post-injury. Then, the
rats were euthanized for histopathological assessment. The results showed that MDA levels
were significantly decreased in EGCG treatment groups. Greater Bcl-2 and attenuated Bax
expression could be detected in the EGCG-treated rats. EGCG significantly reduced TUNELpositive
rate. Also, EGCG significantly reduced the percentage of lesion area and improved
behavioral function than the trauma group. On the basis of these findings, we propose that
EGCG may be effective in protecting rat spinal cord from secondary injury.

Full text

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Research Report
Effects of epigallocatechin gallate on tissue protection and
functional recovery after contusive spinal cord injury in rats
Ali Reza khalatbarya,⁎, Taki Tiraihib, Mandana Beigi Boroujenic, Hasan Ahmadvandd,
Majid Tavafic, Ahmad Tamjidipoorc
aRazi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Khoramabad, Iran
bDepartment of Anatomical Sciences, University of Tarbiat Modarres, School of Medicine, Tehran, Iran
cDepartment of Anatomical Sciences, Lorestan University of Medical Sciences, Khoramabad, Iran
dDepartment of Biochemistry, Lorestan University of Medical Sciences, Khoramabad, Iran
A R T I C L E I N F OA B S T R A C T
Article history:
Accepted 29 September 2009
Available online 6 October 2009
Recent studies revealed the neuroprotective effects of epigallocatechin gallate (EGCG) on a
variety of neural injury .The purpose of this study was to determine the effects of EGCG on
the tissue protection and behavioral improvement after spinal cord injury (SCI). Rats were
randomly divided into four groups of 18 rats each as follows: sham-operated group, trauma
group, and EGCG treatment groups (50 mg/kg, i.p., immediately and 1 hour after SCI). Spinal
cord samples were taken 24 hours after injury and studied for determination of
malodialdehyde (MDA) levels, immunohistochemistry of Bax and Bcl-2, and TUNEL
reaction. Behavioral testing was performed weekly up to 6 weeks post-injury. Then, the
rats were euthanized for histopathological assessment. The results showed that MDA levels
were significantly decreased in EGCG treatment groups. Greater Bcl-2 and attenuated Bax
expression could be detected in the EGCG-treated rats. EGCG significantly reduced TUNEL-
positive rate. Also, EGCG significantly reduced the percentage of lesion area and improved
behavioral function than the trauma group. On the basis of these findings, we propose that
EGCG may be effective in protecting rat spinal cord from secondary injury.
© 2009 Elsevier B.V. All rights reserved.
Keywords:
Epigallocatechin gallate
Spinal cord injury
Immunohistochemistry
1.Introduction
Neurological damages after traumatic spinal cord injury result
from both primary mechanical injury and secondary degene-
ration process (Amar and Levy, 1999). Outcome of spinal cord
injury depends on the extent of secondary damage mediated
by a series of cellular, molecular, and biochemical cascades,
including calcium ion influx (Juurlink and Paterson, 1998),
oxygen free radical-induced lipid peroxidation (Hall, 1993a,b),
inflammatory reaction (Popovich et al., 1997), autoimmune
response (Popovich et al., 1996), and vascular events (Mautes
et al., 2000). Apoptosis is an important mediator of secondary
damage after SCI (Liu et al., 1997), which is triggered by
varieties of mechanisms, including glutamatergic excitotixi-
city, free radical damage, cytokines, and inflammatory injury.
Inrecentyears,muchattentionhasbeenfocusedonsecondary
injury because it appears to be susceptible to therapeutic
interventions that may include using of antiapoptotic drugs,
free radical scavengers, and anti-inflammatory agents.
The chemical composition of green tea contains many
polyphenolic compounds, generally known as catechins.
Catechins have many actions such as free radical
B R A I N R E S E A R C H 1 3 0 6 ( 2 0 1 0 ) 1 6 8 – 1 7 5
⁎ Corresponding author. Fax: +98 o661 6200133.
E-mail address: khalat90@yahoo.com (A.R. khalatbary).
0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2009.09.109
available at www.sciencedirect.com
www.elsevier.com/locate/brainres

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scavenging/antioxidant actions, preventing lipid peroxidation
due to oxidative stress, modulating apoptotic pathways,
prooxidant properties, and anti-inflammatory effects (Suther-
landetal.,2006).(−)-Epigallocatechingallate(EGCG)isthemost
abundantcompositionoftheteacatechinsandisthoughttobe
responsible for the majority of biological activity of green tea
extracts (Kimura et al., 2002). EGCG has been shown to be of
some protective effects against neuronal damage after tran-
sient ischemia (Choi et al., 2004), oxidative damage on
periventricular white matter in hydrocephalic rats (Etus
etal.,2003),suppressionofdiseaseprogressionofamyotrophic
lateral sclerosis (Koh et al., 2006), acute hypoxia (Wei et al.,
2004), iron-induced oxidative stress (Lin et al., 1998), Alzhei-
mer'sandParkinson'sdiseases(Weinrebetal.,2004),andaging
(He et al., 2009). Recently, green tea extract has been demon-
strated to attenuate secondary inflammatory response follow-
ing spinal cord injury in mice (Paterniti et al., 2009).
In spite of some experimental evidence for the neuropro-
tective effects of EGCG in cerebral ischemia and neurodege-
nerative diseases, evidence regarding its effects on SCI is still
limited. Accordingly, in the present study, we investigated the
beneficial effects of EGCG administration on tissue protection
and neurobehavioral recovery after SCI.
2. Results
2.1.Lipid peroxidation levels
The histogram of the MDA levels for all groups at 24 hours
post-injury is shown in Fig. 1. The MDA levels were 4.34±1.54
forthesham-operated group,18.80±2.54 forthetraumagroup,
11.59±1.51 for the EGCG1 treatment group, and 9.88±1.34 for
the EGCG2 treatment group. Induction of SCI in the trauma
(p<0.001) and EGCG treatment groups (p<0.01) produced a
significant elevation in lipid peroxidation level compared to
the sham-operated group. The MDA levels in the EGCG
treatment groups were significantly lower than those in the
trauma group (p<001), while the differences between EGCG1
and EGCG2 were not significant (p>0.05).
2.2.TUNEL
Almost no TUNEL-positive cells could be detected in sham-
operated group (1.128±0.68), whereas many cells were
intensely stained in the tissues obtained from trauma
group (47.56±9.11) (Fig. 2A). In contrast, a small number of
TUNEL-positive cells were detected in tissues obtained from
EGCG-treated rats (EGCG1: 22.07±5.08; EGCG2: 20.60±2.57)
Fig. 1 – Effects of EGCG on MDA level. Histogram shows the
levels of malondialdehyde (MDA) at 24 hours after SCI.
Values are expressed as nanomoles per gram of wet tissue.
*p<0.001 versus sham group; **p<0.01 versus sham and
trauma groups;#p>0.05 versus EGCG1 group.
Fig. 2 – Effects of EGCG on cell apoptosis. Light
photomicrographs show TUNEL-positive cells on the spinal
slides obtained from trauma(A) and EGCG (B) groups24 hours
after injury (counterstained with hematoxylin;
magnification, ×200). The positive staining of TUNEL is
presented by a brown color of nucleus. (For interpretation of
the references to colour in this figure legend, the reader is
referred to the web version of this article.)
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(Fig. 2B). The histogram of the quantitative analysis of
TUNEL-positive cells in experimental groups is shown in
Fig. 3.
2.3. Immunohistochemistry for Bax and Bcl-2
Figs. 4 and 5 show the immunohistochemical staining of Bax
and Bcl-2, respectively. Sections of spinal cord from sham-
operated rats did not stain for Bax, whereas the spinal cord
sections of trauma rats exhibited a positive staining for Bax
(62.89±5.63) (Fig. 4A). EGCG treatment reduced the degree
of positive staining for Bax (EGCG1: 36.22±3.93; EGCG2:
33.29±7.55) (Fig. 4B). In addition, the expression of Bcl-2 was
strong in the sham-operated group (84.43±2.61) and was
moderate in the trauma group (38.93±5.83) (Fig. 5A) compared
with the strong up-regulation in the EGCG treatment groups
(EGCG1: 55.77±6.29; EGCG2: 57.64±4.79) (Fig. 5B). The histo-
grams of the quantitative analysis of Bax- and Bcl-2-positive
cells in the experimental groups are shown in Figs. 6 and 7,
respectively.
Fig. 3 – Percentage of apoptotic cells. Histogram shows the
percentage of TUNEL-positive cells expressed as mean±SD.
*p<0.001 versus sham group; **p<0.001 versus sham and
trauma groups;#p>0.05 versus EGCG1 group.
Fig. 4 – Effects of EGCG on Bax expression. Light
photomicrographs show immunohistochemical expression
of Bax in trauma (A) and EGCG (B) groups 24 hours after SCI
(magnification, ×200). The positive staining of Bax is
presented by a brown color of cytoplasm. (For interpretation
of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Fig. 5 – Effects of EGCG on Bcl-2 expression. Light
photomicrographs show immunohistochemical expression
of Bcl-2 in trauma (A) and EGCG (B) groups 24 hours after SCI
(magnification, ×200). The positive staining of Bcl-2 is
presented by a brown color of cytoplasm. (For interpretation
of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
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2.4.Evaluation of motor function
The BBB locomotor rating scores in the four groups has been
presented as mean value±SD (Fig. 8). All rats of the sham-
operated group had a normal score (21). Rats in the trauma
group had an early weak improvement in neurological
function (3.66±0.8 on day 1; 5.33±0.81 on day 14), which was
plateaued by the fourth week. A significant gradual recovery
was observed in the rats treated with EGCG. There was a
significant difference in the BBB score at the end of the study
(6th week) between EGCG treatment and trauma groups
(p<0.001), while the differences between EGCG1 and EGCG2
were not significant (p>0.05).
2.5.Histopathological assessment
Results of histopathological examination are shown in Fig. 9.
Spinal cord sections obtained from the sham-operated rats
were normal. Characteristic necrosis, complete loss of
neurons, wide demyelination, and extensive vacuolation in
the epicenter of injured spinal cord obtained from the
trauma rats are shown in Fig. 9A. The sections of injured
spinal cord obtained from the EGCG treatment groups (Fig.
9B) retained more myelin tissue and neurons in gray matter
than those in the trauma group. Also, the histogram of
quantification of lesion area in spinal cord is shown in Fig.
10. The area of lesion was calculated as a percentage of the
whole spinal cord area. The injured spinal cords of EGCG-
injected rats had significantly smaller lesion areas (EGCG1:
31.73±5.29, EGCG2: 29.39±4.02) than those of the trauma rats
(68.52±4.77) (p<0.001).
3.Discussion
The main findings of the current study showed that treatment
of SCI with EGCG attenuates (1) MDA levels, as an indicator of
lipid peroxidation, (2) neuronal apoptosis, (3) spinal tissue
loss, and (4) motor dysfunction.
Secondary autodestructive processes of SCI have a highly
debilitating pathology (Collins, 1983), considered to be a
number of interrelated processes such as free radical genera-
tion (Hall, 1993a), lipid peroxidation (Hall, 1993b), and apop-
tosis (Liu et al., 1997). Each treatment, which interrupts the
secondary processes, could improve SCI. Free radicals are
molecules that possess unpaired electrons that make them
highly reactive to lipids, which cause oxidation of fatty acids
(Hall et al., 1994). Lipidperoxidation isan important pathologic
event in post traumatic neuronal degeneration, demonstrated
to reach peak values immediately after SCI (Kwon et al., 2004).
Thus, inhibition of lipid peroxidation is thought to be one of
the principal mechanisms of action for therapeutic agents. In
this study, malondialdehyde (MDA), which is formed from
polyunsaturated fatty acids' breakdown, was measured as an
index of lipid peroxidation (Koudelva and Mourek, 1994). Our
results showed that administration of EGCG immediately and
1 hour after SCI significantly attenuated the level of MDA
compared to those of trauma group. The neuroprotective
properties of EGCG against neurodegenerative diseases and
cerebral ischemia have been well documented (Weinreb et al.,
2004; Sutherland et al., 2006). One of the neuroprotective
mechanisms of EGCG is probably related with its effects
onfreeradical-inducedlipidperoxidation.Green tea
Fig. 6 – Percentage of Bax-positive cells. Histogram shows the
percentage of Bax-positive cells expressed as mean±SD.
*p<0.001 versus sham group; **p<0.001 versus sham and
trauma groups;#p>0.05 versus EGCG1 group.
Fig. 7 – Percentage of Bcl-2-positive cells. Histogram shows
the percentage of Bcl-2-positive cells expressed as
mean±SD. *p<0.001 versus sham group; **p<0.001 versus
sham and trauma groups;#p>0.05 versus EGCG1 group.
Fig. 8 – Effects of EGCG on motor recovery. Histogram shows
motor recovery in all groups. Values are mean±SD. *p<0.001
versus trauma group;#p>0.05 versus EGCG1 group.
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polyphenols, mainly EGCG, due to the hydroxyl groups can
bindtothefree radicalsandneutralizethose(Sutherland etal.,
2006). Moreover, they can indirectly increase the body's
endogenous antioxidants (Skrzydlewska et al., 2002). EGCG
reduced the lipid peroxidation injury in synaptosomes (Guo et
al., 1996). Previousstudies reportedthatlipidperoxidationwas
reducedbyEGCGadministrationaftercerebralischemiainrats
(Choi et al., 2004) and gerbils (Lee et al., 2004). Recently, some
investigators revealed that malondialdehyde (MDA) levels
decreased after EGCG treatment on aging mice model (He
et al., 2009).
Apoptosis is a key mechanism of secondary damages after
SCI (Crowe et al., 1997) that is regulated by the Bcl-2 family
proteins. Among these proteins, Bcl-2 and Bax play antiapop-
totic and proapoptotic roles, respectively (Reed et al., 1998).
The ratio of Bax to Bcl-2 determines the cell fate; excess Bcl-2
leadsto survival of cells, whileBax inducesapoptosis(Oltvai et
al., 1993; Zha et al., 1996). After traumatic spinal cord injury,
neuralcellscontinuetodievia apoptosis,andthisrepresents a
potentially avoidable event by pharmacological interventions
(Kwon et al., 2004). Results of our immunohistochemical
assessment showed that the treatment with EGCG reduced
positive staining for Bax; while on the contrary, it increased
positive staining for Bcl-2 in the EGCG treatment groups.
Conversely, EGCG inhibited the expression of proapoptotic
protein Bax and induced that of the antiapoptotic protein Bcl-
2, thereby provided the molecular evidence for the neuropro-
tective activity of EGCG. To correlate neuronal cell loss to
apoptosis, we carried out TUNEL staining method. The
numbers of TUNEL-positive cells at the spinal lesion site of
EGCG-treated rats were significantly lower than the trauma
group. In vitro and in vivo studies suggest the protective
effects of EGCG on neural apoptosis via altering the expression
of antiapoptotic and proapoptotic genes (Sutherland et al.,
2006). EGCG had a protective effect against 6-hydroxydopa-
mine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro
model of Parkinson's disease (Nie et al., 2002). EGCG sup-
pressed apoptosis induced by oxidative radical stress through
increasing phosphatidylinositol-3 kinase/Akt-dependent anti-
apoptotic signals (Koh et al., 2004). Moreover, recent studies
have shown that EGCG inhibits caspase-3 activation in the
spinal cord in amyotrophic lateral sclerosis model mice (Koh
et al., 2006) and on aging mice that was induced by D-galactose
(He et al., 2009). Other studies showed that EGCG prevented
Bax and Bad expression, while it induced Bcl-2 to protect SH-
SY5Y cells from apoptosis (Levites et al., 2002). On the
contrary, several studies demonstrated that EGCG inhibits
cellular proliferation and induces apoptosis in various cancer
cell lines (Khan et al., 2006) with minimum or without
affecting normal cells. EGCG-induced apoptosis can be
mediated through activation of caspases (Wu et al., 2009),
depolarization of mitochondrial membranes (Chung et al.,
2001), production of reactive oxygen species (Nakazato et al.,
2005), inhibition of NF-kappaB (Ahmad et al., 2000), inhibition
of TNF-alpha gene expression and its release (Okabe et al.,
Fig. 9 – Effects of EGCG on tissue protection.
Photomicrographs of horizontal sections through the lesion
epicenter were taken 6 weeks after SCI in trauma (A) and
EGCG (B) groups (stained with hematoxylin and eosin;
magnification, ×40).
Fig. 10 – Percentage of spared tissue. Histogram shows the
mean lesion area as a percentage of the whole spinal cord.
Values are mean±SD. *p<0.001 versus sham group;
**p<0.001 versus sham and trauma groups;#p>0.05 versus
EGCG1 group.
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1999), induction of death-associated protein kinase2, P53-like
proteins, and cyclin-dependent kinase inhibitors gene expres-
sion (Shammas et al., 2006), inhibition of growth factor's
signaling (Otsuka et al., 1998), and binding to Fas on the cell
surface (Hayakawa et al., 2001). Other studies documented
that EGCG treatment decreases the expression of Bcl-2 and
Bcl-xl, but increases the expression of Bax and Bad (Nishikawa
et al., 2006), meanwhile, inhibits the antiapoptotic function of
Bcl-2 proteins (Lambert et al., 2005). Recently, the 67-kDa
laminin receptor has been identified as a cell surface receptor
for a specific function of EGCG on tumor cells, which is
significantly elevated in cancer cell line relative to the normal
cell (Shammas et al., 2006; Umeda et al., 2008).
The preservation of the spinal tissue from secondary injury
is essential for limiting neurological deficits. Also, the forma-
tion of cystic cavities is the fate of progressive changes of
contusive SCI (Basso et al., 1996). Results of histopathological
assessment showed that significantly more spinal tissue was
spared in EGCG-treated rats. Investigations revealed that
EGCG treatment reduce infarct volume in a variety of cerebral
ischemia models (Sutherland et al., 2006). Another study
showed that EGCG had a neuroprotective effect on hydro-
cephalus-induced periventricular white matter damage (Etus
et al., 2003).
To evaluate the effectiveness of EGCG therapy on beha-
vioral outcomes after SCI, we used the BBB locomotor rating
scale. Our results showed that after EGCG injection, functional
outcome was significantly improved compared to nontreated
rats. All the findings obtained from the present study
demonstrated biochemical, histopathological, and functional
evidences that EGCG treatment after SCI had neuroprotective
effects.
4.Experimental procedures
4.1.Animals
Female adult Spargue-Dawley rats were used (250–300 g)
(Pasteur's Institute, Tehran, Iran) in this study. They were
kept under standard conditions according to the guidelines of
the university's animal care codes to minimize the animal's
suffering. The ovarian cycles of the animals became
synchronized.
4.2. SCI
Contusive SCI was carried out using the weight dropping
technique (Basso et al., 1996). The animals were anesthetized
with ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.).
Laminectomy was performed at T9 level vertebra; the dorsal
surface of the cord was then subjected to weight drop impact
using a 10-g weight dropped from a height of 2.5 cm in order to
produce contusive SCI. Following the surgery, the recovery of
the animals was assisted by administering lactated Ringer's
solution (12–25 ml) subcutaneously immediately after surgery
and cefazolin (Jaber Ibn Hayan, Tehran) (50 μg/kg) which was
administered twice daily for 3 days (Chopp et al., 2000). The
urinary bladders were pressed three times a day until the
function was retained.
4.3.Experimental groups
The rats were randomly allocated in four groups, each
containing 18 rats: (i) sham-operated group, which underwent
laminectomy alone; (ii) trauma group, which underwent
laminectomy followed by SCI and received saline (vehicle);
(iii and iv) EGCG treatment groups, which underwent laminec-
tomy followed by SCI and received a 50-mg/kg single dose of
EGCG (Sigma) intraperiteonally immediately (EGCG1) and
1 hour (EGCG2) after trauma, respectively. Each group of
animals was divided into 3 subgroups: (A) for biochemical
analysis (n=6), (B) for TUNEL staining and immunohistochem-
istry (n=6), and (C) for behavioral and histopathological
assessment (n=6).
4.4.Biochemical analysis
Six rats from each group were euthanized, and 1.5-cm
traumatized spinal cord sample was removed for biochemical
analysis 24 hours after SCI. The obtained samples were
thoroughly cleaned of blood and the meninges were carefully
removed. Then the tissue samples were immediately frozen
and stored in a −70 °C freezer for assays of tissue malondial-
dehyde(MDA)levels(MiharaandUchiyama, 1978) asa product
of lipid peroxidation which reacts with thiobarbituric acid
(TBA). The absorbance of the supernatant was measured by
spectrophotometry at 535 nm. TBA reactant concentration
was expressed as nanomoles per gram of wet tissue.
4.5.TUNEL staining and immunohistochemistry
At 24 hours after SCI, the spinal cords, which contained the
contusion epicenter, were fixed in 10% (wt./vol.) PBS-buffered
formaldehyde and embedded in paraffin. Eight-micrometer
sections were serially cut horizontally from each block. TUNEL
staining was performed using a TUNEL detection kit according
to the manufacturer's instructions (Roche). Briefly, spinal
sections rehydrated, incubated in 3% H2O2for 10 minutes and
incubated with proteinase-K for 15 minutes at 37 °C. TUNEL
reaction mixture was added to the samples and incubated for
60 minutes at 37 °C. In the following, converter POD was added
and samples were incubated for 30 minutes at 37 °C, demon-
strated with diaminobenzidine tetrahydrochloride for 5 min-
utes, and counterstained with hematoxylin. A negative
control was similarly performed except for omitting the
TUNEL reaction mixture. For quantitative analysis, percen-
tages of TUNEL-positive neurons were taken in 10 microscopic
fields for each animal.
For immunohistochemistry, sections were incubated in
goat serum (in order to block nonspecific site), and anti-Bax
rabbit polyclonal antibody (1:50 in PBS, vol./vol., Abcam), or
anti-Bcl-2 rabbit polyclonal antibody (1:100 in PBS, vol./vol.,
Abcam) overnight at 4 °C. Sections were washed with PBS
and then incubated with secondary antibody conjugated
with horseradish peroxidase (goat anti-rabbit IgG peroxi-
dase, Abcam) for 2 hours and demonstrated with diamino-
benzidine tetrahydrochloride for 5 minutes. Afterwards,
they were counterstained with hematoxylin, dehydrated,
and mounted. For negative controls, primary antibodies
were omitted. For quantitative analysis, percentages of Bax-
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and Bcl-2-positive neurons were taken in 10 microscopic
fields for each animal.
4.6.Behavioral assessment
Six rats fromeach experimental groupwereused in behavioral
analysis. The Basso–Beattie–Bresnehan (BBB) behavioral score
test (Basso et al., 1995) was used to evaluate hind limb motor
function, which was carried out weekly up to 6 weeks after
injury. The maximum possible BBB score (normal) was 21 and
a score of 0 was used to represent complete paralysis of the
hind limb after SCI.
4.7. Histopathological assessment
Six weeks after SCI, rats were deeply anesthetized and
intracardiac perfusion was performed with saline, followed
by 4% paraformaldehyde. After perfusion, spinal cords were
removed immediately and immersed in the same fixative
overnight. The spinal cords containing contusion epicenter
were embedded in paraffin. Eight-micrometer sections were
serially cut horizontally from each block and stained with
hematoxylin and eosin. The area of the profile and the lesion
areas in the same section were measured. Then, the percen-
tage of lesion areas of each sample were calculated.
4.8.Statistical analysis
Statistical analysis was carried out using the SPSS package.
Resultswerepresentedasmeanvalues(±SD).TheK–Stestwas
used in order to evaluate the normality of the data. Also,
Tukey's multiple comparison test and the analysis of the
variance were used in order to compare each two groups and
compare the data among the groups, respectively. A value of
P<0.05 was considered significant.
Acknowledgment
This work was supported by Razi Herbal Medicines Research
Center.
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