Curcuma comosa Prevents the Neuron Loss and Affects the Antioxidative Enzymes
in Hippocampus of Ethanol-treated Rats
Curcuma comosa Roxb. is widely used as a gynaecological traditional medicine in South-East Asia and recent behavioral studies have shown that C. comosa extract significantly improved the spatial memory in rats. The present study investigated the protective effects of Curcuma comosa hexane extract on the ethanol (EtOH)-induced oxidation in rat brains. Young female Wistar rats were given 20% of EtOH intraperitoneally to induce the oxidative stress. Subsequently, C. comosa hexane extract was intraperitoneally co-administered at the doses of 100 and 250 mg kg-1 b.wt. to the EtOH-induced rats for 14 days. The neuron densities of CA1, CA3 and CA4 areas of the hippocampus were counted and the activities of hippocampal Catalase (CAT), Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) were determined. EtOH significantly decreased the neuron densities in Cornu Ammonis (CA), including CA1 and CA3 areas; however, the decrease was prevented by C. comosa co-administration. EtOH administration also increased the CAT and GPx activities in the hippocampus which were reversed by C. comosa co-administration. Moreover, C. comosa administration increased the SOD activity in a dose-dependent manner in the EtOH treated groups. C. comosa prevented the neuron loss in the hippocampus caused by EtOH. The possible neural protective mechanism may involve with the changes in activities of the antioxidant enzymes in the hippocampus.
Received: May 11, 2012;
Accepted: July 17, 2012;
Published: September 03, 2012
Oxidative stress is well recognized as an underlying etiology associated with
the aging of the brain and many neurodegenerative diseases (Maracchioni
et al., 2007; Lin and Beal, 2006). The brain
is vulnerable to oxidative stress because of its high consumption of oxygen
and its structural richness in polyunsaturated fatty acids. Many factors could
affect oxidative stress in the brain, especially the presence of some xenobiotics.
Alcohol is one of the notable inducers of oxidative stress. In the brain, alcohol
is generally oxidized by the enzyme alcohol dehydrogenase and cytochrome P450
enzymes and consequently increases the Reactive Oxygen Species (ROS) concentration.
The extra ROS breaks the balance of the oxidative status in the brain and neuronal
diseases occur. Alcohol consumers are generally at higher risk of neuron injury
associated with cognitive deficits compared to non-alcohol consumer (Parson,
1998; Zeigler et al., 2005).
To relieve oxidative stress in the brain, the endogenous ROS scavengers, such
as Catalase (CAT), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx),
are generally considered as the essential enzymes to remove excess ROS. The
activities of these three enzymes are important in maintaining a normal oxidative
status (Bakan et al., 2003).
Curcuma comosa Roxb. is widely used as a gynaecological traditional
medicine in South-East Asia (Piyachaturawat et al.,
1995a). The plants hexane extract has been also reported to have uterotrophic
effects and estrogenic activities in rats (Piyachaturawat
et al., 1995a, b), Other pharmacological
effects, such as decreasing the cholesterol (Piyachaturawat
et al., 1999), enhancing the vascular relaxation (Intapad
et al., 2009) and preventing the bone loss in the estrogen deficient
mice (Weerachayaphorn et al., 2011) have also
been documented. Recent behavioral studies have shown that C. comosa
hexane extract significantly improved the spatial memory in the ovariectomized
rats (Su et al., 2010, 2011).
The hippocampus is the essential area related to spatial memory. Biological
and histological changes can be found in association with behavioral changes.
In this study, the effect of C. comosa hexane extract on EtOH-induced
oxidative stress in rat brains was investigated by measuring hippocampal neuron
density and antioxidative enzyme activities to reveal the conceived neural protective
mechanism of C. comosa.
MATERIALS AND METHODS
Plant material and preparation: Curcuma comosa Roxb. rhizome
was harvested in January, 2008 from Nakon Pathom Province, Thailand, (identified
and provided by Professor Piyachaturawat Pawinee; Mahidol University, Bangkok).
A voucher specimen was filed and kept in our laboratory (BS-C-03). The experiment
was performed at 2009. Dry Curcuma comosa rhizome was crushed to crude
powder and extracted in n-hexane Soxhlet apparatus until the outlet hexane was
colorless. The hexane fraction was evaporated to a brown-yellow oily extract
and then kept at 4°C until use. One of a major pure compounds, 1,7-diphenyl-5-hydroxy-(1E,
3E)-1,3-heptadiene, extracted from this crude extract (kindly provided by Professor
Apichart Suksamrarn from Ramkhamhaeng University) was used as a standard reference
(Fig. 1). A milligram crude extract was equal to 0.311 mg
of 1,7 -diphenyl-5-hydroxy-(1E, 3E)-1,3-heptadiene.
Chemicals: Absolute ethanol was obtained from WNR International Ltd. (England); Potassium thiocyanide (KSCN), ethylenediaminetetraacetic acid (EDTA), nitroblue tetrazolium (NBT), xanthine, xanthine oxidase, glutathione (reduced form) (GSH), glutathione reductase (GR), hydrogen peroxide (H2O2), sodium azide (NaN3) and DL-dithiothreitol (DTT) were obtained from Sigma Ltd. (USA). All other chemicals were analytical grade obtained from local distributors.
Experimental animals: Eighty four female Wistar rats, 8 weeks old, were
obtained from the National Animal Center of Mahidol University, Thailand and
kept in an environment maintained at 25±2°C, relative humidity of
50~70% and a 12 h light/dark cycle. Food and tap water were provided ad libitum.
The animals were allowed 1 week to acclimatize this environment prior to the
start of the experiment.
||The HPLC chromatograph of (a) 1,7-diphenyl-5-hydroxy-(1E,
3E)-1,3-heptadiene (0.2 mg mL-1) and (b) C. comosa hexane
extract (0.1 mg mL-1)
All experiments were conducted under the National Institute of Health Guide
for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised
1996 and approved by the Ethics Committee (Animal Care and Use Committee) of
Khon Kaen University (Reference No. 05220.127.116.11/30).
The rats were randomly divided into 6 groups (n = 14) as follows:
||Group 1: Control group (control): received normal saline
2 g kg-1 b.wt.
||Groups 2: Ethanol group (EtOH): received 20% ethanol
2 g kg-1 b.wt.
||Group 3, 4: C. comosa hexane extract treated
groups (C1, C2): received C. comosa extract at the doses of 100 and
250 mg kg-1 b.wt., respectively
||Group 5, 6: EtOH and C. comosa extract co-treated
groups (C1+E, C2+E): received 20% ethanol 2 g kg-1 b.wt. plus
C. comosa extract at the doses of 100 or 250 mg kg-1 b.wt.,
All the administrations were intraperitoneally injected every day for 14 days. At the 15th day, the rats were sacrificed by cervical dislocation. The brains were immediately removed, rinsed in 0.1 M ice-cold Phosphate Buffer Solution (PBS) and incised longitudinally into two halves. The left halves was used for histological studies and the hippocampi of the right halves were separated on an ice-cold stage and then kept at -80°C for further enzyme assay.
Measurement of the neuron density in the hippocampus
Sample preparation: The left half of the brain used for histological
studies was saturated in 0.1 M PBS containing 30% sucrose at room temperature
for 5 days. The brain was frozen with dry ice and then sectioned by microtome
at 30 μm thickness. Serial one from every six sections was collected and
mounted on the glass slide and air dried overnight. Then the collected sections
were then stained by 1% cresyl violet.
Neuron counting: The neuron numbers of hippocampus in CA1, CA3 and CA4
areas were counted under a light microscope at 40 times magnification (Fig.
2). The neuron counting began from the section that each area was identified
until the longitudinal end of the area and totally 20-30 slides were counted
per animal depending on the brain size. All the neuron cells in the 0.075 mm2
were compared. Overlay neurons were counted twice. The neuron cells at the edges
of the field were included if more than 50% of their size were in the counting
||Morphology of hippocampus demonstrating the CA1, CA3 and CA4
area. The dashed squares show the neuron counting areas
The UTHSCSA Imager Tool computer program (developed at the University of Texas
Health Science Center at San Antonio, Texas and available from the Internet
by anonymous FTP from maxrad6.uthscsa.edu) was used in the counting process.
Assay of antioxidant enzymes activity in the hippocampus
Sample preparation: Hippocampi from two animals were pooled together
for analysis because of their tiny size so that the sample size for enzymatic
study was seven. The pooled hippocampi (around 0.25 g) were separately homogenized
with three volume of 0.1 M PBS and then centrifuged at 10,000 g, 4°C for
10 min. The supernatants were taken into aliquots and kept at -20°C until
the consecutive analysis. Protein assays were carried out for each sample following
the method described in a previous report (Lowry et al.,
Assay of enzyme activity: For superoxide dismutase activity, the method
was modified from a previous study (Ewing and Janero, 1995).
Catalase activity was measured by a modified method of Cohen
et al. (1996). For glutathione peroxidase activity, the method was
modified from Mannervik (1985).
Statistical analysis: The results are shown as Mean±SEM. One-way ANOVA followed by a LSD post hoc test was used to analyze the difference among groups using the SPSS software (version 11.0). The criteria for statistical significance was p<0.05.
Quality control of the C. comosa extract: One of a major pure
compounds, 1,7-diphenyl-5-hydroxy-(1E, 3E)-1,3- heptadiene, extracted from this
crude extract was used as a standard reference.
||The neuron density of (a) CA1 and (b) CA3 areas in hippocampus,
(a1) and (b1): Control groups, (a2) and (b2): Ethanol treated groups and
(a3) and (b3): Ethanol and C. comosa co-treated groups, Bar = 75
Figure 1 showed the HPLC chromatogram of C. comosa
extract and the reference standard. A milligram crude extract was equal to 0.311
mg of 1,7-diphenyl-5-hydroxy-(1E, 3E)-1,3-heptadiene.
Histological study: A randomly selected area from each area of CA1,
CA3 and CA4 in hippocampus were selected for neuron counting and the neuron
densities between groups were compared to show the significant difference (Fig.
2). As shown in Fig. 3, a2 and b2
showed the decreased neuron density of CA1 and CA3 in ethanol treated group
when compared to that of control (Fig. 3a1, b1)
and EtOH plus C. comosa treated (Fig. 3a3, b3)
groups. As the statistical analyze result (Fig. 4) among the
six animal groups, the EtOH-treated group had the significantly lowest neuron
density in the CA1 and CA3, but not the CA4 areas. Administration of C. comosa
in both doses prevented the neuron loss in CA1 and CA3 areas in the EtOH-treated
groups. Administration of C. comosa alone in the normal rats did not
change the neuron density in CA1, CA3 and CA4 areas of the hippocampus.
Antioxidant enzyme activities: As shown in Table 1,
the EtOH administration alone slightly increased the SOD activity in hippocampus
when compared to the control group but with no statistical significance.
||The neuron density in CA1, CA3 and CA4 areas. The values are
expressed in terms of Mean±SEM (n = 14), *Significant decrease of
neuron density of the EtOH group when compared to the other 5 groups in
the CA1 and CA3 areas
|| The enzyme activities in hippocampus
|The values are expressed in terms of Mean±SEM (n=7).
1 1,2, 3, 4 denote the significant difference from
Control and C2 groups; Control, C1 and C2 groups; C1+E and C2+E groups;
the other 5 groups, respectively (p<0.05)
However, when C. comosa hexane extract was administered at the doses
of 100 and 250 mg kg-1 body weight to the EtOH-treated groups, the
SOD activities were significantly higher than the other groups. The EtOH administration
slightly increased the CAT activity when compared to the control group. The
administration of C. comosa significantly reversed the increasing CAT
activity. The EtOH administration significantly increased the GPx activity in
hippocampus when compared to the other groups. However, the administration of
C. comosa in the EtOH-treated groups kept the GPx activities at the
same level as the control group.
The present study used EtOH to induce the toxicity in the central nervous systems.
It is well known that EtOH consumption cause neurodegeneration. Numerous studies
have provided the histological or the biological evidence to reveal the putative
mechanism. Enhanced neuroinflammation and oxidative damage have been observed
in animal models after chronic EtOH consumption. As a consequence, neuronal
loss is observed in alcoholics from postmortem examination of their brain tissues
(Zimatkin and Buben, 2007). In animal study, continually
consumption of EtOH was reported to increase the oxidative stress and decrease
the brain weight (Jayaraman et al., 2008). Similarly,
the present study reported the brain damage by the EtOH administration which
decreased the neuron densities in CA1 and CA3 areas. Of the sub-regions in hippocampus,
CA3 is critical for encoding the information and the CA1 is an important output
pathway of the signal from CA3. Lesion of CA3 and CA1 sub-regions resulted in
the dysfunction of learning and memory processes (Kesner,
2007). Dentate Gyrus (DG) was another sub-region related to the CA3 output
pathway. However, DG was not included in this study because the neuron density
of DG in our sections was too high to be clearly identified for counting.
After absorption, more than 90% of EtOH was metabolized while it passed through
the ventricular system of the brain (Zimatkin and Buben,
2007) and then in the brain, EtOH was metabolized by the three main ethanol
metabolizing enzymes, alcohol dehydrogenase, cytochrome P450 2E1 and catalase
(Zimatkin and Deitrich, 1997). Abundant ROSs are generated
from this metabolizing process and then followed by the activities changes of
the antioxidative enzymes. Several research reported that the EtOH administration
decrease the activities of antioxidative enzymes (Pushpakiran
et al., 2004; Reddy et al., 1999).
However, in the present study we reported that EtOH increase the CAT and GPx
activities while the SOD activity was in the normal level. These might be refer
to the different experimental design, such as the use of animal model, doses
and duration of EtOH administration or the different body regions observed.
In the animals treated with EtOH and C. comosa, the activities of CAT
and GPx came back to the normal levels suggesting that C. comosa prevented
neuron loss. This phenomenon indicated that CAT and GPx may be involved in the
neuron loss process. They were more likely to be indicators of neuron loss in
this study. The SOD activity responded in a different way. In the EtOH-induced
oxidative rat brain, SOD activity was at the control level but it increased
following the administration of C. comosa extract which may help to prevent
the neuron loss in the CA1 and CA3 areas. It was reasonable to assume that the
increasing SOD activity diminished the extra ROS induced by EtOH consumption
thus preventing neuron loss.
In the present study, the activities of antioxidative enzymes were changed
after the animal receiving the C. comosa extract which indicated a modification
to the enzyme activity by the plant extract. One explanation for this could
be the estrogenic-like effect from the extract. Estrogen has been reported to
modulate the antioxidative enzymes in the brain. For example, Schmidt
et al. (2005) reported that estrogen changed the CAT activity in
hippocampus HT22 and C6 glial cells. Sobocanec
et al. (2003) found that in the aging female mice, their brain had
lower oxidant and higher antioxidant capacity than that of male mice and these
differences were mostly related to CAT and GPx activity. Bilateral ovariectomy
increased the SOD activity in the rat brain (Pajovic et
al., 1993). However, whether the effects of C. comosa extract
on the activities of antioxidative enzymes were related to its unique estrogenic-like
effects requires further studies.
In conclusion, neuron loss was found in the rat hippocampus after the ethanol toxicity was induced; and the activities of the antioxidant enzymes were measured. Administration of C. comosa extract prevented the neuron loss and the potential mechanism was by modulating the antioxidant enzymes activities of hippocampus.
This study was supported by the Center for Research and Development of Herbal Health Products, Khon Kaen University and the National Research Council, Thailand. We are heartily thankful to Dr. Jeff John and Mr. James McCloskey for their suggestions in the language.
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