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American Journal of Biomedical Research


Vol. 4, No. 2, 2016, pp 42-45. doi: 10.12691/ajbr-4-2-3 | Research Article

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The Effect of Giving Trigona Honey and Honey Propolis
Trigona to the mRNA Foxp3 Expression in Mice Balb/c
Strain Induced by Salmonella Typhi
Andi Nilawati Usman1, Yuliana Syam2, Rosdiana Natzir3, Sutji Pratiwi Rahardjo4,
Mochammad Hatta5, , Ressy Dwiyanti6, Yuyun Widaningsih7, Ainurafiq8, Prihantono9
1
Department of Epidemiology, Health College of RSU Daya, Makassar, Indonesia
2
Department of Nursing, Hasanuddin University, Makassar, Indonesia
3
Department of Biochemistry, Hasanuddin University, Makassar, Indonesia
4
Department of ENT, Faculty of Medicine, Hasanuddin University, Indonesia
5
Department Molecular Biology and Immunology, Faculty of Medicine, Hasanuddin
University, Makassar, Indonesia
6
Department of Microbiology, Tadulako University, Palu, Indonesia
7
Department of Clinical Pathology, Hasanuddin University, Makassar, Indonesia
8
Department of Chemistry, Hasanuddin University, Makassar, Indonesia
9
Department of Oncology, Hasanuddin University, Makassar, Indonesia

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Abstract
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion
Acknowledgement
Competing Interest
References

Abstract

Immune balance during infection is important to support both the defense of body immune
system and prevent excessive immune response. Protein Foxp3, a transcription factor of
regulatory T cell has pivotal roles in balancing body immune system. Honey and Propolis
have proved their effects to both the proinflamatory and anti inflammatory responses but their
effects to the Foxp3 expression need to be investigated. This study was investigated the effect
of giving Trigona honey and honey propolis Trigona to the mRNA Foxp3 expression in Balb/c
mice induced Salmonella typhi. Results of the study indicated that honey propolis Trigona
had the highest effect to the mRNA Foxp3 expression followed by Trigona honey. Both
Trigona honey and honey propolis had immunomodulatory effects through the Foxp3 mRNA
expression.

Keywords: Trigona, Honey, Honey Propolis, Foxp3

Copyright 2016 Science and Education Publishing. All Rights Reserved.

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Usman, Andi Nilawati, et al. "The Effect of Giving Trigona Honey and Honey
Propolis Trigona to the mRNA Foxp3 Expression in Mice Balb/c Strain Induced by
Salmonella Typhi." American Journal of Biomedical Research 4.2 (2016): 42-45.

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1. Introduction

Several studies have been conducted to analyze the role of regulatory T cells (Treg) during
the infection process because they have important roles in immune homeostasis. Induction of
Treg cells are the response of hosts either to maintain or restore immune homeostasis during
the infection, because body requires the control of immune response that recognize and
control the microbial attack while preventing damage of tissues due to excessive immune
responses [1, 2].

Development and function of Treg cells require a stable and continuous expression of Foxp3
as a transcription factor (Abbas, 2011 [3, 4, 5]. Foxp3 insufficiency affects the development of
Treg and Induction of transcription factor Foxp3 could change nave CD4 + T cells into CD4 +
CD25- CD25 + Treg [6, 7].

Honey and propolis have been shown affecting the immune system, both pro-inflammatory
and anti-inflammatory. Intravenous injection of honey in rats induced LPS showed effects on
TNF-, IL-1, IL-6 and IL-10 [8]. Honey and Propolis, natural chemical products of bees
contain polyphenols and flavonoids, Evidence also indicates that flavonoid content of honey
and propolis could serve as both therapeutic and effective agents against bacterial pathogens
including salmonella [9-14][9] . However their effects on the balance of immune homeostasis
during infection have not been studied previously, particularly on the expression of the
transcription factor of Foxp3.

Flavonoids components can be found in propolis and honey [11]. Existing study regarding the
effects of flavonoids on Foxp3 have different results. Baicalin, flavon components found at
the root of Scutellaria Baicalensis Georgi has proved induced the expression of Foxp3 Treg
[15]
. Polyphenols, naringenin had also proved induced by Foxp3 Treg in the presence of TGF-
through the activation of transcription factor of Aryl hydrocarbon receptor [16]. Contrary
results shown to others studies, giving total flavonoids isolated from the roots of Radix
tetrastigmae T. hemsleyanum Diels et Gilg able to suppress the development of regulatory T
cells in mice [17]. Experiments in mice by giving flavonoids from Scutellaria Ocmulgee leaf
extract (Socl) showed significant inhibition against Treg frequency [18].

This study aimed to determine the effect of honey and honey propolis Trigona on the
expression of Foxp3 mRNA expression in Balb/c mice induced Salmonella typhi.

2. Material and Methods

2.1. Honey and Honey Propolis Trigona


Compilation and processing of both honey and propolis were conducted in collaboration with
Professor Mappatoba Trigona Sila, the Indonesian expert for both bee honey and propolis.
Both honey and propolis produced by bees Trigona types were obtained from Masamba
district, South Sulawesi Province.

Honey was deposited and filtered for 3 days. Propolis was dissolved in 1000 mL of distilled
water and heated, homogeneous solution was cooled to float on the surface of the solid wax,
this solid wax then removed and the propolis solution was filtered. Honey and propolis then
stored in the refrigerator. Honey propolis made by mixing 85% honey with 25% propolis (85
gram honey and 15% propolis Trigona).

2.2. Experimental Animals

Ethical use of Animal in Ethical Commission of Hasanuddin University and Immunology and
Biomolecular Laboratorium have approved the protocol of study. Male Balb/c mice with
weighing 25-27 g were housed under both 12-hour light and 12-hour dark periods, they were
fed a chow diet and water ad libitum. Mice Balb/c were divided into four groups (n=5/group)
after 7 days of adaptation to environmental condition.

Two of groups served as control group, both the negative control and positive controls, while
others as the intervention group. Negative control group was not given any intervention but
only standard diet, positive control was injected Salmonella typhi 103, one of treatment group
was intraperitoneally injected with Salmonella typhi 103 and also treated with Trigona honey
0.27 ml/20 g Bw and other groups also injected Salmonella typhi 103 intraperitoneally and
given honey propolis 0.27 ml/20 gr Bw. Salmonella typhi injected at the first day and honey
and honey propolis were given for 3 days after salmonella induction. All groups were taken
100 L via blood vessels in the tail before injected Salmonella typhi (baseline), 24 hours and
72 hours after injected Salmonella typhi.

2.3. RNA Isolation and Real-time RT-PCR

Total RNA from blood cells was prepared by using the Trizol reagent according to the
manufacturers protocol (Invitrogen). cDNA was synthesized with the first-strand cDNA
synthesis kit and oligo (dT) primers (Fermentas, Hanover, MD), Primer sequences was FW-
TTTACTCGCATGTTCGCCTACTT, RV-TCAAATTCATCTACGGTCCACAC. PCR
reaction used SYBR Green PCR Master Mix (Macrogen) and GADPH gene was chosen as
an internal standard, normalizing by GADPH preceding calculation of mRNA level [15, 19].

2.4. Statistical Analysis

Data presented with figures and tables and expressed as means and standard deviation (SD).
All p-values 0.05 were considered significant.

3. Results

Data indicated no significance difference from baseline until both 24 hours and 72 hours after
Salmonella typhi induction for the negative control group (p=0.300). Data in Table 1 showed
the significance difference of Foxp3 mRNA expression from baseline to 24 and 72 hours after
Salmonella typhi induction for the positive the control group (p=0.020), where the
experimental group was given honey (p=0.002) and another was given honey propolis
(p=006). (Table 1)

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Figure 1. Comparison of mean values for the Foxp3 mRNA Expression among experimental
groups given by both Trigona Honey and Honey Propolis Trigona with the control group.
Mice Balb/c strains were divided into 4 groups (n=5), one group as the reference and the
others as the positive control (Induced Salmonella), Both experimental groups were given
both honey and honey propolis Trigona. All groups were examined for their Foxp3 mRNA
Expression before and after the iduction of Salmonella Typhi for 24 and 72 hours. Data were
presented as meanstandard deviation. (*P 0.05)

Table 1. Analysis of the Foxp3 mRNA Expression from Baseline to 24 Hours and Post
Salmonella Typhi Induction for 24 hours ad 72 hours

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Analysis in Figure 1 showed no significance difference of the Foxp3 mRNA expression


between negative and positive control group at baseline. Positive control group had higher
significance in increasing the Foxp3 mRNA expression than negative control group at 24
hours of post Salmonella typhi induction and group given honey and honey propolis Trigona
had higher significance in increasing the Foxp3 mRNA expression than both negative and
positive control groups. The highest Foxp3 mRNA expression was the group given honey
propolis Trigona.

After 72 hours of post Salmonella typhi induction, the positive control group still had higher
significance in increasing the Foxp3 mRNA expression than the negative control group, and
the group given both honey and honey propolis Trigona had higher significance in increasing
the Foxp3 mRNA expression than both negative and positive control groups. The highest
Foxp3 mRNA expression was the group given honey propolis Trigona. The difference of
Foxp3 mRNA Expression between 24 hours with 72 hours of post Salmonella typhi induction
was higher for the Foxp3 mRNA Expression at 72 hours than 24 hours (Table 1 and Figure 1).

4. Discussion

Data from this study showed that the intervention group was given honey and honey propolis
had the greater increase of the expression of Foxp3 mRNA than the negative control group.
Notwithstanding, both the positive control groups also increased the expression of Foxp3
mRNA but lower than the intervention group.

Bacteria gram-negative including Salmonella, its endotoxin during infection process could be
a determinant of shock and multiple organ dysfunction [20]. As a response to Salmonella typhi
bacteria and other gram-negative bacteria, the activity of pro-inflammatory cytokines
correlated with the systemic Inflammatory Response Syndrome (SIRS), such as the increase
of interleukin-1, tumor necrosis Alpha (TNF-) and interleukin-6 (IL-6) [21]. Honey
treatment in rats induced by LPS could be protected from endotoxemia through the induction
of heme oxygenase-1 and inhibited of cytokines and nitric oxide [22]. Foxp3 expression is
needed during bacterial infection to rise up immune homeostasis.

Potential mechanism for both honey and honey propolis to affect Foxp3 is investigated
through their ability to increase TGF- [23]. Quercetin compound found in both honey and
propolis had contributed to TGF- [24]. The systemic rising of TGF- would increase the
frequency of Treg, its mechanism involves induction of Smad3 (pSmad3). Induced Smad3,
initially binds to the enhancer site Foxp3 in intron 2 and interacts with nuclear factor-kB,
NFATc2 and CREB that would binding with Foxp3 promoter [25].
Our data did not explain the mechanism, so further studies should be conducted to explore
more understandings on honey, propolis and Foxp3 Treg

5. Conclusion

The evidence from this study revealed that honey and honey propolis Trigona could increased
the Foxp3 mRNA expression and the effect of honey propoliss was higher than honey.

The data in this study simply indicate the potentiality of honey and honey propolis Trigona to
increase Foxp3 mRNA expression, but did not study the mechanism. Further study should
include the mechanism, either to TGF- or other potential mechanisms.

Acknowledgement

The authors would like to acknowledge the support of the Indonesian honey expert, Professor
Mappatoba Sila for his valuable discussions.

Competing Interest

The authors declare that they have no competing interests.

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Abstract

Recent data highlight the undeniable role of programmed cell death type I of lymphocytes in
the pathogenesis of certain allergic diseases and autoimmune diseases such as Bronchial
Asthma and hemorrhagic rectocolitis. But little data exist on the enzymatic activity of
secretory granules associated with lymphocytes of patients suffering from these inflammatory
diseases. The aim of the study was to characterize the activity of the DNase in the secretory
granules of T lymphocytes isolated from peripheral blood of patients with Bronchial Asthma
(n = 20) and Hemorrhagic Rectocolitis (n = 20). Thus, the secretory granules were isolated
from lymphocytes by the ultracentrifugation method on percoll density gradient. The
detection of the activity of the protein extracts was performed by zymography and
electrophoresis method. The results reveal the presence of a protein extract with a molecular
weight of 66 kDa both at the level of the granules of the lymphocytes of patients suffering
from hemorrhagic rectocolitis considered as a classical autoimmune disease and in the
granules of lymphocytes of patients with Bronchial Asthma. The study of physicochemical
properties showed an increase in the enzymatic activity of DNase of secretory granules when
1 mM Ca2+ was added to the incubation medium at a pH = 7.5. On the other hand, the
addition of 1 mM Zn2 + causes the inhibition of enzyme activity. These results suggest that the
enzymatic activity of DNase detected in the granule of lymphocytes of patients with
Bronchial Asthma and hemorrhagic rectocolitis occurs not only in the fragmentation of
double stranded DNA but could also play a role in the apoptotic process of these same T-
lymphocytes. The study of the properties of this DNase in the inflammatory diseases could
enable to use this protein as a marker for determining the severity of disease.

Keywords: DNase, secretory granules, Bronchial Asthma, Hemorrhagic Rectocolitis

Copyright 2016 Science and Education Publishing. All Rights Reserved.

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, Bera C., Cyrille A. VODOUNON, Irina D. RESHETNIKOVA, Boris B.


LEGBA, and Zinaida I. ABRAMOVA. "Granule Associated DNase in T-Lymphocytes
from Patients with Inflammatory Disease." American Journal of Biomedical Research
4, no. 4 (2016): 87-93.

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1. Introduction

The apoptosis or programmed cell death type I is the keystone to many biological processes
in multicellular organisms [1, 2]. Among other things, it plays an undeniable role in the final
stages of inflammation, during this period, the removal of active immune cells having
completed their functions takes place. The dysfunction of this process in immunocompetent
cells is one of the causes of development of allergic diseases and autoimmune diseases. For
instance, one can cite Bronchial Asthma [3, 4] and Hemorrhagic Rectocolitis, respectively.
According to data from the literature, Bronchial Asthma is associated with the resistance of
lymphocytes to apoptosis, which leads to a persistence of allergic inflammation [5, 6, 7]. This
resistance leads to a disruption in the removal of lymphocytes from the lung tissue
accompanied by a bronchial hyper-reactivity. On the other hand, the Hemorrhagic
Rectocolitis is considered as an autoimmune disease caused by a disorder of the immune
system that attacks its own cells. The inflammatory immune response in Hemorrhagic
Rectocolitis patients is perpetuated by the maintenance of T-lymphocytes activation and the
production of a cascade of inflammatory mediations. The apoptosis disorder of these
lymphocytes leads to a disruption of the negative selection system. Thus, the auto-reactive
lymphocytes that escaped this process can survive and attack its own cells. The tissues are
infiltrated by lymphocytes and more specifically cytotoxic lymphocytes causing disruption of
some biochemical processes. One of the key stages of apoptosis is the fragmentation of DNA.
The DNA fragmentation is a complex biochemical process, which involves a group of
nucleases with different activities and specificities for the substrate. It was detected in the
secretory granules of patient lymphocytes, granular extracts of which enzymatic activity
increases with the degree of severity of the disease and the balance of the regulatory activity
of the immune response. The characteristics of the enzymatic activity of DNase are different
from the activity of other known endonuclease involved in DNA fragmentation (DNase I,
DNase II). The DNase may be involved in both the apoptosis of its own lymphocytes and
could also serve as cytotoxic factors [8]. The identification of new nucleases and the analysis
of their functions in the apoptotic process and their development should lead to a
comprehensive study of the links between apoptotic degradation of DNA sequences and
autoimmune and allergic diseases [9]. The aim of this work was to characterize the activity of
the DNase in the secretory granules of T lymphocytes of patients with Bronchial Asthma and
Hemorrhagic Rectocolitis.

2. Materials and Methods

2.1. Study Purpose

The aim of the study was the activity of DNase associated with secretory granules of T
lymphocytes isolated from peripheral blood of patients with Bronchial Asthma (BA, n = 20)
and Hemorrhagic Rectocolitis (HR, n = 20). The choice of these diseases (BA and HR) as a
model for the study is related to the fact that during the development of these diseases, an
important role is played by the stability of the lymphocytes in relation to apoptosis, and
moreover hitherto there is still no consensus on the importance of the autoimmune process in
the pathogenesis of these diseases. The HR is referred to serious autoimmune disease with
cell mediation, such as those described for the activity of DNase of granules of T-
lymphocytes.
2.2. Isolation of Peripheral Blood Lymphocytes

The lymphocytes were isolated from the peripheral blood of donors relatively healthy (HD)
and patients with BA and HR on a ficol-verografine density gradient from the standard
method proposed by Patel et al [10] and improved by Boyum [11, 12].

2.3. Isolation of Sub-populations of T- lymphocytes

The subpopulations of T- lymphocytes were obtained by negative immunomagnetic


separation method with the use of a set of magnetic particles Dynabeads Untouched
Human T Cells Kit (Invitrogen, USA). This method consists in isolating 95% of T
lymphocytes and their viability was determined by the method of exclusion with trypan blue
[13]
.

2.4. Obtainment Secretory Granules of Cytotoxic T-lymphocytes

The secretory granules were isolated from lymphocytes by the ultracentrifugation method
proposed by Borregard [14] on Percoll density gradient (Invitrogen, USA) and then optimized
by Podack et al [15] with some modifications [16]. For the preservation of the structure of the
lymphocytes and for the obtaining of secretory granules, the lymphocytes were rinsed in 0.32
sucrose solution prepared in buffer solution 0.01 Tris-HCl at 7.2 and containing
0.003 Cl2. After centrifugation at 5000 rpm/min for 20 min, we added to the pellet
obtained, 1 ml buffer solution of 0.01 Tris-HCl at 7.3. The cells were then suspended in
the buffer solution and were all transferred in a Potter glass tube. The cells were ground in the
homogenizer with the potter piston for 2 min in an ice bath, and then 3 ml buffer solution of
0.01 Tris-HCl was added. They were incubated for 30 min in an ice bath. The substance
obtained after grinding was centrifuged for 15 min at 2300 rpm / min and at 4. The
supernatant was collected and the pellet was washed with a buffer solution of 0.01 buffer
Tris and centrifuged again for 15 min at 2300 rpm / min (-24). The supernatant was
collected again. The procedure was repeated again, after which supernatants were put
together for possible extractions of granules. The granules were obtained by Percoll density
gradient (=1.080 g/cm). And five (5) milliliters of supernatant obtained were placed on the
Percoll gradient and centrifuged at 19500 rpm/ min for 35 min with the Beckman centrifuge
(L90-K, Coulter, USA). After centrifugation, 2 ml fractions 5-7 were collected and which
were visible to the naked eye. For the removal of Percoll gradient these fractions were
centrifuged at 45 000 rpm/ min for 2.5 hours at 4. A whitish supernatant was formed on the
Percoll of 80-90% homogeneous supernatant containing secretory granules. This whitish
supernatant was solubilized in a buffer solution for eventual use.

2.5. Extraction and Purification of Protein Extracts from Granules of T lymphocyte

In order to destroy the granule membrane the granule solution was subjected to several cycles
of freezing and thawing in liquid nitrogen after storage at - 80C overnight. For complete
lysis of the membrane of the granules we added a buffer solution (1 M NaKHPO4, pH 7.4, 1
mM EDTA, 3 mM NaN3, and 2 mM PMSF) at equal volume. The mixture was incubated in a
refrigerator for 30 minutes. The addition of high concentrations of phosphate led to the
precipitation with particles Percoll, which were easily removed by centrifugation for 30
minutes at a speed of 20000 rpm/m at 4C (Rotor SW 55 Ti). To precipitate lymphocyte
granule proteins, the method of separation was used with ammonium sulfate. For purification
of these extracts, the dialysis method was used. Dry polyethylene glycol of molecular weight
40000 g was used to enhance the concentration of protein extracts.

2.6. Disc Electrophoresis of Protein Extracts from Granules of T lymphocytes

The separation of protein extracts contained in the granules of cytotoxic lymphocytes


according to their molecular weight was performed using the method of 12% disk
electrophoresis under the denaturing conditions [17].

2.7. Detection of the Activity of Protein Extracts by Zymography Method

To visualize the enzymatic activity of protein extracts of granules with the simultaneous
determination of the molecular weight of these extracts, zymography, a sensitive method, was
used [18]. It summarize the procedure of electrophoresis on 12% polyacrylamide gel under
denaturing conditions after polymerizing native DNA in the acrylamide gel and initiated an
enzyme reaction by incubation in an activation buffer solution. The gel was washed and
incubated in buffer solution for 30 minutes at 37C containing ethidium bromide at a final
concentration of 1 g/ml. The fluorescence of ethidium bromide was recorded by the gel
documentation system BioRad ChemiDoc XRS + (~ 300 nm excitation, ~600 nm emission).
In this context the bottom of the gel with polymerized DNA showed a black spot at the places
where occurred enzymatic reactions.

2.8. Study of Cation Dependence and the pH Influence on the Activity of Protein
Extracts from Granules of T lymphocytes

We studied the catalytic activity of protein extracts of granules dependent on cations by


power of native DNA degradation in the presence of different concentrations of Ca2 + and Zn2
+
ions. The protein extracts with a concentration of 350 g/ml) were incubated in 50 l of
buffer solution (10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 10 mM MgCl2), containing 10g of
genomic DNA of the hedgehog sea and Ca2 + and Zn2 + ions. During the study of the
influence of pH, the protein extracts of the granules were incubated for 24 hours in a buffer
solution (50 mM NaCl; 10 mM MgCl2) at pH = 5.0, pH 7 and pH 8.0. After incubation for
24h, we caused the migration of 20 l of these aliquots on an agarose gel containing 1 g/ml
1% ethidium Bromide for 3 hours with a voltage of 5V/cm.

2.9. Statistic Analysis

Statistic analysis was performed using the software package STATISTICA 6.0. For
quantitative and qualitative analysis of the activity of granuleextracts, the gels were scanned
and the results were processed with the "Scion image" program. We also used the excel
package to analyze the data.

3. Results

3.1. Extraction of Secretors Granules of T lymphocytes

Because of the special role of T lymphocytes in the development of the immune response to
some diseases such as BA and HR, we chose the negative immunomagnetic separation
method to obtain a homogeneous subpopulation of T lymphocytes from a fraction of
mononuclear cells from peripheral blood. Secretors granules of T lymphocytes were isolated
by ultracentrifugation on a density gradient Percoll. During the design of this protocol, we
were forced to adapt the procedure to the realities of our laboratory. It was necessary for us to
optimize the gradient/supernatant ratio for a successful extraction of secretory granules. We
considered several gradient/supernatant ratios (2: 1; 3: 1; 4: 1). The 4: 1 turns out to be the
most appropriate. With such a ratio, it was observed in the Percoll gradient visible and clear
layer constituted of secretory granule after centrifugation. This is presented in Figure 1. The
ratios 2: 1 and 3: 1 did not show the area containing secretory granule.

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Figure 1. The conditions for an optimum choice of the ratio gradient / supernatant for the
isolation of secretory granules of T lymphocytes. The clear area encircled in red shows the
position of granules in the Percoll gradient for gradient / supernatant ratio 4: 1
3.2. Electrophoresis of Protein Extracts of the Secretory Granules of Lymphocytes

In order to determine the molecular weight of the protein extract contained in the secretory
granules of patient lymphocytes, vertical electrophoresis was performed on a polyacrylamide
gel. The electropherogram has revealed the presence of a protein extract with a molecular
weight of 66 kDa both at the level of the granules of the lymphocytes of patients with HR and
in granules extracted from lymphocytes of patients with BA. In short we have identified a
protein extract contained in the granules with a molecular weight of 66 kDa. The
electropherogram shows in the third lane, apart from one protein band with a molecular
weight of 66 kDa, three additional protein bands which could be other types of protein
extracts. The identification of these proteins was not included in the objective of our research
work (Figure 2).

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Figure 2. Electropherogramme of protein extracts of secretory granules of lymphocytes


showing the molecular weight of these extracts. (Polyacrylamide gel under denaturing
conditions at 12%, - revelation of protein extracts with silver nitrate - lane 1: Serum Albumin
of bovine (68 kda); lane 2 DNase 1 (31kda), lane 3: protein extract from granules of
lymphocytes from patients with CU (30g); lane 4: protein extracts from granules of
lymphocytes from patient with BA (30g)
3.3. Detection and Localization of the Enzymatic Activity of Secretory Granules of T-
lymphocytes
We used zymography method to verify the presence of a DNase in the lymphocyte granules
with a molecular weight of 66 kDa and their enzymatic activities. This highly sensitive
method was used both to separate proteins according to their molecular weight and to
visualize the enzyme activity of this enzyme protein directly into the gel that was previously
polymerized with DNA (due to activation of nucleases) in the gel after electrophoresis. A
commercial preparation of DNase of calf pancreas (Sigma-Aldrich, USA) served as control
for the enzyme activity. The purity of this preparation was 85%, which was insufficient for
zymography, as the zymogram shows in addition to the nuclease activity of DNase 1 (with a
molecular weight of 31 kDa) other bands which might give a false interpretation of results.
To this end, it was important for us to purify the preparation to remove impurities. For
purification, we used the gel filtration chromatography method. The results of the purification
of DNase are presented in the chromatogram (Figure 3). The control of the purification of
protein fractions obtained from impurities after chromatography was verified by the
electrophoresis method in polyacrylamide gel; the results are presented in Figure 4. The
electropherogram shows many contaminants with different molecular weights at the levels of
lanes B2-B4. On the other hand, at the level of lanes C4-C7, traces of impurities were
observed. As a matter of fact, we determined DNase activity found in lanes B8-C3 by
zymography method. The fractions of lanes C1-C2 DNase were used as controls to verify
enzyme activity of the protein fractions of isolated granules from T lymphocytes of patients
with BA and HR (Figure 5). Zymography was performed on 12% gel of SDS-polyacrylamide
polymerized with native DNA (15g/ml) for a period of 18 hours. The zymogram has shown
an enzymatic activity of protein fractions of lymphocyte granules corresponding to proteins
with a molecular weight of 66 kDa (Figure 6).

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Figure 3. chromatogramme of protein after gel filtration, of a commercial preparation of


DNase I

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Figure 4. Electropherogramme of protein fractions after gel filtration of commercial DNase.


Lane K; DNase before purification; B2-C2: DNAse after the chromatography method of gel
filtration; lane B8-C3: DNase free of almost all impurities
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Figure 5. Electropherogramme (A) and zymogramme (B) of purified fractions of commercial


DNase I. lane 1: commercial DNase before purification. Lanes B2-C2: purified DNase
fraction; lanes C1-C2: DNase fraction retained for use as a control for the following
experiments
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Figure 6. Zymogramme showing the enzymatic activity of protein extracts of granules from
T lymphocytes. Lane 1 - markers (BSA, 68 kD; DNase I-31 kDa), revealed with silver
nitrate; lane 2 - purified commercial DNase I; lane 3 - protein extracts of lymphocyte
granules of patients with BA (30g); lane 4 - protein extract of lymphocyte granules of
patients suffering from CS (30 g). The red band shows the demonstration of the enzymatic
activity of DNase corresponding to a protein with a molecular weight of 66 kDa
3.4. Study of Physico-chemical Properties of Protein Extracts of the T-lymphocyte
Secretory Granules

To determine the physical and chemical properties of the protein extracts secreted by granules
of T lymphocytes, we examined the dependence of cations such as Ca2+ and Zn2+ and the
influence of pH of the reaction medium. Therefore, we determined the influence of cations on
the enzyme activity of protein extracts by studying the degree of degradation of the
commercial DNA of sea urchin (10 g) with protein extracts secreted by the granules after an
incubation in a buffer solution (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2)
containing Ca2 + and Zn2 + ions. The incubation was performed at 37C for 24 hours and the
results are presented in Figure 7. We observed that the enzymatic activity of protein extracts
of T lymphocytes of patients suffering from BA and HR increases when it is added to the
incubation medium 1 mM Ca2 + and the activity decreases when 10 mM Ca 2+ was added to
the medium. After addition of 1 nM Zn 2+ the enzymatic activity of the protein extracts of T
lymphocytes decreased in both cases. The influence of the pH of the medium on the
enzymatic activity of the protein extracts of lymphocyte granules was determined after
incubation of native DNA of the sea urchin (10 g with protein extracts of granules in a
buffer solution (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM CaCl2) with pH of the
medium slightly acidic (pH = 5.0), neutral (pH = 7.0) and slightly basic (pH = 8.0 ). the
incubation was carried out at 37 degrees Celsius for 24 hours. There was an effect of pH on
the activity of protein extracts of granules isolated from lymphocytes of patients with BA and
HR. The enzymatic activity became maximal after a slightly acidic medium around 5.0.

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Figure 7. Electropherogram showing the influence of the cations on the protein extracts
lymphocyte granules of patients with BA and patients suffering from CR. The DNA was
incubated in a buffer solution with different concentration of Ca2 + and Zn2 +. The lanes
marked in red shows the activating effect of 1 mM Ca2 + and inhibitor effect of 1 mM Zn2 +

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Figure 8. Electropherogramme showing influence of pH of the medium after incubation of
the DNA of the sea urchin with protein extracts of lymphocyte granules of patients with BA
and CR in a buffer solution (10 mM Tris-HCl; 10 mM Mg2: 50 mM + 1 mM CaCl2)

4. Discussion

The apoptosis and autoimmunity are potentially important factors playing a curable role in
the pathogenesis of some serious diseases [19, 20]. Apart from these factors, one can also think
of the lymphocytes that constitute the keystone and which are involved in almost all immune
reactions and autoimmune as well as allergic reactions [21]. The inflammatory reactions in
asthmatics are characterized by delayed-type hypersensitivity reactions [22]. Therefore, there is
a selective accumulation of T lymphocytes in the respiratory tract. The accumulation of these
lymphocytes in the respiratory tract is due to the malfunction of the apoptotic process of these
cells. Some authors argue that apoptosis of eosinophils and T lymphocytes with cellular
mediation is a key pathogenic event leading to the loss of epithelial cells in asthma patients.
The key stage of apoptosis of these cells is the stage of fragmentation of nuclear DNA. And
DNA degradation is due to the activity of various enzymes. A failure of the activity of these
enzymes may cause malfunction of the apoptotic process and in particular its inhibition. It
has been described the enzymatic activity of DNase associated with cytoplasmic granules of
activated T lymphocytes after incubation of mononuclear cells from peripheral blood of
relatively healthy donors in the presence of immunomodulatory peptides. The induction of
their activity in human cytotoxic lymphocytes, particularly T-lymphocytes during the
development of autoimmune diseases has led us to study the possibility of eventual presence
of granular DNase found in the lymphocytes of peripheral blood from patients with asthma
and HR. Some common elements in the development of asthma and specific autoimmune
diseases of organs (for instance, infiltration of T- lymphocytes in the outbreak of the disease)
led us to study the activity of the nuclease of secretory granules of peripheral blood
lymphocytes of patients with BA and HR. The results of our research allowed us to detect the
presence of DNase with enzymatic activity in the secretory granules of T-lymphocytes of
patients with BA and HR. The method of zymography a very sensitive method was used to
visualize the activity of this DNase in a polymerized polyacrylamide gel with double-
stranded DNA. The protein identified in the granules had a molecular weight of 66 kDa if we
confine ourselves to the nuclease activity of the enzymatic protein. From the study of
physicochemical properties, an increase in the enzymatic activity of DNase of secretory
granules when added 1 mM Ca2+ to the incubation medium and pH = 7.5 was observed. On
the other hand, the addition of 1 mM Zn2+ causes the inhibition of enzyme activity. The
activity of the protein fractions contained in the lymphocyte granules of BA patients and HR
increases after addition of Ca2+ ions to the incubation medium with an acidic pH. Thus, it was
observed in the secretory granules of T lymphocytes the presence of DNase corresponding to
Ca2 + ion-dependent DNase with a molecular weight of 66 kDa, of which the activity is more
important at a slightly acidic pH. This DNase associated with granules corresponds to a
protein band detected by Lopez Moratalla [8]. But the role played by this DNase is not yet
elucidated clearly, and one might assume that this could be another cytolytic factor. This
hypothesis is consistent with the idea that the expression of enzymes in the secretory granules
could be associated with potentially cytolytic T lymphocytes, and the mechanism of cytolytic
T lymphocytes is linked to an active transfer of fragmented DNA by cytotoxic factors in the
target cells. In this respect, one might assume that DNase detected in the granules is not only
involved in the fragmentation of double-stranded DNA of the target cells but also in the
apoptotic process of the same T lymphocytes. This hypothesis coincides with the fact that, in
the thymocytes the increase in the concentration of intracellular Ca2+ and the connection with
the Zn2+ ions [23] cause an increase in the DNA fragmentation due to the acidification of the
medium. On the other hand, it has been shown that cytotoxic T lymphocytes could be
involved in damage to target tissues. For example, the activity of DNase associated with
secretory granules of T lymphocytes [8] which increase in patients with primary biliary
cirrhosis of the liver but whose activity is lower in patients with Basedow disease which
correlates with the highest immunoregulatory disorders. Thus, the detected DNase could play
an exceptionable role not only in the autoimmune diseases but also in the diseases such as
bronchial asthma. Many researchers have studied the role of autoimmunity in the
pathogenesis of bronchial asthma by the functioning of T lymphocytes [19, 24]. It was thus
discovered molecules that can initiate, carry out the apoptosis of lymphoid cells in the course
of certain diseases, but it should be noted that stimulants leading to their action and
mechanisms used in these cases are limited. However, since the induction or inhibition of
apoptosis is targeted as an ideal way of treating certain pathologic diseases, the development
and research in this area should be considered a high priority. In the same vein, the study of
apoptosis of lymphoid cells in the course of bronchial asthma is interesting and relevant.
Henceforth, the discovery of a DNase with a molecular weight of 66 kDa in the granules of T
lymphocytes from patients with BA and HR coincides with these characteristics with that
previously studied by PIO et al in patients with severe autoimmune disease. The presence of
such DNase molecules in asthmatics indicates the role of the autoimmune process in the
pathogenesis of BA. Further study of the functions of this molecule would help to establish a
link between the apoptotic degradation of DNA and disease, and the study of its activity
according to the form and degree of disease severity would permit the use this DNAse as
marker of immune system deregulation. This could help us justify the paradigm of bronchial
asthma is an autoimmune disease.

5. Conclusion

In summary our data show that DNase was present in granules of lymphocytes freshly
obtained from patients with BA and HR and may suggest that this DNAse could play some
role in the apoptosis of T-lymphocytes. All this might be used for the diagnosis of
inflammatory disease with a better accuracy on the prognosis of the severity degree.

Acknowledgements

We thank Prof Myctaffin (Kazan Medical Institute) and Prof BABA-MOUSSA (UAC-Benin)
for supporting our discussion and providing technical expertise.

Funding

"This research was supported by the subsidy of the Russian Government to support the
Program of competitive growth of Kazan Federal University"

Competing Interests

The authors declare that they have no competing interests.

All authors have read and approved the final manuscript.

Ethics Approval
Experimental research has been performed with the approval of an appropriate ethics
committee. The work was done jointly with medical doctor who received approval from the
Local Ethics Committee of the Medical University of Kazan for the conduct of biomedical
research. The work was performed in accordance with the rules of the Ethics Committee in
the laboratory of Clinical Immunology and Allergy of RKB. All patients were informed and
they gave their consent for experiment.

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Abstract

Purpose: The aim of this study was to evaluate phytochemical, analgesic and anticancerous
activities of Cymbopogon citratus. Methods: Phytochemical screening, analgesic test and
anticancerous activities were evaluated by the different chemical tests, writhing test and tail
immersion test, brine shrimp lethality bioassay. Results: In the case of acetic acid induced
writhing test, C. citratus showed highest percent of inhibition at 400 mg/kg, p.o. which is
49.01% whereas standard diclofenac sodium showed 73.76% at 10 mg/kg, i.p. In the case of
tail immersion method, C. citratus exhibited its highest activity at 400 mg/kg, p.o. in 90 min
which is 5.560.88 whereas diclofenac sodium showed 6.710.15 at 10 mg/kg, i.p. in 90 min.
In the case of anticancerous activity, methanolic leaf extract of C. citratus demonstrated
significant activity which is 113.74 g/ml and standard vincristine sulphate showed 12.59
g/ml. Conclusions: The result of phytochemical screening revealed that, methanolic leaf
extract of Cymbopogon citratus contain alkaloids, steroids, flavonoids, tannins, saponins and
carbohydrates. Methanolic leaf extracts of this plant possess moderate analgesic activity as
well as exhibited significant anticancerous activity.

Keywords: cymbopogon citratus, phytochemical screening, analgesic, diclofenac sodium,


acetic acid, writhing test, tail immersion test, brine shrimp

Copyright 2015 Science and Education Publishing. All Rights Reserved.

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Md. Irfan Amin Chowdury, Mitali Debnath, Md. Foyez Ahmad, Mohammad Nazmul
Alam, Arif Mohammad Saleh, Sudipta Chowdhury, Rajesh Barua, Muhammad Moin
Uddin Mazumdar, Abu Hena Mustafa Kamal. Potential Phytochemical, Analgesic and
Anticancerous Activities of Cymbopogon citratus Leaf. American Journal of
Biomedical Research. Vol. 3, No. 4, 2015, pp 66-70.
http://pubs.sciepub.com/ajbr/3/4/2

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1. Introduction

Natural products as an alternative and complementary medicine to many ailments have been
a major interest among researchers. In addition to documenting the traditional knowledge
related to medicinal plants, scientific authentication of these medicinal plants has been an
important path of recent research [1]. Epidemiological evidence suggests that consumption of
a diet rich in vegetables and fruits has optimistic implications for human health. The World
Health Organization reported that 80 % of the world population rely chiefly on indigenous
medicine and that the majority of traditional therapies involve the use of plant extracts or of
their active constituents [2] and over 25% of modern medicines that are commonly used
worldwide contains compounds extracted from medicinal plants [3]. The active principles
differ from plants to plants due to their biodiversity and produce a definite physiological
action on the human body that develops interest on their medicinal properties [4]. In recent
years, there has become a revival in the use of traditional medicinal plants and therefore,
pharmaceutical companies are products extracted from plants [5]. In Bangladesh thousands of
plant species are considered to have medicinal value [6] and ninety percent of the medicinal
plants are wild sourced [7, 8]. Cymbopogon citratus is native from the Southwest Asia and,
now, it grows spontaneously around the world, mainly in the tropical and savannah regions [9].
Leaf wax contains triterpenes, cymbopogonol and cymbopogone. Rhizome contains alkaloids
(hordenine, gramine), saponin glucoside, -sitosterol, hexacosanol and triacontanol, alkaloid,
saponin, glucoside and the phenolic compounds, p-coumaric acid, protocatechuic acid, ferulic
acid and phloroglucinol carboxylic acid. An alkaloid with an indole nucleus has been isolated
from the rhizome of this plant [10].

2. Materials and Methods

2.1. Plant Material


Cymbopogon citratus was collected from local area of Chittagong district (Bhatiary),
Bangladesh and authenticated by the Botanist Dr. Shaikh Bokhtear Uddin, Associate
Professor, Department of Botany, University of Chittagong, Bangladesh.

2.2. Preparation of Extraction

The leaf was indirectly sun dried by shade and ground. The ground (500 g) were soaked in
sufficient amount of methanol (1:4) for one week at room temperature with occasional
shaking and stirring then filtered through a cotton plug followed by Whitman filter paper No.
1. The solvent was evaporated under vacuum at room temperature to yield semisolid.
Methanolic leaf extract was then preserved in a refrigerator at 4 C till further use.

2.3. Experimental Animals

Adult Swiss albino mice (both sex) weighing approximately 30-35 g were used for this
experiment. The mice were purchased from the animal research branch of the International
Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). The animals were
maintained standard laboratory conditions (25 C and light/dark cycles i.e. 12/12 h) and
provided with standard laboratory food and distilled water ad lib.

2.4. Chemicals and Reagents

Diclofenac sodium, acetic acid were obtained from MERCK, India. 0.9% NaCl saline
solution was obtained from Popular Pharmaceuticals Ltd., Bangladesh. All other reagents
were of analytical grade. Two more chemicals were purchased from Sigma Aldrich (Munich,
Germany), which were vincristine sulfate and 99.5% absolute methanol.

Mayers Reagent:

1.36 gm mercuric iodide in 60 ml of water was mixed with a solution contains 5 gm of


potassium iodide in 20 ml of water.

Dragendorffs Reagent

1.7 gm basic bismuth nitrate and 20 gm tartaric acid ware dissolved in 80 ml water. This
solution was mixed with a solution contains 16 gm potassium iodide and 40 ml water.

Fehlings Solution A:

34.64 gm copper sulphate was dissolved in a mixture of 0.50 ml of sulfuric acid and
sufficient water to produce 500 ml.

Fehlings Solution B:

176 gm of sodium potassium tartarate and 77 gm of sodium hydroxide were dissolved in


sufficient water to produce 500 ml. Equal volume of above solution were mixed at the time of
use.

Benedicts Reagent:
1.73 gm cupric sulphate, 1.73 gm sodium citrate and 10 gm anhydrous sodium carbonate
were dissolved in water and the volume was made up to 100 ml with water.

Molisch Reagent:

2.5 gm of pure -naphthol was dissolved in 25 ml of ethanol.

2.5. Phytochemical Screening

The following tests were performed for identifying different chemical groups by Trease et al.,
1983 and Ghani et al., 1998.

Tests for reducing sugar:

Benedicts test

0.5 ml of aqueous extract of the plant material was taken in a test tube. 5 ml of Benedicts
solution was added to the test tube, boiled for 5 minutes and allowed to cool spontaneously. A
red color precipitate of cuprous oxide was formed in the presence of a reducing sugar.

Fehlings Test

2 ml of an aqueous extract of the plant material was added 1ml of a mixture of equal volumes
of Fehlings solutions A and B. Boiled for few minutes. A red or brick red color precipitate
was formed in the presence of a reducing sugar.

Test for Tannins:

Ferric Chloride Test

5 ml solution of the extract was taken in a test tube. Then 1 ml of 5%Ferric chloride solution
was added. Greenish black precipitate was formed and indicated the presence of tannins.

Potassium Dichromate Test

5 ml solution of the extract was taken in a test tube. Then 1 ml of 10% Potassium dichromate
solution was added. A yellow precipitate was formed in the presence of tannins.

Test for Flavonoids:

Added a few drops of concentrated hydrochloric acid to a small amount of an alcoholic


extract of the plant material. Immediate development of a red color indicates the presence of
Flavonoids.

Test for Saponins:

1 ml solution of the extract was diluted with distilled water to 20 ml and shaken in a
graduated cylinder for 15 minutes. One centimeter layer of foam indicates the presence of
saponnins.
Test for Carbohydrates:

5 ml solution of the extract was taken and then Molish reagent and Sulphuric acid were
added. Red-violet ring produced at the junction of two liquids indicate the presence of gums
and carbohydrate.

Test for Steroids:

Sulphuric Acid Test

1 ml solution of chloroform extract was taken and then added1ml Sulphuric acid. Chloroform
layer acquired reddish brown color and acid layer showed green furoescence indicates the
presence of steroid.

Test for Alkaloids:

Mayers test

2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube.
Then 1 ml of Mayers reagent was added. Yellowish buff color precipitate was formed and
that was indicated as the presence of alkaloids.

Dragendorffs test

2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube.
Then 1 ml of Dragendroffs reagent was added. Orange brown precipitate was formed and
that was indicated as the presence of alkaloids.

Wagners test

2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube.
Then 1 ml of iodine solution (Wagners reagent) was added. Reddish brown precipitate was
formed and that was indicated as the presence of alkaloids.

Hagers test

2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube.
Then 1 ml of picric acid solution (Hagers reagent) was added. Yellowish precipitate was
formed and that was indicated as the presence of alkaloids.

2.6. Tests for Analgesic Activity

The study of analgesic activity of the extract was performed in animal models for both central
and peripheral mechanism of pain. For the screening of analgesic activity against peripheral
mechanism of pain acetic acid induced writhing was considered. On the other hand, to
evaluate the analgesic activity against centrally mediated pain tail immersion test was done.
Beside this, to evaluate the possible mechanism related to the analgesic activity is evaluated
by treating with Diclofenac Na. Two methods were employed to study the analgesic effects
by using Swiss Albino Mice.
Acetic Acid Induced Writhing Method

Mice of either sex (n = 5) weighing 25-30 gm were used and divided into 4 groups. Group 1
received normal saline (10 ml/kg, p.o.) as control, Group 2 received standard drug diclofenac
sodium (10 mg/kg, i.p.) while the remaining groups were received 200 and 400 mg/kg, p.o. of
Cymbopogon citratus leaf extract. After 30 min of saline, diclofenac sodium, and plant
extracts received, the animals were treated i.p. with 1 % (v/v) acetic acid. The number of
abdominal constrictions (writhes) was counted after 5 min of acetic acid injection for the
period of 10 min and compared to the response in the control group [11]. Antinociceptive
activity was calculated as the percentage inhibition of writhing.

Tail Immersion Test

Mice were divided into four groups of five animals each. Group 1 received normal saline
(0.9% NaCl, 10 ml/kg, p.o.) as control and group 2 received the standard drug Diclofenac
Sodium (10 mg/kg, i.p.) Group 3 and 4 received 200mg/kg and 400mg/kg of methanol extract
of Cymbopogon citratus orally respectively. The animal withdrawing his tail from hot water
within 5 s were selected for the study. The lower 3 cm portion of the tail of mice was dipped
in a water bath maintaining at temperature of (55.00.5). The time in second (s) for tail
withdrawal from the water was taken as the reaction time and recoded by a stopwatch at
before 0 and 30, 60 and 90 after the administration of test samples. A maximum immersion
time of 15 s was maintained to prevent thermal injury to the animals [12].

Oral Toxicity Test

An acute oral toxicity test was performed according to the Organization for Environmental
Control Development guidelines (OECD: Guidelines 420; Fixed Dose Method). Swiss
Albino mice (n=5) overnight fasted for 18h were used for the study. Different doses of
methanolic plant extract were administered orally into the mice. The maximum given dose
was 600 mg/kg body weight. Then the animals were observed for the first three hours of
administration and mortality recorded within 48 hours.

2.7. Anticancerous Activity

Anticancerous activity of plant extract is evaluated by brine shrimp lethality bioassay, which
is widely used for screening bioactive compounds [13, 14]. In this study, a simple zoological
organism (Artemia salina) was used as a convenient monitor for the experiment. The eggs of
the brine shrimp were collected from an aquarium shop (Dhaka, Bangladesh) and hatched in
artificial seawater (3.8% NaCl solution) for 48 hrs to develop into larval shrimp called
nauplii. The cytotoxicity assay was performed on the brine shrimp nauplii using the Meyer
method. The test samples (extract) were prepared by dissolving them in DMSO (not more
than 50 L in 5 mL solution) plus seawater (3.8% NaCl in water) to attain concentrations of
10, 50, 100, 200, 300 and 500 g/ml. A vial containing 50 L DMSO diluted to 5 mL was
used as a control. Standard vincristine sulfate was used as a positive control. Mature shrimps
were placed into each of the experimental vials. After 24 h, the vials were inspected using a
magnifying glass, and the number of surviving nauplii in each vial was counted. From these
data, the percentage of lethality of the brine shrimp nauplii was calculated for each
concentration using the following formula:
Where Nt = Number of dead nauplii after a 24-h incubation;

N0 = Number of total nauplii transferred i.e., 10.

The LC50 (median lethal concentration) was determined from the log concentration versus
percent mortality curve [15].

3. Results

3.1. Phytochemical Screening

The result of phytochemical screening revealed that, methanolic leaf extract of Cymbopogon
citratus contain alkaloids, steroids, flavonoids, tannins, saponins and carbohydrates

Table 1. Results of Different Chemical Group Test of the Crude Extracts of the Leaf of
Cymbopogon citratus

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3.2. Analgesic Test

Writhing test

Analgesic activity was evaluated by acetic acid induced writhing test and tail immersion test.
In the case of acetic acid induced writhing test, C. citratus showed highest percent of
inhibition at 400 mg/kg, p.o. which is 49.01% whereas standard diclofenac sodium showed
73.76% at 10 mg/kg, i.p.

Table 2. Analgesic Activity of methanolic leaf extract of Cymbopogon citratus on mice


(Mouse Writhing Test)

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Tail Immersion test

In the case of tail immersion method, C. citratus exhibited its highest activity at 400 mg/kg,
p.o. in 90 min which is 5.560.88 whereas diclofenac sodium showed 6.710.15 at 10 mg/kg,
i.p. in 90 min.

Table 3. Analgesic Activity of methanolic leaf extract of Cymbopogon citratus on mice


(Tail Immersion Test)

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3.3. Anti-cancerous Activity

In the case of anticancerous activity, methanolic leaf extract of C. citratus demonstrated


significant activity which is 113.74 g/ml and standard vincristine sulphate showed 12.59
g/ml.

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Figure 1. Effect of methanolic leaf extract of Cymbopogon citratus and vincristine sulphate
on brine shrimp nauplli

Table 4. Percent of mortality of both the extract at six concentrations


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4. Discussion

People on all continents have used hundreds to thousands of indigenous plants for treatment
of ailments since prehistoric times. According to World Health Organization, about 80% of
the worlds population presently uses phytotherapy for some aspect of primary health care
system. Approximately 25 percent of modern drugs used in the United States have been
derived from plant origins [16]. So, research on phytotherapy has got great momentum in
recent years to find out noble pharmaceuticals. The acetic acid induced writhing response is a
sensitive procedure to evaluate peripherally acting analgesic. The response is thought to be
mediated by the prostaglandin pathways, peritoneal mast cells and acid sensing ion channels
[17, 18, 19]
. Therefore, the significant pain reduction of the plant extract may be due to the
presence of analgesic principles acting with the prostaglandin pathways or interfering with
other mediators responsible for peripheral pain. The tail immersion method was used to
evaluate the central mechanism of analgesic activity. Here the painful reactions in animals
were produced by thermal stimulus that is by dipping the tip of the tail in hot water. Analgesic
effect against thermal noxious stimuli may be elicited through opoid receptors or through
modulation of several neurotransmitters involved in relevant phenomena. But the extend of
activity shown by the crude extracts are less than that of the standard drug nalbuphine but
many fold more than that of the control group, which justifies its activity. Narcotic analgesics
inhibit both peripheral and central mechanism of pain, while non-steroidal anti-inflammatory
drugs inhibit only peripheral pain [20, 21]. The extract inhibited both mechanisms of pain,
suggesting that the plant extract may act as a narcotic analgesic. The cytotoxic and antitumor
activity of plant derived product is either through induction of apoptosis or inhibition of
neovascularization [22]. Ideally, any agent useful in the treatment of cancer should not be toxic
to normal cell. However, in reality, anticancer agents are often toxic to normal cells,
particularly towards rapidly growing cells [23]. It is necessary to test this extract in low
concentration to evaluate its potency and also against various cancer cell lines as well as
normal cell line so justify the potential to further investigate this plant for anticancer activity.

5. Conclusion

The result of phytochemical screening revealed that, methanolic leaf extract of Cymbopogon
citratus contain alkaloids, steroids, flavonoids, tannins, saponins and carbohydrates.
Methanolic leaf extracts of this plant possess moderate analgesic activity. Writhing test and
Tail immersion test proved that this plant has moderate analgesic activity and it is also proved
that higher dose is more effective than the lower dose. This plant is also exhibited significant
anticancerous activity.

Ethical Approval

The experimental protocol was approved by the P&D committee, Department of Pharmacy,
International Islamic University Chittagong, Bangladesh according to governmental
guidelines.

Acknowledgement

Authors wish to thank Botanist Dr. Shaikh Bokhtear Uddin, Associate Professor, Department
of Botany, University of Chittagong, Bangladesh, who helped to identify the plant. The
authors are also grateful to the Department of Pharmacy, International Islamic University
Chittagong, Chittagong, Bangladesh, for providing research facilities.

Conflict of Interest Statement

Authors have none to declare.

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