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CONTENTS 5.1.5.2.5.3.
AbstractIntroductionMaterialsandMethods
5.3.1.5.3.2.
PreparationofbenzeneextractofG.acerosaAssessmentofcytotoxiceffectofG.acerosabenzeneextractusinghumanPeripheralBloodMononuclearCells(PBMC)
5.3.2.1.5.3.2.2.5.3.2.3.
5.3.2.4.5.3.2.5.
PBMCisolationExperimentalsetupTrypanblueexclusionassayMTTassayHemolysisassay
5.3.3. Bacterialreversemutationtest 5.3.4. Bacterialreversemutationtestwithrat‐liverS9fraction
andmix 5.3.5. Cometassay 5.3.6. ToxicityevaluationofG.acerosainSwissalbinomice
5.3.6.1.5.3.6.2.5.3.6.3.5.3.6.4.5.3.6.5.
AnimalsAcutetoxicitytestSub‐acutetoxicity testAnalysisofhematologicalandbiochemicalparametersHistopathologicalanalysis
5.3.7. Statisticalanalysis
5.4. ResultsandDiscussion5.4.1. AssessmentofcytotoxiceffectsofG.acerosainhuman
mononuclearcells 5.4.2. Evaluation of membrane disintegrating effects of
G.acerosainhumanerythrocytes. 5.4.3. AssessmentofmutagenicpropertiesofG.acerosausing
Salmonellatyphimuriummutantstrains 5.4.4. GenotoxicevaluationofG.acerosausinghumanPBMC
5.4.5. AcutetoxicitystudiesofG.acerosa5.4.5.1.5.4.5.2.
HematologicalandbiochemicalparametersHistopathologicalanalysis
5.4.6. Sub‐acutetoxicitystudies
5.4.6.1.5.4.6.2.
HeamatologicalandbiochemicalparametersHistopathologicalanalysis
5.5. Conclusion
5.6. Summaryoftheresults
Chapter III 103
5.1.ABSTRACT
Gelidiella acerosa benzene extract (possessing excellent antioxidant and anti-
cholinesterase activity) was subjected to toxicity evaluation. Cytotoxic evaluation of G.
acerosa benzene extract was done by Trypan blue exclusion, MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays using human PBMC
(Peripheral Blood Mononuclear Cells) and RBC (Red Blood Cells) lysis assay using
human erythrocytes. Ames Salmonella mutagenicity test was performed (with the
histidine dependent mutant strains TA 98, TA100 and TA 1538) to evaluate the
mutagenic potential of the benzene extract. The genotoxic effect of G. acerosa benzene
extract was assessed by comet assay. The in vivo toxicity was evaluated by acute and
sub-acute toxicity studies in Swiss albino mice. The results of in vitro cytotoxic assays
suggest that the extract did not possess any cytotoxic activity both in PBMC and RBC.
Ames Salmonella mutagenicity test demonstrated that the seaweed was not found to
induce any mutations both in the presence and absence of S9 microsomal fraction.
Genotoxic analysis of G. acerosa benzene extract revealed that the extract exhibit less
or no damage to cells, thus proving the non-genotoxic effect of the extract. The results
of in vivo toxicity tests suggest that the benzene extract did not cause any adverse
pathological changes in mice treated with benzene extract during both the long-term
and short-term toxicity studies. Since these non-clinical studies clearly demonstrate the
non-toxic nature of the G. acerosa benzene extract, it could be exploited for further
characterization, which would result in development of novel and safe therapeutic
entities.
Chapter III 104
5.2.INTRODUCTION
For the past several decades, natural compounds has been used as a potential
source of medicines, as they possess excellent therapeutic potentials and serves as
excellent drug leads (Huang et al., 2008; Castro et al., 2009). The use of botanical
preparations as medicines has been greatly increased and around 80% of the world
population relies on these natural compounds based treatments (Carabin et al., 2000).
In recent decades, several compounds with diverse pharmacological activities ranging
from anti-viral to anti-cancer have been isolated from various marine sources (Arif et
al., 2004). Among various natural compounds, the seaweeds or macroalga are enriched
with secondary metabolites such as terpenoids, flavonoids, sterols, polysaccharides and
fatty acids, which acts as potential drug leads that facilitates the unraveling of novel
biosynthetic pathways (Choi et al., 2009; Lane et al., 2010). However, certain
metabolites are toxic in nature, which necessitates the toxicological evaluation of
natural products. Generally, the toxicological profile of any natural compound can be
tested by evaluating the cytotoxic, genotoxic and mutagenic potentials (Verschaeve
and Staden, 2008; Varalakshmi et al., 2011). Mutagenicity is the process of
induction of permanent transmissible changes in the genetic material of the cells.
Whereas, genotoxicity is a broader term which is the ability of a compound to interact
with the genetic material and also with other cellular apparatus that maintains the
fidelity of the genome (Maurici et al., 2003). Only non-toxic and non-mutagenic plants
could be considered for relative safe use and also for detailed study of other
pharmacological potentials (Varalakshmi et al., 2011). Since the results of preliminary
work (which demonstrated the antioxidant, metal chelating, anti-cholinesterase and
anti-amyloidogenic activity of benzene extract) were promising, the safety assessment
of the benzene extract is crucial for an in depth understanding of other pharmacological
effects of the extracts and also the mechanism through which it acts. Hence the present
study aims at the evaluation of cytotoxic, mutagenic and genotoxic potential of benzene
extract of Gelidiella acerosa using in vitro and in vivo systems.
Chapter III 105
5.3.MATERIALSANDMETHODS
5.3.1.PreparationofbenzeneextractofG.acerosa
The benzene extract of G. acerosa was prepared by the method as mentioned in the section 3.3.2.
5.3.2.AssessmentofcytotoxiceffectofG.acerosabenzeneextractusinghumanPeripheralBloodMononuclearCells(PBMC)
5.3.2.1.PBMCisolation
Three mL of blood was collected from healthy volunteers in a tube containing
EDTA, to which equal amount of LSM (Lymphocyte Separation Medium) was added.
Human PBMC were separated by centrifugation at 2200 rpm for 15 min. The white
layer formed intermittently was taken carefully and washed by using RPMI1640
(Roswell park memorial institute) medium at 2200 rpm for 15 min. The cell viability
was assessed by trypan blue exclusion assay. PBMC were adjusted to 1×106 cells/mL in
complete medium and used for further experiments. The protocol was approved by the
Institutional Ethics Committee of Alagappa University, Karaikudi, India (No.
IEC/ALU/1-2008)
5.3.2.2.Experimentalsetup
PBMC were incubated with different concentrations (250, 500, 1000 µg/mL) of
G. acerosa benzene extract at 37C with 5% CO2 in water jacketed incubator for 24 h.
The cell viability was checked at various time intervals (12 h, 18 h and 24 h) and the
loss of viability was compared with 1 mM H2O2 by trypan blue exclusion assay and
MTT assay.
5.3.2.3.Trypanblueexclusionassay
Principle
The trypan blue exclusion assay is used to determine the number of viable cells.
It works on the principle that live cells possess intact cell membranes that exclude
trypan blue whereas, the dead cells will have disintegrated membrane and thus uptakes
the dye inside the cells.
Chapter III 106
Percentageofviability=Numberofviablecells
Totalnumberofcells×100
Procedure
Trypan blue exclusion assay was performed according to Strober (1997). Equal
volume of trypan blue solution was added to the cell suspension and incubated for 10
min at room temperature. The number of live and dead cells was counted in light
microscope using Neubauer counting chamber to calculate the percentage of cell
viability. Cells which are stained blue were regarded as dead cells (disrupted
membrane), whereas those cells without staining were considered as live cells (intact
membrane).
Calculation
The percentage of viability was calculated using the formula given below:
5.3.2.4.MTTassay
Principle
MTT is a yellow dye which is reduced to purple colored formazan crystals by
the activity of mitochondrial succinate dehydrogenase enzyme in viable cells. The
increase in absorbance due to the formation of formazan crystals was measured at 540
nm, which corresponds to the viability of cells.
Procedure
MTT assay was performed according to the method of Mosmann (1983) with
slight modifications. Cells after incubation were resuspended in 200 µL of 5 mg/mL
MTT solution. The suspended cells were then incubated at 37°C for 3 h in dark. After
incubation, the cells were centrifuged at 3000 rpm for 7 min and the cells were washed
with PBS to remove the excess MTT solution. 300 µL of DMSO was added to the pellet
and incubated at 37C for 30 min. The cells were then centrifuged at 3000 rpm for 7
Chapter III 107
min and the supernatant was collected and made to 1 mL with DMSO. The absorbance
was read at 540 nm.
Calculation
The viability of the cells was calculated using the formula mentioned below:
5.3.2.5Hemolysisassay
Principle
Erythrocytes are simple blood cell type without any sub-cellular organelles and
they can be exploited for testing in vitro toxicity of selected compounds by measuring
the release of their hemoglobin content, which is generally represented as an index of
cell membrane damage (Akanji et al., 2013).
Procedure
The hemolysis assay was performed according to Fischer et al. (2003) with
minor modifications. Blood was taken from healthy volunteers and RBC was freshly
isolated from the blood. The cells were washed thrice with freshly prepared 150 mM
NaCl. After centrifugation at 2500 rpm for 15 min, the supernatant was removed and
the cells were re-suspended in 100 mM sodium phosphate buffer. 200 µL of RBC
solution was mixed with different concentrations of benzene extract (250, 500, 1000
µg/mL) and made to 1 mL with phosphate buffer. Triton X-100 and sodium phosphate
buffer (with milli Q water) was used as positive and negative control respectively. Then
the tubes were incubated at 37C in a water bath for 30 min. After incubation, the cells
were centrifuged at 2500 rpm for 15 min. The amount of hemoglobin released was
taken as a measure of cell lysis. The supernatant was collected and the absorbance was
measured at 540 nm in a UV-Vis Spectrophotometer.
5.3.3.Bacterialreversemutationtest
Principle
Bacterial reverse mutation test (Ames test) was performed to evaluate the
mutagenic properties of the seaweeds. This test involves the use of histidine dependent
bacteria that can grow on glucose minimal agar plate containing trace amount of
histidine. The cells that revert to histidine independent (His+) will be able to form
% Cell survival = Mean absorbance in test wells Mean absorbance in control wells
×100
Chapter III 108
colonies. The small amount of histidine allows all the plated bacteria to undergo a few
cell divisions; in many cases, this growth is essential for mutagenesis to occur. The
number of spontaneously induced revertant colonies is relatively constant for each
strain. However, when a mutagen is added to the plate, the number of revertant colonies
per plate will be increased in a dose related manner (Mortelmans and Zeiger, 2000).
Reagents
VB salt solution MgSO4 - 1 g Citric acid monohydrate - 10 g K2HPO4 - 50 g Sodium ammonium phosphate
- 17.5 g
All the components were dissolved in d.H2O and made to 100 mL
Glucose minimal agar 50 X VB salt solution - 2 mL 40% Glucose - 5 mL 1.5% Agar - 1.5 g All the components were dissolved in d.H2O and made to 100 mL 0.6% Agar - 15 mg in 2.5 mL of d.H2O
0.6% NaCl - 15 mg in 2.5 mL of d.H2O
0.05 mM Histidine - 125 µL of 1 mM histidine in 2.5 mL reaction
mixture 0.05 mM Biotin - 250 µL of 0.5 mM Biotin in 2.5 mL reaction
mixture Procedure
A single colony of Salmonella typhimurium tester strains (TA 98, TA 100 and
TA 1538) was picked and inoculated into 3 mL of LB broth containing 0.5 mM Biotin
and 0.05 mM Histidine and it was incubated at 37°C for 12 h. The culture was then
centrifuged at 6600 rpm for 15 min at 4°C and the pellet obtained was resuspended in 1
mL of phosphate buffer. Incubation mixture was prepared as follows: 50 µL of 0.015 M
Phosphate buffer mixed with different concentrations of benzene extract (250, 500,
1000, 2000, 4000 µg/plate) and 50 µL of bacterial suspension. The mixture was then
incubated for 90 min at 37°C. 2.5 mL of molten surface agar containing 0.6% agar,
0.6% NaCl, 0.05 mM L-Histidine and 0.05 mM Biotin was mixed with the incubation
mixture and then poured over the glucose minimal agar plates. Sodium azide, a strong
mutagen (1 µg/plate) was used as positive control for the tester strain TA 100 and 4-
Chapter III 109
Nitroquinoline (0.1 µg/plate) was used as positive control for the strains TA 98 and TA
1538. The plates were incubated for 66 h at 37°C.
Calculation
The mutagenicity ratio (MR) was calculated as the number of revertants in the
treated sample to the number of spontaneous revertants. If the value of MR is higher
than 2.0 then the sample was considered as mutagenic.
5.3.4.Bacterialreversemutationtestwithrat‐liverS9FractionandMix
Principle
Certain mutagens remain inactive unless they get metabolized in to active forms.
Unfortunately, the bacterial strains that are used for Ames test does not possess these
systems and hence, the oxidation system has to be introduced externally along with the
substance, which is suspected. In order to achieve this, S9 microsomal fraction (9000 g
supernatant fraction of rat liver homogenate), which is the rodent metabolic activation
system, has been employed in the presence of cofactors such as NADP and NADPH.
Procedure
Preparation of Rat-liver S9 Fraction and Mix
Healthy male Sprague-Dawley rats were treated with 30 mg/kg sodium
phenobarbitone (in 0.9% w/v saline) on day one and 60 mg/kg on day 2, 3 and 4. After
five days, animals were sacrificed by cervical dislocation and the liver was collected,
homogenized in 0.15 M KCl. The homogenate was centrifuged at 9,000 g for 10 min
and the supernatant was aliquoted (2 mL portions) and stored at -80°C until use. The S9
mix prepared includes 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-Phosphate, 4 mM
NADP, 0.1 M Sodium phosphate buffer (pH 7.4) and 10% S9 fraction (v/v)
(Mortelmans and Zeiger, 2000).
The procedure was similar to that of the test described above, but in addition to
the components of incubation mixture, S9 mix was added. A single colony of
Salmonella typhimurium tester strains was picked and inoculated into 3 mL of LB broth
containing 0.5 mM Biotin and 0.05 mM Histidine and it was incubated at 37°C for 12 h.
The culture was then centrifuged at 6600 rpm for 15 min at 4°C and the pellet obtained
was resuspended in 1 mL of phosphate buffer. Incubation mixture was prepared as
follows: 500 µL of S9 mix (8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4
Chapter III 110
mM NADP, 100 mM Sodium phosphate, 10% S9 fraction), 50 µL of 0.015 M
Phosphate buffer mixed with different concentrations of seaweed extract (250, 500,
1000, 2000, 4000 µg/plate) and 50 µL of bacterial suspension. The mixture was then
incubated for 30 min at 37°C. The molten surface agar (2.5 mL) containing 0.6% agar,
0.6% NaCl, 0.05 mM L-Histidine and 0.05 mM Biotin was mixed with the incubation
mixture and then poured over the glucose minimal agar plates. 2- aminoanthracene (2-
AA) (1 µg/plate) was used as positive control.
Calculation
The results were expressed as mutagenicity ratio and the calculation was done as
mentioned earlier.
5.3.5.CometAssay
Principle
Comet assay is a rapid and cell based method widely employed to detect various
genotoxic agents. In this assay, the embedded cells in agarose gels were subjected to
alkaline lysis and the extent of DNA damage caused by the suspected genotoxic agents
can be determined by measuring the tail moment, olive moment and percentage of DNA
in tail.
Reagents
Phosphate buffered saline (PBS) (pH- 7.4) - 200 mL 1% Normal melting agarose (NMA) - 0.5 g of NMA in 50 mL of PBS 0.75% Low melting agarose (LMA) - 0.22 g of LMA in 30 mL of PBS
Lysis solution
1% Sodium lauryl sarcosinate - 1 g 2.5 M NaCl - 14.60 g 100 mM Na2EDTA - 3.72 g 10 mM Tris-Cl - 0.121 g 1% Triton X-100 - 1 mL 10% DMSO - 10 mL Dissolve all the components in d.H2O and made to 100 mL.
Electrophoretic buffer 300 mM NaOH - 7.2 g 1 mM Na2EDTA - 0.22 g
Dissolve all the components in d.H2O and made to 600 mL. Neutralization buffer (100 mL)
0.4 M Tris-Cl (pH - 7.5) - 4.84 g
Chapter III 111
Procedure
PBMC suspension of >95% viability were adjusted to a concentration of
1×106cells/mL with complete medium and incubated for 24 h with various
concentrations of benzene extract (250, 500, 1000 µg/mL). 250 µM of H2O2 was used
as positive control. After exposure, the cells were centrifuged at 2200 rpm for 5 min
and washed with PBS twice. The pellet was suspended in 0.75% low melting agarose
(LMA) and poured over the slides which were pre-coated with normal melting agarose
(NMA). The gel was covered with a glass cover slip and left to set at 4C for 5-10 min.
Gel embedded cells were lysed in lysis buffer for 12 h at 4C. Electrophoresis was
carried out in freshly prepared pre-cooled buffer for 20 min at 25 V. The slides were
washed thoroughly with neutralization buffer and were stained with 10 µL of Ethidium
Bromide (10 µg/mL) and viewed under Confocal Laser Scanning Microscopy. The
scanned images were analyzed in Comet Score 1.5 software (TriTek's revolutionary
technology). Around 100 cells were scored for each individual sample. The parameters
employed for evaluating DNA damage were tail moment, olive moment and percentage
DNA in tail.
5.3.6.ToxicityevaluationofG.acerosainSwissalbinomice
5.3.6.1Animals
Healthy Swiss albino mice weighing 25-30 g were used to undertake the acute
and sub-acute toxicity tests. All the animals were separated according to the gender and
housed in plastic cage in a ventilated room with 12 h cycle of day and night light. They
were fed with a standard pellet diet (Hindustan Lever Ltd, Mumbai). The room
temperature was maintained around 25C. All the procedures followed were approved
by the Institutional Animal Ethics Committee of C.L. Baid Metha College of Pharmacy,
Chennai, India (Approval No: IAEC/XXXIX/01/CLBMCP/2013 dated 29.06.2013)
5.3.6.2.Acutetoxicitytest
Acute toxicity study was carried out as per OECD (Organization for Economic
Co-operation and Development) guidelines - 423. Animals were divided into two
groups (Control and extract treated) of six animals each. All the animals were deprived
of food for 16 h prior to the administration of test drug. The control group received
0.1% CMC (carboxymethyl cellulose) orally, the other groups were treated with G.
acerosa benzene extract aseptically suspended in 0.1% CMC solution and administered
in a single oral dose of 2000 mg/kg of body weight by gavage. The general behavior
Chapter III 112
and presence of toxicity was observed and recorded periodically at 1, 2, 4, 6 up to 24 h
after drug administration. The presence and absence of convulsions and tremors were
monitored and recorded. Presence of any abnormal clinical symptoms was also noted.
5.3.6.3.Sub‐acutetoxicitytest
Sub-acute toxicity test was also performed to evaluate the toxicity of G. acerosa
benzene extract during the repeated dosage for 28 days. The animals were divided in to
three groups of six animals each. G. acerosa benzene extract was suspended in 0.1%
CMC and the mixture was administered orally by gavage at two dose levels 200 and
400 mg/kg of body weight. The extract was administered daily for a period of 28 days.
Animals in the control group were administered with highest volume of 0.1% CMC
used for extract suspension. All the animals were weighed and observed daily for water
and food intake. Presence of physiological and behavioral changes was also monitored.
5.3.6.4.Analysisofhematologicalandbiochemicalparameters
At the end of the study, whole blood sample was collected by cardiac puncture
and used for hematological studies. Hematological and biochemical analysis was
performed for both acute and sub-acute toxicity studies. The parameters analyzed were
RBC, WBC, platelet count, packed cell volume, blood sugar, Blood Urea Nitrogen
(BUN), serum creatinine, alkaline phosphatase, serum total protein, serum albumin,
Serum Glutamic Oxaloacetic Transaminase (SGOT), hemoglobin (Hb), Mean
Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), neutrophils,
lymphocytes, eosinophils, monocytes, basophils, Serum Glutamic Pyruvic
Transaminase (SGPT), calcium, sodium, potassium, bilirubin, serum total cholesterol,
serum triglycerides, serum HDL cholesterol, serum LDL cholesterol, serum VLDL
cholesterol.
5.3.6.5.Histopathologicalanalysis
Histopathological analysis was done using the vital organs like liver, brain,
kidney and heart. All the organs were collected, weighed and visually inspected for
pathological changes in tissues, followed by preservation in 10% formalin for
histopathological analysis. The collected tissues were embedded in paraffin, sliced as
thin layers, fixed in slides and subjected to hematoxylin-eosin staining. The
pathological observations were performed on gross and microscopic basis.
Chapter III 113
0
20
40
60
80
100
120
Per
cen
tage
of
viab
ilit
y
12 h 18 h 24 h
*
* *
5.3.7.StatisticalAnalysis
Experimental results concerning this study were represented as Mean ± S.D of
three parallel measurements. All the statistical analysis was done using the software
SPSS Statistics 17.0. The results of the cytotoxicity and mutagenicity tests were
analyzed by one-way ANOVA. Mann-Whitney U test was performed to analyze the
data in comet assay. P-value <0.05 were regarded as significant.
5.4.RESULTSANDDISCUSSION
5.4.1. Assessment of cytotoxic effects of G. acerosa in humanmononuclearcells
Cytotoxic evaluation of G. acerosa benzene extract was done by trypan blue
exclusion assay, in which the effect of seaweed extracts on cell viability was evaluated
at 12, 18 and 24 h. The results show that benzene extract did not affect the cell viability
at the concentrations of 250 and 500 µg/mL. However, a slight decrease in the
percentage of viability (90.23 ± 1.67, 82.79 ± 1.23 and 78.88 ± 5.97% at 12 h, 18 h and
24 h respectively) was observed at the concentration of 1000 µg/mL (which is not
significant). Treatment with 1 mM H2O2 showed a significant decrease (P<0.05) in cell
viability (40.61 ± 8.48, 27.47 ± 2.82 and 22.78 ± 4.56% at 12 h, 18 h and 24 h
respectively) (Fig. 5.1).
Figure5.1:Cytotoxic evaluationofbenzeneextractofG.acerosaby trypanblue exclusion assay.The valuesareexpressedasMean±SD.
Chapter III 114
0
20
40
60
80
100
120
Per
cen
tage
of
viab
ilit
y
12 h 18 h 24 h
*
* *
MTT assay was performed to further validate the non-cytotoxic effects of the
seaweed extracts. Treatment with 1000 µg /mL of the benzene extract for 12 h did not
affect the viability of the cells (Fig. 5.2). However, treatment with benzene extract for
18 h and 24 h resulted in a slight decrease (not significant) in the viability. 1 mM H2O2
was used as positive control and it reduced the cell viability significantly (P<0.05) to
46.83 ± 9.41, 18.43 ± 0.79 and 16.75 ± 2.45% after incubating at 12 h, 18 h and 24 h
respectively (Fig. 5.2). The results suggest that G. acerosa benzene extract was less
cytotoxic in PBMC cells.
Figure 5.2: Evaluation of cytotoxicity of benzeneextract of G. acerosa by MTT assay. The values areexpressedasMean±S.D.
5.4.2. Evaluation of membrane disintegrating effects ofG.acerosainhumanerythrocytes.
Human erythrocytes were used for the evaluation of membrane disintegrating
activity of seaweed extract. RBC treated with various concentrations of G. acerosa
benzene extract exhibited the least absorbance of 0.071 ± 0.009 and 0.111 ± 0.013 for
250 and 500 µg/mL respectively. However, at 1000 µg/mL, the extract induced a slight
damage and exhibited the absorbance of 0.18 ± 0.01. A complete loss of membrane
integrity was observed in the cells treated with 0.2% Triton X-100, as the absorbance at
540 nm was significantly high (P<0.05) and increased to 1.40 ± 0.05 (Fig. 5.3).
Therefore, the results suggest that G. acerosa benzene extract did not interrupt or
damage the erythrocyte membrane.
Chapter III 115
0
0.2
0.4
0.6
0.8
1
1.2
Ab
sorb
ance
at
540
nm
*
Figure 5.3: Determination of hemolytic activity ofbenzene extract of G. acerosa. The values areexpressedasMean±S.D.
5.4.3.AssessmentofmutagenicpropertiesofG.acerosausingSalmonellatyphimuriummutantstrains
Ames Salmonella mutagenicity test has been recognized as the widely employed
test to evaluate whether the given substance can produce genetic damage resulting in
gene mutations. The present study reveals that benzene extract did not produce any
mutagenic effect in all the three strains employed. At the concentration of 4 mg/mL,
mutagenicity ratio (MR) exhibited by the tester strains of TA 98, TA 100 and TA 1538
were 0.5 ± 0.03, 0.82 ± 0.18 and 1.20 ± 0.17 respectively (Table 5.1). In the case of
strains treated with positive controls (Sodium azide for TA 100 and 4-Nitroquinoline
for TA 98 and TA 1538), a significant increase (P<0.05) in MR was observed. The MR
values obtained were 2.81 ± 0.26, 3.62 ± 0.53 and 11.31 ± 1.23 for the tester strains TA
98, TA 100 and TA 1538 respectively. A similar mutagenic evaluation was done on
aqueous extract of the green alga Spirogyra neglecta by Thumvijit et al. (2013) using
S. typhimurium strains TA 98 and TA 100. The results show that the extract did not
exhibit mutagenic effect both in the presence and absence of S9 fraction. Kim et al.
(2010) showed that the sporophyll of Undaria pinnatifida displayed no mutagenic
effect in S. typhimurium strains irrespective of the presence or absence of S9 mix in the
medium. .
Chapter III 116
Table5.1:Mutagenic evaluation ofG. acerosabenzene extractby Ames test using Salmonella typhimurium tester strains TA98,TA100andTA1538
a Results were expressed as Mean ± SD (n = 3). MR - Mutagenicity ratio; SA - Sodium Azide (Standard mutagen for TA 100); 4-NQ - 4-Nitroquinoline (Standard mutagen for TA 98 & TA 1538); **P<0.05
In addition to the above mentioned test, the non-mutagenic potential of seaweed
extracts were further validated by the bacterial reverse mutation test in the presence of
metabolic activation systems i.e. in the presence of S9 microsomal fraction. The results
show that at the concentration of 4 mg/mL of G. acerosa benzene extract, the tester
strains TA 98, TA 100 and TA 1538 exhibited the MR of 1.06 ± 0.13, 0.93 ± 0.03 and
0.44 ± 0.03 respectively. Whereas, treatment with 2-AA significantly (P<0.05)
increased the frequency of his+ revertants and exhibited higher MR of 5.8 ± 0.93, 2.11
± 0.07 and 2.07 ± 0.08 for TA 98, TA 100 and TA 1538 respectively (Table 5.2). Hence
the results suggest that the benzene extract was non-mutagenic and do not induce gene
mutations.
Treatment Concentration
(µg/plate) MRa
(TA 98) MRa
(TA 100) MRa
(TA 1538)
G. acerosa benzene extract
250 1.24 ± 0.24 0.97 ± 0.12 0.81 ± 0.20
500 1.21 ± 0.12 0.92 ± 0.07 0.81 ± 0.17
1000 0.91 ± 0.27 0.94 ± 0.10 0.91 ± 0.45
2000 0.5 ± 0.24 1.01 ± 0.03 1.13 ± 0.27
4000 0.5 ± 0.03 0.82 ± 0.18 1.20 ± 0.17
Negative control
(distilled water)
40µL 0.64 ± 0.19 0.87 ± 0.02 0.34 ± 0.07
Positive control
SA- 1 µg/plate 4-NQ- 0.11
µg/plate 2.81 ± 0.26** 3.62 ± 0.53**
11.31 ± 1.23**
Chapter III 117
Table 5.2: Mutagenic evaluation of G. acerosa benzeneextract by Ames test using Salmonella typhimurium testerstrainswithratliverS9microsomalfraction.
a Results were expressed as Mean ± SD (n = 3) MR - Mutagenicity ratio; 2-AA - 2-Aminoanthracene **P<0.05
5.4.4.GenotoxicevaluationofG.acerosausinghumanPBMC
The assessment of genetic instability by DNA damage and repair is an important
aspect of safety assessment of specific compounds. Comet assay is a simple and
sensitive method of evaluating DNA damage in individual cell. This assay can be
performed in small number of cells and the results can be obtained in a short period of
time (Jaiswal et al., 1995). The degree of DNA damage can be represented using the
parameters like tail moment, olive moment and percentage of DNA in tail. Fig. 5.4
illustrates the genotoxic effect of G. acerosa benzene extract. The results show that the
tail moment, olive moment and percentage of DNA in tail was significantly high
(P<0.05) in the case of PBMC treated with 1 mM H2O2 when compared to the cells
treated with seaweed extract. The tail moment, olive moment and percentage of DNA in
tail exhibited by the cells treated with H2O2 was 69.89, 42.15 and 51.38 respectively.
Interestingly, the cells treated with 1000 µg/mL of benzene extract exhibited lesser tail
moment, olive moment and percentage of DNA in tail of about 1.22, 1.34 and 7.66
Treatment Concentration
(µg/plate) MRa
(TA 98) MRa
(TA 100) MRa
(TA 1538)
G. acerosa benzene extract
250 0.80 ± 0.13 0.78 ± 0.06 0.024 ± 0.005
500 0.48 ± 0.13 0.64 ± 0.35 0.030 ± 0.008
1000 0.64 ± 0.25 0.82 ± 0.04 0.041 ± 0.027
2000 0.91 ± 0.10 0.55 ± 0.05 0.30 ± 0.008
4000 1.06 ± 0.13 0.93 ± 0.03 0.44 ± 0.03
Negative control
(distilled water)
40µL 0.28 ± 0.10 0.80 ± 0.05 0.036 ± 0.005
Positive control
2-AA - 1 µg/plate 5.8 ± 0.93** 2.11 ± 0.07** 2.07 ± 0.08**
Chapter III 118
respectively. Therefore the results suggest that G. acerosa benzene extract did not
induce any genotoxic effect in the PBMC cells. Similarly, the non-genotoxic property
of the brown seaweed Fucus vesiculosus was evaluated by Leite-Silva et al. (2007) and
the results suggests that the seaweed did not exhibited any genotoxic effect in cultured
human lymphocytes.
(I)
(II)
Figure 5.4: (I) ‐ Confocal image illustrating the effect of G.acerosa on PBMC. A‐ Control, B‐ H2O2 treatment, C, D& E‐Treatmentwith G. acerosa benzene extract (250, 500, 1000µg/mL respectively). (II)‐ Genotoxic effect of G. acerosabenzeneextract.
Chapter III 119
5.4.5.AcutetoxicitystudiesofG.acerosa
Acute toxicity study of a test substance is employed as an excellent tool for
assessing its toxicity risk in human health and environment (Chandrasekaran et al.,
2009). The results of acute toxicity studies indicate that G. acerosa did not cause any
adverse behavioral changes or mortality upon treatment with 2000 mg/kg of BW. At the
end of the study, all the animals were sacrificed and the organs were collected and
analyzed for changes in the organ weight. No significant changes were observed in the
weight of organs upon treatment with G. acerosa benzene extract (Table 5.3).
Table5.3:OrganweightofmicetreatedwithG.acerosabenzeneextract(2000mg/kgofBW)
S. No Organ Controla G. acerosa
(2000 mg/Kg BW of mice) a
1. Kidneysb 1.433 ± 0.3512 1.233 ± 0.152
2. Liverb 5.167 ± 0.3512 5.4 ± 0.26
3. Heart b 0.4333 ± 0.05774 0.433 ± 0.115
4. Lungsb 0.5667 ± 0.05774 0.623 ± 0.351
5. Spleenb 0.5833 ± 0.03512 0.536 ± 0.0152
6. Pancreasb 0.3667 ± 0.05774 0.233 ± 0.057
7. Brainb 0.48 ± 0.02 0.4567 ± 0.047
8. Ovaryb 0.26 ± 0.03464 0.2867 ± 0.030
a Results were expressed as Mean ± S.D. bg/100 g of BW
5.4.5.1.Hematologicalandbiochemicalparameters
Blood is an important index of physiological and pathological condition in
animals and human beings. Upon ingestion of the plant constituents, the normal ranges
of these parameters may tend to alter and therefore analyzing the hematological
parameters has become crucial in toxicity testing (Adeneye et al., 2006). The effect of
G. acerosa benzene extract on blood constituents were verified by analyzing the
Chapter III 120
hematological and biochemical parameters. Table 5.4 illustrates the hematological
parameters of mice treated with 2000 mg/ kg (of BW) of G. acerosa benzene extract
(acute toxicity study). Minor changes such as increase in the percentage of eosinophil
and decrease in the percentage of neutrophils, MCV and monocytes were observed
upon treatment with G. acerosa benzene extract. However, the changes were not
significant in all the parameters analyzed, between control and treated groups.
Table 5.4: Hematological profile of mice treated withG.acerosabenzeneextract(2000mg/kgofBW)
a Results were expressed as Mean ± S.D.
The results of biochemical parameters suggest that a slight decrease in the level
of blood sugar and an increase (P<0.05) in the level of sodium were observed upon
treatment with G. acerosa benzene extract (Table 5.5). It should be noted that there is
no significant change (P<0.05) observed in all the other parameters evaluated.
S. No Parameters Controla G. acerosa
(2000 mg/Kg BW of mice)a
1. Total RBC count (1 × 106
µL) 7.557 ± 0.3636 7.6 ± 0.26
2. Total WBC count (1 × 103
µL) 5.793 ± 0.1607 5.2 ± 0.305
3. Platelet count (1 × 103 µL) 179 ± 6.245 174 ± 4
4. Packed cell volume (%) 41 ± 3 42.67 ± 3.055
5. HB (g/dL) 12.4 ± 0.8 11.37 ± 0.15
6. MCV (fl) 47.33 ± 5.686 43.67 ± 2.0
7. MCH (pg) 6.867 ± 0.2517 6.33 ± 0.25
8. Neutrophils (%) 69.67 ± 3.215 66 ± 2.64
9. Lymphocytes (%) 77.33 ± 5.686 76.67 ± 2.08
10. Eosinophils (%) 0.3333 ± 0.5774 0.666 ± 0.577
11. Monocytes (%) 1.667 ± 0.5774 1.33 ± 0.577
Chapter III 121
Table5.5:Biochemicalprofile ofmice treatedwithG. acerosabenzeneextract(2000mg/kgofBW)
S. No Parameters Controla G. acerosa
(2000 mg/Kg BW of mice)a
1. Blood sugar (mg/dL) 78.33 ± 4.163 71 ± 2
2. BUN (mg/dL) 24.33 ± 3.055 24.33 ± 2.08
3. Serum creatinine (mg/dL) 0.8467 ± 0.0602 0.76 ± 0.12
4. Alkaline phosphatase (U) 100.7 ± 3.512 99.67 ± 3.055
5. Serum total protein (g/dL) 5.4 ± 0.9644 5.16 ± 0.230
6. Serum albumin (g/dL) 2.967 ± 0.2082 2.4 ± 0.2
7. SGOT (AST) (IU/mL) 54 ± 3.606 52.33 ± 3.215
8. SGPT (ALT) (IU/L) 60 ± 2.646 64 ± 1
9. Calcium (mmol/L) 6.933 ± 0.3786 7.433 ± 0.30
10. Sodium (mmol/L) 137.7 ± 4.509 154.3 ± 3.055*
11. Potassium (mmol/L) 5.267 ± 0.2082 5.4 ± 0.45
12. Bilirubin total (mg/dL) 0.7 ± 0.1732 0.733 ± 0.115
13. Serum total cholesterol (mg/dL)
85 ± 4.583 87.33 ± 1.52
14. Serum triglycerides level (mg/dL)
54.67 ± 3.512 53.83 ± 2.079
15. Serum HDL cholesterol (mg/dL)
66.67 ± 5.859 64 ± 1.732
16. Serum LDL cholesterol (mg/dL)
12.33 ± 3.215 11 ± 1
17. Serum VLDL cholesterol (mg/dL)
10.2 ± 1.908 10.3 ± 0.984
a Results were expressed as Mean ± S.D. *P<0.05
5.4.5.2.Histopathologicalanalysis
Fig. 5.5 illustrates the results of histopathological analysis of control and treated
groups of acute toxicity study. Generally, the toxic constituents present in the plants and
other foreign substances get accumulated in liver which are further subjected to
detoxification. Hence analyzing the architecture of liver tissues is essential (Adeneye et
al., 2006). Histopathology of G. acerosa treated mice liver shows that the hepatocyte
architecture was normal and sign of necrosis was not observed. Moreover, the liver
parenchymal cells were normal both in the control and treated groups. In case of heart
Chapter III 122
tissues, the fiber alignment and cardiac muscles were normal. In addition to that, tissue
integrity of heart was also unaltered in the seaweed treated mice. Histopathology of
kidney and brain reveal that tubules and interstitium (in kidney) and marginal alignment
and interneuronal space (in brain) were found to be normal in the mice treated with G.
acerosa benzene extract. Hence the results suggest that G. acerosa benzene extract did
not cause any adverse pathological effects during the short-term treatment in mice.
5.4.6.Sub‐acutetoxicitystudies
The results of sub-acute toxicity studies shows that treatment with 200 and 400
mg/kg BW of G. acerosa benzene extract (for 28 days) did not cause adverse changes
in behavior or mortality. Parameters like body weight, body tone, salivation, urination,
rearing, defecation, locomotion were found to be normal throughout the study period
(results not shown). At the end of the study, all the animals were sacrificed and the
organs were collected and weighed. No significant changes were observed in the organ
weight between the control and treated groups (Table 5.6).
Chapter III 123
Table 5.6: Organ weight of mice treated with G. acerosabenzeneextracts(200and400mg/kgofBW)
a Results were expressed as Mean ± S.D. bg/100 g of BW
5.4.6.1.Hematologicalandbiochemicalparameters
The hematological profile of mice treated with G. acerosa benzene extract (in
sub-acute toxicity study), suggest that no significant changes (P<0.05) were observed in
all the parameters evaluated (Table 5.7).
S. No Organ Controla G. acerosa 200 mg/kg of BWa
G. acerosa 400 mg/kg of BWa
1. Kidneysb 1.383 ± 0.132 1.203 ± 0.07 1.533 ± 0.103
2. Liverb 5.6 ± 0.229 5.6 ± 0.27 5.65 ± 0.372
3. Heart b 0.483 ± 0.147 0.6 ± 0.109 0.633 ± 0.0816
4. Lungsb 0.6533 ± 0.019 0.665 ± 0.025 0.68 ± 0.0275
5. Spleenb 0.553 ± 0.041 0.5683 ± 0.021 0.5733 ± 0.02251
6. Pancreasb 0.2 ± 0.0632 0.166 ± 0.081 0.233 ± 0.051
7. Brainb 0.465 ± 0.019 0.5567 ± 0.022 0.5667 ± 0.0265
8. Ovaryb 0.37 ± 0.02 0.34 ± 0.045 0.37 ± 0.0219
Chapter III 124
Table 5.7: Hematological profile of mice treated withG.acerosabenzeneextract(200and400mg/kgofBW)
S. No Parameters Controla 200 mg/kg of
BWa 400 mg/kg
of BWa
1. Total RBC count (1 × 106
µL) 7.983 ± 0.42 8.45 ± 0.36 8.38 ± 0.41
2. Total WBC count (1 × 103
µL) 5.033 ± 0.47 5.81 ± 0.41 6.31 ± 0.56
3. Platelet count (1 × 103 µL) 178.8 ± 3.65 177 ± 5.83 177.2 ± 5.94
4. Packed cell volume (%) 44.17 ± 3.43 46 ± 1.89 46.5 ± 2.88
5. HB (g/dL) 11.55 ± 0.187 11.55 ± 0.30 11.85 ± 0.36
6. MCV (fl) 46.67 ± 2.5 48.5 ± 2.4 46.5 ± 2.0
7. MCH (pg) 6.48 ± 0.27 6.76 ± 0.25 6.833 ± 0.12
8. Neutrophils (%) 67.5 ± 2.07 69 ± 1.897 70.17 ± 5.41
9. Lymphocytes (%) 75.83 ± 2.85 75.17 ± 3.18 77 ± 2.75
10. Eosinophils (%) 0.333 ± 0.416 0.666 ± 0.526 0.67 ± 0.306
11. Monocytes (%) 1.167 ± 0.408 1.57 ± 0.547 1.333 ± 0.56
12. Basophils (%) 0.166 ± 0.408 0.333 ± 0.051 0.31 ± 0.16 a Results were expressed as Mean ± S.D.
The results of biochemical profile of sub-acute toxicity were analyzed and the
results suggest a mild increase in the level of blood sugar and SGOT and a significant
increase (P<0.05) in sodium (Table 5.8). All the other parameters did not show any
significant changes, when compared to the control group.
Chapter III 125
Table5.8:Biochemicalprofile ofmice treatedwithG. acerosabenzeneextract(200and400mg/kgofBW)
a Results were expressed as Mean ± S.D.*P<0.05
5.4.6.2.Histopathologicalanalysis
The results of histopathological analysis of sub-acute toxicity studies were
illustrated in Fig. 5.6. Microscopic images of G. acerosa treated (200 and 400 mg/kg of
BW) mice liver shows normal hepatocellular arrangement with intact membrane.
Histopathology of heart of mice treated with 200 mg/Kg and 400 mg/Kg of BW shows
normal fibre integrity and cell alignment. Kidney sections of mice treated with G.
acerosa benzene extract shows normal prominent interstitium with no signs of edema.
Histopathology of brain shows normal interneuronal space with well developed
neurons. Also, no signs of inflammation or hemorrhage were observed. Hence the
S. No Parameters Controla 200 mg/kg of
BWa 400 mg/kg of
BWa
1. Blood sugar (mg/dL) 74.5 ± 5.5 82.83 ± 5.49 85.17 ± 2.78
2. BUN (mg/dL) 27.3 ± 2.42 26.5 ± 2.81 26.83 ± 2.63
3. Serum creatinine (mg/dL) 0.8833 ± 0.39 0.901 ± 0.070 0.931 ± 0.64
4. Alkaline phosphatase (U) 104 ± 4 102.3 ± 1.966 101.2 ± 3.54
5. Serum total protein
(g/dL) 5.317 ± 0.174 6.117 ± 0.523 6.133 ± 0.54
6. Serum albumin (g/dL) 2.65 ± 0.18 3.05 ± 0.295 3.4117 ± 0.343
7. SGOT (AST) (IU/mL) 60.83 ± 4.16 62.67 ± 3.882 67.68 ± 3.50
8. SGPT (ALT) (IU/L) 65.33 ± 2.50 66 ± 3.28 66.33 ± 2.94
9. Calcium (mmol/L) 7.25 ± 0.48 7.56 ± 0.265 7.68 ± 0.14
10. Sodium (mmol/L) 152.7 ± 2.066 156.3 ± 2.33 163.5 ± 3.61*
11. Potassium (mmol/L) 5.667 ± 0.265 5.6 ± 3.033 5.917 ± 0.098
12. Bilirubin total (mg/dL) 0.8 ± 0.167 0.6167 ± 0.1169
0.8 ± 0.154
13. Serum total cholesterol
(mg/dL) 85.17 ± 2.137 85 ± 4.817 83.33 ± 2.25
14. Serum triglycerides level
(mg/dL) 55 ± 2.28 55.17 ± 3.25 54.5 ± 3.98
15. Serum HDL cholesterol
(mg/dL) 65.5 ± 2.58 65.67 ± 3.14 67.17 ± 2.99
16. Serum LDL cholesterol
(mg/dL) 10.33 ± 1.21 13.33 ± 2.33 12 ± 1.89
17. Serum VLDL cholesterol
(mg/dL) 10.67 ± 1.211 11 ± 2 10.83 ± 1.83
Chapter III 126
results of histopathological analysis of all the vital organs suggest that G. acerosa did
not exhibit any pathological effects in mice treated with G. acerosa (both 200 mg and
400 mg/Kg of BW).
Chapter III 127
5.5.CONCLUSION
Safety assessment of G. acerosa benzene extract was performed through in vitro and in
vivo toxicity assays.
The results of in vitro tests suggest that the extract was devoid of cytotoxic,
mutagenic and genotoxic effects.
The in vivo acute and sub-acute toxicity tests suggest that benzene extract did
not exhibit any significant alterations in hematological and biochemical
parameters.
Moreover, the histopathological analysis of vital organs like liver, heart, kidney
and brain revealed that the extract did not cause any adverse pathological
effects in mice.
On the whole, the study demonstrates that G. acerosa did not cause toxic effects, which
could be a productive approach in the facet of drug discovery and development.
Chapter III 128
G.acerosabenzeneextract
Safetyevaluation
Invitro
Trypanblue/MTT/Heamolysis
asay
Non‐cytotoxic
Amestest
Non‐mutagenic
Cometassay
Non‐genotoxic
Invivo
Acutetoxicity Sub‐acutetoxicity
Haematological&Biochemicalparameters/Histopathology
Noobservableadverseeffects
5.6.SUMMARYOFTHERESULTS