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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