evaluation of invitro anticancer activity of bulb …

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Sakthi et al. World Journal of Pharmacy and Pharmaceutical Sciences

EVALUATION OF INVITRO ANTICANCER ACTIVITY OF BULB

EXTRACTS OF Allium fistulosum L. AGAINST HEPATOCELLULAR

CARCINOMA CELL LINE (Hep G2)

Monika N.1 and Sakthi Abirami M.*

2

1Institute of Pharmacology, Madras Medical College, Chennai-3.

2Assistant Professor Institute of Pharmacology, Madras Medical College, Chennai-3.

ABSTRACT

Natural phytochemicals derived from medicinal plants have attained a

greater significance in management of cancer. The liver cancer is

ranked in the top 10 human cancer worldwide and among top 5 of

cancers in terms of mortality more than 70% of primary liver cancer is

presented as hepatocellular carcinoma. Allium fistulosum L. was

sequentially extracted with solvents such as Petroleum ether, Ethyl

acetate and Ethanol at increasing polarity using soxhlet apparatus.

Qualitative analysis was done for alkaloids, flavonoids, phenols,

tannins, saponins, carbohydrates, proteins, amino acids, glycosides,

gums, mucilage, steroids, phenols, terpenoids, resins, fixed oils and

fats. The presence of phenolic and flavonoid components possess wide spectrum of biological

activities such as antioxidant, anti-mutagenic, anti-carcinogenic and age related degeneration

of cell components. In order to determine its role of flavonoids and phenolic components in

cancer, quantitative estimation was done. The three extracts were further evaluated for invitro

anticancer activity against HepG2 cell line by MTT assay which was compared with the

Standard drug 5-Fluorouracil. The MTT assay is the best known method for determining

mitochondrial dehydrogenase activity in the living cells. From the obtained results, the IC50

value of the Petroleum ether, Ethyl acetate and Ethanol extract of Allium fistulosum L. were

found to be 65.22 µg, 58.09 µg, 33.27µg respectively and the IC50 value of 5-Fluorouracil

was found to be 17.90 µg. So it was concluded that among the three extracts, the ethanolic

extract was found to be potent and effective.

KEYWORDS: Allium fistulosum, anticancer, MTT assay, Hep G2, phytochemicals.

Article Received on

19 March 2018,

Revised on 09 April 2018,

Accepted on 30 April 2018,

DOI: 10.20959/wjpps20185-11647

*Corresponding Author

Sakthi Abirami M.

Assistant Professor Institute

of Pharmacology, Madras

Medical College, Chennai-3.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.421

Volume 7, Issue 5, 1678-1699 Research Article ISSN 2278 – 4357


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INTRODUCTION

Cancer is a disease of cell proliferation in which normal cells are transformed into cells with

dysregulated growth. Cancer is the second leading cause of death globally and nearly 1 in 6

deaths is due to cancer.[1]

Cancer cells manifest to varying degrees, four characteristics that

distinguish them from normal cells and it compete with normal cells for energy and nutrition

resulting in deterioration of normal organ function. They are uncontrolled proliferation,

dedifferentiation, loss of function, invasiveness and metastasis.[2]

The liver cancer is ranked

in the top 10 human cancer worldwide and among top 5 of cancers in terms of mortality more

than 70% of primary liver cancer is presented as hepatocellular carcinoma. It is a malignant

liver tumour that shows hepatocyte differentiation.[3]

However, many chemotherapeutic drugs

are presently placed in a predicament of reduced therapeutic effect due to the problem of drug

resistance. Chemotherapeutic drugs also exert toxicity to normal cells, which in turn causes

the unpleasant side effects to the patients. For these reasons, research and development of

new classes of anticancer agents which exhibit efficient and selective toxicity in tumour cells

in enticing increased attention. Although many plants are used to treat tumour in Indian

Traditional Medicine, most of these plants are not scientifically evaluated. Allium is a genus

belonging to the family Alliaceae. The Allium genus includes approximately 500 species, the

most widely used, are onions (Allium cepa), garlic (Allium sativum), leeks (Allium porrum),

chives (Allium schoenoprasum) and shallots (Allium ascalonicum). Such plants have been

employed for centuries for the pungency and flavouring value and for their medicinal

properties.[1]

Allium fistulosum L. is a monocot perennial herb which was widely cultivated

throughout the world from tropical Asia to Siberia, particularly in Japan, Korea and China.

This species is known as Welsh onion derived from the German word ‘welshche’ meaning

‘foreign’. It is used as a folk medicine for treating febrile disease, headache, abdominal pain,

diarrhoea, eye related disorders and habitual abortion. The bulb contains an essential oil that

is rich in sulphur compounds. It acts as antibacterial, antiseptic, diaphoretic, diuretics,

galactogogue, stomachic and vermifuge. The bulbs can also be made into a poultice to drain

pus from sores, boils and abscesses. Allium fistulosum L. was rich in antioxidants such as

Flavonoids, Quercetin, Kaempferol, Polyphenols and other Organosulphur compounds which

deactivate the molecules that are harmful to the body cells. It has been evaluated for their

anti-oxidant, anti-inflammatory, anti-fungal, anti-influenza, anti-bacterial, anti-obesity, anti -

atherosclerosis and it has been reported to inhibit platelet aggregation, modulate the aortic

vascular tone, lower blood pressure and hyperglycaemia.[4]

Phytochemical evaluation is used

to determine the nature of phytoconstituents present in the plant by using suitable chemical


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tests. The presence of phenolic and flavonoid components acts as free radicals scavengers and

it possess wide spectrum of biological activities such as antioxidant, antimutagenic,

anticarcinogenic and age related degeneration of cell components. Free radicals have been

implicated in the development of a number of disorders, including cancer, neurodegeneration

and inflammation. Free radicals cause severe oxidative damage to proteins, lipids, enzymes

and DNA by covalent binding and lipid peroxidation, with subsequent tissue injury. Based on

the strong evidence on biological activities, this study was focused on quantitatively

determining flavonoids and phenolic components using Aluminium chloride method and

Folin-Ciocalteu method respectively.[5]

Further, it was analysed for anticancer activity by

enzyme based assay. Among the enzyme based assay, the MTT assay is the best known

method for determining mitochondrial dehydrogenase activity in the living cells. In this

method, MTT (3-(4, 5- Dimethylthiazol-2yl) - 2, 5 – Diphenyl tetrazolium Bromide) is

reduced to purple formazan by NADH. However, formazan is insoluble in water and it forms

purple needle-shaped crystals in the cells.[6]

In this study, it was aimed to find out a new

source with low or non-toxic natural anticancer agent against hepatocellular carcinoma.

MATERIALS AND METHODS

I. Extraction

a. Collection and Authentication of Plants

The plant Allium fistulosum L. was collected from Chennai in the month of August 2017. The

plant was identified and authenticated by DR. JAYARAMAN, Plant Anatomy Research

Centre at Tambaram, Chennai.

b. Preparation of Extracts

The bulbs of the plant were cleaned, shade dried for about 2 weeks. The dried bulbs were

pulverized to a coarse powder by grinding in mixer and stored in an air tight container.

Extraction is the preliminary step involved in the phytochemical studies. It is the separation

of medicinally active portions of plant using selective solvents through standard procedures.

Hot Percolation Method

30g of the dried coarsely powdered plant material of Allium fistulosum L. were successively

extracted with Petroleum ether to remove fatty matter. It was sequentially extracted with

solvents of increasing polarity (Ethyl acetate and Ethanol) using soxhlet apparatus at 60⁰-70⁰

for 18hrs, until the solvent become colourless in the siphon tube. Each time before extracting


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with the next solvent, the material was removed and dried at room temperature. The extracts

were concentrated by rotary evaporator. The semisolid residue obtained was weighed and

stored in desiccators. The yield value of the residue obtained was calculated using the given

formula,

Weight of the residue

Weight of the dried plant

Further studies were conducted with the obtained residue from the above process.

II. Phytochemical Analysis

Phytochemical analysis helps in correlating the nature of the compound present in the extract

by using suitable chemical tests. It can be done by qualitative analysis using specific reagents

followed by confirmation with different identification tests.

A. Qualitative Analysis[7,8]

i. Detection of Alkaloids

a. Mayer’s Test: 0.5ml of the extract was treated with few drops of Mayer’s reagent. The

formation of creamy white precipitate indicates the presence of alkaloids.

b. Dragendroff test: 0.5ml of the extract was treated with few drops of Dragendroff reagent.

The formation of orange red precipitate indicates the presence of alkaloids.

ii. Detection of Flavonoids

Alkaline reagent Test: 1ml of the extract was treated with few drops of sodium hydroxide

solution. The formation of intense yellow colour was observed which becomes colourless on

addition of dilute HCl, it indicates the presence of flavonoids.

iii. Detection of Saponins

Foam Test: 0.5ml of the extract was shaken with the 2ml of water. If foam produced persists

for few minutes, it indicates the presence of saponins.

iv. Detection of Carbohydrates

a.) Molisch’s Test: 0.5ml of the extract was treated with few drops of α-naphthol and

concentrated sulphuric acid was added carefully along the sides of the test tube. The

formation of violet ring at the junction indicates the presence of carbohydrates.

X 100 Yield value =


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b.) Benedict’s test: The extract was treated with Benedict’s reagent and heated on water

bath. The formation of orange red precipitate indicates the presence of reducing sugars.

v. Detection of Proteins And Amino Acids

a. Ninhydrin Test: To the 0.5ml of extract, few drops of 0.25% Ninhydrin solution was

added and boiled for few minutes. The formation of blue colour indicates the presence of

amino acid.

b. Biuret Test: 0.5ml of the extract was treated with 1ml of 10% sodium hydroxide solution

and heated. To this a drop of 0.7% copper sulphate solution was added. The formation of

purplish violet colour indicates the presence of proteins.

vi. Detection of Phenolic Acids and Tannins

a. Ferric Chloride Test: 0.5ml of the extract was treated with few drops of ferric chloride

solution. The formation of blackish violet or pinkish red colour indicates the presence of

tannins or phenolic acids.

b. Gelatin Test: 0.5ml of the extract was treated with few drops of 1% Gelatin solution

containing 10% sodium chloride. The formation of white precipitate indicates the

presence of tannins.

vii. Detection of Glycosides

Legal’s test: 0.5ml of the extract was treated with sodium nitroprusside in pyridine and

methanolic alkali (NaOH). The formation of pink to blood red colour indicates the presence

of cardiac glycosides.

viii. Detection of Gums And Mucilage

0.5ml of the extract was treated with the alcohol; the formation of white precipitate indicates

the presence of gums and mucilage.

ix. Detection of Steroids and Terpenoids

Libermann Burchard’s test: 1ml of the extract was treated with chloroform and a few drops

of acetic anhydride, boiled and cooled. Then, few drops of concentrated sulphuric acid was

added carefully along the sides of the test tube. The formation of green colour indicates the

presence of steroids and pink colour indicates terpenoids.


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x. Detection of Resins

Acetone-Water Test: Extracts were treated with acetone. Small amount of water was added

and shaken. Appearance of turbidity indicates the presence of resin.

xi. Detection of Fixed Oils And Fats

Stain Test: Small quantities of extracts were pressed between two filter papers. An oily stain

on filter paper indicates the presence of fixed oils and fat.

B. Quantitative Estimation of Flavonoid And Phenolic Content[9-12]

Flavonoids and phenols are highly effective free radical scavengers and antioxidants. They

are used for the prevention and cure of various diseases which are originated by formation of

free radicals. The flavonoid and phenolic compounds appear to function as good electron and

hydrogen donors and therefore should be able to terminate free radical chain reaction by

converting the free radicals to more stable compound.

i. Total Flavonoid Content- Aluminium Chloride Colorimetric Method

Principle: Aluminium chloride forms acid stable complexes with C4 keto groups and either

the C-3 or C-5 hydroxyl group of flavones and flavonols and it also forms acid labile

complexes with ortho dihydroxyl groups in the A or B ring of flavonoids.

Materials and Reagents: Petroleum ether, ethyl acetate and ethanolic extracts of A.

fistulosum L., 10% AlCl3 solution, 5% sodium nitrite solution, 1M sodium hydroxide

solution, quercetin and UV spectrophotometer.

Procedure: 0.5ml of aliquots of sample (50µg) and quercetin standard solution of varying

concentration (12.5µg/ml to 200µg/ml) was added into each standard flask of 10ml capacity

containing 4ml of distilled water. To each flask 0.5ml of Sodium nitrite was added. After 5

minutes, 0.3ml of aluminium chloride was added. After 5 minutes, 2ml of Sodium hydroxide

was added to each flask. The volume was finally made up to 10ml with distilled water and

allowed to stand for 15 minutes at room temperature and filtered. The absorbance of the

filtrate was measured at 510nm using UV – Visible spectrometer. Blank was performed using

distilled water for standard and reagent blank without addition of aluminium chloride for

recording sample absorbance. All the determinations were done in triplicate. The calibration

curve of quercetin was plotted with concentration against absorbance. The concentration of

the flavonoid present in 50µg of crude extract is determined using interpolations methods


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from quercetin standard calibration curve. The total flavonoid content of the extracts was

expressed as quercetin equivalents [(QE)/ g] of dried extract.

ii. Total phenolic Content – Folin-Ciocalteu method

Total phenols were estimated according to the Folin-ciocalteu method.

Principle: Folin-ciocalteu reagent is a mixture of phosphomolybdate and phosphotungstate.

The presence of hydroxyl phenol group reduces the phosphomolybdate present in the Folin-

ciocalteu (F/C) reagent to form blue colour which can be measured colorimetrically at 765

nm.

Materials and Reagents: Petroleum ether, ethyl acetate and ethanolic extracts of A.

fistulosum L., Folin-ciocalteu reagent, 7.5% sodium bicarbonate, Gallic acid and UV-Visible

spectrophotometer.

Procedure: Plant extracts were diluted to a concentration of 1mg/ml, and aliquots of 0.5ml

were mixed with 2.5ml of Folin-ciocalteu reagent (previously diluted 10-fold with distilled

water) and 2ml of Sodium bicarbonate (7.5%). After 15minutes of incubation at 45⁰C, the

absorbance was measured at 765nm in a spectrophotometer against a blank sample. Total

phenols were determined as Gallic acid equivalents (mg GA/g of extract) and the values are

represented as means of triplicate analyses.

The concentration of the flavonoid and phenolic content present in 50µg of crude extract of

Allium fistulosum L. was determined from standard calibration curve of quercetin and Gallic

acid by interpolation. From the above obtained concentration, the total flavonoid and

phenolic content of the extracts were determined using the formula,

=C x V/m

C=Concentration of equivalent drug established from the calibration curve in mg/ml

V= volume of extract in ml

m= weight of plant extract in g

The Total flavonoid and phenolic content of the extracts was expressed as standard

equivalents per g of dried extract.


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iii. Determination of Invitro Anticancer Activity Using MTT Assay[13-21]

Cell lines and reagents

Hep G2 cell lines, Vero cell line, Sample to be tested, Minimum essential media (MEM), 5%

Fetal Bovine Serum (Sigma Aldrich), Penicillin, Streptomycin, Phenol red, MTT (3-(4, 5-

dimethylthiazol-2yl)- 2,5 – diphenyl tetrazolium Bromide), Dimethyl sulfoxide (DMSO),

0.45µ filter, Spectrophotometer, 24 well plate, Tissue paper, Marker pen, Spirit, cotton and

Gloves 1 ml, 2ml pipettes, TPVG (Trypsin, phenol, versene, glycol) Micropipette tips,

discarding jar with 1% hypochlorite solution and Fluorouracil (5- FU). Reagents were

purchased from Sigma Aldrich, USA.

Preparation of Media

Procedure

The powder form of MEM was dissolved in pre-sterilized Millipore distilled water, and

sterilized it at 121˚C, for 15 minutes. In a 1000ml flask, 857 ml of sterilized MEM and each

1ml of penicillin, streptomycin, phenol red, amphotericin B were added and mixed well.

Then 10ml of 3% L-glutamine, FBS 100ml, and 30ml of 7.5% NaHCO3 were added to make

the total volume of 1000ml, mixed well and adjusted to pH 7.2 to 7.4. The medium was

stored for two days at 37˚C and check the pH frequently and store it in refrigerator.

Cell Line and Culture

The Human liver Cancer cell line, Hep G2 was obtained from NCCS, Pune. They were

cultured in complete essential growth medium in T25ml cultured vented flask and incubated

at 37˚C in 5% CO2 incubator. After monolayer reaches the 90% confluency (adherent), it was

used for study.

Sub-culturing adherent cells

Passaging is a process of separating the cells from primary adherent cell culture for sub-

culturing purpose.

Procedure

The whole procedure was carried out under sterilized condition. After monolayer formation

in the cultured flask, discard the spent culture media. Pipette in 2ml of wash solution, PBS, to

the side of the flask without disturbing cell monolayer and moved gently to and fro several

times to remove the traces of serum calcium, magnesium, which may inhibit the action of

TPVG. Discard the wash solution and add 2ml of dissociation reagent, TPVG ; move to and


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fro several times. Then, incubate the flask for 5 minutes at room temperature and the cell

detachment were confirmed by microscope. Then 2ml of growth media was added over the

cell monolayer and disperse it. The solution is transferred to sterile tube and centrifuged for

5-10 minutes at 5000 rpm. Discard the supernatant and resuspend the pellet with growth

medium. Pipette 1ml of resuspended solution into seven new vented T-25ml tissue culture

flasks and add 4ml of growth media each and incubated it in 5% CO2 incubator.

Sample dilutions

Stock concentration

1mg of sample is dissolved in 1ml of serum free MEM/DMSO. The stock was prepared fresh

and filtered through 0.45µ filter before each assay. Working concentration of sample ranging

from 1000 to 7.8µg was prepared as follows.

Preparation of working stock - 1mg/ml

To 1ml MEM without serum/ DMSO, add 1ml of sample to give working concentration.

Sample concentration ranging from 1000µg to 7.8µg can be prepared from the working stock

and making up the volume to 1ml in MEM without serum /DMSO. Prepare required volume

of test sample for each concentration.

Procedure

Cells (1X 105 per well) (Hep G2) were plated in 24 well plates and incubated at 37⁰ C with

5% CO2 condition. After the cell reaches the confluence, media was removed from the wells

carefully without disturbing the cells. The various concentrations of the samples were added

and incubated for 24 hrs. After incubation, the sample was removed from the well and

washed with phosphate buffered saline (pH 7.4) or MEM without serum. 100µl / well

(5mg/ml) of 0.5% 3-(4,5 – dimethyl -2- thiazolyl ) -2,5- diphenyl –tetrazolium bromide

(MTT ) was added and incubated for four hours at 37⁰ C in the dark condition. After

incubation, 1ml of DMSO (0.1% v/v) was added in all the wells. The absorbance at 570nm

was measured with ELISA reader or UV Spectrometer using DMSO as the blank. All assays

were carried out in triplicate and their absorbance was expressed as Mean SEM.

Measurements were performed and the concentration required for a 50 % inhibition (IC50)

was determined graphically. The percentage cell viability was calculated using the following

formula:

A570 of treated cells

A570nm of control cells

X 100 % Cell viability =


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Graphs are plotted using the percentage of cell viability at Y-axis and concentration of the

sample in X-axis. Cell control and sample control is included in each assay to compare the

cell viability.[6, 13-16]

The IC50 value of the extract was determined graphically.

Statistical Analysis

Experimental results are expressed as Mean ± SEM. All measurements were replicated three

times. The IC50 values were calculated from linear regression analysis.

IV. RESULTS

i. The percentage yield was calculated and tabulated in table 1.

Table 1: Percentage yield value of the bulb extracts of Allium fistulosum L.

S.no Type of extract Yield Value (% w/w)

1. Petroleum ether 1.66%

2. Ethyl acetate 3 %

3. Ethanol 8.66%

ii. Phytochemical Analysis

a. Qualitative Estimation

The phytochemical constituents present in the bulbs of Allium fistulosum L. are.

Table 2: Phytochemical Analysis – Qualitative.

PHYTOCHEMICAL TESTS PET. ETHER ETHYL ACETATE ETHANOL

Alkaloids - - -

Flavonoids - + +

Saponins - - +

Carbohydrates - + +

Proteins and amino acids - + +

Tannins - + -

Glycosides - + -

Gums and mucilage + - -

Steroids + - -

Phenols - + +

Terpenoids - + +

Resins + - +

Fixed oils and fats + + +

(+): Present ; (-): Absent

b. QUANTITATIVE ESTIMATION:

Estimation of Total phenolic (TPC) and flavonoid content (TFC):

The total Flavonoid and Phenolic content were quantitatively estimated by Aluminium

chloride method and Folin-ciocalteu method. The varying concentration of Quercetin and


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Gallic acid were prepared and the absorbance of different concentration measured using UV

spectrophotometer were recorded and tabulated in Table 3a and 3b.

i) Standard calibration curve of Quercetin & Gallic Acid

Table no: 3a TFC of Quercetin.

Concentration(µg/ml)

Of Quercetin

Absorbance at

510nm

12.5 0.09

25 0.015

50 0.022

100 0.037

200 0.045

Table no: 3b- TPC of Gallic acid.

Concentration(µg/ml)

Of Gallic Acid

Absorbance at

765nm

2 0.185

4 0.340

6 0.493

8 0.659

10 0.815

Figure 1: Standard calibration curve of a) Quercetin (QE) & b) Gallic acid (GA).

By using Microsoft Excel, the calibration curve of Quercetin and Gallic acid were plotted at

different concentrations. The correlation co-efficient (R2) was found to be 0.9740 and 0.9998

which denotes linearity in the values.

a) b)


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Table no. 4: Absorbance of three extracts at 510 nm (TFC) and 765 nm (TPC).

Extract type Mean Absorbance at

510nm (TFC)

Mean Absorbance at

765 nm(TPC)

Petroleum Ether (0.5ml) 0.057±0.012 0.014±0.000

Ethyl acetate (0.5ml) 0.112±0.011 0.324±0.077

Ethanol (0.5ml) 0.181±0.038 0.404±0.045

By interpreting the absorbance value in the standard calibration curve, the concentration of

flavonoids and phenolic content in 0.5ml of extract was determined. Using conversion

formula, Total flavonoid and phenolic content in 1g of the dried extract was determined. It is

tabulated in the table 5.

Table 5: Estimation of Phenolic and Flavonoid Content of by Interpolation method.

Extract Type QE eq. wt / ml of extract QE/g GA eq. wt /ml of extract GA/g

Petroleum Ether 7.34µg 0.0734g 0.13µg 0.013g

Ethyl acetate 13.19µg 1.319g 2.29µg 0.229g

Ethanol 20.53µg 2.053g 2.91µg 0.291g

Figure 2: Graphical representation of Total Flavonoid and Phenolic content of the

extracts of Allium fistulosum L.

iii. Determination Of Invitro Anticancer Activity By MTT Assay

a. Anticancer Activity of petroleum ether Extract

The anticancer evaluation of the different concentration of petroleum ether extract of Allium

fistulosum L. was carried out using MTT assay on Hep G2 cells, their absorbance and

percentage inhibition was represented in the Table 6 and Figure 3 & 4.


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Table 6: Effect of Petroleum Ether Extract of Allium fistulosum L. (PEEAF) on Hep G2

Cell Line.

S.no Concentration

(µg)

Mean absorbance

(nm)

Inhibition of cell growth

(%) IC 50 R

2

1 Cell control 0.829 0±0.00

65.22 0.7581

2 7.8 0.620 25.22±1.60

3 15.6 0.550 33.66±4.01

4 31.2 0.482 41.86±3.10

5 62.5 0.418 49.58±1.73

6 125 0.362 56.34±0.80

7 250 0.308 62.85±1.51

8 500 0.246 70.33±1.22

9 1000 0.169 79.62±1.40

All the values in the table are represented as Mean ± SEM (n=3)

The graph was plotted with the data obtained from the above table with the help of Microsoft

Excel. The dose response relationship of PEEAF on Hep G2 was shown in the Figure 3.

Figure 3: Graphical representation of Cell growth Inhibition of Petroleum ether extract

of Allium fistulosum L. against Hep G2 cell line.

a) Normal Hep G2 Cell line.


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Figure 4: Effect of PEEAF on Hep G2 cells.

b. Anticancer Activity of Ethyl Acetate Extract

The anticancer evaluation of the different concentration of ethyl acetate extract of Allium


percentage inhibition was represented in table 7 and Figure 5 and 6.

Table 7: Effect of Ethyl Acetate Extract of Allium fistulosum L. (EAEAF) on Hep G2

Cell Line.

S.no Concentration

(µg)

Mean

absorbance

Inhibition of cell

growth (%) IC50 R

2

1 Cell control 0.829 0±0.004

58.092 0.7346

2 7.8 0.583 29.68±1.79

3 15.6 0.530 36.07±2.48

4 31.2 0.468 43.55±1.69

5 62.5 0.409 50.67±1.31

6 125 0.350 57.79±0.91

7 250 0.295 64.42±1.67

8 500 0.238 71.34±1.34

9 1000 0.176 78.77±0.50

All the values in the table are represented as Mean ± SEM (n=3).


Excel. The dose response relationship of EAEAF on Hep G2 was shown in the Figure 5.

c) 62.5 µg

µg

d) 1000 µg b) 7.8 µg


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Figure 5: Graphical representation of Cell growth Inhibition of Ethyl Acetate extract of

Allium fistulosum L. against Hep G2 cell line.

a) Normal Hep G2 Cell line.

b. 7.8 µg c. 62.5 µg d. 1000 µg

Figure 6: Effect of EAEAF on Hep G2 cells.

c. Anticancer activity of Ethanolic extract

The anticancer evaluation of the different concentration of ethanolic extract of Allium


percentage inhibition was represented in the Table 8 and Figure 7 & 8.


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Table 8: Effect of Ethanolic extract of Allium fistulosum L. on Hep G2 Cell Line.

S.no Concentration

(µg)

Mean

absorbance

Cell growth

inhibition (%) IC50(µg) R

2


33.27 0.6921

2 7.8 0.542 34.37±1.04

3 15.6 0.475 42.70±1.13

4 31.2 0.420 49.40±1.23

5 62.5 0.358 56.75±3.52

6 125 0.312 62.4±1.49

7 250 0.258 68.9±0.67

8 500 0.206 75.14±1.63

9 1000 0.152 81.65±1.57



Excel. The dose response relationship of EEAF on Hep G2 was shown in the Figure 7.

Figure 7: Graphical Representation of Inhibition of cell Proliferation of Ethanolic

extract of Allium fistulosum L. against Hep G2 cell line.

a) Normal Hep G2 cell line.


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b) 7.8µg c) 31.2 µg d) 1000 µg

Figure 8: Effect of EEAF on Hep G2 cells.

d. Anticancer activity of Standard Drug, 5- Fluorouracil

The anticancer evaluation of the different concentration of Standard drug, 5- Fluorouracil was

carried out using MTT assay on Hep G2 cells, their absorbance and percentage inhibition was

represented in the Table 9 and Figure 9 & 10.

Table 9: Effect of 5- Fluorouracil (5 – FU) on Hep G2 Cell Line.

S.no Concentration

(µg/ml)

Mean

absorbance

Cell growth

inhibition (%) IC 50 R

2


17.90 0.6665

2 7.8 0.482 41.85±1.54

3 15.6 0.426 48.62±1.21

4 31.2 0.367 55.80±3.26

5 62.5 0.311 62.51±1.71

6 125 0.250 69.85±0.73

7 250 0.196 77.59±1.50

8 500 0.149 81.97±1.29

9 1000 0.095 88.57±1.96



Excel. The dose response relationship of 5- Fluorouracil on Hep G2 was shown in the

Figure 9.


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Figure 9: Graphical Representation of Inhibition of Cell growth of 5-Fluorouracil

against Hep G2 cell line.

a) Normal Hep G2 cell line.

b) 7.8 µg c) 15.6 µg d) 1000 µg

Figure 10: Effect of 5-FU on Hep G2 cells.

Comparison of IC50 Values Of Different Solvent Extracts With Standard Drug 5-

Fluorouracil (5-FU)

From the above obtained results, the IC50 values of different extracts were compared to

determine the potent and effective extract and it was tabulated in Table 10 and Figure 11.

Table 10: IC50 values of the bulb Extracts of Allium fistulosum L. and 5-FU.

S.No. Extracts IC50 Values (µg)

1. Petroleum ether extract 65.22

2. Ethyl acetate extract 58.09

3. Ethanolic extract 33.27

4 5- Fluorouracil (5-FU) 17.90


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Figure 11: Graphical Representation of IC50 values of three solvents compared with

Standard Drug 5-Fluorouracil.

The comparison between IC50 of these extracts shows that ethanolic extract has lower IC50

value than the others and could be considered as potential source of anticancer compounds.

The ethanolic extract of Allium fistulosum L. (EEAF) was found to be effective and its

IC50 value was 33.27µg. From the above obtained results, it proves that bulbs extract of

Allium fistulosum L. attenuates the cell viability in a dose dependent manner.

V. DISCUSSION

Based on the traditional use, secondary metabolites and their biological activities, the plant

Allium fistulosum L. has been selected for the study. Based on the polarity of the solvent, it

was successively extracted with petroleum ether to remove fatty matter, followed by ethyl

acetate and ethanol. The yield obtained from ethanolic extract was higher than the other two

extracts of bulbs of Allium fistulosum L.

The phytochemical analysis of the extracts would help in predicting the nature of the

compound responsible for the biological activity. The phytochemical analysis of petroleum

ether extract reveals the presence of steroids, resins, fixed oil, gums and mucilage. The ethyl

acetate extract reveals the presence of flavonoids, carbohydrates, proteins, amino acids,

tannins, glycosides, terpenoids, phenols, fixed oils and fats. The ethanolic extract bulbs of

Allium fistulosum L. revealed the presence of Flavonoids, saponins, carbohydrates, proteins,

amino acids, terpenoids, resins, fixed oils and fats. It was observed that the ethanolic extract

had a wider spectrum of phytochemicals and it has a greater concentration of flavonoids and

phenols on quantification.

The bulbs of Allium fistulosum L. contains phenols and flavonoids which has potent

antioxidant property, there is no scientific data published regarding cytotoxic potential of this


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plant against Hepatocellular carcinoma. Hence, the study was carried out to determine the

anticancer activity of Allium fistulosum L. against Hepatocellular carcinoma.

In order to determine the cytotoxic potential of the plant, preliminary invitro study was

conducted using MTT assay. This assay is based on the principle that the viable cells contain

mitochondrial dehydrogenase which converts the dye into blue coloured formazan crystals.

The absorbance produced is directly proportional to the number of viable cells.

MTT measures cell respiration and the amount of formazan produced is proportional to the

number of living cells present in culture. An increase or decrease in cell number results in a

concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity

caused by the drug. IC50 is the concentration of the extract able to cause the death of 50% of

the cells and can be predictive of the degree of cytotoxic effect. The lower the absorbance

value, the more cytotoxic is the substance.

It is often used as an invitro screening model system to measure cytotoxic effects of plant

extract against cancer cell line. This method also has several advantages with respect to

rapidity, quantitation, management of many samples and cell number required for the assay.

Application of this assay to chemosensitivity testing seems to be valuable and useful.[22]

The MTT assay was carried out to determine the IC50 concentration of effective extract of

Allium fistulosum L. among the three extracts. The ethanolic extract was found to be effective

and potent, its IC50 concentration of 33.27µg/ml which is followed by ethyl acetate extract,

58.02µg/ml and petroleum ether extract, 65.22 µg/ml. The IC50 of Standard drug, 5-

fluorouracil was found to be 17.90µg/ml. Allium fistulosum L. inhibited the cell growth and

significantly decreased the cell survival through induction of cell death at high concentration

in a dose dependent manner. It proves that, the drug concentration is directly proportional to

the inhibition of cell proliferation.

VI. CONCLUSION

The inhibition of cell growth might be due to the presence of total flavonoid and phenolic

content which has direct relationship with the anticancer activity. This study provides only

basic data of screening, further studies are necessary for elucidating the mechanism involved

in the activity.


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CONFLICT OF INTEREST: The authors declare no conflict of interest.

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