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EVALUATION OF ANTI-CANCER ACTIVITY OF
Linum usitatissimum ESSENTIAL OIL ON A549
HUMAN LUNG CANCER CELL LINE.
S. Amal Dhivahar, A. J. Shinny Beulah, S. Srividya, V. M. Berlin Grace*,
Perinba Danisha Jesubatham and S. Viswanathan
Department of Biotechnology, School of Agriculture and Biosciences,
Karunya institute of Technology and Sciences, Coimbatore-641114, India.
*Corresponding Author: berlin@karunya.edu
Essential oils:
- any concentrated,
hydrophobic liquids
containing volatile aroma
compounds from plants.
- Also known
as volatile or ethereal oil
s, or simply as the "oil
of" the plant material
from which they were
extracted.
- An example is oil of
clove.
Natural oils:
- triglycerides in which the
glycerine is esterified with three
fatty acids.
-they are the main constituent
of vegetable oil and animal
fats.
Primary Lung Cancer
Small cell lung cancer
(SCLC)-faster, chest
centre (bronchi)
Non small cell lung
cancer (NSCLC) – slow-
growing -outer area,
lung centre or anywhere
A549
Large cell
carcinoma
Squamous
cell
carcinoma
Adenocarcinoma
Backgrou
nd
ONCOLOGY & INVITRO
STUDIES
INSIGHTS FROM LITERATURE REVIEW
Statistic
s
Usage
Extract
Cell
line
TBA
MTT
Statistic
Usage
Extract
Cell
line
TBA
MTT
Prevailing cause of cancer-related
morality with NSCLC
85%
Accounting for most lethal
cancers worldwide in both
the genders
>30%
A549 – Human Adenocarcinomic
Alveolar Basal Epithelial Cells
(Fan et al., 2006).
1972
LUNG CARCINOMA
“Chloroquine inhibits cell growth and induces cell death in A549 lung
cancer cells. Bioorganic & medicinal chemistry. 14(9):3218-22”
Statistic
Usage
Extract
Cell
line
TBA
MTT
Ayurvedic
traditionally used medicinal
plant possessing many
therapeutic compounds
(Goyal et al., 2014).
Alpha-linolenic fatty acid
ALA is one of the compounds
extracted is known to be enhanced
in preventing and treating diseases
of the heart and blood vessels
(Harper et al., 2006).
Flax Apps
for treating osteoarthritis, anxiety,
benign prostatic hyperplasia or
hypertrophy (BPH), vaginal
infections, dry eyes,
atherosclerosis, high blood
pressure, heart disease, diabetes,
attention deficit-hyperactivity
disorder (ADHD), etc.
Cancer prevention
Some people use flaxseed
oil as a laxative for
constipation, for weight
loss, and to prevent breast
and prostate cancer
Breast Cancer Reduction
Animal studies have shown
that both flaxseed oil and
lignans can reduce breast
tumor growth and spread,
even for ER-cancer cells
ALA Apps
also used to treat rheumatoid
arthritis (RA), multiple
sclerosis(MS), diabetes, renal
disease, ulcerative colitis, and
Crohn’s disease.
Flax
Seeds
Statistic
Usage
Extract
Cell
line
TBA
MTT
EXTRACT
PREPARATION
Grinder
Flax seeds
purchased
from the
market were
powdered
STEP 1
Soxhlet
Apparatus
25g – extracted
using hexane at a
BP of65 - 68°C
STEP 2
Rotary Vacuum
Evaporator
Solvent
Segregation
STEP 3
(Zhang et al., 2008).
Statistic
Usage
Extract
Cell
line
TBA
MTT
CELL LINE
MAINTENANCE
CULTURE: A549
Human non-small cell
lung cancer (NSCLC)
cell line
MEDIA:HAM F12K
media + 10% FBS
+PSN antibiotics
INCUBATION: 5% CO2
incubator (Eppendorf – New
Brunswick, Model: Galaxy 170S,
Hamburg, Germany) at 37ºC
(Koparal , Zeytinoğlu , 2003).
Statistic
Usage
Extract
Cell
line
TBA
MTT
Centrifuged
pellet + 1X
PBS wash +
0.4% Trypan
blue dye
stain (1:5)
Haemo-
cytometer
cell counting
+ inverted
microscope
@ 4X
magnificence
Incubated
cells +
different
conc. of
drug in
DMSO
TRYPAN BLUE
ASSAY
% Viability
= [Average live cells in 4
squares / (Average live
cells in 4 squares +
Average dead cells in 4
squares)] ×100
(Altman et al., 1993)
Statistic
Usage
Extract
Cell
line
TBA
MTT
Adherent cells +
diff. conc. of drug
+ 24hr incubation
1.Drug Administration
PBS wash + 20µl
of 0.5% MTT +
4hr incubation
2.MTT Addition
MTT mixture removal +
100µl DMSO addition +
kept aside for 10 mins.
3.DMSO Addition
ELIZA Plate
reader – 570nm +
OD calculation
4.Absorbance
Measurement
MTT
Assay
(Chung et al., 2015)
Tetrazolium dye (MTT)
NADPH dependent oxidoreductase
(Mitochondria) – ‘Viable cells’
FORMAZON: Purple coloured compound –
Colorimetric assay (ELIZA Reader)
% Inhibition = [(OD control – OD sample) / ODcontrol] ×100
% viability = 100 - % Inhibition
RESULTS
A
B
96.5
89.69
77.72
48.4
26.06
11.04
Untreated 0.04 0.08 0.12 0.16 0.2
Concentration (mg/ml)
TRYPAN BLUE ASSAY
C
TT ASSAY
D
Inference:
❖ Cell viability - analysed
❖ Promising cytotoxic properties – identified
❖ Base for future studies - formed
❖ Further research could shed more light – understood
ACKNOWLEGEMENT:
Sincere gratitude is expressed towards Karunya Institute of Technology and Sciences
(KITS) for providing us with the laboratory and seed money to carry out the study and to
derive a conclusion successfully. The basic laboratory facilities were also partially supported
by the DST, Government of India [SB/YS/LS-252/2013(15 May 2014)] and DBT,
Government of India [BT/PR14632/NNT/28/824/2015], which is greatly acknowledged.
Thank
You

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Evaluation of Anti-cancer Activity of Linum usitatissimum Essential Oil on A549 Human Lung Cancer Cell Line

  • 1. EVALUATION OF ANTI-CANCER ACTIVITY OF Linum usitatissimum ESSENTIAL OIL ON A549 HUMAN LUNG CANCER CELL LINE. S. Amal Dhivahar, A. J. Shinny Beulah, S. Srividya, V. M. Berlin Grace*, Perinba Danisha Jesubatham and S. Viswanathan Department of Biotechnology, School of Agriculture and Biosciences, Karunya institute of Technology and Sciences, Coimbatore-641114, India. *Corresponding Author: berlin@karunya.edu
  • 2. Essential oils: - any concentrated, hydrophobic liquids containing volatile aroma compounds from plants. - Also known as volatile or ethereal oil s, or simply as the "oil of" the plant material from which they were extracted. - An example is oil of clove. Natural oils: - triglycerides in which the glycerine is esterified with three fatty acids. -they are the main constituent of vegetable oil and animal fats.
  • 3. Primary Lung Cancer Small cell lung cancer (SCLC)-faster, chest centre (bronchi) Non small cell lung cancer (NSCLC) – slow- growing -outer area, lung centre or anywhere A549 Large cell carcinoma Squamous cell carcinoma Adenocarcinoma
  • 4. Backgrou nd ONCOLOGY & INVITRO STUDIES INSIGHTS FROM LITERATURE REVIEW Statistic s Usage Extract Cell line TBA MTT
  • 5. Statistic Usage Extract Cell line TBA MTT Prevailing cause of cancer-related morality with NSCLC 85% Accounting for most lethal cancers worldwide in both the genders >30% A549 – Human Adenocarcinomic Alveolar Basal Epithelial Cells (Fan et al., 2006). 1972 LUNG CARCINOMA “Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorganic & medicinal chemistry. 14(9):3218-22”
  • 6. Statistic Usage Extract Cell line TBA MTT Ayurvedic traditionally used medicinal plant possessing many therapeutic compounds (Goyal et al., 2014). Alpha-linolenic fatty acid ALA is one of the compounds extracted is known to be enhanced in preventing and treating diseases of the heart and blood vessels (Harper et al., 2006). Flax Apps for treating osteoarthritis, anxiety, benign prostatic hyperplasia or hypertrophy (BPH), vaginal infections, dry eyes, atherosclerosis, high blood pressure, heart disease, diabetes, attention deficit-hyperactivity disorder (ADHD), etc. Cancer prevention Some people use flaxseed oil as a laxative for constipation, for weight loss, and to prevent breast and prostate cancer Breast Cancer Reduction Animal studies have shown that both flaxseed oil and lignans can reduce breast tumor growth and spread, even for ER-cancer cells ALA Apps also used to treat rheumatoid arthritis (RA), multiple sclerosis(MS), diabetes, renal disease, ulcerative colitis, and Crohn’s disease. Flax Seeds
  • 7. Statistic Usage Extract Cell line TBA MTT EXTRACT PREPARATION Grinder Flax seeds purchased from the market were powdered STEP 1 Soxhlet Apparatus 25g – extracted using hexane at a BP of65 - 68°C STEP 2 Rotary Vacuum Evaporator Solvent Segregation STEP 3 (Zhang et al., 2008).
  • 8. Statistic Usage Extract Cell line TBA MTT CELL LINE MAINTENANCE CULTURE: A549 Human non-small cell lung cancer (NSCLC) cell line MEDIA:HAM F12K media + 10% FBS +PSN antibiotics INCUBATION: 5% CO2 incubator (Eppendorf – New Brunswick, Model: Galaxy 170S, Hamburg, Germany) at 37ºC (Koparal , Zeytinoğlu , 2003).
  • 9. Statistic Usage Extract Cell line TBA MTT Centrifuged pellet + 1X PBS wash + 0.4% Trypan blue dye stain (1:5) Haemo- cytometer cell counting + inverted microscope @ 4X magnificence Incubated cells + different conc. of drug in DMSO TRYPAN BLUE ASSAY % Viability = [Average live cells in 4 squares / (Average live cells in 4 squares + Average dead cells in 4 squares)] ×100 (Altman et al., 1993)
  • 10. Statistic Usage Extract Cell line TBA MTT Adherent cells + diff. conc. of drug + 24hr incubation 1.Drug Administration PBS wash + 20µl of 0.5% MTT + 4hr incubation 2.MTT Addition MTT mixture removal + 100µl DMSO addition + kept aside for 10 mins. 3.DMSO Addition ELIZA Plate reader – 570nm + OD calculation 4.Absorbance Measurement MTT Assay (Chung et al., 2015)
  • 11. Tetrazolium dye (MTT) NADPH dependent oxidoreductase (Mitochondria) – ‘Viable cells’ FORMAZON: Purple coloured compound – Colorimetric assay (ELIZA Reader) % Inhibition = [(OD control – OD sample) / ODcontrol] ×100 % viability = 100 - % Inhibition
  • 12. RESULTS A B 96.5 89.69 77.72 48.4 26.06 11.04 Untreated 0.04 0.08 0.12 0.16 0.2 Concentration (mg/ml) TRYPAN BLUE ASSAY C TT ASSAY D
  • 13. Inference: ❖ Cell viability - analysed ❖ Promising cytotoxic properties – identified ❖ Base for future studies - formed ❖ Further research could shed more light – understood
  • 14. ACKNOWLEGEMENT: Sincere gratitude is expressed towards Karunya Institute of Technology and Sciences (KITS) for providing us with the laboratory and seed money to carry out the study and to derive a conclusion successfully. The basic laboratory facilities were also partially supported by the DST, Government of India [SB/YS/LS-252/2013(15 May 2014)] and DBT, Government of India [BT/PR14632/NNT/28/824/2015], which is greatly acknowledged.