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Cytotoxic Activity of
Linum usitatissimum L. Essential oil
against Lung Adenocarcinoma Cell Line
and its Phytochemical Analysis.
Amal Dhivahar S., Dr. V. M. Berlin Grace and Srividya S.
Department of Biotechnology,
School of Agriculture and Biosciences.
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Abstract
Keywords: Flaxseed Essential Oil;
Hydro-distillation; GC-MS analysis;
In vitro Cancer assays; Migration assay;
Apoptosis induction.
The Essential oils of numerous medicinal plants are
reported to have significant anti-cancer effects due to the presence of
active phytochemicals. Flax seeds (Linum usitatissimum L.) are found
to be a rich source of alpha-linolenic acid (ALA), an essential fatty
acid, which is a precursor to several omega-3 fatty acids, claimed to
have lipid-lowering properties. It is also known to have enriched with
certain unsaturated fatty acids such as oleic, linoleic and linolenic
acids and relatively low glucosinolate content. In this study, we have
extracted the essential oil from flaxseeds (FEO) through the Hydro-
distillation method and analyzed its anti-cancer effects on A549
human lung adenocarcinoma cell line. The GC-MS analysis indicated
the presence of various phytochemical compounds with antitumor
properties such as hexadecanoic acid, cis - vaccenic acid, and
isopropyl myristate. The Trypan blue and MTT assays conducted with
concentrations ranging from 0.04 to 0.20 V/V% provided IC50 values
of 0.11 and 0.16 V/V% respectively for cancer cell line growth
inhibition. The cell line migration was also monitored using the
wound-scratch assay with essential oil concentrations of 0.16 and
0.20 V/V% at the 24th hour. The essential oil could efficiently prevent
the rapid migration of lung cancer cells when observed under an
inverted microscope. Thus, the flaxseed essential oil treatment on
A549 cancer cells exhibited a dose-dependent decrease in their
viability and caused apoptosis induction. Therefore, flaxseeds have a
greater potency to treat lung cancer cells and this work also proposes
further in vivo studies to establish this natural medication as an
eminent anti-cancer drug.
2
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Introduction
Why Flax?
Treatment:
• Rheumatoid arthritis,
• High cholesterol level,
osteoarthritis,
• Anxiety
• Vaginal infections,
• Atherosclerosis,
• BPH
• ADHD.
Why Essential Oil?
• Lignans – surplus
studies exist
• Essential oil - now
considered in treating
many disorders
including cancers
• they contain
significant volatile
phytochemicals
Why A549?
Lung carcinoma:
• accounts for more than
30% of the cancer-related
deaths globally of both
men and women.
• Cancer cell lines - a
convenient representation
of the traits and aspects
of cancer.
3
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Chemical Composition (g/100g of flaxseed)
22.8
5.9
7.3
1.3
2.1
4
Alpha Linolenic Acid (ALA)
Linolenic Acid
Palmitic Acid
Oleic Acid
Stearic Acid
Bernacchia et. al, 2014
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Alpha Linolenic Acid
(ALA)
Essential Fatty Acid
a precursor to a number of
omega-3 fatty acids, claimed to
have lipid-lowering properties
(Williams et al., 2007).
Adds value to Flaxseeds
high anti-inflammatory
and disease-resisting
property.
Prevention & Treatment
Effects
• High blood pressure and cholesterol.
• Multiple sclerosis (MS),
• Rheumatoid arthritis (RA),
• Ulcerative colitis,
• Lupus,
• Diabetes,
• Crohn’s disease, etc.
5
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Overview
Extraction
By Hydro-distillation using
a Clever Apparatus.
Trypan Blue Assay
Dye Exclusion Assay to
Test Cell Viability.
MTT Assay
Colorimetric Assay for
assessing Cell metabolic
activity.
Wound-Scratch
Migration Assay
To observe the impact of
the drug on the cell line
migration.
GC-MS Analysis
An Analytical method to
identify the various
components of a test
sample.
[South India Textile
Research Association
(SITRA), Coimbatore] 6
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Essential Oil Extraction
From Flaxseeds by Hydro-distillation
Step 1
Grinder
• The flax seeds were
powdered using a
grinder.
• 25g of the powdered
seed sample was packed
in a pouch and was set
for extraction.
Step 2
Clevenger Apparatus
• Distillation by
 boiling,
 condensing and
 decantation to separate
the oil.
Step 3
Hydro-distillation
• Essential oil extraction
using elevated pressure.
• The essential oil droplets
were collected and then
stored in a refrigerator.
7
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Calculation
Trypan Blue Assay:
% Viability =
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 4 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
(𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 4 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 + 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 4 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
𝑥 100
MTT Assay:
• % Inhibition = [(OD control – OD sample) / OD control] ×100
• % viability = 100 - % Inhibition.
8
Statistical Analysis: The results are shown as mean ± SD for the all of representative experiments. Multiple
comparisons between the experimental groups were distinguished by one-way ANOVA. The level of significance was
set at p < 0.05.
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Trypan Blue Assay
The %viability values are expressed in mean ± SD for triplicates of the cell line.
S.No. Concentration EEO (V/V%) %Viability ± SD
1 Untreated 96.50±1.73
2 0.04 89.69±4.32
3 0.08 77.72±9.42
4 0.12 48.40±4.19
5 0.16 26.06±2.25
6 0.2 11.04±2.73
0
20
40
60
80
100
120
0.00 0.04 0.08 0.12 0.16 0.20
%Viability
Conc. V/V%
9
Strober, 2015
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MTT Assay
Concentration vs % Inhibition values expressed in mean ± SD for triplicates of the cell line
S.No. Concentration EEO (V/V%) %Inhibition @570nm
1 0.04 21±4.59
2 0.08 22±6.91
3 0.12 27±6.54
4 0.16 46±3.36
5 0.2 69±2.73 0
10
20
30
40
50
60
70
80
0.04 0.08 0.12 0.16 0.20
%Inhibition
Conc. V/V%
10
Chung et al., 2015
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Wound-Scratch Migration Assay
At 20X magnification
captured using a
stereomicroscope.
11
She et al., 2017
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Phytochemical Profile
Various compounds identified in the essential oil
by GC-MS analysis.
12
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SN Compound name Common name Molecular weight Molecular formula Area% % p Medicinal effects
1. Hexadecanoic acid, 2,3-dihydroxy
propyl ester
Glyceryl
Palmitate 330
C19H38O4
0.18 79.95
Anti-proliferative effects
2. 9-Octadecenoic acid (Z) Oleic acid 282 C18H34O2
1.8 10.34 Apoptotic activity,
Anti-inflammatory and cancer preventive
3. Hexadecanoic acid Palmitic acid 256 C16H32O2
0.10 9.7 Antitumor, Antioxidant and
Anti-androgenic
4. Tetradecanoic acid, methyl ester Methyl myristate 242 C15H30O2
0.75 29.58 Anticancer activity
5. 14-á-H-PREGNA - 288 C21H36
0.49 36.15 Antifungal, Antitumor and Antibacterial
6. .psi.,.psi.-Carotene, 1,1',2,2'-
tetrahydro-1,1'-dimethoxy Lycopene 600
C42H64O2
0.06 44.83
Adulticidal, Repellent, Antibacterial,
Antifungal,
Anti-inflammatory and Anticancer
7. Tetradecanoic acid,
ethyl ester
Ethyl myristate 256 C16H32O2
0.57 13.73 Antitumor activity
13
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SN Compound name Common name Molecular weight Molecular formula Area% % p Medicinal effects
8. Tetradecanoic acid Myristic acid 228 C14H28O2
0.06 16.01 Anticancer activity and Antioxidant activity
9. Methyl-9,9,10,10-d4-octa-
decanoate
- 298 C19H34D4O2
0.57 25.49 Antioxidant and Anti-diabetic activity
10. Hexadecanoic acid,
methyl ester
Methyl palmitate 270 C17H34O2
0.57 11.21 Anti-inflammatory, cancer preventive and
antiacne
11. Tridecanoic acid,
12-methyl-, methyl ester
Methyl iso-
myristate
242 C15H30O2
0.75 12.58 Anticancer
12. Octadecanoic acid,
methyl ester
Methyl stearate 298 C19H38O2
0.45 10.63 Anticancer activity
13. Hexadecanoic acid,
2-hydroxy-1,3-propanediyl ester
Glycerol 1,3-
dihexa-decanoate
568 C35H68O6
0.19 21.14 Carbohydrate metabolism and Antitumor
activity.
14. Cis- Vaccenic acid Vaccenic acid 282 C18H34O2
1.70 4.98 Hypolipidaemic, Antihypertensive and
Antitumor activity
15. Palmitoyl chloride Palmitic acid
chloride
274 C16H31ClO 0.22 9.28 Anticancer
14
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SN Compound name Common name Molecular weight Molecular formula Area% % p Medicinal effects
16. Isopropyl myristate Bisomel 270 C17H34O2
1.80 6.08 Antitumor and Anti-trypansomal activity
17. Oleic acid, eicosyl ester Zicosyl oleate 562 C38H74O2
0.10 5.82 Anti-inflammatory,
Cancer preventive and Hypocholesterolemic
18. Dotriacontane n-Dotriacontane 450 C32H66
0.24 5.59 Antimicrobial and Antioxidant activity
19. 2,6,10,15,19,
23-Hexamethyl tetracosa-
2,6,10,14,18,
22 - hexaene
Squalene 410 C30H50
0.23 11.6 Antioxidant, Possesses carcinoma protective
and cardio-protective properties
15
Ads et al., 2017, Bhattacharya et al., 2014, Bo et al., 2014,
Cooper and Yao, 2012, Gnanavel and Mary, 2013, Hoet at al., 2006,
Mebude and Adeniyi, 2017, Omoruyi et al., 2014,
Paramanantham and Murugesan, 2014, Passalacqua et al., 2013,
Qing-Zhi et al., 2008, Rajalakshmi and Mohan, 2016,
Saikala and Chandra, 2014, Sheela and Uthayakumari, 2013.
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ALA – A Plant
derived Essential
FA.
Humans
Other Omega3
FA
EPA DHA
PRECURSOR
Series 3-
thromboxanes
Series 5-
prostaglandins
Series 3-
prostaglandins
Anti-inflammatory
& Anti-
atherogenic
Properties
Other Metabolites
Synthesiz
e
Pro-inflammatory
eicosanoids
Pro-inflammatory
cytokines
PGE2 LTBE TNF-α IL-1β
Inhibits the
Production
Transcriptome
Factors
NF-kappa B
Members of the PPAR
Family
Modulate several Gene
Expressions
FA
Metabolism
Inflammatio
n
Affect
EPA - Eicosapentaenoic Acid; DHA - Docosahexaenoic Acid; PGE2 -
Prostaglandin E2; LTBE - Leukotriene B4 ; TNF-α -Tumor Necrosis
Factor-alpha; IL-1β - Interleukin-1 beta; PPAR - Peroxisome
Proliferator-Activated Receptor
ALA - Mechanism
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ALA &
Metabolites.
Cell Membrane
Affects
Membrane
Fluidity
Anti-
inflammatory
Activity
Inhibition of
Platelet
Aggregation
Anti-Proliferative
Actions
Anti-cancer
Activity
ALA – Anticancer Activity
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Conclusion
“The Study encourages flaxseed supplementation
on diet because of it’s high nutritive content for
the prevention of malignant diseases and
proceeds with further analyses & in vivo studies”.
• We have observed the cytotoxicity of the
flaxseed essential oil on treating human lung
cancer cells based on the results of MTT assay &
Trypan blue assay.
• The cancer cell migration inhibition effect of the
essential oil also was shown in our study in
wound scratch assay.
• The extracted essential oil from Linum
usitatissimum L. - screened by GC-MS for its
phytochemicals, showed the presence of many
unsaturated fatty acids such as oleic acid, with
anticancer properties.
18
Thank You
Amal Dhivahar S.
8524946294
amals@karunya.edu.in

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Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adenocarcinoma Cell Line and its Phytochemical Analysis

  • 1. Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adenocarcinoma Cell Line and its Phytochemical Analysis. Amal Dhivahar S., Dr. V. M. Berlin Grace and Srividya S. Department of Biotechnology, School of Agriculture and Biosciences.
  • 2. Your Logo or Name Here Abstract Keywords: Flaxseed Essential Oil; Hydro-distillation; GC-MS analysis; In vitro Cancer assays; Migration assay; Apoptosis induction. The Essential oils of numerous medicinal plants are reported to have significant anti-cancer effects due to the presence of active phytochemicals. Flax seeds (Linum usitatissimum L.) are found to be a rich source of alpha-linolenic acid (ALA), an essential fatty acid, which is a precursor to several omega-3 fatty acids, claimed to have lipid-lowering properties. It is also known to have enriched with certain unsaturated fatty acids such as oleic, linoleic and linolenic acids and relatively low glucosinolate content. In this study, we have extracted the essential oil from flaxseeds (FEO) through the Hydro- distillation method and analyzed its anti-cancer effects on A549 human lung adenocarcinoma cell line. The GC-MS analysis indicated the presence of various phytochemical compounds with antitumor properties such as hexadecanoic acid, cis - vaccenic acid, and isopropyl myristate. The Trypan blue and MTT assays conducted with concentrations ranging from 0.04 to 0.20 V/V% provided IC50 values of 0.11 and 0.16 V/V% respectively for cancer cell line growth inhibition. The cell line migration was also monitored using the wound-scratch assay with essential oil concentrations of 0.16 and 0.20 V/V% at the 24th hour. The essential oil could efficiently prevent the rapid migration of lung cancer cells when observed under an inverted microscope. Thus, the flaxseed essential oil treatment on A549 cancer cells exhibited a dose-dependent decrease in their viability and caused apoptosis induction. Therefore, flaxseeds have a greater potency to treat lung cancer cells and this work also proposes further in vivo studies to establish this natural medication as an eminent anti-cancer drug. 2
  • 3. Your Logo or Name Here Introduction Why Flax? Treatment: • Rheumatoid arthritis, • High cholesterol level, osteoarthritis, • Anxiety • Vaginal infections, • Atherosclerosis, • BPH • ADHD. Why Essential Oil? • Lignans – surplus studies exist • Essential oil - now considered in treating many disorders including cancers • they contain significant volatile phytochemicals Why A549? Lung carcinoma: • accounts for more than 30% of the cancer-related deaths globally of both men and women. • Cancer cell lines - a convenient representation of the traits and aspects of cancer. 3
  • 4. Your Logo or Name Here Chemical Composition (g/100g of flaxseed) 22.8 5.9 7.3 1.3 2.1 4 Alpha Linolenic Acid (ALA) Linolenic Acid Palmitic Acid Oleic Acid Stearic Acid Bernacchia et. al, 2014
  • 5. Your Logo or Name Here Alpha Linolenic Acid (ALA) Essential Fatty Acid a precursor to a number of omega-3 fatty acids, claimed to have lipid-lowering properties (Williams et al., 2007). Adds value to Flaxseeds high anti-inflammatory and disease-resisting property. Prevention & Treatment Effects • High blood pressure and cholesterol. • Multiple sclerosis (MS), • Rheumatoid arthritis (RA), • Ulcerative colitis, • Lupus, • Diabetes, • Crohn’s disease, etc. 5
  • 6. Your Logo or Name Here Overview Extraction By Hydro-distillation using a Clever Apparatus. Trypan Blue Assay Dye Exclusion Assay to Test Cell Viability. MTT Assay Colorimetric Assay for assessing Cell metabolic activity. Wound-Scratch Migration Assay To observe the impact of the drug on the cell line migration. GC-MS Analysis An Analytical method to identify the various components of a test sample. [South India Textile Research Association (SITRA), Coimbatore] 6
  • 7. Your Logo or Name Here Essential Oil Extraction From Flaxseeds by Hydro-distillation Step 1 Grinder • The flax seeds were powdered using a grinder. • 25g of the powdered seed sample was packed in a pouch and was set for extraction. Step 2 Clevenger Apparatus • Distillation by  boiling,  condensing and  decantation to separate the oil. Step 3 Hydro-distillation • Essential oil extraction using elevated pressure. • The essential oil droplets were collected and then stored in a refrigerator. 7
  • 8. Your Logo or Name Here Calculation Trypan Blue Assay: % Viability = 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 4 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 (𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 4 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 + 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 4 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑥 100 MTT Assay: • % Inhibition = [(OD control – OD sample) / OD control] ×100 • % viability = 100 - % Inhibition. 8 Statistical Analysis: The results are shown as mean ± SD for the all of representative experiments. Multiple comparisons between the experimental groups were distinguished by one-way ANOVA. The level of significance was set at p < 0.05.
  • 9. Your Logo or Name Here Trypan Blue Assay The %viability values are expressed in mean ± SD for triplicates of the cell line. S.No. Concentration EEO (V/V%) %Viability ± SD 1 Untreated 96.50±1.73 2 0.04 89.69±4.32 3 0.08 77.72±9.42 4 0.12 48.40±4.19 5 0.16 26.06±2.25 6 0.2 11.04±2.73 0 20 40 60 80 100 120 0.00 0.04 0.08 0.12 0.16 0.20 %Viability Conc. V/V% 9 Strober, 2015
  • 10. Your Logo or Name Here MTT Assay Concentration vs % Inhibition values expressed in mean ± SD for triplicates of the cell line S.No. Concentration EEO (V/V%) %Inhibition @570nm 1 0.04 21±4.59 2 0.08 22±6.91 3 0.12 27±6.54 4 0.16 46±3.36 5 0.2 69±2.73 0 10 20 30 40 50 60 70 80 0.04 0.08 0.12 0.16 0.20 %Inhibition Conc. V/V% 10 Chung et al., 2015
  • 11. Your Logo or Name Here Wound-Scratch Migration Assay At 20X magnification captured using a stereomicroscope. 11 She et al., 2017
  • 12. Your Logo or Name Here Phytochemical Profile Various compounds identified in the essential oil by GC-MS analysis. 12
  • 13. Your Logo or Name Here SN Compound name Common name Molecular weight Molecular formula Area% % p Medicinal effects 1. Hexadecanoic acid, 2,3-dihydroxy propyl ester Glyceryl Palmitate 330 C19H38O4 0.18 79.95 Anti-proliferative effects 2. 9-Octadecenoic acid (Z) Oleic acid 282 C18H34O2 1.8 10.34 Apoptotic activity, Anti-inflammatory and cancer preventive 3. Hexadecanoic acid Palmitic acid 256 C16H32O2 0.10 9.7 Antitumor, Antioxidant and Anti-androgenic 4. Tetradecanoic acid, methyl ester Methyl myristate 242 C15H30O2 0.75 29.58 Anticancer activity 5. 14-á-H-PREGNA - 288 C21H36 0.49 36.15 Antifungal, Antitumor and Antibacterial 6. .psi.,.psi.-Carotene, 1,1',2,2'- tetrahydro-1,1'-dimethoxy Lycopene 600 C42H64O2 0.06 44.83 Adulticidal, Repellent, Antibacterial, Antifungal, Anti-inflammatory and Anticancer 7. Tetradecanoic acid, ethyl ester Ethyl myristate 256 C16H32O2 0.57 13.73 Antitumor activity 13
  • 14. Your Logo or Name Here SN Compound name Common name Molecular weight Molecular formula Area% % p Medicinal effects 8. Tetradecanoic acid Myristic acid 228 C14H28O2 0.06 16.01 Anticancer activity and Antioxidant activity 9. Methyl-9,9,10,10-d4-octa- decanoate - 298 C19H34D4O2 0.57 25.49 Antioxidant and Anti-diabetic activity 10. Hexadecanoic acid, methyl ester Methyl palmitate 270 C17H34O2 0.57 11.21 Anti-inflammatory, cancer preventive and antiacne 11. Tridecanoic acid, 12-methyl-, methyl ester Methyl iso- myristate 242 C15H30O2 0.75 12.58 Anticancer 12. Octadecanoic acid, methyl ester Methyl stearate 298 C19H38O2 0.45 10.63 Anticancer activity 13. Hexadecanoic acid, 2-hydroxy-1,3-propanediyl ester Glycerol 1,3- dihexa-decanoate 568 C35H68O6 0.19 21.14 Carbohydrate metabolism and Antitumor activity. 14. Cis- Vaccenic acid Vaccenic acid 282 C18H34O2 1.70 4.98 Hypolipidaemic, Antihypertensive and Antitumor activity 15. Palmitoyl chloride Palmitic acid chloride 274 C16H31ClO 0.22 9.28 Anticancer 14
  • 15. Your Logo or Name Here SN Compound name Common name Molecular weight Molecular formula Area% % p Medicinal effects 16. Isopropyl myristate Bisomel 270 C17H34O2 1.80 6.08 Antitumor and Anti-trypansomal activity 17. Oleic acid, eicosyl ester Zicosyl oleate 562 C38H74O2 0.10 5.82 Anti-inflammatory, Cancer preventive and Hypocholesterolemic 18. Dotriacontane n-Dotriacontane 450 C32H66 0.24 5.59 Antimicrobial and Antioxidant activity 19. 2,6,10,15,19, 23-Hexamethyl tetracosa- 2,6,10,14,18, 22 - hexaene Squalene 410 C30H50 0.23 11.6 Antioxidant, Possesses carcinoma protective and cardio-protective properties 15 Ads et al., 2017, Bhattacharya et al., 2014, Bo et al., 2014, Cooper and Yao, 2012, Gnanavel and Mary, 2013, Hoet at al., 2006, Mebude and Adeniyi, 2017, Omoruyi et al., 2014, Paramanantham and Murugesan, 2014, Passalacqua et al., 2013, Qing-Zhi et al., 2008, Rajalakshmi and Mohan, 2016, Saikala and Chandra, 2014, Sheela and Uthayakumari, 2013.
  • 16. Your Logo or Name Here 16 ALA – A Plant derived Essential FA. Humans Other Omega3 FA EPA DHA PRECURSOR Series 3- thromboxanes Series 5- prostaglandins Series 3- prostaglandins Anti-inflammatory & Anti- atherogenic Properties Other Metabolites Synthesiz e Pro-inflammatory eicosanoids Pro-inflammatory cytokines PGE2 LTBE TNF-α IL-1β Inhibits the Production Transcriptome Factors NF-kappa B Members of the PPAR Family Modulate several Gene Expressions FA Metabolism Inflammatio n Affect EPA - Eicosapentaenoic Acid; DHA - Docosahexaenoic Acid; PGE2 - Prostaglandin E2; LTBE - Leukotriene B4 ; TNF-α -Tumor Necrosis Factor-alpha; IL-1β - Interleukin-1 beta; PPAR - Peroxisome Proliferator-Activated Receptor ALA - Mechanism
  • 17. Your Logo or Name Here 17 ALA & Metabolites. Cell Membrane Affects Membrane Fluidity Anti- inflammatory Activity Inhibition of Platelet Aggregation Anti-Proliferative Actions Anti-cancer Activity ALA – Anticancer Activity
  • 18. Your Logo or Name Here Conclusion “The Study encourages flaxseed supplementation on diet because of it’s high nutritive content for the prevention of malignant diseases and proceeds with further analyses & in vivo studies”. • We have observed the cytotoxicity of the flaxseed essential oil on treating human lung cancer cells based on the results of MTT assay & Trypan blue assay. • The cancer cell migration inhibition effect of the essential oil also was shown in our study in wound scratch assay. • The extracted essential oil from Linum usitatissimum L. - screened by GC-MS for its phytochemicals, showed the presence of many unsaturated fatty acids such as oleic acid, with anticancer properties. 18
  • 19. Thank You Amal Dhivahar S. 8524946294 amals@karunya.edu.in