Flax (Linum usitatissimum L.) or the Linseed, a cultivated food cum fibre crop well known in the textile industry, comes from the Linaceae family. Flaxseeds have a major significance in history since their first domestication in the Fertile Crescent region and are well known for their high nutritional and medicinal values. Flax seeds contain more than 70% of alpha-linolenic acid (ALA), an omega3 essential fatty acid that is known to suppress the proliferation of several cancer cell lines, along with certain unsaturated fatty acids such as oleic, linoleic and linolenic acids. Flaxseed oil (FSO) is looked up for the treatment of various ailments including rheumatoid arthritis, high cholesterol level, osteoarthritis, anxiety, vaginal infections, atherosclerosis, benign prostatic hyperplasia (BPH), attention deficit-hyperactivity disorder (ADHD) and so on. It is usually extracted from the flaxseeds by press and solvent extraction methods and was used for research purposes in the past years in the medicinal, pharmaceutical, and food industry. In the present study, the flaxseeds were powdered using a grinder and the FSO was extracted using a Clevenger apparatus by the hydro-distillation method. A comprehensive analysis of the phytochemical composition and alimental properties revealed that flaxseeds may have anti-cancer effects when consumed regularly as part of one's diet.

1. An Overview of Flax Seeds –
Phytochemical Profile,
Extraction &
Dietary Benefits
Amal Dhivahar S. and Dr. V. M. Berlin Grace.
Karunya University
D e p a r t m e n t o f B i o t e c h n o l o g y

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Summary
Keywords: Flax seeds; alpha-linolenic acid (ALA); hydro-distillation method; phytochemical
composition; dietary benefits;anti-cancer effects.
2
Flax (Linum usitatissimum L.) or the Linseed, a cultivated food cum fibre
crop well known in the textile industry, comes from the Linaceae family. Flaxseeds have a
major significance in history since their first domestication in the Fertile Crescent region
and are well known for their high nutritional and medicinal values. Flax seeds contain
more than 70% of alpha-linolenic acid (ALA), an omega3 essential fatty acid that is known
to suppress the proliferation of several cancer cell lines, along with certain unsaturated
fatty acids such as oleic, linoleic and linolenic acids. Flaxseed oil (FSO) is looked up for the
treatment of various ailments including rheumatoid arthritis, high cholesterol level,
osteoarthritis, anxiety, vaginal infections, atherosclerosis, benign prostatic hyperplasia
(BPH), attention deficit-hyperactivity disorder (ADHD) and so on. It is usually extracted
from the flaxseeds by press and solvent extraction methods and was used for research
purposes in the past years in the medicinal, pharmaceutical, and food industry. In the
present study, the flaxseeds were powdered using a grinder and the FSO was extracted
using a Clevenger apparatus by the hydro-distillation method. A comprehensive analysis
of the phytochemical composition and alimental properties revealed that flaxseeds may
have anti-cancer effects when consumed regularly as part of one's diet.

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Introduction
Significance of the study.
3

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Background
» Lignans are known to be the main components in
Linum species and studies have been made by
extracting this phytochemical from flaxseeds to
evaluate their biological effects on breast cancer cell
lines (Al-jumaily et al., 2012, Ansenberger at al., 2010,
Long et al., 2011).
» The oil extracted from flaxseeds were also found to
induce apoptosis and disrupt mitochondrial function in
B16-BL6 murine melanoma and MCF-7 breast cancer
cells (Buckner et al., 2019).
4
» It was looked up for the
treatment of a range of
ailments (Oomah, 2001):
 rheumatoid arthritis,
 high cholesterol level,
 osteoarthritis,
 anxiety,
 vaginal infections,
 atherosclerosis,
 benign prostatic
hyperplasia (BPH),
 attention deficit-
hyperactivity disorder
(ADHD).

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Extracts
» Simple Extraction process.
» Used for other purposes also such as cooking,
perfumes or as a part of a medicine.
» Has more of the carrier solvent like alcohol.
5
Comparison
Essential Oil
» More concentrated in nature and has pure volatile
compounds.
» Complex Extraction Process.
» Generally used for therapeutic purposes.
» Have profound effects on the body.

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ChemicalComposition(g/100gof flaxseed)
22.8
5.9
7.3
1.3
2.1
6
Alpha Linolenic Acid
(ALA)
Linolenic Acid
Palmitic Acid
Oleic Acid
Stearic Acid
Bernacchia et. al, 2014

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AlphaLinolenicAcid(ALA)
7
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.

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Extraction
of the Essential oil
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EssentialOilExtraction
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.
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PreliminaryTests
Phytochemical tests for the presence of fats and oils and
chromatography (Kenwat et al., 2014)
» Grease spot test
» Solubility test
» Hubl’s Test
» Thin Layer Chromatography
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Flaxseeds and the extracted
oil

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» A drop of sample was spotted on the butter paper to
check the translucence.
» In butter paper, the spot with sample was seen
translucent indicating the presence of oil.
Greasespottest
For the presence of fat in essential oil.
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» The sample was added to polar (ethanol and distilled
water) as well as nonpolar (hexane) solvents.
» The Essential oil was soluble in
(A) hexane
but insoluble in
(B) Water and
(C) Ethanol
Solubilitytest
To check the solubility.
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» In a test tube, 3ml of chloroform was added with four
drops of the essential oil sample and it was shaken
well.
» freshly prepared Hubl’s reagent (5% Iodine and 7%
Mercuric Chloride were added to 99% Ethanol) was
added.
» (A) Negative Control (water) doesn’t show fading of
purple color.
» But (B) Sample and (C) Positive Control (Olive oil)
shows the faded purple color indicating the presence of
unsaturated fatty acid.
Hubl’stest
To find presence of saturated and unsaturated oil
determined by the fading of violet color
(Gupta et al., 1994).
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» Stationary phase: A thin strip of aluminum coated
with silica gel.
» Mobile phase: Hexane.
» The solvent was allowed to reach a height of about
3/4th of the strip and then viewed underTLC - UV
cabinet.
ThinLayerChromatography(TLC)
To indicate the presence of phytochemical compounds.
14
Extracted
oil
Commercial
oil

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Phytochemical Analysis 15

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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
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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
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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
18
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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HealthBenefits
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Compound Health Benefits
Omega 3-fatty acids Anti-ulcer activity, anti-secretory effect , reno-protection in lupus nephritis, anti-
anti-atherogenic effect, CVD prevention, Decrease blood pressure.
Proteins Neurodegenerative disease prevention, Control blood pression, Control blood
blood pression, Influence on diabete mellitus, Anti-hypertensive properties.
Dietary fibres Hunger suppression, Decrease total cholesterol in blood.
Micronutrients (vitamin E and minerals) Antioxidant properties, Decrease blood pressure, Promotion of sodium excretion
Lignans Control on hypertension, Protection against cancer and diabetes, Control on
dyslipidemia, Reduce breast cancer growth, Effects on postmenopausal women
women symptons.
Bernacchia et al., 2014

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Why Flax is
Anti-proliferative?
The Molecular Mechanisms
Cancerdevelopmentischaracterizedby:
*Specificcellulartransformations
involving,
* ChangesinProliferationrates
* Inactivationof tumor-suppressorgenes
* Inhibitionof Apoptosis
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Carrillo et al., 2012

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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 LTB4 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; LTB4 - Leukotriene B4 ; TNF-α -Tumor Necrosis Factor-alpha; IL-1β -
Interleukin-1 beta; PPAR - Peroxisome Proliferator-Activated Receptor.
ALA-Mechanism

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Oleicacid-Mechanism
OA – cellular
fuel
Deplete ER Ca2+
stores
Release of Ca2+
Chromatin
fragmentation
Alter the
intracellular Ca2+
homeostasis
Eg: OA reduced
catalase activity
in human
leukemia cell
lines
Reduction in anti-
oxidant Enzyme
activities
Mediated by
Caspase-3
activity
increased
Mito. Membrane
potential
collapse
Mito.
dysfunction
Production of
ROS
Disrupts the redox state of
cells
Apoptosi
s

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23
Phytochemicals
Cell Membrane
Affects
Membrane
Fluidity
Anti-
inflammatory
Activity
Inhibition of
Platelet
Aggregation
Anti-Proliferative
Actions
Anti-cancer
Activity
AnticancerActivity–ThePossibilities

24. Thank You
Amal Dhivahar S.
8524946294
amaldhivahars@gmail.com
Karunya University
D e p a r t m e n t o f B i o t e c h n o l o g y
http://karunya.edu/biotechnology