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1. yassarnalquran.wordpress.com 1
‫الرحيم‬ ‫الرحمن‬ ‫هللا‬ ‫بسم‬
In the name of Allah, the Merciful, the Most Beneficent

2. ‘A thousand mile
journey begins with
a single step’
-Lao Tzu

3. PHYTOCHEMICAL
INVESTIGATION OF LAWSONIA
INERMIS LINN AND ITS
APPLICATIONS AS COLORANT
MAMOONA NIGHA
M.Phil. Applied Chemistry
2013-m.phil-app-chem-7
UNIVERSITY OF ENGINEERING AND TECHNOLOGY,
LAHORE
SUPERVISOR: DR. ABDUL GHAFFAR

4. CONTENTS
• Abstract
• Aims and Objectives
• Background
• Methods
• Results
• Conclusion
• Future Plan

5. Abstract
Lawsonia inermis Linn leaves (Henna Plant)
was investigated for:
• Phytochemical components
• Elemental analysis
• Essential oil
• Dyeing pigment (Lawsone)
And then, their
Proficiency of was
evaluated by antioxidant
and
antimicrobial/antibacterial
action.
Moreover,
Color combinations with
mordants, were made and
their properties was tested
on leather.

6. • To reveal antioxidant
and
antimicrobial/antibacteri
al potencies of essential
oil of Lawsonia Inermis
Linn leaves and their
extracts.
• To estimate whether the
metal content of
Lawsonia Inermis Linn
is within the
recommendation
ranges.
• Characterization of
lawsone, and its
applications on leather.
Aims and objectives

7. Background
• The use of plants for the healing
purposes cited around 3000 B. C, in
China, Egypt and subcontinent.
• In the subcontinent, the herbal medical
system is very popular which dates
about 800 B. C.
• The Ayurveda and Yunnan, which made
use of extract of plants for the curing of
diseases.
• In the background of the tremendous
benefits of medicinal plants, many
research institutes have been shown to
carry out work on plants and reveal the
miraculous powers present in them.

8. Methods
Extraction of Essential Oil
• Powdered leaves were
hydro distilled in the
modified Dean Stark
apparatus.
• The percent yield was
calculated based on the
dried weight of the plant
material and stored until
analysis.
• The oil was then subjected
for GC-MS analysis and for
antioxidant, and
antimicrobial activity.

9. Methods
Characterization of Essential Oil
Antioxidant activity was determined
using DPPH free radical scavenging
assay
Antimicrobial activity of essential oil
was determined by paper disc method.
Vancomycin was used as positive
control.
Test Pathogens
Menthicillin Resistant Styphylococcus
Aureus (MRSA ATTC-43300) and
Menthicillin Sensitive Styphylococcus
Aureus (MSSA ATTC-25923)

10. Methods
Phytochemical Investigation of crude extracts of powdered leaves in hexane,
toluene, THF, ethyl acetate, and ethanol
Quantitative and qualitative test for
Alkaloids
Flavonoids
Saponins
Tannins
Terpenoids
Phenolic Compounds
Steroids
Glycosides
Quantitative test for
Carbohydrates
Resins
Amino Acid
Fats

11. Methods
Elemental analysis
• Preparation of Ash
• Digestion of Ash
• Analysis through FAAS

12. Methods
Isolation and
Characterization
of Lawsone
• Isolation was
done by
maceration.
• Identification of
the Lawsone
was done by
TLC and FT-IR.

13. Methods
Antioxidant and Antibacterial Activity
• Antioxidant activity was evaluated through DPPH
free radical scavenging method
• Antibacterial activity was determined by Paper
Disc method using
Staphylococcus aureus ATCC 6538 and E. coil
ATCC 10240

14. Results (GC-MS spectra)

15. Results (chemical constituents of EO, GC-MS)
Sno# Compound Name Molecular Formula Molecular weight %composition Retention time
1 α- pinene C11H16 136 13.345 4.148
2 Eucalyptol C10H18O 154 18.141 5.453
3 Linalool C10H18O 154 10.527 7.118
4 Trans- pinocarveol C10H15O 151 0.811 7.919
5 α- terpineol C10H18O 154 1.205 8.417
6 2,6-octadien-1-ol C10H18O 154 3.195 9.784
7 Nerolic acid (2,6-octadienoic acid) C11H1802 182 1.408 10.683
8 2,6- octadien-1-ol, 3,7-dimethyl, acetate, (E) C12H20O2 196 5.636 11.678
9 α- caryophyllene C15H24 204 2.867 12.485
10 1,2-dimethoxy-4-[2-propenyl], benzene C11H14O2 178 2.129 12.909
11 1,6-octadien-3-ol, 3,7-dimethyl, formate C11H18O2 182 0.834 13.223
12 9-isopropyl-1-methyl-2-methylene-5-oxatricyclo [5,4,0,0
{3,8}] undecane
C15H24O 220 1.493 13.750
13 Durohydroquinone C10H14O2 166 7.856 14.465

16. Results (anti-oxidant activity of
EO)
20 µL 40 µL 60 µL 80 µL 100 µL
Essential oil 44 58 82 87 88
BHT 51 63 74 89 92
0
20
40
60
80
100
120
Essential oil BHT
10 µL 20 µL 30 µL 40 µL 50 µL
Essential oil 32 45 52 60 75
BHT 36 51 57 63 78
0
10
20
30
40
50
60
70
80
90
Essential oil BHT

17. Results (anti-microbial activity of
EO)
75
80
72
73
74
75
76
77
78
79
80
81
%inhibition
Microbial strains
Antimicrobial Activity
MRSAMSSA
Organ
ism
Concentr
ation of
Vancomy
cin
Inhibition
zone Of
essential oil ±
SD for 10.9µL
pure oil
Inhibition zone
Of essential oil
± SD for 22.6µL
pure oil
Inhibition zone
Of Vancomycin
± SD
MRSA 20µL 22mm ± 5.0 25mm ± 5.0 20mm ± 0.0
MSSA 40µL 11mm ± 5.0 15mm ± 5.0 20mm ± 0.0
Orga
nism
Concentr
ation of
Vancom
ycin
MIC values in
µL/smL
For 10.9µL
MIC values in
µL/mL
For 22.6µL
Vancomycin
MRSA 20µL 3.3 ± 0.6 11.4± 0.6 -
MSSA 40µL 4.6 ± 0.6 14.9± 0.6 -

18. Results (FAAS)
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Sodium Chromium Nickel Copper Potassium Cadmium Manganese Iron Zinc
Concentrationinppm
Metals present in leaves of Lawsonia inermis Linn

19. Results (Phytochemical analysis)
S.No Phytochemical
Constituents
Identification test Hexane Toluene THF Ethyl Acetate Ethanol
1 Alkaloids Wagner’s test _ + + + +
2 Flavonoids Alkaline reagent
test
_ _ + + +
3 Saponins Foam test _ + _ + +
4 Triterpinoids - _ + + _ +
5 Glycosides Killer Kilian’s test + - + + +
6 Steroids Salkowski test + _ + + +
7 Tannins Ferric chloride test _ + + _ +
8 Carbohydrates Benedict test + + + + +
9 Amino acids Biuret test _ _ _ _ _
10 Fats Spot test + + + - +
Saponification test + + +
11 Resins Turbidity test _ _ _ _ _

20. Results (Phytochemical analysis)
Compounds Hexane ± SD Toluene ± SD THF ± SD Ethyl Acetate ± SD Ethanol ± SD
Alkaloids - 18mg/L ± 2.02 22.42mg/L ± 0.28 24mg/L ± 1.44 22.16mg/L ± 0.28
Flavonoids - - 4.6mg/L ± 0.21 4.45mg/L ± 0.01 4.02mg/L ± 0.19
Saponins - 2.139mg/L ± 1.06 - 0.89mg/L ± 1.06
Triterpenoids - 0.1mg/L ± 0.08 - 0.272mg/L ± 0.05
Glycosides 0.158mg/L ± 0.19 - 2.365mg/L ± 0.36 2.818mg/L ± 0.62 1.65mg/L ± 0.05
Steroids 0.862mg/L ± 0.23 - 0.114mg/L ± 0.19 0.003mg/L ± 0.25 0.851mg/L ± 0.23
Tannins - 1.106mg/L ± 0.08 0.121mg/L ± 0.6 1.741mg/L ± 0.53
Total phenolic content 217mg/L 118mg/L 245mg/L 103mg/L 107mg/L

21. Results (FT-IR)

22. Results (FT-IR)

23. Results (TLC)
Solvent system Rf value Reported Rf
values
n – hexane: ethanol (6:4) 0.25 0.23
n – hexane: methanol (3:1) 0.85 0.79-0.85
ethanol: methanol: n – hexane (1:3:9) 0.38 0.39
toluene: ethyl acetate: acetic acid (8:1:1) 0.44 0.43
toluene: ethyl acetate: methanol: acetic acid (3:4:3:1) 0.71 0.73
chloroform: ethanol: acetic acid (4:5:1) 0.55 0.53
ethyl acetate: water: methanol (5:3:4) 0.23 0.23

24. Results (antioxidant activity of
extracts)
0
10
20
30
40
50
60
70
80
90
100
o 2.4 4.4 4.7 5.2
Percentageinhibition
Extracts in solvents of increasing polarities
0
10
20
30
40
50
60
70
80
90
100
20 µL 40 µL 60 µL 80 µL 100 µL
Percentageinhibition
BHT in solvents of increasing polarities

25. Results (antioxidant activity of
extracts)Calculations For hexane extract For toluene extract For THF extract For ethyl acetate extract For ethanol extract
Weight of dry 25mL flask at room temperature 66.90gm 75.46gm 59.76gm 73.72gm 56.65gm
Weight of 25mLflask+sample+solvent 92.14gm 116gm 112.6gm 109gm 98.91gm
Weight of antioxidant in sample 25.24gm 40.54gm 52.84gm 35.28gm 42.26gm
Weight of 25mL flask solvent evaporated 73.22gm 91.42gm 108gm 88gm 86.11gm
Weight of residue+25mL flask 73.66gm 91.45gm 108.04gm 88.03gm 86.16gm
Dry weight of 25mL flask-weight of
residue+25mL flask
6.76gm 15.99gm 48.28gm 14.31gm 29.51gm
%calculations 10% 21% 80% 19% 52%

26. Results (antibacterial activity of
extracts)
0
2
4
6
8
10
12
Against E. coli Blank against E. coli Aganinst S. aureus Blank against S. aureus
Hexane 1 0 4 0
Toluene 5 0 5 3
THF 10 3 3 2
Ethyl Acetate 4 0 9 1
Ethanol 9 4 12 5
INHIBITIONZONE
Hexane Toluene THF Ethyl Acetate Ethanol

27. Results (Formulation of Dye)
formulations Lawsonia Inermis
Linn extract
Beetroot
Extract
Oxalic acid Tartaric acid Citric acid Para- formaldehyde Ferrous sulphate Sodium picramate
L1 4 1 _ _ _ _ _ _
L1a 5 1
L1b 2 1
L1c 1 2
L2 4 1 _ 1 _ _ _ _
L2a 5 1 1
L2b 1 1 1
L2c 2 2 1
L3 4 1 1 _ _ _ _ _
L3a 5 1 1
L3b 1 1 1
L3c 2 2 1
L4 4 1 _ _ 1 _ _ _
L4a 5 1 1
L4b 1 1 1
L4c 2 2 1
L5 4 1 _ 1 _ 2 _ _
L5a 5 1 1 2
L5b 1 1 1 2
L5c 2 2 1 2
L6 4 _ _ _ _ _ _ 1
L6a 4 2
L6b 4 3
L6c 4 4
L6d 4 5
L7 2 _ _ _ _ _ 2 1
L7a 3 2 1
L7b 1 2 1
L7c 2 2 3

28. Results (formulation of dyes, pH
values)
formulations pH Formulations pH Formulations pH Formula
tions
pH
L1 5.90 L2 5.23 L3 5.80 L4 4.62
L1a 5.01 L2a 5.76 L3a 4.81 L4a 4.23
L1b 4.73 L2b 5.26 L3b 4.22 L4b 4.56
L1c 4.50 L2c 5.67 L3c 3.23 L4c 4.22
L5 4.68 L6 3.58 L7 3.98
L5a 4.51 L6a 3.23 L7a 3.64
L5b 4.50 L6b 3.80 L7b 3.30
L5c 4.48 L6c 2.94 L7c 2.95
L6d 2.81 L7d 2.81

29. Results (Fastness properties)
Dye Mordant Light
fastness
Wash
fastness
Rub fastness
Wet rubbing Dry rubbing
10% Without
Mordant
3 4 4 3
20% 3 4 4 3
10% Ferrous
sulfate
5 4-5 4 3-4
20% 5 5 4 4
10% Chrome 4 3-4 3-4 3-4
20% 4 4 4 4
10% Potassium
Permanganate
4-5 4 3 4
20% 5 4 3 4

30. Results (fastness properties)
0
1
2
3
4
5
6
1 2 3 4
Fastnessraye
Fastness Properties
Wash fastness Dry rubbing Wet rubbing Light fastness

31. Conclusion
The study revealed that essential oil and leaves
extract of Lawsonia inermis Linn consist of mainly
terpenoids, flavonoids and phenolic compounds. Due
to the presence of these compounds, it has high
antioxidant and antimicrobial/antibacterial activity.
The Lawsone from Lawsonia inermis Linn was
applied on leather for its coloring effect.

32. Applications and Future path
• The biological potencies of essential oil and
lawsone make them good ingredient to be use in
the future drugs against various diseases probably
skin infections.
• Based on the dyeing method and selection of the
mordants variety of shades can be obtained.
• Besides this dyeing processing cost is also low
than that of the synthetic dyes.
• So the usage of natural dye is of great significance
for the future of commercial dyeing industry.

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