In vitro Anti-oxidant and cytotoxic activities of Lawsonia inermis L. different extracts TWAS 8 th Annual Meeting Poster Session
In vitro Anti-oxidant and
cytotoxic activities of Lawsonia
inermis L. different extracts
TWAS 8th Annual Meeting
Poster Session
What are
they, why?
Free radicals
TWAS 8th Annual Meeting
Poster Session
Free radicals (FRs)
FRs are unstable molecules with an un-paired
electron and highly reactive.
To be stable, they take electron from other
molecules.
TWAS 8th Annual Meeting
Poster Session
Reactive
Oxygen
Species
(ROS)
Classification
Of FRs
Reactive
Nitrogen
Species
(RNS)
TWAS 8th Annual Meeting
Poster Session
Eg. Reactive Oxygen Species (ROS) and Reactive
Nitrogen Species (RNS).
Reactive oxygen species Reactive nitrogen species
Alkoxyl radicals RO• Nitric oxide radical NO•
Peroxyl radicals ROO• Nitrogen dioxide radicalNO2
Hydroxyl radical HO•
Superoxide anion radical O2•–
TWAS 8th Annual Meeting
Poster Session
Sources of free radicals
TWAS 8th Annual Meeting
Poster Session
Effect of free radicals • FRs damage the cell molecules as DNA,
Proteins, Lipids, potentially causing a variety of disorders, including diabetes mellitus,hypertension,cancer,Alzheimer, and aging of body
TWAS 8th Annual Meeting
Poster Session
Oxidative Stress
The oxidative stress leads to the over 200 disorders
including aging of body, degenrative disorders and
cancer .
ROS
Antioxidant
TWAS 8th Annual Meeting
Poster Session
TWAS 8th Annual Meeting
Poster Session
Can we slow down the oxidative process in our bodies?
TWAS 8th Annual Meeting
Poster Session
Anti-Oxidants What are Anti-Oxidants? Anti-oxidants are compounds which neutralize free radicals or prevents free radical.
R•, RO•, ROO•, O-2
1O2, -OH, H2O2,Cu, Fe Antioxidants
R•, RO•,
ROO•,
1O2, O-2,
-OH, H2O2,
Cu, Fe
TWAS 8th Annual Meeting
Poster Session
Antioxidants classification Natural antioxidants, e.g.
Tocopherols
Sesamol
Gossypol
Synthetic antioxidants, e.g.
Butylated Hydroxy Anisole (BHA)
Butylated Hydroxy Toluene (BHT)
Propyl Gallate (PG)
Tertiary Butyl Hydroquinone (TBHQ)
TWAS 8th Annual Meeting
Poster Session
Mechanism of Action
Preventive Antioxidants Antioxidant enzymes:
Superoxide dismutase.
Catalase.
Glutathione peroxidase.
Radical Scavenging Hydrogen donating antioxidants.
Iron chelating antioxidants.
TWAS 8th Annual Meeting
Poster Session
Hydrogen Donating Antioxidants
• Antioxidants donate a hydrogen to peroxy radicals of lipid or protein, which are mainly responsible for free radical chain reaction in biological system.
O H
C(CH3)3
OCH3
O
OCH3
C(CH3)3
.
H
.
TWAS 8th Annual Meeting
Poster Session
O
M
C
O
C H 2
O C
O
C H 2
N C H 2
C H 2
O
O N
C O
C H 2
C
O
C H 2
Ions – EDTA Complex Formation
Iron Chelating antioxidants
Iron chelators deactivate trace metals by the
formation of complex ion.
(EDTA)
TWAS 8th Annual Meeting
Poster Session
PMNS & MNCS
6 hours–few days
weeks to years
TWAS 8th Annual Meeting
Poster Session
VERO CELLS
Vero cells are lineages of cell used in cell culture
was isolated from kidney epithelial cells
extracted from an African green monkey
TWAS 8th Annual Meeting
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The general objectives of the study:
investigate the antioxidant properties of methanol and
chloroform extracts obtained from Lawsonia inermis in
vitro anti-Oxidation using high throughput screening
technique (HTS).
To measure the cyto-toxic effect by using Mono
Nuclear Cells (MNCs), Polymorph Nuclear Cells
(PMNs) and vero cell.
TWAS 8th Annual Meeting
Poster Session
TWAS 8th Annual Meeting
Poster Session
Plants material:
The plants under study were collected from Khartoum,
Sudan),identified and authenticated By the Dr:Waeil
Alsadeig & Dr: Haidar Abd Algadeer who are the
taxonomists of Medicenal and Aromatic Plants
Research Institute (MAPRI).
TWAS 8th Annual Meeting
Poster Session
Lawsonia inermis L.
Family: Lythraceae
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Poster Session
Methodology
Extraction of Plant:
leaves and bark of L.n was extracted by methanol
and chloroform, 50 mg of plant material was masurated
in 250 ml chloroform for 24 hrs at room temperature
with shaking, the supernatant was filtrated and the
solvent removed by the rotary evaporator.
Each residue was weighed and the yield percentage was
calculated.
TWAS 8th Annual Meeting
Poster Session
Antioxidant Assays
TWAS 8th Annual Meeting
Poster Session
Strategy of Antioxidants Screening
(DPPH)
Extract / compounds
Incubation 30 min
Measurement of absorbance 517 nm
96-well plate
CALCULATION RSA%
TWAS 8th Annual Meeting
Poster Session
In vitro antioxidant assays
DPPH radical scavenging Assay:
Diphenyl picryl hydrazyl (DPPH) is un stable free radical
that can accept an electron or hydrogen radical to
become stable molecule
Screening of plant exts, and stands (PG ) in Conc (500µg/mL)
was done in 96-well plate.
Preparation of DPPH in Conc (300 µM) ethanol.
Incubation of 96-well plate for 30 mentis.
Measurement of absorbance at 517nm using Multiskan
spectrum (thermo scientific).
Calculation of radical scavenging activity.
TWAS 8th Annual Meeting
Poster Session
Mechanism of Action of antioxidant
TWAS 8th Annual Meeting
Poster Session
Iron Chelating Assay:
Plant exts, and stand (EDTA) in Conc 500 µg/ml
was added in 96-well plate.
Ferrous sulphate in Conc (0.0625 mM) was added to the all samples. (test, cont and stand)
Absorbance was measured at 562 nm.
Ferrozine in Conc (5 mM) was added to all samples for Initiation of the reaction.
The plate was incubated at r,t for 10 mints
The absorbance was measured at 562 nm.
Calculation of chelating activities.
TWAS 8th Annual Meeting
Poster Session
TWAS 8th Annual Meeting
Poster Session
Cyto-toxicity Study
TWAS 8th Annual Meeting
Poster Session
Isolation of Phagocytes cells from Human Blood • Ten ml of venous Blood was collected
• 50 ml of Falcon tube were added to the Blood, the Ficoll paque and the HBSS-- in equal volumes.
• Incubation, transferred into another tube.
• Centrifugation, (MNCs) was appeared at the junction of two layers.
• resuspend pellet in 1 ml HBSS--.
• Store on ice, Check the viability by Trypan Blue
TWAS 8th Annual Meeting
Poster Session
In vitro susceptibility assays
• 5 mg/ml (5000ppm).
• The final volume in the wells was 100 µl.
• whereas untreated cells used as a negative controls .
Samples were taken for counting at 0, 1,2 and 3 hrs.
• For counting the samples were mixed with Trypan
blue in equal volume.
TWAS 8th Annual Meeting
Poster Session
MTT cytotoxicity test
• The final volume was 200 µl/mL
• 0.5% Tritonx (+ve).
• Cell suspension in a complete culture medium containing 7 × 106/ml.
• Incubate for 3 days.
• MTT stock (5 mg/mL) was prepared.
• read at 540 nm using microplate reader.
TWAS 8th Annual Meeting
Poster Session
Calculation and statistical analysis Calculation of RSA:
Where:
RSA=Radical Scavenging Activity. As=absorbance of DPPH and ferrozine Fe+2 complex in the presence of test sample. Ac=absorbance of control.
Statistical analysis:
All data were presented as means ± SEM (standard error of the means)
n=3. Statistical analysis for all the results was done using Microsoft Excel program.
TWAS 8th Annual Meeting
Poster Session
TWAS 8th Annual Meeting
Poster Session
Results DPPH radical scavenging activity of l. i L
0
10
20
30
40
50
60
70
MeoH L MeoH B CHcL3 L CHcL3 B PG
R S
A
of
DPP
H
.Methanolic extract of leaves and bark (MeoH L, and B. Methanol Leaves extract. Bark) CHcL3 L and B. Chloroform Leaves extract. Bark) PG, Propyl
gallate as standard.
TWAS 8th Annual Meeting
Poster Session
Iron chelating activity of Lawsonia inermis L.
0
20
40
60
80
100
120
MeoH L MeoH B CHcL3 L CHcL3 B EDTA
Iro
n c
hela
tin
g a
ctiv
ity
%
leaves, Bark and EDTA as standard.
TWAS 8th Annual Meeting
Poster Session
The effect of L. inermis on phagocytes
The Part of plant Type of phagocytes Type of solvents
MeoH CHCl3
Leaves
mortality % of PMNs/ppm 64.3/1000 68.2/250
mortality % of MNCs /ppm 40.9/250 79.8/250
Bark
mortality % of PMNs /ppm 74.6/250 74.6/500
mortality % of MNCs/ppm 75.4/250 92.3/250
TWAS 8th Annual Meeting
Poster Session
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Chloroform bark Methanol bark Chloroform leaf Methanol leaf
Ab
sorb
an
ce r
ead
ing
at
54
0 n
m
Part of plants extractet by differient solvents
5000 ppm
2500 ppm
1225 ppm
ctrl -ve
MTT reduction cytotoxic assay for evaluation
of plant extract
TWAS 8th Annual Meeting
Poster Session
The isolation of their most potent compounds for in
vivo pharmacological studies is highly recommended
None of the above extracts indicated no toxic effects
against vero cell & human cells at the doses tested
The methanolic and chloroform leave and bark
extracts of Lawsonia inermis had an antioxidant
activity and protective effect against free radicals.
TWAS 8th Annual Meeting
Poster Session
Medicinal and Aromatic Plants Research Institute(MAPRI)
. Medicinal and Aromatic Plants Research Institute (MAPRI)
TWAS 8th Annual Meeting
Poster Session
ACKNOWLEDGMENT
Medicinal and Aromatic Plants Research Institute (MAPRI)
TWAS 8th Annual Meeting
Poster Session