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

Poster Session

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

TWAS 8th Annual Meeting

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