1.
Identification of apoptotic pathways in Neem extract
gold nanoparticles treated parasites using FACS.
Presented by
Bulet Kumar Gupta
MS pharma
Biotechnology
Supervisor: Dr. Murali Kumarasamy
Co-Supervisor: Dr. Abhishek Sahu

2.
The plant product show an important role in diseases prevention and treatment
through the enhancement of antioxidant activity. Inhibition of bacterial growth and
modulation of genetic pathways the therapeutics role of number of plants in
diseases management is still being enthusiastically researched due to their less side
effect and affordable properties. It has been accepted that drugs based on allopathy
are expensive and also exhibit toxic effect on normal tissues and on various
biological activities.
Neem leaves Neem fruits Neem flowers
Neem (Azadirachta Indica)
Introduction

3.
Nimbidol
Azadirone
Salannin
Gedunin
Azadirachtin
Nimbin
Nimbidol
Chemical constituents of Neem

4.
Antiviral
Anticancer
Anti-
leishmanial
Antidiabetic
Antibacterial
Antifungal
Pharmacological activities of Neem

5.
Review of Literature

6.
Review of Literature

7.
Review of Literature

8.
 Extraction of Neem contents
Preparation of gold nanoparticles using neem extract
Characterization of nanoparticles prepared using neem extract
Antibacterial activity analysis using S. aureus and E. coli
Antileishmanial activity analysis
In vitro assay of apoptosis in parasite
OBJECTIVES

9.
Synthesis of neem gold
nanoparticles
Checking the antibacterial activity
of neem gold nanoparticles at
different concentrations.
Checking the antileishmanial
activity of neem gold
nanoparticles
Identifying the apoptotic pathways of
leishmanial cells by treating with neem
gold nanoparticles

10.
PREPARATION OF NEEM LEAVES EXTRACT (GREEN SYNTHESIS PROCESS)
Take neem leaves
powder
Take 1gm of neem extract powder and
add 20 ml of water then vortex 30min
continuously, after that keep at room
temperature for overnight, centrifuge for
20 min. at 4500 RPM for 3times, after
that filtered and collect in falcon tube.
Take 1gm of neem extract powder and
add 20 ml of methanol then vortex for 30
min continuously, then keep at room
temperature for overnight, after that
centrifuge for 20 min. at 4500 RPM for 3
times, then filtered and collect in falcon
tube
Aqueous neem
extract
Methanolic
neem etract

11.
 It's soft, ductile, malleable, and a good heat and electrical conductor, as well as chemically incredibly tough.
 Metallic gold is immune to strong alkalis and acids, and it does not oxidise or burn in air, even when heated.
 Gold is a noble metal in the periodic table's group 1B, with an atomic number of 79 and a molecular weight of 197.
 Gold has three valence states: Au0, Au+, and Au3+.
Properties of Gold Nano-particles
 High surface area to volume ratio.
 Biocompatibility
 A ease in surface Functionalization with recognition probes(Antibodies and peptides).
 Easy surface modification with biocompatibility and suitable candidates for biosensors.
 Resistance to corrosion and oxidation.
 High electric current thermal conductivity.
 Chemical stability, high catalytic activity.
 Antimicrobial activity against wide diversity of micro-organisms.
This properties made me to choose Gold as a metal for synthesis of Nanoparticles.
Introduction of Gold Nano particles

12.
Application of Gold Nano-Particles
Bio-sensor
Drug
delivery
Catalyst
Antibacterial
activity
Gene
delivery
Bio-assay

13.
200ul Sodium carbonate
1ml Aq. Extract
40µl Chloroauric acid 500mM
1gm neem
powder in 20
ml
methanol/water
Shaking for ½
h & kept it in
room temp. for
6 h
Centrifugation
at 4500 rpm
Supernant is
taken
Synthesis of Neem-Gold Nanoparticles
During the biosynthesis
the color of the reaction
medium changed rapidly
from colorless to dark
brown on formation of the
gold nanoparticles.

14.
Aq. Powder Extract
Methanolic. leaf Extract
Ethanolic. leaf Extract
Methanolic. Powder Extract
Aq. Leaf Extract
UV Spectra of Leaf & Powder Extract

15.
CHARACTERIZATION OF NEEM LEAF AND POWDER EXTRACT BY UV SPECTROSCOPY
 To Perform this first we have taken Neem leaves and we crushed to make paste. Same method is followed to extract neem
contents from neem powder.
 Then we extracted the neem contents by methanol and water. After that we have to take reading by UV Spectrophotometer.
 In Neem leaf paste extract we are getting peak at between 600-700 nm this is because of chlorophyll pigment.
 Neem Powder extract and neem leaf paste are showing similar peak.

16.
Characterization of neem GNP by UV Spectra

17.
Characterization of Neem gold nanoparticles by UV spectroscopy
 For this we have to take 10 ml of water in conical flask and add 200 µl sodium carbonate for adjusting the pH 9.
 Then add 1ml of neem aqueous extract. keep this on the magnetic stirrer up to reaching 90°C temperature.
 Then add 40 µl of chloroauric acid solution of 500mM concentration. keep this for continuous stirring and check the
synthesis in UV.
 At 540 -560 nm the neem nanoparticle is synthesised. Then this synthesised nanoparticles is washed twice with water.

18.
Characterization of Neem aq. Extract, powder, and N-GNP by FT-IR Spectrum
O-H
(3400)
C-O
(2925)
O-H Str
(1647)
N-H
(2359)
C-H
(1395)
C=O
(1070)

19.
Characterization of Neem aq. Extract, powder, and N-GNP by FT-IR Spectrum
 FTIR spectrum was used to identify the functional group of the active components based on the peak value in the region of
Infra-red radiation.
 The results of FTIR peak values and functional groups were represented in the FTIR spectrum profile was illustrated in the
below figure.
 FTIR spectrum confirmed the presence of alcohol, phenol, alkanes, alkyl halide, amino acids, carboxylic acid, aromatic, amines
in the leaves and seeds of the medicinal plants taken.
 The intense bands occurring at 3402.45 cm-1, 2924.35 cm-1, 2359.59 cm-1, 1647.37 cm-1, 1395.52 cm-1, 1070.55 cm-1, and
670.53 cm-1 corresponding to O-H / C-O str/ N-H / O-H str/ C-H/ C=O stretching, bending, vibrations respectively indicate the
presence of alcohol, phenol, amines, amides, carboxylic group, ester, ether, amino acids group in leaves of Azadirachta indica
(Neem).

20.
Characterization of neem GNP by DLS (Size)

21.
 For this I have taken 1 ml diluted sample in the DLS tube and take the readings.
 Then I found neem gold nanoparticle diameter is 92nm. Polydispersity index (PDI) which gives measure of the
distribution of molecular mass in a given sample is found to be 0.20.
Characterization of neem GNP by DLS (Size)
 Dynamic Light Scattering (DLS) is an established and precise measurement technique for
characterizing particle sizes in suspensions and emulsions.
 It is based on the Brownian motion of particles - this states that smaller particles move faster,
while larger ones move slower in a liquid.
 Dynamic light scattering enables the analysis of particles in a size range from 0.3 nm to 10000
nm.
DLS Result

22.
Characterization of Neem GNP by DLS (Zeta Potential)

23.
Characterization of neem GNP by DLS (Zeta Potential)
 Zeta Potential Analyzer is a user-friendly system for colloidal, nanoparticulate and macro molecular
characterization. It can determine particle zeta potential (related to the magnitude of the electrical
charge at the particle surface) and molecular weight of large polymeric substances dispersed in water
 Zeta potential is a measure of the effective electric charge on the nanoparticle's surface,
quantifying the charges.
 The magnitude of the zeta potential provides information about particle stability. The higher the
magnitude of potential exhibits increased electrostatic repulsion and therefore increased stability.
 Zeta potential measure the charge distribution of whole particles; where negative charge indicates the
more stability of nano particles.
 Here we found the -29.2Mv surface charge with single peak which clearly reflex the uniformity of
particles with the high stability.
Zeta Potential Result
Zeta Potential sample cell

24.
 This experiment is conducted for checking whether Neem GNP is inhibiting harmful bacterial species or not.
 For this first we have to culture E. coli and for this culture we have to add neem GNP of different concentrations.
 Then this is kept in incubator, readings are taken for every 4 hrs. till 30 hrs. Here I found 113.3µg/ml is showing more inhibition than
5.6µg/ml of Neem GNP.
Antibacterial activity of neem GNP
Growth curve of E.coli
0 6 12 18 24 30
0.0
0.5
1.0
1.5
control
Kanamycine 30ug/ml
Neem GNP 5.6ug/ml
Neem GNP 11.3ug/ml
Neem GNP 34ug/ml
Neem GNP 56.6ug/ml
Neem GNP 113.3ug/ml
Time
Absorbance

25.
Growth curve of S.aureus
0 6 12 18 24 30
0.0
0.2
0.4
0.6
0.8
1.0
CONTROL
Kanamycin30ug/ml
Neem GNP 5.6ug/ml
Neem GNP 8.5ug/ml
Neem GNP 11.3ug/ml
Neem GNP 17ug/ml
Neem GNP 22.6ug/ml
Neem GNP 34ug/ml
Neem GNP 45.3ug/ml
Neem GNP 56.6ug/ml
Time
Absorbance
Antibacterial activity of neem GNP

26.
MTT Assay after 48 Hrs
C
o
n
t
r
o
l
L

N
E
E
M
A
q
.
e
x
t
r
a
c
t
1
0
L

N
E
E
M
G
N
P
2
0
L

N
E
E
M
G
N
P
2
5
L

N
E
E
M
G
N
P
3
0
L

N
E
E
M
G
N
P
5
0
0
50
100
150
concentration
%
Cell
Survived
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT ]
assay for Neem GNP
IC 50= 56.6µg/ml
IC-50 Determination of neem GNP on leishmania donovani by MTT assay
 Cell viability assay of leishmania donovani by Neem GNP at various concentrations by MTT assay.
 IC-50 was found after 48 hrs. treatment with 56.6µg/ml of Neem GNP.
MTT Assay after 24 Hrs
C
o
n
t
r
o
l
L

N
e
e
m
A
q
.
e
x
t
r
a
c
t
1
0
L

N
e
e
m
G
N
P
2
0
L

N
e
e
m
G
N
P
2
5
L

N
e
e
m
G
N
P
3
0
L

N
e
e
m
G
N
P
5
0
0
50
100
150
concentration
%
Cell
Survived
IC 50= 56.6µg/ml

27.
RNS data after 12 Hr
C
o
n
t
r
o
l
A
m
p
h
o
t
e
r
i
c
i
n
-
B
l

N
e
e
m
G
N
P
2
5
l

N
e
e
m
G
N
P
5
0
0
1
2
3
concentration
Fold
change
in
RNS
RNS data after 6 Hr
C
o
n
t
r
o
l
A
m
p
h
o
t
e
r
i
c
i
n
-
B
N
e
e
m
G
N
P
2
5
m
l
N
e
e
m
G
N
P
5
0
m
l
0.0
0.5
1.0
1.5
2.0
2.5
concentration
Fold
change
in
RNS
Reactive nitrogen species data for GNP
 This data provides information about induced apoptosis in leishmania donovani.
 Amphotericin -B is used as positive control.
 Neem GNP 34µg/ml and 56.6µg/ml producing more RNS which triggers the apoptotic pathway in the leishmanial cells and
producing antileishmanial activity.

28.
Flow cytometry and fluorescence activated cell sorting
 Flow cytometry (FCM) is a method for measuring the chemical and/or
physical characteristics of cells passing through a detection device in a
fluid stream.
 Fluorescence-activated cell sorting (FACS) is a specialized type of
FCM. FACS provides a fast, objective, and quantitative method of
recording of the number of dying cells in a population and is, therefore,
routinely used to study cell death.
 Annexin V is used as a probe to detect cells in which
phosphatidylserine (PS) is exposed at the outer leaflet of the plasma
membrane.
 It provides a method for sorting a heterogeneous mixture of biological
cells into two or more containers, one cell at a time, based upon the
specific light scattering and fluorescent characteristics of each cell.

29.
 Seed cells (1 × 106 cells) in a T25 culture flask (in triplicate for experiments) and three T25 culture flask for control
(unstained, Annexin only, propidium iodide and both only).
 After different time point incubation, collect the supernatant (floating apoptotic cells) and trypsinize the adherent cells (1 ×
106 cells) from each T25 flask (combine respective floating and trypsinized cells resulting in six tubes).
 Wash the collected cells twice with PBS and centrifuge (4500 RPM For 10 min).
 Re-suspend each pellet (1 × 106 cells) in PBS (400 µl). For experimental cells (Triplicate) - (400 µl of cells + 100 µl of
incubation buffer with 5 µl of Annexin [5 mg/ml] and 5µl of propidium iodide [5 mg/ ml]). For control cells Control
FACS Methodology

30.
 1: (unstained) - (without any stain (400 µl of cells + 100 µl of incubation buffer) Control.
 2: (Propidium iodide only) - (400 µl of cells + 100 µl of incubation buffer with 5µl of propidium iodide(5mg/ml) Control.
 3: (Annexin V only) - (400 µl of cells + 100 µl of incubation buffer with 5µl of Annexin (5 mg/ml).
 4: (PI + Annexin)- (400 µl of cells + 100 µl of incubation buffer with 5µl of Annexin and 5 µl propidium iodide,(5 mg/ml).
 Analysed the cells using a flow cytometry without washing the cells that were propidium iodide (PI) negative and
Annexin V negative are considered healthy, cells, PI negative and Annexin V positive cells are considered apoptotic, and
cells that are positive to both PI and Annexin V considered necrotic.
FACS Methodology

31.
13µg/ml
Q1= Necrosis cells = 0.6 %
Q2= Late Apoptosis = 3.1 %
Q3= Normal cells = 95.8%
Q4= Early apoptosis = 0.5%
Q1= Necrosis cells = 0.2 %
Q2= Late Apoptosis = 1.8 %
Q3= Normal cells = 97.7%
Q4= Early apoptosis = 0.3%
FACS Analysis data for L. Donovani after 12hrs. treatment

32.
ROS Assay of leishmania Cells Treated with Neem GNP
Control strained
Neem GNP 34µl/ml treated cell
P3= Treated Cell produce ROS(7.3%)
Am-B 50nM(+ve control)
P3= 20.4%

33.
Cell cycle of leishmania cells by FACS
Methodology
 To conduct this experiment first we have to take 1X106 cells per ml for treatment with drug up to 24 hrs.
 Then we have to wash with 1XPBS for two times at 4500 rpm for 10 mins.
 Add 70% chilled ethanol and incubate for 24 hrs. at 4°C.
 Again we have to wash with 1XPBS for two times at 4500 rpm for 10 mins.
 Prepare 200 µg/ml RNAse and add in each sample tube for removing RNA from the sample.
 Incubate the sample tubes at 37 °C for 1 hr.
 Add propidium iodide 50 µg/ml in each sample tube and keep this tubes in dark for 20 mins.
 After this process run the sample in FACS.
Sub G0/G1= Cell arrest and restricted to enter into further phase.
G0/G1= Initial phase of cell cycle
S = Synthesis of DNA takes place in this phase
G2/M= Cell divide in this phase

34.
Cell cycle of leishmania cells by FACS
Fig.1 Control Fig.2 Am-B (50nM)
Fig.4 L.D treated with Neem
GNP 56.6µg/ml(2x)
Fig.5 L.D treated with Neem
GNP 113.3µg/ml(3x)
Fig.3 L.D treated with Neem
GNP 34µg/ml(1x)

35.
Conclusion
 The present investigation was carried out to the study of the synthesis, characterization of GNPs using Neem aqueous extract
and to evaluate the antibacterial and anti-leishmanial potential of functionalized N-GNPs.
 It is concluded from my study the green synthesis of GNPs, using plant materials (Neem leaves) as a reducing agent, it has
advantage such as- ease in availability, ecofriendly and economically. The conclusion finding of this investigation and
summarized below-
 The gold nano particles with neem extract were synthesized and characterized by UV spectroscopy, N-GNP peak find at
540nm.
 FT-IR analysis stated that several functional group of neem extract was used in stabilization of formed gold nanoparticles.
 By Dynamic light scattering (DLS) I found diameter of the N-GNPs is 92nm and Poly dispersity index (PDI) is 0.21.
 Zeta potential analysis further confirmed the stability of the N-GNPs is (-29mV).
 N-GNPs shows Anti-leishmanial with an IC50 of 56.6μg/ml. Neem aq. extract is more effective than N-GNPs.
 Neem extract is also had anti-bacterial activity.
 I have done apoptosis analysis by using FACS for leishmanial parasites with Neem-GNP at different concentration, but I
didn’t find any apoptosis effect.