GREEN SYNTHESIS OF SILVER NANOPARTICLES USING

DECALEPIS HAMILTONII ROOT EXTRACT AND ITS

CANCER CELLS INHIBITION STUDIES

PROJECT REFERENCE NO.: 38S1543

COLLEGE : M.S RAMAIAH INSTITUTE OF TECHNOLOGY

BRANCH : DEPARTMENT OF BIOTECHNOLOGY
GUIDE : MRS. BHAVYA S.G.
DR. K. N. CHIDAMBARA MURTHY
STUDENTS : MS. KEERTHI V R

1. Keywords: Decalepis hamiltonii, silver nanoparticles, CaCo-2 cell lines, propidium iodide,
Acridine orange.

2. Introduction :
Nanotechnology is one of the fast emerging fields of research because of its application in
various fields such as medicine, agriculture, food packaging, cosmetics, electronics etc. silver
nanoparticles are among most widely used nano-products because of its unique optical
properties which can be incorporated into antimicrobial applications, biosensor materials,
composite fibers, cryogenic superconducting materials, cosmetic products, and electronic
components. But application of silver nanopartilces to treat cancer is still unexplored field.
Silver has its importance in the field of medicine right from history. Hippocrates states the use
of silver in treating wounds in his writings. The colloidal silver was used as disinfectant and
germicide before antibiotics were started in 1940s, silver threads were used as sutures in
1920s by surgeons to stitch the surgical wounds/openings. Currently approx. 14% of present
wound dressings are coated with silver. Green synthesis is the eco-friendly process of
synthesis of Nanoparticles using aqueous solvent medium and environmentally friendly
nontoxic reducing and stabilising agent (Iravani et al., 2014) hence, in this study aqueous
extract of Decalepis hamiltonii is used as reducing agent. Decalepis hamiltonii belongs to the
family Asclepiadaceae. Roots, leaves and follicles have medicinal properties. The root of D.
hamiltonii contains antioxidants which are used for several medicinal purposes particularly as
an anti-inflammatory. The species is endemic to peninsular India. It has been recorded in the
dry and moist deciduous forests of Karnataka, Andhra Pradesh and Tamil Nadu. (Anburaja et
al.,2012). Colon cancer is the third most commonly occurring cancer in the world followed by
breast and lung cancer. Colorectal cancer is a malignant tumor arising from the inner wall of
the large intestine. Based on the ICMR statistics ~10, 15,000 colorectal cancers were
diagnosed in India 2014. Research has shown the benefit of natural molecules & plant based

Page 1 of 8
 food in prevention of colorectal cancer. Hence, the approach of synthesizing silver NPS from
plant extracts having medicinal properties will pave the way for treatment of cancer with
minimal adverse effects.

3. Objectives:
The main objectives of the research proposal are to study the inhibitory effect of silver
nanoparticles produced using the Decalepis hamiltonii on human cancer cell lines.
7.1 Preparation of plant extract using roots of Decalepis hamiltonii and synthesis of silver
nanoparticles from plant extract under optimised conditions.
7.2 Characterization of the silver nanoparticles (Microscopic analysis, SEM, XRD, FTIR)
7.3 Understand the possible proliferation inhibition activity using human colon cancer cell lines.

4. METHODOLOGY:
8.1 Preparation of Plant extract: The fresh roots of authenticated Decalepis hamiltonii will be
purchased from TIRUPATI. Then washed, finely crushed and soaked in boiling distilled
water for 5-10 min and filtered using whattman filter paper no.1 (Prabhu et al., 2012).
8.2 Synthesis of silver nanoparticles under optimised condition: For preparation of silver
nanoparticles, different volumes of plant extract added into 45ml of 0.5Mm, 1.0Mm, 1.5mM
& 2.0mM silver nitrate solution , tested under different pH like 2,5,7 & 8 and kept in dark
for 18-20 hours at varied temperature to find the optimum condition for synthesis of
nanoparticles with maximum yield . The nanoparticles solution thus obtained was purified
by repeated centrifugation at 15,000 rpm for 20 min. Supernatant is discarded and the pellet
is dissolved in deionised water. The silver nanoparticles were confirmed by colour changes
and qualitatively characterized by UV-visible spectrophotometer (Prabhu et al., 2012, Vikas
sarsar et al., 2013).
8.3 Characterization of the silver nanoparticles: Surface morphology of the nanoparticles
will be determined by Scanning electron microscopy. X-ray diffraction studies will
determine the percentage of crystallinity of the sample. The possibility of contamination of
the nanoparticle with any other compound will be determined using FTIR spectrometry (Hu
et al., 2012).
8.4 Testing the inhibitory effect of plant extract and synthesized silver nanoparticles on
CaCo-2 cell lines: Using different concentration of silver nanoparticle, response will be
prepared by against % cytotoxicity to CaCo-2 cell lines. Based on the response optimum
concentration (IC50) will be subjected for further to understand the possible mechanism of
cytotoxicity as per our published protocol. The cytotoxicity of silver nanoparticles is
analysed using MTT Aaay and the possible mechanism for apoptosis studied by
cytomorphology analysis. (Chidambara Murthy et al., 2011)

Page 2 of 8
8.4.1 Cytotoxicity (MTT)assay:
• CaCO-2 cell line can be purchased from NCCS
fiedModi
• The cell line can be grown in Dulbecco’s Eagle’s Medium(DMEM)
supplemented with 2mMl glutamine,100U/ml penicillin, 100g/ml streptomycin and
10% fetalbovineserum(FBS).
• Cells can be cultured in 75cm2 cell culture flasks at 37oC in a 5% CO2 atmosphere
• Cultured CaCO-2 cells are seeded into 96well plates approximately as 5×104 cells in
each well and incubate for 48h.
• CaCO-2 cells treated with different concentrations of synthesized Ag-NPs and plant
extract. After treatment, the plates can be incubated for 24–72h.
• MTT can be added in each well and incubated for 2h. Purple color formazone crystals
can be noticed.
• These crystals can be observed at 570nm in a multiwell ELISA plate reader. Optical
density value subjected to percentage of viability by using the following formula
• Percentage of inhibition = 100- (100* OD value of experimental samples)
(OD value of experimental controls)
8.4.2 CYTOMORPHOLOGY
• CaCO-2 cells were plated at 5×104 cells/well into a six well chamber plate. At >90%
confluence, the cells were treated with AgNPs, plant extract and paclitaxel, for 24h.
• The cells were washed with PBSfixed in methanol:aceticacid (3:1,v/v) for 10min and
stained with 1mg/ml propidium iodide and 10mg/ml acrilamide orange stain for 20min.
• Nuclear morphology of the cells with condensed/fragmented nuclei was examined under
fluorescence microscope.

Page 3 of 8
5. RESULTS & CONCLUSIONS :
9.1 Table no. 1: Optimization of process parameter for silver nanoparticles synthesis.

SL NO. Conc. Of Temp. Vol. Of Vol. of UV-vis spec. Yield

Discussion:AgNO3 (OC) AgNO3 plant extract Wavelength(nm)
5ml 288 -
10ml 277 -
RT 45ml
20ml 277 -
30ml 277 -
5ml 277 -
10ml 277 -
1. 0.5 mM 40 45ml
20ml 280 -
30ml 280 -
5ml 283 -
10ml 280 -
45ml
4 20ml 280 -
30ml 280 -
5ml 448 7.21mg
10ml 277 -
RT 45ml
20ml 277 -
30ml 280 -
5ml 470 -
10ml 452 3.61mg
2. 1.0 mM 40 45ml
20ml 283 -
30ml 277 -
5ml 278 -
10ml 280 -
4 45ml
20ml 277 -
30ml 277 -
5ml 452 0.1mg
10ml 436 0.7mg
RT 45ml
20ml 274 -
30ml 274 -
5ml 478 -
10ml 441 0.6mg
3. 1.5Mm 40 45ml
20ml 274 -
30ml 280 -
5ml 277 -
10ml 280 -
4 45ml
20ml 283 -
30ml 280 -
5ml 303 -
10ml 308 -
RT 45ml
20ml 321 -
30ml 315 -
5ml 315 -
10ml 314 -
4. 2.0mM 40 45ml
20ml 521 -
30ml 491 -
5ml 308 -
10ml 308 -
4 45ml
20ml 314 -
30ml 310 -
Discussion:
 Concentration of silver nitrate: 1mM AgNO3 gave characteristic absorption peak at 440 nm
in UV–vis spectrum, whereas the peak got shifted at 0.5 mM, 1.5 mM & 2mM
concentrations
 Temperature: At 4oC no activity was found, At 40oC with 1.0mM AgNO3 & 10ml plant
extract the yield was 3.61mg however at room temperature with 1.0mM AgNo3 & 5 ml of
plant extract the yield of 7.21mg was observed.
Page 4 of 8
 Volume of plant extract: There was no activity observed for 20 & 30ml of plant extract, at
10ml varying yields of 0.1mg – 3.61mg were obtained. However, maximum yield of 7.21mg
was found for 5ml of plant extract.
 Concentration ratio of plant extracts and silver nitrate solution: different concentration ratio
of plant extracts and silver nitrate solution were also Characterizer for silver nanoparticles
by UV-Visible spectroscopy. The ratio of1ml +9ml showed characteristic absorption peak at
448 nm and maximum yield of 7.21mg.
 pH: There was no reaction at pH 2,5 & 8 However pH 7 is the optimum condition for
completion of reaction.
 Time: The optimum time for the completion of reaction for our study was 22 hrs.
 Microwave assisted: The alternative approach for reducing reaction time by using
microwave assisted was found unsatisfactory as no reaction was observed.

9.2 XRD Result:

Discussion: The comparision of the XRD graph of synthesised silver NPs with a standard
graph on the left hand side shows a prominent peak between 30-40 degrees depicting the
presence of silver. According to the “Match! Phase Analysis Report” sample contains about
60% of silver. The crystal system is monoclinic.

9.3 FTIR spectra of AgNPs:

Page 5 of 8
Discussion:

Wave Number(cm -1) Molecular motion Functional group

2921.75 C- H Stretch Alkanes

1602.98 N-H Bend Amines
1019.13 C-N Stretch Amines
517.10 C-Br Stretch Alkyl halides
479.99 C-Br Stretch Alkyl halides
455.39 C-Br Stretch Alkyl halides
440.17 C-Br Stretch Alkyl halides
424.50 C-Br Stretch Alkyl halides
411.37 C-Br Stretch Alkyl halides

9.4 Scanning Electron Microscopy:

Discussion: scanning electron microscopy images of the synthesized silver nanoparticles

show the morphology and size of the particles. The above figure shows that the particles are
spherical in shape with the particle size ranges from 50-70nm.

9.5 Cytotoxicity (MTT) assay:

120
SILVER NANOPARTICLES
100
80
% of cell inhibition

60
24hrs
40
48hrs
20
72hrs
0
2.5 5 µM 10µM 15µM 20 25µM 50
µM Conc. of silverµM
NPs µM

Page 6 of 8
 90 PLANT EXTRACT 100 paclitaxel
80 90
70 80
% of cell inhibition

% OF Cell inhibition
60 70
50 60
40 50
24h 24h
30 40
20 48h 48h
30
10 72h 20 72h
0 10
0
5ppm 10ppm 15ppm
Conc. of plant extract Conc. of Drug

Discussion:
• Dose dependent increase in the percentage of cell inhibition. Increasing the time of incubation
showed a further increase in cell inhibition. The cell inhibition is more in Ag-NPs when
compared to standard drug paclitaxel as well as plant extract . Approximately 50% inhibition
of cell viability was seen with AgNPs at 10µM upon 24h exposure. Hence the IC50 value of
AgNPs is 10 µM.

9.6 Cytomorphology:
Propidium iodide and acridine orange stained human colon cancer cell lines

Control cells

AgNPs treated cells

Page 7 of 8
 Paclitaxel treated cells

Plant extract treated cells

Discussion:
• In case of control cells, a very negligible number of propidium iodide stained cells were
present representing more cell viability.
• Cells treated with AgNPs were propidium iodide positive indicating apoptosis induction.
Treatment of plant extract and paclitaxel (positive control) have shown the presence of both
live and pre apoptotic cells.

6. Scope Of Future Work: Testing the biosynthesized silver nanoparticles on animal models
induced with colon cancer and there by proceeding for clinical trials on humans and possible
utilization of silver nanoparticles in treatment of human colon cancer.

7. Reference
• D.Prabhu et al., 2013, Biologically synthesised green silver nanoparticles from leaf extract of
vitex negundo induce growth inhibitory effect on colon cancer cell line HCT15, ELSEVIER
process biochemistry
• Vikas sarsar et al., 2013, Significant parameters in the optimization of biosynthesis of silver
nanoparticles using psidium guajava leaf extract and evaluation of their antimicrobial activity
against human pathogenic bacteria, An International Journal of Advances in Pharmaceutical
Sciences.
• Murthy K N Chidambara, Rajasekaran T , Giridhar P ,Ravishankar G A,2006, Antioxidant
property of Decalepis hamiltonii Wight & Arn, Indian Journal of Experimental Biology,
Vol.44(10) , pages 832-837.
• V. Anburaja , V. Nandagopalan , S. Prakash & A. Lakshmi Prabha, 2012, A report of the
threatened plant Decalepis hamiltonii Wight & Arn. (Asclepiadaceae) from the mid elevation
forests of Pachamalai Hills of the Eastern Ghats, Tamil Nadu, India, Journal of Threatened
Taxa, 4(15): 3447–3449.

• Iravani S, Korbekandi H, Mirmohammadi S.V. and Zolfaghari B, 2014, Synthesis of silver

nanoparticles: chemical, physical and biological methods, Research in Pharmaceutical
Sciences, 9(6): 385-406.

Page 8 of 8