GREEN SYNTHESIS OF SILVER NANO-PARTCILES

USING THE LEAF EXTRACT OF

OCIMUM SANCTUM (TULSI)

Project Submitted by:

Students of B.Sc. (MPC) 2016

K.V. Sai Swetha 134207

S. Navya 134212
Supriya Veronica 134206
Anuradha 134202

Project Submitted to:

The Department Of Chemistry

St. Francis Degree College for Women

Begumpet, Hyderabad. 500016

 ACKNOWLEDGEMENT

We would like to take this opportunity to thank St. Francis College and the Department of chemistry
for providing us with an opportunity to gain insight and experience into experimental methods in
chemistry. We express our deepest and most sincere gratitude to our mentor and teacher, Mrs. Y.
Lakshmi Madhuri maam for her constant guidance, support and encouragement throughout the
course of our thesis. We also thank the non-teaching staff of the Department of Chemistry for their
patient assistance and support during the experimental phase of our project. In conclusion, we thank
our fellow team mates for their enthusiastic and active participation at each and every stage, right
up to the culmination of our thesis. The inception and completion of this project would not have
been possible without the infrastructure and laboratory facilities provided by the college and hence,
once again, we extend to them, our sincerest thanks.

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 INDEX

Abstract..........................................................................................................................4
Introduction....................................................................................................................5
Objective........................................................................................................................6
Principle.........................................................................................................................6
Methods..........................................................................................................................7
Procedure........................................................................................................................8
Result..............................................................................................................................9
Discussion....................................................................................................................11
Conclusion....................................................................................................................11
References....................................................................................................................12

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 ABSTRACT

Recent advances in the field of nanotechnology have found interesting and important applications in
clinical medicine and surgery. One promising application is the use of nano-sized particles for the
delivery of drugs, heat, radiation and other substances to specific types of cells (such as cancer
cells). In view of the steep rise in the infections caused by multi drug resistant bacteria in recent
years and the reduced efficacy of employed antibiotics - owing to their overuse or misuse, bacterial
resistance has dramatically increased. In this respect, Silver, which has long been recognized as
having inhibitory effect on microbes, has found a major use in the form of nanoparticles. These
biologically synthesized nanoparticles have proved to be highly toxic against different multi-drug
resistant human pathogens. When synthesized from medicinal plant extracts, these devices can
effectively prevent and combat strains of microbes that have developed resistance to common
antibiotic drugs. Further, nanoparticles can also be coated with suitable substrates and used in
chemotherapy. In certain cases, formal chemotherapeutic methods are proving to be ineffective, as a
result of the development of multiple drug resistance (MDR) in cancer cells by unknown
mechanisms.

A prospective solution herein, involves the design of nanoparticles with proteins that specifically
detect the drug resistant cells and ensure high drug concentration at the designated site, while
limiting side effects to neighbouring, healthy tissues. However, conventional procedures for
synthesis of silver nanoparticles such as ultraviolet irradiation, aerosol technologies are expensive
and sometimes release environmentally hazardous chemicals. To circumvent these problems,
biosynthesis of nanoparticles- a greener alternative- is being employed wherever suitable. This
project deals with the green synthesis of Silver nanoparticles from the leaf extract of common
Indian plant, Ocimum sanctum- the Holy Basil.

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 INTRODUCTION

AgNPs are nanoparticles of silver which are in the size range of 1-100 nm. Their unique optical,
electrical, thermal and surface properties help in molecular diagnostics and therapies. Synthesis of
AgNPs is primarily through physical and chemical methods. The problem with these methods is
that they are expensive and the AgNPs can also have toxic substances absorbed onto them. To
overcome this, the biological method provides a feasible alternative. However, the biosynthesis of
AgNPs using microorganisms like bacteria, fungi, enzymes; in spite of being eco-friendly, require
highly aseptic conditions that are not industrially viable. Further, the rate of synthesis is also
reduced when compared to the plant mediated procedures. Hence, plant-mediated synthesis is
preferred as it is cost-effective, environmentally friendly, and safe for human therapeutic use.

Plants are important sources of drugs and have been credited with medicinal properties since times
immemorial. The earliest mention of medicinal plants is found in the Rigveda, which dates back
to 4500-1600 B.C. Ayurveda, which is a 5000 year old system of Indian medicine, has extensively
documented the Indian basil, Tulsi (Ocimum sanctum) for its therapeutic potential and therein, the
plant has been described as Dashemani Shwasharini (anti-asthmatic) and Kaphaghna (anti-cough
drug). Countless other medicinal properties have been attributed to Tulsi, in other Vedic literature
such as Siddha, and also in Greek, Roman and Unani systems of medicine. The ancient Rishis of
India saw the herb to be so effective for healing that it was declared as the one that is matchless-
Tulsi.

Ocimum Sanctum Linn: Scientifically established

Among the plants known for medicinal value, those belonging to the family Labiatae under the
genus Ocimum have been found to be very important in view of their therapeutic value. Ocimum
sanctum L. (Tulsi), Ocimum gratissium (Ram Tulsi), Ocimum canum (Dulal Tulsi), Ocimum
basilicum (Van Tulsi) are important species that are endemic to the Indian subcontinent. The plants
are fragrant, bushy, perennials growing up to 1.5m in height with profusions of white blooms and
slightly purple tinted foliage. Scientifically established properties of the plants derivatives include
hypo-glycaemic, hypo-lipidemic, immuno-modulatory, antimicrobial, anti-ulcer, anti inflammatory,
chemo-preventive and radio-protective activity.

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 OBJECTIVE

In this project we aim to synthesize silver nanoparticles from the leaf extracts of Ocimum Sanctum
Linn.

PRINCIPLE

Phytofabrication of AgNPs:
It has been shown that many plants can actively uptake and bio-reduce metal ions from soils and
solutions, thereby forming insoluble complexes with the metal ion in the form of nanoparticles. This
is the central principle of plant mediated synthesis of NPs.
AgNO3 Ag+ + NO3-

Ag+ + e- Ag

Factors affecting Phytofabrication of AgNPs:

Certain physiochemical properties such as temperature, time, pH, optical, concentration of the
substrate, and enzyme sources, play an important role in the phytofabrication of nanoparticles,
influencing their shape, size, and distribution.

The particle size is expected to be lesser in basic medium and greater in acidic medium. If the pH is
changed, the electrical charges on the biomolecules may change which might affect their capping
and stabilizing activity and subsequently affect the growth of the AgNPs.

Increase in the concentration of the plant extract decreases the particle size. In higher concentrations
of the extract, the biomolecules cap the nanoparticles surfaces, protecting them from coagulation.
Thus, high concentrations of extract are necessary for the synthesis of symmetrical AgNPs.

Temperature and contact time have also been found to have a direct bearing upon the stability and
rate of growth of the nanoparticles. Room temperature has been found to be suitable for the
synthesis of silver nanoparticles. An increase in temperature is found to increase the rate of particle
growth and also increase the size of the AgNPs formed.

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 METHODS

Generally there are two approaches involved in the syntheses of AgNPs: top to bottom approach
or bottom to up approach. In bottom to top approach, nanoparticles can be synthesized using
chemical and biological methods by self-assembly of atoms to new nuclei which grow into a
particle of nanoscale; while in top to bottom approach, suitable bulk material breaks down into fine
particles by size reduction and physical methods.
In top to bottom approach, nanoparticles are generally synthesized by evaporationcondensation
using a tube furnace at atmospheric pressure. In this method the foundation material; within a boat;
centred inside the furnace is vaporized into a carrier gas. The generation of silver nanoparticles
using a tube furnace has numerous drawbacks as it occupies a large space and munches a great deal
of energy while raising the environmental temperature around the source material, and it also entails
a lot of time to succeed thermal stability. One of the biggest limitations in this method is the
imperfections in the surface structure of the product.

In bottom to top approach, chemical reduction is the most common scheme for syntheses AgNPs.
Different organic and inorganic reducing agents, such as sodium borohydride (NaBH4), sodium
citrate, ascorbate, elemental hydrogen, etc are used for reduction of silver ions (Ag+) in aqueous or
non-aqueous solutions, in the chemical methods. But these methods involve the use of perilous
chemicals that can endanger biological systems. Also, capping agents have to be added separately
for size stabilization of the nanoparticles.

In green synthesis of AgNPs, inorganic reducing agents are replaced by organic plant constituents
such as enzymes and protein contents (reductases) which biologically reduce the substrates, such as
silver nitrate into the corresponding NPs. The term phytofabrication indicates the synthesis of the
nanoparticles with the help of the plant constituents. Also, plant derivatives, as they are nontoxic
chemicals, provide natural and better capping agents of inherent therapeutic value. Through this
method, a large quantity of nanoparticles can be synthesized in a short span of time.

PROCEDURE

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Collection of plant parts:

The leaves of Ocimum Sanctum Linn were collected

from four different locations and washed thoroughly
thrice with tap water and then with double distilled
water to remove adhering impurities and dust, if
any. The clean leaves were then dried in shade for 5 days and then finely ground to a powder.

Preparation of aqueous extract:

10 grams of the finely grounded powder of dried leaves was weighed and then transferred to a 250
ml beaker. This is then boiled with 100 ml of double distilled water for 5 minutes when a brown
colour was imparted to the liquid. The liquid was then stirred and filtered when still hot to get the
extract.

The suggested whatmann no.1 filter paper was

employed after filtering the solution through
ordinary filter paper twice. The resulting
extract was a dark brown, clear solution with a
very pungent and characteristic aroma of Tulsi. It
had the consistency of water. It was then stored at
low temperature for further use.

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Preparation of AgNO3 of requisite molarity:

0.001 M AgNO3 solution was prepared by

combining 1 ml of 0.1 M AgNO3 solution with 100
ml of double distilled water.

Synthesis of Silver nanoparticles:

To the 10 ml of 0.001 M AgNO3 solution taken in a

25 ml glass beaker, varying amounts of the plant
extract from 1 5 ml was added separately and vice
versa. The beakers were marked to indicate the ratio
of extract to AgNO3 solution and were left
undisturbed in a dark chamber for 24 hours. The
colours of the solutions were documented before and
after the 24 hour period.

RESULT:

On comparing the colours of the solutions after a

period of 24 hours, a considerable deepening of
colour was observed in every beaker, indicating the
formation of AgNPs. The nanoparticles formed were
then separated from the solution by filtering through
Whatmann no.1 filter paper using Buchner
funnel. AgNPs were obtained thus.

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OBSERVABLE CHANGE IN COLOUR

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 BEFO
RE

AFTER

BEFO AFTE
RE R

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 DISCUSSION

The synthesized AgNPs are impregnated with therapeutic capping agents of Ocimum sanctum and
can be further capped with more potent drugs for greater activity. The AgNPs thus prepared, when
injected into an infected biological system enter the cells of the microbe. When the positively
charged AgNPs accumulate on the negatively charged cell membrane, it brings about a substantial
conformational change in the membrane and the membrane ultimately loses permeability control
leading to inflow of the AgNPs. The nanoparticles interact with sulphur containing proteins present
in the bacterial membrane as well as with the phosphorus-containing DNA. Ag+ from the AgNPs
then binds to the functional groups of proteins, resulting in protein denaturation which causes
considerable damage to the DNA resulting in cell death. The AgNPs show efficient antimicrobial
property due to their extremely large surface area, which provides better contact with
microorganisms. Smaller particles having a larger surface area available for interaction will have a
stronger bactericidal effect than will larger particles.

TOXICITY OF AgNPs:

During a storage period of several days at room temperature coated silver nanoparticles can undergo
dissolution releasing toxic silver ions. The toxicity of silver ions arises from unfavourable binding
interactions with the DNA/ nucleic acids and cell wall components of the host cells. Small sized
nanoparticles have been reported to have not only lodged but also to have passed through protein
channels and nuclear membrane pores in the size range of 9-10 nm. Such infiltration of AgNPs
presents problems due to their interaction with structures internal to these membranes. Hence,
AgNPs of stipulated size are being used for clinical procedures presently.

CONCLUSION

The rapid synthesis of silver nanoparticles through leaf mediated synthetic procedures has been
demonstrated. As discussed above, these obtained nanoparticles have powerful applications in the
biomedical field and this simple procedure has several advantages such as cost-effectiveness,
compatibility for medical and pharmaceutical applications as well as large scale commercial
production.

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 REFERENCES
[1] Y.Lakshmi Madhuri, Green Synthesis of Silver Nanoparticles: An Ecofriendly Approach
(2015)

[2] K.Mallikarjuna et al, Green synthesis of Silver Nanoparticles using Ocimum leaf extract and
their characterization, Digest Journal of nanomaterials and Biostructures, Vol.6 No1, Jan-
March (2011)

[3] Ram Prasad, Synthesis of Silver Nanoparticles in Photosynthetic Plants, Journal of

nanoparticles, vol.2014. Article ID 963961.

[4] Shakeel Ahmed et al, A Review on plant extract mediated synthesis of silver nanoparticles
for antimicrobial applications: A green expertise, Journal of Advanced research. (2015)

[5] Sumita Chanda, Silver nanoparticles (medicinal plants mediated) : A new generation of
antimicrobials to combat microbial pathogens- a review, Formatex (2013)

[6] Bhateja Sumit, Arora Geetika, Therapeutic benefits of Holy Basil (Tulsi) in general and oral
medicine: A review. IJRAP 3(6) Nov-Dec 2012

[7] Subir Kumar Das, D.M.Vasudevan, Tulsi: The Indian holy power plant. Natural Product
radiance, vol 5(4), 2006

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