GREEN SYNTHESIS AND OPTIMIZATION OF SILVER

NANOPARTICLES USING LEAF EXTRACT OF HOUTTUYNIA

CORDATA LEAF EXTRACT AND ITS ANTIBACTERIAL
PROJECT REPORT
submitted by

Ningsangwabang (110256)
Zubeni Kikon (110259)
Yangerdenla Jamir (110262)
Sikha Morang (110264)
Dimple Saikia (110265)

In partial fulfillment of the requirements for the degree

Of

BACHELOR OF TECHNOLOGY IN BIOTECHNOLOGY

UNDER THE SUPERVISION OF

Mr. Hanumant Singh Rathore
Assistant Professor
Department of Biotechnology
School of Engineering and Technology
Nagaland University, Dimapur-797112 June 2015

ACKNOWLEDGEMENT

It is our great honor and privilege to extend and express our heartfelt sense of gratitude
and sincere appreciation to thank Mr. Hanumant Singh Rathore, Head of Department,
Biotechnology, Nagaland University, Dimapur, Nagaland, for his guidance, support,
constant encouragement, splendid supervision and valuable advice throughout our project
work. It was his valuable discussion and endless endeavors through which we have gained
a lot.
We are also very grateful to Mr. Kekresilie, JRF, Department of Biotechnology, Nagaland
University, Dimapur Nagaland, for being very kind, passionate and supportive during our
project work and also not forgetting our lab attendant. Finally, we thank our Almighty God
for guiding, protecting and giving us strength all through our project work

DECLARATION

We hereby declare that the project entitled Green synthesis and optimization of silver
nanoparticles using leaf extract of Houttuynia cordata leaf extract and its antibacterial
submitted for the B.TECH Degree is our original work and the project has not formed the
basis for the award any degree, associate-ship, fellowship or any other similar titles. We
are wholly responsible for any plagiarism case against this report if it arises.

Ningsangwabang (110256):

Sikha Morang (110264):

Place:

Place:

Date:

Date:

Zubeni Kikon (110259):

Dimple Saikia (110265):

Place:

Place:

Date:

Date:

Yangerdenla Jamir (110262):

Place:
Date:

ABSTRACT
Physical & chemical methods are successful in producing of silver nanoparticle (AgNPs)
but have drawbacks e.g. complex and high energy process, hazardous by-products. So
herewith we developed and optimized an alternative green synthesis of silver
nanoparticles by using Houttuynia cordata leaf extract. The Houttuynia cordata leaves
were extracted and phytochemical assay was done. The main parameters affecting the
formation and size of AgNPs includes the ratio of plant extract and silver nitrate solution
were investigated and optimized. The optimum ratio of plant extract and silver nitrate
solution was 7:10 for producing AgNPs. The developed AgNPs size was measured by
Malvern Zetasizer S90 and shown size range 49-60 nm. The antibacterial potential of
AgNPs was determined against the culture of gram positive Staphylococcus aureus and
gram negative Pseudomonas aeruginosa and Escherichia coli. The result zone of
inhibition showed an enhanced antibacterial efficacy.

CONTENTS
SL.
NO.
1.

CHAPTER PARTICULARS

PAGE
NO.

INTRODUCTION
1.1 Different methods for synthesis of nanoparticles
1.2 Toxicological aspects of synthesizing nanoparticles using physical
and chemical approach
1.3 Green synthesis of nanoparticles

2.

REVIEW OF LITERATURE
2.1 Silver nanoparticle synthesizing plant(H.cordata)
2.2 Phytochemical components
2.3 Health benefits
2.4 Significance of silver nanoparticles
2.5 Silver nanoparticle as an antimicrobial agent
2.6 Mechanism of action of silver nanoparticles on microbes

3.

OBJECTIVE

4.

MATERIALS AND METHOD

4.1 Experimental materials
4.2 Preparation of plant extract from H.cordata leaves
5

4.3 Biosynthesis of nanoparticles

4.4 Optimization of silver nanoparticles
4.5 Phytochemical test
4.6 Antimicrobial activity of synthesized silver nanoparticles and plant
extract

5.

RESULT & DISCUSSION

6. CONCLUSION
7.

BIBLIOGRAPHY

LIST OF FIGURES
FIG
No.
NAME OF THE FIGURES
1.1 Top down process
1.2
2.1

PAGE
NO.
3

Bottom up process

Houttuynia cordata thunb

2.2.1 Aristolactam A

10

2.2.2 Quercetin

11

5.1

phytochemical analysis of Houttuynia cordata plant extract

22

5.2

Color changes of plant extract & AgNO3 solution (a) Before

22

5.5

autoclave (b) After autoclave.

size of Ag NP giving minimum size about 93nm range at
0.8mM concentration along with plant extract(10:1 v/v).
Size of Ag NP giving a minimum size of about 49nm range
when the concentration of plant extract along silver nitrate
solution are in the ratio of 7:10 v/v.
Size of AgNP before and after autoclave

5.6

Ag NP size distribution by intensity

5.7

Antimicrobial activity tests by well diffusion method.

5.1

LIST OF TABLES
Phytochemical analysis

28

5.2

Antimicrobial activities test by well diffusion method

31

5.3
5.4

23
23

24
24

1. INTRODUCTION
Studies in nanotechnology have induced great scientific advancement in the field
of research and technology. Nanotechnology is the study of small object which can be
used across all fields such as chemistry, biology, physics, material science and
engineering. Nanotechnology is emerging as a manufacturing new material at the
nanoscale level (Albrecht et al., 2006). The prefix nano indicates one billionth or
109 units. Nanoparticles are clusters of atoms in the size range of 1100 nm (Williams,
2008). Nanoparticles are mostly prepared from noble metals such as Gold (Gu et al.,
2003), silver, platinum, zinc, copper, magnesium, titanium (Retchkiman-Schabas et al.,
2006), alginate (Ahmed et al., 2005). But among them, silver nanoparticles (AgNPs or
nanosilver) have been exploited or attracted the most because they have proved to be
effective as it has good antimicrobial efficacy against bacteria, viruses and other
eukaryotic microorganisms (Gong et al., 2007).
Silver nanoparticles have become the focus of intensive research owing to their wide
range of applications in areas such as in selective coatings for solar energy absorption and
intercalation material for electrical batteries, as optical receptors, for biolabelling, and as
antimicrobials, in the development of new technologies such as material sciences and
medicine at the nanoscale (Korbekandi H. et al., 2012). There are many consumer
products and applications utilizing nano-silver which have the highest degree of
commercialization for example disinfecting medical devices and home appliances to water
treatments. According to the Project on Emerging Nanotechnologies over 1300
manufacturer-identified, nanotechnology-enabled products have entered the commercial
market place around the world. Among them, there are 313 products utilizing nanosilver,
this has made nanosilver the largest and fastest growing class of NPs in consumer products
applications. Silver nanoparticles find use in many fields, and the major applications
include their use as catalysts, as optical sensors of zeptomole concentration, in textile

engineering, in optics, and most importantly in the medical field as a bactericidal and as a
therapeutic agent.
Nanoparticles of gold, silver, copper, silicon, zinc, titanium, magnetite, palladium
formation by plants have been reported.
Traditionally, researchers generally used two methods for the synthesis of nanoparticles
such as Top-down approach and Bottom-up approach.
i)

In top to bottom approach, suitable bulk material break down into fine particles
by size reduction with various lithographic techniques e.g. grinding, milling,
sputtering and thermal/laser ablation(Samberg et al.,2010).

Fig: 1.1 Top-down processes

ii)

In bottom up approach nano-architectural phenomenon of self assembly of

materials from cluster-to-cluster, molecule-to-molecule or atom-to-atom on top
of a base substrate. The main concern in the bottom-up approach is the
adhesion of the surface layers to the base substrate. The most commonly used
bottom-up methods are wielding & riveting (R.Elghanian et al., 1997).

Fig: 1.2 Bottom-up processes

1.1

DIFFERENT

METHODS

FOR

SYNTHESIS

OF

NANOPARTICLES

1.1.1 PHYSICAL APPROACH

In physical approach, the metallic nanoparticles are synthesized by evaporation
condensation method which could be carried out by using a tube furnace at atmospheric
pressure. Gutav et al., has successfully synthesized Au, Ag and Pb nanoparticles by using
evaporation method. However, using a tube furnace method has some drawbacks such as a
large space of tube furnace, great consumption energy for raising the environmental
temperature around the source material and a lot of time for achieving thermal stability.
Therefore, various methods of synthesis of nanoparticles based on the physical approach
have been developed.
A discharge method was developed to fabricate AgNPs in deionized water with no added
surfactants. In this type, silver wires (Gredmann, 99.99%, 1 mm in diameter) were
submerged in deionized water and used as electrodes. The experimental results show that
Ag-NPs suspension fabricated by means of arc discharge method with no added
surfactants contains metallic Ag-NPs and ionic silver.
Kholoud et al., synthesized silver nanoparticles synthesis by laser ablation method. The
particles synthesized through laser ablation method depends upon the wavelength of the
laser, the duration of the laser pulses the laser fluence, the ablation time duration and the
effective liquid medium which may or may not containing the surfactant. In another work
Jung et al reported an attempt to synthesize NPs via a small ceramic heater that has a local
heating area. The small ceramic heater was used to evaporate source materials. The results
showed that the geometric mean diameter, the geometric standard deviation and the total
number concentration of NPs increase with heater surface temperature.

10

In summary, the physical synthesis process of NPs usually utilizes the physical energies
(thermal, ac power, arc discharge) to produce NPs with nearly narrow size distribution.

1.1.2 CHEMICAL APPROACH

Chemical method is the most commonly used method for the synthesis of silver
nanoparticles. Sun Y et al., synthesized Ag NPS by using polyol process. This method
involves monodisperse samples of silver nanocubes were synthesized in large quantities
by reducing silver nitrate with ethylene glycol in the presence of polyvinyl pyrrolidone
(PVP). In this case, ethylene glycol served as both reductant and solvent. It showed that
the presence of PVP and its molar ratio relative to silver nitrate. Kim D et al., has shown a
spherical shape of Ag-NPs with a controllable size and high mono dispersity which is
synthesized by using polyol process and a modified precursor injection technique.
Another process to synthesize NPs using chemical is Sol-gel technique, which is a wet
chemical technique used for the fabrication of metal oxides from a chemical solution
which acts as a precursor for integrated network (gel) of discrete particles or polymers.
The most commonly used reducing agents are sodium borohydride, hydrazine hydrate,
potassium auro chlorate and sodium citrate. The reduction of various complexes with Ag+
ions leads to the formation of silver atoms (Ag0), which is followed by agglomeration into
oligomeric clusters. These clusters eventually lead to the formation of colloidal Ag
particles. The function of the protective agent is to protect the nanoparticles from
agglomeration (Oliveira et al., 2005; Bai et al., 2007). The most commonly used
protecting agents are poly (vinyl pyrrolidone) (PVP), poly (ethylene glycol) (PEG), poly
(methacrylic acid) (PMAA) and poly (methylmethacrylate) (PMMA)

1.2

TOXICOLOGIC

NANOPARTICLES

ASPECTS

USING

OF

PHYSICAL

SYNTHESIZING
AND

CHEMICAL

APPROACH
11

Although physical & chemical methods are very successful in producing large scale
production of nanoparticles, they have certain drawbacks such as the presence of toxic
organic solvents, release of hazardous by-products and intermediary compounds, high
energy consumption, and long time for synthesis and difficulty in purification (Nagajyothi
and Lee, 2011). Besides, other problems are found in NP synthesis such as toxicity (Quang
Huy Tran et al 2013), reduced rate of particle synthesis, structural particle deformation,
and inhibition of particle growth. Additionally, chemical NP synthesis included in
nanocomposites or metallic NPs are composed by more than one chemical species or
molecules which could increase the particle reactivity and toxicity and might harm human
health and the environment due to the composition ambiguity and lack of predictability
(Ana Cauerhff et al., 2013). Global warming & climate change has induced a worldwide
awareness to reduce the toxic & hazardous waste materials, thus, the green synthesis route
have raised actively the progress in the fields of science & industry (Ahmad et al., 2011).

1.3 GREEN SYNTHESIS OF NANOPARTICLES

Bhattacharya et al., has highlighted that Green synthesis of nanoparticles is the emerging
method of synthesis in the field of nanotechnology and biotechnology which has received
increased attention due to growing need to develop environmentally benign technologies
in material synthesis. Moreover, the process is more biocompatible and cost effective too
(N.Roy et al., 2010). Recently, Many researchers have reported that biosynthetic methods
employing either plants extract (Gardea-Torresdey et al., 2002 and, Shankar et al., 2003)
and microorganisms such as bacteria (Joerger et al., 2000) and fungus (Shankar et al.,
2003) have mushrooming as an economic alternative to more complex chemical and
physical methods of nanoparticles formation. Green synthesis possesses the following
advantages over traditional chemical methods (Youmie Park, 2014). (I) Green synthesis is
simple and usually involves a one-pot reaction; (II) it is amenable to scale up; (III) the
toxicity-associated hazardous chemicals are eliminated, and (IV) green biological entities
can be used as reducing agents.

12

Recently, many studies have already been conducted to synthesize nano particles from
different parts of plants (Sivash Iravani, 2011). An earlier literature has suggested that
nanoparticles are synthesized from leaf extracts of various plants such as Pelargonium
graveolns (S. S. Shankar et al.,2003), Medicagosativa (J. L. Gardea-Torresdey et al.,
2003), Azadirachtaindica ( Shankar et al., 2004), Lemongrass(S.S. Shankar et al.,2005),
Emblicaofficinalis(Ankamwar et al.,2005), Aloe vera ( Chandran et al., 2006),
Cinnamomumcamphora (J. Huang, Q. Li, D. Sun et al.,2007), Capsicum annuum (Y. Shen
et al.,2007), Diopyros kaki (J. Y. Song et al.,2008), Bryophyllum sp., Cycas Leaf.,
Hydrilla sp. ( Jha et al.,2009), Hibiscus rosasinensis, ( Philip, 2010), Ipomoea aquatica,
Enhydrafluctuans, Ludwigiaadscendens ( Roy and Barik, 2010), Psidiumguajava
( Raghunandan et al., 2011), Ocimum sanctum ( Philip, 2010), Krishna tulsi (Ocimum
sanctum) ( Philip and Unni, 2011), Ipomoea indica (Pavani et al.,2013) have been used for
the synthesis of Ag , Au, Cu, Fe, Pt etc. nanoparticles.
In recent years the outbreak of re-emerging and emerging infectious diseases has been a
significant burden on global economies and public health. Transmission of infectious
pathogens to the community has caused outbreaks of diseases such as influenza, diarrhea,
cholera, etc throughout the world. To overcome such infectious pathogens is by using
advanced disinfectant nanoparticles (especially AgNPs) have been proposed by (Quang
Huy Tran et al 2013).However, synthesis of AgNPs using green technology and its
antibacterial potential have gained a lot of attention due to their wide range of
applications.
Dulen Saikia et al., has extracted from Asiatic Pennywort and Bryophyllum which act as
reducing or capping agents were used for the synthesis of silver nanoparticles. The
synthesized AgNPs of average sizes of ~18-21nm were obtained via bio-reduction of
silver nitrate (AgNO3). They have found that Ag nanoparticles obtained from Asiatic
Pennywort was more effective on gram positive bacteria Staphylococcus Epidermidis
while AgNPs obtained from Bryophyllum was more effective on gram negative bacteria
Pseudomonas Fluorescens and suggested that it was so because of its size dependent
interaction of Ag nanoparticles.

13

The antimicrobial effects of silver nanoparticles using Green synthesis method have been
reported by many researchers. To evaluate applications, (Manisha D.R et al., 2014)
synthesized silver nanoparticles and the antimicrobial activity of Catharanthus roseus
flower mediated silver nanoparticles has been studied. Silver nanoparticles are formed by
the reduction of Ag+ to Ag0 and confirmation of formation of Ag nanoparticles was done
by using UV spectral analysis. The UV-vis Spectral analysis of the flower extract mediated
nanoparticles within the range of 300nm - 700nm gave a peak at 460nm. While using
TEM, it showed that the synthesized silver nanoparticles were spherical in shape and the
sizes of nanoparticles were in the range of 11nm-15nm. They had found that it has
potential

antibacterial

activity

against

Escherichia

coli,

Pseudomonas

putida,

Staphylococcus aureus, Klebsiella pneumoniae and Bacillus subtilis.

Pavani, K. V., et al., had revealed that AgNPs were spherical and cuboid in shape, with a
size range of about 5-50nm. Their work proved that silver nanoparticles synthesized from
Ipomea Indica extract can be used in the field of nanomedicine and nano-diagnostics, and
these synthesized particles are eco-friendly and non toxic in nature.

Peter Logeswari et al., synthesized silver nanoparticles (Ag NPs) from silver nitrate
solution using plants extract from various plants such as Ocimum tenuiflorum, Solanum
tricobatum, Syzygium cumini, Centella asiatica and Citrus sinensis and also showed
efficient antimicrobial activity of synthesized nanoparticles and revealed the size of silver
nanoparticles with an average range of 28 nm, 26.5 nm, 65 nm, 22.3 nm and 28.4 nm
respectively. Well diffusion method is used to perform antimicrobial efficacy against
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella
pneumoniae. The Ag nanoparticles obtained from S. tricobatum, O. tenuiflorum extract
showed more effective antimicrobial activity against S. aureus (30 mm) and E. coli
(30 mm) respectively while less effective antimicrobial activity of silver nanoparticles
obtained by S. tricobatum extract was found against P. aeruginosa (12 mm) and E. coli
(12 mm).
Contrary to bactericidal effects of ionic silver, the antibacterial effect of colloid Ag
particles is influenced by the dimensions of the particles the smaller the particles, the
14

greater antimicrobial effect (Shahverdi et al., 2007). M. Umadevi et al., studied on various
concentration of D. carota to synthesized silver nanoparticles. As a result the average size
of 20 nm with spherical shape was confirmed and also found the presence of ascorbic acid
in D. carota extract which is used as reducing agent.

15

2. REVIEW 0F LITERATURE

2.1

SILVER

NANOPARTICLE

SYNTHESIZING

PLANT

(H.CORDATA)
The major advantage of using plant extracts for silver nanoparticles synthesis is that they
are easily available, safe, and nontoxic in most cases, have a broad variety of metabolites
that can aid in the reduction of silver ions, and are quicker than microbes in the synthesis
(Sukumaran et al., 2012).

Scientific classification:
Kingdom: plantae
Order: piperales
Family: saururaceae
Genus: Houttuynia cordata thunb.
Species: H.cordata
Fig: 2.1 Houttuynia cordata plant
Houttuynia cordata Thunb. is a flowering and perennial herb native to China, Japan,
Korea and Southeast Asia (mostly found in India). It is also known by the name as lizard
tail, chameleon plant, heartleaf, fishwort, and bishop's weed. It is often regarded as a food
or herbal medicine which has many benefits for people. It grows optimally on moist,
shady hillside, wayside and ridge of field with an altitude of 300 - 2600 m. It is a perennial
herb with stoloniferous rhizome having two distinct chemotypes. The Chinese chemotype
of the species is found in wild and semi-wild conditions in the North-East of India from
April to September. In the north east region of India especially in Brahmaputra valley of

16

Assam, whole plant of Houttuynia cordata is eaten raw as a medicinal salad for lowering
the blood sugar level (Rathi et al., 2013)
The plant H. cordata is an aromatic medicinal herb with creeping root stock. It grows
about 20-50 cm in height with leaves measuring 4-8 cm in length, 3-6 cm in width and are
broad, ovate-cordate. The stems are green or sometimes purplish red, and either smooth or
pubescent on the nodes. The lower parts of the leaf stalks form a sheath round the stem.
The leaves are usually heart-shaped, 410 cm long and 2.56.0 cm wide, and purple
underneath. Flowers are naked with dense spikes, subtended by four white and petalloid
bracts at the base and flowering occurs in June-July (Bown et al., 1995)

2.2 PHYTOCHEMICAL COMPONENTS

Several test were performed on the plant extract of H. cordata to find its chemical
constituent such as

I)

Alkaloid
Alkaloids are a class of naturally occurring organic nitrogen containing bases.
Alkaloids have diverse and important physiological effects on humans and
other animals. Well known alkaloids include morphine, strychnine, quinine,
ephedrine, and nicotine. Many kinds of alkaloids has been obtained from
H.cordata which possesses anti platelet and cytotoxic activities and include
compounds such as aporphine, pyridine etc (Probstel et al., 1992). They
isolated aristolactam A, aristolactam B, pi- perolactam A and norcepharacdione
B from H. cordata. ( Jong et al,.1993) isolated 7-chloro-6-demethyl-cepharadione, long chain substituted pyridine alkaloids 3,5-dide- canoyl-pyridine,2nonyl-5-decanoylpyridine which are rare in nature and N-methyl-5-methoxypyr-rolidin-2- one from this plant , respectively.

17

Fig 2.2.1 Aristolactum A

II)

Flavonoid

Flavonoids or bioflavonoids are non-nitrogenous plant pigments which gives the

coloration of flower, producing yellow or red/blue pigmentation in petals designed to
attract pollinator animals.They are widely distributed in plants, fulfilling many functions.
A number of Flavonoids has been obtained and identified from H.cordata such as
quercetin which was first to be extracted from the leaves and stems of this plant
( nakamura et al,. 1936) and also involved other compound such as Quercetin-3-O--Dgalac- toside-7-O--D-glucoside, kaempferol 3-O-[-L-rha- mnopyranosyl-(16)--Dglucopyranoside], quercetin 3-O--L-rhamnopyranosyl-7-O--D-glucopyranoside three
flavonoid glycosides , chlorogenic acid methyl ester, 4-[(2E)-3-(-D-glucopyranosyloxy)2-buten-1-yl]-4-hy-droxy-3,5,5-trimethyl-2-cyclohexen-1-one,2-(4-hy-droxyphenyl)ethyl-D-glucopyranoside, 2-(3,4-dihydro- xyphenyl) ethyl--D-glucopyranoside, 4-(-Dglucopy- rano-syloxy)-3-hydroxybenzoic acid five polyphenols , catechin, procyanidin B,
houttuynamide A, houut- tuynoside A were isolated from H. cordata.

Fig2.2.2 Quercetin

18

III)

STEROLS

A number of sterols have been isolated from H. cordata such as Stigmast-4-en-3-one, 3hydroxystigmast-5-en- 7-one, 5-stigmastane-3, 6-dione and stigmast-4-ene-3,6- dione
( Jong et al.,1993). Stigmast-3, 6- dione, sitoindoside I and daucosterol were also isolated
and purified from dried rhizome of H. cordata by solvent extraction, silica gel and
Sephadex LH-20 column chro- matographs (Wang et al., 2007).

IV)

AMINO ACIDS AND MICRO ELEMENTS

H. cordata contains more than 20 amino acids, including alanine, valine, glutamic acid,
aspartic acid, isoleucine, proline, leucine, glycine, serine, lysine, cystine, tyrosine,
methionine, phenylalanine, histidine, threonine, trypto- phane, arginine, hydroxyproline
and citrulline (Choe et al., 1989)( Mori et al.,1995). Among all amino acids, the glutamic
acid content was the highest followed by leucine and aspartic acid. Many micro- elements
including iron, magnesium, manganese, potassium, copper, zinc and calcium, etc., are also
present in H. cordata.

2.3 HEALTH BENEFITS

H.cordata has many benefits for human health. As a fresh herb, it is often eaten, applied to
the skin, or placed in a bath. When it is dried, it is used in tea and as a healing powder. In
the recent research data show that Houttuynia cordata Thunb. helps improve
body metabolism by making the blood clean and promoting the kidneys various functions
as

the

plant

contains

biological

active

compounds

such

as

Decanoilacetadehyde, Quercitrin and Kallum in its leaves, stems and roots. Besides, the
plant has anti-bacteria, anti inflammation, anticancer and anti-oxidation abilities, which
helps remove toxicity in the body (Li et al., 2005). Some of the benefits included are:

I) ALLERGIES

19

In a recent article, researchers examined H.cordata effects against allergies and asthma.
Both issues are the result of the excessive release of histamine, a compound that initiates a
local immune response to toxin exposure. When the body is allergic to a substance, the
substance causes the body to produce an excess of histamine. Often known as anaphylactic
reactions, repeated exposure to an allergic substance can be life threatening if not dealt
with properly. Studies demonstrate that H.cordata has excellent inhibitory effects on
histamine release, possibly blocking the compound and reducing anaphylactic effects (Lu
et al., 2006).

II) REMOVES FREE RADICALS

A free radical is any chemical compound with a free, unpaired electron. Compounds with
a missing electron are often unstable and exert damage on other compounds, cells, and
DNA. Free radicals are thought to artificially age the body and cause cancer. Recent article
has shown that there are many plants native to Korea, including H.cordata, which are
excellent free radical scavengers. In other words, the biological components of
H.cordata search the blood and neutralize free radicals. Houttuynia cordata contains
polyphenols, the active free radical neutralizing chemical (Lu et al., 2006)

III) BACTERIAL PROTECTION

H.cordata fights a variety of harmful bacteria in the digestive system, especially
Propionibacterium acnes and Staphylococcus epidermis. Both types are the primary cause
of acne; some professionals recommend it as natural, helpful skin supporter.

IV) IMMUNE SYSTEM

Houttuynia cordata decoction in vitro can obviously promote the peripheral blood leukocy
te staphylococcus aureus ability. Sodium houttuyfonate can improve the patients with chro
nic bronchitis leukocytee phagocytosis function; improve the rabbit and patients serum pro
perdin level (Lu et al., 2006).
20

V) ANTICANCER/ANTITUMOUR EFFECTS
Extracts and compounds from H. cordata are used as traditional Chinese medicine,
formulae containing H. cordata were reported to have anticancer effect. (X. Kou 2009).
H.cordata extract (HCE) treatment may also lowers the cell viability in various human
cancer cell lines (H.Jung et al., 1996), and (W. Bae et al., 2001) reported that HCE
prevented the increase of mass weight of melanoma BBL16 tumor cells inoculated into
mice.
New houttuynine sodium bisulfite to airy ascites carcinoma may be the inhibition effect of
cancer cells and improve the level of cAMP. Results show that: in different time of mice c
eliac injection of different dosages of the new houttuynia cordata element, the total numbe
r of cancer cells, cancer cells division index, abdomen water were significantly reduced, a
nd the cancer cells within the cAMP level has increased.
T. H. Hoang et al., 2003 has studied the anticancer activity of flavonoid extracts from H.
cordata. The flavonoid extract of H. cordata gave highest rate of death and showed good
inhibitory effect on the growth of ascites tumor by S-180 in mice. The findings suggested
that polyphenol in H. cordata could be an important and necessary factor in the defense
against CYP450-mediated cancers and other chronic diseases (Chen et al., 2005).

VI) ANTIBACTERIAL EFFECT

Kwon et al., revealed that the volatile flavor components of H.cordata showed strong
antibacterial activities against Bacillus cereus, Bacillus subtilis, Vibrio cholerae and
Vibrio parahaemo- lyticus. Meng (J. Meng et al., 2008)

found that water and ethanol

extracts of fresh and dry H. cordata showed antimicrobial activity against Staphylococcus
aureus and Escherichia coli. Houttuynia cordata extract isolated from the rhizome of H.
cordata suppressed the growth of yeasts and molds ( Y. Isogai,1952) and 3-nonylpyrazole
inhibited the growth of Staphylococcus aureus, Bacillus subtilis, Trichophytons,
Zygosaccharomyces salsus, and Aspergillus niger (G. Kim et al.,2008)

VII) ANTIVIRAL EFFECTS

21

H. cordata has progressively shown anti-HIV activity (S. B. Bharate et al., 2003).The
compounds from fresh plants of H. cordata showed dose-dependent virucidal activity
against HIV-1 without showing cytotoxicity in vitro.

VIII) DIURETIC EFFECT

By houttuynia cordata perfusion toad renal or frog web, can make the blood capillary dilat
e, increases the flow of blood and urine secretion, which have the effect of dieresis.

The

components quercetin extracted from the leaves and isoquercitrin from the floral spikes
and fruit spikes of Houttuynia cordata show diuretic action.

2.4 SILVER NANOPARTICLES

Silver nanoparticles have been explored as demandable research of interest in the field of
nanoscience and technology and therefore have been extensively studied (Nair, L. S. et al.,
2007) (Zhang, Y. W et al., 2008) (Sharma, V.K. et al., 2009). AgNPs find use in several
applications such as electrical conducting, catalytic, sensing, optical and antimicrobial
properties (Abou El-Nour et al., 2010) .Silver nanoparticles are the metal of choice as they
hold the promise to kill microbes effectively and effect on both extracellularly as well
intracellularly (Raid Salih Jawaad et al., 2014).

2.4.1 SIGNIFICANCE OF SILVER NANOPARTICLES

Silver is one of the basic and naturally occurring elements that make up our planet. Silver
has been occupied in 47th position in periodic table. It has an atomic weight of 107.8 and
two natural isotopes 106.90 Ag and 108.90 Ag with abundance of 52 and 48%. Silver can
be present in four different oxidation states: Ag0, Ag2+, Ag3+. Metallic silver is insoluble
in water, but metallic salts such as AgNO3 and Silver chloride are soluble in water (WHO,
2002).Soluble silver compounds such as silver slats, have been used in treating mental illness,
epilepsy, nicotine addition, gastroenteritis and infectious diseases including syphilis and
gonorrhea (Ramya et al., 2012).

Pure silver has the highest electrical and thermal

conductivity of all metals and has the lowest contact resistance. Silver nanoparticle are
smaller than 100 nm and it is consist of about 20-15,000 silver atoms (J, C.X.a.S.H. et al.,
22

2008). It has particular interest because of distinctive properties such as good conductivity,
chemical stability, catalytic and antibacterial activity due to the higher surface area per
mass, allowing a larger amount of atoms to interact with their surroundings (Frattini et al.,
2005). Due to its antimicrobial activity silver is used in nano formulation.

2.5 SILVER NANOPARTICLE AS AN ANTIMICROBIAL AGENT

Silver nanoparticles have been widely studied as antimicrobial materials. (MartinezCastanon et al., 2008) studied the effect on nanoparticle size on antibacterial effectiveness.
The increased antimicrobial activity of the smaller nanoparticles could be due to the fact
that smaller particles have an easier time getting through the cell membrane and cell wall
and that relative to larger nanoparticles; smaller particles have a greater surface area to
volume ratio (Martinez-Castanon et al., 2008). Silver nanoparticles have the ability to
anchor to the bacterial cell wall and subsequently penetrate it, thereby causing structural
changes in the cell membrane like the permeability of the cell membrane and death of the
cell (Salopek-Sondi et al., 2004) There have been electron spin resonance spectroscopy
studies suggested that there is formation of free radicals by the silver nanoparticles when
in contact with the bacteria, and these free radicals have the ability to damage the cell
membrane and make it porous which can ultimately lead to cell death. Silver nanoparticles
are non toxic and a safe antibacterial agent; developments for patient use are currently
underway, which present an exciting horizon in the future of antibiotics.

2.6 MECHANISM OF ACTION OF SILVER NANOPARTICLES ON

MICROBES
Danilcauk et al., reported that when silver nanoparticles contact with microbes, there is a
formation of free radicals and these free radicals have the ability to damage the bacterial
cell membrane. Therefore, make it porous which can ultimately lead to cell damage (Kim
et al., 2007) which results generation of oxygen species, produced possibly through the
inhibition of a respiratory enzyme by silver ions and attack the cell itself. Moreover, the
released silver ions by the nanoparticles (Feng et al., 2008) can interact with the thiol
groups of many vital enzymes and therefore inactivate those (Matsumura et al., 2003).

23

Since silver is a soft acid, it has high tendency to react with bases (Morones et al., 2005).
The cells are generally made up of sulfur and phosphorus which is soft bases, the action of
these nanoparticles on the cell can cause the reaction and subsequently lead to cell death
(Morones et al., 2005).

3. OBJECTIVE:

Preparation and extraction from Houttuynia cordata leaf.

Green synthesis of silver nanoparticle by using autoclave method.
Optimization of silver nanoparticle.
Antimicrobial activity of silver nanoparticle.

4. MATERIALS AND METHOD

4.1
4.1.1

EXPERIMENTAL MATERIALS
PLANT MATERIAL: Freshly collected Houttuynia cordata leaves were

obtained from Dimapur, Nagaland and were used in the study.

4.1.2 BACTERIAL STRAINS: Gram positive bacteria (Staphylococcus aureus) and

two gram negative (Escherichia coli and Pseudomonas aeruginosa) bacteria strains were
used for this experiment.

4.2 PREPARATION OF PLANT EXTRACT FROM HOUTTUYNIA

CORDATA LEAVES
Houttuynia cordata leaves were washed and cleaned with tape water and dried for 15 days
under the shade. They were ground into fine powder by using pestle mortar. 1 gram of
sterilized H. cordata leaf powder were mixed with 100 ml. of ddH 2O and kept in boiling
water bath for 1 hour and filtered with what man no.1 filter paper. Filtrates were collected
in brown bottle and stored in refrigerator for further studies.

4.3 BIOSYNTHESIS OF NANOPARTICLES

24

Silver nitrate was used for biosynthesis of nanoparticles using plant extract (H. cordata)
reducing agent. 0.8 mM of AgNO3 was prepared separately with the leaf extract in the
ratio (1:10) v/v and were kept in magnetic stirrer at room temperature for 30 minutes. The
reaction mixture was then kept in autoclave at 15 psi for 15 minutes. The sizes of the
nanoparticles were then measured by Zetasizer (S90) at room temperature.

4.4 OPTIMIZATION OF SILVER NANOPARTICLES

Silver nanoparticles were optimized under different concentration of leaf extract and silver
nitrate solution. 0.1,0.2,0.4,0.5,0.6,0.8,1 and 1.2mM of silver nitrate solution were mixed
with H. cordata leaf extract 1:10 v/v at room temperature and were kept in magnetic
stirrer for 30 minutes and then autoclaved at 15 psi for 15 minutes. The reaction mixture
was then cooled and the sizes of the silver nanoparticles were then measured by Zetasizer
(S90). The data obtained were recorded.
Another reaction mixture was performed using leaf extract and silver nitrate solution with
ratio (1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 10:10) v/v keeping silver nitrate
solution constant.
With the data obtained from different concentration of leaf extract, 7:10 v/v of leaf extract
and silver nitrate solution yielded minimum size of silver nanoparticles. Using this ratio,
another silver nitrate concentration (1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM,
9mM, 10mM) was performed and were autoclaved at 15 psi for 15 minutes and the sizes
of the nanoparticles were measured in the Zetasizer (S90).

4.5 PHYTOCHEMICAL TEST

1. TEST FOR ALKALOID
To 1 ml of extract, add 1 ml of Mayers reagent (Potassium mercuric iodide solution)
(Mercuric chloride-1.36g, potassium iodide-5gram in 100 ml water) .Whitish yellow
colored ppt. indicates the presence of alkaloids.

2. TEST FOR TERPENOID

25

2ml of CHCl3 was added. 0.5ml extract to this few drops of conc.H 2SO4 was added along
the inner walls of the test tube. Reddish brown coloration on the interface indicates the
presence of terpenoid.

3. TEST FOR FLAVONOID

The extract is treated with sodium hydroxide; formation of yellow color indicates the
presence of flavonoid.
4. TEST FOR STEROID Dissolve the extract in CHCl3 and add equal volume of
H2SO4. Formation of bluish red to cherry color in CHCl 3 layer and green
fluorescence in the acid layer represents the steroidal components in tested extract.

5. TEST FOR CARBOHYDRATES AND SUGARS

Fehlings test: To 1ml of the extract, add equal quantities of Fehling solution A and B,
upon heating formation of a brick red precipitate indicates the presence of sugars.
6. TEST FOR SAPONIN:
Test for Saponins a) Foam test 1ml solution of extract was diluted with distilled water
to 20 ml and shaken in a graduated cylinder for 15 minutes. Development of stable
foam suggests the presence of saponins. b) 1ml extract was treated with 1% lead
acetate solution. Formation of white precipitates indicates the presence of saponins.
7. TEST FOR PHENOLIC COMPOUNDS:
Detection of Phenol Compound Ferric chloride test the extract (50 mg) was
dissolved in 5 mL of distilled water. To this, few drops of neutral 5% ferric
chloride solution were added. A dark green color indicated the presence of Lead
acetate test the extract (50 mg) was dissolved in 5 mL of distilled water. To this,
3ml of 10% lead acetate were added. A bulky white precipitate indicated the
presence of phenol compounds.

4.6 ANTIMICROBIAL ACTIVITY OF SILVER NANOPARTICLES

SYNTHESIZED USING H.CORDATA LEAF EXTRACT
4.6.1 TEST MICROORGANISM
26

The test microorganism used were Gram (+ve) Escherichia coli bacteria, Gram (-ve)
Staphylococcus aureus, Pseudomonas aeruginosa bacteria for antimicrobial screening.
The microorganisms were maintained in bacterial culture on nutrient agar slants and were
stored at -4oC.

4.6.2 DETERMINATION OF ANTIBACTERIAL ACTIVITY

Antimicrobial activity of the plant extract, Ag NP synthesized from the respective extract
0.8 mM silver nitrate, 0.8 mM Ampicillin and 0.8 mM Tetracycline were determined by
well diffusion method. Agar medium was poured on to sterile petri plates and the
inoculums containing the cultured bacteria were spread into the solid plates with the help
of a sterile swab and each plate was divided into two equal parts. With the help of a sterile
borer, wells of 5mm diameter were cut on the nutrient agar plates, and on each well the
required suspension was loaded with the help of a micropipette. The plates were incubated
at 37oC for 24 hours. After incubation period, the plates were observed for zone of
inhibition (ZI) and the diameter of the inhibition zone was measured using scale and mean
were recorded.

5. RESULT AND DISCUSSION

The main parameters affecting the formation and size of nanoparticles, includes ratio
of silver nitrate solution and plant extract were investigated and optimized.

27

5.1 PHYTOCHEMICAL ANALYSIS RESULT

FIG: 5.1 phytochemical analysis of Houttuynia cordata plant extract.

Table: 5.1
Componen

Alkaloi

Terpenoi

Flavonoi

Steroi

ts

reducin

Saponi

Phenoli

g
Result

Sugar
+

28

(a)

(b)

Fig: 5.2 Color changes of plant extract & AgNO3 solution (a) Before autoclave (b)
After autoclave.

29

30

Fig: 5.5 Size of AgNP before and after autoclave.

Size Distribution by Intensity

Intensity (Percent)

10
8
6
4
2
0
0.1

10

100

1000

10000

Size (d.nm)

Record 147: 3.)

0.8mM agno3+plant extract after autoclave 1

Figure: 5.6 Ag NP size distribution by intensity

Table: 5.2 Antimicrobial activities test by well diffusion method

Microorganism

Diameter of zone of inhibition (mm)

s
AgN

Plant

extract

0.8mM AgNO3

0.8mM Amp

0.8mMTet

31

Control

E.coli

71.1

40.5

61

233.21

210.8

0.00

S.aureus

80.5

31.1

51

244

261.5

0.00

P.aeruginosa

71.5

40.5

51

222

221.5

0.00

Results were expressed as meanstandard deviation

Fig: 5.7 Antimicrobial activity tests by well diffusion method.

32

6. CONCLUSION
33

The present study reports a cost-effective and eco-friendly method of green technology
approach for synthesizing of silver nanoparticles produced by Houttuynia cordata plant
extract .The resulting nanoparticles shows a size ranged from 49-60nm at 7:10 v/v with
plant extract and silver nitrate solution. The zones of inhibition were formed in the
antimicrobial screening test which indicated, that the AgNPs synthesized in this process
has the efficient antimicrobial activity against pathogenic bacteria when compared to plant
extract and 0.8mM silver nitrate solution. Thus, the biologically synthesized silver
nanoparticles could be of immense use in medical field for their efficient antimicrobial
function.

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