SYNTHESIS OF SILVER AND GOLD NANOPARTICLES USING ZIZIPHUS NUMMULARIA LEAVES EXTRACT AND ITS ANTIMICROBIAL STUDY

Dissertation Submitted to RAJRISHI COLLEGE ALWAR in partial fulfillment of the requirement for the Degree

Of

Bachelor of Science

In

(B.Sc. in biotechnology)

By

KAPIL KUMAR AVASTHI

Department of biotechnology

Raj Rishi College

2013

SYNTHESIS OF SILVER AND GOLD NANOPARTICLES USING

ZIZIPHUS NUMMULARIA LEAVES EXTRACT AND ITS

ANTIBACTERIAL ACTIVITY STUDY

Dissertation submitted to RAJ RISHI COLLEGE ALWAR (RAJASTHAN) INDIA towards partial fulfillment for the award of

Bachelor of Science in Biotechnology

By

Kapil Kumar Avasthi

B.Sc. (Biotechnology), Semester VI

Session 2010-2013

Under the guidance of

Er. Mukesh Sharma

Jaipur Institute of biotechnology

Maharaja Vinayak Global University, Jaipur

CERTIFICATE – I

This is to certify that Kapil Kumar Avasthi a student of B.Sc. Biotechnology has completed dissertation entitled “SYNTHESIS OF SILVER AND GOLD NANOPARTICLES USING ZIZIPHUS NUMMULARIA LEAVES EXTRACT AND ITS ANTIMICROBIAL STUDY” which is being submitted to the Department of biotechnology , Raj Rishi College, Alwar

To the best of my knowledge the work has not been submitted in part or in full to

any other University or Institute for the Award of any degree.

The assistance and help received during the course of investigation is duly

acknowledged.

(Advisor) Dated :

Dedicated toMy teacher

&My family

Synthesis of silver and gold nanoparticles

Using ziziphus nummularia leaf

Extract and its Antimicrobial

Activity Study

List of Abbireviation:-

z.nummularia Ziziphus nummularia

AgNO3 Silver nitrate

AgNPs Silver nanoparticles

CFU Colony forming unit

FTIR Fourier transforms infrared

MIC minimum inhibitory concentration

mM Milli meter

MRSA Methicillin resistant S.aureus

NIPs Nanoscale inorganic particles

Nm Nano meter

NNI National nanotechnology initiative

NPs Nanoparticles

PEN project on emerging nanotechnologies

rpm Rotation per minutes

SAED Selected area electron diffraction

S.aureus Staphylococcus aureus

SEM scanning electron microscopy

STEM Scanning transmission electron

microscopy

TEM transmission electron microscopy

UV-vis spectra UV-visible spectroscopy

XRD X-ray diffraction

ZOI Zone of inhibition

ZP Zeta potential

1. Introduction

- Nanotechnology

- Zizyphus

2. History

- Nanotechnology

- Zizyphus

3. Applications

- Nanoparticles

- Zizyphus

4. Review of literature

- Green synthesis of nanoparticles

- Antibacterial activity of zizyphus

5. Methods of green synthesis

- Materials and methods

- Materials

- Plant collection

- Preparation of chloroauric acid solution

- Preparation of silver nitrate solution

- Preparation of plant extract

- Synthesis of silver nanoparticles

- Synthesis of gold nanoparticles

- UV-Vis spectrometer

6. Antibacterial activity

- MIC

- MBC

7. Results

8. Conclusion

9. References

Introduction

Nanotechnology

Nanotechnology literally means any technology on a nanoscale that has applications in the real world. Nanotechnology encompasses the production and application of physical, chemical, and biological systems at scales ranging from individual atoms or molecules to submicron dimensions, as well as the integration of the resulting nanostructures into larger systems. Nanotechnology is likely to have a profound impact on our economy and society in the early 21st century, comparable to that of semiconductor technology, information technology, or cellular and molecular biology. Science and technology research in nanotechnology promises breakthroughs in areas such as materials and manufacturing, nanoelectronics, medicine and healthcare, energy, biotechnology, information technology, and national security. It is widely felt that nanotechnology will be the next Industrial Revolution. Nanometer-scale features are mainly built up from their elemental constituents. Examples include chemical synthesis, spontaneous self-assembly of molecular clusters (molecular self-assembly) from simple reagents in solution, biological molecules (e.g., DNA) used as building blocks for production of three-dimensional nanostructures, and quantum dots (nanocrystals) of arbitrary diameter (about 10–105 atoms). The definition of a nanoparticle is an aggregate of atoms bonded together with a radius between 1 and 100 nm. It typically consists of 10–105 atoms. A variety of vacuum deposition and nonequilibrium-plasma chemistry techniques are used to produce layered nanocomposites and nanotubes. Atomically controlled structures are produced using molecular-beam epitaxy and organometallic vapor-phase epitaxy. Micro- and nanosystem components are fabricated using top-down lithographic and nonlithographic fabrication techniques and range in sizefrom micro- to nanometers. Continued improvements in lithography for use in the production of nanocomponents have resulted in line widths as small as 10 nm in experimental prototypes. The nanotechnology field, in addition to the fabrication of nanosystems, provides impetus for the development of experimental and computational tools. The discovery of novel materials, processes, and phenomena at the nanoscale and the development of new experimental and theoretical techniques for research provide fresh opportunities for the development of innovative nanosystems and nanostructured materials. The properties of materials at the nanoscale can be very different from those at a larger scale. When his dimension of a material is reduced from a large size, the properties remain the same a t first, and then small changes occur, until finally when the size drops below 100 nm, dramatic changes in properties can occur. If only one length of a three-dimensional nanostructure is of nanodimension, the structure is referred to as a quantum well; if two sides are of nanometer length, the structure is referred to as a quantum wire. A quantum dot has all three dimensions in the nano range. The term quantum is associated with these three types of nanostructures because the changes in properties arise from the quantum-mechanical nature of physics in the domain of the ultrasmall. Materials can be

nanostructured for new properties and novel performance. This field is opening new avenues in science and technology.

Ziziphus nummularia

Zizyphus nummularia is a thorny small bush or a shrub 6-8 m, with widely divaricating, flexuosus, pale-purplish stems and grey velvety stipular prickles in pairs. The branches are widely divaricate, purplish in colour and with a velvety surface. The lateral roots system is deep and extensive. Leaves alternate, simple, ovate or orbicular, 2.5 cm long, deep green and shining above, densely tomatoes beneath and white, serrate, 3- to 5- veined from the base. Stipules frequently spine scent, dark brown; one short, hooked, bent downwards while the other is 1 cm and straight. Flowers small, bisexual, pentamerous, pale yellow, in auxiliary heads, or cymes; petals may be absent; stamens inserted beneath the cone-shaped disc; ovary enveloped by the disc, 2- to 4-chambered. Fruits a red or black fleshy drupe, globosely, less than one cm diameter. Seed smooth, brownish, shinning and soft, usually 2 contained in hard stones of the fruit. It flowers in the rainy season (July to September) while fruiting takes place in November to December. Leaves fall in January-March, and are simultaneously replaced with new ones. It is found on most ecological habitats such as hills, ravines or plains including cultivated fields Altitude: up to 1700 m altitude Mean annual rainfall: 100-1000 mm Temperature range: 15-35°C Soil type: It grows on very shallow and skeletal soils, gravely plains, sand dunes, alluvium and rocky areas. The map above shows countries where the species has been planted. It does neither suggest that the species can be planted in every ecological zone within that country, nor that the species cannot be planted in other countries than those depicted. Species distribution- Afghanistan, India, Iran, Lebanon, Pakistan, Zimbabwe Mauritania, Nigeria, Uganda

History

Nanotechnology

On December 29, 1959 at the California Institute of Technology, Nobel Laureate Richard P. Feynman gave a talk at the Annual Meeting of the American Physical Society that has become one of the 20th century’s classic science lectures, entitled There’s Plenty of Room at the Bottom [1.8]. He presented a technological vision of extreme miniaturization in 1959, several years before the word chip became part of the lexicon. He talked about the problem of manipulating and controlling things on a small scale. Extrapolating from known physical laws, Feynman envisioned a technology using the ultimate toolbox of nature, building nanoobjects atom by atom or molecule by molecule. Since the 1980s, many inventions and discoveries in the fabrication of nanoobjects have been testaments to his vision. In recognition of this reality, the National Science and Technology Council (NSTC) of the White House created the Interagency Working Group on Nanoscience, Engineering, and Technology (IWGN) in 1998. Ina January 2000 speech at the same institute, President W. J. Clinton talked about the exciting promise of nanotechnology and the importance of expanding research in nanoscale science and technology more broadly. Later that month, he announced in his State of the Union Address an ambitious US$ 497 million federal, multiagency National Nanotechnology Initiative (NNI) in the fiscal year 2001 budget, and made the NNI a top science and technology priority [1.9, 10]. The objective of this initiative was to form a broad-based coalition in which academia, the private sector, and local, state, and federal governments work together to push the envelope of nanoscience and nanoengineering to reap nanotechnology’s potential social and economic benefits.Funding in the USA has continued to increase. In January 2003, the US Senate introduced a bill to establish a National Nanotechnology Program. On December 3, 2003, President George W. Bush signed into law the 21st Century Nanotechnology Research and Development Act. This legislation put into law programs and activities supported by the National Nanotechnology Initiative. The bill gave nanotechnology a permanent home in the federal government and authorized US$ 3.7 billion to be spent in the 4 year period beginning in October 2005 for nanotechnology initiatives at five federal agencies. The funds would provide grants to researchers, coordinate research and development (R&D) across five federal agencies [the National Science Foundation (NSF), the Department of Energy (DOE), the National Aeronautics and Space Administration (NASA), the National Institute of Standards and Technology (NIST), and the Environmental Protection Agency (EPA)], establish interdisciplinary research centers, and accelerate technology transfer into the private sector. In addition, the Departments of Defense (DOD), Homeland Security, Agriculture, and Justice as well as the National Institutes of Health (NIH) also fund large R&D activities. They currently for more than one-third of the federal budget for nanotechnology.

Ziziphus nummularia

Ziziphus nummularia belonging to the family Rhamnaceae, commonly known as Jharber in Hindi is a most commonly occurring branched thorny shrub species in the Indian desert with a height of 1-2 m and light colored bark. The leaves are antipyretic and reduce obesity. The fruit is cooling, tonic, digestible, laxative aphrodisiac and removes biliousness, thirst, vomiting and burning sensations. The dried fruits contain alkaloids, triterpenoids and saponins. They are anticancer, anodyne, refrigerant, sedative, pictorials, styptic, stomachic and tonic. They are used to purify the blood and aid digestion. They are also used internally in the treatment of a range of conditions including loss of appetite, chronic fatigue, diarrhea, pharyngitis, bronchitis, burns, anaemia, irritability, hysteria. The drug has been scientifically validated for certain pharmacological effects viz. antitumor, anthelmintic , antibacterial , and antifertility effects. Phytochemical reports on Ziziphus nummularia have revealed the presence of polysaccharides, pectin composed of l-rhamnose, d-galacturonic acid, dgalactose, l-arabinose, peptide alkaloids, cyclopeptides, flavonoides, saponins, triterpenoides, fatty acides, Ziziphin N, O, P & Q and dodecaacetylprodelphinidin B3. The compounds nummularogenin, zizynummin and lapachol have been isolated from the plant. However, there are no reports on the pharmacognostical studies of the plant. Hence, the present work is an attempt in this direction and includes morphological and physical evaluation, determination of physic chemical constants and preliminary phytochemical screening of different extracts of Z. nummularia.

Taxonomy of Ziziphus nummularia:

• Kingdom: Plante

• Division: Magnoliophyta

• Class: Magnoliopsida

• Order: Rosales

• Family: Rhamnaceae

• Genus: Zizyphus

• Species: Z. nummularia

Application

Application of Nanoparticles

Tissue engineering

Natural bone surface is quite often contains features that are about 100 nm across. If the surface of an artificial bone implant were left smooth, the body would try to reject it. Because of that smooth surface is likely to cause production of a fibrous tissue covering the surface of the implant. This layer reduces the bone-implant contact, which may result in loosening of the implant and further inflammation. It was demonstrated that by creating nano-sized features on the surface of the hip or knee prosthesis one could reduce the chances of rejection as well as to stimulate the production of osteoblasts. The osteoblasts are the cells responsible for the growth of the bone matrix and are found on the advancing surface of the developing bone.

The effect was demonstrated with polymeric, ceramic and, more recently, metal materials. More

than 90% of the human bone cells from suspension adhered to the nanostructured metal surface,

but only 50% in the control sample. In the end this findings would allow to design a more

durable and longer lasting hip or knee replacements and to reduce the chances of the implant

getting loose. Titanium is a well-known bone repairing material widely used in orthopaedics and

dentistry. It has a high fracture resistance, ductility and weight to strength ratio. Unfortunately, it

suffers from the lack of bioactivity, as it does not support cell adhesion and growth well. Apatite

coatings are known to be bioactive and to bond to the bone. Hence, several techniques were used

in the past to produce an appetite coating on titanium. Those coatings suffer from thickness non-

uniformity, poor adhesion and low mechanical strength. In addition, a stable porous structure is

required to support the nutrients transport through the cell growth.

It was shown that using a biomimetic approach – a slow growth of nanostructured apatite film

from the simulated body fluid – resulted in the formation of a strongly adherent, uniform

nanoporous layer. The layer was found to be built of 60 nm crystallites, and possess a stable

nanoporous structure and bioactivity.

Cancer therapy

Photodynamic cancer therapy is based on the destruction of the cancer cells by laser generated

atomic oxygen, which is cytotoxic. A greater quantity of a special dye that is used to generate the

atomic oxygen is taken in by the cancer cells when compared with a healthy tissue. Hence, only

the cancer cells are destroyed then exposed to a laser radiation. Unfortunately, the remaining dye

molecules migrate to the skin and the eyes and make the patient very sensitive to the daylight

exposure. This effect can last for up to six weeks.

To avoid this side effect, the hydrophobic version of the dye molecule was enclosed inside a

porous nanoparticle. The dye stayed trapped inside the Ormosil nanoparticle and did not spread

to the other parts of the body. At the same time, its oxygen generating ability has not been

affected and the pore size of about 1 nm freely allowed for the oxygen to diffuse out.

Multicolour optical coding for biological assays

The ever increasing research in proteomics and genomic generates escalating number of

sequence data and requires development of high throughput screening technologies.

Realistically, various array technologies that are currently used in parallel analysis are likely to

reach saturation when a number of array elements exceed several millions. A three-dimensional

approach, based on optical "bar coding" of polymer particles in solution, is limited only by the

number of unique tags one can reliably produce and detect.

Single quantum dots of compound semiconductors were successfully used as a replacement of

organic dyes in various bio-tagging applications. This idea has been taken one step further by

combining differently sized and hence having different fluorescent colours quantum dots, and

combining them in polymeric microbeads. A precise control of quantum dot ratios has been

achieved. The selection of nanoparticles used in those experiments had 6 different colours as

well as 10 intensities. It is enough to encode over 1 million combinations. The uniformity and

reproducibility of beads was high letting for the bead identification accuracies of 99.99%.

Manipulation of cells and biomolecules

Functionalized magnetic nanoparticles have found many applications including cell separation

and probing; these and other applications are discussed in a recent review. Most of the magnetic

particles studied so far are spherical, which somewhat limits the possibilities to make these

nanoparticles multifunctional. Alternative cylindrically shaped nanoparticles can be created by

employing metal electrode position into nonporous alumina template. Depending on the

properties of the template, nanocylinder radius can be selected in the range of 5 to 500 nm while

their length can be as big as 60 μm. By sequentially depositing various thicknesses of different

metals, the structure and the magnetic properties of individual cylinders can be tuned widely.

As surface chemistry for fictionalization of metal surfaces is well developed, different ligands

can be selectively attached to different segments. For example, porphyries with thiol or carboxyl

linkers were simultaneously attached to the gold or nickel segments respectively. Thus, it is

possible to produce magnetic nanowires with spatially segregated fluorescent parts. In addition,

because of the large aspect ratios, the residual magnetization of these nanowires can be high.

Hence, weaker magnetic field can be used to drive them. It has been shown that a self-assembly

of magnetic nanowires in suspension can be controlled by weak external magnetic fields. This

would potentially allow controlling cell assembly in different shapes and forms. Moreover, an

external magnetic field can be combined with a lithographically defined magnetic pattern

("magnetic trapping").

Protein detection

Proteins are the important part of the cell's language, machinery and structure, and understanding

their functionalities is extremely important for further progress in human well being. Gold

nanoparticles are widely used in immunohistochemistry to identify protein-protein interaction.

However, the multiple simultaneous detection capabilities of this technique are fairly limited.

Surface-enhanced Raman scattering spectroscopy is a well-established technique for detection

and identification of single dye molecules. By combining both methods in a single nanoparticle

probe one can drastically improve the multiplexing capabilities of protein probes. The group of

Prof. Mirkin has designed a sophisticated multifunctional probe that is built around a 13 nm gold

nanoparticle. The nanoparticles are coated with hydrophilic oligonucleotides containing a Raman

dye at one end and terminally capped with a small molecule recognition element (e.g. biotin).

Moreover, this molecule is catalytically active and will be coated with silver in the solution of

Ag (I) and hydroquinone. After the probe is attached to a small molecule or an antigen it is

designed to detect, the substrate is exposed to silver and hydroquinone solution. A silver-plating

is happening close to the Raman dye, which allows for dye signature detection with a standard

Raman microscope. Apart from being able to recognize small molecules this probe can be

modified to contain antibodies on the surface to recognize proteins. When tested in the protein

array format against both small molecules and proteins, the probe has shown no cross-reactivity.

Commercial exploration

Some of the companies that are involved in the development and commercialization of

nonmaterial’s in biological and medical applications are listed below. The majority of the

companies are small recent spinouts of various research institutions. Although not exhausting,

this is a representative selection reflecting current industrial trends. Most of the companies are

developing pharmaceutical applications, mainly for drug delivery. Several companies exploit

quantum size effects in semiconductor nanocrystals for tagging biomolecules, or use bio-

conjugated gold nanoparticles for labelling various cellular parts. A number of companies are

applying nano-ceramic materials to tissue engineering and orthopedics.

Examples of Companies commercializing nanomaterials for bio- and medical applications.

Most major and established pharmaceutical companies have internal research programs on drug

delivery that are on formulations or dispersions containing components down to nano sizes.

Colloidal silver is widely used in anti-microbial formulations and dressings. The high reactivity

of titania nanoparticles, either on their own or then illuminated with UV light, is also used for

bactericidal purposes in filters. Enhanced catalytic properties of surfaces of nano-ceramics or

those of noble metals like platinum are used to destruct dangerous toxins and other hazardous

organic materials.

Future directions

As it stands now, the majority of commercial nanoparticles applications in medicine are geared

towards drug delivery. In biosciences, nanoparticles are replacing organic dyes in the

applications that require high photo-stability as well as high multiplexing capabilities. There are

some developments in directing and remotely controlling the functions of nano-probes, for

example driving magnetic nanoparticles to the tumor and then making them either to release the

drug load or just heating them in order to destroy the surrounding tissue. The major trend in

further development of nanomaterials is to make them multifunctional and controllable by

external signals or by local environment thus essentially turning them into nano-devices.

Application of Ziziphus numnmularia

Food: The sweet and acidulous fruit is either eaten fresh, pickled, dried or made into confectionery. The juice can be made into a refreshing drink. In India, the fruit, when fully ripe and less than one centimeter in diameter, are gathered in the beginning of the winter months, dried, ground, and sieved. The powder formed is eaten either alone, mixed with Gur (a sugar condiment) or Bajra (millet) flour.

Fodder: The leaves of Z. nummularia provide excellent fodder for livestock. In India, the average total yield of forage was about 1000 kg ha-1. The leaves are collected dried and stored.

Fuel: It is a source of high calorific value (4400 kcal/kg) fuel and charcoalTimber: The heartwood is yellow to dark brown, hard, 738 kg/m3 and it is used in farm implements and for house Construction.

Medicine: Dried fruit used medicinally as astringent in bilious affliction in India. The leaves are used to treat scabies and other skin diseases.

Pharmacology Activity:Zizyphus nummularia contains Antimicrobial activity, Antibacterial activity, Anti-inflammatory and antispastic effect, Antiulcer activity, Antiallergic.

Services:

Intercropping: Z. nummularia shrubs are often intercropped with millet, legumes and oil seeds

Erosion control: The shrubs have been shown to effectively check wind erosion, help in deposition of soil, and bring about a change in the microhabitat, causing favorable conditions for the appearance of succession species such as perennial grasses

Shade: It provides shade. Boundary or barrier or support: In India, it is commonly erected as ‘brush-wood barriers’ (micro-windbreaks) together with Crotalaria burhia.

Reclamation: It has proved successful in sand dune stabilization in India.

Tree Management: It produces copious coppice shoots and roots suckers forming dense thorny thickets often collecting moulds of leaves and dust.

Germplasm Management: Seed storage behavior is orthodox. There are 1800-2000 seeds/Kg

Pests and Diseases: This species is a host of larvae of butterfly Tarucus balkanica Freyer in Africa, Balkans, Iran, Asia Minor, Lebanon and Mauritania.

Review

Of

Literature

“Biogenic synthesis of silver nanoparticles by leaf extract of Cassia angustifolia T Peter” (Amaladhas, S Sivagami, T Akkini Devi, N Ananthi and S Priya Velammal)

In this study Cassia angustifolia (senna) is used for the environmentally friendly synthesis of silver nanoparticles. Stable silver nanoparticles having symmetric surface plasmon resonance (SPR) band centred at 420 nm were obtained within 10 min at room temperature by treating aqueous solutions of silver nitrate with C. angustifolia leaf extract. The water soluble components from the leaves, probably the sennosides, served as both reducing and capping agents in the synthesis of silver nanoparticles. The nanoparticles were characterized using UV–Vis, Fourier transform infrared (FTIR) spectroscopic techniques and transmission electron microscopy (TEM). The nanoparticles were poly-dispersed, spherical in shape with particle size in the range 9–31 nm, the average size was found to be 21.6 nm at pH 11. The zeta potential was –36.4 mV and the particles were stable for 6 months. The crystalline phase of the nanoparticles was confirmed from the selected area diffraction pattern (SAED). The rate of formation and size of silver nanoparticles were pH dependent. Functional groups responsible for capping of silver nanoparticles were identified from the FTIR spectrum. The synthesized silver nanoparticles exhibited good antibacterial potential against Escherichia coli and Staphylococcus aureus.

1.) “Synthesis of Silver Nanoparticles from the Aqueous Extract of Leaves of Ocimum sanctum for Enhanced Antibacterial Activity”

(Charusheela Ramteke, Tapan Chakrabarti, Bijaya Ketan Sarangi, and Ram-Avatar Pandey)

The field of nanotechnology is the most active area of research in modern materials science. Though there are many chemical as well as physical methods, green synthesis of nanomaterials is the most emerging method of synthesis. We report the synthesis of antibacterial silver nanoparticles (AgNPs) using leaf broth of medicinal herb, Ocimum sanctum (Tulsi). The synthesized AgNPs have been characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), and X-ray diffractometry. The mean particle of synthesized NPs was found to be 18 nm, as confirmed by TEM. The qualitative assessment of reducing potential of leaf extract has also been carried out which indicated presence of significant amount of reducing entities. FTIR analysis revealed that the AgNPs were stabilized by eugenols, terpenes, and other aromatic compounds present in the extract. Such AgNPs stabilized by Tulsi leaf extract were

found to have enhanced antimicrobial activity against well-known pathogenic strains, namely Staphylococcus aureus and E. coli.

2.) “Biocompatible   synthesis   of   silver   and   gold   nanoparticles   using   leaf extract of Dalbergia sissoo”

                                   (Chandan Singh, Ritesh K. Baboota, Pradeep K. Naik, Harvinder Singh)

This report presents a rapid, reproducible and a green biogenic approach for the biosynthesis of gold and silver nanoparticles using leaf extract of Dalbergia sissoo. The biomolecules present in the plant induced the reduction of Au3+ and Ag+ ions from HAuCl4 and AgNO3 respectively, which resulted in the formation of Dalbergia conjugated nanoparticles. The growth of nanoparticles was monitored by UV-vis spectrophotometer that demonstrated a peak at 545 and 425 nm corresponding to Plasmon absorbance of gold and silver nanoparticles respectively. The leaf extract was found to direct different shape and sized gold nanoparticles. Gold nanoparticles were 50-80 nm in size and their shape varied from spherical to few triangular and hexagonal polyshaped. While silver nanoparticle synthesized were spherical, in the range of 5-55 nm in size. X-ray diffraction studies corroborated that the biosynthesized nanoparticles were crystalline gold and silver. Fourier transform infra-red spectroscopy analysis revealed that biomolecules were involved in the synthesis and capping of silver nanoparticles and gold nanoparticles

“Biological synthesis of silver nanoparticles using Glycine max (soybean) leaf extract”(School of Engineering, Thornbrough Building, University of Guelph, Guelph, N1G 2W1 ON,

Canada.)

Biological synthesis of silver nanoparticles using Glycine max (soybean) leaf extract as reducing agent was reported. The effect of different soybean varieties on the synthesis of silver nanoparticles was investigated. The microstructure of silver nanoparticles was identified from transmission electron microscopy (TEM) and the particle size of silver nanoparticles synthesized by this processes were from 25 to 100 nm. X-ray diffraction (XRD) analysis showed that the synthesized silver nanoparticles crystallized in face centered cubic (FCC) symmetry. The purity and the agglomerations of silver nanoparticles were respectively investigated through particle size and FTIR analysis. Soybean leaf extract shows rapid reduction of silver ions and yields organic free silver nanoparticles.

3.) “Synthesis of silver nanoparticles Using extracts of Neem Leaf and Their Antibacterial Activity”

(asmita j. gavhane, P. Padmanabhan, Suresh P. kamble And Suresh, N jangle)

Silver nanoparticles were synthesized using extracts of Neem (Azadirachta indica) leaves and Triphala-a well known herbal mixture, used widely in treatment of various ailments. The characteristics of silver nanoparticles were studied using NTA, TEM and EDX. The EDX

spectrum of the silver nanoparticles confirmed the presence of elemental silver signal. NTA measurements showed that the average size of silver nanoparticles synthesized using Neem leaves extract were 43nm and 59nm for silver nanoparticles synthesized by Triphala. TEM analysis showed that the silver nanoparticles were predominantly spherical in nature. It was found that the growth of gentamycin and ampicillin resistant K. pneuomoniae was inhibited by both Neem and Triphala synthesized silver nanoparticles. Similar observations were noted forgentamicin and piperacillin resistant S. typhi. Fluconazole resistant C. albicans were found to be sensitive to silver nanoparticles. Growth of multiple drug resistant E.coli was inhibited by silver nanoparticles and this effect was augmented by synergistic action of gentamycin and the silver nanoparticles synthesized by aqueous extract of Neem and Triphala.

4.) “Potential antimicrobial activity of Zizyphus nummularia against medically important pathogenic microorganisms”

(Sujata Gautam a, Ashok Kumar Jain,b, Ajay Kumar a)A. IASCA, ITM Universe, Jiwaji University, Gwalior (M.P), IndiaB. Department of Botany, Jiwaji University, Gwalior (M.P), India

Zizyphus nummularia (Rhamnaceae) found to have potential antibacterial and antifungal activity against four medically important bacterial and fungal strains respectively Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus niger, Aspergillus flavus, Candida albicans and Trichophyton rubrum. The antimicrobial activity of aqueous and ethanol extracts was determined by agar disk diffusion method and dry weight method. The ethanol extract was more active than the aqueous extract. The most susceptible bacteria was Staphylococcus aureus, and fungi Trichophyton rubrum, hence this plant can be further subjected to isolation of the therapeutic antimicrobial and further pharmacological evaluation.

5.) “Antibacterial Activity in Herbal Products Used in Pakistan” (Cynthia Walter, Zabta K. Shinwari, Imaran Afzal, and Riffat N. Malik.)

1Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan2Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan

Antibacterial activity was examined in three herbal products and from ten selected medicinal plants: Ziziphus vulgaris, Malva sylvestris, Onosma bracteatum, Hyssopus officinalis, Ephedra gerardiana, Cordia latifolia, Althaea officinalis, Mentha piperita, Glycyrrhiza glabra, Justica adhatoda. Antibacterial activity was determined by the agar well diffusion method; crude extracts were obtained by using methanol as the extraction solvent. Five concentrations (15 mg/ml, 12.5 mg/ml, 10 mg/ml, 7.5 mg/ml and 5 mg/ml) were used to check the antibacterial activity of plant extracts. Each plant sample was tested against one Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. Most of the plant extracts showed antibacterial activity against the Gram-positive bacterium. The order of antibacterial activity was S. aureus>P. aeurginosa>E. coli. Maximum zones of inhibition were seen in Hyssopus officinalis (3.37 ± 0.05 mm) against S. aureus, Glycyrrhiza glabra (3.6 ± 0.3 mm) against E. coli and Justica adhatoda (2.67 ± 0.06 mm) against P. aeruginosa. Herbal product 1 showed antibacterial activity against P. aeruginosa (2.93 ± 0.15 mm), S. aureus (2.2 ± 0.1 mm) and E. coli (1.33 ± 0.21 mm). Herbal product 2 showed

antibacterial activity against S. aureus (2.93 ± 0.15 mm), P. aeruginosa (2.1 ± 0.1 mm) and E. coli (1.33 ± 0.11 mm). Herbal product 3 was also effective against S. aureus (2.6 ± 0.1 mm), P. aeruginosa (2.33 ± 0.51 mm) and E. coli (1.33 ± 0.15 mm). All three herbal products show significant antibacterial activity.

6.) “Antibacterial activity of Ziziphus nummularia and Prosopis cineraria leaves extracts against Staphylococcus aureus and Escherichia coli”

(Sharma, S. K.; Singh, J.; Maherchandani, S.; Kashyap, S. K.)

About 80% of world's rural population relies on herbal traditional medicines as their primary health care. Ziziphus spp. and Prosopis spp., common plants of arid region are used as feed and have been reported to be used in many clinical indications like wounds, abscess, burn, cough, bronchitis, dysentery, diarrhea, stomatitis, gum bleeding, and urinary troubles. Due to these medicinal properties of both the plants, the present study was planned to determine antibacterial properties of leaves extracts of Prosopis cineraria and Ziziphus nummularia in various extraction solvents against S. aureus and E. coli by the agar disc diffusion method (CLSI, 2005). P. cineraria showed highest antibacterial property in benzene extract against S. aureus, followed by petroleum ether, aqueous, chloroform and acetone extracts, respectively. Z. nummularia, ether leaf extract showed highest antibacterial activity for S. aureus, followed by benzene, chloroform, aqueous and acetone extracts, respectively. Overall the benzene leaves extract of P. cineraria showed the maximum antibacterial assay against S. aureus. No antibacterial activity was observed against E. coli with different extracts of both the plants.

7.) “Screening of Zizyphus jujuba for antibacterial, phytotoxic and haemagglutination activities”(Bashir Ahmad1, Ibrar Khan, Shumaila Bashir, Sadiq Azam and Farrukh Hussain)

Pharma biotech Research Lab, Centre for Biotechnology and Microbiology, University of Peshawar, KPK, Pakistan.

Department of Pharmacy, University of Peshawar, KPK, Pakistan.Centre of Biodiversity, University of Peshawar, KPK, Pakistan

Plants are very useful, self-generating machines, producing a variety of useful bioactive products. Keeping in view this idea, the crude methanolic extract and various fractions of Zizyphus jujuba were screened for antibacterial, phytotoxic and haemagglutination activities. The n-hexane and aqueous fractions showed significant activity of 60 and 66.66% against Bacillus pumalis and Pseudomonas aerugenosa, respectively. Activity of ethyl acetate fraction was 65.38, 62.96, 62.96 and 72% against Staphylococcus epidermidis, Salmonella typhi, P. aerugenosa and B. pumalis, respectively. On the contrary, no activity of this fraction was recorded against Streptococcus pneumoniae. The crude, nhexane, chloroform, ethyl acetate and aqueous fractions showed 41.37, 44.82, 41.37, 55.17 and 44.82% activity against Enterobacter aerogenes, respectively. The crude methanolic extract and n-hexane fractions were inactive against Escherichia coli, S. pneumoniae and Klebsella pneumoniae, respectively. The ethyl acetate fraction was moderately phytotoxic against Lemna minor L at 1000 μg/ml. All the other

fractions showed low phytotoxic activity at 1000 μg/ml. At 100 μg/ml, all the fractions showed low phytotoxic activity except crude methanolic extract, which was inactive. All the test samples were inactive at 10 μg/ml. All dilutions of the test samples showed no haemagglutination activity against any blood group.

10. “Crude Extract from Ziziphus Jujuba Fruits, a Weapon against Pediatric Infectious Disease”

(PHD F Daneshmand, Msc H Zare-Zardini *, Msc B Tolueinia, Msc Z Hasani , Msc T Ghanbari)

Abstract Background Pediatric infectious disease is one of the main problems in cancerous children that treat by chemotherapy drugs. Thus, study in this regard is necessary. The aim of this study was to evaluate antimicrobial properties of ethanolic extract of Ziziphus Jujuba fruits against different infectious pathogens. Materials and Methods this study is descriptive. In vitro antimicrobial activity of extract was assessed on gram negative and gram positive bacteria as well as fungi. The antimicrobial activity was tested by Radial Diffusion Assay (RDA) and Minimal Inhibitory Concentration (MIC) methods. Results The results showed a wide antimicrobial activity of the extract against the microbes studied. Escherichia coli was the most susceptible to the extracts among tested microorganisms for which the MIC was 0.65±0.22 mg/ml. Amongst the bacterial strains investigated, Staphylococcus aureus was the most resistant strain with MIC of 2.26±0.68 mg/ml. The ethanolic extract also showed antimicrobial activity on the fungi studied as no growth was observed in 2.35±0.38 and 2.86±0.7 mg/ml concentration for Candida albicans and Aspergillus fumigatus, respectively. The results of qualitive and quantitative test are well indicative of the extract effective activity against the microbes mentioned. Conclusion confirming the potential antimicrobial activities of crude extract of Ziziphus Jujuba fruits, this study suggested that ethanolic extracts of this plant is appropriate candidate for treatment of microbial infections, especially pediatric infectious diseases.

Methods of Green Synthesis

1.) Polysaccharide method:

In this method, reduction of silver ions to Ag NPs is achieved using polysaccharides as reducing agents that also act as capping agent. Reaction, furthermore, was performed in water which is considered as an environmentally being solvent system. A simple method involves synthesis of starch tagged Ag NPs where β-D—glucose act as a reducing agent in a gently heated system. The starch in the solution mixture act as a capping agent avoids use of relatively toxic organic solvents. Furthermore, the binding interactions between starch and Ag NPs are weak and can be reversible at higher temperatures, allowing separation of the synthesized particles. In another study, stable Ag NPs (10-34nm) were synthesized by autoclaving a solution of AgNO3 and starch (capping/reducing agent) at 15 psi and 121°c for 5 min. significance of these starch protected nanoparticles lies in their easy integration into system for biological and

pharmaceutical applications the Ag NPs were also synthesized by using negatively charged heparin as a reducing/stabilizing agent by heating a solution of Ag NO3 and heparin to 70°C for <8h.

2.) Pollens Method:

The Tollens synthesis method gives Ag NPs with a controlled size in a one-step process. The basic tollens reactions involve in the reduction of Ag (NH3)2+ a tollens reagent, by an aldehyde, E q. (1).

Ag (NH3)2 (aq) + RCHO (aq) Ag (s) +RCOOH (q) …………………………………. (1)

In the modified tollens procedure, Ag+ ions are reduced by saccarides in the presence of ammonia, yielding Ag NP films with particles size from 50-200 nm, Ag hydrosols with particles in the order of 20-50 nm, and Ag NPs of different shapes. Ag(NH3)2+ is a stable complex ion resulting from ammonia’s strong affinity for Ag+, therefore the ammonia concentration and nature of the reluctant must play a major role in controlling the Ag NP size. A modified Ag mirror reaction (tollens reaction) is an example of a synthesis route yielding Ag NPs of different shapes. Ag NPs of various morphologies with <10 nm diameters were synthesized in water by adjusting the concentrations of n-hexadecyltrimethlammonium bromide (HTAB) and Tollens reagent, Ag (NH3)2, at 120°c.

3.) Irradiation method:

Various kinds of radiation can be used successfully to synthesize Ag NPs well defined shape and size has been produced by laser irradiation of an aqueous solution of Ag salt containing surfactant. Laser has also been applied in photo-sensitization techniques for the synthesis of Ag NPs using benzophenone. Here, low laser powers at short irradiation times gave Ag NPs of ≈5 nm. The formation of Ag NPs by this photo-sensitization technique was also achieved using a mercury lamp. Synthesis procedures using microwave irradiation has also been employed. Microwave radiation of a solution containing carboxy methyl cellulose sodium and silver nitrates solution produced uniform Ag NPs that were stable for two months at room temperature. The solvated electrons reduced the Ag+ ions and a characteristic Plasmon absorption was detected within 1-10 s after the ionization pulse. Furthermore radiolysis has been applied in the Ag NPs production. in ethylene glycol for the Ag NPs production was studied. Ag NPs supported on silica aerogel were synthesized were using gamma radiolysis and check for stability against pH change. In another work, oligochitosan as a stabilizer was used in preparation of Ag NPs by gamma radiation synthesizing 5-15 nm stable Ag NPs in a 1.8-9.0 pH range. Ag NPs of different size (60-200nm) have also been synthesized by irradiating a solution, prepared by mixing AgNO3 and poly-vinyl-alcohol, with 6 MeV electrons beam. The pulse radiolysis

techniques have been used to study the reactions of inorganic and organic species in Ag NP synthesis.

4.) Biological method:

Extracts from bio-organism may act both as reducing and capping agent in Ag NPs synthesis. The reduction of Ag+ ion by biomolecules found in these extracts such as enzymes/proteins, amino acids, polysaccharides, and vitamins is environmentally benign. One approach that shows immense –potential is based on the synthesis of nanoparticles using microorganism such as bacteria, fungi, yeasts and actinomycities. These biosynthetic methods can be divided into two categories depending on the place microorganisms reduce inorganic materials either intra or extracellularly. For example, bacteria Pseudomonas strutzeri isolated from silver mine materials is able to reduce Ag+ ions and accumulates silver nanoparticles, with the average diameter of 27 nm. The extra cellular production of metal nanoparticles, by several strains of the fungus fusarium oxysporum has been reported. The extra cellular hydrogenase in the F.oxysporum shows excellent redox properties and it can act as an electron shuttle in metal reduction. The first step involves the trapping of Ag+ at the surface of the fungal cells. Ag+ reduction by culture supernatants of Klebsiella pneumonia, Escherichia coli (E. coli) and Enterobacter cloacae (Enterobaceteriacae) produced rapid formations of Ag NPs. An extensive volume of literature reports successful Ag NP synthesis using bioorganic compounds. The carboxyl groups in aspartic and glutamine residues and the hydroxyl group in tyrosine residues of the proteins were suggested to be responsible for the Ag+ ion reduction. Carrying out the reduction process by a simple bifunctional tripe tide Asp-Tyr –Ome further identified the involvement of these residues. This synthesis process gave small Ag nanoparticles with low poly dispersity in good yield (>55%). Glutathione (Glu-Cys-Gly) as a reducing/capping agent can produce water-soluble and size tunable Ag NPs that easily bind to model protein (bovine serum albumin) attractive for medical applications. Plant extract from live alfalfa, the broth of lemongrass, geranium leaves and others have served as green reactants in Ag NP synthesis. The reaction of aqueous AgNo3 with an aqueous extract of leaves of a common ornamental geranium plant, pelargonium graveolens, gave Ag NPs after 24 h. the reaction time was reduced to 2 h by heating the reaction mixture just below the boiling point. Secreted proteins In spent mushroom substrate reduced Ag + to give uniformly distributed Ag-protein (core-shell) NP with an average size of 30.5 nm. A vegetable, capsicum annum L, was used to also synthesize Ag NPs. Studying the Synthesis of Ag NPs with isolated/purified bioorganics may give better insight into the system mechanism.

5.) Polyoxometalates method:

Polyoxometalates, POMs, have potential of synthesizing Ag NPs because they are soluble in water and have the capability of undergoing stepwise, multielectron redox reaction without disturbing their structure. For Example, Ag NPs were synthesized by illuminating a deaerated solution of POM/S/Ag+ in this method POMs serve both as a photo catalyst, a reduction agent, and as a stabilizer. In another study, one-step synthesis and stabilization of Ag nanostructures

with MoV-MoVI mixed-valance POMs in water at room temperature has been demonstrated. This method did not use a catalyst or a selective etching agent. Ag NPs of different shape and size can be obtained using different POMs in which the POMs serve as a reductant and a stabilizer.

Materials

And

Methods

Chemicals-

Materials used for the synthesis of silver nanoparticles were used:

Silver nitrate (AgNo3)

Gold (III) chloride trihydrate (HAuCl4)

M17 broth (use in Himedia Ref M1029-500G, ISO certified Company, Himedia Laboratories Pvt. Ltd.)

Nutrient agar (use in Himedia, Himedia laboratories Pvt. Ltd. (Mumbai). Silver nitrate (AgNo3) central drug House (P) Ltd. Bombay- New Delhi. Central Drug House (P) Ltd.)

Double-distilled deionized water was used.

Plant Collection:-

Fresh Z.   nummularia  leaves   collected   from   the   Maharaj   Vinayak   Global   University (Jaipur, Rajasthan India) were used as a plant source for green nanoparticle synthesis.

Preparation of chloroauric acid solution:-

Synthesis of gold nanoparticles were pre-pared by the reduction HAuCl4 was use without further purification, aqueous solution was prepared by dissolving the required amount in the distilled water and stored in brown bottle. Double distilled co2 free and deionized water was used as solvent to prepare all solutions.

Preparation of silver nitrate solution:-

In a typical one-step synthesis silver nanoparticles were pre-pared by the reduction AgNo3 (silver nitrate, 99%, Merck India product was used without further purification, aqueous solution was prepared by dissolving the required amount in the distilled water and stored in brown bottle. Double distilled, CO2 free and deionized water was used as solvent to prepare all solutions).

Preparation of plant extract:-

Fresh 5g leaves collect from our university campus, thoroughly washed with distilled water to remove dust particles and were then cut into small pieces boiled for 25 min with 100ml distilled

water in a 250-ml Erlenmeyer flask. The mixture was cooled and filtered with Whatman paper no.1. Filtrate was collected, stored at the room temperature and used as the reactant for the preparation of Ag-nanoparticles.

Synthesis of silver nanoparticles:-

There are several ways to synthesize silver nanoparticles in this work I.e. either in aqueous or organic solution, or using different oxidation agent. Each way will result in the different oxidation agent. The stability is very important to be considered for storage and application

purpose. For reduction of Ag+ ions, 2 mL of aqueous extract of Z. nummularia leaves was

added to 10  mL of silver nitrate solution (concentration 10−3 M).The reduction of pure Ag ions was monitored by measuring the UV-vis spectra of the solution at regular intervals after diluting a small aliquot (0.2 ml) of the 20 times. UV-vis spectra were recorded as a function of time of reaction on a UV-Vis spectrophotometer.

Fig.1. Color change in reaction mixture: (a) at 0 minutes, (b) after 30 minutes.

Synthesis of gold nanoparticles:-

In Synthesis of gold nanoparticles 1ml of aqueous extract of Z, nummularia leaves was added to 1 ml of HAuCl4. And incubation time for 1 hr. and after gold nanoparticles is synthesized. UV-vis spectra were recorded as a function of time of reaction on a UV-Vis spectrophotometer.

UV-Vis spectrometer:-

The reaction mixture of Z. nummularia leaf extract with aqueous solutions of silver nitrate started to change its color from yellowish brown to reddish brown. It indicates the formation of

silver nanoparticles with the reduction of silver ions. The reduction of pure Ag+ ions was monitored by measuring the UV-Vis spectrum of reduction medium at 5 hours after diluting a small aliquot of the sample into distilled water. UV-Vis spectral analysis was done by using UV-VIS spectrophotometer. Reduction of Ag ion into silver particles during exposure to the plant extracts could be followed by the color change. Ag nanoparticles exhibit dark yellowish-brown color in aqueous solutions due to the surface Plasmon resonance phenomenon. The result obtained in this investigation is very interesting in terms of identification of potential plants for synthesizing the Ag nanoparticles. UV-Vis spectrograph of the colloidal solution of silver nanoparticles has been recorded as a function of time. Absorption spectra of silver nanoparticles formed in the reaction media at 10 min has absorbance peak at 430-470 nm, broadening of peak indicated that the particles are poly dispersed.

The reduction of metal ions was routinely monitored by visual inspection of the solutions as well as by measuring the UV-Vis spectra of the solution by periodic sampling of the aliquots (2 ml) of the aqueous component. The UV-Vis spectroscopy measurements were recorded on a spectrophotometer operated at a resolution of 1 nm. The fluorescence measurements were carried out on a spectrophotometer.

Particles Conc. Of extract AgNo3/HAuCl4 Conc.

Peak value(300-500nm)

Absorbance

Silver 100 µl 500 µl 443 1.597Gold 100 µl 100 µl 551 0.416

Antibacterial Activity:

1.) Zone of inhibition

Synthesis of ag nanoparticles were studied for antibacterial activity against pathogenic microorganism (clinical isolate) by using standard zone of inhibition (ZOI) microbiology assay, with a well size of 5 mm diameter and 30 μl against all the test organisms. Maximum zone of inhibition was found to be staphylococcus aureus and E. coli.

Fig 2:-Representative results of antimicrobial activity of nanoparticles in Z. nummularia leaf Extract.

2.) Minimal inhibitory concentration (MIC)

Bacterial strain was grown on M 17 plates at 37ºC before being used. The antimicrobial activity of Ag-NPs was examined using the standard broth dilution method (CLSI M07-A8). The MIC was determined in M17 broth (Mumbai, India) using serial two-fold dilutions of Ag-NPs in concentration ranging from 200 to 1.5625 µg/ml, initial bacterial inoculums of 2×108 CFU/ml and the time and temperature of incubation being 24h at 27ºc respectively. The MIC is the lowest concentration of antimicrobial agents that completely visually inhibits 99% growth of the microorganisms. The MIC for each tested Bacteria Minimum inhibitory concentration was determined by medium dilution technique according to standard protocol NCCLs (1997). Serial dilutions were undertaken with a 5-105 cells/ml inoculums from an o.1 g/ml concentration of aqueous Z.nummularia extract.

After 37ºC in 15 hrs).02% resazurin sodium saltare added in all wells and incubate 37ºC in 3hrs. After 3hrs the colour is change some wells are pinkin colour and some is blue in colour.

1.) Colony Forming Unit (CFU)

M17 media was used to count the numbers of bacterial colonies. The above media was autoclaved with various concentration ranges from 10-40µg/ml of silver nanoparticles and then it allowed cooling in different petri-dishes. We have another M17 media which does not have silver nanoparticles, Act as the control for experimental Result (104cells/ml) E.coli and Staphylococcus were incubated for 24 hrs to analyze the colony forming units (CFU). The viable cell number was recorded by counting the number of bacterial colonies grown on the media.

Conclusion

Z. nummularia leaf extract was prepared and successfully used for the single-pot biosynthesis of SNPs. This method is rapid, facile, convenient, less time consuming, environmentally safe and can be applied in a variety of existing applications. The process for the rapid synthesis of stable silver nanoparticles at high concentration using Z. nummulari leaf broth was demonstrated. The flavonoide and terpenoid constituents of the leaf broth are believed to be the surface active molecules stabilizing the nanoparticles.

Interestingly, silver nanoparticles were synthesized rapidly with in 1 h of incubation period. The aqueous silver nitrate solution was turned to yellowish brown colour with in 1 h. intensity of brown colour increased in direct proportion to the incubation period. It was due to the excitation of surface Plasmon resonance (SPR) effect and reduction of AgNO3. The silver surface Plasmon resonance was observed at 420nm which steadily increases in intensity as a function of time of reaction (ranging from 10 min to 6h) without showing any shift.

Achievement of such rapid time scales for synthesis of silver nanoparticles contributes to an increase in the efficiency of synthetic procedures using environmentally benign natural resources as an alternative to chemical synthesis protocols.

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