pharmacological activity of silver nanoparticles using ...joics.org/gallery/ics-3818.pdf ·...

Pharmacological activity of silver nanoparticles using

Moringa Oleifera flower extracts

P. Anitha *1, P. Sakthivel 2, S.Balamurugan 3, N. Muruganantham 4, M. M. Senthamilselvi 5

and R.Govindharaju6 *1 Assistant Professor, Department of Physics, Roever College of Engineering and Technology,

(Affiliated to Anna University), Perambalur - 621220, Tamil Nadu, India. 2Professor, PG & Research Department of Physics, Urumu Dhanalakshmi College,

(Affiliated to Bharathidasan University),Trichy, Tamilnadu, India. 3 Professors in Physics and Director, Roever Group of Institutions, (Affiliated to Anna

University),

Perambalur - 621220, Tamil Nadu, India. 4,6 Assistant Professor, PG & Research Department of Chemistry, Thanthai Hans Roever College

(Autonomous),

(Affiliated to Bharathidasan University), Perambalur - 621220, Tamil Nadu, India. 5Regional Joint Director (Retd), Department of Collegiate Education, Tiruchirappalli (Tamil

Nadu), India.

*Corresponding author: [email protected]

ABSTRACT

Silver nanoparticles are synthesized from aqueous extracts of Moringa Oleifera flower extracts.

Pharmacological activities such as Anti-oxidant, Anti- inflammatory activity and Anti diabetic

activities were studied. The AgNPs have showed significant pharmacological activity on multi

drug resistance in biological fields. The AgNPs had shown strong antioxidant by DPPH

scavenging activity. The AgNPs exhibited strong anti-inflammatory activity by albumin

denaturation activity. The AgNPs had strongly inhibited the aglucosidase to a-amylase. To the

best of our knowledge, this is the first attempt on the synthesis of nanoparticles using Moringa

Oleifera flowers extracts. Hence, to authenticate our results, the invivo studies at molecular level

are needed to develop an antioxidant, anti-diabetic and anti-inflammatory agent. Results of the

present study, highlighted the eco-friendly approach of plant mediated synthesis of nano

particles and its potential application in the field as an alternative to chemical drug for disease

management.

Keywords: AgNPs, Nanoparticles, antioxidant, anti-diabetic and anti-inflammatory agent etc.,

1. INTRODUCTION

Nanotechnology is a science centered on molecular and supramolecular molecules aiming

to create nanostructures with enhanced functionalities [1], and the term nanoparticle describes

particulate matter ranging in size from 1–100 nm [2]. Bearing a nano scale size offers the benefit

of having a significantly large surface area to volume ratio[3]. Increased surface area, in

Journal of Information and Computational Science

Volume 10 Issue 7 - 2020

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mailto:[email protected]

combination with nanoparticle conformation and distribution in solution contribute to their

enhanced physical and chemical properties which are useful in a diversity of fields such as

antimicrobial development [4], bio-molecular detection, diagnostics [5], catalysis [6], micro-

electronics [7], sensing devices and targeting of drugs to cancer cells [8]. These wide ranging

applications and the increasing ability to manipulate nanoparticle form and function have

sparked great interest in the scientific community over the past decade, particularly in developing

a competent and eco- friendly synthetic methods.

Conventional physical and chemical methods currently have limited use in preparing

metal nanoparticles due to toxic chemicals [9]. Moreover, these methods are associated with

high-energy input and costly downstream processing [10]. Green synthesis is defined as the

utilize of eco-friendly compatible materials such as bacteria, fungi and plants in the synthesis of

nanoparticles [11]. These nice-looking green strategies are free of the short falls linked with

conventional synthetic strategies, i.e. they are eco-friendly [12]. on the other hand, synthesis

from organically derived extracts offers several advantages such as hasty synthesis, high yields

and importantly, the lack of costly downstream processing required producing the particles [13–

15]. Hence, nanoparticle synthesis from plant extracts tentatively offers a route for huge scale

production of commercially attractive nanoparticles.

Frequent studies report on the use of plant extracts to synthesis AgNPs with significant

antimicrobial activities: leaf extracts of Acalypha indica [16], Solidago altissima [17], Xanthium

strumerium L [18], Murraya koenigii (curry leaf) [19], Ocimum sanctum (Tulsi leaf) [20, 21],

seed extracts of Acacia farnesiana [22], Macrotyloma uniflorum [23], root extracts of

Trianthema decandra [24], stem extracts of Ocimum sanctum [20], and even fruit extracts of

Musa paradisiacal (banana) peels [25] and Carica papaya [26]. In these studies, silver nitrate

(AgNO3) is used as a precursor in synthesis of silver nanoparticle. Also the phytocompounds

present in the plant extracts serve as reducing and/or capping agents in reaction with AgNO3.

Moringa oleifera Lam (drumstick tree) is a tree species indigenous to north-western India

but is also regarded as a vital crop in several other countries such as the Philippines, Sudan,

Ethiopia and South Africa [27, 28]. It belongs to the genus Moringa and the family Moringaceae

[29], and is highly sought after for its tender pods, flowers and leaves, all of which are confined

for human consumption [30]. The leaves, in particular, are recognized for their natural healing

properties and are popularly consumed in a variety of ways [29, 31]. Research to date has

revealed that extracts prepared from the leaves possess high natural antioxidant properties and

some antibacterial activity against gram-positive and gram-negative bacteria [27, 32].

Nanomaterials have a long list of applicability in civilizing human life and its

environment. The first relation between human life and nano scale was developed as expected in



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ayurveda, which is a 5000-year-old Indian system of medicine. It had some knowledge of

nanoscience and technology before the term ‘nano’ was even formed. Modern science has just

started exploring nanoscience in the 21st century[33]. Research and development in this field is

growing rapidly throughout the world. A chief yield of this activity is the development of new

materials in the nanometer scale, including nanoparticles. These are usually clear as particulate

materials with atleast one dimension less than 100 nanometers (nm), even the particles could be

of zero dimension as in the case of quantum dots. Metal nanoparticles which have a high specific

surface area and a high fraction of surface atoms have been studied extensively because of their

unique physicochemical character including catalytic activity, optical properties, electronic

properties, antibacterial properties and magnetic properties[34]. Synthesis of noble nanoparticles

for the applications such as electronics, environmental and biotechnology is an area of constant

interest [35]. Generally metal nanoparticles are synthesized and stabilized by using element

methods such as chemical reduction [36,37], electrochemical techniques[38], microwave assisted

process[39] and now a day via green chemistry route[40]. Synthesis of nanoparticles using plants

are quite novel leading to innovation over chemical and physical method as it is cost effective

and environment friendly. It is also easily synthesized to a great extent in a large scale as there is

no need to use high pressure, energy, temperature and toxic chemicals in this method. Bacteria

and fungi could be used for the blend of nanoparticles[41,42] but use of leaf extract[43] reduces

the cost and we do not require any special culture preparation and isolation techniques.

Flowers

Fig.1. Moringa Oleifera

In the present study, we report the biosynthesis of AgNPs from M. oleifera flower

extract. There are few published comparisons on nanoparticle yield, quality and bioactivity for

extracts prepared with fresh tissue within individual species. Moringa flowers represent a

promising candidate for green synthesis of bioactive AgNPs that can be produced in an

environmentally friendly manner.

2. MATERIALS AND METHODS

2.1 Collection of root

Fresh root of the samples were collected from Perambalur, during the month of March.



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2.2 Preparation of root extract

The fresh and young flowers of the samples were collected & washed thoroughly with sterile

double distilled water (DDW). Twenty grams of sterilized root samples were taken and cut into

small pieces. Finely cut roots were placed in a 500 ml Erlenmeyer flask containing 50ml of

sterile DDW. After that, the mixture was transferred to Soxhlet apparatus to derive extracts. The

extract was stored in 4 0C.

2.3 Microwave assisted synthesis of metal nanoparticles

Metal nitrate was used as precursor in the synthesis of metal nanoparticles. 100 ml of flower

extract was added to 100 ml of 0.1N metal nitrate aqueous solution in conical flask of 250 ml

content at room temperature. The conical flask was subsequently put into shaker (100 rpm) at 500

C and for a period of 12 hrs the reaction was carried out. Then the mixture is exposed to heat

with the help of microwave oven.

Metals nanoparticles are made by a chemical reduction of a metal salt in the presence of a

stabilizing agent. Rapid microwave heating and agitation gives monodispersed particles. Add

200 ml of extract with 1M metal nitrate in beaker and Cover loosely. Expose the sample in

Microwave radiation for 20 minutes at 100% power. The setting of time is done on the basis of

trial and error method. The color will continue to change with respect to time. The mixture was

completely dried after a period of 20 minutes and hence nanoparticles in form of powders were

obtained.

2.4 Anti-Oxidant Studies

DPPH scavenging assay

The ability to scavenging the stable free radical, DPPH was measured as a decrease in

absorbance at 517 nm by the method.

Reagents

2,2-Diphenyl-1-picryl hydrazyl (DPPH) – 90.25mM in methanol in a dark room.

Procedure

To a methanolic solution of DPPH (90.25 mM), an equal volume of ethanolic Rhizome of

Cyperus rotundus L (250-1500 µg) was added and made up to 1.0 ml with methanolic DPPH. An

equal amount of methanol was added to the control. After 20 min, the absorbance was recorded

at 517 nm in a Systronics UV-visible Spectrophotometer. Ascorbic acid was used as standard for

comparison. The inhibition of free radicals by DPPH in percentage terms (%) was calculated by

using the following equation.

% Scavenging = (OD of Control- OD of Sample/ OD of Control) X 100.

Where, A control is the absorbance of the control reaction (containing all reagents except the test

compound) and A sample is the absorbance of the test compound.



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2.5 Anti- inflammatory activity

Inhibition of Albumen Denaturation

The reaction mixture consists of test extracts and 1% solution of bovine albumin fraction, pH of

mixture was adjusted by adding a small amount of HCl at 37°C. The sample extracts were

incubated at 37°C for 20 minutes and then heated to 51°C for 20 minutes after cooling the

samples the turbidity was measured spectrophotometrically at 660 nm. Diclofenac sodium was

taken as a standard drug. The experiment was performed in triplicates. Percent inhibition of

protein denaturation was calculated as follows:

Percent inhibition (%) = (OD of Control- OD of Sample/ OD of Control) X 100.

2.6 Inhibition Of Alpha-Amylase Enzyme

Starch solution (0.1% w/v) was prepared by stirring 0.1 g of potato starch in 100 ml of 16 mM of

sodium acetate buffer. The enzyme solution was prepared by mixing 27.5 mg of α-amylase in

100 ml of distilled water. The colorimetric reagent is prepared by mixing sodium potassium

tartarate solution and 3,5-di nitro salicylic acid solution 96 mM. The starch solution is added to

the both control and plants extract tubes and left to react with α-amylase solution, under alkaline

conditions at 25ºC. The reaction was allowed for 3 min. The generation of maltose was

quantified by the reduction of 3,5-dinitro salicylic acid to 3-amino-5-nitro salicylic acid. This

reaction is detectable at 540 nm.

% Inhibition = (OD of Control- OD of Sample/ OD of Control) X 100.

3. Result and Discussion

3.1 Anti – Oxidant activity

in Figure [Table 2 and Figure 3]. The albumin denaturation method was carried out for AgNPs

DPPH scavenging assay method

There are several methods available to assess the antioxidant activity of compounds. DPPH free

radical scavenging assay is an easy, rapid, and sensitive method for the antioxidant screening of

plant extracts. In the presence of an antioxidant, DPPH radical obtains one more electron and the

absorbance decreases.

In the present study, the AgNPs using flower extracts of Moringa Oleifera flowers have

high DPPH scavenging capacity, which increased with increasing concentration [Table 1 and

Figure 2]. It is evident from the data presented in Table, that the sample possesses DPPH assay

activity. For the AgNPs the result shows the percentage of cytotoxicity for 250 mg/ml as

13.22%, 500 mg/ml as 27.5%, 750 mg/ml as 48.5% and 1000 mg/ml as 53.1%. These inhibition

values are compared with standard drug of Ascorbic acid for 250 mg/ml as 23.63%, 500 mg/ml

as 29.00% ,750 mg/ml as 45.25% and1000 mg/ml as 52.05 %. Hence, this assay provided

information on the reactivity of test samples with a stable free radical.



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As a part of the investigation on the mechanism of the anti‐oxidant activity, ability of

extract to inhibit DPPH scavenging assay was studied. The in-vitro study of anti‐oxidant activity

indicates that the inhibition percentage of DPPH scavenging assay by Moringa Oleifera flowers

extracts of AgNPs.

S.No Test

Concentration

of the sample

(mg/ml)

% of

inhibition of

the AgNPs

Ascorbic

acid

(Standard)

1

DPPH

250 13.22 23.63

2 500 27.5 29.00

3 750 48.5 45.25

4 1000 53.1 52.05

Table.1 Anti Oxidant activity of AgNPs using flowers extracts of Moringa Oleifera by

DPPH Scavenging assay.

Fig.2 Graphical representation of Anti oxidant activity of AgNPs using flowers extracts of

Moringa Oleifera by DPPH Scavenging assay.

3.2 Anti- inflammatory activity

Inhibition of Albumen Denaturation method

There are certain problems in using animals in experimental pharmacological research,

such as moral issues and the lack of rationale for their use when other suitable methods are

available. Hence, in the present study, the protein denaturation bioassay was selected for in vitro

13.22

27.5

48.553.1

23.63

29

46.25

52.05

250 500 750 1000

0

10

20

30

40

50

60

Concentration

% o

f in

hib

itio

n

DPPH Scavenging assay activity

% of inhibitionof theMoringaOleifera Flowers

Ascorbic acid(Standard)



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assessment of the anti-inflammatory property of Silver nanoparticles synthesized Moringa

Oleifera. The Albumen Denaturation is a well-documented cause of inflammation. Most

biological proteins lose their biological functions when denatured. Production of autoantigen in

certain arthritic disease is due to denaturation of protein. Aspirin was used as a standard anti-

inflammation drug as shown. at different concentrations such as 100µg/ml 200µg/ml, 300 µg/ml,

400 µg/ml, 500 µg/ml.

For the AgNPs, the result shows the percentage of cytotoxicity for 100 mg/ml as 44 %,

200 mg/ml as 49.2%, 300 mg/ml as 58.1%, 400 mg/ml as 68 %, and 500 mg/ml as 76%. For the

These inhibition values are compared with standard drug of Aspirin

for 100 mg/ml as 45%, 200 mg/ml as 56.25% ,300 mg/ml as 66.20%, 400 mg/ml as 72.02%, 500

mg/ml as 82%.

As a part of the investigation on the mechanism of the Anti- inflammatory, ability of

extract to inhibit Inhibition of Albumen Denaturation was studied. The in-vitro study of Anti-

inflammatory activity indicates that the inhibition percentage of Albumen Denaturation by

Moringa Oleifera flowers extracts of AgNPs.

S.No Test

Concentration

of the sample

(mg/ml)

% of Protein

Denaturation

of the AgNPs

Aspirin

(Standard)

1

Albumin

denaturation

100 44 45

2 200 49.2 56.25

3 300 58.1 66.20

4 400 68 72.02

5 500 76 82

Table.2 Anti-inflammatory activity AgNPs using flowers extracts of Moringa Oleifera by

Albumen Denaturation.



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Fig.3 Graphical representation of Anti-inflammatory activity AgNPs using flowers extracts

of Moringa Oleifera by Albumen Denaturation.

3.3 Anti diabetic activity

Inhibition of Alpha-Amylase Enzyme

Diabetes mellitus is a group of metabolic diseases in which there are high blood sugar

levels over an extended period. A therapeutic approach to decrease the hyperglycaemia is to

inhibit the carbohydrate digesting enzymes (α-glucosidase and α-amylase), thereby preventing

the breakdown of carbohydrates into monosaccharides which is a main cause of increasing blood

glucose level. Therefore, developing compounds having inhibitory activities towards

carbohydrate hydrolysing enzymes may be a useful way to manage diabetes. As shown in Figure

4 and Table 3, α-amylase and α-glucosidase were significantly inhibited in a dose-dependent

manner by the AgNPs. The results suggest that with the increased AgNPs concentration, the

activity levels of enzyme were remarkably reduced, Hence, the biomolecules likely enhanced the

antidiabetic potential of the synthesized NPs. α-Amylase inhibitory actions were observed in

increasing order, as Acarbose (Figure 4). Comparable results were observed. However, the

foregoing results suggest that the synthesized AgNPs have potential antidiabetic property and

could prove its effectiveness in the diabetes care.

For the AgNPs, the result shows the percentage of cytotoxicity for 0.05 mg/ml as 33.2 %,

0.1 mg/ml as 41.1%, 0.15 mg/ml as 55%, 0.2 mg/ml as 58.4% and 0.25 mg/ml as 76.2%. These

inhibition values are compared with standard drug of Acarbose for 0.05 mg/ml as 35%, 0.1

mg/ml as 42%, 0.15 mg/ml as 56%, 0.2 mg/ml as 61% and 0.25 mg/ml as 79%. On comparing, it

was observed that when the concentration of the sample increases the inhibition also increases

showing a good sign of Anti-diabetic activity.

4449.2

58.1

68

76

45

56.25

66.272.02

82

0

10

20

30

40

50

60

70

80

90

100 200 300 400 500

% o

f in

hib

itio

n

Concentration

Albumen Denaturation

% of inhibitionof theMoringaOleifera FlowersAspirin(Standard)



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https://www.tandfonline.com/doi/full/10.1080/21691401.2017.1337031#F0009



As a part of the investigation on the mechanism of the Anti diabetic activity,

ability of extract to inhibit Inhibition of Alpha-Amylase Enzyme was studied. The in-vitro study

of Anti diabetic activity indicates that the inhibition percentage of Alpha-Amylase Enzyme by

Moringa Oleifera flowers extracts of AgNPs.

Table.3 Anti diabetic activity of AgNPs using flowers extracts of Moringa Oleifera by Alpha

amylase.

Fig.4 Graphical representation of Anti diabetic activity of AgNPs using flowers extracts of

Moringa Oleifera by Alpha amylase.

4. Conclusion

Scientists have shifted their interest from chemical or physical methods to biological

methods as it does not involve a combination of abusive or toxic chemicals to human health or

any involvement of immense machines or equipment. The biological methods incorporate other

plant or microbial mediated methods that are cheap and easily accessible in daily life. The

33.2

41.1

5558.4

76.2

3542

5661

79

0.05 0.1 0.15 0.2 0.25

0

10

20

30

40

50

60

70

80

90

Concentration

% o

f in

hib

itio

n

Inhibition of Alpha-Amylase Enzyme

% of inhibitionof theMoringaOleifera Flowers

Acarbose(Standard)

S.No Test

Concentration

of the sample

(µg/ml)

% of

inhibition of

the AgNPs

Acarbose

(Standard)

1

Alpha amylase

inhibitory

activity

0.5 33.2 35

2 0.1 41.1 42

3 0.15 55 56

4 0.2 58.4 61

5 0.25 76.2 79



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medicinal plant Moringa Oleifera has been used as a traditional medicinal plant due to the

presence of phytochemicals in it. The various applications of the foot extract have already been

established till date. Now, in this study, the flower extracts have been used for the biogenesis of

the AgNPs. The DPPH assay is the most acceptable, fastest and simplest method for the

calculation of the free radical scavenging activity. As shown in the Table 1 and Figure 2. The

AgNPs shows better antioxidant property when compared with the standard ascorbic acid with

an IC50 values. Denaturation of proteins is a well documented cause of inflammation.

Phenylbutazones, salicylic acid, flufenamic acid (anti‐inflammatory drugs), have shown dose

dependent ability to thermally induced protein denaturation. As a part of the analysis on the

mechanism of the anti‐inflammatory activity, ability of extract to inhibit protein denaturation

was studied. The in-vitro study of a anti-inflammatory activity indicates that the inhibition

percentage of albumin denaturation by Moringa Oleifera flower extracts. It is inferred that the

Anti- inflammatory activity of AgNPs synthesized from Moringa Oleifera flower extracts

indicates a good and higher inhibition percentage than AgNPs from Moringa Oleifera flower

extracts as presented in Table 2 and Figure 3. α-amylase is a key enzyme in carbohydrate

metabolism. Inhibition of α-amylase is one of the strategies for treating diabetes. Amylase

inhibitors are also known as starch blockers because they contain substances that prevent dietary

starches from being absorbed by the body. Amylase inhibitor with starchy meal will reduce the

usual rise in blood sugar levels. The result suggests that Ag Nanoparticle exhibits well α-

amylase inhibition under in vitro conditions (Tables 3 and Fig 4).

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