American Journal of Life Sciences 2015; 3(1-3): 1-4

Published online September 29, 2014 (http://www.sciencepublishinggroup.com/j/ajls)

doi: 10.11648/j.ajls.s.2015030103.11

ISSN: 2328-5702 (Print); ISSN: 2328-5737 (Online)

Biosynthesis of nano-silver by cell free secretions from seeds of Medicago sativa

Gamal Hassan Rabie1, Hegazy Sadek Hegazy

1, Lamis Desoky Shaban

1, Diana Salah Raie

2

1Botany Department, Faculty of Science, Zagazig University, Zagazig City, Sharqya State, Egypt 2Process Design and Development Department, Egyptian Petroleum Research Institute, Nasr City, Cairo, Egypt

Email address: rabiegam@hotmail.com (G. H. Rabie), hegazyshegazy@gmail.com (H. S. Hegazy), lamis_shaaban@yahoo.com (L. D. Shaban),

raiediana@yahoo.com (D. S. Raie)

To cite this article: Gamal Hassan Rabie, Hegazy Sadek Hegazy, Lamis Desoky Shaban, Diana Salah Raie. Biosynthesis of Nano-Silver by Cell Free Secretions

from Seeds of Medicago sativa. American Journal of Life Sciences. Special Issue: Industrial Biotechnology. Vol. 3, No. 1-3, 2015, pp. 1-4.

doi: 10.11648/j.ajls.s.2015030103.11

Abstract: Biosynthesis of silver nanoparticles is an eco-friendly method in the field of nanotechnology. In the current study, we use silver nitrate as a source of silver ions. Cell free secretions from seeds of Medicago sativa is the source of bio-reducers

and bio-stabilizers. The bio-synthesized nanoparticles are spherical in shape with a size ranging from 2.5 to 25 nm. From the

results, the stabilization has mostly been carried out by polyphenols present in the cell-freed exudates. Also, we study the effect

of pH on the size of nano-silver. Ammonium hydroxide and nitric acid are the alkalifying and acidifying agents. A spherical

shape in a size ranges from 2.5 nm to 25 nm occurs at pH 10. The main reducing agent is supposed to be an antioxidant.

Keywords: Silver Nanoparticles, Medicago Sativa, Seed Secretions, Biosynthesis, Ammonium Hydroxide, Antioxidants

1. Introduction

Botanical world is a rich land for exploring active

components. Phyto-constituents can be derived from

different parts of the plant like bark, leaves, flowers, roots,

fruits, seeds. Also, broad classes of metabolites can be

obtained for examples antioxidants and other organics. Some

of them can work as reductants and/or adsorbents either in

purified or crude form. So, they can be used as fabricants for

nanoparticles [1].

Cell free crude secretions from seeds are presented as

promising green fabricators for nano-silver due to the coming

reasons. First, seeds are embryonic plants covered by a

protective outer seed coat, commonly with a quantity of

stored food. In addition, during their germination, seeds

exude a variety of metabolites including carbohydrates,

vitamins, amino acids, and other organic compounds [1].

Thus, they are considered as reservoirs for active constituents.

Second, there are different methods to use bio-mass as

aqueous solutions. For example, they can be used for

nanoparticles synthesis as secretions from whole bio-mass

[1], secretions from powdered seed [2], or broth [3]. The

former has advantages more than being green. It avoids

thermal inactivation of seeds. It gets benefit of living being

metabolism. Last but not least, cell free secretions overcome

the problem of bio-sorption of substrates by cells which

negatively affects the net results.

One of the most famous bio-builder of nano-metals is

Medicago sativa. As a leguminosae member, it is considered

an appropriate contender for this biosynthesis of nano-

materials because it is distributed and available. Moreover, it

is a metallo-phyte [4]. So, it is abundant with active

ingredients which are suitable for metal interaction.

There is a successful trail for using cell free secretions

from Medicago sativa seeds for nano-silver formation. In the

mentioned trail, authors used sodium hydroxide and nitric

acid to study the effect of pH on the bio-process [1]. The bio-

produced nano-items are poly-sized and poly-shaped [1].

Some researchers explain this as a result of an intrinsic

character of silver. As only a portion of ions is reduced. Thus

an entrapment occurs [5, 6]. While, other get it back to

presence of broad spectra of bio-molecules [1]. Hence, the

current work is aiming to assay the influence of pH on nano-

size using ammonium hydroxide as alkalifying and nitric acid

as acidifying agents.

2 Gamal Hassan Rabie et al.: Biosynthesis of Nano-Silver by Cell Free Secretions from Seeds of Medicago Sativa

2. Materials and Methods

To bio-fabricate nano-silver, two reactants were used at

room temperature in dark condition as following: Reactant

(A) was cell free seed exudates of M. sativa. Four grams of

fresh seeds of M. sativa were macerated in de-ionized water

for 8 h. Seeds were discarded from their aqueous secretions

by centrifugation at 3000 rpm for 10 min [1]. The freed

supernatant was processed for colorimetric assay of total

proteins [7], carbohydrates [8], polyphenols [9] and

flavonoids [10] contents. Reactant (B) was 0.1 M of aqueous

silver nitrate. Two ml from both reactant solutions were

mixed. The silver nitrate was adjusted at pH 2, 5, 7, 9, 10,

and 11 using ammonium hydroxide [5, 6] and nitric acid [1,

6]. After 24 h of reaction, aliquots of reactants were subjected

to UV-Vis spectrophotometer to check the reduction of silver

ions. Formation of silver nanoparticles was confirmed by

TEM [1-3, 5, 6]. Nanoparticles were collected by

centrifugation. The pellet was washed then dried for FTIR

analysis [1, 3, 6].

3. Results and Discussion

Figure 1. Quantitative assay of possible anti-oxidants present in cell free

seed secretions

(a) (b)

(c) (d)

Figure 2. Bio-synthesized nano-silver by cell free seed secretions reacted with silver nitrate (a) Yellowish color (b) UV-Vis Spectrum (c) FTIR Spectrum of

possible stabilizing agent (d) TEM Micrograph

Making nano-colloid through bio-routes depends on the

ability of biological systems to produce bio-reducers and bio-

stabilizers [1-3, 6]. In this study, cell free secretions of M.

sativa seeds were subjected to bio-convert silver nitrate into

nano-silver through their secretions. So before reaction, the

most common antioxidants were calorimetrically analyzed as

American Journal of Life Sciences 2015; 3(1-3): 1-4 3

a bio-chemical profile of these secretions (Figure. 1). The

present results were of two cases; pH independent and

dependent study.

In pH independent case, yellowish color appeared

immediately after mixing reactants (Figure. 2. a). This color

pointed for nano-silver presence. It was inspired as a result of

excitation of surface plasmon resonance [1-3, 5, 6]. The

sample was subjected to UV-Vis spectrophotometer. A single

broad absorbance band appears in the visible region. We

predicted that the bio-formed nano-silver was mono-shaped

with a large size distribution (Figure. 2. b). TEM image

confirmed our guess (Figure. 2. d). Spherical shapes with

variable sizes were formed. The size ranged from 2.5 to 25 nm.

This variation in size might be due to problems in nuclei

formation and/or stabilization step. Reducers were supposed to

be responsible for induction of nucleation [1, 5, 6].

Simultaneously, mixing the reducing agent and aqueous salt

formed a large number of nanoparticles. However, only a

portion of silver ions could be reduced to metal, in contrast to

other noble metals. Due to this predictable result, more nuclei

were going to be formed. Initially produced particles would

continue to grow at the same time, supplied by remaining

silver ions in the medium. This process was leading to particles

with a broad size distribution [5, 6]. In addition, presence of

different reducers with different concentration (Figure. 1)

affected the rate on nuclei production as in turn particle growth

and nanoparticle morphology. Also, crude secretions might

have insufficient quantity stabilizers in the secretions which

may influence the nano-morphology [1]. Bio-stabilizers were

expected using FTIR spectrum (Figure. 2. c). It showed peaks

at 3430, 2925, 1628 and 1027 cm-1

. These bands corresponded

to O-H of polyphenol, C-H of alkanes, C=C of alkenes, and C-

N of aliphatic amine [3]. So, we concluded that the compounds

attached with the nano-silver could be polyphenols with bound

amide region. In turn, the bio-produced nano-silver was

supposed to be negatively charged [1, 3, 6].

Towards size control, we designed an experiment to assay

the effect of pH. In this paper, we used ammonia as

alkalifying agent for duplicated function. From one hand, it

formed a complex with silver. So, it could trap all the free

ions after the nucleation step [5, 6]. From anther, it provided

an alkaline medium. In this condition, it improved the

repulsion among negatively charged particles [1, 6]. Also, it

provided the suitable pH for antioxidant activity for

flavanoids; the possible reducing agent [11].

In pH independent study, excluding at pH 2 we observed a

gradual increase in yellowish color with increase in pH,

immediately after combining reactants (Figure. 3. a). The

darkest yellowish color occurs at pH 10. This might be as a

result of presence of flavanoids in addition to nano-silver [1,

6]. At pH 2 even after 24 h of reaction, no change in color

was observed. This referred to that there was no reaction [1,

6]. Notable all of them absorbed in visible region, except at

pH 2 [1, 6]. These spectra return to nano-silver formation

(Figure. 3. b). The smallest absorption band appeared at pH

10 motivated us to focus on its morphology. At pH 10, TEM

image revealed spherical nanoprticles in a size ranged from 2

to 15 nm (Figure. 3. c). Statistically, the mean of size of

nano-objects produced at both cases were significant

different. So, pH played a considerable role in controlling

nano-size.

(a)

(b)

(c)

Figure 3. Effect of pH on bio-synthesis of nano-silver by cell free secretions

(a) Different degree of yellowish color at different pH (b) UV-Vis

Spectrophotometer (c) TEM micrograph of bio-synthesized nano-silver at pH

10.

4. Conclusion

From the current study, cell free secretions from seeds of

Medicago sativa was presented as a green source of bio-

fabricants for nano-silver formation. Adjusting pH was a

significant factor for controlling the nano-size. The main bio-

reducers and bio-stabilizers were supposed to be an

antioxidant and poly-phenol members, respectively.

4 Gamal Hassan Rabie et al.: Biosynthesis of Nano-Silver by Cell Free Secretions from Seeds of Medicago Sativa

Acknowledgement

We want to acknowledge Academy of Scientific Research

and Technology (ASRT) for funding our research through

Scientists for Egypt: Next Generation (SNG) program.

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