Soybeans as a Phytochemical Reservoir for the Production and Stabilization of Biocompatible Gold...

Green Nanoparticles by Using Green Chemistry

Green chemistry

DOI: 10.1002/smll.200800525

Soybeans as a Phytochemical Reservoir for the Productionand Stabilization of Biocompatible Gold NanoparticlesRavi Shukla, Satish K. Nune, Nripen Chanda, Kavita Katti, Swapna Mekapothula,Rajesh R. Kulkarni, Wade V. Welshons, Raghuraman Kannan,* and Kattesh V. Katti*

Keywords:� cytotoxicity

� gold nanoparticles

� green nanotechnology

� nanomedicine

� soybeans

The present study demonstrates an unprecedented green process for the

production of gold nanoparticles by simple treatment of gold salts with

soybean extracts. Reduction capabilities of antioxidant phytochemicals

present in soybean and their ability to reduce gold salts chemically to

nanoparticles with subsequent coating of proteins and a host of other

phytochemicals present in soybean on the freshly generated gold nano-

particles are discussed. The new genre of green nanoparticles exhibit

remarkable in vitro stability in various buffers including saline, histidine,

HSA, and cysteine solutions. MTT assays reveal that the green gold nano-

particles are nontoxic and thus provide excellent opportunities for their

applications in nanomedicine for molecular imaging and therapy. The overall

strategy described herein for the generation of gold nanoparticles meets all 12

principles of green chemistry, as no ‘‘man-made’’ chemicals, other than the

gold salts, are used in the green nanotechnological process.

1. Introduction

The role of plants and plant species for the production of

nanoparticles is directly related to the creation of an important

[�] Prof. K. V. Katti, Prof. Dr. R. Kannan

Room 201, Alton Building Laboratories, 301, Business Loop 70W

University of Missouri-Columbia

Columbia, MO 65212 (USA)

Fax: (þ1) 573-884-5679

E-mail: [email protected]; [email protected]

Dr. R. Shukla, Dr. S. K. Nune, Dr. N. Chanda, K. Katti

Department of Radiology, 301, Business Loop 70W


Columbia, MO 65212 (USA)

S. Mekapothula, Prof. W. V. Welshons

Department of Biomedical Sciences, University of Missouri-Columbia

Columbia MO 65212 (USA)

R. R. Kulkarni

Nuclear Science and Engineering Institute,


Columbia MO 65212 (USA)

: Supporting Information is available on the WWW under http://www.small-journal.com or from the author.

small 2008, 4, No. 9, 1425–1436 � 2008 Wiley-VCH Verl

symbiosis between nanotechnology and green chemistry.[1–11]

As the nanorevolution unfolds, it is imperative to develop

connections between nanotechnology and nature. The pro-

duction of nanoparticles under nontoxic green conditions is of

vital importance to address growing concerns on the overall

toxicity of nanoparticles for medical and technological

applications.[12–23] The power of phytochemicals that initiate

various chemical transformations within biological systems is

well-known.[24–32] For example, genistein found in soybeans

acts both as a phytoestrogen and antioxidant and has been

used extensively to treat conditions affected by estrogen levels

in the body.[33–45] Several investigations have concluded that

people who live in Japan have lower rates of breast, ovarian,

and prostate cancer because of the genistein content in their

diet, which is high in soybeans.[46–48] Genistein is known to

compete effectively with estrogen and to bind in place of

estrogen to receptors in cancerous cells that need this hormone

to grow.[49–59] Several studies have also demonstrated that

genistein helps to regulate blood sugar and to prevent the

development of insulin resistance that can lead to diabetes and a

host of disorders associated with this condition, including

diabetic retinopathy.[60–62] Numerous investigations have

demonstrated the role of genistein in the prevention of the

ag GmbH & Co. KGaA, Weinheim 1425

full papers R. Kannan, K. Katti et al.

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growth of certain tumors by depriving cancer cells of tyrosine

protein kinase, which they need to flourish, and disrupting the

action of certain enzymes that allow tumors to develop their

own blood supply.[63–70] Soybeans are also effective owing to

their antioxidant properties. In particular, the antioxidant

properties of isoflavonoids for radical scavenging, and their

antiestrogenic, antimutagenic, antiproliferative differentia-

tion-inducing and angiogenesis-inhibiting activities have led to

a greater awareness of the importance of soybeans within the

human food chain.[71–82]

We hypothesized that the effective utilization of various

phytochemicals that contain functional groups such as

carboxy, amino, thio, and hydroxy units within the protein

frameworks, in combination with the presence of saccharides

(sucrose and stachyose) embedded within soybeans, will

provide synergistic chemical reduction power for the reduc-

tion of gold salts into their corresponding nanoparticles.[83,84]

We further hypothesized that the proteins glycinin (11s),

b-conglycinin (7s), and trypsin inhibitor (2s) along with the

isoflavones (genistein, diadzein, and glycitein) and phyto-

estrogens of soybeans will provide a coating of phytochemicals

on the gold nanoparticles, thus allowing an unprecedented

green process for the production and stabilization of gold

nanoparticles. The rationale behind this hypothesis is based on

the reduction capabilities of the cocktail of phytochemicals

present in soybeans and their ability to reduce chemically

gold(III) salts to nanoparticles, with subsequent coating of the

freshly generated gold nanoparticles with proteins and a host

of other phytochemicals present in the soybeans. We argue

that validation of this hypothesis would result in a versatile

green nanotechnology and the application of gold nanopar-

ticles in a myriad of applications in nanomedicine and

technology.[85–95] On the technology front, large-scale produc-

tion of nanoparticles through plant species and nontoxic seeds

will minimize/eliminate chemical interventions, thus resulting

in true green and nonpolluting industrial processes for the

production of nanoparticle-based smart materials (Table 1).

Herein we report an unprecedented synthetic route that

involves the production of well-defined spherical gold

nanoparticles by simple mixing of soybeans in an aqueous

solution of sodium tetrachloroaurate. This soybean-mediated

green nanotechnological process allows the production of gold

nanoparticles within 2 h. The gold nanoparticles generated

through the soybean-mediated process did not aggregate, thus

suggesting that the cocktail of phytochemicals, including

proteins, serve as excellent coatings on nanoparticles and thus

provide robust shielding from aggregation. Furthermore, the

Table 1. Physicochemical data parameters of Soy-AuNPs.

Sample Size z

TEM DLS[a] CPS

[nm] [nm] [nm] [mV]

Soy-AuNP-1 15� 4 17� 1

Soy-AuNP-2 15� 4 61� 1 10� 1 �30� 1

[a] Hydrodynamic diameter; [b] S: Stable; [c] U: Unstable.

www.small-journal.com � 2008 Wiley-VCH Verlag Gm

phytochemical coatings on the gold nanoparticles impart

nontoxic properties on these ‘‘green’’ products, as demon-

strated through detailed 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assays performed on

normal fibroblast cells. Results of our studies presenting a new

green method for the production and utility of gold

nanoparticles for potential applications in nanomedicine are

discussed in the following sections.

2. Results and Discussion

Several studies have demonstrated the importance of

soybeans in the diet because the phenolics present in such

crops act as antioxidants, thereby reducing the risk of

atherosclerosis and coronary heart disease.[71–82] In addition

to phenolics, soybeans also contain isoflavonoids, tannins,

proanthocyanidins, and lignin precursors, which have been

shown to be powerful antioxidants serving the role of reactive-

oxygen-species scavengers.[71–82,96–99] As part of our ongoing

efforts toward the design and development of biocompatible

gold nanoparticles for subsequent use in medical applica-

tions,[21–23] we have initiated studies on the direct intervention

of phytochemicals for the production of gold nanoparticles.

The composition of various phytochemicals in soybeans is

outlined in Figure 1. Our initial studies involved the direct

interaction of soybeans with sodium tetrachloroaurate

(NaAuCl4) in aqueous media. Simple mixing of sodium

tetrachloroaurate with soybeans at 25 8C produced purple

colorations within 2–6 h. These reactions went to completion

within 24 h. Detailed UV/Vis spectrophotometric and trans-

mission electron microscopy (TEM) analysis of purple

solutions confirmed the identity and composition of soy-

bean-derived gold nanoparticles (Soy-AuNP-1).[100] Our 1H

NMR spectroscopic investigations[100] of soybean extract

obtained by soaking soybeans in D2O indicated that

phytochemicals from soybeans are extracted at very slow

rates in aqueous media. Therefore, the synthesis of nano-

particles from preformed soybean extract (supernatant

solutions after soaking of soybeans in water) was explored

(Soy-AuNP-2) (Figure 2). Extracts prepared by incubation of

soybeans in doubly ionized water were used within 3 days for

subsequent gold nanoparticle synthesis. Soybean extracts

mixed with NaAuCl4 at 95 8C resulted in the formation of

purple-red colorations within 10 min. Absorption measure-

ments indicated that the plasmon resonance wavelength lmax

and the plasmon line width Dl of Soy-AuNP-2 are �530 nm

and 180 nm, respectively (Figure 3A). The size of the

In Vitro Stability

NaCl Cysteine Histidine pH 7 pH 9

(10%) (0.5%) (0.2%) (PBS) (PBS)

U[b] S S S S

S[c] S S S S

bH & Co. KGaA, Weinheim small 2008, 4, No. 9, 1425–1436


Figure 1. Composition of various phytochemicals in soybeans.

Soy-AuNP-2 is found to be 15� 4 nm as measured from the

TEM images (Figure 3B). Synthetic conditions have been

optimized for the large-scale conversions of NaAuCl4 into the

corresponding AuNPs. Specific details on the nature and

chemical roles of different phytochemicals in soybeans that are

responsible for the production of Soy-AuNPs are summarized

in the following sections.

2.1. Role of Phytochemicals

The main phytochemicals present in soybeans consist of

water-soluble proteins (globulins and albumins), carbo-

hydrates (sucrose, raffinose, and stachyose), saponins, isofla-

vones, and amino acids.[96,101–103] As the new Soy-AuNP

Figure 2. Synthesis of Soy-AuNP-2.

small 2008, 4, No. 9, 1425–1436 � 2008 Wiley-VCH Verlag

production process involves aqueous media, the water-soluble

proteins, including glycinin (11s), b-conglycinin (7s), and

trypsin inhibitor (2s), may play a major role in the overall

reduction reactions of NaAuCl4. Therefore, we have system-

atically investigated the roles of proteins, sugars, antioxidants,

and various phytochemicals for the generation and stabiliza-

tion of AuNPs through independent experiments.

2.1.1. Role of Soybean Proteins

To understand the chemical propensities of various

soybean proteins to reduce NaAuCl4 to the corresponding

gold nanoparticles, our experiments involved separation of

water-soluble soybean proteins into low-molecular-weight

(<5 kDa) and high-molecular-weight (>5 kDa) fractions by

GmbH & Co. KGaA, Weinheim www.small-journal.com 1427


Figure 3. Physicochemical analysis of Soy-AuNP-2: A) UV/Vis absorp-

tion spectrum; B) TEM image. Inset: size-distribution histogram.

Figure 4. Generation and characterization of gold nanoparticles with

high-molecular-weight fraction of soybean extract.

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using Centricon Plus-20 centrifugal filter devices. Both

fractions were used independently to evaluate their ability

to generate AuNPs. Experimental protocols involved mixing

of protein fractions with NaAuCl4 in aqueous media. High-

molecular-weight fractions mixed with NaAuCl4 at 95 8Cproduced gold nanoparticles within 10 min (Figure 4). The

corresponding reactions with the lower-molecular-weight

fractions also resulted in the production of gold nanoparticles

with poor stability toward aggregation (Figure 4). These

results suggest that both the high- and low-molecular-weight

fractions exhibit high kinetic propensity to initiate the

reduction of NaAuCl4 to the corresponding nanoparticles.

However, high-molecular-weight proteins, in addition to their

efficacy for reduction, demonstrated their ability to provide an

efficient coating on the gold nanoparticles. The amino acids

present in both the high- and low-molecular-weight fractions


are presumably responsible for the formation of gold

nanoparticles. The role of amino acids such as L-tryptophan,

L-tyrosine, L-arginine, L-lysine, and L-aspartic acid and also

small peptides such as glycyl-L-tyrosine in the production of

gold nanoparticles has been reported recently.[104–106] A

recent report on the generation of gold nanoparticles with

proteins further corroborates our findings on the role of phyto-

derived proteins in the reduction of gold salts to the

corresponding gold nanoparticles.[84] From these results, we

conclude that higher-molecular-weight soybean proteins have

distinct dual roles in the reduction of AuIII with subsequent

stabilization of gold nanoparticles. It is conceivable that the

disulfide bonds in the trypsin inhibitor (2s) protein along with

the glycinin (11s) and b-conglycinin (7s) provide robust

shielding on the gold nanoparticles against aggregation, thus

allowing excellent in vitro stability.

To gain further insight into the role of specific soybean

proteins, responsible for the generation of the robust coating

on AuNPs, we carried out SDS-PAGE (sodium dodecyl

sulfate-polyacrylamide gel electrophoresis) analysis. These

experiments involved the analysis of the presence of various

proteins in 1) soybean extract and 2) Soy-AuNP-2 super-

natants. Soy-AuNP-2 supernatant was obtained by centrifugal

separation of AuNPs from Soy-AuNP-2. In these experiments,

total proteins from soybean extract and Soy-AuNP-2 super-

natants were resolved on a 4-20% Tris-glycine minigel

followed by coomassie brilliant blue staining. As shown in

Figure 5, lane 1 corresponds to the standard protein marker,

lane 2 corresponds to the total proteins from soybean extract,

and lane 3 represents Soy-AuNP-2 supernatant (soybean

proteins leftover after centrifugal removal of AuNPs).

Comparisons of lane 2 and lane 3 revealed that the bands

corresponding to 51 kDa and 80 kDa in lane 2 are absent in



Figure 5. SDS-PAGE profile of soy proteins. Lane 1: standardmolecular-

weight marker; lane 2: soybean extract soaked for 72 h; lane 3: Soy-

AuNP-2 supernatant.

Figure 7. Venn diagram showing the possible role of phytochemicals in

soybean extract for generation and stabilization of gold nanoparticles.

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

750700650600550500450400λ // nm

A

GA+Soy Protein+IsoflavonesGA+CarbohydratesGA+Carbohydrates+IsoflavonesGA+IsoflavonesSoy Protein+IsoflavonesGA+Soy ProteinSoy ProteinSoy Protein +Sucrose

Figure 6. UV/Vis absorption spectra of gold nanoparticles generated

with various soybean components.

lane 3. Importantly, polypeptides in the molecular weight

range of 51–80 kDa are produced when the soybean protein b-

conglycinin degrades. The disappearance of the bands for the

51-kDa and 80-kDa species from the soybean protein extracts

after the synthesis of the gold nanoparticles suggests that these

polypeptides are involved (and fully consumed) in the

generation and stabilization of the gold nanoparticles.

Notably, Wilson et al.[107] have demonstrated that high-

molecular-weight proteins such as glycinin and b-conglycinin

undergo degradation to low-molecular-weight polypeptides in

the range of 21–31 kDa upon long-term soaking of soybeans.

The above results clearly corroborate our findings that

gold nanoparticles generated from the direct interaction of

soybeans with sodium tetrachloroaurate showed lower in vitro

stability than those generated by the interaction of NaAuCl4with preformed soybean extracts. The reaction conditions

involved direct interaction of soybeans with sodium tetra-

chloroaurate over a period of 4 h at 25 8C. It is conceivable that

high-molecular-weight soybean proteins are not completely

released into water within the 4-h reaction period. Indeed, our1H NMR spectroscopic analysis of aqueous aliquots soaked

with soybeans corroborate the slow release of soybean

proteins in water.[100] The lack of high-molecular-weight

proteins presumably results in limited shielding of the gold

nanoparticles, thus resulting lower in vitro stability. In this

context, it is important to recognize that the 1H NMR

spectroscopic studies of soybean extracts produced by soaking

the soybeans in aqueous media for over 72 hours clearly

showed the presence of high-molecular-weight proteins in

good abundance. These high-molecular-weight proteins are

highly effective in creating a powerful coating over gold

nanoparticles, thus rendering robust layers with consequent

shielding from aggregation. Therefore, gold nanoparticles

generated by using preformed soybean extracts exhibited

unprecedented in vitro stability beyond 3 months.

2.1.2. Role of Isoflavones

To investigate the roles of the isoflavones genistein, and

diadzein, we also investigated the reactions of individual

phytochemical components with NaAuCl4. Poor aqueous

solubility of both genistein and diadzein was a major

experimental difficulty in these reactions. A mixture of

dimethyl sulfoxide and water was used to dissolve genistein


(and diadzein). However, the reactions of genistein (and

diadzein) with NaAuCl4 at above 25 8C did not result in the

formation of AuNPs. These results suggest that genistein (and

diadzein) present in soybeans are not involved in the overall

reduction of NaAuCl4 (Figure 6). It is also important to note

that the 1H NMR spectroscopic studies of the extracts of

soybeans in D2O indicated the presence of genistein and

diadzein. This means that both genistein and diadzein as they

are extracted directly from soybeans into water may be

associated with hydrophilic pockets of proteins/carbohydrates

or other phytochemicals. Therefore, we cannot rule out the

cumulative reduction/stabilizing influence of both genistein

and diadzein on the overall production of gold nanoparticles,

as they are chemically available in soybean extracts (Figure 7).

2.1.3. Role of Sugars

Soybeans contain carbohydrates, including the disaccharide

sucrose and the oligosaccharides raffinose and stachyose

(Figure 1). We have tested the efficacy of individual

carbohydrates for gold nanoparticle production by interac-

tions with NaAuCl4 in aqueous media (Figure 8). Experiments

with raffinose at above 25 8C did not result in gold

nanoparticles. Glucose and sucrose have been known to act

as mild reducing agents for the reduction of NaAuCl4 in

aqueous media. However, such reduction reactions with

sucrose and glucose proceed only at elevated temperatures,



Figure 8. Synthesis of gold nanoparticles with various phytochemicals present in soybeans.

DI¼ doubly ionized.

1430

resulting in incomplete conversions into gold nanoparticles

and considerable amounts of unconverted NaAuCl4. Indeed,

our experiments have demonstrated that the reaction of

NaAuCl4 with sucrose at 25 8C does not result in gold

nanoparticles. However, when this reaction was carried out at

higher temperatures, partial conversion into gold nanoparti-

cles was noticed, and a considerable quantity of unconverted

tetrachloroaurate was observed (Figure 8). These results

unequivocally provide compelling evidence that soybean

proteins are the rate-limiting phytochemicals directly involved

in the reduction of NaAuCl4 and that various carbohydrates

and sugars present in soybeans provide synergistic benefits to

the overall reduction process (Figures 7 and 8). Higher

reaction temperatures presumably result in conformational

changes of soybean proteins and may also aid in opening

up the protein structures, including glycinin (11s) and

b-conglycinin (7s), so that the disulfide bonds become

accessible to nanoparticles. Higher reaction temperatures

are also important to exploit the synergistic reduction powers

of the carbohydrates within the soybean phytochemicals.

2.2. Nanoparticle Characterization and SizeDistribution

Physicochemical properties, such as size, charge, and

morphology of gold nanoparticles generated from soybeans

and soybean extracts, were determined by three independent

techniques. TEM, differential centrifugal sedimentation

(DCS, Disc Centrifuge, CPS Instruments), and dynamic light

scattering (DLS) were used to measure size and distribution.

TEM and DCS were used to determine the core size of the

gold nanoparticles, and DLS was used to evaluate the sizes of

soybean protein-coated gold nanoparticles.

2.2.1. Size and Morphology

TEM measurements of Soy-AuNP-1 (See Supporting

Information) and Soy-AuNP-2 showed that the particles are

spherical and in the size range 15� 4 nm (Table 1). Size-

distribution analysis of Soy-AuNPs confirm that the particles

are nearly monodisperse (Figure 7 and Table 1). DCS allows

www.small-journal.com � 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

the measurement of the sizes of

nanoparticles by determining the

time required for nanoparticles to

traverse a sucrose density gradient

created in a disc centrifuge. Both

TEM and DCS allow the determi-

nation of the sizes of the metallic

gold cores. The gold nanoparticle

sizes measured by TEM and DCS

are in good agreement and in the

range 9–16 nm (Table 1). DLS was

employed to calculate the sizes of

gold nanoparticles coated with pro-

teins (hydrodynamic radius). The

soybean protein coatings on gold

nanoparticles are expected to cause

substantial changes in the hydro-

dynamic radius of Soy-AuNPs. The

hydrodynamic diameter of Soy-

AuNP-2 was determined from DLS measurements to be

61� 1, suggesting that soybean proteins are capped on gold

nanoparticles. The measurement of the charge on the

nanoparticles, the zeta potential z, provides crucial informa-

tion on the stability of the nanoparticle dispersion. The

magnitude of z is an indication of the repulsive forces present

and can be used to predict the long-term stability of the

nanoparticle dispersion. The stability of the nanoparticle

dispersion depends on the balance of the repulsive and

attractive forces between the nanoparticles as they approach

one another. If all the particles have a mutual repulsion, then

the dispersion will remain stable. However, little or no

repulsion between particles leads to aggregation. The negative

value z of �30� 1 for Soy-AuNP-2 indicates that the particles

repel each other and that there is no tendency for the particles

to aggregate (Table 1 and Figure 3).

2.2.2. In Vitro Stability Studies

An issue of critical importance for biomedical applica-

tions is the stability of Soy-AuNPs over a reasonable time

period. In vitro stability of Soy-AuNPs was evaluated by

monitoring the plasmon wavelength (lmax) and plasmon

bandwidth (Dl) in NaCl (10%), histidine (0.2 M), bovine

serum albumin (BSA, 0.5%), or human serum albumin

(HSA, 0.5%). The plasmon wavelength and bandwidth in all

the above formulations shifts by approximately 5 nm. This

indicates that the AuNPs are intact, and hence demonstrate

high in vitro stability of Soy-AuNP-2 in biological fluids at

physiological pH values (Figure 9).

Biomedical applications require lower concentrations of

AuNPs and it is vital that the dilution of AuNP solutions does

not alter the characteristic chemical and photophysical

properties. We undertook a detailed investigation to ascertain

the effect of dilution on the stability of Soy-AuNP-2. To

establish the stability of Soy-AuNP-2 under dilution, the

plasmon resonance wavelength (lmax) and bandwidth (Dl)

were monitored after every successive addition of 0.2 mL of

doubly ionized water to 1 mL of Soy-AuNP-2 solutions (Figure

10). The absorption intensity at lmax is found to be linearly

dependent on the concentration of AuNPs, in accordance with

small 2008, 4, No. 9, 1425–1436


8007006005000.0

0.2

0.4

0.6

0.8

A

Water 10% NaCl 0.2M Histidine 0.5% HSA 0.5% BSA pH 5, Buffer pH 7, Buffer pH 9, Buffer

8007006005000.0

0.2

0.4

0.6

0.8A

λ /nm

λ /nm

Water10% NaCl0.2M Histidine0.5% HSA 0.5% BSApH 5, BufferpH 7, BufferpH 9, Buffer

B)

A)

Figure 9. UV/Vis spectra showing the in vitro stability of Soy-AuNP-2 in

aqueous solutions after 24 h. A) 1.25mM gold atoms; B) 500mM gold

atoms after synthesis of nanoparticles for 15 days.

the Beer–Lambert law as shown in Figure 10. It is important to

recognize that lmax and Dl of the AuNPs did not change at

dilutions in the range of 10�5–10�6M. These are typical

concentrations encountered when working at cellular levels.

Therefore, the data outlined in Figure 9 demonstrates that the

Soy-AuNPs have exceptional in vitro stability in HSA,

histidine, cysteine, saline, and phosphate buffer.

It is conceivable that the isoflavones, tannins, proanthro-

cyanidins, and various proteins in soybeans act synergistically

1401201008060402000.0

0.2

0.4

0.6

0.8

1.0

A

Soy-AuNP-2 [Au] /µµM

Figure 10. Change in the plasmon absorption of Soy-AuNP-2 under

various dilution conditions: &experimental data; linear fit.


to provide a robust coating around gold nanoparticles to

prevent aggregation. It is also remarkable that this ‘‘nano-

compatible’’ structural motif of phytochemicals in soybeans

offer stability to gold nanoparticles in aqueous media for over

three months. These results suggest that the green process

reported herein provides both the production and stabilization

efficacies under environmentally-benign conditions without

the intervention of any man-made chemicals.

2.3. Cytotoxicity of Soy-AuNPs

When new nanomaterials are intentionally synthesized and

used for biomedical applications, their biocompatibility is the

first and foremost important criteria to be evaluated. A key

parameter in evaluating their biocompatibility centers on the

evaluation of their potential cytotoxicity. We have studied the

cytotoxicity of Soy-AuNP-2 in primary human fibroblast

cultures as in vitro experimental models. The cytotoxicity of

Soy-AuNP-2 under in vitro conditions in human fibroblast cells

was examined in terms of the effect of Soy-AuNPs on cell

proliferation by the MTT assay. Fibroblast cells treated with

Soy-AuNPs at concentrations of 0, 25, 50, 125, and 165mM (Au

atoms) for 24 h were subjected to MTT assay for cell-viability

determination. After treatment with Soy-AuNP-2 for 24 h, cells

showed more than 85% viability at concentrations up to 165mM

(Figure 11). It may be noted that a number of AuI and AuIII

complexes have demonstrated significant cytotoxicity.[108,109]

The co-operative reduction capabilities of phytochemicals

in soybeans that effectively reduce sodium tetrachloroaurate

in aqueous media satisfy the fundamental principles that

govern green chemistry.[110–112] This green nanotechnology

process is unique because the Soy-AuNPs can be produced

and stabilized simultaneously (Table 2). It is also important to

recognize that although the cocktail of phytochemicals,

proteins, and carbohydrates ensures the reduction of the gold

salts to their corresponding nanoparticles, the host of soybean

protein matrices along with antioxidant phytochemicals in

soybeans effectively coat around gold nanoparticles to render

excellent robustness against aggregation.

The current discovery demonstrates that the chemical

power of phytochemicals in soybeans is of paramount

importance in the context of the production of gold

nanoparticles for medical and technological applications

under nontoxic conditions. The role of soybeans as a reservoir

Figure 11. Dose-dependent cytotoxicity showing nontoxic profiles of

Soy-AuNP-2 in fibroblast cells after 24 h of exposure.



Table 2. Green chemistry criteria for gold nanoparticle production.

Reducing agents Green Chemistry Nanoparticle Properties

Citrate No � susceptible to aggregation

� need additional stabilizing agents for stability

� interact with serum proteins instantaneously

� stable in solution only

� poor in vivo stability

Borohydride No � highly toxic to humans/animals

� poor in vivo stability

� susceptible to aggregation

� need additional stabilizing agents for stability

� stable in solution only

Bacteria No � intracellular synthesis

� limited control over size

� high purification cost

� increased biohazard risk

Phytochemicals Yes � highly stable in solution

� greater in vivo stability

� better control over size

� noncytotoxic

� shielded by natural targeting agents

� nonhazardous

� follows all 12 principles of green chemistry

1432

of nontoxic phytochemicals is also pivotal, as it serves as a

source of highly effective reducing agents with capabilities for

in vivo administrations in situations that require the genera-

tion of gold nanoparticles under in vivo conditions.

3. Conclusions

Functionalized and hybrid gold nanoparticles play an

indispensable role in the overall design and development of

AuNP-based nanopharmaceuticals. For molecular imaging

and therapy, it is of pivotal importance to generate

nanoparticles in biofriendly media and even in the presence

of chemically sensitive proteins, peptides and receptor-specific

biovectors. These performance requirements will impose

serious constraints on the nature of synthetic routes and the

conditions of reactions that can be used to generate gold

nanoparticles. The studies reported herein serve as a unique

example of the kinetic propensity of the phytochemicals in

soybeans to reduce gold metal at the macro- or in pico-/sub-

nanomolar concentrations to the corresponding gold nano-

particles. The single-step green process has been shown to be

effective for the generation and stabilization of nontoxic gold

nanoparticles for direct applications in a myriad of diagnostic

and therapeutic applications. Occlusion of cancer-fighting

phytochemicals in various plant species and their future utility

in the development of tumor-specific gold nanoparticles will

provide unprecedented opportunities toward the design and

development of functional ‘‘green’’ gold nanoparticles that

can be safely produced, stored, and shipped worldwide.

4. Experimental Section

Materials and characterization methods: All chemicals used in

the synthesis of gold nanoparticles (AuNPs) were procured from


standard commercial vendors: NaAuCl4 (Alfa-Aesar); proteins,

isoflavones, and sugars (Sigma-Aldrich). Size, charge, and

morphology of gold nanoparticles generated from soybeans and

soybean extracts were determined by three independent tech-

niques: TEM, DCS (Disc Centrifuge, CPS Instruments), and DLS

methods (Malvern Instruments Ltd, USA) were employed. TEM

images were obtained on a JEOL 1400 transmission electron

microscope, JEOL, LTD., Tokyo, Japan. TEM samples were prepared

by placing 5mL of gold nanoparticle solution on a 300-mesh

carbon-coated copper grid, and the solution was allowed to stand

for 5min. Excess solution was removed carefully, and the grid was

allowed to dry for an additional 5min. The average size and size

distributions of gold nanoparticles were determined by processing

the TEM images with image-processing software such as Adobe

Photoshop (with Fovea plug-ins). DCS was used to measure size

and was measured on a DC 24000, CPS Instruments Inc. USA. The

hydrodynamic radius and the zeta potential were obtained on a

Zetasizer Nano S90, Malvern Instruments Ltd. USA. The absorption

measurements were carried out in Varian Cary 50UV/Vis spectro-

photometers with 1mL of gold nanoparticle solution in disposable

cuvettes with a 10-mm pathlength.

Synthesis and stabilization of gold nanoparticles by direct

reduction of tetrachloroaurate with soybeans (Soy-AuNP-1): Gold

nanoparticles were prepared by suspending 3 g of soybeans in

10mL of doubly ionized water for 2 h. To this suspension, 0.3mL

(0.289mM) of 0.1 M NaAuCl4 solution was added, and the mixture

was stirred at 25 -C for 4 h. The color of the supernatant solution

changed from light yellow to dark purple, indicating the formation

of gold nanoparticles (Soy-AuNP-1). Soybeans were removed from

the solution by decanting the solution, followed by filtration

through a 5-mm filter. The natural pH value of the Soy-AuNP-1

solution was found to be 4 and was further adjusted to

physiological pH by adding 0.1mL of 10X phosphate buffer

concentrate (pH 7) to the whole volume. The identity of Soy-AuNP-

1 was confirmed through observations of the plasmon resonance

band at 540 nm in UV/Vis absorption measurements. The size



distribution of Soy-AuNP-1 was inferred through detailed TEM and

DCS. In vitro stability studies in various media as described below

have confirmed that these nanoparticles are stable over long

periods of time (over two weeks), suggesting that the proteins in

soybeans have provided effective coating on AuNPs.

Preparation of soybean extract: Organic soybeans purchased

from a local grocery shop were used in all the experiments.

Soybeans (8 g) were washed with doubly ionized water to remove

any contaminants or dust particles. Soybeans were incubated in

50mL of doubly ionized water at 25 -C for 72 h. The supernatant

was decanted and centrifuged at 8000 rpm for 10min at 25 -C. Thesupernatant was stored at 4 -C and was used within 3 days for

subsequent gold nanoparticle synthesis.

Synthesis of gold nanoparticles with soybean extract (Soy-

AuNP-2): A soybean extract as described above was used to

obtain phytochemically derived reducing agents for the generation

and stabilization of gold nanoparticles. In a typical experiment,

4mL of soybean extract was diluted to 8mL in doubly ionized

water and heated to 95 -C for 3min. To this solution, 100mL of

NaAuCl4 (0.1 M) was added and further heated to 95 -C with

constant stirring for 5min. The color of the mixture turned from

pale yellow to purple-red within 5min, indicating the formation of

gold nanoparticles (Soy-AuNP-2), and the reaction mixture was

stirred further for 2 h at 25 -C to ensure complete reaction.

Formation of gold nanoparticles was monitored by UV/Vis

absorption spectroscopy. The plasmon resonance band at

540 nm indicated the formation of gold nanoparticles, and TEM

measurements confirmed their size distribution. In vitro stability

studies in various media as described below confirmed that these

nanoparticles are stable over long periods of time (over two

weeks), suggesting that the protein in soybeans have provided

effective coating on AuNPs.

Fractionation of high- and low-molecular-weight fractions of

soybean extracts: The soybean extract was further purified and

concentrated by using 5-kDa centrifugal filter devices (Centricon

Plus-20). The centrifuge tube filled with 6mL of soybean extract

was centrifuged at 4000 rpm for 10min. The separated low- and

high-molecular-weight fractions were further diluted to a total

volume of 8mL with doubly ionized water and were used as such

for the generation of gold nanoparticles.

Generation of gold nanoparticles by using high-molecular-

weight protein fraction of soybean Extract (Soy-AuNP-3): To a 20-

mL vial was added 8mL of high-molecular-weight soybean protein

fraction (>5 kDa) obtained by using a Centricon Plus-20 centrifugal

filter device. The fraction was heated at 95 -C with continuous

stirring for 3min. To the stirred mixture was added 100mL of 0.1 M

NaAuCl4 solution (in doubly ionized water), and the reaction

mixture was heated for a further 5min, and then stirred for an

additional 15 minutes at 25 -C. The formation of gold nanopar-

ticles was monitored by UV/Vis absorption spectroscopy. The

plasmon resonance band at 540 nm indicated the formation of

gold nanoparticles, and. TEM measurements confirmed their size

distribution.

Generation of gold nanoparticles using low-molecular-weight

fraction of soybean extract (Soy-AuNP-4): To a 20-mL vial was

added 8mL of the low-molecular-weight fraction obtained by

using a Centricon Plus-20 centrifugal filter device. The mixture was

heated at 95 -C with continuous stirring for 3min, and then 100mL


of 0.1 M NaAuCl4 solution (in doubly ionized water) was added.

The mixture was heated for a further 5min and then stirred for an

additional 15min at 25 -C. This reaction resulted in the formation

of a dark-purple precipitate due to aggregation of gold nanopar-

ticles. This observation suggests that low-molecular-weight

fractions of soybean extracts are capable of reducing AuIII to the

corresponding gold nanoparticles; however, they are incapable of

wrapping around the nanoparticles to impart stability against

aggregation.

Attempted synthesis of isoflavone-mediated gold nanoparticles:

To a 20-mL vial was added 0.012 g of gum arabic followed by

0.47mg of genistein, 0.3mg of diadzein, and 8mL of doubly

ionized water. The reaction mixture was heated at 95 -C and

stirred continuously for 3min, after which 100mL of 0.1 M NaAuCl4solution (in doubly ionized water) was added. The reaction

mixture was heated for an additional 5min and then stirred for a

further 15min at 25 -C. UV/Vis spectrophotometric analysis of the

reaction mixture indicated that there was no formation of AuNPs,

suggesting that isoflavones have limited/no role in the reduction

of AuIII to the corresponding nanoparticles.

Attempted synthesis of carbohydrate-mediated gold nanoparticles:

To a 20-mL vial was added 0.0544 g of sucrose, 0.0064 g of

raffinose, 0.0064 g of stachyose, and 8mL of doubly ionized

water. The reaction mixture was heated at 95 -C and stirred

continuously for 3min. To the stirred mixture was added 100mL of

0.1 M NaAuCl4 solution (in doubly ionized water). The solution was

heated further for 5min and then stirred for an additional 15min

at 25 -C. The progress of the reaction was monitored by UV/Vis

absorption spectroscopy. Broad uncharacteristic bands in the

500–550-nm region indicated that the gold nanoparticles are

aggregated and that carbohydrates initiate gold nanoparticle

formation but are incapable of coating around the nanoparticles

to impart stability against aggregation.

In vitro stability studies of Soy-AuNP-2: The in vitro stability of

Soy-AuNP-2 against aggregation was tested by mixing nanoparti-

cle solutions with aqueous solutions of NaCl, histidine, HSA, and

BSA solutions. Briefly, 0.5mL of Soy-AuNP-2 was added to

0.25-mL aliquots of 10% NaCl, 0.2 M histidine, 0.5% BSA, or

0.5% HSA. We also investigated the stability of Soy-AuNP-2 in pH

5, 7, and 9 phosphate buffer solutions. The stability and the

identity of Soy-AuNPs were measured by recording the UV/Vis

absorbance spectrum after 24 h (Figure 9). The plasmon reso-

nance band at 530 nm confirmed the retention of nanoparticulates

in all the above mixtures. In vitro challenging studies, as

described above, were performed on Soy-AuNP-2 solutions stored

at 25 -C over a 15-day period. Detailed UV/Vis spectrophotometric

inferred in vitro stability and the retention of nanoparticle

composition in all the above in vitro studies (Figure 9B).

SDS-PAGE analysis of soybean extract and Soy-AuNP-2 super-

natant: The soybean extract prepared by soaking soybeans for

72 h was used for the synthesis of gold nanoparticles. The

nanoparticles contained in soybean protein extracts were cen-

trifuged at 20000 g. After centrifugation, 1.8mL of supernatant

was concentrated to 0.3mL by using 3-kDa centricon filters. In

separate microcentrifuge tubes, 50mL of each of soybean extract

and gold nanoparticle supernatant were mixed with equal

volumes of 2x laemmli sample loading buffer (Biorad) with

2-mercaptoethanol (Sigma) and were heated at 95 -C for 10min,



1434

and 25mL of each of the samples was resolved on a 4–20% tris-

Glycene minigel (Biorad). One well of the gel was loaded with

10mL of standard protein molecular weight marker (Biorad), and

the gel was subsequently stained with coomassie brilliant blue

stain and scanned with an Epson 4990 photo scanner.

Cell culture: Human fibroblast primary cultures were main-

tained in DMEM with 10 pgmLS1 phenol red, 10mM HEPES,

100 unitsmLS1 penicillin, 100 pgmLS1 streptomycin, and 10%

donor bovine serum (maintenance medium).

MTT cytotoxicity assay: For the cytotoxicity evaluation of Soy-

AuNP-2, an MTT assay was performed as described by the

manufacturer (ATCC, USA). Briefly, 2T104 cells at the exponential

growth phase were seeded in each well of a flat-bottomed 96-well

polystyrene-coated plate and were incubated for 24 h in a CO2

incubator at 5% CO2 and 37 -C. Series of dilutions with 25, 50,

125, and 165mM of these gold nanoparticles were prepared in the

medium. Each concentration was added to the plate in pentaplet

manner. After 24 h of incubation, 10mL per well of MTT (stock

solution 5mgmLS1 PBS) (ATCC, USA) was added for 24 h.

Formosan crystals were dissolved in 100mL detergent. The plates

were kept for 18 h in the dark at 25 -C to dissolve all the crystals,

and the intensity of color progression was measured with a

microplate reader (Dynastic MR 5000, USA) operating at 570 nm.

Wells with complete medium, nanoparticles, and MTT, but without

the cells were used as blanks. Untreated cells were considered as

100% viable.

Acknowledgements

This work was supported by the National Institutes of Health/

National Cancer Institute under the Cancer Nanotechnology

Platform program (grant number: 5R01CA119412-01). RRK

acknowledges ‘‘Clinical biodetectives training grant’’, (grant

number: NIH-R90 DK71510) for support. The authors thank

Prof. Cris Lorson, Bond Life Science Centre, University of

Missouri-Columbia for a gift of noncancerous fibroblast cells.

[1] P. Raveendran, J. Fu, S. L. Wallen, Journal of the American

Chemical Society 2003, 125, 13940.[2] S. S. Shankar, A. Rai, B. Ankamwar, A. Singh, A. Ahmad,M. Sastry,

Nature Materials 2004, 3, 482.[3] M. A. Albrecht, C. W. Evans, C. L. Raston,Green Chemistry 2006, 8,

417.

[4] D.Mandal,M.E.Bolander,D.Mukhopadhyay,G.Sarkar,P.Mukher-

jee, Applied Microbiology and Biotechnology 2006, 69, 485.[5] T. Premkumar, K. E. Geckeler, Journal of Physics and Chemistry of

Solids 2006, 67, 1451.[6] P. Raveendran, J. Fu, S. L. Wallen, Green Chemistry 2006, 8, 34.[7] K. Roy, S. Lahiri, Green Chemistry 2006, 8, 1063.[8] N. Vigneshwaran, R. P. Nachane, R. H. Balasubramanya, P. V.

Varadarajan, Carbohydrate Research 2006, 341, 2012.[9] J. L. Huang, Q. B. Li, D. H. Sun, Y. H. Lu, Y. B. Su, X. Yang, H. X.

Wang, Y. P. Wang, W. Y. Shao, N. He, J. Q. Hong, C. X. Chen,

Nanotechnology 2007, 18, 105104.[10] C. C. Wu, D. H. Chen, Gold Bulletin 2007, 40, 206.


[11] Y. Yang, T. Coradin, Green Chemistry 2008, 10, 183.[12] G. Zhang, B. Keita, A. Dolbecq, P. Mialane, F. Secheresse, F.

Miserque, L. Nadjo, Chemistry of Materials 2007, 19, 5821.[13] P. Mohanpuria, N. K. Rana, S. K. Yadav, Journal of Nanoparticle

Research 2008, 10, 507.[14] J. Cervini-Silva, P. Fernandez, B. Gilbert, M. Hernandez, J. Guz-

man, E. Chavira, Geochimica Et Cosmochimica Acta 2007, 71,A155.

[15] S. Gill, R. Lobenberg, T. Ku, S. Azarmi, W. Roa, E. J. Prenner,

Journal of Biomedical Nanotechnology 2007, 3, 107.[16] C. M. Goodman, C. D. McCusker, T. Yilmaz, V. M. Rotello, Bio-

conjugate Chemistry 2004, 15, 897.[17] Y. H. Jin, S. Kannan, M. Wu, J. X. J. Zhao, Chemical Research in

Toxicology 2007, 20, 1126.[18] N. Lewinski, V. Colvin, R. Drezek, Small 2008, 4, 26.[19] S. Singh, H. S. Nalwa, Journal of Nanoscience and Nanotechnol-

ogy 2007, 7, 3048.[20] R. Kattumuri, International Journal of Std & Aids 2003, 14, 552.[21] R. Kannan, V. Rahing, C. Cutler, R. Pandrapragada, K. K. Katti, V.

Kattumuri, J. D. Robertson, S. J. Casteel, S. Jurisson, C. Smith, E.

Boote, K. V. Katti, Journal of the American Chemical Society 2006,128, 11342.

[22] V.Kattumuri,M.Chandrasekhar,S.Guha,K.Raghuraman,K.V.Katti,

K. Ghosh, R. J. Patel, Applied Physics Letters 2006, 88, 153114.[23] V. Kattumuri, K. Katti, S. Bhaskaran, E. J. Boote, S. W. Casteel, G.

M. Fent, D. J. Robertson, M. Chandrasekhar, R. Kannan, K. V. Katti,

Small 2007, 3, 333.[24] I. T. Johnson, Proceedings of the Nutrition Society 2007, 66, 207.[25] K. H. Kwon, A. Barve, S. Yu, M. T. Huang, A. N. T. Kong, Acta

Pharmacologica Sinica 2007, 28, 1409.[26] H. Ohigashi, A. Murakami, Drugs of the Future 2007, 32, 3.[27] H. Nishino, Y. Satomi, H. Tokuda, M. Masuda, Current Pharma-

ceutical Design 2007, 13, 3394.[28] M. Kaur, R. Agarwal, Current Cancer Drug Targets 2007, 7, 355.[29] A. Deorukhkar, S. Krishnan, G. Sethi, B. B. Aggarwal, Expert

Opinion on Investigational Drugs 2007, 16, 1753.[30] R. J. Molyneux, S. T. Lee, D. R. Gardner, K. E. Panter, L. F. James,

Phytochemistry 2007, 68, 2973.[31] S. Rochfort, J. Panozzo, Journal of Agricultural and Food Chem-

istry 2007, 55, 7981.[32] A. M. Boudet, Phytochemistry 2007, 68, 2722.[33] F. G. E. Perabo, E. C. Von Low, J. Ellinger, A. Von Rucker, S. C.

Muller, P. J. Bastian, Prostate Cancer and Prostatic Diseases

2008, 11, 6.[34] N. J. M. Raynal, L. Momparler, M. Charbonneau, R. L. Momparler,

Journal of Natural Products 2008, 71, 3.[35] H. W. Si, D. M. Liu, Journal of Nutrition 2008, 138, 297.[36] L. Lehmann, L. Jiang, J. Wagner, Carcinogenesis 2008, 29, 363.[37] R. S. MacDonald, J. Przybyszewski, D. Sleper, M. Berhow, Faseb

Journal 2007, 21, A1101.[38] M. Messina, B. Lane, Future Lipidology 2007, 2, 55.[39] I. L. F. Nielsen, G. Williamson, Nutrition and Cancer-an Inter-

national Journal 2007, 57, 1.[40] J. J. Raffoul, S. Banerjee, M. X. Che, Z. E. Knoll, D. R. Doerge, J.

Abrams, O. Kucuk, F. H. Sarkar, G. G. Hillman, International

Journal of Cancer 2007, 120, 2491.[41] M. Yamasaki, S. Fujita, E. Ishiyama, A. Mukai, H. Madhyastha, Y.

Sakakibara, M. Suiko, K. Hatakeyama, T. Nemoto, K. Morishita, H.

Kataoka, H. Tsubouchi, K. Nishiyama, Cancer Science 2007, 98,1740.

[42] P. J. Magee, I. R. Rowland, British Journal of Nutrition 2004, 91,513.

[43] J. L. Limer, V. Speirs, Breast Cancer Research 2004, 6, 119.[44] S. M. Heinonen, A. Hoikkala, K. Wahala, H. Adlercreutz, Journal of

Steroid Biochemistry and Molecular Biology 2003, 87, 285.[45] P. Nakamura, J. Frecka, J. Ross-Viola, N. F. Shay, Faseb Journal

2006, 20, A596.



[46] N. Kurahashi, M. Iwasaki, S. Sasazuki, T. Otani, M. Inoue, S.

Tsugane, J. P. H. C.-B. Prosp, S. Grp, Cancer Epidemiology

Biomarkers & Prevention 2007, 16, 538.[47] K. Takamatsu, Abstracts of Papers of the American Chemical

Society 2001, 221, U31.[48] F. Sakauchi, M. M. H. Khan, M. Mori, T. Kubo, Y. Fujino, S. Suzuki,

S. Tokudome, A. Tamakoshi, J. S. Grp, Nutrition and Cancer-an

International Journal 2007, 57, 138.[49] E. J. Yearley, E. A. Zhurova, V. V. Zhurov, A. A. Pinkerton, Journal of

the American Chemical Society 2007, 129, 15013.[50] T. Misztal, M. Wafikowska, K. Gorski, K. Romanowicz, Acta Neu-

robiologiae Experimentalis 2007, 67, 411.[51] Y. Takahashi, S. D. Hursting, S. N. Perkins, T. C. Wang, T. T. Y.

Wang, Molecular Carcinogenesis 2006, 45, 18.[52] Y. Ng, S. Hanson, J. A. Malison, B. Wentworth, T. P. Barry,

Aquaculture 2006, 254, 658.[53] P. S. Cooke, V. Selvaraj, S. Yellayi, Journal of Nutrition 2006, 136,

704.

[54] C. Borras, J. Gambini, M. C. Gomez-Cabrera, J. Sastre, F. V.

Pallardo, G. E. Mann, J. Vina, Faseb Journal 2006, 20, 2136.[55] S. Hooshmand, D. Y. Soung, S. V. Madihally, L. Devareddy, E. A.

Lucas, B. H. Arjmandi, Journal of Bone and Mineral Research

2005, 20, S199.[56] J. R. Zhou, Z. M. Mai, G. L. Blackburn, Cancer Epidemiology

Biomarkers & Prevention 2004, 13, 1859s.[57] F. Squadrito, D. Altavilla, G. Squadrito, A. Saitta, D. Cucinotta, L.

Minutoli, B. Deodato, M. Ferlito, G. M. Campo, A. Bova, A. P.

Caputi, Cardiovascular Research 2000, 45, 454.[58] T. T. Y. Wang, N. Sathyamoorthy, J. M. Phang, Carcinogenesis

1996, 17, 271.[59] S. Yellayi, A. Naaz, M. A. Szewczykowski, T. Sato, J. A. Woods, J. S.

Chang, M. Segre, C. D. Allred, W. G. Helferich, P. S. Cooke,

Proceedings of the National Academy of Sciences of the United

States of America 2002, 99, 7616.[60] M. S. Choi, U. J. Jung, J. Yeo, M. J. Kim, M. K. Lee, Diabetes-

Metabolism Research and Reviews 2008, 24, 74.[61] M. Nakajima, K. Y. Chang, J. Cao, A. Ando, G. J. An, M. J. Cooney, E.

de Juan, Investigative Ophthalmology & Visual Science 2000, 41,S639.

[62] M. Nakajima, M. J. Cooney, A. H. Tu, K. Y. Chang, J. T. Cao, A. Ando,

G. J. An, M. Melia, E. de Juan, Investigative Ophthalmology &

Visual Science 2001, 42, 2110.[63] K. Lober, A. Alfonso, L. Escribano, L. M. Botana, Journal of Cellular

Biochemistry 2008, 103, 1076.[64] M. Luke, R. Krott, M. Warga, P. Szurman, S. Grisanti, K. U. Bartz-

Schmidt, T. Schneider, C. Luke, Graefes Archive for Clinical and

Experimental Ophthalmology 2007, 245, 242.[65] T. S. Stantchev, I. Markovic, W. G. Telford, K. A. Clouse, C. C.

Broder, Virus Research 2007, 123, 178.[66] L. Thors, K. Alajakku, C. J. Fowler, British Journal of Pharmacology

2007, 150, 951.[67] C. Chen, S. Wong, B. Tay, J. Shen, M. Khan, J. H. Han, Ejc

Supplements 2005, 3, 283.[68] L. J. Liu, T. M. Yang, S. A. Simon, Pain 2004, 112, 131.[69] C. Matsumura, H. Kuwashima, S. Soma, T. Kimura, Journal of

Pharmacological Sciences 2007, 103, 263p.[70] K. T. Papazisis, T. G. Kalemi, D. Zambouli, G. D. Geromichalos, A.

F. Lambropoulos, A. Kotsis, L. L. Boutis, A. H. Kortsaris, Cancer

Letters 2006, 233, 255.[71] K. M. Cesario, F. M. Steinberg, R. L. Prior, Faseb Journal 2003, 17,

A1116.

[72] D. C. Liebler, S. Valcic, A. Arora, J. A. Burr, S. Cornejo, M. G. Nair, B.

N. Timmermann, Biological Reactive Intermediates Vi 2001, 500,191.

[73] R. P. Patel, B. J. Boersma, J. H. Crawford, N. Hogg, M. Kirk, B.

Kalyanaraman, D. A. Parks, S. Barnes, V. Darley-Usmar, Free

Radical Biology and Medicine 2001, 31, 1570.


[74] C. E. Rufer, S. E. Kulling, Journal of Agricultural and Food Chem-

istry 2006, 54, 2926.[75] F. M. Steinberg, N. L. Guthrie, A. C. Villablanca, K. Kumar, M. J.

Murray, American Journal of Clinical Nutrition 2003, 78, 123.[76] J. H. Lee, I. Y. Baek, J. M. Ko, N. S. Kang, S. H. Shin, S. G. Lim, K. W.

Oh, S. O. Shin, K. Y. Park, K. H. Park, T. J. Ha, Food Science and

Biotechnology 2008, 17, 1.[77] J. B. Liu, S. K. C. Chang, D. Wiesenborn, Journal of Agricultural and

Food Chemistry 2005, 53, 2333.[78] R. Takahashi, R. Ohmori, C. Kiyose, Y. Momiyama, F. Ohsuzu, K.

Kondo, Journal of Agricultural and Food Chemistry 2005, 53,

4578.

[79] I. R. Record, I. E. Dreosti, J. K. Mcinerney, Journal of Nutritional

Biochemistry 1995, 6, 481.[80] H.C.Wei, R.Bowen,Q.Y.Cai,S.Barnes, Y.Wang,Proceedingsof the

Society for Experimental Biology and Medicine 1995, 208,

124.

[81] A. Arora, S. Valcic, S. Cornejo, D. C. Liebler, Abstracts of Papers of

the American Chemical Society 2000, 219, U31.[82] J. M. Choi, H. J. Ryu, J. H. Chung, J. C. Park, J. K. Hwang, D. B. Shin,

S. K. Lee, R. Ryang, Food Science and Biotechnology 2005, 14,399.

[83] D. A. Rickert, L. A. Johnson, P. A. Murphy, Journal of Agricultural

and Food Chemistry 2004, 52, 1726.[84] J. P. Xie, J. Y. Lee, D. I. C. Wang, Y. P. Ting, Small 2007, 3, 672.[85] S. M. Moghimi, A. C. Hunter, J. C. Murray, Faseb Journal 2005, 19,

311.

[86] H. W. Liao, C. L. Nehl, J. H. Hafner, Nanomedicine 2006, 1, 201.[87] K. K. Jain, Clinical Chemistry 2007, 53, 2002.[88] X. H. Huang, P. K. Jain, I. H. El-Sayed, M. A. El-Sayed, Nanome-

dicine 2007, 2, 681.[89] X. H. Huang, I. H. El-Sayed, W. Qian, M. A. El-Sayed, Journal of the

American Chemical Society 2006, 128, 2115.[90] M. Hu, J. Y. Chen, Z. Y. Li, L. Au, G. V. Hartland, X. D. Li, M.

Marquez, Y. N. Xia, Chemical Society Reviews 2006, 35, 1084.[91] G. Han, P. Ghosh, V. M. Rotello, Nanomedicine 2007, 2, 113.[92] J. F. Hainfeld, D. N. Slatkin, T. M. Focella, H. M. Smilowitz, British

Journal of Radiology 2006, 79, 248.[93] A. G. Cuenca, H. B. Jiang, S. N. Hochwald, M. Delano, W. G. Cance,

S. R. Grobmyer, Cancer 2006, 107, 459.[94] L. Cognet, C. Tardin, D. Boyer, D. Choquet, P. Tamarat, B. Lounis,

Proceedings of the National Academy of Sciences of the United

States of America 2003, 100, 11350.[95] D. Boyer, P. Tamarat, A. Maali, B. Lounis, M. Orrit, Science 2002,

297, 1160.

[96] G. Sakthivelu, M. K. A. Devi, P. Giridhar, T. Rajasekaran, G. A.

Ravishankar, M. T. Nikolova, G. B. Angelov, R. M. Todorova, G. P.

Kosturkova, Journal of Agricultural and Food Chemistry 2008, 56,2090.

[97] A. Moure, H. Dominguez, J. C. Parajo, Process Biochemistry 2006,41, 447.

[98] Y. L. Lee, J. H. Yang, J. L. Mau, Food Chemistry 2008, 106,

1128.

[99] H. Esaki, R. Watanabe, N. Hishikawa, T. Osawa, S. Kawakishi,

Journal of the Japanese Society for Food Science and Technology-

Nippon Shokuhin Kagaku Kogaku Kaishi 2004, 51, 47.[100] See Supporting, Information.

[101] M. Friedman, D. L. Brandon, Journal of Agricultural and Food

Chemistry 2001, 49, 1069.[102] T. H. Kao, B. H. Chen, Journal of Agricultural and Food Chemistry

2006, 54, 7544.[103] M. I. Genovese, F. M. Lajolo, Journal of Food Science 1993, 58,

148.

[104] X. L. Zhu, Q. L. Yang, J. Y. Huang, I. Suzuki, G. X. Li, Journal of

Nanoscience and Nanotechnology 2008, 8, 353.[105] G. Mihailescu, L. Olenic, S. Pruneanu, I. Bratu, I. Kacso, Journal of

Optoelectronics and Advanced Materials 2007, 9, 756.



1436

[106] S. K. Bhargava, J. M. Booth, S. Agrawal, P. Coloe, G. Kar, Langmuir

2005, 21, 5949.[107] K. A. Wilson, B. R. Rightmire, J. C. Chen, A. L. Tan-Wilson, Plant

Physiol. 1986, 82, 71.[108] C. F. Shaw, Chemical Reviews 1999, 99, 2589.[109] C. Basset, J. Vadrot, J. Denis, J. Poupon, E. S. Zafrani, Liver

International 2003, 23, 89.


[110] M. Poliakoff, P. Licence, Nature 2007, 450, 810.[111] M. Poliakoff, J. M. Fitzpatrick, T. R. Farren, P. T. Anastas, Science

2002, 297, 807.[112] S. L. Y. Tang, R. L. Smith, M. Poliakoff, Green Chemistry 2005, 7,

761.

bH & Co. KGaA, Weinheim

Received: April 13, 2008Published online: July 18, 2008

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