Biosynthesis of Gold Nanoparticles Using Ocimum sanctum Extracts

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Biosynthesis of gold nanoparticles using Ocimum sanctum extracts by solvents with different polarity Shi Yn Lee , Sneha Krishnamurthy, Chul-Woong Cho, and Yeoung-Sang Yun ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00161 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on March 29, 2016

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ACS Sustainable Chemistry & Engineering

Biosynthesis of gold nanoparticles using Ocimum sanctum extracts by solvents with different polarity .

Shi Yn Lee,a Sneha Krishnamurthy*,b Chul-Woong Cho*,c and Yeoung-Sang Yun*,a,b,c a

Department of Bioprocess Engineering, Chonbuk National University, Jeonbuk 561-756, South of

Korea. b

Department of BIN Fusion Technology, Chonbuk National University, Jeonbuk 561-756, South of

Korea. c

School of Chemical Engineering, Chonbuk National University, Jeonbuk 561-756, South of Korea.

ACS Paragon Plus Environment


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ABSTRACT In nanoparticle biosynthesis, identification and/or characterization of extract and its biochemicals, playing a key role to control the morphology of nanoparticle, are important to minimize the synthesis steps, and thus to reduce environmental risks and cost. Ocimum sanctum plant leaves were subject to sequential solvent fractionation with different polarity (hexane, chloroform, n-butanol, and water). In the steps, the four dried fractions were extracted and used for biogenic synthesis of gold nanoparticles. Gold nanoparticles of varying shape and size were synthesized according to the extraction solvents i.e., chloroform fraction (circular discs with rough edges), hexane fraction (spherical nanoparticles smaller than 100 nm), aqueous fraction (large, anisotropic platelets with defined edges) and n-butanol fraction (Au aggregates). In second step, we identified the biochemicals in the extracts using gas chromatography-mass spectrometry (GC-MS). Methyl eugenol and β-caryophyllene were observed in the hexane fraction and eight known compounds i.e., glycerol, phosphoric acid, succinic acid, tartaric acid, d-gluconic acid, and myo-inositol, 2-methoxy-4-vinylphenol and ferulic acid were identified in the aqueous fraction. Further, we checked that some identified biochemicals i.e., ferulic acid, caryophyllene, and 2 methoxy-4-vinylphenol synthesized micro-scale sheets, spherical particles and a branched nano-structure, respectively. Keywords: Gold nanoparticles, plant, biosynthesis, sequential extraction, biochemical identification


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Introduction The fascinating and insightful optical, chemical, mechanical and electrochemical properties of noble metal nanoparticles make them an excellent resource to be used for diversified applications e.g., anticancer, cosmetics, coating and biocatalysis.1 The majority of the conventional methods for nanoparticle fabrication involve toxic chemicals, expensive techniques or inefficient consumption of energy and resources.2 Considering the depleting effect that these processes could cause on the environment risks, researchers have focused on non-toxic, environmentally benign, green chemistry routes for synthesis of metal nanoparticles using biological organisms and plants. During the last 30 years, it has long been recognized that biological organisms such as bacteria, actinomycetes and fungi, and plants are potential “green” nanofactories.3-8 Among applications of the biomaterials, the use of plant extracts in fabrication of nanoparticles has an advantage over other biomaterials since it facilitates easier and less expensive routes to industrial scale-up and is much safer for human use.5,8 Since the morphology and size of metal nanomaterial strongly influence the chemical, mechanical and optical properties of nanoparticles, physiological factors (pH, temperature and metal concentrations) of plant extracts have been monitored to synthesize well dispersed, morphology controlled spherical and anisotropic nanoparticles. The majority of crude plant extracts synthesize a heterogeneous mixture of spherical and non-spherical nanoparticles since they contain concoction a naturally occurring both reducing and stabilizing agent, with an exception of few reports that demonstrated the controlled synthesis of biogenic nanoparticles.5,9,10 Some experiments conducted using lemon grass extract yielded a high percentage of triangular gold nano-sheets when exposed to a solution of chloroauric acid.11 The mechanisms of metal resistance in living plants include cell-wall binding, metal chelation with phytochelatins, metallothioneins and other metal binding proteins, or active transport into cell vacuoles.9,12 Investigations on mechanistic aspects of metal reduction in plant parts/extracts have explained that polyols such as terpenoids, polysaccharides, and flavone participate in the bio-reduction and stabilization of metal ions to their zero valent form. Conversely, few reports believe the water soluble organics and protein to be the main reducing and capping agents.13 Prior literature on Au triangular nanoprism synthesis using lemon grass extract report the role of sugars and aldehydes/ketone in formation of gold platelets.11 The reports suggested polyols, polysaccharides and flavones in Cinnamomum camphora and Eucalyptus hybrida aided the reduction of metal ions.14 The studies on Cinnamon zylanicum, Capsicum annum and Carica papaya suggested the involvement of protein and terpinoids in reduction and stabilization of biogenic nanoparticles. Chandran et al.15 demonstrated the synthesis of zero valent gold using aloe vera extracts and explained that biomolecules less than 3kDa participate in metal reduction. It has been reported that during synthesis using plant extract and dead microbe extract, the redox reactions is mainly mediated by the non-enzymatic process, i.e., in the presence of antioxidants. Thus, in order to



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implement effective biological process of nanometal synthesis, it is necessary to examine the intermediate biochemicals concerned in redox reactions during metal reduction using plant extracts. Again, identifying a specific biochemical will help circumvent the tedious process of optimization studies, and will primarily benefit the engineering of nanoparticles on an industrial scale. Therefore, the aim of this present study was to identify the key factors in O. sanctum leaf extract responsible for morphological control of gold nanoparticles and to understand various chemicals involved in bio-reduction. For the investigation, O. sanctum biochemicals were extracted by using sequential liquid-liquid partition and/or solid phase extraction based on polarity of the solvents - i.e., hexane, chloroform and n-butanol in the present study - which has been most widely used in extraction of different phenolics from crude plant extract.16-18 And then the extracted biochemicals were identified by using chromatography systems. As a part of the work, we also described the relationships between antioxidant activity, total phenolic content and reducing power for each solvent extract with respect to nanoparticles reduction using HAuCl4 as a precursor. To the best of our knowledge, this is the first report on the identification of specific biochemicals involved in reduction and stabilization of plant extract for the synthesis of gold nanoparticles.

Experimental Plant Material Fresh O. sanctum leaves were purchased commercially. The leaves were washed properly and cut into small pieces, before being dried under sunlight for several days. The dried plant leaves were ground to a fine powder, and then boiled for 2 minutes in sterile distilled water. The sample was filtered using a 20-mesh sieve to achieve consistent particles size distribution. The plant powder was then stored in an airtight container at -20°C.

Sequential Extraction of Plant Extract In a typical experiment, the extract of O. sanctum leaf was tested for synthesis of Au nanoparticles. In order to study the characteristics of biochemicals responsible for the formation of different kinds of nanoparticles in plant extract, the leaf extract was subject to extraction using hexane, n-butanol, water and chloroform, respectively. The extracts by different solvents were prepared as shown in Scheme 1. Each fraction by different solvents was dried by vacuum evaporation. The reducing powers for each fraction were evaluated before and after biosynthesis of AuNPs. 200 g of dried plant powder was weighed and boiled with 8 L of sterile distilled water for 2 min. The resulting infusion was left to cool at room temperature and then filtered carefully in order to obtain a plant broth. Here, the first filtered plant extract was used for control test. The plant broth was successively extracted with hexane, nbutanol, water and chloroform. The fractions were collected and then dried using a rotary vacuum


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evaporator (NE-2001, Eyela, Japan) at 55°C, followed by freeze drying in a glass amber bottle and then stored at -20°C. All fractions were tested for synthesis of nanoparticles. Biochemical characterization of various fractions was carried out using reverse phase high performance liquid chromatography and gas chromatography.

Scheme 1. Preparation of O. sanctum extracts by using different solvents for gold nanoparticles synthesis



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Table 1 Total phenolic content, antioxidant activity, and reducing power of different solvent extracts TPC

Antioxidant

(mg GAE/g DE)

activity

Hexane

54.36±0.21

n-Butanol

Fraction

Absorption at 700 nm (ABS) Before

After

Reducing

reduction

reduction

Power

49.03±2.39

0.34±0.003

0.19±0.01

0.15±0.01

87.33±1.32

64.79±2.50

0.58±0.004

0.28±0.01

0.30±0.01

Chloroform

45.82±0.58

40.23±3.13

0.40±0.01

0.28±0.02

0.12±0.02

Aqueous

62.67±0.58

51.77±1.90

0.55±0.02

0.31±0.01

0.24±0.02

Values are presented as mean ±SE (n=3), TPC-Total phenolic content, GAE-Gallic acid equivalent, DE-Dry extract.

AuNP synthesis and characterization Studies (Total phenolic content, antioxidant activity and reducing power of different extracts) Chloroauric acid (HAuCl4), purchased from Sigma-Aldrich, USA was used as a source of chloroaurate ions (AuCl4¯) in all experiments. In a typical synthesis method, 0.01 g of each fraction (hexane, n-butanol, chloroform or aqueous fraction) was added to 10 mL of 1 mM HAuCl4 solution in a 50 mL sterile Erlenmeyer flask. The flasks were then incubated in a shaking incubator at a speed of 160 rpm at 30°C for 24 h, to assure that the reduction reaction reached a saturation level. The reaction mixture was centrifuged at 12,000 (rpm) for 20 min, and the pellet was used for further analysis. Absorption spectra were measured at a resolution of 1 nm on a JASCO V-670 double-beam spectrophotometer. Transmission electron microscopy (TEM) measurements of the AuNPs synthesized using different fractions were carried out on a Hitachi-JP/H7600 instrument operated at an accelerating voltage at 100 kV. TEM samples were prepared by drop-coating the AuNPs solutions onto carbon-coated copper grids. The carbon films were then allowed to dry prior to the TEM measurements. The morphology of the AuNPs in different fractions was also analyzed using Hitachi S-4800 FE-SEM (Tokyo, Japan). The acceleration voltage was kept constant at 1.5 kV. The fraction samples were prepared on a silicon glass slide with platinum sputter coating on the surface of the specimens. Energy dispersive X-ray spectroscopy (EDAX) studies were also carried out in parallel to identify the existence of metallic AuNPs. The Fourier transform infrared (FTIR) measurements were performed to determine the possible biomolecules responsible for the bioreduction of AuCl4 and the stabilization of AuNPs by using different fractions. The infrared adsorption spectra were obtained on a FTIR spectroscopy (Perkin Elmer GX FT-IR System, USA), in the range of 400-4000 cm-1, using pelted samples as KBr pellets. The total phenolic content (TPC) and antioxidant activity (AA) in each fraction were evaluated using modified methods by Slinkard and Singleton,19 Taga et al.,20 and


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Juntachote and Berghofer,21 respectively. The reducing power was calculated according to the following equation.22 Reducing power (Absorbance at 700 nm) = ABS (before bioreduction) – ABS (after bioreduction) (1) Solvent fractions in similar concentration were used for all experiments. Experimental data were collected and expressed as the mean ± SD of three replicates in separate experiments.

Fractionation using RP-HPLC The extracted aqueous fraction was further purified using the reversed phase HPLC with UV-vis detection. Experimental apparatus HP Agilent 1100 series HPLC system consisted of vacuum degasser, quaternary pump, auto sampler, thermostatted column compartment, and diode array detector (HP/Agilent Technologies, Waldbronn, Germany) was used. The separation was performed on a Waters Xterra RP18 column (4.6x150 mm, 5µm) (Waters Corporation, USA). All the prepared solutions were filtered through 0.45 µm membranes (Fisher Scientific) and the mobile phase was degassed before injection onto HPLC. The mobile phase consisted of a mixture of distilled water (A) with 0.1 % v/v formic acid/ acetonitrile (B) with 0.1 % v/v formic acid, at a flow rate of 1 mL/min. Absorption wavelength was selected at 254 nm. The column was operated at 25°C. The solvent gradient was started at 10 % solvent B, followed by a linear incremental of solvent B to 90 % in 30 min, and then to 100 % in 5 min. The mobile phase composition then reinitialized the conditions in 3 min for the subsequent run. Injection volume was 100 µL, and HPLC separated samples were collected every 3 min in sterile amber vial, for a total of 30 min. Total 10 fractions (F1-F10) were labeled according to the incremental of the retention time and collected in volume of 50 mL each tube. 2 mg of each freeze dried fraction powder were added into 5 mL of 1 mM HAuCl4 and incubated for 24 h. Then the post-bioreduction samples were determined by following analytical machines, to analyze the characterization of the AuNPs from each HPLC fractions. Analytical apparatus included of a JASCO V-670 UV-vis NIR adsorption spectroscopy and a Hitachi-JP H7600 TEM instrument. Selected fractions were further analyzed by GC-MS, to identify the presence of molecules in each fraction.



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Results and Discussion Total phenolic content, antioxidant activity and reducing power in different extracts before and after reduction The antioxidant capacity of plant extracts is largely dependent on sort and amount of phenolics compounds,23,24 synthesized by secondary metabolites of plants due to its adaptation in response to biotic and abiotic stresses.25 Previously, it has been extensively reported that the main biochemicals involved in the bioreduction of nanoparticles are antioxidants such as flavanoids, alkaloids, tannins, steroids, and terpenoids, and it is necessary to analyze the relationship in the phenolic content of various extracts and its antioxidant activity.26 Therefore, in this study the total phenolic contents of O. sanctum by different extraction solvents i.e., chloroform, hexane, water, and n-butanol were assessed using the standard Folin-Ciocalteu colorimetric method. Results showed a significant variation of gallic acid equivalents in dry extracts as follows: 45.82±0.58 mg (chloroform fraction), 54.36±0.21 mg (hexane fraction), 62.67±0.58 (aqueous fraction) and 87.33± (n-butanol fraction) (Table 1). Their antioxidant activity was determined by using a β-carotene/linoleic acid system, was found to be 40.23±3.13 % (chloroform fraction) to 49.03±2.39 % (hexane fraction) to 51.77±1.90 % (aqueous fraction) to 64.79±2.50 % (n-butanol fraction), and increased in the order as mentioned above. Table 1 summarizes the total phenolic content, antioxidant activity and reducing power of each fraction (hexane, n-butanol, chloroform, and water. The total phenolics content and antioxidant activity of O. sanctum extract followed the trend chloroform fraction < hexane fraction < aqueous fraction < nbutanol fraction. Correlation coefficients between total phenolics content and antioxidant activity were high (R2=0.976) (Figure 1).

Figure 1 Relationship of total phenolic content, antioxidant activity and reducing power of different solvent extracts. Values are presented as mean ±SE (n=3). The total phenolics content and antioxidant activity of Ocimum sanctum extract followed the trend chloroform fraction < hexane fraction < aqueous fraction < n-butanol fraction. Correlation coefficients between total phenolics content and antioxidant activity were high (R2=0.976)

The presence of antioxidants in each dry extract before bioreduction and the residual antioxidants in dry extract after bioreduction would result in reducing Fe3+/ferricyanide complex to the ferrous form


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(Fe2+). The Fe2+ then can be monitored by measuring UV absorbance at 700 nm. Table 1 shows that the reducing power (as indicated by the absorbance at 700 nm) calculated using Eq. (1), were 0.12±0.02 (chloroform fraction), 0.15±01 (hexane fraction), 0.24±0.02 (aqueous fraction) and 0.30±0.01 (n-butanol fraction) and increased in the trend mentioned above. The antioxidant activity and reducing power of individual solvent extracts increased in the same order aforementioned. The high R2 values in all cases (shown in Table 1 and Figure 1) indicated that the values of antioxidant activity, phenolic content and reducing power are highly correlated. This result is supported by report from Duh et al.27 They also reported that antioxidant activity is always associated with the reducing power. Here, it is expected that the reducing power of each fraction is closely related to the bioreduction of chloroaurate ions, concentration of antioxidants and phenolics compounds. Hence, further experiments were performed in order to analyze the effects of different solvent extracts on the size and morphology of Au nanoparticles.

Characterization of Au nanoparticles in different solvent extracts The incubation of O. sanctum extract (control) and solvent extracts with 1mM chloroauric acid induced a colour change within 6 h of the reaction. The reaction was allowed to continue for 24 h to ensure complete reduction, and the reaction conditions were maintained similar to the control. On mixing different freeze-dried powders of hexane fraction, n-butanol fraction, chloroform fraction and aqueous fraction with 1 mM chloroauric acid, visual observations showed the presence of nanoparticles in all fractions. The synthesis of AuNPs was confirmed visual inspection, followed by spectrometric analysis. The spectrum clearly shows that the magnitude of out-of-plane SPR band intensity increased from aqueous fraction to n-butanol fraction, followed by chloroform fraction and hexane fraction. The aqueous fraction clearly shows two distinct absorption bands: one centered at ca. 535 nm and another band in the NIR region from 800 nm to 1200 nm, due to the difference in the aspect ratio of the particles of different particles. The SPR in the UV-vision is unique for the presence of spherical nanoparticles, whereas plasmon resonance in the NIR region is typical of anisotropic nanoparticles.28-30 It is interesting to note that the plasmon band of AuNPs synthesized in the hexane fraction are rather different from those in the aqueous fraction, wherein the single absorption band at ca. 510 nm is observed with little evidence of any peak in the NIR region. Close observation for the optical properties of chloroform fraction and n-butanol fraction synthesized AuNP showed the presence of a weak plasmon band in the NIR region alongside the sharp peak at approximately 520 nm (Figure 2). However, the net absorption of n-butanol fraction and chloroform fraction in the NIR spectrum is significantly lower than that observed in the aqueous fraction. The UV-vis-NIR pattern of the aqueous fraction is similar to that of control and a clear peak can be seen for the aqueous fraction and control ca. 536 nm and 548 nm, respectively. This indicates that the spherical nanoparticles in the aqueous fraction are smaller than AuNPs in the control. A pronounced difference between the NIR



spectrum of control and the aqueous fraction was seen. The NIR spectrum in the control appeared to red shift beyond 1250 nm. This large increase in the transverse component is probably due to the presence of truncated platelets. Similar observations for truncated anisotropic AuNPs synthesized in betel and Lemon grass extract in the presence of KBr were reported by Rai et al.30 and Sneha et al.8 The surface plasmon resonance of the synthesized nanoparticles depends on factors like the dielectric constant of the medium and particles, shape, temperature, and size of nanoparticles. This explains the differences in the SPB and bandwidth regarding the synthesis AuNPs.31

2.5 Hexane Fraction n-Butanol Fraction Chloroform Fraction Aqueous Fraction Control

2.0

Hexane Fraction

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1.5

1.0

0.5

0.0 400

600

800

1000

1200

Wavelength (nm)

Figure 2 UV-vis-NIR absorption spectra of gold nanoparticles after bioreduction of 1mM HAuCl4 with each fraction (n-butanol, aqueous, chloroform, and hexane) at 30°C and at pH 3.00 ± 0.05. The spectrum shows that the SPR band intensity increased from aqueous fraction to n-butanol fraction, followed by chloroform fraction and hexane fraction.


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Figure 3 TEM images of the gold nanoparticles synthesized by (a) O. sanctum extract, (b) chloroform fraction (inset scale-100 nm), (c) hexane fraction, (d) aqueous fraction, (e) n-butanol (inset scale-50 nm)

TEM analysis confirmed the morphology and size of nanoparticles synthesized in different solvent extracts. Figure 3 shows that O. sanctum extract synthesized a dense and heterogeneous mixture of nanoparticles in size ranging from 10 nm to 300 nm. In addition to spherical nanoparticles, anisotropic nanoparticles such as triangular, hexagonal and truncated platelets were observed in O. sanctum extract synthesized AuNPs. During nanoparticle synthesis, it is expected that the biomolecules act as a capping as well as a reducing agent, which direct the crystal growth and balance the electrostatic force during the growth process. However, it cannot be ignored that the particle morphology is also affected by various other factors like the nature of the reaction medium. Furthermore, the TEM images show a noticeable difference in the morphology of the nanoparticles synthesized in different fractions obtained by liquid-liquid extraction of O. sanctum, in comparison with the control. These results suggest the role of different biochemicals in the fabrication of different kinds of spherical and nonspherical nanoparticles using plant extract. TEM micrographs of hexane fraction synthesized Au nanoparticles revealed that the synthesis of nano-scale particles mainly consisted of spherical nanoparticles along with some anisotropic particles. The hexane fraction synthesized nano-scale particles were 1 nm to 50 nm in size. Also, the hexane fraction synthesized trace amounts of nanorods and the size of the triangular particles synthesized did not exceed 100 nm. The nanoparticles synthesized using n-butanol fraction confirms the presence of clustered nanoparticles of irregular



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shape and size. Clearly, the n-butanol fraction lacks the stabilizing agents to control the structural parameters during AuNP synthesis. Nevertheless, bulk gold was seen settling at the bottom of the reaction vial after 48h. These results indicated that the strong reducing power of biochemicals in nbutanol fraction (table 1) lead to the rapid reduction and subsequent aggregation of the bulk gold in the reaction mixture. These observations contradict the results of Gan et al.,32 wherein the n-butanol extract did not reveal any reducing property. As suggested by the UV-vis-NIR studies, the AuNPs synthesized using the aqueous fraction resembles those synthesized in crude extract (control). Nearly perfect nano-scale anisotropic platelets were synthesized using the aqueous fraction (50 nm to 300 nm in size) and more than 50 % of the particles were anisotropic in nature. The Au platelets synthesized in the aqueous fraction and crude extract displayed a liquid like shiny substance on the surface, which is due to the presence of aldehydes and ketones from the biomolecules. Shankar et al.13 and Sneha et al.8 have also observed similar substance on the surface of gold platelets. It is believed that the fluid like substance or interference fringes displayed on the surface of the plates and discs are bending contours stem due to the differences in the angle between the atomic planes. Chloroform fraction synthesized platelets display irregular wavy edges that resemble disc like platelets. The nanoparticles synthesized using chloroform extract produced mostly large discs (>200 nm). Nonetheless, chloroform alone, did not have an impact on synthesized AuNPs. Our studies on chloroform extract opposed to those by Shankar et al.11 where in chloroform fractions of lemon grass extract failed to synthesize AuNPs. The results obtained in the aqueous fraction were concomitant with those observed by Shankar et al.11 and Gan et al.32 It has been previously reported that the formation of AuNPs is a result of the nucleation in the presence of extract followed by the sintering of AuNPs, which are also in the presence of biochemicals in the extract. Moreover, in the presence of water, large amounts of aggregation and growth are expected to occur. The high polarity of water induces a hydrolytic reaction favouring the growth of nano platelets.34 The hexane fraction is perhaps rich in capping agents which tightly cap the nanoparticles, and prevents crystal growth with interfering the formation of larger nanoparticles.33,34 However, the presence of numerous biochemicals leads to the initial reduction, which followed by strong capping by the capping agents present in the hexane fraction. Also, the residual hexane in the extract adds to the repulsive force between the existing nuclei and free chloroaurate ions. Hence, the polarity of the solvent and the reducing agent can effectively alter the morphology of nanoparticles. The AuNPs synthesized by using the aqueous and hexane fractions were remarkably stable, and they could be separated by centrifugation. Figure 4 illustrates the HR TEM and SAED of a single gold plate and disc. The hexagonal nature of the diffraction spots clearly represent the nature of crystal growth towards [111] the plane (see Figure S1).35,36 However, the diffraction pattern of the Au nanoparticles in the hexane fractions verifies the existence of polycrystalline properties.37,38 All the particles show an interplanar spacing of ca. 2.35 Å. This corresponds to the Miller index of [111] in the Au FCC crystalline. The crystalline nature of nanoscale Au was confirmed


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by X-ray diffraction (Figure S1 in supporting information). Four distinct peaks in all fractions can be assigned to [111], [200], [220] and [311] diffraction peaks of metallic gold, respectively, which is indicating the crystalline nature of the Au nanoparticles by all solvent extracts. The ratio for the intensity between [111] and [200], once again confirms that the platelets are typically dominated by the [111] plane. It is well known that fro FCC, [111] facet is the most densely packed surface, it is energetically most beneficial.39 Hence, the Au growth is preferentially observed in the [111] facet.

Figure 4 HRTEM and SAED micrographs of aqueous fraction (a,b,c) and hexane fraction (d,e,f)

FTIR spectrum of (a) O. sanctum extract (b) hexane fraction (c) n-butanol fraction (d) chloroform fraction (e) aqueous fraction, before and after synthesis of AuNPs is shown in supporting information Figure S2. Energy dispersive X-ray spectra (EDAX) verified that the particles in different fractions contain a high content of gold (high intensity peak ~ 2 keV) (Supporting information Figure S3). Other weak peaks i.e., C and O peaks were observed in the spot profile EDAX spectrum for the anisotropic nanoparticles (Supporting information Figure S3d and S3f), may arise from plant extract (enzymes/proteins) that bound to the surface of AuNPs or were present in the vicinity of the particles for the stabilization of the nanoparticles. However, only a weak carbon peak appeared in the EDAX spectrum in c and e for the nano-platelets. It was explained by Chandran et al.15 that the carbon present on the surface of the gold nanotriangles originates from the biomolecules of plant extract.

Correlation between solvents extract, phenolic content on metal reduction and particle morphology



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We aim to determine the correlation between the phenolic content and the synthesized AuNPs using different extracts obtained by solvent extraction. As previously mentioned that plant phenolics constitute one of the major groups of compounds acting as the principal antioxidants or free radical terminators, a good correlation was obtained between the reducing power, antioxidant activity and total phenolics content. Here in our study, we observed that the chloroform fraction possessed minimum phenolic content and reducing activity whereas the n-butanol fraction possessed the maximum. It is indicating that considerable difference in the morphology of the particles can be caused in the presence of various solvent extracts. As expected, the chloroform fraction synthesized disk shaped nanoparticles. Weak reducing power of chloroform extract results in a slower reaction due to which the Au ions adsorb on the surface in the presence of biomolecules. Owing to the gradual reduction and growth kinetics, it is expected that Au ions and phenolics will not be rapidly exhausted. Hence, it may be due to that the presence of phenolics as antioxidants and the slow or controlled rate of reaction allow the expansion of the particles into the large Au discs. Since the hexane fraction contains abundant amount of capping agents, it inhibits the formation of larger nanoparticles. The aqueous fraction appears to be a suitable medium for the synthesis of thin platelets and sheets because high phenolic content enables the primary nucleation of Au3+ to Au0. Furthermore, the phenolics along with the abundant water domains interact synergistically with the existing nuclei and lay the foundation to create highly structured sheets of zero-valent gold. Also, the surface tension of the medium and the components have a substantial effect on the morphology of the synthesized nanometal.40 The large nano-plates had well defined edges in the aqueous extract. The fluid like finish on the sheets is due to the chelating agents in the biochemicals9,11 and the bending of the atomic angle due to the differences in the atomic plane. Gan et al.32 report that no nanoparticles were synthesized using any solvent extract performed using palm oil mill effluent. They claim that compounds involved in the nano-metal synthesis of AuNPs are polar and water-soluble. According to our observations, the phenolic content and reducing power of fractions (having different polarity) can be highly correlated to the crystal growth; nano-metal structure formed using the fraction. Fractions with low phenolic content supply harmonious conditions for steady reaction kinetics and sufficient biomolecules for a slow reaction in order to form discs. However, they lack biomolecules that directly promote the formation of triangular-plates. Fractions with high phenolic content (n-butanol) are too strong, thereby rapidly reducing the Au(III) ions to bulk gold. The fractions with intermediate reducing power and phenolic content like the hexane fraction (non-polar) are suitable for synthesizing small spherical particles or anisotropic particles less than 50 nm in size, whereas the aqueous fraction contains the biomolecules that synthesize sheets with a distinctive shape and defined edge.


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F4

1.5

Absorbance (a.u.)

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F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

F3 1.0 F2 F1 F5

0.5

400

600

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Wavelength (nm)

Figure 5 UV-Vis-NIR spectra of fractions F6-F10 show no adsorption in UV-vis-NIR region. Fractions F6 to F10 do not show any plasmon band for the presence of gold nanoparticles.

Identification of reducing and stabilizing biochemical in formation of spherical and nonspherical gold nanoparticles in O. sanctum extract The aqueous fractions (F1~10) purified by the reversed phase HPLC with UV-vis detection were added into 5 mL of 1 mM HAuCl4 and incubated for 24 h. Yellowish color of Fractions F1 to F5 changed to yellow-green, purple and red colors after reaction. However, no color change was observed for the rest of the fractions. The UV-Vis-NIR spectra showed that fractions F6-F10 show no adsorption in UV-vis-NIR region (Figure 5). The analyte with a larger hydrophobic surface area present in F6 to F10 do not involve in the reduction of AuCl4¯ activity. Among these five fractions that showed positive reaction, F4 showed the highest intensity of SPR band at ~565.0 nm. F1 showed a weak Plasmon band. According to Mie Theory, intensity and position of SPR band are directly influencing the diameter of particles. Transmission electron micrographs represent the variation of morphologies of the gold nanoparticles obtained from the above fractions. (Supporting information Figure S4) clearly shows that small nanoparticles were formed in F1. Nano-platelets were observed from in F2 to F5 fraction, with increment of the size of the particles. The TEM images showed that the size of the nanoplatelets increased from F2 to F5 plausibly due to the variation in polarity on the surface of biomolecules present in fraction decreased. High content of low polar group (e.g., amide) on the surface of biomolecules may serve as “shape-control agent”, and promote the favored growth direction, which can yield nanoplatelets.41 On the other hand, the biomolecules contained high polar groups (eg. –OH) on their surface, may increase the rate of nucleation and induced more AuNPs formation, but inhibited the assembly of AuNPs to create nanosheets. Hexane fraction was analyzed using GC-MS and we observed two compounds i.e. methyl eugenol and β-caryophyllene. The methyl eugenol (4-allyl-1, 2-dimethoxybenzene) is one of the primary



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chemical constituents of O. sanctum, a volatile essential oil, which used extensively in various application, and β-caryophyllene (Caryophyllene) is also another major component of essential oils. Comparing the retention time showed the 74 % methyl eugenol content and 26 % β-caryophyllene in the fraction (Supporting information Figure S5a). Further investigation was performed by comparison of hexane fraction involved in pre- and post-bioreduction of Au ions reactions. The GC chromatogram identified two new known compounds, 3-methylthiolane and veratradehyde (Supporting information Figure S5b). It is due to that methyl eugenol and β-caryophyllene were oxidized in post-bioreduction reaction. Therefore, eugenol and β-caryophyllene may be reducing agents present in the hexane fractions. F1 and F5 fractions of aqueous fraction separated using RP-HPLC were selected for further verification by GC-MS. From TEM images, we observed that the morphology of F1 was different from others. Among these fractions, F5 had largest size of nano-platelets as compare to F2, F3, and F4. Further identification by GC-MS may lead us to understand the role of each plant biomolecule in bioreduction of Au ions. The obtained GC-MS data showed two compounds in hexane fraction and 8 compounds were identified in F1 and F5 (Supporting information Table S2). The six principal compounds in F1 were identified as glycerol, phosphoric acid, succinic acid, tartaric acid, d-gluconic acid, and myo-inositol, which are antioxidants for plant O. sanctum. However, only two compounds were identified in F5 including 2-methoxy-4-vinylphenol and ferulic acid that are also antioxidants from plant O. sanctum.

Figure 6. Transmission electron micrographs representing the variation using the identified


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biochemicals (a) ferulic acid, (b) caryophyllene, (c) 2-methoxy-phenol, (d) Succinic acid (e) Glyconic acid (f) Glycerol.

Synthesis of nanoparticles using the identified biochemicals Following the biochemical characterization of the plant extract, the identified biochemicals i.e., (a) ferulic acid, (b) caryophyllene, (c) 2-methoxy-phenol, (d) succinic acid (e) glyconic acid (f) glycerol were purchased commercially and tested for the synthesis of Au nanoparticles. The identified chemicals could synthesize Au nanoparticles of different morphologies. Figure 6 represents various shapes of nanoparticles synthesized by using the commercially purchased biochemicals. It was found that ferulic acid synthesized micro-scale nanosheets (200 nm ~ 2 µm), and succinic acid synthesized irregular gold sheets. Both chemicals facilitated the formation of a liquid like structure, similar to the liquid-like sheen observed on the nano-sheets synthesized using the crude extract (Figure 6a and 6d). Methyl eugenol, one of the main chemicals identified in hexane extract, synthesized well-dispersed spherical nanoparticles ranging from 1 nm to 20 nm (Figure 6b). These results are at par with our observations on nanoparticle synthesis using hexane extract. Further, coral shaped gold aggregates synthesized using 2-methoxy-phenol (Figure 6c) and glyconic acid synthesized large irregular mesh type gold aggregates (Figure 6e). Most natural plant extracts synthesize a heterogeneous mixture of spherical and non-spherical nanoparticles since they normally contain concoction of naturally occurring reducing and stabilizing agents. These results clearly show that specific chemicals are responsible for the synthesis of a heterogeneous gold colloid using plant extract. Namely, some identified biochemicals synthesize unique nanoparticles shapes without the presence of capping agent. For instance, ferulic acid, caryophyllene and 2-methoxy-phenol can synthesize different shapes of nanoparticles whereas others biochemicals may require the support of other existing biomolecules in order to cap the reduced nanoparticles. Since the biochemical samples contained a high amount of gold post-bioreduction, we have not been able to identify the biochemical pathway. In detail studies of a particular compound is necessary to determine the oxidized product obtained as a result of nanoparticles formation in plant extracts.

CONCLUSIONS The results reported in this article demonstrate a strong correlation between the phenolic content, antioxidant activity, bioreduction and nano-metal morphology, and different kinds of solvent extracts can lead to the formation of nano-metals of different morphology and the reducing property, phenolic content. Even though both wazfter-soluble extract and non-polar extracts can have reducing properties, the morphology of nanoparticles can be strongly influenced by characteristics of the bio-reducer. Specifically, hexane extract can synthesize nanoparticles of 1 nm to 100 nm in size, whereas phenolics in the aqueous fraction can synthesize highly structured micro-sheets. Concomitantly, methyl eugenol



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identified in hexane fraction can synthesize spherical nanoparticles; 2-methoxy-4-vinylphenol and ferulic acid identified in the aqueous fraction facilitate the synthesis of coral shaped aggregates and micro-scale sheets, respectively. It is clear from the studies that strictly thermodynamic arguments cannot be applied to the synthesis diverse shapes of gold synthesized in using plant extracts. Although crude plant extract contain a mixture of natural compounds, some may directly be responsible for a unique shape of nano-metal formed, while other biomolecules require the presence of other biomolecules as reducing or capping agent. These results constitute a significant step forward in elucidating the mechanism of nanoparticle synthesis using plant extracts, and identified the potential biochemicals involved in the formation of spherical and non-spherical particles within plant extracts. These findings will support to effectively establish the environmental friendly biosynthetic methods by extracting and using activating biochemicals as a reducer for the synthesis of Au nanoparticles. Specifically, it will be helpful to minimize the synthesis steps and to increase synthesis success rate; therefore these advantages will reduce environmental risks and cost for chemical processes. However, it should be further studied for methodological improvements to make the system suitable for the synthesis of functionalized nanoparticles or large scale production.

ASSOCIATED CONTENT Supporting information The detailed on materials, sample preparations (TMS derivatives), GC-MS analysis steps, and Supplemental Figures are given. The material is available free of charge via the Internet at http://pubs.acs.org.

Author information Corresponding Authors *E-mail: [email protected] (Sneha Krishnamurthy) *E-mail: [email protected] (Chul-Woong Cho) *E-mail: [email protected] (Yeoung-Sang Yun)

Funding This work was supported by the Korean Government through NRF (2014R1A2A1A09007378, 2014R1A1A2008337).

Notes


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TOC/Abstract graphic

Title: Biosynthesis of gold nanoparticles using Ocimum sanctum extracts by solvents with different polarity Authors: Shi Yn Lee, Sneha Krishnamurthy, Chul-Woong Cho, and Yeoung-Sang Yun

Synopsis: Based on the results i.e., identification and characterization of biochemical in extract from plant, the biosynthesis process for nanoparticles can be optimized and its productivity for shapecontrolled nanoparticles will increase.


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Biosynthesis of Gold Nanoparticles Using Ocimum sanctum Extracts

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