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Plant-mediated synthesis of Au nanoparticles: Separation and identification of active biomolecule in the water extract of Cacumen Platycladi Hai Liu, Ting Lian, Yang Liu, Yingling Hong, Daohua Sun, and Qingbiao Li Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017 Downloaded from http://pubs.acs.org on April 21, 2017

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Industrial & Engineering Chemistry Research

Plant-mediated synthesis of Au nanoparticles: Separation and identification of active biomolecule in the water extract of Cacumen Platycladi Hai Liu,†, ‡ Ting Lian,† Yang Liu,† Yingling Hong,† Daohua Sun,†,* Qingbiao Li†,* †

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Fujian Provincial Key Laboratory of Chemical Biology, Xiamen University, Xiamen, 361005, PR China.

‡

College of Chemistry and Chemical Engineering, Beifang University of Nationalities, Yinchuan, Ningxia, 750021, PR China.

Email to: [email protected]

ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: In order to investigate the roles of active molecules during the bio-inspired synthesis process of gold nanoparticles by Cacumen platycladi leaf extract, flash column chromatography was employed to separate the Cacumen platycladi leaf extract according to the polarity differences. Fourier transform infrared spectroscopy and high performance liquid chromatography equipped with photodiode array detector were adopted to identify the category of the isolated samples. The results have demonstrated that components in the Cacumen platycladi leaf extract can be sorted into three categories: sugars (the Sloading and S0), polyphenols (S1~S5) and flavonoids (S6~S8). The characterizations of gold nanoparticles, produced by each isolated sample with chloroauric acid, indicate the strong-to-weak sequence of reducing power is polyphenols, flavonoids and sugars. Sugars have no protection strength for the biosynthesis of gold nanoparticles, polyphenols involved in isotropous stabilization while flavonoids are responsible for the anisotropic growth. Keywords: gold nanoparticles; plant-mediated synthesis; active biomolecules; flash column chromatography


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Introduction Noble metal nanoparticles in the respect such as optics, electronics, thermodynamics, catalysis with unusual performance,1, 2 have received tremendous attention recently and become an important branch of nanomaterial. Among the noble metallic nanoparticles, gold nanoparticles (GNPs) are more stable, and their quantum-size effect and applications in optics, catalysis and biology lead to the exponential growth of exploration.3 In contrast to the traditional chemical approaches, the bio-inspired process with mild reaction conditions and environmental friendly feature has gradually become a new bright spot in preparation of nanomaterial. Candidates of biological resources generally include microorganisms (such as bacteria,4 fungi,5 actinomycete,6 yeast,7 and viruses8), plant biomass,9-12 and plant extract from leaf,13-17 peel,18 flower,19 fruit,20 and root.21 Since the specialized facilities, the long incubation time, and the more biohazards on production by microorganism-mediated synthesis, many researchers have diverted their attention to plant-mediated GNPs biosynthesis due to comparatively simple, higher reduction rate, more cost-effective and purification-handy.22 The plant-mediated synthesis of GNPs can date back to 1999 when alfalfa biomass firstly acted as natural reducing and capping agent to prepare five different types of gold particles.9 In recent decades, various plants were used to successfully prepare the GNPs with different dimension and shapes. Philip et al.15 reported that mangifera indica leaf could produce the spherical GNPs with the size of 17-20 nm, and Mohammed Fayaz et al.23 demonstrated the possibility of using madhuca



longifolia extract for the biosynthesis of triangular GNPs with the size ranging from 7 nm to 3 µm at pH 2.0. Our group demonstrated that artocarpus heterophyllus,24 Cinnamomum camphora,25 and Cacumen platycladi leaf extract (CPLE)16, 26 can be utilized to fabricate multiple Au nanomaterial. Meaningfully, since no extra reducing and capping agent were introduced during the biosynthesis of GNPs, the diversity of Au product is assumed to be dependent to the composition and structure of biomolecule in different plants. The active components in plant mainly include protein, sugars, flavonoids, terpenoids, alkaloids, polyphenols, and secondary metabolites etc.27 During the bio-inspired synthesis, the aqueous extracts of plant were extensively adopted, hence the components are usually water-soluble protein, saponins, sugars, flavonoids, and polyphenols.24 Some researchers have tried to identify the main active molecules in plant extract involved in the reduction and protection of metal ions. Up to now, two means have been reported to recognize the functional molecules: (1) Fourier transform infrared spectroscopy (FTIR) analyze before and after the biosynthesis. For example, the FTIR analyses by Ayman A et al28 revealed that the flavones and proteins present in the sago pondweed extract are responsible for the synthesis and stabilization of Au, Ag, and Au-Ag alloy nanoparticles. Nagaraj B et al29 proposed that the flavonol derivatives present in the fruit extract of Hovenia dulcis first bind with Au3+ to form gold complexes, which are reduced to seed particles (Au0). After reduction the oxidized biomolecules were capped on the GNPs. (2) As for the content change of active component during the synthesis, and the compounds with great


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variation maybe play the crucial role of reduction and/or protection. Zhou et al30 showed that flavanoids and reducing sugars are reductants, and protein is protection agent in the bio-inspired synthesis of GNPs by foliar broth from 24 kinds of randomly selected plant leaves by means of the component analysis. Lu et al31 found that flavonoid, polyphenols and chlorogenic acid are important for the formation of metal nanoparticles because their content have significant drop after reaction. Despite the above reports proposed possible biomolecules or function groups of active compounds, we note that the component determination was carried out under no moderate separation for the plant extract, but considering complex plant a whole to study. It maybe cause that active functional groups interfere with each other and completely not expose to metal ions. Cacumen platycladi leaf is a traditional Chinese herb32, and the literature have be documented that Au,16,

26

Ag,33 Pt34 nanoparticles, Au-Ag,35, 36 Au-Pd37 bimetallic

nanoparticles, and flower-shaped Au@Pd38 nanoparticles could be produced by CPLE. Hence in this work, although difficult to retain strong polar macromolecules, flash column chromatography, widely adopted for the pretreatment of natural product due to its advantage of rapid separation, high separation efficiency and low reagent consumption, were used for the separation of CPLE according to the polarity difference.39 The categories of the obtained isolated samples were identified by FTIR and high performance liquid chromatography equipped with photodiode array detector (HPLC-PAD), then to compare the ability as reducing and protecting agent, the isolated samples reacted with gold ions to product GNPs respectively.



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2. Experimental 2.1 Materials Chloroauric acid (HAuCl4·4H2O), sodium borohydride (NaBH4) and trisodium citrate (Na3C6H5O7) was purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Cacumen platycladi leaf was purchased from Xiamen Jiuding Drugstore (China). Ultrapure sphere silica gels packing, reversed phase (RP) C18 (50 µm, 120 Å), was purchased from Silicycle Inc. (Canada). Deionized water was prepared in our laboratory. HPLC-grade methanol was purchased from J&K Scientific Ltd. (China). Acetic acid (HAc), analytical reagent (AR), was provided from Tianjin Kemiou Chemical Company (China). The glucose, gallic acid, and rutin with AR grade were obtained from Aladdin Industrial Corporation (China). 2.2 Preparation of CPLE The Cacumen platycladi leaf was milled, and 2.0 g powder was dispersed in 100mL deionized water. The mixture was thereafter shaken using a water bath shaker for 24 h at a rotation rate of 150 rpm and 30 ℃ to obtain the extract. Then it was filtrated to remove the residual insoluble biomass, and the resulting filtrate was used later for the subsequent separation and synthesis of the GNPs. 2.3 Separation of the biomolecules in CPLE The

separation

systems

were

equipped

with

nitrogen

cylinder

and

chromatographic column (Ø20 mm×300 mm) with reservoir and teflon piston. The 25 g RP C18 filler, 25 g, was exposed to 100 mL of methanol in a beaker of 250 mL capacity, and ultrasonic for 30 minutes and sitting for 24 hours ensure filler soaked


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and air discharged. Then the column made of glass was packed with above-mentioned mixing slurry and compacted through a compressed nitrogen gas cylinder. Subsequently, 100 mL CPLE was added to the reservoir and flowed through the column under the pressure of 0.15 MPa and the eluent, denoted as Sloading, was collected. Next, the laden columns were washed in turn with 100mL deionized water-methanol (100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 30/70, 20/80, v/v) solution under the same pressure and the correspondingly trapped solution after removing the methanol were denoted as S0, S1, S2, S3, S4, S5, S6, S7 and S8, respectively. Afterwards, these samples were frozen dried at -50 ℃, weighted and formulated as a specific concentration for future use. 2.4 Method of HPLC analysis The analytical chromatographic analyses were performed with an HPLC system equipped with a 600 HPLC pump and a 2996 PAD controlled by empower 2 chromatography data workstation. The column used was a YMC-Pack ODS-A column (250×4.6 mm, 5 µm) together with a C18 guard column. The aqueous solutions of samples, filtered through at 0.45 µm syringe filter prior to injection, were separated by gradient elution in the presence of the 0.1% HAc-methanol as eluent A and 0.1% HAc-water as eluent B. The elution profile was: 0-10 min, 95% B (isocratic); 10-15 min, 95-70% B (linear gradient); 15-20 min, 70-65% B (linear gradient); 20-40 min, 65-55% B (linear gradient); 40-45 min, 55% B (isocratic); 45-70 min, 55-30% B (linear gradient); 70-100 min, 30-0% B (linear gradient); 100-120 min, 0% B (isocratic). Flow rate of 0.95 mL/min, column temperature of 30℃, injection volume



of 20 µL and detection wavelength of 254 nm. 40, 41 2.5 Biosynthesis of GNPs In a typical synthesis for the GNPs, 12.5 mL of CPLE was first heated at constant temperature of 30 ℃ in an oil bath. Afterward, some amount of aqueous HAuCl4 (0.04856 mol/L) was added suddenly to the flask in order that the reaction solution possessed the initial HAuCl4 concentrations of 0.5 mM. And the mixture was kept at 30 ℃ under vigorous magnetic stirring for 2 h to gain GNPs. 2.6 Characterization Determination of the biomolecules type was carried out by a Nicoler 6700 FTIR spectrometer (Thermo Fisher, USA). Waters 600 liquid chromatograph with a 2996 PAD (Waters, USA) was used to further ascertain the component. The ultraviolet visible (UV-Vis) spectra were obtained on a Thermal Evolution 200 spectrometer (Thermo Fisher, USA) at a wavelength range of 350-1100 nm. Transmission electron microscope (TEM) characterizations were executed on a Phillips Analytical FEI Tecnai 30 electron microscope (FEI, Netherlands) at a voltage of 300 KV. The GNPs sizes were estimated via the assistance of SigmaScan Pro software on basis of TEM micrographs. 3. Results and Discussion 3.1 Separation of biomolecules in original CPLE Flash column chromatography is a quick and facile way to separate complex mixtures. A reversed-phase flash chromatographic column was employed to isolate the biocomponents of original CPLE. The dry weight of CPLE aqueous solution


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(100mL) and that of all isolated samples are shown in Fig. 1. The dry weight of original CPLE is up to 771.8 mg. As for the isolated samples, the content of Sloading and S0 nearly accounts for two-thirds of total sample weight (64.4%), while that of S1~S5 are basically about 40 mg. The content of S6~S8 dropped sharply to 19.2, 9.2, 5.4 mg, respectively. It’s worth noting that the weight of all trapped samples after separation covers 96.55% of total quality of original CPLE. This result and the residence time of liquid chromatographic peak of CPLE and isolated samples (Fig. S1) fully illustrates that almost all biocompounds of CPLE can be eluted into the isolated samples, making it possible to identify and compare the strength of reducing and protecting power of the entire active components in the CPLE. In particular, the S7 and S8 will be ignored in the following experiments to the negligible amounts to react with gold ions. 800

S8 S7 S6 S5 S4 S3 S2 S1 S0 Sloading

700 Dry weight (mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


600 500 400 300 200 100 0 original CPLE

isolated samples

Figure 1 Dry weight of the original CPLE (100 mL) and isolated samples 3.2 Identification of biomolecules in isolated samples Generally, protein and saponins of CPLE were usually ignored for the low content. Sugars are a kind of polarity-strong biomacromolecule, the main functional groups



relating to the biosynthesis of GNPs are the hydroxyl. The family of polyphenols mainly includes flavonoids, stilbenes, phenolic acid, lignans, and hydrolysable or condensed tannins, which own phenolic hydroxyl groups.24 In this paper, polyphenols refers in particular to phenolic acids and hydrolysable tannins that are of excellent water solubility and embody abundant adjacent phenolic hydroxyl. Hence, CPLE compounds mainly include sugars, polyphenols, and flavonoids. FTIR analysis was firstly carried out to obtain the preliminary identification. The FTIR spectra of the original CPLE and the different isolated samples (Sloading, S0, S1, S2, S3, S4, S5, and S6) are shown in Fig. 2a. As described in Fig. 2a, the isolated samples can be divided into three groups on basis of the similarity of FTIR spectra: (a) Sloading and S0; (b) S1~S5; (c) S6~S8; and we speculate that the samples in the same group should be alike in biomolecules type. The spectra of three groups samples all show bands at about 3400, 2930, 1620, and 1070 cm-1 that are assigned respectively to the stretching vibration of ν(O-H), ν(C-H), ν(C=C), ν(C-O).16 The differences emerge in the range of 1620~1420 cm-1 and 900~800 cm-1 (dotted box in figure 2a). Purposively select S0, S3, and S6 as the representative of three groups for further identification. The spectra of S3 (Fig. 2b) reveal three obvious characteristic peaks in vicinity of 1620-1420 cm-1 (1615, 1516, and 1445 cm-1), indicating the presence of aromatic rings.28 The peak at 3419 cm-1 can be attributed to the stretching vibration of phenolic hydroxyls (O-H bond) in the S3. The absorption peak at 1386 cm-1, arises from the deformation of phenolic hydroxyls (O-H bond). In addition, the peak at 1203 cm-1 is beneficial to recognize adjacent phenolic hydroxyls. Above all, it can be


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concluded that the main component of S3 should be polyphenols. In the S0 spectra, two characteristic absorption peaks at 861 and 790 cm-1 can be assigned to the glycosidic bonds. The bands at 1419 and 1256 cm-1 are deformation vibration of the methyl and stretching vibrations of epoxy bond. Hence, the main component of S0 should be sugars because that it contains O-H, C-H, C-O, epoxy bond, and glycosidic bonds, etc. but have no feature of aromatic ring. For this reason, it can be also understood that the FTIR spectra of the original CPLE (Figure 2a), consisted of greater proportion of sugars, is similar with that of the Sloading and S0. However, from the FITR spectra of S6, it is difficult to identify the molecular category according to the characteristic absorption peak.

a

CPLE

Sloading S0

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S1 S3

S2 S3 S4 S5 S6 S7 S8

3000

1500

1000

Wavelength (cm-1)

b Transmittance (%)

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1256 1065

S0

869 787

3387

1419 1622 1386

2932

S3 2929 3419 2932

814

1615 1445 1516 1203

1067 810

S6

1627 1385 1085

3421 3500

3000

2500

2000

1500

1000

Wavelength (cm-1)

Figure 2 FITR spectra of the (a) original CPLE and isolated samples; (b) S0, S3, and



S6. To further identify the biomolecular categories in the S0, S3 and S6, characteristic HPLC traces of partial plant molecular from the three aqueous solutions are shown in Fig. 3. In addition, UV spectra of these peaks were detected with PAD, and the maximum absorption wavelength (λmax) of each peak are shown in Table 1-3. In general, UV spectra of polyphenols reveal one or two strong characteristic absorption peak in the range of 260~330 nm.40,

42

The λmax of S3 accords with these traits,

therefore, the biomolecular category of S3 is confirmed as polyphenols. As for the sample S0, in spite that it is mostly occupied by abundant sugars, tiny amounts of polyphenols were detected by the PDA. The structure of flavonoids can be divided into A and B two parts. A ring is the derivatives of benzoic acid, and B ring is cinnamyl derivatives. Thus, UV spectrum of polyphenols can be resolved into two absorption bands which respectively originate from the UV-absorption of A and B ring of flavonoids. One stronger absorption band lies in between 300 and 400 nm, and another weaker band frequently emerge within the limits of 240~280 nm.40, 42 So, it is clear the biomolecular category of S6 was considered as flavonoids through the data of Table 3.


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Figure 3 HPLC chromatograms of (a) S0; (b) S3; (c) S6.

Table 1 Retention time (tR) and maximum of the absorption wavelength (λmax) of S0 by HPLC-PDA No.*

tR(min)

λmax(nm)

category

1

23.623

286

polyphenols

2

23.862

269

polyphenols

3

25.799

260, 294

polyphenols

* Refer to peak number in Fig. 3a




tR(min)

λmax(nm)

category

1

29.082

274

polyphenols

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

29.560 30.234 32.790 33.484 34.637 36.067 37.749 38.382 39.249 39.943 41.864 43.341 45.331 49.770 51.499 56.663 65.212 75.808

275 274 276 277 264 277 274 275 271 279 274 274 274 274 310 291, 323 264, 295 310

polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols

* Refer to peak number in Fig. 3b


tR(min)

λmax(nm)

category

1 2 3

81.246 89.702 95.699

299, 349 256, 350 264, 343

polyphenols flavonoids flavonoids

* Refer to peak number in Fig. 3c Hence, based on the FTIR and HPLC-PDA analysis, the components in the isolated samples can be sorted into three categories. Among them, Sloading and S0,


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S1~S5, S6 mainly belong to sugars, polyphenols, and flavonoids, respectively. 3.3 Reducing power of the biomolecules To compare the strength of reducing power, the different isolated samples (Sloading~S6, 0.5 mg/mL) were reacted with chloroauric acid (0.5 mM) in aqueous solution for 2 h at 30 ℃ to synthesize GNPs, respectively. The UV-Vis spectra of the obtained GNPs by the isolated samples are shown in Fig. 4a, and that of original CPLE is also given for contrast. The characteristic surface plasmon resonance (SPR) band of CPLE and S0~S6 all center about 530 nm, which can be confirmed as the spherical GNPs, and the band at long-wave area (>700nm) assigned to the characteristic peak of triangular/hexagonal GNPs emerged in the spectroscopy of S6.26 Generally, the intensity of SPR peak has positive relationship with the reduction degree of gold ions. With the enhancement of the band intensity at about 530 nm, it is obvious that the reduction degree of all the tested samples are ranked in the sequence of group (b), CPLE, group (c), and group (a), from high to low. Furthermore, the time dependence evolution of absorbance (Abs-T) plots for SPR peak (530 nm) are given in Fig. 4b, which can help us compare the reducing rate among the different separated samples. Apparently, the absorbance of GNPs prepared by Sloading and S0 increased rather tardily within 120 min, suggesting the weak reducing power for Sloading and S0. By contrast, the samples S1~S5 showed the stronger reducing rate, with the complete reduction within 20 min. The S6 came second, hence in the view of reducing rate, the tested samples are ranked in the same sequence of group (b), group (c), and group (a) from high to low.



a

Sloading S0 S1 S2 S3 S4 S5 S6 CPLE

0.6 0.4 0.2

4 3

Abs

0.8

Abs

b S3 S2

S5

S4

2

S1

1

CPLE

S6 S0 Sloading

0.0

0 400

600

800

1000

0

20

0.4 0.3

c

40

60

80

100 120

Time (min)

Wavelength (nm)

Abs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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glucose gallic acid rutin

0.2 0.1 0.0 400 500 600 700 800 900 1000

Wavelength (nm) Figure 4 (a) UV-Vis spectra of GNPs synthesized by CPLE and Sloading~S6; (b) Abs-T plots for GNPs synthesized by CPLE and Sloading~S6; and (c) UV-Vis spectra of GNPs synthesized by glucose, gallic acid and rutin. Furthermore, we selected glucose, gallic acid, and rutin, as representative of sugars, polyphenols and flavonoids respectively to reacted with chloroauric acid (0.5M) at the same conditions. The UV-Vis spectra of the GNPs synthesized by three substances are shown in Fig. 4c. No absorption band was observed for the GNPs synthesized by glucose, and the intensity of the characteristic SPR obtained from gallic acid goes far beyond that of rutin. Through the above discussions on the reduction degree and reduction rate, it can be drawn that for the biomolecules in the CPLE, the sequence of reducing power is polyphenols, flavonoids and sugars from high to low.


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3.4 Protective/Stabilizing power of the biomolecules The protecting/stabilizing power of active biomolecules, during the growth process of nanoparticles, is a critical aspect to consider. It need to be investigated to achieve better control over the size distribution and morphology of biosynthesized.27 The TEM images of the GNPs synthesized by the CPLE and Sloading~S6 are shown in Fig. 5. The agglomeration of GNPs is clearly visible in the TEM images obtained from Sloading and S0. The S1~S5-inspired GNPs are almost spherical with the size of 20-50 nm（see the insets of Figure 5c-5g）, while the main morphology of the GNPs produced by the S6 is spherical or triangular/hexagonal. It can be seen that the component had significant effect on the particle size of the obtained GNPs. The particles obtained from S1 showed bigger size of 50±7 nm due to the presence of small quantity of polar carbohydrate molecules. The GNPs obtained from S2 and S3 exhibited the size of 23±6 nm and 22±8 nm, respectively. While the S4 and S5, consisting polyphenols and some weak polar flavonoids, resulted in the GNPs of 33±8 nm and 33±5 nm. 50±7nm

30

40

50

60

70

Size (nm)

a 23±6nm

b 22±8nm


c 33±8nm


5

10

15 20 25 30 35 40 45

10

Size (nm)

20

30

40

50

Size (nm)

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10

20

30

40

e

d

60

50

Size (nm)

f

33±5nm

20

30

Size (nm)

40

50

g

h

Figure 5 TEM images of GNPs bio-synthesized by (a) Sloading; (b) S0; (c) S1; (d) S2; (e) S3; (f) S4; (g) S5; and (h) S6. The insets show the statistical size of GNPs. The chem-synthesized method of GNPs usually employs NaBH4 as reducing agent to react with HAuCl4·4H2O and Na3C6H5O7 as stabilizer.44 To make it clear how the active biomolecules interplay with the GNPs during the growth of particles, we design several experimental schemes: respectively taking glucose, gallic and rutin as stabilizing agents to substitute for Na3C6H5O7 in above method. The TEM images of obtained product are shown in Fig. 6. The TEM image (Fig. 6a) of the GNPs with Na3C6H5O7 as capping agent shows the morphology (sphere) and size (2~5 nm) of the particles, while no stabilizing agent was added, the GNPs (Fig. 6b) were observed the tendency to aggregate. Choosing glucose simulated sugars as capping agent, the coagulation (Fig. 6c) still exists on accounts of the lack of protection. Obviously, given that agglomeration phenomena of the GNPs prepared by Sloading and S0, sugars should not undertake the role of capping agent owing to the weak adsorption


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capacity of biomolecular function groups. The TEM image (Fig. 6d) reveals that gallic acid could unselectively adsorb on the surface of GNPs to form sufficient repulsive forces to overcome coagulation during the slow growth stage of particles. As compared with Na3C6H5O7, the adsorption capacity and rate of gallic acid is relatively slight so that the particles size is larger. However, the feature, flower-like (Fig. 6e), of the GNPs obtained from rutin as capping agent present a trait of anisotropic protection.

a

c

b

d

e

Figure 6 TEM images of GNPs synthesized with 0.5 mM HAuCl4 and 0.6 mL 0.1 M NaBH4 at 30 ℃ for 5 h, 2.5×10-4 M (a) Na3C6H5O7; (b) blank; (c) glucose; (d) gallic acid; and (e) rutin as capping agents. In order to survey the influence of concentration of active components, the CPLE were mixed with the S0, S3, and S6 to improve the relative content of sugars, polyphenols, and flavonoids with the same concentration (0.5 mg/L) of reaction mixture, then were respectively reacted with chloroauric acid (0.5 mM) at the same



conditions. The TEM image in Fig. 7b shows, with the increase of sugars content, the reaction liquid lacks sufficient stabilizing biomolecules to aggravate the agglomeration of particles. Comparing with the original CPLE (Fig. 7a), the mixture added S3 can produce the smaller spherical GNPs for the strong isotropous protection of polyphenols (Fig. 7c), while the mixture added S6 (main content of flavonoids) can selectively adsorb on the {111} plane of GNPs to form nanoplates (Fig. 7d).

a

b

c

d

Figure 7 TEM images of bio-synthesized GNPs by (a) CPLE 3.6 mL; (b) CPLE2.7 mL + S0 0.9 mL; (c) CPLE 2.7 mL + S3 0.9 mL; and (d) CPLE 2.7 mL + S6 0.9 mL. In the previous reports13, 15-17, 20, 28-31, to explore the role of the active components, the plant extract were previously used as whole by the means of FTIR analysis or content determination during the biosynthesis. Dinesh D et al15 revealed that organic functional groups of phytochemicals like flavonoids, polyphenols and proteins are responsible for formation and stabilization of GNPs. Sathishkumar G et al20 reported that total phenol content decrease of extract depicted the role polyphenolic


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antioxidants in reduction and capping of nanomaterials. Comparing with the above results, after the separation of plant extract in this study, the role in the reduction and stabilization of AuNPs were separately investigated, and the sequence in the reducing and stabilizing power were obtained. In addition, the role of sugars, rarely mentioned but the most abundant active component in the plant aqueous extracts, were also involved. 3.5 Illustration of reduction and growth period of GNPs biosynthesized by different active components Due to the competition and cooperation of ionic or electrostatic force between the metal complexes and the different functional groups, the content of active biomolecule is a critical factor on the regulation of size and morphology of GNPs.27 Based on above study and the results from other researchers,22, 27, 43 the illustration of the reduction and growth period of GNPs biosynthesized by different active components can be depicted in Scheme 1. The S0, sugars rich sample, has abundant hydroxyl as reducing group, and its reducing power is relatively weak. With the improvement of the sugars content, the growth of grain size, even agglomeration of particles, is inevitable due to the functional group is difficult to adsorb on the surface of gold nanoclusters. Compared with phenolic-OH, adjacent group can give stronger electron donating ability, reducing and protecting power, so the abundant adjacent phenolic-OH in the S3 could rapidly fabricate gold atoms and interact with the small particles through ionic binding with the o-dihydroxyphenyl to grow the isotropous spherical particles.44 In the absence of o-dihydroxyphenyl group, the phenolic-OH of



the flavonoids, higher redox potential than hydroxyl, could reduce Au (Ⅲ) to Au (0) and be oxidized to carbonyl groups, which purposefully adsorb on the specific surface, such as {111} plane, to lead the nanoplates emerged.27 During the S6-inspired biosynthesis of GNPs, given the stronger reducing and protecting power of little polyphenols, the spherical nanoparticles could coexist with the nanoplates.

Scheme 1 Illustration of reduction and growth period of GNPs bio-synthesized by different active components 4. Conclusion To summarize, we have separated original CPLE according to the polarity differences through flash column chromatography, and identified the molecular categories in the isolated samples based on the FTIR and HPLC-PDA analysis. The components in the isolated samples can be sorted into three categories: sugars (group a: the Sloading and S0), polyphenols (group b: the S1~S5), and flavonoids (group c: the S6~S8). Comparing the process of each isolated samples reacted with HAuCl4, the sequence of reducing power is polyphenols, flavonoids and sugars from high to low in


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the viewpoint of reducing degree and reducing rate. In addition, sugars have no protection for the biosynthesis of GNPs, polyphenols involved in isotropous stabilizing action through chelating between o-dihydroxyphenyl groups and the gold nanocluster, and flavonoids are good anisotropic capping agent due to oriented absorption of phenolic-OH and carbonyl groups. Finally, the reduction and growth period of GNPs biosynthesized by different active components are proposed. To the best of our knowledge, it is the first report to systematically separate, identify and describe the role of main component in the plants extract. The confirmation of the reducing and protective power of the active components such as sugars, polyphenols and flavonoids makes the regulation of the particle size and morphology of GNPs more feasible. It would be valuable to the plant-mediated synthesis of other nanomaterials, providing a possible protocol in understanding and predicting particle’s size and shape from a given set of biosynthetic conditions.

Acknowledgements This work was supported by the NNSF of China (NSFC Project No. 21536010). H. Liu thanks Beifang University of Nationalities of China for financial support (2013XYZ033).

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