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Industrial Crops and Products 50 (2013) 737– 742

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l h om epage: www.elsev ier .com/ locate / indcrop

reen synthesis of gold nanoparticles from leaf extract of Terminaliarjuna, for the enhanced mitotic cell division and pollen germinationctivity

. Gopinatha, K.S. Venkatesha, R. Ilangovana, K. Sankaranarayananb, A. Arumugama,∗

Department of Nanoscience and Technology, Alagappa University, Karaikudi 630 004, Tamil Nadu, IndiaDepartment of Physics, Alagappa University, Karaikudi 630 004, Tamil Nadu, India

r t i c l e i n f o

rticle history:eceived 13 May 2013eceived in revised form 1 August 2013ccepted 17 August 2013

eywords:erminalia arjuna

a b s t r a c t

This present work reports an ecofriendly approach for the synthesis of spherical gold nanoparticles (AuNPs) using aqueous leaf extract of Terminalia arjuna. T. arjuna contains arjunetin, leucoanthocyanidinsand hydrolyzable tannins, which are found to be responsible for the bio-reduction of Au NPs. The formedAu NPs were characterized by UV–vis, FTIR, XRD, AFM and TEM analysis. UV–visible spectra of the aque-ous medium containing gold nanoparticles showed a surface plasmon resonance peak at 530 nm. FT-IRanalysis was performed to analyze the biomolecules responsible for the reduction of Au NPs. XRD results

old nanoparticlesreen synthesisitotic cell division

ollen germination

confirmed the presence of gold nanoparticles with face centered cubic structure. The calculated crys-tallite sizes are in the range of 20 to 50 nm and the spherical nature of the Au NPs was ascertained bytransmission electron microscopy. The efficacy of the synthesized Au NPs was tested for the mitotic celldivision and pollen germination. It is suggested that Au NPs induces the mitotic cell division and pollengermination. There was no cytotoxic effect of Allium cepa root tip cells and Gloriosa superba pollen grains.

. Introduction

Nanostructured materials have been attracting a keen atten-ion owing to their unique properties. It finds application in manyelds such as nanocomputers (Tseng and Ellenbogen, 2001), catal-sis (Kim et al., 2003), optical devices (Kamat, 2002), cell labelingWu et al., 2003), cell tracking (Parak et al., 2002), in vivo imagingDubertret et al., 2002), DNA detection (Taylor et al., 2000), antimi-robial activity (Krishnaraj et al., 2012) and so on. Nanoparticleynthesis is usually carried out by various physical and chem-cal methods such as chemical vapour deposition (Dillon et al.,006), sol–gel technique (Sobhani et al., 2008), aerosol technol-gy (Magnusson et al., 1999), sono chemical method (Kenji et al.,005), photochemical reduction (McGilvray et al., 2006) and so on.he chemicals used for these syntheses are often toxic, costly andon-ecofriendly. However, green synthesis approach for produc-

ng Au NPs is an alternative source of conventional methods andossesses excellent anti-fungal activity (Jayaseelan et al., 2013).

ecently, the synthesis of Au NPs have been reported using extrac-ion of plants such as Anacardium occidentale (Sheny et al., 2011),assia auriculata (Ganesh Kumar et al., 2011), Centella asiatica

∗ Corresponding author: Tel.: +91 4565 225630; fax: +91 4565 225202.E-mail addresses: sixmuga@yahoo.com, kgnanee@gmail.com (A. Arumugam).

926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2013.08.060

© 2013 Elsevier B.V. All rights reserved.

(Kumar Das et al., 2010), Chenopodium album (Dwivedi and Gopal,2010), Coleus amboinicus (Narayanan and Sakthivel, 2010), Crocussativus (Vijayakumar et al., 2011), Macrotyloma uniflorum (AswathyAromal et al., 2012), Terminalia chebula (Kumar et al., 2012),Trigonella foenum-graecum (Aswathy Aromal and Philip, 2012) andMurraya koenigii (Philip et al., 2011).

Metal nanoparticles are used for crop production. An et al.(2008) have reported an increase in ascorbate and chlorophyllcontents in leaves of Asparagus treated with silver nanoparticles(Ag NPs). Arora et al. (2012) have reported that 10 ppm of goldnanoparticles treated of B. juncea seedlings which enhance the netproductivity of seed yield. In another study, Brassica juncea plantstreated with Ag NPs did not seem to accumulate Ag in any form(Haverkamp and Marshall, 2009). Lettuce and cucumber seeds weretreated with different concentration of Au NPs. The results wereobserved in most significant effect of lettuce seed than cucum-ber seeds and did not showed any significant effect (Barrena et al.,2009). Arabidopsis thaliana seeds were treated with 10 �g/ml of AuNPs (24 nm) which enhance the seed yield. Consequently, 80 �g/mldose of Au NPs was recommended for other vegetative crops pro-ductivity (Kumar et al., 2013).

Terminalia arjuna belongs to Combretaceae family and it is alarge evergreen tree with spreading crown, drooping branches(Chopra and Ghosh, 1929; Caius et al., 1930). T. arjuna bark has beenused for the treatment of coronary artery disease (Dwivedi and

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38 K. Gopinath et al. / Industrial Cr

auhari, 1997), heart failure (Antani et al., 1991) and hypercholes-erolemia (Tiwari et al., 1990). In addition, it possesses antibacterialnd anti-mutagenic properties (Perumal Samy et al., 1998; Kaurt al., 2001). T. arjuna leaf extract contains arjunetin, leucoan-hocyanidins and hydrolyzable tannins (Arumugam and Gopinath,011).

In this present study, we have reported the green synthesis andharacterization of gold nanoparticles using T. arjuna leaf extractnd their potential applications for mitotic cell division and pollenermination activity. To the best of our knowledge, this is the firsteport for the synthesis of Au NPs using T. arjuna leaf extract.

. Material and methods

.1. Synthesis of gold nanoparticles using T. arjuna leaf extract

Fresh T. arjuna plant leaves were collected from Karaikudi (Tamiladu, India) and used to retrieve their extraction. First, the leavesere cleaned with tap water, followed by distilled water and thennely cut into small pieces. 10 g of finely cut leaves were added with00 ml of double distilled water and boiled for 5 min. The obtainedxtraction was filtered using Whatman No. 1 filter paper and theltrate was collected in 250 ml Erlenmeyer flask and stored at roomemperature for further usage. Then, 1 ml of T. arjuna leaf extractas added to 100 ml of 1 mM HAuCl4 solution at room temperature

nd rapid reduction of Au NPs was clearly observed within next5 min.

.2. Characterization

The synthesized gold nanoparticles were subjected toV–visible analysis in the wavelength range of 350–800 nmsing Shimadzu spectrophotometer (Model UV-1800) operating at

resolution of 1 nm. Also, Fourier transform infrared spectroscopyFTIR) analysis was carried out in the range of 400–4000 cm−1.-ray diffraction (XRD) analysis for a thin film sample contain leafxtract with Au NPs were prepared on a glass slide (1 × 1 cm) byropping 100 �L of the sample on the slide, and allowed to dryor 30 min, then XRD pattern was recorded using Cu K� radiation� = 1.54060 A) with nickel monochromator in the range of 2� from0◦ to 80◦. The nano-crystallite domain size was calculated fromhe width of the XDR peaks using Scherrer formula D = 0.9�/�cos �Krishnaraj et al., 2012). AFM analysis for a thin film of the sampleas prepared on a glass slide (1 × 1 cm) by dropping 100 �L of the

ample on the slide, and allowed to dry for 30 min. The slides werehen scanned with AFM (APE Research-model no: A100SGS). TheFM characterization was carried out in ambient temperature inon-contact mode using silicon nitrite tips with varying resonance

requencies. TEM measurements were carried out to bring outhe morphology of the green synthesized nanoparticles in termsf size and shape. Samples for TEM analysis were prepared byrop coating the nanoparticle solutions on carbon-coated copperrids at room temperature. The excess nanoparticles solutionas removed with filter paper. The copper grid was finally dried

t room temperature and was subjected to TEM analysis by thenstrument Tecnai F20 model operated at an accelerating voltagef 200 kV.

.3. Mitotic index testing system using Au NPs

The gold nanoparticles were suspended directly in deionizedater and dispersed by ultrasonic vibration (100 W, 30 kHz) for

0 min to produce three different concentrations at 10 �M, 100 �Mnd 1000 �M. Four healthy Allium cepa bulbs (25 g) were grownn 250 ml conical flask in dark atmosphere at room temperature28 ± 1 ◦C) by changing the water for the interval of 24 hours for

d Products 50 (2013) 737– 742

two days. When the roots reached to 2–3 cm in length then theywere treated with different concentrations of 10, 100 and 1000 �MAu NPs suspension for 4 h. Each experiment was performed forfive replicates. Ten new root tips were used for each concentra-tion. The micro slides were prepared for each concentration andcontrolled the sample using Saffranin squash technique. The roottips were kept in 1 M HCl solution for 6 min followed by stainingwith 40% Saffranin. Staining was continued to 5–6 min. Thus pre-pared slides were examined by confocal laser scanning microscopy(CLSM). The total number of cells (1000 cells) were counted andnumber of dividing cells manifesting the different stages of mito-sis i.e. propahse (P), metaphase (M), anophase (A) and telophase(T) were recorded (Fiskesjo, 1985; Kumari et al., 2009). The mitoticindex was calculated using the following formula:

(%) Mitotic Index = Number of cells in mitosisTotal number of cells

× 100

2.4. In vitro pollen grains germination test treated with Au NPs

The green synthesized Au NPs were suspended directly in deion-ized water and dispersed by ultrasonic vibration (100 W, 30 kHz) for30 min to produce three different concentrations at 10 �M, 100 �Mand 1000 �M. The Gloriosa superba pollen grains were collectedfrom dehisced flowers in morning time (7–10 a.m.). The pollen ger-mination basal medium (BM) containing (10% sucrose and 0.01%boric acid, pH 5.8) and supplemented with different concentra-tion of Au NPs ranging from 10, 100 and 1000 �M were used. Thecollected pollen grains were aseptically inoculated in the pollengermination medium. All cultures were maintained at 25 ± 1 ◦C for18 h photoperiod at a photosynthetic flux of 2.6 � mol m−2 s−1, pro-vided by cool daylight fluorescent lamps. Finally, germinated pollengrains were counted from total number of 100 cells and each exper-iment was performed for three times. The G. superba pollen grainswere excited using argon-laser at 25 mW 1.5% exposure. Consecu-tively, the germinated pollen grains of G. superba were analyzed byCLSM at the room temperature of 20 ◦C.

3. Results and discussion

A rapid reduction of Au NPs was clearly observed when T. arjunaleaf extract was added with HAuCl4 solution within 15 min. Thecolor of the solution was immediately changed from yellow to darkred which indicates the rapid formation of the Au NPs possibly inthe range of 20 nm (Sheny et al., 2011; Vijayakumar et al., 2011;Aswathy Aromal and Philip, 2012).

3.1. UV–vis spectroscopy

The mixture of leaf extract and HAuCl4 solution was subjectedto UV–vis spectroscopy analysis during the rapid reduction pro-cess so as to understand the mechanism of Au NPs formation. Therecorded spectra at different time intervals such as 5 min, 10 minand 15 min are shown in (Fig. 1A) along with the spectra recordedfor the blank solutions of the leaf extract and HAuCl4. The UV–visspectra recorded on the blank solutions show no absorption peakin the region of 350–800 nm while the recorded spectra during therapid reduction process at different time intervals showed a absorp-tion peak at 530 nm which corresponds to the wavelength of thesurface plasmon resonance of Au NPs (Sheny et al., 2011; AswathyAromal et al., 2012; Aswathy Aromal and Philip, 2012).

3.2. X-ray diffraction

X-ray Diffraction pattern was recorded for the synthesized AuNPs (Fig. 1B). Three distinct diffraction peaks at 38.26◦, 44.48◦ and

K. Gopinath et al. / Industrial Crops and Products 50 (2013) 737– 742 739

F s solun ous so

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ig. 1. (A) UV–vis spectra of Au NPs synthesized by reacting 1 mM HAuCl4 aqueouanoparticles synthesized by treating the leaf extract of T. arjuna with HAuCl4 aque

6.29◦ were indexed with the planes (1 1 1), (2 0 0) and (2 2 0) forhe face centered cubic gold as per the JCPDS card no. 04-0784. Theell resolved and intense XRD pattern clearly showed that the AuPs formed by the reduction of Au+ ions using T. arjuna leaf extractre crystalline in nature. Similar results were reported for Au NPsn the literature (Sheny et al., 2011; Aswathy Aromal et al., 2012;swathy Aromal and Philip, 2012). The low intense peak at 77.35◦

elongs to (3 1 1) plane.

.3. Fourier transform infrared spectroscopy

FTIR analysis was performed to identify the possibleiomolecules responsible for the reduction of the Au+ ionsnd capping of the reduced Au NPs synthesized using T. arjunaeaf extract. The strong band at 3416 cm−1 corresponds to N Htretching vibration of primary amines, whereas the band at754 cm−1 corresponds to C H stretching vibration of aldehyde.

he band at 2073 cm−1 corresponds to C N stretching of any

N C S, the medium band at 1639 cm−1 corresponds to similaronjugation effects of N H and the low band 688 cm−1 correspondso Cl stretching. Hence, the main components such as arjunetin,

Fig. 2. (A) FTIR spectra of gold nanoparticles synthesized using T. arjuna le

tion with T. arjuna leaf extract at different time intervals. (B) XRD pattern of goldlution.

leucoanthocyanidins and hydrolyzable tannins present in the leafextract of T. arjuna are responsible for the observed reduction andcapping during the synthesis of Au NPs. The two new strong bandsrecorded at 1368 and 1229 cm−1 in the spectra of the synthesizedmaterial were assigned to NO2- stretching and C O stretchingrespectively. These peaks may be raised due to the reduction ofgold chloride to gold nanoparticles (Fig. 2 A).

3.4. Atomic force microscopy and transmission electronmicroscopy

Surface topology of the formulated gold nanoparticles was stud-ied by AFM analysis (Fig. 2B). The micrographs clearly indicate thatthe formulated Au NPs possess spherical shape and has the cal-culated sizes in the range of 20–50 nm. The TEM image (Fig. 3A)further ascertain that the gold nanoparticles are predominantlyspherical in morphology with their sizes ranging from 20–50 nm

and has an average size of about 20 nm. The selected area electrondiffraction (SAED) pattern (Fig. 3B) of Au NPs resulted in the char-acteristics ring pattern of face centered cubic (fcc) and it manifeststhe high degree of crystallinity of Au NPs.

af extract. (B) AFM – 3D- images of Au NPs synthesized by T. arjuna.

740 K. Gopinath et al. / Industrial Crops and Products 50 (2013) 737– 742

F he leaf extract of T. arjuna. and (B) selected area electron diffraction (SAED) pattern of AuN

3

(q71Appe1ompteotiiNa

TE

T

ig. 3. (A) TEM image of gold nanoparticles formed by reduction of Au+ ions using tPs.

.5. Au NPs used for the mitotic cell division

A. cepa root samples treated with different concentration of10, 100 and 1000 �M) Au NPs exhibited the increment in the fre-uency of mitotic index (Table 1) 40.16 ± 0.61, 50.66 ± 0.44 and1.68 ± 0.47 respectively. From the perusal of results, obviously at000 �M Au NPs had most significant effect of mitotic index (Fig. 4). Au NPs interact with intracellular chromatin (DNA + Histonerotein) and mitotic inter-phase, and then enhance the DNA androtein synthesis (Nabiev et al., 2007; Singh et al., 2009; Symenst al., 2012; Kumar et al., 2013; Feldherr, 1966; Feldherr and Akin,990). Subsequently, there was an increase in the mitotic indexf A. cepa root tip cells in different phases such as prophase (P),etaphase (M), anaphase (A) and telophase (T). The mitotic index

ercentage was increased for the treated cell compared with con-rol (Fig. 5A–D). Results showed that increase of the dose of Au NPsnhances the mitotic index without any chromosomal aberrationf the A. cepa root tip cells. Au NPs used in mitotic cell divisionest causes no cytotoxic effect in the cell cycle. Similarly, Au NPs

nduced the cell division without any endocytosis and cytotox-city effect of HeLa and E. coil cells (Cui et al., 2012). Hence, AuPs can be used for plant tissue culture and plant embryologypplications.

able 1ffect of gold nanoparticles induce the mitotic cell division of Allium cepa root tip cells.

Treatments Dividing cell (Total) P

Control (DistilledWater)

280 276

295 290

305 299

308 303

292 285

Au NPs – 10 �M 416 402

405 397

408 401

399 388

380 372

Au NPs – 100 �M 520 495

514 489

498 479

502 498

499 481

Au NPs – 1000 �M 723 698

710 692

721 690

702 682

728 699

he result are the mean ± S.E of 5 replicates. S.E – standard error. MI – mitotic index, P – p

Fig. 4. (A) Au NPs treated in different concentration (10, 100 and 1000 �M) of Alliumcepa root tip cells and (B) Gloriosa superba pollen grains. (The squared area indicatesthe significant points.)

3.6. Au NPs used for G. superba pollen germination

The basal medium was used in the control experiments forin vitro G. superba pollen germination. Pollen grains treated with

M A T MI (%) Mean ± SE (%)

2 1 1 28.01 2 1 29.52 2 2 30.5 29.60 ± 0.502 1 2 30.82 3 2 29.29 4 1 41.65 3 0 40.56 1 0 40.8 40.16 ± 0.617 3 1 39.96 2 0 38.0

13 8 4 52.011 9 5 51.4

8 7 4 49.8 50.66 ± 0.445 4 2 50.29 7 2 49.9

12 8 5 72.310 5 3 71.018 7 6 72.1 71.68 ± 0.4711 5 4 70.218 7 4 72.8

rophase, M – metaphase, A – anaphase, T – telophase.

K. Gopinath et al. / Industrial Crops and Products 50 (2013) 737– 742 741

Fig. 5. Confocal images showed that the Au NPs treated in different concentration A – control (with out Au NPs), B – 10 �M, C – 100 �M and D – 1000 �M) of Allium cepa rootcells.

Table 2Effect of gold nanoparticles induce the pollen germination of Gloriosa superba.

Treatments Total number of cells No. of cells tubes formation Mean ± SE(%)

Basal medium (BM) 100 24100 22 24.00 ± 1.15100 26

BM + Au NPs – 10 �M 100 42100 36 38.33 ± 1.86100 37

BM + Au NPs – 100 �M 100 66100 73 69.33 ± 2.03100 69

BM + Au NPs–1000 �M 100 79100 84 80.33 ± 1.26100 78

The result are the mean ± S.E of 3 replicates. S.E – standard error.

F – conp

di8ctptcn(c

4

Nnsa

ig. 6. Confocal images showed that the Au NPs treated in different concentration Aollen grains.

ifferent concentration of (10, 100 and 1000 �M) Au NPs wouldncreases the pollen germination to (Table 2) 38.33%, 69.33% and0.33% respectively (Fig. 4B). Au NPs are involved in inter-cellularommunication and intracellular signaling with cells. Au NPsreated samples induced the pollen germination and tube growthercentage, compared with the control (Fig. 6A–D). Results showedhat induction of the pollen germination depends on Au NPs con-entration. Malva sylvestris and Yucca filamentosa pollen grains didot show any significant effect of pollen germination in basal mediaDane et al., 2004). It is suggested that use of Au NPs in basal mediaan enhance the pollen germination of other plants.

. Conclusion

A simple, reliable, cost-effective and rapid green synthesis of Au

Ps using T. arjuna leaf extraction is reported. The synthesized goldanoparticles have particle sizes in the range of 20–50 nm with thepherical nature. The efficacy of the Au NPs in mitotic cell divisionnd pollen germination was tested and found to be more signif-

trol (with out Au NPs), B – 10 �M, C – 100 �M and D – 1000 �M) of Gloriosa superba

icant. There was no carcinogenic and cytotoxic effect of the cellsand pollen grains. It is suggested that green synthesis of nanoparti-cles has beneficial application in the field of cell division and pollengermination of rare species.

Acknowledgements

The authors gratefully thank School of Physics, Alagappa Univer-sity, for extending the XRD facility and also thank the Departmentof Animal Health and Management, Alagappa University, for pro-viding the CLSM facility.

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