Introduction

Nanoparticles research is an area of great scientific interest due to their large surface area to volume ratio and diverse physicochemical characteristics. Preparation of silver nanoparticles (AgNPs) has attracted particularly considerable attention due to wide variety of applications including electrical batteries (Wang et al. 2010), optical receptors (Karimzadeh and Mansour 2010), sensors (Dubas and Pimpan 2008), catalysts in chemical reactions (Edison and Sethuraman 2012), bioactive materials (Blaker et al. 2004) and antimicrobial agents (Saxena et al. 2012). AgNPs are most promising as they are possessing antimicrobial properties due to larger surface area to volume ratio which is of great interest to researchers due to the increasing microbial resistance against antibiotics, metal ions and development of resistant strains. It is very well known that silver ions and silver-based compounds are lethal to microorganisms and no organism has ever been reported to develop resistance against silver. This aspect of silver makes it an excellent antimicrobial agent and thus was used in skin ointments and creams to improve wound healing (Zhao and Stevens 1998; Elliott 2010).

Various physical and chemical methods which include gamma irradiation assisted (Huang et al. 2009), ultrasound irradiation assisted (Abbasi et al. 2012), thermal decomposition (Yang et al. 2007), laser ablation (Simakin et al. 2004), electrochemical assisted (Hosseini and Momeni 2010), sonochemical synthesis (Salkar et al. 1999) and chemical reduction (Lee and Meisel 1982) methods were employed to synthesize AgNPs, but most of these methods are expensive and involve the use of toxic chemicals which may pose potential environmental and biological risks. Hence, there is a need to develop eco-friendly procedures. Biosynthesis of AgNPs using microorganisms, enzymes, and plants or plant extracts have been suggested as cost effective, eco-friendly alternatives to chemical and physical methods. Various plant extracts which include Andrographis paniculata (Kotakadi et al. 2014), Catharanthus roseus (Kotakadi et al. 2013), Cassia alata (Gaddam et al. 2014), Cissus quadrangularis (Valli and Vaseeharan 2012), Coleus aromaticus (Vanaja and Annadurai 2012), Tithonia diversifolia (Tran et al. 2013) and Brucea javanica (Salprima et al. 2013) were reported for the biosynthesis of AgNPs.

Centella asiatica, an important medicinal plant belongs to the Umbelliferae family. It is a valued ethnomedicine in traditional Indian ayurvedic system. It is used for improving memory, treat gastric ulcers and kidney troubles (Kumar and Gupta 2002). The bioactive compounds of this plant also possess antitumor (Babu et al. 1995), immunomodulating (Wang 2003), cardioprotective (Pragada et al. 2004) and anti-depressant activities (Chen et al. 2005). Development of callus culture methods from the leaf explants will be alternative approaches for the optimization of bioactive compounds. The callus cultures of C. asiatica are explored for the biosynthesis of AgNPs as another important pharmaceutical or biomedical application of C. asiatica.

In this study, we first report the synthesis of AgNPs using aqueous callus extract of C. asiatica. The synthesized AgNPs were characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM) and X-ray diffraction method (XRD). The antimicrobial activity of the AgNPs was evaluated against S. aureus, B. subtilis, E. coli and P. aeruginosa to check their biomedical importance.

Materials and methods

Plant material collection

Centella asiatica plants were collected from medicinal plants garden and authenticated with taxonomist, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. The voucher specimen was deposited in the herbarium (VM-0912).

Explant sterilization

The young leaves of C. asiatica were excised and washed thoroughly with sterile double distilled water for 3–5 min to remove the dust. Then leaves were treated with 10 % NaOCl for 60–90 s. Surface sterilization was done under aseptic conditions using 0.1 % HgCl2 for 30–45 s followed by a final rinse 2–3 times with sterile double distilled water.

Callus induction and culture conditions

The sterilized leaves were blotted on sterile blotting paper, cut into small parts (0.8 × 1.0 cm) and inoculated on Murashige–Skoog (MS) medium containing 2.0 mg/L of α-naphthalene acetic acid and 1.0 mg/L of kinetin as callus inducing plant growth regulators (Murashige and Skoog 1962). The cultures were incubated at 23 °C under photoperiod of 12-h light with intensity 45–50 μ mol/m2/s.

Preparation of aqueous callus extracts

Fresh biomass of callus was collected and dried in the oven at 40 °C for 24 h. The dried biomass of callus was crushed into fine powder. Five gram of fine powder was taken in 250 ml Erlenmeyer flask, added 50 ml of sterile double distilled water and boiled for 10–15 min at 70 °C. The extract was then allowed to cool to room temperature and then filtered using Whatman No. 1 filter paper. The filtrate named aqueous callus extract was used for biosynthesis of AgNPs. The extract was also stored at 4 °C for future use.

Biosynthesis of AgNPs

Silver nitrate (AgNO3) was purchased from Himedia, Bangalore, India. The reaction mixture was prepared by adding 10 ml of the callus extract to 90 ml aqueous solution of 1 mM AgNO3 (9:1 ratio-optimized concentration) in 250 ml Erlenmeyer flask and incubated in a dark place at 35 °C for about 48 h. The primary detection of reduction of silver ions (Ag+) to AgNPs (Ag°) was carried out by observing the color change of the reaction mixture from light yellow to dark brown. Confirmation of the synthesis and characterization was carried out by spectrophotometric measurements.

Characterization of AgNPs

The characteristic SPR peak of the AgNPs solution was recorded using UV–Visible spectroscopy (Analytical Technologies Ltd, India). The FTIR spectrum was obtained in the range of 500–4000 cm−1 with the resolution of 2 cm−1 by employing alpha interferometer FTIR. TEM micrographs to determine the size and shape of the synthesized AgNPs were obtained using FEI Tecnai F12 (Philips, Holland) operated at 100 kV. XRD patterns to confirm the crystalline nature of AgNPs were recorded using Cu Kα radiation source on an Ultima IV X-ray powder diffractometer (Rigaku, Tokyo, Japan).

Antimicrobial activity

The antimicrobial activity of the AgNPs was evaluated against pathogenic bacteria S. aureus, B. subtilis (Gram-positive) and E.coli, P. aeruginosa (Gram-negative). The antimicrobial activity was carried out with 24 h active cultures by employing disc diffusion method (Ghassan et al. 2013; Cruickshank 1968). Two hundred microlitre of bacterial inoculum was swabbed on the surface of nutrient agar medium plates. Sterile discs impregnated with 20 μl of AgNPs solution at a concentration of 100 µg/ml were then placed on the surface of the inoculated medium. Sterile disc without any treatment was used as negative control. Standard antibiotic was used as positive control. The agar plates were incubated at 37 °C for 24 h. The values of diameter of zone of inhibition (ZOI) were represented.

Results and discussion

Callus was initiated after 14 days of inoculation on MS medium. Light yellow and friable callus (Fig. 1) was harvested after 6th week of inoculation. The callus at this stage considered well developed and matured due to secretion of plant secondary metabolites and proteins and hence is used for the synthesis of AgNPs.

Fig. 1
figure 1

Light yellow and friable callus developed from the leaves of Centella asiatica on MS media supplemented with 2.0 mg/L of α-naphthalene acetic acid and 1.0 mg/L of kinetin

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The synthesis of AgNPs was initially observed by the color change from light yellow to dark brown color (Fig. 2). The color change is due to the excitation of surface plasmon resonance vibrations in AgNPs. Noble metal nanoparticles with bright and fascinating colors owing to their localized surface plasmon resonance effect offer convenient surface bioconjugation using molecular biomarkers which in turn offer wide variety of applications (Noginov et al. 2007; Stamplecoskie and Scaiano 2010).

Fig. 2
figure 2

Yellow color of the Centella asiatica callus extract exposed to AgNO3 (a) and dark brown color of the reaction mixture after 48 h of incubation (b)

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UV–Vis analysis

The UV–Vis absorption spectra of the AgNPs showed (Fig. 3) the absorbance peak at 453 nm, confirmed the synthesis of AgNPs. Owing to the surface plasmon vibrations excited, AgNPs exhibited strong absorption peak in the visible range named surface plasmon resonance (SPR) peak. The SPR peak and different molecules of callus extracts could be responsible for capping and stabilization of AgNPs formed. This characteristic SPR peak may also correspond for the spherical shape of AgNPs (Kotakadi et al. 2014; Noginov et al. 2007; Stamplecoskie and Scaiano 2010).

Fig. 3
figure 3

Characteristic SPR peak of AgNPs shown by UV–Vis absorption spectroscopy

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FTIR analysis

The FTIR spectrum (Fig. 4) showed peaks at 3273, 2922, 2361, 1603, 1515, 1369, 1025 cm−1. The peak at 3273 cm−1 could be assigned to N–H stretching vibrations of the secondary amide of the protein and the peak at 2922 cm−1 could be assigned to C–H stretching of methylene groups of the protein and 2361 cm−1 corresponding to N–H stretching/C–O stretching vibrations (Bozanic et al. 2010; Kumar and Mamidyala 2011; Mahitha 2011). The peak at 1603 cm−1 corresponds to asymmetric C=O stretching vibration and/or aromatic C=C stretching vibration (Aruna et al. 2012; Valentina and Boris 2013; Bellamy 1975). The peak at 1515 cm−1 corresponds to amide II linkage of the proteins (Monali 2009). The peak at 1369 and 1025 cm−1 could be assigned to C–O stretching and O–H deformation of phenolic OH groups (Valentina and Boris 2013; Bellamy 1975; Monali 2009). FTIR peaks and assigned functional groups clearly indicated in the Table 1. Based on the FTIR studies, it is reported that phenolic compounds present in the callus extract could be responsible for the reduction of silver ions (Ag+) into AgNPs (Ag°). Proteins could be responsible for both synthesis and stabilization of AgNPs. But the exhaustive mechanism of the synthesis of nanoparticles in this bio-based reduction by callus extract is to be further elucidated.

Fig. 4
figure 4

FTIR spectra of AgNPs produced by callus extract of Centella asiatica

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Table 1 FTIR peaks and corresponding functional groups
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TEM analysis

From the TEM micrograph (Fig. 5) it is clear that the synthesized AgNPs were well dispersed and their shape is roughly spherical with the size ranging 5–40 nm. A small percentage of AgNPs in solution was partially aggregated but uniform in their size and shape. The TEM results are consistent with many earlier reports (Tran et al. 2013; Salprima 2013).

Fig. 5
figure 5

TEM micrograph of silver nanoparticles by callus extract of Centella asiatica (inset bar 50 nm)

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XRD analysis

A representative XRD pattern (Fig. 6) of the synthesized AgNPs showed four distinct diffraction peaks at 38.26°, 44.58°, 64.51° and 77.62° corresponding to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of the face-centered cubic (fcc) lattice of silver, respectively (JCPDS File No 87-0720), and revealed the crystalline nature of AgNPs. The XRD results are consistent with those reported for face-centered cubic (fcc) lattice of silver (Gaddam et al. 2014; Aruna et al. 2012).

Fig. 6
figure 6

XRD pattern obtained for AgNPs produced by callus extract of Centella asiatica

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Antimicrobial activity

After incubation for 24 h, growth inhibition (ZOI) was observed around discs impregnated with silver nanoparticles and positive control (streptomycin), but the negative control discs could not produce ZOI (Fig. 7). AgNPs exhibited strong antimicrobial activity against both Gram-positive, S. aureus, B. subtilis and Gram-negative bacteria, E. coli, P. aeruginosa and formed the ZOI of diameters 21.3, 19.4, 16.2 and 18.8 mm, respectively (Table 2). AgNPs showed strong inhibition against Gram-positive bacteria compared to Gram-negative bacteria. Higher ZOI was noticed for S. aureus compared with other Gram-positive strain. The results obtained for antimicrobial activity are very effective and consisted with many earlier reports of plant extract assisted synthesis of AgNPs (Kotakadi 2013; Mahitha 2011; Aruna et al. 2012). Many researchers reported the possible mechanism of action behind the inhibitory activity of AgNPs against different bacterial strains. AgNPs could preferentially bind the cell membrane and cause dissipation of proton motive force (PMF) and thus membrane destruction occurs. AgNPs increase the membrane permeability by forming pores or pits on the membranes and finally results in cell death (Morones et al. 2005; Lok et al. 2006; Shahverdi 2007). AgNPs bind with bacterial cell membrane and enter into cytosol of bacteria by dissipating PMF and interrupts bacterial DNA replication system and inactivates synthesis of proteins causing the inhibition of bacterial growth (Kotakadi 2013).

Fig. 7
figure 7

Antimicrobial activity of silver nanoparticles against S. aureus (a), B. subtilis (b), E. coli (c) and P. aeruginosa (d)

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Table 2 Antimicrobial activity of AgNPs produced by callus extract of Centella asiatica against pathogenic bacteria
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Conclusion

In the present study, we have synthesized AgNPs of 5–40 nm in size with spherical shape using aqueous callus extract of Centella asiatica for first time. AgNPs synthesized by aqueous callus extract were very distinct with very small size, well-defined shape, well dispersed, crystalline nature and clearly proved their biomedical importance by exhibiting strong antimicrobial activity against both Gram-positive and Gram-negative bacteria. Thus AgNPs possess important applications in biomedical or pharmaceutical industry for the preparation of antibacterial skin ointments.