Antifungal potential of green synthesized silver nanoparticles (AgNPS) from the stem bark extract of Kigelia pinnata

 

Lokesh Ravi1, Krishnan Kannabiran2*

1Department of Botany, St. Joseph’s College (Autonomous), Bengaluru - 27.

2Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology, Vellore-14.

*Corresponding Author E-mail: kkb@vit.ac.in

 

ABSTRACT:

Aqueous extract of bark of Kigelia pinnata was used as reducing source for the biological green synthesis of AgNPs. The synthesized particles were characterized by UV-Visible spectrum, XRD, SEM, dynamic light scattering (DLS). The anti-fungal activity of AgNPs was evaluated against Aspergillus niger (MTCC:281), Aspergillus flavus (MTCC:277) and Candida albicans (MTCC:227) by agar diffusion method. Particle size of AgNPs was measured as 76.4nm ± 6.3nm and Zeta potential was stable at -43.2mV using DLS analysis. SEM analysis measured the size of AgNPs as ~75nm. XRD analysis confirmed that the synthesized NPs were silver (Ag), based on the JCPDS entry 85-1355 and it is crystalline in nature. The size of the crystal was calculated as 10nm using the Scherrer formula. The AgNPs demonstrated significant anti-fungal activity against C. albicans (MIC:15.6µg/ml), A. niger (MIC:62.4µg/ml) and A. flavus (MIC:31.2µg/ml). These results confirm that AgNPs synthesized using K. pinnata barks could be used as potential antifungal agent against fungal pathogens.

 

KEYWORDS: Kigelia pinnata, Ag nanoparticles (AgNPs), antifungal activity, Candida albicans, Aspergillus flavus, Aspergillus niger.

 

 


INTRODUCTION:

Kigelia pinnata, belongs to Bignoniaceae family found predominantly in African subcontinent. Though the genus Kigelia had several species in the past, recently it became mono-specific and it is called by above 100 different colloquial names in Africa. It is most commonly called as ‘Sausage tree’ due to the enormous size of the fruits which look like a sausage. Traditionally they have many important pharmacological applications. The extract obtained from bark is used to heal abscesses and wounds. K. pinnata, a medicinal plant from Africa has been traditionally used to cure several health issues. It is also used to treat dysentery in addition to sexually transmitted diseases. The fruit of this plant is used to treat various skin diseases, ulcer and rheumatism1. Various researchers have reported the antimalarial, antimicrobial, anti-diabetic, anti-inflammatory and anti-cancer potential of phytochemical compounds extracted from K. pinnata2,3.

 

Nanoparticles are smaller particles with the dimensions from 1nm to 100 nm range in size4. Silver nanoparticles (Ag NPs) in particular have unique properties and so they have found applications in several fields, which include electronics, medical and cosmetics5. In medical field silver nanoparticles are proven to be excellent antimicrobial and anticancer agent6. They have also been successfully used in drug delivery and imaging techniques7. Top down and bottom up are well known two approaches generally followed in the synthesis of nanoparticles. In the bottom up approach the silver nanoparticles are built from small atomic level to the final product. In the case of top down approach, bulk materials are converted to products of nano scale dimensions8. They are manufactured through physical, chemical or biological methods. Physical methods are more energy consuming and the whole manufacturing process is laborious. Chemical methods lead to many chemical contaminations and so they pose serious health issues due to the toxicity9. Biological synthesis of silver (Ag) nanoparticles with microorganisms or plant extracts is of current biological importance worldwide10.

 

Synthesis of AgNPs using the extracts obtained from K. pinnata bark and their antimicrobial potential is not been reported so far. Hence, a study was planned to synthesize AgNPs from the water extract of K. pinnata bark and to study its antifungal activity against selected fungal pathogens.

 

MATERIALS AND METHODS:

Preparation of stem bark extract:

Dry bark samples of K. pinnata were chipped off from the tree stem, from Woodstock, Vellore Institute of Technology, Tamil Nadu, India. The sample was washed, dried and then powdered using mortar and pestle. 10g of powdered bark samples was mixed with 100ml deionized water (10% W/V). The mixture was incubated at 60şC for 1h with occasional stirring. The mixture was filtered using Whatman No.1 paper to obtain the aqueous extract4.

 

Synthesis of silver nanoparticles:

Silver nitrate (AgNO3) was procured from Hi Media, Mumbai, India and used. AgNO3(1mM) was prepared in double-distilled water and mixed with the water extract of bark at 1:1 ratio and placed in room temperature for 48h in dark. The mixture was subjected for centrifugation (10,000rpm for 15 min) and the pellet obtained was dried and the Ag nanoparticles obtained was used for characterization8,11–13.

 

Characterization of Ag nanoparticles:

Presence of silver in the synthesized nanoparticles was confirmed by measuring the characteristic Ag nanoparticles absorbance between 400nm–500nm using the UV- visible spectrophotometer (Double beam, SL-210 UV-vis Spectrophotometer, ELICO, India). The obtained particles was dissolved in water and analyzed for zeta potential and size (HORIBA Scientific Nano PARTICA SZ-100 Dynamic Light Scattering analyzer). Synthesized AgNPs were also subjected to crystallographic phase analysis (X-Ray diffractometer, Bruker D8 Model with Cu Kα (λ=1.5405 Ĺ) radiation). Diffraction pattern recorded as 2Ө range from 10ş to 80ş with scanning speed of 1ş/min. The obtained graphical data was analyzed in PCPDFWIN software. Crystal size was calculated from the XRD analysis, using Scherrer formula. The shape and size of the NPs were observed under scanning electron microscopy (Zeiss EVO 18 Research SEM). The NPs are scanned at an operating voltage of 10 KV with a working distance 12.5 mm from low to high magnification range. Unit-Cell parameters (System, Space Group and Setting) of Ag nanoparticles were retrieved from JCPDS database (i.e., entry that matched to the XRD analysis) using PCPDFWIN software14,15.

 

Antifungal assay of silver nanoparticles by well diffusion method:

Three fungal pathogens Aspergillus niger (MTCC:281), Aspergillus flavus (MTCC:277), and Candida albicans (MTCC:227) were purchased from MTCC (IMTECH, Chandigarh, India) and used for this study. The antifungal activity was determined by agar well diffusion method16. About 25mL of potato dextrose agar (PDA) was used to prepare a media in a sterile Petri plate (HI Media, Mumbai, India). The media was allowed to solidify, 100 µl of fungal cultures (18 h grown; OD adjusted to 0.6) used for making a lawn culture with the help of sterile cotton swab. A 5mm well was bored by using a sterile cork borer. The test sample was dissolved in DMSO and loaded into wells with various concentrations 25, 50, 75 and 100µg/well. Clotrimazole (30µg/ml) was positive control. The C. albicans inoculated plates were incubated at 37°C under 40 W florescent light sources (~ 400 nm) for 24 h and A. niger, A. flavus plates were kept for 72 h. The antifungal activity was measured as the zone of inhibition in millimeters around the well using antibiotic-zone-scale (HiMedia, Mumbai, India). Minimal Inhibitory Concentration (MIC) of the nanoparticles against the three fungal pathogens was studied using the standard broth micro dilution method, according to CLSI guidelines. For A. niger and A. flavus Sabouraud dextrose broth was used and the observations were made after 48h of incubation16–18.

 

RESULTS AND DISCUSSION:

Synthesis of AgNPs:

Synthesis of silver nanoparticles (AgNPs) was monitored by change in color of the incubation mixture (AgNO3: aqueous extract) from colorless to brownish yellow color suggesting formation of Ag nanoparticles. The UV-visible absorbance of AgNPs at 428nm (λmax) corresponds to the characteristic absorption maxima for silver (400~500nm) The UV absorbance spectrum of the incubated mixture at 24h and 48hours is shown in Figure.1.

 

 

Figure.1. UV-visible absorption spectra of synthesised AgNPs

 

Characterization of nanoparticles:

Particle Size Analysis:

Dynamic Light Scattering (DLS) particle size analysis suggested that the AgNPs were of 76.6nm ± 6.3nm in size. As shown in Figure.2 [A] the DLS analysis suggests that the mixture contains particles of uniform size group indicating the presence of monodispersed nanoparticles with a standard deviation of 6.3nm. Also the zeta potential analysis using the DLS analyzer revealed that the AgNPs demonstrated a stable zeta potential of -43.2mV Figure.2 [B]. DLS analysis confirms that the synthesized particles are in nanometer scale and are monodispersed. Additionally, the zeta potential analysis also suggests that the zeta potential of the surface of the nanoparticles are stable.

 

 

Figure. 2. DLS analysis [A] particle size analysis; [B] Zeta potential analysis of AgNPs

 

X-Ray Diffraction Analysis:

The synthesized nanoparticles were subjected for X-Ray Diffraction (XRD) analysis, to understand its crystal properties. Initial observation of the XRD plot, suggested that the nanoparticles are crystalline in nature. The XRD plot is shown in Figure.3. The phase shits at 27.6°, 32.39°, 38.1°, 44.1°, 46.1°, 54.7°, 64.4° and 76.7° had a perfect match with the JCPDS database entry 85-1355, confirming that the synthesized particles were AgNPs. Further, the unit cell crystal size was found to be 10nm in size based on the Scherrer formula analysis. Thus the XRD analysis also confirmed that the synthesized nanoparticles are crystalline in nature and that they are silver (Ag).

 

Figure. 3. XRD analysis of AgNPs

 

Scanning Electron Microscope:

The nanoparticles are further subjected for Scanning Electron Microscope (SEM) to understand the morphology of the individual particles. As typically observed in several other green synthesized nanoparticles, this nanoparticle also demonstrated mild agglomeration/coagulated morphology. The individual particles exhibited almost a semi-spherical nature, while several of those are agglomerated and produces a rosette appearance. The approximate size of synthesized AgNPs were found to be ~75nm with minor deviations as indicated in SEM image Figure.4. It further confirmed the results of DLS analyzer.

 

Figure.4. SEM image of AgNPs

 

Thus the characterization studies on the synthesized particles, confirmed that the particles are in nanometer scale (76.6nm ± 6.3nm) and that they are crystalline in nature, with mild agglomeration, showing a rosette appearance.

 

Antifungal activity of AgNPs:

The synthesized AgNPs demonstrated significant antagonism against the tested fungal pathogens. C. albicans was most susceptible to AgNPs. The zone of inhibition exhibited by AgNPs at varying concentrations against fungal pathogens is given in Table.1. The zone of inhibition (21mm) of AgNPs (100µg/ml) against C. albicans was shown in Figure.5[A] and against A. flavus (14mm) was shown in Figure.5[B]. AgNPs demonstrated significant MIC value of 15.6µg/ml against C. albicans, however it showed comparatively higher MIC values against A. flavus (31.2µg/ml) and A. niger (62.4µg/ml).


 

Table 1: Zone of inhibition of AgNPs at various concentrations against fungal pathogens

Fungal pathogens

AgNPs (25mg/ml)

AgNPs (50mg/ml)

AgNPs (75mg/ml)

AgNPs (100mg/ml)

Clotrimazole (30 µg/ml)

Aspergillus niger

-

7mm

9mm

10mm

15mm

Aspergillus flavus

7mm

10mm

11mm

14mm

19mm

Candida albicans

14mm

16mm

18mm

21mm

22mm

 


 

Figure. 5: Antifungal activity of different concentrations of AgNPs [A] C. albicans and [B] A. flavus. +Ve : Clotrimazole(30 µg/ml).

 

DISCUSSION:

The aqueous and ethanolic extracts of K. africana (Bignoniaceae) stem bark has proven to possess antimicrobial activity19. Extracts prepared from K. africana fruits showed antimicrobial activity against yeast and pathogenic bacteria20. Solvent extracts prepared from leaf of K. pinnatais proven to have antagonism against bacterial pathogens21. These reports proves that the species of Kigelia possess bioactive phytochemicals that serves as antimicrobial agents, against pathogenic fungi and bacteria.

 

The AgNPs synthesized using water extract of leaves of K. Africana is proven to exert antibacterial activity against some common bacterial pathogens22. Many other medicinal plants known for antibacterial activity were used for green synthesis of silver nanoparticles (AgNPs), which include leaves of Cissus quadrangularis Linn23, root extract of Onosmadi chroantha Boiss24 and aqueous extract of flowers in Albizia julibrissin25. Anticandidal activity of AgNPs produced from the Prunus persica L outer peel has been reported by Patara et.al.,26. Ag-AgNPs synthesized using Agrimonia pilosa plant has shown to exhibit antibacterial activity against bacterial pathogens27. Ag NPs synthesized using water extract of K. africana has been shown to exhibit strong anticandidal and antibacterial activity28. These reports prove that, plant phytochemicals, in particular components of Kigelia species have already proven to be reducing agents for synthesizing biologically active AgNPs.

 

In this study the aqueous extract prepared from the bark of K. pinnata has been proven to produce crystalline AgNPs, which demonstrates significant antagonism against fungal pathogens, C. albicans, A. niger and A. flavus. Among these three pathogens, AgNPs demonstrated highest antagonism against C. albicans with a MIC value of 15.6µg/ml.

 

CONCLUSION:

This study suggests that the bark extract of K. pinnata serves as a good source of phytochemicals that have significant reducing potential for nanoparticle green synthesis. The results also conclude that, the AgNPs synthesized using K. pinnata bark extract serves as a significant antifungal agent. Although the mechanism of action, their in-vivo efficacy and in-vitro and in-vivo toxicity of the AgNPs are yet to be studied, the observations of this study conclude that these AgNPs has potential to be used in pharmaceutical applications to treat topical fungal infections.

 

ACKNOWLEDGEMENT:

The management of Vellore Institute of Technology, Vellore and St. Joseph’s College (Autonomous), Bengaluru are gratefully acknowledged for providing facilities to carry out this study.

 

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Received on 08.02.2020            Modified on 23.04.2020

Accepted on 05.06.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):1842-1846.

DOI: 10.52711/0974-360X.2021.00326