Antimicrobial activity of Biosynthesized Silver Nanoparticles of Bauhinia racemosa leaf extracts

 

K. Riazunnisa*

Department of Biotechnology and Bioinformatics, Yogi Vemana University Kadapa, Andhra Pradesh, India

 

ABSTRACT:

Increased wettability or solubility of nanoparticles (NPs) can greatly improve their antibacterial activities. The effect of the stabilising agent on the solubility of silver NPs (AgNPs) and their antibacterial and antifungal properties were examined in this study. Aqueous and methanolic solutions of Bauhinia racemosa plant leaf extracts were used as bioreductants for making silver NPs. The synthesised AgNPs were first identified by a change in colour from pale green to dark brown and further confirmed by surface plasmonic resonance (SPR) band at 430 nm for aqueous and 360 nm for methanolic extracts using UV-Visible spectroscopy. The size distribution of generated AgNPs in solution was examined using a DLS particle size analyzer, and it was found to be 114 and 122 nm for aqueous and methanolic extract respectively. Bauhinia racemosa AgNPs exhibited significant antibacterial and anti-fungal activities against E. coli, K. pneumonia, P. aeruginosa, S. aureus and Aspergillus niger, demonstrating that they may be utilized in the biomedicine field.

 

 

 

INTRODUCTION: 

Nanoparticles have one dimension that ranges from 1-100nm. Paints, waste water treatment, and drug delivery all use nanoparticles. Nanoparticles play a critical role in invitro diagnostics, gene delivery, drug delivery¹, photodynamics and imaging (MRI), particularly in the life sciences². When compared to typical chemical agents, nanoparticles have the advantage of low toxicity, making them suitable for drug/gene delivery.

 

Silver nanoparticles are currently used in diagnostic applications, such as quantitative biosensors, antibacterial applications, such as cosmetics and wound dressings, optical applications, such as metal enhanced fluorescence (MEF) and Surface Enhanced Raman Scattering (SERS) and conductive applications, such as conductive inks. Apart from that, nanoparticles could be used to remove pathogens from water and in the improvement of latent finger prints³.

 

The Caesalpiniaceae family includes the plant Bauhinia racemosa L. It is majorly distributed in India, China, Ceylon and Timor. Jamdar et al.⁴ reported B. racemosa may be used as natural pH indicator. Its bark has shown medicinal properties including antioxidant and antimicrobial activity⁵, antitumor activity against Ehrlich ascites carcinoma⁶ and anti-inflammatory, analgesic and antipyretic effects⁷. And the methanolic leaf extracts have antibacterial efficacy⁸. The presence of flavonoids and alkaloids were found during a phytochemical examination of B. racemosa aqueous leaf extract and saponins, alkaloids and tannins in methanol extract⁹. Abundant literature is available on the antibacterial activity of medicinal plants^10-14.

 

Metal and metal oxide nanoparticles were synthesized from B. variegata¹⁵ B. tomentosa^16,17 and but very few reports are available with the B. racemosa¹⁸. Enhanced antibacterial activity was monitored during lunar eclipse with the ZnO NPs of B. racemose leaves¹⁸. Secondary metabolites found in the leaf extract may act as reducing agents in the creation of silver nanoparticles, converting ionic silver (Ag^o) from AgNO₃ to Ag¹. The present study demonstrates the biogenic synthesis of silver nanoparticles by aqueous and methanolic leaf extracts of B. racemosa and further evaluates the antifungal and antibacterial activity of AgNPs of B. racemosa.

 

MATERIALS AND METHODS:

Collection of plant material:

Healthy leaves of Bauhinia racemosa L. (Caesalpiniaceae) were collected from botanical garden of Yogi Vemana University, Kadapa. The plant material was gathered and washed under running water, then rinsed with distil water and left to completely dry at room temperature. The dried Bauhinia racemosa leaves were ground to fine powder and stored at 4 ^oC for further analysis.

 

Preparation of aqueous and methanolic extract:

Each 17g of leaf powders were mixed with 100ml of 80 % methanol and 200 ml of water and kept in water bath at 40^oC for 24 h. After incubation period, solvent was removed by rotary evaporator at 40^oC. The concentrated extract was then kept at cold temperature until use.

 

Biosynthesis of silver nanoparticles of aqueous and methanolic extract:

2.5ml of aqueous leaf infusion was mixed with 25ml of 0.1mM silver nitrate solution and 2.5ml of methanolic leaf extract was mixed with 50ml of 0.1mM silver nitrate, were kept in a shaker for 36 h at 37^oC.  After incubation period, the color change was observed visually¹⁹.

 

Separation of silver nanoparticles:

Centrifugation was done at 8000rpm for 15 minutes in a REMI centrifuge to separate the silver nanoparticles. The procedure was repeated three times to remove any uncoordinated biomolecules. The liquid from the supernatant was resuspended in sterile double distilled water. The supernatant liquid was discarded after the specified reaction period, and the pellets were collected. The obtained pellet solution was transferred to a sterile petridish in a hot air oven at 60^oC for overnight and stored at 4^oC for further use.

 

Characterization:

a) UV-Visible spectroscopy:

The optical property of bio-reduced silver nanoparticles from the both aqueous and methanolic leaf extracts of Bauhinia racemosa were analyzed by UV-Visible spectrophotometer (Thermoscientific Evolution-201) at room temperature. Optical absorbance of the synthesized AgNPs was monitored between 300-700nm.

 

b) DLS analysis:

Using a Zeta sizer S-90, Malvern, UK, the size of the produced nanoparticles was measured in a dilute dispersion solution of AgNPs in distilled water.

Antimicrobial activity by well diffusion method:

Antibacterial activity of the biosynthesized AgNPs was evaluated against strains of Gram-negative bacteria: K. pneumoniae (MTCC 618), Escherichia coli (MTCC 443), and P. aeruginosa (MTCC 1688): Gram-positive bacteria: Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 441), and antifungal activity against Aspergillus niger by the standard agar well diffusion method. LB and potato dextrose agar media was prepared by mixing all the components in a required volume. 100mg of AgNPs powder was dissolved in 1.0 ml (100ppm) DMSO under sonicator to provide even suspension of AgNPs. Different concentrations such as 25, 50 and 100µg/ml of AgNPs, 50g/ml of leaf extract, 1 µg/ml standard antibiotic (Tetracycline) and 30g/ml fluconazole were added into respective wells and incubated at 37^oC for 24 h. The diameter of the inhibitory zone around each well was measured after the findings were observed.

 

Statistical analysis:

All of the tests were done in triplicate. For all assays, the mean and standard deviation (SD) were analysed. The data was presented as a mean±SEM of three studies.

 

RESULTS:

In the present work, we synthesized the AgNPs from aqueous and methanolic extracts of Bauhinia racemosa. Further we evaluated the comparison of antimicrobial activity of AgNPs and plant extracts against four MTCC bacterial strains and one fungal strain. The color change from light green to dark brown indicated the formation of AgNPs in both the samples (Fig. 1A and 2A). After incubation at 36 h the absorption maximum was observed at wavelength range between 300-700nm for different concentrations of aqueous leaf extract and presented in Fig. 1B. The SPR band was monitored at 430nm for aqueous extract and 360 nm for methanolic extract (Fig. 1B and 2B). Likewise, the methanolic extracts of varied concentrations of absorption maxima and color change were depicted in Fig. 2B.

 

Dynamic light scattering (DLS) technique was used to determine the size of particles in the colloidal suspension for synthesized aqueous and methanolic leaf infusion AgNPs, and results are depicted in Fig. 1C and 2C. The size of nanoparticles were 114nm and 122nm for B. racemosa aqueous and methanolic extracts.

 

 

A

 

Fig. 1: Color transmission from pale green to dark brown (A), Absorption spectrum (B) and DLS analysis (C) of varied concentrations (2.5, 7.5 and 1.0 ml of aqueous leaf extract/25 ml AgNO₃) of silver nanoparticles using aqueous leaf extract of B. racemosa.

 

 

 

Fig. 2: Color transmission from pale green to brown (A) Absorption spectrum (B) DLS analysis (C) of varied concentrations (2.5, 7.5 and 1.0ml of methanolic leaf extract/50 ml AgNO₃) of silver nanoparticles using methanolic leaf extract of B. racemosa.

 

A

 

Table 1. The zone of inhibition of Bauhinia racemosa aqueous leaf extract and its silver nanoparticles on bacterial and fungal species.

S. No	Tested pathogen	Zone of inhibition (mm)
		Concentration (mg/ml)
		Leaf extract	25	50	75	100	Tetracyclin (1µg/ml)
1	E. coli	12 + 0.33	15 + 0.19	17 + 0.43	18 + 0.16	20 + 0.51	28 + 0.19
2	K. pneumoniae	13 + 0.21	15 + 0.25	17 + 0.28	20 + 0.23	24 + 0.12	30 + 0.15
3	P. aeruginosa	12 + 0.14	16 + 0.34	17 + 0.27	18 + 0.38	18 + 0.33	27 + 0.22
4	B. subtilis	13 + 0.17	18 + 0.26	18 + 0.14	17 + 0.29	18 + 0.21	29 + 0.31
5	S. aureus	13 + 0.35	18 + 0.18	18 + 0.12	18 + 0.37	21 + 0.52	36 + 0.36
6.	Aspergillus niger (fungal strain)	4 + 0.51	7 + 0.29	11 + 0.41	11 + 0.21	-	Fluconazole (30 g/ml) 15 + 0.12

 

 

Table 2. The zone of inhibition of Bauhinia racemosa methanolic leaf extract and its silver nanoparticles on bacterial and fungal species.

S. No	Tested pathogen	Zone of inhibition (mm)
		Concentration (mg/ml)
		Leaf extract	25	50	75	100	Tetracyclin (1µg/ml)
1	E. coli	13 + 0.25	14 + 0.15	18 + 0.28	20 + 0.29	22 + 0.40	29 + 0.33
2	K. pneumoniae	14 + 0.19	19 + 0.32	22 + 0.39	25 + 0.33	25 + 0.12	32 + 0.13
3	P. aeruginosa	12 + 0.33	17 + 0.30	17.5 + 0.11	19 + 0.13	21 + 0.19	26 + 0.18
4	B. subtilis	15 + 0.41	16 + 0.23	17 + 0.21	18 + 0.30	19 + 0.15	28 + 0.32
5	S. aureus	14 + 0.28	18 + 0.17	18 + 0.32	20 + 0.11	23 + 0.14	33 + 0.17
6.	Aspergillus niger (fungal strain)	4 + 0.14	7 + 0.36	12 + 0.10	12 + 0.22	-	Fluconazole (30 g/ml) 14+ 0.15

 

In the present work the effect of B. racemosa AgNPs was studied on the antimicrobial activity of the bacterial cultures of E. coli, K. pneumonia, P. aeruginosa, B. subtilis, S. aureus and fungal culture of Aspergillus niger. Significant inhibition of all the cultures was observed, maximum region of inhibition was examined against K. pneumonia and S. aureus i.e., 24mm and 21 mm. It is followed by E. coli, P. aeruginosa and B. subtilis with aqueous extracts. 25 and 23mm zone of inhibition was monitored against K. pneumonia and S. aureus with methanolic extracts. The attained results were analyzed and compared with standard positive control. Tetracyclin was the standard antibacterial agent used.  Maximum zone of inhibition observed against Aspergillus niger was 11mm and 12mm with both the aqueous and methanolic leaf extracts (Table 1 and 2)

 

DISCUSSION:

The phytochemicals present in the B. racemosa leaf extracts acted as reducing agents for the formation of AgNPs⁹. The production of nanoparticles was confirmed by UV Spectrophotometry and DLS analysis.  The synthesized nanoparticles were evaluated against bacterial strains and fungi. The attained results were analyzed and compared with the positive controls tetracyclin and fluconazole. Our results exhibited good antibacterial effect against gram (-) and gram (+) bacteria and antifungal activity. Maximum antibacterial activity was observed against K. pneumonia and S. aureus. Excellent antimicrobial activity of silver nanoparticles was reported by several authors with Bauhinia species such as B. purpurea, B. variegata, B. tomentosa and B. acuminata^20,21,17,22. Potential antibacterial activity of silver, copper, zinc oxide and ruthenium oxide nanoparticles were reported by many researchers^{23,24,25,26,27}.

 

CONCLUSION:

The ecofriendly synthesized silver nanoparticles by using aqueous and methanolic leaf extracts of B. racemosa presented good antibacterial and antifungal activity against all the tested organisms. Therefore, the produced AgNPs may be used as potential antimicrobial agents in treating the diseases and can be further used for drug discovery.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest.

 

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Accepted on 27.05.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):745-749.