Synthesis and Optimization of Silver Nanoparticles using Leaf extract of Tamarindus indica by Box-Behnken Design for Enhanced Antimicrobial Activity

 

Puneeth Deepak Ail¹, Divya Jyothi²*, Rokeya Sultana³, Lenisha Crasta⁴

¹Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences,

Nitte (Deemed to be) University, Deralakatte, Karnataka, India.

²Department of Pharmacognosy, Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences,

Nitte (Deemed to be) University, Deralakatte, Karnataka, India.

³Department of Pharmacognosy, Yenepoya Pharmacy College,

Yenepoya (Deemed to be) University, Deralakatte, Karnataka, India.

⁴Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences,

Nitte (Deemed to be) University, Deralakatte, Karnataka, India.

 

ABSTRACT:

In the present study, eco-friendly synthesis of silver nanoparticles using aqueous leaf extract of Tamarindus indica as a reducing agent were carried out to in order to enhance antimicrobial activity of extract. In addition, different factors involved in silver nanoparticle synthesis was optimized by response surface methodology via Box-Benkhen Design (BBD). Silver nanoparticle synthesis was carried out by using aqueous leaf extract of Tamarindus indica by biological reduction method. The optimisation of different factors involved in synthesis were done by response surface methodology via Box-Benkhen Design (BBD). Using BBD, investigation of effect of different factors (independent variables) such as volume of Tamarindus indica extract, temperature and concentration of silver nitrate on particle size and polydispersiblity index (responses) of silver nanoparticles were carried out. Quadratic polynomial model was used for mathematic modelling and response surface analysis were performed for understanding the factor-response relationship. Further the optimized silver nanoparticles were characterized by UV-Visible spectroscopy, FTIR spectroscopy, Scanning electron microscopy and Transmission electron microscopy imaging studies. Optimised silver nanoparticle formulation was subjected to antibacterial activity studies against Escherichia coli and Staphylococcus aureus. Production of silver nanoparticles using 5ml of aqueous leaves extract of Tamarindus indica, 0.055M silver nitrate concentration at a temperature of 50°C was found to be the optimized condition. Optimised silver nanoparticle formulation showed SPR peak at 423nm and was spherical in shape with particle size of 148±0.11 nm and PDI of 0.2±0.09. Antibacterial activity study indicated that synthesized silver nanoparticles had better antimicrobial activity than aqueous leaf extract of Tamarindus indica. The antimicrobial activity of tamarind extract was improved by formulating into silver nanoparticle by eco-friendly approach and this study highlighted the different associated factors which influences the quality of silver nanoparticles and thereby achieving successful synthesis of silver nanoparticles with the improved antibacterial effect.

 

 

 

INTRODUCTION: 

Metal nanoparticles possess large specific surface area and high surface area to volume ratio. Now a days, novel method of synthesis of metal nanoparticles are gaining lot of attention because of their unique physical and chemical characteristics which includes antibacterial activity, optical properties, electronic properties, catalytic activity and magnetic properties^1,2. Silver nanoparticles (AgNPs) have been popular in comparison with other metal nanoparticles due to its non-toxic, environmentally safe and antimicrobial properties^3,4.

 

Biological synthesis of silver nanoparticles is becoming popular as green chemistry approach, because it is simple, environmentally friendly and less costly in comparison with physical and chemical methods⁵. Biological method involves the plant/plant extracts or micro-organisms intermediated synthesis of metallic nanoparticles. Plant mediated synthesis are more advantages because they eliminate the extensive process of culturing and maintaining of the cell, causes quick reduction metal ions producing faster and stable metallic nanoparticles⁶. In addition, by changing the pH and temperature of the reaction, the size as well the shape of the nanoparticles can be altered.

 

The optimisation of variables involved in nanoparticle synthesis is very essential to obtain high yield, stability with less time, energy and material usage. Optimization can be done by studying the effect of one factor at a time (OFAT), while maintaining other factors constant, but it includes many experiments and estimation of interaction effect between the factors is not possible⁷. Therefore, in order to estimate the effect of each factor more accurately and to study the interaction effect of factors, Design of Experiment (DOE) is used as an alternative method for optimization⁸. In DoE, one of the statistical tool used widely in optimization procedures is Response surface methodology (RSM). It helps in experimental model building and exploitation of model. Through suitable design and analysis, RSM helps in studying most significant variables affecting the responses, interaction between the variables, most suitable experimental conditions and ideal level of variables which ensures maximum yield with a fixed number of experiments⁹.

 

Tamarindus indica L., (Tamarind) belonging to the family Leguminosae is a tree, whose different parts are used as analgesic, diuretic, febrifuge, anthelmintic, anti-inflammatory, antifungal and for gastrointestinal problems in various traditional system of medicines¹⁰. In northern Nigeria, Tamarind fruits or macerate of leaves with potash is used as laxative¹¹. Leaves or bark of Tamarindus indica in the form of decoction or powder or poultice were applied externally for the treatment of cuts, wounds and abscesses^12,13 and leaf decoction were used for cleaning of wound caused by guinea worm infections¹⁴. Tamarind leaves were used in the treatment of Malaria in Ghana¹⁵, Benin¹⁶ and Nigeria¹¹.

 

By considering the above facts, in this study effort has been made to synthesis silver nanoparticles by biological reduction method using the aqueous extract of Tamarindus indica leaves and investigate the optimized parameters by application of Box Benkhen Design (BBD)

 

MATERIALS AND METHODS:

Materials:

Tamarindus indica leaves were collected from local areas of Mangalore, (Dakshina Kannada district, India) in the month of August and authenticated by botanist, St. Aloysius college, Mangalore. From Sigma Aldrich Chemicals, silver nitrate was procured. For synthesis of nanoparticles distilled water was used.

 

Plant extract preparation:

Freshly collected leaves of Tamarindus indica were washed with distilled water to get rid of dust and shade dried. About 10g of finely cut leaves were stirred with 100mL distilled water for 30min. The extracts were filtered to get clear solution.

 

Synthesis of silver nanoparticles:

Synthesis of silver nanoparticles were carried out by using different volume of plant extract (1-3mL), different concentration of AgNO3 (0.01-0.10M) and at different temperature 50-80°C. Plant extracts (1-3mL) were diluted with the distilled water to make up the volume to 10mL. To the above solution, 5mL of AgNO3 solution of various concentrations (0.01M-0.1M) was added with constant stirring using a magnetic stirrer at 2000rpm for 15mins by maintaining the temperature in the range of 50-80°C. Visual inspection of colour change from yellow to brown was done in order to identify the formation of silver nanoparticles. Silver nanoparticles were centrifuged at 10,000rpm and stored till further use at 4°C in dark conditions.

 

Optimization of silver nanoparticle synthesis:

Silver nanoparticle synthesis was optimized by the Box-Behnken design using the Design Expert® version 11 software (Stat-Ease Inc, USA). Three factors include volume of Tamarindus indica extract solution, temperature and concentration of silver nitrate was selected as independent variables. Particle size and Polydispersiblity index of synthesized nanoparticles were determined as responses. Optimisation was done at three levels of the factors viz., low (-1), medium (0) and high (+1). The selected design generated 14 experimental trials which is shown in Table 1 and experiments were performed accordingly. After entry of the obtained data in BBD, to analyse the results mathematical modelling was done. Based on this quadratic second order model was chosen and data fitting with the model was analysed by ANOVA. Based on the graphical optimization technique and numerical desirability function optimised condition for silver nanoparticle synthesis was identified.

 

Table 1: Experimental design matrix showing trial runs by the application of Box-Benkhen design for optimization of tamarind leaf extract-mediated synthesis of silver nanoparticles

Runs	A: Conc of AgNO3 (M)	B: Temperature(°C)	C: Plant Extract(ml)	Particle size(nm)	PDI
1	0.055	65	3	208.37±0.10	0.19±0.05
2	0.055	50	5	148.73±0.11	0.218±0.09
3	0.055	80	5	234.67±0.20	0.298±0.11
4	0.01	50	3	265.7±0.17	0.242±0.15
5	0.1	50	3	103.07±0.16	0.276±0.11
6	0.1	80	3	161.97±0.05	0.336±0.17
7	0.01	65	5	202.25±0.19	0.385±0.09
8	0.1	65	5	160.77±0.27	0.29±0.14
9	0.055	65	3	196.1±0.15	0.194±0.16
10	0.01	65	1	208.57±0.19	0.31±0.15
11	0.055	80	1	104.93±0.11	0.27±0.17
12	0.01	80	3	202.13±0.10	0.358±0.12
13	0.055	50	1	215.4±0.16	0.222±0.11
14	0.1	65	1	125.17±0.17	0.365±0.19

All the values are expressed as mean±SD (n= 3)

 

Characterization of silver nanoparticles:

Determination of particle size and polydispersiblity index was carried out using Malvern Zeta sizer (Nano ZS, Malvern Instruments, UK) by Dynamic light scattering. The presence of surface plasmon resonance peak was observed by using double beam UV-Visible spectrophotometer (UV5704S from Electronics, India ltd) in the range of 200-800nm for confirmation of formation of silver nanoparticles. Surface and morphological characterization of optimized silver nanoparticle formulation was done by Scanning Electron Microscopy and Transmission Electron Microscopy (TEM). Transmission electron microscope JM 2100 (JEOL, Tokyo, Japan) was used for TEM. Chemical characterization of the Tamarindus indica extract and optimised silver nanoparticles was carried by Fourier Transform Infrared spectroscopy using Shimadzu FTIR 8300 spectrophotometer.

 

Investigation of antibacterial activity:

Optimised silver nanoparticle formulation was subjected for evaluation of its antibacterial potential against Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) by agar well diffusion method. For antibacterial activity studies, the bacterial isolates which were maintained on nutrient agar slants at 4°C were used. The bacterial cultures were inoculated on nutrient agar broth and placed in an incubator for a period 24hrs for their growth. Afterwards samples were centrifuged, washed with phosphate buffer saline and standardized with 10⁶ CFU per ml of broth medium. The nutrient agar medium was inoculated with 100µl of each of bacterial isolates and poured on to the agar plates. The wells were made on the agar plate. In each well, 100µl of the samples (synthesised silver nanoparticles, tamarind leaves extract and standard ciprofloxacin) were placed separately for investigation of antibacterial activity. Plates were placed in refrigerator for 2hrs followed by incubation at 37°C for 24hrs. After the period of incubation, the zone of inhibition shown by each sample were determined.

 

RESULTS AND DISCUSSION:

Synthesis of silver nanoparticles:

In recent years, silver nanoparticle synthesis by using the plant extract as reducing agents has gained lot of attention^11-15. In this study, production of silver nanoparticles is carried out using Tamarind leaves aqueous extract by biological reduction method.

 

Optimization of silver nanoparticles:

Silver nanoparticle synthesis process was optimised by BBD using Design Expert® Software Version 11 (State-Ease Inc., Minneapoils, USA). Responses chosen for the optimization are particle size (nm) and polydispersiblity index. The independent variables at three different levels, viz. low (-1), medium (0), high (+1) selected for the study includes silver nitrate concentration, temperature and volume of Tamarindus indica leaves extract. Design Expert software by BBD suggested total 14 experimental trials. Data obtained was fitted to the quadratic polynomial model and fitting analysis was done using statistical parameters.

 

The polynomial equations generated after the data modelling are given in equation (1) and (2) which showed the presence of interaction effect and curvature effect for the response variable particle size and polydispersiblity index respectively. Coefficient of correlation for particle size and polydispersiblity index was found to be 0.9271 and 0.9867 respectively along with good values of adjusted and predicted R² values.

 

Particle size = 181.27-40.96* A – 3.65 * B + 11.54 * C + 30.62* AB + 10.48 * AC +49.10* BC.....-Equation (1)

Polydispersity index = 0.1920 – 0.0035*A + 0.0380*B+0.0030 *C – 0.0140*AB – 0.0375*AC+0.0080* BC +0.0983* A2 + 0.0128* B2 + 0.0472 * C2--------------------------------------Equation (2)

 

In the above equation (1) and (2) the positive values have a synergistic effect with the response (particle size and PDI) and the negative values have an antagonistic effect on the response, where ‘A’ is concentration of silver nitrate, ‘B’ is the temperature and ‘C’ is the volume of Plant extract

 

Factor-response relationship and response surface mapping:

3D response surface plots were used for response surface analysis, which describes the presence of interaction among the variables and their effect on the responses. Fig (1) illustrates the response surface analysis plot for particle size of silver nanoparticles. Fig 1(A) explains the interaction of concentration of silver nitrate and temperature and effect on particle size. There was a complex interaction effect as observed in 3D response surface plot, where rise in silver nitrate concentration at medium level of temperature showed a slight decreasing trend on the particle size of silver nanoparticles. Fig. 1(B) explains the relationship between silver nitrate concentration and volume of tamarind leaves extract. The silver nitrate concentration and effect of plant extract on particle size exhibited a linear trend i.e with rise in silver nitrate concentration and plant extract volume caused the reduction in particle size. The effect of volume of tamarind leaves extract and temperature in combination was highly significant and as the temperature reduces with increase in volume of plant extract showed a slight decreasing trend on size of the synthesized silver nanoparticles.

 

Particles size=181.27-40.96*A–3.65*B+11.54*C +30.62*AB+10.48*AC+49.10*BC-----------Equation (1)

Poly dispersibility index=0.1920-0.0035*A+0.0380*B+0.0030*C-0.0140*AB+ 0.0375*AC+0.0080*BC+0.0983*A2+0.0128*B2+0.0472*C2----------------------------------------------Equation (2)

 

Fig 1: Response surface analysis plots- 3D response surface plots for particle size of nanoparticles; A: Influence of concentration of silver nitrate and temperature; B: Influence of silver nitrate concentration and plant extract volume; C: Influence of temperature and volume of plant extract.

 

Fig. 2 shows the response surface analysis plots for the polydispersiblity index of synthesized nanoparticles. Fig 2(A) explains the relationship between silver nitrate concentration and temperature. 3D response surface plot indicated the curvature effect, where increase in silver nitrate concentration indicated curvilinear trend with an initial declining trend of polydispersiblity index between low to mid-level, followed by sharp increase pattern between mid to high levels. The effect of temperature was prominent on the polydispersiblity index which indicated linearly declining trend i.e as the temperature decreased, there was reduction in polydispersiblity index. The relationship between volume of tamarind leaves extract and silver nitrate concentration and effect on the polydispersiblity index is depicted in Fig. 2(B), where 3D-response surface plot showed inverted tomb shape indicating the curvilinear trend of the increase in the levels of the factors from low to high values on the response. Fig 2(C) illustrates the relationship between temperature and volume of tamarind leaves extract, where there is a prominent influence of temperature on polydispersiblity index showing linearly increasing trend. Polydispersiblity index was found to be low at intermediate concentration of silver nitrate, low temperature and intermediate volume of tamarind leaves extract.

 

    

A                                                                                                                            B

 

C

Fig 2. Response surface analysis plots -3D response surface plots for polydispersiblity index of nanoparticles; A: Influence of concentration of silver nitrate and temperature; B: Influence of concentration of silver nitrate and volume of extract; C: Influence of temperature and volume of extract

 

Selection of optimized nanoparticles:

Based on numerical optimization with desirability function closer to 1, the optimised parameters for synthesis of nanoparticles were identified. Minimizing of both particle size (i.e, less than 150nm) and polydispersiblity index (i.e., less than 0.3) were set as target goals for each response variable. Optimized region with flagged point representing 0.077M silver nitrate concentration, temperature of 50°C and 4.39mL of volume of tamrind leaves extract was observed in overlay plot indicated by yellow region. Overlay plot also showed the predicted response as particle size of 126.2 nm and polydispersiblity index of 0.2 for synthesized nanoparticles. Further, optimized silver nanoparticles were subjected for validation studies and responses were evaluated. The observed values of responses were particle size of 148.0 nm and polydispersiblity index of 0.218 which was close to the predicted responses (i.e within the prediction error ± 10%).

 

Characterization of silver nanoparticles:

UV-Visible Spectroscopy Analysis:

 Silver nanoparticle was synthesized at optimised conditions of concentration of silver nitrate, temperature and volume of tamarind leaves extract and kept at room temperature for 24h. The UV spectrum scanned in the range of 300 to 800 nm for the presence of SPR peak which was observed at 423nm (Fig 3B). This was also evident by visual observation of reaction mixture which showed change in colour Tamarind leaf extract from green to brown colour when exposed to Ag+ ions (AgNO3).

A

 

B

Fig 3A: UV-Visible spectrum of Tamarind leaf extract ; 3B: UV-Visible absorption spectrum of silver nanoparticles

 

Fourier Transform Infrared spectroscopy (FTIR):

FTIR spectroscopy of the tamarind leaves extract and synthesized nanoparticle formulation are shown in Fig 4A and 4B. Results of FT-IR spectra of tamarind leaf extract (Fig. 4A) showed peaks at 3421 cm⁻¹ and 2354 cm⁻¹ which corresponds to aliphatic C-H and OH- stretching respectively. Peak at 1639 cm⁻¹ is for amide I due to C=O stretching. The FTIR spectra of synthesized silver nanoparticle (Fig.4B) showed peak at 2359 cm⁻¹ for OH stretching, peak at 3381 cm⁻¹ for aliphatic CH stretching and 1639 cm⁻¹ for amide I due to C=O stretching. The above results indicate that tamarind leaves extract contain carbonyl and hydroxyl functional groups which are probably involved in the reduction of silver ions during synthesis of silver nanoparticles and help in prevention of agglomeration of silver nanoparticles thereby improving its stability.

 

A

 

B

Fig 4A: FTIR spectrum of Tamarind leaf extract 4B: FTIR of silver nanoparticles

 

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy [TEM]

Spherical shape of silver nanoparticles were evident by the SEM images as shown in Fig.5A.

 

Fig 5A: The scanning electron microscope Fig 5B: TEM images of silver nanoparticles

 

 

Size of silver nanoparticles were nanometric range with spherical shape as visible from TEM images (Fig 5B). DLS showed that particle size distribution of optimized nanoparticle with the average diameter of 148nm.

 

Evaluation of antimicrobial activity:

Silver nanoparticles were proven to have broad spectrum of activity against fungi, bacteria including multidrug resistant bacteria ^17,18. Nano size of silver nanoparticle and larger ratio of surface area to volume make them effective bactericidal agents. Various mechanism of antibacterial action has been suggested by various authors which includes increased permeability of cell membranes, production of reactive oxygen species (ROS), interruption of replication of deoxyribose nucleic acid ^19,20,21. All the mechanism was exhibited due to the slow oxidation and release of silver ions into the surrounding medium from silver nanoparticles.

 

In comparison with the silver nanoparticle synthesized by chemical method, the plant extract mediated synthesis of silver nanoparticle has proven to be more efficacious as antibacterial agents ^22,23,24.

 

In the present study, anti-bacterial activity of synthesised silver nanoparticles, and tamarind leaves extract was investigated by agar well diffusion method against S. aureus (Fig.6A) and E.coli (Fig. 6B) at 50 µg/ml. Synthesized nanoparticle showed more zone of inhibition in comparison with the tamarind leaves extract and equally potent as standard drug. This result indicates that synthesized tamarind leaves extract mediated silver nanoparticles can be utilized as a potent antibacterial agent.

 

A

 

B

Fig 6: Agar plates showing zone of inhibition: (1) Blank (distilled water), (2) Tamarind leaves extract, (3) optimised silver nanoparticle formulation containing tamarind leaves extract 4) Ciprofloxacin standard in different microorganisms :(A) S.aureus and (B) E.coli

 

CONCLUSION:

In the present study, simple, cost effective and greener approach was utilised for silver nanoparticle synthesis by biological method using tamarind leaves extract as reducing agent. Box- Benkhen design was utilized for optimization of silver nanoparticle synthesis by observing the effect of factors i.e. silver nitrate concentration, temperature, tamarind leaves extract volume on the responses like particle size and polydispersiblity index. Optimised silver nanoparticles were spherical in shape with particle size of 148.0 nm and polydispersiblity index of 0.218. The antimicrobial activity of aqueous leaf extract of Tamarindus indica was enhanced by formulating into silver nanoparticles.

 

ACKNOWLEDGEMENTS:

We acknowledge Nitte Deemed to be University, NGSM Institute of Pharmaceutical Sciences (NGSMIPS), Mangaluru, Karnataka for providing the financial support and NGSMIPS for the facilities to perform this work.

 

CONFLICT OF INTEREST:

No conflict of interest.

 

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Accepted on 10.01.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4583-4590.