Green Synthesis, Characterization and Antimicrobial activity of Silver Nanoparticles from the Extract of Lagerstroemia speciosa

Introduction

Advances in synthesis of nanoparticles from various substances and their applications have led no research area to be remained untouched towards new verdicts day by day. Nanoparticles of different materials have shown broad applications in the field of synthesis, chemicals, pharmaceuticals, health care, optics, environmental, food, mechanical, manufacturing, materials industries and many more ^1-4. The shape and size of nanoparticles depends upon working strategies and methods of their synthesis (chemical, physical and biological) ^5-8. The chemical and physical methods are not environment friendly due to the use of toxic organic solvents, reducing/ stabilizing agents; production of hazardous by-products and high energy consumption ^9-11. Green synthesis has been identified as one of the alternate to replace these methods because of its large quantity capability, eco-friendly, cost-effective, simple work procedure^12-14.

Many researchers have proved that plants have effective potential towards green synthesis approach of metal nanoparticles ^15-17 because of secondary metabolites. The secondary metabolites include alkaloids, essential oils, flavonoids, phenols, terpenoidsetc. ^18-20 and these act as reducing and capping agent for the formation of metallic nanoparticles ^{21, 22}. The plant extract and its nanoparticles with metals (silver, gold, platinum, titanium, zinc, cerium, iron, and thallium) have shown several biological activities such as anti-arthritic, antidiabetic, anti-inflammatory, antimicrobial, anti-nociceptive, anti-obesity, anti-oxidant, cytotoxicity and so on ^23-26. However, metal nanoparticle modification of crude extract has resulted into enhanced activity as compared to crude alone ^27-30.

Lagerstroemia speciosa is commonly known as Banaba, Jarul, Queen of flowers, Crepe myrtle, Pride of India, belongs to Lythraceae family having more than 50 species ^31-33. Various parts of L. speciosa viz., fruits, leaves, bark, roots have been used as a traditional medicine to treat several diseases. The synthesis of nanoparticle from the fruits of this plant and study of their antimicrobial activities have not been reported till date. However, synthesis of silver ^{34, 35} and zirconium oxide nanoparticle ^{36, 37} from aqueous extract of L. speciosa leaves and analysis their antimicrobial, biofilm, photocatalytic and cytotoxicity activities ^34-37 have been carried out so far. In the present study, we have synthesized the silver nanoparticles from ethanolic extract of fruits of L. speciosa(contains reducing/ stabilizing agent) through green synthesis method and have compared the activities of Ee-Ls, Ag₂O, and Ls-Ag NPs. Ag NPs are found to be better biologically active to that of Ee-Ls and Ag₂O.

Materials and Methods

All the chemicals (ethanol, silver oxide and silver nitrate) have been purchased from Sigma-Aldrich. TheAgar-agar powder, Nutrient broth, and Potato dextrose agar (Hi-media) were used for microbial culture. The standard drugs, Ciprofloxacin (for antibacterial) and Fluconazole (for antifungal) had purchased from Local Retail Pharmacy Shop.

Plant materials

The fruits of L. speciosa were collected from campus garden of Maharishi Markandeshwar University, Sadopur (Ambala) in October,2018. The authentication of the plant has been confirmed from Department of Natural Products in National Institute of Pharmaceutical Education and Research (NIPER), Punjab, India. A voucher specimen number NIP-H-275 has been preserved for further verification.

Preparation of the extract

The air-dried fruits (1 kg) have been extracted with ethanol (60-70°C) by using a soxhlet apparatus for 72 hours as per standard procedure ³⁸. After the completion of extraction, liquid was concentrated at 40-60°C by rotary evaporator till the saturated mixture obtained and then evaporated on a water bath at 40-50°C to gotten crude extract. The obtained crude extract (46 g) was kept in the refrigerator at 4°C in glass vials for further analysis.

Secondary metabolites analysis of the Ee-Ls

The secondary metabolites analysis of crude extract has been doneby their specific confirmatory tests for alkaloids, carbohydrates, fat-oils, flavonoids, glycosides, phenols, proteins, saponins, steroids, tannins, and terpenoids^39-41.The presence of alkaloids, carbohydrates, flavonoids, glycosides, phenols, steroids, tannins, and terpenoids were shown in Table 1.

Preparation of the Ls-Ag NPs

Green synthesis method has been adopted to the preparation of Ls-Ag NPs. The reaction mixture was prepared by mixing 20 ml of ethanol extract with 180 ml of AgNO₃(1.0 mM) solution in a 250 ml of the conical flask for reduction of Ag⁺ to Ag⁰ions. The mixture has been stirred continuously on the hot plate magnetic stirrer at 60-70°C 1 h. The colour of solution changed from pale yellow to dark brown, indicate the nanoparticles have been prepared ^34,37.

Purification of nanoparticles

To separate the non-metal components from the Ls-Ag NPs, coloured suspension was centrifuged at 6000 rpm for 20 min. The ethanol layer of prepared Ls-Ag NPs was collected carefully and dispersed with ethanol. This process was repeated thrice to separate the entities from metal nanoparticles. The centrifugation process was done by laboratory centrifuge (REMI India). To prevent the agglomeration of the ions, the purified solution of Ls-Ag NPs was kept for sonication after treating with ethanol for 10 min³⁷.

Characterization of the Ls-Ag NPs

High-resolution transmission electron microscopy analysis

The size and shape of the prepared Ls-Ag NPs have been studied using High-resolution transmission electron microscopy. The sample has been carried out on an HRTEM, JEOL-2100 plus microscope, working at an acceleration voltage of 200 kV. It prepared by prior dispersion in ethanol at the appropriate concentration and placing a small drop of solution on a carbon-coated copper grid. After 2 min of deposition of the film on a TEM grid, the excess solution has been removed using a blotting paper and the grid was allowed to dry for overnight at room temperature to measurement.

Field emission scanning electronmicroscope analysis

The surface texture and morphology of the synthesis Ls-Ag NPs have been characterized by using Field emission scanning electron microscope, Hitachi FE 8010.A thin film of the sample has been prepared on a platinum-coated carbon tape by dropping a small amount of the sample on a grid, an extra sample has been removed, and then the film on the SEM grid was allowed to dry under a mercury lamp for 5 min.

Antimicrobial assay

The antimicrobial analysis of theEe-Ls,Ag₂O and Ls-Ag NPs have been confirmed by well-diffusion method against selected gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria as well as pathogenic fungus Aspergillus niger. For the culturing of bacterial and fungal strains, Nutrient broth (NA)and Potato dextrose agar (PDA) were used as media. Loops full of all the bacterial and fungus cultures were inoculated in nutrient at 37 ºC for 24-48 h and in potato dextrose at 27 ºC for 48-72 h^43-45.

Through serial dilution method, different concentrations (100, 200, 400 ppm) of samples Ee-Ls,Ag₂O and Ls-Ag NPs have been prepared in DMSO and compared with standard antibiotic Ciprofloxacin (100 ppm) for bacterial and Fluconazole (100 ppm) for fungal assay. The respective solvent (sterile DMSO) has been used as a negative control. The freshly prepared inoculums (10⁸ CFU/ml) of each test bacterium spread on the sterile petri plates. The plates were allowed to dry, then four wells were bored having 7 mm diameter using sterile cork-borer and were labelled properly ⁴⁶. Subsequently, 40 µL of each dilution were added in triplicate wells using microtiter-pipette and plates were allowed to stand at least 1 h for diffusion to take place. The plates were then incubated in an upright position at 37 ºC for 24 h for bacterial analysis and for fungal assay the plates have been incubated at 27 ºC for 72 h. The results were evaluated by measuring the width of the zone of inhibition growth against the selected microorganisms in comparison with antibiotics (Ciprofloxacin and Fluconazole) and mean values were tabulated.

Results

Secondary metabolites analysis of the Ee-Ls

In the present investigation, the secondary metabolites analysis has been done for Ee-Ls fruits by their specific confirmatory tests^39-41. The presence of secondary metabolites are shownin Table 1.

Table 1: Secondary metabolites analysis of the Ee-Ls.

Phytochemical constituents	Ee-Ls
Alkaloids	–
Carbohydrates	+
Fats/oils	–
Flavonoids	–
Glycosides	–
Phenols	+
Proteins	+
Saponins	+
Steroids	–
Tannins	+
Terpenoids	–

‘+’ indicates presence, ‘-‘ indicates an absence

High-resolution transmission electron microscopy (HRTEM)

HRTEM performed to acquire the size and shape of the synthesized Ls-Ag NPs, shown in Fig. 1 (1a and 1b). The average particle size of Ls-Ag NPs was calculated around50-60 nm. The eclipsed shape of Ls-Ag NPs was appeared in the characterized TEM images.

Figure 1: HRTEM images of Ls-Ag NPs from Ee-Ls.  (a) scale 20 nm, (b) scale 500 nm. Click here to View figure

Field emission scanning electronmicroscope (FESEM)

The FESEM analysis explain the surface texture and morphology of the synthesis Ls-Ag NPs shown in Fig. 2. The microscope image prove that Ls-Ag NPs are in nanoscale range and have uniform distribution.

Figure 2: FESEM image of Ls-Ag NPsfrom Ee-Ls;scale 500 nm. Click here to View figure

Antimicrobial activity

In the presence study, the antimicrobial activity of synthesis Ls-Ag NPs compared with Ag₂Oand Ee-Ls at concentration (100, 200, 400 ppm) by well-diffusion method against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacterial strains and one fungal strain viz. Aspergillusniger. The Ee-Ls, Ag₂O, and Ls-Ag NPs inhibited the growth of all tested microorganisms with a zone of inhibition range from 0.9±0.1 to 5.7±0.2mm, 3.62±0.2 to 7.3±0.2mm and 7.5±0.2 to 10±0.2mm for bacterial strains and fungal strain the range of zone of inhibition was 0.5±0.1 to 3.4±0.2mm, 1.5±0.2 to 4.3±0.2mm and 4±0.1 to 6.1±0.1mm.The Ls-Ag NPs havebeen shown effective results against selected bacterial (E. coli and S. aureus) and fungal strain (A. niger) shown in Table 2 and Fig. 3. The Ls-Ag NPs have beenexhibited the maximumzone of inhibition against gram-positive (S. aureus; 10±0.2 mm) bacterial strains compared withgram-negative (E. coli; 9.2±0.2 mm) bacterial strains at 400 ppm concentration. For fungal strain, Ls-Ag NPs have been observed maximum zone of inhibition (A.niger; 6.1±0.2 mm) at 400 ppm concentration.

Table 2: Antimicrobial activity of Ee-Ls,Ag₂O, and Ls-Ag NPs against different antimicrobial organisms

Test organisms	Different concentrations (ppm)	Ee-Ls(mm)	Ag2O (mm)	Ls-Ag NPs (mm)	Ciprofloxacin (100 ppm)	Fluconazole (100 ppm)	DMSO
Zone of inhibition
Bacterial strains
E. coli	100	0.9­­­­±0.1	4±0.2	7.9±0.2	15±0.3	—-	NO
	200	2.7±0.2	3.8±0.2	8±0.2
	400	3.5±0.2	5.6±0.2	9.2±0.2
S. aureus	100	2±0.2	3.6±0.2	7.5±0.2	15±0.3	—-	NO
	200	3.9±0.2	5±0.2	8.7±0.2
	400	5.7±0.2	7.3±0.2	10±0.2
Fungal strains
A. niger	100	0.5±0.1	1.5±0.2	4±0.1	—-	9.5±0.2	NO
	200	2.5±0.1	3.5±0.1	5.7±0.1
	400	3.4±0.2	4.3±0.2	6.1±0.2

‘NO’- not observed

Figure 3: Histogram of Antimicrobial activity ofEe-Ls, Ag2O, and Ls-Ag NPs against different antimicrobial organisms (3a) E. coli; (3b) S. aureus; (3c) A. niger Click here to View figure

Discussion

In the present study, the prelims phytochemicals i.e., carbohydrates, phenols, proteins, saponins, and tannins have been observed in Ee-Lswhile alkaloids, Fats/oils, flavonoids, glycosides, steroids, terpenoidshave been found to be absent^39-41.The secondary metabolites quantified in this analysis have a great deal of biological effects and pharmacological properties⁴⁹.These prelims phytochemicals have been observed from the different parts (fruits, leaves, and roots) of L. speciosa^53-55. Therefore, the carbohydrates, phenols, proteins, saponins, and tannins present in Ee-Ls are the most effective biomolecule in synthesis of Ls-Ag NPs^50,51.

The morphology of Ls-Ag NPs has been observed byHRTEM analysis. The Ls-Ag NPs have been detected the eclipse in shape with 50-60 nm of average particle size. The prelims constituent’s viz. carbohydrates, phenols, proteins, saponins, and tannins have been reported as potential reducing/ stabilizing agent in the synthesis of Ls-Ag NPs^{47, 48}. According to Saraswathi and Santhakumar (2017), the presence of tannins will help to cap the zirconium oxide nanoparticles and shown photocatalytic activity³⁷.Likewise, the carbohydrates content reflects the capping properties of the extract⁵². Saraswathi et al., (2017) have been reported that the formation of silver nanoparticles by using secondary metabolites (tannins, phenols and flavonoids) which act as a reducing agent during synthesis and shown biofilm activity against selected Pseudomonas aeruginosa clinical strains³⁵.

The surface texture and morphology of synthesis Ls-Ag NPs have been explained by using the FESEM analysis. The SEM images have been gathered on to the surface due to the hydrogen bond and electrostatic interactions between the bio-organic capping molecules bound to the Ls-Ag NPs⁵⁶. Similar spectacle has been reported, where the SEM morphology shows crystalline spherical Ag NPs³⁴.

The antimicrobial activity of synthesis Ls-Ag NPs compared with Ag₂O (uncapped Ee-Ls) and Ee-Ls against selected two bacterial strainsi.e., Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) and one fungal strain viz. Aspergillusniger. Result shows that the synthesisLs-Ag NPs have been enhanced antimicrobial activity than Ag₂O and Ee-Ls, similar result has been observed by Elumalai and Velmurugan (2015)⁵⁷. The possible reason due to the activity depends on the particle size, morphology, specific surface area and presence of phytochemicals components. In green synthesized Ls-Ag NPs, Ee-Ls fruits act as capping agent surround around the Ls-Ag NPs to reduce the size of particles and enriches the antimicrobial properties of particles⁵⁸.Sundararajan and kumara (2014)have been reported that the synthesized Ag NPs showed more effective antimicrobial results than that of the L.speciosa leaves extract ³⁴. Hence, we can say that the synthesis Ls-Ag NPs are the useful and effective agent against bacterial and fungal pathogens, which will be more specific and cost-effective.

Conclusion

In summary, the silver nanoparticles have been synthesised by using the green method. The analysis of secondary metabolites has revealed the presence of carbohydrates, phenols, proteins, saponins, and tannins in ethanolic extract of fruits of L.speciosa(Ee-Ls). These metabolites act as reducing agents, thereby these reduce the metal ions into nanoparticles (Ls-Ag NPs) in environment friendly manner while Ee-Ls stabilises the nanoparticles and hence belongs to green synthesis method. Their morphological studies have revealed particles size of 50-60 nm with eclipsed shape. The biosynthesized Ls-Ag NPs possess much effective antimicrobial activities against two bacterial strains: Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) and one fungal strain: Aspergillus niger as compared to Ee-Ls and Ag₂O. These findings seem to be imperative from environmental, pharmaceutical and therapeutic point of view.

Acknowledgement

We would like to thank Maharishi Markandeshwar Education Trust-Ambala (Haryana), India for their support in all respect.

Conflict of Interest

The authors declare that they have read policy and guidelines of the journal and there is no conflict of interest.

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