*Article* **Green Synthesis and Characterization of Silver Nanoparticles Using** *Spondias mombin* **Extract and Their Antimicrobial Activity against Biofilm-Producing Bacteria**

**Sumitha Samuggam 1, Suresh V. Chinni 1,\* , Prasanna Mutusamy 1, Subash C. B. Gopinath <sup>2</sup> , Periasamy Anbu 3, Vijayan Venugopal <sup>4</sup> , Lebaka Veeranjaneya Reddy <sup>5</sup> and Balaji Enugutti <sup>6</sup>**


**Abstract:** Multidrug resistant bacteria create a challenging situation for society to treat infections. Multidrug resistance (MDR) is the reason for biofilm bacteria to cause chronic infection. Plantbased nanoparticles could be an alternative solution as potential drug candidates against these MDR bacteria, as many plants are well known for their antimicrobial activity against pathogenic microorganisms. *Spondias mombin* is a traditional plant which has already been used for medicinal purposes as every part of this plant has been proven to have its own medicinal values. In this research, the *S. mombin* extract was used to synthesise AgNPs. The synthesized AgNPs were characterized and further tested for their antibacterial, reactive oxygen species and cytotoxicity properties. The characterization results showed the synthesized AgNPs to be between 8 to 50 nm with -11.52 of zeta potential value. The existence of the silver element in the AgNPs was confirmed with the peaks obtained in the EDX spectrometry. Significant antibacterial activity was observed against selected biofilm-forming pathogenic bacteria. The cytotoxicity study with *A. salina* revealed the LC50 of synthesized AgNPs was at 0.81 mg/mL. Based on the ROS quantification, it was suggested that the ROS production, due to the interaction of AgNP with different bacterial cells, causes structural changes of the cell. This proves that the synthesized AgNPs could be an effective drug against multidrug resistant bacteria.

**Keywords:** *Spondias mombin*; AgNP; biofilm bacteria

#### **1. Introduction**

Nanotechnology is a recent new branch of science that has shown a wide range of development of novel technological advancements in environmental, biochemical, biological, and other applications [1]. Silver nanoparticles with the size of 1–100 nm are commonly applied in nanotechnology and science. In recent years, silver nanoparticles (AgNPs) have generated huge interest among scientists because of their impressive protection against numerous infective microorganisms. Several different ways of synthesizing AgNPs have been reported, including physical, biological, and chemical processes [2–5]. These approaches have their own benefits and drawbacks, based on their final applications. For instance, nanoparticles (NPs) synthesized through a chemical method can be immediately available for functionality testing [6]. However, chemically synthesized NPs exhibit many possible

**Citation:** Samuggam, S.; Chinni, S.V.; Mutusamy, P.; Gopinath, S.C.B.; Anbu, P.; Venugopal, V.; Reddy, L.V.; Enugutti, B. Green Synthesis and Characterization of Silver Nanoparticles Using *Spondias mombin* Extract and Their Antimicrobial Activity against Biofilm-Producing Bacteria. *Molecules* **2021**, *26*, 2681. https://doi.org/10.3390/ molecules26092681

Academic Editors: Nagaraj Basavegowda and Annarita Stringaro

Received: 28 March 2021 Accepted: 30 April 2021 Published: 3 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

risks, including cytotoxicity, genotoxicity, carcinogenicity, and general toxicity [7,8]. On the other hand, physical methods are considered to take a longer time and are restricted to special requirements, including certain elevated temperatures or pressures, making the procedure expensive [8]. In contrast with these methods, biological methods (e.g., plant extracts, bacteria, and fungi) are known to be safe as they utilize very fewer toxic reactants or additives. This method is also considered to be rapid, simple, user-friendly, and inexpensive and includes the capability of synthesis in large quantities [9]. The synthesis of NPs using biological sources has gained interest in recent days. The application of plant extracts is highly recommended for the production of AgNPs [10]. Extracts from plant materials are high in secondary metabolites, including enzymes, polysaccharides, alkaloids, tannins, phenols, terpenoids and vitamins, which allow them to display excellent antimicrobial properties [11]. It is assumed that organic components from the leaf extract (flavonoids and terpenoids) help to stabilize the AgNPs [12].

Recently, the WHO published a list of antibiotic-resistant biofilm-producing bacteria including *Acinetobacter*, *Salmonella*, *Pseudomonas*, *Klebsiella*, *E. coli*, and *Proteus*. These bacteria cause deadly infection and are becoming resistant to most of the currently available antibiotics [13]. Multidrug resistance is the reason for biofilm-producing bacteria to contribute to chronic diseases [14]. The increasing occurrence of MDR bacteria against clinically important antibiotics has become the reason for the use of AgNPs to enhance the antibiotic effect, as AgNPs possess antibacterial, antiviral, antifungal, and also anti-inflammatory properties [15]. According to WHO, by 2050, MDR bacterial infection is predicted to kill more people than cancer and cost \$100 trillion for healthcare. High research development costs and lack of profitability have long hindered the investment in novel antibiotic discovery. Consequently, using plant-based antimicrobials as an alternative therapeutic agent for the treatment of infections caused by MDR bacteria has gained popularity recently. Medicinal plants are rich in active compounds that have antimicrobial activity, and they are generally safer to use in terms of side effects, compared to conventional antibiotics. *Spondias mombin (S. mombin)* often gains attention among the researchers, due to its antimicrobial characteristics [16]. *Spondias mombin*, from the family of *Anacardiaceae* is also best-known as ambarella. This species has been used as a traditional medicine to treat diseases like anti-inflammatory and antithrombolytic complaints [17]. Every part of the *S. mombin* plant is reported to have its own medicinal values. For example, the bark of the tree is used as a treatment for diarrhea in countries like Cambodia and the fruit of *S. mombin* is used to cure itchiness, internal ulceration, sore throats, as well as skin inflammation. Evidence suggests that *S. mombin* leaves and fruits possess high antimicrobial, antioxidant, cytotoxic, antidiabetic, and thrombolytic ability [16].

In light of the importance of *S. mombin* and biologically synthesized AgNPs, the present research was designed to study plant-mediated AgNP synthesis and the characterization and predicted antimicrobial activity to counter different infective bacteria.

#### **2. Results and Discussion**

#### *2.1. Silver Nanoparticle-Synthesis*

The color change from colorless to yellowish brown suggested the reduction of AgNO3 had occurred by using the *S. mombin* extract as a bioreducing agent. The color change proved absorption of visible light due to the excitation of the AgNP surface plasmons (Figure 1) [18]. For further confirmation of the reduction of Ag ions to AgNPs, the maximum absorbance of UV−VIS spectra of different wavelengths was obtained (Figure 1). It was observed that there was no peak obtained in the silver nitrate (AgNO3) as it did not contain any reducing agent. However, the AgNO3 with the plant extract showed maximum intensity between the wavelengths of 300 to 400 nm, confirming the role of *S. mombin* leaf extract as a reducing agent to form AgNPs.

**Figure 1.** UV−Visible spectra of *Spondias mombin* leaf extract mediated silver nanoparticles.

A study showed that in the biosynthesis of AgNPs by the reduction of Ag ions, the terpenoids in the leaf extract play a crucial role [19]. A previous study reported this function of terpenoids from *Geranium* leaves in the biosynthesis of AgNPs [20]. A similar process might have worked in the current study, where the flavonoids and phenolic compounds from *S. mombin* extract acted as a capping and stabilizing agent in the formation of NPs.

#### *2.2. Characterization of Synthesized Silver Nanoparticles*

The particle size of the nanoparticle is an important consideration in biological applications and it strongly affects the diffusion rate via biological membranes. Previous reports showed that the smaller the size of the nanoparticle, the higher its permeability, but showed increased toxicity. Thus, a suitable size is highly recommended for specific biological functions. Hence, scanning electron microscopy (SEM) was used to study the surface morphology. Figure 2 clearly shows that the AgNPs were spherical in shape with smooth edges. The mean particle size of AgNPs was 17 nm, which is the appropriate size (8–50 nm) for biological membrane permeation, and this is the tolerable range for inducing toxicity within cells.

The elemental composition was revealed by EDX analysis of the synthesized AgNPs. EDX analysis was also used to determine the amount of each element in the formation of AgNPs. Based on Figure 2E,F, two peaks were observed in the spectrum in between 2 to 4 keV. The peak that formed at 3 keV showed the existence of an elemental Ag signal, as AgNPs have optical peaks at ~3 keV due to major emission energies. Another peak indicated the presence of elemental chlorine which could have been from the plant extract. The zeta potential was found to be −11.52 mV for the synthesized AgNPs from the *S. mombin* leaf extract (Figure 3). Based on the zeta potential value of the AgNPs, it was shown the AgNPs had moderate stability. This could be due to the existence of bioactive contents in the extract. Thus, the particles might aggregate and flocculate due to the absence of a repulsive force. It was also observed that there was the presence of some weak peaks at 28◦, 54◦, 57◦ and 86◦ which might have been from the organic compound in the leaf extract (Figure 4) [21,22].

**Figure 2.** Structural characteristics of produced AgNPs. (**A**) SEM image with the scale of 200 nm; (**B**) spherical AgNPs observed using AFM; (**C**) TEM image with the scale of 50 nm; (**D**) histogram representing AgNP size distribution; (**E**) energy dispersive X-ray spectroscopy analysis with field emission scanning electron microscopy; (**F**) energy dispersive X-ray spectroscopy analysis with TEM.

**Figure 3.** The zeta potential value of synthesized AgNPs.

**Figure 4.** X-ray diffraction pattern of synthesized AgNPs. Ag peaks are marked (\*) and 2θ values are given.

#### *2.3. Antibacterial Activity of Spondias Mombin Leaf Extract*

The antibacterial activity of *S. mombin* ethanolic leaf extract for selected bacteria was studied. A ciprofloxacin commercial antibiotic disc was used as positive control whereas the 10% DMSO was employed as negative control.

A clear zone of inhibition indicates a deterrent to the bacteria from growing. Results that were obtained from this study showed that *S. mombin* leaf extract had its own antimicrobial activity as they produced a clear zone of inhibition against the bacteria tested. From the results obtained, the nanoparticles showed an equal level of antimicrobial activity towards *Enterobacter cloacae*, *Escherichia coli, Klebsiella pneumoniae* and *Salmonella typhi*. Besides that, *Vibrio cholera* showed the lowest zone of inhibition compared to the other bacteria (Figures 5 and 6). However, there was not any zone of inhibition observed when the plant extract was tested with *Lactobacillus*. This showed that the plant extract had no antimicrobial property against *Lactobacillus*. As the bacteria have proven health benefits, this *Lactobacillus* group are classified as 'generally recognized as safe' bacteria [23]. They are categorized as nonpathogenic bacteria and the most common type of lactic acid bacteria in food and feed products [24]. The resistance of *Lactobacillus* towards *S. mombin* leaf extract could serve as an alternative treatment against human pathogenic bacteria as it does not affect the normal human flora population.

**Figure 5.** Antimicrobial analysis against selected Gram-positive bacteria. A: *Staphylococcus haemolyticus*, B: *Staphylococcus epidermidis*, C: *Bacillus subtilis*, D: *Staphylococcus aureus*, E: *Streptococcus pyogens*, F: *Lactobacillus*. Comparative evaluation of selected Gram-positive bacteria vs. zone of inhibition.

**Figure 6.** Antimicrobial activity against selected Gram-negative bacteria. A: Proteus mirabilis, B: Salmonella typhi, C: Vibrio cholera, D: Enterobacter cloacae, E: Klebsiella pneumoniae, F: E. coli, G: Pseudomonas aeruginosa, H: Acinetobacter baumannii. Comparative evaluation of selected Gram-negative bacteria vs. zone of inhibition.

*Acinetobacter baumannii* also showed no zone of inhibition for positive control which was the ciprofloxacin commercial antibiotic disc. This resistance of *A. baumannii* is mainly due to the mutation in the quinolone resistance determining region of DNA gyrase [25].

The known antimicrobial mechanism of the plant extract against various bacteria was inhibiting the cell wall synthesis, accumulating in the bacterial membrane which caused energy depletion or interference with the permeability of the cell membrane. This would eventually result in mutation, cell damage and the death of the bacteria. There is a study reporting that the phenolic and flavonoid content in the plant extract is the reason for the immune-modulator organs killing the bacteria [26].

#### *2.4. Antibacterial Activity of Synthesized Silver Nanoparticles*

The antimicrobial activity of silver nanoparticles (AgNPs) synthesis from *S. mombin* leaf extract using ethanol as solvent with disc diffusion method is tabulated in Tables 1 and 2. For the positive and negative control, ciprofloxacin commercial antibiotic disc and 10% DMSO were used, respectively.

**Table 1.** Antimicrobial activity of ethanolic extract of *S. mombin* and AgNP produced with ethanolic extract of *S. mombin* counter to Gram-positive bacteria. The information is presented in the table with the mean (±SE, standard error), *p* < 0.05.


**Table 2.** Antimicrobial activity of ethanolic extract of *S. mombin* and AgNP synthesized with ethanolic extract of *S. mombin* against Gram-negative bacteria. The information presented in the table with the mean (±SE, standard error), *p* < 0.05.


A significant antimicrobial activity showed in the presence of *S. mombin* capped AgNPs against the selected bacteria. Silver nanoparticles synthesized from *S. mombin* leaf extract showed high antimicrobial activity for *Staphylococcus epidermidis* and *Salmonella typhi*. *Proteus mirabilis*, *Enterobacter cloacae*, *Escherichia coli*, *Pseudomonas aeruginosa* and showed a constitutive level of antimicrobial activity against the synthesized silver nanoparticle. Since there was no antibacterial activity observed in the plant extract against *Lactobacillus* and *Acinetobacter baumanii*, these strains were not tested with synthesized AgNPs.

When AgNPs contact with moisture, Ag<sup>+</sup> ions are released. The Ag+ ions react with nucleic acid mainly with nucleosides forming the complex of the bacteria. AgNPs accumulate and form something called a 'pit' in the bacteria's cell wall and the nanoparticles slowly penetrate the intracellular component of the bacteria. The silver particles cause the plasma membrane to detach from the cell wall. This results in a loss of DNA replication and the protein synthesis process is also inhibited which causes the death of the bacteria. In addition to that, the hindrance of biofilm formation by AgNPs is an important mechanism, as biofilm plays a crucial part in the development of bacterial resistance against common drugs [27].

AgNPs demonstrated mediocre antibacterial action in Gram-positive bacteria compared to Gram-negative bacteria, depending on the result. This is due to the Gram-positive bacteria having a thick peptidoglycan layer. This causes difficulty in spreading AgNPs across the cell wall to disrupt the cell's activity and to inhibit its growth [28]. Gram-positive bacteria are made up of 70–100 peptidoglycans layers. Peptidoglycan consists of two polysaccharides, N-acetyl-glucosamine and N-acetyl-muramic acid, interlinked with peptide side chains and cross bridges [29]. On the other hand, compared to Gram-negative bacteria, the outer membrane of Gram-positive bacteria might cause less silver to reach the cytoplasmic membrane [30]. As a result, Gram-positive bacteria displayed a higher tolerance to synthesized silver nanoparticles relative to Gram-negative bacteria.

The oxidation of AgNP releases Ag<sup>+</sup> ions, and the ions are responsible for circulation in the living organism. The production of reactive oxygen species (ROS) induces oxidative stress that damages the membrane, proteins, DNA/RNA, and lipids, which enhance the cytotoxicity in prokaryotic cells. Thus, the ROS production was analyzed in these selected Gram-positive (*S. haemolyticus*, *S. epidermidis*, *B. subtilis*, *S. aureus*, *S. pyogenes*) and Gramnegative bacterial strains (*P. mirabilis*, *V. cholera, K. pneumoniae*, *E. coli*, *P. aeruginosa*, *E. cloacae, S. typhi.*) by treating with plant extract, AgNP and ciprofloxacin to quantify the amount of ROS production in contrast to the negative control (DMSO), and the findings are represented in Figures 7 and 8. However, Figure 7 shows that the ROS level in Grampositive bacteria and plant extract showed its effect in the following order. i.e., *S. aureus, S. epidermidis, B. subtilis, S. haemolyticus,* and *S. pyogenes*. AgNP showed an excellent ROS level in all the strains as compared with ciprofloxacin. Figure 8 shows the ROS production level in the Gram-negative strains and the plant extract showed a similar level in all the strains. AgNP showed significant ROS level in *S. typhi* and *V. cholera* followed by other strains as compared with ciprofloxacin. Based on the results, it is suggested the ROS production is due to the interaction of AgNP with different bacterial cells. The interaction causes structural changes of the cell by causing toxicity by inducing oxidative stress. This affects the protein synthesis process resulting in cell death.

**Figure 7.** ROS production in selected Gram-positive bacteria. A: Staphylococcus haemolyticus, B: Staphylococcus epidermidis, C: Bacillus subtilis, D: Staphylococcus aureus, E: Streptococcus pyogenes.

#### *2.5. Cytotoxic Study*

The cytotoxicity study revealed that the highest mortality of 70% was obtained at 1.0 mg/mL. Figure 9 shows the plot of mortality percentage against the various concentrations of synthesized AgNPs. The graph showed a direct proportional relationship between the concentration of synthesized AgNPs and the rate of mortality. The LC50 of synthesized AgNPs was observed to be at 0.81 mg/mL. A study was conducted by Samuggam et al. using *Durio zibethinus* AgNPs showed the LC50 was 3.03 mg/mL [28]. Another study conducted by Shriniwas et al., reported the LC50 value of AgNPs synthesized using L. *camara* L. was 0.51 mg/mL [29]. The *A. salina* cytotoxicity depends on the AgNP size. It was stated that the cytotoxicity activity would be stronger when the size of the AgNPs was smaller [30].

**Figure 9.** The cytotoxicity rate of synthesized AgNPs using *A. salina.*

#### **3. Materials and Methods**

#### *3.1. Collecting the Plant Samples*

Healthy, disease free young leaves of *S. mombin* were accumulated from Kulim, Kedah. These samples were shade dried for 2 weeks and crushed into powder form. The fine powdered leaves were stored in an airtight container at room temperature until they were required.

#### *3.2. Preparation of S. mombin Leaf Extract*

Fifty grams of powdered plant materials and 250 mL of solvent (99.98% ethanol) were added to a conical flask. This conical flask was further positioned in the incubator at 180 rpm at 37 ◦C for 7 days. Then, the extracted components of the plant were filtered and concentrated at temperatures around 35 ◦C–40 ◦C with the help of a rotary evaporator. The concentrated extract was air dried and the leaf extract was stored at 4 ◦C.

#### *3.3. Biosynthesis of Silver Nanoparticles*

AgNPs were biosynthesized according to the method mentioned previously (15–17). A millimolar solution of silver nitrate was prepared. The mixture was mixed with the magnetic mixer until it fully dissolved the silver nitrate crystals. The AgNO3 solution was applied with five milliliters of plant extract slowly, until the hue shifted from pale yellow to brown. For 19 h in the dark room, the solution was incubated. After 19 h, the solution was centrifuged, for 15 min at 4000 rpm and the supernatant discarded. The pellet was then cleaned, scattered, and poured out into the glass of the clock with purified water. The pellet was air dried and stored for further use at 4 ◦C.

#### *3.4. Characterization of Synthesized Silver Nanoparticles*

To detect the reduction of the aqueous silver ion by scanning from 300 to 900 nm to obtain the maximum absorption strength of AgNPs, the UV−visible spectrophotometer (Beckman Coulter DU 800 Spectrophotometer, Williamston, SC, USA) was used. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the morphological and structural features of the synthesized AgNPs. SEM and TEM analysis were performed on a Hitachi, S-4300 SE, Japan and a JEM-2100F, JEOL, Japan, respectively. The EDX analysis was performed to study the elemental composition of the AgNPs. Both SEM and TEM were equipped with energy-dispersive X-ray analysis. AgNP FESEM photographs were taken under a high-energy electron beam with a working distance of 15 kV and 4.5 mm. Surface texture analysis was carried using atomic force microscopy (AFM) using Nano Scope, Ica, Vecco, Plainsview, NY, USA. The sample for AFM analysis was conducted by preparing a thin pellet of AgNPs on a glass slide which could dry for 5 min. Crystalline nature of AgNP was determined using an X-ray Diffractometer (DMAX-2500, Rigaku, Tokyo, Japan). The diffraction angle was varied from 10◦ to 90◦ at 40 kV and 100 mA with Cu Ka radiation source. The size distribution and stability of AgNPs were studied using particle size analyzer (PHOTAL OTSUKA ELECTRONICS, ELC-Z model, Osaka, Japan).

#### *3.5. Verification of the Antibacterial Activity of Synthesized Silver Nanoparticles*

Kirby−Bauer antibiotic test (disc diffusion test) was used in this project. In this test, 10 bacteria were used as the testing microorganisms which consisted of Gram positive and Gram negative. They were *Bacillus subtilis*, *Escherichia coli*, *Staphylococcus aureus*, *Enterobacter cloacae*, *Staphylococcus epidermidis*, *Klebsiella pneumoniae*, *Vibrio cholera*, *Salmonella typhi*, *Staphylococcus haemolyticus*, *Proteus mirabilis*, *Streptococcus pyogenes*, *Pseudomonas aeruginosa*, *Lactobacillus* and *Acinetobacter baumannii*. The antimicrobial activity between the AgNPs and leaf extract was analyzed by the zone of inhibition formed on the agar plates.

#### *3.6. Reactive Oxygen Species (ROS) Quantification*

The Choi et al., 2006 method was used to determine the amount of reactive oxidative species (ROS) released by the microbes. To conclude, a total of 200 mL of bacterial strain was applied with 1 mL of plant extract, AgNP, ciprofloxacin (positive control) and DMSO (negative control) and kept in 37 ◦C incubator shaker. Once 6 h of incubation had been achieved, the bacteria suspension was centrifuged at 11,000× *g* for 11 min at low temperature to obtain the pellet. The pellet was applied with 2% Nitro Blue Tetrazolium (NBT) mixture. This pellet was kept at room temperature for 60 min in dark conditions. After centrifugation of the solution, the supernatant was removed, and the pellet was rinsed twice using PBS before another centrifugation at 9000× *g* for 3 min. The obtained pellet containing cells membrane was disrupted by treating with 2 M KOH solution. A sample of 50% DMSO was combined with the solution and followed by 10 min incubation at room temperature to dissolve formazan crystals. The solution was again centrifuged and 100 μL of the supernatant was distributed to 96 well plates. The absorbance was calculated at 620 nm using ELISA reader.

#### *3.7. Cytotoxicity Study*

The cytotoxicity study was done using *Artemia salina* (*A. salina*) according to Samuggam et al. [28]. Total of 10 larvae of *A. salina* were incubated at different concentrations of AgNPs in range of 0.2 to 1.0 mg/mL in 1 milliliter of sterilized seawater. This *A. salina* was incubated for 16 h and 8 h of light and dark, respectively, at 25 ◦C for 24 h. The assay was carried out in triplicate. Based on the larval mortality percentage, the LC50 values were determined.

#### **4. Conclusions**

In conclusion, *Spondias mombin* mediated silver nanoparticles proved their antibacterial ability against biofilm-producing bacteria, *S. haemolyticus*, *S. epidermidis*, *B. subtilis*, *S. aureus*, *S. pyogenes, Enterobacter cloacae*, *Escherichia coli, Klebsiella pneumoniae* and *Salmonella typhi*. The property of antibacterial activity of this plant extract was improved by synthesizing *Spondias mombin* leaf extract with capped silver nanoparticles. The production of ROS and cytotoxicity studies suggested the interaction of AgNPs with the bacterial cells caused structural changes which led to cell death and less cytotoxicity, respectively. Thus, these synthesized silver nanoparticles have the potential to be an effective drug against biofilmproducing bacteria.

**Author Contributions:** Conceptualization, S.V.C., S.S. and S.C.B.G.; methodology, S.V.C., S.S. and S.C.B.G., P.A. and P.M.; software, S.S., B.E. and S.V.C.; validation, V.V., L.V.R., S.S. and S.V.C.; formal analysis, S.V.C., S.S. and S.C.B.G., P.A. and P.M.; investigation, S.V.C., S.S. and S.C.B.G., P.A., V.V., L.V.R. and P.M.; resources, S.V.C., S.S. and S.C.B.G. and P.A.; data curation, S.V.C., S.S. and S.C.B.G., B.E. and P.A.; writing—original draft preparation, S.V.C., S.S. and P.M.; writing—review and editing, S.V.C., S.S., B.E., V.V., L.V.R. and P.M.; visualization, S.V.C., S.S. and S.C.B.G., P.A., V.V., L.V.R. and Prasanna M; supervision, S.V.C., S.S. and S.C.B.G.; project administration, S.V.C., S.S. and S.C.B.G.; funding acquisition, S.V.C.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by FRGS, Malaysia, FRGS/1/2018/STG03/AIMST/02/1.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors are grateful to AIMST University, Malaysia for the support to successfully accomplish this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds or not available from the authors.

#### **References**


### *Article* **Biogenic Synthesis of NiO Nanoparticles Using** *Areca catechu* **Leaf Extract and Their Antidiabetic and Cytotoxic Effects**

**Shwetha U R 1, Rajith Kumar C R 1, Kiran M S 1, Virupaxappa S. Betageri 1,\*, Latha M S 2, Ravindra Veerapur 3, Ghada Lamraoui 4, Abdulaziz A. Al-Kheraif 5, Abdallah M. Elgorban <sup>6</sup> , Asad Syed <sup>6</sup> , Chandan Shivamallu <sup>7</sup> and Shiva Prasad Kollur 8,\***


**Abstract:** Nanoworld is an attractive sphere with the potential to explore novel nanomaterials with valuable applications in medicinal science. Herein, we report an efficient and ecofriendly approach for the synthesis of Nickel oxide nanoparticles (NiO NPs) via a solution combustion method using *Areca catechu* leaf extract. As-prepared NiO NPs were characterized using various analytical tools such as powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and UV-Visible spectroscopy (UV-Vis). XRD analysis illustrates that synthesized NiO NPs are hexagonal structured crystallites with an average size of 5.46 nm and a hexagonalshaped morphology with slight agglomeration. The morphology, size, and shape of the obtained material was further confirmed using SEM and TEM analysis. In addition, as-prepared NiO NPs have shown potential antidiabetic and anticancer properties. Our results suggest that the inhibition of α-amylase enzyme with IC 50 value 268.13 μg/mL may be one of the feasible ways through which the NiO NPs exert their hypoglycemic effect. Furthermore, cytotoxic activity performed using NiO NPs exhibited against human lung cancer cell line (A549) proved that the prepared NiO NPs have significant anticancer activity with 93.349 μg/mL at 50% inhibition concentration. The biological assay results revealed that NiO NPs exhibited significant cytotoxicity against human lung cancer cell line (A549) in a dose-dependent manner from 0–100 μg/mL, showing considerable cell viability. Further, the systematic approach deliberates the NiO NPs as a function of phenolic extracts of *A. catechu* with vast potential for many biological and biomedical applications.

**Keywords:** *Areca catechu*; NiO NPs; TEM; antidiabetic activity; anticancer potential

#### **1. Introduction**

For the past few years, nanotechnology has acquired marvelous impetus by creating new scientific ideas in this rapidly growing technological era [1,2]. Nanomaterials have revealed many technological insights with their tremendous applications and specific

**Citation:** U R, S.; C R, R.K.; M S, K.; Betageri, V.S.; M S, L.; Veerapur, R.; Lamraoui, G.; Al-Kheraif, A.A.; Elgorban, A.M.; Syed, A.; et al. Biogenic Synthesis of NiO Nanoparticles Using *Areca catechu* Leaf Extract and Their Antidiabetic and Cytotoxic Effects. *Molecules* **2021**, *26*, 2448. https://doi.org/10.3390/ molecules26092448

Academic Editor: Nagaraj Basavegowda

Received: 18 December 2020 Accepted: 10 March 2021 Published: 22 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

properties [3,4]. Surface morphology, characteristic size, and shape are key features for nanomaterials, which make them highly attractive and more reactive for researchers [5,6]. Biologically fabricated nanoparticles with their immense applications in various fields are growing continuously through the collaboration of different natural science sectors. The world of nanotechnology may furnish a novel resource for the evaluation and development of safer, newer, and effective drug formulations in the treatment of infectious diseases [7].

Recently, the interest in synthesizing metal oxide nanoparticles has increasingly been employed in various fields due to their potential applications in memory storage devices, photocatalytic sensors, magnetic resonance imaging, drug delivery, catalysis, and biomedicine [8]. Nanoparticles exhibits cytotoxic activity due to their higher adsorption ability over bulk materials [9]. Hence, they are used to treat various tumor and cancer cells [10]. Nickel oxide is a p-type semiconductor metal oxide possessing a band gap from 3.6 to 4.0 eV that has great importance and has received enormous consideration in research owing to its peculiar properties like large surface area, high chemical stability, good electronic conductivity, and super conductance characteristics [11,12]. Its ecofriendly nature and high reactivity makes it a potential candidate for applications in the field of magnetism, electronics, energy technology gas sensors, electrochemical super capacitors, catalysis, battery cathodes, magnetic materials, fuel cells, optical fibers, and biomedicines [13,14]. Moreover, NiO nanostructures have motivated young researchers due to their easy availability with low cost, quantum size confinement, and surface-to-volume effect [15,16]. NiO NPs are synthesized by different physical and chemical methods, namely, Sol-gel, hydrothermal, precipitation, solvothermal, etc. However, the biogenic synthesis approach has drawn the attention of researchers due to its biocompatibility and ecofriendly process, which involves green synthetic routes that are less toxic. Exploiting the potential of medicinal plants is one of the green synthesis routes, which includes algae, microorganisms, plants, etc., and is significant because the current therapeutic approaches have toxicity problems and microbial multidrug resistance issues. Metal nanoparticles have received great attention across the globe, so, in this study, we discuss and focus on metallic nanoparticles obtained by green synthesis using medicinal plants. We also discuss medicinal properties like antidiabetic and anticancer activities of synthesized nanoparticles. The biomolecules, secondary metabolites, and coenzymes present in the plants help with the easy reduction of metal ions to nanoparticles. Such nanoparticles are considered as potential antioxidants and promising candidates in cancer treatment. Thus, the synthesis of ecofriendly nanoparticles from combustion solutions is one of the simplest and easiest synthetic approaches towards uniform mixing of plant extract with precursor/oxidizing agents [17].

Plants are known for their medicinal values in terms easier availability and large number of biologically active components. *A. catechu* is one of the known fruit plants belonging to the Palmaceae family and is cultivated in most Asian countries [18]. Medicinal properties of this plant's extracts are due to the presence of various phytochemicals that are present in the different parts of the plant [19]. Perusal of the literature shows that *Areca* leaves possess more bioactive molecules, namely, arecoline, arecolidine, arecaidine, guvacoline, guvacine, and isoguvacine. Use of plant extracts for the synthesis of nanoparticles is desirable due to the various plant metabolites like polyphenols, alkaloids, phenolic acids, and terpenoids, which play a major role in the bioreduction of metal ions, yielding nanoparticles. Plant act as bioreactors in the binding and reduction of metal ions, thereby influencing the formation of nanoparticles.

In recent years, solution combustion synthesis is emerging as one of the efficient methods to produce nanomaterials with a controlled size and shape. It is also used as a rapid heating method for metal oxides synthesis. Beyond rapid heating, this green synthesis method gives good product yield in less time when compared to other conventional methods. The present study sheds light on the synthesis of highly efficient, cost effective, nanosized NiO nanoparticles by using the solution combustion synthesis method. Solution combustion synthesis is a green, efficient, simple, fast, and high-yield method. The novelty of the study is the use of Areca catechu leaf extract as a reducing and stabilizing agent

for NiO nanoparticles synthesis. Temperature plays a pivotal role here. The solution combustion reaction depends on various process parameters, and it plays a significant role in phase formation, phase stability, and physical characteristics. The reaction temperature is a crucial parameter in the synthesis of materials. The released heat of the combustion reaction fulfils the energy requirement for the formation of oxides. The presence of phytochemical constituents in the plant extract; concentration of plant extract; and reaction conditions like temperature, reducing agent concentration, reaction time, and size of nanoparticles all influence the stability of NiO NPs [20].

The size and morphology of the nanoparticles play a significant role in developing the chemical and physical properties and largely influence their existing applications. Therefore, much effort was dedicated to the fabrication of NiO NPs with different sizes and morphologies. The decrease in dimension leads to an increase in the surface area and this enhances the biological properties.

In the current study, *A. catechu* leaf extract is used as a reducing and stabilizing agent to synthesize NiO NPs. Prepared nanoparticles were characterized using XRD, SEM with EDAX, and HR-TEM. Furthermore, we investigated the cytotoxicity of NiO NPs by examining cell viability and antidiabetic activity. This study provides detailed information about the cytotoxic effects of as-prepared NiO NPs against human lung cancer cells and offers a sound basis for the clarification of its toxicity mechanisms.

#### **2. Materials and Methods**

All the chemicals were analytical grade, procured from SD Fine and Himedia Laboratory Pvt. Ltd., India, and used without further purification. The morphology of asprepared NiO NPs was observed by Transmission Electron Microscopy (TEM-1011, JEOL, Tokyo, Japan). SEM with Energy dispersive X-ray Analysis was utilized to evaluate the elemental study (Hitachi S3400n, Tokyo, Japan). X-ray diffraction examination of NiO NPs was done on a PANalytical X'Pert-PRO (Rigaku Smart Lab). UV-Visible spectrophotometer (Shimadzu UV-2450, Kyoto, Japan) was used to record electronic absorption spectra.

#### *2.1. Preparation of Areca Catechu Leaf Extract and Synthesis of NiO NPs*

*Areca Catechu* leaves were collected from the local areas near Davanagere. Freshly collected leaves were washed with double distilled water, dried, and grinded well to get fine powder. To prepare the leaf extract, 10 g of *A. catechu* leaf powder was boiled in 100 mL distilled water for 30 min at 60 ◦C. Further, the extract was filtered and dried under vacuum using a rotary evaporator.

The solution combustion method was used to synthesis NiO NPs. In a typical experiment, 10 mL of *A. catechu* leaf extract and 1 g of nickel nitrate hexahydrate Ni(NO3)26H2O) were taken in a silica crucible and placed in a preheated muffle furnace maintained at 500 ◦C. An exothermic, vigorous reaction leads to the formation of fine, black colored NiO NPs. The obtained product was kept in an airtight container for further analysis [21].

#### *2.2. Antidiabetic Activity: Inhibition of Alpha Amylase Enzyme Assay*

Pancreatic α-amylase belongs to the class of α-1,4-gluconohydrolases and is one of the important target enzymes for the conventional treatment of diabetes. It catalyzes the initial step in hydrolysis of starch to maltose and maltotriose, which are then acted upon by α-glucosidases, broken down into glucose, and enter the blood stream. Naturally available α-amylase inhibitors from medicinally important plants are shown to be very effective in managing postprandial hyperglycemia, which is a major concern in type 2 diabetes [22].

In a fresh tube, 1 mL of phosphate buffered saline (PBS) solution was mixed with 0.5 mL of different concentrations (100, 200, 300, 400, and 500 μg/mL) of samples or the standard solution, then 200 μL of 0.5 mg/mL α-amylase was added followed by 200 μL of 5 mg/mL starch solution and incubated for 10 min at room temperature. Control was taken as starch with amylase and without α-amylase. Then, the reaction mixture was stopped by adding 400 μL of Dextrose normal saline (DNS) solution, followed by heating the mixture

in a boiling water bath for 5 min, then cooling. The reaction without *A. catechu* leaf extract was used as a control. Metformin was used as a standard drug [23]. Inhibition of enzyme activity was calculated using the following formula:

% Inhibition of enzyme activity = Abs sample − Abs control / Abs sample × 100. (1)

#### *2.3. Anticancer Activity: Cytotoxicity Assay of NiO NPs*

The cytotoxicity assay of biosynthesized NiO NPs was performed against human lung cancer cell line (A549). The cell lines were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum, with antibiotics as supplements. Temperature was maintained around 37 ◦C with humidified 5% CO2 atmosphere for about 24 h. The cells were seeded in 96-well plates at a density 25×103 cells/well. Cytotoxicity of biosynthesized NiO NPs was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Here, human cancer cell lines were treated with different concentrations of NiO NPs (20 to 100 mg/mL from stock). The plate was removed from the incubator and the drug-containing media was aspirated. A total of 100 μL of medium containing 10% MTT reagent was then added to each well to get a final concentration of 0.5 mg/mL, and the plate was incubated at 37 ◦C and 5% CO2 atmosphere for 3 h. The culture medium was removed completely without disturbing the crystals formed. Then, 100 μL of solubilization solution (DMSO) was added and the plate was gently shaken in a gyratory shaker to solubilize the formed formazan [22].

The absorbance was measured using a microplate reader at a wavelength of 570 nm and also at 630 nm. The percentage growth inhibition was calculated, after subtracting the background, the blank, and the concentration of test drug needed to inhibit cell growth by 50% (IC50). Yellow color MTT dye turning to purple color due to the reduction of formazon crystals in the presence of cytotoxic activity shows in the mitochondrial succinate dehydrogenase enzyme in viable cells. The amount of 50% inhibition concentration was obtained by plotting the dose-dependent curve [24].

#### **3. Results and Discussion**

#### *3.1. XRD Analysis*

The XRD pattern of green synthesized NiO NPs from *Areca catechu* leaf extract show strong diffraction peaks at 37.23◦, 43.29◦, 62.88◦, and 75.45◦, which are assigned to the crystal planes (111), (200), (220), and (311), respectively, as shown in Figure 1, and are further well matched with JCPDS card no. 4-835. These planes indicate the formation of FCC cubical structure for NiO NPs. Further, no impurities were observed, which suggests a high purity of monophasic NiO NPs. The average crystalline size found to be 5.63 nm, calculated by the Debye–Scherer formula [25]. Moreover, the EDAX spectra of nanoparticles displayed the peaks of Ni and O, as seen in Figure 2, suggesting the chemical nature of the prepared material. The obtained profile of the synthesized nanoparticles confirmed the presence of nickel and oxygen in the nanoparticles.

#### *3.2. UV-Visible Spectral Analysis*

It is clear from the UV-Visible spectrum of as-prepared NiO NPs (Figure 3) that the maximum absorption band observed at 380 nm reveals the formation of pure NiO NPs. This absorption in the UV region can be attributed to the electronic transition from the valence band to the conduction band in the NiO semiconducting nanocrystals.

**Figure 1.** X-ray diffraction patterns revealing the crystal planes of as-prepared NiO NPs.

**Figure 2.** Energy-dispersive X-ray (EDAX) spectra depicting the chemical composition of the synthesized NiO NPs.

**Figure 3.** UV-Visible Spectrum of as-prepared NiO NPs.

#### *3.3. SEM Analysis*

The surface morphological features of synthesized NiO NPs was studied using scanning electron microscope (SEM). In Figure 4, the SEM micrographs show the agglomeration with irregularly shaped nanoparticles. It can also be seen that the particles have a hexagonal shape with some degree of agglomerations, which may be attributed to the fact that NiO nanoparticles have high surface energy and high surface tension.

**Figure 4.** SEM images showing the morphology of as-prepared NiO NPs with different magnifications.

#### *3.4. TEM Analysis*

The formation of NiO NPs was perceived in the TEM images (Figure 5), which specifies the particle size within the range of 5 to 15 nm. Further, this supports the average crystal size from the XRD pattern. Figure 5b,c represent the HR-TEM micrographs that show particles in the hexagonal and rhombohedral shapes with an interplanar spacing of 0.21 nm. The selected area electron diffraction (SAED) pattern depicted in Figure 5d indicates the presence of the (111), (200), and (220) planes of the synthesized rhombohedral NiO NPs.

#### *3.5. Antidiabetic Studies*

#### In Vitro Alpha Amylase Inhibition Method

In our digestive system, pancreatic α-amylase is a key enzyme that catalyzes the initial step in the hydrolysis of starch. It is the main source of glucose in the diet. α-amylase inhibitors are those that inhibit the amylase activity that results in the delay of carbohydrate digestion and prolongs overall carbohydrate digestion time, causing a reduction in the rate of glucose absorption and consequently reducing the postprandial plasma glucose rise.

The α-amylase inhibitor effectiveness of NiO NPs was compared with standard drug Metformin. The values were presented with graphical representation of the same in Figure 6. Alpha amylase is an enzyme that hydrolyses α-bonds of large α-linked polysaccharides such as glycogen and starch to yield glucose and maltose. α-amylase inhibitors bind to α-bond of polysaccharide and prevent the breakdown of polysaccharides in monoand disaccharide. Standard drug Metformin showed inhibitory effects on the α-amylase activity with an IC50 value of 232.12 μg/mL. Prepared NiO NPs from Areca leaves exhibited α-amylase inhibitory activity with an IC50 value of 268.13 μg/mL. As a result,

as-synthesized NiO NPs showed significant antidiabetic activity compared to Metformin. Moreover, drugs that inhibit carbohydrate hydrolyzing enzymes have been demonstrated to decrease postprandial hyperglycemia and improve impaired glucose metabolism without promoting insulin secretion of noninsulin-dependent diabetic patients. The results of in vitro studies showed that NiO NPs inhibits α-amylase activity [26].

**Figure 5.** (**a**,**b**) TEM images, (**c**) HR-TEM image, and (**d**) SAED of as-prepared NiO NPs.

**Figure 6.** Antidiabetic potential of as-prepared NiO NPs showing inhibition of α-amylase activity at different concentrations.

As-prepared NiO NPs showed a percentage inhibition of 3.35 and 19.77 at 20 μg/mL and 100 μg/mL, respectively. The IC50 value of the extract was found to be 268.13 μg/mL, whereas the IC50 value of metformin was observed to be 232.12 μg/mL (Table 1). The concentration-based inhibition was noticed and the same has been depicted in Figure 6. Metformin is a standard antidiabetic drug and is competitively and reversibly inhibiting the pancreatic α-amylase. The retardation of glucose diffusion is also due to the inhibition of α-amylase, thereby limiting the release of glucose from the starch. The inhibition of

α-amylase activity by medicinal plants might be attributed to several possible factors such as fiber concentration; the presence of inhibitors on fibers; and the encapsulation of starch and enzymes by the fibers present in the sample, thereby reducing accessibility of starch to the enzyme and direct adsorption of the enzyme on fibers, leading to decreased amylase activity. Thus, the inhibition of α-amylase activity is important to control postprandial hyperglycemia in the treatment of diabetes [27].


**Table 1.** Antidiabetic activity of NiO NPs by α-amylase (pancreatic) inhibition assay by DNS method.

#### *3.6. Cytotoxicity Studies*

The evaluation of cytotoxicity of biosynthesized NiO NPs against A549 cell line cancer cells was measured based on cellular reduction of MTT during in vitro analysis. The as-prepared NiO NPs was screened against cell lines with the respective positive control Cisplatin, as shown in the Figure 7. NiO NPs treatment enhanced the cell death and also inhibited A549 cell population in a concentration-dependent manner. After treatment with different concentrations (20, 40, 60, 80, and 100 μg/mL), the plating efficiency of A549 cells declines, as proved by the reduction in the number of cancer cells formed. Exposure of various concentrations NiO NPs shows a decline in cell survival and plating efficiency. When compared with regular cisplatin, minimum inhibition was observed at 20 μg/mL and maximum at 100 μg/mL. The viability assay of cytotoxicity of NiO NPs against the cancer cell line is shown in Figure 8. Further, the IC50 density was found to be 93.349 μg/mL. The healthy and rapidly growing cells exhibit high rates of MTT reduction to formazan while the dead or inactive cells fail to do so. Viability in the MTT assay is connected linearly with enzyme activity and indirectly to the number of viable cells. The decrease in cell viability with the increasing concentration of NiO NPs shows significant cytotoxicity to accumulate in the internal cells and higher stress, ultimately leading to apoptosis [28,29].

**Figure 7.** Graph representing the screening of anticancer activity with respect to the standard control for different concentrations of synthesized NiO NPs.

**Figure 8.** The viability assay of cytotoxicity of NiO NPs against cancer cell line (A549) treated with different concentrations of NiO NPs.

#### **4. Conclusions**

In summary, we have reported the synthesis of NiO NPs by an ecofriendly approach via solution combustion method using the *Areca catechu* leaf extract. Areca is the important plant in Asia both in an agricultural role and as a traditional medicine. Preliminary phytochemicals like phenolic compounds, alkaloids, glycosides, and tannins are wellreported in literature. The X-ray diffractogram revealed the formation of hexagonal NiO NPs with a well crystalline nature and a very fine crystallite size of 5.63 nm. Further, the morphological characteristics determined by SEM and TEM analysis disclosed a size and shape of as-prepared nanostructures. Further, the antidiabetic activity of as-prepared NiO NPs was carried out using glucose uptake by yeast cell and α-amylase inhibition, which demonstrated significant antidiabetic activity. In addition, the prepared material showed potential anticancer activity against human lung cancer cell lines. The chemical constituents of areca plant had proven diverse pharmacological actions and were used as antidiabetic and anticancer agents. Overall, the present study clearly indicated that biosynthesized NiO NPs from *Areca catechu* leaves are a promising avenue for the prevention of diabetes and cancer diseases.

**Author Contributions:** Conceptualization, S.U.R., R.K.C.R. and K.M.S.; methodology, S.U.R. and S.P.K.; software, R.V. and G.L.; validation, V.S.B., C.S. and L.M.S.; formal analysis, S.U.R., S.P.K., C.S. and A.S.; investigation, S.P.K., R.V., A.A.A.-K. and L.M.S.; resources, A.A.A.-K., A.M.E. and V.S.B.; data curation, S.U.R., R.K.C.R. and R.V.; writing—original draft preparation, S.U.R., S.P.K., and V.S.B.; writing—review and editing, V.S.B. and S.P.K.; visualization, R.V. and C.S.; supervision, V.S.B.; project administration, L.M.S., C.S. and A.S.; funding acquisition, A.A.A.-K., A.M.E. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship of Scientific Research, King Saud University through the Vice Deanship of Scientific Research Chairs.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** Authors thank the Director, Indian Institute of Science, Bengaluru, India for analytical facilities. KSP is grateful to the Director, Amrita Vishwa Vidyapeetham, Mysuru campus

for infrastructure support. CS acknowledge the support and infrastructure provided by the JSS Academy of Higher Education and Research (JSSAHER), Mysuru, India. The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs.

**Conflicts of Interest:** The authors declare that there are no conflicts of interest.

#### **References**

