Anticariogenic Activity Of Novel Herbal Formulations (Amla , Neem ) Mediated Silver Nanoparticles - An In vitro study

Keywords

Green Synthesis ; Silver Nanoparticles; Neem Extracts; Phyllanthus Emblica.

Introduction

Azadirachta Indica (neem), Indian Lilac or margosa is one of the most well known subcontinent Indian native species, which is known for its bioactivity .Biogenic synthesis of the silver nanoparticles (AgNPs) using the plants has become one of the promising substitute to conventional chemical synthesis method. As the Silver nanoparticles have received high attention due to their extraordinary biological activities they are used in drug delivery, bio-labeling, sensing, food preservation, wound healings, water purifications and cosmetics[1, 2]. The use of extract from neem leaves has shown synergistic antibacterial effects .

As there are different methods for synthesizing silver nanoparticles , that includes chemical reduction , photochemical reduction vaporization , electrochemical reduction and thermal synthesis thus increasing its interest in developing to produce AgNPs using green synthesis and plant extracts . Several studies have reported that the silver nanoparticles which were made up of different noble metals like Ag, Cu, and Au, which can be applied to kill both resistant and non resistant bacteria [3,4]. The plant mediated green synthesis of AgNps are developing into a new branch of nanotechnology. In this way it has been less toxic and hazardous materials and environmentally benign solvents,[5] simple, rapid and cost effective,[2]consumes less energy and performs under moderate operation conditions,[6] combines the potency of both silver nanoparticles and plant active ingredients.

Plant-mediated nanoparticle synthesis, which is also commonly known as “green synthesis”, as is the most widely acknowledged way of synthesis of silver nanoparticles because of the diversified cellular metabolites present in plant extracts. Plant mediated synthesis of silver nanoparticles is most preferable over microbemediated synthesis. The latter is not feasible and requires high aseptic conditions, time taking process and long incubation periods.[ 7] The biodiversity of plants along with their phytochemical variations is the most accredited factor influencing the physical, chemical, and biological properties of biosynthesized nanoparticles as they generally get fabricated with nanoparticles during the synthesis progression.[8]

Recent research reported that AgNPs have been synthesized using a variety of natural plants from the fruit extract of Emblica officinalis[9], leaves extract of Citrus limon (Vankar and Shukla, 2012), green tea (Camellia sinensis)[10], Coffea Arabica[11], neem (Azadirachta indica)[12], Acalypha indica (Krishnaraj et al., 2012), Aloe vera plant extract[13], latex of Jatropha gossypifolia[14], root extract of Morinda citrifolia[15] etc.

In the synthesis of silver nanoparticles, plant extracts and microbes act as the reducing agents for reducing Ag+ to Ag0 and capping or stabilizing agents for preventing the aggregation of the nanoparticles.[15] Various mechanisms attributed to the antimicrobial activity shown by AgNPs, however, the exact mechanism is yet to be elucidated because the nanoparticles act on different organisms in different ways. The development of antibiotic resistance by bacterial cells upon a frequent use of antibiotics is a serious concern [16]. Recently, the promising antimicrobial potential of AgNPs against both gram-positive and gram-negative bacterial cells and their stability has attracted scientific interest. Furthermore, by the use of AgNPs, the bacterial cells are less prone to develop antibacterial resistance [17].

Aloe vera leaves which have been used as medicinal plants that possess anti-inflammatory activity, promote wound and burn-healing, UV protection, antiarthritic properties and have been noted for their antibacterial properties[16-19]. There are numerous available biologically active constituents of aloe vera leaves. These include lignin, hemicellulose, pectins which can be used in the reduction of silver ions [22]. It has been believed that large enzymes and proteins in aloe vera extract have been weakly bound to silver ions and help in the function as a complexing agent. Azadirachta indica (commonly known as neem) which is a member of the Meliaceae family used for the synthesis of silver nanoparticles. This is a medicinal plant and is used for the treatment of bacterial, fungal, viral, and many types of skin ailments since ancient times. The aqueous neem extract which is used for synthesizing various nanoparticles such as gold, zinc oxide, silver, etc. Terpenoids and flavanones are the two important phytochemicals present in the neem which play a vital role in stabilizing the nanoparticle and also act as capping and reducing agents [23]. Aqueous neem leaf extract reduces silver salt to silver nitrate, this capped nanoparticle with neem extract exhibit antibacterial activity.[24]

In the present study, the green synthesized silver nano-particle and antibacterial effect as its role in caries activity was studied by analysing the bacterial count using the disc diffusion method. Pertaining to this study the concentrations of silver nanoparticles was effective against the growth of gram-positive and gram -negative bacteria .Previously our team has a rich experience in working on various research projects across multiple disciplines [20-34] Now the growing trend in this area motivated us to pursue this project.

Materials and Methods

It was an in vitro study conducted in the month of November 2020 – December 2020 in the city of Chennai, Tamil Nadu. Plant Material And Characterisation Of The Silver Particles Leaves of Phyllanthus emblica and Azadirachta indica were collected from University Campus in the month of November from chennai, Tamil Nadu, India. Leaves of Phyllanthus emblica and Azadirachta indica were thoroughly washed under running water to remove the dirt and dust on the surface of the leaves. The leaves were air dried for 10 days and then kept in the hot air oven at 600C for 24-48 hours. These leaves were then grounded to a fine powder. 1 mM silver nitrate was dissolved in 80 ml of distilled water , to that 20 ml of filtered Amla , Neem plant extracts were added and kept in an orbital shaker for 2 days . Readings were taken using a U.V Spectrophotometer ( 250 - 750 nm ) .

The supernatants were heated at 50 to 950C. A change in the colour of the solution was observed during the heating process within 10-15 minutes. The colour changes indicate the formation of silver nanoparticles (SNPs).The reduction of pure Ag2+ ions were monitored by measuring the UV spectrum of the reduction media at 5 hours after diluting a small sample in distilled water by using systronic 118 UV Spectrophotometer as standardised in the study by Savithramma et al[35]

Antimicrobial Analysis

The antibacterial activity was assessed using a disc diffusion method. Nutrient agar medium plates were prepared, sterilized and solidified. After solidification, bacterial cultures of S. Aureus , C.Albicans , S. Mutants and E.. Faecalis were swabbed on the agar plates. The sterile discs were then dipped in silver nanoparticles solution (10 mg/ml) and placed in the nutrient agar plate and kept for incubation at 370C for 24 hours. Zones of inhibition were measured. The experiments were repeated thrice and mean values of zone diameter were presented.[36]

Antifungal Activity

The dextrose agar plates were then prepared, sterilized and solidified. After solidification fungal cultures were also swabbed on these plates. Then sterile discs were then dipped in the silver nanoparticles solution (10mg/ml) and placed in the agar plate and kept for incubation for 7 days. The zone of inhibition was measured after 7 days .

Statistical Analyses

The collected data was tabulated into Microsoft office Excel 2013 transferred to SPSS version 26.0 software (SPSS, Chicago, IL, USA) for statistical analysis. Descriptive data analysis was done to find the mean and standard deviation for the data. This data was analyzed statistically using the chi square test to see if the results were statistically significant. The confidence interval was set at 95%. Data are represented as the average values with standard error of at least three values of each independent experiment.

Results

As shown in Table 1, a minimum of 10mm zone of inhibition was observed for both C.albicansand S.mutans, whereas E. faecalis showed a minimum of 22mm and S.aureusshowed only 9mm in diameter. Study appeared to have higher results for C.albicans as compared to the antibiotic group at all concentrations and at higher concentrations for S. aureus. For E. faecalis and S. mutansthe formulations appeared to be half as effective as the antibiotic used. These preliminary data indicated that AgNPs, as well as Ag+,have antibacterial activity.

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Figure 1: Sample Preparation For Groups A,B And C. Mounted Samples On Die Stone.

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Figure 2: Brushing Simulator.

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Figure 3:Vickers Hardness Tester.

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Figure 4:Vickers Hardness Tester (Measurement Of Sample).

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Figure 5: Vickers Hardness Tester (Measurement Of Sample).

Discussion

Biologically synthesized AgNPs have been reported to be promising therapeutic molecules with significant antimicrobial and antiviral activities[37]. These are environmentally friendly processes that avoid the toxicity of chemicals. Compared to toxicity of chemicals, plant extracts contain functional substances, including cyclic peptides, sorbic acid, citric acid, euphol, polyhydroxy limonoids, ascorbic acid, retinoic acid, tannins, ellagic acid, and gallic acid, among others, are strongly believed to play a crucial role in the bioreduction and stabilization of nanoparticles[38].

Overall, this study reported green-synthesized AgNPs mediated by Azadirachta indica and Aloe barbadensis plant extracts and demonstrated good antibacterial activity.

Biosynthesis of nanoparticles employing the use of plant extract is a noble strategy for biosynthesis reaction because of their nontoxic properties and thus provide natural capping agents[39,40]. One of the probable mechanisms is that silver nanoparticles attach to the surface of the cell membrane, the respiratory function and permeability of the bacterial cells become unstable[41]. It was speculated that the hydroquinones in the aloe vera plant extract act as the reducing agents. Reactive Oxygen species (ROS) such as singlet oxygen O2, hydroxyl radical OH- and peroxide radical O2-, are produced by silver nanoparticles which are toxic to the bacteria[42]. It has been noted that the susceptibility of different types of bacteria was attributed to the structure of their bacterial cell walls.

We have investigated the zone of inhibition of AgNPs as compared against the reference antibiotics drug . This study clearly indicates that the stabilized AgNPs has excellent antimicrobial activity against gram positive organisms of S. aureus and C.albicans . In the case of C.albicans, the result shows that the antibacterial activity at 25 µL of the AgNPs is equal to antibacterial activity at 100 µL . For the case of S. aureus, the result shows an equal of 100% of the antibacterial activity of the antibiotic at 100 µL as compared to 25 µL. For E.faecalis and S.mutans the formulations appeared to be either half or slightly less effective as the reference antibiotic used even at 100 µL concentrations. Previous studies indicated that the silver ion released from AgNPs was responsible for antibacterial activity[19]. The free silver ion can then bind with the thiol groups of enzymes[43]. In a previous study[44], the antimicrobial activity of silver nanoparticles for Gram-positive bacteria was less compared to Gram-negative bacteria. Similar results have been reported earlier for neem as well as other plant extracts. This is attributed to the peptidoglycan layer which is negatively charged and prevents the free entry of Ag ions into the cell wall[45,46].

The present work supports the medicinal values of these plants also reveals a simple, rapid and economical route to synthesis of silver nanoparticles; and their capability of rendering the antimicrobial efficacy. The synthesized Silver nanoparticles enhance the therapeutic efficacy and strengthen the medicinal values of these plants.

Our institution is passionate about high quality evidence based research and has excelled in various fields [47-57].

Conclusion

Biosynthesis of silver nanoparticles using Amla , Neem herbal formulations were done. They were effective against the strains of S.Mutansat all concentrations and followed by S.Aureusat 100 microliter concentration and it has been found that it was not effective against E.Faecalis.

Acknowledgement

The authors thank the institute for providing the materials required for the study.