Comparison of antibacterial properties of zinc-oxide nanoparticles synthesized from aqueous root extracts of Rubia Cordifolia

Introduction

Nanotechnology is an evolving field of science and technology with diverse applications in various field like biomedical, industrial, drug delivery and anti-microbial treatment. The potential uses of nanoparticles (NPs) in a variety of fields are based on their unique characteristics, which include their size, shape, charge, high surface area to mass ratio and high reactivity (Kim et al., 2007) [15] . Because of the distinctive properties they possess oxide nanoparticles such as gold, silver, platinum and others have found broad use in the field of nano-medicine (Jeyaraj et al., 2013) [12] . According to Ingle et al., (2013), nanoparticles high surface area to volume ratio makes them remarkable antibacterial agents and a special kind of delivery system. Among these zinc oxide (ZnO) nanoparticles are thought to be non-toxic, bio-safe and biocompatible in a variety of applications, including semiconductors, materials with magnetic properties, electroluminescent materials, piezoelectric sensors and actuators, nano-structure varistors, field emission displaying materials, thermoelectric materials, gas sensors and cosmetic components. Nanoparticles can be synthesized by physical, chemical and biological methods. One such effective method for synthesizing nanoparticles is green synthesis. The creation of nanoparticles using green chemistry depends on the bioactive substances originating from natural resources including plants, algae and a variety of other sources. More and more studies are being conducted these days on the synthesis of nanoscale metals using physical, chemical and green synthesis techniques (Horwat et al., 2011) [7] . According to Alsammarraie et al. (2018) [3] , physical and chemical methods are gradually being replaced by green synthesis techniques. This is because of things like excessive energy consumption (Horwat et al., 2011) [7] , toxic and hazardous chemical releases. Comparing green synthesis to chemical and physical approaches provides multiple advantages: it is more affordable (Kataria and Garg, 2018) [13] , nonpoisonous (Devi et al., 2019) [6] , non-polluting (Alsammarraie et al., 2018) [3] , more ecologically friendly and more sustainable (Nasrollahzadeh and Mohammad Sajadi, 2016) [22] . Plant components such as roots, leaves, stems, seeds and fruits have also been used for the production of NPs because their extract contains an abundant number of phytochemicals that function as stabilizing and reducing agents (Agarwal et al, 2017) [2] . The Rubia cordifolia (Rubiaceae family) is a perennial climbing vine known as a medicinal plant. R. cordifolia is commonly referred to as India Madder or Madder in India. India's locals use it as "Manjistha. " (Adwankar et al 1980) [1] . In Ayurveda, R. cordifolia is externally applied to painful regions, ulcers and fractures. The root of this plant has been used to treat several kinds of prolonged swellings. It has additionally been widely used as a coloring agent for therapeutic oils. (Patil et al., 2009) [20] . Multiple pharmacological actions of R. cordifolia including immunosuppression, anti-inflammatory, anti-tumor, neuroprotective, antibacterial and antioxidant effects (Balachandran et al., 2021) [4] . ZnO's antibacterial activity has been extensively studied using a variety of pathogenic and non-pathogenic microorganisms including klebsiella, pseudomonas and S. saprophyticus. The current study is focused on green synthesis of zinc oxide nanoparticles from root extract of Rubia cordifolia and examined for its antibacterial properties against gram positive and negative bacteria.

Experimental details Materials and Methods

All chemical reagents utilized for this research work were of high purity standards (AR grade). Antibiotics discs, LB broth and LB agar medium were procured from Hi Media Laboratories Pvt. Ltd. zinc acetate dihydrate, sodium hydroxide pellets, ethanol and all other chemicals and solvents of reagent grade were procured from Sisco Research Laboratories Pvt. Ltd. (SRL).

Preparation of root extract of Rubia cordifolia

Rubia cordifolia roots were obtained from the general shop of the Rohtak market and were well cleaned under tap water. The roots of Rubia cordifolia were dried in shade and homogenized to fine powder using a mortar and pestle or electric grinder and then sieve the powder through muslin cloth. 20 grams of powder was weighed and mixed in 200ml of distilled water, then we take this mixture in water bath at 60℃ temperature for 24 hours. Next day the mixture filtered into plastic bottle with the help of Whatman filter paper and stored in freeze at -4℃till further use (Chandrashekar et al., 2018) [10] .

Biosynthesis of R. cordifolia-aqueous zinc oxide nanoparticles (RCAE-ZnONPs)

Biosynthesis of R. cordifolia aqueous Zinc oxide nanoparticles done according to the protocol modified from Naiel et al. 2022. Twenty gram of plant extract was mixed into 200 ml of double distilled water. Now 1. 31 grams of zinc acetate was added to 600 ml of double distilled water to make the zinc acetate dihydrate solution. 190ml of zinc acetate dihydrate solution was mixed with 10ml of aqueous root extract and kept on heated magnetic stirrer for two hours at 300rpm. The pH of the solution was maintained at 12 by adding 2M NaOH dropwise to achieve complete reduction and the appearance of a white precipitate. Sonicate and then centrifuge the mixture at 10, 000 rpm for 10 minutes. The final product was then collected by decantation, cleaned with deionized water to get rid of any leftovers, and oven-dried overnight at 60 °C to produce powdered ZnO nanoparticles. The dried material was kept at 4℃ temperatures in an airtight container for further use and analysis.

Characterization of ZnO NPs

Using a UV-Visible spectroscopy, the optical transmission/absorption spectra of ZnO dispersed in water was examined. The absorbance spectrum was obtained at the range of 200-800nm. The absorbance was used to measure the concentration of a solution using Beer-Lambert law. The functional groups of ZnONPs were identified using the FTIR analysis. No special FTIR sample preparation was done. The spectra were acquired in the 400-4000cm-1 wave number range in an attenuated total reflection (ATR) mode using a BRUKER FTIR Spectrometer. The synthesized samples FTIR confirmed presence of several reducing agents functional group present in the Rubia cordifolia root extract as well as production of zinc nanoparticles. Zeta Potential measurements were performed using (Zeta sizer Nano-ZS, ZEN3600, Malvern) analyzer to investigate particles' surface charge of prepared green-synthesized RCAE-ZnONPs'. The electrostatic potential of the particle was determined in room temperature deionized water ultrasonic dispersion at 0. 01 g per 100 ml. TEM analysis was done to determine the morphology, size and foam of Zn NPs.

Anti-bacterial activity

To check the antibacterial activity of zinc oxide nanoparticles, a zone of inhibition was observed around the wells. The larger the diameter of the zone of inhibition, higher the antibacterial activity of ZnO nanoparticles. RCAE-ZnO-NPs in DMSO at different concentrations (50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml) showed antibacterial activity against K. pneumonia, S. saprophyticus and P. aeruginosa. The higher concentration of ZnO caused higher bacterial inhibition (Sirelkhatim et al. 2015) [27] . Our results are in accordance with the earlier studies. In control groups, no antibacterial activity was observed but significant antibacterial activity was observed in aqueous root extract and RCAE-ZnO nanoparticles in different concentrations with a maximum zone of inhibition of 8mm and 6 mm at 200 mg/ml with Agar well diffusion assay, for K. pneumoniae and P. aeruginosa respectively. After that, a decrease/saturation in the inhibition zone was observed for other bacteria. The ZnO-NPs made in other solvents like methanol or ethanol and at some other concentrations may show more antibacterial activity against K. pneumoniae, S. saprophyticus and P. aeruginosa. Many researchers have noted that ZnO-NPs exhibit a broad range of antibacterial activity against different microbes (Sirelkhatim et al. 2015) [27] . Further, the RCAE-ZnO-NPs were coated with polyvinyl alcohol (PVA) and nanocomposites were made and their antibacterial activity was also studied by Agar well diffusion assay PVA nanocomposites also showed antibacterial activity with bacterial strains at lower concentration i. e. at 5mg/ml to 20mg/ml (table 4) compared to RCAE-ZnO nanoparticles which showed antibacterial activity at higher concentration i. e. 50 to 200 mg/ml

Result and Discussion

Visible Observations Before the addition of Zinc acetate, the colour of Rubia cordifolia solution was dark brown which changes to light brown after the addition of zinc acetate solution. The colour change indicates the formation of Rubia cordifolia zinc oxide nanoparticle (RC-ZnO NPs). The colour was changed due to a size dependent property of nanoparticles.

UV-Visible spectroscopy analysis

Nanoparticles possess exceptional optical qualities that are sensitive to the shape, agglomeration state, size, refractive index near the nanoparticle surface and concentration which makes UV-Vis an invaluable technique for the identification and characterization of nanoparticles. In the present study, UV-visible spectroscopy was used to determine the size and form of the zinc oxide nanoparticles in the aqueous extract of Rubia cordifolia. To confirm the generation of RCAE-ZnONPs', the UV-vis spectroscopy method was used. The absorbance peak of RCAE -ZnONPs was at 263 nm. The UV absorption peak of Zinc nanoparticles generally lies in the 280-380 nm range (Kebede Urge et al. 2023) [14] . This confirms that the proper shape and sized RCAE-ZnO NPs were prepared.

Zeta potential

RC-ZnO NPs were found to have a Zeta potential of -17. 5 mV in distilled water (DW), which indicates that they are strongly anionic. Particle stability of NPs is determined by the magnitude of their Zeta potential, which in turn determines the variety of applications they can be used for. Particles having a high ZP have a high level of stability (Iqbal et al., 2021) [9] .

TEM (Transmission electron microscope) analysis

Transmission electron microscopy (TEM) was performed to know morphology (size and shape) of biosynthesized RC-ZnO NPs. TEM results had shown that the diameter of prepared nanoparticles in the solution was about 12. 3 nm to 28 nm. The RC-ZnO NPs were of varied shapes like spheres, dumbbell shaped, polyhedrons, flowers, boxes, hexagonal prismatic rods and plates etc. Our results are similar to other investigations (Wahab et al. 2010) [28] who used the similar methods for preparing ZnO nanoparticles and obtained structure of spherical surface that showed high antibacterial activity against the tested pathogens. Similarly, spherical shaped ZnO-NPs in another investigation (Wahab et al. 2014) [28] were obtained and it was used for the treatment of bacteria (S. saprophyticus, P. aeruginosa and B. subtilis,) and cancer cells (MCF-7 and HepG2 cell lines).

FTIR analysis

Major functional groups that are involved in the reduction, stabilization, and synthesis of NPs can be found using Fourier transform infrared (FT-IR) spectroscopy in a variety of phytoconstituents (Maheswari et al., 2021) [17] . The FTIR spectrum of Rubia cordifolia mediated Zinc oxide nanoparticles showed that peaks were observed at 659. [27] . These results of FTIR spectra analysis of RC -ZnO NPs are in accordance with other previous studies (Iqbal et al., 2021) [9] Conclusion and Future prospectus Over the years, the "green" production of metal and metal oxide nanoparticles has become a very popular field of study in the previous ten years. A [21] So nanoparticles of zinc were prepared from plant extract of Rubia cordifolia and various techniques were used for the characterization of nanoparticles like UV-Spectroscopy, FTIR(Fourier transform infrared), Zeta potential, TEM (Transmission electron microscopy), XRD(X-Ray diffraction). These techniques gives complete information about the nanoparticles like size, shape, stability of nanoparticles. To check the antibacterial activity of nanoparticles three strains were used staphylococcus, klebsiella, pseudomonas. These three strains are pathogenic in nature causes various diseases. Petri plates were prepared and antibacterial activity was observed. Antibacterial activity was observed when zinc nanoparticles were mixed with DMSO. The diameter of zone of inhibition was increased as concentration of nanoparticles was increased. So, we can conclude that nanoparticles of zinc show antibacterial activity on the strains. Researchers revealed that nanoparticles prepared using different plant extract have variety of properties including antimicrobial, antibacterial, antioxidant. According to Salem et al., 2015 [23] the antibacterial activity was checked on Vibrio cholerae and enterotoxin Escherichia coli using zinc and silver nanoparticles using plant extract of C. procera. size of the nanoparticles was between 90nm-100nm. Antibacterial was more shown by zinc nanoparticles then silver nanoparticles. Their investigation demonstrated that zinc nanoparticles, which lower the amount of secondary messenger, a known regulator of biofilm formation, decreased adenylyl cyclase activity. subsequently, they demonstrated how a single injection of silver nanoparticles can reduce the rate of colonization by 75-100 times in baby mice containing colonized ETEC or V. cholerae. This antibacterial activity will be very useful to the medical field in developing medicine for various diseases. There are various applications used regarding antibacterial activity are: In coating of implanted devices.  In wound dressing.  In Dental material and in bone cementing. So, we can conclude that based on our experiment ZnNPs are highly effective in antibacterial activity and various other metals and strains can be used to check antibacterial activity. Antibiotics can be successfully substituted by nanoparticles, which also hold enormous potential in treating the problem of bacterial multi-drug resistance. The current in-depth analysis of antibacterial processes may aid in the creation of effective antibacterial. Nanoparticles and the avoidance of nanoparticle cytotoxicity (Wang et al., 2017). Due to limited period of time the experiment was done to some extent but this study can be further elaborated.

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