Green Synthesis and Bioactivity of Copper Nanoparticles from Asparagus aethiopicus and Asparagus densiflorus

An aqueous extract of the shade-dried parts of Asparagus aethiopicus L. and Asparagus densiflorus (Kunth) Jessop was used to synthesize copper (CuO) nanoparticles (Cu NPs). UV-visible spectroscopy was performed to assess the formation of Cu NPs. Maximum absorbance was obtained between 310–360 nm, confirming the formation of Cu NPs. CuO NPs were effective against Pseudomonas aeruginosa (ATCC 27853), a gram-negative human pathogenic bacterium. The bacterial growth curve showed that the NPs inhibited bacterial growth. This study was conducted using different parts of the plant, such as the roots, shoots, fruits, and seeds of A. aethiopicus and the roots and shoots of A. densiflorus . The fruit and seeds of A. aethiopicus showed better results than the roots and shoots. Therefore, employing green synthetic processes inhibits the release of harmful by-products. Compared to traditional techniques of NP synthesis, the transition to the extracellular production of CuO NPs utilizing dried biomass appears to be economical and environmentally beneficial.


INTRODUCTION
Particles 1-100 nm in size are referred to as nanoparticles (NPs).These NPs may be formed from carbon, metal, metal oxides, or organic components.NPs show physical, chemical, and biological properties that are different from those of their parent particles on a large scale. 1 In the contemporary research, nanotechnology plays a dominant role in exhibiting anticancer and antimicrobial properties against bacteria.The development of environmentally acceptable processes for the synthesis of NPs without the use of hazardous chemicals is becoming increasingly important.Compared to larger particles, NPs display entirely new and better features.Biological techniques involving microorganisms and plant extracts have been proposed as viable alternatives to chemical methods of metal NP production.Because complex procedures such as intracellular synthesis are avoided, using plant resources in several purification processes and upkeep of microbial cell cultures for the creation of NPs may be more beneficial. 2iological techniques use microorganisms and medicinal plants to create NPs.The benefit of using medicinal plants is that they impart therapeutic characteristics to NPs during manufacturing.Plant phytocompounds give the NPs antioxidant properties, whereas plants with antibacterial activity give them additional antioxidant and antibacterial properties. 2 Metal NPs are among the types of NPs that are beneficial in biomedical applications, including imaging the delivery of biological activity, drug transport, catalysis and photo dynamics, and photothermal treatment. 3n recent years, the green production of metallic NPs has emerged as a new and exciting area of study.Creation of NPs using green methods has become more popular in recent years owing to its many benefits, including ease of use, low cost, high stability, quick production, lack of harmful by-products, and capacity to be scaled up for large-scale synthesis. 4Green synthesis to produce different metal and metal oxide NPs has drawn attention because chemical synthesis techniques result in the presence of harmful chemical species absorbed on the surface of NPs.These metal NPs are shown to be more efficient and dependable when produced using green synthesis methods. 4ecause of their unique features and use in numerous domains of science and technology, metallic NPs synthesized using green methods have attracted enormous interest over the past decades.It is superior to chemical and physical syntheses because it is more affordable, environmentally friendly, and easily scaled-up for large-scale syntheses.It also eliminates need for toxic chemical, high temperature, energyintensive processes, and synthetic production; is inexpensive; and have numerous scientific uses. 5The purpose of the study is to describe the benefit of various types of biomolecules as dependable, long-lasting, and environmentally acceptable building blocks for the synthesis of metal and metal oxide NPs. 4 For medical and biological applications, where the purity of NPs is crucial, biogenic reduction in metal precursors to create matching NPs is more cost effective, chemically free, and environmentally benign.NPs show effective antibacterial activity against harmful bacteria, including Klebsiella aerogenes and Staphylococcus aureus. 6anomaterials produced via green syntheses are crucial for the use of nanotechnology in various industries.Green nanoproducts, and their use in promoting sustainable development, are referred to as "green technologies."Chemical processes are most commonly used for creating NPs.However, chemical techniques cannot prevent the presence of harmful compounds in the synthetic pathway.Because metal NPs are frequently used in areas where people come in contact with them, there is an increasing need to develop ecologically safe NPs synthesis techniques that do not include hazardous chemicals.Environmentally friendly alternatives to chemical and physical processes for creating NPs have been proposed, including microbes, enzymes, and plant extracts. 4ompared to other biological processes, using plants for NP synthesis is advantageous because it does not require a laborious process to maintain cell cultures and can be appropriately scaled up for large-scale NP creation. 4Plant extracts are used in the quick, low-cost, and environmentally friendly biogenic production of metallic NPs.Because of their non-toxic nature and special qualities, copper NPs have been employed in several cutting-edge biomedical applications, such as in vitro and in vivo cancer treatment, gene transport, tissue engineering, and biological halflife investigations.In preclinical cancer models, nanomedicine has shown exceptional success, yet only a few anticancer nano-drugs have received an approval from regulatory agencies. 3Although just a few studies have examined the antibacterial abilities of copper NPs, they have great potential as bactericidal agents. 4tudies have shown that copper NPs outperform silver NPs in tests comparing their antibacterial properties using Escherichia coli and Bacillus subtilis.In this study, a straightforward and environmentally friendly method was used to create copper oxide NPs that included aqueous extracts of Asparagus aethiopicus and Asparagus densiflorus.Spectral and bioactivity studies were used to describe the products of CuO NPs. 3 Chemical processes such as chemical reduction, electrochemical methods, microwaveaided processes, and green chemistry are currently used to create and stabilize metal NPs.The use of plants for NP synthesis is a novel approach.This method is superior to chemical and physical methods because it is economical and environmentally friendly and can be easily scaled up for large-scale synthesis.It also avoids the use of toxic chemicals, high pressure, high energy, and high temperatures.Although bacteria and fungi can be utilized to make NPs, using leaf extract lowers the cost and eliminates the need for specialized culture preparation and separation methods. 3

Use of copper and copper oxide
Metallic NPs play an important role in plant health and nutrition as excellent antimicrobial agents and catalysts.Cu particles in plants stimulate the photosynthetic rate at low concentrations.DNA damage and lesions are treated with CuO NPs.The phytotoxic effect increases, which influences plant growth and plays a significant role in chlorophyll synthesis for the metabolism of carbohydrates and proteins.It is ubiquitously used as an antimicrobial agent cost-effective compared to other particles and is highly effective against microorganisms, such as Pseudomonas aeruginosa, S. aureus, and Candida albicans. 7sparagus based on Asparagus officinalis L. was recognized as a genus by Linnaeus in "Species Plantarum" (1753).This genus comprises more than 200 species, but the use of Asparagus species in humans is limited.Some species of Asparagus are used as ornamental plants (Asparagus asparagoides, Asparagus scandens, Asparagus plumosus, and Asparagus falcatus).A. officinalis is an important vegetable cultivated worldwide.Asparagus racemosus (known as Shatavari in Sanskrit) and Asparagus cochinchinensis are extensively used in Ayurvedic and Oriental medicine.With more than 200 species and limited usage in humans, a large number of untapped genetic resources exist within this genus.8 Asparagus aethiopicus L. is a flowering perennial monocot herb that belongs to the Asparagaceae family.An ornamental plant that grows both indoors and outdoors has solid green aerial stems, partial spines with fasciculate clustered roots, bright green needle-like leaflets, and small white or pink flowers arranged in clusters on the stems.Its fruit is a berry, spherical, red in color with slight pungent smell; each fruit contains two to a few seeds that are black in color and tough.
Asparagus densiflorus (Kunth) Jessop, commonly called the foxtail fern, is a woody perennial herb with a fern-like habit that belongs to the Asparagaceae family.It has thick fasciculate roots; narrow, flat branches; and dark green, needle-like leaves.Its axillary, bell-shaped, yellowgreen flowers droop.It forms green fruits mature to brilliant red berries contains one to three black seeds.
A. aethiopicus and A. densiflorus were differentiated and identified based on the morphological characteristics of their shoots and roots.

Shoot structure
Leaf spurs on the stem are cladode and leaves are spiny and highly reduced in both species.In A. aethiopicus, the leaf spur is mostly solitary, and the distance between the spurs is greater than that in A. densiflorus, where the leaf spur is in fascicles and the distance between the spurs is smaller.A. densiflorus stem is tapering towards the axils, which gives it a tail-like appearance, whereas the stems of A. aethiopicus are long and droopy in nature.

Roots
Thick, fibrous, and fascicular roots.Fascicular root structures are short, fleshy, watery, bulb-like structures.These roots are characteristic of both A. aethiopicus and A. densiflorus.

Collection of plant samples
The plant samples were collected from the Sri Subramanya Swamy Nursery in Vidyaranyapura, Bengaluru, India.The identification of the collected plants was cross-verified and confirmed with the help of eflora. 9,10Procured plants were stored in a greenhouse until used in the experiment.

Chemicals used
High-purity analytic grade chemicals were employed for the green synthesis of NPs.

Preparation of A. aethiopicus and A. densiflorus root and shoot extracts
The roots and shoots of A. aethiopicus and A. densiflorus were collected, surface-cleaned under running tap water, and then cleaned with distilled water several times.The plant parts were then dried in a shaded room for 21 d (A.aethiopicus) or 60 d (A.densiflorus).A. densiflorus root parts were placed in a hot air oven at 50°C for 72 h.The wet and dry weights of the plant parts were recorded.The dried shoots and roots were chopped using a secateur and powdered using a mixer grinder.Powdered samples were transferred and stored in polythene Ziplock pouches.The extraction was performed by adding 10 g of powdered root and shoot into a 500-mL beaker, followed by the addition of 200 mL of deionized water.The contents in the beaker were allowed to boil at 50°C for 1 h on a magnetic stirrer, after which it was allowed to cool down to room temperature overnight.The mixture of plant samples and water was filtered using Whatman No.1 filter paper to obtain clear solutions. 11This extract was then subjected to green synthesis.

Green synthesis of CuO NPs from root and shoot extracts
Aqueous solution of 0.2 M Cu(NO 3 ) 2 .3H 2 O was prepared and stored in brown bottles.Shoot and root extract (50 mL) was mixed with 200 mL of 0.2 M Cu(NO 3 )2.3H 2 O solution (1:4) dropwise with constant stirring.A brown precipitate of CuO NPs was obtained at the end of this procedure.The NP product obtained in the aqueous form was subjected to further studies. 12

Preparation of A. aethiopicus fruit and seed extract
A. aethiopicus fruits were collected from St. Joseph's College Campus (SJCC) garden and dried in the shade for two weeks.The fruits were washed thoroughly under running tap water, followed by washing with deionized water.The washed fruits were then squashed to separate the seeds from the fruit pulp.The separated seeds and pulp were stored in different containers.The obtained seeds were thoroughly washed to remove excess fruit pulp and dried over blotting sheets.The weights of the fruits, pulp, and seeds were recorded at each step.
Fruit pulp was boiled in 100 mL distilled water at 80°C using water bath for 10 min and was allowed to cool to room temperature.The extract mixture was filtered through Whatman No.1 filter paper and stored at room temperature until further use. 11he seeds were ground using a mortar and pestle.The seed powder (1 g) was boiled in 100 mL of deionized water for 5 min and allowed to cool to room temperature.The extract mixture was filtered through a filter paper (Whatman No.1) and stored at room temperature. 11

Green synthesis of CuO NPs from A. aethiopicus fruit and seed extract
Aqueous fruit extract (100 mL) was mixed with 4 g of CuSO4.5H2O using magnetic stirrer at room temperature for 4 h.Aqueous seed extract (20 mL) was mixed with 80 mL CuSO 4 .5H 2 O with constant stirring using magnetic stirrer at 45-50°C for 6-7 h.At the end of this step, a brownish-black product was obtained (Figure 1).The obtained product was subjected to further studies. 11

UV-Visible Spectrophotometer studies
The NP end samples obtained were subjected to UV-Visible Spectrophotometer (Thermo Scientific UV-Visible Spectrophotometer Genesys 10S) and readings of the samples were obtained. 13The readings were taken in the range of 200-500 nm.UV Visible spectroscopy was also used to confirm the formation of Cu NPs based on their optical properties (Figure 2).

Bioactivity tests
The bioactivity of the obtained NPs was assessed against bacterium Pseudomonas aeruginosa (ATCC 27853) by studying their growth curves.P. aeruginosa, a gram-negative bacterium, is an opportunistic pathogen.It causes nosocomial (hospital-borne) and communityacquired infections in humans. 14reparation of stock bacterial culture: Stored-plated cultures of P. aeruginosa were used to obtain a stock liquid culture of bacteria.For which, 100 mL of nutrient broth was prepared, autoclaved, and cooled.To this broth, 200 µL fluconazole antifungal agent was added.The stored culture was inoculated into the broth using an inoculation loop under aseptic conditions in a laminar airflow (LAF) chamber.The inoculated bacteria were incubated at 37°C for 24 h.
The nutrient broth was prepared again for the bioactivity test.To a conical flask, 15 mL of nutrient broth and 1 mL of an aqueous NP sample were added.Conical flasks containing the nutrient broth and NP samples were prepared similarly.To these conical flasks, 1 mL of P. aeruginosa culture was added under LAF with aseptic conditions.UV-Visible spectrophotometer readings were obtained.After each hour, spectrometer readings were recorded for six generations.The reading was noted and a graph was plotted.

UV-Visible Spectrophotometer studies
Absorbance readings were obtained using UV-Vis spectroscopy.The spectra were plotted against wavelength to obtain graphs for all the NPs samples.The highest absorbance of the NP samples was observed in the range of 310-360 nm (Figure 2).P. aeruginosa, as confirmed by bacterial growth curve studies at 660 nm and thus proved to be a viable alternative to contemporary antimicrobial agents.Increased oxidative stress induced by NPs and lytic degradation are the reasons for bacterial inhibition.][18] Similarly, Cu NPs synthesized from Asparagus adscendens, a native species from Pakistan to western Himalayas and northwestern India, showed antimicrobial activity against. 19owever, bimetallic alloy nanoparticles (Ag-Au alloy NPs) of A. racemosus, a wild tropical biome species, showed highest zone of inhibition against P. aeruginosa and S. aureus strains when compared to single metal nanoparticles and plant extract. 20hereas the CuO nano-rods synthesized using root extract of A. racemosus showed effective antibacterial activity against tested bacterial pathogens like Bacillus subtilis, S. aureus, E. coli, Klebsiella pneumonia, Aeromonas hydrophila, Pseudomonas fluorescence, Flavobacterium branchiophilum, Edwardsiella tarda, and Yersinia rukeri. 21u NPs play a major role in physiological function as copper is involved in immune system of animals via lymphocyte proliferation, 22 also increases skin cell migration, multiplication, and neovascularization in rapid wound healing process. 23Since Cu NPs are involved in body functions, these particles can be used in antibiotics, drug delivery, and other pharmacological formulations. 24Cu NPs are proven to have high antimicrobial activity against E. coli, S. aureus, and C albicans. 25,26Cu NPs along with polypyrrole formed nanocomposite through in situ polymerization method in water and inhibited against E. coli and S. aureus. 27Green-synthesized NPs not only performs antimicrobial functions, but is also useful in environmental remediations. 28

CONCLUSION
It was concluded that aqueous extracts of Asparagus densiflorus root and shoot, and A. aethiopicus leaves, shoots, roots, fruits, and seeds can produce CU NPs that are quite stable in solution.The green synthesis of Cu NPs was performed using aqueous extracts of A. densiflorus and A. aethiopicus.Cu NP synthesis using plant samples was confirmed by the color change of the reactions and UV spectra absorption in the range of 500-700 nm wavelength.supported the synthesis of the NPs.The bioactivity and usefulness of the systematics were ascertained.This is the first report of the synthesis of Cu NPs from A. densiflorus and A. aethiopicus.Further characterization of NPs and their potential in plant systematics are future prospects for this study.This study also opens a new avenue for the convenient synthesis Cu NPs using cultivated Asparagus spp., which could be useful for various potential applications, including drug formulation, drug delivery, and biomedical applications.