Comparative evaluation of biomedical and phytochemical applications of zinc nanoparticles by using Fagonia cretica extracts

The use of the green approach for nanoparticle synthesis yielded noticeable concern due to its eco-friendliness, cost-effectiveness, and reduced production of toxic chemicals. The current study was designed to formulate Zinc oxide nanoparticles (ZnO NPs) by using Fagonia cretica extracts, evaluating its phytochemical content, and different biological activities. Four different solvents; methanol (MeOH), n-Hexane (n–H), aqueous (Aq), and ethyl acetate (EA), had been utilized in the extracting method. ZnO NPs were successfully synthesized and characterized by UV–vis spectroscopy and scanning electron microscopy (SEM). The UV–vis spectra showed absorbance peaks between 350–400 nm range and SEM analysis revealed spherical morphology with particle sizes ranging from 65–80 nm. In phytochemical analysis, crude extracts exhibited the highest phytochemical content as they contain enriched secondary metabolites. n-hexane extract showed the highest phenolic contents while aqueous extracts showed the highest flavonoid content. Maximum free radicle scavenging activity was observed in NPs synthesized from ethyl-acetate extract with an IC50 value of 35.10 µg/ml. Significant antibacterial activity was exhibited by NPs polar solvents against K. pneumonae, E. coli, and B. subtilis. Polar solvents showed considerable antifungal potential against A. flavus and F. solani. NPs synthesized from nH extract showed potential cytotoxic activity with an LC50 value of 42.41 µg/ml against brine shrimps. A noteworthy antidiabetic activity was exhibited by nanoparticles synthesized from methanol extract i.e., 52.61 ± 0.36%. Significant bald zones were observed in nanoparticles synthesized from methanol extract rendering protein kinase inhibition. The present study highlights the significance of F. indica as a natural source for synthesizing functional nanoparticles with substantial antioxidant, antimicrobial, cytotoxic, protein kinase inhibitory, and antidiabetic properties.

Scanning electron microscopy (SEM). The SEM results are considered of great help to determine the morphology, size, and particle separation images of ZnO nanoparticles. It has been confirmed that the particles were within 100 nm for all four extracts of the aerial parts of the plants. The size was within the range of 65-80 nm at 30 kV for the synthesized ZnO NPs (Fig. 2). The shapes varied at some points but spherical was dominant and SEM analysis depicted the well scattered and combination of particles.
Phytochemical analysis. Total phenolic contents. Total phenolic content (TPC) showed the highest values in crude extracts as compared to nanoparticles (Fig. 3). A substantial effect was seen in the crude extracts.
Total flavonoid contents. The highest TFC was observed in crude extracts of the plant as compared to its synthesized ZnO NPs. The highest TFC was measured in aqueous extract i.e. 93.74 followed by methanol (84.49), n-hexane (78.87), and ethyl acetate (61. 20). The synthesized nanoparticles showed minimum TFC as compared  www.nature.com/scientificreports/ to crude extracts. For nanoparticles, the highest value was quantified in n-hexane (36.91) followed by aqueous (31.74), methanol (24.25), and ethyl acetate (18.52) (Fig. 4).
Biological activities. Antioxidant assay. The free radicle scavenging activity was highest in nanoparticles as compared to crude extracts. For nanoparticles, n-hexane showed most promising activity with an IC 50         www.nature.com/scientificreports/ n-hexane and aqueous extracts showed clear zones i.e., 6 and 7 mm respectively. A moderate protein kinase inhibition was exhibited by the nanoparticles. Maximum bald zone of inhibition was observed by methanol extract mediated ZnO NPs (15 mm), followed by aqueous extract mediated ZnO NPs (11 mm), n-hexane extract mediated ZnO NPs (9 mm) and ethyl acetate extract mediated ZnO NPs (9 mm) (Fig. 7, Table 5). Table 3. Antifungal activity of F.cretica crude extracts and its ZnO nanoparticles. *DMSO = negative control.

Plant name Extract name Sample
Antifungal activity

Discussion
Throughout history, traditional medicine is considered the preferred primary health care system globally. Approximately 60% of the total world's population and about 80% of developing countries rely on medicinal plants as a basic health care system. Herbal medicines have gained importance due to certain reasons including accessibility, efficacy, and affordability 24 . The therapeutic value of medicinal plants is relatively high because of the existence of bioactive phytochemical constituents. Fortunately, nature has gifted Pakistan with rich flora and its varied climate condition supporting the growth of nearly 6000 species of higher plants. Twelve and a half percent of plant species are reported for their medicinal values and their number is constantly increasing based on the interest of local researchers in natural products 6 .
Nanoparticles especially metallic nanoparticles have drawn attention in various fields like medicine, electronics, diagnostics, photonics, environment, and agriculture. Nanoparticle synthesis mediated by physical and chemical approaches is subjected to the toxic use of chemicals that have a higher risk of causing environmental toxicity and inducing carcinogenicity 25 . The synthesis of nanoparticles using biological entities as an effective method; gained significant importance by the use of microorganisms and plants residing medicinal value. www.nature.com/scientificreports/ However, the green approach for the synthesis of ZnO NPs is nontoxic, cost-reducing, and biocompatible 26 . ZnO NPs are extensively used in several commercial products as well as in biological and medical applications 27 .
Fagonia cretica is commonly known as 'Dhamaasaa'and possesses a well-acknowledged medicinal stature. The subject plant turned out to be effective against fever, toothache, asthma, scabies, stomach troubles, tumors, and urinary discharges 28 and also reported for its antimicrobial, anti-inflammatory, anti-hemorrhage, thrombolytic and antioxidant properties 9 . Very limited data is available on the synthesis of zinc nanoparticles using F.cretica plant extracts.
In the present study, ZnO NPs were synthesized first time using F.cretica extracts (aerial parts). A change in color i.e., to off-white, indicated the synthesis of ZnO NPs. UV and SEM characterized the synthesized nanoparticles. It is reported that UV-Vis spectroscopy can be used to examine the form and length of nanoparticles 29 . The absorption peaks for ZnO NPs were observed between 350-400 nm which are characteristics of ZnO nanoparticles. SEM analysis was used to determine the morphological attributes of the ZnO NPs. SEM analysis revealed the formation of fine, clear ZnO NPs, as well as an agglomeration of the particles, spherical in shape and particle size, ranging from 65-80 nm. These results are in accordance with the previous reports of 30 and 31 on the plant-based synthesis of ZnO NPs.
Phytochemicals are very important for the treatment of several degenerative abnormalities 32 . The results of phytochemical analysis and biological activities of F. cretica (aerial parts) crude extracts and nanoparticles revealed that high levels of flavonoids were found in n-hexane crude extracts as compared to nanoparticles (Fig. 3). The highest TFC values were measured in crude aqueous extracts in comparison with ZnO NPs (Fig. 4).
The results of the present study are supported by 33,34 .
DPPH free radical scavenging activity results revealed that nanoparticles synthesized from ethyl acetate extract showed maximum scavenging activity i.e. 67.79% with IC 50 of 35.10 µg/ml (Fig. 4) as compared to crude extracts. The crude extracts were unable to show notable free radical scavenging potential at optimized concentrations. Similar results were observed by [35][36][37][38][39][40] while working with NPs of different plant species.
Alpha-amylase inhibition assay was performed for screening the anti-diabetic activity of F.cretica crude extracts and nanoparticles. α-amylase inhibition assay results showed that nanoparticles of methanolic extract showed high α-amylase inhibitory activity with 52% inhibition as compared to the crude extracts (Fig. 8). Zinc plays a prominent role in insulin action and carbohydrate metabolism Moreover, the antidiabetic activity of ZnO NPs synthesized from plants has also been reported by 41 who found increased antidiabetic activity in nanoparticles. The crude extracts and synthesized ZnO nanoparticles of F.cretica were also screened for protein kinase inhibition potential. The results showed that nanoparticles synthesized from methanolic extract showed a remarkable bald zone of inhibition (Table 5, Fig. 7) in comparison with the crude extracts. The anticancer activity of ZnO nanoparticles has already been reported by 42

in plants.
Antibacterial activities against K. pneumoniae, E. coli, B. subtilis, S. aureus, and P. aeruginosa have been evaluated. Samples that exhibited a significant zone ≥ 12 mm were subjected to MIC determination. The present study results showed that NPs displayed potential antibacterial activity against K. pneumoniae and B. subtilis ( Table 2, Fig. 6). Our results are in accordance with the previously reported results of 42 . The crude plant extracts and synthesized nanoparticles were assessed for antifungal activity. Antifungal activity against A. flavus, A fumigatus, Mucor, and F.solani was investigated. The results showed that ZnO nanoparticles synthesized from polar solvent extracts showed significant antifungal activity against A. flavus and F. solani (Table 3) as compared to crude extracts. These findings are in contrast with the statement that plant extracts from non-polar solvents exhibit strong antimicrobial potential as compared to polar extracts 43 . Antifungal activities of ZnO NPs from different plants have been reported widely 42,44 .
Brine shrimp lethality assay has been thought of as an effective method for the evaluation of safety and toxicity profile of plant extracts and also determine their pharmacological activities 45 . The cytotoxic effect of F. cretica crude extracts and synthesized nanoparticles indicated that nanoparticles synthesized from n-hexane showed a substantial cytotoxic effect in comparison to the crude extracts (Table 4). These results are supported by a similar study on different green synthesized nanoparticles by 46 .

Plant sample collection and identification. Fresh plants were collected from the Punjab province of
Pakistan. An expert taxonomist Professor Dr. Mushtaq Ahmad at the Department of Plant Sciences, Quaidi-Azam University, Islamabad, authenticated the plant as Fagonia cretica and its specimen preserved in the Department's herbarium for future reference (ACC 543220). The aerial parts were separated and washed to clean debris and dried in shade. These dried parts were crushed with a pestle and mortar. The fine powder was stored separately for further use.

Plant extract formulation.
Extract of dehydrated F. cretica aerial parts was formulated through a simplified maceration process as explained by 47 . Four solvents, non-polar to the polar range, were used i.e., n-hexane (nH), ethyl acetate (EA), methanol (MeOH), and aqueous (Aq). For three days, 100 g of the powdered plant was soaked in 600 ml of each solvent. The soaked plant material was periodically sonicated at a 25 kHz frequency. After the specified period, filtration was done and re-extraction was done with the same solvent. Using a rotary evaporator, all filtrates from the respective solvents were mixed and left to dry. After being thoroughly dried, these crude extracts were kept at − 80 °C. Similar procedure was adopted for each solvent. www.nature.com/scientificreports/ heated extract. The mixture was constantly stirred for two hours at 50-60 °C on a hot plate. The color change was observed after 2 h which was the first visual confirmation of the synthesis of ZnO NPs and the extract was left to cool. The extract was transferred to Petri plates and spread as a very thin layer. The plates were left to dry overnight in a drying oven at 60 °C. The fine and dried powder was ready for the characterization procedure. The same process was repeated for each extract.

Characterization of zinc oxide nanoparticles. UV-visible spectroscopy. UV-Vis spectroscopy is a
widely utilized method for characterizing nanoparticles 49 . It adheres to the Beer-Lambert law 50 . The characterization of zinc oxide nanoparticles was done using a wavelength of 350-400 nm. The material was examined using a spectroscope, and the spectra were monitored between 300 and 700 nm with a 1-nm resolution.
Scanning electron microscopy analysis (SEM). SEM (KYKY-EM6900) examination was utilized to examine the shape and size of nanoparticles on a micrometer to nanometer level 51 . Zinc oxide nanoparticles were evaluated by putting a droplet of sample solution onto a grid evenly covered with carbon, then dehydrating it beneath the mercury lamp for 15 min at 30 kV. It was examined and photographed. Finally, the instrument was equipped with an energy dispersive spectrum (EDS) to ensure nanoparticle presence.
Phytochemical analysis. Total flavonoid concentration assessment. The complete flavonoid concentration was assessed as per the methodology of 52 . The extracts/samples (20 µl) were mixed with 10 µl potassium acetate, 10 µl aluminum chloride, and 160 µl of distilled water in 96 well plates. This mix was subsequently incubated for half an hour at room temperature. The absorbance was observed at a 405-nm wavelength on a microplate reader. To evaluate total flavonoid concentrations in equivalence to quercetin, the standard curve was constructed using quercetin solutions at values of 2.5-40 µg/ml. 20 µl of respective solvents were used as a negative control.
Total phenolic concentration assessment. Utilizing the Folin-Ciocalteu reagent, the complete phenolic content was measured according to the methodology of 52 . Plant extracts and standard solutions were made at a concentration of 1 mg/µl. A portion of 200 µl was loaded in a 96-well plate, along with 90 µl of Folin-Ciocalteu reagent, which had been stirred thoroughly. The solution was incubated at room temperature for 5 min before adding 90 µl of sodium carbonate and mixed thoroughly using a plate shaker. This resultant mixture was then incubated for a 60-min at room temperature before being measured using a microplate reader at a 630-nm wavelength. Gallic acid (3.125-25 µg/µl) was used for plotting the standard calibration curve. Gallic acid equivalents in percentage weight by weight were used to express the total phenolic content. As a negative control, 20 µl of the respective solvents were utilized.
Biological activities. The following biological activities of F. cretica were performed.
Antibacterial assay. The disc diffusion technique was used to analyze the antibacterial properties of each test extract in vitro as explained by 53 . Five bacterial strains i.e., two gram-positive bacteria namely Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633), and three gram-negative bacteria namely Pseudomonas aeruginosa (ATCC-15442), Escherichia coli (ATCC 15224), and Klebsiella pneumoniae (ATCC-1705) were used to analyze the antibacterial activity of plants extracts. On nutrient agar plates, a bacterial lawn was created using a fresh culture of strains of bacteria with a seeding density of 1 106 CFU/ml. Each test extract (5 µl from 20 mg per milliliter DMSO) was impregnated on sterile filter paper discs, with Cefaxime and roxithromycin (5 µl from 4 mg per milliliter DMSO) serving as positive controls and DMSO (5 µl) serving as the negative control. After 24 h of incubation at 37 °C, these discs were put on appropriately labeled seeded agar plates, and inhibition zones surrounding every disc were determined by measuring. This test was performed three times and the mean value was determined with the standard deviation. The MIC was obtained using the technique described by 53 . The MIC of samples with significant inhibition zones, i.e. 12 mm, was determined using the micro broth dilution technique. Each strain of bacterial inoculum was made using a density (5104 CFU/ml) which was adjusted beforehand. In a 96-well plate, threefold sequential dilutions of every experiment sample were made utilizing nutrient broth up to final concentrations of 100, 33.33, 11.11, and 3.70 µg per milliliter. Bacterial cultures were rehydrated in broth culture for 11 h before being kept at 4 °C in the refrigerator.
Antifungal assay. The antifungal test was performed according to the description of 53 . Aspergillus fumigatus (FFBP 66), Mucor species (FFBP 0300), Fusarium solani (FFBP 0291), and Aspergillus flavis (FFBP 0064) were all tested for antifungal activity. All fungal strains were cultured at 28 °C on 6.5 percent SDA (Sabouraud dextrose agar, pH 5.7) then stored in the refrigerator at 4 °C. The standard treatment was clotrimazole (4 mg/ ml), while the negative control was DMSO. SDA plates holding 25 ml media were infected with 100 µl of fungal inoculum that had been renewed. On seeded SDA plates, sterile filter paper discs containing test extracts (5 μl, 20 mg/ml DMSO), DMSO (5 µl), and clotrimazole (5 μl, 4 mg/ml DMSO), had been inserted. These inoculated plates were placed for 24-h incubation at 30 °C, and the inhibition zones surrounding each disc were calculated in millimeters (mm).
Brine shrimp cytotoxicity assay. In a narrow rectangle pan ( www.nature.com/scientificreports/ plastic separator with several holes were placed inside the pan. The eggs (about 25 mg) were dispersed inside the bigger section, which was shaded with aluminum foil while the other section was lighted. Phototropic nauplii (brine shrimp larvae) were collected by pipetting from the lighted side after being detached from their shells through the separator after one of the emergences. The cytotoxicity experiment was carried out on a 96-well plate with different alphabets (A-H). 44 µl of seawater was poured into wells A and E of the microwell plate. Twenty-five microliters of seawater were poured into B, C D, F, G, H, and 6 µl of sample in A and E. 25 µl of the sample was taken from A and poured into well B, and from well B, 25 µl was added in well C and the same process was repeated for D, and 25 µl from D was discarded. This was done to ensure uniform dilution values. The same steps were repeated for E, F, G, and H and from H to discard. Ten shrimps were transferred into each well of the microplate and the quantity was completed by adding 300 µl seawater in all wells and kept for 24 h. The survival of larvae was observed under a microscope. The test was done three times, and Abbott's method was used to compute the percentages of dead larvae.
Free radical scavenging property. The 2, 2, diphenyl-1-picrylhydrazyl (DPPH) test was used to determine the free radical scavenging property. The DPPH free radical test was performed using the methodology of 52 . DPPH of 9.6 mg was dissolved in 100 ml of methanol to make a solution of DPPH. The tested samples were prepared as 4 mg per milliliter in Dimethyl sulfoxide. Standard ascorbic acid was formed in DMSO as 1 mg/ml. In each well of a 96-well plate, an aliquot of 10 µl of the test material was introduced, accompanied by 190 µl of DPPH reagent. The mixtures were subsequently stirred and put for incubation at 37 °C for an hour in no light. Absorbance was calculated at 515 nm by an ELISA plate reader. DMSO was used as a negative control and ascorbic acid (ASA) as a positive control. The experiment was repeated three times for each test sample with IC 50 values derived using table curves software, and percentage inhibition was computed using the following equation.
where; "Ac" is Absorbance of negative control and "As" is Absorbance of the experimental sample.
Enzyme inhibition assays. Protein kinase assay. In this assay, hyphae formation was observed in the purified strain of Streptomyces 85E according to the methodology designated by 54 . By distributing spores (mycelia fragments) from a fresh Streptomyces culture on sterilized plates with limited ISP4 media, a bacteria lawn was cultured. On sterilized 6-mm filter paper discs, about 5 μl of every extract (20 mg per milliliter of dimethyl sulfoxide) was poured. The impregnated paper discs were put directly on top of the plates inoculated with Streptomyces 85E at the peak ratio of 100 µg per disc. Discs injected with dimethyl sulfoxide and Surfactin were employed as negative and positive controls, correspondingly. These plates were put for a 3-day incubation at 30 °C (This is the time taken by Streptomyces 85E to produce hyphae), and the findings were assessed as a bald inhibition zone surrounding the samples and controls inserted discs.
α-amylase inhibition assay. The anti-diabetic capacity of sample extracts was assessed using the standard -amylase inhibition assay with slight modifications 55 . In a 96 well plate, a reaction mix comprising 25 µl of amylase (0.14 U/ml), 150 µl of phosphate buffer (pH 6.8), 40 µl of starch solution (2 mg per liter in potassium phosphate buffer), and 10 μl sample (4 mg per milliliter Dimethyl sulfoxide) was put for incubation at 50 °C for half an hour before being inhibited by 20 µl of 1 molar solution of hydrochloric acid. Following this, the individual well was filled with 90 µl of iodine solution (5 mM iodine, 5 mM potassium iodide). There were no extracts of plants in the negative control, while the blank was produced with no amylase and plant extract and both were substituted with equal amounts of the buffer. As a positive control, 250 μM acarbose was employed. After incubating, the absorbance of this reaction plate was assessed at 540 nm. The performance was measured in percent α-amylase inhibition per milligram dry extract. It was subsequently determined using this formula: where Ob = Blank well Absorbance, Os = Sample Absorbance, and On = Negative Control Absorbance.
Ethics approval and consent to participate. No ethical considerations apply to this paper.
Experimental research on plants, including the collection of plant material complied with the relevant institutional, national, and international guidelines and legislation.

Conclusion
The present study reported a simple and successful synthesis of ZnO nanoparticles by using Fagonia cretica extracts. The resultant nanoparticles were characterized using UV-vis spectroscopy and Scanning electron microscopy (SEM). The UV-visible spectrum showed the characteristic peaks for ZnO nanoparticles ranging from 350-400 nm. SEM analysis disclosed that nanoparticles were spherical in appearance with particle dimensions ranging from 65-80 nm. Results of the present study revealed that the phytochemical content (phenols and flavonoids) of pure plant extract is higher than those of synthesized nanoparticles. Moreover, the study demonstrates that ZnO nanoparticles synthesized from F. cretica extract exhibit strong antibacterial, antifungal, antioxidant, antitumor, antidiabetic, and cytotoxic activities. Biocompatible nanoparticles synthesized by employing plant (enriched pharmacological active compounds) have diverse applications as nanomedicines in pharmaceuticals, targeted drug delivery, food, cosmetics, and agriculture, thus becoming major candidates in www.nature.com/scientificreports/ biomedical research. In conclusion, the results of the undertaken study show that eco-friendly ZnO NPs synthesized from ethno-medicinally valued F. indica could be used biomedically for the treatment of anomalies allied with oxidative stress, infections, and as an antidiabetic. Further studies on the isolation of active compounds of this plant can be planned to discover new drugs.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.