Digera muricata (L.) Mart. mediated synthesis of antimicrobial and enzymatic inhibitory zinc oxide bionanoparticles

Herein, we report a simple and ecofriendly synthesis of ZnO nanoparticles (ZnO NPs) employing Digera muricata along with bioassay studies of synthesized NPs. The ZnO NPs obtained were indicated by a colour change from yellow to almost faint yellow giving whitish tinge and supported by the appearance of UV-Vis band at 373 nm and were characterized by using Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray (EDX), and scanning electronmicroscopy (SEM). The FT-IR spectrum confirmed the presence of biomolecules fabricated on ZnO NPs as indicated by the absorption bands at 1,378 for C–O cm, and ZnO NPs were also evident from the absorption bands at 440 and 670 cm, the former being the result of symmetric vibration of hexagonal ZnO and the latter belonged to a very weak vibration of ZnO. Its surfacemorphologywas confirmed by SEM, and the zinc and oxygen bonds were confirmed by EDX analysis giving sharp signals for Zn and oxygen with At% of 17.58 and 30.49, respectively. The antimicrobial activity of ZnO nanoparticles was determined by the agar well diffusion method against pathogenic bacterial and fungal strains using imipenem and miconazole as standards. The results reflected that ZnO NPs enhanced the activity of plant extracts against all employed algal (E. coli, S. faecalis, P. aeruginosa, K. pneumonia, S. aureus, and B. subtilis) and fungal (T. mentogrophytes, E. floccosum, A. niger, M. canis, and F. culmorum) strains. The antibacterial and antifungal activities of extracts were enhanced by the formation of ZnO NPs. The results indicated that Digera muricata extract contains effective reducing agents for green synthesis of Digera muricata fabricated ZnO NPs, which are more potent antimicrobial than the plant extract and showed almost similar inhibition against lipoxygenase, i.e., the IC50 value of 83.82 ± 1.15, comparable to the standard.


Introduction
Nanoparticles have diverse applications in catalysis, purification of water, biological and chemical sensors, wireless electronic equipment, and memory schemes [1][2][3]. Among the metals, zinc is present in all types of enzymes, i.e., transferases, oxidoreductases, ligases, lyases, hydrolases, and isomerases. Moreover, zinc oxide nanoparticles (ZnO NPs) exhibit properties like pyroelectric, semiconducting, and piezoelectric materials [4]. ZnO NPs are employed as preservatives towards the protection of wood and food items from pathogenic agents [5,6]. These act as photocatalysts, which are being used for the removal of organic pollutants [7]. Nanostructured ZnO show extraordinary uses in the area of agriculture, antimicrobial effect, and medicine, and environmental remediation [8]. ZnO NPs also have many uses in gas sensors, cosmetics, optoelectronics, pharmaceuticals, and solar cells [9]. The antimicrobial action of nanoparticles depends on different factors like size, bacterial strain, type of phytochemicals, conditions, shapes, and medium [10]. The enzyme inhibition studies by employing ZnO NPs have been extensively reported in the literature.
Among many other methods available for the synthesis of nanoparticles, plants are employed for the synthesis of nanoparticles, and such a method, called a green approach, is considered more significant than the others [11,12]. The phytochemicals present in plants contain functional groups capable of reducing metal ions and such methods are environmentally friendly and an inexpensive way for the formation of metal nanoparticles. Keeping in view the potential of phytochemicals, plantbased methods of synthesizing nanoparticles has gained momentum at present [13]. The first plant used for the synthesis of nanoparticles was Medicago sativa (alfalfa) [14]. The proposed mechanism for plant-mediated synthesis is the electrostatic interaction between functional groups of phytochemicals and metal salts [15]. In this perspective, Digera muricata (False Amaranth) is an important plant, which is spread worldwide and belongs to the family Amaranthaceae [16,17]. It is a wild herb plant, used as a laxative, edible, and usually known as tandla or tandula. Different primary metabolites (protein, lipids, phenols, and amino acids) and secondary metabolites (flavonoids, saponins, coumarins, tannins, terpenoids, alkaloids, anthraquinones, and cardiac glycosides) have been isolated from Digera muricata [18]. Its ethanolic extract is a diuretic and is widely used in the herbal treatment for various diseases like constipation, diabetes, and to cure kidney stones in the urinary tract [16,19]. In the present work, Digera muricata has been employed for the synthesis and biofabrication of ZnO NPs. The major benefit of employing Digera muricata for nanoparticles synthesis is the ease of availability of the plant material [20] with its broad range of primary and secondary metabolites along with its medicinal impact. The synthesized metal nanoparticles were characterized by UV-Vis spectroscopy, Fouriertransform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray analysis (EDX). In addition to the synthesis of ZnO NPs, the phytochemical analysis of the plant has been accomplished to investigate the type of secondary metabolites associated with this plant. Moreover, the plant extract and the synthesized ZnO NPs were screened against various bacterial and fungal strains to check their antimicrobial potential along with enzymatic inhibition potential. The comparative analysis revealed that ZnO NPs have more antimicrobial and enzyme inhibition potential than the plant extract.

General
All analytical grade chemicals, reagents, enzymes, and activity standards were purchased from Sigma-Aldrich and were used without any purification. Double-distilled water was used in all experiments. All glassware was washed with deionized water and dried before use.

Collection of plant material
The plant specimen (2 kg) including flowers, stems, leaves, and roots was collected from rural areas of Dunyapur in October 2017 and identified by Dr. Zafar Ullah Zafar, Associate Professor of Botany, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan. A voucher specimen was deposited in the herbarium of Bahauddin Zakariya University, Multan (R. R. Stewart F. W. Pak. 232 (1)).

Preparation of the plant extract
The plant material was washed, crushed dried, and soaked in a conical flask containing methanol for 15 days. After that, the supernatant was concentrated on a rotary evaporator. Moreover, the same process was repeated three times and the extract thus obtained was dried. The dry weight of plant extract was 40 g. This plant extract was used for different chemical tests.

Phytochemical screening
Phytochemical tests were used for the identification of various classes of naturally occurring compounds by employing standard procedures. The tests for alkaloids, tannins, flavones and flavonols, flavonoids, glycosides, triterpenoids, saponins, and carbohydrates were conducted, and the results indicated the presence of most of the above-mentioned compounds in the extract of Digera muricata. For alkaloids, in a 3 mL sample solution in water, a few drops of Wagner's reagent were added. and the formation of a brownish precipitate indicated the presence of alkaloids. For tannins, a few drops of 1% lead acetate solution was added and the appearance of a yellow-coloured precipitate indicated its presence. For flavones, to the plant extract was added concentrated H 2 SO 4 dropwise; a deep yellow colour appeared that indicated the presence of flavones and flavonols. The Shinoda test showed the presence of flavonoid, Liebermann's test showed the presence of glycosides, and the Salkowski test indicated the presence of triterpenoids. The Molisch test indicated the absence of carbohydrates, and the foam test was used to test for saponins, indicating their presence in Digera muricata.

Synthesis of ZnO nanoparticles
The method reported by Fakhari et al., with slight modifications, was used for the preparation of ZnO NPs [21]. Zinc acetate (0.25 g) was dissolved in 50 mL of distilled water to which was added dropwise to the plant extract solution (5 mL). This mixture was then stirred for 10 min by using a magnetic stirrer and NaOH (0.02 M) was added dropwise into the solution with stirring to adjust the pH to 12. Zinc oxide crystals (off-white) were formed, which were washed with distilled water, filtered, and ovendried to obtain ZnO nanoparticles.

UV-Vis and Fourier transform infrared (FTIR) spectroscopy
The formation of zinc oxide nanoparticles was confirmed by obtaining UV-Vis spectra of the solution in quartz cuvettes with an optical path length of 10 mm and a resolution of 1 nm using a UV-Vis. spectrophotometer (1,800) at absorption wavelengths between 200 and 800 nm. The FTIR spectra of ZnO nanoparticles were obtained using an AT-FTIR spectrophotometer. Both samples were placed in the sample compartment of the instrument for analysis, and a spectrum in the range 4,000-500 cm −1 was plotted.

Scanning electron microscopy (SEM) analysis
The particle shape, morphology, and sizes of nanoparticles at micro and nanoscales are determined by SEM. In this technique, a high-energy electron beam was scanned over the nanoparticles sample, and then this electron beam was scattered back to obtain the characteristic features [22]. The elemental analysis of single particles was carried out using an energy dispersive X-ray spectroscope (EDX) supplemented with a Jeol 5800 LV scanning electron microscope at the instrument conditions: accelerating voltage of 20 keV and counting time of 100 s.

Energy-dispersive X-ray (EDX) spectroscopy
When the electron beam is bombarded onto the sample, it ejects electrons and creates vacant space, which is filled by electrons coming from a high energy shell; to balance the energy gap between these two electrons the X-rays are emitted. The EDX counts the number of X-rays (characteristic of element) emitted against their energy. A spectrum having energy on one axis and X-rays emitted on the other axis was obtained. This gives quantitative and qualitative information about the elements found in the sample.

In vitro antibacterial and antifungal activity
The in vitro antibacterial activities of plant extract and ZnO NPs was determined against bacterial strains E. coli (ATCC 25922), S. faecalis (ATCC 700802), P. aeruginosa (ATCC 27853), K. pneumoniae (ATCC 700721), S. aureus (ATCC 25923), and B. subtilis (ATCC 6051) using the agar well diffusion method [23] and the results are shown in Table 3. For screening, the same concentrations of the standard antibacterial agent (imipenem) and test sample solutions were used. The molten agar was cooed to 45°C, mixed with bacterial inoculum, and poured into the sterile Petri dishes. Using a sterile metallic borer, the wells (6 mm in diameter) were made in the agar media at a distance of almost 22-24 mm away from each other. Different cork borers were used for different bacterial strains. Then, 100 µL of the test sample was introduced on the agar surface with the help of a sterile pipette. After that, the Petri dishes were kept for incubation for 24 h at 37°C. In the end, the antibacterial activity of the reference drug and each sample was determined by measuring the diameter of inhibition zones in mm. The antifungal activity was determined against the fungal strains; T. mentagrophytes (TIMM 2789), E. floccosum (NBRC 9045), A. niger (ATCC 1015), M. canis (NBRC 9182), and F. culmorum (NRRL 25475) according to the previous procedure [24]. The dextrose agar medium mixed with fungal inoculum was evenly spread by means of a sterile cotton swab on sterilized Petri dishes. Using sterilized metallic borers, the wells (6 mm in diameter) were dug in the agar media spaced at a distance of almost 22-24 mm. The same concentrations (100 µL) of the sample solutions and reference antifungal agent (miconazole) were poured into the Petri dishes. The Petri dishes were incubated at 37°C for 48 h. After incubation, confluent fungal growth was obtained. Inhibition zones of the fungal growth were measured in mm for all the test samples and the antifungal agent.

Enzymatic (lipoxygenase and α-glucosidase) assay
Inhibitions of the crude extract and ZnO nanoparticles by lipoxygenase were assayed according to the spectrophotometric (absorbance at 234 nm) method established by Tappel [25] by using quercetin (0.5 mM well −1 ) as a positive control. For the lipoxygenase activity, a final volume of 200 µL of the assay mixture was prepared, which consisted of 20 µL of lipoxygenase enzyme, 160 µL of sodium phosphate buffer (pH 8.0, 100 mM), and 10 µL of test samples (in 100 mM Tris buffer, pH 7.4). The assay mixture was preincubated at 25°C for 10 min. Then, the reaction was initiated by adding 10 µL of linoleic acid solution that acted as substrate. The change in the absorbance reading was recorded at 234 nm after 6 min. The IC 50 values were calculated using EZ-Fit Enzyme Kinetics Software [26]. The inhibitory activity of the compounds was determined against α-glucosidase according to the previous method [27]. A 250 µL of acarbose was incubated with α-glucosidase solution (500 µL) in phosphate buffer (pH 6.9, 100 mM) for 20 min at 37°C. Then, p-NPG (p-nitrophenyl-β-D-glucopyranoside solution (1 mM, 250 µL) and 250 µL of starch (1%) were added to the reaction mixture and incubated for 1 h at 37°C. After that, the absorbance of the resulting mixture was recorded at 400 nm. A blank solution was used for correcting the absorbance readings.

Results and discussion
Digera muricata contains a rich number of secondary metabolites and has attracted great interest due to its extraordinary medicinal value, therapeutic uses, and edible properties. Therefore, phytochemical analysis of Digera muricata along with the synthesis of zinc oxide nanoparticles was performed from the methanolic extract of Digera muricata.

Phytochemical screening of the plant extract
For phytochemical analysis, standard tests for various classes of compounds like alkaloids, tannins, flavonoids, terpenoids, saponins, glycosides, carbohydrates, flavones and flavonols, and triterpenoids were performed. These tests were positive for alkaloids, tannins, flavonoids, terpenoids, glycosides, flavones and flavonols, triterpenoids, and saponins and negative for carbohydrates as summarized in Table 1. In previous reports, these phytochemicals have been responsible for the reduction and then capping of nanoparticles [28,29]. The presence of these phytochemicals in Digera muricata was therefore responsible for reducing zinc metal to zinc oxide nanoparticles, which were later surrounded by the plant extract, also called capping of nanoparticles.

Biosynthesis of Digera muricate-ZnO nanoparticles
ZnO nanoparticles were obtained as light grey precipitates The precipitates were then thoroughly washed with distilled water to obtain white ZnO nanoparticles. The mechanism reported for the chemical synthesis of ZnO NPs is shown in Eqs. 2 and 3. A similar colour of ZnO nanoparticles was reported by Varghese et al. [28]: Zn CH COO OH NaOCOCH Keeping in view the above mechanisms, an analogous mechanism for green synthesis has been proposed. The proposed mechanism consists of three steps. In the first step, sodium hydroxide abstracts a proton from the phenolic or flavonoid moiety containing OH groups on the benzene ring. Then, this negatively charged phenolate ion replaces one of the acetate ions by a simple replacement reaction. Later, the organometallic zinc compound disintegrates in such a way to give ZnO along with a phenolic or flanoidal compound containing acetate groups.

Characterization of ZnO NPs: UV-Vis spectroscopy
The green, eco-friendly, low-cost synthesis of ZnO-NPs was performed using Digera muricata extract. The biochemicals like alkaloids, terpenoids, flavonoids, phenols, etc. present in the Digera muricata leaf extract reduced Zn ions to ZnO nanoparticles, which was visible by the colour change, a first indication of the ZnO-NPs synthesis. The change in the colour corresponding to the single peak of absorption in the UV-Vis spectrum was because of characteristic changes in the energy levels of electrons in the nanoparticles. The maximum absorption peaks of ZnO were observed at 373 nm in the 200-800 nm range.

FTIR analysis
The reducing and stabilizing or capping were predictable by comparing the different functional groups present in FTIR spectra of the extract and ZnO nanoparticles. Metal nanoparticles were frequently centrifuged and redispersed in deionized water in order to eliminate the possibility of unbound residual organic moieties. The FTIR spectra of the plant extract showed a very close resemblance to the FTIR spectra of Digera muricata-ZnO NPs, excluding some differences in a few peaks. This indicated that some metabolites of the Digera muricata extract remained on the surface of ZnONPs. The possible biomolecules responsible for ZnO reduction and then its

Scanning electron microscopy (SEM) analysis
The SEM images of ZnO-NPs are shown in Figure 1, which confirmed the surface morphologies and their shape.
The SEM image revealed that zinc oxide nanoparticles are spherical, irregular, and spongy, and their individual particle [28] size ranges from 55 to 80 nm with some deviations. The metal oxides were uniformly distributed in the three-dimensional polymeric network structure; the accumulation could be induced by densification resulting in the narrow space between particles, which may be due to the flavonoids, alkaloids, tannins, saponins, and glycosides present in the Digera muricata and were responsible for the formation of ZnO NPs.

Energy-dispersive X-ray spectroscopy (EDX) analysis
The EDX results confirmed the presence of Zn-NPs in the oxide form. It was used to find the elemental composition of nanoparticles and the pattern indicated that the sample contains zinc oxide. The presence of weak signals of carbon and oxygen atoms displayed that biomolecules within the extract are involved in the synthesis of ZnO-NPs. The EDX spectrum for the newly synthesized ZnO-NPs in Figure 2 shows the elemental profile of the Digera muricata-ZnO NPs. The EDX analysis showed sharp signals of Zn and oxygen with At% of 17.58 and 30.49. The atomic and mass % obtained from the EDX spectrum is presented in Table 2.

Antimicrobial activity
The antimicrobial activities of the plant extract and ZnO-NPs were determined against selected bacterial and fungal strains according to the standard method [30]. The bacterial strains (E. coli, S. aureus, S. faecalis, K. pneumoniae, P. aeruginosa, and B. subtilis) used imipenem as a standard, whereas fungal strains (T. mentagrophytes, E. floccosum, A. niger, M. canis, and F. culmorum) used miconazole as a standard; the results are tabulated in Tables 3 and 4, respectively. The results showed that the microbicidal activity improved during the ZnO-NP synthesis and indicated the dual role of phytochemicals, i.e., metal salt reduction as well as NP stabilization. The zone of inhibition was found to be in the range 7-22 mm against bacterial strains, while that for fungal strains, was recorded in the range of 17-33 mm.

Lipoxygenase and α-glucosidase inhibitory activities
The plant extract and ZnO-NPs were evaluated for their lipoxygenase inhibitory activities; they showed the strongest potential with IC 50 values of 61.07 and 57.01 µg/mL, respectively, compared with the positive control quercetin with an IC 50 value of 23.82 µg/mL, as shown within   Each value are average of three assays and represented as mean ± standard error mean (SEM). Table 4. The plant extract and ZnO-NPs were also evaluated for α-glucosidase enzymatic inhibitory activities and displayed IC 50 (μM) values of 106.5 and 98.1, respectively ( Table 5). Deoxynojirimycin (IC 50 = 301.4 μM) was used as a positive control for the α-glucosidase enzyme inhibitory activity. As these compounds showed moderate to significant enzymatic inhibition, in the future, these nanoparticles and plant extract phytochemicals may be used for the treatment of COVID-19 based main protease (M pro ) protein inhibition [31,32].

Conclusions
Phytochemical screening of the methanolic extract of Digera muricata showed the presence of alkaloids, tannins, flavonoids, terpenoids, glycosides, flavones and flavonols, triterpenoids, and saponins. A fast, easy, and green synthesis of zinc oxide nanoparticles using Digera muricata extracts was successfully completed by the reduction of zinc acetate solutions. The synthesized ZnO-NPs were confirmed by characterization using spectroscopic techniques like UV-Vis spectroscopy, FTIR spectroscopy, and microscopic techniques like SEM and EDX. The EDX analysis showed sharp signals between 0 and 2 and Zn peaks between 8 and 10. In addition, the antimicrobial potential of nanoparticles and their comparison with that of Digera muricata extract indicated that ZnO-NPs were more promising antimicrobial agents than that of plant extract. The extract and ZnO-NPs demonstrated lipoxygenase and α-glucosidase inhibitory activities using quercetin and deoxynojirimycin as standards, respectively. It can be assumed that the plant extract and ZnO NPs contain potent enzymes (lipoxygenase and α-glucosidase) inhibiting phytochemicals that may be used for the treatment of inflammation, COVID-19, asthma, and cancer. Each value are average of three assays and represented as mean ± standard error mean (SEM). Hussain: writingoriginal draft, formal analysis, visualization, project administration; Muhammad Naeem Qaisar: writingoriginal draft, visualization; Muhammad Imran: writingreview and editing, visualization, project administration; Ahmad Irfan: visualization, project administration.

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: All the data generated or analyzed during this study are included in this published article. The datasets are also available from the corresponding author on reasonable request.