Biogenic Synthesis of Copper Oxide Nanoparticles Using Pimpinella anisum Seed Extract Characterization and Antibacterial Activity

Introduction

Nanoparticles Metal and metal oxides have gotten considerable consideration from researchers owing to their special visual, conductor , and catalytic characteristics . Due to their prospective uses in various disciplines, such as industrial, medical, pharmaceutical drug delivery, and catalytic applications, scientists have recently placed a significant emphasis on metal nanoparticle synthesis[1, 2]. Numerous chemical and physical processes are utilized to produce varied types of metal nanoparticles. However, these techniques  are costly , need high pressure and radiation , grand negate hazardous and dangerous by products that are environmentally risky[3]. Hence, there is a need for the development of new,  mild, harmless,  and a natural product for metal/metal oxides production in an aqueous environment[4– 6]. Using herbs attract the of researchers consideration  the as most appropriate biological synthesis methods in a comparison with other synthesis methods [7-9]. Due to the wide abundant of the medicinal plants and its costly effective, there is a considerable growth in the synthesis of copper oxide nanoparticles utilizing green chemicals. [10–12].

Copper oxide nanoparticles have attracted interest among numerous metal nanoparticles because they exhibit mechanical and biological features using modern technologies [13]. Copper oxide nanoparticles have been discovered to be particularly effective in gas sensors, photocatalytic applications, and the biomedical sciences for the treatment of numerous disorders [14].

Pimpinanella anisum L. (Apiaceae), is an annual medicinal plant commonly known as anise, it is widely distributed in Europe, Turkey Central Asia, and Egypt. Pimpinella anis is a well-known culinary plant, utilized in the manufacture of renowned liqueurs and also valued for its therapeutic qualities [15]. Anise seed extract is a rich source of flavonoids, phenols, proteins, and numerous B-complex vitamins, such as pyridoxine, niacin, riboflavin, and thiamin, in addition to possessing antioxidant properties  [16].  Aqueous and ethanolic extracts of seeds have also been reported to have antibacterial action. [17]. In this study, we synthesized and characterized CuO NPs using Pimpinella anisum seed extract as a stabilizer and reducing agents.

Experimental

Chemicals

All chemicals (CuCl₂ and NaOH) were obtained from Sigma-Aldrich in St. Louis. Agar powder, yeast extract, and Peptone were Purchased from Merk KGaA, Darmstadt, Germany. Pathogenic microorganisms were supplied by the medicine  faculty,  King Faisal University , Saudi Arabia. The remaining reagents utilized in this study are laboratory-grade. To prepare all sample solutions, deionized water was used.

Preparation of Pimpinella anisum extract:

In order to create Pimpinella anisum extract, 5 g of dried fine seeds were combined with volume of (100 ml ) distilled water. The mixture was then brought to a boil for 10 minutes, a  color change of solution was observed from  transparent to brown yellow. The mixture was subsequently cooled and filtered using Whatman No. 1 filter paper. The extract was  kept at room temperature for future use in. experiments.

Synthesis of copper oxide nanoparticles

In a 250 ml beaker, 0.1 M CuCl2 2H2O and 100 ml deionized water are mixed. Specific volume of  the extract and  copper chloride dihydrate solution were mixed and heated  the to 80 °C with steady stirring. In a burette, 40mL of 2 M sodium hydroxide NaOH was drawn to be added drop by drop to the mixture. The mixture color changed rapidly from blue to dark brown. The quick formation of the black precipitate at the bottom of the beaker. Black precipitate was obtained after three washes with deionized water and filter paper Whatman No.1. The washed precipitate was then dried at 120 °C for 3hrs. The product was calcined for four hours at 500 °C.

Characterization

Fourier-transform infrared spectroscopy (FTIR) Cary (360)  California, United States of America, was utilized to conduct FT-IR measurements on sample material. Morphological analysis was preformed using (FE-SEM) technique (FEI, QUANTA FEG, 250 high-resolution field emission scanning electron microscope) equipped with a high-angle, angular darkfield sensor and an x-ray energy dispersive spectroscopy system (EDX) to determine the particle crystallinity and size. To identify the particles crystallinity X-ray diffractometer was used (EMPYREAN via radiation of Cu Ka at wavelength of 1.54A), the crystalline phases were identified via XRD analysis. BET surface area (BET SSA), used to determine the surface area and porosity. The pore length and volume were calculated using an adsorption-desorption isotherm of nitrogen at 77 K boiling point.

Assessment of the antibacterial inhibitory potentials of CuO nanoparticles

Biosynthesized CuO nanoparticles’ antibacterial efficacy was evaluated against Four species of dangerous germs, including two Gram-negative bacteria (E. coli) and Salmonella, and two Gram-positive bacteria (Marsa, Staphylococcus aureus, bacillus subtilis). Various concentrations (0, 2, 6, 10, 14, 18, and 22 ppm) of produced CuO nanoparticles were employed to investigate the antibacterial activity against harmful microbes. In addition, a comparative assessment was conducted using a commercial amoxicillin concentration of 22 ppm. A suspension of nanoparticles was made using (5 ml of the broth of nutrient (NB) medium and CuO nanoparticles ) and peptone, yeast extract(10 ml) , and NaCl (5 ml ) [18, 19]. Pathogenic inoculum (100 l) was introduced into the medium of nutrient broth supplemented with nanoparticles (5 ml) during the logarithmic phase. under sterile environments for microorganisms. The absorbance was carried out spectrophotometrically at 600 nm after incubated for 24 hours of at 30 ^◦C for all samples.

Results and Discussion

Recent research has focused heavily on the green production of metal nanoparticles due to their limitless possibilities [4]. The creation of copper oxide nanoparticles was confirmed by UV-visible spectroscopy and visually noticed as a color shift. When the extract of Pimpinella anisum seeds is applied to a solution of copper chloride, a clear change in the color instantly was observed from light blue to a brown[20]. Strong absorbance between 250 and 300 nm in Figure 1 indicates the production of copper oxide nanoparticles. The occurrence of SPR absorption provides information on the nanoparticles’ structure and size [13],[21].

Figure 1: UV-Vis spectra of green synthesized CuO NPs using extract of Pimpinella anisum seeds Click here to View figure

To identify the biomolecules in Pimpinella anisum seeds extract used to reduce or synthesize CuO NPs, FTIR analysis was carried out. Fig.2 shows the FTIR spectrum of green synthesized CuO nanoparticles. The presence of the OH group of absorbed moisture on the copper oxide NPs surface is characterized by the broadband at 3440 cm^-1. Stretching vibrations of carboxylic bonds had an absorbed band at 1670 cm^-1 ; additionally,  at 1370 cm^-1 a peak for  O-H bending vibrations in combination with Cu atoms was observed , while peaks at 591 cm^-1 are owing to stretching vibrations of Cu-O [22]. These results showed that the biomolecules of Pimpinella anisum seeds extract are responsible for the surface stabilization of CuO NPs.

Figure 2: FTIR spectra of synthesized CuO nanoparticles. Click here to View figure

As shown in Fig.3, the morphology of biosynthesized CuO nanoparticles was evaluated using FE-SEM. The synthesized CuO NPs have a size in nanometers with a spherical morphology, consistent with previous studies.[9][12]. A SEM image showed that the synthesized nanoparticles were monodispersed and spherical, with a diameter ranging from 46.9-72.8 nm.

Figure 3:  SEM images of CuO NPs Click here to View figure

CuO-NPs were subjected to a 15 keV EDX assay, which revealed the presence of copper (Cu) and oxygen (O). According to EDX analysis, the percentages mass of copper and oxygen were 28.80 and 71.20, respectively. The EDX spectrum did not reveal any further elemental impurities. EDX examination revealed that CuO had a uniform distribution of copper-to-oxygen with a 3:1 atomic ratio. An elemental analysis confirms the existence of copper oxide.

Figure 4: EDX analysis of CuO nanoparticles Click here to View figure

Nanomaterials can be identified by their specific surface area, which is one of the primary indicators of their nanoparticle content. CuO NPs were characterized by their surface area (BET). The approach is based on constant-temperature single-gas adsorption. As an adsorbate on the surface of CuO NPs, nitrogen gas was utilized. To assess the surface texture parameters of CuO, Brunauer Emmett Teller (BET) and Barrett Joyner Halenda (BJH) diagrams were utilized. Based on the N2 sorption isotherm, the isotherm is of Langmuir type IV (Fig. 4a), with a relative pressure (P/P o = 0.0-0.99). In addition, the hysteresis loop is type H3 (Fig. 4b), confirming that the nanoparticles with slit-shaped pores are mesoporous. The results indicate that CuO NPs have a surface area of 14,034 m2/g, which is a considerable value. In addition, the pore volume and radius are 0.02 cc/g and 15.897A0, respectively. The antibacterial capabilities of CuO NPs were influenced by their high surface area. Due to its enhanced ability to absorb UV light [17] and generate radical species, the presence of nano CuO with a wide surface area can boost antibacterial activity. Owing to the enhanced production of Cu²⁺ ions and the reactivity of the oxygen  , the CuO NPs were able to destroy bacterial cells (ROS).

Figure 5: (a) (BET) and N2 adsorption –desorption isotherm (b) (BJH) pore size distribution of the CuO nanoparticles samples Click here to View figure

To examine the crystallinity of CuO nanoparticles, XRD diffraction analysis was performed. Figure.6 depicts the CuO nanoparticles’ XRD pattern. The formation of tiny, well-crystallized copper oxide nanoparticles is permitted for this result. The obtained results demonstrated discrete diffraction peaks at 2 theta = 32.816, 38.844, 66.08, and 68, which are assigned   to 002, 111, 311, and 113 planes of  a monoclinic phase of copper oxide nanoparticles. The XRD spectrum revealed the crystalline nature of the biosynthesized CuO NPs. In accordance with (ICSD087122), the peak positions indicated that CuO possesses a monoclinic structure [21].  The CuO nanoparticles average  size was determined by the Scherer equation :

D = 0.9 λ/β cos θ                       (1)

where λ is X-ray radiation wavelength, β is the full width at half maximum (FWHM)  for the peaks at the angle θ. It was determined and the it was around 22 nanometers in diameter.

Figure 6: XRD Pattern of CuO nanoparticles. Click here to View figure

Table 1: XRD parameters of CuO NPs

No.	B obs. [°2Th]	B std. [°2Th]	Peak pos. [°2Th]	B struct. [°2Th]	Crystallite size [nm]
1	0.263	0.008	32.422	0.255	32.4
2	0.433	0.008	35.421	0.425	19.6
3	0.393	0.008	38.69	0.385	21.9
4	0.354	0.008	48.744	0.346	25.2
5	0.742	0.008	53.349	0.734	12.1
6	0.354	0.008	58.304	0.346	26.3
7	0.393	0.008	61.461	0.385	24.0
8	0.472	0.008	66.215	0.464	20.4
9	0.629	0.008	72.324	0.621	15.8
10	0.472	0.008	74.918	0.464	21.6
					Average= 21.93

 

Antibacterial Activity of CuO nanoparticles

The results of the antibacterial assessment of CuO nanopowder in contrast to five species of human pathogenic bacteria; are Escherichia coli, Marsa, Pseudomonas aeruginosa Klebsiella Pseudomonas, and Candiia. Figure.7. Our results showed that CuO nano powder are highly active in the tests of the pathogenic bacteria and fungi employed in this experiment. The presence of biomolecules capped the synthesized nanopowder the bacterial efficacy was high which can explained by the linking of CuO with the terpenoids  from the extract during the process of capping. [23]. The increasing of CuO nanopowder concentration inhibits the virulence of microbial pathogens.

Figure 7: Antibacterial activity of biosynthesized CuO nanoparticles Click here to View figure

Conclusion

Based on this study, we found that biogenic synthesis of CuO nanopowder from Pimpinella anisum seed extract is simple, economical, and environmentally friendly. Nanopowder synthesized was initially confirmed by the presence of SPR bands at 340 nm in UV-Vis spectra. A spherical particle with a diameter of 22 nm was detected. Copper nanoparticles with crystalline nanostructure are confirmed by the XRD spectrum. Finally, the green CuO NPs generated exhibited stronger antibacterial infections; this property makes the CuO NPs more relevant in domains such as medicine.

Acknowledgement

The author thanks Department of Chemistry, Imam Mohammad Ibn Saud Islamic University.

Conflict of Interest

The authors declare no conflict of interest.

Funding Sources

There is no funding source.

References

1. Dizaj, S. M.; Lotfipour, F.; Barzegar-Jalali, M.; Zarrintan, M. H.; Adibkia, K. Materials Science and Engineering C. 2014, 44. 278–284.
   CrossRef
2. Nguyen, N. H. A.; Padil, V. V. T.; Slaveykova, V. I.; Černík, M.; Ševců, A. Nanoscale Res. Lett. 2018, 13.
   CroossRef
3. Cuevas, R.; Durán, N.; Diez, M. C.; Tortella, G. R. ; Rubilar, O.  J. Nanomater. 2015, 2015.
   CrossRef
4. Hekmati, M. Catal. Letters. 2019, 149, 2325–2331.
   CrossRef
5. Al-Qasmi, N.  Processes. 2021, 9.
   CrossRef
6. Philip. D. Acta – Part A Mol. Biomol. Spectrosc. 2009, 73, 374–381.
   CrossRef
7. Zangeneh, M. M.; Bovandi, S.; Gharehyakheh, S.; Zangeneh, A.; Irani, P. Appl. Organomet. Chem. 2019, 33.
   CrossRef
8. Bhattacherjee, A. ; Ghosh, T.; Datta, A. J. Exp. Nanosci. 2018, 13, 50–61.
   CrossRef
9. Liu, H.; Wang, G.; Liu, J.; Nan, K.; Zhang, J.; Guo, L.; Liu, Y. J. Exp. Nanosci. 2021. 16, 411–423.
   CrossRef
10. Zangeneh, M. M.; Zangeneh, A.; Pirabbasi, E.; Moradi, R.; Almasi, M. Appl. Organomet. Chem. 2019, 33, 5246.
    CrossRef
11. Liu, Y.; Zeng, Z.; Jiang, O.; Li, Y.; Xu, Q.; Jiang, L.; Xu, D. Mater. Res. Express. 2021, 8.
    CrossRef
12. Wu, S.; Rajeshkumar, S. ; Madasamy, M.; Mahendran, V.  Artif. Cells, Nanomedicine Biotechnol. 2020, 48, 1153–1158.
    CrossRef
13. Sankar, R.; Manikandan, P.;  Malarvizhi, V.;  Fathima, T.;  Shivashangari, K. S.; Ravikumar,  V. Spectrochim. Acta – Part A Mol. Biomol. Spectrosc. 2014, 121, 746–750,
    CrossRef
14. Ananda Murthy, H. C. ; Abebe, B.; C H, P.; Shantaveerayya, K. Mater. Sci. Res. India. 2018, 15, 279–295.
    CrossRef
15. Initial, F. Community herbal monograph on Pimpinella anisum L ., fructus.  2013, 44, 0–6,
16. Rashnaei, N.; Siadat, S. D.; Akhavan Sepahi, A.; Mirzaee, M.; Bahramali, G.; Arab Joshaghani, A. Vaccine Res. 2020, 7, 17–24.
    CrossRef
17. AlSalhi, M. S.; Devanesan, S.; Alfuraydi, A. A.; Vishnubalaji, R.; Munusamy, M. A.; Murugan, K.; Benelli, G. Int. J. Nanomedicine. 2016, 11, 4439–4449.
    CrossRef
18. Hassanin, H. A.; Taha, A.; Afkar, E. Ceram. Int., , 2021, 47, 3099–3107.
    CrossRef
19. Da’na, E.; Taha, A.; Afkar, E. Appl. Sci. 2018, 8.
    CrossRef
20. Narasaiah, P.; Mandal, B. K.; Sarada, N. C. IOP Conf. Ser. Mater. Sci. Eng. 2017, 263.
    CrossRef
21. Kumar, P. P. N. V.; Shameem, U.; Kollu, P. ; Kalyani, R. L.; Pammi, S. V. N. Bionanoscience. 2015, 5, 135–139,.
    CrossRef
22. Alhalili, Z.  Arab. J. Chem., 2022, 15, 103739.
    CrossRef
23. Akintelu, S. A. ; Folorunso, A. S.; Folorunso, F. A.; Oyebamiji, A. K. Heliyon, 2020, 6.
    CrossRef

---

This work is licensed under a Creative Commons Attribution 4.0 International License.