Elsevier

Phytochemistry Letters

Volume 31, June 2019, Pages 170-180
Phytochemistry Letters

Phytochemical investigation of Medicago sativa L. extract and its potential as a safe source for the synthesis of ZnO nanoparticles: The proposed mechanism of formation and antimicrobial activity

https://doi.org/10.1016/j.phytol.2019.04.009Get rights and content

Highlights

  • The phytochemical investigation of Medicago sativa L. aqueous extract.

  • Medicago sativa L. as a new and safe source for the effective synthesis of ZnO bio-NPs.

  • Physicochemical characterization and determination of ZnO NPs antimicrobial activity.

  • Mechanism description of ZnO NPs formation by M. sativa L. was proposed.

  • A new insights into the processes involved in the plant-mediated ZnO NPs synthesis.

Abstract

The main goal of present work was the investigation of Medicago sativa L. aqueous extract as a new, safe and accessible agricultural waste source for the synthesis of zinc oxide bio-nanoparticles (ZnO bio-NPs). Secondly, the phytochemical investigation and determination of M. sativa biologically active compounds content before and during the synthesis has led to the proposal of a possible ZnO NPs plant- mediated synthesis mechanism. In this fact, the present research show the implementation of the interdisciplinary approach involving a wide range of modern and complementary instrumental techniques which have confirmed the effective plant-mediated synthesis and antimicrobial potential of ZnO nanoparticles. X-ray diffraction (XRD) data characterized final product as a highly crystalline and hexagonal ZnO. Results of transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) indicated the formation of nanoparticles and confirmed the chemical composition of samples. Moreover, our work reported that carboxyl group derived from alfalfa proteins and zinc-flavonoids/polyphenols complexes can be mostly involved in the nanoparticles formation. The antimicrobial potential of obtained nanomaterial was tested against bacteria (Staphylococcus epidermidis ATCC49461, Lactococcus lactis ATCC49032 and Lactobacillus casei ATCC334 and yeast (Candida albicans ATCC10231 and Saccharomyces cerevisiae MG012794) strains using minimum inhibitory concentration (MIC) and fluorescence microscopy approaches. The ZnO NPs showed an inhibitory effect against all tested microorganisms. Data from the fluorescent microscopy indicated the vacuolization and deformation of yeast cells after the nano-ZnO treatment.

Introduction

Nowadays, nanotechnology is an important part of modern science and play a crucial role in providing innovative solutions in the field of manufacture, optics and electronics as a nano-devices, in food packaging and in biomedicine (Sanchez and Sobolev, 2010). Zinc oxide (ZnO) became a one of the most studied nanomaterials nowadays, mainly because of its appealing properties. ZnO found the application not only in industry, but also as a e.g. a good nanoplatforms for drug or gene delivery (Nie et al., 2007; Yuan et al., 2010). Moreover, due to their strong antimicrobial activity against both bacteria and fungi (Jones et al., 2008; Sawai and Yoshikawa, 2004), they can be a promising tool in the development of the effective antimicrobial agent in medicine and are increasingly used to target bacteria as an alternative to antibiotics. The antimicrobial action of ZnO NPs depends on the many factors such as particle size or concentration (Yamamoto, 2001). The various antibacterial mechanisms of nanomaterials are mostly connected with their high surface to volume ratio and unique physicochemical properties, but the precise mechanism is still under debate (Wang et al., 2017). The growing focus on nano-ZnO resulted in the development of many different methods of synthesis. There are known such approaches as physical, chemical or biological, but currently the development of “green chemistry” seems to be a one generating much interest. Synthesis involving the use of biological systems provides many advantages in comparison to traditional chemical methods, such as not-using toxic and expensive organic solvents, being environmental friendly and allowing to control the NPs size and shape. What is more, synthesis of nanoparticles using biological approach can be a promising technology to reduce the cytotoxicity of ZnO NPs commonly associated with chemical and physical synthesis approaches as well as improving the antibacterial activity of obtained nanomaterials. In case of the biological methods, the selection of the used source for the synthesis is crucial - the NPs synthesized by different sources such as plants or microorganisms exhibit a different antimicrobial effects against pathogens (Buszewski et al., 2017; Railean-Plugaru et al., 2016a). Therefore, the implementation of the interdisciplinary approach for the biological synthesis of nano-ZnO including use of complementary instrumental and microbiological techniques is pivotal for proper understanding of nanomaterials properties.

Recently, many researcher groups have been focused on the development of new method for MeO NPs synthesis through using a plants among which crop plants seems to be attracting much attention. The leaves and stalks of most crop plants are considered to be an agricultural waste and are available in quantities of thousands of tons (Loehr, 1974). Therefore, it is necessary to develop a new methodology for the further use of the crops residues. One of the promising approach can be using those plants for the synthesis of nanomaterials such as zinc oxide NPs. Plant extracts consist of a wide range of bio-compounds such as e. g. flavonoids and polyphenols which can act both as reducing and capping agents in the process of nanoparticles synthesis (Mittal et al., 2013). Synthesis of zinc oxide NPs from plants such as Aloe vera, Pongamia pinnata, Physalis alkekengi L., Cassia fistula and others have been reported (Qu et al., 2011; Sangeetha et al., 2011; Sundrarajan et al., 2015; Suresh et al., 2015). Sundrarajan et al. (Sundrarajan et al., 2015) have performed a synthesis of spherical nanoparticles about 100 nm in size with hexagonal structure and of high purity using Pongamia pinnata extract which contains a large amount of compounds such as e.g. flavonoids or terpenoids. It was also reported (Qu et al., 2011) that Physalis alkekengi, a plant able to grow in soils with high zinc level, is a good plant for production of crystalline ZnO nanoparticles at 72.5 nm size. According to the current papers, exotic plants, which are not native to Europe, are used for ZnO NPs synthesis often. Therefore, the attention should be paid to the potential use of crop plants widely grown in native and local environments - one of the plants is Medicago sativa L (called also Lucerne or alfalfa). Lucerne is a plant from the Fabaceae family and is cultivated as a one of the most important forage crop around the world (Rafińska et al., 2017). This plant is easy to grow and can be harvested from 2 to 3 times per year (Rashmi et al., 1997). Due to the high secondary metabolites content, alfalfa is considered as a cheap and valuable source of animal fodder. Furthermore, M. sativa has a lot of a precious therapeutic value and has been used in the folk medicine for the long time as an excellent cure for the kidney problems oral central nervous system disorders (Rafińska et al., 2017). Recent studies have demonstrated that lucerne is rich in essential aminoacids (Rafińska et al., 2017), minerals, vitamins (Iii, 2008) and secondary metabolites such as saponins, flavonoids and phenolic compounds (Karimi et al., 2013; Rafińska et al., 2017). The high content of listed composites make alfalfa a good source of bioactive compounds which can play a crucial role in synthesis of zinc oxide nanoparticles. It is believed that the presence of phytochemicals in the plant extracts helps in the synthesis of metal oxide NPs by inducing oxidation and reduction reaction.

In this paper, the use of Medicago sativa L. aqueous extract for the zinc oxide nanoparticles production as a new, eco-friendly and agricultural waste source and its application in the field of nanotechnology is described for the first time. Moreover, the phytochemical investigation of Medicago sativa aqueous extract were performed and, based on it, the effective synthesis of ZnO nanoparticles by using zinc nitrate as a precursor is reported.Furthermore, a complementary approach for the physicochemical characterization and antimicrobial activity assays of obtained nanomaterial was performed. Based on the determination of M. sativa biologically active compounds content before and during the synthesis process the ZnO NPs plant- mediated synthesis mechanism is proposed.

Section snippets

Preparation of plant extract

The plant material were sourced from the RWE-TRADE company (Poland). The aqueous extract of Medicago sativa was prepared by mixing a slightly cut plant material with a ultrapure water in 10:100 (w/v) ratio. Then, it was heated at 60⁰C for 2 h and after that residues were filtered by Whatman No.1 filter paper and membrane filter (MediaKap-2 Plus). The filtered extract was used for the further process.

Determination of Medicago sativa bio-active compounds content and antioxidant activity

The total phenolic and flavonoid content of M. sativa aqueous extract was examined by the

Determination of M. sativa bio-active compounds content

The results of quantitative analysis of bio-active compounds and the percentage content of them in the aqueous extract of M. sativa plant before and during the ZnO NPs synthesis are shown at the Fig. 1. The efficiency of the process was 85.08 ± 0.23%.

The total content of phenolic compounds (mg/g of dry extract) in the aqueous extract were determined from regression equation of calibration curve y = 0.469x – 0.015 with the R2 = 0.993 and expressed in rutin equivalents (RE). The total amount of

Discussion

The synthesis of ZnO nanoparticles can be performed by using a wide range of plant extracts such as Aloe vera, Pongamia pinnata, Physalis alkekengi L., Cassia fistula, Parthenium hysterophorus L., Passiflora caerulea and other (Qu et al., 2011; Rajiv et al., 2013; Sangeetha et al., 2011; Santhoshkumar et al., 2017; Sundrarajan et al., 2015; Suresh et al., 2015). In our study, the Medicago sativa L. aqueous extract, as a new, safe and agricultural waste source, was chosen for the production of

Conclusions

In this study, the use of Medicago sativa L. aqueous extract for the zinc oxide nanoparticles production as a new, eco-friendly and industrial approach as well as its application in the field of nanotechnology is described for the first time. Moreover, the possible mechanism of the novel plant-mediated ZnO nanoparticles synthesis was proposed. In this fact, a wide range of modern and powerful instrumental techniques, such as spectroscopic, spectrometric, diffraction and microscopic methods were

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by Plantarum, BIOSTRATEG2/298205/9/NCBR/2016 (2016-2019) and by Foundation for Polish Science “START” No. 068.2017; subsidy (2017/2018).

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