In vitro DNA damage prevention, antioxidant and antidiabetic activities of achillea biebersteinii/millefolium extracts and synthesized ZnO nanoparticles

Authors

Abstract

AimIn this study, DNA damage preventive effects, antioxidant and antidiabetic activities of extracts of Achillea species growing endemic in the Lapta region of Kyrenia district of Cyprus, and zinc oxide-nanoparticles/Achillea (ZnO-NPs/Ach) were investigated.
MethodsWe performed green synthesis of ZnO-Nps using the extract obtained from Achillea species (ZnO-NPs/Ach). We performed green synthesis of ZnO-Nps using the extract obtained from Achillea species (ZnO-NPs/Ach). Additionally, we determined the anti-DNA damage, antioxidant, and antidiabetic activities of these nanoparticles.
ResultsThe highest concentration of 500 μg/mL of ZnO-NPs/Ach had an excellent protective effect. It was determined that the activity of ZnO-NPs/Ach was relatively vigorous in preventing lipid peroxidation. The lipid peroxidation inhibitory activities of ZnO-NPs/A. biebersteinii and ZnO-NPs/A. millefolium were calculated as 90.87% and 89.11%, respectively. Additionally, both demonstrated high antidiabetic effects compared to the positive control acarbose.
ConclusionAs zinc oxide nanoparticles obtained through green synthesis have been found to have preventive effects on DNA damage, as well as antioxidant and antidiabetic properties, this study is expected to make a valuable contribution to the medical and technological literature.

Keywords

Introduction

Studies in the health field worldwide have shown that nanoscience has made significant contributions to medicine, particularly in the diagnosis, treatment, and prevention of diseases, as well as drug development. Recently, nanoparticles have been utilized in the diagnosis and treatment of various diseases, including as antimicrobial, antioxidant, antifungal, anticancer, and antidiabetic agents. Green synthesis is the preferred method for obtaining nanoparticles due to its cost-effectiveness, environmental friendliness, ease of preparation, and controllability, as opposed to the use of decaying reaction conditions and hazardous intermediate components.
Zinc oxide nanoparticles are particularly advantageous compared to other metal oxide nanoparticles due to their electrical and optical properties, biocompatibility, low cost, and low toxicity. Additionally, they have the potential to exhibit antibacterial and cytotoxic effects against cancer cells. Zinc oxide nanoparticles are frequently used by researchers due to their wide range of applications in medicine and health science.
The genus Achillea is cultivated in temperate zone countries, including Cyprus, Türkiye, Iran, and Pakistan. Extracts from Achillea species, such as Achillea biebersteinii and Achillea millefolium, have been traditionally used in folk medicine in those countries for their diuretic, appetizing, gas-digesting, wound healing, astringent (especially in haemorrhoids), urinary antiseptic, and antitussive properties. It is also known to be successfully applied in treating inflammatory conditions and pain, such as menstruation and postpartum discomfort.^1,2
This study aims to perform green synthesis of zinc oxide nanoparticles using the extract obtained from Achillea species. Additionally, the study will investigate the anti-DNA damage, antioxidant, and antidiabetic activities of these nanoparticles. The results of this analysis will contribute to the field of green synthesis studies in nanotechnology.

Materials and Methods

Preparation Of Achillea Species ExtractsAchillea biebersteinii and Achillea millefolium were collected from Lapta Hill in Kyrenia, Cyprus. The plant samples were identified, and a voucher specimen was recorded and stored in the Herbarium of Girne American University for reference. The Achillea species studied were dried and powdered using an electric plant grinder. All chemicals used in the study, zinc acetate, were purchased from Merck Company (Germany). The powdered Achillea biebersteinii/millefolium (200 g) was extracted using 2 L of 70% ethanol as a solvent in a 1:10 ratio for 24 hours with regular shaking. The resulting mixture was filtered using a Whatman 1 filter paper. The ethanolic extracts of Achillea biebersteinii/millefolium plant were stored at +4°C in a refrigerator for further research.³
Synthesis Of Zinc Oxide Nanoparticles/AchilleaFirstly, 100 mL of Achillea biebersteinii/millefolium extract and 500 mL of 1 mM zinc acetate solution were left at room temperature for 1 hour. The colour of the mixture changed from yellow to brown, indicating the formation of zinc oxide nanoparticles/Achillea. The solution was then repeatedly centrifuged (10,000 rpm × 10 minutes) and distilled water was added. The solid particles obtained from centrifugation were filtered and dried in the oven at 50°C for 24 hours. The absorbance of zinc oxide nanoparticles/Achillea was measured using a UV spectrophotometer (Shimadzu UV 1800, Japan) in the wavelength range of 200–790 nm.⁴ The structural characterisation and particle size examination of zinc oxide nanoparticles/Achillea were done by Transmission Electron Microscopy and Scanning Electron Microscopy (Hitachi, Japan). In addition, the spectra properties were carried out by Fourier Transform Infrared Spectroscopy (Bruker, Germany) in the spectra range of 4000–400 cm⁻¹. X-ray diffraction of the zinc oxide nanoparticles/Achillea was analysed using an XPert PRO diffractometer (Holland) in the 2Ɵ range of 20–80°.
The Preventive Effect Of Zinc Oxide Nanoparticles/Achillea On DNA DamageTo investigate the protective effect of zinc oxide nanoparticles/Achillea against DNA damage, electrophoresis was performed using PBR322 plasmid DNA (4361 base pairs).⁵ A 1% agarose gel was loaded into five wells. The first well contained plasmids and dye, while the remaining wells contained plasmids and loading dye exposed to hydrogen peroxide and UV rays for DNA damage. Plasmids, loading dye, hydrogen peroxide, and zinc oxide nanoparticles/Achillea solutions were loaded from the third to seventh well in different concentrations (50–100–150–250 and 500 µg/mL). Electrophoresis was then performed to obtain images (Table 1).
Antioxidant Activity Of Zinc Oxide Nanoparticles/Achilleahe lipid peroxidation inhibitory activity of zinc oxide nanoparticles/Achillea was determined using the thiobarbituric acid test.⁶ To prepare the zinc oxide nanoparticles/Achillea solution, the concentration series (50, 100, 150, 250, and 500 mg/L [w/v]) were dissolved in 97% ethanol. Next, 200 μL of zinc oxide nanoparticles/Achillea, 200 μL of FeCl₃, 200 μL of EDTA, 200 μL of H₂O₂, and 200 μL of ascorbic acid were added and mixed with a vortex. The tubes were then incubated at 37°C for 1.5 h. After incubation, 1.2 mL of 28% thiobarbituric acid was added and the mixture was centrifuged at 3000 rpm for 15 minutes. The remaining pellet was then filtered and 1.2 mL of thiobarbituric acid was added again. The tubes containing the samples were boiled for 10 minutes and then cooled to room temperature. The absorbances of the samples were measured at 532 nm using a spectrophotometer. The lipid peroxidation inhibition was calculated using the formula: inhibition = [(Acont – Asamp) / Acont] × 100.
Antidiabetic Effects Of Zinc Oxide Nanoparticles/AchilleaTo investigate the antidiabetic effects of Achillea species, we assessed the enzyme inhibition capacities of the carbohydrate digestive enzymes α-amylase and α-glucosidase.⁷ For the α-amylase test, the enzyme solution was prepared by mixing 27.5 mg of alpha-amylase into 100 mL of distilled water. 1 mL of the α-amylase enzyme (1 U/mL), 0.1 g/mL zinc oxide nanoparticles/Achillea and 10 mL of 6.85 mM NaCl (pH 6.9 adjusted using a phosphate buffer) were mixed and centrifuged at 8500 rpm for 15 minutes. The liquid was separated, and a 1% starch solution of 500 μL in 0.02 M sodium phosphate buffer (pH 6.9) was added to tubes containing five different concentrations (100–500 µg/mL). The reaction was stopped using a colour reagent, 3.5-dinitrosalicylic acid, and the samples were incubated in a water bath at 100°C for 5 minutes. The tubes were then incubated at 25°C for 10 minutes. After cooling to 20°C, 10 mL of distilled water was added to each sample. Absorbance measurements were taken at a wavelength of 540 nm. The α-glucosidase inhibition capacity was also determined. Then, we incubated 1 mL of starch substrate (2% maltose or sucrose), 1 U/mL of α-glucosidase enzyme, and zinc oxide nanoparticles/Achillea samples (50, 100, 150, 250, and 500 mg/L [w/v]) at 37°C for 5 minutes. The mixture was then placed in a boiling water bath for 2 minutes and cooled to 20°C. Absorbance values were measured at 420 nm. We calculated the α-amylase and α-glucosidase inhibitor activity using the same formula.
Ethical ApprovalAll procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article. The ethics committee of Girne American University has stated that there is no need to obtain ethics approval for this reason (Date: 06.06.2023, Decision No: 2023/032).

Results

Synthesis Of Zinc Oxide Nanoparticles/AchilleaThe study synthesised zinc oxide nanoparticles/Achillea from Achillea biebersteinii/millefolium extracts using the green chemistry method. UV spectroscopy, Fourier Transform Infrared Spectroscopy, X-ray diffraction, and Scanning Electron Microscopy data were analysed. Zinc oxide nanoparticles/Achillea formation was analysed using a UV spectrophotometer in the 380–410 nm absorbance range, as previous studies have shown.⁸ In this study, peak values were determined at 405 nm, supporting the synthesis of zinc oxide nanoparticles/Achillea. The Fourier Transform Infrared Spectroscopy results indicate that zinc oxide nanoparticles/Achillea exhibit tensile vibrations in the bands between 440 and 540 cm⁻¹. It is common for metal nanoparticles, including zinc oxide oxides, to exhibit vibration frequencies below 1000 cm⁻¹. Additionally, peaks in the 450 to 600 cm⁻¹ range were observed for zinc oxide nanoparticles/Achillea. Previous studies have reported that C-H molecules (e.g. CH₃, CH₂) in chemical compounds produce peaks in the range of 3500–2800 cm⁻¹. Additionally, stress vibrations of the aromatic groups C=O and C=C in Achillea biebersteinii/millefolium plants are in the range of 1000–1700 cm⁻¹.⁹ (Figure 1)
X-ray diffraction analysis was conducted to determine the crystal structure of the zinc oxide nanoparticles/Achillea synthesized in our study. The peaks in Figure 2 were identified as 34.38, 37.22, 44.79, 49.82, 55.91, 63.04, 65.57, 68.32, and 77.34 for Achillea biebersteinii and 35.02, 42.21, 50.08, 57.33, 62.56, 69.26, and 76.92 for Achillea millefolium. The X-ray diffraction peaks identified in our study were consistent with the results found in previous studies,¹⁰ verifying the hexagonal structure for the nanoparticles synthesized in cross-lattice planes (002), (100), (101), (102), (103), (110), (112), (200), and (202).
The synthesis of zinc oxide nanoparticles/Achillea is confirmed by the zinc peak observed in the energy-dispersive X-ray spectrum. The carbon, potassium, and magnesium peaks, however, are attributed to bioactive components attached to the surface of zinc oxide nanoparticles. The Scanning Electron Microscopy analysis determined that the synthesized zinc oxide nanoparticles/Achillea are evenly distributed and not clustered in specific regions.¹⁰ (Figure 2)
Anti-DNA Damage Activities Of Zinc Oxide Nanoparticles/AchilleaThe study investigated the protective effect of zinc oxide nanoparticles/Achillea against DNA damage using agarose gel electrophoresis and PBR322 plasmid DNA as target DNA. Different concentrations of zinc nanoparticles (50–100–250–150–500 μg/mL zinc oxide nanoparticles/Achillea) were compared in wells. The DNA progressed in the first well but was damaged by hydrogen peroxide and UV from the second well onwards. The gel electrophoresis results indicate that the addition of 50 mg/L nanoparticles in the third well prevented DNA damage, and as the zinc oxide nanoparticles/Achillea ratio increased, the DNA moved more significantly. Furthermore, the addition of zinc oxide nanoparticles/Achillea after the third well prevented DNA damage as the concentration increased. The highest concentration of 500 μg/mL of zinc oxide nanoparticles/Achillea in the seventh well showed an excellent protective effect. Figure 3 shows the agarose gel electrophoresis.
Antioxidant Activity Of Zinc Oxide Nanoparticles/AchilleaWe found that the lipid peroxidation prevention activities of zinc oxide nanoparticles/Achillea increased in direct proportion to the concentration. The highest inhibition value was observed at the nanoparticle level of 500 µg/mL, as determined by the thiobarbituric acid assay. The malondialdehyde levels of zinc oxide nanoparticles/Achillea biebersteinii and zinc oxide nanoparticles/Achillea millefolium were found to be 90.87% and 89.11%, respectively (see Figure 6).
Antidiabetic Effects Of Zinc Oxide Nanoparticles/AchilleaThe antidiabetic effect of zinc oxide nanoparticles/Achillea was compared to that of acarbose (Sigma Aldrich Chemical Co, USA), which was a standard. Acarbose inhibited the α-amylase and α-glucosidase at 64% and 88.52%, respectively. Zinc oxide nanoparticles/Achillea biebersteinii and zinc oxide nanoparticles/Achillea millefolium had effects of 58.12% and 52.55%, and 94.37% and 92.57%, respectively (Table 2).

Discussion

Reactive oxygen species formed in the body can cause rapid oxidation of target molecules, leading to lipid peroxidation in cells, among other biochemical reactions. This process can contribute to various diseases caused by oxidative stress, including neurological diseases, aging, cancer, and heart disease.^11,12 Lipid peroxidation can result in the formation of complex compounds containing reactive carbonyl compounds, such as malondialdehyde. The malondialdehyde measurement is commonly used as a marker of lipid peroxidation in studies related to oxidative stress and redox signalling, particularly in those investigating the antioxidant effects of plants. According to a study,¹³ medicinal plants can prevent lipid peroxidation through their interaction with zinc oxide. The study found that the lipid peroxidation prevention activities of zinc oxide nanoparticles/Achillea increased in direct proportion to the concentration. The highest inhibition value was observed at the nanoparticle level of 500 µg/mL.
Our study aimed to determine the potential antidiabetic effect of Achillea species, a medicinal plant used in the treatment of various diseases, including diabetes, in Türkiye and Cyprus. These plants exert their antidiabetic effects by controlling hyperglycaemia through the inhibition of enzymes such as α-amylase and α-glucosidase, as well as delaying glucose absorption.^14,15 Acarbose was used as a standard, and it was found to have a strong inhibitory activity against α-amylase and α-glucosidase when compared to zinc oxide nanoparticles/Achillea.
Cancer is caused by mutations in cells. Preventing diffraction and mutation of the DNA molecule is crucial, particularly in the treatment of pathological processes such as cancer. Recent studies have focused on synthesising nanocomponents and investigating their effects on preventing DNA damage.¹⁶ In this study, we observed that the addition of 50 mg/L nanoparticles prevented DNA damage in the third well. Additionally, we found that the DNA mobility increased as the zinc oxide nanoparticles/Achillea ratio increased. Furthermore, we observed that the addition of zinc oxide nanoparticles/Achillea after the third well prevented DNA damage as the concentration increased. Finally, we found that the highest concentration of 500 μg/mL of zinc oxide nanoparticles/Achillea had a perfect protective effect. A study was conducted using electrophoresis of agarose gel to examine the biogenic zinc oxide nanoparticles formed by green synthesis mediated by Thymbra spicata L. The study investigated the effectiveness of DNA cleavage ability and found that the protective effect of zinc oxide nanoparticles against DNA damage increased with concentration.¹⁷

Conclusion

Our study investigated the antioxidant effects of ZnO-NPs/A. biebersteinii and ZnO-NPs/A. millefolium, synthesized using the green method. The results showed that the nanoparticles exhibited radical quenching activity and reduced biomolecular agents in plant structure. ZnO-NPs/Ach demonstrated high antioxidant activity, particularly at a concentration of 500 µg/mL. ZnO-NPs/Ach have been found to be potent inhibitors of α-amylase and α-glucosidase, as well as having antidiabetic activity. Additionally, these nanoparticles provide high levels of protection against DNA damage. It has been suggested that ZnO-NPs/A. biebersteinii and ZnO-NPs/A. millefolium could be used as antioxidant or antidiabetic agents to protect cells against DNA damage. Zinc oxide nanoparticles may have potential in the treatment of cancer, diabetes, and other chronic diseases caused by oxidative stress. Our study’s results could be a precursor to further scientific research in the health field. More comprehensive studies should investigate the effects of Achillea plant extracts and the nanoparticles synthesized from them.

Declarations

References

1. Ahmad KS, Hamid A, Nawaz F, et al. Ethnopharmacological studies of indigenous plants in Kel village, Neelum Valley, Azad Kashmir, Pakistan. J Ethnobiol Ethnomed. 2017;13(1):68. doi:10.1186/s13002-017-0196-1
   Article PubMed Google Scholar
2. Bina F, Daglia M, Santarcangelo C, et al. Phytochemical profiling and ameliorative effects of Achillea cretica L. on rat model of endometriosis. J Ethnopharmacol. 2020;254:112747. doi:10.1016/j.jep.2020.112747
   Article PubMed Google Scholar
3. Gul Z, Akbar A, Leghari SK. Elucidating therapeutic and biological potential of Berberis baluchistanica Ahrendt bark, leaf, and root extracts. Front Microbiol. 2022;13:823673. doi:10.3389/fmicb.2022.823673
   Article PubMed Google Scholar
4. Joseph S, Mathew B. Microwave-assisted green synthesis of silver nanoparticles and the study on catalytic activity in the degradation of dyes. J Mol Liq. 2015;204:184-191. doi:10.1016/j.molliq.2015.01.027
   Article PubMed Google Scholar
5. Silva S, Costa EM, Vicente S, et al. DNA agarose gel electrophoresis for antioxidant analysis: development of a quantitative approach for phenolic extracts. Food Chem. 2017;233:45-51. doi:10.1016/j.foodchem.2017.04.075
   Article PubMed Google Scholar
6. Lo KM, Cheung CK. Antioxidant activity of extracts from the fruiting bodies of Agrocybe aegerita var alba. Food Chem. 2005;89:533-539. doi:10.1016/j.foodchem.2004.03.006
   Article PubMed Google Scholar
7. Meydan I, Burhan H, Gür T, Seçkin H, Tanhaei B, Sen F. Characterization of Rheum ribes with ZnO nanoparticle and its antidiabetic, antibacterial, DNA damage prevention, and lipid peroxidation prevention activity in vitro. Environ Res. 2022;204(Pt D):112363. doi:10.1016/j.envres.2021.112363
   Article PubMed Google Scholar
8. Rajeshkumar S, Parameswari RP, Sandhiya D, et al. Green synthesis, characterization, and bioactivity of Mangifera indica seed-wrapped zinc oxide nanoparticles. Molecules. 2023;28(6):2818. doi:10.3390/molecules28062818
   Article PubMed Google Scholar
9. Yuvakkumar R, Suresh J, Saravanakumar B, Joseph Nathanael A, Hong SI, Rajendran V. Rambutan peels promoted biomimetic synthesis of bioinspired zinc oxide nanochains for biomedical applications. Spectrochim Acta A Mol Biomol Spectrosc. 2015;137:250-258. doi:10.1016/j.saa.2014.08.022
   Article PubMed Google Scholar
10. Khodadadi B, Bordbar M, Nasrollahzadeh M. Achillea millefolium L. extract-mediated green synthesis of waste peach kernel shell-supported silver nanoparticles: application of the nanoparticles for catalytic reduction of a variety of dyes in water. J Colloid Interface Sci. 2017;493:85-93. doi:10.1016/j.jcis.2017.01.012
    Article PubMed Google Scholar
11. Caylak E, Halifeoglu I, Aydin S, Telo S, Bulmus O, Celik H. The effects of sulfur-containing compounds on total antioxidant capacity levels of liver, kidney, and brain in lead-exposed rats. Turkiye Klinikleri J Med Sci. 2007;27(6):823-828.
    PubMed Google Scholar
12. Caylak E, Aytekin M, Halifeoglu I. Antioxidant effects of methionine, α-lipoic acid, N-acetylcysteine, and homocysteine on lead-induced oxidative stress to erythrocytes in rats. Exp Toxicol Pathol. 2008;60(4-5):289-294. doi:10.1016/j.etp.2007.11.004
    Article PubMed Google Scholar
13. Reddy Pullagurala VL, Adisa IO, Rawat S, et al. ZnO nanoparticles increase photosynthetic pigments and decrease lipid peroxidation in soil-grown cilantro (Coriandrum sativum). Plant Physiol Biochem. 2018;132:120-127. doi:10.1016/j.plaphy.2018.08.037
    Article PubMed Google Scholar
14. Chávez-Silva F, Cerón-Romero L, Arias-Durán L, et al. Antidiabetic effect of Achillea millefolium through multitarget interactions: α-glucosidases inhibition, insulin sensitization, and insulin secretagogue activities. J Ethnopharmacol. 2018;212:1-7. doi:10.1016/j.jep.2017.10.005
    Article PubMed Google Scholar
15. Ramírez G, Zavala M, Pérez J, Zamilpa A. In vitro screening of medicinal plants used in Mexico as antidiabetics with glucosidase and lipase inhibitory activities. Evid Based Complement Alternat Med. 2012;2012:701261. doi:10.1155/2012/701261
    Article PubMed Google Scholar
16. Srivastava AK, Bhatnagar P, Singh M, et al. Synthesis of PLGA nanoparticles of tea polyphenols and their strong in vivo protective effect against chemically induced DNA damage. Int J Nanomedicine. 2013;8:1451-1462.
    PubMed Google Scholar
17. Gur T, Meydan I, Seckin H, Bekmezci M, Sen F. Green synthesis, characterization, and bioactivity of biogenic zinc oxide nanoparticles. Environ Res. 2022;204(Pt A):111897. doi:10.1016/j.envres.2021.111897
    Article PubMed Google Scholar

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About This Article

Received:

November 22, 2023

Accepted:

January 30, 2024

Published Online:

January 31, 2024

Printed:

February 1, 2024