Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities
Abstract
:1. Introduction
2. Results
2.1. Identification of Volatile Components in Hydrosols from T. majus Seeds
2.2. Cytotoxic Activity
2.3. Scratch Assay
2.4. Antibacterial Activity
2.5. Bacterial Growth Kinetics
2.6. Antiadhesion Activity
2.7. Antiphytoviral Activity
3. Discussion
4. Materials and Methods
4.1. Plant Material and Reagents
4.2. Preparation of the Samples and Analyses of Hydrosols
4.3. Gas Chromatography and Mass Spectrometry
4.4. Cytotoxic Activity
4.5. Scratch Assay
4.6. Antibacterial Activity
4.7. Antibacterial Susceptibility
4.8. Bacterial Growth Kinetics
4.9. Antiadhesion Assay
4.10. Antiphytoviral Activity Assay
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Niizu, P.Y.; Rodriguez-Amaya, D.B. Flowers and Leaves of Tropaeolum majus L. as Rich Sources of Lutein. J. Food Sci. 2006, 70, S605–S609. [Google Scholar] [CrossRef]
- Vrca, I.; Ramić, D.; Fredotović, Ž.; Možina, S.S.; Blažević, I.; Bilušić, T. Chemical Composition and Biological Activity of Essential Oil and Extract from the Seeds of Tropaeolum majus L. Var. Altum. Food Technol. Biotechnol. 2022, 60, 533–542. [Google Scholar] [CrossRef] [PubMed]
- Calil Brondani, J.; Cuelho, C.; Marangoni, L.; Lima, R.; Guex, C.; Bonilha, I.; Manfron, M. Traditional Usages, Botany, Phytochemistry, Biological Activity and Toxicology of Tropaeolum majus L. A Review. Bol. Latinoam. Caribe Plantas Med. Aromat. 2016, 15, 264–273. [Google Scholar]
- Blažević, I.; Đulović, A.; Burčul, F.; Popović, M.; Montaut, S.; Bilušić, T.; Vrca, I.; Markić, J.; Ljubenkov, I.; Ruščić, M.; et al. Stability and Bioaccessibility during Ex Vivo Digestion of Glucoraphenin and Glucoraphasatin from Matthiola incana (L.) R. Br. J. Food Comp. Anal. 2020, 90, 103483. [Google Scholar] [CrossRef]
- Romeo, L.; Iori, R.; Rollin, P.; Bramanti, P.; Mazzon, E. Isothiocyanates: An Overview of Their Antimicrobial Activity against Human Infections. Molecules 2018, 23, 624. [Google Scholar] [CrossRef]
- Wagner, A.E.; Boesch-Saadatmandi, C.; Dose, J.; Schultheiss, G.; Rimbach, G. Anti-Inflammatory Potential of Allyl-Isothiocyanate–Role of Nrf2, NF-ΚB and MicroRNA-155. J. Cell Mol. Med. 2012, 16, 836–843. [Google Scholar] [CrossRef]
- Li, H.; Ming, X.; Wang, Z.; Li, J.; Liang, Y.; Xu, D.; Liu, Z.; Hu, L.; Li, H.; Ming, X.; et al. Encapsulation of Benzyl Isothiocyanate with β-Cyclodextrin Using Ultrasonication: Preparation, Characterization, and Antibacterial Assay. Foods 2022, 11, 3724. [Google Scholar] [CrossRef]
- Vrca, I.; Burčul, F.; Blažević, I.; Bratanić, A.; Bilušić, T. Comparison of Gastrointestinal Stability of Isothiocyanates from Tropaeolum majus L. Altum Using In Vitro and Ex Vivo Digestion Methods. Croat. J. Food Sci. Technol. 2021, 13, 160–166. [Google Scholar] [CrossRef]
- Mostafa, A.A.; Al-Askar, A.A.; Almaary, K.S.; Dawoud, T.M.; Sholkamy, E.N.; Bakri, M.M. Antimicrobial Activity of Some Plant Extracts against Bacterial Strains Causing Food Poisoning Diseases. Saudi J. Biol. Sci. 2018, 25, 361–366. [Google Scholar] [CrossRef]
- Uppal, S.; Sharma, P.; Kumar, R.; Kaur, K.; Bhatia, A.; Mehta, S.K. Effect of Benzyl Isothiocyanate Encapsulated Biocompatible Nanoemulsion Prepared via Ultrasonication on Microbial Strains and Breast Cancer Cell Line MDA MB 231. Colloids Surf. A Physicochem. Eng. Asp. 2020, 596, 124732. [Google Scholar] [CrossRef]
- Fouché, M.; Willers, C.; Hamman, S.; Malherbe, C.; Steenekamp, J. Wound Healing Effects of Aloe Muth-Muth: In Vitro Investigations Using Immortalized Human Keratinocytes (HaCaT). Biology 2020, 9, 350. [Google Scholar] [CrossRef] [PubMed]
- Goels, T.; Eichenauer, E.; Tahir, A.; Prochaska, P.; Hoeller, F.; Heiß, E.H.; Glasl, S. Exudates of Picea abies, Pinus nigra, and Larix decidua: Chromatographic Comparison and Pro-Migratory Effects on Keratinocytes In Vitro. Plants 2022, 11, 599. [Google Scholar] [CrossRef] [PubMed]
- Vuko, E.; Radman, S.; Jerković, I.; Kamenjarin, J.; Vrkić, I.; Fredotović, Ž. A Plant Worthy of Further Study—Volatile and Non-Volatile Compounds of Portenschlagiella ramosissima (Port.) Tutin and Its Biological Activity. Pharmaceuticals 2022, 15, 1454. [Google Scholar] [CrossRef] [PubMed]
- Vuko, E.; Rusak, G.; Dunkic, V.; Kremer, D.; Kosalec, I.; Rada, B.; Bezic, N. Inhibition of Satellite RNA Associated Cucumber Mosaic Virus Infection by Essential Oil of Micromeria Croatica (Pers.) Schott. Molecules 2019, 24, 1342. [Google Scholar] [CrossRef]
- Moeini, A.; van Reenen, A.; Van Otterlo, W.; Cimmino, A.; Masi, M.; Lavermicocca, P.; Valerio, F.; Immirzi, B.; Santagata, G.; Malinconico, M.; et al. α-Costic Acid, a Plant Sesquiterpenoid from Dittrichia viscosa, as Modifier of Poly (Lactic Acid) Properties: A Novel Exploitation of the Autochthone Biomass Metabolite for a Wholly Biodegradable System. Ind. Crops Prod. 2020, 146, 112134. [Google Scholar] [CrossRef]
- Vrca, I.; Čikeš Čulić, V.; Lozić, M.; Dunkić, N.; Kremer, D.; Ruščić, M.; Nazlić, M.; Dunkić, V. Isolation of Volatile Compounds by Microwave-Assisted Extraction from Six Veronica Species and Testing of Their Antiproliferative and Apoptotic Activities. Plants 2023, 12, 3244. [Google Scholar] [CrossRef]
- Vrca, I.; Šćurla, J.; Kević, N.; Burčul, F.; Čulić, V.Č.; Bočina, I.; Blažević, I.; Bratanić, A.; Bilušić, T. Influence of Isolation Techniques on the Composition of Glucosinolate Breakdown Products, Their Antiproliferative Activity and Gastrointestinal Stability of Allyl Isothiocyanate. Eur. Food Res. Technol. 2022, 248, 567–576. [Google Scholar] [CrossRef]
- Shafie, M.H.; Kamal, M.L.; Razak, N.A.A.; Hasan, S.; Uyup, N.H.; Rashid, N.F.A.; Zafarina, Z. Antioxidant and Antimicrobial Activity of Plant Hydrosol and Its Potential Application in Cosmeceutical Products. Jundishapur J. Nat. Pharm. Prod. 2022, 17, 124018. [Google Scholar] [CrossRef]
- Jakubczyk, K.; Tuchowska, A.; Janda-Milczarek, K. Plant Hydrolates–Antioxidant Properties, Chemical Composition and Potential Applications. Biomed. Pharmacother. 2021, 142, 112033. [Google Scholar] [CrossRef]
- Nazlić, M.; Dunkić, V.; Dželalija, M.; Maravić, A.; Mandić, M.; Srečec, S.; Vrca, I.; Vuko, E.; Kremer, D. Evaluation of Antiphytoviral and Antibacterial Activity of Essential Oil and Hydrosol Extracts from Five Veronica Species. Agriculture 2023, 13, 1517. [Google Scholar] [CrossRef]
- Butnariu, M.; Bostan, C. Antimicrobial and Anti-Inflammatory Activities of the Volatile Oil Compounds from Tropaeolum majus L. (Nasturtium). Afr. J. Biotechnol. 2011, 10, 5900–5909. [Google Scholar]
- Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing: Carol Stream, IL, USA, 2017; ISBN 978-1-932633-21-4. [Google Scholar]
- Bloem, E.; Berk, A.; Haneklaus, S.; Selmar, D.; Schnug, E.; Landbauforschung; Bloem, E.; Berk, A.; Haneklaus, S.; Selmar, D.; et al. Influence of Tropaeolum majus Supplements on Growth and Antimicrobial Capacity of Glucotropaeolin in Piglets. Forestry Res. 2008, 3, 203–210. [Google Scholar]
- Benyelles, B.; Allali, H.; Belkaid, A.B.; Fekih, N.; Muselli, A. Chemical Composition of the Volatile Components of Tropaeolum majus L. (Garden Nasturtium) from North Western Algeria. PhytoChem BioSub J. 2015, 9, 92–97. [Google Scholar]
- Kissen, R.; Bones, A.M. Nitrile-Specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana. J. Biol. Chem. 2009, 284, 12057–12070. [Google Scholar] [CrossRef]
- Matusheski, N.V.; Juvik, J.A.; Jeffery, E.H. Heating Decreases Epithiospecifier Protein Activity and Increases Sulforaphane Formation in Broccoli. Phytochemistry 2004, 65, 1273–1281. [Google Scholar] [CrossRef] [PubMed]
- Wielanek, M.; Urbanek, H.; Majorowicz, H. Endogenous Hydrolysis of Glucotropaeolin to Benzyl Isothiocyanate in Hairy Root Cultures of Tropaeolum majus L. Biotechnologia 2004, 86, 210–220. [Google Scholar]
- Lee, W.; Song, G.; Bae, H. Glucotropaeolin Promotes Apoptosis by Calcium Dysregulation and Attenuates Cell Migration with FOXM1 Suppression in Pancreatic Cancer Cells. Antioxidants 2023, 12, 257. [Google Scholar] [CrossRef] [PubMed]
- Lawson, A.P.; Long, M.J.C.; Coffey, R.T.; Qian, Y.; Weerapana, E.; El Oualid, F.; Hedstrom, L. Naturally Occurring Isothiocyanates Exert Anticancer Effects by Inhibiting Deubiquitinating Enzymes. Cancer Res. 2015, 75, 5130–5142. [Google Scholar] [CrossRef] [PubMed]
- Wah Han, K.W.; Wah Po, W.; Dong Sohn, U.; Hyun-Jung, K. Benzyl Isothiocyanate Induces Apoptosis via Reactive Oxygen Species-Initiated Mitochondrial Dysfunction and DR4 and DR5 Death Receptor Activation in Gastric Adenocarcinoma Cells. Biomolecules 2019, 9, 839. [Google Scholar] [CrossRef]
- Jang, M.; Hong, E.; Kim, G.H. Evaluation of Antibacterial Activity of 3-Butenyl, 4-Pentenyl, 2-Phenylethyl, and Benzyl Isothiocyanate in Brassica Vegetables. J. Food Sci. 2010, 75, M412–M416. [Google Scholar] [CrossRef]
- Owis, A.I.; Sherif, N.H.; Hassan, A.A.; EL-Naggar, E.M.B.; EL-Khashab, I.H.; EL-Ghaly, E.S. Tropaeolum Majus L. and Low Dose Gamma Radiation Suppress Liver Carcinoma Development via EGFR-HER2 Signaling Pathway. Nat. Prod. Res. 2023, 37, 1030–1035. [Google Scholar] [CrossRef] [PubMed]
- Espinoza Silva, C.R. Evaluación Invitro de La Citotoxicidad de Extractos Fenólicos de Pétalos de Flores Anaranjadas de Tropaeolum majus L. En Líneas Celulares Cancerígenas de Mama; Universidad Peruana Los Andes: Huancayo, Peru, 2019. [Google Scholar]
- Pintao, A.M.; Pais, M.S.S.; Coley, H.; Kelland, L.R.; Judson, I.R. In Vitro and in Vivo Antitumor Activity of Benzyl Isothiocyanate: A Natural Product from Tropaeolum majus. Planta Med. 1995, 61, 233–236. [Google Scholar] [CrossRef]
- Silva-Correa, C.R.; Pazo-Medina, G.I.; Villarreal-La Torre, V.E.; Calderón-Peña, A.A.; Aspajo-Villalaz, C.L.; Cruzado-Razco, J.L.; Rosario-Chávarri, J.D.; González-Siccha, A.D.; Guerrero-Espino, L.M.; González-Blas, M.V.; et al. Wound Healing Activity of Tropaeolum tuberosum-Based Topical Formulations in Mice. Vet. World 2022, 15, 390–396. [Google Scholar] [CrossRef]
- Ojeh, N.; Stojadinovic, O.; Pastar, I.; Sawaya, A.; Yin, N.; Tomic-Canic, M. The Effects of Caffeine on Wound Healing. Int. Wound J. 2016, 13, 605–613. [Google Scholar] [CrossRef] [PubMed]
- Alishahedani, M.E.; Yadav, M.; McCann, K.J.; Gough, P.; Castillo, C.R.; Matriz, J.; Myles, I.A. Therapeutic Candidates for Keloid Scars Identified by Qualitative Review of Scratch Assay Research for Wound Healing. PLoS ONE 2021, 16, e0253669. [Google Scholar] [CrossRef] [PubMed]
- Atef, N.M.; Shanab, S.M.; Negm, S.I.; Abbas, Y.A. Evaluation of Antimicrobial Activity of Some Plant Extracts against Antibiotic Susceptible and Resistant Bacterial Strains Causing Wound Infection. Bull. Nat. Res. Cent. 2019, 43, 1–11. [Google Scholar] [CrossRef]
- Hemeg, H.A.; Moussa, I.M.; Ibrahim, S.; Dawoud, T.M.; Alhaji, J.H.; Mubarak, A.S.; Kabli, S.A.; Alsubki, R.A.; Tawfik, A.M.; Marouf, S.A. Antimicrobial Effect of Different Herbal Plant Extracts against Different Microbial Population. Saudi J. Biol. Sci. 2020, 27, 3221–3227. [Google Scholar] [CrossRef]
- Gonelimali, F.D.; Lin, J.; Miao, W.; Xuan, J.; Charles, F.; Chen, M.; Hatab, S.R. Antimicrobial Properties and Mechanism of Action of Some Plant Extracts against Food Pathogens and Spoilage Microorganisms. Front. Microbiol. 2018, 9, 389103. [Google Scholar] [CrossRef]
- Manandhar, S.; Luitel, S.; Dahal, R.K. In Vitro Antimicrobial Activity of Some Medicinal Plants against Human Pathogenic Bacteria. J. Trop. Med. 2019, 2019, 1895340. [Google Scholar] [CrossRef]
- Kaiser, S.J.; Mutters, N.T.; Blessing, B.; Günther, F. Natural Isothiocyanates Express Antimicrobial Activity against Developing and Mature Biofilms of Pseudomonas Aeruginosa. Fitoterapia 2017, 119, 57–63. [Google Scholar] [CrossRef]
- Kuete, V. Potential of Cameroonian Plants and Derived Products against Microbial Infections: A Review. Planta Med. 2010, 76, 1479–1491. [Google Scholar] [CrossRef]
- Taglienti, A.; Donati, L.; Ferretti, L.; Tomassoli, L.; Sapienza, F.; Sabatino, M.; Di Massimo, G.; Fiorentino, S.; Vecchiarelli, V.; Nota, P.; et al. In Vivo Antiphytoviral Activity of Essential Oils and Hydrosols From Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis to Control Zucchini Yellow Mosaic Virus and Tomato Leaf Curl New Delhi Virus in Cucurbita pepo L. Front. Microbiol. 2022, 13, 840893. [Google Scholar] [CrossRef]
- Nie, L.X.; Wu, Y.L.; Dai, Z.; Ma, S.C. Antiviral Activity of Isatidis Radix Derived Glucosinolate Isomers and Their Breakdown Products against Influenza A in Vitro/Ovo and Mechanism of Action. J. Ethnopharmacol. 2020, 251, 112550. [Google Scholar] [CrossRef] [PubMed]
- Hegazy, A.; Mostafa, I.; Elshaier, Y.A.M.M.; Mahmoud, S.H.; Abo Shama, N.M.; Shehata, M.; Yahya, G.; Nasr, N.F.; El-Halawany, A.M.; Ali, M.A.; et al. Robust Antiviral Activity of Santonica Flower Extract (Artemisia cina) against Avian and Human Influenza A Viruses: In Vitro and Chemoinformatic Studies. ACS Omega 2022, 7, 41212–41223. [Google Scholar] [CrossRef]
- Shattuck, V.I. Glucosinolates and Glucosinolate Degradation in Seeds from Turnip Mosaic Virus-Infected Rapid Cycle Brassica campestris L. Plants. J. Exp. Bot. 1993, 44, 963–970. [Google Scholar] [CrossRef]
- Nazlić, M.; Fredotović, Ž.; Vuko, E.; Fabijanić, L.; Kremer, D.; Stabentheiner, E.; Ruščić, M.; Dunkić, V. Wild Species Veronica officinalis L. and Veronica saturejoides Vis. Ssp. Saturejoides—Biological Potential of Free Volatiles. Horticulturae 2021, 7, 295. [Google Scholar] [CrossRef]
- Vuko, E.; Dunkić, V.; Ruščić, M.; Nazlić, M.; Mandić, N.; Soldo, B.; Šprung, M.; Fredotović, Ž. Chemical Composition and New Biological Activities of Essential Oil and Hydrosol of Hypericum perforatum L. Ssp. Veronense (Schrank) H. Lindb. Plants 2021, 10, 1014. [Google Scholar] [CrossRef]
- Nazlić, M.; Akrap, K.; Kremer, D.; Dunkić, V. Hydrosols of Veronica Species—Natural Source of Free Volatile Compounds with Potential Pharmacological Interest. Pharmaceuticals 2022, 15, 1378. [Google Scholar] [CrossRef]
- Dunkić, V.; Kosalec, I.; Kosir, I.; Potocnik, T.; Cerenak, A.; Koncic, M.; Vitali, D.; Muller, I.; Kopricanec, M.; Bezic, N.; et al. Antioxidant and Antimicrobial Properties of Veronica spicata L. (Plantaginaceae). Curr. Drug Targets 2015, 16, 1660–1670. [Google Scholar] [CrossRef]
- Mass Spectrometry Data Center|NIST. Available online: https://www.nist.gov/mml/biomolecular-measurement/mass-spectrometry-data-center (accessed on 12 September 2022).
- Fredotović, Ž.; Soldo, B.; Šprung, M.; Marijanović, Z.; Jerković, I.; Puizina, J. Comparison of Organosulfur and Amino Acid Composition between Triploid Onion Allium cornutum Clementi Ex Visiani, 1842, and Common Onion Allium cepa L., and Evidences for Antiproliferative Activity of Their Extracts. Plants 2020, 9, 98. [Google Scholar] [CrossRef]
- Klančnik, A.; Piskernik, S.; Jeršek, B.; Možina, S.S. Evaluation of Diffusion and Dilution Methods to Determine the Antibacterial Activity of Plant Extracts. J. Microbiol. Methods 2010, 81, 121–126. [Google Scholar] [CrossRef] [PubMed]
- Schön, T.; Werngren, J.; Machado, D.; Borroni, E.; Wijkander, M.; Lina, G.; Mouton, J.; Matuschek, E.; Kahlmeter, G.; Giske, C.; et al. Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis Complex Isolates–The EUCAST Broth Microdilution Reference Method for MIC Determination. CMI 2020, 26, 1488–1492. [Google Scholar] [CrossRef] [PubMed]
- Šikić Pogačar, M.; Klančnik, A.; Bucar, F.; Langerholc, T.; Smole Možina, S. Anti-Adhesion Activity of Thyme (Thymus vulgaris L.) Extract, Thyme Post-Distillation Waste, and Olive (Olea europea L.) Leaf Extract against Campylobacter jejuni on Polystyrene and Intestine Epithelial Cells. J. Sci. Food. Agric. 2016, 96, 2723–2730. [Google Scholar] [CrossRef] [PubMed]
Volatile Compound in Hydrosols of T. majus | RI | MAE (%) | MHG (%) |
---|---|---|---|
α-Thujene | 924 | 1.71 | 5.25 |
Benzaldehyde | 952 | 9.91 | 1.86 |
Benzene acetaldehyde | 1036 | 0.22 | 0.43 |
Benzyl cyanide (BCN) | 1140 | 15.02 | 65.33 |
Carvone | 1241 | 0.25 | 2.31 |
Benzyl isothiocyanate (BITC) | 1371 | 62.29 | 17.89 |
Methyl eugenol * | 1403 | 1.59 | 1.11 |
Caryophyllene oxide * | 1581 | 2.51 | – |
Total identification (%) | 93.5 | 94.18 |
T. majus L. HY | Concentration (µg/mL) | % Cell-Free Area | SEM | p Value |
---|---|---|---|---|
HY isolated using MAE | 5 | 63.40 | 4.69 | p > 0.05 |
HY isolated using MAE | 20 | 61.41 | 6.53 | p > 0.05 |
HY isolated using MHG | 15 | 77.10 | 3.02 | * p < 0.05 |
T. majus Samples | S. aureus ATCC 25,923 | E. coli ATCC 11,229 |
---|---|---|
γ/(mg/mL) MIC | γ/(mg/mL) MIC | |
HY isolated using MAE | >0.5 | >0.5 |
HY isolated using MHG | >2 | >2 |
dpi | LLN ± SD | ||
---|---|---|---|
C | PT-HYMAE | PT-HYMHG | |
4th | 10.63 ± 0.98 | 0.89 ± 1.08 *** | 0 ± 0.0 *** |
7th | 9.83 ± 1.24 | 1.39 ± 1.16 *** | 0 ± 0.0 *** |
14th | 10.51 ± 1.27 | 1.67 ± 0.92 *** | 0 ± 0.0 *** |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Vrca, I.; Jug, B.; Fredotović, Ž.; Vuko, E.; Brkan, V.; Šestić, L.; Juretić, L.; Dunkić, V.; Nazlić, M.; Ramić, D.; et al. Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities. Plants 2023, 12, 3897. https://doi.org/10.3390/plants12223897
Vrca I, Jug B, Fredotović Ž, Vuko E, Brkan V, Šestić L, Juretić L, Dunkić V, Nazlić M, Ramić D, et al. Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities. Plants. 2023; 12(22):3897. https://doi.org/10.3390/plants12223897
Chicago/Turabian StyleVrca, Ivana, Blaž Jug, Željana Fredotović, Elma Vuko, Valentina Brkan, Loriana Šestić, Lea Juretić, Valerija Dunkić, Marija Nazlić, Dina Ramić, and et al. 2023. "Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities" Plants 12, no. 22: 3897. https://doi.org/10.3390/plants12223897