In Vivo Evaluation of Genotoxicity and Antioxidant Capacity of Ajuga Orientalis L. (Lamiaceae) Leaf Extracts

Mohammad M. Al-Gharaibeh; Ahmed O.  Maslat; Noor Alyaqin A. Kanakr; Yazun B. Jarra

doi:10.35516/jjps.v17i4.2466

Authors

Mohammad M. Al-Gharaibeh Department of Plant Production, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, Jordan
Ahmed O. Maslat Department of Biology, Faculty of Science, Yarmouk University, Irbid, Jordan.
Noor Alyaqin A. Kanakr Department of Biology, Faculty of Science, Yarmouk University, Irbid, Jordan.
Yazun B. Jarra Department of Basic Medical Sciences, Faculty of Medicine, Al-Balqa Applied University, Al-Salt, Jordan.

DOI:

https://doi.org/10.35516/jjps.v17i4.2466

Keywords:

medicinal plant, micronucleus, oriental bugle, total antioxidant capacity

Abstract

Ajuga orientalis L. (Lamiaceae) is a fragrant herb native to the Eastern Mediterranean region, widely used in traditional healing practices in Jordan and neighboring countries. Despite its extensive use, there is a lack of toxicological studies on its leaf extracts. This study aims to address this gap by evaluating the genotoxic potential of ethanolic and aqueous leaf extracts using a micronucleus (MN) assay on mice DNA, alongside assessing their antioxidant status. The median lethal dose 50% (LD50) was tested in ten groups of sixty male Balb/c mice to determine the acute toxicity of A. orientalis leaf extracts. Four groups of male Balb/c mice (n=6) were used to evaluate micronucleus (MN) formation and total antioxidant capacity for each extract. Each group received daily intraperitoneal injections of one of the following concentrations: 4000, 2000, 1000, and 500 mg/kg over 28 days. Additionally, three control groups were included for comparison purposes. Peripheral blood samples were screened for MN formation, and liver samples were assessed for total antioxidant capacity. Results revealed an LD50 of 4000 mg/kg for both extracts, alongside a significant dose-dependent increase in MN formation and lower antioxidant capacity compared to controls. The findings indicate the genotoxicity of A. orientalis leaf extracts in Balb/c mice, urging caution in human consumption. Further research is warranted to comprehensively assess their safety and toxicity, especially considering their traditional medicinal use.

References

Oran S.A., Althaher A.R. and Shhab M.A. Al Chemical Composition , in Vitro Assessment of Antioxidant Properties and Cytotoxicity Activity of Ethanolic and Aqueous Extracts of Ajuga Orientalis L . ( Lamiaceae ). J. Pharm. Pharmacogn. Res. 2022; 10, 486–495. DOI: https://doi.org/10.56499/jppres22.1344_10.3.486

Mensah M.L.K., Komlaga G., Forkuo A.D., Firempong C., Anning K., Komlaga G., Dickson R.A., Forkuo D., Firempong C., Anning K., et al. Toxicity and Safety Implications of Herbal Medicines Used in Africa Toxicity and Safety Implications of Herbal Medicines Used in Africa. In Herbal Medicine; Builders, P.F., Ed.; IntechOpen, 2019.

Kahaliw W., Hellman B. and Engidawork E. Genotoxicity Study of Ethiopian Medicinal Plant Extracts on HepG2 Cells. BMC Complement. Altern. Med. 2018; 18: 1–9. doi: 10.1186/s12906-017-2056-x. DOI: https://doi.org/10.1186/s12906-017-2056-x

V K. Toxicological Survey of African Medicinal Plants; V, K., Ed.; 1st ed.; London: Elsevier Inc. 2014.

Ferreira-machado S.C., Rodrigues M.P., Nunes A.P.M., Bezerra R.J.A.C., Caldeira-de-araujo A. Genotoxic Potentiality of Aqueous Extract Prepared from Chrysobalanus Icaco L . Leaves. Toxicol. Lett. 2004.

: 481–487, doi: 10.1016/j.toxlet.2004.03.014. DOI: https://doi.org/10.1016/j.toxlet.2004.03.014

Bardoloi A. and Soren A.D. Genotoxicity Induced by Medicinal Plants. Bull. Natl. Res. Cent. 2022. doi: 10.1186/s42269-022-00803-2. DOI: https://doi.org/10.1186/s42269-022-00803-2

Melo-Reis P., Bezerra L., Vale M., Canhête R., Chen-Chen L. Assessment of the Mutagenic and Antimutagenic Activity of Synadenium Umbellatum Pax Latex by Micronucleus Test in Mice. Brazilian J. Biol. 2011; 71: 169–174. doi: 10.1590/s1519-69842011000100024. DOI: https://doi.org/10.1590/S1519-69842011000100024

Verschaeve L. NPC Natural Product Communications. Nat. Prod. Commun. 2015; 10: 1489–1493. doi: 10.1177/1934578X1501000843. DOI: https://doi.org/10.1177/1934578X1501000843

Yonekubo B.T., Miranda H. D.e., Alves C., Marques D.S., Perazzo F.F., César P., Rosa P., Gaivão O.N.D.M., Maistro E.L., Marques S., et al. The Genotoxic Effects of Fruit Extract of Crataegus Oxyacantha ( Hawthorn ) in Mice. J. Toxicol. Environ. Heal. Part A. 2018; 81: 974–982. doi: 10.1080/15287394.2018.1503982. DOI: https://doi.org/10.1080/15287394.2018.1503982

Shin S., Yi J.M., Kim N.S., Chan-Sung Park Kim S.H., Ok-Sun Bang Aqueous Extract of Forsythia Viridissima Fruits: Acute Oral Toxicity and Genotoxicity Studies. J. Ethnopharmacol. 2020; 249: 112381. doi: 10.1016/j.jep.2019.112381. DOI: https://doi.org/10.1016/j.jep.2019.112381

Harutyunyan K., Balayan K., Tadevosyan G., Hayrapetyan M., Musayelyan R., Grigoryan R., Khondkaryan L., Sarkisyan N. and Babayan N. Genotoxic Potential of Selected Medicinal Plant Extracts in Human Whole Blood Cultures. J. HerbMed Pharmacol. 2019; 8: 160–162, doi: 10.15171/jhp.2019.25. DOI: https://doi.org/10.15171/jhp.2019.25

Adwan G. In Vitro Assessment of Antibacterial Activity and Potential Genotoxic Effect of Fruit Extracts of Capparis Spinosa L . Plant. Jordan J. Pharm. Sci. 2023; 16: 322–329. DOI: https://doi.org/10.35516/jjps.v16i2.517

Oran S.A. The Status of Medicinal Plants in Jordan. J. Agric. Sci. Technol. A. 2014; 4, 461–467.

Abu-Irmaileh B.E. and Afifi F.U. Herbal Medicine in Jordan with Special Emphasis on Commonly Used Herbs. J. Ethnopharmacol. 2003; 89: 193–197. doi: 10.1016/S0378-8741(03)00283-6. DOI: https://doi.org/10.1016/S0378-8741(03)00283-6

El-alali A., Zoubi A. A.l., Gharaibeh M., Tawaha K. and Alali F.Q. Phytochemical and Biological Investigation of Nitraria Retusa Asch. Jordan J. Pharm. Sci. 2012; 5: 155–163.

Uritu C.M., Mihai C.T., Stanciu G.D., Dodi G., Alexa-Stratulat T., Luca A., Leon-Constantin M.M., Stefanescu R., Bild V., Melnic S., et al. Medicinal Plants of the Family Lamiaceae in Pain Therapy: A Review. Pain Res. Manag. 2018; 2018. doi: 10.1155/2018/7801543. DOI: https://doi.org/10.1155/2018/7801543

Abu-Odeh A., Fino L., Al-Absi G., Alnatour D., Al-Darraji M., Shehadeh M. and Suaifan G. Medicinal Plants of Jordan: Scoping Review. Heliyon. 2023; 9: e17081. doi: 10.1016/j.heliyon.2023.e17081. DOI: https://doi.org/10.1016/j.heliyon.2023.e17081

Brahmi F., Khaled-khodja N., Bezeghouche R., Bouharis S., Elsebai M.F., Madani K. and Boulekbache-makhlouf L. Ethnobotanical Study of the Most Lamiaceae Used as Medicinal and Culinary Plants by the Population of Bejaia Province , Algeria. Jordan J. Pharm. Sci. 2023; 16: 268–281. DOI: https://doi.org/10.35516/jjps.v16i2.1330

Taha-Salaime L., Davidovich-Rikanati R., Sadeh A., Abu-Nassar J., Marzouk-Kheredin S., Yahyaa Y., Ibdah M., Ghanim M., Lewinsohn E., Inbar M., et al. Phytoecdysteroid and Clerodane Content in Three Wild Ajuga Species in Israel. ACS Omega. 2019; 4: 2369–2376. doi: 10.1021/acsomega.8b03029. DOI: https://doi.org/10.1021/acsomega.8b03029

Göger F., Köse Y.B., Göger G. and Demirci F. Phytochemical Characterization of Phenolics by LC-MS/MS and Biological Evaluation of Ajuga Orientalis from Turkey. Bangladesh J. Pharmacol. 2015; 10: 639–644.

doi: 10.3329/bjp.v10i3.23500. DOI: https://doi.org/10.3329/bjp.v10i3.23500

Luan F., Han K., Li M., Zhang T., Liu D., Yu L. and Lv H. Ethnomedicinal Uses, Phytochemistry, Pharmacology, and Toxicology of Species from the Genus Ajuga L.: A Systematic Review. Am. J. Chin. Med. 2019; 47: 959–1003.

doi: 10.1142/S0192415X19500502. DOI: https://doi.org/10.1142/S0192415X19500502

Sajjadi S.E. and Ghannadi A. Volatile Oil Composition of the Aerial Parts of Ajuga Orientalis L . from Iran §. Zeitschrift für Naturforsch. 2004; 59: 166–168. DOI: https://doi.org/10.1515/znc-2004-3-404

Gautam R., Jachak S.M. and Saklani A. Anti-Inflammatory Effect of Ajuga Bracteosa Wall Ex Benth. Mediated through Cyclooxygenase (COX) Inhibition. J. Ethnopharmacol. 2011; 133: 928–930.

doi: 10.1016/J.JEP.2010.11.003. DOI: https://doi.org/10.1016/j.jep.2010.11.003

Zengin G., Ceylan R., Katani J., Aktumsek A., Mati S., Boroja T., Mihailovi V., Seebaluck-sandoram R., Mollica A. and Fawzi M. Exploring the Therapeutic Potential and Phenolic Composition of Two Turkish Ethnomedicinal Plants – Ajuga Orientalis L . and Arnebia Densi Fl Ora (Nordm .). Ind. Crop. Prod. 2018; 116: 240–248. doi: 10.1016/j.indcrop.2018.02.054. DOI: https://doi.org/10.1016/j.indcrop.2018.02.054

Shabbar I.M.S., Maslat A., Shabbar I.M.S., Genotoxicity A.M., Rich L.A., Fruit C. and Using J. Genotoxicity of Ecballium Elaterium (L) A Rich Cucurbitaceae Fruit Juice Using Micronucleus Assay and DNA Single Strand Break Techniques. Internet J. Heal. 2012; 6: 1–12. doi: 10.5580/1b75. DOI: https://doi.org/10.5580/1b75

Heddle J.A., Hite M., Kirkhart B., Mavournin K., MacGregor J.T., Newell G.W. and Salamone M.F. The Induction of Micronuclei as a Measure of Genotoxicity. A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. Genet. Toxicol. 1983; 123: 61–118. doi: 10.1016/0165-1110(83)90047-7. DOI: https://doi.org/10.1016/0165-1110(83)90047-7

Schmid W. The Micronucleus Test. Mutat. Res. Mutagen. Relat. Subj. 1975; 31: 9–15.

doi: 10.1016/0165-1161(75)90058-8. DOI: https://doi.org/10.1016/0165-1161(75)90058-8

Khalil A.M., Salman W.K. and Al-Qaoud K.M. Evaluation of Genotoxic Potential of 2-(Bromoacetamido) Phenylboronic Acid on Balb/C Mice Peripheral Blood Cells Using in Vivo Micronucleus Assay. Jordan J. Pharm. Sci. 2017; 10: 103–112. doi: 10.12816/0040698. DOI: https://doi.org/10.12816/0040698

Petrovska B.B. Historical Review of Medicinal Plants’ Usage. Pharmacogn. Rev. 2012; 6: 1–5. doi: 10.4103/0973-7847.95849. DOI: https://doi.org/10.4103/0973-7847.95849

Ahmad R., Ahmad N., Naqvi A.A., Shehzad A. and Al-Ghamdi M.S. Role of Traditional Islamic and Arabic Plants in Cancer Therapy. J. Tradit. Complement. Med. 2017; 7: 195–204. doi: 10.1016/j.jtcme.2016.05.002. DOI: https://doi.org/10.1016/j.jtcme.2016.05.002

Oloya B., Namukobe J., Ssengooba W., Afayoa M. and Byamukama R. Phytochemical Screening, Antimycobacterial Activity and Acute Toxicity of Crude Extracts of Selected Medicinal Plant Species Used Locally in the Treatment of Tuberculosis in Uganda. Trop. Med. Health. 2022; 50.

doi: 10.1186/s41182-022-00406-7. DOI: https://doi.org/10.1186/s41182-022-00406-7

El Hilaly J., Israili Z.H. and Lyoussi B. Acute and Chronic Toxicological Studies of Ajuga Iva in Experimental Animals. J. Ethnopharmacol. 2004; 91: 43–50. doi: 10.1016/j.jep.2003.11.009. DOI: https://doi.org/10.1016/j.jep.2003.11.009

Hao D., cheng and Xiao P. gen Pharmaceutical Resource Discovery from Traditional Medicinal Plants: Pharmacophylogeny and Pharmacophylogenomics. Chinese Herb. Med. 2020; 12: 104–117. doi: 10.1016/j.chmed.2020.03.002. DOI: https://doi.org/10.1016/j.chmed.2020.03.002

Rohit Sahu, Goli Divakar, K.D. IN VIVO RODENT MICRONUCLEUS ASSAY OF GMELINA ARBOREA ROXB (GAMBHARI) EXTRACT. J. Adv. Pharm. Technol. Res. 2010; 1: 22–29. DOI: https://doi.org/10.4103/2231-4040.70516

Kwasniewska J. and Bara A.W. Plant Cytogenetics in the Micronuclei Investigation—The Past, Current Status, and Perspectives. Int. J. Mol. Sci. 2022; 23.

doi: 10.3390/ijms23031306. DOI: https://doi.org/10.3390/ijms23031306

Luzhna L., Kathiria P. and Kovalchuk O. Micronuclei in Genotoxicity Assessment : From Genetics to Epigenetics and Beyond. Front. Genet. 2013; 4: 1–17. doi: 10.3389/fgene.2013.00131. DOI: https://doi.org/10.3389/fgene.2013.00131

Phillips D. and Arlt V. Genotoxicity: Damage to DNA and Its Consequencese. In Molecular, Clinical and Environmental Toxicology. 2009; 87–110. DOI: https://doi.org/10.1007/978-3-7643-8336-7_4

Zhou J., Ouedraogo M., Qu F. and Duez P. Potential Genotoxicity of Traditional Chinese Medicinal Plants and Phytochemicals: An Overview. Phyther. Res. 2013; 27: 1745–1755.doi: 10.1002/ptr.494 DOI: https://doi.org/10.1002/ptr.4942

Kayani W.K., Dilshad E., Ahmed T., Ismail H. and Mirza B. Evaluation of Ajuga Bracteosa for Antioxidant, Anti-Inflammatory, Analgesic, Antidepressant and Anticoagulant Activities. BMC Complement. Altern. Med. 2016; 16: 1–13.

doi: 10.1186/s12906-016-1363-y. DOI: https://doi.org/10.1186/s12906-016-1363-y

Suryavanshi A., Kumar S., Kain D. and Arya A. Chemical Composition, Antioxidant and Enzyme Inhibitory Properties of Ajuga Parviflora Benth. Biocatal. Agric. Biotechnol. 2021; 37: 102191. doi: 10.1016/j.bcab.2021.102191. DOI: https://doi.org/10.1016/j.bcab.2021.102191

Esposito T., Sansone F., Auriemma G., Franceschelli S., Pecoraro M., Picerno P., Aquino R.P. and Mencherini T. Study on Ajuga Reptans Extract: A Natural Antioxidant in Microencapsulated Powder Form as an Active Ingredient for Nutraceutical or Pharmaceutical Purposes. Pharmaceutics. 2020; 12: 1–23.

doi: 10.3390/pharmaceutics12070671. DOI: https://doi.org/10.3390/pharmaceutics12070671

Venkatesan T., Choi Y.W. and Kim Y.K. Effect of an Extraction Solvent on the Antioxidant Quality of Pinus Densiflora Needle Extract. J. Pharm. Anal. 2019; 9: 193–200. doi: 10.1016/j.jpha.2019.03.005. DOI: https://doi.org/10.1016/j.jpha.2019.03.005

Bonnefont-Rousselot D., Rouscilles A., Bizard C., Delattre J., Jore D. and Gardès-Albert M. Antioxidant Effect of Ethanol toward in Vitro Peroxidation of Human Low-Density Lipoproteins Initiated by Oxygen Free Radicals. Radiat. Res. 2001; 155: 279–287. doi: 10.1667/0033-7587(2001)155[0279: AEOETI]2.0.CO;2. DOI: https://doi.org/10.1667/0033-7587(2001)155[0279:AEOETI]2.0.CO;2

Khlebnikov A.I., Schepetkin I.A., Domina N.G., Kirpotina L.N. and Quinn M.T. Improved Quantitative Structure-Activity Relationship Models to Predict Antioxidant Activity of Flavonoids in Chemical, Enzymatic, and Cellular Systems. Bioorganic Med. Chem. 2007; 15: 1749–1770.

doi: 10.1016/j.bmc.2006.11.037 DOI: https://doi.org/10.1016/j.bmc.2006.11.037