Preclinical Evaluation of the Innovative MK-7 NE: Toxicity Assessment and Safety Optimization in In Vivo Models

Authors

  • Sanaa Ismael Abduljabbar Microbial & Pharmaceutical Biotechnology Laboratory (MPBIL), Department of Pharmacognosy & Phytochemistry, School of Pharmaceutical Education & Research, Jamia Hamdard, New Delhi, India https://orcid.org/0000-0002-4936-3289
  • Jalaluddin Khan Microbial & Pharmaceutical Biotechnology Laboratory (MPBIL), Department of Pharmacognosy & Phytochemistry, School of Pharmaceutical Education & Research, Jamia Hamdard, New Delhi, India
  • Farhan Jalees Ahmed Department of Pharmaceutics, School of Pharmaceutical Education & Research, Jamia Hamdard, New Delhi, India https://orcid.org/0000-0002-0740-8573
  • Bibhu Prasad Panda Microbial & Pharmaceutical Biotechnology Laboratory (MPBIL), Department of Pharmacognosy & Phytochemistry, School of Pharmaceutical Education & Research, Jamia Hamdard, New Delhi, India https://orcid.org/0000-0001-5110-2945

Abstract

Vitamin K is crucial in blood clotting and bone health, yet its bioavailability is limited. NEs (NE) have emerged as promising delivery systems for fat-soluble vitamins like vitamin K because they enhance solubility, control drug release, prevent enzymatic degradation, and improve therapeutic efficacy. This study investigates a novel, cold-processed, acoustically stable Menaquinon-7 Nanoemulsion (MK-7 NE) derived from both fermented and standard MK-7. Preclinical optimization is essential for understanding potential toxicities and improving safety. A single oral dose of a placebo and MK-7 NE at varying concentrations, based on body weight, was administered to 35 Wistar albino rats, along with a 2 mg/kg dose of a standard MK-7 solution. Plasma MK-7 concentrations were monitored for 72 hours using HPLC analysis, while histopathological examination assessed tissue degradation in the rats' organs. To evaluate acute toxicity, an adult Danio rerio (Zebrafish) model was exposed to a 20 mg/kg concentration of the NE. The results indicated that MK-7 NE supplementation did not cause tissue or organ damage in either model, demonstrating its safety. These findings support the potential of MK-7 NE as an effective and safe formulation for enhancing vitamin K2 bioavailability and therapeutic benefits while minimizing potential risks.

Keywords:

High-performance liquid chromatography (HPLC), Histopathology, Menaquinone-7, Nanoemulsion, Zebrafish, Vitamin K2

DOI

https://doi.org/10.25004/IJPSDR.2025.170301

References

Shah P, Bhalodia D, Shelat P. NE: A pharmaceutical review. Syst Rev Pharm. 2010;1(1):24–32.

Lovelyn C, Attama AA, Lovelyn C, Attama AA. Current State of NE in Drug Delivery. J Biomater Nanobiotechnol [Internet]. 2011 Dec 9 [cited 2023 Aug 15];2(5):626–39. Available from: http://www.scirp.org/Html/9112.html

Jadhav N, Ajgaonkar S, Saha P, Gurav P, Pandey A, Basudkar V, et al. Molecular Pathways and Roles for Vitamin K2-7 as a Health-Beneficial Nutraceutical: Challenges and Opportunities. Front Pharmacol. 2022;13(June).

Chime SA, Kenechukwu FC, Attama AA, Chime SA, Kenechukwu FC, Attama AA. NE — Advances in Formulation, Characterization and Applications in Drug Delivery. Appl Nanotechnol Drug Deliv [Internet]. 2014 Jul 25 [cited 2025 Mar 18]; Available from: https://www.intechopen.com/chapters/47116

Meng J, Yan Z, Wu Y, Gao M, Li W, Gao F, et al. Preclinical safety evaluation of IFNα2a-NGR. Regul Toxicol Pharmacol. 2008;50(3):294–302.

Muthulakshmi S, Maharajan K, Habibi HR, Kadirvelu K, Venkataramana M. Zearalenone induced embryo and neurotoxicity in zebrafish model (Danio rerio): Role of oxidative stress revealed by a multi biomarker study. Chemosphere [Internet]. 2018;198:111–21. Available from: https://doi.org/10.1016/j.chemosphere.2018.01.141

Bhangare D, Rajput N, Jadav T, Sahu AK, Tekade RK, Sengupta P. Systematic strategies for degradation kinetic study of pharmaceuticals: an issue of utmost importance concerning current stability analysis practices. J Anal Sci Technol. 2022;13(1):7. Available from: https://doi.org/10.1186/s40543-022-00317-6

Inaba N, Sato T, Yamashita T. Low-Dose Daily Intake of Vitamin K(2) (Menaquinone-7) Improves Osteocalcin γ-Carboxylation: A Double-Blind, Randomized Controlled Trials. J Nutr Sci Vitaminol (Tokyo) [Internet]. 2015 [cited 2025 Jan 31];61(6):471–80. Available from: https://pubmed.ncbi.nlm.nih.gov/26875489/

Molnar A, Lakat T, Hosszu A, Szebeni B, Balogh A, Orfi L, et al. Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques. Amino Acids [Internet]. 2021 Jun 1 [cited 2023 Jul 13];53(6):917. Available from: /pmc/articles/PMC8128086/

Sireeratawong S, Jaijoy K, Khonsung P, Lertprasertsuk N, Ingkaninan K. Acute and chronic toxicities of Bacopa monnieri extract in Sprague-Dawley rats. BMC Complement Altern Med [Internet]. 2016;16(1):1. Available from: http://dx.doi.org/10.1186/s12906-016-1236-4

Domingues I, Oliveira R, Lourenço J, Grisolia CK, Mendo S, Soares AMVM. Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults. Comp Biochem Physiol - C Toxicol Pharmacol [Internet]. 2010;152(3):338–45. Available from: http://dx.doi.org/10.1016/j.cbpc.2010.05.010

Su LJ, Zhang JH, Gomez H, Murugan R, Hong X, Xu D, et al. Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxid Med Cell Longev [Internet]. 2019 [cited 2023 Jul 30];2019. Available from: /pmc/articles/PMC6815535/

Sochor J, Ruttkay-Nedecky B, Babula P, Adam V, Hubalek J, Kizek R, et al. Automation of Methods for Determination of Lipid Peroxidation. Lipid Peroxidation [Internet]. 2012 Aug 29 [cited 2023 Jul 30]; Available from: https://www.intechopen.com/chapters/38473

Cong B, Liu C, Wang L, Chai Y. The Impact on Antioxidant Enzyme Activity and Related Gene Expression Following Adult Zebrafish (Danio rerio) Exposure to Dimethyl Phthalate. Anim 2020, Vol 10, Page 717 [Internet]. 2020 Apr 20 [cited 2025 Mar 25];10(4):717. Available from: https://www.mdpi.com/2076-2615/10/4/717/htm

Muniz MS, Halbach K, Alves Araruna IC, Martins RX, Seiwert B, Lechtenfeld O, et al. Moxidectin toxicity to zebrafish embryos: Bioaccumulation and biomarker responses. Environ Pollut. 2021;283.

Pucaj K, Rasmussen H, Møller M, Preston T. Safety and toxicological evaluation of a synthetic vitamin K2, menaquinone-7. Toxicol Mech Methods. 2011 Sep;21(7):520–32.

Hwang SB, Choi MJ, Lee HJ, Han JJ. Safety evaluation of vitamin K2 (menaquinone-7) via toxicological tests. Sci Reports 2024 141 [Internet]. 2024 Mar 5 [cited 2025 Mar 25];14(1):1–19. Available from: https://www.nature.com/articles/s41598-024-56151-w

Jacob S, Kather FS, Boddu SHS, Shah J, Nair AB. Innovations in NE Technology: Enhancing Drug Delivery for Oral, Parenteral, and Ophthalmic Applications. Pharm 2024, Vol 16, Page 1333 [Internet]. 2024 Oct 17 [cited 2025 Feb 26];16(10):1333. Available from: https://www.mdpi.com/1999-4923/16/10/1333/htm

Wang Y, Yang W, Liu L, Liu L, Chen J, Duan L, et al. Vitamin K2 (MK-7) attenuates LPS-induced acute lung injury via inhibiting inflammation, apoptosis, and ferroptosis. PLoS One [Internet]. 2023 Nov 1 [cited 2025 Mar 25];18(11). Available from: https://pubmed.ncbi.nlm.nih.gov/38011192/

Yang W, Wang Y, Liu L, Liu L, Li S, Li Y. Protective Effect of Vitamin K2 (MK-7) on Acute Lung Injury Induced by Lipopolysaccharide in Mice. Curr Issues Mol Biol 2024, Vol 46, Pages 1700-1712 [Internet]. 2024 Feb 22 [cited 2025 Mar 25];46(3):1700–12. Available from: https://www.mdpi.com/1467-3045/46/3/110/htm

Bresson JL, Flynn A, Heinonen M, Hulshof K, Korhonen H, Lagiou P, et al. Vitamin K2 added for nutritional purposes in foods for particular nutritional uses, food supplements and foods intended for the general population and Vitamin K2 as a source of vitamin K added for nutritional purposes to foodstuffs, in the context of Regulation (EC) N° 258/97 - Scientific Opinion of the Panel on Dietetic Products, Nutrition and Allergies. EFSA J [Internet]. 2008 Nov 1 [cited 2024 Sep 27];6(11):822. Available from: https://onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2008.822

El-Sherbiny M, Atef H, Helal GM, Al-Serwi RH, Elkattawy HA, Shaker GA, et al. Vitamin K2 (MK-7) Intercepts Keap-1/Nrf-2/HO-1 Pathway and Hinders Inflammatory/Apoptotic Signaling and Liver Aging in Naturally Aging Rat. Antioxidants (Basel, Switzerland) [Internet]. 2022 Nov 1 [cited 2025 Mar 25];11(11). Available from: https://pubmed.ncbi.nlm.nih.gov/36358523/

Vera MC, Lorenzetti F, Lucci A, Comanzo CG, Ceballos MP, Pisani GB, et al. Vitamin K2 supplementation blocks the beneficial effects of IFN-α-2b administered on the early stages of liver cancer development in rats. Nutrition [Internet]. 2019 Mar 1 [cited 2025 Mar 25];59:170–9. Available from: https://pubmed.ncbi.nlm.nih.gov/30496957/

Sultana H, Komai M, Shirakawa H. The Role of Vitamin K in Cholestatic Liver Disease. Nutrients [Internet]. 2021 Aug 1 [cited 2025 Mar 25];13(8):2515. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC8400302/

[Malondialdehyde (MDA) as a lipid peroxidation marker] - PubMed [Internet]. [cited 2025 Mar 25]. Available from: https://pubmed.ncbi.nlm.nih.gov/15765761/

Renuka RR, Ravindranath RRS, Raguraman V, Yoganandham ST, Kasivelu G, Lakshminarayanan A. In Vivo Toxicity Assessment of Laminarin Based Silver Nanoparticles from Turbinaria ornata in Adult Zebrafish (Danio rerio). J Clust Sci [Internet]. 2020 Jan 1 [cited 2025 Mar 25];31(1):185–95. Available from: https://link.springer.com/article/10.1007/s10876-019-01632-6

Huang X, Li Y, Wang T, Liu H, Shi J, Zhang X. Evaluation of the Oxidative Stress Status in Zebrafish (Danio rerio) Liver Induced by Three Typical Organic UV Filters (BP-4, PABA and PBSA). Int J Environ Res Public Health [Internet]. 2020 Jan 2 [cited 2025 Mar 25];17(2). Available from: https://pubmed.ncbi.nlm.nih.gov/31963911/

Cassar S, Adatto I, Freeman JL, Gamse JT, Iturria I, Lawrence C, et al. Use of Zebrafish in Drug Discovery Toxicology. Chem Res Toxicol. 2020;33(1):95–118.

Borges RS, Pereira ACM, Souza GC de, Carvalho JCT, Borges RS, Pereira ACM, et al. Histopathology of Zebrafish (Danio rerio) in Nonclinical Toxicological Studies of New Drugs. Zebrafish Biomed Res [Internet]. 2019 Oct 25 [cited 2023 Jul 29]; Available from: https://www.intechopen.com/chapters/69752

Farhana A, Lappin SL. Biochemistry, Lactate Dehydrogenase. StatPearls [Internet]. 2023 May 1 [cited 2025 Mar 26]; Available from: https://www.ncbi.nlm.nih.gov/books/NBK557536/

Ayala A, Muñoz MF, Argüelles S. Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxid Med Cell Longev [Internet]. 2014 [cited 2025 Mar 26];2014:360438. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4066722/

Fujii J, Homma T, Osaki T. Superoxide Radicals in the Execution of Cell Death. Antioxidants 2022, Vol 11, Page 501 [Internet]. 2022 Mar 4 [cited 2025 Mar 26];11(3):501. Available from: https://www.mdpi.com/2076-3921/11/3/501/htm

Published

30-05-2025
Statistics
Abstract Display: 73
PDF Downloads: 15
Dimension Badge

How to Cite

“Preclinical Evaluation of the Innovative MK-7 NE: Toxicity Assessment and Safety Optimization in In Vivo Models”. International Journal of Pharmaceutical Sciences and Drug Research, vol. 17, no. 3, May 2025, pp. 216-2, https://doi.org/10.25004/IJPSDR.2025.170301.

Issue

Section

Research Article

How to Cite

“Preclinical Evaluation of the Innovative MK-7 NE: Toxicity Assessment and Safety Optimization in In Vivo Models”. International Journal of Pharmaceutical Sciences and Drug Research, vol. 17, no. 3, May 2025, pp. 216-2, https://doi.org/10.25004/IJPSDR.2025.170301.