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Original Article

J Pharmacopuncture 2022; 25(4): 364-368

Published online December 31, 2022 https://doi.org/10.3831/KPI.2022.25.4.364

Copyright © The Korean Pharmacopuncture Institute.

Bacterial Reverse Mutation Test of Verbenalin

Hye Jeong Shin1 , Yi Gun Lim1 , Ji Su Ha2 , Gabsik Yang1 , Tae Han Yook1*

1Department of Korean Medicine, College of Korean Medicine, Woosuk University, Jeonju, Republic of Korea
2Department of Acupuncture & Moxibustion Medicine, National Medical Center, Seoul, Republic of Korea

Correspondence to:Tae Han Yook
Department of Korean Medicine,College of Korean Medicine, Woosuk University, 61 Seonneomeo 3-gil, Wansan-gu, Jeonju 54986, Republic of Korea
Tel: +82-63-220-8625
E-mail: nasiss@naver.com

Received: August 18, 2022; Revised: August 24, 2022; Accepted: September 6, 2022

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objectives: Verbenalin is a compound found in herbs such as Cornus officinalis and Verbena officinalis. This study investigated whether verbenalin is safe by analyzing its mutagenicity.
Methods: To examine the mutagenic potential of verbenalin, a bacterial reverse mutation test (Ames test) was conducted with Salmonella typhimurium and Escherichia coli strains. Experiments with and without metabolic activity were performed.
Results: The mean colony number was less than double that of the control. Growth inhibition and precipitation of verbenalin were not apparent in all strains at different concentrations regardless of metabolic activity.
Conclusion: Verbenalin did not show any signs of mutagenicity in this study. Additional toxicity studies including repeated oral toxicity, reproductive toxicity, and carcinogenicity tests are needed.

Keywords: dementia, mutagenicity test, verbenalin

INTRODUCTION

Dementia causes behavioral abnormalities and personality changes including cognitive decline due to biological aging [1]. It is estimated that there are around 55 million people with dementia, which is predicted to reach 78 million in 2030 [2]. In Korean medicine, the recognized causes of dementia include aging, poor movement of qi and blood due to stress, damage to qi and blood due to inability to control food intake, trauma to the head, and chronic consumptive disease [3].

Various studies have reported the therapeutic efficacy of herbal extracts for dementia. Standard extracts of Ginkgo biloba leaves [4], an alkaloid compound constituting the medicinal herb Huperzia serrata (huperzine A) [5], standard ethanolic extracts of Angelica gigas Nakai (INM-176) [6], dehydroevodiamine hydrochloride (DHED), and the active ingredients of Evodia officinalis [7], Perilla frutescens [8], and Gastrodiae Rhizoma [9] were demonstrated to have potential therapeutic efficacy against dementia.

Verbenalin is an alkaloid present in herbs such as Verbena officinalis, Cornus officinalis, and Symplocos glauca [10-12]. Verbenalin was found to improve brain microcirculation and protect brain tissue and neurons in a study using a cerebral ischemia rat model. Other studies reported that it exhibited sleep-promoting and antioxidant effects [13, 14] and the potential to reduce apoptosis [15].

To confirm the therapeutic effect of verbenalin on dementia, studies have been conducted to determine whether verbenalin inhibits the production of amyloid-β (Aβ) peptides. Various effects of verbenalin have been identified; however, its toxicity has not been sufficiently investigated. To use verbenalin for dementia treatment, it is important to identify any toxic and mutagenic properties. Therefore, an oral dose toxicity study using ICR mice is currently ongoing to evaluate the toxicity of verbenalin. In this study, the Ames test was performed to determine whether verbenalin is mutagenic. The Ames test can sensitively, quickly, and accurately identify mutant activity [16]. This study was based on the Organization for Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, No. 471 ‘Bacterial Reverse Mutation Test’ (1997).

MATERIALS AND METHODS

Verbenalin (purity > 99.3%, molecular formula: C17H24O10, molecular weight: 388.37 g/mol) was obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). Dimethylsulfoxide (DMSO) and other solvents and chemicals were purchased from Merck (Darmstadt, Germany). Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2uvrA were obtained from Molecular Toxicology Inc. (Boone, NC, USA). DMSO was used as a solvent for verbenalin as well as a negative control.

The Ames test was performed with 4 Salmonella typhimurium strains and 1 Escherichia coli strain, both with and without metabolic activity [17].

Verbenalin was dissolved in DMSO and mixed with a vortex mixer. Solvents were added to achieve the desired concentration level. The maximum concentration of verbenalin was set to 5,000 μg/plate and diluted to obtain 2,500, 1,000, 500, 100, 50, 10, and 5 μg/plate concentrations. A negative control group was also established. The test was performed using the pre-incubation method, and a total of 2 plates were used for all concentration levels with and without metabolic activity.

In experiments without metabolic activity, 100 μL of verbenalin and the negative control were placed in separate tubes. Then, a mixture of 500 μL of 0.1 M sodium phosphate buffer and 100 μL of strain suspension was incubated in a shaking incubator (90 rpm, 37℃). Heated agar (2 mL) was added and mixed in a vortex mixer. The mixtures were solidified on glucose agar plates at room temperature.

In experiments with metabolic activity, instead of sodium phosphate buffer, 500 μL of S9 mixture was added. After solidification, the inverted plates were cultured at 37℃ for 48 h in an incubator (DK-LI020-P; Daiki Scientific Co., Ltd., Seoul, Korea).

Precipitation was observed visually and recorded during the treatment with verbenalin. The number of revertant colonies was automatically calculated by a colony counter (ProtoCOL3; Synbiosis, Cambridge, UK) after incubation. When the number obtained by automatic counting was incorrect, the colonies were counted manually.

To detect growth inhibition, the background lawn was examined with a stereoscopic microscope (45-fold magnification, SZ61; Olympus, Tokyo, Japan). Growth inhibition was judged based on the extent to which the number of revertant colonies was decreased or the background lawn was reduced compared with those of the control group.

RESULTS

If a dose-related increase was observed over the tested range and/or the increase reflected the average number of revertant colonies for at least one strain regardless of metabolic activity, the result was considered positive. Cytotoxicity was defined as the reduction of the background lawn or colony number by more than 50% compared with those of the vehicle control.

Overall, the number of revertant colonies was less than double that of the control at all concentrations for all strains regardless of metabolic activity. Growth inhibition and precipitation of verbenalin were not observed at all concentrations in all strains regardless of metabolic activity (Table 1).

VC, vehicle control (dimethyl sulfoxide)..

&md=tbl&idx=1' data-target="#file-modal"">Table 1

The number of revertant colonies.

StrainTest substanceDose (μg/plate)Absence of metabolic activationPresence of metabolic activation


Individual revertant colony countsMeanIndividual revertant colony countsMean
TA98VC0141012273330
Verbenalin5111111252425
10151616293432
50101211273230
100141012243027
500131012302729
1,00013911272928
2,500131112252726
5,000131213332730
TA100VC099110105111123117
Verbenalin5101111106119120120
10100110105120110115
5097109103122121122
100112123118112121117
500119110115116120118
1,000116115116117133125
2,50094113104118128123
5,000988391119125122
TA1535VC0587698
Verbenalin51079978
10857486
5061081089
100587857
500587746
1,000555777
2,500555587
5,00076711810
TA1537VC0576101513
Verbenalin55667119
10555111011
506108151515
10059712911
50057691010
1,00057611910
2,500888968
5,00087891010
WP2uvrAVC0283230353133
Verbenalin5312629293231
10303533353736
50243128333936
100313533323735
500323835313634
1,000272928323433
2,500303533403236
5,000263229373235

VC, vehicle control (dimethyl sulfoxide)..


DISCUSSION

Dementia is a syndrome with behavioral and psychological symptoms such as cognitive and memory deterioration, emotional abnormalities, and hallucinations [18].

Recently, herbal extracts have attracted attention due to their medicinal properties including rapid action, low frequency of side effects, and potential synergistic effects [19, 20].

The active ingredients of herbal extracts may be used for the treatment of dementia. For example, galantamine and huperzine A, which are derived from herbal sources, have been developed clinically to treat mild-to-moderate dementia [21, 22]. Extracts of Ginkgo biloba leaves were reported to protect brain tissue against hypoxic damage [4] and partially prevent oxidative damage in the brain of aged animals [23]. Huperzine A from the medicinal herb Huperzia serrata was demonstrated to treat cognitive deficits [5] and reduce glutamate-induced impairment in the brain [24]. INM-176, an extract of Angelica gigas Nakai, showed memory-improving effects against scopolamine-induced memory damage [6]. DHED, the active ingredient of Evodia officinalis, demonstrated memory-improving and neuroprotective effects in experiments with various models [7]. The positive effects of Gastrodiae Rhizoma on learning and memory were confirmed in experiments using APP/PS1 mice [9]. The effects of Perilla frutescens extract on a mouse model with memory disorder were demonstrated through Y-maze and other types of tests [8]. In addition, studies using various herbal extracts are being conducted to develop new drugs that can treat the cause of dementia.

Verbenalin, also known as cornin, is an iridoid glucoside component of the herbs Cornus officinalis and Verbena officinalis (a plant of the Verbenaceae family) [10-12]. Verbenalin protects brain tissue, improves brain microcirculation, and reduces cerebral ischemic damage. It has also been reported to have antioxidant activity and promote sleep [13, 14]. Previous studies showed that human amniotic epithelial cells treated with verbenalin might exert therapeutic activity against Alzheimer’s disease by regulating gene expression associated with neurometabolic aging, lysosomal dysfunction, pathological angiogenesis, and 24 h periodic rhythm. Furthermore, verbenalin has been reported to significantly reduce cell death [15].

Studies are currently being conducted to investigate the efficacy of verbenalin in inhibiting Aβ peptide production, the effect of verbenalin on the behavioral characteristics of APPswe mice with dementia, and the anti-inflammatory effect of verbenalin on lipopolysaccharide-activated BV2 microglial cells. To evaluate the safety of verbenalin, a single oral dose toxicity study using ICR mice is also ongoing. This study was conducted to investigate the genetic toxicity of verbenalin.

To investigate the potential mutagenicity of verbenalin, the Ames test was performed with Escherichia coli and Salmonella typhimurium strains that required specific amino acids. The study attempted to determine whether specific chemicals could mutate the DNA of the test organisms [25]. Based on the number of revertant colonies, there was no mutagenic activation by verbenalin in all strains compared with the control group regardless of metabolic activity.

CONCLUSION

All bacterial strains (TA98, TA100, TA 1535, TA1537, and WP2uvrA) showed a negative result over the tested dose range. Therefore, verbenalin is a non-genotoxic substance.

A bacterial reverse mutation test for verbenalin was conducted in this study. Furthermore, the findings of an ongoing single oral toxicity study will be reported in the future. Nevertheless, safety tests including carcinogenicity, repeated oral toxicity, reproductive toxicity, and local toxicity tests should be conducted. Additional toxicity studies with different doses of verbenalin are needed to confirm its safety.

ACKNOWLEDGMENT

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; Ministry of Science, ICT & Future Planning) (No. NRF-2018R1D1A1B07044595).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Table 1 . The number of revertant colonies.

StrainTest substanceDose (μg/plate)Absence of metabolic activationPresence of metabolic activation


Individual revertant colony countsMeanIndividual revertant colony countsMean
TA98VC0141012273330
Verbenalin5111111252425
10151616293432
50101211273230
100141012243027
500131012302729
1,00013911272928
2,500131112252726
5,000131213332730
TA100VC099110105111123117
Verbenalin5101111106119120120
10100110105120110115
5097109103122121122
100112123118112121117
500119110115116120118
1,000116115116117133125
2,50094113104118128123
5,000988391119125122
TA1535VC0587698
Verbenalin51079978
10857486
5061081089
100587857
500587746
1,000555777
2,500555587
5,00076711810
TA1537VC0576101513
Verbenalin55667119
10555111011
506108151515
10059712911
50057691010
1,00057611910
2,500888968
5,00087891010
WP2uvrAVC0283230353133
Verbenalin5312629293231
10303533353736
50243128333936
100313533323735
500323835313634
1,000272928323433
2,500303533403236
5,000263229373235

VC, vehicle control (dimethyl sulfoxide)..


References

  1. American Psychiatric Association. Diagnostic and statistical manual of mental disorders: DSM-5. 5th ed. Washington, D.C.: American Psychiatric Association; 2013.
    CrossRef
  2. World Health Organization. Fact Sheets of Dementia [Internet]. Geneva: WHO; 2021 [cited 2022 Jul 7]. Available from: https://www.who.int/health-topics/dementia#tab=tab_1.
  3. Oriental Neuropsychiatry Editorial Committee of Korean Medicine schools. Oriental neuropsychiatry: revised ed. Gyeonggi: Jipmoondang; 2011. p. 332-41.
  4. Loew D. [Value of Ginkgo biloba in treatment of Alzheimer dementia]. Wien Med Wochenschr. 2002;152(15-16):418-22. German.
    Pubmed CrossRef
  5. Xu SS, Gao ZX, Weng Z, Du ZM, Xu WA, Yang JS, et al. Efficacy of tablet huperzine-A on memory, cognition, and behavior in Alzheimer's disease. Acta Pharmacol Sin. 1995;16(5):391-5.
  6. Park SJ, Jung JM, Lee HE, Lee YW, Kim DH, Kim JM, et al. The memory ameliorating effects of INM-176, an ethanolic extract of Angelica gigas, against scopolamine- or Aβ(1-42)-induced cognitive dysfunction in mice. J Ethnopharmacol. 2012;143(2):611-20.
    Pubmed CrossRef
  7. Choi SH, Park CH, Seo JH, Suh YH. Dehydroevodiamine.HCl protects neuron from cytotoxicity induced by Aβ or oxidative stress: its antioxidant. J Neurochem. 2002;81(s1):101-5.
    CrossRef
  8. Lee JH, Lee EH, Jung EM, Kim DH, Kim SK, Park MH, et al. Perilla Frutescens extract protects against Scopolamine-induced memory deficits in mice. J Physiol Pathol Korean Med. 2021;35(3):97-103.
    CrossRef
  9. Gao YM, Wang XL, Deng YX, Reng JM, Shen XC, Guan ZZ, et al. Effect of Gastrodiae Rhizoma powder on prevention of dementia and oxidation resistance in mice. Chin J Exp Tradit Med Formulae. 2020;24:52-8.
  10. Bahramsoltani R, Rostamiasrabadi P, Shahpiri Z, Marques AM, Rahimi R, Farzaei MH. Aloysia citrodora Paláu (Lemon verbena): a review of phytochemistry and pharmacology. J Ethnopharmacol. 2018;222:34-51.
    Pubmed CrossRef
  11. Bilia AR, Giomi M, Innocenti M, Gallori S, Vincieri FF. HPLC-DAD-ESI-MS analysis of the constituents of aqueous preparations of verbena and lemon verbena and evaluation of the antioxidant activity. J Pharm Biomed Anal. 2008;46(3):463-70.
    Pubmed CrossRef
  12. Schönbichler SA, Bittner LK, Pallua JD, Popp M, Abel G, Bonn GK, et al. Simultaneous quantification of verbenalin and verbascoside in Verbena officinalis by ATR-IR and NIR spectroscopy. J Pharm Biomed Anal. 2013;84:97-102.
    Pubmed CrossRef
  13. Cao L, Miao M, Qiao J, Bai M, Li R. The protective role of verbenalin in rat model of focal cerebral ischemia reperfusion. Saudi J Biol Sci. 2018;25(6):1170-7.
    Pubmed KoreaMed CrossRef
  14. Makino Y, Kondo S, Nishimura Y, Tsukamoto Y, Huang ZL, Urade Y. Hastatoside and verbenalin are sleep-promoting components in Verbena officinalis. Sleep Biol Rhythms. 2009;7(3):211-7.
    CrossRef
  15. Ferdousi F, Kondo S, Sasaki K, Uchida Y, Ohkohchi N, Zheng YW, et al. Microarray analysis of verbenalin-treated human amniotic epithelial cells reveals therapeutic potential for Alzheimer's disease. Aging (Albany NY). 2020;12(6):5516-38.
    Pubmed KoreaMed CrossRef
  16. Diehl MS, Willaby SL, Snyder RD. Comparison of the results of a modified miniscreen and the standard bacterial reverse mutation assays. Environ Mol Mutagen. 2000;36(1):72-7.
    Pubmed CrossRef
  17. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983;113(3-4):173-215.
    Pubmed CrossRef
  18. Korean Neuropsychiatric Association. Textbook of neuropsychiatry. 3rd ed. Seoul: iMiS Company; 2019. p. 579-89.
    CrossRef
  19. Kumar A, Singh A; Ekavali. A review on Alzheimer's disease pathophysiology and its management: an update. Pharmacol Rep. 2015;67(2):195-203.
    Pubmed CrossRef
  20. Lane RF, Dacks PA, Shineman DW, Fillit HM. Diverse therapeutic targets and biomarkers for Alzheimer's disease and related dementias: report on the Alzheimer's Drug Discovery Foundation 2012 International Conference on Alzheimer's Drug Discovery. Alzheimers Res Ther. 2013;5(1):5.
    Pubmed KoreaMed CrossRef
  21. Wang R, Yan H, Tang XC. Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine. Acta Pharmacol Sin. 2006;27(1):1-26.
    Pubmed CrossRef
  22. Takata K, Kitamura Y, Saeki M, Terada M, Kagitani S, Kitamura R, et al. Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors. J Biol Chem. 2010;285(51):40180-91.
    Pubmed KoreaMed CrossRef
  23. Sastre J, Millán A, García de la Asunción J, Plá R, Juan G, Pallardó FV, et al. A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress. Free Radic Biol Med. 1998;24(2):298-304.
    Pubmed CrossRef
  24. Wang BS, Wang H, Wei ZH, Song YY, Zhang L, Chen HZ. Efficacy and safety of natural acetylcholinesterase inhibitor huperzine A in the treatment of Alzheimer's disease: an updated meta-analysis. J Neural Transm (Vienna). 2009;116(4):457-65.
    Pubmed CrossRef
  25. OECD. est No. 471: bacterial reverse mutation test. Paris: OECD; 1997.