Articles

Bacterial Reverse Mutation Test of Verbenalin
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
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.
J Pharmacopuncture 2022; 25(4): 364-368
Published December 31, 2022 https://doi.org/10.3831/KPI.2022.25.4.364
Copyright © The Korean Pharmacopuncture Institute.
Abstract
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
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
Verbenalin is an alkaloid present in herbs such as
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).
The Ames test was performed with 4
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).
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
Verbenalin, also known as cornin, is an iridoid glucoside component of the herbs
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
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.
References
- American Psychiatric Association. Diagnostic and statistical manual of mental disorders: DSM-5. 5th ed. Washington, D.C.: American Psychiatric Association; 2013.
- 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.
- Oriental Neuropsychiatry Editorial Committee of Korean Medicine schools. Oriental neuropsychiatry: revised ed. Gyeonggi: Jipmoondang; 2011. p. 332-41.
- Loew D. [Value of Ginkgo biloba in treatment of Alzheimer dementia]. Wien Med Wochenschr. 2002;152(15-16):418-22. German.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983;113(3-4):173-215.
- Korean Neuropsychiatric Association. Textbook of neuropsychiatry. 3rd ed. Seoul: iMiS Company; 2019. p. 579-89.
- Kumar A, Singh A; Ekavali. A review on Alzheimer's disease pathophysiology and its management: an update. Pharmacol Rep. 2015;67(2):195-203.
- 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.
- 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.
- 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.
- 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.
- 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.
- OECD. est No. 471: bacterial reverse mutation test. Paris: OECD; 1997.
Related articles in JoP

Article
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
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
Verbenalin is an alkaloid present in herbs such as
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).
The Ames test was performed with 4
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 1The number of revertant colonies.
Strain Test substance Dose (μg/plate) Absence of metabolic activation Presence of metabolic activation Individual revertant colony counts Mean Individual revertant colony counts Mean TA98 VC 0 14 10 12 27 33 30 Verbenalin 5 11 11 11 25 24 25 10 15 16 16 29 34 32 50 10 12 11 27 32 30 100 14 10 12 24 30 27 500 13 10 12 30 27 29 1,000 13 9 11 27 29 28 2,500 13 11 12 25 27 26 5,000 13 12 13 33 27 30 TA100 VC 0 99 110 105 111 123 117 Verbenalin 5 101 111 106 119 120 120 10 100 110 105 120 110 115 50 97 109 103 122 121 122 100 112 123 118 112 121 117 500 119 110 115 116 120 118 1,000 116 115 116 117 133 125 2,500 94 113 104 118 128 123 5,000 98 83 91 119 125 122 TA1535 VC 0 5 8 7 6 9 8 Verbenalin 5 10 7 9 9 7 8 10 8 5 7 4 8 6 50 6 10 8 10 8 9 100 5 8 7 8 5 7 500 5 8 7 7 4 6 1,000 5 5 5 7 7 7 2,500 5 5 5 5 8 7 5,000 7 6 7 11 8 10 TA1537 VC 0 5 7 6 10 15 13 Verbenalin 5 5 6 6 7 11 9 10 5 5 5 11 10 11 50 6 10 8 15 15 15 100 5 9 7 12 9 11 500 5 7 6 9 10 10 1,000 5 7 6 11 9 10 2,500 8 8 8 9 6 8 5,000 8 7 8 9 10 10 WP2uvrA VC 0 28 32 30 35 31 33 Verbenalin 5 31 26 29 29 32 31 10 30 35 33 35 37 36 50 24 31 28 33 39 36 100 31 35 33 32 37 35 500 32 38 35 31 36 34 1,000 27 29 28 32 34 33 2,500 30 35 33 40 32 36 5,000 26 32 29 37 32 35 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
Verbenalin, also known as cornin, is an iridoid glucoside component of the herbs
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
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.
Strain Test substance Dose (μg/plate) Absence of metabolic activation Presence of metabolic activation Individual revertant colony counts Mean Individual revertant colony counts Mean TA98 VC 0 14 10 12 27 33 30 Verbenalin 5 11 11 11 25 24 25 10 15 16 16 29 34 32 50 10 12 11 27 32 30 100 14 10 12 24 30 27 500 13 10 12 30 27 29 1,000 13 9 11 27 29 28 2,500 13 11 12 25 27 26 5,000 13 12 13 33 27 30 TA100 VC 0 99 110 105 111 123 117 Verbenalin 5 101 111 106 119 120 120 10 100 110 105 120 110 115 50 97 109 103 122 121 122 100 112 123 118 112 121 117 500 119 110 115 116 120 118 1,000 116 115 116 117 133 125 2,500 94 113 104 118 128 123 5,000 98 83 91 119 125 122 TA1535 VC 0 5 8 7 6 9 8 Verbenalin 5 10 7 9 9 7 8 10 8 5 7 4 8 6 50 6 10 8 10 8 9 100 5 8 7 8 5 7 500 5 8 7 7 4 6 1,000 5 5 5 7 7 7 2,500 5 5 5 5 8 7 5,000 7 6 7 11 8 10 TA1537 VC 0 5 7 6 10 15 13 Verbenalin 5 5 6 6 7 11 9 10 5 5 5 11 10 11 50 6 10 8 15 15 15 100 5 9 7 12 9 11 500 5 7 6 9 10 10 1,000 5 7 6 11 9 10 2,500 8 8 8 9 6 8 5,000 8 7 8 9 10 10 WP2uvrA VC 0 28 32 30 35 31 33 Verbenalin 5 31 26 29 29 32 31 10 30 35 33 35 37 36 50 24 31 28 33 39 36 100 31 35 33 32 37 35 500 32 38 35 31 36 34 1,000 27 29 28 32 34 33 2,500 30 35 33 40 32 36 5,000 26 32 29 37 32 35 VC, vehicle control (dimethyl sulfoxide)..
References
- American Psychiatric Association. Diagnostic and statistical manual of mental disorders: DSM-5. 5th ed. Washington, D.C.: American Psychiatric Association; 2013.
- 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.
- Oriental Neuropsychiatry Editorial Committee of Korean Medicine schools. Oriental neuropsychiatry: revised ed. Gyeonggi: Jipmoondang; 2011. p. 332-41.
- Loew D. [Value of Ginkgo biloba in treatment of Alzheimer dementia]. Wien Med Wochenschr. 2002;152(15-16):418-22. German.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983;113(3-4):173-215.
- Korean Neuropsychiatric Association. Textbook of neuropsychiatry. 3rd ed. Seoul: iMiS Company; 2019. p. 579-89.
- Kumar A, Singh A; Ekavali. A review on Alzheimer's disease pathophysiology and its management: an update. Pharmacol Rep. 2015;67(2):195-203.
- 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.
- 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.
- 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.
- 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.
- 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.
- OECD. est No. 471: bacterial reverse mutation test. Paris: OECD; 1997.