Original Article

Split Viewer

Journal of Acupuncture Research 2023; 40(4): 344-350

Published online November 30, 2023

https://doi.org/10.13045/jar.2023.00164

© Korean Acupuncture & Moxibustion Medicine Society

The Evaluation of the Single-Dose Toxicity and Safety of 4-Carvomenthenol in ICR Mice

Yigun Lim1 , Jihoon Kong1 , Jiwon Lee2 , Gabsik Yang3 , Taehan Yook1

1Department of Acupuncture and Moxibustion Medicine, Woosuk University Hospital of Korean Medicine, Jeonju, Korea
2Department of Korean Medicine Rehabilitation, Woosuk University Hospital of Korean Medicine, Jeonju, Korea
3Department of Pharmacology, College of Korean Medicine, Woosuk University, Jeonju, Korea

Correspondence to : Taehan Yook
Department of Acupuncture and Moxibustion Medicine, Woosuk University Hospital of Korean Medicine, 46 Eoeun-ro, Wansan-gu, Jeonju 54987, Korea
E-mail: nasiss@naver.com

Received: July 24, 2023; Revised: August 17, 2023; Accepted: August 23, 2023

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 non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: 4-carvomenthenol[4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol] is a main component of Origanum vulgare L., Zanthoxylum piperitum (L.) DC., and other plants. It has been reported to exhibit anti-inflammatory, antibacterial, and anti-tumor effects. Furthermore, it is necessary to conduct a toxicity test on 4-carvomenthenol to ensure its safety.
Methods: This study included 5-week-old Institute of Cancer Research mice that were categorized into 3 treatment groups (12, 25, and 50 mg/kg 4-carvomenthenol dose levels) and a control group (10% dimethyl sulfoxide, 40% polyethylene glycol 300, 5% Tween 80, and 45% normal saline injection of the final volume), with 5 male mice and 5 female mice per group. All groups were observed for clinical symptoms and body weight in a period of 14 days and were subjected to gross necropsy after euthanasia.
Results: No deaths were recorded. No test substance-related clinical signs in the female mice of the 12 mg/kg dose group were observed. Abnormal gait was observed in 1 male from day 1 to day 3 in the 12 mg/kg dose group; 1−3 males from day 1 to day 7 and 1−5 females from day 1 to day 15 in the 25 mg/kg dose group; and 2−5 males and 2–5 females from day 1 to day 15 in the 50 mg/kg dose group. No test substance-related effect on the body weight and necropsy findings was observed.
Conclusion: The results of this study suggested that the lethal dose of 4-carvomenthenol could be greater than 50 mg/kg. However, further research is needed, especially repeated-dose toxicity studies, to confirm the efficacy and safety of 4-carvomenthenol.

Keywords 4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol; Medicinal herb; Toxicity test; Toxicology

Herbal medicine has consistently played an important role in health management. A great majority of the global population (approximately 80%) depends on the use of herbal medicine for their healthcare [1]. However, only a few studies have focused on the safety of herbal medicine [2], which raises a growing concern about the potential side effects of herbal medicine [3,4].

The appropriate drug dosage is highly crucial in the treatment process. This is because even though a drug has an anticipated beneficial effect at a particular dose, it can be toxic when administered at higher doses [5]. Therefore, conducting a toxicity test is crucial to ensure the safe use of herbal medicine.

4-carvomenthenol[4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol] is a main component of Origanum vulgare L. and Zanthoxylum piperitum (L.) DC. (Z. piperitum DC.) [6,7]. Origanum vulgare and Z. piperitum are used in various fields, including the food, medical, and cosmetic sectors [8,9].

A comprehensive investigation has been conducted to understand the effects of 4-carvomenthenol. Based on the findings of the literature, it has been shown that 4-carvomenthenol exhibits anti-inflammatory and immunomodulatory properties. Bezerra Barros et al. [10] found that 4-carvomenthenol alleviated the symptoms of allergic rhinitis and asthma by suppressing interleukin-13 and mucus production in mice. Previous studies also revealed its significant antibacterial effectiveness against both Gram-positive and Gram-negative bacteria [6,11]. Moreover, it also possesses anticancer properties by triggering apoptosis in the colorectal tumor cells of mice [12].

Considering the various effects and the wide range of uses of plants containing 4-carvomenthenol, it is important to ensure the safe use of 4-carvomenthenol. However, toxicity related to the dosage of 4-carvomenthenol has not been investigated. Therefore, the aim of this study is to evaluate the potential toxicity and approximate lethal dose of 4-carvomenthenol.

1. Test substance

4-carvomenthenol (purit, 99.0%; conten, p-Menth-1-en-4-ol and 95–100%; molecular weight, 154.25 g/mol; Sigma-Aldrich) was suspended sequentially with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich), 40% polyethylene glycol (PEG) 300 (Sigma-Aldrich), 5% Tween 80 (Sigma-Aldrich) of the final volume. The dosing formulation was prepared to the desired concentration with the normal saline injection (JW Pharmaceutical Co., Ltd.). The dose was prepared on the same day of administration.

2. Experimental animals

This study employed Institute of Cancer Research (CRlOri: CD1 [ICR]) mice (Orient Bio Inc.) as they have abundant historical control data and are commonly used in toxicity studies. A total of 48 5-week-old mice were included in this study, which comprised 24 male mice weighing between 26.3 and 30.8 g and 24 female mice weighing between 22.1 and 25.1 g.

Throughout the quarantine-acclimation period, these mice were observed daily for any clinical signs. No abnormalities were observed in any of them. The experiment conditions are as follows: temperature, 20.2–23.4℃; relative humidity, 50.1–63.4%; ventilation, 10−15 times/h; and light, 7 AM to 7 PM (150–300 lux). Food and water were supplied without restrictions. On the last day of the quarantine-acclimation period, 20 males and 20 females with body weights close to the mean body weight were selected. Subsequently, they were randomly assigned to four groups, and each group had five subjects of the same sex.

This experiment was carried out at Biotoxtech Co., Ltd., complying with the regulations of the Animal Protection Act of the Republic of Korea (the Guide for the Care and Use of Laboratory Animals) [13]. Biotoxtech Co., Ltd., obtained full certification from the Association for Assessment and Accreditation of Laboratory Animal Care International in 2010.

Based on the Animal Protection Act of the Republic of Korea, the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech Co., Ltd., thoroughly reviewed and approved this study (Enactment May 31, 1991, No. 4379, Revision Feb. 11, 2020, No.16977) (Approval No.: 220304).

3. Single-dose intramuscular toxicity study

This study was conducted in accordance with the Standards for Toxicity Studies of Drugs [14]. The dosing route was an intramuscular injection. The route was chosen to evaluate the toxicity of intramuscular exposure to the test substance. The dosage of each animal was determined by rounding up the third decimal of their body weight, which was recorded prior to the day of administration. The dosing formulation was administered once into the left thigh muscle via intramuscular injection using a disposable syringe.

The study comprised four groups: a control group and three treatment groups administered at dose levels of 12, 25, and 50 mg/kg, wherein each group consisted of 5 males and 5 females. The control group was administered with the vehicle (10% DMSO, 40% PEG 300, 5% Tween 80, and 45% normal saline injection of final volume) that has the same dose volume as the treatment group (Table 1).

Table 1 . Group designation

GroupDose weight (mg/kg)Dose volume (mg/kg)No. of animals (ID)
MaleFemale
G1 (control)025 (1101–1105)5 (2101–2105)
G2 (low dose)1225 (1201–1205)5 (2201–2205)
G3 (mid dose)2525 (1301–1305)5 (2301–2305)
G4 (high dose)5025 (1401–1405)5 (2401–2405)

G1, 0 mg/kg dose group; G2, 12 mg/kg dose group; G3, 25 mg/kg dose group; G4, 50 mg/kg dose group.



4. Evaluated parameters

All subjects were monitored for clinical signs (type, severity, time of onset and recovery, etc.) and mortality at 30 minutes, 1, 2, 4, and 6 hours after administration (day 1) and once daily thereafter for 14 days (day 2 to day 15). The body weight measurements were taken once on the day of administration (prior to dosing) and subsequently on days 4, 8, and 15. On day 15, CO2 gas inhalation was used to anesthetize all the subjects, followed by the exsanguination from the abdominal aorta. Subsequently, complete gross postmortem examinations were conducted on each subject in the study. Since no gross findings during the necropsy were recorded, a histopathological examination was not performed.

5. Statistical analysis

The body weight data were analyzed using the SAS program version 9.4 (SAS Institute). The homoscedasticity of the measurement data was measured using Bartlett test (α = 0.05). When the homoscedasticity assumptions were met, one-way analysis of variance was conducted, and the level of significance was set at an α value of 0.05.

1. Mortality

During the observation period, no deaths of animals in both sexes were recorded in the control group and the 12, 25, and 50 mg/kg dose groups (Table 2).

Table 2 . Summary of mortality

SexGroup/dose (mg/kg)No. of animalsDays after treatmentMortality (dead/total)
123456789101112131415
MaleG1/050000000000000000/5
G2/1250000000000000000/5
G3/2550000000000000000/5
G4/5050000000000000000/5
FemaleG1/050000000000000000/5
G2/1250000000000000000/5
G3/2550000000000000000/5
G4/5050000000000000000/5


2. Clinical signs

During the observation period, no abnormalities of clinical signs were recorded in both sexes in the control group and in the female mice of the 12 mg/kg dose group. In the 12 mg/kg dose group, abnormal gait (left hindlimb) was observed in one male from 30 minutes after treatment to day 3. In the 25 mg/kg dose group, abnormal gait (left hindlimb) was observed in 1−3 males from 30 minutes after treatment to day 7 and in 1−5 females from 30 minutes after treatment to day 15. In the 50 mg/kg dose group, abnormal gait (left hindlimb) was consistently observed in 2−5 males and 2–5 females from 30 minutes after treatment to day 15 (Table 3).


3. Body weights

Throughout the observation period, no significant changes were observed in the body weights of animals in both sexes in the 12, 25, and 50 mg/kg dose groups and the control group (Table 4).


4. Necropsy findings

No abnormal gross findings were observed in both sexes in the 12, 25, and 50 mg/kg dose groups and control group at necropsy.

As the global demand for herbal medicine continues to rise remarkably, numerous studies have been conducted to emphasize its safe application [15]. However, only a few studies have confirmed the safety of traditional medicine [2], leading to misinformation [15]. Furthermore, several studies have revealed the toxic side effects resulting from the arbitrary and excessive use of herbal medicines without a prescription from medical professionals [3]. Using herbal medicine with the appropriate dosage is important. Furthermore, it is important to conduct toxicity studies regarding treatment dosage to ensure the safety and efficacy of the treatment method.

4-carvomenthenol is a main component of O. vulgare, Z. piperitum, and other plants [7,16]. According to the results of previous studies, it has a notable ability to inhibit the growth of colorectal, pancreatic, prostate, and gastric cancer cells [17]. It also possesses anti-inflammatory and antibacterial functions [6,10,11]. However, research data on the dose toxicity of 4-carvomenthenol is insufficient [16].

Zanthoxylum piperitum is found in traditional herbal medicine and is reported to possess antibacterial, anti-inflammatory, and antioxidant effects [18,19]. Previous studies also confirmed its therapeutic effects on osteoarthritis and rheumatoid arthritis [20,21].

Due to the lack of toxicity test conducted on 4-carvomenthenol, this study aims to gather baseline data for long-term and repeated-dose toxicity, determine effective clinical dosages and appropriate administration volumes, and provide a basis for research on the maximum dosage by conducting a single intramuscular injection toxicity test.

A preliminary study investigated the plasma and dermal pharmacokinetics of 4-carvomenthenol in rats following intravenous administration at a dose of 2 mg/kg [22]. To investigate toxicity levels up to 25 times the dosage used in the preliminary study, 50 mg/kg was selected as the high dose of this study. The mid and low doses were selected at 25 and 12 mg/kg, respectively.

No fatalities were recorded in any groups. The test substance showed no significant impact on body weight and necropsy. The only clinical sign that was monitored during the observation period was the abnormal gait in the left hindlimb where the 4-carvomenthenol was administered. Since the clinical sign was observed only in the test substance dose groups, it is considered to be caused by the test substance.

Our findings revealed that the lethal dose of 4-carvomenthenol could be greater than 50 mg/kg in both male and female mice. Since these results are only based on a short-term, single-dose acute toxicity test, further research, such as long-term and multiple-dose toxicity studies, is needed to ensure the safety and efficacy of 4-carvomenthenol.

The results of this study revealed that the lethal dose of 4-carvomenthenol could be greater than 50 mg/kg in both male and female mice following a single intramuscular injection.

Conceptualization: LYG, KJH, LJW. Data curation: LYG. Funding acquisition: YTH. Investigation: LYG, KJH, LJW. Methodology: LYG, KJH, LJW, YGS, YTH. Project administration: YTH. Resources: KJH, LJW. Supervision: YGS, YTH. Validation: YGS, YTH. Writing – original draft: LYG. Writing – review & editing: LYG, YGS.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (No. NRF-2023R1A2C2005333).

This experiment was carried out at Biotoxtech Co., Ltd., complying with the regulations of the Animal Protection Act of the Republic of Korea (the Guide for the Care and Use of Laboratory Animals) [13]. Biotoxtech Co., Ltd., obtained full certification from the Association for Assessment and Accreditation of Laboratory Animal Care International in 2010.

Based on the Animal Protection Act of the Republic of Korea, the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech Co., Ltd., thoroughly reviewed and approved this study (Enactment May 31, 1991, No. 4379, Revision Feb. 11, 2020, No.16977) (Approval No.: 220304).

  1. Woo CSJ, Lau JSH, El-Nezami H. Herbal medicine: toxicity and recent trends in assessing their potential toxic effects. Adv Bot Res 2012;62:365-384. doi: 10.1016/B978-0-12-394591-4.00009-X.
    CrossRef
  2. Pittler MH, Ernst E. Systematic review: hepatotoxic events associated with herbal medicinal products. Aliment Pharmacol Ther 2003;18:451-471. doi: 10.1046/j.1365-2036.2003.01689.x.
    Pubmed CrossRef
  3. Jang IS, Yang CS, Lee SD, Han CH. A review of herbal medicinal products associated with toxic events in Korea. J Korean Med 2007;28:1-10.
  4. Park HM, Jang IS, Lee SD. Hepatotoxic events associated with herbal medicinal products, folk remedies and food supplements in Korea. J Korean Med 2005;26:152-165.
  5. Cock IE. The safe usage of herbal medicines: counterindications, cross-reactivity and toxicity. Pharmacogn Commun 2015;5:1-38.
    CrossRef
  6. Hammer KA, Carson CF, Riley TV. Effects of Melaleuca alternifolia (tea tree) essential oil and the major monoterpene component terpinen-4-ol on the development of single- and multistep antibiotic resistance and antimicrobial susceptibility. Antimicrob Agents Chemother 2012;56:909-915. doi: 10.1128/AAC.05741-11.
    Pubmed KoreaMed CrossRef
  7. Jung HS. Studies on the Zanthoxylum piperitum De Candolle - pungent principles and Essential oil composition. J Korean Soc Food Nutr 1987;16:123-127.
  8. Premarathne MDGP, Fukutome N, Yamasaki K, Hayakawa F, Nagano AJ, Mizuno H, et al. Elucidation of Japanese pepper (Zanthoxylum piperitum De Candolle) domestication using RAD-Seq. Sci Rep 2021;11:6464. doi: 10.1038/s41598-021-85909-9.
    Pubmed KoreaMed CrossRef
  9. Moghrovyan A, Sahakyan N, Babayan A, Chichoyan N, Petrosyan M, Trchounian A. Essential oil and ethanol extract of oregano (Origanum vulgare L.) from Armenian flora as a natural source of terpenes, flavonoids and other phytochemicals with antiradical, antioxidant, metal chelating, tyrosinase inhibitory and antibacterial activity. Curr Pharm Des 2019;25:1809-1816. doi: 10.2174/1381612825666190702095612.
    Pubmed CrossRef
  10. Bezerra Barros GC, Paiva Ferreira LKD, Ferreira LAMP, Mozzini Monteiro T, Alves AF, Pereira RA, et al. 4-Carvomenthenol ameliorates the murine combined allergic rhinitis and asthma syndrome by inhibiting IL-13 and mucus production via p38MAPK/NF-κB signaling pathway axis. Int Immunopharmacol 2020;88:106938. doi: 10.1016/j.intimp.2020.106938.
    Pubmed CrossRef
  11. Mondello F, De Bernardis F, Girolamo A, Cassone A, Salvatore G. In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species. BMC Infect Dis 2006;6:158. doi: 10.1186/1471-2334-6-158.
    Pubmed KoreaMed CrossRef
  12. Nakayama K, Murata S, Ito H, Iwasaki K, Villareal MO, Zheng YW, et al. Terpinen-4-ol inhibits colorectal cancer growth via reactive oxygen species. Oncol Lett 2017;14:2015-2024. doi: 10.3892/ol.2017.6370.
    Pubmed KoreaMed CrossRef
  13. Clark JD, Gebhart GF, Gonder JC, Keeling ME, Kohn DF. Special report: the 1996 guide for the care and use of laboratory animals. ILAR J 1997;38:41-48. doi: 10.1093/ilar.38.1.41.
    Pubmed CrossRef
  14. Standard for toxicity study of pharmaceuticals. Ministry of Food and Drug Safety [Internet].
    Available from: https://www.mfds.go.kr/eng/brd/m_18/down.do?brd_id=eng0003&seq=71523&data_tp=A&file_seq=1. Accessed , 2022. cited 2023 Jun 29.
  15. Zhang J, Onakpoya IJ, Posadzki P, Eddouks M. The safety of herbal medicine: from prejudice to evidence. Evid Based Complement Alternat Med 2015;2015:316706. doi: 10.1155/2015/316706.
    Pubmed KoreaMed CrossRef
  16. Api AM, Belsito D, Botelho D, Browne D, Bruze M, Burton GA Jr, et al. RIFM fragrance ingredient safety assessment, 4-Carvomenthenol, CAS Registry Number 562-74-3. Food Chem Toxicol 2017;110 Suppl 1:S403-S411. doi: 10.1016/j.fct.2017.07.040.
    Pubmed CrossRef
  17. Shapira S, Pleban S, Kazanov D, Tirosh P, Arber N. Terpinen-4-ol: a novel and promising therapeutic agent for human gastrointestinal cancers. PLoS One 2016;11:e0156540. doi: 10.1371/journal.pone.0156540.
    Pubmed KoreaMed CrossRef
  18. Jang MJ, Rhee SJ, Cho SH, Woo MH, Choi JH. A study on the antioxidative, anti-inflammatory and anti-thrombogenic effects of Zanthoxylum piperitum DC. Extract. J Korean Soc Food Sci Nutr 2006;35:21-27. doi: 10.3746/jkfn.2006.35.1.021.
    CrossRef
  19. Kusuda M, Inada K, Ogawa TO, Yoshida T, Shiota S, Tsuchiya T, et al. Polyphenolic constituent structures of Zanthoxylum piperitum fruit and the antibacterial effects of its polymeric procyanidin on methicillin-resistant Staphylococcus aureus. Biosci Biotechnol Biochem 2006;70:1423-1431. doi: 10.1271/bbb.50669.
    Pubmed CrossRef
  20. Hwang KA, Kwon JE, Noh Y, Park B, Jeong YJ, Lee SM, et al. Effects of Zanthoxylum piperitum ethanol extract on osteoarthritis inflammation and pain. Biomed Pharmacother 2018;105:481-490. doi: 10.1016/j.biopha.2018.05.109.
    Pubmed CrossRef
  21. Yun SW, Seo YJ, Kwon JE, Park DW, Lee YG, Choe TH, et al. Preclinical evaluation of Zanthoxylum piperitum Benn., traditional muscle pain remedy, for joint inflammation. J Ethnopharmacol 2022;286:114921. doi: 10.1016/j.jep.2021.114921.
    Pubmed CrossRef
  22. Chooluck K, Singh RP, Sathirakul K, Derendorf H. Plasma and dermal pharmacokinetics of terpinen-4-ol in rats following intravenous administration. Pharmazie 2013;68:135-140. doi: 10.1691/ph.2013.2116.

Article

Original Article

Journal of Acupuncture Research 2023; 40(4): 344-350

Published online November 30, 2023 https://doi.org/10.13045/jar.2023.00164

Copyright © Korean Acupuncture & Moxibustion Medicine Society.

The Evaluation of the Single-Dose Toxicity and Safety of 4-Carvomenthenol in ICR Mice

Yigun Lim1 , Jihoon Kong1 , Jiwon Lee2 , Gabsik Yang3 , Taehan Yook1

1Department of Acupuncture and Moxibustion Medicine, Woosuk University Hospital of Korean Medicine, Jeonju, Korea
2Department of Korean Medicine Rehabilitation, Woosuk University Hospital of Korean Medicine, Jeonju, Korea
3Department of Pharmacology, College of Korean Medicine, Woosuk University, Jeonju, Korea

Correspondence to:Taehan Yook
Department of Acupuncture and Moxibustion Medicine, Woosuk University Hospital of Korean Medicine, 46 Eoeun-ro, Wansan-gu, Jeonju 54987, Korea
E-mail: nasiss@naver.com

Received: July 24, 2023; Revised: August 17, 2023; Accepted: August 23, 2023

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 non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: 4-carvomenthenol[4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol] is a main component of Origanum vulgare L., Zanthoxylum piperitum (L.) DC., and other plants. It has been reported to exhibit anti-inflammatory, antibacterial, and anti-tumor effects. Furthermore, it is necessary to conduct a toxicity test on 4-carvomenthenol to ensure its safety.
Methods: This study included 5-week-old Institute of Cancer Research mice that were categorized into 3 treatment groups (12, 25, and 50 mg/kg 4-carvomenthenol dose levels) and a control group (10% dimethyl sulfoxide, 40% polyethylene glycol 300, 5% Tween 80, and 45% normal saline injection of the final volume), with 5 male mice and 5 female mice per group. All groups were observed for clinical symptoms and body weight in a period of 14 days and were subjected to gross necropsy after euthanasia.
Results: No deaths were recorded. No test substance-related clinical signs in the female mice of the 12 mg/kg dose group were observed. Abnormal gait was observed in 1 male from day 1 to day 3 in the 12 mg/kg dose group; 1−3 males from day 1 to day 7 and 1−5 females from day 1 to day 15 in the 25 mg/kg dose group; and 2−5 males and 2–5 females from day 1 to day 15 in the 50 mg/kg dose group. No test substance-related effect on the body weight and necropsy findings was observed.
Conclusion: The results of this study suggested that the lethal dose of 4-carvomenthenol could be greater than 50 mg/kg. However, further research is needed, especially repeated-dose toxicity studies, to confirm the efficacy and safety of 4-carvomenthenol.

Keywords: 4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol, Medicinal herb, Toxicity test, Toxicology

INTRODUCTION

Herbal medicine has consistently played an important role in health management. A great majority of the global population (approximately 80%) depends on the use of herbal medicine for their healthcare [1]. However, only a few studies have focused on the safety of herbal medicine [2], which raises a growing concern about the potential side effects of herbal medicine [3,4].

The appropriate drug dosage is highly crucial in the treatment process. This is because even though a drug has an anticipated beneficial effect at a particular dose, it can be toxic when administered at higher doses [5]. Therefore, conducting a toxicity test is crucial to ensure the safe use of herbal medicine.

4-carvomenthenol[4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol] is a main component of Origanum vulgare L. and Zanthoxylum piperitum (L.) DC. (Z. piperitum DC.) [6,7]. Origanum vulgare and Z. piperitum are used in various fields, including the food, medical, and cosmetic sectors [8,9].

A comprehensive investigation has been conducted to understand the effects of 4-carvomenthenol. Based on the findings of the literature, it has been shown that 4-carvomenthenol exhibits anti-inflammatory and immunomodulatory properties. Bezerra Barros et al. [10] found that 4-carvomenthenol alleviated the symptoms of allergic rhinitis and asthma by suppressing interleukin-13 and mucus production in mice. Previous studies also revealed its significant antibacterial effectiveness against both Gram-positive and Gram-negative bacteria [6,11]. Moreover, it also possesses anticancer properties by triggering apoptosis in the colorectal tumor cells of mice [12].

Considering the various effects and the wide range of uses of plants containing 4-carvomenthenol, it is important to ensure the safe use of 4-carvomenthenol. However, toxicity related to the dosage of 4-carvomenthenol has not been investigated. Therefore, the aim of this study is to evaluate the potential toxicity and approximate lethal dose of 4-carvomenthenol.

MATERIALS AND METHODS

1. Test substance

4-carvomenthenol (purit, 99.0%; conten, p-Menth-1-en-4-ol and 95–100%; molecular weight, 154.25 g/mol; Sigma-Aldrich) was suspended sequentially with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich), 40% polyethylene glycol (PEG) 300 (Sigma-Aldrich), 5% Tween 80 (Sigma-Aldrich) of the final volume. The dosing formulation was prepared to the desired concentration with the normal saline injection (JW Pharmaceutical Co., Ltd.). The dose was prepared on the same day of administration.

2. Experimental animals

This study employed Institute of Cancer Research (CRlOri: CD1 [ICR]) mice (Orient Bio Inc.) as they have abundant historical control data and are commonly used in toxicity studies. A total of 48 5-week-old mice were included in this study, which comprised 24 male mice weighing between 26.3 and 30.8 g and 24 female mice weighing between 22.1 and 25.1 g.

Throughout the quarantine-acclimation period, these mice were observed daily for any clinical signs. No abnormalities were observed in any of them. The experiment conditions are as follows: temperature, 20.2–23.4℃; relative humidity, 50.1–63.4%; ventilation, 10−15 times/h; and light, 7 AM to 7 PM (150–300 lux). Food and water were supplied without restrictions. On the last day of the quarantine-acclimation period, 20 males and 20 females with body weights close to the mean body weight were selected. Subsequently, they were randomly assigned to four groups, and each group had five subjects of the same sex.

This experiment was carried out at Biotoxtech Co., Ltd., complying with the regulations of the Animal Protection Act of the Republic of Korea (the Guide for the Care and Use of Laboratory Animals) [13]. Biotoxtech Co., Ltd., obtained full certification from the Association for Assessment and Accreditation of Laboratory Animal Care International in 2010.

Based on the Animal Protection Act of the Republic of Korea, the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech Co., Ltd., thoroughly reviewed and approved this study (Enactment May 31, 1991, No. 4379, Revision Feb. 11, 2020, No.16977) (Approval No.: 220304).

3. Single-dose intramuscular toxicity study

This study was conducted in accordance with the Standards for Toxicity Studies of Drugs [14]. The dosing route was an intramuscular injection. The route was chosen to evaluate the toxicity of intramuscular exposure to the test substance. The dosage of each animal was determined by rounding up the third decimal of their body weight, which was recorded prior to the day of administration. The dosing formulation was administered once into the left thigh muscle via intramuscular injection using a disposable syringe.

The study comprised four groups: a control group and three treatment groups administered at dose levels of 12, 25, and 50 mg/kg, wherein each group consisted of 5 males and 5 females. The control group was administered with the vehicle (10% DMSO, 40% PEG 300, 5% Tween 80, and 45% normal saline injection of final volume) that has the same dose volume as the treatment group (Table 1).

Table 1 . Group designation.

GroupDose weight (mg/kg)Dose volume (mg/kg)No. of animals (ID)
MaleFemale
G1 (control)025 (1101–1105)5 (2101–2105)
G2 (low dose)1225 (1201–1205)5 (2201–2205)
G3 (mid dose)2525 (1301–1305)5 (2301–2305)
G4 (high dose)5025 (1401–1405)5 (2401–2405)

G1, 0 mg/kg dose group; G2, 12 mg/kg dose group; G3, 25 mg/kg dose group; G4, 50 mg/kg dose group..



4. Evaluated parameters

All subjects were monitored for clinical signs (type, severity, time of onset and recovery, etc.) and mortality at 30 minutes, 1, 2, 4, and 6 hours after administration (day 1) and once daily thereafter for 14 days (day 2 to day 15). The body weight measurements were taken once on the day of administration (prior to dosing) and subsequently on days 4, 8, and 15. On day 15, CO2 gas inhalation was used to anesthetize all the subjects, followed by the exsanguination from the abdominal aorta. Subsequently, complete gross postmortem examinations were conducted on each subject in the study. Since no gross findings during the necropsy were recorded, a histopathological examination was not performed.

5. Statistical analysis

The body weight data were analyzed using the SAS program version 9.4 (SAS Institute). The homoscedasticity of the measurement data was measured using Bartlett test (α = 0.05). When the homoscedasticity assumptions were met, one-way analysis of variance was conducted, and the level of significance was set at an α value of 0.05.

RESULTS

1. Mortality

During the observation period, no deaths of animals in both sexes were recorded in the control group and the 12, 25, and 50 mg/kg dose groups (Table 2).

Table 2 . Summary of mortality.

SexGroup/dose (mg/kg)No. of animalsDays after treatmentMortality (dead/total)
123456789101112131415
MaleG1/050000000000000000/5
G2/1250000000000000000/5
G3/2550000000000000000/5
G4/5050000000000000000/5
FemaleG1/050000000000000000/5
G2/1250000000000000000/5
G3/2550000000000000000/5
G4/5050000000000000000/5


2. Clinical signs

During the observation period, no abnormalities of clinical signs were recorded in both sexes in the control group and in the female mice of the 12 mg/kg dose group. In the 12 mg/kg dose group, abnormal gait (left hindlimb) was observed in one male from 30 minutes after treatment to day 3. In the 25 mg/kg dose group, abnormal gait (left hindlimb) was observed in 1−3 males from 30 minutes after treatment to day 7 and in 1−5 females from 30 minutes after treatment to day 15. In the 50 mg/kg dose group, abnormal gait (left hindlimb) was consistently observed in 2−5 males and 2–5 females from 30 minutes after treatment to day 15 (Table 3).


3. Body weights

Throughout the observation period, no significant changes were observed in the body weights of animals in both sexes in the 12, 25, and 50 mg/kg dose groups and the control group (Table 4).


4. Necropsy findings

No abnormal gross findings were observed in both sexes in the 12, 25, and 50 mg/kg dose groups and control group at necropsy.

DISCUSSION

As the global demand for herbal medicine continues to rise remarkably, numerous studies have been conducted to emphasize its safe application [15]. However, only a few studies have confirmed the safety of traditional medicine [2], leading to misinformation [15]. Furthermore, several studies have revealed the toxic side effects resulting from the arbitrary and excessive use of herbal medicines without a prescription from medical professionals [3]. Using herbal medicine with the appropriate dosage is important. Furthermore, it is important to conduct toxicity studies regarding treatment dosage to ensure the safety and efficacy of the treatment method.

4-carvomenthenol is a main component of O. vulgare, Z. piperitum, and other plants [7,16]. According to the results of previous studies, it has a notable ability to inhibit the growth of colorectal, pancreatic, prostate, and gastric cancer cells [17]. It also possesses anti-inflammatory and antibacterial functions [6,10,11]. However, research data on the dose toxicity of 4-carvomenthenol is insufficient [16].

Zanthoxylum piperitum is found in traditional herbal medicine and is reported to possess antibacterial, anti-inflammatory, and antioxidant effects [18,19]. Previous studies also confirmed its therapeutic effects on osteoarthritis and rheumatoid arthritis [20,21].

Due to the lack of toxicity test conducted on 4-carvomenthenol, this study aims to gather baseline data for long-term and repeated-dose toxicity, determine effective clinical dosages and appropriate administration volumes, and provide a basis for research on the maximum dosage by conducting a single intramuscular injection toxicity test.

A preliminary study investigated the plasma and dermal pharmacokinetics of 4-carvomenthenol in rats following intravenous administration at a dose of 2 mg/kg [22]. To investigate toxicity levels up to 25 times the dosage used in the preliminary study, 50 mg/kg was selected as the high dose of this study. The mid and low doses were selected at 25 and 12 mg/kg, respectively.

No fatalities were recorded in any groups. The test substance showed no significant impact on body weight and necropsy. The only clinical sign that was monitored during the observation period was the abnormal gait in the left hindlimb where the 4-carvomenthenol was administered. Since the clinical sign was observed only in the test substance dose groups, it is considered to be caused by the test substance.

Our findings revealed that the lethal dose of 4-carvomenthenol could be greater than 50 mg/kg in both male and female mice. Since these results are only based on a short-term, single-dose acute toxicity test, further research, such as long-term and multiple-dose toxicity studies, is needed to ensure the safety and efficacy of 4-carvomenthenol.

CONCLUSION

The results of this study revealed that the lethal dose of 4-carvomenthenol could be greater than 50 mg/kg in both male and female mice following a single intramuscular injection.

AUTHOR CONTRIBUTIONS

Conceptualization: LYG, KJH, LJW. Data curation: LYG. Funding acquisition: YTH. Investigation: LYG, KJH, LJW. Methodology: LYG, KJH, LJW, YGS, YTH. Project administration: YTH. Resources: KJH, LJW. Supervision: YGS, YTH. Validation: YGS, YTH. Writing – original draft: LYG. Writing – review & editing: LYG, YGS.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare.

FUNDING

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (No. NRF-2023R1A2C2005333).

ETHICAL STATEMENT

This experiment was carried out at Biotoxtech Co., Ltd., complying with the regulations of the Animal Protection Act of the Republic of Korea (the Guide for the Care and Use of Laboratory Animals) [13]. Biotoxtech Co., Ltd., obtained full certification from the Association for Assessment and Accreditation of Laboratory Animal Care International in 2010.

Based on the Animal Protection Act of the Republic of Korea, the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech Co., Ltd., thoroughly reviewed and approved this study (Enactment May 31, 1991, No. 4379, Revision Feb. 11, 2020, No.16977) (Approval No.: 220304).

Table 1 . Group designation.

GroupDose weight (mg/kg)Dose volume (mg/kg)No. of animals (ID)
MaleFemale
G1 (control)025 (1101–1105)5 (2101–2105)
G2 (low dose)1225 (1201–1205)5 (2201–2205)
G3 (mid dose)2525 (1301–1305)5 (2301–2305)
G4 (high dose)5025 (1401–1405)5 (2401–2405)

G1, 0 mg/kg dose group; G2, 12 mg/kg dose group; G3, 25 mg/kg dose group; G4, 50 mg/kg dose group..


Table 2 . Summary of mortality.

SexGroup/dose (mg/kg)No. of animalsDays after treatmentMortality (dead/total)
123456789101112131415
MaleG1/050000000000000000/5
G2/1250000000000000000/5
G3/2550000000000000000/5
G4/5050000000000000000/5
FemaleG1/050000000000000000/5
G2/1250000000000000000/5
G3/2550000000000000000/5
G4/5050000000000000000/5

References

  1. Woo CSJ, Lau JSH, El-Nezami H. Herbal medicine: toxicity and recent trends in assessing their potential toxic effects. Adv Bot Res 2012;62:365-384. doi: 10.1016/B978-0-12-394591-4.00009-X.
    CrossRef
  2. Pittler MH, Ernst E. Systematic review: hepatotoxic events associated with herbal medicinal products. Aliment Pharmacol Ther 2003;18:451-471. doi: 10.1046/j.1365-2036.2003.01689.x.
    Pubmed CrossRef
  3. Jang IS, Yang CS, Lee SD, Han CH. A review of herbal medicinal products associated with toxic events in Korea. J Korean Med 2007;28:1-10.
  4. Park HM, Jang IS, Lee SD. Hepatotoxic events associated with herbal medicinal products, folk remedies and food supplements in Korea. J Korean Med 2005;26:152-165.
  5. Cock IE. The safe usage of herbal medicines: counterindications, cross-reactivity and toxicity. Pharmacogn Commun 2015;5:1-38.
    CrossRef
  6. Hammer KA, Carson CF, Riley TV. Effects of Melaleuca alternifolia (tea tree) essential oil and the major monoterpene component terpinen-4-ol on the development of single- and multistep antibiotic resistance and antimicrobial susceptibility. Antimicrob Agents Chemother 2012;56:909-915. doi: 10.1128/AAC.05741-11.
    Pubmed KoreaMed CrossRef
  7. Jung HS. Studies on the Zanthoxylum piperitum De Candolle - pungent principles and Essential oil composition. J Korean Soc Food Nutr 1987;16:123-127.
  8. Premarathne MDGP, Fukutome N, Yamasaki K, Hayakawa F, Nagano AJ, Mizuno H, et al. Elucidation of Japanese pepper (Zanthoxylum piperitum De Candolle) domestication using RAD-Seq. Sci Rep 2021;11:6464. doi: 10.1038/s41598-021-85909-9.
    Pubmed KoreaMed CrossRef
  9. Moghrovyan A, Sahakyan N, Babayan A, Chichoyan N, Petrosyan M, Trchounian A. Essential oil and ethanol extract of oregano (Origanum vulgare L.) from Armenian flora as a natural source of terpenes, flavonoids and other phytochemicals with antiradical, antioxidant, metal chelating, tyrosinase inhibitory and antibacterial activity. Curr Pharm Des 2019;25:1809-1816. doi: 10.2174/1381612825666190702095612.
    Pubmed CrossRef
  10. Bezerra Barros GC, Paiva Ferreira LKD, Ferreira LAMP, Mozzini Monteiro T, Alves AF, Pereira RA, et al. 4-Carvomenthenol ameliorates the murine combined allergic rhinitis and asthma syndrome by inhibiting IL-13 and mucus production via p38MAPK/NF-κB signaling pathway axis. Int Immunopharmacol 2020;88:106938. doi: 10.1016/j.intimp.2020.106938.
    Pubmed CrossRef
  11. Mondello F, De Bernardis F, Girolamo A, Cassone A, Salvatore G. In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species. BMC Infect Dis 2006;6:158. doi: 10.1186/1471-2334-6-158.
    Pubmed KoreaMed CrossRef
  12. Nakayama K, Murata S, Ito H, Iwasaki K, Villareal MO, Zheng YW, et al. Terpinen-4-ol inhibits colorectal cancer growth via reactive oxygen species. Oncol Lett 2017;14:2015-2024. doi: 10.3892/ol.2017.6370.
    Pubmed KoreaMed CrossRef
  13. Clark JD, Gebhart GF, Gonder JC, Keeling ME, Kohn DF. Special report: the 1996 guide for the care and use of laboratory animals. ILAR J 1997;38:41-48. doi: 10.1093/ilar.38.1.41.
    Pubmed CrossRef
  14. Standard for toxicity study of pharmaceuticals. Ministry of Food and Drug Safety [Internet]. Available from: https://www.mfds.go.kr/eng/brd/m_18/down.do?brd_id=eng0003&seq=71523&data_tp=A&file_seq=1. Accessed , 2022. cited 2023 Jun 29.
  15. Zhang J, Onakpoya IJ, Posadzki P, Eddouks M. The safety of herbal medicine: from prejudice to evidence. Evid Based Complement Alternat Med 2015;2015:316706. doi: 10.1155/2015/316706.
    Pubmed KoreaMed CrossRef
  16. Api AM, Belsito D, Botelho D, Browne D, Bruze M, Burton GA Jr, et al. RIFM fragrance ingredient safety assessment, 4-Carvomenthenol, CAS Registry Number 562-74-3. Food Chem Toxicol 2017;110 Suppl 1:S403-S411. doi: 10.1016/j.fct.2017.07.040.
    Pubmed CrossRef
  17. Shapira S, Pleban S, Kazanov D, Tirosh P, Arber N. Terpinen-4-ol: a novel and promising therapeutic agent for human gastrointestinal cancers. PLoS One 2016;11:e0156540. doi: 10.1371/journal.pone.0156540.
    Pubmed KoreaMed CrossRef
  18. Jang MJ, Rhee SJ, Cho SH, Woo MH, Choi JH. A study on the antioxidative, anti-inflammatory and anti-thrombogenic effects of Zanthoxylum piperitum DC. Extract. J Korean Soc Food Sci Nutr 2006;35:21-27. doi: 10.3746/jkfn.2006.35.1.021.
    CrossRef
  19. Kusuda M, Inada K, Ogawa TO, Yoshida T, Shiota S, Tsuchiya T, et al. Polyphenolic constituent structures of Zanthoxylum piperitum fruit and the antibacterial effects of its polymeric procyanidin on methicillin-resistant Staphylococcus aureus. Biosci Biotechnol Biochem 2006;70:1423-1431. doi: 10.1271/bbb.50669.
    Pubmed CrossRef
  20. Hwang KA, Kwon JE, Noh Y, Park B, Jeong YJ, Lee SM, et al. Effects of Zanthoxylum piperitum ethanol extract on osteoarthritis inflammation and pain. Biomed Pharmacother 2018;105:481-490. doi: 10.1016/j.biopha.2018.05.109.
    Pubmed CrossRef
  21. Yun SW, Seo YJ, Kwon JE, Park DW, Lee YG, Choe TH, et al. Preclinical evaluation of Zanthoxylum piperitum Benn., traditional muscle pain remedy, for joint inflammation. J Ethnopharmacol 2022;286:114921. doi: 10.1016/j.jep.2021.114921.
    Pubmed CrossRef
  22. Chooluck K, Singh RP, Sathirakul K, Derendorf H. Plasma and dermal pharmacokinetics of terpinen-4-ol in rats following intravenous administration. Pharmazie 2013;68:135-140. doi: 10.1691/ph.2013.2116.
JAR
Feb 29, 2024 Vol.41 No.1, pp. 1~73

Stats or Metrics

Share this article on

  • line

Journal of Acupuncture Research

pISSN 2586-288X
eISSN 2586-2898
qr-code Download