전체메뉴

Journal of Pharmacopuncture

Article Search

JoP

Original Article

Split Viewer

Single-Dose Intramuscular Toxicity Study of SU-Eohyeol Pharmacopuncture in Rats

Ji Hye Hwang1 , Jaseung Ku2 , Chul Jung3*

1Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Gachon University, Seongnam, Republic of Korea
2Bogwang Korean Medicine Clinic, Seoul, Republic of Korea
3Namsangcheon Korean Medicine Clinic, Seoul, Republic of Korea

Correspondence to: Chul Jung
Namsangcheon Korean Medicine Clinic, 109 Banpo-daero, Seocho-gu, Seoul 06656, Republic of Korea
Tel: +82-2-583-0326
E-mail: jcnu2000@hanmail.net

Received: April 23, 2022; Revised: May 6, 2022; Accepted: June 16, 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.

J Pharmacopuncture 2022; 25(3): 268-275

Published September 30, 2022 https://doi.org/10.3831/KPI.2022.25.3.268

Copyright © The Korean Pharmacopuncture Institute.

Abstract

Go to
Objectives: This toxicological study was performed to assess for potential toxicity and to determine the approximate lethal dose of SU-Eohyeol pharmacopuncture (SUEP) following a single intramuscular injection of SUEP into male and female Sprague-Dawley (SD) rats.
Methods: The groups in our experiment consisted of an experimental group treated with SUEP at a dose of 1.0 mL/animal and a control group injected with a normal saline solution, and five male and female rats were placed in each group. Each animal was administered a single intramuscular injection. We monitored all rats for clinical signs and body weight changes for 14 days after administration. At the end of the observation period, the rats were euthanized and autopsied, and localized tolerance examinations were conducted at the site of administration of the test substance.
Results: There were no deaths in either sex in the SUEP-treated group. There was no significant difference between the SUEP-treated group and the control group in the clinical signs and weight changes among the rats. In addition, no significant SUEP-related changes were observed on autopsy findings or local tolerance examinations at the injection site by histopathological examination.
Conclusion: Our results suggest that the approximate lethal dose of a single intramuscular administration of SUEP in female and male rats under the conditions of this study is greater than 1.0 mL/animal. To determine the safety of the use of SUEP in Korean medical clinical practice, additional toxicity studies will be needed.

Keywords

safety, acupuncture, single intramuscular toxicity, jungsongouhyul pharmacopuncture, cervi parvum cornu, su-eohyeol

INTRODUCTION

Go to

Pharmacopuncture (acupoint injection, herbal acupuncture, aqua-acupuncture) used in traditional Chinese and Korean medicine (TCM and TKM, respectively) is a new form of acupuncture involving the injection of various herbal medicines based on both the meridian and Qi flavor theory. Unlike oral administration through the digestive system, pharmacopuncture can deliver herbal medicine directly to the target site; therefore, it is known to have a larger and faster effect than oral administration [1, 2]. Currently, in Korea, many types of pharmacopuncture agents are being used as staples in clinical practice [3, 4], and new pharmacopuncture agents are actively being developed based on literature and clinical experience.

Jungsong-ouhyul pharmacopuncture (JOP), which consists of eight medicinal herbs such as Gardeniae Fructus, Olibanum, Myrrha, Corydalis Tuber, Persicae Semen, Salviae Miltiorrhizae Radix, Paeoniae Radix, and Sappan Lignum, is known to have effects such as such as promoting blood circulation, relieving pain, and reducing inflammation [5, 6]. Cervi Parvum Cornu (CPC), a predominant nontoxic agent in TCM and TKM has also been reported to have the effect of replenishing Blood and Essence, tonifying Kidney Yang, and strengthening bones and muscles [7, 8]. In addition, CPC has been pharmacologically demonstrated to improve immunity and possess antifatigue, antioxidant, stress relief, anti-inflammatory, anti-cancer, and analgesic effects [7, 9]. Thus, CPC pharmacopuncture is a type of immune (meridian, meridian-field) pharmacopuncture that can be used to treat a wide range of diseases by compensating for deficiencies in Qi, Blood, Yin and Yang. However, early reports have suggested that when CPC is prepared by alcohol immersion, it becomes fibrous when exposed to air and causes burning pain when injected into patients [1, 5].

SU-Eohyeol pharmacopuncture (SUEP) was recently developed by adding CPC to JOP to supplement essential body fluids in human tissues. SU is the English transcription of the Korean pronunciation of the Chinese character 秀, which means excellent. This was developed by changing the existing preparation method to include CPC to improve the durability of the treatment effect. By reducing the size of particles through nano-extraction and microfiltration and increasing the concentration of the immersion solution under reduced pressure concentration, SUEP can also be applied more efficiently to weak patients.

However, newly developed pharmacopuncture agents require toxicity evaluation data to be recognized as safe pharmacopuncture agents. Although there have been many studies on the effectiveness of JOP as a widely used agent in clinical practice [4, 10], no toxicological evaluation studies have been conducted. In terms of SUEP, a clinical study was reported in which a mixed pharmacopuncture of Jungsong-ouhyul and CPC was used to treat lower back pain [11]; however, no other clinical or experimental studies have been performed. In this study, to determine the scientific basis for the safety of the newly developed SUEP, the safety of SUEP was evaluated through a single-dose intramuscular toxicity test in male and female Sprague-Dawley (SD) rats.

MATERIALS AND METHODS

Go to

1. Preparation of SUEP and control materials

SUEP is a pharmacopuncture agent with nine herbs. SUEP used in this study was manufactured at an external herbal dispensary (EHD) facility (Namsangcheon EHD, Yongin, Korea) that meets the Korean Good Manufacturing Practice standards. The amount of dry raw herbs used for extraction based on 1 L water was as follows: CPC (50 g), Gardeniae Fructus (75 g), Olibanum (30 g), Myrrha (30 g), Corydalis Tuber (30 g), Persicae Semen (22.5 g), Salviae Miltiorrhizae Radix (22.5 g), Paeoniae Radix (22.5 g), and Sappan Lignum (22.5 g). Two extraction methods were used in this study: 1) In the case of CPC, the tips of Russian deer velvet antlers were powdered, water was added to the distillation extractor for injection, and the extract was recovered by circulation extraction. After adding alcohol, stirring, paper filtering, and removing the alcohol from the filtrate using a reduced pressure concentrator, the extract was subjected to ultrafiltration with a molecular weight fraction of 10,000 Da. 2) In the case of other herbal medicines, water for injection was added to the distillation extractor, and the distillate was recovered after circulation. 3) CPC concentrates and other herbal distillates that had undergone ultrafiltration were mixed and filtered, and 0.9% NaCl was added, dissolved by stirring, and then titrated to pH 7.4. The filtrate was then filtered a final time through a 0.45-0.2-µm filter and stored in a sealed container.

In this single-dose intramuscular toxicity test, 2 mL of pale-yellow liquid SUEP sealed in a transparent vial was used, and normal saline was set as the control.

2. Experimental animals

SD rats are widely used in drug safety testing [12] and were selected because of the abundance of basic data available for comparison. We purchased SD rats (5-week-old males and females, n = 6 per group) from Orient Bio Inc. (Seongnam, Korea) for the single-dose intramuscular toxicity testing. On the first arrival of the animals, visual inspection was performed; then, their weights were measured and recorded. The body weight was in the range of 120.9-138.4 g and 110.1-118.7 g for male and female rats, respectively. During the quarantine and acclimatization periods, their general symptoms such as appearance, posture, attitude, behavior, nervous system, respiration, body temperature, and excretion were observed once a day. At the end of these periods, all SD rats were weighed again, and general symptoms and lack of changes in weight confirmed that there were no abnormalities in any of the animals. At the end of the acclimatization period, 10 males and females each of an average weight (186.1-204.9 g and 150.6-168.5 g for male and female rats, respectively) were selected and grouped into two groups (5 males and 5 females per group). The selected animals were randomly placed such that the average body weight of each group was equal. SD rats were housed in controlled environmental conditions of 20.3-24.4℃ ambient temperature, 44.9-60.9% relative humidity, 12-h/12-h light/dark cycle, 10-15 ventilation times/hour, illuminance of 150-300 Lux, and free access to food (Envigo RMS Inc., Huntingdon, UK) and water.

The animals were cared for and treated in accordance with the guidelines of the Korean National Institute of Health (KNIH) and the Korean Academy of Medical Sciences (KAMS). This study was conducted at Biotoxtech (Cheongwon, Korea) (experiment number: B211217). Biotoxtech obtained full certification from the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) in 2010.

Our toxicity evaluation study was conducted in accordance with the following Good Laboratory Practice (GLP) Regulations: “GLP Regulation for Nonclinical Laboratory Studies” (Notification No. 2018-93, Ministry of Food and Drug Safety [MFDS] [November 21, 2018]) [13], and “OECD Principles of GLP” (OECD, ENV/MC/CHEM(98)17 [as revised in 1997]) [14].

This test was approved by the Animal Ethics Experimentation Committee of Biotoxtech Co., Ltd. based on the Animal Protection Act (Act No. 4379 of May 31, 1991, partially revised Act No. 16977 of February 11, 2020) (Approval No.: 210819).

3. Single-dose intramuscular toxicity test in SD rats

1) Experimental group designation and administration method

The expected clinical application route of SUEP is through the muscle; therefore, in this study, the muscle route was selected. The expected clinical application dose of SUEP is 0.1-1.0 mL/human (0.1 mL/time). Thus, in this experiment, a dose of 1.0 mL/animal and 0.5 mL/site was administered once each into the left and right thigh muscles of the rats using a disposable syringe (Table 1).

Table 1 . Group designation of a single-dose intramuscular toxicity test for SU-Eohyeol pharmacopuncture in Sprague-Dawley (SD) rats.

GroupDose of SUEP
(mL/animal)		Injection dose amount (mL/animal)Number of animals (object number)
MaleFemale
G1Control (normal saline)01.05 (1101-1105)5 (2101-2105)
G2Test substance (SUOP)1.01.05 (1201-1205)5 (2201-2205)


As a preliminary test for this study (Biotoxtech Study No.: B211217P1), one male and female rat underwent a single intramuscular administration of 1.0 mL/animal. Since no deaths were observed, the doses of SUEP and control saline were set to 1.0 mL/animal, and a total amount of 1.0 mL/animal was intramuscularly injected into the left and right thigh muscles.

2) Methods of observation and examination of animals

On the day of administration, the general state of the rats (type of toxic signs, time of onset, recovery period, etc.) and any potential deaths were noted at 30 min and 1, 2, 4, and 6 h after administration. Thereafter, general symptoms were observed in SD rats once a day for 14 days (from the 2nd day to the 15th day of administration). The body weight of rats was measured on the day of administration (before administration) and on days 4, 8, and 15 (necropsy day).

3) Histopathology

All SD rats used in the main experiment were euthanized by inhalation of CO2 gas on the day of autopsy, exsanguinated from the abdominal aorta, and then autopsied. For all animals subjected to autopsy, the extracted tissues from the injection site were fixed in 10% neutral-buffered formalin (NBF). Organs and tissues subjected to histopathological examination were prepared according to the standard operating procedure for histopathological specimen preparation, and the remaining organs and tissues were preserved in 10% NBF. For histopathological examination, the organ and tissue samples were examined through a localized tolerance examination of the injection site of all animals.

4. Statistical analysis

The body weights obtained in the experiment were statistically analyzed using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA), and the measured values were tested for equality of variance with the Folded-F test (p < 0.05). Equal variance was recognized, and significance was confirmed by Student’s t-test (p < 0.05, p < 0.01).

RESULTS

Go to

1. Observations of general health and weight changes

During the 14-day observation period, no general health-related abnormalities or deaths were observed in the control or SUEP-treated group in all male and female SD rats (Table 2).

Table 2 . SU-Eohyeol pharmacopuncture-related effects on clinical sign changes in a single-dose intramuscular toxicity study in Sprague-Dawley (SD) rats.

SexGroupDay 1 (hour)Day
Dose
(mL/animal)		Animal ID0.5124623456789101112131415
MaleG1 (n = 5)
0		1101-------------------
1102-------------------
1103-------------------
1104-------------------
1105-------------------
G2 (n = 5)
1.0		1201-------------------
1202-------------------
1203-------------------
1204-------------------
1205-------------------
FemaleG1 (n = 5)
0		2101-------------------
2102-------------------
2103-------------------
2104-------------------
2105-------------------
G2 (n = 5)
1.0		2201-------------------
2202-------------------
2203-------------------
2204-------------------
2205-------------------

-: No observable abnormalities in clinical signs..

G1: Control group administered with normal saline (1.0 mL/animal)..

G2: SU-Eohyeol pharmacopuncture administration group (1.0 mL/animal)..

For the change in body weight during our experiment, the body weight of male rats in the SUEP-treated and control groups changed from 200.3 ± 3.7 g (G1) and 196.9 ± 6.9 g (G2), respectively, to 330.4 ± 17.5 g (G1) and 327.1 ± 16.1 g (G2), respectively, whereas that of female rats changed from 161.5 ± 4.6 g (G1) and 156.3 ± 4.2 g (G2), respectively, to 220.9 ± 7.7 g (G1) and 212.8 ± 15.1 g (G2), respectively. No significant difference was observed in the body weight changes in male and female rats treated with SUEP 1.0 mL/animal compared with those in the control group (Fig. 1, Table 3).

Table 3 . Effects of SU-Eohyeol pharmacopuncture on changes in mean body weights in a single-dose intramuscular toxicity test in Sprague-Dawley (SD) rats.

SexGroup/Dose
(mL/animal)		DayDay 1-15
Gain (g)
14815
MaleG1
0		Mean200.3226.7264.1330.4130.1
S.D.3.75.39.717.515.9
N (g)55555
G2
1.0		Mean196.9226.2261.9327.1130.2
S.D.6.98.510.116.110.8
N (g)55555
FemaleG1
0		Mean161.5174.2190.2220.959.4
S.D.4.63.35.37.77.6
N (g)55555
G2
0		Mean156.3173.0189.9212.856.6
S.D.4.27.18.815.111.8
N (g)55555

Significantly different from control by Student’s test..

S.D., standard deviation; N, number of animals..
Figure 1.Effects of SU-Eohyeol Pharmacopuncture on the body weight of male (A) and female (B) Sprague-Dawley (SD) rats.

2. Macroscopic examination following necropsy

There were no abnormalities in the male and female SD rats in the control group or SUEP-treated group on macroscopic examination following necropsy.

3. Histopathological examination

The results of the local tolerance test of SUEP were observed by microscopic examination of the tissues obtained from the administration sites of all male and female SD rats. As a result of microscopic histopathological examination, no SUEP-related changes were observed at the injection site. Treatment with SUEP was considered to be tolerated by all animal groups (Fig. 2).

Figure 2.Histopathological results of a single-dose intramuscular toxicity test for SU-Eohyeol Pharmacopuncture (SUEP) in male and female Sprague-Dawley (SD) rats Histopathological examination images of injection sites in Sprague-Dawley (SD) rats were captured at ×200 (scale bar = 200 μM).

DISCUSSION

Go to

After Nam Sang-cheon officially disclosed pharmacopuncture to the TKM community in 1967, pharmacopuncture has continued to develop and has subsequently become one of the most used TKM treatment methods in Korea [1-5]. In a survey study on the usage status and satisfaction of pharmacopuncture conducted by TKM doctors in Korea, 88% of respondents had experience using pharmacopuncture in the previous year, pharmacopuncture was mainly used for the treatment of musculoskeletal, nervous system, and gastrointestinal diseases, and the two main types used in TKM clinical practice were bee venom and ouhyul pharmacopuncture [4].

While medicinal products are usually administered by the MFDS, pharmacopuncture is implemented in Korea according to prescriptions issued to EHDs by TKM and TCM institutions [15, 16]. An EHD is a type of pharmacy that offers various types and formulations of herbal medicines to TKM institutions according to TKM doctors’ prescriptions based on each patient’s condition and was legalized by the Ministry of Health and Welfare (MoHW) in Korea in 2008. In September 2018, the MoHW announced that the Accreditation System of EHDs would be applied in TKM to evaluate and certify herbal and pharmacopuncture preparations [15-17].

With the MoHW certification mark, EHDs are said to provide safely formulated pharmacopuncture preparations; however, in order for these preparations to be scientifically recognized, the efficacy and safety of newly developed pharmacopuncture drugs needs to be evaluated both non-clinically and clinically.

Conversely, the MFDS requires non-clinical data on safety, such as genotoxicity, single-dose toxicity, repeated dose toxicity, and efficacy, to approve herbal medicines for clinical trials [18-22]. JOP, one of the most widely used pharmacopuncture techniques in TKM, has many reports of its clinical efficacy [4, 10], but no toxicity evaluation reports have been conducted to date. Therefore, SUEP, which was developed by adding CPC to JOP, should be scientifically verified for safety through toxicity evaluation. It is expected that public trust will be gained through these efforts.

Single-dose toxicity studies are conducted to describe the qualitative and quantitative data of time-related toxic phenomena and occurrences after administration of a single dose of a substance or a combination of substances [23]. There are species and phylogenetic differences in biological responses to test substances. In the selection of an animal species, an animal species with a similar metabolic profile to that of a human is preferred. However, during the stage of single-dose toxicity testing, it may be difficult to select an animal species or lineage that satisfies the above criterion; therefore, an animal that is easy to handle, is qualitatively uniform, and has abundant background data is usually selected.

The most commonly preferred rodent is the rat, and the most preferred animal for non-rodent experiments is the dog. In addition, depending on the drug, there may be sex-specific differences in the appearance of toxicity. Thus, toxicity should be evaluated in at least one male and female animal [21, 24].

We performed a SUEP single-dose toxicity study in female and male SD rats. Our toxicological study was performed to assess for potential toxicity and to determine the approximate lethal dose of SUEP after a single intramuscular injection of SUEP into the thigh of 6-week-old male and female SD rats. Our findings showed that there were no deaths in either sex in the SUEP-treated group. There was no significant difference between the SUEP-treated group and the control group in the general condition, weight change, or clinical signs of male or female SD rats. In addition, no significant SUEP-related changes were observed in autopsy findings or local tolerance examinations at the injection site by histopathological examination. According to our toxicity study results, the lethal dose of SUEP is approximately greater than 1.0 mL/animal (approximately 1,525 mg/kg) in both male and female SD rats following a singular intramuscular injection of SUEP.

The expected clinical application dose of SUEP is 1.0 mL at a time based on an adult weight (60 kg), and the dose of 1.0 mL/animal used in this experiment is approximately 500 times higher than the actual clinical dose of SUEP used (1.0 mg/kg). The appropriateness and safety of the SUEP dose used in clinical practice has thus been determined. Therefore, the findings of this study allow us to infer that acupuncture using SUEP agents can be safely administered to humans at a dose of 1.0 mL or less.

However, since our study only investigated the toxic effects of a single dose of SUEP only for an observation period of 14 days, in the future, additional intramuscular toxicity tests, such as multiple-dose and long-term toxicity observations, and various toxicological studies will be needed to establish the effectiveness and safety of SUEP.

Fortunately, pharmacopuncture-related adverse reactions have not been described in clinical reports using a combination of JOP and CPC pharmacopuncture [11]; however, no official clinical study has been conducted on the side effects of SUEP to date. Therefore, many experimental and clinical studies will need to provide scientific evidence regarding the efficacy and safety of SUEP.

CONCLUSION

Go to

According to the results of our toxicity study, the approximate lethal dose of SUEP was greater than 1.0 mL/animal (approximately 1,525 mg/kg) in both male and female SD rats following a single intramuscular injection under the conditions of this study. This finding suggests that, in TKM clinical trials, SUEP can be considered safe at doses below 1.0 mL. To determine the safety of the use of SUEP in TKM clinical practice, additional toxicity studies will be needed.

CONFLICT OF INTEREST

Go to

The authors declare no conflict of interest.

FUNDING

Go to

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2022R1A2C1013518).

References

Go to
  1. Jung C, Jung JH, Lee MS. A clinical study of immune pharmacopuncturology. Yeongi: Kyungrak Medical Publishing Co.; 2011. p. 127-33.
  2. Korean Acupuncture and Moxibustion Society. Acupuncture medicine. Seoul: Hanmi Medical Publishing Co.; 2020. p. 204-28.
  3. Park J, Lee H, Shin BC, Lee MS, Kim B, Kim JI. Pharmacopuncture in Korea: a systematic review and meta-analysis of randomized controlled trials. Evid Based Complement Alternat Med. 2016;2016:4683121.
    Pubmed PMC CrossRef
  4. Park JE, Kim KH, Kang S, Lee EK, Kim JC, Jang BH, et al. Usage status and satisfaction with pharmacopuncture in Korea: a survey among Korean medicine doctors. Eur J Integr Med. 2019;27:121-30.
    CrossRef
  5. Korean Pharmacopuncture Institute Science Committee, Pharmacopuncturology Institute. Pharmacopuncturology. 2nd ed. Seoul: Elsevier Korea; 2008. p. 3-8, 163-80, 259-420.
    CrossRef
  6. Yun JM, Hur NY, Kim KH. The analysis of Korean clinical studies on the effect of pharmacopuncture for whiplash injury after traffic accidents. J Pharmacopuncture. 2020;23(4):262-72.
    Pubmed PMC CrossRef
  7. Lee DJ, Hwangbo M, Kwon K, Seo HS. Review of cervi cornu parvum pharmacopuncture in korean medicine. J Pharmacopuncture. 2013;16(2):7-14.
    Pubmed PMC CrossRef
  8. Bensky D, Gamble A. Chinese herbal medicine: materia medica. Revised ed. Seattle: Eastland Press; 1993. p. 32-44.
    CrossRef
  9. Wu F, Li H, Jin L, Li X, Ma Y, You J, et al. Deer antler base as a traditional Chinese medicine: a review of its traditional uses, chemistry and pharmacology. J Ethnopharmacol. 2013;145(2):403-15.
    Pubmed CrossRef
  10. Yoon JW, Kim SJ. A review of clinical study on Jungsongouhyul pharmacopuncture treatment published in Korea. J Korean Med Rehabil. 2017;27(4):75-84.
    CrossRef
  11. Ryu HS, Kim SP, Chun HS, Ryu MS, Shin JC, Wei TS. The comparative study of effects between acupuncture treatment and cotreatment with mixed pharmacopuncture of Jungsongouhyul and Cervi Pantotrichum Cornu on low back pain patients. J Korean Acupunct Moxib Soc. 2011;28(6):149-57.
  12. Xu Z, Kang Q, Yu Z, Tian L, Zhang J, Wang T. Research on the species difference of the hepatotoxicity of medicine based on transcriptome. Front Pharmacol. 2021;12:647084.
    Pubmed PMC CrossRef
  13. Ministry of Food and Drug Safety. Good Laboratory Practice Regulation for Nonclinical Laboratory Studies (Notification No. 2018-93) [Internet]. Cheongju: Ministry of Food and Drug Safety; 2018 [cited 2018 Nov 21]. Available from: https://www.law.go.kr/LSW/admRulLsInfoP.do?admRulSeq=2100000170349.
  14. Organisation for Economic Co-operation and Development (OECD). ENV/MC/CHEM(98)17: OECD series on principles of good laboratory practice and compliance monitoring number 1. Paris: OECD; 1998. p. 41.
  15. Sung SH, Shin BC, Park MJ, Kim KH, Kim JW, Ryu JY, et al. Current status of management on pharmacopuncture in Korea through introduction of an accreditation system. J Pharmacopuncture. 2019;22(2):75-82.
    Pubmed PMC CrossRef
  16. Ministry of Health and Welfare. Guideline on Installation and Utilization of External Herbal Dispensaries of Traditional Korean Medicine Clinics and Share-use of Herbal Dispensaries [Internet]. Sejong: Ministry of Health and Welfare; 2017 [cited 2018 Nov 21]. Available from: http://www.mohw.go.kr/react/jb/sjb0406vw.jsp?PAR_MENU_ID=03&MENU_ID=030406&CONT_SEQ=342727&page=1.
  17. Ministry of Health and Welfare. Introduction of External Herbal Dispensaries of Traditional Korean Medicine Clinics [Internet]. Sejong: Ministry of Health and Welfare; 2018 Available from: http://www.mohw.go.kr/react/al/sal0301vw.jsp?PAR_MENU_ID=04&MENU_ID=0403&page=1&CONT_SEQ=344877&SEARCHKEY=TITLE&SEARCHVALUE=%ED%95%9C%EC%95%BD.
  18. Ministry of Food and Drug Safety. Good Laboratory Practice (GLP) [Internet]. Cheongju: Ministry of Food and Drug Safety; 2017 [cited 2022 Apr 1]. Available from: https://www.law.go.kr/LSW/admRulInfoP.do?admRulSeq=2100000086689.
  19. Ministry of Food and Drug Safety. Partial amendment official announcement about toxicity test for drug. Cheongju: Ministry of Food and Drug Safety; 2015.
  20. Lee MY, Seo CS, Kim JY, Shin HK. Genotoxicity evaluation of Guibi-Tang extract using an in vitro bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus test. BMC Complement Altern Med. 2014;14:215.
    Pubmed PMC CrossRef
  21. Sung H, Lee E. Single-dose intramuscular toxicity of Mahwangcheonoh pharmacopuncture in a rat model: toxicity of Mahwangcheonoh Pharmacopuncture in SD rats. J Pharmacopuncture. 2016;19(4):336-43.
    Pubmed PMC CrossRef
  22. Hwang JH, Ku J, Jung C. Evaluation of the single-dose toxicity of capsaicin pharmacopuncture in rats. J Acupunct Res. 2020;37(3):167-72.
    CrossRef
  23. Rousseaux CG, Bracken WM. Overview of Drug Development. In: Haschek WM, Rousseaux CG, Wallig MA, editors. Haschek and Rousseaux's handbook of toxicologic pathology. 3rd ed. London: Academic Press; 2013. p. 647-685.
    CrossRef
  24. Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, et al. Report from the in vitro micronucleus assay working group. Mutat Res. 2003;540(2):153-63.
    Pubmed CrossRef

Related articles in JoP

More Related Articles

Article

Original Article

J Pharmacopuncture 2022; 25(3): 268-275

Published online September 30, 2022 https://doi.org/10.3831/KPI.2022.25.3.268

Copyright © The Korean Pharmacopuncture Institute.

Single-Dose Intramuscular Toxicity Study of SU-Eohyeol Pharmacopuncture in Rats

Ji Hye Hwang1 , Jaseung Ku2 , Chul Jung3*

1Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Gachon University, Seongnam, Republic of Korea
2Bogwang Korean Medicine Clinic, Seoul, Republic of Korea
3Namsangcheon Korean Medicine Clinic, Seoul, Republic of Korea

Correspondence to:Chul Jung
Namsangcheon Korean Medicine Clinic, 109 Banpo-daero, Seocho-gu, Seoul 06656, Republic of Korea
Tel: +82-2-583-0326
E-mail: jcnu2000@hanmail.net

Received: April 23, 2022; Revised: May 6, 2022; Accepted: June 16, 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: This toxicological study was performed to assess for potential toxicity and to determine the approximate lethal dose of SU-Eohyeol pharmacopuncture (SUEP) following a single intramuscular injection of SUEP into male and female Sprague-Dawley (SD) rats.
Methods: The groups in our experiment consisted of an experimental group treated with SUEP at a dose of 1.0 mL/animal and a control group injected with a normal saline solution, and five male and female rats were placed in each group. Each animal was administered a single intramuscular injection. We monitored all rats for clinical signs and body weight changes for 14 days after administration. At the end of the observation period, the rats were euthanized and autopsied, and localized tolerance examinations were conducted at the site of administration of the test substance.
Results: There were no deaths in either sex in the SUEP-treated group. There was no significant difference between the SUEP-treated group and the control group in the clinical signs and weight changes among the rats. In addition, no significant SUEP-related changes were observed on autopsy findings or local tolerance examinations at the injection site by histopathological examination.
Conclusion: Our results suggest that the approximate lethal dose of a single intramuscular administration of SUEP in female and male rats under the conditions of this study is greater than 1.0 mL/animal. To determine the safety of the use of SUEP in Korean medical clinical practice, additional toxicity studies will be needed.

Keywords: safety, acupuncture, single intramuscular toxicity, jungsongouhyul pharmacopuncture, cervi parvum cornu, su-eohyeol

INTRODUCTION

Pharmacopuncture (acupoint injection, herbal acupuncture, aqua-acupuncture) used in traditional Chinese and Korean medicine (TCM and TKM, respectively) is a new form of acupuncture involving the injection of various herbal medicines based on both the meridian and Qi flavor theory. Unlike oral administration through the digestive system, pharmacopuncture can deliver herbal medicine directly to the target site; therefore, it is known to have a larger and faster effect than oral administration [1, 2]. Currently, in Korea, many types of pharmacopuncture agents are being used as staples in clinical practice [3, 4], and new pharmacopuncture agents are actively being developed based on literature and clinical experience.

Jungsong-ouhyul pharmacopuncture (JOP), which consists of eight medicinal herbs such as Gardeniae Fructus, Olibanum, Myrrha, Corydalis Tuber, Persicae Semen, Salviae Miltiorrhizae Radix, Paeoniae Radix, and Sappan Lignum, is known to have effects such as such as promoting blood circulation, relieving pain, and reducing inflammation [5, 6]. Cervi Parvum Cornu (CPC), a predominant nontoxic agent in TCM and TKM has also been reported to have the effect of replenishing Blood and Essence, tonifying Kidney Yang, and strengthening bones and muscles [7, 8]. In addition, CPC has been pharmacologically demonstrated to improve immunity and possess antifatigue, antioxidant, stress relief, anti-inflammatory, anti-cancer, and analgesic effects [7, 9]. Thus, CPC pharmacopuncture is a type of immune (meridian, meridian-field) pharmacopuncture that can be used to treat a wide range of diseases by compensating for deficiencies in Qi, Blood, Yin and Yang. However, early reports have suggested that when CPC is prepared by alcohol immersion, it becomes fibrous when exposed to air and causes burning pain when injected into patients [1, 5].

SU-Eohyeol pharmacopuncture (SUEP) was recently developed by adding CPC to JOP to supplement essential body fluids in human tissues. SU is the English transcription of the Korean pronunciation of the Chinese character 秀, which means excellent. This was developed by changing the existing preparation method to include CPC to improve the durability of the treatment effect. By reducing the size of particles through nano-extraction and microfiltration and increasing the concentration of the immersion solution under reduced pressure concentration, SUEP can also be applied more efficiently to weak patients.

However, newly developed pharmacopuncture agents require toxicity evaluation data to be recognized as safe pharmacopuncture agents. Although there have been many studies on the effectiveness of JOP as a widely used agent in clinical practice [4, 10], no toxicological evaluation studies have been conducted. In terms of SUEP, a clinical study was reported in which a mixed pharmacopuncture of Jungsong-ouhyul and CPC was used to treat lower back pain [11]; however, no other clinical or experimental studies have been performed. In this study, to determine the scientific basis for the safety of the newly developed SUEP, the safety of SUEP was evaluated through a single-dose intramuscular toxicity test in male and female Sprague-Dawley (SD) rats.

MATERIALS AND METHODS

1. Preparation of SUEP and control materials

SUEP is a pharmacopuncture agent with nine herbs. SUEP used in this study was manufactured at an external herbal dispensary (EHD) facility (Namsangcheon EHD, Yongin, Korea) that meets the Korean Good Manufacturing Practice standards. The amount of dry raw herbs used for extraction based on 1 L water was as follows: CPC (50 g), Gardeniae Fructus (75 g), Olibanum (30 g), Myrrha (30 g), Corydalis Tuber (30 g), Persicae Semen (22.5 g), Salviae Miltiorrhizae Radix (22.5 g), Paeoniae Radix (22.5 g), and Sappan Lignum (22.5 g). Two extraction methods were used in this study: 1) In the case of CPC, the tips of Russian deer velvet antlers were powdered, water was added to the distillation extractor for injection, and the extract was recovered by circulation extraction. After adding alcohol, stirring, paper filtering, and removing the alcohol from the filtrate using a reduced pressure concentrator, the extract was subjected to ultrafiltration with a molecular weight fraction of 10,000 Da. 2) In the case of other herbal medicines, water for injection was added to the distillation extractor, and the distillate was recovered after circulation. 3) CPC concentrates and other herbal distillates that had undergone ultrafiltration were mixed and filtered, and 0.9% NaCl was added, dissolved by stirring, and then titrated to pH 7.4. The filtrate was then filtered a final time through a 0.45-0.2-µm filter and stored in a sealed container.

In this single-dose intramuscular toxicity test, 2 mL of pale-yellow liquid SUEP sealed in a transparent vial was used, and normal saline was set as the control.

2. Experimental animals

SD rats are widely used in drug safety testing [12] and were selected because of the abundance of basic data available for comparison. We purchased SD rats (5-week-old males and females, n = 6 per group) from Orient Bio Inc. (Seongnam, Korea) for the single-dose intramuscular toxicity testing. On the first arrival of the animals, visual inspection was performed; then, their weights were measured and recorded. The body weight was in the range of 120.9-138.4 g and 110.1-118.7 g for male and female rats, respectively. During the quarantine and acclimatization periods, their general symptoms such as appearance, posture, attitude, behavior, nervous system, respiration, body temperature, and excretion were observed once a day. At the end of these periods, all SD rats were weighed again, and general symptoms and lack of changes in weight confirmed that there were no abnormalities in any of the animals. At the end of the acclimatization period, 10 males and females each of an average weight (186.1-204.9 g and 150.6-168.5 g for male and female rats, respectively) were selected and grouped into two groups (5 males and 5 females per group). The selected animals were randomly placed such that the average body weight of each group was equal. SD rats were housed in controlled environmental conditions of 20.3-24.4℃ ambient temperature, 44.9-60.9% relative humidity, 12-h/12-h light/dark cycle, 10-15 ventilation times/hour, illuminance of 150-300 Lux, and free access to food (Envigo RMS Inc., Huntingdon, UK) and water.

The animals were cared for and treated in accordance with the guidelines of the Korean National Institute of Health (KNIH) and the Korean Academy of Medical Sciences (KAMS). This study was conducted at Biotoxtech (Cheongwon, Korea) (experiment number: B211217). Biotoxtech obtained full certification from the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) in 2010.

Our toxicity evaluation study was conducted in accordance with the following Good Laboratory Practice (GLP) Regulations: “GLP Regulation for Nonclinical Laboratory Studies” (Notification No. 2018-93, Ministry of Food and Drug Safety [MFDS] [November 21, 2018]) [13], and “OECD Principles of GLP” (OECD, ENV/MC/CHEM(98)17 [as revised in 1997]) [14].

This test was approved by the Animal Ethics Experimentation Committee of Biotoxtech Co., Ltd. based on the Animal Protection Act (Act No. 4379 of May 31, 1991, partially revised Act No. 16977 of February 11, 2020) (Approval No.: 210819).

3. Single-dose intramuscular toxicity test in SD rats

1) Experimental group designation and administration method

The expected clinical application route of SUEP is through the muscle; therefore, in this study, the muscle route was selected. The expected clinical application dose of SUEP is 0.1-1.0 mL/human (0.1 mL/time). Thus, in this experiment, a dose of 1.0 mL/animal and 0.5 mL/site was administered once each into the left and right thigh muscles of the rats using a disposable syringe (Table 1).

Table 1

Group designation of a single-dose intramuscular toxicity test for SU-Eohyeol pharmacopuncture in Sprague-Dawley (SD) rats.

GroupDose of SUEP
(mL/animal)		Injection dose amount (mL/animal)Number of animals (object number)
MaleFemale
G1Control (normal saline)01.05 (1101-1105)5 (2101-2105)
G2Test substance (SUOP)1.01.05 (1201-1205)5 (2201-2205)


As a preliminary test for this study (Biotoxtech Study No.: B211217P1), one male and female rat underwent a single intramuscular administration of 1.0 mL/animal. Since no deaths were observed, the doses of SUEP and control saline were set to 1.0 mL/animal, and a total amount of 1.0 mL/animal was intramuscularly injected into the left and right thigh muscles.

2) Methods of observation and examination of animals

On the day of administration, the general state of the rats (type of toxic signs, time of onset, recovery period, etc.) and any potential deaths were noted at 30 min and 1, 2, 4, and 6 h after administration. Thereafter, general symptoms were observed in SD rats once a day for 14 days (from the 2nd day to the 15th day of administration). The body weight of rats was measured on the day of administration (before administration) and on days 4, 8, and 15 (necropsy day).

3) Histopathology

All SD rats used in the main experiment were euthanized by inhalation of CO2 gas on the day of autopsy, exsanguinated from the abdominal aorta, and then autopsied. For all animals subjected to autopsy, the extracted tissues from the injection site were fixed in 10% neutral-buffered formalin (NBF). Organs and tissues subjected to histopathological examination were prepared according to the standard operating procedure for histopathological specimen preparation, and the remaining organs and tissues were preserved in 10% NBF. For histopathological examination, the organ and tissue samples were examined through a localized tolerance examination of the injection site of all animals.

4. Statistical analysis

The body weights obtained in the experiment were statistically analyzed using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA), and the measured values were tested for equality of variance with the Folded-F test (p < 0.05). Equal variance was recognized, and significance was confirmed by Student’s t-test (p < 0.05, p < 0.01).

RESULTS

1. Observations of general health and weight changes

During the 14-day observation period, no general health-related abnormalities or deaths were observed in the control or SUEP-treated group in all male and female SD rats (Table 2).

-: No observable abnormalities in clinical signs..

G1: Control group administered with normal saline (1.0 mL/animal)..

G2: SU-Eohyeol pharmacopuncture administration group (1.0 mL/animal)..

&md=tbl&idx=2' data-target="#file-modal"">Table 2

SU-Eohyeol pharmacopuncture-related effects on clinical sign changes in a single-dose intramuscular toxicity study in Sprague-Dawley (SD) rats.

SexGroupDay 1 (hour)Day
Dose
(mL/animal)		Animal ID0.5124623456789101112131415
MaleG1 (n = 5)
0		1101-------------------
1102-------------------
1103-------------------
1104-------------------
1105-------------------
G2 (n = 5)
1.0		1201-------------------
1202-------------------
1203-------------------
1204-------------------
1205-------------------
FemaleG1 (n = 5)
0		2101-------------------
2102-------------------
2103-------------------
2104-------------------
2105-------------------
G2 (n = 5)
1.0		2201-------------------
2202-------------------
2203-------------------
2204-------------------
2205-------------------

-: No observable abnormalities in clinical signs..

G1: Control group administered with normal saline (1.0 mL/animal)..

G2: SU-Eohyeol pharmacopuncture administration group (1.0 mL/animal)..

For the change in body weight during our experiment, the body weight of male rats in the SUEP-treated and control groups changed from 200.3 ± 3.7 g (G1) and 196.9 ± 6.9 g (G2), respectively, to 330.4 ± 17.5 g (G1) and 327.1 ± 16.1 g (G2), respectively, whereas that of female rats changed from 161.5 ± 4.6 g (G1) and 156.3 ± 4.2 g (G2), respectively, to 220.9 ± 7.7 g (G1) and 212.8 ± 15.1 g (G2), respectively. No significant difference was observed in the body weight changes in male and female rats treated with SUEP 1.0 mL/animal compared with those in the control group (Fig. 1, Table 3).

Significantly different from control by Student’s test..

S.D., standard deviation; N, number of animals..

&md=tbl&idx=3' data-target="#file-modal"">Table 3

Effects of SU-Eohyeol pharmacopuncture on changes in mean body weights in a single-dose intramuscular toxicity test in Sprague-Dawley (SD) rats.

SexGroup/Dose
(mL/animal)		DayDay 1-15
Gain (g)
14815
MaleG1
0		Mean200.3226.7264.1330.4130.1
S.D.3.75.39.717.515.9
N (g)55555
G2
1.0		Mean196.9226.2261.9327.1130.2
S.D.6.98.510.116.110.8
N (g)55555
FemaleG1
0		Mean161.5174.2190.2220.959.4
S.D.4.63.35.37.77.6
N (g)55555
G2
0		Mean156.3173.0189.9212.856.6
S.D.4.27.18.815.111.8
N (g)55555

Significantly different from control by Student’s test..

S.D., standard deviation; N, number of animals..
Figure 1. Effects of SU-Eohyeol Pharmacopuncture on the body weight of male (A) and female (B) Sprague-Dawley (SD) rats.

2. Macroscopic examination following necropsy

There were no abnormalities in the male and female SD rats in the control group or SUEP-treated group on macroscopic examination following necropsy.

3. Histopathological examination

The results of the local tolerance test of SUEP were observed by microscopic examination of the tissues obtained from the administration sites of all male and female SD rats. As a result of microscopic histopathological examination, no SUEP-related changes were observed at the injection site. Treatment with SUEP was considered to be tolerated by all animal groups (Fig. 2).

Figure 2. Histopathological results of a single-dose intramuscular toxicity test for SU-Eohyeol Pharmacopuncture (SUEP) in male and female Sprague-Dawley (SD) rats Histopathological examination images of injection sites in Sprague-Dawley (SD) rats were captured at ×200 (scale bar = 200 μM).

DISCUSSION

After Nam Sang-cheon officially disclosed pharmacopuncture to the TKM community in 1967, pharmacopuncture has continued to develop and has subsequently become one of the most used TKM treatment methods in Korea [1-5]. In a survey study on the usage status and satisfaction of pharmacopuncture conducted by TKM doctors in Korea, 88% of respondents had experience using pharmacopuncture in the previous year, pharmacopuncture was mainly used for the treatment of musculoskeletal, nervous system, and gastrointestinal diseases, and the two main types used in TKM clinical practice were bee venom and ouhyul pharmacopuncture [4].

While medicinal products are usually administered by the MFDS, pharmacopuncture is implemented in Korea according to prescriptions issued to EHDs by TKM and TCM institutions [15, 16]. An EHD is a type of pharmacy that offers various types and formulations of herbal medicines to TKM institutions according to TKM doctors’ prescriptions based on each patient’s condition and was legalized by the Ministry of Health and Welfare (MoHW) in Korea in 2008. In September 2018, the MoHW announced that the Accreditation System of EHDs would be applied in TKM to evaluate and certify herbal and pharmacopuncture preparations [15-17].

With the MoHW certification mark, EHDs are said to provide safely formulated pharmacopuncture preparations; however, in order for these preparations to be scientifically recognized, the efficacy and safety of newly developed pharmacopuncture drugs needs to be evaluated both non-clinically and clinically.

Conversely, the MFDS requires non-clinical data on safety, such as genotoxicity, single-dose toxicity, repeated dose toxicity, and efficacy, to approve herbal medicines for clinical trials [18-22]. JOP, one of the most widely used pharmacopuncture techniques in TKM, has many reports of its clinical efficacy [4, 10], but no toxicity evaluation reports have been conducted to date. Therefore, SUEP, which was developed by adding CPC to JOP, should be scientifically verified for safety through toxicity evaluation. It is expected that public trust will be gained through these efforts.

Single-dose toxicity studies are conducted to describe the qualitative and quantitative data of time-related toxic phenomena and occurrences after administration of a single dose of a substance or a combination of substances [23]. There are species and phylogenetic differences in biological responses to test substances. In the selection of an animal species, an animal species with a similar metabolic profile to that of a human is preferred. However, during the stage of single-dose toxicity testing, it may be difficult to select an animal species or lineage that satisfies the above criterion; therefore, an animal that is easy to handle, is qualitatively uniform, and has abundant background data is usually selected.

The most commonly preferred rodent is the rat, and the most preferred animal for non-rodent experiments is the dog. In addition, depending on the drug, there may be sex-specific differences in the appearance of toxicity. Thus, toxicity should be evaluated in at least one male and female animal [21, 24].

We performed a SUEP single-dose toxicity study in female and male SD rats. Our toxicological study was performed to assess for potential toxicity and to determine the approximate lethal dose of SUEP after a single intramuscular injection of SUEP into the thigh of 6-week-old male and female SD rats. Our findings showed that there were no deaths in either sex in the SUEP-treated group. There was no significant difference between the SUEP-treated group and the control group in the general condition, weight change, or clinical signs of male or female SD rats. In addition, no significant SUEP-related changes were observed in autopsy findings or local tolerance examinations at the injection site by histopathological examination. According to our toxicity study results, the lethal dose of SUEP is approximately greater than 1.0 mL/animal (approximately 1,525 mg/kg) in both male and female SD rats following a singular intramuscular injection of SUEP.

The expected clinical application dose of SUEP is 1.0 mL at a time based on an adult weight (60 kg), and the dose of 1.0 mL/animal used in this experiment is approximately 500 times higher than the actual clinical dose of SUEP used (1.0 mg/kg). The appropriateness and safety of the SUEP dose used in clinical practice has thus been determined. Therefore, the findings of this study allow us to infer that acupuncture using SUEP agents can be safely administered to humans at a dose of 1.0 mL or less.

However, since our study only investigated the toxic effects of a single dose of SUEP only for an observation period of 14 days, in the future, additional intramuscular toxicity tests, such as multiple-dose and long-term toxicity observations, and various toxicological studies will be needed to establish the effectiveness and safety of SUEP.

Fortunately, pharmacopuncture-related adverse reactions have not been described in clinical reports using a combination of JOP and CPC pharmacopuncture [11]; however, no official clinical study has been conducted on the side effects of SUEP to date. Therefore, many experimental and clinical studies will need to provide scientific evidence regarding the efficacy and safety of SUEP.

CONCLUSION

According to the results of our toxicity study, the approximate lethal dose of SUEP was greater than 1.0 mL/animal (approximately 1,525 mg/kg) in both male and female SD rats following a single intramuscular injection under the conditions of this study. This finding suggests that, in TKM clinical trials, SUEP can be considered safe at doses below 1.0 mL. To determine the safety of the use of SUEP in TKM clinical practice, additional toxicity studies will be needed.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

FUNDING

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2022R1A2C1013518).

Fig 1.

Download
Figure 1.Effects of SU-Eohyeol Pharmacopuncture on the body weight of male (A) and female (B) Sprague-Dawley (SD) rats.
Journal of Pharmacopuncture 2022; 25: 268-275https://doi.org/10.3831/KPI.2022.25.3.268

Fig 2.

Download
Figure 2.Histopathological results of a single-dose intramuscular toxicity test for SU-Eohyeol Pharmacopuncture (SUEP) in male and female Sprague-Dawley (SD) rats Histopathological examination images of injection sites in Sprague-Dawley (SD) rats were captured at ×200 (scale bar = 200 μM).
Journal of Pharmacopuncture 2022; 25: 268-275https://doi.org/10.3831/KPI.2022.25.3.268

Table 1 . Group designation of a single-dose intramuscular toxicity test for SU-Eohyeol pharmacopuncture in Sprague-Dawley (SD) rats.

GroupDose of SUEP
(mL/animal)		Injection dose amount (mL/animal)Number of animals (object number)
MaleFemale
G1Control (normal saline)01.05 (1101-1105)5 (2101-2105)
G2Test substance (SUOP)1.01.05 (1201-1205)5 (2201-2205)

Table 2 . SU-Eohyeol pharmacopuncture-related effects on clinical sign changes in a single-dose intramuscular toxicity study in Sprague-Dawley (SD) rats.

SexGroupDay 1 (hour)Day
Dose
(mL/animal)		Animal ID0.5124623456789101112131415
MaleG1 (n = 5)
0		1101-------------------
1102-------------------
1103-------------------
1104-------------------
1105-------------------
G2 (n = 5)
1.0		1201-------------------
1202-------------------
1203-------------------
1204-------------------
1205-------------------
FemaleG1 (n = 5)
0		2101-------------------
2102-------------------
2103-------------------
2104-------------------
2105-------------------
G2 (n = 5)
1.0		2201-------------------
2202-------------------
2203-------------------
2204-------------------
2205-------------------

-: No observable abnormalities in clinical signs..

G1: Control group administered with normal saline (1.0 mL/animal)..

G2: SU-Eohyeol pharmacopuncture administration group (1.0 mL/animal)..

Table 3 . Effects of SU-Eohyeol pharmacopuncture on changes in mean body weights in a single-dose intramuscular toxicity test in Sprague-Dawley (SD) rats.

SexGroup/Dose
(mL/animal)		DayDay 1-15
Gain (g)
14815
MaleG1
0		Mean200.3226.7264.1330.4130.1
S.D.3.75.39.717.515.9
N (g)55555
G2
1.0		Mean196.9226.2261.9327.1130.2
S.D.6.98.510.116.110.8
N (g)55555
FemaleG1
0		Mean161.5174.2190.2220.959.4
S.D.4.63.35.37.77.6
N (g)55555
G2
0		Mean156.3173.0189.9212.856.6
S.D.4.27.18.815.111.8
N (g)55555

Significantly different from control by Student’s test..

S.D., standard deviation; N, number of animals..

References

  1. Jung C, Jung JH, Lee MS. A clinical study of immune pharmacopuncturology. Yeongi: Kyungrak Medical Publishing Co.; 2011. p. 127-33.
  2. Korean Acupuncture and Moxibustion Society. Acupuncture medicine. Seoul: Hanmi Medical Publishing Co.; 2020. p. 204-28.
  3. Park J, Lee H, Shin BC, Lee MS, Kim B, Kim JI. Pharmacopuncture in Korea: a systematic review and meta-analysis of randomized controlled trials. Evid Based Complement Alternat Med. 2016;2016:4683121.
    Pubmed KoreaMed CrossRef
  4. Park JE, Kim KH, Kang S, Lee EK, Kim JC, Jang BH, et al. Usage status and satisfaction with pharmacopuncture in Korea: a survey among Korean medicine doctors. Eur J Integr Med. 2019;27:121-30.
    CrossRef
  5. Korean Pharmacopuncture Institute Science Committee, Pharmacopuncturology Institute. Pharmacopuncturology. 2nd ed. Seoul: Elsevier Korea; 2008. p. 3-8, 163-80, 259-420.
    CrossRef
  6. Yun JM, Hur NY, Kim KH. The analysis of Korean clinical studies on the effect of pharmacopuncture for whiplash injury after traffic accidents. J Pharmacopuncture. 2020;23(4):262-72.
    Pubmed KoreaMed CrossRef
  7. Lee DJ, Hwangbo M, Kwon K, Seo HS. Review of cervi cornu parvum pharmacopuncture in korean medicine. J Pharmacopuncture. 2013;16(2):7-14.
    Pubmed KoreaMed CrossRef
  8. Bensky D, Gamble A. Chinese herbal medicine: materia medica. Revised ed. Seattle: Eastland Press; 1993. p. 32-44.
    CrossRef
  9. Wu F, Li H, Jin L, Li X, Ma Y, You J, et al. Deer antler base as a traditional Chinese medicine: a review of its traditional uses, chemistry and pharmacology. J Ethnopharmacol. 2013;145(2):403-15.
    Pubmed CrossRef
  10. Yoon JW, Kim SJ. A review of clinical study on Jungsongouhyul pharmacopuncture treatment published in Korea. J Korean Med Rehabil. 2017;27(4):75-84.
    CrossRef
  11. Ryu HS, Kim SP, Chun HS, Ryu MS, Shin JC, Wei TS. The comparative study of effects between acupuncture treatment and cotreatment with mixed pharmacopuncture of Jungsongouhyul and Cervi Pantotrichum Cornu on low back pain patients. J Korean Acupunct Moxib Soc. 2011;28(6):149-57.
  12. Xu Z, Kang Q, Yu Z, Tian L, Zhang J, Wang T. Research on the species difference of the hepatotoxicity of medicine based on transcriptome. Front Pharmacol. 2021;12:647084.
    Pubmed KoreaMed CrossRef
  13. Ministry of Food and Drug Safety. Good Laboratory Practice Regulation for Nonclinical Laboratory Studies (Notification No. 2018-93) [Internet]. Cheongju: Ministry of Food and Drug Safety; 2018 [cited 2018 Nov 21]. Available from: https://www.law.go.kr/LSW/admRulLsInfoP.do?admRulSeq=2100000170349.
  14. Organisation for Economic Co-operation and Development (OECD). ENV/MC/CHEM(98)17: OECD series on principles of good laboratory practice and compliance monitoring number 1. Paris: OECD; 1998. p. 41.
  15. Sung SH, Shin BC, Park MJ, Kim KH, Kim JW, Ryu JY, et al. Current status of management on pharmacopuncture in Korea through introduction of an accreditation system. J Pharmacopuncture. 2019;22(2):75-82.
    Pubmed KoreaMed CrossRef
  16. Ministry of Health and Welfare. Guideline on Installation and Utilization of External Herbal Dispensaries of Traditional Korean Medicine Clinics and Share-use of Herbal Dispensaries [Internet]. Sejong: Ministry of Health and Welfare; 2017 [cited 2018 Nov 21]. Available from: http://www.mohw.go.kr/react/jb/sjb0406vw.jsp?PAR_MENU_ID=03&MENU_ID=030406&CONT_SEQ=342727&page=1.
  17. Ministry of Health and Welfare. Introduction of External Herbal Dispensaries of Traditional Korean Medicine Clinics [Internet]. Sejong: Ministry of Health and Welfare; 2018 Available from: http://www.mohw.go.kr/react/al/sal0301vw.jsp?PAR_MENU_ID=04&MENU_ID=0403&page=1&CONT_SEQ=344877&SEARCHKEY=TITLE&SEARCHVALUE=%ED%95%9C%EC%95%BD.
  18. Ministry of Food and Drug Safety. Good Laboratory Practice (GLP) [Internet]. Cheongju: Ministry of Food and Drug Safety; 2017 [cited 2022 Apr 1]. Available from: https://www.law.go.kr/LSW/admRulInfoP.do?admRulSeq=2100000086689.
  19. Ministry of Food and Drug Safety. Partial amendment official announcement about toxicity test for drug. Cheongju: Ministry of Food and Drug Safety; 2015.
  20. Lee MY, Seo CS, Kim JY, Shin HK. Genotoxicity evaluation of Guibi-Tang extract using an in vitro bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus test. BMC Complement Altern Med. 2014;14:215.
    Pubmed KoreaMed CrossRef
  21. Sung H, Lee E. Single-dose intramuscular toxicity of Mahwangcheonoh pharmacopuncture in a rat model: toxicity of Mahwangcheonoh Pharmacopuncture in SD rats. J Pharmacopuncture. 2016;19(4):336-43.
    Pubmed KoreaMed CrossRef
  22. Hwang JH, Ku J, Jung C. Evaluation of the single-dose toxicity of capsaicin pharmacopuncture in rats. J Acupunct Res. 2020;37(3):167-72.
    CrossRef
  23. Rousseaux CG, Bracken WM. Overview of Drug Development. In: Haschek WM, Rousseaux CG, Wallig MA, editors. Haschek and Rousseaux's handbook of toxicologic pathology. 3rd ed. London: Academic Press; 2013. p. 647-685.
    CrossRef
  24. Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, et al. Report from the in vitro micronucleus assay working group. Mutat Res. 2003;540(2):153-63.
    Pubmed CrossRef