Safety assessment of Gami-daebo-tang gagambang used for treating stroke sequelae
Article information
Abstract
Objectives
This study aimed to investigate wheter Gami-daebo-tang gagambang, commonly used for treating stroke sequelae, induces acute toxicity or genetoxicity.
Methods
Gami-daebo-tang gagambang contains that the root of Angelica gigas Nakai inducing acute headache and weakness, and the rhizome of Cnidium officinale MAKINO, an herb with potential teratogenicity. Teratogenicity is closely associated with genotoxicity. We analyzed whether Gami-daebo-tang gagambang induces acute toxicity and genotoxicity in accordance with Korean non-clinical test standards and OECD test guidelines TG471, TG473, TG474.
Results
When male and female rats were given a single dose of Gami-daebo-tang gagambang, no toxic effects or organ damage were observed at dosages reaching 2,500 mg/kg. The bacterial reverse mutation assay showed no evidence of DNA mutations at concentrations up to 5,000 μg/plate. In vitro studies indicated that Gami-daebo-tang gagambang did not cause structural or numerical chromosomal aberrations at concentrations as high as 2,000 μg/mL. Moreover, animal studies demonstrated that the substance did not induce bone marrow toxicity or the formation of micronuclei in erythrocytes at doses up to 2,000 mg/kg.
Conclusions
The studies conducted with different models showed that Gami-daebo-tang gagambang did not cause acute toxicity or genotoxicity. This study provides evidence for the safety of Gami-daebo-tang gagambang in terms of acute toxicity and genotoxicity, supporting its potential safe application in treating stroke sequelae.
Introduction
The prescription Gami-daebo-tang (加味大補湯, GDT), composed of herbs such as Notopterygii Rhizoma (羌活), Angelicae Sinensis Radix (當歸), Araliae Continentalis Radix (獨活), and Rehmanniae Radix Preparata (熟地黃), is recorded in several medical texts including the Bangyak Hapbyeon (方藥合編). In Korean medicine, GDT has been passed down as being effective for tonifying energy (Qi, 氣) and blood (大補氣血), calming the mind and soothing the spirit (寧心安神), strengthening the weakened function of the stomach and generating fluids (益胃生津), as well as tonifying the liver and kidneys (補肝腎)1).
Stroke is defined as a condition in which brain dysfunction persists for more than a day or results in death, and may be accompanied by muscle stiffness, sensory impairment, pain, and visual or speech impairment after treatment4,5). Over time, the severity of the aftereffects intensifies and physical activity declines, adversely affecting the patient. Therefore, appropriate treatment for the aftereffects is required based on the patient's condition6,7). In clinical settings, GDT is known to have therapeutic effects in patients with acute ischemic stroke accompanied by bleeding as well as in patients with Wallenberg syndrome due to lateral medullary infarction and cerebellar infarction2,3). Moreover, the co-administration of GDT, aspirin, clopidogrel, and atrovastatin reportedly reduced the intima-media thickness (IMT) of the carotid artery in patients with carotid artery atherosclerosis8). In the preclinical field, studies have been conducted on the effects of orally administered GDT in mice, which showed an increase in phagocytic activity by promoting the production of tumor necrosis factor-alpha and nitric oxide in macrophages, thereby influencing non-specific immune responses
Korean medicine prescriptions (decoctions) in clinical settings are based on compositions derived from various Korean medicine books; however, most are used with changes in composition based on the patient’s condition, preferences, and Korean medicine principles4,9,10). Generally, Western medicine offers personalized treatment based on genomic technology, whereas Korean medicine provides patient-centered customized treatment based on pattern identification11). GDT is often used as Gagambang that adds or excludes various hurbs including the scion root of Aconitum carmichaeli Debeaux (附子), the seed of Coix lacryma-jobi Linne var. ma-yuen Stapf (薏苡仁) and the resin of Aquilaria agallocha Roxburgh (沉香)1–4).
Korean medicine minimizes side effects by using other herbs together or by reducing toxicity through preparations when the toxicity of a medicinal herb is observed based on clinical symptoms12–13). As medicines have been developed and their safety has come to the fore, various safety studies have been conducted on medicinal herbs. For example, although Buja (附子) is controversial because of its analgesic effects and toxicity when taken in excess, many studies have accumulated evidence for its safe use14–16). The rhizome of Cnidium officinale MAKINO (川芎), one of the components of Gami-daebo-tang gagambang, is known to delay the development and growth of rodent embryos and cause mild limb deformities in non-clinical studies, but the mechanism by which this is induced is unclear17–18), and the root of Glycyrrhiza uralensis Fisch. ex Dc. (甘草) has been reported to cause acute headaches or weakness in clinical settings19).
Among the various side effects of medicinal herbs, malformations can occur when genotoxicity affects the reproductive cells of parents, affecting genes in their offspring. This is because malformations are closely related to genetic mutations and chromosomal abnormalities within cells. Genotoxicity is not only associated with malformations, but can also damage cellular functions, potentially leading to cancer development or accelerated aging20–24).
As science advances, there is a growing demand for evidence-based medicine, rather than relying solely on experience and tradition. While Korean medicine prescriptions handed down through traditional Korean medicine texts have long been regarded as safe owing to their historical basis in clinical experience, Korean medicine-related researchers are actively conducting research on the effectiveness and safety of Korean medicine in various fields to accumulate scientific evidence25–26). Safety evaluations are being conducted not only by extracting and isolating individual components of herbal medicines, but also to assess the potential side effects and risks arising from interactions between the components of compound herbal medicines27–29).
In this study, we aimed to evaluate the acute toxicity and genotoxicity of Gami-daebo-tang gagambang (GDTG), including herbs with potential side effects, to provide evidence of GDTG.
Materials and Method
1. Gami-daebo-tang gagambang(GDTG)
The GDTG was prepared using herbal materials sourced from a GMP (Good Manufacturing Practice) certified manufacturer and purchased from the Hocburi (The facility standard of herbal dispensaries, Hwaseong, gyeonggi-do, Korea). GDTG was prepared using medicinal herbs excluding the root of Aconitum carmichaeli Debx and resin of Aquilaria agallochum (Lour.) Roxb. ex Finl. In the GDT composition.
The results of the analysis for the formula’s composition, residual pesticides, and heavy metals are presented in Table 1, confirming that the herbal materials used met the standards of the Korean Pharmacopoeia and the Korean Pharmacopoeia: Herbal Medicine (Crude Drugs) Standards.
Composition of Gami-daebo-tang Gagambang
For extraction, 10 times the total weight of the herbal materials was used in distilled water, and the mixture was extracted at 115°C for 4 hours. The extract, filtered through a non-woven fabric filter, was then concentrated to 10 brix at 120°C and sterilized at 100°C for 30 minutes. The concentrated extract was converted into a powder using a spray dryer and reconstituted with distilled water for use in the experiments. (Table 1)
2. Animal facility environment
The experimental animals were housed in a facility with a 12-hour light/dark cycle, maintaining an indoor temperature of 19–25°C and a relative humidity of 30–70%. Noise levels were kept below 60 dB, ammonia levels were maintained at 20 ppm, and illumination was limited to 150–300 lux. HEPA-filtered air was supplied to the animal housing facility at a rate of 10–20 exchanges per hour. Sterilized cages and water bottles, sterilized by high-pressure steam, were replaced at least twice a week, and the animals were provided with ad libitum access to food and water.
3. In vivo Acute toxicity
The acute toxicity evaluation was approved by the Good Laboratory Practice (GLP) Steering Committee of the National Institute of Korean Medicine (Study Number: N22005) and the Animal Ethics Committee (Ethics Approval Number: NIKOM-2022-11), and was conducted according to the toxicity testing standards set by the Ministry of Food and Drug Safety.
Six-week-old Sprague-Dawley rats, both males and females, were purchased from Orient Bio (Seongnam, KOREA) and acclimated for 5 days. The rats were then divided into groups of five. The control group received distilled water, while the GDTG was administered at doses of 625, 1,250, or 2,500 mg/kg. After a 12-hour fast, each group received a single oral dose of either distilled water or the GDTG, and food was provided 4 hours later.
Body weight measurements were taken before administration and on days 1, 3, 7, and 14 post-administration. To assess toxic reactions from the GDTG, general observations such as appearance, pain, and respiration of the rats were recorded 4 times on the day of administration and daily for the following 14 days. On the 14th day post-administration, the rats were euthanized using carbon dioxide overdose, and a visual inspection of systemic organs, including the liver, kidneys, and heart, was performed.
4. Bacterial reverse mutation assay
The bacterial reverse mutation assay was approved by the Biosafety Committee and the GLP Steering Committee (Study Number: T22053), and was conducted according to the OECD Test Guideline (TG 471). The experiment utilized histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and tryptophan-requiring strains of Escherichia coli(WP2 uvrA), with the strains' amino acid requirements, rfa mutations, and other genetic characteristics confirmed before use.
The experimental groups consisted of a negative control group, a positive control group, and a GDTG treatment group. The negative control group was treated with distilled water, while the positive control group was treated with substances known to induce genetic mutations depending on the metabolic activation of the strains. The GDT modified formula treatment group was initially tested at concentrations of 6.86, 20.6, 61.7, 185.2, 555.6, 1,666.7, and 5,000 μg/plate. Since no precipitation or toxicity was observed, the secondary experiment was conducted with concentrations of 61.7, 185.2, 555.6, 1,666.7, and 5,000 μg/plate.
Following treatment, the samples were pre-incubated for 20 minutes in a 37°C shaking incubator, then overlaid with top agar containing 10% amino acids and incubated at 37°C for 48 hours. After incubation, the basic growth layer was examined for any abnormalities using a microscope, and the number of revertant colonies was observed visually.
5. In vitro Mammalian chromosome aberration assay
The in vitro mammalian chromosome aberration test was approved by the Biosafety Committee and the GLP Steering Committee (Study Number: T22054) and was conducted in accordance with the OECD Test Guideline (TG 473). The experimental conditions were divided into short-term treatment with metabolic activation, short-term treatment without metabolic activation, and long-term treatment with metabolic activation. The short-term treatment involved exposure for 6 hours, while the long-term treatment involved exposure for 24 hours. Each treatment was further categorized into negative control, positive control, and GDTG treatment groups.
The negative control group received distilled water, while the positive control group was treated with substances known to induce chromosomal aberrations depending on the series. The GDTG treatment groups were exposed to concentrations of 500, 1,000, and 2,000 μg/mL.
Cells were treated with colcemid for 2 hours to induce metaphase, and then specimens were prepared using KCl and acetic acid/methanol solution. The specimens were stained with 5% Giemsa solution, and 300 cells were analyzed for chromosomal aberrations.
6. In vivo Micronucleus assay using rodent hematopoietic cells
The in vivo micronucleus assay using rodent hematopoietic cells was approved by the GLP Steering the National Institute of Korean Medicine (Study Number: T22055) and the Animal Experimentation Ethics Committee (Ethics Approval Number: NIKOM-2022-34), and was conducted in accordance with the OECD Test Guideline (TG 474).
Six-week-old male ICR mice were purchased from Orient Bio (Seongnam, Korea) and acclimated for 5 days. The experimental groups were divided into a negative control group, a positive control group, and a GDTG treatment group. The negative control group received distilled water orally once daily for 2 days, while the positive control group was administered mitomycin C at a dose of 2 mg/kg intraperitoneally 24 hours before sample collection, as it is known to induce micronuclei in hematopoietic cells. The GDTG treatment group was given the formula orally once daily for 2 days at doses of 500, 1,000, and 2,000 mg/kg.
To assess the toxicity of the GDTG treatment, general symptoms such as appearance, pain response, and respiration were observed immediately after dosing, 2 hours after dosing, and on the day of sample collection. The animals were euthanized using carbon dioxide, and bone marrow was collected from the femurs after injection with fetal bovine serum. The bone marrow was smeared on slides, fixed with methanol, and stained with 5% Giemsa solution. Stained slides were analyzed for the number of polychromatic erythrocytes with micronuclei by counting 4,000 polychromatic erythrocytes per animal, and the ratio of polychromatic to normochromatic erythrocytes was calculated to evaluate any bone marrow toxicity induced by GDTG.
7. Statistical analysis
Statistical analysis was performed using SPSS Statistics 25. Statistical significance for body weight and micronucleus frequency was tested using one-way analysis of variance (ANOVA) followed by Dunnett’s test, while the incidence of chromosomal abnormalities was analyzed using Fisher’s exact test. When significant results were observed compared to the negative control group, regression analysis was conducted.
Results
1. In vivo Acute toxicity
To determine whether a single oral administration of the GDTG induces acute toxicity in female and male rats, changes in body weight, clinical signs, and necropsy findings were observed. The changes in body weight before and after administration of the GDTG are presented in Table 2, and no statistically significant differences in weight changes between groups were observed. No abnormal reactions were noted during the 14-day observation period for general symptoms such as appearance, posture, and consciousness. Details of general symptoms are presented in Table 7. Necropsy revealed no differences in the condition of major organs, including the liver and heart, compared to the negative control group. (Table 2)
The Body Weight Changes in Acute Toxicity Assessment
General symptoms
2. Bacterial reverse mutation assay
The results of whether the GDTG induces genetic mutations in microorganisms are recorded in Table 3 and Table 4. When substances known to induce genetic mutations were applied to each strain depending on their metabolic activation status, an increase in the number of revertant colonies confirmed the appropriateness of the experiment. When the GDTG was applied to each strain with or without metabolic activation, no precipitation or toxicity to microorganisms was observed up to 5,000 μg/plate. Additionally, the number of revertant colonies did not show a significant increase compared to the negative control group. (Table 3, 4)
The Revertant Colony in Bacterial Reverse Mutation Test without Metabolic Activation
The Revertant Colony in Bacterial Reverse Mutation Test with Metabolic Activation
3. In vitro Mammalian chromosome aberration assay
In order to determine whether the GDTG induces structural or numerical chromosomal aberrations in cells, an in vitro chromosomal aberration test was conducted, and the results are summarized in Table 5. Treatment with the GDTG did not affect the pH or osmolality of the cell culture medium. The positive control group showed a statistically significant increase in the proportion of cells with structural abnormalities compared to the negative control group, confirming the validity of the experiment. The treatment with the GDTG did not result in a statistically significant difference in the proportion of cells with structural or numerical chromosomal aberrations compared to the negative control group. Therefore, the GDTG did not induce structural or numerical chromosomal abnormalities in cells. (Table 5)
The Results of In vitro Mammalian Chromosomal Aberration Test
4. In vivo Micronucleus assay using rodent hematopoietic cells
An experiment was conducted to determine whether the GDTG induces micronucleus formation in mouse bone marrow hematopoietic cells, and the results are summarized in Table 6. Treatment with the GDTG not only had no effect on the mouse body weight but also did not result in any toxicologically significant clinical signs. The investigation of the ratio of normocytes to polychromatic erythrocytes revealed no evidence of marrow toxicity due to the GDTG. The micronucleus induction rate in the positive control group was statistically significant compared to the negative control group, confirming the validity of the experiment. However, the proportion of polychromatic erythrocytes with micronuclei in the GDTG treatment group was not statistically different from the negative control group. Therefore, under the test conditions, the GDTG did not induce micronuclei in hematopoietic cells. (Table 6, 7)
The Results of Mammalian Erythrocyte Micronucleus Test
Discussion
A review of the research status of 56 types of Korean medicine formulations covered by health insurance showed that among 813 papers, 7.5% focused on biochemical analysis, 9.8% on toxicity testing, and 82.7% on studies involving cells, animals, and humans. This indicates that the proportion of toxicity testing research is relatively low compared with other fields30). GDTG also lacks scientific evidence on safety.
In non-clinical studies, acute toxicity assessment is conducted to identify toxic reactions and target organs resulting from overdosing during the drug development process and to observe the reversibility of toxicity and approximate lethal dose31). In this study, the highest concentration expected to induce clear toxic responses according to the Korean non-clinical test standards.
As no animals died following GDTG administration, the lethal dose and LD50 (50% lethal dose) of GDTG could not be determined. Moreover, the no-observed-adverse-effect level (NOAEL, a safe exposure level) of the acute toxicity test was judged to be 2,500 mg/kg, as no general symptoms or organ damage were observed following GDTG administration.
Various genotoxicity tests are used to determine whether the components of pharmaceuticals, chemicals, or medical devices can cause genotoxicity using commonly used methods, including the bacterial reverse mutation test, in vitro chromosome aberration test, and in vivo micronucleus test32).
In the bacterial reverse mutation test, a substance is judged to have mutagenic potential if it causes a statistically significant increase in the number of revertant colonies compared to the negative control, or typically shows a 2- to 3-fold increase over the historical negative control data and demonstrates a dose-dependent effect33).
The highest concentration is determined based on the results of the preliminary test, which includes evaluating cytotoxicity, the presence of precipitates, and the number of reverse mutations. If no significant issues are found in the preliminary test, the concentration is set according to the maximum concentration limit specified in the OECD guidelines. Based on the results of the preliminary experiment, no significant issues were observed up to the maximum dose of 5,000 μg/plate, as specified in the OECD guidelines 471. Therefore, the highest dose was set at 5000.
In the case of GDTG, the number of revertant colonies did not increase 2- to 3- fold up to 5,000 μg/plate, regardless of metabolic activation, compared to the negative control. Benzo[a]pyrene is known to cause genotoxicity only when metabolized by hepatic enzymes34). Since GDTG did not significantly increase the number of revertant colonies, even with metabolic activation by the rat liver fraction, it was considered that there was no mutagenic effect caused by rat hepatic enzymes. Further studies using liver fractions from other species such as humans and hamsters may clarify the mutagenic activity of GDTG following its metabolic activation.
Chromosomal aberrations that occur in cells can cause cell death by causing loss of cell function, or can be involved in the development of cancer cells, and are known to be the cause of genetic diseases such as Down syndrome or intellectual disability35–36). The highest concentration is determined based on the maximum dose specified in the OECD guidelines 473, and cytotoxicity is assessed by evaluating the cell proliferation rate.
In the in vitro chromosome aberration test, GDTG did not induce structural or numerical chromosome aberration up to 2,000 μg/mL, regardless of metabolic activation. Furthermore, no effects of metabolic activation were observed, indicating that GDTG did not cause chromosomal abnormalities in the cells.
Micronuclei are induced by cell division via chromosome aberrations and damaged spindle fiber37). Clinically, micronuclei are associated with smoking, alcohol consumption, and exposure to genotoxic substances, and are found in several diseases such as cancer, rheumatoid arthritis, and chronic kidney disease. This study was conducted based on the maximum dose specified in the OECD guidelines 474. Following a preliminary experiment to assess the ratio of polychromatic erythrocytes to normochromatic erythrocytes, which are indicators of cytotoxicity, the experiment was carried out.
Since the administration of GDTG to mice did not result in micronucleus formation or myelotoxicity compared to the negative control, it was concluded that GDTG is not a micronucleus inducing substance.
GDTG contains the rhizome of Cnidium officinale MAKINO (川芎), which is known to pose potential risks such as teratogenicity at the preclinical level. However, various experimental models used to assess the different endpoints of genotoxicity have shown that GDTG does not induce gene mutations, chromosomal abnormalities, or micronuclei. Therefore, it can be concluded that GDTG does not exhibit genotoxicity.
In addition, the root of Glycyrrhiza uralensis Fisch. ex Dc. (甘草) included in GDTG has been reported to cause acute headaches or weakness. It is known that the administration of nitroglycerin, a well-known headache trigger in humans, induces hindpaw allodynia, light aversion and conditioned place aversion in mice. Multiple spontaneous behaviors including head-directed grooming, lateralized eye blink and whole-body shuddering are considered suggestive of spontaneous head pain in mice38). However no general symptoms related to headaches were observed, nor were any other general symptoms indicating toxic reactions observed in acute toxicity tests using rodents. Therefore, it can be concluded that under the conditions of this study, acute toxicity is not induced.
This study is meaningful in that it evaluates the safety of a compound herbal medicine rather than individual herbal ingredients, and provides safety data for subsequent research.
In addition to the methods used in this study, various other testing methods are available for non-clinical safety evaluations. To improve the perception of safety and promote the use of Korean medicines, it is necessary to provide safety information through a range of testing methods.
Conclusion
GDTG contains the root of Glycyrrhiza uralensis Fisch. ex Dc. (甘草), which can cause acute headaches in clinical settings, and the rhizome of Cnidium officinale MAKINO (川芎), which is known to cause malformations in rodents at a non-clinical level. The acute toxicity and genotoxicity of GDTG were evaluated using pharmaceutical toxicity testing standards and OECD test guidelines.
GDTG did not show any toxicity up to 2,500 mg/kg in rats, regardless of sex, and did not induce genotoxicity, including gene mutations, in various experimental models. Although some traditional medicinal herbs included in the formula are known to have side effects, GDTG did not cause acute toxicity or genotoxicity under the test conditions. However, to provide comprehensive safety information on GDTG, additional non-clinical safety evaluations are necessary.
Acknowledgment
This research was supported by the Ministry of Health and Welfare, the Korea Health Industry Development Institute [RS-2023-KH139577], Korea Biotechnology research and innovation association.