Medicinal plants

Korean medicine prescriptions eligible for medical insurance benefits must be prepared using medicinal plants for pharmaceutical use at a facility certified under the Korean standard for herbal dispensaries. The medicinal plants were purchased from a GMP-certified pharmaceutical company, meeting the essential criteria required for pharmaceutical use according to the Korean Pharmacopoeia of the Ministry of Food and Drug Safety, and the analytical results are presented in Table 1.

Table 1 A summary of the analytical certificate for GMP-certified herbal plants used for medicinal purposesAsh analysis

The ash content of 14 medicinal plants was analyzed by a GMP-certified pharmaceutical company according to the general test methods of the Korean Pharmacopoeia. To evaluate whether the 14 medicinal plants constituting Mangeum-tang comply with the standards of the Korean Pharmacopoeia, the homogenized and pulverized dried samples were placed in a crucible and incinerated at 500–550 °C for over 4 h until no carbonized residue remained. The samples were then allowed to cool naturally, after which their masses were measured. The ash content (%) was subsequently calculated and is presented in Table 1.

Sulfur dioxide analysis

The analysis of sulfur dioxide in medicinal plants was conducted by a GMP-certified pharmaceutical company in accordance with the MFDS notification. Following the Monier-Williams method, 50 g of ground sample was mixed with 100 mL of 5% ethanol and 4 mol/L hydrochloric acid and subjected to distillation for 105 min. Sulfur dioxide was collected in a receiver containing 30 mL of 3% hydrogen peroxide solution and titrated with 0.01 mol/L sodium hydroxide until a yellow color persisted for at least 20 s. The sulfur dioxide content was calculated using the sample weight, sodium hydroxide consumption, and titration factor, and the results are presented in Table 1.

Heavy metal analysis

The heavy metal content of 14 medicinal plants comprising Mangeum-tang was analyzed by a GMP-certified pharmaceutical company according to the heavy metal test method in the general test methods of the Korean Pharmacopoeia. The dried plant materials were pulverized, and 2 g of the powder was digested in nitric acid using microwave digestion. The digested solution was then filtered and analyzed using an inductively coupled plasma spectrometer (ICP) and a mercury analyzer. The concentrations of lead (Pb), arsenic (As), cadmium (Cd), and mercury (Hg) were summed and are presented in Table 1.

Aflatoxin analysis

Aflatoxin analysis of Glycyrrhizae radix was conducted at a GMP-certified pharmaceutical company in accordance with the legal regulations of South Korea and was performed following the general test methods of the Korean Pharmacopoeia. To analyze aflatoxin in Glycyrrhizae radix, 5 g of the powdered sample was added to 10 mL of methanol and ultrasonically extracted for 30 min, followed by filtration. The filtrate was then diluted to 100 mL with methanol, and 10 mL of this solution was mixed with 70 mL of water. The mixture was passed through an immunoaffinity column for aflatoxin purification. Subsequently, the aflatoxins adsorbed on the column were eluted with methanol, and the aflatoxin B1, B2, G1, and G2 standards were analyzed using photochemical reactor enhanced detection with high performance liquid chromatography.

Residual pesticide analysis

The pesticide residue analysis of each medicinal plant was conducted by a GMP-certified pharmaceutical company in accordance with the “Analytical methods for contaminants in agricultural products” (MFDS notification No. 2016 − 148) in South Korea, covering 320 pesticides. The samples were extracted and purified using a salt-containing acetonitrile/partitioned solid-phase extraction method (QuEChERS) and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS).

A total of 207 pesticides, including abamectin B1, acephate, and acetamiprid, were analyzed using LC-MS/MS, while 113 pesticides, including acrinathrin, alachlor, and aldrin, were analyzed using GC-MS/MS to ensure compliance with legal residue limits.

Quantitative analysis

The quantitative analysis required for the recognition of medicinal plants was conducted at a GMP-certified pharmaceutical company in accordance with the quantitative analysis methods specified in the Korean Pharmacopoeia. The criteria for each plant are as follows: Glycyrrhizae radix (glycyrrhizic acid > 2.5% and liquiritigenin > 0.7%), Angelicae Gigantis Radix (total content of nodakenin, decursin, and decursinol angelate > 6%), Aralia cordata Thunb (total content of kaurenoic acid and continentalic acid > 0.4%), Eucommiae Cortex (pinoresinol diglucoside > 0.05%), Cinnamomi Cortex (cinnamic acid > 0.03%), and PGinseng Radix (ginsenoside Rg1 > 0.1% and ginsenoside Rb1 > 0.2%). The quantitative analysis results obtained from the GMP-certified pharmaceutical company are presented in Table 1.

Extraction

Mangeum-tang was extracted from Hocburi (a facility certified under the Korean standard for herbal dispensaries) and consisted of 14 medicinal plants, including Achyranthis Radix, Glycyrrhizae Radix, and Cnidii Rhizoma. Only medicinal plants that met the standards of the Korean Pharmacopoeia were used for extraction.

The total weight of the medicinal plants was measured according to the ratios indicated in Table 1 and then extracted with 10 times their weight in sterilized water at 115 °C for 4 h. The extract was filtered through a non-woven filter and concentrated to 10 Brix at 120 °C. The concentrates were sterilized at 100 °C for 30 min and subsequently spray-dried to obtain a powder. The powder was dissolved in a vehicle solution and used in each experiment.

Condition of animal facilities

The specific pathogen-free (SPF) animal facilities were managed in accordance with the guidelines for animal experiments (Animal and plant quarantine agency, directive No. 75, south Korea) and ISO 14644-1 guidelines (ISO 2015).

Animals that passed quarantine were housed in rooms meeting the following conditions: a 12-h light-dark cycle, room temperature of 19–25 °C, relative humidity of 30–70%, noise level below 60 dB, ammonia level below 20 ppm, illuminance between 150 and 300 lx, and ventilation of 10–20 air changes per hour using HEPA-filtered air. The cages and water bottles were changed at least twice a week. Breeding equipment was washed with a sterilizing detergent, sterilized in a high-pressure steam sterilizer (121 °C for 20 min), and brought into the animal room for use.

The housing of animals was managed in accordance with the drinking water quality standards and inspection regulations (Ministry of environment, decree No. 1061, South Korea), the standards and specifications for feed (Ministry of agriculture, food and rural affairs, notification No. 2024–27 m South Korea), and the FDA guidelines (FDA 2000).

Rodents were fed ad libitum, and purified water disinfected with ultraviolet rays and reverse osmosis was placed in water bottles for free drinking. For acute toxicity assessment, male and female rats were housed individually in stainless-steel cages. Male mice used for the in vivo micronucleus test were housed in polycarbonate cages with five animals per group. From the date of isolation to the end of the experiment, including the acclimatization period, the animal room was cleaned and disinfected daily using three types of disinfectants that were alternated weekly at concentrations that did not affect the animals.

Acute toxicity assessment

The acute toxicity assessment was approved by the Institutional Animal Care and Use Committee (IACUC) of the Korean Medicine Non-Clinical Study Center (approval number: NIKOM-2022-12) and the Steering Committee of GLP (good laboratory practice, study number: N22006).

The acute toxicity assessment was conducted in compliance with toxicity testing standards for pharmaceuticals and related substances (Ministry of food and drug safety, notification No. 2022-18) and the international council for harmonization of technical requirements for pharmaceuticals for human use (ICH) M3 (European medicines agency 2009) and was performed under GLP conditions.

Six-week-old Sprague Dawley (SD) rats were purchased from Orient Bio, acclimatized in the animal room for five days, and then used in the experiment. Five animals were randomly assigned to each group, both male and female (a total of 50 animals). The negative control group received distilled water, while the test substance was dissolved in distilled water at concentrations of 625, 1,250, or 2,500 mg/kg for the treated groups. SD rats fasted for 12 h were orally administered a single dose of distilled water and Mangeum-tang into their stomachs through a plastic sonde attached to a disposable syringe. Immediately after administration, general symptoms were observed to check for toxic reactions due to the administration of Mangeum-tang at 0.5, 1, 2, and 4 h, and feed was provided again 4 h later. From the day after administration to the end of the experiment, general symptoms, moribund animals, and dead animals were assessed once a day, and body weights were measured on days 1, 3, 7, and 14 of administration. SD rats were euthanized with CO2, and the condition of organ tissues, including the liver, kidney, and heart, was visually inspected.

Bacterial reverse mutation test

The bacterial reverse mutation test was approved by the GLP Steering Committee (study number: T22056) and was performed using Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2 uvrA, according to the organization for economic co-operation and development (OECD) TG 471 guidelines (OECD 2020).

All strains were purchased from MOLTOX (North Carolina, USA), and their properties, including auxotrophy, antibiotic resistance, spontaneous reversion mutations, and sensitivity to positive control, were evaluated and verified prior to their use in the experiments.

The experimental groups were divided into negative control, positive control, and test substance treatment groups. The negative control group was treated with distilled water, and the positive control group was treated with substances known to cause mutations, depending on the presence or absence of metabolic activation in each strain.

In the primary experiment, the test substance treatment group was treated with Mangeum-tang at concentrations of 6.86, 20.6, 61.7, 185.2, 555.6, 1,666.7, and 5,000 g/plate. The secondary experiment was conducted to confirm the reproducibility of the results, wherein the test substance treatment group was re-treated with Mangeum-tang at concentrations of 6.86, 20.6, 61.7, 185.2, 555.6, 1,666.7, and 5,000 µg/plate. Some low doses were excluded from the secondary experiment because toxicity was not observed in the primary experiment.

Test tubes were treated with 0.1 mL distilled water, test substance (Mangeum-tang) or positive control material (0.5 mL of S9 mixture or 0.1 M phosphate buffer solution), depending on the presence or absence of metabolic activation. Each test tube was treated with 0.1 mL of strain culture medium at a concentration of approximately 1 × 109 cells/mL and then incubated in a shaking incubator at 37 ℃ and 120 rpm for 20 min. After shaking, 2 mL of the molten top agar was added to the test tube, mixed, and plated on a minimal glucose agar plate. After the agar was completely solidified, the plate was turned over and cultured in an incubator at 37 ℃ for 48 h. Subsequently, the background lawn where the microorganisms grew was observed under a microscope to confirm the microbial toxicity caused by Mangeum-tang, and two researchers counted the revertant colonies with the naked eye.

In vitro chromosomal aberration test

The in vitro chromosomal aberration test was approved by the GLP Steering Committee (study number: T22057) and performed in accordance with the OECD TG 473 guidelines (OECD 2016a).

The Chinese hamster lung cell line (CHL/IU) was purchased from ATCC (Virginia, USA) and was used after verifying its karyotype, cell cycle, modal number, and contamination status prior to the experiment.

The experimental groups were divided into a negative control group, positive control group, and test substance group to confirm the influence of metabolic activation or treatment time. We divided the series into short-term treatment series without metabolic activation, short-term treatment series with metabolic activation, or long-term treatment series without metabolic activation.

The negative control group was treated with distilled water, while the positive control group was treated with substances known to cause chromosomal aberrations, depending on the metabolic activation for each series. The test substance group was treated with Mangeum-tang at concentrations of 500, 1,000, and 2,000 µg/mL.

Cells were seeded at a concentration of 5 × 105 cells/mL in a 60-mm dish and cultured for 24 h until the next day. The next day, in the short-term treatment series, the test material was applied for 6 h, after which the medium was replaced with fresh medium, followed by an 18-h recovery period. In the long-term treatment series, the test material was applied for 24 h before the medium was replaced with fresh medium. To obtain metaphase cells, the cells were treated in a culture dish for 2 h and then cultured in KCl solution for 30 min. The KCl solution was removed by centrifugation, fixed with an acetic acid/methanol solution (1:3), and stained with 5% Giemsa stain. We counted 300 cells per group and calculated the percentage of cells with chromosomal aberration.

In vivo micronucleus test

The test was conducted in accordance with the OECD TG 474 guidelines (OECD 2016b) and approved by the Institutional Care and Use Committee for Oriental Medicine Non-Clinical Animal Experiments (IACUC, approval number: NIKOM-2022-34) and the GLP Steering Committee (Research Number: T22058).

Six-week-old male Institute of Cancer Research mice were purchased from Orient Bio (Gyeonggi-do, Korea) and acclimatized for five days. Since toxicity due to sex differences was not found in the preliminary experiments, only males were used. Five mice were assigned to each group, divided into a negative control group, positive control group, and three concentration groups of test substance (total of 25 animals).

In the positive control group, mitomycin C, which induces micronuclei formation, was administered intraperitoneally once 24 h before euthanasia. The negative control group was administered distilled water, and the test substance group was orally administered Mangeum-tang at concentrations of 500, 1,000, and 2,000 mg/kg once a day for two days. General symptoms due to Mangeum-tang administration were observed at twice daily.

All animals were sacrificed with CO2 24 h after the last dose, and the bone marrow was harvested by perfusing the femur with fetal bovine serum. The bone marrow obtained was spread on a glass slide and fixed with methanol. The glass slides that underwent fixation were stained with a 5% Giemsa solution and sealed using an encapsulant. Among 4,000 polychromatic erythrocytes (PCEs) per animal, the number of micronucleated polychromatic erythrocytes (mnPCEs) was counted using a microscope, and the polychromatic red blood cells/normochromatic erythrocytes ratio (PCE/NCE ratio) was calculated to assess myelotoxicity caused by the oral administration of Mangeum-tang.

Statistical analysis

All statistical processing was performed using a statistical program (SPSS Statistics 25 for analysis, IBM, USA). The significance of body weight and mnPCE between the groups was analyzed using a one-way analysis of variance and Dunnett’s test (Significance level: 0.05). The significance of the chromosomal aberration rate between the negative control, positive control, and test substance groups was confirmed using Fisher’s Exact Test.