Medicinal herbs and herbal formulas are generally considered to be safer than conventional drugs. However, there is no evidence for the quality, safety, and efficacy of most commonly used herbal formulas including YMJ. In addition, very little information exists on the safety of YMJ for clinical use.
To establish evidence-based toxicity data for YMJ, we analyzed the literature, efficacy, safety, and chemical constituents of YMJ. As a part of the safety evaluation of YMJ, acute oral dose toxicity and a subchronic oral dose toxicity studies were conducted to investigate the potential toxicity after a single or 13-week repeated oral dose administration of YMJ in Sprague-Dawley rats. The present study was carried out in compliance with good laboratory practice (GLP) and the test guidelines of the Organization for Economic Cooperation and Development (OECD) [
The constituents of YMJ are listed in Table
Composition of Yukmijihwang-tang (YMJ).
Herbs | Scientific names | Ratio |
---|---|---|
Rehmanniae Radix Preparat | 8 | |
Dioscoreae Rhizoma | 4 | |
Corni Fructus | 4 | |
Hoelen | 3 | |
Moutan Cortex | 3 | |
Alismatis Rhizoma | 3 |
The chemicals used for the identification and quantification of compounds within the YMJ extract include the following: 5-hydroxymethyl-2-furaldehyde (5-HMF, MW: 126.11) as a component of Rehmanniae Radix Preparata; loganin (MW: 390.38) of Corni Fructus; paeoniflorin (MW: 480.46) and paeonol (MW: 166.17) of Moutan Cortex.
YMJ extract (10 mg) was dissolved in 1 mL of 50% methanol and filtered through a 0.45-
Chromatograms of standard components and
In order to make standard calibration curves, 5-HMF (1 to 500
For the acute and subchronic toxicity studies, specific pathogen-free Sprague-Dawley Crl : CD (SD) rats of both genders (five weeks old) were obtained from OrientBio Co., Ltd. (Seoungnam, Korea). The animals were allowed to acclimatize for one week before the initiation of experiments. Animals were housed in a stainless wire cage (255 mm W × 465 mm L × 200 mm H) at ≤5 animals/cage for the quarantine period and at ≤2 animals/cage for the observation period. Pelleted food was purchased from PMI Nutrition International (USA). The pellet food was gamma-ray irradiated and given
YMJ is widely used as a traditional medicine and was not anticipated to cause animal death following a single dose. The highest dose of 2000 mg/kg, with a common ratio of 2, was selected for this study. YMJ extract was dissolved in distilled water for injection (Choongwae Pharmaceutical, Ltd., Korea). Distilled water was given to the animals in the vehicle control group. The animals were grouped into four groups with five rats/sex/group at random, and YMJ was dissolved in purified water and administered by oral gavage at doses of 0, 500, 1000, or 2000 mg/kg body weight. The observations of general condition, toxic symptoms, and mortality in rats were monitored for 14 days after YMJ administration. The grouping of animals in good health during the acclimation period was carried out using the A-module of Path/Tox System (Ver. 4.2.2, Xybion Medical Systems Corporation, USA).
In the subchronic study, rats (10 rats/sex/group) were randomly divided into four groups. A previous four-week repeated dose study showed no abnormal changes even at the highest dose, 2000 mg/kg. Therefore, 2000 mg/kg was selected as the highest dose, with a common ratio of 2.
Mortality and clinical signs were continually observed for 0, 1, 2, 3, 4, 5, and 6 hr after dosing on day 1 and at one time per day from day 2 to day 15. The animals were weighed before dosing (day 1) and after dosing (days 2, 4, 8, and 15) using an electronic balance (Sartorious Co., Germany). On day 15, all animals were euthanized by exsanguination from the abdominal aorta and abdominal vena cava under CO2 gas overdose and examined for internal organ abnormalities.
Mortality and clinical signs were recorded once a day during the study period using the Path/Tox Program (Ver. 4.2.2). Animals were weighed on animal arrival, randomization, the first day of dosing, once a week thereafter, and at the termination of the experiment. The amounts of food and water given and their remnants on the next day were measured to calculate the difference, which was regarded as daily consumption. External eye examination was investigated during the pretest period; both external and fundus examinations were completed using an indirect binocular ophthalmoscope (Vantage Plus Digital, Keeler Ltd., UK).
During the last week of dosing, urinalysis was carried out with urine collected over approximately 16 hr from all animals. Urinalysis measurements included urine volume, specific gravity, pH, protein, ketone body, occult blood, glucose, bilirubin, nitrite, urobilinogen, color, and sediment (cast, epithelial cell, erythrocyte, and leucocyte). Urine volume was measured using a mass cylinder, color with macroscopic examination, and sediment using a microscope. The other parameters in the urinalysis were analyzed using Multistix 10 SG strips (Bayer, USA) and a CliniTek-100 (Bayer, USA).
Animals were fasted over night prior to necropsy and blood sampling. All living animals were anesthetized by isoflurane inhalation, blood samples were taken, and then the animals were sacrificed by exsanguination from the aorta. Complete gross observation was performed on all terminated animals. The blood samples were collected into tubes containing EDTA-2K and analyzed for complete blood cell counts. Complete blood cells were measured by an ADVIA120 Hematology System (Bayer, USA). Prothrombin time (PT) and activated partial thromboplastin time (APTT) were analyzed with blood samples treated with 3.2% sodium citrate using an ACL 9000 (Instrumentation Laboratory, Italy).
Blood samples for serum biochemistry and hematology were drawn from the posterior vena cava of all animals. Serum samples were prepared after blood centrifugation and analyzed using a Toshiba 200FR NEO (Toshiba Co., Japan). The following parameters were measured: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), glucose (GLU), total cholesterol (TCHO), albumin/globulin ratio (A/G), total protein (TP), albumin (ALB), creatine kinase (CK), triglyceride (TG), total bilirubin (TBIL), phospholipid (PL), gamma glutamyl transferase (GGT), calcium (Ca), inorganic phosphorus (IP), chloride (Cl), sodium (Na), and potassium (K) [
Absolute organ weights were measured, and relative organ weights (organ/body weight) were calculated for the organs of all animals when sacrificed.
Data collected during the study were examined for variance homogeneity using the Bartlett’s test. When the Bartlett’s test indicated no significant deviations from variance homogeneity, a one-way ANOVA was performed at
There were no animal deaths in any groups. Therefore, the approximate lethal doses of YMJ extract in male and female rats are higher than 2000 mg/kg. Clinical signs observed included loss of fur in 1/5 males from day 11 until the end of the analysis and 2/5 females from day 4 until the end of the analysis in the YMJ 2000 mg/kg group. Normal body weight gains were observed in males and females of all dose groups (Figure
Changes in body weight during a two-week acute toxicity study. (a) Male rats and (b) female rats.
There were no animal deaths during the study period. In males, clinical signs observed during the study period included loss of fur (1/10) and swollen pinna (1/10) in the vehicle control group; loss of fur (1/10), swollen pinna (1/10), and scratch wound (1/10) in the 500 mg/kg group; loss of fur (4/10) and scratch wound (2/10) in the 1000 mg/kg group; loss of fur (2/10) and scratch wound (2/10) in the 2000 mg/kg group. In the females, clinical signs included loss of fur (1/10) in the vehicle control group; loss of fur (4/10) and scratch wound (1/10) in the 500 mg/kg group; loss of fur (2/10) in the 1000 mg/kg group; loss of fur (3/10) in the 2000 mg/kg group.
No treatment-related body weight changes were observed during the study period (Figure
Increase in body weight during the 13-week subchronic toxicity study. (a) Male rats and (b) female rats.
Food consumption of rats administrated
In ophthalmological examination, no abnormalities were noted in any animals. No treatment-related changes were observed in urinalysis during the study period. Hematological examination performed at necropsy showed an increase in NEU% (50%) and a decrease in LYM% (9%) in the male 1000 mg/kg group (Table
Hematological parameters of male and female rats on subchronic toxicity study.
(a) | (b) | |||||||
Parameter | Vehicle control | YMJ-500 mg | YMJ-1000 mg | YMJ-2000 mg | Vehicle control | YMJ-500 mg | YMJ-1000 mg | YMJ-2000 mg |
WBC (×103/ | ||||||||
Reticulocytes (%) | ||||||||
Neutrophils (%) | ||||||||
Lymphocytes (%) | ||||||||
Eosinophils (%) | ||||||||
Monocytes (%) | ||||||||
Basophils (%) | ||||||||
LUC (%) | ||||||||
RBC (×106/ | ||||||||
Hemoglobin (g/dL) | ||||||||
Hmatocrit (%) | ||||||||
MCV (fL) | ||||||||
MCH (pg) | ||||||||
MCHC (g/dL) | ||||||||
Platelet (×103/ | ||||||||
PT (sec) | ||||||||
APTT (sec) |
Values are mean ± SEM for 10 rats in each group.
Serum biochemical parameters of male and female rats on subchronic toxicity study.
(a) | (b) | |||||||
Parameter | Vehicle control | YMJ-500 mg | YMJ-1000 mg | YMJ-2000 mg | Vehicle control | YMJ-500 mg | YMJ-1000 mg | YMJ-2000 mg |
Glucose (mg/dL) | ||||||||
BUN (mg/dL) | ||||||||
Creatinine (mg/dL) | ||||||||
Total protein (g/dL) | ||||||||
Albumin (g/dL) | ||||||||
A/G (ratio) | ||||||||
TCHO (mg/dL) | ||||||||
Triglyceride (mg/dL) | ||||||||
Phospholipid (mg/dL) | ||||||||
AST (IU/dL) | ||||||||
ALT (IU/dL) | ||||||||
TBIL (mg/dL) | ||||||||
ALP (IU/dL) | ||||||||
CK (IU/dL) | ||||||||
Ca (mg/dL) | ||||||||
IP (mg/dL) | ||||||||
Na (mg/dL) | ||||||||
K (mg/dL) | ||||||||
Cl (mg/dL) | ||||||||
GGT (mg/dL) |
Values are mean ± SEM for 10 rats in each group.
Gross findings at male necropsy included swollen pinna (1/10) and irregular surface of lung (1/10) in the vehicle control group, swollen pinna (1/10) in the 500 mg/kg group, small testes and epididymis (1/10) in the 1000 mg/kg group, and a scratched wound on face (1/10) in the 2000 mg/kg group. In females, gross findings included focus/foci in the adrenal glands (1/10) in the vehicle control group and nodule in the kidneys (1/10), adhesion in the diaphragm (1/10), hemorrhage in the thoracic cavity (1/10), and discoloration of the thymus (1/10) in the 2000 mg/kg group.
Organ weights measured at female necropsy showed a decrease (33%) in the absolute weight of the uterus/cervix in the 500 mg/kg group and an increase (9%) in the relative weight of the liver in the 2000 mg/kg group (Table
Absolute organ weights (g) of male and female rats on subchronic toxicity study.
(a) | (b) | |||||||
Parameter | Vehicle control | YMJ-500 mg | YMJ-1000 mg | YMJ-2000 mg | Vehicle control | YMJ-500 mg | YMJ-1000 mg | YMJ-2000 mg |
Brain | ||||||||
Pituitary | ||||||||
Liver | ||||||||
Spleen | ||||||||
Adrenal gland | ||||||||
Kidneys | ||||||||
Heart | ||||||||
Thymus | ||||||||
Salivary glands | ||||||||
Lung | ||||||||
Thyroid/Parathyriod | ||||||||
Epididymides | — | — | — | — | ||||
Seminal vesicle | — | — | — | — | ||||
Prostate | — | — | — | — | ||||
Testes | — | — | — | — | ||||
Ovaries | — | — | — | — | ||||
Uterus/Cervix | — | — | — | — |
Values are mean ± SEM for 10 rats in each group. *: significant differences from the vehicle control (0) group (
In histopathological examination, lesions such as inflammatory cell foci in the liver and kidneys, mineralization in the kidneys, and cardiomyopathy in the heart in males and females in the 2000 mg/kg groups were also detected in the vehicle control group with similar incidence rates. The acanthosis and chondropathy in the ear, chronic dermatitis of the skin, M-nephroblastoma in the kidney, pigmented macrophages in the thymus, tubular atrophy in the testes, and aspermia in the epididymides were not dose dependent and occurred at low incidence rates. There were other lesions that were considered to be spontaneous or nontreatment related.
Globally, the increasing use of complementary and alternative medicine (CAM) such as herbal remedies may be due to a variety of factors, including the limits of current therapy and adverse effects of conventional drugs [
YMJ is one of the most popular herbal formulas in Asian countries; however, evidence-based information is limited. Many studies have reported pharmacological efficacies and benefits of YMJ, but there is no information on its risk and safety such as its acute and subchronic toxicity.
To evaluate the acute toxicity, YMJ extract was orally given at doses 0, 500, 1000, or 2000 mg/kg to male and female Crl:CD (SD) rats. Mortalities, clinical signs, body weight changes, and gross findings were monitored for 14 days (days 1~15). The loss of fur observed in some animals at the highest dose group (2000 mg/kg) has been observed in control SD rats also and considered to be due to individual variation.
No treatment-related toxicity was observed in the study carried out to evaluate the subchronic 13-week repeated oral dose toxicity of YMJ extract. Some changes noted in the clinical observation, food consumption, hematology, serum biochemistry, gross findings, and organ weights were considered to be not treatment-related but incidental and within normal ranges. Histopathological findings, such as the inflammatory cell foci in the liver and kidneys, mineralization in the kidneys, and cardiomyopathy in the heart were also observed in the vehicle control group at similar levels and incidence rates, indicating no toxicological significance. Lesions such as acanthosis and chondropathy in the ear, chronic dermatitis of the skin, tubular atrophy in the testes, and aspermia in the epididymides were considered to be associated with gross findings but appear to be incidental because of a lack of dose dependency and low incidence rates. There were other lesions that were considered to be spontaneous or not related to the treatments.
Consequently, no acute toxicity was found in rats treated with YMJ extract, and approximate lethal doses for males and females were higher than 2000 mg/kg. The 13-week repeated oral administration of YMJ in SD rats showed no toxicity and a no-observed-adverse-effect-level (NOAEL) of 2000 mg/kg/day.
In humans, the reported single dose of YMJ is about 46.875 g dried herb [
In conclusion, the present studies demonstrate that at doses consumed in traditional medicine, the aqueous extract of YMJ may be relatively safe because it did not cause any lethality or changes in the general behavior in rats in both the acute and subchronic toxicity studies.
This research was supported by a grant, “The evidence-based medicine for herbal formulae,” from Korea Institute of Oriental Medicine (KIOM). H. Ha and J. K. Lee contributed equally to this work.