Acute and subacute toxicity of an ethanolic extract of Melandrii Herba in Crl:CD sprague dawley rats and cytotoxicity of the extract in vitro

Melandrii Herba was purchased from Kwangmyungdang (Ulsan, Korea) in July 2014 and identified by Dr. Jung Hoon Kim, a pharmacognosist at the K-herb Research Center, Korea Institute of Oriental Medicine (KIOM; Daejeon, Korea). A voucher specimen (2014-GO-23) has been deposited at the K-herb Research Center, KIOM.

Schaftoside, homoorientin, and isovitexin were purchased from Shanghai Sunny Biotech (Shanghai, China). Cytisoside was provided by Prof. Jong-Keun Son, College of Pharmacy, Yeungnam University, Korea. The purity of the four reference standards was at least 95 %. Methanol, acetonitrile, and water were HPLC-grade solvents and purchased from J.T. Baker (Phillipsburg, NJ). Trifluoroacetic acid was analytical reagent grade and purchased from Sigma-Aldrich (St Louis, MO).

Dried Melandrii Herba (3.0 kg) was extracted with 70 % ethanol (30 L × 3) by ultrasonication for 60 min at room temperature. The extracted solution was filtered using filter paper, the ethanol was removed at 40 °C under vacuum (N-11; Eyela, Tokyo, Japan) and the concentrated extract lyophilized (PVTFD10RS; ilShinBioBase, Yangju, Korea). Extract lyophilized 345.5 g with yield (11.5 %) was obtained.

High-performance liquid chromatography (HPLC) for the quantitative analysis of the four flavonoids, schaftoside, homoorientin, cytisoside, and isovitexin, was conducted on a Shimadzu Prominence LC-20A series system (Kyoto, Japan). This system consisted of a solvent delivery unit (LC-20AT), online degasser (DGU-20A3), column oven (CTO-20A), automatic sample injector (SIL-20 AC), and photodiode array detector (PDA, SPD-M20A). The data were acquired and processed using LC solution software (version 1.24; Shimadzu, Kyoto, Japan). Separation of the four flavonoids was achieved on a Gemini C18 column (250 mm × 4.6 mm, 5 μm, Phenomenex, Torrance, CA) maintained at 50 °C. The mobile phases consisted of 0.1 % (v/v) aqueous trifluoroacetic acid (A) and acetonitrile (B). The gradient flow was as follows: 5–10 % B for 0–10 min, 10–50 % B for 10–30 min, 50–100 % B for 30–40 min, 100 % B for 40–45 min, and 100–5 % B for 45–50 min. Re-equilibration time was 10 min. The flow rate was 1.0 mL/min and injection volume was 10 μL. For quantitative determination, 250 mg of lyophilized sample was dissolved in 50 mL of distilled 70 % methanol and then ultrasonicated for 20 min. After extraction, the solution was filtered through a 0.2 μm syringe filter (Pall Life Sciences, Ann Arbor, MI) before injection into the HPLC system.

The animal studies were conducted according to the guidance of the Institutional Animal Care and Use Committee in the Korea Institute of Toxicology (KRICT) (accredited by AAALAC International, 1998) under the current Good Laboratory Practice regulations for nonclinical laboratory studies and approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (Daejeon, Korea). We obtained five (5)-week-old specific pathogen-free Crl:CD Sprague Dawley rats (n = 22/sex) from Orient Bio Co. (Seoul, Korea) and were used for the experiments after one week of acclimatization. The rats were housed in a room temperature at 22 ± 3 °C and a relative humidity of 50 ± 20 % with artificial lighting from 08:00 to 20:00 and 10–20 air changes per hour as described previously [10].

Group assignment and treatment were as described previously [10]. Briefly, healthy male and female rats were assigned to four groups (n = 5/group) using a Path/Tox System (version 4.2.2; Xybion Medical Systems Corporation, Cedar Knolls, NJ). MHEE was dissolved in distilled water for oral administration (Choong-Wae Pharmaceutical, Korea).

To study its acute toxicity, MHEE was administered to the rats by oral gavage at doses of 0, 500, 1000, and 2000 mg/kg and the rats were monitored for mortality, body weight, and clinical signs for 14 days after a single dose. To study its subacute toxicity, MHEE was administered once daily by oral gavage at doses of 0, 500, 1000, and 2000 mg/kg for 4 weeks. MHEE was prepared freshly on each treatment day and the vehicle control group received an equal volume of distilled water. The daily dose (10 mL/kg body weight) of MHEE was calculated based on the most recently recorded body weights of individual rats.

Gross postmortem examinations were performed after an overdose of anesthetic as described previously [11]. Absolute organ weights were measured and relative organ weights (organ-to-body weight ratios) were calculated for the following: brain, pituitary gland, adrenal gland, liver, spleen, kidneys, heart, thymus, lung, salivary gland, thyroids, testes, ovaries, epididymides, seminal vesicle, prostate, and uterus.

All analyses were performed as described previously [10]. Briefly, blood samples for hematology were analyzed to determine red blood cell (RBC) count, white blood cell (WBC) count, differential WBC count, hemoglobin concentration (HGC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), and reticulocyte (RET) count using an ADVIA120 Hematology System (Bayer).

Serum biochemistry analysis included measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), creatinine (CREA), creatine kinase (CK), glucose (GLU), total cholesterol (TCHO), albumin (ALB), albumin/globulin ratio (A/G), total protein (TP), triglyceride (TG), total bilirubin (TBIL), phospholipids (PL), sodium (Na), potassium (K), calcium (Ca), chloride (Cl), and inorganic phosphorus (IP).

Urinalysis included measurement of urine volume, glucose, ketone bodies (KET), bilirubin (BIL), specific gravity (SG), pH, and urobilinogen (URO).

Normal prostate stromal WPMY-1 cells and normal prostate epithelial RWPE-1 cells were obtained from the American Type Culture Collection (Rockville, MD), and benign prostatic hyperplasia epithelial BPH-1 cells were obtained from Creative Bioarray (Shirley, NY). WPMY-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY) supplemented with 5 % fetal bovine serum (FBS; Gibco). RWPE-1 cells were maintained in keratinocyte Serum-Free Medium (Gibco) supplemented with 5 ng/mL of human epidermal growth factor and 0.05 mg/mL of bovine pituitary extract. BPH-1 cells were maintained in RPMI 1640 medium (Gibco) supplemented with 20 % FBS. The cells were cultured at 37 °C under an atmosphere of 5 % CO₂.

Cytotoxicity was measured using a nonradioactive Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) viability assay according to the manufacturer’s instructions. WPMY-1 and BPH-1 cells were cultured in phenol red-free medium containing 5 % charcoal-treated FBS. WPMY-1 (3 × 10³ cells/well), RWPE-1 (8 × 10³ cells/well), and BPH-1 (5 × 10³ cells/well) cells were seeded into 96-well plates and incubated with various concentrations (0, 3.13, 6.25, 12.5, 25, 50, or 100 μg/mL) of MHEE for 24 h. Cell viability was calculated as described previously [10].

Statistical analyses were based on methods described previously [10]. Data collected during the study were examined for homogeneity of variance using Bartlett’s test. When Bartlett’s test indicated no significant deviation from homogeneity, a one-way analysis of variance (ANOVA) was performed at α = 0.05. When significant difference was noted using the ANOVA, a multiple comparison test (Dunnett’s post hoc test) was conducted to determine which pairs of groups were significantly different. When significant deviations from homogeneity of variance were noted, a nonparametric comparison (Kruskal–Wallis test) was conducted. The Dunn’s Rank Sum test was conducted to determine the specific pairs when a significant difference was observed in the Kruskal–Wallis test. P < 0.05 was considered significant. Values are presented as means ± SD.