Acute oral toxicity of the ethyl acetate fraction of Orostachys japonicus in mice

Abstract

Context: Orostachys japonicus (Crassulaceae) is referred to as Wa-song in Korea. It is used as an anti-inflammatory, antifebrile, hemostatic, and anti cancer agent, and as an antidote.

Objective: The purpose of this study was to evaluate the acute toxicity of the ethyl acetate fraction of O. japonicus (OJE) after the oral administration in Balb/c mice of both sexes.

Materials and methods: Mice were oral administered a single doses of 500, 1000, and 2000 mg/kg of body weight and were monitored for 14 d. Biochemical parameters [aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total protein (TP), globulin (GB), total cholesterol (TC), triglyceride (TG), blood urea nitrogen (BUN), and creatinine (CR)] and histopathological examination of liver were performed.

Results and conclusion: No animals died and no toxic changes were observed in clinical signs, body weight, and organ weight. The LD₅₀ of orally administered OJE was higher than 2000 mg/kg/d in both sexes. No toxicological findings were found in biochemical parameters. In histophathological examination, neutrophilic infiltration was observed at a dose of 2000 mg/kg group in both sexes. These finding suggest that oral administration of OJE does not produce acute toxicity. Therefore, these results could provide satisfactory preclinical evidence of safety to launch clinical trials on standardized formulation of OJE to be a biohealth product.

• Acute toxicity
• histopathology
• serum biochemistry
• single dose

Orostachys japonicus (Crassulaceae) is referred to as Wa-song in Korea. It is used as an anti-inflammatory, antifebrile, hemostatic, and anti-cancer agent, as well as an antidote (Ma et al., 2009; Ryu et al., 2009). Dried whole plants of this species have been used as an herbal medicine for the treatment of fever, hepatitis, arthritis, eczema, and intoxication, and are useful for maintaining hemostasis (Ryu et al., 2009). A previous study revealed the presence of flavonoids, triterpenoids, 3,4-dihydroxybenzoic acid, gallic acid, fridelin, epi-friedlanol, grutinone, glutinol, triterpenid, β-sitosterol, campesterol, fatty acid ester, kaempferol, quercetin, and aromatic acids in O. japonicus (Choi et al., 2006; Jeong et al., 2011; Kim et al., 2011; Lee et al., 2011a; Park et al., 2005). It has been reported that this plant has anti-inflammatory, antitumor, hypolipidemic, and hypoglycemic properties (Lee et al., 2011b, 2012; Ryu et al., 2009, 2012). In a previous study, dried O. japonicus powder was extracted and fractionated using a series of organic solvents, including n-hexane (hexane), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (BuOH), and water (H₂O). These extracts were examined for their anticancer activities on various cancer cell lines, including human AGS gastric, A549 lung, HepG2 liver, and HT-29 colon cancer cells. Among the O. japonicus fractions tested, the EtOAc-soluble fraction showed the highest anti-cancer activity against the four cancer cell lines (Ryu et al., 2012).

Natural plants have been used in various folk medications for many years and have been shown to be effective by modern medical science. However, few studies have addressed the toxicity of natural plants, although many questions have been raised regarding their safety (Stein et al., 2001; Wirth et al., 2005). To determine the safety of drugs and plant products for human use, toxicological evaluations are conducted in experimental animals in order to predict toxicity and establish guidelines (Gao et al., 2012; Hamid et al., 2008). According to OECD guidelines (OECD, 2007), acute toxicity is defined as the toxicity produced by a pharmaceutical when it is administered in one or more doses during a single period. Results of acute toxicity tests can be used to screen for the toxicity of a pharmaceutical or to determine whether a compound is toxic or not. Therefore, acute toxicity studies in animals are generally necessary before human use.

The EtOAc fraction of O. japonicus (OJE) is likely to be developed as a functional food or drug. Even though much is known regarding the biological activity of O. japonicus, the toxicological effect of OJE remains unknown. The study of OJE toxicity is important given the likelihood of its development as a bio-health product. Therefore, this study demonstrates the biochemical and histopathological changes associated with acute toxicity of OJE administered orally in BALB/c mice and provides experimental evidence of its safety.

Materials and methods

Preparation of ethyl acetate fraction of O. japonicus

The dried O. japonicus provided by Geobugiwasong Ltd. (Miryang, Korea) was sliced and powdered. The plant was compared with voucher specimen (voucher specimen no. NNMBS302) preserved by New Natural Material Bank of Korea National Research Resources Centers (Iksan, Korea). The powdered O. japonicus (200 g) was extracted by boiling three times with 95% ethyl alcohol (EtOH) for 3 h each time. The EtOH extracts were filtered by Whatman No. 1 filter paper and concentrated by rotary evaporation at 40 °C. The concentrate was suspended in water and fractioned with same amount of following sequence of organic solvents: n-hexane (hexane), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (BuOH), and water (H₂O). The solvent of the supernatant was removed by rotary evaporator and speed vacuum concentrator (Biotron, Inc., Puchon, Korea).

Experimental animals

Each of the 24 female and male Balb/c mice (6-week) was obtained from Hyochang Science. Animals were allocated three per polycarbonate cage with stainless steel tops in the animal care facility, where room temperature (20–25 °C), humidity (45–50%), and ventilation were controlled according to international standards. The animals were maintained in a 12 h light-cycle, and feed (Hyochang Science, Korea) and water were supplied free to access. This study was approved by the Animal Ethical Committee of the Inje University (Gimhae, Korea) and conducted in test guidelines of the Organization for Economic Cooperation and Development (OECD, 2007) and the Korea Food and Drug Administration (KFDA, 2009).

Administration of EtOAc fraction of O. japonicus

During the acclimatization period of 7 d, clinical observations and body weight measurements were conducted to confirm these animals health. The animals were randomly divided into four groups taking six in each group. The test drug (OJE) was administered orally at dosage levels of 500, 1000, and 2000 mg/kg with a volume of 10 mL/kg using Corn oil (Sigma, St. Louis, MO) as a vehicle.

Mortality and clinical observation

Mortality and clinical sign were observed every hour until 6 h after administration of test drug, and continued to next 15 d experimental period. Abnormal signs were recorded daily for each animal. The acute toxic effects of OJE were assessed on the basis of mortality, which was expressed as Lethal Dose (LD₅₀). The LD₅₀ was calculated using Turner’s equation (Turner, 1965).

Body weight

Body weight of individual mice was measured before oral administration (day 0) and days 1, 2, 4, 8, and 15 after administration.

Biochemical analysis in serum

Blood was collected into serum separation tube (Becton Dickinson, Phymouth, UK) during necropsy. Blood samples were centrifuged at 3000 rpm for 5 min using centrifuge after coagulation by maintaining it at room temperature for 20 min. The following biochemical assays were performed using Modular analytics (Roche, Germany): aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total protein, globulin, total cholesterol, triglyceride (TG), blood urea nitrogen (BUN), and creatinine.

Organ weights

The heart, lungs, kidneys, liver, thymus, spleen, and stomach were weight after extirpation as soon as possible. Mean organ-to-terminal body weight ratios were calculated against fasting body weight of final day.

Histology

Immediately after removal from the animals, the liver and kidney tissues were fixed in 10% formalin. The tissues are immersed in paraffin and stained with hematoxylin and eosin (H&E stain), and then observed by microscope.

Statistical analysis

The results are expressed as mean ± standard deviation (SD). Statistical difference was established using Student’s t-test to compare with control. Values of p < 0.05 were considered significant. All statistical analyses were carried out using SPSS 12.0 (SPSS Inc., Chicago, IL).

Results

Clinical signs and mortality

Death of the animals in either gender was observed after the oral administration of OJE during the experimental period. No physical signs of toxicity such as abnormal breathing, movement, and abnormal stool were observed.

Body weight

The changes in weight of the animals during the experimental period are shown in Figures 1 and 2. There is no statistically significant difference between the control group and treated group.

Figure 1. Changes of body weights in female mice after single oral administration of EtOAc fraction of O. japonicus at dose levels of 0 (▪), 500 (▴), 1000 (Χ), or 2000

mg/kg. There were no significant differences in body weight compared with control.

Figure 2. Changes of body weights in male mice after single oral administration of EtOAc fraction of O. japonicus at dose level of 0 (▪), 500 (▴), 1000 (Χ), or 2000

mg/kg. There were no significant differences in body weight compared with control.

Serum biochemistry

The data in Tables 1 and 2 showed the effect of single dose oral administration of OJE on the serum biochemical parameters. The treated female mice showed significant decrease from the control group in BUN. The 1000 mg/kg treated group of male showed significant difference from control in AST and ALT. There was no significant difference in the AST, ALT, ALP, total protein, albumin, globulin, total cholesterol, TG, and creatinine between the control group and treated group at any dose in female mice. In male mice, ALP, total protein, albumin, globulin, total cholesterol, TG, creatinine, and BUN showed no significant difference.

Table 1. Biochemical parameters of the female mice after single oral administration of EtOAc fraction of O. japonicus at dose levels of 0, 500, 1000, or 2000 mg/kg.

Parameter		0	500	1000	2000
AST	U/L	145.0 ± 53.8	107.5 ± 22.9	121.3 ± 48.3	128.2 ± 18.3
ALT	U/L	65.0 ± 36.8	41.3 ± 13.9	51.3 ± 38.3	48.8 ± 7.39
ALP	U/L	122.5 ± 13.2	12.7.8 ± 12.0	138.5 ± 14.4	131.6 ± 7.80
Protein, total	g/dL	4.97 ± 0.21	5.08 ± 0.38	5.28 ± 0.43	5.16 ± 0.05
Albumin	g/dL	3.53 ± 0.21	3.35 ± 0.21	3.80 ± 0.28	3.63 ± 0.15
Globulin	mg/dL	1.52 ± 0.26	1.60 ± 0.29	1.68 ± 0.38	1.64 ± 0.33
Total cholesterol	mg/dL	94.7 ± 10.0	88.0 ± 16.0	86.0 ± 12.4	95.2 ± 8.67
Triglyceride	mg/dL	191.3 ± 24.5	197.3 ± 12.9	178.0 ± 28.2	186.4 ± 24.3
Creatinine	mg/dL	0.46 ± 0.08	0.44 ± 0.01	0.46 ± 0.03	0.49 ± 0.06
BUN	mg/dL	25.10 ± 0.36	17.80 ± 2.58*	20.40 ± 9.16*	21.00 ± 1.88*

AST, aspartate amino transferase; ALT, alanine amino transferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. Values are expressed as mean ± SD of six mice.

*Significantly different from control at p < 0.05

Table 2. Biochemical parameters of the male mice after single oral administration of EtOAc fraction of O. japonicus at dose levels of 0, 500, 1000, or 2000 mg/kg.

Parameter		0	500	1000	2000
AST	U/L	60.4 ± 9.10	59.3 ± 5.47	79.5 ± 11.4*	64.7 ± 16.1
ALT	U/L	28.0 ± 5.52	25.2 ± 4.71	43.6 ± 10.6*	31.0 ± 11.2
ALP	U/L	135.0 ± 12.1	144.3 ± 13.9	135.0 ± 10.6	146.8 ± 21.6
Total protein,	g/dL	5.17 ± 0.37	5.02 ± 0.24	5.20 ± 0.41	5.23 ± 0.22
Albumin	g/dL	3.23 ± 0.15	3.26 ± 0.09	3.25 ± 0.07	3.47 ± 0.15
Globulin	mg/dL	1.85 ± 0.31	1.72 ± 0.16	2.00 ± 0.40	1.72 ± 0.17
Total cholesterol	mg/dL	143.2 ± 20.5	157.33 ± 16.1	132.0 ± 24.0	140.7 ± 19.2
Triglyceride	mg/dL	181.0 ± 15.1	190.4 ± 15.4	157.3 ± 26.0	180.7 ± 24.5
Creatinine	mg/dL	0.32 ± 0.02	0.32 ± 0.05	0.29 ± 0.08	0.33 ± 0.05
BUN	mg/dL	21.3 ± 2.00	21.7 ± 1.64	28.0 ± 9.42	21.0 ± 1.95

AST, aspartate amino transferase; ALT, alanine amino transferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. Values are expressed as mean ± SD of six mice.

*Significantly different from control at p < 0.05.

Organ weight

Final organ weights and organ/body weight ratios are shown in Tables 3 and 4. Stomach weight of 500 and 1000 mg/kg group of female mice was increased when compared with those in the control group. And in 2000 mg/kg group of male mice, liver and spleen weight were increased significantly. No significant changes were observed in any of the organ weight between the control group and treated groups at the end of the experimental period in both genders.

Histology

Histopathology results of liver tissue are shown in Figure 3. No meaningful changes on the histopathological findings were observed in 500 and 1000 mg/kg groups as compared with the control group of each gender. Hepatic cells are round and contain a spherical nucleus. In 2000 mg/kg group of both genders, polymorphonuclear leukocytes were observed compared with the control group. However, no fatty changes, dilation of blood vessels were observed. There are no significant morphological changes in kidney in all groups (data not shown).

Figure 3. Liver tissue stained with hematoxylin and eosin (H&E-stained 200×) showing the effect of EtOAc fraction of O. japonicus at dose levels of 0 (A), 500 (B), 1000 (C), or 2000 (D) mg/kg in female mice, and dose level of 0 (E), 500 (F), 1000 (G), or 2000 (H) mg/kg in male mice.

Discussion

The purpose of this study was to investigate the toxicity of a single oral dose of OJE in female and male mice. In this 15 d acute toxicity study, OJE was orally administered once to female and male mice at doses of 500, 1000, and 2000 mg/kg, according to the OECD guidelines (OECD, 2007).

Acute oral toxicity refers to those adverse effects that occur following the oral administration of a single dose of a substance or multiple doses administered within 24 h. The purpose of acute toxicity studies is to evaluate the toxicity of an unknown substance, and in these studies, a substance is administered orally to a group of experimental animals at a defined dose. Toxicity studies in animal models are commonly used to assess the potential health risk of a substance to humans (Asare et al., 2011). The variations in an acute toxicity study may be due to differences in the route of entry, duration, and frequency of exposure, age, and sex, as well as variations between different species (interspecies) and among members of the same species (intraspecies) (Black, 2002). In this study, therefore, both female and male mice were used in order to demonstrate the gender-related pharmacokinetic differences of OJE. Subsequent toxicity testing of drugs and xenobiotics involves the use of LD₅₀ as an index. LD₅₀ is the amount of an orally administered substance that can be expected to cause death in 50% of animals (Dietrich, 1983). In this study, OJE administration neither demonstrated any clinical signs nor caused mortality. Thus, a single oral dose of OJE did not induce any harmful effects, and the LD₅₀ of orally administered OJE was higher than 2000 mg/kg/d in both sexes. Although there were no significant differences between the body weights of OJE-treated male mice and control group mice, there were significant increases in the liver and spleen weights of the OJE-treated males compared with the corresponding weights of the mice in the control group. The stomach weights of the female mice in the 500 and 1000 mg/kg groups increased, but there were no differences between the body weights of OJE-treated female mice and control group mice.

Various biochemical parameters were used to evaluate the toxicity of OJE. AST, ALT, ALP, total protein, albumin, globulin, total cholesterol, and TG levels were measured in the liver function test, which used serum samples. The liver is the major organ involved in the synthesis of serum proteins and it is the only site in which albumin synthesis occurs (Johnston, 1999). AST and ALT are considered specific indicators of hepatocellular necrosis. AST is present in both the mitochondria and cytosol of hepatocytes, whereas ALT is present only in the cytosol. Therefore, increases in mitochondrial AST levels in the serum reflect extensive tissue necrosis and hepatic cell damage, whereas increased ALT levels reflect hypertrophy and other conditions of the liver. Furthermore, high ALP levels indicate obstruction of the bile ducts, and low levels of albumin are associated with cirrhosis and ascites (Lagarto et al., 2011; Thapa & Walia, 2007). The total protein level increases under conditions of dehydration or increased synthesis by the liver (Singh et al., 2013). Albumin is synthesized by the liver and mainly functions as a general binding and transport protein. Albumin is identifiable as a single discrete molecular species, but the globulins are a mix of proteins of various types such as α-, β-, or γ-globulins (Kaneko, 1997). In our study, AST and ALT levels increased significantly only in male mice of the 1000 mg/kg group, and ALP, total protein, albumin, and globulin levels did not change significantly relative to the corresponding levels in the control group. In female mice, none of the liver function parameters changed significantly. Total cholesterol and TG were measured as indicators of hyperlipidemia. The total cholesterol and TG results of the present study did not show any significant changes. These biochemical parameters associated with liver function did not show any worrisome results since all the changes were within the normal ranges expected for the mice used in this study.

Table 3. Changes on final absolute organ weight (g) and final relative organ weight (%) of female mice after oral administration of EtOAc fraction of O. japonicus.

	Control	500	1000	2000
Mean weight (g)
Heart	0.098 ± 0.021	0.112 ± 0.012	0.112 ± 0.008	0.100 ± 0.006
Lung(L)	0.048 ± 0.010	0.060 ± 0.009	0.055 ± 0.012	0.112 ± 0.142
Lung(R)	0.068 ± 0.015	0.100 ± 0.011*	0.078 ± 0.010	0.080 ± 0.020
Kidney(L)	0.130 ± 0.011	0.133 ± 0.010	0.127 ± 0.014	0.138 ± 0.017
Kidney(R)	0.142 ± 0.010	0.145 ± 0.010	0.143 ± 0.016	0.147 ± 0.016
Liver	0.880 ± 0.100	0.883 ± 0.077	0.860 ± 0.098	0.918 ± 0.070
Thymus	0.063 ± 0.021	0.065 ± 0.012	0.070 ± 0.014	0.060 ± 0.014
Spleen	0.062 ± 0.028	0.075 ± 0.010	0.068 ± 0.012	0.078 ± 0.012
Stomach	0.157 ± 0.037	0.203 ± 0.027*	0.205 ± 0.015*	0.160 ± 0.028
Mean organ-to-terminal body weight ratios (%)
Heart	0.502 ± 0.100	0.556 ± 0.058	0.552 ± 0.037	0.485 ± 0.031
Lung(L)	0.247 ± 0.046	0.299 ± 0.045	0.272 ± 0.061	0.541 ± 0.691
Lung(R)	0.349 ± 0.069	0.498 ± 0.055*	0.387 ± 0.049	0.338 ± 0.097
Kidney(L)	0.663 ± 0.051	0.664 ± 0.051	0.626 ± 0.068	0.671 ± 0.084
Kidney(R)	0.723 ± 0.046	0.722 ± 0.052	0.708 ± 0.081	0.711 ± 0.079
Liver	4.489 ± 0.466	4.399 ± 0.383	4.250 ± 0.484	4.453 ± 0.338
Thymus	0.323 ± 0.096	0.324 ± 0.061	0.346 ± 0.070	0.291 ± 0.069
Spleen	0.315 ± 0.130	0.374 ± 0.052	0.338 ± 0.058	0.380 ± 0.057
Stomach	0.799 ± 0.171	1.013 ± 0.136*	1.013 ± 0.075*	0.776 ± 0.134

L, left side; R, right side. Values are expressed as mean ± SD of six mice.

*Significantly different from control at p < 0.05.

Table 4. Changes on final absolute organ weight (g) and final relative organ weight (%) of male mice after oral administration of EtOAc fraction of O. japonicus.

	Control	500	1000	2000
Mean weight (g)
Heart	0.148 ± 0.019	0.132 ± 0.008	0.118 ± 0.012	0.110 ± 0.020
Lung(L)	0.060 ± 0.015	0.050 ± 0.006	0.047 ± 0.012	0.052 ± 0.015
Lung(R)	0.090 ± 0.017	0.097 ± 0.008	0.098 ± 0.031	0.088 ± 0.015
Kidney(L)	0.188 ± 0.008	0.177 ± 0.012	0.180 ± 0.009	0.172 ± 0.019
Kidney(R)	0.210 ± 0.020	0.187 ± 0.012	0.200 ± 0.019	0.190 ± 0.013
Liver	1.023 ± 0.056	1.037 ± 0.062	1.000 ± 0.102	1.067 ± 0.021*
Thymus	0.050 ± 0.011	0.053 ± 0.016	0.050 ± 0.013	0.060 ± 0.018
Spleen	0.067 ± 0.014	0.063 ± 0.018	0.068 ± 0.008	0.078 ± 0.012*
Stomach	0.213 ± 0.034	0.165 ± 0.029	0.170 ± 0.020	0.202 ± 0.028
Mean organ-to-terminal body weight ratios
Heart	0.628 ± 0.075	0.570 ± 0.033	0.494 ± 0.049	0.459 ± 0.083
Lung(L)	0.254 ± 0.060	0.216 ± 0.027	0.195 ± 0.051	0.216 ± 0.061
Lung(R)	0.381 ± 0.065	0.418 ± 0.035	0.411 ± 0.128	0.369 ± 0.061
Kidney(L)	0.798 ± 0.029	0.764 ± 0.052	0.752 ± 0.037	0.717 ± 0.081
Kidney(R)	0.890 ± 0.077	0.808 ± 0.052	0.836 ± 0.079	0.793 ± 0.053
Liver	4.336 ± 0.217	4.486 ± 0.269	4.178 ± 0.425	4.453 ± 0.086*
Thymus	0.212 ± 0.042	0.231 ± 0.071	0.209 ± 0.053	0.250 ± 0.075
Spleen	0.282 ± 0.053	0.274 ± 0.076	0.286 ± 0.031	0.327 ± 0.049*
Stomach	0.904 ± 0.131	0.714 ± 0.128	0.710 ± 0.084	0.842 ± 0.116

L, left side; R, right side. Values are expressed as mean ± SD of six mice.

*Significantly different from control at p < 0.05.

Creatinine and blood urea nitrogen (BUN) levels are used as indicators of renal function. Creatinine is formed by the hydrolysis of creatine and phosphocreatine in muscles, and its levels increase in conditions of impaired kidney function and decrease during pregnancy or upon the use of certain drugs, such as cimetidine or trimethoprim. BUN is mostly synthesized in the liver and is the end product of protein metabolism. Low levels of BUN are related to liver disease, and high levels of BUN imply extensive impairment of renal function (Wallach, 2007). In our study, BUN levels in female mice decreased significantly but remained in the normal range (13–33 mg/dL) (Jacob et al., 1991). The other parameters related to renal function did not change significantly in both female and male mice. These results show that OJE was not toxic to the kidneys.

Histological examination was performed to evaluate any potential damage to the liver and kidney caused by the oral administration of OJE. Anatomical–morphological studies are conducted for the anatomical localization of the action of a toxin (Singh et al., 2013). Hepatic cells in the normal liver are round and contain a spherical nucleus. As shown by Hong et al. (2013), treatment with tert-butyl hydroperoxide (t-BHP) induces neutrophilic infiltration and cell death. Therefore, this change was used as an index of necrosis with inflammatory cell infiltration. According to the acute toxicity study conducted by Harizal et al. (2010), a single oral administration of Mitragyna speciosa (Rubiaceae) extract caused significant damage to liver tissue, including sinusoid congestion, hepatocyte rupture, fat accumulation, and centrilobular necrosis. In our study, polymorphonuclear leukocytes were observed in the liver tissue of male and female mice in the 2000 mg/kg group, indicating neutrophilic infiltration. This change in liver tissue was related to the inflammation reaction induced by OJE acute toxicity. No severe toxicity symptom was observed in the liver tissue. No toxic changes were observed up to a dose level of 2000 mg/kg. Hence, these results suggest that, although OJE did not cause significant damage to the liver, a high dose of OJE burdened the liver. The kidneys did not show any specific symptoms associated with toxicity.

In conclusion, this study shows that a single oral administration of OJE in female and male mice did not induce any clinical signs of toxicity or cause mortality. The LD₅₀ of OJE was higher than 2000 mg/kg/d in both sexes. These results indicate that oral administration of OJE does not produce acute toxicity. However, histology results revealed that oral administration at 2000 mg/kg/d burdened the liver.

Declaration of interest

The authors declared no conflict of interest. This work was supported by a Basic Science Research Program grant through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2010-0023450).