Acute and subacute toxicity evaluation of ethanol extract from aerial parts of Epigynum auritum in mice

Food and Chemical Toxicology 131 (2019) 110534

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Acute and subacute toxicity evaluation of ethanol extract from aerial parts of Epigynum auritum in mice

T

Meilian Yanga, Zihuan Wua, Yudan Wangb, Guoyin Kaic, Guy Sedar Singor Njatengd, Shengbao Caia, Jianxin Caoa,∗∗, Guiguang Chenga,∗ a

Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, 650500, People's Republic of China Engineering Research Center of Biopolymer Functional Materials of Yunnan, Yunnan Minzu University, Kunming, 650500, People's Republic of China c College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, 310053, People's Republic of China d State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650204, People's Republic of China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Epigynum auritum Acute and subacute oral toxicities Hematology Histopathology Serum biochemistry analysis

Acute and subacute toxicities of the ethanol extract from Epigynum auritum (EAE) wereperformed by oral administration in pathogen-free mice. Acute toxicity study was performed at a single dose of 5000 mg/kg for 14 consecutive days, while subacute toxicity test was conducted by daily oral administration of EAE at doses of 312, 625, 1250, and 2500 mg/kg for 28 days. Acute toxicity study showed that LD50 of EAE was over 5000 mg/kg. The results of subacute toxicity showed no significant adverse effect of EAE at 312 mg/kg. Moreover, EAE exhibited toxicities to liver, spleen and kidney in mice determined by hematological, serum biochemical and histological analyses during daily oral administration of 1250 mg/kg and 2500 mg/kg EAE. The results revealed that the dose of EAE lower than 625 mg/kg can be regarded as safe.

1. Introduction Medicinal plants are an important part in our daily life and are often used as therapeutic sources for the treatment of various diseases and ailments (Silva et al., 2014). It is estimated that around 80% of the world's population depends on traditional medicine for primary health care, especially in undeveloped countries and regions (Xiang et al., 2015). In recent decades, the interest of natural therapies has increased remarkably in American and several Europe counties (Dutra et al., 2016). The extensive acceptance of the traditional medicine can be attributed to their accessibility, affordability, and historical experimental basis (Fabricant and Farnsworth, 2001). The dependence on medicinal plants with potential therapeutic value gives impetus to the pharmacological and phytochemical studies for assessing their pharmaceutical value, chemical components, and drug-discovery potentials. Based on the indigenous ethical knowledge and scientific pharmacology on medical plants, a number of clinical medicines and functional foods like herbal drinks, teas, infusions, and beverages, are discovered from their extracts or secondary metabolites in recent years (Ahmad et al., 2016; Begas et al., 2017; Kogiannou et al., 2013; Pereira et al., 2017). However, there is a shortage of scientific and experimental toxicity

∗

studies on traditional medicines, which could provide a safe and effective profile for practical application within local communities (Bhowmik et al., 2009). For comprehensive application of medicinal plants, there is an increasing need to evaluate their safety and ensure a constant and adequate quality and efficacy (Thelingwani and Masimirembwa, 2014). Epigynum is a small genus including 5 species in the Family Apocynaceae, and distributed in Southeast Asia. They are lianas woody climbers bearing white latex and opposite leaves (Middleton, 2005). Previous phytochemical studies on Epigynum plants led to the isolation of various secondary metabolites, including pregnane glycosides, triterpenoid saponins, and phenolic glycosides (Cao et al., 2005; Gao et al., 2016, 2017; Wang et al., 2018). Pregnane glycoside is an important class of natural products in modern drug-discovery, which has immunosuppressive (Gao et al., 2017), anti-inflammatory (Jeong et al., 2014), anti-epileptic (Li et al., 2015), neuroprotective (Zhao et al., 2013), anti-hyperglycemic (Tsoukalas et al., 2016), and cytotoxic activities (De Leo et al., 2005). Our previous investigations revealed that epigynosides E–G and epigycosides A-B exhibited significant inhibitory effects in a dose-dependent manner against concanavalin A stimulated proliferation of mice splenocytes (Gao et al., 2017; Shao et al., 2018;

Corresponding author. Corresponding author. E-mail addresses: [email protected] (J. Cao), [email protected] (G. Cheng).

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https://doi.org/10.1016/j.fct.2019.05.042 Received 7 January 2019; Received in revised form 8 May 2019; Accepted 27 May 2019 Available online 28 May 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved.

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the present study to determine the median lethal dose (LD50). The mice were given a certain amount of food (water), and the amount of remaining food (water) was measured at the same time in the next day. The food (water) consumption was calculated by the difference. Body weight, water and food intakes were measured daily during treatment and then weekly changes were recorded. Meanwhile, mortality (if any) and abnormity of mice were also recorded.

Wan et al., 2017). E. auritum is the only species found in China, and has been historically used as a “dai” ethnopharmacy with clearing away heat, detoxifying and analgesic effects (Jin and Mu, 1991). The physiological and phytochemical studies on E. auritum have been investigated (Cao et al., 2003, 2005; Jin and Mu, 1990, 1991). To date, however, the safety or the potential toxicity of E. auritum is not studied. Thus, the present study focuses on safety assessment of ethanol extract from aerial parts of E. auritum through acute and subacute toxicity evaluations by oral administration using mice as animal models.

2.4. 28-Day subacute oral toxicity The 28-days subacute toxicity experiment was performed according to the OECD Guideline 407 programme (OECD 407, 2008). Fifty mice were randomly separated into 5 groups, each group comprising 10 animals of 5 males and 5 females. In this study, LD50 was determined more than 5000 mg/kg. Based on that dose, the experimental groups were given EAE daily at graded doses of 2500, 1250, 625 and 312 mg/ kg by gavage for 28 consecutive days, while the control group was treated with vehicle (0.9% saline). The body weight, food consumption, water intake, abnormal behavior, and adverse signs of toxicity were observed daily during treatment. The weight was recorded weekly. At the end of the treatment, all mice were anaesthetized with chloral hydrate, and blood samples were immediately collected for hematological and biochemical analysis. The organs (liver, lung, heart, spleen, kidney, testis and ovary) were collected and weighed to calculate organ coefficients, while part of the tissues was used for histopathological analysis.

2. Materials and methods 2.1. Plant material and extract preparation The aerial parts of E. auritum were collected in April 2018 from pu'er of Yunnan Province, China, and identified by Prof J.X. Cao, Kumming University of Science and Technology. A voucher specimen (No. cheng20180412-01) is preserved at the Laboratory of Functional Foods, Yunnan Institute of Food Safety, Kunming University of Science and Technology. The dried and powdered sample was extracted with 70% ethanol by ultrasonic-assisted extraction for three times, half an hour for each time in a material-to-solution ratio of 1:10. After centrifugation at 4000g for 10 min, the supernatant was collected. All supernatants were combined and evaporated under reduced pressure at 40 °C using a rotary evaporator (Hei-VAP, Heidolph, Germany) to afford a condensed ethanol extract. The condensed sample was finally lyophilized to obtain the ethanol extract (EAE) using a lyophilizer (Alpha 1–2 LD plus, Christ, Germany). The extraction ratio of the ethanol extract in plant sample was 13.12%.

2.4.1. Histological analysis After euthanasia, the tissues (liver, heart, lung, spleen, kidney, testis and ovary) were fixed in 10% buffered formalin and underwent a conventional histological process for paraffin embedding and light microscopic examination. All tissues were then sectioned and stained with hematoxylin-eosin (H&E), Thereafter, all tissue sections were examined using a microscope.

2.2. Animals In the present study, seventy Special pathogen-free (SPF) ICR mice (18–22 g), 4–8 weeks old of either sex (45 females and 25 males) were used. The experimental mice were purchased from Kunming Medical University (License number: SYXK, 2011–0004). The basic animal feed was provided by Kunming Medical University (60% carbohydrate, 20% protein, 5% fat, 5% fiber). All the animals were housed at room temperature (22 ± 2 °C) and constant humidity (40%–70%) under a 12 h light-dark cycle from 08:00 to 20:00 in SPF grade laboratory. Male and female mice were housed separately and individually in sterile polypropylene cages, and then fed with basic feed and cold boiled water. Animals were acclimated for 7 days before treatment. All animal procedures were strictly in accordance with the National Institutes of Guide for the Care and Use of Laboratory Animals (NRC , 1996), and approved by the Ethical Committee for Animal Experimentation of Kunming University of Science and Technology.

2.4.2. Hematological and biochemical analysis For the hematological analysis, tubes containing heparin lithium were used to collect blood. The blood samples were subjected to evaluation of white blood cell (WBC), lymphocyte (LYM), percentage of lymphocytes (LYM%), monocytes (MON), percentage of granulocytes (GRA%), red blood cell (RBC), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red blood cell volume (HCT), platelet (PLT), platelet pressure (PCT), and mean platelet volume (MPV), by using an automatic blood cell analyzer (HF3800; HANFANG Ltd., Jinan, China). Regarding the biochemical analysis, the solidified blood samples were centrifuged at 4000 rpm in 4 °C for 10 min to obtain sera. The serum biochemical parameters such as total protein (TP), albumin (ALB), aspartate transaminase (AST), aminotransferase (ALT), total bilirubin (TBIL), triglyceride (TG), total cholesterol (TC), creatinine (CRE), alanine blood urea nitrogen (BUN), glucose (GLU), sodium (Na), potassium (K), and chlorine (Cl) were analyzed using kits purchased from Nanjing Jiancheng Biological Co., Ltd.

2.3. Acute oral toxicity The assay of acute toxicity was performed with respect to the Organization for Economic Cooperation and Development (OECD) guideline 407 and 423 (OECD 407, 2008; OECD 423, 2001). A total of 20 female mice, weighing between 18 and 22 g, were randomly divided into four experimental groups (control, 1000, 2000 and 5000 mg/kg groups) with 5 mice each. The EAE dose was selected by the Guidance according to the toxic potential of the substance. Those doses prepared in deionized water were orally administered by gastric intubation. The control group was treated parallel with the same volume of distilled water to establish a comparative. All animals were observed periodically for mortality and changes in general behavior at 30 min, 2 h, 4 h, 6 h, 10 h and 24 h in the first day after the sample administration, and then daily for a total of 14 days. The general behaviors of the mice, including hypo-activity, breathing difficulty, tremors, and convulsion, were observed daily. The “Acute Toxic Class Method” was applied in

2.5. Statistical analysis Data were expressed as mean ± standard deviation (SD). The values were analyzed with one-way ANOVA to evaluate the significant differences (p < 0.05). All of the analyses were performed with Origin 8.5 software (OriginLab, Northampton, MA, USA). 3. Results 3.1. Acute toxicity study The results of acute toxicity testing of EAE revealed that oral 2

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Table 1 Body weight gain, food and water consumption of mice treated orally with ethanol extract of E. auritum. Parameters

Subacute toxicity

Female Initial weight (g) One week (g) Two week (g) Three week(g) Final weight(g) BWG# Food intake (g/day) Water intake (mL/day) Male Initial weight (g) One week (g) Two week (g) Three week (g) Final weight (g) BWG# Food intake (g/day) Water intake (mL/day)

Subacute toxicity

Control

5000

Control

312

625

1250

2500

27.45 ± 0.44 32.72 ± 1.03 33.73 ± 1.00 – – 6.28 9.47 ± 0.72 10.06 ± 1.19

28.65 ± 0.65 32.13 ± 0.73 34.38 ± 0.54 – – 5.73 7.25 ± 0.96 9.68 ± 1.24

24.15 ± 0.43 27.47 ± 0.83 29.98 ± 0.95 31.07 ± 0.64 32.18 ± 0.75 8.03 7.03 ± 0.52 11.38 ± 1.36

24.26 ± 0.96 25.12 ± 1.83 26.90 ± 2.18 29.94 ± 1.97 31.12 ± 1.70 6.86 6.62 ± 0.61 9.89 ± 1.48

23.88 ± 0.83 24.43 ± 1.86 24.97 ± 2.52 26.20 ± 2.00 28.01 ± 2.20* 4.13 5.35 ± 1.31* 8.96 ± 0.73

24.31 ± 1.26 22.65 ± 2.05 23.92 ± 2.23 24.52 ± 2.35 25.40 ± 1.08* 1.09 5.17 ± 0.39* 6.18 ± 0.66*

24.16 ± 1.38 23.76 ± 2.01 23.75 ± 1.85 24.22 ± 1.57 24.96 ± 1.94* 0.80 4.87 ± 0.82* 5.31 ± 2.04*

– – – – – – – –

– – – – – – – –

31.91 ± 0.74 35.65 ± 1.24 38.02 ± 0.95 40.06 ± 2.02 40.94 ± 1.86 9.03 8.14 ± 0.72 12.63 ± 1.80

32.12 ± 0.61 32.38 ± 2.77 33.21 ± 3.04 34.98 ± 4.97 38.65 ± 4.18 6.53 6.94 ± 0.90 10.97 ± 0.88

31.29 ± 0.59 32.52 ± 2.55 32.86 ± 1.09 33.89 ± 2.47 36.98 ± 2.76 5.69 6.45 ± 0.91 9.42 ± 0.37

32.92 ± 0.40 30.95 ± 3.12 31.02 ± 2.81 32.63 ± 1.80 35.53 ± 1.64* 2.61 5.86 ± 0.58* 7.76 ± 1.04*

31.57 ± 1.03 30.06 ± 1.17 30.59 ± 2.47 31.09 ± 3.71 32.51 ± 2.17* 0.94 5.27 ± 0.39* 6.95 ± 0.55*

625

1250

Values expressed as mean ± SD.* Significantly different from the control group, p < 0.05. # BWG: body weight gain (g). Table 2 The organ coefficient results of mice treated orally with ethanol extract of E. auritum. Parameters

Acute toxicity Control

Female Heart (g/100 g) Liver (g/100 g) Spleen (g/100 g) Lung (g/100 g) Kidney (g/100 g) Ovary (g/100 g) Male Heart (g/100 g) Liver (g/100 g) Spleen (g/100 g) Lung (g/100 g) Kidney (g/100 g) Testis (g/100 g)

0.45 5.14 0.31 0.62 1.14 0.05 – – – – – –

± ± ± ± ± ±

Subacute toxicity 5000

0.03 0.03 0.01 0.01 0.05 0.01

0.48 5.13 0.27 0.61 1.19 0.06

Control

± ± ± ± ± ±

0.00 0.02 0.01 0.08 0.02 0.01

– – – – – –

312

2500

0.57 5.10 0.42 0.65 1.33 0.13

± ± ± ± ± ±

0.02 0.15 0.02 0.03 0.03 0.01

0.53 5.13 0.40 0.67 1.32 0.11

± ± ± ± ± ±

0.02 0.24 0.03 0.09 0.14 0.01

0.55 4.99 0.37 0.69 1.31 0.11

± 0.04 ± 0.12 ± 0.01* ± 0.06 ± 0.13 ± 0.02

0.53 4.91 0.40 0.74 1.24 0.10

± 0.03 ± 0.32 ± 0.04* ± 0.07* ± 0.02 ± 0.02

0.56 4.97 0.35 0.77 1.21 0.10

± 0.04 ± 0.25 ± 0.03* ± 0.03* ± 0.07 ± 0.01

0.54 4.90 0.34 0.64 1.59 0.84

± ± ± ± ± ±

0.01 0.06 0.01 0.01 0.01 0.04

0.54 4.57 0.31 0.64 1.61 0.89

± ± ± ± ± ±

0.04 0.25 0.01 0.01 0.04 0.03

0.59 4.50 0.25 0.68 1.56 0.92

± 0.03 ± 0.22 ± 0.01* ± 0.04 ± 0.04 ± 0.04

0.56 4.63 0.26 0.66 1.47 0.91

± 0.04 ± 0.18 ± 0.02* ± 0.08 ± 0.05 ± 0.03

0.53 4.64 0.24 0.67 1.45 0.90

± 0.02 ± 0.12 ± 0.03* ± 0.02 ± 0.05 ± 0.05

Results are expressed as mean ± SD (male/female n = 5). * Significantly different from the control group, p < 0.05.

the control group, irregular hair was more common in female and male mice that received doses of 1250 and 2500 mg/kg/day during the experimental period. No other behavioral changes were found in the EAEtreated groups as well as control animals. As shown in Table 1, the body weight decreased slightly in the first two weeks of the experiment compared with the control group, but after three weeks, the body weight returned to normal for animals treated with 312 mg/kg. At the end of experiment, there were significant decreases (p < 0.05) in body weight between the control and the groups that received 1250 mg/kg and 2500 mg/kg of EAE. The food consumption significantly decreased both in males and females at doses of 1250 mg/kg and 2500 mg/kg compared to control (p < 0.05). Moreover, the water intake in the groups of 1250 and 2500 mg/kg (both males and females) were lower than those of the control group (p < 0.05).

administration of a single dose (1000, 2000, and 5000 mg/kg) did not show any signs of morbidity or mortality in treated animals during the 14 days. We recorded the behavioral changes in mice. The female mice exposed showed no behavioral changes at the doses given during the treatment period; however, food and water consumption were decreased compared to that of the control group, the weight gain of the treated groups was slightly lower than that of the control group with no significant difference (Table 1). At the end of the experiment, the anatomy revealed that the organ coefficients of heart, liver, kidney, lung, and ovary were all within the normal range except that of spleen which was slightly reduced (Table 2). In addition, no histopathological changes were observed in those organs of the control and EAE-treated groups. No animal death was registered during the 14 days of observation. Therefore, it is assumed that the LD50 of EAE was above 5000 mg/kg.

3.2.2. Organ coefficient The results of organ coefficient are presented in Table 2. The organ coefficient of heart, liver, kidney, testis and ovary between males and females in experimental groups ranging from 312 to 2500 mg/kg had no significant difference (p > 0.05) compared with their control groups. However, the changes in the relative weights of the spleens in

3.2. Subacute toxicity study 3.2.1. Body weight, food consumption, and water intake No treatment-related mortality was observed in animals treated with EAE throughout the experiment (data not shown). Compared with 3

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Table 3 Hematological analysis results of mice treated orally with ethanol extract of E. auritum. for 28 days. Parameters Female WBC109/L LYM 109/L LYM % MON 109/L GRA % RBC 1012/L HGB g/L MCHPg MCV fL HCT % PLT 109/L PCT % MPVfL Male WBC 109/L LYM 109/L LYM % MON 109/L GRA % RBC 1012/L HGB g/L MCHPg MCV fL HCT % PLT 109/L PCT % MPVfL

Control

312 mg/kg

625 mg/kg

1250 mg/g

2500 mg/kg

6.81 ± 0.51 6.34 ± 0.28 68.50 ± 1.50 0.68 ± 0.19 29.30 ± 0.60 10.26 ± 0.05 134.40 ± 2.15 10.10 ± 0.75 43.25 ± 2.05 42.40 ± 1.05 483.00 ± 48.50 0.52 ± 0.04 5.53 ± 0.35

6.62 ± 0.47 6.27 ± 0.63 66.90 ± 3.75 0.65 ± 0.46 28.80 ± 2.10 9.99 ± 0.25 134.00 ± 3.05 11.30 ± 0.75 41.16 ± 3.45 41.41 ± 1.40 510.00 ± 43.05 0.57 ± 0.10 5.46 ± 0.46

5.73 ± 0.50* 6.01 ± 0.24 64.60 ± 2.60 0.54 ± 0.22* 28.17 ± 1.55 11.23 ± 0.11 135.24 ± 2.28 12.10 ± 0.90 40.90 ± 2.25 39.60 ± 0.55 525.00 ± 49.05 0.63 ± 0.05 5.60 ± 0.86

5.23 ± 1.00* 5.84 ± 0.54* 60.60 ± 0.20* 0.51 ± 0.10* 27.87 ± 0.65 11.95 ± 0.06* 144.20 ± 4.45* 12.00 ± 1.05 41.18 ± 0.86 42.80 ± 0.35 575.00 ± 38.10* 0.82 ± 0.07* 5.96 ± 0.31*

4.82 ± 0.25* 4.71 ± 0.45* 54.50 ± 2.70* 0.42 ± 0.15* 24.74 ± 1.60* 13.87 ± 0.09* 168.50 ± 3.18* 13.60 ± 1.05* 39.84 ± 3.55* 43.00 ± 0.75 672.00 ± 57.30* 0.97 ± 0.08* 6.09 ± 0.20*

6.75 ± 0.25 6.63 ± 0.10 69.30 ± 2.70 0.75 ± 0.19 24.60 ± 0.95 9.15 ± 0.33 131.28 ± 2.55 12.27 ± 1.75 46.82 ± 0.70 42.90 ± 1.80 459.00 ± 25.50 0.84 ± 0.14 6.10 ± 0.58

6.60 ± 0.55 6.56 ± 0.86 66.90 ± 1.50 0.73 ± 0.06 25.47 ± 1.30 9.31 ± 0.41 137.90 ± 4.25 12.60 ± 0.60 45.50 ± 1.05 43.60 ± 0.90 505.00 ± 25.00 0.87 ± 0.05 5.96 ± 0.45

6.18 ± 0.82* 6.23 ± 0.21* 63.30 ± 3.82* 0.68 ± 0.07* 25.80 ± 1.25 9.24 ± 0.24 140.40 ± 6.85 12.32 ± 0.84 44.38 ± 2.05 42.80 ± 0.65 553.00 ± 42.50 0.84 ± 0.10 6.10 ± 0.24

5.69 ± 0.40* 6.01 ± 0.57* 60.40 ± 2.55* 0.62 ± 0.10* 22.15 ± 2.40 10.08 ± 0.24* 162.80 ± 2.65* 12.40 ± 0.30 45.40 ± 2.15 44.70 ± 0.42 588.00 ± 42.20* 0.99 ± 0.08 6.03 ± 0.20

5.17 ± 0.50* 5.31 ± 0.52* 57.30 ± 4.56* 0.57 ± 0.07* 19.84 ± 2.45* 11.59 ± 0.25* 170.20 ± 4.95* 13.60 ± 0.95 44.90 ± 1.56 44.50 ± 0.90 612.00 ± 20.50* 1.14 ± 0.09* 6.68 ± 0.25

Results are expressed as mean ± SD (male/female n = 5). * Significantly different from the control group, p < 0.05.

testis and ovary of the mice in the control group and the groups treated with the lowest and highest doses (312 and 2500 mg/kg) were analyzed. As shown in Fig. 1, the microscopic observation revealed that some pathological changes in liver, spleen, lung, and kidney were observed in the experimental animals after administration of the highest dose of EAE, while heart, testis and ovary showed no remarkable pathological changes. The detailed results of seven organ sections are as follows: The liver architecture of animals that received the lowest dose ((Fig. 1 A2) was comparable to the control (Fig. 1 A1), and no sign of apoptosis was observed. However, in animals to which the highest dose was administered (Fig. 1 A3), hepatocyte apoptosis and few pores were found around the hepatic sinusoid (focal necrosis) when compared to the control group. The kidney histological assessment is shown in Fig. 1 (B1-B3). Normal glomeruli, tubules, and interstitium were observed in the low dose treatment and control groups. In the high dose treatment group, the vacuolar degeneration and interstitial edema were observed in renal tubular epithelial cells, the mesangial cells proliferation was promoted, the renal glomerulus was atrophied, and inflammatory cell infiltration was observed in renal interstitium. Spleen architecture of both control and low dose treated mice displayed normal red and white pulps, but some nuclear aggregations and focal necrosis were observed in the highest dose treated mice (Fig. C1-C3). In lung histology, a number of pathological changes were observed in the high dose treated group, such as increased thickness of the alveolar capillary wall, atrophy or collapse of pulmonary alveoli, dissolution of partial structure, disruption of cell membrane structure, and edematous capillary endothelium cells (Fig. 1 D3). No significant differences were observed in the ovary and testicle in mice of the treated and control groups (Fig. 1 F1-F3 and G1-G3).

males and females were significantly lower than in control group (p < 0.05). In contrary to males, the females lung coefficient was statistically higher at 1250 and 2500 mg/kg compared to control group (p < 0.05). 3.2.3. Hematological parameters The hematological results were shown in Table 3. In the group of animals that received 625 mg/kg of EAE, the WBC and MON values were significantly lower in both males and females, while only males presented a significant decrease in LYM and LYM% values. The administration of 1250 and 2500 mg/kg of EAE in both male and female mice had significantly lowered WBC, LYM, LYM%, and MON values (p < 0.05), whereas RBC HGB, and PLT values were significantly higher (p < 0.05) when compared to those of control group. In addition, PCT and MPV levels significantly increased in females (p < 0.05) while GRA% significantly decreased in both males and females (p < 0.05) that received the dose of 2500 mg/kg. A significant increase of MCH and MCV were noted in females (p < 0.05), whereas the PCT values were significantly increased in males in comparison with those of control group. There was no significant difference of other hematology parameters in EAE treated groups compared to control. 3.2.4. Serum biochemistry analysis Serum biochemical analysis is reported in Table 4. In the males and females after administration of the higher doses of 1250 and 2500 mg/ kg EAE, levels of TP and ALB significantly decreased (p < 0.05) while AST, ALT, TBIL, BUN, and CRE levels markedly increased (p < 0.05), in comparison to the control group. Na values in males and females treated with 2500 mg/kg EAE significantly reduced (p < 0.05), while K concentrations in females treated with the same dose reduced significantly. The values of TG, TC, GLU, and Cl presented no significant changes (p > 0.05), in both sexes of experimental animals at different doses as compared to the control group.

4. Discussions E. auritum is rich in steroidal compounds and historically used as folk medicine in the prevention of some chronic diseases. However, up to date, there have been no reports on its toxicity evaluation. Toxicological studies are required to determine the safety,

3.2.5. Histopathological analysis Histological sections of heart, liver, heart, lung, spleen, kidney, 4

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Table 4 Serum biochemistry results of mice treated orally with the ethanol extract from E. auritum for 28 days. Parameters Female TP (g/L) ALB (g/L) AST (U/L) ALT (U/L) TBIL (μmol/L) TG (mmol/L) TC (mmol/L) CRE (mg/dL) BUN(mmol/L) GLU (mg/dL) Na (mmol/L) K (mmol/L) Cl (mmol/L) Male TP (g/L) ALB (g/L) AST (U/L) ALT (U/L) TBIL (μmol/L) TG (mmol/L) TC (mmol/L) CRE (mg/dL) BUN(mmol/L) GLU (mg/dL) Na (mmol/L) K (mmol/L) Cl (mmol/L)

Control

312 mg/kg

625 mg/kg

1250 mg/g

2500 mg/kg

46.88 ± 0.22 28.49 ± 1.06 129.26 ± 11.03 40.75 ± 3.08 7.89 ± 0.57 1.07 ± 0.03 2.09 ± 0.08 36.40 ± 0.83 6.45 ± 0.17 3.63 ± 0.13 148.56 ± 10.96 7.60 ± 0.57 100.72 ± 5.38

45.64 ± 0.57 29.48 ± 1.41 129.57 ± 8.98 46.51 ± 4.11 7.78 ± 0.61 1.10 ± 0.01 2.38 ± 0.07 35.84 ± 1.95 6.59 ± 0.48 3.77 ± 0.20 146.76 ± 11.09 7.34 ± 0.64 100.69 ± 3.58

48.41 ± 0.84 27.25 ± 0.64 135.87 ± 10.08 48.30 ± 2.94 8.31 ± 0.43 1.05 ± 0.07 2.38 ± 0.08 39.01 ± 2.31 7.04 ± 0.51 3.91 ± 0.10 150.48 ± 9.88 7.17 ± 0.61 101.73 ± 2.11

42.20 ± 0.51 24.96 ± 0.81* 142.78 ± 7.96* 64.55 ± 3.57* 10.85 ± 0.83* 1.05 ± 0.06 2.05 ± 0.01 45.98 ± 0.48* 7.89 ± 0.13* 3.98 ± 0.14 152.71 ± 12.22 7.10 ± 0.54 98.97 ± 1.84

39.05 ± 0.27* 21.55 ± 0.21* 168.75 ± 11.58* 69.87 ± 4.08* 14.81 ± 1.048* 1.04 ± 0.01 2.07 ± 0.02 51.74 ± 0.57* 9.93 ± 0.22* 3.92 ± 0.15 124.28 ± 10.71* 6.42 ± 0.63* 95.7 ± 3.02

48.64 ± 1.41 29.68 ± 1.02 143.23 ± 10.28 43.53 ± 2.86 8.15 ± 0.57 0.96 ± 0.11 1.94 ± 0.02 47.33 ± 1.03 5.55 ± 0.51 4.84 ± 0.09 150.28 ± 10.28 7.01 ± 0.47 102.22 ± 3.28

48.41 ± 0.84 30.22 ± 0.17 141.02 ± 12.75 45.73 ± 3.62 8.52 ± 0.78 1.01 ± 0.04 1.92 ± 0.03 46.33 ± 2.34 5.39 ± 0.46 4.76 ± 0.12 151.85 ± 9.19 7.42 ± 0.59 101.92 ± 4.21

49.23 ± 0.41 28.96 ± 0.47 153.68 ± 8.71 48.30 ± 3.77 9.01 ± 0.65 0.96 ± 0.07 1.97 ± 0.03 49.54 ± 0.96 6.34 ± 0.32 4.66 ± 0.10 159.78 ± 11.07 7.21 ± 0.48 102.16 ± 7.02

43.97 ± 0.31* 25.75 ± 1.02* 165.56 ± 6.21* 54.73 ± 4.19* 12.04 ± 0.89* 0.98 ± 0.02 1.96 ± 0.06 55.82 ± 1.71* 6.91 ± 0.45* 4.59 ± 0.13 156.56 ± 12.75 7.39 ± 0.75 100.13 ± 2.18

40.54 ± 0.95* 22.12 ± 0.51* 181.56 ± 13.08* 62.05 ± 3.99* 13.73 ± 1.22* 1.03 ± 0.03 1.91 ± 0.05 59.92 ± 0.61* 7.83 ± 0.59* 4.72 ± 0.08 137.78 ± 11.83* 6.45 ± 0.56 97.73 ± 4.55

Results are expressed as mean ± SD (male/female n = 5). * Significantly different from the control group, p < 0.05.

extract and determine major toxic effects on organs (Traesel et al., 2014). The hematopoietic parameters are considered to be the most sensitive to the toxic effects of the substances, and could be used for assessing physiological and pathological status in humans and animals (Li et al., 2010).Significant decreases in WBC (p < 0.05) at doses of 625, 1250, 2500 mg/kg (both males and females) groups were most likely due to the immune function suppression, which could be partly proved by the significant reduction of LYM, MON and GRA% and the decrease in spleens coefficient in the groups with doses higher than 625 mg/kg. The significant increase of RBC and HGB values in mice (both female and male) of both 1250 mg/kg and 2500 mg/kg treated groups suggested that the EAE affected erythropoiesis, or morphology and osmotic fragility of RBC, indicating that liver and kidney tissues may have been damaged (Fabio et al., 2010; Wu et al., 2018). This latter could be supported by biochemical analysis and histopathological examination results. PLT was significantly increased in the 2500 mg/kg group (p < 0.05), while GRA was slightly decreased, indicating that the reduction of granulocytes led to the suppression of immune function. This may be related to spleen damage (Chen et al., 2017), and that could also be obtained from a decrease in the spleen index. The increases of PCT and MPV could demonstrate this from another point of view. MCH was significantly increased in the 2500 mg/kg group of female mice, and the possible cause is the decrease of protein synthesis, which may be related to the decrease of Fe2+ in blood, and may result in thrombosis and cardiovascular diseases such as coronary atherosclerosis (Porto et al., 2013). This latter could also be testified by the increase of HGB. The significant decrease of MVC in female mice at high doses may be due to liver damage (p < 0.05). Liver function can be assessed by glucose metabolism, protein synthesis, biliary secretion, or by evaluating abnormal protein levels in the blood to indicate whether the liver cells are damaged (Peng et al., 2016). Some enzymes and proteins, including ALT, AST, gamma-glutamyltransferase, and bilirubin, are known as sensitive biomarkers of hepatocellular function (Traesel et al., 2014). When there is liver damage, serum levels of AST and ALT rise (Josef et al., 2008). In addition, the elevation of these enzymes is associated with liver necrosis,

demonstrating the need to evaluate the toxicological profile for selecting a safe dose (Elham et al., 2013). Oral acute and sub-acute toxicities of E. auritum were firstly investigated. The mouse has been one of the main mammalian species used in preclinical studies ranging from pharmacology and safety assessment. The use of mice as models in safety evaluations is currently required in international guidelines for both chemicals and pharmaceuticals (Hedrich and Bullock, 2004). Mice are relatively small in size and ease of maintenance reduces the costs of research and their accelerated lifespan (1 mouse year = ∼30 human years) allows all life stages to be studied (Hsu et al., 2011). Thus, mouse is an ideal animal model organism for various experiments due to their small size, low life span, easy availability, and low cost. The acute toxicity tests showed that, at the tested doses, neither toxic symptoms nor death and no obvious behavioral changes were observed in all mice. Therefore, the plant extract can be considered non-toxic based on the method of acute toxicity classification (Duan and Liang, 2011). Toxicological evaluations after repeated administrations provide dose-response evidence on possible health risks after a 28 day subacute toxicity test. Thus, four different EAE doses (312, 625, 1250, and 2500 mg/kg) were administered to two sexes of animals in present study. The middle and high doses (1250 mg/kg and 2500 mg/kg) significantly inhibited the weight gain, food consumption and water intake during the experiment period (p < 0.05) in both sexes, suggesting that high-doses of the extract lead to adverse side effects. The changes in body weight may be due to either the decreases in the food and water consumption or the organ injuries caused by the test substance (Rivas et al., 2013). A similar phenomenon about the subacute toxicity results was also reported in a previous study (Lin et al., 2011), which demonstrated that the remarkable decrease in body weight of the experimental mice was probably due to the increase of the dose and exposure time of test substances. At the end of the subacute period, the organs were removed and weighed. There were significant decreases in the relative organ weight of spleen between the control and EAE (625, 1250, and 2500 mg/kg) treated groups for both sexes (p < 0.05). Therefore, hematological, serum biochemical and histopathological parameters are necessary to evaluate the toxicity of E. auritum ethanol 5

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Fig. 1. Histopathological results of seven organs in mice after oral administration for 28days. Liver: A1(Control, HE 200×); A2 (312 mg/kg of EAE, HE 200×); A3 (2500 mg/kg of EAE, HE 200×). Kidney: B1 (Control, HE 200×); B2 (312 mg/kg of EAE, HE 200×); B3 (2500 mg/kg of EAE, HE 200×). Spleen: C1 (Control, HE 200×); C2 (312 mg/kg of EAE, HE 200×); C3 (2500 mg/kg of EAE, HE 200×). Lung: D1 (Control, HE 200×); D2 (312 mg/kg of EAE, HE 200×); D3 (2500 mg/kg of EAE, HE 200×). Heart: E1 (Control, HE 200×); E2 (2500 mg/kg of EAE, HE 200×); E3 (2500 mg/kg of EAE, HE 200×). Testis: F1 (Control, HE 200×); F2 (312 mg/kg of EAE, HE 200×); F3 (2500 mg/kg of EAE, HE 200×). Ovary: G1 (Control, HE 200×); G2 (312 mg/kg of EAE, HE 200×); G3 (2500 mg/kg of EAE, HE 200×).

ethanol extract may possess toxicity to the liver tissue and result in liver injuries. ALB is part of serum, which is synthesized in liver. Changes in ALB levels can lead to a series of pathological secondary syndromes (Lin et al., 2011). And TP is a kind of substance with the most content in the

hepatitis, and liver toxicity, which is an adjunct to the diagnosis of liver disease (Utohnedosa et al., 2009). In the present work, the ALT, AST and TBIL of the 1250 mg/kg and 2500 mg/kg (both male and female) groups increased remarkably (p < 0.05), suggesting that high doses of 6

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kidney, and lung of mice. Moreover, EAE did more damage to female than to male mice. Therefore, the development of an EAE-based phytomedicine must take into account the choice of the appropriate dose for better activity without adverse side effects.

solid components of serum. Protein synthesis due to liver damage was reduced at doses of 1250 and 2500 mg/kg (both females and males), and TP and ALB were significantly lower than those of the control group indicating that the function of liver to synthesize protein decreased. Total protein is composed of albumin and globulin. And the decrease of ALB content indicated that osmotic pressure is affected. The decrease of GLB may be due to the suppression of immune function. The possible reason why ALB/GLB also showed a downward trend is that the liver's ability to synthesize protein decreases, while the kidney's ability to excrete protein increases (Yun et al., 2018). The decline of WBC and LYM could prove this hypothesis. The significant decrease in food and water intake in mice at high doses may have led to decreased protein synthesis. Renal function can be assessed by changes in urea nitrogen and creatinine, and an increase in these parameters indicated injury of renal function (Ezeja et al., 2014). The BUN and CRE contents of mice (both female and male) in the group of 1250 mg/kg and 2500 mg/kg presented a significant difference in comparison to those of the control group (p < 0.05). Significant increases in CRE and BUN indicated impairment of renal function, which could be confirmed by the histological analysis which showed kidney injury in the mice treated with 2500 mg/kg of EAE, such as tissue edema, congestion of the vessels. Sodium and potassium ions showed a stable trend at low doses, but decreased significantly at 2500 mg/kg. In the group with doses less than 1250 mg/kg, the glomeruli still had filtration function. But at high dose (2500 mg/kg), the glomeruli and renal tubules were severely damaged, and may result in impairment of renal tubular reabsorption and glomerular filtration, or excessive secretion of aldosterone hormones, leading to inability to absorb various ions and severe tissue edema. These were consistent with the decrease of TP and ALB and also confirmed by histopathological analysis. It is noteworthy that the decrease of TP and MCV in females was significantly higher than that in males at high doses, and the rise of MCH, MPV in females was also significant, indicating that EAE did more damage to female mice than to male mice. However, there was no significant difference between TC and TG at all doses, suggesting that EAE had no effect on glucose and lipid metabolism in mice. The histopathological studies serve as a supportive evidence for hematological and biochemical analysis (Traesel et al., 2016). The histopathology changes mainly occurred in the spleen, liver and kidney. Meanwhile, the lungs at 2500 mg/kg also showed hyperemia. According to the result of blood and biochemical analysis, high doses of EAE can cause the changes of various indices in mice, and the most obvious changes are that the WBC, MON, LYM decreased significantly, and biochemical indexes of TP and ALB were also significantly lower. The ALT, AST and BUN, CRE were significantly higher, leading to the disorder of various ions in the serum biochemistry (Zhou et al., 2017). These results suggested that high doses of the EAE may damage mice organs causing liver damage, glomerular swelling, partial spleen cell apoptosis, and alveolar congestion. All lesions can be confirmed from the histopathological slices. However, the specific mechanism of toxic action still needs to be further studied.

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Conflicts of interest The authors declare no conflict of interest. Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant numbers 31600274 and 31460083), the Applied Basic Research Project of Yunnan Province (Grant numbers 2018FB036 and 2017FD121), and the Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Traditional Chinese Pharmacology), Zhejiang Chinese Medical University (Grant number ZYAOX2018031). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.fct.2019.05.042. References Ahmad, M., Khan, M.P.Z., Mukhtar, A., Zafar, M., Sultana, S., Jahan, S., 2016. Ethnopharmacological survey on medicinal plants used in herbal drinks among the traditional communities of Pakistan. J. Ethnopharmacol. 184, 154–186. Begas, E., Tsioutsiouliti, A., Kouvaras, E., Haroutounian, S.A., Kasiotis, K.M., Kouretas, D., Asprodini, E., 2017. Effects of peppermint tea consumption on the activities of CYP1A2, CYP2A6, xanthine oxidase, n-acetyltranferase-2 and UDP-glucuronosyltransferases-1A1/1A6 in healthy volunteers. Food Chem. Toxicol. 100, 80–89. Bhowmik, Debjit, Chiranjib, Pawan D., Margret, C., Kumar, K.P.S., 2009. Herbal drug toxicity and safety evaluation of traditional medicines. Arch. Appl. Sci. Res. 2, 32–56. Cao, J.X., Lai, G.F., Wang, Y.F., Yang, L.B., Luo, S.D., 2003. A new triterpenoid saponin and a new glycoside from Epigynum auritum. Chin. J. Chem. 21 (12), 1665–1668. Cao, J.X., Pan, Y.J., Lu, Y., Wang, C., Zheng, Q.T., Luo, S.D., 2005. Three novel pregnane glycosides from Epigynum auritum. Tetrahedron 61 (27), 6630–6633. Chen, Y., Wu, M.X., Liu, J., Ma, X.J., Shi, J.L., Wang, S.N., Zheng, Z.Q., Guo, J.Y., 2017. Acute and sub-acute oral toxicity studies of the aqueous extract from radix, radix with cortex and cortex of Psammosilene tunicoides in mice and rats. J. Ethnopharmacol. 213, 199–209. De Leo, M., De Tommasi, N., Sanogo, R., Autore, G., Marzocco, S., Pizza, C., Morelli, I., Braca, A., 2005. New pregnane glycosides from Caralluma dalzielii. Steroids 70, 573–585. Duan, W.L., Liang, X.M., 2011. Technical Guidelines Assembly of Veterinary Medicine Research. Chemical Industry Press, Beijing. Dutra, R.C., Campos, M.M., Santos, A.R.S., Calixto, J.B., 2016. Medicinal plants in Brazil: pharmacological studies, drug discovery, challenges and perspectives. Pharmacol. Res. 112, 4–29. Elham, Farsi, Shafaei, A., Hor, S.Y., Mohamed, B.K.A., Yam, M.F., Asmawi, M.Z., Zhari, I., 2013. Genotoxicity and acute and subchronic toxicity studies of a standardized methanolic extract of Ficus deltoidea leaves. Clinics 68 (6), 865–875. Ezeja, M.I., Anaga, A.O., Asuzu, I.U., 2014. Acute and sub-chronic toxicity profile of methanol leaf extract of Gouania longipetala in rats. J. Ethnopharmacol. 151 (3), 1155–1164. Fabio, Talamo, D'Ambrosio, C., Arena, S., Del, V.P., Ledda, L., Zehender, G., Ferrara, L., Scaloni, A., 2010. Proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver, kidney, muscle, plasma and red blood cells. Proteomics 3 (4), 440–460. Fabricant, D.S., Farnsworth, N.R., 2001. The value of plants used in traditional medicine for drug discovery. Environ. Health Perspect. 109 (s1), 69–75. Gao, F., Yao, Y.C., Cai, S.B., Zhao, T.R., Yang, X.Y., Fan, J., Li, X.N., Cao, J.X., Cheng, G.G., 2017. Novel immunosuppressive pregnane glycosides from the leaves of Epigynum auritum. Fitoterapia 118, 107–110. Gao, F., Yao, Y.C., Wan, Z., Cai, S.B., Fan, J., Zhao, T.R., Cao, J.X., Cheng, G.G., 2016.

5. Conclusions In this study, acute toxicity and sub-acute toxicity study of EAE was performed by oral administration using mice as animal model. Results showed that oral single administration of the tested doses did not lead to any mortality. Therefore, the LD50 of EAE extract for mice was greater than 5000 mg/kg BW, and the extract can be considered as practically non-toxic. However, in the subacute toxicity test, it was observed that the extract led to changes in some parameters in a doseresponse manner. Therefore, at low doses (312 mg/kg), the extract show little toxicity though not significant during daily oral administration, and should therefore be used with caution. While at doses greater than 625 mg/kg the extract lead to toxic effects on liver, spleen, 7

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