Chemical composition of the volatile oil of Chenopodium ambrosioides L. from Mianyang in Sichuan Province of China and its sub-chronic toxicity in mice

Purpose: To determine the chemical constituents of the volatile oil of Chenopodium ambrosioides L. from Mianyang in Sichuan Province of China, and assess the sub-chronic toxicity of the volatile oil in mice. Methods: The volatile chemical components were analyzed by gas chromatography-mass spectrometry (GC-MS). Sixty Kunming mice were divided into six groups of ten mice each. One group served as control (no treatment), two groups were orally administered ρ-cymene at doses of 3 and 7 mg/kg, respectively, for 27 days, while three groups received the volatile oil at doses of 10, 25 and 40 mg/kg, respectively, for the same duration. Sub-chronic toxicity in the mice was evaluated by observing their general behavior, measuring serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), evaluating liver, heart, kidney and thymus indices, and assessing the histological morphology of the organs. Results: The volatile oil contained 14 chemical components, of which α-terpinene and ρ-cymene accounted for 32.89 and 24.25 %, respectively. The volatile oil caused significant (p < 0.05) increase in liver index, and serum AST and ALT levels, and also induced distinct morphological changes in mouse liver, heart and kidney. Conclusion: The main volatile components of the oil are α-terpinene and ρ-cymene. The volatile oil showed dose-dependent toxicity in mice, thus lending some support for the safe use of C. ambrosioides in traditional medicine.


INTRODUCTION
Chenopodium ambrosioides L. is a perennial plant native to tropical America and widespread in many provinces of China [1,2]. Existing studies showed that chemical composition of the volatile oil derived from C. ambrosioides varied with countries and areas where the plants grow [3][4][5][6][7][8][9]. For example, the volatile oil of the plants harvested in Egypt and Nigeria contained three main chemical components (-cymene, terpinene and ascaridole) [4,5], whereas that of those in Chengdu China had two main chemical components (-cymene and -terpinene) [6].
C. ambrosioides has been used for centuries as herb medicine for anti-helmintics and healer [10,11]. Previous studies demonstrated that the volatile oil of C. ambrosioides in Chengdu of Sichuan Province of China had anti-tumor effect: it effectively inhibited proliferation of human breast cancer cells MCF-7 and hepatocellular carcinoma cells SMMC-7721 in vitro [6,12], and the inhibition mechanism may be related to blocking cell cycle and inducing caspasedependent apoptosis of cancer cells [12]. Evaluations of in vivo effects of the volatile oil derived from C. ambrosioides are necessary to ensure safe use of the volatile oil as herb medicine in practice. However, such information is lacking.
Therefore, the aim of this study was to analyze chemical components of volatile oil of C. ambrosioides from Mianyang city in Sichuan Province of China. Toxicity of the volatile oil was evaluated in mice, which would provide experimental data for safe use of C. ambrosioides in vivo.

Extraction of the volatile oil from C. ambrosioides
According to a slightly modified steam distillation method [13], 500 g of the aboveground parts of the dried plants was boiled in 3 L water to obtain the volatile oil. The volatile oil was dried over anhydrous Na2SO4, filtered, sealed in a brown reagent bottle and kept at -20 °C as reserve.

GC-MS analysis
Agilent 6890-5973 gas chromatography-mass spectrometer (GC-MS, Agilent Technologies Inc. USA) equipped with a flame ionization detector and an Agilent DB-Wax capillary column (60 m×0.25 mm, 0.25 μm film thickness), was used in analysis of the volatile chemical components. GC conditions: oven temperature was initially 80 °C for 2 min, increased at 5 °C/min to 200 °C and at 20 °C/min to 280 °C, at which it was held for 5 min. Injector temperature was held at 270 °C. 1 μL of the sample was injected, with split ratio of 20:1. Carrier gas was helium (99.999 %), and the flow rate was 1.0 mL/min. MS conditions were as followings. The temperature of electron impact ion source was set at 230 °C. The four-stage bar temperature was 150 °C and the interface temperature was 280 °C, with solvent delay of 8 min. Mass spectra were obtained by scanning 35 to 450 m/z at 70 eV.

Determination of chemical components of the volatile oil
The volatile chemical components were identified by comparing their mass spectra with the database of NIST version 11 (USA), and their relative amount was determined by calculating their GC peak areas.

Animals
Healthy male Kunming mice weighing 20 to 26 g were bought from Dashuo Experimental Animals Co. (Chengdu China). Use of male mice in the present study is to avoid any probability of estrogen interference with the volatile oil. Standard laboratory food bought from Dashuo Experimental Animals Co. (Chengdu China) and distilled water were applied to the mice. After the mice lived in the laboratory for 7 days and adapted to the environment, the experiments were performed according to the recommendations of the International Guidelines for the use of Animals for Scientific Purposes [14]. The experiments were approved by the animal ethics committee of Sichuan Normal University (approval no. 20180209).

Toxicological test of the volatile oil in mice
Sixty mice were randomly selected and divided into 6 groups, each group consisting of 10 mice. One group was used as control (no treatment), two groups were orally administered for 27 days with -cymene at doses of 3 and 7 mg /kg body weight, respectively, and three groups with the volatile oil at doses of 10, 25 and 40 mg /kg body weight, respectively, for the same duration. The doses of -cymene used in this test were determined according to its relative amount (24.25 %) of the volatile oil. Body weight of each mouse was measured every 7 days. At day 28, each mouse was weighed and then anesthetized by intraperitoneal injection with 10 % chloral hydrate at the dose of 0.5 mL/100 g body weight. Blood samples were drawn from abdominal aorta. Four organs (heart, thymus, kidney and liver) were excised and weighed. Their organ indexes were calculated according to Eq 1.

Measurement of serum AST and ALT levels
Blood samples were held at room temperature for blood clotting, and then centrifuged at 3000 rpm for 10 min to isolate serum. Serum AST and ALT levels were measured using AST and ALT activity measurement kits (Jiancheng Shengwu Gongcheng Co., Nanjing China), in accordance with the suggestions of the manufacturer.

Histological examination
Heart, thymus, kidney and liver of each mouse were fixed in 10 % formalin, and then dehydrated in an ethanol series of increasing concentrations (v/v) (50, 60, 70, 80, 90, 95 and 100 %) and embedded in paraffin. The organs were cut into slices with 5 μm thickness, and then stained with hematoxylin and eosin, and imaged under light microscope using BA200 Digital camera (Mike Audi Industrial Co., China).

Data analysis
Data were analyzed using SPSS 19.0 (SPSS Inc, USA). Least significant differences (LSD) were calculated using Microsoft Excel 2010 (Microsoft, USA). Differences were considered significant at p < 0.05.

Chemical composition of volatile oil from C. ambrosioides
The yield of the volatile oil in extractions from C. ambrosioides was 0.10 -0.16 %. Fourteen chemical components of the volatile oil were separated ( Figure 1) and identified (Table 1), which accounted for 64.73 % of the total (Table  1). -Terpinene and -cymene accounted for 32.89 and 24.25 %, respectively, and they were main components of the volatile oil.

Toxicological effects of volatile oil from C. ambrosioides on mice
The 27-day oral administration with the volatile oil from C. ambrosioides at low, medium and high doses affected mice behaviors ( Table 2). Ten of the total 30 mice tested became listless, 15 mice reduced in drinking water and eating food, and mice body weights decreased, in comparison with control and -cymene treated groups ( Table 2). All doses of the volatile oil tested significantly (p < 0.05) increased liver indexes but did not affect the indexes of heart, thymus and kidney, compared with control and cymene treated groups ( Figure 2). In addition, all doses of the volatile oil significantly (p < 0.05) increased serum AST and ALT levels, in comparison with control and -cymene treated groups ( Figure 3). The significant increases in serum AST and ALT levels and the liver indexes caused by the volatile oil suggest that the volatile oil may injure the liver of the mouse. ambrosioides on the indexes of mice organs in the 27day sub-chronic toxicological test. * represents p < 0.05 "Cymene low" and "Cymene medium" are two mice groups orally treated with -cymene at doses of 3 and 7 mg /kg body weight, respectively. "Volatile low", "Volatile medium" and "Volatile high" are three mice groups orally treated with the volatile oil at doses of 10, 25 and 40 mg /kg body weight, respectively.   The kidneys of mice in control group had normal morphology of renal parenchyma, with welldefined tubules and glomeruli (Figure 6 A). However, the increasing doses of the volatile oil caused increasing degrees of glomerular hypertrophy, tubular cell vacuolization and degeneration (e.g. cloudy swelling) in mice kidneys, with occasional tubular atrophy ( Figure  6 D, E and F). However, significant histological changes of the thymuses were not observed in mice orally treated with the volatile oil, compared with control and -cymene treated groups ( Figure  7).
In this study, the volatile oil significantly increased the liver index and the serum levels of liver transaminases (ALT and AST) in mice, indicating that it damages the livers. This result is consistent with literatures that increased serum ALT and AST levels can be directly linked to damage of hepatocytes [19,20]. Obvious histopathological changes of livers, hearts and kidneys seen in mice orally treated with the volatile oil confirmed that the volatile oil damaged these organs. The toxicity of the volatile oil in mice was dose-dependent, increasing doses of the volatile oil caused increases in serum levels of ALT and AST, the liver index and degrees of histopathological changes of mice livers, hearts and kidneys. It suggests that the volatile oil should be carefully used at adequate dose when evaluating its effect in vivo. The result that cymene, as one main component of the volatile oil, had less toxicity in mice than the volatile oil, is because the volatile oil is a complex of many chemical components [9,21].