Revista Brasileira de Farmacognosia 26 (2016) 438–445 ww w.elsevier.com/locate/bjp Original Article A subchronic toxicity study of ethanol root extract of baked Aconitum flavum in rats a,1 a,1 a,1 a a a a Yuanbin Zhang , Enshuang Guan , Xin Zhao , Bing Wang , Lei Yin , Liming Zhang , Jun Huang , a,b,c,∗ Xueyan Fu a School of Pharmacy, Ningxia Medical University, Yinchuan, China b Ningxia Engineering and Technology Research Center for Modernization of Hui Medicine, Yinchuan, China c Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education (Ningxia Medical University), Yinchuan, China a b s t r a c t a r t i c l e i n f o Article history: The genus Aconitum has strong toxicity, but the acute toxicity of baked Aconitum flavum Hand.- Received 23 September 2015 Mazz., Ranunculaceae, was reduced significantly on the premise of keeping anti-inflammatory and Accepted 1 March 2016 anti-nociceptive activities. However, the risk associated with long-term use is unknown. In a sub- Available online 31 March 2016 chronic toxicity study, rats were orally administered A. flavum at doses of 0.76–3.03 g/kg for 90 days and further recovered for 14 days. Our results showed that oral treatment with A. flavum for 90 days Keywords: caused significant changes in some hematological indicators at doses of 3.03 and 1.52 g/kg, such as red Rats blood cell, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular Sub-chronic toxicity hemoglobin concentration. These results indicated that the A. flavum affects the structure and function Recovering study of red blood cell. Furthermore, significant changes were observed in the white blood cell at dose of Tissue damage Pathological change 3.03 g/kg in male rats, which confirmed tissue damage or toxicity. The liver function tests exhibited non- Routine blood test significant alterations in aspertate aminotransferase, alanine aminotransferase and avenin-like storage proteinsgene. But other parameters, such as total protein and albumin were obviously decreased at all doses. A. flavum also caused a significant decrease in glucose, cholesterol and triacylglyceride at all doses. For kidney function, there were significant elevations in urea and creatinine at doses of 3.03 and 1.52 g/kg. + + − The levels of certain electrolytes (Na , K and Cl ) were significantly different after 90 days of treatment with A. flavum (3.03 and 1.52 g/kg). Organs were observed by light microscopy after hematoxylin-eosin staining. Hemosiderin depositions in the spleen were observed in the A. flavum group. These data demon- strated that the subtoxicity of A. flavum was reduced considerably by baked, but the subchronic toxicity effects on the liver, kidney and spleen should not be ignored. © 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction 2012). However, with increasing popularity, Aconitum poisoning can occur in any part of the world because of the highly toxic The genus Aconitum consists of over 300 species distributed in and narrow margin of safety between therapeutic and toxic doses Asia, Europe and North America. Most of them grow in high alti- of these herbs. Many studies have demonstrated that the high tudes in the northern hemisphere. There are 200 species in China, toxicity of Aconitum herbs is primarily attributed to diester diter- such as Aconitum kusnezoffii Reichb., A. carmichaelithe Debx. and A. pene alkaloids (DDA) (Chan, 2009, 2012; Singhuber et al., 2009). flavum Hand.-Mazz. (Xiao, 2006). In traditional Chinese medicine, Aconitine, mesaconitine, hypaconitine and other DDA are potent the genus Aconitum has similar pharmacological actions and has cardiotoxins and neurotoxins found in all parts of the Aconitum been commonly used for the treatment of various diseases, such as species, especially in the tubers and roots. Patients with Aconitum rheumatic fever, painful joints, gastroenteritis, edema, bronchial poisoning often present with a combination of cardiovascular, neu- asthma and various tumors (Singhuber et al., 2009; Qin et al., rological, gastrointestinal and other signs and symptoms (Chan, 2012; Qin et al., 2012). Although patients face a huge risk, the enthusiasm for the use of Aconitum has not diminished. Thus, it ∗ has become a popular research topic to determine how to reduce Corresponding author. the toxicity and retain the therapeutic effect of the genus Aconi- E-mail: [email protected] (X. Fu). 1 These authors contributed equally to this manuscript. tum. http://dx.doi.org/10.1016/j.bjp.2016.03.004 0102-695X/© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Y. Zhang et al. / Revista Brasileira de Farmacognosia 26 (2016) 438–445 439 “Paozhi”, a traditional Chinese medicine (TCM) herbal cages, in moderate humidity (50% ± 5%), at constant temperature ◦ processing methods might exert much influence on the con- (22 ± 1 C) in a 12-h light-dark cycle room. All animals had free version or transformation of plant contents and especially access to food and water during the experimental period. The secondary plant metabolites to change the toxicity or effect of experimental protocol was approved by the Ethics Committee of herbs. To meet the safety of clinical use, the genus Aconitum Ningxia Medical University (Ningxia, China, Ethics approval: 2013- are applied after careful processing (usually soaking, steaming, 146). boiling, baking and processing with Chebulae Fructus decoction) in TCM (Wu et al., 1990; Kim et al., 2012), which could greatly Subchronic oral toxicity study reduce the toxicity of the drug by decomposing the DDA to the less toxic monoester diterpene alkaloids (MDA). The content of DDA A subchronic toxicity test was performed following the reported becomes relatively lower after the application of these traditional technique (Zhang and Du, 2012). Rats of both sexes were ran- processing techniques, which means that the toxicity of the drug domly divided into four groups: control groups and three treatment drops remarkably (Singhuber et al., 2009). groups (n = 40; 20 males and 20 females). The LD50 of rats for a Aconitum flavum Hand.-Mazz., Ranunculaceae, belongs to the period of 14 days was 18.2 g/kg (this value was calculated by the genus Aconitum and is widely used in western China (Shanxi, Bliss method), meaning that the doses of 3.03, 1.52 and 0.76 g/kg Ningxia, Gansu, Inner Mongolia, Qinghai and Tibet). It has been used were selected in this subchronic toxicity study. The BA extract was for the treatment of rheumatism, traumatic injury and toothache in suspended in water with 0.5% (w/v) sodium carboxyl methyl cellu- folk and clinical medicine (Wang et al., 2008). Water decoction and lose (Na-CMC) and administered orally on a daily basis for 90 days, powders of A. flavum were used in traditional medicine for internal while the control group received only 0.5% sodium carboxyl methyl and external use. cellulose (Na-CMC). Twenty rats (ten males and ten females) of each This herb is allowed to be sold on the herbal commercial market group were sacrificed at the end of the 90 days. The remaining rats by the government, although it has notable toxicity in oral applica- in each group were continuously observed in the next 14 days and tion. Like other herbs of the genus Aconitum, the main chemicals sacrificed at the end. The extract was freshly prepared with vehicle of A. flavum are diester diterpene alkaloids. Thus, A. flavum has on a daily basis. strong toxicity. Our laboratory used a variety of traditional and The rats were weighed and visually observed for mortality, modern processing methods to process A. flavum and studied the behavioral patterns (salivation, coat, lethargy, and sleep), changes oral acute toxicity, anti-inflammatory and anti-nociceptive activ- in physical appearance, injury, pain and signs of illness were con- ity of differently processed products. The research showed that the ducted once daily during that period by one researcher. At the end ethanol extract of baked A. flavum reduced its acute toxicity (LD50 of the experiment, blood samples were collected from all animals 22 g/kg) on the premise of maintaining anti-inflammatory and anti- via abdominal aorta puncture for biochemical and hematological nociceptive activities compared to the toxicity of A. flavum (LD50 analyses. The organs were excised, weighed, and examined macro- 1.02 g/kg) in mice (Fu et al., 2013). This study laid a solid founda- scopically. The relative organ weight was calculated. Principal vital tion for the safety of A. flavum applications, but the evidence of organs (liver, kidney, lung, heart and spleen) were preserved in risk from long-term use is unknown. In order to ensure the clinical a fixation medium of 10% buffered formalin for histopathological safety of baked A. flavum (BA), a study of its long-term toxicity is study. necessary. Clinical biochemistry analysis Materials and methods The blood samples were centrifuged for 15 min to collect the serum. Serum was analyzed for alanine aminotransferase (ALT), Preparation of ethanol root extract of baked Aconitum flavum aspartate aminotransferase (AST), albumin (ALB), alkaline phos- phatase (ALP), total protein (TP), triacylglycerol (TG), glucose (GLU), The roots of Aconitum flavum Hand.-Mazz., Ranunculaceae, were + creatinine (CREA), urea, cholesterol (CHO), sodium (Na ), potas- purchased from an Herbal Medicinal Materials Company of the + − sium (K ), chloride (Cl ). Mingde Pharmaceutical Co., Ltd. (Ningxia, China). The plant grows in the Liupan mountains, Longde, Guyuan, and Ningxia, in China. The roots were harvested from August to September. The plant Hematology analysis samples were authenticated by Lin Dong in the Pharmacognosy Department, College of Pharmacy, Ningxia Medical University, and The blood samples that were collected complete blood count a voucher specimen was deposited in the same unit (Herbarium using the automatic hematology system to evaluate red blood cell number: 20130924).