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Research Article Chimonanthus Nitens Var. Salicifolius Aqueous Extract Protects Against 5-Fluorouracil Induced Gastrointestinal Mucositis in a Mouse Model

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Research Article Chimonanthus Nitens Var. Salicifolius Aqueous Extract Protects Against 5-Fluorouracil Induced Gastrointestinal Mucositis in a Mouse Model Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 789263, 12 pages http://dx.doi.org/10.1155/2013/789263 Research Article Chimonanthus nitens var. salicifolius Aqueous Extract Protects against 5-Fluorouracil Induced Gastrointestinal Mucositis in a Mouse Model Zhenze Liu,1,2 Jun Xi,3 Sven Schröder,4 Weigang Wang,3 Tianpei Xie,5 Zhugang Wang,3 Shisan Bao,2,6 and Jian Fei1,2,3 1 School of Life Science and Technology, Tongji University, Shanghai 200092, China 2 The Sino-Australia Joint Laboratory, Lishui Institute of Traditional Chinese Medicine, Tongji University, Lishui 323000, China 3 Shanghai Research Centre for Model Organisms, Shanghai 201203, China 4 HanseMerkur Centre for Traditional Chinese Medicine at the University Medical Centre Hamburg-Eppendorf, Haus Ost 55, UKE Campus, Martinistraße 52, 20246 Hamburg, Germany 5 Shanghai Standard Biotech Co., Ltd., Shanghai 201203, China 6 Discipline of Pathology, Bosch Institute and School of Medical Sciences, University of Sydney, NSW 2006, Australia Correspondence should be addressed to Shisan Bao; [email protected] and Jian Fei; [email protected] Received 19 June 2013; Revised 8 September 2013; Accepted 16 September 2013 Academic Editor: Lorenzo Cohen Copyright © 2013 Zhenze Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gastrointestinal mucositis is a major side effect of chemotherapy, leading to life quality reduction in patients and interrupting the therapy of cancer. Chimonanthus nitens var. salicifolius (CS) is a traditional Chinese herb for enteral disease. Considering the protective effect of CS on intestine, we hypothesize that the aqueous extract of CS could be benefcial to gastrointestinal mucositis. To verify this, a mouse mucositis model was induced by 5-Fluorouracil (5-Fu). Male Balb/C mice were treated with CS aqueous extract (5, 10, and 20 g/kg) or loperamide (0.2 mg/kg) intragastrically for 11 days, and the severity of mucositis was evaluated. Furthermore, the chemical compounds of CS aqueous extract were also analysed by high-performance liquid chromatography (HPLC). Our results demonstrated that CS aqueous extract improved mice body weight, diarrhoea, and faecal blood, maintained the liver function and intestinal length, alleviated villus shortening, and suppressed the apoptosis and inflammation in small intestine. We concluded that CS could protect mice against 5-Fu induced mucositis by inhibiting apoptosis and inflammation, and this protective effect might be associated with the 3 flavonoids (rutin, quercetin, and kaempferol) identified in CS aqueous extract. 1. Introduction for treating common cold, influenza, dyspepsia, gastritis, enteral disease, and insects bites in part of China [3, 4]. Dry Chimonanthus nitens var. salicifolius (also named Chimonan- leaves of CS are also boiled as tea by the indigenous people, thus salicifolius, abbreviation CS) belongs to Calycanthaceae andtheyarethoughttohaveaprotectiveeffectongastroin- family and is considered as a unique plant species in China. testinal track [4]. This plant is generally distributed in the mountain areas of Most chemotherapeutic agents target rapidly dividing southeast China and it is a semievergreen shrub with solitary cells indiscriminately, either malignant or healthy, including and small yellowish flowers. The leaf blade of CS is linear- intestinal basal stem cells in crypts [5, 6]. Mucositis is the lanceolate or oblong-lanceolate and has bristle on veins and resultant damage to gut induced by chemotherapy, and this margin [1]. It is reported that leaves of CS are rich in protein, side effect limits its clinical applications7 [ ]. Patients with crude fat, fibers, minerals, vitamin2 B ,andC[2]. Tradition- gastrointestinal mucositis usually show abnormal pain, mal- ally, the leaves and branchlets of CS are considered as herbs absorption, and a range of gastrointestinal symptoms, like 2 Evidence-Based Complementary and Alternative Medicine vomiting, diarrhoea, ulceration, and bleeding [8–11]. Gas- water into 3 different concentrations (0.25, 0.5, and 1 g/mL of trointestinal mucositis has been reported to occur in 40% of crude drug). Mice were administrated intragastrically with patients receiving standard dose and almost in all patients CS aqueous extract twice a day by 10 mL/kg body weight receivinghighdosechemotherapy[12], thus represents a (equivalent to 5, 10, and 20 g/kg). significant clinical and economic burden in oncology [13]. The chemotherapy induced gastrointestinal mucositis 2.3. High Performance Liquid Chromatography (HPLC) Anal- involves not only the direct injury to intestinal basal stem ysis. Main compounds in CS aqueous extract (0.1 g/mL) were cells, but also a consequence of complex biological events, analysed using HPLC method. The apparatus system (Agilent such as reactive oxygen species (ROS) generation, immune Technologies, Germany) was equipped with a binary pump, cells infiltration, and proinflammatory cytokines oversecre- a diode array detector, and an autosampler. An extend-C18 tion [14]. Therefore, drugs with antiapoptosis, antioxidative, column (150 nm ∗ 4.6 mm, Agilent, Germany) was used for ∘ or anti-inflammatory properties were thought to benefit separation and the temperature was maintained at 25 C. mucositis patients [15]. Over these few decades, a variety of 10 Lofsamplewasinjectedintothesystemandtheflow strategies have been developed for mucositis cure; however, rate was set at 1 mL/min. The mobile phase consisted of few of these approaches have shown sufficient safety and methanol (B) and 0.1% phosphoric acid in water (A). The efficacy16 [ ]. With a growing interest in nature medicine, linear gradient elution started at 30% (B), changed to 40% herb is being adopted as a potential strategy for treating (B) after 15 min, and changed to 60% after 35 min. UV-Vis this adverse effect of chemotherapy [17, 18]. More recently, absorption spectra were monitored by diode array detector CShasbeenprovedtohaveanantidiarrhoeaeffectin at 265 nm. For analysis, the standard substances of rutin, mice [19]. However, its function in chemotherapy induced quercetin, and kaempferol were used (Sigma-Aldrich, USA). gastrointestinal mucositis is unknown. Compounds in aqueous extract of CS were quantified on In the current study, we utilized 5-fluorouracil (5-Fu), a the basis of their peak areas and calibration curves of the commonly used chemotherapeutic agent, to induce intestinal corresponding standard substance. injury in Balb/C mice, which mimics human chemotherapy induced mucositis. Three different doses of CS aqueous 2.4. Mucositis Inducement and Treatment. 5-Fu was pur- extract were administered to mice for 11 days, and their chased from XuDong HaiPu Pharmaceutical Co., Ltd (Shang- possible effect was examined by clinical symptoms, blood hai, China). Loperamide (from Xi’an Jangsen Pharmaceutical biochemical tests, intestinal histopathology, and expression Ltd, China) was employed to compare the efficacy with CS analysis of genes relevant to apoptosis, proliferation, and aqueous extract. Mucositis in mice was induced by 3-day 5- inflammation. In addition, loperamide, which is used for FU administration (50 mg/kg, intraperitoneally) on days 5, 6, managing chemotherapy induced mild-moderate diarrhoea and7,whilethecontrolsweretreatedwithsaline.Loperamide [20, 21], was chosen as a symptomatic drug to compare the (0.1 mg/kg) or CS aqueous extract (2.5, 5, and 10 g/kg) was efficacy with CS aqueous extract. Such data may provide use- given intragastrically from day 1 to 11, twice daily at 9:00 a.m. ful information for the prevention or treatment of mucositis and9:00p.m.Thecontroland5-Fugroupsweretreatedwith in patients undergoing chemotherapy. vehicle only (normal drinking water). 2. Materials and Methods 2.5. Tissue Collection and Mucositis Assessment. Disease severity was assessed by monitoring body weight and scoring 2.1. Animals. Adult (9 weeks) male BALB/c mice, obtained for diarrhoea and faecal blood. Diarrhoea score was based from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, on the consistency of stools, using the modified parameters China), were housed under specific pathogen-free environ- as described before [15, 22]:0,normal;1,slightlywet;2, ment with food and water ad libitum.Theenvironment ∘ moderatewet;3,loose;4,waterystool.Thefaecalbloodwas was maintained at 22 Cwitha12hlight/darkcycle.Animal measured by a commercial testing paper (BASO diagnostics welfare and experimental procedures were carried out strictly Inc., China) with the following scores: 0, normal; 1, slight inaccordancewiththeguidanceforcareanduseoflaboratory bleeding; 2, moderate bleeding; 3, severe bleeding; and 4, animals (National Research Council of USA, 1996) and visible bleeding. approved by Institutional Animal Care and Use Committee Serum was collected on day 11 for blood biochemical of Shanghai Research Centre for Model Organisms. assays. The small and large intestines were harvested and measured for their lengths immediately following mice sacri- 2.2. Preparation of CS Aqueous Extract. CS was obtained fice. Then small intestines were flushed with cold phosphate from Tongji University, Lishui Institute of Traditional Chinese bufferedsaline.Partofsmallintestinewasfixedin4% Medicine (Lishui, Zhejiang, China). In brief, fresh leaves of paraformaldehyde overnight
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