a 90day subchronic oral toxicity study of triterpeneenriched extract from alismatis rhizoma in rats：泽泻浓缩90天大鼠亚慢性经口毒性研究三萜提取物.pdf

a 90-day subchronic oral toxicity study of triterpene-enriched extract from alismatis rhizoma in rats ming-qing huang a,1, wen xua,1, shui-shen wua, jin-jian lub,c, xiu-ping chenb acollege of pharmacy, fujian university of traditional chinese medicine, fuzhou 350108, china bstate key laboratory of quality research in chinese medicine, institute of chinese medical sciences, university of macau, av. padre toms pereira taipa, macao, china ccollege of life sciences, zhejiang chinese medical university, hangzhou 310053, china a r t i c l ei n f o article history: received 20 january 2013 accepted 7 may 2013 available online 16 may 2013 keywords: triterpenes alismatis rhizoma water plantain root rats subchronic toxicity a b s t r a c t alismatis rhizoma has been used in east asia as a traditional treatment for various illnesses and symp- toms, and the presence of protostane-type triterpenes has been claimed to provide health benefi ts. to investigate the subchronic toxicity of triterpene-enriched extract from alismatis rhizoma (tear), a 90- day oral toxicity study was conducted in rats. spraguedawley rats were randomly divided into four groups (10 rats/sex/group) and received doses of 0, 360, 720, and 1440 mg/kg/d of tear for 90 days. daily clinical observations as well as weekly measurement of body weight and food consumption were con- ducted. blood samples were obtained on day 91 to measure changes in hematology and biochemistry. urine samples were collected on days 0 and 91 for urinalysis. at necropsy, selected organs were weighed and recorded, and histological examination was performed. no mortality or obvious treatment-related clinical signs, hematology, urinalysis parameters, and macroscopic or microscopic examinations were observed. differences in weight gain, food consumption, biochemistry, and relative organ weight between the treated group and the control group were not considered treatment-related. on the basis of these fi ndings, the no-observed-adverse-effect level for tear was 1440 mg/kg/d in both sexes. ? 2013 elsevier ltd. all rights reserved. 1. introduction alismatis rhizoma (water plantain root, also known as ze xie in chinese), the dried rhizomes of alisma orientalis (sam.) juzep., is a well-known east asian medicine and has been commonly used forthetreatmentofvariousdiseases,includinghyperlipidemia,dia- betes, hypertension, and urological diseases (imai et al., 1970; lee et al., 2001; li and qu, 2012). it is also widely used as an important ingredient in a number of traditional chinese medicine (tcm) for- mulations, such as long dan xie gan wan and liu wei di huang wan (china, 2010; xie et al., 2012). most benefi cial effects of alismatis rhizoma are attributed to the presence of terpenoids (hur et al., 2007). these terpenoids are mainly comprised of protos- tane-type triterpenes, guaiane-type sesquiterpenes, and kaurane- type diterpenes (fukuyama et al., 1988; liu et al., 2010; murata et al., 1970; penget al., 2003; yoshikawa et al., 1994b). in particular, protostane-type triterpenes such as alisol a, alisol b, and alisol c, exist only in alisma plants and are considered chemotaxonomic markers of the genus (liu et al., 2010). these triterpenes have particularly been the focus of research in recent years because of their relatively high levels of alismatis rhizoma and various phar- macological activities (fong et al., 2007; lee et al., 2010; li and qu, 2012; wang et al., 2004; zhao et al., 2008). alisol a, alisol a 24-acetate, alisolb 23-acetate, andalisolc 23-acetate exhibitedsig- nifi cant hypolipidemic effects in high-fat-fed rats (imai et al., 1970; murata et al., 1970). these alisol derivatives inhibited an allergic re- sponse and experimental atopic dermatitis (kubo et al., 1997; lee et al., 2012). in addition, alisol b and alisol b 23-acetate induced endoplasmic reticulum stress, autophagy, and apoptosis in several cancer cell lines (huang et al., 2006; law et al., 2010; xu et al., 2009). most tcm are lack of systematic safety evaluation using mod- ern technology due to the academic and historical reasons. in re- centyears,withtheprocessofinternationalizationand modernization of tcm, its safety has aroused peoples attention. experts both in china and aboard have realized the importance and necessity to re-evaluate the safety of tcm using modern tech- nology. alismatis rhizoma has been generally considered safe. however, overdosage may induce hepatotoxicity and/or nephro- toxicity (xie et al., 2012). the safety of its water-soluble fraction has been confi rmed by some recent studies (duan et al., 2004; zhu et al., 2007). the safety of the lipid-soluble fraction of alisma- tis rhizoma containing various triterpenes (murata et al., 1970) has not been scientifi cally studied. therefore, a conscientious and careful safety evaluation on the extract is necessary for the 0278-6915/$ - see front matter ? 2013 elsevier ltd. all rights reserved. /10.1016/j.fct.2013.05.009 corresponding author. address: college of pharmacy, fujian university of traditional chinese medicine, no. 1 huatuo road, shangjie university town, fuzhou 350108, china. tel.: +86 591 22861043; fax: +86 591 22861135. e-mail address: labtcm (s.-s. wu). 1 these authors contributed equally to this work. food and chemical toxicology 58 (2013) 318323 contents lists available at sciverse sciencedirect food and chemical toxicology journal homepage: development of new drug and better understanding on the use of the lipid-soluble fraction from alismatis rhizoma. in this study, a triterpene-enriched extract from alismatis rhizoma (tear) was prepared and its safety was evaluated by using a subacute toxicity study design. female and male spraguedawley rats were orally administered tear at doses of 360, 720, or 1440 mg/kg/d for 90 consecutive days. physical changes, hematology, clinical biochem- istry, urinalysis, and histopathological changes were measured to monitor treatment-related adverse effects in rats. 2. materials and methods 2.1. extract preparation and quantifi cation alismatis rhizoma was purchased from chinese herbal medicine company of fuzhou (fuzhou, china) and identifi ed by one of the authors (prof. shui-shen wu). according to the pharmacopoeia of the peoples republic of china (2010), this plant was authenticated as a. orientalis (sam.) juzep. by morphology and thin-layer chromatography, comparing with authenticated reference herb and reference com- pound (alisol b 23-acetate) purchased from the national institute for the control of pharmaceutical and biological products (beijing, china), respectively (data not shown). a voucher specimen (no. cph2011023) was deposited in the herbarium of the college of pharmacy, fujian university of traditional chinese medicine. according to the procedures previously described by our laboratory (qiu et al., 2012) with some modifi cations, the dried rhizomes of alismatis rhizoma were ground into powder (24 mesh). the powder (80 kg) was extracted twice by decoc- tion with 80% ethanol (640 l) for 1 h. the fi ltrate was concentrated to about 400 l under reduced pressure at 65 ?c and then chromatographed on an ab-8 macroretic- ular resin (nankai university chemical co., tianjin, china) column by using deion- ized water (1600 l), 30% ethanol (800 l), 50% ethanol (800 l), and 75% ethanol (3200 l) as eluant. in order to get rid of tannins, the 75% ethanol fraction was concentrated to about 800 l under reduced pressure at 65 ?c and then chromato- graphed on a polyamide resin (6080 mesh, sinopec, hunan, china) column by using deionized water (3200 l) and 70% ethanol (4800 l) as eluant. the 70% ethanol fraction was concentrated in vacuo (65 ?c) and lyophilized using a freeze-drying system. total acquired tear extract was 1.024 kg (yield 1.28%). in order to analyze the total triterpenes in tear, the extract was analyzed by high-performance liquid chromatography (hplc). a dionex lc-20a series hplc sys- tem (dionex, sunnyvale, usa) with a reversed-phase ultimate xb-c18 column (150 mm ? 4.6lm i.d., 5lm) (welch materials, maryland, usa) was used. approx- imately 25 mg of the extract was dissolved in 50 ml methanol and fi ltered through a 0.45l m nylon syringe fi lter. the mobile phase consisted of acetonitrile (a) and methanol solution (5% (v/v), b) (merck, darmstadt, germany) using a linear gradi- ent program of 55% a from 0 min to 10 min, 5565% a in 1040 min, and 65% a in 4060 min. the fl ow rate was 1 ml/min, and the diode array detector was set at 208 nm. 2.2. animals specifi c pathogen-free spraguedawley rats were received from the center of laboratory animal science of guangdong province (foshan, china) and divided into two groups consisting of 50 male and 50 female rats. mean body weights mean standarddeviation(sd)uponreceiptwere160.2 g 12.6 gand 148.3 g 9.8 g for males and females, respectively. all animals were examined for clinical signs of ill health upon receipt and observed within 5 days of arrival. the rats were housed individually in stainless steel cages (w 215 ? l 355 ? h 200 mm) under standard environmental conditions (23 ?c 2 ?c, 4060% relative humidity, and 12 h light/12 h dark cycle) and allowed free access to tap water and food. each cage was distinguished by a label with the study number and animal id. quality analysis and microbiological assessment of the tap water was performed once per week and once per month, respectively. the rats were fed with a standard rodent food (containing protein, 230 g/kg; fat, 35 g/kg; fi ber, 50 g/kg; carbohydrate, 600 g/kg; and water) obtained from the center of laboratory animal science of guangdong province (foshan, china). the experimental protocols for this study were approved by the institutional animal care and use committee and the ani- mals were kept according to the institutional ethical guideline at fujian university of traditional chinese medicine. 2.3. experimental design a total of 80 healthy male and female rats were selected and randomly divided into four groups after a 5-day acclimatization. one group served as the control groups and was given a 0.5% sodium carboxyl methyl cellulose (cmcna) solution (containing 10% peg 400) as vehicle by gavage. the three remaining groups were given three descending doses of tear in 0.5% cmcna solution (containing 10% peg 400) for 90 consecutive days. the selection of the dosages (1440, 720, and 360 mg/kg/d) was according to our previous data on its acute oral toxicity. in our previous acute oral toxicity study in rats, the doses of 1440 and 3600 mg/kg tear did not produce mortality or signifi cant changes in the general behavior and gross appearance of the internal organs except for the decrease of daily food consump- tion. therefore, the dose of 1440 mg/kg/d was selected as the highest dose level, and 720 mg/kg/d and 360 mg/kg/d were chosen as another two levels in our exper- iment. the gavage volume was 2 ml/100 g body weight. the body weights were measured weekly, and the gavage volume was adjusted based on the weekly body weight of rats. body weight, food consumption, and clinical signs of toxicity were recorded throughout the experiment. urine samples were collected at day 0 and day 91 for analysis. following fasting for 1618 h, the rats were anesthetized with 3% sodium pentobarbital solution, and blood was collected in 10% ethylenediamine- tetraacetic acid (edta) tubes for hematology and non-oxalate tubes for separation of serum on days 91. this study was in accordance with the guidelines for method- ologies on long-term toxicity research of traditional chinese medicine and natural medicine (2005) approved by the state food and drug administration of china. these regulations conform to the oecd guidelines for testing of chemicals, sec- tion 408 (1998). 2.4. signs of toxicity and mortality signs of toxicity, including salivation, lacrimation, piloerection, diarrhea, dysp- nea, tremor, convulsion, paralysis, and death were observed once daily throughout the period of exposure. 2.5. hematological parameters blood collected in 10% edta was analyzed for hemoglobin (hb), mean cell vol- ume (mcv), red blood cell count (rbc), hematocrit (hct), white blood cell count (wbc), reticulocyte count (rc), differential leukocyte count, and platelet count through an automated cell counter (mek-7222 k, tokyo, japan). prothrombin time (pt) was determined using an automated hematocoagulation parameter (mc-4000 plus, karlsruhe, germany). 2.6. biochemical parameters aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), glucose, creatinine, triglycerides (tg), total cholesterol (tc), to- tal protein, albumin, total bilirubin, blood urea nitrogen (bun), creatine phosphoki- nase (cpk), sodium, potassium, and chloride in serum were measured with a fully automated biochemical analyzer (echo, rome, italy). 2.7. urine examination on days 0 and 91, urine samples of individual rats after fasting for 1618 h were collected for the qualitative analysis of ph, specifi c gravity, glucose, protein, biliru- bin, urobilinogen, ketone, presence of blood, and wbc by using bayer multistix 10 sg (siemens, munich, germany). 2.8. gross pathology and organ weights all rats were humanely sacrifi ced at the end of the test, and a complete nec- ropsy was performed. the criteria of gross pathological examination were based on the position, shape, size, color, and consistency of the organs. some organs, including adrenal glands, brain, epididymis, heart, liver, spleen, lung, kidneys, ova- ries, testes, thymus, and uterus, were weighed individually, and the organ body weight ratio was calculated. 2.9. histopathological studies in addition to the above-mentioned organs, tissues such as aorta, cecum, colon, cervix, duodenum, ileum, jejunum, rectum, esophagus, eyes, lacrimal gland, lymph node, mammary gland, pancreas, thyroids, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord, femur with bone marrow, sternum with bone marrow, trachea, and urinary bladder, were fi xed in 10% formal saline solution. after routine processing, a paraffi n section of each tissue was cut at 5lm thicknesses and stained with hematoxylin and eosin for microscopic examination. 2.10. statistical analysis all statistical analyses were performed using spss version 12.0 (spss, chicago, usa) with one-way analysis of variance. the homogeneity of variance was exam- ined by bartletts test. when variance between the groups was confi rmed to be homogenous, the difference between the control group and each treated group was examined by duncans multiple range test. when variance between the groups was not homogenous, the normal distribution of the date was determined by kolmogorovsmirnov test. when a non-normal distribution was found, variance between the groups was examined by kruskalwallis one-way analysis. when a m.-q. huang et al./food and chemical toxicology 58 (2013) 318323319 normal distribution was found, the difference between the control group and each treated group could be examined by mannwhitney u-test. p values less than 0.05 were considered signifi cant. all data were presented as mean sd. 3. results 3.1. determination of triterpenes in tear table 1 showed the triterpene composition of tear used in this study. the amount of total triterpenoids, containing 12 triterpenes, in tear was 721.41 mg/g. alisol a, alisol a 24-acetate, alisol b 23- acetate and alisol c 23-acetate were the major triterpenes, com- prising 61.33% of total triterpenes in tear. other major triterpenes including 16-di-hydroxy-alisol a 23-acetate, 16-di-hydroxy-alisol a 24-acetate, alisol c, alisol l, alisol a 23-acetate, alisol g, alisol b, and 11-deoxyalisol b were also identifi ed (fig. 1). 3.2. mortality and clinical signs neither treatment-related mortality nor obvious clinical signs, including hair loss, scabbing, soft or mucoid feces, decreased defe- cation or feces smaller than normal, wet yellow material in the urogenital area or vocalization upon handling, were found in any of the treated groups throughout the experimental period. animals from all treatment groups appeared healthy at the conclusion of the study period. 3.3. body weights and food consumption figs. 2 and 3 showed the mean body weights of female and male rats. compared with the control group, female rats of 360 mg/kg/d treatment group had signifi cantly higher weight gain from week 3 to week 5, and male rats of the same dose at week 13 also had higher body weight gain. no other signifi cant differences were indicated between the treatment groups and the control group in both genders throughout the experimental period. there were no signifi cant differences in daily food consumption for all male treatment groups compared with the control group (data not shown). females of 360 mg/kg/d treatment group had signifi cantly higher food consumption than that of the control group at weeks 5 (21.7 1.6 vs. 18.6 1.1 g) and 7 (20.7 0.3 vs. 18.2 0.2 g). females of 720 mg/kg/d treatment group at week 8 table 1 triterpene contents in triterpene-enriched extract from alismatis rhizoma (tear). compoundcontent (mg/g) total triterpene (sum of below mentioned 12 triterpenes)721.41 16-di-hydroxy-alisol a 23-acetate7.55 0.44 16-di-hydroxy-alisol a 24-acetate15.09 0.89 alisol c40.07 0.62 alisol c 23-acetate137.36 0.34 alisol l51.46 0.76 alisol a82.62 1.67 alisol a 23-acetate31.06 0.16 alisol a 24-acetate68.33 0.35 alisol g47.27 0.40 alisol b54.11 0.53 alisol b 23-acetate154.15 0.49 11-deoxyalisol b32.33 1.14 01020304050 min 0 100 200 300 mv 12 3 4 5 6 7 8 9 10 11 12 01020304050 min 0 100 200 300 mv 12 3 4 5 6 7 8 9 10 11 12 (a) (b) fig. 1. hplc chromatograms of triterpene-enriched extract from alismatis rhizoma (tear) analysis. (a) tear; (b) standard mixture of 16-di-hydroxy-alisol a 23-acetate (peak 1), 16-di-hydroxy-a