preclinical safety of the root exract of polygala tenuifolia

Research ArticlePreclinical Safety of the Root Extract of Polygala tenuifoliaWilldenow in Sprague-Dawley Rats and Beagle Dogs

Ki Young Shin12 Beom Young Won1 Hyun Jee Ha1 Yeo Sang Yun1 and Hyung Gun Lee1

1 Research amp Development Center Braintropia Co Ltd Anyang-si Gyeonggi-do 431-716 Republic of Korea2Department of Microbiology College of Natural Science Dankook University Cheonan-si Chungnam 330-714 Republic of Korea

Correspondence should be addressed to Ki Young Shin newsky73braintropiacom

Received 30 July 2014 Revised 13 October 2014 Accepted 13 October 2014 Published 5 November 2014

Academic Editor Khalid Rahman

Copyright copy 2014 Ki Young Shin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The root of Polygala tenuifolia Willdenow has been used for the treatment of insomnia depression and amnesia However thetoxicological properties of the herb have been overlooked because it has been used for a long time for various purposes In thisstudy we evaluated the preclinical safety of the root extract in rats and beagle dogs First the acute oral toxicity was tested inboth rats and dogs In the rats only one female of 2 gkg died but no treatment-related death or clinical and gross findings wereobserved after the administration No toxicological changes or mortalities related to the test substance were also observed afterthe administration in the dogs Although vomiting discoloration or hemorrhage was found in some dogs there were no seriousabnormalities Second the subchronic toxicity was investigated in the rats Two animals were found dead in the female group of1000mgkgday but there were no abnormal findings associated with the test substance There also were no adverse effects on theclinical signs body weight and hematological and biochemical findingsTherefore our results showed that the acute or subchronictoxicity of the root extract of Polygala tenuifoliamight not be toxic to rats and dogs

1 Introduction

Traditional oriental herbal prescriptions have regained popu-larity over the past decadeThey are used widely for the treat-ment and prevention of various diseases [1 2] because theirlong history of clinical application and natural origin seem toguarantee that the prescriptions will be effective and nontoxic[3] Each plant used in such herbal prescriptions can provideopportunities for the development of herbal food productsdietary supplements and functional foods [4] Howeverthe toxicological aspects of medicinal herbs or naturallyoccurring functional foods have been neglected due to theirlong history of use It has been demonstrated that medicinalplants may have undesirable properties [5] because someindividuals taking herbal medicines have reported certainside effects Therefore the use of any plant for medicinalpurposes by no means guarantees the safety of such a plantThis fact raises concerns about the potential toxic effectsresulting from short-term and long-term use of such medic-inal plants Data from acute and subchronic toxicity studieson medicinal plants or naturally occurring functional foods

should be obtained to increase confidence in the safety oftheir use particularly in the development of pharmaceuticals[6] Polygala tenuifolia Willdenow is a perennial herbaceousplant distributed widely in China and Korea In Asia it is awell-known traditional medicine for the treatment of phlegmand detumescence Traditional medicines are prepared fromthe roots of P tenuifolia because of its expectorant tonictranquilizing and antipsychotic properties [7ndash9] In particu-lar the herb is used against insomnia neurasthenia amnesiadepression anxiety-related palpitations restlessness disori-entation dementia and memory failure [10 11] Previouslywe reported that the root extract of P tenuifolia was shownto enhance memory and cognitive function in two animalmodels and two human models [12ndash15] The extract has beenshown to contain C-glycosides triterpene saponins sucroseesters and oligosaccharide esters [16 17] It also containsvarious substances such as tenuigenin tenuifolin DISS (361015840-disinapoyl sucrose) and TMCA (345-trimethoxycinnamicacid) which have been shown to have proliferative andprotective effects on hippocampal neurons [18 19] Several

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 570134 15 pageshttpdxdoiorg1011552014570134

2 Evidence-Based Complementary and Alternative Medicine

researchers have demonstrated the effects of P tenuifolia butinformation on its safety is lacking Therefore systematicevaluation of the safety of the root extract of this herbis necessary for the development of new foods or drugsIn this study an alcohol extract from the dried root of Ptenuifolia was prepared and its safety was evaluated usingan acute oral toxicity test and a subchronic oral toxicity testin Sprague-Dawley rats and beagle dogs in accordance withtwo standards the Korea Food and Drug AdministrationNotification numbers 2005-60 and 2005-79

2 Materials and Methods21 Preparation of the Root Extract of Polygala tenuifolia Thedried root extract of Polygala tenuifolia (500 g) was refluxedwith 75 ethanol for 4 hr in a boiling water bath Thisprocedure was repeated twice and the ethanol solution wasconcentrated under a vacuum The resulting concentratedethanol fraction (125 g) of the plant root was used for thisstudy [12]

22 Experimental Animals and Animal Husbandry Six-week-old specific pathogen-free Sprague-Dawley (HSD SD)rats were used Six animals of each sex for the acute oral toxi-city study and 40 animals of each sex for the subchronic oraltoxicity study were received from Koatech Co (Pyeongtaek-si Gyeonggi-do Korea) Ltd For the acute oral toxicity studysix beagle dogs of each sex at 6 months of age were obtainedfromtheHanlim Laboratory Animals Company (Hwaseong-si Gyeonggi-do Korea)The animal room was maintained ata temperature of 23 plusmn 3∘C with a relative humidity of 55 plusmn15 air ventilation of 10ndash20 timesh and ambient light (150ndash300 Lux) controlled to produce a 12 h lightdark cycle Ani-mals were given irradiation-sterilized pellet feed and groundwater disinfected by an ultraviolet sterilizer and ultrafilteredad libitumThe dogs were reared in stainless steel cages (8000times 900 times 750mm) and identification cards showing theirstudy number and animal number were attached to the cages

23 Study Design Overview This study was conducted incompliance with the Good Laboratory Practice (GLP) andTest Guidelines of the Organization for Economic Coopera-tion and Development [20 21] and the Korea Food and DrugAdministration [22 23] at the GLP Institute approved by theKFDA (Korea Food and Drug Administration) The studyprotocol was approved by the Institutional Animal Care andUse Committee of the institute which is accredited by theAssociation for Assessment and Accreditation of LaboratoryAnimal Care International 2010

231 Acute Oral Toxicity Study in Rats After a 7-day quaran-tine and acclimatization period ten healthy animals of eachgender at 6 weeks of age were used The study included twogroups a control group given no treatment and an exper-imental group given 2000mgkg based on a preliminarystudy (data not shown) Each group consisted of five ratsof each sex and body weight ranges at dosing were 16161ndash16838 g for males and 14498ndash15817 g for females The testsubstance was administered orally to each group Thereafter

clinical signs and body weight were observed throughout the15-day experimental period and gross findingswere observedon the scheduled necropsy day This study was carriedout in accordance with the standard operating proceduresof SNUH-GLP (Non-Clinical Research Center BiomedicalResearch Institute Seoul National University Hospital)

232 Acute Oral Toxicity Study in Beagle Dogs Six maleand six female dogs were divided into three groups of fouranimals then two dogs of each gender were allocated Thestudy included three groups a control group given no treat-ment and experimental groups exposed to 1000mgkg and2000mgkg The body weight ranges at dosing were 7269ndash8785 kg for males and 6531ndash7449 kg for females The testsubstance was administered orally to each group Thereafterclinical signs mortality and body weight were observedthroughout the 15-day experimental period and gross find-ings were observed on the scheduled necropsy dayThis studywas carried out in accordance with the standard operatingprocedures of Preclinical ResearchCenter ChemOn (YonginGyeonggi-do Korea) Inc

233 Subchronic Oral Toxicity Study in Rats After a 7-dayquarantine and acclimatization period forty healthy animalsof each gender at 6 weeks of age were used A dose of1000mgkgday was set as the high dose considering theresults of a preliminary 1-week subchronic oral toxicity study(data not shown) and the characteristics of the test substanceDoses of 600 and 360mgkgday were set as the middle andlow doses respectively using a common ratio of 167 Thevehicle-treated group was administrated with sterile waterfor injection only Each group consisted of 10 rats of eachsex and 10 rats of each sex and group were killed after a 90-day treatment period Body weight ranges at the beginningof dosing were 16956ndash19056 g for males and 12530ndash14200 gfor females The test substance was administered repeatedlyby the oral route to each group for 90 days The followingitemswere examined during the experimental period clinicalsigns body weight food and water consumption ophthalmicexamination urinalysis hematology blood clotting timeserum biochemistry necropsy findings organ weight andhistopathologyThis studywas carried out in accordance withthe standard operating procedures of Preclinical ResearchCenter ChemOn Inc

24 Observation and Examination Items of the Acute OralToxicity Study in Rats

241 Clinical Signs In the rat studies clinical signs andmortality were observed continuously for the first 1 h afteradministration and every hour for 6 h Each animal wasobserved daily throughout the entire 15-day experimentalperiod Abnormal type and severity of signs as well asthe observation day and time were recorded The day ofadministration was set as day 1

242 Body Weight Each rat was measured on days 1 3 7and 14 using an electronic balance (Sartorius Co GottingenLower Saxony Germany)

Evidence-Based Complementary and Alternative Medicine 3

243 Necropsy On the scheduled necropsy day (day 15) allsurviving animals were euthanized by exsanguination fromthe abdominal aorta and abdominal vena cava under a CO

2

gas overdose and examined grossly for abnormalities of thebody surface and internal organs

25 Observation and Examination Items of the Acute OralToxicity Study in Dogs

251 Clinical Signs In the dog studies the clinical signsand mortalities were checked every hour for 6 hours afteradministration Each animal was observed daily throughoutthe entire 15-day experimental period Abnormal type andseverity of signs as well as the observation day and time wererecorded The day of administration was set as day 1

252 Body Weight All dogs were individually weighedbefore administration and on days 1 3 7 and 14 after theadministration

253 Necropsy In the dog studies the animals were anes-thetized with ketamine (Yuhan Yanghaeng) and rompun(Bayer Korea) and euthanized by exsanguinations from theaxillary artery and vein All organs in the thoracic andabdominal cavities were observed grossly and the grossfindings were recorded

26 Observation and Examination Items of the SubchronicOral Toxicity Study in Rats

261 Clinical Signs All animals were observed daily forclinical signs andmortalityThe type date of occurrence andthe severity of the signswere recorded individuallyThe initialday of administration was set as day 1

262 Body Weight Animals were weighed on the initial dayof administration once per week during the experimentalperiod and on the day of the necropsy The body weight atthe necropsy was measured after an overnight fast

263 Food and Water Consumption Food and water con-sumption were measured on the initial day of administrationand then once per week during the experimental period Theamounts of food and water were measured before they weresupplied to each cage and the food and water remaining thenext dayweremeasured to calculate the difference whichwasregarded as daily food and water consumption (gratday)

264 Ophthalmic Examination In the last week of obser-vation a mydriatic (Ocuhomapin Lot number 013118 SamilPharmCo Korea) was dropped into both eyes of each animalto facilitatemydriasis after observing the external appearanceof the eyes of five males and five females per group Theanterior parts of the eyes the optic media and the ocularfundus were then observed with an ophthalmoscope and afundus camera (Genesis Kowa Co Tokyo Japan) No abnor-mal signs were observed during the ophthalmic examinationTherefore no eye photographs or further examinations wereperformed

265 Urinalysis Five males and five females per group werehoused inmetabolic cages for urine collection in the last weekof observation and fresh urine samples (about 1mL) werecollected for 3 h and used for urinalysis and urine sedimenttests Additionally the total urine volume was measuredfrom urine samples collected for 24 h Test strips (Multistix10 SG Siemens Washington DC USA) were dipped inabout 03mL of urine and the specific gravity pH proteinglucose ketone bodies occult blood bilirubin urobilinogenand nitrite levels were analyzed with an automatic analyzer(CliniTek 100 Siemens Ames Division Miles LaboratoryUSA) Urine color was observed with the naked eye and theresult was input into the automatic analyzer Approximately07mL of urine was centrifuged (Hanil MF300 Seoul Korea)for 5min The sediment was stained using the Sternheimer-Malbin method (Sternheimer and Malbin 1951) and redblood cells (RBCs) white blood cells (WBCs) epithelial cellsand casts were observed under a microscope

266 Hematology Approximately 1mL of the blood takenduring the necropsywas placed into aCBCbottle (Vacutainer3mL Becton Dickinson Franklin Lakes NJ USA) contain-ing EDTA-2K anticoagulant The following parameters weremeasured with a Coulter counter (ADVIA 2120 SiemensAmes Division Miles Laboratory USA) white blood cellcount red blood cell count hemoglobin hematocrit meancorpuscular volume mean corpuscular hemoglobin meancorpuscular hemoglobin concentration red cell distribu-tion width hemoglobin distribution width platelets meanplatelet volume and WBC differential count (neutrophilslymphocytes monocytes eosinophils basophils and largeunstained cells) 18mL of the blood taken during thenecropsy was dispensed into a microtube containing 02mL32 sodium citrate and plasma was obtained from cen-trifugation (Eppendorf Hamburg Germany) for 10min toassess the blood clotting time The prothrombin time andactivated partial thromboplastin time were measured in sec-onds from plasma using the nephelometric analysis method(Woziwodzki 1970) with a coagulation time analyzer (ACL100 Instrumentation Laboratory Bedford MA USA)

267 SerumBiochemistry More than 2mLof the blood takenduring the necropsy was added to a 5mL Vacutainer tube(IMPROBE Germany) containing a clot activator The bloodwas coagulated by maintaining it at room temperature for15ndash20min and was then centrifuged (3000 rpm 1500 RCFMF300 Hanil Korea) for 10min The following parameterswere measured with a serum biochemistry analyzer (AU400Olympus Tokyo Japan) aspartate aminotransferase alanineaminotransferase alkaline phosphatase creatine phosphoki-nase total bilirubin glucose total cholesterol triglyceridestotal protein albumin albuminglobulin ratio blood ureanitrogen creatinine inorganic phosphorus and calciumions The electrolytes were measured with the electrolyteautoanalyzer (644 Na K Cl Analyzer Ciba-Corning USA)

268 Necropsy and Organ Weight Before the schedulednecropsy all surviving animals were fasted overnight (for


16ndash20 h) and euthanized with isoflurane (Ifran liquid HanaPharm Co Korea) inhalation on the day of the necropsyAfter anesthesia was confirmed blood was taken from theposterior vena cava for hematology and serum biochemistryanalyses The abdominal aorta and posterior vena cava werecut to euthanize the animals All organs of the body surfacesubcutis head and all internal organs of the abdominal andthoracic cavities were observed grossly Next the pituitarygland ovaries uterus adrenal glands thymus prostate glandtestes epididymides spleen kidneys heart lung brain andliver were removed and weighed with an electronic balance(BP221S Sartorius Co Gottingen Lower Saxony Germany)and all paired organs were measured separately The absoluteorgan weights were converted to relative organ weights basedon the organ-to-fasted body weight ratios

269 Histopathology Microscopic examinations were per-formed on the preserved organs and tissues taken fromall animals in the vehicle-treated and high dose-treatedgroups All gross lesions as defined by the study pathologistwere also included in the examination The eyes and theoptic nerves were preserved in Davidsonrsquos fixative and thetestes and epididymides were preserved in Bouinrsquos fixativeThe following organs and tissues were fixed in 10 neutralbuffered formalin solution the thymus spleen pancreasstomach duodenum jejunum ileum cecum colon rectummesenteric lymph nodes mandibular lymph nodes salivarygland thyroid gland (including parathyroid gland) Harde-rian gland heart lung kidneys adrenal glands liver aortabrain pituitary gland tongue trachea esophagus sternumthoracic spinal cord femorotibial joint peripheral nerve(sciatic) skeletal muscle (femoral) prostate gland seminalvesicles ovaries uterus vagina urinary bladder and skin(including mammary gland)

27 Statistical Analysis In the rat studies data are presentedas mean plusmn standard deviation Body weight food and waterconsumption total volume of urine hematology and serumbiochemistry and absolute and relative organ weights wereassumed to be normally distributed and were analyzed by aone-way analysis of variance (ANOVA) [24]The assumptionof homogeneity was tested using Levenersquos test [25] If theoverall ANOVAwas significant and the assumption of homo-geneity of variance was met Duncanrsquos multiple-range testwas used as a post hoc test to identify significantly differentgroups from the vehicle control group [26] Scheffersquos test wasused if the sample size was unequal between the groups [27]If the assumption of homogeneity of variance was not metDunnettrsquos T3 test was used as the post hoc test [28] Studentrsquos119905-test was used to analyze the differences in means betweenthe two independent groups The urinalysis data were rank-transformed and analyzed by the nonparametric Kruskal-Wallis 119867-test [29] If a statistically significant difference wasobserved between groups theMann-Whitney119880 test was usedto identify the groups that were significantly different fromthe vehicle control group [30] SPSS 101K was used for allstatistical analyses (Chicago IL USA) A 119875 lt 005 wasconsidered significant

In the dog studiesdata are presented as mean plusmn standarddeviation Body weight food and water consumption totalvolume of urine hematology and serum biochemistry andabsolute and relative organ weights were assumed to benormally distributed andwere analyzed by a one-way analysisof variance (ANOVA) [24] SPSS 101K was used for allstatistical analyses (Chicago IL USA) A 119875 lt 005 wasconsidered significant

3 Results

31 Acute Oral Toxicity Study in Rats The test substancewas orally administered to a single group of five male andfive female SD rats at 7 weeks of age in dosage levelsof 2 gmLkg BW Only one female rat given 2 gmLkgdisplayed piloerection and activity decrease 1 day after thetreatment and died within the same day However the grossnecropsy results showed no treatment-related changes andrevealed no evidence of specific toxicity related to the testsubstance Also there was a statistically significant decreasein the body weights at 1 day in all test group rats (Table 1)However the body weights of the test groups recoveredTherefore this was a temporary side effect from the testsubstance But all rats except the aforementioned femalerat were not noted in the mortality clinical and grossfindings

32 Acute Oral Toxicity in Beagle Dogs

321 Clinical Signs and Mortality No dogs died duringthe experimental period After the administration of thetest substance vomiting was observed In the male groupgiven 1000mgkg (Table 2) very slight vomiting of the testsubstance at 3 hr and vomiting of the food at 5 hr wereobserved in one male In addition in the male group given2000mgkg severe vomiting was observed in two males at30min and further vomiting of the food was observed in amale on day 5 As shown in Table 3 vomiting of the food wasobserved in one vehicle-treated female on day 1 moderatevomiting of the test substance was observed in one femalegiven 1000mgkg at 1 hr and severe vomiting of the testsubstance was observed in two females given 2000mgkgat 30min Vomiting of the food was observed in all femalegroups on day 7

322 BodyWeights Thebody weight was observed through-out the 15-day experimental period (Table 4) In the males aloss of body weight was observed in amale given 1000mgkgon days 1 and 7 and in a male given 2000mgkg on day 3In the females a loss of the body weight was observed in avehicle-treated female on day 3 in a female given 1000mgkgon days 1 and 7 and in a female given 2000mgkg on days 1and 7

323 Necropsy As shown in Table 5 hemorrhage of themucosa of the duodenum and dark red discoloration inthe right lobule of a lung were observed in one maleHowever no abnormalities were observed in other males andfemales


Table 1 Body weight values of Sprague-Dawley rats orally treated with Polygala tenuifolia extracts in acute oral toxicity study

Group summary of body weight

Dosage in days (Vehicle-treated group 0 g10mLkgBW) (Test substance-treated group 2 g10mLkgBW)Mean plusmn SD 119873 Mean plusmn SD 119873

Maleminus1 21270 plusmn 2955 5 24020 plusmn 2953 50 19353 plusmn 2767 5 19171 plusmn 3125 51 21814 plusmn 1530 5 19255lowast plusmn 13589 57 27723 plusmn 8416 5 25549lowast plusmn 15501 514 32820 plusmn 19827 5 31322 plusmn 13880 5

Femaleminus1 19717 plusmn 4587 5 19716 plusmn 4621 50 16389 plusmn 5228 5 15958 plusmn 4439 51 18193 plusmn 86178 5 16381lowast plusmn 10634 47 21562 plusmn 14040 5 20359 plusmn 11938 414 23538 plusmn 21433 5 23121 plusmn 6933 4

SD Standard deviation119873 Number of animalslowastP lt 005 compared to the vehicle-treated group(Unit g)

33 Subchronic Oral Toxicity Study in Rats

331 Clinical Signs and Mortality A rat was found deadon day 50 and another on day 67 after administration inthe female group with 1000mgkgday In both dead ratsabnormal fur dirty noses and hypothermia were observedon the day before death There were no abnormal findingsthat were associated with the administration of the testsubstance other than minor observed signs such as scratchedwounds crust formation scarring and the loss of teethwhich occurred at a low frequency with no dose-relationshipduring the study period (data not shown)

332 Body Weight and Food and Water Consumption As aresult of the observations of body weight changes and bodyweight gains for 13 weeks during the administration andobservation periods there were no statistically significantchanges in all treatment groups of both sexes compared tothe vehicle-treated groupThe food consumption in the malegroup of 360mgkgday was significantly increased at weeks4 and 10 after the administration compared to the vehicle-treated group (Tables 6 and 7) (119875 lt 005 or 119875 lt 001) Therewas no significant change in water consumption in all groupsof both sexes compared to the vehicle-treated group

333 Ophthalmic Examination In all groups there were noparticular signs observed from external eye examinationsat grouping and ophthalmic examinations using a funduscamera in the final week of administration (data not shown)

334 Urinalysis There were no particular signs associatedwith the administration of the test substance as a result ofurinalysis performed in the final administration week (datanot shown)

335 Hematology The RDW (red cell distribution width)in the male group administered with 1000mgkgday sig-nificantly decreased compared to the vehicle-treated group(Tables 8 and 9) (119875 lt 001) but no consistent changes wereobserved in the male or female groups in association withthe administration of the test substance in other examinationitems There were no significant changes between the groupsas a result of measurement of PT (prothrombin time) andAPTT (activated partial thromboplastin time) (data notshown)

336 Serum Biochemistry As a result of serum biochemicalexaminations using the serum of collected blood duringthe necropsy (Tables 10 and 11) dose-related decreasingtendencies of ALT BUN GLU AST ALP CHO PRO andCPK and dose-related increases of AG Na+ and Clminus wereobserved in the males In addition statistically significantdose-dependent decreases of ALT BUN and GLU wereobserved compared to the vehicle-treated group (119875 lt 005or 119875 lt 001) and significant dose-dependent increases ofAG Na+ and Clminus were observed compared to the vehicle-treated group (119875 lt 001) In the female groups no staticallysignificant changes were observed in all measurement items

337 Necropsy As a result of the scheduled necropsy of liveanimals at 13 weeks after the administration no notable dose-related changes were observed in either males or females Formales enlargement of the spleen was observed in one case inthe vehicle-treated group and one case in the 600mekgday-treated group a solid brown nodule of the prostate glandwas observed in one case in the vehicle-treated group anddiffuse red spots or a flare-up of the thymus was observedin one case in the vehicle-treated group and one case in thetest substance-treated groups For the females the retention


Table 2 Incidence of clinical signs ofmale beagle dogs orally treatedwith Polygala tenuifolia extracts in single dose oral toxicity study

Sex male

Day Signs observed Group0 1000 2000

0-1 (05 hours) Appears normal 22lowast 22 02Vomiting (severe) 02 02 22

0ndash2 (1 hour) Appears normal 22 22 220ndash3 (2 hours) Appears normal 22 22 22

0ndash4 (3 hours) Appears normal 22 12 22Vomiting (mild) 02 12 02

0ndash5 (4 hour) Appears normal 22 22 22

0ndash6 (5 hours) Appears normal 22 12 22Vomiting (food) 02 12 02

0ndash7 (6 hours) Appears normal 22 22 221 Appears normal 22 22 222 Appears normal 22 22 223 Appears normal 22 22 224 Appears normal 22 22 22

5 Appears normal 22 22 12Vomiting (food) 02 02 12

6 Appears normal 22 22 227 Appears normal 22 22 228 Appears normal 22 22 229 Appears normal 22 22 2210 Appears normal 22 22 2211 Appears normal 22 22 2212 Appears normal 22 22 2213 Appears normal 22 22 2214 Appears normal 22 22 22lowastNumber of animals with the signnumber of animals examined

of a clear fluid in the uterus was observed in the vehicle-treated group and the 360 600 and 1000mgkgday-treatedgroups (2 4 3 and 2 cases resp) and diffuse red spots in thethymus and dark yellowish brown discoloration of the lungwere observed in the 600mgkgday-treated group in twocases and one case respectively Additionally enlargement ofthe submandibular lymph node was observed in one case inthe 1000mgkgday-treated groupAs a result of the necropsyof two dead females of the 1000mgkgday-treated groupdiffuse red spots or dark red coloration in the lung and diffusered spots in the anterior stomach and in the thymus wereobserved along with gas retention due to postmortal changes(data not shown)

338 Organ Weight For males the absolute and relativeweights of the thymus showed significant increases in the 360and 600mgkgday-treated groups compared to the vehicle-treated group (Tables 12 and 13) (119875 lt 005 or 119875 lt 001) andthe relative weight of the left epididymis showed a significantdecrease in the 600mgkgday-treated group compared to thevehicle-treated group (119875 lt 005) but these changes werenot dose-related For females the relative weight of the liver

Table 3 Incidence of clinical signs of female beagle dogs orallytreated with Polygala tenuifolia extracts in single dose oral toxicitystudy

Sex female



0ndash2 (1 hour) Appears normal 22 12 22Vomiting (moderate) 02 12 02

0ndash3 (2 hours) Appears normal 22 22 220ndash4 (3 hours) Appears normal 22 22 220ndash5 (4 hours) Appears normal 22 22 220ndash6 (5 hours) Appears normal 22 22 220ndash7 (6 hours) Appears normal 22 22 22


2 Appears normal 22 22 223 Appears normal 22 22 224 Appears normal 22 22 225 Appears normal 22 22 226 Appears normal 22 22 227 Vomiting (food) 22 22 228 Appears normal 22 22 229 Appears normal 22 22 2210 Appears normal 22 22 2211 Appears normal 22 22 2212 Appears normal 22 22 2213 Appears normal 22 22 2214 Appears normal 22 22 22lowastNumber of animals with the signnumber of animals examined

showed a significant increase in the 1000mgkgday-treatedgroup compared to the vehicle-treated group (119875 lt 005)

339 Histopathology As a result of histopathological exami-nation of rats with a scheduled necropsy in the males in thevehicle-treated group severe granulomatous inflammation ofthe prostate gland moderate pyelonephritis in the kidneysight cysts in the pituitary gland and moderate granulomain the liver were observed in one case along with moderateinflammatory cell infiltration in the Hadrian gland and slightinflammatory cell infiltration in the heart (one case each)For the females two cases of slight tubular regeneration inthe kidney were observed in one case each In the maleswith a scheduled necropsy from the 1000mgkgday-treatedgroup slight tubular remnants in the pituitary gland slightgranuloma in the liver and a slight dilation of the lumen inthe uterus were observed in one case in each group In thelow- and medium-dose groups pale yellowish brown discol-oration of the left lobe of the lungs (identified as focal alve-olitis) was observed in one female from the 600mgkgday-treated group and diffuse red spots in the thymus wereobserved in two cases (identified as focal hemorrhages) Noabnormal findings were observed in the abnormal organs


Table 4 Body weight values of beagle dogs orally treated with Polygala tenuifolia extracts in single dose oral toxicity study

Sex maleGroup Body weights (kg)(mgkg) Animal ID 0 d 1 d 3 d 7 d 14 d Gain

0(119899 = 2)

1 7283 7350 7694 7741 8226 09432 8075 8043 8097 8185 8448 0373

Mean 7679 plusmn 0560 7697 plusmn 0490 7896 plusmn 0285 7963 plusmn 0314 8337 plusmn 0157 0658 plusmn 0403

1000(119899 = 2)

3 8785 8549 8666 8551 8938 01534 7961 7863 8128 8068 8352 0391


2000(119899 = 2)

5 8282 8225 8437 8511 8918 06366 7269 7595 7375 7588 7959 0690

Mean 7776 plusmn 0716 7910 plusmn 0445 7906 plusmn 0751 8050 plusmn 0653 8439 plusmn 0678 0663 plusmn 0038Sex female

Group Body weights (kg)(mgkg) Animal ID 0 d 1 d 3 d 7 d 14 d Gain

0(119899 = 2)

7 7145 7412 7160 7308 7567 04228 6531 6656 6291 6875 7159 0628


1000(119899 = 2)

9 7433 7465 7417 7395 7307 minus012610 7046 6474 6658 6660 6930 minus0116

Mean 7240 plusmn 0274 6970 plusmn 0701 7038 plusmn 0537 7028 plusmn 0520 7119 plusmn 0267 minus0121 plusmn 0007

2000(119899 = 2)

11 7449 7138 7238 7245 7534 008512 7096 6898 7111 7007 7156 0060


Table 5 Gross findings of beagle dogs orally treated with Polygala tenuifolia extracts in single dose oral toxicity study

Sex maleGroup Gross observation Frequency(mgkg) Location Gross findings Death Survivors0 No gross findings lowast00 221000 No gross findings 00 22

No gross findings 00 022000 Duodenum Hemorrhage on mucous membrane 00 12

Lung Dark red discoloration of right lobule 00 12Sex female

Group Gross observation Frequency(mgkg) Location Gross findings Death Survivors0 No gross findings lowast00 221000 No gross findings 00 222000 No gross findings 00 22lowastNumber of animals with the signNumber of animals examined

As a result of the histopathological examinations on the twodead female rats from the 1000mgkgday-treated groupit was found that the rats had suffered severe postmortalchanges and therefore an accurate evaluation of the lesionswas difficult However in rats that died on day 67 afterthe administration diffuse vacuolation in the adrenal glandatrophy of the spleen and inflammatory cell infiltration inthe heart were observed to a minor extent and no abnormalfindings were observed in a dead animal on day 50 after theadministration

4 DiscussionPolygala tenuifolia root is a famous traditional medicineAlthough many studies have reported the pharmacologicalefficacy of the root extract there is no information on itssafety such as its acute and subchronic oral toxicity First weinvestigated the acute and subchronic oral toxicity of the rootextract in rats The root extract was administered orally at 0or 2 gkg body weight for the acute oral toxicity test and at0 360 600 or 1000mgkg body weight for the subchronicoral toxicity test In the acute oral toxicity study one female


Table 6 Food consumption of male rats

Food consumption (g)Study 08-RR-011 Dose (mgkgday) Sex male

Weeks 0 360 600 1000Mean plusmn SD 119873 Mean plusmn SD 119873 Mean plusmn SD 119873 Mean plusmn SD 119873

0 1969 plusmn 065 10 1925 plusmn 181 10 1990 plusmn 117 10 2065 plusmn 154 101 2098 plusmn 149 10 2096 plusmn 126 10 2120 plusmn 131 10 2078 plusmn 177 102 1967 plusmn 152 10 2109 plusmn 151 10 2100 plusmn 074 10 2113 plusmn 123 103 2013 plusmn 127 10 2177 plusmn 106 10 2170 plusmn 186 10 2044 plusmn 113 104 1860 plusmn 080 10 2084 plusmn 130lowastlowast 10 1965 plusmn 084 10 1906 plusmn 033 105 1934 plusmn 059 10 2027 plusmn 080 10 2026 plusmn 166 10 1978 plusmn 122 106 2032 plusmn 078 10 2041 plusmn 118 10 2015 plusmn 095 10 1902 plusmn 141 107 1972 plusmn 044 10 1985 plusmn 066 10 1977 plusmn 087 10 1872 plusmn 148 108 1763 plusmn 069 10 1861 plusmn 156 10 1835 plusmn 174 10 1827 plusmn 164 109 1912 plusmn 179 10 1995 plusmn 099 10 1905 plusmn 056 10 1839 plusmn 089 1010 2020 plusmn 123 10 2153 plusmn 101lowast 10 1990 plusmn 085 10 1897 plusmn 061 1011 1817 plusmn 041 10 1911 plusmn 144 10 1840 plusmn 154 10 1762 plusmn 094 1012 1967 plusmn 131 10 2064 plusmn 135 10 1885 plusmn 119 10 1996 plusmn 202 1013 2046 plusmn 127 10 2180 plusmn 077 10 2061 plusmn 273 10 2173 plusmn 098 10lowastP lt 005 and lowastlowastP lt 001 compared to the vehicle-treated group

Table 7 Food consumption of female rats

Food consumption (g)Study 08-RR-011 Dose (mgkgday) Sex female


0 1484 plusmn 052 10 1434 plusmn 159 10 1523 plusmn 064 10 1308 plusmn 294 101 1275 plusmn 117 10 1301 plusmn 133 10 1346 plusmn 136 10 1251 plusmn 059 102 1337 plusmn 085 10 1228 plusmn 135 10 1214 plusmn 133 10 1184 plusmn 240 103 1396 plusmn 045 10 1395 plusmn 141 10 1391 plusmn 106 10 1210 plusmn 330 104 1270 plusmn 182 10 1274 plusmn 064 10 1237 plusmn 131 10 1152 plusmn 182 105 1372 plusmn 189 10 1414 plusmn 071 10 1296 plusmn 260 10 1235 plusmn 199 106 1311 plusmn 121 10 1340 plusmn 155 10 1359 plusmn 138 10 1143 plusmn 175 107 1387 plusmn 087 10 1268 plusmn 113 10 1216 plusmn 051 10 1107 plusmn 329 108 1158 plusmn 108 10 1281 plusmn 123 10 1268 plusmn 133 10 1275 plusmn 106 99 1244 plusmn 124 10 1319 plusmn 042 10 1265 plusmn 172 10 1169 plusmn 168 910 1433 plusmn 106 10 1349 plusmn 081 10 1181 plusmn 221 10 1242 plusmn 149 811 1306 plusmn 128 10 1276 plusmn 070 10 1164 plusmn 112 10 1241 plusmn 101 812 1335 plusmn 065 10 1310 plusmn 130 10 1333 plusmn 225 10 1315 plusmn 091 813 1433 plusmn 119 10 1444 plusmn 143 10 1368 plusmn 247 10 1549 plusmn 155 8

rat given 2 gkg of the root extract exhibited piloerectionand decrease in activity levels 1 day after treatment and diedwithin the same dayThe gross findings of the autopsy showedno evidence of specific toxicity related to the test substance Inaddition there was a statistically significant decrease in bodyweight at day 1 in all test-group rats but the body weightslater recovered Hence this finding may show a temporaryeffect of the test substance However all rats except the afore-mentioned female rat were not noted in themortality clinicaland gross findingsThese results suggest that the approximatelethal dose for the root extract of P tenuifoliamay be ge2 gkgin male rats and le2 gkg in female rats under the conditions

used in this study In the subchronic oral toxicity studythere were no abnormal signs associated with administrationof the test substance No specific findings for body weightconsumption of food and water ophthalmic examinationurinalyses and blood clotting time were observed in relationto administration of the test substance In the female groupof 1000mgkgday rats that died on day 50 and day 67showed normal clinical signs body weight changes andconsumption of food and water during the study periodbut demonstrated abnormal fur dirty noses or mouths andhypothermia the day before death Autopsies revealed darkred coloration or diffuse red spots in the lungs as well


Table 8 Hematological values (mean plusmn standard deviation) of male rats in the subchronic oral toxicity study of the root of Polygala tenuifolia

Parameter Dose (mgkgday)0 360 600 1000

WBC (103120583L) 983 plusmn 283 819 plusmn 129 891 plusmn 214 940 plusmn 132RBC (106120583L) 875 plusmn 031 881 plusmn 024 869 plusmn 033 865 plusmn 029Hemoglobin (gdL) 157 plusmn 05 157 plusmn 04 154 plusmn 03 153 plusmn 05Hematocrit () 460 plusmn 14 455 plusmn 10 450 plusmn 10 449 plusmn 15MCV (fL) 525 plusmn 16 517 plusmn 13 518 plusmn 16 520 plusmn 14MCH (pg) 179 plusmn 05 178 plusmn 05 178 plusmn 04 177 plusmn 04MCHC (g120583L) 341 plusmn 03 345 plusmn 04 344 plusmn 03 341 plusmn 05RDW () 120 plusmn 03 118 plusmn 03 118 plusmn 02 116 plusmn 03lowastlowast

HDW (gdL) 235 plusmn 005 235 plusmn 008 235 plusmn 009 232 plusmn 012Platelet (103120583L) 10611 plusmn 1065 10304 plusmn 599 10438 plusmn 384 11030 plusmn 647MPV (fL) 70 plusmn 07 68 plusmn 07 68 plusmn 07 70 plusmn 07Reticulocyte () 200 plusmn 028 184 plusmn 031 190 plusmn 031 183 plusmn 018119873 10 10 10 10WBC white blood cell count RBC red blood cell count MCV mean corpuscular volume MCH mean corpuscular hemoglobin concentration MCHC meancorpuscular hemoglobin concentration RDW red cell distribution width HDW hemoglobin distribution width MPV mean platelet volumelowastlowastSignificant difference at P lt 001 compared with the vehicle-treated group

Table 9Hematological values (meanplusmn standard deviation) of female rats in the subchronic oral toxicity study of the root ofPolygala tenuifolia


WBC (103120583L) 460 plusmn 063 475 plusmn 094 552 plusmn 192 511 plusmn 079RBC (106120583L) 789 plusmn 023 798 plusmn 036 787 plusmn 038 778 plusmn 016Hemoglobin (gdL) 143 plusmn 06 147 plusmn 07 141 plusmn 06 146 plusmn 03Hematocrit () 412 plusmn 13 421 plusmn 25 409 plusmn 19 419 plusmn 08MCV (fL) 523 plusmn 20 528 plusmn 11 520 plusmn 16 539 plusmn 11MCH (pg) 182 plusmn 09 185 plusmn 03 180 plusmn 07 188 plusmn 06MCHC (g120583L) 347 plusmn 07 350 plusmn 04 346 plusmn 05 349 plusmn 05RDW () 108 plusmn 04 106 plusmn 03 108 plusmn 04 106 plusmn 03HDW (gdL) 228 plusmn 019 222 plusmn 013 221 plusmn 016 233 plusmn 015Platelet (103120583L) 11044 plusmn 1030 11666 plusmn 897 11941 plusmn 1664 11340 plusmn 883MPV (fL) 69 plusmn 09 65 plusmn 044 70 plusmn 09 65 plusmn 06Reticulocyte () 205 plusmn 060 191 plusmn 020 197 plusmn 032 208 plusmn 046119873 9 10 10 8WBC white blood cell count RBC red blood cell count MCV mean corpuscular volume MCH mean corpuscular hemoglobin concentration MCHC meancorpuscular hemoglobin concentration RDW red cell distribution width HDW hemoglobin distribution width MPV mean platelet volume

as diffuse red spots in the anterior stomach and thymusgland A more accurate evaluation of the lesions was madedifficult by severe postmortem changes in two rats duringthe histopathological studies In the rat that died 67 daysafter administration of the root extract diffuse vacuolationin the adrenal glands atrophy of the spleen and infiltrationof the inflammatory cells in the heart were observed Inthe rat that died on day 50 abnormal findings were notobserved Asmentioned above the clinical signs bodyweightchanges food and water consumption necropsy findingsand histopathological findings obtained for the dead rats didnot indicate any toxicity of the test substance Therefore thedeaths of the rats were not associated with the administrationof the test substance and were judged to be accidental dueto administration errors This hypothesis is supported by the

fact that no abnormal findings were observed in relation tothe administration of the test substance in the remainingrats in the same dose group We demonstrated that thetest substance could be used as a food supplement becausesome famous food ingredients were toxic at high doses (eglemongrass (Cymbopogon citratus) essential oil Roystonearegia fruit and 120572-glycerylphosphorylcholine (AGPC)) [31ndash33]The results of the serum biochemical examinations of therat sera revealed reductions in the levels of AST and ALT inthe male groups AST and ALT are coenzymes of pyridoxalphosphate (PALP) PALP is a derivative of vitamin B

6and

AST and ALT are activated by union with PALP but non-linked aminotransferases are not activatedHence reductionsin the levels of ALT and AST occur if there is low generationof aminotransferases or PALP or if the linkage between the


Table 10 Serum biochemical values (mean plusmn standard deviation) of male rats in the subchronic oral dose toxicity study of the root of Polygalatenuifolia


Number of animals examined 10 10 10 10AST (IUL) 1050 plusmn 266 934 plusmn 246 879 plusmn 185 805 plusmn 117ALT (IUL) 601 plusmn 127 466 plusmn 75 446 plusmn 68lowast 400 plusmn 50lowastlowast

ALP (IUL) 1212 plusmn 328 1044 plusmn 297 1051 plusmn 152 995 plusmn 175BUN (mgdL) 189 plusmn 35 150 plusmn 18lowast 146 plusmn 14lowast 143 plusmn 16lowast

CRE (mgdL) 064 plusmn 009 056 plusmn 003 054 plusmn 004 060 plusmn 006GLU (mgdL) 1398 plusmn 181 1229 plusmn 118lowastlowast 1259 plusmn 111lowast 1241 plusmn 88lowast

CHO (mgdL) 1272 plusmn 386 1074 plusmn 123 952 plusmn 117 980 plusmn 111PRO (gdL) 751 plusmn 112 656 plusmn 018 650 plusmn 026 64 plusmn 016CPK (IUL) 1954 plusmn 1279 1414 plusmn 963 982 plusmn 788 933 plusmn 686ALB (gdL) 351 plusmn 043 325 plusmn 008 324 plusmn 012 324 plusmn 009BIL (mgdL) 022 plusmn 002 020 plusmn 002 020 plusmn 002 020 plusmn 001TG (mgdL) 608 plusmn 168 572 plusmn 98 537 plusmn 67 632 plusmn 150IP (mgdL) 842 plusmn 156 692 plusmn 021 718 plusmn 063 749 plusmn 034Ca2+ (mgdL) 1181 plusmn 175 1011 plusmn 015 1018 plusmn 023 1013 plusmn 028AG (ratio) 089 plusmn 007 099 plusmn 003lowastlowast 100 plusmn 004lowastlowast 102 plusmn 004lowastlowast

Na+ (mmolL) 1405 plusmn 422 1445 plusmn 108lowastlowast 1444 plusmn 107lowastlowast 1439 plusmn 074lowastlowast

K+ (mmolL) 439 plusmn 015 447 plusmn 027 450 plusmn 025 449 plusmn 015Clminus (mmolL) 1042 plusmn 297 1074 plusmn 084lowastlowast 1076 plusmn 07lowastlowast 1073 plusmn 082lowastlowast

119873 10 10 10 10lowastlowastlowastSignificant difference at P lt 005001 compared with the vehicle-treated group

Table 11 Serum biochemical values (mean plusmn standard deviation) of female rats in the subchronic oral dose toxicity study of the root ofPolygala tenuifolia


Number of animals examined 10 10 10 10AST (IUL) 911 plusmn 140 940 plusmn 178 859 plusmn 167 868 plusmn 140ALT (IUL) 355 plusmn 58 345 plusmn 41 329 plusmn 51 326 plusmn 53ALP (IUL) 641 plusmn 149 748 plusmn 99 738 plusmn 208 712 plusmn 101BUN (mgdL) 193 plusmn 30 172 plusmn 19 174 plusmn 19 184 plusmn 16CRE (mgdL) 065 plusmn 007 064 plusmn 006 066 plusmn 011 067 plusmn 009GLU (mgdL) 1162 plusmn 127 1192 plusmn 116 1117 plusmn 73 1104 plusmn 110CHO (mgdL) 972 plusmn 177 1063 plusmn 167 1129 plusmn 194 1204 plusmn 151PRO (gdL) 623 plusmn 015 636 plusmn 029 623 plusmn 032 611 plusmn 019CPK (IUL) 1348 plusmn 609 1202 plusmn 843 1159 plusmn 821 1219 plusmn 634ALB (gdL) 323 plusmn 009 331 plusmn 015 326 plusmn 015 320 plusmn 012BIL (mgdL) 020 plusmn 002 021 plusmn 002 021 plusmn 003 021 plusmn 002TG (mgdL) 439 plusmn 52 429 plusmn 75 528 plusmn 163 478 plusmn 83IP (mgdL) 600 plusmn 085 615 plusmn 122 642 plusmn 099 646 plusmn 045Ca2+ (mgdL) 961 plusmn 013 978 plusmn 046 971 plusmn 017 963 plusmn 015AG (ratio) 108 plusmn 005 109 plusmn 008 110 plusmn 007 111 plusmn 008Na+ (mmolL) 1433 plusmn 106 1436 plusmn 097 1427 plusmn 106 1425 plusmn 120K+ (mmolL) 424 plusmn 019 457 plusmn 125 415 plusmn 027 424 plusmn 018Clminus (mmolL) 1083 plusmn 116 1091 plusmn 120 1096 plusmn 084 10988 plusmn 164119873 10 10 10 8


Table 12 Absolute and relative organ weight of males in the subchronic oral dose toxicity study of the root of Polygala tenuifolia


Body weight 44451 plusmn 2261 46088 plusmn 3959 45034 plusmn 3011 43799 plusmn 1981Adrenal gland left 00304 plusmn 00048 00282 plusmn 00039 00295 plusmn 00038 00300 plusmn 00045Per body weight () 00069 plusmn 00011 00061 plusmn 00007 00065 plusmn 00006 00068 plusmn 00010Adrenal gland right 00292 plusmn 00045 00285 plusmn 00039 00288 plusmn 00035 00284 plusmn 00042Per body weight () 00066 plusmn 00011 00062 plusmn 00009 00064 plusmn 00006 00065 plusmn 00009Pituitary gland 00143 plusmn 00031 00131 plusmn 00013 00126 plusmn 00018 00130 plusmn 00032Per body weight () 00032 plusmn 00006 00028 plusmn 00002 00028 plusmn 00003 00030 plusmn 00007Thymus 02515 plusmn 00423 03159 plusmn 00400lowastlowast 03294 plusmn 00524lowastlowast 02891 plusmn 00583Per body weight () 00570 plusmn 00115 00686 plusmn 00067lowast 00734 plusmn 00120lowastlowast 00660 plusmn 00132Prostate 05877 plusmn 01284 05855 plusmn 01785 05839 plusmn 01195 06297 plusmn 01285Per body weight () 01330 plusmn 00325 01278 plusmn 00404 01289 plusmn 00196 01436 plusmn 00280Testis left 29152 plusmn 01365 20512 plusmn 01353 20208 plusmn 01221 20452 plusmn 01618Per body weight () 04546 plusmn 00383 04473 plusmn 00410 04495 plusmn 00245 04667 plusmn 00251Testis left 20112 plusmn 01409 20550 plusmn 01629 20063 plusmn 01262 20337 plusmn 01905Per body weight () 04539 plusmn 00414 04484 plusmn 00486 04464 plusmn 00278 04636 plusmn 00269Epididymis left 06852 plusmn 00348 06768 plusmn 00397 06403 plusmn 00492 06961 plusmn 00583Per body weight () 01546 plusmn 00108 01474 plusmn 00093 01423 plusmn 00085 01590 plusmn 00119Epididymis right 07013 plusmn 00830 06667 plusmn 00476 06459 plusmn 00381 06834 plusmn 00461Per body weight () 01580 plusmn 00177 01452 plusmn 00113 01436 plusmn 00057 01561 plusmn 00103Spleen 08944 plusmn 02307 08399 plusmn 00698 08576 plusmn 01300 08627 plusmn 00864Per body weight () 02019 plusmn 00562 01828 plusmn 00154 01899 plusmn 00215 01969 plusmn 00173Kidney left 13475 plusmn 01109 13537 plusmn 01786 13185 plusmn 01704 12721 plusmn 01128Per body weight () 03032 plusmn 00174 02930 plusmn 00189 02923 plusmn 00259 02903 plusmn 00190Kidney right 13439 plusmn 01324 13803 plusmn 01793 13786 plusmn 01808 12969 plusmn 01078Per body weight () 03020 plusmn 00167 02989 plusmn 00216 03057 plusmn 00274 02960 plusmn 00180Heart 13823 plusmn 01325 15006 plusmn 01825 15012 plusmn 01357 14132 plusmn 01456Per body weight () 03017 plusmn 00197 03252 plusmn 00241 03333 plusmn 00200 03225 plusmn 00271Lung 19149 plusmn 01630 18571 plusmn 01483 18949 plusmn 01652 18769 plusmn 01039Per body weight () 04314 plusmn 00363 04043 plusmn 00327 04211 plusmn 00302 04288 plusmn 00210Brain 20127 plusmn 00602 19798 plusmn 00874 20014 plusmn 00659 19522 plusmn 00843Per body weight () 04539 plusmn 00237 04313 plusmn 00254 04457 plusmn 00248 04459 plusmn 00135Liver 117210 plusmn 10504 115963 plusmn 11787 114193 plusmn 11750 112844 plusmn 00850Per body weight () 26348 plusmn 01369 25149 plusmn 01118 25319 plusmn 01342 25752 plusmn 01174119873 10 10 10 10Body weight before necropsy and after fastinglowastlowastlowastSignificant difference at P lt 005001 compared with the vehicle-treated group

aminotransferases and PALP is disturbed Cephalosporin-like antibiotics are known to induce reductions in ALT levelsbut in toxicity studies reductions have been mentioned ingeneral and toxicological significance has not been attributed[34 35] The blood urea nitrogen (BUN) levels in the bloodrepresent the nitrogen content in relation to urea whichis a metabolite of nitrogen and is closely related to heartdiseases The BUN value might show a decrease due to areduction in urea synthesis during toxic hepatitis severe hep-atic impairment and consuming a low protein diet Howeverin the present study no abnormal findings related to dosewere observed In general the decrease in BUN level is not acritical factor in toxicity studies [34]Moreover these changeswere not regarded as adverse effects because they remained

within normal ranges [36 37] Hematological examinationsrevealed reductions in red blood cell volume distributionwidth (RDW) in the male group given 1000mgkgdayDecreasing tendencies in the levels of GLU ALP CHO PROALB and CPK and increments in the levels of AG Na+and Clminus in male treatment groups were within the normalrange [38 39] and also remained within the normal rangewhen compared with the historical data [21] available atChemOn Inc In the vehicle-treated group increments orreductions in some measurement items were also observedaccidentally and were thought to be somewhat contributoryto the above results [40] therefore it was considered thatthese changeswere toxicological andwere not associatedwiththe administration of the test substance RDW decreased


Table 13 Absolute and relative organ weight of females in the subchronic oral dose toxicity study of the root of Polygala tenuifolia


Body weight 26011 plusmn 1583 25959 plusmn 680 25395 plusmn 1741 25061 plusmn 787Ovary left (g) 00505 plusmn 00119 00547 plusmn 00081 00467 plusmn 00138 00537 plusmn 00067Per body weight () 00193 plusmn 00040 00211 plusmn 00031 00183 plusmn 00045 00214 plusmn 00025Ovary right 00532 plusmn 00072 00526 plusmn 00064 00515 plusmn 00092 00538 plusmn 00058Per body weight () 00204 plusmn 00025 00203 plusmn 00026 00203 plusmn 00032 00215 plusmn 00024Adrenal gland left 00332 plusmn 00073 00339 plusmn 00030 00324 plusmn 00052 00370 plusmn 00041Per body weight () 00128 plusmn 00025 00131 plusmn 00010 00128 plusmn 00020 00148 plusmn 00019Adrenal gland right 00279 plusmn 00044 00309 plusmn 00046 00301 plusmn 00036 00319 plusmn 00068Per body weight () 00107 plusmn 00016 00119 plusmn 00017 00119 plusmn 030015 00128 plusmn 00029Pituitary gland 00148 plusmn 00053 00137 plusmn 00011 00134 plusmn 00015 00141 plusmn 00019Per body weight () 00056 plusmn 00016 00053 plusmn 00004 00053 plusmn 00004 00056 plusmn 00009Thymus 02235 plusmn 00323 02292 plusmn 00349 02243 plusmn 00232 02290 plusmn 00320Per body weight () 00863 plusmn 00142 00882 plusmn 00127 00883 plusmn 00068 00915 plusmn 00128Uterus 06040 plusmn 01695 05195 plusmn 01438 04848 plusmn 01573 06109 plusmn 03313Per body weight () 02347 plusmn 00730 02002 plusmn 00548 01929 plusmn 00678 02436 plusmn 01313Spleen 05954 plusmn 0596 05786 plusmn 00512 05402 plusmn 01754 06280 plusmn 03671Per body weight () 02287 plusmn 00147 02228 plusmn 00174 02111 plusmn 00673 02506 plusmn 01340Kidney left 07683 plusmn 00657 07544 plusmn 00510 07713 plusmn 01081 07513 plusmn 04212Per body weight () 02953 plusmn 00162 02905 plusmn 00164 03049 plusmn 00495 03000 plusmn 01856Kidney right 07547 plusmn 00548 07553 plusmn 00511 07900 plusmn 01248 07618 plusmn 04748Per body weight () 02898 plusmn 00117 02909 plusmn 00178 03124 plusmn 00572 03041 plusmn 01993Heart 09126 plusmn 00678 08911 plusmn 00619 09117 plusmn 01012 09030 plusmn 06912Per body weight () 03511 plusmn 00197 03433 plusmn 00225 03588 plusmn 00280 03607 plusmn 03053Lung 14077 plusmn 00881 14371 plusmn 00983 14229 plusmn 02161 14301 plusmn 09812Per body weight () 05418 plusmn 00275 05535 plusmn 00329 05628 plusmn 01006 05704 plusmn 03008Brain 18129 plusmn 00649 18308 plusmn 00685 18026 plusmn 00750 18440 plusmn 05932Per body weight () 06982 plusmn 00274 07058 plusmn 00358 07123 plusmn 00485 07367 plusmn 03932Liver 60596 plusmn 02314 63790 plusmn 06585 64577 plusmn 05182 64618 plusmn 04515Per body weight () 23339 plusmn 00996 24553 plusmn 02191 25459 plusmn 01755 25778 plusmn 01469lowast

119873 10 10 10 8Body weight before necropsy and after fastinglowastSignificant difference at P lt 005 compared with the vehicle-treated group

significantly but the changes remained within the limitsof normal biological variations [41 42] Significant changesin the absoluterelative weights of the thymus gland inthe male groups given 360 and 600mgkgday the relativeweights of the left epididymis in the male groups given600mgkgday and the relative weights of the liver in thefemale group given 1000mgkgday were not accompaniedby abnormal autopsy or histopathological findings A rela-tionship with sex or dose was not observed so these changeswere not considered to be a toxicological feature of the testsubstance Even though the weights of the thymus glandand left epididymis changed significantly they were withinthe limits of normal biological variations this finding wasin accordance with results reported by Wang et al [43]Regarding relative organweight an increase in the liver in thefemale rats compared with Banpungtongseong-san reportedorgan weights analysis showed that there were statisticallysignificant changes in the absolute weights of the thymus in

the male group of 2000mgkgday However these changeswere not regarded as adverse effects because they wereminorremained within normal historical control ranges for Fischer344 rats and were not correlated with pathological lesions inthe respective organs [44 45] Autopsy and histopathologicalexaminations revealed abnormal findings in some organs butthe prevalence of these findings was low and these changeswere spontaneous and nonspecific [46 47] Therefore thetoxicological importance of these findings was negligibleNo abnormal findings were observed in association withadministration of the test substance upon histopathologicalexamination of abnormal organs in low- and medium-dosegroups

Second acute oral toxicity of the root extract P tenuifo-lia in beagle dogs was investigated The root extract wasadministered orally at 1000 or 2000mgkg body weightsthe vehicle-treated group was only given gelatin capsules forthe single dose oral toxicity test As a result of the present


study vomiting and inhibition of body weight gain wereobserved after the administration of the test substance butother abnormalities in clinical signs deaths and abnormalfindings in the necropsy results were not observed in relationto administration of the test substance The results of clinicalsign examinations of the dogs observed vomiting in onemalegiven 1000mgkg at 5 hr and in one male given 2000mgkgon day 5 This was after the administration was consideredto be unrelated to the test substance because vomiting wasalso observed in the vehicle-treated female group on day1 and vomiting like this is commonly observed in dogswhich have a well-developed vomiting system Howeverthese changes were not considered serious when comparedwith the previous study as some famous oriental medicineand food compositions were found to be toxic includingPaecilomyces sinclairii [48] SH21-B [49] epigallocatechingallate (EGCG) [50] novel thiazolidinedione (MCC-555)[51] and nelumbinis semen (NS the seeds of Nelumbonucifera) [52] Vomiting of the food occurred sporadicallyin some dogs in all groups including the controls Thisfrequency was typical for this colony of dogs [53] In generalthe incidence of vomiting did not suggest an effect of thetest substance [54] Vomiting is a common presenting signin small animal practice [55] Moreover many peripheralstimuli of abdominal structures will initiate vomiting in dogs[56 57] The ldquochemoreceptor trigger zonerdquo of the brainstemhas been identified as the area postrema that is located onthe dorsal surface of the medulla oblongata adjacent to thecaudal end of the fourth ventricle [58] Therefore it wasnot considered to be related to the treatment with the testsubstanceThe loss of body weight was observed inmales andfemales but because vomiting of the test substance occurredmore frequently at 2000mgkg than at 1000mgkg theabsorption of test substance seemed to be relatively higherat 1000mgkg than at 2000mgkg Also the loss of bodyweight showed a temporary serious tendency on days 1 and3 after the administration However the body weights ofthe male 1000mgkg and 2000mgkg groups recoveredIn addition the vehicle-treated group of females showed atendency to decrease in body weight from approximately14 days In all groups increment and reduction tendencieswere also observed and these changes were observed [59ndash62] The changes in body weights were probably the resultof irritation of the alimentary tract [61] We also consideredgastroenteric issues from vomiting as accordingly inducinga disorder of the nutriments absorption [63] A hemorrhageof the mucosa of the duodenum and the dark discolorationof the lung observed in a male given 2000mgkg were notconsidered as test substance-related changes because thesewere occasionally found in domestic beagle dogs withoutadministration of any of the test substances Dog experimentfindings were generally observed as environmental causesbecause these results are ignored [64] In the necropsy resultsa dark red spot on the lung and a light gray color changewere observed However this change was not dose-related[49] but it was an accidental change Also the lungs showeddiscoloration (dark mottled and focus) and the stomachshowed discoloration [65 66] The gross lesion of the massor nodule corresponded to the microscopic lesion of the

thrombus These were direct effects of the drug or weresecondary to the vascular damage caused by dehydration(with hemodynamic shock) from vomiting and diarrhea [66]Histopathology changes and hemorrhages in the lung wereobserved byWerley et al [67]However theywere consideredto be typical of spontaneously arising background findingsand no other biologically significant effects were observedby histopathology studies on the tissues and organs Thediscolorations in the other various organs and tissues werewithin the range of normal background lesions in dogs of thisstrain and age and were therefore not considered to be relatedto the test substance [68]

In this experiment the administration of the root extractof Polygala tenuifoliaWilldenow (2 gkgday) did not result inacute oral toxicity in the SD rats The minimum lethal dose(MLD) was also considered to be higher than 1000mgkgsince vomiting of the test substance occurred at 2000mgkgIn the subchronic oral toxicity test no obvious toxic changesdue to administration of the root extract of P tenuifoliawere observed in any of the parameters tested (clinicalsigns mortality body weight changes food and water con-sumption ophthalmic examination urinalyses hematologyserumbiochemistry organweights autopsy and histopathol-ogy) Therefore under the experimental conditions the no-observed-adverse-effect level (NOAEL) of the root extract ofP tenuifolia was determined to be 1000mgkgday for bothsexes but the target organ was not established In conclusionthe present study demonstrated that the acute or subchronictoxicity of the root extract of Polygala tenuifoliawas not toxicin rats and beagle dogs

Conflict of Interests

The authors of this paper have no conflict of interests

Acknowledgments

The collection analysis and interpretation of data of theacute toxicity and the subchronic toxicity were supportedby SNUH-GLP (Non-Clinical Research Center BiomedicalResearch Institute Seoul National University Hospital) andPreclinical Research Center ChemOn Inc respectively

References

[1] W-Y Jiang ldquoTherapeutic wisdom in traditional Chinesemedicine a perspective from modern sciencerdquo Trends in Phar-macological Sciences vol 26 no 11 pp 558ndash563 2005

[2] S Liu L-Z Yi and Y-Z Liang ldquoTraditional Chinese medicineand separation sciencerdquo Journal of Separation Science vol 31no 11 pp 2113ndash2137 2008

[3] J-L Tang B-Y Liu and K-W Ma ldquoTraditional ChinesemedicinerdquoThe Lancet vol 372 no 9654 pp 1938ndash1940 2008

[4] C-F Chau and S-H Wu ldquoThe development of regulations ofChinese herbal medicines for both medicinal and food usesrdquoTrends in Food Science amp Technology vol 17 no 6 pp 313ndash3232006

[5] K Chan and L Cheung ldquoExamples of interactions betweenChinese herbal medicinal products and orthodox drugrdquo in


Interactions between Chinese Herbal Medicinal Products andOrthodox Drugs pp 57ndash97 Harwood Academic PublishersAmsterdam The Netherlands 2000

[6] A N Ukwuani M G Abubakar S W Hassan and B MAgaie ldquoToxicological studies of hydromethanolic leaves extractof Grewia crenatardquo International Journal of PharmaceuticalSciences and Drug Research vol 4 pp 245ndash249 2012

[7] T Nagai Y Suzuki H Kiyohara et al ldquoOnjisaponins fromthe root of Polygala tenuifoliaWilldenow as effective adjuvantsfor nasal influenza and diphtheria-pertussis-tetanus vaccinesrdquoVaccine vol 19 no 32 pp 4824ndash4834 2001

[8] L C Klein Jr S Faloni de Andrade andV C Filho ldquoA pharma-cognostic approach to the polygala genus phytochemical andpharmacological aspectsrdquoChemistry and Biodiversity vol 9 no2 pp 181ndash209 2012

[9] Z L Jin N Gao D Zhou M G Chi X M Yang and J P XuldquoThe extracts of Fructus Akebiae a preparation containing 90of the active ingredient hederagenin Serotonin norepinephrineand dopamine reuptake inhibitorrdquo Pharmacology Biochemistryand Behavior vol 100 no 3 pp 431ndash439 2012

[10] K C Huang The Pharmacology of Chinese Herbs CRC PressBoca Raton Fla USA 1993

[11] P Liu Y Hu D-H Guo et al ldquoPotential antidepressantproperties of Radix Polygalae (Yuan Zhi)rdquo Phytomedicine vol17 no 10 pp 794ndash799 2010

[12] C H Park S H Choi J-W Koo et al ldquoNovel cognitiveimproving and neuroprotective activities of Polygala Tenuifoliawilldenow extract BT-11rdquo Journal of Neuroscience Research vol70 no 3 pp 484ndash492 2002

[13] J-Y Lee K Y Kim K Y Shin B Y Won H Y Jung andY-H Suh ldquoEffects of BT-11 on memory in healthy humansrdquoNeuroscience Letters vol 454 no 2 pp 111ndash114 2009

[14] K Y Shin B Y Won C Heo et al ldquoBT-11 improves stress-induced memory impairments through increment of glucoseutilization and total neural cell adhesion molecule levels in ratbrainsrdquo Journal of Neuroscience Research vol 87 no 1 pp 260ndash268 2009

[15] K Y Shin J-Y Lee B Y Won et al ldquoBT-11 is effectivefor enhancing cognitive functions in the elderly humansrdquoNeuroscience Letters vol 465 no 2 pp 157ndash159 2009

[16] Y Ikeya S Takeda M Tunakawa et al ldquoCognitive improvingand cerebral protective effects of acylated oligosaccharides inPolygala tenuifoliardquo Biological and Pharmaceutical Bulletin vol27 no 7 pp 1081ndash1085 2004

[17] Y Ling Z Li M Chen Z Sun M Fan and C HuangldquoAnalysis and detection of the chemical constituents of RadixPolygalae and their metabolites in rats after oral administrationby ultra high-performance liquid chromatography coupledwith electrospray ionization quadrupole time-of-flight tandemmass spectrometryrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 85 pp 1ndash13 2013

[18] Y J Chen X B Huang Z X Li et al ldquoTenuigeninprotescultured hippocampal neurons against methlglyoxal-inducedneurotoxicityrdquo European Journal of Pharmacology vol 645 pp1ndash8 2010

[19] Y Chen X Huang W Chen N Wang and L Li ldquoTenuigeninpromotes proliferation and differentiation of hippocampal neu-ral stem cellsrdquo Neurochemical Research vol 37 no 4 pp 771ndash777 2012

[20] OECD OECD Principles of Good Laboratory PracticeENVMCCHEM (98) 17 OECD Paris France 1998

[21] OECD Guidelines for Testing of Chemicals Guidline 408Repeated Dose 90-day Oral Toxicity Study in Rodents OECDParis France 1998

[22] KFDA ldquoGood Laboratory Practice Regulation for Non-ClinicalLaboratory Studiesrdquo (Notification No 2009-102) KFDA 2009

[23] KFDA The Standards of Toxicity Study for Medicinal Products(Notification No 2009-116) KFDA Seoul Republic of Korea2009

[24] G W Snedecor and W G Cochran ldquoOne way classifica-tions analysis of variancerdquo in Statistical Methods pp 215ndash237Thelowa state University Press Ames Iowa USA 7th edition1980

[25] H Levene ldquoIn contributions to probability and statisticsrdquo inEssays in Honor of Harold Hotelling I Olkin S G Ghurye WHoeffding et al Eds pp 278ndash292 Stanford University Press1960

[26] D B Duncan ldquoMultiple range and multiple 119865 testsrdquo Biometricsvol 11 pp 1ndash42 1955

[27] H Scheffe ldquoAmethod for judging all contrasts in the analysis ofvariancerdquo Biometrika vol 40 no 1-2 pp 87ndash104 1953

[28] C W Dunnett ldquoNew tables for multiple comparisons with acontrolrdquo Biometrics vol 20 pp 482ndash491 1964

[29] W H Kruskal and W A Wallis ldquoUse of ranks in one-criterion analysis of variancerdquo Journal of the American StatisticalAssociation vol 47 pp 583ndash621 1952

[30] H B Mann and D R Whitney ldquoOn a test of whether one oftwo randomvariables is stochastically larger than the otherrdquoTheAnnals of Mathematical Statistics vol 18 pp 50ndash60 1947

[31] A M Brownawell E L Carmines and F Montesano ldquoSafetyassessment of AGPC as a food ingredientrdquo Food and ChemicalToxicology vol 49 no 6 pp 1303ndash1315 2011

[32] C A R A Costa L T Bidinotto R K Takahira D M FSalvadori L F Barbisan and M Costa ldquoCholesterol reductionand lack of genotoxic or toxic effects in mice after repeated 21-day oral intake of lemongrass (Cymbopogon citratus) essentialoilrdquo Food and Chemical Toxicology vol 49 no 9 pp 2268ndash22722011

[33] A Gutierrez R Gamez M Noa et al ldquoOne year oral Toxicityof D-004 a lipid extract from Roystonea regia fruits in SpragueDawley ratsrdquo Food and Chemical Toxicology vol 49 no 11 pp2855ndash2861 2011

[34] G N Lee and O H Kwon Clinical Pathology File MedicalCultural Press Seoul Republic of Korea 2003

[35] T Yoshifumi ldquoCollection of questions and answers in toxicol-ogyrdquo Scientist Inc 2003

[36] Z Z Han H D Xu K H Kim et al ldquoReference data of themain physiological parameters in control Sprague-Dawley ratsfrom pre-clinical toxicity studierdquo Laboratory Animal Researchvol 26 pp 153ndash164 2010

[37] S-H Shin K-H Koo J-S Bae et al ldquoSingle and 90-dayrepeated oral dose toxicity studies of fermentedRhus vernicifluastem bark extract in Sprague-Dawley ratsrdquo Food and ChemicalToxicology vol 55 pp 617ndash626 2013

[38] B H Kang H Y Son C S Ha H S Lee and S W SongldquoReference values of hematology and serum chemistry in KtcSprague-Dawley ratsrdquo Korean Journal of Laboratory AnimalScience vol 11 pp 141ndash145 1995

[39] S T Wolford R A Schroer F X Gohs et al ldquoReferencerange data base for serum chemistry and hematology values inlaboratory animalsrdquo Journal of Toxicology and EnvironmentalHealth vol 18 no 2 pp 161ndash188 1986


[40] W F Loeb and F W Quimby The Clinical Chemistry ofLabolatory Animals Pergamon Press 1989

[41] B M Schmidt N Ilic A Poulev and I Raskin ldquoToxicologicalevaluation of a chicory root extractrdquo Food and ChemicalToxicology vol 45 no 7 pp 1131ndash1139 2007

[42] D Wang J Meng K Xu et al ldquoEvaluation of oral subchronictoxicity of Pu-erh green tea (camellia sinensis var assamica)extract in Sprague Dawley ratsrdquo Journal of Ethnopharmacologyvol 142 no 3 pp 836ndash844 2012

[43] M Wang J Liu B Zhou et al ldquoAcute and sub-chronic toxicitystudies of Danshen injection in Sprague-Dawley ratsrdquo Journalof Ethnopharmacology vol 141 no 1 pp 96ndash103 2012

[44] M-Y Lee I-S Shin C-S Seo J-H Kim S-R Han and H-KShin ldquoSubchronic oral toxicity studies of the traditional herbalformula Bangpungtongseong-san in Crl CD (SD) ratsrdquo Journalof Ethnopharmacology vol 144 no 3 pp 720ndash725 2012

[45] Y-H Lee D Kim M J Lee et al ldquoSubchronic toxicity study ofCoptidis Rhizoma in ratsrdquo Journal of Ethnopharmacology vol152 no 3 pp 457ndash463 2014

[46] G A Boorman S L Eustis M R Elwell et al Pathology of theFischer Rat New York NY USA Academic Press 1990

[47] P Greves Histopathology of Preclinical Toxicity Studies Inter-pretation and Relevance in Drug Safety Evaluation Elsevier2000

[48] M Y Ahn S C Kang N J Jung et al ldquoAcute oral toxicity ofPaecilomyces sinclairii in beagle dogsrdquo Journal of Toxicology andPublic Health vol 19 no 3 pp 241ndash245 2003

[49] S H Kim S J Park and Y S Yun ldquoAcute oral toxicity testof oriental medical preseription SH21-Brdquo Journal of OrientalMedicine vol 9 pp 131ndash148 2003

[50] R A Isbrucker J A Edwards E Wolz A Davidovich andJ Bausch ldquoSafety studies on epigallocatechin gallate (EGCG)preparations Part 2 Dermal acute and short-term toxicitystudiesrdquo Food and Chemical Toxicology vol 44 no 5 pp 636ndash650 2006

[51] N Sun G Lu M Lin G Fan and Y Wu ldquoSubchronic toxicityand toxicokinetics of MCC-555 a novel thiazolidinedione after270-day repeated oral administration in dogsrdquo EnvironmentalToxicology and Pharmacology vol 27 no 2 pp 237ndash246 2009

[52] H-S Chung H J Lee I Shim andH Bae ldquoAssessment of anti-depressant effect of nelumbinis semen on rats under chronicmild stress and its subchronic oral toxicity in rats and beagledogsrdquo BMC Complementary and Alternative Medicine vol 12article 68 2012

[53] G A Burdock andW G Flamm ldquoA review of the studies of thesafety of polydextrose in foodrdquo Food and Chemical Toxicologyvol 37 no 2-3 pp 233ndash264 1999

[54] R M McClain E Wolz A Davidovich F Pfannkuch and JBausch ldquoSubchronic and chronic safety studies with genisteinin dogsrdquo Food and Chemical Toxicology vol 43 no 10 pp 1461ndash1482 2005

[55] C Elwood P Devauchelle J Elliott et al ldquoEmesis in dogs areviewrdquo Journal of Small Animal Practice vol 51 no 1 pp 4ndash222010

[56] I M Lang S K Sarna and R E Condon ldquoGastrointestinalmotor correlates of vomiting in the dog quantification andcharacterization as an independent phenomenonrdquo Gastroen-terology vol 90 no 1 pp 40ndash47 1986

[57] J Xu and J D Z Chen ldquoIntestinal electrical stimulationimproves delayed gastric emptying and vomiting induced byduodenal distension in dogsrdquoNeurogastroenterology ampMotilityvol 20 no 3 pp 236ndash242 2008

[58] C L Chernicky K L Barnes J P Conomy and C M FerrarioldquoAmorphological characterization of the canine area postremardquoNeuroscience Letters vol 20 no 1 pp 37ndash43 1980

[59] M Tobe T Furuya Y Kawasaki et al ldquoSix-month toxicity studyof butylated hydroxyanisole in beagle dogsrdquo Food and ChemicalToxicology vol 24 no 10-11 pp 1223ndash1228 1986

[60] H P Til and A Bar ldquoSubchronic (13-week) oral toxicitystudy of 120574-cyclodextrin in dogsrdquo Regulatory Toxicology andPharmacology vol 27 no 2 pp 159ndash165 1998

[61] C D Hebert H D Giles J E Heath D B Hogan J PModderman and R E Conn ldquoToxicity of lactide in dogsafter 2 and 13 weeks of daily oral dosingrdquo Food and ChemicalToxicology vol 37 no 4 pp 335ndash342 1999

[62] YMao X Zhang L Li B Yuan andG Lu ldquoSubchronic toxicityand toxicokinetics of LZB a new proton pump inhibitorafter 13-week repeated oral administration in dogsrdquo RegulatoryToxicology and Pharmacology vol 50 no 1 pp 75ndash86 2008

[63] H Koop ldquoReview article metabolic consequences of long-term inhibition of acid secretion by omeprazolerdquo AlimentaryPharmacology andTherapeutics vol 6 no 4 pp 399ndash406 1992

[64] K Yamashita S Nakano M Kuwata et al ldquoSingle dosetoxicity studies of suplatast tosilate (IPD-1151T)rdquoThe Journal ofToxicological Sciences vol 17 no 2 pp 1ndash9 1992

[65] Cargill Acute Oral Toxicity Study of Lacide 2097-99-2 in Rats(EPA Guidelines) 1993

[66] I M Kapetanovic J A Crowell R Krishnaraj A Zakharov MLindeblad and A Lyubimov ldquoExposure and toxicity of greentea polyphenols in fasted and non-fasted dogsrdquo Toxicology vol260 no 1ndash3 pp 28ndash36 2009

[67] M S Werley P McDonald P Lilly et al ldquoNon-clinical safetyand pharmacokinetic evaluations of propylene glycol aerosol inSprague-Dawley rats and Beagle dogsrdquo Toxicology vol 287 no1ndash3 pp 76ndash90 2011

[68] M Kaszkin-Bettag A Richardson R Rettenberger and PW Heger ldquoLong-term toxicity studies in dogs support thesafety of the special extract ERr 731 from the roots of Rheumrhaponticumrdquo Food and Chemical Toxicology vol 46 no 5 pp1608ndash1618 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


researchers have demonstrated the effects of P tenuifolia butinformation on its safety is lacking Therefore systematicevaluation of the safety of the root extract of this herbis necessary for the development of new foods or drugsIn this study an alcohol extract from the dried root of Ptenuifolia was prepared and its safety was evaluated usingan acute oral toxicity test and a subchronic oral toxicity testin Sprague-Dawley rats and beagle dogs in accordance withtwo standards the Korea Food and Drug AdministrationNotification numbers 2005-60 and 2005-79

2 Materials and Methods21 Preparation of the Root Extract of Polygala tenuifolia Thedried root extract of Polygala tenuifolia (500 g) was refluxedwith 75 ethanol for 4 hr in a boiling water bath Thisprocedure was repeated twice and the ethanol solution wasconcentrated under a vacuum The resulting concentratedethanol fraction (125 g) of the plant root was used for thisstudy [12]

22 Experimental Animals and Animal Husbandry Six-week-old specific pathogen-free Sprague-Dawley (HSD SD)rats were used Six animals of each sex for the acute oral toxi-city study and 40 animals of each sex for the subchronic oraltoxicity study were received from Koatech Co (Pyeongtaek-si Gyeonggi-do Korea) Ltd For the acute oral toxicity studysix beagle dogs of each sex at 6 months of age were obtainedfromtheHanlim Laboratory Animals Company (Hwaseong-si Gyeonggi-do Korea)The animal room was maintained ata temperature of 23 plusmn 3∘C with a relative humidity of 55 plusmn15 air ventilation of 10ndash20 timesh and ambient light (150ndash300 Lux) controlled to produce a 12 h lightdark cycle Ani-mals were given irradiation-sterilized pellet feed and groundwater disinfected by an ultraviolet sterilizer and ultrafilteredad libitumThe dogs were reared in stainless steel cages (8000times 900 times 750mm) and identification cards showing theirstudy number and animal number were attached to the cages

23 Study Design Overview This study was conducted incompliance with the Good Laboratory Practice (GLP) andTest Guidelines of the Organization for Economic Coopera-tion and Development [20 21] and the Korea Food and DrugAdministration [22 23] at the GLP Institute approved by theKFDA (Korea Food and Drug Administration) The studyprotocol was approved by the Institutional Animal Care andUse Committee of the institute which is accredited by theAssociation for Assessment and Accreditation of LaboratoryAnimal Care International 2010

231 Acute Oral Toxicity Study in Rats After a 7-day quaran-tine and acclimatization period ten healthy animals of eachgender at 6 weeks of age were used The study included twogroups a control group given no treatment and an exper-imental group given 2000mgkg based on a preliminarystudy (data not shown) Each group consisted of five ratsof each sex and body weight ranges at dosing were 16161ndash16838 g for males and 14498ndash15817 g for females The testsubstance was administered orally to each group Thereafter

clinical signs and body weight were observed throughout the15-day experimental period and gross findingswere observedon the scheduled necropsy day This study was carriedout in accordance with the standard operating proceduresof SNUH-GLP (Non-Clinical Research Center BiomedicalResearch Institute Seoul National University Hospital)

232 Acute Oral Toxicity Study in Beagle Dogs Six maleand six female dogs were divided into three groups of fouranimals then two dogs of each gender were allocated Thestudy included three groups a control group given no treat-ment and experimental groups exposed to 1000mgkg and2000mgkg The body weight ranges at dosing were 7269ndash8785 kg for males and 6531ndash7449 kg for females The testsubstance was administered orally to each group Thereafterclinical signs mortality and body weight were observedthroughout the 15-day experimental period and gross find-ings were observed on the scheduled necropsy dayThis studywas carried out in accordance with the standard operatingprocedures of Preclinical ResearchCenter ChemOn (YonginGyeonggi-do Korea) Inc

233 Subchronic Oral Toxicity Study in Rats After a 7-dayquarantine and acclimatization period forty healthy animalsof each gender at 6 weeks of age were used A dose of1000mgkgday was set as the high dose considering theresults of a preliminary 1-week subchronic oral toxicity study(data not shown) and the characteristics of the test substanceDoses of 600 and 360mgkgday were set as the middle andlow doses respectively using a common ratio of 167 Thevehicle-treated group was administrated with sterile waterfor injection only Each group consisted of 10 rats of eachsex and 10 rats of each sex and group were killed after a 90-day treatment period Body weight ranges at the beginningof dosing were 16956ndash19056 g for males and 12530ndash14200 gfor females The test substance was administered repeatedlyby the oral route to each group for 90 days The followingitemswere examined during the experimental period clinicalsigns body weight food and water consumption ophthalmicexamination urinalysis hematology blood clotting timeserum biochemistry necropsy findings organ weight andhistopathologyThis studywas carried out in accordance withthe standard operating procedures of Preclinical ResearchCenter ChemOn Inc

24 Observation and Examination Items of the Acute OralToxicity Study in Rats

241 Clinical Signs In the rat studies clinical signs andmortality were observed continuously for the first 1 h afteradministration and every hour for 6 h Each animal wasobserved daily throughout the entire 15-day experimentalperiod Abnormal type and severity of signs as well asthe observation day and time were recorded The day ofadministration was set as day 1

242 Body Weight Each rat was measured on days 1 3 7and 14 using an electronic balance (Sartorius Co GottingenLower Saxony Germany)



2




















3 Results























Sex male













Sex female












0(119899 = 2)

1 7283 7350 7694 7741 8226 09432 8075 8043 8097 8185 8448 0373


1000(119899 = 2)

3 8785 8549 8666 8551 8938 01534 7961 7863 8128 8068 8352 0391


2000(119899 = 2)

5 8282 8225 8437 8511 8918 06366 7269 7595 7375 7588 7959 0690



0(119899 = 2)

7 7145 7412 7160 7308 7567 04228 6531 6656 6291 6875 7159 0628


1000(119899 = 2)

9 7433 7465 7417 7395 7307 minus012610 7046 6474 6658 6660 6930 minus0116


2000(119899 = 2)

11 7449 7138 7238 7245 7534 008512 7096 6898 7111 7007 7156 0060






























6and



































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2




















3 Results























Sex male













Sex female












0(119899 = 2)

1 7283 7350 7694 7741 8226 09432 8075 8043 8097 8185 8448 0373


1000(119899 = 2)

3 8785 8549 8666 8551 8938 01534 7961 7863 8128 8068 8352 0391


2000(119899 = 2)

5 8282 8225 8437 8511 8918 06366 7269 7595 7375 7588 7959 0690



0(119899 = 2)

7 7145 7412 7160 7308 7567 04228 6531 6656 6291 6875 7159 0628


1000(119899 = 2)

9 7433 7465 7417 7395 7307 minus012610 7046 6474 6658 6660 6930 minus0116


2000(119899 = 2)

11 7449 7138 7238 7245 7534 008512 7096 6898 7111 7007 7156 0060






























6and



































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results























Sex male













Sex female












0(119899 = 2)

1 7283 7350 7694 7741 8226 09432 8075 8043 8097 8185 8448 0373


1000(119899 = 2)

3 8785 8549 8666 8551 8938 01534 7961 7863 8128 8068 8352 0391


2000(119899 = 2)

5 8282 8225 8437 8511 8918 06366 7269 7595 7375 7588 7959 0690



0(119899 = 2)

7 7145 7412 7160 7308 7567 04228 6531 6656 6291 6875 7159 0628


1000(119899 = 2)

9 7433 7465 7417 7395 7307 minus012610 7046 6474 6658 6660 6930 minus0116


2000(119899 = 2)

11 7449 7138 7238 7245 7534 008512 7096 6898 7111 7007 7156 0060






























6and



































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































Sex male













Sex female












0(119899 = 2)

1 7283 7350 7694 7741 8226 09432 8075 8043 8097 8185 8448 0373


1000(119899 = 2)

3 8785 8549 8666 8551 8938 01534 7961 7863 8128 8068 8352 0391


2000(119899 = 2)

5 8282 8225 8437 8511 8918 06366 7269 7595 7375 7588 7959 0690



0(119899 = 2)

7 7145 7412 7160 7308 7567 04228 6531 6656 6291 6875 7159 0628


1000(119899 = 2)

9 7433 7465 7417 7395 7307 minus012610 7046 6474 6658 6660 6930 minus0116


2000(119899 = 2)

11 7449 7138 7238 7245 7534 008512 7096 6898 7111 7007 7156 0060






























6and



































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















0(119899 = 2)

1 7283 7350 7694 7741 8226 09432 8075 8043 8097 8185 8448 0373


1000(119899 = 2)

3 8785 8549 8666 8551 8938 01534 7961 7863 8128 8068 8352 0391


2000(119899 = 2)

5 8282 8225 8437 8511 8918 06366 7269 7595 7375 7588 7959 0690



0(119899 = 2)

7 7145 7412 7160 7308 7567 04228 6531 6656 6291 6875 7159 0628


1000(119899 = 2)

9 7433 7465 7417 7395 7307 minus012610 7046 6474 6658 6660 6930 minus0116


2000(119899 = 2)

11 7449 7138 7238 7245 7534 008512 7096 6898 7111 7007 7156 0060






























6and



































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































6and



































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















preclinical safety of the root exract of polygala tenuifolia

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