research article antioxidant and toxicity studies of 50%...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 351602 10 pageshttpdxdoiorg1011552013351602

Research ArticleAntioxidant and Toxicity Studies of 50 Methanolic Extractof Orthosiphon stamineus Benth

Mun Fei Yam1 Chung Pin Lim1 Lee Fung Ang1 Lip Yee Por2 Siew Tung Wong3

Mohd Zaini Asmawi1 Rusliza Basir4 and Mariam Ahmad1

1 School of Pharmaceutical Sciences Universiti Sains Malaysia 11800 Pulau Penang Malaysia2 Faculty of Computer Science and Information Technology University of Malaya 50603 Kuala Lumpur Malaysia3 International Medical University Jalan Jalil Perkasa 19 Taman Esplanade 57000 Kuala Lumpur Malaysia4 Faculty of Medicine and Health Sciences Universiti Putra Malaysia 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Mariam Ahmad mariamusmmy

Received 5 October 2013 Revised 23 November 2013 Accepted 24 November 2013

Academic Editor Isabel C F R Ferreira

Copyright copy 2013 Mun Fei Yam 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 present study evaluated the antioxidant activity and potential toxicity of 50 methanolic extract of Orthosiphon stamineus(Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats Superoxide radical scavenging hydroxyl radicalscavenging and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract In acute toxicitystudy single dose of MEOS 5000mgkg was administered to rats by oral gavage and the treated rats were monitored for 14days While in the subchronic toxicity study MEOS was administered orally at doses of 1250 2500 and 5000mgkgday for 28days From the results MEOS showed good superoxide radical scavenging hydroxyl radical scavenging ferrous ion chelatingand antilipid peroxidation activities There was no mortality detected or any signs of toxicity in acute and subchronic toxicitystudies Furthermore there was no significant difference in bodyweight relative organ weight and haematological and biochemicalparameters between bothmale and female treated rats in any doses tested No abnormality of internal organs was observed betweentreatment and control groupsThe oral lethal dose determined wasmore than 5000mgkg and the no-observed-adverse-effect level(NOAEL) of MEOS for both male and female rats is considered to be 5000mgkg per day

1 Introduction

Herbal medicines have received a great deal of attention asalternative medicines in recent years inMalaysia and are soldas dietary supplements One of the Malaysian local herbsscientifically known as Orthosiphon stamineus Benth (Lami-aceae) or locally called Misai Kucing has attracted muchattention for research purposesOrthosiphon stamineus is alsofound in other countries such as Thailand Indonesia andEurope It is widely used for the treatment of many diseasesespecially those affecting the urinary tract diabetes mellitushypertension rheumatism tonsillitis and menstrual disor-ders [1] The benefits of the traditional use of Orthosiphonstamineus have also been supported by the isolation andidentification of several possible active chemical constituentsfrom this plant including flavonoids [2 3] terpenoids [45] saponins hexoses organic acids caffeic acid derivatives

chromene and myo-inositol [4ndash6] Among the reportedcompounds the most important components ofOrthosiphonstamineus leaves are the polyphenols polymethoxylatedflavonoids such as sinensetin and eupatorine caffeic acidderivatives which include rosmarinic acid cichoric acid andcaffeic acid [6]

Notwithstanding the widespread and long time usageof this plant little toxicological information is availableregarding the safety following chronic consumption of Ostamineus especially the bioactive 50 methanolic extractwhich has been reported to be effective in protection againstalcohol-induced gastropathy CCl

4-induced liver damage

antipyretic anti-inflammatory and analgesic effects [7ndash10]Currently Malaysian authorities are paying more and moreattention on the safety and potential toxicity of botanicalsincluding medicinal plants and edible materials Thereforethe aims of the present study were to evaluate the antioxidant

2 BioMed Research International

and provide scientific data on the safety of O stamineusfocusing on the 14-day acute and 28-day subchronic toxicityof bioactive 50 methanolic extract of O stamineus usingSprague-Dawley (SD) rats and hence providing guidance forselecting a safe dose of O stamineus in its use as a traditionalmedicine

2 Materials and Methods

21 Chemical Ferrous chloride (FeCl2) hematoxylin

ferrozine 2-deoxyribose ethylenediaminetetraacetic acid(EDTA) ferrous sulfate (FeSO

4) trichloroacetic acid (TCA)

nitroblue tetrazolium (NBT) xanthine xanthine oxidasesodiumphosphate (dibasic) sodiumphosphate (monobasic)butylated hydroxytoluene (BHT) butylated hydroxyanisole(BHA) manganese chloride xylenol orange manganesechloride and sodium dihydrogen phosphate (NaH

2PO4)

were purchased from Sigma (St Louis MO USA) Absolutealcohol and eosin were purchased from Riedel-de Haen(Seelze Germany) Paraplast was purchased from OxfordLabware (St Louis MO USA) Xylene was purchased fromFisher Scientific (Leics UK) Disposable microtome blades818 were purchased from LEICA (Germany) Thiobarbituricacid (TBA) was purchased from AppliChem (DarmstadtGermany) 31015840-hydroxy-56741015840-tetramethoxyflavone sinen-setin and eupatorin were obtained from Indofine ChemicalCompany (NJ USA) HPLC-grade acetonitrile and isopropylalcohol were purchased fromMerck (Darmstadt Germany)

22 Plant Materials and Extract Orthosiphon stamineus wasgrown from cuttings using standard agronomic practices atKepala Batas Pulau Pinang Malaysia The leaves of the plantwere collected after flowering The leaves were identified byMr Adenan Jaafar School of Biological Sciences UniversitiSains Malaysia A voucher specimen (number 10810) wasdeposited at the Herbarium of School of Biological SciencesUniversiti Sains Malaysia The leaves were rinsed and driedin oven at 40∘C for 2 daysThe dried leaves were then groundby an electric grinder to a coarse powder and weighedSubsequently 300 g of powdered leaves of O stamineuswas extracted with 5 L methanol water (50 50 vv) at 60∘C(for 8 hours) by maceration method The resulting 50methanolic extract of O stamineus (MEOS) was concen-trated using a Buchi-RE121 evaporator (Buchi Laboratorium-Technik Switzerland) equipped with a Buchi-B169 vacuumsystem and then lyophilized in a Hetovac VR-1 (HETO LabEquipment Denmark) freeze dryerThe weight of the MEOSwas recorded and the final plant-to-extract ratio was about6 The MEOS was then kept in desiccators in a refrigerator(0ndash4∘C) The MEOS was freshly prepared daily by dissolvingin distilled water

23 HPLC Study

231 HPLC Instrumentation HPLC analysis was performedusing a Shimadzu-LC system (Shimadzu Japan) equippedwith a CBM-20A controller LC-20AT pump DGU-20A5degasser SIL-20A autosampler SPD-20AV detector andCTO-10ASvp column oven

232 Chromatographic Conditions Chromatographic sepa-rations were achieved using an Agilent Eclipse Plus C18 (250times 46mm id 5 120583m) A Zorbax guard fittings kit packed withreplaceable Eclipse Plus C18 Guard column (125 times 46mmid 5 120583m) was used to protect the analytical column Areverse-phase HPLC assay was carried out using an isocraticsystem with a flow rate of 1mLmin a column temperatureof 25∘C and a mobile phase of acetonitrile isopropyl alco-hol 20mM phosphate buffer (NaH

2PO4) (30 15 55 vv)

with pH adjusted to 35 using 85 phosphoric acid TheUV detection was set at 340 nm The injection volume was20120583L of solution Total run time was less than 20min foreach injection Data was acquired and processed with LC-Solution software The peaks were detected at 340 nm andidentified using reference standards of 31015840-hydroxy-56741015840-tetramethoxyflavone sinensetin and eupatorin Sinensetinand eupatorin were selected as standards because these twocompounds were found to be the bioactive ingredients in theplant [11ndash13]

24 Antioxidant

241 Ferrous Ion Chelating Activity Ferrous chelating activ-ity was determined according to the method of Dinis etal [14] MEOS was added to a solution of 005mL ferrouschloride (FeCl

2) (2mM) The reaction was initiated by the

addition of 02mL ferrozine (5mM) and the mixture wasshaken vigorously and left standing at room temperature(24 plusmn 2∘C) for 10min Absorbance of the solution was thenmeasured spectrophotometrically (U-2000Hitachi Japan) atwavelength 562 nmThepercentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the followingformula

Ferrous ion chelating activity = [(1198600minus 1198601)

1198600

] times 100

(1)

where 1198600was the absorbance of the control and 119860

1was

the absorbance in the presence of extracts The experimentwas run together with butylated hydroxytoluene (BHT) andbutylated hydroxyanisole (BHA) as positive controls

242 Hydroxyl Radical Scavenging Activity Competitionbetween deoxyribose and the extract against hydroxyl radicalgenerated from the Fe3+ascorbateethylenediaminetetraace-tic acid (EDTA)hydrogen peroxide (H

2O2) system was mea-

sured to determine the hydroxyl radical scavenging activityof MEOS [15] The reaction mixture consisted of 030mLof 002M sodium phosphate buffer (pH 70) 015mL of10mM 2-deoxyribose 015mL of 10mM FeSO

4 015mL of

10mMEDTA 015mL of 10mMH2O2 0525mL ofH

2O and

0075mL of extract The reaction mixture was incubated at37∘C for 2 h and the formed TBARS were measured Seventyfive milliliter of 28 trichloroacetic acid and 075mL of 10TBA in 50mMNaOHwere added to test tubes and boiled for20min After cooling the mixture absorbance was measuredat 520 nm The percentage of inhibition of inhibition rate of

BioMed Research International 3

2-deoxyribose oxidation by hydroxyl radical was given in thefollowing formula

Hydroxyl radical scavenging activity =[(1198600minus 1198601)

1198600

]times 100

(2)


1was the

absorbance in the presence of extracts

243 Superoxide Anion Radical Scavenging Activity Super-oxide anion radical generated by the xanthinexanthineoxidase system was determined spectrophotometrically bymonitoring the product of nitroblue tetrazolium (NBT) [16]The reactionmixture consisted of 10mL of 005M phosphatebuffer (pH 74) 004mL of 3mM xanthine 004mL of 3mMEDTA 004mL of 015 bovine serum albumin 004mL of15mM NBT and 004mL of sample solution After incuba-tion at 25∘C for 10min the reaction was initiated by adding004mL of 15 UmL xanthine oxidase and carried out at25∘C for 20min After 20min the absorbance of the reactionmixture was measured at 560 nm Decreased absorbance ofthe reaction mixture indicates increased superoxide anionradical scavenging activity

244 Lipid Peroxidation Inhibition Study Fresh livers wereobtained from healthy male adult SD rats They were thenhomogenized according to the method [17] for the prepara-tion of 40 (wv) liver homogenates in methanol 80 120583L ofliver homogenate was mixed with 10 120583L of Fentonrsquos reagent(containing 5120583L of 5mM manganese chloride and 5 120583L of50mM hydrogen peroxide) and 10120583L of various concentra-tions of MEOS (for control the extract was replaced withdistilled water) The mixture was incubated for 30min at37∘C to produce lipid peroxidation The mixture was thenadded with 900mL FOX reagent (containing 49mg ammo-nium ferrous sulfate in 50mL of 250mM H

2SO4 0397 g of

BHT and 0038 g of xylenol orange in 950mL methanol)Absorbance of themixture was read at 560 nm after 30min ofincubation at room temperatureThe percentage of inhibitionwas calculated with the following equation

of inhibition = [(1198600minus 1198601)

1198601

] times 100 (3)

where 1198600was the absorbance in the presence of extracts and

1198601was the absorbance of the control

25 Toxicology Studies

251 Experimental Animals Eight-week-old male andfemale Sprague-Dawley rats (160ndash190 g)were purchased fromthe Animal House School of Pharmaceutical Sciences Uni-versiti Sains Malaysia The rats were acclimatised to labo-ratory conditions for 7 days prior to performing the experi-ments All rats were kept at 26 plusmn 3∘C with a lightdark cycleof 12 hours The rats were housed in a single polycarbonatecage (3 rats per cage) with free access to food (normal

laboratory chow Gold Coin) and tap water ad libitum Allthe procedures were performed according to the AnimalEthics Guidelines of Universiti Sains Malaysia

252 Acute Toxicity Study The experiment was performedaccording to the Organisation for Economic Cooperationand Development (OECD) revised up and down procedurefor acute toxicity testing [18] All the rats were fastedovernight and weighed before the extract was administeredA maximum dose of 5000mgkg of MEOS was administeredby oral gavage to 5 healthy female adult Sprague-Dawleyrats The administration volume was adjusted between 1and 2mL for each and every rat After administration ofMEOS to the first rat the rat was observed for clinical signsof toxicity for the first hour then hourly for three hoursand then periodically throughout 48 hours Other rats wereadministrated sequentially at 48-hour intervals if the first ratsurvived after 48 hours of treatment All the experimental ratswere maintained under close observation throughout the 14days and the number of mortality was recorded The LD

50

was predicted to be above 5000mgkg if three or more ratssurvived after the experimental period [19]

253 Subchronic Toxicity Study Experimental Sprague-Dawley rats of either sex were randomly assigned to 4groups (119899 = 12 six males and six females per group)and their weights were recorded Different doses of MEOS(1250 2500 or 5000mgkg) were prepared in distilled waterand administered daily as single doses to different groupsof rats group 1 (5000mgkg) group 2 (2500mgkg) andgroup 3 (1250mgkg) while group 4 (control) receivedonly distilled water Toxic manifestations and mortality weremonitored daily for 28 days The bodyweights of all therats were measured and recorded at the end of every weekand the treated rats were anesthetised using CO

2after 28

days of treatment Blood samples were collected via cardiacpuncture and transferred into nonheparinised and EDTA-containing tubes for both biochemical and haematologicalanalyses respectively [20] Thereafter the rats were killed bycervical dislocation All organs brain heart lungs thymusliver kidneys adrenal glands sex organs (ovaries and uterusfor female rats testes for male rats) spleen stomach andgut (begining from small intestine until the end of largeintestine) of all experimental rats were excised weighedand examined macroscopically Vital organs such as lungskidneys livers and stomach were then preserved in 10formalin for histopathological study [19]

254 Relative Organ Weight The excised organs wereweighed individually The relative organ weight index ofeach organ to its bodyweight was calculated as (weight oforganbodyweight of rats on the day of sacrifice) times 100 [20]

255 Blood Analyses Haematological and biochemical anal-yses were performed at the Pathology Laboratory LamWah Ee Hospital Penang Complete blood cell counts weredetermined using a fully automated haematological analyserAbbott Cell-Dyn 3500 (Abbott Laboratories IL USA) while


05

1015202530354045

0 05 1 15 2

Chel

atin

g eff

ect (

)

Concentration (mgmL)

BHTBHA

MEOS

Figure 1 Ferrous ion chelating activity of MEOS (119899 = 3) Data areexpressed as mean plusmn SD

serum biochemistry tests were performed using a COBASIntegra 800 (Roche Germany) [20]

256 Histopathological Study Vital organs (kidney and liver)were processed using a Citadel 1000 Histokinette (ShandonScientific Ltd Cheshire UK) These tissues were embeddedin paraffinusing aHisto-Center II-N (BarnsteadThermolyneCorp Dubuque IA USA) and cut into 5 120583m thick sectionswith a Reichert-Jung Histocut 820 II (Cambridge InstrumentGmbH Nussloch Germany) All the sections were stainedwith haematoxylin and eosin and examined microscopically[20]

26 Statistical Analysis Statistical analysis was carried outusing the Statistical Package for Social Sciences (SPSS) Allthe data are indicated as mean plusmn standard error of mean(SEM) and were analysed using one-way analysis of variance(ANOVA) Significant differences between the groups weredetermined using a Dunnett-comparison test with 119875 lt 005taken as significant

3 Results

31 HPLC Analysis The objective of HPLC analysis in thisexperiment was to standardize the MEOS using sinensetineupatorin and31015840-hydroxy-56741015840-tetramethoxyflavone Fromthe results the percentages of sinensetin eupatorin and 31015840-hydroxy-56741015840-tetramethoxyflavone determined in MEOSwere 112 plusmn 0026 094 plusmn 0025 and 046 plusmn 0016 (ww)respectively

32 Antioxidant Activities

321 Ferrous Ion Chelating Activity Ferrozine can quantita-tively form the complexes with Fe2+ Chelating activity of theMEOSwith ferrous ion was 16 at 0125mgmL In additionthere was an increase of chelating activity to 30 at 2mgmL

0

10

20

30

40

50

60

70

80

90

0 05 1 15 2

Hyd

roxy

radi

cal s

cave

ngin

g ac

tivity

()


BHTBHA

MEOS

Figure 2 Hydroxyl radical scavenging activity of MEOS (119899 = 3)Data are expressed as mean plusmn SD

0102030405060708090

0 02 04 06 08 1 12 14 16 18 2Supe

roxi

de ra

dica

l sca

veng

ing

activ

ity (

)


BHTBHA

MEOS

Figure 3 Superoxide radical scavenging activity of MEOS (119899 = 3)Data are expressed as mean plusmn SD

which was slightly lower than positive control (BHA andBHT) (Figure 1)

322 Hydroxyl Radical Scavenging Activity The hydroxylradical scavenging activity was investigated by using theFenton reaction As shown in Figure 2 addition of less than2mgmL of MEOS effectively inhibited the formation ofhydroxyl radicals linearly up to 72

323 Superoxide Anion Radical Scavenging Activity Super-oxide anion radical scavenging activity of MEOS was deter-mined by the xanthine oxidase system Figure 3 showedthe percentage inhibition of superoxide radical gener-ated by BHT BHA and MEOS The inhibition activityof MEOS reached 63 when 2mgmL of extract wasused


140

160

180

200

220

240

260Bo

dy w

eigh

t (g)

Before Week 1 Week 2 Week 3 Week 4

Control1250mgkg

2500mgkg5000mgkg

(a)

140

160

180

200

220

240

260

Body

wei

ght (

g)


Control1250mgkg

2500mgkg5000mgkg

(b)

Figure 4 The effect of daily oral administration of MEOS on the bodyweight of (a) male and (b) female rats (119899 = 6) Data are expressed asmean plusmn SEM

324 Lipid Peroxidation Inhibition Study Antilipid peroxi-dation activity ofMEOSwas determined by the FOXmethodIC50

of liver homogenate lipid peroxidation of MEOS was034 plusmn 0024mgmL


331 Acute Toxicity Study In this experiment oral admin-istration of 5000mgkg of MEOS did not cause any visiblesigns of toxicity to the rats No changes were observed in thebehavior and mortality of the animals over the 14 days Allfive female SD rats survived until the end of the experimentwith no mortality were recorded The LD

50determined was

greater than 5000mgkg

332 Subchronic Toxicity Study In subchronic toxicity studythere were no observable changes in the general behaviourof all the treated rats (groups 1 2 and 3) as compared to thecontrol group No significant changes were detected in eitherthe bodyweights (Figure 4) or relative organweights (Table 1)of all the treated rats In addition no death was recorded inboth sexes of the treatment groups as well as their respectivecontrol group during or after the course of the experimentThe controls and the treated rats appeared uniformly healthyat the end of the experiment and throughout the 28 daystreatment period

333 Blood Analyses There were no significant differencesobserved in any of the haematological parameters tested(red blood cell count (RBC) haemoglobin concentration(Hgb) haematocrit (Ht) mean corpuscular volume (MCV)mean corpuscular haemoglobin (MCH) mean corpuscularhaemoglobin concentration (MCHC) total white blood cellcount (WBC) or white blood cell differential count) in anyof the treated rats as compared to control rats (Table 2)Meanwhile analyses of biochemical parameters (alaninetransaminase (ALT) aspartate transaminase (AST) alka-line phosphatase (ALP) creatinine bilirubin urea sodium

potassium and chlorine) also showed no significant differ-ences in any of the parameters tested between the control andthe treated groups of both sexes (Table 3)

334 Histopathological Study No lesions or pathologicalchanges were observed in the organs of either sex of theMEOS treated rats as compared to their respective controlgroups

4 Discussion

Orthosiphon stamineus Benth (Lamiaceae) is an importantmedicinal plant in Malaysia It has been widely used fortreatment of various ailments Many studies which involvedboth in vivo and in vitro experiments have reported that theOrthosiphon stamineus leaves possess a wide range of phar-macological properties such as anti-inflammatory analgesicgastroprotective antipyretic and hepatoprotective effectsThe present study was conducted to determine the antiox-idant potential which is responsible for the wide range ofpharmacological properties of this plant and to investigate thepossible harmful effects of MEOS in experimental animals atthe specific doses chosen since the safety use of the plant is ofconcern to the Malaysian authorities The toxicity of MEOSin rats was evaluated by both acute 14-day and subchronic28-day toxicological studies Methanol was chosen as oursolvent since many literature reviews showed that this extractwas very effective in many pharmacological activities such asantipyretic anti-inflammatory and analgesic effects as well asa good protective agent against alcohol-induced gastropathyand CCl

4-induced liver damage [7ndash10]

Antioxidant capacity is one of the commonly used param-eters to determine the bioactive components which exhibitedpharmacological activities Orthosiphon stamineus has beenshown to be a potent scavenger of a variety of reactiveradical species such as DPPH and ABTS [7]Therefore in thepresent study the antioxidant potential of MEOS was testedby Ferrous ion chelating activity hydroxyl radical scavenging


Table 1 (a) Relative organs weight of male rats orally treated daily with 50 methanolic extract of Orthosiphon stamineus for 28 days (b)Relative organs weight of female rats orally treated daily with 50 methanolic extract of Orthosiphon stamineus for 28 days (contrsquo)

(a)

organ weightbodyweightTreatment for 28 days

Control 50 methanolic extract of Orthosiphon stamineus1250mgkg 2500mgkg 5000mgkg

MaleBrain 042 plusmn 002 039 plusmn 001 043 plusmn 002 042 plusmn 002

Heart 154 plusmn 003 144 plusmn 013 143 plusmn 008 137 plusmn 006

Liver 234 plusmn 010 242 plusmn 016 248 plusmn 015 246 plusmn 014

Thymus 014 plusmn 001 016 plusmn 001 015 plusmn 001 015 plusmn 001

Spleen 027 plusmn 002 027 plusmn 001 025 plusmn 001 027 plusmn 002

Kidney (right) 035 plusmn 001 037 plusmn 002 033 plusmn 001 035 plusmn 001

Kidney (left) 032 plusmn 001 033 plusmn 001 032 plusmn 001 033 plusmn 001

Adrenal gland 001 plusmn 000 002 plusmn 000 001 plusmn 000 001 plusmn 000

Lungs 067 plusmn 005 071 plusmn 006 063 plusmn 002 063 plusmn 003

Testis (right) 056 plusmn 002 054 plusmn 001 055 plusmn 001 055 plusmn 002

Testis (left) 054 plusmn 003 055 plusmn 001 054 plusmn 002 056 plusmn 001

Stomach 274 plusmn 031 263 plusmn 006 293 plusmn 008 256 plusmn 011

Stomach (empty) 057 plusmn 001 059 plusmn 001 051 plusmn 002 054 plusmn 002

Gut 664 plusmn 021 638 plusmn 014 645 plusmn 014 632 plusmn 030

Gut (empty) 327 plusmn 003 344 plusmn 011 317 plusmn 004 324 plusmn 013

Data are expressed as mean plusmn SEM

(b)



FemaleBrain 040 plusmn 000 039 plusmn 000 040 plusmn 001 038 plusmn 001









Ovaries 013 plusmn 001 009 plusmn 001 010 plusmn 001 011 plusmn 001

Uterus 021 plusmn 000 016 plusmn 001 019 plusmn 002 015 plusmn 001






activity and superoxide anion radical scavenging activityOur study showed that the antioxidant activity of MEOS wascomparable to the positive controls BHA and BHT In theantioxidant activities we reported that MEOS can stronglyreduce the hydroxyl and supercritical onion radicals This

finding is comparable to the previous study which reportedthat MEOS inhibited lipid peroxidation in different animalmodels [7 10] In addition it has been long reported thatthere is a strong association between the termination of freeradical propagation in biological systems with the reduction


Table 2 (a) Haematological values ofmale rats treated with 50methanolic extract ofOrthosiphon stamineus for 28 days (b) Haematologicalvalues of female rats treated with 50 methanolic extract of Orthosiphon stamineus for 28 days (contrsquo)

(a)

UnitTreatment for 28 days


MaleWhite blood cell count 109L 957 plusmn 032 1105 plusmn 048 1078 plusmn 059 1073 plusmn 021

Neutrophils 109L 246 plusmn 018 309 plusmn 022 315 plusmn 031 318 plusmn 043

Lymphocytes 109L 667 plusmn 024 694 plusmn 046 672 plusmn 029 667 plusmn 041

Monocytes 109L 024 plusmn 003 062 plusmn 009 053 plusmn 015 054 plusmn 005

Eosinophils 109L 002 plusmn 000 004 plusmn 001 005 plusmn 001 005 plusmn 001

Basophils 109L 018 plusmn 003 035 plusmn 004 033 plusmn 008 030 plusmn 002

Red blood cell count 1012L 855 plusmn 012 836 plusmn 017 843 plusmn 020 824 plusmn 003

Hemoglobin gL 1480 plusmn 021 1476 plusmn 014 1502 plusmn 032 1449 plusmn 008

Hematocrit 075 plusmn 001 074 plusmn 002 075 plusmn 002 071 plusmn 000

Mean red blood cell volume fL 8803 plusmn 074 8815 plusmn 127 8851 plusmn 174 8647 plusmn 081

Mean corpuscular Hb pg 1734 plusmn 019 1770 plusmn 031 1775 plusmn 022 1758 plusmn 007

Mean corpuscular Hb concentration gL 1971 plusmn 025 2016 plusmn 040 2012 plusmn 040 2039 plusmn 016

Platelets 109L 120561 plusmn 2653 114919 plusmn 2980 114528 plusmn 6730 112536 plusmn 1804

Mean platelet cell volume fL 812 plusmn 012 846 plusmn 017 826 plusmn 019 811 plusmn 013


(b)



FemaleWhite blood cell count 109L 766 plusmn 096 677 plusmn 043 631 plusmn 072 724 plusmn 035















of chronic diseases DNA damage mutagenesis carcinogene-sis and inhibition of pathogenic bacterial growth [21] Henceas a potent antioxidant it is not surprising that Orthosiphonstamineus is effective in treating various oxidative stress-related chronic diseases such as diabetes alcohol-inducedstomach ulcer and CCl


In acute 14-day toxicity study a limited dose was selectedand performed on the experimental rats Its purpose wasto determine a proper range of doses to be used in the

subsequent subchronic 28-day toxicological study [19] In thisexperiment a single dose of 5000mgkg MEOS was givenorally to five female Sprague-Dawley rats No significantmortality or alteration in the behaviour patterns of the treatedrats was observed compared to their respective controls

During the subchronic 28-day toxicity study the ratswere treated orally with different doses of MEOS (12502500 and 5000mgkgday) for 28 days The results obtainedwere comparable to those in the acute toxicity study Both


Table 3 Biochemical values of male and female rats treated with 50 methanolic extract of Orthosiphon stamineus for 28 days



MaleAspartate transaminase (AST) UL 12274 plusmn 219 13194 plusmn 353 12679 plusmn 940 12181 plusmn 245

Alanine aminotransferase (ALT) UL 6451 plusmn 298 6243 plusmn 259 6450 plusmn 386 6522 plusmn 273

Urea UL 631 plusmn 034 665 plusmn 034 650 plusmn 040 674 plusmn 014

Creatinine 120583molL 4056 plusmn 081 3959 plusmn 030 4108 plusmn 116 3933 plusmn 045

Alkaline phosphatase (ALP) 120583molL 30056 plusmn 718 30963 plusmn 1008 30950 plusmn 1149 31686 plusmn 1342

Bilirubin mmolL 180 plusmn 000 180 plusmn 000 180 plusmn 000 180 plusmn 000

Sodium gL 14253 plusmn 036 14253 plusmn 022 14196 plusmn 058 14222 plusmn 031

Potassium gL 469 plusmn 012 464 plusmn 006 472 plusmn 007 465 plusmn 008

Chlorine gL 10272 plusmn 060 10271 plusmn 033 10375 plusmn 028 10353 plusmn 061

FemaleAspartate transaminase (AST) UL 12783 plusmn 251 12772 plusmn 498 12261 plusmn 1041 11728 plusmn 810










control and treated rats of both sexes appeared generallyhealthy during and throughout the experimental period Nomortality was recorded and no toxicity signs were detectedin any of the treated rats Both evaluation of the acute andsubchronic toxicity of MEOS had indicated that a singleoral administration of MEOS up to 5000mgkg dose causedneither visible signs of toxicity nor mortality to experimentalrats Hence the LD

50of MEOS determined is more than

5000mgkgGenerally an increase or decrease in bodyweight of an

animal has been used as an indicator of adverse effect of drugsand chemicals [22] In our study all the treated animals sur-vived beyond the observation periodThebodyweight of bothcontrol and treatment groups increased gradually throughoutthe experimental period with no significant difference Theincrease in bodyweight was not significantly different ascompared to the control group in both acute and subchronictoxicity studies All the rats at each dosage level continued togain weight throughout the experimental period suggestingthat growth inhibition did not occur during this course ofrepeated dosage

In addition to bodyweight relative organ weight has alsobeen used as another basic indicator to determine whetherthe rats have been exposed to harmful agents [19 22] Theirorgans will tend to swell or damage if they were subjected totoxic substances This will subsequently alter their organ-to-bodyweight ratios as compared to the respective controls Inthe present study the relative organ weights of liver lungsspleen sex organs heart and kidneys in all the treated male

and female rats were not significantly different (119875 lt 005)from those of the control groups

Analysis of blood parameters in animal studies could bevery useful when evaluating the risk of human toxicity as thechanges in the haematological system provide a predictivevalue for toxicity [18]These blood parameters are often calledhaematological parameters Several important haematolog-ical parameters were selected in this study to evaluate thetoxicity ofMEOS From the results the haematological profileof all the treated rats showed no significant differencewith thecontrol group (Table 2)This indicated that theMEOS did notaffect the haematopoiesis system of the treated rats

Several important biochemical parameters were alsoincluded in this toxicity study These biological parametersare indicators for organ toxicity For example kidney func-tions were evaluated by means of serum urea and creatinineIncrease of blood creatinine has been shown to be a goodindicator of negative impact in kidney functions [23] Whilethe increase levels of AST andALT in the blood are associatedwith damage of hepatic cells [24 25] The results of theexperiment suggested that the kidney and liver functionswere not altered for both the treated male and femalerats There were no statistically significant differences increatinine AST and ALT levels between controls and treatedanimals at any dosage levels Hence these findings suggestthat MEOS does not cause any hepatotoxicity and kidneydamage to the rats Both these organs were selected becausekidneys and liver are the first organs to show toxicity whenthe rats are exposed to potential toxic substances Based on


the statistical analyses of these biochemical parameters nosignificant difference was observed in any of the parameterstested for either the treated or normal rats (Table 3)

Histopathological studies were conducted on vital organsof all the rats According to Wang et al impaired organsoften have abnormal atrophy [26] However from ourresults no related histopathological changes were observedGross examination in necropsy andmicroscopic examinationrevealed no changes that might be due to the administrationof MEOS (data not shown) No significant changes ordamages were observed in the morphology of all the isolatedvital organs either from the rats treated with MEOS orcontrol rats (data not shown) The isolated organs showednormal architecture These results again suggested that dailyrepeated oral administration of MEOS did not cause anydetrimental changes or morphological disturbances to therats or to their vital organs The no-observed-adverse-effectlevel (NOAEL) of MEOS determined was 5000mgkg body-weightday (gt3000mgkg bodyweightday) and consideredto be a non-hazard compound

5 Conclusion

This finding is comparable with Mohamed et alrsquos [19] andAbdullah et alrsquos (2009) in which O stamineus alcoholicextract did not show any toxic effect in rats during bothacute and subchronic studies Daily oral administration of50 methanolic extract of O stamineus (MEOS) at dosesof 1250 2500 and 5000mgkg to both male and femaleSprague-Dawley rats for 28 days did not result in mortalityand was not associated with adverse effects as reflected inthe observation of general condition growth body and organweights and hematological and biochemical values Thus itsoral lethal dose for both male and female rats is in excessof 5000mgkg There were no abnormalities in necropsyand histopathological findings An NOAEL from the presentstudy was determined to be 5000mgkg per day for ratsunder the condition tested Further investigation on thepreclinical and clinical studies of the extract will be necessaryto determine a safe dose before it becomes a potential drugand to protect the population from possible toxic effectsof MEOS It would be interesting to assess the toxic effectof the compounds found in MEOS such as eupatorin andsinensetin

Conflict of Interests

Mariam Ahmad declares that no competing interests existedfor the authors or the institute before during and afterpreparing and summiting this paper for review

Acknowledgments

This study was funded by Fundamental Research GrantScheme (FRGS) 203PFARMASI6711306 and FRGSFP31-2012A UMRG Program (Sub-Program 3RP003C-13ICT)andHigh Impact ResearchGrant (UMC6251HIRMOHE

FCSIT15) from Universiti Sains Malaysia University ofMalaya and Ministry Education Malaysia

References

[1] L M Perry Medicinal Plants of East and Southeast AsiaAttributed Properties and Uses MIT Press Cambridge MassUSA 1998

[2] K E Malterud I M Hanche-Olsen and I Smith-KiellandldquoFlavonoids from Orthosiphon spicatusrdquo Planta Medica vol 55no 6 pp 569ndash570 1989

[3] W Sumaryono P Proksch V Wray L Witte and T Hart-mann ldquoQualitative and quantitative analysis of the phenolicconstituents fromOrthosiphon aristatusrdquo PlantaMedica vol 57no 2 pp 176ndash180 1991

[4] T Masuda K Masuda and N Nakatani ldquoOrthosiphol A ahighly oxygenated diterpene from the leaves of Orthosiphonstamineusrdquo Tetrahedron Letters vol 33 no 7 pp 945ndash946 1992

[5] Y Tezuka P Stampoulis A H Banskota et al ldquoConstituentsof the Vietnamese medicinal plant Orthosiphon stamineusrdquoChemical and Pharmaceutical Bulletin vol 48 no 11 pp 1711ndash1719 2000

[6] N-K Olah L Radu C Mogosan D Hanganu and S GocanldquoPhytochemical and pharmacological studies on Orthosiphonstamineus Benth (Lamiaceae) hydroalcoholic extractsrdquo Journalof Pharmaceutical and Biomedical Analysis vol 33 no 1 pp 117ndash123 2003

[7] M F Yam R Basir M Z Asmawi and Z Ismail ldquoAntioxidantand hepatoprotective effects of Orthosiphon stamineus BenthStandardized extractrdquo American Journal of Chinese Medicinevol 35 no 1 pp 115ndash126 2007

[8] M F Yam M Z Asmawi and R Basir ldquoAn investigationof the anti-inflammatory and analgesic effects of Orthosiphonstamineus leaf extractrdquo Journal of Medicinal Food vol 11 no 2pp 362ndash368 2008

[9] M F Yam L F Ang R Basir I M Salman O Z Ameerand M Z Asmawi ldquoEvaluation of the anti-pyretic potentialof Orthosiphon stamineus Benth standardized extractrdquo Inflam-mopharmacology vol 17 no 1 pp 50ndash54 2009

[10] M F Yam L F Ang I M Salman et al ldquoOrthosiphonstamineus leaf extract protects against ethanol-induced gas-tropathy in ratsrdquo Journal of Medicinal Food vol 12 no 5 pp1089ndash1097 2009

[11] I Doleckova L Rarova J Gruz M Vondrusova M Strnadand V Krystof ldquoAntiproliferative and antiangiogenic effects offlavone eupatorin an active constituent of chloroform extractof Orthosiphon stamineus leavesrdquo Fitoterapia vol 83 no 6 pp1000ndash1007 2012

[12] H-S Shin S-I Kang S-A Yoon H-C Ko and S-J KimldquoSinensetin attenuates LPS-induced inflammation by regulatingthe protein level of I120581B-120572rdquo Bioscience Biotechnology and Bio-chemistry vol 76 no 4 pp 847ndash849 2012

[13] M Laavola R Nieminen M F Yam et al ldquoFlavonoids eupa-torin and sinensetin present in orthosiphon stamineus leavesinhibit inflammatory gene expression and STAT1 activationrdquoPlanta Medica vol 78 no 8 pp 779ndash786 2012

[14] T C P Dinis V M C Madeira and L M Almeida ldquoActionof phenolic derivatives (acetaminophen salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidationand as peroxyl radical scavengersrdquo Archives of Biochemistry andBiophysics vol 315 no 1 pp 161ndash169 1994


[15] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[16] T Nagai M Sakai R Inoue H Inoue and N Suzuki ldquoAntiox-idative activities of some commercially honeys royal jelly andpropolisrdquo Food Chemistry vol 75 no 2 pp 237ndash240 2001

[17] E Perez A J Rodrıguez-Malaver and P Vit ldquoAntioxidantcapacity of venezuelan honey in wistar rat homogenatesrdquoJournal of Medicinal Food vol 9 no 4 pp 510ndash516 2006

[18] OECD ldquoOECD guideline 425 acute oral toxicity-up-and-downprocedurerdquo inOECDGuidelines for the Testing of Chemicals vol2 Organization for Economic Cooperation and DevelopmentParis France 1998

[19] E A H Mohamed C P Lim O S Ebrika M Z Asmawi ASadikun and M F Yam ldquoToxicity evaluation of a standardised50 ethanol extract of Orthosiphon stamineusrdquo Journal ofEthnopharmacology vol 133 no 2 pp 358ndash363 2011

[20] R Rosidah M F Yam A Sadikun M Ahmad G A Akowuahand M Z Asmawi ldquoToxicology evaluation of standardizedmethanol extract ofGynura procumbensrdquo Journal of Ethnophar-macology vol 123 no 2 pp 244ndash249 2009

[21] Q Y Zhu R M Hackman J L Ensunsa R R Holt andC L Keen ldquoAntioxidative activities of oolong teardquo Journal ofAgricultural and Food Chemistry vol 50 no 23 pp 6929ndash69342002

[22] S Teo D Stirling S Thomas A Hoberman A Kiorpesand V Khetani ldquoA 90-day oral gavage toxicity study of D-methylphenidate and DL-methylphenidate in Sprague-Dawleyratsrdquo Toxicology vol 179 no 3 pp 183ndash196 2002

[23] H Rhiouani J El-Hilaly Z H Israili and B Lyoussi ldquoAcuteand sub-chronic toxicity of an aqueous extract of the leaves ofHerniaria glabra in rodentsrdquo Journal of Ethnopharmacology vol118 no 3 pp 378ndash386 2008

[24] C Burger D R Fischer D A Cordenunzzi A P de BorbaBatschauer V C Filho and A R dos Santos Soares ldquoAcute andsubacute toxicity of the hydroalcoholic extract from Wedeliapaludosa (Acmela brasiliensis) (Asteracea) in micerdquo Journal ofPharmacy and Pharmaceutical Sciences vol 8 no 2 pp 370ndash373 2005

[25] P Witthawaskul A Panthong D Kanjanapothi T Taesothikuland N Lertprasertsuke ldquoAcute and subacute toxicities of thesaponin mixture isolated from Schefflera leucantha ViguierrdquoJournal of Ethnopharmacology vol 89 no 1 pp 115ndash121 2003

[26] T C Wang Y P Su T Y Hsu C C Yang and C C Lin ldquo28-day oral toxicity study of the aqueous extract from spider brake(Pteris multifida Poiret) in ratsrdquo Food and Chemical Toxicologyvol 45 no 9 pp 1757ndash1763 2007

[27] J F Copplestone ldquoThe development of the WHO recom-mended classification of pesticides by hazardrdquo Bulletin of theWorld Health Organization vol 66 no 5 pp 545ndash551 1988

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


and provide scientific data on the safety of O stamineusfocusing on the 14-day acute and 28-day subchronic toxicityof bioactive 50 methanolic extract of O stamineus usingSprague-Dawley (SD) rats and hence providing guidance forselecting a safe dose of O stamineus in its use as a traditionalmedicine

2 Materials and Methods

21 Chemical Ferrous chloride (FeCl2) hematoxylin

ferrozine 2-deoxyribose ethylenediaminetetraacetic acid(EDTA) ferrous sulfate (FeSO

4) trichloroacetic acid (TCA)

nitroblue tetrazolium (NBT) xanthine xanthine oxidasesodiumphosphate (dibasic) sodiumphosphate (monobasic)butylated hydroxytoluene (BHT) butylated hydroxyanisole(BHA) manganese chloride xylenol orange manganesechloride and sodium dihydrogen phosphate (NaH

2PO4)

were purchased from Sigma (St Louis MO USA) Absolutealcohol and eosin were purchased from Riedel-de Haen(Seelze Germany) Paraplast was purchased from OxfordLabware (St Louis MO USA) Xylene was purchased fromFisher Scientific (Leics UK) Disposable microtome blades818 were purchased from LEICA (Germany) Thiobarbituricacid (TBA) was purchased from AppliChem (DarmstadtGermany) 31015840-hydroxy-56741015840-tetramethoxyflavone sinen-setin and eupatorin were obtained from Indofine ChemicalCompany (NJ USA) HPLC-grade acetonitrile and isopropylalcohol were purchased fromMerck (Darmstadt Germany)

22 Plant Materials and Extract Orthosiphon stamineus wasgrown from cuttings using standard agronomic practices atKepala Batas Pulau Pinang Malaysia The leaves of the plantwere collected after flowering The leaves were identified byMr Adenan Jaafar School of Biological Sciences UniversitiSains Malaysia A voucher specimen (number 10810) wasdeposited at the Herbarium of School of Biological SciencesUniversiti Sains Malaysia The leaves were rinsed and driedin oven at 40∘C for 2 daysThe dried leaves were then groundby an electric grinder to a coarse powder and weighedSubsequently 300 g of powdered leaves of O stamineuswas extracted with 5 L methanol water (50 50 vv) at 60∘C(for 8 hours) by maceration method The resulting 50methanolic extract of O stamineus (MEOS) was concen-trated using a Buchi-RE121 evaporator (Buchi Laboratorium-Technik Switzerland) equipped with a Buchi-B169 vacuumsystem and then lyophilized in a Hetovac VR-1 (HETO LabEquipment Denmark) freeze dryerThe weight of the MEOSwas recorded and the final plant-to-extract ratio was about6 The MEOS was then kept in desiccators in a refrigerator(0ndash4∘C) The MEOS was freshly prepared daily by dissolvingin distilled water

23 HPLC Study

231 HPLC Instrumentation HPLC analysis was performedusing a Shimadzu-LC system (Shimadzu Japan) equippedwith a CBM-20A controller LC-20AT pump DGU-20A5degasser SIL-20A autosampler SPD-20AV detector andCTO-10ASvp column oven

232 Chromatographic Conditions Chromatographic sepa-rations were achieved using an Agilent Eclipse Plus C18 (250times 46mm id 5 120583m) A Zorbax guard fittings kit packed withreplaceable Eclipse Plus C18 Guard column (125 times 46mmid 5 120583m) was used to protect the analytical column Areverse-phase HPLC assay was carried out using an isocraticsystem with a flow rate of 1mLmin a column temperatureof 25∘C and a mobile phase of acetonitrile isopropyl alco-hol 20mM phosphate buffer (NaH

2PO4) (30 15 55 vv)

with pH adjusted to 35 using 85 phosphoric acid TheUV detection was set at 340 nm The injection volume was20120583L of solution Total run time was less than 20min foreach injection Data was acquired and processed with LC-Solution software The peaks were detected at 340 nm andidentified using reference standards of 31015840-hydroxy-56741015840-tetramethoxyflavone sinensetin and eupatorin Sinensetinand eupatorin were selected as standards because these twocompounds were found to be the bioactive ingredients in theplant [11ndash13]

24 Antioxidant

241 Ferrous Ion Chelating Activity Ferrous chelating activ-ity was determined according to the method of Dinis etal [14] MEOS was added to a solution of 005mL ferrouschloride (FeCl

2) (2mM) The reaction was initiated by the

addition of 02mL ferrozine (5mM) and the mixture wasshaken vigorously and left standing at room temperature(24 plusmn 2∘C) for 10min Absorbance of the solution was thenmeasured spectrophotometrically (U-2000Hitachi Japan) atwavelength 562 nmThepercentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the followingformula

Ferrous ion chelating activity = [(1198600minus 1198601)

1198600

] times 100

(1)


1was

the absorbance in the presence of extracts The experimentwas run together with butylated hydroxytoluene (BHT) andbutylated hydroxyanisole (BHA) as positive controls

242 Hydroxyl Radical Scavenging Activity Competitionbetween deoxyribose and the extract against hydroxyl radicalgenerated from the Fe3+ascorbateethylenediaminetetraace-tic acid (EDTA)hydrogen peroxide (H

2O2) system was mea-

sured to determine the hydroxyl radical scavenging activityof MEOS [15] The reaction mixture consisted of 030mLof 002M sodium phosphate buffer (pH 70) 015mL of10mM 2-deoxyribose 015mL of 10mM FeSO

4 015mL of

10mMEDTA 015mL of 10mMH2O2 0525mL ofH

2O and

0075mL of extract The reaction mixture was incubated at37∘C for 2 h and the formed TBARS were measured Seventyfive milliliter of 28 trichloroacetic acid and 075mL of 10TBA in 50mMNaOHwere added to test tubes and boiled for20min After cooling the mixture absorbance was measuredat 520 nm The percentage of inhibition of inhibition rate of




1198600

]times 100

(2)


1was the




2SO4 0397 g of



1198601

] times 100 (3)







50



2after 28





05

1015202530354045

0 05 1 15 2

Chel

atin

g eff

ect (

)


BHTBHA

MEOS





3 Results




0

10

20

30

40

50

60

70

80

90

0 05 1 15 2

Hyd

roxy

radi

cal s

cave

ngin

g ac

tivity

()


BHTBHA

MEOS


0102030405060708090

0 02 04 06 08 1 12 14 16 18 2Supe

roxi

de ra

dica

l sca

veng

ing

activ

ity (

)


BHTBHA

MEOS






140

160

180

200

220

240

260Bo

dy w

eigh

t (g)


Control1250mgkg

2500mgkg5000mgkg

(a)

140

160

180

200

220

240

260

Body

wei

ght (

g)


Control1250mgkg

2500mgkg5000mgkg

(b)






50determined was






4 Discussion






(a)



















(b)























(a)


















(b)

























































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




1198600

]times 100

(2)


1was the




2SO4 0397 g of



1198601

] times 100 (3)







50



2after 28





05

1015202530354045

0 05 1 15 2

Chel

atin

g eff

ect (

)


BHTBHA

MEOS





3 Results




0

10

20

30

40

50

60

70

80

90

0 05 1 15 2

Hyd

roxy

radi

cal s

cave

ngin

g ac

tivity

()


BHTBHA

MEOS


0102030405060708090

0 02 04 06 08 1 12 14 16 18 2Supe

roxi

de ra

dica

l sca

veng

ing

activ

ity (

)


BHTBHA

MEOS






140

160

180

200

220

240

260Bo

dy w

eigh

t (g)


Control1250mgkg

2500mgkg5000mgkg

(a)

140

160

180

200

220

240

260

Body

wei

ght (

g)


Control1250mgkg

2500mgkg5000mgkg

(b)






50determined was






4 Discussion






(a)



















(b)























(a)


















(b)

























































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


05

1015202530354045

0 05 1 15 2

Chel

atin

g eff

ect (

)


BHTBHA

MEOS





3 Results




0

10

20

30

40

50

60

70

80

90

0 05 1 15 2

Hyd

roxy

radi

cal s

cave

ngin

g ac

tivity

()


BHTBHA

MEOS


0102030405060708090

0 02 04 06 08 1 12 14 16 18 2Supe

roxi

de ra

dica

l sca

veng

ing

activ

ity (

)


BHTBHA

MEOS






140

160

180

200

220

240

260Bo

dy w

eigh

t (g)


Control1250mgkg

2500mgkg5000mgkg

(a)

140

160

180

200

220

240

260

Body

wei

ght (

g)


Control1250mgkg

2500mgkg5000mgkg

(b)






50determined was






4 Discussion






(a)



















(b)























(a)


















(b)

























































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


140

160

180

200

220

240

260Bo

dy w

eigh

t (g)


Control1250mgkg

2500mgkg5000mgkg

(a)

140

160

180

200

220

240

260

Body

wei

ght (

g)


Control1250mgkg

2500mgkg5000mgkg

(b)






50determined was






4 Discussion






(a)



















(b)























(a)


















(b)

























































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



(a)



















(b)























(a)


















(b)

























































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



(a)


















(b)

























































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



































5 Conclusion




Acknowledgments



References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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