research article acute toxicity study of zerumbone-loaded...

Research ArticleAcute Toxicity Study of Zerumbone-Loaded NanostructuredLipid Carrier on BALB/c Mice Model

Heshu Sulaiman Rahman,1,2,3 Abdullah Rasedee,1,2 Hemn Hassan Othman,1,3

Max Stanley Chartrand,4 Farideh Namvar,5 Swee Keong Yeap,2 Nozlena Abdul Samad,2

Reena Joys Andas,2 Nabilah Muhammad Nadzri,2 Theebaa Anasamy,2

Kuan Beng Ng,2 and Chee Wun How2

1 Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia2 Institute of Bioscience, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia3 Faculty of Veterinary Medicine, University of Sulaymaniyah, Sulaymaniyah, Kurdistan, Iraq4DigiCare Behavioral Research, Casa Grande, AZ, USA5 Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia (UPM),43400 Serdang, Selangor, Malaysia

Correspondence should be addressed to Heshu Sulaiman Rahman; [email protected] Abdullah Rasedee; [email protected]

Received 7 June 2014; Accepted 1 August 2014; Published 8 September 2014

Academic Editor: Lisa A. Delouise

Copyright © 2014 Heshu Sulaiman Rahman et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Zerumbone- (ZER-) loaded nanostructure lipid carrier (NLC) (ZER-NLC) prepared for its antileukemia effect in vitrowas evaluatedfor its toxicological effects by observing changes in the liver, kidney, spleen, lung, heart, and brain tissues, serum biochemicalparameters, total haemogram, and bone marrow stem cells.The acute toxicity study for ZER-NLC was conducted by orally treatingBALB/c mice with a single dose with either water, olive oil, ZER, NLC, or ZER-NLC for 14 days. The animals were observed forclinical and behavioral abnormalities, toxicological symptoms, feed consumption, and gross appearance. The liver, kidney, heart,lung, spleen, and brain tissues were assessed histologically. Total haemogramwas counted by hemocytometry andmicrohematocritreader. Bone marrow examination in terms of cellular morphology was done by Wright staining with bone marrow smear.Furthermore, serum biochemical parameters were determined spectrophotometrically. Grossly all treated mice, their investigatedtissues, serum biochemical parameters, total haemogram, and bonemarrow were normal. At oral doses of 100 and 200mg/kg ZER-NLC there was no sign of toxicity or mortality in BALB/c mice. This study suggests that the 50% lethal dose (LD

50) of ZER-NLC is

higher than 200mg/kg, thus, safe by oral administration.

1. Introduction

Obviously, one of the most important elements of a drugdiscovery program is the prediction of a new compound’stoxicity. Conducting effective toxicology studies can helpresearchers proceed with more certainty and confidence toclinical trials, and ultimately, lead to the introduction ofmoreeffective and safer therapies for patients in need.

Zerumbone (ZER) is a sesquiterpene that was first iso-lated from the essential volatile oil of rhizome of Zingiberzerumbet by L. Smith [1]. Other plant species also known to

contain ZER include Zingiber amaricans [2], Zingiber ottensiiValeton [3], Zingiber cassumunar Roxb [4], Zingiber aro-maticum [5], Curcuma amada Roxb. [6], Alpinia galanga [7],Zingiber montanum [8], andXylopia aethiopica [9]. Biologicaland therapeutic effects of ZER include anti-inflammatory[10], anticancer [11, 12], antioxidant [13], antinociceptive[14], antihypercholesterolemia [15], antimicrobial [4], andimmunomodulatory [16] activities.

ZER is plagued with low water solubility that com-promises oral bioavailability and limits transportation incirculation, which impair delivery to target organs and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 563930, 15 pageshttp://dx.doi.org/10.1155/2014/563930

2 BioMed Research International

Ginger Nanostructure lipid carrierRhizome Oil Zerumbone ZER-NLC

+HPO

Thimerosal

Olive oil

SorbitolLipoid S 100

ddH2O

Figure 1: Steps of ZER-NLC formulation.

tissues. However, the solubility of ZER can be improved byloading in lipid nanoparticles [17]. One of themajor concernswith using nanoparticle drug carriers is toxicity. Generally,lipid nanoparticle carriers are ideal, because they not onlyimprove deliverywith targeting potential, but also reduce tox-icity and improve therapeutic effectiveness of loaded drugsand compounds. However, these carriers, although verypromising drug delivery systems and broadly applicable inmedicine, suffer from serious stability problems that can leadto aggregation, fusion, and phospholipid hydrolysis, whichcan compromise shelf life and limit parenteral applications[18, 19].

Recently, we developed ZER-NLC (Figure 1), which isa modified formula composed of ZER, hydrogenated palmoil (HPO), double-distilled water (ddH

2O), olive oil, lipoid-

S 100, lecithin, thimerosal, and sorbitol [20, 21]. The ZER-NLCwasmeant for treatment of diseases via oral application.The oral route of drug administration is generally preferred,because of its versatility, safety, and relative comfort for thepatient. Nanostructured lipid carrier alone is not toxic tohuman peripheral blood mononuclear cells (PBMC) andBALB/c 3T3 cells in vitro [22, 23], thus presumably safe fororal and parenteral applications.

However, as a drug carrier in ZER-NLC, its safety, biolog-ical fate, and potential toxicity of ZER-NLC were not known.A rigorous toxicity evaluation of ZER-NLC would ensure thesafety of its use as a therapeutic compound and nutritionalsupplement. Thus, the aim of this study was to evaluatethe potential toxicity of ZER-NLC in BALB/c mice modelthrough its effect on the serum biochemical parameters,histopathology of tissues, total haemogram count, and bonemarrow smear.

2. Materials and Methods

2.1. Zerumbone-Loaded Nanostructured Lipid Carrier. Purecolorless zerumbone crystals were prepared from essential oilof fresh Zingiber zerumbet (L.) Smith rhizomes extracted bysteam distillation according to the method described earlier[24]. The ZER-NLC prepared by high-pressure homoge-nization method and characterized by zetasizer, reversephase high performance liquid chromatography (RP-HPLC),transmission electron microscopy (TEM), wide angle X-ray diffraction (WAXR), differential scanning colorimeter(DSC), and Franz diffusion cell (FDC) system ZER-NLCwas shown to be physically stable, with particle size (PS) of52.68 ± 0.1 nm, zeta potential (ZP) of −25.03 ± 1.24mV andpolydispersity index (PDI) of 0.29 ± 0.0041 𝜇m [21, 23].

2.2. Animal Groups. BALB/c mice aged six to eight weekswere purchased from the Animal House of Faculty ofMedicine andHealth Sciences, Universiti PutraMalaysia.Themice were housed in polypropylene plastic cages with woodchips as bedding. The mice were acclimatized to the labora-tory environment at 24 ± 1∘C under a 12-hour dark-light cyclefor at least 5 days, before commencement of the experiment.Themicewere provided pellet andwater ad libitumduring theperiod of study. This study was approved by the InstitutionalAnimal Care and Use Committee (IACUC), Universiti PutraMalaysia (UPM/FPV/PS/3.2.1.551/AUP-R152).

2.3. Evaluation of Acute Toxicity. Forty-eight male and 48female BALB/c mice (18 to 22 g) were divided randomly into8 groups of 6 males and 6 females each. Group 1 served ascontrols and received water only, group 2 received 0.2mLolive oil, groups 3 and 4 were given 100 and 200mg/kgZER dissolved in 0.2mL olive oil, respectively. Groups 5 and6 were given 100 and 200mg/kg NLC, respectively, whilegroups 7 and 8 were given 100 and 200mg/kg ZER-NLC,respectively, following the method described earlier [25].Theanimals were deprived of feed 12 hours prior to treatment.Oral intubation was done using a ball-tipped stainless steelgavage needle attached to a syringe.

2.4. Clinical Observations and Body Weight Measurements.The animals were observed for clinical and behavioral abnor-malities, toxicological symptoms, feed consumption, andgross appearance twice each day over a period of 14 daysposttreatment. Body weights of live mice were recordedbefore treatment and on days 7 and 14.

2.5. Histopathological Study. Histopathology provides a rapidmethod to detect effects of irritants in various organs.Further confirmation of this finding should be supportedby histopathological analysis, to verify the toxicity of thetest compound and formula. For this purpose, liver, kidney,spleen, heart, lung, and brain tissue samples were collected,cut into sections of approximately 0.5 cm2 sizes, and fixedin 10% formalin for at least 48 hours. The fixed sampleswere placed in plastic cassettes and dehydrated using anautomated tissue processor (Leica ASP300, Germany). Theprocessed tissues were embedded in paraffin wax (LeicaEG1160, Germany) and the blocks trimmed and sectioned toabout 5 × 5 × 4 𝜇m size using a microtome (Leica RM2155).The tissue sections were mounted on glass slides using a hotplate (Leica HI1220, Germany) and subsequently treated in

BioMed Research International 3

Table 1: Body weight of mice treated with ZER, nanostructured lipid carrier (NLC), and zerumbone-loaded NLC.

Animal groupsFemale Male

Body weight (g) Body weight (g)

Day 0 Day 7 Day 14 Weight gain(g) on day 14 Day 0 Day 7 Day 14 Weight gain

(g) on day 14Control 19.3 ± 1.05 23.6 ± 1.0 28.2 ± 0.8∗ 8.9 ± 0.26 21.0 ± 0.8 25.6 ± 1.0 30.3 ± 0.8∗ 9.3 ± 0.65Vehicle 20.1 ± 1.7 23.14 ± 1.5 29.11 ± 0.77∗ 9.01 ± 0.95 21.49 ± 1.4 25.7 ± 1.9 30.20 ± 0.95∗ 8.71 ± 1.45ZER (100mg/kg) 20.5 ± 0.5 24.15 ± 0.9 28.75 ± 1.1∗ 8.25 ± 0.63 20.9 ± 1.3 24.85 ± 1.5 29.7 ± 1.6∗ 8.8 ± 1.25ZER (200mg/kg) 20.8 ± 0.7 24.65 ± 1.4 28.9 ± 3.2∗ 8.1 ± 1.75 21.3 ± 1.5 24.4 ± 0.9 28.9 ± 1.45∗ 7.6 ± 1.8NLC (100mg/kg) 19.6 ± 1.3 23.3 ± 2.1 27.6 ± 2.2∗ 7.9 ± 1.7 21.7 ± 0.2 26.3 ± 0.6 31.2 ± 1.1∗ 9.4 ± 1.0NLC (200mg/kg) 20.0 ± 1.7 24.5 ± 2.2 29.9 ± 2.0∗ 9.9 ± 0.3 19.7 ± 1.6 25.1 ± 0.9 30.4 ± 1.0∗ 10.6 ± 0.7ZER-NLC (100mg/kg) 19.6 ± 0.2 23.2 ± 1.1 26.0 ± 1.1∗ 6.4 ± 0.8 21.0 ± 0.9 24.2 ± 1.0 28.2 ± 0.8∗ 8.2 ± 1.4ZER-NLC (200mg/kg) 19.5 ± 0.8 23.3 ± 0.5 26.8 ± 0.9∗ 7.3 ± 1.5 19.1 ± 1.0 24.4 ± 0.9 26.6 ± 0.7∗ 7.5 ± 0.7Values are mean ± SD (𝑛 = 6) and have been analysed using post hoc comparison test one way ANOVA. Data revealed significant 𝑃 < 0.05. ∗Increasing ofbody weight after 14 days in all treated groups when compared to that of untreated control group.

order with 100, 90, and 70% ethanol for two minutes each.Finally, the sections were rinsed with tap water and stainedwith Harris’s haematoxylin and eosin for light microscopy[26].

2.6. Serum Biochemistry. Blood samples were collected fromtail veins in plain tubes and centrifuged (Hettich zent-EBA20, Germany) at 3500 rpm for 10 minutes, the serumwasobtained and stored at −20∘C until analyzed. The concentra-tions of serum enzymes, proteins, bilirubin, electrolytes, andrenal function parameters were determined using standarddiagnostic kits (Roche) in an automatic biochemistry ana-lyzer (Hitachi 902, Japan).

2.7. Haematological Study. On experimental day 15, animalswere sacrificed under anaesthesia and peripheral blood wascollected from tail veins with ethylenediaminetetraaceticacid (EDTA) vacuumed blood collection tubes, was shakenimmediately to mix well, and was analyzed directly withoutdelay. The total and differential white blood cells (WBC),total red blood cells (RBC), hemoglobin (Hb), and plateletsin each sample were measured by automatic hematologyanalyzer (Cell Dyn, 3700, Abbot, USA). On the other hand,packed cell volume (PCV) was donemanually using capillarymicrohematocrit tubes.

2.8. Bone Marrow Smear. Drug toxicity often manifests inbone marrow, causing a range of serious side effects, includ-ing leucopenia (especially neutropenia),mitochondrial toxic-ity, bone marrow suppression (especially myelosuppression),or immunotoxicity.Thus, a key objective during drug discov-ery is the assessment of the potentially toxic impact of newcompounds on bone marrow.

Near the end of the study, all mice were dissected andbone marrow was washed with 100 𝜇L phosphate buffer solu-tion (PBS) via the flashing method on the femur of sacrificedmice [27]. Bone marrow toxicity in terms of cell morphologywas done by Wright’s staining with bone marrow smear andwas examined under normal light microscope (Leica, Japan).

Moreover, blood samples were taken to perform total anddifferential white blood cell count, as well as platelet countin all animal groups of either sex as mentioned in previoussection to determine bone marrow toxicity.

2.9. Statistical Analysis. The experiments were done in trip-licate, and the results were expressed as mean ± SD andanalyzed statistically using SPSS version 20.0 (SPSS Inc.,Chicago, USA). Post hoc comparison test one way ANOVAwas done using the Tukey’s b test. Probability values of lessthan alpha 0.05 (𝑃 < 0.05) were considered statisticallysignificant.

3. Results

3.1. Clinical Observations and Body Weight Measurements.In this study, extreme care was taken to ensure these fun-damentals were strictly observed. The most notable clinicalabnormalities observed in a few mice treated with a singlehigh oral dose of ZER and ZER-NLC for 14 days were roughcoat and decreased activity.Themajority ofmice did not showtoxicological effect, behavioral change, or significant bodyweight loss. In fact slight body weight gain was noted in theseanimals during the test period (Table 1).

3.2. Histopathological Observation. Histopathological studieswere carried to confirm biochemical findings. Generally, noalterations were observed inmost of the tested organs of bothsexes of control animals, as well as in animals treated withvarious doses of ZER, NLC, and ZER-NLC. The photomi-crographs of the vital organs of the control, and all treatedgroups, both male and female, had normal morphologicalarchitecture. In this study, histopathological observation wasdone on six major organs, which are liver, kidney, heart, lung,spleen, and brain. Under microscopic examination, the liverin the majority of treated animals showed normal cellulararchitecture and binucleation and was without any distor-tions. There were no signs of injury, necrosis, congestion,fatty acid accumulation, or hemorrhagic areas around the


central vein or sinusoids of the liver. The hepatocytes werestill arranged in cords and clearly visible.The cross-section ofthe liver showed no lyses in the blood cells, neutrophil, lym-phocyte, or macrophage infiltration (Figure 2). The currentstudy showed that histologically there were nomorphologicalchanges in the kidneys of mice from all treated groups. Theglomerular architecture showed normal appearance similarto the control groups. The cross-sections of the glomeruli,distal, and proximal tubules in the kidney appeared to benormal in both male and female mice. There was alsono interstitial and intraglomerular congestion or tubularatrophies. All the nephron cells showed normal and clearlyvisible nucleoli with no degeneration, bleeding, or necrosis(Figure 3). As shown in Figure 4, all treatment groups showedno signs of cardiac necrosis, haemorrhage, inflammatory exu-dates, myocardial infarction, infiltration, vacuolization, fattychanges, or other damage. Cardiac myofibrils and myocyteswere normally arranged with no morphological change.Likewise, the lung tissues of all treated mice groups exhibitedno obvious morphological and histological differences whencompared with the control group. There was no bronchioleor alveoli collapse, no alveolar epithelial denaturation, andno inflammatory cell infiltration surrounding the bronchiof the treated groups (Figure 5). From the cross-section ofthe spleen, the tissue structure of the spleens was normal.There was no hemorrhage and no pathological change inthe spleen sinus (Figure 6). Moreover, no histopathological,degenerative, congestive, haemorrhagic, or vacuolar changeswere observed in brain tissues of both dosed and controlgroups after dosing of ZER, NLC, and ZER-NLC (Figure 7).Thus, the microscopic evaluations of the selected organsdid not reveal morphological abnormalities that could beattributed to the oral administration of ZER-NLC to themice.

3.3. Serum Biochemistry. Several biochemical tests are usedto determine potential adverse effect of chemicals, drugs,formulations, and synthetic materials on the renal and hep-atic functions of test animals. Abnormalities in the liver andkidneys can be detected by estimating serum biochemistryassociated with liver and kidney integrity and functions[18]. In this study the serum liver enzymes, bilirubin, andprotein concentrations in the ZER-NLC treated were similarto control mice (𝑃 > 0.05). Similarly, the concentrations ofelectrolytes associated with kidney function, namely, sodiumand potassium, as well as chloride, urea, and creatinine levels,did not differ between ZER-NLC treated and control groups(𝑃 > 0.05). Based on the serum biochemical parametersZER-NLC, treatment even at the dosage level of 200mg/Lproduced no abnormalities in liver or kidneys (Tables 2(a)and 2(b)).Therefore, the study suggests that the LD

50of ZER-

NLC value in female and male mice would have to be greaterthan 200mg/kg.

3.4. Haematological Study. Although bone marrow suppres-sion is characterized by predictable macrocytosis, raisedmean corpuscular haemoglobin concentration, thrombocy-topenia, and sometimes pancytopenia, leucopenia is themostcommon and most important haematological complicationin case of bone marrow toxicity; thus regular monitoring

of the full blood count is recommended during treatment.In this study, the haematological results of all treated miceexhibited normal levels of total and differential white bloodcells, red blood cells, haemoglobin, and platelet counts as wellas packed cell volume in the peripheral blood (Tables 3(a) and3(b)). Thus, thrombocytopenia, leucopenia, pancytopenia(indications of bone marrow suppression and toxicity), oranaemia was not predicted in treated ZER, NLC, and ZER-NLC mice.

3.5. Bone Marrow Smear. The blood/bone marrow systemis one of the largest organs in the body and is an impor-tant potential target of chemical exposure, in particular,chemotherapeutic agents. Consequently, analysis of bloodand bone marrow has become a routine procedure in theinvestigation of haematological and bone marrow disordersin chemotherapy assessments. Microscopic evaluations ofstained bone marrow smear did not manifest any morpho-logical abnormalities of bone marrow stem cells (blast cells,reticulocytes, or megakaryocytes) or pronounced toxicity ofbone marrow of mice in all groups of both sexes. All cells hada normal histological appearance without deformations innucleus, cytoplasm, or outer membrane after treatment withdifferent doses of ZER, NLC, or ZER-NLC. Normal shapedfat cells were also observed (Figure 8).

4. Discussion

Our previous studies have reported the potential of NLCto turn the lipophilic antitumor natural compound, Zerum-bone, as a sustained-release antileukemic drug in vitro [21,23]. Although our in vitro cytotoxicity study of ZER-NLCon peripheral blood mononuclear cell isolated from healthyhuman subjects has proven that ZER-NLC possessed no toxi-city against normal human cell [23], it is important to evaluatethe safety of ZER-NLC in vivo before further preclinical andeven clinical studies of ZER-NLC. Acute toxicity test usingsmall animal models is imperative in order to determinesafety and efficacy and to identify active components oftraditional medicine and herbal products [28]. This studyprovides information on toxic doses and therapeutic indices(LD50) of drugs and xenobiotics [29]. ZER is the antitumor

sesquiterpenoid that can be isolated from rhizome ofZingiberzerumbet Smith [10]. In this study, 4 different groups ofcontrol were evaluatedwhere group 1 is themice that receivedwater only, group 2 is the mice that received olive oil whilegroups 5 and 6 are the mice that received 100 and 200mg/kgof NLC. No significant difference was observed in all thetested experiments among all these control groups. Previousstudy has reported that zerumbone did not possess in vitroand in vivo genotoxic effects [30]. Furthermore, zerumbonewas also reported not to cause any significant changes in clin-ical condition, growth, organ weights, haematology, serumbiochemistry, and histopathological analysis after single orrepeated intraperitoneal injection in mice [31]. In this study,similar results were obtained where oral administration ofZER did not induce significant acute toxicity on mice.


50𝜇m 50𝜇m 50𝜇m

50𝜇m50𝜇m50𝜇m

50𝜇m 50𝜇m

(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)



50𝜇m 50𝜇m

(A) (B) (C)

(D) (E) (F)

(G) (H)

(b)

Figure 2: (a) Liver of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER) atconcentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G)and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the liver of these mice(400x magnification). (b) Liver of male BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone(ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg,and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the liver ofthese mice (400x magnification).




50𝜇m 50𝜇m

(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)

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(A) (B) (C)

(D) (E) (F)

(G) (H)

(b)

Figure 3: (a) Kidney of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER)at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G)and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the kidney of thesemice (400x magnification). (b) Kidney of male BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D))zerumbone (ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and200mg/kg, and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed inthe kidney of these mice (400x magnification).


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(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)

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(A) (B) (C)

(D) (E) (F)

(G) (H)

(b)

Figure 4: (a) Heart of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER)at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G)and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the heart of these mice(400x magnification). (b) Heart of male BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone(ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg,and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the heart ofthese mice (400x magnification).


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(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)

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(A) (B) (C)

(D) (E) (F)

(G) (H)

(b)

Figure 5: (a) Lung of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER) atconcentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G)and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the lung of these mice(400x magnification). (b) Lung of male BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone(ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg,and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the lung ofthese mice (400x magnification).


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(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)

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(A) (B) (C)

(D) (E) (F)

(G) (H)

(b)

Figure 6: (a) Spleen of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER) atconcentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G) and(H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the spleen of these mice(400xmagnification). (b) Spleen of male BALB/cmice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone(ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg,and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the spleenof these mice (400x magnification).


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(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)

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(A) (B) (C)

(D) (E) (F)

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Figure 7: (a) Brain of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER)at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G)and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the brain of these mice(400x magnification). (b) Brain of male BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D)) zerumbone(ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and 200mg/kg,and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observed in the brain ofthese mice (400x magnification).


Table 2: (a) Serum liver function parameters of mice treated with ZER, nanostructured lipid carrier (NLC), and zerumbone-loaded NLC.(b) Serum renal function parameters of mice treated with ZER, nanostructured lipid carrier (NLC), and zerumbone-loaded NLC.

(a)

(𝑛 = 6) TP (g/L) Albumin(g/L)

Globulin(g/L)

TB(𝜇mol/L)

CB(𝜇mol/L) ALP (U/L) ALT

(U/L) AST (U/L) GGT(U/L)

ControlFemale 57.5 ± 0.5 29.5 ± 0.5 27.9 ± 0.5 2.3 ± 0.1 <0.1 112.3 ± 2.5 61.3 ± 1.4 143.2 ± 1.1 4.1 ± 0.17Male 66.9 ± 2.0 27.2 ± 2.1 37.6 ± 1.9 2.1 ± 1.7 <0.1 122 ± 1.8 43.9 ± 2.0 81.2 ± 1.7 4.4 ± 2.7

VehicleFemale 58.9 ± 0.4 30.7 ± 0.5 28.2 ± 0.8 1.7 ± 0.5 <0.1 119.6 ± 1.0 52.6 ± 1.3 91.7 ± 1.9 4.3 ± 0.3Male 66.3 ± 1.2 27.1 ± 1.8 35.4 ± 3.2 1.9 ± 1.1 <0.1 121.2 ± 2.1 48.9 ± 3.5 87.1 ± 1.5 4.2 ± 2.5

ZER(100mg/kg)

Female 57.7 ± 0.4 27.7 ± 0.5 28.6 ± 1.7 2.01 ± 0.2 <0.1 121.4 ± 1.7 54.1 ± 1.0 93.9 ± 1.6 4.35 ± 0.2Male 68.9 ± 1.9 28.7 ± 1.5 35.4 ± 1.0 2.4 ± 3.0 <0.1 124.6 ± 2.2 49.1 ± 1.9 90.3 ± 2.3 4.8 ± 0.6

ZER(200mg/kg)

Female 60.5 ± 0.4 30.9 ± 0.5 29.1 ± 0.4 2.1 ± 0.1 <0.1 121.6 ± 0.4 56.7 ± 1.2 95.4 ± 1.4 4.5 ± 0.5Male 67.2 ± 2.3 28.3 ± 3.4 38.8 ± 0.9 2.9 ± 2.4 <0.1 125.9 ± 3.4 50.2 ± 0.9 91.6 ± 2.7 5.1 ± 2.8

NLC(100mg/kg)

Female 57.9 ± 1.6 29.4 ± 1.01 25.1 ± 5.4 2.4 ± 0.2 <0.1 111.2 ± 1.1 56.6 ± 3.5 142 ± 3.6 4.4 ± 0.9Male 67.3 ± 0.6 26.2 ± 0.6 38.5 ± 2.7 1.7 ± 0.6 <0.1 119.3 ± 1.5 42.6 ± 2.0 83 ± 1.7 4.3 ± 0.5

NLC(200mg/kg)

Female 57.3 ± 2.2 30.4 ± 0.9 26.8 ± 1.3 2.6 ± 0.1 <0.1 113.4 ± 1.6 56.8 ± 4.4 131.1 ± 8.8 4.5 ± 0.4Male 67.8 ± 0.4 27.5 ± 1.9 36.9 ± 0.8 2.1 ± 0.32 <0.1 121.3 ± 1.1 46.1 ± 2.0 75.7 ± 2.8 3.6 ± 0.51

ZER-NLC(100mg/kg)

Female 57.0 ± 2.4 27.7 ± 0.3 29.2 ± 2.6 2.2 ± 0.1 <0.1 105.4 ± 4.5 53.3 ± 7.3 109.4 ± 2.3 4.1 ± 0.2Male 66.6 ± 0.5 26.5 ± 0.4 37.7 ± 1.9 2.0 ± 0.1 <0.1 121.3 ± 1.5 46.4 ± 1.6 82.5 ± 2.5 3.9 ± 0.8

ZER-NLC(200mg/kg)

Female 57.7 ± 0.4 29.5 ± 1.06 28.22 ± 0.9 2.4 ± 0.2 <0.1 109.8 ± 5.2 2.34 ± 2.8 138.4 ± 3.1 4.0 ± 0.14Male 65.0 ± 4.4 26.0 ± 1.1 34.2 ± 0.9 3.2 ± 0.7 <0.1 131.0 ± 4.4 42.2 ± 3.8 82.3 ± 2.5 4.0 ± 0.8

Treatment was performed for 14 days. Values are mean ± SD (𝑛 = 6) and have been analyzed using post hoc comparison test one way ANOVA. Data revealednonsignificant (𝑃 > 0.05) difference in liver function test after 14 days in all treated groups when compared to that of untreated control group. TP: total protein;TB: total bilirubin; CB: conjugated bilirubin; ALP: alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: 𝛾-glutamyltransferase.

(b)

(𝑛 = 6) Sodium(mmol/L)

Potassium(mmol/L)

Chloride(mmol/L)

Urea(mmol/L)

Creatinine(𝜇mol/L)

Control Female 148.6 ± 0.4 39.6 ± 1.6 106.4 ± 2.3 7.9 ± 0.1 31.8 ± 0.6Male 142.5 ± 1.5 54.8 ± 2.2 111.7 ± 2.1 7.2 ± 1.0 32 ± 2.9

Vehicle Female 143.2 ± 1.2 39.8 ± 0.8 109.4 ± 1.6 7.2 ± 1.2 32.8 ± 1.1Male 145.2 ± 1.7 59.8 ± 2.02 115.7 ± 1.6 7.7 ± 1.1 33.2 ± 1.8

ZER (100mg/kg) Female 151.5 ± 0.5 41.7 ± 0.3 108.2 ± 0.7 8.4 ± 1.0 35.6 ± 0.8Male 144.7 ± 2.1 57.3 ± 1.9 112.1 ± 2.7 7.3 ± 2.3 35.8 ± 3.1

ZER (200mg/kg) Female 153.2 ± 0.3 44.4 ± 1.6 111.06 ± 2.1 8.8 ± 2.2 37.2 ± 3.2Male 145.8 ± 1.9 56.4 ± 3.3 117.05 ± 3.1 7.9 ± 3.5 35.6 ± 2.7

NLC (100mg/kg) Female 152 ± 0.9 43.7 ± 0.5 107.7 ± 0.6 7.35 ± 0.1 33.53 ± 0.8Male 142.6 ± 3.0 56.2 ± 3.3 107.8 ± 0.3 6.3 ± 1.7 31.3 ± 1.5

NLC (200mg/kg) Female 145.4 ± 0.6 43.2 ± 0.4 106.7 ± 0.6 6.4 ± 0.4 34.6 ± 1.5Male 141.1 ± 1.0 550.4 ± 1.4 108.5 ± 1.3 6.3 ± 1.1 36.7 ± 2.5

ZER-NLC (100mg/kg) Female 146.4 ± 0.8 44.5 ± 0.4 106 ± 0.2 6.6 ± 0.3 33.8 ± 0.1Male 140.2 ± 3.0 55.2 ± 2.5 107.4 ± 3.1 6.9 ± 0.4 34.6 ± 2.5

ZER-NLC (200mg/kg) Female 146.9 ± 0.7 47 ± 0.2 107.8 ± 0.3 6.5 ± 0.3 36 ± 0.2Male 141.9 ± 2.1 56.8 ± 3.8 112.3 ± 2.5 7.8 ± 1.2 29.7 ± 2.6

Treatment was performed for 14 days. Values are mean ± SD (𝑛 = 6) and have been analyzed using post hoc comparison test one way ANOVA. Data revealednonsignificant (𝑃 > 0.05) difference in liver function test after 14 days in all treated groups when compared to that of untreated control group.


50𝜇m50𝜇m



(A) (B) (C)

(D) (E) (F)

(G) (H)

(a)

50𝜇m50𝜇m



(A) (B) (C)

(D) (E) (F)

(G) (H)

(b)

Figure 8: (a) Bone marrow smear of female BALB/c mice treated orally with (A) water (control), (B) olive oil (vehicle), ((C) and (D))zerumbone (ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructured lipid carrier (NLC) at concentrations 100 and200mg/kg, and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kg for 14 days. No sign of toxicity was observedin the bone marrow smear of these mice (400x magnification). (b) Bone marrow smear of male BALB/c mice treated orally with (A) water(control), (B) olive oil (vehicle), ((C) and (D)) zerumbone (ZER) at concentrations 100 and 200mg/kg, ((E) and (F)) blank nanostructuredlipid carrier (NLC) at concentrations 100 and 200mg/kg, and ((G) and (H)) zerumbone-loaded NLC at concentrations 100 and 200mg/kgfor 14 days. No sign of toxicity was observed in the bone marrow smear of these mice (400x magnification).


Table 3: (a) Total RBC, Hb, PCV, and platelets count in mice treated with ZER, nanostructured lipid carrier (NLC), and zerumbone-loadedNLC. (b) Total and differential white blood cell count in mice treated with ZER, nanostructured lipid carrier (NLC), and zerumbone-loadedNLC.

(a)

Animal groupsFemale Platelets

(×105/𝜇L)

Male Platelets(×105/𝜇L)Total RBC

(×1012/L) Hb (g/L) PCV (L/L) Total RBC(×1012/L) Hb (g/L) PCV (L/L)

Control 8.49 ± 0.7 138.3 ± 1.1 0.37 ± 0.05 6.3 ± 0.35 8.9 ± 0.17 140.3 ± 2.1 0.38 ± 0.75 6.5 ± 0.37Vehicle 8.51 ± 0.11 140.0 ± 1.2 0.38 ± 0.04 6.6 ± 0.1 8.75 ± 0.15 141.0 ± 5.2 0.36 ± 0.35 6.55 ± 0.15ZER(100mg/kg) 8.34 ± 0.21 137.2 ± 2.3 0.35 ± 1.1 6.6 ± 0.11 8.35 ± 0.25 139.2 ± 0.9 0.36 ± 1.9 6.75 ± 0.21

ZER(200mg/kg) 7.79 ± 0.32 138.4 ± 2.1 0.37 ± 2.2 6.4 ± 0.25 7.99 ± 0.35 139.5 ± 1.1 0.37 ± 2.5 6.46 ± 0.29

NLC(100mg/kg) 8.10 ± 0.4 135.05 ± 1.7 0.37 ± 1.7 6.7 ± 0.31 8.5 ± 0.41 136.15 ± 1.5 0.38 ± 0.7 6.87 ± 0.55

NLC(200mg/kg) 8.01 ± 0.34 136.6 ± 0.9 0.36 ± 0.5 6.1 ± 0.5 8.35 ± 0.33 135.1 ±

0.95 0.35 ± 0.9 6.95 ± 0.45

ZER-NLC(100mg/kg) 8.22 ± 0.55 137.5 ± 1.5 0.35 ± 0.09 6.0 ± 0.07 8.29 ± 0.5 138.5 ± 1.01 0.36 ± 0.4 6.3 ± 0.09

ZER-NLC(200mg/kg) 7.98 ± 0.06 138.44 ±

1.3 0.31 ± 0.31 6.25 ± 0.19 8.85 ± 0.75 139.02 ± 1.7 0.35 ± 0.11 6.45 ± 0.4

Values are mean ± SD (𝑛 = 6) and have been analysed using post hoc comparison test one way ANOVA. Data revealed nonsignificant (𝑃 > 0.05) differencein all hematological parameters after 14 days in all treated groups when compared to that of untreated control group.

(b)

(𝑛 = 6) TWBC (×103/𝜇L) Neutrophil (%) Basophil (%) Eosinophil (%) Lymphocyte (%) Monocyte (%)

Control Female 2.75 ± 0.9 15 ± 0.15 0.00 ± 0.00 5 ± 0.5 70 ± 0.11 10 ± 0.75Male 2.66 ± 0.15 16 ± 0.22 0.00 ± 0.00 3 ± 0.65 69 ± 0.17 12 ± 0.56

Vehicle Female 2.55 ± 0.42 14 ± 0.7 0.00 ± 0.00 4 ± 0.13 70 ± 0.15 12 ± 0.5Male 2.8 ± 0.5 15 ± 0.65 0.00 ± 0.00 3 ± 0.17 69 ± 0.7 13 ± 1.0

ZER(100mg/kg)

Female 2.79 ± 0.13 14 ± 0.5 0.00 ± 0.00 5 ± 0.75 71 ± 0.91 10 ± 1.1Male 2.8 ± 0.17 13 ± 0.23 0.00 ± 0.00 3 ± 0.45 73 ± 0.55 11 ± 1.1

ZER(200mg/kg)

Female 2.79 ± 0.3 14 ± 0.43 0.00 ± 0.00 3 ± 0.15 75 ± 0.45 9 ± 0.45Male 2.85 ± 0.32 12 ± 0.55 0.00 ± 0.00 5 ± 0.25 72 ± 0.67 11 ± 0.2

NLC(100mg/kg)

Female 2.81 ± 1.4 15 ± 1.0 0.00 ± 0.00 4 ± 0.2 73 ± 0.95 8 ± 0.7Male 2.83 ± 1.1 15 ± 0.67 0.00 ± 0.00 2 ± 0.3 72 ± 1.0 11 ± 0.9

NLC(200mg/kg)

Female 2.9 ± 0.9 14 ± 0.45 0.00 ± 0.00 4 ± 0.9 71 ± 0.45 11 ± 1.0Male 2.72 ± 0.81 15 ± 0.89 0.00 ± 0.00 3 ± 0.89 70 ± 0.25 12 ± 0.55

ZER-NLC(100mg/kg)

Female 2.65 ± 0.55 16 ± 0.9 0.00 ± 0.00 3 ± 0.5 69 ± 0.6 12 ± 0.64Male 2.89 ± 0.45 15 ± 1.2 0.00 ± 0.00 5 ± 0.75 69 ± 0.11 11 ± 0.79

ZER-NLC(200mg/kg)

Female 2.63 ± 0.35 13 ± 0.5 0.00 ± 0.00 5 ± 0.61 70 ± 0.2 12 ± 0.85Male 2.77 ± 0.85 15 ± 0.7 0.00 ± 0.00 4 ± 0.2 69 ± 0.35 12 ± 1.2

Treatment was performed for 14 days. Values are mean ± SD (𝑛 = 6) and have been analyzed using post hoc comparison test one way ANOVA. Data revealednonsignificant (𝑃 > 0.05) difference in total and differential leucocyte count after 14 days in all treated groups when compared to that of untreated controlgroup.

Furthermore, no significant changes on behaviour, growth,serum biochemical profile, and histopathology (liver, heart,spleen, kidney, brain, and lung) were observed in ZER andcontrol mice. Furthermore, similar to the previous study onrat [30], administration of ZER did not cause abnormalityto bone marrow. Thus, this study was aimed to evaluateand compare the acute toxicity of ZER-NLC with ZER.Besides ZER, acute toxicity of ZER-NLC was first evaluated

in this study. Similar to ZER, ZER-NLC also did not possessacute toxicity on mice as shown by the above results. Allof the tested parameters on the ZER-NLC treated micewere also similar to the control (group 1, group 2, andgroups 5 and group 6). These results suggested that bothZER and ZER-NLC were nontoxic but ZER-NLC carriesthe potential advantages as a sustainable release antitumordrug.


5. Conclusion

In conclusion, overall results of this study clearly demonstratethat oral administration of both ZER and ZER-NLC at acutedoses induced no behavioral alterations, toxicological signs,or any other adverse effects on the experimental animalsduring the fourteen-day test period, indicating that ZER-NLC offers significant potential as a novel sustainable releaseantitumor drug for cancer treatment without acute toxic sideeffect. Further in vivo antitumor studies of the ZER-NLCshould be carried out to further confirm its antitumor effectespecially on its antileukemic effect.

Conflict of Interests

All the authors declare no conflict of interests.

Acknowledgments

The authors would like to express their appreciation tothe Institute of Bioscience (IBS), Universiti Putra Malaysia(UPM), for providing facilities and materials for this study.They are also grateful for the analytical services providedby the Haematology and Clinical Biochemistry and theHistopathology laboratories, Faculty of Veterinary Medicine,UPM. This project was funded by Ministry of Science,Technology and Innovations, Malaysia (MOSTI) (Grant no.5495308).

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