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Article

A 90-day subchronic toxicity studyof neem oil, a Azadirachta indica oil,in mice

C. Wang1, M. Cao2, D-X. Shi1, Z-Q. Yin1, R-Y. Jia1,K-Y. Wang1, Y. Geng1, Y. Wang1, X-P. Yao1,

Z-R. Yang3 and J. Zhao3

Abstract

To determine the no-observed-adverse-effect level (NOAEL) of exposure and target organs of neem oil forestablishing safety criteria for human exposure, the subchronic toxicity study with neem oil in mice wasevaluated. The mice (10 per sex for each dose) was orally administered with neem oil with the doses of 0

(to serve as a control), 177, 533 and 1600 mg/kg/day for 90 days. After the treatment period, observationof reversibility or persistence of any toxic effects, mice were continuously fed without treatment for the fol-lowing 30 days. During the two test periods, the serum biochemistry, organ weight and histopathology wereexamined. The results showed that the serum biochemistry and organ coefficient in experimental groups hadno statistical difference compared with those of the control group. At the 90th day, the histopathologicalexaminations showed that the 1600 mg/kg/day dose of neem oil had varying degrees of damage on each organexcept heart, uterus and ovarian. After 30-day recovery, the degree of lesions to the tissues was lessened oreven restored. The NOAEL of neem oil was 177 mg/kg/day for mice and the target organs of neem oil weredetermined to be testicle, liver and kidneys.

Keywords

Azadirachta indica oil; neem oil; subchronic toxicity; mice

Introduction

Neem (Azadirachta indica) has been accepted univer-

sally as a wonder tree in India.1 Neem oil, also

called Margosa oil, is an extract from seeds or fruits

of A. indica obtained through pressing or solvent

extraction. Neem oil was widely used as a traditionalmedicine by Indians in India, Sri Lanka, Burma,

Thailand, Malaysia and Indonesia and already has

more than 2000 years of history. It is used mainly forexternal applications and was often administered orally

for deworming, leprosy, constipation, rheumatism,

ulcer, relieve itching and chronic skin diseases.2,3 It

contains mostly long- and medium-chain fatty acids

(8095%) and volatile sulfur compounds (420%) as

well as a number of bioactive compounds, such as nim-

bin, nimbidin and nimbinin,4,5 that have been demon-

strated to have biocidal activity against nearly 200

medical and veterinary arthropods, without any

adverse effects toward most nontarget organisms.6,7

It was also found to have acaricidal, antibacterial, anti-

fungal, antimalarial, antiparasitic, anti-inflammatory,

promotion of wound healing and immunomodulatory

properties in different animal species.1,5,713

Our previous research showed that the median lethal

dose (LD50) of extract chloroform isolated from neem

oil in Sprague Dawley rats was above 10,000 mg/

1 College of Veterinary Medicine, Sichuan Agricultural University,

Yaan, China2 Core Laboratory, Sichuan Academy of Medical Sciences &

Sichuan Provincial Peoples Hospital, Chengdu, China3 Key Laboratoryof Biological Resource and Ecological Environment

of Chinese Education Ministry, College of Life Science, Sichuan

University, Chengdu, China

Corresponding author:

Zhong-Qiong Yin, College of Veterinary Medicine, Sichuan

Agricultural University, Yaan 625014, China.Email: [email protected]

Human and Experimental Toxicology

32(9) 904913

The Author(s) 2013

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DOI: 10.1177/0960327113475677

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kg,14 the LD50 of neem oil in mice was 31,950 mg/kg,

theaccumulative coefficient (K)wasabove5,ithadlow

toxic effects.15 The present study was thereby per-

formed to evaluate the subchronic toxicity of neem oil

according to the OECD test guideline 408 for

Repeated dose 90-day oral toxicity study in rodents

except for the lack of ophthalmological examinationand functional observations during feeding experi-

ment.16 The study will provide information on the

major toxic effects, indicate target organs, and can pro-

vide an estimate of a no-observed-adverse-effect level

(NOAEL) of neem oil, and provide information on the

possible health hazards likely to arise from people

repeated exposure over a prolonged period of time.

Materials and methods

Plant materialNeem oil that was extracted from the seeds of the

neem (A. indica) using carbon dioxide supercritical

fluid extraction was supplied by a pesticide company

(Green Gold Biological Science & Technology Co.

Ltd, Chengdu, PR China). All chemicals we used in

the test were of analytical grade (>99.7%).

Animals and treatments

Kunming strain male and female mice (a closed

strain coming from Kunming, Yunnan Province,

PR China) were obtained from the Chengdu Dossy

Experimental Animals Co., Ltd (Sichuan provence,

Chengdu, P.R. China, License No.SCXK (Sichuan)

2008-24), weighing 1315 g, kept at room tempera-

ture of 22C. Mice were fed with a standard diet

from Nuvital Nutrientes (Colombo/PR, Brazil) and

allowed free access to water and have been accli-

mated to laboratory conditions for 7 days.

Three groups of 20 mice, each containing 10

females and 10 males, received a daily dose of

177 mg/kg of body weight (b.w.; group II), 533 mg/

kg b.w. (group III) and 1600 mg/kg b.w. (group IV)of neem oil mixed with 1% carboxymethyl cellulose

sodium, a vehicle-control group (group I) formed by

20 mice received a daily dose of 533 mg/kg b.w. of

1% carboxymethyl cellulose sodium during a

90-day period. In each case, the product volume admi-

nistered by gavage was 2 mL/100 g b.w. Each mouse

was marked with a unique identification number

using trinitrophenol. Body weight was measured once

a week and the behavior was observed daily during

the trial period. At the end of the treatment, mice were

continuously fed without treatment for 30 days to

detect persistence of or recovery from toxic effects.

Clinical biochemistry analyses

At the end of experimentation (the 90th and 120thday), half of the total amount of mice per sex, respec-

tively, underwent overnight fasting prior to collection

of the blood sample. Blood of each mouse wascollected by retro-orbital bleeding and subjected to

clinical biochemical tests. For the hepatic function,

serum alkaline phosphatase, alanine aminotransferase

and aspartate aminotransferase were determined,

while for the renal function, serum urea nitrogen and

serum creatinine were evaluated. Serum glucose was

accessed for carbohydrate metabolism analysis. Total

protein, albumin, globulin, albumin/globulin ratio (A/

G), total bilirubin and cholesterol were also measured.All these biochemical parameters were determined as

described previously by Lincopan et al.17

Organic coefficient and histopathological analysis

On the 90th day and 120th day after blood collection

for biochemical analysis, all the animals were eutha-

nized, detailed gross necropsy was carefully exam-

ined. Extracted heart, liver, spleen, lungs and doublekidneys were trimmed of any adherent tissue, their

wet weight taken as soon as possible after dissectionto avoid drying to cipher organic coefficient (ratio

of organ weight to body weight was calculated). The

principal vital organs (heart, liver, spleen, lung,

kidney, testis, ovary and uterus) were preserved in

fixation medium of 10% solution of buffered formalin

(pH 7.4) and enclosed in paraffin-intended subsequent

histopathological examination. A section of each

organ tissue of 5 mm was stained with hematoxylin

and eosin (H&E). Each section was examined under

an optical microscope.

Statistic evaluation

All results were expressed as mean + SD (x s) for

the indicated number of experiments. The signifi-

cance of the difference among groups was analyzed

using one-way analysis of variance followed by the

StudentNewmanKeuls test. All statistic analyses

were made using the statistical analysis software

SPSS 17.0. The significant values at either p < 0.05

(*) or p < 0.01 (**) were represented as asterisks.

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Results

General observation, effects on clinical signs and

food consumption

There was no treatment-related mortality in animals

treated with neem oil for 90 days at any dose tested.

The group at the dose of 1600 mg/kg/day showed

treatment-related clinical signs, such as rough fur and

loss of appetite in the last 2 weeks. No treatment-

related clinical signs were observed in other experi-

mental groups.

The food consumption result is shown in Table 1.

The food consumption of all the test groups had no

statistical difference compared with that of the con-

trol group in month 1 and recovery month (p > 0.05),

while that of the dose 1600 mg/kg/day group was

very significantly decreased in months 2 and 3

(p < 0.01).

Table 1. Food consumption (g/100 g) of mice treated with neem oil.a

Groups Doses (mg/kg)

Food consumption of mice within 4 months postadministration

Month 1 Month 2 Month 3 Recovery month

Group I 0 11.56 + 1.71 9.20 + 2.53 8.43 + 1.67 8.81 + 1.72

Group II 177 10.88 + 1.34 8.13 + 2.31 7.29 + 2.80 7.80 + 2.09Group III 533 10.51 + 1.30 8.45 + 2.88 7.31 + 0.76 7.70 + 1.44Group IV 1600 9.09 + 2.53 5.43 + 0.64bc 4.90 + 0.52bc 7.08 + 0.87

ANOVA: analysis of variance.aData shown as mean + SD were analyzed by ANOVA followed by SPSS 17.0.bSignificantly different from control p < 0.01.cSignificantly different from control p < 0.05.

Table 2. Serum biochemistry parameters of mice treated with neem oil.a

Groups Doses (mg/kg) TP (g/L) ALB (g/L) GLO (g/L) A/G TBIL (mmol/L) ALT (U/L)

Administration for 90 days

Group I 0 74.87 + 5.43 47.20 + 4.99 27.67 + 0.45 1.73+ 0.15 12.63 + 4.64 135 + 2.15Group II 177 71.17 + 2.04 43.93 + 2.90 27.23 + 2.22 1.43+ 0.42 16.85 + 3.70 214 + 1.78Group III 533 76.80 + 0.80 44.67 + 5.67 32.13 + 4.87 2.20+ 0.11 18.74 + 6.87 147 + 3.77Group IV 1600 80.07 + 9.07 50.60 + 8.28 29.47 + 1.86 1.73+ 0.15 14.78 + 6.45 160 + 7.64After 30-day recoveryGroup I 0 66.57 + 3.10 41.53 + 3.73 25.03 + 1.21 1.63+ 0.23 15.12 + 4.35 103 + 7.52Group II 177 73.07 + 5.04 45.17 + 7.06 27.90 + 2.52 1.63+ 0.38 14.85 + 3.55 121 + 7.64Group III 533 71.37 + 3.25 41.73 + 1.34 29.63 + 3.34 1.40+ 0.17 18.96 + 6.40 95 + 7.07Group IV 1600 67.00 + 3.34 38.93 + 5.34 28.07 + 5.69 1.43+ 0.51 19.11 + 7.04 86 + 9.40

Groups Doses (mg/kg) AST (U/L) GLU (mmol/L) CHO (mmol/L) AST/ALT BUN (mmol/L) CRE (mmol/L)

Administration for 90 days

Group I 0 570 + 49.96 8.34+ 1.15 3.37 + 0.94 4.60+ 1.68 26.84 + 1.71 65.19 + 1.04Group II 177 300 + 22.63 9.05+ 1.72 3.05 + 0.37 4.90+ 1.02 19.85 + 0.82 55.70 + 1.78Group III 533 473 + 87.17 7.29+ 0.90 2.82 + 0.08 3.47+ 1.56 22.74 + 0.60 62.77 + 2.04Group IV 1600 570 + 57.09 8.78+ 0.85 3.53 + 0.53 4.07+ 2.25 26.95 + 2.21 74.26 + 0.85After 30-day recoveryGroup I 0 449 + 92.36 7.72+ 0.74 2.74 + 0.88 4.67+ 1.68 20.79 + 0.71 54.62 + 4.10Group II 177 526 + 32.18 6.40+ 1.00 2.08 + 0.37 4.07 + 2.24 22.70 + 0.15 61.89 + 2.80Group III 533 475 + 37.21 7.58+ 0.27 2.26 + 0.43 4.97 + 1.03 25.94 + 0.54 55.70 + 3.04Group IV 1600 411 + 31.52 9.73+ 1.89 2.48 + 0.43 4.70+ 0.85 23.49 + 1.98 55.69 + 2.00

ALT: alanine aminotransferase; AST: aspartate aminotransferase; BUN: urea nitrogen***; CRE: creatinine; GLU: glucose; TP: totalprotein; ALB: albumin; GLO: globulin; TBIL: total bilirubin; CHO: cholesterol; ANOVA: analysis of variance.aData shown as mean + SD were analyzed by ANOVA followed by SPSS 17.0.No significant difference from the control group at p > 0.05.

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Table 3. Organic coefficient (g/100 g) of mice treated with neem oil.a

Groups Doses (mg/kg) Heart Live Spleen Lung Kidney

Administration for 90 daysGroup I 0 0.54 + 0.15 4.67 + 0.86 0.28 + 0.03 0.88 + 0.25 1.34 + 0.21Group II 177 0.47 + 0.14 4.18 + 0.57 0.29 + 0.04 0.89 + 0.76 1.32 + 0.42

Group III 533 0.50 + 0.16 4.54 + 0.75 0.34 + 0.12 0.81 + 0.19 1.21 + 0.16Group IV 1600 0.49 + 0.18 4.31 + 0.47 0.23 + 0.06 0.76 + 0.15 1.23 + 0.41After 30-day recoveryGroup I 0 0.46 + 0.30 4.50 + 0.71 0.25 + 0.02 0.76 + 0.13 1.21 + 0.35Group II 177 0.47 + 0.37 4.18 + 0.53 0.30 + 0.03 0.68 + 0.15 1.25 + 0.16Group III 533 0.51 + 0.24 4.99 + 0.68 0.26 + 0.07 0.62 + 0.05 1.12 + 0.13Group IV 1600 0.46 + 0.10 4.99 + 0.45 0.27 + 0.07 0.73 + 0.32 1.37 + 0.46

ANOVA: analysis of variance.aData shown as mean + SD were analyzed by ANOVA followed by SPSS 17.0.No significant difference from the control group at P > 0.05.

(a)

Si

BDHA

H

P V

(b)

Si

(d)

CV

CV

K

(c)

CV

Si

Figure 1. Effect of neem oil on the microstructures of liver of mice after administration for 90 days. Panel A: group I(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); Panel D: group IV(1600 mg/kg, HE 400). Photomicrographs of the liver from mice treated with 0, 177, 533 and 1600 mg/kg in a 90-daysubchronic oral toxicity evaluation of neem oil. Cross-sections were stained with hematoxylin and eosin. Observationwas made at different amplified levels. Group I, the cross-section showed the normal appearance of liver, hepatic artery,portal vein, bile duct, Sis, Hs, all clearly conserved. Groups II and III, showed central venous (CV) and Sis congestion in liverlobule (!); group IV, central venous congestion (!), granular and vacuolar degeneration ("), karyorrhexis (K) wereobserved in Hs. Si: sinusoid; H: hepatocyte.

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Biochemical examination

The serum biochemistry parameters were examined

after 90 days of oral administration with neem oil.

No significant differences were noted between the

mice-treated groups with the 177, 533 and 1600 mg/

kg/day dose of neem oil and the vehicle-control

group. Similar results were obtained 30 days afterrecovery (Table 2).

Effects on organ coefficient

The results of organ coefficient are summarized in

Table 3, the organ coefficient of heart, liver, spleen,

lung and kidneys in the experimental groups with

177, 533 and 1600 mg/kg/day dose of neem oil for

90 days had no statistical difference compared with

that of the vehicle-control group, the same result was

also shown 30 days after recovery (p > 0.05).

Histopathological findings

At the 90th day, the histopathological examination

showed that only the 1600 mg/kg/day dose of neemoil had varying degrees of damage on each organ

except heart, uterus and ovary. The consistent

treatment-related histopathological changes were

found in both sexes.

In the vehicle-control group (group I), the cross-

section of liver showed normal appearance, hepatic

artery, portal vein, bile duct, sinusoids (Sis) and hepato-

cytes (Hs), all clearly conserved (Figure 1(a)). Doses

(177 and 533 mg/kg/day) of groups II and III, respec-

tively, only showed central venous and Sis congestion

(d)(c)

(b)(a)ST RP

WP

GC

Figure 2. Effect of neem oil on the microstructures of spleen of mice after administration for 90 days. Panel A: group I(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV

(1600 mg/kg, HE 400). Group I, the cross-section showed normal appearance of the spleen, spleen trabecula, RP, whitepulp, germinal centers, all clearly conserved. Groups II and III, showed the normal characteristic of spleen, infiltration ofmultinucleate giant cells in spleen (!); group IV, severe hyperemia of RP (") and infiltration of multinucleate giant cells (!)in spleen were observed. RP: red pulp.


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in liver lobule, Hs occurred granular and slight vacuolar

degeneration (Figure 1(b) and (c)), while at the dose

(1600 mg/kg/day dose) of group IV, central venous

congestion, granular and vacuolar degeneration, karyor-

rhexis were observed in Hs (Figure 1(d)).

In group I, the cross-section of the spleen showednormal appearance, spleen trabecula, red pulp (RP),

white pulp and germinal centers, all clearly conserved

(Figure 2(a)). Groups II and III showed the normal

characteristic of spleen associated with infiltration

of multiple giant cells (Figure 2(b) and (c)), while

in group IV severe hyperemia of RP and infiltration

of multinucleate giant cells in spleen were observed

(Figure 2(d)).

In group I, the cross-section of the lung showednormal appearance (Figure 3(a)). In group II, the

alveolar walls were thickened, the capillaries in the

alveolar walls and interstitial were congested with

many red blood cells (Figure 3(b)), while in groups III

and IV, the alveolar walls were thickened, the capil-

laries in the alveolar walls and interstitial were

severely congested, a serious alveolar cavity hemor-rhage was observed (Figure 3(c) and (d)).

In group I, the cross-section of the kidneys showed

normal appearance, glomerulus and renal tubule

structure was normal (Figure 4(a)). Groups II and III

showed capillary of glomerulus and interstitial

angiectasis hyperemia, renal tubular epithelial cells

swelling, granular degeneration and some of them

were separated from the basement membrane (Figure

4(b) and (c)). In group IV, massive inflammatory cells

especially neutrophilic granulocyte infiltrated in the

(d)

(b)

(c)

(a)

A

AS

AD

Figure 3. Effect of neem oil on the microstructures of lung of mice after administration for 90 days. Panel A: group I(0 mg/kg, HE 400); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV

(1600 mg/kg, HE 400). Group I, the cross-section showed the normal appearance of lung. Alveolus (A), alveolar ducts(AD), alveolar sac (AS), all clearly conserved. Group II, showed the alveolar walls were thickened ("), the capillaries in thealveolar walls and interstitial were congested with many red blood cells (!); groups III and IV, the alveolar walls werethickened ("), the capillaries in the alveolar walls and interstitial were severe congested, alveolar cavity hemorrhage seri-ous were observed (!).

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glomeruli and nephric tubules, RETC degeneration

and necrosis, segregated with basilar membrane. Therenal tubule revealed protein cast (Figure 4(d)).

In group I, the cross-section showed the normal char-

acteristic of testicle, the seminiferous tubules structurewas intact, all levels of spermatogenic cells (SCs) were

arranged in order (Figure 5(a)). In Groups II andIII show-

ing the decrease of SCs and blister degeneration, in some

more serious cases, SCs occurs ballooning degeneration

(Figure 5(b) and (c)). Group IV, the basic structure of

seminiferous tubule was destroyed, SCs were seriously

dissolved and disappeared, sperm within the seminifer-

ous lumen almost completely disappeared (Figure 5(d)).

Thirty days after recovery, the degree of injury to

the tissues was lessened or even restored.

Discussion

Food consumption is a key parameter for determining

the dose of feeding and an important indicator of toxic

effects of chemical substances.18 The results obtained

from the study demonstrated that the food consumptionof mice at the dose of 1600 mg/kg/day decreased very

significantly in months 2 and 3, while those from the

other experimental groups had no significant changes

compared with the control group. And this was consis-

tent with the general clinical manifestations observed

and recorded for group IV, so the clinical dosage of

neem oil should be lower than 1600 mg/kg/day.

The results from the subchronic oral toxicity study

with neem oil on male and female young adult mice

Figure 4. Effect of neem oil on the microstructures of kidneys of mice after administration for 90 days. Panel A: group I(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV(1600 mg/kg, HE 400). Group I, the cross-section showed the normal appearance of kidney, glomerulus (G), proximaltubule (PT) and distal tubule (DT), all conserved. Groups II and III, capillary of renal interstitium was hemolysis (!), renaltubular epithelial cells swelling, granular degeneration, separated from basement membrane; group IV, massive inflamma-tory cells (#) infiltrated in the glomeruli and nephric tubules, RETC degeneration and necrosis, segregated with basilarmembrane. The renal tubule revealed protein cast.


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showed that all the serum biochemistry parameters of

experimental groups had no statistically significant

difference compared with those of the control group

(p > 0.05) after oral administration with neem oil for

90 days. Similar results were obtained 30 days after

recovery. It indicated that oral administration of neem

oil in mice for 90 days at and below 1600 mg/kg/daydose had no damage on the serum biochemistry para-

meters. And this was consistent with the result of the

studies of Rukmini et al. and Lakshminarayana.19,20

Organ coefficient can reflect the degree of organs

damages. Excluding the loss of water, age, gender, the

effect of malnutrition and other factors before being

weighed, the increase in the organ coefficient indi-

cates that there are changes in congestion, edema,

hyperplasia, hypertrophy in organs, while there are

changes in shrink and degeneration when it is

decreased.21 In the present study, all the examined

organ coefficients had no statistical difference com-

pared with those of the vehicle group, which indicated

that neem oil had mild or no damages on organs of

mice. And this was consistent with the result that the

oral administration with neem oil for 90 days had

varying degrees of damages on organs, but this waslessened or even to be restored 30 days after recovery.

To determine the NOAEL and target organ toxicity

of neem oil, the pathological examination of principal

vital organs in mice at different doses of neem oil were

observed under a microscope. The results of oral admin-

istration with neem oil for 90 days from the study

showed that neem oil had no damage on heart. The

177 and 533 mg/kg/day doses of neem oil had mild

damages on liver, spleen, lung, kidneys and testicle,

such as slight vascular congestion, while 1600 mg/kg/

(a)

IC

SzST

St

SC

(b)

SC

ST

(c)

ST

SC

(d)

ST

SC

Figure 5. Effect of neem oil on the microstructures of testicle of mice after administration for 90 days. Panel A: group I(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV

(1600 mg/kg, HE 400). Group I, the cross-section showed the normal appearance of testicle, the seminiferous tubules,SCs at all levels, spermatozoon (Sz), testicular interstitial cells (IC) in stroma (St) surround the seminiferous tubules, allconserved. Groups II and III, the SCs abscission, quantity decrease, blister degeneration, serious turned into ballooningdegeneration; group IV, the basic structure of seminiferous tubule was destroyed, SCs were seriously dissolved and dis-appeared, sperm within the seminiferous lumen was almost completely disappeared. SC: spermatogenic cell.

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day dose of neem oil had varying degrees of damages on

each organ, mainly granular and vacuolar degeneration

in cells and vascular congestion, in addition, in the lung,

alveolar walls were thickened and hemorrhage damage

was shown. All the damages on organs were lessened or

even to be restored 30 days after recovery. So the patho-

logic damage on mice with neem oil was reversible, thedamage may be gradually restored after thediscontinua-

tion of treatment in the long run.

In the present study, neem oil had mild damages on

organs at a dose of 177 mg/kg/day, and the damages

were restored after the discontinuation of treatment for

30 days, so the NOAEL for males and females was con-

sidered to be 177 mg/kg/day. The effect of neem oil on

the liver, kidneys andtesticle was showing a good dose

response relationship, indicating that the target organs

of neem oil toxicity were the liver, kidneys and testicle.

The testicle has been proved to be the target organ,which was consistent with the findings of Yin

et al.22,23 The mechanism of the antifertility effect

of extract chloroform from neem oil on mice maybe that the replacement process of testicular sperm

nuclear protein was blocked, which led to abnormal

epididymal sperm nucleoprotein and sperm not suffi-

ciently differentiated to mature. Further study needed

to do to explore the toxic mechanism of neem oil on

liver and kidney.

FundingThis work was supported by the Special Fund for Agro-

scientific Research in the Public Interest (201203041);

National Natural Science Foundation of China (Grant no.

31272612); National Science & Technology Program in

Rural Areas During the 12th Five Year Plan Period

(2011BAD34B03-4); the Doctoral Program of Higher

Education Research Fund (Instructor Dr Class

20105103110001).

Authors Note

The first three authors contribute equally to this work and

should be considered as first author. CW and MC contrib-uted equally to this work.

Acknowledgments

The authors thank Green Gold Biological Science & Tech-

nology (Chengdu, PR China) for supplying neem oil.

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C o p y r i g h t o f H u m a n & E x p e r i m e n t a l T o x i c o l o g y i s t h e p r o p e r t y o f S a g e P u b l i c a t i o n s , L t d .

a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v w i t h o u t

t h e c o p y r i g h t h o l d e r ' s e x p r e s s w r i t t e n p e r m i s s i o n . H o w e v e r , u s e r s m a y p r i n t , d o w n l o a d , o r

e m a i l a r t i c l e s f o r i n d i v i d u a l u s e .

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