Parkinson’s disease (PD) is a progressive, age-related, neu-rodegenerative disorder characterized by bradykinesia, rigid-ity, resting tremor and gait disturbance1); it occurs in 1—2%of the population over the age of 60 years.2) Typical neu-ropathologic features of the disease are dopaminergic neurondegeneration in the substantia nigra and the presence ofeosinophilic intracytoplasmic inclusions (Lewy bodies) inthe residual dopaminergic neurons. The most widely usedmouse model of PD is produced by systemically administer-ing the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine (MPTP).3) MPTP is converted by monoamine oxidase B(MAO-B) to 1-methyl-4-phenylpyridinium (MPP�), which isa neurotoxic metabolite and could block cellular respiration,promote reactive oxygen species (ROS) formation, and causeneuronal death.4,5)

Oxidative stress has been implicated either as the cause oras a consequence of the pathogenetic mechanisms responsi-ble for neurodegenerative disease, including PD. Indeed, thebrain is prone to producing ROS and oxidative stress, due toits high metabolic rate, combined with the high content ofoxidizable molecules, such as dopamine and neuromelanin,whose metabolism generates ROS.6—8) Superoxide dismutase(SOD) is one of the key enzymes that provide the first line ofdefense against pro-oxidants. Glutathione peroxidase (GSH-PX) is thought to play an important role in protecting mem-branes from damage due to lipid peroxidation. The majordetoxification function of GSH-PX is the termination of radi-cal chain propagation by quick reduction to yield further rad-icals.9) The levels of SOD and GSH-PX activity reflect theability of organism to eliminate free radicals. Malondialde-hyde (MDA) is the end product of lipid peroxidation, and itslevel could indirectly reflect the metabolic degree of oxygenradicals, thus serving as an index of oxidative damage.10)

Kaempferol is a prototype member of the flavonol sub-class of flavonoids, which is widely found in tea, broccoli,grapefruit, brussel sprouts and apple and is claimed to havestrong antioxidant and anti-inflammatory properties.11)

Kaempferol, can be given orally, is absorbed well and the

bioavailability is proportionate to its dose.12) Kaempferol hadearlier been shown to afford efficient neuroprotection againstseveral apoptosis and necrosis-inducing insults, such as oxidized low-density lipoproteins13,14) and L-glutamate.15,16)

Ishige et al.15) demonstrated that kaempferol efficientlyblocked the increase in ROS associated with the oxidativestress caused by glutamate in the mouse hippocampal cellline HT-22. Filomeni et al.6) demonstrated that kaempferolexerted a strong and prolonged protective effect againstrotenone toxicity, which was a classical toxin inducing PD.Striatal glutamatergic response of rat brain slices was alsopreserved by kaempferol, posing a more general protectionof kaempferol in PD.

Preliminary studies in our laboratory have shown thatkaempferol derivatives prevent oxidative stress-induced celldeath in a DJ-1-dependent manner in vitro, and that DJ-1 wasa causative gene product of a familial form of PD,17) indicat-ing that kaempferol might be neuroprotective in PD. How-ever there has been no systematic research on this topic invivo so far. The purpose of the present study was to evaluatethe potential neuroprotective effects of kaempferol in themouse model of MPTP-induced dopaminergic neuronal dam-age, and explore its possible mechanism.

MATERIALS AND METHODS

Materials MPTP and kaempferol (Fig. 1) were pur-chased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Assaykits for SOD, GSH-PX, MDA and MAO-B were purchasedfrom Jiancheng Bioengineering Institute (Nanjing, China).Goat polyclonal antibody against tyrosine hydroxylase (TH)

August 2011 1291Regular Article

Neuroprotective Effect of Kaempferol against a 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Mouse Model of Parkinson’s Disease

Shen LIa,b and Xiao-Ping PU*,a,b

a National Key Research Laboratory of Natural and Biomimetic Drugs, Peking University; and b Department of Molecularand Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University; Beijing 100191, P. R. China.Received March 12, 2011; accepted May 14, 2011; published online May 27, 2011

In the present study, we investigated the neuroprotective effects of kaempferol in the mouse model ofParkinson’s disease, which was induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Weconfirmed that MPTP led to behavioral deficits, depletion of dopamine and its metabolites, reduction in super-oxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and the elevation of malondialdehyde(MDA) levels in the substantia nigra. When administered prior to MPTP, kaempferol improved motor coordina-tion, raised striatal dopamine and its metabolite levels, increased SOD and GSH-PX activity, and reduced thecontent of MDA compared with mice treated with MPTP alone. Immunohistochemical studies using anti-tyro-sine hydroxylase (TH) antibody showed that medication of kaempferol could prevent the loss of TH-positive neu-rons induced by MPTP. Taken together, we propose that kaempferol has shown anti-parkinsonian properties inour studies. More work is needed to explore detailed mechanisms of action.

Key words kaempferol; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; neuroprotection; oxidative stress; Parkinson’s disease

Biol. Pharm. Bull. 34(8) 1291—1296 (2011)

© 2011 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: pxp123@bjmu.edu.cn

Fig. 1. Chemical Structure of Kaempferol

was purchased from Chemicon International (Temecula, CA,U.S.A.). Histostain™-SP and diaminobenzidine (DAB) kitswere purchased from Zhongshan Goldenbridge Biotechnol-ogy (Beijing, China).

Animal Grouping and Treatment Adult male C57BL/6mice with an initial experimental weight of 20—25 g (8weeks old) were purchased from the Laboratory AnimalCenter of Peking University Health Science Center (Beijing,China). These mice met the approval of the local animalcommittee with confirmation number SCXK (Jing) 2007—0001. Animals were housed in a controlled environment(22�2 °C) with food and water available ad libitum. All ex-periments were performed under the Guidelines of the Ex-perimental Laboratory Animal Committee of Peking Univer-sity Health Science Center and were in strict accordance withthe principles and guidelines of the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.

Animals were acclimatized for 1 week prior to experimen-tation, then randomly divided into the following six groups(12 mice per group): group A (vehicle control group, anequal volume of normal saline); group B (MPTP modelgroup, pre-treatment with an equal volume of saline); groupsC, D and E (low, moderate and high doses of kaempferol,pre-treatment with kaempferol at doses of 25, 50 and100 mg/kg, respectively); group F (positive-control group,pre-treatment with selegiline at dose of 15 mg/kg). Allgroups were administered the respective pre-treatment com-pounds orally (per os (p.o.)) every 24 h for 14 consecutivedays, with the first day of administration designated as day 1.The PD mouse models for groups B, C, D, E, F were gener-ated by five consecutive injections of MPTP at a dose of30 mg/kg every 24 h from day 10 to day 14.18) For all groups,MPTP was injected intraperitoneally 1 h following oral ad-ministration of the pre-treatment compound. An equal vol-ume of saline instead of MPTP was injected into mice ingroup A.

Behavior Test. Rotarod Performance Test The micetook part in the rotarod performance test to evaluate motorcoordination 1 d after MPTP treatment.19—21) A rotarod ma-chine (Experimental Factory of Peking University HealthScience Center, Beijing, P. R. China) with a rotating spindle(diameter 7.3 cm) and five individual compartments was usedto test five mice at a time. The surface of the rotating bar wasdesigned to prevent mice gripping onto the surface. In theformal test, the rotation speed was set to 25 rpm. The timethat the mice remained on the rotating bar was recorded forthree tests for each mouse at 5-min intervals. Data are pre-sented as mean time on the rotating bar over the three tests.

Spontaneous Motor Activity Test Spontaneous motoractivity was assessed with an infared motion activity system(Experimental Factory of Chinese Academy of Medical Sci-ences, Beijing, China) that consisted of four Plexiglas cages(23 cm�30 cm, diameter�height) 2 d after MPTP treatment.Each cage was equipped with three infrared beams that con-tinuously detected all vertical and horizontal movements per-formed by the mouse. The activity was assessed by countingthe number of infrared beam crossing the photocell apparatusper 5 min by an automated counting system.22) The sponta-neous motor activity was measured for 5 min.

High Pressure Liquid Chromatography (HPLC) Assayfor Analysis of Dopamine and Its Metabolite Brain Tis-

sue Preparation: Six mice from each group were sacrificed bycervical dislocation 3 d after MPTP treatment. The brainswere rapidly removed and dissected on an ice-cold plate. Foreach mouse, the striata from the left and right hemisphereswere together transferred into a 1.5 ml plastic vial, weighedand homogenized in iced-cold HClO4 (0.4 M) using an ultra-sonicator. After storage for 1 h in ice, the homogenates werecentrifuged at 12000 g for 15 min at 4 °C. The supernatantwas then incubated with a mixed buffer (20 mM sodium citrate, 300 mM K2HPO4, 2 mM sodium ethylenediamine-tetraacetic acid [Na2EDTA]) at the ratio (v/v) of 1 : 2 for 1 hin ice and centrifuged at 12000 g for 15 min at 4 °C. The supernatant was collected and filtered through a 0.22-mm filter and subsequently analyzed by HPLC.23)

HPLC Assay: The levels of dopamine and 3,4-dihydroxy-phenylacetic acid (DOPAC) were determined by HPLC,which was equipped with an electrochemical (EC) detectorused for quantification.24) Briefly, the striatum was homoge-nized in 0.1 mol/l HClO4 containing 0.1 mM EDTA. The lev-els of dopamine and DOPAC were determined by referenceto standard curves. Results were calculated and expressed asmg/g tissue weight.

Tyrosine Hydroxylase (TH) ImmunohistochemistryFour days after MPTP treatment, mice were anaesthetizeddeeply by intraperitoneal (i.p.) injection of 10% chloral hy-drate and perfused through the left ventricle with normalsaline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Brains were removed andpost-fixed in 4% paraformaldehyde for 8 h. Next, they werecytoprotected by soaking in 10% and then 30% sucrose untilsinking.25) Serial coronal sections were cut through the sub-stantia nigra pars compacta (SNPc) at 20 mm by a freezingmicrotome. The sections were mounted on slides and storedat �20 °C until use.

Sections were rinsed several times in PBS. Tissue endo-genous peroxidase was inactivated by incubating in 10%methanol and 3% hydrogen peroxide in PBS for 10 min.After three washes in PBS, the sections were pre-incubatedin blocking buffer (PBS containing 10% goat serum) to re-duce non-specific binding, and then were incubated overnightat 4 °C in a humidified chamber with rabbit TH primary anti-body at dilution of 1 : 500. Sections were rinsed in PBS, thenincubated with goat anti-rabbit secondary antibody for30 min at 37 °C following the manufacturer’s instructions.The sections were stained with a dimethylaminoazobenzene(DAB) kit, dehydrated in graded alcohols, cleared with xy-lene and coverslipped. Control sections were treated with thesame protocol but omitting the primary antibody. For mor-phological analysis, the images were recorded with an in-verted microscope (OLYMPUS) connected to a camera. Cellcounts were determined from 4 anatomically matched sec-tions from each of the animals, and 3 animals were used forcell counts.

Assay of SOD Activity, GSH-PX Activity, MAO-B Ac-tivity and MDA Content Four days after MPTP treatment,the brain was quickly removed, and the substantia nigra wasisolated on an ice-cold glass plate. Samples were weighed ac-curately and prepared with 0.86% normal saline to give 10%tissue homogenate by super-audible cell disintegrator (Son-ics), which was then centrifuged at 3000 rpm/min for 15 minat 4 °C. The supernatant was collected and kept at �80 °C

1292 Vol. 34, No. 8

until use. The protein concentration of the substantia nigrawas determined by the method of Lowry et al. using bovineserine albumin as a standard.26)

With the respective detection kits (Nanjing Jiancheng Bio-engineering Institute, China), the activities of SOD, GSH-PXand MAO-B, and the content of MDA in the substantia nigrawere determined following the kit specifications. Results arepresented as units of activity per mg of protein (wet weight)or content.

Statistical Analysis Data are expressed as mean�stan-dard deviation (S.D.). A one-way analysis of variance(ANOVA) followed by Dunnett’s post-hoc analysis was per-formed to determine whether individual doses were signifi-cantly different relative to controls. A p-value less than 0.05was considered statistically significant.

RESULTS

Motor Behavioral Test In the rotarod test, the latent pe-riod that represented the time for mice to remain on the barreduced significantly in the PD experimental model group,relative to the control group (p�0.01). The groups pre-treated with moderate and high doses of kaempferol and thepositive control group (selegiline) rescued this reduction

relative to the PD experimental model group (p�0.01 for all comparisons, F5,30�58.615). Moreover, the effect ofkaempferol in the rotarod test was dose dependent. Resultsare shown in Fig. 2.

In the spontaneous motor activity test, the number ofmovements by the mice in 5 min reduced significantly in thePD experimental model group, relative to the control group(p�0.01). The groups pre-treated with moderate and highdoses of kaempferol and the positive control group (selegi-line) rescued this reduction relative to the PD experimentalmodel group (p�0.05, p�0.01, p�0.01, respectively,F5,30�11.170). Moreover, the effect of kaempferol in thespontaneous motor activity test was dose dependent. Resultsare shown in Fig. 3.

Striatal Dopamine and DOPAC Levels The effects ofkampferol on the levels of dopamine and DOPAC in the stri-ata of MPTP-induced PD model mice are shown in Table 1.The basal striatal levels of dopamine and DOPAC decreasedin the MPTP experimental group relative to the control group(p�0.01 for both comparisons). Compared with the MPTP-treatment group, both the kaempferol pre-treatement groupsand the selegiline pre-treatment group attenuated MPTP-induced dopamine depletion (p�0.01, p�0.05, p�0.01,p�0.01, respectively, F5,30�26.408). The groups pre-treated

August 2011 1293

Fig. 2. The Time Period on the Rotarod

The duration of time on the rotating rod was significantly decreased by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (15.2�5.7 s) when compared with thecontrol group (102.0�16.7 s), but this difference was reversed by kaempferol pre-treatment (21.0�6.1 s, 50.8�10.2 s, 82.5�9.8 s, respectively) and selegiline (84.8�15.8 s). Val-ues are presented as mean�S.D. (n�6). ∗∗ p�0.01, compared with the MPTP group. ## p�0.01, compared with the control group.

Fig. 3. The Number of Total Movements in 5 min in the Spontaneous Locomotion Test

Locomotion counts were drastically reduced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (145.3�31.0) when compared with the control group(275.3�41.4). However, this reduction in motor activity was prevented by kaempferol (182.7�39.5, 230.8�31.7, 242.0�35.8 respectively) and selegiline (256.0�34.0). Values arepresented as means�S.D. (n�6). ∗ p�0.05, ∗∗ p�0.01, compared with the MPTP group. ## p�0.01, compared with the control group.

with high doses of kaempferol showed reduced MPTP-in-duced DOPAC depletion (p�0.01, F5,30�30.975). Moreover,in the kaempferol pre-treatment groups, the results were dosedependent.

The ratio DOPAC/dopamine, which is indicative ofdopamine turnover in dopaminergic terminals, was increasedin the experimental group (p�0.05), while it was altered byhigh doses of kaempferol and selegiline (p�0.05, p�0.01,respectively, F5,30�20.115) markedly. Results are shown inFig. 4.

Tyrosine Hydroxylase (TH) ImmunohistochemistryTyrosine hydroxylase staining was performed to evaluate thesurvival of dopaminergic neurons. Morphological observa-tion was shown in Fig. 5A. In the control group, TH-positivecells were plentiful, cytoplasm and neurofibers of which wereclear and intensively stained. Mice in the model groupshowed a pronounced reduction in the number of TH-positivecells with their neurofibers fractured and smaller cell size incomparison with the control group; no effect was seen inmice given kaempferol and selegiline, this administration re-sulted in an increase in TH-positive cells with similar cellmorphology with the control group, thus suggestingkaempferol could protect dopaminergic neurons from MPTPneurotoxicity in mice.

In agreement with the above cellular morphological obser-vation, in the control group, the average cell count was 86�10 per section. In MPTP group, the mean was 39�14 persection (p�0.01). In kaempferol (100 mg/kg) pre-treatmentMPTP group, the mean was 76�10 per section (p�0.01). In

the selegiline group pre-treatment MPTP group, the meanwas 79�9 per section (p�0.01, F3,44�46.666).

Effects of Kaempferol on the Activity of SOD, GSH-PXand on the MDA Levels The effects of kaempferol on theactivity of SOD, GSH-PX and the content of MDA in thesubstantia nigra of mice are shown in Table 2. MPTP admin-istration in the PD model group mice resulted in a significantreduction in SOD and GSH-PX activity and increased levelof MDA relative to the control group (p�0.01 for all com-parisons). However, this condition was partially rescued inthe kaempferol-pre-treatment and selegiline groups. The activities of SOD in either kaempferol groups or the selegi-line group were increased (p�0.01 for all comparisons,F5,30�52.192). The activities of GSH-PX were increased inthe groups given moderate and high doses of kaempferol(p�0.01 for both comparisons, F5,30�33.060). The level ofMDA was decreased in the moderate and high doses ofkaempferol groups and selegiline group (p�0.01 for allcomparisons, F5,30�65.256).

Effects of Kaempferol on the Activity of MAO-B Asshown in the Table 3, MPTP treatment induced a marked in-crease in MAO-B activity (p�0.01). Selegiline, the wellknown MAO-B inhibitor, exerted a strong effect on MAO-Binhibition (p�0.01,). However, pre-treatment with kaempfe-rol at either dose failed to significantly affect the activity ofMAO-B (p�0.05, F5,30�7.325), therefore suggesting that theneuroprotective effects of kaempferol was not due to theMAO-B inhibition.

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Table 1. Effects of Kaempferol on the Levels of Dopamine (DA) and 3,4-Dihydroxyphenylacetic Acid (DOPAC) in the Striata of Mouse 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model

Group DA (mg/g) DOPAC (mg/g)

Control 11.53�1.70 5.08�0.43Model (MPTP 30 mg/kg) 4.48�0.86## 3.11�0.49##

Kaempferol (25 mg/kg)�MPTP 6.81�0.75** 3.70�0.67Kaempferol (50 mg/kg)�MPTP 7.51�1.26* 3.91�0.15Kaempferol (100 mg/kg)�MPTP 9.25�1.36** 4.44�0.38**Selegiline (15 mg/kg)�MPTP 9.74�0.85** 2.18�0.40

Values are mean�S.D. (n�6), ∗ p�0.05, ∗∗ p�0.01, compared with the MPTPgroup. ## p�0.01, compared with the control group.

Fig. 4. Effects of Kaempferol on Dopamine Metabolism

The 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine (DA) ratio was significantly elevated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (0.71�0.11)in comparison with the control group (0.45�0.09). While it was reduced in the kaempferol treatment groups (0.55�0.09, 0.53�0.08, 0.49�0.08) and selegiline treatment group(0.23�0.06). ∗ p�0.05, ∗∗ p�0.01, compared with the MPTP group. # p�0.05, compared with the control group.

Table 2. Effects of Kaempferol on the Activity of Superoxide Dismutase(SOD), Glutathione Peroxidase (GSH-PX) and the Content of Malondialde-hyde (MDA)

GroupSOD GSH-PX MDA

(U/mg prot) (U/mg prot) (nmol/mg prot)

Control 160.33�15.31 24.17�3.31 6.12�0.29Model (MPTP 30 mg/kg) 83.00�7.40## 9.00�1.78## 13.23�0.85##

Kaempferol (25 mg/kg)�MPTP 106.33�8.58** 11.33�2.07 11.15�1.32Kaempferol (50 mg/kg)�MPTP 133.67�6.89** 15.50�1.52** 9.70�0.54**Kaempferol (100 mg/kg)�MPTP 152.83�9.64** 16.17�1.60** 8.75�0.36**Selegiline (15 mg/kg)�MPTP 146.17�10.83** 12.17�2.79 9.17�0.43**

Values are mean�S.D. (n�6), ∗∗ p�0.01, compared with the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) group. ## p�0.01, compared with the control group.

DISCUSSION

Our studies showed that treatment with kaempferol im-proved motor abnormalities and increased striatal dopamine

and DOPAC content in PD mice. Moreover, as a possiblemechanism of its neuroprotection, kaempferol increased theactivity of SOD and GSH-PX, reduced the content of MDA,which indicated its raised anti-oxidative capacity.

The neurotoxin MPTP is known to cause C57BL/6 mice todevelop parkinsonism. The behavioral manifestation, neuro-chemical features and primary pathological condition in-duced by MPTP in mice are similar to that shown by PD pa-tients. Therefore, MPTP-treated C57BL/6 mice make excel-lent conventional models and this regime is widely used forstudies on PD.1,3,23) In the present study, MPTP-lesioned miceshowed a behavioral deficit, but no mice were dead duringthe MPTP administration. During the first two or 3 d, the av-erage body weight was slightly decreased, but recovery laterin the MPTP treatment. Kaempferol-pre-treated mice exhib-ited increased motor coordination and spontaneous locomo-tion compared with MPTP-treated mice alone.

Selegiline (L-deprenyl) is believed to render protectionagainst MPTP neurotoxicity to a signficant extent via a freeradical scavenging mechanism and the ability to inhibitMAO-B in the brain. Its neuroprotective action was attrib-uted to the inhibition of the metabolism of dopamine in thebrain by dopamine reuptake inhibition, stimulation of antiox-idant enzyme, such as SOD and catalase and increasedturnover of dopamine, etc.27—29) Therefore, we use selegilineas a positive control to evaluate the neuroprotective effect ofkaempferol.

Dopamine is the primary neurotransmitter involved inmotor functions, its loss directly impacts physical move-ments and contributes to the clinical symptoms,2) depletionof which is also considered a cardinal feature in the cause ofPD in humans or in animal models of the disease.30,31) MPTPcauses a partial lesion of the substantia nigra and a signifi-cant reduction in striatal dopamine levels.23.32) Drugs that areable to ameliorate MPTP-induced neuronal damage are con-sidered to be neuroprotective. The results of our presentstudy show that the pre-intake of kaempferol markedly im-proved MPTP-induced dopamine and DOPAC depletion inthe striatum, and reduced the DOPAC/dopamine ratio, whichwas significantly altered by MPTP. The metabolite/neuro-transmitter ratio is an index of the rate of neurotransmittermetabolism. A decrease in the ratio indicates a decrease inneurotransmitter renovation rate.23,33) The enhancement ofdopamine content by kaempferol might have restored thechanges in locomotor activity. The neuroprotective effect ofkaempferol may be due, in part, to a decrease in dopaminemetabolism in the striata.

Dopamine is synthesized in two steps. Firstly, tyrosine isconverted to L-dihydroxyphenylalanine (L-DOPA) by TH,and then L-DOPA is then converted to dopamine by L-DOPAdecarboxylase. Tyrosine hydroxylase is, therefore, a key en-zyme for dopamine biosynthesis and is used as a marker fordopaminergic neurons.34,35) Our results show that administra-tion of kaempferol reduced the MPTP-induced loss of tyro-sine hydroxylase-positive neurons in the mouse substantianigra, which suggest that kaempferol may enhance the sur-vival of dopamine neurons in MPTP-lesioned mice.

To further investigate the mechanism of action ofkaempferol, we measured the anti-oxidative capacity and theactivity of MAO-B. Oxidative stress refers to the cytologicconsequences of a mismatch between the production of free

August 2011 1295

(A)

(B)

Fig. 5. Immunohistochemical Staining of Tyrosine Hydroxylase (TH) inSubstantia Nigra of Mice

Photomicrographs were taken at a magnification of 200�. (A) Control group. b)Model group (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 30 mg/kg). c)Kaempferol (100 mg/kg)�MPTP group. d) Selegiline (15 mg/kg)�MPTP group. (B)Values are presented as means�S.D. of 3 mice per group and 4 sections per mouse.# p�0.01, compared with the control group. ∗ p�0.01, compared with the MPTP group.

Table 3. Effects of Kaempferol on the Activity of Monoamine Oxidase B(MAO-B)

GroupMAO-B

(U/mg prot)

Control 6.12�0.42Model (MPTP 30 mg/kg) 7.81�0.40##

Kaempferol (25 mg/kg)�MPTP 6.98�1.26Kaempferol (50 mg/kg)�MPTP 7.52�0.92Kaempferol (100 mg/kg)�MPTP 7.06�1.50Selegiline (15 mg/kg)�MPTP 4.93�0.69**

Values are mean�S.D. (n�6), ∗∗ p�0.01, compared with the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) group. ## p�0.01, compared with the control group.

radicals and the ability of the cell to defend against them.36)

A defect in one or more of the naturally occurring antioxi-dant defenses could lead to neurodegeneration in PD.37) Thisimbalance results in apoptosis of neurons and auto-oxidationof dopamine.38) Our studies show a decrease in the activitiesof SOD and GSH-PX in the MPTP group. However, the de-crease of antioxidant enzyme activities caused by MPTP wasmarkedly restored by pre-treatment with kaempferol. Mean-while, the level of MDA, which serves as an index for deter-mining the extent of lipid peroxidation, was reduced bykaempferol which was altered by MPTP. Restoration of theactivities of SOD and GSH-PX and reduction of the contentof MDA due to pre-treatment with kaempferol demonstratethe protective role of kaempferol.

As previously reported, kaempferol behaves as a potentMAO inhibitor in vitro.39) It also has been shown that theMAO-B activity is elevated with age, which is directly re-lated to PD, and MAO-B inhibitor has been used as a classicdrug for clinical treatment. In the PD mouse model, MPTPwas metabolized selectively by MAO-B to the active toxinMPP�.1) Sundstrom and Jonsson reported that MAO-B inhibitor could attenuate the MPTP-induced increase ofdopamine turnover through inhibition of dopamine metabo-lization in the mouse.40) The activity of MAO-B, therefore, isof critical importance. We evaluated the activity of MAO-Bin the mouse striata by exploring the action of kaempferol.Data obtained in the present experiments show that pre-treat-ment with kaempferol could not effectively inhibit MAO ac-tivity, but selegiline could, suggesting that kaempferol doesnot exert a MAO-B inhibiting effect in vivo. The two studieswere different in methods and its sensitivity. Furthermore,kaempferol in the study of the reference cited produced moreinhibition of MAO-A, not MAO-B. While in our study, weonly detected the effect of kaempferol on MAO-B inhibition.Considering these differences, kaempferol didn’t have aMAO-B inhibition in our present study, unlike the results inreference. Or the number of samples was a little small. Thus,the excellent anti-oxidative capacity, but not MAO-B inhibi-tion, contributed more to the neuroprotective mechanism ofkaempferol.

In conclusion, our data provide evidence that kaempferolhas neuroprotective effects in MPTP-induced PD mice,which may have contributed to its anti-oxidative capacity toscavenge free radicals and resulted in the survival of moredopamine neurons. We propose that kaempferol has shownanti-parkinsonian properties, pending future studies to eluci-date further detailed mechanisms of action.

Acknowledgements We are grateful to Dr. Xin Zhao forher assistance in experiments and her comments and sugges-tions during the preparation of this manuscript. This workwas supported by a Grant from the National Natural Scienceof China (No. 30973889).

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