Food Sci. Biotechnol. 23(4): 1341-1348 (2014)

DOI 10.1007/s10068-014-0184-3

Antioxidant, Antimutagenic, and In vivo Buccal Mucosa Cancer

Preventive Effects of Fructus Malvae

Guijie Li, Qiang Wang, Yu Qian, and Xin Zhao

Received June 16, 2013; revised October 8, 2013; accepted October 9, 2013; published online August 31, 2014

© KoSFoST and Springer 2014

Abstract Fructus Malvae is functional food known for

antioxidant, anti-mutagenic, and in vivo anticancer effects.

Fructus Malvae extracts demonstrated high antioxidant

activities in DPPH and hydroxyl radical scavenging activity

assays. In an Ames mutagenicity test, Fructus Malvae

exhibited antimutagenicty in association with MNNG (N-

methyl-N'-nitro-N-nitrosoguanidine) in Salmonella Typhimurium

TA100 cells. U14 squamous carcinoma cells were injected

into the buccal mucosa of KM (Kunming) mice. The

wound at the injection site was smeared with a Fructus

Malvae solution, which was also administered to mice by

gavage. Tumor volumes were reduced and tissue section

analysis of buccal mucosa cancer and cervical lymph node

tissues showed anti-cancer effects in Fructus Malvae

treated groups. Fructus Malvae possesses good antioxidant

and antimutagenic activities and exerts a preventive effect

against buccal mucosa cancer in vivo.

Keywords: antimutagenicity, Fructus Malvae, antioxidant,

anticancer, mice

Introduction

Fructus Malvae is a Mongolian medical herb functional

food from the recurring, unisexual plant Malva verticillata

L., a winter dry ripe fruit. The thin, short stalks of the fruit

are 1.4-2.5 mm in diameter, the surface is yellow-white or

yellowish brown, and uplift of the fine veins and the seed

kidney is tan or dark brown (1,2). Fructus Malvae is used

for making health foods and products, such as Fructus

Malvae dietic tea and Fructus Malvae skin care power, and

is also used for cooking in a stew. In ancient times, Fructus

Malvae was used as a Chinese traditional medicine to treat

edema, thirst, and urinary infections. In recent studies,

Fructus Malvae polysaccharides have displayed a reticulo-

endothelial system activity and a strong antioxidant effect

related to removal of oxygen free radicals (3,4).

Buccal mucosa cancer is the most common cancer of the

oral cavity (5). The U14 mouse tumor is a squamous cell

carcinoma that is ectopically induced by treating the

uterine cervix with 20-methylcholanthrene (6). U14 cell

transplantation into mice caused buccal mucosa cancer (7).

In the present study, the antioxidant and antimutagenic

activities of Fructus Malvae were investigated. The cancer

preventive effect of Fructus Malvae was also evaluated

using a mouse model of buccal mucosa cancer. Fructus

Malvae was shown to have antioxidant, antimutagenic, and

anticancer effects. As a functional medicine, Fructus

Malvae demonstrated oral health benefits in mice (3).

Materials and Methods

Fructus Malvae extract preparation Medicinal Fructus

Malvae was purchased from Neimenggu Mongolian

Medicine Corporation (Tongliao, China) in the Inner

Mongolia Autonomous Region of China. Fructus Malvae

samples were freeze-dried and powdered to prepare boiled

water extracts. A 10× volume of boiling water was added

to powdered samples, followed by extraction twice by

shaking. The water extract was evaporated using a rotary

evaporator (N-1100; Eyela, Tokyo, Japan).

DPPH free radical assay The DPPH radical scavenging

activity was determined according to the method of Blois

(8). An amount of 4 mL of different concentrations of

Guijie Li, Qiang Wang, Yu Qian, Xin Zhao (�)Department of Biological and Chemical Engineering, ChongqingUniversity of Education, Chongqing 400067, ChinaTel, Fax: +86-23-62658256E-mail: foods@live.cn

RESEARCH ARTICLE

1342 Li et al.

sample solutions was added to 1.0 mL of a DPPH methanol

solution (1.5/104 M). After storage at room temperature for

30 min, the absorbance of the solution was determined at

520 nm using a spectrophotometer (iMark; Bio-Rad,

Hercules, CA, USA), and the remaining DPPH was

measured. Results are expressed as the mean values of

triplicates.

Hydroxyl radical assay Hydroxyl radical scavenging

activities were determined as described by Banerijee et al.

(9). The reaction system (1.4 mL) contained extracts,

deoxyribose (6 mM, 0.2 mL), 0.2 mL of a sodium

phosphate buffer solution (20 mM, pH 7.4), 0.2 mL of iron

chloride, anhydrous (FeCl3) (400 µM), 0.2 mL of FeSO4,

EDTA (400 µM), 0.2 mL of H2O2 (3 mM), 0.2 mL of

ascorbic acid (400 µM), and 0.2 mL of the extracts. After

incubation in a 37oC water bath for 60 min, the reaction

was stopped by adding 1 mL of trichloroacetic acid and

1 mL of 2-thiobarbituric acid to the 1.4 mL reaction

system. The solution was then boiled for 20-25 min at 90oC.

The absorbance was measured at 532 nm. All analyses were

performed in triplicate and mean values are reported.

Antimutagenic analysis The Salmonella Typhimurium

strain TA100, a histidine-requiring mutant bacterium, was

maintained as described by Maron and Ames (10). In brief,

0.5 mL of phosphate buffer containing the direct mutagen

MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) was

distributed in sterilized, capped tubes, then 0.1 mL of a test

bacterial suspension from an overnight culture (1-2×109

cells/mL) and 0.1 mL of a test sample compound (50 µL of

the mutagen and/or 50 µL of a test sample) was added.

After gentle vortexing (Vortex-Genie 2 Digital; Scientific

Industries Inc., Springfield, MA, USA) and preincubation

at 37oC for 30 min, 2 mL of top agar supplemented with L-

histidine and D-biotin kept at 45oC was added to each tube

and vortexed for 3 s. The entire resulting mixture was

overlaid on a minimal agar plate, followed by incubation at

37oC for 48 h, then revertant bacterial colonies on each

plate were counted.

Animals Seven-week-old female KM (Kunming) mice

were purchased from the Experimental Animal Center of

Chongqing Medical University (Chongqing, China). Mice

were maintained in a temperature-controlled (temperature

23±1oC, relative humidity 50±5%) facility with a 12-h

light/dark cycle and unlimited access to a standard mouse

chow diet and water.

Cell preparation U14 squamous carcinoma cells obtained

from the Chinese Academy of Medical Sciences (Beijing,

China) were used. Cells were cultured in RPMI-1640

medium (Gibco Co., Birmingham, MI, USA) supplemented

with 10% fetal bovine serum (FBS) and 1% penicillin-

streptomycin (Gibco-BRL, Grand Island, NY, USA) at

37oC in a humidified atmosphere with 5% CO2 (incubator

model 311 S/N29035; Forma, Waltham, MA, USA). The

medium was changed 2 or 3 times a week. In vitro cultured

U14 cells (5×106/mouse) were injected into the abdominal

cavity of 7-week-old female KM mice. After 1 week,

carcinoma ascites were collected and diluted in sterile

saline to a concentration of 1×107/mL.

Induction of buccal mucosa cancer To investigate the

preventive effects of Fructus Malvae against buccal

mucosa cancer that is induced by injection of U14 cells,

mice were divided into 2 treatment groups and 2 control

groups with 10 mice in each group. The experimental

design included 2 treatment groups, called A and B, and 2

control groups, one for group A and one for group B.

Fructus Malvae solutions were administered to group A at

500 mg/kg and to group B at 1,000 mg/kg via gavage. The

corresponding control groups for A and B did not receive

Fructus Malvae solution administration. The control and

Fructus Malvae treatment group mice were then inoculated

in the buccal mucosa with 0.05 mL of suspended cancer

cells (1×107/mL). The buccal mucosa tissue injection

wound site of mice in the treatment groups was also

smeared with Fructus Malvae solutions (group A, 200 mg/

mL; group B, 400 mg/mL) every 12 h for 14 days. Mice

were sacrificed 14 days after the beginning of treatment

and tumor volumes and lymph node metastasis rates were

determined, as previously described (11). All experimental

procedures followed protocols approved by the Animal

Ethics Committee of Chongqing Medical University

(Chongqing, China).

Histological grading of buccal mucosa cancer Buccal

mucosa tissues were removed and embedded in paraffin

for histological analysis using hematoxylin and eosin

(H&E) staining. Buccal mucosa cancer was graded as i)

well-differentiated carcinoma; cells resemble adjacent

benign squamous epithelium, ii) moderately differentiated

carcinoma; cells form large anastomosing areas in which

keratin pearls are formed but are not numerous, and the

main component consists of cells with pronounced

cytonuclear atypia, and iii) poorly differentiated carcinoma;

cells have lost the majority of their squamous epithelial

characteristics and architecture (12).

Reverse transcription-polymerase chain reaction (RT-

PCR) analysis Total RNA from buccal mucosa tissues

of mice was isolated using Trizol reagent (Invitrogen,

Carlsbad, CA, USA) according to the manufacturer’s

recommendations. RNA was digested using RNase-free

DNase (Roche, Basel, Switzerland) for 15 min at 37oC and

Anticancer preventive effects of Fructus Malvae 1343

purified using an RNeasy kit (Qiagen, Hilden, Germany)

according to the manufacturer’s protocol. cDNA was

synthesized using 2 µg of total RNA via incubation at 37oC

for l h with avian myeloblastosis virus reverse transcriptase

(GE Healthcare, Little Chalfont, UK) with random hexa-

nucleotides, according to the manufacturer’s instructions.

Sequences of primers used to specifically amplify the

genes of interest are shown in Table 1. Amplification was

performed in a thermal cycler (Eppendorf, Hamburg,

Germany). PCR products were separated in 1.0% agarose

gel and visualized using ethidium bromide staining (13).

RT-PCR results were quantified, and mean values were

subjected to statistical analysis.

Protein extraction and western blot analysis Total cell

lysates were obtained using an extraction buffer as previously

described (14). Protein concentrations were determined

using a protein assay kit (Bio-Rad, Hercules, CA, USA).

For Western blot analysis, cell lysates were separated via

12% SDS-PAGE, transferred to a polyvinylidene fluoride

membrane (GE Healthcare), blocked using 5% skim milk,

and incubated with primary antibodies (1:1,000 dilution).

Antibodies against Bax, Bcl-2, caspase-3, caspase-9, NF-

κB, IκB-α, iNOS, and COX-2 were obtained from Santa

Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). After

incubation with horseradish peroxidase-conjugated secondary

antibody at room temperature, immunoreactive proteins

were detected using a chemiluminescent enhanced chemilu-

minescence assay kit (GE Healthcare) according to the

manufacturer’s instructions. Bands in blots were visualized

using a LAS3000 luminescent image analyzer (Fujifilm

Life Science, Tokyo, Japan).

Statistical analysis Data are presented as mean±standard

deviation. Differences between mean values for individual

groups were assessed using a one-way analysis of variance

(ANOVA) with Duncan’s multiple range test. Statistical

significance was defined as p<0.05. The SAS version 9.1

statistical software package (SAS Institute Inc., Cary, NC,

USA) was used for analysis.

Results and Discussion

DPPH radical and OH radical scavenging activities of

Fructus Malvae The radical scavenging effects of Fructus

Malvae on DPPH radicals was tested (Fig. 1). Fructus

Malvae showed scavenging activities of 42.2% and 77.9%

at concentrations of 100 and 200 mg/mL, respectively,

indicating that the radical scavenging activity of Fructus

Malvae increased at higher concentrations.

The effects of Fructus Malvae on the hydroxyl scavenging

activity were determined based on deoxyribose damage

induced using a Fe3+/ascorbate/EDTA/H2O2 system, and

measured using the TBA method. Deoxyribose degrades

into fragments that react with TBA upon heating at a low

pH to form a pink color. The inhibitory effects of Fructus

Malvae on deoxyribose damage are shown in Fig. 1.

Inhibition with the 200 mg/mL extract was 85.4%, higher

than inhibition with the 100 mg/mL (58.8%) extract.

Table 1. Sequences of RT-PCR primers used in this study

Gene name Sequence

BaxForward: 5'-AAG CTG AGC GAG TGT CTC CGG CG-3'

Reverse: 5'-CAG ATG CCG GTT CAG GTA CTC AGT C-3'

Bcl-2Forward: 5'-CTC GTC GCT ACC GTC GTG ACT TGG-3'

Reverse: 5'-CAG ATG CCG GTT CAG GTA CTC AGT C-3'

Caspase-3Forward: 5'-CAA ACT TTT TCA GAG GGG ATC G-3'

Reverse: 5'-GCA TAC TGT TTC AGC ATG GCA-3'

Caspase-9Forward: 5'-GGC CCT TCC TCG CTT CAT CTC-3'

Reverse: 5'-GGT CCT TGG GCC TTC CTG GTA T-3'

NF-κBForward: 5'-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3'

Reverse: 5'-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3'

IκB-αForward: 5'-GCT GAA GAA GGA GCG GCT ACT-3'

Reverse: 5'-TCG TAC TCC TCG TCT TTC ATG GA-3'

iNOSForward: 5'-AGA GAG ATC GGG TTC ACA-3'

Reverse: 5'-CAC AGA ACT GAG GGT ACA-3'

COX-2Forward: 5'-TTA AAA TGA GAT TGT CCG AA-3'

Reverse: 5'-AGA TCA CCT CTG CCT GAG TA-3'

GAPDHForward: 5'-CGG AGT CAA CGG ATT TGG TC-3'

Reverse: 5'-AGC CTT CTC CAT GGT CGT GA-3'

1344 Li et al.

Damage from free radicals can cause cancer. Antioxidants

interact with and stabilize free radicals and may prevent

some of the damage that free radicals might otherwise

cause (15). DPPH and OH radical assays are the basis of

common antioxidant assays (8,16). DPPH and OH radical

studies can be used to check the antioxidant effects of

samples.

Antimutagenic effect of Fructus Malvae Fructus Malvae

showed an inhibitory effect on spontaneous mutations in

Salmonella Typhimurium TA100 cells (Table 2). At 1.25

mg of Fructus Malvae/plate the spontaneous mutation

inhibitory rate was 52%. At 2.5 mg/plate, the inhibition

rate was 82%. These results indicate that the inhibitory

effect of Fructus Malvae on spontaneous mutation increased

with an increased concentration.

Fructus Malvae showed an anti-mutagenic effect in

Salmonella Typhimurium TA100 cells (Table 3) treated

with MNNG. At 1.25 mg of Fructus Malvae/plate, the

mutagenic inhibition rate was 34%, indicating an anti-

mutagenic effect. When the Fructus Malvae concentration

was increased to 2.5 mg/plate, a significantly increased

anti-mutagenic effect with an inhibition rate of 71% was

observed.

An Ames test can be used to rapidly screen for chemical

carcinogens (10). Biological genetic mutations are regarded

as key factors that lead to cancer (17), and use of

Salmonella Typhimurium as a test strain is common in

biological assays to assess the mutagenic potential of

chemical compounds (18).

Tumor volumes and lymph node metastasis rates in

Fructus Malvae treated mice U14 cervical squamous

cell carcinoma (U14) (6) is an ectopic cervical cancer that

is induced using a methylcholanthrene thread placed in the

cervix of young mice that is then implanted in the

hypodermic tissues of adult mice (19). In early stages, the

U14 structure is similar to carcinosarcoma. U14 has been

classified as an undifferentiated carcinoma since the 1980s,

with a metastasis rate of 95% in the lymph nodes and 80%

in the lungs. Moreover, it is a bidirectional metastatic

tumor strain and, as an optimum model, is widely used for

study of tumor metastasis, invasion, recurrence, and drug

screening (6).

Buccal mucosa cancer was induced by injecting U14

cells into mice via oral gavage, which is the most frequent

method for checking the functional effects of foods in

mice. Fructus Malvae is often used to make tea and soup

that is administered via oral gavage and is also smeared on

buccal mucosa tissues. Smearing is a method used to test

the medicinal effect of a treatment in oral cancer models

(20). Oral gavage and smearing together can increase the

anticancer effect (11). Fructus Malvae is typically administered

to adults at a dosage of 9-15 g/day (21). The corresponding

mouse dosage would be 1.35-2.25 g/kg. The extraction

percentage for Fructus Malvae is approximately 40%.

Therefore, extract concentrations of 500 and 1,000 mg/kg

were used in this study. After 14 days, mice in all groups

exhibited carcinogenesis, and tumor volumes in buccal

mucosa tissues were measured. Tumor volumes for the

Fig. 1. Effects of Fructus Malvae on DPPH radical scavengingand hydroxyl radical (OH) scavenging activities.

Table 2. Effect of Fructus Malvae on spontaneous mutagenicity1)

Treatment(level of sample, mg/plate)

Revertants/Plate

Treatment(level of sample, mg/plate)

1.25 2.5

Spontaneous 117±11a2)

Fructus Malvae 56±12b (52) 21±5c (82)

1)Values are mean±SD of revertants/plate.Values in parentheses areinhibition rates (%).

2)a-cMean values with different letters in the same column aresignificantly different (p<0.05) according to Duncan’s multiplerange test.

Table 3. Effect of Fructus Malvae on mutagenicity inducedusing MNNG (0.4 µg/plate) in Salmonella TyphimuriumTA100 cells1)

Treatment(level of sample, mg/plate)

Revertants/Plate

Treatment(level of sample, mg/plate)

1.25 2.5

Spontaneous 117±110

MNNG (control)2) 1018±35a3)

Fructus Malvae 712±31b (34) 378±28c (71)

1)Values are mean±SD of revertants/plate. Values in parentheses areinhibition rates (%), inhibition rate=[(MNNG-spontaneous)−(FructusMalvae−spontaneous)]/MNNG- Spontaneous.

2) MNNG, N-methyl-N'-nitro-N-nitrosoguanidine3)a-cMean values with different letters in the same column aresignificantly different (p<0.05) according to Duncan’s multiplerange test.

Anticancer preventive effects of Fructus Malvae 1345

Fructus Malvae control groups A and group B were 8.7

and 4.4 mm3, respectively (Table 4). A total of 6 mice

demonstrated lymph node metastasis in control groups, 3

in Fructus Malvae control group A, and 1 in Fructus

Malvae control group B. Consequently, the lymph node

metastasis rates were 60, 30, and 10%, respectively, for

control, A and B groups. These results demonstrate that

Fructus Malvae is effective in impeding carcinogenesis,

proliferation, and metastasis.

With growth of tumor tissue, open and expanding peri-

cancer lymphatic vessel growth increases. Vessel walls

were thinner, and gaps or nicks appeared in the thin vessel

walls. This phenomenon is usually observed in areas where

tumor cells are concentrated and is favorable in helping

tumor cells enter the lymphatic lumen (22).

Histopathology of buccal mucosa tissues of Fructus

Malvae treated mice Histological changes in the buccal

mucosa of mice injected with U14 cells were examined

using H&E staining. Histological tissue sections of mice in

the control groups demonstrated normal squamous epithelium

tissue morphology. Histopathological evaluation revealed

indications of buccal mucosa cancer in both control and

Fructus Malvae treatment groups (Fig. 2). Tissue sections

from control group mice showed that tissues lost typical

squamous epithelial characteristics and architectures (grade

iii). Tissue sections from both Fructus Malvae treatment

groups A and B looked much like sections from adjacent

benign squamous epithelium (grade i). Sections from

Fructus Malvae treatment group B relieved cancer in tissue

lesions. Based on results from tissue section analysis,

Fructus Malvae showed a preventive effect against buccal

mucosa cancer. Lymph node sections from the control

groups exhibited a large area of liquefaction necrosis of

lymph nodes (Fig. 3). Treatment group A and B sections

showed only a small amount of liquefaction necrosis.

Fig. 2. Histology of buccal mucosa tissues after induction via injection of U14 squamous cell carcinoma cells in mice (H&Estaining, 100× magnification). Group A, mice were administered 500 mg/kg Fructus Malvae by gavage and the buccal mucosa wassmeared with 200 mg/mL of Fructus Malvae; group B, mice were administered 1,000 mg/kg Fructus Malvae by gavage and the buccalmucosa was smeared with 400 mg/mL of Fructus Malvae.

Table 4. Tumor size and lymph node metastasis rates for Fructus Malvae treated mice

Normal group1) Control groupFructus Malvae

Group A Group B

Tumor volume (mm3) ND 9.8±0.5a2) 7.7±0.4b 4.4±0.2c

Lymph node/metastasis rate3) ND 6/101) (60%) 3/10 (30%) 1/10 (10%)

1)Normal group, mice were no treated with the U14 cells; group A, mice were administered 500 mg/kg Fructus Malvae by gavage and thebuccal mucosa tissue was smeared with 200 mg/mL of Fructus Malvae; group B, mice were administered 1,000 mg/kg Fructus Malvae bygavage and the buccal mucosa tissue was smeared with 400 mg/mL of Fructus Malvae.

2)a-cMean values with different letters in the same column are significantly different (p<0.05) according to Duncan’s multiple range test.3)Number of lymph node metastasis/total number

1346 Li et al.

These results demonstrate that Fructus Malvae is effective

in preventing lymph node metastasis.

Histopathology is an important clinical method for

diagnosis of oral cancer (23). Using histopathological assays,

the cancer preventive effects of Fructus Malvae can be

identified directly. Establishment of a cultured tumor cell

line, that forms tumors in vivo, is helpful for study of tumor

biology on cellular and molecular levels. U14 cells were

ectopically induced via treatment of the uterine cervix with

a methylcholanthrene thread. This method induces cancer

via implantation into the hypodermic tissues of adult mice

(24). The most important components for functional effects

of Fructus Malvae are the polysaccharides, and there are

two kinds of polysaccharides in Fructus Malvae, a neutral

polysaccharide and an acidic polysaccharide (25). These active

polysaccharides interfere with normal immunocompetence,

increase cancer cell phagocytosis in the reticulo-endothelial

system, promote cell transformation, activate T and B cells,

and increase the amounts of antibodies (4).

Apoptosis related mRNA and protein expression levels

of Bax, Bcl-2, and caspases in the buccal mucosa tissues

of Fructus Malvae treated mice In order to elucidate the

protective mechanisms of Fructus Malvae against buccal

mucosa cancer, expressions of Bax, Bcl-2, and caspases in

buccal mucosa tissues were analyzed using RT-PCR and

western blot assays after treatment with Fructus Malvae

solutions. In the presence of Fructus Malvae, levels of the

pro-apoptotic Bax product and the anti-apoptotic Bcl-2

product showed significantly changes (Fig. 4), suggesting

that Fructus Malvae strongly induces apoptosis in buccal

mucosa cancer via both Bax and Bcl-2 dependent pathways.

Apoptosis induction after a high concentration Fructus

Malvae treatment was related to increased Bax expression

and decreased Bcl-2 expression, compared to a low

Fructus Malvae concentration. The mRNA expressions of

caspase-3 and caspase-9 were slight in untreated U14 cell

control mice, but were strongly detected in mice treated

with Fructus Malvae. The mRNA and protein expressions

of caspase-3 and caspase-9 were significantly increased

with an increasing treatment concentration. These results

indicate that Fructus Malvae offers promising anticancer

effects in buccal mucosa cancer.

Apoptosis is a fundamental cellular event, understanding

the mechanisms of which will help harness this process for

use in tumor diagnosis and therapy (26). The Bcl-2 family

of genes is pivotal to regulating apoptosis. Defective

regulation of apoptosis is central to cancer pathogenesis

and progression, and has been associated with resistance to

standard therapies (27). The Bcl-2 family includes pro-

apoptotic Bax and anti-apoptotic Bcl-2 proteins. Activation

of downstream signals caused by interactions between

these anti and pro-apoptotic proteins is critical to the

ultimate sensitivity of cells to various apoptotic stimuli

(28). Caspases, a family of cysteine proteases, play essential

roles in apoptosis (29). Caspase 3 is a protein that interacts

with both caspase 8 and 9 (30). Caspases are essential for

apoptosis.

Fig. 3. Histology of buccal mucosa cancer cervical lymph node metastasis after induction via injection of U14 squamous cellcarcinoma cells in mice (H&E staining, 100× magnification). Group A, mice were administered 500 mg/kg Fructus Malvae by gavageand the buccal mucosa was smeared with 200 mg/mL of Fructus Malvae; group B, mice were administered 1,000 mg/kg Fructus Malvae

by gavage and the buccal mucosa was smeared with 400 mg/mL of Fructus Malvae.

Anticancer preventive effects of Fructus Malvae 1347

Inflammation related mRNA and protein expression

levels of NF-κB, IκB-α, iNOS, and COX-2 in buccal

mucosa tissues of Fructus Malvae treated mice RT-PCR

and western blot analyses were used to investigate whether

the inhibitory effect of Fructus Malvae on inflammation is

due to transcriptional regulation of inflammatory mediators

in the buccal mucosa. U14 cell treatment significantly

increased the mRNA and protein levels of these inflammatory

mediators (Fig. 5). However, a high concentration of

Fructus Malvae also significantly increased the mRNA

and protein expression levels of IκB-α. Fructus Malvae

also decreased the expression levels of NF-κB, iNOS, and

COX-2, and buccal mucosa tissues from Fructus

Malvae-treated mice showed decreased expression of these

genes, compared with the control groups.

Anti-cancer mechanisms underlying the effect of Fructus

Malvae on buccal mucosa cancer involve induction of

apoptosis by increasing the number of apoptotic bodies,

regulating the mRNA and protein expressions of Bax and

Bcl-2, and promoting anti-inflammatory effects by down-

regulating iNOS and COX-2 gene expression. COX-2 has

been suggested to play an important role in colon

carcinogenesis, and NOS, along with iNOS, may be a

potential target for chemoprevention of cancer (31). NF-κB

is a ubiquitous transcription factor that regulates expressions

of genes that are required for cellular proliferation,

inflammatory responses, and cell adhesion. NF-κB is

present in the cytosol bound to the IκB inhibitory protein

(16). These mechanisms may be involved in the anticancer

effects of Fructus Malvae in buccal mucosa cancer. Based

on expression patterns of pro-apoptotic genes observed in

the present study, cancer tissues in mice treated with

Fructus Malvae underwent apoptosis.

Both in vitro and in vivo experimental methods,

including DPPH and hydroxyl radical assays, the Ames

test, histopathology analyses, RT-PCR, and western blot

assays were used to evaluate the cancer preventive effects

of Fructus Malvae. Fructus Malvae displays a strong

antioxidant activity and can decrease the number of

spontaneous revertants in Salmonella Typhimurium TA100

cells, while also decreasing the mutagenic effect of

MNNG. A mouse model bearing tumors produced by U14

Fig. 4. Effects of Fructus Malvae on the mRNA and protein expression of Bax, Bcl-2, and caspases in buccal tissues. Nor, normalgroup mice; Con, control group mice; A, group A mice; B, group B mice

Fig. 5. Effects of Fructus Malvae on the mRNA and protein expression of NF-κB, IκB-α, iNOS, and COX-2 in buccal tissues. Nor,normal group mice; Con, Control group mice; A, group A mice; B, group B mice

1348 Li et al.

squamous cell carcinoma cells was used to study the in

vivo effects of Fructus Malvae. A strong anticancer activity

against buccal mucosa cancer was observed. Overall,

Fructus Malvae showed in vitro anti-mutagenic effects and

in vivo anticancer activities. In conclusion, increased

Fructus Malvae concentrations should be used to increase

the oral cancer preventive effect.

Acknowledgments The Chongqing Innovative Research

Team in University (KJTD201325), China provided support

for this research.

Disclosure The authors declare no conflict of interest.

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