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SCIENCE CHINA
Life Sciences
© Science China Press and Springer-Verlag Berlin Heidelberg 2010 life.scichina.com www.springerlink.com
†Contributed equally to this work
*Corresponding author (email: bjtaoran@126.com; zhaobl@sun5.ibp.ac.cn)
$Correspondence concerning tea filter techniques (email: sdyt_lyl318@yahoo.com.cn)
• COVER ARTICLE • May 2010 Vol.53 No.5: 533–541
doi: 10.1007/s11427-010-0097-1
The cessation and detoxification effect of tea filters on cigarette
smoke
YAN JingQi1,2†, DI XiaoJing1,4†, LIU CaiYi3, ZHANG HuiMin3, HUANG XiouQin3,
ZHANG JunJing1,2, ZHAO Yan5, ZHANG LongZe1, CHANG YanZhong4,
LIANG YongLin2$, TAO Ran3* & ZHAO BaoLu1,2*
1 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;
2 Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing 100101, China;
3 Addiction Branch, Beijing Military Region General Hospital, Beijing 100700, China;
4 Institute of Molecular Neurobiology, Hebei Normal University, Shijiazhuang 050016, China;
5 Department of Food Sciences and Technology, Harbin Institute of Technology at Weihai, Weihai 264209, China
Received December 29, 2009; accepted March 4, 2010
To treat tobacco addiction, a tea filter was developed and studied for smoking cessation. This work reports the smoking cessa-
tion effect of tea when it was used as a component of cigarette filters. In one trial it was found that after using the tea filters for
2 months, the volunteer smokers decreased their cigarette consumption by 56.5%, and 31.7% of them stopped smoking. This
work identified a new method and material, tea filter and theanine, which inhibit tobacco and nicotine addiction and provide an
effective strategy for treating tobacco addiction.
cigarette smoking addiction, cigarette cessation, tea filter, theanine, nicotine dependence, nicotine acetylcholine recep-
tor (nAChR), dopamine, public health, free radical
Citation: Yan J Q, Di X J, Liu C Y, et al. The cessation and detoxification effect of tea filters on cigarette smoke. Sci China Life Sci, 2010, 53: 533–541,
doi: 10.1007/s11427-010-0097-1
Cigarette smoking has been linked to many life threatening
diseases including heart disease, cancer and chronic ob-
structive pulmonary disease [1–5]. There are about 1.25
billion smokers in the world and 5 million die every year
because of smoking-related diseases [6], exceeding many
other diseases combined. It is estimated that the global cost
for smoking-related disease is about 1.2 trillion yuan each
year, resulting in one of the world’s largest public human
health problems. Many methods have been developed for
smoking cessation by researchers and clinicians [7–14].
However, due to the addictive nature of nicotine, quitting
smoking remains extremely difficult. Despite all efforts,
currently available smoking cessation methods produce only
modestly successful rates with frequent relapse. In addition,
they are often perceived as being inconvenient and lead to a
wide variety of side effects [7–13]. Therefore, the need for
developing alternative remains a high public health priority,
smoking cessation strategies with improved efficacy and
fewer side effects.
Epidemiological and experimental evidence suggests that
drinking tea is adversely associated with cancer [15],
hyperglycemia [16] and other diseases [17]. In addition, our
work has shown that tea components protect cells from
cigarette smoke-induced toxicity [18–23]. We developed a
cigarette filter containing tea. Human tests showed that
smoking using tea filters significantly decreased the
cigarette consumption of the volunteer smokers without any
apparent side effects. Further study showed that an amino
534 Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5
acid derivative of tea, theanine, exerted an effect similar to
the nicotine acetylcholine receptor inhibitor.
1 Materials and methods
1.1 Preparation of cigarettes with tea filters
“Honghe” cigarettes purchased from a market (made by the
Honghe Tobacco Ltd Company, Yunnan, China) with
regular filters (cellulose acetate filter 2.5 cm) were used as
the control cigarettes. A complex tea filter consisting of half
of a tea filter (1.25 cm) and half of a regular filter (1.25 cm)
was used in the human test for the tea filter group. The tea
filter half was attached to the cigarette and the cellulose
acetate filter half was attached to the tea filter. The total
length of the complex tea filter was 2.5 cm, with a similar
appearance to a regular cellulose acetate filter (2.5 cm). The
theanine in the tea filter was about 1%.
1.2 Human test
A human test was approved by the authorized committee
and conducted in the Addiction Branch, Beijing Military
Region General Hospital (ClinicalTrials.govIdentifier:
NCT00971529). In one trial, one hundred healthy male
cigarette smokers, aged 18 to 30 years, were screened using
the standard exclusion/inclusion criteria [11]. Thirty of the
volunteers were excluded and 70 of them were double-
blinded, placebo-controlled and randomized into 2 groups
(smoking with tea filters or regular filters). In another trial,
70 healthy male cigarette smokers, aged 30 to 65 years,
were screened and 59 volunteers with a longer smoking
history and a stronger desire to quit smoking were tested
using the tea filter for 3 months. Smoking history, including
assessment of nicotine dependence, was evaluated at the
screening visit.
Subjects received brief smoking cessation counseling (up
to 10 min) at the baseline visit and at each visit afterwards.
Self-reported smoking status since the last visit and exhaled
carbon monoxide measurement were assessed at each
weekly visit. Vital signs, weight, and adverse event infor-
mation were collected at each visit. Physical examinations
were performed prior to randomization and at the final visit.
During the follow-up period, use of nicotine replacement
therapy did not disqualify subjects from being considered
abstinent. Subjects who withdrew or were lost to follow-up
were assumed to be smokers for the remainder of the study.
The exhaled carbon monoxide was detected by a carbon
monoxidemeter (1209-1 CO, DWYER, USA). Cotinine in
urine was analyzed using a HPLC (Hitachi, Japan) as re-
ported in the literature [24].
1.3 Animal treatment
Animal experiments were carried out in accordance with the
NIH Guide for the Care and Use of Laboratory Animals and
were approved by the local animal care committee. Six to 8
week old female C57BL/6J mice were purchased from the
Institute of Laboratory Animal Science, Chinese Academy
of Medical Science and housed in a specific parasite free
(SPF) environment at 22°C with a 12 h light-dark cycle.
Food (Mouse Diet, Beijing Experiment Animal Center) and
water were available ad libitum. Chemicals were dissolved
in saline and subcutaneously injected. Animals were ran-
domly divided into the following groups (n=10) for treat-
ment with different chemicals: Control: mice were injected
with 0.9% physiological saline, s.c.; Nicotine: Mice were
injected with nicotine (0.5 mg kg–1 d–1 , s.c.); Nicotine+GTP:
mice were lavaged with green tea polyphenols (250 mg kg–1
d–1) and injected with nicotine; Nicotine+CF: mice were
injected with caffeine (2 mg kg–1 d
–1, s.c.) and nicotine;
Nicotine+TH1: mice were injected with L-theanine-low
(250 mg kg–1 d
–1, s.c.) and nicotine; Nicotine+TH2: mice
were injected with L-theanine-high (500 mg kg–1 d–1, s.c.)
and nicotine; Nicotine+DHβE: mice were injected with
Dihydro-β-erythroidine hydrobromide (DHβE) (2.0 mg kg–1
d–1, s.c.) and nicotine [25]. Mice were daily injected with
nicotine (0.5 mg kg–1 d–1) or physiological saline for 1 week.
Different compounds were administered 15 min before
nicotine injection.
1.4 Conditioned place preference (CPP) test
CPP is a behavioral test and a measure of nicotine rein-
forcement, that is typically used to study the rewarding
properties of nicotine and other drugs [26–28]. This proce-
dure consisted of 3 phases: preconditioning, conditioning,
and post conditioning. In our procedure, days 1 and 2 were
preconditioning days and mice were allowed to roam freely
among the 2 identically sized compartments (20 cm×15
cm×15 cm), which were separated by a narrow compart-
ment (10 cm×15 cm×15 cm), for 900 s and the time spent in
each compartment was recorded. According to the test data,
mice were divided into 7 groups so that all groups showing
an unconditioned preference (n=10 mice per group). On
days 3–9, the mice were injected s.c. with saline or chemi-
cals and immediately placed in one of the pairing compart-
ments for 30 min. Five hours later, the mice were injected
with the alternate chemicals or saline and immediately
placed in the opposite chamber for 30 min. Control groups
received saline on both sides of the chamber. On day 10, the
animals once again were allowed to freely roam among the
3 compartments for 900 s and the time spent on each side
was recorded. The animals were drug-free on precondition-
ing days and the post conditioning day. The mice were
killed by cervical dislocation, and the brains were quickly
removed and treated in different ways for analysis.
1.5 Western blotting assay
The mice brain tissues were homogenized on ice in a buffer
Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5 535
(50 mmol L–1 Tris-Cl, 150 mmol L–1 NaCl, 0.02 NaN2, 100
µg mL–1 PMSF, 1 µg mL–1 Aprotitin and 1% Triton X-100)
at 0°C for 30 min. Tissue homogenates were centrifuged at
12000×g for 25 min at 4°C. Supernatants were collected
and analyzed by Western blotting using the standard pro-
tocol. Band intensities were quantified using an image ana-
lyzing software (NIH Image). Antibodies of AChRα4 (sc-
74519), AChRβ2 (sc-11372), AChRα7 (sc-11372) and β-
Actin (sc-1616-R) were used to detect the expression of the
proteins.
1.6 Dopamine measured by high-performance liquid
chromatography (HPLC) with electrochemical detection
Thirty min after subcutaneous injection of saline or different
chemicals, mice were killed by cervical vertebra dislocation
and both sides of the striatum were carefully isolated. Sam-
ples were weighed and homogenized in 1 mL 0.2 mol L–1
perchloric acid. Tissue homogenates were centrifuged at
20000 r min–1 for 15 min at 4°C and the supernatants were
collected for further analysis. The level of the striatal dopa-
mine (DA) was determined using a previously described
method by the HPLC ESA-5600A Coularray system (ESA,
USA).
1.7 Exposure of rodents to cigarette smoke
To study acute and chronic toxicity, the rodents were ex-
posed to cigarette smoke in a polyacryl glass chamber (35.6
cm×35 cm×20 cm) with two 1.5 cm2 holes, one located on
top of the chamber for ventilation and the other at the bottom
as entrance for the gas phase [29]. The gas phase of smoke
was delivered with a special pump to the chamber contain-
ing one group of 10 rodents. Rodents in group 3 served as a
control and were not treated with cigarette smoke.
During the experiments pO2, pNOx and pCO2 were moni-
tored and it was found that the changes of pO2 and pCO2
were less than 5%; the level of pNOx was increased from 0
to about 60 ppm. All of these changes did not reach lethal
levels. These results indicated that the differences at death
between the different groups were not caused by the
changes of pO2, pNOx and pCO2.
1.8 Acute toxicity of cigarette smoke in mice
Twenty four rodents were randomly divided into 3 groups.
Rodents in group 1 were treated with the gas phase of
smoke from cigarettes with standard filters. Rodents in
group 2 were treated with the gas phase of smoke from
cigarettes with tea filters. Rodents in group 3 served as a
control and were not treated with cigarette smoke. All mice
(n=8) were exposed to the gas phase of cigarette smoke as
described above, recording the time and number of ciga-
rettes until the lethal endpoint was reached. The deceased
mice were examined for histopathological changes and the
congestion and haemorrhage in lung tissue were quantified
as markers of damage.
1.9 Chronic toxicity of cigarette smoke in rats
Three groups of rats were exposed to the gas phase of ciga-
rette smoke as described above. Each group of 5 rats was
exposed to the gas phase of 7 cigarettes during 30 min. This
procedure was carried out twice a day, with an intermission
of 4 h, over a total time period of 75 d. This protocol was
approved by the Animal Experimentation Committee of the
China Academy of Traditional Chinese Medicine. Mutageni-
city of chronic smoking was established by micronucleus
assay. Bone marrow of the sacrificed rats was flushed out of
femurs, homogeneously mixed with an equal volume of
fetal bovine serum, centrifuged, resuspended and spread on
a slide. The smear was air-dried and stained with May-
Grünwald/Giemsa. 1000 polychromatic erythrocytes (PCEs)
were analyzed per animal for micronuclei. To describe a
cytotoxic effect the ratio between polychromatic and nor-
mochromatic erythrocytes (NCE) was determined in the
same sample and expressed in NCE per 1000 PCEs.
Specimens from lungs, heart, liver, spleen, kidneys and
adrenal glands were taken from organs of all rats in the
same positions, fixed with 10% formalin, embedded and
sectioned in paraffin and stained with HE. Pathological
changes were examined under a light-microscope and the
toxic effects were quantified.
1.10 Measurement of carboxyhaemoglobin (COHb) in
blood
Carboxyhaemoglobin (COHb) is a stable complex of carbon
monoxide and hemoglobin that forms in red blood cells
when carbon monoxide is inhaled. Tobacco smoking through
carbon monoxide inhalation raises the blood levels of
COHb [30,31]. The COHb levels in the blood were mea-
sured at 450 nm with a kit (Shanghai Yope Biotechnology
Co. LTD).
1.11 Statistical analysis
ANOVA was used to estimate overall significance followed
by post hoc Tukey’s tests corrected for multiple compari-
sons [32]. Data was presented as mean±SEM. A probability
level of 5% (P<0.05) was considered to be significant.
2 Results
2.1 Human test
A human test for the cessation effect of a tea filter on smok-
ing was performed in the Addiction Branch, Beijing Mili-
tary Region General Hospital. In one trial, healthy male
cigarette smokers who consumed approximately 14 ciga-
536 Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5
rettes per day on average were recruited and randomly di-
vided into 2 groups (double-blinded, placebo-controlled):
smoking with tea filters or with regular filters. After using
the tea filter for 1 month, the average daily cigarette con-
sumption decreased about 43% in the tea filter group. By
contrast, no change in average daily cigarette consumption
was detected in the control group using regular filters (Fig-
ure 1A). As a consequence of the reduction of cigarette
consumption, the levels of exhaled carbon monoxide and
urine cotinine content in the volunteers who smoked with
tea filters were respectively significantly decreased by about
52.6% and 26.3% (Figure 1B). The test was discontinued
for the control group, and the tea filter group was followed
for an additional month. After using the tea filter for 2
months, the average daily cigarette consumption was de-
creased by about 56.5% (31.7% of the smokers quit smok-
ing, 13.9% of the smokers reduced their cigarette consump-
tion from 14 d–1 to 1-5 d–1; 8.9% and 31.7% of the smokers
respectively reduced their daily cigarette consumption by
60% and 30% and 13.9% of them did not change their ciga-
rette consumption) (Figure 2A).
In another trial, we tested the effect of the tea filter on
heavier smokers who had a stronger desire to quit smoking.
59 healthy male cigarette smokers were recruited and tested
to smoke with tea filters for 3 months. The result showed
that their average daily cigarette consumption respectively
decreased by about 48%, 83% and 91% after using the tea
filter for 1, 2 and 3 months and the average daily cigarettes
consumed decreased to about 3 d–1 in the last month (Figure
2B), (the levels of exhaled carbon monoxide and urine co-
tinine content in the volunteers were significantly decreas-
ed), suggesting that the tea filter is effective for smoking
cessation. The efficacy of the tea filter on smoking cessation
is better than many other methods reported [7–14]. In addi-
tion, most subjects described that sputum and their smok-
ing-related symptoms were reduced compared to the control
group. Physical examinations of the subjects did not reveal
any apparent side-effects.
2.2 Effect of theanine on the rewarding effect induced
by nicotine
The conditioned place preference (CPP) paradigm is a
measure of nicotine reinforcement. To find which materials
in the tea filter are responsible for smoking cessation and to
elucidate the smoking cessation mechanisms of the tea filter,
we examined the effect of various components in the tea
filter on nicotine induced reinforcement using the CPP
method in a mouse model. Nicotine induced reinforcement
was induced by daily injection of the mice with nicotine
(0.5 mg kg–1 d
–1) for 7 d, while different compounds iso-
lated from the tea filter were administrated 15 min before
each nicotine injection. The results revealed that theanine
(500 mg kg–1), an amino acid derivative component of tea,
had a similar effect in mice to DHβE, an inhibitor of
nAChR, but green tea polyphenols (250 mg kg–1) and caf-
feine (2 mg kg–1) had no effect on nicotine induced rein-
forcement in the animals (Figure 3). The inhibition effects
of theanine appeared to be time and dose dependent. While
administration of theanine (250 mg kg–1 and 500 mg kg–1)
for 7 d respectively inhibited nicotine induced reinforce-
ment about 25% and 50%, the inhibition of nicotine
induced reinforcement was respectively about 90% and
95% after 2 weeks of theanine treatment for both doses.
2.3 Effects of theanine on the expression of the nicotine
receptor (nAChR) in mouse brains
It has been shown that nicotine treatment increases the ex-
pression of nAChR while inhibition of nAChR and its re-
lated processes causes smoking cessation [33–36]. To study
the cessation mechanisms of theanine on nicotine depend-
ence, we next investigated whether or not theanine caused
nicotine cessation by affecting nicotine-induced expression
of nAChR using a mouse model. After daily injection with
nicotine for 2 weeks, the protein levels of nAChR subunits
Figure 1 Smoking using tea filters significantly reduced the number of cigarettes consumed by volunteer smokers for one month. A, Average number of
cigarettes consumed daily (CCD) by each volunteer smoker before (week 0) and after (week 1–4) using a regular or a tea filter; B, Effect of a tea filter on
exhaled CO and urine cotinine excretion of volunteer smokers; *, P<0.05 when compared to week 0. Details are described in “Methods”.
Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5 537
Figure 2 Smoking using tea filters significantly reduced the number of
cigarettes consumed by volunteer smokers for 2 and 3 months. A, The
percentage of the smoker population and their average daily cigarette con-
sumption after using a tea filter for 2 months; B, the average number of
cigarettes consumed daily (CCD) by each volunteer smoker before (week 0)
and after (week 1–14) using a tea filter. *, P<0.05 when compared to week
0; **, P<0.01 when compared to week 0. Details are described in
“Methods”.
Figure 3 Effects of different compounds on nicotine dependence. The
mice were treated with nicotine or different compounds in physiological
saline every day for 9 d. The nicotine dependence of the mice was exam-
ined on the 10th day by a conditioned place preference (CPP) test. Con,
mice were treated with physiological saline; Nic, mice were treated with
nicotine (0.5 mg kg–1 d–1); GTP(N), mice were treated with nicotine (0.5
mg kg–1 d–1) and green tea polyphenols (250 mg kg–1); CF(N), mice were
treated with nicotine (0.5 kg–1 d–1) and caffeine (2 mg kg–1); TH-L(N), mice
were treated with nicotine (0.5 mg kg–1 d–1) and theanine (250 mg kg–1);
TH-H(N), mice were treated with nicotine (0.5 mg kg–1 d–1) and theanine
(500 mg kg–1); DHβE(N), mice were treated with nicotine (0.5 mg kg–1 d–1)
and DhβE (2.0 mg kg–1). The results are presented as mean±SEM,
n=8. *, P<0.05, compared with control; #, P<0.05 compared with the nico-
tine group. Details are described in “Methods”.
α4, α7 and β2 were increased in mouse brains. Theanine
pretreatment significantly inhibited the induction of nAChR
subunits in the brain (Figure 4). Therefore, the cessation
effect of theanine on nicotine dependence may be attributed
to its inhibition on nicotine-induced expression of nAChR
subunits.
2.4 Effects of theanine on dopamine release in mouse
brains
The increase of dopamine (DA) release is a significant re-
ward process caused by nicotine [37]. We examined
whether or not theanine had any effect on the DA release
induced by nicotine in mice. After nicotine injection, the
levels of DA were significantly increased in the striatum of
the mouse brains. Pre-treating animals with theanine before
nicotine injection significantly reduced the elevation of DA
(Figure 5) induced by nicotine.
2.5 Detoxification effect of a tea filter on acute toxicity
caused by cigarette smoking
Previous studies showed that the tea components, green tea
Figure 4 Effects of theanine on the expression of the nicotine receptor
(nAChR) in mouse brain. Mice were daily treated with nicotine (0.5 mg
kg–1 d–1) for 2 weeks with or without theanine administered 15 min before
nicotine injection. Protein extracts prepared from mouse brains were ana-
lyzed using Western blotting (A, B). The expression of nAChR was exam-
ined by Western blotting. Details of the procedures are described in
“Methods”. Control, mice were treated with physiological saline; Nic, mice
were treated with nicotine; T(L), mice were treated with theanine (250 mg
kg–1); T(H), mice were treated with theanine (500 mg kg–1); Nic+T(L),
mice were treated with nicotine and theanine (250 mg kg–1); Nic+T(H),
mice were treated with nicotine and theanine (500 mg kg–1). Data was
analyzed by the ratio of band intensity of nAChRs over that of -actin, ex-
pressed as ratio±SEM, n=4. *, P<0.05, compared with the control; #, P<
0.05 compared with the nicotine group.
538 Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5
Figure 5 Effects of theanine (0.5 mg kg–1 d–1) on nicotine-induced dopa-
mine release. The mice were injected with nicotine (0.5 mg kg–1 d
–1) or
physiological saline every day for 9 d. Different concentrations of theanine
were injected alone or 15 min before nicotine injection. The levels of do-
pamine in the mouse brain were measured by HPLC with electrochemical
detection. The details of the procedure are described in “Methods”. Con,
mice were treated with physiological saline; Nic, mice were treated with
nicotine; Th(N), mice were treated with nicotine and theanine; DHβE(N),
mice were treated with nicotine and an inhibitor of nAChR, DHβE. The
results are presented as the mean±SEM, n=3. *, P<0.05, compared with the
control; #, P<0.05 compared with the nicotine group.
polyphenols, and a tea filter scavenged the tar, free radicals,
nitrosamine, [a] pyrene, benzo [a] anthracene, chrysene and
total polycyclic aromatic hydrocarbons (PAHs) generated in
cigarette smoking [18–23]. We further studied the efficacy
of tea filters on the acute toxicity of cigarette smoke and
found that the survival time of animals respectively in-
creased by 32.2% and 60% by complex (half tea filter/half
cellules acetate) and full tea filters and the acute toxicity of
cigarette smoke was significantly reduced (Table 1). In the
control group (cigarette with normal filters) marked conges-
tion and haemorrhage in lung tissue was observed in 80% of
mice. The tea filters reduced the number of mice with these
pathological changes to 40% (results not shown).
2.6 Detoxification effect of a tea filter on mutagenicity
caused by cigarette smoking
We investigated the effect of a tea filter on the incidence of
micronuclei in polychromatic erythrocytes (PCE) as a
measure of the mutagenicity ratio and as an indicator of
toxicity in rats exposed to cigarette smoke for 75 d. The
incidence of micronuclei in PCE significantly increased
when rats were chronically exposed to cigarette smoke, in
accordance with a mutagenic activity of cigarette smoke.
When a cigarette with a tea filter was used, the incidence of
micronuclei in PCE was inhibited by about 46% as com-
pared to the rats which inhaled smoke from cigarettes with
normal filters (Table 2). Hence there was a significant re-
duction in the mutagenicity of cigarette smoke by tea filters.
2.7 Effects of a tea filter on carboxyhaemoglobin
(COHb) in mouse blood
Carboxyhaemoglobin (COHb) is a stable complex of carbon
monoxide and hemoglobin that forms in red blood cells
when carbon monoxide is inhaled. Tobacco smoking
through carbon monoxide inhalation raises the blood levels
of COHb [30,31] causing cardiovascular and cerebrovas-
cular damage and diseases, e.g. neurasthenia, myocardium
damage and atherosclerosis. In order to study the effect of a
tea filter on the toxicity of CO generated from cigarette
smoking, we measured the COHb levels in the blood. It was
found that the COHb levels in the blood of the mice ex-
posed to smoke from cigarettes with a normal filter were
increased about 561% compared with the mice without such
exposure, while the COHb levels in the blood of the mice
exposed to smoke from cigarettes with a tea filter were de-
creased about 53% compared with the mice exposed to
smoke from cigarettes with normal filters (Table 2). These
Table 1 Inhibition effect of a tea filter on the acute toxicity of cigarette smoke for mice
Groups n survival time (min) effect
Smoking (normal filter) 10 11.25±0.56
Smoking (complex tea filter) 10 14.85±0.77* +32.2(%)
Smoking (whole tea filter) 10 18.00±0.68* +60(%)
*, P<0.05 compared with smoking with a normal filter.
Table 2 Protective effect of a tea filter against mutation and COHb increase in blood caused by cigarette smoking
Groups n PCE/NCE effect HbCO (mg/mL) effect
Control (no smoking) 5 1.85±1.42 1.27±0.66
Smoking (normal filter) 5 14.55±7.06# +687% 8.40±0.42# +561%
Smoking (tea filter) 5 5.68±2.10* −60% 3.98±0.99* −53%
#, P<0.05 compared with a control (no smoking); *, P<0.05 compared with smoking with a normal filter.
Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5 539
results suggest that a tea filter inhibited the COHb levels
generated in cigarette smoking and prevented cardiovas-
cular and cerebrovascular diseases caused by cigarette
smoking.
2.8 The detoxification effect of a tea filter on lung
damage caused by cigarette smoking
Specimens of lungs from each animal from all groups were
examined under the optical microscope for pathological
changes. A variety of pathological alterations were discov-
ered in the lungs of the control group. Tea filters signifi-
cantly reduced these pathological changes. An increased
broken alveol, thickness of bronchi capillary walls with
signs of neutrophil and lymphocyte infiltration was ob-
served. There was an increase of inflammatory exudate and
lympho-proliferation in the bronchial lumen (Figures 6B
and C). Abscess and low fiber proliferation were found in
the lung tissue in 4 rats and 1 rats showed minor pathologi-
cal changes from the normal filter group. Only 1 rat showed
the pathological alterations and 1 rat showed minor patho-
logical changes in the lungs (moderate neutrophil and lym-
phocyte infiltration of alveoli) (Figure 6D), and in 3 animals
almost normal lung tissue was observed from the tea filter
group.
3 Discussion
Cigarette smoking addiction is caused by the interaction of
nicotine with the nAChR in the brain [33–36]. Our animal
experiments showed that theanine in the filter exerted an
inhibition effect similar to the nicotine acetylcholine recep-
tor (nAChR) inhibitor. To further improve the mechanism of
the inhibition effect of theanine on nicotine dependence, we
studied the effect of theanine on the expression of nAChR.
The results showed that theanine significantly inhibited the
nicotine-induced expression of nAChR (Figure 4). The in-
crease of dopamine (DA) release is a fundamental reward
process caused by nicotine [37]. Our results showed that
pre-treating animals with theanine before nicotine injection
significantly reduced the elevation of the DA level in mouse
brains.
To determine whether or not theanine in tea filters is
readily inhaled into the lungs, the hot gas steam from a
burning cigarette with a tea filter was collected using a spe-
cially designed smoking instrument and the amount of
theanine was measured. It was found that approximately 65
μg of theanine was brought out and entered into the hot gas
stream after smoking one cigarette with the filter, about 10
μg mL–1 in the plasma of animals after inhaling the smoke
from 7 cigarettes with tea filters, suggesting that theanine is
readily inhaled into the lungs and probably enters the
pulmonary circulation of the smoker. Once in the blood,
theanine passes through the blood-brain barrier and reaches
targets in the brain [38], preventing the development of
nicotine dependence. Because there are more than 450 kinds
of compounds in tea, it is possible that components other
than theanine in the tea filter also have a tobacco cessation
effect or they may act synergistically with theanine to pro-
mote tobacco cessation. Further studies on other tea com-
ponents may reveal additional tea components which are
factors in the tobacco cessation effect.
The animal experiments also showed that the tea filter
significantly reduced the acute and chronic toxicities as
shown in tables 1, 2 and Figure 6. Our previous work has
shown that tea components, the green tea polyphenols,
scavenge the tar, free radicals, nitrosamine, [a] pyrene,
benzo [a] anthracene, chrysene and total PAHs and protect
cells from cigarette smoke-induced toxicity [8–23], so the
detoxification effects of tea filters may mainly come from
green tea polyphenols. These results indicated that tea filters
not only helped smokers to quit smoking but also reduced
the toxicity induced by cigarette smoking.
Cigarette smoking is the major risk factor for a series of
life threatening diseases including cancer and heart attack,
which causes millions of deaths each year worldwide [1–5].
Different cigarette filters have been developed with the
purpose of reducing such harmful chemicals as tar and nico-
tine in tobacco smoke. However, a smoker may smoke more
cigarettes using these filters, inhale more deeply or decrease
the time between puffs to compensate for the desired nico-
tine intake, leading to exposure to equal or greater doses of
the toxic and cancer-causing substances present in cigarette
smoke [39]. Therefore, smoking using these filters is not an
alternative for lowering the risk of smoking-related dis-
eases.
Smoking cessation is the ultimate method for reducing
smoking-related diseases. However, quitting smoking is
extremely difficult due to the addictive nature of nicotine.
Such smoking cessation methods as nicotine replacement
therapy (NRT) and nAChR partial agonists and antagonists
have been shown to help some smokers quit, but they are
also reported to have high relapse rates [7–14]. In addition,
some of these methods are perceived as being inconvenient.
For example, nicotine products such as nicotine spray and
gum have to be frequently replaced, while nAChR partial
agonists are prescribed medications and are administrated as
drugs. These methods are not easily psychologically ac-
cepted by the smokers who want to quit, affecting the effi-
cacy of their smoking cessation.
The novel tea filter treatment might avoid the pitfalls
mentioned above and effectively promote smoking absti-
nence. Because it uses the smoking process to help quit
smoking, it may be accepted by smokers with less psycho-
logical obstacles and side effects. When a smoker is smok-
ing using the tea filter, the inhibitors of the nicotine receptor
in the tea filter are absorbed through the respiratory system
and travel to the brain where they exert cessation effects.
This appears as a circulatory process moving towards
smoking cessation, which continues until the smoker quits
smoking (Figure 7).
540 Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5
Figure 6 Effect of a tea filter on the histopathology of the lung tissue of rats treated with cigarette smoke. A, Normal animals without being treated with
cigarette smoke; B and C, animals treated with normal cigarette smoke and D, animals treated with smoke generated from cigarettes with tea filters for 75 d.
A variety of pathological alterations were discovered in the lungs. Most significantly, many of the alveoli were found broken when animals were treated with
normal cigarette smoke (arrows in B and C), while using a tea filter almost reversed this effect. Specimens were taken from the same position of the lung
tissue of the rats, fixed with 10% formalin, embedded in paraffin, sectioned and stained with HE. Pathological changes were examined under a
light-microscope. Details are described in “Methods”.
Figure 7 Tea filter inhibits cigarette smoking addiction through a circula-
tive process moving towards smoking cessation.
Smoking is one of the largest international public health
problems. For example, in China, the population of smokers
is about 350 million and is increasing. Similar situations are
found in India and other developing countries [3,40]. The
tea filter is produced by a slight and inexpensive modifica-
tion of the existing filter which is suitable for developing
countries.
In conclusion, this work proposed a tea filter and
theanine, for inhibiting smoking addiction through inhibi-
tion of nAChR and provides an effective method for treating
tobacco addiction.
This work was supported by the National Natural Science Foundation of
China (Grant No. 30870587) and the National Basic Research Program of
China (Grant No. 2006CB500700). This work was partly supported by the
Key Laboratory of Mental Health, Chinese Academy of Sciences. We ap-
preciate Dr. John Fassett from the School of Medicine, University of Min-
nesota for his suggestions and fine work in the correction of the English text.
1 Chung Y F, Khoo M L, Heng M K, et al. Epidemiology of Warthin’s
tumor of the paroted galnd in an Asian population. Br J Surg, 1999,
86: 661–664
2 Zhao B. Cigarette, free radicals and cancer. Nature J, 1989, 12:
453–460.
3 Gu D F, Kelly T N, Wu X, et al. Mortality attributable to smoking in
China. N Engl J Med, 2009, 360: 150–159
4 Goddard E. Smoking and drinking among adults 2006. General
Household Survey 2006. Newport: Office for National Statistics,
2008
5 Mizoue T, Tokui N, Nishisaka K, et al. Prospective study on the rela-
tion of cigarette smoking with cancer of the liver and stomach in an
endemic region. Intern J Epidemiol, 2000, 29: 232–237
6 World Health Organization. Updated status of the WHO framework
convention on tobacco control [OL]. [2007-1–11]. http: //www.who.
int/tobacco/framework//
7 Balbani A P S, Montovani J C. Methods for smoking cessation and
treatment of nicotine dependence. Rev Bras Otorrinolaringol, 2005,
71: 820–826
8 Ray R, Schnoll R A, Lerman C. Nicotine dependence: Biology, be-
havior, and treatment. Annu Rev Med, 2009, 60: 247–260
9 Rigotti A N. Treatment on tobacco use and dependence. N Engl J
Med, 2002, 346: 506–512
10 Peng R L, Wang S L. Nicotine sublingual tablet for smoking cessa-
Yan JingQi, et al. Sci China Life Sci May (2010) Vol.53 No.5 541
tion in 115 cases: A double-blind randomized placebo-controlled
clinical trial. J Clin Rehabilit Tissue Engin Res, 2007, 11: 10443–
10446
11 Wagena E J, Knipschild P G, Huibers M J H, et al. Efficacy of bu-
propion and nortriptyline for smoking cessation among people at risk
for or with chronic obstructive pulmonary disease. Arch Intern Me,
2005, d 16: 2286–2292
12 Oncken C, Gonzales D, Nides M, et al. Efficacy and safety of the
novel selective nicotinic acetylcholine receptor partial agonist,
varenicline, for smoking cessation. Arch Intern Med, 2006, 166:
1571–1577
13 Hays J T, Ebbert J O. Varenicline for tobacco dependence. N Engl J
Med, 2008, 359: 2018–2024
14 Franklin T R, Harper D, Kampman K, et al. The GABA B agonist
baclofen reduces cigarette consumption in a preliminary double-blind
placebo-controlled smoking reduction study. Drug Alcohol Depend,
2009, 103: 30–36
15 Jankan J, Selman S H, Swiecz R. Why drinking green tea could pre-
vent cancer. Nature, 1997, 387: 661–662
16 Mackenzie T, Leary L, Brooks W B. The effect of an extract of green
and black tea on glucose control in adults with type 2 diabetes melli-
tus: double-blind randomized study. Metabolism, 2007, 56: 1340–
1344
17 Guo S H, Yan J, Bezard E, et al. Protective effects of green tea poly-
phenols in the 6-OHDA rat model of Parkinson’s disease through
inhibition of ROS-NO pathway. Biol Psychia, 2007, 62: 1353–1362
18 Yang F, Zhao B, Xin W. Studies on toxicological mechanisms of gas
phase of cigarette smoke and protective effects of GTP. Environm
Chem, 1992, 11: 39–50
19 Yang F, Zhao B, Xin W. An ESR study on lipid peroxidation of rat
liver microsome induced by gas-phase cigarette smoke. Environ
Chem, 1993: 12: 116–120
20 Gao J, Tang H, Zhao B. The toxicological damagement of gas phase
cigarette smoke on cells and protective effect of green tea polyphe-
nols. Res Chem Intermed, 2001, 29: 269–279
21 Zhao B, Li X, He R, et al. Scavenging effect of extracts of green tea
and natural antioxidants on active oxygen radicals. Cell Biophys,
1989, 14: 175–184
22 Yao E, Zhang J, Tai J, et al. Reducing tobacco-specific nitrosamine
in cigarette main stream smoke by using tea filter. Chinese Agricul
Sci Bull, 2007, 23: 125–128
23 Yao E, Zhang J, Mao Z, et al. Application of cellulose containing tea
powder acetate dual filter in cigarette. Tobac Sci Technol, 2007, 49:
49–50
24 Henningfield J E, Stapleton J M, Benowitz N L, et al. Higher levels
of nicotine in arterial than in venous blood after cigarette smoking.
Drug Alcohol Depend, 1993, 33: 23–29
25 Nguyen H N, Rasmussen B A, Perry D C. Subtypeselective
up-regulation by chronic nicotine of high-affinity nicotinic receptors
in rat brain demonstrated by receptor autoradiography. J Pharmacol
Experim Therap, 2003, 307: 1090–1095
26 Laviolette S R, Nader K, van der Kooy D. Motivational state deter-
mines the functional role of the mesolimbic dopamine system in the
mediation of opiate reward processes. Behav Brain Res, 2002, 129:
17–29
27 Laviolette S R, van der Kooy D. Blockade of mesolimbic dopamine
transmission dramatically increases sensitivity to the rewarding ef-
fects of nicotine in the ventral tegmental area. Molec Psychia, 2003, 8:
50–55
28 Huang E Y, Liu T C, Tao P L. Co-administration of dextromethor-
phan with morphine attenuates morphine rewarding effect and related
dopamine releases at the nucleus accumbens. Naunyn Schmiedebergs
Arch Pharmacol, 2003, 368: 386–392
29 Zhang D L, Tao Y, Duan S J, et al. Pycnogenol in cigarette filters
scavenges free radicals and reduces mutagenicity and toxicity of to-
bacco smoke in vivo. Toxicol Ind Health, 2002, 18: 215–224
30 Cardoso W V, Saldiva P H N, Criado P M P, et al. A comparison
between the isovolume and the end-inflation occlusion methods for
measurement of lung mechanics in rats. J Applied Toxicol, 1991, 11:
79–84
31 Cendon S P, Battlehner C, Lorenzi-Filho G, et al. Pulmonary emphy-
sema induced by passive smoking: An experimental study in rats.
Braz J Med Biol Res, 1997, 30: 1241–1247
32 Miller R G. Simultaneous statistical inference. New York: Springer,
1981. 230
33 Xiu X, Puskar N L, Shanata J A P, et al. Nicotine binding to brain
receptors requires a strong cation-pi interaction. Nature, 2009, 458:
534–538
34 Lape R, Colquhoun D, Sivilotti1 L G. On the nature of partial ago-
nism in the nicotinic receptor superfamily. Nature, 2008, 454:
722–728
35 Tapper A R, McKinney S L, Nashmi R, et al. Nicotine activation of
α4 receptors: Sufficient for reward, tolerance, and sensitization. Sci-
ence, 2004, 306: 1029–1032
36 Picciotto M R, Zoli M, Rimondini R, et al. Acetylcholine receptors
containing the β2 subunit are involved in the reinforcing properties of
nicotine. Nature, 1998, 391: 173–177
37 Benwell M E, Balfour D J. The effects of acute and repeated nicotine
treatment on nucleus accumbens dopamine and locomotor activity.
Br J Pharmacol, 1992, 105: 849–856
38 Bryan J. Psychological effects of dietary components of tea: Caffeine
and L-theanine. Nutrition Rev, 2007, 66: 82–90
39 Rigotti N A, Tindle H A. The fallacy of “light” cigarettes. BMJ, 2004,
328: E278–279
40 Thankappan K R, Mini G K. Case-control study of smoking and
death in India. N Engl J Med, 2008, 358: 2842–2842
