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A forest bathing trip increases human natural killer activity and expression of anti-cancer
proteins: a comparison with a trip to a place without forest
Qing Li1*, Kanehisa Morimoto2, Maiko Kobayashi1, Hirofumi Inagaki1, Masao Katsumata1,
Yukiyo Hirata1, Kimiko Hirata1, Hiroko Suzuki1, Yingji Li1, Yoko Wakayama1, Tomoyuki
Kawada1, Yoshifumi Miyazaki3, Takahide Kagawa3, Tatsuro Ohira3, Norimasa Takayama3,
Alan M. Krensky4
1 Department of Hygiene and Public Health, Nippon Medical School, Tokyo, Japan
2 Department of Social and Environmental Medicine, Osaka University Graduate School of
Medicine, Osaka, Japan
3 Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan
4 Stanford University School of Medicine, Palo Alto, CA, USA
*Corresponding author
Qing Li, MD, Ph.D
Department of Hygiene and Public Health, Nippon Medical School,
1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
e-mail: qing-li@nms.ac.jp
Tel: +81-3-3822-2131
Fax: +81-3-5685-3065
1
Abstract
We previously have reported that a forest bathing trip (Shinlinyoku) enhanced human natural
killer (NK) activity; number of NK cells, and perforin, granzymes and granulysin-expression
in peripheral blood lymphocytes (PBL). In the present study, we investigated how long the
increased NK activity will be lasted and also compared the effect of a forest bathing trip on
NK activity with a trip to a place without forest (general trip). 12 healthy male subjects, age
35-56 years, were selected with informed consent from four large companies in Tokyo, Japan.
The subjects experienced a three-day/two-night trip at forest fields and a place without forest.
On the first day, subjects walked for two hours in the afternoon in a forest field; and on the
second day, they walked for two hours in the morning and afternoon, respectively, at two
different forest fields. Blood was sampled on the second and third days during the trip, and on
days 7 and 30 after the trip, and NK activity; numbers of NK, T cells, granulysin, perforin,
and granzymes A/B-expressing cells in PBL were measured. Similar measurements were
made before the trip on a normal working day as the control. The forest bathing trip
significantly increased NK activity, the numbers of NK, perforin, granulysin, and granzymes
A/B-expressing cells. The increased NK activity was lasted for more than 7 days after the trip.
On the other hand, a general trip did not increase NK activity, the numbers of NK, and
intracellular anti-cancer proteins. Taken together, these findings indicate that a forest bathing
trip can increase NK activity, the number of NK cells and intracellular anti-cancer proteins,
and that this effect at least lasts more than 7 days after the trip.
Key words: anti-cancer proteins, forest bathing, NK activity, granulysin, granzyme, perforin
2
Introduction
A forest bathing trip, called “Shinrinyoku” in Japanese, involves a visit to a forest field for
the purpose of relaxation and recreation by breathing in the volatile substances, called
phytocide released from tree, (Li et al., 2007). It was first proposed in the 1980s and has
become a recognized relaxation activity in Japan (Ohtsuka et al., 1998; Yamaguchi et al.
2006; Morita et al., 2007; Li et al., 2007). Since forests occupy 67% of the land in Japan
(Forestry Agency of Japan, 2002), participation in forest bathing trip is easily accessible.
According to a public opinion poll conducted in Japan in 2003, 25.6% of respondents had
participated in forest bathing trip, indicating the popularity of forest bathing trip in Japan
(Morita et al., 2007). Moreover, forest bathing trip is possible in forest environments in the
world. We previously have reported that phytoncytes enhanced human natural killer (NK)
activity and intracellular levels of perforin, granulysin and granzyme A in NK cells in vitro (Li
et al. 2006a). Komori et al. (1995) also reported that citrus fragrance found in the forest
affects the human endocrine and immune systems as analyzed by measurement of urinary
cortisol and dopamine levels, NK activity and CD4/8 ratios. These findings strongly suggest
that forest bathing trip may have beneficial effects on human immune function, thus, we
previously have investigated the effect of forest bathing trip on human NK activity, and found
that a forest bathing trip increased human NK activity, number of NK cell, and intracellular
levels of perforin, granulysin and granzymes A/B in peripheral blood lymphocytes (PBL) (Li
et al., 2007). However, there are two questions remained to be resolved: one is that whether a
trip to a place without forest (a general trip) also can increase NK activity? Another is that
3
how long the increased NK activity will be lasted after a forest bathing trip? In the present
study, we conducted two investigations to resolve the above-mentioned two questions.
Subjects and methods
Subjects
Twelve healthy male subjects, aged 35-56 years (45.1±6.7), were selected from four large
companies in Tokyo, Japan in the present study. The information gathered from a self-
administered questionnaire including age, and lifestyle habits that asked about cigarette
smoking, alcohol drinking habits, eating breakfast, sleeping hours, working hours, physical
exercise, nutritional balance and mental stress, which have been reported previously (Li et al.,
2006b; 2007). Written informed consent was obtained from all subjects after a full
explanation of the study procedures. None of the subjects had any signs or symptoms of
infectious disease, used drugs that might affect immunological analysis, or were taking any
medications at the time of the study. The ethics committee of the Nippon Medical School
approved this study (approval No. 16-1).
Forest bathing trip and a trip to a place without forest (general trip)
In the forest bathing trip, the subjects experienced a three-day/two-night trip at three different
forest fields in early September, 2006. On the first day, subjects walked for two hours in the
afternoon in a forest field, and then stayed at a nearby hotel within the forest. On the second
day, subjects walked for 2 hours in the morning and afternoon, respectively, at two different
forest fields. Whereas, in the general trip, the subjects experienced a three-day/two-night trip
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at a city in middle May, 2006. On the first day, subjects walked for two hours in the afternoon
in a tourist rout in a city, and then stayed at a hotel in the city. On the second day, subjects
walked for 2 hours in the morning and afternoon, respectively, at two different tourist routs in
the same city. Each course in the both trips was 2.5 km, closely resembling normal physical
activity for the subjects on normal working days. Daily physical activity of the subjects was
monitored with a pedometer and the duration of sleep was measured with a piezo-electric
accelerometer, Actiwatch(R) (Mini Mitter Co. Inc., Sunriver), worn on the wrist of the non-
dominant arm. The validation study was previously reported (Kawada et al. 2001). Blood was
sampled on the second and third days during the trips, and days 7 and 30 after the forest
bathing trip and three days prior to the trips as a control. Since it has been reported that
human NK cell activity shows circadian rhythms (Angeli 1992), all samples were obtained at
8:00 am. All blood samples were placed in an ice/water box at 4°C and assays performed
within four hours of the blood draw. NK activity; proportions of NK, T cells, granulysin,
perforin, and granzymes A/B-expressing cells in peripheral blood lymphocytes (PBL), counts
of white blood cells were measured. Adrenaline concentration in urine also was determined.
Reagents
RPMI 1640 was purchased from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine
serum (FBS) was purchased from JRH Biosciences (Lenexa, KS), and heat-inactivated at
56°C for 30 min prior to use. Sodium 51Cr-chromate was obtained from PerkinElmer (Boston,
MA). Fluorescein isothiocynate (FITC)-mouse anti-human perforin, granzyme A (GrA),
granzyme B (GrB) and FITC/phycoerythrin (PE)-CD16, PerCP-Cy5.5-CD3, FITC/PE-
5
negative isotypic control antibodies, and Cytofix/cytoperm solution were purchased from BD
Pharmingen (San Diego, CA). Rabbit anti-human granulysin (GRN) polyclonal antibody was
described previously (Hanson et al. 1999). PE-goat-anti rabbit IgG were purchased from
Vector Laboratories Inc. (Burlingame, CA).
NK activity
Human peripheral blood lymphocytes (PBL) were separated from peripheral blood with
BD Vacutainer CPT (Becton Dickinson, Franklin Lakes, NJ), and then adjusted to 4x106
cells/ml for the assay of NK activity. The viability of the cells as determined by trypan blue
dye exclusion was more than 95%. Human NK activity was assayed according to the
traditional method (Li et al., 2007). Briefly, K-562 target cells were labeled with a sodium
51Cr-chromate solution for 60 min at 37°C in 5% CO2 and washed 4 times in RPMI 1640
containing 10% FBS. The target cells were plated into round-bottomed 96-well microplates,
then the effector cells (PBL) at 4x106, 2x106 and 1x106 cells/ml in 100 µl were added to the
wells in triplicate at E:T ratios of 40:1, 20:1 and 10:1. Following a 4-hr incubation at 37°C in
5% CO2, the microplates were centrifuged and 100 µl of supernatant from each well was
collected and measured in a gamma counter. Then, the NK activity was calculated as
described previously (Li et al., 2007).
Cell staining and flow cytometric analysis
The surface markers of PBL were stained with PE/FITC-CD16 for NK cells and PerCP-
Cy5.5-CD3 for T cells for 30 min in the dark. Then, the cells were fixed/permeablized with
Cytofix/cytoperm solution for 20 min at 4°C, and then the intracellular perforin and GrA/B
6
were stained with FITC- anti-human perforin and GrA/B, respectively, for 30 min at 4°C
according to the manufacturer’s instructions (BD PharMingen, San Diego, CA). Intracellular
GRN was stained with rabbit anti-human GRN polyclonal Ab after fixation/permeablization
with Cytofix/cytoperm solution, and then stained with PE-goat anti-rabbit IgG for 30 minutes
at 4°C in the dark. After staining, the cells were washed twice with the fixative solution and
once with PBS containing 1% FBS. Flow cytometric analysis was performed with a FACScan
flow cytometer (Becton Dickinson, San Jose, CA) as described previously (Li et al. 2005;
2006b; 2007). Lymphocytes were identified by their characteristic appearance on a dot plot of
FSC versus SSC and electronically gated to exclude dead cells and granulocytes.
Measurements for adrenaline in urine
The levels of adrenalin in urine were measured by HPLC method using an analyzer HLC-
725CAII. The instrument features a column-switching system composed of two pretreatment
columns and one separation column and a high-sensitive detection unit based on a post-
column reaction using a fluorogenic reagent, 1,2-diphenylethyleneamine. The detection limit
of adrenaline in urine was 8 fmol/ml (Hirowatari et al., 1999).
White blood cell (WBC) count
WBC, RBC and platelet counts, the percentages of granulocyte, lymphocyte and
macrophage, concentration of Hb, Hct, MCV, MCH and MHCH were determined by an
automatic cell counter (LC-550, Horiba Co., LTD. Kyoto, Japan) as described previously (Li
et al. 2007).
Measurements of phytoncide, and environmental temperature/ humidity in the forest fields
7
during the investigation
The volatile organic compounds (phytoncide) in forest air, and temperature and humidity in the
forest fields were measured as previously reported (Li et al., 2007).
Statistical analysis
Multiple comparisons were made with the paired t-test if the analysis of variance was
significant. The analysis was performed with the SPSS 11.5J software package for Windows.
The significance level for p values was set at < 0.05.
Results
Effect of a forest bathing trip and the general trip on NK activity
Because the difference among individuals in NK activity is very big, we do not compare the
difference in NK activity between the forest bathing trip and general trip, but compared the
difference in NK activity between before and after the trips in the present study.
As shown in Fig. 1A, forest bathing trip significantly increased human NK activity, and this
increase lasted more than 7 days after the trip. On the other hand, the general trip did not
increase human NK activity (Fig. 1B).
Effect of a forest bathing trip and the general trip on CD16+ NK cells
As shown in Fig. 2A, forest bathing trip significantly increased the number of CD16+ NK
cells, and this increase lasted more than 7days after the trip. On the other hand, although the
general trip also slightly increased CD16+ NK cells, this increase was not significant (Fig.
2B).
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The forest bathing trip did not affect lymphocytes and WBC counts.
Effect of forest bathing trip on the percentage of cells expressing cytolytic molecules
The forest bathing trip significantly increased the percentages of GRN, perforin, and GrA/B-
expressing cells in PBL (Fig 3A). Whereas, although the general trip also slightly increased
the percentages of GRN, perforin, and GrA/B-expressing cells in PBL, these increases were
not significant (Fig. 3B).
Effect of forest bathing trip on T (CD3+) cells
Both forest bathing trip (Fig. 4A) and general trip (Fig. 4B) did not affect the number of T
cells.
Effect of forest bathing trip on adrenalin concentration in urine
The forest bathing trip significantly decreased the concentration of adrenaline in urine,
whereas, a general trip did not affect the level of adrenaline in urine, indicating that forest
bathing trip has a relaxation effect (Fig. 5A, 5B).
There were no significant differences in daily physical activity before and during the trips
(Fig. 6A, 6B). The hours of sleep were, increased during the trips compared with the control
days (Table 2), however, the difference was not significant.
Lastly, phytoncides, such as alpha-pinene, beta-pinene and limonene were detected in the
forest fields during the investigation (Table 1), and not detected in the urban area of Tokyo,
and in the city for general trip. Weather during the forest bathing trip was excellent with
9
average temperatures and humidity in the forest fields during the walking of 16.31± 0.23°C,
99.77±0.44% on day 1 in the afternoon; 19.55± 0.61°C, 78.62±3.24% on day 2 in the
morning; and 20.78± 0.42°C, 74.16±61.94% on day 2 in the afternoon, The average
temperature and humidity in urban area of Tokyo on the control day was 28.5°C, 62%,
respectively.
Discussion
We previously have found that a forest bathing trip increased human NK activity, number
of NK cells, and intracellular levels of perforin, granulysin and granzymes A/B in PBL (Li et
al., 2007). However, there are two questions remained to be resolved: one is that whether a
trip to a place without forest (general trip) also can increase NK activity? Another is that how
long the increased NK activity will be lasted after a forest bathing trip? The present study
reconfirmed that a forest bathing trip can enhance the immune response as measured by
human NK activity, and the numbers of NK cells, which is similar to our previous report (Li
et al., 2007), whereas, a trip to a place without forest (general trip) has no effect on NK
activity, indicating that forest bathing trip indeed can enhance human NK activity. Moreover,
we also found that the increased NK activity and NK cells induced by forest bathing trip
lasted more than 7 days, even 30 days after the trip. This suggests that if people go to a forest
bathing trip once a month, they can always keep NK activity in a higher level. This has a very
important significance in the health promotion from the viewpoint of preventive medicine.
NK cells kill tumor or virus infected cells by release of perforin, granzymes (Gr) (Shinkai
10
et al. 1988; Smyth et al. 2001; Beresford et al. 1997; Li et al. 2002), and granulysin (GRN)
(Okada et al. 2003; Hanson et al. 1999; Stenger et al. 1998) via the granule exocytosis
pathway. Cytotoxicity mediated by NK cells is greatly impaired in perforin-deficient mice
(Kagi et al. 1994b; Li et al. 2004). GrA plays a critical role in triggering apoptosis in target
cells either directly or via the activation of cellular caspases, and also cleaves IL-1 , theβ
nucleosome assembly protein called putative HLA-associated protein II, TAF-I , histones andβ
lamins (Smyth et al. 2001; Zhang et al. 2001ab). GrB directly cleaves the downstream caspase
substrates, nuclear matrix antigen, catalytic subunit of DNA-associated DNase inhibitor and
lamins (Smyth et al. 2001; Zhang et al. 2001a). GRN, a lytic molecule expressed by human
CTL and NK cells, is active against tumor cells and a variety of microbes. GRN can enter
target cells in the absence of perforin and induce apoptosis, although GRN and perforin
together are required to kill intracellular microbes like Mycobacteria tuberculosis (Okada et
al. 2003; Hanson et al. 1999; Stenger et al. 1998). GRN is associated with diverse activities of
NK cells and CTL in physiological and pathological settings and may be a useful marker to
evaluate the status of host cellular immunity (Ogawa et al. 2003).
In order to explore the mechanism of enhancement of NK activity by forest bathing, we
investigated the effect of forest bathing on the intracellular levels of perforin, GRN, and
GrA/B in PBL. We found that the forest bathing trip significantly increased the proportion of
perforin, GRN, GrA/B-expressing cells in PBL, which are similar to our previous report (Li et
al., 2007), whereas, general trip had no effect on perforin, GRN, GrA/B-expressing cells,
indicating that forest bathing trip indeed can increase perforin, GRN, GrA/B-expressing cells.
11
Moreover, we also found that increased perforin, GRN, GrA/B-expressing cells induced by
forest bathing trip lasted more than 7 days, even 30 days after the trip. These cytolytic
molecules contribute to NK and anti-tumor activity.
Activity of sympathetic nervous system causes adrenaline to be released into the
circulation from the adrenal cortex. Therefore, concentration of adrenaline in urine has also
been used as measures of autonomic nervous activity in response to mental demands
(Hjemdahl et al. 1984). We found that forest bathing trip significantly decreased the
adrenaline concentration in urine, however, the general trip had no effect on the adrenaline
concentration in urine, suggesting that the parasympathetic nervous system of subjects was
dominant, associated with relaxation and decreased stress (Mori et al., 2002). It has been
reported that adrenaline inhibits human NK activity (Garland et al. 2003). The increased NK
activity in forest bathing trip may be related to an attenuated stress hormone response
(adrenaline) associated with forest bathing trip. Previous studies have reported that forest
bathing reduces the concentration of cortisol in saliva, reduces prefrontal cerebral activity,
reduces blood pressure and stabilize autonomic nervous activity in humans (Miyazaki and
Motohashi 1996; Park et al., 2005, Yamaguchi et al. 2006).
Many factors, including circadian variation (Angeli 1992), physical exercise (Nieman
2000; Miles et al. 2002, Li et al., 2006b) and alcohol consumption (Ochshorn-Adelson et al.
1994, Li et al., 2006b) can affect human NK activity. In order to control the effect of circadian
rhythms on NK activity, we sampled blood at 8 am on all days. To control for the effect of
physical exercise on NK activity, we limited the walking steps during the trip to the normal
12
workday distances as monitored by a pedometer. To control the effect of alcohol on NK
activity, the subjects did not consume alcohol during the study. The sleeping hours during the
trips were a little longer than the average working days (Table 2), however, the difference was
not significant in both forest bathing trip and general trip. There are several reports addressing
the effect of sleeping hours on NK cell activity. Many reports suggest that sleep deprivation
increases human NK activity (Dinges et al. 1994; Matsumoto et al. 2001), while others
suggest that sleep deprivation decreased human NK activity (Moldofsky et al. 1989; Irwin et
al. 1994); still other studies by Kusaka et al. (1992) and Inoue et al. (1996) reported that
sleeping hours did not affect NK or LAK activity, or NK cell numbers under physiologic
conditions. In fact, we also found that there was no difference in the numbers of NK cells, nor
perforin, GRN, GrA/B-expressing cells in PBL among the subjects who slept 5, 6 or 7 hours,
respectively (Li et al. 2006b). In addition, although the sleeping hours during the general trip
were a little longer than the average working day (Table 2), however, the NK activities during
trip were almost the same with the working day, indicating that the longer sleeping hours did
not contribute to NK activity in the general trip. Taken together, although the sleeping hours
during the trips were a little longer than that on the average working day, this difference did
not affect either NK activity or numbers in the present study.
As detailed in Table 1, we detected several phytoncides such as alpha-pinene, beta-pinene
and limonene in the forest fields during the trip. We previously found that phytoncides, such
as alpha-pinene, d-limonere significantly enhanced human NK activity and increased
expression of intracellular cytolytic molecules, perforin, GrA and GRN in vitro (Li et al.
13
2006a), suggesting that phytoncide may partially contribute to the enhanced NK activity
during the forest bathing trip.
Taken together, these findings indicate that forest bathing trip can increase human NK
activity, the number of NK cells and intracellular perforin, GrA/B and GRN, and that
increased NK activity, number of NK cells and intracellular perforin, GrA/B and GRN at least
can last more than 7 days after a trip.
Acknowledgements: This work was supported partly by a research project for utilizing
advanced technologies in agriculture, forestry and fisheries of Japan (2006).
14
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Figure legends
Figure 1. Effect of the forest bathing trip (A) and a general trip (B) on NK activity. Data are
presented as the mean+SE ((n=12 in A and n=11 in B). ANOVA indicated that the forest
bathing trip significantly affected the NK activity (p<0.01). *: p<0.05, **: p<0.01,
significantly different from before the trip by the paired t-test. The activity values for an E/T
ratio of 20/1 are shown, and the similar results were also obtained with E/T ratios of 40/1 and
10/1.
Figure 2. Effect of the forest bathing trip (A) and a general trip (B) on the number of NK cells.
Data are presented as the mean+SE (n=12 in A and n=11 in B). ANOVA indicated that the
forest bathing trip significantly affected the percentage and number of NK cells (all p<0.01). *:
p<0.05, **: p<0.01, #: p=0.054 significantly different from before the trip by the paired t-test.
Figure 3. Effect of the forest bathing trip (A) and a general trip (B) on GRN, perforin,
GrA/B-expressing cells in PBL. Data are presented as the mean+SE (n=12 in A and n=11 in
B). ANOVA indicated that the forest bathing trip significantly affected the GRN, perforin,
GrA/B-expressing cells in PBL (all p<0.01). *: p<0.05, **: p<0.01, significantly different
from before the trip by the paired t-test.
Figure 4. Effect of the forest bathing trip (A) and a general trip (B) on the total number of T
cells. A: Data are presented as the mean+SE (n=12 in A and n=11 in B). ANOVA indicated that
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both the forest bathing trip and general trip did not affect the number of T cells.
Figure 5. Effect of the forest bathing trip (A) and a general trip (B) on adrenaline
concentration in urine. Data are presented as the mean+SE (n=12 in A and n=11 in B).
ANOVA indicated that the forest bathing trip significantly affected the adrenaline
concentration in urine (p<0.05). *: p<0.05, significantly different from before the trip by the
paired t-test.
Figure 6. Consumption of energy of the subjects before and during the forest bathing trip (A)
and general trip (B). Data are presented as the mean+SE (n=12 in A and n=11 in B). ANOVA
indicated that there was no significant difference before and during the trips in the
consumption of energy of the subjects.
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Table 1. Concentration of volatile substances in the air of forest fields calculated as alpha-
pinene (ng/m3)
Measuring points Field 1
Day 1 pm Field 2
Day 2
Kind of Trees Chamaecyparis Chamaecyparis
White cedar
Tricyclene 299.7 805.5
-Pineneα2,886.7 1,281.7
Camphene 375.6 486.8
-Pineneβ137.5 66.9
Myrcene 109.4 71.8
-3-Careneδ66.6 25.3
-Terpineneα43.8 26.9
p-Cymene 109.4 67.3
Limonene 111.1 48.4
-Terpineneγn.d 33.2
Terpinolene 87.5 13.4
Camphor 32.8 14.8
Bornyl acetate 54.7 43.4
n.d: not detected.
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Table 2. Sleeping hours of the subjects before and during the forest bathing trip and general
trip (Mean±SD)
Control day
(Before trip)
Day 1
(During trip)
Day 2
(During trip)
1 Week
(After trip)
1 Month
(After trip)
Forest bathing trip 6.18±1.63 7.29±1.69 7.03±1.44 5.38±0.88 5.63±0.96
General trip 5.98±1.79 7.22±1.52 6.59±1.41
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