Although these studies demonstrated that forest bathing trips were able to enhance human NK activity in male subjects, the question remaining to be resolved was whether or not forest bathing trips would also increase NK activity in female subjects. It has been reported that menstrual cycle significantly affects NK activity [17
]; consequently, the influence of the menstrual cycle on NK activity should be controlled for in experiments with female subjects.
In this study [4
], 13 healthy nurses, aged 25–43 years (mean 28.8 ± 4.6 years) and active in their profession for 4–18 years (mean 6.7 ± 3.8), were selected with informed consent. None of the subjects had any signs or symptoms of infectious disease, used drugs that may affect immunological analysis, or were taking any medication at the time of the study. The subjects experienced a 3-day/2-night trip to forest fields around Shinano town, Nagano prefecture, located in northwest Japan in early September 2007. The schedule of the forest bathing trip and blood sampling was similar to that described for the earlier studies [2
], and the same blood/urine tests were carried out (WBC counts, NK activity, numbers of NK and T cells, and GRN-, perforin-, and GrA/B-expressing lymphocytes in the blood samples; concentrations of adrenaline and noradrenaline in urine) as well as tests measuring the concentrations of estradiol and progesterone in serum. The same control measurements were made before the trip on a normal working day. Blood was sampled at 0800 hours on all days. The concentrations of phytoncides in the forests were measured.
The forest bathing trip significantly increased NK activity (Fig. ) and the positive rates of NK (Fig. ) and perforin-, GRN-, and GrA/B-expressing cells (Fig. ). The increased NK activity (Fig. ) and the positive rates of NK (Fig. ) and perforin-, GRN-, and GrA/B-expressing cells (Fig. ) lasted for more than 7 days after the trip [4
], which confirmed the previous findings in male subjects [3
]. Phytoncides, such as alpha-pinene and beta-pinene, were detected in forest air. These findings indicate that a forest bathing trip also increased NK activity, number of NK cells, and the levels of intracellular anti-cancer proteins in female subjects and that this effect lasted for at least 7 days after the trip. Phytoncides released from trees may partially contribute to the increased NK activity [1
]. A forest bathing trip was found to significantly decrease the percentage of T cells in female subjects (Fig. ) [4
]. It has been reported that mental stress increases T cell levels among PBLs [17
] and that people with a poor lifestyle have a higher percentage of T cells than people with a good lifestyle [16
]; therefore, it can be speculated that the proportion of T cells in PBLs may reflect stress status.
Fig. 5 Effect of a forest bathing trip on NK activity in female subjects. Data are presented as the mean ± SE (n = 13). *P < 0.05, **P < 0.01, significantly different from (more ...)
Fig. 6 Effect of a forest bathing trip on the percentage of NK cells. Data are presented as the mean ± SE (n = 13). **P < 0.01, significantly different from before the trip by the paired t test. (more ...)
Fig. 7 Effect of a forest bathing trip on the levels of GRN-, perforin-, and GrA/B-expressing cells in PBLs. Data are presented as the mean ± SE (n = 13). *P < 0.05, **P < 0.01, (more ...)
Fig. 8 Effect of the forest bathing trip on the percentage of T cells. Data are presented as the mean ± SE (n = 13). *P < 0.05, **P < 0.01, significantly different from before (more ...)
Souza et al. reported that NK activity is significantly higher in the follicular phase of the menstrual cycle than in the luteal phase and that the levels of NK activity in postmenopausal women are similar to those of women in the follicular phase but significantly higher than those of women in the luteal phase [19
]. Conversely, Yovel et al. [20
] reported that the menstrual cycle had no significant effect on the activity levels of NK cells. Roszkowski et al. [21
] found that patients with low (<50 pg/ml) and high (>200 pg/ml) estradiol levels showed an increase and a decrease of NK cell activity, respectively. Progesterone at 100–400 nM
(31.45–125.8 ng/ml) inhibits NK activity in healthy pregnant women, whereas 100-fold higher concentrations are required for reducing NK activity in non-pregnant women [22
]. These suggest that the menstrual cycle and the levels of estradiol and progesterone in serum may affect human NK activity.
To control for the influence of menstrual cycle on NK activity, a questionnaire was administered to obtain information on the menstrual cycle of the subjects. The ratios of subjects who were in the follicular phase during the experiment were 5/13, 6/13, 6/13, 7/13, and 6/13 on the day before the trip, days 1 and 2 during the trip, and days 7 and 30 after the trip, respectively, indicating that there was no significant difference in the proportion of women in different phases of the menstrual cycle between the different days. This observation suggests that the menstrual cycle had a similar influence on the average of NK activity on the different days. Therefore, statistical analyses to compare the mean values of NK activity on the different days by a paired t
test should be appropriate. In addition, the concentrations of estradiol and progesterone in the serum of the subjects were also measured to confirm the influence of estradiol and progesterone on NK activity. In this study, there was no significant difference in the concentration of estradiol in serum between the days before, during, and after the forest bathing trip, indicating that, in this case, estradiol had a similar effect in all subjects on NK activity on the different days. Although the levels of progesterone on days 1 and 2 were higher than that before the study, the difference in serum progesterone concentration between days 1 or 2 and before the trip was not significant, suggesting that progesterone had a similar effect on NK activity on the different days [4
Many factors, including circadian variation [23
], physical exercise [16
], and alcohol consumption [16
], can affect human NK activity. In order to control for the effect of circadian rhythm on NK activity, blood was sampled at 0800 hours on all days [2
]. To control for the effect of physical exercise on NK activity, the number of steps taken during the walking trips were limited to average normal workday distances, as monitored by a pedometer. The levels of physical activity among all trips were also matched. To control for the effect of alcohol on NK activity, the subjects did not consume alcohol for 2 days before providing the blood sample during the study period for both trips, including before the trips and after the trips on days 7 and 30. The number of hours spent sleeping during the trips were slightly more than those on average working days; however, the difference was not significant in either type of trip. Kusaka et al. [26
] reported that the number of sleeping hours did not affect NK activity or NK cell numbers under physiological conditions. Li et al. [16
] also found that there was no difference in the number of NK cells or the levels of perforin-, GRN-, or GrA/B-expressing cells in PBL among subjects who slept for 5, 6, or 7 h, respectively. In addition, although the number of hours slept during the city tourist visit were slightly more than those on an average working days, the NK activities during the trip were almost the same as for working days, indicating that the longer sleeping hours did not affect NK activity in the city tourist visit [3
]. Taken together, it can be concluded that although the number of sleeping hours during the trips was slightly higher than those on average working days, this difference did not affect NK activity or cell numbers in the city tourist trip.