The essential oils used are listed in Table 1 with their manufacturer and main constituents. Table 1 also indicates literature references that show clinical use related to inflammatory symptoms 111213. Essential oils were purchased from Hyperplants, Ltd. (Tokyo, Japan). The constitution of the geranium oil was determined by gas chromatography in this laboratory 14 using a GC apparatus (Model 353B, GL Sciences, Tokyo) equipped with a DB-5 column (0.5 mm × 30 m; J&W Scientific, Folsom, LA, USA), and was shown to contain about 24 % β-citronellol, 10 % citronellyl formate and 7 % geraniol and others.

For intraperitoneal injection, these essential oils were diluted to 2.5, 5 % solution by 2.5 % dimethyl sulfoxide (DMSO) in saline and 50 μl of Tween 20 was added to 4 ml of the essential oil solution. For cutaneous application, each essential oil was diluted to 5, 10, 20 and 50 % in DMSO.

Curdlan, a (1→3)-β-glucan preparation purified from the culture fluid of Alcaligenes faecalis, was purchased from Wako Pure Chemical Industries, Ltd.(Osaka, Japan), and suspended in 10 mg/ml in saline for intradermal injection and in 5 mg/ml for intraperitoneal injection. Hexadecyltrimethylammonium bromide (HTAB), human myeloperoxidase (MPO), and tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich Japan (Tokyo). Polyoxyethylene(20) sorbitan monolaurate (Tween 20) was from Wako Pure Chemical Industries, Ltd.. Prednisolone injection (10 mg/ml) was from Mitaka Pharmaceutical, Ltd. (Tokyo). Dulbecco's phosphate-buffered saline (PBS) was from Invitrogen Corp. (Carlsbad, CA, USA) and stored at 4°C. Diff-Quik was from International Reagents Corp. (Hyogo, Japan). Geraniol and linalool were from Wako Pure Chemical Industries, Ltd.. Terpinen-4-ol and β-citronellol were from Tokyo Kasei Kogyo Co., Ltd. (Tokyo). Hair remover, anchone^® was from Imju Co., Ltd. (Tokyo).

All animal experiments were performed according to the guidelines for the care and use of animals approved by Teikyo University. Six week-old female ICR mice (Charles River Japan, Inc., Kanagawa, Japan) were used for all animal experiments except the one using 6 week-old female HR-1 hairless mice (Hoshino Laboratory Animals, Saitama, Japan). The photoperiods were adjusted to 12 hr of light and 12 hr darkness daily, and the environmental temperature was constantly maintained at 21°C. The mice were kept in cages housing 4–6 animals and were given ad libitum access to food and water.

Fur in the dorsal region of mice (n = 5), approximately 20 × 50 mm square, was removed on day -3. The animals wore neck collars on day -2 to prevent their licking of the essential oils from the skin. On day 0, 200 μl of curdlan suspension (5 mg/ml) was injected intraperitoneally. A negative control group of mice was injected with 200 μl of saline instead of curdlan suspension. Immediately and 3 hr later, 100 μl of 20 % geranium oil in DMSO was dropped on the dorsal skin and gently spread over the fur-removed area using a glass spreader. To determine the number of leukocytes, mice were sacrificed with carbon dioxide 6 hr after curdlan injection. Three ml of PBS was then injected into their peritoneal cavity, and 2 ml of exudates were taken from the cavity to collect leukocytes. After centrifugation at 350 × g at 4°C for 5 min, the precipitate was suspended in 2 ml of PBS containing 10% heat-inactivated fetal calf serum (PBS solution). The numbers of leukocytes were measured by an electric cell counter; Celltac (Nihon Kohden Corporation, Tokyo). This leukocyte suspension was used for Diff-Quik staining and MPO assay as described below.

Neutrophils recovered from the peritoneal cavity were fixed on slide glass by cytocentrifugation and stained by Diff-Quik as described previously 15. Briefly, the leukocyte suspension was diluted to about 1 × 10⁶ cells/ml. Two hundred μl of the suspension was poured into a plastic tube attached to a slide glass and cytocentrifuged at 75 × g for 5 min, the slide glasses were then stained by Diff-Quik. Percentage ratio and the number of neutrophils were calculated by counting the neutrophil number of more than 50 leukocytes/sample.

The MPO assay was based on the method of De Young et al. 16 and partly modified. One ml of the leukocyte suspension was centrifuged at 620 × g at 4°C for 2 min. The precipitate was suspended in 1 ml of 80 mM sodium phosphate buffer, pH5.4, containing 0.5% HTAB (0.5% HTAB solution), freeze-thawed 3 times and centrifuged at 1400 × g at 4°C for 5 min. Triplicate 30 μl samples of resulting supernatant were poured into 96 well microtiter plates. For assay, 200 μl of a mixture containing 100 μl phosphate buffered saline, 85 μl 0.22 M sodium phosphate buffer, pH5.4, and 15 μl of 0.017 % hydrogen peroxide were added to the wells. The reaction was started by the addition of 20 μl of 18.4 mM TMB•2HCl in 8 % aqueous dimethylformamide. Plates were stirred and incubated at 37°C for 3 min and then placed on ice where the reaction was stopped by addition to each well of 30 μl of 1.46 M sodium acetate, pH3.0. The MPO value was evaluated by measuring the absorbance of samples at 620 nm (OD value) and being converted it into MPO values per mouse. The MPO activity was expressed by relative values calculated by the following formula: (MPO value recovered from oil-treated mice)/(MPO value recovered from control mice) × 100 (%)

Fur in the abdominal region of mice (n = 5–6) was removed on day -3. On day 0, 50 μl of curdlan suspension (10 mg/ml) in saline was injected intradermaly in the abdominal skin of mice. Immediately and 3 hr after curdlan injection, 200 μl of the diluted geranium oil solution was injected intraperitoneally. A dose of 2.5 % solution corresponded to 5 μl of pure oil. The control group of mice was received 200 μl of 2.5 % DMSO solution. One hundred μl/flank × 2 of prednisolone was injected subcutaneously to another active control group of mice 1 hr before curdlan injection, instead of essential oil. All mice were sacrificed with carbon dioxide 6 hr after curdlan injection. Skin was cut off in a 6 mm diameter area, weighed and placed in 1.05 ml of 0.5 % HTAB solution, and stored at -20°C until assay. The average weight of the skin was calculated as a parameter of edema.

Fur in the abdominal and dorsal regions of mice (n = 15 for 5–20 μl geranium oil in experiment shown in Fig 3(b), n = 3–6 for another experiments) except hairless mice (n = 4–5) was removed on day -3. The animals wore neck collars on day -2 to prevent their licking essential oils from the skin. On day 0, 50 μl of curdlan suspension (10 mg/ml) in saline was injected intradermaly to mice. Immediately and 3 hr later, 100 μl solution of a designated concentration of the essential oil or its components was dropped on the dorsal skin and gently spread over the fur-removed area using a glass spreader. A dose of 20 % solution corresponded to 20 μl of pure oil. The control group of mice was applied 100 μl of DMSO. Their skin preparations were obtained 6 hr after curdlan injection as described above.

Frozen samples were thawed at room temperature and homogenized for 45 sec at 0°C by Polytron (Kinematica AG, Lucerne, Switzerland). The homogenates were poured into sampling tubes and centrifuged at 12000 × g at 4°C for 15 min. The resulting supernatants were used for MPO assay as described above. The MPO value per each skin sample was calculated.

The results were expressed by the mean ± standard deviation. All statistical analysis was calculated using the StatView software. Statistical analysis was performed as follows; Students t-test for Fig 3(c), Dunnett after ANOVA for Fig 2(a), 3(a,b), 4 and 5, and Tukey-Kramer after ANOVA for Fig 1. Pearson's correlation coefficient was calculated for Fig 6.

Skin inflammatory response induced by intradermaly injected curdlan (0.5 mg/50 μl), was investigated first by using two parameters, the MPO value of skin homogenates and skin weight. The MPO value and skin weight of the skin lesion 6 hr after curdlan injection were 4.54 ± 2.43 units/skin lesion and 22.5 ± 8.3 mg (n = 49), which were significantly higher than those of saline injection, 0.21 ± 0.14 units/skin lesion and 8.9 ± 1.4 mg, respectively (n = 4). This indicates that curdlan injection caused neutrophil accumulation, which was monitored by increase in the MPO activity, and skin edema, which was observed by increase in skin weight.

We examined the neutrophil accumulation in the peritoneal cavity after curdlan injection microscopically and enzymatically using MPO activity. The effect of cutaneous application of geranium oil on these changes was observed.

Figure 1(a) shows the number of leukocytes and cell differentials in peritoneal exudates, which were determined using Diff-Quik staining. About 3.66 ± 0.49 × 10⁶ leukocytes were recovered from the peritoneal cavity of saline-induced mice, and the intraperitoneal injection of 200 μl curdlan solution increased this number to 9.48 ± 1.78 × 10⁶ cells. Neutrophils were rarely observed in peritoneal cells of saline-injected mice and the increased content of peritoneal leukocytes in curdlan-injected mice was mostly neutrophils. The MPO value of leukocyte preparation obtained 6 hr after curdlan intraperitoneal injection was 5.36 ± 1.86 units/mouse, which was significantly higher than that of the mice without curdlan (0.064 ± 0.026 units/mouse) (Fig. 1(b)).

Figure 1 also shows that compared to the curdlan control, cutaneous application of geranium oil to these mice significantly lowered the number of leukocytes and neutrophils (Fig. 1(a)) as well as the MPO value (Fig. 1(b)).

These results indicated that intraperitoneal injection of curdlan caused both the accumulation of neutrophils in the peritoneal cavity and increase of the MPO value, and that cutaneous application of geranium oil suppressed both of them. Therefore, we confirmed that the MPO activity corresponds to the number of neutrophils and that MPO activity can be used as a parameter for this number.

The effects of geranium oil administered intraperitoneally or cutaneously on the inflammation induced by curdlan intradermal injection were examined.

At first, geranium oil was injected intraperitoneally to mice. Prednisolone was used as an active control. As shown in Fig. 2(a), administration of 2 mg per mouse of prednisolone suppressed the MPO activity to 7 ± 3 %. Similarly but to a lesser degree, 5 and 10 μl of geranium oil significantly lowered the MPO activity to 30 ± 15 and 14 ± 10 %, respectively.

From the histological examination (Fig. 2(b)), it was observed that prednisolone clearly suppressed neutrophils accumulated following curdlan injection. Geranium oil also suppressed this accumulation, however, the suppression was not as strong as by prednisolone.

In the second experiment, we examined the effect of the cutaneous administration of geranium oil. As shown in Fig. 3(a), 20, 50 and 100 μl per mouse of geranium oil application lowered the MPO activity significantly (24 ± 14, 13 ± 1 and 19 ± 13 %, respectively). In this experiment, we observed that the mice receiving 50 and 100 μl of geranium oil exhibited an unusual behavior (sedated condition with loss of normal active movement) after the second administration. Therefore, the dose of geranium oil tested was reduced to 5, 10 and 20 μl (Fig. 3(b)) and they showed significant suppression of the MPO activity (63 ± 40, 56 ± 32, and 37 ± 23 % respectively). These data depicted in Fig 3(a) and 3(b) suggest that geranium oil suppress MPO activity in a dose-dependent manner.

In the third experiment, the similar effect of geranium oil on the hairless mice was examined. In this experiment, treatment by hair remover was omitted. Figure 3(c) shows that the MPO activity in hairless mice was significantly reduced by geranium application (32 ± 8 %) as in the case of fur-removed mice. This indicated that the geranium application suppressed the fur-removed skin and normal hairless skin similarly, and the effect of the remover was negligible.

We compared the effects of 10 μl of various essential oils (geranium, lavender, tea tree, eucalyptus, and juniper) against the MPO activity. Although all oils except juniper oil lowered the activity significantly (Fig. 4), the inhibitory activity of geranium oil was estimated to be strongest (34 ± 27 %). On the other hand, juniper oil did not significantly suppress the activity (66 ± 7 %).

We compared the activities of the main constituents of geranium, lavender and tea tree oils. As shown in Fig. 5, geraniol and terpinen-4-ol lowered the MPO activity (65 ± 23 and 68 ± 32 %, respectively), but not significantly, and linalool and β-citronellol did not lower the activity (126 ± 48 and 89 ± 37 %, respectively).

We measured the skin weight as a parameter of edema for each experiment. The correlation between skin weight and the MPO activity was examined for all control mice and all mice applied with 20 μl of geranium oil. As shown in Fig. 6, the skin weight of each mouse closely correlated with the MPO activity (r = 0.757, p < 0.0001).

The average skin weight and the MPO value were 22.5 ± 8.3 mg and 4.54 ± 2.43 units/skin lesion for control (n = 49), and 12.0 ± 3.7 mg and 1.16 ± 0.75 units/skin lesion for the geranium group (n = 26), respectively. This indicates that geranium oil suppressed both the neutrophil accumulation and edema induced by curdlan.