Inhaled essential oil from Chamaecyparis obtuse ameliorates the impairments of cognitive function induced by injection of β-amyloid in rats

Figures & data

Figure 1.  Chromatogram of EOCO. 1) α-thujene; 2) α-pinene; 3) camphene; 4) β-phellandrene; 5) β-myrcene; 6) α-terpinene; 7) ρ-cymene; 8) limonene; 9) γ-terpinene; 10) α-terpinolene; 11) α-terpineol; 12) bornyl ester; 13) 2-bornyl aetate; 14) α-terpinyl acetate; 15) thujopsene; 16) (+)-δ-cadinene; 17) (+)-epi-bicyclosesquiphellandrene; 18) (+)-ρ-himachalene; 19) (+)-α-longipinene; 20) elemol; 21) α-cedrol; 22) γ-eudesmol; 23) β-eudesmol; 24) beyerene. (Compounds occupying more than 0.4% peak area are shown.)

Table 1.  Chemical composition of the essential oil extracted from Chamaecyparis obtusa.

Terpenes	Compounds	RT, min	Peak area, %
Monoterpenes	α-thujene	11.039	1.07
	α-pinene	11.386	2.18
	camphane	12.074	0.41
	β-phellandrene	12.859	13.11
	β-pinene	13.172	0.31
	β-myrcene	13.288	5.68
	α-phellandren	14.203	0.18
	α-terpinene	14.619	1.80
	ρ-cymene	14.92	1.21
	limonene	15.132	6.49
	γ-terpinene	16.265	4.28
	4-thujanol	16.796	0.05
	α-terpinolene	17.409	1.37
	camphor	20.11	0.10
	borneol	21.036	0.31
	α-terpineol	21.298	4.33
	lynalyl acetate	23.56	0.22
	bornylacetate	25.174	7.48
	isobornylaetate	25.311	0.57
	α-terpinyl acetate	27.310	16.82
Sesquiterpenes	(+)-epi-bicyclosesquiphellandrene	31.578	2.04
	β-elemene	28.970	0.06
	β-cedrene	30.493	0.15
	thujopsene	30.815	4.50
	(+)-δ-cadinene	30.970	1.67
	γ-muurolen	31.434	0.23
	(-)-germacrene	32.208	0.20
	longifolene	32.362	0.22
	(+)-ρ-himachalene	32.99	0.80
	(-)-calamenene	33.391	0.25
	(+)-α-longipinene	33.903	0.88
	elemol	36.417	6.22
	α-cedrol	37.512	1.72
	γ-eudesmol	36.864	2.23
	(-)-guaiol	36.992	0.11
	α-cadinol	34.163	0.27
	β-eudesmol	37.638	4.13
	α-bisabolol	38.210	0.11
	iso-longifolol	39.252	0.07
	δ-guaiene	39.516	0.11
Diterpenes	rimuene	44.521	0.29
	beyerene	45.691	3.35
	kaur-15-ene	45.859	0.09
	β-selinene	47.333	0.23
Sum of components occupying less than 0.05%			2.10
Total			100

Figure 2.  Effects of EOCO on Aβ_1–40-induced impairments of memory acquisition and retention in the Morris water maze test. Before receiving intrahippocampally injection of either ACSF or Aβ_1–40 (4 μL), the rats inhaled EOCO (1 mL/cage) in a VSC for 30 days. Donepezil (1 mg/kg b.w./day) served as a positive control. The rats were given four trials each day for 4 consecutive days. (A) The effects of EOCO on the memory of rats impaired by injection of Aβ_1–40 in the fourth day of the Morris water maze test. (B) The effects of EOCO on the memory of rats impaired by injection of Aβ_1–40 in the first trial and fourth trials of the Morris water maze test over 4 days. The values shown are the mean latency ± S.D (n = 7); *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the Aβ_1–40-treated group. Statistical significance was tested with the unpaired Student’s t-test.

Table 2.  Effects of EOCO on Aβ_1–40-induced impairment of memory acquisition and memory retention in the passive avoidance test.

Group	Control	Aβ1–40	Donepezil	EOCO
Retention latencies (s)	268.69 ± 10.97***	114.32 ± 21.28	200.34 ± 23.01*	192.89 ± 24.41*

Data represent the mean ± S.E (n = 7).Values with different letters in a column are significantly different by ANOVA test (p < 0.05). *p < 0.05, significantly different from Aβ_1-40 group, **p < 0.01,***p < 0.001.

Figure 3.  Effects of EOCO on Aβ_1–40-induced impairments of memory acquisition and retention in probe trials. (A) Effects of EOCO on probe trial sessions of the Morris water maze test. The time in the figure represents the cumulative time in the target quadrant of the pool in the 90 s probe trial. (B) Swimming pattern on probe trial sessions of the Morris water maze test. The values shown are the mean latency ± S.D (n = 7); *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from Aβ_1–40-treated group. Statistical significance was tested with the unpaired Student’s t-test.

Figure 4.  Effects of EOCO on Aβ_1–40-induced behavioral deficits. (A) Effects of EOCO on Aβ_1–40-induced sensorimotor deficits in the vertical pole test. (B) Effects of EOCO on Aβ_1–40-induced motor coordination and balance deficits in the rotarod test. The values shown are the mean latency ± S.E. (n = 7); *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from Aβ_1–40-treated group. Statistical significance was tested with the unpaired Student’s t-test.

Figure 5.  Effects of EOCO on AchE activity (A) and ACh content (B) in Aβ_1–40-injured rats. Results are expressed as the means ± S.D. (n = 7); *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from only Aβ_1–40-treated group. Statistical significance was tested with the unpaired Student’s t-test.

Figure 6.  Effects of EOCO on Aβ_1–40-induced neuronal degeneration and DNA strand breakage. (A) Effects of EOCO on Aβ_1–40-induced neuronal degeneration assessed by TTC staining. Representative brain sections (2 mm thick) from EOCO-treated rats stained with 2% TTC showing neuronal degeneration. Red colored regions in the TTC-stained sections indicate nondegenerated regions and pale colored regions indicate degeneration. (B) Effects of EOCO on Aβ_1–40-induced neuronal apoptosis assessed by TUNEL assay. Detection of hippocampal apoptotic cells was carried out using the TUNEL method. TUNEL-positive pyramidal neurons undergoing apoptosis were observed in the hippocampus (fluorescence). (C) Representative quantification of the number of TUNEL-positive cells. Results are expressed as the means ± S.E. (n = 7); *p < 0.05, **p < 0.01,***p < 0.001, significantly different from Aβ_1–40-treated group. Statistical significance was tested with the unpaired Student’s t-test.