药学学报

t_R/min	Name	Chemical formula	Relative content/%
6.77	2-Methyl-1-octen-3-yne	C₉H₁₄	0.39%
7.31	Camphene	C₁₀H₁₆	0.39%
8.41	(1S)-(-)-beta-Pinene	C₁₀H₁₆	0.29%
9.05	beta-Pinene	C₁₀H₁₆	0.35%
10.67	Isopulegol	C₁₀H₁₈O	1.14%
11.13	Menthol	C₁₀H₁₈O	0.18%
14.18	Linalool	C₁₀H₂₀O	4.78%
14.69	1-Octen-3-yl acetate	C₁₀H₁₈O₂	0.67%
16.59	L-Menthone	C₁₀H₁₈O	5.39%
17.01	Isomenthone	C₁₀H₁₈O	1.36%
17.92	DL-Menthol	C₁₀H₁₈O	14.89%
18.44	alpha-Terpineol	C₁₀H₁₈O	0.92%
20.40	Dorzolamide	C₁₀H₁₆O	1.02%
21.16	Menthone	C₁₀H₁₆O	0.30%
21.36	Citronellol	C₁₂H₂₀O	1.09%
22.10	Cinnamaldehyde	C₉H₈O	7.92%
22.60	beta-Ocimene	C₁₀H₁₆O	0.44%
23.03	Geranyl acetate	C₁₀H₂₀O₂	4.34%
26.10	Neryl acetate	C₁₀H₂₀O₂	0.08%
26.50	(+)-α-Pinene	C₁₅H₂₄	0.45%
27.00	Bornyl acetate	C₁₀H₂₀O₂	0.27%
28.30	Methyleugenol	C₁₁H₁₄O₂	0.76%
29.41	Cinnamyl acetate	C₁₁H₁₂O₂	0.42%
30.03	cis-Methylisoeugenol	C₁₁H₁₄O₂	0.12%
30.85	Caryophyllene	C₁₅H₂₄	2.05%
31.60	Methyl isoeugenol	C₁₁H₁₄O₂	0.29%
32.20	Shyobunone	C₁₅H₂₄O	0.36%
32.60	delta-Cadinene	C₁₅H₂₄O	0.19%
32.87	2'-Methoxycinnamaldehyde	C₁₀H₁₀O₂	0.45%
34.60	γ-Asarone	C₁₂H₁₆O₃	1.11%
34.81	(+)-α-Funebrene	C₁₅H₂₆	0.79%
36.90	beta-Asarone	C₁₂H₁₆O₃	33.47%
38.70	α-Asarone	C₁₂H₁₆O₃	4.27%
39.19	Aristolone	C₁₅H₂₂O	0.49%
40.80	Isocalamendiol	C₁₅H₂₆O₂	0.59%

t_R/min

Name

Chemical formula

Relative content/%

6.77

2-Methyl-1-octen-3-yne

C₉H₁₄

0.39%

7.31

Camphene

C₁₀H₁₆

0.39%

8.41

(1S)-(-)-beta-Pinene

C₁₀H₁₆

0.29%

9.05

beta-Pinene

C₁₀H₁₆

0.35%

10.67

Isopulegol

C₁₀H₁₈O

1.14%

11.13

Menthol

C₁₀H₁₈O

0.18%

14.18

Linalool

C₁₀H₂₀O

4.78%

14.69

1-Octen-3-yl acetate

C₁₀H₁₈O₂

0.67%

16.59

L-Menthone

C₁₀H₁₈O

5.39%

17.01

Isomenthone

C₁₀H₁₈O

1.36%

17.92

DL-Menthol

C₁₀H₁₈O

14.89%

18.44

alpha-Terpineol

C₁₀H₁₈O

0.92%

20.40

Dorzolamide

C₁₀H₁₆O

1.02%

21.16

Menthone

C₁₀H₁₆O

0.30%

21.36

Citronellol

C₁₂H₂₀O

1.09%

22.10

Cinnamaldehyde

C₉H₈O

7.92%

22.60

beta-Ocimene

C₁₀H₁₆O

0.44%

23.03

Geranyl acetate

C₁₀H₂₀O₂

4.34%

26.10

Neryl acetate

C₁₀H₂₀O₂

0.08%

26.50

(+)-α-Pinene

C₁₅H₂₄

0.45%

27.00

Bornyl acetate

C₁₀H₂₀O₂

0.27%

28.30

Methyleugenol

C₁₁H₁₄O₂

0.76%

29.41

Cinnamyl acetate

C₁₁H₁₂O₂

0.42%

30.03

cis-Methylisoeugenol

C₁₁H₁₄O₂

0.12%

30.85

Caryophyllene

C₁₅H₂₄

2.05%

31.60

Methyl isoeugenol

C₁₁H₁₄O₂

0.29%

32.20

Shyobunone

C₁₅H₂₄O

0.36%

32.60

delta-Cadinene

C₁₅H₂₄O

0.19%

32.87

2'-Methoxycinnamaldehyde

C₁₀H₁₀O₂

0.45%

34.60

γ-Asarone

C₁₂H₁₆O₃

1.11%

34.81

(+)-α-Funebrene

C₁₅H₂₆

0.79%

36.90

beta-Asarone

C₁₂H₁₆O₃

33.47%

38.70

α-Asarone

C₁₂H₁₆O₃

4.27%

39.19

Aristolone

C₁₅H₂₂O

0.49%

40.80

Isocalamendiol

C₁₅H₂₆O₂

0.59%

t_R/min	Name	Chemical formula	Relative content/%
6.77	2-Methyl-1-octen-3-yne	C₉H₁₄	0.39%
7.31	Camphene	C₁₀H₁₆	0.39%
8.41	(1S)-(-)-beta-Pinene	C₁₀H₁₆	0.29%
9.05	beta-Pinene	C₁₀H₁₆	0.35%
10.67	Isopulegol	C₁₀H₁₈O	1.14%
11.13	Menthol	C₁₀H₁₈O	0.18%
14.18	Linalool	C₁₀H₂₀O	4.78%
14.69	1-Octen-3-yl acetate	C₁₀H₁₈O₂	0.67%
16.59	L-Menthone	C₁₀H₁₈O	5.39%
17.01	Isomenthone	C₁₀H₁₈O	1.36%
17.92	DL-Menthol	C₁₀H₁₈O	14.89%
18.44	alpha-Terpineol	C₁₀H₁₈O	0.92%
20.40	Dorzolamide	C₁₀H₁₆O	1.02%
21.16	Menthone	C₁₀H₁₆O	0.30%
21.36	Citronellol	C₁₂H₂₀O	1.09%
22.10	Cinnamaldehyde	C₉H₈O	7.92%
22.60	beta-Ocimene	C₁₀H₁₆O	0.44%
23.03	Geranyl acetate	C₁₀H₂₀O₂	4.34%
26.10	Neryl acetate	C₁₀H₂₀O₂	0.08%
26.50	(+)-α-Pinene	C₁₅H₂₄	0.45%
27.00	Bornyl acetate	C₁₀H₂₀O₂	0.27%
28.30	Methyleugenol	C₁₁H₁₄O₂	0.76%
29.41	Cinnamyl acetate	C₁₁H₁₂O₂	0.42%
30.03	cis-Methylisoeugenol	C₁₁H₁₄O₂	0.12%
30.85	Caryophyllene	C₁₅H₂₄	2.05%
31.60	Methyl isoeugenol	C₁₁H₁₄O₂	0.29%
32.20	Shyobunone	C₁₅H₂₄O	0.36%
32.60	delta-Cadinene	C₁₅H₂₄O	0.19%
32.87	2'-Methoxycinnamaldehyde	C₁₀H₁₀O₂	0.45%
34.60	γ-Asarone	C₁₂H₁₆O₃	1.11%
34.81	(+)-α-Funebrene	C₁₅H₂₆	0.79%
36.90	beta-Asarone	C₁₂H₁₆O₃	33.47%
38.70	α-Asarone	C₁₂H₁₆O₃	4.27%
39.19	Aristolone	C₁₅H₂₂O	0.49%
40.80	Isocalamendiol	C₁₅H₂₆O₂	0.59%

t_R/min

Name

Chemical formula

Relative content/%

6.77

2-Methyl-1-octen-3-yne

C₉H₁₄

0.39%

7.31

Camphene

C₁₀H₁₆

0.39%

8.41

(1S)-(-)-beta-Pinene

C₁₀H₁₆

0.29%

9.05

beta-Pinene

C₁₀H₁₆

0.35%

10.67

Isopulegol

C₁₀H₁₈O

1.14%

11.13

Menthol

C₁₀H₁₈O

0.18%

14.18

Linalool

C₁₀H₂₀O

4.78%

14.69

1-Octen-3-yl acetate

C₁₀H₁₈O₂

0.67%

16.59

L-Menthone

C₁₀H₁₈O

5.39%

17.01

Isomenthone

C₁₀H₁₈O

1.36%

17.92

DL-Menthol

C₁₀H₁₈O

14.89%

18.44

alpha-Terpineol

C₁₀H₁₈O

0.92%

20.40

Dorzolamide

C₁₀H₁₆O

1.02%

21.16

Menthone

C₁₀H₁₆O

0.30%

21.36

Citronellol

C₁₂H₂₀O

1.09%

22.10

Cinnamaldehyde

C₉H₈O

7.92%

22.60

beta-Ocimene

C₁₀H₁₆O

0.44%

23.03

Geranyl acetate

C₁₀H₂₀O₂

4.34%

26.10

Neryl acetate

C₁₀H₂₀O₂

0.08%

26.50

(+)-α-Pinene

C₁₅H₂₄

0.45%

27.00

Bornyl acetate

C₁₀H₂₀O₂

0.27%

28.30

Methyleugenol

C₁₁H₁₄O₂

0.76%

29.41

Cinnamyl acetate

C₁₁H₁₂O₂

0.42%

30.03

cis-Methylisoeugenol

C₁₁H₁₄O₂

0.12%

30.85

Caryophyllene

C₁₅H₂₄

2.05%

31.60

Methyl isoeugenol

C₁₁H₁₄O₂

0.29%

32.20

Shyobunone

C₁₅H₂₄O

0.36%

32.60

delta-Cadinene

C₁₅H₂₄O

0.19%

32.87

2'-Methoxycinnamaldehyde

C₁₀H₁₀O₂

0.45%

34.60

γ-Asarone

C₁₂H₁₆O₃

1.11%

34.81

(+)-α-Funebrene

C₁₅H₂₆

0.79%

36.90

beta-Asarone

C₁₂H₁₆O₃

33.47%

38.70

α-Asarone

C₁₂H₁₆O₃

4.27%

39.19

Aristolone

C₁₅H₂₂O

0.49%

40.80

Isocalamendiol

C₁₅H₂₆O₂

0.59%

Group	Total distance of movement/mm	Dwell time in central compartment/s	Number of passes through the central compartment/times
Control (n = 8)	2 698.27 ± 1 036.44	7.28 ± 7.39	5.00 ± 3.31
Model (n = 7)	619.12 ± 284.90^##	1.56 ± 1.75^##	1.80 ± 2.17^#
LDG (n = 7)	3 038.95 ± 1 034.58^***	2.18 ± 4.78	1.80 ± 2.39
MDG (n = 7)	3 503.57 ± 533.88^**	3.15 ± 2.75	3.40 ± 1.14
HDG (n = 7)	3 149.21 ± 866.88^***	4.63 ± 4.53	3.20 ± 3.11
Diazepam (n = 8)	3 740.52 ± 697.54^**	5.87 ± 3.75	3.80 ± 2.17

Group

Total distance of movement/mm

Dwell time in central compartment/s

Number of passes through the central compartment/times

Control (n = 8)

2 698.27 ± 1 036.44

7.28 ± 7.39

5.00 ± 3.31

Model (n = 7)

619.12 ± 284.90^##

1.56 ± 1.75^##

1.80 ± 2.17^#

LDG (n = 7)

3 038.95 ± 1 034.58^***

2.18 ± 4.78

1.80 ± 2.39

MDG (n = 7)

3 503.57 ± 533.88^**

3.15 ± 2.75

3.40 ± 1.14

HDG (n = 7)

3 149.21 ± 866.88^***

4.63 ± 4.53

3.20 ± 3.11

Diazepam (n = 8)

3 740.52 ± 697.54^**

5.87 ± 3.75

3.80 ± 2.17

Group	Total distance of movement/mm	Dwell time in central compartment/s	Number of passes through the central compartment/times
Control (n = 8)	2 698.27 ± 1 036.44	7.28 ± 7.39	5.00 ± 3.31
Model (n = 7)	619.12 ± 284.90^##	1.56 ± 1.75^##	1.80 ± 2.17^#
LDG (n = 7)	3 038.95 ± 1 034.58^***	2.18 ± 4.78	1.80 ± 2.39
MDG (n = 7)	3 503.57 ± 533.88^**	3.15 ± 2.75	3.40 ± 1.14
HDG (n = 7)	3 149.21 ± 866.88^***	4.63 ± 4.53	3.20 ± 3.11
Diazepam (n = 8)	3 740.52 ± 697.54^**	5.87 ± 3.75	3.80 ± 2.17

Group

Total distance of movement/mm

Dwell time in central compartment/s

Number of passes through the central compartment/times

Control (n = 8)

2 698.27 ± 1 036.44

7.28 ± 7.39

5.00 ± 3.31

Model (n = 7)

619.12 ± 284.90^##

1.56 ± 1.75^##

1.80 ± 2.17^#

LDG (n = 7)

3 038.95 ± 1 034.58^***

2.18 ± 4.78

1.80 ± 2.39

MDG (n = 7)

3 503.57 ± 533.88^**

3.15 ± 2.75

3.40 ± 1.14

HDG (n = 7)

3 149.21 ± 866.88^***

4.63 ± 4.53

3.20 ± 3.11

Diazepam (n = 8)

3 740.52 ± 697.54^**

5.87 ± 3.75

3.80 ± 2.17

Group	Open arm dwell time ratio (toc = to/to+tc)	Open arm entry ratio (Noc = Noe/Noe+Nce)	Total number of arm feeds /times
Control (n = 8)	0.33 ± 0.3	0.33 ± 0.3	22.2 ± 7.9
Model (n = 7)	0.12 ± 0.1^#	0.16 ± 0.2^#	9.2 ± 3.7^#
LDG (n = 7)	0.21 ± 0.1	0.18 ± 0.1	13.5 ± 1.3^*
MDG (n = 7)	0.39 ± 0.1^*	0.24 ± 0.2^*	14.5 ± 3.4^*
HDG (n = 7)	0.41 ± 0.4^*	0.30 ± 0.3^*	14.0 ± 3.0
Diazepam (n = 8)	0.46 ± 0.2^*	0.34 ± 0.2^*	16.3 ± 2.5^*

Group

Open arm dwell time ratio (toc = to/to+tc)

Open arm entry ratio (Noc = Noe/Noe+Nce)

Total number of arm feeds /times

Control (n = 8)

0.33 ± 0.3

22.2 ± 7.9

Model (n = 7)

0.12 ± 0.1^#

0.16 ± 0.2^#

9.2 ± 3.7^#

LDG (n = 7)

0.21 ± 0.1

0.18 ± 0.1

13.5 ± 1.3^*

MDG (n = 7)

0.39 ± 0.1^*

0.24 ± 0.2^*

14.5 ± 3.4^*

HDG (n = 7)

0.41 ± 0.4^*

0.30 ± 0.3^*

14.0 ± 3.0

Diazepam (n = 8)

0.46 ± 0.2^*

0.34 ± 0.2^*

16.3 ± 2.5^*

Group	Open arm dwell time ratio (toc = to/to+tc)	Open arm entry ratio (Noc = Noe/Noe+Nce)	Total number of arm feeds /times
Control (n = 8)	0.33 ± 0.3	0.33 ± 0.3	22.2 ± 7.9
Model (n = 7)	0.12 ± 0.1^#	0.16 ± 0.2^#	9.2 ± 3.7^#
LDG (n = 7)	0.21 ± 0.1	0.18 ± 0.1	13.5 ± 1.3^*
MDG (n = 7)	0.39 ± 0.1^*	0.24 ± 0.2^*	14.5 ± 3.4^*
HDG (n = 7)	0.41 ± 0.4^*	0.30 ± 0.3^*	14.0 ± 3.0
Diazepam (n = 8)	0.46 ± 0.2^*	0.34 ± 0.2^*	16.3 ± 2.5^*

Group

Open arm dwell time ratio (toc = to/to+tc)

Open arm entry ratio (Noc = Noe/Noe+Nce)

Total number of arm feeds /times

Control (n = 8)

0.33 ± 0.3

22.2 ± 7.9

Model (n = 7)

0.12 ± 0.1^#

0.16 ± 0.2^#

9.2 ± 3.7^#

LDG (n = 7)

0.21 ± 0.1

0.18 ± 0.1

13.5 ± 1.3^*

MDG (n = 7)

0.39 ± 0.1^*

0.24 ± 0.2^*

14.5 ± 3.4^*

HDG (n = 7)

0.41 ± 0.4^*

0.30 ± 0.3^*

14.0 ± 3.0

Diazepam (n = 8)

0.46 ± 0.2^*

0.34 ± 0.2^*

16.3 ± 2.5^*

Group	CRH	ACTH	MT
Control (n = 8)	50.1 ± 2.5	40.7 ± 11.3	55.8 ± 2.2
Model (n = 7)	61.6 ± 3.3^#	49.5 ± 5.0^##	48.0 ± 2.8^##
LDG (n = 7)	51.7 ± 4.7^*	45.3 ± 4.9	58.6 ± 11.8^*
MDG (n = 7)	51.5 ± 10.9^*	42.9 ± 3.2^*	58.5 ± 10.2^*
HDG (n = 7)	53.6 ± 1.9	46.7 ± 5.9	60.4 ± 7.0^*
Diazepam (n = 8)	51.7 ± 8.8^*	48.2 ± 4.7	60.7 ± 13.2^*

Group

CRH

ACTH

Control (n = 8)

50.1 ± 2.5

40.7 ± 11.3

55.8 ± 2.2

Model (n = 7)

61.6 ± 3.3^#

49.5 ± 5.0^##

48.0 ± 2.8^##

LDG (n = 7)

51.7 ± 4.7^*

45.3 ± 4.9

58.6 ± 11.8^*

MDG (n = 7)

51.5 ± 10.9^*

42.9 ± 3.2^*

58.5 ± 10.2^*

HDG (n = 7)

53.6 ± 1.9

46.7 ± 5.9

60.4 ± 7.0^*

Diazepam (n = 8)

51.7 ± 8.8^*

48.2 ± 4.7

60.7 ± 13.2^*

Group	CRH	ACTH	MT
Control (n = 8)	50.1 ± 2.5	40.7 ± 11.3	55.8 ± 2.2
Model (n = 7)	61.6 ± 3.3^#	49.5 ± 5.0^##	48.0 ± 2.8^##
LDG (n = 7)	51.7 ± 4.7^*	45.3 ± 4.9	58.6 ± 11.8^*
MDG (n = 7)	51.5 ± 10.9^*	42.9 ± 3.2^*	58.5 ± 10.2^*
HDG (n = 7)	53.6 ± 1.9	46.7 ± 5.9	60.4 ± 7.0^*
Diazepam (n = 8)	51.7 ± 8.8^*	48.2 ± 4.7	60.7 ± 13.2^*

Group

CRH

ACTH

Control (n = 8)

50.1 ± 2.5

40.7 ± 11.3

55.8 ± 2.2

Model (n = 7)

61.6 ± 3.3^#

49.5 ± 5.0^##

48.0 ± 2.8^##

LDG (n = 7)

51.7 ± 4.7^*

45.3 ± 4.9

58.6 ± 11.8^*

MDG (n = 7)

51.5 ± 10.9^*

42.9 ± 3.2^*

58.5 ± 10.2^*

HDG (n = 7)

53.6 ± 1.9

46.7 ± 5.9

60.4 ± 7.0^*

Diazepam (n = 8)

51.7 ± 8.8^*

48.2 ± 4.7

60.7 ± 13.2^*

No.	Metabolite	Chemical shift/ppm
1	LDL/VLDL	0.88 (m)
2	Isoleucine	0.95 (t), 1.32 (s), 1.33 (s)
3	3-HB	1.19 (d), 2.30 (dd)
4	N-Acetylglucosamine	1.91 (s)
5	O-Acetylglucosamine	2.07 (s)
6	Glutamic acid	2.35 (m), 2.45 (m)
7	Choline	3.04 (s)
8	Trimetlylamine oxide	3.34 (s)
9	β-Glucose	3.52 (dd), 3.43 (m), 4.64 (d)
10	Allantoic acid	5.20 (d)

No.

Metabolite

Chemical shift/ppm

LDL/VLDL

0.88 (m)

Isoleucine

0.95 (t), 1.32 (s), 1.33 (s)

3-HB

1.19 (d), 2.30 (dd)

N-Acetylglucosamine

1.91 (s)

O-Acetylglucosamine

2.07 (s)

Glutamic acid

2.35 (m), 2.45 (m)

Choline

3.04 (s)

Trimetlylamine oxide

3.34 (s)

β-Glucose

3.52 (dd), 3.43 (m), 4.64 (d)

Allantoic acid

5.20 (d)

No.	Metabolite	Chemical shift/ppm
1	LDL/VLDL	0.88 (m)
2	Isoleucine	0.95 (t), 1.32 (s), 1.33 (s)
3	3-HB	1.19 (d), 2.30 (dd)
4	N-Acetylglucosamine	1.91 (s)
5	O-Acetylglucosamine	2.07 (s)
6	Glutamic acid	2.35 (m), 2.45 (m)
7	Choline	3.04 (s)
8	Trimetlylamine oxide	3.34 (s)
9	β-Glucose	3.52 (dd), 3.43 (m), 4.64 (d)
10	Allantoic acid	5.20 (d)

No.

Metabolite

Chemical shift/ppm

LDL/VLDL

0.88 (m)

Isoleucine

0.95 (t), 1.32 (s), 1.33 (s)

3-HB

1.19 (d), 2.30 (dd)

N-Acetylglucosamine

1.91 (s)

O-Acetylglucosamine

2.07 (s)

Glutamic acid

2.35 (m), 2.45 (m)

Choline

3.04 (s)

Trimetlylamine oxide

3.34 (s)

β-Glucose

3.52 (dd), 3.43 (m), 4.64 (d)

Allantoic acid

5.20 (d)

No.	Metabolite	Chemical shift/ppm
1	3-HB	1.32 (d), 2.66 (dd)
2	Alanine	1.47 (d)
3	GABA	1.88 (m), 2.29 (t), 3.00 (t)
4	N-Acetylaspartylglutamic acid	1.91 (s)
5	Glutamic acid	2.01 (m), 2.05(m), 2.35 (m)
6	Creatine/creatine phosphate	3.03 (s), 3.93 (s)
7	L-Glycerophosphorylcholine	3.20 (s)
8	Taurine	3.26 (t), 3.42 (t)
9	Succinate acid	3.35 (s)
10	β-Glucose	3.53 (dd), 3.75 (m)
11	Glycine	3.55 (s)
12	Tryptophan	7.65 (s)

No.

Metabolite

Chemical shift/ppm

3-HB

1.32 (d), 2.66 (dd)

Alanine

1.47 (d)

GABA

1.88 (m), 2.29 (t), 3.00 (t)

N-Acetylaspartylglutamic acid

1.91 (s)

Glutamic acid

2.01 (m), 2.05(m), 2.35 (m)

Creatine/creatine phosphate

3.03 (s), 3.93 (s)

L-Glycerophosphorylcholine

3.20 (s)

Taurine

3.26 (t), 3.42 (t)

Succinate acid

3.35 (s)

β-Glucose

3.53 (dd), 3.75 (m)

Glycine

3.55 (s)

Tryptophan

7.65 (s)

No.	Metabolite	Chemical shift/ppm
1	3-HB	1.32 (d), 2.66 (dd)
2	Alanine	1.47 (d)
3	GABA	1.88 (m), 2.29 (t), 3.00 (t)
4	N-Acetylaspartylglutamic acid	1.91 (s)
5	Glutamic acid	2.01 (m), 2.05(m), 2.35 (m)
6	Creatine/creatine phosphate	3.03 (s), 3.93 (s)
7	L-Glycerophosphorylcholine	3.20 (s)
8	Taurine	3.26 (t), 3.42 (t)
9	Succinate acid	3.35 (s)
10	β-Glucose	3.53 (dd), 3.75 (m)
11	Glycine	3.55 (s)
12	Tryptophan	7.65 (s)

No.

Metabolite

Chemical shift/ppm

3-HB

1.32 (d), 2.66 (dd)

Alanine

1.47 (d)

GABA

1.88 (m), 2.29 (t), 3.00 (t)

N-Acetylaspartylglutamic acid

1.91 (s)

Glutamic acid

2.01 (m), 2.05(m), 2.35 (m)

Creatine/creatine phosphate

3.03 (s), 3.93 (s)

L-Glycerophosphorylcholine

3.20 (s)

Taurine

3.26 (t), 3.42 (t)

Succinate acid

3.35 (s)

β-Glucose

3.53 (dd), 3.75 (m)

Glycine

3.55 (s)

Tryptophan

7.65 (s)

基于¹H NMR代谢组学与网络药理学的复方宁神精油抗失眠组分物质基础及作用机制研究

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柴青 ¹ , 张宏斌 ² , 吴立东 ³ , 王婧毅 ² , 李海超 ² , 刘玉红 ¹^,⁵^,⁶ , 刘红燕 ²^,⁵^,⁶ , 蒋海强 ²^,⁵^,⁶^,^* , 田振华 ⁴^,⁵^,⁶^,^*

药学学报 | 研究论文 2024,59(8): 2313-2325

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药学学报 | 研究论文 2024, 59(8): 2313-2325

基于¹H NMR代谢组学与网络药理学的复方宁神精油抗失眠组分物质基础及作用机制研究

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柴青¹, 张宏斌², 吴立东³, 王婧毅², 李海超², 刘玉红¹^,⁵^,⁶, 刘红燕²^,⁵^,⁶, 蒋海强²^,⁵^,⁶^,^*, 田振华⁴^,⁵^,⁶^,^*

作者信息

1.山东中医药大学药学院, 山东济南 250355

2.山东中医药大学中医药创新研究院, 山东济南 250355

3.山东欣悦健康科技有限公司, 山东滨州 256600

4.山东中医药大学实验中心, 山东济南 250355

5.山东省中医药基础研究重点实验室, 山东济南 250355

6.山东中医药大学, 教育部中医药经典理论重点实验室, 山东济南 250355

通讯作者:

^*蒋海强, Tel: 86-531-89628014, E-mail: jhq12723@163.com;

田振华, E-mail: tianzhenhuatina@163.com

Study on the material basis and mechanism of anti-insomnia mechanism of Ning Shen Essential Oil based on ¹H NMR metabolomics and network pharmacology

Qing CHAI¹, Hong-bin ZHANG², Li-dong WU³, Jing-yi WANG², Hai-chao LI², Yu-hong LIU¹^,⁵^,⁶, Hong-yan LIU²^,⁵^,⁶, Hai-qiang JIANG²^,⁵^,⁶^,^*, Zhen-hua TIAN⁴^,⁵^,⁶^,^*

Affiliations

1. School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

2. Institute of Traditional Chinese Medicine Innovation, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

3. Shandong Xinyue Health Science and Technology Co., Ltd, Binzhou 256600, China

4. Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

5. Shandong Provincial Key Laboratory of Basic Research of Traditional Chinese Medicine, Jinan 250355, China

6. Key Laboratory of Classical Theory of Chinese Medicine, Ministry of Education, Shandong University of Traditional Chinese Medicine, Jinan 250355, China

出版时间: 2024-08-12 doi: 10.16438/j.0513-4870.2023-1436

文章导航

摘要

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本文应用GC-MS、网络药理学与¹H NMR代谢组学技术相结合, 研究复方宁神精油抗失眠的物质基础与作用机制。通过GC-MS分析复方宁神精油的主要挥发性成分, 利用中药系统药理学数据库与分析平台(TCMSP) 及GeneCards、OMIM、Drugbank等数据库预测失眠相关靶点。腹腔注射对氯苯丙氨酸(PCPA) 复制大鼠失眠模型。动物实验经山东中医药大学动物伦理委员会批准(伦理号: SDUTCM20221025010)。以行为学相关指标评价复方宁神精油对大鼠焦虑行为的调节作用; 采用酶联免疫法检测大鼠血清中促肾上腺激素释放激素(CRH)、促肾上腺激素(ACTH)、褪黑素(MT) 含量; 取大鼠血清、海马进行¹H NMR代谢组学检测大鼠血清海马中内源性代谢物变化, 指认差异代谢物并构建代谢通路。结果显示, 模型组大鼠旷场实验运动距离、高架十字迷宫实验进入开放臂次数及时间均显著降低(P < 0.05, P < 0.01); 复方宁神精油干预后, 大鼠行为学指标均不同程度改善。模型组大鼠血清CRH、ACTH含量均显著升高(P < 0.05, P < 0.01), MT含量显著降低(P < 0.01); 干预后, 血清CRH、ACTH含量均不同程度降低, 且能回调MT含量。¹H NMR代谢组学共筛选10种与失眠相关的潜在生物标志物, 涉及到6条潜在的代谢通路。通过GC-MS共检测出复方宁神精油35个成分, 复方宁神精油主要成分靶点与失眠疾病靶点取交集, 共筛选出172个交集基因, 核心靶点26个。研究表明复方宁神精油干预PCPA所致失眠大鼠的作用机制可能与调节下丘脑-垂体-肾上腺轴轴相关激素, 调控机体氨基酸代谢、脂质代谢和胆碱代谢有关。

关键词

复方宁神精油 / 失眠 / 旷场实验 / 高架十字迷宫 / 核磁共振 / 代谢组学 / 网络药理学

Abstract

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This paper applied gas chromatography-mass spectrometry (GC-MS), network pharmacology and nuclear magnetic resonance hydrogen spectroscopy (¹H NMR) metabolomics techniques to study the material basis and mechanism of action of Ning Shen Essential Oil in anti-insomnia. The main volatile components of Ning Shen Essential Oil were analyzed by gas chromatography-mass spectrometry (GC-MS), and the insomnia-related targets were predicted using the Traditional Chinese Medicine Systematic Pharmacology Database and Analytical Platform (TCMSP) and the databases of GeneCards, OMIM and Drugbank. The insomnia model of rats was replicated by intraperitoneal injection of 4-chloro-DL-phenylalanine (PCPA). Animal experiments were approved by the Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (Ethics No.: SDUTCM20221025010). The modulating effect of Compound Ning Shen Essential Oil on anxiety behavior of rats was evaluated by behavioral related indexes. The serum levels of corticotropin-releasing hormone (CRH), adrenotropic corticotropic hormone (ACTH) and melatonin (MT) were measured by enzyme-linked immunoassay (ELISA). Rat serum and hippocampus were taken for nuclear magnetic resonance (¹H NMR) metabolomics to detect the changes of endogenous metabolites in rat serum hippocampus, to designate the differential metabolites and to construct metabolic pathways. The results showed that the exercise distance in the open field experiment and the number of times and time to enter the open arm in the elevated cross maze experiment of the rats in the model group were significantly reduced (P < 0.05, P < 0.01). The behavioral indexes of rats improved to different degrees after the administration of Ning Shen Essential Oil. The serum CRH and ACTH levels of rats in the model group increased significantly (P < 0.05, P < 0.01), and the MT level decreased significantly (P < 0.01); After the intervention, serum CRH and ACTH levels were reduced to different degrees, and MT levels could be regressed. ¹H NMR metabolomics screened 10 potential biomarkers related to insomnia, which involved in 6 potential metabolic pathways. A total of 35 components of Ning Shen Essential Oil were detected by GC-MS, the main component targets of Ning Shen Essential Oil and insomnia disease targets were intersected, a total of 172 intersecting genes were screened, and 26 core targets were identified. The study demonstrated that Ning Shen Essential Oil had protective effects against PCPA-induced insomnia in rats, which was probably correlated with regulation of the hypothalamic-pituitary-adrenal axis (HPA) related hormones and metabolism of amino acids, lipids and choline.

Key words

Ning Shen Essential Oil / insomnia / open field experiment / elevated cross maze / NMR / metabolomics / network pharmacology

引用本文

柴青, 张宏斌, 吴立东, 王婧毅, 李海超, 刘玉红, 刘红燕, 蒋海强, 田振华. 基于¹H NMR代谢组学与网络药理学的复方宁神精油抗失眠组分物质基础及作用机制研究. 药学学报, 2024 , 59 (8) : 2313 -2325 . DOI: 10.16438/j.0513-4870.2023-1436

Qing CHAI, Hong-bin ZHANG, Li-dong WU, Jing-yi WANG, Hai-chao LI, Yu-hong LIU, Hong-yan LIU, Hai-qiang JIANG, Zhen-hua TIAN. Study on the material basis and mechanism of anti-insomnia mechanism of Ning Shen Essential Oil based on ¹H NMR metabolomics and network pharmacology[J]. Acta Pharmaceutica Sinica, 2024 , 59 (8) : 2313 -2325 . DOI: 10.16438/j.0513-4870.2023-1436

正文

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失眠发生在全球约达1/3的成年人中^[1], 指睡眠的始发和睡眠维持发生障碍, 致使睡眠质量不能满足个体生理需要而显著影响患者白天社会功能的一种睡眠障碍综合征^[2]。失眠引起焦虑、抑郁或恐惧, 免疫功能下降、植物神经功能紊乱以及阿尔茨海默病, 帕金森综合征、高血压、糖尿病等严重疾病^[3]。目前用于治疗失眠的西药以中枢神经系统镇静催眠药物为主, 长时间应用会引起药物依赖性和耐药性等不良反应^[4]。相比西药而言, 中药以其不良反应小的优势受到关注。

芳香疗法治疗失眠在我国历史悠久^[5], 唐代孙思邈《千金方》载有37个芳香类药物治疗不寐的医方, 应用石菖蒲、肉桂、干姜、生姜和陈皮等气味芳香药物^[6]。张凯华等^[7]用中药香囊治疗肝郁脾虚型失眠, 结果香囊组有效降低了失眠患者的SPIEGEL评分及中医证候积分, 证明其香囊内的玫瑰花、合欢花、薰衣草、陈皮、薄荷、冰片等芳香药物有疏肝解郁、健脾安神之功。张晗羚等^[8]将冰片、安息香、甘松、酸枣仁、豆蔻、五味子、香橼、天竺黄、香附、柴胡等药制成安神香, 观察失眠大鼠睡眠潜伏期、睡眠时间及5-羟色胺(5-hydroxytryptamine, 5-HT) 等神经递质的变化, 安神香吸入对改善失眠有积极作用。许多学者对精油活性成分进行了药理研究。石菖蒲挥发油中的有效成分α-细辛醚能够提高小鼠脑内单胺类神经递质5-HT的含量^[9], 还可通过维持兴奋性/抑制性神经元的兴奋性/抑制性传递和衰减神经元过度兴奋性之间的平衡发挥抗焦虑作用^[10]。β-细辛醚可通过增加抑郁小鼠中脑及纹状体中5-HT和多巴胺(dopamine, DA) 的含量, 发挥抗抑郁作用^[11]。罗坤垛等^[12]发现ICR小鼠吸嗅玫瑰精油能延长ICR小鼠的戊巴比妥钠睡眠时间。左旋芳樟醇、桂皮醛皆能作用于单胺能神经传导系统的多种受体, 小鼠口服土肉桂精油和右旋芳樟醇能够减少大脑额皮质和海马体释放5-HT、DA和去甲肾上腺素(norepinephrine, NE)^[13]。有报道薄荷醇能加强戊巴比妥钠的中枢抑制作用, 且具有一定的量效关系^[14]。以上研究为复方中药精油改善睡眠药理作用提供了实验基础和依据。

本课题组利用中国知网(CNKI)、Web of Science、PubMed等数据库, 以“镇静催眠精油、安神精油、Sedative hypnotic essential oils、Anshen essential oils”等为关键词检索抗失眠中药精油的相关研究报道, 共检索14种与镇静催眠有关的中药精油, 根据出现频次选出排名前五的中药, 分别是薰衣草^[15-17]、薄荷^{[18, 19]}、玫瑰^{[20, 21]}、肉桂^[22]、石菖蒲^[23-25]。拟出本方配比为石菖蒲∶薄荷∶玫瑰∶薰衣草∶肉桂= 4∶4∶2∶2∶0.5。

本研究采用腹腔注射对氯苯丙氨酸(4-chloro-DL-phenylalanine, PCPA) 建立失眠大鼠模型, 通过检测大鼠血清中促肾上腺激素释放激素(corticotropin releasing hormone, CRH)、促肾上腺激素(adreno corticotropic hormone, ACTH)、褪黑素(melatonin, MT) 含量及行为学等指标检测复方宁神精油对PCPA致失眠大鼠下丘脑-垂体-肾上腺轴(hypothalamic-pituitary-adrenal axis, HPA) 轴的影响。基于¹H NMR代谢组学从整体探讨复方宁神精油治疗失眠的代谢通路, 进而通过中药系统药理学数据库及分析平台(traditional Chinese medicine systems pharmacology database and analysis platform, TCMSP)、在线人类孟德尔遗传(online Mendelian inheritance in man, OMIM)、有机小分子生物活性数据(Pubchem)、小分子数据信息挖掘(Drugbanks) 等数据库构建复方安神精油活性成分-作用靶点网络。采用代谢组学技术结合网络药理学, 分析复方宁神精油主要活性成分抗失眠功效的作用机制。

材料与方法

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实验动物 SPF级Wistar雄性大鼠共48只, 体质量190~220 g, 购自北京维通利华实验动物技术有限公司, 许可证号: SCXK (京) 2021-0006。动物实验经山东中医药大学动物伦理委员会批准(伦理号: SDUTCM20221025010)。动物饲养屏障内饲养室采光按照白昼循环模式, 安静环境下, 自由饮水、摄食, 室温18~23 ℃, 相对湿度45%~55%。自然昼夜节律光照, 自由进食进水。

药材与试剂 石菖蒲、薄荷、玫瑰、薰衣草、肉桂, 均购自安徽亳州药材市场。经山东中医药大学实验中心刘红燕教授鉴定为天南星科植物石菖蒲Acorus tatarinowii Schott的干燥根茎、唇形科植物薄荷Mentha haplocalyx Briq.干燥地上部分、蔷薇科植物玫瑰Rosa rugosa Thunb.的干燥花蕾、唇形科植物狭叶薰衣草Lavandula angustifolia Mill.的干燥地上部分、樟科植物肉桂Cinnamomum cassia Presl的干燥树皮。

乙酸乙酯(南京化学试剂股份有限公司, 批号: 210907420K); 大鼠CRH、ACTH、MT酶联免疫检测试剂盒(上海酶联生物科技有限公司, 批号: 20221025-3064A, 20221025-3024A, 20221025-3520A); 重水(批号DLM-4TPC-25) 购自美国Cambridge Isotope Laboratories公司; HPLC级甲醇和氯仿购自美国Thermo Fisher公司。

仪器 AVANCE Ⅲ 600 MHz型超导傅里叶变换核磁共振波谱仪(德国布鲁克科技有限公司); 7697A气相-质谱联用仪及HP-5MS气相色谱柱(30 m × 250 μm × 0.25 μm), 美国安捷伦科技有限公司; SuperMaze动物行为分析系统、旷场实验系统、高架十字迷宫实验系统(上海欣软信息科技有限公司)。

复方宁神精油的提取与供试品溶液的制备 称取石菖蒲、薄荷、玫瑰、薰衣草、肉桂5味药材, 按照中国药典(2020年版) 四部通则2204挥发油测定法提取挥发油^[26], 浸泡2 h, 加入12倍量水, 提取5 h。将提取得到的挥发油4 ℃保存。精密吸取1 mL挥发油, 加乙酸乙酯稀释并定容至5 mL量瓶中, 摇匀即得供试品溶液。将供试品溶液储存于4 ℃, 进行GC/MS分析。

复方宁神精油化学成分色谱方法 GC条件: 柱型HP-5MS (30 m × 250 μm × 0.25 μm), 进样量: 1 μL, 载气高纯氦气, 流速1 mL·min^-1, 进样口温度为250 ℃, 分流比50∶1; 色谱条件为60 ℃ (保持5 min), 以3 ℃·min^-1升温到180 ℃, 保持时间10 min, 然后以300 ℃运行5 min。MS条件: 电离方式为EI源, 电离能量为70.0 eV, 离子源温度为230 ℃, 离子传输温度为250 ℃, 质量扫描范围为m/z 40~550。

大鼠失眠模型建立、分组及复方宁神精油干预方法 大鼠适应性饲养1周后, 随机选取8只作为对照组, 腹腔注射生理盐水, 其余大鼠按每只450 mg·kg^-1的用量腹腔注射PCPA混悬液, 每日注射一次, 连续3天。末次给药后, 大鼠眼底静脉丛取血, 4 ℃分取血清。采用ELISA检测CRH、ACTH、MT含量。采用旷场实验测定运动距离、进入中央格次数等指标, 采用高架十字迷宫测定进入开放臂次数及进臂次数等指标检测造模是否成功。将造模成功的大鼠随机分成5组, 每组8只, 分别为模型组(等量生理盐水, 灌胃)、地西泮组(2 mg·kg^-1, 灌胃)、复方宁神精油低剂量组(LDG, 60 mg·kg^-1灌胃)、复方宁神精油中剂量组(MDG, 150 mg·kg^-1灌胃)、复方宁神精油高剂量组(HDG, 300 mg·kg^-1灌胃), 连续灌胃21天, 并于最后3天给予模型组腹腔注射PCPA再次复制失眠模型^[27]。

行为学测试

旷场实验分别于造模后第4天, 复方宁神精油干预后第22天, 进行旷场实验, 评估大鼠活动情况及焦虑样行为。大鼠提前适应环境5 min后, 将每只大鼠放置于旷场实验箱的中央格, 观察5 min, 记录大鼠运动总距离、穿过中央格时间及穿过中央格次数, 每只大鼠于实验结束后取出, 用75%酒精擦拭实验箱防止气味等因素干扰。

高架十字迷宫实验分别于造模后第5天, 复方宁神精油干预后第23天, 进行高架十字迷宫实验, 评估大鼠活动情况及焦虑样行为。大鼠提前适应环境5 min后, 快速将大鼠放在高架十字迷宫的正中心区域, 另其头部正对随机一个开放臂, 释放后记录进入开放臂次数、进入开放臂时间, 进臂次数, 计算进入开放臂次数比, 进入开放臂时间比。每只大鼠于实验结束后取出, 用75%酒精擦拭实验箱防止气味等因素干扰^[28]。

样本收集及处理 大鼠于末次给予复方宁神精油干预后, 禁食禁水12 h, 以5%戊巴比妥钠麻醉, 待深度麻醉后, 采用注射器腹主动脉取血置于10 mL EP管中, 室温静置30 min, 4 000 r·min^-1离心15 min, 获得血清, -80 ℃保存, 备用。收集血液后迅速取出海马, 并置于-80 ℃保存备用。

ELISA检测大鼠血清CRH、ACTH、MT含量 造模第6天和复方宁神精油干预第24天, 大鼠眼底静脉丛取血, 4 ℃分取血清。采用ELISA检测大鼠血清CRH、ACTH、MT含量^[29]。

¹H NMR样品制备

血清样品利用甲醇沉淀蛋白法, 以提取低浓度代谢物^[30]。取400 μL解冻后的血清与预冷甲醇(按照样品∶甲醇为1∶2) 在EP管中混合, 涡旋混匀, 置于4 ℃离心机13 000 r·min^-1离心15 min, 取上清液转移到新的EP管中, 氮吹浓缩至干。将样品溶于600 μL重水中, 移取550 μL上清液至核磁管中, 待测。

海马样品采用甲醇/氯仿/水的两相提取法, 取海马(约50 mg) 置于2 mL EP管中, 精密称定, 按比例加入预冷的甲醇(4 mL·g^-1)、氯仿(4 mL·g^-1) 和蒸馏水(2 mL·g^-1), 涡旋混匀, 冰上匀浆, 4 ℃静置15 min。4 ℃, 12 000 r·min^-1离心10 min, 取上清移至EP管中, 氮吹浓缩至干。把样品溶于600 μL重水中, 移取550 μL上清液至核磁管中, 待测^[31]。

¹H NMR测定及条件 ¹H NMR测定频率为600.13 MHz, 检测温度为298 K, 调用zg30脉冲序列进行¹H NMR图谱采集, 谱宽为12 019.23 Hz, 扫描次数为128次, 空扫次数为2次, 接收器增益因子为226, 采样点数为64 K, 采样时间为2.7 s, 弛豫时间为2 s, 自由感应衰减(free induction decay, FID) 分辨率为0.37 Hz, FID信号经过64 K傅里叶变换转为¹H NMR图谱。

数据处理及分析 采用软件MestReNova (西班牙Mestrelab Research公司) 对所获谱图进行手动相位和基线校正后, 对血清和海马样本参照重水(δ 4.79) 对 ¹H NMR谱的化学位移进行定标。在δ 0.5~9.5区域按δ 0.01等间隔分段积扣除δ 4.7~5.0处重水中残余H₂O的影响^[32], 将图谱积分值输出到Excel 2010 (Microsoft Inc, Bellevue, WA) 中, 然后对各分段积分值进行归一化处理。将导入Excel中的数据用SIMCA-P 13.0软件(瑞典Umetrics公司) 进行主成分分析(principal components analysis, PCA) 和偏最小二乘-判别分析(partial least squares-discriminant analysis, PLS-DA)。根据PLS-DA相应的VIP值(variable importance in the projection, VIP) 结果寻找对分类贡献较大的变量(化学位移), 确定各组的共性代谢物和差异的生物标志物。根据差异代谢物的化学位移信息, 查阅文献并参考人类代谢组数据库(human metabolome database, HMDB) 数据库(http://www.hmdb.ca/), 根据化合物化学位移、峰裂分情况和耦合常数对图谱进行化合物指认。通过对对照组与模型组的S-plot图进行分析, 结合VIP > 1的变量及SPSS 16.0进行t检验, 确定潜在的生物标志物^[33]。

复方宁神精油活性成分抗失眠的相关靶点筛选 通过中药系统药理学数据库与分析平台(TCMSP, https://tcmspe.com/tcmsp.php) 输入关键词“石菖蒲”“薄荷”“玫瑰花”“肉桂”, 根据口服利用度(OB) ≥ 30%且类药性(DL) ≥ 0.18进行活性成分初步筛选, 以获得活性化合物及蛋白质靶点^[34]。将GC-MS鉴定得到的挥发性成分及上述活性成分按照CAS号及分子量导入Pubchem (https://pubchem.ncbi.nlm.nih.gov) 数据库, 得到各个成分SMILES号, 再将各个SMILES号导入到Swiss Targetprediction (http://www.swisstargetprediction.ch) 数据库得到复方宁神精油成分靶点^[35]。

失眠症相关靶点筛选 以“insomnia”为关键词, 分别在GeneCards (https://www.genecards.org)、OMIM (http://www.omim.org) 及Drugbank (https://go.drugbank.com/) 检索失眠的相关靶点。为确定复方宁神精油治疗失眠症相关机制的潜在靶点, 将复方宁神精油活性成分靶点及失眠症疾病靶点取交集, 以获得交集基因。

蛋白质相互作用(PPI) 网络的构建及核心靶点筛选 将交集基因导入STRING数据库(https://string-db.org, Version 11.0) 进行蛋白质相互作用(protein-protein interactions, PPI) 分析。将数据以TSV格式保存, 导入Cytoscape 3.9.1, 根据节点度值大小决定节点直径以及颜色深浅, 并筛选核心靶点。

成分-靶点网络的构建及核心靶点基因富集分析 将复方宁神精油活性成分、核心靶点导入到Cytoscape3.9.2构建“活性成分-核心靶点”网络图, 以明确复方宁神精油治疗失眠症疾病的潜在机制。利用Metascape (http://metascape.org/) 对复方宁神精油治疗失眠的核心靶点进行分析。设置物种为“Homo sapiens”, 进行基因本体(GO) 注释及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG) 通路富集分析^[35]。

统计学分析 实验数据以

$ \overline{x} $

± s表示, 使用SPSS 26.0软件进行数据处理。组间数据比较采用单因素方差分析(ANOVA), 以P < 0.05表示差异显著, P < 0.01表示差异极显著, 具有统计学意义。

结果与讨论

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1 复方宁神精油成分分析

利用气相色谱-质谱联用仪对精油挥发性成分进行分析, 得到总离子流图(图 1)。通过NIST标准谱图数据库进行分析, 共鉴定出35种主要挥发性成分, 占精油总成分的92.02%。各挥发性成分的出峰时间及相对质量分数见表 1。分析表明复方宁神精油中挥发性成分的相对质量分数在1.0%以上的主要成分有13种化学成分, 分别为: 芳樟醇(4.78%)、DL-薄荷醇(14.89%)、异胡薄荷醇(1.14%)、左旋薄荷酮(5.39%)、蒲勒酮(1.02%)、香茅醇(1.09%)、桂皮醛(7.92%)、乙酸香叶酯(4.34%)、石竹烯(2.05%)、β-细辛脑(33.47%)、α-细辛脑(4.27%)、γ-细辛醚(1.11%)、异薄荷酮(1.36%), 占挥发性成分总量的82.83%。

2 复方宁神精油对失眠大鼠行为学的影响

与对照组相比, 模型组大鼠旷场实验运动距离、高架十字迷宫实验进入开放臂次数及时间均显著降低(P < 0.05, P < 0.01)。此外, 除对照组外, 其余大鼠出现白天不断移动, 性情焦躁易怒, 睡眠状态显著减少, 饮水量增加, 表明失眠模型造模成功。

各组的旷场实验中, 总活动距离、穿过中央格次数、中央格停留时间的统计结果见表 2。复方宁神精油干预失眠大鼠后, 与模型组相比, 复方宁神精油低剂量组和高剂量组的总活动距离极显著性升高(P < 0.001), 复方宁神精油中剂量组总活动距离和地西泮组与模型组相比显著升高(P < 0.01)。虽然穿过中央格次数及中央格停留时间与模型组相比差异无统计学意义(P > 0.05), 但均有增加的趋势。

各组高架十字迷宫统计结果见表 3。与对照组相比, 模型组开放臂停留时间比、进臂次数比和总进臂次数均显著降低(P < 0.05), 复方宁神精油干预失眠大鼠后, 与模型组相比, 中剂量组和地西泮组开放臂停留时间比、进臂次数比和总进臂次数均显著增加(P < 0.05); 高剂量组开放臂停留时间比和进臂次数比显著增加。

3 ELISA试剂盒检测大鼠血清CRH、ACTH、MT含量

与对照组相比, 模型组大鼠血清CRH、ACTH含量均显著升高(P < 0.05, P < 0.01), MT含量显著降低(P < 0.01), 表明PCPA失眠模型制备成功。与模型组相比, 各个给药组大鼠血清激素CRH、ACTH、MT含量检测结果见表 4。复方宁神精油干预后, 低剂量组、中剂量组与地西泮组大鼠血清CRH含量与模型组相比, 均具有显著降低的趋势(P < 0.05); 各组大鼠血清ACTH含量, 中剂量组与模型组相比, 具有显著降低的趋势(P < 0.05), 低剂量组、高剂量组、地西泮组与模型组相比均具有降低趋势; 各组大鼠血清MT含量与模型组相比, 均具有显著升高的趋势(P < 0.05)。本实验表明失眠会引起HPA轴紊乱, 进而引起相关激素发生变化。

4 生物样本模式识别分析及潜在生物标志物的发现

用多元统计分析方法对¹H NMR数据进行多变量数据分析。首先采用无监督的PCA得分散点图(图 2A、C), 从图中可以看出, 正常组和模型组聚类趋势尚不明显, 血清PCA得分图R²X和Q²值分别为0.947和0.872, 海马PCA得分图R²X和Q²值分别为0.935和0.698。图 2B所示, 第一次拟合得到的PCA图中有一个在红线值上两倍的异常点, 去掉异常值的血清样本, 拟合后未见异常值存在, 各组样本都处于95%置信区间内。为进一步发现PCPA诱导的失眠大鼠的潜在生物标志物, 通过有监督的PLS-DA模型, 图 3A、4A所示, 正常组和PCPA诱导的失眠大鼠模型组的血清、海马样本在PC1维可以完全分开, 说明PCPA诱导失眠后大鼠机体生理及物质代谢情况已经发生了明显的改变。血清PLS-DA模型的R²X、R²Y和Q²分别为0.826、0.993、0.964; 海马PLS-DA模型的R²X、R²Y和Q²分别为0.598、0.998、0.983。采用200次置换检验, 如图 3C所示, 血清置换检验后的R²和Q²的回归截距分别为0.634、-0.194。海马置换检验后的R²和Q²的回归截距分别为0.234、-0.332, 如图 4B所示。Q²截距均为负值, 表明模型未过拟合, 模型有效。在OPLS-DA模式下, 生成能预测差异代谢物的S-plot图及VIP图。如图 3B、4C所示, 在S-plot图中, 越远离图中心的样本, 被认为对模型的分离贡献越大。如图 3D、4D所示, VIP > 1的变量在OPLS模型中区分度较大, 有较高的预测能力, 将其对应的代谢物作为潜在的差异代谢物。结合S-plot图(|Pcorr| > 0.58)^[33]和t检验(P < 0.05) 对候选变量进行分析, 最终筛选出10种潜在生物标志物, 分别为低密度脂蛋白/极低密度脂蛋白(low density lipoprotein/very low density lipoprotein, LDL/VLDL)、丙氨酸、N-乙酰天冬氨酰谷氨酸、甘氨酸、谷氨酸、γ-氨基丁酸(gamma-aminobutyric acid, GABA)、胆碱、牛磺酸、异亮氨酸、色氨酸。

5 大鼠血清和海马¹H NMR代谢物图谱分析及归属

根据化学位移、峰裂分情况、耦合常数等信息, 通过查阅文献^[32-34]并查询HMDB (http:/www.hmdb.ca) 数据库, 最终在血清样本中共筛选得到10种潜在生物标志物(表 5), 分别为低密度脂蛋白/极低密度脂蛋白、3-羟基丁酸(3-hydroxybutyrate, 3-HB)、异亮氨酸、N-乙酰糖蛋白、O-乙酰糖蛋白、谷氨酸、胆碱、D-葡萄糖、氧化三甲胺、尿囊素等。其化学位移及谱峰归属, 代谢物图谱见图 5A。在海马样本中筛选得到12种潜在生物标志物(表 6), 分别为丙氨酸、N-乙酰天冬氨酰谷氨酸、甘氨酸、谷氨酸、GABA、肌酸/磷酸肌酸、甘油磷酸胆碱、牛磺酸、D-葡萄糖、琥珀酸、氧化三甲胺、色氨酸等。其差异代谢物化学位移及谱峰归属, 代谢物图谱见图 5B。

6 复方宁神精油对PCPA诱导的失眠大鼠模型的干预作用

为了进一步分析复方宁神精油对10种潜在生物标志物的回调与下调趋势, 对潜在生物标志物的相对含量进行t检验分析。如图 6所示, 与对照组比较, 模型组血清和海马中GABA、丙氨酸、甘氨酸、牛磺酸、色氨酸含量均有不同程度的降低(P < 0.05、P < 0.01、P < 0.001); 异亮氨酸、谷氨酸、胆碱、低密度脂蛋白/极低密度脂蛋白含量均有不同程度的升高(P < 0.05、P < 0.01); 复方宁神精油干预后, 与模型组比较, 血清和海马中GABA、丙氨酸、甘氨酸、牛磺酸、色氨酸含量均有不同程度回调(P < 0.05、P < 0.01); 异亮氨酸、谷氨酸、胆碱、低密度脂蛋白/极低密度脂蛋白含量均有不同程度的降低(P < 0.05、P < 0.01、P < 0.001)。

7 代谢通路和相关靶点分析

将10种潜在生物标志物输入到MetPA (http://www.metaboanalyst.ca/) 数据库中构建代谢通路, 把Impact > 0.1的代谢通路作为潜在的靶标通路, 各通路Impact值如图 7A所示, 综合KEGG及相关富集分析方法得到潜在生物标志物相关代谢通路, 前25条通路见图 7B。结合P值(图 7B) 共筛选得到PCPA诱导的失眠模型大鼠有6条代谢通路发生显著变化(图 7A), 分别为丙氨酸、天冬氨酸和谷氨酸代谢, 乙醛酸和二羧酸代谢, 精氨酸生物合成, 谷胱甘肽代谢, 精氨酸和脯氨酸代谢, D-谷氨酰胺和D-谷氨酸代谢。

8 复方宁神精油活性成分与疾病靶点的筛选

在GeneCards、OMIMDrugbank筛选出失眠的相关靶点共2 503个。复方宁神精油治疗失眠症相关靶点共544个, 将复方宁神精油活性成分靶点及失眠症疾病靶点取交集, 获得交集基因共176个, 如图 8A。

9 PPI网络及核心靶点的筛选

将176个交集基因导入个交集靶点导入STRING平台, 物种限定为“Homo sapiens”, 最低交互分数设定为“medium confidence (0.400)”, 得到PPI网络。利用Cytoscape 3.9.1进一步处理, 以节点度值closeness ≥ 0.004 5, degree > 4.85为参数, 共得到26个核心靶点。其中度值、介值大的靶点预测为失眠症的关键靶点, 分别为活化蛋白激酶(mitogen-activated protein kinase, MAPK)、微管相关蛋白(microtubule-associated proteins, MAPT)、淀粉样前体蛋白(amyloid precursor protein, APP)、单胺氧化酶A (monoamine oxidase-A, MAOA)、丁酰胆碱酯酶(butyrylcholinesterase, BCHE)、乙酰胆碱酯酶(acetylcholinesterase, ACHE)、低密度脂蛋白相关受体蛋白1 (low density lipoprotein receptor adaptor protein 1, LDLRP1)、促肾上腺皮质激素释放激素受体1 (corticotropin releasing hormone receptor 1, CRHR1)、酪氨酸蛋白激酶受体(anexelekto, AXL)、肿瘤坏死因子(tumor necrosis factor, TNF)、一氧化氮合酶2 (nitric oxide synthase 2, NOS2) 等, 筛选流程图如图 8B。

10 GO和KEGG富集分析

将26个核心靶点导入至Metascape平台完成GO及KEGG分析。GO分析主要包括生物过程(BP)、细胞组分(CC) 和分子功能(MF) 3项, 共涉及1 391项BP, 53项CC, 105项MF。BP、CC、MF前10项GO富集通路结果如图 8C所示。KEGG富集分析显示复方宁神精油通过118条信号通路对失眠症发挥治疗作用。前10条KEGG富集通路如图 8D所示, 涉及到神经活性配体-受体相互作用、钙信号通路、脂质代谢、酪氨酸酶代谢、MAPK信号通路等多条通路。

讨论

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本文采用PCPA制备失眠大鼠模型, 探讨了复方宁神精油对失眠大鼠行为学指标及HPA轴相关激素的调节作用, 并通过网络药理学与代谢组学结合共同验证复方宁神精油治疗失眠症的作用机制。PCPA诱导的大鼠失眠模型是目前被广泛接受的用来研究失眠机制和药效评价的模型之一^[36]。PCPA可以选择性作用于色氨酸羟化酶, 抑制大鼠大脑5-HT合成, 造成睡昼夜节律消失, 当5-HT含量降低时, 机体内HPA轴随之亢奋, 与HPA轴相关的一些中枢神经递质如ACTH、CRH、GABA等的含量均会受到影响, 与失眠相关的其他神经递质如MT等的含量也会发生相应的变化, MT是5-HT的代谢产物, 它在松果体内以光调节的形式与环境信号同步, 以此来维持机体昼夜节律^[37]。造模成功后, 模型组大鼠活动度降低, 精神萎靡, 旷场实验结果显示模型组大鼠运动总距离显著减少, 高架十字迷宫实验大鼠进入开放臂次数及时间均显著减少, 说明失眠会引起大鼠对外界探索能力下降的焦虑样行为, 宁神精油干预后大鼠运动总距离、高架十字迷宫实验大鼠进入开放臂次数及时间均有不同程度的增加; ELISA结果检测发现ACTH和CRH含量降低, 提示HPA轴激活, 宁神精油干预后, ACTH和CRH含量升高, 提示复方宁神精油能够调节PCPA引起的失眠大鼠HPA轴紊乱。

代谢组学研究发现, 失眠可引起机体氨基酸代谢紊乱, 表现在模型组较对照组大鼠血清中谷氨酸、异亮氨酸水平升高, 海马组织中N-乙酰天冬氨酸水平升高, 丙氨酸、GABA水平降低。谷氨酸对中枢神经系统神经元起兴奋作用, 是主要的兴奋性递质, 其释放的增加导致奋性神经毒性, 在失眠中起重要作用^[38], 谷氨酸升高可以刺激HPA轴CRH, ACTH释放^[39]。大鼠α-酮戊二酸升高, 使机体谷氨酸分泌增加, 促进ACTH, CRH分泌, 这与ELISA实验结果一致。异亮氨酸属于支链氨基酸, 可促进蛋白质合成、降低分解, 同时通过减少色氨酸入脑的含量来降低5-HT的浓度, 进而抑制、减缓中枢疲劳^[40]。有研究显示异相睡眠剥夺会导致大脑皮质谷氨酸水平明显升高^[41]。GABA可以抑制神经系统中的神经元, 具有神经保护作用, 并具有催眠、镇静、抗焦虑等功能^[42]。牛磺酸能和GABA彼此作用, 激活GABA受体和甘氨酸受体^[43], 进一步加强对中枢神经系统的抑制作用。胆碱是细胞磷脂和神经鞘磷脂的重要组成部分, 是乙酰胆碱和磷脂酰胆碱的前体物质, 乙酰胆碱与学习记忆和认知功能密切相关, 而磷脂酰胆碱参与细胞膜的构成^[44]。本研究中复方宁神精油能显著下调失眠大鼠血清与海马中异亮氨酸、谷氨酸、胆碱、低密度脂蛋白/极低密度脂蛋白水平, 上调GABA、丙氨酸、甘氨酸、牛磺酸、色氨酸水平, 说明宁神精油干预失眠的机制与调节机体氨基酸代谢、脂质代谢、胆碱代谢有关。此外本研究还发现, 失眠大鼠血清中低/极低密度脂蛋白水平明显升高, 说明失眠可引起机体脂质代谢紊乱, 经宁神精油干预后, 血清中低/极低密度脂蛋白回调趋近正常水平, 说明复方宁神精油可通过调节脂质代谢来干预失眠。

通过网络药理学探讨复方宁神精油活性成分-靶点相互作用, 发现复方宁神精油中的主要活性成分主要作用于MAPK、MAPT、APP、MAOA、BCHE、ACHE、LRP1、CRHR1、AXL、TNF、NOS2等靶点, 涉及到神经活性配体-受体相互作用、钙信号通路、脂质代谢、酪氨酸酶代谢、MAPK信号通路等多条通路。

综上所述, 对PCPA所致失眠大鼠的¹H NMR代谢组学及网络药理学显示失眠涉及HPA轴、能量代谢、谷氨酸、牛磺酸、甘氨酸、色氨酸、丙酮酸代谢。¹H NMR代谢组学显示复方宁神精油干预PCPA所致失眠大鼠模型后能上调GABA、丙氨酸、甘氨酸、牛磺酸、色氨酸水平; 下调异亮氨酸、谷氨酸、胆碱、低密度脂蛋白/极低密度脂蛋白水平。ELISA法显示复方宁神精油干预PCPA所致失眠大鼠模型后, 能显著降低失眠大鼠血清中CRH、ACTH含量, 显著升高MT含量, 从而改善失眠大鼠的HPA轴、脂质代谢和氨基酸代谢紊乱。本研究为复方中药精油治疗失眠作用机制的深入研究奠定了一定基础。

作者贡献: 柴青负责实验设计、动物实验研究及数据处理, 并完成论文初稿撰写; 蒋海强负责文章整体设计及文章修改; 吴立东负责文章整体实验框架设计及实验进度; 刘红燕负责药材购买及鉴定; 田振华、刘玉红负责指导实验及文章修改; 张宏斌、王婧毅协助完成动物实验; 李海超协助提取挥发油。

利益冲突: 所有作者均声明不存在利益冲突。

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山东省青创人才引育计划

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2024年第59卷第8期

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doi: 10.16438/j.0513-4870.2023-1436

接收时间：2023-12-15
首发时间：2025-11-26
出版时间：2024-08-12

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收稿日期：2023-12-15
修回日期：2024-06-07

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山东省青创人才引育计划

作者信息

1.山东中医药大学药学院, 山东济南 250355

2.山东中医药大学中医药创新研究院, 山东济南 250355

3.山东欣悦健康科技有限公司, 山东滨州 256600

4.山东中医药大学实验中心, 山东济南 250355

5.山东省中医药基础研究重点实验室, 山东济南 250355

6.山东中医药大学, 教育部中医药经典理论重点实验室, 山东济南 250355

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^*蒋海强, Tel: 86-531-89628014, E-mail: jhq12723@163.com;

田振华, E-mail: tianzhenhuatina@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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t_R/min	Name	Chemical formula	Relative content/%
6.77	2-Methyl-1-octen-3-yne	C₉H₁₄	0.39%
7.31	Camphene	C₁₀H₁₆	0.39%
8.41	(1S)-(-)-beta-Pinene	C₁₀H₁₆	0.29%
9.05	beta-Pinene	C₁₀H₁₆	0.35%
10.67	Isopulegol	C₁₀H₁₈O	1.14%
11.13	Menthol	C₁₀H₁₈O	0.18%
14.18	Linalool	C₁₀H₂₀O	4.78%
14.69	1-Octen-3-yl acetate	C₁₀H₁₈O₂	0.67%
16.59	L-Menthone	C₁₀H₁₈O	5.39%
17.01	Isomenthone	C₁₀H₁₈O	1.36%
17.92	DL-Menthol	C₁₀H₁₈O	14.89%
18.44	alpha-Terpineol	C₁₀H₁₈O	0.92%
20.40	Dorzolamide	C₁₀H₁₆O	1.02%
21.16	Menthone	C₁₀H₁₆O	0.30%
21.36	Citronellol	C₁₂H₂₀O	1.09%
22.10	Cinnamaldehyde	C₉H₈O	7.92%
22.60	beta-Ocimene	C₁₀H₁₆O	0.44%
23.03	Geranyl acetate	C₁₀H₂₀O₂	4.34%
26.10	Neryl acetate	C₁₀H₂₀O₂	0.08%
26.50	(+)-α-Pinene	C₁₅H₂₄	0.45%
27.00	Bornyl acetate	C₁₀H₂₀O₂	0.27%
28.30	Methyleugenol	C₁₁H₁₄O₂	0.76%
29.41	Cinnamyl acetate	C₁₁H₁₂O₂	0.42%
30.03	cis-Methylisoeugenol	C₁₁H₁₄O₂	0.12%
30.85	Caryophyllene	C₁₅H₂₄	2.05%
31.60	Methyl isoeugenol	C₁₁H₁₄O₂	0.29%
32.20	Shyobunone	C₁₅H₂₄O	0.36%
32.60	delta-Cadinene	C₁₅H₂₄O	0.19%
32.87	2'-Methoxycinnamaldehyde	C₁₀H₁₀O₂	0.45%
34.60	γ-Asarone	C₁₂H₁₆O₃	1.11%
34.81	(+)-α-Funebrene	C₁₅H₂₆	0.79%
36.90	beta-Asarone	C₁₂H₁₆O₃	33.47%
38.70	α-Asarone	C₁₂H₁₆O₃	4.27%
39.19	Aristolone	C₁₅H₂₂O	0.49%
40.80	Isocalamendiol	C₁₅H₂₆O₂	0.59%

t_R/min

Name

Chemical formula

Relative content/%

6.77

2-Methyl-1-octen-3-yne

C₉H₁₄

0.39%

7.31

Camphene

C₁₀H₁₆

0.39%

8.41

(1S)-(-)-beta-Pinene

C₁₀H₁₆

0.29%

9.05

beta-Pinene

C₁₀H₁₆

0.35%

10.67

Isopulegol

C₁₀H₁₈O

1.14%

11.13

Menthol

C₁₀H₁₈O

0.18%

14.18

Linalool

C₁₀H₂₀O

4.78%

14.69

1-Octen-3-yl acetate

C₁₀H₁₈O₂

0.67%

16.59

L-Menthone

C₁₀H₁₈O

5.39%

17.01

Isomenthone

C₁₀H₁₈O

1.36%

17.92

DL-Menthol

C₁₀H₁₈O

14.89%

18.44

alpha-Terpineol

C₁₀H₁₈O

0.92%

20.40

Dorzolamide

C₁₀H₁₆O

1.02%

21.16

Menthone

C₁₀H₁₆O

0.30%

21.36

Citronellol

C₁₂H₂₀O

1.09%

22.10

Cinnamaldehyde

C₉H₈O

7.92%

22.60

beta-Ocimene

C₁₀H₁₆O

0.44%

23.03

Geranyl acetate

C₁₀H₂₀O₂

4.34%

26.10

Neryl acetate

C₁₀H₂₀O₂

0.08%

26.50

(+)-α-Pinene

C₁₅H₂₄

0.45%

27.00

Bornyl acetate

C₁₀H₂₀O₂

0.27%

28.30

Methyleugenol

C₁₁H₁₄O₂

0.76%

29.41

Cinnamyl acetate

C₁₁H₁₂O₂

0.42%

30.03

cis-Methylisoeugenol

C₁₁H₁₄O₂

0.12%

30.85

Caryophyllene

C₁₅H₂₄

2.05%

31.60

Methyl isoeugenol

C₁₁H₁₄O₂

0.29%

32.20

Shyobunone

C₁₅H₂₄O

0.36%

32.60

delta-Cadinene

C₁₅H₂₄O

0.19%

32.87

2'-Methoxycinnamaldehyde

C₁₀H₁₀O₂

0.45%

34.60

γ-Asarone

C₁₂H₁₆O₃

1.11%

34.81

(+)-α-Funebrene

C₁₅H₂₆

0.79%

36.90

beta-Asarone

C₁₂H₁₆O₃

33.47%

38.70

α-Asarone

C₁₂H₁₆O₃

4.27%

39.19

Aristolone

C₁₅H₂₂O

0.49%

40.80

Isocalamendiol

C₁₅H₂₆O₂

0.59%

Group	Total distance of movement/mm	Dwell time in central compartment/s	Number of passes through the central compartment/times
Control (n = 8)	2 698.27 ± 1 036.44	7.28 ± 7.39	5.00 ± 3.31
Model (n = 7)	619.12 ± 284.90^##	1.56 ± 1.75^##	1.80 ± 2.17^#
LDG (n = 7)	3 038.95 ± 1 034.58^***	2.18 ± 4.78	1.80 ± 2.39
MDG (n = 7)	3 503.57 ± 533.88^**	3.15 ± 2.75	3.40 ± 1.14
HDG (n = 7)	3 149.21 ± 866.88^***	4.63 ± 4.53	3.20 ± 3.11
Diazepam (n = 8)	3 740.52 ± 697.54^**	5.87 ± 3.75	3.80 ± 2.17

Group

Total distance of movement/mm

Dwell time in central compartment/s

Number of passes through the central compartment/times

Control (n = 8)

2 698.27 ± 1 036.44

7.28 ± 7.39

5.00 ± 3.31

Model (n = 7)

619.12 ± 284.90^##

1.56 ± 1.75^##

1.80 ± 2.17^#

LDG (n = 7)

3 038.95 ± 1 034.58^***

2.18 ± 4.78

1.80 ± 2.39

MDG (n = 7)

3 503.57 ± 533.88^**

3.15 ± 2.75

3.40 ± 1.14

HDG (n = 7)

3 149.21 ± 866.88^***

4.63 ± 4.53

3.20 ± 3.11

Diazepam (n = 8)

3 740.52 ± 697.54^**

5.87 ± 3.75

3.80 ± 2.17

Group	Open arm dwell time ratio (toc = to/to+tc)	Open arm entry ratio (Noc = Noe/Noe+Nce)	Total number of arm feeds /times
Control (n = 8)	0.33 ± 0.3	0.33 ± 0.3	22.2 ± 7.9
Model (n = 7)	0.12 ± 0.1^#	0.16 ± 0.2^#	9.2 ± 3.7^#
LDG (n = 7)	0.21 ± 0.1	0.18 ± 0.1	13.5 ± 1.3^*
MDG (n = 7)	0.39 ± 0.1^*	0.24 ± 0.2^*	14.5 ± 3.4^*
HDG (n = 7)	0.41 ± 0.4^*	0.30 ± 0.3^*	14.0 ± 3.0
Diazepam (n = 8)	0.46 ± 0.2^*	0.34 ± 0.2^*	16.3 ± 2.5^*

Group

Open arm dwell time ratio (toc = to/to+tc)

Open arm entry ratio (Noc = Noe/Noe+Nce)

Total number of arm feeds /times

Control (n = 8)

0.33 ± 0.3

22.2 ± 7.9

Model (n = 7)

0.12 ± 0.1^#

0.16 ± 0.2^#

9.2 ± 3.7^#

LDG (n = 7)

0.21 ± 0.1

0.18 ± 0.1

13.5 ± 1.3^*

MDG (n = 7)

0.39 ± 0.1^*

0.24 ± 0.2^*

14.5 ± 3.4^*

HDG (n = 7)

0.41 ± 0.4^*

0.30 ± 0.3^*

14.0 ± 3.0

Diazepam (n = 8)

0.46 ± 0.2^*

0.34 ± 0.2^*

16.3 ± 2.5^*

Group	CRH	ACTH	MT
Control (n = 8)	50.1 ± 2.5	40.7 ± 11.3	55.8 ± 2.2
Model (n = 7)	61.6 ± 3.3^#	49.5 ± 5.0^##	48.0 ± 2.8^##
LDG (n = 7)	51.7 ± 4.7^*	45.3 ± 4.9	58.6 ± 11.8^*
MDG (n = 7)	51.5 ± 10.9^*	42.9 ± 3.2^*	58.5 ± 10.2^*
HDG (n = 7)	53.6 ± 1.9	46.7 ± 5.9	60.4 ± 7.0^*
Diazepam (n = 8)	51.7 ± 8.8^*	48.2 ± 4.7	60.7 ± 13.2^*

Group

CRH

ACTH

Control (n = 8)

50.1 ± 2.5

40.7 ± 11.3

55.8 ± 2.2

Model (n = 7)

61.6 ± 3.3^#

49.5 ± 5.0^##

48.0 ± 2.8^##

LDG (n = 7)

51.7 ± 4.7^*

45.3 ± 4.9

58.6 ± 11.8^*

MDG (n = 7)

51.5 ± 10.9^*

42.9 ± 3.2^*

58.5 ± 10.2^*

HDG (n = 7)

53.6 ± 1.9

46.7 ± 5.9

60.4 ± 7.0^*

Diazepam (n = 8)

51.7 ± 8.8^*

48.2 ± 4.7

60.7 ± 13.2^*

No.	Metabolite	Chemical shift/ppm
1	LDL/VLDL	0.88 (m)
2	Isoleucine	0.95 (t), 1.32 (s), 1.33 (s)
3	3-HB	1.19 (d), 2.30 (dd)
4	N-Acetylglucosamine	1.91 (s)
5	O-Acetylglucosamine	2.07 (s)
6	Glutamic acid	2.35 (m), 2.45 (m)
7	Choline	3.04 (s)
8	Trimetlylamine oxide	3.34 (s)
9	β-Glucose	3.52 (dd), 3.43 (m), 4.64 (d)
10	Allantoic acid	5.20 (d)

No.

Metabolite

Chemical shift/ppm

LDL/VLDL

0.88 (m)

Isoleucine

0.95 (t), 1.32 (s), 1.33 (s)

3-HB

1.19 (d), 2.30 (dd)

N-Acetylglucosamine

1.91 (s)

O-Acetylglucosamine

2.07 (s)

Glutamic acid

2.35 (m), 2.45 (m)

Choline

3.04 (s)

Trimetlylamine oxide

3.34 (s)

β-Glucose

3.52 (dd), 3.43 (m), 4.64 (d)

Allantoic acid

5.20 (d)

No.	Metabolite	Chemical shift/ppm
1	3-HB	1.32 (d), 2.66 (dd)
2	Alanine	1.47 (d)
3	GABA	1.88 (m), 2.29 (t), 3.00 (t)
4	N-Acetylaspartylglutamic acid	1.91 (s)
5	Glutamic acid	2.01 (m), 2.05(m), 2.35 (m)
6	Creatine/creatine phosphate	3.03 (s), 3.93 (s)
7	L-Glycerophosphorylcholine	3.20 (s)
8	Taurine	3.26 (t), 3.42 (t)
9	Succinate acid	3.35 (s)
10	β-Glucose	3.53 (dd), 3.75 (m)
11	Glycine	3.55 (s)
12	Tryptophan	7.65 (s)

No.

Metabolite

Chemical shift/ppm

3-HB

1.32 (d), 2.66 (dd)

Alanine

1.47 (d)

GABA

1.88 (m), 2.29 (t), 3.00 (t)

N-Acetylaspartylglutamic acid

1.91 (s)

Glutamic acid

2.01 (m), 2.05(m), 2.35 (m)

Creatine/creatine phosphate

3.03 (s), 3.93 (s)

L-Glycerophosphorylcholine

3.20 (s)

Taurine

3.26 (t), 3.42 (t)

Succinate acid

3.35 (s)

β-Glucose

3.53 (dd), 3.75 (m)

Glycine

3.55 (s)

Tryptophan

7.65 (s)