Low Pressure Liquid Chromatography

Minor phenolic acids which were tedious to be isolated were applied on LPLC using Lichoprep RP-18 column and MeOH-H2O-HCOOH (45:55:1) as solvent. The regioisomeric compounds salvianolic acid H and I were successfully isolated by this method.

UV SPECTRA OF SALVIANOLIC ACIDS AND RELATED COMPOUNDS

The UV spectra of salvianolic acids and related compounds exhibited characteristic absorptions for a cinnamoyl chromophore at 230, 290 and 320 nm. A shoulder at 220 nm was observed in the spectra of some compounds. A highly conjugated system in the structure increased the intensity at 310 nm and caused an additional absorption maximum at 340 nm. Salvianolic acid B and lithospermic acid which possess an aryl dihydrobenzofuranoid skeleton revealed absorption at 253 nm.

Figure 2 MS fragmentation of methylated salvianolic acids.

MS OF SALVIANOLIC ACIDS AND RELATED COMPOUNDS

The fact that salvianolic acids are highly polar compounds the molecular ion could only be obtained by FDMS or FAB MS. However the methylated product revealed diagnostic fragmentation pattern of a ^-(3, 4-dimethoxyphenyl) lactic ester at mlz 222, (M±222), 191, 181, 163, and 151 (Fig. 2).

NMR SPECTRA OF SALVIANOLIC ACIDS AND RELATED COMPOUND

The 'H NMR spectra of salvianolic acids and related compounds revealed signals of trans disubstituted double bond at 87.60-8.00 (a-H,/=16 Hz) and ¿6.20-6.30 (S-H, /=16 Hz). The 3, 4-dihydroxyphenyl lactic moiety showed signals of two methylene proton doublets at 8 3.0-3.5, an oxygen bearing methine proton at 8 4.43-4.50 (dd, J=4/7Hz) and three aromatic protons at 8 6.70-6.90. A pair of doublets at 8 4.434.50 (J=5.4, C-8H) and 8 5.90-6.00 (J=5.4, C-7H) indicated the presence of a trans disubstituted dihydrobenzofuranoid skeleton for salvia nolic acid B and lithospermic acid. A relative downfield olefinic proton singlet at 8 8.0±0.1 for salvianolic acid E suggested that the two phenyl groups of the carbo xystilbene should be cis oriented. In the trans isomer this proton signal was less deshielded and appeared at 8 7.2. The dibenzooxepin skeleton of isosalvianolic acid C was deduced from the fact that the two olefinic signals at 8 6.94 and 8 7.00 possessed a coupling constant of J=11.5 Hz. A pair of oxygen bearing methine proton doublets at 84.81 and 5.31 with a coupling

Table 1 Specifie rotation and occurrence of salvianolic acids and related compounds.

Compound an (EtOH) Plant

Table 1 Specifie rotation and occurrence of salvianolic acids and related compounds.

Compound an (EtOH) Plant

Salvianolic acid A

+41

Sm, Scs, Sc, Sf, Sb, Sp

Salvianolic acid B

+92

Sm, Scs, Sc, Sf, Sch, Sb, Sp, Sy

Salvianolic acid C

+70

Sm, Scs, Sc, Sb, Sp

Salvianolic acid D

+68

Sm, Sch

Salvianolic acid E

+59

Sm

Salvianolic acid F

Sm

Salvianolic acid G

-100

Sm

Salvianolic acid H

+63

Scs

Salvianolic acid I

+71

Sc, Scs

Salvianolic acid J

+26

Sf

Isosalvianolic acid C

+39

Sch, Scs, Sc, Sb

Rosmarinic acid

+67

Sm, Scs, Sc, Sf, Sch, Sb, Sp

Lithospermic acid

+ 12

Sm, Scs, Sc, Sch

Przewalskinic acid

Sprz

Salviaflaside

+18.5

Sf

Sm: Salvia miltiorrhiza Bunge, Scs: Salvia cavaleriei Levl.var. simplicifolia Peter- Stibal Sc: Salvia cavaleriei Levi., Sf: Salvia flava Forrest ex Dieles, Sch: Salvia chinensis Benth, Sb: Salvia bowleyana Dunn, Sp: Salvia prionitis Hance, Sy: Salvia yunnanensis C.H. Wright, Spr: Salvia przewalskii Maxim

Sm: Salvia miltiorrhiza Bunge, Scs: Salvia cavaleriei Levl.var. simplicifolia Peter- Stibal Sc: Salvia cavaleriei Levi., Sf: Salvia flava Forrest ex Dieles, Sch: Salvia chinensis Benth, Sb: Salvia bowleyana Dunn, Sp: Salvia prionitis Hance, Sy: Salvia yunnanensis C.H. Wright, Spr: Salvia przewalskii Maxim

Table 2 UV spectral data of Salvianolic acids and related compounds.

Compound

Salvianolic acid A

205 (4.27),

218

Salvianolic acid B

203 (4.95),

253

Salvianolic acid C

202 (4.79),

215

Salvianolic acid D

203 (4.73),

220

Salvianolic acid E

205 (4.92),

299

Salvianolic acid G

203 (5.57),

257

403 (4.76)

Salvianolic acid H

205 (4.76),

220

Salvianolic acid I

205 (4.76),

220

318(4.38)

Salvianolic acid J

203 (4.87),

218

Isosalvianolic acid C

202 (4.77),

222

(4.24)

Rosmarinic acid

203 (4.52),

220

Lithospermic acid

203 (4.52),

220

Table 3.1 Selected 1H NMR spectral data of salvianolic acids and related compounds.

H

la"

2

2a

2b21

3

3a

3b

4

4a

5

6.90

6.91

6.90

6.93

6.84

6.81

6.84

6.88

6.90

;=s

7=8

7=8

7=8

/=8

7=8

7=8

7=8

7=8

6

7.30

7.28

7.20

7.27

6.99

6.95

7.00

7.24

7.40

J=8

7=8

7=8

7=8

7=8

7=8

7=8

7=8

7=8

7

8.10

7.66

7.61

7.79

7.97

8.00

7.96

7.96

7.88

/=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

8

6.32

6.30

6.21

6.31

6.55

6.52

6.32

6.32

6.32

/=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

2'

6.92

7.41

7.44

7.44

5'

6.90 1

6.80

7=8

6'

7.10

7.00

6.90

6.84 7=2/8

7.54 7=2/8

7.54 7=2/8

7.56 7=2/8

7'

7.16 /=16

5.90 7=5.2

6.05 7=5.4

6.07 7=5.4

8'

6.64

4.47

4.43

4.51

7.55

7.20

7.40

7=16

7=5.2

7=5.4

7=5.4

OCH,

3.72

3.72

3.80

3.77

3.97

3.68

3.80

(x3)

3.82

3.83

4.04

3.76

3.84

3.88

3.88

3.84

4.12

3.83

(x2)

(x3)

(x2)

3.96

(x2)

3.92

3.90

3.96

4.01

3.86

3.96

(x2)

4.10

3.91

(x2)

3.98

2-CH2

3.90

3.86

solvent5'

C

A

C

C

A

C

C

A

C

Table 3.2 Selected 1H NMR spectral data of salvianolic acids and related compounds.

H

4b

5

5a

6

6a

7

7a

8

8a

2

7.08 7=2

6.91 7=2

5

7.04

7.01

7.10

6.80

6.88

6.95

6.97

6.81

6.96

7=8

7=8

7=8

7=8

7=8

/=8

7=8

7=8

7=8

6

7.51

7.40

7.60

7.20

7.42

6.95

6.95

7.13

7.15

7=8

7=8

7=8

7=8

7=8

M

7=8

7=2/8

7=2/8

7

7.85

7.56

7.65

8.00

8.11

2.31

2.33

7.50

7.53

7=16

7=16

7=16

2.49

2.54

7=9/15

7=16

7=16

8

6.31

6.28

6.30

6.14

6.31

5.52

5.43

6.20

6.20

7=16

7=16

7=16

7=16

7=16

7=6/9

7=6/9

7=16

7.02

7.20

7.73

6.60

7.10

6.80

7.10

3.78

7.02

7.20

7.73

7.14

7.38

7.78

7.34

7.30

7.39

3.86

solvent* C

constant of J=3 Hz indicated the presence of a cis disubstituted benzodioxane skeleton for salvianolic acid J. The location of the aryl and carboxyl groups on the dioxane ring was determined by selective DEPT experiments. The calculated chemical shift value of the aryl bearing methine proton (8 5.31, C-7H) should be at lower field compared to that of the carboxyl bearing methine proton (84.81, C -8H). When the methine proton signal at 8 5.31 and the aromatic proton doublet at 8 7.28 (J=2, C-2H) were selectively irradiated, an enhancement of the C-4 signal at 8 142.9 was observed in both cases. Thus this carbon (C-4) is coupled individually through three bonds to C-7H and C-2H.

Table 3.3 Selected NMR spectral data of salvianolic acids and related compounds.

H

8b

9

9a

10

lib

12

12a

13

15

2

7.08

7.18

7.12

7.28

7.21

7.40

7.41

7=2

7=2

7=2

7=2

7=2

7=2

s,br

5

7.13

6.79

6.75

6.88

7.11

6.88

6.84

7.00

6.83

7=8

7=8

7=8

7=8

7=8

7=8

7=8

7=8

7=8.3

6

7.28

7.01

6.97

7.14

7.53

7.04

7.08

7.32

7.18

7=2/8

7=2/8

7=2/8

7=2/8

7=8

7=2/8

7=2/8

7=8

7=8.3

7

7.50

7.60

7.61

7.48

7.80

7.60

7.68

7.72

7.41

7=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

8

6.23

6.39

6.33

6.40

6.30

6.32

6.32

6.36

6.33

7=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

7=16

T

7.49

7.30

7.37

6.74

6.90

;=2

7=2

7=2

7=2

6.80

5'

6.92

6.80

6.82

6.61

6.80

7=8

7=8

7=8

7=8

6'

7.28

7.12

7.19

6.68

7.00

/=2/8

/=2/8

7=2/8

7=2/8

7'

7.41

7.37

7.38

5.31

6.94

5.90

7=3

7=11.5

7=5.2

8'

4.81

7.00

4.81

7=3

7=11.5

7=5.2

OCH,

3.68

3.74

3.72

3.75

3.79

(x2)

3.77

3.87

3.98

3.77

3.91

(x2)

3.84

3.97

3.86

(x2)

(x2)

Glu-1"

j ,yo

4.79

7=7.2

solvent*

A

A

c

D

D

A

C

A

D

a1): methylated compound b2): hydrolysis product of methylated compound.

a1): methylated compound b2): hydrolysis product of methylated compound.

CHEMICAL TRANSFORMATION

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