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Secoiridoids and hydroxycoumarins in Bulgarian Fraxinus species
) Pergamon
Biochemical Sysrematics and Ecology, Vol.25, No. 3, pp. 271-274,1997 © 1997 ElsevierScienceLtd All rights reserved.Printedin GreatBritain 0305-1978/97 $17.00+0.00
PII: S0305-1978(97)00006-9
Secoiridoids and Hydroxycoumarins in Bulgarian Fraxinus Species TANIA IOSSIFOVA,* IVANKA KOSTOVA*$ and LJUBA N. EVSTATIEVAI *Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria; tlnstitute of Botany, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria
Key W o r d Index--Fraxinus omus; Fraxinus excelsior, Fraxinus oxycarpa; Fraxinus pallisiae; Oleaceae; secoiridoids; hydroxycoumarins; chemotaxonomy.
Subject and Source Four Fraxinus L. species grow in Bulgaria: Fraxinus ornus L., Fraxinus excelsior L., Fraxinus oxycarpa Birb ex Willd (=F. angustifolia Vahl. ssp. oxycarpa [Birb ex Willd] Franco et Rocha Afonso) and Fraxinus pallisiae WUmott. Samples of F. ornus and F. excelsior bark and leaves were collected at the beginning of October 1994 in the region of Sofia. Samples of F. pallisiae and F. oxycarpa were collected at the end of September 1994 near the town of Tzarevo. Voucher specimens have been deposited in the Herbarium of the Inst. of Botany, BAS, Sofia.
Previous W o r k Secoiridoids: F. ornus bark and leaves--ornoside, ligstroside, hydroxyornoside (Iossifova et al., 1993, 1995; Kostova et al., 1993); F. excelsior leaves--10-hydroxyligstroside, excelsioside, oleuropein, ligstroside, 7-~-Dglucopyranosyl oleoside, 7,11 -oleoside dimethyl ester (Jensen and Nielsen, 1976; Damtoft et aL, 1992); F. oxycarpa leaves and bark--ligstroside, oleuropein, ligstral, angustifolioside A, angustifolioside B, 10-hydroxyligstroside ((~alis et al., 1993; Kostova et al., 1995). Hydroxycoumarins: F. omus, F. oxycarpa and F. excelsior bark and leaves--a number of 6,7-dihydroxy-, 6,7,8-trihydroxy- and 5,6,7-trihydroxycoumarins were reported (Artemevea et al., 1975; Jensen and Nielsen, 1976; Kostova, 1992; Nykolov et aL, 1993; Kostova et aL, 1994, 1995). There are no literature data on the chemical constituents of F. pallisiae. According to the classification of Engler (1920) F. excelsior, F. pallisiae and F. oxycarpa belong to the section Fraxinaster, while F. ornus belongs to the section Ornus.
Present Study The air-dried leaves of F. pallisiae were extracted successively with CHCI3 and MeOH and the extracts were evaporated under reduced pressure. The methanol extract was further worked up by vacuum liquid chromatography over silica gel and selected fractions were subjected to CC and TLC over silica gel. Four secoiridoids---oleuropein 1 (as a major component), ligstroside 2, lO-hydroxyligstroside 3 and 7,11-oleoside dimethyl ester 4, were isolated, as well as the coumarins esculin 5 and fraxin 6. All compounds were identified by a comparison of their IR, 1H and 13C NMR spectra with literature data. This is the first report of the presence of 1-6 in F. pallisiae. In the case of F. oxycarpa leaves the same working procedure was followed and the same four secoiridoids 1-4 and two coumarins 5 and 6 were isolated. The occurrence of the 7,11 -oleoside dimethyl ester 4 in this species is reported for the first time here. The TLC comparison of chloroform extracts of F. pallisiae and F. oxycarpa leaves using authentic samples and two different solvent systems (A: toluene-Et20, 1:2-saturated with 10% aq. CH3COOH; B: EtOAc-tolueneEtOH, 60:40:8) showed the presence of esculetin 7, fraxetin 8, isoscopoletin 9 and traces of scopoletin 10 in both of them. TLC investigation of the chloroform and methanol extracts of F. excelsior leaves using authentic samples 1 10 and two different solvent systems for each of the extracts (A, B for chloroform and C, D for methanol $Corresponding author. (Received 13 September 1996; accepted 6 January 1997) 271
272
T. IOSSIFOVA ETAL. 7
11
R#)oc
CO01~
R3e / ~ - ~
RI
R2
R3
CH]
H
Oleuropein
OH
CH3
H
Ligstrosid¢
OH
CH3
OH
10-Hydroxyligstrosid¢
CH3
H
7,1 i-Oleosidedimethylester
H
Omosid¢
H
Hydroxyomosicle
H
3~c~c~ 4
II
~ ~ CH~
I " 2"' // --CH2CH2-'==~ ",
6 OGh
\\,..
~-==OH
.
O~CHzCH2~ 7" 8" 12 --CH2CH 2
Q--O~___~ OH
extract--C: EtOAc-HCOOH-CH3COOH-H20, 100:15:15:27; D: CHCI3-MeOH-H20, 30:10:3, to 25 ml of the lower layer 0.5 ml of HCOOH is added) confirmed the presence of these compounds in our sample. The results of TLC comparison of chloroform and methanol extracts of F. omus, F. oxycarpe, F. pallisiae and F. excelsior bark and leaves are given in Table 1. The quantity of secoiridoids 1-4 and coumarins 5-10 was established to be similar in the extracts of the three Fraxinus species belonging to the section Fraxinaster. However, a significant difference was observed between the secoiridoid and coumarin content of Fraxinaster's species and that of F. ornus (section Ornus). In our previous work on F. o m u s we described the occurrence of secoiridoids---ligstroside 2, ornoside 11, hydroxyornoside 12 (Iossifova et al., 1993, 1995; Kostova et aL, 1993) and a number of coumarins (Kostova, 1992; Nykolov et al., 1993), including coumarins 5-10. Our present comparative studies have shown that the macrocyclic secoiridoid diesters 11 and 12 are not present in F. pallisiae, F. oxycarpa and /E excelsior, while oleuropein 1 and lO-hydroxyligstroside 3 are not found in F. ornus. Another big difference between F. ornus on the one hand and F. pallisiae, F. oxycarpa and F.
S E C O I R I D O I D S A N D H Y D R O X Y C O U M A R I N S IN B U L G A R I A N
R2 ~
' ~
273
SPECIES
FRAXINUS
-O" ~'O
/
Rs
RI
R2
R3
5
OGlu
OH
H
Esculin
6
OMe
OH
OGlu
Fraxin
7
OH
OH
H
Esculetin
8
OMe
OH
OH
Fraxetin
9
OH
OMe
H
Isoscopoletin
I0
OMe
OH
H
Scopoletin
T A B L E 1. D I S T R I B U T I O N OF S O M E S E C O I R I D O I D S A N D H Y D R O X Y C O U M A R I N S IN F. A N D F.
PALLISIAE
Secoiridoids Section
ORNUS,
F. E X C E L S I O R ,
F. O X Y C A R P A
B A R K A N D LEAVES
Species
1
Hydroxycoumarins
2
3
4
11
12
5
6
7
8
9
10
(+)
A. Bark
Ornus Fraxinaster
F. o m u s
-
++
-
-
+++
++
+/,10"
+++
+++
++
(+)
F. e x c e l s i o r
++
+++
++
+
-
-
++
+++
++
+++
(+)
(+)
F. o x y c a r p a
+
+++
++
+
-
-
++
++4-
++
++4-
(+)
(+)
F. p a / l i s i a e
+
+ + +
+ +
+
-
-
+ +
+ + +
+ +
+ + +
(+)
(+)
F. o r n u s
-
+
-
-
4-+
4-4-4-
4-/5*
4-4-
4-4-4-
4-+
-
-
F. e x c e l s i o r
+/,5"
4-
+
+
-
-
+
+ +
+
+ +
-
-
F. o x y c a r p a
+ + +
+
+
+
-
-
+
+ +
+
4- + +
-
-
F. p a l l i s i a e
+ + +
+
+
+
-
-
+
+ + +
+
+ +
--
-
B. Leaves
Ornus Fraxinaster
(+) = presence of a trace amount. + = presence of the compound. The number of" + " reflects the quantity of the respective compound. - = absence of the compound. "The designation + / . 1 0 means that the content of this compound is 1 0 times more than " + ".
excelsior on the other hand is the ratio of esculin 5 and fraxin 6 content. Esculin 5 dominates in F. ornus, while fraxin 6 and its aglucone 8 predominate in all the above-mentioned species of section Fraxinaster, although their quantity is always less than that of esculin 5 in F. ornus.
Chemotaxonomical Significance The presence of various hydroxycoumarins and oleoside type secoiridoids is a characteristic feature of Fraxinus species. Our present investigations on the distribution of the main secoiridoids and hydroxycoumarins in the four Bulgarian Fraxinus species, have revealed a close chemical relationship between F. excelsior, F. oxycarpa and F. pallisiae. Fraxinus ornus exhibited some qualitative and quantitative difference in the chemical composition. These observations support the classification of Engler (1920), based on morphological grounds in
274
T. IOSSIFOVAETAL.
which these three species have been set apart in the section Fraxinaster, while F. ornus has been detached in the section Ornus. Our results indicate clearcut differences between the members of the two sections. For now we have no chemical evidence for the differentiation of the Bulgarian species in the section Fraxinaster. Acknowledgements--The financial support of this work by the National Foundation "Scientific Investigations" is gratefully acknowledged.
References Artemevea, M. V., Karryev, M, O. and Nykonov, G. K. (1975) Rastit. Resur. 11, 368-371; (~alis, I'., Hosny, i . , Khalifa, T. and Nishibe, S. (1993) Phytochemistry 33, 1453-1456; Damtoft, S., Franzyk, H. and Jensen, S. R. (1992) Phytochemistry 31, 4197-4201; Engler, A. (1920) Oleaceae-Oleoidae-Fraxinae. In Das Pflanzenreich Regnii vegetabilis Conspectus, 9. Vien; Iossifova, T., i i k h o v a , B. and Kostova, I. (1993) Phytochemistry 34, 1373-1376; Iossifova, T., Mikhova, B. and Kostova, I, (1995) Mortars. f Chemie 126, 1 257-1264; Jensen, S. R. and Nielsen, B. J. (1976) Phytochernistry 15, 221-223; Kostova, I. (1992) Planta Med. 58, 484; Kostova, I., Yossifova, T., Vassileva, E. and Mikhova, B. (1993) Planta Med. 59, A705; Kostova, I., Sidjimov, A., Kostova, V. and Dimov, S. (1994) Compt. rend. Acad. bu/g. Sci. 47, 45-48; Kostova, I., Dinchev, D., Mikhova, B. and Iossifova, T. (1995) Phytochernistry 38, 801802; Nykolov, N., Iossifova, T., Vassileva, E., Kostova, I. and Stoev, G. (1993) PhytochemicalAnalysis 4, 86-88.
Biochemical Sysrematics and Ecology, Vol.25, No. 3, pp. 271-274,1997 © 1997 ElsevierScienceLtd All rights reserved.Printedin GreatBritain 0305-1978/97 $17.00+0.00
PII: S0305-1978(97)00006-9
Secoiridoids and Hydroxycoumarins in Bulgarian Fraxinus Species TANIA IOSSIFOVA,* IVANKA KOSTOVA*$ and LJUBA N. EVSTATIEVAI *Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria; tlnstitute of Botany, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria
Key W o r d Index--Fraxinus omus; Fraxinus excelsior, Fraxinus oxycarpa; Fraxinus pallisiae; Oleaceae; secoiridoids; hydroxycoumarins; chemotaxonomy.
Subject and Source Four Fraxinus L. species grow in Bulgaria: Fraxinus ornus L., Fraxinus excelsior L., Fraxinus oxycarpa Birb ex Willd (=F. angustifolia Vahl. ssp. oxycarpa [Birb ex Willd] Franco et Rocha Afonso) and Fraxinus pallisiae WUmott. Samples of F. ornus and F. excelsior bark and leaves were collected at the beginning of October 1994 in the region of Sofia. Samples of F. pallisiae and F. oxycarpa were collected at the end of September 1994 near the town of Tzarevo. Voucher specimens have been deposited in the Herbarium of the Inst. of Botany, BAS, Sofia.
Previous W o r k Secoiridoids: F. ornus bark and leaves--ornoside, ligstroside, hydroxyornoside (Iossifova et al., 1993, 1995; Kostova et al., 1993); F. excelsior leaves--10-hydroxyligstroside, excelsioside, oleuropein, ligstroside, 7-~-Dglucopyranosyl oleoside, 7,11 -oleoside dimethyl ester (Jensen and Nielsen, 1976; Damtoft et aL, 1992); F. oxycarpa leaves and bark--ligstroside, oleuropein, ligstral, angustifolioside A, angustifolioside B, 10-hydroxyligstroside ((~alis et al., 1993; Kostova et al., 1995). Hydroxycoumarins: F. omus, F. oxycarpa and F. excelsior bark and leaves--a number of 6,7-dihydroxy-, 6,7,8-trihydroxy- and 5,6,7-trihydroxycoumarins were reported (Artemevea et al., 1975; Jensen and Nielsen, 1976; Kostova, 1992; Nykolov et aL, 1993; Kostova et aL, 1994, 1995). There are no literature data on the chemical constituents of F. pallisiae. According to the classification of Engler (1920) F. excelsior, F. pallisiae and F. oxycarpa belong to the section Fraxinaster, while F. ornus belongs to the section Ornus.
Present Study The air-dried leaves of F. pallisiae were extracted successively with CHCI3 and MeOH and the extracts were evaporated under reduced pressure. The methanol extract was further worked up by vacuum liquid chromatography over silica gel and selected fractions were subjected to CC and TLC over silica gel. Four secoiridoids---oleuropein 1 (as a major component), ligstroside 2, lO-hydroxyligstroside 3 and 7,11-oleoside dimethyl ester 4, were isolated, as well as the coumarins esculin 5 and fraxin 6. All compounds were identified by a comparison of their IR, 1H and 13C NMR spectra with literature data. This is the first report of the presence of 1-6 in F. pallisiae. In the case of F. oxycarpa leaves the same working procedure was followed and the same four secoiridoids 1-4 and two coumarins 5 and 6 were isolated. The occurrence of the 7,11 -oleoside dimethyl ester 4 in this species is reported for the first time here. The TLC comparison of chloroform extracts of F. pallisiae and F. oxycarpa leaves using authentic samples and two different solvent systems (A: toluene-Et20, 1:2-saturated with 10% aq. CH3COOH; B: EtOAc-tolueneEtOH, 60:40:8) showed the presence of esculetin 7, fraxetin 8, isoscopoletin 9 and traces of scopoletin 10 in both of them. TLC investigation of the chloroform and methanol extracts of F. excelsior leaves using authentic samples 1 10 and two different solvent systems for each of the extracts (A, B for chloroform and C, D for methanol $Corresponding author. (Received 13 September 1996; accepted 6 January 1997) 271
272
T. IOSSIFOVA ETAL. 7
11
R#)oc
CO01~
R3e / ~ - ~
RI
R2
R3
CH]
H
Oleuropein
OH
CH3
H
Ligstrosid¢
OH
CH3
OH
10-Hydroxyligstrosid¢
CH3
H
7,1 i-Oleosidedimethylester
H
Omosid¢
H
Hydroxyomosicle
H
3~c~c~ 4
II
~ ~ CH~
I " 2"' // --CH2CH2-'==~ ",
6 OGh
\\,..
~-==OH
.
O~CHzCH2~ 7" 8" 12 --CH2CH 2
Q--O~___~ OH
extract--C: EtOAc-HCOOH-CH3COOH-H20, 100:15:15:27; D: CHCI3-MeOH-H20, 30:10:3, to 25 ml of the lower layer 0.5 ml of HCOOH is added) confirmed the presence of these compounds in our sample. The results of TLC comparison of chloroform and methanol extracts of F. omus, F. oxycarpe, F. pallisiae and F. excelsior bark and leaves are given in Table 1. The quantity of secoiridoids 1-4 and coumarins 5-10 was established to be similar in the extracts of the three Fraxinus species belonging to the section Fraxinaster. However, a significant difference was observed between the secoiridoid and coumarin content of Fraxinaster's species and that of F. ornus (section Ornus). In our previous work on F. o m u s we described the occurrence of secoiridoids---ligstroside 2, ornoside 11, hydroxyornoside 12 (Iossifova et al., 1993, 1995; Kostova et aL, 1993) and a number of coumarins (Kostova, 1992; Nykolov et al., 1993), including coumarins 5-10. Our present comparative studies have shown that the macrocyclic secoiridoid diesters 11 and 12 are not present in F. pallisiae, F. oxycarpa and /E excelsior, while oleuropein 1 and lO-hydroxyligstroside 3 are not found in F. ornus. Another big difference between F. ornus on the one hand and F. pallisiae, F. oxycarpa and F.
S E C O I R I D O I D S A N D H Y D R O X Y C O U M A R I N S IN B U L G A R I A N
R2 ~
' ~
273
SPECIES
FRAXINUS
-O" ~'O
/
Rs
RI
R2
R3
5
OGlu
OH
H
Esculin
6
OMe
OH
OGlu
Fraxin
7
OH
OH
H
Esculetin
8
OMe
OH
OH
Fraxetin
9
OH
OMe
H
Isoscopoletin
I0
OMe
OH
H
Scopoletin
T A B L E 1. D I S T R I B U T I O N OF S O M E S E C O I R I D O I D S A N D H Y D R O X Y C O U M A R I N S IN F. A N D F.
PALLISIAE
Secoiridoids Section
ORNUS,
F. E X C E L S I O R ,
F. O X Y C A R P A
B A R K A N D LEAVES
Species
1
Hydroxycoumarins
2
3
4
11
12
5
6
7
8
9
10
(+)
A. Bark
Ornus Fraxinaster
F. o m u s
-
++
-
-
+++
++
+/,10"
+++
+++
++
(+)
F. e x c e l s i o r
++
+++
++
+
-
-
++
+++
++
+++
(+)
(+)
F. o x y c a r p a
+
+++
++
+
-
-
++
++4-
++
++4-
(+)
(+)
F. p a / l i s i a e
+
+ + +
+ +
+
-
-
+ +
+ + +
+ +
+ + +
(+)
(+)
F. o r n u s
-
+
-
-
4-+
4-4-4-
4-/5*
4-4-
4-4-4-
4-+
-
-
F. e x c e l s i o r
+/,5"
4-
+
+
-
-
+
+ +
+
+ +
-
-
F. o x y c a r p a
+ + +
+
+
+
-
-
+
+ +
+
4- + +
-
-
F. p a l l i s i a e
+ + +
+
+
+
-
-
+
+ + +
+
+ +
--
-
B. Leaves
Ornus Fraxinaster
(+) = presence of a trace amount. + = presence of the compound. The number of" + " reflects the quantity of the respective compound. - = absence of the compound. "The designation + / . 1 0 means that the content of this compound is 1 0 times more than " + ".
excelsior on the other hand is the ratio of esculin 5 and fraxin 6 content. Esculin 5 dominates in F. ornus, while fraxin 6 and its aglucone 8 predominate in all the above-mentioned species of section Fraxinaster, although their quantity is always less than that of esculin 5 in F. ornus.
Chemotaxonomical Significance The presence of various hydroxycoumarins and oleoside type secoiridoids is a characteristic feature of Fraxinus species. Our present investigations on the distribution of the main secoiridoids and hydroxycoumarins in the four Bulgarian Fraxinus species, have revealed a close chemical relationship between F. excelsior, F. oxycarpa and F. pallisiae. Fraxinus ornus exhibited some qualitative and quantitative difference in the chemical composition. These observations support the classification of Engler (1920), based on morphological grounds in
274
T. IOSSIFOVAETAL.
which these three species have been set apart in the section Fraxinaster, while F. ornus has been detached in the section Ornus. Our results indicate clearcut differences between the members of the two sections. For now we have no chemical evidence for the differentiation of the Bulgarian species in the section Fraxinaster. Acknowledgements--The financial support of this work by the National Foundation "Scientific Investigations" is gratefully acknowledged.
References Artemevea, M. V., Karryev, M, O. and Nykonov, G. K. (1975) Rastit. Resur. 11, 368-371; (~alis, I'., Hosny, i . , Khalifa, T. and Nishibe, S. (1993) Phytochemistry 33, 1453-1456; Damtoft, S., Franzyk, H. and Jensen, S. R. (1992) Phytochemistry 31, 4197-4201; Engler, A. (1920) Oleaceae-Oleoidae-Fraxinae. In Das Pflanzenreich Regnii vegetabilis Conspectus, 9. Vien; Iossifova, T., i i k h o v a , B. and Kostova, I. (1993) Phytochemistry 34, 1373-1376; Iossifova, T., Mikhova, B. and Kostova, I, (1995) Mortars. f Chemie 126, 1 257-1264; Jensen, S. R. and Nielsen, B. J. (1976) Phytochernistry 15, 221-223; Kostova, I. (1992) Planta Med. 58, 484; Kostova, I., Yossifova, T., Vassileva, E. and Mikhova, B. (1993) Planta Med. 59, A705; Kostova, I., Sidjimov, A., Kostova, V. and Dimov, S. (1994) Compt. rend. Acad. bu/g. Sci. 47, 45-48; Kostova, I., Dinchev, D., Mikhova, B. and Iossifova, T. (1995) Phytochernistry 38, 801802; Nykolov, N., Iossifova, T., Vassileva, E., Kostova, I. and Stoev, G. (1993) PhytochemicalAnalysis 4, 86-88.