Chemical components of Fraxinus ornus bark — Structure and biological activity

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 26 © 2002 Elsevier Science B.V. All rights reserved.

313

CHEMICAL COMPONENTS OF FRAXINUS ORNUS BARK - STRUCTURE AND BIOLOGICAL ACTIVITY IVANKA N. KOSTOVA AND TANYA lOSSIFOVA Institute of Organic Chemistry with Centre ofPhytochemistry, Bulgarian Academy of Sciences, Bg-1113 Sofia, Bulgaria ABSTRACT: This review describes the investigations carried out by the authors and coworkers on the chemical composition and biological activity of Fraxinus ornus. Its stem bark has been used in the traditional medicine for treatment of wounds, inflammation, arthritis and dysentery. Our results support the claims of the folk medicine by presenting scientific proofs for the antimicrobial, antiinflammatory, immuno-modulatory, skinregenerating, antioxidant, photo-dynamic damage prevention and antiviral properties of the bark extract and its components. The ethanolic extract of the stem bark and its main constituent esculin were found practically non-toxic. They inhibited the classical and alternative pathways of complement activation. The extract and esculin displayed antiinflammatory activity in both zymosan- and carrageenan-induced paw edema in mice. The extract exhibited a pronounced antioxidative activity and caused intense wound epithelization. The antimicrobial and photodynamic damage prevention properties of the extract and its fractions were dependable on their hydroxycoumarin composition. The isolated secondary metabolites belong to the groups of hydroxycoumarins, secoiridoids, phenylethanoids and lignans. Most of the coumarins and secoiridoids possess the ability to affect the complement activity. A clear correlation between the structure and the biological activity (antimicrobial, antioxidative and photodynamic damage prevention) of the studied hydroxycoumarins was observed. Isolation of new biologically active compounds and finding of new biological properties of already known bioactive substances has been achieved.

INTRODUCTION Throughout the ages plants have been the source of medicinal agents. Today a revival of thousand-years-old herbal remedies and a return to the ancient form of medicine is observed. Herbal remedies are common in Asia and Europe, particularly in Germany, France and Italy. More and more Americans are supplementing and replacing prescription medicines with various medicinal herbs. However, in many cases the claims of the folk medicine are still to be scientifically proved. Some claims may not be accurate. The herbal medicines should also meet the contemporary requirements for safety and effectiveness. The expansion of the market for herbs demands strict standards for ingredients and manufacturing. The standardization of the herbal preparations requires a detailed study of their chemical composition and finding of the active components. Herb remedies that

314 enjoy the greatest popularity are generally those that have been the most thoroughly investigated. Fraxinus ornus (L.) is a small tree belonging to the Oleaceae family widely found in Bulgaria. Its stem bark is used in the Bulgarian folk medicine for treatment of infected wounds, inflammation, arthritis and dysentery [1,2]. However, the claims of the traditional medicine were not scientifically confirmed, the toxicity of the extract was not examined and the chemical composition of the bark was not completely investigated. In order to explain and confirm the biological activities claimed by the traditional medicine, and to search for new biologically active compounds we studied the antimicrobial, antioxidative, immunomodulatory, antiinflammatory, skin-regenerating and antiviral properties of Fraxinus ornus bark extract and its components. In a parallel detailed phytochemical investigation of the extract we isolated and determined the structures of many hydroxycoumarins, secoiridoid glucosides, caffeoyl esters of phenylethanoid glycosides, lignans and other phenolic compounds. In this review we present the results from our phytochemical and biological investigations. PREVIOUS INVESTIGATIONS Previous investigations on bark, flowers and leaves of Fraxinus ornus have shown the presence of the hydroxycoumarins esculin (1), esculetin (2), fraxin (3 ), fraxetin (4), cichoriin (5) and of some phenolic acids [36]. The isolation of the flavonoids rutin, quercetin, quercetin-3-glucoside, quercetin-3-galactoside, quercetin-3,7-digalactoside and rhamnetin from leaves and flowers has also been reported [7-9]. GENERAL APPROACH The bark of Fraxinus ornus was subjected to systematic phytochemical investigation. The isolated compounds and some bark extracts were fiirther investigated for their biological activities. In some cases synthetic derivatives of the natural compounds were prepared and their biological properties studied.

315

CHEMICAL STUDIES Extraction Extraction of the dried and well ground plant material was carried out with hot ethanol. The extract was concentrated to a small volume and the deposited solid (a mixture of the coumarin glucosides 1 and 3) filtered. The mother liquor was concentrated to obtain the total ethanol extract 1 (TEl). In some cases the ethanolic extract was concentrated without removal of the deposited solid to give the total ethanol extract 2 (TE2). TEl and TE2 were further used in the biological studies and for isolation of pure components. Hydroxycoumarins A characteristic feature of Fraxinus species is the presence of simple hydroxycoumarins having OH or/and OMe groups only in the benzene ring. In Fraxinus ornus we found derivatives of 6,7-dihydroxy-, 6,7,8trihydroxy- and 5,6,7-trihydroxycoumarins. Isolation of Hydroxycoumarins

For isolation of hydroxycoumarins the TEl was subjected to liquid vacuum chromatography (LVC) with PE, CHCI3, EtOAc and MeOH to yield the corresponding fractions [10]. No coumarins were found in the PE fraction. The CHCI3 fraction was chromatographed over a silica gel column with a dichloroethane (DCE) - MeOH gradient. A TLC study of the DCE fractions on silica gel yielded the coumarins esculetin (2), fraxetin (4), scoparone (6), isoscopoletin (7), scopoletin (8), fraxidin (9) and fraxinol (10). Identification of all coumarins was achieved by UV, IR, ^H NMR and mass spectra, and direct comparison with authentic samples. NOE experiments confirmed the structures of 9 and 10. RP' HPLC Analysis of Hydroxycoumarins in Fraxinus ornus Bark Extract

The hydroxycoumarin content of plants varies significantly depending on the stage of the plant growth and development, and on the climatic conditions. Investigations of these variations are of scientific and practical interest. High pressure liquid chromatography (HPLC) is the most promising method for separation and detection of coumarins because it allows high resolution and a rapid and reproducible determination even of

316 trace compounds [11-13]. However, the very complex hydroxycoumarin composition of many plants requires further improvement and development of the HPLC procedure.

R'

R^

R^

R'

1

H

OGlc

OH

H

la

H

OGlc 4Ac

OAc

H

2

H

OH

OH

H

2a

H

OAc

OAc

H

3

H

OMe

OH

OGlc

4

H

OMe

OH

OH

4a

H

OMe

OAc

OAc

5

H

OH

OGlc

H

6

H

OMe

OMe

H

7

H

OH

OMe

H

8

H

OMe

OH

H

9

H

OMe

OMe

OH

OMe

OH

OMe

H

10 11

H

OGIc

OMe

H

12

H

OMe

OMe

OMe

13

H

H

H

H

Glc == glucose Ac = acetate

In this connection we attempted a reverse phase - high pressure liquid chromatography (RP-HPLC) determination of 11 naturally occurring hydroxycoumarins 1-8 and 11-13 [14]. Conditions were found for best resolution of the standard mixture - mobile phase H20-MeOH, detection at A.= 220nm, and appropriate gradient profile and flow rate. These conditions were used for the HPLC analysis of the total extract of Fraxinus ornus bark.

317 2

3

30

w. 40 Mm.

Fig. (1). HPLC profile of the ethanolic bark extract of F. ornus collected from region 1 (see Table 1): peaks are labelled with the corresponding compound numbers.

The HPLC profile of the extract showed a good resolution of the main constituents. Esculin (1), esculetin (2), and fraxin (3) are the major components of this species, while the others are present in smaller amounts, Fig (1). Coumarin (13) was not detected in the extract. In addition 7-methylesculin (11) and 6,7,8-trimethoxycoumarin (12 ) were detected. This was the first report of the occurrence of 11 and 12 in the Oleaceae family. In Bulgaria Fraxinus ornus bark is a major source for the industrial preparation of esculin (1), an antiinflammatory and vitamin-P-like agent. The selection of appropriate plant material, i.e. of higher esculin (1) and of lower esculetin (2), fraxin (3) and fraxetin (4) content is economically important. For this reason a quantitative determination of 1-4 in commercial samples of Fraxinus ornus bark from five different regions in Bulgaria was carried out and the results presented in Table 1. The table shows that the samples from regions 1-4 are characterized by higher esculin (more than 8%) and total coumarin content (more than 9%). These values are also higher than those reported for the Chinese species F. chinensis, F. bungeana and F. stylosa [12]. Region 5 exhibited lower

318 esculin (6.3%) and higher esculetin, fraxin and fraxetin content (total coumarins 7.8%). Table 1. Concentrations of Hydroxycoumarins 1-4 in Samples of F. ornus Bark from Different Regions of Bulgaria

Regions 1 2 3 4

1

5

Esculin (I) 8.06 8.48 8.83 8.53 6.27

Esculetin (2) 0.25 0.26 0.29 0.29 0.50

Concentration % w/w) Fraxin Fraxetin (4) (3) 0.05 0.79 1.02 0.04 0.05 1.03 0.94 0.05 0.07 1.25

1

Total

1

9.15 9.80 10.20 9.81

7.79

1

The proposed method is applicable to the analytical control of 1-4 for scientific and industrial purposes. This method has been applied for determination of the hydroxycoumarin composition of all extracts in our further biological studies. It could be successfully used for phytochemical investigation of many hydroxycoumarin-bearing plants belonging to different families. Secoiridoids and Phenylethanoids Isolation of Secoiridoids and Phenylethanoids

The general scheme which we have employed for isolation of pure secoiridoids and phenylethanoids is schematically described below. Solvent-solvent partitioning of the TEl with PE and EtOAc yielded the corresponding PE, EtOAc and MeOH-H20 extracts. The EtOAc extract was further subjected to LVC with DCE-MeOH gradient yielding residues Rl and R2 (DCE-MeOH, 10:1); R3, R4 and R5 (DCE-MeOH, 5:1), and R6 and R7 (DCE-MeOH, 3:1). From Rl ligstroside (14), omoside (15) and caffeic acid (16) were isolated by LVC and TLC. R3 was subjected to CC over silica gel and HPLC to give hydroxyomoside (17). R4 after repeated CC and RP-HPLC afforded secoiridoids 14, 17-20a,b and the lignan 21. RP-HPLC of R5 gave the coumarin glucosides esculin (1) and fraxin (3), and the caffeoyl esters of phenylethanoid glucosides 22 and 23. RP-HPLC of R6 and R7 yielded the caffeoyl esters of phenylethanoid glycosides 24- 27.

319 Secoiridoid Glucosides

Secoiridoid glucosides of oleoside (28) type frequently occur in Oleaceae family. Usually, in Fraxinus species they are present as mono- or diesters of p-hydroxyphenylethanol (29) derivatives. Our phytochemical studies on Fraxinus ornus resulted in the isolation of ligstroside (14), oleuropein (18), framoside (19) and of the new compounds ornoside (15), hydroxyornoside (17), hydroxyframoside A (20a) and hydroxyframoside B (20b) [15-17]. The FAB spectrum of ornoside (15) exhibited [M+H]^ at m/z 629. Its ^H and ^"^C NMR spectra (Table 2) revealed the typical signals of an oleoside (28) nucleus and suggested the presence of one 1,4-disubstituted benzene ring, one 1,2,4-trisubstituted benzene ring and two sets of OCH2CH2Ph moieties. The ^H NMR spectrum of its acetate 15a exhibited signals for only four alcoholic and one phenolic acetyl group in the trisubstituted benzene ring, and suggested no free PhCH2 CH2 OH groups. Alkaline hydrolysis of ornoside (15) afforded glucose and the new phenolic compound, named omosol, whose structure was elucidated as 30 on the basis of its spectral data and chemical behavior. These findings implied that in ornoside, the ornosol (30) moiety is linked to the oleoside (28) through the nonphenolic CH2CH2OCO ester bonds at C-7 and C-11. The position of attachment was unambiguously established by detailed NOE experiments on ornoside. The most characteristic NOEs are presented in Fig. (2). On irradiation of CH2-2", an enhancement of the H-3 singlet, the doublet for H-4" and H-8" and CH21" was observed. Irradiation of CHa- T" leads to enhancement of CH2-2*" and H-4'". This confirms the assignment of CH2-I", 2", 1'" and 2'" and proves, that the disubstituted (tyrosol) moiety is linked to C-11. Therefore, the structure 15 was assigned to ornoside.

0-Glc

Fig. (2). Most important NOEs observed for 15

320

14

14a

CH2CH2—(

. 7 R^OOC

11 , C00R2

8

\ OGIcR^

>—OH

-OAc

CH2CH2-

R^

R^

R^

Me

OH

H

Me

OAc

8" T

CH2CH2-¥ T-OH \ / 5" 4' ^ _ ^ ^ C ' H / H 2

15

OH

r~8" CH2CH2—/

\—OAc

15a

OAc Cn2Cn2

CH2CH2—f

17

^

>—OH ^

CHjCHz-

/

\

^O—
OH

-2"

x:

y-CHCHz

-OAc

17a

O—/

OAc

V-CHCH2

=/ i,

>AC

OH

18

19

Me

-OH

CH2CH2"

CH2CH2—{

)—OH

HO—(f

OH

\—CH2CH2

OH

OH

20a

20b

CH2CHJ

-OH

CH2CH2—(

\—OH

Hu

\

CH2CH2—\

/ -OH

C H 2C H 2

OH

HO^ HO—/

H Me

/

V-CH2CH2

OH

H Me

OH OH

H H

Me

OH

OY

321

Table 2. ^H and ^^C NMR Data of Insularoside (15) and Hydroxyornoside (17)inCD30D(ppm) 17

15 1 Positions 1 3 4 5 6a 6b 7 8 9 10 11

r

2' 3' 4* 5' 6'a 6'b I'a I'b 2"a 2'b 3" 4" 5' 6" 7" 8" r"a r"b 2'" 3.,, 4'" 5"' 6"' 7'"

1 ^Z 1

(J in HZ) 5.87 brs 7.55 s

1

6H

!1 3.80 dd (10.9, 3.6) 2.17 dd (15.1, 10.9) 2.32 dd (15.1, 3.6)

172.6s 124.9d 129.9s 13.7q 167.9s 100.9d 74.2d 77.9d 71.4d 78.3d 62.7t

6.06 qd (7.2, 0.8)

1.62 dd (7.2, 1.5)

4.79 d (7.8) 3.28 dd (9.1, 7.8) 3.401 (9.1) 3.2-3.4 obscured by MeOH 3.2-3.4 obscured by MeOH 3.65 dd (11.8, 5.7) 3.87 dd (11.8, 2.0) 4.49 ddd (11.2, 8.5, 3.1) 4.53 ddd (11.2, 5.9,4.4) 2.91ddd (14.5, 5.9, 3.1) 3.02 ddd (14.5, 8.5,4.4)

65.9t 35.8t

-

7.21 d (8.6) 6.94 d (8.6)

-

6.94 d (8.6) 7.21 d (8.6) 4.03 ddd (10.8, 6.5,4.3) 4.27 ddd (10.8, 6.0, 4.0) 2.761 (5.3) 2H

1

-

6.53 d (2.0)

6.85 d (8.1) 6.77 dd (8.1,2.0)

1 |

135.4s 131.5d 120.9d 157.4s 120.9d 131.5d 66.3t 34.7 132.2s 120.0d 147.0s 147.6s 117.7d 125.1d

1

-

-

1

1

|6H(/inHz) 5.85 brs 7.55 s

5c 95.1d 155.3d 109.7s 31.3d 40.8t

3.80 dd (10.9, 3.9) 2.13 dd (15.1, 10.9) 2.23 dd (15.1,3.9)

172.6s 125.2d 129.9s 13.6q 167.6s 100.9d 74.7d 77.9d 71.4d 78.3d 62.7t

6.05 brq(7.0)

1.61 d (7.00)

4.78 d (7.8) 3.29 dd (9.1, 7.8) 3.421 (9.1) 3.2-3.4 obscured by MeOH 3.2-3.4 obscured by MeOH 3.66 dd (11.9, 5.7) 3.88 dd (11.9, 1.5) 4.28 dd (10.8, 7.8) 4.56 dd (10.8, 3.8) 4.90 dd (7.8, 3.8)

71.6d

1

138.3s I29.2d 120.5d 158.5s 120.5d 129.2d 66.3t

7.36 d (8.7) 6.97 d (8.7)

-

'

1

6.86 d (8.2) 1 7.79 dd (8.2, 1.8)

1

68.7t

-

6.58 d (1.8)

1

95.2d 155.6d 109.5s 31.3d 40.8t

-

-

j 6.97 d (8.7) j 7.36 d (8.7) 4.04 ddd (10.7, 6.5,4.0) 14.27 m 2.77 m2H

Sc

|

347t 132.3s 120.3d 146.7s 147.7s 117.8d 125.0d

1

1

Our paper on isolation and structure elucidation of omoside was already in press, when the publications of Tanahashi et al. [18] and Shen et al. [19] appeared in Phytochemistry. Tanahashi and co-authors were the first to report the isolation and structure determination of a novel secoiridoid glucoside from F. insularis, named insularoside, and to assign structure 15 to this compound. Later, Shen et al. described the occurrence of insularoside in Fraxinus uhdei under the name uhdoside [19,20].

322

Therefore, almost simultaneously and independently the secoiridoid glucoside 15 has been isolated from three different Fraxinus species under three different names. Our approach in its structure elucidation provided some additional data and more chemical evidence about this unusual secoiridoid. Insularoside (uhdoside, ornoside) belongs to the group of the rare secoiridoid glucosides in which a flexible macrocyclic ring is formed between one phenolic compound and the oleoside aglucone. Most probably, in the preferred conformation protons H-6a and H-6b are influenced by the ring current of the aromatic rings, which would explain the observed highfield shift of the corresponding protons in 15 versus 14 without a significant change of the coupling constants. This fact and the observed NOE enhancement of H-5 and H-1 on irradiation of H-6a and H6b, respectively, indicate that the configuration at C-5 and C-1 in insularoside is the same as that described for oleoside derivatives, Fig. (2) [21]. A molecular formula of C32H36O14 was established for hydroxyornoside (17) by its ^H and ^^CNMR and FAB spectra ([M+Na]"^ at m/z 667). Its ^H NMR spectrum clearly indicated that it is an analog of insularoside (15). However, instead of the two protons at 2" position as in 15, only one proton was observed at 6 4.80. The ^"^C NMR spectrum (Table 2) of 17 was in full agreement with that of 15 for all carbons except for C- 1", 2", 4" and 8". The most striking difference was observed for C-2" - a doublet at 8 71.5 for 17 instead of the triplet at 6 35.8 observed for insularoside. Upon acetylation, hydroxyornoside provided a hexaacetate (17a) which showed six acetyl singlets in its ^H NMR spectrum instead of five for insularoside. It was suggested that 17 is a hydroxyderivative of 15 with an additional OH group located at position 2". Extensive NOE studies on 17a revealed the close relationship in space between the acetate group at C-2" and H-3, H-l", H-2", H-4", and H.8", Fig. (3).

H „f^. AcO

Fig. (3). Important NOEs observed for 17a

323

On alkaline hydrolysis, 17 afforded a new phenolic compound whose structure was unambiguously shown to be 2"- hydroxyomosol (31) by its spectral data. Acid hydrolysis of hydroxyomoside (17) using 16.0% methanolic H2SO4 resulted in a complex reaction mixture from which 32a,b could be isolated as a minor product and 33a,b as a major one. Under the same reaction conditions insularoside (15) afforded 32a,b and 34a,b, while ligstroside produced only 32a,b.

(CH30)2HC

R^ 32a,b

CH3

33a,b

CH3

CH3 HOCH2CH2-

I V

CHCHo

in HOCH2CH2-

34a,b

CH3

36a,b

CH3

OH 2" / V •CH2CH2

ACOCH2CH2-

•OAc

/"V

2"

CH2CH2

RtlOC ^ C00R2

OH

35a

R' =

CHjCH

CH3

35b

R' =

CHjCHj

CH3

r\.

OH

324

The ^H NMR (Table 3) and MS data of the less polar compound supported the proposed structure 32a9b and were in agreement with those described for the natural compounds 35a and 35b [22]. However, no CHO signal was visible and the presence of two CH protons and four OMe signals was observed. This suggested that compound 32a,b is a derivative of 35a and 35b in which the CHO group at position 1 has been acetalized. To account for the presence of two diastereomers we accept that under the applied reaction conditions a rearrangement of the secoiridoid nucleus takes place via the mechanism already proposed by Gariboldi et al. for the formation of 35a and 35b from oleuropein (18 ) in plants [22]. Evidently, under the conditions of acid hydrolysis this rearrangement is followed by acetalization of the CHO group. The stereochemistry at C-8 and C-9 in 32a,b could not be assigned with the help of NMR experiments because of the overlapping of ^H NMR signals. Table 3. *H NMR Data for Compounds 32a,b, 33a,b, 34a,b and 36a,b in CDCI3 (ppm); Coupling Constants (Hz) Are Given in Parentheses 1 Positions H-1 d H-3s H-5m Ha-6 dd Hb-6 dd H-8m H-9m H-lOd CH2-l"td CH2-2" td H-4", H-8" d H-5", H-7" d CH2-r"t CH2-2"'t H-4"' d H-7'" d H-8"' dd COOCH3 OCH3

1 AcO a.b

32a,b 4.41 (3.4) 4.29 (7.8) 7.58 7.54 3.30-3.10 2H 2.83(15.3,3.5)^ 2.39(16.0,4.4)^ 2.23(15.5,11.0)* 2.57(16.0,8.0)'' 4.20-4.10 2H 2.00-1.90 2H 1.43(7.0) 1.41 (7.0)

3.71 3H, 3.69 3H 3.68 3H, 3.67 3H 3.36 3H, 3.34 3H 3.31 6H

-

7.56 7.52 3.30-3.10 2H 2.68(16.0,3.5)^ 2.27(16.0,4.0)" 2.17(16.0,11.0)* 2.58(16.0,8.0)" 4.40-4.20 2H 2.00-1.90 2H 1.41(7.00) 1.39(7.00) 4.40-4.20 4H 4.40-4.20 2H 7.30 (8.5) 4H 6.98 (8.5) 4H 3.78 (6.5) 4H 2.75 (6.5) 4H 6.79 (1.8) 2H 6.99 (8.0) 2H 6.92(8.0, 1.8) 2H 3.68 3H, 3.66 3H

36a,b 34a,b "1:41 (3.4^ 4.39 (3.4) 4.22 (7.0) 4.26 (7.7) 7.55 7.54 7.50 7.50 3.30-3.10 2H 3.30-3.10 2H 2.76(16.0,3.4)* 2.66(16.0,3.5)* 2.34(16.0,4.4)" 2.29(16.0,4.2)" 2.23(16.0, 11.0)* 2.17(16.0,11.0)* 2.60(16.0,7.8)" 2.57(16.0,7.9)" 4.40-4.20 2H 4.20-4.10 2H 2.00-1.90 2H 2.00-1.90 2H 1.42(7.8) 1.41 (7.0) 1.39(6.5) 1.39(7.0) 4.301 (6.0) 4H 4.32 (7.0, 2.0) 4H 2.921 (6.0) 4H 2.93 (7.0, 2.0) 4H 7.17 (8.5) 4H 7.18 (7.0) 4H 6.92 (8.5) 4H 6.92 (7.0) 4H 4.22 (7.0) 4H 3.76 (6.5) 4H 2.85 (7.0) 4H 2.71 (6.5) 4H 6.82 (1.8) 2H 6.75 (1.9) 2H 7.07 (8.0) 2H 6.97 (7.0) 2H 6.96(8.0, 1.8) 2H 6.89(7.9. 1.8) 2H 3.67 3H, 3.66 3H 3.67 3H, 3.66 3H

3.35 3H, 3.33 3H 3.31 3H, 3.29 3H

3.35 3H, 3.33 3H 3.29 3H, 3.30 3H

33a,b 4.40-4.20 2H

3.34 3H, 3.32 3H 3.30 3H, 3.29 3H 2.16 6H, 1.99 6H

Signals with identical indexes belong to the same isomer; the other signals are not assigned.

1

|

325 The H NMR spectrum (Table 3) of the more polar compound 33a,b showed the absence of the sugar unit and revealed the protons of hydroxyomosol moiety and the rearranged secoiridoid nucleus. The doubling of all signals in the spectra suggested the existence of two diastereomers in nearly 1:1 ratio. The presence of two COOMe signals in addition to the four OMe singlets from the acetalized CHO group indicated that after hydrolysis of one of the ester linkages in hydroxyomoside a transesterification with MeOH has taken place. A similar information regarding the rearranged secoiridoid nucleus was forthcoming and from the ^H NMR spectrum of 34a,b, where the protons of ornosol moiety were also clearly visible. Acetylation of 34a,b afforded the acetate 36a,b, whose ^H NMR spectrum showed one aliphatic and one aromatic OAc signal (Table 3). The NOE experiments performed on 36a,b resulted in an enhancement of H-3, H-8", H-4", and CU2-T upon irradiation of CH2- 1" which suggested that the p-substituted phenethoxy unit is attached to C-11 of the secoiridoid nucleus. Therefore, under the applied conditions the acid methanolysis of 15 and 17 leads to opening of the macrocyclic ring at C-7. This is in accordance with the results of Tanahashi and coworkers [18] for the mild alkaline hydrolysis of insularoside (15) and the previous observations of LaLonde [23] that saturated esters undergo easier solvolysis compared to a,p - unsaturated ones. The new secoiridoid glucosides hydroxyframoside A (20a) and hydroxyframoside B (20b) were isolated as an unresolved mixture 20a,b in a ratio 2:1 as suggested by the ^H and ^^C NMR spectra. The molecular formula C32H38O14 was established for 20a and 20b based on the NMR spectra (Table 4) and the negative ESIMS, where only one peak at m/z 645 was found for the [M-H]" ion in both compounds. The ^H NMR spectrum of 20a,b suggested that each of the isomeric compounds had one 4-hydroxyphenethoxy and one 3,4-dihydroxy-phenethoxy unit. The exact position of their attachment to the oleoside nucleus of 20a and 20b was deduced on the basis of HMBC, HH-LR-COSY and NOESY spectra. The heteronuclear long range correlations from the methylene protons at 6 4.10 and 6 4.21 of isomer 20a to the carbon signal at 5 172.45 (assigned to C-7), and from the methylene protons at 8 4.09 and 4.22 of isomer 20b to the carbon signal at 6 172.5 (C-7) placed the corresponding CH2 groups at position 1'" in 20a and 20b, respectively. In the COSY spectrum of 20a,b the CH2-I'" protons of 20a and 20b showed coupling cross peaks to the protons resonances of the methylene groups at 5 2.77 for 20a and 6 2.82 for 20b, assigned to the respective CH2-2'" in both compounds. The presence of the cross peaks 52.77(CH22'")/56.67(lH, d, y=2.09Hz) and 52.77(CH2-2'")/6.55(lH, dd, J= 8.02 and 2.09 Hz) for 20a and 62.82 (CH2-2"')/67.05 (2H, d,J= 8.55Hz) for 20b in the HH-LR-COSY spectrum of 20a,b indicated the linkage of one 3,4-

326 dihydroxybenzene ring to CH2-2'" in 20a and one 4-hydroxybenzene ring to the same position in 20b. These data unambiguously proved the substitution at C-7 in the two compounds: one 3,4-dihydroxyphenethoxy unit in 20a and one 4-hydroxyphenethoxy unit in 20b. Table 4. ^H and ^^C NMR Data of Hydroxyframoside A (20a) and Hydroxyframoside B (20b) in CD3OD (ppm) 20a 1 Positions 1 3 4 5 6a 6b 7 8 9 10 11

r

2' 3' 4' 5' 6'a 6'b l"a l"b 2" 3" 4" 5" 6" 7" 8"

r"a

1

1

l'"b 9'" 3.,, 4"' 5'"

6 H (7 in Hz) 5.90 br s 7.47 s

3.94 dd (9.40, 4.45) 2.39 dd (14.22, 9.40) 2.64 dd (14.22,4.45)

6.08 qd (7.10, 1.00)

1.66 dd (7.10, 1.48)

4.80 d (7.82) 3.2-3.4 obscured by MeOH 3.411 (8.83) 3.2-3.4 obscured by MeOH 3.2-3.4 obscured by MeOH 3.68 dd (11.87, 5.50) 3.89 dd (11.87, 1.70) 4.27 dt (10.80, 6.74) 4.31 dt (10.80, 6.74) 2.87 t 2H (6.74)

7.07 d (8.60) 6.71 d (8.60)

6.71 d(8.60) 7.07 d (8.60) 4.10 dt (10.71, 7.07) 4.21 dt (10.71, 7.07) 2.77 t2H (7.07)

20b 8C 94.51 154.39 108.84 31.04 40.47

172.45 124.14 130.02 12.88 167.47 100.19 74.06 77.71 70.77 77.25 62.03

65.67

34.57 129.72" 130.25 115.54 156.34 115.54 130.25 66.17

-

6H(/inHz) 5.90 brs 7.48 s

3.94 dd (9.40,4.61) 2.39 dd (14.22, 9.40) 2.65 dd (14.22,4.61)

6.07 qd (7.05, 1.00)

1.64 dd (7.05, 1.48)

4.80 d (7.82) 3.2-3.4 obscured by MeOH 3.411 (8.83) 3.2-3.4 obscured by MeOH 3.2-3.4 obscured by MeOH 3.68 dd (11.87, 5.50) 3.89 dd (11.87, 1.70) 4.27 dt (10.80, 6.74) 4.31 dt (10.80, 6.74) 2.81 t2H (6.71)

6.68 d (2.02)

6.69 d (8.02) 6.56 dd (8.02, 2.02) 4.09 dt (10.70, 7.17) 4.22 dt (10.70, 7.17) 2.82 t2H (7.17)

34.70 129.52'^ 7.05 d (8.55) 6.67 d (2.09) 116.36 6.72 d (8.55) 145.53 144.23 6'" i 7'" 1 6.70 d (8.02) 115.75 6.72 d (8.55) 120.62 7.05 d (8.55) 6.55 dd (8.02,2.09) 8"' *' ^ Values with the same superscript are interchangeable.

dC 94.45 154.36 108.86 31.07 40.47 172.50 124.14 130.02 12.86 167.47 100.15 74.06 77.74 71.80 77.25 62.06 65.63 34.46 129.76'' 116.29 145.53 144.22 115.66 120.58 66.17 34.82' 129.33" 130.29 115.60 156.34 115.60

1

1 1

1

130.29 1

327

Furthermore, the HMBC, COSY and HH-LR-COSY spectra of 20a,b gave evidence for the attachment of one 4-hydroxyphenethoxy unit in 20a and one 3,4-dihydroxyphenethoxy unit in 20b to C-11 of the oleoside moieties. The proposed arrangements of 20a and 20b were further confirmed by the following cross peaks in the NOESY spectrum of 20a,b - for 20a: H3/H-2" and H-27H-4",8"(5 7.07, 2H, d,J= 8.60 Hz), and for 20b: H-3/H2" and H-27H-4"(8 6.68, IH, dd, J =2,02 Hz), and H-2 7H-8" (6 6.56, dd, J = 8.02 and 2.02 Hz), Fig. (4). Therefore, 20a and 20b are hydroxyderivatives of framoside (19) isolated from the same extract.

H OGIc

20a 20b

OH H

H OH

Fig. (4). Important NOEs observed for 20a and 20b

Tanahashi and co-authors [18] postulated that insularoside (15) is biosynthesized by oxidative C-0 coupling of the two p-hydroxyphenethyl moieties from framoside (19) found at that time in Fraxinus formosana [24]. The presence of 15, hydroxyframoside A (20a) and framoside (19) in Fraxinus ornus is in support of their postulate. However, the cooccurrence of 15 and omosol (30) in Fraxinus ornus allows to speculate that 30 could be also involved in the biosynthesis of insularoside (15). Caffeic Acid Esters of Phenylethanoid Glycosides

It is known that the Oleaceae family is a rich source of phenylethanoid glycosides (PhGs). Many cinnamic esters of PhGs of diverse structures have been isolated from the genera Syringa, Forsythia, Ligistrum, Jasminum and Osmanthus [25]. However, only the occurrence of verbascoside (24) and calceolarioside A and B in Fraxinus species have been reported [19,26,27].

328 Our investigations revealed the presence of calceolarioside B (23) in Fraxinus ornus leaves [28] and of six caffeic acid esters of PhGs in the polar part of the EtOH extract of the bark [29]. Five of them were identified as the known 2-(4-hydroxyphenyl)-ethyl-(6-0-caffeoyl)-p-Dglucopyranoside (22), calceolarioside B (23), verbascoside (24), isoacteoside (25) and lugrandoside (26). The occurrence of 22 and 26 in Oleaceae has not been reported so far. 0R5

r.R2 0R1

R^ 22 H 23 H 24 H 25 H 26 H 27 H 27a Ac Caff=Caffeoyl Ester Unit

H OH OH OH OH OH OAc

H H Rha Rha H Caff Caff 2 Ac

H H Caff H Caff H Ac

Caff Caff H Caff Glc Glc Glc 4Ac

The structure of the novel PhG-ester, named isolugrandoside, was deduced as 27 by concerted application of ID and 2D NMR methods and MS studies. A molecular formula of C29H36O16 was established on the basis of its negative HRFAB-MS ([M-H]" at m/z 639.1950) and the ^H and ^^C NMR spectra (Table 5). The ^H NMR spectrum revealed the signals typical of a /ra«5-caffeoyl ester unit, a 3,4-dihydroxyphenethyl unit and two P-glucose units. The detailed analysis of its ^H NMR, COSY, ID TOCSY, ^^C NMR, GHSQC and HMBC spectra fixed the position of the ester and glucosidic linkages to one central Pglucopyranosyl moiety (Glc-1). The HMBC correlation from H-1 to C-8" gave evidence for the attachment of the 3,4-dihydroxyphenethoxy moiety to C-1 of the central glucose unit, as usual in the PhGs [25]. The position of linkage of the terminal glucopyranosyl moiety (Glc-2) at C-6 of the central glucose unit was deduced from the ^*^C NMR spectrum where the resonances of C-6 and C-5 were observed at 5 69.9 and 76.9, respectively [25]. The HMBC correlation from the anomeric proton T" to C-6 indicated that the interglucosidic linkage is between C-T" of Glc-2 and C-6 of Glc-1. The position of the rraw^-caffeoyl unit at C-3 of the central glucose unit was confirmed by the HMBC correlation from H-3 to C-9'.

329 Table 5. ^H and ^^C NMR Data of Isolugrandoside (27) and Isolugrandoside Acetate (27a) 27" 1 Moieties

[ Positions

1 1 GIc-1

2 3 4 5 6a 6b

r

Caffeoyl

1 Phenethyl 1 Alcohol

2' 3' 4' 5' 6' ?• 8' 9' 1" 2" 3" 4" 5" 6" 7" 8"a 8"b j,„ 9'"

Glc-2

3'" 4'" 5'" 6'"a 6'"b AcO-Ph

1

6H(yinHz) 4.51 d (7.8) 3.44 t-like (9.0) 5.061 (9.0) 3.64 m 3.62 m 3.86 dd (11.3, 4.6) 4.18brd(11.3)

[

1

'

-

-

128.3 115.9 146.8 149.6 117.9 123.7 147.7 115.8 170.0 132.2 117.3 144.6 146.1 117.2 122.2 36.6 72.9

7.12 d (1.9)

6.85 d (8.2) 7.02 dd (8.2,1.9) 7.61 d (15.9) 6.37 d (15.9)

6.76 (2.0)

6.75 d (8.1) 6.64 dd (8.1, 2.0) 2.82 t, (7.1) 2H 3.79 dt (9.7, 7.1) 4.05 dt (9.7, 7.1) 4.43 d (7.8) 3.28 t-like (9.0) 3.42 t (9.0) 3.35m 3.30m 3.70 dd (11.6, 5.2) 3.89 brd (11.9, 1.9)

-

1 1

bC 104.5 73.6 79.7 69.8 76.9 69.9

7.22 d (8.5) 7.38 d (8.5, 2.0) 7.58 d (16.0) 6.28 d (16.0)

7.08 br s

j

104.9 75.2 78.1 71.7 78.0 62.8

AcO-Glc 1 ^ Measured in CD3OD (ppm). ^ Measured in CDCI3 (ppm).

1

-

1

7.34 d (2.0)

-

1

^^

6H(7inHz) 4.50 d (7.9) 5.08 t (9.0) 5.301 (9.0) 4.97 t (9.0) 3.72 m 3.64 m 3.89 brd (10.9)

1

1

1 1 1 1 1 1

7.06 d (8.0) 7.06 br d (8.0) 2.90 m 3.65 m 1 4.12 m 4.58 d (8.14) 5.00 m 5.211 (9.0) 5.08 m 3.67 m 4.14 brd (12.6) 4.28 dd (12.6,4.8) 2.32,2.31,2.29,2.28 2.10, 2.04, 2.02, 2.00, 1.98,1.91 1

Acid hydrolysis of 27 produced caffeic acid, 3,4-dihydroxyphenylethyl alcohol and D-glucose. The identification of D-glucose including its absolute configuration was conducted according to the procedure of Oshima et al. [30]. The proposed structure of 27 was in full agreement with the ^H NMR spectrum of its diacetate 27a. Our experimental data excluded the isomerization of 26 to 27 during the isolation and purification procedure and confirmed the natural occurrence of isolugrandoside.

330

Minor Phenolic Compounds The isolation of the minor components caffeic acid (16) and the lignan (+)-l-hydroxypinoresinol-4'-P-D-gIucoside (21) is described in the section "Isolation of secoiridoids and phenylethanoids". GC-MS analysis of the ethanolic extract revealed the presence of tyrosol (29) and of the new phenylethanoid ornosol (30) in trace amounts.

HO^

in

HO-"^ ^ ^ ^ ^ ^ ^ C O O H

16

_f

\—CH2CH2OH

29

ROCH2CH2—{

\-0—/

y-CH2CH20R

30 R = H 30aR = Ac

BIOLOGICAL STUDIES Antimicrobial Properties of Hydroxycoumarins, Phenylethanoids and Fraxinus ornus Bark Extracts

Secoiridoids,

The weak bacteriostatic effect of ethanolic extracts of Fraxinus ornus bark against Staphylococcus aureus has been reported [31]. The antimicrobial properties of the natural dihydroxycoumarins esculin (1), esculetin (2) and daphnetin have been also investigated using different test systems [32]. However, there was no detailed investigation on the antimicrobial activity of Fraxinus ornus extract and the compounds responsible for have not been identified. We studied the antimicrobial properties [33] of three Fraxinus ornus bark extracts and their main components 1-4 against Staphylococcus aureus, Candida species, Escherichia coli and Pseudomonas aeruginosa using the procedure of Heiss [34]. Evaluation of microbial growth on the contact surface was performed by measuring the Contact Growth Index (CGI). The crude extract (extract A) of the bark was separated into

331

petroleum ether (extract B), ethyl acetate (extract C) and water-methanol (extract D) applying the solvent-solvent partition method. Extract B was not further investigated because of its low solubility and absence of coumarins 1-4. The content of coumarins 1-4 in extracts A, C and D determined by HPLC is presented in Table 6 [14]. Table 6.

Coumarin Composition of Extracts A, C and D

Extracts 1 1

Extract A Extract C Extract D

Escuiin (1) 36.50 32.17 43.50

Content of the main coumarins in % w/w Fraxin (3) Esculetin(2) 5.00 2.33 1.83 5.33 7.00 0.17

Fraxetin (4) 0.33 0.80

0.12

| |

1

The propylene glycol solutions of all tested compounds and extracts were not active against E, coli and P. aeruginosa. Against Candida species fraxin (3) at concentration of 0.62% and fraxetin (4) at concentration of 0.84% exhibited some activity (CGI = 3). Against S. aureus, a 2.80 % solution of escuiin (1) showed lower activity (CGI=4) than 0.62% solution of fraxin (3 ) (CGI =3), while a 0.72% solution of esculetin (2) and a 0.84%) solution of fraxetin (4) revealed full inhibition (CGI=0). Extract C exhibited the highest antimicrobial activity among the tested extracts: full inhibition (CGI=0) of S. aureus and Candida species at a concentration of 8.80% and weak inhibition (CGI=3) of Candida species at a concentration of 2.10%). The antibacterial activity of the 0.2% ethanolic solutions of the studied compounds (for fraxin 0.1%) and bark extracts on S. aureus, compared with that of famesol, is presented in Table 7. The data allow a better comparison of the antibacterial power of the products. It follows from the results that the antimicrobial properties of the studied pure compounds depend on their structure and substitution: a. fraxetin (4), a 6-OMe-7,8-dihydroxycoumarin is a stronger inhibitor of S, aureus than esculetin (2), a 6,7- dihydroxycoumarin; b. glucosidation decreases the inhibitory power of both types of dihydroxycoumarins; c. the coumarins fraxin (3) and fraxetin (4) inhibit Candida species, while escuiin (1) and esculetin (2) do not. The activity of extracts A, C and D (Table 7) indicates a clear correlation with their hydroxycoumarin composition. The extract C enriched with the coumarins esculetin (2), fraxin (3) and fraxetin (4) shows the highest antimicrobial activity of all studied extracts. The inhibitory power of extracts C and D is comparable with that of famesol. The HPLC profiles of the tested extracts indicated the presence of some other bark constituents, different from 2-4, that could also contribute to their activity [14].

332

Table 7.

Sample Esculin(l) Esculetin(2) 1 Fraxin (3) 1 Fraxetin (4) 1 Extract A Extract C Extract D 1 Farnesol

Contact Growth Index (CGI) of 5. aureus Concentration (w/v%) in 96% EtOH 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2

10^ 4 1 3 0 3 0 0 0

Suspension for inoculation (CFUy ml) 10^ 4 2 4 0 4 0 0 0

10* 4 3 4 0 4 0 1

2

1

This gave us a reason to study and compare [35] the antimicrobial properties of a series of Fraxinus ornus bark constituents - the hydroxycoumarins 1-4, 6-7, 11-12, and acetates 2a and 4a; the secoiridoids 14, 14a, 15, 15a, and the tyrosol derivatives 29 and 30 applying the method described by Bankova et al. [36]. In this case the antibacterial properties of the studied compounds were also dependable on their structure and substitution (Table 8). In the group of coumarins, both glucosides esculin (1) and fraxin (3) showed negligible activity (Minimum inhibitory concentration (MIC) > lOOO^g/ml). This in contrast to the higher potency of the corresponding aglucones esculetin (2) and fraxetin (^4) (MIC = 500 and 125 |^g/ml, respectively) and indicates, that glucoside bonding of one of the phenolic hydroxyls leads to a significant weakening of the inhibitory properties. As already established by the method of Heiss, fraxetin (4), a 6-OMe,7,8dihydroxycoumarin, is a more potent inhibitor of the growth of S. aureus than esculetin (2), a 6,7-dihydroxycoumarin derivative. Compound 2 inhibits both the gram-positive and gram-negative organisms, while 4 is an inhibitor only of the gram-positive. This suggests that the position of the phenolic hydroxyls is essential for the activity. Isoscopoletin (7), a 7- methyl derivative of esculetin is less inhibitory than esculetin itself, while scoparone (6), a 6,7-dimethyl derivative of esculetin, is completely inactive. The 7-0-methylesculin (11) and 6,7,8trimethoxycoumarin (12) are totally deprived of activity. Evidently, a methylation of the phenolic OH decreases the activity. The MIC of the acetates esculetin 2 Ac (2a) and fraxetin 2 Ac (4a ) were found to be equal to those of the parent compounds 2 and 4. Most probably, acetylation of the phenolic OH does not alter the antibacterial activity of the hydroxycoumarins. Compounds 4 and 4a are the most potent inhibitors of S. aureus among the tested Fraxinus ornus components.

333

Tabie 8. Antibacterial Activity of Hydroxycoumarins, Secoiridoids and Tyrosol Derivatives

1 Compound 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Inhibitory zone (d jmrnl) S. aureus E. coli

1 Hydroxycoumarins 16 18 Esculin (1) 17 Esculetin (2) 19 16 17 Esculetin 2Ac (2a) 17 20 Fraxin (3) 21 Fraxetin(4) 0 20 0 Fraxetin 2AC (4a) 0 0 Scoparone(6) 14 0 Isoscopoletin (7) 0 0 Methylesculin(ll) 0 0 6,7,8-Trimethoxy-coumarin (12) Secoiridoids 28 Ligstroside (14) 26 26 0 Ligstroside 5 Ac (14a) 24 16 Insularoside (15) 23 0 Insularoside 5 Ac (15a) Tyrosol derivatives 6 0 Tyrosol (29) 24 0 Omosol(30) ' MIC was determined only for compounds with inhibitory zone more than

MIC

1

S. aureus

£. coli

> 1000 500 500 >1000 125 125 -

> 1000 500 500 > 1000

500 500 500 1000

500

1 1 1 1 1 1 500

1 500 16mm.

1

-

1 1 1 I

The resistance of some plants to attacks of insects and microbes has been attributed [37] to the presence of the bitter secoiridoid ligstroside (14), which predominantly occurs in Fraxinus species. In this investigation ligstroside (14), its acetate (14a), insularoside (15) and the tyrosol derivative omosol (30) exhibited equal inhibition (MIC = 500 jag/ml) on the growth of S. aureus. The natural secoiridoids 14 and 15 inhibited both the gram-positive and the gram-negative microorganisms (MIC = 500 |Lig/ml), while their respective acetates 14a and 15b, and tyrosol derivatives 29 and 30 inhibited only the gram-positive. Our preliminary screening of the secoiridoids 14, 14a, 15, 15a and omosol 3 Ac (30a), using the method of Romans et al. [38], showed clearly visible zones of inhibition of the growth of Cladosporium cucumerinum and suggested them to be fungitoxic. The antibacterial activity of the secoiridoids 14,15, 17-19, 20a, b, 28a and 38, and the caffeoyl esters of phenylethanoid glycosides 22 -27 has also been tested using the direct bioautographic TLC assay as published by Hamburger and Cordell [39]. Bacillus subtilis spp. and Pseudomonas fluorescens were the representatives of the gram-positive and gramnegative bacteria, respectively. The minimum inhibition amount (MIA) was determined. Cefotaxime was used as a positive control.

334

Most of the tested secoiridoids displayed an inhibition of the growth of B. subtilis and P, fluorescens (Table 9). Insularoside (15) and hydroxyornoside (17) are more active against B. subtilis. The mixture of hydroxyframoside A, B (20a, b) showed the best activity against Pseudomonas fluorescens. Table 9. Minimum Inhibition Amount (MIA) of Secoiridoids Against B, subtilis and P. fluorescens Compound Ligstroside (14) Insularoside (15) Hydroxyornoside (17) 1 Oleuropein (18) Framoside (19) Hydroxyframoside A,B (20a,b) 7,11 -Dimethyloleoside (28a) 10-Hydroxyligstroside (38) 1 Cefotaxime ^NA- not active at a concentration of 50 |ig/spot

B. subtilis [|ag/spot] 0.5 0.2 0.2 1.0 1.0 2.5 NA 40 0.01

P. fluorescens [|ag/spot]

10

1

10 20 10 10 2.5 40 NA

0,01

1

The caffeoyl esters 22-27 showed no activity against Pseudomonas fluorescens at a concentration of 50 |uig/spot. The MIA of 22-27 against Bacillus subtilis is the following: 22- 20; 23 - 10; 24 - 2.5; 25 - 2.5; 26 20; 27 - 50 |ig/spot [40]. Skin - regenerating Properties of Esculin and Fraxinus ornus Bark Extract We investigated the skin regenerating properties of esculin (1) and Fraxinus ornus bark extract on male white Wistar rats having standard oval wounds [41]. The rats were divided into four groups of 6 animals and treated as follows: I. (control) group, destined for spontaneous recovery; II. (control) group, treated with propylene glycol; III. (test) group, treated with extract. A solution (18.2%) of the total ethanol extract of the bark (TE2) in propylene glycol was applied. RPHPLC analysis of the extract revealed the following hydroxycoumarin composition: esculin (27.1%), esculetin (0.1%), fraxin (0.3%), fraxetin (0.4%) and minor components scoporone (6), isoscopoletin (7), scopoletin (9), methylesculin (11) and 6,7,8- trimethoxycoumarin (12). The presence of coumarins 10 and 11, and of compounds 14 - 27 was confirmed by TLC and HPLC.

335

IV. (test) group, treated with a 3.45% solution of esculin in propylene glycol. The percent of epithelization with respect to the beginning of the experiment (zero day) was calculated (Table 10). Table 10. 1

Group

i II III

1

IV

Epithelization (%) of Wounds with Respect to the Zero Day

V

3^*^ day

•^th

Day

day

lO*" day

10.0 9.4 39.8 38.6

30.7 31.1 55.8 50.4

43.9 45.3 84.9 67.8

89.9 88.8 96.9 87.4

day

i^

11

963

1

96.5 100.0

98.9

1

The III group of animals exhibited a more intense epithelization of the wounds in comparison with the control groups at every stage of the investigation. A weaker regenerating effect was found in the IV group of animals treated with esculin. The application of propylene glycol alone ( II group) did not result in an epithelizing effect. On the 7^^ day the biopsy of the I and II (control) groups established mostly a massive leucocytic necrotic swellings on the wound surface. A less pronounced leucocytic necrotic elevation was established in the III group, where definite zones of the wounds were almost completely filled with granular tissue. The amount of the riper collagen fibrils exceeded the amount of young fibrils. The epithelial regenerate consisted of ten and more cells. The basal prismatic layer, which was rich in mitoses, was separated from the overlying layers consisting of larger cells with round light nuclei. The III and IV groups of animals exhibited similar developments. These investigations have shown that the alcoholic extract and esculin obtained from Fraxinus ornus bark exercise moderate skin regenerating effects, no toxicity or local irritation being observed. The experimental results are in line with the antimicrobial properties of the extract and its constituents, and with the use of the bark in the traditional medicine for wound treatment and against inflammation. In addition, we studied the acute toxicity of the total ethanolic extract TE2 and its main component esculin (1) and found that applied p.o. to white mice and white Wistar rats in doses from 50 to 8000 mg/kg they were practically non-toxic. No lethality was observed up to 21 day and with the highest doses used. No significant changes were found both in the behavior and the reflexes of the animals. No pathological deviations from the physiological values were found in all hematological and clinical -chemical indices studied.

336 Antioxidative Action of the Ethanolic Extract Hydroxycoumarins from Fraxinus ornus Bark

and

some

The need to prepare lipids and lipid-containing products that are stable with respect to the effect of atmospheric oxygen has increased the interest in finding suitable natural sources of harmless antioxidants. The high biological activity of extracts from such sources is often due to the presence of phenolic compounds, many of them exercising a pronounced antioxidative effect on lipids [42,43]. In the literature there was no information about the antioxidative action of Fraxinus ornus extract and the data on hydroxycoumarins were scarce [44]. We examined the antioxidative action of the total ethanolic extract (TE2) of Fraxinus ornus bark as well as of its main hydroxycoumarin components esculin (1), esculetin (2), fraxin (3) and fraxetin (4) [45]. Quantitative reverse phase HPLC analysis [14] of the extract used in this study established the following hydroxycoumarin content: esculin 53.7%, esculetin 0.5%, fraxin 8.7% and fraxetin 0.3%. The investigations were performed at 100^ with kinetically pure triacylglycerols of lard and sunflower oil (TGL and TGSO), which represent models of two types of natural lipid unsaturation. The stabilization factor F as a measure of the effectivity and the oxidation rate ratio (ORR) as a measure of the strength of the tested antioxidants were estimated. The parameters F and ORR for TGL and TGSO oxidation in the presence of 0.05 and 0.10% ethanolic extracts oi Fraxinus ornus bark are presented in Table 11. The data indicate that the ethanolic extract of Fraxinus ornus bark has a pronounced antioxidative activity during the oxidation of both lipid substrates. This activity is commensurate with the inhibiting effect of the same concentration of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) during TGL oxidation [46]. In order to elucidate the contribution of the main phenolic components [esculin (1), esculetin (2), fraxin (3) and fraxetin (4)] in the extract to its stabilizing action, we studied the autoxidation kinetics of the two natural lipid systems (TGL and TGSO) in the presence of different concentrations of 1-4. For TGL autoxidation in the presence of 0.04% esculin and 0.04% fraxin the following data for F and ORR were obtained: esculin, F = 2.5, ORR = 0.7; fraxin, F = 2.1, ORR = 1.0. In TGL addition of 0.02% esculin and 0.02% fraxin resulted in practically no stabilizing action. In TGSO these glucosides exhibited no antioxidative action. The kinetic parameters characterizing the inhibitor action of esculetin and fraxetin are also presented in Table IL The experimental data demonstrate that the fraxetin possesses a higher effectivity and greater strength than does the esculetin. Both substances are less effective

337

inhibitors in the lipid system with a higher oxidizability (TGSO). Both hydroxycoumarins are antioxidants of a relatively high effectivity and of a great strength because, under the same oxidation conditions for 9.1x 10' "•M B H T in TGL F = 4.8, ORR = 0.33; for 4.6 x 10"* M a-tocopherol F = 6.5, ORR = 0.13; for 1.0 x 10"^M ferulic acid F = 3.1, ORR = 0.59; for 1.3 x lO'^M 3,4-dihydroxybenzoic acid F = 13.4, ORR = 0.07 and for 1.1 x lO'^M caffeic acid F = 62.1. For 1.0 x 10"^ M ferulic acid in TGSO F = 2.6, ORR = 0.60, ; for 1.3 x lO'^M 3,4-dihydroxybenzoic acid F = 3.6, ORR = 0.60 and for 1.1 x 10"^M caffeic acid F = 33.6, ORR = 0.07 [46,47]. Table 11. Stabilization Factor F and Oxidation Rate Ratio ORR for the Inhibited Oxidation of TGL and TGSO at 100°C in the Presence of Ethanolic Extract, Esculetin (2) and Fraxetin (4) from Fraxinus omus bark 1 Antioxidant 1 Extract

Esculetin (2)

Fraxetin (4)

Inhibitor concentration M % 0.05 0.10 0.56x10-^ 0.01 1.12x10-^ 0.02 0.05 2.81x10-' 0.10 5.56x10-' 0.48x10-' 0.01 0.02 0.95x10"' 0.05 2.38x10-' 0.10 4.76x10-'

TGSO

TGL F 4.8 6.1 22.7 42.7 64.5 73.1 38.2 86.3 208.0 340.0

ORR 0.28 0.28 0.07 0.06 0.05 0.05 0.05 0.03 0.02 0.01

F 3.6 4.0 14.8 20.8 37.6 41.2 13.2 27.2 72.0 125.0

1 ORR 1 0.60 0.50 0.15 0.09 0.06 0.05 0.15 0.09 0.04

0.02 1

It is worth mentioning that when ORR is larger than 1, the oxidation proceeds faster in the presence of an inhibitor than in its absence. The lower the ORR, the stronger the inhibitor. Comparison of the F and ORR values for the glucosides with the values of the same parameters for the aglucones at almost the same molar concentrations (0.02% aglucones) shows that the glucoside bonding of one of the phenolic OH groups leads to a significant weakening of the inhibiting properties. The results obtained indicate blocking of the more active phenol group in fraxin, due to which the difference in antioxidative activities of glucosides esculin and fraxin is, in contrast to aglucones esculetin and fraxetin, negligible. Considering the antioxidative effects of coumarins 1-4 in TGL and TGSO and the content of these compounds in the extract, and eliminating the possibility of antagonism or synergism between them, it was calculated that the four compounds under consideration determined twothirds of the antioxidative effectivity of the extract in TGL and half of the effectivity in TGSO.

338 TLC analysis of the antioxidatively acting compounds [48] in the ethanoHc extract from Fraxinus ornus bark revealed the presence of additional antioxidative acting compounds. Among them calceolarioside B (23) demonstrated a significant activity. Most probably, caffeic acid (16) and the caffeoyl esters of phenylethanoid glycosides 22, 24-27 also contribute to the antioxidative properties of the extract. Photodynamic Damage Prevention by Extracts Hydroxycoumarins from Fraxinus ornus Bark

and

Some

The antioxidants or quenchers of free radicals are knovm to minimize skin photoaging and the protective action of sun screens correlates closely to their free radical scavenging activity [49,50]. This prompted us to use the prevention of photodynamic yeast cell damage to comparatively investigate the protective activity of the four hydroxycoumarins esculin, esculetin, fraxin and fraxetin, and a widely used sun screen paminobenzoic acid (FABA). We applied the same test to characterize the protective activity of four Fraxinus ornus bark preparations containing these coumarins in different concentrations and to examine the activity of caffeic acid (16), a minor constituent of these preparations [51]. Table 12. Preparation 1 Total extract 1 Fraction A 1 Fraction B 1 Fraction C

Protective Effect of the Fraxinus ornus Preparations Protectio n factor in% 9.8 2.3 54.6 6.1

Hydroxycoumarin composition in % EsculinCl) 40.5 0.0 28.4 45.3

Esculetin (2) 1.0 0.0 6.3 0.3

Fraxin (3) 6.9 0.0 2.5 9.0

Fraxetin (4) 0.2 0.0 1.6

|

0.2

1

The total extract of Fraxinus ornus bark v^as prepared and further subjected to solvent-solvent partition to obtain the fractions A, B, and C w^ith the hydroxycoumarin composition given in Table 12. The test for protective effect evaluation was performed using yeast cells (strain Kluyveromyces fragilis 129-1) according to the procedure described by Lazarova and Ignatova [52] and the protection factor determined. All of the tested pure compounds showed protective activity (Table 13). The protection achieved was higher for esculetin (2) and fraxetin (4) as compared to the corresponding glucosides esculin (1) and fraxin (3). This finding is in accordance with the data obtained by Bakalova et al [53] concerning the effect of the same coumarins on the lipid peroxidation of liver microsomes, as well as with our own results on their antioxidative action presented above.

339 The protective effects of the four Fraxinus ornus preparations depend on their hydroxycoumarin composition (Table 12). Fraction B, which is enriched in aglucones, exerts the highest protective activity. Fraction C, with lower aglucone and higher glucoside concentration, demonstrated a protective activity lower than that of the total extract. The negligible protective activity of fraction A correlates very well with its composition, i.e. no measurable concentration of the coumarins under investigation. Protective Effect of the Pure Compounds

Table 13. 1

Compound Esculin (1) Esculetin (2) Fraxin (3) Fraxetin (4) PABA

Caffeic acid (16)

Concentration in mg/1

Protection factor in %

20 50 20 50 20 50 20 50 2.5 5 25 2.5 5 25

38.7 26.3 97.6 99.8 23.1 72.8 94.7 98.2 47.2 943 92.6 49 1 91.0

98,2

1

Our results suggest that the caffeic acid (16) and the aglucones esculetin (2) and fraxetin (4) approximate the skin protective effect exerted by the conventional sun screen PABA. The Fraxinus ornus bark preparations enriched in the compounds 2 and 4 seem to be effective protectors too. Complement Inhibition and Antiinflammatory Activity of Hydroxycoumarins, Secoiridoids and Extracts from Fraxinus ornus Bark The use of Fraxinus ornus bark in the Bulgarian folk medicine for treatment of inflammation, arthritis and dysentery [1,2] suggests the presence of some active principles with anti-inflammatory activity. Our phytochemical investigations have shown that the bark contains hydroxycoumarins, secoiridoid glucosides, caffeoyl esters of phenylethanoid glycosides and other phenolic compounds [10,14-17,28]. RP-HPLC analysis of commercial samples of Fraxinus ornus bark revealed high esculin (1) content (6-9%) [14].

340

Since the complement system is highly involved in an inflammatory response [54,55] many substances exhibiting anticomplementary activity have proved to be effective antiinflammatory agents [56]. In this connection we studied and compared the effects of the ethanolic extract of Fraxinus ornus bark and its main component esculin (1) on some in vitro and in vivo reactions related to acute inflammatory processes [57]. Quantitative RP-HPLC analysis of the total extract used in this study showed the following hydroxycoumarin composition: esculin 40.0%, esculetin 2.4%, fraxin 7.8% and fraxetin 0.4% [14]. The inhibitory effects of TE2 and 1 on the classical pathway (CP) and the alternative pathway (AP) of the complement activation in mouse serum were estimated at final concentrations varying from 1 to 50)ag, Fig. (5). The TE2 caused a more pronounced reduction of CP hemolysis compared to esculin. In the AP assay they exhibited nearly equal dosedependent inhibition of complement - mediated lysis.. inhibition (%)

Concentration (|ig)

Inhibition (%)

Concentration (|ig)

Fig. (5). Inhibition of CP (A) and AP (B) complement activity in mouse serum by different concentrations of TE2 (o) and esculin (•)

341 The comparison between the effects obtained with TE2 and 1 in the hemolytic inhibitory assay indicates that the anticomplementory action of TE2 is not due only to esculin. The effect of esculin on CP activity was less pronounced than that of the total extract, although it represents 40% of the content of TE2 and in these experiments it was tested in equal concentrations with TE2. The full inhibition of AP activity was achieved at concentration of 50)ig for both esculin and TE2. This also suggested that, excepting esculin, some other extract constituents contribute to its anticomplementary action. The esculin concentration causing 50% inhibition of complement activity in vitro is about 10"^ M which appears to be similar to that established for chemically similar compounds. Carrageenan- and zymosan- induced paw edema were chosen as suitable models for evaluation of the antiinflammatory activities of TE2 and esculin (Table 14). The results showed that both TE2 and esculin significantly reduced formation of the zymosan-induced paw edema in mice. In the case of carrageenan-induced edema, only TE2 at a dose of 15 mg/kg significantly reduced the inflammation, while esculin was ineffective. This indicated that the extract contains active components with different mode of action. Evidently, TE2 and esculin possess the ability to influence complement activity in vitro and to suppress some complement-mediated reactions after in vivo application. Coumarin derivatives are known to possess antiinflammatory and antimetastatic properties. The mode of action of coumarins is mainly attributed to their direct action on cells participating in the inflammatory process [58]. The influence of coumarins on the complement system, which is involved in the different stages of inflammatory response, has not been thoroughly investigated. Table 14. Effect of Esculin (1) and Total Extract from F. ornus Bark on Zymosan- and Carrageenan- Induced Paw edema in Mice Carrageenan- induced Zymosan-induced oedema oedema Paw volume" | Paw volume' 1 Control 42.6 ±5.5 89.0 ±9.5 15 30.3 ±5.4* 15.7 ±1.3* Total extract 5 28.6 ±10.0* 21.0 ±2.0 15 38.0 ±6.2* 36.0 ±2.6 Esculin (1) 5 1 38.2 ±2.0 40.6 ±3.5* ® Difference (mg) between the weight of a zymosan or carrageenan treated paw and the concentrated saline treated paw. Significant from respective control: * P< 0.05. Test material

Dose (mg/kg)

For this reason we examined the in vitro effect of 12 hydroxycoumarins on classical and alternative complement activity in

342

normal human serum and the consumption of the key components CI and C3 [59]. The coumarins 1-4, as well as their acetylated and methylated derivatives la, 2a, 4a, 6-8,11 and 12 have been investigated at different concentrations. The effect of the substances at concentrations of 1.2x10"'^ and 5.0 xlO'^^M, as the most representative, are shown in Table 15. All the substances tested had a moderate or weak ability to affect at least one of the complement pathways. The effect was not strictly dose-dependent. Esculin 5Ac (la), esculetin 2Ac (2a) and 7-methylesculin (11) exhibited good inhibition on CP activity. Scoparone (6) strongly reduced AP activity in normal human serum (NHS). Scopoletin (8), esculin (1) and esculetin (2) enhanced complement mediated hemolysis. Some of the compounds exhibited combined effect - activated one of the pathways and inhibited the other. Table 15. Inhibition (-) or Activation (+) of CP and AP Activity by Some Hydroxycoumarins

Hydroxycoumarins 1 1

1 1 1 1 1 1 1

Esculin (1) Esculin 5Ac (la) Esculetin (2) Esculetin 2Ac (2a) Fraxin (3) Fraxetin (4) Fraxetin 2Ac (4a) Scoparone (6) Isoscopoletin (7) Scopoletin (8) Methylesculin(ll) Trimethoxycoumarin (12) NA = not active

CP + 28.2 -21.5 + 7.1 + 8.2 + 28.2 -8.2 -8.2 + 5.9 + 8.2 + 22.4 -28.2 -16.5

Concentration 1.2xlO-^M Activity (%) AP NA -28.2 -5.9 -28.2 NA NA -20.6 -45.9 NA NA -34.1 -25.9

CP + 27.1 -40.0 + 24.3 -32.9 NA -17.5 -28.6 -11.4 + 24.3 + 10.0 -51.4 + 13.6

Concentration 5.0xlO"*M Activity (%)

|

AP

1

+ 10.7 + 17.9 + 15.7 -28.6 + 15.0 -15.0 -12.1 -57.1 NA + 20.0 -8.6

-28.6

1

Seven hydroxycoumarins were further tested at a single concentration (5.0x10"^ M) for their ability to influence CI and C3 functional activities after preincubation with undiluted NHS. 7-Methylesculin (11) had a good effect on reducing total, CI, and C3 hemolysis via both pathways. Scoparone (6) strongly inhibited C3 alternative activity but in the case of the classical pathway only the total hemolysis was diminished without influence on CI and C3. Esculin (1) slightly increased C3 classical activity but caused exhaustion of alternative C3 activity. In subsequent experiments 1 and 8 altered the effect of other complement activators (heat aggregated IgG, suramin and zymosan) when applied with them simultaneously in vitro.

343

The experimental data presented above suggest that hydroxycoumarins may counteract with some of the complement proteins and thus inhibit their functional activity. Also, it is possible that the substances form complexes with the serum proteins which are able to activate complement system. It is difficult to make conclusions about the relationship between the structure of the coumarins and their action on complement mediated reactions. It might be concluded, however, that the methylated hydroxycoumarins 6 and 11 are the most potent inhibitors of AP and CP activities and deserve attention as a possible antiinflammatory agents. A series of pure secoiridoid glucosides isolated from different Fraxinus species was compared in vitro for anticomplement action as well as for their ability to prevent cobra venom-induced complement activation in normal human serum [60]. Table 16 shows that most of the secoiridoids possess the ability to suppress CP and AP activities. The most effective inhibitors of CP in guinea-pig serum (GPS) were ligstroside (IC50 33 |Lig/ml) and insularoside (IC50 62 |iig/ml). With regard to NHS the most pronounced decrease of CP was caused by 7,11-dimethyloleoside (28a) at a concentration of 250 |ag/mL Altemative pathway hemolysis was slightly altered, although the substances were used in a higher concentration (Img/ml) than in the CP assay (250 |ag/ml). Evidently, secoiridoid glucosides exhibit a greater effect on CP activity. It makes them interesting for further investigation as there is a need for selective inhibitors of the complement system for possible therapeutic use. Table 16. Effect of Some Secoiridoids and Fraxinus ornus Bark Extracts on CP and AP Activity in GPS and NHS 1 1

Product

GPS ICP5o(Mg/ml)*

?3HS Inhibition (%)

CP**

AP'

1

Extract TEl 302 ±10 1 Extract TE2 1438 ±20 12.7 15.5 1 Ligstroside (14) 33 ±4 38.2 18.2 1 Insularoside (15) 62 ± 8 28.2 1 Hydroxyornoside (17) 5.5 185 ±10 23.6 18.2 1 Oleuropein (18) 130 ±8 29.1 1 Framoside (19) 4.5 160 ±6 96.4 7,11 -Dimethyloleoside (28a) 8.2 180±4 1 10-Hydroxyligstroside (38) 34.5 >1000 4.5 1 ' The concentration giving 50 % inhibition of CP (ICP50). ^ Measured at concentration of 250 ^g/ml. Measured at concentracion of l^ig/ml 1

1

In our further experiments the secoiridoids 15, 17-19 expressed the ability to prevent CP and AP activation, caused by cobra venom [61]. This

344

suggested that among the active secoiridoid constituents may exit one with C3- convertase inhibitory property. Our investigations revealed the contribution of secoiridoid glucosides to the antiinflammatory action of the extract. Antiviral Activity of Some Hydroxycoumarin Derivatives Studies on the antiviral effects of 6,7-dihydroxy- and 6,7,8trihydroxycoumarin derivatives were very limited [62,63]. This prompted us to study the antiviral properties of the structurally related compounds 1, 2, 4, 6, 7,11,12 and acetates la, 2a, 4a [64]. Primary screening for antiviral activity was carried out using the agardiffusion plaque-inhibition method with cylinders [65]. The compounds were tested against one representative in each of the four taxonomic viral groups; namely, picoma-, orthomyxo-, paramyxo- and herpes viruses, which represent a few of the most important families of the human patogens. The viruses used were poliovirus 1 (PVl), influenza virus A (FPV), Newcastle disease virus (NDV) and pseudorabies virus (PsRV). Two of the ten hydroxycoumarin derivatives tested showed activity against NDV, namely esculetin (2) and its diacetate (2a), when applied in cylinders at a dose of 2.8 and 1.9 mM/0.1 ml, respectively. Their activity was significant, although inferior when compared to that of ribavarin (used as a reference paramyxovirus inhibitor) at a dose of 2.0 mM /O.l ml. The remaining compounds tested were without effect on the replication of the four viruses studied. Evidently, no definitive conclusion could be drawn regarding structure-activity correlations. It could be noticed, however, that methylation and glucosidation of esculetin (2) lead to a loss of activity. SUMMARY AND CONCLUSIONS The work presented in the preceding pages described the antimicrobial, antioxidative, antiinflammatory, immunomodulatory, skin regenerating, photodynamic damage prevention and antiviral properties of Fraxinus ornus bark extract and its constituents. The total ethanolic extract of the stem bark and its main constituent esculin (1) were found practically non-toxic. They inhibited the classical pathway and alternative pathway of complement activation. The total extract and 1 displayed antiinflammatory activity in both zymosan- and carrageenan-induced paw edema in mice. The extract exhibited a pronounced antioxidative activity and caused intense wound epithelization. The antimicrobial and photodynamic damage prevention properties of the extract and its fractions were dependable on their

345

hydroxycoumarin composition. These results to some extent explained the traditional use of the bark for treatment of wounds, inflammation, dysentery and arthritis. The isolated secondary metabolites belong to the groups of hydroxycoumarins, secoiridoid glucosides, phenylethanoids and lignans. Of them 6 are new compounds. Isolation of new biologically active compounds and finding of new biological properties of already known bioactive substances has been achieved. Most of the secoiridoids and the coumarins possess the ability to affect at least one of the complement pathways. A clear correlation between the structure and the biological activity (antimicrobial, antioxidative and photodynamic damage prevention) of the studied hydroxycoumarins was observed. Our investigations provide more data on the chemical composition and biological properties of Fraxinus ornus and support the claims of the traditional medicine by presenting scientific proofs for the biological activity of the bark extract and its components. It was concluded that the total ethanolic extract of the bark can be included in the group of the practically non-toxic substances and may be used in the therapeutic practice for treating wounds, bums etc., as well as in the perfumery and cosmetics [66]. ABBREVIATIONS Ac AP BHT BHA Caff CGI CP DCE FPV Glc GPS HPLC IR LVC MIA MIC MS NA NAS NDV

Acetate (COCH3) Alternative Pathway Butylated Hydroxytoluene Butylated Hydroxyanisole Caffeoyl Ester Unit Contact Growth Index Classical Pathway Dichloroethane Influenza Virus A Glucose Guinea-pig Serum High Pressure Liquid Chromatography Infra-red Liquid Vacuum Chromatography Minimum Inhibition Amount Minimum Inhibitory Concentration Mass Spectrometry Not Active Normal Human Serum Newcastle Disease Virus

346 NMR ORR PABA PhG PsRV PVl Rha RP TEl TE2 TGL TGSO TLC UV

= = = = = = = = = = = = = =

Nuclear Magnetic Resonance Oxidation Rate Ratio p-Amino Benzoic Acid Phenylethanoid Glykoside Pseudorabies Virus Poliovirus 1 Rhamnose Reverse Phase Total Ethanol Extract 1 Total Ethanol Extract 2 TriacylglycerolsofLard Triacylglycerols of Sun Flower Oil Thin Layer Chromatography Ultra-violet

ACKNOWLEDGEMENTS The phytochemical investigations described in this review were carried out at the Institute of Organic Chemistry, Bulgarian Academy of Sciences and partially in a collaboration with Prof. W. Kraus, Dr. B. Vogler and Mrs. L Klaiber from the University of Hohenheim, Stuttgart. We are grateful for their participation. The work carried out at the Institute of Organic Chemistry was made possible thanks to the skillful efforts of Dr. B. Mikhova, Dr. G. Stoev, Dr. E. Vassileva, Mr. N. Nykolov, and for the antioxidative action thanks are due to Dr. N. Yanishlieva and Dr. E. Marinova. The biological investigations are a result of an effective collaboration with our following co-workers from the Institute of Microbiology, Bulgarian Academy of Sciences: Prof A. Galabov, Dr. N. Ivanovska, Dr. A. Kuyumgiev, Dr. G. Lazarova, Dr. H. Neychev, Dr. Z. Stefanova and Mrs. A. Ignatova. We gratefully acknowledge their valuable contribution. We also acknowledge the collaboration with Dr. L. Chipilska from the National Centre of Hygiene and Medical Ecology and Dr. E. Klouchek and Dr. A. Popov from the Higher Medical Institute. Dr. I. Kostova thanks Mrs. A. Ivanova for the technical assistance in the preparation of the manuscript.

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