AntiComplementary Activity of Crataegus sinaica

Anti-Complementary Activity of Grataegus sinaica AbdelaatyA. Shahat"2'3, Fa iza Hammouda', Shams I. Ismail', SafaaA. Azzam1, TessDeBruyne2, Aleidis Lasure2, Ban Van Pod2, Luc Pieters2, and Arnold J. Vlietinck2 2

Pharmaceutical Sciences Departments, NRC, 12311 Dokki, Cairo, Egypt Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium Address for correspondence

Received: April 21, 1995; Revision accepted: July 1, 1995

Abstract The 80% and 70% acetone extracts from fruits and leaves of Crataegus sinaica Boiss (Rosaceae)

and the ethyl acetate-, butanol-, and water-fractions obtained from these initial extracts as well as the isolated compounds, quercetin (1) (1), hyperoside (quercetin 3-O-galactoside) (2) (2), rutin (quercetin 3-0rutinoside) (3) (2), vitexin (4) (1), rhamnosylvitexin (5) (3), monoacetyirhamnosylvitexin (6) (3), epicatechin (7) (4), procyanidin B-5 (8) (4), proanthocyanidin A-2 (9) (5), procyanidin B-2 (10) (4), and procyanidin c-i (11) (4), were tested for their influence on the classical (CP) and alternative (AP) pathways of complement-mediated hemolysis. All extracts and fractions showed a strong

anti-complementary effect in a dose-dependent way which was more pronounced on the CP than on the AP.

The results indicated that the pure proanthocyanidins were active on the Cp. Procyanidin C-i and proanthocyanidin A-2 were the most active on the

CP and also showed activity on AP, whereas the flavonoids isolated were generally less active. However, rutin, showed a strong activity, quercetin and rhamnosylvitexin a moderate activity on the CP. This is the first

report on the chemical constituents and complementmodulating activity of C. sinaica and on the occurrence of proanthocyanidin A-2 (9) in hawthorn.

Key words

Crataegus sinaica Boiss, Rosaceae, complement inhibition, flavonoids, proanthocyanidins.

decrease of arterial blood pressure, increase of skeletal muscle blood flow and decrease of heart rate in vitro for coronary heart disease, and a minor increase of contractility of heart muscle in vitro and in vivo for heart failure (8).

The flavonoids such as hyperoside and rhamnosylvitexin and the oligomeric procyanidins which have been isolated from many Crataegus. sp. such as C. monogyna Jaeg. emend. Lindm or C. laevigata (Poir.) D. C. (synonym: C. oxyacantha L.) or, less frequently, from other European C. species (Rosaceae) are considered to be the main active constituents. The flavonoids as well as the procyanidins have been found to inhibit 3',5'-cycic adenosine monophosphate phosphodiesterase (9—il). Schüssler et al. (12) reported the inhibition of 3',5'-cydic adenosine monophosphate phosphodiesterase in a guinea-pig heart by flavonoids such as hyperoside, vitexine, rhamnosylvitexin, and monoacetylrhamnosylvitexin isolated from hawthorn C. monogyna. C. laevigata and other species such as C. pentagyna Waldst. et Kit. ex Willd., C. nigra Waldst. et Kit., and C. azarolus L. Activity of the extracts on

skin microcirculation disturbances has also been studied (13). On the other hand, weak genotoxic potencies were observed in the Salmonella/microsome assay (14) and in the sister chromatid exchange assay (15). Rewerski could demonstrate that the oligo-

meric procyanidins isolated from C. oxyacantha lower both blood pressure and body temperature (16). Ammon and Handel reported that acute toxic side effects are not to

be expected when Crataegus preparations are applied therapeutically (8). These pharmacological investigations of Crataegus. sp. demonstrate that flavonoids and oligomeric procyanidins are the main active constituents responsible for the biological activity of Crataegus. sp. (16, 17).

Introduction Hawthorn is widely used in phytotherapy due to its improvement of the heart function in declining cardiac performance equivalent to stages I and II of the NYHA classification (6, 7). Preparations of Crataegus are therefore used in minor forms of coronary heart disease, heart failure, and cardiac arrythmia. In animal tests preparations of Crataegus exhibited the following pharma-

cological effects which may be related to their therapeutical use: increase of coronary blood flow in vitro and in vivo, Planta Med. 62 (1996) 10—13 © Georg Thieme Verlag Stuttgart. New York

Flavolloids (18, 19, 20) and proanthocyanidins (5) are reported to have an influence on the complement system. Crataegus sp. have a high content of these

compounds, therefore C. sinaica was included in our screening of higher plants for complement modulation and of its isolated active constituents. The complement system

plays an important role in the host defence system, inflammation and allergic reactions. Hence, the activators and inhibitors of the complement system are suggested to be modulators of the immune system. Activation of the complement system can proceed via two different pathways. The classical pathway (CP) is activated by immune complexes containing IgG od 1gM antibodies, while the

Planta Med. 62(1996)

Anti-Complementary Activity of Crataegus sinaica

alternative one (AP) is activated by a variety of substances, for instance polysaccharides.

Materials and Methods Crataegas sinaica Boiss leaves and fruits were collected from St. Catherin (South Sinai, Egypt) in October 1992 and voucher specimens were kindly identified by Prof. Dr. K. H. El-Batanouny, Botany Dept. Faculty of Science, Cairo University,

by PTLC (sihca gel, 1mm) with solvent A (Hf = 0.11 and 0.23, respectively). Fraction VI was subjected to further chromatography on Sephadex LH-20 (35 x 2.5 cm) with EtOH as eluant (fractions lOml/each). The first ten fractions gave compound 10 (red colour with reagent (b), H1 = 0.82). The next ten fractions were subjected

to further column chromatography on Fractogel (30 x 2.5 cm) with MeOH resulting in the isolation of compound 11 (red colour with reagent (b), H1= 0.65).

Hemolytical assay

Egypt.

For CC, Sephadex® LH20 (Pharmacia), Fractogel® TSK HW-40 (5) (Merck) and silica gel 60 (230—400 mesh ASTM) (Merck) were used.

Analytical method TLC: silica gel 60 F245 (Merck); Solvent system:

EtOAc-HOAc-HCOOH-H20, 30:0.8:1.2:8 v/v (system A); detection: reagent (a) diphenylboric acid-ethanolamine complex (Naturstoffreagenz A) (flavonoids); reagent (b) vanillin-H2504 lvanillin 1 % in methanol and H2504 5% in EtOHI (procyanidins). UV detection at 254 and 366nm. FAB-mass spectral analysis were

The inhibition of complement activity was determined as described by Klerx et al. (21). The test was performed in V-well microtiter plates. Human pooled serum (HPS) was used as source of complement. Briefly, the samples were dissolved with DMSO in the appropriate buffer (DMSO conc. < 1 %). Then they

were diluted 1/2 or 1/3 in the plate with BBS (CP) or with APCFTD (AP) (final volume 50 p1/well for CF and 100 p1/well for AP). Thereafter, SOpi of a dilution of HPS in BBS (140 pl/lO ml) (CP) or

25 p1 of dilution of HPS in AP-CFTD (1/1) (AP) were added per well. After a standard incubation at 37°C for 30mm, SOp1 of a suspension of sensitixed sheep erythrocytes (CP) or 25 p1 of a suspension of uncoated rabbit erythrocytes (AP) were added.

performed on a VG 70-SEQ Hybrid mass spectrometer. NMR spectra were recorded on Varian Unity 400 spectrometer.

Extraction and isolation The air-dried and powdered leaves (500 g) and fresh fruits (1000 g) were extracted exhaustively with 70% and 80% acetone, respectively, at room temperature. The acetone was removed by evaporation under reduced pressure. The resulting

aqueous solution was defatted with ether, and extracted successively with EtOAc and n-BuOH. The ethyl acetate and butanol

fractions were evaporated under reduced pressure and the aqueous layer was lyophilized. The EtOAc extract of fruits was subjected to column chromatography on Sephadex LH-20 (60 x 3 cm) using EtOH as eluant. Fractions (15m1 each) were collected and combined after monitoring by TLC using solvent A. The spots were detected with UV (254 and 366 nm) before and after spraying with reagent (a) followed by reagent (b). Fractions giving positive results with reagent (a) and which came off first were combined in

two fractions I and II. Fractions giving positive results with reagent (b) were combined in two fractions III and IV. Fraction II was further chromatographed on sihca gel (35 x 2.5 cm) eluting with chloroform and containing with successive 5 % increase of methanol (flow 2.5 mi/mm). The initial fractions eluted with 20 % MeOH gave compound 1 (orange colour with reagent (a) Rf = 0.93). The fractions eluted with 40% MeOH gave two spots under UV; PTLC (silica gel 1 mm) with solvent A yielded compounds 2, and 3. Both gave an orange colour under UV after spraying with reagent (a) (R1= 0.29 and 0.25, respectively). Fraction III was subjected to further column chromatography on Sephadex LH 20(35 x 2.5 cm) with EtOH (flow 1 mi/mm) (fractions 10 ml each). Fractions from 10— 15 contained pure compound 7 (red colour with reagent (b), fif = 0.87). Fractions from 18— 30, containing two compounds, were subjected to further column chromatography on Fractogel (30 x 1.5 cm) with MeOH (10 ml each). Fractions from 5— 10 yielded compound 9 (pink colour with reagent (b), H1 = 0.86). Fractions from 12—18 rechromatographed on Sephadex LH 20 with EtOH yielded compound 8 (red colour with reagent (b), Hf = 0.82). The n-BuOH

fraction of leaves was subjected to column chromatography on Sephadex LH-20 (50 x 3 cm) using EtOH as eluant. Fractions (15 ml each) were collected and grouped after monitoring by TLC using solvent A. Fractions giving positive results with reagent (a) were combined in one fraction V and fractions giving positive resuits with reagent (b) were combined in two fractions VI and VII. Further chromatography of fraction V on Sephadex LH-20 with EtOH (fractions lOmi each): The fractions from 5—10 contained compound 4 (green colour with reagent (a), H1 = 0.36). Fractions from 12—20 contained compounds 5 and 6, which were isolated

The plates were incubated at 37°C for 60mm (CP) or 30 mm (AP). Subsequently the plates were centrifuged for 10 mm at 2000 r/min. To quantify hemolysis, 50 p1 of the super-

natant were mixed with 200 p1 water in flat-bottom microtiter

plates and the absorption at 414nm was measured with a Labsystems Multiskan® Mcc/340.

Controls in this assay consisted of erythrocytes incubated in water (100% hemolysis), in buffer (0% hemolysis) or in buffer supplemented with the appropriate HPS dilution (0% inhibition or 50% hemolysis). Data were collected as mean from 2 samples. 1C55-values were calculated.

Results and Discussion

—

Repeated column chromatography of the ethyl acetate extracts of fruits and the butanohc fraction of the leaves yielded six flavonoids and five procyanidins. The isolated compounds were identified by spectrophotometric methods (UV, FAB-MS, NMR) and by comparison with reported physical and spectral data. The isolated flavonoids were quercetin (1) and its O-glycosides hyperoside (2) and rutin (3) for the fruits and the apigenin C-glycosides vitexin (4) rhamnosylvitexin (5), and monoacetylrhamnosylvitexin (6) for the leaves. The fruits yielded the flavan-3-ol epicatechin (7), proanthocyanidin A2 (8) and procyanidin B 5 (9). The leaves yielded the procyanidins B-2 (10) and C 1

(11).

The different extracts and isolated compounds were tested for their influence on complementmediated hemolysis. Results are shown in Table 1. Both leaves and fruits extracts showed a strong inhibition of the CP and a less pronounced inhibition of the AP. The ethyl acetate and the butanol fractions of the leaves and fruits have the same high activity, while the water extract is less active. The O-glycoside rutin was the most active of the flavonoids tested (CP IC50 = 10.3 pg/mi), the aglycone quercetin was less active (IC50 = 53.8 pg/ml). The C-glycosides such as vitexin and its derivatives were not active; only rhamnosylvitexin showed a moderate inhibition of the CP. Concerning the procyanidins, proanthocyanidin A-2 and procyanidin Cl showed a very strong inhibition of the

CP. The B-dimers procyanidins B-2 and B-S were less

11

Planta Med. 62 (1996)

AbdelaatyA. Shahatetal.

Table 1 Anti-complementary activity of fractions and pure compounds of C. sinaica. (na: not active). AP

CP

Fractions/Compounds

IC53 (1ug/ml)

Fruits

EtOAc (fraction) BuOH (fraction) H20 (fraction) Quercetin Hyperoside Rutin Epicatechin Procyanidin B5 Proanthocyanidin A2

Leaves

IC50 (1ug/ml)

18.0

179.1

41.4 50.0

na na

53.8 na 10.3

na

EtOAc (fraction) BuOH (fraction) H20 (fraction) Vitexin Rharnnosylvitexin Monoacetylrhamnosylvitexin Procyanidin B2 Procyanidin Cl

extracts was not decreased when preincubation was performed at 4°C, indicating that the interference does not occur at the enzymatic level. A variation in the length of preincubation (0, 15, 30mm) at 37°C had no effect on the inhibition either. This means that depletion of complement is not the mechanism involved. The mode of action of the

na na na na

297.6 46.9 8.0

82.5

13.5 18.8 14.9

214.8

phenolic compounds may be based on a chelation of bivalent ions essential for the complement activation (23).

na na na na na na

na

56.5 na

55.7 < 5.2

To investigate the mode of action of anticomplement effects, logarithmic dilutions of the ethyl acetate extracts of leaves and fruits were tested in the assay for CP complement activation, using different preincubation conditions as shown in (Fig. 1). The inhibitory effect of both

Acknowledgements A. Lasure is research assistant of the National Fund for Scientific Research (Belgium). T. De Bruyne received a fellowship from the University of Antwerp (UIA) (1994—1996). A. A. Shahat received a fund from Egyptian Government. This work was supported by the Flemish Government Grant no. (92/94-09).

31.5

References

active (IC50 = 55.7 and 46.9 pm/mi, respectively). On the

AP the procyanidin C 1 was active while proanthocyanidin A-2 was less active.

These activities are in agreement with those reported in literature (19, 22). Crataegic acid, which

has been isolated from other C. species, also showed a strong inhibition of the CP (22). The inhibition of the complement mediated hemolysis occurred for the different extracts and isolated compounds in a dose-dependent way.

1 2

Kashnikova, M. V. (1984) Khim. Prirodn. Soedin. 1, 108. Nikolov, N,, Wagner, H., Chopin, J., Dellamonica, G., Chari, V.,

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Fig. 1 Effect of preincubation contitions on the inhibitory activity of leaf and fruit extracts on classical pathway activation.

Leaves EtOAc extract 100 80 >

—---37°C, Ommni —0—37°C, 15min

60 40

—8—37°C. 30mm

Ct

o

20 0 -20 0.5

—0—4°C, 3OmmnJ

1.4

4.1

12.3

37

111

333

concentration pg/mi

Fruit EtOAc extract 120 100

['-—37°C. 0 mm

>, 80

:

—o—37°C, 15mm

60

—6—37°C, 30mm

40

L9T±°c,

20 0 -20

0.5

1.4

4.1

12.3

37

concentration pg/mi

111

333

30mm

Planta Med. 62 (1996)

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20 Wagner, H., Knaus, W., Jordan, E. (1987) Z. Phytother. 8, 148—149. 21 Klerx, J. P. A. M., Beukelman, C. J., Van Dijk, H., Willers, J. M. N. (1983) J. lmmunol. Moth. 63, 215— 220. 22 Knaus, U. (1989) Ph. D. Thesis, MUnchen, Germany.

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