Journal Elsevier
of Ethnopharmacology, Scientific
Publishers
30 (1990) 205-214 Ireland Ltd.
205
A HYPOTENSIVE PROCYANIDIN-GLYCOSIDE LYCIOIDES SSP. LYCIOIDES
M.C. TERENCIO,
M.J. SANZ
FROM
RHAMNUS
and M. PAYA
Farmacognosia y Farmacodinamia, Departamento de Farmacologia y Farmaxotecnia, de Farmaciu, Universitat de Va’a9ncia. Avda. Blawo Ibdiiez lpi, 46010 Val&nciu (Spain) (Accepted
Facultad
May 14, 1990)
Summary
A lyophilized hot water extract of the aerial parts of Rhamnus lycioides L. (Rhamnaceael produced a lowering of systemic arterial blood pressure in normotensive anaesthetized Wistar rats. An activity-guided fractionation of the methanolic extract led to the isolation of a tetrameric procyanidin-glycoside which produced a clear dose-dependent hypotensive response (1.5-6 mg/kg i.v.l. This principle was characterized using acid hydrolysis, thiolytic degradation and spectroscopic methods. It consisted of four flavanol units with a 2,3-c& configuration and with a O-/J-D-glucosylpyranoside function on the epicatechin terminal unit. The interflavan linkage was (4-88).
Introduction
i Since ancient times, hypertensive patients have been treated orally with medicinal plant crude extracts based on folk medicine (Funayama et al., 1981; Villar et al., 1986al. In this context, an aqueous extract of the aerial part of Rhamnus lycioides L. ssp. lycioides (Rhamnaceael has been widely used for the treatment of hypertensive disorders in the Comunidad Valenciana (Spain). Traditionally, the plant material is taken by drinking an infusion made with hot water. The aerial parts of R. lycioides has been shown to contain appreciably large amounts of flavonoids, steroid-triterpenes, catechin tannins, sugars and resins, and have been shown to lack the anthraquinone compounds which are considered characteristic of the Rhamnus genus. PreCorrespondence
to: Dr. M. Pay&
0 1990 Elsevier 0378-874Ut03.50 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
206
liminary screening showed that the lyophilized aqueous extract produced a definite hypotensive effect at a dose of 50 mglkg i.v. in normotensive anaesthetized Wistar rats (Villar et al., 1986bl. In this paper, the isolation, structure determination and pharmacological activity of a proeyanidin-glycoside from R. lycioides is described. Materials and Methods Plant material
Authentic aerial parts of R. lycioides ssp. lycioides (5 kg1 were collected on Vedat mountain (Valencial in June 1984. A voucher specimen was deposited in the herbarium of the Botany Department, Faculty of Pharmacy, Valencia. Analytical
materials
and methods
Column chromatography: cellulose microcrystalline (Avicel, Merck), Sephadex LH-20 (Pharmacial, Polyamide CC6 (Macherey Nagell, Lobar B (RP-8, 40-63 mesh, Merck); TLC for sugars: silica gel 60, acetone/water 9:l; spray reagent: a-naftolsulphuric acid, after development heating at 110 “C; TLC for anthocyanidins: cellulose (Merck), HOAclHCl con./H,O 30:3:10 (Forestall; 2D-TLC: cellulose (Merck), HOAc 6%. (sytem Al, n-BuOHIHOAc/H,O 14: 1:5 (system Bl; UV spectra were recorded on a Lambda 15 Perkin Elmer spectrophotometer and IR spectra were performed with KBr pellets on a Perkin Elmer 853 spectrophotometer; HPLC was conducted with a two-pump Model LC 55B Perkin Elmer liquid chromatograph using the Lichrocart RP18 system. 13C-NMR spectra (d scale in ppml were recorded on a Brucker WP 80 SY 20 MHz spectrometer using Me,CO-d,/H,O (1:ll as solvent. Sugar analysis
Sugar analysis was performed by acid hydrolysis of 5 mg of 1 in 0.25 ml HCl 2N under reflux for 2 h. Enzymatic hydrolysis was carried out with 1 mg of 1 in 2 ml of pH 5 buffer (an aqueous 0.5 M NaOAc solution adjusted to pH 5 with HOAcl and 1 mg of /3-glucosidase (Sigma). The reaction was maintained for 12 h at 37OC (Mabry et al., 19701. A sample of both reactions was concentrated under high vacuum, and it was identified as D-ghCOSe by TLC comparison with authentic samples. Bate-Smith
hydrolisis/PC:PD
ratio
The sample (1 mgl was heated for 2 h at 95OC with 5 ml of n-BuOHIHCI cont. (95:5) and the absorbance of the anthocyanidin produced was measured on a recording spectrophotometer at 547 nm (cyanidinl or 558 nm (delphinidin). The relative amounts of each could then be calculated from the observed maximum wavelength (Bate-Smith, 19811. By TLC comparison with authentic samples it was identified as cyanidin-HCl. ( - l-Epicatechin: R,(A)
0.42, R,(B) 0.55 (violet); (+ l-catechin: R,(A) 0.46, R,(B) 0.67 (violet) (Vennat et al., 19871. Analytical dearadation with toluene-a-thiol A lOO-mg of sample was dissolved in EtOH (8 ml) containing toluene-athiol (2 ml) and HOAc (1 ml). The mixture was refluxed in N, for 50 h (Thompson et al., 19721. The remaining toluene-a-thiol was removed from the reaction products by chromatography on a silica gel column using n-hexane as eluent and the mixture of degradation products was eluted with MeOH. The degradation products were then separated on a Sephadex LH-20 column in CHClJEtOH (4:l) and the thiol product fractions were combined. After evaporation, the residue was dissolved in 5 ml MeOH and used for TLC and HPLC analysis under the following conditions: Lichrosorb RP-18 (300 x 3.8 mm). Eluents: MeOH/H,PO, (0.01 Ml: KH,PO, (0.01 Ml 30:17:53 (Al and 60:13: 27 (Bl. Elution profile: 0 to 5 min. A isocratic, 5 to 8 min from 100% A to 100% B, 8 to 30 min 100% B isocratic. Flow rate 1 ml/min. Detection 280 nm. The retention time for epicatechin and the thioether were, respectively, 6.5 min and 17.4 min. The mean polymerization degree and average molecular weight of this compound were also calculated on the basis of quantitative determination of its degradation products by HPLC. Isolation and identification The air-dried and powdered plant material was percolated with 70% methanol and concentrated under reduced pressure until completely eliminated of methanol. The aqueous residue was fractionated with EtOAc and nBuOH. As shown in Scheme 1, the butanolic extract was chromatographed over cellulose microcrystalline and BuOH/HOAc/H,O (4:1:5 upper phase) to obtain the active fraction C (45 g) which was eluted in a Sephadex LH-20 column with 800/b methanol. The pharmacologically active fraction Cm (3 g) was dissolved in water and precipitated after centrifugation to obtain a supernatant (1.8 gl which was chromatographed over polyamide CC6 using the following solvent systems: (Al water, (Bl methanol 60%. The active fraction b (546 mgl obtained was purified through a Lobar Lichroprep RP-8 column (Merck) with aqueous-acetonitrile 50% as eluent to obtain the pure Compound 1 (372 mgl. This compound was then characterized using acid hydrolysis, thiolytic degradation and spectroscopic methods. Epicatechin-4-8-epicatechin-4-8-epicatechin-4-8-epicatechin-3-O-B-D-glucopyranoside (11 was obtained as a brown amorphous powder (372 mgl. UV (MeOH)lmaXnm: 273,238 IR for 1:
of Ethnopharmacology, Scientific
Publishers
30 (1990) 205-214 Ireland Ltd.
205
A HYPOTENSIVE PROCYANIDIN-GLYCOSIDE LYCIOIDES SSP. LYCIOIDES
M.C. TERENCIO,
M.J. SANZ
FROM
RHAMNUS
and M. PAYA
Farmacognosia y Farmacodinamia, Departamento de Farmacologia y Farmaxotecnia, de Farmaciu, Universitat de Va’a9ncia. Avda. Blawo Ibdiiez lpi, 46010 Val&nciu (Spain) (Accepted
Facultad
May 14, 1990)
Summary
A lyophilized hot water extract of the aerial parts of Rhamnus lycioides L. (Rhamnaceael produced a lowering of systemic arterial blood pressure in normotensive anaesthetized Wistar rats. An activity-guided fractionation of the methanolic extract led to the isolation of a tetrameric procyanidin-glycoside which produced a clear dose-dependent hypotensive response (1.5-6 mg/kg i.v.l. This principle was characterized using acid hydrolysis, thiolytic degradation and spectroscopic methods. It consisted of four flavanol units with a 2,3-c& configuration and with a O-/J-D-glucosylpyranoside function on the epicatechin terminal unit. The interflavan linkage was (4-88).
Introduction
i Since ancient times, hypertensive patients have been treated orally with medicinal plant crude extracts based on folk medicine (Funayama et al., 1981; Villar et al., 1986al. In this context, an aqueous extract of the aerial part of Rhamnus lycioides L. ssp. lycioides (Rhamnaceael has been widely used for the treatment of hypertensive disorders in the Comunidad Valenciana (Spain). Traditionally, the plant material is taken by drinking an infusion made with hot water. The aerial parts of R. lycioides has been shown to contain appreciably large amounts of flavonoids, steroid-triterpenes, catechin tannins, sugars and resins, and have been shown to lack the anthraquinone compounds which are considered characteristic of the Rhamnus genus. PreCorrespondence
to: Dr. M. Pay&
0 1990 Elsevier 0378-874Ut03.50 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
206
liminary screening showed that the lyophilized aqueous extract produced a definite hypotensive effect at a dose of 50 mglkg i.v. in normotensive anaesthetized Wistar rats (Villar et al., 1986bl. In this paper, the isolation, structure determination and pharmacological activity of a proeyanidin-glycoside from R. lycioides is described. Materials and Methods Plant material
Authentic aerial parts of R. lycioides ssp. lycioides (5 kg1 were collected on Vedat mountain (Valencial in June 1984. A voucher specimen was deposited in the herbarium of the Botany Department, Faculty of Pharmacy, Valencia. Analytical
materials
and methods
Column chromatography: cellulose microcrystalline (Avicel, Merck), Sephadex LH-20 (Pharmacial, Polyamide CC6 (Macherey Nagell, Lobar B (RP-8, 40-63 mesh, Merck); TLC for sugars: silica gel 60, acetone/water 9:l; spray reagent: a-naftolsulphuric acid, after development heating at 110 “C; TLC for anthocyanidins: cellulose (Merck), HOAclHCl con./H,O 30:3:10 (Forestall; 2D-TLC: cellulose (Merck), HOAc 6%. (sytem Al, n-BuOHIHOAc/H,O 14: 1:5 (system Bl; UV spectra were recorded on a Lambda 15 Perkin Elmer spectrophotometer and IR spectra were performed with KBr pellets on a Perkin Elmer 853 spectrophotometer; HPLC was conducted with a two-pump Model LC 55B Perkin Elmer liquid chromatograph using the Lichrocart RP18 system. 13C-NMR spectra (d scale in ppml were recorded on a Brucker WP 80 SY 20 MHz spectrometer using Me,CO-d,/H,O (1:ll as solvent. Sugar analysis
Sugar analysis was performed by acid hydrolysis of 5 mg of 1 in 0.25 ml HCl 2N under reflux for 2 h. Enzymatic hydrolysis was carried out with 1 mg of 1 in 2 ml of pH 5 buffer (an aqueous 0.5 M NaOAc solution adjusted to pH 5 with HOAcl and 1 mg of /3-glucosidase (Sigma). The reaction was maintained for 12 h at 37OC (Mabry et al., 19701. A sample of both reactions was concentrated under high vacuum, and it was identified as D-ghCOSe by TLC comparison with authentic samples. Bate-Smith
hydrolisis/PC:PD
ratio
The sample (1 mgl was heated for 2 h at 95OC with 5 ml of n-BuOHIHCI cont. (95:5) and the absorbance of the anthocyanidin produced was measured on a recording spectrophotometer at 547 nm (cyanidinl or 558 nm (delphinidin). The relative amounts of each could then be calculated from the observed maximum wavelength (Bate-Smith, 19811. By TLC comparison with authentic samples it was identified as cyanidin-HCl. ( - l-Epicatechin: R,(A)
0.42, R,(B) 0.55 (violet); (+ l-catechin: R,(A) 0.46, R,(B) 0.67 (violet) (Vennat et al., 19871. Analytical dearadation with toluene-a-thiol A lOO-mg of sample was dissolved in EtOH (8 ml) containing toluene-athiol (2 ml) and HOAc (1 ml). The mixture was refluxed in N, for 50 h (Thompson et al., 19721. The remaining toluene-a-thiol was removed from the reaction products by chromatography on a silica gel column using n-hexane as eluent and the mixture of degradation products was eluted with MeOH. The degradation products were then separated on a Sephadex LH-20 column in CHClJEtOH (4:l) and the thiol product fractions were combined. After evaporation, the residue was dissolved in 5 ml MeOH and used for TLC and HPLC analysis under the following conditions: Lichrosorb RP-18 (300 x 3.8 mm). Eluents: MeOH/H,PO, (0.01 Ml: KH,PO, (0.01 Ml 30:17:53 (Al and 60:13: 27 (Bl. Elution profile: 0 to 5 min. A isocratic, 5 to 8 min from 100% A to 100% B, 8 to 30 min 100% B isocratic. Flow rate 1 ml/min. Detection 280 nm. The retention time for epicatechin and the thioether were, respectively, 6.5 min and 17.4 min. The mean polymerization degree and average molecular weight of this compound were also calculated on the basis of quantitative determination of its degradation products by HPLC. Isolation and identification The air-dried and powdered plant material was percolated with 70% methanol and concentrated under reduced pressure until completely eliminated of methanol. The aqueous residue was fractionated with EtOAc and nBuOH. As shown in Scheme 1, the butanolic extract was chromatographed over cellulose microcrystalline and BuOH/HOAc/H,O (4:1:5 upper phase) to obtain the active fraction C (45 g) which was eluted in a Sephadex LH-20 column with 800/b methanol. The pharmacologically active fraction Cm (3 g) was dissolved in water and precipitated after centrifugation to obtain a supernatant (1.8 gl which was chromatographed over polyamide CC6 using the following solvent systems: (Al water, (Bl methanol 60%. The active fraction b (546 mgl obtained was purified through a Lobar Lichroprep RP-8 column (Merck) with aqueous-acetonitrile 50% as eluent to obtain the pure Compound 1 (372 mgl. This compound was then characterized using acid hydrolysis, thiolytic degradation and spectroscopic methods. Epicatechin-4-8-epicatechin-4-8-epicatechin-4-8-epicatechin-3-O-B-D-glucopyranoside (11 was obtained as a brown amorphous powder (372 mgl. UV (MeOH)lmaXnm: 273,238 IR for 1:
