Chemical characterization of pharmacologically active compounds from Synadenium pereskiifolium

KERSTIN HERMANSSON*
LENNARD KENNE*, GEOFREY M. RUKUNGA**
GUNNAR SAMUELSSON*** and W. M. KOFI-TSEKPO**.

* Department of Organic Chemistry, Arrhenius Laboratory,
University of Stockholm, S-106 91 Stockholm, Sweden.

** Kenya Medical Research Institute
Traditional Medicines and Drugs Research Centre
P.O. Box 54840, Nairobi, Kenya.

*** Department of Pharmacognosy
University of Uppsala, Biomedicum
P.O. Box 579, S-751 Uppsala, Sweden.

ABSTRACT

Synadenium pereskiifolium (Baill.) Guill (Euphorbiaceae) is the key plant among the six plants which are used in the preparation of an anti- asthmatic drug regimen by traditional doctors. Although this plant belongs to the family of poisonous plants, traditional doctors have used it effectively in the treatment of asthma for decades with no adverse effects. Phytochemical screening of the aqueous extract of this plant revealed the presence of glycosides, terpenoids, flavonoids and other phenolic compounds. In order to characterize the pharmacologically active compounds from the aqueous extract of S. pereskiifolium, a method was adopted that was based on ion exchange, gel nitration on sephadex and extraction with organic solvents.

Introduction

Synadenium pereskiifolium (Baill.) Guill, belongs to the family Euphorbiaceae. The plant is used in the preparation of various traditional medicines, the most important preparations being an asthma remedy. S. pereskiifolium has been reported in the literature (Verdicourt and Trump, 1969; Watt and Breyer-Brandwijk, 1962) as a poisonous plant and no therapeutic value has to-date been ascribed to it. There are several publications which have mentioned other plants used traditionally for the treatment of asthma (Adjanohoun, 1983; Oliver, 1960; Nad Karni, 1976; Kokwaro, 1976; Watt and Breyer-Brandwijk, 1962). None of the publications have mentioned S. pereskiifolium as a drug plant for asthma. Yet in the preliminary study in our laboratories, medicines prepared from this plant by a traditional medicineman have shown very promising therapeutic effects on man. Preliminary phytochemical screening revealed that the leaves and stems of the plant contained glycosides, flavonoids and terpenoids.

An aqueous extract of the stems and leaves of S. pereskiifolium showed both contracting and inhibition activity of the isolated Guinea pig ileum. The aqueous extract was thus subjected to a bioassay-guided fractionation according to the scheme for preliminary chemical characterization of pharmacologically active compounds in aqueous plant extracts (Samuelsson et al., 1985).

Experimental

Solutions were concentrated under reduced pressure at temperatures not exceeding 40°C. Proton nuclear magnetic resonance (¹H-NMR) spectra were obtained at 270 MHz, and Carbon-13 nuclear magnetic resonance (¹³C-NMR) spectra were taken at 67.8 MHz on a JOEL GSX-270 spectrometer using sodium 3-trimethysilyl-propanoate-d₄ i (TSP, ¹H-NMR, D₂O) and 1, 4-dioxane (¹³C-NMR, D₂O; 67.40) as internal references. Spectra were obtained at 70°C. Separation of 2-butyl glucosides was performed on Hp-54 fused-silica capillary columns (30 m × 0.3 mm) at 190-250°C, 3°/min. A Hewlett Packard 5970 MSD gas chromatograph - mass spectrometer (GC-MS) was used for GC-MS analysis. Positive FAB-MS spectra were obtained on a JEOL Dx-303 spectrometer.

Plant material

Fresh aerial parts of S. pereskiifolium were collected from South Nyanza, Kenya and transported to Sweden by airfreight. The identity of the plant was established by Dr. Mats Thulin, Department of Systematic Botany, University of Uppsala, Sweden.

Extraction

The fresh material (1.2 kg) was cut to small pieces in a blender with rotating knives and extracted in water (81) by stirring at room temperature overnight. The extract was filtered, concentrated in vacuo in a cyclone evaporator and lyophilized, yielding crude material (41.9 g).

Isolation of glucosides

Crude extract (10 g) was dissolved in water (100 ml) and acetone (1 l) was added with stirring. The precipitate which formed was recovered and lyophilized, yielding 8.0 g of material. An aqueous solution of this material was applied on Dowex 50 (H⁺) (160 ml) and eluted with water until the effluent was colourless. The eluate was neutralized with ammonia, concentrated in vacuo and lyophilized, yielding 5.0 g of material. The water eluate was partitioned between water (300 ml) and n-butanol (5x200 ml). The aqueous phase was concentrated in vacuo and lyophilized, yielding 4.8g of material. Part of this material (2.5g) was subjected to flash chromatography on silica gel (180 g) eluating with methanol: acetic acid: chloroform (85:10:5v/v). The separation was monitored by thin layer chromatography (TLC) using ethanol: acetic acid: propanol (50:30:10 v/v) and the compounds were visualized by spraying with anisaldehyde-sulphuric acid. One fraction contained a component which gave a green spot on TLC. The solvent from this fraction was evaporated and the material was lyophilized (0.9 g). Further purification of the material (100 g) was performed on Sephadex LH 20. Eluation was performed with water and the separation was monitored by TLC. Fractions containing the compound giving a green spot were combined and lyophilized (56 mg). The yield corresponded to 8.9% of the original aqueous extract of the plant and 0.3% of the fresh plant material. Part of the material was transformed to the acid form by passing it through Dowex 50 (H⁺). The sodium salt was obtained by evaporating part of the material with sodium bicarbonate followed by purification on a column of Bio-gel P-2. The material was analysed by MS and NMR spectroscopy.

Figure

Acid hydrolysis of the glucoside

The glucoside (43 mg) was treated with 2M trifluoroacetic acid for two hours at 120°C. The reaction mixture was purified over Bio-Gel P-2, eluted with water. A fraction containing pure aglycone was obtained and the latter was shown to be malic acid by NMR and MS spectroscopy and comparison with authentic L-malic acid. Glucose was also isolated from the reaction mixture and identified by sugar analysis and ¹H-NMR spectoscopy.

Determination of the absolute configuration

The glucose (2.7 mg) was treated with 2M hydrochloric acid in (+)- 2- butanol (0.2 ml) at 80° for eight hours in a sealed tube (Gerwig, et al, 1978). The mixture was neutralized with silver carbonate and then evaporated to dryness over phosphorus pentoxide. Part of the material was analysed by GC-MS and another part was silylated with a mixture of trimethylchlorosilane-hexamethydisilane (1:3) in pyridine at 22° for thirty minutes, concentrated to dryness, dissolved in ethyl acetate and then analysed by GC-MS. Authentic D-glucose and L-malic acid were treated with racemic and (+)-2-butanol in the same way and injected as references.

Results and discussion

The compound giving green colour with anisaldehyde-sulphuric acid was isolated from S. pereskiifolium as described in the experimental section. Analysis of the ¹H and ¹³C-NMR spectra (Table 1 and 2) showed that the substance consisted of one sugar residue and an aglycone which had one CH₂ group, one CH group and two carbonyl carbons. ¹H-NMR chemical shifts and coupling constants of the signal from the sugar residue indicated a D- glucopyranoside. The ¹H- and ¹³C-NMR chemical shifts of the CH-signal indicated the presence of a CH-O group (Table 1). Positive FAB-MS produced an ion at m/z 319 (M+Na⁺) which corresponds to a molecular weight of 296 for the compound. These data, together with the sugar analysis and the determination of the absolute configuration, demonstrated that the substance consists of a b-D-glucopyranosyl group, linked to a hydroxylated dicarboxylic acid. The latter was isolated after acid hydrolysis of the glucoside and separation of the products by chromatography on Bio-Gel P-2.

The ¹H-and ¹³C-NMR spectra of the dicarboxylic acid were compared and found to be identical with spectra of malic acid.

Determination of the absolute configuration of the acid by GC-MS after reaction with 2-(+)-butanol demonstrated it to be L-malic acid. No separation of the D-and L- forms of malic acid could be obtained after the hydroxyl group of the butyl ester was silylated. On the basis of these results, structure 1(2-O-b-D-glucopyranosyl-l-malic acid) was proposed for the isolated compound. This compound inhibited electrically stimulated contractions of the Guinea pig ileum eight times more than the original total aqueous extract. To our knowledge this compound has never been found in higher plants, but itself and the similar D-tartaric acid glucoside have been synthesized by Helferich and Arndt (1965).

Acknowledgements

This work-was partly sponsored by the International Program in the Chemical Sciences at the University of Uppsala, Sweden, which is hereby gratefully acknowledged. Preliminary work was done at the Department of Traditional Medicines and Drugs Research Centre, Kenya Medical Research Institute. A traditional doctor, Mr. C. Obuya, is also gratefully acknowledged for the basic information he gave on the use of the plant.

Table 1: ¹H-NMR. Chemical shifts (d values) of 1 isolated from Synadenium pereskiifolium and of L-malic acid (coupling constants Hz in parentheses)

Notes

1. The coupling constant could not be obtained from the spectrum. 9 Hz gave the best result in spin simulation experiments.

2. Unresolved signals.

Table 2: 13C-NMR Chemical Shifts (d values) of 1 isolated from Synadenium pereskiifolium and L-malic acid

References

Adjanohoun. (ed.) (1983): Traditional Medicine and pharmacopoeia. Agence de Cooperation Culturelle et Technique (Agency for Cultural and Technical cooperation), Paris.

Gerwig, G.J., Kamerling, J. P. and Vliegenthart, J.F.G. (1978): Carbohydr. Res. 62: 349.

Helferich, B. and Arndt, O. (1965): Ann. Chem. 686: 206.

Kokwaro, J. O. (1976): Medicinal Plants of East Africa. East African Literature Bureau. Nairobi.

Nodharni, A. K. (1976): Dr. K. M. Nadkarni's Indian Materia Medica. Popular Prakashan Private Ltd. Bombay.

Oliver, B. (1960): Medicinal Plants of Nigeria, Nigerian College of Arts, Science and Technology, Ibadan.

Samuelsson, G., Kyerematen, G. and Farah, M.H. (1985): J. Ethnopharmacol. 14: 193.

Watt, H.M. and Breyer-Brandwijk, M.G. (1962): The Medicinal and poisonous plants of Southern and Eastern Africa. E. & S Livingstone Ltd. London: 437.