14 Planta Med. 58(1992)
Pharmacologically Active Phenylpropanoids from
Mohamed Hussein Farah' and Gunnar Sa,nuelsson"2 1 2
Department of Pharmacognosy, Uppsala University, Biomedical Center, Box 579, S-751 23 Uppsala, Sweden Address for correspondence
Received: February 11, 1991
Coniferaldehyde, scopoletin, sinapaldehyde, and syringaldehyde were isolated from an aqueous extract of Senra incana. All four compounds inhibited prostaglandin synthetase in a dose-dependent way. Compared to aspirin, the potency of coniferaldehyde and scopoletin was about five times higher, whereas syringaldehyde and sinapaldehyde had about half the potency of this reference compound. On topical application, sinapaldehyde and scopoletin dose-dependently inhibited ethyl phenylpropiolate-induced edema of the rat ear. The active dose range was 1—1O/1g/ear. Higher doses had a lower effect. Syringaldehyde was active in the range 20—100 jig/ear, whereas the effect of coniferaldehyde was inconclusive. Coniferaldehyde and sinapaldehyde inhibited electrically induced contractions of the guinea pig ileum in a dose-dependent way. Syringaldehyde showed a weak inhibition at a concentration of 550 jiM.
Key words Senra incana, phenylpropanoids, coniferaldehyde, scopoletin, sinapaldehyde, syringaldehyde, prostaglandin synthetase inhibition.
Introduction Senra incana Cavan. (Malvaceae) is used in Somali traditional medicine for treatment of swollen parts of the body (e. g. bruises), snake bites and wounds. The vernacular name of the plant is Balanbaal-Madow. For treatment of swellings and snake bites one whole plant is crushed and mixed with half a glass of water. The mixture is applied to the affected part of the body once a day for three days. For treatment of wounds the plant is roasted and ground to a moderately coarse powder, which is applied to the wound.
When tested on the isolated guinea pig ileum, an aqueous 2 % acetic acid extract of the plant was found to cause both contractions and inhibition of aritificially induced contractions of the ileum. These antagonistic
effects could be separated by extraction with butanol, when the contracting compounds remained in the aqueous phase and the inhibitors were extracted into the butanol.
When the butanol extract was subjected to a test programme for preliminary chemical characterization (1), the active compounds appeared to be of low relative molecular mass, moderately polar, and acidic. The activity was stable
to treatment with acid, but was partly lost on treatment with alkali. Based on these findings a separation scheme was devised, which permitted the isolation of four pharmacologically active compounds. The isolation procedure and
some studies of the pharmacological properties of the compounds is described in this paper.
Materials and Methods Reagents and apparatus Chemicals Chemicals were of analytical or puriss. grade unless otherwise specified. Puriss. grade solvents were distilled prior to use. Solvents for HPLC were of analytical or special grade. Silica
gel for column chromatography had an average particle size of 40—60 mesh and was obtained from E. Merck, Darmstadt, Germany. Reference samples of scopoletin and syringaldehyde were obtained from Aidrich-Chemie GmbH & Co. KG, Steinheim, Germany or Sigma Chemical Co., St. Louis MO, U.S.A. Coniferaldehyde was synthesized from ferulic acid (Sigma) according to (2). Melt-
ing point, MS, and 1H-NMR data were in agreement with published data (2, 3). Sinapaldehyde was synthesized from sinapic acid (Fluka Chemie AG, Buchs, Switzerland) as described in (4) and characterized by MS and 1H-NMR (5, 6).
Plant material Entire plants of Senra incana were collected in the neighbourhood of Shalambod village, Marka district, in Soma-
lia. The plant material was dried and shipped to Sweden for further studies. The plant was identified by Dr Mats Thulin, Department of Systematic Botany, Uppsala University, Sweden, in collaboration with Dr. A. M. Warfa, Department of Botany, Faculty of Agriculture, Somali National University, Mogadishu, Somalia. An herbarium specimen, labelled SMP 56, has been deposited at the
Herbarium of Uppsala University. Duplicate sets have also been deposited at the National Herbarium, Mogadishu, Somalia, the Herbarium of the Royal Botanic Gardens, Kew, England, and at the Herbarium of the Faculty of Agriculture, Mogadishu, Somalia.
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Planta Mccl. 58(1992) 15
Pharmacologically Active Phenylpropanoids from Senra incana
Reversed phase TLC Plates: ftP-is, F-254, 20 x 10cm, 0.25 mm (E. Merck, Darmstadt). Solvent: Water-acetonitrile-methanol (8 : 2 : 3). Spots were detected by spraying with vanillin-sulphuric acid, followed by heating at 110°C for five minutes [reagent no. 247 in (7)].
tion was started with chloroform (450 ml), followed by chloroform-ethyl acetate (8:2, lOOml), (3:2, 75 ml), 1:1, 35 ml), ethyl acetate (10 ml), and methanol (100 ml). Fractions of 5 ml were collected and the separation followed by reversed phase TLC of lOyl aliquots of the fractions. The contents of tubes with identical composition were pooled and the solvent removed; 9 fractions, labelled A—I were obtained (Table 1).
Preparative HPLC The column (10 x 500 mm) was of stainless steel, packed with Lichrosorb RP-18, 7pM (E. Merck, Darmstadt). The solvent (water-acetonitrile-methanol, 8 : 2 3) was pumped with a Waters Model 6000 chromatography pump at a rate of 2 ml/min. The sample solution was injected with a Waters Model U6K injector and an LDC spectrometer II was used as detector at 254 nm. :
Preparative HPLC Fraction A yielded two major and two minor peaks. The material from the two major peaks were identified as syringaldehyde and coniferaldehyde. The material obtained from the minor peaks was too small for identification.
Fraction B gave three major and one minor M.p.'s were determined with a Leitz Mikroheiztisch 350.
Electron impact mass spectra El-MS were recorded with an LKB 9000 instrument with direct inlet.
'H-NMR 1H-NMR spectra were recorded with a JEOL 90 FT-NMR spectrometer. The solvent was CD3OD and TMS was used
as internal standard.
Methods Extraction The plant material (550g) was ground to a moderately coarse powder and extracted twice with 2 % aqueous acetic acid (3 1) by stirring over night. The combined extracts were concentrated in vacuo and lyophilized. Yield: 80 g (= 14.5% w/w).
Precipitation of mucilage An aqueous solution of the extract was very viscous, presumably due to the presence of mucilage. The mucilage was removed by precipitation with ethanol as follows: The extract (80 g) was dissolved in water to make 500 ml and the solution added slowly with stirring to 11 of 95% ethanol. Following centrifugation, the mucilage was redissolved in 500 ml of water and the solution added with stirring to 500 ml 95% ethanol. The combined supernatants were taken to dryness in vacuo at 40°C.
Extraction with butanol and chloroform The dried supernatant from the precipitation with ethanol was dissolved in water to make 500 ml and the solution extracted three times with 500 ml n-butanol. The combined extracts were taken to dryness in vacuo at 40°C. The residue was dissolved in a small quantity of water and lyophilized. Yield 0.22 g (= 0.04% w/w of the dried plant material). The butanol extract
was dissolved in water and extracted three times with 10 ml of chloroform. The combined chloroform extracts were dried over anhydrous sodium sulphate and taken to dryness in vacuo. Yield: 0.17 g (= 0.03% w/w of the dried plant material).
Flash chromatography The above described procedures were repeated several times to yield 1.52 g of chloroform extract, which was dissolved in chloroform and the solution mixed with 2 g of silica gel. After removal of the solvent, the silica gel was placed on top of a column (4.5 x 50cm) of silica gel, packed in chloroform. Flash elu-
peak. The materials from the major peaks were identified as respectively syringaldehyde, scopoletin, and sinapaldehyde.
Physical data of isolated compounds Coniferaldehyde M.p. 80—82°C [Literature: 80—81°C (2)]. Mass and 1H-NMR spectra were identical to those of the reference compound.
Scopoletin M.p. 203—204°C [Literature: 203—204°C (8)]. Mass and 1H-NMR spectra were identical to those of the reference compound and to published data (8).
Sinapaldehyde M.p. 108°C [Literature: 108°C (4)]. Mass and 1H-NMR spectra were identical to those of the reference compound.
Syringaldehyde M.p. 110—113°C [Literature: 113°C (9)]. Mass and 1H-NMR spectra were identical to those of the reference compound and to published data (10. 11).
Isolated guinea pig ileum test Isolated guinea pig ileum test was performed as described in (12).
Test for inhibition of prostaglandin
synthesis This test was performed according to (13). Ten p1
of a microsome fraction of bovine vesicles (= 10—30 pg protein) were preincubated with SOpl of cofactor solution (reduced glutathione and l-epinephrine, 0.3 mg/mI each in Tris buffer, pH 8.2) in an ice/water bath for 15 minutes. Twenty p1 of vehicle (or solution
of the test substance) and 20 p1 of [14C]-arachidonic acid (l6pCi/pmol, 3OpM) were added and the mixture incubated at 37°C for 10 minutes. A blank was kept in the ice/water bath. After incubation, 5 p1 of a carrier colution of unlabelled prostaglandins
(0.2 mg/ml) was added and the reaction terminated by the addition of 5 p1 of 2N HC1. The reaction mixture was applied to a Biosil column and unmetabolized arachidonic acid eluted with a mixture of hexane-dioxane-glacial acetic acid (70 : 30: 1). The prostaglan-
din products were then eluted with ethyl acetate-methanol (85: 15) and the radioactivity of the sample determined with a Packard scintillation spectrometer. Samples were dissolved in water or 10% ethanol. Indomethacin and aspirin were used as reference compounds.
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Mohained Hussein Farah and Gunnar Sainuelsson
Planta Med. 58(1992)
Test for inhibition ofEPP-induced edema of the rat ear The method described in (14) was used with a few modifications. Male Sprague-Dawley rats (50—70 g) in groups of 5—7 animals were used. Ethyl phenylpropiolate (EPP) was dissolved in acetone at a concentration of 50mg/nil. Samples and ref-
erence substances were dissolved in acetone to appropriate concentrations. A pair of Oditest calipers (Mitutoyo, Japan) was used to determine ear thickness. Ear edema was induced by topical application of 10 l of the EPP solution to each side of the rat ear. The total amount of EPP applied was thus 1mg/ear. Ear thickness was determined before and at various time intervals after induction of the edema. The increase in ear thickness (in m) was calculated by subtraction of the thickness of the ear before induction of the edema from the thickness at the time of measurement. The result is a measure of the edema. Acetone solutions of the compounds to be tested (10 l to each side of the ear) were administered just before
induction of the edema. Mepyramine and oxyphenylbutazone in doses of 1 mg/ear were used as reference compounds. The edema of the control group reached its maximum 60 minutes after induction. The percent inhibition of the edema, caused by the tested substance, was calculated compared to the control group.
showed a great number of spots (ranging from 4—15). There
was also a partial overlap between the fractions. Fractions A and B had the highest activity in the guinea pig ileum test. Preparative HPLC of these fractions resulted in isolation of coniferaldehyde (1), scopoletin (2), sinapaldehyde (3), and syringaldehyde (4). These compounds were identi-
fied from spectral data and comparison with reference substances. Attempts to isolate smooth muscle relaxing compounds from the other fractions of the flash chromatography experiment failed, due to their extremely complex composition, which resulted in very small yields of pure compounds, the amounts of which were not sufficient for identification and tests for pharmacological activity.
Table 2 Inhibition by coniferaldehyde and sinapaldehyde of electrically induced contractions of the guinea pig ileum. Sample
Concn. in bath (jtM)
6 6 6
Student's t-test was used to determine the stasticical significance. IC50 values were calculated by regression methods.
Results Isolation of pharmacologically active compounds
chromatography of the chloroform soluble part of the aqueous extract of Senra incana (For details see Materials and Methods).
42±4 70±6 46±3
Table 3 Inhibition of prostaglandin synthetase by coniferaldehyde, scopoletin, sinapaldehyde, syringaldehyde, and selected reference substances. Substance
5 x i0 1 x 10
1x 5x Scopoletin
5 x lO 4x104 3 2x 1 x
Decrease of twitch response in the guinea pig ileum testa Syringaldehyde
101 77 68
100 100 75
83 127 140 182 500
With reference to the contraction amplitude of the guinea pig ileum before addition of the samples. All samples were tested at a concentration in the bath of 0.2 mg/ ml. The figures are the means of two determinations.
55.0 35.3 13.8
85.3 0.1 61.0 0.2
0.8 0.3 99.4 0.2
80.4 61.0 39.5 12.4
5 x iO 2.5 x i0
1 x iO 1 x 106
6 6 6
3.2 x 2.4 x
1.4 x i0 6.4 x i0
3.2 x i0 2.4 x io
1.6 x 1.2 x Aspirin
6.4 x 10 1.8 x
F G H
88±4 26 2
Table 1 Yields and smooth relaxant activity of fractions from flash
Number of experiments. Each experiment was performed on a separate preparation of ileum.
Compared to the twitch response of the leum before addition of the test sub-
The isolation of pharmacologically active compounds from the aqueous extract of Senra incana was monitored by tests on the isolated guinea pig ileum. When distributed between water and n-butanol, the butanol phase was found to contain compounds with relaxing activity on the guinea pig ileum, whereas the aqueous phase had contracting activity. Following removal of the solvent, the butanol fraction was further purified by distribution between water and chloroform. The chloroform phase (yield 0.03 % w/w of the dried plant material) contained the total pharmacological activity of the butanol extract. Flash chromatography on silica gel of the chloroform fraction, monitored by reversed phase TLC, gave 9 fractions, all of which had smooth muscle relaxing activity (Table 1). All fractions
Decrease of twitch
0.2 0.4 0.2 0.6 1.3 1.1
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Planta Mcd. 58(1992) 17
Pharmacokinetics of Vinyldithiins. Transformation Products ofAllicin
Pharmacological properties of isolated
— EPP lmg/ear+Vehicle
compounds EPP (lmg/ear)+Scopoletin lOpg/ear EPP (lmg/ear)+Sinapaldehyde 5pg/ear -- - EPP (lmg/ear)i-Syringaldehyde
Relaxation of smooth muscle Coniferaldehyde and sinapaldehyde inhibited electrically induced contractions of the guinea pig ileum
in a dose-dependent fashion (Table 2). Syringaldehyde showed a weak inhibition (about 10%) at a concentration
of 550 jiM. The smooth muscle relaxing properties of scopoletin have been reported previously (18—20). Inhibition of prostaglandin synthetase
scopoletin, sinapaldehyde, and syringaldehyde showed a dose dependent inhibition of prostaglandin synthetase. IC50 was calculated to 1.98 x iO, 2.31 x iO-', 2.96 x io-, and 2.01 x 103M respectively (Table 4). Coniferaldehyde and scopoletin are thus about five, respectively four times more potent than aspirin (IC50 = 9.47 x 104M), whereas sinapaldehyde and syringaldehyde have about half the po-
tency of this reference compound. The IC50 values obtained 60 Table 4 IC50 values and potency of coniferaldehyde, scopoletin, sinapaldehyde, syringaldehyde, and selected reference agents as inhibitors of prostagland in synthetase.
Coniferaldehyde Scopoletin Sinapaldehyde Syringaldehyde Aspirin Indomethacin
24 24 18 11
0.938 0.983 0.993 0.974 0.982
Time minutes Fig. 1 Inhibition by scopoletin, sinapaldehyde, and syringaldehyde of EPP-induced edema of the rat ear as a function of time. Significantly (p < 0.01) different from vehicle. Significantly (p < 0.001) different from vehicle.
1.98 x i0 2.31 x iO
2.96 x i0
4.8 4.1 0.3
for the reference compounds agree well with values report-
2.01 x iO
ed in the literature (13, 25).
9.53 x i0
Inhibition of EPP-induced edema of
the rat ear
n = number of observations after discarding inhibition values < 10% or> 95%. r = correlation coefficient.
A dose dependent inhibition of edema of Table 5 Effect of topical application of scopoletin, sinapaldehyde, and syringaldehyde on EPP-indu ced ear edema in rats. Substance
Edema thickness (jim) 60 mm after induction of edema and application of
substance SEM Vehicle
9 10 8 10 10 8 8 10 10 9
1000 5.0 7.0 10.0 12.0 1.3 2.5 5.0 10.0
the rat ear caused by ethyl phenylpropiolate (EPP) was observed (Table 5) for scopoletin, sinapaldehyde, and syringaldehyde. The inhibition as a function of time is presented in Fig. 1. The effect was biphasic. The ranges for dose dependence were 1—5 jig/ear for sinapaldehyde, 5—lOjig/ear,
for scopoletin, and 20—100 jig/ear, for syringaldehyde. Higher doses were less effective. For coniferaldehyde the results were inconclusive. Weak inhibition (< 20%) of the edema was observed in the dose range 1—50 jig/ear but the effect was not dose dependent. Higher doses (> 100 jig! ear) increased the edema.
The whole chloroform-soluble fraction
11.5 9.9 28.6
from which these compounds were obtained also inhibited
108 152 162