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Preliminary phytochemical studies of Combretodendron macrocarpum (P. beauv) keay with reference to its hypotensive principles
Journal of Ethnopharmacology, 9 (1983) 337-345 Elsevier Scientific Publishers Ireland Ltd.
337
Short Communication
PRELIMINARY
MACROCARPUM HYPOTENSIVE
PHYTOCHEMICAL STUDIES OF COMBRETODENDRON (P. BEAUV) KEAY WITH REFERENCE TO ITS PRINCIPLES
A.O. OGUNDAINa, WOLDE-AB YISAKa’* and J.A.O. OJEWOLEb aDepartment of Pharmaceutical Chemistry and bDepartment of Pharmacy, University of Ife, Ile-Ife (Nigeria)
of Pharmacology,
Faculty
(Accepted April 15, 1983)
The aqueous extract of the bark of Combretodendron macrocarpum is traditionally used in the treatment of constipation, haemorrhoids, venereal diseases and as an abortifacient. In this preliminary phytochemical and pharmacological investigation of the bark extracts we have isolated and identified fi-sitosterol, stigmasterol, octacosan-l-01 and an unsaturated dicarboxylic fatty acid with molecular formula of ClbHz604 from the petroleum ether extract. The methanol extract was fractionated by chromatography into five fractions (BBF1- BBFS) and from BBF4 a pure yellow compound with m.p. 315-316°C was isolated. Intravenous administration of 1-3 mg/kg body wt of the various extracts into normotensive male rats showed that the methanol extract and especially the chromatographic fraction BBF4 had significant hypotensive activity with little or no effect on heart rate. Introduction
C. macrocarpum (P. Beauv) Keay belongs to the Lecythidaceae family and is a woody tree which is usually 30-40 m tall. Its deeply furrowed stem bark smells foetid when slashed. It bears papery fruits which are conspicuous on both the tree and the ground. The plant grows extensively in lowland rain forest areas of West and Central Africa extending from the Ivory Coast to Angola (Keay et al., 1960). The aqueous extract of the stem bark is used in the treatment of constipation and also for haemorrhoids in Mbali and Bungali regions of the Congo (Sandberg, 1965). In Gabon it is used in the treatment of venereal diseases (Walker and Sillans, 1961) and is a&o widely *To whom all correspondence
should be addressed.
037%8741/83/$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
338
used as an abortifacient in various parts of West Africa (Dalziel, 1955; Aubreville, 1959; Hutchinson and Dalziel, 1966). Bouquet et al. (1967) showed that the aqueous extract of the bark of C. macrocarpum increases the amplitude of contractions of isolated guinea pig uterus. The extracted tannins and tannin-saponoside complexes increased both tonus and contractions while free saponosides decreased the tonus and abolished contractions. During a study involving the pharmacological screening of a large number of plants from Equatorial Africa, Sandberg and Cronlund (1982) observed that the ethanolic extract of the stem bark of C. macrocarpum produced hypotensive effects. We now wish to report our preliminary results on the phytochemical and pharmacological studies of the bark extract with special reference to the hypotensive principles. Materials and methods Plant material
The stem bark of C. macrocarpum was collected in March 1979 from within the University of Ife Campus at Be-Ife (Nigeria). The plant was authenticated by both the Botany Department of the University of Ife and the Forestry Research Institute, Ibadan. The bark was first chopped into smaller pieces while wet and dried in the oven at 40°C for 72 h. The dried bark was ground into a fine powder using an electric mill. Chromatography
Thin-layer chromatography (TLC) was carried out on silica gel G (supplied by MFC Hopkins and Williams) 5 X 10 cm and 0.5 mm in thickness. Columns were run with either neutral aluminia Brockman activity I or silica gel (Kieselgel60, 70-230 mesh ASTM, Merck) as adsorbent with a ratio of 1 g sample to 25.0 g of adsorbent. Spectral
and other physical
constants
Melting points were determined using an Electra-therm01 Melting Point Apparatus and recorded values are uncorrected. Infrared (IR) spectra were run on a Unicam SP 1100 spectrophotometer. A Perkin Elmer R 12 A 60 MHz and a Jeol90 MHz spectrophotometer were used for the proton magnetic resonance (PMR) measurements. The spectra were run in deuterochloroform (CDCl,) with tetramethylsilane (TMS) as the internal standard. The mass spectral recordings were taken on a Varian gas chromatograph- mass spectrometer, Model Mat 122s and ionization was by electron impact at 70 eV unless otherwise stated. Bioassay for cardiovascular
activity
Albino rats (200- -300 g) were used. All the crude extracts and prepurified chromatographic fractions were tested for cardiovascular activity. The extracts that were water soluble were tested as solutions in 0.9% w/v saline.
339
All other extracts were emulsified in Tween 80. Known weights of the samples (0.2-0.5 mg) and Tween 80 (1: 2) were dissolved separately in a minimum amount of methanol and/or acetone. The two solutions were mixed thoroughly and concentrated in vacua to remove the organic solvent. The resulting residue was emulsified by the gradual addition of 0.9% w/v solution of sodium chloride with concomitant shaking. Blood (BP) and Electrocardiogram (ECG) readings were taken on a 4-channel Nihon-Kohden Model RM-46 Polygraph. Six animals were used in testing each extract, fraction or subfraction. The animals were anaesthetized with an intraperitoneal injection of pentobarbitone sodium (Saga&l@, 60 mg/kg). The petroleum ether extract, the methanol extract and its ether and butanol fractions (Fig. 1) were tested at a single dose of 1 mg/kg body wt, while the butanol subfractions from
C.MACROCARPUM BARK extraction with methanol concentrated in \acuo -~
and
EXTRACT dissol\ed
AQUEOUS
in warm
water
SOLUTION extraction
with
ether
ETHER
AQUEOUS
LAYER
LAYER
extraction
with
saturated
n-butanol
with
water
BLJTANOL FRACTION I
AQUE6JS FRACTION
column
chromatography
I
BBF (5.90)
1
BBF g)
Fig. 1. Summary
(2.10
2 g)
BBF 3 (0.116 g)
of the partitioning
BBF 4
BBF
(9.58
(I.20
g)
and fractionation
5 g)
of the methanol
extract.
340
column chromatography, BBF,, BBF2, BBFJ and BBFS were tested at doses varying from 1 mg/kg to 3 mg/kg body wt. BBF4 was tested at dose ranges of 0.2-1.6 mg/kg body wt (Table 1). The various extracts, fractions and prepurified subfractions were intravenously administered into animals through polyethylene cannulae inserted into their femoral veins and washed in with O.l---0.2 ml of normal saline. Control solutions were separately administered into the animals by the same route before the administration of the extracts. Results and discussion Phytochemical
The powdered bark (2 kg) was extracted in a Soxhlet extractor for 24 h with 5 II of petroleum ether (60- 80°C) and with 5 II of methanol consecutively. The residue was then soaked in water and stirred with a mechanical stirrer for 24 h after which it was filtered off and lyophilised. The petroleum TABLE
I
ARTERIAL BLOOD SUBFRACTIONS Dose (mg/kg
body
wt.)
PRESSURE
AND
HEART
RATE
READINGS
OF THE BUTANOL
Heart rate (beatslmin)
Systolic blood pressure
Diastolic blood pressure
(mm/Hg)
(mm/Hg)
115-15 100 + 5 90 + 5
85 rf: 6 75 + 5 65 + 5
470 rl: 5 410 + 5 470 + 5
105 + 5 95+5 75+3 75+3
85+-4 85+4 60 +4 65 + 3
380 390 390 390
BBF, Control 1 2
BBF, Control 1 2 3
+ + f +
5 5 5 5
BBF, Control 1 2 3
135 130 135 135
+_ 5 + 5 + 5 f 5
110 105 110 115
+ * * +
5 5 5 5
140 110 90 85 100
f 5 + 5 + 5 f 5 +_ 8
110 95 a5 70 80
+ 5 * 4 +4 * 5 + 5
410 f 4 390 rt 4 360+4 31024
BBF, Control 0.185 0.370 0.740 1.580
430 430 430 430 430
+ * + * *
4 4 4 4 4
341
ether extract gave 220 g of a light brownish yellow oil, the methanol extract gave 345 g of a dark brown residue and the aqueous extract yielded 85 g of a greyish powder. A part of the petroleum ether extract (82 g) was saponified by refluxing with alcoholic potassium hydroxide for 12 h and after the removal of the alcohol in vacua the neutral portion was taken up in diethyl ether (3 X 300 ml) and yielded 32.0 g of a neutral fraction (CMN). The residue after ether extraction was acidified with concentrated hydrochloric acid and extracted with diethyl ether (3 X 300 ml) to give 47.5 g of an acidic fraction (CMA). The neutral fraction was subjected to repeated column chromatography on neutral alumina and eluted with a gradient solvent of increasing polarity (petroleum ether, ether and methanol) and gave four fractions. After preparative TLC on silica gel, three compounds were isolated namely CMNl,, CMNlb and CMNz (Fig. 2). CMNI, and b were obtained as an inseparable mixture of a white solid, m.p. 131- 134”C, Rf value of 0.28 in benzene/ ethyl acetate (9 : 1) and was soluble in dichloromethane. The IR spectrum (KBr pellet) showed absorbance at yrnax 960 cm-‘, 1050 cm-’ (C- 0 stretch) and 3500 cm-’ (O--H stretch). The PMR gave signals at 6 5.4 ppm (broad, vinyl protons), 5.16 ppm (doublet, 2H, J = 4 Hz), 3.56 ppm (broad 0- H) and 2.4- 0.6 ppm (complex multiplets). CC- MS, however, was able to resolve the mixtures and CMNl, was shown to be the major component with molecular ion (M+) at m/z 414 (28%) and other ions at m/z 399 (8.6), 396 (9.0), 329 (2.3), 303 (2.7), 273 (27.5), 255 (50.5), 232 (7.3), 213 (28), 163 (14.7), 145 (36.1) 120 (16.4), 85 (13.9), 69 (60.7), 55 (100) and 43 (68.7). Whereas the minor component (contaminant) has its M’ at m/z 412 (28.5), the base peak at m/z 55 and other ions at m/z 369 (9), 351 (8.5), 300 (16.8), 271 (67), 231 (20.3), 99 (4), 97 (35), 83 (60.1) and 67 (44.8). On the basis of these spectral data and also by comparison to literature data (Singh, 1969) CMNI, was identified as p-sitosterol whereas CMNlb was shown to be stigmasterol. CMNz was obtained as white (plate) crystals, m.p. 81-- 82°C Rf of 0.25 in
CMNla
CH3~n2)*P20~ CMN2 Fig. 2.
CMNlb
cti--(c~~~cH*-c02”
“““-““‘~y4-,“” 2
CMA
342
chloroform/petroleum ether (1: 1) and soluble in chloroform, ether and insoluble in acetone and methanol. The IR spectrum (KBr pellets) showed 7max at 719-720 cm-‘, 1050 cm-’ (C--O stretch) and 3450 cm-’ (e-H stretch). The PMR showed absorbance at 6 0.86 ppm (triplet, 3H), 1.28 1.56 ppm (multiplet, 51H) and 3.62 ppm (triplet, 2H, J = 4 Hz). In the mass spectrum the M’ could not be observed at 70 eV but the base peak appeared at m/z 57 and other ions were observed at m/z 410, 392, 364, 281, 267, 253, 239, 225, 223, 211, 209, 197, 195, 183, 181, 127, 125, 114,112,99, 97, 85, 83,71,69,57, 55,43 and 41. Both the PMR and MS of CMNz show strong characteristics of a long chain (unbr~ched) primary alcohol. On the basis of its spectral data and also by comparison to the literature (Pollard and Piper, 1933) CMN,! has been identified as octacosanl-01. Purification of the acidic fraction of the petroleum ether extract (CMA) was also achieved by repeated column chromatography on silica gel followed by prepa~tive TLC. The TLC were developed in toluene/ace~ne/acetic acid (100: 3: 0.07) system. A pure yellow oil (CMA) was thus isolated from CMA, it was soluble in all organic solvents, soluble in dil. NaOH and NaHC13solutions but insoluble in dil. HCl solution, and had an Rf of 0.2 in toluene/ acetone/acetic acid (100: 3 : 0.07). The IR showed absorbance at ?max 960 cm-’ (trans substituted double bond), 1470 cm-‘, 2940 cm-’ (C--H stretch), 1715 cm-’ (carbonyl stretch) 3000- 3300 cm-’ (O- .H stretch). The PMR gave signals at 6 9.41 ppm (carboxylic OH) 5.38 ppm (multiplet, vinyl protons, 2H), 2.80 ppm (triplet, ally& methyne group, lH), 2.40 ppm (triplet, 2H) 2.09 ppm (doublet, 2H), 1.8- 1.1 ppm (multiplet, 12H) and 0.94 (triplet, 3H). About 40 mg of CMAl was esterified using an ethereal solution of diazometh~e to provide CMAE whose PMR showed signals at 6 5.38 ppm (multipIet, vinyl protons), 3.68 ppm (singlet, 3H), 3.20 ppm {singlet, 3H), 2.80 ppm (triplet, lH), 2.40 ppm (triplet, 2H), 2.09 ppm (doublet, 2H), 1.8-1.1 ppm (multiplet, 12H) and 0.97 ppm (triplet, 3H). In the MS at 15 eV CMA, gave M’ at m/z 280 with other fragment ions at m/z 262,237,222,210,196,182,162,152,124,110,96,82 and 68 whereas the ester CMAE showed M’ at m/z 308 with other prom~ent ions at m/z 277, 249,221,194,185,167,153,151,139,125,113,111, 109, 99, 97, 85, 83, 74, 69, 57, 55 and 43. The IR absorbance at 960 cm-‘, 1715 cm-” and 3006-3300 cm-’ suggest CMAl to be an unsaturated carboxylic acid. The poor signals at 6 5.38 ppm for vinyl protons and the two distinct methyl ester singlets at 63.68 and 3.20 ppm of CMAE as well as the molecular ions of CMAl (m/z 280) and CMAE (m/z 308) lend strong support to the fact that CMAl may be an unsaturated dicarboxylic acid in which the environments of the two carboxylic acids are non-equivalent. The proton integration in the spectrum tends to suggest that CMA, has about 26 total protons, furthermore the substance contains two carboxyhc acids and its M’ is m/z 280; this then suggests that the molecular formula of CMAl is C16H2604_On the basis of these data a tentative structure for CMA1 is provided (Fig. 2).
343
The methanol extract which was pharmacologically the most interesting was shown to contain mainly saponins and tannins. Attempts to precipitate the saponins out by forming a cholesteride complex (Walter, 1978) or by simple addition of acetone (Odebiyi, 1978) proved unsuccesful. The fractionation and partial purification of the methanol extract was achieved by the scheme outlined in Fig. 1. The methanol extract was dissolved in warm water and was first extracted with an equal volume of diethyl ether in divided portions followed by a similar extraction with n-butanol. After evaporation it was observed that the ether took lo%, n-butanol 50% and the remaining stayed in the aqueous phase. The aqueous fraction contained mainly tannins. The butanol fraction (BBF) was subjected to column chromatography on silica gel and eluted with gradient solution of varying polarity (petroleum ether, 50% petroleum ether in dichloromethane, dichloromethane, 60% dichloromethane in ether, ether, 60% ether in acetone, acetone, 80% acetone in butanol, butanol, 70% butanol in water and butanol/water/acetic acid (60: 15: 25)). After column chromatography, the fractions were reasonably free from tannins and on the basis of their TLC pattern five fractions, namely BBF1-BBF,, were obtained. The distribution of the material in the various fractions was 30%, lo%, 0.5%, 50% and 9.5%, respectively. A portion of BBF, was subjected to further purification by combination of column and TLC chromatography. The column system was as described for the butanol fraction but the preparative TLC plates were developed in an acetone/ethyl acetate (9: 1) solvent system. From a 2.10-g sample of BBF4 only 25 mg of pure, yellow, crystalline material (BBFJ1) was obtained. BBFql, melted with decomposition at 315~-316°C had an Rf of 0.79 in chloroform/methanol (1: 1) and Rf 0.81 in ethyl acetate/acetone (1: 9). It was insoluble in chloroform, ether, acetone, dimethyl sulphoxide and very slightly soluble in methanol. It was insoluble in dil. HCl but was soluble in dil. NaOH. The PMR data were not obtained for lack of a suitable solvent. The MS at 70 eV and 120” C provided fragment ions at m/z 330 (base peak) 315 (55.5), 287 (24.9), 259 (8.7), 231 (12.3), 203 (12.2), 188 (6.5), 160 (10.5), 132 (6.4), 104 (9.8), 86 (11.2), 96 (4.7), 75 (24.1), 74 (21), 69 (10.2), 53 (10.3), 50 (10.7) and 43 (9.6). The spectral data thus far available on BBFhl is considered insufficient to enable a conclusion or even a suggestion to be made with regard to its structure. Pharmacological Most of the extracts and fractions obtained from the stem bark of C. macrocarpum were active on the circulating system of the experimental animals used in viva (Fig. 3). Intravenous injections of the methanolic extract, BBF and the subfractions from the butanol extract BBF4 induced strong depressor (hypotensive) effects in anaesthetized normotensive male rats while the aqueous extract, ether fraction and subfractions BBF, and BBF2 produced mild to moderate depressor activities. It is also interesting to note that in all the active fractions, the blood pressure was depressed
344
C.MACROCARPUM (stem bark)
I
PETROLEUM SPIRIT EXTRACT
METHANOL EXTRACT
7i: BBF 1
BUTANOL
FRACTION
FRACTION
2
BBF
(+)
3
(-)
BBF 4 (++)
active
(+) = Active
(++k Very
(+I
ETHER
BBF
(+I (-) = Not
AQUEOUS EXTRACT
(++)
(-1
[i.e. produced
depressor
(hypotensive)
effects
I
active
Fig. 3. Summary of the hypotensive activities of extracts, fractions and subfractions of C. macrocarpum stem bark.
without any significant effect on the heart rate (Table 1). On the other hand the petroleum ether extract and the butanol subfraction BBF3 did not produce any notable effect on the cardiovascular system, although BBF3 provoked a significant negative chronotropic response (Table 1) in the anaesthetized rats examined. The results of this study, though preliminary, do confirm that the butanol fraction of the methanol extract of the bark of C. macrocarpum does possess significant hypotensive activity and this may not be entirely unconnected to its traditional use as an abortifacient. Acknowledgement We wish to thank Professors F. Sandberg of Uppsala University and ‘A.A. Olaniyi of University of Ibadan for many useful discussions. Financial support through research grants from the University of Ife grant no. 1425/ FC and the International Foundation of Science grant no. 525 and through a short term fellowship from the International Seminar for Chemistry of the University of Uppsala is kindly acknowledged.
345
References Aubreville, A. (1959) La Flare Forestkre De La CGte D’lvoire, 2nd edn., Vol. 3, Nogen Sur-Marne, France. Bouquet, A., Debray, M.M., Daguet, J.C., Patey, R., Girre, A. and Leclair, J.F. (1967) Therapie, 22, 325-332. Hutchinsson, J. and Dalziel, J.M. (1966) Flora of West Tropical Africa, 2nd edn., Vol. 1, Crown agents for Overseas Government and Administration, London. Keay, R.W.J., Onochie, C.F.A. and Stanfield, D.P. (1960) Nigerian Trees, Vol. 1, Federal Government Printer, Lagos. Odebiyi, 0.0. (1978) Nigerian Journal ofpharmacy, 7, 2-7. Pollard, A. and Piper, C. (1933) Biochemical Journal, 27, 1889-1896. Sandberg, F. (1965) Cahiers de la Maboke, Tome III. Fasicule, 2, 19. Sandberg, F. and Cronlund, A. (1982) An ethnopharmacological inventory of medicinal and toxic plants from equatorial Africa. Journal of Ethnopharmacology, 5, 187-204. Singh, K.C. (1969) Journal of Scientific and Industrial Research, 28, 339-345. Walker, A. and Sillans, R. (1961) Les Plant Utiks Du Gabon, Paul Lechevalier, Paris. Walter, E.D., Van Atta, G.R., Thompson, C.R. and Maclay, W.D. (1978) Journal of the American Chemical Society, 76, 2-7.
337
Short Communication
PRELIMINARY
MACROCARPUM HYPOTENSIVE
PHYTOCHEMICAL STUDIES OF COMBRETODENDRON (P. BEAUV) KEAY WITH REFERENCE TO ITS PRINCIPLES
A.O. OGUNDAINa, WOLDE-AB YISAKa’* and J.A.O. OJEWOLEb aDepartment of Pharmaceutical Chemistry and bDepartment of Pharmacy, University of Ife, Ile-Ife (Nigeria)
of Pharmacology,
Faculty
(Accepted April 15, 1983)
The aqueous extract of the bark of Combretodendron macrocarpum is traditionally used in the treatment of constipation, haemorrhoids, venereal diseases and as an abortifacient. In this preliminary phytochemical and pharmacological investigation of the bark extracts we have isolated and identified fi-sitosterol, stigmasterol, octacosan-l-01 and an unsaturated dicarboxylic fatty acid with molecular formula of ClbHz604 from the petroleum ether extract. The methanol extract was fractionated by chromatography into five fractions (BBF1- BBFS) and from BBF4 a pure yellow compound with m.p. 315-316°C was isolated. Intravenous administration of 1-3 mg/kg body wt of the various extracts into normotensive male rats showed that the methanol extract and especially the chromatographic fraction BBF4 had significant hypotensive activity with little or no effect on heart rate. Introduction
C. macrocarpum (P. Beauv) Keay belongs to the Lecythidaceae family and is a woody tree which is usually 30-40 m tall. Its deeply furrowed stem bark smells foetid when slashed. It bears papery fruits which are conspicuous on both the tree and the ground. The plant grows extensively in lowland rain forest areas of West and Central Africa extending from the Ivory Coast to Angola (Keay et al., 1960). The aqueous extract of the stem bark is used in the treatment of constipation and also for haemorrhoids in Mbali and Bungali regions of the Congo (Sandberg, 1965). In Gabon it is used in the treatment of venereal diseases (Walker and Sillans, 1961) and is a&o widely *To whom all correspondence
should be addressed.
037%8741/83/$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
338
used as an abortifacient in various parts of West Africa (Dalziel, 1955; Aubreville, 1959; Hutchinson and Dalziel, 1966). Bouquet et al. (1967) showed that the aqueous extract of the bark of C. macrocarpum increases the amplitude of contractions of isolated guinea pig uterus. The extracted tannins and tannin-saponoside complexes increased both tonus and contractions while free saponosides decreased the tonus and abolished contractions. During a study involving the pharmacological screening of a large number of plants from Equatorial Africa, Sandberg and Cronlund (1982) observed that the ethanolic extract of the stem bark of C. macrocarpum produced hypotensive effects. We now wish to report our preliminary results on the phytochemical and pharmacological studies of the bark extract with special reference to the hypotensive principles. Materials and methods Plant material
The stem bark of C. macrocarpum was collected in March 1979 from within the University of Ife Campus at Be-Ife (Nigeria). The plant was authenticated by both the Botany Department of the University of Ife and the Forestry Research Institute, Ibadan. The bark was first chopped into smaller pieces while wet and dried in the oven at 40°C for 72 h. The dried bark was ground into a fine powder using an electric mill. Chromatography
Thin-layer chromatography (TLC) was carried out on silica gel G (supplied by MFC Hopkins and Williams) 5 X 10 cm and 0.5 mm in thickness. Columns were run with either neutral aluminia Brockman activity I or silica gel (Kieselgel60, 70-230 mesh ASTM, Merck) as adsorbent with a ratio of 1 g sample to 25.0 g of adsorbent. Spectral
and other physical
constants
Melting points were determined using an Electra-therm01 Melting Point Apparatus and recorded values are uncorrected. Infrared (IR) spectra were run on a Unicam SP 1100 spectrophotometer. A Perkin Elmer R 12 A 60 MHz and a Jeol90 MHz spectrophotometer were used for the proton magnetic resonance (PMR) measurements. The spectra were run in deuterochloroform (CDCl,) with tetramethylsilane (TMS) as the internal standard. The mass spectral recordings were taken on a Varian gas chromatograph- mass spectrometer, Model Mat 122s and ionization was by electron impact at 70 eV unless otherwise stated. Bioassay for cardiovascular
activity
Albino rats (200- -300 g) were used. All the crude extracts and prepurified chromatographic fractions were tested for cardiovascular activity. The extracts that were water soluble were tested as solutions in 0.9% w/v saline.
339
All other extracts were emulsified in Tween 80. Known weights of the samples (0.2-0.5 mg) and Tween 80 (1: 2) were dissolved separately in a minimum amount of methanol and/or acetone. The two solutions were mixed thoroughly and concentrated in vacua to remove the organic solvent. The resulting residue was emulsified by the gradual addition of 0.9% w/v solution of sodium chloride with concomitant shaking. Blood (BP) and Electrocardiogram (ECG) readings were taken on a 4-channel Nihon-Kohden Model RM-46 Polygraph. Six animals were used in testing each extract, fraction or subfraction. The animals were anaesthetized with an intraperitoneal injection of pentobarbitone sodium (Saga&l@, 60 mg/kg). The petroleum ether extract, the methanol extract and its ether and butanol fractions (Fig. 1) were tested at a single dose of 1 mg/kg body wt, while the butanol subfractions from
C.MACROCARPUM BARK extraction with methanol concentrated in \acuo -~
and
EXTRACT dissol\ed
AQUEOUS
in warm
water
SOLUTION extraction
with
ether
ETHER
AQUEOUS
LAYER
LAYER
extraction
with
saturated
n-butanol
with
water
BLJTANOL FRACTION I
AQUE6JS FRACTION
column
chromatography
I
BBF (5.90)
1
BBF g)
Fig. 1. Summary
(2.10
2 g)
BBF 3 (0.116 g)
of the partitioning
BBF 4
BBF
(9.58
(I.20
g)
and fractionation
5 g)
of the methanol
extract.
340
column chromatography, BBF,, BBF2, BBFJ and BBFS were tested at doses varying from 1 mg/kg to 3 mg/kg body wt. BBF4 was tested at dose ranges of 0.2-1.6 mg/kg body wt (Table 1). The various extracts, fractions and prepurified subfractions were intravenously administered into animals through polyethylene cannulae inserted into their femoral veins and washed in with O.l---0.2 ml of normal saline. Control solutions were separately administered into the animals by the same route before the administration of the extracts. Results and discussion Phytochemical
The powdered bark (2 kg) was extracted in a Soxhlet extractor for 24 h with 5 II of petroleum ether (60- 80°C) and with 5 II of methanol consecutively. The residue was then soaked in water and stirred with a mechanical stirrer for 24 h after which it was filtered off and lyophilised. The petroleum TABLE
I
ARTERIAL BLOOD SUBFRACTIONS Dose (mg/kg
body
wt.)
PRESSURE
AND
HEART
RATE
READINGS
OF THE BUTANOL
Heart rate (beatslmin)
Systolic blood pressure
Diastolic blood pressure
(mm/Hg)
(mm/Hg)
115-15 100 + 5 90 + 5
85 rf: 6 75 + 5 65 + 5
470 rl: 5 410 + 5 470 + 5
105 + 5 95+5 75+3 75+3
85+-4 85+4 60 +4 65 + 3
380 390 390 390
BBF, Control 1 2
BBF, Control 1 2 3
+ + f +
5 5 5 5
BBF, Control 1 2 3
135 130 135 135
+_ 5 + 5 + 5 f 5
110 105 110 115
+ * * +
5 5 5 5
140 110 90 85 100
f 5 + 5 + 5 f 5 +_ 8
110 95 a5 70 80
+ 5 * 4 +4 * 5 + 5
410 f 4 390 rt 4 360+4 31024
BBF, Control 0.185 0.370 0.740 1.580
430 430 430 430 430
+ * + * *
4 4 4 4 4
341
ether extract gave 220 g of a light brownish yellow oil, the methanol extract gave 345 g of a dark brown residue and the aqueous extract yielded 85 g of a greyish powder. A part of the petroleum ether extract (82 g) was saponified by refluxing with alcoholic potassium hydroxide for 12 h and after the removal of the alcohol in vacua the neutral portion was taken up in diethyl ether (3 X 300 ml) and yielded 32.0 g of a neutral fraction (CMN). The residue after ether extraction was acidified with concentrated hydrochloric acid and extracted with diethyl ether (3 X 300 ml) to give 47.5 g of an acidic fraction (CMA). The neutral fraction was subjected to repeated column chromatography on neutral alumina and eluted with a gradient solvent of increasing polarity (petroleum ether, ether and methanol) and gave four fractions. After preparative TLC on silica gel, three compounds were isolated namely CMNl,, CMNlb and CMNz (Fig. 2). CMNI, and b were obtained as an inseparable mixture of a white solid, m.p. 131- 134”C, Rf value of 0.28 in benzene/ ethyl acetate (9 : 1) and was soluble in dichloromethane. The IR spectrum (KBr pellet) showed absorbance at yrnax 960 cm-‘, 1050 cm-’ (C- 0 stretch) and 3500 cm-’ (O--H stretch). The PMR gave signals at 6 5.4 ppm (broad, vinyl protons), 5.16 ppm (doublet, 2H, J = 4 Hz), 3.56 ppm (broad 0- H) and 2.4- 0.6 ppm (complex multiplets). CC- MS, however, was able to resolve the mixtures and CMNl, was shown to be the major component with molecular ion (M+) at m/z 414 (28%) and other ions at m/z 399 (8.6), 396 (9.0), 329 (2.3), 303 (2.7), 273 (27.5), 255 (50.5), 232 (7.3), 213 (28), 163 (14.7), 145 (36.1) 120 (16.4), 85 (13.9), 69 (60.7), 55 (100) and 43 (68.7). Whereas the minor component (contaminant) has its M’ at m/z 412 (28.5), the base peak at m/z 55 and other ions at m/z 369 (9), 351 (8.5), 300 (16.8), 271 (67), 231 (20.3), 99 (4), 97 (35), 83 (60.1) and 67 (44.8). On the basis of these spectral data and also by comparison to literature data (Singh, 1969) CMNI, was identified as p-sitosterol whereas CMNlb was shown to be stigmasterol. CMNz was obtained as white (plate) crystals, m.p. 81-- 82°C Rf of 0.25 in
CMNla
CH3~n2)*P20~ CMN2 Fig. 2.
CMNlb
cti--(c~~~cH*-c02”
“““-““‘~y4-,“” 2
CMA
342
chloroform/petroleum ether (1: 1) and soluble in chloroform, ether and insoluble in acetone and methanol. The IR spectrum (KBr pellets) showed 7max at 719-720 cm-‘, 1050 cm-’ (C--O stretch) and 3450 cm-’ (e-H stretch). The PMR showed absorbance at 6 0.86 ppm (triplet, 3H), 1.28 1.56 ppm (multiplet, 51H) and 3.62 ppm (triplet, 2H, J = 4 Hz). In the mass spectrum the M’ could not be observed at 70 eV but the base peak appeared at m/z 57 and other ions were observed at m/z 410, 392, 364, 281, 267, 253, 239, 225, 223, 211, 209, 197, 195, 183, 181, 127, 125, 114,112,99, 97, 85, 83,71,69,57, 55,43 and 41. Both the PMR and MS of CMNz show strong characteristics of a long chain (unbr~ched) primary alcohol. On the basis of its spectral data and also by comparison to the literature (Pollard and Piper, 1933) CMN,! has been identified as octacosanl-01. Purification of the acidic fraction of the petroleum ether extract (CMA) was also achieved by repeated column chromatography on silica gel followed by prepa~tive TLC. The TLC were developed in toluene/ace~ne/acetic acid (100: 3: 0.07) system. A pure yellow oil (CMA) was thus isolated from CMA, it was soluble in all organic solvents, soluble in dil. NaOH and NaHC13solutions but insoluble in dil. HCl solution, and had an Rf of 0.2 in toluene/ acetone/acetic acid (100: 3 : 0.07). The IR showed absorbance at ?max 960 cm-’ (trans substituted double bond), 1470 cm-‘, 2940 cm-’ (C--H stretch), 1715 cm-’ (carbonyl stretch) 3000- 3300 cm-’ (O- .H stretch). The PMR gave signals at 6 9.41 ppm (carboxylic OH) 5.38 ppm (multiplet, vinyl protons, 2H), 2.80 ppm (triplet, ally& methyne group, lH), 2.40 ppm (triplet, 2H) 2.09 ppm (doublet, 2H), 1.8- 1.1 ppm (multiplet, 12H) and 0.94 (triplet, 3H). About 40 mg of CMAl was esterified using an ethereal solution of diazometh~e to provide CMAE whose PMR showed signals at 6 5.38 ppm (multipIet, vinyl protons), 3.68 ppm (singlet, 3H), 3.20 ppm {singlet, 3H), 2.80 ppm (triplet, lH), 2.40 ppm (triplet, 2H), 2.09 ppm (doublet, 2H), 1.8-1.1 ppm (multiplet, 12H) and 0.97 ppm (triplet, 3H). In the MS at 15 eV CMA, gave M’ at m/z 280 with other fragment ions at m/z 262,237,222,210,196,182,162,152,124,110,96,82 and 68 whereas the ester CMAE showed M’ at m/z 308 with other prom~ent ions at m/z 277, 249,221,194,185,167,153,151,139,125,113,111, 109, 99, 97, 85, 83, 74, 69, 57, 55 and 43. The IR absorbance at 960 cm-‘, 1715 cm-” and 3006-3300 cm-’ suggest CMAl to be an unsaturated carboxylic acid. The poor signals at 6 5.38 ppm for vinyl protons and the two distinct methyl ester singlets at 63.68 and 3.20 ppm of CMAE as well as the molecular ions of CMAl (m/z 280) and CMAE (m/z 308) lend strong support to the fact that CMAl may be an unsaturated dicarboxylic acid in which the environments of the two carboxylic acids are non-equivalent. The proton integration in the spectrum tends to suggest that CMA, has about 26 total protons, furthermore the substance contains two carboxyhc acids and its M’ is m/z 280; this then suggests that the molecular formula of CMAl is C16H2604_On the basis of these data a tentative structure for CMA1 is provided (Fig. 2).
343
The methanol extract which was pharmacologically the most interesting was shown to contain mainly saponins and tannins. Attempts to precipitate the saponins out by forming a cholesteride complex (Walter, 1978) or by simple addition of acetone (Odebiyi, 1978) proved unsuccesful. The fractionation and partial purification of the methanol extract was achieved by the scheme outlined in Fig. 1. The methanol extract was dissolved in warm water and was first extracted with an equal volume of diethyl ether in divided portions followed by a similar extraction with n-butanol. After evaporation it was observed that the ether took lo%, n-butanol 50% and the remaining stayed in the aqueous phase. The aqueous fraction contained mainly tannins. The butanol fraction (BBF) was subjected to column chromatography on silica gel and eluted with gradient solution of varying polarity (petroleum ether, 50% petroleum ether in dichloromethane, dichloromethane, 60% dichloromethane in ether, ether, 60% ether in acetone, acetone, 80% acetone in butanol, butanol, 70% butanol in water and butanol/water/acetic acid (60: 15: 25)). After column chromatography, the fractions were reasonably free from tannins and on the basis of their TLC pattern five fractions, namely BBF1-BBF,, were obtained. The distribution of the material in the various fractions was 30%, lo%, 0.5%, 50% and 9.5%, respectively. A portion of BBF, was subjected to further purification by combination of column and TLC chromatography. The column system was as described for the butanol fraction but the preparative TLC plates were developed in an acetone/ethyl acetate (9: 1) solvent system. From a 2.10-g sample of BBF4 only 25 mg of pure, yellow, crystalline material (BBFJ1) was obtained. BBFql, melted with decomposition at 315~-316°C had an Rf of 0.79 in chloroform/methanol (1: 1) and Rf 0.81 in ethyl acetate/acetone (1: 9). It was insoluble in chloroform, ether, acetone, dimethyl sulphoxide and very slightly soluble in methanol. It was insoluble in dil. HCl but was soluble in dil. NaOH. The PMR data were not obtained for lack of a suitable solvent. The MS at 70 eV and 120” C provided fragment ions at m/z 330 (base peak) 315 (55.5), 287 (24.9), 259 (8.7), 231 (12.3), 203 (12.2), 188 (6.5), 160 (10.5), 132 (6.4), 104 (9.8), 86 (11.2), 96 (4.7), 75 (24.1), 74 (21), 69 (10.2), 53 (10.3), 50 (10.7) and 43 (9.6). The spectral data thus far available on BBFhl is considered insufficient to enable a conclusion or even a suggestion to be made with regard to its structure. Pharmacological Most of the extracts and fractions obtained from the stem bark of C. macrocarpum were active on the circulating system of the experimental animals used in viva (Fig. 3). Intravenous injections of the methanolic extract, BBF and the subfractions from the butanol extract BBF4 induced strong depressor (hypotensive) effects in anaesthetized normotensive male rats while the aqueous extract, ether fraction and subfractions BBF, and BBF2 produced mild to moderate depressor activities. It is also interesting to note that in all the active fractions, the blood pressure was depressed
344
C.MACROCARPUM (stem bark)
I
PETROLEUM SPIRIT EXTRACT
METHANOL EXTRACT
7i: BBF 1
BUTANOL
FRACTION
FRACTION
2
BBF
(+)
3
(-)
BBF 4 (++)
active
(+) = Active
(++k Very
(+I
ETHER
BBF
(+I (-) = Not
AQUEOUS EXTRACT
(++)
(-1
[i.e. produced
depressor
(hypotensive)
effects
I
active
Fig. 3. Summary of the hypotensive activities of extracts, fractions and subfractions of C. macrocarpum stem bark.
without any significant effect on the heart rate (Table 1). On the other hand the petroleum ether extract and the butanol subfraction BBF3 did not produce any notable effect on the cardiovascular system, although BBF3 provoked a significant negative chronotropic response (Table 1) in the anaesthetized rats examined. The results of this study, though preliminary, do confirm that the butanol fraction of the methanol extract of the bark of C. macrocarpum does possess significant hypotensive activity and this may not be entirely unconnected to its traditional use as an abortifacient. Acknowledgement We wish to thank Professors F. Sandberg of Uppsala University and ‘A.A. Olaniyi of University of Ibadan for many useful discussions. Financial support through research grants from the University of Ife grant no. 1425/ FC and the International Foundation of Science grant no. 525 and through a short term fellowship from the International Seminar for Chemistry of the University of Uppsala is kindly acknowledged.
345
References Aubreville, A. (1959) La Flare Forestkre De La CGte D’lvoire, 2nd edn., Vol. 3, Nogen Sur-Marne, France. Bouquet, A., Debray, M.M., Daguet, J.C., Patey, R., Girre, A. and Leclair, J.F. (1967) Therapie, 22, 325-332. Hutchinsson, J. and Dalziel, J.M. (1966) Flora of West Tropical Africa, 2nd edn., Vol. 1, Crown agents for Overseas Government and Administration, London. Keay, R.W.J., Onochie, C.F.A. and Stanfield, D.P. (1960) Nigerian Trees, Vol. 1, Federal Government Printer, Lagos. Odebiyi, 0.0. (1978) Nigerian Journal ofpharmacy, 7, 2-7. Pollard, A. and Piper, C. (1933) Biochemical Journal, 27, 1889-1896. Sandberg, F. (1965) Cahiers de la Maboke, Tome III. Fasicule, 2, 19. Sandberg, F. and Cronlund, A. (1982) An ethnopharmacological inventory of medicinal and toxic plants from equatorial Africa. Journal of Ethnopharmacology, 5, 187-204. Singh, K.C. (1969) Journal of Scientific and Industrial Research, 28, 339-345. Walker, A. and Sillans, R. (1961) Les Plant Utiks Du Gabon, Paul Lechevalier, Paris. Walter, E.D., Van Atta, G.R., Thompson, C.R. and Maclay, W.D. (1978) Journal of the American Chemical Society, 76, 2-7.