strongly suggests that its consumption by the population may be hazardous. Introduction. Species of the genus Taluuma (Magnoliaceae) are used as traditional ...
Journul of Ethnophannacology, Elsevier Scientific Publishers
26 (19891277-286 Ireland Ltd.
CHEMICAL AND PHARMACOLOGICAL OVATA ST. HIL. (MAGNOLIACEAE)
STUDIES
ON TALA UMA
GINA S. MORATO, JOAO B. CALIXTO, LUIZ CORDEIRO”, THEREZA C.M. de LIMA, EDELTON F. MORATO, MAURO NICOLAU, GILES A. RAE, REINALDO N. TAKAHASHI, ROSA M.R. VALLE and ROSENDO A. YUNES’ Departamentos Florianbplis, (Accepted
de Farmacologia
e OQuimica, Universidade
Federal
de Santa Catarina,
SC lBrasiU February
6, 19891
Summary
Antidiabetic activity of crude extract of leaves of Taluuma owata St. Hil. (Magnoliaceael was analysed as part of a general pharmacological screening of this plant. Chemical analysis demonstrated the presence of phytosteroids, saponins, alkaloids and tannins in the crude extract. Pharmacological studies failed to demonstrate hypoglycemic effect of this plant in normoglycemic, hyperglycemic or alloxan-diabetic rats. The low LD,, obtained for this plant strongly suggests that its consumption by the population may be hazardous.
Introduction
Species of the genus Taluuma (Magnoliaceae) are used as traditional medicine for treating a variety of ailments. For example, in Mexico, treatment with Taluuma mexicana St. Hil. is indicated to overcome bowel disfunctions, regulate the heart, increase fertility and relieve epileptic fits. In Guatemala, however, the same specie is taken to treat arterial hypertension (Morton, 1981). Leaf infusion of Taluuma ova@ a tree found in Southern Brazil where it is named “Baguayu”, is used as a popular remedy for treating diabetes. Although several plant species have been studied for their antidiabetic properties, to our knowledge T. ovata has not yet been submitted to either chemical or pharmacological characterization. The present study was undertaken to provide an initial profile of the pharmacological properties and chemical constituents of the crude extract of T. ovata leaves, including an evaluation of its possible antidiabetic efficacy. Correspondence to: Gina S. Morato. Departamento de Farmacologia, Centro de Ciencias Biolbgicas, Universidade Federal de Santa Catarina, 88015 Florianopolis SC, Brasil. 01989 Elsevier and Printed in Ireland
0378.8741/89/.$03.85
Published
Scientific Publishers Ireland Ltd.
Material and methods Plant material
The plant used in this study was collected near the city of Florianbpolis, Santa Catarina, by Drs. Ademir Reis and Antonio Bresolin of the Department of Botany of the Federal University of Santa Catarina. Preparation
of extracts
Leaves and young shoots of T. ovata (250 g dry wtl were milled and crude extractions were carried out at 22OC with 1300 ml of 95% ethanol under reflux for 30 min. The extract was filtered, left to stand in a refrigerator (4OCl overnight, refiltered and evaporated to dryness in vacua. The residue was dissolved in water to obtain a 15% stock solution which was then stored at - 20°C until use in the pharmacological experiments. To obtain the alkaloidal fraction, dried and milled leaves (20 gl were extracted in 100 ml of 95% ethanol under reflux for 4 h. The filtrate was evaporated to dryness, washed with petroleum ether and ethyl ether, and acidified with 10 ml of 1% HCl. Following refiltration, the extract was adjusted to pH 9.0 with NH, and mixed twice with 20 ml CHCl,. Both chloroformic phases were combined to give the alkaloidal fraction, which was then dried over Na,SO, and evaporated, furnishing an oily mixture of alkaloids. Separation of the alkaloids was performed by TLC, on silica gel G-60 plates using CHClJmethanol (1: 11 as the solvent and visualized under short and long UV wavelengths or following spray with Draggendorf’s reagent. Spots with Rf values of 0.77, 0.45 and 0.05 indicated the presence of three different alkaloids. Drugs
The following drugs were used: carrageenan (Marine Coloids, Inc.), sodium pentobarbital (Abbott), heparin (Liquemine @, Roche), urethane, noradrenaline bitartrate, acetylcholine hydrochloride, estradiol benzoate, atropine sulfate, d-tubocurarine chloride, diphenhydramine hydrochloride, histamine diphosphate (all from Sigma, USA). Animals
Pharmacological experiments were conducted with adult male and female Wistar rats, male Swiss mice and male guinea-pigs, kept in a temperature and illumination controlled environment for at least a week before testing. Toads hfo ma&us) were kept in a moistened vivarium until use. In vivo experiments (al Acute toxicity.
Toxicity
was assessed
in mice (20-30
g) through
deter-
279
mination of the lethal dose 50% (LD,,). Groups of 10 animals received intraperitoneal (i.p.) injections of the crude extract (250, 300, 350, 400, 450 and 500 mg/kg) and the number of deaths was registered 24 h later. The LD,, was calculated from the dose-mortality curve as proposed by Bliss (1935). lb) Locomotor activity. Mice were treated intraperitoneally with saline or crude extract (40 and 80 mg/kg) and placed individually in cages lined with three photocell units each for 60 min. Locomotion was recorded automatically as the number of light beam interruptions which occurred during four consecutive periods of 15 min. fc) Convulsive threshold. Male rats (2’70- 320 g) were given an intraperitoneal injection of saline or crude extract (20, 100 and 300 mglkg) and 30 min later submitted to audiogenic stimulation for 2 min. Reactions were ranked according to the audiogenic convulsion index proposed by Jobe et al. (1973). (d) Effects on glycemia. Normoglycemic male rats were .fasted for 18 h prior to testing but given free access to water. Crude extract was administered (150 mglkg, i.p. or 150 and 300 mglkg, p.o.) and blood was collected from the orbital sinus 1, or 2, and 6 h after treatment for determination of glucose levels. An additional group of rats were given an oral glucose overload (1 g/kg) just after treatment with the extract (150 mg/kg, i.p.) and glycemia was evaluated 1 h later. Control groups were treated with saline. Blood glucose levels were determined by the method of Fings et al. (1970). fel Anti-edematogenic activity. Young male rats (loo-150 g) were treated with the crude extract (100 mg/kg, i.p.) and 60 min later received an intraplantar injection of carrageenan (200 pg in 0.1 ml) into one of the hind paws. The contralateral paw was injected with an equal volume of saline and served as control. Carrageenan-induced paw edema was evaluated plethysmographically 0.5, 1, 2 and 4 h after injection of the irritant and was corrected for changes in volume measured in the saline-treated paw. If, Blood pressure. Normotensive male rats (250-350 g) were anesthetized with a mixture (0.4 ml/kg, i.p.) of urethane (20%) and sodium pentobarbital (1%). Blood pressure and heart rate were recorded from a common carotid artery by means of a pressure transducer coupled to a polygraph. Both parameters were measured before and during the effect of increasing doses of the crude plant extract (l- 100 mg/kg, i.v.) or alkaloidal fraction (l-30 mg/ kg, i.v.). A concentration-response curve to crude extract and alkaloidal fraction was obtained with an interval of at least 10 min between administration. Each animal received heparin (25 IU, i.v.) to ensure patency of the arterial catheter throughout the experiment. Isolated tissue experiments (al Guinea-pig ileum. Strips 15-20 mm long, taken from a portion situated 30-10 cm proximal to the ileo-cecal junction, were set up for isotonic recording of contractions in organ baths containing 10 ml of aerated Tyrode solution at 30% under a 1 g load. After equilibration for 40-60 min cumulative and consecutive dose-response curves to either the crude extract, acetylcholine or histamine were obtained at 30 min intervals. Some experi-
280
ments were performed to assess the influence of atropine and diphenhydramine on response to the extract. Ib) Rat uterus. Uterine strips 15 mm long, taken from rats (180-250 gl treated with estradiol (0.5 mg/kg, s.c.1 24 h prior to sacrifice, were set up in 10 ml of aerated De Jalon solution at 30°C under a 1 g load. Consecutive isotonic cumulative concentration-response curves to oxytocin or acetylcholine were constructed at 30 min intervals, beginning 60 min after set up, in the absence or presence of increasing concentrations of the crude or alkaloidal extracts. lc) Rat vas deferens. Desheathed vasa deferentia from adult rats (250-350 gl were set up for recording of isotonic contractions in 10 ml of aerated Tyrode solution at 30 OC under a 1 g load. After equilibration for 40 - 60 min, consecutive and cumulative concentration response curves to acetylcholine or noradrenaline were constructed in the absence or presence of increasing concentrations of the crude extract at 30 min intervals. ld) Rat left atrium. Left atria were dissected from female rats (180-250 gl and mounted in 10 ml of gassed (95% 0, and 5% CO,) Krebs-Henseleit solution at 37OC under a tension of 1 g. Immediately after set up, each preparation was driven by electrical square pulses of 1 ms duration and 1.5 times threshold voltage delivered by a stimulator via platinum electrodes at a frequency of 2-3 Hz. Following a 60 min equilibration period and stabilization of isometric twitches, a single cumulative concentration-response curve was obtained to the crude extract. (e) Toad rectus abdominii. Following decerebration, both rectus abdominii muscles were isolated, stripped of adherent fascia and set up for recording of isotonic contractions in 10 ml of aerated Ringer solution at 24 + 2OC, under a 1 g load. After equilibration for 40-60 min, successive concentration-response curves were obtained for acetylcholine or for the crude extract at 30 min intervals in the absence or presence of d-tubocurarine. Statis tics Results are presented as means + S.E.M. When appropriate, statistical analysis was performed using the Student t-test. Differences below the probability level of 0.05 were considered statistically significant. Results Phytochemical
analysis
Different tests conducted with the crude extract of T. ovata detected the occurrence of alkaloids, saponins, phytosteroids and tannins. The alkaloidal extract presented a UV spectral band of 276 nm and another shorter band, which could suggest that the three alkaloids (Rr 0.77, 0.45 and 0.011 are aporphinic.
281
In vivo experiments
The crude extract exhibited high toxicity in mice, yielding an LD,, of 350 mg/kg. In animals given 500 mg/kg i.p. of the extract, death occurred within 10 min and was preceded by convulsion. However, the extract up to 300 mgl kg i.p. failed to affect the susceptibility of rats to audiogenic convulsions (data not shown). Doses of the extracts up to 80 mglkg, i.p. did not modify locomotor activity in mice (data not shown). The results contained in Table 1 show that the crude extract of T. ovata did not exhibit hypoglycemic activity when given p.o. or i.p. to rats. Indeed, glycemia was significantly elevated 1 h after administration of 150 mg/kg of the extract p.o. P < 0.051. In rats rendered hyperglycemic by oral glucose overload, the extract (150 mg/kg, i.p.1 did not modify glycemia measured 1 h later, although there was a trend towards higher blood glucose levels in extract-treated animals (0.1 > P > 0.051 (Table 21. Development of paw edema in response to intraplantar injection of carrageenan was unaffected by pretreatment of the rat with the crude extract (up to 100 mglkg, i.p.1. When administered i.v. to anesthetized rats, both the crude extract and the alkaloidal fraction caused significant and dose-dependent hypotensive response. Only at the highest doses tested did the crude extract (100 mg/kgl or the alkaloidal fraction (30 mglkgl depress heart rate. Figure 1 shows the results obtained with the alkaloidal fraction. Hypotension induced by the alkaloidal fraction was blocked by pretreatment with atropine (1 mg/kg, i.v.1 but not with an association of anti-histaminergic drugs, pyrilamine (4 mg/kg, i.v.l plus cimetidine (10 mglkg, i.v.1. The administration of an anticholinesterase drug increased the hypotensive effect of the crude extract.
TABLE
1
EFFECT GLUCOSE Values
OF CRUDE EXTRACT FROM LEAVES LEVELS k? f S.E.M.) of NORMOGLYCEMIC
are expressed
as mg/lOO ml. Number
Treatment
Time after
T. ovata (150 mglkg, i.p.) T. ovata (150 mglkg, p.0.) T. ovata (300 mglkg, p.0.) Saline (i.p.)
*P < 0.05 compared
of animals injection
TALAUMA RATS
are shown
OVATA
(h) 2
6
81.6 -c 2.9 (14) 92.6 2 3.6* (5) -
86.9 f 4.2 (9) 80.3 2 2.3 (6) 81.3 -t 4.6 (6)
73.4 (10) 83.0 (5) 68.0 (5) 80.6 (9)
(saline),
2 3.1
Student
t-test.
ON
in parentheses.
1
82.1 (6) to control
OF
* 3.9 -c 2.9 f
3.5
2 3.5
BLOOD
282 TABLE
2
EFFECT OF CRUDE EXTRACT FROM LEAVES OF TALAUMA GLUCOSELEVELSOFGLUCOSE-INDUCEDHYPERGLYCEMICRATS Values are expressed
as mg/lOO ml @ f
OVATA
ON BLOOD
S.E.M.1. Number of animals are in parentheses.
Treatment
Basal values
1 h after glucose administration
Saline (i.p.1 + saline lp.o.1
85.1 2 2.1 (111
81.5 f (111
5.7
Saline (i.p.1 + glucose (p.o.1
87.6 f (141
4.6
101.1 f (141
4.2*
T. ouata (150 mg/kg, i.p.1 + glucose (p.0.)
94.6 f (17)
6.5
113.8 f 070
6.6*
*P < 0.05compared to basal values (Student t-test for paired data).
Isolated
tissue experiments
The crude extract caused concentration-dependent contractions of the guinea-pig ileum,. As shown in Fig. 2 the extract was about 2000-fold less potent than acetylcholine or histamine and produced a maximal response which was 60-70% of those caused by the two agonists. Extract-induced
ALKALOIDAL FRACTION
DOSE
l mg/Kg)
Fig. 1. Effect of intravenous administration of increasing doses of alkaloidal fraction of T. ovata on mean blood pressure and heart rate of urethane/sodium pentobarbital anesthetized rats. Each point is the mean of four observations and the vertical lines represent the S.E.M.
283
lo-4 18
16’ Id’ 1
lo
IO2103lo4
CONCENTRATION
ld4 16’ x5* 16'
1
10
lo* 12 lo4
(Aug/ml)
Fig. 2. Mean cumulative concentration-response curves on the isolated guinea-pig ileum for: (A) acetylcholine (01, crude extract (0) and crude extract + atropine 0.5 rg/ml tm); (B) histamine (O), crude extract !a), crude extract + diphenhydramine 0.25 pglml (V) and crude extract + diphenhydramine 0.50 rg/ml (W. Each point represents the mean of seven experiments and vertical lines are the S.E.M.
contractions were completely suppressed by atropine (0.25 pglmll or diphenhydramine (0.5 pglml). Contractions of rat uterine strips produced by oxytotin or acetylcholine were inhibited in a concentration-dependent and noncompetitive fashion by the crude extract of T. ovata (0.25-2.0 mglml, results not shown). Moreover, the extract failed to alter the tone of the preparations when given alone. In contrast, exposure of the rat vas deferens to the extract resulted in the appearance of spontaneous concentration-dependent phasic contractions. Prior incubation with the extract (0.5-10 mglml) caused concentration-dependent inhibition of the maximal responses of the vas deferens to both noradrenaline and acetylcholine (data not shown). However, the concentration-response curve to acetylcholine, but not to noradrenaline, was shifted to the left, i.e. the extract incrased the sensitivity to acetylcholine. Similar experiments using the alkaloidal fraction (40 - 160 pg/ ml) evidenced a similar potentiation of response to low concentration of acetylcholine only, although depression of the maximal response to either agonist were somewhat less expressive than those obtained with the crude extract (Fig. 31. Exposure of the toad rectus abdominii muscles to the crude extract resulted in concentration-dependent contractions. The maximal response to the extract was similar to that obtained using acetylcholine. Prior addition of d-tubocurarine (2 pglml) to the bathing solution significantly inhibited responses to the extract (Fig. 41. In electrically driven left atria of the rat,
284
100
60
60
40
20
0 6
7
6
NORADRENALINE
5
4
6
1 -LOCI M 1
5
4
ACETYLCHOLINE
3
2
(- LOG M )
Fig. 3. Mean concentration-response curves obtained for (A) noradrenaline (01, and (B) acetylcholine (A) in the isolated rat vas deferens in the absence and presence of alkaloidal fraction from T ovata (40 wg/ml, 0; 80 pg/ml, Yj; 160 pg/ml, W). Each point represents the mean of seven determinations and vertical lines indicate the S.E.M.
concentrations of the crude extract greater than 30 pg/ml elicited a concentration-dependent but transient inhibition of contraction, which peaked within 1 min and gradually waned off in 2 - 10 min. Although the transitory effects could reflect desensitization of the preparation, 1 mglml of the extract caused an initial inhibition of inotropism of about 50% which slowly reversed into an actual potentiation of electrically-evoked twitches by about 20% after 20-30 min (results not shown). In all preparations the effects of the crude extract (up to 1 mglml) and/or
Ach x m5tv!
T. ovoto
{mg/ml)
d-?-C
T. owoto
Img/ml)
2pg/ml Fig. 4. Typical recording of the effects produced by cumulative ndditlon oi acetykholine iAchi or crude extract from 2’. ovatn in the absence and presence of d-tubocurarine WTC) on the toad rectus abdominii. Concentrations are indicated in the figure.
285
the alkaloidal extract (up to 160 pg/mll were reversed completely within 3040 min following washout. Effects produced by higher concentrations were reversed only in part even after several renewals of the bathing solution for over 60 min. Discussion The crude extract of T. ovata exhibited relatively high toxicity in mice a previous report using this plant (LD, = 350 mg/kg), thus corroborating (Hoehne, 19391. The occurrence of convulsions followed by death in animals receiving large doses of the crude extract strongly supports the presence of constituents with stimulant activity on the central nervous system. However, subtoxic doses of the extract did not increase locomotor activity or susceptibility to audiogenic convulsions. Several aspects of the present data suggest that the crude extract exhibits cholinergic activity. It is well established that large doses of cholinergic muscarinic agonists precipitate convulsions (Turski et al., 1983). Contractions of the guinea-pig ileum by the crude extract were susceptible to blockade by atropine and, to a lesser extent, by diphenhydramine, an antagonist of H,-histaminergic receptor which also exhibits anti-muscarinic activity. The frog rectus abdominii muscle was also contracted by the extract in a dtubocurarine sensitive manner, suggesting that the cholinergic-like constituents also stimulates nicotinic receptors. Moreover, the negative inotropic effect observed in the isolated atrium and the hypotension produced in anesthetized rats could both reflect muscarinic receptor activation. It appears likely that at least part of the cholinergic activity of the crude extract of T. ovata is related to the presence of one or more alkaloids. The alkaloidal extract produced hypotension in the anesthetized rat, which was blocked by pretreatment with atropine. Also, the crude and the alkaloidal extracts both potentiated contractions of the rat vas deferens induced by low concentrations of acetylcholine, but not of noradrenaline. Part of the present data clearly indicates that the crude extracts of T. ovata also displays activities unrelated to cholinergic mechanisms. This view is based on the non-selective and non-competitive inhibition of agonistinduced contractions of the rat uterus and vas deferens and on the late positive inotropic effect seen in isolated atria. Since oral or intraperitoneal administration of T. ovata extract failed to lower blood glucose levels, the present findings do not lend support to the popular belief that this species displays antidiabetic properties. Indeed, the results show that the crude extract increased glycemia in normal rats and tended to do the same in hyperglycemic animals. Therefore, at least in rats, the crude extract of T. ovata exhibits hyperglycemic activity. It may be that this possible misuse of T. ovata by the population of Southern Brazil for the treatment of diabetes stems from the fact that its popular name, “Baguaqu” is also applicable to other species such as Sizigium jambohum and Eugenia umbelliflora (Myrtaceael which are also used to treat diabetes.
286
Preliminary chemical tests indicated that the crude extract of T. ovata contains alkaloids, saponins, tannins and phytosteroids. The structural characterization of these constituents remains to be established. However, it is likely that the alkaloids are of aporphinic nature, as has been demonstrated to occur in T. mexicuna (Raffauf, 1976). Whatever the structure of the phytosteroids, the present results in the rat paw-edema test would suggest that such compounds lack significant anti-edematogenic activity. In conclusion, the crude extract of T. ovata exhibits significant cholinergic activity which may be correlated with the presence of alkaloids. Moreover, the lack of a significant hypoglycemic effect of the extract in rats allied to its high toxicity argue against the use of this species by the population for the treatment of diabetes. Acknowledgement This work was supported
by Central de Medicamentos
(CEME).
References Bliss, C.I. (19351 The calculation of the dosage-mortality curve. American Applied Biology 22, 134. Fings, C.S., Tatliff, C.R., Dunn, R.T., Toro, C. and Ackerman, P.G. (19701 Clinical Chemistry, Little, Brown & Co., Boston, MA. Hoehne, F.C. (19391 PZuntas e subst&cias wegetais t6xicas e medi cinais. Graphicars, SBo Paula, p. 122. Jobe, P.C., Picchioni, A. and Chin, L. (19731 Role of brain norepinephrine in audiogenic seizure in the rat. Journal of Pharmacology and Experimental Therapeutics 184, 1- 10. Morton, J.F. (1981) Atlas of Medicinal Plants of Middle America Charles C. Thomas, Springfield, IL, p. 220. Raffauf, R.F. (19’76) A Handbook of Alkaloids and Alkaloid-Containing Plants, John Wiley, New York. Turski, W.A., Cavalheiro, E.A., Schiwarz, M., Czuczwar, S.J., Kleinrok, Z. and Turski, L. (1983) Limbic seizures produced by pilocarpine in rats: a behavioral, electroencephalographic and neuropathological study. Behavioral Brain Research 9, 315-336.
