Journal oj Ethnopharmacology, 37 (1992) 71-76 Elsevier Scientitic Publishers Ireland Ltd.

71

Preliminary pharmacologic evaluation of crude whole plant extracts of E~epha~~~p~s scaber. Part I: in vivo studies

Anicleto Polia, Mauro Nicolaua, Clzhdia Maria OIiveira Sim6esb, Rosa Maria Ribeiro-do-Valle Nicolaua and Marlene Zanina

“Department of Pharmacology, Center of Biological Sciences and ‘Department of Pharmacognosy, Center of Health Sciences, Vniversi~de Federal de Santa Catarina, 88049 Florian~poIis, Santa Catarina (Rrazifi

(Received December 4, 1990; revision received April 22, 1992; accepted May 26, 1992)

Elephantopus scaber has been used in Brazil as a traditional remedy to cause diuresis, antipyresis and to eliminate bladder stones. In the current study, aqueous and hydroalcohohc extracts of whole plants were tested for acute toxicity, analgesic, antipyretic, anti- infla~atory, cardiovascuiar, diuretic and constipating activities. Both extracts (0.3-6 gikg i.p.) induced writhing, loss of muscle tone, ataxia, prostration and death in mice. No analgesic effects of these extracts were detected using mouse hot-plate and acetic acid- induced writhing tests. Both extracts failed to modify diuresis or carrageenan-induced rat paw edema. In contrast, given intraperitoneally, both reduced brewer’s yeast-induced hyperthermia in rats, but when given orally did not affect it. Moreover, the aqueous extract decreased the intestinal transit time in mice while the hydroalcoholic extract increased it. Finally, these extracts, given intravenously, reduced blood pressure and heart rate in rats; these effects could be btocked by atropine but not by co-administration of pyrilamine and cimetidine.

Key words: Elephantopus scaber, Elephantopus mollis; in vivo pharmacological assays; Compositae; phytochemical analyses

Introduction

Elephantop~ scaber L. (family Compositac), a perennial herb named elephant’s foot, ironweed, bull’s tongue and dog’s tongue, is a plant distributed worldwide in all tropical regions (Pio Corrga, 1926-1978; Cabrera and Klein, 1980). In Brazil, the infusion and the decoction of the whole plant are used in traditional medicine to stimulate diuresis, reduce fever and to eliminate bladder stones (Hoehne, 1939; Cabrera and Klein, 1980).

The genus Elephantopus is known to be rich in cytotoxic and antitumoral sesquiter~ne lactones (Hayashi et al., 1987). The species under study, E. scaber L. (synonmy: E. mollis H.B.K. and E. toment~sus L.) has been reported to contain the hydroxylated germacranolides, molephantin and molephantinin, which are also endowed with cyto- toxic and antitumoral properties (Lee et al., 1973, 1975). The latter sesquiterpene lactone has also been reported to possess antileukemic activity (Lee et al., 1980). This plant also contains phantomolin and its cis-epoxide, which exhibit potent inhibitory

Correspondence to: A. Poli, Department of Pharmacology, Center of Biotogical Sciences, Campus Universitario, Trin- dade, 88049 Florianopolis SC, Brazil.

actions on Ehrlich ascitis carcinoma and on Walker 256 carcinosarcoma cells (McPhail et al., 1974; Lee et al., 1980), in addition to elephantopin and deoxyelephantopin (Kurokawa et al., 1970), isodeoxyelephantopin (Govindachari et al., 1972) and 1 I, 12-dihydrodeoxyelephantopin (De Silva et al., 1982). Other triterpene constituents of E. scaber have been characterized as alpha- curcumene, beta-amyrin, lupeol, epifriedelanol (Bohlmann and Zdero, 1976) and stigmasterol (De Silva et al., 1982).

The present study was undertaken to provide an in vivo evaluation of the pha~acolo~cal proper- ties of whole plant crude extracts of E. scaber.

Materials and Methods

Plant materials Whole plant specimens of E. scaber were col-

lected at Belem (Para, Brazil) in March 1987 and supplied to us by Dr. M.E. Van den Berg of Museum Emilio Goeldi, BelCm, Par& Brazil, where a voucher specimen is deposited. They were dried at ~30°C and pulverized.

To obtain the aqueous extract (AQ), whole plant material was added to a predeterminated volume of distilled water (l/10 w/v) and was boiled

0378-8741/92~05.~ 0 1992 Elsevier Scientiiic Publishers Ireland Ltd. Printed and Published in Ireland

12

for 10 min. The hydroalcoholic extract (HA) was obtained by extracting with 50% ethanol (l/5 w/v), using a Soxhlet apparatus for 8 h. Both extracts were filtered and evaporated to dryness under reduced pressure at 50” f 5°C. The residues were dissolved in saline and the concentrated stock solutions were stored at -20°C until use in the pha~acological experiments. Yields were 2.3% and 1 .OO/o for AQ and HA, respectively, in terms of dried starting material.

Preliminary phytochemical analyses Several reactions for the identification of

chemical constituents were carried out on the same extracts used in the pharmacological tests. These assays were performed according to the methodology proposed by Farnsworth (1966), Dominguez (1973), Marini-Bettolo et al. (1981) and Harborne (1984).

Pharmacological experiments All the in vivo tests were performed as previous-

ly described (Sirnoes et al., 1989). The following methodologies were employed: acute toxicity (Malone et al., 1983); analgesic activity as evaluated by hot-plate assay (Takahashi and Kar- niol, 1975) and the acetic acid-induced writhing test (Collier et al., 1968); antipyretic activity (Adams et al., 1968); antiinflammatory activity (Winder et al., 1957; Winter et al., 1962); car- diovascular effects using urethan-anesthetized rats (Simoes et al., 1989); diuretic effects (Cummings et al., 1960) and effects on the gastrointestinal pro- pulsion of a charcoal sus~nsion (Janssen and Jageneau, 1957; Wong and Wai, 1981).

Adult male Wistar rats (3 months of age, 270-320 g) and adult male Swiss Webster mice (2 months of age, 20-30 g) were used in these experiments, with the exception of the antiinflam- matory screening in which the rats were 2 months of age (150-180 g). All animals were obtained from our own vivarium and were kept on a 12-h light/dark cycle with the temperature maintained at 23” f 1°C. Food and tap water were available ad libitum up to the beginning of the experiment. All experiments were carried out during the light cycle.

Statistical analysis The data are given as mean f S.E.M. Dif-

ferences between groups were evaluated statistical- ly using analysis of variance (ANOVA) followed by Newman-Keuls post hoc test, and P < 0.05 was set as the critical level for signi~cance.

Results and Discussion

Preliminary phyto~hem~cai analyses Table 1 shows the results obtained with the AQ

and HA of E. scaber in a preliminary phyto- chemical analysis. The tests revealed the presence of alkaloids, aurones, chalcones, steroids/triter- penoids and sesquiterpene lactones in both ex- tracts. Flavonoids were only observed in the AQ.

Anthocyanins, anthranoids, cardiac glycosides, coumarins, leucoanthocyanidins, saponins and tannins were not detected in either extract.

Acute toxicity Oral administration of either AQ or HA up to

6 g/kg, p.o. did not produce significant modifica- tions of the general behavior in mice.

Intraperitoneal injections of AQ (0.03-3 g/kg) or of HA (0.1-3 g/kg) induced writhing, loss of muscle tone, ataxia, prostration and death. These effects were more evident at doses z 1 g/kg. It was impossible to estimate the median lethal dose (LD,,) in mice accurately because there was no positive correlation between doses and deaths. However, the acute toxicity tests indicated that the LD,, values for the extracts were higher than 2 g/kg and 6 g/kg when given i.p. and p.o., respec- tively, thus revealing a low acute toxicity. It is noteworthy, though, that several authors have demonstrated cytotoxic effects for a number of the sesquiter~ne lactones isolated from the E~ephan- topus genus (Hsieh and Yang, 1969; Geran et al., 1972; Lee et al., 1980).

TABLE 1

PRELIMINARY PHYTOCHEMICAL ANALYSIS OF THE AQUEOUS (AQ) AND HYDROALCOHOLIC (HA) EXTRACTS OF E. SCABER

Chemical test AQ HA

Alkaloids Anthocyanins Anthranoids Aurones Cardiac glycosides Chalcones Coumarins Steroids/triterpenoids Flavonoids Leucoanthocyanidins Saponins Sesquiterpene lactones Tannins

+ + - -

+ +

+ + - -

+ + c - _

+ + -

73

TABLE 2

EFFECT OF ORAL (p.0.) AND INTRAPERITONEAL (ip.) ADMINISTRATION OF THE AQUEOUS (AQ) AND THE HYDROALCOHOLIC (HA) EXTRACTS OF E. SCABER AND DIPYRONE ON BREWER’S YEAST-INDUCED HYPER- THERMIA IN RATS

Treatment Route Mean f S.E.M. Rectal temperature (“C)

(mglkg) Before +16 h after After extract yeast yeast

+I h +2 h +3 h

Saline p.0. 37.1 f 0.1 38.5 f 0.1*** 38.8 l 0.2 (+0.3) 38.7 f 0.2 (+0.2) 38.6 f 0.1 (+O.l)

AQ (300) p.o. 37.4 f 0.1 38.5 f 0.1** 38.4 f 0.1 (-0.1) 38.2 f 0.3 (-0.3) 38.3 f 0.3 (-0.2) AQ W@) P.O. 37.4 f 0.2 38.6 j: 0.2** 38.8 zt 0.2 (+0.2) 38.7 f 0.2 (iO.1) 38.6 f 0.3 (0.0) HA (300) p.o. 31.4 f 0.1 38.5 f 0.1** 38.3 f 0.2 (-0.2) 38.7 f 0.2 (+0.2) 38.4 * 0.2 (-0.1) HA (608) p.0. 37.5 f 0.1 38.4 f O.l*** 38.4 ziz 0.2 (0.0) 38.4 f 0.1 (0.0) 38.3 f 0.2 (-0.1)

Saline i.p. 37.3 f 0.1 38.5 f O.l*** 38.4 zt 0.1 (-0.1) 38.5 f 0.1 (0.0) 38.3 f 0.2 (-0.2)

AQ (300) ip. 37.6 f 0.1 38.5 f 0.2** 36.9 i 0.2 (-1.6)t 36.8 f 0.3 (-1.7)t 37.0 f 0.3 (-1.5)? AQ (600) Lp. 37.5 f 0.1 38.7 f O.l*** 36.9 ztz 0.1 (-1.8)t 36.9 f 0.2 t-1.8)? 36.5 f 0.3 (-2.2)t HA (300) i-p. 37.2 f 0.1 38.3 f O.l*** 36.1 l 0.3 (-2.2)? 35.8 f 0.2 (-2.5)t 35.8 f 0.2 (-2.5)$ HA (600) i.p. 37.4 * 0.1 38.5 f O.l** 36.7 f 0.2 (-1.8)t 36.4 f 0.3 (-2.l)t 36.4 f 0.3 f-2.1)?

Dipyrone (100) i.p. 37.6 f 0.1 38.5 zt O-l** 36.5 + 0.3 (-2.0)t 36.5 i 0.2 (-2.0)t 37.1 * 0.1 f-1.4)t

Figures in parentheses indicate the change relative to the values +I6 h after yeast injection and just before extract ad~nistration: tP < 0.001. Statistically significant relative to the before yeast values: **P < 0.01, ***P < 0.001. Analysis of variance (ANOVA) followed by Newman-Keuls post hoc test was used in both cases; nitabular figure = 8.

Analgesic activity Oral administration of 30, 60, 100, 300 and 600

mg/kg and intraperitoneal injection of 3, 10 and 30 m&g of the two crude extracts obtained from E. scaber failed to modify the reaction time of mice in the hot-plate test (data not shown). Oral treatment of mice with 100 and 300 mg/kg of both extracts did not reduce the 0.6% acetic acid-induced writhing (data not shown). In these experiments morphine (30 mg/kg, i.p.) and dipyrone (200 m&g, i.p.) were used as active reference drugs.

L)iuretic screening Oral treatment of adult rats with 100 and 300

mg/kg of the two extracts did not influence hydration-induced diuresis (data not shown).

A~ti~yr~tic activity Oral administration of 300 and 600 mg/kg of

both extracts failed to affect the hyperthermia induced by brewer’s yeast in rats. However, when given intraperitoneally, these extracts and dipyr- one (100 mg/kg) signiftcantly reduced brewer’s yeast-induced hy~~he~ia + 1, +2 and +3 h after a~nistration (Table 2). It thus seems that the active antipyretic substances contained in the extracts may either not be absorbed from or are

inactivated by enzymes of the gastrointestinal tract.

Antiin~~mmatory screening Both extracts of E. scaber (300 and 600 mg/kg,

TABLE 3

EFFECTS OF ORAL ADMINlSTRATION OF AQUEOUS AND HYDROALCOHOLIC EXTRACTS OF E. SCABER AND TANNIC ACID AND LOPERAMIDE ON INTESTINAL TRANSIT IN MICE.

Treatment Oral dosea Intestinal transit

(m&kg) (%)h

Control: saline - 69.6 f 2.6 Aqueous extract 300 45.5 f 2.8**

600 39.6 j: 2.6** Hydro~coholic 300 72.1 i 2.6

extract 600 81.0 f 2.t* Tannic acid 200 40.6 i 2.4** Loperamide 5 37.3 f 3.9**

‘Administered p.o. to 24-h fasted mice, 60 min before dehver- ing charcoal meal.

bExpressed as the percentage of the distance travetled in 45 min by the charcoal meal with respect to the total length of the small intestine.

Significant relative to saline control: *P < 0.05, **P < 0.001; n/group = 8-10; analysis of variance (ANOVA) followed by Newman-Keuls post hoc test.

10 r

0.

-10 .

-20 -

-30.

-40

-50'

13 * ** 0 25 so 100200

- 40

- 0 c

f

f --40 .

0

--SO (r( 2 Q

--120 &

--I60 z

a

"-200

4QUEOUS EXTRACT OF

& SCASER ( ma/Kg 1

Fig. 1. Effects of 10-s intravenous administration of increasing doses of the aqueous extract of E. scaler (B-200 mgikg) and saline on mean blood pressure and heart rate of urethan- anesthetized rats. Each point represents the mean of 4-6 exper- iments and the vertical bars indicate the calculated S.E.M.

Significant mean blood pressure (*P < 0.001) and heart rate (**Z’ < 0.01) effects relative to saline control using ANOVA and Newman-Keuls post hoc test.

p.o.) failed to reduce edema induced by carra- geenan in the rat hind paw +0.5, + 1, +2 and +4 h after carrageenan injection (data not shown).

Effects on gastrointestinal propulsion AQ (300 and 600 mgikg, po.) signi~~ant~y

decreased intestinal transit in mice. In contrast, administration of HA (600 mg/kg) significantly increased intestinal transit (Table 3). Furthermore, as expected, both ~o~ramide (5 mglkg) and tannic acid (200 mgkg) significantly decreased intestinal transit when compared with control values. These results together with preliminary phytochemical analyses suggest that different substances may be present in the extracts which are antagonistic. Although both extracts contain chalcones and aurones, flavonoids were only detected in the AQ (Shinoda reaction).

Cardiovascular effects Intravenous injection of AQ (25-200 mgkg)

produced dose-dependent decreases in rat blood

200-

150 -

? f loo-

SO-

o-

ZOO-

lx)-

f LOO-

M-

O-

A

BEFORE AFTER 1. see. lose%

Fig. 2. Typical recording of btood pressure and heart rate of urethan-anesthetized rats. (A) Effects of 10-s intravenous administration of saline (SAL) and the aqueous extract (AQ) of E. S&W (50 m&g) before and after atropine injection (I mgkg, i.v.); (B) effects of 10-s intravenous administration of the same extract before and after injection of pyrilamine (4 mgikg, i.v.) plus cimetidine (10 mg/kg, i.v.).

10

f

40 1 -

-30 -120 k

-40 - --160

Q

-x1- t 0 25 50 & --200

HVDf?OALCOHoUC EXTRIUTT OF

& SCARER (mg /Kg 1

Fig, 3. Effects of 10-s intravenous administration of increasing doses of the hydroalcohohc extract of E. scuber (25-1OQ m&kg) and saline on mean blood pressure and heart rate of urethan- anesthetized rats. Each point represents the mean of 4-6 exper- iments and the vertical bars indicate the calculated S.E.M.

pressure and heart rate (Fig. 1). The depressor response to this extract was significantly inhibited by pretreatment with a&opine (1 mglkg, i.v.) but not by combined pretreatment with the an- tihistamines pyrilamine (4 mg/kg, i.v.) and

i-5

2 2 -_5

z E -10 cl

8 ii -15

5 w B

-20

d -W

A +5r

0 ”

-s _

-lo

-15 _

-20 _

-2s i.

Fig. 4. Effects of 10-s intravenous administration of the hydroalcoholic extract of E. scaler (50 mgikg) on mean blood pressure of urethan-anesthetized rats. (A) Before and after cimetidine p&us pyrilamine 110 and 4 mgi kg, i.v., respeetiveiy); (B) before and after atropine treatment (1 mg/kg, i.v.). Each point represents the mean of 4-6 experiments and vertical bars indicate the calculated S.E.M. Significant relative to control: *P < 0.001; ANOVA and Newman-Keuis post hoc test.

75

cimetidine (10 m&kg, i.v.) (Fig. 2). Like AQ, in- travenous administration of HA (25-100 mg/kg) produced a dose-dependent decrease in mean blood pressure and heart rate (Fig. 3). However, it was not possible to evaluate the effects of higher doses of this extract since these doses induced a high number of deaths. Pretreatment with atropine inhibited the hypotensive response to a single dose of 50 mgikg of HA (Fig. 4B). On the other hand, combined intravenous pretreatment with pyril- amine and cimetidine (4 mg/kg and 10 mg/kg, i.v., respectively) did not inhibit sig~i~cantly the hypotensive effect with the same dose of the ex- tract (Fig. 4A). Thus, the depressor effects of both extracts on cardiovascular parameters most likely involve chofinergic stimulation.

In conclusion, the assays carried out with whole plant extracts of Elephantopus scaber did not con- firm their reputed folk therapeutic uses. However, further experiments with isolated smooth muscles are underway to characterize the pharmacological profiles of these extracts better.

The authors are grateful to the technicians and to the trainee students of the Department of Phar- macology far their assistance and to Dr. Ciles A. Rae for his stylistic review. This work was sup- ported by grants from CEME (Brazilian Health Administration) and CNPo (National Research Council).

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