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Analgesic activity of Psychotria colorata (Willd. ex R. & S.) Muell. Arg. alkaloids
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Journal of
ETHNO PHARMACOI,OGY ELSEVIER
Journal of Ethnopharmacology 48 (1995) 77-83
Analgesic activity of Psychotria colorata (Willd. ex R. & S.) Muell. Arg. alkaloids Elaine Elisabetsky *c, T~nia A. Amador c, Ruti R. Albuquerque a, Domingos S. Nunes b, Ana do C.T. Carvalho u aLaboratdrio de Etnofarmacologia, Departamento de Fisiologia, Centro de Ci~ncias Biol6gicas, Universidade Federal do Pard, 66.000 Bel~m, Pard, Brazil bMestrado em Quimica de Produtos Naturais, Centro de Ci~ncias Exatas e Naturais, Universidade Federal do Pard, 66035-360 Belkm, PA, Brazil CDepartamento de Farmacologia, lnstituto de Bioci~ncias. Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite 500, 90050-170 Porto Alegre, RS, Brazil
Received 24 May 1995; revision received I0 July 1995; accepted 11 July 1995
Abstract
An ethnopharmacological survey showed that home remedies prepared with flowers, fruits and roots of Psychotria colorata (Willd. ex R. & S.) Muell. Arg. (RUBIACEAE) are used by Amazonian caboclos as pain killers. These data led to the evaluation of analgesic activity of extracts of P. colorata, using the formalin, writhing and tail-flick methods. This paper reports the Naloxone reversible opioid-like analgesic activity of alkaloids present in leafs and flowers of P. colorata. Keywords." Analgesic activity; Psychotria colorata; Amazonian caboclos; Naloxone; Alkaloids
I. Introduction
The search for new analgesic compounds has been a priority in the pharmaceutical industries (Mattison et al., 1988) since the 1960s. Two main factors have been determinants of this interest: first, analgesic compounds (or formulations) available in the market, still present a wide range of undesired effects, leaving an open door for new and better compounds (Miller et al., 1978); and, * Corresponding author Caixa Postal 5072, 90041-970 Porto Alegre, RS, Brazil
secondly, the international market of analgesics is estimated to be worth several billion dollars (Flower et al., 1985). Natural products in general, and medicinal plants in particular, are believed to be an important source of new chemical substances with potential therapeutic applicability (Elisabetsky, 1986; Farnsworth, 1989; Eisner, 1990). Well known bioactive compounds, such as atropine, A9-tetra hydrocannabinol, cocaine and morphine, were discovered in plant species traditionally used as analgesics. Taking into account that the most important analgesic prototypes (e.g. salicylic acid
0378-8741/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-8741 (95)01287-N
78
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
and morphine) were originally derived from the plant kingdom, the study of plant species traditionally used as pain killers should still be seen as a fruitful research strategy in the search for new analgesic drugs. Several Psychotria species are used medicinally for pain-related purposes (Leal and Elisabetsky, 1995). This paper reports additional pharmacological data (Elisabetsky et al., 1990, 1991) on leaf extracts of P. colorata. It is concluded that alkaloids present in leafs and flowers of this species have marked opioid-like analgesic activity. 2. Materials and methods
2.1. Ethnopharmacology A survey of medicinal plants used a analgesics was made among Amazonian caboclos of the State of Pardi, Brazil (Elisabetsky and Castilhos, 1990). Three therapeutical uses were previously reported in Brazil for P. colorata, known as 'Perp6tua do mato': for treatment of earache (Elisabetsky and Castilhos, 1990), for calming abdominal pain (Amorozo and Gely, 1988), and simply as of 'medicinal use' (Correa, 1931). P. poeppigiana Muell. Arg. (flowers) is used by the Cr6ole and the Wayapi in the French Guyana, as an analgesic for earache and by the Palikur as a cough suppressor. P. ulviformes Steyerm. (whole plant) is used by Cr6ole and Saramaka to apply over snake bites; the Wayapi use this species as a febrifuge bath. P. platypoda DC. (leaves) is used by the Wayapi as a poultice to apply over furuncles (Grenand et al., 1987). Schultes and Raffauf (1990) commented on the use of various Psychotria spp, amongst which P. alboviridula Krause is used by Tikunas to rub over bites of fire ants. Amazonian Caboclos prepare P. colorata-based remedies as follows: (a) to treat earache, a handful of small pieces of flowers are packed in banana leaves and left over warm ashes ('rescaldado'); after a while the warmed flowers are mixed with milk (preferably mother's milk), filtered (usually through a piece of cloth) and topically applied as drops; (b) to treat abdominal pain, roots and fruits are mixed with water and left to boil; the decoction is taken by mouth. Clinically, external otitis is commonly seen in
adults, whereas medium otitis is more frequently found amongst infants. External otitis is treated with locally acting anti-inflammatory drugs (e.g. cortisone) and local analgesics and medium otitis can be only cured with systemic anti-inflammatory and/or antibiotics. It is likely that indigenous populations, due to frequent contact with water, are more subjected to external otitis.
2.2. Plant collection and identification Plant material was collected in several places around the city of Bel6m (Pardi, Brazil). A voucher specimen is deposited at the the New York Botanical Garden (ZC 23, NYBG) and identification was confirmed by Dr. Brian Boom, the New York Botanical Garden. 2.3. Phytochemical procedures Preliminary chemical tests: Flowers and leaves are alkaloid-positive in precipitation tests with Dragendorff and Mayer reagents (Stahl, 1988). The roots are alkaloid-negative in the same tests. Alkaloid content is -0.8% in flowers and -0.2% in leaves. Preliminary analysis demonstrated that leaves and flowers contain the same major alkaloids, varying in the content of some minor components. Due to greater availability of leaves, only leaf extracts/fractions were used in the study reported here. Aqueous extract: Dried milled leaves (119 g) were extracted in a Soxhlet apparatus with hexane (3.2%), Et20 (1.2%), EtOAc (1.6%), EtOH (12.3%) and H20 (6.7%). The organic extracts were concentrated in a vacuum evaporator, whereas the aqueous one was liophilized. Spot test for alkaloids (Stahl, 1988) were positive for ethanol and aqueous extracts. Alkaloid extracts: Dried milled leaves (2.7 kg) were wetted with NH4OH 6N and extracted with EtOH in a Soxhlet apparatus. After distillation of solvent, the crude ethanol extract (290.0 g) was poured into a 5% HC1 solution, left for 12 h in a refrigerator and filtered. The clear acidic solution was extracted, with CH2CI2 being the residue discarded. The aqueous phase was adjusted to pH 7 with NaOH 6N and extracted with CH2C12, the organic layer washed, dried (Na2SO4) and evaporated (pH 7 alkaloid extract, 0.996 g). The
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
strong emulsion formed at pH 7 during the extraction with CHzCI 2 was separated, resolved by raising it to pH 10 with diluted NaOH, and extracted with CH2CI2 in the same way, resulting in 4.727 g of a dry foam (pH 10 alkaloid extract) after evaporation of the organic layer in vacuo. TLC analysis shows a very similar composition for both alkaloid extracts. Flash-chromatography: The alkaloid extract (pH 10, 4.7 g) was flash-chromatographed (Still et al., 1978) over 75 g of silica gel with MeOH/H20/ NH4OH (7:3:0.1) as eluent and a flow rate of I1 ml/min (air, 71b/in2). One hundred twenty-three fractions (20ml/fraction) were collected and evaporated. F20 is the mixture of five analogous fractions (0.554 g) and F26 the mixture of seven ones (0.632 g). The two main alkaloids of F26 were further purified through flash-chromatography (CHCI3/MeOH/NH4OH - - 10:3:0.1) to obtain fraction F36 (0.109 g). TLC-analysis: Pre-coated silica gel plates (F254) and eluent systems composed of MeOH/H20/ NH4OH, as well as alumina plates with EtzO/ C6HI2/MeOH were found to be useful in comparing alkaloid extracts and fractions. Chromatograms were sprayed with Dragendorff and Ehrlich reagents for alkaloids and FeCI3 2% methanol solution for phenols (Stahl, 1988). Positive alkaloid reactions were found with both alkaloid reagents; no positive reaction for phenol was observed. UV-spectra." Very similar spectra were found for all alkaloid extracts and fractions: ~kmax (MeOH) 248 nm and 307 nm. Addition of basic or acidic solution did not shift these absorption maxima.
2.4. Pharmacological evaluation Antinociceptive activity." In all analgesic experiments, male mice (food and water ad libitum) were used. Forty-five minutes before testing, the animals were placed individually in acrylic cages (20 × 20 × 20 cm), which also served as observation chambers. Extracts were given i.p. 30 min before the algogenic procedures. Aqueous extract was solubilized in water and the fractions in Tween 80 (100t~l/ml). Writhing: The method of Turner (1965) was used. Mice are made to writhe by an intraperitoneal injection of 0.6% aqueous acetic acid (0.1
79
ml/10 g). Test substances were administered (i.p.) 30 min before acetic acid. Animals were kept under observation immediately after acetic acid injection, for two consecutive 5-min periods. The number of writhes (full extension of hind paws) were recorded. Formalin." Acetic acid-induced writhing is a highly sensitive but not a very selective pain test, with false positives occurring with sedatives, muscle relaxants and other pharmacological activities. The formalin test in mice is sensitive to non-steroid anti-inflammatory drugs and other mild analgesics. The test possesses two distinctive phases, possibly reflecting different types of pain. The earlier phase reflects direct effect of formalin on nociceptors (non-inflammatory pain), whereas the late phase reflects inflammation (Hunskaar and Hole, 1987). The method of Hunskaar and Hole (1987) was used. Twenty microliters of 1% formalin were injected into the dorsal surface of the left hindpaw. Mice were observed in the chambers with a mirror mounted on three sides to allow an unobstructed view of the paws; time spent licking the injected paw (licking time) was recorded. Animals were observed for the first 5 min postformalin (early phase) or for 10 min starting at the 20th min post-formalin (late phase). Tail-flick." Analgesia was assessed with a tailflick apparatus (Albasch Equipamentos Eletr6nicos). The method of Ramabadran et al. (1989) was adapted as follows. Baseline latency (reaction time) was obtained with three measurements; after each measure animals were returned to the observation chambers for 2 min. The mean of these three measurements is the pre-drug latency time. At this point, animals presenting two measures _>6 s were discarded. Extracts or drugs were administered (i.p.) immediately after the third predrug measure. Thirty minutes afterwards, another set of three measures were taken and the mean considered as the post-drug reaction time. A cutoff time of 10 s was used to prevent tissue damage. Reversibility by Naloxone (i.p., 3.0 mg/kg) was tested by administering it 8 min before extracts or fractions. Acute toxicity: Lethality was assessed using death within 48 h as an index of toxicity following i.p. and p.o. administration of the extract.
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
80
Table 1 Effects o f P. colorata leaves a q u e o u s e x t r a c t o n w r i t h i n g i n d u c e d by acetic acid Treatment
Dose (mg/kg)
N
Writhing ( 5 - 1 0 rain)
Writhing ( 1 0 - 1 5 min)
Writhing (total)
Inhibition (%)
Saline
--
10
13.8 ± 5.1
8.3 ± 3.1
22.1 ± 6.4
--
0.4 0.8 1.6
5 6 6
12.2 + 6.1 3.8 ± 3.3 0.8 4- 0.9
5.2 4- 2.0 2.5 4- 3.5 1,3 ± 1.6
17.4 4- 7.8 6.3 ± 5.8 2.2 ± 2.4
21.3 71.5 a 90.2 b
15.6 31.2 125.0 500.0
6 6 6 6
Morphine
Aq. extr.
3.2 2.5 0.2 1.0
± 4± ±
2.8 4.2 0.4 1.4
2.3 3.2 0.2 0.0
± -~ 4±
2.1 3.9 0.4 0.0
5.5 5.7 0.3 1.0
± + ± ±
5.0 8.0 0.5 1.4
75.1 b 74.3 b 98.5 b 95.5 b
W r i t h i n g : m e a n ± S.D. a p < 0.05, b p < 0.01, A N O V A .
3. Results
tivity at the late phase is close to the maximum effect at 15.6 mg/kg
3.1. Writhing Results of aqueous extract are shown in Table 1 (expressed as number of writhes, mean + S.D.). Aqueous extract was strongly active in this test, although not in a clear dose-effect fashion.
3.4. Tail-flick Results for the pH 7 alkaloid extract and fractions F20, F26 and F36 are presented in Table 3 (reaction time in s, mean + S.D.). Alkaloid extract and fractions were clearly active in this test and the activity was reversed by previous administration of Naloxone. F20 is roughly twice as active as pH 7 and F26. F20 activity at 10.0mg/kg is comparable to Morphine at 3.5mg/kg F36 is twice as active as F26. F36 is significantly
3.2. Formalin Results of aqueous extract are shown in Table 2. The extract was active in both early and late phases of formalin-induced pain. Results obtained at the early phase show a dose-effect activity; ac-
Table 2 Effects o f P. colorata leaves a q u e o u s e x t r a c t o n the f o r m a l i n test (early a n d late phases) Treatment
Dose (mg/kg)
N
Licking (0-5min)
Inhibition (%)
Saline
--
17
130.4 ± 36.4
--
0.4 0.8 1.6
18 12 12
114.4 -4- 28.0 78.3 ± 15.0 69.7 4- 24.4
15.6 62.5 250.0 500.0
12 4 6 5
113.3 76.0 38.8 9.0
Morphine
Aq. extr.
Licking : m e a n * S.D. a p < 0.05, A N O V A , versus saline.
4444-
39.3 32.5 18.7 4.9
N
Licking ( 2 0 - 3 0 min)
Inhibition (%)
8
104.5 + 45.9
--
12.2 40.0 a 46.5 a
11 12 11
64.6 ± 35.3 61.1 4- 40.0 30.3 4- 34.1
38.1 a 41.5 a 71.0 a
13.2 41.7 70.3 a 93.1 a
4 -5 .
1,9 ± 3.3
98.2 a
0,0 4- 0.0 . .
100.0 a
-.
E. Elisabetsky et aL / Journal of Ethnopharmacology 48 (1995) 77-83
81
Table 3 Effects of pH 7 alkaloid extract, fractions F20, F26 and F36 in the tail flick test Treatment
Dose (mg/kg)
N
Reaction time Pre
Saline
Delta Post
9
4.8 4- 1.3
5.8 4. 1.3
1.4 4. 1.0
Morphine
2.0 3.5
8 5
4.7 4- 1.3 4.3 4- 2.0
7.2 4. 1.8 b 9.8 4- 0.4 c
2.4 4. 1.3 5.5 4- 1.7
Morphine + Naloxone
3.5
5
4.5 4- 1.0
5.9 4. 1.2
1.4 4. 1,1
6
4.9 4- 0.5
6.1 4. 2.4
1.2 4. 2,3
Tween 80 AIk. pH 7 a
10.0 20.0
5 5
4.4 4- 0.2 4.3 4- 1.3
6.7 4. 2.1 9.2 4. 1.5 c
2.3 4. 2.1 4.9 4. 0.7
Alk. pH 7 a + Naloxone
20.0 3.0
6
3.8 4. 0.8
5.3 4. 1.1
1.5 4. 1.5
6
3.64. 1.1
4.6 4. 1.9
0.9 4. 1.6
Tween 80 F20"
5.0 7.5 10.0
6 6 6
6.0 4. 1.5 4.1 4-0.5 5.2 4. 0.5
5.4 4. 0.9 5.3 4. 1.0 9,3 4- 0.8 c
-0.7 4. 1,9 1.2 4. 1.1 4.3 4- 0.9
F20 a + Naloxone
20.0
6
4.2 4. 1.0
5.9 4- 1.8
1.7 4- 1.6
F26 a
10.0 20.0
12 6
4.1 4- 0.9 5.1 4- 1.1
6.4 4. 1.6 c 9.2 4. 2.0 c
2.3 4. 1.6 4.0 4. 1.9
F26 a + Naloxone
20.0
6
4.74- 1.1
5.2 q- 2.1
0.5 4. 1.9
6
4.64. 0.7
5.3 4. 1.3
0.7 4. 1.7
Tween 80 F36 a
5.0 10.0 20.0
6 6 6
4.64. 0.7 4.34- 1.0 4.24. 0.9
8.9 4. 0.6 c 9.0 4. 1.0 ¢ 10.0 4. 0.7 c
4.2 4. 0.5 4.6 + 1.1 5.8 4. 0.9
F36 a + Naloxone
5.0 10.0
6 9
4.14-0.7 4.14. 0.7
6.9 4. 1.9 c 6.9 4- 1.9 c
2.8 ± 1.7 2.8 + 1.7
maximum
effect at
a F26 and F36 solubilized in Tween 80. Reaction time, delta : mean a- S.D. Wilcoxon bp < 0.5, cp < 0.05.
active at 5.0 mg/kg, reaching 20.0 mg/kg. Probably
due to the fact that even the
lowest dose studied shows activity close to maximum
effect, a statistically-significant
relationship diminished
was
not
found.
but not completely
mg/kg of Naloxone.
F36
3,5. Acute toxicity Aqueous
extract produced
(70%) when administered
dose-effect
1.0 g / k g a n d n o d e a t h s
activity
ministered
prevented
is
by 3.0
significant lethality
i.p. a t d o s e s h i g h e r t h a n were observed
v.o. up to 4.0 g/kg. Extract
when
ad-
at pH
7
(i.p.) d i d n o t i n d u c e d e a t h s a t 7 5 . 0 m g / k g ( i n t e n s e tremors
were
observed).
Fraction
F20
(i.p.) in-
82
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
duced no deaths at 40.0 mg/kg (sedations and ptosis were observed), 30% lethality at 75.0 mg/kg (sedations, tremors and ptosis were observed), and 60% lethality at 100.0 mg/kg (with clonic convulsions). Fraction F26 has a toxicity profile similar to F20. No toxic signs were observed for F36 at the doses studied. 4. Discussion
Plants used in South America to prepare the hallucinogenic beverage ayahuasca such as P. viridis and P. psychotriaefolia were found to contain tryptamine and/3-carbolin alkaloids (Schultes and Raffauf, 1990). Presence of alkaloids was reported for P. poeppigiana, P. ulviformis and P. platypoda, the former two used traditionally for pain related problems (Grenand et al., 1987). Outside South America, several Psychotria species containing pyrrolidinoindoline alkaloids have been studied (Hart et al., 1974; Roth et al., 1985; Libot et al., 1987). The previously reported cooccurrence of pyrrolidinoindolines and their rearranged calycanthine-forms in Calycodendron milnei (Libot et al., 1987) were also recently found in P. oleoides (Sevenet et al., 1990). Nevertheless, it has been suggested that alkaloids of the calycanthine type may be formed during isolation procedures (Libot et al., 1988). Interestingly, there are structural resemblances between morphine and pirrolidineindolines. For instance, the four six-membered rings in morphine have the same stereochemistry as four rings in meso-chimonanthine (although two carbons of morphine are substituted for nitrogen in mesochimonanthine). It is fair to assume that the stereospecificity of the opiate receptor is compatible with meso-chimonanthine molecule, taking into account the structural features needed for opioid-mediated analgesia (Korolkovas and Burckhalter, 1988). In this study, the antinociceptive activity of P. colorata extracts was evaluated using the formalin (early and late phases), acetic acid-induced writhing, and tail-flick tests. These procedures are used to detect narcotic and non-narcotic analgesics. Furthermore, the body of data allow for the detection of central and/or peripheral analgesia
and to distinguish analgesic from antiinflammatory properties. It is known that thermal nociceptive tests are more sensitive to opioid #-agonists and nonthermal tests to opioid x-agonists (Abbott, 1988; Furst et al., 1988). Our data suggest the involvement of both # and Kopioid receptors in the analgesic activity of alkaloids from P. colorata. Morphine effects in formalin are supposedly mediated by r-agonists (Abbott and Young, 1988). Morphine effects in tail-flick is in part mediated by descending brainstem and bulbar serotoninergic systems (Abbott and Young, 1988). At this point, we can not rule out the possibility that tryptamine alkaloids found in Psychotria may interact with serotoninergic systems; such interaction would be relevant to analgesic effects. The toxicity observed in mice might explain why these remedies are traditionally used by topical application. As a matter of fact, it cannot be concluded if the alkaloids are the only active principle or if the alkaloids have other pharmacological activities. 5. Conclusion
The data reported here show that alkaloids present in leaves and flowers of P. colorata have marked opioid-like analgesic activity. As in other cases, it is necessary to fully isolate and identify the active compound(s) and obtain a comprehensive pharmacological profile. At this point, it seems that there is a rational base to the use of P. colorata as a local analgesic to treat earache in adults. We suggest that the study of alkaloids present in Psychotria spp can be regarded as a fruitful research effort. This study also confirms the validity of the ethnopharmacological approach in the search for new drugs. Acknowledgments
This work was supported by CNPq and IFS. The authors wish to thank Mar~al de Souza Luna (phytochemistry) and Zuleica C. Castilhos (Formalin and Writhing) for technical assistance; Dr. Jos6 Roberto Leite for morphine hydrochloride; Dr. Marco Elisabetsky and Dr. Thierry Sevenet for discussing the original manuscript.
E. Elisabetsky et al./Journal of Ethnopharmacology 48 (1995) 77-83
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Journal of
ETHNO PHARMACOI,OGY ELSEVIER
Journal of Ethnopharmacology 48 (1995) 77-83
Analgesic activity of Psychotria colorata (Willd. ex R. & S.) Muell. Arg. alkaloids Elaine Elisabetsky *c, T~nia A. Amador c, Ruti R. Albuquerque a, Domingos S. Nunes b, Ana do C.T. Carvalho u aLaboratdrio de Etnofarmacologia, Departamento de Fisiologia, Centro de Ci~ncias Biol6gicas, Universidade Federal do Pard, 66.000 Bel~m, Pard, Brazil bMestrado em Quimica de Produtos Naturais, Centro de Ci~ncias Exatas e Naturais, Universidade Federal do Pard, 66035-360 Belkm, PA, Brazil CDepartamento de Farmacologia, lnstituto de Bioci~ncias. Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite 500, 90050-170 Porto Alegre, RS, Brazil
Received 24 May 1995; revision received I0 July 1995; accepted 11 July 1995
Abstract
An ethnopharmacological survey showed that home remedies prepared with flowers, fruits and roots of Psychotria colorata (Willd. ex R. & S.) Muell. Arg. (RUBIACEAE) are used by Amazonian caboclos as pain killers. These data led to the evaluation of analgesic activity of extracts of P. colorata, using the formalin, writhing and tail-flick methods. This paper reports the Naloxone reversible opioid-like analgesic activity of alkaloids present in leafs and flowers of P. colorata. Keywords." Analgesic activity; Psychotria colorata; Amazonian caboclos; Naloxone; Alkaloids
I. Introduction
The search for new analgesic compounds has been a priority in the pharmaceutical industries (Mattison et al., 1988) since the 1960s. Two main factors have been determinants of this interest: first, analgesic compounds (or formulations) available in the market, still present a wide range of undesired effects, leaving an open door for new and better compounds (Miller et al., 1978); and, * Corresponding author Caixa Postal 5072, 90041-970 Porto Alegre, RS, Brazil
secondly, the international market of analgesics is estimated to be worth several billion dollars (Flower et al., 1985). Natural products in general, and medicinal plants in particular, are believed to be an important source of new chemical substances with potential therapeutic applicability (Elisabetsky, 1986; Farnsworth, 1989; Eisner, 1990). Well known bioactive compounds, such as atropine, A9-tetra hydrocannabinol, cocaine and morphine, were discovered in plant species traditionally used as analgesics. Taking into account that the most important analgesic prototypes (e.g. salicylic acid
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E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
and morphine) were originally derived from the plant kingdom, the study of plant species traditionally used as pain killers should still be seen as a fruitful research strategy in the search for new analgesic drugs. Several Psychotria species are used medicinally for pain-related purposes (Leal and Elisabetsky, 1995). This paper reports additional pharmacological data (Elisabetsky et al., 1990, 1991) on leaf extracts of P. colorata. It is concluded that alkaloids present in leafs and flowers of this species have marked opioid-like analgesic activity. 2. Materials and methods
2.1. Ethnopharmacology A survey of medicinal plants used a analgesics was made among Amazonian caboclos of the State of Pardi, Brazil (Elisabetsky and Castilhos, 1990). Three therapeutical uses were previously reported in Brazil for P. colorata, known as 'Perp6tua do mato': for treatment of earache (Elisabetsky and Castilhos, 1990), for calming abdominal pain (Amorozo and Gely, 1988), and simply as of 'medicinal use' (Correa, 1931). P. poeppigiana Muell. Arg. (flowers) is used by the Cr6ole and the Wayapi in the French Guyana, as an analgesic for earache and by the Palikur as a cough suppressor. P. ulviformes Steyerm. (whole plant) is used by Cr6ole and Saramaka to apply over snake bites; the Wayapi use this species as a febrifuge bath. P. platypoda DC. (leaves) is used by the Wayapi as a poultice to apply over furuncles (Grenand et al., 1987). Schultes and Raffauf (1990) commented on the use of various Psychotria spp, amongst which P. alboviridula Krause is used by Tikunas to rub over bites of fire ants. Amazonian Caboclos prepare P. colorata-based remedies as follows: (a) to treat earache, a handful of small pieces of flowers are packed in banana leaves and left over warm ashes ('rescaldado'); after a while the warmed flowers are mixed with milk (preferably mother's milk), filtered (usually through a piece of cloth) and topically applied as drops; (b) to treat abdominal pain, roots and fruits are mixed with water and left to boil; the decoction is taken by mouth. Clinically, external otitis is commonly seen in
adults, whereas medium otitis is more frequently found amongst infants. External otitis is treated with locally acting anti-inflammatory drugs (e.g. cortisone) and local analgesics and medium otitis can be only cured with systemic anti-inflammatory and/or antibiotics. It is likely that indigenous populations, due to frequent contact with water, are more subjected to external otitis.
2.2. Plant collection and identification Plant material was collected in several places around the city of Bel6m (Pardi, Brazil). A voucher specimen is deposited at the the New York Botanical Garden (ZC 23, NYBG) and identification was confirmed by Dr. Brian Boom, the New York Botanical Garden. 2.3. Phytochemical procedures Preliminary chemical tests: Flowers and leaves are alkaloid-positive in precipitation tests with Dragendorff and Mayer reagents (Stahl, 1988). The roots are alkaloid-negative in the same tests. Alkaloid content is -0.8% in flowers and -0.2% in leaves. Preliminary analysis demonstrated that leaves and flowers contain the same major alkaloids, varying in the content of some minor components. Due to greater availability of leaves, only leaf extracts/fractions were used in the study reported here. Aqueous extract: Dried milled leaves (119 g) were extracted in a Soxhlet apparatus with hexane (3.2%), Et20 (1.2%), EtOAc (1.6%), EtOH (12.3%) and H20 (6.7%). The organic extracts were concentrated in a vacuum evaporator, whereas the aqueous one was liophilized. Spot test for alkaloids (Stahl, 1988) were positive for ethanol and aqueous extracts. Alkaloid extracts: Dried milled leaves (2.7 kg) were wetted with NH4OH 6N and extracted with EtOH in a Soxhlet apparatus. After distillation of solvent, the crude ethanol extract (290.0 g) was poured into a 5% HC1 solution, left for 12 h in a refrigerator and filtered. The clear acidic solution was extracted, with CH2CI2 being the residue discarded. The aqueous phase was adjusted to pH 7 with NaOH 6N and extracted with CH2C12, the organic layer washed, dried (Na2SO4) and evaporated (pH 7 alkaloid extract, 0.996 g). The
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
strong emulsion formed at pH 7 during the extraction with CHzCI 2 was separated, resolved by raising it to pH 10 with diluted NaOH, and extracted with CH2CI2 in the same way, resulting in 4.727 g of a dry foam (pH 10 alkaloid extract) after evaporation of the organic layer in vacuo. TLC analysis shows a very similar composition for both alkaloid extracts. Flash-chromatography: The alkaloid extract (pH 10, 4.7 g) was flash-chromatographed (Still et al., 1978) over 75 g of silica gel with MeOH/H20/ NH4OH (7:3:0.1) as eluent and a flow rate of I1 ml/min (air, 71b/in2). One hundred twenty-three fractions (20ml/fraction) were collected and evaporated. F20 is the mixture of five analogous fractions (0.554 g) and F26 the mixture of seven ones (0.632 g). The two main alkaloids of F26 were further purified through flash-chromatography (CHCI3/MeOH/NH4OH - - 10:3:0.1) to obtain fraction F36 (0.109 g). TLC-analysis: Pre-coated silica gel plates (F254) and eluent systems composed of MeOH/H20/ NH4OH, as well as alumina plates with EtzO/ C6HI2/MeOH were found to be useful in comparing alkaloid extracts and fractions. Chromatograms were sprayed with Dragendorff and Ehrlich reagents for alkaloids and FeCI3 2% methanol solution for phenols (Stahl, 1988). Positive alkaloid reactions were found with both alkaloid reagents; no positive reaction for phenol was observed. UV-spectra." Very similar spectra were found for all alkaloid extracts and fractions: ~kmax (MeOH) 248 nm and 307 nm. Addition of basic or acidic solution did not shift these absorption maxima.
2.4. Pharmacological evaluation Antinociceptive activity." In all analgesic experiments, male mice (food and water ad libitum) were used. Forty-five minutes before testing, the animals were placed individually in acrylic cages (20 × 20 × 20 cm), which also served as observation chambers. Extracts were given i.p. 30 min before the algogenic procedures. Aqueous extract was solubilized in water and the fractions in Tween 80 (100t~l/ml). Writhing: The method of Turner (1965) was used. Mice are made to writhe by an intraperitoneal injection of 0.6% aqueous acetic acid (0.1
79
ml/10 g). Test substances were administered (i.p.) 30 min before acetic acid. Animals were kept under observation immediately after acetic acid injection, for two consecutive 5-min periods. The number of writhes (full extension of hind paws) were recorded. Formalin." Acetic acid-induced writhing is a highly sensitive but not a very selective pain test, with false positives occurring with sedatives, muscle relaxants and other pharmacological activities. The formalin test in mice is sensitive to non-steroid anti-inflammatory drugs and other mild analgesics. The test possesses two distinctive phases, possibly reflecting different types of pain. The earlier phase reflects direct effect of formalin on nociceptors (non-inflammatory pain), whereas the late phase reflects inflammation (Hunskaar and Hole, 1987). The method of Hunskaar and Hole (1987) was used. Twenty microliters of 1% formalin were injected into the dorsal surface of the left hindpaw. Mice were observed in the chambers with a mirror mounted on three sides to allow an unobstructed view of the paws; time spent licking the injected paw (licking time) was recorded. Animals were observed for the first 5 min postformalin (early phase) or for 10 min starting at the 20th min post-formalin (late phase). Tail-flick." Analgesia was assessed with a tailflick apparatus (Albasch Equipamentos Eletr6nicos). The method of Ramabadran et al. (1989) was adapted as follows. Baseline latency (reaction time) was obtained with three measurements; after each measure animals were returned to the observation chambers for 2 min. The mean of these three measurements is the pre-drug latency time. At this point, animals presenting two measures _>6 s were discarded. Extracts or drugs were administered (i.p.) immediately after the third predrug measure. Thirty minutes afterwards, another set of three measures were taken and the mean considered as the post-drug reaction time. A cutoff time of 10 s was used to prevent tissue damage. Reversibility by Naloxone (i.p., 3.0 mg/kg) was tested by administering it 8 min before extracts or fractions. Acute toxicity: Lethality was assessed using death within 48 h as an index of toxicity following i.p. and p.o. administration of the extract.
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
80
Table 1 Effects o f P. colorata leaves a q u e o u s e x t r a c t o n w r i t h i n g i n d u c e d by acetic acid Treatment
Dose (mg/kg)
N
Writhing ( 5 - 1 0 rain)
Writhing ( 1 0 - 1 5 min)
Writhing (total)
Inhibition (%)
Saline
--
10
13.8 ± 5.1
8.3 ± 3.1
22.1 ± 6.4
--
0.4 0.8 1.6
5 6 6
12.2 + 6.1 3.8 ± 3.3 0.8 4- 0.9
5.2 4- 2.0 2.5 4- 3.5 1,3 ± 1.6
17.4 4- 7.8 6.3 ± 5.8 2.2 ± 2.4
21.3 71.5 a 90.2 b
15.6 31.2 125.0 500.0
6 6 6 6
Morphine
Aq. extr.
3.2 2.5 0.2 1.0
± 4± ±
2.8 4.2 0.4 1.4
2.3 3.2 0.2 0.0
± -~ 4±
2.1 3.9 0.4 0.0
5.5 5.7 0.3 1.0
± + ± ±
5.0 8.0 0.5 1.4
75.1 b 74.3 b 98.5 b 95.5 b
W r i t h i n g : m e a n ± S.D. a p < 0.05, b p < 0.01, A N O V A .
3. Results
tivity at the late phase is close to the maximum effect at 15.6 mg/kg
3.1. Writhing Results of aqueous extract are shown in Table 1 (expressed as number of writhes, mean + S.D.). Aqueous extract was strongly active in this test, although not in a clear dose-effect fashion.
3.4. Tail-flick Results for the pH 7 alkaloid extract and fractions F20, F26 and F36 are presented in Table 3 (reaction time in s, mean + S.D.). Alkaloid extract and fractions were clearly active in this test and the activity was reversed by previous administration of Naloxone. F20 is roughly twice as active as pH 7 and F26. F20 activity at 10.0mg/kg is comparable to Morphine at 3.5mg/kg F36 is twice as active as F26. F36 is significantly
3.2. Formalin Results of aqueous extract are shown in Table 2. The extract was active in both early and late phases of formalin-induced pain. Results obtained at the early phase show a dose-effect activity; ac-
Table 2 Effects o f P. colorata leaves a q u e o u s e x t r a c t o n the f o r m a l i n test (early a n d late phases) Treatment
Dose (mg/kg)
N
Licking (0-5min)
Inhibition (%)
Saline
--
17
130.4 ± 36.4
--
0.4 0.8 1.6
18 12 12
114.4 -4- 28.0 78.3 ± 15.0 69.7 4- 24.4
15.6 62.5 250.0 500.0
12 4 6 5
113.3 76.0 38.8 9.0
Morphine
Aq. extr.
Licking : m e a n * S.D. a p < 0.05, A N O V A , versus saline.
4444-
39.3 32.5 18.7 4.9
N
Licking ( 2 0 - 3 0 min)
Inhibition (%)
8
104.5 + 45.9
--
12.2 40.0 a 46.5 a
11 12 11
64.6 ± 35.3 61.1 4- 40.0 30.3 4- 34.1
38.1 a 41.5 a 71.0 a
13.2 41.7 70.3 a 93.1 a
4 -5 .
1,9 ± 3.3
98.2 a
0,0 4- 0.0 . .
100.0 a
-.
E. Elisabetsky et aL / Journal of Ethnopharmacology 48 (1995) 77-83
81
Table 3 Effects of pH 7 alkaloid extract, fractions F20, F26 and F36 in the tail flick test Treatment
Dose (mg/kg)
N
Reaction time Pre
Saline
Delta Post
9
4.8 4- 1.3
5.8 4. 1.3
1.4 4. 1.0
Morphine
2.0 3.5
8 5
4.7 4- 1.3 4.3 4- 2.0
7.2 4. 1.8 b 9.8 4- 0.4 c
2.4 4. 1.3 5.5 4- 1.7
Morphine + Naloxone
3.5
5
4.5 4- 1.0
5.9 4. 1.2
1.4 4. 1,1
6
4.9 4- 0.5
6.1 4. 2.4
1.2 4. 2,3
Tween 80 AIk. pH 7 a
10.0 20.0
5 5
4.4 4- 0.2 4.3 4- 1.3
6.7 4. 2.1 9.2 4. 1.5 c
2.3 4. 2.1 4.9 4. 0.7
Alk. pH 7 a + Naloxone
20.0 3.0
6
3.8 4. 0.8
5.3 4. 1.1
1.5 4. 1.5
6
3.64. 1.1
4.6 4. 1.9
0.9 4. 1.6
Tween 80 F20"
5.0 7.5 10.0
6 6 6
6.0 4. 1.5 4.1 4-0.5 5.2 4. 0.5
5.4 4. 0.9 5.3 4. 1.0 9,3 4- 0.8 c
-0.7 4. 1,9 1.2 4. 1.1 4.3 4- 0.9
F20 a + Naloxone
20.0
6
4.2 4. 1.0
5.9 4- 1.8
1.7 4- 1.6
F26 a
10.0 20.0
12 6
4.1 4- 0.9 5.1 4- 1.1
6.4 4. 1.6 c 9.2 4. 2.0 c
2.3 4. 1.6 4.0 4. 1.9
F26 a + Naloxone
20.0
6
4.74- 1.1
5.2 q- 2.1
0.5 4. 1.9
6
4.64. 0.7
5.3 4. 1.3
0.7 4. 1.7
Tween 80 F36 a
5.0 10.0 20.0
6 6 6
4.64. 0.7 4.34- 1.0 4.24. 0.9
8.9 4. 0.6 c 9.0 4. 1.0 ¢ 10.0 4. 0.7 c
4.2 4. 0.5 4.6 + 1.1 5.8 4. 0.9
F36 a + Naloxone
5.0 10.0
6 9
4.14-0.7 4.14. 0.7
6.9 4. 1.9 c 6.9 4- 1.9 c
2.8 ± 1.7 2.8 + 1.7
maximum
effect at
a F26 and F36 solubilized in Tween 80. Reaction time, delta : mean a- S.D. Wilcoxon bp < 0.5, cp < 0.05.
active at 5.0 mg/kg, reaching 20.0 mg/kg. Probably
due to the fact that even the
lowest dose studied shows activity close to maximum
effect, a statistically-significant
relationship diminished
was
not
found.
but not completely
mg/kg of Naloxone.
F36
3,5. Acute toxicity Aqueous
extract produced
(70%) when administered
dose-effect
1.0 g / k g a n d n o d e a t h s
activity
ministered
prevented
is
by 3.0
significant lethality
i.p. a t d o s e s h i g h e r t h a n were observed
v.o. up to 4.0 g/kg. Extract
when
ad-
at pH
7
(i.p.) d i d n o t i n d u c e d e a t h s a t 7 5 . 0 m g / k g ( i n t e n s e tremors
were
observed).
Fraction
F20
(i.p.) in-
82
E. Elisabetsky et al./ Journal of Ethnopharmacology 48 (1995) 77-83
duced no deaths at 40.0 mg/kg (sedations and ptosis were observed), 30% lethality at 75.0 mg/kg (sedations, tremors and ptosis were observed), and 60% lethality at 100.0 mg/kg (with clonic convulsions). Fraction F26 has a toxicity profile similar to F20. No toxic signs were observed for F36 at the doses studied. 4. Discussion
Plants used in South America to prepare the hallucinogenic beverage ayahuasca such as P. viridis and P. psychotriaefolia were found to contain tryptamine and/3-carbolin alkaloids (Schultes and Raffauf, 1990). Presence of alkaloids was reported for P. poeppigiana, P. ulviformis and P. platypoda, the former two used traditionally for pain related problems (Grenand et al., 1987). Outside South America, several Psychotria species containing pyrrolidinoindoline alkaloids have been studied (Hart et al., 1974; Roth et al., 1985; Libot et al., 1987). The previously reported cooccurrence of pyrrolidinoindolines and their rearranged calycanthine-forms in Calycodendron milnei (Libot et al., 1987) were also recently found in P. oleoides (Sevenet et al., 1990). Nevertheless, it has been suggested that alkaloids of the calycanthine type may be formed during isolation procedures (Libot et al., 1988). Interestingly, there are structural resemblances between morphine and pirrolidineindolines. For instance, the four six-membered rings in morphine have the same stereochemistry as four rings in meso-chimonanthine (although two carbons of morphine are substituted for nitrogen in mesochimonanthine). It is fair to assume that the stereospecificity of the opiate receptor is compatible with meso-chimonanthine molecule, taking into account the structural features needed for opioid-mediated analgesia (Korolkovas and Burckhalter, 1988). In this study, the antinociceptive activity of P. colorata extracts was evaluated using the formalin (early and late phases), acetic acid-induced writhing, and tail-flick tests. These procedures are used to detect narcotic and non-narcotic analgesics. Furthermore, the body of data allow for the detection of central and/or peripheral analgesia
and to distinguish analgesic from antiinflammatory properties. It is known that thermal nociceptive tests are more sensitive to opioid #-agonists and nonthermal tests to opioid x-agonists (Abbott, 1988; Furst et al., 1988). Our data suggest the involvement of both # and Kopioid receptors in the analgesic activity of alkaloids from P. colorata. Morphine effects in formalin are supposedly mediated by r-agonists (Abbott and Young, 1988). Morphine effects in tail-flick is in part mediated by descending brainstem and bulbar serotoninergic systems (Abbott and Young, 1988). At this point, we can not rule out the possibility that tryptamine alkaloids found in Psychotria may interact with serotoninergic systems; such interaction would be relevant to analgesic effects. The toxicity observed in mice might explain why these remedies are traditionally used by topical application. As a matter of fact, it cannot be concluded if the alkaloids are the only active principle or if the alkaloids have other pharmacological activities. 5. Conclusion
The data reported here show that alkaloids present in leaves and flowers of P. colorata have marked opioid-like analgesic activity. As in other cases, it is necessary to fully isolate and identify the active compound(s) and obtain a comprehensive pharmacological profile. At this point, it seems that there is a rational base to the use of P. colorata as a local analgesic to treat earache in adults. We suggest that the study of alkaloids present in Psychotria spp can be regarded as a fruitful research effort. This study also confirms the validity of the ethnopharmacological approach in the search for new drugs. Acknowledgments
This work was supported by CNPq and IFS. The authors wish to thank Mar~al de Souza Luna (phytochemistry) and Zuleica C. Castilhos (Formalin and Writhing) for technical assistance; Dr. Jos6 Roberto Leite for morphine hydrochloride; Dr. Marco Elisabetsky and Dr. Thierry Sevenet for discussing the original manuscript.
E. Elisabetsky et al./Journal of Ethnopharmacology 48 (1995) 77-83
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