Anexos 70
Trabalhos publicados com a participação do autor em periódicos.
Acta Farm. Bonaerense16 (3): 169-72 (1997) Trabajos originaies Recibido el 6 de junio de 1997
Aceptado el 30 de junio de 1997
A Triterpene and a Flavonoid C-Glycoside from
Aleurites moluccana L. Willd. (Euphorbiaceae)
Christiane MEYRE-SILVA \ Ticiana C. MORA \ Adair R. SOARES SANTOS ^ Jacir DAL MACRO Rosendo Augusto YUNES
Franco DELLE MONACHE ^ & Valdir CECfflNEL-FELHO ‘ *
^ Núcleo de Investigações Quúnico-Farmacêuticas (NIQFAR)IFAQFAR, Universidade do Vale do Itajai- UNIVALI, 88302-202, Itajai-SC, Brazil. ^ Departamento de Ciências Biológicas e da Saúde, Universidade do Oeste de Santa Catarina-UNO ESC, Chapecó-SC, Brazil. ^ Departamento de Química,
Universidade Federal de Santa Catarina UFSC, 88040-900, Florianópolis-SC, Brazil.
^ Centro de Chimica dei Recettori, CNR, Universita Cattolica S. Cuore, Largo F. Vito 1, 00168, Rome, Italy.
SUMMARY. This paper describes the isolation of two phytoconstituents from the leaves and barics of Aleurites moluccana, a medicinal plant widely employed in folk medicine for the treatment of several ailments. On the basis of spectroscopic evidence, these compounds were identified as swertisin (leaves), a flavonoid C-glycoside yet not described for the family Euphorbiaceae and acetil aleuritoiic acid (bark), a triterpene which was recently reported as an antimicrobial agent
RESUMEN. “Un triterpeno y un flavonoide glicosidico de Aleurites moluccana L. Willd.
(Euphorbiaceae)”. El presente trabajo describe el aislamiento de dos componentes de hojas y corteza de Aleurites moluccana, una planta medicinal ampliamente empleada en medici na popular para el tratamiento de varias dolências. Sobre la bases de la evidencia espec- troscópica existente, estos compuestos fueron identificados como swertisina (hojas), un fla vonoide glicosidico aún no descrito para la familia Euphorbiaceae y el ácido acetil-aleuri- tlico (corteza), un triterpeno que ha sido recientemente citado como agente antimicrobiano.
INTRODUCTION
Aleurites m o lu cca n a L.Willd. (Euphorbiaceae), known as “Nogueira-da-
India” or “Nogueira-de-Iguape” is a medicinal plant frequently employed in folk medicine for the treatment of tumours, ulcers, headache, fevers, diarrhea and gon orrhea 1, and also have been used as antiinflammatory 2.
Previous phytochemical studies with this plant have revealed the presence of triterpenes 3 and a new coumarinolignoid, denominated moluccanin whereas bi ological investigations have shown antimicrobial 5 and antiviral 6 activities.
Preliminary pharmacological studies conducted by our laboratories have demonstrated that hydroalcoholic extract obtained from A. moluccana leaves ex hibited potent analgesic effect in mice, which could be related with the presence of hydrocarbons, sterols and triterpenes
In the present report, we have extended our previous chemical findings and analysed other constituents present in the leaves and barks of A. moluccana.
KEY WORDS: Acetil aleuritoiic acid, Aleurites moluccana, Chromatography, Swertisin
PALABRAS CLAVE: Acido acetil-aleuritólico. Aleurites moluccana, Cromatografia, swer
tisina
* Author to whom correspondence should be addressed.
MATERIAL & METHODS
Plant material
The plant was collected, in Itajai (State of Santa Catarina, Brazil), in January 1996 (leaves) and July 1996' (barks) and classified by Dr. Ademir Reis (HER). A voucher specimen was deposited in Herbarium Barbosa Rodrigues. (HBR), Itajai (V.C. Filho, 001).
Isolation o f constituents
The air-dried leaves (6OO g) and barks (570 g) were powdered and separatel- ly extracted with ethanol and methanol, respectively, at room terriperature for ap- proximatelly two weeks. After solvent removal, the extracts were concentrated (re duced pressure) and successively partitioned with hexane, CH2CI2, AcOEt and bu tanol, respectively. However, we have initially examined the extracts obtained from leaves. Thus, hexane fraction furnished n-hentriacontane, a and ^-amyrin, p-sitos terol and stigmasterol, according previously d e s c r ib e d 7. The AcOEt and butanol fractions exhibited a similar chromatographic profile (TLC) and were then com bined. This material (3.38 g) was chromatographed on a silica gel column eluted with a CHCljiMeOH gradient. Elution with CHCl3:MepH ,5:5 yielded a yellow solid (80 m ^ , identified as a glycoside flavonoid, 6-(3-D-Glucopyranosyl-5-hydroxy-2(4- hydroxyphenyl-)-7-methoxy-4H-l-benzopyran-4-one (swertisin) (1). The spectro scopic data, especially ^H- and ^3C-NMR, are identical to those reported in the lit
erature 8.9. -■ - ' ' .
The CH2CI2 fraction (3.9 g) obtained from yl; mo/MCCflni3 barks was ^ matographed as in previous case, eluted , with hexane:AcOEt, gradient. After rnoni- toring by TLC, the similar fractions were combined and rechromatographed using the same procedure to afford a white crystalline solid, identified as acetyl aleuri tolic acid (19 mg) (2), direcdy compared with authentic sample and spectral data (IR, ^H- and i^C-NMR), which were found to be identical to those previously ; de-' scribed
Meyre-Silva, C., T.C. Mora, A.R. Soares Santos, J. Dal Magro, R.A. Yunes, F. Delle Monache & V. Cechinel-Filho
RESULTS AND DISCUSSION
In order to isolate and identify other constituents responsible for the anal gesic effects previously shown by some extracts and non polar compounds pre sent in A. m o lu c ca n a , w e have now reinvestigated both ethyl acetate and dichlorometha-ne fractions obtained from leaves and barks of this plant, respec tively. Chromatographic procedures carried out with ethyl acetate fraction gave a yellow solid which gave positive reaction with FeCl3-reagent. The spearal data, especially NMR-^H and ^^C were compatible with those of swertsin (1), a flavonoid glycoside, isolated by the first time from Swertia japonica Although such com pound have been earlier isolated from other plants, it is an uncommon natural product, being here the first report of them from the family Euphorbiaceae.
On the order hand, swertisin was not detected in the barks of this plant. However, the preliminar analysis by using TLC in the conventional manner re vealed the presence of some terpenes in this part of A. moluccana. One of them was isolated from the dichlorometane extract and identified as acetil aleuritolic
acid (2). The spectroscopic characteristics were in accordance with literature data and Co-TLC with authentic sample confirmed such structure.
This compound have been earlier isolated by two of us (RAY and FDM) from
Croton u ru cu ra n a , which have shown antimicrobial effects against different
pathogenic bacteria ” •
Presently, the phytochemical and pharmacological studies are in progress in order to examine which compounds contributes for explain, in addition to com pounds previously described, the potent analgesic effects demonstrated for the hy droalcoholic extract of A. moluccana.
acta farmacêutica bonaerense - vol. 16 n° 3 - ano 1997
A c k n o w le d g e m e ttts . The authors are grateful to Mrs. Carlos PicoUi and Paulo José da Silva, for
c o llec tio n o f the plant material. This w ork w a s supported by grants from CNPq and P roB lC / ProPPEx/UNIVALl, BrazÜ.
REFERENCES
1. Pio Correia, M. (1984) “D i c i o n á r i o d a s P l a n t a s Ú t e i s d o B r a s i l e d a s E x t i c a s C u l t i v a d a s " , IBDF, Brasilia-DF, Vol. V, pp. 294-5
2. Duke, J.A. (199D " H a n d b o o k o f M e d i c i n a l H e r b s ” , U.S.A.; CRC Press, 29 3. Hui, W.H. & C.T. Ho (1968) A u s t . J . C h e m . 2 1 . 1675-7
4. Shamsuddin, T., W. Rahman, S.A. Khan, K.M. Shamsuddin, J.B. lüntzinger (1988) P h y t o c h e m i s t r y 2 7 - . 1908-9
5. Locher, C.P., M.T. Burch, H.F. Mower, J. Berestecky, H. Davis, B. Van Poel, A. Lasure, D.A Vanden Berghe, & A.J. Vlietinck (1995) / E t h n o p h a r m a c o l . 49: 23-32
6. Locher, C.P., M. Witvrouw, M.P. Béthune, M.T. Burch, H.F. Mower, H. Davis, A. Lasure, R. Pauwels, E. Clercq, & A.J. Vlietinck (1996) P h y t o m e d i c i n e 2: 259-64
7. Meyre-Silva, C., T.C. Mora, M.W. Biavatti, A.R.S. Santos, J. Dal Magro, R.A. Yunes & V. Cechinel Filho (1997) Phytom edicine fm press)
8. Komatsu, M., & T. Tomimori (19Ó7) Chem. Pbarm . Buli. 15: 263-9
9. Agrawal, P.K., & M.C. Bansal (1989) Carbon-13 NMR o f Flavonoids (Agrawal, P.K., Editor), Elsevier, New York, pp. 264-83
10. Mahato, S.B., A.K. Nandy, & G. Roy (1992) Phytochemistry 31: 2199-249
11. Peres, M.T.L.P., F. Delle Monache, A. Bella Cruz, M.G. Pizzolatti, & R.A. Yunes (1997)
J. Ethnopharm acol. 56: 223-6
Meyre-Silva, C., T.C. Mora, A.R. Soares Santos, J. Dal Magro, R.A. Yunes, F. Delle Monache & V. Cechinel-Filho
Phytom edicine, Vol. 5(2), pp. 1 0 9 -1 1 3 © G ustav Fischer Verlag 1998 ________
Preliminary phytochemicai and pharmacological studies
of Aleurites moluccana leaves [LJ Willd.
C. Meyre-Silva\ T. C. M ora\ M. W. Biavatti^ A. R. S. Santos^ J. Dal-Magro^, R. A. Yunes^ and V. Cechinel-Filho^
'N ú cleo de Investigações Q uím ico-Farm acêuticas (NIQ FA R)/FAQ FAR , U niversidade do Vale do Itajai-UNIVALI, Itajai-SC, Brazil
^Departamento de Ciências B iológicas e da Saúde, U niversidade do O este de Santa C atarina-UN O ESC, C hapecó-SC , Brazil ^Departamento de Q uím ica, U niversidade Federal de Santa Catarina - UFSC, Florianópolis-SC , Brazil.
Summary
In the present w ork w e have investigated tfie analgesic activity o f various extracts prepared from
A leu rites m oluccana leaves by using the w rithing test in mice. T he results show ed that the hydroalcohol-
ic extract and the hexane fraction exhibit poten t antinociceptive action. We have also isolated and iden tified som e chemical constituents from the hexane fraction. In addition, the nature o f substances present in ethyl acetate and butanol fractions w ere evaluated by TLC. Such fractions revealed the presence o f phenohc com pounds. Chrom atographic procedures carried out w ith hexane fraction revealed the pres ence o f n-hentriacontane, a-am yrin, p-amyrin, stigm asterol, P-sitosterol and cam pesterol, w h ich were identified by spectroscopic data and H R G C or H R G C /M S techniques. T he com pounds considerably in hibited acetic acid-induced abdom inal constrictions, being more efficacious than aspirin and paraceta m ol. In addition, preliminary HPLC analysis indicated, using a U V detector, one m ain constituent in al coholic extract.
Keywords: Aleurites m oluccana, analgesia, w rithing test, hydrocarbon, phytosterols, triterpenes.
a Introduction
The fam ily Euphorbiaceae comprises o f several genus treatm ent o f tum ors, ulcers, headache, fevers, diarrhea,
and num erous species, and a great number o f papers dysentery and gonorrhea (Duke, 1991).
have been published on their chemical constituents and Previous reports have show n that this plant contains
pharm acological activities. triterpenes (Hui and H o , 1968) and a new coum arino-
We have previously investigated som e members o f lignoid, denoted m oluccanin (Shansuddin et al., 1988).
this fam ily and have identified some im portant classes To our know ledge, no pharm acological investigation
o f active com pounds including acetophenones, sterols, has so far been published on this plant. H ow ever,
flavonoids and tannins (C alixto et al., 1990; Cechinel A leurites m oluccana was recently reported to have
Filho et al., 1996; M iguel et al., 1996; Santos et al., antibacterial activity against Staphylococcus aureus
1995a; N iero et al, 1996). and Pseudom onas aeruginosa (Locher et al., 1995) and
In the present study, we have selected another Eupho- antiviral property (Locher et al., 1996).
biaceae for investigation, A leurites m oluccana, a tree In the present study w e have analysed the analgesic
know n as “N ogueira-da-índia” or “N ogueira-do- effects o f A/ewnies mo/Mccawa leaves by using acetic ac-
Iguape”, widely-distributed in the south o f Brazil. Its id-induced writhing in mice, as w ell as isolated and
leaves and stem bark are frequendy em ployed in folk identified som e active constituents,
1 1 0 c . M eyre-Silva e t al.
Materials and Methods
Plant material
A . m oluccana was collected in Itajaí, State o f Santa
Catarina, Brazil, in January 19 9 6 , and identified by Dr. Ademir Reis (HBR). A voucher specim en has been de posited at the Barbosa Rodrigues Herbarium , Itajaí (V.C. Filho, 001).
Isolation and identification of active constituents Dried leaves were cut in to small pieces and tw o differ ent extracts, hydroalcoholic and ethanolic, w ere pre pared for pharm acological and phytochem ical analysis. The hydroalcoholic extract w as obtained by extrac tion w ith 50% ethanol-water, after m aceration at room temperature for 10 days. The extract w as then co n cen trated (reduced pressure) to desired volum e and stored at - 2 0 °C. The resulting extract was dissolved in 0.9% N aC l solution to desired concentration just before use.
Ethanolic extract w as prepared as described above (600 g leaves). This extract w as how ever concentrated
and then successively partitioned w ith hexane, C H2CI2,
EtOAc and butanol, respectively. The hexane extract (16.0 g) w as chrom atographed on a silica gel colum n eluted w ith a hexane: EtO Ac gradient. After m on itor ing by TLC, fractions w ere com bined and re-chrom at- ographed as described above, affording three solids, w hich were first denom inated I (30 m g), II (80 mg) and III (710 mg).
Solids II and III gave positive reaction w ith sulphuric anisaldehyde and other reagents, and appeared one spot on TLC (system: hexane-EtOAc 7:3). Further analysis o f spectral data (IR, ’H- and ^^C-NMR) and high resolution gas chrom atoghaphy (H R G C ) or H R G C /EM techniques by co-injection w ith authentic samples, allow ed us to conclude that com pound I is n- hentriacontane whereas II is a mixture o f phytosterols
containing P-sitosterol (78.39% ), stigm asterol
(16.35% ), cam pesterol (3.77% ) and an additional un- dentified sterol (1.49% ). Solid III w as identified as a mixture o f triterpenes, a-am yrin (48.93% ) and P-amy- rin (51.07% ). All spectroscopic data are identical to those reported in the literature (Seo et al., 1975; C han dler et al., 1982).
The constituents present in EtOAc and butanol frac tions were analysed by TLC, according to the previous ly described m ethodology (M arini-Bettolo et al., 1981; Ugaz, 1994).
HPLC-fingerprInt analysis
The alcoholic extract o f A. m oluccana w as analysed by HPLC-fingerprint, using a Beckm an-166 apparatus. The extract w as dissolved in m ethanol (10 m g/m l), fil
tered and analysed (injection volume o f 1 0 |xl) through
a reversed phase, C -18 colum n w ith w ater-m ethanol 80:20 v/v at a flow rate o f 1 ml/min using U.V. detector at 3 0 0 nm (Fig. 1). (U> 0.9000 0.8000 0.7000 0.6000 0.5000 0.4000 0.3000 0.2000 O.lOOft r u _ j J a O.D 2.0 4.0 6.0 8.0 10,0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 <MÍn)
Preliminary phytochemical and pharmacological studies of Aleurites moluccana leaves 111
Pharmacological analysis: Abdominal constriction response caused by intraperitoneai injection of dilute acetic acid
M ale Swiss mice, 2 5 - 3 0 g, w ere kept in a tem pera ture controlled environm ent (23 ±. 2 °C) w ith a 12-h light-dark cycle. Food and water were freely avail able. The abdom inal constriction resulting from intra
peritoneal injection o f acetic acid (0.6%), consisting
o f a contraction of the abdom inal m uscle together w ith a stretching o f hind Hmbs, w as carried out ac cording to procedures described previously (Collier et ai., 1968; Santos et al., 1994). Anim als were pretreat ed w ith the extracts or com pounds through intraperit oneal adm inistration ( 1 - 3 0 mg/kg) 30 m in prior to acetic acid injection. Control anim als received a sim i lar volum e o f 0.9% N aC l (10 m l/kg). All experim ents were carried out at 20 - 22 °C. A fter challenge, pairs o f m ice were placed in separate b oxes and the num ber o f abdom inal constrictions w as cum ulatively
counted over a period o f 2 0 min.
Statistical Analysis
The results are presented as m ean ± s.e.m ., and the sta tistical significance between the groups w as analysed by m eans o f analysis o f variance follow ed by D unnett’s m ultiple com parison test. P values less than 0 .0 5 were considered as indicative o f significance. W hen appro
priate, the ID50 values (the dose o f extract that reduced
responses by 50% relative to control value) were esti mated by graphical interpolation from individual e x periments.
□ Results and Discussion
In order to investigate the analgesic activity o f A. m o
luccana, a hydroalcoholic extract (HE) prepared from
the leaves w as investigated for inhibitory effect on ace tic acid-induced abdom inal constriction. As can be seen in Fig. 2, this extract, given intraperitoneally (3 to 60 mg/kg) exhibited potent and dose-dependent antinoci ceptive effect against acetic acid-induced writhing re
sponse in mice. The m ean ID50 value w as 9.5
(5.9 - 1 5 .4 ) mg/kg w ith a m axim al inhibition o f
8 8 ± 4% . These prom ising results led us to investigate
this plant in m ore detail, in order to isolate and identi fy the phytoconstituents responsible for the indicated pharm acological effects.
Thus, w e have prepared another extract (ethanolic) o f A. m oluccana leaves, w hich w as successively parti tioned w ith solvents o f increasing polarity, ie hexane, dichlorom ethane, ethyl acetate and butanol. Since the hexane soluble fraction yielded approxim ately 15-fold more than all other extracts, and exhibited a significant
analgesic activity (results n ot show n), it w as selected for phytochem ical analysis.
The extract w as chrom atographed on a silica gel co l umn eluted w ith increasing am ount o f EtOAc in h ex ane. After fractionation, three w hite solids were o b tained and denom inated as I, II and III. The purity o f each was first verified TLC.
Spectral data (IR, H^-NM R) o f com pound I indicat ed only the presence o f carbon and hydrogen atom s and the use o f GC/MS confirm ed to be a saturated hy
drocarbon, n-hentriacontane (1), which is a w ell-
know n alkane present in several plants o f the family Euphorbiaceae, particularly in species o f E uphorbia,
Colliguaja and A rgytam n ia (G necco et al., 1996). This
com pound w as recently reported to be a. potent anti-tu- mor agent (Takahashi et al., 1995).
60 W LU X t: flC
o
cc UJ CD => 30 10 30 60Hydroalcoholic extract o f A. moluccana (mg/kg, Lp.)
Fig. 2. Effect o f the hydroalcoholic extract obtained from A.
m o lu cca n a leaves, against acetic acid-induced abdom inal constrictions in m ice. Each colu m n represents m ean ± s.e.m . o f eight to tw elve experim ental values. Significantly differ ent from controls; P < 0 .0 1 .
Table 1. Effect o f com pounds isolated from A . m o lu cca n a leaves, aspirin and paracetam ol, against acetic acetic-induced abdom inal constrictions in m ice.
C om pound Inhibition (%) n-hentriacontane 94 ± 6 a and P-amyrin 84 ± 3 P-sitosterol 5 4 ± 2 stigm asterol 4 2 ± 1 aspirin 35 ± 2 paracetam ol 38 ± 1
1 1 2 C . M eyre-Silva et al.
Solids II and III showed, positive reactions w ith sul phuric anisaldehyde and Liebermann-Burchard re agents, suggesting them to be sterols or terpenes (Mari- ni-Bettolo et al., 1981; U gaz, 1994). Further spectral analysis, especially '^C-NM R, indicated the presence o f at least tw o com pounds. The use o f H R G C or H RG C/M S techniques revealed a m ixture containing m ainly P-sitosterol, stigm asterol and cam pesterol. M oreover, spectral data w ere identical to those report ed previously (Aragao et al., 1990; Ikan, 1991)). A l though these phythosterols are com m on constituents o f higher plants, we have recently reported their analgesic properties in several m odels o f pain in m ice (N iero, 1993; Santos et al., 1995b). These com pounds, in addi tion to tannins and flavonoids, are responsible for the analgesic effects o f other members o f Euphorbiaceae, particularly those belonging to the genus Phyllanthus (Santos et al., 1994, 1995a,b; M iguel et a l , 19 9 5 , 1996; C echinel Filho et al., 1996). Other studies have show n that such phytosterols, besides antinociceptive actions, exert potent antiinflam m atory action against carrageenan and cotton pellet induced oedem a in rats, the effect being com parable to that o f hydrocortisone and phenylbutazone (Handa et al, 1992).
O n the other hand, spectral data (IR, ^H, ^^C-NMR and EM) o f solid III were com patible w ith those o f a and p-amyrin (Seo et al., 1975; Chandler et al., 1982), tw o w ell-know n antiinflam m atory triterpenes (Loggia et al., 1994; Recio et al., 1 9 9 5 ). The use o f H R G C in dicated similar amounts o f both com pounds.
In order to investigate if n-hentriacontane and triter penes contribute to the analgesic activity, w e have ana lysed them for inhibition o f acetic acid-induced abdom inal constrictions. The results show n in Table 1 suggest
a ID50 low er than 10 m g/kg. Hentriacontane and the
triterpenes inhibited the constrictions by 94 ± 6 and 84
± 3 % , respectively. In contrast, aspirin and paraceta m ol, tw o w ell-know n nonsteroidal antiinflam m atory drugs caused an inhibition o f 35 ± 2 % and 38 ± 1%, respectively (Cechinel Filho et al., 1995). These results, together w ith results previously reported (N iero, 1993; Santos et al., 1995a) strongly suggest that n-hentria contane as w ell as phytosterols and triterpenes may be responsible, at least in part, for the analgesic activity dem onstrated by hydroalcoholic and hexane extracts o f A. m oluccana leaves.
It is interesting to m ention that m oluccanin (Sham suddin et al., 1988) and the triterpenes m oretenone and m oretenol (Hui and H o , 1 9 6 8 ), substances previously isolated from A. m oluccana, were not observed in our investigation. This suggests that environmental factors m ay be acting on the plant.
We have also exam ined the chrom atographic profile of the ethyl acetate and butanol fractions o f this plant by TLC, using specific reagents (M arini-Bettolo et al..
1981; Ugaz, 1994). The results indicated the presence o f phenolic com pounds, particularly flavonoids, and the absence of alkaloids. Preliminary HPLC analysis show ed one main peak, but it w as n ot yet identified (Fig. 1).
Finally, our results show ed that extracts obtained from A. m oluccana leaves exhibit analgesic properties, w hich are mainly related w ith the presence o f n-hentri- acontane, phytosterols and triterpenes in hexane frac tion . Further studies are n o w in progress in order to isolate and identify other possible active principles present in the plant.
Acknowledgements
T he authors are grateful to Dr. Franco D elle M onache for d o n a tio n o f authentic sam ples o f a and P-amyrin, to M rs. Car lo s Picolli and Paulo José da Silva, for collection o f the plant m aterial and to Dr. A ngelo A. Ruzza, for HPLC analysis. The authors are also indebted to Dr. A dem ir R eis for botanical classification o f A . m o lu cca n a and to M s. Joanésia M . J. R otnstein (Central B iotério, UFSC) providing the anim als. T h is w ork w as supported by grants from C N Pq and ProB- IC/ProPPExAJNIVALI, Brazil.
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Collier, H . O. J., D innen, L. C ., Johnson, C. A ., Schneider, C.: T he abdom inal constriction response and its suppression