Departamento de Bioquimica Vegetal, Facultad de Farmacia y Bioquirnica, Junin 956, Buenos Aires, Argentina
PHENOLIC COMPOUNDS OF TERMlNALlA TRIFLORA' By V.S . MARTINO, M. N. GRAZIANO, 0. HNATYSZYN and J. D.Coussro
Abstract
Terminalia triflora (GRISEB.)LILLO(Combretaceae) is an Argentine tree which is common throughout the North and Northeastern regions of Argentina. It is known with the, common name of ,,lanzaU; ,,lama amarilla", ,,amarillo de rio" o r ,,palo amari!lo" (DIGILIO and LEGNAME 1966). Phytochemical investigations on members of thegenus Terminalia by IDEMUDIA (1967, 1970) has led t o the isolation of ellagic, trimethylellagic from T. iuorensis and T . avicennoides; ellagic, trimethylellagic and tetramethylellagic from T . glaucescens and T . laxiflora. RAMACHANDRA ROW and SUBBA RAO (1962) isolated 3,3dimethylellagic 4-glucoside from T . paniculata. Phytochemical screenings on T. triflora have established the presence of saponins and oxidases in the stems, a colorant matter in the wood (DOMINGUEZ et a]., 1919), and phenolic compounds in the leaves (RONDINA eta]., 1970). There is no record of the uses of this plant in folk medicine. H I E R ~ N Y(1882) M~~ mentions its use in making posts, furniture, lances for soldiers and for combustible. Nevertheless we have found data on the medicinal use of other exotic species of the genus Terminalia. HARTWELL (1968) mentions the use of T. arjuna (RoxB.) WIGHTet ARN. by natives of Ceylon against calluses; T . bellerica ROXB.and T. chebula RETZ. in India and Japan is said to have antitumor activity; T . sericea BURCH.in Rhodesia is used in native medicine against plantar warts. PLANESde BANCHEROand SOUTO(1969) describe the antimicrobial, antifago and antiviral ,,in vitro" activity o f the aqueous extracts of leaves of Terminalia triflora against Foot and Mouth disease, this .activity being attributed to the
Part IX in the series ,Flavonoids from Argentine Medicinal Plants". For Part VIII see Phytochemistry 12,1825 (1973).
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T h e phytochemical examination of the methanolic extract of the leaves of Terminalia triflora resulted in the isolation and identification of quercitrin, afzelin, ellagic and gallic acids, methyl gallate and two tannins named D and E, whose structure is being determined.
Phenolic Compounds of Terminalia triflora
227
tannins in the plant. Recently R I V Aand ~ DISTEFANO (1971, 1973) demonstrate the antiviral activity of the same extracts against RNA viruses of different types: Rabies, Fot and Muth disease and Influenza.
Experimental Material The leaves of Temiinalia triflora were collected during October 1971 a t the Province of Tucumin, Argentina. A voucher specimen is deposited in the University Herbarium.
Ether fraction The ether extract furnished on standing a pale yellow precipitate (4 g). Column chromatography of this precipitate was carried out on polyamide eluted with ethanol and water in a gradient from (3 : 7 ) to (7.5:2.5) yielding ellagic acid and a mixture of flavonoids. The latter was resolved by preparative T L C with chloroform-methanol-methylacetone-acetylacetone (20 : 10 : 5 : 1) into: Band I, Quercitrin (Quercetin-3-rhamnoside) and Band 11, Afzelin (Kaempferol-3-rhamnoside). The individual compounds were identified as follows: Band I (Quercetin-3-rhamnoside). The first band (Rf = 0.34) was cristallized from aqueous ethanol, m.p. 182-183O C. ljV spectrum in methanol of the compound showed the following peaks: 254, 266 sh, 302 sh, 352 mp. UV Shiffs with Al CI,: 276, 304 sh, 333, 430 my. AL C1,IHCI: 272, 303 sh, 353, 401 mli. NaOMe: 270,326,393 mp. NaOAc: 272,322 sh, 372 my. IR spectrum (KBr disc) showed peaks a t 3400, 2950, 1730, 1665, 1610, 1570, 1510, 1460, 1370, 1295,1240,1205,1170,1125,1085,1015,960,945,875,810 and 725 cm-I. T h e compound (25 mg) was dissolved in 1ml of methanol and hydrolyzed with 6% HCI at 100' C 45 min. T h e aglucon precipitated on cooling and was filtered. UV, IR and T L C comparison against an authentic sample showed it to be quercetin; the sugar was found to be rhamnose by paper chromatographic analysis. From UV, IR and T L C against an authentic sample the compound was identified as quercetin3-rhamnoside. Band I1 (Kaempferol-3-rhamnoside). This band (Rf = 0.49) was cristallized from aqueous ethanol n1.p. 172-174O C. UV spectrum (MeOH): 275, 290 sh, 341 mk. AICI,: 285, 302 sh, 350, 410 mp. AICI,/HCI: 285, 302 sh, 342, 393 mp. NaOMe: 287, 322 sh, 390 rnp. NaOAc: 371, 300 sh, 272 mu. 1R spectrum (KBr disc) showed peaks a t 3450,2975,1660,1620,1510,1460,1370,1210,975, 840 and 720 cm-1. T h e compound (1Omg) was dissolved in 1 ml of methanol and hydrolyzed with 6% HCI at 100° C 45 min. T h e aglucon precipitated o n cooling and was filtered. Mixed m.p. UV and IR comparison as well as T L C against a n authentic sample proved the aglucon t o be kaempferol. The sugar was identified by paper chromatographic comparison as rhamnose. This compound through UV, IR and TLC comparison against an authentic sample was identified as kaempferol-3-rhamnoside.
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Extraction of the plant T h e coarsely milled dried plant material (1.500 g) was extracted continuously in a soxhlet apparatus using successively chloroform' and methanol. A yellow substance named D (100 g) precipitated from the methanolic extract by the addition of chloroform. T L C of the isolate showed a single spot. T h e remaining solution was taken to dryness by means of a flash evaporator in vacuo a t 4S0 C, and the residue (214 g) was dissolved with hot water, and this phase partitioned succesively with ether and ethylacetate.
228
Martino, Graziano, Hnatyszyn and Coussio
Planta medica Vol. 27 1975
+ ?4d)H
ext
.
ICC15 added
\1
MeOH s o l n .
precipitate
t o dryness
Tannin I )
I
I
F
JI
extd. Et20
l
Quercitrin A f zelin Ellagic acid Callic acid Wethyl e l l a t e
EtOAc extd.
Tannin E
.
I
( discard)
Fig. 1. Extraction procedure of the phenolic compounds from the leaves of Terminalia tripora Ellagic acid This compound did not melt below 350° C. Paper chromatography using n BuOH: AcOH : H,O (4: 1:5), 6% AcOH atid water gave a single spot at Rf = 0.33, 0.02 and 0.00 respectively. UV spectrum in methanol gave maxima at 257 and 367 mp. With NaOAc a new band appeared at 280 with a weakeningof the band at 255 mu. IR spectrum (KBr disc) showed absorption bands a t 3550 cm-1 (OH), 1700 cm-I (unsaturated lactone) and 1580 cm-I (aromatic character). Comparison by paper chromatography, UV and IR showed the isolate and a reference sample of ellagic acid to be identical. The ethereal solution yielded gallic acid and methyl gallate. The identity of the isolated compounds was confirmed by direct comparison with authentic samples (mixed m.p., TLC, UV and IR).
Downloaded by: NYU. Copyrighted material.
H20 added
Phenolic Compounds of Terminalia triflora
229
Ethylacetate fraction This fraction gave on standing a pale yellow compound named E (45g) which by T L C showed a single spot. Substance D Hydrolysis. Acid hydrolysis of compound D (50 mg) in 2N H,SO, under reflux for 10 hr gave a beige precipitate (20mg) identified as ellagic acid. An examination of the acid layer by descending paper chromatography with butanol-pyridine-water (6 :4 : 3) showed glucose as the only sugar present. T h e acid laver was extracted with ethylacetate, and T L C analysis of the organic layer on Silicagel G plates using chloroform-ethylacetate-formic acid (5 : 4 : 1) revealed the presence of gallic acid. Both gallic acid and glucose were confirmed by GLC. Substance E Hydrolysis. Acid hydrolysis of tannin E carried out in the same way as for tannin D provided ellagic and gallic acids and glucose. T h e complete structure of this tannins will be subject of a future investigation.
T h e methanolic extract of the dried powdered leaves of T. triflora was submitted to fractionation (Fig. 1) to give quercitrin (quercetin 3-rhamnoside), afzelin (kaempferol 3-rhamnoside), ellagic and gallic acids and methylgallate and two unidentified tannins named D and E. The purification of the identified compounds was carried out by preparative chromatography (column and TLC) and their identity was established by TLC, spectral data (UV and IR) and acid hydrolysis by comparison with authentic reference material. T h e tannins named D and E gave ellagic and gallic acids and glucose after hydrolysis. RIVASand MERCADO (1973) prove that the antiviral activity of the aqueous extracts of T. triflora is due to tannins D and E. Acknowledgements This investigation was supported by Consejo Nacional d e Investigaciones Cientificas y Tkcnicas, Grants N o 3520 a171 and 3520 c173. T h e authors are grateful to Dr. FEDERICOFELFORT,Ins ~ i t u t oMiguel A. Lillo, Universidad Nacional de, Tucurnln for collecting plant material and Dr. P. R. LEGNAME for botanical identification. References P. R.: "Los Arboles Indigenas d e la Provincia de TucumBn", DICILIO,A. P. L. and LEGNAME, Universidad Nacional de Tucumin, Instituto Miguel A. Lillo, Tucumin (1966) J. A.: "Contribuciones a la Materia Mtdica Argentina", J. Peuser Ltda, Buenos DOMINGUEZ, Aires (1928)
Downloaded by: NYU. Copyrighted material.
Results and Discussion
230
Martino, Graziano, Hnaryszyn and Coussio
Planta medica Vol. 27 1975
HARTWELL, J. L.: Lloydia 31,71 (1968) HIERONYMUS, J.: Bol. Acad. Cienc. C6rdoba 4,199 (1882) 0.G. and EKONG,D. E. U.: J:Chem. Soc., 863 (1967) IDEMUDIA, IDEMUDIA, 0.G.: Phytochem. 9,2401 (1970) PLANES DE BANCHERO, E. and SOUTO;J.: Rev. Invest. Agropec. 6,155 (1969) RAMACHANDRA ROW,L. and SUBBA RAO,G. S. R.: Tetrahedron 18,357 (1962) A. and GRAZIANO, M. N.: V°Congreso Lacinoamericano de MicrobioRIVAS,M., DISTEFANO, logia Punia del Este, Abstr. p. 21 (1971) RIVAS,M. and DISTEFANO, A.: Rev. Asoc. Argent. Microbiol. 5,55 (1973) RIVAS,M. and MERCADO,E.: 111 Jornadas Argentinas d e Microbiologia, Tucumhn, Abstr. p. 9 (1973) M. E. and Cousslo, I. D.:Invest. Agropec 7, 271 (1970) .RONDINA, R. V. D., MENDIONDO,
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Address: Prof. Dr. Jorge Daniel Coussio, Departamento de Bioquimica Vegetal, Cdtedra de Farmacognosia, Facultad de ,Farmaciay Bioquimica. Universidad de Buenos Aires. Junin 956, Buenos Aires, Argentina
PHENOLIC COMPOUNDS OF TERMlNALlA TRIFLORA' By V.S . MARTINO, M. N. GRAZIANO, 0. HNATYSZYN and J. D.Coussro
Abstract
Terminalia triflora (GRISEB.)LILLO(Combretaceae) is an Argentine tree which is common throughout the North and Northeastern regions of Argentina. It is known with the, common name of ,,lanzaU; ,,lama amarilla", ,,amarillo de rio" o r ,,palo amari!lo" (DIGILIO and LEGNAME 1966). Phytochemical investigations on members of thegenus Terminalia by IDEMUDIA (1967, 1970) has led t o the isolation of ellagic, trimethylellagic from T. iuorensis and T . avicennoides; ellagic, trimethylellagic and tetramethylellagic from T . glaucescens and T . laxiflora. RAMACHANDRA ROW and SUBBA RAO (1962) isolated 3,3dimethylellagic 4-glucoside from T . paniculata. Phytochemical screenings on T. triflora have established the presence of saponins and oxidases in the stems, a colorant matter in the wood (DOMINGUEZ et a]., 1919), and phenolic compounds in the leaves (RONDINA eta]., 1970). There is no record of the uses of this plant in folk medicine. H I E R ~ N Y(1882) M~~ mentions its use in making posts, furniture, lances for soldiers and for combustible. Nevertheless we have found data on the medicinal use of other exotic species of the genus Terminalia. HARTWELL (1968) mentions the use of T. arjuna (RoxB.) WIGHTet ARN. by natives of Ceylon against calluses; T . bellerica ROXB.and T. chebula RETZ. in India and Japan is said to have antitumor activity; T . sericea BURCH.in Rhodesia is used in native medicine against plantar warts. PLANESde BANCHEROand SOUTO(1969) describe the antimicrobial, antifago and antiviral ,,in vitro" activity o f the aqueous extracts of leaves of Terminalia triflora against Foot and Mouth disease, this .activity being attributed to the
Part IX in the series ,Flavonoids from Argentine Medicinal Plants". For Part VIII see Phytochemistry 12,1825 (1973).
Downloaded by: NYU. Copyrighted material.
T h e phytochemical examination of the methanolic extract of the leaves of Terminalia triflora resulted in the isolation and identification of quercitrin, afzelin, ellagic and gallic acids, methyl gallate and two tannins named D and E, whose structure is being determined.
Phenolic Compounds of Terminalia triflora
227
tannins in the plant. Recently R I V Aand ~ DISTEFANO (1971, 1973) demonstrate the antiviral activity of the same extracts against RNA viruses of different types: Rabies, Fot and Muth disease and Influenza.
Experimental Material The leaves of Temiinalia triflora were collected during October 1971 a t the Province of Tucumin, Argentina. A voucher specimen is deposited in the University Herbarium.
Ether fraction The ether extract furnished on standing a pale yellow precipitate (4 g). Column chromatography of this precipitate was carried out on polyamide eluted with ethanol and water in a gradient from (3 : 7 ) to (7.5:2.5) yielding ellagic acid and a mixture of flavonoids. The latter was resolved by preparative T L C with chloroform-methanol-methylacetone-acetylacetone (20 : 10 : 5 : 1) into: Band I, Quercitrin (Quercetin-3-rhamnoside) and Band 11, Afzelin (Kaempferol-3-rhamnoside). The individual compounds were identified as follows: Band I (Quercetin-3-rhamnoside). The first band (Rf = 0.34) was cristallized from aqueous ethanol, m.p. 182-183O C. ljV spectrum in methanol of the compound showed the following peaks: 254, 266 sh, 302 sh, 352 mp. UV Shiffs with Al CI,: 276, 304 sh, 333, 430 my. AL C1,IHCI: 272, 303 sh, 353, 401 mli. NaOMe: 270,326,393 mp. NaOAc: 272,322 sh, 372 my. IR spectrum (KBr disc) showed peaks a t 3400, 2950, 1730, 1665, 1610, 1570, 1510, 1460, 1370, 1295,1240,1205,1170,1125,1085,1015,960,945,875,810 and 725 cm-I. T h e compound (25 mg) was dissolved in 1ml of methanol and hydrolyzed with 6% HCI at 100' C 45 min. T h e aglucon precipitated on cooling and was filtered. UV, IR and T L C comparison against an authentic sample showed it to be quercetin; the sugar was found to be rhamnose by paper chromatographic analysis. From UV, IR and T L C against an authentic sample the compound was identified as quercetin3-rhamnoside. Band I1 (Kaempferol-3-rhamnoside). This band (Rf = 0.49) was cristallized from aqueous ethanol n1.p. 172-174O C. UV spectrum (MeOH): 275, 290 sh, 341 mk. AICI,: 285, 302 sh, 350, 410 mp. AICI,/HCI: 285, 302 sh, 342, 393 mp. NaOMe: 287, 322 sh, 390 rnp. NaOAc: 371, 300 sh, 272 mu. 1R spectrum (KBr disc) showed peaks a t 3450,2975,1660,1620,1510,1460,1370,1210,975, 840 and 720 cm-1. T h e compound (1Omg) was dissolved in 1 ml of methanol and hydrolyzed with 6% HCI at 100° C 45 min. T h e aglucon precipitated o n cooling and was filtered. Mixed m.p. UV and IR comparison as well as T L C against a n authentic sample proved the aglucon t o be kaempferol. The sugar was identified by paper chromatographic comparison as rhamnose. This compound through UV, IR and TLC comparison against an authentic sample was identified as kaempferol-3-rhamnoside.
Downloaded by: NYU. Copyrighted material.
Extraction of the plant T h e coarsely milled dried plant material (1.500 g) was extracted continuously in a soxhlet apparatus using successively chloroform' and methanol. A yellow substance named D (100 g) precipitated from the methanolic extract by the addition of chloroform. T L C of the isolate showed a single spot. T h e remaining solution was taken to dryness by means of a flash evaporator in vacuo a t 4S0 C, and the residue (214 g) was dissolved with hot water, and this phase partitioned succesively with ether and ethylacetate.
228
Martino, Graziano, Hnatyszyn and Coussio
Planta medica Vol. 27 1975
+ ?4d)H
ext
.
ICC15 added
\1
MeOH s o l n .
precipitate
t o dryness
Tannin I )
I
I
F
JI
extd. Et20
l
Quercitrin A f zelin Ellagic acid Callic acid Wethyl e l l a t e
EtOAc extd.
Tannin E
.
I
( discard)
Fig. 1. Extraction procedure of the phenolic compounds from the leaves of Terminalia tripora Ellagic acid This compound did not melt below 350° C. Paper chromatography using n BuOH: AcOH : H,O (4: 1:5), 6% AcOH atid water gave a single spot at Rf = 0.33, 0.02 and 0.00 respectively. UV spectrum in methanol gave maxima at 257 and 367 mp. With NaOAc a new band appeared at 280 with a weakeningof the band at 255 mu. IR spectrum (KBr disc) showed absorption bands a t 3550 cm-1 (OH), 1700 cm-I (unsaturated lactone) and 1580 cm-I (aromatic character). Comparison by paper chromatography, UV and IR showed the isolate and a reference sample of ellagic acid to be identical. The ethereal solution yielded gallic acid and methyl gallate. The identity of the isolated compounds was confirmed by direct comparison with authentic samples (mixed m.p., TLC, UV and IR).
Downloaded by: NYU. Copyrighted material.
H20 added
Phenolic Compounds of Terminalia triflora
229
Ethylacetate fraction This fraction gave on standing a pale yellow compound named E (45g) which by T L C showed a single spot. Substance D Hydrolysis. Acid hydrolysis of compound D (50 mg) in 2N H,SO, under reflux for 10 hr gave a beige precipitate (20mg) identified as ellagic acid. An examination of the acid layer by descending paper chromatography with butanol-pyridine-water (6 :4 : 3) showed glucose as the only sugar present. T h e acid laver was extracted with ethylacetate, and T L C analysis of the organic layer on Silicagel G plates using chloroform-ethylacetate-formic acid (5 : 4 : 1) revealed the presence of gallic acid. Both gallic acid and glucose were confirmed by GLC. Substance E Hydrolysis. Acid hydrolysis of tannin E carried out in the same way as for tannin D provided ellagic and gallic acids and glucose. T h e complete structure of this tannins will be subject of a future investigation.
T h e methanolic extract of the dried powdered leaves of T. triflora was submitted to fractionation (Fig. 1) to give quercitrin (quercetin 3-rhamnoside), afzelin (kaempferol 3-rhamnoside), ellagic and gallic acids and methylgallate and two unidentified tannins named D and E. The purification of the identified compounds was carried out by preparative chromatography (column and TLC) and their identity was established by TLC, spectral data (UV and IR) and acid hydrolysis by comparison with authentic reference material. T h e tannins named D and E gave ellagic and gallic acids and glucose after hydrolysis. RIVASand MERCADO (1973) prove that the antiviral activity of the aqueous extracts of T. triflora is due to tannins D and E. Acknowledgements This investigation was supported by Consejo Nacional d e Investigaciones Cientificas y Tkcnicas, Grants N o 3520 a171 and 3520 c173. T h e authors are grateful to Dr. FEDERICOFELFORT,Ins ~ i t u t oMiguel A. Lillo, Universidad Nacional de, Tucurnln for collecting plant material and Dr. P. R. LEGNAME for botanical identification. References P. R.: "Los Arboles Indigenas d e la Provincia de TucumBn", DICILIO,A. P. L. and LEGNAME, Universidad Nacional de Tucumin, Instituto Miguel A. Lillo, Tucumin (1966) J. A.: "Contribuciones a la Materia Mtdica Argentina", J. Peuser Ltda, Buenos DOMINGUEZ, Aires (1928)
Downloaded by: NYU. Copyrighted material.
Results and Discussion
230
Martino, Graziano, Hnaryszyn and Coussio
Planta medica Vol. 27 1975
HARTWELL, J. L.: Lloydia 31,71 (1968) HIERONYMUS, J.: Bol. Acad. Cienc. C6rdoba 4,199 (1882) 0.G. and EKONG,D. E. U.: J:Chem. Soc., 863 (1967) IDEMUDIA, IDEMUDIA, 0.G.: Phytochem. 9,2401 (1970) PLANES DE BANCHERO, E. and SOUTO;J.: Rev. Invest. Agropec. 6,155 (1969) RAMACHANDRA ROW,L. and SUBBA RAO,G. S. R.: Tetrahedron 18,357 (1962) A. and GRAZIANO, M. N.: V°Congreso Lacinoamericano de MicrobioRIVAS,M., DISTEFANO, logia Punia del Este, Abstr. p. 21 (1971) RIVAS,M. and DISTEFANO, A.: Rev. Asoc. Argent. Microbiol. 5,55 (1973) RIVAS,M. and MERCADO,E.: 111 Jornadas Argentinas d e Microbiologia, Tucumhn, Abstr. p. 9 (1973) M. E. and Cousslo, I. D.:Invest. Agropec 7, 271 (1970) .RONDINA, R. V. D., MENDIONDO,
Downloaded by: NYU. Copyrighted material.
Address: Prof. Dr. Jorge Daniel Coussio, Departamento de Bioquimica Vegetal, Cdtedra de Farmacognosia, Facultad de ,Farmaciay Bioquimica. Universidad de Buenos Aires. Junin 956, Buenos Aires, Argentina
