Institute of History of Medicine and Medical Research, Tughlakabad, New Delhi-62' and Department of Chemistry, University of Delhi, Delhi?
PHENOLIC CONSTITUENTS OF TAXUS BACCATA LEAVES
M. S. Y. KHAN^, ISHWARKUMARI, J. SIVAPR AS AD^, G. R. NAGARAJAN~, M. R. PARTHASAUTHY~ and H. G. KRISHNAMURTY~
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Abstract Betuloside (1) and three biflavanoids, viz., sciadopitysin (III), ginkgetin (IV) and sequoiaflavone (V) are isolated and identified from T a x u s b a c c a t a leaves.
The toxicology of Taxus baccara leaf alkaloids has been described (CHARLES Jr. 1970). The extracts of the leaves are used in the treaunent of various ailments in et al. 1958). Of particular interest is the the Indian system of medicine (CHOPRA report by VOHORAand KUMAR(1971) that a water extract of the leaves possesses good tranquilising activity. We began a systematic investigation of the chemical constituents of the leaves in order to locate the active principles responsible for this activity. In the course of this work, we examined the leaf phenolic constituents and this paper reports their identification. We found that the water extracts gave strong phenolic tests and negative alkaloid reactions. The penolic compounds could be extracted into ethyl acetate and the extract yielded a compound 'A', m.p. 187-90°. In order to isolate it in substantial amounts the powdered leaves were exhaustively extracted directly with hot ethyl acetate and the concentrated extract on cooling deposited a crystalline yellow powder 'B' (300 mg). The mother liquor gave the crystalline compound A. Compound A is optically active, soluble in hot water and has a bitter taste. I t formed an acetate, m.p. 185-86O, whose NMR indicated one phenolic acetoxyl (6 2.15), four alcoholic acetoxyls (6 1-95), secondary methyl group (ô 1.1, J=7 Hz), a triplet for 2 protons (6 2.7, 5=7 Hz) and a A2B2 doublets for a para disubstituted benzene ring (ô 6.8 and 7.1, J = 9 Hz) and signals between ô 3.5-5.0. It thus appeared to be a glycoside. Acid hydrolysis of A gave glucose. The aglycone was recovered from the aqueous solution by repeated extraction with ether and crystallized from petroleum, m.p. 80°. It is phenolic in nature and optically active. The NMR of the aglycone showed a secondary methyl (6 1.3), a two proton multiplet (6 1.8), benzylic two proton triplet (ô 2.7), one proton multiplet (ô 4.0), besides the A2B2aromatic protons. Based on these data, the aglycone is formulated
,
Phenolic Constituents of Taxus baccata Leaves
83
as in (II). The physical properties of the glucoside and the aglycone and their NMR spectra are in complete agreement with betuloside (1) and betuligenol (II), respectively. Thus the compound A is betuloside (1), first reported from Betula alba (ANTONIA SOSA,1933).
Hoecni-cH.-F*-cH3 OR HO
O
III. R I = R 2 = R 4 = C H 3 ; R ~ : H IV. R I = R 2 = C H 3 ; R 3 . R q = H Y. RI:CH3: R z = R 3 = R 4 = H W. R I = R 2 = R 3 = R 4 = n
The yellow powder (fraction B) showed typical colour tests for flavonoids (Mg-HCI; NH,). Preliminary examination showed it to be a mixture of three compounds (TB-1, TB-2 and TB-3) belonging to biflavones. Complete methylation of the mixture using N a O H and excess (CH,), SO, gave amentoflavone hexamethylether only. The khree biflavanoids were separated by PTLC using toluenepyridine-acetic acid (10:l:l vlv) on silica gel and crystallized from pyridinemethanol. The homogenity of each compound was checked by TLC in three different solvent systems. TB-1, TB-2 and TB-3 were identified as sciadopitysin (III), ginkgetin (IV) and sequoiaflavone (V) respectively based on complete methylation and identification of the product, UV spectra using shift reagents, preparation of acetates and careful analysis of the methoxyl and acetoxyl positions in their NMR spectra and finally by direct comparison of the respective acetates with the appropriate authentic compounds by TLC and mixed m.p. The alcoholic extract of the leaves yielded more of betuloside and the biflavanoid mixture as the chief phenolic fraction. Taxtts baccata leaves were previously examined by Dr MODICAet al. (1959) who showed the presence of three biflavones, one of which was identified as sciadopitysin. The results of Our work completes the identification of the biflavones in this plant. They are the C-C linked biflavones of the amentoflavone (VI) type. No C-O-C linked biflavones were detected by LIS. Experimental The powdered and dried leaves (1 kg) were exhaustively extracted with hot ethyl acetate, the extract was concentrated to about 200 ml and left overnight. A crystalline yellow powder (Fraction B) separated (300 mg). The mother liquor was evaporated to dryness and the 'residue washed with dry petroleum. The residue was crystallized from ethyl acetate giving compound A (2
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1. R % G l u c o s e II. R . H
84
Khan et al.
Planta medica Vol. 30 1976
Compound A (Betuloside) I t was recrystallised from ethyl acetate; colourless solid, m.p. 187-90' [a]g0=-48 (1010, H,O). It is freely soluble in hot water and ethanol and moderately in ether and ethyl acetate.
Hydrolysis of compound A and identification of products A solution of compound A (0.5 g) in 5010 aqueous HCI was heated on steam bath for 2 hr. The aglycone was isolated by repeated extraction with ether and crystallized from petroleum, m.p. 80°, [a]g0=-20' (1.2S0/o, EtOH); NMR (CDCI,, 6): 1.3 (3H, d, secondary methyl, J=6 Hz), 1.8 (2H, m), 2.7 (2H, t, Ar-CH,), 4.0 ( l H , m, -CHOH) ,6.9 (2H, d, Ar-H, J=9 Hz) and 7.25 (2H, d, Ar-H, J = 9 Hz). The m.p. and the NMR data of the aglycone are in agreement with betulogenol (ANTONIOSOSA,1933). T h e aqueous solution was evaporated to dryness in vacuum and the dried residue dissolved in 950/0 alcohol and the sugar was identified as glucose by paper chromatography. Fraction B (Biflavones) This fraction was separated into its constituents TB-1, TB-2 and TB-3 by preparative TLC on silica gel using toluene-pyridine-acetic acid (10:l:l v/v). The yellow bands were scraped and eluted with hot pyridine-MeOH, concentrated and the compounds recovered by pouring into dil.HC1. Each of the fractions thus obtained was crystallized from pyridine-methanol. TB-1 (sciadopitysin, 0.15 g), m.p. 315O; IR (KBr): 1665 cm-'; UV max(Me0H): 270 and 325 nm. The acetate was prepared by heating a mixture of TB-1, pyridine and acetic anhydride a t 110° for 6 hr, m.p. 265-68O. NiMR (CDCI,, 6) (Acetoxyl and Methoxyl signals only): singlets at 2.08, 2.45 and 2.5 (one acetoxyl each) and singlets a t 3.85, 3.9 and 3.95 (one methoxyl each). Mixed m.p. of the acetate with sciadopitysin triacetate was undepressed. TB-2 (gingketin, 0,08 g), m.p. 320°; IR (KBr).: 1665 cm-'; UV max(Me0H): 270 and 335 nm. The acetate of TB-2 was prepared as for TB-1, m.p. 26S0; NMR (CDCI,, 6) (Acetoxyl and Methoxyl signals only): singlets at 2.08, 2.26, 2.46 and 2.5 (one acetoxyl ench) and singlets a t 3.7 and 3.8 (one methoxyl each). Mixed m.p. of the acetate with authentic ginkgetin tetraacetate was undepressed. TB-3 (sequoiaflavone, 0.02 g), m.p. 320°; IR (KBr): 1665 cm-'; UV max(Me0H): 272 and 340 nm. The acetate has m.p. 242-44O; NMR (CDCI, 6) (Acetoxyl and Methoxyl signals only): singlets a t 2.04, 2.06, 2.25, 2.43 and 2.47 (one acetoxyl each) and singlet at 3.84 (one methoxyl). Mixed m.p. of the acetate and sequoiaflavone pentaacetate was not depressed. Methylation of the biflavanoid mixture T h e biflavanoid mixture (10 mg) in 1 ml of ethanol was inethylated with excess dimethyl sulphate (0.2 ml) and aqueous sodium hydroxide (3 ml) (NATARAJAN, MURTI and SESHADRI, 1970). After 30 rnts. a t room temperature, the reaction mixture was extracted with chloroform and the chloroform layer separated, washed with water and concentrated. The concentrated chloroform extract was spotted on TLC plates (silica gel G) along with amentoflavone hexamethyl ether, hinokiflavone pentamethyl ether and cupressuflavone hexamethyl ether, using toluene-pyridine-acetic acid 10:l:l v/v as the irrigant. Amentoflavone hexamethyl ether only was detected.
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Acetate of compound A The compound 4 (100 mg) was acetylated using pyridine (1 ml) and acetic anhydride (2 ml) a t 100° for 3 hr. Usual work up yielded the acetate, m.p. 185-86O; N M R (CDCI,, 6): 1.1 (3H, d, secondary methyl, J = 7 Hz), 1.95 (12H, s, 4 X -OCOCH,), 2.15 (3H, s, -OCOCH,), 2.7 (2H, t, Ar-CH,, J = 7 Hz), 6.8 and 7.1 (4H, d, p-disubstituted benzene protons, J = 9 Hz) and signals between 6 3.5-5.0.
Phenolic Constituents of Taxus baccara Leaves
85
The authors are thankfui to HAKIMABDULSAHIB,president, Institutee of History of Medicine and Medical Research, New Delhi for his keen interest and encouragement in the work, to Prof. N. KAWANO, Nagasaki University, Japan for recording the NMR and comparing the acetate of TB-3 with the authentic sample and to (late) Prof. T. R. SESHADRI for providing us with biflavonoid samples for comparison.
ANTONIOSOSA:Compt. Rend, 196, 1827 (1933); Chem. Abstr. 27, 4235 (1933) CHARLES, H., Jr.: Diss. Abstr., 29, 4072 (B 1969) CHOPRA,R. N., CHOPRA,1. C., HANDA,K. L. and KAPOOR,L. D.: "Chopra's Indigenous Drugs of India", (2nd Edition (1958), U. N. Dhur & Sons Ltd., Calcutta), 526 (1958) Dr MODICA,G., ROSSI,P. F. and RIVERO,A. M.: Atti Accad. Naz. Lincei Rc. 27, 127 (1959); Chem. Abstr. 54,14235 (1960) NATARAJAN, S., MURTI,V. V. S. and SESHADRI,T. R.: Phytochernistry, 9, 575 (1970) 1.: Planta Medica, 2, 100 (1971) VOHORA,S. B. and KUMAR, Address: Dr. H . G. Krishnamurty, Department o f Chemistry, University of Delhi, Delhi-1 10007, India
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References
PHENOLIC CONSTITUENTS OF TAXUS BACCATA LEAVES
M. S. Y. KHAN^, ISHWARKUMARI, J. SIVAPR AS AD^, G. R. NAGARAJAN~, M. R. PARTHASAUTHY~ and H. G. KRISHNAMURTY~
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Abstract Betuloside (1) and three biflavanoids, viz., sciadopitysin (III), ginkgetin (IV) and sequoiaflavone (V) are isolated and identified from T a x u s b a c c a t a leaves.
The toxicology of Taxus baccara leaf alkaloids has been described (CHARLES Jr. 1970). The extracts of the leaves are used in the treaunent of various ailments in et al. 1958). Of particular interest is the the Indian system of medicine (CHOPRA report by VOHORAand KUMAR(1971) that a water extract of the leaves possesses good tranquilising activity. We began a systematic investigation of the chemical constituents of the leaves in order to locate the active principles responsible for this activity. In the course of this work, we examined the leaf phenolic constituents and this paper reports their identification. We found that the water extracts gave strong phenolic tests and negative alkaloid reactions. The penolic compounds could be extracted into ethyl acetate and the extract yielded a compound 'A', m.p. 187-90°. In order to isolate it in substantial amounts the powdered leaves were exhaustively extracted directly with hot ethyl acetate and the concentrated extract on cooling deposited a crystalline yellow powder 'B' (300 mg). The mother liquor gave the crystalline compound A. Compound A is optically active, soluble in hot water and has a bitter taste. I t formed an acetate, m.p. 185-86O, whose NMR indicated one phenolic acetoxyl (6 2.15), four alcoholic acetoxyls (6 1-95), secondary methyl group (ô 1.1, J=7 Hz), a triplet for 2 protons (6 2.7, 5=7 Hz) and a A2B2 doublets for a para disubstituted benzene ring (ô 6.8 and 7.1, J = 9 Hz) and signals between ô 3.5-5.0. It thus appeared to be a glycoside. Acid hydrolysis of A gave glucose. The aglycone was recovered from the aqueous solution by repeated extraction with ether and crystallized from petroleum, m.p. 80°. It is phenolic in nature and optically active. The NMR of the aglycone showed a secondary methyl (6 1.3), a two proton multiplet (6 1.8), benzylic two proton triplet (ô 2.7), one proton multiplet (ô 4.0), besides the A2B2aromatic protons. Based on these data, the aglycone is formulated
,
Phenolic Constituents of Taxus baccata Leaves
83
as in (II). The physical properties of the glucoside and the aglycone and their NMR spectra are in complete agreement with betuloside (1) and betuligenol (II), respectively. Thus the compound A is betuloside (1), first reported from Betula alba (ANTONIA SOSA,1933).
Hoecni-cH.-F*-cH3 OR HO
O
III. R I = R 2 = R 4 = C H 3 ; R ~ : H IV. R I = R 2 = C H 3 ; R 3 . R q = H Y. RI:CH3: R z = R 3 = R 4 = H W. R I = R 2 = R 3 = R 4 = n
The yellow powder (fraction B) showed typical colour tests for flavonoids (Mg-HCI; NH,). Preliminary examination showed it to be a mixture of three compounds (TB-1, TB-2 and TB-3) belonging to biflavones. Complete methylation of the mixture using N a O H and excess (CH,), SO, gave amentoflavone hexamethylether only. The khree biflavanoids were separated by PTLC using toluenepyridine-acetic acid (10:l:l vlv) on silica gel and crystallized from pyridinemethanol. The homogenity of each compound was checked by TLC in three different solvent systems. TB-1, TB-2 and TB-3 were identified as sciadopitysin (III), ginkgetin (IV) and sequoiaflavone (V) respectively based on complete methylation and identification of the product, UV spectra using shift reagents, preparation of acetates and careful analysis of the methoxyl and acetoxyl positions in their NMR spectra and finally by direct comparison of the respective acetates with the appropriate authentic compounds by TLC and mixed m.p. The alcoholic extract of the leaves yielded more of betuloside and the biflavanoid mixture as the chief phenolic fraction. Taxtts baccata leaves were previously examined by Dr MODICAet al. (1959) who showed the presence of three biflavones, one of which was identified as sciadopitysin. The results of Our work completes the identification of the biflavones in this plant. They are the C-C linked biflavones of the amentoflavone (VI) type. No C-O-C linked biflavones were detected by LIS. Experimental The powdered and dried leaves (1 kg) were exhaustively extracted with hot ethyl acetate, the extract was concentrated to about 200 ml and left overnight. A crystalline yellow powder (Fraction B) separated (300 mg). The mother liquor was evaporated to dryness and the 'residue washed with dry petroleum. The residue was crystallized from ethyl acetate giving compound A (2
€9.
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1. R % G l u c o s e II. R . H
84
Khan et al.
Planta medica Vol. 30 1976
Compound A (Betuloside) I t was recrystallised from ethyl acetate; colourless solid, m.p. 187-90' [a]g0=-48 (1010, H,O). It is freely soluble in hot water and ethanol and moderately in ether and ethyl acetate.
Hydrolysis of compound A and identification of products A solution of compound A (0.5 g) in 5010 aqueous HCI was heated on steam bath for 2 hr. The aglycone was isolated by repeated extraction with ether and crystallized from petroleum, m.p. 80°, [a]g0=-20' (1.2S0/o, EtOH); NMR (CDCI,, 6): 1.3 (3H, d, secondary methyl, J=6 Hz), 1.8 (2H, m), 2.7 (2H, t, Ar-CH,), 4.0 ( l H , m, -CHOH) ,6.9 (2H, d, Ar-H, J=9 Hz) and 7.25 (2H, d, Ar-H, J = 9 Hz). The m.p. and the NMR data of the aglycone are in agreement with betulogenol (ANTONIOSOSA,1933). T h e aqueous solution was evaporated to dryness in vacuum and the dried residue dissolved in 950/0 alcohol and the sugar was identified as glucose by paper chromatography. Fraction B (Biflavones) This fraction was separated into its constituents TB-1, TB-2 and TB-3 by preparative TLC on silica gel using toluene-pyridine-acetic acid (10:l:l v/v). The yellow bands were scraped and eluted with hot pyridine-MeOH, concentrated and the compounds recovered by pouring into dil.HC1. Each of the fractions thus obtained was crystallized from pyridine-methanol. TB-1 (sciadopitysin, 0.15 g), m.p. 315O; IR (KBr): 1665 cm-'; UV max(Me0H): 270 and 325 nm. The acetate was prepared by heating a mixture of TB-1, pyridine and acetic anhydride a t 110° for 6 hr, m.p. 265-68O. NiMR (CDCI,, 6) (Acetoxyl and Methoxyl signals only): singlets at 2.08, 2.45 and 2.5 (one acetoxyl each) and singlets a t 3.85, 3.9 and 3.95 (one methoxyl each). Mixed m.p. of the acetate with sciadopitysin triacetate was undepressed. TB-2 (gingketin, 0,08 g), m.p. 320°; IR (KBr).: 1665 cm-'; UV max(Me0H): 270 and 335 nm. The acetate of TB-2 was prepared as for TB-1, m.p. 26S0; NMR (CDCI,, 6) (Acetoxyl and Methoxyl signals only): singlets at 2.08, 2.26, 2.46 and 2.5 (one acetoxyl ench) and singlets a t 3.7 and 3.8 (one methoxyl each). Mixed m.p. of the acetate with authentic ginkgetin tetraacetate was undepressed. TB-3 (sequoiaflavone, 0.02 g), m.p. 320°; IR (KBr): 1665 cm-'; UV max(Me0H): 272 and 340 nm. The acetate has m.p. 242-44O; NMR (CDCI, 6) (Acetoxyl and Methoxyl signals only): singlets a t 2.04, 2.06, 2.25, 2.43 and 2.47 (one acetoxyl each) and singlet at 3.84 (one methoxyl). Mixed m.p. of the acetate and sequoiaflavone pentaacetate was not depressed. Methylation of the biflavanoid mixture T h e biflavanoid mixture (10 mg) in 1 ml of ethanol was inethylated with excess dimethyl sulphate (0.2 ml) and aqueous sodium hydroxide (3 ml) (NATARAJAN, MURTI and SESHADRI, 1970). After 30 rnts. a t room temperature, the reaction mixture was extracted with chloroform and the chloroform layer separated, washed with water and concentrated. The concentrated chloroform extract was spotted on TLC plates (silica gel G) along with amentoflavone hexamethyl ether, hinokiflavone pentamethyl ether and cupressuflavone hexamethyl ether, using toluene-pyridine-acetic acid 10:l:l v/v as the irrigant. Amentoflavone hexamethyl ether only was detected.
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Acetate of compound A The compound 4 (100 mg) was acetylated using pyridine (1 ml) and acetic anhydride (2 ml) a t 100° for 3 hr. Usual work up yielded the acetate, m.p. 185-86O; N M R (CDCI,, 6): 1.1 (3H, d, secondary methyl, J = 7 Hz), 1.95 (12H, s, 4 X -OCOCH,), 2.15 (3H, s, -OCOCH,), 2.7 (2H, t, Ar-CH,, J = 7 Hz), 6.8 and 7.1 (4H, d, p-disubstituted benzene protons, J = 9 Hz) and signals between 6 3.5-5.0.
Phenolic Constituents of Taxus baccara Leaves
85
The authors are thankfui to HAKIMABDULSAHIB,president, Institutee of History of Medicine and Medical Research, New Delhi for his keen interest and encouragement in the work, to Prof. N. KAWANO, Nagasaki University, Japan for recording the NMR and comparing the acetate of TB-3 with the authentic sample and to (late) Prof. T. R. SESHADRI for providing us with biflavonoid samples for comparison.
ANTONIOSOSA:Compt. Rend, 196, 1827 (1933); Chem. Abstr. 27, 4235 (1933) CHARLES, H., Jr.: Diss. Abstr., 29, 4072 (B 1969) CHOPRA,R. N., CHOPRA,1. C., HANDA,K. L. and KAPOOR,L. D.: "Chopra's Indigenous Drugs of India", (2nd Edition (1958), U. N. Dhur & Sons Ltd., Calcutta), 526 (1958) Dr MODICA,G., ROSSI,P. F. and RIVERO,A. M.: Atti Accad. Naz. Lincei Rc. 27, 127 (1959); Chem. Abstr. 54,14235 (1960) NATARAJAN, S., MURTI,V. V. S. and SESHADRI,T. R.: Phytochernistry, 9, 575 (1970) 1.: Planta Medica, 2, 100 (1971) VOHORA,S. B. and KUMAR, Address: Dr. H . G. Krishnamurty, Department o f Chemistry, University of Delhi, Delhi-1 10007, India
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References
