Pharmacognosy Dept., Faculty of Pharmacy, Cairo University, Cairo
THE ISOLATION AND CHARACTERISATION O F EMETINE ALKALOID FROM HEDERA HELIX By G. H. MAHRAN,S. H. HILALand T. S. EL-ALFY
Emetine alkaloid was successively separated for the first time from the alcoholic extract of the four varieties of Hedera helix L. growing in Egypt. The identification of the alkaloid was achieved by applying I . R., U.V., N . M . R. and Mass spectral analysis, as well as, preparation of derivatives.
Alkaloids o r nitrogenous bases have never been reported in Hedera helix I.. During the course of preliminary chemical screening of the different organs of H. helix the four varieties growing in Egypt. viz., Hedera helix var. baltica HORT, var. hibernica KIRCHN.,H. helix var. marginata and H. helix var. erecta SCHULZE, their alcoholic extracts were found to give positive tests for alkaloids and/or. 1951). It was deemed of interest, nitrogenous bases (MUNIERand MACHEBOEUF, therefore, to try the isolation and identification of these compounds.
Experimental Preparation of the alcoholic extract. The powdered whole plant material (5 Kg) was exhaustively extracted with 96% alcohol (20 I). The alcoholic extract was concentrated under vacuum at a temperature not exceeding 40°, to a syrupy consistancy (500 ml). This extract was found to give 1951). positive alkaloidal tests (MUNIER and MACHEBOEUF,
T. L. C. of the alcoholic extract. By chromatographing the alcoholic extract on T. L. C. plates of Silica gel G (E. Merck), and the plates were developed with the solvent systems recorded in table I, visualised with modified DragendorWs reagent, after drying, it was evident that the alcoholic extract contained four chromatographically separated substances. Fractionation of the alcoholic extract. The concentrated alcoholic extract was diluted with twice its volume of water, when a green precipitate was separated by filtration (A). The clear aqueous filtrate was vigorously shaken with half its volume of benzene (saturated with water). On standing, the aqueous phase was repeatedly shaken with benzene and the combined benzene extracts -
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Abstract
128
Mahran, Hilal and El-Alfy
Planta medica Vol. 27 1975
after concentration - were treated with 1% hydrochloric acid. The aqueous acidic layer was made distinctly alkaline with concentrated ammonium hydroxide solution and extracted several times with chloroform. The chloroform extract, on concentration, gave a residue (C), which gave positive test for alkaloids and/or nitrogenous bases. T h e green precipitate (A) was successively extracted with petroleum ether (b.r. 40-60' C), then with 1% hydrochloric acid. The acidic extract was rendered alkaline with ammonium hydroxide, then extracted several times with chloroform; the chloroform extract, on concentration, gave a residue (D) which gave positive alkaloid tests (Scheme I).
Column d~romatographyof the combined chloroform extracts. The combined chloroform extracts were concentrated after dehydration with anhydrous sodium sulphate, then fractionated on a column of Kieselgel (E. Merck) 1X 15 cm. Elution was carried out with acetone : methanol 95 :.5 V/V,which was found to give an efficient separation (Table 1). 20 rnl fractions were collected; Table I
T. L. C. systems and Rr values for spots of the chromatograms of the alcohol extract Solvent systems 1. n-Butanol-Acetic Acid-Water 4 : 1 : 1 (v/v/v)
2. Acetone-Methanol 9s : 5 (v/v) 3. Benzene-Methanol '
3 : 1(v/v) 4 : 1 (v/v)
4. Chloroform-Methanol
9 : 1 (vlv) 5. Chloroform-Methanol-Acetic Acid 90 : 10 : 0.5 (v/v/v) 90 : 10 : 0.25 (v/v/v) 6.70% Ethanol-25% Ammonia 99 : I (v/v)
7. Benzene-Ether-Diethylamine 20 : 10 : 5 (v/v/v)
No. of spots
Rfvalues
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T. L. C. of the two chloroform extracts C and D. The two extracts C and D were chromatographed on T. L. C. using the previous method. The chromatograms after visualisation, showed that both extracts gave the same four spots; having the characteristic orange colour, corresponding to those given by the alcoholic extract. The two extracts were combined, then were fractionated adopting column chromatographic technique for the isolation of these constituents.
Pet. ether
B I1
cone
MY-
7 Ether
1 1%HCI
A
Bases
+ CHCI,
+ NH40H
Green resinous precipitate
Residue
+
C
Bases
Benzene Aq. HCI 1- N H 4 0 H CHCI,
I
CHCI,
1
I
+
I
PPt.
aq. layer
Saponin mixture
Butanol Dehydrated twice vol. of ether
I
I
.1
Aq. filrrate
+ equal vol. of 4Water.
Supernatent
D
I
PPt.
Ethylacetate Flavonoid
B1
Aq. phase Interphase
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BI
Alcohol
4
Benzene Layer
+ benzene (saturated with water)
+ 1%HCI
~ q u e o u phase s
+ twice its volume water I
96% Alcohol
Concentrated Alcoholic Extract
I
(5 Kg)
Dry powdered drug
Scheme I Schematic isolation of Hederasaponinsfrom Hedera helix var. baltica Hort.
130
~ a h i a nHilal , and El-Alfy
Planta medica Vol. 27 1975
T a b l e I1 Rfvalues and tests of the fractions of the column
r 2 3 4
No. of spots
Rr
Colour of spots
Cold chloride
Platinic chloride
Picric acid
Test for nitrogen
-
-
-
-
-
-
-
1 1 1
0.86 0.86 0.86
orange orange orange
needles needles needles
-
-
-
PPtPPr. PPt. -
-
-
-
-
-
-
-
-
1
0.54
fai~t orange
faint faint opalescence opalescence
1
0.49 orange
opalescence opalescence
faint orange orange
5 6 7
-
9
-
10 11 12
13 14 15
-
-
-
-
-
18
1
0.49
19
1
0.49
20 21
-
-
-
1
0.41
22
1
0.41
23
-
-
- = negative,
-
PPt. PPt. PPt-
-
-
-
-
-
-
-
-
-
-
+ +
faint faint opalescence opalescence
-
+
opalescence opalescence
-
+ + +
-
-
-
faint orange
faint turbidity
faint turbidity
faint turbidity
orange
turbidity
turbidity
turbidity
-
++ ++ ++
-
-
-
+ = positive (faint blue), + + = positive (blue), ppt. = precipitate
each fraction was separately concentrated under reduced pressure to 1ml., tested for alkaloids and chromatographed on T.L.C. using the same techniques adopted before. Results obtained are summarised in table 11. From table 11, it may be concluded: 1. Fractions No. 3,4,5 gave only one spot; Rf 0.89 and gave strongly positive tests for alkaloids. 2. Fractions No. 6 to 13 did not show any spots. 3. Fractions No. 14,15 showed one spot; Rf 0.54. 4. Fractions No. 16,17 showed no spots. 5. Fractions No. 18,19 showed one spot; Rf 0.49.
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Fraction No.
Isolation of Ernetine from Hedera helix
131
80 60 40 20
3 000
2000 1700 1400 1100 Fig. 1. IR spectrum of alkaloid
800
500
Fig. 2. UV-spectruni
I
I
I
I
I
I
I
I
8
7
6
5
I
3
2
I
Fig. 3. NMR-spectrum
200 Downloaded by: Chinese University of Hong Kong. Copyrighted material.
4 000
132
Mahran, Hilal and El-Alfy
Planta medica Vol. 27 1975
6. Fraction No. 20 was free from any spots. 7. Fractions No. 21,22 showed one spot; Rf 0.41. 8. Further elution with the same solvent, then with stronger polar solvents showed no spots. As fractions No. 3; 4; 5 contained the same substance in larger proportion while the other fractions contained different substances in very small amounts (faint spot colour, slightly positive tests), isolation of the first substance was tried and the other fractions were reserved for further study.
Characters of the isolated crystals. The crystals were collected, washed with distilled water, dried in a vacuum desiccator. The crystals dissolve freely in methanol, ethanol, ether, benzene and acidulated water, but are insoluble in water and petroleum ether; m.p. 74" C. (KOFLER). By T.L.C., using the previous system, it showed to be one substance, being separated in one definite spot. The picrate derivative was obtained in yellow needles; m.p. 161° C and the auric chloride derivative in orange cubes; m.p. 184' C. Microanalysis. C% 74.69; H%, 8.59; N%, 5.05. Molecular Weight 486.06 (determined by new Mayers apparatus). Calculated empirical formula C2,H,,N202. T h e infra-red spectrum (Fig. 1) showed band for O H group a t 3200 cm-I, band at 1520 cm-1, for NH group (bending), a t 3500 cm-I (stretch and band 1500 cm-I for aromatic system, WILLIAMS and FLEMING,1966). The ultraviolet spectrum (Fig. 2) showed that the substance had an absorption maximum at 205 mu; indicating the aromatic structure. The N.M.R. and Mass spectra (Fig. 3,4) were found to'be identical to those of the alkaloid emetine published by SPITELLER and SPITELLER-FRIEDMANN, 1963.
Fig. 4. Mass spectrum of alkaloid
References MUNIER, R. and MACHEBOEUF, M.: ,Bull. Soc. Chim. Biol.", 33,846 (1951) WILLIAMS, D. H., and FLEMING, I.: "Spectroscopic methods in organic Chemistry*, McGrawHill Publishing Co. Ltd., London (1966) G. and SPITELLER-FRIEDMANN, M.: ,Tetrahedron Letters", 3, 157 (1963) SPITELLER, Address: Prof. Dr. G. H. Mahran, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo, Egypt
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isolation of the first alkaloidal substance. Fractions No. 3, 4 and 5 were combined together and the solvent was removed leaving an oily residue. The residue was dissolved in absolute methanol, decolourised with activated charcoal and filtered. The filtrate, on concentration, gave a white microcrystalline needle-like substance.
THE ISOLATION AND CHARACTERISATION O F EMETINE ALKALOID FROM HEDERA HELIX By G. H. MAHRAN,S. H. HILALand T. S. EL-ALFY
Emetine alkaloid was successively separated for the first time from the alcoholic extract of the four varieties of Hedera helix L. growing in Egypt. The identification of the alkaloid was achieved by applying I . R., U.V., N . M . R. and Mass spectral analysis, as well as, preparation of derivatives.
Alkaloids o r nitrogenous bases have never been reported in Hedera helix I.. During the course of preliminary chemical screening of the different organs of H. helix the four varieties growing in Egypt. viz., Hedera helix var. baltica HORT, var. hibernica KIRCHN.,H. helix var. marginata and H. helix var. erecta SCHULZE, their alcoholic extracts were found to give positive tests for alkaloids and/or. 1951). It was deemed of interest, nitrogenous bases (MUNIERand MACHEBOEUF, therefore, to try the isolation and identification of these compounds.
Experimental Preparation of the alcoholic extract. The powdered whole plant material (5 Kg) was exhaustively extracted with 96% alcohol (20 I). The alcoholic extract was concentrated under vacuum at a temperature not exceeding 40°, to a syrupy consistancy (500 ml). This extract was found to give 1951). positive alkaloidal tests (MUNIER and MACHEBOEUF,
T. L. C. of the alcoholic extract. By chromatographing the alcoholic extract on T. L. C. plates of Silica gel G (E. Merck), and the plates were developed with the solvent systems recorded in table I, visualised with modified DragendorWs reagent, after drying, it was evident that the alcoholic extract contained four chromatographically separated substances. Fractionation of the alcoholic extract. The concentrated alcoholic extract was diluted with twice its volume of water, when a green precipitate was separated by filtration (A). The clear aqueous filtrate was vigorously shaken with half its volume of benzene (saturated with water). On standing, the aqueous phase was repeatedly shaken with benzene and the combined benzene extracts -
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Abstract
128
Mahran, Hilal and El-Alfy
Planta medica Vol. 27 1975
after concentration - were treated with 1% hydrochloric acid. The aqueous acidic layer was made distinctly alkaline with concentrated ammonium hydroxide solution and extracted several times with chloroform. The chloroform extract, on concentration, gave a residue (C), which gave positive test for alkaloids and/or nitrogenous bases. T h e green precipitate (A) was successively extracted with petroleum ether (b.r. 40-60' C), then with 1% hydrochloric acid. The acidic extract was rendered alkaline with ammonium hydroxide, then extracted several times with chloroform; the chloroform extract, on concentration, gave a residue (D) which gave positive alkaloid tests (Scheme I).
Column d~romatographyof the combined chloroform extracts. The combined chloroform extracts were concentrated after dehydration with anhydrous sodium sulphate, then fractionated on a column of Kieselgel (E. Merck) 1X 15 cm. Elution was carried out with acetone : methanol 95 :.5 V/V,which was found to give an efficient separation (Table 1). 20 rnl fractions were collected; Table I
T. L. C. systems and Rr values for spots of the chromatograms of the alcohol extract Solvent systems 1. n-Butanol-Acetic Acid-Water 4 : 1 : 1 (v/v/v)
2. Acetone-Methanol 9s : 5 (v/v) 3. Benzene-Methanol '
3 : 1(v/v) 4 : 1 (v/v)
4. Chloroform-Methanol
9 : 1 (vlv) 5. Chloroform-Methanol-Acetic Acid 90 : 10 : 0.5 (v/v/v) 90 : 10 : 0.25 (v/v/v) 6.70% Ethanol-25% Ammonia 99 : I (v/v)
7. Benzene-Ether-Diethylamine 20 : 10 : 5 (v/v/v)
No. of spots
Rfvalues
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T. L. C. of the two chloroform extracts C and D. The two extracts C and D were chromatographed on T. L. C. using the previous method. The chromatograms after visualisation, showed that both extracts gave the same four spots; having the characteristic orange colour, corresponding to those given by the alcoholic extract. The two extracts were combined, then were fractionated adopting column chromatographic technique for the isolation of these constituents.
Pet. ether
B I1
cone
MY-
7 Ether
1 1%HCI
A
Bases
+ CHCI,
+ NH40H
Green resinous precipitate
Residue
+
C
Bases
Benzene Aq. HCI 1- N H 4 0 H CHCI,
I
CHCI,
1
I
+
I
PPt.
aq. layer
Saponin mixture
Butanol Dehydrated twice vol. of ether
I
I
.1
Aq. filrrate
+ equal vol. of 4Water.
Supernatent
D
I
PPt.
Ethylacetate Flavonoid
B1
Aq. phase Interphase
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
BI
Alcohol
4
Benzene Layer
+ benzene (saturated with water)
+ 1%HCI
~ q u e o u phase s
+ twice its volume water I
96% Alcohol
Concentrated Alcoholic Extract
I
(5 Kg)
Dry powdered drug
Scheme I Schematic isolation of Hederasaponinsfrom Hedera helix var. baltica Hort.
130
~ a h i a nHilal , and El-Alfy
Planta medica Vol. 27 1975
T a b l e I1 Rfvalues and tests of the fractions of the column
r 2 3 4
No. of spots
Rr
Colour of spots
Cold chloride
Platinic chloride
Picric acid
Test for nitrogen
-
-
-
-
-
-
-
1 1 1
0.86 0.86 0.86
orange orange orange
needles needles needles
-
-
-
PPtPPr. PPt. -
-
-
-
-
-
-
-
-
1
0.54
fai~t orange
faint faint opalescence opalescence
1
0.49 orange
opalescence opalescence
faint orange orange
5 6 7
-
9
-
10 11 12
13 14 15
-
-
-
-
-
18
1
0.49
19
1
0.49
20 21
-
-
-
1
0.41
22
1
0.41
23
-
-
- = negative,
-
PPt. PPt. PPt-
-
-
-
-
-
-
-
-
-
-
+ +
faint faint opalescence opalescence
-
+
opalescence opalescence
-
+ + +
-
-
-
faint orange
faint turbidity
faint turbidity
faint turbidity
orange
turbidity
turbidity
turbidity
-
++ ++ ++
-
-
-
+ = positive (faint blue), + + = positive (blue), ppt. = precipitate
each fraction was separately concentrated under reduced pressure to 1ml., tested for alkaloids and chromatographed on T.L.C. using the same techniques adopted before. Results obtained are summarised in table 11. From table 11, it may be concluded: 1. Fractions No. 3,4,5 gave only one spot; Rf 0.89 and gave strongly positive tests for alkaloids. 2. Fractions No. 6 to 13 did not show any spots. 3. Fractions No. 14,15 showed one spot; Rf 0.54. 4. Fractions No. 16,17 showed no spots. 5. Fractions No. 18,19 showed one spot; Rf 0.49.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Fraction No.
Isolation of Ernetine from Hedera helix
131
80 60 40 20
3 000
2000 1700 1400 1100 Fig. 1. IR spectrum of alkaloid
800
500
Fig. 2. UV-spectruni
I
I
I
I
I
I
I
I
8
7
6
5
I
3
2
I
Fig. 3. NMR-spectrum
200 Downloaded by: Chinese University of Hong Kong. Copyrighted material.
4 000
132
Mahran, Hilal and El-Alfy
Planta medica Vol. 27 1975
6. Fraction No. 20 was free from any spots. 7. Fractions No. 21,22 showed one spot; Rf 0.41. 8. Further elution with the same solvent, then with stronger polar solvents showed no spots. As fractions No. 3; 4; 5 contained the same substance in larger proportion while the other fractions contained different substances in very small amounts (faint spot colour, slightly positive tests), isolation of the first substance was tried and the other fractions were reserved for further study.
Characters of the isolated crystals. The crystals were collected, washed with distilled water, dried in a vacuum desiccator. The crystals dissolve freely in methanol, ethanol, ether, benzene and acidulated water, but are insoluble in water and petroleum ether; m.p. 74" C. (KOFLER). By T.L.C., using the previous system, it showed to be one substance, being separated in one definite spot. The picrate derivative was obtained in yellow needles; m.p. 161° C and the auric chloride derivative in orange cubes; m.p. 184' C. Microanalysis. C% 74.69; H%, 8.59; N%, 5.05. Molecular Weight 486.06 (determined by new Mayers apparatus). Calculated empirical formula C2,H,,N202. T h e infra-red spectrum (Fig. 1) showed band for O H group a t 3200 cm-I, band at 1520 cm-1, for NH group (bending), a t 3500 cm-I (stretch and band 1500 cm-I for aromatic system, WILLIAMS and FLEMING,1966). The ultraviolet spectrum (Fig. 2) showed that the substance had an absorption maximum at 205 mu; indicating the aromatic structure. The N.M.R. and Mass spectra (Fig. 3,4) were found to'be identical to those of the alkaloid emetine published by SPITELLER and SPITELLER-FRIEDMANN, 1963.
Fig. 4. Mass spectrum of alkaloid
References MUNIER, R. and MACHEBOEUF, M.: ,Bull. Soc. Chim. Biol.", 33,846 (1951) WILLIAMS, D. H., and FLEMING, I.: "Spectroscopic methods in organic Chemistry*, McGrawHill Publishing Co. Ltd., London (1966) G. and SPITELLER-FRIEDMANN, M.: ,Tetrahedron Letters", 3, 157 (1963) SPITELLER, Address: Prof. Dr. G. H. Mahran, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo, Egypt
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
isolation of the first alkaloidal substance. Fractions No. 3, 4 and 5 were combined together and the solvent was removed leaving an oily residue. The residue was dissolved in absolute methanol, decolourised with activated charcoal and filtered. The filtrate, on concentration, gave a white microcrystalline needle-like substance.
