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Alkaloids of Thalictrum angustifolium
Tawfeq A. Al-Howiriny, PhD
Michael A. Zemaitis, PhD
Fu-Tyan Lin, PhD
Jack L. Beal, PhD
Paul L. Schiff, Jr., PhD
ABSTRACT. Fractionation and chromatography of the ethanolic ex-tract of the roots of Thalictrum angustifolium L. (Ranunculaceae) af-forded five alkaloids: noroxyhydrastinine (1), O-methylthalicberine (2),berberine iodide (3), jatrorrhizine iodide (4), magnoflorine iodide (5). Inaddition, one simple aromatic compound, methyl-4-hydroxybenzoate(6), was also isolated. These compounds were identified via comparisonof their spectral data with authentic compounds or spectra availablewithin our laboratory. This is the first reported isolation of these com-pounds from this species. [Article copies available for a fee from The HaworthDocument Delivery Service: 1-800-HAWORTH. E-mail address: <[email protected]> Website: <http://www.HaworthPress.com> 2002 byThe Haworth Press, Inc. All rights reserved.]
Tawfeq A. Al-Howiriny, Michael A. Zemaitis, and Paul L. Schiff, Jr. are affiliatedwith the Department of Pharmaceutical Sciences, School of Pharmacy, University ofPittsburgh, Pittsburgh, PA 15261.
Fu-Tyan Lin is affiliated with the Department of Chemistry, Faculty of Arts andSciences, University of Pittsburgh, Pittsburgh, PA 15260.
Jack L. Beal (deceased) was affiliated with the College of Pharmacy, The OhioState University, Columbus, OH 43210.
Address correspondence to: Paul L. Schiff, Jr. at the above address (E-mail:[email protected]).
The authors gratefully acknowledge the gift of this plant material from ProfessorRaymond W. Doskotch, Division of Medicinal Chemistry and Pharmacognosy, Col-lege of Pharmacy, The Ohio State University, Columbus, OH 43210. In addition, theauthors gratefully acknowledge the partial financial support of King Saud University(TAA).
Journal of Herbal Pharmacotherapy, Vol. 2(2) 2002 2002 by The Haworth Press, Inc. All rights reserved. 1
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KEYWORDS. Thalictrum angustifolium L., alkaloids, noroxyhydra-stinine (1), O-methylthalicberine (2), berberine iodide (3), jatrorrhizineiodide (4), magnoflorine iodide (5), methyl-4-hydroxybenzoate (6)
INTRODUCTION
Thalictrum is an ancient name of doubtful origin which may derivefrom the Greek thallo (to grow green). Thalictrum (meadow rue), a ge-nus of plant of the Ranunculaceae (Crowfoot family), includes a widerange of tall, erect, perennial herbs that are mostly dispersed in temper-ate and cold countries at altitudes ranging from 200 to 2000 metersabove sea level.1 Crude extracts of Thalictrum species have been usedas folk medicinals throughout the world for centuries. The literatureabounds with references to the use of these extracts in the treatment ofvarious conditions, some of which include snakebite, jaundice, leprosy,rheumatism, external and internal infections, and pregnancy-inducedvomiting in countries as culturally diverse as China, Russia, Japan, theUnited States, and Canada.2-4 The genus Thalictrum is a rich source ofbenzylisoquinoline-derived alkaloids, with no less than approximately250 alkaloids having been isolated from some 60 different species.2,3 Inour continuing investigation of the alkaloids of this genus,2,3,5-7 it wasdecided to examine an extract of the herb Thalictrum angustifolium L.(Ranunculaceae) because there have been no literature reports concerningits constituents and to expand our knowledge of the chemotaxonomiccharacter of this genus.
METHODOLOGY
General Experimental Procedures. Melting points were determinedon a Fisher-Johns hot-stage apparatus and are uncorrected. Specificrotations were measured on a Perkin-Elmer Model 241 Automaticpolarimeter. UV spectra were obtained in MeOH on a Hewlett-PackardHP-845 UV-VIS spectrophotometer, and the IR spectra were recordedas a thin film on a KBr window using a Nicolet Impact 410 spectro-photometer. 1H and 13C NMR spectra were recorded in CDCl3 orCD3OD on a Bruker model WH-300 spectrometer operating at 300MHz and 75 MHz, respectively. EIMS were performed on an ExtrelELQ400 quadrupole instrument equipped with a DCI Probe HP directprobe from Vacumetrics, Inc. or a Hewlett-Packard 5971A. HREIMS
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were recorded on a Fisons VG Autospec Spectrometer or a Fison VGAnalytical 70-G Spectrometer. Thin-layer chromatography (TLC) wasperformed using 5 cm 3 20 cm precoated TLC sheets of Si gel 60 F254,0.2 mm layer thickness (E. Merck). Preparative thin-layer chromatogra-phy was performed using 20 cm 3 20 cm glass plates precoated with 1.0mm of Si gel GF254 (Analtech). Column chromatography was per-formed using Si gel (60-200 Mesh) (Baker Analyzed Reagents). Ion-ex-change chromatography was performed using Amberlite® IRA-400 (Cl)resin (Aldrich Chemical Company, Inc., Milwaukee, WI). Solvents (Et2Oand CH2Cl2) used in fractionation were dried over anhydrous Na2SO4and filtered prior to evaporation. Evaporation of solvents was done at40°C under reduced pressure, and all solvents used were analyticalgrade.
Plant Material. Powdered, dried roots of Thalictrum angustifoliumL. (Ranunculaceae) were supplied by Professor Raymond W. Doskotch,Division of Medicinal Chemistry and Pharmacognosy, College of Phar-macy, The Ohio State University, Columbus, OH 43210. A herbariumspecimen is on deposit at that site.
Extraction and Fractionation. Roots of Thalictrum angustifolium L.(5 kg) were extracted via percolation with petrol (22 L) and the solventevaporated to leave a greenish white residue (11.5 g) (Fraction A). Theplant material was dried, extracted with EtOH (125 L), and the solventevaporated to leave a dark residue (215 g) (Fraction B). The ethanolicextract was treated with aqueous citric acid (1%) (9 L) and filtered. Theinsoluble portion was treated with Et2O (2.5 L) and filtered to leave in-soluble material (9.3 g) (Fraction C). The filtrate was added to the citricacid solution, partitioned with Et2O (700 mL 3 10), and the solventevaporated to provide brownish yellow residue (13.35 g) (Fraction D)of nonalkaloidal components (Dragendorff negative). The aqueousphase was basified with NH4OH to pH 8-9, extracted with Et2O (600mL 3 8), and the combined Et2O extracts were evaporated to leave abrown residue (9.5 g) (Fraction E). The remaining ammoniacal solutionwas acidified to pH 3-4 with HCl (1%) and treated with ammoniumreineckate (Reinecke salt) solution (1%) until precipitation ceased. Theresulting precipitate was filtered via suction, washed with H2O, dis-solved in MeOH (2.8 L) and passed through a column of anion ex-change resin (iodide form) (450 g). The resin (iodide form) was preparedby passing an aqueous solution of NaOH (5%) through a columnpacked with resin (chloride form) until the solution became alkaline,and then successively rinsing the column with H2O to neutrality, aque-ous KI (5%), H2O, and then MeOH. The column eluent was evaporated
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to afford a dark brown residue (70.3 g) (Fraction F) (Quaternary Alka-loid Fraction). Fraction E (9.5 g) was dissolved in Et2O (500 mL) andpartitioned with NaOH (1%) (500 mL). The Et2O phase was separatedand the alkaline solution partitioned with Et2O (500 mL 3 5). The Et2Oextracts were combined, washed several times with water, and evapo-rated to leave a brown residue (4.20 g) (Fraction G) (Nonquaternary,Nonphenolic Alkaloid Fraction). The pH of the remaining ammoniacalsolution was adjusted to 8-9 via the addition of NH4Cl, followed by par-titioning with Et2O (500 mL 3 5). The Et2O extracts were combinedand evaporated to leave a light brown residue (0.48 g) (Fraction H)(Nonquaternary Phenolic Alkaloid Fraction).
Chromatography and Isolation of Compounds from the QuaternaryAlkaloid Fraction (Fraction F). TLC of fraction F (70.3 g) revealed thepresence of three Dragendorff reagent positive spots (Rf 0.55, 0.45, and0.1) [MeOH-NH4OH-H2O (8:1:1)]. Fraction F was dissolved in MeOH(150 mL), adsorbed onto Si gel (100 g), and chromatographed over acolumn of Si gel (430 g) prepared via slurry in CH2Cl2. The column waseluted using increasing concentrations of MeOH in CH2Cl2 and 500 mLfractions were collected. The collected fractions were pooled accordingto their TLC behavior into nine fractions (F1-F9). Fraction F6 (ColumnA) (13.37 g) was dissolved in MeOH (35 mL), adsorbed onto Si gel (18g), and chromatographed over a column of Si gel (Column B) (250 g)prepared via slurry in CH2Cl2. The column was eluted using increasingconcentrations of MeOH in CH2Cl2 and 300 ml fractions were col-lected. These fractions were pooled according to their TLC behavior togive Fractions F6A-F6F.
Methyl-4-Hydroxybenzoate (6). Fraction F6A (60 mg) (Column B)was chromatographed over a column of Si gel (7 g) using petrol:CH2Cl2(1:1). Elution with CH2Cl2 resulted in isolation of the nonalkaloidalsubstance as an amorphous white residue (9 mg). Recrystallizationfrom CH2Cl2 afforded white crystals of methyl-4-hydroxybenzoate (6)
(5 mg), mp 126-128°C dec.; identical by direct comparison (mp, UV, IR,1H NMR, EIMS) with an authentic sample (Compound #M5, 010-9),purchased from Aldrich Chemical Company ([email protected]; Mil-waukee, WI).
Berberine Iodide (3). Fractions F6C and F6D (Column B) were com-bined (8.53 g) because of TLC similarity, adsorbed onto Si gel (12 g),and subjected to chromatography on a column (Column C) of Si gel(150 g). Elution of this column with 1% MeOH in CH2Cl2 provided yel-low crystalline material (4.55 g), which upon recrystallization from
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MeOH afforded yellow needles of berberine iodide (3) (3.84 g), mp260-261°C dec.; identical by direct comparison (mp, UV, IR, and 1HNMR) with an authentic sample8 available in our laboratory.
Jatrorrhizine Iodide (4). Continued elution of column C with 3%MeOH in CH2Cl2 resulted in the isolation of an orange crystalline resi-due (235 mg), which was recrystallized from MeOH to afford orangeneedles of jatrorrhizine iodide (4) (103 mg), mp 209-211° dec.; identi-cal by direct comparison (mp, UV, IR, and 1H NMR) with an authenticsample8 available in our laboratory.
Magnoflorine Iodide (5). TLC of fraction F6E (1.93 g) (Column B)revealed the presence of one bright blue UV fluorescent spot that waspositive with Dragendorff reagent (Rf0.45) [MeOH-NH4OH-H2O (8:1:1)].Fraction F6E was dissolved in MeOH (15 mL) adsorbed onto Si gel (3.5g), and chromatographed over a column of Si gel (50 g) prepared viaslurry of CH2Cl2. Elution of this column with 10% MeOH in CH2Cl2yielded a pale brown residue (130 mg) that upon recrystallization fromMeOH furnished brown crystals of magnoflorine iodide (5) (43 mg),
mp 249-250°C dec., [α]24D + 208° (c 0.5, MeOH); identical by direct com-
parison (mp, [α]D, UV, IR, 1H NMR, EIMS) with an authentic sample8
available in our laboratory.Chromatography and Isolation of Alkaloids from the Nonquater-
nary, Nonphenolic Alkaloid Fraction (Fraction G). Fraction G (4.20 g)was dissolved in CH2Cl2 and applied to a column (Column F) of Si gel(80 g) prepared via slurry in CH2Cl2. Elution was begun with CH2Cl2,and the polarity was gradually increased via the addition of MeOH.Fractions (200 mL) were collected and combined according to theirTLC behavior to give five additional fractions (G1-G5). Fraction G1(0.330 g) was dissolved in CH2Cl2 (8 mL) and applied to a column (Col-umn G) of Si gel (10 g) prepared via slurry in CH2Cl2. The polarity wasgradually increased via the addition of MeOH and 150 mL fractionswere collected. The fractions were pooled according to their TLC be-havior to yield seven main fractions (G1A-G1G).
Noroxyhydrastinine (1) (Figure 1). Fraction G1B (34 mg) was chrom-atographed over a column of Si gel (6 g) using CH2Cl2. Elution withCH2Cl2-MeOH (99.9-0.1) afforded noroxyhydrastinine (56) as an amor-phous white residue (2.5 mg). The spectral data (UV, IR, 1H NMR,EIMS) were indistinguishable from those previously reported for thisalkaloid.9,10
O-Methylthalicberine (2) (Figure 2). Fraction G2 (0.943 g) (ColumnF) was chromatographed over a column of Si gel (35 g) using CH2Cl2.
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Elution with CH2Cl2-MeOH (99:1) afforded a residue (195 mg). Furtherpurification of this residue via preparative TLC using CHCl3-MeOH-NH4OH (9:1:0.1) resulted in the isolation of O-methylthalicberine (2)(38 mg) (light brown residue), whose spectral data (UV, IR, 1H NMR,EIMS) were indistinguishable from those previously reported forO-methylthalicberine.2,10 Furthermore, the specific rotation of the iso-
late ([α]24D + 174° [c 2.1, MeOH]) compared very favorably with litera-
ture values ([α]25D + 175.5° [c 0.2, MeOH]).2,10
RESULTS AND DISCUSSION
The potential use of any Thalictrum as a traditional medicinal is in-herently linked with the individual bioactivities of the various constitu-ents. Toward that end, a brief discussion of the bioactivities of theseisolated compounds follows.
Berberine (3) (Figure 3) is a commonly occurring protoberberine baseand has been isolated from various genera of the families Annonaceae,
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Berberidaceae, Menispermaceae, Papaveraceae, Ranunculaceae, andRutaceae.11 The first apparent isolation of berberine from a Thalictrumspecies was as the iodide salt from Thalictrum foliolosum DC. in1941.2,3 Although berberine has been isolated from at least 37 otherThalictrum species and/or varieties/races/subspecies,2,3 this is the firstreported isolation of this quaternary alkaloid from T. angustifolium.Berberine is well-known to possess a diverse profile of interesting bio-logical activities, including a wide range of antimicrobial activity againstsuch varied organisms as bacteria (including mycobacteria), amoebicand flagellated protozoa, fungi, and helminths.2,3,12,13 The alkaloid hasbeen administered orally as an antiamoebic (Entamoeba histolytica)and has been employed as an antimicrobial ophthalmic. Berberine isknown to intercalate with DNA, to inhibit cellular respiration, and to in-hibit the activities of esterase, DNA polymerase, and RNA polymeraseenzymes.2,3,12,13
Jatrorrhizine (4) (Figure 4) was apparently first isolated from Jateorhzacolumba (Menispermaceae) in 1906, but was not found in a Thalictrumspecies (Thalictrum foliolosum DC.) until 1952, when the alkaloid wasisolated as its tetrahydro-derivative.2,3,13 The alkaloid was subsequently
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reisolated from the same species several decades later,14 and has beenisolated from 19 Thalictrum species to date.12,13 Jatrorrhizine was ob-served to inhibit the growth of Mycobacterium smegmatis in in vitroantimicrobial testing and to possess some inhibitory action against thegrowth of Saccharomyces carlsbergensis.2,3,13 The alkaloid was evalu-ated for antimalarial activity against Plasmodium falciparum (in vitro)and Plasmodium berghei (in vivo) (mice), and found to exhibit anantimalarial potency equivalent to that of quinine in vitro, but was inac-tive in vivo.3 Jatrorrhizine was also found to possess anti-inflammatoryactivity in the cobra venom factor-induced rat paw edema assay.3
Magnoflorine (5) (Figure 5) is the most widely distributed naturallyoccurring quaternary aporphine alkaloid in the genus Thalictrum.2,3 Itwas first isolated from a Thalictrum species, Thalictrum thumbergiiDC. in 1956, and has subsequently been isolated from more than 39Thalictrum species.2,3 Magnoflorine has undergone bioevaluation in nu-merous systems. The alkaloid has been observed to suppress the localgraft-versus-host reactions in mice, and to suppress the induction phaseof the cellular immune response in various animals. The compound hasalso decreased arterial blood pressure in rabbits, produced hypothermiain mice, and induced contractions in the isolated pregnant rat uterus andthe isolated guinea pig ileum.2,3,13
Methyl-4-hydroxybenzoate (6) (Figure 6) has been isolated fromPueraria lobata (Leguminosae), Aristolochia kankauensis (Aristolochi-aceae), Tetradium globrifolium (Rutaceae), and Gonocaryum calleryanum(Icacinaceae).13 This is the first reported isolation of methyl-4-hydroxy-benzoate from the genus Thalictrum and the family Ranunculaceae.There have been few reports concerning the bioactivities of this simplearomatic ester. The compound was reported to be poorly active in inhib-iting the growth of Gram-negative bacteria and to possess only weak
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nematicidal activity.13 Administration of the compound to normoten-sive anaesthetized rats (30 mg/kg) resulted in a hypotensive response asnoted via a 47% fall in mean arterial blood pressure.13
Noroxyhydrastinine (1) was first isolated from Thalictrum minus L.var. adiantifolium Hort. in 1969, although the compound had long beenknown in the literature as an oxidation product of berberine.9 This sim-ple isoquinolone has been isolated from 5 Thalictrum species to date.2,3
There is little known of its bioactivity, but the alkaloid was found to bedevoid of antimicrobial activity when tested against a routine bank ofmicroorganisms.2
The alkaloid thalmidine was first isolated from Thalictrum minus L.in 1950, and later shown to be identical with O-methylthalicberine (2)in 1956.2 O-methylthalicberine has been isolated from at least 20Thalictrum species and/or varieties to date.2,3 The alkaloid has beendemonstrated to produce restlessness, dyspnea, and paralysis of the ex-tremities when administered to mice.2,3 In addition, the compound wasalso observed to induce a relaxation of an isolated segment of rabbit in-testine, to decrease acetylcholine-induced contractions, and to exhibitanti-inflammatory activity in experimentally induced mouse paw in-flammation.2,3
REFERENCES
1. Bailey LH. The Standard Cyclopedia of Horticulture, Vol. 3. New York:Macmillan, 1943:3326.
2. Schiff Jr. PL. The Thalictrum Alkaloids: Chemistry and Pharmacology. In: Al-kaloids: Chemical and Biological Perspectives, Vol. 5, Pelletier SW, Ed. New York:John Wiley & Sons, Inc., 1987:271-637.
3. Schiff Jr. PL. The Thalictrum Alkaloids: Chemistry and Pharmacology (1985-1995). In: Alkaloids: Chemical and Biological Perspectives, Vol. 11, Pelletier SW, Ed.Oxford: Elsevier Science Ltd., 1996:1-236.
4. Moerman DE. Medicinal Plants of Native America, Vol. 1. Ann Arbor: Univer-sity of Michigan Museum of Anthropology Technical Reports, No. 19, 1986:480-481.
5. Al-Rehaily AJ, Gao CY, Martin GE, Lin FT, Zemaitis MA, and Schiff Jr. PL.Thalpetaline: A New Alkaloid of Thalictrum petaloideum var. supradecompositum.Planta Med. 1998; 64:681.
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6. Al-Rehaily AJ, Sharaf MHM, Zemaitis MA, Gao CY, Martin GE, Hadden CE,Thamann TJ, Lin FT, and Schiff Jr. PL. Thalprzewalskiinone, A New Oxobenzyl-isoquinoline Alkaloid of Thalictrum przewalskii. J Nat Prod. 1999; 62(1):146-148.
7. Al-Howiriny TA, Zemaitis MA, Gao CY, Hadden CE, Martin GE, Lin FT, andSchiff Jr. PL. Thalibealine, A Novel Tetrahydroprotoberberine-Aporphine DimericAlkaloid from Thalictrum wangiii. J Nat Prod. 2001; 64(6):819-822.
8. Lou ZC, Gao CY, Lin FT, Lin MC, Zhang J, Slatkin DJ, and Schiff Jr PL. Qua-ternary Alkaloids of Thalictrum glandulosissimum. Planta Med. 1987; 53(5):498-499.
9. Doskotch RW, Schiff Jr PL, and Beal JL. Alkaloids of Thalictrum. X. Two NewAlkaloids from T. minus var. adiantifolium: Noxoxyhydrastinine and Thalifoline. Tet-rahedron. 1969; 25:469-475.
10. Doskotch RW, Schiff Jr. PL, and Beal JL. Alkaloids of Thalictrum. XI. Isola-tion of Alkaloids from Thalictrum minus var. adiantifolium. Lloydia. 1969; 32:29-35.
11. Harborne JB and Baxter H. Phytochemical Dictionary. London: Taylor & Fran-cis, 1993:197.
12. Roberts MF and Wink M. Alkaloids: Biochemistry, Ecology, and MedicinalApplications. New York: Plenum, 1998:438.
13. Al-Howiriny, TA. PhD Dissertation, University of Pittsburgh: Part I–AlkaloidalConstituents of Selected Fractions of Thalictrum angustifolium L. and Thalictrumwangii Boivin (Ranunculaceae); Part II–A Revision of the Structure of Thalprze-walskiinone–The Synthesis of 2-Methyl-6,7-Dimethoxy-39-Methoxy-49-Hydroxy-oxobenzylisoquinoline and 2-Methyl-6,7-Dimethoxy-39-Hydroxy-49-Methoxyoxoben-zylisoquinoline. Pittsburgh, 2000:275 pp.
14. Chattopadhyay SK, Ray AB, Slatkin DJ, and Schiff Jr. PL. Quaternary Alka-loids of Thalictrum foliolosumi. Phytochem. 1983; 22(11):2607-2610.
SUBMITTED: 05/09/01REVISED: 06/20/01
ACCEPTED: 08/13/01
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