PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 10, 6–11, (1999)
Alkaloids and Organic Acids Content of Eight Fumaria Species J. Sousˇek,1* D. Gue´don,2 T. Adam,3 H. Bochorˇa´kova´,4 E. Ta´borska´,4 I. Va´lka5 and V. Sˇima´nek5 1
Laboratory of Bioanalytical Research, Palacky´ University, Hneˇvotı´nska´ 3, 775 15 Olomouc, Czech Republic Arkopharma, Laboratoires Pharmaceutiques, B.P. 28, 06511, Carros Cedex, France Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, Medical Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic 4 Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, Masaryk University, Komenske´ho na´m. 2, 662 43 Brno, Czech Republic 5 Institute of Medical Chemistry, Faculty of Medicine, Palacky´ University, Hneˇvotı´nska´ 3, 775 15 Olomouc, Czech Republic 2 3
The isoquinoline alkaloids adlumiceine, adlumidiceine, coptisine, corytuberine, cryptopine, fumaricine, fumariline, fumaritine, fumarophycine, O-methylfumarophycine, palmatine, parfumine, protopine, sinactine, stylopine, and N-methylstylopine were determined by reversed phase high performance liquid chromatography in Fumaria agraria, F. capreolata, F. densiflora, F. muralis, F. officinalis, F. parviflora, and F. vaillantii. Organic acids, namely citric, coumaric, ferulic, fumaric, malic, 3-hydroxybenzoic, protocatechuic acid and caffeic acid (and its methylester) were identified by gas chromatography—mass spectrometry. The total content of phenolic constituents was quantified by a colorimetric method using a phosphomolybdic–phosphotungstic reagent. The chemotaxonomic significance of the results is discussed. # 1999 John Wiley & Sons, Ltd. Keywords: Fumaria; isoquinoline alkaloids; organic acids; total phenols; chemotaxonomy; reversed-phase high performance liquid chromatography; gas chromatography–mass spectrometry.
INTRODUCTION The number of taxons of the genus Fumaria (Fumariaceae) ranges from 40 (Hegi, 1931) to 215 (Jackson, 1893). These differences are probably due to the use of several synonyms for plants having very close morphological properties. The identification of Fumaria plants is thus subjective. The results of chemotaxonomic investigations could be valuable for the systematic evaluation of this genus (Sousˇek and Vales-ova´, 1995), the species of which have been used for many centuries in folk medicine. Today, Fumaria extracts are components of several phytotherapeutic preparations, which are used mostly in cases of minor hepatobiliary disfunction, gastrointestinal diseases and skin disorders (Martindale, 1996). The biological activity of Fumaria is mostly associated with the presence of isoquinoline alkaloids and several techniques have been used for their determination in plant extracts, including thin layer chromatography (TLC) and high performance TLC (Freytag, 1980), gas chromatography (Yoshikawa and Furuya, 1985), enzyme immunoassay, radioimmunoassay (Yoshikawa and Fur-
* Correspondence to: Dr. J. Sousˇek, Laboratory of Bioanalytical Research, Palacky´ University, Hneˇvotı´nska´ 3, 775 15 Olomouc, Czech Republic. Email:
[email protected]. Contract/grant sponsor: Ministry of Education, Youth and Sport; Contract/ grant number: VS 96 021. Contract/grant sponsor: Ministry of Health of the Czech Republic; Contract/ grant number: 2709-3.
CCC 0958–0344/99/010006–06 $17.50 # 1999 John Wiley & Sons, Ltd.
uya, 1985; Wieczorek et al., 1986), capillary isotachophoresis (Va´lka and Sˇima´nek, 1988) and high performance liquid chromatography (HPLC) (Evidente et al., 1983; Stahl and Schild, 1985; Va´lka and Sˇima´nek, 1988). Only a limited number of alkaloids can be determined using most of the methods published to date, and the identification of minor alkaloids has remained a problem. Furthermore, non-alkaloid components have only been studied in Fumaria officinalis (Torck et al., 1971; Hahn and Nahrstedt, 1993) and in F. capreolata (Boegge et al., 1995). This paper describes a reversed-phase (RP)-HPLC method for the determination of isoquinoline alkaloids and a GC–mass spectrometric (MS) method for the identification of organic acids in eight species of Fumaria.
EXPERIMENTAL Materials. HPLC grade acetonitrile was provided by Riedel de Haen Seelze (Hannover, Germany) and triethylamine by Fluka (Buchs, Switzerland). Reference standards of alkaloids were obtained from collections of the Institute of Medical Chemistry (Olomouc, Czech Republic) and the Institute of Medical Chemistry and Biochemistry (Brno, Czech Republic). The purities and identities of the alkaloid standards were determined by comparison of their melting points and infrared spectra with those reported in the literature (Southon and Buckingham, 1989). Organic acids were purchased from Received 4 June 1997 Revised 31 October 1997 Accepted 3 November 1997
# 1999 John Wiley & Sons, Ltd.
Table 1. The alkaloid composition of eight species of Fumaria determined by RP-HPLC Alkaloid (tR - min)
Adlumiceine (11.0) Adlumidiceine (8.1) Coptisine (18.7)
Cryptopine (11.5) Fumaricine (7.2) Fumariline (13.8) Fumaritine (3.9) Fumarophycine (9.2) O-Methylfumarophycine (15.0)
Parfumine (6.0) Protopine (12.8) Sinactine (12.4)
3.7 0.2 5.1 0.3 4.8 0.2 nf nf nf 12.6 0.5 nf nf 4.0 0.2 0.6 0.1 2.6 0.1 7.2 0.3 4.8 0.2 4.7 0.2 nf nf nf 10.7 0.4 12.7 0.5 4.8 0.2 11.2 0.5 5.2 0.2 2.6 0.1 4.2 0.2 3.9 0.2 6.4 0.3 nf nf nf nf nf nf 11.7 0.5 12.1 0.5 12.1 0.5 35.7 1.2 34.2 1.2 23.9 0.9 nf nf nf
F. capreolata
6.2 0.3 6.3 0.3 6.9 0.3 11.5 0.5 9.9 0.4 nf nf nf nf nf nf nf 1.8 0.1 nf 3.1 0.2 1.6 0.1 2.0 0.1 2.6 0.1 10.5 0.4 13.6 0.5 13.2 0.5 4.1 0.2 4.5 0.2 5.0 0.2 5.3 0.2 2.2 0.1 5.6 0.2 nf nf nf nf nf nf 11.9 0.5 12.8 0.5 13.0 0.5 23.4 1.0 29.7 1.1 30.4 1.1 nf nf nf
F. densi¯ora
nfb Ð 2.0 0.1 nf Ð nf nf Ð nf nf Ð nf 1.1 0.1 Ð 3.1 0.2 0.8 0.1 Ð 3.0 0.2 11.5 0.4 Ð 11.6 0.4 7.7 0.3 Ð 7.6 0.3 8.0 0.3 Ð 8.1 0.3 23.8 1.1 Ð 23.7 1.1 nf Ð nf 8.2 0.3 Ð 10.3 0.4 30.3 1.1 Ð 25.0 1.0 0.2 Ð 0.2
F. muralis
nfc nfc 1.0 0.1c nfc 2.5 0.1c nfc 8.2 0.3c 2.8 0.1c 21.8 1.0c 23.1 1.1c nfc 5.6 0.2c 27.5 1.1c 2.1 0.1c
F. of®cinalis
F. parvi¯ora
1.9 0.1 nf nf nf nf nf 0.7 0.1 0.9 0.1 0.3 1.0 0.1 0.6 0.1 2.6 0.1 2.8 0.1 4.5 0.2 7.3 0.3 3.0 0.1 0.3 nf 2.0 0.1 3.8 0.2 3.9 0.2 2.7 0.1 3.3 0.2 3.4 0.2 19.3 1.0 37.4 1.3 33.2 1.2 29.8 1.2 17.1 0.8 22.2 1.1 nf nf nf 4.0 0.2 5.4 0.2 4.5 0.2 22.7 1.1 18.0 1.0 17.4 1.0 6.6 0.2 7.7 0.3 6.2 0.2
2.8 0.1 3.2 0.2 2.1 0.1 10.7 0.4 8.9 0.4 9.1 0.4 17.3 0.7 14.6 0.6 9.3 0.5 nf nf nf 4.8 0.2 7.2 0.3 5.3 0.2 nf nf nf 6.6 0.3 7.0 0.3 3.8 0.1 2.6 0.1 2.0 0.1 3.6 0.2 11.5 0.5 18.5 0.8 24.1 1.0 6.2 0.3 8.9 0.4 10.4 0.4 0.7 0.1 0.5 0.3 10.5 0.4 7.6 0.3 6.6 0.3 21.4 1.1 17.2 0.9 15.4 0.8 nf nf 3.4 0.1
F. spicata
F. vaillantii
2.8 0.1c
0.7c
nfc
0.3c
0.4c
0.6c
nfc
nfc
nfc
8.8 0.3c
nfc
1.7 0.1c
81.1 3.2c
3.0 0.1c
nfc
2.4 0.1c
13.3 0.6c
36.5 1.5c
nfc
15.6 0.7c
nfc
nfc
nfc
5.1 0.2c
1.1 0.1c
21.1 1.1c
nfc
0.4c
7
Phytochem. Anal. 10: 6–11 (1999)
Palmatine (21.1)
F. agraria
March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991
ALKALOIDS AND ACIDS OF FUMARIA SPP
Corytuberine (4.5)
Percentage alkaloid composition ( standard deviation)a
Collection date of plant spp.
J. SOUSˇEK ET AL.
Plant material. The aerial parts of Fumaria species were collected in France (dates of collection are given in Table 1). F. agraria, F. capreolata, F. densiflora, F. officinalis and F. parviflora originated from five sites in the Alpes– Maritimes; F. muralis, F. spicata, and F. vaillantii originated from sites in Arde´che, Puy-de-Dome and Pyre´ne´es-Orientales. Each sample was a mixture of material from several plants. The botanical identification was made by the coauthor (D.G.) and Dr. J.-L. Lamaison (Laboratoire de Pharmacognosie et Phytophe´rapie, Clermont-Ferrand, France). Voucher specimens are deposited with the Institute of Medical Chemistry, Faculty of Medicine, Palacky´ University in Olomouc, Czech Republic.
c
Individual alkaloids are expressed as a percentage of the total alkaloid content (sample standard deviation n = 3). nf = none found. Collected in June 1991. b
Total Alkaloids (mg/100g dry weight)
N-methylstylopine (16.5)
# 1999 John Wiley & Sons, Ltd.
a
2100.7 57.0c
1.2 0.1c
F. vaillantii F. spicata F. parvi¯ora
Aldrich (Prague, Czech Republic) and ICN Biomedicals (Basingstoke, UK).
1.8 0.1 nf nf 2.1 0.1 1.7 0.1 1.6 0.1 1386.1 41.4 740.2 24.6 624.4 22.7
F. of®cinalis F. muralis
5.5 0.2 Ð 2.9 0.1 2.9 0.1 Ð 2.5 0.1 479.3 16.0 Ð 563.5 18.7
4.2 0.1c
F. densi¯ora
nf nf nf 23.7 1.0 19.0 0.8 10.3 0.4 1009.2 31.3 652.7 23.1 732.7 24.0 nf nf nf 9.0 0.4 21.4 0.9 23.3 0.9 503.3 17.1 464.9 16.1 536.0 18.7
F. capreolata F. agraria
March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 March 1991 April 1991 May 1991 Stylopine (15.5)
Alkaloid (tR - min)
Collection date of plant spp.
Percentage alkaloid composition ( standard deviation)a
Table 1. Continued
2.3 0.1 nfc nfc 2.4 0.1 4.8 0.2 2.2 0.1 1.3 0.1c 1.4 0.1c 2.0 0.1 1.7 0.1 724.9 23.1 3666.0 76.2c 1744.4 52.3c 904.2 27.2 438.4 15.5
8
Extraction of alkaloids and organic acids. Ground plant material (5.0 g) was extracted (Soxhlet) with methanol for 8 h and then evaporated in vacuo. The residue was dissolved in 0.02 M HCl (100 mL). The lipophilic constituents and chlorophyll were removed with petroleum ether, the aqueous layer was filtered through Whatman grade 3 paper and the alkaloids were precipitated with 5 mL of Mayer reagent (4.9% KI and 1.35% HgCl2 in water). The precipitate was collected by filtration, washed with water, and dissolved in 20 mL of acetone:methanol:water (6:2:1, v/v/v). The solution was allowed to pass through an Amberlite IRA 400 or IRA 401 column to convert the salts into chlorides. The eluate was evaporated in vacuo and the alkaloid fraction was analyzed using HPLC. For the GC-MS analyses, ground plant material (1.0 g) was extracted with methanol and evaporated in vacuo. The residue was dissolved in 10 mL of 0.02 M HCl and was extracted with petroleum ether. The aqueous phase was then extracted five times with 20 mL of diethyl ether, evaporated and the extract was used for the GC-MS analysis (see below). RP-HPLC analysis. An Hypersil (Runcorn, UK) ODS C18 column (250 4.6 mm i.d.; 5 mm), a Spectra-Physics (San Jose, CA, USA) liquid chromatograph (model SP 8700 pump and SpectraFocus UV fast scanning detector) equipped with a Rheodyne 7125 injection valve (10 mL sample loop) were employed. Solvents for the preparation of the mobile phases were: I 10.1 mL of triethylamine added to 1 L redistilled water, adjusted with H3PO4 to pH 2.5; and II acetonitrile. The mobile phases used were: A 80% of I and 20% of II (v/v); and B 40% of I and 60% of II (v/v). The mobile phase program was: 0–2 min 100% A, 2–15 min 77% A and 23% B, 15–30 min 60% A and 40% B. For analyses, the alkaloid standards and the alkaloid fractions were precisely weighed (in the range of 0.01–0.03 mg for the alkaloids, and 2.0 mg for the alkaloid fractions), dissolved in 1 mL of mobile phase A, and then a 10 mL aliquot of this solution was injected into the HPLC system. All measurements were performed at room temperature and the mobile phase flowrate was 1.2 mL/min. Each determination was repeated three times. GC-MS analysis. GC-MS analyses were carried out using a Trio 1000 system (Fisons Instruments, Danvers, MA, USA). Samples of extracts prepared as described above (1 mg) were derivatized using 100 mL of bis(triPhytochem. Anal. 10: 6–11 (1999)
ALKALOIDS AND ACIDS OF FUMARIA SPP
9
Figure 1. Reversed-phase HPLC chromatogram (UV detection at 280 nm) of a standard mixture of the alkaloids which have been identi®ed in species of Fumaria. (For chromatographic protocol see Experimental section.)
methylsilyl) trifluoroacetamide:trimethylchlorosilane: pyridine (1:0.04:1 v/v/v). GC-MS analyses were performed using a DB5MS column (30 m 0.25 mm i.d.; film thickness 0.25 mm; J&W Scientific, Folsom, CA, USA). The temperature of the column was increased from 150°C to 300°C at 12°C/min and then held at 300°C for 10 min. The ion source of the ms was operated at 250°C. Electron impact ionisation was carried out with an electron energy of 70 eV. Samples of 0.3–0.5 mL were introduced using an on-column injector. The identities of the compounds studied were confirmed using MS of standards, retention times and a MS Library (NIST). Quantification of total phenolic constituents. Photometric measurements were performed on a Shimadzu 1601 spectrophotometer (Kyoto, Japan). Total phenolics were determined using Folin–Ciocalteu reagent and gallic acid as a standard (Singleton and Rossi, 1965). Plant material (0.2 g) was extracted for 30 min with 10 mL of hot methanol. The cooled mixture was filtered through Whatman grade 3 paper into a 500 mL volumetric flask, and the remaining material was rinsed with the amount of water necessary to bring the extract to volume. An aliquot (2 mL) of the diluted sample and 10 mL of an aqueous dilution (1:10) of Folin–Ciocalteu reagent were mixed, and after 5 min, 8 mL of a solution containing Na2CO3 (75 g/L) were added and the whole mixed again. After 2 h the absorbance at 765 nm was # 1999 John Wiley & Sons, Ltd.
measured: the total phenolic content was calculated in terms of gallic acid equivalents by comparison with a standard curve.
RESULTS AND DISCUSSION The eight species of the genus Fumaria were analysed for their alkaloid and organic acid profiles. The amount of individual alkaloids as determined by RP-HPLC are reported in Table 1. The acetonitrile: triethylammonium phosphate gradient provided good resolution of both tertiary and quaternary alkaloids. The peaks of all of the alkaloids were resolved and only sinactine and protopine were not baseline separated (Fig. 1). The 16 alkaloids were identified in assayed extracts by means of their retention times (tR) with those of standards and by using a SpectraFocus UV fast scanning detector. As expected, the type of substituents had an influence on the tR of the alkaloids: the tR value decreased as a function of the hydroxyl group substitution. A difference in tR was also observed for quaternary alkaloids in comparison to tertiary bases: the former had larger tR value (Table 1). The elution order of the quaternary alkaloids was probably due to their higher hydrophobicity than the parent tertiary bases (Ulrichova´ et al., 1983). Among the main alkaloids (Table 1), protopine, OPhytochem. Anal. 10: 6–11 (1999)
J. SOUSˇEK ET AL.
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Table 2. Alkaloids found for the first time in the various Fumaria species investigated Species
F. agraria F. capreolata F. muralis
F. of®cinalis F. parvi¯ora F. spicataa F. vaillantii a
Alkaloids
Adlumiceine, coptisine, cryptopine, fumariline, fumaritine, fumarophycine, parfumine, N-methylstylopine Adlumiceine, coptisine, cryptopine, fumariline, fumaritine, fumarophycine, parfumine, N-methylstylopine Coptisine, cryptopine, fumariline, fumaritine, fumarophycine, O-methylfumarophycine, parfumine, sinactine, stylopine, N-methylstylopine Adlumiceine, adlumidiceine, corytuberine, parfumine, N-methylstylopine Adlumiceine, coptisine, fumaritine, sinactine, N-methylstylopine Adlumiceine, coptisine, fumariline, fumarophycine, protopine, N-methylstylopine Fumaricine, fumarophycine, O-methylfumarophycine
This species has not been previously investigated.
methylfumarophycine and fumarophycine accounted for about 60–70% of the total alkaloid content in F. officinalis and F. muralis. In F. agraria, protopine, Nmethylstylopine, fumariline and parfumine were the major alkaloids (65–80% of the total alkaloid content); O-methylfumarophycine was not detected in this plant. The composition of the major alkaloids in F. parviflora was similar to that of F. officinalis but differed in the higher content of adlumidiceine found in the former (4– 12% of the total alkaloids in F. parviflora; trace amounts in F. officinalis; not detected in F. agraria). In F. capreolata, protopine, fumarophycine, N-methylstylopine, parfumine and adlumidiceine accounted for 78– 90% of the total alkaloids; O-methylfumarophycine was not found. In F. densiflora, protopine and O-methylfumarophycine made up about 55% of the total alkaloids: adlumidiceine was not detected. The major alkaloids of F. vaillantii analysed were protopine and O-methyl-
fumarophycine. F. spicata differed from all of the other plants studied because only six alkaloids were found (fumariline and fumarophycine as major alkaloids making up 94% of total alkaloid content, and adlumiceine, protopine and coptisine as minor alkaloids). F. spicata was the only species studied that contained protopine as a minor alkaloid. Many of the Fumaria examined had only been studied superficially in the past. The present phytochemical research of eight Fumaria species adds information about alkaloid composition: in all of the species studied, various alkaloids were identified and quantified for the first time and they are listed in Table 2. Currently, there is substantial interest in the nonalkaloidal constituents of Fumaria, particularly phenolic compounds, owing to their various biological effects. However, these types of compounds have only been studied rarely in Fumaria plants and hence the application of GC-MS allowed us to identify organic and phenolic acids not investigated before. Table 3 shows the organic and phenolic acids identified in all of the plants studied. It can be seen that the GC-MS analyses of the diethyl ether extracts of Fumaria showed components widely distributed in the plant kingdom. Glycosides and flavonoids, described previously in F. officinalis, were not detected in this study because the diethyl ether extraction procedure is not suitable for their isolation. In order to quantify phenolic constituents in the investigated plants, a photometric method using Folin–Ciocalteu reagent and gallic acid as standard was applied. Again, F. spicata differed from all of the other plants studied in having the lowest content of phenolics. The content of phenolics in F. muralis was, on the contrary, approximately two times greater than in other Fumaria plants. Acknowledgements We are grateful to Dr. Jean-Louis Lamaison (Laboratoire de Pharmacognosie et Phytothe´rapie, Clermont-Ferrand, France) for assistance with collections of Fumaria plants. We thank the Ministry of Education, Youth and Sport (grant No. VS96 021) and the Internal Grant Agency (Ministry of Health of the Czech Republic: grant No. 2709-3) for financial support.
Table 3. Distribution of organic acids among the eight species of Fumaria investigated Organic acids
F. agraria
F. capreolata
F. densi¯ora
F. muralis
F. of®cinalis
F. parvi¯ora
F. spicata
F. vaillantii
Caffeic acid a Methyl ester of Caffeic acid ÿb ÿ ÿ c ÿ ÿ Citric acid ÿ ÿ ÿ ÿ ÿ ÿ ÿ Coumaric acid ÿ ÿ ÿ Ferulic acid ÿ ÿ ÿ ÿ Fumaric acid Malic acid 3-Hydroxybenzoic acid ÿ ÿ ÿ ÿ ÿ ÿ ÿ Protocatechuic acid ÿ ÿ ÿ Total phenolics 2095 34d 2533 42d 2425 41d 5171 68g 2938 42d 2037 27d 1672 19g 2041 24g (mg/100 g of dry weight) 1884 32e 2143 29e 2539 44f 2820 38e 2275 38e f f 1962 37 2102 34 2616 40f 2180 36f a
Component present. ÿ Component absent. c Component not found in all plant specimens. d Collected March 1991. e Collected April 1991. f Collected May 1991. g Collected June 1991. b
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