Abstract
In the present study, the alkaloid composition of the aerial parts of Genista vuralii A. Duran & H. Dural (Fabaceae) was investigated by capillary GC-MS. Ten quinolizidine alkaloids were identified by capillary GC-MS, namely, N-methylcytisine, cytisine, tetrahydrorhombifoline, 17-oxosparteine, 5,6-dehydrolupanine, lupanine, 17-oxolupanine, anagyrine, baptifoline, and 13α-tigloyloxylupanine. Among them, anagyrine (93.04%) was the most abundant alkaloid. Furthermore, antibacterial and antifungal activities of the alkaloid extract of G. vuralii were tested against standard strains of bacteria (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus) as well as fungi (Candida albicans, Candida krusei). The alkaloid extract of G. vuralii presented good activity against S. aureus, B. subtilis, and C. krusei, with minimum inhibitory concentrations (MIC) of 62.5 μg/mL. The remaining MIC values were found to range between 125 and 500 μg/mL. To the best of our knowledge, the current work is the first to report the alkaloid profile and antimicrobial activity of G. vuralii L. growing in Turkey.
Introduction
The genus Genista L. (Fabaceae) includes chiefly deciduous shrubs or small trees in the Mediterranean area and Western Asia. There are 14 Genista (Fabaceae) species in the flora of Turkey and the East Aegean Islands; among these species, G. aucheri, G. burdurensis, G. sandrasica, G. involucrate, and G. vuralii are endemic (CitationGibbs, 1970; CitationDavis et al., 1988; CitationDuran & Dural, 2003). G. vuralii A. Duran & H. Dural, a procumbent or ascending small shrublet, grows in the transition territory of Central and North Anatolia, Euro-Siberian element. G. vuralii grows in rocky areas, steppes, and forest clearings, flowering in June–July (CitationDuran & Dural, 2003).
Quinolizidine alkaloids (QA) are characteristic secondary metabolites of the Fabaceae family and are especially abundant in the tribes Genisteae, Sophoreae, and Thermopsideae (CitationKinghorn & Balandrin, 1984; CitationWink, 1993). A wide range of biological activities is attributed to pure QA or mixtures of QA (CitationKinghorn & Balandrin, 1984; CitationWink, 1984; CitationWippich & Wink, 1985; CitationOhmiya et al., 1995; CitationErdemoglu et al., 2007). In addition, QA play a chemical defensive role in plants against herbivores, and inhibit the growth of bacteria and pathogen microorganisms (CitationWink, 1984, Citation1988, Citation1992; CitationWippich & Wink, 1985).
In our previous studies on Genista species, we isolated alkaloids from 11 Genista species growing in Turkey, as well as flavonoids from G. aucheri and G. involucrata (Tosun et al., 1986, Citation1987a,Citationb,Citationc, Citation1988, Citation1994; CitationNasution et al., 1991; CitationTosun & Akyuz, 1998, Citation2000). In our other studies, the aerial parts of 11 Genista species were analyzed for their total and free genistein and daidzein contents by use of LC-MS (CitationTosun et al., 2003; CitationErdemoglu et al., 2006). Both the alkaloids and the flavonoids are significant chemotaxonomic markers of the genus Genista (CitationHarborne, 1994). In the present work, the alkaloid composition and antimicrobial activity of the alkaloid extract of the aerial parts of G. vuralii were investigated.
Materials and methods
Plant material
The aerial parts of G. vuralii A. Duran & H. Dural (Fabaceae) were collected at the flowering stage from çankırı, Ilgaz Mountain, Derbent facility, at an altitude of 1800 m in July 2004 in Turkey. Plant material was identified by one of us (Associate Professor Dr Ahmet Duran). Voucher specimens (A. Duran 6735) are kept at the herbarium in Selcuk University, Faculty of Education, Department of Biology, in Konya, Turkey.
Extraction of alkaloids
Alkaloid extraction was carried out as described in CitationWink (1993): 2 g plant material were homogenized in 30 mL of 0.5 N HCl. After 30 min at room temperature, the homogenate was centrifuged for 10 min at 5000g. For quantitative work, the pellet was re-suspended in 0.5 N HCl and centrifuged again. Both supernatants were then pooled and adjusted to pH 12–14 with NH4OH (25%). Alkaloids were extracted by solid-phase extraction using an Extrelut column (Merck, Darmstadt, Germany). Total alkaloids were eluted with CH2Cl2 and the solvent evaporated in vacuo.
Analysis of alkaloids
The alkaloid extract was dissolved in CH2Cl2 and injected into a GC-MS apparatus (Hewlett Packard model 6890 series) equipped with a mass selective detector. Experimental conditions for capillary GC-MS analysis were developed under the following conditions. Capillary column HP-5 (crosslinked 5% phenylmethylsiloxane, 50 m×0.32 mm (i.d.), with 0.17 μm film thickness, model no. HP 19091J-015), detector temperature 280ºC, injector temperature 250ºC, carrier gas He (1 mL/min), split ratio 1/20, injection volume 0.2 μL, and mass range (m/z) 20–440. GC oven temperature was kept at 120ºC for 2 min, programmed to 300ºC at a rate of 6ºC/min, and kept constant at 300ºC for 10 min.
Antimicrobial activity
Microorganisms
Standard strains of four bacteria, namely, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633), and Staphylococcus aureus (ATCC 25923), were used for determination of antibacterial activity, along with standard strains of Candida albicans (ATCC 10231) and Candida krusei (ATCC 14243) used for determination of antifungal activity.
Antibacterial and antifungal tests
The minimum inhibitory concentrations (MIC) of the extracts and references (ciprofloxacin and flucanozole) were determined by broth microdilution techniques according to the Clinical Laboratory Standards Institute (CitationCLSI, 1996, Citation2002). Mueller–Hinton broth (Merck) and Mueller–Hinton agar (Oxoid Ltd, Basingstoke, UK) were applied for growing and diluting of the bacteria. Sabouraud liquid medium (Oxoid Ltd) and Sabouraud dextrose agar (Oxoid Ltd) were applied for growing and diluting of the fungi. The medium RPMI-1640 (Sigma Chemical Co., St. Louis, MO, USA) with l-glutamine was buffered at pH 7 with MOPS. The extracts were dissolved in DMSO. Extract concentrations ranging from 1.000 to 3.75 μg/mL were prepared. Microorganism inoculums were standardized to a turbidity equivalent to that of a 0.5 McFarland standard (106 yeasts or 108 bacterial cells), and diluted for the broth microdilution procedure. Final concentrations were approximately 1–5×103 cells/mL for yeasts and 1–5×104 for bacteria. Microtiter plates were incubated under normal atmospheric conditions at 37°C for 24 h for bacteria and at 30°C for 48 h for yeast. The microorganisms and pure media (positive and negative controls) were placed in the wells of a microtiter plate. The MIC was defined as the lowest concentration of extracts that produced an 80% reduction in visible growth compared with control. The bacterial growth was indicated by the presence of a white ‘pellet’ on the well bottom. Each extract was tested in triplicate.
The in vitro antimicrobial results of the extracts were classified as follows: the antibacterial activity was considered as significant when the MIC was 100 μg/mL or less; moderate, when the MIC was 100–500 μg/mL; weak, when the MIC was 500–1000 μg/mL; and inactive, when the MIC was above 1000 μg/mL.
Results and discussion
In the present study, we aimed to investigate the alkaloid profile of G. vuralii by capillary GC-MS analysis and the antimicrobial activity of its alkaloid extract. Ten alkaloids were identified in the alkaloid extract of the aerial parts of G. vuralii. The structures of the alkaloids were identified based on comparison of their Kovats retention indices and mass spectral fragmentation with those of reference data in the literature (CitationWink, 1993; CitationWink et al., 1995) as well as by a library search (Wiley GC-MS library databank) and comparison with authentic alkaloids such as anagyrine and cytisine. Relative contents of % alkaloids were determined via areas under the peaks from total ion chromatography using Hewlett Packard software. The quantitative pattern of the minor alkaloids is given in . Anagyrine (93.04%) was determined as the major alkaloid in the aerial parts of G. vuralii. In addition, N-methylcytisine, cytisine, tetrahydrorhombifoline, 17-oxosparteine, 5,6-dehydrolupanine, lupanine, 17-oxolupanine, baptifoline, and 13α-tigloyloxylupanine were detected as the minor alkaloids in the plant.
Table 1. Alkaloid composition and alkaloid content of the aerial parts of Genista vuralii.
There are a number of reports on the alkaloid patterns of Genista species by capillary GC-MS (CitationMontllor et al., 1990; CitationGreinwald et al., 1992; CitationWink & Witte; 1993; CitationKirch et al., 1995; CitationPistelli et al., 2001; CitationMartins et al., 2005). In the CitationMontllor et al. (1990) study, dehydroaphyllidine and N-methylcytisine were detected as the major alkaloids in G. monspessulana, together accounting for 74% of the total alkaloids. CitationGreinwald et al. (1992) investigated the alkaloid pattern of G. cinerea subsp. ausetana, G. cinerascens, and G. majorica, and detected cytisine and anagyrine as the main alkaloids in these plants. In CitationWink and Witte’s study (1993), 12 QA were identified in G. acanthoclada, with sparteine, cytisine, retamine, and 17-oxosparteine found to be the main compounds. In another study, the alkaloid profiles of G. lobelia and G. salzmannii were studied by GC-MS, and sparteine, lupanine, 5,6-dehydrolupanine, anagyrine, cytisine, N-methylcytisine, and ammodendrine were identified as the major alkaloids (CitationKirch et al., 1995). In the study of CitationPistelli et al. (2001) on QA patterns of Genista species, anagyrine, lupanine, retamine, 17-oxoretamine, and 12α-hydroxylupanine were found in the aerial parts of G. ephedroides. CitationMartins et al. (2005) reported ten alkaloids, anagyrine, lupanine, cytisine, N-methylcytisine, and N-formylcytisine as the major components, and dehydrocytisine, 5,6-dehydrolupanine, rhombifoline, aphylline, and thermopsine in only trace amounts, in the aerial parts of G. tenera. Comparing the present results with the above-mentioned literature findings, the alkaloid compositions were similar in these species, but the relative amounts of the alkaloids showed a higher diversity. For example, while anagyrine (93.04%) was the most abundant alkaloid in the present study, sparteine (46.1%) was the main alkaloid and anagyrine (0.7%) was a minor amount in G. acanthoclada (CitationWink & Witte, 1993). Moreover, in G. tenera, anagyrine (34.5%) was the major compound (CitationMartins et al., 2005).
In our previous research on Turkish Genista species, including G. acanthoclada, G. anatolica, G. sessilifolia, G. aucheri, G. carinalis, G. involucrata, G. albida, G. tinctoria, G. burdurensis, G. lydia var. lydia, G. lydia var. antiochia, and G. libanotica, 21 QA, calycotomine (tetrahydroisoquinoleine), and N-methylamodendrine (dipiperidine) were isolated. In these studies, lupanine, anagyrine, cytisine, and N-methylcytisine were found in almost all of the above-mentioned Genista species. Besides, for the first time, genisteine, pusilline, 13-hydroxysparteine, 13-epimethoxylupanine, N-acetylcytisine, N-formylcytisine, N-methylammodendrine, calycotomine, oxymatrine, and 10α-hydroxymethylsparteine were obtained from Genista species in addition to other known QA compounds (Tosun et al., 1986, Citation1987a,b,c, Citation1988, Citation1994; CitationNasution et al., 1991). In agreement with our previous findings in Turkish Genista species, N-methylcytisine, cytisine, tetrahydrorhombifoline, 17-oxosparteine, 5,6-dehydrolupanine, lupanine, 17-oxolupanine, anagyrine, and baptifoline were found in the alkaloid profile of the aerial parts of G. vuralii in the present study. Although the esters of hydroxylupanine are not usual components encountered in Genista genus (CitationWink, 1993; CitationOhmiya et al., 1995), 13α-tigloyloxylupanine was determined for the first time from a Turkish Genista species. 13α-Tigloyloxylupanine was previously detected in G. cinerea subsp. ausetana, G. cinerascens, and G. majorica (CitationGreinwald et al., 1992).
In addition, the antibacterial and antifungal activities of G. vuralii alkaloid extract against standard strains of bacteria (E. coli, P. aeruginosa, B. subtilis, S. aureus) as well as fungi (C. albicans, C. krusei) were also investigated in the present work. Results of the antibacterial and antifungal activities are given in . The alkaloid extract of G. vuralii presented significant activity against the Gram-positive bacteria S. aureus and B. subtilis (MIC=62.5 μg/mL), moderate activity against the Gram-negative bacterium P. aeruginosa (MIC=125 μg/mL), and weak activity against the Gram-negative bacterium E. coli (MIC=500 μg/mL). In the anti-yeast assay, the extract displayed significant activity against C. krusei (MIC=62.5 μg/mL), but this extract had only moderate activity against C. albicans (MIC=250 μg/mL).
Table 2. Antimicrobial activity of Genista vuralii alkaloid extract.
A number of biological activities of QA such as hypoglycemic activity, inhibition of edema, nicotine-like activity, inhibition of β-glucuronidase, inhibition of acetylcholinesterase, nematocidal, and antimicrobial activities have been reported (CitationWink, 1984; CitationWippich & Wink, 1985; CitationTyski et al., 1988; CitationMatsuda et al., 1989; CitationOhmiya et al., 1995; CitationErdemoglu et al., 2007). In addition, QA have a deterrent activity by way of repel feeding of herbivores, or a directly toxic or mutagenic effect (CitationWink, 1988, Citation1992). In CitationWink’s study (1984), sparteine was reported to possess antimicrobial activity against bacteria and phytopathogenic fungi. In addition, CitationWippich and Wink (1985) reported that sparteine, lupanine, and 13-tigloyloxylupanine inhibited the germination of conidia Erysiphe graminis f. sp. hordei. CitationTyski et al. (1988) reported that pure QA, namely, lupanine, 13α-hydroxylupanine, angustifoline, and sparteine, showed bacteriostatic effects on S. aureus, B. subtilis, E. coli, P. aeruginosa, and Bacillus thuringiensis. In addition, various biological effects of anagyrine such as nematocidal and acetylcholinesterase inhibitory activity as well as teratogenic effect have been reported in the literature (CitationMatsuda et al., 1989; CitationOhmiya et al., 1995). Our antimicrobial results support the idea that QA may be involved in the antimicrobial defense system of plants (CitationWink, 1984; CitationWippich & Wink, 1985). Also, our GC-MS analysis demonstrated that the alkaloid extract of G. vuralii contained anagyrine as the main alkaloid. Although no antimicrobial activity for anagyrine has been reported to date, anagyrine might be responsible for the antimicrobial activity of the alkaloid extract of G. vuralii which has been found herein to have significant antimicrobial activity against S. aureus, B. subtilis, and C. krusei.
Acknowledgment
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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