Abstract
Three alkaloids, cheilantifoline, mecambrine, and laudanosine, and two flavonoids, luteoline and tricine, have been isolated from two samples of Papaver macrostomum. Boiss. & Huet ex Boiss. (Papaveraceae) of Turkish origin. Antimicrobial tests have been performed on the extracts obtained from these species. It has been found that diethyl ether and acetone extracts of two samples obtained from the aerial parts of the plant have antimicrobial activity against almost all bacteria tested. The existence of flavonoids and the antimicrobial activity of this species are reported for the first time.
Introduction
In the Flora of Turkey., annual Papaver. L. (Papaveraceae) species are grouped into four sections; Mecones Bernh., Carinatae Fedde, Papaver. L., and Argemonorhoeades Fedde (Cullen, Citation1965; Güner et al., 2001). Of these sections, Carinatae is represented by only one species, Papaver macrostomum. Boiss. & Huet ex Boiss, which is widely distributed in Turkey. This species is used as an antitussive and sedative together with Papaver rhoeas. L. in the folk medicine of Turkey (Baytop, Citation1984). Previous investigations on the alkaloids of this species revealed the existence of the benzylisoquinoline (macrostomine, dehydromacrostomine, sevanine), aporphine (isocorydine), protopine (protopine), and rhoeadine (rhoeadine, papaverrubine A, B, D, E), types (Preininger, Citation1986). Recently, we reported the isolation of isopavine (amurensine, amurensinine) and protoberberine (cheilantifoline) types from Papaver macrostomum. collected from Northwestern Turkey (Sarıyar, Citation2002).
In this work, we aimed to determine the alkaloid and flavonoid contents of two samples of P. macrostomum. of Turkish origin collected from two different regions of eastern Turkey (M1 and M2) to determine the existence of chemotypes and to investigate the antimicrobial activities of the extracts prepared from the aerial parts of the plant using different solvents.
Materials and Methods
General
UV spectra were taken with a Jasco 530V spectrophotometer (UK). 1H NMR spectra were measured on a Varian Unity Inova (500 MHz) instrument (USA) at ITL, University of Istanbul. 13C NMR and Heteronuclear Multiple Quantam Correlation (HMQC) spectra were taken with a Varian Mercury-VX (400 MHz) spectrometer at Boğaziçi University. Mass spectra were obtained on a JEOL GC Mate II instrument (USA) at Research Resources Center, University of Illinois. IR spectra were run on a Perkin-Elmer 1600 Series FTIR instrument (USA).
Plant material
The aerial parts of P. macrostomum. were collected from the eastern part of Turkey in Malatya in June 2001 (M1) and in Van in June 2002 (M2). Voucher specimens are deposited in the herbarium of the Faculty of Pharmacy, Istanbul University (M1 ISTE 79426 and M2 ISTE 81463). The plant identification was verified by Prof. G. Sarıyar.
Extraction and isolation
Extraction and isolation of alkaloids
The dried and powdered material (M1, 6 kg; M2, 8.796 kg) was percolated with ethanol at room temperature. The ethanol extract was concentrated under vacuum, and the residue was taken up in 5% hydrochloric acid. The acid extract was first washed with light petroleum and then with diethyl ether. The aqueous layer was made alkaline with NH4OH to pH 7–8 and extracted successively with CHCl3. The combined CHCl3 extracts were dried over anhydrous Na2SO4, filtered, and concentrated to dryness under vacuum to yield the tertiary alkaloid extracts of M1 (2.9392 g) and M2 (6.5328 g).
The tertiary alkaloid extracts were each separated on a column of silica gel eluting with CHCl3, CHCl3:MeOH (95:5, 90:10, 80:20). Fractions were evaporated and purified by preparative thin-layer chromatography on silica gel to afford the pure alkaloids.
The M1 column fractions 67–73 eluted with CHCl3:MeOH (90:10) were purified by an aluminum oxide column and gave mecambrine (11 mg). The M2 column fractions 5–9 eluted with CHCl3 and fractions 26–30 eluted with CHCl3:MeOH (95:5) were separated by preparative TLC on silica gel with toluene: Me2CO:MeOH:NH4OH (45:45:7:3) and yielded cheilanthifoline (90 mg) and laudanosine (9 mg), respectively.
Extraction and isolation of flavonoids
The dried aerial parts of P. macrostomum. (300 g) were extracted with petroleum ether in a Soxhlet apparatus. The petroleum ether extract (A) was concentrated and extracted with 60% EtOH, which was treated with CHCl3 (B). The petroleum ether exhausted material was extracted with EtOH. The extract was concentrated, diluted with H2O, and extracted with toluene (C) and CHCl3 (D). Extract (D) was investigated for flavonoids.
Extract (D) of M1 and M2 were applied to a polyamide column (H2O and increasing concentrations of MeOH as eluent). From M1 column, fractions 63–69 eluted with H2O:MeOH (20:80) and, from M2 column, fractions 100–106 eluted with H2O:MeOH (30:70), tricine (3 mg), and luteoline (4 mg) were obtained after a Sephadex LH20 purification with MeOH.
Antimicrobial activity tests
The antimicrobial activity tests were performed on the extracts obtained from the aerial parts of P. macrostomum. (25 g) using solvents petroleum ether, diethyl ether, chloroform, acetone, and ethanol in a Soxhlet apparatus. The antibacterial and antifungal tests were carried out using the agar diffusion method followed by the dilution method for extracts that presented a bioactivity.
Extracts were tested against six strains of bacteria; Staphylococcus aureus. (ATCC 6538), S. epidermidis. (ATCC 12228), Escherichia coli. (ATCC 11229), Pseudomonas aeruginosa. (ATCC 1539), Proteus mirabilis. (ATCC 14153) and Klebsiella pneumoniae. (ATCC 4352). Muelleor-Hinton agar (MHA) and Mueller-Hinton broth (MHB) were used for the bacteria.
Extracts were tested against six fungal strains; Candida albicans. (ATCC 10231), C. glabrata. (ATCC 90030), C. guilliermondii. KUEN 998, C. tropicalis. KUEN 1021, C. pseudotropicalis (kefyr.). KUEN 1012, and C. krusei. (ATCC 6258). Sabouraud dextrose broth (SDB) and Sabouraud dextrose agar (SDA) were used for yeast. Minimum inhibitory concentration (MIC) was determined by the dilution method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (Citation1997Citation1999) (May et al., Citation1997). Results are shown in Tables and .
Table 1.. Antimicrobial activity of first sample of P. macrostomum., which was collected from Malatya.
Table 2.. Antimicrobial activity of second sample of P. macrostomum., which was collected from Van.
Microorganisms were obtained from the Department of Microbiology, Faculty of Pharmacy, Marmara University, Istanbul.
Meropenem (512 µg/mL) served as the positive control for the tested bacteria, whereas fluconazole (5120 µg/mL) served as the positive control yeast. Petroleum ether, diethyl ether, chloroform, acetone, and ethanol were tested as solvent controls.
Results and Discussion
The structures of known alkaloids and flavonoids were elucidated through spectrocopic analysis and TLC by direct comparison with authentic samples. Mecambrine was isolated from the sample (M1) collected at Malatya, whereas a sample collected at Van (M2) yielded cheilantifoline and laudanosine. The presence of mecambrine and laudanosine has been shown in P. macrostomum. for the first time. However, the amount of alkaloids in two samples has been found in very low quantity. The two samples also differ in their flavonoid contents, yielding tricine (M1) and luteoline (M2), which is the first report on the presence of these flavonoids in a Papaver. species. This study revealed that these two samples of P. macrostomum. were two new chemotypes having proaporphine and benzylisoquinoline-protoberberine types of alkaloids when compared with the findings of previous work that reported the existence of three chemotypes containing benzylisoquinoline, rhoeadine-protopine, and isopavine-protoberberine-aporphine types.
Although they differ chemically, the diethyl ether and acetone extracts prepared from the aerial parts of the two samples have similarities in their antimicrobial activity. It has been found that diethyl ether and acetone extracts of P. macrostomum. have antimicrobial activity against almost all microorganisms tested.
As a result of our investigations on the alkaloids of samples of P. macrostomum. growing in different regions of Turkey, the presence of three chemical races containing proaporphine, benzylisoquinoline-protoberberine, and isopavine-protoberberine-aporphine types has been found so far.
Acknowledgments
This work was supported by The Scientific Research Projects of Istanbul University (project no. T-1256/01112001). We express our sincere thanks to Prof. Dr. A. Douglas Kinghorn (College of Pharmacy, University of Illinois at Chicago) for mass measurements.
References
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