Antiviral and Antimicrobial Profiles of Selected Isoquinoline Alkaloids from Fumaria and Corydalis Species Ilkay Orhana, Berrin Özc¸elikb, Taner Karaog˘luc, and Bilge S¸ enera,* a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey. E-mail: [email protected] b Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey c Department of Virology, Faculty of Veterinary, Ankara University, 06110 Ankara, Turkey * Author for correspondence and reprint requests Z. Naturforsch. 62c,19Ð26 (2007); received July 28, 2006 In the current study, 33 isoquinoline alkaloids belonging to protopine-, benzylisoquinoline-, benzophenanthridine-, spirobenzylisoquinoline-, phthalideisoquinoline-, aporphine-, proto- berberine-, cularine-, and isoquinolone-types as well as 7 derivatives of them obtained from some Fumaria and Corydalis species growing in Turkey have been evaluated for their in vitro antiviral and antimicrobial activities. Both DNA virus Herpes simplex (HSV) and RNA virus Parainfluenza (PI-3) were employed for antiviral assessment of the compounds using Mad- ine-Darby bovine kidney and Vero cell lines and their maximum non-toxic concentrations (MNTC) and cytopathogenic effects (CPE) were determined using acyclovir and oseltamivir as the references. Antibacterial and antifungal activities of the alkaloids were tested against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acineto- bacter baumannii, Staphylococcus aureus, Bacillus subtilis, and Candida albicans by the mi- crodilution method and compared to ampicilline, ofloxacine, and ketocanazole as the referen- ces. The alkaloids did not present any notable antibacterial effect, while they had significant antifungal activity at 8 μg/ml concentration. On the other hand, the alkaloids were found to have selective inhibition against the PI-3 virus ranging between 0.5 and 64 μg/ml as minimum and maximum CPE inhibitory concentrations, whereas they were completely inactive to- wards HSV. Key words: Isoquinoline Alkaloids, Antiviral Activity, Antimicrobial Activity Introduction Fumaria L. and Corydalis Medik., which are known to produce isoquinoline alkaloids derived Most of the world’s cultures have centuries of from phenylalanine and tyrosine (Davis and Cul- tradition in the use of crude plant materials to con- len, 1984). trol infectious diseases. Many studies have been Our focus in the present work was to investigate conducted on antiviral and antimicrobial activities the antiviral activity of 33 isoquinoline alkaloids of such materials, although relatively few studies and 7 derivatives of them, which are classified as have been done on pure chemicals. The subject of biological evaluation of compounds from plant protopine-type [protopine (1) and -allocrypto- species is highly relevant to the identification of pine (2)], benzylisoquinoline-type [(+)-reticuline lead candidates for drugs. (3) and (+)-norjuziphine (4)], benzophenanthri- Alkaloids are bioactive secondary metabolites dine-type [sanguinarine (5), norsanguinarine (6), widely found in nature. A number of isoquino- and chelidimerine (7)], spirobenzylisoquinoline- lines, a large and well-known class of alkaloids, type [fumarophycine (8), (Ð)-fumarophycine ace- was isolated previously in a project which had tate (9), (Ð)-corpaine (10), (ð)-sibiricine (11), si- been initiated to search for alkaloids of the Fuma- biricine acetate (12), (ð)-dihydrosibiricine (13), riaceae plant species growing in Turkey (Blasko (+)-fumariline (14), (Ð)-dihydrofumariline (15), et al., 1981, 1982; S¸ ener et al., 1983; S¸ ener, 1984, (+)-parfumine (16), parfumine acetate (17), and 1985, 1986, 1988, 1989; S¸ ener and Temizer, 1990; (Ð)-dihydroparfumine diacetate (18)], phthalide- Küc¸ükboyaci et al., 1998). This family is repre- isoquinoline-type [α-hydrastine (19), (+)-bicucul- sented by 2 genera in the flora of Turkey, namely line (20), (Ð)-bicuculline (21), and (Ð)-adlumidine 0939Ð5075/2007/0100Ð0019 $ 06.00 ” 2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 20 I. Orhan et al. · Isoquinoline Alkaloids from Fumaria and Corydalis (22)], aporphine-type [(+)-bulbocapnine (23) and (RSKK 574), and Acinetobacter baumannii (+)-isoboldine (24)], protoberberine-type [berber- (RSKK 02026; Culture Collection of Refik Say- ine (25), (Ð)-stylopine (26), (Ð)-canadine (27), dam Central Hygiene Institute, Ankara, Turkey), (Ð)-sinactine (28), (Ð)-ophiocarpine (29), ophio- for determination of antibacterial activity and carpine-N-oxide (30), corydalmine (31), palmatine standard strain of the yeast-like fungus Candida (32), (ð)-corydalidzine (33), dehydrocorydaline albicans (ATCC 10231) for evaluation of the anti- (34), and dehydrocavidine (35)], cularine-type fungal activity were employed. In order to deter- [(+)-cularicine (36), oxocularine (37), oxosarco- mine the antiviral activity, Herpes simplex virus capnine (38), and oxosarcocapnidine (39)], and (HSV) and Parainfluenza-3 virus (PI-3) obtained isoquinolone-type [corydaldine (40)], against Her- from the Department of Virology, Faculty of Vet- pes simplex virus (HSV) and Parainfluenza-3 virus erinary, Ankara University (Turkey) were em- (PI-3) using Madine-Darby bovine kidney ployed. (MDBK) and Vero cell lines. Moreover, the alka- loids were also tested against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Kleb- Antibacterial and antifungal activities siella pneumoniae, Acinetobacter baumannii, The isoquinoline alkaloids tested (1Ð40) were Staphylococcus aureus, and Bacillus subtilis,as dissolved in ethanol/hexane (1:1, v/v) by using 1% well as the fungus Candida albicans by the micro- Tween 80 solution at a final concentration of dilution method for their antibacterial and anti- 1024 μg/ml, sterilized by filtration using a 0.22 μm fungal activities. Millipore filter (MA, USA), and used as the stock solutions. Standard antibacterial powders of ampi- Materials and Methods cilline (AMP; Fako Pharmaceutical Company, Is- tanbul, Turkey) and ofloxacine (OFX; Hoechst Tested alkaloids Marion Roussel, Istanbul, Turkey) along with standard antifungal powders of ketoconazole The extraction, isolation, and purification proce- (KET; Bilim Pharmaceutical Company, Istanbul, dures were beforehand described in our previous Turkey) were obtained from the respective manu- papers (Blasko et al., 1981, 1982; S¸ ener et al., 1983; facturers and dissolved in phosphate buffer solu- S¸ ener, 1984, 1985, 1986, 1988; Küc¸ükboyaci et al., tion (AMP, pH 8.0, 0.1 m), dimethylsulphoxide 1998). The isoquinoline alkaloids (1Ð40) tested (DMSO) (KET), and water (OFX). The stock were obtained from the respective species among solutions of the agents were prepared in medium fourteen Fumaria [F. asepala Boiss., F. bastardii, F. according to the NCCLS recommendations capreolata L., F. cilicica Hausskn., F. densiflora (NCCLS, 1996). DC, F. gaillardotii Boiss., F. judaica Boiss., F. kra- The microdilution method was employed for likii Jordan, F. macrocarpa Parlatore, F. micro- antibacterial and antifungal activity tests. Media carpa Boiss., F. officinalis L., F. parviflora Lam., were placed into each well of the microplate. F. petteri Reichb. ssp. thuretii (Boiss.) Pugsley, F. Compound solutions at 1024 μg/ml were added vaillantii] and six Corydalis species [C. caucasica into the first raw of the microplates and 2-fold di- DC, C. rutifolia (Sibth and Sm.) DC ssp. erdelii lutions of the compounds (512Ð0.25 μg/ml) were (Zucc.) Cullen and Davis, C. rutifolia (Sibth and made by dispensing the solutions to the remaining Sm.) DC ssp. kurdica Cullen and Davis, C. solida wells. 10 μl of culture suspensions were inoculated (L.) Swartz ssp. brachyloba (Boiss.) Cullen and into all the wells. The sealed microplates were in- Davis, C. solida (L.) Swartz ssp. solida, and C. sol- cubated at 35 ∞C for 24 h and 48 h in a humid ida ssp. Tauricola] growing in Turkey. chamber. The lowest concentrations of the com- pounds that completely inhibit macroscopic Microorganisms growth and minimum inhibitory concentrations (MICs) were determined (NCCLS, 2002; Özc¸elik Standard strains of bacteria, namely Escherichia et al., 2005). coli (ATCC 35218), Pseudomonas aeruginosa Mueller-Hinton Broth (Difco) and Mueller-Hin- (ATCC 10145), Proteus mirabilis (ATCC 7002), ton Agar (Oxoid) were applied for growing and Staphylococcus aureus (ATCC 25923), Bacillus diluting the bacteria. As for growing and diluting subtilis (ATCC 6633), Klebsiella pneumoniae of the fungus, Sabouraud liquid medium (Oxoid) I. Orhan et al. · Isoquinoline Alkaloids from Fumaria and Corydalis 21 and Sabouraud dextrose agar (SDA) (Oxoid) were Results and Discussion applied. The medium RPMI-1640 with l-gluta- The results of the antibacterial and antifungal mine was buffered to pH 7 with 3-(N-morpho- activities evaluation of the tested alkaloids are lino)-propanesulfonic acid (MOPS). Prior to the presented in Table I. For determining the antibac- tests, strains of bacteria and fungus were cultured terial effect, the alkaloids were tested against five on media and passaged at least twice to ensure Gram-negative bacteria (E. coli, P. aeruginosa, P. purity and viability at 35 ∞C for 24 to 48 h. Culture mirabilis, K. pneumoniae, and A. baumannii) and suspensions were prepared according to NCCLS two Gram-positive bacteria (S. aureus and B. sub- M27-A (Özc¸elik et al., 2004). The bacterial suspen- 5 tilis) using AMP and OFX as the reference com- sions