Natural Aronia melanocarpa fruit juice (AMFJ) is rich in polyphenols most of which are anthocyanins. These compounds and other natural plant products are recently evaluated for their anti-infectious activity. The aim of the present study was to investigate the antibacterial and antiviral activity of AMFJ. AMFJ bacteriostatic activity on referent strains of S. aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 was assessed by counting the number of colonies after 24h incubation at 37°C using the microbial number method. AMFJ was not bacteriostatic for P. aeruginosa. The microbial number for E. coli was 0 ,3 6xl02 CFU/ml AMFJ and for N. aureus 0,87x10s CFU/ml AMFJ. Gram-positive bacteria should mainly be used as a test for further investigation of the bacteriostatic activity of AMFJ. AMFJ antiviral activity directed to the reproduction in ovo of influenza virus type A (H3N2) was investigated. AMFJ inhibited the reproduction of influenza virus in its initial stages, most probably, due to the formation of complex compounds between the virion, on one hand, and the polyphenols, on the other hand, which influenced the adsorption of the influenza virus on the cell surface

Belcheva, A.

Bojkova, K.

Russev, V.

Valcheva-Kuzmanova, S.

English

Varna Medical University Press: Journals

Scripta Scientifica Medica, vol. 35 (2002), pp 21-23 Copyright © Medical University, VarnaSTUDY OF NATURAL ARONIA MELANOCARPA FRUIT JUICE FOR ANTIBACTERIAL AND ANTIVIRAL ACTIVITYS. Valcheva-Kuzmanova, V. Russev1, K. Bojkova1, A. BelchevaDepartment o f  Preclinicai and Clinical Pharmacology and Biochemistry, 1Department o f  Microbiology> and Virology, P rof Paraskev Soyanov Medical University o f  VarnaA B ST R A C TNatural Aronia melanocarpa fruit juice (AM FJ) is rich in polyphenols m ost o f which are anthocyanins. These compounds and other natural plant products are recently evaluated for their anti-infectious activity. The aim  of the present study was to investigate the antibacterial and antiviral activity o f AM FJ. AM FJ bacteriostatic activity on referent strains o f S. aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 was assessed by counting the number o f colonies after 24h incubation at 37°C using the m icrobial num ber method. AM FJ was not bacteriostatic for P. aeruginosa. The m icrobial num ber for E. coli was 0 ,3 6x l02 CFU/m l AM FJ and for N. aureus 0,87x10s CFU/m l AM FJ. Gram-positive bacteria should mainly be used as a test for further investigation o f the bacteriostatic activity o f  AM FJ. AM FJ antiviral activity directed to the reproduction in ovo o f influenza virus type A (H3N2) was investigated. AM FJ inhibited the reproduction o f influenza virus in its initial stages, most probably, due to the formation o f com plex com pounds between the virion, on one hand, and the polyphenols, on the other hand, which influenced the adsorption o f the influenza virus on the cell sur­face.K ey words: A ronia m elanocarpa, Staphylococcus aureus, E scherichia coli, Pseudom onas aeruginosa, influenza virusThe flavonoids have long been recognized to possess antiinflammatory, antioxidative, antiallergic, antiviral, hepatoprotective, antithrombotic and anticancerogenic ac­tivities (8).Since the flavonoids are known to be synthesized in plants in response to microbial infection it should not be surprising that they have been found in vitro to be effective antimicrobial substances against a wide variety o f microor­ganisms (4). Such microorganisms are Staphylococcus aureus, Corynebacterium diphtheriae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Neisseria meningitides, Vibrio cholerae 01, Streptococcus mutans, Shigella, Bordetella pertussis and Mycoplasma pneumoniae (3). Plant extracts rich in the flavonoids apigenin and luteolin which proved to be effective against methicillin-resistant S. aureus (15,17) are an example of new substances that could be effectively applied in infec­tions nowadays difficult to treat.It was demonstrated that the pomegranate peel extract rich in anthocyanins has a strong antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomorias aeruginosa and Candida albicans (10).Naturally occurring flavonoids with antiviral activity have been known since the 1940s (16). Some viruses against which flavonoids exhibit inhibitory effects are the herpes simplex virus, respiratory syncytial virus, parainfluenza vi­rus and adenoviruses, picomaviruses along with the vesicu-E-mail: pharmacol'ayisclep.muvar.acadbgIN T R O D U C T IO NAronia melanocarpa is a bush grown in the woody regions of the Eastern part o f North America from where it was car­ded to East Europe and RussiaThe fruits are rich in cyanidin and its glycosides (cyanidin-3-galactoside, cyanidin-3-arabinoside, cyanidin- 3-xyloside and cyanidin-3-glucoside) (13). Cyanidin is a flavonoid from the athocyanin subclass (Fig. 1).Fig. I. C yanidinAddress for correspondence:5! Valcheva-Kicmanova, Dept, o f Preclinicai and Clinical Pharmacology and Biochemistry, Prof. P. Stoyanov Medical University o f Varna 55 Marin Drinov St, BG-9002 Varna21S. Valcheva-Kuzmanova, V. Russev, K. Bojkova .lar stomatitis virus, pouoviruses, influenza A ana В viruses, and human immunodeficiency virus (HIV) (3).Most antiviral studies were carriea out in vitro and little is known about the antiviral effects o f the flavonoids in vivo. Antiviral activity was demonstrated for the anthocyanin-rich extract from black currants (Ribes nigrum L.) against influenza A and В viruses (6). Anthocyanins in the fruits of elderberry (Sambacus nigra) inhibit the replica­tion o f the human influenza virus (19).The aim o f the present study was to test the natural Amnia melanocarpa fruit juice (AMFJ) for bacteriostatic activity in vitro and for antiviral activity in ovo.M A T E R IA L  A N D  M A T H O D SBacteriostatic activityThe bacteriostatic activity was detemiined after the serial dilution method (5). Referent strains o f microorganisms were used: 24h broth cultures o f Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudo­monas aeruginosa ATCC 27583 with a concentration of 109 colonies forming units/ml (CFU/ml). AMFJ was di­luted from 10 1 до 1 fr10 in a sterile broth medium. One ml of the bacterial suspension was added to each tube with the di­luted AMFJ. After 2h cultivation at 37°C 0,005 ml from each tube were plated to three plates with solid meat-peptone agar. The plates were cultivated for 24h at 37°C. The number o f colonies was counted (CFU/ml). The mean value from three dilutions was calculated and was multiplied times the corresponding reciprocal value. The lowest concentration that still inhibited the bacterial growth was accepted for bacteriostatic.Antiviral activityTwo virulent iniluenza virus strains were used: subtype A /Vama/3/003 and A/Vama/5/003 with an antigen formula H3N2 with an infectious activity 5,00-6,00 IgLDso. Sterile AMJF was used. AMFJ virusocidal activity was measured on 9-11-days old chicken embryos. One ml o f AMFJ was added to 1 ml virus containing suspension (5,00 lgLD50 with a hem agglutination titer 1:256). The so prepared sus­pension (pH 7,0) was incubated at 37°C for l-3h. Before the embryos were infected the hem agglutination titer o f the virus strains was checked. The titration o f the suspension was made on chicken embryos. Embryos infected with the corresponding viruses and with AMFJ were used as con­trols.The results were presented as mean values from three ex­periments earned out with both virus strains.R E SU L T S A N D  D ISC U SSIO NBacteriostatic activityFor the Escherichia coli ATCC 25922 strain it was estab­lished that CFU were still observed in dilutions from 1 fr1 до lfr3 and the mean value related to the dilution of 1(>2 was 36. The microbial number for the bacterium was 0,36x 102.For the Staphylococcus aureus ATCC 25923 strain the counted CFU were in dilutions o f AMFJ from lfr7 to lfr9 and the mean value related to the dilution o f lfr8 was 87. The microbial number for the bacterium was 0,87x108.For the Pseudomonas aeruginosa ATCC 27583 strain no bacteriostatic activity of AMFJ was detected.It is difficult to outline the probable mechanism of the flavonoid action because o f controversial data concerning the structure activity relationships o f flavonoids. There are data that the flavonoids without a hydroxyl group in their В rings are more effective against microorganisms than those with OH groups (2); this finding supports the idea that the membrane is the target o f the flavonoid action in the micro­organisms. The more lipophylic substances should be more devastating for this structure. Other authors, however, have established the opposite effect, i.e. the greater hydroxylation the greater antimicrobial activity (14).Some authors have found a correlation between the antimicrobial and antioxidant activity of the plant products. Osato et al. ( 12) have established that the seeds and the fruits o f the tropical medicinal plant Carica papaya Linn. are bacteriostatic for several enteric pathogens: Bacillus subtilis, Enterobacter cloacae, Escherichia coli, Salmo­nella typhi, Staphylococcus aureus, Proteus vulgaris, Pseu­domonas aeruginosa, and Klebsiella pneumoniae. The same parts o f the plant scavenge free radicals - hydroxyl and superoxide, which aie probably a part o f the cellular metabolism of these bacteriaProbably, similar mechanisms underlie AMFJ inhibitory activity on Staphylococcus aureus and Escherichia coli which was demonstrated in the present investigation. Escherichia coli possesses adhesins which prominate on the bacterial cell surface like hairs and thus allow the bacte­ria to adhere on the walls of the urinary tract and other inter­nal organs. It was shown that the extract from blueberry (Vaccinium) containing cyanidin-3-galactoside, cyanidin- 3-arabinoside, and cyanidin-3-glucoside possesses an antiadhesin activity (11). It is similar to other findings (7) that the blueberry anthocyanins can effectively cure and prevent light chronic and recurrent infections of the urinary bladder and urinary tract; they prevent the bacterial adhe­sion on the urinary tract walls.Antiviral activityThe present study showed that the hem agglutination activ­ity o f both influenza virus subtypes decreased - from 1:256 to 1:16 for A /Vama/3/ООЗ and to 1:32 for A/Vama/5/003. The studies o f AMFJ antiviral activity in ovo showed that the infectivity o f both strains o f influenza viruses decreased to OJgLDso. The structural basis for the antiviral activity of naturally occurring flavonoids was studied (18). It was demonstrated that the hydroxylation in positions 3', 4', 3, 5 and 7 was linked with the highest inhibition o f HSV-1 repli­cation. According to Selway (16), the hydroxylation in po­sition 3 was predisposing for the antiviral activity. Accord­ing to Bae et al. ( 1), the flavonoids in their glycone form have a greater inhibitory activity on the infectivity of rotaviruses than in their aglycone form.22*Study of natural Aronia melanocarpa fruit juice for antibacterialAMFJ contains cyanidin mainly in aglycone form (13). All anthocyanins have an OH-group in position 3 of the С ring, and cyanidin has hydroxyl groups in position 3', 41 o f the В ппц. These structural characteristics explain the good anti­viral activity of AMFJ. In our study, AMFJ inhibited the re­production of influenza virus in its initial stages most proba­bly due to the fonuation of complexes between the virion and the phenolic substances as a result o f which the adsorp­tion of influenza virus on the cell surface was prevented. Similar mechanisms of the antiviral effect o f flavonids are discussed in the literature. The green tea flavonoids inhibit the infectivity of the Influenza A and В viruses. They agglu­tinate the viruses by binding to hem agglutinine and thus in­hibit their adsorption on the host cells (9).Knox et al. (6) have investigated the antiviral activity of anthocynanin containing extract from black currant (Ribes nigrum L.) against Influenza A and В viruses. According to them, the anthocyanins do not directly inactivate the influ­enza viruses but inhibit their adsorption on the cells and their release from the infected cell. There are data from clin­ical investigations that anthocyanins alleviate the symptoms of the influenza infection and lead to a faster improvement of patients in comparison with placebo ( 19). The beneficial effect o f the anthocyanins in vivo can be explained with their ability not only to inhibit the viral replication but also to stimulate the immune system via the induction of cytokine production either in healthy individuals, or in pa­tients with influenza (19) and in immune-suppressed pa­tients such as those with cancer and HIV.C O N C L U SIO NAMFJ is bacteriostatic against Staphylococcus aureus and Escherichia coli in vitro and has no inhibitory activity against Pseudomonas aeruginosa. The bacteriostatic activ­ity against Staphylococcus aureus is greater than that against Escherichia coli. Gram-positive bacteria should mainly be used as a test for a further investigation o f AMFJ. AMFJ inhibits in ovo the reproduction o f Influenza virus type A in its initial stages, most probably, due to inhibition of the virus adsorption on the cell surface. In viral infections even better effect might be expected because of AMFJ stimulation of the immune system.R E F E R E N C E S1. B a e ,  E. A . ,  M . J. H a n ,  M . L e e ,  e t  a l .  In vitro inhibitory effect o f  some flavonoids on rotavirus infectivity.- Biol. Pharm. Bull., 23, 1997, 1122-1124.2. C h a b o t ,  S . ,  R. B e l - R h l i d ,  R. C h e n e v e r t ,  Y. P i c h e .  Hyphal growth promotion in vitro o f  the VA mycorrhizal fungus, Gigaspora margarita Becker and Hall, by the activity o f  structurally specific flavonoid compounds under C02-enriched conditions.- New Phytol., 122, 1992,461-467.3. C o w a n ,  M . M . Plant products as antimicrobial agents.- Clin. Microbiol. Rev., 12, 1999, 564-582.4. D i x o n ,  R . A . ,  P. M . D e y ,  C .  J. L a m b .  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Study of Natural Aronia Melanocarpa Fruit Juice for Antibacterial and Antiviral Activity

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Study of Natural Aronia Melanocarpa Fruit Juice for Antibacterial and Antiviral Activity

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