Antimikrobno dejstvo ceđenih sokova i ekstrakata plodova odabranog voća porodice Rosaceae

Abstract

Nesavesna upotreba antibiotika izazvala je razvoj antimikrobne rezistencije zabrinjavajućih razmera. Porast rezistencije prema konvencionalnim antimikrobicima nametnuo je potrebu pronalaska novih supstanci, koje će biti delotvorne protiv otpornih sojeva. U poslednje četiri decenije, dominira trend da se lekovi i lekovita sredstava prave na bazi prirodnih proizvoda, a naročito biljnog materijala. Hemijski sastav plodova porodice ruža (Rosaceae) upućuje na to da bi određene vrste ove porodice biljaka mogle ispoljavati antimikrobno dejstvo. Plodovi voća porodice Rosaceae istraživani u okviru ove disertacije su: malina (Rubus idaeus), kupina (Rubus fruticosus), trešnja (Prunus avium), višnja (Prunus cerasus) i aronija (Aronia prunifolia). Predmet istraživanja ove doktorske disertacije je antimikrobno tj. antibakterijsko, antigljivično, antialgalno i antivirusno dejstvo sokova i ekstrakata plodova odabranog voća porodice Rosaceae gajenog i prikupljenog na teritoriji Srbije na širok spektar humanih i animalnih patogenih mikroorganizama.Sokovi su dobijeni ceđenjem svežih plodova voća. Ekstrakti su dobijeni iz ostataka voća nakon ceđenja (tropa) metodom dvostruke etanolne ekstrakcije. Nakon ceđenja i ekstrakcije sprovođeno je uparavanje do suva sokova i ekstrakata. HPLC metodom je ispitano prisutstvo 23 hemijske komponente u svakom od sokova i ekstrakata. Antimikrobna analiza je sprovedena na 21 mikroorganizmu, od čega su 15 bile bakterije, 2 gljivice, 2 alge i 2 virusi. Od navedenih mikroorganizama 14 je bilo autohtonih izolata, a 7 referentnih sojeva. Ispitivanje antibakterijskog, antifungalnog i antialgalnog dejstva plodova odabranog voća je sprovedeno difuzionom i mikrodilucionom metodom. Analiza antivirusne aktivnosti plodova odabranog voća je sprovedena posmatranjem inhibicije citopatogenog efekta (CPE) virusnih čestica na ćelijsku kulturu. Sve studije su sprovedene u triplikatu. Rezultati hemijske analize su obrađeni statistikom metodom srednje vrednosti, uz računanje standardne devijacije. Eksperimentalno dobijeni podaci u okviru ispitivanja antibakterijskog, antigljivičnog i antialgalnog dejstva su uvršteni u statističku analizu. Metodom aproksimacije je utvrđena tačna koncentracija soka ili ekstrakta koja daje željeni efekat na ispitivani mikroorganizam. Rezultati analize antivirusnog dejstva su statistički obrađeni metodom srednje vrednosti. Kako bi se utvrdio stepen povezanosti rezultata dobijenih u okviru antimikrobnih analiza sa hemijskim supstancama prisutnim u plodovima odabranog voća sprovedena je regresiona analiza za vaki pojedinačni slučaj. Antibakterisjko, antiungalno i antialgalno dejstvo sokova i ekstrakata je upoređeno sa istim dejstvima konvencionalnih antimikrobnih lekova. Najveću ukupnu količinu ispitivanih supstanci je sadržao ekstrakt kupine (3965,56 mg/ml), a najmanju ekstrakt trešnje (161,44 mg/ml). Od svih ispitivanih hemijskih supstanci, samo su: antocijani, katehin, galna kiselina i vitamin C, prisutni u svim ispitivanim sokovima i ekstraktima. Sve ispitivane biljke sadrže veću količinu supstanci u ekstraktu, osim kod trešnje, kod koje se veća količina supstanci nalazi u soku. U najvećoj meri su u sokovima i ekstraktima plodova biljaka prisutni antocijani i to najviše kod kupine od ekstrakata, a kod maline od sokova. Antimikrobno (antibakterijsko, antifungalno, antialgalno i antivirusno) dejstvo je dokazano za svaki od sokova i/ili ekstrakata plodova odabranog voća. Po prosečnim vrednostima rezultata dilucione analize antimikrobnog i dejstva na MDBK ćelije, protiv najvećeg broja mikroorganizama najdelotvorniji je bio ekstrakt kupine, a neaktivan ili najslabije aktivan (aktivan u najvećoj koncentraciji) sok trešnje. Prosečne vrednosti antimikrobnog i dejstva na MDBK ćelije su bile u opsegu od 0,71 mg/ml do 100 mg/ml. Sve ispitivane supstance su pokazale antibakterijsku aktivnost u ispitivanim koncentracijama na sve ispitivane bakterije, osim soka maline na Escherichia-u coli i soka aronije na Klebsiella-u pneumoniae. Ekstrakti su bolje delovali u većini slučajeva, uz nekoliko izuzetaka. Sve ispitivane supstance su pokazale antfiungalnu aktivnost na Candida-u albicans ATCC 24433, osim soka aronije. Na autohtoni soj Candida-e albicans su delovali samo ekstrakti kupine i trešnje. Ekstrakti su bolje delovali u većini slučajeva, osim kupine na Candida-u albicans ATCC 24433, u kojem je dejstvo soka bilo jednako sa dejstvom ekstrakta. Iako su skoro svi ispitivani sokvi i ekstrakti ispoljili antialgalno dejstvo, na Prototheca-u wickerhamii nisu delovali višnja i sok maline, dok sok aronije nije delovao antialgalno ni na jednu od ispitivanih Prototheca. Ekstrakti su bolje delovali u većini slučajeva, osim maline kod koje je, u oba slučaja, bolje delovao sok. Sve ispitivane supstance su pokazale antivirusnu aktivnost na Bovine herpesvirus i Bovine viral diarrhea virus, kao i toksičnost na MDBK ćelije, osim sokova maline i trešnje, koji su bili neaktivni protiv Bovine herpesvirus-a. Ekstrakti su bolje delovali u većini slučajeva, osim trešnje na MDBK ćelije i Bovine viral diarrhea virus, na koje je jače delovao sok. U potpunosti jednako delovanje soka i ekstrakta je uočeno kod aronije za sva tri dejstva i maline za toksičnost na MDBK ćelije. Pri ispitivanju difuzionom metodom antibiotik je ispoljio jače dejstvo u odnosu na sokove i ekstrakte, osim ekstrakata kupine, maline i višnje na Trueperella-u pyogenes. Pri ispitivanju dilucionom metodom, dejstvo ekstrakata i sokova na Gram pozitivne bakterije je bilo uporedivo sa dejstvom Streptomicina, dok je dejstvo antibiotika na Gram negativne bakterije bilo jače u odnosu na sokove i ekstrakte, izuzetev jačeg dejstva ekstrakta maline protiv Escherichia-e coli. Nistatin je sve antifungalne i antialgalne efekte na gljivice i alge obuhvaćene istraživanjem ispoljio u manjim koncentracijama u odnosu na ispitivane sokove i ekstrakte.Najpovezanije sa antibakterijskim dejstvom sokova i ekstrakata na odgovarajuće mikroorganizme su bile: kumarinska, ferulna i galna kiselina, katehin. Najveći procenat povezanosti sa antimikrobnom aktivnosti ploda imali su antocijani na Enterococcus faecalis (71,94%). Najmanje povezani sa antimikrobnom aktivnosti na odgovarajuće mikroorganizme su bili: hlorogenska kiselina, kempferol, kumarinska i siringinska kiselina, vitamin C, vanilinska kiselina, epikatehin, katehin, rutin, krisin, elaginska i p-hidroksibenzoeva kiselina. Saznanja dobijena u okviru ove disertacije proširuju obim znanja o antimikrobnom dejtvu voća i predstavljaju osnovicu za svrsishodniju primenu voća kako u ishrani tako i u terapiji. Međutim, dalja istraživanja u smislu proširenja spektra ispitivanih mikroorganizama, antimikrobnih studija pojedinačnih komponenti iz plodova i mehanizama dejstva su potrebna.

Nonconscientious use of antibiotics has caused the development of antimicrobial resistance of worrying proportions. The increase in resistance to conventional antimicrobials has imposed a need to find new substances that will be effective against resistant strains. Over the last four decades, the trend is to medicines and medicinal products be made on the basis of natural products, and especially plant material. The chemical composition of the Rose family (Rosaceae) fruits suggests that certain species of this family of plants may exhibit an antimicrobial effect. The fruits of the Rosaceae family studied in the framework of this dissertation are: Raspberry (Rubus idaeus), Blackberry (Rubus fruticosus), Cherry (Prunus avium), Cherry (Prunus cerasus) and Aronia (Aronia prunifolia). The subject of the research of this doctoral dissertation is antimicrobial ie. antibacterial, antifungal, antialgal and antiviral effects of juices and fruit extracts, of the selected fruit of the Rosaceae family collected on the territory of Serbia, on a wide range of human and animal pathogenic microorganisms. Juices were obtained by the process of squeezing fresh fruits. The extracts were obtained from residues of fruits after extraction (pomace) by the double ethanol extraction method. After squeezing and extraction, evaporation to dryness of juices and extracts was carried out. The presence of 23 chemical components in each of the juices and extracts was examined by the HPLC method. Antimicrobial analysis was carried out on 21 microorganisms, of which 15 were bacteria, 2 fungi, 2 algae and 2 viruses. 14 of these microorganisms were autochthonous isolates and 7 reference strains. The antibacterial, antifungal and antialgal effect of the selected fruit was tested by the diffusion and microdilution method. The analysis of the fruits antiviral activity was carried out by observing the inhibition of cytopathogenic effect (CPE) of viral particles per cell culture. All studies were conducted in triplicate. The results of the chemical analysis were processed by means of a mean value method, with calculation of the standard deviation. The experimentally obtained data within the antibacterial, antifungal and antialgal examination were included in the statistical analysis. The method of approximation was used in order to determine the exact concentration of the juice or extract, which gives the desired effect on the examined microorganisms. The results of the antiviral analysis were statistically processed using the mean value method. In order to determine the degree of correlation of the results obtained in the framework of antimicrobial analysis with the chemical substances present in the fruits of the selected fruit, regression analysis was carried out for each individual case. Antibacterial, antifungal and antialgic effects of juices and extracts are compared with he same effects of conventional antimicrobial drugs, Streptomycin and Nystatin. The largest total amount of test substances was contained by blackberry extract (3965.56 mg/ ml), and the smallest by extract of sweet cherry (161.44 mg/ ml). Of all the investigated chemicals, only: anthocyanins, catechins, gallic acids and vitamin C were present in all examined juices and extracts. All investigated plants contain a higher amount of substances in the extract, except for sweet cherries, in which a greater amount of substances is present in the juice. In the juices and extracts of fruits anthocyanins are present to the greatest extent, mostly in the blackberry extract, and in raspberry juices. According to the average values of the results of the dilution analysis of the antimicrobial and the effect on the MDBK cell, the most effective was the blackberry extract and the most active inactive (active in the highest concentration) cherry juice against the most numerous microorganisms. The average antimicrobial values and effects on the MDBK cells were in the range of 0.71 mg/ ml to 100 mg/ ml. All investigated substances showed antibacterial activity in the tested concentrations on all examined bacteria, except raspberry juice on Escherichia coli and aronia juice on Klebsiella pneumoniae. Extracts acted better in most cases, with few exceptions. All investigated substances showed antifungal activity on Candida albicans ATCC 24433, except aronia juice. On the autochthonous Candida albicans, only blackberry and sweet cherry extracts were active. Extracts worked better in most cases, except for blackberry on Candida albicans ATCC 24433, in which the effect of the juice was equally efficient as the extract. Although almost all of the examined juices and extracts exhibited an antialgal effect, the sour cherry and raspberry juice did not work on the Prototheca wickerhamii, while the aronia juice did not express antialgal activity to any of tested Prototheca isolates. Extracts worked better in most cases, apart from raspberries, wich juice was stronger in both cases. All investigated substances showed antiviral activity on Bovine herpesvirus and Bovine viral diarrhea virus, as well as the toxicity to MDBK cells, except for raspberry and cherry juices, that were inactive against Bovine herpesvirus. The extracts worked better in most cases, except sweet cherries on MDBK cells and Bovine viral diarrhea virus, whose juice had stronger effect. The equally effective action of juice and extract was observed in aronia for all three effects and raspberries for toxicity to MDBK cells. In the diffusion test, antibiotic showed a stronger effect than juices and extracts, except for extracts of blackberry, raspberry and sour cherries on Trueperella pyogenes. In a dilution study, the effect of extracts and juices on Gram-positive bacteria was comparable to that of Streptomycin, while the effect of the antibiotic on Gram negative bacteria was stronger than juices and extracts, with exception of the stronger effect of raspberry extract against Escherichia coli. Nystatin has expressed its all antifungal and antialgal effects on fungi and algae included in the study in smaller concentrations compared to the examined juices and extracts. The most consistent with the antibacterial effect of juices and extracts on the appropriate microorganisms were: coumarin, ferulan and gallic acid, catechin. The highest percentage of association with antimicrobial activity of the fruits was given by anthocyanins to Enterococcus faecalis (71.94%). The least related to the antimicrobial activity on the appropriate microorganisms were: chlorogenic acid, champferol, coumarin and saringic acid, vitamin C, vanillic acid, epicatechin, catechin, rutin, chrysin, elaginic and p-hydroxybenzoic acid. The knowledge gained through this dissertation expands the scope of knowledge on antimicrobial activity of fruits and forms the basis for more effective use of fruits in both therapy and nutrition. However, further research in terms of extending the range of examined microorganisms, antimicrobial studies of individual fruits components and mechanisms of action are required.