Bratko Filipič

Vpliv matičnega mlečka in humanega interferona-alfa (HuIFN-αN3) na proliferacijo, nivo glutationa in na preoksidacijo lipidov v humanih kolorektalnih adenokarcinomskih celicah in vitro

Among royal jelly’s (RJ) various biological activities, its possible antitumour activity deserves particular attention. The purpose of this study was to investigate the influence of RJ, its bioactive component 10-hydroxy-2-decenoic acid (10- HDA), and human interferon-alpha (HuIFN-αN3) on the proliferation of human colorectal adenocarcinoma cells (CaCo- 2), and ascertain their effect on intracellular glutathione (GSH) level and lipid peroxidation. We studied the antiproliferative (AP) activity of RJ [(0.1 g/10 mL phosphate buffer saline (PBS)], HuIFN-αN3 (1000 I.U. mL-1), 10-HDA at 100.0 μmol L-1, and their different combinations, in the ratio 1:1, 1:2, and 2:1 on CaCo-2 cells. The GSH level was measured by glutathione assay. The lipid peroxidation was measured by malondialdehyde (MDA) assay. Single RJ had a low AP activity: 2.0 (0.5 mg mL-1). HuIFN-αN3 had an AP activity of 2.5 (208.33 I.U. mL-1), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL-1). The highest AP activity of 3.8 was obtained when RJ and HuIFN-αN3 were applied at the ratio 2:1. In that combination the level of GSH was 24.9±2.4 nmol g-3 of proteins (vs. 70.2±3.2 nmol g-3 in the control) and the level of MDA was 72.3±3.1 nmol g-3 (vs. 23.6±9.1 nmol g-3 in the control). It is generally assumed that 10-HDA, an important constituent of RJ, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cells proliferation in vitro. In our study, however, RJ and HuIFN-αN3 applied at 2:1 decreased the level of GSH the most and significantly increased lipid peroxidation via MDA in CaCo-2 cells. Future studies should show whether these GSH- and MDA-related activities of RJ, HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of various tumour cells in vitro. Kot del biološke aktivnosti MM (Matičnega mlečka) so avtorji preučevali njegovo protitumorsko delovanje kot tudi možno interakcijo s humanim interferonom alfa (HuIFN-αN3). Cilj opravljenih poskusov je bil preučiti vpliv kombinacije med MM in HuIFN-αN3 na proliferacijo celic Humanega kolorektalnega adenokarcinoma (CaCo-2) in njun vpliv na znotrajcelični nivo glutationa (GSH) in peroksidacijo lipidov. Avtorji so preučevali AP (Antiproliferativno) delovanje MM (0.1 g/10 mL fosfatnega pufra) (PBS), HuIFN-αN3, (1000 I.U. mL-1), 10-hidroxy-2-decenoične kisline (10-HDA) (100.0 μmol L-1) in različne kombinacije med njimi (1:1, 1:2 in 2:1) na celice CaCo-2 in vitro. Njihov vpliv na znotrajcelični nivo GSH so merili s pomočjo komercialnega kita. Peroksidacijo lipidov so merili s pomočjo meritve vrednosti malondialdehida (MDA). MM sam kaže AP aktivnost 2.0 (0.5 mg mL-1 ). HuIFN-αN3 ima AP aktivnost 2.5 (208.33 I.U. mL-1) medtem ko ima 10-HDA AP aktivnost 1.5 (37.5 μmol mL-1). AP aktivnost kombinacije MM:HuIFN-αN3 (2:1) je bila 3.8. Pri tej kombinaciji je bil viden vpliv na nivo GSH: 24.9±2.4 nmol g-3 proteinov (70.2±3.2 nmol g-3 pri kontroli). Nivo MDA je bil 72.3±3.1 nmol g-3 pri kontroli). 10-HDA je glavna sestavina MM, ki v kombinaciji s HuIFN-αN3 deluje antiproliferativno na CaCo-2 celice. MM in HuIFN-αN3 v kombinaciji 2:1 pospešujeta peroksidacijo lipidov (MDA) in zmanjšujeta nivo glutationa (GSH). Nadaljni poskusi bodo pokazali ali z GSH- in MDA- povezane aktivnosti MM, HuIFN-αN3, 10-HDA in kombinacij med njimi, zmanjšujejo indeks tumorigenosti in s tem tumorigeni potencijal različnih tumorskih celic in vitro.

The influence of royal jelly and human interferon-alpha (HuIFN-αN3) on proliferation, glutathione level and lipid peroxidation in human colorectal adenocarcinoma cells in vitro.

Among royal jelly’s (RJ) various biological activities, its possible antitumour activity deserves particular attention. The purpose of this study was to investigate the influence of RJ, its bioactive component 10-hydroxy-2-decenoic acid (10- HDA), and human interferon-alpha (HuIFN-αN3) on the proliferation of human colorectal adenocarcinoma cells (CaCo- 2), and ascertain their effect on intracellular glutathione (GSH) level and lipid peroxidation. We studied the antiproliferative (AP) activity of RJ [(0.1 g/10 mL phosphate buffer saline (PBS)], HuIFN-αN3 (1000 I.U. mL-1), 10-HDA at 100.0 μmol L-1, and their different combinations, in the ratio 1:1, 1:2, and 2:1 on CaCo-2 cells. The GSH level was measured by glutathione assay. The lipid peroxidation was measured by malondialdehyde (MDA) assay. Single RJ had a low AP activity: 2.0 (0.5 mg mL-1). HuIFN-αN3 had an AP activity of 2.5 (208.33 I.U. mL-1), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL-1). The highest AP activity of 3.8 was obtained when RJ and HuIFN-αN3 were applied at the ratio 2:1. In that combination the level of GSH was 24.9±2.4 nmol g-3 of proteins (vs. 70.2±3.2 nmol g-3 in the control) and the level of MDA was 72.3±3.1 nmol g-3 (vs. 23.6±9.1 nmol g-3 in the control). It is generally assumed that 10-HDA, an important constituent of RJ, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cells proliferation in vitro. In our study, however, RJ and HuIFN-αN3 applied at 2:1 decreased the level of GSH the most and significantly increased lipid peroxidation via MDA in CaCo-2 cells. Future studies should show whether these GSH- and MDA-related activities of RJ, HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of various tumour cells in vitro. Kot del biološke aktivnosti MM (Matičnega mlečka) so avtorji preučevali njegovo protitumorsko delovanje kot tudi možno interakcijo s humanim interferonom alfa (HuIFN-αN3). Cilj opravljenih poskusov je bil preučiti vpliv kombinacije med MM in HuIFN-αN3 na proliferacijo celic Humanega kolorektalnega adenokarcinoma (CaCo-2) in njun vpliv na znotrajcelični nivo glutationa (GSH) in peroksidacijo lipidov. Avtorji so preučevali AP (Antiproliferativno) delovanje MM (0.1 g/10 mL fosfatnega pufra) (PBS), HuIFN-αN3, (1000 I.U. mL-1), 10-hidroxy-2-decenoične kisline (10-HDA) (100.0 μmol L-1) in različne kombinacije med njimi (1:1, 1:2 in 2:1) na celice CaCo-2 in vitro. Njihov vpliv na znotrajcelični nivo GSH so merili s pomočjo komercialnega kita. Peroksidacijo lipidov so merili s pomočjo meritve vrednosti malondialdehida (MDA). MM sam kaže AP aktivnost 2.0 (0.5 mg mL-1 ). HuIFN-αN3 ima AP aktivnost 2.5 (208.33 I.U. mL-1) medtem ko ima 10-HDA AP aktivnost 1.5 (37.5 μmol mL-1). AP aktivnost kombinacije MM:HuIFN-αN3 (2:1) je bila 3.8. Pri tej kombinaciji je bil viden vpliv na nivo GSH: 24.9±2.4 nmol g-3 proteinov (70.2±3.2 nmol g-3 pri kontroli). Nivo MDA je bil 72.3±3.1 nmol g-3 pri kontroli). 10-HDA je glavna sestavina MM, ki v kombinaciji s HuIFN-αN3 deluje antiproliferativno na CaCo-2 celice. MM in HuIFN-αN3 v kombinaciji 2:1 pospešujeta peroksidacijo lipidov (MDA) in zmanjšujeta nivo glutationa (GSH). Nadaljni poskusi bodo pokazali ali z GSH- in MDA- povezane aktivnosti MM, HuIFN-αN3, 10-HDA in kombinacij med njimi, zmanjšujejo indeks tumorigenosti in s tem tumorigeni potencijal različnih tumorskih celic in vitro.

Chemical analysis and antioxidant content of various propolis samples collected from different regions and their impact on antimicrobial activities

Objective: To assess the antioxidant content, antimicrobial and antioxidant activities of various propolis samples. Methods: Seven propolis samples were collected from different locations in Morocco, which are characterized by different plant predominant vegetations. The resin, wax and balsam of hydroalcoholic extract of propolis content were identified, and the antioxidant content was analyzed with the use of HPLC and colorimetric methods. The antioxidant activity was assessed by DPPH, ABTS.+ and ferric reducing power assays. The antimicrobial activity was assessed against bacterial species, including methicillin resistant Staphylococcus aureus and Candida albicans, and expressed as the minimal inhibitory concentration. Results: The propolis samples showed significant variations in the chemical composition and in the antioxidant or antimicrobial activities even when the samples were collected from the same location. Propolis with high resin and low wax content had high level of antioxidant compounds, and strong antioxidant and antimicrobial activities. Gram-positive bacteria, especially, methicillin resistant Staphylococcus aureus were more sensitive to all propolis samples than Gram-negative bacteria and Candida albicans. Conclusions: The chemical composition and the antioxidant and antimicrobial activities of various propolis samples are different and rely on the geographic and plant origin of propolis collection. Propolis samples with low wax and high resin content might be more suitable to be used in future preclinical or clinical investigations.

Additive Effects of Water-Soluble Propolis (Greit 120) and Human Interferon Alfa (HuIFN-αN3) against Influenza Viruses in Vitro

Influenza virus affects the respiratory tract in humans causing a range of distinct manifestations including fever, nasal secretions, cough, headaches, muscle pain and pneumonia, which could become violent and severe. It was found that influenza A viruses remain resistant to amantadine and rimantadin with high level of oseltamvir resistance. Therefore, there is a need for constant improvement of drugs active against resistant influenza viruses. Propolis has anti-influenza activity both in vitro and in vivo. Human leukocyte interferon (HuIFN-αN3) is a multi- subtype protein that displays an antiviral activity against influenza virus. In this study we elucidated the anti-influenza activity of the mixes of water-soluble propolis (WSP) (Greit 120) and HuIFN-αN3 at different ratios. Greit 120 polyphenols, total phenol acids and bioflavonoid were characterized by HPLC-UV-ESI- MS504971 and HuIFN-αN3 by reverse-phase high- performance liquid chromatography (RP-HPLC). Influenza A and B viruses were separately added to the LLC-MK2 cells treated with WSP (Greit 120) and HuIFN-αN3 alone or in proportions 1:1, 1:2 and 2:1. Plates were incubated and cytopathic effect was determined. The best results (ID50) were obtained with the mix of 10% WSP and HuIFN-αN3 in proportion 1:2, showing ID50 at 12 ± 0.2 μg/mL and 19 ± 0.6 μg/mL for influenza A and B viruses, respectively. When comparing anti- influenza activity of WSP (Greit 120)/HuIFN-αN3 with that of ribavirin, it was found that 1:2 was the optimal ratio for WSP (Greit 120)/HuIFN-αN3 (0.5 and 0.6 for influenza A and B, respectively). This new formulation of WSP (Greit 120) and HuIFN-αN3, showing better anti-Influenza activity, will definitely improve its application in flu infections.

Bee Product Apiderm Royal Gel® in Pressure Injuries and Wound Care - Case Report

Bee products are known for thousands years for its beneficial effect on wound healing process [1,2]. Its effectiveness is partly explained only in recent years explaining the multiple bioactivities involved in healing process. It seems that the most important activity of bee products is acidification that promotes healing of deep wounds [3]. Similar report was given by M phande et al. [4] who reported on positive effects of honey and sugar dressing on wound healing. Part of the bee product activity in healing process could be addressed to its strong antibacterial activity directed even to multiresistant strains of bacteria [5]. Honey was also used in treatment of decubitus ulcers [6]. It is obvious that different bee products can act very positive to the healing process in both mechanical wounds as well as to the pressure wounds [7].

Water Soluble Propolis and Royal Jelly Enhance the Antimicrobial Activity of Honeys and Promote the Growth of Human Macrophage Cell Line

Due to the overuse and misuse of antibiotic, an increase in antibiotic resistance of pathogenic bacteria is evolving. Attention should be focused on natural alternatives to antibiotics, like propolis, royal jelly (RJ) and honeys. They all have strong antibacterial properties due to the active substances they contain. This study investigated the effect of combination of water soluble propolis (WSP) Greit120 or fresh royal jelly (F-RJ) (Mižigoj) and Forest honeys as antibacterial against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter baumanii, Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Streptococcus agalactiae and Candida albicans. These substances are also cell growth promoters for human macrophage (TLT) cell line. WSP Greit120, F-RJ (M) and different Forest honeys were prepared in saline as 10% solutions. The antimicrobial activity was expressed as the minimal inhibitory concentration (MIC) in mg/mL. The growth promotion activity was measured at optical density (OD) 595 nm. The combination of WSP Greit120 with different Forest honeys is better than F-RJ (M) in same combination with different Forest honeys. The best antibacterial/antifungal activity was found with the combination of 10% WSP Greit120 in the Forest honey (1:10) from Italy or Spain. When measuring the growth promoting activity of TLT cell line, the best activity was detected at the combination of 10% WSP Greit120 in the Forest honey from Italy (GI3 = 0.796 ± 0.014 and GI5 = 1.133 ± 0.022). Antimicrobial and growth promoting activities are correlated and WSP-dependent.

Multiplication of HVT FC-126 (Herpesvirus turkey) virus in the kidney cell lines of no avian origin

The presented experiments were aimed to cultivate and multiplicate HVT FC-126 in the PLA (Adult pig kidney) and GL-4 (Gerbil kidney) cell lines. Two different HVT FC-126 vaccine strains were used: Marikal SPF (Veterina d.o.o., Croatia) and Lyomarex (Merial, USA). They were adapted to the PLA and GL-4 cell lines. After adaptation, they were titrated on PLA (TCID50 2^4.23) and GL-4 (TCID50 2^4.96). On both cell lines they show similar CPE (cytophatic effect). The difference between them was detected using Real Time PCR, which was also positive by agarose gel analysis for the virus contained in Lyomarex, but not in the Marikal SPF. It can be concluded that both cell lines are sensitive to HVT FC-126 and the virus can be multiplied in high titers though much lower than in the calf intestinal epithelial cell line (CIEB) cells (TCID50 2^7.93).