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The Polyphenol Content and Antimicrobial Activity of Selected Propolis’ Extracts

Journal of Agricultural Science and Technology(B) 2020年第6期10卷 350-364页

作者： Bratko Filipič Lidija Gradišnik dunja Gregorič Adriana Pereyra Eva Ružić-Sabljić Hrvoje Mazija Croatian Institute for Experimental and Translation oncology(CIETo) Koledinečka 03Zagreb 10040Croatia Institutes of Biomedical Sciences Medical Faculty of MariborTaborska 8Maribor 2000Slovenia Retired Glavarjeva 19Ljubljana 1000Slovenia Research and developement Medex D.o.o.Linhartova 049aLjubljana 1000Slovenia Institutes of Microbiology and Immunology Medical FacultyZaloška 4Ljubljana 1105Slovenia

Propolis is a resinous natural product,produced by bees(Apis mellifera),from vegetable parts and plant ***’samples A,B,C and d were extracted with phosphate buffer saline(PBS)or with 70%EtoH at pH values 8.0,7.2 and 6.4 followed by:(1)reverse-phase high-performance liquid chromatography(RP-HPLC)on Purospher®Star RP-18 column,the quantity of caffeic acid,chrysin,pinocembrin and galangin was determined;(2)determination of total flavonoids in both extracts;(3)antimicrobial tests of both extracts against(a)Gram-positive bacteria:methicillin-resistant Staphylococcus aureus(MRSA),***,Streptococcus pyogenes,***,(b)Gram-negative bacteria:Escherichia coli,Pseudomonas aeruginosa,Proteus mirabilis,Acinetobacter baumanii and(c)yeast:Candida *** antimicrobial activity of propolis’PBS extracts against Gram-positive bacteria shows the lowest minimal inhibitory concentration(MIC,mg/mL)at pH 8.0 in sample C,followed by A,B and *** sample C,MICs at pH 8.0 were 0.007(***),0.015(MRSA),0.015(***)and 0.007(***).The polyphenol content of sample C is:flavonoid content 5.47±0.62 mg/mL,caffeic acid 1.33±0.92 mg/mL,chrysin 41.02±4.22μg/mL,pinocembrin 2.93±0.33 mg/mL and galangin 41.87±4.23 mg/*** extracts against Gram-negative bacteria show the lowest MIC(mg/mL)at pH 8.0 in sample d,followed by B,C and *** sample d,MICs at pH 8.0 were 0.003(***,***,***)and 0.007(***).Polyphenol content of sample d is:flavonoids 8.28±0.92 mg/mL,caffeic acid 3.56±0.32 mg/mL,chrysin 677.42±68.42μg/mL,pinocembrin 146.49±13.89 mg/mL and galangin 59.81±5.86 mg/*** strongest anti *** activity,with the lowest MIC(mg/mL),at pH 8.0 was in the sample C,followed by samples d,A and *** sample C,the MIC at pH 8.0 is 0.001(PBS extract).The antimicrobial activities of selected propolis samples correlate with their polyphenol content,more precisely,flavonoid,caffeic acid,chrysin,pinocembrin and galangin content.

关键词： Propolis pH phosphate buffer saline extract 70%EtoH extract antimicrobial activity

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Effect of electroacupuncture at Neiguan(PC6)at different time points on myocardial ischemia reperfusion arrhythmia in rats

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Journal of Traditional Chinese Medicine 2024年第1期44卷 113-121页

作者： SUN Qianhui CHENG Kai dAI Xingye YANG Zhiwen WU Xiaoling XU Chang QIU Xinghua GAo Xiaofeng LIU daonan YANG Qirui School of Acupuncture and Tuina Beijing University of Chinese MedicineBeijing 100029China Beijing medex Technology Co. Ltd.Beijing 100029China

oBJECTIVE:To observe the effects of electroacupuncture at Neiguan(PC6)at different time points on reperfusion arrhythmia(RA)after myocardial ischemia and reperfusion in rats,and to investigate the correlation of this protective effect with nerve growth factor(NGF),tyrosine kinase A(Trk A),tyrosine hydroxylase(TH),and norepinephrine(NE).METHodS:A total of 72 Sprague-dawley male rats were randomly divided into six groups(n=12 rats/group):normal group(Norm),sham operation group(Sham),ischemia reperfusion group(I/R),pre-ischemic electroacupuncture group(EAI),pre-reperfusion electroacupuncture group(EAII),post-reperfusion electroacupuncture group(EAIII).The myocardial ischemia-reperfusion injury(MIRI)model was induced by occlusion of left anterior descending coronary artery for 20 min followed by reperfusion for 40 min in *** no intervention in the Norm group and only threading without ligation in the Sham *** pretreatment at 20 min/d for 7 d before ligation in the EAⅠgroup,20 min of electroacupuncture before reperfusion in the EAII group and 20 min of electroacupuncture after reperfusion in the EAIII *** electrocardiogram(ECG)of each group was recorded throughout the whole process,and the success of the MIRI model was determined based on the changs of J-point and T-wave in the *** arrhythmia score was used to record premature ventricular contractions,ventricular tachycardia and ventricular fibrillation during the reperfusion period to assess the reperfusion induced *** expression levels of NGF,Trk A,TH protein were measured by Western ***,the expression levels of plasma and myocardial NE levels were detected by enzyme linked immunosorbent ***:The differences between Norm group and Sham group were not statistically significant in all *** score,myocardial NGF,Trk A,TH,and NE expression were significantly higher in the I/R group compared with the Sham *** score,myocardial NGF,T

关键词： myocardial reperfusion injury reperfusion arrhythmia electroacupuncture point PC6(Neiguan) nerve growth factor protein-tyrosine kinases norepinephrine

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Propolis Extracts Enhance the Antimicrobial Activity of Slovenian:Chestnut,Forest and Flower Honeys

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Journal of Agricultural Science and Technology(B) 2019年第3期9卷 203-219页

作者： Bratko Filipic Klemen Rihar dunja Exel Gregoric Lidija Gradisnik Adriana Pereyra Eva Ruzic-Sabljic Fabio Galeotti Alfredo Fachini Hrvoje Mazija Croatian Institute for Experimental and Translational oncology(CIETo) Koledinecka 03Zagreb 10040Croatia Retired Chengdujska 4Ljubljana 1000Slovenia Retired Glavarjeva 19Ljubljana 1000Slovenia Institute of Biomedical Sciences Medical Faculty of MariborTaborska 8Maribor 2000Slovenia medex d.o.o. Linhartova 49aLjubljana 1000Slovenia Institute for Microbiology and Immunology Medical Faculty of LjubljanaZloska 04Ljubljana 1105Slovenia department of Life Sciences University of Modena&Regio EmiliaModena 41125Italy BNatural Via Gran SassoCorbettaMI 20011Italy

Because of 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 and honey. They all have strong antibacterial properties because of the active substances they contain. In the present study, the influence of P1 (water-soluble propolis (WSP) Greit 120) or P2 (phosphate buffer saline (PBS) propolis extract Lj. Center pH = 8.0) on antimicrobial activity of different Slovenian: chestnut honey (CH), forest honeys (FoH), and flower honey (FlH) against various Gram-positive or Gram-negative bacteria and yeast Candida albicans are shown. Gram-positive bacteria used in the experiments were: methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus mutans. The yeast was Candia albicans. Gram-negative bacteria used were: Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumanii. P1, P2, FoH, CH, FlH and their combinations were prepared in saline as 10% solutions. The composition of both propolis extracts and the three kinds of honey was determined by reverse-phase high-performance liquid chromatography (RP-HPLC). It expressed the antimicrobial activity as a minimal inhibitory concentration (MIC) in mg/mL. Each of the two propolis extracts showed antimicrobial activity when applied alone. Also, the application of each of honey yielded an antimicrobial activity in decreasing order: CH, FoH and FlH honey. The combination of any of the propolis extract with any of the honey showed the additive antimicrobial effect on either Gram-positive or Gram-negative bacteria and on yeast Candida albicans. The effectiveness of P1 or P2 depends on their nature and from the sort of honey and is the highest in the case of CH followed by FoH and FlH.

关键词： Honey propolis flavonoids antimicrobial activity enhancement

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Efficacy on rabbits with arrhythmia: needling acupoint of Neiguan(PC6) at shallow or deep depth, and retaining needles for 10, 20, or 30 min

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Journal of Traditional Chinese Medicine 2021年第6期41卷 968-973页

作者： GU Chunling CHENG Kai YANG Shouqin RAN Shaofeng TAI Hengyap GAo Xiaofeng PANG dandan School of Acupuncture and Tuina Beijing University of Chinese MedicineBeijing 100029China Beijing medex Technology Co. Ltd.Beijing 100029China China Academy of Chinese Medical Sciences Beijing 100700China

oBJECTIVE:To compare the effects of Neiguan(PC6)acupuncture at different depths and retention time on arrhythmia duration,myocardial tissue morphology,mRNA expression level of L-type calcium channelα1C subunit and Ca^(2+)-Mg^(2+)-At Pase activity in tachirrhythmia model of ***:The tachyarrhythmia model was made by intravenous injection of barium chloride into the ears of rabbits.A total of 56 healthy adult male New Zealand big-eared white rabbits,apply the random number table method,divided into normal control group(group A),model group(group B),shallow needling Neiguan(PC6)10 min group(group C),shallow needling Neiguan(PC6)20 min group(group d),shallow needling Neiguan(PC6)30 min group(group E),deep needling Neiguan(PC6)10 min group(group F),deep needling Neiguan(PC6)20 min group(group G),deep needling Neiguan(PC6)30 min group(group H),7 animals in each *** were used to collect the duration of arrhythmia;hematoxylin-eosin staining method was performed on myocardial tissue,RT-PCR tested the expression ofα1C subunit mRNA,and the activity of Ca^(2+)-Mg^(2+)-ATPase were quantified by phosphorus determination ***:The duration of arrhythmia in each acupuncture treatment group was shortened to varying *** to the model group,the tissue damage from barium chloride inducing was improved in the acupuncture *** to the model group,except for group E,most treatment groups had varying degrees of improvement with significantly down-regulated L-type calcium channelα1C subunit mRNA expressions level and increased Ca^(2+)-Mg^(2+)-ATPase ***:The effect of acupuncture at Neiguan(PC6)with different depths and retention time can reduce the duration of arrhythmia induced by barium chloride relatively,improve the induced pathological changes,down regulate L-type calcium channelα1C subunit mRNA expressions level and increase Ca^(2+)-Mg^(2+)-ATPase *** the shallow and deep tissues of Neiguan(PC6)may

关键词： Point PC6(Neiguan) arrhythmias,cardiac calcium channels,L-type Ca^(2+)-Mg^(2+)-ATPase needle retention time

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

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Journal of Agricultural Science and Technology(B) 2016年第1期6卷 35-47页

作者： Bratko Filipic Lidija Gradisnik Eva Ruzic-Sabljic Brabara Trtnik Adriana Pereyra domen Jaklic Rok Kopinc Jana Potokar Almin Puzic Hrvoje Mazija Institute for Microbiology and Immunology Medical Faculty Ljubljana University of Ljubljana Zalogka 4 Ljubljana 1105 Slovenia Institute for Biomedical Sciences Medical Faculty Maribor University of Maribor Taborska 8 Maribor 2000 Slovenia Research and Control department MEDEX d o.o. Linhartova 49a Ljubljana 1000 Slovenia Veterinary Stations of Mostar Bigke Polje 106 Mostar 88000 Bosnia and Herzegovina Croatian Institute for Experimental and Translation oncology Koledinedka 3 Zagreb 10040 Croatia

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 （R J） 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） Greitl20 or fresh royal jelly （F-RJ）（MiZigoj） 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 Greitl20, 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 ofWSP Greitl20 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 Greit 120 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 Greitl20 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.

关键词： Propolis royal jelly Forest honey antimicrobial activity human macrophage cell line cell growth promoting activity.

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

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Journal of Agricultural Science and Technology(A) 2018年第1期8卷 36-47页

作者： Bratko Filipic Lidija Gradisnik Adriana Pereyra Rok Kopino Klemen Rihar Eva Ruzic-Sabljic Snezana Kramar damir dermie Eugen Soos Nicola Volpi Alfredo Fachini and Hrvoje Mazija Croatian Institute of Experimental and Translation oncology Koledinecka 3Zagreb 10040Croatia Institute of Biomedical Sciences Medical Faculty of MariborTaborska 8Maribor 2000Slovenia medex d.o.o. Linhartova 49bLjubljana 1000Slovenia Chengdujska 4 Ljubljana 1000Slovenia Institute of Microbiology and Immunology Medical FacultyZaloska 4Ljubljana 1105Slovenia division of Molecular Biology Ruaer Boskovic InstituteBijenicka 54Zagreb 10000Croatia Trg Sv.Ivana 5 Klostar Ivanic 10312Croatia department of Life Sciences University of Modena&Reggio EmiliaModena 41125Italy BNatural Via Gran Sasso 33Corbetta-MI 20011Italy

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.

关键词： Water-soluble propolis Greit 120 HuIFN-αN3 anti-influenza activity ribavirin Id_(50)

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