Study of medicinal potential of red fruits

Study of Medicinal Potential of Red Fruits

Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde

Tatiana Fernandes Soriano Louçano

Orientador: Professora Doutora Fernanda Maria Madaleno Rei Tomás Leal

Santos

Co-Orientador: Professora Doutora Ana Cláudia Correia Coelho

Universidade de Trás-os-Montes e Alto Douro

Study of Medicinal Potential of Red Fruits

Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde

Tatiana Fernandes Soriano Louçano

Orientador: Professora Doutora Fernanda Maria Madaleno Rei Tomás Leal

Santos

Co-Orientador: Professora Doutora Ana Cláudia Correia Coelho

Composição do Júri:

This original research was developed to achieve the Master Degree in

Biotechnology for Health Sciences (Decreto-Lei nº 74/2006 with the modifications introduced by the Decreto-Lei nº 107/2008,

Decreto-Lei nº 230/2009,Decreto-Lei n.º 113/2014, and Declaração de retificação n.º 774/2016)

This dissertation is dedicated to those who never stopped believing in me! “A winner is a dreamer who never gives up”

Acknowledgments

This work was developed in the Plant Biotechnology Lab of Genetics and Biotechnology Department and the Medical Microbiology Laboratory at the University of Tras-os-Montes and Alto Douro (UTAD). This would not be possible without the help and support of many people and institutions, which I want to sincerely thank:

To the Rector of the UTAD, Professor António Fontainhas Fernandes, Ph.D., the possibility of the realization of my dissertation at this University.

To the Direction of the Master’s degree in Biotechnology for Health Sciences, for the accepting of this dissertation.

To the Director of the Master’s degree in Biotechnology for Health Sciences, Professor Paula Filomena Martins Lopes, Ph.D, for the availability to any clarification.

To Professor Fernanda Maria Madaleno Rei Tomás Santos, Ph.D, my supervisor and to Professor Ana Cláudia Correia Coelho, Ph.D, my co-supervisor, for the orientation of this dissertation, the teachings; the availability for any clarification and for the critical review o f the work.

To Professor Maria Manuela do Outeiro Correia de Matos, Ph.D, for all the help and for all time spent in corrections and clarifications.

Totechnicians of Medical Microbiology Laboratory, Mrs Fatima Fraga, Mrs Sonia Dias, Mrs Fátima Peixoto and Mrs Lourdes Campos, for the kindness and help in the laboratory. To Cátia Castro and Emilie Alves, my trainees, for their help in this work.

To Daniela Alves, Dr., and Sofia Saraiva, Dr., for their help and cooperation in the laboratory. To Ana Sofia Soares, MSc., for the friendship and cooperation inside and outside the laboratory.

To Maria João Fernandes, MSc., and Sandra Alves, Dr., friendship, patience, availability, and for several hours inside and outside the laboratory.

I thank my mother, for being a mother, friend and confidente! Mom, I'll never have enough words or gestures to thank all you have done and are doing for me!

Thank you to all who in any way contributed to the realization of this work, thank you so much!

Publications resulting from the experimental work:

Review Paper:

Soriano T, Soares AS, Matos M, Leal F and Coelho AC. Mirtilos: Potenciais Benefícios para a Saúde, AGROTEC - Pequenos Frutos, 3º Trimestre 2015; vol. 12; pp. 23-25.

Communications resulting from the experimental work

Soriano T., Soares, A. S., Matos, M., Coelho A. C., Leal, F., 2015. Capacidade antifúngica de extratos aquosos de frutos vermelhos, sobre o género Aspergillus. VII Jornadas de Genética e Biotecnologia. 26-28 de Março, Vila Real, p. 39.

Soriano T, Soares AS, Alves S, Matos M, Coelho AC, Leal F,. 2016. Capacidade Antifúngica de extratos aquosos de frutos vermelhos, sobre Alternaria sp. VIII Jornadas de Genética e Biotecnologia. 10-12 Março. Vila Real, p.48.

T Soriano, A S Soares, S Alves, D Alves, S Saraiva, M Matos, A C Coelho, F Leal,. Ação Antifúngica do Extrato Aquoso de uma Mistura de Frutos Vermelhos, Sobre Penicillium marneffei.2016. IX Jornadas de Bioquímica da UTAD. 13 – 14 Abril, Vila Real, p.24.

Panel Communication:

Soriano T., Soares A. S., Alves E., Matos M., Coelho A. C., Leal F., 2015. Capacidade Antifúngica do Extrato Aquoso do Fruto Vaccinium corymbosum, Sobre Aspergillus niger. VII Jornadas de Genética e Biotecnologia. 26-28 de March, Vila Real, p. 78.

T Soriano; AS Soares; E Alves; AC Rocha; S mTeixeira; I Silva; M Matos; AC Coelho; F Leal, 2015. Potencial fungicida de extratos aquosos obtidos de Ribes rubrum e Vaccinium corymbosum, sobre Aspergillus sp. VIII Jornadas de Bioquímica da UTAD. 15-16 Abril, Vila Real, p. 35.

Soriano T., Soares A. S., Alves E., Silva I., Rocha A. C., Teixeira S., Matos M., Coelho A.C., Leal F., 2015. Antifungal capacity of aqueous extract of Ribes rubrum, on Aspergillus fumigatus. XXXIX Jornadas Portuguesas da Genética., 25-27 May, Braga, p.59.

Soriano T, Soares AS, Alves S, Matos M, Coelho AC, Leal F., 2016.Potencial antifúngico do extrato aquoso do fruto Vacinium corymbosum sobre Penicillium marneffei VIII Jornadas de Genética e Biotecnologia. 10-12 Março. Vila Real, p.80.

Study of Medicinal Potential of Red Fruits

Resumo

Os frutos vermelhos, também conhecidos por frutos do bosque, são designações sob as quais se agrupam estas pequenas bagas de cor vermelha, azulada ou preta. A opinião de muitos cientistas sobre as suas qualidades nutricionais e os seus benefícios, é a mesma: estas bagas, são concentrados de saúde.

Em particular os mirtilos (Vaccinium corymbosum), mirtilos vermelhos (Vaccinium oxycoccos), groselha vermelha (Ribes rubrum vermelha), groselha branca (Ribes rubrum branca), groselha preta (Ribes nigrum) e as amoras (Morus nigra), têm vindo a ser estudados nos últimos anos, pelas suas atividades anticancerígenas, anti-inflamatórias, antibacterianas, antifúngicas, antidiabéticas, protetoras do sistema imunitário, entre outras.

Vírus, bactérias e fungos são microrganismos que tanto podem ser completamente inofensivos para as plantas e para os animais, como podem ser agentes causadores de diferentes tipos de infeções, e em alguns casos ocasionar a morte.

O principal objetivo deste trabalho, foi conhecer melhor os potenciais benefícios para a saúde dos frutos vermelhos, e qual o seu efeito em diferentes microrganismos.

Neste estudo pretendeu-se: avaliar a atividade de Vaccinium corymbosum contra Aspergillus niger; verificar a capacidade fungicida de Ribes rubrum sobre Aspergillus fumigatus; comparar a atividade de antifúngicos comerciais compostos por Itraconazol (10 mg/mL) e Terbinafina (10 mg/mL) com a atividade antifúngica de extratos aquosos de mirtilo e mirtilo vermelho sobre diferentes fungos; comprovar a atividade fungicida e /ou fungistática de extratos aquosos de diferentes variedades de groselhas sobre Aspergillus spp.; Averiguar as propriedades antifúngicas de diferentes frutos vermelhos sobre Penicillium marneffei.

A atividade antifúngica dos diferentes extratos aquosos e dos antifúngicos comerciais, foi possível, através do método de crescimento micelial, permitindo comparar qual o extrato e qual a concentração mais eficaz contra os diferentes fungos testados.

Os resultados obtidos "in vitro" nos diferentes ensaios durante este trabalho, mostraram que os extratos aquosos de mirtilo (V. corymbosum), mirtilo vermelho (V. oxycoccos), groselhas vermelhas (Ribes rubrum red), groselhas brancas (Ribes rubrum white), groselhas

bacteriostático em concentrações menores de 5 mg / mL. O crescimento micelial de diferentes fungos patogénicos (Aspergillus spp., Alternaria spp., Penicillium spp., Rhizopus spp., Trichophyton spp., and Talaromyces (Penicillium) marneffei) é inibido por extratos aquosos dos frutos acima referidos, com concentrações inferiores a 30 mg / mL. No entanto, é necessário continuar a realizar mais estudos com mais microrganismos e diferentes concentrações antes de se poderem utilizar "in vivo" os extratos destes frutos no tratamento de doenças provocadas por estes agentes.

Palavras-chave: Frutos vermelhos; potencial medicinal; microrganismos; atividade fungicida;

Abstract

The berries, also known as wild berries, are designations under which are grouped these little berries of red, blue or black colors. The opinion of many scientists on their nutritional qualities and its benefits, is the same: these berries are concentrated health.

In particular blueberries (Vaccinium corymbosum), cranberries (Vaccinium oxycoccos), red currant (Ribes rubrum red), white currant (Ribes rubrum white), black currant (Ribes nigrum) and blackberries (Morus nigra) have been studied in recent years for its anti-cancer activity, anti-inflammatory, antibacterial, antifungal, antidiabetic, protective immune system, among others.

Viruses, bacterias and fungi are microorganisms that can be either completely harmless to plants and animals, as can be causative agents of different types of infections, and in some cases leading to death.

The main objective of this study was to better understand the potential health benefits of red fruits, and what is its effect on different microorganisms.

This study was intended to: evaluate the Vaccinium corymbosum activity against Aspergillus niger; verify the Ribes rubrum red fungicide capacity of Aspergillus fumigatus; comparing the activity of a commercial antifungal compounds Itraconazole (10 mg / mL) and terbinafine (10mg / mL) with the antifungal activity of aqueous extracts of blueberry and cranberries on various fungi; confirm the fungicidal activity and / or fungistatic of aqueous extracts of different varieties of currants on Aspergillus spp.; investigating the antifungal properties of different berries on Penicillium marneffei.

Antifungal activity of different aqueous extracts and commercial antifungals, was possible through the mycelial growth method, allowing you to compare which extract and what the most effective concentration against different fungi tested.

The results "in vitro" in the various trials during this work, showed that, the aqueous extracts of Blueberry (V. corymbosum), cranberry (V. oxycoccos), red currants (Ribes rubrum red), white currants (Ribes rubrum white), black currants (Ribes nigrum L.), and mulberry (Morus nigra), have bactericidal effect against Staphylococcus aureus at concentrations between 40 mg / mL and 5 mg / mL and bacteriostatic effect at concentrations below 5 mg / mL. Mycelial growth of different pathogenic fungi (Aspergillus spp., Alternaria spp., Penicillium spp., Rhizopus spp., Trichophyton spp., and Talaromyces (Penicillium) marneffei) is inhibited by aqueous extracts of the above fruits, with concentrations below 30 mg / mL. However, is necessary to continue further studies with more microorganisms and different concentrations before they can use "in vivo" the extracts of these fruits in the treatment of diseases caused by these agents

Keywords: Red fruits; medicinal potencial; microorganisms; fungicidal activity

Index

Acknowledgments ...v

Publications resulting from the experimental work: ... vi

Communications resulting from the experimental work ... vi

Resumo ... vii

Abstract ... viii

Index ...x

Index of Figures ... xiii

Index of Tables ...xvi

List of Abbreviations : ...xvii

Layout of this dissertation ...xix

Chapter I:General Introduction and objectives ... 1

I.1 – Background ... 2

I.2 – Red Fruits ... 5

I.2.1 – Vaccinium spp. ... 6

I.2.1.1 – Vaccinium corymbosum...6

I.2.1.2 – Vaccinium oxycoccos ...7

I.2.2– Ribes spp. ... 8

I.2.2.1 – Ribes rubrum red ...9

I.2.2.2 – Ribes rubrum white ...9

I.2.2.3 – Ribes nigrum ... 10

I.2.3 – Morus nigra ... 11

I.2.4 – Red fruits Benefits ... 12

I.3 – Bacterias ... 14

I.3.1 – Staphyloccocus aureus ... 14

I.4. – Fungi ... 15

I.4.1 – Alternaria spp. ... 16

I.4.1.1 – Alternaria alternata ... 16

I.4.2 – Aspergillus spp. ... 17

I.4.2.1 – Aspergillus fumigatus ... 18

I.4.2.2 – Aspergillus niger ... 18

I.4.3 – Trichophyton mentagrophytes ... 19

I.4.4 – Penicillium spp. ... 20

I.5 – Mycoses ... 22

I.6 – Antifungals ... 25

I.7 – Objectives: ... 26

I.8 – References... 27

Chapter II: Antibacterial Potential of Berries Extracts Against Staphylococcus aureus ... 39

II.1 – Abstract ... 40

II.2 – Introduction ... 41

II.3 – Materials and methods ... 42

II.3.3 – Antimicrobial activity ... 43

II.4 – Results and Discussion ... 44

II. 5 – Conclusion ... 49

II. 6 –References ... 50

Chapter III:Antifungal Activity of Vaccinium corymbosum against Aspergillus niger ... 53

III.1 – Letter to Editor: ... 54

III.2 – References ... 55

Chapter IV:Fungicide Capacity of Ribes rubrum againstAspergillus fumigatus ... 57

IV.1 – Research Note ... 58

IV.2 – References ... 59

Chapter V: Effectiveness of Blueberry and Cranberry Aqueous Extract on different Pathogenic Fungi ... 61

Abstract ... 62

V.1. Introduction ... 63

V.2. Materials and Methods... 64

V.2.1. Plant material ... 64

V.2.2. Method of mycelial growth ... 65

V.3.1. Results ... 66

V.3.2. Discussion ... 70

V. 4. Conclusion... 71

V. 5. References ... 72

Chapter VI:Antifungal properties of different cultivars of Currants against Aspergillus spp. ... 75

VI. 3.1. Plant Material ... 78

VI. 3.2. Mycelial growth method ... 78

VI. 4.1 – Results ... 79

VI. 5 – Conclusion ... 82

VI. 6 – References ... 82

Chapter VII: Fungicide Capacity of Different Berries Against Talaromyces (Penicillium) marneffei ... 85

VII. 1 – Abstract... 86

VII. 2 – Introduction... 87

VII. 3 – Materials and methods... 88

VII. 3.1 – Plant Material ... 88

VII. 3.2 – Mycelial growth method ... 88

VII.4 – Results and Discussion ... 89

VII. 4.1 Results ... 89

VII. 4.2 – Discussion ... 92

VII. 5 – Conclusion ... 93

VII. 6 – References... 93

Chapter VIII:Final Considerations ... 97 Supplemental Material ... XXI Supplement 1 ... XXII Supplement 2 ... XXIII Supplement 3 ... XXIV Supplement 4 ... XXV Supplement 5 ... XXVI

Index of Figures

Chapter I:

FIGURE I.1.BIOACTIVE COMPONENTS IN BERRIES ... 3

FIGURE I.2.A.BERRY PHENOLICS AND FLAVONOIDS AND THEIR BIOLOGICAL ACTIVITIES ... 5

FIGURE I.2.1.1.B.V. CORYMBOSUM FRUITS IN DIFFERENT MATURATION. ... 7

FIGURE I.2.1.1.A.V. CORYMBOSUM SHRUB. ... 7

FIGURE I.2.1.2.B.VACCINIUM OXYCOCCOS – RIPE FRUIT ... 8

FIGURE I.2.1.2A.VACCINIUM OXYCOCCOS SHRUB. ... 8

FIGURE I.2.2.A.CURRANTS... 8

FIGURE I.2.2.B.CURRANTS"STRIGS". ... 8

FIGURE I.2.2.1.A.RIBES RUBRUM RED. ... 9

FIGURE I.2.2.1.B.RIBES RUBRUM RED "STRIGS". ... 9

FIGURE I.2.2.2.A.RIBES RUBRUM WHITE. ... 10

FIGURE I.2.2.2.B.RIBES RUBRUM WHITE "STRIGS". ... 10

FIGURE I.2.2.3.A.RIBES NIGRUM.. ... 10

FIGURE I.2.2.3.B.RIBES NIGRUM FRUITS. ... 10

FIGURE I.2.3.A.MORUS NIGRA TREE. ... 11

FIGURE I.2.3.B.MORUS NIGRA FRUIS... 11

FIGURE I.3.1.A.STAPHYLOCOCCUS AUREUS ... 15

FIGURE I.3.1.B. CHILD WHITH SUBCUTANEOUS ABSCESS WING METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS ... 15

FIGURE I.4.1.A.ALTERNARIA SP.LACTOPHENOL COTTON BLUE PREPARATION SHOWING DARKLY PIGMENTED CHAINS OF MURIFORM CONIDIA ... 17

FIGURE I.4.1.B.PHOTOGRAPH OF INVASIVE A. ALTERNATA INFECTION AFTER FIRST RESECTION.NOTE HYPEREMIC AND NECROTIC TISSUE ... 17

FIGURE I.4.2.1.A.ASPERGILLUS FUMIGATUS LACTOPHENOL COTTON BLUE PREPARATION SHOWING CONIDIAL HEADS ... 18

FIGURE I.4.2.1.B. ACUTE INVASIVE PULMONARY ASPERGILLOSIS, CAUSED BY ASPERGILLUS FUMIGATUS, POST TRANSPLANT ... 18

Figure I.4.2.2.A Aspergillus niger in a cavitary lung lesion showing both hyphae and conidial head……….18

Figure I.4.2.2.B. External auditory canal with Aspergillus niger overgrowth manifesting as a cottony matrix topped by small black balls………...……….19

FIGURE I.4.3.A.TRICHOPHYTON MENTAGROPHYTES,LACTOPHENOL COTTON BLUE. ... 20

Figure I.4.3.B. Multiple inflammatory, itching ringworm lesions, caused by Trichophyton mentagrophytes……….……….20

FIGURE I.4.4.1.A.GOMORI METHENAMINE SILVER–STAINED YEAST FORMS OF P. MARNEFFEI, INCLUDING FORMS WITH SINGLE, WIDE, TRANSVERSE SEPTA (CENTER) ... 21 FIGURE I.4.4.1.B.SKIN LESIONS ASSOCIATED WITH P. MARNEFFEI INFECTION.CHRONIC ULCERS A, PUSTULAR

FIGURE I.4.5.A. RHIZOPUS SP. SHOWING SPORANGIUM AND RHIZOIDS ... 22

FIGURE I.4.5.B.AN ULCERATIVE LESION AT THE RIGHT CHEST OF A FORMERLY HEALTHY 2-YEAR-OLD BOY.THE RARE FUNGUS RHIZOPUS ORYZAE CAUSED THIS INFECTION ... 22

Chapter II:

FIGURE II.3.3.1.MICROPLATE 1 AND 2 WITH DIFFERENT EXTRACTS OF FRUITS, BACTERIA AND CULTURE MEDIUM. ... 44 FIGURE II.3.3.2.MICROPLATE 1 AND 2 WITH DIFFERENT EXTRACTS OF FRUITS, BACTERIA, CULTURE MEDIUM

AND RESARZURIN. ... 44 FIGURE II.4.1.MICROPLATE 1,14 HOURS AFTER INOCULATION, THE WELLS MARKED WITH A CIRCLE ARE THE

WELLS WHERE THE SAMPLES WERE WITHDRAWN FOR INOCULATION IN PETRI DISHES... 46 FIGURE II.4.2.[2.5 MG/ML]V. CORYMBOSUM AQUEOUS EXTRACT, THERE IS THE GROWTH OF VARIOUS COLONIES OF S. AUREUS. ... 46 FIGURE II.4.3.[0.625 MG/ML]R. NIGRUM EXTRACT, THERE IS THE GROWTH COLONY OF S. AUREUS. ... 46 FIGURE II.4.4.[0.625 MG/ML]R. RUBRUM RED AQUEOUS EXTRACT, THERE IS GROWTH OF SMALL COLONIES OF S.

AUREUS. ... 46 FIGURE II.4.5.MICROPLATE 2,14 HOURS AFTER INOCULATION, THE WELLS MARKED WITH A CIRCLE ARE THE WELLS WHERE THE SAMPLES WERE WITHDRAWN FOR INOCULATION IN PETRI DISHES... 47 FIGURE II.4.6.[2.5 MG /ML] OF AQUEOUS EXTRACT OF MORUS NIGRA, THERE IS ONLY ONE COLONY OF S. AUREUS. ... 47 FIGURE II.4.7.[2.5 MG / ML] OF R. RUBRUM WHITE EXTRACT, IS OBSERVED GROWTH OF VARIOUS COLONIES OF

S. AUREUS. ... 47 FIGURE II.4.8.[1.25 MG/ML]V. OXICOCCUS EXTRACT, IS OBSERVED THE GROWTH OF VARIOUS COLONIES OF S.

AUREUS. ... 47

Chapter V:

FIGURE V.3.1.MYCELIAL GROWTH AFTER 7 DAYS OF FOUR DIFERENT FUNGI:A–PENICILLIUM SP. MYCELIAL GROWTH (CONTROL); B –PENICILLIUM SP. MYCELIAL GROWTH (V. CORYMBOSUM AQUEOUS EXTRACT [30MG/ML]);C–PENICILLIUM SP. MYCELIAL GROWTH (V. OXYCOCCOS AQUEOUS EXTRACT [10MG/ML]);D – PENICILLIUM SP. MYCELIAL GROWTH (ITRACONAZOLE [10MG/ML]); E– PENICILLIUM SP. MYCELIAL GROWTH (TERBINAFINE HYDROCHLORIDE [10MG/ML]); F – ALTERNARIA SP. MYCELIAL GROWTH

(CONTROL);G–ALTERNARIA SP. MYCELIAL GROWTH (V. CORYMBOSUM AQUEOUS EXTRACT [30MG/ML]);H –ALTERNARIA SP. MYCELIAL GROWTH (V. OXYCOCCOS AQUEOUS EXTRACT [30MG/ML]);I–ALTERNARIA SP.

MYCELIAL GROWTH (ITRACONAZOLE [10MG/ML]);J–ALTERNARIA SP. MYCELIAL GROWTH (TERBINAFINE HYDROCHLORIDE [10MG/ML]);K–TRICHOPHYTON MENTAGROPHYTES MYCELIAL GROWTH (CONTROL);L–

T. MENTAGROPHYTES MYCELIAL GROWTH (V. CORYMBOSUM AQUEOUS EXTRACT [20MG/ML]); M – T.

MENTAGROPHYTES MYCELIAL GROWTH (V. OXYCOCCOS AQUEOUS EXTRACT [10MG/ML]); N – T.

MENTAGROPHYTES MYCELIAL GROWTH (ITRACONAZOLE [10MG/ML]);O–T. MENTAGROPHYTES MYCELIAL

GROWTH (TERBINAFINE HYDROCHLORIDE [10MG/ML]); P–RHIZOPUS SP. MYCELIAL GROWTH CONTROL); Q–RHIZOPUS SP. MYCELIAL GROWTH (V. CORYMBOSUM AQUEOUS EXTRACT [20MG/ML]);R–RHIZOPUS SP.

MYCELIAL GROWTH (V. OXYCOCCOS AQUEOUS EXTRACT [10MG/ML]); S – MYCELIAL GROWTH

(ITRACONAZOLE [10MG/ML]);T– MYCELIAL GROWTH (TERBINAFINE HYDROCHLORIDE [10MG/ML]).THE BLUE CIRCLE CORRESPONDS TO THE DIAMETER OF THE MYCELIAL GROWTH OF THE FUNGUS IN COTROL PLATE. ... 69

Chapter VI:

FIGURE VI.4.1.1. A – ASPERGILLUS NIGER MYCELIAL GROWTH AFTER 7 DAYS (CONTROL); B – A. NIGER

MYCELIAL GROWTH (RIBES RUBRUM RED EXTRACT [30 MG/ML]) AFTER 7 DAYS;C–A. NIGER MYCELIAL

GROWTH (R. RUBRUM WHITE EXTRACT [30 MG/ML]) AFTER 7 DAYS;D–A. NIGER MYCELIAL GROWTH (R.

NIGRUM EXTRACT [30 MG/ML]) AFTER 7 DAYS.THE BLUE CIRCLE CORRESPONDS TO THE DIAMETER OF THE MYCELIAL GROWTH OF THE FUNGUS IN CONTROL PLATE. ... 80 FIGURE VI.4.1.2A–ASPERGILLUS FUMIGATUS MYCELIAL GROWTH AFTER 7 DAYS (CONTROL);B–A. FUMIGATUS

MYCELIAL GROWTH (RIBES RUBRUM RED EXTRACT [30 MG/ML]) AFTER 7 DAYS; C – A. FUMIGATUS

MYCELIAL GROWTH (R. RUBRUM WHITE EXTRACT [30 MG/ML]) AFTER 7 DAYS;D–A. FUMIGATUS MYCELIAL

GROWTH (R. NIGRUM EXTRACT [30 MG/ML]) AFTER 7 DAYS.THE BLUE CIRCLE CORRESPONDS TO THE DIAMETER OF THE MYCELIAL GROWTH OF THE FUNGUS IN CONTROL PLATE. ... 81

Chapter VII:

FIGURE VII.4.1.A–TALAROMYCES (PENICILLIUM) MARNEFFEI MYCELIAL GROWTH AFTER 7 DAYS (CONTROL);

B–T. MARNEFFEI MYCELIAL GROWTH (VACCINIUM CORYMBOSUM AQUEOUS EXTRACT [30 MG/ML]);C–T.

MARNEFFEI MYCELIAL GROWTH (V. OXYCOCCOS AQUEOUS EXTRACT [30 MG/ML]);D–T. MARNEFFEI (RIBES RUBRUM RED AQUEOUS EXTRACT [30 MG/ML]);E–T. MARNEFFEI. MYCELIAL GROWTH (R. RUBRUM WHITE

AQUEOUS EXTRACT [30 MG/ML]);F–T. MARNEFFEI MYCELIAL GROWTH (R. NIGRUM AQUEOUS EXTRACT [30 MG/ML]);G–T. MARNEFFEI MYCELIAL GROWTH (MORUS NIGRA AQUEOUS EXTRACT [30 MG/ML]) AFTER

7 DAYS.THE BLUE CIRCLE CORRESPONDS TO THE DIAMETER OF THE MYCELIAL GROWTH OF THE FUNGUS IN CONTROL PLATE. ... 91

Index of Tables

TABLE I.2.4.1.COMPARATIVE ACCOUNT OF AND BIOLOGICAL PROPERTIES OF BERRIES. ... 13

TABLE I.4.6.1. CLASSIFICATION OF HUMAN MYCOSES AND REPRESENTATIVE ETIOLOGIC AGENTS ... 24 Chapter II:

TABLE II.4.1.THE ANTIBACTERIAL EFFECTS OF THE TESTED PLANT AQUEOUS EXTRACTS WERE ALSO CLASSIFIED ACCORDING TO THE FOLLOWING SCHEME: NO BACTERIAL GROWTH (-); WITH BACTERIAL GROWTH (+); TURNING ZONE BEGINS BACTERIAL GROWTH (-/+). ... 45 TABLE II.4.2.THE ANTIBACTERIAL EFFECTS OF THE TESTED PLANT AQUEOUS EXTRACTS WERE ALSO CLASSIFIED

ACCORDING TO THE FOLLOWING SCHEME: NO BACTERIAL GROWTH (-); WITH BACTERIAL GROWTH (+);

TURNING ZONE BEGINS BACTERIAL GROWTH (-/+). ... 46 Chapter V:

TABLE V.3.1. PERCENT INHIBITION OF AQUEOUS EXTRACTS OF TWO DIFFERENT PLANTS (B, C) AND TWO COMMERCIAL ANTIFUNGAL COMPOUNDS (D,E) ON FOUR DIFFERENT FUNGI (PENICILLIUM SP.,ATERNARIA

SP.,TRICHOPHYTON MENTAGROPHYTES AND RHIZOPUS SPP.), ON THE 3RD,5TH AND 7TH DAY OF READING. ... 67

Chapter VI:

TABLE VI.4.1.PERCENT INHIBITION OF AQUEOUS EXTRACTS OF THREE DIFFERENT CURRANTS (RED CURRANT –

RIBES RUBRUM RED; WHITE CURRANT –RIBES RUBRUM WHITE; BLACKCURRANT –RIBES NIGRUM) ON TWO DIFFERENT FUNGI (ASPERGILLUS NIGER AND ASPERGILLUS FUMIGATUS), ON THE 3RD,5TH AND 7TH DAY OF READING. ... 79 Chapter VII:

TABLE VII.4.1.PERCENT INHIBITION OF AQUEOUS EXTRACTS OF DIFFERENT BERRIES (BLUEBERRY –VACCINIUM CORYMBOSUM, CRANBERRY –V. OXYCOCCOS,RED CURRANT –RIBES RUBRUM RED; WHITE CURRANT –RIBES RUBRUM WHITE; BLACKCURRANT –RIBES NIGRUM AND MULBERRY –MORUS NIGRA) ON TALAROMYCES (PENICILLIUM) MARNEFFEI, ON THE 3RD_,5TH _{AND 7}TH _{DAY OF READING. ... 90}

List of Abbreviations :

A. alternata – Alternaria alternata A. flavus – Aspergillus flavus

A. fumigatus – Aspergillus fumigatus A. nidulans – Aspergillus nidulans A. niger – Aspergillus niger BHI – Brain Heart Infusion CO2 – Carbon dioxide

C. neoformans – Cryptococcus neoformans DNA – Deoxyribonucleic acid

E. coli – Escherichia coli

FAO – Food and Agriculture Organization of the United Nations F. verticillioides – Fusarium verticillioide

H. pylori – Helicobacter pylori

HIV – Human Immunodeficiency Virus M. nigra – Morus nigra

MRSA – S. aureus methicillin-resistant N – Nitrogen

PDA – Potato dextrose agar

P. boydii – Pseudallescheria boydii P. jirovecii – Pneumocystis jirovecii P. marneffei – Penicillium marneffei ROS – reactive oxygen species R. rubrum red – Ribes rubrum red R. rubrum white – Ribes rubrum white R. nigrum – Ribes nigrum

RNA – Ribosomal ribonucleic rRNA – Ribosomal ribonucleic acid spp. –species

T. mentagrophytes - Trichophyton mentagrophytes UTAD – University of Trás-os-Montes and Alto Douro UV – ultra violet

V. corymbosum – Vaccinium corymbosum V. oxycoccos – Vacciniumoxycoccos WHO – World Health Organization

Layout of this dissertation

This dissertation is constituted by seven chapters: Chapter I – General introduction and Objectives; Chapter II to Chapter VII corresponding to scientific articles (see below) submitted to international journals with peer review and belonging to the Scientific International Index; Chapter VIII corresponds to the final considerations of the research developed.

Soriano T, Fernandes M J, Alves S, Soares A S, Matos M, Leal F, Coelho A C (2016) Antibacterial Potential of Berries Extracts Against Staphylococcus aureus. Paper not Submitted (Chapter II).

Soriano T, Soares A S, Matos M, Leal F, Coelho A C (2016) Antifungal Activity of Vaccinium corymbosum against Aspergillus niger. Letter Submitted “Journal Agricultural and Food Chemistry” (Chapter III).

Tatiana Soriano, Ana Sofia Soares, Manuela Matos, Fernanda Leal, Ana Cláudia Coelho (2016) Fungicide Capacity of Ribes rubrum against Aspergillus fumigatus. Research Note Submitted “Journal of Food and Drug Analysis” (Chapter IV).

Soriano T, Alves S, Soares A S, Fernandes M J, Matos M, Leal F, Coelho A C (2016) Effectiveness of Blueberry and Cranberry Aqueous Extract Extract on Different Pathogenic Fungi. Paper Submitted to “Journal of Herbal Medicine” (Chapter V).

Soriano T, Soares A S, Alves S, Castro C, Matos M, Coelho A C, Leal F, (2016) Antifungal properties of different cultivars of Currants against Aspergillus spp. Paper Submitted to “Plant Foods for Human Nutrition” (Chapter VI).

Soriano T, Alves S, Soares A S, Alves D, Saraiva S, Matos M, Leal F, Coelho A C, (2016) Fungicide Capacity of Different Berries Against Talaromyces (Penicillium) marneffei. Paper Submitted to “Journal of Berry Research” (Chapter VII).

Chapter I:

I.1 – Background

Nature has been a source of medicinal agents for thousands of years and an important number of modern drugs have been isolated from natural sources and many of them are based on their use in traditional medicine [1, 2].

Plants contain a great variety of phytonutrients, of which many have antioxidant properties. Antioxidant compounds include vitamins, phenols carotenoids and flavonoids. Phenolic are secondary plant metabolites found in the majority of herbs, vegetables, and tea [3, 4].

The constituents identified as secondary metabolites, secondary products, or natural products, have no generally recognized, direct roles in the processes of photosynthesis, respiration, solute transport, translocation, protein synthesis, nutrient assimilation, differentiation or metabolism processes as the formation of carbohydrates, proteins and lipids [5].

Plants are a great source of antioxidant compounds, such as phenolic acids, flavonoids (including anthocyanins and tannins), vitamins and carotenoids, that may be used as pharmacologically active products [6, 7]. The great wealth of chemical medicinal plants that have microbiocidal active ingredients, making it a potential source of molecules that can be used by plants for defense against microorganisms [8, 9]. Produce a variety of groups of bioactive compounds in plant tissues as secondary metabolites that have antifungal activity to stop or inhibit the advance of mycelia growth, inhibition of germination or reduce sporulation of fungal pathogens, each these groups presented variable mechanisms of action, like the toxicity of polyphenols in microorganisms is attributed to enzyme inhibition by oxidation of compounds [5].

The culture of plant tissue can be a potential alternative for the production of secondary metabolites. The main advantages of tissue culture over the traditional method is the synthesis of bioactive metabolites in a controlled environment, independent of climate and soil conditions, negative biological influences that affect the production of secondary metabolites in nature are eliminated (microorganisms and insects), control the cell growth and regulate the production of metabolites [10].

Diet rich in vegetables and fruits is the starting point for good health [9,11,12,13, 14]. Fruits like berries are consumed fresh, frozen, as processed products and derivatives, including yogurts, juices, jams, dried and canned fruits [9, 15].

The red fruits are a rich source of non-nutritive, nutritional and bioactive compounds, such as the flavonoids, phenolics, anthocyanins, phenolic acids, stilbenes, and tannins as well as nutritional compounds (Figure I.1.), such as sugars, essential oils, carotenoids, vitamins, and minerals [9, 12, 16, 17, 18].

Anthocyanins are the largest group of pigments (red, purple, violet, blue) soluble in water polyphenolic constituent of red fruits such as mulberries, currants, cranberries or blueberries [14, 19].

In recent years, several studies have shown how, red fruits bioactive compounds can exhibit a wide range of biological activities, like, antioxidant, antinflammatory, antifungal [20], antimicrobial, anticancer, prevention of degenerative cardiovascular, diseases [9, 13, 14, 21, 22, 23] and treatment of diabetes mellitus [19].

Current lifestyle including diet, is causing the over production of free radicals and reactive oxygen species (ROS) in our organism and decreasing the physiological antioxidant capacity [6, 24]. Unhealthy diet plays a significant role with body fatness and a risk factor for several cancers. Experimental studies have indicated that, diet may influence the cancer process in several ways [25].

Many scientists have reported the benefits of berry consumption in relation to human health [9, 26, 27, 28, 29, 30, 31]. This is the reason, to these fruits can be used in varying proportions as ingredients in so called “Functional Foods” [15, 32].

Bacterial infections are a global health problem associated with high levels of mortality and morbidity [33, 34].

Staphylococcus aureus is an opportunistic pathogen often asymptomatic human body but can also be the main cause of purulent infections in humans with the potential to contaminate all tissues and all the anatomical sites [35, 36]. When S. aureus strains acquire the gene mecA, which is transported in a large mobile genetic element called the staphylococcal chromosomal cassette mec (SCCmec), becoming S. aureus methicillin-resistant (MRSA) and is resistant to all beta-lactam antibiotics [36, 37]. The resistance of pathogens to antibiotics complicates treatment of the infection being common administration of multiple drugs to improve the therapeutic effects [33].

Fungi are one of the known biological factors that have a negative impact on human health and constitute particular threat to health. Airborne spores of these microorganisms are ubiquitous, and under favorable conditions begin development cycle. Molds have the ability to produce toxins that have harmful effects on human body and on animals [38, 39].

Systemic mycoses are a heterogeneous group of infections caused by different species of fungi that mainly affect individuals with primary or secondary alterations of immunity [40, 41], Alternaria spp. [42], Aspergillus spp., Penicillium spp. [43], Rhizopus spp. [44] and Trichophyton spp. are an example of fungi, which cause mycoses in animals and humans [45]. The use of plant extracts can be an alternative to antifungics [46] antibiotics, and immunoprophylactic [47]. Essential oils from red fruits have shown significant antimicrobial activity against pathogenic bacteria [48, 49]. Thus, the berries may be a natural alternative for the treatment of infectious diseases [49].

I.2 – Red Fruits

Berries are called, small fruits that can be eaten whole without large seeds [14]. Since always, Man picked ripe berries [50]. Nowadays, berry fruits are eaten fresh, frozen and in processed products and derivatives including dry, canned fruits, yogurts, beverages, and jams [11, 51].

Different studies indicate that the red fruits provide significant health benefits due to high levels of polyphenols, antioxidants, vitamins, minerals and fibers. Polyphenols (Figure I.2.A.), include a wide variety of compounds, divided into various classes, such as hydroxybenzoic acids, hydroxycinnamic acid, anthocyanins, proanthocyanidins, flavonols, flavones, flavanones, flavonoids, isoflavones, lignans and stilbenes that occur in these small fruit [9, 52, 53, 54, 30].

The World Health Organization (WHO) emphasizes the importance of antioxidant activity of phenolic compounds for the prevention of major health problems including cardiovascular disease, diabetes, cancer and obesity. Many berries phenolic compounds are responsible for the

color (anthocyanins) and flavor (tannins). Phenolic compounds protect plants against adverse conditions, such as infection, physical damage, UV radiation and other factors. The primary function of resveratrol is to protect plants against fungal infections [9, 11, 28, 51].

Blueberry (V. corymbosum), cranberry (V. oxycoccos), red currants,(Ribes rubrum L.), black currants (Ribes nigrum L.) [26], and mulberry (Morus nigra) belong to the group of berries more functional properties [9, 11].

I.2.1 – Vaccinium spp.

The Vaccinium genus comprises about 450 species dispersed throughout the world. However, it is the United States who play a greater role in the development of new cultivars, production and processing industry, as well as blueberries consumption. Probably more than 95% of existing cultivars are complex hybrids more or less North American species [55]. Two of the best-known species are Vaccinium corymbosum and V. oxycoccos [27, 56].

These small berries, with shapes and dimensions of the most varied, were harvested and consumed by both animals and humans since prehistory. The recollection of fruits of wild species for their own use or for sale, remains an important activity [57].

Vaccinium genus berries are known for the high anthocyanin content [58]. Many researchers tested extracts of berries against several common diseases achieving significant results, suggesting that, consumption of these fruits is associated with a reduced risk neurodegenerative diseases, vascular and different types of cancer are some examples [10, 18, 59, 60, 61].

I.2.1.1 –

corymbosum

Vaccinium corymbosum (V. corymbosum), blueberry (Figures I.2.1.1), are flowering plants and deciduous, which belong to the genus Vaccinium [58]. Traditionally used for both fresh consumption and processed into jams, jellies and juices [10].

The leaves of blueberries, have been used medicinally, for generations [62]. Fruits are a good source of chlorogenic acid, quercetin, kaempferol, myricetin, procyanidins, catechin, epicatechin, resveratrol, and vitamin C to contribute to antioxidant activity [9,58, 60, 62].

Anthocyanins, polyphenols are recognized by their ability to deliver and activate antioxidant cell protection, to inhibit expression of inflammatory gene and, consequently, protect against

However, not many research exists on the antimicrobial potential [64], and antifungal of blueberry extracts [20].

Recently, several studies have shown that the polysaccharides possess antiviral activity, anti-aging properties, resistance to bone marrow suppression, anti-tumor, and immune-regulatory activities, side effects or limited adverse reactions, increasing research into the effects polysaccharide immunomodulators [61, 65].

Advertised as a "superfood" because of the rich nutrients and bioactive substances [64], FAO (Food and Agriculture Organization of the United Nations), recommends, it as one of the top five healthy fruits [61].

I.2.1.2 –

oxycoccos

Vaccinium oxycoccos (Figure I.2.1.2), belongs to the Ericaceae family, genus Vaccinium oxycoccos L. species. It is a small shrub, the fruit is small subspherical and dark red shape when mature [28].

Cranberry is a native American plant, that has been widely investigated [56, 66], because, as the blueberry is very rich in bioactive compounds capable of improving immune function, prevention of cardiovascular disease, tumor suppression, antibacterial properties [58], among others [9, 27, 51] Extracts of cranberry contain high molecular weight materials that inhibit viral adhesion and infectivity of the A and B influenza viruses, can suppress urinary infections, H. pylori infections in epidemically affected populations, and influenza virus infections [11].

Its use in cosmetics and pharmaceutical products has been growing in recent years [61, 66]. Figure I.2.1.1.A. V. corymbosum shrub. Figure I.2.1.1.B. V. corymbosum fruits in different maturation.

I.2.2– Ribes spp.

The Ribes genus has more than 150 known species and hundreds of cultivated varieties (cultivars) [67], Ribes spp., belong to the family Grossulariaceae are berries grown in European and commercial plantations gardens [68].

The currant fruit may be colored red, white, pink (Ribes rubrum L.), and black (Ribes nigrum L.), and are produced and harvested grape bunches called "strigs" (Figures I.2.2.) [67],), as its flavor is acidic, they are rarely eaten fresh, but processed into juices, jams, jellies and syrup s [67, 68, 24]. Currants fruits are a source of natural antioxidant compounds, contain high levels of phenolics (flavonoids and anthocyanins) [69], fiber, vitamins, and minerals [24], for use in the food industry, cosmetic and pharmaceutical industries [70, 71].

Currants have been used for centuries as a food and medicine mainly in Western and Northern Europe and North America [26], different studies indicate that these fruits have anticancer and anti-inflammatory properties [72] and fungicidal [73].

Figure I.2.1.2 A. Vaccinium oxycoccos shrub. Figure I.2.1.2.B. Vaccinium oxycoccos – ripe fruit [149].

I.2.2.1 – Ribes rubrum red

The color red of Ribes rubrum fruits (Figure I.2.2.1.) can be assigned to different anthocyanins, a subclass of phenolic compounds, located in the soft tissue vacuoles, located mainly in the fruit peel [70]. Besides these compounds, many other phenolic compounds are present in currants, however, they have no influence on the color of the fruit. Phenolic compounds have different functions such as UV protection, act as antioxidants, preventing the pathogens, and influence the taste of fruit [67, 70].

Cascade, Jonkeers van, Detvan, Red Lake, Wilder, Rovada and Tatran are examples of different redcurrants cultivars [67]. Z

I.2.2.2 – Ribes rubrum white

The white and pink currants, are the most difficult to find, they grow as red varieties (Figure I.2.2.2.A.), but it has a unique taste, sweeter. The fruits are small, and their color can be yellow or pink white, and opaque to translucent (Figure I.2.2.2.B.) [67].

The most common varieties are Imperial White, Pink Champagne, Blanka, Primus [67]. Figure I.2.2.1.B. Ribes rubrum red. Figure I.2.2.1.A. Ribes rubrum red "strigs".

I.2.2.3 – Ribes nigrum

Blackcurrant (Figure I.2.2.3) (Ribes nigrum L.), with such white currants and red (Ribes rubrum L.) is a small evergreen shrub that reaches a height of up to 1.5 / 2 meters [73].

The berries are dark blue or almost black, with intense aroma and sweet-sour taste [67], is widely used for drinks and jams [73]. Due to their essential nutrients, such as antioxidant vitamins, folic acid, potassium and fibers [74], have been tested in different clinical trials [9, 13, 73]. Ben Sarek, Consort, Coronet and Crusader, Ben Lomond, Titania, Tisel and Black September, are the most common varieties [67].

Figure I.2.2.2.A. Ribes rubrum white. Figure I.2.2.2.B. Ribes rubrum white "strigs".

I.2.3 – Morus nigra

The mulberry (Morus nigra) tree belongs to the order Rosales to the genus Morus of Moraceae family. This genus has a worldwide distribution,

This genus has a worldwide distribution, there are currently recognized and accepted 10-16 species of Morus [75]. Black mulberry, M. nigra (Figure I.2.3.) is the Morus species with the highest chromosome number [76].

In most European countries, mulberry trees are grown for fruit production [77, 78], in India and China, blackberry is used by foliage to feed the silkworm (Bombyx mori L.) [77, 78,79].

The fruit is attractive scent as well as a high content of organic acids and fiber together with high polyphenol and vitamin C [80, 81].

In China, is one of the oldest fruit trees, used in traditional medicine as a diuretic, laxative, antitussive, expectorant, sedative, anxiolytic [82, 83], fruits, roots and Morus nigra peels have been used to treat diabetes, hypertension, anemia and arthritis [84, 85].

Recent investigations showed that, mulberry fruit and leaves of plants have several bioactive components such as alkaloids, flavonoids and anthocyanins. Resveratrol (trans-3,4',5-trihydroxystilbene) and oxyresveratrol (trans-2',3,4',5-tetrahydroxystilbene) hydroxystilbenes are found in berries [86]. Given the importance of phenolic antioxidant role of protection against many diseases, it appears that the increase diet berries can affect heart disease risk factors such as lipid profile, blood pressure, and inflammatory markers [87].

I.2.4 – Red fruits Benefits

Most medications that exist, have side effects (short, medium or long term), which can become serious if the dosage is high and / or prolonged. The search for new alternative treatments, who can be used alone or combinated with conventional drugs to minimize the side effects of conventional treatments has increased in recent years [65, 88].

Medicinal plants are used for thousands of years for therapeutic purposes in many diseases due to their low or no side effects [28, 88].

After the discovery of bioactive properties of red fruits, research on the usefulness of berries health has been increasing [9, 26].

Berries such as blueberries, cranberry, currants and mulberries contain many bioactive compounds with different beneficial properties to health [9,13], as can see a resume in Table I.2.4.1.

Table I.2.4.1. Comparative account of and biological properties of berries.

Berries Phitochemicals Biological properties

V. Corymbosum (Blueberrie) Vitamin A, B complex, C, E, Selenium, Zn, Fe, Mn, b-carotene, Lutein, Zeaxanthin Resveratrol [11, 89]; Proanthocyanidins, Anthocyanins [28, 51, 58]; Ascorbic acid, Folic acid, Pterostilbene, Piceatannol [41].

Neuroprotective: [52, 59, 90], protective effects

in Parkinson´s and Alzheimer´s diseases [ 9, 11];

Anticancer properties:

Chemopreventive and chemotherapeutic [11, 13, 18, 29, 51, 61, 89, 91, 92, 93];

Effects: inflammatory [12, 13, 29, 51];

anti-diabetic [9, 12, 13, 28];

Protection against: cardiovascular diseases,

atherosclerosis [13, 28, 51];

Natural antimicrobial: [13, 28, 94].

V. oxicoccus

(Cranberries)

Vitamins A, C, Ca, Fe, Folate, Mg, and Mn. Flavonoids, Tannin [9]; Proanthocyanidins [11]; Antioxidants, Phenolic Acids Anthocyanins [66, 79].

Antibacterial [9, 11, 58]; antiseptic, antiviral,

diuretic, promotes cardiovascular health [9, 13, 27, 58];

Anticancer properties: antimetastasis and

chemoprotective properties, tumor and anti-radiation properties [ 9, 11, 13]. R. rubrum – red R. Rubrum white R. nigrum (Currants) Minerals (Ca, Zn, Mg, K) [11]; Vitamins A, C, [82]; Phenolic compounds: Ellagitannins; Anthocyanins [89]; Flavonoids [52]; Tannins, Stilbenes, Gibberellic acid, Phytochemicals, Carotenoids [11, 18, 30, 53, 54, 88].

Anti-inflammatory properties: mitigate

digestive diseases, diabetes [9, 51, 62],

Neurodegenerative disorders: [52], [90];

scavenging ability of reactive oxygen species (ROS) [7, 13];

Anti-carcinogenesis and stimulate the apoptosis

of cancer cells [11, 18, 88, 89];

Protection against cardiovascular diseases

[49]; and diabetes [9, 11, 53], antimicrobials [9, 11] microbial infections [18]. Morus nigra (Mulberrie) Minerals (P, Ca, Mg, Mn, Zn) [79] .Flavonoids [95]; Carotenoids [82, 83]; Vitamins C, A, And B, Alkaloids, Phenols [96]; Anthocyanins [31, 97]; Oxyresveratrol [86]; Resveratrol [98]

Protection against: Oxidative damage to

membranes and biomolecules, hepatoprotective activity, anti-inflammatory, [96];

Reduced risks of cancer, cardiovascular disease and other chronic diseases [95, 97];

Potential in improving cognitive deficits in mice useful to suppress aging [99]; hypoglycemic activities with non-toxic side effects [19, 76, 100].

I.3 – Bacterias

Infections are become a public health problem due to the increase in resistant microorganisms [33, 101].

The main bacteria that cause hospital infections are a problem for the World Health Organization (WHO), the treatment of diseases caused by these microorganisms is increasingly difficult due to the emergence of resistance to drugs of the last generation [102]. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter sp., Acinetobacter sp. and Enterococcus faecalis are the main species that have developed a high degree of resistance to antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus which are responsible for much of nosocomial infections [102, 103].

The microorganisms may be wound into a bacterium such as communities or surface attachment called biofilm, surrounded by a polymeric matrix comprising protein, DNA and polysaccharides, making difficult diagnosis and treatment strategies [35, 104].

Morbidity and mortality rates in patients with infections has increased, which increases the costs of care [28],and there is a need to develop new drugs with antibiotic activity to combat these infections. Natural products from plants can be a source of many compounds with potential antibiotic activity or to modulate resistance to antibiotics [105].

I.3.1 – Staphyloccocus aureus

Staphylococcus aureus (Figure I.3.1.A) is a ubiquitous Gram-positive commensal, which is present in about one third of the world's human population [34, 35, 106]. It is a bacterial species found in various natural environments and as part of the normal microbial flora of the skin and mucosa of different animals. Some strains of Staphylococcus aureus are etiological agents of opportunistic infections, causing many infections [105] (Figure I.3.1.B). It is isolated pathogen more frequently in infections of the skin and soft tissue [107, 108], highly versatile can cause a variety of clinical manifestations of varying severity, from superficial infections, the inflammation with risk life and sepsis, often caused by strains resistant to multiple antibiotics [34].

Staphylococcus aureus make infections in people of all ages and backgrounds, the most serious in young children, the elderly, immunocompromised [107].

Bacterial antibiotic resistance is a serious problem, morbidity and mortality levels is growing in patients with such infections, increasing the cost of health care. It is important to develop new drugs with antibiotic activity to fight these infections [28, 105, 109].

Plants can provide countless compounds of high biological activity, contributing to the discovery of new therapeutic agents in combating multidrug-resistant microorganisms [28].

I.4. – Fungi

The bioaerosols are constituted by microorganisms of different species scattered in the air, generated during the mechanical disturbance, contain many organic compounds capable of provoked awareness of the vital respiratory organs [38]. The most important in triggering respiratory problems are fungi, according the number of spores that inhales [39].

Fungi are ubiquitous on Earth, occupying more niches than any other eukaryotic kingdom [110].

Different studies have shown that molds are a threat to health, the spores of these microorganisms are ubiquitous, and under favorable conditions can start the life cycle [78]. Molds have the ability to produce secondary metabolites that have toxic effects on human body and animals and can cause different diseases [38, 39].

Recent research showed the presence of fungal hyphae and cells in all patients with Alzheimer's disease (AD) analyzed. Fungal material was found not only in sections of the brain AD patients as well as in blood vessels [111].

Figure I.3.1.B. Staphylococcus aureus [150].

Figure I.3.1.A. Child whith subcutaneous abscess wing methicillin-resistant Staphylococcus aureus [151].

Fungal infections can range from nail infections and superficial skin for invasive fungal infections (IFIs) depending on the immune status of the host [112].

With the increase of these diseases, it is important to study the development of these microorganisms to promote an effective figth against them [113].

I.4.1 – Alternaria spp.

The Alternaria genus of fungi, are ubiquitous, saprobic, endophytic, appearing on different substrates, including seeds, plants, agricultural products, animals, soil and air. Alternaria species are known as serious plant pathogens, causing great losses on different crops [114].

Alternaria cause of significant negative effects on humans [115], the spores of these fungi are one of the most common and potent allergens. Different studies indicated that Alternaria sensitivity is linked with the development of asthma in varying degrees [114], and other respiratory diseases [42].

I.4.1.1 – Alternaria alternata

Alternaria alternata (Figure I.4.1), is the most common species of Alternaria genus, the spores of this fungus are all over the world, throughout the year, between spring and autumn, the spores spread in the air, so, are ubiquitous [116], contaminants saprophytic [42].

Colonies growth fast, have dark colors ranging from gray to brown or olive. The surfaces of the mature colonies may appear "diffuse", "fluffy wool", or "how chamois" due to the presence of numerous hyphae. Microscopically, hyphae and conidiophores are septate and brown, conidia single or forming chains [116].

Alternaria alternata has been clinically associated with asthma, allergic rhinosinusitis, hypersensitivity pneumonitis, oculomycosis, onychomycosis, skin infections and allergic bronchopulmonary and invasive mycosis. This fungus produce a wide variety of clinical manifestations [117].

I.4.2 – Aspergillus spp.

Aspergillus spp. are mainly saprophytic fungi that occur commonly in soil and other organic and inorganic substrates [118]. Conidia (asexual spores) are hydrophobic airborne with the ability to germinate in different conditions. Organisms are thermotolerant, they grow at temperatures ranging from 12 °C to over 50 °C contributing to its success as opportunistic pathogens in vertebrates [119].

The genus Aspergillus is associated with hyalohyphomycetes, among other fungi, such as Penicillium hyalohyphomycosis, a term used to describe infections caused by fungi of hyaline septate, branched hyphae without pigment [40].

Aspergillus fumigatus is the most frequently isolated in humans, followed by A. flavus, A. niger [43, 120].

Aspergillus spp., pathogens have worldwide distribution. They are present from tropical, temperate regions to polar regions [40].

The incidence of infections by Aspergillus species is increasing, due to the action of new drugs and therapeutic tools that induce different degrees of immunosuppression, it is considered important nosocomial disease causing serious fungal infections, often fatal, depending immune status of the host [121].

Figure I.4.1.A. Alternaria sp. Lactophenol cotton blue preparation showing darkly pigmented chains of muriform conidia [142].

Figure I.4.1.B. Photograph of invasive A.

alternata infection after first resection. Note

I.4.2.1 – Aspergillus fumigatus

Aspergillus fumigatus (Figure I.4.2.1.A), the most pathogenic species is able to grow at different temperatures between 15 °C and 55 °C (thermotolerant) [122]. It has a simple life cycle, one of the main features is the enormous capacity of sporulation, resulting in the ubiquitous presence of high spore concentrations in the environment [123].

Microorganisms have colonies with rapid growth, flat and compact. The appearance is white in the beginning, and then passes to a blue-green color and velvety aspect consistent [124]. The surface has a few kinks and white tufts, acquires yellowish tint to grow old. Its growth is faster at 37 °C. A. fumigatus is an opportunistic pathogen as well as a major allergen, its conidia production is prolific, and so human respiratory tract exposure is almost constant. In immunocompromised individuals, the incidence of invasive infection (Figure I.4.2.1.B) can be as high as 50% and the mortality rate is often about 50% [122].

I.4.2.2 – Aspergillus niger

Aspergillus niger (Figure I.4.2.2.A.) an opportunistic filamentous fungus who develops colonies in less than seven days, initially the colonies are white or yellow but quickly turn black [125].

Aspergillus niger is a secondary etiologic agent of bacterial ear infections and is common in lung diseases in immunocompromised patients [126]. It is associated with otomycosis [127]

Figure I.4.2.1.A. Aspergillus fumigatus Lactophenol cotton blue preparation showing conidial heads [142].

Figure I.4.2.1.B. Acute invasive pulmonary aspergillosis, caused by Aspergillus fumigatus, post

I.4.3 – Trichophyton mentagrophytes

Trichophyton mentagrophytes is one of the species belonging to dedermatophytes, there are the filamentous fungal group is the most common cause of cutaneous mycoses [128]. These fungi are classified into three genera, Epidermophyton, Microsporum and Trichophyton [129]. Dermatophytoses are skin infections caused by the invasion of keratinized mucosa by dermatophytes [130]. Typically, they are superficial infections, but in immunocompromised patients may become serious. Although dermatophyte infections are treatable reinfection probability is high [128].

Trichophyton mentagrophytes (Figure I.4.3.A) is a zoophilic fungus with a worldwide distribution and a wide range of host animals. It is the most isolated dermatophyte infections with Trichophyton, are usually superficial and restricted to the epidermis (Figure I.4.3.B), but these fungi can be invasive and causing severe and disseminated infections in immunocompromised patients, such as those associated with AIDS, diabetes mellitus, organ transplantation with development of granulomas [131].

Typically, the colonies are flat, with colors from white to cream, with a granular surface and take a few days to grow. They have many unicellular microconidia, often in dense clusters [131].

Figure I.4.2.2.A. Aspergillus niger in a cavitary lung lesion showing both hyphae and conidial head (Gomori methenamine silver, ×1000 [142].

Figure I.4.2.2.B. External auditory canal with

Aspergillus niger overgrowth manifesting as a

I.4.4 – Penicillium spp.

Penicillium genera is one of the best known fungi, containing 354 species occurring in different environments from the ground vegetation, air, indoors and various food products, among others [43]. It has distribution is worldwide, the species can contaminate diffrent substrates and produce mycotoxins. Its main function in nature is the decomposition of organic materials, where the species can be devastating, such as pre- and post-harvest pathogens on food crops, as well as the production of a wide range of mycotoxins [44]. Some species are used in the food industry, production of specialty cheeses such as Camembert or Roquefort and fermented sausages received particular attention due to production of penicillin, which has revolutionized the medical approaches to the treatment of bacterial diseases [40].

There pathogenic species for humans and animals, opportunistic infections caused by species of this genus may lead to death, depending on the host immune state [132].

I.4.4.1 – Talaromyces (Penicillium) marneffei

Penicillium marneffei (Figure I.4.4.1.A.) was recently transferred from the Penicillium genus to genus Talaromyces based on phylogenetic analysis [133]. However, the disease caused by this fungus, remain calls penicelioses, and the fungus may appear as Penicillium marneffei

Figure I.4.3.A. Trichophyton mentagrophytes, Lactophenol cotton blue [142].

Figure I.4.3.B. Multiple inflammatory, itching ringworm lesions, caused by Trichophyton

Talaromyces marneffei (formerly Penicillium) is a dimorphic pathogen that presents temperature dependent growth [135]. At 25 °C, it grows in the mycelial form with vegetative conidia (saprophytic form), since the conidia are inhaled, it suffers from dimorphic switching producing yeast cells at 37 °C (parasitic form), the normal temperature of humans and hot blood animals. The pathogenicity appears to be associated with dimorphic transition, although the mechanism that governs this change, is still unknown [40, 136].

Talaromyces marneffei is a pathogenic thermal dimorphic fungus most important in China and Southeast Asia [137]. This fungus can cause fatal systemic mycoses in both immunocompetent and immunocompromised patients with or without HIV infection [138], (Figure I.4.4.1.B). The penicilliosis pathology in different organs varies depending upon the host immunity, early diagnosis and prompt initiation of treatment are crucial for patient survival [132].

Due to human migration and travel, this fungal infection has been diagnosed in individuals in Europe [40].

I.4.5 – Rhizopus spp.

Rhizopus (Figure I.4.5.A.) genus is a common saprophyte of specialized plants fungi and parasites in animals. The fungi belonging to this genus, characterize by being filamentous, with cenocytic hyphae (have transverse walls), have asexual and sexual reproduction through spores [139].

Some species Rhizopus are causative agents of opportunistic fungal infections who can be fatal, depending on the immune status of the host. Infections caused by fungi of Rhizopus are

Figure I.4.4.1.A. Gomori methenamine silver–stained yeast forms of P. marneffei, including forms with single, wide, trans-verse septa (center) [142].

Figure I.4.4.1.B. Skin lesions associated with P. marneffei infection. Chronic ulcers A, pustular psoriasis B, and subcutaneous abscesses C. HIV negative patients [138].

I.5 – Mycoses

Infections caused by fungi are called mycoses, these can be classified as the infected tissues and in accordance with the features of fungi that cause infection [141] . The mycoses can be superficial, skin and subcutaneous, endemic, opportunistic (Table I.5.1) [142].

Superficial mycoses are infections of hair and more skin surface areas are essentially aesthetics. Fungi associated with these superficial infections include Malassezia furfur, Hortae werneckii, Piedraia hortae and Trichosporon spp. [141, 142].

Dermatomycoses (dermal fungal infections) may occur when the fungus infects or colonizes skin or hair follicles. The cutaneous lesions induced by fungi depend on the location and structure of the skin (surface layer of the skin, hair or nails) are mainly caused by Microsporum, Trichophyton, Candida and Malassezia spp.) [129].

Dermatophytosis is a infectious disease of animals caused by Microsporum and Trichophyton species affecting hair, nails and epidermal keratin [130]. These fungi common in nature, are a public health problem and veterinary reported from different parts of the world are causing major economic losses) [129]. Yeasts of the genus Malassezia inhabit the skin in a variety of mammals and birds which grow easily, but are also capable of acting as opportunistic pathogens in animals. They have been implicated in various skin disorders in animals, especially otitis externa and dermatitis [141].

Subcutaneous mycoses can reach the cornea, muscle and connective tissue are caused by Figure I.4.5.A. Rhizopus sp. showing sporangium

and rhizoids [142].

Figure I.4.5.B. An ulcerative lesion at the right chest of a formerly healthy 2-year-old boy. The rare fungus Rhizopus oryzae caused this infection [147].

Fungi which gain access to deeper tissue usually by traumatic inoculation, the host immune system recognizes the fungi causing the variable destruction of tissues and often hyperplasia. The infections can be caused by Hyaline molds, such as Acremonium spp. and Fusarium spp. or dematiaceous and pigmented fungi such as Alternaria spp., Cladosporium spp., and Exophiala spp. (Phaeohyphomycosis, chromoblastomycosis) [142].

Endemic mycoses are caused by dimorphic pathogenic fungi H. capsulatum, B. dermatitidis, Emmonsia pasteurian, Coccidioides immitis, Coccidioides posadasii, Paracoccidioides brasiliensis, and Talaromyces (Penicillium) marneffei. These fungi present thermal dimorphism (exist as yeast or beads at 37 °C and mold at 25 °C) and are generally confined to geographical regions which occupy specific environmental or ecological niche. As these organisms are pathogens capable of causing infection in healthy individuals, endemic mycoses are often referred to as systemic mycoses [40]. Typically, these agents produce a primary infection in the lungs, with subsequent spread to other organs and tissues [142].

The fungi that are normally found as human commensals, animals or the environment can cause opportunistic mycoses. Virtually any fungus can be pathogenic opportunistic (depending on the immune status of the host) [126], and the list of opportunistic mycoses becomes larger every year. The most common opportunistic pathogenic fungi are Candida spp., and C. neoformans, Aspergillus spp., and P. jirovecii [118, 142].

Table I.5.1 Classification of Human Mycoses and Representative Etiologic Agents [142]. Superficial

Mycoses

Cutaneous and

Subcutaneous Mycoses Endemic Mycoses

Opportunistic Mycoses Black piedra Piedraia hortae Dermatophytosis Microsporum spp. Trichophyton spp. Epidermophyton floccosum Blastomycosis Blastomyces dermatitidis Aspergillosis Aspergillus fumigatus A. flavus A. niger A. terreus Tinea nigra Hortae werneckii Tinea unguium Trichophyton spp. E. floccosum Histoplasmosis Histoplasma capsulatum Candidiasis Candida albicans C. glabrata C. parapsilosis C. tropicalis Pityriasis versicolor Malassezia furfur Onychomycosis Candida spp. Aspergillus spp. Trichosporon spp. Geotrichum spp. Coccidioidomycosis Coccidioides immitis/posadasii Cryptococcosis Cryptococcus neoformans White piedra Trichosporon spp. Mycotic keratitis Fusarium spp. Aspergillus spp. Candida spp. Penicilliosis Talaromyces (Penicillium) marneffei Trichosporonosis Trichosporon spp. Chromoblastomycosis Fonsecaea spp. Phialophora spp. Paracoccidioidomycosis Paracoccidioides brasiliensis Hyalohyphomycosis Acremonium spp. Fusarium spp. Paecilomyces spp. Scedosporium spp. Emmonsiasis Emmonsia pasteuriana Mucormycosis Rhizopus spp. Mucor spp. Lichtheimia corymbifera Phaeohyphomycosis Alternaria spp. Curvularia spp. Bipolaris spp. Wangiella spp. Pneumocystosis Pneumocystis jirovecii