Antifungal Activity of Botanical Extracts against Malassezia furfur Implicated in Common Skin Conditions Pubblico
Pintas, Stephanie (Spring 2018)
Published
Abstract
Malassezia furfur is a commensal lipophilic fungus found on the surface of the skin that is implicated in the pathogenesis of several prevalent skin conditions, including Atopic Dermatitis (AD), Seborrheic Dermatitis (SD), and Tinea Versicolor (TV). With antifungal resistance becoming an increasing concern, it is important to identify new therapeutic options. While several studies have demonstrated anti-Malassezia activity with botanical extracts, the concentration tested is often high (>500 μg/mL) and not evaluated for mammalian cytotoxicity.
The purpose of this study was to screen for potential anti-Malassezia activity of botanical extracts using a unique chemical library, the Quave Natural Products Library (QNPL), based on plants used in the traditional treatment of infectious and inflammatory skin disease. Initial screen of the QNPL (~1000 extracts) was conducted at 16 μg/mL, but failed to demonstrate anti- Malassezia activity. A second screen of the library was conducted at 128 μg/mL by broth microtiter plate dilution method in triplicate.
The screen revealed potential activity for crude extracts 31, 55, 66, 637, and 1251 and pure compounds 448, 449, and 451. Further analysis via two-fold serial dilution in quadruplicate demonstrated 48-hour MIC90 at 128 μg/mL for extract 1251, Allium amethystinum, and 16 μg/mL for pure compound 451 as compared to 2 μg/mL for Ketoconazole control. Minimum fungicidal concentration (MFC) was determined by agar plate test. MFC was 256 μg/mL for both pure compound 451 and extract 1251. We also conducted two-fold serial dilution on extracts 158 and 237, ethanolic and methanolic crude extracts of Allium cepa (onion) in the same genus as A. amethystinum, but failed to observe any growth inhibition at either 24 or 48-hour optical density reads.
To our knowledge, this is the first report of antifungal activity of Allium amethystinum. Mammalian cytotoxicity as determined by testing of human keratinocytes (HaCaTs) via LDH assay revealed that extract 1251 did not achieve an IC50 in HaCaTs at 512 μg/mL, twice the concentration that exhibited fungicidal activity. This provides promising evidence for potential topical treatments that include Allium amethystinum.
Chemical inhibition of Malassezia furfur at lesional skin sites could support reduced allergenic responses and inflammation, improving symptoms of disease and decreasing risk of fungal resistance from current topical treatments. Future efforts to isolate the active compound(s) of extract 1251 via iterative bioassay guided fractionation are warranted.
Table of Contents
TABLE OF CONTENTS
Chapter 1
Introduction........................................................................................................... 1
1.1 Skin Microbiome.................................................................................................. 1
1.2 Characteristics of Malassezia spp......................................................................... 7
1.3 Atopic Dermatitis................................................................................................ 9
1.4 The Role of Malassezia species in Atopic Dermatitis ....................................... 11
1.5 Seborrheic Dermatitis ......................................................................................... 16
1.6 Tinea Versicolor................................................................................................... 20
Chapter 2 Background ........................................................................................... 22
2.1 Antifungal Resistance and Toxicity: The Need for New Solutions.................................... 22
2.2 Implications of Other Treatments ....................................................................................... 28
2.3 Current Research on Botanical Extracts ............................................................................. 31
2.4 In Vitro Research against Malassezia species .................................................................... 32
2.5 Clinical Research Utilizing Botanical Extracts................................................................... 42
2.6 Relevance of Studying Botanicals ...................................................................................... 52
Chapter 3 Materials and Methods....................................................................................... 53
3.1 Collection and Extraction ................................................................................... 53
3.2 Growing Malassezia species: Agar and Broth Microdilution Method ............... 56
3.3 Microbiological Assay: Initial Screen ................................................................ 57
3.4 Serial Dilution and Fungicidal Plate Test ........................................................... 59
3.5 XTT Assay ........................................................................................................ 62
3.6 Cytotoxicity Assay.............................................................................................. 66
Chapter 4 Results.................................................................................................... 67
4.1 Screening Outcomes ........................................................................................... 67
4.2 Antifungal Activities—MIC Results ................................................................. 92
4.3 Fungicidal Plate Results...................................................................................... 98
4.4 LDH Cytotoxicity Results................................................................................. 102
Chapter 5 Discussion ............................................................................................ 104
References Cited................................................................................................... 117
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