Anna Dzierżęga-Lęcznar

Hypericum perforatum L. (St. John’s wort) is one of the most popular medicinal plants in the world. Due to its documented antimicrobial and antioxidant properties, it is used in the treatment of bacterial and viral infections as well as inflammations. It is also used to treat gastrointestinal diseases and mild to moderate depression. In recent years, there has been an increase in the popularity of herbal medicine. Many people collect their own herbs and dry them at home. A common choice for quick drying of fruits, vegetables and herbs at home are food dehydrator machines. There are not many publications in the scientific literature examining the quality of dried herbal material obtained in such dryers. We characterized St. John’s wort harvested in southern Poland and investigated the effect of specific drying methods on the volatile component profile. The herbal raw material was dried using three methods: indoors at room temperature, in an incubator at 37 °C and in a food dehydrator machine. Volatile components were analysed by HS-SPME GC/MS. The herb dried in a food dehydrator, compared to other drying methods, retained similar or slightly smaller amounts of the compounds from the mono- and sesquiterpenes group, aromatic monoterpenes, aromatic monoterpenoids, sesquiterpenoids, aromatic sesquiterpenes and alkanes. However, monoterpenoids and compounds coming from decomposition reactions, such as alcohols, short-chain fatty acids and esters, were noticed in larger quantities. Usage of a food dehydrator at home can be a convenient alternative to drying herbs. However, due to a different profile of volatile components depending on the drying method, the amount of biologically active substances needs to be considered. By using various methods of drying, the medical effects of herbs can be enhanced or weakened; therefore, further research in this direction should be continued.

Echinacea is a widely used plant medicine, valued especially for its well-documented ability to stimulate the immune system. It has been suggested that melanin could be one of the bioactive factors responsible for the immunostimulatory properties of the plant. The biological functions of melanin pigments are closely related to their chemical composition and structural features. The aim of this study was to characterize the melanin from Echinacea purpurea based on the analysis of thermal degradation products of the well-purified pigment extracted from the dried herb. The melanin was pyrolyzed, and the resulting products were separated by gas chromatography and identified using a triple quadrupole mass spectrometer operating in full scan and multiple reaction monitoring modes. Three groups of marker products were detected in the melanin pyrolysate: polyphenol derivatives, nitrogen-containing heterocycles devoid of sulfur, and benzothiazines/benzothiazoles. This suggests that E. purpurea produces three structurally different melanin pigments: allomelanin, eumelanin, and pheomelanin, which in turn may affect the biological activity of the herb. Our results provide the first-ever evidence that plants are capable of synthesizing pheomelanin, which until now, has only been described for representatives of the animal and fungal kingdoms.

Introduction: There is some evidence that pheomelanin produced in skin melanocytes may be involved in the development of cutaneous melanoma, particularly in individuals with a light skin/red hair phenotype. However, nothing is known about possible correlation between the type and/or clinical stage of melanoma and the content of pheomelanin in the tumor tissue. We believe that archival formalin-fixed and paraffin-embedded (FFPE) melanoma tissues could be a good source of melanin pigment for future large-scale research on that issue. Aim: The aim of this work was to develop a method for isolation and purification of melanin from FFPE samples of human melanoma. To test the suitability of the isolation protocol for planned structural studies, the obtained melanin was analyzed for pheomelanin content by the method based on pyrolysis (Py) coupled with gas chromatography and tandem mass spectrometry (GC/MS/MS). Material and methods: For melanin isolation, microtome sections of FFPE tissue of primary lesion and lymph node metastases were subjected to a multistep procedure of paraffin removal, tissue rehydration, homogenization, and digestion with the set of proteolytic enzymes. The pigment samples were then pyrolyzed at 500 °C, and the GC-separated thermal degradation products were identified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode. Results: GC/MS/MS analysis of the pyrolysis products revealed the presence of pheomelanin markers, which allowed quantitation of a pheomelanin component of each of the isolated pigments. Melanin from the FFPE primary melanoma was found to contain 6.6%, and the pigment from FFPE metastatic lymph node 7.5% of pheomelanin. Conclusions: The developed protocol allows for the isolation of melanin from FFPE melanoma specimens. The pigment can be successfully studied for pheomelanin content by Py-GC/MS/MS method. The results of our study indicate that archival FFPE tumor tissues can be used as a good source of melanin for future structural studies aimed at shedding more light on the role of pheomelanin in the pathomechanism of cutaneous melanoma.

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll‐like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA‐binding activity of NF‐κB p50 and p65. We found substantial differences in the LPS‐stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL‐16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL‐6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.

Here, we describe the reliable method for the detection and quantitation of a pheomelanin component in melanin pigments. Synthetic melanins with various contents of pheomelanin‐type structural units were thermally degraded, and the multiple reaction monitoring mode was applied to detect the pheomelanin markers in the pyrolysates by GC/MS/MS. The method allowed the specific detection and quantitation of a pheomelanin component in melanin with the incorporation of pheomelanin‐type units as low as 0.05%. Considering highly universal character of the pheomelanin markers, the method could be applied for structural studies of natural melanin pigments being mixtures of eumelanin and pheomelanin. Copyright © 2012 John Wiley & Sons, Ltd.

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.

Melanin formation in pigmented melanoma cells is considered as a target for the tumor therapy. The evaluation of potential correlation between melanin structure and the tumor type could be also of diagnostic and prognostic importance. One of the major problems in structural investigations of natural melanins is the lack of appropriate methods, which allow isolation of pure intact pigment. In this study the thermochemolysis technique was used to assess the purification grade of melanin isolated from the human melanoma malignum cells by two different enzymatic methods. Melanin samples were thermally degraded in the presence of tetramethylammonium hydroxide and the thermochemolysis products were analyzed by gas chromatography/mass spectrometry (GC/MS). Compounds of lipid origin, especially fatty acid methyl esters and aliphatic and cyclic hydrocarbons, were predominant among pyrolysis products of melanin isolated from the tumor cells by method I. In contrast, during thermochemolysis of the pigment sample isolated by the method II, mainly eumelanin markers (pyrrole and its methyl derivatives, toluene, styrene, phenol, benzyl nitrile and indole) were formed. The comparison of pyrolysis profiles of the analyzed samples indicate that method II is more efficient for melanoma pigment purification.