Chemische Analyse von amorphen, pharmazeutisch relevanten Ausgangsmaterialien unter besonderer Berücksichtigung von Mumijo, Torf und Assa-foetida
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Philipps-Universität Marburg
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Abstract
Amorphous substances are used in pharmacy due to their good water solubility and bioavailability. mumijo, peat and assa-foetida are pharmaceutical raw materials that occur naturally in amorphous form and have been used in medicine for thousands of years.
Mumijo is a brown-black amorphous substance that is embedded in rocks in the high mountains, primarily in Central Asia. After the snow melts mumijo emerges in caverns from fissures in the rocks. It consists of up to 85 % humus, and contains lipids, steroids, hydrocarbons, alkaloids, amino acids, proteins and other organic compounds. The formation process of mumijo is controversial in the literature. The objective of the dissertation is to clarify the specific genesis of Mumijo on the one hand and to identify pharmacologically active compounds on the other.
In order to gain an overview of the polarities of the material, the aqueous extract of four samples from the Eastern Pamir region was subjected to thin-layer chromatography (TLC). Thereafter, analytical High-performance liquid chromatography (HPLC) tests were carried out with the objective of developing an optimized program for HPLC-HRMS tests. These tests showed that one sample contained urolithin A and urolithin B. These 3,4-dibenzo-α-pyrones are known for their antiproliferative effects. Alternariol, a mycotoxin that also has antiproliferative properties, was detected in three other samples. In order to gain a more comprehensive overview of the genesis of the material, a cylohexane extract was obtained from the samples, which were then analyzed for their constituent components using GC-MS. Besides to relatively unselective markers such as long-chain n-alkanes (>C23), as well as oleic acid and palmitic acid, more specific compounds were also discovered that provide information about the genesis of the material. The compounds identified in this study include cis-vaccenic acid, which was present in all samples. As a bacterial marker, it is indicative particularly of bacteria belonging to the genus Lactobacillus. These bacteria are found in the stomachs of mammals and are involved in the digestive process. One hypothesis regarding the origin of mumijo proposes that it is primarily composed of the excretions of animals. This hypothesis is thus reinforced. Furthermore, one of the samples was tested positive for p-cresol, a compound commonly found in urine, which serves to reinforce the hypothesis.
A further aim of this dissertation is a comparison between mumijo and peat with the question of whether both materials are so similar in terms of their chemical composition that they can each be substituted by the other for medical applications. Peat is an organic decomposition product of plants and animals that is primarily formed in boreal peatlands when the productivity of the biomass exceeds its decomposition. Peat contains n-alkanes, n-alcohols, n-aldehydes, saturated and unsaturated fatty acids, methyl ketones, sterols, hopanes, vitamin E and other compounds.
In this study, a peat sample from the Woeste nature reserve in Germany was subjected to analysis using the same analytical methods previously employed for Mumijo, namely TLC, HPLC-HRMS and GC-MS. The key difference was the use of a serial soxhlet extraction with cyclohexane, dichloromethane, ethyl acetate, and methanol, in addition to a shortened HPLC-HRMS method. HPLC-HRMS analysis of dichloromethane, ethyl acetate and methanol extracts revealed pharmacologically active triterpenic acid, hederagenin.. Furthermore, fungal metabolites, e.g., pulvic acid and terrein, were identified. Additionally, vitamin K1 and riboflavin (vitamin B2) were detected. Based on the GC-MS analyses, specific biomarkers for mosses of the genus Sphagnum were detected with heptacosan-2-one, pentacosan-2-one and nonacosan-2-one. In addition, γ-sitosterol and lup20(29)-en-3-one were identified as typical biomarkers for higher plants. This composition of substances is characteristic of a transition peat bog.The comparison revealed that both peat and mumijo contain palmitic, oleic and cis-vaccenic acids and that there is a match in the n-alkane composition, ranging from a chain length from C21 to C31. Nevertheless, there are also notable distinctions. In addition to the biomarkers previously mentioned, n-alcohols, n-aldehydes, which are degradation products of fatty acids, and vitamin E together with its degradation products, as well as a hopane derivative, were also detected in the peat sample. These are typical components of peat, but do not occur in Mumijo.
Despite the similarities mentioned, the differences in chemical composition are so extensive that it is not possible to conclude with certainty whether mumijo and peat can be substituted for one another.
The GC method developed in this dissertation shows the potential to detect and examine amorphous substances and gain insights into the genesis of the material in question. A number of groups of substances can be analyzed in parallel with high precision, allowing the creation of profiles of an equivalent standard, which can then be used as the basis for a robust comparison of different samples. Building on this, it is possible that amorphous substances such as mumijo or peat may be analyzed with high quality in future studies.
The third amorphous material examined in this work was Assa-foetida, an exudate obtained from the roots of the medicinal plant Ferula assa-foetida. This plant is native to Iran, Afghanistan, and parts of the former USSR. Assa-foetida consists of resin (40-64 %), a water-soluble fraction (25 %) and essential oils (10-17 %). The resin fraction contains ferulic acid and its esters, coumarins, sesquiterpene coumarins and other terpenoids.
The present study aims to create a profile of the sesquiterpene coumarins and guaianolides contained in the resin fraction of Assa-foetida. This will enable conclusions to be drawn about its place of origin. A serial Soxhlet extraction was carried out analogous to the analysis of the peat sample. Of the extracts obtained, only the dichloromethane extracts of four samples underwent thin-layer chromatography analysis. The analysis revealed that the spots characteristic of coumarins or sesquiterpene coumarins under UV light at 366 nm.
Analogous to the investigation of the peat, optimized HPLC-HRMS analysis were carried out. The results were compared with a list of substances compiled as part of this dissertation, which focuses on sesquiterpene coumarins and guaianolides.
Farnesiferol A, a compound found in Ferula assa-foetida, was detected in both samples. In addition to this compound, talasin A, fegvolide, malaphyllinin, malaphyll and diversin are also present. These compounds are only found in this composition in Ferula diversivittata, a plant from the Uzbek-Afghan border region. Other compounds are cauferidin and cauferin, which are found in Ferula conocaula, a plant native to Uzbekistan. The results obtained indicate that this sample only partly comes from Afghanistan and partly from Uzbekistan.
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Created: 2024Issued: 2024-12-17Updated: 2024-12-17
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
pharmacological active compoundsHPLC-HRMS-Analysismumijoassa-foetidapharmakologisch wirksame VerbindungenGC-MS-AnalysisTLC-Analysispeat
DFG-subjects
MumijoHPLC-HRMS-AnalyseDC-AnalyseTorfGC-MS-AnalyseAssa-foetida
DDC-Numbers
540
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Götz, Simon Vincent: Chemische Analyse von amorphen, pharmazeutisch relevanten Ausgangsmaterialien unter besonderer Berücksichtigung von Mumijo, Torf und Assa-foetida. : Philipps-Universität Marburg 2024-12-17. DOI: https://doi.org/10.17192/z2024.0502.