Optimal conditions for extraction of potentially therapeutic compounds from Pelargonium sidoides

Abstract

Pelargonium sidoides (P. sidoides) DC., a member of the Geraniaceae family, is of significant importance in both traditional herbal practices and modern phytotherapy. It is widely used to treat bronchitis and infections of the upper respiratory tract. This herb has gained substantial recognition due to its scientifically established effectiveness and safety, as supported by clinical trials and previous patents. Its successful presence in the German and international markets underscores its popularity. The core of this herbal remedy is EPs® 7630, a unique aqueous-ethanolic (11 % (m/m) ethanol) root extract of P. sidoides. This extract is notably documented in the European Pharmacopoeia (EP). However, the existing pharmacopoeial method, which involves assessing tannin content in roots, is not ideal for evaluating final products that are ethanol extracts or dried root extracts. This limitation arises from the potential impact of the extraction solvent on various constituents. In particular, P. sidoides is reported to encompass a diverse array of compounds beyond tannins.

Currently, the roots used for commercial purposes are sourced through wild harvesting in South Africa. This practice introduces significant variability, as existing studies have focused mainly on fluctuations in umckalin levels within the roots. Umckalin serves as a coumarin marker but is not necessarily reflective of the pharmacological effectivity (or bioactivity). Organisations that oversee wild harvesting only assess umckalin levels to gauge root quality. Despite extensive research, the precise active constituent within P. sidoides remains elusive. Interestingly, even today, only 80 % of the root composition has been characterised, leaving approximately 20 % unidentified. The Willmar Schwabe Pharma group of companies has conducted numerous clinical studies on Pelargonium, and particularly on their trademarked active ingredient EPs® 7630. As a result of these efforts, the European Medicines Agency (EMA) has endorsed an 11 % ethanolic extraction (1:8-10) and its equivalent dried root extract (4-25:1) to treat colds and flu. This has emerged as the industry standard, often referred to as the gold standard. Schwabe initially held a patent on the 11 % ethanol extraction; however, they withdrew it in 2010 to ensure fairness within the Pharma industry.

A notable challenge arises from the absence of established quality control parameters to compare these roots extracts with each other or the market leader. Furthermore, the Schwabe product lacks standardisation to specific constituents, complicating matters even more. The specifications provided in the EP sole concern is the ethanol extraction percentage and the drug extraction ratio. A literature survey revealed that the choice of 11 % ethanol extraction lacks scientific justification. The approval from the EMA comes from the traditional use of the product and its market presence for more than three decades. Historically in South Africa, prior to the 2014 Complementary Medicine regulations, a common practice was to employ a 60 % ethanol extraction due to higher yields, reduced microbial contamination, and a streamlined process. With the regulatory shift, an exploration was initiated to assess the differences between various ethanol percentage extractions. Furthermore, an attempt was made to create an industry specification to help pharmacists evaluate P. sidoides root extract or products. With the introduction of the new regulations, it was decided to first evaluate what the difference would be between different percentage extractions of ethanol and then also to try and compile a standardised final product specification that can be used in the industry for pharmacists when evaluating the options of the root products of P. sidoides.

Several critical issues have been identified within the context of P. sidoides products and their manufacturing. First, the practice of wild harvesting roots introduces significant variability due to the differing conditions, altitudes, and rainfall in various regions. This variability is further compounded by the maturity of the roots, which are typically harvested after four years. Second, there is a limitation in umckalin testing, as it primarily serves for species verification and does not provide insights into the root's activity. Third, leading trademarked extracts and dried root extracts lack standardisation with respect to specific constituents, relying mainly on the percentage of ethanol and the drug extraction ratio. This problem is exacerbated by the absence of specific regulatory guidance for P. sidoides products in South Africa, even after the introduction of complementary medicines guidelines by the South African Health Products Regulatory Authority (SAHPRA), forcing industry pharmacists to resort to European guidelines for guidance. Fourth, there is a notable absence of publicly available information concerning the distinctions between different percentages of ethanol used in extractions. Finally, quality control research has touched on various aspects, including distinguishing P. sidoides from related species and the significance of sulfated coumarins, especially in the context of COVID-19 research. In response to these challenges, there is an urgent need for a more robust approach to quality control and regulation that takes into account the diversity of products, regional variations, and the sustainable management of this valuable botanical resource.

This thesis encompasses three distinct studies. The first study evaluated the impact of different concentrations of ethanol on the extraction of Pelargonium roots, focusing on yield, umckalin levels, and total polyphenols. The second study investigated the effects of 11 % and 60 % ethanol concentrations on the chemical fingerprint of the root extract and its bioactivity, including antimicrobial, anti-inflammatory, and immune modulating properties. This study also included an examination of the antiviral activity of a P. sidoides root extract against SARS-CoV-2. The final part of the thesis formulated a comprehensive quality control specification, integrating insights from previous research, with the goal of defining essential criteria for a dried P. sidoides root extract in line with the study findings for practical applications. The primary objective of this research was to systematically explore optimal sources and conditions for extracting potentially therapeutic compounds from P. sidoides, a vital medicinal plant indigenous to southern Africa, known for its efficacy against respiratory infections. This work aims to contribute to the development of formal specifications and criteria necessary for the registration of P. sidoides-based products in South Africa.

The research in question was designed to achieve specific objectives aimed at advancing our understanding of P. sidoides and improving the quality control and regulation of its products. First, an exhaustive review of the existing knowledge surrounding P. sidoides was conducted, with a focus on its mechanisms of action, quality control methods, extraction techniques, chemical composition, in vitro activity, clinical applications, and considerations related to legal frameworks, procurement processes, and regulatory approvals. This served as a foundational knowledge base for subsequent investigations. Subsequently, representative root samples were obtained from two distinct geographic regions, the Eastern Cape and the Free State, where P. sidoides is traditionally sourced and harvested. These samples were crucial for subsequent analyses and comparisons. To further explore the chemical composition of P. sidoides, ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technology was utilised to identify and quantify umckalin and umckalin sulfate in various batches of P. sidoides, with reference to a standard umckalin sample. The influence of various extraction methods was another key objective of investigation, with a specific focus on different concentrations of ethanol and extraction temperatures, as these parameters can significantly affect the chemical makeup of the root extracts. Using the same UPLC-QTOF-MS technology, a comprehensive chemical fingerprint unique to P. sidoides was established, adding to the knowledge of its constituents. To gauge the practical implications of these findings, the study delved into the biological activity of selected dried root extracts, comparing those extracted with 11 % and 60 % ethanol concentrations. The assessment included statistical analyses to identify differences between the chemical fingerprint and biological activity in various sample sets and to find potential correlations among the levels of selected constituents. Finally, one of the critical objectives was to develop quality control criteria that could be adopted by SAHPRA for the standardisation and regulation of P. sidoides root products. This step was designed to provide a framework to ensure the quality and consistency of P. sidoides-based products, contributing to the overall enhancement of their safety and efficacy within the market.

The key findings and conclusions derived from this research include various aspects of the root extracts of P. sidoides. First, the high sensitivity of the UPLC-MS-QTOF method for quality control was demonstrated, with umckalin sulfate consistently identified as the predominant peak. Electrospray ionisation in negative mode proved to be more efficient than in positive mode, enhancing the method's reliability. Accurate umckalin quantification was found to be best achieved after hydrolysis, enabling precise values for stability studies and robust quality control. Distinctions were revealed in the roots of P. sidoides from different regions, with the Eastern Cape roots exhibiting higher umckalin levels but a lower polyphenol content, while those of the Free State showed the opposite trend. Despite relatively consistent coumarin levels in root samples, these differences were not statistically significant, necessitating further exploration with larger sample sets. Higher concentrations of ethanol and elevated extraction temperatures were highlighted to lead to increased levels of umckalin and polyphenols extracted from roots. The 30 % heated extraction method and the 80 % heated ethanol extraction resulted in the highest umckalin and total polyphenol levels. Furthermore, all the same peaks were identified in the 11 % and 60 % ethanol extracts, with only differing intensities. A comparison of the biological activity of root extracts with ethanol concentrations of 11 % and 60 % indicated advantages for the 60 % root extracts, including total minimum inhibitory concentration (MIC) activity, gram-positive and gram-negative activity, umckalin sulfate levels, and antifungal activity. Positive correlations were discovered between MIC values and total polyphenol levels, highlighting the significance of polyphenols in the root extract, which have associations with immunomodulation, anti-inflammatory effects, chemoprevention, neuroprotection, cardioprotection, and treatment for various diseases, as well as antibacterial and antiviral properties.

In terms of specification for the dried root extract of P. sidoides, it was recommended to determine umckalin concentration after acid hydrolysis as a critical marker for differentiation between P. sidoides DC. and P. reniforme (Andrews) Curtis. Quantifying the total polyphenolic content was considered essential, with a minimum reference value of 15 % of total constituents. Additionally, methodically selecting the root harvest location could significantly enhance the potency of the root extract by increasing the proportion of polyphenolic compounds within the root extract. The hypothesis could also be proposed that umckalin is stored in the roots in a more water-soluble sulfate format, with evidence from acid hydrolysis indicating the detachment of the sulfate moiety from the umckalin molecule during the process. For precise umckalin quantification, umckalin levels were recommended to be quantified before and after acid hydrolysis. Lastly, identifying peaks and calculating total umckalin and polyphenol levels were advocated as a more accurate and comprehensive approach to quality control procedures, rather than relying solely on marker ratios. Additionally, a hypothesised synergistic interaction between coumarins and polyphenols suggested that polyphenols may enhance the bioavailability of coumarins, further emphasising the significance of considering the overall picture of both in quality control assessments of P. sidoides containing products.