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9EXTRACTION AND IDENTIFICATION OF ARTEMISININ AND ITS INTERMEDIATES FROM NATURAL AND MODIFIED
SOURCES
1Rakhmanov B., 2Imamkhodjaeva A., 3Usmanov D., 4Ubaydullaeva Kh, 5Shermatov
Sh., 6Buriev Z., 7Abdurakhmonov I.
1,2,3,4,5,6,7Center of Genomics and Bioinformatics, AS RUz https://doi.org/10.5281/zenodo.13150964
Abstract. The discovery of artemisinin has revolutionized malaria treatment and saved millions of lives worldwide. Its unique mechanism of action and rapid parasite elimination make it an indispensable tool in the fight against malaria. In addition, artemisinin's potential to treat cancer, viral infections and inflammatory diseases opens new avenues for research and therapy. Its diverse therapeutic potential underscores the compound's versatility and effectiveness, positioning it as an important component of modern medicine.
In the field of oncology, artemisinin has shown promising results in preclinical studies. Its ability to specifically target cancer cells while sparing normal cells offers a distinct advantage over traditional chemotherapy, which is often associated with severe side effects. Artemisinin and its derivatives have been shown to be effective against a wide range of cancers, including leukemia, breast cancer and colon cancer. By inducing apoptosis and inhibiting cancer cell proliferation, artemisinin could become a cornerstone of combination therapies and improve the efficacy of existing cancer treatments.
Keywords: oncology, artemisinin, SARS-CoV-2 virus, COVID-19, inflammatory diseases.
Artemisinin's antiviral properties also offer exciting possibilities. Research has shown that artemisinin can inhibit the replication of several viruses, including hepatitis B and C and possibly the SARS-CoV-2 virus responsible for COVID-19. Its immunomodulatory effects may improve the body's ability to fight off viral infections, making it a valuable addition to antiviral treatment regimens.
Artemisinin's anti-inflammatory and immunomodulatory effects open up new possibilities in the treatment of autoimmune and inflammatory diseases. Conditions such as rheumatoid arthritis and inflammatory bowel disease could benefit from artemisinin's ability to modulate the immune response and reduce inflammation. These potential positions artemisinin as a versatile therapeutic capable of addressing various health challenges.
The future of artemisinin in medicine depends on addressing current challenges. The threat of resistance requires further research to develop new artemisinin-based combination therapies (ACTs) and alternative treatment strategies. Ensuring a stable supply of artemisinin is critical, with advances in synthetic biology and semi-synthetic production offering potential solutions to supply chain issues.
Safety and efficacy in non-malarial conditions remain areas of active research. While preliminary studies are encouraging, large clinical trials are essential to confirm the therapeutic potential of artemisinin in various medical areas. Collaboration between researchers, health professionals and policymakers will be critical to translate these findings into clinical practice.
As we continue to explore and understand this remarkable compound, artemisinin remains a beacon of hope in both medicine and global health. Its impact on malaria treatment is undeniable and its potential applications in oncology, virology and immunology are expanding the horizons of medical science. The evolution of artemisinin from a traditional Chinese remedy to a modern medical marvel illustrates the power of natural compounds in tackling some of humanity's most pressing health problems.
Therefore, research into alternative therapeutic options, particularly those derived from natural products, has gained considerable attention [1-9].
Artemisia annua contains both volatile and nonvolatile chemicals. Terpenoids, coumarins, and flavonoids are among the most common nonvolatile compounds. Artemisinin, in particular, belongs to the class of terpenoids.
Artemisia annua L. produces bioactive chemicals such as artemisinin, which is mainly found in the aerial parts of the plant such as leaves, stems, buds and flowers. Artemisinin from Artemisia annua L. is usually extracted using organic solvents such as propylene glycol methyl ether, butanol, dichloromethane, hexane, petroleum ether, toluene, isopropanol and chloroform.
This review presents the extraction and identification methods for artemisinin and highlights their advances, advantages, and limitations. It describes the chemical composition of Artemisia plants, focusing on its nonvolatile compounds, including terpenoids such as artemisinin. Artemisinin, which is predominantly found in the aerial parts of Artemisia annua L., is extracted using various organic solvents. New techniques such as ultrasonic-assisted, microwave-assisted, and low-pressure solid-liquid extractions, as well as supercritical carbon dioxide, offer higher efficiency compared to traditional Soxhlet extraction. The studies highlight the importance of extraction methods that preserve the bioactivity of artemisinin while reducing the risk of degradation by volatile solvents.
The purpose of our study
The purpose of this study is to investigate the metabolomic changes between genetically transformed plant lines and control plant lines. These transformed plant lines were generated by Agrobacterium-mediated transformation using genetic constructs based on artemisinin biosynthesis genes. After transformation, the plant explants were cultured under aseptic conditions using tissue culture techniques.
Figure 1. Scheme of artemisinin extraction with hexane-ethyl acetate mixed solvent [10].
To achieve our objective, we initiated a comprehensive review of various research works and protocols for the extraction and identification of artemisinin, its related compounds and
intermediates. This literature review is crucial as it provides a solid foundation for our subsequent experiments and ensures that we obtain accurate and high-quality results in the extraction, identification and analysis of artemisinin and its derivatives. By synthesizing the existing knowledge and methods, we aim to refine our experimental procedures and improve the efficiency and effectiveness of our metabolomic analyses. Ultimately, this study will contribute to a deeper understanding of the biochemical pathways involved in artemisinin biosynthesis and potentially lead to improved production methods for this valuable compound.
Figure 2. Evaluation of the Thin layer chromatography plates using methanol/isopropyl alcohol (30/70) as an example.
On the left side of the plate is a standard artemisinin solution, on the right side - the extract sample. Part (a) shows the normal chromatogram, part (b) the chromatogram under ultraviolet (UV) light (at 254 nm), part (c) under 366 nm and part (d) after treatment with vanillin-phosphoric acid reagent [11].
Materials and Methods for Extraction and Identification
In this study, the phytochemical and transcriptomic differences between high artemisinin producing (HAP) and low artemisinin producing (LAP) chemotypes of Artemisia annua, an important source of the antimalarial drug artemisinin, were investigated. The plant material was grown in a greenhouse and Artemis, an F1 hybrid of A. annua, was generated by crossing C4 and C1 parental material of East Asian origin. Monoterpenes, sesquiterpenes and other volatile compounds in fresh and dried leaf samples were quantified and analyzed using gas chromatography-mass spectrometry and ultra-performance liquid chromatography-mass
spectrometry. Extraction and identification were performed by extraction of 5 kg of plant material with chloroform (20 L), followed by rotary evaporation to obtain an extract (2.5% w/w), which was subjected to gradient column chromatography on silica with ethyl acetate in hexane (10%, 20% and 30% EtOAc/hexane). The structure of the new compounds was elucidated using nuclear magnetic resonance spectroscopy. Metabolite analysis involved dilution in ethanol and injection into an Acquity UPLC system with detection via a LTQ Orbitrap mass spectrometer. Artemisinin quantification was performed using a standard curve of the artemisinin reaction ratio to Partemether and metabolite identification was achieved using authentic standards or NMR-resolved structures with empirical mass formulas. The HAP chemotype, particularly the Artemis variant, exhibited significantly higher artemisinin levels compared to the LAP chemotype (NCV variant). Transcriptome analysis revealed upregulation of genes in the artemisinin biosynthesis pathway in the HAP chemotype, with principal component analysis highlighting distinct metabolic profiles between the chemotypes. In addition, novel amorphane and cadinane sesquiterpenes were identified. These findings advance the understanding of artemisinin biosynthesis and provide insights into improving artemisinin yield through breeding and biotechnological methods [12, 13].
Explored green extraction technologies were for artemisinin extraction, including ultrasound-assisted, supercritical CO2, deep eutectic solvent, and subcritical water extractions. Artemisinin was present up to 3.21 |ig/mg dry weight, with supercritical CO2 (SCO2) extraction yielding the highest amount due to the solvent's non-polar properties. The volatile compounds profile of SCO2 extract included camphor (12.23%), arteannuin B (15.29%), and artemisia ketone (10.97%). These results support using green extraction techniques for artemisinin separation, showing potential for pharmaceutical, cosmetic, and food applications. Artemisia annua L. material was processed at 4°C. Ethanol and CO2 (99.97% purity) were used for SCO2 extraction. SCO2E involved a custom system processing 100 g of plant material at 300 bar and 40°C. Ultrasound-assisted extraction (UAE) used an ultrasonic bath at varying temperatures and times, while subcritical water extraction (SWE) utilized a high-pressure extractor. Deep eutectic solvent extraction involved choline chloride-based DESs. Analysis was performed with gas chromatography-mass spectrometry and high-performance liquid chromatography. The study shows Artemisia annua is a valuable artemisinin source, with SCO2 extraction being the most effective method. SCO2 and UAE were more efficient than DES, despite SCO2's higher equipment cost and potential for contaminant co-extraction. Future research should optimize extraction parameters and purification methods for sweet wormwood extract [14].
Comparison of Extraction Methods
In the traditional solvent extraction process, the leaves and flowers of Artemisia annua are dried and ground into a fine powder, which is then subjected to solvent extraction using organic solvents such as hexane, petroleum ether, or ethanol. The solvent dissolves the artemisinin, which is later separated by evaporation of the solvent. This is a simple and inexpensive method suitable for large-scale extraction, but also presents challenges such as solvent residues, environmental concerns, and variable yields depending on solvent choice and quality of the plant material. The supercritical fluid extraction (SFE) process uses supercritical CO2 as the extraction solvent. It involves exposing the plant material to supercritical CO2, which penetrates the plant matrix and dissolves artemisinin, which is then precipitated by depressurization of the CO2. This is an environmentally friendly method with no solvent residues, high purity, and selective extraction, but requires high initial investment in equipment and expertise to operate. Similarly, the
microwave assisted extraction (MAE) process uses microwave energy to heat the plant material in the presence of a solvent, disrupting the cells and facilitating the release of artemisinin into the solvent. This allows for rapid extraction, lower solvent usage, and higher yields, but requires specialized equipment and optimization of microwave parameters. The ultrasound assisted extraction (UAE) process uses ultrasonic waves to create cavitation bubbles in the solvent, which implode, leading to cell disruption and enhanced release of artemisinin, increasing extraction efficiency, shortening extraction times, and reducing solvent usage, but potentially degrading sensitive compounds due to the intense ultrasonic energy. Alternatively, the hydrodistillation process involves boiling the plant material in water, condensing and collecting the vapor containing artemisinin, from which artemisinin is then separated. This is a simple and safe method suitable for small-scale extractions but has lower yields, longer extraction times and possible thermal degradation of artemisinin.
To identify artemisinin, thin layer chromatography (TLC) involves applying a small amount of the extract to a TLC plate and developing it with an appropriate solvent system. The plate is then visualized under UV light or a chemical reagent is used to identify artemisinin by its Rf value. This is a simple, rapid and inexpensive method for preliminary identification but has limited sensitivity and specificity compared to other techniques. In high performance liquid chromatography (HPLC), artemisinin is separated from other components of the extract using a high pressure liquid chromatograph and the eluted compounds are detected using a UV detector or mass spectrometer. This offers high sensitivity, specificity and accuracy and is suitable for quantitative analysis but requires expensive equipment and technical expertise. Gas chromatography-mass spectrometry (GC-MS) involves evaporating the extract and separating the compounds using a gas chromatograph. The separated compounds are then identified using their mass spectra. This provides high sensitivity and specificity and can identify trace amounts of artemisinin, but requires derivatization of artemisinin and expensive instrumentation. Nuclear magnetic resonance (NMR) spectroscopy uses magnetic fields and radio waves to determine the molecular structure of artemisinin in the extract. It provides detailed structural information and confirms the identity of artemisinin, but is associated with high equipment costs and requires a significant amount of the pure compound. Fourier transform infrared spectroscopy (FTIR) measures the absorption of infrared radiation by the extract, creating a fingerprint of the compounds present. It is a non-destructive, rapid method that requires minimal sample preparation, but is less sensitive and specific compared to HPLC and GC-MS.
The study aimed to determine artemisinin content using liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS), investigate the in vitro biological activity of artemisinin from A. annua plants grown in Turkey with various extraction methods, and elaborate on its in silico activity against SARS-CoV-2 using molecular modelling; twenty-one different extractions, including direct and sequential extractions, ultrasonic-assisted maceration, Soxhlet, and ultra-rapid methods, were compared, and the inhibition of spike protein and main protease (3CL) enzyme activity of SARS-CoV-2 was assessed by time-resolved fluorescence energy transfer (TR-FRET) assay; artemisinin content ranged from 0.062% to 0.066%, showed significant inhibition of 3CL protease activity [15].
The wild species studied include Artemisia herba-alba (AH), Artemisia campestris subsp. glutinosa (AC) and Artemisia judaica subsp. sahariensis (AJ). HPLC analyses of hexane extracts revealed different artemisinin contents between species, with yields of 0.64% for AC, 0.34% for
AH and 0.04% for AJ. Notably, the artemisinin content in A. campestris was higher than that previously reported for A. sieberi and A. annua, representing the first documentation of such a yield. In addition, the study investigated the antiradical activities of methanolic extracts of these plants, which showed significant antioxidant capacities in all samples. These findings highlight the potential of these Algerian Artemisia species as artemisinin sources and their promising antioxidant properties.
Figure 3. Extraction method and HPLC analysis [16].
Seasonal variations in artemisinin and its precursors in Artemisia annua from various regions were investigated by identifying different chemotypes. Significant differences were found in artemisinin, artemisinic acid, and dihydroartemisinic acid levels among the plants. One chemotype with high artemisinin also had high dihydroartemisinic acid and low artemisinic acid, while another with low artemisinin and dihydroartemisinic acid had high artemisinic acid. This suggests that the conversion of artemisinic acid to dihydroartemisinic acid is a rate-limiting step. After night frost, Vietnamese A. annua showed increased artemisinin and decreased dihydroartemisinic acid, indicating stress-induced transformation. High dihydroartemisinic acid levels may protect the plant by converting to stable artemisinin under stress [17].
Artemisinin and its derivatives are key antimalarial drugs, however, the early steps of biosynthesis in Artemisia annua L. remain largely unknown. Analysis of leaves and secretory cells of A. annua revealed oxygenated derivatives of amorpha-4,11-diene, including artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde, and dihydroartemisinic acid. The study also detected biosynthetic enzymes such as amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, artemisinic alcohol dehydrogenase, and dihydroartemisinic aldehyde dehydrogenase in both leaves and glandular trichomes. The obtained data indicate that artemisinin biosynthesis begins with the hydroxylation of amorpha-4,11-diene
to artemisinic alcohol, oxidation to artemisinic aldehyde, reduction to dihydroartemisinic aldehyde and further oxidation to dihydroartemisinic acid [18].
Genetic Engineering For Production of Artemisinin
Artemisinin production in genetically modified tobacco plants was studied. Expression of Artemisia annua genes led to amorphadiene and artemisinic alcohol accumulation, while additional genes produced dihydroartemisinic alcohol. These results indicate that transgenic tobacco favors reduction to alcohols over oxidation to acids, with significant implications for artemisinin production. For phytochemical analysis by GC-MS, leaf material was collected, processed or frozen at -80°C, homogenized in pentane with octadecane as an internal standard, centrifuged, and the pentane layer concentrated for GC-MS analysis [19].
To address the need for affordable artemisinin production, a new synthetic biology approach, combinatorial super transformation of transplastomic recipient lines (COSTREL), was developed and applied to tobacco. The complete artemisinic acid biosynthesis pathway was introduced into the tobacco chloroplast genome. Subsequent super transformation with additional enzyme cassettes resulted in plants producing over 120 mg artemisinic acid per kg biomass. This work demonstrates an efficient strategy to engineer complex biochemical pathways in plants and optimize metabolic performance. For GC-MS profiling of lipophilic saponification products from N. tabacum leaves containing artemisinic acid and biosynthesis intermediates, 150 ± 5 mg of frozen powdered leaves were saponified with 500 ^L of 2 N KOH/methanol for 1 h at 70 °C. After acidification with HCl and extraction with hexane, the hexane layer was concentrated and trimethylsilylated with BSTFA. Metabolite profiling was performed by GC-EI/TOF-MS using an Agilent 6890N24 gas chromatograph coupled to a Pegasus III time-of-flight mass spectrometer [20].
Physcomitrella patens can produce the anti-malaria drug artemisinin by expressing artemisinin pathway genes. In experiments UPLC-MRM-MS analysis method was performed for fresh moss samples. Moss (3000 mg) was harvested, frozen, ground, and extracted with citrate phosphate buffer (3 mL, pH 5.4) and ethyl acetate. The extracts were concentrated, stored, dried under nitrogen, and resuspended in methanol-water. Liquid culture extracts (300 mL of 75% MeOH:H2O (v:v)) were processed similarly. This method ensures precise preparation for subsequent analysis. The moss exhibits endogenous enzyme activity similar to Artemisia annua, affecting artemisinin and arteannuin B levels. Knocking down certain oxidizing enzymes could increase artemisinin yield. These findings highlight the potential of P. patens for metabolic manipulation of artemisinin and other high-value terpenes. Physcomitrella patens can produce artemisinin with partial or full expression of pathway genes and exhibits enzyme activity similar to Artemisia annua. Knocking down certain oxidizing enzymes could increase artemisinin yield. This work highlights the potential of P. patens for manipulating valuable terpenes and its versatile enzymes for synthetic biology [21, 22].
Conclusion
The extraction and identification of artemisinin has evolved significantly over the years. Traditional solvent extraction methods, while still commonly used, are being supplemented and in some cases replaced by more advanced techniques such as supercritical fluid extraction and microwave-assisted extraction. Identification methods have also evolved, with HPLC and gas chromatography-mass spectrometry (GC-MS) providing the high sensitivity and specificity essential to ensure the purity and potency of artemisinin.
These continuous improvements not only increase the quality of artemisinin, but also
support the global fight against malaria by ensuring a reliable supply of this life-saving compound.
As technology advances, even more efficient and sustainable methods are likely to emerge, further
cementing artemisinin's role in global health.
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