Fast Isolation and Purification of the Antidepressant Alkaloids Mesembrine and Mesembrenone From Mesembryanthemum tortuosum

Fast Isolation and Purification of the Antidepressant Alkaloids Mesembrine and Mesembrenone From Mesembryanthemum tortuosum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Fast Isolation and Purification of the Antidepressant Alkaloids Mesembrine and Mesembrenone From Mesembryanthemum tortuosum Suzanne Elaine Van Niekerk, Dorothea Malan, Josias Hendrik Hamman, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5413491/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Mesembryanthemum tortuosum L., Aizoaceae, is a medicinal plant that has traditionally been used as mood elevator, stress reducer, analgesic, anxiolytic, and even a narcotic. The main activity is attributed to the mesembrine-type alkaloids, which have shown promise to provide pharmacologically and clinically antidepressant and anxiolytic effects. Due to the limited availability and cost of pure reference compounds of the main active chemical compounds of M. tortuosum , this study aimed to develop a fast purification method for mesembrine and mesembrenone from M. tortuosum . A simple acid-base extraction was used as the first semi-purification step, which was directly followed by semi-preparative HPLC purification as final step. This reduced the number of steps as previously reported. Analytical quantities of mesembrine and mesembrenone (chemical structures confirmed by NMR and MS) could be purified up to roughly 98% purity (as determined by HPLC) with the whole purification process from extraction to obtaining the pure compounds completed at the relatively short period of only four hours. A quick and simple method to purify the mesembrine-type alkaloids, mesembrine and mesembrenone, was developed which produced sufficient quantities for use in analytical and biological studies. This provides a cost-effective method to overcome the lack of pure reference compounds for M. tortuosum . Alkaloids Anxiety HPLC Isolation Mesembryanthemaceae Sceletium tortuosum Introduction Mesembryanthemum tortuosum L. Mesembryanthemaceae (Aizoaceae), also known as Sceletium tortuosum (L.) N.E.Br., has received significant scientific interest due to its clinical potential in treating anxiety and depression. This specific pharmacological action is attributed to its active chemical constituents referred to as mesembrine-type alkaloids (Olatunji et al. 2022; Maphanga et al. 2022). Traditionally, this medicinal plant has been used as mood elevator, stress reducer, analgesic, anxiolytic or even a narcotic. It has also been used for its euphoric/intoxicating effects, as a hunger suppressant or to treat toothache and abdominal pain. Aerial parts of M. tortuosum are mostly chewed, but it is also smoked, ingested as a tincture or tea and even used as a snuff (Gericke and Viljoen 2008; Loria et al. 2014; Olatunji et al. 2022). The major alkaloids in M. tortuosum are (−)-mesembrine ( 1 ), and (+)-mesembrenone ( 2 ) with mesembranol, and (+)-mesembrenol being present at lower concentrations. Mesembrine is, however, the major chemical constituent and is considered to be the main active component of M. tortuosum (Gericke and Viljoen 2008; Shikanga et al. 2011; Krstenansky 2017). The mesembrine-type alkaloids have received a lot of attention for the possible treatment of anxiety and depression (Olatunji et al. 2022). The mesembrine - alkaloids function in the brain by dual inhibition of phosphodiesterase-4 (PDE4) and serotonin (5-HT) reuptake, which has a synergistic therapeutic action (Gericke and Viljoen 2008). Mesembrine has been shown to be a potent 5-hydroxytryptamine (5-HT) reuptake inhibitor, which is essential for effective treatment of depression, whereas mesembrenone was found to have greater activity on PDE4A and PDE4B than the other alkaloids (Harvey et al. 2011). Due to the scarcity and cost of pure reference standards of the mesembrine-type alkaloids, such as mesembrine and mesembrenone, in-depth pre-clinical and clinical research in this field is severely hampered. There are currently very few suppliers and those that do supply the alkaloids produce them synthetically at a very high cost. Further inconvenience is created by a very long lead time as the compounds are usually only synthesised on demand. The synthesis of mesembrine was first achieved in 1991 and subsequently, various improved methods of total synthesis have been reported, but it remains a tedious multistep process (Parkinson and Pinhey 1991; Van Otterlo and Green 2018). Purification of mesembrine-type alkaloids from M. tortuosum plant material has also been reported. Only one study focussed solely on the purification of the alkaloids and reported on a method using gravity-fed column chromatography (CC) to separate a crude extract into four fractions, followed by high-speed counter-current chromatography as a final purification step (Shikanga et al. 2011). Purification employing several CC steps has also been reported by other researchers mainly due to the unavailability of reference compounds necessitating purification for analytical, chemotaxonomic, pharmacological, pharmacokinetic and toxicological studies (Patnala and Kanfer 2010; Harvey et al. 2011; Patnala and Kanfer 2013; Meyer et al. 2015; Manda et al. 2016). The difficulty in obtaining pure reference standards for the pharmacologically active phytochemicals of M. tortuosum therefore warrants a quick and efficient method to purify the main alkaloids, mesembrine and mesembrenone. Here we report on a fast method using semi-preparative HPLC to directly purify mesembrine and mesembrenone from a crude extract of M. tortuosum to yield highly pure compounds for analytical studies. Materials and methods HPLC grade acetonitrile (ACN) was obtained from Thermo Fisher (Cape Town, South Africa) and CP grade CH 2 Cl 2 from MCL (Johannesburg, South Africa). Pure water was obtained from a Rephile direct pure UP Ultrapure & RO Lab water system (Boston, MA, USA). A mesembrine reference standard (98.6% purity, lot number 5888-078A8) was purchased from TLC Pharmaceutical standards (Ontario, Canada). Commercial cultivated M. tortuosum was obtained from GeoGreen Health (Klerksdorp, South Africa) which supplied fermented powder (TKP/240123/M) produced on 15/01/2024. According to the certificate of analysis, the sample contained 1.550 and 0.306% of mesembrine and mesembrenone, respectively. An Ultimate 3000 semi-preparative high-performance liquid chromatography (HPLC) system consisting of an HPG-3200BX Biocompatible binary semi-preparative pump, VWD-3100 variable wavelength detector and a Rheodyne manual injector with a 1 ml injection loop was used for separation and purification. The fractions were collected with a fraction collector model F. The system was operated with Chromeleon 7 software and a Fortis C18 column was used for separation (21.2 × 250 mm, 5 µm). An isocratic mobile phase consisting of 0.1% ammonia (A) and acetonitrile (ACN) (B) was used at a flow rate of 14 ml/min. 50% B was used for a total run time of 12 min with mesembrenone and mesembrine eluting at 7.0 and 9.0 min, respectively. For determining the purity of mesembrenone and mesembrine a Shimadzu i-Nexera HPLC system equipped with a quaternary pump, autosampler, and a photodiode array detector was used. The system was fitted with a GL sciences C-18, 2.1 × 150 mm, 3 µm column with the column oven set to 40°C. The solvent system consisted of 0.1% ammonia (A) and ACN (B), and an isocratic system was employed in a 60% (A) 40% (B) ratio. The flow rate was 0.25 ml/min and 1 µl of each sample was injected. The DAD detector was set to 228 nm. NMR spectra were recorded on a Bruker 600 Avance II NMR at 600MHz for 1 H NMR using CDCl 3 as solvent. MS data was generated on an Agilent Ultivo TQ system using the same analytical setup as described for HPLC. A flow diagram of the process is provided in Fig. S1 . In short, CH 2 Cl 2 extraction was performed by adding 5 g of M. tortuosum plant material to 100 ml CH 2 Cl 2 and sonicating the solution for 15 min. The solution was filtered with Whatman® GF/C 55 mm filter paper (BN:1822055). The extraction procedure was repeated three times in total. The combined filtrates were liquid:liquid partitioned with 0.25M sulfuric acid and separated from the CH 2 Cl 2 fraction (repeated three times). The pH of the combined acidified solution was adjusted to 9 with the addition of 20% (v/v) ammonia. CH 2 Cl 2 was added to the basified solution, the solution was then shaken vigorously and allowed to separate (3 × 50 ml). The final CH 2 Cl 2 solutions were combined and evaporated to dryness. The final CH 2 Cl 2 extract was redissolved in HPLC eluent to a concentration of 50 mg/ml. The solution was filtered before injection into the chromatograph, which was operated at room temperature. Fraction collection times were set to 7.0-8.5 and 9.0-10.5 min with a total run time of 12 min. Results and Discussion The method developed and described here offers a fast way to purify the important alkaloids mesembrine and mesembrenone. The most time-consuming step remains the acid-based extraction and semi-purification of the alkaloids. This step which was repeated three times for each stage took approximately two-three hours in total. This time can be reduced by only repeating each step once, but this can lead to some loss in total yield. Drying the final CH 2 Cl 2 extract yielded 176 mg (3.56% yield). According to the supplier of the Fortis column the loading capacity on the column is between 10–200 mg depending on several factors such as how well retained the target compounds are (longer R t = higher capacity), the complexity of the sample (higher capacity for simple mixtures), and the solubility of the sample. Therefore, the crude extract was reconstituted to provide a 50 mg/ml solution for injection. However, due to the limited solubility of the CH 2 Cl 2 extract, a filtering step was required which removed approximately 40% of the extract resulting in only 30.1 mg/ml that could be injected per run (determined by separately filtering and drying 1 ml of the solution). The purification step is quick, with a total run time of 12 min per 1 ml injection with collection of the two target compounds at times 7.0-8.5 min (mesembrenone) and 9.0-10.5 min (mesembrine). Drying of these two fractions yielded 3.4 and 8.4 mg for mesembrenone and mesembrine, respectively. This represents a yield of 80% and 39.5%, respectively (the theoretical yields based on the certificate of analysis provided with the M. tortuosum sample are 4.25 and 21.25 mg per 1 ml injected). The purity of the compounds based on HPLC analysis was 97.2 and 98.9% for mesembrenone and mesembrine, respectively (Fig. S2 ), with 1 H NMR, MS and UV analysis confirming the chemical structure and identity which corresponded well with literature (Jeffs et al. 1970; Patnala and Kanfer 2010; Meyer et al. 2015; Krstenansky 2017). The purchased authentic mesembrine standard was spiked with the purified mesembrine and analysed by HPLC. Only one peak was observed which provided further confirmation of its identity. Chemical data of the two purified compounds (i.e. mesembrenone and mesembrine) matched well with the literature (spectra available in the Supplementary Information). Mesembrine ( 1 ) : 1 H NMR (600 MHz, CDCl 3 ) δ: 6.91 (dd, J = 8.4 and 1.8 Hz, H-6′, 1H), 6.89 (d, J = 1.8 Hz, H-2′, 1H), 6.87 (d, J = 8.4 Hz, H-5′, 1H), 3.88 (s, 3H, 3′-OCH 3 , 3H), 3.86 (s, 3H, 4′-OCH 3 , 3H), 3.12–3.16 (m, 1H), 2.95 (m, 1H), 2.59 (m, 2H), 2.32–2.45 (m, 2H) 2.33 (s, N-Me, 3H), 2.04–2.26 (m, 5H). UV λ max 229 and 280 nm; MS (+ H) 290.3. Mesembrenone ( 2 ) : 1 H NMR (600 MHz, CDCl 3 ) δ: 6.89 (dd, J = 8.0 and 2.0 Hz; H-6′), 6.85 (d, J = 2.0 Hz, H-2′), 6.84 (d, J = 8.0 Hz, H-5′), 6.72 (dd, J = 10.2 and 1.8 Hz, H-4), 6.11 (d, J = 10.2, H-5), 3.88 (s, 3H, 3′-OCH3), 3.87 (s, 3H, 4′-OCH3), 3.32 (m, 1H), 2.68 (m, 1H), 2.42–2.61 (m, 5H), 2.32 (s, 3H, NCH3), 2.18–2.25 (m, 1H). UV λ max 225 and 279nm; MS (+ H) 288.3. Depending on the specific requirements of a study, a single injection of 1 ml on the semi-preparative HPLC will yield enough purified material for analytical studies such as chemotaxonomic and/or metabolomics analysis. With a run time of only 12 min, multiple injections will yield relatively large quantities (i.e. 100’s of milligrams) within a matter of hours and with the use of a larger column and injector loop, gram quantities could be purified using this method. Declarations Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contributions SEN: Investigation, data curation, writing - review & editing, visualization. DM: Investigation, writing - review & editing. JH: Conceptualization, resources, writing, review & editing, supervision. FK: Conceptualization, investigation, resources, writing original draft, supervision. All authors have read and agreed to the published version of the manuscript. Acknowledgements The authors are thankful to Pharmacen™, (Centre of Excellence for Pharmaceutical Sciences), North-West University for financial support. We thank GeoGreenhealth (Klerksdorp, South Africa) for donating the M. tortuosum material used in this study. Graphical abstract and Fig. 1 partially created with BioRender.com, photographic illustrations of M. tortuosum taken from Gericke and Viljoen (2008) (permission granted from Senior Copyrights Specialist) and chemical structures were created with MestReNova. Availability of data and material Not applicable References Gericke N, Viljoen AM (2008) Sceletium - A review update. J Ethnopharmacol 19: 653-663. https://doi.org/10.1016/j.jep.2008.07.043 Harvey AL, Young LC, Viljoen AM, Gericke NP (2011) Pharmacological actions of the South African medicinal and functional food plant Sceletium tortuosum and its principal alkaloids . J Ethnopharmacol 137:1124-1129. https://doi.org/10.1016/j.jep.2011.07.035 Jeffs PW, Hawks RL, Farrier DS (1970) Structures of the mesembranols and the absolute configuration of mesembrine and related alkaloids. J Am Chem Soc 91:3831–3839. https://doi.org/10.1021/jo00835a071 Krstenansky JL (2017) Mesembrine alkaloids: Review of their occurrence, chemistry, and pharmacology. J Ethnopharmacol 4:10-19. https://doi.org/10.1016/j.jep.2016.12.004 Loria MJ, Ali Z, Abe N, Sufka KJ, Khan IA (2014) Effects of Sceletium tortuosum in rats. J Ethnopharmacol 155:731-735. https://doi.org/10.1016/j.jep.2014.06.007 Manda VK, Avula B, Ashfaq MK, Abe N, Khan IA, Khan SI (2016) Quantification of mesembrine and mesembrenone in mouse plasma using UHPLC-QToF-MS: Application to a pharmacokinetic study. Biomed Chromatogr 31:1-7. https://doi.org/10.1002/bmc.3815 Maphanga VB, Skalicka-Wozniak K, Budzynska B, Skiba A, Chen W, Agoni C, Enslin GM, Viljoen AM (2022) Mesembryanthemum tortuosum L. alkaloids modify anxiety-like behaviour in a zebrafish model. J Ethnopharmacol 290:115068. https://doi.org/10.1016/j.jep.2022.115068 Meyer GMJ, Wink CSD, Zapp J, Maurer HH (2015) GC-MS, LC-MS n , LC-high resolution-MS n , and NMR studies on the metabolism and toxicological detection of mesembrine and mesembrenone, the main alkaloids of the legal high “Kanna” isolated from Sceletium tortuosum . Anal Bioanal Chem 407:761–778. https://doi.org/10.1007/s00216-014-8109-9 Olatunji TL, Siebert F, Adetunji AE, Harvey BH, Gericke J, Hamman JH, Van der Kooy F (2022) Sceletium tortuosum : A review on its phytochemistry, pharmacokinetics, biological, pre-clinical and clinical activities. J Ethnopharmacol 287:114711. https://doi.org/10.1016/j.jep.2021.114711 Parkinson CJ, Pinhey JT (1991) Use of the electrophilic arylation reaction of aryllead triacetates in syntheses of (±)-O-methyljoubertiamine and (±)-mesembrine. J Chem Soc Perkin Trans 1:1053-1057. https://doi.org/10.1039/P19910001053 Patnala S, Kanfer I (2010) HPLC analysis of mesembrine-type alkaloids in Sceletium plant material used as an African traditional medicine. J Pharm Pharm Sci 13:558–570. https://doi.org/10.18433/J3DK5F Patnala S, Kanfer I (2013) Chemotaxonomic studies of mesembrine-type alkaloids in Sceletium plant species. S Afr J Sci 109:5. https://doi.org/10.1590/sajs.2013/882 Shikanga EA, Viljoen A, Combrinck S, Marston A (2011) Isolation of Sceletium alkaloids by high-speed countercurrent chromatography. Phytochem Lett 4:190-193. http://dx.doi.org/10.1016/j.phytol.2011.03.003 Van Otterlo WAL, Green IR (2018) A review on recent syntheses of Amaryllidaceae alkaloids and isocarbostyrils (time period mid-2016 to 2017). Nat Prod Commun 13:255-277. http://dx.doi.org/10.1177/1934578X1801300305 Supplementary Files GraphicalabstractMesembrinePurification.tif Supplementarydata.docx Cite Share Download PDF Status: Under Revision Version 1 posted Reviewers agreed at journal 08 Jan, 2025 Reviewers invited by journal 08 Jan, 2025 Editor invited by journal 06 Jan, 2025 First submitted to journal 05 Jan, 2025 Editorial decision: Major revisions 25 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5413491","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":399418444,"identity":"b58c892b-d403-482b-bcaa-a64a0a39027c","order_by":0,"name":"Suzanne Elaine Van Niekerk","email":"","orcid":"","institution":"North-West University Potchefstroom Campus: North-West University","correspondingAuthor":false,"prefix":"","firstName":"Suzanne","middleName":"Elaine Van","lastName":"Niekerk","suffix":""},{"id":399418445,"identity":"0b9b9c63-d550-45e6-8ec5-5c37b1a7fcd6","order_by":1,"name":"Dorothea Malan","email":"","orcid":"","institution":"North-West University Potchefstroom Campus: North-West University","correspondingAuthor":false,"prefix":"","firstName":"Dorothea","middleName":"","lastName":"Malan","suffix":""},{"id":399418446,"identity":"79d027ea-19ec-4a79-9e9a-9bf42d5296f0","order_by":2,"name":"Josias Hendrik Hamman","email":"","orcid":"","institution":"North-West University Potchefstroom Campus: North-West University","correspondingAuthor":false,"prefix":"","firstName":"Josias","middleName":"Hendrik","lastName":"Hamman","suffix":""},{"id":399418447,"identity":"019778fc-d666-41dc-a2ae-17e777c318ef","order_by":3,"name":"Frank van der Kooy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYBAC9gYGBsYGEOsAAzOIkgOzH+DRwnMATYsxmJ1AipZEMAevFunDzx7ObLPL4zve+9jgQ8Xh9Plhhx8CbbGT023AoYUvzdxwY1tyseSZ48aJM84czt14O80AqCXZ2OwAdi32PAxmkg/OMCduuJHGfJi3DahldgJIy4HEbTi08PCwfwNqqU/ccP8ZWEu64ez0DwS08JhJbqg4DLSFjTkZqCVBXjqHkC08ZZIzKo4nzjyTxmw440y64QbpnIIDCQa4/QJ02DbJHoPqxL7jx5glPlRYy8vPTt/84UOFnRwuLeigmcEArNKAOOUgUMcg30C86lEwCkbBKBgZAAAaIWV18iWF3gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0565-5132","institution":"North-West University Potchefstroom Campus: North-West University","correspondingAuthor":true,"prefix":"","firstName":"Frank","middleName":"van der","lastName":"Kooy","suffix":""}],"badges":[],"createdAt":"2024-11-08 04:17:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5413491/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5413491/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73454145,"identity":"801ad6f0-4493-4944-ac1a-38ca527987c6","added_by":"auto","created_at":"2025-01-10 06:52:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":380561,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5413491/v1/a8132c46-f198-41b8-b3ca-77566cc1b246.pdf"},{"id":73454051,"identity":"ebb70b4d-16b5-450b-ac62-b771c6cf4e10","added_by":"auto","created_at":"2025-01-10 06:44:40","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":420202,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractMesembrinePurification.tif","url":"https://assets-eu.researchsquare.com/files/rs-5413491/v1/361ad42e9ddc9edf1a4dc962.tif"},{"id":73453144,"identity":"35eb9c92-3a84-483b-81a5-2f81607356d0","added_by":"auto","created_at":"2025-01-10 06:36:40","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":558033,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5413491/v1/6b6f9c679c9deef1bdeb7154.docx"}],"financialInterests":"","formattedTitle":"Fast Isolation and Purification of the Antidepressant Alkaloids Mesembrine and Mesembrenone From Mesembryanthemum tortuosum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMesembryanthemum tortuosum\u003c/em\u003e L. Mesembryanthemaceae (Aizoaceae), also known as \u003cem\u003eSceletium tortuosum\u003c/em\u003e (L.) N.E.Br., has received significant scientific interest due to its clinical potential in treating anxiety and depression. This specific pharmacological action is attributed to its active chemical constituents referred to as mesembrine-type alkaloids (Olatunji et al. 2022; Maphanga et al. 2022). Traditionally, this medicinal plant has been used as mood elevator, stress reducer, analgesic, anxiolytic or even a narcotic. It has also been used for its euphoric/intoxicating effects, as a hunger suppressant or to treat toothache and abdominal pain. Aerial parts of \u003cem\u003eM. tortuosum\u003c/em\u003e are mostly chewed, but it is also smoked, ingested as a tincture or tea and even used as a snuff (Gericke and Viljoen 2008; Loria et al. 2014; Olatunji et al. 2022).\u003c/p\u003e \u003cp\u003eThe major alkaloids in \u003cem\u003eM. tortuosum\u003c/em\u003e are (\u0026minus;)-mesembrine (\u003cb\u003e1\u003c/b\u003e), and (+)-mesembrenone (\u003cb\u003e2\u003c/b\u003e) with mesembranol, and (+)-mesembrenol being present at lower concentrations. Mesembrine is, however, the major chemical constituent and is considered to be the main active component of \u003cem\u003eM. tortuosum\u003c/em\u003e (Gericke and Viljoen 2008; Shikanga et al. 2011; Krstenansky 2017). The mesembrine-type alkaloids have received a lot of attention for the possible treatment of anxiety and depression (Olatunji et al. 2022). The mesembrine\u003cem\u003e-\u003c/em\u003ealkaloids function in the brain by dual inhibition of phosphodiesterase-4 (PDE4) and serotonin (5-HT) reuptake, which has a synergistic therapeutic action (Gericke and Viljoen 2008). Mesembrine has been shown to be a potent 5-hydroxytryptamine (5-HT) reuptake inhibitor, which is essential for effective treatment of depression, whereas mesembrenone was found to have greater activity on PDE4A and PDE4B than the other alkaloids (Harvey et al. 2011).\u003c/p\u003e\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1736490475.png\"\u003e\u003cbr\u003e\u003c/p\u003e\u003cp\u003eDue to the scarcity and cost of pure reference standards of the mesembrine-type alkaloids, such as mesembrine and mesembrenone, in-depth pre-clinical and clinical research in this field is severely hampered. There are currently very few suppliers and those that do supply the alkaloids produce them synthetically at a very high cost. Further inconvenience is created by a very long lead time as the compounds are usually only synthesised on demand. The synthesis of mesembrine was first achieved in 1991 and subsequently, various improved methods of total synthesis have been reported, but it remains a tedious multistep process (Parkinson and Pinhey 1991; Van Otterlo and Green 2018).\u003c/p\u003e \u003cp\u003ePurification of mesembrine-type alkaloids from \u003cem\u003eM. tortuosum\u003c/em\u003e plant material has also been reported. Only one study focussed solely on the purification of the alkaloids and reported on a method using gravity-fed column chromatography (CC) to separate a crude extract into four fractions, followed by high-speed counter-current chromatography as a final purification step (Shikanga et al. 2011). Purification employing several CC steps has also been reported by other researchers mainly due to the unavailability of reference compounds necessitating purification for analytical, chemotaxonomic, pharmacological, pharmacokinetic and toxicological studies (Patnala and Kanfer 2010; Harvey et al. 2011; Patnala and Kanfer 2013; Meyer et al. 2015; Manda et al. 2016).\u003c/p\u003e \u003cp\u003eThe difficulty in obtaining pure reference standards for the pharmacologically active phytochemicals of \u003cem\u003eM. tortuosum\u003c/em\u003e therefore warrants a quick and efficient method to purify the main alkaloids, mesembrine and mesembrenone. Here we report on a fast method using semi-preparative HPLC to directly purify mesembrine and mesembrenone from a crude extract of \u003cem\u003eM. tortuosum\u003c/em\u003e to yield highly pure compounds for analytical studies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eHPLC grade acetonitrile (ACN) was obtained from Thermo Fisher (Cape Town, South Africa) and CP grade CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e from MCL (Johannesburg, South Africa). Pure water was obtained from a Rephile direct pure UP Ultrapure \u0026amp; RO Lab water system (Boston, MA, USA). A mesembrine reference standard (98.6% purity, lot number 5888-078A8) was purchased from TLC Pharmaceutical standards (Ontario, Canada).\u003c/p\u003e \u003cp\u003eCommercial cultivated \u003cem\u003eM. tortuosum\u003c/em\u003e was obtained from GeoGreen Health (Klerksdorp, South Africa) which supplied fermented powder (TKP/240123/M) produced on 15/01/2024. According to the certificate of analysis, the sample contained 1.550 and 0.306% of mesembrine and mesembrenone, respectively.\u003c/p\u003e \u003cp\u003eAn Ultimate 3000 semi-preparative high-performance liquid chromatography (HPLC) system consisting of an HPG-3200BX Biocompatible binary semi-preparative pump, VWD-3100 variable wavelength detector and a Rheodyne manual injector with a 1 ml injection loop was used for separation and purification. The fractions were collected with a fraction collector model F. The system was operated with Chromeleon 7 software and a Fortis C18 column was used for separation (21.2 \u0026times; 250 mm, 5 \u0026micro;m). An isocratic mobile phase consisting of 0.1% ammonia (A) and acetonitrile (ACN) (B) was used at a flow rate of 14 ml/min. 50% B was used for a total run time of 12 min with mesembrenone and mesembrine eluting at 7.0 and 9.0 min, respectively.\u003c/p\u003e \u003cp\u003eFor determining the purity of mesembrenone and mesembrine a Shimadzu i-Nexera HPLC system equipped with a quaternary pump, autosampler, and a photodiode array detector was used. The system was fitted with a GL sciences C-18, 2.1 \u0026times; 150 mm, 3 \u0026micro;m column with the column oven set to 40\u0026deg;C. The solvent system consisted of 0.1% ammonia (A) and ACN (B), and an isocratic system was employed in a 60% (A) 40% (B) ratio. The flow rate was 0.25 ml/min and 1 \u0026micro;l of each sample was injected. The DAD detector was set to 228 nm. NMR spectra were recorded on a Bruker 600 Avance II NMR at 600MHz for \u003csup\u003e1\u003c/sup\u003eH NMR using CDCl\u003csub\u003e3\u003c/sub\u003e as solvent. MS data was generated on an Agilent Ultivo TQ system using the same analytical setup as described for HPLC.\u003c/p\u003e \u003cp\u003eA flow diagram of the process is provided in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. In short, CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e extraction was performed by adding 5 g of \u003cem\u003eM. tortuosum\u003c/em\u003e plant material to 100 ml CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e and sonicating the solution for 15 min. The solution was filtered with Whatman\u0026reg; GF/C 55 mm filter paper (BN:1822055). The extraction procedure was repeated three times in total. The combined filtrates were liquid:liquid partitioned with 0.25M sulfuric acid and separated from the CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e fraction (repeated three times). The pH of the combined acidified solution was adjusted to 9 with the addition of 20% (v/v) ammonia. CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e was added to the basified solution, the solution was then shaken vigorously and allowed to separate (3 \u0026times; 50 ml). The final CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e solutions were combined and evaporated to dryness.\u003c/p\u003e \u003cp\u003eThe final CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e extract was redissolved in HPLC eluent to a concentration of 50 mg/ml. The solution was filtered before injection into the chromatograph, which was operated at room temperature. Fraction collection times were set to 7.0-8.5 and 9.0-10.5 min with a total run time of 12 min.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe method developed and described here offers a fast way to purify the important alkaloids mesembrine and mesembrenone. The most time-consuming step remains the acid-based extraction and semi-purification of the alkaloids. This step which was repeated three times for each stage took approximately two-three hours in total. This time can be reduced by only repeating each step once, but this can lead to some loss in total yield. Drying the final CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e extract yielded 176 mg (3.56% yield).\u003c/p\u003e \u003cp\u003eAccording to the supplier of the Fortis column the loading capacity on the column is between 10\u0026ndash;200 mg depending on several factors such as how well retained the target compounds are (longer \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e = higher capacity), the complexity of the sample (higher capacity for simple mixtures), and the solubility of the sample. Therefore, the crude extract was reconstituted to provide a 50 mg/ml solution for injection. However, due to the limited solubility of the CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e extract, a filtering step was required which removed approximately 40% of the extract resulting in only 30.1 mg/ml that could be injected per run (determined by separately filtering and drying 1 ml of the solution).\u003c/p\u003e \u003cp\u003eThe purification step is quick, with a total run time of 12 min per 1 ml injection with collection of the two target compounds at times 7.0-8.5 min (mesembrenone) and 9.0-10.5 min (mesembrine). Drying of these two fractions yielded 3.4 and 8.4 mg for mesembrenone and mesembrine, respectively. This represents a yield of 80% and 39.5%, respectively (the theoretical yields based on the certificate of analysis provided with the \u003cem\u003eM. tortuosum\u003c/em\u003e sample are 4.25 and 21.25 mg per 1 ml injected). The purity of the compounds based on HPLC analysis was 97.2 and 98.9% for mesembrenone and mesembrine, respectively (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), with \u003csup\u003e1\u003c/sup\u003eH NMR, MS and UV analysis confirming the chemical structure and identity which corresponded well with literature (Jeffs et al. 1970; Patnala and Kanfer 2010; Meyer et al. 2015; Krstenansky 2017). The purchased authentic mesembrine standard was spiked with the purified mesembrine and analysed by HPLC. Only one peak was observed which provided further confirmation of its identity. Chemical data of the two purified compounds (i.e. mesembrenone and mesembrine) matched well with the literature (spectra available in the Supplementary Information).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMesembrine (\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e \u003cem\u003e)\u003c/em\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ: 6.91 (dd, J\u0026thinsp;=\u0026thinsp;8.4 and 1.8 Hz, H-6\u0026prime;, 1H), 6.89 (d, J\u0026thinsp;=\u0026thinsp;1.8 Hz, H-2\u0026prime;, 1H), 6.87 (d, J\u0026thinsp;=\u0026thinsp;8.4 Hz, H-5\u0026prime;, 1H), 3.88 (s, 3H, 3\u0026prime;-OCH\u003csub\u003e3\u003c/sub\u003e, 3H), 3.86 (s, 3H, 4\u0026prime;-OCH\u003csub\u003e3\u003c/sub\u003e, 3H), 3.12\u0026ndash;3.16 (m, 1H), 2.95 (m, 1H), 2.59 (m, 2H), 2.32\u0026ndash;2.45 (m, 2H) 2.33 (s, N-Me, 3H), 2.04\u0026ndash;2.26 (m, 5H). UV λ\u003csub\u003emax\u003c/sub\u003e 229 and 280 nm; MS (+\u0026thinsp;H) 290.3.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMesembrenone (\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e \u003cem\u003e)\u003c/em\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ: 6.89 (dd, J\u0026thinsp;=\u0026thinsp;8.0 and 2.0 Hz; H-6\u0026prime;), 6.85 (d, J\u0026thinsp;=\u0026thinsp;2.0 Hz, H-2\u0026prime;), 6.84 (d, J\u0026thinsp;=\u0026thinsp;8.0 Hz, H-5\u0026prime;), 6.72 (dd, J\u0026thinsp;=\u0026thinsp;10.2 and 1.8 Hz, H-4), 6.11 (d, J\u0026thinsp;=\u0026thinsp;10.2, H-5), 3.88 (s, 3H, 3\u0026prime;-OCH3), 3.87 (s, 3H, 4\u0026prime;-OCH3), 3.32 (m, 1H), 2.68 (m, 1H), 2.42\u0026ndash;2.61 (m, 5H), 2.32 (s, 3H, NCH3), 2.18\u0026ndash;2.25 (m, 1H). UV λ\u003csub\u003emax\u003c/sub\u003e 225 and 279nm; MS (+\u0026thinsp;H) 288.3.\u003c/p\u003e \u003cp\u003eDepending on the specific requirements of a study, a single injection of 1 ml on the semi-preparative HPLC will yield enough purified material for analytical studies such as chemotaxonomic and/or metabolomics analysis. With a run time of only 12 min, multiple injections will yield relatively large quantities (i.e. 100\u0026rsquo;s of milligrams) within a matter of hours and with the use of a larger column and injector loop, gram quantities could be purified using this method.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eSEN: Investigation, data curation, writing - review \u0026amp; editing, visualization. DM: Investigation, writing - review \u0026amp; editing. JH: Conceptualization, resources, writing, review \u0026amp; editing, supervision. FK: Conceptualization, investigation, resources, writing original draft, supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors are thankful to Pharmacen\u0026trade;, (Centre of Excellence for Pharmaceutical Sciences), North-West University for financial support. We thank GeoGreenhealth (Klerksdorp, South Africa) for donating the \u003cem\u003eM. tortuosum\u003c/em\u003e material used in this study. Graphical abstract and Fig.\u0026nbsp;1 partially created with BioRender.com, photographic illustrations of \u003cem\u003eM. tortuosum\u003c/em\u003e taken from Gericke and Viljoen (2008) (permission granted from Senior Copyrights Specialist) and chemical structures were created with MestReNova.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGericke N, Viljoen AM (2008) Sceletium - A review update. J Ethnopharmacol 19: 653-663. https://doi.org/10.1016/j.jep.2008.07.043\u003c/li\u003e\n \u003cli\u003eHarvey AL, Young LC, Viljoen AM, Gericke NP (2011) Pharmacological actions of the South African medicinal and functional food plant \u003cem\u003eSceletium tortuosum\u003c/em\u003e and its principal alkaloids\u003cem\u003e.\u0026nbsp;\u003c/em\u003eJ Ethnopharmacol 137:1124-1129. https://doi.org/10.1016/j.jep.2011.07.035\u003c/li\u003e\n \u003cli\u003eJeffs PW, Hawks RL, Farrier DS (1970) Structures of the mesembranols and the absolute configuration of mesembrine and related alkaloids. J Am Chem Soc 91:3831\u0026ndash;3839. https://doi.org/10.1021/jo00835a071\u003c/li\u003e\n \u003cli\u003eKrstenansky JL (2017) Mesembrine alkaloids: Review of their occurrence, chemistry, and pharmacology. J Ethnopharmacol 4:10-19. https://doi.org/10.1016/j.jep.2016.12.004\u003c/li\u003e\n \u003cli\u003eLoria MJ, Ali Z, Abe N, Sufka KJ, Khan IA (2014) Effects of \u003cem\u003eSceletium tortuosum\u003c/em\u003e in rats. J Ethnopharmacol 155:731-735. https://doi.org/10.1016/j.jep.2014.06.007\u003c/li\u003e\n \u003cli\u003eManda VK, Avula B, Ashfaq MK, Abe N, Khan IA, Khan SI (2016) Quantification of mesembrine and mesembrenone in mouse plasma using UHPLC-QToF-MS: Application to a pharmacokinetic study. Biomed Chromatogr 31:1-7. https://doi.org/10.1002/bmc.3815\u003c/li\u003e\n \u003cli\u003eMaphanga VB, Skalicka-Wozniak K, Budzynska B, Skiba A, Chen W, Agoni C, Enslin GM, Viljoen AM (2022) \u003cem\u003eMesembryanthemum tortuosum\u003c/em\u003e L. alkaloids modify anxiety-like behaviour in a zebrafish model. J Ethnopharmacol 290:115068. https://doi.org/10.1016/j.jep.2022.115068\u003c/li\u003e\n \u003cli\u003eMeyer GMJ, Wink CSD, Zapp J, Maurer HH (2015) GC-MS, LC-MS\u003csup\u003en\u003c/sup\u003e, LC-high resolution-MS\u003csup\u003en\u003c/sup\u003e, and NMR studies on the metabolism and toxicological detection of mesembrine and mesembrenone, the main alkaloids of the legal high \u0026ldquo;Kanna\u0026rdquo; isolated from \u003cem\u003eSceletium tortuosum\u003c/em\u003e. Anal Bioanal Chem 407:761\u0026ndash;778. https://doi.org/10.1007/s00216-014-8109-9\u003c/li\u003e\n \u003cli\u003eOlatunji TL, Siebert F, Adetunji AE, Harvey BH, Gericke J, Hamman JH, Van der Kooy F (2022) \u003cem\u003eSceletium tortuosum\u003c/em\u003e: A review on its phytochemistry, pharmacokinetics, biological, pre-clinical and clinical activities. J Ethnopharmacol 287:114711. https://doi.org/10.1016/j.jep.2021.114711\u003c/li\u003e\n \u003cli\u003eParkinson CJ, Pinhey JT (1991) Use of the electrophilic arylation reaction of aryllead triacetates in syntheses of (\u0026plusmn;)-O-methyljoubertiamine and (\u0026plusmn;)-mesembrine. J Chem Soc Perkin\u003cem\u003e\u0026nbsp;\u003c/em\u003eTrans 1:1053-1057. https://doi.org/10.1039/P19910001053\u003c/li\u003e\n \u003cli\u003ePatnala S, Kanfer I (2010) HPLC analysis of mesembrine-type alkaloids in \u003cem\u003eSceletium\u003c/em\u003e plant material used as an African traditional medicine. J Pharm Pharm Sci 13:558\u0026ndash;570. https://doi.org/10.18433/J3DK5F\u003c/li\u003e\n \u003cli\u003ePatnala S, Kanfer I (2013) Chemotaxonomic studies of mesembrine-type alkaloids in \u003cem\u003eSceletium\u003c/em\u003e plant species. S Afr J Sci 109:5. https://doi.org/10.1590/sajs.2013/882\u003c/li\u003e\n \u003cli\u003eShikanga EA, Viljoen A, Combrinck S, Marston A (2011) Isolation of \u003cem\u003eSceletium\u003c/em\u003e alkaloids by high-speed countercurrent chromatography. Phytochem Lett 4:190-193. http://dx.doi.org/10.1016/j.phytol.2011.03.003\u003c/li\u003e\n \u003cli\u003eVan Otterlo WAL, Green IR (2018) A review on recent syntheses of Amaryllidaceae alkaloids and isocarbostyrils (time period mid-2016 to 2017). Nat Prod Commun 13:255-277. http://dx.doi.org/10.1177/1934578X1801300305 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alkaloids, Anxiety, HPLC, Isolation, Mesembryanthemaceae, Sceletium tortuosum","lastPublishedDoi":"10.21203/rs.3.rs-5413491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5413491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMesembryanthemum tortuosum\u003c/em\u003e L., Aizoaceae, is a medicinal plant that has traditionally been used as mood elevator, stress reducer, analgesic, anxiolytic, and even a narcotic. The main activity is attributed to the mesembrine-type alkaloids, which have shown promise to provide pharmacologically and clinically antidepressant and anxiolytic effects. Due to the limited availability and cost of pure reference compounds of the main active chemical compounds of \u003cem\u003eM. tortuosum\u003c/em\u003e, this study aimed to develop a fast purification method for mesembrine and mesembrenone from \u003cem\u003eM. tortuosum\u003c/em\u003e. A simple acid-base extraction was used as the first semi-purification step, which was directly followed by semi-preparative HPLC purification as final step. This reduced the number of steps as previously reported. Analytical quantities of mesembrine and mesembrenone (chemical structures confirmed by NMR and MS) could be purified up to roughly 98% purity (as determined by HPLC) with the whole purification process from extraction to obtaining the pure compounds completed at the relatively short period of only four hours. A quick and simple method to purify the mesembrine-type alkaloids, mesembrine and mesembrenone, was developed which produced sufficient quantities for use in analytical and biological studies. This provides a cost-effective method to overcome the lack of pure reference compounds for \u003cem\u003eM. tortuosum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Fast Isolation and Purification of the Antidepressant Alkaloids Mesembrine and Mesembrenone From Mesembryanthemum tortuosum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-10 06:36:35","doi":"10.21203/rs.3.rs-5413491/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-01-08T12:43:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-08T12:28:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2025-01-06T18:58:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-01-06T00:46:21+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2024-12-26T01:54:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"457599f4-23b6-4a51-a5b7-f189eddc7f19","owner":[],"postedDate":"January 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T16:41:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-10 06:36:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5413491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5413491","identity":"rs-5413491","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}