Mar 13, 2025
Prefractionating Cyanobacterial Extracts by Solid-Phase Extraction
- Leonardo Santos de Jesus1,
- Marcio B. Weiss1,
- Jaewon Y1,
- Samuel Cavalcante do Amaral1,
- Camila Manoel Crnkovic1
- 1Universidade de São Paulo
Protocol Citation: Leonardo Santos de Jesus, Marcio B. Weiss, Jaewon Y, Samuel Cavalcante do Amaral, Camila Manoel Crnkovic 2025. Prefractionating Cyanobacterial Extracts by Solid-Phase Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wdkrvo5/v1
Manuscript citation:
Weiss, M.B. Chemoprospection of Brazilian Cyanobacteria Using Metabolomics and Biological Assays. Master’s Dissertation. MSc in Biochemical and Pharmaceutical Technology, University of São Paulo, 2023
Figueiredo, L.F. Prefractionation Strategy and Bioassays for Screening Natural Products from Brazilian Cyanobacteria. Undergraduate thesis. B.Sc. in Pharmacy & Biochemistry, School of Pharmaceutical Sciences, University of São Paulo, 2023.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 20, 2025
Last Modified: March 13, 2025
Protocol Integer ID: 118741
Keywords: Prefractionation, Solid-Phase Extraction, HP20SS, Cyanobacteria
Funders Acknowledgements:
FAPESP
Grant ID: 2020/07710-7
Abstract
This protocol presents a prefractionation method for cyanobacterial organic extracts by solid-phase extraction, using a isopropanol-water step gradient and HP20SS as stationary phase. Extract prefractionation allows for improved results in bioassays and metabolomic analyses.
Materials
Solvents and solutions:
- Dichloromethane;
- Methanol (100%);
- HPLC-grade water;
- Acetone;
- Ethyl Acetate;
- Isopropanol (IPA):water solutions (20%, 40%, 70%, 90%, and 100% IPA).
Resin:
- DIAIONTM HP20SS.
Equipment:
- SPE column with frits;
- Manifold and vaccum pump;
- 20 mL test tubes;
- Ultrasonic bath;
- 4 mL vials;
- N2 flow (eqquiped with glass microfiber polypropylene filter, 0.3 µm);
- Vacuum concentrator (optional).
Abstract
Abstract
Cyanobacteria are producers of chemically diverse secondary metabolites with pharmaceutical potential (Weiss et al., 2025). The creation of extract and fraction libraries help systematize the process of natural product discovery (Wilson et al., 2020). Most biological extracts do not allow for an efficient assessment of the bioactivity, often resulting in false positive or false negative results (Wagenaar, 2008). Prefractionation is a widely used method for separating mixtures aiming to reduce the chemical complexity of organic extracts (Stobaugh; Fague; Jorgenson, 2013). Separating extracts into a few prefractions increases the concentration of bioactive metabolites in the sample and allows for improved results in bioassays and metabolomic analyses (Clish, 2015; Rague et al., 2018; Tu; Yan, 2012).
This protocol aims to present a prefractionation method for cyanobacterial organic extracts, using a isopropanol-water step gradient and HP20SS as the stationary phase. It was adapted from Bugni et al., 2008 and Orjala et al., 2011.
Materials and Equipment
Materials and Equipment
Solvents and solutions:
- Dichloromethane;
- Methanol (100%);
- HPLC-grade water;
- Acetone;
- Ethyl Acetate;
- Isopropanol (IPA):water solutions (20%, 40%, 70%, 90%, and 100% IPA).
Resin:
- DIAIONTM HP20SS.
Equipment:
- SPE column with frits;
- Manifold and vaccum pump;
- 20 mL test tubes;
- Ultrasonic bath;
- 4 mL vials with caps;
- N2 flow (eqquiped with glass microfiber polypropylene filter, 0.3 µm);
- Vacuum concentrator (optional).
Dry load preparation
Dry load preparation
Weigh the dry organic extract. A dry extract is shown in Figure 1. In a vial, dissolve the dry organic extract previously prepared from a dichloromethane-methanol 1:1 solution in the smallest possible amount of the same solvent. Use the ultrasonic bath, if needed.
Figure 1: Cyanobacterial extract. Source: Authors (2023).
Add to the vial the same amount of chromatographic resin (HP20SS) as the initial dry extract. Allow the mixture to dry under a nitrogen (N2) flow. Ensure that the flow is correctly regulated, as the resin is very powdery and can easily spread.
Prefractionation procedure
Prefractionation procedure
Packing the solid-phase extraction column:
- Place one of the frits at the bottom of the SPE column. Since new columns usually contain 2 frits at the bottom, remove one of them;
- Add the HP20SS resin. Tap it a few times to ensure the resin is more evenly arranged on the frit. The amount of resin should be calculated according to the amount of dry extract. A minimum of 1 g of resin should be packed. In case >0,2 mg of dry extract, add 5-7 times the weight of the initial dry extract;
- In this step, the resin tends to stick to the walls of the column. To avoid it, prepare a funnel with weighing paper, fold the bottom, and secure it with tape, so that it becomes a small cup. It must fit inside the SPE column. Weigh the HP20SS resin using the adapted cup and then fit it inside the column, upside down, as deep as possible (holding it with tweezers may help). Turning it over, releasing the resin directly at the bottom of the column;
- Place the second frit. The frit should always be placed horizontally inside the column, trying to push any particles from the walls of the SPE column to its bottom, ensuring that the resin layer is as homogeneous as possible. Using a glass tube and pushing it from the edges, very slowly, helps with this process.
Lable the 20-mL test tubes and add them to rack inside the vacuum manifold.
Connect the packed SPE column to the manifold and wash it with 10 mL of ethyl acetate and 10 mL of IPA.
Equilibrate the packed column with 10 mL of water for HPLC (2x).
Add the dry load to the SPE column. Suggestion: use the weighing paper cup, following the same procedure described in step 6.3.
Place a third frit on top of the dry load with the same care described in step 6.4. The vacuum manifold and packed SPE column are shown in Figure 2.
Figure 2: Vacuum manifold with a packed SPE column. Source: Authors (2024).
Use the IPA solutions to prefractionate the extract using the step-gradient shown in Table 1. The total volume of 20 mL per fraction should be distributed in 2 test tubes (2x10mL).
Table 1: Isopropanol (IPA) solutions for the fractionation method.
* HPLC-grade water 100%
Source: Authors (2023).
After the 6th fraction (F6), wash the column with 2x10 mL 100% ethyl acetate (7th fraction) followed by 2x10 mL of 100% acetone (8th fraction). An example of fractions obtained by this procedure is demonstrated in Figure 3.
Figure 3: Prefractionated cyanobacterial extract. Source: Authors (2023).
- Wash fractions usually contain a significant amount of chlorophyll and fatty acids. However, it is recommended to store them for potential future investigations, for example in cases where the extract displays biological activity, which is not detected in F1-F6.
Dry the fractions under N2 flow or using a vacuum concentrator.
Resuspending fractions and calculating yields
Resuspending fractions and calculating yields
Resuspend the dry fractions with a small volume of HPLC-grade methanol or the solvent solution used for producing the fraction. Use the ultrasonic bath.
Transfer the fractions from the tubes to labeled and pre-weighed 4 mL vials, as shown in Figure 4. Vials must be weighed without the caps.
Figure 4: Fractions in 4 mL vials. Source: Authors (2023).
- Fractions 7 and 8 (wash) may be stored in the same 4 mL vial.
Dry the fractions under N2 flow or using a vacuum concentrator.
Weigh the fraction vials (without caps) and calculate the prefractionation yield (%): sum of fraction mass divided by the mass of the initial extract.
References
References
Bugni, T.S. et al. Fractionated Marine Invertebrate Extract Libraries for Drug Discovery. Molecules 2008, 13, 1372-1383. DOI: 10.3390/molecules13061372.
Clish, C.B. Metabolomics: an emerging but powerful tool for precision medicine. Cold Spring Harb Mol Case Stud. 2015, 1(1), a000588. DOI: 10.1101/mcs.a000588.
Orjala, J. et al. Discovery of potential anticancer agents from aquatic cyanobacteria, filamentous fungi, and tropical plants. In Bioactive Compounds from Natural Sources: Natural Products as Lead Compounds in Drug Discovery, Second Edition, CRC Press 2011, p. 37-64.
Rague, A.L.; Parker, S.-A.J.; Tidgewell, K.J. Evaluating Marine Cyanobacteria as a Source for CNS Receptor Ligands. Molecules 2018, 23, 2665. DOI: 10.3390/molecules23102665.
Stobaugh, J.; Fague, K.; Jorgenson, J. Prefractionation of Intact Proteins by Reversed-Phase and Anion-Exchange Chromatography for the Differential Proteomic Analysis of Saccharomyces cerevisiae. Journal of Proteome Research 2013, 12 (2), 626-636. DOI: 10.1021/pr300701x.
Tu Y.; Yan B. High-throughput fractionation of natural products for drug discovery. Methods Mol Biol. 2012, 918:117-26. DOI: 10.1007/978-1-61779-995-2_9. PMID: 22893289.
Wagenaar, M.M. Pre-fractionated Microbial Samples – The Second Generation Natural Products Library at Wyeth. Molecules 2008, 13, 1406-1426. DOI: 10.3390/molecules13061406.
Weiss, M.B. et al. Chemical diversity of cyanobacterial natural products, Natural Product Reports. Nat. Prod. Rep., 2025, 42, 6-49. DOI: 10.1039/D4NP00040D.
Wilson, B.A.P. et al. Creating and screening natural product libraries, Natural Product Reports. Nat. Prod. Rep., 2020, 37, 893-918. DOI: 10.1039/C9NP00068B.
Protocol references
Bugni, T.S. et al. Fractionated Marine Invertebrate Extract Libraries for Drug Discovery. Molecules 2008, 13, 1372-1383. DOI: 10.3390/molecules13061372.
Clish, C.B. Metabolomics: an emerging but powerful tool for precision medicine. Cold Spring Harb Mol Case Stud. 2015, 1(1), a000588. DOI: 10.1101/mcs.a000588.
Rague, A.L.; Parker, S.-A.J.; Tidgewell, K.J. Evaluating Marine Cyanobacteria as a Source for CNS Receptor Ligands. Molecules 2018, 23, 2665. DOI: 10.3390/molecules23102665.
Stobaugh, J.; Fague, K.; Jorgenson, J. Prefractionation of Intact Proteins by Reversed-Phase and Anion-Exchange Chromatography for the Differential Proteomic Analysis of Saccharomyces cerevisiae. Journal of Proteome Research 2013, 12 (2), 626-636. DOI: 10.1021/pr300701x.
Tu Y.; Yan B. High-throughput fractionation of natural products for drug discovery. Methods Mol Biol. 2012, 918:117-26. DOI: 10.1007/978-1-61779-995-2_9. PMID: 22893289.
Wagenaar, M.M. Pre-fractionated Microbial Samples – The Second Generation Natural Products Library at Wyeth. Molecules 2008, 13, 1406-1426. DOI: 10.3390/molecules13061406.
Weiss, M.B. et al. Chemical diversity of cyanobacterial natural products, Natural Product Reports. Nat. Prod. Rep., 2025, 42, 6-49. DOI: 10.1039/D4NP00040D.
Wilson, B.A.P. et al. Creating and screening natural product libraries, Natural Product Reports. Nat. Prod. Rep., 2020, 37, 893-918. DOI: 10.1039/C9NP00068B.
Acknowledgements
We thank Prof. Jimmy Orjala and his laboratory members at the University of Illinois at Chicago for developing earlier versions of this protocol.