Method for extracting and detecting polyamine substances in nori species biomass
Through acidic extraction solution and LC-ELSD method, the foliar and filamentous cells of the species Alanaceae species were broken and detected, which solved the problem of insufficient research on polyamines in large seaweeds, and achieved accurate and quantitative analysis of polyamine components.
Patent Information
- Application Number
- CN202510681454.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-08-22
AI Technical Summary
At present, there are few studies on polyamines of large seaweed species, and it is difficult for the existing technology to effectively extract and qualitatively quantify its polyamine components.
The foliar and filamentous cells of the species Nari are crushed with acid extraction solution, and the qualitative and quantitative detection of polyamines are performed using liquid chromatography combined with evaporative light scattering detector (LC-ELSD). The specific steps include preparing disodium hydrogen phosphate-citric acid solution, cell breakage, centrifugation of the supernatant extracted and LC-ELSD detection.
Effective extraction and quantitative analysis of polyamine components of the genus Nari species are achieved, and an accurate detection method for polyamine species and content is provided.
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Figure CN120522331A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of marine biotechnology, and specifically is a method for qualitatively and quantitatively detecting polyamines in Porphyra species by utilizing biomass of haploid thallus and / or diploid filaments of Porphyra species, disrupting the cells, and using an extraction solution to extract polyamine components in the thallus and filaments respectively, and using liquid chromatography (LC) combined with an evaporative light scattering detector (ELSD). Background Art
[0002] Polyamines are a class of small, positively charged aliphatic compounds found in most organisms. In addition to being free within cells, they often bind to negatively charged RNA, DNA, and proteins, and are essential for cell growth, proliferation, and tissue regeneration.
[0003] Polyamines in bacteria and animals include putrescine, spermidine, spermine and cadaverine. In addition to putrescine, spermidine and spermine, the polyamine components of plant cells also include non-spermines and thermospermine (Hosoya et al. 2005, Sagor et al. 2013. Hosoya, R.; Hamana, K.; Isobe, M.; Yokota, A. Polyamine Distribution Profiles within Cyanobacteria. Microbiol. Cult. Collect. 2005, 21, 3–8. Sagor, GH; Liu, T.; Takahashi, H.; Niitsu, M.; Berberich, T.; Kusano, T. Longer uncommon polyamines have a stronger defense gene-induction activity and a higher suppressing activity of Cucumber mosaicvirus multiplication compared to that of spermine in Arabidopsis thaliana. Plant Cell Rep. 2013, 32, 1477–1488.). Polyamines are involved in a variety of biological processes, including gene expression, translation, cell proliferation, regulation of cell signaling, and membrane stability. They also regulate the activity of certain ion channels (Pegg 2009, Igarashi and Kashiwagi 2006. Handa et al. 2018. Pegg AE. Mammalian polyamine metabolism and function. IUBMB Life. 2009; 61: 880–894. Igarashi K, Kashiwagi K. Polyamine modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines. J Biochem (Tokyo). 2006; 139: 11–16. Handa, AK; Fatima, T.; Mattoo, AK. Polyamines: Bio-Molecules with Diverse Functions in Plant and Human Health and Disease. Front. Chem. 2018, 6, 1–18.).Since polyamines have multiple functions in cells, maintaining their homeostasis is crucial. Intracellular polyamine balance can be ensured by regulating biosynthesis, catabolism, and transport.
[0004] Compared to bacteria, mammals, higher plants, and microalgae, relatively little research has been conducted on the types and contents of polyamines in macroalgae, particularly those found in intertidal zones. Currently studied macroalgae include species of the genus Ulva (Chlorophyta), the genus Dictyota dichotoma (Bryophyta), and the red algae Gracilaria cornea, Gelidium canariensis, and Grateloupia doryphora. Thin-layer chromatography (TLC) or high-pressure liquid chromatography (HPLC) has been used to identify the types and contents of polyamines in these macroalgae (Marián et al. 2000a, 2000b, Schweikert et al. 2011).
[0005] Porphyra is an important large seaweed. Its multicellular life cycle consists primarily of haploid thalluses (thallus) and diploid filaments (filaments). Wild thalluses are found in intertidal environments, and large-scale marine aquaculture of Porphyra thalluses has been established. Wild filaments are primarily found in calcium carbonate materials such as shells and can now be artificially preserved in a free-standing filamentous state. However, no studies have yet been published on polyamines in Porphyra species, an economic seaweed.
[0006] The invention relates to a method of firstly crushing thallus and filamentous cells of Porphyra species, extracting intracellular polyamines with an acidic solution, and qualitatively and quantitatively analyzing intracellular polyamine components of Porphyra species using LC-ELSD. Summary of the Invention
[0007] The purpose of the present invention is to provide a method for extracting and detecting polyamine substances in the biomass of Porphyra species. The method uses an acidic extraction solution to extract polyamine components from the thallus and filamentous cells of the polyamine-rich large economic seaweed Porphyra species, and uses LC-ELSD to separate and identify the types and contents of polyamines.
[0008] To achieve the above object, the present invention adopts the following technical solutions:
[0009] A method for extracting and detecting polyamines in Porphyra species biomass, firstly, adding an acidic extract to a Porphyra species biomass sample, crushing the biomass sample, then extracting the supernatant, and performing qualitative and quantitative detection of the supernatant using liquid chromatography coupled with an evaporative light scattering detector (LC-ELSD). The specific process is as follows:
[0010] 1) Preparation of acidic extraction solution: First, prepare 0.5-1M and 1-2M mother liquors of citric acid monohydrate and disodium hydrogen phosphate, respectively. Then, mix the mother liquors in a volume ratio of 3-1:1 of citric acid monohydrate to disodium hydrogen phosphate, and dilute them 8-10 times with water to obtain a disodium hydrogen phosphate-citric acidic extraction solution.
[0011] 2) Biomass material disruption: Wipe dry the surface moisture of a Porphyra species biomass sample, weigh 0.04-0.1 g, add 500-1000 μl of the above-mentioned acidic extraction solution, and disrupt in a cell disruptor at a frequency of 70 Hz for 50 seconds, then rest for 10 seconds, for a total of 3-5 times.
[0012] 3) Extracting the supernatant: Centrifuge the cell disruptor material at 1000-2000g, 0-4°C for 10-30 min to extract the supernatant. Add 200-500 μl of the acidic extract and an equal volume of 10-12 M hydrochloric acid to the precipitate. Mix well and incubate in an 80°C water bath for 10-12 hours. Centrifuge at 1000-2000g, 0-4°C for 10-30 min to obtain the supernatant. Combine the two supernatants.
[0013] 4) LC-ELSD detection: 20-40 μl of the extract was loaded for detection to obtain a chromatogram showing the peak time and signal intensity of the polyamine components in the extract;
[0014] 5) LC-ELSD detection of polyamine standard samples to obtain chromatograms of the peak elution time and signal intensity of the standard samples, taking different amounts of the polyamine standard samples for LC-ELSD detection, and establishing a standard curve of polyamine content and peak area. The LC-ELSD detection conditions are the same as those in step 4);
[0015] 6) Determine the polyamine components in the biomass sample based on the peak time of the polyamine standard, and calculate the content of the polyamine components in the biomass sample based on the standard curve.
[0016] Wherein, the pH value of the sodium hydrogen phosphate-citric acid extraction solution in step 1) is 3-6.
[0017] In step 4), the LC-ELSD detection conditions were as follows: mobile phase A: acetonitrile: water = 20:80 (v / v), with 0.05% trifluoroacetic acid (v / v) in phase A; mobile phase B: acetonitrile: water = 20:80 (v / v), with 0.35% trifluoroacetic acid (v / v) in phase B. The elution procedure was as follows: starting from A:B = 100:0 (v / v), after 10 minutes, A:B = 70:30 (v / v), after 8 minutes, A:B = 40:60 (v / v), followed by isocratic elution for 2 minutes, and then for 5 minutes to bring A:B to 0:100 (v / v). Finally, 10 minutes were required to restore the starting conditions (A:B = 100:0, v / v), and the flow rate was kept constant at 0.5-1 mL min. -1 , the total analysis time was 35 min, the time interval between two consecutive injections was 10-15 min, the flow rate of nebulizer gas nitrogen was maintained at 3.3 bar, and the drift tube temperature was 40-60 °C.
[0018] Furthermore, the Porphyra species include one or more of Pyropia yezoensis, Pyropia haitanensis, Porphyra umbilicalis, Porphyra suborbiculata, and Pyropia kinositae.
[0019] The biomass material of Porphyra species is the thallus and filaments of Porphyra species, in particular the filaments thereof, and the thallus includes one or both of wild type and / or artificially cultivated type.
[0020] The polyamine substances include all types of polyamines, specifically one or more of putrescine, spermidine, spermine or thermospermine, cadaverine and non-spermine.
[0021] The acid extraction solution is a disodium hydrogen phosphate-citric acid solution with a pH of 3-6.
[0022] The polyamine components were detected by liquid chromatography coupled with an evaporative light scattering detector (LC-ELSD).
[0023] Advantages of the present invention
[0024] 1. Use a cell disruptor to break up the seaweed thallus and filaments rich in polysaccharides and colloids.
[0025] 2. Extract polyamines using disodium hydrogen phosphate-citric acid solution;
[0026] 3. Use LC-ELSD method to qualitatively and quantify the polyamine components and contents in the thallus and filaments of Porphyra species. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The peak times of putrescine, spermidine, spermine and thermospermine were detected by LC-ELSD. A is putrescine, B is spermidine, C is spermine and D is thermospermine.
[0028] Figure 2 The standard curves of putrescine, spermidine, spermine and thermospermine established by LC-ELSD method, where A is the standard curve of putrescine, B is the standard curve of spermidine, C is the standard curve of spermine, and D is the standard curve of thermospermine.
[0029] Figure 3 The polyamine component spectrum obtained by LC-ELSD detection of Porphyra yezoensis conchoceles. A is the peak time of putrescine, B is the peak time of spermidine, and C is the peak time of thermospermine.
[0030] Figure 4 The polyamine component spectrum obtained by LC-ELSD detection of Porphyra yezoensis thallus. A is the peak time of putrescine, B is the peak time of spermidine, and C is the peak time of thermospermine.
[0031] Figure 5 This is the polyamine component spectrum obtained by LC-ELSD detection of Porphyra haitanensis conchoceles. A shows no putrescine detected, B shows the peak time of spermidine, and C shows the peak time of thermospermine.
[0032] Figure 6 This is the polyamine component spectrum obtained by LC-ELSD detection of Porphyra haitanensis thallus. A: No putrescine was detected, B: The peak time of spermidine, and C: The peak time of thermospermine.
[0033] Figure 7 The component spectrum of polyamine extract from Porphyra conchocelis was detected by HPLC fluorescence.
[0034] Figure 8 Spectra obtained for the LC-ELSD analysis of Porphyra yezoensis conchoceles. DETAILED DESCRIPTION
[0035] Example 1:
[0036] The polyamine extraction method used in the embodiment refers to the literature (Schweikert, K., Sutherland, JES, Hurd, CL et al. UV-B radiation induces changes in polyamine metabolism in thered seaweed Porphyra cinnamomea. Plant Growth Regul 65, 389–399 (2011). https: / / doi.org / 10.1007 / s10725-011-9614-x. Liu Y, Zhao WH, Dong B, Li B, and Yang XH. Changes in intracellular and extracellular free polyamines during the growth cycle of Prorocentrum donghaiense. Earth and Environmental Science 344 (2019) 012060 doi: 10.1088 / 1755-1315 / 344 / 1 / 012060), except that the acid solution is replaced by trichloroacetic acid, perchloric acid, etc. with disodium hydrogen phosphate-citric acid solution. The materials used are the thallus and filaments of Porphyra yezoensis.
[0037] Specific operations:
[0038] 1) Preparation of extraction solution: Prepare 2 mol / L sodium hydrogen phosphate aqueous solution and 1 mol / L citric acid aqueous solution, then mix 10 mL of the sodium hydrogen phosphate aqueous solution and 10 mL of the citric acid aqueous solution, and dilute tenfold with water to obtain a sodium hydrogen phosphate-citric acid extraction solution with a pH of 5.0.
[0039] 2) Material Disintegration: Wipe dry the surface moisture of Porphyra yezoensis thallus and filaments, weigh 0.05 g of each, add 500 μl of the above-mentioned sodium hydrogen phosphate-citric acid extract, and add two steel balls with a particle size of 2 mm to 5 mm. Disintegrate in a cell disruptor at a frequency of 70 Hz for 50 seconds and a rest period of 10 seconds, for a total of four times.
[0040] 3) Extract the supernatant: Centrifuge the cell disruptor material at 1000g, 4°C for 10 minutes, and separate the supernatant from the precipitate. Store the supernatant at -20°C. Add 400μl of the above-mentioned sodium dihydrogen phosphate-citric acid extract to the precipitate, followed by an equal volume of 12M hydrochloric acid. Mix thoroughly, incubate in an 80°C water bath for 12 hours, and centrifuge at 1000g, 4°C for 10 minutes. Collect the supernatant. Combine the two supernatants to obtain the extract samples of the Porphyra yezoensis thallus and conchocelis to be tested.
[0041] 4) LC-ELSD Detection: The HPLC instrument used was a DGU-20AR (Shimadzu Instruments Co., Ltd.), the detector was an ELSD-16 (Shimadzu Instruments Co., Ltd.), and the column was an XDB-C18 (4.6 x 250 mm, 5 µm). Detection conditions were as follows: Mobile phase A was acetonitrile:water = 20:80 (v / v), with 0.05% trifluoroacetic acid (v / v); Mobile phase B was acetonitrile:water = 20:80 (v / v), with 0.35% trifluoroacetic acid (v / v). Elution was performed as follows: Starting with an A:B ratio of 100:0 (v / v), after 10 minutes, the ratio reached 70:30 (v / v). After 8 minutes, the ratio was 40:60 (v / v). Isocratic elution was then performed for 2 minutes, followed by 5 minutes to reduce the A:B ratio to 0:100 (v / v). Finally, 10 min were required to restore the starting conditions (A:B 100:0, v / v). The flow rate was kept constant at 0.7 mL min -1 The total analysis time was 35 minutes and the flow rate was kept constant at 0.7 mL min -1 The total analysis time was 35 min, with a 10-min interval between two consecutive injections. The flow rate of nitrogen carrier gas in the nebulizer was maintained at 3.3 bar, and the drift tube temperature was 40 °C.
[0042] 5) Detection of polyamines putrescine, spermidine, spermine and thermospermine standards and establishment of standard curves,
[0043] Preparation of standard solution: Putrescine, spermidine and spermine are all water-soluble. The final concentration of putrescine in ultrapure water is 1.289 mg / ml, the final concentration of spermidine in ultrapure water is 2.037 mg / ml, and the final concentration of spermine in ultrapure water is 1.619 mg / ml. Hot spermine is prepared with dimethyl sulfoxide (DMSO) to a solution with a concentration of 1.619 mg / ml.
[0044] Use the detection conditions in step 4 to detect the peak time and signal intensity of the standard putrescine, spermidine, spermine and thermospermine (such as Figure 1 The loading volume of the standard solution was 40 μl.
[0045] Standard curve establishment process: Taking putrescine as an example, 5 μl, 10 μl, 15 μl, 20 μl, and 40 μl of putrescine standard solution (1.289 mg / ml) were sampled and tested by LC-ELSD. According to the detection conditions in step 4, chromatograms of the peak time and signal intensity of putrescine standards with different contents were obtained. The signal intensity was converted into the corresponding peak area using conventional methods. With different putrescine contents (mg) as the abscissa and the corresponding peak area as the ordinate, a trend line of the corresponding relationship between putrescine content (mg) and peak area was established, which was the standard curve of putrescine (such as Figure 2 The standard curves of the other three polyamine standards were established in the same way as putrescine to obtain the standard curves of each standard (as shown in Figure 2 shown).
[0046] 6) The above-mentioned Porphyra yezoensis thallus and filamentous body extract samples were tested using the test conditions in step 4, with a sample volume of 40 μl. The test results are as follows: Figure 3 、 Figure 8 and Figure 4 As shown, Figure 3 The peak time and signal intensity spectra of polyamine components obtained for the detection of Porphyra yezoensis conchocelis were obtained. Figure 4 To detect the thallus of Porphyra yezoensis, we obtained the peak time and signal intensity spectra of polyamine components. Figure 8 Spectra obtained for the detection of Porphyra yezoensis conchoceles.
[0047] 7) The peak time of polyamine components in the thallus and filaments of Porphyra yezoensis obtained by the detection in step 6) Figure 3 and Figure 4 Peak time spectrum of the polyamine component standard obtained by detection in step 5) Figure 1 The results of the control showed that putrescine, spermidine and thermospermine were detected in the thallus and filaments of Porphyra yezoensis, but no spermine was detected. The standard curves of putrescine, spermidine, spermine and thermospermine established by LC-ELSD method (such as Figure 2 The contents of polyamine components in the thallus and filaments of Porphyra were calculated.
[0048] The contents of putrescine, spermidine and thermospermine detected in the thallus and filaments of Porphyra yezoensis are shown in Table 1, among which thermospermine is the main component.
[0049] Table 1 Contents of putrescine, spermidine and thermospermine detected in the thallus and filaments of Porphyra yezoensis
[0050]
[0051] Example 2:
[0052] The process and conditions are the same as those in Example 1, except that:
[0053] 1) The materials used are the thallus and filaments of Porphyra haitanensis, and the remaining steps are the same.
[0054] 2) The peak time and peak diagram of each polyamine component of Porphyra haitanensis conchocelis were detected as follows Figure 5 As shown; the peak time and peak diagram of each polyamine component detected in the thallus of Porphyra haitanensis are as follows Figure 6 As shown, Figure 5 and Figure 6 Peak time spectrum of putrescine, spermidine, spermine and thermospermine Figure 1 The results of the control showed that spermidine and thermospermine were detected in the thallus and filaments of Porphyra haitanensis, while spermine and putrescine were not detected.
[0055] 3) According to the standard curves of putrescine, spermidine, spermine and thermospermine established by LC-ELSD method, the contents of thermospermine and spermidine in the thallus and conchoceles of Porphyra haitanensis were calculated as shown in Table 2.
[0056] Table 2 Contents of putrescine, spermidine and thermospermine detected in the thallus and filaments of Porphyra haitanensis
[0057]
[0058] Comparative Example 1: HPLC fluorescence detection of polyamine components and contents in conchoceles of Porphyra yezoensis
[0059] Since polyamines do not contain fluorescent groups, when using HPLC fluorescence detection, the laver extract must be derivatized before HPLC detection. The method is as follows:
[0060] 1) The method for extracting polyamines from Porphyra yezoensis conical biomass is the same as that in Example 1 above.
[0061] 2) Dansylation of the extract. First, dissolve dansyl chloride in acetone to prepare a 5 mg / ml dansyl chloride-acetone solution. Mix the sodium hydrogen phosphate-lemon extract of Porphyra yezoensis conicals with an equal volume of saturated sodium carbonate. This mixture is then mixed with the dansyl chloride-acetone solution (5 mg / ml) at a volume ratio of 1:2. Derivatize at 40°C for 30 minutes.
[0062] 3) HPLC Detection: The HPLC instrument used was a DGU-20AR (Shimadzu Instrument Co., Ltd.), and the chromatographic column was an XDB-C18 (4.6 x 250 mm, 5 m). 40 μl of the dansylated Porphyra yezoensis conical extract was loaded for detection. Detection procedure: Mobile phase A: 0.1% formic acid in water; mobile phase C: acetonitrile. The elution procedure was as follows: Starting with an A:C ratio of 95:5 (v / v), after 2 minutes, the ratio reached A:C 10:90 (v / v). After 18 minutes, the ratio was adjusted to 0:100 (v / v). After 19 minutes, the ratio was adjusted to 95:5 (v / v) until the 30th minute, followed by isocratic elution for 2 minutes and 11 minutes. The flow rate was maintained at 1 mL / min. -1 The instruments and chromatographic columns used were the same as those in Examples 1 and 2. The test results were as follows: Figure 7 shown.
[0063] 4) Since Porphyra biomass is rich in protein (the protein content of Porphyra yezoensis thallus accounts for 40-50% of the dry weight, and the protein content of the conchocelis accounts for 10-25% of the dry weight). Proteins and amino acids can also be acylated by dansylation. Therefore, compared with the spectrum of Porphyra yezoensis conchocelis detected by LC-ELSD method ( Figure 8 The HPLC fluorescence detection method has a messy background, which makes it difficult to distinguish the polyamine component from other dansyl-substituted substances.
Claims
1. A method for extracting and detecting polyamines in Porphyra species biomass, characterized by: An acidic extract was added to a biomass sample of a Porphyra species, the biomass sample was disrupted, and the supernatant was extracted. The supernatant was then qualitatively and quantitatively detected using liquid chromatography coupled with an evaporative light scattering detector (LC-ELSD).
2. The method for extracting and detecting polyamines in Porphyra species biomass according to claim 1, characterized in that: The specific process is: 1) Preparation of acidic extraction solution: First, prepare 0.5-1.5M and 1-2M mother liquors of citric acid monohydrate and disodium hydrogen phosphate, respectively. Then, mix the mother liquor of citric acid monohydrate and disodium hydrogen phosphate in a volume ratio of 3-1:1 and dilute 8-10 times with water to obtain a disodium hydrogen phosphate-citric acidic extraction solution. 2) Biomass material disruption: Wipe dry the surface moisture of a Porphyra species biomass sample, weigh 0.04-0.1 g, add 500-1000 μl of the above-mentioned acidic extraction solution, and disrupt in a cell disruptor at a frequency of 70 Hz for 50 seconds, then rest for 10 seconds, for a total of 3-5 times. 3) Extracting the supernatant: Centrifuge the cell disruptor material at 1000-2000g, 0-4°C for 10-30 min to extract the supernatant. Add 200-500 μl of the acidic extract and an equal volume of 10-12 M hydrochloric acid to the precipitate. Mix well and incubate in an 80°C water bath for 10-12 hours. Centrifuge at 1000-2000g, 0-4°C for 10-30 min to obtain the supernatant. Combine the two supernatants. 4) LC-ELSD detection: 20-40 μl of the extract was loaded for detection to obtain a chromatogram showing the peak time and signal intensity of the polyamine components in the extract; 5) LC-ELSD detection of polyamine standard samples to obtain a chromatogram of the peak time and signal intensity of the standard samples, taking different amounts of polyamine standard samples for LC-ELSD detection, and establishing a standard curve of polyamine content and peak area; 6) Determine the polyamine components in the biomass sample based on the peak time of the polyamine standard, and calculate the content of the polyamine components in the biomass sample based on the standard curve.
3. The method according to claim 2, characterized in that: The pH value of the sodium hydrogen phosphate-citric acid extraction solution in step 1) is 3-6.
4. The method according to claim 2, characterized in that: The LC-ELSD detection conditions in steps 4) and 5) are as follows: mobile phase A is acetonitrile: water = 20:80 (v / v), and phase A contains 0.05% trifluoroacetic acid (v / v); mobile phase B is acetonitrile: water = 20:80 (v / v), and phase B contains 0.35% trifluoroacetic acid (v / v). The elution program is as follows: starting from A:B = 100:0 (v / v), reaching A:B = 70:30 (v / v) after 10 minutes, and then A:B = 40:60 (v / v) after 8 minutes, then isocratic elution for 2 minutes, and then for 5 minutes to make A:B reach 0:100 (v / v). Finally, it takes 10 minutes to restore the starting conditions (A:B = 100:0, v / v), and the flow rate is kept constant at 0.5-1 mL·min -1 , the total analysis time was 35 min, the time interval between two consecutive injections was 10-15 min, the flow rate of nebulizer gas nitrogen was maintained at 3.3 bar, and the drift tube temperature was 40-60 °C.
5. The method according to claim 1 or 2, characterized in that: The Porphyra species include one or more of Pyropia yezoensis, Pyropia haitanensis, Porphyra umbilicalis, Porphyra suborbiculata, and Pyropia kinositae.
6. The method according to claim 1 or 2, characterized in that: The biomass sample of the Porphyra species is the filaments and / or thallus of the Porphyra species, in particular the filaments thereof, and the thallus includes one or both of wild type and / or artificially cultivated type.
7. The method according to claim 1 or 2, characterized in that: The polyamine substances include all types of polyamines, specifically one or more of putrescine, spermidine, spermine or thermospermine, cadaverine and non-spermine.