A extraction method of heparan sulfate from porcine internal mucosa

Abstract

The present invention relates to a method for extracting heparan sulfate, comprising the steps of: (S1) adding purified water and sodium metabisulfite to porcine intestinal mucosal tissue and adjusting the pH to obtain a mucosal tissue slurry; (S2) adding a proteolytic enzyme to the mucosal tissue slurry and enzymatically hydrolyzing it at 50–55°C for 12–18 hours to obtain an enzymatic hydrolysate; (S3) reacting the enzymatic hydrolysate at a temperature of 75–95°C for 5–15 minutes to inactivate the proteolytic enzyme, then adjusting the temperature to 60–70°C to adjust the pH to 3–4, allowing it to stand, and then filtering to obtain a filtrate; (S4) contacting the filtrate with an anion exchange resin to adsorb heparan sulfate, separating the solution, washing with purified water to remove impurities adsorbed on the anion exchange resin, and then eluting the heparan sulfate with NaCl having a salinity of 0.6–1.0 M to obtain an eluent. and (S5) a 40-55% ethanol mixture prepared by mixing ethanol with the above eluent is left at room temperature to precipitate heparan sulfate, and then the precipitate is recovered and dried to obtain heparan sulfate powder.

Description

Technology Field

[0001] The present invention relates to a method for extracting heparan sulfate from porcine intestinal mucosa. Background Technology

[0002] Heparan sulfate (HS) is a glycosaminoglycan (GAG) polymer that plays diverse and important roles in animal cell function and has the function of ensuring cell survival through the regulation of division, differentiation, migration, and adhesion (Fedarko, Ishihara, & Conrad, 1989; Lin, 2004; Moon et al., 2005; Nurcombe, Ford, Wildschut, & Bartlett, 1993). The biological activity of heparan sulfate stems from interactions with cell surface receptors, growth factors, matrix proteins, and cytokines. Heparan sulfate plays a critical role in regulating developmental biology, cell differentiation, angiogenesis, bone formation and wound repair, and related processes including cancer (Jochmann, Bachvarova, & Vortkamp, ​​2014; Knelson, Nee, & Blobe, 2014; Olczyk, Mencner, & Komosinska-Vasev, 2015; van Wuffelt, 2014). Heparan sulfate is a regulator of immune responses and inflammation, and dysfunction of heparan sulfate-growth factor signaling has been identified as a contributing factor to Alzheimer's disease, epilepsy, atherosclerosis, and diabetes (Farrugia, Lord, Melose, & Whiteock, 2018; Simon Davis & Parish, 2013). Accordingly, research aimed at controlling the biological processes related to this has focused on heparan sulfate in the context of stem cells (Ravikumar, Smith, Nurcombe, & Cool, 2020) and tissue repair (Houlton, Abumaria, Hinkley, & Clarkson, 2019).

[0003] Commercially available heparan sulfate is highly valuable for various research activities, but heparan sulfate isolated from bovine kidneys has the limitation of being expensive. Therefore, porcine intestinal mucosa is a major source of heparan sulfate used in medicine, and heparan sulfate can be extracted from a mixture of glycosaminoglycans (GAGs) remaining after heparin extraction. Meanwhile, due to the surge in demand for heparan sulfate resulting from various research activities, there is a need for a method to manufacture heparan sulfate commercially in large quantities with low cost and high yield. The problem to be solved

[0005] Until now, heparan sulfate has been obtained from impurities generated during the purification process of crude heparin sodium, but the present invention aims to provide a method for directly producing heparan sulfate from pig small intestine mucosa and mucous threads. means of solving the problem

[0007] The present invention relates to a method for extracting heparan sulfate, comprising the steps of: (S1) adding purified water and sodium metabisulfite to porcine intestinal mucosal tissue and adjusting the pH to obtain a mucosal tissue slurry; (S2) adding a proteolytic enzyme to the mucosal tissue slurry and enzymatically hydrolyzing it at 50–55°C for 12–18 hours to obtain an enzymatic hydrolysate; (S3) reacting the enzymatic hydrolysate at a temperature of 75–95°C for 5–15 minutes to inactivate the proteolytic enzyme, then adjusting the temperature to 60–70°C to adjust the pH to 3–4, allowing it to stand, and then filtering to obtain a filtrate; (S4) contacting the filtrate with an anion exchange resin to adsorb heparan sulfate, separating the solution, washing with purified water to remove impurities adsorbed on the anion exchange resin, and then eluting the heparan sulfate with NaCl having a salinity of 0.6–1.0 M to obtain an eluent. and (S5) a 40-55% ethanol mixture prepared by mixing ethanol with the above eluent is left at room temperature to precipitate heparan sulfate, and then the precipitate is recovered and dried to obtain heparan sulfate powder.

[0008] In one embodiment of the present invention, in step S1, the pH may be adjusted to 5 to 6 and the mucosal tissue slurry may be left at room temperature for 4 to 8 weeks.

[0009] In one embodiment of the present invention, in step S1, sodium metabisulfite may be included in an amount of 2 to 4 weight percent in the mucosal tissue slurry.

[0010] In one embodiment of the present invention, in step S2, one or more proteolytic enzymes selected from alcalase, flavorzyme, neutrase, protamex, bromelain, and papain may be used.

[0011] In one embodiment of the present invention, in step S2, a proteolytic enzyme may be added to the mucosal tissue slurry at 4 to 5% of the total weight of the porcine intestinal mucosal tissue to enzymatically hydrolyze it. Effects of the invention

[0013] Conventional technology separates heparan sulfate from impurities generated during the purification process of crude heparin sodium, but the present invention is a technology that directly extracts heparan sulfate from the intestinal mucosa containing mucus, thereby extracting heparan sulfate more easily and supplying heparan sulfate in a more diverse form for use as a domestic pharmaceutical material. Brief explanation of the drawing

[0015] Figure 1 illustrates a process flow diagram for manufacturing heparan sulfate according to the present invention. Figure 2 illustrates the HPLC analysis results of one embodiment of the present invention. Figure 3 illustrates the results of the anticoagulant activity analysis of one embodiment of the present invention. Figure 4 illustrates the H-NMR analysis results of one embodiment of the present invention. FIG. 5 shows the 1H NMR analysis spectrum results of one embodiment of the present invention. Specific details for implementing the invention

[0016] The present invention relates to a method for directly extracting heparan sulfate from porcine intestinal mucosa, comprising: (S1) a step of obtaining a mucosal tissue slurry by adding purified water and sodium metabisulfite to porcine intestinal mucosal tissue and adjusting the pH; (S2) a step of obtaining an enzymatic hydrolysate by adding a proteolytic enzyme to the mucosal tissue slurry and enzymatically hydrolyzing it at 50–55°C for 12–18 hours; (S3) a step of inactivating the proteolytic enzyme by reacting the enzymatic hydrolysate at a temperature of 75–95°C for 5–15 minutes, then adjusting the temperature to 60–70°C to adjust the pH to 3–4, allowing it to stand, and then filtering to obtain a filtrate; (S4) a step of contacting the above liquid with an anion exchange resin to adsorb heparan sulfate, then separating and washing with purified water to remove impurities adsorbed on the anion exchange resin, and then eluting the heparan sulfate with NaCl having a salinity of 0.6 to 1.0 M to obtain an eluent; and (S5) a step of mixing ethanol with the above eluent to prepare a 40 to 55% ethanol mixture, leaving it at room temperature to precipitate heparan sulfate, then recovering the precipitate and drying it to obtain heparan sulfate powder.

[0018] The process for each step is explained in detail below.

[0020] S1: Preparation of porcine intestinal mucosal tissue slurry

[0021] This step involves adding purified water and sodium metabisulfite to porcine intestinal mucosal tissue and adjusting the pH of the mixture to obtain a mucosal tissue slurry. At this time, the sodium metabisulfite in the mucosal tissue slurry may be included in an amount of 1 to 5 weight%, and preferably 2 to 4 weight%.

[0022] The above mucosal tissue slurry can be adjusted to have a pH range of 5 to 6 and then left at room temperature for 4 to 8 weeks.

[0024] S2 and S3: Enzymatic protein degradation and filtration

[0025] In this step, a proteolytic enzyme is added to the mucosal tissue slurry and enzymatically hydrolyzed at 50 to 55°C for 12 to 18 hours to obtain an enzymatic hydrolysate. At this time, one or more enzymes selected from alcalase, flavorzyme, neutrase, protamex, bromelain, and papain may be used as the proteolytic enzyme, and preferably, alcalase may be used. When adding a proteolytic enzyme to the mucosal tissue slurry in step S2, the proteolytic enzyme is added in an amount of 4 to 5% of the total weight of the porcine intestinal mucosal tissue and enzymatically hydrolyzed for 12 to 18 hours to obtain an enzymatic hydrolysate.

[0026] Afterwards, the above enzyme hydrolysate is reacted at a temperature of 75 to 90°C for 5 to 15 minutes to inactivate the proteolytic enzyme, then the temperature is adjusted to 60 to 70°C to adjust the pH to 3 to 4, and after standing, the solution is filtered through a 60 mesh sieve to obtain the filtrate.

[0028] S4: Resin adsorption and elution step

[0029] The step of obtaining an eluent involves adjusting the pH of the filtrate obtained in step S3, adding an ion exchange resin, stirring and adsorbing the solution, filtering it through a sieve, washing the collected resin with purified water, and then eluting it to obtain an eluent. In this step, the ion exchange resin adsorbed with heparan sulfate is immersed in brine to obtain the eluent. Preferably, the temperature of the filtrate obtained in step S3 is lowered to 60~70℃ and the pH is adjusted to 8~9. After that, an FPA98 Cl-type ion exchange resin is added to the filtrate and stirred and adsorbed for 4 hours, then passed through a 100-mesh sieve. The recovered resin is then washed with purified water to remove impurities adsorbed on the anion exchange resin, and the heparan sulfate is eluted with NaCl with a salinity of 0.6~1.0M to obtain the eluent.

[0031] S5: Preparation of Heparan Sulfate Powder by Drying

[0032] As a step to obtain heparan sulfate by drying the eluent obtained in step S4, ethanol is uniformly stirred into the eluent obtained in step S4, and then the final obtained ethanol mixture with a concentration of 40-50% is allowed to settle for at least 8 hours, after which the precipitate is collected and vacuum dried to obtain the final product, which is powder.

[0034] The above final product contains glycosaminoglycan (GAG) polymers including heparan sulfate.

[0036] The contents of the present invention will be explained in detail below through examples and test examples. However, these are intended to explain the present invention in more detail and do not limit the scope of the rights of the present invention.

[0037] Example 1:

[0038] Porcine intestinal mucosal tissue was immersed in a solution mixed with purified water and sodium metabisulfite, and the pH of the solution was adjusted to 5 to 6 to obtain a mucosal tissue slurry. At this time, the final concentration of sodium metabisulfite in the mucosal tissue slurry was set to 2% by weight, and the mucosal tissue slurry was left for 8 weeks.

[0039] Alcalase was added to the mucosal tissue slurry as a proteolytic enzyme in a weight ratio of 2:1 relative to the slurry, and the mucosal tissue slurry was heated to 50–55°C and enzymatically hydrolyzed for 12–18 hours. After enzymatic hydrolysis, the added proteolytic enzyme and undigested mucosal proteins were denatured and inactivated by heat treatment at 75–95°C for 5–15 minutes. Then, the temperature was adjusted to 60°C to adjust the pH to 3–4, and after standing, the solution was filtered to remove insoluble substances and the filtrate was obtained.

[0040] The temperature of the above filtrate was lowered to 62℃, and the pH was adjusted to 8.5. FPA98 Cl-type ion exchange resin was added and stirred and adsorbed for 4 hours, after which it was separated and washed with purified water to remove impurities adsorbed on the anion exchange resin. The eluent was obtained by eluting the heparan slate with NaCl with a salinity of 0.6–1.0 M.

[0041] After that, ethanol was mixed with the above eluent, and the resulting 50% concentration ethanol mixture was left at room temperature for 24 hours to precipitate, after which the precipitate was recovered and dried to obtain an analysis sample.

[0043] Experimental Example

[0044] The analysis sample obtained in Example 1 above was analyzed by the following method, and the results were described.

[0046] (1) HPLC analysis

[0047] The heparan content and heparinoid substances contained in the sample of Example 1 were analyzed by the HPLC method. The HPLC analysis was performed by modifying and supplementing the USP to meet our company's standards.

[0049] Detection system: SHIMADZU product, UV detector

[0050] Column and Guard Column: IonPac AS11-HC (2mm×250mm), IonPac AS11-HC (2mm×5mm)

[0051] Column temperature: 40℃

[0052] Mobile phase: Mobile phases A and B were both prepared with 2.6 mM sodium phosphate (pH 3).

[0053] Prepared by adding 1M sodium perchlorate to solvent B.

[0054] Separation flow rate: 0.22 ml / min

[0056] As controls, 2 mg and 20 mg of heparan sulfate (Celsus) standard were used, respectively, and 2 mg of the analytical sample of Example 1 was used. As can be seen from Fig. 2, Example 1 shows a peak similar to the control group on the chromatograph (see Fig. 2).

[0058] (2) Anticoagulant activity analysis

[0059] Anticoagulant activity was measured using the HYPHEN™ ANTI-IIa Assay Kit from HYPHEN BioMed. Crude heparin was diluted to a concentration of 1 mg / ml in distilled water, thoroughly dissolved using a vortex, and then filtered using a syringe filter. The control heparin standard and the analytical sample from Example 1 were prepared by diluting them to various concentrations (0, 0.5, 1, 2, 4, and 6 IU / ml) in 0.9% saline and 1% BSA solutions. The prepared samples were analyzed as follows, according to the manual provided by the manufacturer. 20 μl of the standard and analytical sample were dispensed into a 96-well microplate, followed by the sequential addition of 20 μl of R1 (Human Antithrombin), 100 μl of R4 (Assay reaction buffer), and 40 μl of R2 (Chromogenic substrate specific for Thrombin), and the reaction was carried out at 37°C for 2 minutes. Subsequently, 40 µl of R3 (Human thrombin) was added, and the reaction was carried out at 37°C for 5 minutes to induce a color reaction. After the color reaction occurred, 80 µl of citric acid was added to the sample to terminate the reaction, and the OD value was measured at a wavelength of 405 nm using a microplate reader. The concentration of the analytical sample relative to the standard was calculated using the CombiStats program based on the measured wavelength values. Heparan sulfate must not have anticoagulant activity to be utilized as a pharmaceutical material. The results of the anticoagulant activity analysis are shown in Figure 3.

[0061] (3) Substrate reaction analysis method by Heparinase III

[0062] The commercially available Sigma Heparin lyase from Flavobacterium heparinum (EC 4.2.2.8) was used as the heparin-degrading enzyme. Distilled water was added to the freeze-dried heparin lyase powder to completely dissolve it, and the enzyme concentration was adjusted for disaccharide analysis, after which activity was measured. Heparin lyase III was analyzed by processing it as follows, using sodium heparin (Sigma) as the control group, heparan sulfate (Celsus) as the comparison group, and the analytical sample from Example 1 as substrates for glycosaminoglycan standards. First, the internal temperature of the microplate reader was maintained at 37 ℃. 10 mM sodium acetate (60 μL) and 10 mM calcium acetate (60 μL) were added as buffer solutions to 200 μg of heparin, heparan sulfate, and unfractionated heparin as reaction substrates. Then, 0.1 U of Heparin lyase III enzyme was added to make the final reaction volume 200 μL. Using a microplate reader, the change in absorbance at 232 nm was measured at 1-minute intervals over 30 minutes.

[0063] As shown in Fig. 4, in the case of the control group, heparin sodium (Sigma), no substrate reaction occurred upon treatment with the Heparinase III enzyme, whereas in the case of the comparison group, heparan sulfate (Celsus), a substrate reaction occurred. Thus, it can be seen that the analytical sample of Example 1 contains heparan sulfate, as a substrate reaction was confirmed upon treatment with the Heparinase III enzyme.

[0065] (4) H-NMR analysis method

[0066] Deuterated sodium trimethylsilylpropionate for nuclear magnetic resonance spectrum measurement was prepared by dissolving it in a deuterium oxide solution at a concentration of 20 μg / mL. 20 mg of the sample was dissolved in 0.7 mL of the prepared solution and injected into an NMR tube, and the H-NMR spectra were measured using an NMR spectrometer (Jeol JNM-ECP 500 MHz).

[0067] The device frequency was set to 300 MHz or higher, the temperature to 25℃ or higher, the spectrum width to 10~12 ppm, the centerad to about 4.5 ppm, and the pulse width to 30˚~90˚.

[0069] Figure 5 is a figure showing the 1H NMR analysis spectrum results for heparan sulfate obtained in Example 1.

[0071] As described above, although the present invention has been described with reference to preferred embodiment 1, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention as described in the following claims.

Claims

Claim 1 (S1) A step of obtaining a mucosal tissue slurry by adding purified water and sodium metabisulfite to porcine intestinal mucosal tissue and adjusting the pH; (S2) A step of obtaining an enzymatic hydrolysate by adding a proteolytic enzyme to the mucosal tissue slurry and enzymatically hydrolyzing it at 50–55°C for 12–18 hours; (S3) A step of inactivating the proteolytic enzyme by reacting the enzymatic hydrolysate at a temperature of 75–95°C for 5–15 minutes, then adjusting the temperature to 60–70°C to adjust the pH to 3–4, allowing it to stand, and then filtering to obtain a filtrate; (S4) A step of obtaining an eluent by contacting the filtrate with an anion exchange resin to adsorb heparan sulfate, separating it, washing it with purified water to remove impurities adsorbed on the anion exchange resin, and then eluting the heparan sulfate with NaCl with a salinity of 0.6–1.0 M; A method for extracting heparan sulfate, comprising the step of (S5) mixing ethanol with the above-mentioned eluent to prepare a 40-55 wt% (w / w) ethanol mixture, leaving it at room temperature to precipitate heparan sulfate, recovering the precipitate, and drying it to obtain heparan sulfate powder. Claim 2 A method according to claim 1, wherein in step S1, the pH is adjusted to 5 to 6 and the mucosal tissue slurry is left for 4 to 8 weeks. Claim 3 A method according to claim 1, wherein the mucosal tissue slurry contains 2 to 4 weight percent of sodium metabisulfite in step S1. Claim 4 A method according to claim 1, wherein in step S2, the proteolytic enzyme is one or more selected from alcalase, flavorzyme, neutrase, protamex, bromelain, and papain. Claim 5 A method according to claim 1, wherein in step S2, a proteolytic enzyme is added to the mucosal tissue slurry at 4 to 5% of the total weight of the porcine intestinal mucosal tissue to enzymatically hydrolyze it.

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