Method for extracting D-chiro-inositol from carob beans and application of D-chiro-inositol

Abstract

The invention relates to carbocyclic compounds, in particular to the field of cyclohexanehexol, and particularly relates to a method for extracting D-chiro-inositol from carob beans and application of the D-chiro-inositol. According to the method, an acid hydrolysis method is used, TFA is used as a catalyst, L-(-) camphorsulfonic acid is used as a specific recrystallization selective agent, high-purity DCI is obtained from carob beans, and the method has the advantages of being high in extraction efficiency, DCI yield and purity, low in cost, environmentally friendly and the like.

Description

Technical Field

[0001] This invention relates to carbocyclic compounds, and more particularly to the field of cyclohexanehexol, and especially to a method and application for extracting D-chiral inositol from Mediterranean carob. Background Technology

[0002] Carob ( Ceratonia siliqua Carob (Carob) is a plant native to the eastern Mediterranean region, belonging to the Carob family (family Carobaceae). It is rich in inositol molecules, primarily existing as D-pinitol and D-chiroinositol (DCI) covalently bound to glycosides. D-pinitol is the methyl ether of DCI and is the most abundant in carob inositol molecules. Studies have found that DCI plays an important role in the treatment of diabetes and is now widely used as a next-generation insulin receptor sensitizer.

[0003] Currently, dichlorodimethyl chloride (DCI) is mainly extracted, separated, and purified from carob beans. Common extraction methods include maceration, finning, decoction, reflux extraction, continuous reflux extraction, fermentation, and enzymatic hydrolysis. However, conventional extraction methods such as maceration and decoction not only have low extraction efficiency but also yield unsatisfactory DCI yields. Prolonged high temperatures can also easily lead to the oxidative degradation of DCI, further reducing the yield. While fermentation and enzymatic hydrolysis offer milder reaction conditions, biological extraction methods present challenges in scaling up production, reaction control, and the production of high levels of biological byproducts, significantly hindering subsequent purification and separation. Improving the yield and extraction efficiency of DCI remains a pressing issue in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for extracting D-chiral inositol from Mediterranean carob. The DCI obtained by the method provided by this invention has the characteristics of high optical purity, high yield and short extraction time, and is suitable for large-scale industrial production.

[0005] In view of this, on the one hand, the present invention provides a method for extracting D-chiral inositol from Mediterranean carob, comprising the following steps: S1. Pretreatment: Pretreatment of carob beans by drying, defatting and / or grinding; S2. Hydrolysis: The pretreated carob beans and hydrolysate are mixed evenly at a material-to-liquid ratio of 1:(8~10). Under inert gas protection, the hydrolysis reaction is carried out in a high-pressure reactor to obtain an extract. The hydrolysate contains 5%~10% formic acid, 5%~10% acetic acid, 0.5%~0.8% TFA, and 0.01%~0.1% EDTA-2Na by weight. The reaction temperature in the high-pressure reactor is not less than 104℃ and the pressure is not less than 0.35MPa. S3, Neutralization and Impurity Removal: The extract is subjected to neutralization, acid removal, impurity removal and EDTA removal treatment to obtain a crude solution; S4. Crystallization: D-chiral inositol crystals are obtained by crystallization from the crude solution.

[0006] DCI in carob is often covalently bound to galactose via β-1,4 glycosidic bonds. Conventional impregnation or reflux extraction methods are insufficient to break these covalent bonds, resulting in extremely low yields of DCI obtained through these methods. This invention innovatively employs acid hydrolysis to extract DCI from carob, effectively breaking the covalent bonds between DCI and galactose while simultaneously hydrolyzing D-pineol via acid catalysis, thereby increasing the DCI yield. This invention utilizes a formic acid / acetic acid mixture to acid-hydrolyze the lignin-hemicellulose complex in the carob cell wall, followed by the innovative addition of TFA to catalyze the breakdown of β-1,4 glycosidic bonds, thus improving the DCI yield. Furthermore, the addition of EDTA-2Na and the placement of the hydrolysis reaction under an inert gas atmosphere effectively protect the chiral structure of DCI, successfully yielding D-chiral inositol with high optical purity.

[0007] In some embodiments, the volume ratio of formic acid in the hydrolysate is 5%.

[0008] In some embodiments, the volume ratio of acetic acid in the hydrolysate is 5%.

[0009] In some embodiments, the volume percentage of TFA in the hydrolysate is 0.8%.

[0010] In some embodiments, the weight percentage of EDTA-2Na in the hydrolysate is 0.1%.

[0011] Neutralization and impurity removal are common post-treatment steps in acid hydrolysis reactions. Those skilled in the art are familiar with various neutralization, impurity removal, and EDTA removal methods. Different neutralization, impurity removal, and EDTA removal methods are not decisive for DCI yield; those skilled in the art can select appropriate neutralization and impurity removal methods according to actual process requirements to obtain pure DCI. Preferably, this invention uses a method of adjusting the pH of the extract to approximately 4.8-5.3, employing silica gel column chromatography for impurity removal and anion exchange resin adsorption for acid removal, and finally targeted removal of EDTA to neutralize and remove impurities to obtain a crude solution, thereby obtaining DCI with higher purity, thus simplifying the subsequent crystallization process. Preferably, the pH of the extract is adjusted to 4.8, 4.9, 5.0, 5.1, 5.2, or 5.3. More preferably, the pH of the extract is adjusted to 5.0.

[0012] In some embodiments, the method for adjusting the pH of the extract in the neutralization and impurity removal step includes the following steps: lowering the temperature inside the vessel to no higher than 40°C, slowly adding 10% ammonia water to the vessel until the pH value is about 2.4 to 2.8, then continuing to lower the temperature to no higher than 25°C, and slowly adding 5% NaHCO3 solution to the vessel until the pH value is about 4.8 to 5.3.

[0013] This invention uses a stepwise pH adjustment method to first neutralize the residual TFA in the solution, and then neutralize the formic acid and acetic acid in the solution, which helps to further reduce the residual amount of TFA.

[0014] In some embodiments, the crystallization includes the following steps: adding L-(-)camphorsulfonic acid to the crude solution, slowly cooling to no higher than 0°C to precipitate crude crystals; washing the crude crystals with cold ether, dissolving the crude crystals in ultrapure water, adsorbing and removing L-(-)camphorsulfonic acid using an anion exchange resin, collecting the effluent, concentrating it, adding 80% acetone solution and heating to 55°C~60°C to redissolve it, and slowly cooling to -25°C~-20°C to precipitate crystals to obtain purified D-chiral inositol crystals.

[0015] L-(-)camphorsulfonic acid can specifically bind with DCI to form DCI·L-(-)camphorsulfonate, thereby separating DCI from the other 8 isomers. Then, L-(-)camphorsulfonic acid is removed using anion exchange resin to obtain free pure DCI.

[0016] Pretreatment of raw materials is a conventional technique in this field, including drying and pulverizing. Since this invention uses acid hydrolysis to extract DCI, and the lipid components in carob beans are easily acid-hydrolyzed to produce free fatty acids that bind to DCI, defatting pretreatment of the carob beans is necessary. Defatting methods are also well-known to those skilled in the art. In some embodiments of this invention, n-hexane is used as the defatting solvent to defatt the dried carob beans.

[0017] On the other hand, the present invention provides a D-chiral inositol prepared by the above method.

[0018] The present invention has the following advantages and effects: 1. This invention uses acid hydrolysis to extract DCI from carob beans, which has the advantages of high extraction efficiency, high DCI yield, high DCI optical purity, low cost, and green environmental protection, and is conducive to continuous industrial production.

[0019] 2. In this invention, L-(-)camphorsulfonic acid is used to selectively form salts with DCI in various inositols, thereby separating DCI from the remaining inositols and obtaining crystals with high optical purity. Detailed Implementation

[0020] The extraction method of D-chiral inositol of the present invention belongs to the technical field of cyclohexanehexyl alcohol preparation. To make the objectives, features, and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, all experimental reagents and materials used in this invention are commercially available. Example

[0022] DCI is prepared through the following steps: S1. Take fresh carob beans, wash, chop, and dry them. Add n-hexane at a material-to-liquid ratio of 1:6, reflux at 80℃ for 2 hours, filter, evaporate the solvent from the residue, mix with coarse quartz sand, grind in a ball mill for 30 minutes, and pass through a 100-mesh sieve to obtain defatted bean flour.

[0023] S2. After mixing defatted soybean flour and hydrolysate at a certain material-to-liquid ratio in a high-pressure reactor, nitrogen gas is introduced into the reactor, and the reaction is carried out in a sealed environment at a pressure of 0.35 MPa for 2.5 hours to obtain the extract. The material-to-liquid ratio of defatted soybean flour and hydrolysate, the composition of the hydrolysate, and the reaction temperature are shown in Table 1.

[0024] S3. After cooling the extract to 40℃, slowly add 10% ammonia water until the solution pH is 2.5. Then continue cooling to 25℃ and slowly add 5% NaHCO3 solution until the solution pH is 5.0. The pH-adjusted solution is then sequentially eluted and adsorbed onto a silica gel column and a 201×7 anion exchange resin. The eluent is collected. In silica gel column chromatography, the eluent is an ethyl acetate / methanol solution (95:5 volume ratio). A Dowex 50WX4 eluent is used. ® The column was used as the chromatographic column, and ultrapure water was used as the mobile phase for elution. The eluent was connected to the liquid chromatography column, and after confirming that the residual EDTA was <1ppm under UV245nm, the eluent was collected to obtain the crude solution.

[0025] S4. Add excess L-(-)camphorsulfonic acid to the crude solution (the approximate DCI content in the crude solution can be calculated under UV245nm). After stirring and dissolving evenly, cool the solution to 0℃ at a rate of 0.2℃ / min to precipitate crude crystals. Filter the solution and wash the crude crystals three times with cold ether. Dissolve the crude crystals in ultrapure water and remove L-(-)camphorsulfonic acid by adsorption using a 201×7 type anion exchange resin. Collect the effluent, concentrate it to supersaturation, and then add 80% acetone solution at 60℃ to completely dissolve the crystals. Then cool the solution to -20℃ at a rate of 0.2℃ / min to precipitate crystals. Filter the solution to obtain DCI.

[0026] Table 1 Hydrolysis reaction parameters for each embodiment

[0027] Comparative Example 6 DCI is prepared through the following steps: S1. Take fresh carob beans, wash, chop, and dry them. Add n-hexane at a material-to-liquid ratio of 1:6, reflux at 80℃ for 2 hours, filter, evaporate the solvent from the residue, mix with coarse quartz sand, grind in a ball mill for 30 minutes, and pass through a 100-mesh sieve to obtain defatted bean flour.

[0028] S2. Defatted soybean flour and hydrolysate are mixed evenly in a high-pressure reactor at a material-to-liquid ratio of 1:10. Nitrogen gas is then introduced into the reactor, and the mixture is reacted under sealed conditions at a pressure of 0.35 MPa and a reaction temperature of 105°C for 2.5 hours to obtain the extract. The hydrolysate is an aqueous solution containing 5% formic acid (by volume), 5% acetic acid (by volume), 0.8% TFA (by volume), and 0.1% EDTA-2Na (by weight).

[0029] S3. Cool the extract to 25℃, and slowly add 10% NaOH solution until the pH reaches 5.0. After pH adjustment, the solution is sequentially eluted and adsorbed onto a silica gel column and a 201×7 anion exchange resin. Collect the eluent. In silica gel column chromatography, the eluent is an ethyl acetate / methanol solution (95:5 volume ratio). A Dowex 50WX4 eluent was used. ® The column was used as the chromatographic column, and ultrapure water was used as the mobile phase for elution. The eluent was connected to the liquid chromatography column, and after confirming that the residual EDTA was <1ppm under UV245nm, the eluent was collected to obtain the crude solution.

[0030] S4. Add excess L-(-)camphorsulfonic acid to the crude solution (the approximate DCI content in the crude solution can be calculated under UV245nm). After stirring and dissolving evenly, cool the solution to 0℃ at a rate of 0.2℃ / min to precipitate crude crystals. Filter the solution and wash the crude crystals three times with cold ether. Dissolve the crude crystals in ultrapure water and remove L-(-)camphorsulfonic acid by adsorption using a 201×7 type anion exchange resin. Collect the effluent, concentrate it to supersaturation, and then add 80% acetone solution at 60℃ to completely dissolve the crystals. Then cool the solution to -20℃ at a rate of 0.2℃ / min to precipitate crystals. Filter the solution to obtain DCI.

[0031] Comparative Example 7 DCI is prepared through the following steps: S1. Take fresh carob beans, wash, chop, and dry them. Add n-hexane at a material-to-liquid ratio of 1:6, reflux at 80℃ for 2 hours, filter, evaporate the solvent from the residue, mix with coarse quartz sand, grind in a ball mill for 30 minutes, and pass through a 100-mesh sieve to obtain defatted bean flour.

[0032] S2. Defatted soybean flour and hydrolysate are mixed evenly in a high-pressure reactor at a material-to-liquid ratio of 1:10. Nitrogen gas is then introduced into the reactor, and the mixture is reacted under sealed conditions at a pressure of 0.35 MPa and a reaction temperature of 105°C for 2.5 hours to obtain the extract. The hydrolysate is an aqueous solution containing 5% formic acid (by volume), 5% acetic acid (by volume), 0.8% TFA (by volume), and 0.1% EDTA-2Na (by weight).

[0033] S3. After cooling the extract to 40℃, slowly add 10% ammonia water until the solution pH is 2.5. Then continue cooling to 25℃ and slowly add 5% NaHCO3 solution until the solution pH is 5.0. The pH-adjusted solution is then sequentially eluted and adsorbed onto a silica gel column and a 201×7 anion exchange resin. The eluent is collected. In silica gel column chromatography, the eluent is an ethyl acetate / methanol solution (95:5 volume ratio). A Dowex 50WX4 eluent is used. ®The column was used as the chromatographic column, and ultrapure water was used as the mobile phase for elution. The eluent was connected to the liquid chromatography column, and after confirming that the residual EDTA was <1ppm under UV245nm, the eluent was collected to obtain the crude solution.

[0034] S4. After concentrating the crude solution to supersaturation, add 80% acetone solution at 60℃ to completely dissolve it. Then cool the solution to -20℃ at a rate of 0.2℃ / min to precipitate crystals. Filter to obtain DCI.

[0035] Test Example 1 DCI Yield Based on the weight of DCI obtained in each embodiment and comparative example and the weight of defatted soybean flour used, the DCI yield of each embodiment and comparative example was calculated, and the results are shown in Table 2.

[0036] Yield (%) = (Weight of DCI / Weight of defatted soybean flour) * 100% Table 2 DCI Yield

[0037] As shown in Table 2, the DCI yields of Examples 1, 2, 3, Comparative Example 6, and Comparative Example 7 all exceeded 10%. However, the DCI yields of Comparative Examples 1-5 were significantly lower than 10%. Specifically, compared to Example 1, Comparative Example 1, which did not include TFA, had a DCI yield close to 0%, demonstrating that acidolysis of TFA is crucial for the chemical preparation of DCI, and that TFA has a significant catalytic effect on the hydrolysis of β-1,4 glycosidic bonds. Compared to Example 1, Comparative Example 2, which did not include EDTA-disodium, had a DCI yield of only 3.32%, demonstrating that metal ions have a significant degradation effect on DCI, necessitating the addition of metal ion chelating agents such as EDTA-disodium to protect the DCI. Compared with Example 1, Comparative Examples 3, 4, and 5 differed only in reaction temperature, which was 98°C, 102°C, and 103°C, respectively. The DCI yields of Comparative Examples 3 and 4 were close to 0%, indicating that acid hydrolysis to produce DCI could not be completed at this reaction temperature. This may be because the Gibbs free energy required for the cleavage of β-1,4 glycosidic bonds is high, and the lower reaction temperature cannot meet the energy requirements of the chemical reaction. The DCI yield of Comparative Example 5 reached 6.87%, which is speculated to be because the temperature control during the reaction was unstable or it was at the critical temperature of the reaction, resulting in the cleavage of some β-1,4 glycosidic bonds and the acquisition of some DCI.

[0038] The above demonstrates that the acid hydrolysis method for producing DCI requires TFA catalysis and the protection of a metal ion chelating agent, as well as a reaction temperature of at least 104°C. The preparation method disclosed in this invention can improve the yield of DCI, thereby reducing production costs and increasing production capacity.

[0039] Test Example 2: Determination of Residual TFA Each sample was dissolved in methanol, and the residual TFA was determined by HPLC under the following chromatographic conditions: Chromatographic column: ZORBAX 300SB-C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: Phase A: 0.08% phosphoric acid solution (pH 3.0); Phase B: 5% methanol solution; A:B = 1:1; Flow rate: 0.8 mL / min; Column temperature: 35℃; Detection wavelength: 210nm.

[0040] The results are shown in Table 3.

[0041] Table 3 TFA Residue

[0042] As shown in Table 3, except for Comparative Example 6, the residual TFA levels in all other samples were less than 10 ppm, meeting safety standards. Compared to Example 1, Comparative Example 6 directly used 10% NaOH solution for pH adjustment during the neutralization step, resulting in incomplete TFA reaction. The above demonstrates that the staged pH adjustment method of the present invention is beneficial for the efficient removal of residual TFA.

[0043] Test Example 3: Determination of Optical Purity of DCI Based on the HPLC method for DCI determination described in Hu Junjun, Yi Xin, Hu Hongjuan, et al., Pre-column derivatization high performance liquid chromatography method for determining D-chiral inositol content in buckwheat [J]. Food Industry Technology, 2018, 39(13):248-252, the DCI content in the samples of each example was determined, and the purity of DCI crystals was calculated. The results are shown in Table 4.

[0044] Table 4 DCI Purity

[0045] The results showed that the purity of DCI in the DCI crystals of Examples 1-3 and Comparative Examples 1-6 was higher than 99%, while the purity of DCI in the DCI crystals of Comparative Example 7 was only 43.2%. Compared with Example 1, Comparative Example 7 did not use L-(-)camphorsulfonic acid for specific separation of DCI in the crystallization step, resulting in a significant decrease in its purity. This demonstrates that L-(-)camphorsulfonic acid can selectively bind to DCI, and can effectively separate DCI from other configurations of inositol, thereby obtaining high-purity DCI crystals.

[0046] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0047] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0048] The present invention has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for extracting D-chiral inositol from Mediterranean carob, characterized in that, Includes the following steps: S1. Pretreatment: Pretreatment of carob beans by drying, defatting and / or grinding; S2. Hydrolysis: The pretreated carob beans and hydrolysate are mixed evenly at a material-to-liquid ratio of 1:(8~10). Under inert gas protection, the hydrolysis reaction is carried out in a high-pressure reactor to obtain an extract. The hydrolysate contains 5%~10% formic acid, 5%~10% acetic acid, 0.5%~0.8% TFA, and 0.01%~0.1% EDTA-2Na by weight. The reaction temperature in the high-pressure reactor is not less than 104℃ and the pressure is not less than 0.35MPa. S3, Neutralization and Impurity Removal: The extract is subjected to neutralization, acid removal, impurity removal and EDTA removal treatment to obtain a crude solution; S4. Crystallization: D-chiral inositol crystals are obtained by crystallization from the crude solution.

2. The method according to claim 1, characterized in that, The volume ratio of formic acid in the hydrolysate is 5%.

3. The method according to claim 1, characterized in that, The volume ratio of acetic acid in the hydrolysate is 5%.

4. The method according to claim 1, characterized in that, The volume ratio of TFA in the hydrolysate is 0.8%.

5. The method according to claim 1, characterized in that, The weight percentage of EDTA-2Na in the hydrolysate is 0.1%.

6. The method according to any one of claims 1-5, characterized in that, The neutralization and impurity removal process includes the following steps: adjusting the pH of the extract to 4.8-5.3, removing impurities by silica gel column chromatography, adsorbing and removing acid by anion exchange resin, and then targeting and removing EDTA to obtain a crude solution.

7. The method according to claim 6, characterized in that, In the neutralization and impurity removal step, the method for adjusting the pH of the extract includes the following steps: lowering the temperature inside the vessel to no higher than 40°C, slowly adding 10% ammonia water to the vessel until the pH value is 2.4~2.8, then continuing to lower the temperature to no higher than 25°C, and slowly adding 5wt% NaHCO3 solution to the vessel until the pH value is 4.8~5.

3.

8. The method according to any one of claims 1-5, characterized in that, The crystallization process includes the following steps: adding L-(-)camphorsulfonic acid to the crude solution, slowly cooling to no higher than 0°C to precipitate crude crystals; washing the crude crystals with cold ether, dissolving the crude crystals in ultrapure water, adsorbing and removing L-(-)camphorsulfonic acid using an anion exchange resin, collecting the effluent, concentrating it, adding 80% acetone solution and heating to 55°C~60°C to redissolve it, and slowly cooling to -25°C~-20°C to precipitate crystals, obtaining purified D-chiral inositol crystals.

9. The method according to any one of claims 1-5, characterized in that, In step S1, carob beans are defatted using n-hexane.

10. A D-chiral inositol, prepared by the method according to any one of claims 1-9.

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