A method for extracting d-chiro-inositol from ceratonia siliqua and applications thereof

Abstract

The present application relates to carbocyclic compounds, more particularly to the field of cyclohexanehexol, and particularly to a method for extracting D-chiro-inositol from Ceratonia siliqua and application thereof. The present application uses an acid hydrolysis method, takes TFA as a catalyst, and takes L-(-) camphor sulfonic acid as a specific recrystallization selection agent to obtain high-purity DCI from Ceratonia siliqua, and has the advantages of high extraction efficiency, high DCI yield, high purity, low cost, green environmental protection 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, the present invention provides a method for extracting D-chiral inositol from Mediterranean carob, comprising the following steps:

[0006] S1. Pretreatment: Pretreatment of carob beans by drying, defatting and / or grinding;

[0007] S2, hydrolysis: the pretreated Ceratonia siliqua is mixed with a hydrolysis solution in a ratio of 1:8-10, and the hydrolysis reaction is carried out in a high-pressure reaction kettle under inert gas protection, to obtain an extraction solution; the hydrolysis solution contains formic acid in a volume ratio of 5%-10%, acetic acid in a volume ratio of 5%-10%, TFA in a volume ratio of 0.5%-0.8%, and EDTA-2Na in a weight percentage of 0.01%-0.1%; the reaction temperature in the high-pressure kettle is not less than 104 DEG C, and the pressure is not less than 0.35 MPa;

[0008] S3, neutralization and impurity removal: the extraction solution is subjected to neutralization and acid removal, impurity removal, and EDTA removal, to obtain a crude solution;

[0009] S4, crystallization: D-chiro-inositol crystals are obtained by crystallization from the crude solution.

[0010] DCI in Ceratonia siliqua is often covalently combined with galactose in the form of a β-1,4 glycosidic bond, and it is difficult to break the covalent bond between DCI and galactose by ordinary immersion or reflux extraction, so the yield of DCI obtained by ordinary immersion or reflux extraction is extremely low. The acid hydrolysis method is used in the application to extract DCI from Ceratonia siliqua, which not only effectively breaks the covalent bond between DCI and galactose, but also hydrolyzes D-pinitol to obtain DCI through acid catalysis, thereby increasing the yield of DCI. The lignin-hemicellulose complex of the cell wall of Ceratonia siliqua is subjected to acid hydrolysis and destruction by a formic acid / acetic acid mixture, and then TFA is added to catalytically break the β-1,4 glycosidic bond, thereby achieving the effect of increasing the yield of DCI. In addition, EDTA-2Na is added, and the hydrolysis reaction is carried out under inert gas protection, thereby effectively protecting the chiral structure of DCI, and successfully obtaining D-chiro-inositol with high optical purity.

[0011] In some embodiments, the volume ratio of formic acid in the hydrolysis solution is 5%.

[0012] In some embodiments, the volume ratio of acetic acid in the hydrolysis solution is 5%.

[0013] In some embodiments, the volume ratio of TFA in the hydrolysis solution is 0.8%.

[0014] In some embodiments, the weight percentage of EDTA-2Na in the hydrolysis solution is 0.1%.

[0015] The neutralization and impurity removal is a common post-treatment step for acid hydrolysis reaction, and various neutralization methods, impurity removal methods and EDTA removal methods are well known to those skilled in the art. Different neutralization methods, impurity removal methods and EDTA removal methods are not decisive for the output of DCI, and those skilled in the art can select appropriate neutralization and impurity removal methods according to the actual process needs to obtain pure DCI. In the present application, the neutralization and impurity removal are preferably carried out by adjusting the pH of the extract to about 4.8-5.3, removing impurities by silica gel column chromatography and removing acid by anion exchange resin adsorption, and finally removing EDTA to obtain a crude solution, so as to obtain DCI with higher purity, thereby 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. Further preferably, the pH of the extract is adjusted to 5.0.

[0016] In some embodiments, in the neutralization and impurity removal step, the method for adjusting the pH of the extract comprises the following steps: reducing the temperature in the kettle to not higher than 40℃, slowly adding 10% ammonia water to the kettle to a pH of about 2.4-2.8, and then continuing to reduce the temperature to not higher than 25℃, slowly adding 5% NaHCO3 solution to the kettle to a pH of about 4.8-5.3.

[0017] By the method of adjusting the pH step by step, the present application first neutralizes the residual TFA in the solution, and then neutralizes the formic acid and acetic acid in the solution, which is beneficial to further reduce the residual amount of TFA.

[0018] In some embodiments, the crystallization comprises the following steps: after adding L-(-) camphorsulfonic acid to the crude solution, slowly reducing the temperature to not higher than 0℃ to precipitate crude crystals; washing the crude crystals with cold ether, dissolving the crude crystals in ultrapure water, adsorbing the L-(-) camphorsulfonic acid on an anion exchange resin, collecting the effluent, concentrating, adding 80% acetone solution and heating to 55-60℃ to redissolve, and slowly reducing the temperature to -25℃ to -20℃ to precipitate crystals, thereby obtaining purified D-chiro-inositol crystals.

[0019] L-(-) camphorsulfonic acid can specifically bind with DCI to form DCI·L-(-) camphorsulfonic acid salt, thereby separating DCI from the remaining 8 isomers, and then removing the L-(-) camphorsulfonic acid by anion exchange resin to obtain free and pure DCI.

[0020] The pretreatment of the extraction raw material is a conventional technical means in the art, including drying, crushing, etc. Since the acid hydrolysis method is used to extract DCI in the present application, and the lipid components contained in the longpod cowpea are also prone to acid hydrolysis to produce free fatty acids and combine with DCI, therefore, the pretreatment of defatting the longpod cowpea is necessary. The method of defatting is also a method that is skilled by those skilled in the art, and in some embodiments of the present application, n-hexane is used as a defatting solvent to defat the dried longpod cowpea.

[0021] In another aspect, the present application provides a D-chiro-inositol obtained by the above method.

[0022] The present application has the following advantages and effects:

[0023] 1. The present application uses acid hydrolysis method to extract DCI from longpod cowpea, which has the advantages of high extraction efficiency, high DCI yield, high optical purity of DCI, low cost, green environmental protection, etc., and is conducive to industrialized continuous production.

[0024] 2. The present application uses L-(-) camphorsulfonic acid to selectively salt with DCI in a plurality of inositol, thereby separating DCI from the remaining inositol, and obtaining a crystalline body with high optical purity. DETAILED DESCRIPTION

[0025] The extraction method of D-chiro-inositol of the present application belongs to the technical field of cyclohexanehexol preparation. In order to make the purpose, characteristics and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below in combination with examples. Obviously, the described examples are part of the examples of the present application, not all. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0026] Unless otherwise specified, the experimental reagents and materials used in the present application can be obtained by market purchase. EXAMPLE

[0027] DCI is prepared by the following steps:

[0028] S1, take fresh longpod cowpea, wash, cut, dry, add n-hexane according to the ratio of 1:6, reflux at 80℃ for 2h, filter, dry the residue after solvent, mix with coarse quartz sand, put into the ball mill and grind for 30min, pass through 100 mesh sieve, and get defatted bean powder.

[0029] S2, mix the defatted bean powder and hydrolysis liquid according to a certain ratio in a high-pressure reaction kettle, then fill nitrogen into the high-pressure reaction kettle, and seal the reaction under the condition of 0.35MPa for 2.5h to obtain the extraction liquid. The ratio of defatted bean powder to hydrolysis liquid, the composition of hydrolysis liquid and the reaction temperature are shown in Table 1.

[0030] S3, after the extract solution is cooled to 40°C, 10% ammonia water is slowly added until the solution pH is 2.5, then the solution is continuously cooled to 25°C, and 5% NaHCO3 solution is slowly added until the solution pH is 5.0; the solution after pH adjustment is sequentially eluted and adsorbed by a silica gel column and a 201x7 type anion exchange resin, and the eluate is collected. In the silica gel column chromatography, the eluate is an ethyl acetate / methanol solution (volume ratio 95:5). The eluate is connected to a liquid chromatograph using Dowex 50WX4 ® <1 ppm, the eluate is collected to obtain a crude solution.

[0031] S4, an excess of L-(-) camphorsulfonic acid (the approximate content of DCI in the crude solution can be calculated at UV 245 nm) is added to the crude solution, after stirring and dissolving uniformly, the solution is cooled to 0°C at a rate of 0.2°C / min, and the crude crystals are precipitated, filtered, and washed with cold ether 3 times, the crude crystals are dissolved in ultrapure water, and the L-(-) camphorsulfonic acid is removed by adsorption on a 201x7 type anion exchange resin, the effluent is collected, concentrated to supersaturation, and then 80% acetone solution at 60°C is added to completely dissolve the crystals, and then the solution is cooled to -20°C at a rate of 0.2°C / min, and the crystals are precipitated, filtered, and DCI is obtained.

[0032] Table 1 Hydrolysis reaction parameters of each example

[0033]

[0034] Comparative Example 6

[0035] DCI is prepared by the following steps:

[0036] S1, fresh long beans are washed, chopped, and dried, and then added to n-hexane at a material to liquid ratio of 1:6, and refluxed at 80°C for 2h, filtered, and the residue is dried, mixed with coarse quartz sand, and put into a ball mill for grinding for 30min, and then passed through a 100 mesh sieve to obtain defatted bean powder.

[0037] S2, the defatted bean powder and the hydrolysis solution are mixed uniformly in a high-pressure reaction kettle at a material to liquid ratio of 1:10, nitrogen is filled into the high-pressure reaction kettle, and the reaction is carried out at a pressure of 0.35MPa and a reaction temperature of 105°C for 2.5h to obtain an extract solution. The hydrolysis solution is an aqueous solution containing formic acid at a volume ratio of 5%, acetic acid at a volume ratio of 5%, TFA at a volume ratio of 0.8%, and EDTA-2Na at a weight percentage of 0.1%.

[0038] S3, the extract solution is cooled to 25°C, and 10% NaOH solution is slowly added until the pH of the solution is 5.0; the solution after pH adjustment is eluted and adsorbed in sequence through a silica gel column and a 201x7 type anion exchange resin, and the eluate is collected. In the silica gel column chromatography, the eluate is an ethyl acetate / methanol solution (volume ratio 95:5). The eluate is connected to a liquid chromatograph using Dowex 50WX4 ® The chromatographic column is eluted with ultrapure water as the mobile phase, and the eluate is connected to a liquid chromatograph. After confirming that the residual EDTA is <1 ppm at UV 245 nm, the eluate is collected to obtain a crude solution.

[0039] S4, an excess of L-(-) camphorsulfonic acid is added to the crude solution (the approximate content of DCI in the crude solution can be calculated at UV 245 nm), the solution is stirred and dissolved uniformly, and then cooled to 0°C at a rate of 0.2°C / min to precipitate crude crystals. The crude crystals are filtered, washed with cold ether three times, dissolved in ultrapure water, adsorbed on a 201x7 type anion exchange resin to remove L-(-) camphorsulfonic acid, and the effluent is collected. The solution is concentrated to supersaturation, and then 80% acetone solution at 60°C is added to completely dissolve the crystals. The solution is then cooled to -20°C at a rate of 0.2°C / min to precipitate crystals, which are filtered to obtain DCI.

[0040] Comparative Example 7

[0041] DCI is prepared by the following steps:

[0042] S1, fresh long beans are washed, chopped, dried, and added with n-hexane at a material-to-liquid ratio of 1:6, and refluxed at 80°C for 2 h. After filtering and drying the solvent from the residue, the residue is mixed with coarse quartz sand, put into a ball mill for grinding for 30 min, and passed through a 100-mesh sieve to obtain defatted bean powder.

[0043] S2, the defatted bean powder and the hydrolysis solution are mixed uniformly in a high-pressure reaction kettle at a material-to-liquid ratio of 1:10, nitrogen is filled into the high-pressure reaction kettle, and the reaction is carried out at a pressure of 0.35 MPa and a reaction temperature of 105°C for 2.5 h to obtain an extract solution. The hydrolysis solution is an aqueous solution containing formic acid at a volume ratio of 5%, acetic acid at a volume ratio of 5%, TFA at a volume ratio of 0.8%, and EDTA-2Na at a weight percentage of 0.1%.

[0044] S3, the extract solution is cooled to 40°C, 10% ammonia water is slowly added until the pH of the solution is 2.5, and then the solution is further cooled to 25°C and 5% NaHCO3 solution is slowly added until the pH of the solution is 5.0; the solution after pH adjustment is eluted and adsorbed in sequence through a silica gel column and a 201x7 type anion exchange resin, and the eluate is collected. In the silica gel column chromatography, the eluate is an ethyl acetate / methanol solution (volume ratio 95:5). The eluate is connected to a liquid chromatograph using Dowex 50WX4 ®The chromatographic column was eluted with ultrapure water as the mobile phase, and the eluate was connected to a liquid chromatograph. After confirming that the residual EDTA was <1 ppm at 245 nm, the eluate was collected to obtain a crude solution.

[0045] S4. After the crude solution was concentrated to supersaturation, 80% acetone solution at 60°C was added to completely dissolve the solution. The solution was then cooled to -20°C at a rate of 0.2°C / min to precipitate crystals, which were filtered to obtain DCI.

[0046] Test Example 1 DCI Yield

[0047] The DCI yield of each example and the comparative example was calculated according to the weight of the DCI obtained from each example and the weight of the defatted soybean powder used, and the results are shown in Table 2.

[0048] Yield (%) = DCI weight / defatted soybean powder weight * 100%

[0049] Table 2 DCI Yield

[0050]

[0051] As can be seen from the results in Table 2, the DCI yield of Example 1, Example 2, Example 3, Comparative Example 6 and Comparative Example 7 all reached more than 10%, while the DCI yield of Comparative Examples 1-5 were all significantly lower than 10%. In specific analysis, compared with Example 1, Comparative Example 1 did not add TFA, and the DCI yield thereof was close to 0%, proving that the acidolysis of TFA is the key to the chemical preparation of DCI, and TFA has a significant catalytic effect on the hydrolysis of β-1,4 glycosidic bonds. Compared with Example 1, the DCI yield of Comparative Example 2 was only 3.32% without adding EDTA-disodium, proving that the degradation of metal ions on DCI is extremely significant, and it is necessary to add metal ion chelating agents such as EDTA-disodium to protect DCI. Compared with Example 1, Comparative Examples 3, 4 and 5 only differ in reaction temperature, which are 98°C, 102°C and 103°C, respectively, and then the DCI yield of Comparative Examples 3 and 4 is close to 0%, proving that complete acid hydrolysis cannot be achieved to prepare DCI at this reaction temperature, which 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 presumably because the temperature control during the reaction is unstable, or it is at the critical temperature of the reaction, so that part of the β-1,4 glycosidic bonds are cleaved to obtain part of the DCI.

[0052] It is proved that the preparation of DCI by acidolysis method needs catalysis of TFA and protection of metal ion chelating agent, and the reaction temperature is not less than 104℃. The preparation method disclosed in the application can improve the yield of DCI, thereby reducing the production cost and improving the production capacity.

[0053] Residual TFA determination of test example 2

[0054] Each sample was dissolved in methanol, and the residual amount of TFA was determined by HPLC, and the chromatographic conditions were as follows:

[0055] Chromatographic column: ZORBAX 300SB-C18 chromatographic column (4.6 mm x 250 mm, 5 μm);

[0056] Mobile phase: A phase 0.08% phosphoric acid solution (pH 3.0); B phase 5% methanol solution; A:B = 1:1;

[0057] Flow rate: 0.8 mL / min;

[0058] Column temperature: 35℃;

[0059] Detection wavelength: 210 nm.

[0060] The results are shown in Table 3.

[0061] Table 3 TFA residual amount

[0062]

[0063] As can be seen from Table 3, the residual amount of TFA in each sample is less than 10 ppm except for Comparative Example 6, which meets the safety standard. Compared with Example 1, Comparative Example 6 directly uses 10% NaOH solution for pH adjustment in the neutralization step, resulting in incomplete TFA reaction. The above proves that the stepwise pH adjustment method of the application is beneficial to efficiently remove residual TFA.

[0064] DCI optical purity determination of test example 3

[0065] According to the HPLC determination method of DCI recorded in Hu Junjun, Yi Xin, Hu Hongjuan, etc. Determination of D-chiro-inositol content in buckwheat by pre-column derivatization high performance liquid chromatography [J]. Food Industry Science and Technology, 2018, 39(13): 248-252. The content of DCI in each example sample was determined, and the purity of DCI crystal was calculated. The results are shown in Table 4.

[0066] Table 4 DCI purity

[0067]

[0068] The results show that the purity of DCI in the DCI crystals of Examples 1-3 and Comparative Examples 1-6 are all higher than 99%, while the purity of DCI in the DCI crystals of Comparative Example 7 is only 43.2%. Compared with Example 1, Comparative Example 7 does not use L-(-) camphorsulfonic acid to specifically separate DCI in the crystallization step, resulting in a significant drop in purity. The above proves that L-(-) camphorsulfonic acid can selectively bind to DCI, and DCI can be effectively separated from other configurations of inositol by L-(-) camphorsulfonic acid, thereby obtaining high-purity DCI crystals.

[0069] Although the preferred embodiments of the present application have been described, those skilled in the art who are familiar with the basic inventive concept can make further changes and modifications to the embodiments. Therefore, the appended claims are intended to cover the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present application.

[0070] Finally, it should also be noted that, in this document, relational terms such as first and second and the like can only be used to distinguish one entity or action from another entity or action, without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal device including a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or terminal device. Without more limitations, an element defined by the statement "comprising a" does not exclude the existence of additional identical elements in the process, method, article, or terminal device including the element.

[0071] The above has described the present application in detail, and the principles and implementation modes of the present application have been described by applying specific examples in this document. The above description of the examples is only for the purpose of helping to understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, there will be changes in specific implementation modes and application ranges; in view of the above, the content of the specification should not be understood as a limitation of the present application.

Claims

1. A method for extracting D-chiro-inositol from Ceratonia siliqua, characterized by, The method comprises the following steps: S1, pretreatment: pretreatment of drying, n-hexane degreasing and / or crushing of Ceratonia siliqua; S2, hydrolysis: the pretreated Ceratonia siliqua is mixed with a hydrolysis solution at a material-liquid ratio of 1: (8-10), and hydrolysis reaction is carried out in a high-pressure reaction kettle under inert gas protection to obtain an extraction liquid; the hydrolysis solution contains formic acid at a volume ratio of 5-10%, acetic acid at a volume ratio of 5-10%, TFA at a volume ratio of 0.5-0.8%, and EDTA-2Na at a weight percentage of 0.01-0.1%; the reaction temperature in the high-pressure kettle is not less than 104 DEG C, and the pressure is not less than 0.35 MPa; S3, neutralization and impurity removal: the pH of the extraction liquid is adjusted to 4.8-5.3, and after silica gel column chromatography and impurity removal, the acid is removed by adsorption on an anion exchange resin, and then EDTA is removed to obtain a crude solution; S4, crystallization: after L-(-) camphorsulfonic acid is added to the crude solution, slow cooling is carried out to not higher than 0 DEG C, and crude crystals are precipitated; After the crude crystals are washed with cold ether, the crude crystals are dissolved in ultrapure water, the L-(-) camphorsulfonic acid is removed by adsorption on an anion exchange resin, the effluent is collected, concentrated, 80% acetone solution is added and heated to 55-60 DEG C to redissolve, and slow cooling is carried out to -25 DEG C to -20 DEG C to precipitate crystals, and the purified D-chiro-inositol crystals are obtained.

2. The method of claim 1, wherein, The volume ratio of formic acid in the hydrolysis solution is 5%.

3. The method of claim 1, wherein, The volume ratio of acetic acid in the hydrolysis solution is 5%.

4. The method of claim 1, wherein, The volume ratio of TFA in the hydrolysis solution is 0.8%.

5. The method of claim 1, wherein, The weight percentage of EDTA-2Na in the hydrolysis solution is 0.1%.

6. The method of claim 1, wherein, In the neutralization and impurity removal step, the method for adjusting the pH of the extraction liquid comprises the following steps: the kettle temperature is reduced to not higher than 40 DEG C, 10% ammonia water is slowly added to the kettle to a pH of 2.4-2.8, then the temperature is continuously reduced to not higher than 25 DEG C, and 5wt% NaHCO3 solution is slowly added to the kettle to a pH of 4.8-5.3.

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