Intestinal absorption hemin

Abstract

The invention relates to intestinal absorption hemin, and belongs to the technical field of biological medicine, the intestinal absorption hemin comprises hemin and gamma-cyclodextrin; the molar ratio of the hemin to the cyclodextrin is 1: 2; the preparation method of the intestinal absorption hemin comprises the following steps: (1) mixing hemin with ammonia water to obtain a hemin solution; mixing cyclodextrin with water to obtain a cyclodextrin aqueous solution; (2) mixing the hemin solution with the cyclodextrin aqueous solution to obtain a mixed solution; and (3) stirring the mixed solution, and drying for 5 hours to obtain the intestinal absorption hemin. The prepared intestinal absorption hemin has the advantages of being high in water solubility and stability, high in bioavailability after gastrointestinal digestion and the like, and can be used as a raw material for preparing a nutrition enhancer for supplementing iron.

Description

[0001] This application is a divisional application. The application number of the original application is: 202311721161.5, the application date is 2023.12.14, and the name of the invention is: A kind of intestinal absorption hemin and its preparation method and application. Technical Field

[0002] The invention relates to the technical field of biomedicine, and in particular to intestinal absorption of hemin. Background Art

[0003] Cyclodextrin is a cyclic oligosaccharide with a hydrophobic inner cavity and a hydrophilic outer wall, which has good loading properties for hydrophobic molecules. Many natural active ingredients and drug molecules have poor water solubility and low tolerance to stimuli such as light, heat, and pH, which makes their utilization in functional foods or medicines very low. Studies have found that embedding these active molecules with special functions in the cyclodextrin cavity can effectively enhance their water solubility and stability, thereby further improving the storage period and bioavailability of the active molecules.

[0004] Hemin is a purified form of natural heme in vitro. It is usually made from heme extracted from animal blood. Protoporphyrin is first reacted with Fe 3+ Coordination, Fe 3+ It then combines with Cl- to form hemin. Studies have shown that hemin is the biological iron agent with the highest absorption rate known so far, and can be used as an iron fortifier and an anemia drug. However, hemin has extremely poor water solubility, which greatly limits its practical application. Summary of the invention

[0005] The purpose of the present invention is to provide an intestinal absorption hemin and a preparation method and application thereof, so as to solve the problems of poor water solubility, low intestinal absorption rate and limited application of related products of hemin in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an intestinal absorption hemin, comprising hemin and cyclodextrin;

[0008] The molar ratio of the hemin to cyclodextrin is 1-2:1-2.

[0009] Preferably, the cyclodextrin is methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin or γ-cyclodextrin.

[0010] The present invention also provides a method for preparing the intestinal absorption hemin, comprising the following steps:

[0011] (1) mixing hemin with ammonia water to obtain a hemin solution; mixing cyclodextrin with water to obtain a cyclodextrin aqueous solution;

[0012] (2) mixing the hemin solution with the cyclodextrin aqueous solution to obtain a mixed solution;

[0013] (3) The mixed solution is stirred and dried for 4 to 6 hours to obtain intestinal absorption hemin.

[0014] Preferably, the molar volume ratio of hemin to ammonia water in step (1) is 0.01 to 0.02 mol:1L;

[0015] The molar volume ratio of the cyclodextrin to water is 0.01-0.02 mol:2L;

[0016] The initial concentration of the ammonia water is 9-15 wt %.

[0017] Preferably, the volume ratio of the hemin solution to the cyclodextrin aqueous solution in step (2) is 1-5:10-20.

[0018] Preferably, the stirring temperature in step (3) is 30 to 50° C., and the stirring time is 2 to 4 hours.

[0019] Preferably, the drying in step (3) is oven drying, and the oven drying temperature is 40 to 80°C.

[0020] The present invention also provides the use of the intestinal absorption hemin in the preparation of a nutritional enhancer for iron supplementation.

[0021] The present invention also provides the use of the enterically absorbed hemin prepared by the preparation method in the preparation of a nutritional enhancer for iron supplementation.

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

[0023] The present invention is based on the inclusion effect of water-soluble carriers on active molecules, uses different types of cyclodextrins to embed hemin, and prepares intestinal absorption hemin by a solution stirring method. The preparation method of the present invention is simple, rapid, green, and pollution-free. The solubility, stability, and bioavailability of the prepared intestinal absorption hemin in water are significantly improved, and it can be used as a raw material for preparing a nutritional enhancer for iron supplementation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The encapsulation efficiency of intestinal absorption hemin prepared in Examples 1 to 4 and Comparative Example 1;

[0025] Figure 2The water solubility of the enteric absorption hemin saturated solution prepared by using the enteric absorption hemin of Examples 1 to 4 and Comparative Example 1 and the original hemin saturated solution;

[0026] Figure 3 The solubilization multiple of the intestinal absorption hemin prepared in Examples 1 to 4 and Comparative Example 1;

[0027] Figure 4 The Fourier transform infrared spectrum of the intestinal absorption hemin prepared in Example 4;

[0028] Figure 5 The X-ray diffraction pattern of intestinal absorption hemin prepared in Example 4;

[0029] Figure 6 The stability and bioavailability of the enterically absorbed hemin and original hemin prepared in Example 4 during gastrointestinal digestion. DETAILED DESCRIPTION

[0030] The present invention provides an intestinal absorption hemin, comprising hemin and cyclodextrin;

[0031] The molar ratio of hemin to cyclodextrin is 1-2:1-2, preferably 1:2.

[0032] In the present invention, the chemical formula of the hemin is C 34 H 32 ClFeN4O4, relative molecular mass is 654.94;

[0033] The cyclodextrin is methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin or γ-cyclodextrin;

[0034] The chemical formula of the methyl-β-cyclodextrin (Me-β-cyclodextrin) is C 54 H 94 O 35 , the relative molecular mass is 1303.29;

[0035] The chemical formula of the hydroxypropyl-β-cyclodextrin (HP-β-cyclodextrin) is C 63 H 112 O 42 , the relative molecular mass is 1541.54;

[0036] The chemical formula of the sulfobutyl-β-cyclodextrin (SBE-β-cyclodextrin) is C 50 H 84 Na2O 41 S2, relative molecular mass is 1451.29;

[0037] The chemical formula of the γ-cyclodextrin is C 48 H 80 O 40 , relative molecular mass 1297.11.

[0038] The present invention also provides a method for preparing the intestinal absorption hemin, comprising the following steps:

[0039] (1) mixing hemin with ammonia water to obtain a hemin solution; mixing cyclodextrin with water to obtain a cyclodextrin aqueous solution;

[0040] (2) mixing the hemin solution with the cyclodextrin aqueous solution to obtain a mixed solution;

[0041] (3) The mixed solution is stirred and dried for 4 to 6 hours to obtain intestinal absorption hemin.

[0042] In the present invention, the molar volume ratio of hemin to ammonia water in step (1) is 0.01-0.02 mol:1L, preferably 0.0165 mol:1L;

[0043] The molar volume ratio of cyclodextrin to water is 0.01-0.02 mol:2L, preferably 0.0176 mol:2L;

[0044] The initial concentration of the ammonia water is 9-15 wt %, preferably 13 wt %.

[0045] In the present invention, the volume ratio of the hemin solution to the cyclodextrin aqueous solution in step (2) is 1-5:10-20, preferably 4:15.

[0046] In the present invention, the stirring temperature in step (3) is 30-50° C., preferably 40° C.; the stirring time is 2-4 h, preferably 3 h.

[0047] In the present invention, the drying in step (3) is oven drying, the oven drying temperature is 40 to 80° C., preferably 60° C.; the oven drying time is 4 to 6 hours, preferably 5 hours.

[0048] In the present invention, the drying in step (3) further includes washing, the detergent used in the washing is acidic acetone, and the number of washing times is 1 to 3 times, preferably 2 times.

[0049] The present invention further comprises drying after washing, wherein the drying after washing is oven drying, the oven drying temperature is 40 to 80° C., preferably 60° C., and the oven drying time is 4 to 6 hours, preferably 5 hours.

[0050] The present invention also provides the use of the intestinal absorption hemin in the preparation of a nutritional enhancer for iron supplementation.

[0051] The present invention also provides the use of the enterically absorbed hemin prepared by the preparation method in the preparation of a nutritional enhancer for iron supplementation.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1: Preparation of intestinal absorption hemin by hemin and methyl-β-cyclodextrin

[0054] (1) 43 mg of hemin was mixed with 4 mL of 13 wt% ammonia water to obtain a hemin solution; 172 mg of methyl-β-cyclodextrin was mixed with 15 mL of water to obtain a methyl-β-cyclodextrin aqueous solution;

[0055] (2) mixing the hemin solution obtained in step (1) with a methyl-β-cyclodextrin aqueous solution to obtain a mixed solution;

[0056] (3) stirring the mixed solution obtained in step (2) at 40° C. for 3 h to allow inclusion complexation to occur, and then drying in an oven at 60° C. for 5 h to obtain a dry product;

[0057] (4) The dried product obtained in step (3) is washed twice with acidic acetone, and after the acidic acetone is volatilized, the dried product is dried in the oven as described in step (3) again to obtain intestinal absorption hemin.

[0058] Example 2: Preparation of intestinal absorption hemin by hemin and hydroxypropyl-β-cyclodextrin

[0059] (1) 43 mg of hemin was mixed with 4 mL of 13 wt% ammonia water to obtain a hemin solution; 203 mg of hydroxypropyl-β-cyclodextrin was mixed with 15 mL of water to obtain a hydroxypropyl-β-cyclodextrin aqueous solution;

[0060] (2) mixing the hemin solution obtained in step (1) with a hydroxypropyl-β-cyclodextrin aqueous solution to obtain a mixed solution;

[0061] (3) stirring the mixed solution obtained in step (2) at 40° C. for 3 h to allow inclusion complexation to occur, and then drying in an oven at 60° C. for 5 h to obtain a dry product;

[0062] (4) The dried product obtained in step (3) is washed twice with acidic acetone, and after the acidic acetone is volatilized, the dried product is dried in the oven as described in step (3) again to obtain intestinal absorption hemin.

[0063] Example 3: Preparation of intestinal absorption hemin by hemin and sulfobutyl-β-cyclodextrin

[0064] (1) 43 mg of hemin was mixed with 4 mL of 13 wt% ammonia water to obtain a hemin solution; 191 mg of sulfobutyl-β-cyclodextrin was mixed with 15 mL of water to obtain a sulfobutyl-β-cyclodextrin aqueous solution;

[0065] (2) mixing the hemin solution obtained in step (1) with a sulfobutyl-β-cyclodextrin aqueous solution to obtain a mixed solution;

[0066] (3) stirring the mixed solution obtained in step (2) at 40° C. for 3 h to allow inclusion complexation to occur, and then drying in an oven at 60° C. for 5 h to obtain a dry product;

[0067] (4) The dried product obtained in step (3) is washed twice with acidic acetone, and after the acidic acetone is volatilized, the dried product is dried in the oven as described in step (3) again to obtain intestinal absorption hemin.

[0068] Example 4: Preparation of intestinal absorption hemin by hemin and γ-cyclodextrin

[0069] (1) 43 mg of hemin was mixed with 4 mL of 13 wt% ammonia water to obtain a hemin solution; 171 mg of γ-cyclodextrin was mixed with 15 mL of water to obtain a γ-cyclodextrin aqueous solution;

[0070] (2) mixing the hemin solution obtained in step (1) with an aqueous solution of γ-cyclodextrin to obtain a mixed solution;

[0071] (3) stirring the mixed solution obtained in step (2) at 40° C. for 3 h to allow inclusion complexation to occur, and then drying in an oven at 60° C. for 5 h to obtain a dry product;

[0072] (4) The dried product obtained in step (3) is washed twice with acidic acetone, and after the acidic acetone is volatilized, the dried product is dried in the oven as described in step (3) again to obtain intestinal absorption hemin.

[0073] Comparative Example 1: Preparation of intestinal absorption hemin by hemin and β-cyclodextrin

[0074] (1) 43 mg of hemin was mixed with 4 mL of 13 wt% ammonia water to obtain a hemin solution; 150 mg of β-cyclodextrin (chemical formula: C 42 H 70 O 35 , relative molecular mass is 1134.98) was mixed with 15 mL of water to obtain a β-cyclodextrin aqueous solution;

[0075] (2) mixing the hemin solution obtained in step (1) with an aqueous solution of β-cyclodextrin to obtain a mixed solution;

[0076] (3) stirring the mixed solution obtained in step (2) at 40° C. for 3 h to allow inclusion complexation to occur, and then drying in an oven at 60° C. for 5 h to obtain a dry product;

[0077] (4) The dried product obtained in step (3) is washed twice with acidic acetone, and after the acidic acetone is volatilized, the dried product is dried in the oven as described in step (3) again to obtain intestinal absorption hemin.

[0078] Experimental Example 1: Determination of the encapsulation capacity of cyclodextrin for hemin

[0079] The encapsulation efficiency of the cyclodextrins described in Examples 1 to 4 and Comparative Example 1 for hemin was determined by ultraviolet spectrophotometry. The specific method is as follows: 20 mg of hemin was diluted to 100 mL with 0.1 mol / L sodium hydroxide to obtain a hemin solution, and then 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the hemin solution were diluted to 100 mL with 0.1 mol / L sodium hydroxide, respectively, and 0.1 mol / L sodium hydroxide was used as a blank control. The ultraviolet spectrophotometer was used to measure the encapsulation efficiency of the hemin. 385nm The absorbance of each solution was measured to obtain the hemin standard curve y = 90.65x-0.0049 (R 2 =0.9999). 185 mg of the intestinal absorption hemin prepared in Examples 1 to 4 and Comparative Example 1 was diluted to 100 mL with 0.1 mol / L sodium hydroxide to obtain an intestinal absorption hemin solution. 1 mL of the intestinal absorption hemin solution was diluted to 50 mL with 0.1 mol / L sodium hydroxide. The intestinal absorption hemin solution was measured by ultraviolet spectrophotometer at A. 385nm The absorbance was measured at 40 °C, and the concentration and encapsulation efficiency of hemin were calculated according to the hemin standard curve. The results are shown in Table 1 and Figure 1 .

[0080] The formula for calculating the encapsulation efficiency is:

[0081] Where: W 包Hmn is the content of hemin absorbed by intestine, W 总Hmn is the total content of hemin added.

[0082] The results show that there is no significant difference in the encapsulation efficiency of hemin by methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, γ-cyclodextrin and β-cyclodextrin, that is, the cyclodextrins described in Examples 1 to 4 and the β-cyclodextrin described in Comparative Example 1 have the same encapsulation capacity for hemin.

[0083] Experimental Example 2: Determination of the solubilization ability of cyclodextrin on hemin

[0084] The solubilization multiple of hemin by the cyclodextrin described in Examples 1 to 4 and Comparative Example 1 was determined by the saturated solution method. The specific method is as follows: the intestinal absorption hemin obtained in Examples 1 to 4 and Comparative Example 1 was used as a raw material to prepare an intestinal absorption hemin saturated solution, and the original hemin saturated solution was used as a control. 10 mL of each was vortexed and placed in the dark at room temperature for 24 h to fully dissolve. After centrifugation at 5000 rpm for 5 min, the supernatant was diluted 25 times with 0.1 mol / L sodium hydroxide, and the supernatant was measured by ultraviolet spectrophotometer at A 385nm The absorbance was measured at 40 °C and the solubility and solubilization factor were calculated. The results are shown in Table 1 and Figures 2-3 .

[0085] The solubility calculation formula is:

[0086] Where: m Hmn is the content of dissolved hemin, and v is the volume of the solution.

[0087] The calculation formula of solubilization multiple is:

[0088] Where: S 包Hmn is the solubility of hemin absorbed by the intestine, S 原Hmn is the solubility of prohemin.

[0089] Table 1 Encapsulation and solubilization capacity of intestinal absorption of hemin

[0090] Examples and Comparative Examples Cyclodextrin Types Encapsulation rate (%) Solubilization multiple (times) Example 1 Methyl-β-cyclodextrin 70.24％ 111.22 Example 2 Hydroxypropyl-β-cyclodextrin 71.67％ 200.57 Example 3 Sulfonbutyl-β-cyclodextrin 68.17％ 223.29 Example 4 γ-Cyclodextrin 72.11％ 240.60 Comparative Example 1 β-Cyclodextrin 70.91％ 30.90

[0091] The results show that compared with the control, the color of the enteric absorption hemin saturated solution prepared with the enteric absorption hemin obtained in Examples 1 to 4 as the raw material is significantly darker, and the solubilization multiple is significantly increased; while the color of the enteric absorption hemin saturated solution prepared with the enteric absorption hemin obtained in Comparative Example 1 as the raw material is darker, and the solubilization multiple is increased to a certain extent. This shows that methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin and γ-cyclodextrin can all improve the solubility of hemin, among which γ-cyclodextrin has the strongest solubilization ability for hemin. In contrast, β-cyclodextrin has a smaller cavity structure and lower water solubility, so its effect on improving the solubility of hemin is not outstanding.

[0092] Experimental Example 3: Infrared characteristics of intestinal absorption of hemin

[0093] The intestinal absorption hemin prepared in Example 4 was characterized by infrared spectroscopy using iS10 FT-IR spectrometer (Thermo Nicolet Corporation, USA). The characterization results are shown in the figure. Figure 4 shown.

[0094] The results showed that the intestinal absorption of hemin prepared in Example 4 was 1700 cm -1 The C=O stretching vibration peak at 1500cm -1 The latter mainly shows the characteristic peaks of γ-cyclodextrin, which is completely different from the characteristic peaks shown by the physical mixture of γ-cyclodextrin and hemin, indicating that hemin has been embedded in the cavity of γ-cyclodextrin, and its molecular vibration is restricted and cannot fully show its original infrared characteristics.

[0095] Experimental Example 4: X-ray diffraction characteristics of intestinal absorption of hemin

[0096] The intestinal absorption hemin prepared in Example 4 was characterized by X-ray diffraction using a D8 ADVANCE X-ray diffractometer (Bruker Corporation, Germany). The characterization results are shown in FIG. Figure 5 shown.

[0097] The results showed that the characteristic peaks of the intestinal absorbed hemin prepared in Example 4 disappeared, the diffraction peaks decreased, and the crystallinity was lost.

[0098] Example 5: Stability and bioavailability of intestinal absorption hemin in the gastrointestinal tract

[0099] The in vitro simulated gastrointestinal digestion method was used to determine the stability and bioavailability of the intestinal absorbed hemin prepared in Example 4 in the gastrointestinal tract. The specific method was as follows: 2 mg of the intestinal absorbed hemin prepared in Example 4 and 2 mg of the original hemin were taken as a control, respectively dissolved in 5 mL of water, and ultrasonically dispersed for 5 minutes, then 10 mL of artificial gastric juice was added and vortexed, and then placed in a constant temperature oscillator at 37°C and shaken at 160 rpm for 2 hours to obtain gastric digestion products.

[0100] Take 15 mL of gastric digestion product, adjust the pH to 7.0 with 2 mol / L sodium hydroxide, add 10 mL of artificial intestinal fluid and vortex, then place it in a 37°C constant temperature oscillator and shake at 160 rpm for 4 h to obtain gastrointestinal digestion product.

[0101] 2 mL of gastric digestion product and gastrointestinal digestion product were taken to determine the hemin content and evaluate the stability of hemin; 2 mL of gastric digestion product and gastrointestinal digestion product were taken, centrifuged at 9754 rpm for 10 minutes, and the supernatant was collected to determine the hemin content in the aqueous phase and evaluate the bioavailability of hemin. The results are as follows Figure 6 shown.

[0102] The results showed that after simulated digestion, more than 90% of the original hemin and intestinal absorbed hemin were still not degraded, indicating that hemin has high stability during the entire gastrointestinal digestion process.

[0103] After the original hemin is digested by artificial gastric juice, the hemin content in the aqueous phase is only 0.29%, and after further digestion by artificial intestinal juice, the hemin content in the aqueous phase is 9.52%, indicating that the bioavailability of the original hemin after being ingested by the human body is extremely low; and after the intestinal-absorbed hemin is digested by artificial gastric juice, the hemin content in the aqueous phase is only 0.46%, but after further digestion by artificial intestinal juice, the hemin content in the aqueous phase reaches 77.24%, indicating that cyclodextrin can significantly improve the bioavailability of hemin.

[0104] As can be seen from the above embodiments, the present invention provides an enterically absorbed hemin and its preparation method and application. The enterically absorbed hemin prepared according to the method of the present invention has the advantages of strong water solubility and stability, high bioavailability after gastrointestinal digestion, and γ-cyclodextrin has the strongest solubilization ability for hemin.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An intestinal absorption hemin, characterized in that: Includes hemin and gamma-cyclodextrin; The molar ratio of hemin to cyclodextrin is 1:2; The preparation method of intestinal absorption hemin comprises the following steps: (1) mixing hemin with ammonia water to obtain a hemin solution; mixing cyclodextrin with water to obtain a cyclodextrin aqueous solution; (2) mixing the hemin solution with the cyclodextrin aqueous solution to obtain a mixed solution; (3) stirring the mixed solution and drying it for 5 hours to obtain intestinal absorption hemin; The molar volume ratio of hemin to ammonia water in step (1) is 0.016 mol:1 L; The molar volume ratio of the cyclodextrin to water is 0.008:1L; The initial concentration of the ammonia water is 13wt%; The volume ratio of the hemin solution to the cyclodextrin aqueous solution in step (2) is 4:15; The stirring temperature in step (3) is 40° C. and the stirring time is 3 h.

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