Intestinal absorption of hematin chloride
By preparing intestinal-absorbable heme chloride with a molar ratio of heme chloride to cyclodextrin of 1-2:1-2, the problem of poor water solubility of heme chloride is solved, and its efficient absorption and stability in the intestine are achieved, making it suitable as an iron supplement fortifier.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-14
- Publication Date
- 2026-03-20
AI Technical Summary
Heme chloride has poor water solubility, which limits its practical application, especially its low intestinal absorption rate as an iron fortifier and anti-anemia drug.
Intestinal-absorbable heme chloride was prepared by solution stirring using a molar ratio of heme chloride to cyclodextrin of 1–2:1–2, and the water solubility and stability were improved by utilizing the encapsulation effect of cyclodextrin.
It significantly improves the water solubility and bioavailability of heme chloride, enabling it to be used as a raw material for iron supplementation and enhancing its stability and absorption in the intestine.
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Abstract
Description
[0001] This application is a divisional application. The original application has the application number 202311721161.5, the application date is December 14, 2023, and the invention title is: An intestinal absorption heme chloride and its preparation method and application. Technical Field
[0002] This invention relates to the field of biomedical technology, and in particular to an intestinal-absorbable heme chloride. Background Technology
[0003] Cyclodextrin is a cyclic oligosaccharide with a hydrophobic inner cavity and a hydrophilic outer wall, exhibiting excellent loading properties for hydrophobic molecules. Many natural active ingredients and drug molecules suffer from poor water solubility and low tolerance to stimuli such as light, heat, and pH, resulting in low utilization rates in functional foods or pharmaceuticals. Studies have found that encapsulating these functionally specific active molecules within the cavity of cyclodextrin can effectively enhance their water solubility and stability, thereby further improving their shelf life and bioavailability.
[0004] Heme chloride is an in vitro purified form of natural heme, typically obtained from heme extracted from animal blood. The process involves first reacting protonated iron with Fe... 3+ Coordination, Fe 3+ It then combines with Cl- to form heme chloride. Studies have shown that heme chloride is currently the most readily absorbed bioavailable iron, and can be used as an iron fortifier and anti-anemia drug. However, heme chloride has extremely poor water solubility, which greatly limits its practical application. Summary of the Invention
[0005] The purpose of this invention is to provide an intestinal absorbable heme chloride, its preparation method and application, so as to solve the problems of poor water solubility, low intestinal absorption rate and limited application of related products in the prior art.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides an intestinal-absorbable heme chloride, comprising heme chloride and cyclodextrin;
[0008] The molar ratio of heme chloride 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-absorbable heme chloride, comprising the following steps:
[0011] (1) mixing hematin chloride with ammonia water to obtain a hematin chloride solution; mixing cyclodextrin with water to obtain a cyclodextrin aqueous solution;
[0012] (2) mixing the hematin chloride solution with the cyclodextrin aqueous solution to obtain a mixed solution;
[0013] (3) stirring the mixed solution and drying for 4-6 hours to obtain enteric absorption hematin chloride.
[0014] Preferably, the molar volume ratio of the hematin chloride to the ammonia water in step (1) is 0.01-0.02 mol:1 L;
[0015] The molar volume ratio of the cyclodextrin to the water is 0.01-0.02 mol:2 L;
[0016] The initial concentration of the ammonia water is 9-15 wt%.
[0017] Preferably, the volume ratio of the hematin chloride solution to the cyclodextrin aqueous solution in step (2) is 1-5:10-20.
[0018] Preferably, the temperature of the stirring in step (3) is 30-50℃, and the stirring time is 2-4 hours.
[0019] Preferably, the drying in step (3) is oven drying, and the temperature of the oven drying is 40-80℃.
[0020] The application also provides the use of the enteric absorption hematin chloride in the preparation of a nutritional fortifier for iron supplementation.
[0021] The application also provides the use of the enteric absorption hematin chloride prepared by the preparation method in the preparation of a nutritional fortifier for iron supplementation.
[0022] The application has the following technical effects and advantages:
[0023] The application is based on the encapsulation of active molecules by water-soluble carriers, and different types of cyclodextrin are used to encapsulate hematin chloride to prepare enteric absorption hematin chloride by a solution stirring method. The preparation method is simple, fast, green and pollution-free. The solubility, stability and bioavailability of the enteric absorption hematin chloride prepared by the method are significantly improved, and the enteric absorption hematin chloride can be used as a raw material for the preparation of a nutritional fortifier for iron supplementation. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 The encapsulation rate of the enteric absorption hematin chloride prepared for Examples 1-4 and Comparative Example 1;
[0025] Figure 2Water solubility of the enteric absorption hematin saturated solution prepared by using the enteric absorption hematin of Examples 1 to 4 and Comparative Example 1 and the original hematin saturated solution;
[0026] Figure 3 Solubility enhancement fold of the enteric absorption hematin prepared by Examples 1 to 4 and Comparative Example 1;
[0027] Figure 4 Fourier infrared spectrum of the enteric absorption hematin prepared by Example 4;
[0028] Figure 5 X-ray diffraction spectrum of the enteric absorption hematin prepared by Example 4;
[0029] Figure 6 Stability and bioavailability of the enteric absorption hematin prepared by Example 4 and the original hematin in the process of gastrointestinal digestion. DETAILED DESCRIPTION
[0030] The present application provides an enteric absorption hematin comprising hematin and cyclodextrin.
[0031] The molar ratio of the hematin to the cyclodextrin is 1 to 2: 1 to 2, preferably 1:2.
[0032] In the present application, the chemical formula of the hematin is C 34 H 32 ClFeN4O4, and the 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 , and 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 , and 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, and the relative molecular mass is 1451.29.
[0037] The chemical formula of the γ-cyclodextrin is C 48 H 80 O 40 The relative molecular mass is 1297.11.
[0038] The application further provides a preparation method of the intestinal absorption hematin chloride, comprising the following steps:
[0039] (1) mixing hematin chloride with ammonia water to obtain a hematin chloride solution; mixing cyclodextrin with water to obtain a cyclodextrin aqueous solution;
[0040] (2) mixing the hematin chloride solution with the cyclodextrin aqueous solution to obtain a mixed solution;
[0041] (3) stirring the mixed solution and drying for 4-6 hours to obtain intestinal absorption hematin chloride.
[0042] In the application, the molar volume ratio of the hematin chloride to the ammonia water in step (1) is 0.01-0.02 mol:1 L, preferably 0.0165 mol:1 L;
[0043] The molar volume ratio of the cyclodextrin to water is 0.01-0.02 mol:2 L, preferably 0.0176 mol:2 L;
[0044] The initial concentration of the ammonia water is 9-15 wt%, preferably 13 wt%.
[0045] In the application, the volume ratio of the hematin chloride solution to the cyclodextrin aqueous solution in step (2) is 1-5:10-20, preferably 4:15.
[0046] In the application, the temperature of the stirring in step (3) is 30-50 DEG C, preferably 40 DEG C; the stirring time is 2-4 hours, preferably 3 hours.
[0047] In the application, the drying in step (3) is oven drying, the temperature of the oven drying is 40-80 DEG C, preferably 60 DEG C; the oven drying time is 4-6 hours, preferably 5 hours.
[0048] In the application, the drying in step (3) further comprises washing, the washing agent used in the washing is acidic acetone, the washing frequency is 1-3 times, preferably 2 times.
[0049] In the application, the drying after the washing is oven drying, the temperature of the oven drying is 40-80 DEG C, preferably 60 DEG C; the oven drying time is 4-6 hours, preferably 5 hours.
[0050] The application also provides the use of the intestinal absorption hemin in the preparation of a nutritional supplement for iron supplement.
[0051] The application also provides the use of the intestinal absorption hemin prepared by the preparation method in the preparation of a nutritional supplement for iron supplement.
[0052] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.
[0053] Example 1: preparation of intestinal absorption hemin from 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) the hemin solution obtained in step (1) was mixed with the methyl-β-cyclodextrin aqueous solution to obtain a mixed solution;
[0056] (3) the mixed solution obtained in step (2) was stirred at 40°C for 3 h to occur the inclusion, and then was oven dried at 60°C for 5 h to obtain a dry product;
[0057] (4) the dry product obtained in step (3) was washed with acidic acetone for 2 times, and after the acidic acetone was volatilized, the oven drying in step (3) was performed again to obtain the intestinal absorption hemin.
[0058] Example 2: preparation of intestinal absorption hemin from 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) the hemin solution obtained in step (1) was mixed with the hydroxypropyl-β-cyclodextrin aqueous solution to obtain a mixed solution;
[0061] (3) the mixed solution obtained in step (2) was stirred at 40°C for 3 h to occur the inclusion, and then was oven dried at 60°C for 5 h to obtain a dry product;
[0062] (4) the dry product obtained in step (3) was washed with acidic acetone for 2 times, and after the acidic acetone was volatilized, the oven drying in step (3) was performed again to obtain the intestinal absorption hemin.
[0063] Example 3: Preparation of enteric absorbed hematin chloride with sulfobutyl-β-cyclodextrin
[0064] (1) 43 mg of hematin chloride was mixed with 4 mL of 13 wt% ammonia water to obtain a hematin chloride solution; 191 mg of sulfobutyl-β-cyclodextrin was mixed with 15 mL of water to obtain a sulfobutyl-β-cyclodextrin aqueous solution;
[0065] (2) The hematin chloride solution obtained in step (1) was mixed with the sulfobutyl-β-cyclodextrin aqueous solution to obtain a mixed solution;
[0066] (3) The mixed solution obtained in step (2) was stirred at 40°C for 3 h to occur inclusion, and then oven dried at 60°C for 5 h to obtain a dry product;
[0067] (4) The dry product obtained in step (3) was washed with acidic acetone twice, and after the acidic acetone was volatilized, oven drying as described in step (3) was performed again to obtain enteric absorbed hematin chloride.
[0068] Example 4: Preparation of enteric absorbed hematin chloride with γ-cyclodextrin
[0069] (1) 43 mg of hematin chloride was mixed with 4 mL of 13 wt% ammonia water to obtain a hematin chloride solution; 171 mg of γ-cyclodextrin was mixed with 15 mL of water to obtain a γ-cyclodextrin aqueous solution;
[0070] (2) The hematin chloride solution obtained in step (1) was mixed with the γ-cyclodextrin aqueous solution to obtain a mixed solution;
[0071] (3) The mixed solution obtained in step (2) was stirred at 40°C for 3 h to occur inclusion, and then oven dried at 60°C for 5 h to obtain a dry product;
[0072] (4) The dry product obtained in step (3) was washed with acidic acetone twice, and after the acidic acetone was volatilized, oven drying as described in step (3) was performed again to obtain enteric absorbed hematin chloride.
[0073] Comparative Example 1: Preparation of enteric absorbed hematin chloride with β-cyclodextrin
[0074] (1) 43 mg of hematin chloride was mixed with 4 mL of 13 wt% ammonia water to obtain a hematin chloride solution; 150 mg of β-cyclodextrin (chemical formula is 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) Mix the heme chloride solution obtained in step (1) with the β-cyclodextrin aqueous solution to obtain a mixed solution;
[0076] (3) Stir the mixed solution obtained in step (2) at 40°C for 3 hours to achieve inclusion effect, and then dry it in an oven at 60°C for 5 hours to obtain the dried product;
[0077] (4) The dried product obtained in step (3) is washed twice with acidic acetone. After the acidic acetone evaporates, it is dried in the oven as described in step (3) again to obtain intestinal heme chloride.
[0078] Experimental Example 1: Determination of the encapsulation ability of cyclodextrin for heme chloride
[0079] The encapsulation efficiency of cyclodextrin for heme chloride described in Examples 1-4 and Comparative Example 1 was determined by ultraviolet spectrophotometry. Specifically, 20 mg of heme chloride was diluted to 100 mL with 0.1 mol / L sodium hydroxide to obtain a heme chloride solution. Then, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the heme chloride solution were diluted to 100 mL with 0.1 mol / L sodium hydroxide, respectively, using 0.1 mol / L sodium hydroxide as a blank control. The encapsulation efficiency of cyclodextrin for heme chloride in Examples 1-4 and Comparative Example 1 was determined by ultraviolet spectrophotometry. 385nm The absorbance of each solution was measured to obtain the heme chloride standard curve: y = 90.65x - 0.0049 (R²). 2 =0.9999). Take 185 mg of the intestinal-absorbable heme chloride prepared in Examples 1-4 and Comparative Example 1, and dilute to 100 mL with 0.1 mol / L sodium hydroxide to obtain an intestinal-absorbable heme chloride solution. Then take 1 mL of the intestinal-absorbable heme chloride solution and dilute to 50 mL with 0.1 mol / L sodium hydroxide. Use an ultraviolet spectrophotometer to analyze the solution at A. 385nm The absorbance was measured, and the concentration and encapsulation efficiency of heme chloride were calculated based on the heme chloride standard curve. The results are shown in Table 1 and... Figure 1 .
[0080] The formula for calculating the encapsulation ratio is:
[0081] In the formula: W 包Hmn The amount of heme chloride absorbed in the intestine, W 总Hmn This represents the total amount of heme chloride added.
[0082] The results showed that there was no significant difference in the encapsulation efficiency of heme chloride 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 had the same encapsulation ability for heme chloride.
[0083] Experimental Example 2: Determination of solubilizing ability of cyclodextrin on hematin chloride
[0084] The solubilizing multiples of the cyclodextrins on hematin chloride described in Examples 1 to 4 and Comparative Example 1 were determined by saturated solution method. Specifically, the enteric absorbed hematin chloride obtained in Examples 1 to 4 and Comparative Example 1 was used as raw material to prepare enteric absorbed hematin chloride saturated solution, and the original hematin chloride saturated solution was used as control. Each 10 mL was vortexed and placed at room temperature for 24 h to fully dissolve in the dark. After centrifugation at 5000 rpm for 5 min, the supernatant was diluted 25 times with 0.1 mol / L sodium hydroxide, and the absorbance was determined at A 385nm Figures 2-3 .
[0085] The solubility calculation formula is:
[0086] In the formula, m Hmn is the content of dissolved hematin chloride, and v is the volume of the solution.
[0087] The solubilizing multiple calculation formula is:
[0088] In the formula, S 包Hmn is the solubility of enteric absorbed hematin chloride, and S 原Hmn is the solubility of the original hematin chloride.
[0089] Table 1: Embedding ability and solubilizing ability of enteric absorbed hematin chloride
[0090] Examples and comparative examples Cyclodextrin species Encapsulation efficiency (%) Solubilisation fold Example 1 Methyl-β-cyclodextrin 70.24％ 111.22 Example 2 Hydroxypropyl-β-cyclodextrin 71.67％ 200.57 Example 3 Sulfobutyl-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 enteric absorbed hematin chloride saturated solution prepared from the enteric absorbed hematin chloride obtained in Examples 1 to 4 has a significantly darker color and a significantly increased solubilizing multiple; while the enteric absorbed hematin chloride saturated solution prepared from the enteric absorbed hematin chloride obtained in Comparative Example 1 has a darker color and a certain increase in solubilizing multiple. This indicates that methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin and γ-cyclodextrin can all improve the solubility of hematin chloride, among which γ-cyclodextrin has the strongest solubilizing ability on hematin chloride. In comparison, β-cyclodextrin has a small cavity structure and low water solubility, which makes its improvement effect on the solubility of hematin chloride not outstanding.
[0092] Experimental Example 3: Infrared characteristics of enteric absorbed hematin chloride
[0093] The intestinal absorption hematin chloride prepared in Example 4 was characterized by infrared spectroscopy using an iS10 FT-IR spectrometer (Thermo Nicolet Corporation, USA), and the characterization results are shown in Figure 4. Figure 4
[0094] The results show that the C=O stretching vibration peak at 1700 cm -1 of the intestinal absorption hematin chloride prepared in Example 4 disappeared, and the characteristic peaks mainly showed γ-cyclodextrin characteristics after 1500 cm -1 , which were completely different from those of the physical mixture of γ-cyclodextrin and hematin chloride, indicating that the hematin chloride was embedded in the cavity of γ-cyclodextrin, and the molecular vibration was restricted and could not fully show the original infrared characteristics.
[0095] Experimental Example 4: X-ray diffraction characteristics of intestinal absorption hematin chloride
[0096] The intestinal absorption hematin chloride prepared in Example 4 was characterized by X-ray diffraction spectroscopy using a D8 ADVANCE X-ray diffractometer (Bruker Corporation, Germany), and the characterization results are shown in Figure 5. Figure 5
[0097] The results show that the characteristic peaks of the intestinal absorption hematin chloride prepared in Example 4 disappeared, the diffraction peaks decreased, and the crystalline characteristics were lost.
[0098] Example 5: Stability and bioavailability of intestinal absorption hematin chloride in the gastrointestinal tract
[0099] The stability and bioavailability of the intestinal absorption hematin chloride prepared in Example 4 in the gastrointestinal tract were determined by in vitro simulated gastrointestinal digestion method. The specific method was as follows: 2 mg of the intestinal absorption hematin chloride prepared in Example 4 was dissolved in 5 mL of water, and 2 mg of original hematin chloride was used as a control. The mixture was ultrasonically dispersed for 5 min, then 10 mL of artificial gastric juice was added and vortexed, and then placed in a 37°C constant temperature oscillator at 160 rpm for 2 h to obtain the gastric digestion product.
[0100] 15 mL of the gastric digestion product was adjusted to pH 7.0 with 2 mol / L sodium hydroxide, 10 mL of artificial intestinal juice was added and vortexed, and then placed in a 37°C constant temperature oscillator at 160 rpm for 4 h to obtain the gastrointestinal digestion product.
[0101] Take 2 mL of the gastric digestion product and the gastrointestinal digestion product to determine the content of hematin chloride, and evaluate the stability of hematin chloride; take 2 mL of the gastric digestion product and the gastrointestinal digestion product, centrifuge at 9754 rpm for 10 min, collect the supernatant, determine the content of hematin chloride in the aqueous phase, and evaluate the bioavailability of hematin chloride. The determination results are shown in Table 1. Figure 6
[0102] The results show that more than 90% of the original hematin chloride and the intestinal absorption hematin chloride are not degraded after simulated digestion, indicating that the stability of hematin chloride in the whole gastrointestinal digestion process is high.
[0103] After the original hematin chloride is digested by the artificial gastric juice, the content of hematin chloride in the aqueous phase is only 0.29%, and after the artificial intestinal juice digestion, the content of hematin chloride in the aqueous phase is 9.52%, indicating that the bioavailability of the original hematin chloride is very low after being taken into the human body; although the content of hematin chloride in the aqueous phase is also only 0.46% after the intestinal absorption hematin chloride is digested by the artificial gastric juice, but after the artificial intestinal juice digestion, the content of hematin chloride in the aqueous phase is 77.24%, indicating that cyclodextrin can significantly improve the bioavailability of hematin chloride.
[0104] From the above examples, it can be seen that the present application provides an intestinal absorption hematin chloride and a preparation method and application thereof. The intestinal absorption hematin chloride prepared according to the method of the present application has the advantages of strong water solubility and stability, high bioavailability after gastrointestinal digestion, and the solubilizing ability of γ-cyclodextrin to hematin chloride is the strongest.
[0105] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. An intestinal-absorbable heme chloride, characterized in that, Including heme chloride and γ-cyclodextrin; The chemical formula of the γ-cyclodextrin is C 48 H 80 O 40 , relative molecular mass 1297.11; The method for preparing intestinal-absorbable heme chloride includes the following steps: (1) Mix 43 mg of heme chloride with 4 mL of ammonia water to obtain a heme chloride solution; mix 171 mg of γ-cyclodextrin with 15 mL of water to obtain a cyclodextrin aqueous solution; (2) The heme chloride solution is mixed with the cyclodextrin aqueous solution to obtain a mixed solution; (3) The mixed solution was stirred at 40°C for 3 hours to induce an inclusion reaction, and then dried in an oven at 60°C for 5 hours to obtain the dried product; (4) Wash the dried product obtained in step (3) twice with acidic acetone. After the acidic acetone evaporates, dry it again in the oven as described in step (3) to obtain intestinal-absorbable heme chloride. The initial concentration of the ammonia solution was 13 wt%.
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