Method and preparation for maintaining activity of allicin

Abstract

The invention relates to the technical field of food, health care products and medicines, in particular to a method and a preparation for keeping the activity of allicin. The preparation has a core-shell structure, and comprises an active core containing alliin, alliinase, tocopherol and phytic acid, a pH responsive enteric polymer inner layer wrapping the active core, and a hydrophobic protective outer layer wrapping the inner layer. According to the invention, an on-site preparation and on-site use strategy is designed, that is, alliin and alliinase are respectively protected and are mixed to generate allicin during use, and tocopherol is used as a free radical trapping agent and phytic acid is used as a metal ion chelating agent to carry out immediate protection on allicin generation, so that the problems that allicin is easy to decompose and difficult to store are effectively solved; the method is suitable for preparing health-care foods, medicines and animal feed additives.

Description

Technical Field

[0001] This invention relates to the fields of food, health products and pharmaceutical technology, and specifically to a method and preparation for maintaining the activity of allicin. Background Technology

[0002] Allicin (diallyl thiosulfinate) is the main bioactive substance in garlic, possessing a variety of physiological functions including broad-spectrum antibacterial, antiviral, antioxidant, antitumor, and immune-enhancing effects. However, allicin is chemically extremely unstable, easily affected by temperature, light, pH, metal ions, and its own volatility, rapidly degrading into inert sulfides, leading to a significant loss of its biological activity. This instability severely restricts the high-value-added application of allicin in functional foods, health products, and pharmaceuticals.

[0003] Currently, common stabilization techniques include microencapsulation (such as cyclodextrin encapsulation and spray drying), liposome encapsulation, and emulsification. However, these methods generally have the following drawbacks: (1) Incomplete encapsulation: During the encapsulation process, allicin is exposed to adverse environments (such as high temperature and aqueous media) and partially degrades; (2) Insufficient protection: Single packaging materials are difficult to resist complex inactivation factors, such as gastric acid decomposition and enzymatic hydrolysis; (3) Lack of active stabilization mechanism: Relying only on physical isolation, without regulating the internal microenvironment of the formulation, it is impossible to remove factors that accelerate degradation (such as free radicals and metal ions).

[0004] Therefore, developing a method and formulation that can systematically and multi-level protect the activity of allicin is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a strategy for on-the-spot production and use, namely, separately protecting alliin and alliinase, and mixing them to generate allicin upon use. However, this approach faces two major technical challenges: First, how to ensure absolute isolation between alliin and the enzyme during storage, while ensuring precise and efficient contact and reaction at the moment of use; second, because allicin has the highest activity but is also the most unstable at the moment of generation, it will immediately undergo side reactions such as disproportionation and polymerization, resulting in inactivation. Therefore, it is necessary to protect it immediately at the generation point (reaction microenvironment) to prevent degradation during generation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a formulation for maintaining the activity of allicin, the formulation having a core-shell structure, comprising:

[0008] (1) The active core contains physically isolated alliin and alliinase, as well as tocopherol as a free radical scavenger and phytic acid as a metal ion chelator.

[0009] (2) A pH-responsive enteric polymer inner layer encapsulating the active core, wherein the inner layer remains structurally stable under pH ≤ 5.5 conditions and undergoes swelling or dissolution and release under pH ≥ 6.5 conditions;

[0010] (3) A hydrophobic protective outer layer covering the inner layer;

[0011] In this process, alliin and alliinase react in situ to generate allicin under the triggering conditions of the pH-responsive enteric polymer inner layer during the use of the formulation or after entering the intestine.

[0012] Furthermore, the alliin is contained in a first carrier, and the alliinase is contained in a second carrier, wherein the first carrier and the second carrier are physically mixed within the active core but isolated from each other.

[0013] Furthermore, the first carrier is a lipid matrix, and the second carrier is a hydrophilic or disintegrating solid carrier.

[0014] Furthermore, the combination of tocopherol and phytic acid, without significantly inhibiting alliinase activity, synergistically inhibits the sulfur-oxygen free radical intermediates and metal ion-catalyzed oxidation reactions generated during in-situ generation, thereby improving the stability of the in-situ generated allicin.

[0015] Furthermore, the tocopherol is a fat-soluble α-tocopherol, and the phytic acid is inositol hexaphosphate.

[0016] Furthermore, the mass ratio of tocopherol to phytic acid is 1:(0.2-0.8).

[0017] Furthermore, both tocopherol and phytic acid coexist in the reaction microenvironment for the in-situ generation of allicin within the active core, and the combination of tocopherol and phytic acid prevents the allicin generated in situ from undergoing disproportionation, polymerization, or further oxidative inactivation shortly after generation. However, when the free radical scavenger is replaced with tea polyphenols or ascorbic acid, or when the metal ion chelating agent is replaced with ethylenediaminetetraacetic acid, the improvement in the stability of the in-situ allicin generated under the same conditions is not significant, or it inhibits the activity of alliinase.

[0018] Furthermore, the pH-responsive enteric polymer inner layer material is selected from at least one of sodium alginate, pectin, hydroxypropyl methylcellulose succinate (HPMCAS), and polyacrylic acid resin (Eudragit® L / S series).

[0019] Furthermore, the hydrophobic protective outer layer material is selected from at least one of glyceryl monostearate, chitosan-fatty acid grafts, and polylactic acid-glycolic acid copolymer (PLGA).

[0020] In a second aspect, the present invention provides a method for preparing the formulation as described herein, comprising the following steps:

[0021] S1: Allicin, tocopherol and phytic acid are dispersed or dissolved in a molten lipid matrix, and then solidified and pulverized to obtain allicin lipid particles; allicinase is mixed with a solid carrier to obtain enzyme particles; the allicin lipid particles and enzyme particles are physically mixed to obtain an active core mixture;

[0022] S2: A pH-responsive enteric polymer layer and a hydrophobic material layer are sequentially coated on the surface of the active core mixture;

[0023] S3: Perform low-temperature drying to obtain a solid dosage form.

[0024] Furthermore, the dispersion or dissolution described in step S1 is carried out under light-protected conditions at a temperature below 40°C.

[0025] Furthermore, the inlet temperature of the low-temperature drying process in step S3 is below 50°C.

[0026] Furthermore, in step S2, when coating the pH-responsive enteric polymer layer, an ionic crosslinking method is used for curing.

[0027] In a third aspect, the present invention provides the use of the formulations described herein in the preparation of health foods, pharmaceuticals or animal feed additives.

[0028] Beneficial effects of the present invention

[0029] Compared with the prior art, the present invention has the following outstanding advantages and unexpected technical effects:

[0030] (1) The present invention adopts an innovative on-the-spot strategy, namely, stabilizing the precursor to generate and protect allicin immediately, rather than stabilizing allicin, thus avoiding the degradation problem of allicin itself during storage from the source.

[0031] (2) The formulation of the present invention achieves a systematic timing protection design:

[0032] Storage period: The hydrophobic outer layer is moisture-proof and oxygen-proof; the enteric inner layer isolates the stomach acid; and the core precursor is physically isolated, thus achieving multiple levels of stability.

[0033] After administration: The enteric inner layer enables precise intestinal-targeted release;

[0034] Instantaneous formation: A specific combination of α-tocopherol and phytic acid provides immediate and targeted protection for newly formed allicin in the reaction microenvironment, solving the problem of simultaneous formation and degradation of allicin.

[0035] (3) This invention has discovered a specific combination of α-tocopherol (lipid-soluble) and phytic acid (water-soluble / interfacially active), which, without significantly inhibiting alliinase activity, synergistically inhibits the sulfur-oxygen free radical intermediates and metal ion-catalyzed oxidation reactions generated during in situ generation, thereby improving the stability of the in situ generated allicin; the embodiments of this invention have shown that replacing them with other common antioxidants / chelating agents cannot achieve the same effect;

[0036] (4) The preparation method of the present invention has mild conditions and simple and clear steps, and is suitable for industrial production. Detailed Implementation

[0037] In some embodiments, the present invention provides a formulation for maintaining allicin activity, the formulation having a core-shell structure, comprising:

[0038] (1) An active core comprising alliin and alliinase physically isolated from each other, as well as tocopherol as a free radical scavenger and phytic acid as a metal ion chelating agent; the physical isolation can be achieved by embedding alliin into a lipid matrix, loading alliinase onto a hydrophilic carrier, and physically mixing the two within the core but separating them by their respective carriers; in a specific embodiment, the core may also contain a pH buffer salt to adjust the local microenvironment to the optimal pH range of alliinase (approximately 6.5-7.5) when the reaction is triggered.

[0039] (2) A pH-responsive enteric polymer inner layer encapsulating the active core, wherein the inner layer material (such as sodium alginate, HPMCAS) remains structurally stable under acidic gastric conditions (pH≤5.5) and swells, dissolves or ruptures under near-neutral intestinal conditions (pH≥6.5), thereby achieving gastric protection and targeted release into the intestine;

[0040] (3) A hydrophobic protective outer layer covering the inner layer, the outer layer material (such as glyceryl monostearate, chitosan-fatty acid graft) provides a physical barrier to effectively block the penetration of moisture and oxygen during storage and ensure the stability of the formulation during its shelf life.

[0041] After the preparation enters the intestine, alliin and alliinase, triggered by intestinal fluid (pH change, water osmosis), overcome their physical separation and come into contact, undergoing an enzymatic reaction to generate allicin in situ. α-Tocopherol and phytic acid, pre-positioned in the microenvironment where the reaction occurs, can synergistically inhibit the sulfur-oxygen free radical intermediates generated during the reaction and the oxidation reaction catalyzed by trace metal ions, without significantly inhibiting alliinase activity. This provides immediate protection for the highly active allicin at the moment of generation, improving its stability.

[0042] In some embodiments, the present invention also provides a method for preparing the formulation as described herein, comprising the following steps:

[0043] S1: Construction of the active core:

[0044] a. Allicin, α-tocopherol and phytic acid are dispersed or dissolved together in a molten lipid matrix, cooled and solidified, and then pulverized to obtain allicin lipid particles containing a protective agent;

[0045] b. Alliinase is mixed with a hydrophilic or disintegrating carrier (such as microcrystalline cellulose), and optionally a pH buffer salt is added, to prepare enzyme particles;

[0046] c. Physically mix the allicin lipid particles and enzyme particles in a certain proportion to form an active core mixture;

[0047] In a preferred embodiment, this step is performed in an anhydrous or low-water environment, with the temperature controlled near the lipid melting point but below the enzyme inactivation temperature (typically <40°C).

[0048] S2: Multi-layer coating:

[0049] a. Using fluidized bed coating, spray coating, or iontophoresis, a pH-responsive enteric polymer is coated onto the surface of the active core mixture to form an inner layer;

[0050] b. Continue to coat the inner surface with a hydrophobic material solution or dispersion to form an outer layer;

[0051] S3: Perform low-temperature drying (such as fluidized bed drying, inlet temperature <50℃) to obtain a dried solid granular formulation.

[0052] Furthermore, the mass ratio of α-tocopherol to phytic acid is 1:(0.2-0.8), which achieves a balance between optimal protective effect and minimal interference with enzyme activity.

[0053] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0054] Example 1: Preparation of a formulation that maintains the activity of allicin

[0055] (1) Take 100mg of allicin, 20mg of α-tocopherol and 10mg of phytic acid, add 500mg of molten hydrogenated palm kernel oil, mix homogenously, cool and solidify, and then grind through a 100-mesh sieve to obtain allicin lipid particles.

[0056] (2) Take 50mg of alliinase (2000 U / g), 150mg of microcrystalline cellulose, and 10mg of dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 7.0), mix them, granulate them with 5% polyvinyl alcohol ethanol solution, and dry them at 40℃ to obtain enzyme granules.

[0057] (3) Mix the two types of particles at a mass ratio of 2:1 to obtain the active core;

[0058] (4) Coating:

[0059] a. In a fluidized bed, the active core is coated by bottom spraying with a 2% sodium alginate solution (containing 0.5% CaCl2) to form a cross-linked enteric inner layer;

[0060] b. Continue coating with a 5% glyceryl monostearate ethanol solution to form a hydrophobic outer layer;

[0061] c. Fluidize and dry at 40℃ for 30 minutes to obtain a formulation that retains the activity of allicin.

[0062] Example 2:

[0063] It is basically the same as Example 1, except that the mass ratio of α-tocopherol to phytic acid is adjusted to 1:0.3 (i.e. 20mg:6mg).

[0064] Comparative Example 1 (Replacing the Antioxidant):

[0065] The experiment was essentially the same as in Example 1, except that 20 mg of α-tocopherol was replaced with an equal mass of tea polyphenols.

[0066] Comparative Example 2 (replacing the chelating agent):

[0067] It is basically the same as Example 1, except that 10 mg of phytic acid is replaced with an equal amount of disodium ethylenediaminetetraacetate (EDTA-2Na).

[0068] Comparative Example 3 (using antioxidants only):

[0069] It is basically the same as Example 1, except that phytic acid is not added.

[0070] Comparative Example 4 (using chelating agent only):

[0071] It is basically the same as Example 1, except that α-tocopherol is not added.

[0072] Comparative Example 5 (without stabilizer):

[0073] It is basically the same as Example 1, except that α-tocopherol and phytic acid are not added.

[0074] Comparative Example 6 (low phytic acid ratio):

[0075] The method is basically the same as in Example 1, except that the mass ratio of α-tocopherol to phytic acid is adjusted to 1:0.1 (i.e., 20 mg of α-tocopherol and 2 mg of phytic acid).

[0076] Comparative Example 7 (High Phytic Acid Ratio):

[0077] The example is basically the same as Example 1, except that the mass ratio of α-tocopherol to phytic acid is adjusted to 1:1 (i.e., 20 mg of α-tocopherol and 20 mg of phytic acid).

[0078] Test Example 1: Allicin Production and Stability Test under Simulated Intestinal Environment

[0079] The formulations prepared in Examples 1-2 and Comparative Examples 1-5, containing an equivalent amount of alliin, were placed in phosphate buffer (simulating intestinal fluid) at pH 6.8 and shaken at 37°C. Samples were taken at 0, 5, 10, 20, and 30 minutes after the start of the reaction, and the concentration of allicin in the solution was determined by HPLC. The retention rate 30 minutes after formation was calculated (with the concentration formed at 0 minutes as 100%). The results are shown in Table 1.

[0080] Table 1: Results of Allicin Formation and Stability Tests

[0081] sample α-Tocopherol Phytic acid Other replacements Relative concentration of formation at 0 minutes (%) 30-minute retention rate (%) Example 1 have have none 100.0±1.5 85.2±2.1 Example 2 Yes (in different proportions) have none 98.5±1.7 82.1±2.3 Comparative Example 1 none have Tea polyphenols 78.3±2.5 65.4±3.0 Comparative Example 2 have none EDTA-2Na 62.1±3.0 70.8±2.9 Comparative Example 3 have none none 99.1±1.8 58.7±3.2 Comparative Example 4 none have none 97.8±2.0 55.2±3.4 Comparative Example 5 none none none 100.0±2.0 21.5±4.0 Comparative Example 6 Yes (in different proportions) have none 99.8±1.6 68.3±2.8 Comparative Example 7 Yes (in different proportions) have none 93.8±2.0 82.3±2.4

[0082] As shown in Table 1, the 30-minute retention rates of Examples 1 and 2 exceeded 82%, which was significantly higher than all comparative examples, demonstrating that the combination of α-tocopherol and phytic acid achieved excellent protective effects against allicin.

[0083] In contrast, the concentrations generated at 0 minutes of Comparative Example 1 (tea polyphenols replacing α-tocopherol) and Comparative Example 2 (EDTA-2Na replacing phytic acid) were significantly reduced, indicating that these replacement components inhibited alliinase activity, leading to a decrease in allicin production efficiency.

[0084] When only one of α-tocopherol and phytic acid is used (Comparative Examples 3 and 4), it can be seen that the 30-minute retention rate of allicin is much lower than that in Example 1, demonstrating that the protective effects of α-tocopherol and phytic acid alone are limited, and that the combination of the two produces a synergistic effect.

[0085] Without the use of α-tocopherol and phytic acid (Comparative Example 5), allicin was rapidly degraded, demonstrating the necessity of using the combination of α-tocopherol and phytic acid for immediate protection.

[0086] When the mass ratio of α-tocopherol to phytic acid used was 1:0.1 (Comparative Example 6), the 30-minute retention rate plummeted to 68.3%, far lower than the 85.2% in Example 1. This indicates that the amount of phytic acid used was insufficient to provide enough metal ion chelation to synergistically inhibit free radical chain reactions, resulting in a significant decrease in the protective effect.

[0087] When the mass ratio of α-tocopherol to phytic acid used was 1:1 (Comparative Example 7), the concentration generated at 0 minutes decreased to 93.8%, and the retention rate (82.3%) was also lower than in Example 1. This may be because the excess phytic acid (strong acid) slightly altered the local pH of the reaction microenvironment, or because its strong complexing properties subtly interfered with enzyme activity, leading to a disruption of the balance between generation and protection.

[0088] Test Example 2: Accelerated Stability Test

[0089] The formulations prepared in Examples 1-2 and Comparative Examples 1-5 were placed in a constant temperature and humidity chamber at 40°C and 75% RH. Samples were taken at 0, 1, 2, and 3 months to determine the residual alliin content in the formulations (calculating the alliin retention rate). The stored samples were then reacted in simulated intestinal fluid, and the peak concentration of allicin generated was measured and compared with the peak concentration of the sample at month 0 (calculating the allicin generation efficiency retention rate). The results are shown in Tables 2 and 3.

[0090] Table 2: Results of accelerated stability test (alliin retention rate, %)

[0091] sample describe October January February March Example 1 α-Tocopherol + Phytic Acid (1:0.5) 100.0±1.2 98.5±1.5 97.2±1.8 95.8±2.1 Example 2 α-Tocopherol + Phytic Acid (1:0.3) 100.0±1.0 98.8±1.3 96.9±1.6 94.5±2.3 Comparative Example 1 Tea polyphenols + phytic acid 100.0±1.5 95.2±2.0 90.1±2.5 83.7±3.0 Comparative Example 2 α-Tocopherol + EDTA 100.0±1.3 97.1±1.7 93.4±2.2 88.9±2.8 Comparative Example 3 α-Tocopherol only 100.0±1.3 96.5±1.8 92.0±2.2 86.8±2.8 Comparative Example 4 Phytic acid only 100.0±1.3 97.0±1.7 92.8±2.1 87.5±2.7 Comparative Example 5 No stabilizer 100.0±1.6 93.4±2.3 85.7±3.0 76.5±3.5 Comparative Example 6 The ratio is unfavorable (1:0.1). 100.0±1.3 97.0±1.8 93.5±2.2 88.9±2.7 Comparative Example 7 The ratio is unfavorable (1:1). 100.0±1.4 97.8±1.6 94.8±2.0 90.2±2.5

[0092] Table 3: Results of accelerated stability test (retention rate of allicin production efficacy, %)

[0093] sample describe October January February March Example 1 α-Tocopherol + Phytic Acid (1:0.5) 100.0±2.0 97.1±2.2 94.5±2.5 90.3±3.0 Example 2 α-Tocopherol + Phytic Acid (1:0.3) 100.0±1.8 96.5±2.4 93.2±2.7 88.7±3.2 Comparative Example 1 Tea polyphenols + phytic acid 100.0±2.5 88.3±3.0 79.6±3.5 70.1±4.0 Comparative Example 2 α-Tocopherol + EDTA 100.0±2.2 90.5±2.8 82.4±3.2 73.8±3.8 Comparative Example 3 α-Tocopherol only 100.0±2.2 94.0±2.7 87.5±3.2 79.8±3.8 Comparative Example 4 Phytic acid only 100.0±2.3 94.8±2.8 88.6±3.3 80.9±3.9 Comparative Example 5 No stabilizer 100.0±2.8 85.1±3.5 72.3±4.2 61.4±4.8 Comparative Example 6 The ratio is unfavorable (1:0.1). 100.0±2.1 94.8±2.5 89.6±3.0 82.1±3.5 Comparative Example 7 The ratio is unsuitable (1:1). 100.0±2.2 95.9±2.6 91.2±3.1 85.4±3.6

[0094] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A formulation for maintaining the activity of allicin, characterized in that, The formulation has a core-shell structure, comprising: (1) The active core contains physically isolated alliin and alliinase, as well as tocopherol as a free radical scavenger and phytic acid as a metal ion chelator. (2) A pH-responsive enteric polymer inner layer encapsulating the active core, wherein the inner layer remains structurally stable under pH ≤ 5.5 conditions and undergoes swelling or dissolution and release under pH ≥ 6.5 conditions; (3) A hydrophobic protective outer layer covering the inner layer; In this process, alliin and alliinase react in situ to generate allicin under the triggering conditions of the pH-responsive enteric polymer inner layer during the use of the formulation or after entering the intestine.

2. The formulation according to claim 1, characterized in that, The alliin is contained in a first carrier, and the alliinase is contained in a second carrier. The first carrier and the second carrier are physically mixed within the active core but isolated from each other.

3. The formulation according to claim 2, characterized in that, The first carrier is a lipid matrix, and the second carrier is a hydrophilic or disintegrating solid carrier.

4. The formulation according to claim 1, characterized in that, The tocopherol is a fat-soluble α-tocopherol, and the phytic acid is inositol hexaphosphate; The mass ratio of tocopherol to phytic acid is 1:(0.2-0.8).

5. The formulation according to claim 1, characterized in that, The pH-responsive enteric polymer inner layer material is selected from at least one of sodium alginate, pectin, hydroxypropyl methylcellulose acetate succinate (HPMCAS), and polyacrylic acid resin (Eudragit® L / S series).

6. The formulation according to claim 1, characterized in that, The hydrophobic protective outer layer material is selected from at least one of glyceryl monostearate, chitosan-fatty acid grafts, and polylactic acid-glycolic acid copolymer (PLGA).

7. A method for preparing the formulation according to any one of claims 1-6, characterized in that, Includes the following steps: S1: Allicin, tocopherol and phytic acid are dispersed or dissolved in a molten lipid matrix, and then solidified and pulverized to obtain allicin lipid particles; allicinase is mixed with a solid carrier to obtain enzyme particles; the allicin lipid particles and enzyme particles are physically mixed to obtain an active core mixture; S2: A pH-responsive enteric polymer layer and a hydrophobic material layer are sequentially coated on the surface of the active core mixture; S3: Perform low-temperature drying to obtain a solid dosage form.

8. The method according to claim 7, characterized in that, The dispersion or dissolution described in step S1 is carried out under light-protected conditions at a temperature below 40°C; the inlet temperature of the low-temperature drying described in step S3 is below 50°C.

9. The method according to claim 7 or 8, characterized in that, In step S2, when coating the pH-responsive enteric polymer layer, an ionic crosslinking method is used for curing.

10. The use of the formulation according to any one of claims 1-6 in the preparation of a health food, dietary supplement or medicine for oral administration.