Peach gum - zein-based lycopene nanoparticles and preparation method and application thereof

Abstract

The present application belongs to the field of food science and engineering and biological medicine technology, and provides peach gum-corn alcohol soluble protein based lycopene nanoparticles and application thereof, in particular, application of the nanoparticles in preparation of health products, dietary nutrients, drugs and other related products for preventing or relieving alcoholic liver injury. The present application uses peach gum and corn alcohol soluble protein as raw materials, obtains peach gum-corn alcohol soluble protein grafts through Maillard reaction, loads lycopene under the crosslinking action of genipin, and prepares peach gum-corn alcohol soluble protein based lycopene nanoparticles with good stability, embedding rate and slow release, and taking the nanoparticles before drinking can significantly improve liver injury caused by long-term alcohol intake.

Description

Technical Field

[0001] This invention belongs to the fields of food science and engineering and biomedicine, specifically relating to a gum-zein-based lycopene nanoparticle, its preparation method and application, and particularly to the application of the nanoparticle in the preparation of health products, dietary supplements, drugs and other related products for preventing or alleviating alcoholic liver damage. Background Technology

[0002] Alcohol has a significant toxic effect on the liver, and long-term heavy drinking often leads to alcoholic liver damage. Alcoholic liver disease has become a serious public health problem, making the search for a safe, stable, and effective dietary supplement to alleviate alcoholic liver damage of great importance. Lycopene (Lyc) is a highly lipophilic carotenoid with multiple biological activities, including scavenging free radicals, enhancing immune activity, and alleviating oxidative stress. Therefore, it has a certain intervention effect on liver damage. For example, patents CN104800182A and CN103948028A respectively provide lycopene tablets for liver protection and hangover relief, and a composition with liver-damaging effects, thus incorporating lycopene into liver-protective products.

[0003] However, lycopene is highly sensitive to light and oxygen. The existing technologies described above all involve directly physical blending lycopene or lycopene oil with excipient powders, which offers limited protection for lycopene. Furthermore, ordinary film-coated tablets lack stomach acid tolerance, causing rapid disintegration after entering the stomach. This exposes lycopene directly to stomach acid and pepsin, making it easily degraded and inactivated, thus affecting its utilization.

[0004] In recent years, in order to further improve the utilization rate of lycopene and avoid its oxidative loss caused by external factors such as light and heat during application, nanocarrier delivery technology has become a popular research direction.

[0005] Protein-based natural polymers are biocompatible and completely biodegradable. Their molecules contain both hydrophobic and hydrophilic regions, allowing them to self-assemble into nanocarriers, making them ideal substrates for encapsulating hydrophobic active ingredients. However, pure protein nanocarrier systems have poor hydrophilicity and are prone to particle aggregation in aqueous systems. Furthermore, under the influence of strong stomach acid and pepsin, the protein shell is easily degraded by enzymes, causing premature release of encapsulated lycopene in the stomach, resulting in leakage and inactivation of the active ingredient, thus preventing controlled release from the intestines.

[0006] To overcome the shortcomings of single-protein carriers, current research often employs a protein-polysaccharide composite modification strategy, which involves using natural polysaccharides to modify the surface of protein nanoparticles and constructing a protein-polysaccharide composite nanodelivery system. However, the degree of binding between proteins and polysaccharides is greatly affected by processing parameters such as the types of proteins and polysaccharides, pH, and ratio. Therefore, different complexes exhibit significant differences in their loading capacity and bioavailability of lycopene.

[0007] Zein is the main energy storage protein in corn, possessing advantages such as wide availability, renewability, biocompatibility, and antioxidant properties. It contains a large number of nonpolar amino acids and exhibits unique hydrophobic characteristics, making it an ideal candidate for manufacturing nanobiopolymer materials for food and nutritional applications. Peach gum (PG) molecules contain a large number of hydroxyl groups, uronic acids, and a small amount of bound polyphenols, exhibiting excellent emulsifying, film-forming, and weak gelling properties, making it a high-quality natural edible microcapsule wall material. For example, patent CN113229504A discloses a peach gum polysaccharide-zein composite system; however, this disclosed technology uses a self-assembly process to combine zein and peach gum, resulting in limited system stability. Furthermore, this technology only mentions the preparation of the composite and does not address its related applications.

[0008] In addition, previous reports have also used hawthorn pectin-zein protein composite systems to deliver lycopene (Wei Xueyan et al., Preparation of hawthorn pectin-zein protein composite nanoparticles and their effect on lycopene delivery). However, because it also uses a protein-polysaccharide self-assembly composite process, the stability of the composite system is limited. In addition, the large amount of galacturonic acid in pectin and zein rely on strong electrostatic interaction to combine, and the complex is prone to aggregation and damage under acidic conditions. Therefore, the resulting composite system is relatively sensitive to pH. Ultimately, the composite system obtained by this technology also has poor stability in gastric juice, with a lycopene release rate of more than 40% within 2 hours.

[0009] It is worth mentioning that, to date, there are no publicly available reports on the application of the zein-gum complex system as a lycopene delivery system for alcoholic liver injury.

[0010] In conclusion, developing a stable and efficient lycopene complex carrier delivery system using zein and gum arabic as raw materials to achieve efficient utilization of lycopene and expand its practical application in the protection against alcoholic liver injury has significant research value. Summary of the Invention

[0011] To address the above technical problems, this invention proposes a gum-zein-based lycopene nanoparticle, its preparation method, and its application.

[0012] This invention uses peach gum and zein as raw materials, employing a wet Maillard reaction to covalently graft peach gum onto zein to form an amphiphilic protein-polysaccharide conjugate for lycopene delivery. The peach gum used in this invention is rich in highly branched arabinogalactan, which can be grafted onto zein via a carbonyl-amine reaction to form a more stable complex with higher structural integrity in simulated gastric juice. This helps reduce the release of lycopene in the stomach, achieving a stable and sustained release of lycopene in the intestinal environment.

[0013] The technical solution provided by this invention is:

[0014] The first aspect of this invention is to provide a method for preparing gum-zein-based lycopene nanoparticles, comprising the following steps:

[0015] Preparation of S1 gum-zein covalent graft

[0016] A zein-ethanol solution was prepared by dissolving zein in ethanol solution, and a gum arabic solution was prepared by dissolving gum arabic in deionized water. The two solutions were mixed, stirred evenly, and the pH was adjusted to 7.5-8.5. The mixture was then heated at 40-70℃ to obtain a gum arabic-zein covalently grafted product. The mass ratio of zein to gum arabic was 1:1-2.5.

[0017] Preparation of S2 organic phase

[0018] Using 70%~80% ethanol as solvent, the gum-zein covalent graft obtained by S1 was used as a carrier, and medium-chain triglycerides were used as the oil phase to encapsulate lycopene. The mixture was homogenized and dispersed evenly to obtain the organic phase.

[0019] Lycopene accounts for 2% to 10% of the zein protein mass, the mass ratio of gum gum-zein covalent graft to lycopene is 5 to 15: 20 to 200, and medium-chain triglycerides account for 5% to 8% of the organic phase volume;

[0020] S3 crosslinking to prepare nanoparticles

[0021] The organic phase of S2 was slowly added dropwise into water, and genipin solution was added for cross-linking in the dark. After the cross-linking reaction was completed, the mixture was centrifuged, the supernatant was discarded, and the resulting product was washed and freeze-dried to obtain peach gum-zein-based lycopene nanoparticles.

[0022] In the preparation method provided by the present invention, preferably, the volume fraction of the ethanol solution in S1 is 70%~80%, more preferably 70%. The main purpose of using this volume fraction of ethanol solution is to ensure that zein can be effectively dissolved. If the ethanol concentration is too low, such as below 60%, zein will precipitate, leading to experimental failure.

[0023] Preferably, the heating temperature in S1 is 50~60℃. The heating temperature directly determines the Maillard reaction effect of zein and gum arabic, most directly manifested in a significant difference in grafting rate. At lower temperatures, such as 30℃, the grafting rate is very low, only 6.29%, resulting in poor stability of the obtained complex and poor subsequent encapsulation of lycopene. Conversely, if the temperature is too high, the Maillard reaction is too vigorous, leading to the production of more black byproducts, which seriously affects the taste and appearance of the product, and is also detrimental to the preparation of related products.

[0024] Preferably, in S2, lycopene accounts for 2% to 6% of the zein mass, the mass ratio of gum-zein covalent graft to lycopene is 8 to 12: 20 to 60, and medium-chain triglycerides account for 5% to 7% of the organic phase volume.

[0025] Preferably, in S3, the volume ratio of the organic phase to water is 1~5:1, the molar ratio of genipin to the graft is 1:10~40, the crosslinking reaction temperature is 25~45℃, and the reaction time is 2~5 h.

[0026] A second aspect of the present invention is to provide a pharmaceutical composition for preventing or alleviating alcoholic liver injury, said pharmaceutical composition comprising gum arabic-zein-based lycopene nanoparticles prepared by the aforementioned method.

[0027] Preferably, the pharmaceutical composition further includes conventional pharmaceutical excipients, such as sweeteners, excipients, fillers, pH adjusters, etc.

[0028] A third aspect of the present invention is to provide the application of the gum-zein-based lycopene nanoparticles prepared by the above-described preparation method, specifically in the preparation of any one of the following products: health products, dietary supplements, or pharmaceuticals for the prevention or relief of alcoholic liver damage.

[0029] Preferably, the health supplement, dietary supplement, or medicine must be taken before drinking alcohol.

[0030] Preferably, the health product, dietary supplement, or medicine is taken 1 to 5 hours before drinking alcohol.

[0031] As a further preferred option, the health product, dietary supplement, or medicine is taken 1-2 hours before drinking alcohol.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] (1) This invention uses peach gum and zein as raw materials. The highly branched arabinogalactan in peach gum is grafted onto zein via wet Maillard reaction to obtain a composite graft with higher stability. The results show that when the mass ratio of zein to peach gum is 1:1.5~2.5 and the reaction temperature is 40~50℃, the grafting rate of the obtained composite is as high as 22.25%~28.23%;

[0034] (2) Based on the gum-zein graft, the present invention loads lycopene and uses genistein as a cross-linking agent to prepare a gum-zein-based lycopene nanodelivery system. The obtained nanodelivery system can achieve an encapsulation rate of up to 80.25% for lycopene, which is 22.35% and 10.51% higher than that of the gum-zein physical mixing loading system and the genistein-free cross-linking delivery system, respectively.

[0035] (3) The peach gum-zein-based lycopene nanoparticles provided by the present invention have good sustained-release properties. The results of the in vitro simulated gastrointestinal fluid experiment show that the release rate of lycopene in gastric fluid after 2 hours of digestion is less than 15%, and more lycopene can be fully released in the intestine, which improves the bioavailability of lycopene.

[0036] (4) The present invention also provides a gum-zein-based lycopene nanoparticle formulation for relieving alcoholic liver damage, and clarifies the timing of its administration. In particular, the nanoparticles can significantly improve liver damage caused by alcohol consumption when taken before drinking alcohol. Attached Figure Description

[0037] Figure 1 The infrared spectra of each sample in Experimental Example 3 of this invention are shown below.

[0038] Figure 2 This is a diagram illustrating the in vitro sustained-release effect of peach gum-zein-based lycopene nanoparticles in Example 1 of the present invention.

[0039] Figure 3 The figure shows the effect of the timing of PG-Zein / Lyc nanoparticle intake on ALT activity in mouse serum in Example 2 of this invention.

[0040] Figure 4 This is a graph showing the effect of the timing of PG-Zein / Lyc nanoparticle intake on AST activity in mouse serum in Application Example 2 of this invention.

[0041] Figure 5 This is a graph showing the effect of the timing of PG-Zein / Lyc nanoparticle intake on the liver index in mouse serum in Example 2 of this invention.

[0042] Figure 6 The figure shows the effect of the timing of PG-Zein / Lyc nanoparticle intake on SOD activity in mouse serum in Application Example 2 of this invention.

[0043] Figure 7 The figure shows the effect of the timing of PG-Zein / Lyc nanoparticle intake on GSH activity in mouse serum in Application Example 2 of this invention. Detailed Implementation

[0044] To enable those skilled in the art to better understand the present invention, specific embodiments will now be described in further detail. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0045] It should be noted that the specific ingredient values ​​listed in the embodiments of this invention are only for illustrative purposes to demonstrate the technical solution and effects of this invention, and do not represent a limitation on the scope of protection. For example, the mass ratio of zein to gum arabic is 1:1.5. In practical applications, ingredient ratios such as 1 g:1.5 g, 2 g:3 g, 10 g:15 g, and 1 kg:1.5 kg can be used. Those skilled in the art can scale up or down proportionally according to the production scale. As long as the mass ratio of zein to gum arabic satisfies this mass ratio relationship, the target product of this invention can be obtained, and all such products fall within the scope of protection of this invention.

[0046] Furthermore, the term "water" as used in this invention is a general term that includes any form of water that can be purchased from the market or made by those skilled in the art, including but not limited to deionized water, distilled water, and purified water.

[0047] Example 1

[0048] This embodiment provides a peach gum-zein covalent graft, which is prepared by the following method:

[0049] 1 g of zein was dissolved in 100 mL of 70% ethanol to prepare a 10 mg / mL zein ethanol solution. Separately, 1.5 g of gum arabic (PG) was dissolved in deionized water to prepare a 15 mg / mL gum arabic solution. The two solutions were mixed and stirred evenly. The pH of the mixed solution was adjusted to 8.0 with 0.1 mol / L NaOH, and stirring was continued for 30 min. The mixture was heated at 60℃ for 2 h. After the reaction was completed, the mixture was removed and quickly cooled with ice water to terminate the Maillard reaction. The resulting solution was placed in a dialysis bag (molecular weight cutoff 8-14 kDa) and dialyzed in deionized water for 48 h, with the water changed 6 times. After dialysis purification, the gum arabic-zein covalent graft was obtained. The grafting rate was determined by the o-phthalaldehyde method (OPA).

[0050] Example 2

[0051] This embodiment provides a peach gum-zein covalent graft, the only difference in its preparation method from that of Example 1 is that the mass of peach gum is 0.5 times that of zein.

[0052] Example 3

[0053] This embodiment provides a peach gum-zein covalent graft, the only difference in its preparation method from that of Example 1 is that the amount of peach gum added is the same as the mass of zein.

[0054] Example 4

[0055] This embodiment provides a peach gum-zein covalent graft, the only difference in its preparation method from that of Example 1 is that the mass of peach gum is twice the mass of zein.

[0056] Example 5

[0057] This embodiment provides a peach gum-zein covalent graft, the only difference in preparation method from that of Example 1 is that the mass of peach gum is 2.5 times that of zein.

[0058] Example 6

[0059] This embodiment provides a gum-zein covalent graft, the only difference between its preparation method and that of Example 1 is that the reaction temperature is 30°C.

[0060] Example 7

[0061] This embodiment provides a gum-zein covalent graft, the only difference between its preparation method and that of Example 1 is that the reaction temperature is 40°C.

[0062] Example 8

[0063] This embodiment provides a gum-zein covalent graft, the only difference between which is the preparation method and that of Example 1, except that the reaction temperature is 50°C.

[0064] Example 9

[0065] This embodiment provides a gum-zein covalent graft, the only difference between which is the preparation method and that of Example 1, the reaction temperature is 70°C.

[0066] Example 10

[0067] This embodiment provides a peach gum-zein-based lycopene nanoparticle, and the specific preparation method is as follows:

[0068] Preparation of S1 organic phase

[0069] First, prepare the graft ethanol solution: Dissolve the peach gum-zein graft from Example 1 in 70% ethanol to prepare a graft ethanol solution with a concentration of 10 mg / mL.

[0070] Next, the lycopene oil phase was prepared: lycopene was dissolved in medium-chain triglyceride oil to obtain the lycopene oil phase, wherein the added mass of lycopene accounted for 6% of the mass of zein, and the volume of medium-chain triglycerides accounted for 6% of the volume of the organic phase;

[0071] Add the lycopene oil phase to the above-mentioned ethanol solution of the graft, stir magnetically for 30 min in the dark, and homogenize with a homogenizer until the lycopene is completely dispersed to obtain the organic phase;

[0072] S2 crosslinking preparation of nanoparticles: The organic phase of S1 was slowly added dropwise to deionized water at a dropping rate of 1 mL / min, with a volume ratio of deionized water to organic phase of 3:1. The mixture was stirred continuously under light protection throughout the process. 2 mg / mL genipin solution was added (the molar ratio of genipin to zein was 1:20). The mixture was crosslinked at 30℃ under light protection for 3 h. After centrifugation at 10000 r / min for 15 min, the supernatant was discarded, and the mixture was washed once with deionized water. The mixture was then freeze-dried under light protection throughout the process to obtain gum-zein-based lycopene nanoparticles.

[0073] Example 11

[0074] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 is that the grafting material of Example 2 is used instead of the grafting material of Example 1.

[0075] Example 12

[0076] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 is that the grafting material of Example 3 is used instead of the grafting material of Example 1.

[0077] Example 13

[0078] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 is that the grafting material of Example 4 is used instead of the grafting material of Example 1.

[0079] Example 14

[0080] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 is that the grafting material of Example 5 is used instead of the grafting material of Example 1.

[0081] Example 15

[0082] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the amount of lycopene added is 2%.

[0083] Example 16

[0084] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the amount of lycopene added is 4%.

[0085] Example 17

[0086] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the amount of lycopene added is 8%.

[0087] Example 18

[0088] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the amount of lycopene added is 10%.

[0089] Example 19

[0090] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the ratio of the antisolvent aqueous phase to the organic phase is 1:1.

[0091] Example 20

[0092] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the ratio of the antisolvent aqueous phase to the organic phase is 2:1.

[0093] Example 21

[0094] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the ratio of the antisolvent aqueous phase to the organic phase is 4:1.

[0095] Example 22

[0096] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the ratio of the antisolvent aqueous phase to the organic phase is 5:1.

[0097] Example 23

[0098] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the genipin / graft molar ratio is 1:40.

[0099] Example 24

[0100] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the genipin / graft molar ratio is 1:30.

[0101] Example 25

[0102] This embodiment provides a peach gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the genipin / graft molar ratio is 1:15.

[0103] Example 26

[0104] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the genipin / graft molar ratio is 1:10.

[0105] Example 27

[0106] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the graft concentration is 5 mg / mL.

[0107] Example 28

[0108] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the graft concentration is 8 mg / mL.

[0109] Example 29

[0110] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the graft concentration is 12 mg / mL.

[0111] Example 30

[0112] This embodiment provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the graft concentration is 15 mg / mL.

[0113] Comparative Example 1

[0114] This comparative example differs from Example 1 in that peach gum was not added; instead, zein was used to treat the grafting effect under the conditions of Example 1.

[0115] Comparative Example 2

[0116] In this comparative example, zein and gum arabic were simply physically mixed at room temperature to obtain a mixture, which was then freeze-dried and used directly. The amount of zein and gum arabic added was the same as in Example 1.

[0117] Comparative Example 3

[0118] This comparative example provides a gum-zein-based lycopene nanoparticle, the only difference from Example 10 being that the grafting material of Comparative Example 2 is used instead of the grafting material of Example 1.

[0119] Comparative Example 4

[0120] This comparative example provides a peach gum-zein-based lycopene nanoparticle. The only difference from Example 10 is that the cross-linking agent genipin was not added during the preparation process; otherwise, it is the same as Example 10.

[0121] Experimental Example 1

[0122] This experiment measured the grafting rate of the gum-zein grafts prepared under different conditions in Examples 1-9 and Comparative Examples 1-2. The results are shown in Table 1.

[0123] Table 1 Grafting rates of peach gum-zein grafts prepared under different conditions Example 1 1:1.5 22.25 Example 6 30 6.29 Comparative Example 1 3.12 Example 2 1:0.5 9.51 Example 7 40 12.98 Comparative Example 2 1.01 Example 3 1:1 14.56 Example 8 50 18.07 Example 4 1:2 28.23 Example 9 70 25.12 Example 5 1:2.5 25.87

[0124] Table 1 shows that the ratio of zein to gum and temperature significantly affect the grafting rate of the product. Specifically, with increasing gum content, the grafting rate after the Maillard reaction initially increases significantly and then gradually decreases. The highest grafting rate, reaching 28.23%, is achieved when the mass ratio of zein to gum is 1:2. Furthermore, the grafting rate generally increases with increasing reaction temperature. However, at 70℃, dark brown substances begin to appear in the reaction system, and the rate of increase in grafting rate weakens. This indicates that the reaction has entered the middle and later stages, free amino acids are approaching saturation, and byproducts are beginning to appear. Therefore, to obtain the optimal Maillard reaction product, a reaction temperature of 60℃ is recommended.

[0125] Experimental Example 2

[0126] This experiment measured the encapsulation efficiency of the gum-zein-based lycopene nanoparticles prepared in Examples 10 to 30, and the results are shown in Table 2.

[0127] Table 2. Encapsulation efficiency of different peach gum-zein-based lycopene nanoparticles Example 10 1:1.5 80.25 Example 15 2 75.36 Example 11 1:0.5 49.51 Example 16 4 77.89 Example 12 1:1 68.56 Example 17 8 70.23 Example 13 1:2 76.23 Example 18 10 65.31 Example 14 1:2.5 65.87 Group number Antisolvent aqueous phase / organic phase ratio Encapsulation rate / % Group number Genipin / graft molar ratio Encapsulation rate / % Example 19 1:1 70.26 Example 23 1:40 69.86 Example 20 2:1 73.29 Example 24 1:30 72.69 Example 21 4:1 74.25 Example 25 1:15 79.01 Example 22 5:1 68.91 Example 25 1:10 70.51 Group number Graft concentration / mg / mL Encapsulation rate / % Group number Encapsulation rate / % Example 27 5 55.86 Comparative Example 3 57.90 Example 28 6 65.25 Comparative Example 4 69.74 Example 29 12 72.36 Example 30 15 60.95

[0128] Table 2 records the encapsulation rates of the peach gum-zein-based lycopene prepared in Examples 10 to 30. The results show that adjustments to parameters such as the type of graft, the amount of lycopene added, the ratio of aqueous to organic phases of the antisolvent, the amount of crosslinking agent added, and the concentration of the graft all significantly affect the encapsulation effect of the nanoparticles on lycopene.

[0129] In Examples 10-14, when different grafts (i.e., prepared under the conditions of Examples 1-5) were used to load lycopene, the encapsulation efficiency of the resulting gum-zein-based lycopene nanoparticles was significantly affected by the grafting rate, but the trend was not entirely the same as the increasing trend of the grafting rate. In Example 4, the grafting rate of the graft obtained when the mass ratio of zein to gum was 1:2 was the highest, but its encapsulation effect on lycopene was not the best. That is, the encapsulation efficiency of the nanoparticles in Example 13 was only 76.23%. On the contrary, the encapsulation efficiency of the nanoparticles prepared by the graft of Example 1 (the mass ratio of zein to gum was 1:1.5) was the best, reaching 80.25%.

[0130] In addition, the results of Examples 23-25 ​​show that as the amount of genipin added increases, the encapsulation rate of lycopene by nanoparticles first increases and then decreases, and the encapsulation rate is the highest at 1:20. However, as the amount of crosslinking agent added further increases, excessive crosslinking agent may cause self-crosslinking of the grafted material, which is not conducive to improving the encapsulation rate of lycopene.

[0131] Experimental Example 3

[0132] This experiment characterized the gum-zein-based lycopene nanoparticles prepared in Example 10 using infrared spectroscopy, with the products of Example 1, Comparative Example 3, and Comparative Example 4 serving as controls. Figure 1 As shown, 3200~3600 cm -1 The stretching vibrations attributed to the hydroxyl groups of gum arabic and the amino groups of zein, 1630 cm⁻¹ -1 The characteristic peak of protein amide I is observed at 1000–1200 cm⁻¹. -1 This is the fingerprint region of peach gum polysaccharide.

[0133] Among them, the sample in Comparative Example 3 was at a depth of 3200–3600 cm. -1The presence of extremely strong and broad absorption valleys, a sharp peak in the amide I band, and clear separation of polysaccharide peaks indicates that zein and gum arabic are simply doped via molecular hydrogen bonds. In the sample of Example 1, the absorption intensity of the characteristic peaks of hydroxyl and amide is significantly reduced, and the polysaccharide peaks are weakened by fusion, proving that zein and gum arabic form a covalently grafted complex through the Maillard reaction. In the sample of Comparative Example 4, the absorption of hydrogen bond and amide peaks rebounds, and no new peaks are observed for lycopene reaction, confirming that lycopene is non-covalently embedded within the carrier through hydrophobic interactions. In Example 10, all characteristic absorptions are further weakened, indicating that the crosslinking agent constructs a denser three-dimensional covalent network, effectively improving the structural stability of the nanoparticles. Based on the above infrared spectroscopy results, the Maillard grafted and crosslinked modified gum arabic-zein composite carrier is more suitable for the encapsulation and delivery of lycopene.

[0134] Application Example 1

[0135] This application example uses an in vitro simulated gastrointestinal sustained-release experiment to reflect the drug release behavior of the gum-zein-based lycopene nanoparticles (PG-Zein / Lyc) prepared in Example 10 in the digestive tract, while using oil-soluble lycopene (oil-soluble Lyc) as a control. The in vitro release results are as follows: Figure 2 As shown.

[0136] In a simulated gastric juice environment, the lycopene release of both groups of samples showed a slow increasing trend with the extension of digestion time. After 120 min of digestion, the release rate of oil-soluble lycopene was less than 10%, and the release rate of lycopene in the PG-Zein / Lyc system was less than 15%. The main reason for this phenomenon is the lack of lipase in the gastric juice environment, making it difficult for oil-soluble lycopene to be effectively transferred to the aqueous phase. In the PG-Zein / Lyc system, acidic conditions induce the aggregation of zein, while the gum arabic shell can shield pepsin, inhibiting excessive degradation of the protein matrix. Only a small amount of lycopene diffuses and is released through the particle pores, while most of the core material is confined inside the carrier and transferred to the intestinal juice. After entering the intestinal juice system, the lycopene release rate of both groups of samples significantly increased within the digestion range of 120-150 min, and at each time point, the lycopene release level of the PG-Zein / Lyc system was significantly higher than that of the oil-soluble group. Oil-soluble systems rely on pancreatic lipase to hydrolyze oils, causing lycopene to partition from the oil phase into bile salt mixed micelles. However, a significant amount of lycopene remains trapped in incompletely digested oil droplet structures. In contrast, under neutral intestinal conditions, trypsin hydrolyzes the zein backbone, causing the gum arabic polysaccharide shell to gradually swell and dissociate, releasing a large amount of embedded lycopene. Simultaneously, the spatial exclusion effect of the carrier imparts a sustained-release characteristic to the system. In summary, PG-Zein / Lyc nanoparticles can endow lycopene with superior digestive stability while achieving more complete intestinal release, thus improving the bioavailability of lycopene.

[0137] Application Example 2 This application example focuses on providing an application of gum zein-based lycopene nanoparticles (PG-Zein / Lyc, prepared in Example 10), namely, its use as a protective drug against alcoholic liver injury. Unlike the traditional application method, this application must be taken 1 hour before drinking alcohol to achieve better results. If it is taken at the same time as or after drinking alcohol, the drug will hardly have any effect or the effect will be very poor.

[0138] The specific grouping and experimental procedures for this application example are as follows:

[0139] Normal group: fed with feed + equal volume of distilled water;

[0140] Model group: fed with feed + 0.2 mL (50% ethanol);

[0141] Positive control group: fed with feed + 0.2 mL (50% ethanol), 1 h later, 3.5 mg / kg bw Kexilai;

[0142] Lycopene and alcohol intake group: feed + 0.2 mL (50% ethanol) and 80 mg / kg bw of PG-Zein / Lyc nanoparticles;

[0143] Pre-drinking group: Feed feeding + 80 mg / kg bw Kexilai PG-PG-Zein / Lyc nanoparticles + 0.2 mL (50% ethanol) 1 hour before drinking;

[0144] (f) Group after drinking alcohol: Feed + equal volume of water + 0.2 mL (50% ethanol), 1 h later, 80 mg / kg bw Kexilai PG-Zein / Lyc nanoparticles;

[0145] The mice in the above groups were fed for 30 days, and their weight was measured regularly. After slaughter at the end of the feeding period, ocular ischemia and liver tissue were examined. Physicochemical data such as the content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), mouse liver index, superoxide dismutase (SOD), reduced glutathione (GSH), and malondialdehyde (MDA) in the mouse serum were measured.

[0146] (1) The effect of the timing of PG-Zein / Lyc nanoparticle intake on the activity of alanine aminotransferase (ALT) in mouse serum, the results are shown in Figure 3.

[0147] ALT is the most sensitive biomarker of hepatocellular injury. Compared with the normal group, serum ALT activity in the model group mice increased by 40.6%. Compared with the model group, serum ALT in mice in all groups treated with PG-Zein / Lyc nanoparticles decreased by more than 10% on average. The ALT activity in the positive control group was not significantly different from that in the PG-Zein / Lyc nanoparticle intervention group, but it was significantly lower than that in the model group. Serum ALT activity in mice administered by gavage before drinking alcohol was not significantly different from that in the normal group, while ALT activity in both the simultaneous intake group and the post-drinking intake group was significantly higher than that in the normal group. This indicates that pre-drinking intake of PG-Zein / Lyc nanoparticles can effectively maintain serum alanine aminotransferase activity and effectively reduce hepatocellular damage.

[0148] (2) The effect of the timing of PG-Zein / Lyc nanoparticle intake on serum aspartate aminotransferase (AST) in mice, the results are as follows: Figure 4 .

[0149] Compared with the normal group, serum AST activity increased in all groups of mice after alcohol consumption, with the model group showing the largest increase. Compared with the model group, serum AST activity decreased to varying degrees in all intervention groups of PG-Zein / Lyc nanoparticles, and there was no significant difference in AST levels between the pre-drinking intake group and the normal group. Although the positive control group reduced AST activity, the effect was slightly less than that of the pre-drinking lycopene intake group. The results indicate that supplementing with PG-Zein / Lyc nanoparticles before drinking alcohol can more effectively inhibit the increase in serum AST induced by alcohol and reduce hepatocellular damage, with a protective effect superior to other intake time points.

[0150] (3) The effect of PG-Zein / Lyc intake timing on liver index in mice, the results are as follows: Figure 5 As shown.

[0151] The liver index (liver body index) is the percentage of liver wet weight to mouse body weight. Typically, alcohol consumption causes an increase in the liver index due to liver inflammation, edema, and fat accumulation. Figure 5 It was found that the normal group had the lowest liver index, and the liver organ coefficient was at a normal physiological level. The model group showed a significant increase in liver index, and the proportion of liver weight was significantly higher than that of the normal group, indicating successful modeling. Liver indexes decreased after ingestion of PG-Zein / Lyc nanoparticles, with a decrease of more than 5.86% before alcohol consumption compared to the model group, which was similar to the positive control group. This shows that pre-drinking PG-Zein / Lyc nanoparticle intake can significantly reduce alcohol-induced liver enlargement and alleviate the increase in liver organ coefficient.

[0152] (4) The effect of the timing of PG-Zein / Lyc nanoparticle intake on the activity of superoxide dismutase (SOD) in mouse liver tissue is as follows: Figure 6 As shown.

[0153] Superoxide dismutase (SOD) is a core antioxidant enzyme in the body, capable of scavenging oxygen free radicals produced by alcohol metabolism and reducing oxidative stress damage to hepatocytes. Its activity is negatively correlated with liver organ indices. As shown in the figure, the normal group of mice exhibited the highest SOD enzyme activity in their liver tissue. After alcohol consumption, the SOD enzyme activity in all groups showed a decreasing trend. There were no significant differences in SOD enzyme activity among the model group, the simultaneous intake group, the post-drinking intake group, and the control group. However, the SOD enzyme activity in the liver tissue of mice that ingested lycopene before drinking alcohol was significantly higher than that in all other groups except the normal group. This indicates that ingesting PG-Zein / Lyc nanoparticles before drinking alcohol can better maintain the activity level of the body's antioxidant enzymes, reduce oxidative stress responses induced by alcohol metabolism, and decrease the attack and damage of oxygen free radicals on hepatocytes.

[0154] (5) Effects of different PG-Zein / Lyc intake times on the content of reduced glutathione (GSH) in mouse liver tissue. Results are shown in […]. Figure 7 .

[0155] Reduced glutathione (GSH) is an important antioxidant in hepatocytes, which helps to remove peroxides produced by metabolism and maintain intracellular redox homeostasis. Figure 7 It is evident that, compared to the normal group, the GSH content in the liver tissue of the model group mice was significantly reduced, indicating that alcohol intake had damaged the antioxidant system of hepatocytes, causing oxidative damage. Among the lycopene intervention groups, only the pre-drinking intake group had a significantly higher GSH content than the model group, and there was no significant difference compared to the normal group; while the GSH content in the simultaneous intake group and the post-drinking intake group was slightly higher than that in the model group, the difference was not significant. Therefore, pre-drinking intake of PG-Zein / Lyc nanoparticles can better maintain the content of reduced glutathione in liver tissue and protect the antioxidant capacity of hepatocytes.

[0156] (6) The effect of the timing of PG-Zein / Lyc nanoparticle intake on the malondialdehyde (MDA) content in mouse blood and liver tissue. The results are shown in Table 3.

[0157] Table 3. Effects of the timing of PG-Zein / Lyc nanoparticle intake on MDA content in mouse blood and liver tissue. normal group 8.45±0.82 0.69±0.05 Model group <![CDATA[12.38±0.96 a ]]> <![CDATA[1.88+0.06 a ]]> Positive control group <![CDATA[9.18+0.65 b ]]> <![CDATA[0.90+0.07 ab ]]> Pre-drinking group <![CDATA[9.09±0.75 b ]]> <![CDATA[0.77+0.05 bc ]]> Simultaneous intake group <![CDATA[9.81+0.81 bc ]]> <![CDATA[0.81+0.06 ab ]]> Group after drinking <![CDATA[10.59±0.54 ac ]]> <![CDATA[0.92+0.08 abd ]]>

[0158] MDA is an end product of lipid peroxidation, directly reflecting the degree of cell damage; higher MDA levels indicate more severe lipid peroxidation damage to hepatocytes. Table 3 shows that MDA levels in both serum and liver increased after alcohol consumption. Compared to the model group, the group that ingested lycopene before drinking alcohol showed the most significant decrease in liver MDA content (59.21%) and serum MDA content (31.42%), demonstrating superior reduction compared to the simultaneous intake group, the group ingested after drinking alcohol, and the positive control group. This indicates that ingesting lycopene encapsulated in PG-Zein / Lyc nanoparticles before drinking alcohol can more effectively inhibit lipid peroxidation, reduce malondialdehyde (MDA) production, and alleviate oxidative damage to cell membranes.

[0159] The experimental results show that the protective effect of PG-Zein / Lyc nanoparticles against alcoholic liver injury differs significantly depending on the timing of administration before and after alcohol consumption. Overall, the intervention effect is best when administered 1 hour before alcohol consumption, with better improvement in various indicators than simultaneous administration or administration after alcohol consumption. The improvement effect on some indicators is even better than that of the positive control drug, Kexilai. This invention, by comparing the intervention effect of the timing of PG-Zein / Lyc nanoparticle intake on alcoholic liver injury, not only clarifies the improvement effect of PG-Zein / Lyc nanoparticles on alcoholic liver injury but also determines the optimal administration time, providing a clear application basis for the prevention of alcoholic liver injury using PG-Zein-encapsulated lycopene.

[0160] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for preparing peach gum-zein-based lycopene nanoparticles, characterized in that, The steps include the following: Preparation of S1 gum-zein covalent graft A zein-ethanol solution was prepared by dissolving zein in ethanol solution, and a gum arabic solution was prepared by dissolving gum arabic in deionized water. The two solutions were mixed, stirred evenly, and the pH was adjusted to 7.5-8.

5. The mixture was then heated at 40-70℃ to obtain a gum arabic-zein covalently grafted product. The mass ratio of zein to gum arabic was 1:1-2.

5. Preparation of S2 organic phase Using 70%~80% ethanol as solvent, the gum-zein covalent graft obtained by S1 was used as a carrier, and medium-chain triglycerides were used as the oil phase to encapsulate lycopene. The mixture was homogenized and dispersed evenly to obtain the organic phase. Lycopene accounts for 2% to 10% of the zein protein mass, the mass ratio of gum gum-zein covalent graft to lycopene is 5 to 15: 20 to 200, and medium-chain triglycerides account for 5% to 8% of the organic phase volume; S3 crosslinking to prepare nanoparticles The organic phase of S2 was slowly added dropwise into water, and genipin solution was added for cross-linking in the dark. After the cross-linking reaction was completed, the mixture was centrifuged, the supernatant was discarded, and the resulting product was washed and freeze-dried to obtain peach gum-zein-based lycopene nanoparticles.

2. The preparation method according to claim 1, characterized in that, The volume fraction of the ethanol solution described in S1 is 70%~80%.

3. The preparation method according to claim 1, characterized in that, The heating temperature described in S1 is 50~60℃.

4. The preparation method according to claim 1, characterized in that, In S2, lycopene accounts for 2% to 6% of the zein mass, the mass ratio of gum gum-zein covalent graft to lycopene is 8 to 12: 20 to 60, and medium-chain triglycerides account for 5% to 7% of the organic phase volume.

5. The preparation method according to claim 1, characterized in that, The volume ratio of the organic phase to water in S3 is 1~5:1, the molar ratio of genipin to graft is 1:10~40, the crosslinking reaction temperature is 25~45℃, and the reaction time is 2~5 h.

6. A pharmaceutical composition for preventing or alleviating alcoholic liver injury, characterized in that, It includes gum-zein-based lycopene nanoparticles prepared by the preparation method according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further includes conventional pharmaceutical excipients, including any one of sweeteners, excipients, fillers, and pH adjusters.

8. The use of the gum-zein-based lycopene nanoparticles prepared by the method of claim 1 in the preparation of any one of the following products: health products, dietary supplements, or drugs for preventing or alleviating alcoholic liver damage.

9. The application as described in claim 8, characterized in that, The health products, dietary supplements, or drugs mentioned are taken before drinking alcohol.

10. The application as described in claim 9, characterized in that, The health products, dietary supplements, or drugs mentioned are to be taken 1 to 5 hours before drinking alcohol.

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